Anti-Alzheimer's multitarget-directed ligands with serotonin 5-HT6 antagonist, butyrylcholinesterase inhibitory, and antioxidant activity

Article abstract of DOI:10.1002/ardp.201900041

Serotonin 5-HT₆ receptors, butyrylcholinesterase (BuChE) and oxidative stress are related to the pathophysiology of Alzheimer's disease. Inhibition of BuChE provides symptomatic treatment of the disease and the same effect was demonstrated for

Full text of DOI:10.1002/ardp.201900041

Received: 6 February 2019
Revised: 4 April 2019
Accepted: 7 April 2019
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DOI: 10.1002/ardp.201900041
F U L L P A P E R
Anti‐Alzheimer's multitarget‐directed ligands with serotonin
5‐HT6 antagonist, butyrylcholinesterase inhibitory, and
antioxidant activity
Monika Marcinkowska1
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Adam Bucki1
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Dawid Panek1
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Agata Siwek2
Justyna Godyń1
Marcin Kołaczkowski1
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Nikola Fajkis1 Marek Bednarski3
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Małgorzata Zygmunt3
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Alvaro Del Rio Valdivieso1
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Magdalena Kotańska3
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Anna Więckowska1
1Chair of Pharmaceutical Chemistry, Faculty
of Pharmacy, Jagiellonian University Medical
College, Kraków, Poland
Abstract
Serotonin 5‐HT6 receptors, butyrylcholinesterase (BuChE) and oxidative stress are
related to the pathophysiology of Alzheimer's disease. Inhibition of BuChE provides
symptomatic treatment of the disease and the same effect was demonstrated for
5‐HT6 antagonists in clinical trials. Oxidative stress is regarded as a major and
primary factor contributing to the development of Alzheimer's disease; therefore,
antioxidant agents may provide a disease‐modifying effect. Combining BuChE
inhibition, 5‐HT6 antagonism, and antioxidant properties may result in multitarget‐
directed ligands providing cognition‐enhancing properties with neuroprotective
activity. On the basis of the screening of the library of 5‐HT6 antagonists against
BuChE, we selected two compounds and designed their structural modifications that
could lead to improved BuChE inhibitory activity. We synthesized two series of
compounds and tested their affinity and functional activity at 5‐HT6 receptors,
BuChE inhibitory activity and antioxidant properties. Compound 12 with Ki and Kb
values against 5‐HT6 receptors of 41.8 and 74 nM, respectively, an IC50 value of 5 µM
against BuChE and antioxidant properties exceeding the activity of ascorbic acid is a
promising lead structure for further development of anti‐Alzheimer's agents.
2Department of Pharmacobiology, Faculty of
Pharmacy, Jagiellonian University Medical
College, Kraków, Poland
3Chair of Pharmacodynamics, Faculty of
Pharmacy, Jagiellonian University Medical
College, Kraków, Poland
Correspondence
Monika Marcinkowska and Anna Więckowska,
Chair of Pharmaceutical Chemistry, Faculty of
Pharmacy, Jagiellonian University Medical
College, Medyczna 9, 30‐688 Kraków, Poland.
Email: monika.marcinkowska@uj.edu.pl (M.M.);
anna.wieckowska@uj.edu.pl (A.W.)
K E Y W O R D S
5‐HT6 antagonists, Alzheimer's disease, antioxidant properties, butyrylcholinesterase inhibitors,
multimodal agents, multitarget‐directed ligands, serotonin receptors
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1
INTRODUCTION
affect each other at various levels, leading to neuronal
loss.[3,4]
Alzheimer's disease (AD) is a predominant neurodegenerative
disorder, which affects approximately 35 million people world-
wide.[1] This number is constantly increasing and it has been
projected that by 2050, the number of individuals suffering from
AD will reach 115 million.[2] The pathogenesis of AD is multi-
factorial in origin, and it results from complex processes that
The brain areas primarily affected by progressive neurodegen-
eration involve cholinergic neurons in the cerebral cortex and
hippocampus.[5] As the cholinergic system is associated with memory
and cognition, impaired cholinergic neurotransmission induces
symptoms of cognitive decline and memory impairment in AD
patients. Therefore, alleviating the level of acetylcholine (ACh) in
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Arch Pharm Chem Life Sci. 2019;e1900041.
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cholinergic synapses has been proposed to improve the symptoms of
AD.[6] It is possible to elevate ACh levels in the synaptic cleft by
inhibiting enzymes involved in ACh hydrolysis: acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE).[7] The activity of both
enzymes changes along with the AD progression. In an advanced
stadium of AD, activity of BuChE prevails, and thus may account for
the persistence of symptoms and disease progression.[8] In fact, first
therapeutic agents for AD that appeared on the market act through
inhibition of cholinesterases (donepezil, rivastigmine, and galanta-
mine).[9] However, their clinical efficacy is rather disappointing
considering the fact that improvement in cognitive symptoms is
modest and temporary.[10] Therefore, development of novel ther-
apeutic agents for Alzheimer's disease is urgently needed.
a potential therapeutic target for the treatment of AD, as its inhibition
may provide both symptomatic and the disease‐modifying effects in the
treatment of AD.
ROS have been considered as one of the contributing factors related
to the onset and advancement of AD.[15] The oxidative stress hypothesis
of AD states that mitochondrial dysfunction mediates increased release
of ROS, which contributes to the enhanced expression of transmembrane
amyloid‐β protein precursor (APP) and initiates accumulation of amyloid
β.[16] In fact, numerous clinical studies found elevated levels of oxidative
markers as well as antioxidant enzymes in AD patients.[17] In addition,
enhanced release of ROS and disrupted antioxidant protection may
directly impair synaptic function and neurotransmission resulting in
cognitive decline. These data have suggested that AD patients may
benefit from the treatment with antioxidant agents.[18]
Recently, many novel molecular targets, which might open new
therapeutic opportunities for the treatment of AD, have been suggested.
A growing body of evidence highlights a promising therapeutic potential
of serotonin 5‐HT6 receptor antagonists.[11] Serotonin 5‐HT6 receptors
are expressed in the brain areas (hippocampus and cerebral cortex)
responsible for cognitive functions, and it has been proved in animal
studies that 5‐HT6 antagonists enhance memory and learning processes.
Molecular studies demonstrated that this effect is related to an indirect
enhanced release of ACh in the cortex.[12] Results of phase II clinical trials
showed that combined therapy using 5‐HT6 receptor antagonist
(idalopirdine) and AChE inhibitor (donepezil) results in a superior
therapeutic effect in AD patients compared with monotherapy.[13]
Further studies revealed that 5‐HT6 antagonists enhance neuroplasticity
and provide neuroprotective effect against amyloid β‐induced neurotoxi-
city, by lowering reactive oxygen species (ROS) levels and maintaining the
proper neurite outgrowth.[14] Moreover, among many interesting
pharmacological activities of 5‐HT6 antagonists, antidepressant, and
anxiolytic activities might be particularly beneficial for AD patients,
because approximately 80% of AD patients exhibit comorbid behavioral
disturbances.[12] Consequently, the 5‐HT6 receptor has been regarded as
Given the complex etiology of AD, targeting exclusively one selected
biological target may not result in finding a sufficient treatment for
AD.[19] As a consequence, scientists follow a recent trend focused on the
design of multitarget‐directed ligands (MTDLs), which can simultaneously
interact with several crucial biological targets and offer potentially more
effective treatment of AD.[20,21] Herein, we describe the design, synthesis
and in vitro evaluation of a series of MTDLs combining inhibitory activity
against BuChE, 5‐HT6 receptor antagonism and antioxidant activity. The
biological studies comprise evaluation of inhibitory activity toward
BuChE as well as AChE, binding affinity and intrinsic activity on 5‐HT6
receptors and antioxidant evaluation using ferric reducing antioxidant
power assay (FRAP).
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2
RESULTS AND DISCUSSION
Design
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2.1
In the course of our work on MTDLs, we have screened a series of
5‐HT6 receptor antagonists synthesized in our laboratory against AChE
FIGURE 1 Design strategy of multitarget‐directed ligands combining butyrylcholinesterase inhibitory activity, 5‐HT6 antagonism and
antioxidant properties

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FIGURE 2 The predicted binding mode of compound 5 (orange) and 12 (blue) in the active site of butyrylcholinesterase (a) and the binding
site of the 5‐HT6 receptor (b). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks, whereas
residues identified as crucial for ligand binding, for example, forming H‐bonds (dotted yellow lines), π–π stacking interactions (dotted cyan lines)
and aromatic H‐bonds (dotted pale blue lines) are represented as thick sticks
and BuChE. Among the tested compounds we have selected two:
compounds 4 and 5 (Scheme 1) that displayed high nanomolar affinity
toward 5‐HT6 receptor (Ki = 17 and 3 nM, respectively) and micromolar
activity against BuChE (IC50 = 6.82 and 12.37 μM, respectively).
Following the “designing in” strategy[21] and our recent success on the
development of highly active multifunctional ligands based on these
pharmacophores[22,23] we have designed a novel series of MTDLs with
N‐substituted 4‐(piperazin‐1‐yl)‐1H‐indole core (Figure 1).
negative impact on affinity for the 5‐HT6 receptor. The binding mode of
compound 5 presents interactions in the orthosteric binding site (helices
3, 5, and 6), namely the charged‐reinforced hydrogen bond between the
basic amine and Asp3.32, the π–π stacking between indole moiety and
Phe6.51/6.52, as well as the aromatic interaction of benzyl ring with
Phe5.38 (Figure 2b). Its derivative, compound 12, displays the same
binding pattern, expanding its interactions to the accessory pocket
between helices 2 and 7. Moreover, values of the MM‐GBSA scoring
function used to compare the free energies of the receptor's complexes
with compound 5 and its phthalimide counterpart compound 12
(−65,411 vs. −93,328 kcal/mol, respectively) indicate a beneficial effect
of the modifications planned.
To improve the activity of the compounds against BuChE we have
designed a series of derivatives with piperazine moiety modified by a
phthalimide fragment attached with alkyl linkers of a different length
(Figure 1). We have previously used this fragment in the design of
selective BuChE inhibitors.[24]
A number of studies confirmed that indole‐based compounds
capture free radicals and thus protect lipids, proteins, and other
biological systems from peroxidation.[25,26] We have postulated that
free radical scavenger activity of indole moiety combined with
neuroprotective activity resulting from interaction with the 5‐HT6
receptor may protect neurons from further degeneration.
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2.2
Chemistry
We have synthesized a series of 12 compounds (6–17) according to the
synthetic pathway presented in Scheme 1. The key intermediates for the
synthesis (4 and 5) were prepared according to the procedure that we
have used and described before.[27] Briefly, commercially available
tert‐butyl 4‐(1H‐indol‐4‐yl)piperazine‐1‐carboxylate was alkylated with
benzyl bromide or 1‐(bromomethyl)‐3‐chlorobenzene in the presence of
potassium tert‐butoxide and 18‐crown‐6. The obtained tertiary amines
2 and 3 were subsequently deprotected in acidic conditions to give
compounds 4 and 5 as hydrochloride salts. In the last step, both
compounds were alkylated in acetonitrile reflux with ω‐bromoalkylphtha-
limides in the presence of K2CO3 as a base, to yield final compounds
6–17.
The design of new molecules was supported by docking studies,
using optimized crystal structure of BuChE and homology model of the
5‐HT6 receptor. In the active site of BuChE, as presented for
representative compound 5, the molecule forms π–π interactions with
Trp82 (benzyl group) and Tyr332 (indole moiety) (Figure 2a). Among the
newly designed structures, the best‐performing compound 12, contain-
ing the phthalimide fragment with propylene linker, favorably fills the
volume and interacts with amino acid residues of a hydrophobic pocket
composed of i.a. Val280, Pro281, Pro285 and forms an additional
hydrogen bond with Asn289 (Figure 2a). The proposed structural
modifications were expected to increase inhibitory potency against
BuChE, as the predicted ligand binding energy (MM‐GBSA methodol-
ogy) dropped from −58,476 to −80,280 kcal/mol (compound 5 vs. 12,
respectively). Furthermore, the planned introduction of spatial phtha-
limide substituent to the indole moiety does not appear to have a
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2.3
In vitro studies
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2.3.1
Affinity and functional activity on 5‐HT6
receptor
We assessed the affinity of the final compounds 6–17 for the 5‐HT6
receptor, in a radioligand binding assay with methiothepin as a

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SCHEME 1 Synthesis of compounds 4–17. Reagents and conditions: (i) Benzyl bromide or 1‐(bromomethyl)‐3‐chlorobenzene, THF,
potassium tert‐butoxide, 18‐crown‐6, RT, 24 hr, (ii) 1 M HCl in ethyl acetate, RT, 24 hr, (iii) ω‐bromoalkylphthalimide derivative, MeCN, K2CO3,
KI, reflux, 24 hr
reference compound.[28] The results of this study are summarized in
Table 1. All of the compounds displayed an affinity for 5‐HT6 receptor
ranging from 21 to 252 nM. We could see that the length of an alkyl
linker clearly affects the affinity of the compounds for the 5‐HT6
receptor. Among 3‐chlorobenzyl derivatives, the highest affinity was
detected for compound 13 with butylene linker (Ki = 27 nM). Elongation
of the linker resulted in significant decrease of affinity for compound 14
with pentylene linker (Ki = 238 nM). Further extension of the linker
resulted in Ki values ranging from 127 to 207 nM. Among nonsub-
stituted benzyl derivatives, compound 9 with hexylene linker showed
the highest affinity (Ki = 91 nM) while the lowest activity was detected
for compound 10 with heptylene linker (Ki = 250 nM).
potency against eqBuChE with IC50 values of 3.44 and 5.07 μM,
respectively. Both compounds are derivatives with 3‐carbon atom linker
connecting phthalimide moiety with 4‐piperazineindole core. Com-
pounds with longer linkers (7–9, 11, 14, and 15) displayed rather
modest inhibitory potency against eqBuChE ranging from 18–40%.
Compounds 10, 13, and 16 were not active at the screening
concentration. Regarding the influence of meta‐chlorine substituent at
benzyl moiety on eqBuChE inhibition, we noticed higher activities for
unsubstituted compounds (4 vs. 5, 6 vs. 12, 7 vs. 13, and 11 vs. 17) or no
differences in activities between them (8 vs. 14, 9 vs. 15, and 10 vs. 16).
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2.3.3
Determination of the antioxidant activity by
To determine the antagonistic mode of action, we have selected
three compounds: 6, 12, and 13 for cell‐based functional studies.[28]
The Kb values measured for these compounds: 1670 (6), 74 (12), and
193 nM (13), confirm that the compounds are 5‐HT6 antagonists.
FRAP assay
The FRAP aims to determine potential reducing/antioxidant
activity[30] of the tested compounds. The antioxidant activity is
measured by the ability of the compound to reduce 2,4,6‐tripyridyl‐s‐
triazine (Fe3+‐TPTZ salt) to its blue colored product (Fe2+‐TPTZ
salt).[31] The assay enables to detect molecules with redox potential,
which is crucial for maintaining the redox status in cells. For FRAP
assay we have selected the most promising compounds acting as
BuChE inhibitors/5‐HT6 antagonists, namely: 6, 12, and 13.
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2.3.2
Cholinesterase (AChE/BuChE) inhibitory
activity
We evaluated the ability of the synthesized compounds to inhibit
eeAChE and eqBuChE using Ellman's method.[29] These enzymes are
routinely used as a cheaper alternative and give a good prediction for
potency against expensive human enzymes. After an initial screening at
10 μM, for the compounds with inhibitory potency higher than 50%, we
determined IC50 values. We used tacrine and donepezil as reference
compounds in this assay. Additionally, to confirm that the results of the
assay on eqBuChE correspond well with human BuChE we have tested
compound 6 on the human enzyme. The results are collected in Table 1.
The compounds and a reference drug—ascorbic acid (vitamin C)—
were tested at three concentrations: 10, 100, and 1000 μM. The
results are presented in Figure 3 as a concentration of Fe2+ ions
formed by the reduction of Fe3+ by the tested compounds. All the
compounds displayed antioxidant activity similar or exceeding the
activity of ascorbic acid at the corresponding concentration.
Compound 6 showed significantly higher antioxidant potential in all
three concentrations. These data confirm the validity of the applied
structural design and are in agreement with previous studies, which
Comparing with the potency of their precursors,
4 and 5,
compounds 6 and 12 displayed approximately twofold higher inhibitory

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TABLE 1 Cholinesterase inhibitory potency and 5‐HT6 receptor affinity of compounds 4 and 5 and new multifunctional ligands 6–17
b
EeAChEa
% inh.d
EqBuChE
h5‐HT6Rc
Ki (nM)f
Compounds
R
n
–
–
1
IC50 (µM)e /% inhd
4
5
6
H
Cl
H
<10%
<10%
<10%
6.82 0.23
17.0 1.5
3.0 0.2
12.37 0.35
3.44 0.16
8.27 0.20i
149.8 8.4
1670.0 1.5g
7
H
H
H
H
H
Cl
2
3
4
5
6
1
10.9%
<10%
<10%
<10%
<10%
<10%
34.8%
n. a.
8
40.0%
189.3 18.1
90.6 9.8
252.2 23.1
180.6 12.7
9
14.5%
10
11
12
<10%
28.5%
5.07 0.20
41.8 4.8
74.0 0.3g
13
Cl
2
<10%
<10%
27.2 2.5
193.0 3.5g
14
Cl
Cl
Cl
Cl
3
4
5
6
<10%
37.7%
238.1 6.1
127.0 12.1
207.6 58.1
174.0 11.3
n.d.h
15
<10%
18.2%
16
<10%
<10%
17
<10%
< 10%
Donepezil
Tacrine
Methiothepin
0.011 0.0002e
0.023 0.0004e
1.83 0.04e
0.015 0.0001e
n.d.h
0.5 0.007
Abbrevations: AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; n.a., not available.
a
AChE from the electric eel.
b
BuChE from equine serum.
c
Human 5‐HT6 receptor.
Inhibition % at 10 μM.
d
e
IC50 values are expressed as mean standard error of the mean (SEM) of at least three experiments.
f
Ki values expressed as mean standard error of the mean (SEM) of two experiments.
g
h
i
Kb value.
Not determined.
Human BuChE.
showed that indole‐based compounds exhibit high antioxidant
potential.[25,26]
development of AD. Thus, compounds with antioxidant properties
are thoroughly studied in terms of their applicability in the treatment
of AD. Based on mitochondrial cascade hypothesis and cholinergic
hypothesis we have designed, synthesized, and evaluated a new
series of indole‐based MTDLs combining 5‐HT6 antagonistic activity,
BuChE inhibition, and antioxidant properties. Our study confirms our
previous findings that N‐substituted 4‐(piperazin‐1‐yl)‐1H‐indole core
is a suitable pharmacophore for the development of MTDLs. The
applied structural modifications that resulted in enhanced interac-
tions with BuChE did not affect the affinity at the 5‐HT6 receptor and
the obtained compounds retained high potency. Compound 12
displayed favorable dual activity against selected targets: 5‐HT6
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3
CONCLUSIONS
Currently, available anti‐Alzheimer's drugs offer, at most, sympto-
matic treatment for some patients. Although relief of symptoms is
important, it is crucial to find effective disease‐modifying drugs.
Mitochondrial cascade hypothesis points to oxidative stress as a
primary factor that triggers a neurodegenerative cascade leading to
Aβ accumulation, tau phosphorylation, synaptic loss and, finally,

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The BuChE model was developed on the basis of the experi-
mental structure of the enzyme (PDB ID: 1P0P) was used.[33] The
initial structure was refined using the Protein Preparation Wizard.
Water molecules and hetero groups other than the ligand were
deleted, the missing protein side chain atoms were predicted using
Prime and the energy of the whole system was minimized (OPLS3).
Grid boxes for docking in BuChE models were placed in a centroid of
cocrystallized ligand. The model was tested throughout docking
studies involving BuChE inhibitors of experimentally proven affinity.
The obtained consistent binding modes of the reference compounds
verified the accuracy of the crystal‐based model.
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4.2
Chemistry
General
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4.2.1
1H nuclear magnetic resonance (NMR) and 13C NMR spectra were
recorded on Varian Mercury 300 MHz (Varian, Inc., Palo Alto, CA) or Jeol
500 MHz (Jeol Inc., Peabody, MA). The chemical shifts for 1H NMR are
referenced to tetramethylsilane (TMS) via residual solvent signals (1 H,
CDCl3 at 7.26 ppm, DMSO‐d6 at 2.50 ppm). Mass spectra (MS) were
recorded on UPLC‐MS/MS system consisting of a Waters ACQUITY
UPLC (Waters Corporation, Milford, MA) coupled to a Waters TQD mass
spectrometer (electrospray ionization mode ESI‐tandem quadrupole).
Column chromatography was performed on Merck (Darmstadt, Ger-
many) silica gel 60 (63–200 μm). For the TLC and column chromato-
graphy following solvents were used: dichloromethane (DCM), methanol
(MeOH), petroleum ether, hexane (Hex), diethyl ether (Et2O), chloroform
(CHCl3), ethyl acetate (EtOAc), petroleum ether (PET), 25% ammonia–
water solution. The purity of the final compounds was determined using
an analytical RPLC‐MS on Waters Acquity TQD using an Aquity UPLC
BEH C18 column (1.7 μm, 2.1 × 100 mm) at 214 and 254 nm. CH3CN/
H2O gradient with 0.1% HCOOH was used as the mobile phase at a flow
rate of 0.3 ml/min. All the compounds showed purity above 95%. All of
the reagents were purchased from commercial suppliers and were used
without further purification.
FIGURE 3 Antioxidant effect of compounds 6, 12, 13 and
ascorbic acid in ferric reducing antioxidant power assay (FRAP). The
reducing power (FRAP value) is represented as mM/l of Fe2SO4. The
values are represented as mean standard deviation (n = 3).
***p < 0.001, the significant differences in the antioxidant activity of
the tested compound in comparison to ascorbic acid at the same
concentration (the Student t test)
(Ki = 41.8 nM) and BuChE (IC50 = 5.07 µM). When tested in FRAP
assay, compound 12 displayed favorable antioxidant properties,
exceeding the activity of the reference ascorbic acid. The antioxidant
activity is likely to be derived from an indole moiety, which
previously showed powerful antioxidant properties. Compound 12
might be considered as a promising starting point for further
optimization toward the development of multifunctional ligands
against Alzheimer's disease.
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4
EXPERIMENTAL
Molecular modeling
The following compounds: tert‐butyl 4‐(1‐benzyl‐1H‐indol‐4‐yl)‐
piperazine‐1‐carboxylate (2), tert‐butyl 4‐(1‐(3‐chlorobenzyl)‐1H‐in-
dol‐4‐yl)piperazine‐1‐carboxylate (3), 4‐(1‐benzyl‐1H‐indol‐4‐yl)pi-
perazin‐1‐hydrogen chloride (4), and 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐
4‐yl)piperazin‐1‐hydrogen chloride (5) were reported previously.[27]
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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4.1
The structures of ligands were built in Maestro 2D Sketcher and
optimized using LigPrep tool, generating protonation states according to
Epik. Glide SP flexible docking procedure was carried out using default
parameters and OPLS3 force field. Post‐docking minimization was
performed to retrieve five low energy complexes for each ligand. The
obtained poses were then evaluated based on Glide gscore values and
visual inspection. The selected complexes were optimized using Prime
MM‐GBSA tool. The free energy of ligand binding (MMGBSA dG Bind)
was calculated using VSGB solvation model and OPLS3 force field. A
homology model of serotonin 5‐HT6 receptor and a crystal structure‐
based BuChE model served as molecular targets.
Homology modeling procedure was reported previously[32] and
was applied for the preparation of a model based on the serotonin
5‐HT1B receptor crystal structure (PDB ID: 4IAR).[22] H‐bond
constraints, as well as the centroid of a grid box (25 Å) for docking
studies were located on Asp3.32.
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4.2.2
General procedure for the synthesis of
compounds 6–17 (procedure A)
The appropriate 4‐(1‐benzyl‐1H‐indol‐4‐yl)piperazin‐1‐hydrogen
chloride derivative (1 equiv.) with the 2‐(ω‐bromoalkyl)isoindoline‐
1,3‐dione (1 equiv.) in the presence of K2CO3 (1 equiv.) and KI
(1 equiv.) in MeCN was stirred at 70°C for 24 hr. After that time, the
reaction mixture was filtered and concentrated under reduced
pressure. The solvent was evaporated and the crude mixture was
purified over column chromatography.

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2‐(3‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)propyl)‐
isoindoline‐1,3‐dione (6)
2‐(6‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)hexyl)‐
isoindoline‐1,3‐dione (9)
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)‐
piperazin‐1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(3‐
bromopropyl)isoindoline‐1,3‐dione (111 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg,
0.414 mmol) in 20 ml MeCN was performed. Purification: column
chromatography (DCM/MeOH, 9.8:0.2, v/v); yield: 149 mg (75%);
MW = 478.60; formula C30H30N4O2; MS m/z 479.46 (M+H+); 1H
NMR (300 MHz, CDCl3) δ 7.96–7.79 (m, 2 H), 7.74–7.62 (m, 2 H),
7.33–7.17 (m, 3 H), 7.15–7.01 (m, 4 H), 6.94 (d, J = 8.2 Hz, 1 H),
6.58–6.45 (m, 2 H), 5.28 (s, 2 H), 3.81 (t, J = 6.8 Hz, 2 H), 3.17
(m, 4 H), 2.71 (t, J = 4.6 Hz, 4 H), 2.58 (t, J = 7.1 Hz, 2 H), 2.09, 1.86
(m, 2 H); 13C NMR (75 MHz, CDCl3) δ 168.5, 145.6, 137.6, 137.5,
133.9, 132.3, 128.7, 127.5, 126.7, 123.2, 122.4, 121.9, 106.6,
104.5, 100.2, 56.0, 53.3, 50.9, 50.2, 36.6, 25.1.
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)‐
piperazin‐1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(6‐
bromohexyl)isoindoline‐1,3‐dione (128 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg,
0.414 mmol) in 20 ml MeCN was performed. Purification: column
chromatography (DCM/MeOH, 95:5, v/v); yield: 170 mg (79%);
MW = 520.68; formula C33H36N4O2; MS m/z 521.48 (M+H+); 1H
NMR (300 MHz, CDCl3) δ 7.88–7.83 (m, 2 H), 7.81–7.76 (m, 3 H),
7.27–7.21 (m, 3 H), 7.12 (d, J = 1.8 Hz, 2 H), 7.07–7.02 (m, 2 H),
6.63–6.57 (m, 1 H), 6.52 (d, J = 3.3 Hz, 1 H), 5.35 (s, 2 H), 3.73–3.66
(m, 2 H), 3.34 (m, 4 H), 3.23 (m, 4 H), 3.00–2.82 (m, 2 H), 1.72 (m
2 H), 1.49–1.39 (m, 2 H), 1.31–1.21 (m, 4 H); 13C NMR (75 MHz,
CDCl3) δ 168.4, 145.8, 137.7, 137.5, 133.7, 132.2, 129.3, 127.5,
126.6, 123.5, 122.4, 121.9, 106.5, 104.4, 100.3, 58.9, 53.7, 51.3,
50.2, 38.1, 29.6, 29.5, 28.5, 27.5.
2‐(4‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)butyl)‐
isoindoline‐1,3‐dione (7)
2‐(7‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)heptyl)‐
isoindoline‐1,3‐dione (10)
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)
piperazin‐1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(4‐
bromobutyl)isoindoline‐1,3‐dione (117 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg,
0.414 mmol) in 20 ml MeCN was performed. Purification: column
chromatography (DCM/MeOH, 9.8:0.2, v/v); yield: 167 mg (82%);
MW = 492.62; formula C31H32N4O2; MS m/z 493.36 (M+H+); 1H
NMR (300 MHz, CDCl3) δ 7.85–7.81 (m, 2 H), 7.72, 7.67 (m, 2 H),
7.29–7.22 (m, 3 H), 7.10–7.04 (m, 4 H), 6.99–6.93 (m, 1 H), 6.63–
6.55 (m, 1 H), 6.51 (dd, J = 0.8, 3.3 Hz, 1 H), 5.28 (s, 2 H), 3.73
(t, J = 6.5 Hz, 2 H), 3.44–3.34 (m, 4 H), 2.91–2.82 (m, 4 H), 2.68,
2.59 (m, 2 H), 1.82, 1.66 (m, 4 H); 13C NMR (75 MHz, CDCl3)
δ 168.4, 145.2, 137.5, 137.5, 133.9, 132.1, 128.7, 127.6, 126.9,
126.7, 123.7, 122.4, 121.9, 106.8, 104.9, 100.1, 57.7, 53.3, 50.5,
50.3, 37.5, 26.5, 23.3.
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)piperazin‐
1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(7‐bromoheptyl)
isoindoline‐1,3‐dione (134 mg, 0.414 mmol) in the presence of K2CO3
(57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol) in 20 ml MeCN was
performed. Purification: column chromatography (Hex/Et2O/DCM/
MeOH, 40:30:29:1, v/v/v/v); yield: 117 mg (53%); MW = 534.70;
formula C34H38N4O2; MS m/z 535.51 (M+H+); 1H NMR (300 MHz,
CDCl3) δ 7.88–7.81 (m, 2 H), 7.73–7.66 (m, 2 H), 7.31–7.22 (m, 3 H),
7.13–7.05
(m,
4 H),
6.95
(d,
J = 8.2 Hz,
1 H),
6.60
(d, J = 7.4 Hz, 1 H), 6.55 (d, J = 3.1 Hz, 1 H), 5.29 (s, 2 H), 3.69
(t, J = 7.3 Hz, 2 H), 3.36–3.26 (m, 4 H), 2.76–2.66 (m, 4 H), 2.48–2.39
(m, 2 H), 1.77–1.63 (m, 2 H), 1.63–1.51 (m, 2 H), 1.43–1.23 (m, 6 H); 13C
NMR (75 MHz, CDCl3) δ 168.4, 145.8, 137.7, 137.6, 133.8, 132.2, 129.3,
127.5, 126.7, 123.5, 122.4, 121.9, 106.5, 104.4, 100.3, 58.9, 53.7, 51.3,
50.2, 38.0, 29.7, 29.5, 28.5, 27.5, 26.8.
2‐(5‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)pentyl)‐
isoindoline‐1,3‐dione (8)
2‐(8‐(4‐(1‐Benzyl‐1H‐indol‐4‐yl)piperazin‐1‐yl)octyl)isoindo-
line‐1,3‐dione (11)
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)-
piperazin‐1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(5‐
bromopentyl)isoindoline‐1,3‐dione (123 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg,
0.414 mmol) in 20 ml MeCN was performed. Purification: column
chromatography (Hex/Et2O/DCM/MeOH, 40:30:29:1, v/v/v/v);
yield: 208 mg (99%); MW = 506.65; formula C32H34N4O2; MS m/z
507.39 (M+H+); 1H NMR (300 MHz, CDCl3) δ 7.79–7.73 (m, 2 H),
7.66–7.59 (m, 2 H), 7.24–7.13 (m, 3 H), 7.05–6.96 (m, 4 H), 6.87
(d, J = 8.2 Hz, 1 H), 6.51 (d, J = 7.4 Hz, 1 H), 6.46 (d, J = 3.1 Hz, 1 H),
5.20 (s, 2 H), 3.63 (t, J = 7.3 Hz, 2 H), 3.26–3.17 (m, 4 H), 2.65
(d, J = 3.8 Hz, 4 H), 2.44–2.34 (m, 2 H), 1.73–1.62 (m, 2 H),
1.60–1.49 (m, 2 H), 1.41–1.29 (m, 2 H); 13C NMR (75 MHz, CDCl3)
δ 168.4, 145.7, 137.7, 137.5, 133.9, 132.0, 128.6, 127.5, 126.7,
123.1, 122.4, 121.8, 106.5, 104.5, 100.2, 58.4, 53.5, 50.8, 50.1,
37.8, 28.4, 26.2, 24.8.
Following procedure A, reaction of 4‐(1‐benzyl‐1H‐indol‐4‐yl)piper-
azin‐1‐hydrogen chloride (135 mg, 0.414 mmol) with 2‐(8‐bromooc-
tyl)isoindoline‐1,3‐dione (140 mg, 0.414 mmol) in the presence of
K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol) in 20 ml
MeCN was performed. Purification: column chromatography (Hex/
Et2O/DCM/MeOH, 40:30:29:1, v/v/v/v); yield: 48 mg (21%); MW =
548.73; formula C35H40N4O2; MS m/z 549.47 (M+H+); 1H NMR
(300 MHz, CDCl3) δ 7.87–7.79 (m, 2 H), 7.74–7.65 (m, 2 H), 7.31–7.21
(m, 4 H), 7.12–7.02 (m, 3 H), 6.95 (d, J = 8.5 Hz, 1 H), 6.62–6.57 (m,
1 H), 6.53 (dd, J = 0.6, 3.2 Hz, 1 H), 5.29 (s, 2 H), 3.74–3.63 (m, 2 H),
3.32 (d, J = 4.4 Hz, 4 H), 2.75 (m, 4 H), 2.53–2.41 (m, 2 H), 1.75–1.62
(m, 2 H), 1.57 (m, 2 H), 1.39–1.27 (m, 8 H); 13C NMR (75 MHz, CDCl3)
δ 168.5, 145.7, 137.6, 137.5, 133.9, 132.2, 128.7, 127.5, 126.7, 123.1,
122.4, 121.9, 106.6, 104.6, 100.2, 58.8, 53.5, 51.0, 50.2, 38.0, 29.7,
29.4, 29.1, 28.6, 27.5, 26.8.

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MARCINKOWSKA ET AL.
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2‐(3‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)pro-
pyl)isoindoline‐1,3‐dione (12)
2‐(6‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)hexyl)‐
isoindoline‐1,3‐dione (15)
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐4‐
yl)piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with 2‐(3‐
bromopropyl)isoindoline‐1,3‐dione (111 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol)
in 20 ml MeCN was performed. Purification: column chromatography
(DCM/MeOH, 9.8:0.2, v/v); yield: 155 mg (73%); MW = 513.04;
formula C30H29ClN4O2; MS m/z 513.50 (M+H+); 1H NMR
(300 MHz, CDCl3) δ 7.90–7.79 (m, 2 H), 7.75–7.66 (m, 2 H), 7.27–
7.16 (m, 2 H), 7.12–7.02 (m, 3 H), 6.96–6.85 (m, 2 H), 6.65–6.51 (m,
2 H), 5.26 (s, 2 H), 3.74 (t, J = 7.1 Hz, 2 H), 3.29 (m, 4 H), 2.72 (m, 4 H),
2.55–2.43 (m, 2 H), 1.76 (quin, J = 7.1 Hz, 2 H); 13C NMR (75 MHz,
CDCl3) δ 168.5, 145.8, 139.7, 137.3, 134.7, 133.9, 132.1, 130.0,
127.8, 126.7, 126.6, 124.8, 123.2, 122.7, 121.9, 106.7, 104.3, 100.7,
58.1, 53.6, 51.2, 49.6, 37.9, 24.2.
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐4‐
yl)piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with 2‐(6‐
bromohexyl)isoindoline‐1,3‐dione (128 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol)
in 20 ml MeCN was performed. Purification: column chromatography
(CHCl3/Hex/MeOH, 78:20:2, v/v/v); yield: 207 mg (90%); MW =
555.12; formula C33H35ClN4O2; MS m/z 555.38 (M+H+); 1H NMR
(300 MHz, CDCl3) δ 7.82 (dd, J = 3.1, 5.4 Hz, 2 H), 7.68 (dd, J = 3.1,
5.4 Hz, 2 H), 7.23–7.15 (m, 2 H), 7.12–7.03 (m, 3 H), 6.96–6.85 (m,
2 H), 6.68–6.52 (m, 2 H), 5.23 (s, 2 H), 3.69 (t, J = 7.3 Hz, 2 H), 3.30 (m,
4 H), 2.71 (m, 4 H), 2.52–2.38 (m, 2 H), 1.70 (m, 2 H), 1.57 (m, 2 H),
1.47–1.33 (m, 4 H); 13C NMR (75 MHz, CDCl3) δ 168.4, 145.9, 139.7,
137.4, 134.6, 133.8, 132.1, 130.0, 127.7, 126.7, 126.6, 124.8, 123.1,
122.7, 121.9, 106.8, 104.3, 100.7, 58.7, 53.6, 51.2, 49.6, 38.0, 28.5,
27.2, 26.8, 26.7.
2‐(4‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)butyl)‐
isoindoline‐1,3‐dione (13)
2‐(7‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)hep-
tyl)isoindoline‐1,3‐dione (16)
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐4‐
yl)piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with 2‐(4‐
bromobutyl)isoindoline‐1,3‐dione (117 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol)
in 20 ml MeCN was performed. Purification: column chromatography
(DCM/MeOH, 9.8:0.2, v/v); yield: 175 mg (80%); MW = 527.07;
formula C31H31ClN4O2; MS m/z 527.50 (M+H+); 1H NMR
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐4‐
yl)piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with 2‐(7‐
bromoheptyl)isoindoline‐1,3‐dione (134 mg, 0.414 mmol) in the
presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol)
in 20 ml MeCN was performed. Purification: column chromatography
(Hex/Et2O/DCM/MeOH, 40:30:29:1, v/v/v/v); yield: 183 mg (78%);
MW = 569.15; formula C34H37ClN4O2; MS m/z 569.41 (M+H+); 1H
NMR (300 MHz, CDCl3) δ 7.87–7.81 (m, 2 H), 7.70 (dd, J = 3.2, 5.3 Hz,
2 H), 7.22–7.17 (m, 2 H), 7.12–7.03 (m, 3 H), 6.97–6.87 (m, 2 H), 6.60
(d, J = 7.4 Hz, 1 H), 6.56 (d, J = 3.1 Hz, 1 H), 5.26 (s, 2 H), 3.69
(t, J = 7.2 Hz, 2 H), 3.36–3.26 (m, 4 H), 2.77–2.67 (m, 4 H), 2.50–2.38
(m, 2 H), 1.69 (td, J = 6.7, 13.5 Hz, 2 H), 1.63–1.50 (m, 2 H), 1.44–1.31
(m, 6 H); 13C NMR (75 MHz, CDCl3) δ 168.4, 145.9, 139.7, 137.4,
134.7, 133.8, 132.1, 130.0, 127.8, 126.7, 126.5, 124.8, 123.1, 122.7,
121.7, 106.8, 104.3, 100.7, 58.8, 53.6, 51.2, 49.6, 38.0, 29.8, 29.1,
28.5, 27.5, 26.7.
(300 MHz, CDCl3)
δ 7.94–7.78 (m, 2 H), 7.75–7.62 (m, 2 H),
7.31–7.14 (m, 2 H), 7.06 (dd, J = 3.7, 9.1 Hz, 3 H), 6.96–6.82
(m, 2 H), 6.57–6.42 (m, 2 H), 5.25 (s, 2 H), 3.81 (t, J = 6.9 Hz, 2 H),
3.24–3.02 (m, 4 H), 2.73–2.58 (m, 4 H), 2.54 (t, J = 6.9 Hz, 2 H), 1.94
(quin, J = 6.7 Hz, 2 H), 160–1.52 (m, 2 H); 13C NMR (75 MHz, CDCl3)
δ 168.5, 145.8, 139.7, 137.3, 134.6, 133.9, 132.3, 130.0, 127.8, 126.7,
126.6, 124.8, 123.2, 122.6, 121.9, 106.6, 104.2, 100.2, 56.2, 53.5,
51.2, 49.3, 36.7, 25.3, 24.1.
2‐(5‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)pen-
tyl)isoindoline‐1,3‐dione (14)
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐
4‐yl)piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with
2‐(5‐bromopentyl)isoindoline‐1,3‐dione (123 mg, 0.414 mmol) in
the presence of K2CO3 (57 mg, 0.414 mmol) and KI (69 mg,
0.414 mmol) in 20 ml MeCN was performed. Purification: column
chromatography (CHCl3/PET/MeOH, 77:20:3, v/v/v); yield:
177 mg (79%); MW = 541.09; formula C32H33ClN4O2; MS m/z
541.36 (M+H+); 1H NMR (300 MHz, CDCl3) δ 7.87–7.79 (m, 2 H),
7.72–7.64 (m, 2 H), 7.23–7.15 (m, 2 H), 7.09–7.03 (m, 3 H), 6.96–
6.88 (m, 2 H), 6.62–6.57 (m, 1 H), 6.52 (dd, J = 0.6, 3.2 Hz, 1 H), 5.24
(s, 2 H), 3.70 (t, J = 7.2 Hz, 2 H), 3.38 (d, J = 4.4 Hz, 4 H), 2.86 (m,
4 H), 2.64–2.51 (m, 2 H), 1.72 (qd, J = 7.1, 13.9 Hz, 4 H), 1.48–1.35
(m, 2 H); 13C NMR (75 MHz, CDCl3) δ 168.1, 145.3, 139.7, 137.3,
134.6, 133.9, 132.1, 130.0, 127.8, 126.8, 126.7, 124.8, 123.2,
122.7, 121.6, 107.0, 104.7, 100.5, 58.2, 53.2, 50.4, 49.6, 37.7, 28.3,
25.5, 24.6.
2‐(8‐(4‐(1‐(3‐Chlorobenzyl)‐1H‐indol‐4‐yl)piperazin‐1‐yl)octyl)‐
isoindoline‐1,3‐dione (17)
Following procedure A, reaction of 4‐(1‐(3‐chlorobenzyl)‐1H‐indol‐4‐yl)-
piperazin‐1‐hydrogen chloride (150 mg, 0.414 mmol) with 2‐(8‐bro-
mooctyl)isoindoline‐1,3‐dione (140 mg, 0.414 mmol) in the presence of
K2CO3 (57 mg, 0.414 mmol) and KI (69 mg, 0.414 mmol) in 20 ml MeCN
was performed. Purification: column chromatography (DCM/Hex/
acetone/MeOH, 25:19:5:1, v/v/v/v); yield: 167 mg (69%); MW = 583.17;
formula C35H39ClN4O2; MS m/z 583.44 (M+H+); 1H NMR (300 MHz,
CDCl3) δ 7.88–7.77 (m, 2 H), 7.72–7.59 (m, 2 H), 7.22–7.01 (m, 5 H),
6.97–6.84 (m, 2 H), 6.66–6.51 (m, 2 H), 5.23 (s, 2 H), 3.76–3.59 (m, 2 H),
3.39–3.23 (m, 4 H), 2.80–2.63 (m, 4 H), 2.51–2.37 (m, 2 H), 1.74–1.62
(m, 2 H), 1.55 (m, 2 H), 1.43–1.28 (m, 8 H); 13C NMR (75 MHz, CDCl3) δ
168.4, 145.9, 139.8, 137.4, 134.6, 133.8, 132.1, 130.0, 127.7, 126.8,
126.5, 124.8, 123.3, 122.7, 122.0, 106.7, 104.2, 100.8, 58.9, 53.7, 51.3,
49.6, 38.0, 29.4, 29.1, 28.6, 27.6, 26.8, 26.7.

MARCINKOWSKA ET AL.
9 of 10
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|
4.3
Biological assays
iodide (ATC), and butyrylthiocholine iodide (BTC) were pur-
chased from Sigma‐Aldrich, human plasma BuChE was a gift from
Vivonics. The enzymes were prepared at a final concentration of
0.384 U/ml, aqueous solutions DTNB at 0.0025 M and ATC/BTC
at 0.00375 M. At first, 25 μl of the test compound (or water; i.e.,
blank samples) was incubated (5 min) in 200 μl of 0.1 M
phosphate buffer (pH 8.0) with DTNB (20 μl) and the enzyme
(20 μl) at 25°C. After incubation 20 μl of acetylthiocholine iodide
or butyrylthiocholine iodide solutions were added to start the
reaction. Finally, after 5 min of the reaction, changes in
absorbance were measured at 412 nm, using the microplate
reader (EnSpire Multimode; PerkinElmer). The IC50 values were
calculated from seven concentrations of compounds using non-
linear regression (GraphPad Prism 5; GraphPad Software).
Serotonin 5‐HT6 receptor binding assay[22]
|
4.3.1
The assay was performed on membranes from CHO‐K1 cells stably
transfected with the human 5‐HT6 receptor (PerkinElmer, Waltham,
MA). All assays were carried out in duplicates. Fifty microliters dimethyl
sulfoxide (DMSO) solution (10−5 to 10−12 M) of the tested compounds,
50 µl [3H]‐LSD (final concentration 1.3 nM) and 150 µl diluted mem-
branes (8 µg protein per well) prepared in assay buffer (50 mM Tris, pH
7.4, 10 mM MgCl2, 0.1 mM EDTA) were transferred to polypropylene
96‐well microplate using 96‐wells pipetting station Rainin Liquidator
(Mettler‐Toledo, Columbus, OH). Methiothepin (10 μM) was used to
define nonspecific binding. Microplate was covered with a sealing tape,
mixed and incubated for 60 min at 37°C. The reaction was terminated
by rapid filtration through GF/B filter mate presoaked with 0.5%
polyethyleneimine for 30 min. Ten rapid washes with 200 µl 50 mM Tris
buffer (4°C, pH 7.4) were performed using automated harvester system
Harvester‐96 MACH III FM (Tomtec, Chicago, IL). The filter mates were
dried at 37°C in forced air fan incubator and then solid scintillator
MeltiLex was melted on filter mates at 90°C for 5 min. Radioactivity
was counted in MicroBeta2 scintillation counter (PerkinElmer). Data
were fitted to a one‐site curve‐fitting equation with Prism 6 (GraphPad
Software, San Diego, CA) and Ki values were estimated from the Cheng
−Prusoff equation.
|
4.3.4
Determination of the antioxidant activity by
FRAP assay
The FRAP assay was done according to Benzie and Strain[35] with
some modifications. The FRAP reagent was prepared by mixing
10 parts of 300 mmol/l acetate buffer pH 3.6 (3.1 g CH3COONa × 3
H2O and 16 ml CH3COOH), 1 part of 10 mmol/l 2,4,6‐tripyridyl‐s‐
triazine (TPTZ) solution in 40 mmol/l HCl, and 1 part of 20 mmol/l
FeCl3 × 6H2O solution. Next, 300 μl of the FRAP reagent was mixed
with 10 μl of tested compound (solution in ethanol) and incubated at
room temperature for 10 min in the dark. The absorbance of the
resulting colored product, the ferrous tripyridyltriazine complex, was
recorded at 593 nm. Results are presented as the concentration of
Fe2+ generated in the test. Ascorbic acid was used as reference
compound.
Functional assays for the 5‐HT6 receptor[28]
|
4.3.2
A
cellular aequorin‐based functional assay was performed on
recombinant CHO‐K1 cells expressing mitochondrially targeted
aequorin, human GPCR and the promiscuous G protein α16 for
5‐HT6. After thawing, cells were transferred to assay buffer (DMEM/
HAM's F12 with 0.1% protease free bovine serum albumin [BSA]) and
centrifuged. The cell pellet was resuspended in assay buffer and
coelenterazine h was added at final concentrations of 5 μM. The cells
suspension was incubated at 16°C, protected from light with
constant agitation for 16 hr and then diluted with assay buffer to
the concentration of 100,000 cells/ml. After 1 hr of incubation, 50 μl
of the cells suspension was dispensed using automatic injectors built
into the radiometric and luminescence plate counter MicroBeta2
LumiJET (PerkinElmer) into white opaque 96‐well microplates
preloaded with test compounds (eight concentrations in DMSO).
Immediate light emission generated following calcium mobilization
was recorded for 60 s. In antagonist mode, after 30 min of incubation
the reference agonist was added to the above assay mix and light
emission was recorded again. Final concentration of the reference
agonist was equal to EC80 (40 nM serotonin).
ACKNOWLEDGMENTS
This study received financial support from National Science Center
Poland (2016/23/D/NZ7/01328), and from Jagiellonian University
Collegium Medicum (K/ZDS/007865 and K/ZDS/007862).
ORCID
Monika Marcinkowska
http://orcid.org/0000-0002-4948-6617
Adam Bucki
http://orcid.org/0000-0003-0451-9814
Dawid Panek
Marek Bednarski
http://orcid.org/0000-0002-3211-7188
http://orcid.org/0000-0002-4639-9913
Małgorzata Zygmunt
Magdalena Kotańska
Marcin Kołaczkowski
Anna Więckowska
http://orcid.org/0000-0003-2046-4155
http://orcid.org/0000-0002-3946-1192
http://orcid.org/0000-0001-8402-1121
http://orcid.org/0000-0003-2549-170X
|
4.3.3
In vitro AChE and BuChE inhibition assay
REFERENCES
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AChE from Electrophorus electricus, BuChE from equine serum,
5,5′‐dithiobis‐(2‐nitrobenzoic acid; DTNB), acetylthiocholine
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https://doi.org/10.1002/ardp.201900041