Optimization and preclinical evaluation of novel histamine H3 receptor ligands: Acetyl and propionyl phenoxyalkyl piperazine derivatives

Article abstract of DOI:10.1016/j.bmc.2018.11.010

As a continuation of our search for novel histamine H₃ receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H₃ receptors (hH₃R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH₃R affinity (hH₃R K_i = 5.2–115 nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22 vs. 4 and 10; hH₃R K_i = 5.2 and 15.4 nM vs. 10.2 and 115 nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.

Full text of DOI:10.1016/j.bmc.2018.11.010

Accepted Manuscript
Optimization and preclinical evaluation of novel histamine H3receptor ligands:
acetyl and propionyl phenoxyalkyl piperazine derivatives
Katarzyna Szczepańska, Tadeusz Karcz, Magdalena Kotańska, Agata Siwek,
Kamil J. Kuder, Gniewomir Latacz, Szczepan Mogilski, Stefanie Hagenow,
Annamaria Lubelska, Michał Sobolewski, Holger Stark, Katarzyna Kieć-
Kononowicz
PII:
S0968-0896(18)31528-1
https://doi.org/10.1016/j.bmc.2018.11.010
BMC 14614
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
29 August 2018
26 October 2018
9 November 2018
Please cite this article as: Szczepańska, K., Karcz, T., Kotańska, M., Siwek, A., Kuder, K.J., Latacz, G., Mogilski,
S., Hagenow, S., Lubelska, A., Sobolewski, M., Stark, H., Kieć-Kononowicz, K., Optimization and preclinical
evaluation of novel histamine H3receptor ligands: acetyl and propionyl phenoxyalkyl piperazine derivatives,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.11.010
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Bioorganic & Medicinal Chemistry
Optimization and preclinical evaluation of novel histamine H3 receptor ligands: acetyl
and propionyl phenoxyalkyl piperazine derivatives
Katarzyna Szczepańska a, Tadeusz Karcz a, Magdalena Kotańska b, Agata Siwek c, Kamil J. Kuder a,
Gniewomir Latacz a, Szczepan Mogilski b, Stefanie Hagenow d, Annamaria Lubelska a, Michał Sobolewski
a, 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 Duesseldorf, Germany
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 new acetyl
and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to
four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine
moieties were evaluated for their binding properties at human histamine H3 receptors (hH3R). In
vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH3R affinity
(hH3R Ki = 5.2 – 115 nM). Moreover introduction of carbonyl group containing residues in the
lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity
in correlation to previously described series, whereas propionyl derivatives showed slightly
higher affinities than those of acetyl (16 and 22 vs. 4 and 10; hH3R Ki = 5.2 and 15.4 nM vs. 10.2
and 115 nM, respectively). These findings were confirmed by molecular modelling studies,
demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test
results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus
act as an anorectic compound. Likewise, its protective action against hyperglycemia and the
development of overweight has been shown. In order to estimate drug-likeness of compound 4,
in silico and experimental evaluation of metabolic stability in human liver microsomes was
performed.
Available online
Keywords:
Histamine H3 receptor
Histamine H3 receptor ligands
Non-imidazole histamine H3R ligands
Piperazine derivatives
Anti-obesity agents
Molecular docking
Metabolic stability
2009 Elsevier Ltd. All rights reserved.
1. Introduction
synthesized both, by academic and pharmaceutical company
researchers [10-12]. From the very beginning, structures were
The histamine H3 receptor (H3R), belonging to the
superfamily of G-Protein coupled receptors (GPCRs), is
predominantly expressed in the central nervous system [1-3].
Since its discovery by Arrang et al. [4] it has been related to
several central nervous system diseases by playing a key role as
modulator of neurotransmitter release of e.g. dopamine,
acetylcholine, noradrenaline or serotonin [1-3]. Therefore, a huge
number of H3R antagonists have been investigated on their
potential therapeutic applicability in obesity, depression, mood
disorders, neuropathic pain, and sleep-wake disorders (including
narcolepsy) as well as cognitive and CNS-linked sensorimotor
deficit disorders. Those include Parkinson’s disease, attention
deficit hyperactivity disorder (ADHD), Alzheimer’s disease,
schizophrenia, alcohol addiction, energy homeostasis, epilepsy,
obstructive sleep apnea, diabetic neuropathic pain, Tourette’s
syndrome and catalepsy [1,5-9].
mostly based on imidazole moiety and a number of such have
found utility as pharmacological tools [13]. For this early
generation of H3R antagonists, it later appeared, that due to
several possible pharmacokinetic (PK) liabilities like CYP450
inhibition, low brain penetration, incidence of off-target activity
or lack of subtype selectivity (especially over H4R) [6], such
compounds might not serve as further clinical candidates.
Therefore, the imidazole ring replacement with other heterocyclic
moieties was a milestone in the search for new histamine H3R
ligands. A piperazine moiety is such a replacement, being a
significant versatile scaffold in rational drug design for most of
the GPCR ligands. The two secondary nitrogen atoms within the
piperazine core, have an appropriate pKa to exert the
improvement in pharmacokinetic features of novel drug
candidates [14]. These nitrogen sites lead to the essential increase
in water solubility of the drug-like molecules, thereby playing a
crucial role in bioavailability of the compounds. Maintaining the
balance between pharmacodynamic and pharmacokinetic profiles
In recent years many potent and selective anti-H3R ligands
(either H3R antagonists or inverse agonists) have been

of drug-like molecules is one of the key factors in new drugs
design and development. To design molecules with high affinity
to their targets is one of the goals in contemporary drug
discovery process. Appropriate physicochemical properties of the
piperazine template makes this molecular subunit a useful and
well-positioned system in the rational drug design [15].
emerging during synthesis, were then coupled with
corresponding N-mono-substituted piperazines 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.
2.2. Pharmacology
Paying attention to the results obtained in our previous study,
the 4-pyridyl-piperazino moiety has been established as a new
bioisosteric piperidine replacement in H3R ligands [16].
A position of nitrogen atom in an aromatic ring attached to
piperazine moiety has turned out to be a key structural element
for suitable interaction with its biological target. The most active
compounds are shown in the Figure 1.
2.2.1. Histamine H3 receptor affinity
All compounds in their oxalate forms were tested in H3R in
vitro binding studies, using slightly modified methods to those
described previously [20]. 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.
In order to determine the influence of substituents located in
the „eastern part” of the molecule, structural modifications of
previously obtained compounds including replacement of
branched alkyl benzene substituents, with either acetyl or
propionyl groups, were undertaken (Figure 1). Such carbonyl
consisting moieties, present in various active compounds, e.g.
Ciproxifan, might serve as additional H-bond acceptors to
increase affinity at the desired target. On the other hand, our
previous findings suggested that slight structural modifications
involving nitrogen position change in the basic part of molecule
might highly influence H3R ligands affinity. Therefore in this
study we decided to confirm these findings on structurally related
compounds.
In vitro affinity data are assembled in Table 1. According to
our assumptions, 4-pyridyl derivatives 4, 10, 16 and 22 showed
the highest affinity among tested compounds, much higher than
in our previous study (with exception of compound 10 vs.
previously described t-butyl analogue; hH3R Ki = 115 nM vs.
37.8 nM respectively), indicating the importance of lipophilic
part involvement in interaction with the H3R binding site. Among
4-pyridyl derivatives, compound 16 (hH3R Ki = 5.2 nM) is the
most active one described in this work. In this subgroup of
compounds, affinities tend to be higher for propionyl compared
to acetyl derivatives (16 and 22 vs. 4 and 10; hH3R Ki = 5.2 and
15.4 nM vs. 10.2 and 115 nM, respectively), however, lacking
statistical significance.
It has been shown in recent studies, that Pitolisant , an H3R
inverse agonist, might reduce body weight in obese mice [17]. To
determine potential anti-obesity properties of selected
compounds, preliminary pharmacological in vivo tests have been
performed. In these studies, the influence on body weight, food
and water intake, glucose and triglyceride plasma levels, and
spontaneous activity on rats in the model of excessive eating of
preferential feed was tested.
3-Pyridyl piperazine derivatives appear to be similarity active
than 2-pyridyl derivatives, with the exception of compounds 8 vs.
9, where 3-pyridyl derivative 9 was three orders of magnitude
more potent than its 2-pyridyl analogue 8. Moreover, in case of
dihetero aromatic substituents, no significant difference in
antagonistic properties between 2-(1-piperazinyl)pyrazine and 2-
piperazinopyrimidine derivatives was observed. Comparable to
our previous results, tetrahydrofuroyl derivatives (7, 13, 19, 25)
showed no H3R affinity.
2. Results and discussion
2.1. Chemistry
Paying attention to the alkyl linker length, derivatives with
a four methylene linker tend to be less active than their three
methylene homologues, with the exception of compounds 5 vs.
11 and 6 vs. 12 (hH3R Ki = 2316 vs. 1230 nM and 2453 vs. 1634
nM, respectively).
The synthesis of desired ether derivatives 2–25 was achieved
through the efficient synthetic route outlined in Scheme 1. Para
acetyl and propionyl phenoxyalkyl bromides (1a–1d, synthesis
according [18], modified by authors [16]) were obtained in one-
step alkylation of para hydroxyaceto- and hydroxypropiophenons
with α,ω-dibromoalkanes refluxed in propan-1-ol [19]. Obtained
precursor bromides, after the separation from bis products
2.2.2. Locomotor activity
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).
No.
n
R
hH3R Ki nM [CI 95%]
No.
n
R
hH3R Ki nM [CI 95%]
2
1
1
1
1
1
1
2
2
2
2
2
2
pyridin-2-yl
315 [132, 754]
499 [261, 953]
10.2 [3.6, 29.0]
2316 [940, 5707]
2453 [461, 13047]
4889 [800, 29862]
3697 [2563, 5332]
908 [230, 3582]
115 [26.8, 493]
1230 [377, 4021]
1634 [619, 4318]
> 10000
14
15
16
17
18
19
20
21
22
23
24
25
1
1
1
1
1
1
2
2
2
2
2
2
pyridin-2-yl
377 [116, 1226]
192 [42.4, 871]
5.2 [0.7, 41.5]
3
pyridin-3-yl
pyridin-3-yl
4
pyridin-4-yl
pyridin-4-yl
5
pyrazin-2-yl
pyrimidin-2-yl
tetrahydrofuroyl
pyridin-2-yl
pyrazin-2-yl
pyrimidin-2-yl
tetrahydrofuroyl
pyridin-2-yl
3390 [1528, 7521]
1720 [802, 3687]
1392 [470, 4128]
1444 [172, 12101]
1392 [264, 7346]
15.4 [0.6, 429]
6
7
8
9
pyridin-3-yl
pyridin-3-yl
10
11
12
13
pyridin-4-yl
pyridin-4-yl
pyrazin-2-yl
pyrimidin-2-yl
tetrahydrofuroyl
pyrazin-2-yl
pyrimidin-2-yl
tetrahydrofuroyl
4040 [1099, 14850]
3627 [736, 17879]
> 10000
In order to determine the influence on locomotor activity,
compounds were tested in two doses 3 and 5 mg/kg b.w. The
results showed that single administration of compounds 4 and 16
(5 mg/kg b.w., i.p.) caused a significant decrease in locomotor
activity when compared to vehicle-treated animals (Figure 2).
However, there were no significant changes in activity at dose of
3 mg/kg b.w. Disorders of spontaneous activity (significant
increase in activity as well as sedation) induced by the test
compound could disturb the assessment of the test compound
impact on body weight. Therefore, a dose of 3 mg/kg b.w. for
chronic tests was chosen.
than rats from the control group consuming a preferential feed.
From the 9th day of the experiment, a statistically significant
difference in body weight between the groups was observed (e.g.
day 9 with 8.64% vs. day 14 with 7.89%). Most importantly, the
body weight of rats with access to preferential feed and treated
with compound 4 treatment, did not differ significantly from that
of the control group fed with a standard feed (Figure 3A). There
was no significant decrease in weight gain when compound 4
was administrated at dose 1 mg/kg b.w./day. The weight of these
animals grew in a similar manner to animals consuming
preferential feed and receiving a vehicle.
2.2.3. Body weight and food intake in rats fed with
palatable diet
The animals fed with palatable feed and receiving compound
4 (3 mg/kg b.w. per day) showed significantly less weight gain
Compared to control group consuming a preferential feed, the
rats fed with palatable feed and treated with compound 16 (3
mg/kg b.w. per day) showed no significant changes in weight
gain. Animals from the test group gained weight similarly to
animals from the control group during the experiment (Figure
3B).
Compound 4 administered intraperitoneally at the dose of 3
mg/kg b.w. per day significantly reduced the amount of
consumed calories by the animals on a palatable diet in
comparison to the ones of the control group (Figure 4).
Compound 4 acts much better in conducted research, although
both compounds are characterized by similar affinities to H3R.
Their kinetics, absorption, biotransformation, half-life,
metabolism etc. might indeed play important role. However H3R
besides its presynaptic auto- and heteroreceptor activity in
the central nervous system, is active in peripheral tissues, e.g.
adipocytes and the capillary network in brown adipose tissue
[21]. Therefore, further studies are needed to elucidate the
mechanism of both compounds action in this regard.
Figure 1. KSK29, KSK19, KSK30, KSK31 and general structures of
described ligands 2–25; hH3R – human histamine H3 receptor.

triglyceride and glucose level
Figure 2. Locomotor activity after one administration of tested compounds;
Results are expressed by mean ± SEM, n = 6. Comparisons were performed
by one-way ANOVA, Dunnet’s post hoc; *p < 0.05, ***p < 0.001 significant
to control group – 0.2 ml 1% Tween 80.
The level of glucose and triglyceride in blood was higher in
rats on the palatable vs. standard diet. Rats treated with
compound 4 for two weeks and fed with a palatable diet had
significantly lower level of glucose in plasma in comparison to
the control group fed with the palatable diet (Figure 5A). On the
other hand, no significant effect on triglyceride level of rats fed
with palatable diet was observed in comparison with the control
group. Interestingly, the group that received compound 4, had
significantly lower amount of fat in the peritonea compared to the
obese rats (Figure 5B).
It can be assumed that decreased glucose level may be directly
related to weight loss. However, our research was of a
preliminary character and did not indicate the mechanism of this
action. It might be possible that this compound affects the
secretion of insulin. As it was shown earlier by Nakamura et al.
[22], histamine H3 receptors are e pressed in mouse β cells and
could play a role in insulin secretion and possibly β cell
proliferation. Histamine H3 receptor inverse agonist Pitolisant
administrated for 14 days in obese mice significantly improved
glucose tolerance [17]. Other inverse agonist JNJ-5207852
induces insulin secretion from pancreatic β cells and corrects
glucose level [22]. Further studies are needed to elucidate the
mechanism of tested compounds action in this regard.
2.2.5. Spontaneous activity in chronic dosing
The group chronically treated with compound 4 showed no
significant differences in spontaneous activity compared to rats
fed only with palatable feed (Figure 6). Disorders of spontaneous
activity (significant increase in activity as well as sedation) and
the stress induced by the test compound could be very
disadvantageous and could disturb an impact assessment of the
test compound on body weight [23]. The test compound 4 did not
cause sedation or hyperlocomotion. Therefore, the observed
anorectic effect was definitely not associated with a reduction or
increment in the spontaneous activity of animals.
2.3. Metabolic stability
The metabolic stability of compound 4 was initially examined
using MetaSite computational tool in order to determine the
potential sites of metabolism as well as structures of potential
metabolites [24]. Prediction results of 4 by a computational liver
model, and in vitro metabolic stability studies using human
(HLMs) liver microsomes are given in Supplementary Data.
In silico data indicated the nitrogen atom of the pyridine
moiety as the most probable site of compound 4 metabolism.
Moreover, the aliphatic linker and the methyl group at acetyl
substituent were also shown to be susceptible for metabolic
biotransformations. The provided in silico the most likely
structures of compound 4 metabolites showed that the metabolic
oxidation/hydroxylation are expected, which occurred either at
nitrogen of the pyridine moiety or at the methyl group of acetyl
substituent (see Supplementary Data).
Figure 4. The energy value of intaken food during administration of
compound 4. Results are expressed by mean ± SEM, n = 6. Multiple
comparisons were performed by two-way ANOVA, Bonferroni post-hoc or
one-way ANOVA Tukey post-hoc. *p < 0.05, **p < 0.01, ***p < 0.001
significant between control rats fed with palatable diet vs. control rats fed
with standard diet; ^p < 0.05 significant between control rats fed with
palatable diet vs. rats treated with tested compounds.
In vitro test results suggested very good metabolic stability of
compound 4, as only ~9% was converted into two metabolites. In
MS analysis, the observed molecular masses and ion fragments
of 4, as well as its main metabolite M1 (+2 units) and M2 (+16
units) suggest the reduction of the carbonyl group and the
reaction of oxidation/hydroxylation as metabolic routes of
compound 4. Surprisingly, the determined in vitro reduction of
the carbonyl group in M1 was in opposite to the in silico results.
However, two oxidized or hydroxyled metabolites with
molecular masses similar to obtained in vitro M2 were predicted
by MetaSite software. The site of M2 metabolic
Figure 3. Effect of administration of tested compounds on body weight. A)
Body weight throughout the administration of compound 4. B) Body weight
throughout the administration of compound 16. Multiple comparisons were
performed by two-way ANOVA, Bonferroni post-hoc or one-way ANOVA
Tukey post-hoc. *p < 0.05, **p < 0.01, ***p < 0.001 significant between
control rats fed with palatable diet vs. control rats fed with standard diet; ^p <
2.2.4. The amount of peritoneal adipose tissue or
0.05 significant between control rats fed with palatable diet vs. rats treated
with tested compounds.

CCCP and DX, which decreased more than 50% of control
HepG2 viability at 10 µM and 1 µM, respectively (Figure 7).
2.5. Risk for Phospholipidosis
Many of the marketed cationic amphiphilic drugs (CADs) are
responsible for side effects connected with
a lysosomal
accumulation of phospholipids (phospholipidosis, PLD). The
CADs are characterized by a hydrophobic ring structure and
a hydrophilic side chain with a charged amine group. The CADs-
induced PLD is associated with number of unwanted clinical
toxicities, such as hepatotoxicity, QT prolongation, kidney and
lung injury, and myopathy [25].
During this study the LYSO-ID Red Detection Kit (Enzo,
Farmingdale, NY, USA) was used for prediction of potential
PLD effect induced by compound 4 in HepG2 cells. The used kit
consists of the LYSO-ID Red fluorescent dye, which
accumulates in lysosomes, and Hoechst 33342 nuclear stain.
HepG2 cells were treated for 72h with the reference PLD-
inducing drugs Verapamil, Ketoconazole and tested compound 4
at a concentration of 50 µM. Next, the compounds were removed
and the cells were incubated for 30 min with the mixture of
LYSO-ID Red and Hoechst 33342 and analyzed by fluorescence
microscopy. The reference drugs considerably increased the
number of cellular lysosomes in hepatoma cells in comparison to
the untreated control. However, much lower number of
lysosomes were observed for compound 4 (see Supplementary
Data).
2.6. Molecular modeling
Among four known and described histamine receptors, only
the histamine H1 receptor crystal structure was resolved so far
(PDB ID: 3RZE [26]). Therefore, in search for novel histamine
H3 receptor ligands, homology modeling methods are used for
the H3R binding site. In this study, for docking purposes,
previously described histamine H3 receptor homology model was
used (with Pitolisant as reference ligand in the binding pocket
[16]).
Figure 5. Effects of administration of compound 4 on plasma glucose and
plasma triglyceride level in rats (A) and the amount of peritoneal fat in rats
(B). Results are expressed by mean ± SEM, n = 6. Comparisons were
Figure 6. Spontaneous activity after first (A) and thirteenth (B)
All of the docked compounds were characterized by relatively
high docking score values between -8.391 and -5.11. Both the
highest and lowest values were observed for p-acetyl derivatives.
Supposed key histamine H3 receptor antagonist/inverse agonist
interactions are salt bridge and/or hydrogen bond formation
between protonated piperazine nitrogen and GLU2065.46 (upper
case numeration according to Balesteros-Weinstein [27]) for
most of the compounds.
performed by one-way ANOVA Dunnett post-hoc. *p < 0.05, **p < 0.01
administration of compound 4. Results are expressed by mean ± SEM, n=6.
significant between control rats fed with palatable diet vs. control rats fed
Comparisons were performed by Multiple t-test.
standard diet; ^p < 0.05, ^^p < 0.01 significant between control rats fed with
palatable diet vs. rats treated with compound 4.
biotransformation was determined next due to the fragment ion
with m/z = 176 produced by M2, which excluded the N-oxidation
at the pyridine moiety. Thus, considering both in silico and in
vitro results the metabolite M2 of compound 4 is a hydroxyl
derivative with hydroxyl group located most likely at the methyl
group of acetyl substituent (see Supplementary Data).
Similar to our previous findings, H-bond formation between
TYR3746.51 and ether oxygen was observed for compounds with
alkyl chain composed of 4-methylene groups. Additionally,
heteroaromatic substituents at the terminal nitrogen were
stabilized through π-π interactions with indole and/or benzene
ring of TRP 3716.48 (Figure 8), one of the highly conserved
residues, so-called molecular switches [28], for which the change
of orientation causes a change in the conformation of the
receptor. Again, due to the electron structure of tetrahydrofuroyl
moiety, such interactions were not observed for those piperazine
substituents.
2.4. Hepatotoxicity – the effect on HepG2 cells
The potential antiproliferative activity of 4 was examined in
vitro by using hepatoma HepG2 cells to predict its potential
hepatotoxicity. The statistically significant decrease in the cells
viability was observed at 10 µM (p < 0.01) and 100 μM (p <
0.001) concentrations. However, the effect was weak (~10%
decrease in HepG2 viability), compared to the reference toxins

Figure 7. The effect of compound 4 and the reference toxins DX and CCCP
on the hepatoma HepG2 cells viability. The statistical significance was
evaluated by a one-way ANOVA, followed by Bonferroni’s Comparison Test
(**p < 0.01, ***p < 0.001 compared with negative control).
Pharmacological in vivo test results of compound 4 (3 mg/kg)
clearly indicate that it may affect the amount of calories
consumed, thus act as an anorectic compound. Likewise, its
protective action against hyperglycemia and the development of
overweight has been shown. In the effective dose, compound 4
did neither affect the activity, nor acted seditiously. Moreover,
studies of 4 showed its safety and very good metabolic stability.
Thus, compound 4 has been chosen as a new lead structure for
development of anti-obese H3R ligands and the mechanism of its
action would be a subject of further studies.
4. Experimental protocols
Substituted benzene rings in the lipophilic part of ligands were
stabilized through π-π stacking with at least one of aminoacids
TYR 1153.33, TYR 3947.35 , PHE 193 (ECL2). For most of the
compounds, additional formation of a hydrogen bond between
carbonyl group and ARG3816.58 nitrogens was found. These
interactions might have a positive influence on H3R affinity for
the set of tested ligands, in comparison to previously described t-
amyl and t-butyl derivatives. Although, with the elongation of the
alkyl chain length, the position of basic parts of the ligands was
mostly fixed, acetyl and propionyl substituents reached
progressively higher in the extracellular direction. In most cases
this led to loss of π-π stacking stabilization and/or H-bond with
TYR3746.51, yet still maintaining its interactions with ARG3816.58
(Figure 9).
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
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;
Ac, acetyl; Prop, propionyl; 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). 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: CH2Cl2:MeOH (9:1).
3. Conclusions
A novel series of acetyl and propionyl phenoxyalkylamine
derivatives investigated proved to be potent histamine H3R
ligands with some compounds with the H3R affinities in the
nanomolar concentration range. In vitro test results proved the 4-
pyridylpiperazine moiety to be a crucial element for high hH3R
affinity (hH3R Ki = 5.2 – 115 nM). Moreover, introduction of
carbonyl moiety containing residues in the lipophilic part of
molecules instead of branched alkyl substituents resulted in
increased affinity. This is in accordance with our previously
described series, whereas propionyl derivatives showed slightly
higher affinities compared to those of acetyl derivatives (16 and
22 vs. 4 and 10; hH3R Ki = 5.2 and 15.4 nM to 10.2 and 115 nM,
respectively). These findings were confirmed by molecular
modelling studies, where additional ligand-receptor interactions
were found. Yet again, dinitrogen piperazine substituents showed
significantly lower affinity and tethrahydrofuroyl were
completely inactive.
4.1.1. General synthetic procedure for compounds 1a–1d
To a solution of freshly prepared sodium propanolate, 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
then refluxed for another 3 h. After cooling down to RT, mixture
was filtrated and evaporated. To a rough product, 100 ml of 10%
Figure 9. Compound 10 in histamine H3 homology receptor binding pocket (left) and its ligand-interaction diagram (right).
Figure 8. Ligand interaction diagrams for compounds 4, 16, 7 (left to right).

4.2. Pharmacology
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.
4.2.1. [3 H]Nα -Methylhistamine hH3 R 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 [29].
Affinity values (Ki) were expressed as mean (with 95%
confidence interval) from at least three experiments, each
performed at least in duplicates.
4.1.1.1. 1-(4-(3-Bromopropoxy)phenyl)ethan-1-one (1a)
Synthesis from 1-(4-hydroxyphenyl)ethan-1-one (13.62 g, 0.1
mol), 1,3-dibromopropane (40.38 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 18.51 g of raw product was
obtained, purified by CC (I) with yield of 72%.
The displacement binding assay was carried out as described
by Kottke et al. with slight modifications [20]. 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).
4.1.1.2. 1-(4-(4-Bromobutoxy)phenyl)ethan-1-one (1b)
Synthesis from 1-(4-hydroxyphenyl)ethan-1-one (13.62 g, 0.1
mol), 1,4-dibromobutane (43.18 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 19.25 g of raw product was
obtained, purified by CC (I) with yield 71%.
4.1.1.3. 1-(4-(3-Bromopropoxy)phenyl)propan-1-one (1c)
Synthesis from 1-(4-hydroxyphenyl)propan-1-one (15.02 g,
0.1 mol), 1,3-dibromopropane (40.38 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 18.43 g of raw product was
obtained, purified by CC (I) with yield 68%.
4.1.1.4. 1-(4-(4-Bromobutoxy)phenyl)propan-1-one (1d)
Synthesis from 1-(4-hydroxyphenyl)propan-1-one (15.02 g,
0.1 mol), 1,4-dibromobutane (43.18 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 17.68 g of raw product was
obtained, purified by CC (I) with yield 62%.
4.2.2. In vivo studies
4.2.2.1. Drugs and compounds
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).
Heparin was delivered by Polfa Warszawa S.A. (Warsaw,
Poland), while thiopental sodium was from Sandoz International
(France).
4.2.2.2. Animals
The experiments were carried out on male CD-1 mice, body
weight 20 - 22 g (locomotor activity) or Wistar rats, initial body
weight 160
- 180 g (body weight, biochemical assays,
spontaneous activity). Animals were housed in pairs in plastic
cages in constant temperature facilities exposed to light-dark
cycle, water and food available ad libitum. Control and
experimental groups consisted of six to eight animals each. All
experiments were conducted according to the Guidelines of the
Animal Use and Care Committee of the Jagiellonian University.
Detailed synthetic procedure and analytical data for
compounds 2–25 could be found in Supplementary Data.
4.1.2.1. 1-(4-(3-(4-(Pyridin-2-yl)piperazin-1-
yl)propoxy)phenyl)ethan-1-one hydrogen oxalate
(2)
4.2.2.3. The influence of test compounds on locomotor activity
Synthesis from 1-(pyridin-2-yl)piperazine (0.82 g, 5 mmol),
and compound 1a (1.29 g, 5 mmol) in ethanol (50 ml) in the
presence of K2CO3 (1.17 g, 8.5 mmol) and catalytic amount KI in
water (10 ml). Reaction was refluxed for 20 h and then purified.
Obtained 1.44 g of oil with yield of 72%. Raw product was
transformed into oxalic acid salt yielding 0.88 g of final
The locomotor activity was recorded with an Opto M3
multichannel activity monitor (MultiDevice Software v1.3,
Columbus Instruments, USA). Locomotor activity was evaluated
as the distance travelled by the animals while attempting to climb
upward [30]. Mice were inserted into the parameter-counting
cage immediately after the intraperitoneal administration of the
test compound, although the activity measurement started 30
minutes after administration of the test compound over a 20
minutes period.
1
compound. Mp: 182–183 °C. H NMR (300 MHz, DMSO-d6) δ
ppm: 8.13 (dd, J=4.69, 1.17 Hz, 1H, 2Pyr-6H), 7.91 (m, J=8.79
Hz, 2H, Ph-3,5H), 7.57 (ddd, J=8.79, 7.03, 1.76 Hz, 1H, 2Pyr-
4H), 7.03 (m, J=8.79 Hz, 2H, Ph-2,6H), 6.90 (d, J=8.21 Hz, 1H,
2Pyr-3H), 6.70 (dd, J=6.74, 4.98 Hz, 1H, 2Pyr-5H), 4.13 (t,
J=5.86 Hz, 2H, CH2-O), 3.70 (br. s., 4H, Pip-3,5H), 3.04-3.15
(m, 6H, Pip-2,6H + NCH2), 2.50 (s, 3H, CO-CH3), 2.07-2.18 (m,
2H, CH2-CH2-O). 13C NMR (DMSO-d6): 196.74, 164.52, 162.63,
158.69, 148.03, 138.26, 130.93, 130.45, 114.73, 114.30, 108.00,
66.00, 53.73, 51.47, 42.98, 26.87, 24.36. LC–MS: purity 100% tR
= 2.72, (ESI) m/z [M+H]+ 341.33. Anal. Calcd for C20H25N3O2 x
C2H2O4: C, 61.53, N, 9.78, H, 6.34%. Found: C, 60.95, N, 9.82,
H, 6.28.
4.2.2.4. The influence of test compounds on body weight and food
and water intake in rats fed with palatable diet (Western-style
diet)
In order to determine the anorectic activity of compounds, its
effect on calories and water intake in the model of excessive
eating was assessed [31]. Four groups composed of six rats were
fed during two weeks with a diet consisting of milk chocolate
with nuts, cheese, salted peanuts and 7% condensed milk.

Animals had access to standard food (Labofeed B, Morawski
Manufacturer Feed, Poland) and water ad libitum. Rats were
treated intraperitoneally with compounds once a day in the
morning for 14 days. The palatable control group received a
vehicle intraperitoneally (1% Tween 80) whereas test palatable
groups received: 3 mg/kg b.w. of compound 16 or 3 mg/kg b.w.
of compound 4 or 1 mg/kg b.w. of compound 4 in 1% Tween 80.
Palatable diet contained: 100 g peanuts – 614 kcal; 100 ml
condensed milk – 131 kcal; 100 g milk chocolate with hazelnuts
– 195 kcal; 100 g Greek cheese – 270 kcal. The other group of
rats was on a standard diet (100 g feed – 280 kcal). A vehicle
(1% Tween 80) was administered intraperitoneally to this control
group. The consumption of food and water was evaluated three
times per week and body weight of animals was measured daily,
immediately before administration of substances. On the 15th day
of the experiment, 20 minutes after intraperitoneal administration
of heparin (1000 j/rat) and thiopental (70 mg/kg b.w.), plasma
was collected from the left carotid artery and peritoneal fat was
weighed.
WI, USA). All experiments were performed as described before
[32,33]. 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).
4.4. Antiproliferative assay
Hepatoma HepG2 (ATCC HB-8065) cell line was kindly
donated by the Department of Pharmacological Screening,
Jagiellonian University Medical College. The cell cultures’
growth conditions were applied as described before [33,34]. The
cytostatic drug doxorubicin (Sigma-Aldrich) and hepatotoxin -
carbonyl cyanide 3-chlorophenylhydrazone, CCCP (Sigma-
Aldrich) were used as references. CellTiter 96 AQueous Non-
Radioactive Cell Proliferation Assay (MTS) (Promega) was used
to determine cells’ viability. The absorbance of the samples was
measured
using
a microplate reader EnSpire (PerkinElmer) at 490 nm.
4.5. Phospholipidosis
4.2.2.5. Influence of compound 4 on glucose and triglyceride
levels
To determine the drug-induced phospholipidosis the HepG2
cells were seeded first in 96-wells white, solid plate with
transparent bottom (Corning, Tewksbury, MA, USA) at
concentration of 1.5 × 104 cells/well and were incubated at 37°C
for 24 hours in a humidified atmosphere at 5% CO2. Next,
Verapamil, Ketoconazole and compound 4 were diluted in
glucose (4.5 g/L) and FBS (10%) supplemented Minimum
Essential Medium Eagle (MEM) media (Gibco, Carlsbad, CA,
USA) and added to the cells in the concentration of 50 µM. The
cells were incubated for 72 h. Thereafter, the medium with
compounds was removed and the cells were incubated for 30 min
with the mixture of LYSO-ID Red and Hoechst 33342 (Enzo,
Farmingdale, NY, USA). The cells were next washed twice with
200 μl of PBS Buffer and analyzed by fluorescence microscopy
DMi8 (Leica, Wetzlar, Germany).
To determine the glucose and triglyceride levels in plasma,
standard enzymatic and spectrophotometric tests (Biomaxima
S.A. Lublin, Poland) were carried out [17]. The substrate was
decomposed with enzymes suitable to the relevant product,
which was then converted to a colored compound. The coloration
was proportional to the concentration. The absorbance was
measured at a wavelength of 500 nm (glucose, triglycerides).
4.2.2.6. Influence of compound 4 on spontaneous activity after
chronic treatment in rats fed palatable diet housed in pairs in
home cages
The spontaneous activity of rats treated with compounds was
measured on the 1st and 13th day of the treatment using special
RFID-system – TraffiCage (TSE-Systems, Germany). Rats were
housed in pairs in domestic cages with feed and water available
ad libitum. The animals had transmitter identification (RFID)
implanted subcutaneously, which enabled to count the presence
and time spent in different areas of the cage, and then to collect
the data with special computer application. Spontaneous activity
was monitored for 24 hours after administration of the
compound.
4.6. Molecular modeling
For docking purposes, Schrödinger Maestro Suite (v.
11.5.011, Release 2018-01) was used [35]. Ligands were built in
their ionized forms (protonated N4 piperazine nitrogen, structure
charge +1) and their bioactive conformations were generated
using ConfGen module [36,37] (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 [38-42] (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 [43].
4.2.2.7. Statistical analysis
Statistical calculations were carried out with the GraphPad
Prism 6 [32] program. Results are given as the arithmetic means
with standard error of the mean (SEM). Statistical significance
was calculated using one of the following tests: two-way
ANOVA post-hoc Bonferroni test (body weight changes); one-
way variance analysis (ANOVA), followed by a Dunnett post-
hoc test (triglyceride and glucose levels, energy intake); t-Student
test (locomotor activity); Multiple t-test, under the assumption,
that all of the rows were sampled from populations with the same
scatter (spontaneous activity). Differences were considered
statistically significant at: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Acknowledgements
Authors sincerely acknowledge Prof. Budziszewska and Dr
Bartosz Pomierny, Department of Toxicological Biochemistry,
Jagiellonian University Medical College for enabling
phospholipidosis tests evaluation on fluorescent microscopy
DMi8 (Leica, Wetzlar, Germany). We are pleased to
acknowledge the generous support of National Science Center,
4.3. 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 [24]. Human liver microsomes (HLMs) for
in vitro metabolic stability determination were purchased from
Sigma-Aldrich (St. Louis, MO, USA). The NADPH
Regeneration System was purchased from Promega (Madison,
Poland
granted
on
the
basis
of
decision
No.
2016/23/N/NZ7/00469, 2016/23/B/NZ7/01063, K/DSC/004310,
and K/ZDS/007121. Support by DFG INST 208/664-1 (HS) and
COST CA15135 (HS, KK) is also acknowledged.
Appendix A. Supplementary data

Supplementary data associated with this article can be found in
the online version, at. These data include MOL files and
InChiKeys of the most important compounds described in this
article.
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Stark H. Histamine H3R antagonists: from scaffold hopping
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Histamine Receptors. The Receptors vol. 28, Humana Press,
Cham. 2016; https://doi.org/10.1007/978-3-319-40308-35.
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