Synthesis and biological activity of novel tert-butyl and tert-pentylphenoxyalkyl piperazine derivatives as histamine H3R ligands

Article abstract of DOI:10.1016/j.ejmech.2018.04.043

As a continuation of our search for novel histamine H₃ receptor ligands, a series of twenty four new tert-butyl and tert-pentyl phenoxyalkylamine derivatives (2?25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer were evaluated for their binding properties at human histamine H₃ receptor (hH₃R). The highest affinities were observed for 4-pyridyl derivatives 4, 10, 16 and 22 (K_i = 16.0–120 nM). As it has been shown in docking studies, those specific heteroaromatic 4-N piperazine substituents might interact with one of the key receptor interacting amino acids. Moreover, the most promising compounds exhibited anticonvulsant activity in the maximal electroshock-induced seizure (MES) model in mice. Furthermore, the blood-brain barrier penetration, the functional H₃R antagonist potency as well as the pro-cognitive properties in the passive avoidance test were demonstrated for compound 10. In order to estimate drug-likeness of compound 10, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed. In addition, paying attention to the results obtained within this study, the 4-pyridyl-piperazino moiety has been established as a new bioisosteric piperidine replacement in H₃R ligands.

Full text of DOI:10.1016/j.ejmech.2018.04.043

Accepted Manuscript
Synthesis and biological activity of novel tert-butyl and tert-pentylphenoxyalkyl
piperazine derivatives as histamine H R ligands
3
Katarzyna Szczepańska, Tadeusz Karcz, Szczepan Mogilski, Agata Siwek, Kamil J.
Kuder, Gniewomir Latacz, Monika Kubacka, Stefanie Hagenow, Annamaria Lubelska,
Agnieszka Olejarz, Magdalena Kotańska, Bassem Sadek, Holger Stark, Katarzyna
Kieć-Kononowicz
PII:
S0223-5234(18)30377-5
10.1016/j.ejmech.2018.04.043
EJMECH 10392
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 January 2018
Revised Date: 19 April 2018
Accepted Date: 21 April 2018
Please cite this article as: K. Szczepańska, T. Karcz, S. Mogilski, A. Siwek, K.J. Kuder, G. Latacz,
M. Kubacka, S. Hagenow, A. Lubelska, A. Olejarz, M. Kotańska, B. Sadek, H. Stark, K. Kieć-
Kononowicz, Synthesis and biological activity of novel tert-butyl and tert-pentylphenoxyalkyl piperazine
derivatives as histamine H R ligands, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
3
j.ejmech.2018.04.043.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological activity of novel tert-butyl and tert-pentylphenoxyalkyl
piperazine derivatives as histamine H3R ligands
Katarzyna Szczepańska a, Tadeusz Karcz a, Szczepan Mogilski b, Agata Siwek c, Kamil J. Kuder a,
Gniewomir Latacz a, Monika Kubacka b, Stefanie Hagenow d, Annamaria Lubelska a, Agnieszka Olejarz a,
Magdalena Kotańska b, Bassem Sadek e, Holger Stark d, 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 Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
c Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
d Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf , Universitaetsstr. 1, 40225 Düsseldorf, Germany
e Department of Pharmacology & Therapeutics, College of Medicine & Health Sciences, United Arab Emirates University, P.O. Box 17666, Al Ain, United Arab
Emirate
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
As a continuation of our search for novel histamine H3 receptor ligands, a series of twenty four
new tert-butyl and tert-pentyl phenoxyalkylamine derivatives (2–25) was synthesized.
Compounds with three to four carbon atoms alkyl chain spacer were evaluated for their binding
properties at human histamine H3 receptor (hH3R). The highest affinities were observed for 4-
pyridyl derivatives 4, 10, 16 and 22 (Ki = 16.0–120 nM). As it has been shown in docking
studies, those specific heteroaromatic 4-N piperazine substituents might interact with one of the
key receptor interacting amino acids. Moreover, the most promising compounds exhibited
anticonvulsant activity in the maximal electroshock-induced seizure (MES) model in mice.
Furthermore, the blood-brain barrier penetration, the functional H3R antagonist potency as well
as the pro-cognitive properties in the passive avoidance test were demonstrated for compound
10. In order to estimate drug-likeness of compound 10, in silico and experimental evaluation of
metabolic stability in human liver microsomes was performed. In addition, paying attention to
the results obtained within this study, the 4-pyridyl-piperazino moiety has been established as a
new bioisosteric piperidine replacement in H3R ligands.
Available online
Keywords:
Histamine H3 receptor
Histamine H3 receptor ligands
Non-imidazole histamine H3R ligands
Piperazine derivatives
Anticonvulsants
Pro-cognitives
Molecular docking
Metabolic stability
1. Introduction
compounds (antagonists/inverse agonists) an interesting target in
the search for new drugs. Pharmacological data reveal potentially
Histamine acts as a neurotransmitter via four described
histamine receptors (H1-H4), belonging to G-protein coupled
receptors superfamily (GPCRs) [1]. H1 and H4 receptors
modulate inflammation and allergic responses, while H2
receptors regulate the secretion of gastric acid. Histamine H3
receptors (H3R) provide a wide spectrum of neuromodulatory
functions in the central nervous system (CNS) [2]. H3Rs have
been described as both, presynaptic autoreceptors regulating the
synthesis and release of histamine, and heteroreceptors
modulating the release of neurotransmitters such as dopamine,
acetylcholine, serotonin, norepinephrine, γ-aminobutyric acid,
glutamate and substance P [3]. H3R is expressed predominantly
in the CNS, especially in the region associated with cognition,
sleep, wakefulness and homeostatic regulation [4]. The
distribution of H3R in the body as well as its influence on the
level of important neurotransmitters, makes H3R-inhibiting
beneficial outcomes of H3R antagonists/inverse agonists use in
the treatment of schizophrenia, Alzheimer’s and Parkinson’s
disease, obesity, narcolepsy and attention-deficit hyperactivity
disorder (ADHD) [5], also as multi-targeting ligands [6].
Among a number of active compounds obtained in both
academia and industry [4,7], only one H3R antagonist, Pitolisant
(Bioprojet Biotech) completed phase III of clinical trials with
positive results in narcolepsy and daytime sleepiness in
Parkinson’s disease [8], and was accepted by the European
Medicines Agency as an orphan drug under the name of Wakix®
(Pitolisant hydrochloride) by Bioprojet Pharma [9]. Despite the
fact, that safety pharmacology, as well as recent comprehensive
studies [10] showed some ability of Pitolisant to affect the QT
interval in humans, as well as its slight pro-convulsant potential,
the European Medicines Agency’s Committee for Medicinal

with powdered potassium carbonate and a catalytic amount of
Products for Human Use decided, that Wakix’sAbeCneCfitEs aPreTmEuDch MANUSCRIPT
greater than its risks, and thus recommended its approval for use
in the EU.
potassium iodide. Final products were obtained as free bases and
isolated as oxalic acid salts.
2.2. Pharmacology
In recent years, many potent and selective anti-H3R agents
(H3R antagonists/inverse agonists) have been synthesized, both
by academia and pharmaceutical companies [11-13]. In general,
these compounds fit the traditional pharmacophore model for
histamine H3R antagonists/inverse agonists, that contain a tertiary
basic amine, an aliphatic linker (a linear, usually propyloxy or
structurally constrained chain), a central core (an aromatic one)
and ‘the eastern part’ with acidic, basic, lipophilic or other polar
groups, even an organometallic element [14]. While the amine-
aliphatic part is mostly responsible for the ligand’s affinity, the
arbitrary region might influence activity and pharmacokinetic
properties [15-17].
2.2.1. Histamine H3 receptor affinity
All compounds in their oxalate forms were then tested in H3R
in vitro binding studies, as described previously with slight
modifications. 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 [22].
Antagonistic characterization of compounds is assembled in
Table 1. It could be clearly seen, that most of the tested
compounds showed relatively low histamine H3 receptor affinity.
Interestingly though, 4-pyridyl derivatives: 4, 10, 16 and 22
showed high H3R affinity. Compared to other introduced
modifications at position 4-N of piperazine, 4-pyridyl moiety was
that one tolerated best and was present i.a. in compound 4 (hH3R
Ki = 16.0 nM), being the most active amongst the series
described in the present work. As it has been indicated in docking
studies, those specific heteroaromatic 4-N piperazine substituents
appear to interact with one of the key receptor interacting amino
acids. Among two other pyridyl substituents, 3-pyridyl
piperazine derivatives appear to be of similar affinity as those of
2-pyridyl, with the exception of compounds 20 vs. 21, where 2-
pyridyl derivative 20 was two orders of magnitude more potent
than its 3-pyridyl analogue 21. Moreover, in case of dihetero
aromatic substituents 2-piperazinopyrimidine derivatives (with
exception of compounds 23 and 24; hH3R Ki = 2264 and 683 nM,
respectively) were of higher H3R affinity than 2-(1-
piperazinyl)pyrazine derivatives (i.e. compound 5 vs. 6 and 11
vs. 12). On the other hand, tetrahydrofuroyl derivatives (7, 13,
19, 25) show no H3R affinity (Ki > 10 000), thus might be too
bulky and/or flexible for the histamine H3 receptor binding
pocket.
For purpose of this study we selected two previously
described compounds from our H3R ligands library - DL76 and
DL77, as lead structures for further modifications. Considered
compounds represented very promising, high H3R antagonist
potency and selectivity towards H3R, as well as interesting
anticonvulsant and pro-cognitive pharmacological profile in
several rodents models [18,19,40,41].
Structural modifications performed within this work included:
• Replacement of the basic piperazine ring with different
heterocyclic groups to modify the basic structural part of the
molecule, and
• Extension of the alkyl chain length to include 3–4 methylene
groups (Figure 1).
In 1992, Schunack and co-workers proposed the general
pharmacophore for histamine H3R antagonists, where the
nitrogen-containing ring is connected via aliphatic linker with
a polar moiety [23]. Customarily, the length of such linker was
three methylene groups. One of the first to describe active ligands
with longer linker (consisted of 3–6 methylene groups) was
Ganellin et al. in 1998, where the in vitro affinity of the
compounds found to be high (hH3R Ki = 0.019–0.46 µM),
independently from the linker length (3–5 methylene groups
alkyl spacer) [24]. Based on our data, we can also conclude that
there is no significant difference in receptor affinity between
structures containing three and four methylene groups linker,
with the exception of the most potent 4-pyridyl-containing
structures, e.g. compound 4 vs. 10 (hH3R Ki = 16.0 vs. 37.8 nM
respectively) and 16 vs. 22 (hH3R Ki = 30.6 vs. 120 nM
respectively).
Figure 1. Pitolisant (Wakix®), DL76, DL77 and general structure of described
ligands 2–25; hH3R – human histamine H3 receptor; hH4R – human histamine
H4 receptor; rH3R – rat histamine H3R
2. Results and discussion
In the investigated subgroup of 4-pyridyl-piperazine, t-butyl
substituent of the benzene ring also seems to have positive
influence on H3R affinity, e.g. compound 4 vs. 16 (hH3R Ki =
16.0 vs. 30.6 nM respectively). Interestingly, this feature is not in
agreement with the results previously described by our group for
compounds DL76 (hH3R Ki = 22 nM) and DL77 (hH3R Ki =
8.4 nM), composed of piperidine in their basic part, and t-
butylphenyl and t-pentylphenyl substituents, respectively.
2.1. Chemistry
The synthesis of desired ether derivatives 2–25 was achieved
through the efficient synthetic route outlined in Scheme 1. Para
tert-butyl and tert-pentylphenoxyalkyl bromides (1a–1d,
synthesis according to [20], modified by authors) were obtained
in one-step alkylation of para tert-butyl and tert-pentylphenol
with α,ω-dibromoalkanes refluxed in propan-1-ol [21]. Obtained
precursor bromides, after the separation from bis products
emerging during synthesis, were then coupled with
corresponding cycloalkylamines in the mixture of ethanol/water
2.2.2. Functional bioassay

ACCEPTED MANUSCRIPT
Scheme 1. General synthetic pathway for compounds 2-25
Table 1. Structures of compounds 2–25 and their in vitro histamine H3 receptor (hH3R) affinities. Given data represent mean values within the 95%
confidence interval (CI).
hH3R Ki [nM]
hH3R Ki [nM]
No.
2
n
1
1
1
1
1
1
2
2
2
2
2
2
R
No.
14
15
16
17
18
19
20
21
22
23
24
25
n
1
1
1
1
1
1
2
2
2
2
2
2
R
x [CI 95%]
x [CI 95%]
̅
̅
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
pyrimidin-2-yl
pyrazin-2-yl
tetrahydrofuroyl
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
pyrimidin-2-yl
pyrazin-2-yl
tetrahydrofuroyl
> 10 000
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
pyrimidin-2-yl
pyrazin-2-yl
tetrahydrofuroyl
pyridin-2-yl
pyridin-3-yl
pyridin-4-yl
pyrimidin-2-yl
pyrazin-2-yl
tetrahydrofuroyl
5196 [3187, 8471]
3654 [2085, 6406]
30.6 [25.2, 37.2]
5405 [2197, 13294]
6212 [3345, 11534]
> 10 000
3
8156 [3913, 16998]
16.0 [8.06, 31.7]
3827 [752, 19484]
8740 [5921, 12902]
> 10 000
4
5
6
7
8
2957 [688, 12720]
3420 [1540, 7598]
37.8 [24.0, 59.4]
883 [221, 3527]
5477 [1034, 29027]
> 10 000
482 [137, 1696]
>10 000
9
10
11
12
13
120 [63, 230]
2264 [787, 6510]
683 [305, 1532]
> 10 000
The effect of compound 10 on histamine- and carbachol-
induced contraction was measured in guinea-pig ileum in order to
assess its modulation for histamine H1R and for muscarinic
receptors, respectively. Compound 10 administrated alone did
not induce ileal contractions, proving no agonist properties.
Compound 10 inhibited contractile response to histamine and
carbachol in concentration-dependent manner (Figure 2).
However, the pKB value calculated for 10 was 6.15 ± 0.06,
indicating a negligible competitive interaction of the compound
with histamine H1 receptors. In the carbachol-induced contraction
the pKB value was 6.39 ± 0.12 revealing also weak antagonistic
properties for muscarinic receptors.
in histamine- and carbachol-stimulated ileum, respectively.
Detailed results are presented in Table 2.
Since compound 10 demonstrated weak antagonistic
properties for histamine H1R and muscarinic receptors in guinea-
pig ileum, it might be assumed that its potential therapeutic effect
is the result of acting through H3R.
2.2.3. Rat dipsogenia model
In order to assess the in vivo functional antagonism/inverse
agonism of compound 10 for centrally located H3R, the (R)-α-
methylhistamine (RAMH)-induced dipsogenia model in rats was
applied. The results showed that treatment with 10 mg/kg i.p.
RAMH caused a significant increase in drinking (3.95 ± 0.54 g)
compared to vehicle-treated animals (0.77 ± 0.22 g; p < 0.0001).
The effect was dose-dependently inhibited by pretreatment with
the investigated compound 10 (Figure 3) with an ED50 value of
Under the same experimental conditions, reference
compounds antazoline (pA2 = 7.14 ± 0.08) and atropine (pA2 =
9.00 ± 0.29) showed significantly stronger antagonistic potency

10.69 mg/kg (95% CI, 4.99 - 22.91 mg/kg). Notably, the
reference compound Pitolisant at the dose of 1.25 mg/kg
inhibited RAMH-induced drinking by 87.4% (p < 0.0001).
ACCEPTED MANUSCRIPT
Table 2. Functional affinities of tested and reference compounds for
different types of receptors expressed in guinea-pig ileum. Antagonist
potency expressed as pA2 ± SEM or pKB ± SEM.
The results observed for compound 10 prove the involvement
and might indicate that compound 10 is capable of penetrating
into the CNS where it reversed the RAMH-induced dipsogenia in
tested rats.
Receptor
Histamine (H1)
receptor
Muscarinic (M)
receptor
2.2.4. Step through passive avoidance task
pA2/pKB ± SEM
pKB = 6.15 ± 0.06
pA2 = 7.14 ± 0.08
-
pA2/pKB ± SEM
pKB = 6.39 ± 0.12
-
In the acquisition trial of the passive avoidance task, neither
scopolamine nor compound 10 significantly affected latency
times. However, significant differences in the retention trial were
observed (Figure 4). Accordingly, scopolamine (1 mg/kg i.p.)
significantly shorted latency times compared to control group
(21.06 ± 4.57 vs. 177.00 ± 2.13; ****p < 0.0001) (Figure 4). The
results showed that the scopolamine-induced memory
impairment was significantly ameliorated by acute systemic
pretreatment with compound 10.
Compound 10
Antazoline
Atropine
pA2 = 8.985 ± 0.29
2.3. Anticonvulsant activity of selected ligands in maximal
electroshock-induced convulsion model
The protective activities of acute systemic administration of
the selected H3R antagonists 4, 10, 16 and 22 on maximal
electroshock seizures (MES)-induced convulsions in mice were
investigated (Figure 5). The results showed that pretreatment
with phenytoin (PHT) and H3R antagonists 4, 10, 16 and 22
(10 mg/kg, i.p.) exhibited a significant protective action against
MES-induced convulsions as confirmed by applying one-way
analysis of variance (F(5,36)= 13.39; p < 0.001). Among the H3R
antagonists tested, compound 10 at a dose of 10 mg/kg showed
significant and the most promising protection in MES-model in
mice when compared with the saline-, 4-, 16-, 22-treated groups
(p < 0.05) (Figure 5). Moreover, the results suggest that animals
pretreated with 5 mg/kg of H3R antagonist 10 might be protected
against convulsions, however, the protective effect was lower and
significant when treated with 10 mg/kg, i.p. of the same
compound 10 (p < 0.05) (Figure 5). Substantial amplifications of
protective effects were observed upon acute systemic injection
with 15 or 20 mg/kg of H3R antagonist 10 when compared with
10 mg/kg with [F(1,12)= 4.88, p < 0.05] and [F(1,12)= 5.34, p <
0.05], respectively, with no significant difference between
15 mg/kg and 20 mg/kg induced protection (p = 0.73) (Figure 5).
Figure 2. Concentration-response curves to histamine (HIS) A and Carbachol
(CCH) B in the guinea-pig ileum in the absence (□) or presence of compound
10 (HIS: ■ 0.3, ▲1, ▼3 µM; CCH: ■ 1 µM). Results are expressed as
percentage of the maximal response to histamine in the first concentration-
response curve. Each point represents the mean ± SEM (n = 4-6).
Interestingly, acute systemic administration (10 and 15 mg/kg,
i.p.) of H3R antagonist 10 exhibited a significant increase in the
latency time in scopolamine-induced memory deficits in passive
avoidance paradigm in mice (Figure 4). Consequently, these
combined results for H3R antagonist 10 indicate that it might be
of potential use as antiepileptic drug with memory-enhancing
effects, considering the limitations of currently available
antiepileptic drugs. However, additional preclinical experiments
are still required to be conducted in a series of different seizure as
well as behavioral test models and with different rodent species
to increase the translational value of observed results for possible
applicability of H3R antagonist 10 in the modulation of memory
impairment in several neurological diseases, e.g. epilepsy.
2.4. Metabolic stability
The metabolic stability of compound 10 was initially
examined using computational tool MetaSite to determine the
potential sites of metabolism [25]. The obtained data suggested
the pyridine moiety and t-butyl substituent as the most probable
sites of 10 metabolism (Figure 6).
Figure 3. Effect of the compound 10 and Pitolisant on RAMH-induced
drinking. Animals were injected i.p. with the investigated compounds at the
indicated doses 30 min prior to treatment with 10 mg/kg RAMH i.p. Results
are given as average ± S.E.M. of 6–8 animals. Statistical significance
compared to RAMH-treated animals: *pꢀ<ꢀ0.05, **p <ꢀ0.01,****p < 0.0001.
Statistical analysis: one-way ANOVA followed by Dunnett's Multiple
Comparison Test
Next, the metabolic stability was evaluated in vitro using
human liver microsomes (HLMs). UPLC-MS analysis of the
reaction mixture after 120 minutes incubation of 10 with HLMs

ACCEPTED MANUSCRIPT
Figure 4. The effect of the compound 10 on learning and memory in the mouse step-
through passive avoidance task. The studied compounds or saline were administered
intraperitoneally to mice, 30 min before the acquisition trial of the step - through passive
avoidance test. Scopolamine was injected subcutaneously. Control groups received vehicle
(Veh; 1% tween 80) and 0.9% NaCl (saline). Statistical analysis: one-way ANOVA
(Tukey’s post hoc), ***pꢀ<ꢀ0.001,****p < 0.0001 vs group treated with vehicle and
scopolamine (0); n = 8–10 mice per group
Figure 5. Protective effect of an intraperitoneal injection of H3R antagonists 4, 10, 16 and 22
on MES-induced seizure in mice. A) shows the protective effects of phenytoin (PHT, 10
mg/kg, i.p.) and test compounds 4, 10, 16, and 22 (10 mg/kg) on the duration of THLE induced
in the MES-model in mice. B) shows dose-dependent protective effects of 10 (5, 10, 15, and 20
mg/kg, i.p.) against MES-induced seizure model in mice. Values are mean ± SEM (n = 7). *p <
#
0.05 vs. the saline-treated group. **p < 0.001 vs. the saline-treated group. p < 0.05 vs. 10 (10
mg, i.p.)-treated group
led to the identification of five metabolites with the following
metabolite M5 was also found, which confirmed very high
susceptibility of compound 10 for reactions of hydroxylation or
oxidation at the t-butyl substituent and at the pyridine moiety. In
summary, compound 10 seems to be a moderately stable
structure with high susceptibility for reactions of hydroxylation
or oxidation. Metabolic pathways result mostly in compound’s
degradation.
molecular masses of their quasimolecular ions [M+H]+: m/z =
239.25 (M1, main metabolite), m/z = 384.33 (M2), m/z = 209.14
(M3), m/z = 253.14 (M4) and m/z = 426.33 (M5). Moreover, as
shown in Figure 7 about 30% of 10 was metabolized in the
presence of HLMs.
The exact ion fragment analysis of 10 (Figure 7) and its
metabolites (Figure 6), supported in silico prediction of most
probable metabolism sites and most probable structures of
metabolites (see supplementary data), and allowed to determine
all metabolism pathways. As shown in Figure 6, the main
metabolite (M1) of 10 was formed by compound’s degradation
followed by double hydroxylation at the aliphatic linker and at
the pyridine moiety. One of the main biotransformation reactions
of 10 includes also the hydroxylation at the pyridine moiety
without compound’s degradation. Metabolites M3 and M4 are
examples of another possibility of degradation, either with
double hydroxylation at the aliphatic linker and at the pyridine
moiety, or with triple hydroxylation. The trace amount of
2.5. Molecular modeling
Among four known and described histamine receptor
subtypes, crystal structure only for the histamine H1 receptor was
resolved up to date (PDB ID: 3RZE [26]). Therefore, due to the
lack of crystal structure, in search for novel histamine H3R
ligands, homology modeling methods are used. In this study a
histamine H3R homology model built on crystal structure of
muscarinic M2 aceylcholine receptor was applied (PDB ID:
3UON [27]). This structure has been chosen as a template, due to
similar sequence identity and slightly higher crystal structure
resolution. Also this model showed higher degree of active/non-
active ligands docking differentiation (data not shown). As a

All docked compounds were characterized by relatively high
reference ligand, Pitolisant was placed inAbCinCdinEgPpToEckDet, MANUSCRIPT
docking score values between -7.828 and -4.464. Both, the
Figure 6. MetaSite prediction of the most probable metabolism sites for compoud 10 (highest probability site was marked in blue, other
probable sites were marked in red. The darker the red the higher probability to be involved in the metabolism pathway; top
panel) and most probable structures of metabolites M1-M5 based on in silico prediction and MS fragmentation analysis (panels 2-6,
downwards)
according to data described in literature [28].
highest and lowest values were observed for t-amyl derivatives.
For most of the compounds, a supposedly key histamine H3

receptor antagonist/inverse agonist interactions, i.e. salt bridge
direction, yet still were surrounded by hydrophobic amino acids
ACCEPTED MANUSCRIPT
and/or hydrogen bond formation between protonated piperazine
W1103.28, TYR 189 (ECL2) and TYR 3947.35
.
nitrogen and GLU2065.46 were found (upper case numeration
3. Conclusions
Figure 7. The UPLC spectrum after 2 h reaction of 10 with HLMs (upper panel) and MS fragmentation analysis
of 10 (lower panel)
according to Ballesteros-Weinstein [27]).
A
novel series of tert-butyl and tert-pentyl phenoxy
alkylamine derivatives investigated in the present study proved to
be potent histamine H3R ligands with the H3R affinities in the
nanomolar concentration range. Analyzing the basic fragment of
the compounds, 4-pyridyl-containing derivatives 4, 10, 16 and 22
occurred to show the highest H3R affinity (Ki = 16.4–120 nM)
whilst tetrahydrofuroyl derivatives showed no H3R affinity.
Docking studies to histamine H3R homology model allowed the
observation of previously proposed ligand-receptor interactions.
Moreover, best active compounds were evaluated for
anticonvulsant activity at maximal electroshock (MES) test. The
H3R antagonist 10 provided the most encouraging protection
against MES-induced convulsions. Functional characterization of
compound 10 suggested its potential therapeutic effect through
H3R only. In the RAMH-induced dipsogenia model the blood-
brain barrier penetrating and antagonistic/inverse agonistic
properties of compound 10 for centrally located histamine H3
receptor could be demonstrated. Additional, pro-cognitive
properties of 10 were confirmed in the passive avoidance test,
where this H3R antagonist significantly diminished memory
impairment induced by scopolamine. In order to estimate its
“drug-likeness”, in silico and experimental evaluation of
metabolic stability was performed. Consequently, compound 10
was chosen as a new, promising lead structure and therefore
proceeded to further studies, including i.a. pharmacokinetics
profile, proposed metabolites synthesis and their pharmacological
evaluation. Therefore, with respect to promising biological
activity and docking results of described herein ligands, further
directions of research include modification of the “eastern part”
of the molecule (e.g. introduction of bulky aromatic groups such
as naphthyl, biphenyl, isolated functional groups) in order to test,
whether larger substituents are also tolerated. Besides,
Moreover, for compounds with alkyl chain composed of four
methylene groups, previously described H-bond formation
between TYR3746.51 and the ether oxygen was observed.
Additionally, the heteroaromatic N1 piperazine nitrogen
substituents were stabilized through π-π interactions with indole
and/or benzene ring of TRP 3716.48 (Figure 8). In fact, this amino
acid is one of the so-called molecular switches [30], highly
conserved residues for which the change of orientation causes
a change in the conformation of the receptor. Such interactions
were not observed for the non-affine tetrahydrofuroyl piperazine
derivatives, due to their electron structure, as well as due to the
fact, that in most cases tetrahydrofuroyl moiety was 90 degrees
bend and placed in T-manner in relation to pyridyl moieties. Also
for the most active 1-(4-pyridyl)piperazine derivatives in this
series (e.g. compounds 10 and 22 – Figure 9), an additional
stabilization through π-π stacking between heteroaromatic moiety
and another molecular switch, conserved PHE2075.47 was
observed. A hydrogen bond formation between 1-(4-pyridyl)
nitrogen and conserved ASN4047.45 for compound 4 was also
found, although this resulted in loos of the bond formation with
GLU2065.46 and might be considered as a docking artifact. Those
stabilizing interactions, not seen for other derivatives might be
one of the reasons of highest histamine H3 receptor affinities in
this subgroup of compounds.
On the other hand, benzene rings of the lipophilic part of
ligands were stabilized through π-π stacking with at least one of
amino acids: TYR 1153.33, TYR 3947.35 , PHE 193 (ECL2).
Although, with the elongation of the alkyl chain, the position of
basic parts of the ligands was fixed, t-butyl and t-pentyl
substituents reached progressively higher in the extracellular

4.1.1. General synthetic procedure for compounds 1a–1d
subsequent extension of alkyl linker in theAraCngCe EofPT5 EtoD7 MANUSCRIPT
methylene groups is also planned. Moreover, since metabolic
stability has shown the pyridyl-piperazine scaffold as a labile
part, further modifications regarding this part of structures are
also considered.
Starting compounds were obtained according the procedure
described in Ref. [18,19]. Analytical data of compounds 1a–1e
could be also found in abovementioned references
To a solution of freshly prepared sodium propylate, proper
substituted phenols were added and stirred at room temperature
for 5 min. α,ω-Dibromoalkanes were then added dropwise in the
time of 1 h. The reaction mixture was stirred at 60°C for 3 h, and
Finally, based on the results obtained within this study, the 4-
pyridyl-piperazino moiety has been established as a new
bioisoteric piperidine replacement in H3R ligands.
Figure 8. Ligand interaction diagrams for compounds 3, 10, 19 (left to right)
then refluxed for another 3 h. After cooling down to RT, mixture
was filtrated and evaporated. To a rough product, 100 ml of 10%
NaOH was added and left overnight in cold. To a resulting white
oil CH2Cl2 was added, mixed and layers were then separated.
Organic layer was dried over sodium sulphate, filtered and
evaporated. Resulting product was used for further reactions after
CC purification.
4. Experimental protocols
4.1. Chemistry
All reagents were purchased from commercial suppliers and
were used without further purification. Melting points (m.p.)
were determined on a MEL-TEMP melting point apparatus II
(LD Inc., USA) and are uncorrected. Mass spectra (LC/MS) were
1
4.1.1.1. 1-(3-Bromopropoxy)-4-(tert-butyl)benzene (1a).
performed on Waters TQ Detector mass spectrometer. H NMR
spectra were recorded on a Varian Mercury 300 MHz PFG
spectrometer in DMSO-d6. Chemical shifts were expressed in
parts per million (ppm) using the solvent signal as an internal
standard. Data are reported in the following order: multiplicity (s,
singlet; d, dublet; t, triplet; qi, quintet; m, multiplet; br, broad; t-
b, tert-butyl; t-p, tert-pentyl; Ph, phenyl; Pip, piperazine; Pyr,
pyridine; Pym, pyrimidine; Pyz, pyrazine; Thf, tetrahydrofuran),
approximate coupling constants J expressed in Hertz (Hz),
number of protons. 13C NMR spectra were recorded on Varian-
Mercury-VX 300 MHz PFG or Brukker 400 MHz spectrometer
at 75 MHz in DMSO-d6. LC–MS were carried out on a system
consisting of a Waters Acquity UPLC, coupled to a Waters TQD
mass spectrometer. Retention times (tR) are given in minutes.
Elemental analyses (C, H, N) were performed on an Elemental
AnalyserVario El III (Hanau, Germany) and agreed with
theoretical values within ± 0.4%. For CC purification (column
chromatography using silica gel 60 (0.063–0.20 mm; Merck)
solvent systems were used: I: Petroleum ether:EtOAc (9:1); II:
Synthesis from tert-butylphenol (15.02 g, 0.1 mol), 1,3-
dibromobutane (40.38 g, 0.2 mol) in sodium propylate (0.1 mol,
100 ml). Obtained 21 g of raw product, purified by CC (I). Yield
78%.
4.1.1.2. 1-(4-Bromobutoxy)-4-(tert-butyl)benzene (1b).
Synthesis from tert-butylphenol (15.02 g, 0.1 mol), 1,4-
dibromobutane (42.78 g, 0.2 mol) in sodium propylate (0.1 mol,
100 ml). Obtained 19.68 g of raw product, purified by CC (I).
Yield 69%.
4.1.1.3. 1-(3-Bromopropoxy)-4-(tert-pentyl)benzene (1c).
Synthesis from tert-pentylphenol (16.43 g, 0.1 mol), 1,3-
dibromobutane (40.38 g, 0.2 mol) in sodium propylate (0.1 mol,
100 ml). Obtained 23.39 g of raw product, purified by CC (I).
Yield 82%.
Figure 9. Compound 22 in histamine H3 homologue receptor binding pocket (left) and its ligand-interaction diagram (right)
4.1.1.4. 1-(4-Bromobutoxy)-4-(tert-pentyl)benzene (1d).
CH2Cl2:MeOH (9:1).

4.2.2. In vivo studies
4.2.2.1. Animals (functional bioassay, rat dipsogenia model and
passive avoidance test)
Synthesis from tert-pentylphenol (16.43 g, 0.1 mol), 1,4-
dibromobutane (42.78g, 0.2 mol) in sodium propylate (0.1 mol,
100 ml). Obtained 23.34 g of raw product, purified by CC (I).
Yield 78%.
ACCEPTED MANUSCRIPT
The experiments were carried out on adult male Albino Swiss
mice (CD-1, 18–25 g), male Wistar rats (Krf:(WI) WU), 180 –
250 g) and male guinea-pigs (Outbred CV, 300-400 g). Animals
were housed in plastic cages in room at a constant temperature of
20 ± 2oC, under light/dark (12:12) cycle and had free access to
standard pellet diet and water. Experimental groups consisted of
6–12 animals, all of the animals were used only once and were
killed by cervical dislocation immediately after the assay.
Behavioural measures were scored by trained observers, who
were blind to experimental conditions. Treatment of laboratory
animals in the present study was in full accordance with the
respective Polish regulations. All procedures were conducted
according to guidelines of ICLAS (International Council on
Laboratory Animal Science) and approved by the Local Ethics
Committee on Animal Experimentation (ZI/105/2016).
4.1.2. General synthetic procedure for compounds 2–25
To a suspension of potassium carbonate and catalytic amount
of potassium iodide in water, a mixture of proper cyclic amine
and compound 1a–1d in ethanol was added. Mixture was then
refluxed for 8–20 h (TLC controlled). After cooling down to
room temperature, reaction mixture was filtrated, evaporated and
purified. To a resulting oil, 100 ml of CH2Cl2 was added and
washed with: 0.5% HCl solution, 0.5% NaOH solution and
water. After drying over anhydrous Na2SO4 and evaporation of
organic layer product was further purified using CC (II).
Detailed synthetic procedure and analytical data for
compounds 2–25 could be found in Supplementary Data.
4.1.2.1. 1-(3-(4-(tert-Butyl)phenoxy)propyl)-4-(pyridin-2-
yl)piperazine (2).
4.2.2.2. Animals (anticonvulsant activity)
Synthesis from 1-(pyridin-2-yl)piperazine (0.82 g, 5 mmol), and
compound 1a (1.36 g, 5 mmol) in ethanol (50 ml) in the presence
of K2CO3 (1.17 g, 8.5 mmol) and catalytic amount of KI in water
(10 ml). Refluxed for 20 h and purified. Obtained 0.97 g of oil.
Yield 55%. Raw product was transformed into oxalic acid salt
Inbred adult (age of 8-10 weeks) male Tuck-Ordinary “TO”
mice and weighing 30-35 g (Harlan, UK) were used in the
current study [32-34]. The mice were group-housed in standard
Plexiglas observation cages in the central animal facility of the
College of Medicine and Health Sciences on a 12-h light/dark
cycle (lights on at 6:00 a.m.). Food and water were available ad
libitum. The experiments of the current study were carried out
between 9:00 and 12:00 h, and all procedures were performed
according to the guidelines of the European Communities
Council Directive of 24 November 1986 (86/609/EEC) and were
previously approved for epilepsy study by the College of
Medicine and Health Sciences/United Arab Emirates University
(Institutional Animal Ethics Committee, approval number; ERA-
2017-5535).
1
yielding 0.59 g of final compound. Mp: 168–169°C. H NMR
(300 MHz, DMSO-d6) δ ppm: 8.13 (dd, J=4.69, 1.17 Hz, 1H,
2Pyr-6H), 7.52-7.60 (m, 1H, 2Pyr-4H), 7.27 (d, J=8.79 Hz, 2H,
Ph-3,5H), 6.90 (d, J=8.79 Hz, 1H, 2Pyr-3H), 6.84 (d, J=8.79 Hz,
2H, Ph-2,6H), 6.69 (dd, J=6.74, 4.98 Hz, 1H, 2Pyr-5H), 3.99 (t,
J=5.86 Hz, 2H, CH2-O), 3.67 (br. s., 4H, Pip-3,5H), 2.98-3.11
(m, 6H, Pip-2,6H + NCH2), 2.01-2.12 (m, 2H, CH2-CH2-O), 1.23
(s, 9H, t-a). 13C NMR (DMSO-d6): 164.69, 158.65, 156.51,
148.03, 143.30, 138.29, 126.52, 114.38, 108.01, 65.48, 53.89,
51.39, 42.89, 34.21, 31.78, 24.44. LC–MS: purity 100% tR
=
5.06, (ESI) m/z [M+H]+ 355.48. Anal. Calcd for C22H31N3O x
C2H2O4: C, 64.99, N, 9.47, H, 7.50%. Found: C, 64.95, N, 9.51,
H, 7.52.
4.2.2.3. Drugs and chemicals
For behavioural experiments, investigated compounds and
reference compound were suspended in 1% aqueous solution of
Tween 80 and administered by the intraperitoneal (i.p.) route
30 min before the test. Control animals (negative control) were
given an appropriate amount of vehicle (1% aqueous solution of
Tween 80; i.p.) 30 min before the test.
4.2. Pharmacology
4.2.1. [3H]Nα-Methylhistamine hH3R displacement assay
Competition
binding
data
were
analyzed
using
GraphPadPrism (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 [31].
Affinity values (Ki) were expressed as mean (with 95%
confidence interval) from at least three experiments, each
performed at least in duplicates .
The following drugs and reagents were used: antazoline
(Sigma-Aldrich, Germany), atropine hydrochloride (Sigma-
Aldrich, Germany), carbachol (Sigma-Aldrich, Germany),
histamine hydrochloride (Sigma-Aldrich, Germany), pitolisant
(synthesized in Department of Technology and Biotechnology of
Drugs, JUMC). Other chemicals used in Krebs solution were
obtained from POCH (Polish Chemical Reagents, Poland).
The displacement binding assay was carried out as described
by Kottke et al. with slight modifications [22]. Frozen crude
membrane preparations of HEK-293 cells stably expressing the
recombinant hH3R in full length were thawed, homogenized,
incubated for 90 min at RT (continuous shaking) with [3H]Nα-
methylhistamine (2 nM) and different concentrations of the test
compounds (five to eleven appropriate concentrations between
0.01 nM and 10 µM were used) in a final assay volume of 200 µl
per well. Non-specific binding was determined in the presence of
Pitolisant (10 µM). The bound radioligand was separated from
free radioligand by filtration through GF/B filters (pretreated
with 0.3% (m/v) polyethyleneimine) using an Inotech cell
harvester (Dottikin, Switzerland). Unbound radioligand was
removed by three washing steps with 0.3 ml/well of ice-cold
water. Scintillation cocktail was added and the liquid scintillation
counting was performed with a Perkin Elmer TriluxBeta counter
(Perkin Elmer, Germany).
Phenytoin (PHT) was purchased from Sigma-Aldrich (St
Louis, Missouri, USA). For the in vivo anticonvulsant studies in
MES model, PHT and H3R antagonists 4, 10, 16 and 22 were
dissolved in isotonic saline and administered intraperitoneally
(i.p.)
at
a volume of 1 ml/kg. Doses of all test compounds were expressed
in terms of the free base. For each test compound, a group of
seven animals was used for the anticonvulsant study.
4.2.2.4. Functional bioassay
Isolated guinea-pig ileum was employed to test antagonistic
activity of the investigated compounds for histamine H1 receptors
and muscarinic (M) receptors. A 15 cm ileum segment was
excised from the small intestine of male guinea-pigs and

immersed into a Krebs solution (NaCl 120 mM, KCl 5.6 mM,
7.5 cm × 14 cm, 10 lx) equipped with an electric grid floor
(stainless steel rods through which an electric footshock is
delivered). After 30 s the door to the smaller compartment was
opened. Immediately after the mouse entered the smaller dark
compartment, the door closed and the mouse was punished by an
unavoidable electric foot shock (0.2 mA for 2 s). The mice,
which did not enter the dark compartment within 60 s were
excluded from the study. On the following day (24 h later) the
retention session was carried out. The pre-trained animals were
placed again into the illuminated compartment and observed up
to 180 s. In the retention session, mice did not receive the electric
shock after the entrance to the dark compartment. Mice, which
avoided the dark compartment for 180 s were considered to
remember the task.
ACCEPTED MANUSCRIPT
MgCl2 2.2 mM, CaCl2 2.4 mM, NaHCO3 19 mM, glucose 10
mM). After the first 5 cm length closest to the ileo–caecal
junction had been discarded, 2 cm-long fragments were cut. Each
segment of the ileum was placed in 20 ml chamber of tissue
organ bath system (Tissue Organ Bath System – 750 TOBS,
DMT, Denmark) filled with the Krebs solution at 37°C, pH 7.4,
with constant oxygenation (O2/CO2, 19:1), stretched by means of
closing clips between the metal rod and the force-displacement
transducer. The preparations were allowed to stabilize in organ
baths for 60 min under a resting tension of 0.5 g, washing every
15 min with fresh Krebs solution. After the equilibration period a
cumulative concentration-response curve was constructed for
agonists such as histamine (10 nM – 10 µM) or carbachol (3 nM
– 3 µM). Following the first curve, tissues were incubated with
one of the concentrations of tested compounds for 15 min and the
next cumulative concentration curve to the agonist was obtained.
Only one concentration of the potential antagonist was tested in
each piece of tissue. The experiment was repeated four to eight
times [35].
Data are presented as means ± standard error of the mean
(SEM) and were analysed using GraphPad Prism Software (v.6).
Statistically significant differences between groups were
calculated using one-way analysis of variance (ANOVA) and the
post-hoc Dunnett's or Tukey's multiple comparison test when
appropriate. The criterion for significance was set at p < 0.05.
The log-probit method was applied to statistically determine the
ED50 values, which are accompanied by their respective 95%
confidence limits.
Contractile responses to the stimulation by the agonists (in the
presence or absence of tested compounds) are expressed as
a percentage of maximal histamine or carbachol effect (Emax
=
100%), reached in the concentration-response curves obtained
before incubation with the tested compounds. Data is expressed
by the means ± SEM of at least three separate experiments.
Schild analysis was performed, and when the slope was not
significantly different from unity, the pA2 value was determined.
When the slope was significantly different from unity or when
only one concentration of the tested compound was used, the
affinity was estimated with the equation pKB = log(concentration
ratio–1)-log(molar antagonist concentration), where the
concentration ratio is the ratio of equi-effective agonist
concentrations in the absence and in the presence of the
antagonist [36].
4.2.2.7. Maximal electroshock (MES)-induced seizure
As previously described, [40-47] the convulsions were
induced in TO mice with a 50-Hz alternating current of 120 mA
intensity. The current was applied through ear electrodes for 1 s.
Protection against the spread of MES-induced convulsion was
defined as the abolition of the tonic hind limb extension (THLE)
component of the convulsion [41,44,37-38]. The animals were
divided into groups of seven mice. In the positive control group,
mice were injected with PHT at a dose of 10 mg/kg, this being
the minimal dose of PHT that protected animals against the
spread of MES-induced convulsions without mortality in mice.
Test compounds (10 mg/kg, i.p.) were administered to the
animals in the experimental groups 30-45 min before the MES
challenge. In a further experiment, the most promising H3R
antagonist 10 was selected for further analysis. In three separate
groups of seven mice, different doses (5, 15 and 20 mg/kg, i.p.)
were injected 30-45 min before the MES challenge and were
tested for dose-dependency of protection provided with test
compound 10 at 10 mg/kg, i.p. In regard to the MES test, the
current used in the study was according to previously published
protocols and it produced convulsions in 100% of animals
without mortality [46,47,49,50].
4.2.2.5. Rat dipsogenia model
The procedure used was essentially the same as that described
previously [37,38]. The experiment was carried out on male
Wistar rats (Krf:(WI) WU), < 250 g). The water intake was
induced by the intraperitoneal (i.p.) administration of R-α-
methylhistamine (RAMH) at the dose of 10 mg/kg and measured
for 30 min, starting 20 min after RAMH administration. The
investigated compound, pitolisant or vehicle (1% Tween 80
solution) were injected (i.p.) 30 min before RAMH
administration. Inhibition of RAMH-induced drinking was
calculated using the following equation: [100 – (Dr/DgRAMH) ×
100]; where Dr = the amount of water that individual rat drank;
DgRAMH = the amount of water consumed by the RAMH-treated
group. Statistically significant differences between groups were
calculated using one-way analysis of variance (ANOVA) and the
post-hoc Dunnett's multiple comparison test using GraphPad
Prism Software (v.6). The criterion for significance was set at
p<0.05. The log-probit method was applied to statistically
determine the ED50 values, which are accompanied by their
respective 95% confidence limits.
For statistical comparisons, the software package SPSS 24.0
(IBM Middle East, Dubai, UAE) was used. All data were
expressed as the means ± standard error of mean (SEM). The
effects of H3R antagonists 4, 10, 16 and 22 on duration of THLE
induced in male adult mice were analyzed using one-way
analysis of variance, followed by the Bonferroni post hoc test for
multiple comparisons. In all the tests, criterion for statistical
significance was p < 0.05.
4.2.2.8. Metabolic stability
4.2.2.6. Step-through passive avoidance task
In vitro metabolic stability of 10 was determined as described
previously [51]. In silico study on compound 10 metabolism was
performed using MetaSite 5.1.1 provided by Molecular
Discovery Ltd. The sites with highest metabolism probability, as
well as prediction of metabolites’ structures were analyzed by
a computational liver model.
Step-through passive avoidance task was performed according
the previously described method [39]. The apparatus for step-
through passive avoidance task consisted of two compartments,
separated by an automated sliding door (LE872, Bioseb, France).
For acquisition session, mice were placed individually in an
illuminated white compartment (20 cm × 21 cm × 20 cm, 1000
lx) with the closed door to a smaller dark compartment (7.3 cm ×
4.2.2.9. Molecular modeling

[13] Sadek B, Łażewska D, Hagenow S, Kieć-Kononowicz K, Stark H
For docking purposes, Schrödinger
Suite (v.
MAaesCtrCo EPTED MANUSCRIPT
(2016) Histamine H3R Antagonists: From Scaffold Hopping to Clinical
Candidates. In: Blandina P., Passani M. (eds) Histamine Receptors. The
Receptors, Vol. 28. Humana Press, Cham. DOI 10.1007/978-3-319-
40308-3_5.
11.1.012) was used [52]. Ligands were built in their ionized
forms (protonated N4 piperazine nitrogen, structure charge +1)
and their bioactive conformations were generated using ConfGen
module [40,41] (water environment, target number of conformers
– 20). Binding site was centered on ligand placed in homology
model (Pitolisant). Docking to rigid form of receptor was
performed using Glide module [53-57] (precision standard,
flexible ligand sampling, max 5 poses per conformer). Ligands
were rated according 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 [58].
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Zsilavecz M, Schwartz JC, Stark H. Acidic elements in histamine H3
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5. Acknowledgements
[18] Łażewska D: The search for non-imidazole histamine H3 receptor
ligands. PhD Thesis, Jagiellonian University Medical College, Kraków,
2004.
[19] Łażewska D, Ligneau X, Schwartz JC, Schunack W, Stark H, Kieć-
Kononowicz K. Ether derivatives of 3-piperidinopropan-1-ol as non-
imidazole histamine H3 receptor antagonists. Bioorg Med Chem. 2006;
We thank K. Grau and J. S. Schwed for excellent technical
assistance. We are pleased to acknowledge the generous support
of National Science Center, Poland granted on the basis of
decision
No.
2016/23/B/NZ7/01063,
K/DSC/004310,
K/ZDS/007131 and K/ZDS/007121. Also, support provided to
BS from intermural research grants sponsored by the Research
Office of United Arab Emirates University is acknowledged.
Support by DFG INST 208/664-1 (HS) and COST CA15135
(HS, KK) is also acknowledged.
14: 3522-3529.
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ACCEPTED MANUSCRIPT
Highlights:
•
Synthesis of 4N-(hetero)(aryl)substituted piperazine alkyl ethers was performed
•
•
Human histamine H3 receptor affinity was estimeted
The most promising compounds exhibited anticonvulsant activity in the maximal
electroshock-induced seizure (MES) model in mice
•
•
•
BBB penetration, functional H3R antagonist potency and the pro-cognitive properties
for most promising compound was demonstrated.
As well as its in silico and experimental tests of metabolic stability in human liver
microsomes was performed.
4-pyridyl-piperazino moiety has been established as a new bioisoteric piperidine
replacement in H3R ligands