A dual-acting 5-HT6 receptor inverse agonist/MAO-B inhibitor displays glioprotective and pro-cognitive properties

Article abstract of DOI:10.1016/j.ejmech.2020.112765

The complex etiology of Alzheimer's disease has initiated a quest for multi-target ligands to address the multifactorial causes of this neurodegenerative disorder. In this context, we designed dual-acting 5-HT₆ receptor (5-HT₆R) anta

Full text of DOI:10.1016/j.ejmech.2020.112765

European Journal of Medicinal Chemistry 208 (2020) 112765
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
A dual-acting 5-HT6 receptor inverse agonist/MAO-B inhibitor displays
glioprotective and pro-cognitive properties
Vittorio Canale a, Katarzyna Grychowska a, Rafał Kurczab b, Mateusz Ryng b,
Abdul Raheem Keeri a, Grzegorz Satała b, Agnieszka Olejarz-Maciej c,
Paulina Koczurkiewicz d, Marcin Drop a, Klaudia Blicharz a, Kamil Piska d,
d
e
f
e
_
Elzbieta Pe˛kala , Paulina Janiszewska , Martyna Krawczyk , Maria Walczak ,
Severine Chaumont-Dubel g, Andrzej J. Bojarski b, Philippe Marin g, Piotr Popik f,
,
*
Paweł Zajdel a
a
ꢀ
Jagiellonian University Medical College, Department of Medicinal Chemistry, 9 Medyczna Str., 30-688, Krakow, Poland
b
c
d
e
f
ꢀ
Maj Institute of Pharmacology, Polish Academy of Sciences, Department of Medicinal Chemistry, 12 Sme˛tna Str., 31-324, Krakow, Poland
ꢀ
Jagiellonian University Medical College, Department of Technology and Biotechnology of Drugs, 9 Medyczna Str., 30-688, Krakow, Poland
ꢀ
Jagiellonian University Medical College, Department of Pharmaceutical Biochemistry, 9 Medyczna Str., 30-688, Krakow, Poland
ꢀ
Jagiellonian University Medical College, Department of Toxicology, 9 Medyczna Str., 30-688, Krakow, Poland
ꢀ
Maj Institute of Pharmacology, Polish Academy of Sciences, Department of New Drug Development, 12 Sme˛tna Str., 31-324, Krakow, Poland
g
ꢀ
ꢀ
Institut de Genomique Fonctionelle, Universite de Montpellier, CNRS INSERM, 34094, Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The complex etiology of Alzheimer’s disease has initiated a quest for multi-target ligands to address the
multifactorial causes of this neurodegenerative disorder. In this context, we designed dual-acting 5-HT6
receptor (5-HT6R) antagonists/MAO-B inhibitors using pharmacophore hybridization strategy. Our
approach involved linking priviliged scaffolds of 5-HT6R with aryloxy fragments derived from reversible
and irreversible MAO-B inhibitors. The study identified compound 48 that acts as an inverse agonist of 5-
HT6R at Gs signaling and an irreversible MAO-B inhibitor. Compound 48 showed moderate metabolic
stability in rat microsomal assay, artificial membrane permeability, no hepatotoxicity, and it was well
distributed to the brain. Additionally, 48 showed glioprotective properties in a model of cultured as-
trocytes using 6-OHDA as the cytotoxic agent. Finally, compound 48 (MED ¼ 1 mg/kg, p.o.) fully reversed
memory deficits in the NOR task induced by scopolamine in rats. A better understanding of effects
exerted by dual-acting 5-HT6R/MAO-B modulators may impact the future development of
neurodegenerative-directed treatment strategies.
Received 8 July 2020
Received in revised form
3 August 2020
Accepted 15 August 2020
Available online 22 August 2020
Keywords:
Neurodegenerative disorders
Alzheimer’s disease
MAO-B inhibitors
5-HT6R antagonists
Multi-target directed ligands
Constitutive activity
Glia
© 2020 Elsevier Masson SAS. All rights reserved.
Cognition
1. Introduction
prevalence of AD, together with the lack of effective therapy that
could slow down the progression of the disease, makes it one of the
Neurodegenerative diseases are chronic conditions which
involve progressive loss of neuronal and glial cells, demyelination,
and overall structural and functional brain deficits. Among these
diseases, Alzheimer’s disease (AD) is the most common disorder
characterized by continuous deterioration of cognitive functions,
resulting in complete disability in the elderly life. The increasing
biggest global health challenges [1].
Unfortunately, therapeutically useful disease-modifying and
symptomatic drug candidates for AD developed in the two past
decades have shown disappointing results [2e4]. Nonetheless, our
understanding of G protein-coupled receptors (GPCRs) signaling
networks, and compensatory mechanisms related to enzyme in-
hibition [5] and disease pathology steadily grew, thereby opening
new opportunities for drug discovery. In particular, more focus was
given to develop new strategies using multi-target directed ligands
(MTDL) to address the etiology and symptoms of such a complex
* Corresponding author. Jagiellonian University Medical College, Department of
Medicinal Chemistry, Poland.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
https://doi.org/10.1016/j.ejmech.2020.112765
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
and debilitating disease. Accumulating evidence revealed that
connecting and/or merging structural fragments, which simulta-
neously affect different biological targets, is one of the most
promising areas of anti-AD drug development [6,7]. Several MTDLs
have been reported, with the most prominent examples being dual
enzymatic inhibition of acetylcholinesterase (AChE) and glycogen
synthase kinase (GSK-3
oxidase B (MAO-B) [9,10] and inhibition of
and GSK-3 [11]. The MTDL strategy is further completed with
b
) [8], inhibition of AChE and monoamine
b
-secretase (BACE-1)
b
GPCRs dual-acting compounds as 5-HT6/D3 and 5-HT2A/5-HT6 re-
ceptor antagonists [12e15] as well as 5-HT4R agonist/5-HT6R
antagonist [16]. However, simultaneous targeting of enzyme and
receptor activities constitutes a more challenging area. This stems
from limitations in finding common binding modes and structural
features for targets with different location and structural re-
quirements [17]. Among examples of such an approach, the dual-
acting compounds described thus far include AchE inhibitors and
NMDA receptor antagonists [18], AChE and histamine H3 receptor
(H3R) antagonists [19], AChE inhibitors and sigma s1 receptor li-
gands [20], AChE inhibitors and serotonin 5-HT4R agonists [21] or
AChE inhibitors and 5-HT6R antagonists [22].
Serotonin type 6 receptor (5-HT6R) is a GPCR which has
emerged as a promising target for the treatment of pro-cognitive
deficits of neurodegenerative and psychiatric disorders. It is
almost exclusively expressed in the central nervous system (CNS),
particularly in brain regions involved in cognitive processes, i.e.,
prefrontal cortex, hippocampus and striatum. 5-HT6R is primarily
located in the cilia of neurons which are sensory organelles that
participate in neurodevelopment and neuronal differentiation
[23e26].
Pharmacological blockade of 5-HT6R, located on GABA-ergic and
glutamatergic neurons, increases cholinergic transmission [27,28].
Consequently, 5-HT6R antagonists show beneficial effects on
cognition, as shown by a various preclinical studies and preliminary
clinical studies [29e31] that revealed pro-cognitive effects in
humans [32]. However, phase III clinical trials for these compounds
showed disappointing results.
Fig. 1. The design strategy of dual-acting 5-HT6R/MAO-B modulators.
length. Here, we evaluated the structure-activity relationship (SAR)
within novel dual-acting compounds exhibiting inhibitory activity
for 5-HT6R and MAO-B. We selected a lead compound (compound
48), for which we performed more detailed mechanistic studies.
We assessed its action on both targets and examined its distribu-
tion to the CNS following peripheral administration. Finally, we
showed that this dual 5-HT6R inverse agonist/MAO-B inhibitor
protects astrocytes against 6-hydroxydopamine (6-OHDA)-induced
toxicity and reverses scopolamine-induced cognitive impairment
in the novel object recognition (NOR) task in rats.
The recent findings on the elevated level of MAO-B in AD brains,
corroborated by data showing a MAO-B-dependent control of
amyloid-b production in neurons via g-secretase, suggest a role of
MAO-B in AD [33]. MAO-B is mainly found in the outer membrane
of the mitochondria in non-neuronal cells, such as glial cells
[34,35]. It is a membrane-bound FAD-containing enzyme, involved
in the degradation of monoamines, including dopamine. It has been
demonstrated that the production of reactive oxygen species,
resulting from the activity of MAO-B, increases with age. Further-
more, an elevated level of this enzyme has been found in astrocytes
and pyramidal neurons of AD brains [33,36]. Based on these ob-
servations, MAO-B inhibitors have been investigated as potential
neuroprotective agents for the treatment of neurodegenerative
disorders [37].
To determine how to target two distinct proteins with different
pharmacophore requirements, we analyzed the binding mode in
the 5-HT6R homology model and sought opportunities to target
MAO-B catalytic center using induced fit-docking (IFD) and quan-
tum mechanics/molecular mechanics (QM/MM) calculations. This
prospective in silico analysis, guided the design and synthesis of a
new series of hybrid compounds obtained by linking pharmaco-
phore fragments of 5-HT6R antagonists with molecular frameworks
of known MAO-B inhibitors in a unique molecular entity (Fig. 1).
Structural modifications comprised functionalization of N1 atom of
(indol-4-yl)piperazine with 3-chlorobenzyl and benzenesulfonyl
moieties in the 5-HT6R-aiming substructure and diversification of
aryloxy fragments derived from reversible and irreversible MAO-B
inhibitors, both connected via an alkylene spacer of different
2. Chemistry
The designed compounds were synthesized in two stages. First,
the respective building blocks, namely functionalized (indol-4-yl)
piperazine 5 and 6 (Scheme 1A) and alkylating agents 9e17
(Scheme 1B), were obtained using parallel synthetic routes.
Commercially available 4-bromoindole
1 was subjected to
Buchwald-Hartwig N-arylation to yield 4-(4-Boc-piperazin-1-yl)
indole. Simple heating of the obtained product in ethanol at 150 ꢀC
using microwave irradiation eliminated Boc protecting function
and provided 1H-indole intermediate 2. This was subsequently
reacted with 3-chlorobenzyl bromide or benzenesulfonyl chloride,
in the presence of phosphazene base (BTPP) to afford indoles 3 and
4. Treatment of the latter compounds with TFA furnished secondary
amines 5 and 6, respectively.
In parallel, commercially available phenols 7 and 8 were reacted
with an excess of different a,u-dibromoalkane in biphasic condi-
tions, to give respective alkylating agents 9e17 (Scheme 2). In-
termediates 9e17 were subsequently combined with secondary
amines 5 and 6, yielding derivatives 18e30, by stirring in acetone at
60 ꢀC, in the biphasic system, for 16 h.
Final compounds 31e48 were obtained by reductive amination,
using sodium triacetoxyborohydride (STAB) in THF for primary
amines or STAB in CH2Cl2 for secondary amines.
2

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Table 1
Affinity for 5-HT6R and potency for MAO-B inhibition of synthesized compounds 31e48 and reference 5-HT6R ligands and MAO-B inhibitors.
Compd
R
X
N
R1
Ki [nM]a h5-HT6R
IC50 [nM]b and (%inh)c hMAO-B
31
32
33
34
35
36
37
Cl
Cl
Cl
Cl
Cl
H
CH2
CH2
CH2
CH2
CH2
SO2
SO2
1
2
3
4
5
3
4
I
I
I
I
I
I
I
100
77
113
223
259
50
> 1000 (35)
> 1000 (31)
270 43 (100)
460 51 (80)
> 1000 (34)
> 1000 (44)
163 25 (90)
H
51
38
39
40
41
42
43
Cl
Cl
Cl
Cl
H
CH2
CH2
CH2
CH2
SO2
SO2
2
3
4
5
3
4
II
II
II
II
II
II
158
162
232
340
68
> 1000 (47)
> 1000 (24)
> 1000 (43)
> 1000 (23)
> 1000 (20)
> 1000 (17)
H
65
44
45
46
47
48
Cl
Cl
Cl
H
CH2
CH2
CH2
SO2
SO2
3
4
5
3
4
III
III
III
III
III
195
140
137
54
323 42 (85)
30 2 (100)
> 1000 (32)
> 1000 (51)
154 19 (91)
H
38
A
7
(3)
B
0.3
(3)
Intepirdined
Rasagiline
Safinamide
1.4
N.T.e
15.4 0.6 (100)
N.T.e
N.T.e
7
1.2 (100)
a
Mean Ki values (SEM 15%) based on three independent binding experiments
IC50 SEMs values were determined using rasagiline [1 mM] as positive control
b
c
% of inhibition of control (rasagiline, 1
Data taken from [30]
mM) at 1 mM
d
e
not tested.
derived from rasagiline, safinamide and pargyline were linked to
the 5-HT6R pharmacophore via different length alkylene linker.
Because the molecular framework of the designed hybrids is
different from that of classical MAO-B inhibitors (they are very long,
with two basic centers and a flexible alkylene linker), the possibility
of interaction with the catalytic domain of MAO-B was supported
by in silico experiments. Indeed, molecular dynamics studies indi-
cated the existence of two entrance channels to the MAO-B binding
site for inhibitors (more details in SI), i.e., membrane accessed (A),
and solvent accessed (B) [39]. An analysis of ligand-enzyme com-
plexes for MAO-B crystals showed that entrance A is closed for
several compounds, whereas entrance B forms a narrow channel.
IFD for the designed compounds provided a static view of the
conformation of the enzyme, showing that the only way to enter
the catalytic center is through entrance B, which is devoid of sig-
nificant steric hindrances. It should be emphasized here that such
large compounds would be unable to enter the catalytic MAO-B
through entrance A, because it would be sterically disturbed by
3. Results and discussion
3.1. Structure-activity relationship studies
The molecular framework of the dual-acting derivatives was
based on the pharmacophore hybridization strategy supported
with prospective in silico analysis of homology model of 5-HT6R and
crystal structure of MAO-B. The strategy involved connecting
pharmacophore structures of known selective 5-HT6R antagonists
and MAO-B inhibitors in a single molecule. The selection of phe-
nylsulfonyl-4-(piperazin-1-yl)-1H-indole
(A)
and
N-3-
chlorobenzyl-4-(piperazin-1-yl)indole (B) as 5-HT6R-targeting
fragments, was based on literature survey and unpublished data.
Our results indicated that compounds containing unsubstituted
phenylsulfonyl moiety are highly potent 5-HT6R ligands, whereas
their benzyl analogs require substitution with a chlorine atom in
the meta position [38].
To achieve inhibition of MAO-B activity, aryloxy fragments
3

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Fig. 2. The binding mode of 48 (cyan) in 5-HT6R (A), and comparison of binding modes of 45 (green) and 48 (cyan) in the MAO-B catalytic site (B). (For interpretation of the
references to color in this figure legend, the reader is referred to the Web version of this article.)
formed a salt bridge with D3.32, the indole ring was positioned in
the conserved aromatic cluster of F6.51 and F6.52, and the terminal
phenyl ring was accommodated in a hydrophobic cavity formed by
transmembrane domains 3e5 and extracellular loop 2. It should be
Table 2
Binding data of selected compound 48 and intepirdine for 5-HT6, 5-HT1A, 5-HT2A, 5-
HT7, and D2Rs and inhibitory activity (IC50 values) for MAO-B and MAO-A.
Ki [nM]a
IC50
b
Compd
noted that the indole and phenyl rings form an angle of approxi-
mately 90ꢀ, resulting from the tetrahedral geometry (sp3) of sul-
fonyl linkers. In turn, the propargyl moiety at the aryloxy fragment
was directed toward the extracellular part of the receptor, with the
phenyl ring positioned between N2.63 and W7.39, and protonated
the N-methyl-propargylamine forming a salt bridge with D7.35.
Exploration of SAR toward MAO-B revealed that sulfonamide
derivatives were less preferred for the interaction with the enzy-
matic target than their benzyl analogs (33 vs 36, 40 vs 43, and 44 vs
47). More detailed analysis of the aryloxy terminal fragment,
indicated that compounds 38e43 bearing safinamide-derived ala-
ninamide moiety, showed low inhibitory potency against enzyme.
On the other hand, the presence of propargyl- and N-methyl-
propargylamine (originated from rasagiline and pargyline, respec-
tively) increased the inhibitory effect (31e100% inhibition), thus
highlighting the pivotal role of covalent bond formation of com-
pounds 33, 37, 45 and 48 with FAD cofactor in the enzymatic target.
With regard to the length of the alkylene chain, five methylene
linked hybrides (37, 45, 48) exhibited the highest inhibition activity
for MAO-B. This observation was further confirmed by in silico
studies. The binding modes of the most potent MAO-B inhibitors 45
and 48 (Fig. 2B) were coherent and showed accommodation of the
molecules in an “aromatic cage” formed by Y435, Y398, Y60, and
F343, which are considered crucial in the ligand positioning for the
reaction with FAD [40e42]. Additionally, the hydrogen bond be-
tween the side chain of Q206 and the nitrogen atom of the prop-
argyl functional group seems to play a role in placing this fragment
in an optimal position [42].
5-HT6 5-HT1A 5-HT2A 5-HT7
D2
MAO-B MAO-A
48
38
684
2370
420
26
7996
813 154
1037
NTd
Intepirdinec 1.4
14 230 997 NTd
a
Mean Ki values (SEM 25%) based on three independent binding experiments.
IC50 SEMs values was determined using rasagiline [1 mM] or clorgyline [1 mM]
as positive control for MAO-B and MAO-A, respectively.
b
c
Data taken from [30].
d
Not Tested.
the mitochondrial membrane.
The newly synthesized compounds 31e48 showed moderate
affinity for 5-HT6R in 3[H]-LSD binding experiments and low-to-
high inhibitory potency for MAO-B, with IC50 values ranging from
30 to >1000 nM as assessed using the fluorimetric method
(Table 1).
Generally, linking 5-HT6R pharmacophores with MAO-B-
targeting fragments decreased affinity for 5-HT6R when
compared with the parent compounds A and B. However, this
modification did not totally preclude the interaction of the obtained
derivatives with the binding cavity of 5-HT6R (Ki ¼ 38e340 nM).
Compounds bearing N1-sulfonyl moiety were more potent 5-HT6R
ligands than their congeners with methylene bridge, regardless of
the length of the alkylene linker and the kind of MAO-B fragment
(34 vs 37 and 44 vs 47). Analysis of the obtained ligand-receptor
complexes for compound 48 exhibiting the highest affinity for 5-
HT6R (Fig. 2A), showed that the protonated piperazine fragment
3.2. Determination of selectivity profile for compound 48
Table 3
The antagonist property of compound 48 and intepirdine in 1321N1 cells and their
functional activity at 5-HT6R-dependent Gs signaling in NG108-15-based model.
The SAR analysis allowed to identify the most potent dual-acting
compound 48 possessing a phenylsulfonyl moiety at (indol-4-yl)
piperazine scaffold in tandem with aryloxy fragments derived from
Compd
Kb [nM]a
IC50 [nM]b
Functional profile
pargyline, connected via
a four methylene spacer (Ki 5-
5-HT6
HT6R ¼ 38 nM, IC50 MAO-B ¼ 154 nM). Subsequently, compound
48 was profiled in vitro for selectivity over structurally-related
GPCRs and MAO-A enzyme isoform (Table 2).
48
6
1
860 0.68
97 0.21
Inverse Agonist
Inverse Agonist
Intepirdine
1.3 0.2
a
Mean Kb
SEMs of values obtained in three independent experiments in
SEMs of values obtained in three independent experiments in
Compound 48 showed moderate to high selectivity over 5-HT1A
,
1321N1 cells.
b
5-HT2A, 5-HT7, and D2 receptors. Although it contains aryloxy
fragments derived from pargyline (a known non-selective MAO
inhibitor), it showed moderate inhibitory activity toward the MAO-
Mean IC50
NG108-15 cells.
A
enzyme isoform, thus reducing the possibility to elicit
4

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
opens up the possibility to classify 5-HT6R ligands as inverse ago-
nists or neutral antagonists [30,45].
Hence, the ability of compound 48 to modulate agonist-
independent 5-HT6R-operated Gs signaling was tested in NG108-
15 cells transiently expressing 5-HT6R, a cellular model in which
the receptor displays high constitutive activity [44,46]. We found
that compound 48 decreased basal cAMP level in a concentration-
dependent manner (IC50 ¼ 860 0.68 nM) but its apparent affinity
was much lower than that of the prototypic 5-HT6R inverse agonist
intepirdine (Table 3, Fig. 3).
To investigate the inhibitory mechanism of compound 48 on
hMAO-B, time-dependent inhibition studies were performed using
in vitro method. Rasagiline and safinamide, with known irreversible
and reversible inhibition toward MAO-B were used as reference
drugs (Fig. 4). In the case of reversible inhibitor safinamide, the
competing excess of p-tyramine, used as an enzyme substrate,
significantly reactivated the enzyme activity. In contrast, rasagiline,
depending on the pre-incubation time used, has either slightly
restored MAO-B activity or maintained only residual activity of the
enzyme. A higher than expected signal for rasagiline without pre-
incubation (ca. 30% reactivation) might be explained by the
longer time required to create the covalently bound inhibitor-
enzyme complex by irreversible inhibitors. Although compound
48 was able to restore some activity of MAO-B, the difference be-
tween the enzyme reactivation rate, obtained with and without
pre-incubation, suggests that compound 48 covalently modify the
enzyme.
Fig. 3. Influence of compound 48 and intepirdine on basal cAMP production in NG108-
15 cells transiently expressing 5-HT6Rs. Data are the means SEM of the values ob-
tained in three independent experiments performed in quadruplicate in different sets
of cultured cells. ***p
dentꢂNewmanꢂKeuls test).
<
0.001 vs. vehicle (ANOVA followed by Stu-
cardiovascular side effects (i.e., hypertensive crisis).
To exclude potential risks related to undesired cardiovascular or
CNS side effects, the affinity of compound 48 for selected “off-
target” receptors was assessed by Eurofins Scientific. The tested
compound displayed low affinity for a1A adrenoreceptor (% inhi-
bition at 1
inhibition at 1
m
M ¼ 37%) and did not bind to b1 adrenoreceptor (%
m
M ¼ 5%), H1 histaminic receptor (% inhibition at
3.4. Preliminary evaluation of ADME/Tox properties
As ligands that combine two pharmacophore fragments within
a single molecule usually have physicochemical properties burdens
(high molecular weight or clog P value), which might limit their
bioavailability, compound 48 was subsequently evaluated for its
drug-likeness in in vitro screening. The ability to cross the blood-
brain barrier (BBB) by passive diffusion was assessed by the
commonly used parallel artificial membrane permeability assay
(PAMPA-BBB) [47]. Compound 48 showed an efficient permeability
(Pe) value greater than 4.0 in the tested conditions, thus suggesting
a high CNS penetration (Table 4). The reference drugs verapamil
(Pe ¼ 1.85 ꢁ 10ꢂ5 cm/s) and doxorubicin (not detected in the
acceptor compartment) were used as positive and negative control,
respectively.
Subsequently, the in vitro metabolic stability of compound 48
was determined using rat liver microsomes (RLMs). After 60-min
incubation period, compound 48 showed an average intrinsic
clearance (Clint) of 85 mL/min/kg (Table 4).
Compound 48 was also submitted to preliminary hepatotoxicity
assessment using human liver cancer cell (HepG2). As revealed by
the MTT assay compound 48 did not affect the viability of HepG2
cells after 24-h period (Table 4). Doxorubicine used as reference
cytotoxic drug showed more than 20-fold higher cytotoxic activity
Fig. 4. Reactivation of MAO-B activity upon blockade with compound 48 and reference
inhibitors used at a concentrations corresponding to their IC80 values in the presence
of the substrate p-tyramine. All compounds were tested with 30 min of pre-incubation
(rings) and without pre-incubation (lines). Data are presented as mean values from
two independent experiments run in duplicate. The results were normalized to the
effect induced by 1 mM of p-tyramine (100% value) and solvent (0% value) in the last
measurement of the experiment.
1
1
m
M ¼ 13%) and M1 muscarinic receptor (% inhibition at
mM ¼ 4%).
(IC50 ¼ 2.3
mM).
3.3. Impact of compound 48 on 5-HT6R-operated signaling and on
MAO-B activity
Next, the preliminary pharmacokinetic profile of compound 48
was estimated in male Wistar rats injected with a single dose
(3 mg/kg p.o.). Compound 48 was rapidly absorbed and was able to
cross the BBB with a Cmax value reaching 72.7 ng/mL in the brain
after 30 min (Tmax, Table 4). The molecule was eliminated very
slowly from the rat body, with along half-life of ca. 28 h.
The impact of compound 48 on adenylate cyclase activity was
first examined in 1321N1 cells expressing 5-HT6R and treated with
5-carboxamidotryptamine (5-CT), a 5-HT6R agonist. Compound 48
behaved as a potent antagonist of 5-HT6R in this cellular assay
(Kb ¼ 6 nM, Table 3).
3.5. Glioprotective effects
The high level of constitutive activity of 5-HT6R, defined as the
ability of the receptor to adopt an active conformation able to
transduce signal in the absence of an agonist, has been demon-
strated in both in vitro and ex vivo models [43,44]. This property
Given the supporting role of glial cells in the CNS, protection of
astrocytes might be a relevant strategy for treating neurodegener-
ative disorders in addition to direct action on neuronal cell
5

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Table 4
signaling pathway and an irreversible MAO-B inhibitor. Besides
showing the most efficient combination of 5-HT6R modulation and
MAO-B inhibitory activity, compound 48 was metabolically stable
and brain penetrant. Moreover, compound 48 reduced the gliotoxic
effect of 6-OHDA in C8-D1A astrocytes in two complementary as-
says (MTT and LDH) assessing cell survival. Importantly, this effect
was not observed after treatment with intepirdine (5-HT6R antag-
onist) and MAO-B inhibitor selegiline. Finally, compound 48 also
reversed cognitive decline induced by scopolamine in the NOR test.
These properties highlight 48 as an original example of dual 5-
HT6R/MAO-B modulator and as an interesting prototype in the
development of new treatment strategies for AD.
Summary of the ADME/Tox profile evaluation for compound 48.
Assay type
48
PAMPA permeability
Microsomal stability
Hepatotoxicity
Pe ¼ 4.84 ꢁ 10ꢂ6 cm/sa
Clint ¼ 85 mL/min/kgb
IC50 ¼ 52.7
m
Mc
Preliminary in vivo pharmacokineticsd
Cmax ¼ 72.7 ng/mL
Tmax ¼ 30 min
a
Measured at 100 mM initial concentration at pH 7.4 in a phosphate buffer at
room temperature measured at pH 7.4 in a phosphate buffer at room temperature.
b
Determined at a protein concentration of 0.4 mg/mL in RLM assay.
Concentration that inhibits 50% of HepG2 cell growth, when treated with tested
c
compounds for 24-h period; IC50 was obtained from three independent experi-
ments, using GraphPad Prism software.
d
Measured after p.o. administration of dose 3 mg/kg.
5. Experimental procedures
functions or survival [48]. We thus explored the glioprotective ef-
fects of compound 48 in a model of cultured astrocytes (C8-D1A
astrocytes) exposed to the neurotoxin 6-OHDA. 6-OHDA-induced
cell death is attributed to the oxidative damage by reactive oxygen
species (ROS) derived from 6-OHDA autooxidation or possible
direct effect of 6-OHDA on the mitochondrial respiratory chain
complex [49].
As revealed by the MTT assay (which assesses mitochondrial
metabolism), compound 48 did not induce significant toxicity in
C8-D1A astrocyte cultures at concentrations up to 25 mM but pro-
5.1. Chemistry
5.1.1. General methods
The synthesis was carried out at ambient temperature, unless
indicated otherwise. Organic solvents (from Aldrich and Chempur)
were of reagent grade and were used without purification. All re-
agents (Sigma-Aldrich, Fluorochem, Across and TCI) were of the
highest purity. Column chromatography was performed using silica
gel Merck 60 (70e230 mesh ASTM).
tected C8-D1A astrocytes against 6-OHDA-induced cytotoxicity
(Fig. 5A). These observations were confirmed in lactate dehydro-
genase (LDH) assay, which assesses cell membrane integrity
(Fig. 5B). Notably, intepirdine exhibited no glioprotective properties
[13]. Similarly, although neuroprotective activity of selegiline was
established in several in vitro models [50], this irreversible MAO-B
inhibitor did not reduce gliotoxicity induced by 6-OHDA in both
cell-based assays (Fig. 5A and B). These findings suggest that
blockade of MAO-B alone is not sufficient to protect C8-D1A as-
trocytes against 6-OHDA toxicity, and that the glioprotective effect
of compound 48 results either from 5-HT6 receptor antagonist/in-
verse agonist activity or from its dual inhibitory activities on 5-
HT6R and MAO-B.
Mass spectra were recorded on a UPLC-MS/MS system consisted
of a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA,
USA) coupled to a Waters TQD mass spectrometer (electrospray
ionization mode ESI-tandem quadrupole). Chromatographic sepa-
rations were carried out using the Acquity UPLC BEH (bridged ethyl
hybrid) C18 column; 2.1 ꢁ 100 mm, and 1.7
mm particle size,
equipped with Acquity UPLC BEH C18 Van Guard pre-column;
2.1 ꢁ 5 mm, and 1.7
mm particle size. The column was maintained
at 40 ꢀC, and eluted under gradient conditions from 95% to 0% of
eluent A over 10 min, at a flow rate of 0.3 mL minꢂ1. Eluent A:
water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid
(0.1%, v/v). Chromatograms were made using Waters el PDA de-
tector. Spectra were analyzed in 200e700 nm range with 1.2 nm
resolution and sampling rate 20 points/s. MS detection settings of
Waters TQD mass spectrometer were as follows: source tempera-
ture 150 ꢀC, desolvation temperature 350 ꢀC, desolvation gas flow
rate 600 L h-1, cone gas flow 100 L h-1, capillary potential 3.00 kV,
cone potential 40 V. Nitrogen was used for both nebulizing and
drying gas. The data were obtained in a scan mode ranging from 50
to 2000 m/z in time 1.0 s intervals. Data acquisition software was
MassLynx V 4.1 (Waters). The UPLC/MS purity of all the final
compounds was confirmed to be 95% or higher.
3.6. Pro-cognitive properties
Given the interesting multipotent activity of compound 48, its
ability to rescue cognitive deficit induced by scopolamine (SCOP) in
rats was then investigated using the NOR test. Scopolamine
administration (1.25 mg/kg, p.o.) abolished the ability of rats to
discriminate between novel and familiar objects when compared to
the vehicle-treated animals. These deficits were significantly
reversed by p.o. administration of compound 48 at the doses of 1
and 3 mg/kg (Fig. 6).
1H NMR and 13C NMR spectra were obtained JOEL JNM-ECZR500
RS1 (ECZR version) at 500 and 126 MHz, respectively and are re-
ported in ppm using deuterated solvent for calibration (CDCl3 or
DMSO‑d6). The J values are reported in Hertz (Hz), and the splitting
patterns are designated as follows: br. s. (broad singlet), s (singlet),
d (doublet), t (triplet), q (quartet), dd (doublet of doublets), m
(multiplet).
Elemental analysis for C, H, N and S was carried out using the
elemental Vario EI III Elemental Analyser (Hanau, Germany). All
values are given as percentages, and were within 0.4% of the
calculated values.
Compound 48 selected for behavioral evaluation was converted
into the hydrochloride salt.
All synthetic procedures and spectroscopic data for selected
final compounds are summarized below, while characterization
data for all intermediates and remained final compounds are re-
ported in the Supporting Information.
4. Conclusions
Using the strategy of dual-acting compounds, we designed and
synthesized a series of hybrids that combine a (indol-4-yl)-piper-
azine fragmentda well-established core of 5-HT6R antagonistsd-
with frameworks of known MAO-B inhibitors, connected through
an alkylene linker. As revealed by in silico studies, despite the size of
the molecular framework, designed derivatives could enter the
binding site of MAO-B by a solvent accessed channel B, which is
devoid of significant steric hindrances. In agreement with the
design rationale, the majority of novel hybrid compounds showed
affinity for 5-HT6R and inhibitory activity for the enzyme target.
The study identified compound 48, a dual 5-HT6R/MAO-B modu-
lator, which behaved as an inverse agonist of 5-HT6R at the Gs
6

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Fig. 5. Effect of selegiline, intepirdine, and 48 on 6-OHDA-induced astrocytes cytotoxicity. (A) MTT and (B) LDH assays were performed on astrocytes pre-treated with the tested
compounds (0.25 M) for 24 h and injured by 6-OHDA (25 M) for the next 24-h period. p < 0.05 vs 6-OHDA.
m
m
treated with 3-chlorobenzyl bromide (0.98 mL, 7.47 mmol, 1.5 eq)
or benzenesulfonyl chloride (0.95 mL, 7.47 mmol,1.5 eq) and stirred
for 2 h at rt. After removal of organic solvents, the crude products
were purified on silica gel using a mixture of AcOEt/Hex as eluting
system.
5.1.4. General procedure for Boc deprotection at piperazine moiety
(5e6)
Intermediate 3 (1.45 g, 3.4 mmol) was solubilized in CH2Cl2
(12 mL) and cooled to 0 ꢀC followed by addition of TFA (3 mL). The
reaction mixture was stirred for 2 h at rt. Then, the reaction was
diluted with CH2Cl2 (5 mL) and the acidic excess was carefully
neutralized with cold NH3 aqueous solution till pH ¼ 10. The
organic fraction was washed with brine (1 x), dried over anhydrous
Na2SO4, filtered and concentrated under vacuum. The resulting
product 5 was submitted to the next step without further purifi-
cation. According to the same procedure intermediate 4 was con-
verted into secondary amine 6.
Fig. 6. Effects of compound 48 on SCOP-induced inhibition of novelty discrimination
in the NOR test in rats. The data illustrated represent discrimination index (DI) and are
the mean
standard error of the mean of N ¼ 6e10 animals per group. Symbols:
***p < 0.001 vs vehicle-treated group; ##p < 0.01 vs SCOP-treated group.
5.1.5. General procedure for alkylation of phenols (9e13 and
14e17)
5.1.2. General procedure for the N-arylation of N-Boc-4-bromo-
indole and selective Boc-deprotection (2)
A suspension of 4-hydroxy-indanone (0.75 g, 5.06 mmol, 1 eq),
freshly grinded K2CO3 (1.75 g, 12.66 mmol, 2.5 eq) and KOH (0.14 g,
2.53 mmol, 0.5 eq) in 15 mL of acetone. The proper di-bromoalkane
(20.24 mmol, 4 eq) was slowly dropped to the reaction mixture and
then left stirring at 60 ꢀC overnight. Then, inorganic residues were
filtered off, followed by removal of organic solvent under vacuum.
The crude products were purified on silica gel using a mixture of
AcOEt/Hex as eluting system. to obtain intermediates 9e13. The
same procedure was employed for the alkylation 4-
hydroxybenzaldehyde with different di-bromoalkane to achieve
intermediates 14e17.
N-Boc-4-bromoindole derivative (5 g, 16.88 mmol, 1 eq), Boc-
piperazine (6.28 g, 33.77 mmol, 2 eq), tris(dibenzylideneacetone)
dipalladium (310 mg, 0.34 mmol, 0.02 eq). BINAP (420 mg,
0.68 mmol, 0.04 eq) and NaOtBu (1.77 g, 18.38 mmol, 1.5 eq) were
solubilized in toluene anhydrous (30 mL) in a 250 mL reaction flask.
The flask was evacuated and back filled with argon and the reaction
was left stirring at 110 ꢀC for 5 h. Then, the mixture was diluted
with AcOEt (50 mL) and filtered off on Celite pad and finally
concentrated. The crude mixture was purified on silica gel using a
mixture of AcOEt/Hex (1/9, v/v) as eluting system. Next, the pure
intermediate (4.3 g,10.71 mmol) was dissolved in EtOH (80 mL) and
portioned in 4 seal-closed microwave reactors. Each reactor was
irradiated for 2 h at 150 ꢀC. Then, organic solvent was removed
under vacuum and the resulting product (3.07 g, 95% yield) was
pure enough for the next synthetic step.
5.1.6. General procedure for the alkylation of N1-functionalized-4-
indol-piperazine (18e30)
1-(3-Chlorobenzyl)-4-(piperazin-1-yl)-1H-indole
5 (150 mg,
0.46 mmol 1 eq) and freshly grinded K2CO3 (190 mg 1.38 mmol, 3
eq) were suspended in 5 mL of acetone, followed by the dropwise
addition of the corresponding alkylating agent 9e17 (0.55 mmol,
1.2 eq). The reaction mixture was stirred at 60 ꢀC overnight. Then,
inorganic residues were filtered off, followed by removal of organic
solvent under vacuum. The crude products were purified on silica
gel using a mixture of CH2Cl2/MeOH as eluting system, yielding
intermediates 18e22 and 25e28. The same procedure was
repeated to obtain intermediates 23e24 and 29e30 by alkylation of
5.1.3. General procedure for benzylation or sulfonilation of the N1-
indole moiety (3e4)
A
solution of tert-butyl 4-(1H-indol-4-yl)piperazine-1-
carboxylate (1.5 g, 4.98 mmol, 1 eq) in CH2Cl2 (20 mL) was cooled
to 0 ꢀC followed by addition of phosphazene base P1-t-Bu-tris(te-
tramethylene) (2.27 mL, 7.47 mmol, 1.5 eq). Then, the mixture was
7

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Scheme 1. A: synthesis of 4-piperazine-indole cores 5 and 6. Reagents and conditions: i) Boc-piperazine, Pd2(dba)3, BINAP, t-BuONa, toluene, 110 ꢀC, 5 h; ii) EtOH, 150 ꢀC, MW, 2 h;
iii) 3-chlorobenzyl bromide or benzylsulfonyl chloride, BTPP, CH2Cl2, 0 ꢀC/rt, 2 h; iv) TFA/CH2Cl2, 2/8 v/v, rt, 1 h; B: synthesis of alkylating agents 9e17; i)
a,u-dibromoalkane,
K2CO3, KOH, KI, acetone, 60 ꢀC, 16 h.
Scheme 2. Synthesis of final compounds 31e48. Reagents and conditions: i) alkylating agent 9e13 or 14e17, K2CO3, KI, acetone, 60 ꢀC, 16 h; ii) for primary amine: AcOH, THFanh, rt,
5 h then STAB, rt, 2 h; for secondary amine, AcOH, CH2Cl2anh, 0.5 h then STAB, rt, 2 h.
1-((3-chlorophenyl)sulfonyl)-4-(piperazin-1-yl)-1H-indole
synthesized alkylating agents 11e12 and 15e16, respectively.
with
the reducing agent STAB (158 mg, 0.75 mmol, 3 eq) was added and
the mixture was left stirring for additional 2 h. Subsequently, the
reaction was quenched with a saturated solution of NaHCO3,
washed with H2O (2 ꢁ ), brine (1 ꢁ ), dried over Na2SO4, filtered and
concentrated. The obtained crude product was purified using silica
gel with CH2Cl2/MeOH as an eluting system to obtain final com-
pound 38. The same procedure was applied to intermediates 26e30
for yielding final compounds 39e43.
5.1.7. General procedure for reductive amination of ketone with
propargyl amine (31e37)
Ketone derivative 18 (120 mg, 0.2 mmol, 1 eq), and propargyl
amine (26 L, 0.4 mmol, 2 eq) were solubilized in anhydrous THF
m
(3 mL) and the mixture was stirred for 5 h. The, the reducing agent
STAB (127 mg, 0.6 mmol, 3 eq) was added and the mixture was left
stirring for additional 2 h. Subsequently, the reaction was quenched
with a saturated solution of NaHCO3, washed with H2O (2 ꢁ ), brine
(1 ꢁ ), dried over Na2SO4, filtered and concentrated. The obtained
crude product was purified using silica gel with CH2Cl2/MeOH as an
eluting system to obtain final compound 31. The same procedure
was applied to intermediates 19e24 and for yielding final com-
pounds 32e37.
5.1.9. General procedure for reductive amination of benzaldehyde
with N-methyl-propargyl amine (44e48)
Benzaldehyde derivative 26 (120 mg, 0.24 mmol, 1 eq), and N-
methylpropargyl amine (40 mL, 0.48 mmol, 2 eq) were solubilized in
CH2Cl2 (3 mL) and the mixture was stirred for 30 min. The, the
reducing agent STAB (152 mg, 0.72 mmol, 3 eq) was added and the
mixture was left stirring for additional 1 h. Subsequently, the re-
action was quenched with a saturated solution of NaHCO3, washed
with H2O (2 ꢁ ), brine (1 ꢁ ), dried over Na2SO4, filtered and
concentrated. The obtained crude product was purified using silica
gel with CH2Cl2/MeOH as an eluting system to yield final compound
44. The same procedure was applied to intermediates 26e30 for
yielding final compounds 45e48.
5.1.8. General procedure for reductive amination of benzaldehyde
with
L
-alanamide (38e43)
Benzaldehyde derivative 25 (120 mg, 0.25 mmol, 1 eq), and
alanamide hydrochloride (76 mg, 0.5 mmol, 2 eq) were solubilized
in anhydrous THF (3 mL) and the mixture was stirred for 5 h. The,
L-
8

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
5.1.10. Characterization data for selected final compounds
108.0, 110.9, 114.6, 124.2, 124.9, 125.6, 126.9, 129.3, 131.6, 133.9,
5.1.10.1. 4-(4-(4-(1-(3-Chlorobenzyl)-1H-indol-4-yl)piperazin-1-yl)
136.0, 138.3, 146.1, 158.4, 178.2 (C]O).
butoxy)-N-(prop-2-yn-1-yl)-2,3-dihydro-1H-inden-1-amine
(33).
Brown oil, 76 mg (isolated yield 59%) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/1, v/v); UPLC/MS
purity 98%, tR ¼ 5.14, C35H39ClN4O, MW 567.17, Monoisotopic Mass
5.1.10.4. N-(4-((5-(4-(1-(3-Chlorobenzyl)-1H-indol-4-yl)piperazin-
1-yl)pentyl)oxy)benzyl)-N-methylprop-2-yn-1-amine (45). Pale yel-
low oil, 99 mg (isolated yield 75%) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7, v/v); UPLC/MS
purity 100%, tR ¼ 5.73, C35H41ClN4O, MW 569.19, Monoisotopic
566.28, [MþH]þ 567.4.1H NMR (500 MHz, CDCl3)
d ppm 1.27 (m, 2H,
NeCH2eCH2), 1.38e1.44 (m, 1H, indane), 1.72e1.80 (m, 2H,
OeCH2eCH2), 1.81e1.91 (m, 3H, indane), 2.25 (t, J ¼ 2.4 Hz, 1H,
CH^CeCH2), 2.55 (t, J ¼ 7.3 Hz, 2H, NeCH2eCH2), 2.70e2.82 (m,
4H, piperazine), 3.29e3.34 (m, 4H, piperazine), 3.51 (dd, J ¼ 5.3,
2.4 Hz, 2H, NHeCH2eC^CH), 4.02 (t, J ¼ 6.2 Hz, 2H, OeCH2eCH2),
4.42 (dd, J ¼ 6.9, 5.2 Hz, 1H, CHeNHeCH2), 5.26 (s, 2H, NeCH2-Ar),
6.56 (dd, J ¼ 2.6, 0.9 Hz, 1H, AreH), 6.61 (d, J ¼ 7.4 Hz, 1H, AreH),
6.73 (d, J ¼ 8.3 Hz, 1H, AreH), 6.88e6.97 (m, 3H, AreH), 7.04e7.12
(m, 3H, AreH), 7.14e7.23 (m, 3H, AreH). 13C NMR (126 MHz, CDCl3)
Mass 568.30, [MþH]þ 569.4.1H NMR (500 MHz, CDCl3)
d ppm
1.47e1.56 (m, 2H, OeCH2eCH2eCH2), 1.61e1.70 (m, 2H,
NeCH2eCH2), 1.79e1.86 (m, 2H, OeCH2eCH2), 2.23e2.28 (m, 1H,
CH^CeCH2), 2.33 (s, 3H, NeCH3), 2.46e2.54 (m, 2H, NeCH2eCH2),
2.74 (br. s, 4H, piperazine), 3.28 (d, J ¼ 2.3 Hz, 2H, NeCH2eC^CH),
3.32 (br. s, 4H, piperazine), 3.50 (s, 2H, CH3eNeCH2-Ar), 3.96 (t, J ¼
6.4 Hz, 2H, OeCH2eCH2), 5.25 (s, 2H, NeCH2-Ar), 6.56 (d, J ¼ 3.2 Hz,
1H, AreH), 6.59e6.62 (m, 1H, AreH), 6.85 (d, J ¼ 8.6 Hz, 2H, AreH),
6.91 (dd, J ¼ 10.9, 7.2 Hz, 2H, AreH), 7.04e7.11 (m, 3H, AreH),
d
ppm 23.6 (NeCH2eCH2), 27.3 (CH2-indane), 27.5 (OeCH2eCH2),
33.1 (CH2-indane), 36.2 (NHeCH2eC), 49.7 (NeCH2-Ar), 51.3
(piperazine), 53.6 (piperazine), 58.5 (NeCH2eCH2), 62.4 (CH-
indane), 67.7 (OeCH2eCH2), 71.5 (CH2eC^CH), 82.6 (CH2eC^CH),
100.8, 104.4, 106.9, 110.2, 116.5, 122.0, 122.8, 124.9, 126.7, 126.8,
127.9, 130.1, 131.8, 134.8, 137.5, 139.8, 145.9, 146.5, 155.7.
7.16e7.27 (m, 4H, AreH). 13C NMR (126 MHz, CDCl3)
d ppm 24.2
(NeCH2eCH2eCH2), 26.7 (NeCH2eCH2), 29.3 (piperazine), 29.8
(piperazine), 30.4 (OeCH2eCH2), 41.8 (NeCH3), 44.7 (NeCH2eCH2),
49.7 (NeCH2-Ar), 51.3 (piperazine), 53.7 (piperazine), 58.8
(OeCH2eCH2), 59.4 (Ar-CH2-N-CH3), 67.9 (CH2eC^CH), 73.4
(CH2eC^CH), 78.7 (CH2eC^CH), 100.8, 104.4, 106.9, 114.4, 122.1,
122.8, 124.9, 126.7, 126.8, 127.9, 130.1, 130.3, 130.5, 134.8, 137.5,
139.8, 145.9, 158.4.
5.1.10.2. (S)-2-((4-((6-(4-(1-(3-Chlorobenzyl)-1H-indol-4-yl)piper-
azin-1-yl)hexyl)oxy)benzyl)amino)propanamide (41). Pale yellow
oil, 87 mg (isolated yield 64%) following chromatographic purifi-
cation over silica gel with CH2Cl2/MeOH (9/1, v/v); UPLC/MS purity
97%, tR ¼ 4.61, C35H44ClN5O2, MW 602.22, Monoisotopic Mass
5.1.10.5. N-methyl-N-(4-(4-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)
piperazin-1-yl)butoxy)benzyl)prop-2-yn-1-amine (47). Pale yellow
oil, 100 mg (isolated yield 75%) following chromatographic purifi-
cation over silica gel with CH2Cl2/MeOH (9/0.7, v/v); UPLC/MS pu-
rity 97%, tR ¼ 4.49, C33H38N4O3S, MW 570.75, Monoisotopic Mass
601.32, [MþH]þ 602.3.1H NMR (500 MHz, DMSO‑d6)
d ppm 1.08 (d,
J ¼ 6.9 Hz, 3H, NHeCHeCH3), 1.28e1.36 (m, 2H, NeCH2eCH2eCH2),
1.36e1.42 (m, 2H, OeCH2eCH2eCH2), 1.42e1.49 (m, 2H,
NeCH2eCH2), 1.63e1.71 (m, 2H, OeCH2eCH2), 2.32 (t, J ¼ 7.2 Hz,
2H, NeCH2eCH2), 2.54 (br. s., 4H, piperazine), 2.96 (q, J ¼ 6.9 Hz, 1H,
NHeCHeCH3), 3.07 (br. s., 4H, piperazine), 3.43 (d, J ¼ 13.2 Hz, 1H,
Ar-CH1a-NH), 3.56 (d, J ¼ 13.2 Hz, 1H, Ar-CH1b-NH), 3.89 (t, J ¼
6.6 Hz, 2H, OeCH2eCH2), 5.35 (s, 2H, NeCH2-Ar), 6.40 (d, J ¼ 3.4 Hz,
1H, AreH), 6.43 (d, J ¼ 7.5 Hz, 1H, AreH), 6.80e6.84 (m, 2H,
COeNH2), 6.91e6.97 (m, 2H, AreH), 6.99e7.03 (m, 1H, AreH), 7.07
(d, J ¼ 6.9 Hz,1H, AreH), 7.15e7.21 (m, 3H, AreH), 7.24e7.31 (m, 3H,
AreH), 7.39 (d, J ¼ 2.9 Hz, 1H, AreH). 13C NMR (126 MHz, DMSO‑d6)
570.27, [MþH]þ 571.4.1H NMR (500 MHz, CDCl3)
d ppm 1.67e1.75
(m, 2H, NeCH2eCH2), 1.79e1.85 (m, 2H, OeCH2eCH2), 2.25 (t, J ¼
2.3 Hz, 1H, CH^CeCH2), 2.31 (s, 3H, NeCH3), 2.49 (t, J ¼ 7.4 Hz, 2H,
NeCH2eCH2), 2.58e2.74 (m, 4H, piperazine), 3.15 (t, J ¼ 4.3 Hz, 4H,
piperazine), 3.27 (d, J ¼ 2.3 Hz, 2H, NeCH2eC^CH), 3.49 (s, 2H,
CH3eNeCH2-Ar), 3.97 (t, J ¼ 6.3 Hz, 2H, OeCH2eCH2), 6.67 (dd, J ¼
3.7, 0.9 Hz, 1H, AreH), 6.71 (d, J ¼ 7.7 Hz, 1H, AreH), 6.82e6.86 (m,
2H, AreH), 7.18e7.26 (m, 3H, AreH), 7.37e7.45 (m, 2H, AreH),
7.48e7.53 (m, 2H, AreH), 7.64 (d, J ¼ 8.3 Hz, 1H, AreH), 7.83e7.88
d
ppm 19.8 (CH3eCHeNH), 26.1 (NeCH2eCH2eCH2), 26.8
(m, 2H, AreH). 13C NMR (126 MHz, CDCl3)
d ppm 23.5
(OeCH2eCH2eCH2), 27.3 (NeCH2eCH2), 29.3 (OeCH2eCH2), 49.0
(NeCH2-Ar), 51.0 (NeCH2eCH2), 51.4 (piperazine), 53.7 (piperazine),
56.9 (NHeCHeCH3), 58.5 (OeCH2eCH2), 67.9 (Ar-CH2-NH), 100.7,
104.9,106.6,114.6,121.8, 122.6,126.2,127.2,127.8, 129.7,131.0, 132.7,
133.7, 137.3, 141.5, 146.1, 158.1, 177.4 (C]O).
(NeCH2eCH2), 27.4 (OeCH2eCH2), 41.8 (NeCH3), 44.7
(NeCH2eCH2), 51.6 (piperazine), 53.5 (piperazine), 58.4
(OeCH2eCH2), 59.4 (Ar-CH2-N-CH3), 67.7 (CH2eC^CH), 73.4
(CH2eC^CH), 78.7 (CH2eC^CH), 107.6, 107.9, 110.9, 114.4, 124.3,
124.8, 125.6, 126.9, 129.3, 130.4, 133.9, 136.0, 138.3, 146.2, 158.4.
5.1.10.3. (S)-2-((4-(4-(4-(1-(Phenylsulfonyl)-1H-indol-4-yl)piper-
azin-1-yl)butoxy)benzyl)amino)propanamide (42). Pale yellow oil,
82 mg (isolated yield 60%) following chromatographic purification
over silica gel with CH2Cl2/MeOH (9/1, v/v); UPLC/MS purity 96%,
tR ¼ 4.41, C32H39N5O4S, MW 589.76, Monoisotopic Mass 589.27,
5.1.10.6. N-methyl-N-(4-((5-(4-(1-(phenylsulfonyl)-1H-indol-4-yl)
piperazin-1-yl)pentyl)oxy)benzyl)prop-2-yn-1-amine (48). Pale yel-
low oil, 103 mg (isolated yield 78%) following chromatographic
purification over silica gel with CH2Cl2/MeOH (9/0.7, v/v); UPLC/MS
purity 100%, tR ¼ 4.61, C33H38N4O3S, MW 584.78, Monoisotopic
[MþH]þ 590.4.1H NMR (500 MHz, CDCl3)
d
ppm 1.35 (d, J ¼ 6.9 Hz,
Mass 584.28, [MþH]þ 585.5.1H NMR (500 MHz, CDCl3)
d ppm
3H, NHeCHeCH3), 1.67e1.76 (m., 2H, NeCH2eCH2), 1.78e1.86 (m,
2H, OeCH2eCH2), 2.51 (t, J ¼ 6.3 Hz, 2H, NeCH2eCH2), 2.68 (br. s.,
4H, piperazine), 3.15 (br. s., 4H, piperazine), 3.22 (q, J ¼ 6.3 Hz, 1H,
NHeCHeCH3), 3.63¡3.74 (m, 2H, Ar-CH2-NH), 3.97 (t, J ¼ 6.0 Hz,
2H, OeCH2eCH2), 5.59 (br.s., 1H, COeNH1a), 6.66 (d, J ¼ 4.0 Hz, 1H,
AreH), 6.71 (d, J ¼ 7.4 Hz, 1H, AreH), 6.82e6.87 (m, 2H, AreH), 7.09
(br.s., 1H, COeNH1b), 7.15e7.23 (m, 3H, AreH), 7.38e7.43 (m, 2H,
AreH), 7.46e7.55 (m, 2H, AreH), 7.63 (d, J ¼ 8.0 Hz, 1H, AreH), 7.85
1.48¡1.56 (m, 2H, OeCH2eCH2eCH2), 1.60¡1.67 (m, 2H,
NeCH2eCH2), 1.79¡1.86 (m, 2H, OeCH2eCH2), 2.28 (t, J ¼ 2.6 Hz,
1H, CH^CeCH2), 2.33 (s, 3H, NeCH3), 2.47 (t, J ¼ 7.4 Hz, 2H,
NeCH2eCH2), 2.68 (br. s., 4H, piperazine), 3.18 (t, J ¼ 4.6 Hz, 4H,
piperazine), 3.29 (d, J ¼ 2.3 Hz, 2H, NeCH2eC^CH), 3.51 (s, 2H,
CH3eNeCH2-Ar), 3.96 (t, J ¼ 6.3 Hz, 2H, OeCH2eCH2), 6.68¡6.70
(m, 1H, AreH), 6.73 (d, J ¼ 7.5 Hz, 1H, AreH), 6.83¡6.87 (m, 2H,
AreH), 7.20¡7.26 (m, 3H, AreH), 7.40¡7.44 (m, 2H, AreH),
7.50¡7.53 (m, 1H, AreH), 7.53 (d, J ¼ 3.4 Hz, 1H, AreH), 7.66 (d, J ¼
8.6 Hz, 1H, AreH), 7.86¡7.87 (m, 1H, AreH), 7.88¡7.89 (m, 1H,
(d, J ¼ 7.4 Hz, 2H, AreH). 13C NMR (126 MHz, CDCl3)
d ppm 19.8
(CH3eCHeNH), 23.4 (NeCH2eCH2), 27.3 (OeCH2eCH2), 29.8
(piperazine), 51.5 (piperazine), 52.1 (NeCH2eCH2), 53.5 (piperazine),
57.7 (NHeCHeCH3), 58.3 (OeCH2eCH2), 67.8 (Ar-CH2-NH), 107.5,
AreH). 13C NMR (126 MHz, CDCl3)
d
ppm 24.2 (NeCH2eCH2eCH2),
26.6 (NeCH2eCH2), 29.3 (OeCH2eCH2), 41.7 (NeCH3), 44.7
9

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
(NeCH2eCH2), 51.5 (piperazine), 53.5 (piperazine), 58.7
(OeCH2eCH2), 59.4 (Ar-CH2-N-CH3), 67.8 (CH2eC^CH), 73.4
(CH2eC^CH), 78.7 (CH2eC^CH), 107.6, 108.0, 110.9, 114.4, 124.3,
124.9, 125.6, 126.9, 129.3, 130.3, 130.5, 133.9, 136.0, 138.3, 146.2,
158.4. Anal. calcd for dihydrochloride dihydrate salt
use of Lipofectamine 2000) or CHOeK1 cells with plasmid con-
taining the sequence coding for the human serotonin 5-HT2A re-
ceptor (PerkinElmer) were maintained at 37 ꢀC in a humidified
atmosphere containing 5% CO2 and grown in Dulbecco’s Modified
Eagle’s Medium containing 10% dialyzed fetal bovine serum and
C
33H38N4O3S$2HCl$2H2O: C: 58.87, H: 6.68, N: 8.08, S: 4.62; Found
500 mg/mL G418 sulfate. For membrane preparation, cells were
C: 59.03, H: 6.44, N: 7.72, S: 4.39. M.p. for C33H38N4O3S$2HCl$2H2O:
cultured in 150 cm2 flasks, grown to 90% confluence, washed twice
with pre-warmed to 37 ꢀC phosphate buffered saline (PBS) and
centrifuged (200ꢁg) in PBS containing 0.1 mM EDTA and 1 mM
dithiothreitol. Prior to membrane preparation, pellets were stored
at ꢂ80 ꢀC.
215.1e219.3 ꢀC.
5.2. In silico simulations
5.2.1. Molecular docking
Cell pellets were thawed and homogenized in 10 vol of assay
buffer using an Ultra Turrax tissue homogenizer and centrifuged
twice at 35 000ꢁg for 15 min at 4 ꢀC, with incubation for 15 min at
37 ꢀC between the centrifugations. The composition of the assay
buffers was experimentally selected to achieve the maximum
signal window (more details in SI). All assays were carried out in a
The 5-HT6R homology models were obtained according to the
procedure described before on the b2 receptor template and suc-
cessfully used in our earlier studies of different groups of 5-HT 6R
ligands simulations [51,52]. The structure of monoamine oxidases B
(PDB code 2C65)41 in complex with irreversible inhibitor rasagilline
analog (4-(N-methyl-N-ethyl-carbamoyloxy)-N-methyl-N-prop-
argyl-1(R)-aminoindan) was fetched from the Protein Data Bank.
The structures of the 5-HT6 receptor and MAO-B enzyme were
optimized using the induced-fit docking (IFD) procedure from
total volume of 200 m
L in 96-well plates for 1 h at 37 ꢀC, except 5-
HT1AR and 5-HT2AR which were incubated at room temperature
and 27 ꢀC, respectively. The process of equilibration was terminated
by rapid filtration through Unifilter plates with a 96-well cell
harvester and radioactivity retained on the filters was quantified on
a Microbeta plate reader (PerkinElmer, USA). For displacement
studies the assay samples contained as radioligands (PerkinElmer,
USA): 2.5 nM [3H]-8-OH-DPAT (135.2 Ci/mmol) for 5-HT1AR; 1 nM
[3H]-ketanserin (53.4 Ci/mmol) for 5-HT2AR; 2 nM [3H]-LSD
(83.6 Ci/mmol) for 5-HT6R; 0.8 nM [3H]-5-CT (39.2 Ci/mmol) for 5-
HT7R or 2.5 nM [3H]-raclopride (76.0 Ci/mmol) for D2LR. Non-
€
Schrodinger Suite. The IFD combines flexible ligand docking with
target structure prediction and side chain refinement. The structure
of 48 was used as input to IFD. Centroid of the grid box was
anchored on D3.32 and I199 in 5-HT6 and MAO-B, respectively. Due
to the size of the input ligand, it allowed on residue refinement
within 12 Å and 33 Å of ligand poses for 5-HT6 and MAO-B,
respectively. The Extended Sampling Protocol was used, which
generates up to 80 poses per ligand by automated docking. All
obtained LeR complexes were inspected visually to select those
that displayed the closest compliance with the common binding
mode for studied protein targets. Final validation of the selected
conformations was performed by docking (Glide SP mode) the
designed library, retaining only those with a coherent binding
mode [53]. The 3-dimensional structures of the ligands were ob-
tained using LigPrep v3.6,54 and the appropriate ionization states
at pH ¼ 7.4 1.0 were assigned using Epik v3.4 [55].
specific binding was defined in the presence of 10
5-HT1AR and 5-HT7R binding experiments, whereas 20
mianserin, 10 M of methiothepine or 10 M of haloperidol were
m
M of 5-HT in
mM of
m
m
used in 5-HT2AR, 5-HT6R and D2L assays, respectively. Each com-
pound was tested in triplicate at 7 concentrations (10ꢂ10-10ꢂ4 M).
The inhibition constants (Ki) were calculated from the Cheng-
Prusoff equation [60].
Additionally, the affinity of compound 48 for a1 and b1-adren-
ergic receptors, histaminic H1 and muscarinic M1 receptors were
evaluated at Eurofins. The results were expressed as the % inhibi-
tion of the control binding according to experimental protocols
described online at https://www.eurofins.com/.
5.2.2. QM/MM optimization
The LeR complexes selected in IFD procedure were next opti-
mized using QM/MM approach using QSite. The QM region con-
tained ligand and amino acid side chain directly interacting with
the ligand was described by a combination of DFT hybrid functional
B3LYP and LACVP* basis set, while the rest of the system was
optimized using OPLS2005 force field.
5.3.2. Monoamine oxidase assays
Inhibition activity of evaluated compounds was measured using
human recombinant MAO-B and MAO-A (Sigma Aldrich M7441 and
M7316) in the fluorometric method for detecting monoamine oxi-
dase activity. The assay was carried out in 96-well plate. 2
appropriate concentration of tested compounds in DMSO were
added to wells that contained 98 L of enzyme dilution (0.53 U/ml)
in phosphate buffer (50 mM, pH 7.4). After the 30 min of pre-
incubation in room temperature 50 L of the solution of 800 M 10-
Acetyl-3,7-dihydroxyphenoxazine (Cayman Chemical Company
10010469) and 4 U/mL horse radish peroxidase (HRP, Sigma Aldrich
P6782) was added and enzymatic reaction was started by addition
mL of
5.2.3. Covalent docking
The CovDock workflow [56] from Schrodinger Suite was used for
pose prediction and scoring of covalently bound ligands to a flavin
adenine dinucleotide cofactor (FAD) presence in catalytic site of
MAO-B enzyme. The reaction between FAD and propargyl group
was not pre-defined in CovDock library, thus we used custom re-
action editor to describe the reaction based on the mechanism
m
m
m
ꢀ
proposed by Tandaric et al. [42] At first, we defined in SMARTS
of 50 mL of 800 mM p-tyramine (Alfa Aesar A12220) solution. The
language the fragments of the ligand [C#CC[N,n]] and FAD [[nH]
c(¼O)cNc] between which the reaction occurs. The grid center was
set to I199, assuming its size to 33 Å. The rest setting was used as
default.
signal was measured after 1h (excitation at 570 nm and emission at
585 nm) using EnSpire® multimode plate reader (PerkinElmer,
Inc.). Rasagiline (1 mM) or clorgyline (1 mM) were tested as refer-
ence compounds for MAO-B and MAO-A inhibitions, respectively
[61,62].
5.3. In vitro pharmacological evaluation
5.3.3. Reversibility studies
5.3.1. Radioligand binding assays
To investigate reversibility of MAO-B inhibition compound 48,
rasagiline and safinamide were tested in concentration corre-
sponding to their IC80 values. Experiment was carried out in 96-
well plate divided into two parts. In the first portion hMAO-B was
All experiments were carried out according to the previously
published procedures [57e59]. HEK293 cells stably expressing
human 5-HT1A, 5-HT6, 5-HT7b and D2L receptors (prepared with the
10

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
incubated with inhibitors for 30 min. In the second portion of in-
48 (100
m
M) was incubated on plates in phosphate-buffer saline
hibitors were added to hMAO-B directly before the next step. Then
(pH 7.4) for 5 h at room temperature. Concentrations of 48 in donor
and acceptor solutions were determined using UPLC-MS with the
internal standard based on pentoxifylline method. Membrane
permeability expressed as logPe was calculated as follows:
10
mM p-tyramine and the solution of 800 mM 10-acetyl-3,7-
dihydroxyphenoxazine and 4 U/mL HRP were added to both parts
of the plate. Fluorescence signal had been measured in the micro-
plate reader for 22 min then the concentration of p-tyramine was
increased to 1 mM. After addition of p-tyramine fluorescence was
measured every 5 min for 5 h [62].
ꢀ
ꢁ
ꢂ
0
1
CDꢁVDþCAꢁVA
ꢂln 1 ꢂ CA
VDþVA
B
B
@
C
C
A
ꢃ
ꢄ
logPe ¼ log
1
1
A
A ꢁ
þ V ꢁ t
VD
5.3.4. Determination of cAMP production in 1321N1 cells
The properties of compound 48 to inhibit cAMP production
induced by 5-CT (1000 nM), a 5-HT6R agonist, was evaluated.
where CD and CA are the final concentrations after incubation and
VD, VA - volumes of the donor and the acceptor solutions (0.3 mL,
0.2 mL), respectively. A - membrane surface area (0.3 cm2),t - in-
cubation time (18 000 s). Two reference standards were used in the
study e verapamil (Pe ¼ 1.85 * 10-5 cm/s) e a well permeable drug
and doxorubicin (Pe ¼ ND, not detected in acceptor compartment)
e no membrane penetration properties.
Compounds were tested in triplicate at
8 concentrations
(10ꢂ11e10ꢂ4 M). The level of cAMP was measured using frozen
recombinant 1321N1 cells expressing the Human Serotonin 5-HT6R
(PerkinElmer). Total cAMP was measured using the LANCE cAMP
detection kit (PerkinElmer), according to the manufacturer’s rec-
ommendations. For quantification of cAMP levels, 2000 cells/well
(5
mL) were incubated with mixture of compounds (5 mL) for
5.4.2. In vitro metabolic stability studies
30 min at room temperature in 384-well white opaque microtiter
plate. After incubation, the reaction was stopped and cells were
Metabolic stability of tested compound was analyzed using in-
cubation systems, composed of: tested compound (10 mM), rat liver
lysed by the addition of 10
mL of working solution (5 mL Eu-cAMP
microsomes (RLMs, microsomes from rat male liver, pooled;
0.4 mg/mL; Sigma Aldrich), NADPH-regenerating system (NADPþ,
glucose-6-phosphate and glucose-6-phosphate dehydrogenase in
100 mM potassium buffer, pH 7.4; all from Sigma Aldrich) and
potassium buffer, pH 7.4. Stock solution of tested compounds was
prepared in methanol (the final methanol concentration in incu-
bation mixture does not exceed 0,1%). Firstly, all samples contained
incubation mixture (without NADPH-regenerating system) were
pre-incubated in thermoblock at 37 ꢀC, for 10 min. Then reaction
was initiated by the addition of NADPH-regenerating system. In
control samples NADPH-regenerating system was replaced with
potassium buffer. Probes were incubated for 30 and 60 min at 37 ꢀC.
and 5 L ULight-anti-cAMP) for 1 h at room temperature. Time-
m
resolved fluorescence resonance energy transfer (TR-FRET) was
detected by an Infinite M1000 Pro (Tecan) using instrument set-
tings from LANCE cAMP detection kit manual. Kb values were
calculated from ChengePrusoff equation specific for the analysis of
functional inhibition curves: Kb ¼ IC50/(1þA/EC50) where A repre-
sents agonist concentration, IC50 the concentration of antagonist
producing a 50% reduction in the response to agonist, and EC50 the
agonist concentration which causes a half of the maximal response
[60].
5.3.5. Determination of cAMP production in NG108-15 cells
After addition of internal standard (pentoxifylline, 10
mM)
NG108-15 cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% dialyzed fetal calf serum,
2% hypoxanthine/aminopterin/thymidine (Life technologies), and
antibiotics. cAMP measurement was performed in cells transiently
expressing 5-HT6R using the bioluminescence resonance energy
transfer (BRET) sensor for cAMP, CAMYEL (cAMP sensor using YFP-
Epac-RLuc) [63]. NG108-15 cells were co-transfected in suspension
biotransformation process was stopped by addition of perchloric
acid. Next, samples were centrifuge and supernatants were
analyzed using UPLC/MS (Waters Corporation, Milford, MA). All
experiments were run in duplicates. Half-life time was evaluated
using non-linear regression model using Graph Pad Prism software
and intristinic clearence was calculated from the equation Clint
(volume of incubation [ml]/protein in the incubation [mg]) 0,693/t1/
¼
with 5-HT6R (0.5
mg DNA/million cells) and CAMYEL constructs
[64].
(1 g DNA/million cells), using Lipofectamine 2000, according to
m
2
the manufacturer’s protocol, and plated in white 96-well plates
(Greiner), at a density of 50 000 cells per well. Twenty-four h after
transfection, cells were washed with PBS containing calcium and
magnesium. To test the inverse agonist properties of compound 48
and intepirdine, cells were treated with vehicle or with the tested
compound at incremental concentrations of these compounds
5.4.3. Evaluation of hepatotoxicity
5.4.3.1. Cell culture. Human liver cancer cells HepG2 (ATCC 59195)
were cultured in EMEM media supplemented with 10% FBS at 37 ꢀC,
in a humidified atmosphere with 5% CO2 and antibiotics. Cells were
incubated in the presence of compounds for 24 h. The anti-
proliferative drug DOX was used as a positive control in concen-
(from 0.1 nM to 10
mM). Coelanterazine H (Molecular Probes) was
tration range of 0.1e25 mM.
added at a final concentration of 5
m
M, and left at room tempera-
ture for 5 min. BRET was measured using a Mithras LB 940 plate
reader (Berthold Technologies). Expression of 5-HT6R in NG108-
15 cells induced a strong decrease in CAMYEL BRET signal,
compared with cells transfected with an empty vector instead of
the plasmid encoding the 5-HT6R. This decrease in CAMYEL BRET
signal was thus used as an index of 5-HT6R constitutive activity at
Gs signaling.
5.4.3.2. MTT assay. Cell viability was estimated by using the MTT
assay, which assesses the ability mitochondrial dehydrogenases to
reduce tetrazolium salts into a colored formazan compound [65]. At
the end of the incubation period, 10 mL of MTT (5 mg/mL) were
added to each well. After 4 h, formazan crystals were solubilized
with DMSO and optical density was measured at 570 nm by using a
SpectraMax iD3 Multi-Mode Microplate Reader (Molecular De-
vices). Each experiment was performed in triplicate and repeated
three times.
5.4. Assessment of preliminary ADME/Tox properties
5.4.1. PAMPA e parallel artificial membrane permeability assay
Parallel artificial membrane permeability assay (PAMPA) was
performed using Corning® Gentest™ Pre-coated PAMPA Plate
System, according to the manufacturer’s instructions. Compound
5.4.4. Preliminary in vivo pharmacokinetics experiments
5.4.4.1. Instrumentation and operating conditions. The LC/ESI-MS/
MS experiments were performed on an ABSciex (Concord,
11

V. Canale, K. Grychowska, R. Kurczab et al.
European Journal of Medicinal Chemistry 208 (2020) 112765
Ontario, Canada) API 3200 triple quadrupole mass spectrometer
equipped with an electrospray (ESI) ionization interface. This in-
strument was coupled to Shimadzu (Shimadzu, Japan) LC system.
Data acquisition and processing were accomplished using ABSciex
Analyst 1.5.2 data collection and integration software.
centrifuged at 1000ꢁg for 10 min, and plasma was collected. The
plasma and brain samples were immediately frozen at ꢂ80 ꢀC. All
experimental procedures were carried out in accordance with EU
Directive 2010/63/EU and approved by the I Local Ethics Committee
for Experiments on Animals of the Jagiellonian University in Kra-
kow, Poland.
5.4.4.2. Chromatographic and mass spectrometric conditions.
Acclaim Polar Advantage II (3.0 mm ꢁ 74 mm, 3
mm, 120A, Dionex,
5.5. Assessment of glioprotecting properties
USA) analytical column was used for compound separation. The
temperatures of the column thermostat and the autosampler were
set at 40 ꢀC and 10 ꢀC, respectively. The mobile phase consisted of a
mixture of acetonitrile with addition of 0.1% formic acid (solvent A)
and water with addition of 0.1% formic acid (solvent B) and was set
at a flow rate of 0.4 mL minꢂ1. Starting amount of solvent A is 10%,
isocratic elution from 0 to 5 min, and then gradient up to 90% of
solvent A, and maintained to 10 min, and then gradient down to
10% of solvent A, and maintained to 15 min. To find the optimal
parameters of ion path and ion source for studied compound the
quantitative optimization was done by direct infusion of the
5.5.1. Cell culture
Astrocytes (C8-D1A cells) were cultured in 25 cm2 flask with
DMEM supplemented with 10% FBS at 37 ꢀC, in a humidified at-
mosphere with 5% CO2 until the cells reached 80e90% confluence.
Cells were harvested and seeded in 96-well plates at a density of
1 ꢁ 104 cells per well. They were pre-incubated with tested com-
pounds analyzed compounds for 12 h, then 6-OHDA (25 mM) was
added for next 24 h. The ability of compounds to prevent 6-OHDA-
induced cytotoxicity was assessed by using the MTT and LDH
assays.
molecule at a concentration of 1 mg/mL, and at a flow rate of 10 mL/
min using a Hamilton syringe pump (Hamilton, Reno, Nevada). The
ion source parameters were as follows: ion spray voltage: 4500 V;
nebulizer gas (gas 1): 20 psi; turbo gas (gas 2): 20 psi; temperature
of the heated nebulizer: 400 ꢀC; curtain gas: 40 psi; collision gas: 6
psi. Mass spectra were acquired by SRM with precursor/predomi-
nant product ion transitions for the analyte. The mass spectral
Q1/Q3 transitions monitored for compound 48 and for IS were m/
z 585.1 / 516.2 and m/z 305 / 248, respectively. The peak widths
of precursor and product ions were set to 0.7 full width half-height.
Quantification was done via peak area ratio.
5.5.2. MTT assay
Cell viability was estimated by using the MTT assay, which as-
sesses the ability mitochondrial dehydrogenases to reduce tetra-
zolium salts into a colored formazan compound [65]. At the end of
the incubation period, 10 mL of MTT (5 mg/mL) were added to each
well. After 4 h, formazan crystals were solubilized with DMSO and
optical density was measured at 570 nm by using a SpectraMax iD3
Multi-Mode Microplate Reader (Molecular Devices). Each experi-
ment was performed in triplicate and repeated three times.
5.5.3. LDH assay
5.4.4.3. Sample pretreatment. The plasma and brain sample pre-
Cells were seeded at density 3 ꢁ 104 cells/per well in 96 well
treatment procedure involved acetonitrile precipitation. A 5
aliquot of the internal standard (IS, PH003437, Merck, Darmstadt,
Germany). working solution (5 g/mL) was added to 100 L of the
collected rat plasma sample, which was then vortex-mixed for 10 s.
Thereafter, 200 L of acetonitrile was added, vortexed during
20 min, and then centrifuged (10 000 rpm, 10 min). The superna-
tant (200 L) was then transferred to insert placed in an auto-
sampler vial, and a 20 L volume of this was injected onto the LC
mL
plates. After 24 h selegiline, intepirdine, and compound 48 were
added to final concentrations of 0,25
6-OHDA (25 M) was added to the cultures for an additional 24-h
period to induce astrocyte injury. Then, plates were centrifuged
(200ꢁg, 2 min) and 50 L of the supernatant were transferred into
the corresponding 96-well plate. Subsequently, 50 L of LDH-
mM. After 24 h pre-incubation,
m
m
m
m
m
m
m
reaction mixture prepared according to the manufacturer’s in-
structions (Invitrogen) were added to each well. Incubation was
conducted in darkness for 30 min at room temperature. Next, stop
solution was added and absorbance was measured at 490 nm
(A490) using a plate reader (Spectra Max iD3, Molecular Devices).
m
column. Brain samples were thawed before use, and whole brain
were weighted and placed in a glass mortar and pestle tissue
grinder, and homogenized with an appropriate amount of phos-
phate buffer (pH 7.4) in 1:2.5 ratio. Afterward, 100
homogenates were transferred to new Eppendorf tubes and spiked
with 5 L of the internal standard working solution. All samples
were stored on ice during the preparation process and followed by
procedures similar to those described above.
m
L of tissue
Cytotoxicity
was
determined
as
follows:
cytotoxicity
(%) ¼ [(compound LDH activity e spontaneous LDH activity)/
(maximum LDH activity e spontaneous LDH activity)] ꢁ 100. The
maximum LDH activity was prepared by treating cells with lysis
buffer (control þ). The medium used in the LDH assay contained 1%
FBS. Three independent experiments were performed for each
condition.
m
5.4.4.4. Animals. A group of 32 adult male rats (Wistar, 200e220 g)
were used in the experiment. The animals were purchased from the
Animal House at the Faculty of Pharmacy, Jagiellonian University
Medical College, Krakow, Poland. During the habituation period the
groups of 4 rats were kept in a plastic cage at a controlled room
5.6. In vivo pharmacological evaluation
The experiments were performed according to the previously
reported procedures [66,67] and were conducted in accordance
with the NIH Guide for the Care and Use of Laboratory Animals and
were approved by the Ethics Committee for Animal Experiments,
Institute of Pharmacology. Male SpragueeDawley rats (Charles
River, Germany) weighing ~250 g at the arrival were housed in the
standard laboratory cages, under standard colony A/C controlled
conditions: room temperature 21 2 ꢀC, humidity (40e50%), 12-hr
light/dark cycle (lights on: 06:00) with ad libitum access to food
and water. Rats were allowed to acclimatize for at least 7 days
before the start of the experimental procedure. During this week
animals were handled for at least 3 times. Behavioral testing was
temperature (22
2
ꢀC), humidity (55 10%), full spectrum cold
white light (350e400 lx), on 12 h light/12 dark cycles (the lights on
at 7:00 a.m., and off at 19:00 p.m.), and had free access to standard
laboratory pellet and tap water. For pharmacokinetic study com-
pound 48 dissolved in saline were administered by intragastric
gavage at a dose of 3 mg/kg. Blood samples were collected at 0 min
(predose), 5 min, 15 min, 30 min, 60 min, 120 min, 240 min and
480 min after compound administration. The blood and brain
samples were collected under general anesthesia induced by i.p.
injection of 50 mg/kg ketamine plus 8 mg/kg xylazine. The blood
samples were taken into heparinized tubes, immediately
12

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European Journal of Medicinal Chemistry 208 (2020) 112765
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carried out during the light phase of the light/dark cycle. At least 1 h
before the start of the experiment, rats were transferred to the
experimental room for acclimation. Rats were tested in a dimly lit
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a dull gray plastic
(66 ꢁ 56 ꢁ 30 cm). After each measurement, the floor was cleaned
and dried.
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Scopolamine hydrobromide used to attenuate learning, was
purchase from Sigma Aldrich (Germany). Scopolamine and tested
compound 48 were solubilized in distilled water and then admin-
istered at the dose of 1.25 mg/kg (i.p.) and 1e3 mg/kg (p.o.) 30 and
120 min before familiarization phase (T1), respectively.
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5.6.2. Experimental procedure
Procedure consisted of habituation to the arena (without any
objects) for 5 min, 24 h before the test and test session comprised of
two trials separated by an inter trial interval (ITI). For scopolamine
(SCOP)-induced memory impairment paradigm, 1 h ITI was chosen.
During the first trial (familiarization, T1) two identical objects (A1
and A2) were presented in opposite corners, approximately 10 cm
from the walls of the open field. In the second trial (recognition, T2)
one of the objects was replaced by a novel one (A ¼ familiar and
B ¼ novel). Both trials lasted 3 min and animals were returned to
their home cage after T1. The objects used were the glass beakers
filled with the gravel and the plastic bottles filled with the sand. The
heights of the objects were comparable (~12 cm) and the objects
were heavy enough not to be displaced by the animals. The
sequence of presentations and the location of the objects was
randomly assigned to each rat. The animals explored the objects by
looking, licking, sniffing or touching the object while sniffing, but
not when leaning against, standing or sitting on the object. Any rat
spending less than 5 s exploring the two objects within 3 min of T1
or T2 was eliminated from the study. Exploration time of the ob-
jects and the distance travelled were measured using the Any-
maze® video tracking system. Based on exploration time (E) of two
objects during T2, discrimination index (DI) was calculated ac-
cording to the formula: DI ¼ (EBeEA)/(EA þ AB).
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Declaration of competing interest
[18] E. Simoni, S. Daniele, G. Bottegoni, D. Pizzirani, M.L. Trincavelli, L. Goldoni,
G. Tarozzo, A. Reggiani, C. Martini, D. Piomelli, et al., Combining galantamine
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nonimidazole histamine H3 receptor antagonists combining anticholines-
terase activity, ChemMedChem 5 (7) (2010) 1143e1149.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
[20] J. Lalut, G. Santoni, D. Karila, C. Lecoutey, A. Davis, F. Nachon, I. Silman,
J. Sussman, M. Weik, T. Maurice, et al., Novel multitarget-directed ligands
targeting acetylcholinesterase and s1 receptors as lead compounds for
treatment of Alzheimer’s disease: synthesis, evaluation, and structural char-
acterization of their complexes with acetylcholinesterase, Eur. J. Med. Chem.
162 (2019) 234e248.
The authors acknowledge the financial support from the Na-
tional Science Centre, Poland (grant no. 2016/21/B/NZ7/01742). SCD
and PM were supported by grants from CNRS, INSERM, Montpellier
University of Excellence (iSITE MUSE), the French Foundation for
Medical Research (FRM) and ANR (ANR-17-CE16-0013-01 and ANR-
17-CE16-0010-01).
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C. Ballandonne, S. Corvaisier, A. Malzert Freon, et al., Design of donecopride, a
dual serotonin subtype 4 receptor agonist/acetylcholinesterase inhibitor with
potential interest for Alzheimer’s disease treatment, Proc. Natl. Acad. Sci. Unit.
States Am. 111 (36) (2014) E3825eE3830.
Appendix A. Supplementary data
ꢀ
[22] A. Wie˛ckowska, M. Kołaczkowski, A. Bucki, J. Godyn, M. Marcinkowska,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112765.
K. Wie˛ckowski, P. Zare˛ba, A. Siwek, G. Kazek, M. Głuch-Lutwin, et al., Novel
multi-target-directed ligands for Alzheimer’s disease: combining cholines-
terase inhibitors and 5-HT6 receptor antagonists. design, synthesis and bio-
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