Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives as an active and selective histamine H3 receptor ligands

Article abstract of DOI:10.1016/j.bioorg.2019.103071

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H₃ receptor (hH₃R) ligands in the nanomolar concentration range. While paying attention to

Full text of DOI:10.1016/j.bioorg.2019.103071

Accepted Manuscript
Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-
piperazine derivatives as an active and selective histamine H3 receptor ligands
Katarzyna Szczepańska, Tadeusz Karcz, Agata Siwek, Kamil J. Kuder,
Gniewomir Latacz, Marek Bednarski, Małgorzata Szafarz, Stefanie Hagenow,
Annamaria Lubelska, Agnieszka Olejarz-Maciej, Michał Sobolewski, Kamil
Mika, Magdalena Kotańska, Holger Stark, Katarzyna Kieć-Kononowicz
PII:
S0045-2068(19)30774-6
https://doi.org/10.1016/j.bioorg.2019.103071
103071
DOI:
Article Number:
Reference:
YBIOO 103071
Bioorganic Chemistry
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Revised Date:
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12 June 2019
14 June 2019
Please cite this article as: K. Szczepańska, T. Karcz, A. Siwek, K.J. Kuder, G. Latacz, M. Bednarski, M. Szafarz,
S. Hagenow, A. Lubelska, A. Olejarz-Maciej, M. Sobolewski, K. Mika, M. Kotańska, H. Stark, K. Kieć-
Kononowicz, Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives
as an active and selective histamine H3 receptor ligands, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/
j.bioorg.2019.103071
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Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-
piperazine derivatives as an active and selective histamine H3 receptor ligands
Katarzyna Szczepańska a, Tadeusz Karcz a, Agata Siwek b, Kamil J. Kuder a, Gniewomir Latacz a, Marek
Bednarski c, Małgorzata Szafarz d, Stefanie Hagenow e, Annamaria Lubelska a, Agnieszka Olejarz-Maciej a,
Michał Sobolewski a, Kamil Mika c, Magdalena Kotańska c, Holger Stark e, Katarzyna Kieć-Kononowicz a,*
a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
b Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
c Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
d Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
e Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part
substituents proved to be potent histamine H3 receptor (hH3R) ligands in the nanomolar
concentration range. While paying attention to their alkyl linker length, derivatives with a six
methylene linker tend to be more potent than their five methylene homologues. Moreover, in the
case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess
lower activity than their seven methylene homologues. However, in global analysis of collected
data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest
hH3R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In
the case of biphenyl and benzophenone derivatives, compounds with para- substituted second
aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone
derivative 18 showed the highest affinity among all tested compounds (hH3R Ki = 3.12 nM). The
likely protein-ligand interactions, responsible for their high affinity were demonstrated using
molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H3R, as well as drug-
like properties of selected ligands were evaluated using in vitro methods.
Available online
Keywords:
Histamine H3 receptor
Histamine H3 receptor ligands
Non-imidazole histamine H3R ligands
Piperazine derivatives
Selective ligands
Molecular docking
Metabolic stability
1. Introduction
the most researched areas in the vast field of GPCR ligands –
starting from imidazole containing ligands, through various non-
Function of histamine as neurotransmitter has been proven with
the discovery of the H3 receptor [1,2]. It is presynaptically located
as autoreceptor controlling the synthesis and release of histamine.
As heteroreceptor it modulates with presynaptical localization the
release of numerous other neurotransmitters, e.g. acetylcholine,
norepinephrine, dopamine, serotonin, glutamate, γ-aminobutyric
acid [3]. The histamine H3 receptor is anatomically localized
primarily to the Central Nervous Sysytem (CNS) with prominent
expression in basal ganglia, hippocampus, cortex and striatal area
[4]. In the periphery H3R can be found with low density in
gastrointestinal, bronchial and cardiovascular system. As H3
autoreceptor activation stimulates the negative feed-back
mechanism, reduced central histaminergic activity is observed.
Involvement in cognition, sleep-wake status, energy homeostatic
regulation, inflammation etc. has attracted pharmaceutical
research for numerous so far unmet therapeutic approaches in
different peripheral, but mainly central diseases [5,6]. The wide
spectrum of possible therapeutic implications makes H3Rs one of
imidazole derivatives, with recent introduction of successful
Wakix® to pharmaceutical market.
One such replacement for an imidazole is a piperazine moiety,
an important versatile scaffold in rational drug design for most of
the GPCR ligands. It is one of the most inherent structural
elements in drug finding, marked with a large variety of successes
in biological applications. Appropriate physicochemical properties
of the piperazine template make this molecular subunit a useful
and well-positioned system in the search for new drugs.
Furthermore, it acts as efficient and particular subject for a range
of miscellaneous scientific objectives in medicinal chemistry and
thus, piperazine moiety is regarded as privileged framework [7].
This scaffold is mainly observed in molecules targeting various
CNS receptors, such as different subtypes of adrenergic,
dopaminergic and serotoninergic receptors [8-10]. Therefore,
paying attention to its versatile properties and pointing out that
piperazine is also observed as a key structural feature in many

histamine H3R ligands (as a successful imidazole replacement), we
focused on such derivatives in this work.
Therefore, we selected eight previously described compounds
KSK29, KSK19, KSK30, KSK25, KSK3, KSK31, KSK9 and
Figure 1. KSK29, KSK19, KSK30, KSK25, KSK3, KSK31, KSK9, KSK32 and general structures of described ligands; hH3R – human histamine H3 receptor.
In general, all compounds presented herein fit the traditional
pharmacophore model for histamine H3R blocking compounds,
that contain a basic moiety linked by a spacer to a central, mostly
aromatic core structure which then is connected to further affinity
enhancing elements, e.g. another basic moiety or
hydrophilic/lipophilic groups or a combination thereof [11].
KSK32, presented in the Figure 1, that served as lead structures
for further modifications [12,13]. Considered compounds
represent promising, very high H3R antagonist potency, as well as
interesting anticonvulsant, precognitive and anorectic
pharmacological profile in several rodent models.
Structural modifications of leads performed within this work
were divided into two phases. Phase 1 included:
Structure-activity studies of previously described ligands
allowed for the establishment of the 4-pyridyl-piperazine moiety
a new bioisosteric piperidine replacement in H3R ligands [12]. The
results of the in vitro tests proved this scaffold as a crucial element
for high hH3R affinity. Furthermore, modeling studies showed its
role in the formation of suitable interactions with the hH3R.


extension of the alkyl chain to 5 – 6 methylene groups,
as well as (in the case of acetyl and propionyl derivatives
generally more active than tert-butyl and tert-pentyl
analogues) both: extension of linker up to 8 and subtraction
to 2 methylene groups.
Scheme 1. General synthetic pathway for compounds 2–21.

Table 1. Structures of compounds 2–21 and their in vitro histamine H3 receptor (hH3R) affinities. Given data represent mean values within the 95% confidence
interval (CI).
O
n
R2
R1
N
N
N
No.
2
n
1
R1
R2
H
hH3R Ki [nM] x
̅
[CI 95%]
No.
n
6
R1
R2
H
hH3R Ki [nM] x̅ [CI 95%]
O
O
1957 [1212, 3158]
1007 [622, 1630]
29.9 [1.26, 708]
26.7 [15.9, 44.7]
62.1 [7.02, 549]
12
13
14
15
16
20.0 [13.6, 29.4]
19.3 [6.57, 56.4]
40.5 12.3, 134]
38.9 [9.52, 159]
21.1 [3.83, 116]
O
O
3
4
5
6
1
4
4
4
H
H
H
H
6
7
7
2
H
H
H
H
O
O
O
O
7
8
4
5
H
H
115 [26.8, 493]
12.7 [4.35, 36.9]
17
18
2
2
H
53.6 [16.6, 174]
3.12 [0.66, 14.6]
O
O
H
O
O
9
5
H
16.9 [7.95, 36.0]
19
2
H
22.2 [3.30, 149]
10
11
5
5
H
H
397 [220, 715]
69.2 [29.6, 162]
20
21
2
2
CH2CH3
CH3
H
H
40.4 [17.1, 95.9]
42.7 [12.4, 147]
2.2. Pharmacology
2.2.1. Histamine H3 receptor affinity
After the selection of optimal linker length (based on in vitro
studies), in phase 2, various moieties (methyl, ethyl, phenyl,
benzoyl) were introduced to the aromatic ring in eastern part of
compounds.
All compounds (as oxalate salts) were tested in H3R in vitro
binding studies, using slightly modified methods to those
described previously [15]. Briefly, compounds were tested at five
to eleven appropriate concentrations in a [3H]Nα-Methylhistamine
(KD = 3.08 nM) radioligand depletion assay to determine the
affinity at human recombinant histamine H3R stably expressed in
HEK-293 cells.
Paying attention to structural similarity of concerned
compounds to other GPCR ligands, determination of affinity to
histamine H1, dopamine D2, muscarinic M1 and α1 adrenergic
receptors was also carried out.
2. Results and discussion
In vitro affinity data are assembled in Table 1. Paying attention
to the alkyl linker length, derivatives with either a five or a six
methylene linker show similar affinity with a Ki value below
100 nM with the exception of compound 7 and 10 (hH3R Ki = 115
and 397 nM, respectively). According to our assumptions, acetyl
and propionyl derivatives tend to show a higher affinity at H3R
than tert-butyl and tert-pentyl analogues (6, 10, 7, 11 vs. 4, 8, 5
and 9).
2.1. Chemistry
The synthesis of desired final compounds 2–21 was achieved
through the synthetic route presented in Scheme 1. According to
the procedure [14] modified by us [12,13], the phenoxy alkyl
bromides 1a–u were obtained by one-step alkylation of
commercially available phenols with α,ω-dibromoalkanes in
propan-1-ol under reflux conditions. Obtained precursor bromides
were then coupled with 1-(pyridin-4-yl)piperazine in the mixture
of ethanol/water with powdered potassium carbonate and a
catalytic amount of potassium iodide. Final products were
obtained as free bases and isolated as oxalic acid salts.
In general, both phenoxyacetyl and -propionyl derivatives show
high hH3R affinity in low nanomolar concentration ranges with
minor influences on affinity by extension of alkyl linker (4, 5, 8,

Table 2. Radioligand binding assay for selected GPCRs. In the case of H1, M1 and D2 receptors results are presented as a percentage of control specific binding
at 1 and 0.1 µM concentration. Affinity to α1 receptors are presented as Ki values.
Receptor
H1
M1
D2
α1
% of control
specific
binding at 1
µM conc.
% of control
specific
binding at 0.1
µM conc.
% of control
specific
binding at 1
µM conc.
% of control
specific
binding at 1
µM conc.
% of control
specific binding
at 0.1 µM conc
% of control
specific binding
at 0.1 µM conc
No.
Ki [nM] ± SEM
2
0
0
0
0
0
2
0
0
0
0
1
6
0
0
0
0
0
2
2
0
0
0
9
0
8
1
0
0
0
1
97
99
-
8
4
7
1
0
0
0
0
0
2
0
4
1
0
2
4
0
0
0
1
0
10
0
0
0
0
0
0
3
0
2
0
0
1
-
no affinity
3
no affinity
4
26
48
8
12
24
12
15
4
9
0
-
5
14
10
9
6
-
6
0
-
7
23
0
10
0
-
8
0
-
9
3
12
6
0
4
-
10
11
12
13
14
15
16
17
18
19
20
21
6
0
8
no affinity
30
1
11
2
0
14
0
no affinity
3
> 1000
10
19
14
18
15
15
7
18
6
11
0
0
> 5000
1
no affinity
16
1
0
0
no affinity
0
0
no affinity
15
8
8
15
0
> 5000
0
> 5000
11
0
4
10
0
> 5000
5
0
> 5000
11
14
3
0
3
0
3270.0 ± 320.0
KSK29
22
2
8
0
-
KSK19
0
3
> 5000
KSK30
15
6
28
5
12
0
0
-
KSK25
14
27
0
no affinity
KSK3
0
0
0
no affinity
KSK31
0
0
0
-
KSK9
0
3
0
0
no affinity
KSK32
0
0
0
3
-
Mepyramine
Triprolidine
Atropine
Scopolamine
Pirenzepine
Methoctramine
Haloperidol
Phentolamine
99
100
-
-
-
-
-
-
-
-
-
-
99
100
89
36
-
92
98
45
7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
98
-
93
-
-
-
-
22.0 ± 1.5
9, 12-15). However, a short ethylene linker results in reduction of
affinity (2 and 3, hH3R Ki = 1957 and 1007 nM, respectively).
Global analysis of collected data referring to influence of alkyl
linker length, allowed for the selection of three methylene
homologues as phase 2 templates, due to their highest H3R affinity
values among all described 4-pyridylpiperazine derivatives (see
Figure 1).
of smaller alkyl substituents attached to the aromatic ring able to
interact with the H3R binding site.
2.2.2. Affinity to other GPCRs
In order to confirm selectivity of obtained ligands,
determination of affinity to histamine H1, dopamine D2,
muscarinic M1 and α1 adrenergic receptors was carried out
(Table 2). In the case of H1, D2 and M1R, radioligand binding was
performed using membranes from CHO-K1 cells stably
transfected with proper receptors. Adrenergic (α1) receptor binding
assay was performed using rat cortex tissue. All assays were
carried out in duplicates. They clearly indicate high selectivity of
herein and previously described 4-pyridylpiperazine derivatives
for human H3R.
In order to determine the influence of substituents located in the
“eastern part” of the molecule, various moieties were introduced
to the aromatic ring of ligands. In the case of biphenyl and
benzophenone derivatives, compounds with para position of the
second aromatic ring tend to be of higher affinity than their meta
analogues (16 and 18 vs. 17 and 19, hH3R Ki = 21.1 and 3.12 nM
vs. 53.6 and 22.2 nM). Interestingly, 18 showed the highest affinity
among all tested 4-pyridylpiperazine compounds from our group
up to date (see Figure 1). High affinities of 20 and 21 (hH3R Ki =
40.4 and 42.7 nM, respectively), might also indicate the tolerance
2.2.3. Intrinsic activity at histamine H3 receptor
In order to identify compounds that are antagonists, inverse
agonists and/or partial agonists, efficacy of selected structures was

Table 4. PAMPA results (permeability coefficient Pe) for selected compounds.
CFN – well permeable standard Caffeine, NFX – low permeable standard
Norfloxacin. ND*: No data, compound precipitated under assay conditions.
Table 3. The IC50 values of tested compounds in antagonist dose-response
assay, with reference agonist (R-α-Methylhistamine) used at
a final
concentration equivalent to its EC80 value.
assessed by luminescence detection of calcium mobilization in
response to H3R stimulation. None of the tested compounds
No.
Pe
(10-6 cm/s) ± SD
11
ND*
No.
IC50 [nM] ± SEM
no fit curve
no fit curve
3.5 ± 0.5
12
7.29 ± 1.6
7.11 ± 3.3
6.22 ± 2.3
6.55 ± 0.8
7.36 ± 1.7
4.16 ± 0.7
15.1 ± 0.4
0.56 ± 0.1
1
2
13
10
14
11
no fit curve
299.7 ± 39.4
2600 ± 0.3
2430 ± 0.3
no fit curve
no fit curve
no fit curve
54.5 ± 4.1
15
12
16
13
14
18
15
CFN
NFX
16
17
reasonable to acknowledge studied molecules as substances with
efficient passive transport through biological membranes.
18
19
386.1 ± 25.9
152.8 ± 20.1
78.8 ± 6.3
20
2.3.2. Influence on cytochrome P450 3A4 activity.
21
In order to assess the potential risk of drug-drug interactions of
most active ligand, the luminescence-based CYP3A4 P450-Glo™
assay was used. Compound 18 was incubated with recombinant
CYP3A4 isoform for 30 min. The measurement of CYP3A4
activity showed slight inhibition of enzyme activity by 18, but only
at high micromolar concentrations (10 and 25 µM) (Figure 2).
Clobenpropit
3.4 ± 0.2
showed agonistic activity towards the desired biological target
(data not shown).
Of all compounds, only 10 showed comparable antagonist
activity to the reference compound Clobenpropit (Table 3). The
IC50 value for the antagonistic intrinsic activity of compound 10 is
approximately 100 fold greater than the IC50 value for affinity at
H3R. The test used to determine the antagonistic activity is based
on the evaluation of calcium mobilization in the cell. Therefore,
this result may indicate an additional mechanism of compound 10,
which affects the mobilization of intracellular calcium.
Explanation of this effect is subjected to our further studies.
Compounds 18, 20 and 21 showed antagonist activity, but with
effective concentrations 16 to 37 times lower than the reference
compound. Lowest antagonist potencies were demonstrated by 12,
13, 14 and 19 with the IC50 values in the range between 250 and
2600 nM. For the remaining tested compounds, it was not possible
to fit a dose-response curve in that type of assay.
2.3.3. Hepatotoxicity – the effect on HepG2 cells
To predict potential hepatotoxicity of 16 and 18, their
antiproliferative activity at hepatoma HepG2 cells was evaluated.
Statistically significant decrease in the cell viability was observed
at 10 and 100 μM concentrations. However, this effect was weak,
compared to reference compounds: doxorubicin (DX) and
carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which
decreased viability of HepG2 by > 50% at 1 μM and 10 μM,
respectively (Figure 3).
2.3.4. Metabolic stability
Initially, computational tool MetaSite was used to determine
the potential sites of metabolism of compound 18 [18]. The
obtained data suggested the pyridine moiety as the most probable
site of metabolism. Moreover, the aliphatic linker was also shown
to be susceptible for metabolic biotransformation (see
Supplementary Data).
2.3. Results of selected ADMET parameters
2.3.1. Permeability profile
The ability of selected H3R ligands to penetrate across lipid
membranes was estimated by parallel artificial membrane
permeability assay (PAMPA). In medicinal chemistry, PAMPA is
a method which determines the passive diffusion of substances
from a donor into acceptor compartment, through a lipid – infused
artificial membrane. Passive diffusion it is the predominant
mechanism for absorption of most commercial drugs [16]. The
results were expressed as permeability coefficient Pe calculated
according to the formulas provided by manufacturer [17]. The Pe
values of tested compounds varies between 4.16 and 7.36 × 10-6
cm (Table 4). Considering gathered data and comparing it to two
standards: well-permeable Caffeine (Pe = 15.1 × 10-6 cm/s) and
low-permeable Norfloxacin (Pe = 0.56 × 10-6 cm/s), it seems
Next, the metabolic stability was evaluated in vitro using mouse
liver microsomes (MLMs). The LC/MS analysis of the reaction
mixture after 120 min incubation of 18 with MLMs led to the
identification of two metabolites with the following molecular
masses of their quasimolecular ions [M+H]+: m/z = 404.27 (M1,
main metabolite) and m/z = 418.29 (M2, Figure 4). In MS
analysis, the observed molecular masses and ion fragments of 18,
as well as its main metabolite M1 (+ 2 units) and M2 (+ 16 units)
suggest the ketone reduction and the reaction of hydroxylation as
metabolic routes of compound 18. However, according to the in
silico data, either N-oxidation or hydroxylation were proposed,
which occurred most probably at the 4-pyridyl moiety (see
Supplementary Data). In summary, 18 showed moderate
metabolic stability, as ~42% remained in the reaction mixture,
with ketone reduction as the main metabolic pathway (Figure 4).
2.4. Molecular modeling
Figure 2. The effect of 18 and the reference ketoconazole on CYP3A4 activity.

other hand, in the case of biphenyl compound 16, terminal benzene
ring formed a π-cation interaction with ARG3816.58 (Figure 5),
along with previously mentioned π-π stacking stabilization, which
was not found for the slightly less affine meta substituted
derivative 17. Similar findings were found for benzophenone
derivatives 18 and 19, where meta derivative was derived of π-π
stacking stabilization of terminal benzene ring, when compared to
its para analogue, thus yielding in slightly lower affinity (Figure
6).
In some cases for compounds of 6–8 methylene groups alkyl
chain, a slightly rotated along Y-axis poses were obtained,
resulting in H-bond formation between TYR912.61 (instead of
ARG3816.58) hydroxyl moiety and carbonyl group oxygen.
However, paying attention to overall high H3R affinity in tested
Figure 3. HepG2 cell viability after 72h–long incubation with compounds 16, 18, Doxorubicin (DX) and CCCP. The statistical significance was
evaluated by a one-way ANOVA, followed by Bonferroni’s Comparison Test ( ***p < 0.001 compared with negative control).
Figure 4. The UPLC spectrum of reaction mixture after 2 h incubation of 18 with MLMs.
Although the number of GPCRs crystal structure is
continuously growing, structure of only one member of histamine
receptor family was resolved up to date: histamine H1 receptor
(PDB ID: 3RZE [19]). Therefore, in this study previously built and
described H3R homology model (with reference ligand Pitolisant
in the binding pocket) was used [12].
All of the docking poses presented binding mode perpendicular
to membrane plane, in agreement with findings of Levoin et al.
[20], with p-propionyl and p-acetyl derivatives scored with highest
and lowest scores respectively. For most of the compounds,
necessary hH3R antagonist/inverse agonist interactions were
found: hydrogen bond and /or salt bridge formation between
protonated piperazine nitrogen and GLU2065.46 (upper case
numeration according to Balesteros-Weinstein [21]). Moreover,
formation of H-bond between TYR3746.51 and the ether oxygen for
compounds with alkyl chain up to five methylene groups (e.g. 4
and 16) was observed.
group and overall observed ligand-receptor interactions such poses
and interactions might have no pharmacological coverage.
3. Conclusions
The investigated series of novel 4-pyridylpiperazine derivatives
proved to be potent histamine H3R ligands (hH3R Ki = 3.12 nM –
1.96 µM). Focusing on the alkyl linker length (five to eight
methylene linker), independently of bulky, eastern part
substituents, derivatives show similar H3R affinity with exception
of 7 and 10. However, a short ethylene linker led to reduction of
the hH3R affinity. Overall, three methylene homologues showed
the highest affinity values among all described 4-
pyridylpiperazine derivatives.
Similarly to our previous findings, π-π stabilization of a 4-
pyridyl moiety by either indole or benzene ring of TRP3716.48 was
observed. Since this residue appears as one of the so-called
molecular switches of the GPCR’s [22], herefound interactions
might influence high affinity of the described ligands. In turn,
benzene rings of the lipophilic part of ligands were also stabilized
through π-π stacking with at least one of aminoacids: TYR1153.33
,
TYR3947.35, TYR18945.51, PHE193 (ECL2). For most of the
compounds, formation of hydrogen bond(s) between carbonyl
group oxygen and ARG3816.58 amine groups was found. On the

Figure 5. Left panel: calculated binding pose for ligands 16 (light blue) and 17 (dark green) in histamine H3 homology receptor binding pocket; middle panel &
right panel: ligand interaction diagrams for compounds 16 and 17 respectively.
Figure 6. Left panel: calculated binding pose for ligands 18 (light blue) and 19 (dark green) in histamine H3 homology receptor binding pocket; middle panel &
right panel: ligand interaction diagrams for compounds 18 and 19 respectively.
In order to determine the influence of substituents located in the
“eastern part” of the molecule, various moieties were introduced
to the aromatic ring of ligands resulting in only slight variations
regarding H3R affinity. Acetyl and propionyl as well as tert-butyl
and tert-pentyl showed generally high affinity H3R with few
exceptions. Even larger substituents, namely biphenyl and
benzophenone derivatives, were tolerated by the H3R.
Interestingly, benzophenone derivative 18 showed the highest
affinity among all tested compounds so far (hH3R Ki = 3.12 nM).
Docking studies to histamine H3R homology model allowed the
observation of previously proposed ligand-receptor interactions.
Selectivity of obtained compounds was confirmed in radioligand
binding assays with histamine H1, dopamine D2, muscarinic M1
and α1 adrenergic receptors. In order to determine functional
activity of the compounds, efficacy of selected structures was
assessed by luminescence detection of calcium mobilization in
response to H3R stimulation. In fact, of all compounds, 10, 18, 20
and 21 showed antagonist activity with nanomolar effective
concentrations. Finally, a series of novel 4-pyridylpiperazine
derivatives proved to be active and selective histamine H3R
blocking compounds. Determination of their pharmacological
profile is subjected to our further research.
4. Experimental protocols
4.1. Chemistry
4.1.1. General synthetic procedure for compounds
1a–1u
All compounds were obtained using methods described
previously [12,13]. Detailed synthetic procedure could be found in
Supplementary Data.
4.1.2. General synthetic procedure for compounds 2–21
All compounds were obtained using methods described
previously [12,13]. Detailed synthetic procedure and analytical
data could be found in Supplementary Data.
4.2. Pharmacology

4.2.1. [3 H]Nα -Methylhistamine hH3 R displacement
assay
minutes. Five rapid washes with 300 µl 50 mM Tris buffer (4°C,
pH 7.4) were performed using 96-well FilterMate harvester
(PerkinElmer, USA). 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 4 minutes. Radioactivity was counted
in MicroBeta2 scintillation counter (PerkinElmer). Data were
fitted to a one-site curve-fitting equation with Prism 5 (GraphPad
Software) and Ki values were estimated from the Cheng−Prusoff
equation.
Competition binding data were analyzed using GraphPad Prism
(V6.01, San Diego, CA, USA) software, using non-linear least
squares/regression fit. Ki values were calculated from IC50 values
according to Cheng-Prusoff equation [23]. Statistic analysis was
performed on pKi values from at least three experiments, each
performed at least in duplicates. Mean affinity values (Ki) were
transferred into nanomolar concentrations (with 95% confidence
interval). The displacement binding assay was carried out as
described by Kottke et al. with slight modifications [12,13,15].
4.2.2.3. Affinity at D2 receptors
10 mM stock solutions of tested compounds were prepared in
DMSO. Serial dilutions of compounds were prepared in 96-well
microplate in assay buffers using automated pipetting system
epMotion 5070 (Eppendorf). Each compound was tested in
a screening assay at the final concentrations of 1 µM and 0.1 µM.
Results were expressed as percent inhibition of [3H]-
Methylspiperone binding. Radioligand binding was performed
using membranes from CHO-K1 cells stably transfected with the
human D2 receptor (PerkinElmer). All assays were carried out in
duplicates. 50 µl working solution of the tested compounds, 50 µl
[3H]-Methylspiperone (final concentration 0.4 nM) and 150 µl
diluted membranes (3 µg protein per well) prepared in assay buffer
(50 mM Tris, pH 7.4, 50 mM HEPES, 50 mM NaCl, 5 mM MgCl2,
0.5 mM EDTA) were transferred to polypropylene 96-well
microplate using 96-wells pipetting station Rainin Liquidator
(MettlerToledo). Haloperidol (10 μM) was used to define
nonspecific binding. Microplate was covered with a sealing tape,
mixed and incubated for 60 minutes at 37°C. The reaction was
terminated by rapid filtration through GF/B filter mate presoaked
with 0.5% polyethyleneimine for 30 minutes. 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). 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 minutes. Radioactivity was counted in
MicroBeta2 scintillation counter (PerkinElmer).
4.2.2. Affinity at other GPCRs
4.2.2.1. Affinity at H1 receptors
10 mM stock solutions of tested compounds were prepared in
DMSO. Serial dilutions of compounds were prepared in 96-well
microplate in assay buffers using automated pipetting system
epMotion 5070 (Eppendorf). Each compound was tested in
a screening assay at the final concentrations of 1 µM and 0.1 µM.
Results were expressed as percent inhibition of [3H]-Pyrilamine
binding. Reference compounds were tested in eight concentrations
from 10–5 to 10–12 M (final concentration). Radioligand binding
was performed using membranes from CHO-K1 cells stably
transfected with the human histaminergic H1 receptor. All assays
were carried out in duplicates. 50 µl working solution of the tested
compounds, 50 µl [3H]-Pyrilamine (spec. act. 20.0 Ci/mmol, final
concentration 1.5 nM) and 150 µl diluted membranes (5 µg protein
per well) prepared in assay buffer (50 mM Tris-HCl, pH 7.4; 5 mM
MgCl2) were transferred to polypropylene 96-well microplate
using
96-wells
pipetting
station
Rainin
Liquidator
(MettlerToledo). Mepyramine (10 μM) was used to define
nonspecific binding. Microplate was covered with a sealing tape,
mixed and incubated for 60 minutes at 27°C. The reaction was
terminated by rapid filtration through GF/B filter mate presoaked
with 0.5% polyethyleneimine for 30 minutes. Five rapid washes
with 300 µl 50 mM Tris buffer (4°C, pH 7.4) were performed using
96-well FilterMate harvester (PerkinElmer, USA). 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
4 minutes. Radioactivity was counted in MicroBeta2 scintillation
counter (PerkinElmer). Data were fitted to a one-site curve-fitting
equation with Prism 5 (GraphPad Software) and Ki values were
estimated from the Cheng−Prusoff equation.
4.2.2.4. Affinity at α1 receptors
10 mM stock solutions of tested compounds were prepared in
DMSO. Serial dilutions of compounds were prepared in 96-well
microplate in assay buffers using automated pipetting system
epMotion 5070 (Eppendorf). Each compound was tested in
6 concentrations from 10–5 to 10–10 M (final concentration).
Radioligand binding was performed using tissue rat cortex. All
assays were carried out in duplicates. 50 µl working solution of the
tested compounds, 50 µl [3H]-Prazosin (final concentration 0.2
nM) and 150 µl tissue suspension prepared in assay buffer (50 mM
Tris-HCl, pH 7.6) were transferred to polypropylene 96-well
microplate using 96-wells pipetting station Rainin Liquidator
(MettlerToledo). Phentolamine (10 μM) was used to define
nonspecific binding. Microplate was covered with a sealing tape,
mixed and incubated for 30 minutes at 30°C. The reaction was
terminated by rapid filtration through GF/B filter mate. Ten rapid
washes with 200 µl 50 mM Tris buffer (4°C, pH 7.6) were
performed using 96-well FilterMate harvester (PerkinElmer,
USA). 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 4 minutes. The radioactivity on the filter was
measured in MicroBeta TriLux 1450 scintillation counter
(PerkinElmer, USA). Data were fitted to a one-site curve-fitting
equation with Prism 6 (GraphPad Software) and Ki values were
estimated from the Cheng−Prusoff equation.
4.2.2.2. Affinity at M1 receptors
10 mM stock solutions of tested compounds were prepared in
DMSO. Serial dilutions of compounds were prepared in 96-well
microplate in assay buffers using automated pipetting system
epMotion 5070 (Eppendorf). Each compound was tested in
a screening assay at the final concentrations of 1 µM and 0.1 µM.
Results were expressed as percent inhibition of [3H]-Nα-
Scopolamine binding. Reference compounds were tested in
8 concentrations from 10–5 to 10–12 M (final concentration).
Radioligand binding was performed using membranes from CHO-
K1 cells stably transfected with the human M1 receptor. All assays
were carried out in duplicates. 50 µl working solution of the tested
compounds, 50 µl [3H]-Scopolamine (spec. act. 84.1 Ci/mmol,
final concentration 0.2 nM) and 150 µl diluted membranes (35 µg
protein per well) prepared in assay buffer (PBS, pH 7.4) were
transferred to polypropylene 96-well microplate using 96-wells
pipetting station Rainin Liquidator (MettlerToledo). Atropine (10
μM) was used to define nonspecific binding. Microplate was
covered with a sealing tape, mixed and incubated for 120 minutes
at 27°C. The reaction was terminated by rapid filtration through
GF/A filter mate presoaked with 0.5% polyethyleneimine for 30
4.2.3. Intrinsic activity at histamine H3 receptor

The intrinsic activity at H3R was assessed by luminescence
detection of calcium mobilization using the recombinant jellyfish
photoprotein, aequorin. Measurements were performed with
Histamine H3 AequoScreen cell line (PekinElmer cat. no.ES-392-
A). The cell density in 96-well format measurements was 5,000
cells per well. Cell harvesting, coelenterazine h (Invitrogen, cat.
no. C 6780) loading and preparation were done according to
instructions presented in the AequoScreen Starter Kit Manual .
Compounds stock solutions were diluted in assay buffer (D-
MEM/F-12, Invitrogen cat. no. 11039) containing 0.1% BSA
(Intergen, cat. no. 3440-75) and pipetted (50 μL/well) into white
½ Area Plate – 96 well microplates (PerkinElmer, cat. no.
6005560). In the next step cell suspension was dispensed into plate
wells using the POLARstar optima reader injectors.
described before [24-26]. Test and reference compounds were
analyzed in triplicates. The luminescent signal was measured
using a microplate reader EnSpire PerkinElmer (Waltham, MA,
USA).
4.3.5. Hepatotoxicity – the effect on HepG2 cells
The H3R antagonists’ influence on HepG2 cells was evaluated
according to the procedures described before [13, 24-26]. Cell
viability was determined after 72 h incubation (37°C, 5% CO2)
with tested compounds in serial dilutions (0.1 – 100 µM) by
CellTiter 96® AQueous One Solution Cell Proliferation Assay
(MTS) obtained from Promega® (Madison, WI, USA). The
absorbance was recorded at 490 nm using a 96-well plate reader
(EnSpire, Perkin Elmer). As positive controls, cells were treated
with Doxorubicin (1μM) and mitochondrial toxin CCCP (10μM).
Detection of agonist activity. R-α-Methylhistamine
dihydrochloride (Sigma, Cat. no. H128) was used as an agonist for
histamine H3 receptor. Compounds dilution series in four
replicates were prepared as instructed in the AequoScreen Starter
Kit Manual. Emitted light was recorded for 20 seconds.
4.3.6. Metabolic stability
In silico investigation was performed by MetaSite 6.0.1
provided by Molecular Discovery Ltd. The most probable sites of
metabolism were predicted during this study by liver
computational model [18]. All experiments were performed as
described before [12, 13, 24, 25]. Mass spectra were recorded on
LC/MS system consisted of a Waters Acquity UPLC, coupled to a
Waters TQD mass spectrometer (electro spray ionization mode
ESI-tandem quadrupole).
Detection of antagonist activity. For the antagonist assay, cells
were injected (50 μL) into the assay plate with antagonists (50 μL)
using the POLARstar optima reader. The antagonist dilution series
in four replicates were prepared as instructed in the AequoScreen
Starter Kit Manual in concentrations from 10–11 to 10–6 M.
Antagonist used for the histamine H3 cell line was Clobenpropit
(Sigma, cat. no. C209). Agonist (R-α-Methylhistamine
dihydrochloride) at a single concentration was injected (50 μL,
final concentration EC80) on the preincubated (15–20 min) mixture
of cells + antagonist and the emitted light was recorded for 20
seconds.
4.4. Molecular modeling
For docking purposes, Schrödinger Maestro Suite (v. 11.5.011,
Release 2018-01) was used [27]. Ligands were built in their
ionized forms (protonated N4 piperazine nitrogen, structure charge
+1) using crystal structure of compound KKB-4 [28] as a template.
Bioactive conformations were generated using ConfGen module
[29,30] (Force field: OPLS3, water environment; minimization
method: PRCG with max. iterations of 2500 and convergence
threshold of 0.05; conformational search of 100 steps per
rotational bond). For all of the compounds 5 lowest energy
conformers were selected for docking studies. Binding site was
centered on ligand placed in homology model (Pitolisant).
Docking to rigid form of receptor was performed using Glide
module [31-33] (precision standard, flexible ligand sampling,
max. 5 poses per conformer). Ligands were rated according to their
position in binding pocket, interactions with binding pocket amino
acids, as well as the docking score value. Ligand interaction
diagrams were generated using Schrödinger Maestro, ligand-
receptor visualizations were generated using UCSF Chimera [34].
4.3. Analysis of selected ADMET parameters
4.3.1. Reference compounds
The compounds used as the references: Caffeine (CFN),
carbonyl cyanide 3-chlorophenylhydrazone (CCCP), Doxorubicin
(DX), Ketoconazole (KE), Norfloxacin (NFX) were obtained from
Sigma-Aldrich (St. Louis, MO, USA).
4.3.2. Cell line
Hepatoma HepG2 (ATCC® HB-8065™) cell line used in the
hepatotoxicity study was kindly donated by the Department of
Pharmacological Screening, Jagiellonian University Medical
College. The cell culture growth conditions were as described
before [13, 24-26].
Acknowledgements
4.3.3. Permeability across lipid membranes
We are pleased to acknowledge the generous support of the
grant DFG INST 208/664-1 FUGG (HS), COST Action CA15135
(KKK, HS) and National Science Center, Poland granted on the
Pre-coated PAMPA Plate System Gentest™ was obtained from
Corning, (Tewksbury, MA, USA). It consists of 96-well receiver
filter plate that has been pre-coated with structured layers of
phospholipids and a matched donor microplate. The stock
solutions of H3R antagonists and reference drugs were diluted in
the PBS buffer (pH 7.4) to the final concentration 200 µM. The
compounds were applied into the donor wells (200 µl) and
incubated (5 h) at RT. By using the UPLC-MS spectrometry
(Waters ACQUITY™ TQD system with the TQ Detector, Waters,
Milford, USA) with an internal standard the exact quantity of
molecules that penetrated from donor to acceptor wells through
phospholipid membrane was estimated. The permeability
coefficients (Pe, cm/s) were calculated using the formula provided
by the PAMPA Plate System manufacturer [17].
basis
of
decision
No.
2016/23/N/NZ7/00469
and
2016/23/B/NZ7/01063.
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Highlights
 Synthesis of 4-pyridylpiperazine alkyl ethers was performed.
 Human histamine H3 receptor affinity was estimated.
 Three methylene homologues appeared to be of highest hH3R affinity among all described
4-pyridylpiperazine derivatives from our group up to date.
 Benzophenone derivative 18 showed the highest affinity among all tested compounds
(hH3R Ki = 3.12 nM).
 Selectivity, intrinsic activity at H3R and drug-like properties of selected ligands were
evaluated using in vitro methods.