The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2018.02.083

Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubstituted tacrine forming three different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is consistent with our docking experiments. Moreover, selected ligands were assessed for their potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase inhibitor (hAChE IC₅₀ = 74.5 nM; hBChE IC₅₀ = 83.3 nM) with micromolar antagonistic activity towards M1 mAChR (IC₅₀ = 4.23 μM). A non-selective pattern of cholinesterase inhibition is likely to be valuable during the onset as well as later stages of AD.

Full text of DOI:10.1016/j.ejmech.2018.02.083

Accepted Manuscript
The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid
(BQCA) as multifunctional agents for Alzheimer's disease
V. Hepnarova, J. Korabecny, L. Matouskova, P. Jost, L. Muckova, M. Hrabinova, N.
Vykoukalova, M. Kerhartova, T. Kucera, R. Dolezal, E. Nepovimova, K. Spilovska, E.
Mezeiova, N.L. Pham, D. Jun, F. Staud, D. Kaping, K. Kuca, O. Soukup
PII:
S0223-5234(18)30226-5
10.1016/j.ejmech.2018.02.083
EJMECH 10260
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 August 2017
Revised Date: 9 February 2018
Accepted Date: 27 February 2018
Please cite this article as: V. Hepnarova, J. Korabecny, L. Matouskova, P. Jost, L. Muckova, M.
Hrabinova, N. Vykoukalova, M. Kerhartova, T. Kucera, R. Dolezal, E. Nepovimova, K. Spilovska,
E. Mezeiova, N.L. Pham, D. Jun, F. Staud, D. Kaping, K. Kuca, O. Soukup, The concept of
hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional
agents for Alzheimer's disease, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.02.083.
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ACCEPTED MANUSCRIPT
The Concept of Hybrid Molecules of Tacrine and Benzyl Quinolone Carboxylic Acid (BQCA) as
Multifunctional Agents for Alzheimer´s Disease
Hepnarova V#, Korabecny J#, Matouskova L, Jost P, Muckova L, Hrabinova M, Vykoukalova N, Kerhartova
M, Kucera T, Dolezal R, Nepovimova E, Spilovska K, Mezeiova E, Pham N L, Jun D, Staud F, Kaping D, Kuca
K, Soukup O

ACCEPTED MANUSCRIPT
The Concept of Hybrid Molecules of Tacrine and Benzyl Quinolone Carboxylic Acid (BQCA) as
Multifunctional Agents for Alzheimer´s Disease
Hepnarova V1,2#, Korabecny J1,2#, Matouskova L1, Jost P1, Muckova L1, Hrabinova M1,2, Vykoukalova N4,
Kerhartova M4, Kucera T1, Dolezal R2, Nepovimova E1,2,5, Spilovska K2,3, Mezeiova E2,3, Pham N L1,2, Jun
D1,2, Staud F4, Kaping D3, Kuca K2,5, Soukup O2,*
1 Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of
Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
2
Biomedical Research Centre, University Hospital, Sokolska 581, 500 05 Hradec Kralove, Czech
Republic
3 National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic
4
Department of Pharmacology, Department of Pharmaceutical Chemistry and Drug Control, Faculty
of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
5 Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 500 03
Hradec Kralove, Czech Republic
# contributed equally
* corresponding author e-mail address: osoukup83@gmail.com
ABSTRACT
Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-
target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human
acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is
represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubsituted tacrine forming three
different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive
modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on
M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell
line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in
micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is

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consistent with our docking experiments. Moreover, selected ligands were assessed for their
potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain
barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety
was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase
inhibitor (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3 nM) with micromolar antagonistic activity towards
M1 mAChR (IC50 = 4.23 µM). Non-selective pattern of cholinesterase inhibition is likely to be valuable
during the onset as well as later stages of AD.
Key
words:
7-methoxytacrine,
acetylcholinesterase,
acetylcholinesterase
inhibitor,
butyrylcholinesterase, Alzheimer´s disease, BQCA, multi-target directed ligands, positive allosteric
modulator of muscarinic receptor, tacrine
1. INTRODUCTION
Alzheimer´s disease (AD) is an irreversible, progressive neurodegenerative brain disorder known to
affect adults 65 years and older. Although no AD disease-modifying therapy is currently available,
diverse drug candidates are intensively investigated to slow the worsening of dementia symptoms
and improve quality of life for Alzheimer's patients.
One approach is targeting misfolded proteins such as β-amyloid or τ-protein representing the major
pathological changes of the disease [1]. Other approaches extensively pursue the reduction of
oxidative damage in cell structures, chelation therapy to deal with abnormal metal ion homoeostasis
or drugs affecting divergent enzymatic systems such monoamine oxidase, histamine receptors,
N-methyl-D-aspartate receptors etc. [2–5]. All of the above outlined new therapeutics target the
abundant pathology to play a significant role in AD etiology; notwithstanding, that the major cause of
AD is not fully understood.
An increasing amount of evidence highlight the loss of cholinergic innervation in the basal forebrain
and hippocampus to cause the decline in memory and learning functions in AD patients. In the so-
called cholinergic hypothesis firstly postulated by Bartus and colleagues in 1982, the emerging role of
acetylcholine (ACh) neurotransmitter laid the foundation for AD treatment exploiting
acetylcholinesterase (AChE, E.C. 3.1.1.7) inhibitors (AChEIs) [6]. Accordingly, several AChEIs such as
tacrine, donepezil, rivastigmine and galantamine (Fig. 1) have been marketed [7]. Besides, N-methyl-
D-aspartate receptor (NMDAR) antagonist memantine (Fig. 1) has been approved to repel AD via
decrease in excessive glutamate activation of NMDARs [8].
In 1993, tacrine (9-amino-1,2,3,4,-tetrahydroacridine, THA) was approved by the US Food and Drug
Administration as the first AChEI for AD treatment [9]. However, THA’s hepatotoxicity limited its
further use. Nowadays, THA is still largely used as a versatile tool for designing multi-target ligands

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(MTDLs) to better cope with the multifactorial nature of AD [10–12] When built in MTDLs, THA
confers AChE inhibition properties to the final hybrids. Surprisingly, THA hybridization mostly results
in the reduction of toxicity and amplification of the biological profile.
In the context of the multifactorial AD , the M1 subtype of muscarinic ACh receptors (M1 mAChRs) is
viewed as a suitable target, since the M1 mAChR is an important subtype for memory and attention
mechanisms [13–15]. M1 mAChR stimulation does not only improve cognitive deficit, but also can
decrease τ-protein hyperphosphorylation via the activation of protein kinase C and inhibition of
glycogen synthase kinase 3β (GSK-3β) [16]. M1 agonists could not only provide symptomatic relief by
enhancing cholinergic transmission but also have disease modifying outcomes by influencing amyloid
precursor protein (APP) processing [17–19]. The latter effect can be ascribed to mediation of APP
processing via non-amyloidogenic pathway releasing soluble APP alpha (sAPPα) [20], thus, deterring
the formation of Aβ peptides and slowing AD progression [21]. Unfortunately, difficulties associated
with the development of highly selective M1 receptor agonist are due to the highly conserved
sequence homology among the orthosteric ACh binding site of mAChR subtypes [21]. The proposed
solution for M1 selective ligands appears in targeting less conserved allosteric binding sites hereby
providing the specificity for the M1 subtype.
Merck Research Laboratories developed in this context benzylquinolone carboxylic acid (BQCA; 1-(4-
methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Fig. 1). While BQCA does not interact
with the ACh site of M1 mAChRs it is well-known as a highly selective positive allosteric modulator of
the given receptor subtype. It has subtle effect on other four mAChR subtypes at concentrations up
to 100 µM [22]. In vivo research showed that BQCA acts pro-cognitively by reversing scopolamine-
induces memory deficits [21].

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Fig. 1. Design of novel tacrine-BQCA hybrids as potential AChE inhibitor with M1 mAChR agonist.
2. DESIGN OF NOVEL TACRINE-BQCA HYBRIDS
All the aforementioned results directed us to design, synthesize and evaluate novel tacrine-benzyl
quinolone carboxylic acid (tacrine-BQCA) hybrids to repel AD (Fig. 1). In this work, we utilized our
experiences from MTDLs field in AD [11,23,24]. The rational for combining tacrines with BQCA results
from the rational that tacrine moieties would be responsible for AChE inhibition whereas BQCA
scaffold would deliver M1 receptor agonist properties [21].
These two pharmacologically distinct entities have been connected by the alkyl spacer of varying
lengths into one molecule. As a result, based on tacrine core variants, we have obtained three
different families. The first group is represented by THA-BQCA hybrids. The second subset contains
6-chlorotacrine (6-Cl-THA) that has proven 25-fold higher affinity over THA to AChE [25]. Lastly, we
have implemented 7-methoxytacrine (7-MEOTA) into the third set yielding 7-MEOTA-BQCA hybrids.
The rational for 7-MEOTA implementation is that it exhibits reduced hepatotoxicity while preserving
the THA pharmacological profile [26,27]. However, THA shows multimodal and complex action itself

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[28]. Beyond cholinesterase inhibition, THA or its derivatives have been found to interact with
several other targets related to AD like blockade of M2 subtype of mAChRs, potentiation of brain
nicotinic receptors at low doses and inhibition of NMDA receptors [4]. Further, THA has been
reported to enhance neurotransmitter release of 5-hydroxytryptamine, noradrenaline, dopamine
and GABA [29]. In addition, it shows non-selective monoamine oxidase A/B (MAO-A/B) inhibition
[30].
The concept for embodying AChEI and M1 mAChR agonist into a single molecule have already been
reported [31]. In this case, hybrids linking M1 mAChR receptor agonist xanomeline with AChEI tacrine
via 10 to 17 carbon spacer showed interesting pharmacological profile. Indeed, THA-xanomeline
hybrids retained similar or even higher ability to inhibit AChE compared to THA. In addition, these
hybrids displayed enhanced M1 allosteric affinity explored using orthosteric radioligand [3H]N-
methylscopolamine as a probe. Furthermore, THA-xanomeline hybrids´ effect was determined in vivo
in scopolamine-induced amnesia model in rats resulting in memory improvement out-balancing
solely cholinesterase inhibition.
Taken together, in this article we report on the anticholinesterase properties of novel tacrine-BQCA
hybrids; paying attention to their behavior pattern towards M1 mAChR. Moreover, their cytotoxicity,
in silico binding to AChE/butyrycholinesterase (BChE) and CNS availability have been tested.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The synthetic route of the proposed tacrine-BQCA hybrids is illustrated in Schemes 1 and 2. In order
to obtain one of the parent compounds, we initially underwent a four step synthesis affording BQCA
in 19 % overall (Scheme 1). This included slightly modified reaction conditions of the earlier described
synthetic procedure [32]. The first step consisted in reaction of aniline with diethyl
ethoxymethylenemalonate by the so-called Gould-Jacobs condensation yielding 1,3-diethyl 2-
[(phenylamino)methylidene]propanedioate (1) in 90 %
[33]. Cyclo-condensation of 1 in
diphenylether afforded ethyl 4-oxo-1,4-dihydroquinoline-3-carboxylate (2) in 26 %. Benzylation of 2
with 1-(bromomethyl)-4-methoxybenzene resulted in ethyl ester of BQCA (ethyl 1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxylate; 3 in 84 %) which in subsequent
step, basic ester hydrolysis, turned to 1-[(4-methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (BQCA, 99 %).
Pursuing the MTDLs strategy to obtain the desired compounds, we fused two frameworks of
differently substituted tacrines with BQCA, employing linking approach. A set of diaminotacrines (4a-
4u) were obtained following previously reported procedure [23]. Coupling reaction of 4a-4u with

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BQCA catalyzed by benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) afforded final products 5a-5u (Scheme 2). The identity and purity of 5a-5u was confirmed by
high-resolution mass spectrometry and NMR (given in Experimental section and Supporting
Information).
Scheme 1. Synthesis of BQCA as a building block. Reaction conditions: i) neat, RT to 165°C for 6h; ii)
1M HCl, RT to 115°C; iii) diphenylether, 228-232°C, overnight; iv) K2CO3 anh., 95°C, KI in DMF, 24h; v)
LiOH, water/MeCN, RT, overnight.

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Scheme 2. Synthesis of novel tacrine-BQCA hybrids. Reaction conditions: i) DMF, inert atmosphere,
RT, TEA, BOP.
3.2. Biochemical evaluation
3.2.1. In vitro AChE and BChE inhibition assay
The inhibitory activities against human AChE (hAChE) and human BChE (hBChE) of new tacrine-BQCA
hybrids, together with those of the THA, 7-MEOTA and 6-Cl-THA, taken as references and BQCA, are
reported in Table 1. The activities are expressed as IC50 values. All three families were found to be
potent inhibitors of cholinesterases with IC50 values ranging from micromolar to two digit nanomolar
concentration scale. The most active AChE inhibitors in each family were found as follows, 5f, 5i and
5p with the IC50 values 1.5 µM, 0.13 µM and 42 nM from 7-MEOTA-BQCA, THA-BQCA and 6-Cl-THA-
BQCA subsets, respectively. The general observation can be made between series with descending
hAChE affinity from 7-MEOTA-BQCA ˃ THA-BQCA ˃ 6-Cl-THA-BQCA. This trend is in agreement with
previously published literature for tacrine-trolox hybrids or tacrine-naphthoquinone derivatives
[23,34]. Moreover, these findings are consistent with the tested template tacrines, where 6-Cl-THA
was found to be the most active hAChE inhibitor followed by THA and 7-MEOTA [25,26,35,36]. Note
that, BQCA did not show AChE inhibition ability in the tested concentration scale.
The linker between two basic pharmacophores plays a crucial role in contacting both anionic sites
which are 20 Å distanced from each other [37]. The firstly described dual-binder, namely bis-7-

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tacrine, showed optimal tether size consisting of seven methylene groups [38]. Whereas literature
offers many examples of more shortly-linked dimers such as tacrine-naphthoquinone with n = 2-3
[34], or longer-bridged hybrids such as THA-tianeptines hybrids where n = 10-11 [39] or 7-MEOTA-p-
anisidine hybrids with optimal linker spacer of n= 9 [40]. This may vary depending on the ligand
distribution in the enzyme cavity. Considering this, we inspected the optimal linker length between
appropriate tacrine and BQCA moieties. In the group of 7-MEOTA-BQCA (5a-5g) the highest activity
was maintained in two and three carbon tethered hybrids (5a and 5b). Elongation of the chain
yielded in hybrids with lower inhibition capability with exception displayed by 5f. This is plausibly
produced by the inversion of the binding mode in the hAChE active site where 7-MEOTA resides at
the rim of the hAChE gorge and BQCA is accommodated proximally (see Molecular Modeling
Studies). In THA-BQCA series (5h-5n), the most pronounced hAChE inhibitory activity is associated
with the derivatives bearing two or three methylene units in the linker (5h and 5i). Similarly, for 6-Cl-
THA-BQCA family (5o-5u), 5p bearing three methylenes proved to be the most active against hAChE.
It has to be noted that some derivatives with n = 6 and n = 7 from THA-BQCA (5l and 5m) as well as 6-
Cl-THA-BQCA (5s and 5t) families displayed inhibitory activity in the same order of magnitude as the
highlighted compounds 5i and 5p. These differences might be explained by the plasticity of hAChE
active site and suitable lodging of BQCA in contacting either mid-gorge region or PAS region of hAChE
[10,41].
Several lines of evidence suggest that BChE is able to hydrolyze ACh in AChE-knockout mice,
displaying a compensatory role when AChE levels are depleted [42,43]. In the brain of patients
suffering from AD, BChE co-localizes with AChE contributing to cerebral cortex neurodegeneration in
the form of insoluble fibrils, known as senile plaques[44]. In view of these facts, we inspected hBChE
inhibition by novel tacrine-BQCA hybrids. All the ligands under the survey demonstrated low
micromolar to two digits nanomolar hBChE inhibition power highlighting 5a (IC50 = 0.41 µM), 5h (IC50
= 59 nM) and 5o (IC50 = 83 nM) from 7-MEOTA-BQCA, THA-BQCA and 6-Cl-THA-BQCA, respectively, as
the most potent. These findings are in line with the activity of template tacrine structures where the
decreasing order in hBChE affinity can be outlined as THA ˃ 6-Cl-THA ˃ 7-MEOTA. Again, the size of
the linker between pharmacophores affected the inhibitory activity, revealing that two-carbon
tethered hybrids have the highest inhibition power in all three subsets. This findings agree with
previously published results of the tacrine-naphthoquinones family [34]. The slightly suppressed
affinity of 6-Cl-THA-BQCA family compared to THA is presumably explained by the docking
experiments (see below) [45].
From a pharmacological point of view, the AChE/BChE selectivity issue needs to be clarified. Based on
the line of reasoning outlined above, prevailing AChE inhibition may support the AD patients at the

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onset stages of the disease. In contrast, it is plausible that late stage AD patients have a higher
benefit from non-selective or BChE-selective inhibitors. However, to date there is no clear consensus
whether it is better to use AChE-selective, BChE-selective or non-selective inhibitors [46].
Accordingly, some hybrids from tacrine-BQCA family preferentially inhibit hBChE over hAChE (Table
1). Indeed, the top-three ranked hAChE selective inhibitors were 5u (SI for hAChE = 312), 5s (SI for
hAChE = 142) and 5p (SI for hAChE = 89) all being the members of 6-Cl-THA-BQCA family. Surprisingly,
the selectivity inversion can be found predominantly in 7-MEOTA-BQCA subset where hybrids 5c (SI
for hAChE = 0.114), 5a (SI for hAChE = 0.155) and 5g (SI for hAChE = 60.167) yielded as the most
hBChE selective.
Table 1 Inhibitory potencies of tested compounds towards cholinesterases and M1 mAChR expressed
as IC50 ± SEM (n=3)
Selectivity ratio
M1 mAChR IC50 ±
Compound
hAChE IC50 ± SEM (µM)a hBChE IC50 ± SEM (µM)a
for hAChEb
SEM (µM)
7-MEOTA
THA
6-Cl-THA
BQCA
5a
10.0 ± 1.0
0.320 ± 0.013
0.0176 ± 0.0005
>1000
3.71 ± 0.16
13.0 ± 1.2
17.6 ± 0.8
0.0881 ± 0.0013
1.73 ± 0.10
>1000
1.76
0.275
98.3
n.d.c
0.155
0.460
0.114
0.768
1.66
32.2
0.167
0.315
5.23
2.69
2.81
1.44
6.13
1.67
1.12
88.8
34.8
43.0
142
6.09 ± 0.57
n.d.d
2.64 ± 0.14
3.71 ± 0.31
2.48 ± 0.24
1.07 ± 0.26
1.37 ± 0.18
8.61 ± 1.40
11.4 ± 3.06
7.86 ± 0.84
0.658 ± 0.087
0.455 ± 0.061
1.59 ± 0.19
1.48 ± 0.14
1.49 ± 0.11
2.14 ± 0.27
2.05 ± 0.14
4.23 ± 0.66
4.01 ± 0.26
4.11 ± 0.31
3.27 ± 0.22
2.78 ± 0.30
5.35 ± 0.63
9.45 ± 0.76
0.408 ± 0.023
1.03 ± 0.03
1.08 ± 0.07
6.16 ± 0.49
16.6 ± 2.09
49.3 ± 10.4
12.4 ± 0.9
5b
2.24 ± 0.09
5c
9.50 ± 0.89
5d
8.02 ± 0.90
5e
10.0 ± 1.4
5f
1.53 ± 0.16
5g
74.3 ± 17.2
5h
0.187 ± 0.007
0.129 ± 0.004
0.491 ± 0.027
0.293 ± 0.015
0.194 ± 0.007
0.194 ± 0.008
0.768 ± 0.041
0.0745 ± 0.0031
0.0419 ± 0.0011
0.0896 ± 0.0029
0.180 ± 0.011
0.0941 ± 0.0040
0.0517 ± 0.0034
0.119 ± 0.007
0.0589 ± 0.0012
0.674 ± 0.030
1.32 ± 0.04
0.823 ± 0.018
0.280 ± 0.014
1.19 ± 0.06
1.28 ± 0.04
0.0833 ± 0.0050
3.72 ± 0.13
3.12 ± 0.26
7.74 ± 0.57
13.4 ± 1.0
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
3.34 ± 0.25
64.6
5u
37.2 ± 2.16
312
a Compound´s concentration required to decrease enzyme activity by 50%; the values are the mean ±
b
SEM of three independent measurements, each performed in triplicate; Selectivity for hAChE is

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c
d
determined as ratio hBChE IC50/hAChE IC50; Not determined; Not determined, BQCA showed
agonist profile toward M1 mAChR (see Supplementary information)
3.2.2. Kinetic analysis of hAChE inhibition
To assess the interactions of 5p (as the most potent hAChE inhibitor) of the series, a kinetic study was
performed. Enzyme velocity curves were recorded at various concentrations of substrate
(acetylthiocholine) and tested compound.
The type of hAChE inhibition was derived from the non-linear regression analysis, where results for
each type model of inhibition (competitive, non-competitive, uncompetitive and mixed) were
compared with sum-of-squares F-test. Analysis indicated mixed type of inhibition (p ˂ 0.05),
consistent with graphical representation of Lineweaver–Burk plot (Fig. 2). The same mode of
inhibition was described for its parent compound - tacrine [47].
3.0 107
5p
5.62 nM
2.0 107
3.16 nM
1.78 nM
1.0 107
1.00 nM
-10000 -7500 -5000 -2500
-1.0 107
2500 5000 7500
[S]-1 (M-1)
Figure 2. Steady-state inhibition of hAChE hydrolysis of substrate acetylthiocholine by compound 5p
at different concentrations. Lineweaver−Burk plots of initial velocity at increasing substrate
concentrations (0.156 - 1.250 mM) are presented. Lines were derived from a linear regression of the
data points.
The intersection of lines below the x-axis shows higher affinity of 5p to the enzyme-substrate
complex to free enzyme (Ki > Ki’). The value of Vmax decreased with increasing concentration of tested
compound, while Km slightly decreased. It means that 5p binds reversibly to both free enzyme and
enzyme-substrate complex. The result represents conformational changes of the hAChE and
following changes in the active site. Corresponding Ki value of 3.28 ± 1.22 nM and Ki’ of 1.57 ± 0.28
nM were determined. Compound 5p affects binding of substrate into the enzyme active site and
interacts with peripheral active site (PAS) as well. This type of interaction plausibly broaden the
profile of 5p possessing Aβ anti-aggregating effect mediated through inhibition of hAChE PAS [48,49].

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The same type of inhibition was revealed by donepezil that falls into category of dual-binding site
AChEIs capable to counteract hAChE-accelerated Aβ aggregation (22 % at 100 μM) [50,51].
3.2.3. Determination of M1 mAChR activity
In order to explore whether novel tacrine-BQCA hybrids are able modulate the response mediated
via M1 mAChR receptors, a calcium mobilization assay was used. Obtained activities were compared
with BQCA, highly selective and effective positive allosteric modulator of M1 mAChRs (see
Supporting Information) [21]. Unfortunately, from the whole series BQCA was the only one
compound able to potentiate M1 mAChR–mediated calcium release. All newly prepared
tacrine-BQCA hybrids exerted an antagonistic profile. Such discrepancy might be interpreted in light
of preferential binding of different tacrine units into M1 mAChR allosteric site since such receptor-
blocking interaction between tacrine itself and M1 mAChR has been previously demonstrated
[52,53]. Not encouraged by the obtained data, the IC50 values demonstrating the antagonist effect of
novel drug candidates were determined. All the hybrids displayed affinity towards M1 mAChR in the
micromolar range highlighting 5i as the most active antagonist (IC50= 0.455 ± 0.061 µM) and 5f as the
least potent (IC50= 11.4 ± 3.06 µM). Notably, the influence of the linker length on antagonistic effect
was observed where compounds with 7-8 carbons in the linker exhibited the lowest inhibitory
potency. Thus, the elongation of the spacer could lead to suppression of antagonistic effect towards
M1 mAChRs [54]. Taken into account the potent AChE inhibition, AChE-selective profile and the
lowest M1 mAChR antagonistic profile, 5f can be highlighted and considered as worthy of further
investigation.
3.2.4. Cytotoxicity evaluation
The main drawback disqualifying THA is coined with its severe hepatotoxicity [55]. THA
administration is bound to liver function monitoring such as alanine aminotransferases (ALT) or
lactate dehydrogenase (LDH) [56]. In this context, the scientific community eagerly searches for novel
THA derivatives with reduced side effects linked to hepatotoxicity [26]. In this connection, we tested
the potential in vitro hepatotoxic effect of nine selected compounds (selected pursuant to their IC50
values towards hAChE and hBChE) by measuring the HepG2 cell viability, using BQCA, tacrine, 6-Cl-
THA and 7-MEOTA as standards (Table 2). Intriguingly, our results highlighted THA to be a the less
toxic reference compound followed by 7-MEOTA, 6-Cl-THA and BQCA. In vitro toxicity of the agents
may differ from in vivo toxicity due to the differences in pharmacokinetics and metabolism.
Biotransformation processes potentially cause the toxicity [27]. All the tested tacrine-BQCA

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heterodimers revealed one to two order of magnitude higher cytotoxic effect. This might be
attributed to higher lipophilicity of compounds and molecular structure enlargement as observed
previously in related studies [23,57]. On the contrary, highly lipophilic 5t derivative bearing 6-Cl-THA
scaffold proved to be the least toxic hybrid within the study with cytotoxicity higher than 40 µM. It is
important to note that all the obtained data must be taken with a precaution as they do not reflect
the real in vivo toxicity profile. For instance, it is well-known that during long-term clinical
administration of THA its increased incidence of hepatotoxicity compared to 7-MEOTA was observed
[26].
Table 2 HepG2 cell viability of selected tacrine-BQCA hybrids and reference compounds (n=3).
Compound HepG2 IC50 ± SEM (µM)a
BQCA
> 40
THA
168.47 ± 3.91
44.37 ± 3.35
43.20 ± 1.17
1.63 ± 0.06
2.42 ± 0.13
0.60 ± 0.09
1.44 ± 0.04
2.17± 0.17
2.72 ± 0.14
3.48 ± 0.15
4.99 ± 0.30
> 40
7-MEOTA
6-Cl-THA
5a
5b
5f
5h
5i
5l
5o
5p
5t
a Means ± SEM of triplicates from three different measurements
3.2.5. In vitro BBB permeation assays
Parallel artificial membrane permeation assays (PAMPA) is a tool for predicting passive BBB
permeation which is a fundamental parameter for drug acting in the CNS [58]. In this assay, the brain
penetration of nine novel tacrine-BQCA hybrids was predicted. We optimized, the method due to
limited compounds solubility. To improve the solubility of the compounds we used 1% solution of
poloxamer pluronic F-127 validating the solubility data via the HPLC method (data not shown).
However, the use of different solvents may affect the polar-brain-lipid (PBL) membrane. Indeed, it
has been observed that the Pe values of the reference compounds differ according the solvent used
(phosphate buffer (PBS) or 1% F-127 in PBS) (Table S1 –Supplementary data). Even though, a clear
correlation (Figure S2 – Supplementary data, R2=0,9619) of Pe values in both solvents could hardly be
found in literature, the commonly reported thresholds for CNS available (Pe > 4 x 10-6 cm s-1) and for
CNS unavailable (Pe < 2 x 10-6 cm s-1) in PBS may be shifted when using 1% F-127. Thus, we correlated
the Pe values obtained in the 1% F-127 for novel compounds with those obtained for standard

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compounds, where CNS bioavailability is generally known. By a simple comparison of Pe values we
can estimate the probability of BBB penetration for novel compounds (Table 3).
The obtained data predicts that novel compounds from 7-MEOTA-BQCA subset, namely 5b and 5f,
are able to cross the BBB via passive diffusion. Other derivatives fall into uncertain permeation
interval or their BBB penetration is not probable at all (5a and 5h). However, since our results
indicated that lipid-mediated transport is uncertain, other mechanisms engaged in compounds
delivery could be involved such as carrier-mediated, or receptor-mediated transports for large
molecules that have a high affinity for an endogenous BBB receptor-mediated transport system [59].
Table 3 Prediction of the BBB permeation of novel compounds expressed as the Pe value ± SEM (n =
3).
Compound Pe ± SEM (x 10-6 cm s-1) 1% F-127
CNS a
+/-
+
5t
5f
2.35 ± 0.25
9.25 ± 1.80
2.28 ± 0.58
3.08 ± 0.07
2.37 ± 0.47
2.60 ± 0.61
1.80 ± 0.10
4.85 ± 0.05
1.65 ± 0.25
5l
+/-
+/-
+/-
+/-
-
5p
5o
5i
5h
5b
5a
+
-
a Prediction of BBB permeation is estimated for experimental conditions without F-127. CNS+ stands
for the drugs in the CNS available, CNS- for unavailable and CNS+/- for the drugs with limited or
uncertain access to the CNS.
3.3.
Molecular modeling studies
To better investigate the structure-function relationships of tacrine-BQCA compounds at a molecular
level, docking simulations were performed for the most potent compounds under the study into
hAChE (PDB ID: 4EY7) and hBChE (PDB ID: 4BDS) active sites [60,61]. The characterization of 5f-, 5i-
and 5p-hAChE complexes were carried out, while 5a, 5h and 5o were included to study ligand
accommodation within hBChE active site.
Regarding hAChE, our results clearly delineate the obtained results from in vitro proposing that
calculated binding affinities via AutoDock Vina 1.1.2 for 5f, 5i and 5p were -14.4, - 15.3 and -16.7
kcal/mol, respectively. The orientation of 5f (Fig. 3 A and B) in the hAChE active site placed the
7-MEOTA moiety outward the cavity gorge while BQCA scaffold was situated in the CAS region. In
this case, Tyr72 (3.8 Å; cation-π interaction), Thr75, Tyr341 (2.2 and 2.7 Å, respectively; hydrogen
bonds to methoxy-group from 7-MEOTA), Tyr124 (2.5 Å; hydrogen bond to exocyclic amino group),

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Trp86 (3.5 Å; distorted parallel π-π interaction with 1,4-dihydroquinoline) are deemed to be crucial
players for enzyme-ligand stability. Glu202 (3.2 Å) and Ser203 (3.3 Å) from catalytic triad showed
favorable interactions by providing hydrogen bonds to ketone group from 1,4-dihydroquinoline.
Consistency of our results is further emphasized by our previous findings where 7-MEOTA was shown
to reside 180° rotated in relationship to THA [26]. This occupancy is less favorable yielding to lower
AChE inhibitory potency. Indeed, the combination of 7-MEOTA with BQCA highlighted the latter
moiety as more optimal CAS binder. On the contrary, 5i-hAChE (Fig. 3 C and D) complex revealed
opposite ligand anchoring with THA core placed proximally and BQCA moiety in distal direction. THA
scaffold displayed parallel π-π/cation-π bonding with Trp86 (3.6 Å) in the CAS subsite. Interestingly,
the catalytic triad residues remained unaffected. 1,4-Dihydroquinoline moiety is stacked against
Tyr124 (4.3 Å; parallel π-π interaction). Importantly, Trp286 as a key PAS residue seems to have dual
binding role when surrounding concomitantly 1,4-dihydroquinoline (4.1 Å) as well as 4-
methoxybenzyl (4.3 Å) moieties. Completely different orientation can be found within 5p-hAChE (Fig.
3 E and F) complex. Accordingly, 6-Cl-THA unit faces to Tyr337 (3.2 Å) by π-π stacking in parallel
manner within CAS region. 1,4-Dihydroquinoline provide favorable stacking to Tyr341 (3.8 Å), while
Tyr124 (4.0 Å) is engaged in anchoring 4-methoxybenzyl moiety of BQCA. Similarly, to 5i-hAChE, 5p-
hAChE did not exert any interaction to catalytic triad residues. The importance of chlorine atom in 6-
position of THA core is underlined by the presence of several halogen-alkyl/aryl interactions to
Val340, Leu76, Trp439 and Tyr341. In general, the latter together with the different BQCA moiety
lodging from 5f- and 5i-hAChE complexes can be denoted as the culprits for high affinity towards
hAChE.

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Figure 3. Docking results for the novel tacrine-BQCA hybrids (5f, 5i and 5p) within hAChE active site
(PDB ID: 4EY7). A, C and E—Superimposed analogue 5f, 5i and 5p (salmon carbon atoms) as 3D
figures; B, D and F—2D representation of 5f, 5i and 5p, respectively. Generally to (A, C, E)—important
amino acid residues involved in the ligand-enzyme interactions are displayed as marine blue carbon
atoms; catalytic triad residues (Glu202, Ser203, His447) are shown in yellow, rest of the enzyme is
represented as light-grey cartoon. Figures (B, D and F) were created with Discovery Studio 2016
Client software; Figures (A, C and E) were generated with PyMol 1.5.0.4 (The PyMOL Molecular
Graphics System, Version 1.5.0.4 Schrödinger, LLC, Mannheim, Germany).

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From a structural point of view, BChE active site contains less aromatic residues within PAS region
proposing that it can accommodate more bulkier substrates [62,63]. With this in mind, we have
embedded the 5a, 5h and 5o as the most active hybrids from each subset into the hBChE cavity
highlighting several interesting findings. The scoring function clearly correlates well with the
inhibition power from in vitro since the lowest binding energies calculated are as follows -13.3
kcal/mol, -14.0 kcal/mol and -13.4 kcal/mol for 5a-, 5h- and 5o-hBChE complexes, respectively. Note
that, we are aware of the fact that Autodock Vina software possesses comparatively low standard
error of 2.85 kcal/mol which also has to be considered when docking the ligands [64,65]. 5a-hBChE
(Fig. 4 A and B) complex revealed manifold interactions with similar attributes as in 5f-hAChE
complex in terms of 7-MEOTA distal arrangement and proximal BQCA accommodation. Tyr332 (3.8
Å) is the major aromatic amino acid residue responsible for anchoring 7-MEOTA moiety of 5a. BQCA
moiety demonstrated several π-π interactions with Trp231 (1,4-dihydroquinoline moiety; 3.7 Å),
Phe329 (1,4-dihydroquinoline moiety; 3.4 Å) and Trp82 (4-methoxybenzyl moiety; 3.7 Å). Some van
der Waals forces can be found for catalytic triad residues, namely for His438 and Ser198. 5h (Fig. 4 C
and D) adapted very close spatial orientation as 5a within hBChE active site. 6-Cl-THA preserved π-π
stacking with Tyr332 (3.9 Å). In similar Trp231, Phe329 and Trp82 as well as van der Waals
hydrophobic interacted with catalytic triad residues His438 and Ser198. Hydrogen bonds between
Thr120 (2.4 Å) and oxygen from amidic function in combination with Gln119-ketone group from
1,4-dihydroquinoline (2.6 Å) are plausibly the key mediators responsible for higher affinity to hBChE
compared to 5a and 5o complexes (see below). Generally, 5o (Fig. 4 E and F) is likewise engaged in
the same web of interactions as previously described 5a and 5h hybrids in the hBChE active site.
Subtle differences can be seen such as missing π-π interaction to Trp231 and hydrogen bonds to
Thr120 and Gln119. On the contrary, presence of chlorine atom at THA unit displayed favorable
halogen-alkyl/aromatic interaction which somewhat counterbalance the latter mentioned missing
interactions.

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Figure 4. Docking results for the novel tacrine-BQCA hybrids (5a, 5h and 5o) within hBChE active site
(PDB ID: 4BDS). A, C and E—Superimposed analogue 5a, 5h and 5o (salmon carbon atoms) as 3D
figures; B, D and F—2D representation of 5a, 5h and 5o, respectively. Generally to (A, C, E)—
important amino acid residues involved in the ligand-enzyme interactions are displayed as blue
carbon atoms; catalytic triad residues (Glu197, Ser198, His438) are shown in yellow, rest of the
enzyme is represented as light-grey cartoon. Figures (B, D and F) were created with Discovery Studio

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2016 Client software; Figures (A, C and E) were generated with PyMol 1.5.0.4 (The PyMOL Molecular
Graphics System, Version 1.5.0.4 Schrödinger, LLC, Mannheim, Germany).
4. CONCLUSION
In summary, we developed novel tacrine-BQCA hybrids presumably to counteract AD on double front
following MTDL strategy i) indirectly by enhancing cholinergic transmission, i.e. by cholinesterase
inhibition and ii) directly by supporting cholinergic transmission via M1 mAChR activation. However,
the latter premise was not fulfilled since our concerns raised by preferential tacrine unit orientation
into M1 mAChR which further exerted opposite (i.e. antagonistic) effect of the ligands. We believe
that our findings do not rule out tacrine-BQCA from further testing. There is a need to test tacrine-
BQCA potential from a broader perspective, for instance, under in vivo conditions where metabolism
of the parent compound has to be taken into account. We can pinpoint compound 5o possessing
nanomolar, non-selective cholinesterase inhibition profile (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3
nM) with micromolar affinity towards M1 mAChR (IC50 = 4.23 µM). Non-selective pattern of
cholinesterase inhibition is likely to be valuable during the onset as well as at later stages of AD.
5. EXPERIMENTAL PROTOCOL
5.1. Chemistry
All used chemical reagents were purchased from Sigma-Aldrich (Prague, Czech Republic). Solvents for
synthesis were obtained from Penta chemicals Co. The course of the reactions was monitored by thin
layer chromatography (TLC) on aluminium plates precoated with silica gel 60 F254 (Merck, Czech
Republic) and then visualized by UV 254. Melting points were determined on a melting point
apparatus M-565 (Büchi, Switzerland) and are uncorrected. NMR spectra of target compounds were
1
13
recorded on Varian Mercury VX BB 300 (operating at 300 MHz for H and 75 MHz for C) or on
Varian S500 spectrometer (operating at 500 MHz for 1H and 126 MHz for 13C; Varian Comp. Palo Alto,
USA). Chemical shifts are reported in parts per million (ppm). Spin multiplicities are given as s
(singlet), bs (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), or m
(multiplet). The coupling constants (J) are reported in Hertz (Hz). High-resolution mass spectra
(HRMS) were determined by Q Exactive Plus hybrid quadrupole-orbitrap spectrometer.
BQCA synthesis followed previously published procedure [32]. Similarly, 4a-4u intermediates were
prepared according to the literature [23,40,66,67]. The spectroscopic data for all of these
intermediates were in good agreement with the literature.

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5.1.1. General procedure for synthesis of tacrine-BQCA Hybrids (5a-5u)
BQCA (0.5 mmol), and appropriate N1-(1,2,3,4-tetrahydroacridin-9-yl)alkane-1,ω-diamine (4a-4u, 0.5
mmol) were dissolved in 5 mL of dry N,N-dimethylformamide (DMF) under argon atmosphere
followed by addition of triethylamine (TEA; 1.5 mmol). Reaction was stirred for 1h, after that
benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP; 0.5 mmol) was
added. Stirring continued for further 48h at RT. The reaction mixture was then extracted with water
(100 mL) and ethyl-acetate (3 × 100 mL), dried with anhydrous sodium sulphate and solvent was
removed in vacuo. The crude product was purified using column chromatography with chloroform-
methanol as mobile phase (elution from 40:1 to 25:1; air pressured) to afford the tacrine-BQCA
hybrids 5a-5u.
5.1.1.1.
N-{2-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]ethyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5a)
Intermediate 4a and BQCA reacted according to the general procedure to give the desired product as
brown solid (41 % yield): m.p. 128.4 – 130.1 °C; 1H NMR (500 MHz, Acetone-d6) δ 10.57 (t, J = 6.3 Hz,
1H), 9.01 (s, 1H), 8.43 (d, J = 8.1 Hz, 1H), 7.88 – 7.81 (m, 1H), 7.80 – 7.68 (m, 2H), 7.61 – 7.47 (m, 2H),
7.41 (d, J = 9.0 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 8.2 Hz, 2H), 5.70 (s, 2H), 4.20 (t, J = 5.1 Hz,
2H), 4.00 (s, 3H), 3.92 (t, J = 5.4 Hz, 2H), 3.77 (s, 3H), 3.07 – 2.96 (m, 2H), 2.96 – 2.88 (m, 2H), 1.99 –
1.83 (m, 4H); 13C NMR (126 MHz, Acetone-d6) δ 176.92, 167.99, 160.60, 157.81, 155.65, 152.02,
149.26, 140.44, 136.23, 133.76, 129.17, 128.70, 127.97, 127.36, 125.97, 124.26, 123.70, 119.32,
118.60, 115.22, 113.82, 111.57, 103.91, 57.14, 56.35, 55.56, 51.39, 40.48, 30.29, 25.68, 23.08, 21.90;
HRMS [M+H]+: 563.2637 (calculated for: [C34H35N4O4]+ 563.2614)
5.1.1.2.
N-{3-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5b)
Intermediate 4b and BQCA reacted according to the general procedure to give the desired product as
yellow solid (78 % yield): m.p. 162.7-164.5 °C; 1H NMR (500 MHz, Chloroform-d) δ 10.46 (t, J = 6.5 Hz,
1H), 8.86 (s, 1H), 8.49 (dd, J = 8.0, 1.5 Hz, 1H), 8.23 (d, J = 9.3 Hz, 1H), 7.69 – 7.64 (m, 1H), 7.63 (d, J =
2.6 Hz, 1H), 7.54 (d, J = 8.5 Hz, 1H), 7.51 – 7.45 (m, 1H), 7.29 (dd, J = 9.2, 2.4 Hz, 1H), 7.25 (t, J = 7.6
Hz, 1H), 7.15 – 7.11 (m, 2H), 6.90 – 6.85 (m, 2H), 5.48 (s, 2H), 3.94 – 3.91 (m, 5H), 3.77 (s, 3H), 3.70 –
3.65 (m, 2H), 3.19 (t, J = 6.1 Hz, 2H), 2.80 (t, J = 5.9 Hz, 2H), 2.04 – 1.97 (m, 2H), 1.92 – 1.82 (m, 4H);
13C NMR (126 MHz, Chloroform-d) δ 176.76, 166.95, 159.72, 157.09, 155.27, 149.84, 148.03, 139.29,
133.14, 128.95, 128.14, 127.79, 127.02, 125.79, 125.21, 123.70, 121.94, 117.51, 117.04, 114.64,
111.28, 110.87, 102.82, 57.28, 55.81, 55.27, 43.32, 36.80, 35.38, 28.37, 24.75, 22.19, 21.39, 20.66;
HRMS [M+H]+: 577.2779 (calculated for: [C35H37N4O4]+ 577.2770)

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5.1.1.3.
N-{4-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]butyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5c)
Intermediate 4c and BQCA reacted according to the general procedure to give the desired product as
yellow solid (91 % yield): m.p. 220.5-221.8 °C; 1H NMR (300 MHz, Chloroform-d) δ 10.26 (t, J = 5.9 Hz,
1H), 8.78 (s, 1H), 8.40 (dd, J = 8.1, 1.5 Hz, 1H), 7.70 (dd, J = 9.3, 0.9 Hz, 1H), 7.67 – 7.60 (m, 1H), 7.53 –
7.39 (m, 4H), 7.07 (d, J = 8.5 Hz, 2H), 6.85 – 6.79 (m, 1H), 6.04 (bs, 1H), 5.39 (s, 2H), 4.03 – 3.94 (m,
2H), 3.86 (s, 3H), 3.74 (s, 3H), 3.59 – 3.49 (m, 2H), 2.98 – 2.90 (m, 2H), 2.65 – 2.58 (m, 2H), 2.03 – 1.75
(m, 8H); 13C NMR (126 MHz, Chloroform-d) δ 176.64, 165.37, 159.64, 156.92, 155.46, 149.28, 147.92,
139.19, 133.07, 132.96, 127.77, 127.72, 126.79, 125.91, 125.28, 124.21, 121.09, 117.06, 116.93,
114.55, 111.43, 111.31, 103.61, 57.10, 55.81, 55.25, 47.67, 47.34, 38.47, 28.21, 28.14, 26.73, 23.67,
21.78, 20.49; HRMS [M+H]+: 591.2942 (calculated for: [C36H39N4O4]+ 591.2927)
5.1.1.4.
N-{5-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]pentyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5d)
Intermediate 4d and BQCA reacted according to the general procedure to give the desired product as
1
yellow solid (58 % yield): m.p. 66.7-68.0 °C; H NMR (300 MHz, Chloroform-d) δ 10.08 (t, J = 5.5 Hz,
1H), 8.86 (s, 1H), 8.47 (dd, J = 8.1, 1.6 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.60 (ddd, J = 8.6, 6.9, 1.6 Hz,
1H), 7.47 – 7.38 (m, 2H), 7.28 (d, J = 2.7 Hz, 1H), 7.21 (dd, J = 9.1, 2.7 Hz, 1H), 7.12 – 7.02 (m, 2H),
6.86 – 6.78 (m, 2H), 5.35 (s, 2H), 3.88 (s, 3H), 3.74 (s, 3H), 3.58 – 3.42 (m, 4H), 3.09 – 2.98 (m, 2H),
2.76 – 2.64 (m, 2H), 1.92 – 1.49 (m, 10H); 13C NMR (75 MHz, Chloroform-d) δ 176.73, 164.96, 159.63,
156.06, 154.43, 151.10, 148.06, 139.18, 132.78, 127.98, 127.95, 127.65, 127.61, 127.03, 125.86,
125.06, 121.14, 120.17, 116.78, 115.67, 114.57, 111.85, 102.01, 57.15, 55.52, 55.23, 48.70, 38.75,
32.27, 31.16, 29.32, 24.51, 24.34, 22.71, 22.18; HRMS [M+H]+: 605.3085 (calculated for: [C37H41N4O4]+
605.3083
5.1.1.5.
N-{6-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]hexyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5e)
Intermediate 4e and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (37 % yield): m.p. 76.7-78.7 °C; H NMR (300 MHz, Chloroform-d) δ 10.03 (t, J = 5.8 Hz,
1H), 8.79 (s, 1H), 8.37 (dd, J = 8.1, 1.5 Hz, 1H), 7.78 (d, J = 9.1 Hz, 1H), 7.60 – 7.50 (m, 1H), 7.45 – 7.28
(m, 2H), 7.24 – 7.14 (m, 1H), 7.05 – 6.97 (m, 2H), 6.80 – 6.70 (m, 2H), 5.31 (s, 2H), 3.81 (s, 3H), 3.66 (s,
2H), 3.45 – 3.34 (m, 2H), 3.02 – 2.90 (m, 2H), 2.65 – 2.51 (m, 2H), 1.91 – 1.12 (m, 14H).; 13C NMR (75
MHz, Chloroform-d) δ 176.79, 165.08, 159.94, 159.67, 156.75, 156.64, 148.84, 148.06, 139.25,
134.09, 132.93, 127.94, 127.75, 126.99, 125.94, 125.17, 123.08, 117.48, 116.93, 114.81, 114.60,

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111.79, 103.02, 57.16, 55.78, 55.28, 47.97, 38.70, 31.00, 29.68, 29.36, 26.32, 26.03, 24.06, 22.20,
21.22; HRMS [M+H]+: 619.3253 (calculated for: [C38H43N4O4]+ 619.3240
5.1.1.6.
N-{7-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5f)
Intermediate 4f and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (53 % yield): m.p. 90.8-92.8 °C; H NMR (500 MHz, Chloroform-d) δ 10.06 (t, J = 5.6 Hz,
1H), 8.89 (s, 1H), 8.48 (dd, J = 8.1, 1.6 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.60 (ddd, J = 8.6, 7.0, 1.6 Hz,
1H), 7.48 – 7.38 (m, 2H), 7.29 (d, J = 2.7 Hz, 1H), 7.23 (dd, J = 9.2, 2.7 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H),
6.85 – 6.81 (m, 2H), 5.37 (s, 2H), 3.90 (s, 3H), 3.75 (s, 3H), 3.54 (t, J = 7.2 Hz, 2H), 3.46 (td, J = 6.9, 5.6
Hz, 2H), 3.06 – 2.99 (m, 2H), 2.72 – 2.66 (m, 2H), 1.88 (p, J = 4.5 Hz, 4H), 1.76 – 1.60 (m, 4H), 1.49 –
13
1.37 (m, 6H); C NMR (126 MHz, Chloroform-d) δ 176.74, 164.84, 159.63, 156.09, 154.05, 151.44,
148.07, 148.06, 139.19, 132.73, 127.96, 127.62, 127.44, 127.02, 125.91, 125.00, 121.38, 119.95,
116.76, 115.35, 114.56, 111.94, 102.16, 57.12, 55.52, 55.23, 48.78, 39.05, 32.01, 31.42, 29.38, 28.90,
26.85, 26.70, 24.35, 22.63, 22.10; HRMS [M+H]+: 633.3401 (calculated for: [C39H45N4O4]+ 633.3396
5.1.1.7.
N-{8-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5g)
Intermediate 4g and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (26 % yield): m.p. 76.7-78.0 °C; H NMR (300 MHz, Chloroform-d) δ 10.07 (t, J = 5.6 Hz,
1H), 8.87 (s, 1H), 8.44 (dd, J = 8.1, 1.6 Hz, 1H), 7.81 (d, J = 9.3 Hz, 1H), 7.65 – 7.55 (m, 1H), 7.46 (d, J =
8.6 Hz, 1H), 7.43 – 7.36 (m, 2H), 7.32 – 7.26 (m, 1H), 7.13 – 7.02 (m, 2H), 6.87 – 6.76 (m, 2H), 5.37
(s,2H), 3.88 (s, 3H), 3.83 – 3.74 (m, 2H), 3.73 (s, 3H), 3.51 – 3.34 (m, 2H), 3.05 – 2.95 (m, 2H), 1.99 –
1.51 (m, 8H), 1.49 – 1.18 (m, 10H); 13C NMR (75 MHz, Chloroform-d) δ 176.74, 164.91, 159.59,
156.62, 154.14, 150.73, 148.05, 139.20, 135.57, 132.84, 127.89, 127.67, 126.91, 125.92, 125.07,
123.30, 123.19, 117.95, 116.88, 114.53, 112.62, 111.80, 103.19, 57.11, 55.69, 55.22, 48.33, 39.10,
36.79, 36.73, 31.00, 29.43, 28.89, 26.75, 26.48, 23.79, 22.05, 21.08; HRMS [M+H]+: 647.3577
(calculated for: [C40H47N4O4]+ 647.3553
5.1.1.8.
1-[(4-methoxyphenyl)methyl]-4-oxo-N-{2-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]ethyl}-1,4-dihydroquinoline-3-carboxamide (5h)
Intermediate 4h and BQCA reacted according to the general procedure to give the desired product as
brown solid (63 % yield): m.p. 125.0-126.3 °C; 1H NMR (500 MHz, Chloroform-d) δ 10.44 (t, J = 5.3 Hz,
1H), 8.91 (s, 1H), 8.50 (dd, J = 8.1, 1.6 Hz, 1H), 8.07 (dd, J = 8.6, 1.3 Hz, 1H), 7.92 (dd, J = 8.5, 1.2 Hz,
1H), 7.61 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.52 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.47 – 7.42 (m, 2H), 7.31
(ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.11 – 7.06 (m, 2H), 6.87 – 6.83 (m, 2H), 5.39 (s, 2H), 3.83 – 3.77 (m,

ACCEPTED MANUSCRIPT
13
4H), 3.76 (s, 3H), 3.04 (t, J = 6.0 Hz, 2H), 2.77 (t, J = 5.9 Hz, 2H), 1.93 – 1.82 (m, 4H); C NMR (126
MHz, Chloroform-d) δ 176.72, 166.52, 159.78, 157.87, 151.17, 148.20, 146.58, 139.28, 132.95,
128.49, 128.02, 127.67, 127.24, 125.87, 125.29, 123.72, 122.94, 119.82, 116.84, 115.68, 114.70,
114.05, 111.45, 57.25, 55.31, 50.33, 40.28, 33.54, 25.08, 23.05, 22.61; HRMS [M+H]+: 533.2549
(calculated for: [C33H33N4O3]+ 533.2508
5.1.1.9.
1-[(4-methoxyphenyl)methyl]-4-oxo-N-{3-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]propyl}-1,4-dihydroquinoline-3-carboxamide (5i)
Intermediate 4i and BQCA reacted according to the general procedure to give the desired product as
brown solid (23 % yield): m.p. 148.1-150.7 °C; 1H NMR (500 MHz, Chloroform-d) δ 10.31 (t, J = 6.3 Hz,
1H), 8.91 (s, 1H), 8.51 (dd, J = 8.1, 1.6 Hz, 1H), 8.16 – 8.10 (m, 2H), 7.63 (ddd, J = 8.7, 7.0, 1.6 Hz, 1H),
7.56 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.37 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.14 – 7.08
(m, 2H), 6.89 – 6.84 (m, 2H), 5.44 (s, 2H), 3.76 (s, 3H), 3.72 (t, J = 6.2 Hz, 2H), 3.68 – 3.63 (m, 2H), 3.17
– 3.12 (m, 2H), 2.81 – 2.75 (m, 2H), 2.00 – 1.93 (m, 2H), 1.88 (m, 4H); 13C NMR (126 MHz, Chloroform-
d) δ 176.77, 166.18, 159.72, 155.54, 152.75, 148.14, 139.24, 132.95, 129.71, 127.95, 127.64, 127.12,
125.77, 125.44, 125.27, 124.21, 123.01, 118.45, 116.86, 114.65, 114.14, 111.34, 57.29, 55.26, 44.77,
35.88, 31.57, 29.61, 24.71, 22.67, 21.89; HRMS [M+H]+: 547.2670 (calculated for: [C34H35N4O3]+
547.2664
5.1.1.10. 1-[(4-methoxyphenyl)methyl]-4-oxo-N-{4-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]butyl}-1,4-dihydroquinoline-3-carboxamide (5j)
Intermediate 4j and BQCA reacted according to the general procedure to give the desired product as
1
yellow amorphous solid (19 % yield): H NMR (500 MHz, Chloroform-d) δ 10.16 (t, J = 5.8 Hz, 1H),
8.88 (s, 1H), 8.51 (dd, J = 8.2, 1.6 Hz, 1H), 8.00 (dd, J = 8.6, 1.3 Hz, 1H), 7.93 (dd, J = 8.4, 1.2 Hz, 1H),
7.63 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.53 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.50 – 7.42 (m, 2H), 7.34 (ddd, J
= 8.3, 6.8, 1.3 Hz, 1H), 7.13 – 7.08 (m, 2H), 6.88 – 6.83 (m, 2H), 5.40 (s, 2H), 3.77 (s, 3H), 3.61 (t, J =
6.8 Hz, 2H), 3.57 – 3.51 (m, 2H), 3.10 – 3.03 (m, 2H), 2.74 – 2.67 (m, 2H), 1.88 – 1.76 (m, 4H), 1.28 –
13
1.22 (m, 4H); C NMR (126 MHz, Chloroform-d) δ 176.78, 165.04, 159.71, 157.37, 151.31, 148.09,
146.12, 139.25, 132.81, 128.79, 128.01, 127.65, 127.46, 127.13, 125.88, 125.11, 123.84, 123.00,
119.58, 116.79, 115.32, 114.64, 111.86, 57.22, 55.26, 48.96, 38.73, 33.17, 29.64, 29.63, 29.14, 27.09,
24.59, 22.83, 22.42; HRMS [M+H]+: 561.2853 (calculated for: [C35H37N4O3]+ 561.2821
5.1.1.11. 1-[(4-methoxyphenyl)methyl]-4-oxo-N-{5-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]pentyl}-1,4-dihydroquinoline-3-carboxamide (5k)
Intermediate 4k and BQCA reacted according to the general procedure to give the desired product as
1
yellow amorphous solid (25 % yield): H NMR (500 MHz, Chloroform-d) δ 10.18 (t, J = 5.8 Hz, 1H),

ACCEPTED MANUSCRIPT
8.83 (s, 1H), 8.41 (dd, J = 8.1, 1.6 Hz, 1H), 8.19 (d, J = 8.7 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.67 – 7.58
(m, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.46 – 7.39 (m, 2H), 7.10 – 7.05 (m, 2H), 6.83 – 6.78 (m, 2H), 5.38 (s,
2H), 3.97 – 3.87 (m, 2H), 3.73 (s, 3H), 3.55 – 3.40 (m, 2H), 2.95 (t, J = 6.3 Hz, 2H), 2.56 (t, J = 6.2 Hz,
13
2H), 1.95 – 1.87 (m, 2H), 1.87 – 1.75 (m, 4H), 1.75 – 1.68 (m, 2H), 1.62 – 1.54 (m, 2H); C NMR (126
MHz, Chloroform-d) δ 176.70, 165.20, 159.63, 156.14, 150.44, 148.04, 139.20, 138.24, 133.02,
133.00, 127.79, 127.73, 126.82, 125.93, 125.33, 125.22, 124.83, 119.74, 117.04, 115.44, 114.55,
111.55, 111.28, 57.12, 55.25, 48.50, 38.64, 30.06, 29.64, 28.96, 28.21, 23.91, 21.60, 20.48; HRMS
[M+H]+: 575.2985 (calculated for: [C36H39N4O3]+ 575.2977
5.1.1.12. 1-[(4-methoxyphenyl)methyl]-4-oxo-N-{6-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]hexyl}-1,4-dihydroquinoline-3-carboxamide (5l)
Intermediate 4l and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (60 % yield): m.p. 54.2-56.2 °C; H NMR (500 MHz, Chloroform-d) δ 10.07 (t, J = 5.7 Hz,
1H), 8.88 (s, 1H), 8.49 (dd, J = 8.1, 1.6 Hz, 1H), 8.01 – 7.93 (m, 2H), 7.60 (ddd, J = 8.6, 7.0, 1.6 Hz, 1H),
7.53 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.46 – 7.40 (m, 2H), 7.33 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 7.11 – 7.05
(m, 2H), 6.87 – 6.81 (m, 2H), 5.35 (s, 2H), 3.75 (s, 3H), 3.53 (t, J = 7.2 Hz, 2H), 3.48 (td, J = 6.9, 5.7 Hz,
2H), 3.09 – 3.04 (m, 2H), 2.72 – 2.65 (m, 2H), 1.94 – 1.86 (m, 4H), 1.75 – 1.63 (m, 4H), 1.54 – 1.43 (m,
13
4H); C NMR (126 MHz, Chloroform-d) δ 176.75, 164.85, 159.64, 157.49, 151.20, 148.06, 146.33,
139.19, 132.69, 128.62, 127.99, 127.69, 127.60, 127.08, 125.89, 124.99, 123.67, 122.94, 119.61,
116.70, 115.15, 114.57, 111.97, 57.12, 55.22, 49.17, 38.90, 33.30, 31.46, 29.41, 26.69, 26.47, 24.57,
22.83, 22.45; HRMS [M+H]+: 589.3156 (calculated for: [C37H41N4O3]+ 589.3134
5.1.1.13. 1-[(4-methoxyphenyl)methyl]-4-oxo-N-{7-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]heptyl}-1,4-dihydroquinoline-3-carboxamide (5m)
Intermediate 4m and BQCA reacted according to the general procedure to give the desired product
1
as brown amorphous solid (55 % yield): H NMR (500 MHz, Chloroform-d) δ 10.06 (t, J = 5.7 Hz, 1H),
8.90 (s, 1H), 8.50 (dd, J = 8.1, 1.6 Hz, 1H), 7.99 (dd, J = 8.5, 1.3 Hz, 1H), 7.92 (dd, J = 8.5, 1.3 Hz, 1H),
7.60 (ddd, J = 8.6, 7.0, 1.7 Hz, 1H), 7.54 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 7.47 – 7.40 (m, 2H), 7.34 (ddd, J
= 8.4, 6.8, 1.3 Hz, 1H), 7.11 – 7.06 (m, 2H), 6.87 – 6.81 (m, 2H), 5.36 (s, 2H), 3.75 (s, 3H), 3.57 – 3.51
(m, 2H), 3.51 – 3.45 (m, 2H), 3.08 – 3.03 (m, 2H), 2.71 – 2.66 (m, 2H), 1.93 – 1.86 (m, 4H), 1.74 – 1.62
13
(m, 4H), 1.49 – 1.38 (m, 6H); C NMR (126 MHz, Chloroform-d) δ 176.76, 164.83, 159.64, 157.25,
151.43, 148.09, 146.14, 139.21, 132.69, 128.79, 128.01, 127.61, 127.38, 127.08, 125.92, 124.98,
123.72, 123.08, 119.49, 116.72, 115.05, 114.58, 112.01, 57.12, 55.22, 49.29, 39.07, 33.14, 31.48,
29.61, 29.42, 28.91, 26.86, 26.69, 24.49, 22.79, 22.41; HRMS [M+H]+: 603.3291 (calculated for:
[C38H43N4O3]+ 603.3290

ACCEPTED MANUSCRIPT
5.1.1.14. 1-[(4-methoxyphenyl)methyl]-4-oxo-N-{8-[(1,2,3,4-tetrahydroacridin-9-
yl)amino]octyl}-1,4-dihydroquinoline-3-carboxamide (5n)
Intermediate 4n and BQCA reacted according to the general procedure to give the desired product as
1
yellow amorphous solid (30 % yield): H NMR (500 MHz, Chloroform-d) δ 10.08 – 10.01 (m, 1H), 8.89
(t, J = 2.3 Hz, 1H), 8.51 – 8.44 (m, 1H), 8.19 – 8.04 (m, 2H), 7.64 – 7.55 (m, 2H), 7.49 – 7.33 (m, 2H),
7.13 – 7.05 (m, 2H), 6.88 – 6.80 (m, 2H), 5.38 (s, 2H), 3.75 (s, 3H), 3.73 – 3.65 (m, 2H), 3.51 – 3.42 (m,
2H), 3.18 – 3.11 (m, 2H), 2.67 – 2.60 (m, 2H), 1.94 – 1.83 (m, 4H), 1.79 – 1.69 (m, 2H), 1.69 – 1.57 (m,
2H), 1.49 – 1.30 (m, 8H); 13C NMR (126 MHz, Chloroform-d) δ 176.76, 164.82, 159.66, 153.10, 148.08,
139.21, 139.18, 132.72, 130.25, 127.99, 127.63, 127.04, 125.91, 125.00, 124.69, 124.28, 123.52,
117.88, 116.76, 114.58, 113.98, 112.01, 109.92, 57.15, 55.24, 53.37, 49.00, 39.08, 33.74, 31.84,
31.35, 31.28, 30.11, 29.61, 29.45, 29.27, 29.07, 28.96, 26.80, 26.60, 24.06, 22.61, 22.39, 21.64; HRMS
[M+H]+: 617.3458 (calculated for: [C39H45N4O3]+ 617.3447
5.1.1.15. N-{2-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]ethyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5o)
Intermediate 4o and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (31 % yield): m.p. 162.5-164.0 °C; H NMR (500 MHz, Chloroform-d) δ 10.51 – 10.42 (m,
1H), 8.91 (s, 1H), 8.50 (dd, J = 8.1, 1.5 Hz, 1H), 8.02 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 2.2 Hz, 1H), 7.64
(ddd, J = 8.7, 7.0, 1.6 Hz, 1H), 7.51 – 7.43 (m, 2H), 7.24 (dd, J = 9.1, 2.2 Hz, 1H), 7.15 – 7.08 (m, 2H),
6.91 – 6.85 (m, 2H), 5.42 (s, 2H), 3.83 – 3.78 (m, 4H), 3.78 (s, 3H), 3.02 (t, J = 5.9 Hz, 2H), 2.79 – 2.73
13
(m, 2H), 1.92 – 1.83 (m, 4H); C NMR (126 MHz, Chloroform-d) δ 176.68, 166.66, 159.77, 158.97,
151.23, 148.11, 139.25, 134.17, 132.96, 127.97, 127.63, 127.22, 126.70, 125.73, 125.32, 124.53,
124.27, 117.98, 116.79, 115.53, 114.70, 111.29, 57.27, 55.28, 40.21, 36.81, 36.78, 25.02, 22.91,
22.45; HRMS [M+H]+: 567.2146 (calculated for: [C33H32ClN4O3]+ 567.2118
5.1.1.16. N-{3-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5p)
Intermediate 4p and BQCA reacted according to the general procedure to give the desired product as
1
brown solid (39 % yield): m.p. 95.4-96.1 °C; H NMR (500 MHz, Chloroform-d) δ 10.26 (t, J = 6.3 Hz,
1H), 8.92 (s, 1H), 8.51 (dd, J = 8.0, 1.6 Hz, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.90 (d, J = 2.2 Hz, 1H), 7.63
(ddd, J = 8.6, 7.0, 1.6 Hz, 1H), 7.50 – 7.43 (m, 2H), 7.29 – 7.22 (m, 1H), 7.14 – 7.07 (m, 2H), 6.91 – 6.82
(m, 2H), 5.43 (s, 2H), 3.77 (s, 3H), 3.67 – 3.55 (m, 4H), 3.03 (t, J = 6.2 Hz, 2H), 2.75 (t, J = 5.2 Hz, 2H),
13
1.95 – 1.84 (m, 6H); C NMR (126 MHz, Chloroform-d) δ 176.78, 165.93, 159.73, 151.21, 148.14,
139.25, 134.24, 132.91, 127.98, 127.79, 127.63, 127.15, 126.60, 125.79, 125.24, 124.45, 124.40,

ACCEPTED MANUSCRIPT
118.15, 116.80, 115.77, 114.82, 114.66, 111.50, 57.27, 55.26, 45.19, 36.79, 36.76, 35.97, 31.58,
29.62, 24.97, 22.87, 22.43; HRMS [M+H]+: 581.2294 (calculated for: [C34H34ClN4O3]+ 581.2275
5.1.1.17. N-{4-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]butyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5q)
Intermediate 4q and BQCA reacted according to the general procedure to give the desired product as
1
yellow pale solid (45 % yield): m.p. 65.3-66.7 °C; H NMR (500 MHz, Chloroform-d) δ 10.15 (t, J = 5.8
Hz, 1H), 8.88 (s, 1H), 8.50 (dd, J = 8.1, 1.6 Hz, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.62
(ddd, J = 8.6, 7.0, 1.6 Hz, 1H), 7.49 – 7.42 (m, 2H), 7.24 (dd, J = 9.1, 2.2 Hz, 1H), 7.12 – 7.07 (m, 2H),
6.88 – 6.83 (m, 2H), 5.39 (s, 2H), 3.76 (s, 3H), 3.59 – 3.50 (m, 4H), 3.03 – 2.98 (m, 2H), 2.70 – 2.66 (m,
13
2H), 1.92 – 1.86 (m, 4H), 1.85 – 1.74 (m, 4H); C NMR (126 MHz, Chloroform-d) δ 176.75, 165.02,
159.69, 159.21, 150.75, 148.06, 147.73, 139.21, 133.97, 132.79, 127.98, 127.63, 127.18, 127.10,
125.85, 125.11, 124.57, 124.23, 118.25, 116.75, 115.73, 114.62, 111.82, 57.20, 55.24, 49.08, 38.69,
33.79, 29.20, 27.04, 24.55, 22.82, 22.50; HRMS [M+H]+: 595.2447 (calculated for: [C35H36ClN4O3]+
595.2431
5.1.1.18. N-{5-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]pentyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5r)
Intermediate 4r and BQCA reacted according to the general procedure to give the desired product as
1
brown amorphous solid (52 % yield): H NMR (500 MHz, Chloroform-d) δ 10.11 (t, J = 5.8 Hz, 1H),
8.89 (s, 1H), 8.53 – 8.48 (m, 1H), 7.96 – 7.91 (m, 2H), 7.62 (ddd, J = 8.6, 7.0, 1.6 Hz, 1H), 7.49 – 7.43
(m, 2H), 7.27 – 7.24 (m, 1H), 7.11 – 7.06 (m, 2H), 6.88 – 6.82 (m, 2H), 5.39 (s, 2H), 3.77 (s, 3H), 3.62 –
3.44 (m, 4H), 3.06 – 3.01 (m, 2H), 2.68 – 2.65 (m, 2H), 1.93 – 1.84 (m, 4H), 1.81 – 1.68 (m, 4H), 1.61 –
13
1.52 (m, 2H); C NMR (126 MHz, Chloroform-d) δ 176.80, 165.00, 159.72, 151.22, 148.11, 139.24,
139.22, 132.80, 128.02, 127.64, 127.14, 125.87, 125.10, 124.69, 124.38, 116.77, 114.64, 114.01,
111.94, 57.22, 55.27, 49.27, 38.76, 36.82, 36.78, 31.22, 29.64, 29.61, 29.31, 29.28, 24.48, 24.29,
22.75, 22.35; HRMS [M+H]+: 609.2593 (calculated for: [C36H38ClN4O3]+ 609.2588
5.1.1.19. N-{6-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]hexyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5s)
Intermediate 4s and BQCA reacted according to the general procedure to give the desired product as
1
yellow amorphous solid (80 % yield): H NMR (500 MHz, Chloroform-d) δ 10.06 (t, J = 5.7 Hz, 1H),
8.88 (s, 1H), 8.48 (dd, J = 8.1, 1.7 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 2.2 Hz, 1H), 7.60 (ddd, J =
8.6, 7.0, 1.7 Hz, 1H), 7.47 – 7.39 (m, 2H), 7.23 (dd, J = 9.0, 2.2 Hz, 1H), 7.10 – 7.05 (m, 2H), 6.86 – 6.80
(m, 2H), 5.36 (s, 2H), 3.75 (s, 3H), 3.52 – 3.45 (m, 4H), 3.03 – 2.98 (m, 2H), 2.68-2.64 (m, 2H), 1.92 –
13
1.86 (m, 4H), 1.73 – 1.63 (m, 4H), 1.52 – 1.43 (m, 4H); C NMR (126 MHz, Chloroform-d) δ 176.75,

ACCEPTED MANUSCRIPT
164.85, 159.65, 159.04, 150.91, 148.06, 147.64, 139.19, 134.01, 132.71, 127.98, 127.60, 127.07,
125.88, 125.01, 124.62, 124.15, 118.12, 116.70, 115.43, 114.58, 111.97, 57.13, 55.22, 49.29, 38.87,
36.78, 33.71, 29.37, 26.65, 26.43, 24.47, 22.79, 22.47; HRMS [M+H]+: 623.2768 (calculated for:
[C37H40ClN4O3]+ 623.2744
5.1.1.20. N-{7-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5t)
Intermediate 4t and BQCA reacted according to the general procedure to give the desired product as
1
yellow pale solid (40 % yield): m.p. 82.0-84.7 °C; H NMR (500 MHz, Chloroform-d) δ 10.04 (t, J = 5.7
Hz, 1H), 8.89 (s, 1H), 8.49 (dd, J = 8.1, 1.6 Hz, 1H), 7.93 – 7.86 (m, 2H), 7.60 (ddd, J = 8.6, 7.0, 1.6 Hz,
1H), 7.47 – 7.39 (m, 2H), 7.24 (dd, J = 9.0, 2.2 Hz, 1H), 7.13 – 7.04 (m, 2H), 6.87 – 6.80 (m, 2H), 5.37 (s,
2H), 3.75 (s, 3H), 3.52 – 3.43 (m, 4H), 3.04 – 2.98 (m, 2H), 2.66 – 2.64 (m, 2H), 1.94 – 1.85 (m, 4H),
1.73 – 1.60 (m, 4H), 1.51 – 1.37 (m, 6H); 13C NMR (126 MHz, Chloroform-d) δ 176.83, 164.88, 159.71,
159.16, 150.98, 148.15, 147.78, 139.27, 134.07, 132.76, 128.07, 127.66, 127.21, 127.15, 125.96,
125.05, 124.68, 124.20, 118.21, 116.76, 115.47, 114.65, 112.08, 57.21, 55.28, 49.51, 39.12, 36.84,
36.81, 33.81, 31.63, 29.67, 29.46, 28.96, 26.91, 26.73, 24.53, 22.87, 22.56; HRMS [M+H]+: 637.2903
(calculated for: [C38H42ClN4O3]+ 637.2901
5.1.1.21. N-{8-[(6-chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-1-[(4-
methoxyphenyl)methyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide (5u)
Intermediate 4u and BQCA reacted according to the general procedure to give the desired product as
1
yellow solid (40 % yield): m.p. 54.6-56.7 °C; H NMR (500 MHz, Chloroform-d) δ 10.05 (t, J = 5.7 Hz,
1H), 8.91 (s, 1H), 8.50 (dd, J = 8.1, 1.6 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.88 (d, J = 2.2 Hz, 1H), 7.64 –
7.58 (m, 1H), 7.43 (ddd, J = 8.1, 7.2, 6.1 Hz, 2H), 7.25 (dd, J = 9.1, 2.2 Hz, 1H), 7.12 – 7.07 (m, 2H), 6.88
– 6.82 (m, 2H), 5.38 (s, 2H), 3.77 (s, 3H), 3.51 – 3.44 (m, 4H), 3.05 – 2.99 (m, 2H), 2.69 – 2.64 (m, 2H),
13
1.93 – 1.88 (m, 4H), 1.70 – 1.61 (m, 4H), 1.48 – 1.30 (m, 8H); C NMR (126 MHz, Chloroform-d) δ
176.84, 164.88, 159.72, 159.36, 150.91, 148.19, 148.17, 139.27, 133.98, 132.75, 128.09, 127.67,
127.40, 127.21, 127.18, 125.98, 125.06, 124.66, 124.18, 118.32, 116.75, 114.66, 112.12, 57.22, 55.30,
49.62, 39.19, 36.85, 33.95, 31.72, 29.54, 29.15, 26.91, 24.54, 22.62; HRMS [M+H]+: 651.3081
(calculated for: [C39H44ClN4O3]+ 651.3057
5.2. Cholinesterase Inhibition
The human erythrocyte acetylcholinesterase (hAChE; EC 3.1.1.7) and human plasmatic
butyrylcholinesterase (hBChE; EC 3.1.1.8) inhibitory activity of the tested drugs was determined using
Ellman´s method and is expressed as IC50, i.e. concentration that reduces the cholinesterase activity
by 50% [68]. 5,5´-Dithiobis(2-nitrobenzoic acid) (Ellman´s reagent, DTNB), KH2PO4, K2HPO4,

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acetylthiocholine (ATCh), and butyrylthiocholine (BTCh), were purchased from Sigma-Aldrich, Prague,
Czech Republic. For measuring purposes – polystyrene Nunc 96-well microplates with flat bottom
shape (ThermoFisher Scientific, USA) were utilized. All the assays 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 (100 µL) consisted of 40 µL of 0.1 M phosphate buffer (pH 7.4), 20 µL of 0.01 M DTNB,
10 µL of enzyme, and 20 µL of 0.01 M substrate (ATCh iodide solution). Assay solutions with inhibitor
(10-3 – 10-9 M) were pre-incubated 5 min. The reaction was started by immediate addition of 20 µL of
substrate. The activity was determined by measuring the increase in absorbance at 412 nm at 37 °C
at 2 min intervals - using a Multi-mode microplate reader Synergy 2 (Vermont, USA). Each
concentration was assayed in triplicate. In vitro hBChE assay was similar with the method described
above using BTCh as substrate instead of ATCh. Software GraphPad Prism 5 (San Diego, USA) was
used for the statistical data evaluation.
5.3. Kinetic analysis
The kinetic study of hAChE was performed by using above mentioned Ellman’s method [69–71].
The concentrations of 5p used for the measurements were as follows: 1.00, 1.78, 3.16 and 5.62 nM.
The values of Vmax and Km of the Michaelis-Menten kinetics as well as the values of Ki and Ki’ were
calculated by non-linear regression from the substrate velocity curves. Linear regression was used for
calculation of Lineweaver-Burk plots. All calculations were performed using GraphPad Prism software
version 5.02 for Windows (San Diego, CA, USA).
5.4. Calcium mobilization assay
5.4.1. Cell culturing
Chinese hamster ovary cells (CHO-M1WT2, CRL-1984) stably expressing human recombinant
M1 mAChR (70 fmol/mg) were obtained from ATCC (Virginia, USA). Cells were cultured in the Ham´s
F-12 medium with fetal bovine serum (10%) and geneticin (50 ng/mL). Cells were maintained at 37°C
in the atmosphere of 5 % CO2.
5.4.2. Fluo-4 NW Assay
Cells were plated out at density of ≈ 70,000 cells per well in 100 µL of medium, in a black-
walled, clear-bottomed 96-well plate (Biotech, Czech Republic) and were grown overnight at 37 °C in
the atmosphere of 5 % CO2. The next day, the medium was removed leaving the cells adhering to the
bottom. 100 μL of Fluo-4NW solution was added. Fluo-4NW solution was prepared according to
manufacturer protocol, i.e. 10 mL of Hank´s balanced salt solution (HBSS buffer) and 100 μL of

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probenecid solution were added to the dye mixture. Cells were incubated with the dye for 30 min at
37 °C/ 5 % CO2 in the dark and 30 min at the room temperature.
The tested compounds were dissolved in DMSO and diluted 150 times by distilled water on
the day of experiment. Further dilutions were prepared in the HBSS buffer and injected to a well,
after 5s measurement of the baseline, where achieved the final tested concentration. The increase in
fluorescence caused by the activation of the receptor was monitored for another 30s. The final
concentration of DMSO in the well did not exceed 0.3 % (v/v).
The effect of novel compounds was compared to BQCA, a positive allosteric modulator
standard. Cells were pre-incubated with the tested compound (10 μL/well) for 10 min and then EC20
of Oxo-M (30 nmol/l) was applied. Since the compounds inhibited the EC20 Oxo-M response, their
inhibitory potency was then measured at EC80 Oxo-M (1 µmol/L). Ca2+ influx was measured by
Synergy HT (Biotek, USA) at excitation and emission wavelength 485/20 nm and 528/20 nm
respectively. All measurements were made at the room temperature ≈ 21 °C.
Oxo-M - evoked responses were quantified as the maximum response and expressed as a percentage
of the average baseline values and normalized to the control. The first and the last wells received
Oxo-M control and the mean was considered as 100% response in order to minimize time-depended
errors (fading of responses), which were observed in the long- lasting experiments. Data were fitted
using a standard four-parameter equation GraphPad Prism 5.0 (San Diego, USA) to generate graphs,
IC50 and SEM.
5.5. Evaluation of cytotoxicity (MTT assay)
A hepatotoxicity of tested compounds was evaluated using the HepG2 cell line originally from
human liver hepatocellular carcinoma (ATCC, USA). These cells were plated at density 17 × 103 per
well in 96-well plate in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) with 10% FBS
(Gibco, USA) and incubated overnight. The incubation was performed under condition 37°C, 5% CO2
and 80 – 95% air humidity. Stock solutions of tested compounds were prepared in dimethyl sulfoxide
(DMSO) (Sigma –Aldrich, USA) and then diluted in DMEM medium. The final concentration of DMSO
was less than 0.25%.
The cell viability was detected using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (MTT) assay [72] after 24 h of incubation with compounds. Then, medium was aspirated
and 100 µL of MTT solution (0.5 mg/ml) in serum free of DMEM was added to each well. The cells
were then incubated for one hour. The medium was then aspirated again and purple crystals of MTT
formazan were dissolved in 100 μl DMSO under shaking. The absorbance was measured with
a microplate reader (BioTek Instruments, USA) at a wavelength of 570 nm.