7-methoxytacrine-adamantylamine heterodimers as cholinesterase inhibitors in Alzheimer's disease treatment - Synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.3390/molecules18022397

A structural series of 7-MEOTA-adamantylamine thioureas was designed, synthesized and evaluated as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). The compounds were prepared based on the multi-target-directed ligand strategy with different linker lengths (n = 2-8) joining the well-known NMDA antagonist adamantine and the hAChE inhibitor 7-methoxytacrine (7-MEOTA). Based on in silico studies, these inhibitors proved dual binding site character capable of simultaneous interaction with the peripheral anionic site (PAS) of hAChE and the catalytic active site (CAS). Clearly, these structural derivatives exhibited very good inhibitory activity towards hBChE resulting in more selective inhibitors of this enzyme. The most potent cholinesterase inhibitor was found to be thiourea analogue 14 (with an IC₅₀ value of 0.47 μM for hAChE and an IC₅₀ value of 0.11 μM for hBChE, respectively). Molecule 14 is a suitable novel lead compound for further evaluation proving that the strategy of dual binding site inhibitors might be a promising direction for development of novel AD drugs.

Full text of DOI:10.3390/molecules18022397

Molecules 2013, 18, 2397-2418; doi:10.3390/molecules18022397
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
7-Methoxytacrine-Adamantylamine Heterodimers as
Cholinesterase Inhibitors in Alzheimer’s Disease
Treatment — Synthesis, Biological Evaluation and Molecular
Modeling Studies
Katarina Spilovska 1, Jan Korabecny 1,2, Jan Kral 3, Anna Horova 1, Kamil Musilek 1,2,4
,
Ondrej Soukup 2, Lucie Drtinova 1, Zuzana Gazova 5, Katarina Siposova 5,6 and Kamil Kuca 2,7,
*
1
Department of Toxicology, Trebesska 1575, Faculty of Military Health Sciences,
University of Defence, 500 01 Hradec Kralove, Czech Republic; E-Mails: spilovska@pmfhk.cz (K.S.);
korabecny.jan@gmail.com (J.K.); horova.a@entrum.cz (A.H.); kamil.musilek@uhk.cz (K.M.);
luciedrtinova@centrum.cz (L.D.)
2
3
University Hospital, Biomedicinal Research Centre, Sokolska 581, 500 05 Hradec Kralove,
Czech Republic; E-Mail: soukup.ondrej07@gmail.com (O.S.)
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy,
Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic;
E-Mail: kral.jan@email.cz
4
5
Department of Chemistry, Faculty of Sciences, University of Hradec Kralove, Rokitanskeho 62,
500 03 Hradec Kralove, Czech Republic
Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Watsonova 47, 040 01 Kosice, Slovakia; E-Mails: gazova@saske.sk (Z.G.);
katkasiposova@gmail.com (K.S.)
6
7
Faculty of Sciences, Institute of Chemistry, P. J. Safarik University, Srobarova 2, 041 54 Kosice,
Slovakia
Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defence,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; E-Mail: kucakam@pmfhk.cz
* Author to whom correspondence should be addressed; E-Mail: kucakam@pmfhk.cz;
Tel.: +420-973-253-028; Fax: +420-495-518-094.
Received: 14 January 2013; in revised form: 24 January 2013 / Accepted: 4 February 2013 /
Published: 20 February 2013
Abstract: A structural series of 7-MEOTA-adamantylamine thioureas was designed,
synthesized and evaluated as inhibitors of human acetylcholinesterase (hAChE) and
human butyrylcholinesterase (hBChE). The compounds were prepared based on the

Molecules 2013, 18
multi-target-directed ligand strategy with different linker lengths (n = 2–8) joining the
2398
well-known NMDA antagonist adamantine and the hAChE inhibitor 7-methoxytacrine
(7-MEOTA). Based on in silico studies, these inhibitors proved dual binding site character
capable of simultaneous interaction with the peripheral anionic site (PAS) of hAChE and
the catalytic active site (CAS). Clearly, these structural derivatives exhibited very good
inhibitory activity towards hBChE resulting in more selective inhibitors of this enzyme.
The most potent cholinesterase inhibitor was found to be thiourea analogue 14 (with an
IC50 value of 0.47 µM for hAChE and an IC50 value of 0.11 µM for hBChE, respectively).
Molecule 14 is a suitable novel lead compound for further evaluation proving that the
strategy of dual binding site inhibitors might be a promising direction for development of
novel AD drugs.
Keywords: 7-MEOTA; amantadine; inhibitor; Alzheimer’s disease; acetylcholinesterase;
butyrylcholinesterase
1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia. It is the fourth leading cause of
mortality in the US alone [1]. In Asia, AD is the principal cause of dementia and accounts for 50–60%
of all cases, lasting for about 3–20 years from diagnosis to death. It was first documented in 1906
when Alois Alzheimer, a German psychiatrist and neuropathologist, reported the curious case of one of
his patients, who suffered from memory problems, speaking impairment and difficulty with
comprehensive understanding [2]. AD is a neurodegenerative disorder that results in the progressive
and irreversible cognitive impairment, memory loss, and decline in language [3–5]. Several diverse
hallmarks, such as deposits of aberrant proteins (β-amyloid and τ-protein), oxidative stress,
dyshomeostasis of biometals, and low levels of acetylcholine (ACh) appear to play significant roles in
the pathophysiology of the disease [6,7]. Moreover, AD also represents an economic burden, which
causes profound social problems to both society and families [8,9].
The current standard of care for mild to moderate AD, based on the so-called cholinergic hypothesis,
includes treatment with cholinesterase inhibitors (ChEIs) to improve cognitive functions [5,10,11]. Several
ChEIs have been approved by the US Food and Drug Administration (FDA), including tacrine,
donepezil, rivastigmine and galantamine (Figure 1) [12–16]. Furthermore, memantine (1-amino-3,5-
dimethyladamantane hydrochloride, Figure 1), an uncompetitive antagonist of N-methyl-D-aspartate
(NMDA) receptors has been found to provide symptomatic benefits in clinical trials in AD
patients [17–19]. Evidence of memantine’s neuroprotective effects (e.g., the ability to slow
neurodegeneration) has been gathered primarily from preclinical models of the disease [20].
Memantine is a derivative of amantadine (1-adamantylamine hydrochloride, Figure 1), an antiviral
agent that has long been used clinically to treat Parkinson’s disease (PD) in the US and in Europe. It
also possesses antiepileptic properties, and is currently used in the AD treatment [21]. As an
uncompetitive NMDA receptor antagonist, it proved low occurrence of side effects and showed good
clinical tolerability in more than 200,000 treated patients [22]. It exhibited anti-neuroinflammatory

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effect with ability to act on glial cells. Memantine increased astroglial release of neurotrophic factors
and inhibited inflammatory activation of microglia [23,24]. Amantadine is an established anti-PD
agent with an inhibitory mechanism of action at NMDA receptors [25]. Indeed, evidence suggests that
amantadine may delay the onset and severity of dementia related to PD similarly to its analogue
memantine, which is regularly used in AD therapy [26,27].
In 1993, tacrine (9-amino-1,2,3,4-tetrahydroacridine, THA, Figure 1) was approved by the US Food
and Drug Administration agency (FDA) as the first ChEI for the management of AD [28]. However, it
was later withdrawn from the pharmaceutical market due to hepatotoxicity issues. The 7-methoxy
derivative, 7-MEOTA (9-amino-7-methoxy-1,2,3,4-tetrahydroacridine, Figure 1) was found to be also
an active ChEI with significantly lower side effects compared to THA [29–34].
Figure 1. Structures of ChEIs, tacrine derivatives and polycyclic amines.
Considering the complexity of AD, the standard, one molecule-one target solution may not be
effective enough [35,36]. The novel “multi-target-directed strategy” has received attention, since a
single molecule simultaneously interacts with multiple targets in the complex neuronal cascades. The
multi-targeted analogues could achieve better efficacy by a complementary manner [36,37]. The
studies following this strategy led to the synthesis of several chemically diverse structures with dual or
multiple biological profiles [38], including acetylcholinesterase (AChE) and monoamine oxidase B
(MAO-B) dual inhibitors [39–41], AChE and serotonin transporter (SER) dual inhibitor [42,43] and
AChE, BACE, Aβ aggregation inhibiting and antioxidant multiple functional agents [44–49].
In this presented work, the attention was focused on dual-binding site heterodimers. A series of
7-MEOTA-adamantylamine thioureas designed to simultaneously interact with the active and
peripheral binding sites of both human AChE (hAChE, EC 3.1.1.7) and human butyrylcholinesterase
(hBChE, EC 3.1.1.8) were synthesized. Novel analogues could be effective in the AD treatment with
respect to their ability to interact with multiple targets. The synthesis, biological evaluation and
molecular modeling studies of the new 7-MEOTA-adamantylamine thioureas is reported. The design
strategy of 7-MEOTA-amantadine heterodimers is displayed in Figure 2.

Molecules 2013, 18
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Figure 2. Design strategy of novel dual inhibitors.
2. Results and Discussion
The general synthetic procedure for 7-MEOTA-adamantylamine heterodimers is shown in Scheme 1.
The starting fused ring 7-methoxy-1,2,3,4-tetrahydroacridin-10H-9-one (1) was prepared in good yield
(80%) by a condensation reaction of 4-methoxyaniline with ethyl 2-oxocyclohexanecarboxylate in
refluxing toluene using addition of catalytic p-toluenesulfonic acid. Then, 1 was stirred with
phosphorus oxychloride to give 9-chloro-7-methoxy-1,2,3,4-tetrahydroacridine (2) in quantitative
yield. Spectral data were in good agreement with the literature characterization [28].
The treatment of 2 with appropriate 1,ω-diamines in the presence of phenol yielded the desired
N-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)alkane-1,ω-diamine intermediates 3–9 (70–95%).
1-Adamantyl isothiocyanate (10) was prepared in quantitative yield from 1-adamantyl amine via
desulfurylated dithiocarbamate using carbon disulfide and di-tert-butyl dicarbonate. Spectral data were
in good agreement with the literature [50–52].
Two synthons 10 and diamines 3–9 were utilized for formation of desired 7-MEOTA-adamantylamine
thioureas 11–17. All targeted compounds 11–17 were converted into tartaric salts to increase
solubility. The final compounds were obtained as white-yellow powders in satisfactory yield
(41–84%). Structural determination and signal assignments of thioureas were accomplished by the
application of the usual combination of 1H- and 13C-NMR spectra.

Molecules 2013, 18
Scheme 1. Synthetic route for preparation 7-MEOTA-adamantylamine thioureas 11–17.
2401
The in vitro activity of new 7-MEOTA-diamines 3–9 and 7-MEOTA-amantadine heterodimers 11–17
was determined on the model of human recombinant AChE (hAChE; E.C. 3.1.1.7) and human
plasmatic BChE (hBChE; E.C. 3.1.1.8) using Ellman’s method [53,54] with THA, 7-MEOTA,
amantadine and 10 as reference compounds (Table 1). THA was a more potent inhibitor of hAChE and
hBChE in comparison to 7-MEOTA, amantadine and 10. THA is a two orders of magnitude better
inhibitor of hAChE than 7-MEOTA and 10 and a one order of magnitude better inhibitor of hAChE in
comparison to amantadine. Additionally, 7-MEOTA, amantadine and 10 were weaker hBChE
inhibitors compared to THA and showed higher selectivity. All intermediate diamines 3–9 were potent
inhibitors of both cholinesterases, producing hAChE IC50 values in the range of 5.32–0.21 µM.
Inhibitory activity of hBChE was less influenced by intermediate diamines 3–9 ranging from
64.64–7.26 µM. New prepared 7-MEOTA-diamines 3–9 did not exceed THA inhibitory activity
toward hBChE, but diamine 7 showed higher inhibitory activity to hAChE. Moreover, all amines 3–9
are more potent inhibitors of hAChE than 7-MEOTA. From the series of 7-MEOTA-diamines, the best
IC50 value was shown by diamine 7 bearing six carbons in the spacer. The selectivity index (SI,
determined as the IC50hBChE/IC50hAChE ratio) was calculated for all newly evaluated compounds.
The intermediates amines 3–9 displayed higher selectivity for hAChE than THA, 7-MEOTA,

Molecules 2013, 18
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amantadine or 10, while compound 7 expressed the highest selectivity index for hAChE. The amines
were used as syntons for the synthesis of 7-MEOTA-amantadine heterodimers 11–17. Surprisingly, the
best inhibitory activity of thioureas 11–17 was demonstrated in thiourea analogue 14 with five carbons
in the linker. Considering standards, inhibition activities towards both tested enzymes were exceeded
only by compound 14. Interestingly, compound 7 with six methylenes displayed a better IC50 value
than derivative 14, which had only five carbons between 7-MEOTA and adamantine moieties in the
spacer (not counting the thiourea group). The novel thiourea derivatives proved to be better inhibitors
of both cholinesterases compared with 7-MEOTA. However, some of the heterodimers exhibited
slightly poorer hAChE and hBChE inhibitory activities in comparison with THA. The IC50 values
suggested that compound 14 exerted similar IC50 in sub-µM range for hAChE/hBChE to reference
compound THA. All of the novel compounds have lower SI values compared to 7-MEOTA,
amantadine and 10, so they can be considered as more selective agents for hBChE. This could provide
an advantage for AD course as hBChE inhibition has recently been regarded therapeutically beneficial
for the treatment of AD. Concentration of hBChE, contrary to hAChE, increases during the course of
the AD and may compensate the role of hAChE [55]. Enzyme activity plots of THA, 7-MEOTA and
14 are displayed (Figures 3 and 4). Hence, molecule 14 with the length of five methylene units in the
linker remains the most potent hAChE/hBChE inhibitor among all the newly synthesized thioureas.
Table 1. IC50 values of amines, standards and tested thioureas.
Compound
IC50 (µM) ± SD b
SI c
h AChE
hBChE
0.023 ± 0.003
21.0 ± 3.4
THA
0.5 ± 0.1
10.50 ± 2.40
16.05 ± 3.13
5.32 ± 1.04
1.93 ± 0.38
1.42 ± 0.28
3.44 ± 0.67
0.21 ± 0.04
0.86 ± 0.17
0.47 ± 0.09
24.96 ± 4.87
5.02 ± 0.98
0.53 ± 0.10
2.04 ± 0.39
0.47 ± 0.09
2.09 ± 0.40
3.47 ± 0.67
1.62 ± 0.31
0.05
2.0
7-MEOTA
amantadine
102.60 ± 17.13
64.45 ± 10.76
49.77 ± 8.31
9.22 ± 1.54
29.63 ± 4.95
10.84 ± 1.81
7.26 ± 1.21
10.08 ± 1.68
96.90 ± 16.18
6.02 ± 1.01
1.39 ± 0.23
0.98 ± 0.16
0.11 ± 0.02
0.33 ± 0.05
0.15 ± 0.02
0.26 ± 0.04
6.4
3 a
4 a
5 a
6 a
7 a
8 a
9 a
10
11
12
13
14
15
16
17
12.1
25.8
6.5
8.6
51.6
8.4
21.4
3.9
1.2
2.6
0.5
0.2
0.2
0.04
0.2
a
b
Compounds 3–9 were tested as dihydrochloride salts. The in vitro concentration of tested compound
required to produce 50% inhibition of hAChE or hBChE. Results are the mean of three independent
determinations ± standard deviation. c Selectivity index (SI) for hAChE is determined as ratio of IC50 hBChE
towards IC50 hAChE.

Molecules 2013, 18
Figure 3. Enzyme activity plot of THA, 7-MEOTA and 14 for hAChE.
2403
THA
7-MEOTA
14
100
80
60
40
20
0
-10
-8
-6
-4
-2
log C
Figure 4. Enzyme activity plot of THA, 7-MEOTA and 14 for hBChE.
THA
7-MEOTA
14
100
80
60
40
20
0
-10
-8
-6
-4
-2
log C
To shed light onto the putative orientation of the most promising cholinesterase inhibitor 14 in both
cholinesterases, molecular modeling studies were carried out using the Autodock Vina software. The
molecular modelling results are shown in Figures 5–7. A structure of hAChE complexed with
fasciculin (PDB ID: 1B41) was used for the in silico studies and exploited in the in vitro biochemical
assays presented above. The proposed binding mode of 14, THA and 7-MEOTA with the interacting
key amino acid residues (in blue) in hAChE active site is shown in Figure 5. In complex with THA

Molecules 2013, 18
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(−9.9 kcal/mol; in yellow), the three-ring structure is stacked against the phenyl ring of Tyr337 (3.4 Å)
as well as showing T-shaped π-π bonding with Trp86 (1.0 Å). THA amino moiety is stabilized by
hydrogen bond to carbonyl group of His447 (4.3 Å). Phe338 (4.3 Å) is weakly involved into the direct
aliphatic-π interaction. All these findings fully correspond with that of previously published for THA
in the active site of Torpedo californica AChE (TcAChE) [56]. Similar spatial conformation was found
for the top-scored docking pose of 7-MEOTA (−9.8 kcal/mol, in magenta). Interestingly, 7-MEOTA-
hAChE complex is additionally stabilized with hydrogen bonds of the methoxy moiety and Ser203
(2.2 Å) and the methoxy group is also attached to Gly122 (2.4 Å). Moreover, 7-MEOTA resulted
180 degrees rotated from THA with the same position of 9-amino moiety. Despite the fact of forming
additional hydrogen bonds with the methoxy group of 7-MEOTA, this different spatial conformation
compared to THA might explain its lower inhibition properties in vitro.
Figure 5. Putative binding mode of 14 (green), THA (yellow) and 7-MEOTA (magenta).
Some important amino acid residues (blue) are shown, the rest of hAChE is superimposed
in cartoon conformation.
The binding mode for 14 (−11.1 kcal/mol, in green) suggested that the 7-MEOTA fragment was
bound to near the bottom of the gorge in a slightly different arrangement compared to the parent
compounds THA and 7-MEOTA. The 7-MEOTA scaffold of 14 exerted strong parallel π-π stacking to
Tyr337 (4.2 Å) as well as T-shaped π-π stacking to Trp86 (2.8 Å) within the cation-π site of hAChE.
The methoxy moiety contributed to stabilization of 14-hAChE complex by hydrogen bonding with
Ser125 (2.3 Å). The linkers showed moderate importance on cholinesterase inhibition as the analogue
14 emerged as the most promising with the length of five methylenes between 7-MEOTA scaffold and
adamantyl moiety with small difference to other thioureas. Similar results can be observed for different
series of multi-target directed ligands based on tacrine scaffold including bis-7-tacrine, tacrine-
piperazine derivatives, tacrine-hupyridone dimers, tacrine-melatonin heterodimers, pyrano[3,2-c]-

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quinoline-6-chlorotacrine hybrids and tacrine-ferulic acid nitric-oxide donors [45,47,57–60]. In the
middle of the gorge, the aliphatic alkyl chain of 14 was surrounded with phenyl rings of Tyr124
(3.7 Å), Phe297 (3.5 Å) and Phe338 (4.3 Å) providing 14-hAChE complex stabilization and
constriction. Thiourea group forms very weak hydrogen bonding to hydroxyl group of Tyr72 (3.4 Å).
At the rim of the gorge within peripheral anionic site, adamantyl moiety may have aliphatic-π contact
with Trp286 (2.7 Å) and several weak van der Waals interactions (e.g., Leu289—3.3 Å, Ser293—3.7 Å).
Lower affinity of 14 for hAChE inhibition might be explained by non-aromatic character of
interactions with adamatyl moiety between the key residues in peripheral anionic site. Finally, the
distance between 7-MEOTA and adamantyl moieties had a length of 18.2 Å which correlates with the
ideas of dual binding site heterodimers, where the distance between peripheral anionic site and
catalytic site of hAChE is estimated to about 20 Å [61].
Figure 6. Docked complex of 14 (green) in the hBChE active site. Interaction with key
amino acid residues (blue) are highlighted by black dashed lines. The rest of hBChE is
illustrated as cartoon for clarity.
The crystal structure of THA with hBChE is not available for molecular modeling. To gain insight
into the molecular determinants that modulate the inhibitory activity of the novel 7-MEOTA-adamantine
hybrids, the crystal structure of hBChE (PDB ID: 1P01) that was previously modeled with bound
butyrate was chosen. Flexible docking studies for hBChE showed different spatial orientation for
standard compounds (THA, 7-MEOTA) and 14 (Figures 6 and 7). Focused on reference compounds,
THA (−9.5 kcal/mol, in yellow) and 7-MEOTA (−9.3 kcal/mol, in magenta) showed very similar
orientation with several apparent π-π interactions, that were for THA mainly in T-shaped orientation
(Tyr332—3.3 Å, Trp430—4.6 Å, Trp82—2.4 Å) as well as parallel oriented (Tyr440—3.7 Å).

Molecules 2013, 18
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Hydrogen bonds are formed between THA amino group and Trp430 (3.7 Å) and Ser79 (3.6 Å).
Interestingly, 7-MEOTA provided almost identical putative orientation with additional hydrogen bond
of Trp82 (3.5 Å) towards the methoxy moiety. According to in silico results, 7-MEOTA could be
considered a stronger hBChE inhibitor than THA, but the obtained in vitro results highlighted THA as
a three orders of magnitude better BChE inhibitor. The three-ring core of analogue 14 (−10.3 kcal/mol,
in green) establishes π-π interactions with Trp231 (4.0 Å) and Phe329 (3.9 Å). The nitrogen atom of
the tacrine moiety is bridged to His438 via hydrogen bonding (2.2 Å), the secondary amino group
preserves a weak H-bond to the hydroxyl group of Thr120 (4.2 Å). The dual binding character of
Tyr332 is depicted. This amino acid residue allows H-bonds with its OH moiety to the thiourea linker
(3.9 Å). Aliphatic-π contact can be observed within adamantyl skeleton and Tyr332 (3.8 Å) as well as
with Trp82 (3.4 Å). Finally, the in silico calculated binding energies for hBChE suggested similar or
even higher affinity towards this enzyme, but in vitro 14 resulted one order of magnitude weaker
inhibitor of hBChE reflected to THA.
Figure 7. Top-scored docking poses representation within hBChE active site for 14
(green), THA (yellow) and 7-MEOTA (magenta).
Structural determination and signal assignment of thioureas 11–17 were accomplished by
the application of the usual combination of 1H and 13C-NMR spectra. The unequivocal
assignments were performed by homo- and hetero-correlated two-dimensional NMR experiments
(H,H-COSY, H,C-HSQC, H,C-HMBC). Infrared spectroscopy was conducted only for 10 to observe
1
the isothiocyanate group vibrations. The H- and 13C-NMR spectra, synthesis analyses and mass
spectra are collected in the Experimental section. For the aromatic part of 7-MEOTA moiety
correlations between signals at 7.74–7.79 ppm and 7.35–7.47 ppm allow the assignment of H-5 and
H-6, respectively. The signal at 7.53–7.63 ppm is assigned to H-8. For the alicyclic part of 7-MEOTA
skeleton correlations between signals of the multiplet at 1.79–1.82 ppm allow the assignment of H-2
and H-3, and H-1 and H-4 were assigned based on the correlation between the triplets

Molecules 2013, 18
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at 2.69–2.76 ppm and 2.93–2.97 ppm, respectively. The OCH3 carbon signal was conclusively
assigned on the basis of the correlation between 3.88–3.91 ppm. As for the adamantyl-moiety
correlation between the multiplet at 1.53–2.00 allow the assignment of H-4′′, H-7′′, H-9′′ to be
unequivocally assigned. For the part of the adamantyl-moiety correlation at 1.97–2.00 allows assignment of
H-3′′, H-5′′, H-8′′. The multiplet at 2.05–2.13 is assigned to H-2′′, H-6′′, H-10′′. Thioureas 11–17
displayed a typical C=S carbon resonance at 180.8–181.1 ppm. The thioureas 11–17 were each
converted into the thioureas dihydroxysuccinates using tartaric acid. These thiourea
dihydroxysuccinates were determined on the correlation between the multiplet at 3.98–4.09 ppm to
H-2′′′, H-3′′′ and the carbon signal of C-1′′′, C-4′′′ allowing assignment of signals at 174.0–174.6 ppm,
and 71.8–72.0 ppm to C-2′′′, C-3′′′, respectively.
3. Experimental
3.1. Chemistry
7-MEOTA was prepared at our department according to the method described earlier [15]. All
reagents were reagent grade quality and obtained from Sigma-Aldrich (Prague, Czech Republic). All
experiments were carried out under nitrogen atmospheres. Thin layer chromatography (TLC) was
performed on aluminium sheets precoated with silica gel 60 F254 (Merck, Prague, Czech Republic).
Column chromatography was performed at normal pressure on silica gel 100 (particle size
0.063–0.200 mm, 70–230 mesh ASTM, Fluka, Prague, Czech Republic). Elemental analysis was
measured at Perkin-Elmer CHN Analyser 2,400 Serie II apparatus. Mass spectra were recorded using a
combination of high performance liquid chromatography and mass spectrometry. The HP1100 HPLC
system was obtained from Agilent Technologies (Waldbronn, Germany). It consisted of a G1322A
vacuum degasser, G1311A quaternary pump, G1313A autosampler and a MSD1456 VL quadrupole
mass spectrometer equipped with an electrospray ionization source. Nitrogen for mass spectrometer
was supplied by a Whatman 75–720 nitrogen generator. Data were collected in positive ion mode
with an ESI probe voltage of 4000 V. The pressure of nebulizer gas was set up to 35 psig. Drying
1
gas temperature was operated at 335 °C and flow at 13 L/min. H-NMR and 13C-NMR spectra
were recorded with a Varian S500 spectrometer operating at 500 and 125 MHz, respectively,
in deuteriochloroform (CDCl3; 7.27 (D), 77.2 (C) ppm) or hexadeuteriodimethylsulfoxide
(DMSO-d6; 2.50 (D), 39.7 (C) ppm) using tetramethylsilane (TMS) as internal reference (=0 ppm for
both nuclei). Chemical shifts are reported in parts per milion (ppm, δ) relative to TMS. The assignment
1
1
of chemical shifts is based on standard NMR experiments (1H, 13C, H-1H COSY, H-13C HSQC,
HMBC, DEPT). Melting points were measured on a micro heating stage PHMK 05
(VEB Kombinant Nagema, Radebeul, Germany) and are uncorrected.
3.2. In Vitro Evaluation
A Sunrise multichannel spectrophotometer (Tecan, Salzburg, Austria) was used for all
cholinesterase activity measurements. A previously optimized Ellman procedure was slightly modified
in order to estimate anticholinergic properties [53,54]. 96-well photometric microplates made from
polystyrene (Nunc, Rockilde, Denmark) were used for measuring purposes. Human recombinant

Molecules 2013, 18
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AChE or human plasmatic BChE (Aldrich; commercially purified by affinity chromatography) were
suspended into phosphate buffer (pH 7.4) up to final activity 0.002 U/μL. Cholinesterase (5 μL),
freshly mixed solution of 0.4 mg/mL 5,5′-dithio-bis(2-nitrobenzoic) acid (40 μL), 1 mM
acetylthiocholine chloride in phosphate buffer (20 μL) and appropriate concentration of inhibitor
(1 mM–0.1 nM; 5 μL) were injected per well. Absorbance was measured at 412 nm after 5 min
incubation using automatic shaking of the microplate. The obtained data were used to compute
percentage of inhibition [I; Equation (1)]:
Ai
A0
[%]
I  1
(1)
where ΔAi indicates absorbance change provided by cholinesterase exposed to hAChE inhibitors and
ΔA0 indicates absorbance change caused by intact cholinesterase (phosphate buffer was applied instead
of hAChE inhibitor). IC50 values were calculated using Origin 6.1 (Northampton, MA, USA).
Percentage of inhibition for the given anticholinergic compound was overlaid by proper curve chosen
according to optimal correlation coefficient. IC50 as well as upper limit of inhibition (maximal
inhibition provided by given compound) was computed.
3.3. General Procedure for Synthesis of N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)alkane-1,
ω-diamines (3–9)
Phenol (10.0 g) and 2 (0.5 g, 2.0 mmol) was heated and stirred at 80–90 °C until a homogenous
solution was obtained. The appropriate 1,ω-diaminoalkane (0.49 g, 8.1 mmol) was added and the
temperature of the reaction was raised to 125–130 °C and maintained until starting material 2
disappeared (2–4 h). After cooling the mixture was poured into 20% sodium hydroxide and extracted
with dichloromethane. The organic layer was washed with brine and water, dried over sodium sulphate
and evaporated to dryness under reduced pressure. The oily residue was purified via flash
chromatography EtOAc/MeOH/NH3 (25% aq.) (6:2:0.2) as eluent to give intermediates 3–9.
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)ethane-1,2-diamine (3). Yellow oil, yield: 90%;
1H-NMR (CDCl3) δ 1.91 (m, 4H, 2 × CH2, H-2, H-3), 2.80 (m, 2H, CH2, H-1), 2.95 (t, 2H, CH2, H-2′,
J = 5.6 Hz), 3.04 (m, 2H, CH2, H-4), 3.43 (t, 2H, CH2, H-1′, J = 5.6 Hz), 3.91 (s, 3H, OCH3), 7.25(ddd,
1H, CH, H-6, J = 9.2, 6.0, 2.4 Hz), 7.32 (d, 1H, CH, H-8, J = 2.8 Hz), 7.84 (d, 1H, CH, H-5,
J = 9.2 Hz); 13C-NMR (CDCl3) δ 22.9, 23.1 (C-2, C-3), 24.9 (C-1), 33.7 (C-4), 42.4 (C-2′), 50.8 (C-1′),
55.5 (OCH3), 101.7 (C-8), 117.9 (C-9a), 120.3 (C-6), 121.5 (C-8a), 130.2 (C-5), 143.2 (C-10a), 150.0
(C-9), 156.0 (C-7), 156.2 (C-4a); Elemental analysis: calculated 70.82% C, 7.80% H, 15.49% N; found
70.99% C, 6.55% H, 15.60% N; ESI-MS: m/z 270.1 [M]+ (calculated for: [C16H22N3O]+ 270.2).
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)propane-1,3-diamine (4). Yellow oil, yield: 88%;
1H-NMR (CDCl3) δ 1.82 (m, 2H, CH2, H-2′, J = 6.4 Hz), 1.89 (m, 4H, 2 × CH2, H-2, H-3), 2.74 (m,
2H, CH2, H-1), 2.92 (m, 2H, CH2, H-3′, J = 6.8 Hz), 3.04 (m, 2H, CH2, H-4), 3.55 (t, 2H, CH2, H-1′,
J = 6.4 Hz), 3.90 (s, 3H, OCH3), 5.44 (bs, 1H, NH), 7.23 (dd, 1H, CH, H-6, J = 9.2, 2.8 Hz), 7.28 (d,
1H, CH, H-8, J = 2.8 Hz), 7.85 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (CDCl3) δ 22.6, 22.9 (C-2, C-3),
25.0 (C-1), 33.2 (C-2′), 33.8 (C-4), 40.4 (C-3′), 47.6 (C-1′), 55.4 (OCH3), 101.8 (C-8), 116.8 (C-9a),

Molecules 2013, 18
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120.4 (C-6), 120.9 (C-8a), 129.4 (C-5), 142.5 (C-10a), 150.3 (C-9), 155.7 (C-7), 155.9 (C-4a);
Elemental analysis: calculated 71.55% C, 8.12% H, 14.72% N; found 71.85% C, 8.25% H, 14.85% N;
ESI-MS: m/z 286.2 [M]+ (calculated for: [C17H24N3O]+ 286.2).
1
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)butane-1,4-diamine (5). Yellow oil, yield: 85%; H-NMR
(CDCl3) δ 1.57 (m, 2H, CH2, H-3′), 1.71 (m, 2H, CH2, H-2′), 1.90 (m, 4H, 2 × CH2, H-2, H-3), 2.74
(m, 4H, 2 × CH2, H-1,4′), 3.03 (m, 2H, CH2, H-4), 3.43 (t, 2H, CH2, H-1′, J = 6.4 Hz), 3.91 (s, 3H,
OCH3), 5.75 (bs, 1H, NH), 7.24 (m, 2H, 2 × CH, H-6, H-8), 7.83 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR
(CDCl3) δ 22.8, 23.1 (C-2, C-3), 24.8 (C-1), 29.1 (C-2′), 30.8 (C-3′), 33.6 (C-4), 41.7 (C-4′), 48.9
(C-1′), 55.5 (OCH3), 101.7 (C-8), 117.2 (C-9a), 120.4 (C-6), 121.2 (C-8a), 130.0 (C-5), 143.0 (C-10a),
150.0 (C-9), 156.0 (C-7,4a); Elemental analysis: calculated 72.21% C, 8.42% H, 14.03% N; found
72.15% C, 8.19% H, 14.35% N; ESI-MS: m/z 300.2 [M]+ (calculated for: [C18H26N3O]+ 300.2).
1
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)pentane-1,5-diamine (6). Yellow oil, yield: 93%; H-NMR
(CDCl3) δ 1.47 (m, 4H, 2 × CH2, H-3′, H-4′), 1.68 (m, 2H, CH2, H-2′), 1.91 (m, 4H, 2 × CH2, H-2, H-3),
2.71 (m, 4H, 2 × CH2, H-1, H-5′), 3.04 (m, 2H, CH2, H-4), 3.41 (t, 2H, CH2, H-1′, J = 6.4 Hz), 3.91 (s,
3H, OCH3), 7.23 (m, 2H, 2 × CH, H-6, H-8 Hz), 7.83 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (CDCl3)
δ 22.8, 23.1 (C-2, C-3), 24.4 (C-3′), 24.8 (C-1), 31.7 (C-2′), 33.5 (C-4′), 33.8 (C-4), 42.0 (C-5′), 49.1
(C-1′), 55.5 (OCH3), 101.7 (C-8), 117.4 (C-9a), 120.3 (C-6), 121.3 (C-8a), 130.3 (C-5), 143.4 (C-10a),
149.8 (C-9), 155.9 (C-7), 156.2 (C-4a); Elemental analysis: calculated 72.81% C, 8.68% H, 13.41% N;
found 72.5% C, 8.35% H, 13.62% N; ESI-MS: m/z 314.2 [M]+ (calculated for: [C19H28N3O]+ 314.2).
1
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)hexane-1,6-diamine (7). Yellow oil, yield: 81%; H-NMR
(CDCl3) δ 1.41 (m, 6H, 3 × CH2, H-3′, H-4′, H-5′), 1.67 (m, 2H, CH2, H-2′), 1.91 (m, 4H, 2 × CH2, H-2,
H-3), 2.68 (t, 2H, CH2, H-6′, J = 7.2 Hz), 2.73 (m, 2H, CH2, H-1), 3.04 (m, 2H, CH2, H-4), 3.41 (t, 2H,
CH2, H-1′, J = 7.2 Hz), 3.90 (s, 3H, OCH3), 7.24 (m, 2H, 2 × CH, H-6, H-8), 7.84 (d, 1H, CH, H-5,
J = 9.2 Hz); 13C-NMR (CDCl3) δ 22.8, 23.1 (C-2, C-3), 24.7 (C-1), 26.7, 26.9 (C-3′, C-4′), 31.8 (C-2′),
33.4 (C-5′), 33.7 (C-4), 42.0 (C-6′), 49.1 (C-1′), 55.5 (OCH3), 101.7 (C-8), 117.2 (C-9a), 120.3 (C-6),
121.2 (C-8a), 130.1 (C-5), 143.2 (C-10a), 150.0 (C-9), 155.9 (C-7), 156.1 (C-4a); Elemental analysis:
calculated 73.36% C, 8.93% H, 12.83% N; found 73.51% C, 8.68% H, 12.85% N; ESI-MS: m/z 328.2
[M]+ (calculated for: [C20H30N3O]+ 328.2).
1
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)heptane-1,7-diamine (8). Yellow oil, yield: 91%; H-NMR
(CDCl3) δ 1.32 (m, 6H, 3 × CH2, H-3′, H- 4′, H-5′), 1.42 (m, 2H, CH2, H-6′), 1.66 (m, 2H, CH2, H-2′),
1.91 (m, 4H, 2 × CH2, H-2, H-3), 2.67 (t, 2H, CH2, H-7′, J = 7.2 Hz), 2.73 (m, 2H, CH2, H-1), 3.04 (m,
2H, CH2, H-4), 3.40 (t, 2H, CH2, H-1′, J = 7.2 Hz), 3.91 (s, 3H, OCH3), 7.25 (m, 2H, 2 × CH, H-6,
H-8), 7.84 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (CDCl3) δ 22.8, 23.1 (C-2, C-3), 24.7 (C-1), 26.8,
27.0, 29.3 (C-3′, C-4′, C-5′), 31.7 (C-2′), 33.4 (C-6′), 33.7 (C-4), 42.0 (C-7′), 49.2 (C-1′), 55.5 (OCH3),
101.7 (C-8), 117.2 (C-9a), 120.3 (C-6), 121.2 (C-8a), 130.1 (C-5), 143.2 (C-10a), 150.0 (C-9), 155.9
(C-7), 156.1 (C-4a); Elemental analysis: calculated 73.86% C, 9.15% H, 12.30% N; found 73.75% C,
9.10% H, 12.45% N; ESI-MS: m/z 342.2 [M]+ (calculated for: [C21H32N3O]+ 342.3).

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1
N-(7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)octane-1,8-diamine (9). Yellow oil, yield: 84%; H-NMR
(CDCl3) δ 1.35 (m, 6H, 3 ×CH2, H-3′, H-4′, H-5′), 1.51 (m, 2H, CH2, H-6′), 1.73 (m, 2H, CH2, H-2′),
1.91 (m, 4H, 2 × CH2, H-2, H-3), 2.50 (m, 2H, CH2, H-7′), 2.62 (t, 2H, CH2, H-8′, J = 7.0 Hz), 2.73
(m, 2H, CH2, H-1), 3.04 (m, 2H, CH2, H-4), 3.42 (t, 2H, CH2, H-1′, J = 7.2 Hz), 3.89 (s, 3H, OCH3),
7.27 (m, 2H, 2 × CH, H-6, H-8), 7.80 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (CDCl3) δ 22.9, 23.4 (C-2,
C-3), 24.7 (C-1), 26.9, 27.0, 29.4 (C-3′, C-4′, C-5′), 31.5 (C-2′), 33.6 (C-6′), 33.8 (C-4), 42.0 (C-7′),
43.2 (C-8′), 49.2 (C-1′), 55.9 (OCH3), 101.6 (C-8), 116.9 (C-9a), 120.1 (C-6), 121.2 (C-8a), 130.5 (C-5),
141.0 (C-10a), 150.8 (C-9), 155.9 (C-7), 156.0 (C-4a); Elemental analysis: calculated 74.32% C,
9.36% H, 11.82% N; found 74.38% C, 9.15% H, 12.00% N; ESI-MS: m/z 356.2 [M]+ (calculated for:
[C22H34N3O]+ 356.3).
3.4. General Procedure for Synthesis of 1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-
amino)alkane)thiourea 2,3-dihydroxysuccinates 11–17
N-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)alkane-1,ω-diamines
(3–9)
(10
mmol)
and
1-adamantyl isothiocyanate (10, 12 mmol) were dissolved in CHCl3 and stirred 24 h at room
temperature. The crude products were evaporated to dryness and purified via flash chromatography
CHCl3/MeOH (9:1). Pure basis were converted to tartaric salts by addition of equimolar tartatic acid
and stirred in absolute ethanol (10 ml) for 24 h. Thiourea dihydroxysuccinates 11–17 were obtained as
white-yellow powders in good yields.
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)ethyl)thiourea 2,3-dihydroxysuccinate
1
(11). White-yellow powder, yield: 81.2%; m.p. = 90.1–93.2 °C; H-NMR (DMSO-d6) δ 1.53 (m, 6H,
3 × CH2, H-4′′, H-7′′, H-9′′), 1.79 (m, 4H, 2 × CH2, H-2, H-3), 1.97 (m, 3H, 3 × CH, H-3′′, H-5′′,
H-8′′), 2.05 (m, 6H, 3 × CH2, H-2′′, H-6′′, H-10′′), 2.76 (t, 2H, CH2, H-1, J = 5.7 Hz), 2.95 (t, 2H, CH2,
H-4, J = 6.0 Hz), 3.73 (m, 2H, CH2, H-2′′), 3.84 (m, 2H, CH2, H-1′′), 3.91 (s, 3H, OCH3), 4.09 (m, 2H,
2 × CH, H-2′′′, H-3′′′), 7.01 (bs, 1H, NH), 7.22 (bs, 1H, NH), 7.42 (dd, 1H, CH, H-6, J = 9.2, 2.4 Hz),
13
7.63 (m, 1H, CH, H-8), 7.78 (d, 1H, CH, H-5, J = 9.2 Hz); C-NMR (DMSO-d6) δ 21.1, 22.2 (C-2,
C-3), 25.3 (C-1), 29.2 (C-3′′, C-5′′, C-8′′), 29.5 (C-4), 36.1 (C-4′′, C-7′′, C-9′′), 41.2 (C-2′′, C-6′′,
C-10′′), 43.2 (C-2′), 47.6 (C-1′), 52.9 (C-1′′), 56.0 (OCH3), 72.0 (C-2′′′, C-3′′′), 103.2 (C-8), 113.3
(C-9a), 118.7 (C-8a), 123.0 (C-6), 123.6 (C-5), 135.5 (C-10a), 151.6 (C-4a), 153.9 (C-9), 156.4 (C-7),
174.1 (C-1′′′, C-4′′′),181.1 (C=S); Elemental analysis: calculated 60.57% C, 6.89% H, 9.11% N, 5.22%
S; found 60.32% C, 6.83% H, 8.95% N, 5.20% S; ESI-MS: m/z 465.2 [M]+ (calculated for:
[C27H37N4OS]+ 465.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)propyl)thiourea
2,3-dihydroxy-
succinate (12). White-yellow powder, yield: 41.0%; m.p. = 95.2–97.8 °C; 1H-NMR (DMSO-d6) δ 1.58
(m, 6H, 3 × CH2, H-4′′, H-7′′, H-9′′), 1.82 (m, 6H, 2 × CH2, H-2, H-3, H-2′), 1.99 (m, 3H,
3 × CH, H-3′′, H-5′′, H-8′′), 2.07 (m, 6H, 3 × CH2, H-2′′, H-6′′, H-10′′), 2.73 (m, 2H, CH2, H-1), 2.95
(t, 2H, CH2, H-4, J = 5.7 Hz), 3.44 (m, 2H, CH2, H-3′), 3.68 (t, 2H, CH2, H-1′, J = 6.3 Hz), 3.90 (s, 3H,
OCH3), 4.07 (m, 2H, 2 × CH, H-2′′′, H-3′′′), 7.00 (bs, 1H, NH), 7.47 (dd, 1H, CH, H-6, J = 9.2,
2.4 Hz), 7.59 (m, 1H, CH, H-8), 7.77 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (DMSO-d6) δ 21.2, 22.2
(C-2, C-3), 25.1 (C-1), 29.2 (C-3′′, C-5′′, C-8′′), 29.7 (C-4), 30.7 (C-3′), 36.1 (C-4′′, C-7′′, C-9′′), 40.2

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(C-2′′, C-6′′, C-10′′), 41.3 (C-2′), 44.7 (C-1′), 52.8 (C-1′′), 56.0 (OCH3), 71.9 (C-2′′′, C-3′′′), 103.0
(C-8), 113.3 (C-9a), 118.7 (C-8a), 122.9 (C-6), 123.9 (C-5), 135.9 (C-10a), 151.7 (C-4a), 153.4 (C-9),
156.4 (C-7), 174.0 (C-1′′′, C-4′′′), 180.8 (C=S); Elemental analysis: calculated 61.13% C, 7.05% H,
8.91% N, 5.10% S; found 61.02% C, 6.98% H, 8.98% N, 5.13% S; ESI-MS: m/z 479.2 [M]+
(calculated for: [C28H39N4OS]+ 479.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)butyl)thiourea 2,3-dihydroxysuccinate
1
(13). White-yellow powder, yield: 69.2%; m.p. = 80.3–84.2 °C; H-NMR (DMSO-d6) δ 1.45 (m, 2H,
CH2, H-3′), 1.58 (m, 8H, 4 × CH2, H-2′, H-4′′, H-7′′, H-9′′), 1.80 (m, 4H, 2 × CH2, H-2, H-3), 1.98 (m,
3H, 3 × CH, H-3′′, H-5′′, H-8′′), 2.08 (m, 6H, 3 × CH2, H-2′′, H-6′′, H-10′′), 2.71 (t, 2H, CH2, H-1,
J = 5.8 Hz), 2.94 (t, 2H, CH2, H-4, J = 5.8 Hz), 3.33 (m, 2H, CH2, H-4′), 3.62 (t, 2H, CH2, H-1′,
J = 6.9 Hz), 3.89 (s, 3H, OCH3), 4.02 (m, 2H, 2 × CH, H-2′′′, H-3′′′), 6.72 (bs, 1H, NH), 6.91 (bs, 1H,
NH), 7.38 (dd, 1H, CH, H-6, J = 9.2, 2.4 Hz), 7.56 (m, 1H, CH, H-8), 7.76 (d, 1H, CH, H-5,
13
J = 9.2 Hz); C-NMR (DMSO-d6) δ 21.4, 22.3 (C-2, C-3), 25.0 (C-1), 26.3 (C-4′), 28.2 (C-3′′, C-5′′,
C-8′′), 29.2 (C-4), 30.0 (C-3′), 36.1 (C-4′′, C-7′′, C-9′′), 41.4 (C-2′′, C-6′′, C-10′′), 42.7 (C-2′), 47.0
(C-1′), 52.7 (C-1′′), 55.9 (OCH3), 71.8 (C-2′′′, C-3′′′), 102.9 (C-8), 113.7 (C-9a), 118.9 (C-8a), 122.5
(C-6), 124.6 (C-5), 136.7 (C-10a), 152.1 (C-4a), 153.0 (C-9), 156.2 (C-7), 174.2 (C-1′′′, C-4′′′), 180.8
(C=S); Elemental analysis: calculated 61.66% C, 7.21% H, 8.72% N, 4.99% S; found 61.30% C,
7.32% H, 8.60% N, 5.20% S; ESI-MS: m/z 493.2 [M]+ (calculated for: [C29H41N4OS]+ 493.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)pentyl)thiourea
2,3-dihydroxy-
succinate (14). White-yellow powder, yield: 54.1%; m.p. = 79.5–82.1 °C; 1H-NMR (DMSO-d6) δ 1.33 (m,
2H, CH2, H-4′), 1.43 (m, 2H, CH2, H-2′), 1.58 (m, 8H, 4 × CH2, H-3′, H-4′′, H-7′′, H-9′′), 1.80 (m, 4H,
2 × CH2, H-2, H-3), 1.98 (m, 3H, 3 × CH, H-3′′, H-5′′, H-8′′), 2.09 (m, 6H, 3 × CH2, H-2′′, H-6′′,
H-10′′), 2.70 (t, 2H, CH2, H-1, J = 5.4 Hz), 2.93 (t, 2H, CH2, H-4, J = 5.7 Hz), 3.29 (m, 2H, CH2,
H-5′), 3.59 (t, 2H, CH2, H-1′, J = 7.0 Hz), 3.88 (s, 3H, OCH3), 4.00 (m, 2H, 2 × CH, H-2′′′, H-3′′′),
6.63 (bs, 1H, NH), 6.89 (bs, 1H, NH), 7.37 (dd, 1H, CH, H-6, J = 9.2, 2.4 Hz), 7.55 (m, 1H, CH, H-8),
13
7.76 (d, 1H, CH, H-5, J = 9.2 Hz); C-NMR (DMSO-d6) δ 21.4, 22.3 (C-2, C-3), 23.9 (C-4′), 25.0
(C-1), 28.6 (C-3′′, C-5′′, C-8′′), 29.2 (C-4), 30.2 (C-3′), 30.4 (C-5′), 36.2 (C-4′′, C-7′′ , C-9′′), 41.4
(C-2′′, C-6′′, C-10′′), 42.9 (C-2′), 47.3 (C-1′), 52.7 (C-1′′), 55.9 (OCH3), 71.8 (C-2′′′, C-3′′′), 102.9
(C-8), 113.8 (C-9a), 119.0 (C-8a), 122.4 (C-6), 124.8 (C-5), 137.0 (C-10a), 152.3 (C-4a), 152.8 (C-9),
156.2 (C-7), 174.3 (C-1′′′, C-4′′′), 180.8 (C=S); Elemental analysis: calculated 62.17% C, 7.37% H,
8.53% N, 4.88% S; found 61.95% C, 7.52% H, 8.43% N, 5.02% S; ESI-MS: m/z 507.2 [M]+
(calculated for: [C29H43N4OS]+ 479.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)hexyl)thiourea
2,3-dihydroxy-
succinate (15). White-yellow powder, yield: 66.1%; m.p. = 91.8–94.4 °C; 1H-NMR (DMSO-d6) δ 1.25
(m, 4H, 2 × CH2, H-3′, H-4′), 1.38 (m, 2H, CH2, H-2′), 1.58 (m, 8H, 4 × CH2, H-5′, H-4′′, H-7′′, H-9′′),
1.79 (m, 4H, 2 × CH2, H-2, H-3), 1.98 (m, 3H, 3 × CH, H-3′′, H-5′′, H-8′′), 2.10 (m, 6H, 3 × CH2, H-
2′′, H-6′′, H-10′′), 2.70 (t, 2H, CH2, H-1, J = 5.4 Hz), 2.93 (t, 2H, CH2, H-4, J = 5.6 Hz), 3.27 (m, 2H,
CH2, H-6′), 3.56 (t, 2H, CH2, H-1′, J = 7.0 Hz), 3.88 (s, 3H, OCH3), 3.98 (m, 2H, 2 × CH, H-2′′′,
H-3′′′), 6.51 (bs, 1H, NH), 6.87 (bs, 1H, NH), 7.35 (dd, 1H, CH, H-6, J = 9.2, 2.4 Hz), 7.53 (m, 1H,

Molecules 2013, 18
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CH, H-8), 7.74 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (DMSO-d6) δ 21.5, 22.4 (C-2, C-3), 25.1
(C-4′), 26.2 (C-1), 26.3 (C-6′), 28.8 (C-3′′, C-5′′, C-8′′), 29.2 (C-4), 30.5 (C-3′), 30.6 (C-5′), 36.2
(C-4′′, C-7′′, C-9′′), 41.4 (C-2′′, C-6′′, C-10′′), 42.9 (C-2′), 47.3 (C-1′), 52.7 (C-1′′), 55.8 (OCH3), 71.8
(C-2′′′, C-3′′′), 102.8 (C-8), 114.1 (C-9a), 119.2 (C-8a), 122.1 (C-6), 125.3 (C-5), 137.5 (C-10a), 152.5
(C-4a), 152.6 (C-9), 156.1 (C-7), 174.3 (C-1′′′, C-4′′′),180.8 (C=S); Elemental analysis: calculated
62.66% C, 7.51% H, 8.35% N, 4.78% S; found 62.40% C, 7.62% H, 8.50% N, 4.90% S; ESI-MS: m/z
521.2 [M]+ (calculated for: [C31H45N4OS]+ 521.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)heptyl)thiourea
2,3-dihydroxy-
1
succinate (16). White-yellow powder, yield: 74.5%; m.p. = 100.1–102.9 °C; H-NMR (DMSO-d6) δ
1.27 (m, 6H, 3 × CH2, H-3′, H-6′, H-7′), 1.38 (m, 2H, CH2, H-4′), 1.58 (m, 8H, 4 × CH2, H-5′, H-4′′,
H-7′′, H-9′′), 1.80 (m, 4H, 2 × CH2, H-2, H-3), 1.98 (m, 3H, 3 × CH, H-3′′, H-5′′, H-8′′), 2.10 (m, 6H,
3 × CH2, H-2′′, H-6′′, H-10′′), 2.69 (t, 2H, CH2, H-1, J = 5.6 Hz), 2.94 (t, 2H, CH2, H-4, J = 5.7 Hz),
3.23 (m, 2H, CH2, H-7′), 3.58 (t, 2H, CH2, H-1′, J = 7.0 Hz), 3.88 (s, 3H, OCH3), 3.98 (m, 2H, 2 × CH,
H-2′′′, H-3′′′), 6.52 (bs, 1H, NH), 6.88 (bs, 1H, NH), 7.35 (dd, 1H, CH, H-6, J = 9.2, 2.5 Hz), 7.54 (m,
1H, CH, H-8), 7.74 (d, 1H, CH, H-5, J = 9.2 Hz); 13C-NMR (DMSO-d6) δ 21.8, 22.7 (C-2, C-3), 25.4
(C-4′), 26.7 (C-1), 26.8 (C-6′), 29.0 (C-3′′, C-5′′, C-8′′), 29.1 (C-4), 29.5 (C-7′), 30.8 (C-3′), 30.9
(C-5′), 36.4 (C-4′′, C-7′′, C-9′′), 41.7 (C-2′′, C-6′′, C-10′′), 43.3 (C-2′), 47.6 (C-1′), 53.0 (C-1′′), 56.1
(OCH3), 72.0 (C-2′′′, C-3′′′), 103.1 (C-8), 114.3 (C-9a), 119.5 (C-8a), 122.5 (C-6), 125.6 (C-5), 137.8
(C-10a), 152.8 (C-4a), 152.9 (C-9), 156.4 (C-7), 174.6 (C-1′′′, C-4′′′), 181.1 (C=S); Elemental analysis:
calculated 63.13% C, 7.65% H, 8.18% N, 4.68% S; found 63.40% C, 7.38% H, 8.01% N, 4.75% S;
ESI-MS: m/z 535.2 [M]+ (calculated for: [C32H47N4OS]+ 535.3).
1-Adamantyl-3-(2-(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl-amino)octyl)thiourea
2,3-dihydroxy-
succinate (17). White-yellow powder, yield: 84.0%; m.p. = 75.1–77.8 °C; 1H-NMR (DMSO-d6) δ 1.24
(m, 8H, 4 × CH2, H-2′, H-3′, H-4′, H-6′), 1.40 (m, 2H, CH2, H-7′), 1.62 (m, 8H, 4 × CH2, H-5′, H-4′′,
H-7′′, H-9′′), 1.82 (m, 4H, 2 × CH2, H-2, H-3), 2.00 (m, 3H, 3 × CH, H-3′′, H-5′′, H-8′′), 2.13 (m, 6H, 3
× CH2, H-2′′, H-6′′, H-10′′), 2.71 (m, 2H, CH2, H-1), 2.97 (m, 2H, CH2, H-4), 3.28 (m, 2H, CH2, H-8′),
3.68 (m, 2H, CH2, H-1′), 3.90 (s, 3H, OCH3), 4.07 (m, 2H, 2 × CH, H-2′′′, H-3′′′), 6.91 (bs, 1H, NH),
7.43 (dd, 1H, CH, H-6, J = 9.2, 2.4 Hz), 7.61 (m, 1H, CH, H-8), 7.79 (d, 1H, CH, H-5, J = 9.2 Hz);
13C-NMR (DMSO-d6): 21.2, 22.2 (C-2, C-3), 25.0 (C-1), 26.3, 26.5 (C-4′, C-5′), 28.8 (C-3′, C-6′,
C-7′), 29.2 (C-3′′, C-5′′, C-8′′), 29.6 (C-4), 30.6 (C-2′), 36.2 (C-4′′, C-7′′, C-9′′), 41.4 (C-2′′, C-6′′,
C-10′′), 43.0 (C-8′), 47.2 (C-1′), 52.7 (C-1′′), 55.9 (OCH3), 71.9 (C-2′′′, C-3′′′), 103.2 (C-8), 113.1
(C-9a), 118.5 (C-8a), 122.9 (C-6), 123.8 (C-5), 135.5 (C-10a), 151.6 (C-4a), 153.5 (C-9), 156.3 (C-7),
174.0 (C-1′′′, C-4′′′), 180.8 (C=S); Elemental analysis: calculated 63.58% C, 7.79% H, 8.02% N,
4.59% S; found 63.42% C, 7.62% H, 7.94% N, 4.70% S; ESI-MS: m/z 549.3 [M]+ (calculated for:
[C32H47N4OS]+ 549.4).
3.5. Molecular Docking
Molecular modelling calculations were performed using AutoDock Vina [62]. The molecular
models were built and minimized with UCSF chimera 1.3 (Amber Force Filed) [63]. The structure of
both enzymes, human AChE (hAChE, PDB ID: 1B41) and human BChE (hBChE, PDB ID: 1P0I)

Molecules 2013, 18
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were prepared using Pymol 1.1 from the crystal structures [64,65]. Compounds used in this study and
both enzymes were prepared using AutoDock Tools 1.5.2. in charged form [56]. Molecules of water
with other nonenzymatic molecules were removed (removing the fasciculin 2 from hAChE and
molecules of water from both enzymes) and hydrogens were added. The 3D affinity grid box in the x-,
y- and z- axes were 66, 66 and 66 with spacing 0.253 Å for hAChE, within the hBChE grid box
dimensions were set to x = 46, y = 60, z = 46 with spacing 0.375 Å. For the hAChE docking the grid
for energy was set in the coordinates x = 119.775, y = 117.597 and z = −128.964, within hBChE the
coordinates were adjusted to x = 137.871, y = 115.156 and z = 38.652. The hAChE residues Trp86,
Tyr72, Trp286, Asp74, Tyr341 and Phe297 were set to be flexible by AutoDock Tools 1.5.2, for
hBChE amino-acid residues Glu325, His438, Trp82, Asp70 and Tyr332 were selected as flexible.
Flexible ligand docking was performed for the selected compound 14 and reference compounds
(THA, 7-MEOTA). The docking calculations were made on a Mac Pro 4.1 Quad-Core Intel Xeon 2.93
GHz system. At the end of the calculations, AutoDock Vina was used to perform cluster analysis. The
visualization was carried out in Pymol 1.1. Hydrogens were finally removed to improve figure clarity.
4. Conclusions
In summary, a series of dual binding site cholinesterase inhibitors was designed and investigated.
This new class of 7-MEOTA-adamantyl amine heterodimers with different linker sizes was prepared
and tested for their ability to inhibit both targeted cholinesterases that are involved in proposed
cholinergic hypothesis of AD. As shown in Table 1, all new compounds have good inhibitory activity
to hBChE with IC50 values in the sub-μM range, and some of them also showed promising inhibitory
activity towards hAChE. Our results highlighted compound 14 with a five methylene linker. This
compound possessed the highest inhibitory activity for hAChE as well as for hBChE. Based on
molecular modeling studies for molecule 14, we have developed a novel agent that can directly interact
with both the binding sites of hAChE. This study provided potentially important information for
further development of THA- or 7-MEOTA-adamantyl amine analogues as valuable compounds for
AD treatment. The inhibition of these series compounds to the channel activity of NMDA receptors
will be tested.
Acknowledgments
This study was supported by the specific research (SV/FVZ201201), by the Grant Agency of the
Czech Republic (No. P303/11/1907), by Post-doctoral project (No. CZ.1.07/2.3.00/30.0044), by Long
Term Development plan – 1011, by VEGA 0079, CEX of SAS Nanofluid, APVV 0171-10, by project
26220220005 in the framework of the Structural Funds of European Union and by the project
(Ministry of Health, Czech Republic) for conceptual development of research organization 00179906.
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