Synthesis, design and biological evaluation of novel highly potent tacrine congeners for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2012.06.051

New tacrine derivatives 5a-d, 6a-d with piperazino-ethyl spacer linked with corresponding secondary amines and tacrine homodimer 8 were synthesized and tested as cholinesterase inhibitors on human acetylcholinesterase (hAChE) and human plasmatic butyrylcholinesterase (hBChE). In most cases the majority of synthesized derivatives exhibit a high AChE and BChE inhibitory activity with IC₅₀ values in the low-nanomolar range, being clearly more potent than the reference standard tacrine (9-amino-1,2,3,4-tetrahydroacridine, 1) and 7-MEOTA (7-methoxy-9-amino-1,2,3,4-tetrahydroacridine). Among them, inhibitors 8 and 5c, showed a strong inhibitory activity against hAChE, with an IC ₅₀ value of 4.49 nM and 4.97, nM resp., and a high selectivity to hAChE. The compound 5d acted as the most potent inhibitor against hBChE with an IC₅₀ value of 33.7 nM and exhibited also a good selectivity towards hBChE. The dissociation constants K_i of the selected inhibitors were compared with their IC₅₀ values. Molecular modeling studies were performed to predict the binding modes between individual derivatives and hAChE/hBChE.

Full text of DOI:10.1016/j.ejmech.2012.06.051

European Journal of Medicinal Chemistry 55 (2012) 23e31
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, design and biological evaluation of novel highly potent tacrine
congeners for the treatment of Alzheimer’s disease
,
a
b
a
Slavka Hamulakova a , Ladislav Janovec , Martina Hrabinova , Pavol Kristian ,
*
Kamil Kuca b c, Maria Banasova a, Jan Imrich a
,
a Institute of Chemistry, Faculty of Science, P. J. Safarik University, Moyzesova 11, SK-04167 Kosice, Slovak Republic
b Center for Advanced Studies, Faculty of Military Health Sciences, University of Defence, CZ-50001 Hradec Kralove, Czech Republic
c Center for Biomedical Research, University Hospital, Hradec Kralove, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
New tacrine derivatives 5aed, 6aed with piperazino-ethyl spacer linked with corresponding secondary
amines and tacrine homodimer 8 were synthesized and tested as cholinesterase inhibitors on human
acetylcholinesterase (hAChE) and human plasmatic butyrylcholinesterase (hBChE). In most cases the
majority of synthesized derivatives exhibit a high AChE and BChE inhibitory activity with IC50 values in
the low-nanomolar range, being clearly more potent than the reference standard tacrine (9-amino-
1,2,3,4-tetrahydroacridine, 1) and 7-MEOTA (7-methoxy-9-amino-1,2,3,4-tetrahydroacridine). Among
them, inhibitors 8 and 5c, showed a strong inhibitory activity against hAChE, with an IC50 value of 4.49
nM and 4.97, nM resp., and a high selectivity to hAChE. The compound 5d acted as the most potent
inhibitor against hBChE with an IC50 value of 33.7 nM and exhibited also a good selectivity towards
hBChE. The dissociation constants Ki of the selected inhibitors were compared with their IC50 values.
Molecular modeling studies were performed to predict the binding modes between individual deriva-
tives and hAChE/hBChE.
Received 19 March 2012
Received in revised form
15 June 2012
Accepted 25 June 2012
Available online 4 July 2012
Keywords:
Alzheimer’s disease
Tacrine
AChE/BChE inhibitors
Amyloid
Molecular modeling
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
hypothesis is the cholinergic hypothesis [4,5,9] which has become
the leading strategy for the development of cholinesterase inhibi-
tors (ChEIs) aimed to increasing of levels of acetylcholine (ACh)
through inhibition of cholinesterases (ChEs) [10,11]. The most
prevailing hypothesis, the amyloid hypothesis posits that an
Alzheimer’s disease (AD)2 is a progressive and neurodegenera-
tive disease characterized by progressive memory impairment,
related to a disordered cognitive function [1]. Histological changes
underlying this disorder give rise to a presence of numerous
increased production of b-amyloid peptide and its aggregation and
amyloid plaques of
b
-amyloid peptide (A
b
), neurofibrillary tangles
accumulation in a brain lead to a neuronal cell death [6,12]. Two
classes of drugs, namely tacrine (1, CognexÒ), donepezil (AriceptÒ),
rivastigmine (ExelonÒ), galantamine (ReminylÒ) as AChE inhibitors
(NFT) and a dramatic loss of synapses, a decrease in a number of
neurons [2,3]. Scientists have proposed several theories explaining
the mechanism of AD development [4e8]. The oldest of the AD
(AChEIs) and memantine as ann N-methyl-D-aspartate (NMDA)
receptor antagonist, were approved to treat AD [11e13]. Tacrine
(THA, 9-amino-1,2,3,4-tetrahydroacridine) with a major selectivity
towards BChE instead of AChE, was the first ChEI approved for the
treatment of AD [14,15]. The use of tacrine in AD has been limited
by serious side effects such as hepatotoxicity, gastrointestinal
disturbance and hypotension [16e18]. The multifactorial nature of
AD supports new therapeutic strategies based on a multipotent
approach (one molecule, multiple targets) paradigm [9,19,20]. Such
a strategy has been described in a review that summarized the
research development of tacrine derivatives with a cholinesterase
and amyloid aggregation inhibiting activity from a bioorganic
aspect [21]. As potent acetylcholinesterase inhibitors with
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b-protein; NFT, neurofi-
brillary tangles; AChEIs, acetylcholinesterase inhibitors; AChE, acetylcholinesterase;
ACh, acetylcholine; THA, 9-amino-1,2,3,4-tetrahydroacridine; BChE, butyr-
ylcholinesterase; ChEI, cholinesterase inhibitors; NMDA, N-methyl-
D-aspartate
receptor; BACE, -secretase; APP, amyloid precursor protein; PAS, peripheral
b
anionic site; CAS, catalytic active site; MTDLs, multi-target-directed ligands; M2,
muscarinic receptors; CPC, chloropropionylchloride; DTNB, 5,50-dithiobis(2-
nitrobenzoic acid); ATC, acetylthiocholine; BTC, butylthiocholine; PB, phosphate
buffer.
* Corresponding author. Tel.: þ421 55 2342334; fax: þ421 55 2341197.
E-mail address: slavka.hamulakova@upjs.sk (S. Hamulakova).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.051

24
S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
antioxidant properties, tacrine-melatonin hybrids [16], tacrine-
ferulic acid hybrids [22], tacrine-carvedilol heterodimers [23]
have been synthesized. Some other tacrine heterodimers,such as
tacrine-cystamine dimer [24], tacrine-oxoisoaporphine congeners
[25], heterobivalent tacrine derivatives containing aromatic groups
[26] and tacrine-lipoic acid dimers [27] exhibiting acetylcholines-
of tacrine, piperazino-ethyl side chain and appropriate secondary
amines 5aed, 6aed (Fig. 2) and tacrine homodimer 8. An interaction
with both CAS and PAS of AChE are confirmed by docking simulation.
2. Results and discussion
terase and acetylcholinesterase-induced Ab aggregation inhibition
2.1. Chemistry
activity. Tacrine-gallamine hybrids which act as AChEI and
M2 muscarinic receptor modulators [28], tacrineeNOedonors,
designed as hepatoprotective anti-Alzheimer drug candidates
[29], tacrine-propidium heterodimers with an inhibition effect on
cholinesterase/Ab aggregation and BACE-1 [30] present a further
class of multi-target-directed ligands (Fig. 1).
In the view of the above mentioned reasons, we focused on the
development of more active and selective ligands which are capable
of interacting with a catalytic anionic side (CAS) and also with
a peripheral anionic side (PAS) of AChE. In our previous paper we
reported a synthesis of a novel AChEIs, 2-[(1,2,3,4-tetrahydroacridin-
9-yl)imino]-3-substituted 1,3-thiazolidin-4-ones [31] and tacrine
ligands with piperazine and thiourea linkers, most of them with
a remarkable hAChE/hBChE inhibition activity [32]. Given the fact
that amines are widely known as biologically active compounds or
the important building blocks for the synthesis of biologically active
compounds [33,34] we decided to incorporate appropriate
secondary aminesinto thetacrine moiety.Here, we report the
synthesis, biological evaluation and molecular modeling of ChEIs,
represented by tacrine derivatives (Fig. 2) designed by a combination
Tacrine derivatives 4, 5aed, 6aed and tacrine homodimer 8 were
obtained by the method summarized in Scheme 1. As a starting
compound for the synthesis of a new tacrine ligands, 9-chloro-
1,2,3,4-tetrahydroacridine (2) was used. The synthetic route of N-
(piperazinoethyl)-N-(1,2,3,4-tetrahydroacridin-9-yl)amine (3) was
reported in our previous paper [32]. Piperazinoethyl-
tetrahydroacridinylamine 3 was further acylated by the corre-
sponding 3-chloropropionylchloride (CPC) in excess of triethyl-
amine
[35,36],
to
give
halogenated
intermediate
chloropropionamide 4 (Scheme 1). Subsequent aminolysis of 4 with
appropriate secondary amines (piperidine, methylcyclohexylamine,
ethylcyclohexylamine and N-methylaniline) in acetone/ethanol
afforded the final products 5aed in 50e65% yield. Compounds 6aed
were prepared from derivatives 5aed by a reduction of their amide
group with LiAlH4 (Scheme 1). The reaction of 9-chloro-1,2,3,4-
tetrahydroacridine (2) with 1,2-diaminoethane in phenol gave
known compound 7 [37,38]. For the synthesis of tacrine homodimer
8, N-(1,2,3,4-tetrahydroacridin-9-yl)-1,2-ethanediamine (7) was
reacted with chloropropionamide
4
in the presence of N,
H
R
OH
N
O
NH2
H
N
H
N
H
N
( )n
( )n
N
H
OMe
N
O
N
R´
N
b
a
Tacrine
N
O
Cl
O
H
N
H
N
H
H
N
O
N
( )n
( )3
( )m2 ( )n
N
H
N
NH
N
( )m1
d
c
R
N
O
O
O
S
S
( )
( )4
HN
N
m N
( )n
HN
N
H
H
Cl
N
Cl
e
f
Fig. 1. Selected multi-target-directed ligands bearing tacrine moiety: a) tacrine-melatonin, b) tacrine-ferulic acid, c) tacrine-carvedilol, d) tacrine-oxoisoaporphine, e) tacrine-lipoic
acid, f) tacrine-propidium.

S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
25
N
R1
X
X
( )n
N
N
N
N
H
N
H
R2
N
N
HN
N
HN
N
5a-d: X = CO(CH2)2
6a-d: X = (CH2)3
8: X = CO(CH2)2, n = 2
a
b
c
d
R1
=
N
N
N
N
N
R2
CH3
CH2
CH3
H3C
Fig. 2. Chemical structure of synthesized tacrine derivatives 5aed, 6aed and tacrine homodimer 8.
Scheme 1. Synthesis of designed inhibitors 5aed, 6aed, 8. Reagents and conditions: (i) 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-buthyl ester, phenol, reflux, 4 h; (ii)
AcOH/HCl, 1.5 h; (iii) NH4OH, 15 min; (iv) CPC, Et3N, CH2Cl2, O ꢀC, 1 h; (v) corresponding secondary amine, K2CO3, acetone/ethanol, reflux, 9e13 h; (vi) LiAlH4, THF, reflux, 2 h; (vii) 1,
2-diaminoethane, phenol, reflux, 2h; (viii) 4, N, N-diisopropylethylamine, CH2Cl2, reflux, 72 h.

26
S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
N-diisopropylethylamine (Hunig’s base) in CH2Cl2 (Scheme 1).
Target product 8 was obtained in 63% yield.
6b, 6d inhibited hAChE non-competitively. The Ki1 values of
0.38 nM for the ligand 5b indicate a strongest affinity towards
hAChE that is 600-times higher compared to tacrine and 5500-
times higher related to 7-MEOTA. The Ki1 values of 1.35 nM for
the ligand 5c with affinity towards hAChE was 4-times higher
compared to methyl derivative 5b.
2.2. Inhibition studies on hAChE and hBuChE
To determine a therapeutic potential of the new series of tacrine
derivatives for the treatment of AD, the inhibition constants, IC50 of
compounds 5aed, 6aed and 8 was determined against human
erythrocytal AChE and human plasmatic BChE using the method of
Ellman et al. [39]. For comparison, tacrine 1 and 7-MEOTA were
used as a reference compounds. 7-MEOTA is a weaker inhibitor
compared with tacrine but was found to be less toxic and phar-
macologically equally active to tacrine [40]. IC50 values and selec-
tivity index [SI ¼ IC50(hBChE)/IC50(hAChE)] of tacrine derivatives
5aed, 6aed, 8 and dissociation constants Ki1 and Ki2 of selected
derivatives 5aed, 6b, 6d are summarized in Table 1.
The synthesized tacrine compounds showed an inhibitory
activity against hAChE in the lowenanomolar range of IC50 values.
They are two orders of magnitude more potent than tacrine 1, and
four orders of magnitude more effective in inhibiting AChE than 7-
MEOTA. The derivatives 8 and 5c exhibited the most promising
2.3. Molecular modeling studies
The compound 5c and its structurally related compounds 5b, 6b
were treated in silico towards hAChE in order to find an explanation
for their inhibition potential. A similar study was done for hBChE
with 5bed and 6b. Docking simulations were performed to reveal
possible intermolecular interactions that might drive an inhibition
activity of novel tacrine derivatives (for more details see Experi-
mental section). To overcome a software limitation, we simulated
flipping of spacer’s terminal nitrogen by building two structures for
each derivate with the opposite configuration of substituents (see
Supplementary data, Fig. S1). Subsequently, we solved the orien-
tation of a carbonyl group attached to a piperazine ring. We did not
choose to set up a rotable amide bond within a software option, but
rather we built two structures with an opposite carbonyl orienta-
tion to restrain a planar conformation of an amide group (see
Supplementary data, Fig. S1). Thus, we obtained four structures for
compounds 5bed and two for derivative 6b. In order to find
a degree of protonization of our compounds, we applied software
options within Marvin software packed [http://www.chemaxon.
com]. As a consequence of the prediction with bis/tris protoniza-
tion falling into the ratio 1:1, we decided to study bis and tris
protonated compounds separately (see Experimental section).
Thus, each structure was once treated as bis protonated in a dock-
ing run and then in a tris protonated charged molecule with
protonated positions in accordance to a software prediction. So, we
gained the library of eight items for compound 5bed and four items
inhibition towards hAChE (8, IC50
¼
4.49 nM and 5c,
IC50 ¼ 4.97 nM). The changes of substituents on the terminal side
chain e.g. (5b/5c) evidently influence the increase of their AChE
inhibitory effect (see Table 1). The derivatives with the amide group
(5bed) exhibit a higher activity compared to the compounds in
which the amide group was reduced to a methylene group (6bed).
It is notable that some of the tested compounds exhibited a 1.5- to
338.5-fold higher inhibitory activity towards AChE than towards
BChE. The best selectivity for hAChE was shown by tacrine homo-
dimer 8 (SI ¼ 338.5) and for hBChE by 5d (SI ¼ 0.48). The IC50 values
were compared with two dissociation constants Ki1 and Ki2 which
were measured for selected ligands 5aed and 6b, 6d (Table 1). The
LineweavereBurk plot revealed that tacrine congeners 5aed and
Table 1
Inhibition of hAChE and hBChE by target compounds their selectivity index and dissociation constants Ki1 and Ki2
.
1
X
R
N
N
R2
N
HN
N
R1
R2
c
Compound
X
IC50(hAChE)a ꢁ SD (nM)
IC50(hBChE)a ꢁ SD (nM)
SIb
Ki1 (nM)
Ki2d (nM)
N
5a
5b
5c
5d
6a
6b
6c
6d
Piperidine-1-yl
Methylcyclohexylamino
Ethylcyclohexylamino
Methylphenylamino
Piperidine-1-yl
Methylcyclohexylamino
Ethylcyclohexylamino
Methylphenylamino
e
CO(CH2)2
CO(CH2)2
CO(CH2)2
CO(CH2)2
(CH2)3
(CH2)3
(CH2)3
(CH2)3
CO(CH2)2
e
51.7 ꢁ 10.3
14.9 ꢁ 2.9
4.97 ꢁ 1
77.8 ꢁ 13.5
63.7 ꢁ 11.0
41.2 ꢁ 7.16
33.7 ꢁ 5.86
nd
1.51
4.28
8.29
0,48
nd
3.37
0.65
2.49
338.5
0,05
1,4
16.8
0.38
1.35
13.7
nd
76.1
0.318
3.73
85.5
nd
70.3 ꢁ 14.06
nd
40.7 ꢁ 8.14
194 ꢁ 0.11
84.8 ꢁ 16.9
4.49 ꢁ 0.34
500 ꢁ 100
15 000 ꢁ 2900
137 ꢁ 23.8
126 ꢁ 0.07
211 ꢁ 36.7
1520 ꢁ 0.14
23 ꢁ 4
10.3
e
47.6
e
80.0
e
24.7
e
8
1
e
225
2090
101
6340
7-MEOTA
e
e
21 000 ꢁ 3400
nd: not determined.
a
IC50, inhibitor concentration (means ꢁ SD of three experiments) for 50% inhibition of enzyme.
b
c
Selectivity index for AChE ¼ IC50(hBChE)/IC50(hAChE).
Ki1: Dissociation constant for AChE - inhibitor complex.
Ki2: Dissociation constant for AChE - inhibitoresubstrate complex.
d

S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
27
set for 6b (see Supplementary data, Fig. S1). Conformation of
a piperazine ring remained the same as in an x-ray structure for
a donepezil inhibition complex with TcAChE (PDB: 1EVE). The poses
with the lowest binding energy within hAChE for derivative 5b, 5c,
6b are depicted in Fig. 3.
There are five hydrogen bonds between 5b and TRP86, TYR124,
TYR337 (Fig. 4). A similar binding orientation could be found for
compound 6b (Fig. 4). There are predicted formations of three
hydrogen bonds with TRP86, TYR124, TYR337 amino acids. Deri-
vate 5c adopts a pose directly when interacting with amino acids of
catalytic triad, SER203 and HIS447, which might be the reason for
an overall higher biological activity over other derivatives. An
additional stabilization might be gained by a formation of hydrogen
bonds with TYR124 and TYR337 (Fig. 4).
a methylene group 6bed. The reduction of amide groups in tether
significantly reduced an anti-hBChE activity as indicated by
a comparison of 5bed with 6bed. Among all tested compounds,
derivative 8 showed the best selectivity against hAChE (338.5) and
compound 5d to hBChE (0.48). Presumed binding modes between
individual derivatives and ChEs were predicted by docking calcu-
lations. Molecular modeling studies confirmed that these hybrids
target both, the catalytic active site and peripheral anionic site of
AChE.
4. Experimental section
4.1. Synthesis
The most active derivative 5d with related 5b, 5c, 6b were
studied by a docking simulation toward hBChE. An overall view of
docking poses with the lowest binding energy proposed by
a simulation is depicted in Fig. 5. There we can see a very close
orientation of derivatives 5bed in the enzymatic cavity with
a different orientation of piperazine of 6b. All derivatives, except 6b,
form hydrogen bonds with ASP70, TYR337 and HIS438 as it is
shown in Fig. 4. A higher inhibition of 5d could be prescribed to
a presence of an additional aromatic system, phenyl group, that is
All solvents, chemicals, and reagents were obtained commer-
cially 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 tet-
ramethylsilane as an internal standard. Chemical shifts, d, 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 MachereyeNagel Alu-
gram ÒSil G/UV254 plates, and spots were visualized with UV light.
Chromatographic separations were performed on silica gel 60
(0.063e0.040 mm, Merck) column chromatography. Melting points
were recorded on a Boetius hot-plate apparatus and are uncor-
rected. Yields refer to isolated pure products and were not maxi-
able to provide an additional stabilization by a
pep interaction
with PHE329. This interaction might be missing in derivates 5b, 5c,
6b where phenyl is substituted by cyclohexyl.
3. Conclusion
mized. CHN analysis was performed on
a CHN analyzer
PerkineElmer 2400.
In summary, we have reported the synthesis and the results for
an acetylcholinesterase and butyrylcholinesterase inhibition
activity of eight new tacrine ligands (5aed, 6aed) and tacrine
homodimer 8. Our results showed that synthetized compounds had
a high cholinesterases inhibitory activity with IC50 values less than
15 nM against hAChE, and less than 40 nM toward hBChE, being
clearly more potent than tacrine 1 and 7-MEOTA. The most
promising inhibition activity of two orders of magnitude better
than tacrine and four orders of magnitude better than 7-MEOTA
was found for derivatives 8 and 5c against hAChE (IC50 ¼ 4.49,
4.97 nM). Derivatives 5bed with an amide group in tether showed
better inhibitory activities against hAChE than derivatives with
4.1.1. Synthesis of 3-chloro-1-{4-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
ethyl]piperazino}-1-propanone (4)
A solution of N-(piperazinoethyl)-N-(1,2,3,4-tetrahydroacridin-
9-yl)amine (3, 100 mg, 0.33 mmol) and Et3N (0.11 ml, 0.82 mmol) in
CH2Cl2 (5 ml) was added dropwise to a mixture of chloropropionyl
chloride (0.078 ml, 0.82 mmol) in CH2Cl2 (2 ml) at 0 ꢀC. The mixture
was stirred at 0 ꢀC for 1 h and then at rt for 10 min. A saturated
solution of NH3 in CH3OH (0.3 ml) was added. The mixture was
filtered, and the filtrate was concentrated under reduced pressure.
The residue was dissolved in CH2Cl2 (9 ml) and then washed with
brine (6 ꢂ 3 ml). After drying over anhydrous MgSO4, the organic
layer was concentrated. The residue was purified by column chro-
matography, eluent CHCl3/MeOH (9:1). Compound 4 (0.1 g, 77%
yield) was obtained as a yellow powder, m.p. ¼180e183 ꢀC. 1H NMR
(CDCl3, 400 MHz)
d
1.83e2.0 (m, 4H, 2 ꢂ CH2, H-2,3), 2.48e2.56 (m,
4H, 2 ꢂ CH2, H-40,80), 2.66 (t, 2H, CH2, H-20, J ¼ 5.4 Hz), 2.72e2.76
(m, 2H, CH2, H-1), 2.82 (t, 2H, CH2, H-100, J ¼ 7.2 Hz), 3.08e3.13
(m, 2H, CH2, H-4), 3.51e3.55 and 3.66e3.74 (m, 4H, 2 ꢂ CH2, H-
50,70), 3.64 (t, 2H, CH2, H-10, J ¼ 5.6 Hz), 3.84 (t, 2H, CH2, H-110,
J ¼ 7.0 Hz), 5.30 (bs, 1H, NH), 7.35 (dd, 1H, CH, H-7, J1 ¼ 1.2 Hz,
J2 ¼ 8.4 Hz), 7.60 (dd,1H, CH, H-6, J1 ¼1.2 Hz, J2 ¼ 8.0 Hz), 7.98e8.04
(m, 2H, 2 ꢂ CH, H-5,8). 13C NMR (CDCl3)
d 22.5, 22.9 (C-2, 3), 24.7 (C-
1), 33.1 (C-4), 36.0 (C-100), 39.8 (C-110), 42.0, 45.5 (C-50, 70), 45.0 (C-
10), 52.3, 52.6 (C-40, 80), 57.4 (C-20), 115.4 (C-9a), 119.6 (C-8a), 122.6
(C-8), 123.8 (C-7), 128.1 (C-5), 128.8 (C-6), 146.5 (C-10a), 151.3 (C-9),
157.6 (C-4a), 168.1 (C-90). Anal. Calcd for C22H29ClN4O (400.96): C,
65.90; H, 7.29; N, 13.97. Found: C, 66.02; H, 7.37; N, 14.06.
4.1.2. General procedures for the synthesis of 3-substituted-1-{4-[2-
(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl]piperazino}-1-
propanones 5a-5d
Secondary amine (0.25 mmol), K2CO3 (70 mg, 0.5 mmol) and 1:1
mixture of acetone/ethanol (8 ml) were added to a solution of 3-
chloro-1-{4-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl]
Fig. 3. Top-score docking pose of derivatives 5b (red), 5c (cyan), 6b (green) depicted
their putative structural orientations in the active-site gorge of the hAChE in a ribbon
style [41]. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)

28
S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
Fig. 4. Column A: Top-score docking pose of derivative 5b, 6b, 5c depicted its putative hydrogen bonds formed with amino acid residues in the active-site gorge of the hAChE.
Column B: Top-score docking pose of derivative 5d, 5b, 6b, 5c depicted its putative hydrogen bonds formed with amino acid residues in the active-site gorge of the hBChE. Atoms’
color palette: Cegray; Hewhite; Neblue, Oered [41]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
29
8), 123.7 (C-7), 128.4 (C-6), 128.8 (C-5), 147.3 (C-10a), 151.0 (C-9),
158.5 (C-4a), 167.8 (C-90). Anal. Calcd for C29H43N5O (477.70): C,
72.92; H, 9.07; N, 14.66. Found: C, 73.1; H, 9.13; N, 14.86.
4.1.2.3. Synthesis of 3-[cyclohexyl(ethyl)amino]-1-{4-[2-(1,2,3,4-
tetrahydroacridin-9-ylamino)ethyl]piperazino}-1-propanone (5c).
Intermediate 4 was reacted with ethylcyclohexylamine following
the general procedure to give 61 mg (50%) of 5c as oil after column
chromatograpghy (CHCl3/MeOH/Et3N, 4/1/0.05). 1H NMR (CDCl3,
400 MHz)
d
0.9 (t, 3H, CH3, H-200, J ¼ 7.2 Hz), 1.18e1.28 (m, 10H,
5 ꢂ CH2, H-140,150,160,170,180), 1.89e1.96 (m, 4H, 2 ꢂ CH2, H-2,3),
2.30e2.40 (m, 1H, CH, H-130), 2.45e2.55 (m, 6H, 3 ꢂ CH2, H-
40,80,100), 2.56e2.62 (m, 2H, CH2, H-100), 2.63e2.66 (m, 2H, CH2, H-
20), 2.74e2.80 (m, 2H, CH2, H-1), 2.83 (t, 2H, CH2, H-110, J ¼ 6.8 Hz),
3.07 (t, 2H, CH2, H-4, J ¼ 5.8 Hz), 3.52e3.56 and 3.66e3.70 (m, 4H,
2 ꢂ CH2, H-50,70), 3.56e3.62 (m, 2H, CH2, H-10), 7.34 (ddd,1H, CH, H-
7, J1 ¼ 1.2 Hz, J2 ¼ 6.8 Hz, J3 ¼ 8.4 Hz), 7.55 (ddd, 1H, CH, H-6,
J1 ¼1.2 Hz, J2 ¼ 7.0 Hz, J3 ¼ 8.4 Hz), 7.91 (d, 1H, CH, H-5, J ¼ 8.4 Hz),
8.0 (d, 1H, CH, H-8, J ¼ 8.4 Hz). 13C NMR (CDCl3)
d
14.1 (C-200), 22.8,
Fig. 5. Top-score docking pose for derivatives 5b (yellow), 5c (blue), 5d (cyan), 6b (red)
depicted putative structural orientations in the active-site gorge of the hBChE. For
clarity only enzyme active site is shown as a hydrophobic surface pocket [41,42]. (For
interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)
23.1 (C-2,3), 24.9 (C-1), 26.1 (C-150,170), 26.3 (C-160), 29.1 (C-140,180),
32.0 (C-100), 33.9 (C-4), 41.6, 45.7 (C-50,70), 45.1 (C-10), 45.6 (C-100),
50.6 (C-110), 52.4, 52.8 (C-40,80), 57.6 (C-20), 60.1 (C-130), 116.1 (C-9a),
120.3 (C-8a), 122.6 (C-8), 123.7 (C-7), 128.7 (C-6), 128.8 (C-5), 147.3
(C-10a), 150.9 (C-9), 158.5 (C-4a), 167.8 (C-90). Anal. Calcd for
C30H45N5O (491.73): C, 73.28; H, 9.22; N, 14.24. Found: C, 73.33; H,
9.29; N, 14.34.
piperazino-1-yl}-1-propanone (4, 100 mg, 0.25 mmol),. The
mixture was refluxed and stirred for 9e13 h, then cooled to rt,
filtered, and evaporated.
4.1.2.4. Synthesis of 3-(methylanilino)-1-{4-[2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethyl]-piperazino}-1-propanone (5d). Intermediate
4 was
4.1.2.1. Synthesis of 3-(piperidino)-1-{4-[2-(1,2,3,4-tetrahydroacridin-
9-ylamino)ethyl]-piperazino}-1-propanone (5a). Intermediate 4 was
reacted with piperidine following the general procedure to give
60 mg (60%) of 5a as oil after column chromatograpghy (CHCl3/
reacted with N-methyl-N-phenylamine following the general
procedure to give 72 mg (61%) of 5d as oil after column chroma-
tograpghy (CHCl3/MeOH/Et3N, 4/1/0.05). 1H NMR (CDCl3, 400 MHz)
d
1.88e1.98 (m, 4H, 2 ꢂ CH2, H-2,3), 2.47e2.57 (m, 6H, 3 ꢂ CH2, H-
MeOH/Et3N, 4/1/0.05). 1H NMR (CDCl3, 400 MHz)
d
1.51e1.70 (m, 6H,
40,80,110), 2.62e2.66 (m, 4H, 2 ꢂ CH2, H-20,100), 2.77 (t, 2H, CH2, H-1,
J ¼ 6.0 Hz), 2.83 (s, 3H, CH3, H-100), 3.07 (t, 2H, CH2, H-4, J ¼ 5.8 Hz),
3.53e3.66 and 3.73e3.77 (m, 6H, 3 ꢂ CH2, H-10,50,70), 5.06 (bs, 1H,
NH), 6.60 (dd, 2H, 2 ꢂ CH, H-140,180, J1 ¼ 2.0 Hz, J2 ¼ 8.0 Hz), 6.70
(ddd, 1H, CH, H-160, J1 ¼ 2.0 Hz, J2 ¼ 7.2 Hz, J3 ¼ 8.0 Hz), 7.20 (ddd,
2H, 2 ꢂ CH, H-150,170, J1 ¼ 2.0 Hz, J2 ¼ 7.2 Hz, J3 ¼ 8.0 Hz), 7.34 (dd,
1H, CH, H-7, J1 ¼1.2 Hz, J2 ¼ 7.2 Hz), 7.55 (dd,1H, CH, H-6, J1 ¼1.2 Hz,
J2 ¼ 7.2 Hz), 7.91 (d, 1H, CH, H-5, J ¼ 8.4 Hz), 8.0 (d, 1H, CH, H-8,
3 ꢂ CH2, H-140,150,160), 1.88e2.0 (m, 4H, 2 ꢂ CH2, H-2,3), 2.40e2.55
(m, 8H, 4 ꢂ CH2, H-40,80,130,170), 2.56e2.62 (m, 2H, CH2, H-100),
2.64 (t, 2H, CH2, H-20, J ¼ 5.8 Hz), 2.68e2.82 (m, 4H, 2 ꢂ CH2, H-1,110),
3.05e3.15 (m, 2H, CH2, H-4), 3.50e3.60 and 3.66e3.70 (m, 4H,
2 ꢂ CH2, H-50,70), 3.63 (t, 2H, CH2, H-10, J ¼ 5.6 Hz), 7.35 (dd, 1H, CH,
H-7, J1 ¼ 1.2 Hz, J2 ¼ 7.2 Hz), 7.57 (dd, 1H, CH, H-6, J1 ¼ 1.2 Hz,
J2 ¼ 7.2 Hz), 7.97 (d, 2H, CH2, H-5, J ¼ 8.4 Hz), 8.0 (d, 2H, CH2, H-8,
J ¼ 8.4 Hz). 13C NMR (CDCl3)
d
22.7, 23.0 (C-2, 3), 24.4 (C-1,150), 25.7
J ¼ 8.4 Hz). 13C NMR (CDCl3)
d 22.8, 23.1 (C-2,3), 24.9 (C-1), 33.9 (C-
(C-140,160), 31.0 (C-100), 33.4 (C-4), 41.7, 45.6 (C-50,70), 45.0 (C-10),
52.4, 52.8 (C-40,80,110), 54.6 (C-130,170), 57.5 (C-20), 115.7 (C-9a), 119.9
(C-8a), 122.7 (C-8),123.8 (C-7),128.0 (C-6),128.7 (C-5),146.6 (C-10a),
151.3 (C-9), 158.0 (C-4a), 167.8 (C 90). Anal. Calcd for C27H39N5O
(449.64): C, 72.12; H, 8.74; N,15.58. Found: C, 72.18; H, 8.89; N,15.64.
4,100), 38.0 (C-100), 42.0, 45.7 (C-50,70), 45.0 (C-10), 52.3, 52.8 (C-40,80),
53.7 (C-110), 57.6 (C-20), 112.3 (C-140,180), 116.2 (C-9a), 117.1 (C-160),
120.3 (C-8a), 122.5 (C-8), 123.6 (C-7), 128.3 (C-6), 128.8 (C-5), 129.1
(C-150,170), 147.4 (C-10a), 149.3 (C-130), 150.7 (C-9), 158.6 (C-4a),
167.8 (C-90). Anal. Calcd for C29H37N5O (471.65): C, 73.85; H, 7.91; N,
14.85. Found: C, 73.92; H, 7.98; N, 15.05.
4.1.2.2. Synthesis of 3-[cyclohexyl(methyl)amino]-1-{4-[2-(1,2,3,4-
tetrahydroacridin-9-ylamino)ethyl]piperazino}-1-propanone (5b).
Intermediate 4 was reacted with methylcyclohexylamine following
the general procedure to give 77 mg (65%) of 5b as oil after column
chromatograpghy (CHCl3/MeOH/Et3N, 4/1/0.05). 1H NMR (CDCl3,
4.1.3. General procedure for the synthesis of N-(2-{4-[3-(substituted)
propyl]piperazino}ethyl)-(1,2,3,4-tetrahydroacridin-9-yl) amines
6ae6d
Propanone 5 (0.39 mmol) in THF (10 ml) was added to LiAlH4
(230 mg, 1.56 mmol) suspended in THF (26 ml). The reaction
mixture was stirred under reflux for 2 h. The excess of LiAlH4 was
decomposed by a careful subsequent addition of water (5 drops)
and NaOH (1N, 5 drops) and stirred for further 0.5 h. Then Na2SO4
was added and we continued to stir for final 2 h. After cooling to rt
the product was filtered.
400 MHz)
d
1.20e1.30 (m, 10H, 5 ꢂ CH2, H-140,150,160,170,180),
1.87e1.97 (m, 4H, 2 ꢂ CH2, H-2,3), 2.3 (s, 3H, CH3, H-100), 2.35e2.44
(m, 1H, CH, H-130), 2.46e2.56 (m, 6H, 3 ꢂ CH2, H-40,80,100), 2.63 (t,
2H, CH2, H-20, J ¼ 6.0 Hz), 2.75e2.80 (m, 2H, CH2, H-1), 2.82 (t, 2H,
CH2, H-110, J ¼ 6.8 Hz), 3.07 (t, 2H, CH2, H-4, J ¼ 5.8 Hz), 3.53e3.55
and 3.67e3.72 (m, 4H, 2 ꢂ CH2, H-50,70), 3.55e3.63 (m, 2H, CH2, H-
10), 7.34 (dd,1H, CH, H-7, J1 ¼1.2 Hz, J2 ¼ 8.2 Hz), 7.56 (dd,1H, CH, H-
6, J1 ¼1.2 Hz, J2 ¼ 8.2 Hz), 7.92 (d, 1H, CH, H-5, J ¼ 8.2 Hz), 8.0 (d, 1H,
4.1.3.1. Synthesis of N-(2-{4-[3-(piperidinopropyl)]piperazino}ethyl)-
(1,2,3,4-tetrahydroacridin-9-yl)amine (6a). Starting with 5a and
LiAlH4, followed by the general procedure and column chroma-
tograpghy (eluent CHCl3/MeOH/Et3N, 4/1/0.05), led to 42 mg (25%)
CH, H-8, J ¼ 8.2 Hz). 13C NMR (CDCl3)
d 22.8, 22.9 (C-2,3), 25.0 (C-1),
26.0 (C-150,170), 26.2 (C-160), 29.0 (C-140, 180), 32.0 (C-100), 33.9 (C-
4), 37.8 (C-100), 41.6, 45.7 (C-50,70), 45.0 (C-10), 49.6 (C-110), 52.5, 52.8
(C-40,80), 57.7 (C-20), 63.3 (C-130), 116.1 (C-9a), 120.3 (C-8a),122.6 (C-
of 6a as oil. 1H NMR (CDCl3, 400 MHz)
d
1.51e1.63 (m, 6H, 3 ꢂ CH2,

30
S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
H-140,150,160), 1.67e1.74 (m, 2H, CH2, H-100), 1.88e1.98 (m, 4H,
2 ꢂ CH2, H-2,3), 2.28e2.25-2.46 (m, 8H, 4 ꢂ CH2, H-90,110,130,170),
2.46e2.58 (m, 8H, 4 ꢂ CH2, H-40,80,50,70), 2.60 (t, 2H, CH2, H-20,
J ¼ 6.0 Hz), 2.74e2.82 (m, 2H, CH2, H-1), 3.04e3.12 (m, 2H, CH2, H-
4), 3.53e3.60 (m, 2H, CH2, H-10), 7.33 (ddd, 1H, CH, H-7, J1 ¼ 1.2 Hz,
J2 ¼ 6.8 Hz, J3 ¼ 8.0 Hz), 7.54 (ddd, 1H, CH, H-6, J1 ¼ 1.2 Hz,
J2 ¼ 6.8 Hz, J3 ¼ 8.0 Hz), 7.91 (d, 1H, CH, H-5, J ¼ 8.0 Hz), 8.02 (d, 1H,
J2 ¼ 7.2 Hz, J3 ¼ 8.0 Hz), 7.20 (ddd, 2H, 2 ꢂ CH, H-150,170, J1 ¼ 2.0 Hz,
J2 ¼ 7.2 Hz, J3 ¼ 8.0 Hz), 7.34 (ddd, 1H, CH, H-7, J1 ¼ 1.2 Hz,
J2 ¼ 6.8 Hz, J3 ¼ 8.2 Hz), 7.55 (ddd, 1H, CH, H-6, J1 ¼ 1.2 Hz,
J2 ¼ 6.8 Hz, J3 ¼ 8.2 Hz), 7.94 (d, 1H, CH, H-5, J ¼ 8.2 Hz), 8.05 (d, 2H,
CH2, H-8, J ¼ 8.2 Hz). 13C NMR (CDCl3)
d 22.7, 23.0 (C-2,3), 24.7 (C-1),
29.8 (C-100), 33.4 (C-4), 38.2 (C-100), 45.0 (C-10), 52.5, 53.4 (C-
40,50,70,80), 52.6 (C-90,110), 57.4 (C-20), 112.4 (C-140,180), 115.6 (C-9a),
117.2 (C-160), 120.0 (C-8a), 122.9 (C-8),123.6 (C-7), 128.4 (C-6), 128.6
(C-5), 129.1 (C-150,170), 147.4 (C-10a), 149.3 (C-130), 151.4 (C-9), 158.5
(C-4a). Anal. Calcd for C29H39N5 (457.67): C, 76.11; H, 8.59; N, 15.30.
Found: C, 76.15; H, 8.84; N, 15.37.
CH, H-8, J ¼ 8.0 Hz). 13C NMR (CDCl3)
d 22.8, 23.1 (C-2,3), 24.4 (C-
150), 24.9 (C-1), 26.0 (C-140,160), 30.0 (C-100), 33.8 (C-4), 45.2 (C-10),
52.6, 53.4 (C-40,50,70,80,90,110), 54.6 (C-130,170), 57.5 (C-20), 115.9 (C-
9a), 120.2 (C-8a), 122.8 (C-8), 123.5 (C-7), 128.3 (C-6), 128.5 (C-5),
147.2 (C-10a), 151.2 (C-9), 158.3 (C-4a). Anal. Calcd for C27H41N5
(435.66): C, 74.44; H, 9.49; N, 16.08. Found: C, 74.56; H, 9.64; N,
16.09.
4.1.4. Synthesis of 3-{[2-(1,2,3,4-tetrahydroacridin-9-ylamino)
ethyl]amino}-1-{4-[2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl]
piperazino}-1-propanone (8)
4.1.3.2. Synthesis of N-(2-{4-[3-(cyclohexyl(methyl)amino)propyl]
piperazino}ethyl)-(1,2,3,4-tetrahydroacridin-9-yl)amine (6b). Starting
with 5b and LiAlH4, followed by the general procedure and column
chromatograpghy (eluent CHCl3/MeOH/Et3N, 4/1/0.05), led to 45 mg
A solution of N-(2-aminoethyl)-1,2,3,4-tetrahydroacridin-9-
ylamine (7, 90 mg, 0.37 mmol) in CH2Cl2 (2 ml), 3-chloro-1-{4-[2-
(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl]-piperazino}-1-
propanone (4, 160 mg, 0.40 mmol) in CH2Cl2 (2 ml), and DIPEA
(70 mg, 0.55 mmol) was stirred at rt for 72 h. The reaction mixture
was cooled and evaporation of the solvent afforded a residue which
was dissolved in CH2Cl2 (5 ml) and washed with water (10 ml) and
NaCl (10 ml). The collected organic fractions were dried over
Na2SO4 and the solvent was removed under reduced pressure. The
residue was purified by column chromatography over silica gel in
eluent EtOAc/MeOH/NH4OH, 6/2/0.2. Compound 7 (150 mg, 63%
yield) was obtained as yellow oil. 1H NMR (CDCl3, 400 MHz)
(25%) of 6b as oil. 1H NMR (CDCl3, 400 MHz)
d
1.18e1.28 (m, 10H,
5
ꢂ
CH2, H-140,150,160,170,180), 1.68e1.71 (m, 2H, CH2, H-100),
1.89e1.95 (m, 4H, 2 ꢂ CH2, H-2,3), 2.30 (s, 3H, CH3, H-100), 2.35e2.42
(m, 1H, CH, H-130), 2.45e2.58 (m, 12H, 6 ꢂ CH2, H-40,50,70,80,90,110)
2.61 (t, 2H, CH2, H-20, J ¼ 6.0 Hz), 2.76 (t, 2H, CH2, H-1, J ¼ 5.6 Hz),
3.07 (t, 2H, CH2, H-4, J ¼ 6.0 Hz), 3.56 (t, 2H, CH2, H-10, J ¼ 6.0 Hz),
7.33 (ddd, 1H, CH, H-7, J1 ¼1.2 Hz, J2 ¼ 7.2 Hz, J3 ¼ 8.4 Hz), 7.54 (ddd,
1H, CH, H-6, J1 ¼1.2 Hz, J2 ¼ 7.2 Hz, J3 ¼ 8.4 Hz), 7.91 (d, 1H, CH, H-5,
J ¼ 8.2 Hz), 8.02 (d, 1H, CH, H-8, J ¼ 8.2 Hz). 13C NMR (CDCl3)
d
22.8,
d
1.89e1.96 (m, 8H, 4 ꢂ CH2, H-2,3,200,300), 2.47e2.55 (m, 8H,
23.1 (C-2,3), 25.2 (C-1), 26.0 (C-150,170), 26.3 (C-160), 28.5 (C-140,180),
30.0 (C-100), 33.8 (C-4), 37.8 (C-100), 45.1 (C-10), 51.6 (C-110), 52.6, 53.5
(C-40,50,70,80,90), 57.5 (C-20), 63.0 (C-130), 115.9 (C-9a), 120.2 (C-8a),
122.8 (C-8), 123.5 (C-7), 128.3 (C-6), 128.5 (C-5), 147.2 (C-10a), 151.2
(C-9), 158.4 (C-4a). Anal. Calcd for C29H45N5 (463.72): C, 75.12; H,
9.78; N, 15.10. Found: C, 75.29; H, 9.85; N, 15.30.
4 ꢂ CH2, H-40,80,110,130), 2.63 (t, 2H, CH2, H-20, J ¼ 6.0 Hz), 2.75e2.83
(m, 4H, 2 ꢂ CH2, H-1,100), 3.01e3.09 (m, 4H, 2 ꢂ CH2, H-4,400),
3.54e3.63 (m, 6H, 3 ꢂ CH2, H-10,100,140), 3.67e3.79 (m, 4H, 2 ꢂ CH2,
H-50,70), 4.76 (bs, 1H, NH), 5.04 (bs, 2H, 2 ꢂ NH), 7.31e7.38 (m, 2H,
2 ꢂ CH, H-7,700), 7.52e7.59 (m, 2H, 2 ꢂ CH, H-6,600), 7.88e7.95 (m,
2H, 2 ꢂ CH, H-5,500), 7.97e8.20 (m, 2H, 2 ꢂ CH, H-8,800). 13C NMR
(CDCl3)
d
22.8, 23.1 (C-2,3,200,300), 25.0 (C-1,100), 34.0 (C-4,400), 41.7,
4.1.3.3. Synthesis of N-(2-{4-[3-(cyclohexyl(ethyl)amino)propyl]piper-
azino}ethyl)-(1,2,3,4-tetrahydroacridin-9-yl)amine (6c). Starting with
5c and LiAlH4, followed by the general procedure and column
chromatograpghy (eluent CHCl3/MeOH/Et3N, 4/1/0.05), led to 39 mg
42.0 (C-50,70), 45.0 (C-10), 45.5 (C-100), 45.8 (C-140), 52.3 (C-110,130),
52.7, 52.8 (C-40,80), 57.6 (C-20), 116.1 (C-9a,9a00), 120.3 (C-8a,8a00),
122.5 (C-8,800), 123.6 (C-7,700),128.3 (C-6,600), 128.7 (C-5,500),147.4 (C-
10a,10a00), 150.7 (C-9,900), 158.5 (C-4a,4a00), 168.1 (C-90). Anal. Calcd
for C37H47N7O (605.83): C, 73.36; H, 7.82; N, 16.18. Found: C, 73.56;
H, 7.88; N, 16.38.
(21%) of 6c as oil. 1H NMR (CDCl3, 400 MHz) 0.90 (m, 3H, CH3, H-200),
d
1.18e1.28 (m, 10H, 5 ꢂ CH2, H-140,150,160,170,180), 1.64e1.73 (m, 2H,
CH2, H-100), 1.81e1.96 (m, 10H, 4 ꢂ CH2, H-2,3), 2.23e2.28 (m, 2H,
CH2, H-100), 2.35e2.42 (m, 1H, CH, H-130), 2.47e2.62 (m, 12H,
6 ꢂ CH2, H-40,50,70,80,90,110), 2.63e2.73 (m, 4H, 2 ꢂ CH2, H-1,20),
3.16e3.23 (m, 2H, CH2, H-4), 3.62e3.72 (m, 2H, CH2, H-10), 7.38 (dd,
1H, CH, H-7, J1 ¼1.2 Hz, J2 ¼ 7.2 Hz),7.62 (dd, 1H, CH, H-6, J1 ¼1.2 Hz,
J2 ¼ 7.2 Hz), 8.08 (d, 1H, CH, H-5, J ¼ 8.2 Hz), 8.21 (d, 1H, CH, H-8,
4.2. Pharmacological studies
4.2.1. Determination of inhibitory potency on hAChE and hBChE
An AChE and BChE inhibitory activity of the tested drugs was
determined using Ellman’s method [39] and was expressed as IC50
,
J ¼ 8.2 Hz). 13C NMR (CDCl3)
d
14.1 (C-200), 22.5, 22.6 (C-2,3), 24.2 (C-
i.e. concentration that reduces the cholinesterase activity by 50%.
Human recombinant AChE (AChE; EC 3.1.1.7), human plasmatic
BChE (BChE; EC 3.1.1.8), 5,50-dithiobis(2-nitrobenzoic acid) (Ell-
man’s reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylth-
iocholine (ATC), and butylthiocholine (BTC), and tacrine
hydrochloride were purchased from SigmaeAldrich, Praque, Czech
Republic. For measuring purposes e polystyrene cuvette (-Brand
GmbH þ Co. KG, Denmark) was utilized. All the assays were carried
out in a 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), 26.1 (C-150,170), 26.4 (C-160), 29.7 (C-140,180), 30.0 (C-100), 32.9 (C-
4), 44.4 (C-10), 45.3 (C-100), 52.4, 53.3 (C-40,50,70,80,90,110), 57.6 (C-20),
60.7 (C-130), 116.2 (C-9a), 120.3 (C-8a), 123.3 (C-8) 124.2 (C-7), 125.4,
(C-6), 132.3 (C-5), 147.3 (C-10a), 151.3 (C-9), 158.2 (C-4a). Anal. Calcd
for C30H47N5 (477.74): C, 75.42; H, 9.92; N, 14.66. Found: C, 75.46; H,
10.07; N, 14.71.
4.1.3.4. Synthesis of N-(2-{4-[3-(methylanilino)propyl]piperazino}
ethyl)-(1,2,3,4-tetrahydroacridin-9-yl)amine (6d). Starting with 5d
and LiAlH4, followed by the general procedure and column chro-
matograpghy (eluent CHCl3/MeOH/Et3N, 4/1/0.05), led to 35 mg
(1 ml) consisted of 650
mL of 0.1 M phosphate buffer (pH 7.4), 200
mL
of 0.01 M DTNB, 25 L of enzyme, and 100
m
mL of 0.01 M substrate
(20%) of 6d as oil. 1H NMR (CDCl3, 400 MHz)
d
1.65e1.70 (m, 2H,
(ATC chloride solution). Assay solutions with inhibitor
CH2, H-100), 1.88e1.94 (m, 4H, 2 ꢂ CH2, H-2,3), 2.45e2.58 (m, 10H,
(10ꢃ3e10ꢃ10 M) were preincubated for 5 min. The reaction was
5
ꢂ
CH2, H-40,50,70, 80,90,110), 2.59e2.67 (m, 2H, CH2, H-20),
started by an immediate addition of 100 mL of substrate. The
2.72e2.77 (m, 2H, CH2, H-1), 2.83 (s, 3H, CH3, H-100), 3.04e3.09 (m,
2H, CH2, H-4), 3.55e3.61 (m, 2H, CH2, H-10), 6.61 (dd, 2H, 2 ꢂ CH, H-
140,180, J1 ¼ 2.0 Hz, J2 ¼ 8.0 Hz), 6.70 (ddd, 1H, CH, H-160, J1 ¼ 2.0. Hz,
activity was determined by measuring the increase in absorbance
at 412 nm at 1 min intervals using a spectrophotometer Helios-
Zeta (Thermospectronic, Cambridge UK). Each concentration was

S. Hamulakova et al. / European Journal of Medicinal Chemistry 55 (2012) 23e31
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31
assayed in triplicate. In vitro BChE assay was similar with the
method described above. In addition we calculated the corre-
sponding selectivity index (Ratio ¼ [IC50 (BChE)]/[IC50 (AChE)]).
This method has been optimized to determine the constants Ki1 and
Ki2. Software Origin 6.1 (Northamption USA) was used for the
statistical data evaluation.
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4.2.2. Molecular modeling studies
Molecular models of the derivatives 5bed and 6b were
computer-built by the means of building options in the Marvin 5.1.4
2008, ChemAxon [http://www.chemaxon.com]. The same software
was used to determine the overall protonization of compounds. In
order to prepare the input files, docking simulations were carried
out using AUTODOCK ver. 4.2. MGL TOOLS 1.4.5 (revision 30)
[43,44]. Molecules of water with other nonenzymatic molecules
were removed and hydrogens were added. For ligands and
enzymes, a united atom representation was used. Gasteiger partial
atomic charges for proteins and ligands were added. For the initial
docking, the grid for energy was set in the coordinates x ¼ 116.4,
y ¼ 104.3, z ¼ ꢃ130.6 within the hAChE (PDB ID: 1B41) and
x ¼ 138.7, y ¼ 116.3, z ¼ 41.0 within the hBChE (PDB ID: 1P01) active
site with dimensions 80 points x 80 points x 80 points and with
spacing of 0.375 Å. Thus, a ligand pose with the lowest energy was
chosen as the space for the construction of the redocking energy
grid with coordinates x ¼ 117.0, y ¼ 109.5, z ¼ ꢃ132.2 and with
dimensions 42 points ꢂ 42 points ꢂ 42 points within hAChE (PDB
ID: 1B41). Likewise for hBChE (PDB ID: 1P01), coordinates x ¼ 136.5,
y ¼ 111.1, z ¼ 40.8 with dimensions 49 points ꢂ 55 points ꢂ 43
points were set. Spacing of 0.375 Å was set for both enzymes, which
was used to define rotatable bonds in ligands. A flexible ligand
docking was performed for the compounds. Docking runs were
performed using the Lamarckian genetic algorithm. Docking began
with a population of random ligand conformations in random
orientation and at random translation. Each docking experiment
was derived from 100 different runs that were set to terminate after
a maximum of 5,000,000 energy evaluations or 27,000 generations.
The population size was set to 500. Other parameters were used as
default. Pictures were prepared using Chimera software [41].
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Acknowledgments
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We thank the Slovak Grant Agency VEGA (1/0672/11 and 1/
0179/11), the State NMR Program (grant No. 2003SP200280203)
and the Czech Grant Agency (grant P303/11/1907) for financial
support of this study. 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 (supported by NIH P41 RR-01081).
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Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the onlineversion, at http://dx.doi.org/10.1016/j.ejmech.2012.06.051.
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