Synthesis and biological evaluation of novel tacrine derivatives and tacrine-coumarin hybrids as cholinesterase inhibitors

Article abstract of DOI:10.1021/jm5008648

A series of novel tacrine derivatives and tacrine-coumarin heterodimers were designed, synthesized, and biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Of these compounds,

Full text of DOI:10.1021/jm5008648

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Novel Tacrine Derivatives and
Tacrine−Coumarin Hybrids as Cholinesterase Inhibitors
Slavka Hamulakova,† Ladislav Janovec,† Martina Hrabinova,‡ Katarina Spilovska,†,∥ Jan Korabecny,§,∥
Pavol Kristian,† Kamil Kuca,*,‡,§ and Jan Imrich†
†Institute of Chemistry, Faculty of Science, P. J. Safarik University, SK-041 67 Kosice, Slovak Republic
‡Center for Advanced Studies, Faculty of Military Health Sciences, University of Defence, CZ-500 01 Hradec Kralove, Czech
Republic
§Center for Biomedical Research, University Hospital, CZ-500 05 Hradec Kralove, Czech Republic
∥Department of Toxicology, Faculty of Military Health Sciences, University of Defence, CZ-500 01 Hradec Kralove, Czech Republic
S
* Supporting Information
ABSTRACT: A series of novel tacrine derivatives and tacrine−coumarin heterodimers were designed, synthesized, and
biologically evaluated for their potential inhibitory effect on both acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE). Of these compounds, tacrine−coumarin heterodimer 7c and tacrine derivative 6b were found to be the most potent
inhibitors of human AChE (hAChE), demonstrating IC50 values of 0.0154 and 0.0263 μM. Ligands 6b, 6c, and 7c exhibited the
highest levels of inhibitory activity against human BuChE (hBuChE), demonstrating IC50 values that range from 0.228 to 0.328
μM. Docking studies were performed in order to predict the binding modes of compounds 6b and 7c with hAChE/hBuChE.
penetration into the central nervous system (CNS), are the
structure, lipophilicity, molecular weight, and the presence of
charge in AChEIs.6 The penetration into CNS is typically
confirmed by their therapeutic effect (improved cognitive and
memory functions, improved behavioral deficits) or by their
potency to inhibit cholinesterase in the brain.7 Therefore, all
drugs that were used successfully in the therapy of AD should
be considered as CNS+ targeted.6 In recent years, major
research efforts have been devoted to the development of
compounds that are able to bind simultaneously to both the
peripheral anionic site (PAS) and the catalytic anionic site
(CAS) of the enzyme. The neuroprotective effect of the
compounds that have resulted from these studies is based on
their ability to react with the peripheral site and to block the
interaction of AChE with Aβ (β-amyloid peptide).8,9
Compounds that demonstrate a dual binding affinity with
INTRODUCTION
■
Alzheimer’s disease (AD) is a neurodegenerative disorder that
results in the progressive and irreversible loss of higher brain
functions, including memory, cognition, and reason.1−3 The
pathological hallmarks of AD include widespread neuronal
degeneration, neuritic plaques containing β-amyloid (Aβ), and
τ-rich neurofibrillary tangles (NFT).4 Currently, most ther-
apeutic treatments for AD are drugs that aim to inhibit enzyme
acetylcholinesterase (AChE), thereby increasing acetylcholine
(ACh) concentration in cholinergic synaptic clefts.5 The only
two classes of drugs currently available for AD treatment are
the group of acetylcholinesterase inhibitors (AChEIs) (tacrine
(Figure 1), donepezil, rivastigmine, galantamine) and the N-
methyl-D-aspartate receptor (NMDAR) antagonist memantine,
although it is important to note that the inhibition of the
catalytic activity of AChE is not the only mechanism that is
responsible for the neuroprotective effect of AChEIs.5 The
effectiveness of ChE inhibitors in AD treatment is limited by
their ability to penetrate through the blood−brain barrier
(BBB).6 The important factors, which may influence passive
Received: June 8, 2014
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of tacrine, 7-MEOTA, ensaculine, coumarin derivatives A, B, tacrine−quinoline heterodimers C, and tacrine−4-oxo-
4H-chromene hybrids D.
AChE have also been used in the development of a new type of
therapeutic agent that prevents Aβ aggregation.10,11
been reported to possess a wide variety of biological effects
such as antioxidant,25,26 antimicrobial,27 antifugal,28 anticoagu-
lant,29 anti-inflammatory,30−32 anticancer,33 antitubercular34
activities. Recent studies have also shown that coumarin
analogues exhibit potent AChE activity, a finding that has led
to these compounds being seen as potential drugs in the
treatment of AD.35−37 Many compounds containing a
coumarin scaffold, such as ensaculin (Figure 1), have been
successfully used as AChEIs.38,39
While there is evidence of stable or slowly falling levels of
AChE activity in the brains of patients with AD, slight increases
in BuChE activity have also been reported. Both enzymes
would therefore appear to be suitable targets for the
development of ChE inhibitors in the treatment of AD.12
These two enzymes differ in terms of substrate specificity and
kinetics and also show varying levels of activity in different
regions of the brain.13a−c The two molecules are also
structurally different; the three aromatic residues present in
the PAS of AChE are absent from the PAS of BuChE. One
result of this is that typical PAS ligands exhibit a weaker affinity
toward BuChE than to AChE.14
Tacrine (1,2,3,4-tetrahydroacridine, Figure 1) is noncompe-
titive reversible acetyl/butyrylcholinesterase inhibitor that
selectively binds to the catalytic side of AChE. The main
issue associated with the use of tacrine was the significant side
effects, in particular the hepatotoxicity and cholinergic effects
upon the gastrointestinal tract.15,16 Tacrine is not effective in all
cases of AD because its metabolization to different hydroxy
metabolites depends on individual activities of the cytochrome
P450 isozyme family;17 some of these are pharmacologically
active but are also slightly toxic. In order to find compounds
with reduced side effects, a series of THA derivatives and
analogues was synthesized. 7-MEOTA (9-amino-7-methoxy-
1,2,3,4-tetrahydroacridine, Figure 1) was found to be a potent
and less toxic ChEI that is free of the serious side effects related
to tacrine.15
Zhou et al. have designed and synthesized a series of
ensaculin analogues with phenylpiperazine functional groups38
at position 3, 4, or 6 (Figure 1, coumarin derivatives B). The
analogues substituted at position 3 and/or position 4 of the
coumarin ring demonstrated higher levels of anti-AChEI
activity than 6-substituted coumarins (IC50 of 6.7−9.3 and
4.5−7.9 μM). Piazzi et al. had earlier synthesized a series of
coumarin hybrids with halophenylalkylamidic functional groups
at position 6 or 7 of the coumarin moiety (Figure 1, coumarin
derivatives A) and reported their high potential as multipotent
anti-AD drug candidates. These compounds were found to
exhibit considerable acetylcholinesterase inhibitory activity
(IC50 = 0.181−0.551 μM) and BACE-1 inhibitory activity
(IC50 = 0.099−0.151 μM).35
́
Fernandez-Bachiller et al. recently designed and synthesized
a series of novel multifunctional compounds with antioxidant
and metal-binding properties which also demonstrated an
inhibitory effect on Aβ aggregation and the dual inhibition of
AChE and BuChE40,41 (Figure 1). These tacrine−8-hydrox-
yquinoline (Figure 1, derivatives C) and tacrine−4-oxo-4H-
chromene hybrids (Figure 1, derivatives D) showed excellent
inhibitory activity against hAChE and hBuChE and β-secretase
1 (BACE-1) at both nano and picomolar concentrations.
On the basis of previous work published by Rodriguez-
Franco’s group in the field of AD,40,41 Spuch et al. investigated
the neurotoxic effects of a new tacrine−melatonin hybrid, N-(2-
More importantly, tacrine actually prevents the aggegation
and deposition of Aβ-amyloid plaques, an ability that is related
to its interaction with the PAS of AChE.18,19 The development
of tacrine based dimers and hybrids with improved
pharmacological properties has been the focus of a great deal
of research in recent years.20−24 Coumarin derivatives have
B
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Figure 2. Design strategy for heterodimers 7, 11, and 12.
a
Scheme 1. Synthesis of Tacrine Derivatives 4a−c, 5a−c, 6a−c
a
Reagents and conditions: (i) diaminoalkane (n = 2−4), phenol, reflux, 2 h; (ii) CPC, Et3N, CH2Cl2, 0 °C, 2 h; (iii) secondary amine, DIPEA,
CH3CN, rt, 4 h.
(1H-indol-3-yl)ethyl)-7-(1,2,3,4-tetrahydroacridin-9-ylamino)-
heptanamide.42 This new hybrid is a potent inhibitor of hAChE
and shows a high oxygen radical absorbance capacity which has
been proven to cause a reduction in Aβ deposits.42 Antequera
et al.43 investigated the modulatin effect of a multifunctional
tacrine−8-hydroxyquinoline hybrid (IQM-622) with evidence
of cholinergic, antioxidant, coopper-complexing, and neuro-
protective properties. The authors found that the IQM-622
hybrid controls pathological processes at the cellular and
neuronal level and holds considerable potential for the
treatment of AD-associated brain damage.
order to capitalize on the ChE inhibitory qualities of the former
at the CAS, and the aromatic characteristics of the latter with
AChE at the PAS. On the basis of our knowledge of the well-
known structure of AChE, we decided to connect the tacrine
and coumarin fragments using alkylenediamine tethers of
different lengths, thiosemicarbazides tethers, and linkers with
thiazolidinone heterocycle (Figure 2). Such linkers could be
lodged in the narrow enzymatic cavity, thereby allowing
simultaneous interaction between the heteroaromatic fragments
and both the CAS and PAS of AChE.
As a continuation of our previous research,44,45 this study
describes the synthesis, docking studies, and biological
evaluation of a series of novel tacrine derivatives and tacrine−
coumarin hybrids. The aim of the research presented in this
study was to combine tacrine with the coumarin scaffold in
RESULTS AND DISCUSSION
■
Synthesis of Tacrine Derivatives and Tacrine−
Coumarin Hybrids. Two starting compounds, 9-chloro-
1,2,3,4-tetrahydroacridine46 (1) and (7-hydroxy-2-oxo-2H-
chromen-4-yl)acetic acid (2), were employed in the synthesis
C
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a
Scheme 2. Synthesis of Tacrine−Coumarin Heterodimers 7a−c
a
Reagents and conditions: (i) (n = 2−4), CDI, CH2Cl2, rt, 24 h.
a
Scheme 3. Synthesis of Tacrine−Coumarin Hybrids 11 and 12
a
Reagents and conditions: (i) AgSCN, toluene, 125−130 °C, 6 h; (ii) CH3COCl, methanol, 2 h; (iii) NH2−NH2·H2O, dry ethanol, reflux, 24 h; (iv)
CH3CH2OH, rt, 24 h; (v) BrCH2COOCH3, TEA, CHCl3, 25 h, rt.
of desired derivatives, the synthetic route of which is outlined in
Schemes 1−3. The reaction of 9-chloro-1,2,3,4-tetrahydroacri-
dine (1) with ethylene-, propane-, and butane-1,4-diamine,
respectively, in phenol47 produced three distinct intermediate
compounds, N-(1,2,3,4-tetrahydroacridin-9-yl)alkanediamines
3a−c. These derivatives were then reacted with 3-chloropro-
pionyl chloride (CPC)48,49 to produce the halogenated
intermediates chloropropionamides 4a−c (Scheme 1). A
further reaction of chloropropionamides 4a−c with secondary
amines (methylcyclohexylamine/ethylcyclohexylamine) in the
presence of N,N-diisopropylethylamine (DIPEA) in CH3CN
produced the target compounds: derivatives 5a−c and 6a−c
(Scheme 1).
(7-Hydroxy-2-oxo-2H-chromen-4-yl)acetic acid (2) was
prepared by condensing resorcinol with citric acid in the
presence of concentrated sulfuric acid following a previously
published procedure.50 The reaction between (7-hydroxy-2-
oxo-2H-chromen-4-yl)acetic acid (2) and synthons 3a−c,
which had been obtained in the earlier process, in the presence
of 1,1′-carbonyldiimidazole (CDI) in CH2Cl2 for 24 h resulted
in the synthesis of the target compounds: novel tacrine−
coumarin heterodimers 7a−c with extended alkyl linkers
(Scheme 2).
In order to synthesize tacrine−coumarin hybrids 11 and 12,
(7-hydroxy-2-oxo-2H-chromen-4-yl)acetic acid methyl ester
(9) and (7-hydroxy-2-oxo-2H-chromen-4-yl)acetic acid hydra-
zide (10) were first prepared according to a previously
published method.51
The reaction of hydrazide 10 with 9-isothiocyanato-1,2,3,4-
tetrahydroacridine (8) in ethanol at room temperature
produced N-[2-(1,2,3,4-tetrahydroacridin-9-yl)-2-[2-(7-hy-
droxy-2-oxo-2H-chromen-4-yl)acetyl]-1-hydrazinecarbothioa-
mide (11). The cyclization of thiosemicarbazide 11 with methyl
bromoacetate in CHCl3 in the presence of TEA resulted in
thiazolidinone derivative 12 at a 70% yield in the form of a
yellow powder (Scheme 3).
Inhibition of Human AChE and BuChE. The extent of
inhibition was expressed as the chemical concentration at which
50% of enzyme activity was inhibited (IC50). The IC50 values of
compounds 5a−c, 6a−c, 7a−c, 11, and 12 were determined
against human erythrocytal AChE (hAChE, EC 3.1.1.7) and
human plasmatic butyrylcholinesterase (hBuChE, E.C. 3.1.1.8)
using the method of Ellman et al.52 The IC50 values and
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Table 1. In Vitro hAChE and hBuChE Inhibitory Activity and Selectivity Index of Tacrine Derivatives, 5a−c, 6a−c, and
Tacrine−Coumarin Heterodimers 7a−c, 11, and 12
a
b
c
compd
5a
X
R1/R2
n
IC50 SD (μM) hAChE
IC50 SD (μM) hBuChE
SI
CO(CH2)2
CO(CH2)2
CO(CH2)2
CO(CH2)2
CO(CH2)2
CO(CH2)2
COCH2
Me/Cy
Me/Cy
Me/Cy
Et/Cy
Et/Cy
Et/Cy
2
3
4
2
3
4
2
3
4
0.58 0.26
0.398 0.40
0.104 0.09
0.233 0.19
0.0263 0.22
0.0942 0.11
2.91 0.20
0.277 0.27
0.0154 0.17
8.17 1.63
15.7 3.14
0.5 0.1
2.03 0.14
2.38 0.29
2.23 0.04
0.765 0.82
0.267 0.34
0.228 0.11
12.6 0.11
0.827 0.77
0.328 0.21
1.42 0.24
5.62 0.97
0.023 0.004
21 3.4
3.50
5b
5.98
5c
21.40
3.28
6a
6b
10.15
2.42
6c
7a
4.33
7b
COCH2
2.99
7c
COCH2
21.30
0.174
0.358
0,046
1,4
11
12
tacrine
7-MEOTA
15 2.9
a
b
IC50: 50% inhibitory concentration (mean SD of three experiments) of AChE. IC50: 50% inhibitory concentration (mean SD of three
c
experiments) of BuChE. Selectivity index for AChE is defined as IC50(hBuChE)/IC50(hAChE).
selectivity indices (SI) of tacrine derivatives 5a−c and 6a−c,
tacrine−coumarin heterodimers 7a−c, 11, 12 and the control
compounds of tacrine and 7-MEOTA53 are summarized in
Table 1. The synthesized derivatives demonstrated inhibitory
activity against both hAChE and hBuChE with IC50 values
ranging from micromolar to submicromolar concentrations.
The results listed in Table 1 clearly show that the variation of
the alkyl chain length influences both the anti-AChE and the
anti-BuChE activities of the compounds.
Tacrine derivatives 5a−c and 6a−c were found to be
between 3 and 530 times more potent than thiosemicarbazide
11 and between 6 and 1000 times more potent than tacrine−
coumarin heterodimer 12. The best selectivity for hAChE was
demonstrated by compounds 5c and 7c (SI = 21.40 and 21.30)
and for hBuChE by compound 11 (SI = 0.174). The inhibition
type was determined for selected ligands 6b, 6c, 7c, 11, and 12
using the Lineweaver−Burk plot. The Lineweaver−Burk plot
revealed that ligands 6b, 6c, 7c noncompetitively inhibited
hAChE, and IC50 values of 0.0263, 0.0942, 0.00154 μM,
respectively, were recorded (Table 1). Tacrine−coumarin
heterodimers 11, 12 were competitive inhibitors, showing
IC50 values of 8.17 and 15.7 μM.
The IC50 values of the compounds suggest that the inhibitory
effect of tacrine derivatives 5a−c, 6a−c and tacrine−coumarin
heterodimers 7a−c was far stronger on AChE than on BuChE.
Among these compounds, tacrine−coumarin heterodimer 7c
displayed excellent inhibition of hAChE; the IC50 value of
0.0154 μM is approximately 32 times higher than that of tacrine
and 974 times higher than that of 7-MEOTA. The most potent
inhibitor of hBuChE, tacrine derivative 6c, showed an IC50
value of 0.228 μM; however, while this value is approximately
92 times higher than 7-MEOTA, it is still around 10 times less
effective than tacrine itself. Compounds 5c, 6c, and 7c with
four methylene groups between the tacrine and amide group,
and compound 6b with three methylene groups were the best
inhibitors of AChE in their series. Derivatives 6a−c and 7b,c
demonstrated a similar trend in the inhibition of BuChE. From
these results it can be seen that the length of the alkyl spacer
and the amine moiety at the end of the chain have some
influence on the levels of inhibition. When the methyl group
(Me) in cyclohexylamine (5a−c) was replaced with an ethyl
group (Et) (6a−c), an increase in anti-AChE and anti-BuChE
activity was observed. When the alkyl chain was replaced with a
thiosemicarbazide linker 11 or thiazolidinone moiety 12, the
AChE inhibitory activity dramatically decreased (IC50 = 8.17,
15.7 μM).
The dissociation constants Ki1 of selected ligands, describing
the stability of the enzyme−inhibitor complex, are listed in
Table 2.
The Ki1 value of 0.0041 μM for ligand 7c indicates that this
compound demonstrates the strongest affinity toward hAChE, a
value that is 55 times higher than that of tacrine and 510 times
higher than that of 7-MEOTA.
Table 2. Dissociation Constants Ki1 and Ki2 of Some
Selected Ligands and Reference Compounds
a
b
compd
6b
Ki1 (μM)
Ki2 (μM)
0.0804
0.199
0.0041
1.94
0.0789
0.328
0.0234
92.8
6c
7c
11
12
2.36
65.3
tacrine
7-MEOTA
0.225
2.09
0.101
6.34
a
b
Ki1: dissociation constant for AChE−inhibitor complex. Ki2:
dissociation constant for AChE−inhibitor−substrate complex.
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Molecular Modeling. In order to reveal the possible
intermolecular interactions behind the inhibitory activities of
novel tacrine derivatives, a molecular modeling study was
carried out using docking programs AUTODOCK 4.2 and
DOCK 6.5. The results of the modeling are shown in Figures
2−5.
The first stage of the process was the localization of ligand
binding poses using Autodock. After a visual inspection of the
poses with the highest negative binding energy, the structures
that fill the catalytic cavity of the enzyme were chosen as the
inputs for Amber rescoring. This led to the next stage, the
redocking linked with Amber force field molecular dynamics.
The main advantage of Amber rescoring is that the positions of
both the ligand and the active site of the enzyme can be flexible,
allowing small structural rearrangements to reproduce the so-
called “induced fit”.
Information data, Figure S02). The existence of weak binding
interactions might also be proposed between the endocyclic
tacrine nitrogen and the amino acids of the inner part of the
catalytic cavity of the enzyme. The docking run of derivative 7c
also proposed plausible π−π stacking intermolecular inter-
actions within the Trp86−tacrine−Tyr337 and Trp286−
coumarine core (Figure 4).
Docking simulations were performed with the compounds
that had been found to be the most active against hAChE and
hBuChE, derivatives 6b and 7c (for more details see
Experimental Section).
In order to allow sufficient space sampling for each
derivative, all possible ligand structural combinations were
built into the simulation. R/S configurations on atoms of
nitrogen were combined with s-cis and s-trans conformations
on the amide −NH−CO− bond. The result of these molecular
combinations was a set of eight distinct structures for derivate
6b and four different structures for derivative 7c (see
Supporting Information data, Figure S01). All of these
structures were used as input geometries for docking
simulations in order to reveal all of the possible interactions
between the ligand and the active site of the enzyme. The
protonation level of the ligands in physiological pH was also
studied using the Marvin software pack. [http://www.
chemaxon.com]. Autodock results were visually evaluated,
and the top pose of the most negative cluster with a proper
catalytic cavity orientation was selected for rescoring in
AMBER molecular mechanics force field using DOCK
software. Results were summarized according to the binding
ability of the ligands.
Figure 4. Top-score docking pose of derivative 7c with molecular
surface depicting putative structural orientation in the active-site gorge
of the hAChE in a ribbon style.41
As is depicted in Figure 5, the ternary inhibition complex of
compound 6b with hBuChE shows evidence of direct
The pose of derivative 6b with the lowest binding energy
toward hAChE is depicted in Figure 3. Plausible π−π binding
interactions might be found in the CAS of the enzyme between
the tacrine core and the aromatic residue of Trp86. More
complex stabilization might result from the hydrogen bonds
between the ligand spacer and Tyr337, Tyr124 (see Supporting
Figure 5. Top-score docking pose of derivative 6b with molecular
surface depicting putative structural orientation in the active-site gorge
of the hBuChE in a ribbon style.41
interaction with the catalytic triad via His438. Additional
hydrogen bonds formed between the amino acids of the
catalytic cavity and derivative 6b are depicted in Figure S04
(see Supporting Information). The binding of compound 7c
within the catalytic site of hBuChE is proposed in the ligand’s
folded state locked by the intramolecular interaction of the
coumarine carbonyl oxygen and tacrine endocyclic nitrogen
(Figure 6). It is possible to suggest that derivative 7c could
interact with enzyme via the hydrogen bond with carbonyl
group and also through the histidine core of His438 (see
Supporting Information, Figure S05).
CONCLUSION
■
This study describes the synthesis and biological evaluation of a
series of new tacrine derivatives (5a−c, 6a−c) and tacrine−
coumarin heterodimers (7a−c, 11, 12). Some of the studied
compounds demonstrated higher levels of inhibition of AChE
and BuChE in comparison to those of the control compounds
Figure 3. Top-score docking pose of derivative 6b with molecular
surface depicting putative structural orientation in the active-site gorge
of the hAChE in a ribbon style.41
F
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2.56 (t, 2H, CH2, H-5′, J = 6.2 Hz), 2.62−2.74 (m, 2H, CH2, H-1),
2.95−3.07 (m, 2H, CH2, H-4), 3.47 (t, 2H, CH2, H-2′, J = 5.6 Hz),
3.75 (t, 2H, CH2, H-6′, J = 6.0 Hz), 3.97 (t, 2H, CH2, H-1′, J = 5.6
Hz), 7.55 (dd, 1H, CH, H-7, J = 7.0, 8.4 Hz), 7.85 (dd, 1H, CH, H-6, J
= 6.8, 8.4 Hz), 7.95 (d, 1H, CH, H-5, J = 8.4 Hz), 8.46 (d, 1H, CH, H-
8, J = 8.8 Hz). 13C NMR (DMSO-d6) δ 20.2, 21.4 (C-2,3), 23.6 (C-1),
28.3 (C-4), 37.9 (C-5′), 39.8 (C-2′), 40.8 (C-6′), 47.8 (C-1′), 112.3
(C-9a), 115.3 (C-8a), 119.0 (C-5), 125.0 (C-7), 125.2 (C-8), 132.5
(C-6), 139.9 (C-10a), 151.4 (C-9), 155.2 (C-4a), 170.3 (C-4′). Anal.
Calcd for C18H22N3O (331.85): C, 65.15; H, 6.68; N, 12.66. Found:
C, 65.12; H, 6.63; N, 12.64.
N1-[3-(1,2,3,4-Tetrahydroacridin-9-ylamino)propyl]-3-chlor-
opropanamide (4b). Compound 3b was treated with chloropro-
pionyl chloride according to a commonly used procedure to give the
1
desired product 4b in the form of a yellow oil (47%). H NMR (400
Figure 6. Top-score docking pose of derivative 7c with molecular
surface depicting putative structural orientation in the active-site gorge
of the hBuChE in a ribbon style.41
MHz, DMSO-d6) δ 1.75−1.93 (m, 6H, 3 × CH2, H-2,3,2′), 2.57 (t,
2H, CH2, H-6′, J = 6.4 Hz), 2.65−2.74 (m, 2H, CH2, H-1), 2.96−3.03
(m, 2H, CH2, H-4), 3.13−3.24 (m, 2H, CH2, H-3′), 3.72−3.88 (m,
2H, 2 × CH2, H-1′,7′), 7.53 (ddd, 1H, CH, H-7, J = 1.2, 6.8, 8.4 Hz),
7.81 (dd, 1H, CH, H-6, J = 6.8, 8.4 Hz), 7.89 (dd, 1H, CH, H-5, J =
1.2, 8.8 Hz), 8.36 (d, 1H, CH, H-8, J = 8.8 Hz). 13C NMR (DMSO-d6)
δ 20.5, 21.6 (C-2,3), 24.0 (C-1), 28.8 (C-4), 30.0 (C-2′), 35.6 (C-3′),
38.7 (C-6′), 41.0 (C-7′), 44.6 (C-1′), 112.0 (C-9a), 116.2 (C-8a),
124.5 (C-8), 124.7 (C-7), 120.6 (C-5), 131.7 (C-6), 139.3 (C-10a),
151.8 (C-9), 154.7 (C-4a), 169.3 (C-5′). Anal. Calcd for C19H24N3O
(345.88): C, 65.98; H, 6.99; N, 12.15. Found: C, 65.95; H, 6.96; N,
12.12.
of tacrine 1 and 7-MEOTA. The most potent inhibitors of
hAChE were compounds 7c and 6b which showed IC50 values
of 0.0154 and 0.0263 μM, while compounds 6c, 6b, and 7c
demonstrated the highest efficiency against hBuChE with a
range of IC50 values from 0.228 to 0.328 μM. The highest
selectivity indices were found for compounds 5c and 7c (SI =
21.40, 21.30) and 11 (SI = 0.174). From the results, it can be
concluded that the length of the alkyl spacer and the amine
moiety at the end of the chain have a considerable effect on the
inhibitory effect of the compounds. There was a clear decrease
in inhibitory activity when the alkyl chain was replaced with a
thiosemicarbazide linker or thiazolidinone moiety. Molecular
modeling studies confirmed that these hybrids target both the
CAS and PAS of AChE.
N1-[4-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl]-3-chloro-
propanamide (4c). Compound 3c was treated with chloropropionyl
chloride according to a commonly used procedure to give the desired
1
product 4c in the form of a yellow oil (45%); H NMR (400 MHz,
DMSO-d6) δ 1.41−1.52 (m, 2H, CH2, H-3′), 1.62−1.72 (m, 2H, CH2,
H-2′), 1.79−1.88 (m, 4H, 2 × CH2, H-2,3), 2.50−2.60 (m, 2H, CH2,
H-7′), 2.63−2.72 (m, 2H, CH2, H-1), 2.96−3.02 (m, 2H, CH2, H-4),
3.03−3.11 (m, 2H, CH2, H-4′), 3.62−3.84 (m, 4H, 2 × CH2, H-1′,8′),
7.49 (dd, 1H, CH, H-7, J = 7.0, 8.2 Hz), 7.74 (dd, 1H, CH, H-6, J =
6.8, 8.4 Hz), 7.85 (d, 1H, CH, H-5, J = 8.4 Hz), 8.31 (d, 1H, CH, H-8,
J = 8.4 Hz). 13C NMR (DMSO-d6) δ 20.8, 21.7 (C-2,3), 24.1 (C-1),
26.1 (C-3′), 27.3 (C-2′), 29.6 (C-4), 37.9 (C-4′), 38.1 (C-7′), 40.9
(C-8′), 46.9 (C-1′), 112.5 (C-9a), 116.9 (C-8a), 122.0 (C-5), 124.2
(C-8), 124.3 (C-7), 130.8 (C-6), 140.2 (C-10a), 151.8 (C-9), 154.7
(C-4a), 169.6 (C-6′). Anal. Calcd for C20H26N3O (359.90): C, 66.75;
H, 7.28; N, 11.68. Found: C, 66.73; H, 7.25; N, 11.63.
General Procedure for the Synthesis of N1-[n-(1,2,3,4-
Tetrahydroacridin-9-ylamino)alkyl]-3-[cyclohexyl(methyl)-
amino]propanamides 5a−c. A mixture of corresponding N1-[n-
(1,2,3,4-tetrahydroacridin-9-ylamino)alkyl]-3-chloropropanamide (4,
0.17 mM), N-methylcyclohexylamine (0.022 ml, 0.17 mM), and
N,N-diisopropylethylamine (0.04 mL, 0.231 mM) was stirred under
N2 in acetonitrile (2 mL) for 4 h at room temperature. The solvent
was removed under reduced pressure. CH2Cl2 (1 mL) and water (1
mL) were added to the crude product. The water layer was extracted
with CH2Cl2 (3 × 1 mL). The combined organic extracts were dried
over MgSO4. The residue was purified using column chromatography,
eluent CH2Cl2−MeOH (4:1).
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)ethyl]-3-
[cyclohexyl(methyl)amino]propanamide (5a). Compound 4a was
treated with methylcyclohexylamine according to a commonly used
procedure to give the desired product 5a in the form of a yellow oil
(77%). 1H NMR (400 MHz, CDCl3) δ 1.16−1.36 (m, 10H, 5 × CH2,
H-9′,10′,11′,12′,13′), 1.87−1.97 (m, 4H, 2 × CH2, H-2,3), 2.30 (s,
3H, CH3, H-1″), 2.40−2.50 (m, 1H, CH, H-8′), 2.62 (t, 2H, CH2, H-
5′, J = 6.4 Hz), 2.69−2.78 (m, 2H, CH2, H-1), 3.03−3.10 (m, 2H,
CH2, H-4), 3.59 (t, 2H, CH2, H-2′, J = 5.6 Hz), 3.62−3.72 (m, 2H,
CH2, H-1′), 3.82 (t, 2H, CH2, H-6′, J = 6.4 Hz), 7.36 (ddd, 1H, CH,
H-7, J = 1.2, 7.2, 8.4 Hz), 7.55 (ddd, 1H, CH, H-6, J = 1.2, 6.8, 8.0
Hz), 7.89−7.96 (m, 2H, 2 × CH, H-5,8). 13C NMR (CDCl3) δ 22.7,
23.0 (C-2,3), 25.1 (C-1), 24.8 (C-10′,11′,12′) 29.7 (C-9′,13′), 33.9
(C-4), 37.8 (C-1″), 39.6 (C-5′), 40.1 (C-6′), 40.8 (C-2′), 49.1 (C-1′),
58.5 (C-8′), 116.1 (C-9a), 120.3 (C-8a), 122.8 (C-8), 124.0 (C-7),
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All solvents, chemicals, and
reagents were obtained commercially and used without purification.
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Varian Mercury Plus NMR spectrometer using CDCl3
or DMSO-d6 as solvents with tetramethylsilane as an internal standard.
Chemical shifts, δ, are given in parts per million (ppm), and spin
multiplicities are given as s (singlet), br s (broad singlet), d (doublet),
t (triplet), q (quartet), or m (multiplet). Coupling constants, J, are
expressed in hertz (Hz). Thin-layer chromatography was performed
on Macherey-Nagel Alugram Sil G/UV254 plates, and spots were
visualized with UV light. Chromatographic separations were
performed on silica gel 60 (0.063−0.040 mm, Merck) column
chromatography. Melting points were recorded on a Boetius hot-plate
apparatus and are uncorrected. Yields refer to isolated pure products
and were not maximized. CHN analysis was performed on a CHN
analyzer PerkinElmer 2400.
General Procedure for the Synthesis of N1-[n-(1,2,3,4-
Tetrahydroacridin-9-ylamino)alkyl]-3-chloropropanamides
4a−c. A solution of corresponding N1-(1,2,3,4-tetrahydroacridin-9-
yl)diamine (3, 0.74 mM) in anhydrous CH2Cl2 (4 mL) and Et3N
(0.74 mM) was added dropwise to a solution of chloropropionyl
chloride (0.07 mL, 0.74 mM) in anhydrous CH2Cl2 (2 mL) over 40
min at −5 °C. The mixture was stirred for 30 min at 0 °C and for 1 h
at room temperature. After completion of the reaction, the residue was
washed with 5% aqueous Na2CO3 solution (2 mL). The organic layer
was separated and dried over Na2SO4. Removal of the solvents
produced a residue which was purified using column chromatography,
eluent CH2Cl2−MeOH (4:1).
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)ethyl]-3-chloro-
propanamide (4a). Compound 3a was treated with chloropropionyl
chloride according to a commonly used procedure to give the desired
1
product 4a in the form of a yellow solid (42%). Mp 87−92 °C; H
NMR (400 MHz, DMSO-d6) δ 1.74−1.90 (m, 4H, 2 × CH2, H-2,3),
G
dx.doi.org/10.1021/jm5008648 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
127.3 (C-5), 128.5 (C-6), 147.0 (C-10a), 152.2 (C-9), 158.8 (C-4a),
169.3 (C-4′). Anal. Calcd for C25H36N4O (408.59): C, 73.49; H, 8.88;
N, 13.71. Found: C, 73.50; H, 8.85; N, 13.68.
C26H38N4O (422.62): C, 73.89; H, 9.06; N, 13.26. Found: C, 73.86;
H, 9.01; N, 13.23.
N1-[3-(1,2,3,4-Tetrahydroacridin-9-ylamino)propyl]-3-
[cyclohexyl(ethyl)amino]propanamide (6b). Compound 4b was
treated with ethylcyclohexylamine according to a commonly used
procedure to give the desired product 6b in the form of a yellow oil
(50%). 1H NMR (400 MHz, CDCl3) δ 1.08−1.33 (m, 13H, 5 × CH2,
CH3, H-10′,11′,12′,13′,14′,2″), 1.78−1.85 (m, 2H, CH2, H-2′), 1.87−
1.96 (m, 4H, 2 × CH2, H-2,3), 2.44−2.55 (m, 1H, CH, H-9′), 2.63−
2.81 (m, 6H, 3 × CH2, H-1,6′,1″), 3.01−3.12 (m, 2H, CH2, H-4),
3.41−3.50 (m, 2H, CH2, H-3′), 3.50−3.57 (m, 2H, CH2, H-1′), 3.83
(t, 2H, CH2, H-7′, J = 6.4 Hz), 7.34 (ddd, 1H, CH, H-7, J = 1.2, 7.2,
8.4 Hz), 7.53 (ddd, 1H, CH, H-6, J = 1.2, 7.2, 8.4 Hz), 7.90 (d, 1H,
CH, H-5, J = 8.4 Hz), 8.03 (d, 1H, CH, H-8, J = 8.8 Hz). 13C NMR
(CDCl3) δ 15.1 (C-2″), 22.7, 23.1 (C-2,3), 25.1 (C-1,11′,13′), 26.1
(C-12′), 29.7 (C-10′,14′), 31.3 (C-2′), 33.7 (C-4), 36.7 (C-3′), 39.6
(C-6′), 40.3 (C-7′), 41.0 (C-1″), 45.2 (C-1′), 56.8 (C-9′), 116.4 (C-
9a), 120.3 (C-8a), 122.7 (C-8), 123.9 (C-7), 128.2 (C-5), 128.5 (C-6),
146.8 (C-10a), 150.9 (C-9), 158.2 (C-4a), 170.4 (C-5″). Anal. Calcd
for C27H40N4O (436.65): C, 74.27; H, 9.23; N, 12.83. Found: C,
74.26; H, 9.19; N, 12.79.
N1-[4-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl]-3-
[cyclohexyl(ethyl)amino]propanamide (6c). Compound 4c was
treated with ethylcyclohexylamine according to a commonly used
procedure to give the desired product 6c in the form of a yellow oil
(51%). 1H NMR (400 MHz, CDCl3) δ 1.07−1.36 (m, 13H, 5 × CH2,
CH3, H-11′,12′,13′,14′,15′,2″), 1.58−1.80 (m, 4H, 2 × CH2, H-2′,3′),
1.86−1.98 (m, 4H, 2 × CH2, H-2,3), 2.43−2.55 (m, 1H, CH, H-10′),
2.59 (t, 2H, CH2, H-7′, J = 6.4 Hz), 2.65−2.77 (m, 4H, 2 × CH2, H-
1,1″), 3.00−3.07 (m, 2H, CH2, H-4), 3.29−3.40 (m, 2H, CH2, H-4′),
3.45−3.52 (m, 2H, CH2, H-1′), 3.76−3.85 (m, 2H, CH2, H-8′), 7.34
(ddd, 1H, CH, H-7, J = 1.2, 6.8, 8.0 Hz), 7.53 (ddd, 1H, CH, H-6, J =
1.2, 6.8, 8.4 Hz), 7.90 (d, 1H, CH, H-5, J = 8.0 Hz), 7.98 (d, 1H, CH,
H-8, J = 7.6 Hz). 13C NMR (CDCl3) δ 15.2 (C-2″), 22.8, 23.1 (C-2,3),
25.1 (C-1,2′,12′,14′), 26.1 (C-13′), 27.2 (C-3′), 29.7 (C-11′,15′), 34.1
(C-4), 39.2 (C-4′), 39.6 (C-7′), 40.3 (C-8′), 40.9 (C-1″), 48.9 (C-1′),
56.8 (C-10′), 116.4 (C-9a), 120.4 (C-8a), 122.7 (C-8), 123.8 (C-7),
128.3 (C-6), 128.7 (C-5), 147.4 (C-10a), 150.5 (C-9), 158.6 (C-4a),
169.8 (C-6′). Anal. Calcd for C28H42N4O (450.67): C, 74.62; H, 9.39;
N, 12.43. Found: C, 74.59; H, 9.36; N, 12.39.
General Procedure for the Synthesis of N1-[n-(1,2,3,4-
tetrahydroacridin-9-ylamino)alkyl]-2-(7-hydroxy-2-oxo-2H-
chromen-4-yl)acetamides 7a−c. A mixture of (7-hydroxy-2-oxo-
2H-chromen-4-yl)acetic acid (2, 0.153 g, 0.7 mM) in anhydrous
CH2Cl2 (6 mL) and carbonyldiimidazole (0.124 g, 0.765 mmol) was
stirred for 1.5 h at room temperature. A solution of corresponding N-
(n-aminoalkyl)-N-(1,2,3,4-tetrahydroacridin-9-yl)amine (3, 0.7 mM)
in anhydrous CH2Cl2 (2 mL) was added. The mixture was stirred for
24 h. Water (6 mL) was added to the mixture. The organic layer was
separated, dried over Na2SO4, and the solvents were evaporated. The
residue was purified using column chromatography, eluent EtAC−
MeOH−NH3OH (6:2:0.2).
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)ethyl]-2-(7-hy-
droxy-2-oxo-2H-chromen-4-yl)acetamide (7a). Compound 3a
was treated with (7-hydroxy-2-oxo-2H-chromen-4-yl)acetic acid
according to a commonly used procedure to give the desired product
7a in the form of a yellow oil (34%). 1H NMR (400 MHz, DMSO-d6)
δ 1.80−1.92 (m, 4H, 2 × CH2, H-2,3), 2.80−2.84 (m, 2H, CH2, H-1),
3.02−3.08 (m, 2H, CH2, H-4), 3.45−3.65 (m, 4H, 2 × CH2, H-1′,2′),
3.73 (s, 2H, CH2, H-5′), 6.24 (s, 1H, CH, H-3″), 6.75 (s, 1H, CH, H-
8″), 6.81 (dd, 1H, CH, H-6″, J = 2.4, 8.8 Hz), 7.44−7.62 (m, 3H, 3 ×
CH, H-6,7,5″), 7.85 (d, 1H, CH, H-5, J = 8.4 Hz), 7.95 (d, 1H, CH,
H-8, J = 8.4 Hz). 13C NMR (DMSO-d6) δ 22.3 (C-2,3), 24.3 (C-1),
33.2 (C-4), 38.0 (C-5′), 52.1 (C-1′,2′), 102.3 (C-8″), 112.0 (C-
3″,4a″), 113.0 (C-6″), 115.7 (C-9a), 119.4 (C-8a), 122.6 (C-8), 124.2
(C-7), 126.6 (C-5″), 128.3 (C-5), 128.7 (C-6), 146.7 (C-10a), 149.5
(C-4″), 150.9 (C-9), 154.9 (C-8a″), 158.2 (C-4a), 160.1 (C-2″), 161.2
(C-7″), 171.7 (C-4′). Anal. Calcd for C26H25N3O4 (443.51): C, 70.41;
H, 5.68; N, 9.47. Found: C, 70.39; H, 5.65; N, 9.43.
N1-[3-(1,2,3,4-Tetrahydroacridin-9-ylamino)propyl]-3-
[cyclohexyl(methyl)amino]propanamide (5b). Compound 4b
was treated with methylcyclohexylamine according to a commonly
used procedure to give the desired product 5b in the form of a yellow
1
oil (59%). H NMR (400 MHz, CDCl3) δ 1.10−1.35 (m, 10H, 5 ×
CH2, H-10′,11′,12′,13′,14′), 1.80−2.03 (m, 9H, 3 × CH2, CH3, H-
2,3,1″,2′), 2.38−2.52 (m, 1H, CH, H-9′), 2.69 (t, 2H, CH2, H-6′, J =
6.0 Hz), 2.72−2.82 (m, 2H, CH2, H-1), 2.98−3.10 (m, 2H, CH2, H-
4), 3.40−3.50 (m, 2H, CH2, H-3′), 3.55−3.65 (m, 2H, CH2, H-1′),
3.82 (t, 2H, CH2, H-7′, J = 6.0 Hz), 7.35 (dd, 1H, CH, H-7, J = 7.2, 8.0
Hz), 7.52 (dd, 1H, CH, H-6, J = 7.2, 8.0 Hz), 7.91 (d, 1H, CH, H-5, J
= 8.0 Hz), 8.07 (d, 1H, CH, H-8, J = 8.8 Hz). 13C NMR (CDCl3) δ
22.4, 22.9 (C-2,3), 24.9, 25.0 (C-1,11′,13′), 25.9 (C-12′), 29.7 (C-
10′,14′), 31.2 (C-2′), 33.9 (C-4), 36.5 (C-3′), 37.8 (C-1″), 39.4 (C-
6′), 40.5 (C-7′), 44.9 (C-1′), 58.5 (C-9′), 115.6 (C-9a), 119.7 (C-8a),
123.0 (C-8), 124.1 (C-7), 126.9 (C-5), 129.0 (C-6), 145.7 (C-10a),
151.7 (C-9), 157.1 (C-4a), 170.6 (C-5′). Anal. Calcd for C26H38N4O
(422.62): C, 73.89; H, 9.06; N, 13.26. Found: C, 73.87; H, 9.03; N,
13.24.
N1-[4-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl]-3-
[cyclohexyl(methyl)amino]propanamide (5c). Compound 4c was
treated with methylcyclohexylamine according to a general procedure
1
to give the desired product 5c as yellow oil (50%). H NMR (400
MHz, CDCl3) δ 1.18−1.38 (m, 10H, 5 × CH2, H-11′,12′,13′,14′,15′),
1.60−1.68 (m, 2H, CH2, H-3′), 1.68−1.80 (m, 2H, CH2, H-2′), 1.82−
1.95 (m, 4H, 2 × CH2, H-2,3), 2.30 (s, 3H, CH3, H-1″), 2.47−2.50
(m, 1H, CH, H-10′), 2.62 (t, 2H, CH2, H-7′, J = 6.8 Hz), 2.63−2.72
(m, 2H, CH2, H-1), 3.00−3.08 (m, 2H, CH2, H-4), 3.26−3.37 (m, 2H,
CH2, H-4′), 3.53 (t, 2H, CH2, H-1′, J = 6.8 Hz), 3.80 (t, 2H, CH2, H-
8′, J = 6.4 Hz), 7.34 (dd, 1H, CH, H-7, J = 7.2, 8.0 Hz), 7.53 (dd, 1H,
CH, H-6, J = 7.2, 8.4 Hz), 7.92 (d, 1H, CH, H-5, J = 8.0 Hz), 7.96 (d,
1H, CH, H-8, J = 8.0 Hz). 13C NMR (CDCl3) δ 22.7, 22.9 (C-2,3),
24.8 (C-1,2′), 25.1 (C-12′, 14′), 27.1 (C-3′), 29.2 (C-13′), 31.3 (C-
11′,15′), 33.4 (C-4), 37.8 (C-1″), 39.2 (C-4′), 39.6 (C-7′), 40.4 (C-
8′), 48.8 (C-1′), 58.5 (C-10′), 115.8 (C-9a), 119.9 (C-8a), 122.9 (C-
8), 123.9 (C-7), 127.8 (C-5), 128.8 (C-6), 146.5 (C-10a), 151.1 (C-9),
157.8 (C-4a), 169.7 (C-6′). Anal. Calcd for C27H40N4O (436.65): C,
74.27; H, 9.23; N, 12.83. Found: C, 74.24; H, 9.21; N, 12.80.
General Procedure for the Synthesis of N1-[n-(1,2,3,4-
Tetrahydroacridin-9-ylamino)alkyl]-3-[cyclohexyl(ethyl)-
amino]propanamides 6a−c. A mixture of corresponding N1-[n-
(1,2,3,4-tetrahydroacridin-9-ylamino)alkyl]-3-chloropropanamide (4,
0.12 mM), N-ethylcyclohexylamine (0.018 mL, 0.12 mM), and N,N-
diisopropylethylamine (0.027 ml, 0.155 mM) was stirred under N2 in
acetonitrile (2 mL) for 4 h at room temperature. The solvent was
removed under reduced pressure. CH2Cl2 (1 mL) and water were
added to the crude product. The water layer was treated with CH2Cl2
(3 × 1 mL). The organic layer was dried over MgSO4. A residue was
purified using column chromatography, eluent CH2Cl2−MeOH (4:1).
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)ethyl]-3-
[cyclohexyl(ethyl)amino]propanamide (6a). Compound 4a was
treated with ethylcyclohexylamine according to a commonly used
procedure to give the desired product 6a in the form of a yellow oil
1
(62%). H NMR (400 MHz, CDCl3) δ 0.82−0.98 (m, 3H, CH3, H-
2″), 1.10−1.43 (m, 10H, 5 × CH2, H-9′,10′,11′,12′,13′), 1.83−1.97
(m, 4H, 2 × CH2, H-2,3), 2.42−2.60 (m, 1H, CH, H-8′), 2.63−2.72
(m, 2H, CH2, H-1), 2.75 (t, 2H, CH2, H-5′, J = 6.2 Hz), 2.80−2.88
(m, 2H, CH2, H-1″), 2.97−3.13 (m, 1H, CH, H-4), 3.70−3.78 (m,
2H, CH2, H-2′), 3.79−3.93 (m, 4H, 2 × CH2, H-1′,6′), 7.26 (dd, 1H,
CH, H-7, J = 7.2, 8.0 Hz), 7.47 (dd, 1H, CH, H-6, J = 7.2, 8.0 Hz),
7.96 (d, 1H, CH, H-5, J = 8.4 Hz), 8.02 (d, 1H, CH, H-8, J = 8.8 Hz).
13C NMR (CDCl3) δ 15.1 (C-2″), 22.5,23.1 (C-2,3), 25.0 (C-1), 25.6
(C-10′,11′,12′), 29.7 (C-9′,13′), 33.8 (C-4), 39.2 (C-5′), 40.2 (C-2′),
40.3 (C-6′), 41.0 (C-1″), 49.6 (C-1′), 56.7 (C-8′), 116.3 (C-9a), 120.4
(C-8a), 123.1 (C-8), 124.3 (C-7), 128.2 (C-5), 128.8 (C-6), 147.0 (C-
10a), 151.0 (C-9), 158.8 (C-4a), 169.5 (C-4′). Anal. Calcd for
H
dx.doi.org/10.1021/jm5008648 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)propyl]-2-(7-hy-
droxy-2-oxo-2H-chromen-4-yl)acetamide (7b). Compound 3b
was treated with (7-hydroxy-2-oxo-2H-chromen-4-yl)acetic acid
according to a commonly used procedure to give the desired product
7b in the form of a yellow oil (40%). 1H NMR (400 MHz, CDCl3) δ
1.77−2.0 (m, 6H, 3 × CH2, H-2,3,2′), 2.78−2.83 (m, 2H, CH2, H-1),
3.05−3.10 (m, 2H, CH2, H-4), 3.49−3.56 (m, 2H, CH2, H-1′), 3.56−
3.67 (m, 2H, CH2, H-3′), 3.72 (s, 2H, CH2, H-6′), 6.18 (s, 1H, CH,
H-3″), 6.75 (s, 1H, CH, H-8″), 6.83 (dd, 1H, CH, H-6″, J = 2.4, 8.8
Hz), 7.30−7.45 (m, 2H, 2 × CH, H-7,5″), 7.45−7.60 (m, 1H, CH, H-
6), 8.0 (d, 1H, CH, H-5, J = 8.4 Hz), 8.00 (d, 1H, CH, H-8, J = 8.0
Hz). 13C NMR (CDCl3) δ 22.2, 22.3 (C-2,3), 24.3 (C-1), 33.5 (C-4),
31.2 (C-2′), 37.9 (C-3′,6′), 47.6 (C-1′), 103.4 (C-8″), 111.5 (C-
3″,4a″), 114.1 (C-6″), 115.7 (C-9a), 120.4 (C-8a), 122.8 (C-8), 124.2
(C-7), 126.6 (C-5″), 128.4 (C-5), 129.1 (C-6), 146.9 (C-10a), 150.0
(C-4″), 151.9 (C-9), 155.2 (C-8a″), 157.2 (C-4a), 160.5 (C-2″), 161.5
(C-7″), 171.9 (C-5′). Anal. Calcd for C27H27N3O4 (457.53): C, 70.88;
H, 5.95; N, 9.18. Found: C, 70.86; H, 5.92; N, 9.17.
CH2, H-9′), 4.24 (s, 2H, CH2, H-5′), 6.35 (s, 1H, CH, H-3″), 6.45
(dd, 1H, CH, H-6″, J = 2.4, 8.8 Hz), 6.70 (s, 1H, CH, H-8″), 7.50−
7.57 (m, 1H, CH, H-7), 7.58−7.68 (m, 2H, 2 × CH, H-6,5″), 7.86 (d,
1H, CH, H-8, J = 8.8 Hz), 7.98 (d, 1H, CH, H-5, J = 8.0 Hz), 10.50 (s,
1H, NH). 13C NMR (DMSO-d6) δ 21.6, 22.5 (C-2,3), 23.8 (C-1),
31.1 (C-5′), 31.4 (C-4), 36.6 (C-9′), 102.2 (C-8″), 111.1 (C-4a″),
112.2 (C-3″), 112.7 (C-6″), 119.6 (C-9a), 123.3 (C-7), 123.6 (C-8),
124.2 (C-8a), 126.4(C-5), 126.9 (C-6,5″), 131.1 (C-9), 143.0 (C-10a),
150.2 (C-4″), 154.9 (C-8a″), 158.4 (C-4a), 160.2 (C-2″), 161.3 (C-
7″), 167.6 (C-2′), 168.4 (C-8′), 171.9 (C-4′). Anal. Calcd for
C27H22N4O5S (514.56): C, 63.02; H, 4.31; N, 10.89. Found: C, 62.90;
H, 4.29; N, 10.86.
Biochemical Studies. Cholinesterase Inhibitory Activities.
The AChE and BuChE inhibitory activity of the tested drugs was
determined using Ellman’s method52 and is expressed as IC50, meaning
the concentration at which cholinesterase activity is reduced by 50%.
Human recombinant AChE (hAChE; EC 3.1.1.7), human plasmatic
BuChE (hBuChE; EC 3.1.1.8), 5,5′-dithiobis(2-nitrobenzoic acid)
(Ellman’s reagent, DTNB), phosphate buffer (PB, pH 7.4),
acetylthiocholine (ATC), butylthiocholine (BTC), and tacrine hydro-
chloride were purchased from Sigma-Aldrich, Praque, Czech Republic.
Polystyrene cuvettes (Brand GmbH + Co. KG, Denmark) were used
for measuring purposes. All experiments were carried out in 0.1 M
KH2PO4/K2HPO4 buffer, pH 7.4. Enzyme solutions were prepared at
2.0 units/mL in 2 mL aliquots. The assay medium (1 mL) consisted of
650 μL of 0.1 M phosphate buffer (pH 7.4), 200 μL of 0.01 M DTNB,
25 μL of enzyme, and 100 μL of 0.01 M substrate (ATC chloride
solution). Assay solutions with inhibitor (10−3−10−10 M) were
preincubated for 5 min. The reaction was initiated by an immediate
addition of 100 μL of substrate. The activity was determined by
measuring the increase in absorbance at 412 nm at 1 min intervals
using a spectrophotometer Helios-Zeta (Thermospectronic, Cam-
bridge, U.K.). Each experiment was carried out in triplicate. The study
using in vitro BuChE was carried out using a method similar to that
described above. The corresponding selectivity indices of the
compounds were also calculated (ratio = [IC50(hBuChE)]/
[IC50(hAChE)]) according to a method optimized to allow the
constants Ki1 and Ki2 to be determined. Software Origin 6.1
(Northamption, MA, USA) was used for statistical data evaluation.
Docking Simulation. Molecular models of derivatives 6b and 7c
were created using the building options included in the Marvin 5.1.4
2008 software pack, ChemAxon [http://www.chemaxon.com]. The
same software was used to determine the overall protonization of the
compounds. Docking simulations were carried out using AUTO-
DOCK, version 4.2. MGL TOOLS 1.4.5 (revision 30) was used to
prepare the input files.54,55
Molecules of water with other nonenzymatic molecules were
removed, and missing hydrogens were added. A united atom
representation for ligands and enzymes was used, and Gasteiger
partial atomic charges for proteins and ligands were added. For the
initial docking simulation, the energy grid was set to the coordinates x
= 116.4, y = 104.3, z = −130.6 for hAChE (PDB code 1B41) and x =
138.7, y = 116.3, z = 41.0 for hBuChE (PDB code 1P01), with an
active site with the dimensions of 80 points × 80 points × 80 points
and a spacing of 0.375 Å. Flexible ligand docking was performed for
the compounds. Autotors was used to define the rotatable bonds in the
ligands. Docking runs were performed using the Lamarckian genetic
algorithm. Docking began with a population of random ligand
conformations in random orientations and at random translations. The
results of each docking experiment were derived from 100 different
runs which were set to terminate after a maximum of 5 000 000 energy
evaluations or 27 000 generations. The population size was set to 500,
and the other parameters were used as default. Thus, the ligand pose
with the lowest energy was chosen as the space for the construction of
the rerun energy grid with the coordinates x = 116.40, y = 108.3, z =
−132.9 and with dimensions of 46 points × 46 points × 46 points for
hAChE (PDB code 1B41). For hBuChE (PDB code 1P01), the
coordinates were set as x = 136.0, y = 114.3, z = 38.7 and with
dimensions of 46 points × 46 points × 46 points. Spacing was set at
0.375 Å for both enzymes. The subsequent redocking run used the
N1-[2-(1,2,3,4-Tetrahydroacridin-9-ylamino)butyl]-2-(7-hy-
droxy-2-oxo-2H-chromen-4-yl)acetamide (7c). Compound 3c
was treated with (7-hydroxy-2-oxo-2H-chromen-4-yl)acetic acid
according to a commonly used procedure to give the desired product
1
7c in the form of a yellow oil (33%). H NMR (400 MHz, CDCl3) δ
1.66−1.80 (m, 4H, 2 × CH2, H-2′,3′), 1.80−1.92 (m, 4H, 2 × CH2,
H-2,3), 2.60−2.70 (m, 2H, CH2, H-1), 2.98−3.06 (m, 2H, CH2, H-4),
3.38−3.48 (m, 2H, CH2, H-4′), 3.54−3.62 (m, 2H, CH2, H-1′), 3.70
(s, 2H, CH2, H-7′), 6.22 (s, 1H, CH, H-3″), 6.70 (s, 1H, CH, H-8″),
6.83 (d, 1H, CH, H-6″, J = 8.8 Hz), 7.30−7.42 (m, 2H, 2 × CH, H-
7,5″), 7.49−7.55 (m, 1H, CH, H-6), 7.84 (d, 1H, CH, H-5, J = 8.4
Hz), 7.95 (d, 1H, CH, H-8, J = 8.0 Hz). 13C NMR (CDCl3) δ 22.5,
22.7 (C-2,3), 24.5 (C-1), 26.8, 28.8 (C-2′,3′), 32.4 (C-4), 37.9 (C-7′),
40.5 (C-4′), 48.7 (C-1′), 103.4 (C-8″), 111.5 (C-4a″), 111.8 (C-3″),
114.3 (C-6″), 115.7 (C-9a), 119.4 (C-8a), 123.2 (C-8), 124.0 (C-7),
126.6 (C-5″), 128.2 (C-5), 128.8 (C-6), 145.6 (C-10a), 149.4 (C-4″),
151.8 (C-9), 155.4 (C-8a″), 157.2 (C-4a), 160.5 (C-2″), 161.9 (C-7″),
171.8 (C-6′). Anal. Calcd for C28H29N3O4 (471.56): C, 71.32; H,
6.20; N, 8.91. Found: C, 71.29; H, 6.18; N, 8.89.
N1-[2-(1,2,3,4-Tetrahydroacridin-9-yl)-2-[2-(7-hydroxy-2-
oxo-2H-chromen-4-yl)acetyl]-1-hydrazinecarbothioamide
(11). 9-Isothiocyanato-1,2,3,4-tetrahydroacridine (8, 0.18 g, 0.75 mM)
was added to a solution of (7-hydroxy-2-oxo-2H-chromen-4-yl)acetic
acid hydrazide (10, 0.125 g, 0.5 mM) in ethanol (5 mL). The mixture
was stirred for 24 h at room temperature. The resulting precipitate was
collected by filtration and dried in order to produce compound 11 at a
1
yield of 84%. Mp 170−175 °C. H NMR (400 MHz, DMSO-d6) δ
1.68−1.94 (m, 4H, 2 × CH2, H-2,3), 2.73−2.87 (m, 2H, CH2, H-1),
2.96−3.09 (m, 2H, CH2, H-4), 3.77 (s, 2H, CH2, H-5′), 6.26 (s, 1H,
CH, H-3″), 6.70 (s, 1H, CH, H-8″), 6.77 (d, 1H, CH, H-6″, J = 2.4,
8.8 Hz), 7.42−7.48 (m, 1H, CH, H-7), 7.58−7.70 (m, 2H, 2 × CH, H-
6,5″), 7.76 (d, 1H, CH, H-8, J = 8.4 Hz), 7.90 (d, 1H, CH, H-5, J = 8.4
Hz), 9.33 (s, 1H, NH). 13C NMR (DMSO-d6) δ 21.9, 22.5 (C-2,3),
24.5 (C-1), 33.4 (C-4), 36.8 (C-5′), 102.3 (C-8″), 111.3 (C-4a″),
112.9 (C-3″), 113.2 (C-6″), 123.5 (C-8), 125.4 (C-8a), 125.6 (C-7),
126.7 (C-9a), 126.8 (C-5″), 128.7 (C-5), 129.2 (C-6), 141.3 (C-9),
146.6 (C-10a), 150.5 (C-4″), 155.1 (C-8a″), 158.5 (C-4a), 160.3 (C-
2″), 161.3 (C-7″), 168.2 (C-4′), 181.5 (C-1′). Anal. Calcd for
C25H22N4O4S (474.54): C, 63.28; H, 4.67; N, 11.81. Found: C, 63.25;
H, 4.64; N, 11.78.
N′1-[4-Oxo-3-(1,2,3,4-tetrahydroakridin-9-yl)-1,3-thiazolan-
2-yliden]-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)-
ethanohydrazide (12). Methyl bromoacetate (0.024 mL, 0.25 mM)
and triethylamine (0.025 mL, 0.25 mM) were added to a solution of
N1-[2-(1,2,3,4-tetrahydroacridin-9-yl)-2-[2-(7-hydroxy-2-oxo-2H-4-
chromenyl)acetyl]-1-hydrazinecarbothioamide (11, 0.12 g, 0.25 mM)
in CHCl3 (2 mL). The mixture was stirred for 25 h at room
temperature. The solvent was removed, and the crude solid product
was purified using chromatography, eluent EtAc/MeOH/NH3
1
(6:2:0.2), to give 12 at a yield of 70%. Mp 118−120 °C. H NMR
(400 MHz, DMSO-d6) δ 1.80−1.92 (m, 4H, 2 × CH2, H-2,3), 2.62−
2.72 (m, 2H, CH2, H-1), 3.06−3.17 (m, 2H, CH2, H-4), 4.00 (s, 2H,
I
dx.doi.org/10.1021/jm5008648 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
same parameters as those described above.56 DOCK Amber rescoring
was performed using DOCK, version 6.5.57 A generalized Born/
surface area (GB/SA) continuum model was used for solvation. In
order to reduce the computational cost with no penalty to accuracy,
the atoms 3 Å distant from the site of the ligand binding were kept
frozen with 100 minimization and 3000 MD steps. Other input
parameters were set as defaults in the program. The enzymes were
modeled in their physiologically active forms with neutral His and
deprotonated Glu, which, together with Ser, form the catalytic triad.
The charge distribution of the inhibitors was determined using a
MOPAC semiempirical modul at the AM1-bcc level, a parm99.dat
parameter set for protein atoms, and GAFF atom types for the
ligand.58,59
(5) Munoz-Torrero, D. Acetylcholinesterase inhibitors as disease-
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Kuca, K. Acetylcholinesterase inhibitors used or tested in Alzheimer's
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̃
of the multifunctional profile of dual binding site acetylcholinesterase
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ASSOCIATED CONTENT
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Chem. 2005, 40, 1307−1315.
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Sakhteman, A.; Grandi, M.; Shafiee, A. Novel coumarin derivatives
bearing N-benzyl pyridinium moiety: potent and dual binding site
acetylcholinesterase inhibitors. Bioorg. Med. Chem. 2012, 20, 7214−
7222.
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nesterase: an important new target in Alzheimer’s disease therapy. Int.
Psychogeriatr. 2002, 14, 77−91. (b) Giacobini, E. Cholinesterases: new
roles in brain function and in Alzheimer’s disease. Neurochem. Res.
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Holloway, H. W.; Perry, T.; Lee, B.; Ingram, D. K.; Lahiri, D. K. A new
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(14) Nachon, F.; Masson, P.; Nicolet, Y.; Lockridge, O.; Fontecilla-
Camps, J. C. Comparison of the structures of butyrylcholinesterase
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Inhibitors; Giacobini, E., Ed.; Martin Dunitz: London, 2003; pp 39−54.
(15) Ames, D. J.; Bhathal, P. S.; Davies, B. M.; Fraser, J. R. E.
Hepatotoxicity of tetrahydroacridine. Lancet 1988, 1, 887.
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Lewis, K. W. Hepatotoxic effects of tacrine administration in patients
with Alzheimer-disease. JAMA, J. Am. Med. Assoc. 1994, 271, 992−998.
(17) Patocka, J.; Jun, D.; Kuca, K. Possible role of hydroxylated
metabolites of tacrine in drug toxicity and therapy of Alzheimer’s
disease. Curr. Drug Metab. 2008, 9, 332.
(18) Davis, K. L.; Pochwik, P. Tacrine. Lancet 1995, 345, 625−630.
(19) Giacobini, E. Invited review: Cholinesterase inhibitors for
Alzheimer’s disease therapy: from tacrine to future applications.
Neurochem. Int. 1998, 32, 413−419.
(20) Bornstein, J. J.; Eckroat, T. J.; Houghton, J. L.; Jones, Ch. K.;
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■
S
* Supporting Information
Structures applied in docking runs; top-score docking poses of
derivatives. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +420 973 253 028. Fax: 495 513 018. E-mail:
kucakam@pmfhk.cz.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support for this work from the Slovak Grant Agency
VEGA (Grant 1/0001/13) and the State NMR Program
(Grant 2003SP200280203) is gratefully acknowledged. This
work was also supported by Czech Grant Agency, Grant P303/
11/1907 and by Postdoctoral Project of University Hospital
(No. CZ.1.07/2.3.00/30.0044). Molecular graphics images
were produced using a UCSF Chimera package from the
Resource for Biocomputing, Visualization, and Informatics at
the University of California, San Francisco, CA (supported by
Grant NIH P41 RR-01081).
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; NFT, neurofibrillary tangles; AChE,
acetylcholinesterase; ACh, acetylcholine; AChEI, acetylcholi-
nesterase inhibitor; NMDAR, N-methyl-D-aspartate receptor;
PAS, peripheral anionic site; CAS, catalytic anionic site; Aβ, β-
amyloid peptide; BuChE, butyrylcholinesterase; hAChE,
human acetylcholinesterase; hBuChE, human butyrylcholines-
terase; CPC, chloropropionyl chloride; IC50, 50% inhibitory
concentration; SI, selectivity index
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