Discovery of potential, dual-active histamine h3 receptor ligands with combined antioxidant properties

Article abstract of DOI:10.3390/molecules26082300

In an attempt to find new dual acting histamine H₃ receptor (H₃ R) ligands, we designed a series of compounds, structurally based on previously described in our group, a highly active and selective human histamine H₃ receptor (hH₃ R) ligand KSK63. As a result, 15 obtained compounds show moderate hH₃ R affinity, the best being the compound 17 (hH₃ R K_i = 518 nM). Docking to the histamine H₃ R homology model revealed two possible binding modes, with key interactions retained in both cases. In an attempt to find possible dual acting ligands, selected compounds were tested for antioxidant properties. Compound 16 (hH₃ R K_i = 592 nM) showed the strongest antioxidant properties at the concentration of 10^?4 mol/L. It significantly reduced the amount of free radicals presenting 50–60% of ascorbic acid activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, as well as showed antioxidative properties in the ferric reducing antioxidant power (FRAP) assay. Despite the yet unknown antioxidation mechanism and moderate hH₃ R affinity, 16 (QD13) constitutes a starting point for the search of potential dual acting H₃ R ligands-promising tools for the treatment of neurological disorders associated with increased neuronal oxidative stress.

Full text of DOI:10.3390/molecules26082300

molecules
Article
Discovery of Potential, Dual-Active Histamine H Receptor
3
Ligands with Combined Antioxidant Properties
1
,
2
1
2
3
Kamil J. Kuder * , Magdalena Kota n´ ska
, Katarzyna Szczepa n´ ska , Kamil Mika , David Reiner-Link
,
Holger Stark 3 and Katarzyna Kie c´ -Kononowicz 1
1
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy,
Jagiellonian University Medical College, Medyczna 9, 30-699 Kraków, Poland;
k.szczep@if-pan.krakow.pl (K.S.); mfkonono@cyf-kr.edu.pl (K.K.-K.)
Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College,
Medyczna 9, 30-699 Kraków, Poland; magda.dudek@uj.edu.pl (M.K.); kamil.mika@doctoral.uj.edu.pl (K.M.)
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf,
Universitaetsstr. 1, 40225 Duesseldorf, Germany; david.reiner@sund.ku.dk (D.R.-L.); stark@hhu.de (H.S.)
Correspondence: kamil.kuder@uj.edu.pl; Tel.: +48-12-620-5583
2
3
*
Abstract: In an attempt to find new dual acting histamine H receptor (H R) ligands, we designed a
3
3
series of compounds, structurally based on previously described in our group, a highly active and
selective human histamine H receptor (hH R) ligand KSK63. As a result, 15 obtained compounds
3
3
show moderate hH R affinity, the best being the compound 17 (hH R K = 518 nM). Docking to
3
3
i
the histamine H R homology model revealed two possible binding modes, with key interactions
retained in both cases. In an attempt to find possible dual acting ligands, selected compounds
3
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
were tested for antioxidant properties. Compound 16 (hH R K = 592 nM) showed the strongest
3
i
Citation: Kuder, K.J.; Kota n´ ska, M.;
Szczepa n´ ska, K.; Mika, K.;
−4
antioxidant properties at the concentration of 10 mol/L. It significantly reduced the amount of
free radicals presenting 50–60% of ascorbic acid activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH)
assay, as well as showed antioxidative properties in the ferric reducing antioxidant power (FRAP)
Reiner-Link, D.; Stark, H.;
Kie c´ -Kononowicz, K. Discovery of
Potential, Dual-Active Histamine H3
Receptor Ligands with Combined
Antioxidant Properties. Molecules
assay. Despite the yet unknown antioxidation mechanism and moderate hH R affinity, 16 (QD13)
3
constitutes a starting point for the search of potential dual acting H R ligands-promising tools for the
3
treatment of neurological disorders associated with increased neuronal oxidative stress.
2
021, 26, 2300. https://doi.org/
Keywords: histamine H receptor; histamine H receptor ligands; piperazine derivatives; molecular
1
0.3390/molecules26082300
3
3
modeling; antioxidative agents
Academic Editor: Simona Rapposelli
Received: 12 March 2021
Accepted: 13 April 2021
Published: 15 April 2021
1. Introduction
Histamine H receptor (H R) is an outstanding member of the histamine receptor
3
3
family. Since its discovery and gene cloning, it has served as a widely explored drug
target due to its broad spectrum of neuromodulatory effects in the central nervous system
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
(
CNS) [
results in regulation of synthesis and release of the natural ligand histamine. On the other
hand, activation of H R as a heteroreceptor influences the whole range of other neurotrans-
1]. On the one hand, activation of presynaptic receptors on histaminergic neurons
3
mitters, including acetylcholine, dopamine, serotonin, norepinephrine,
γ-aminobutyric
acid, glutamate, and substance P, to name a few [ ]. Anatomically speaking, H Rs are
2–4
3
prominently expressed in humans in the basal ganglia, globus pallidus, hippocampus,
and cortex–regions associated with cognition, sleep, wakefulness, and homeostatic regu-
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
lation [5]. Consequently, H R ligands, either antagonist or inverse agonists, as they also
3
demonstrate constitutive activity, contribute to one of the largest groups of contempo-
rary GPCR ligand research, with implications to treat a number of CNS-based diseases,
including Alzheimer’s (AD) and Parkinson’s disease (PD), schizophrenia, ADHD, obesity,
and narcolepsy [6,7]. However, while several big pharma companies were working on
development of H R inhibiting ligand and reached the Phase III studies in these indications,
3
4
.0/).
Molecules 2021, 26, 2300. https://doi.org/10.3390/molecules26082300
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2300
2 of 18
all of them failed. The only compound, pitolisant, reached the market under the name of
®
Wakix (Figure 1), with an indication for narcolepsy treatment [
8]. One exception worth
mentioning is the LML134 compound (Figure 1), which completed the Phase I study by
means of safety and tolerability, and went for further trials, with promising results for
the treatment of excessive sleep disorders. However, the studies were terminated due to
business issues [9,10].
At the same time, increasing evidence indicates that oxidative stress plays an important
role in the pathogenesis of many diseases. H receptor-induced activation of Gi/o-proteins
3
seems to stimulate phospholipase A (PLA ) to induce the release of arachidonic acid,
2
2
which, under pathological conditions, act as precursors for various inflammatory com-
pounds, which in turn are crucial in mediating the oxidative and inflammatory responses
in CNS pathologies, such as stroke, AD, PD and multiple sclerosis [11]. In fact, histamine
H receptor ligands that might inhibit oxidative stress by reducing free radical concentra-
3
tions have been shown to have potential therapeutic applications in several neurological
disorders, such as Parkinson’s [12], Alzheimer’s disease [13], cerebral ischemia/stroke [14],
depressive illness [15], epilepsy [16
,17], schizophrenia [18], autism [19], and cardiovascular
disorders, such as myocardial infarction [20]. All of these studies show that the antioxidant
activities of histamine H receptor ligands play a significant role in reducing oxidative
3
stress that accompany the above mentioned diseases.
Throughout the years, an abundant number of H R ligands have been synthesized,
3
based on various heterocyclic moieties, starting from histamine’s native imidazole moiety
to (substituted) piperidines, pyrrolidines, and piperazines, among others. Yet, the overall
structure remained unchanged: basic cores connected via (rigidified) alkyl chain with polar
moiety, which in turn is connected, either directly or via a second linker, with a so-called
“
eastern arbitrary region” [21]. While the latter ones are believed to modulate potency and
pharmacokinetic properties of H R antagonists/inverse agonists, the heterocyclic moiety
3
maintains the necessary interactions with binding pocket amino acids, essential for the
receptor binding.
Recent publications from our group [22–24] allowed for the establishment of unique
heterocyclic moiety, namely 4-pyridylpiperazine as a new bioisosteric replacement of
piperidine. Molecular modeling studies confirmed its ability to form additional interac-
tions, besides those of piperazine protonated nitrogen, with highly conserved H R amino
3
acids. These findings were tested in vitro, proving this scaffold to be a crucial element for
high H R affinity. In turn, bulky substituents of the so-called “eastern region”, such as
3
benzophenones, have also shown to act as possible additional H-bond acceptor moiety,
strengthening ligand-receptor interactions. It was again proved in vitro, resulting in one of
the highest affine H R ligands from our group, KSK63 (hH R K = 3.12 nM, Figure 1).
3
3
i
Figure 1. Structure and properties of reference histamine H receptor ligands [9,24–26] (K -inhibitory
i
3
constant, IC -half maximal inhibitory concentration).
50

Molecules 2021, 26, 2300
3 of 18
In light of the presented findings, we designed the series of ligands with modifica-
tions in the hydrophilic and distal regulatory regions, namely by introducing different
1
2
substituents in the piperazine (R ) and benzophenone (R ) moieties, respectively, based
on the general structure depicted in Figure 2. The high activity associated with the basic
part of the molecule, prompts to look for new scaffolds, even if many attempts lead to
inactive compounds. For this series, we have chosen five different (cyclo)alkyl carbonyl and
4-arylpiperazine substituents. Using direct (cyclo)alkyl carbonyl piperazine N4 nitrogen
substituents, we wanted to test whether such reduction in piperazine N4 nitrogen negative
charge would still be acceptable for the retention of H R affinity [27]. On the other hand,
3
in order to avoid the possible overlap of inductive and mesomeric effects of p-substituted
methoxy group, we have chosen m-substituted derivatives, so as to take only the former
one into account. On the eastern end, we tested whether the increase of the hydrophobicity,
by introduction of halogen atoms to benzophenone moiety, is also tolerated.
Figure 2. General structure of designed ligands.
Hence, it is relevant to search for novel bioactive compounds able to combine in one
molecule multiple action properties. The search for possible dual active H R ligands, which
3
might act as neuroprotectives, both directly (by expressing anti-oxidative properties) and
indirectly (through the effect of H R antagonism), seems necessary, as their applications
3
might be beneficial in future therapies. Even though the obtained compounds lack the
phenolic protons believed to be essential for the antioxidant activity, paying attention to,
e.g., the structure of the known antioxidant melatonin [28], with respect to recent findings
on the tetratargeting Contilisant [26
antioxidative properties. Last, but not least, biological test results were complemented by
the docking studies to the histamine H receptor homology model.
,29], selected compounds were tested for possible
3
2. Results and Discussion
2.1. Chemistry
The desired final compounds 4–18 were obtained through the synthetic route pre-
sented in Scheme 1, according to the previously reported methods [23,30]. Starting phenoxy-
alkyl bromides were obtained in one-step O-alkylation of commercially available (sub-
stituted) p-hydroxy benzophenones with 1,3-dibromopropane, refluxed either in sodium
propanolate (
of potassium iodide (
1
) or acetone with powdered potassium carbonate, and a catalytic amount
). Obtained precursor bromides were then coupled with com-
2, 3
mercially available 4-substituted piperazinyl derivatives in the mixture of ethanol/water
with powdered potassium carbonate and a catalytic amount of potassium iodide. Final
products were obtained either as quick crystallizing free bases (
as oxalic acid salts ( 16 18). For detailed information, please refer to the Materials
and Methods section.
5, 10–15), or were isolated
4,
6–
9
,
–

Molecules 2021, 26, 2300
4 of 18
Scheme 1. General synthetic pathway for compounds 4–18.
2.2. Biological Evaluation
All compounds, in either basic or oxalate forms, were then tested in H R binding
3
studies in vitro, as described previously [31
eleven appropriate concentrations in a [ H]N -methylhistamine (K = 3.08 nM) radioli-
,
32]. In brief, compounds were tested at five to
3
α
D
gand depletion assay to determine the affinity for the human recombinant histamine H R
3
stably expressed in HEK-293 cells. For poorly soluble compounds, 16 and 17, an addition
to 0.025% HCl in the binding test buffer was used.
Human H R binding characterization of the tested compounds are presented in
3
Table 1. Most of the tested compounds show relatively low to no histamine H receptor
3
affinity, with the exception of p-nitro- (14, 15) and m-methoxyphenyl derivatives (16–18),
which display affinities at moderate levels, with compound 17 being the most affine (hH R
3
K = 518 nM) of the group. Regarding the piperazine substituents, one could observe that
i
direct substitution of piperazine N4 nitrogen with simple (cyclo)alkyl carbonyl moieties
has negative influence on hH R affinity. Slightly higher values were demonstrated for
3
p-acetylphenyl derivatives; however, still at an unacceptable level. It appears that such
a group is not suitable for histamine H receptor binding. The reasoning behind these
3
observations might be the low pK
a
value of N4 nitrogen of the obtained compounds.
6, it might be assumed that the majority of the
With, e.g., pK 6.0 for compounds 4–
a
=
−
compounds at physiological pH are in non-ionized form. This might result in a lack of
crucial interactions with binding pocket amino acids, resulting in very low or no affinity at
all. It appears that only m-methoxy- (16
the pK at a level that allows for abundance of ionized forms. Calculated pK
be found in Table S1 of the Supplementary Material.
–
18) and p-nitrophenyl derivatives (14
,
15) express
values can
a
a

Molecules 2021, 26, 2300
5 of 18
Table 1. Structures of compounds
4–18 and their in vitro human histamine H receptor (hH R)
3 3
affinities. Given data represent mean values within the 95% confidence interval (CI).
Compound
R1
R2
hH R K [nM] x¯ [CI 95%]
3
i
4
3653
[2024, 6594]
H
QD11
5
1257
[599, 2638]
Cl
F
QD15
6
1100
[535, 2263]
QD4
7
1334
[274, 6505]
H
QD14
8
Cl
F
>1000
QD19
9
1669
[596, 4676]
QD8
1
0
633
[206, 1942]
H
Cl
F
QD10
1
1
>1000
QD17
1
2
1008
[618, 1646]
QD3
1
3
H
Cl
F
>1000
QD12
1
4
622
[211, 1832]
QD20
1
5
580
[177, 1902]
QD6
1
6
592 a
[394, 889]
518 a
[201, 1336]
H
Cl
F
QD13
1
7
QD18
1
8
715
[352, 1453]
QD9
a
Values were obtained using 0.025% HCl in binding buffer. Values in square brackets indicate the confidence
interval of 95%. Ki-inhibitory constant.
The overall affinity row for 4-piperazinyl substituents can be summarized as m-
methoxyphenyl > p-nitrophenyl > p-acetylphenyl > cyclopropylmethanone > acyl (best to
worst) for the herein presented series. Moreover, no clear structure–affinity relationship
among the three benzophenone modified derivatives can be drawn.
2.3. Antioxidative Properties
In order to determine the possible antioxidant properties, selected compounds were
tested in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power

Molecules 2021, 26, 2300
6 of 18
(FRAP) assay with ascorbic acid as a reference compound (Table 2). For details, please
refer to the Materials and Methods Section. Graphical representation of tests results can be
found in Supplementary Material.
Table 2. Absorbance changes in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing
antioxidant power (FRAP) assays. The highest obtained values for the tested compounds were
marked in bold.
DPPH Assay
λ = 517 nm)
FRAP Assay
(λ = 593 nm)
(
Compound
Absorbance Decrease in
mol/L Concentration [%]
Absorbance Increase in
10 mol/L Concentration [%]
−
4
−4
1
0
Mean ± SEM
Mean ± SEM
Ascorbic acid
20.52 ± 0.51
8.82 ± 0.12
6.23 ± 0.53
6.79 ± 0.51
7.70 ± 0.41
2.44 ± 0.97
3.33 ± 0.45
4.42 ± 0.33
6.31 ± 1.14
12.37 ± 1.31
4.88 ± 0.18
6.35 ± 0.55
344.20 ± 4.65
0.79 ± 0.39
1.43 ± 0.32
0.48 ± 0.48
1.75 ± 0.22
3.66 ± 0.59
3.34 ± 0.22
2.70 ± 1.15
0.22 ± 0.16
32.91 ± 0.81
40.86 ± 0.98
29.73 ± 0.39
6
7
8
9
1
1
1
1
1
1
1
0
1
2
4
6
7
8
Antioxidant activity at the level above 50% of ascorbic acid activity at a concentration
of 10 4 mol/L might be considered noteworthy. In the DPPH assay, compound 16 (QD13)
−
−
4
at a concentration of 10 mol/L caused a decrease in absorbance by 12.37
compared to the sample containing the solvent + reaction mixture (blank, maximum
concentration of the DPPH radical). Moreover, 16, at a concentration of 10 , had 60.28%
of the ascorbic acid activity (10 mol/L, calculated from Table 2). In fact, of all the tested
compounds, 16 expressed the strongest antioxidant properties.
±
1.31% when
−
4
−
4
Eight of the other compounds showed a slightly smaller but pronounced decrease
−
4
in absorbance (antioxidant activity > 20% of ascorbic acid activity at 10 mol/L); thus,
scavenging DPPH free radicals (Table 2, left panel).
On the other hand, in the FRAP assay, compounds 16
–18 showed highest activity of all
−
4
the tested compounds. The level of absorbance at a concentration 10 mol/L was above
−
4
8
% of ascorbic acid activity at a concentration of 10 mol/L (Table 2, right panel).
Considering both tests results, compound 16 showed the strongest antioxidant proper-
−
4
ties. At a concentration of 10 mol/L, it significantly reduced the number of free radicals
DPPH test) and exhibited antioxidative properties in general (FRAP test) despite yet un-
known mechanism. Furthermore, other compounds from the m-methoxyphenyl subgroup,
and 18, also showed antioxidant activity in FRAP assay. Among three various phenyl
Ph) substituents, only methoxy derivatives appeared to possess antioxidant properties.
This might be due to the fact that the p-NO –Ph, p-COCH –Ph are capable of attracting
(
17
(
2
3
electrons, instead of giving them, while p-OCH –Ph are proficient in giving electrons rather
3
than capturing them, which in turn is an indication of their antioxidant activity. However,
with such an electron withdrawing substituent as m-OCH , antioxidant activity appears in
3
the studied group of compounds.
Overall, these results prompt for future investigation of compounds 16–18 antioxidant
properties in various disease entities. Despite its moderate affinity for histamine H3
receptors, 16 (QD13) could be investigated as an adjuvant to known therapies in many
diseases, to which contributes an oxidative stress and where modulation of the brain levels
of histamine and other neurotransmitters provide improvement.

Molecules 2021, 26, 2300
7 of 18
2
.4. Docking Studies
Three out of four known and described histamine receptors still remain a Gordian
knot by means of crystallography. To date, only histamine H receptor crystal structure was
1
resolved (PDB ID: 3RZE [33]). Therefore, we used a previously described histamine H R
3
homology model in our study, which was constructed on the template of crystal structure of
M muscarinic acetylcholine receptor (PDB ID: 3UON) [22]. Considering that, due to very
2
low basicity, the abundance of compounds at physiological pH might appear in non-ionized
forms, for docking studies, we prepared both protonated and non-protonated conformers.
All of the compounds were docked and, independently of the form, fit the H R
3
binding pocket. In case of non-protonated conformers, compounds were placed shallower
in the binding pocket, and mostly stabilized through π–π stacking or additional H-bonds
with Y374 (3.51) or R381 (6.58). However, no interactions with either of the key amino
acids, E206 (5.46) and D114 (3.32), were found. For possible non-ionized forms, this might
explain very low or no affinity for the histamine H receptor. Calculated binding poses for
3
non-protonated conformers can be found in the Supplementary Material (Table S2).
Nonetheless, two different binding modes were surprisingly observed for protonated
conformers: “standard (ST)”, with the eastern region pointing to the extracellular space,
and “upside-down (UD)” (Figure 3). The positions of protonated basic moieties of the
ligands were mostly similar, independently of the mode. In both cases, a crucial histamine
H R antagonist/inverse agonist interaction-salt bridge and/or hydrogen bond formation
3
between protonated amine nitrogen and E206 (5.46) was, however, preserved due to the
approximate location of the protonated nitrogen (superscripts denote Ballesteros–Weinstein
numbering [34]).
Figure 3. Putative binding poses of protonated conformers. Upper panel: KSK63 (left) 15 (middle) and 16 (right); lower
panel: 17 (middle), 18 (right) in histamine H3 homology receptor.

Molecules 2021, 26, 2300
8 of 18
In the ST mode, benzophenone fragments occupied the space fenced by aromatic
features of F193, Y189 (ECL2), and Y394 (7.35) on the sides, and Y91 (2.61) and Y94 (2.64)
on the top. In most cases, a hydrogen bond between the carbonyl group and R381(6.58)
was found. The latter one was not observed for inactive compounds
stabilization through halogen bond formation with Y91 (2.61) and
Y394 (7.35) and W371 (6.48), in the case most active for this series of m-methoxyphenyl
4–
9
. Additional
π–π stacking with
derivatives, was also present. These interactions might explain the overall higher H R
3
affinity of the ligands from this subgroup.
On the other hand, the UD mode was observed for p-acetylphenyl and p-nitrophenyl
derivatives, with the exception of 18, for which both modes were observed. Although, the
ST mode expressed slightly lower glide energy (
−
58.78 vs
−
56.08 kcal/mol). In this mode,
the benzophenone fragments reached deeper towards the narrow sub pocket localized
among the transmembrane regions (TM) 1, TM3, and TM7. The latter is formed by lateral
L117 (3.35) and W371 (6.48), and S121 (3.39) and A122 (3.40) from the bottom. Additional
pose stabilization through cation–π interactions between protonated piperazine nitrogen
and F193 (ECL2), as well as the hydrogen bond between the carbonyl/nitro group and
R381 (6.58) was found.
Along with the novel compounds, reference structure KSK63 was also docked. In this
case the pyridyl moiety nitrogen is protonated instead of the piperazine one. This allows
for a shift of the ligand towards TM3 and formation of ionic interaction with key aminergic
GPCR anchor, D114 (3.32) (Figure 3). Lack of such interactions observed for the novel,
described compounds, might also be responsible for their much lower affinity towards
histamine H receptor.
3
The stability of the calculated poses for this groups’ most affine compounds 15–17 and
both orientations of 18 along with the reference compound KSK63 was further evaluated
by means of molecular dynamics (MD) simulations. From each simulation, 7 poses were
selected (starting pose, and after each 100ps up to 600ps). In the case of 15–17 (Figure 4)
and KSK63, complexes appeared stable through the whole 600ps simulation, retaining
the key interactions, and the potential energy (U) of the atomic system at the level of
~
1000 kcal/mol.
In all cases, starting poses (marked in grey, Figure 4) were very similar to its orientation
at the end of simulation (marked yellow). However, a shift in the binding site with retained
conformation appeared in the first 100ps of the simulation. On the other hand, the analysis
of KSK63 behavior allowed for possible explanation of its’ high affinity. The conformation
remained quite stable during the simulation with consistent set of interactions. What
was not observed for the remaining structures, the protonated pyridyl nitrogen retained
the interactions with not only the key ionic anchor D114 (3.32) but also with E206 (5.46)
through the whole simulation. Detailed analysis of compounds behavior during MD
,
simulations is provided by examining the changes in their interactions with H R (Figure 5).
3
The results indicate a relatively consistent set of ligand-protein interactions occurring
during the whole MD simulation. Most of the interactions occur within the TM3, TM5,
TM6 and TM7 and ECL2, with consistent interactions with Y115 (3.33), Y189 (45.51), E206
(
5.46), Y394 (7.35). In the case of 18, the standard orientation seems more stable during the
simulation, with most of the starting key interactions retained in the last frame.

Molecules 2021, 26, 2300
9 of 18
Figure 4. Orientations of: upper panel-KSK63 (left), 15 (middle), and 16 (right); lower panel-17 (middle), 18 (right) during the
molecular dynamics simulations in 0ps–grey, 100ps–green, 200ps–teal, 300ps–green, 400ps–violet, 500ps–orange, 600ps–yellow.
Figure 5. Interaction fingerprints of compounds KSK63, 15–18 with H R model during molecular dynamics (MD) simulation
3
for particular frames. Colored boxes indicate any interaction between the ligand and corresponding amino acid. Key
interaction amino acids were marked violet bold.

Molecules 2021, 26, 2300
10 of 18
3. Materials and Methods
3.1. Chemistry
All reagents were purchased from commercial suppliers (Alfa Aesar, Sigma-Aldrich,
CHESS GmbH, Chempur) and were used without further purification. Melting points
mp) were determined on a Büchi Melting Point M560 apparatus (BÜCHI Labortechnik
AG, Flawil, Switzerland) and were uncorrected. Liquid chromatography/mass spectra
LC/MS) were obtained using Waters TQ Detector mass spectrometer (Milford, MA, USA).
(
(
1
H NMR spectra were recorded on a Varian Mercury 300 MHz PFG (Palo Alto, CA, USA)
spectrometer in DMSO-d . Chemical shifts were expressed in parts per million (ppm) using
6
the solvent signal as an internal standard. Data have been reported in the following order:
multiplicity (s, singlet; d, doublet; t, triplet; qi, quintet; m, multiplet; br, broad; Ac, acetyl;
Prop, propionyl; t–b, tert–butyl; t–p, tert–pentyl; Ph, phenyl; Pip, piperazine), approximate
13
coupling constants J expressed in Hertz (Hz), number of protons.
were recorded on Varian-Mercury-VX 300 MHz PFG or Bruker 400 MHz spectrometer
Berlin, Germany) at 75 MHz in DMSO-d . LC–MS separation was carried out on a system
C NMR spectra
(
6
consisting of a Waters ACQUITY UPLC, coupled to a Waters TQD mass spectrometer.
Retention times (t ) were stated in minutes. For flash chromatography (FC) purification,
R
silica gel 60 (0.063–0.20 mm; Merck) and the following solvent systems were used: I-
petroleum ether:EtOAc (97.5:1.25); II-CH Cl :MeOH (95:5).
2
2
3.1.1. Procedure 1
To a solution of proper hydroxy benzophenone (0.01 mol) in acetone (60 mL), anhy-
drous potassium carbonate (1.12 g, 0.008 mol) and catalytic amount of potassium iodide
were added. The reaction mixture was stirred for 10 min at room temperature, after which
1
,3-dibromopropane (12 g, 0.06 mol) was added. The reaction mixture was then heated at
reflux for 24 h, after which the inorganic solids were filtered, and acetone was evaporated.
Remaining oils were then purified by flash chromatography (system I).
4-(3-Bromopropoxyphenyl)(phenyl)methanone (1). Synthesis from (4-hydroxyphenyl) (phenyl)
methanone (1.98 g, 0.01 mol). Obtained 2.78 g of white quickly crystalizing oil. C H BrO ,
1
6
15
2
Molecular Weight (MW) = 319.20, Yield = 87%.
-(3-Bromopropoxyphenyl)(4-chlorophenyl)methanone (
nyl)(4-chlorophenyl)methanone (2.32 g, 0.01 mol). Obtained 2.13 g of white quickly crystal-
izing oil. C H BrClO , MW = 351.99, Yield = 60%)
4
2). Synthesis from (4-hydroxyphe-
16
14
2
3.1.2. Procedure 2
Procedure was performed according to the literature reference [30]. To a solution
of freshly prepared sodium propanolate (25 mL propanol; 0.575 g, 0.025 mol Na), (4-
fluorophenyl) (4-hydroxyphenyl)methanone (5.4 g, 0.025 mol) was added and stirred at
room temperature (RT) for 15 min; 1,3-dibromopropane (15.14 g, 0.075 mol) was then added
◦
dropwise over one hour. The reaction mixture was stirred at 60 C for 3 h, and then refluxed
for another 3 h. After cooling down to RT reaction mixture was filtrated and evaporated. To
a resulting brown oil, MeOH was added, and purified by flash chromatography (system II);
obtaining 6 g of yellowish, quickly crystallizing oil.
4
-(3-Bromopropoxyphenyl)(4-fluorophenyl)methanone (3). C H BrFO , MW = 337.18, yield = 71%
16 14 2
3
.1.3. Procedure 3
Final products were obtained according to the procedure described in [22] and purified
by flash chromatography (CH Cl /MeOH, 95:5). Resulting oils were either: transformed
2
2
into oxalic acid salts, using 10% excess of oxalic acid solution in absolute ethanol in RT, and
then precipitated by addition of ethyl ether or, in the case of self-crystalizing oils, remained
free bases.

Molecules 2021, 26, 2300
11 of 18
1
-(4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)ethan-1-one hydrogen oxalate (
4, QD11). Syn-
thesis from
1 (0.47 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.47 g, 2.5 mmol).
Purified by FC. Obtained 550 mg of white oil, transferred into oxalic acid salt. Yield = 63%;
◦
mp = 136.8–140 C; UPLC/MS purity 98.20%, t = 5.05, C H N O
×
C H O , MW = 366.46,
R
22 26
2
3
2 2 4
+
1
[
M + H] 367.25. H NMR (500 MHz, DMSO-d )
δ
ppm: 7.69–7.72 (m, 2H, Ph), 7.60–7.66
6
(
m, 3H, Ph), 7.49–7.54 (m, 2H, Ph), 7.03–7.07 (m, 2H Ph), 4.11 (t, J = 6.01 Hz, 2H, CH -O),
2
3
.47–3.57 (m, 4H, Pip-2,6H), 2.75–2.83 (m, 4H, Pip-3,5H), 2.68–2.74 (m, 2H, NCH ), 1.98–2.04
2
13
(m, 2H, CH ), 1.97 (s, 3H, COCH ). C NMR (DMSO-d6): 194.96, 168.86, 164.29, 162.71,
2 3
1
38.29, 132.74, 129.78, 129.00, 114.89, 66.32, 54.13, 52.56, 52.14, 44.73, 25.18, 21.62.
-(4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)ethan-1-one ( , QD15). Synthesis
(0.53 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.19 g, 1.5 mmol). Purified
1
5
from
2
◦
by FC. Obtained 330 mg of light yellow free-base solid. Yield = 37%; mp = 123.9–126.7 C;
UPLC/MS purity 100%, t = 4.63, C H ClN O , MW = 400.90, [M + H] 401.14. H NMR
+
1
R
22 25
2
3
(500 MHz, DMSO-d6) δ ppm: 7.65–7.72 (m, 4H, Ph), 7.57 (d, J = 8.02 Hz, 2H, Ph), 7.04
(
d, J = 8.59 Hz, 2H, Ph), 4.09 (t, J=6.30 Hz, 2H, CH -O), 3.34–3.41 (m, 4H, Pip-2,6H), 2.41
2
(
1
t, J = 7.16 Hz, 2H, NCH ), 2.31–2.36 (m, 2H, Pip-3,5H), 2.27 (t, J = 4.87 Hz, 2H, Pip-3,5H),
2
13
.94 (s, 3H, COCH ), 1.88 (quin, J = 6.73 Hz, 2H, CH ). C NMR (DMSO-d ): 193.80, 168.60,
3 2 6
1
2
63.08, 137.48, 137.00, 132.76, 131.68, 129.12, 114.94, 66.80, 54.68, 53.55, 53.02, 46.18, 41.36,
6.48, 21.71.
1
-(4-(3-(4-(4-Fluorobenzoyl)phenoxy)propyl)piperazin-1-yl)ethan-1-one hydrogen oxalate (
6, QD4).
Synthesis from (0.50 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.32 g, 2.5 mmol). Puri-
3
fied by FC. Obtained 620 mg of yellow-green oil, transferred into oxalic acid salt. Yield = 70%;
◦
mp = 171.3–173.1 C; UPLC/MS purity 100%, t = 4.26, C H FN O
×
C H O , MW = 384.45
R
22 25
2
3
2 2 4
+
1
(
+90.04), [M + H] 385.19. H NMR (500 MHz, DMSO-d )
δ
ppm: 7.69–7.75 (m, 4H, Ph),
6
7
4
2
.32–7.37 (m, 2H, Ph), 7.04–7.07 (m, 2H, Ph), 4.12 (t, J = 6.01 Hz, 2H, CH -O), 3.57–3.66 (m,
2
H, Pip-3,5H), 3.01–3.07 (m, 4H, Pip-2,6H), 2.97 (br. s., 2H, NCH ), 2.05–2.14 (m, 2H, CH2),
2
.00 (s, 3H, COCH3). 13C NMR (DMSO-d ): 193.63, 169.01, 163.88, 163.68, 162.59, 134.76,
6
1
32.68, 129.95, 116.16, 115.98, 114.93, 66.06, 53.77, 51.95, 51.59, 43.83, 24.37, 21.56.
4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)(cyclopropyl)methanone hydrogen oxalate (
Synthesis from (0.47 g, 1.5 mmol) and cyclopropyl(piperazin-1-yl)methanone (0.23 g,
(
7, QD14).
1
1
.5 mmol). Purified by FC. Obtained 520 mg of white oil, transferred into oxalic acid salt.
◦
Yield = 63%; mp = 167.3–168.6 C; UPLC/MS purity 100%, t = 4.30, C H N O
MW = 392.21, [M + H] 393.41. H NMR (500 MHz, DMSO-d )
×
C H O ,
2 2 4
R
24 28
2
3
+
1
δ
ppm: 7.69–7.74 (m, 2H,
6
Ph), 7.59–7.66 (m, 3H, Ph), 7.49–7.54 (m, 2H, Ph), 7.03–7.08 (m, 2H, Ph), 4.12 (t, J = 6.30 Hz,
2
2
H, CH -O), 3.84 (br. s., 2H, Pip-3,5H), 3.60 (br. s., 2H, Pip-3,5H), 2.92–3.03 (m, 6H, Pip-
2
1
3
,6H + NCH ), 2.03–2.11 (m, 2H, CH ), 1.93–1.99 (m, 1H, Cp), 0.66–0.74 (m, 4H, Cp).
C
2
2
NMR (DMSO-d ): 194.96, 171.76, 164.19, 162.63, 138.28, 132.73, 129.78, 129.00, 114.90, 66.18,
6
53.92, 24.69, 10.73, 7.70
(4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)(cyclopropyl)methanone hydrogen oxalate
(8, QD19). Synthesis from 2 (0.28 g, 0.78 mmol) and cyclopropyl(piperazin-1-yl)methanone
(
0.12 g, 0.78 mmol). Purified by FC. Obtained 220 mg of light-yellow oil solid, transferred
◦
into oxalic acid salt. Yield = 26%; mp = 176.9–179.2 C; UPLC/MS purity 100%, t = 4.99,
C H ClN O , MW = 426.94, [M + H] 427.41. H NMR (500 MHz, DMSO-d )
R
+
1
δ ppm:
24
27
2
3
6
7
4
2
.69–7.73 (m, 2H, Ph), 7.64–7.69 (m, 2H, Ph), 7.56–7.60 (m, 2H, Ph), 7.03–7.08 (m, 2H, Ph),
.12 (t, J = 6.01 Hz, 2H, CH -O), 3.83 (br. s., 2H, Pip-3,5H), 3.60 (br. s., 2H, Pip-3,5H),
2
.91–3.03 (m, 4H, Pip-2,6H), 2.88 (br. s., 2H, NCH ), 2.02–2.10 (m, 2H, CH ), 1.92–2.00 (m,
2
2
1
1
H, Cp), 0.65–0.73 (m, 4H, Cp).). 13C NMR (DMSO-d ): 193.83, 171.75, 164.12, 162.79, 137.52,
6
36.95, 132.75, 131.69, 129.13, 114.98, 66.22, 53.91, 24.71, 10.73, 7.70
(4-(3-(4-(Cyclopropanecarbonyl)piperazin-1-yl)propoxy)phenyl)(4-fluorophenyl)methanone hydro-
gen oxalate ( , QD8). Synthesis from (0.50 g, 1.5 mmol) and cyclopropyl(piperazin-1-
9
3
yl)methanone (0.23 g, 1.5 mmol). Purified by FC. Obtained 530 mg of light oil, transferred
◦
into oxalic acid salt. Yield = 64%; mp = 181.1–182.7 C; UPLC/MS purity 100%, t = 4.48,
R

Molecules 2021, 26, 2300
12 of 18
+
1
C H FN O
DMSO-d6)
×
C H O , MW = 410.48 (+90.04), [M + H] 411.18. H NMR (500 MHz,
2 2 4
24
27
2
3
δ ppm: 7.68–7.76 (m, 4H, Ph), 7.32–7.37 (m, 2H, Ph), 7.03–7.07 (m, 2H, Ph), 4.12
(
t, J = 6.01 Hz, 2H, CH -O), 3.83 (br. s., 2H, Pip-2,6H), 3.59 (br. s., 2H, Pip-2,6H), 2.90–3.00
2
(
1
m, 4H, Pip-3,5H), 2.87 (br. s., 2H, NCH ), 2.03–2.11 (m, 2H, CH ), 1.96 (tt, J = 7.73, 4.87 Hz,
2
2
H, Cp), 0.66–0.74 (m, 4H, Cp). 13C NMR (DMSO-d ): 193.62, 171.75, 165.87, 164.08, 162.65,
6
134.80, 132.69, 129.90, 116.15, 115.98, 114.93, 66.20, 53.94, 24.74, 10.72, 7.70.
1-(4-(4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (10, QD10). Synthesis
from
1
(0.47 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.51 g, 2.5 mmol). Pu-
◦
rified by FC. Obtained 300 mg of yellow free-base solid. Yield = 62%; mp = 148.4–151.4 C
UPLC/MS purity 100%, t = 4.94, C H N O , MW = 442.56, [M + H] 443.22. H NMR
;
+
1
R
28 30
2
3
(
(
4
500 MHz, DMSO-d6)
m, 3H, Ph), 7.49–7.53 (m, 2H, Ph), 7.03–7.07 (m, 2H, Ph), 6.92 (d, J = 9.17 Hz, 2H, Ph),
.10 (t, J = 6.30 Hz, 2H, CH -O), 3.26–3.31 (m, 6H, Pip-2,6H + NCH ), 2.45–2.47 (m, 4H,
δ ppm: 7.76 (d, J = 9.17 Hz, 2H, Ph), 7.69–7.72 (m, 2H, Ph), 7.59–7.66
2
2
13
Pip-3,5H), 2.41 (s, 3H, COCH ), 1.91 (quin, J = 6.73 Hz, 2H, CH ). C NMR (DMSO-d6):
3
2
1
96.08, 194.94, 162.95, 154.38, 138.33, 132.75, 130.60, 129.77, 128.98, 114.88, 113.58, 66.81,
54.77, 52.99, 47.18, 26.62.
1-(4-(4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (11, QD17). Syn-
thesis from
2
(0.53 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.3 g, 1.5 mmol).
◦
Purified by FC. Obtained 260 mg of yellowish free-base solid. Yield = 35%; mp = 180.6–183.4 C
UPLC/MS purity 100%, t = 5.49, C H ClN O , MW = 477.00, [M + H] 477.18. H NMR
;
+
1
R
28 29
2
3
(500 MHz, DMSO-d6)
δ ppm: 7.76 (m, J = 8.88 Hz, 2H, Ph), 7.65–7.71 (m, 4H, Ph), 7.56–7.59
(m, 2H, Ph), 7.04–7.08 (m, 2H, Ph), 6.93 (d, J = 9.16 Hz, 2H, Ph), 4.11 (t, J = 6.30 Hz, 2H,
CH -O), 3.29–3.31 (m, 10H, Pip-3,5H + Pip-2,6H + NCH ), 2.41 (s, 3H, COCH ), 1.88–1.95
2
2
3
13
(
m, 2H, CH2). C NMR (DMSO-d ): 196.30, 163.10, 158.53, 132.79, 131.70, 130.61, 129.45,
6
129.13, 114.96, 113.59, 63.87, 53.00, 26.64.
1
-(4-(4-(3-(4-(4-Fluorobenzoyl)phenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (12, QD3). Syn-
thesis from
3 (0.50 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.51 g,
2
.5 mmol). Purified by FC. Obtained 230 mg of yellowish free-base solid. Yield = 31%;
◦
mp = 162–163.6 C; UPLC/MS purity 98.12%, t = 5.11, C H FN O , MW = 460.55,
R
28 29
2
3
+
1
[M + H] 461.23. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.78 (m, 6H, Ph), 7.34
(
(
t, J = 8.88 Hz, 2H, Ph), 7.06 (m, J = 9.17 Hz, 2H, Ph), 6.93 (m, J = 9.16 Hz, 2H, Ph), 4.11
t, J = 6.30 Hz, 2H, CH -O), 2.44–2.47 (m, 10H, Pip-3,5H + Pip-2,6H + NCH2), 2.41 (s, 3H,
2
13
COCH ), 1.92 (m, J = 6.87 Hz, 2H, CH ). C NMR (DMSO-d ): 196.12, 162.97, 132.72,
3
2
6
130.60, 129.70, 116.14, 115.97, 114.91, 113.59, 66.83, 54.77, 53.00, 47.18, 26.63, 26.54.
(
4-(3-(4-(4-Nitrophenyl)piperazin-1-yl)propoxy)phenyl)(phenyl)methanone (13, QD12). Synthesis
from
1
(0.47 g, 1.5 mmol) and 1-(4-nitrophenyl)piperazine (0.31 g, 1.5 mmol). Purified by
◦
FC. Obtained 130 mg of yellow, quick crystalizing oil. Yield = 18%; mp = 182.3–183.9 C;
UPLC/MS purity 100%, t = 5.28, C H N O , MW = 445.51, [M + H] 446.40. H NMR
+
1
R
26 27
3
4
(500 MHz, DMSO-d6) δ ppm: 8.00–8.03 (m, 2H, Ph), 7.69–7.72 (m, 2H, Ph), 7.63–7.66 (m, 2H,
Ph), 7.60–7.62 (m, 1H, Ph), 7.49–7.53 (m, 2H, Ph), 7.04–7.07 (m, 2H, Ph), 7.04–7.07 (m, 2H,
Ph), 4.11 (t, J = 6.44 Hz, 2H, CH -O), 3.40–3.44 (m, 8H, Pip-3,5H + Pip-2,6H), 3.27 (m, 2H,
2
13
NCH ), 1.92 (m, J = 7.02 Hz, 2H, CH ). C NMR (DMSO-d ): 196.19, 168.03, 164.95, 156.22,
2
2
6
132.77, 129.78, 129.00, 126.26, 114.89, 113.13, 66.80, 52.87, 46.84, 25.76.
(4-Chlorophenyl)(4-(3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)phenyl)methanone (14, QD20). Syn-
thesis from
2
(0.53 g, 1.5 mmol) and 1-(4-nitrophenyl)piperazine (0.31 g, 1.5 mmol). Purified
◦
by FC. Obtained 500 mg of yellow, quick crystallizing oil. Yield = 67%; mp = 154.5–155.8 C
UPLC/MS purity 100%, t = 5.83, C H ClN O , MW = 479.96, [M + H] 480.39. H NMR
;
+
1
R
26 27
3
4
(
500 MHz, DMSO-d6)
δ
ppm: 8.01 (d, J = 9.74 Hz, 2H, Ph), 7.66 (d, J = 8.02 Hz, 2H, Ph),
.70 (d, J = 9.17 Hz, 2H, Ph), 7.57 (d, J = 8.59 Hz, 2H, Ph), 7.05 (d, J = 8.59 Hz, 2H, Ph),
.99 (d, J = 9.17 Hz, 2H, Ph), 4.11 (t, J = 6.01 Hz, 2H, CH -O), 3.41 (br. s., 4H, Pip-3,5H),
7
6
3
2
13
.23–3.37 (m, 6H, Pip-2,6H + NCH ), 1.88–1.96 (m, 2H, CH ). C NMR (DMSO-d ): 193.81,
2
2
6

Molecules 2021, 26, 2300
13 of 18
1
63.09, 155.28, 137.48, 137.37, 137.00, 132.77, 131.68, 129.12, 126.24, 114.95, 113.12, 66.83,
54.65, 52.86, 46,85, 26.51.
(4-Fluorophenyl)(4-(3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)phenyl)methanone (15, QD6). Syn-
thesis from
3
(1 g, 3 mmol) and 1-(4-nitrophenyl)piperazine (0.62 g, 3 mmol). Purified by
◦
FC. Obtained 245 mg of yellow, quick crystallizing oil. Yield = 34%; mp = 173.3–174.2 C
UPLC/MS purity 100%, t = 5.52, C H FN O , MW = 463.51, [M + H] 464.15. H NMR
;
+
1
R
26 27
3
4
(
(
(
(
500 MHz, DMSO-d6) δ ppm: 8.01 (m, J = 9.74 Hz, 2H, Ph), 7.67–7.76 (m, 4H, Ph), 7.34
t, J = 8.88 Hz, 2H, Ph), 7.06 (m, J = 9.16 Hz, 2H, Ph), 6.99 (d, J = 9.74 Hz, 2H, Ph), 4.11
t, J = 6.30 Hz, 2H, CH -O), 3.39–3.44 (m, 4H, Pip-3,5H), 3.29 (s, 6H, Pip-2,6H + NCH ), 1.92
2
2
13
quin, J = 6.59 Hz, 2H, CH2). C NMR (DMSO-d ): 193.62, 162.96, 155.28, 137.36, 132.71,
6
1
29.70, 126.25, 116.14, 115.97, 114.91, 113.12, 66.80, 54.66, 52.86, 46.85, 26.51.
4-(3-(4-(3-Methoxyphenyl)piperazin-1-yl)propoxy)phenyl)(phenyl)methanone hydrogen oxalate
16, QD13). Synthesis from (0.47 g, 1.5 mmol) and 1-(3-methoxyphenyl)piperazine
(
(
(
1
0.28 g, 1.5 mmol). Purified by FC. Obtained 650 mg of yellow oil, transferred into
◦
oxalic acid salt. Yield = 87%; mp = 143.6–146.1 C; UPLC/MS purity 100%, t = 5.32,
C H N O
R
+
1
×
C H O , MW = 430.55 (+ 90.04), [M + H] 431.44. H NMR (500 MHz,
27
30
2
3
2 2 4
DMSO-d6)
.05–7.12 (m, 3H, Ph), 6.53 (dd, J = 8.02, 2.00 Hz, 1H, Ph), 6.47 (t, J = 2.29 Hz, 1H, Ph), 6.38
dd, J = 8.16, 1.86 Hz, 1H, Ph), 4.13 (t, J=6.01 Hz, 2H, CH -O), 3.68 (s, 3H, CH ), 3.31 (br. s.,
δ ppm: 7.70–7.73 (m, 2H, Ph), 7.60–7.66 (m, 3H, Ph), 7.50–7.54 (m, 2H, Ph),
7
(
2
3
4
H, Pip-3,5H), 3.10 (br. s., 4H, Pip-2,6H), 3.02–3.07 (m, 2H, NCH ), 2.08–2.14 (m, 2H, CH ).
2 2
1
3
C NMR (DMSO-d ): 194.98, 164.54, 162.63, 160.80, 160.78, 151.87, 138.29, 132.75, 132.73,
6
129.80, 129.01, 114.93, 108.81, 105.45, 102.47, 66.18, 55.49, 53.79, 51.78, 46.69, 24.54.
(4-Chlorophenyl)(4-(3-(4-(3-methoxyphenyl)piperazin-1-yl)propoxy)phenyl)methanone hydrogen
oxalate (17, QD18). Synthesis from (0.53 g, 1.5 mmol) and 1-(3-methoxyphenyl)piperaz-
2
ine (0.28 g, 1.5 mmol). Purified by FC. Obtained 350 mg of yellow oil, transferred into
◦
oxalic acid salt. Yield = 46%; mp = 100.2–102.4 C; UPLC/MS purity 100%, t = 5.95,
R
+
1
C H ClN O , MW = 464.98, [M + H] 465.44. H NMR (500 MHz, DMSO-d )
δ
ppm:
.65–7.71 (m, 4H, Ph), 7.55–7.59 (m, 2H, Ph), 7.03–7.07 (m, 3H, Ph), 6.47 (dd, J = 8.02, 1.72 Hz,
H, Ph), 6.39 (t, J = 2.29 Hz, 1H, Ph), 6.39 (t, J = 2.29 Hz, 1H, Ph), 4.10 (t, J = 6.30 Hz, 2H, CH2-
O), 3.30 (s, 3H, CH ), 3.05–3.11 (m, 4H, Pip-3,5H), 2.42–2.47 (m, 6H, Pip-2,6H + NCH ), 1.90
27
29
2
3
6
7
1
3
2
13
(
quin, J = 6.73 Hz, 2H, CH2). C NMR (DMSO-d ): 193.80, 163.11, 160.71, 152.94, 137.48,
6
1
37.00, 132.76, 131.68, 129.11, 114.95, 108.52, 104.58, 101.94, 66.88, 55.37, 53.29, 48.70, 26.58.
4-Fluorophenyl)(4-(3-(4-(3-methoxyphenyl)piperazin-1-yl)propoxy)phenyl)methanone hydrogen
oxalate (18, QD9). Synthesis from (0.4 g, 1 mmol) and 1-(3-methoxyphenyl)piperazine
0.19 g, 1 mmol). Purified by FC. Obtained 430 mg of light oil, transferred into oxalic acid
(
3
(
◦
salt. Yield = 57%; mp = 127.4–129.7 C; UPLC/MS purity 100%, t = 5.47, C H FN O ,
MW = 448.54, [M + H] 449.20. H NMR (500 MHz, DMSO-d ) δ ppm: 7.69–7.75 (m, 4H,
R
27 29
2
3
+
1
6
Ph), 7.32–7.37 (m, 2H, Ph), 7.05–7.12 (m, 3H, Ph), 6.53 (dd, J = 8.31, 2.00 Hz, 1H, Ph), 6.47 (t,
J = 2.00 Hz, 1H, Ph), 6.38 (dd, J = 8.02, 2.29 Hz, 1H, Ph), 4.13 (t, J = 6.30 Hz, 2H, CH -O),
2
3
.68 (s, 3H, CH ), 3.31 (br. s., 4H, Pip-3,5H), 3.09 (br. s., 4H, Pip-2,6H), 3.01–3.06 (m, 2H,
3
13
NCH ), 2.07–2.14 (m, 2H, CH ). C NMR (DMSO-d ): 193.63, 165.87, 164.60, 162.64, 160.78,
2
2
6
151.90, 134.80, 132.69, 130.33, 129.92, 116.16, 114.94, 108.79, 105.42, 102.45, 66.20, 55.47, 53.81,
51.83, 46.77, 24.61
3
[3
.2. Pharmacology
α
H]N -Methylhistamine hH R Displacement Assay
3
The displacement binding assay was carried out as described by Kottke et al. with
slight modifications [30
,31]. Frozen crude membrane preparations of HEK-293 cells stably
expressing the recombinant full length hH R were thawed, homogenized, and incubated
3
3
α
for 90 min at RT (continuously shaking) with [ H]N -Methylhistamine (2 nM) and dif-
ferent concentrations of the test compounds (five to eleven appropriate concentrations
−
11
−4
and 10 M, dilutions prepared robot-assisted with a Tecan Freedom EVO
between 10

Molecules 2021, 26, 2300
14 of 18
(Männedorf, Switzerland) or HP D300 Dispenser (Männedorf, Switzerland) in a final as-
say volume of 200
µL per well. Non-specific binding was determined in the presence
of pitolisant (10 M). The bound radioligand was separated from the free radioligand
µ
by filtration through GF/B filters (pretreated with 0.3% (m/v) polyethylenimine) using
an Inotech cell harvester (Dottikin, Switzerland). Unbound radioligand was removed by
three washing steps with ice-cold water. Scintillation cocktail was added, and the liquid
scintillation counting was performed with a Perkin Elmer TriluxBeta counter (Perkin Elmer,
Rodgau, Germany).
Scintillation data (c.p.m.) corrected for non-specific binding were analyzed using
GraphPad Prism (V6.01, San Diego, CA, USA) software, using non-linear least squares/regr-
ession fit. K values were calculated from IC values according to the Cheng–Prusoff equa-
i
50
tion [35]. Statistical analyses were performed on pK values from at least three experiments,
i
each performed, at least, in duplicates. Mean affinity values (K ) were transferred into
i
nanomolar concentrations (with 95% confidence interval).
3.3. In Vitro Antioxidant Activity
Antioxidant properties of compounds were tested in vitro in two different assays:
the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [36
,37] and the FeCl reduction activity
3
assay (FRAP, ferric reducing antioxidant power) [37]. The tested compounds and ascorbic
−
2
acid were dissolved in DMSO at the 10 mol/L concentration, and in ethanol at the
−
4
concentration of 10 mol/L respectively. Moreover, 30% and 50% ascorbic acid activity
cut-off for DPPH and FRAP assays respectively were set ad hoc.
3.3.1. DPPH Assay
DPPH is a molecule containing a stable free radical. The reduction of DPPH (purple)
in ethanol solution takes place in the presence of a hydrogen-donating antioxidant due to
the formation of the non-radical form of DPPH-H (yellow). This transformation results in
color change from purple to yellow, which can be measured spectrophotometrically. Thus,
the decrease in absorbance is proportional to the decrease in concentration of DPPH (free
radical) and discoloration of reaction mixture indicates the scavenging of free radicals by a
tested antioxidant.
In order to determine antioxidant capacity, 20
µL of the compound solutions (dissolved
in 96% ethanol) was mixed with 180 L of 0.3 mM ethanolic DPPH solution. The change in
µ
the absorbance was detected at 517 nm after 30 min of incubation. Results were expressed as
percentage decrease in absorbance of the tested sample, compared to the sample containing
the solvent + reaction mixture (blank with maximum concentration of the DPPH radical).
L-ascorbic acid was used as a reference compound. Calculations were made using MS
Excel, from the proportion where the change of absorbance in the vitamin C (vit. C) group
was treated as 100%, according to Equation (1):
∆
∆
A × 100
b
%
activity of Vit.C =
(1)
A
Vit. C
Calculation of % Vit. C activity: ∆A -the change in the absorbance of the test sample
b
and ∆AVit. C is change in the absorbance of reference sample, or with vit. C.
3.3.2. FRAP Assay
A modified method of Benzie and Strain [38] was adopted for the FRAP assay. The
stock solutions included 300 mM acetate buffer (3.1 g of C H NaO
×
3H O and 16 mL
2
3
2
2
of C H O ), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and
2
4
2
2
0 mM FeCl3
of acetate buffer, 1 part of TPTZ solution, and 1 part of FeCl3
of 180 L of the FRAP solution was mixed with 20 L of the tested compound solution
and incubated at room temperature for 10 min in the dark. Readings of the color product
ferrous tripyridyl triazine complex) were then taken at 593 nm against ethanol. Results are
×
6H O solution. The working solution was prepared by mixing 10 parts
2
×
6H O solution. A portion
2
µ
µ
(

Molecules 2021, 26, 2300
15 of 18
expressed as an increase in absorbance of the test sample compared to a sample containing
the solvent. L-ascorbic acid was used as reference compound.
3.4. Molecular Modeling
For docking purposes, Schrödinger Maestro Suite (v. 11.5.011, Release 2019-01) was
used [39]. Ligands were built in either their forms (protonated N1 piperazine nitrogen,
structure charge +1) or non-ionized forms. Bioactive conformations were generated using
ConfGen module [40,41] (force field: OPLS3, water environment; minimization method:
Polak-Ribier Conjugate Gradient (PRCG) with maximum iterations of 2500 and a conver-
gence threshold of 0.05; conformational search of 100 steps per rotational bond). For all
compounds, the five lowest energy conformers were selected for docking studies. Possi-
ble binding pocket adaptation in the presence of certain ligands was examined using an
induced fit refinement protocol [42] with 17. In order to validate the methods used, the
ligand was then re-docked with high confidence. The site was then centered on the ligand.
Docking to rigid the form of the receptor was performed using the Glide module [43
,44]
(extra precision, flexible ligand sampling, and a maximum of five poses per conformer).
Ligands were rated according to their position in the binding pocket, interactions with
the binding pocket amino acids, as well as the docking score value. Ligand–receptor
visualizations were generated using Schrödinger Maestro.
Theoretical pKa values were calculated using the Jaguar module [45,46].
Dynamic simulations (in time of 600 ps, T = 300 K) were performed using the Nosé–
Poincaré–Andersen equations of motion, forcefield AMBER10: EHT; R-Field 1:80, cutoff
(
8,10), and performed in MOE v. 2019.1 [47].
4. Conclusions
Looking for a new, active structures, is not an easy task. It even becomes harder when
the reference structure is of one of the highest affinity hH R ligands obtained to date; thus,
3
increasing the possibility of ending up with lower affinity compounds. However, obtaining
such compounds teaches us a lesson on structural uniqueness.
In an attempt to find new possibly dual acting H R ligands, in this study we ob-
3
tained a series of compounds of moderate hH R affinity, ranging around K of 500 nM
3
i
(
17), on the one hand, and on the other hand, expressing antioxidative properties (e.g., 16,
−
4
hH R K = 592 nM, showing 50–60% of vitamin C activity at a 10 mol/L). To our knowl-
3
i
edge, alongside a recently published Tetratarget ligand Contilisant and its predecessor
ASS234 [27], obtained herein derivatives are some of the few histamine H R ligands with
3
described antioxidative properties. From a structural point of view, it appeared that simple
(cyclo)alkyl substituents at the 4-position of piperazine negatively impact the ligand’s
activity at the desired target, when compared to KSK63 (Figure 1). Nonetheless, in the
case of simple acyl moiety, a benzene ring separating the acyl from the basic core, through
additional interactions, might force slightly higher affinity within our novel series (e.g., 10
vs. 4), yet still on the low level. Interestingly, docking into the histamine H R homology
3
model revealed two putative binding modes, with ionic key interactions retained in both
cases. The stability of such obtained complexes was demonstrated by MD simulations.
This also allowed for possible explanation of KSK63 high affinity. Last, but not least,
in an attempt to find possible dual acting ligands, selected compounds were tested for
antioxidant properties. Among others, compound 16 (hH R K = 592 nM) showed the
3
i
−
4
strongest antioxidant properties at a 10 mol/L concentration. Compound 16 not only
significantly reduced the amount of free radicals showing 50–60% of AA activity, but had
also exhibited antioxidative properties in general. Despite yet unknown mechanisms and
moderate hH R affinity, compound 16 (QD13) constitutes a novel, potential dual acting
3
H R ligand-promising starting point towards treatment of neurologic disorders associated
3
with increased neuronal oxidative stress.
In conclusion, although the obtained series of compounds appeared to be weak
histamine H receptors ligands, they might be valuable pharmacological tools in the
3

Molecules 2021, 26, 2300
16 of 18
search for novel molecules, targeting oxidative stress-based diseases. Identification of
strong histamine H receptor ligands, with antioxidative properties, will drive our further,
3
planned studies.
Supplementary Materials: The following are available online, The Supplementary Material contain
Table S1: Calculated pKa values for piperazine N1 and N4 nitrogen, Table S2: Putative binding poses
for non-protonated conformers, Table S3: Putative binding poses for protonated conformers, Table
S4: Molecular dynamics frames alignment for selected ligands and Figure S1: Antioxidant activity of
the tested compounds in DPPH and FRAP assay.
Author Contributions: Conceptualization, K.J.K. and M.K.; methodology, K.J.K., M.K. and D.R.-L.;
validation, K.S., K.M. and D.R.-L.; formal analysis, K.S.; investigation, K.J.K., K.M. and D.R.-L.; data
curation, K.J.K., M.K. and K.S.; writing—original draft preparation, K.J.K.; writing—review and
editing, K.K.-K., M.K., D.R.-L., H.S. and K.S.; visualization, K.K.-K. and M.K.; supervision, K.K.-K.
and H.S.; project administration, M.K.; funding acquisition, M.K. All authors have read and agreed
to the published version of the manuscript.
Funding: This study was partly supported by statutory funds from the Faculty of Pharmacy Jagiel-
lonian University Medical College, Krakow, Poland (No: N42/DBS/000041 and N42/DBS/000068).
This work was partly supported by National Science Center, Poland, granted on the basis of decision
no: 2016/23/B/NZ7/01063. Further support by COST Actions CM1103, CA15135, CA18133, and
CA18240 as well as DFG INST 208/664e1 FUGG (Germany) is also kindly acknowledged (to HS).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
References
1
2
3
4
.
.
.
.
Arrang, J.M.; Garbarg, M.; Schwartz, J.C. Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine
receptor. Nature 1983, 302. [CrossRef]
Berlin, M.; Boyce, C.W.; De Lera Ruiz, M. Histamine H3 receptor as a drug discovery target. J. Med. Chem. 2011, 54, 26–53.
[CrossRef] [PubMed]
Schlicker, E.; Malinowska, B.; Kathmann, M.; Göthert, M. Modulation of neurotransmitter release via histamine H3 heteroreceptors.
Fundam. Clin. Pharmacol. 1994, 8, 128–137. [CrossRef] [PubMed]
Sadek, B.; Saad, A.; Sadeq, A.; Jalal, F.; Stark, H. Histamine H3 receptor as a potential target for cognitive symptoms in
neuropsychiatric diseases. Behav. Brain Res. 2016, 312, 415–430. [CrossRef] [PubMed]
5
6
.
.
Haas, H.L.; Sergeeva, O.A.; Selbach, O. Histamine in the nervous system. Physiol. Rev. 2008, 88, 1183–1241. [CrossRef]
Ła z˙ ewska, D.; Kie c´ -Kononowicz, K. Progress in the development of histamine H3 receptor antagonists/inverse agonists: A
patent review (2013-2017). Expert Opin. Ther. Pat. 2018, 28, 175–196. [CrossRef] [PubMed]
7
.
Tiligada, E.; Zampeli, E.; Sander, K.; Stark, H. Histamine H3 and H4 receptors as novel drug targets. Expert Opin. Investig. Drugs
2
009, 18, 1519–1531. [CrossRef]
8
9
.
.
Syed, Y.Y. Pitolisant: First Global Approval. Drugs 2016, 76, 1313–1318. [CrossRef]
Troxler, T.; Feuerbach, D.; Zhang, X.; Yang, C.R.; Lagu, B.; Perrone, M.; Wang, T.L.; Briner, K.; Bock, M.G.; Auberson, Y.P.
The Discovery of LML134, a Histamine H3 Receptor Inverse Agonist for the Clinical Treatment of Excessive Sleep Disorders.
ChemMedChem 2019, 14. [CrossRef]
1
1
1
1
0. A Study to Assess the Wakefulness Promoting Effect, Safety, Tolerability, and Pharmacokinetics (PK) of LML134 in Shift Work
Disorder. Available online: http://clinicaltrials.gov/ct2/show/NCT03141086 (accessed on 13 April 2021).
1. Bhowmik, M.; Khanam, R.; Vohora, D. Histamine H3 receptor antagonists in relation to epilepsy and neurodegeneration: A
systemic consideration of recent progress and perspectives. Br. J. Pharmacol. 2012, 167, 1398–1414. [CrossRef]
2. Badenhorst, H.E.; Maharaj, D.S.; Malan, S.F.; Daya, S.; van Dyk, S. Histamine-3 receptor antagonists reduce superoxide anion
generation and lipid peroxidation in rat brain homogenates. J. Pharm. Pharmacol. 2005, 57, 781–785. [CrossRef] [PubMed]
3. Mani, V.; Jaafar, S.M.; Azahan, N.S.M.; Ramasamy, K.; Lim, S.M.; Ming, L.C.; Majeed, A.B.A. Ciproxifan improves cholinergic
transmission, attenuates neuroinflammation and oxidative stress but does not reduce amyloid level in transgenic mice. Life Sci.
2
017, 180, 23–35. [CrossRef] [PubMed]

Molecules 2021, 26, 2300
17 of 18
1
1
1
4. Akhtar, M.; Pillai, K.K.; Vohora, D. Effect of thioperamide on oxidative stress markers in middle cerebral artery occlusion model
of focal cerebral ischemia in rats. Hum. Exp. Toxicol. 2008, 27, 761–767. [CrossRef] [PubMed]
5. Akhtar, M.; Pillai, K.K.; Yohora, D. Effect of thioperamide on modified forced swimming test-induced oxidative stress in mice.
Basic Clin. Pharmacol. Toxicol. 2005, 97, 218–221. [CrossRef]
6. Alachkar, A.; Azimullah, S.; Ojha, S.K.; Beiram, R.; Łazewska, D.; Kie c´ -Kononowicz, K.; Sadek, B. The neuroprotective effects
of histamine h3 receptor antagonist E177 on pilocarpine-induced status epilepticus in rats. Molecules 2019, 24, 4106. [CrossRef]
[PubMed]
1
7. Alachkar, A.; Azimullah, S.; Lotfy, M.; Adeghate, E.; Ojha, S.K.; Beiram, R.; Ła z˙ ewska, D.; Kie c´ -Kononowicz, K.; Sadek,
B. Antagonism of histamine H3 receptors alleviates pentylenetetrazole-induced kindling and associated memory deficits by
mitigating oxidative stress, central neurotransmitters, and c-fos protein expression in rats. Molecules 2020, 25, 1575. [CrossRef]
8. Mahmood, D.; Khanam, R.; Pillai, K.K.; Akhtar, M. Reversal of oxidative stress by histamine H 3 receptor-ligands in experimental
models of schizophrenia. Arzneim. Forsch. Drug Res. 2012, 62, 222–229. [CrossRef]
9. Eissa, N.; Jayaprakash, P.; Azimullah, S.; Ojha, S.K.; Al-Houqani, M.; Jalal, F.Y.; Ła z˙ ewska, D.; Kie c´ -Kononowicz, K.; Sadek, B. The
histamine H3R antagonist DL77 attenuates autistic behaviors in a prenatal valproic acid-induced mouse model of autism. Sci.
Rep. 2018, 8. [CrossRef]
1
1
2
0. Hass, C.; Panda, B.P.; Khanam, R.; Najmi, A.K.; Akhtar, M. Histamine H3 Receptor Agonist Imetit Attenuated Isoproterenol
Induced Renin Angiotensin System and Sympathetic Nervous System Overactivity in Myocardial Infarction of Rats. Drug Res.
2
016, 66, 324–329. [CrossRef]
1. Walter, M.; Stark, H. Histamine receptor subtypes: A century of rational drug design. Front. Biosci. Sch. 2012, 4, 461–488.
CrossRef]
2. Szczepa n´ ska, K.; Karcz, T.; Mogilski, S.; Siwek, A.; Kuder, K.J.; Latacz, G.; Kubacka, M.; Hagenow, S.; Lubelska, A.; Olejarz, A.;
et al. Synthesis and biological activity of novel tert-butyl and tert-pentylphenoxyalkyl piperazine derivatives as histamine H3R
ligands. Eur. J. Med. Chem. 2018, 152, 223–234. [CrossRef] [PubMed]
2
2
[
2
2
3. Szczepa n´ ska, K.; Karcz, T.; Kota n´ ska, M.; Siwek, A.; Kuder, K.J.; Latacz, G.; Mogilski, S.; Hagenow, S.; Lubelska, A.; Sobolewski,
M.; et al. Optimization and preclinical evaluation of novel histamine H3 receptor ligands: Acetyl and propionyl phenoxyalkyl
piperazine derivatives. Bioorg. Med. Chem. 2018, 26, 6056–6066. [CrossRef] [PubMed]
4. Szczepa n´ ska, K.; Karcz, T.; Siwek, A.; Kuder, K.J.; Latacz, G.; Bednarski, M.; Szafarz, M.; Hagenow, S.; Lubelska, A.; Olejarz-Maciej,
A.; et al. Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives as an active and
selective histamine H3 receptor ligands. Bioorg. Chem. 2019, 91. [CrossRef]
2
5. Schwartz, J.C. The histamine H3 receptor: From discovery to clinical trials with pitolisant. Br. J. Pharmacol. 2011, 163, 713–721.
[CrossRef] [PubMed]
2
6. Bautista-Aguilera, Ó.M.; Hagenow, S.; Palomino-Antolin, A.; Farré-Alins, V.; Ismaili, L.; Joffrin, P.L.; Jimeno, M.L.; Soukup, O.;
Jano cˇ ková, J.; Kalinowsky, L.; et al. Multitarget-Directed Ligands Combining Cholinesterase and Monoamine Oxidase Inhibition
with Histamine H3R Antagonism for Neurodegenerative Diseases. Angew. Chem. Int. Ed. 2017, 56. [CrossRef] [PubMed]
7. Szczepanska, K.; Kuder, K.; Kiec-Kononowicz, K. Histamine H3 receptor ligands in the group of (homo)piperazine derivatives.
Curr. Med. Chem. 2017, 25, 1609–1626. [CrossRef] [PubMed]
2
2
2
8. Mayo, J.C.; Sainz, R.M. Melatonin from an antioxidant to a classic hormone or a tissue factor: Experimental and clinical aspects
2
019. Int. J. Mol. Sci. 2020, 21, 3645. [CrossRef]
9. Bautista-Aguilera, Ó.M.; Budni, J.; Mina, F.; Medeiros, E.B.; Deuther-Conrad, W.; Entrena, J.M.; Moraleda, I.; Iriepa, I.;
López-Muñoz, F.; Marco-Contelles, J. Contilisant, a Tetratarget Small Molecule for Alzheimer’s Disease Therapy Combin-
ing Cholinesterase, Monoamine Oxidase Inhibition, and H3R Antagonism with S1R Agonism Profile. J. Med. Chem. 2018, 61.
[CrossRef]
3
0. Kuder, K.; Łazewska, D.; Latacz, G.; Schwed, J.S.; Karcz, T.; Stark, H.; Karolak-Wojciechowska, J.; Kie c´ -Kononowicz, K.
Chlorophenoxy aminoalkyl derivatives as histamine H3<R ligands and antiseizure agents. Bioorg. Med. Chem. 2016, 24.
[CrossRef]
3
3
3
3
3
3
1. Kottke, T.; Sander, K.; Weizel, L.; Schneider, E.H.; Seifert, R.; Stark, H. Receptor-specific functional efficacies of alkyl imidazoles as
dual histamine H3/H4 receptor ligands. Eur. J. Pharmacol. 2011, 654, 200–208. [CrossRef] [PubMed]
2. Khanfar, M.A.; Reiner, D.; Hagenow, S.; Stark, H. Design, synthesis, and biological evaluation of novel oxadiazole- and thiazole-
based histamine H3R ligands. Bioorg. Med. Chem. 2018, 26, 4034–4046. [CrossRef]
3. Shimamura, T.; Shiroishi, M.; Weyand, S.; Tsujimoto, H.; Winter, G.; Katritch, V.; Abagyan, R.; Cherezov, V.; Liu, W.; Han, G.W.;
et al. Structure of the human histamine H 1 receptor complex with doxepin. Nature 2011, 475. [CrossRef] [PubMed]
4. Ballesteros, J.A.; Weinstein, H. [19] Integrated methods for the construction of three-dimensional models and computational
probing of structure-function relations in G protein-coupled receptors. Methods Neurosci. 1995, 25, 366–428. [CrossRef]
5. Yung-Chi, C.; Prusoff, W.H. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes
5
0 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099–3108. [CrossRef]
6. Dziubina, A.; Szkatuła, D.; Gdula-Argasi n´ ska, J.; Kota n´ ska, M.; Filipek, B. Antinociceptive, antiedematous, and antiallodynic
activity of 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives in experimental models of pain. Naunyn. Schmiedebergs. Arch.
Pharmacol. 2019, 3, 813–827. [CrossRef]

Molecules 2021, 26, 2300
18 of 18
3
3
7. Kota n´ ska, M.; Mika, K.; Szafarz, M.; Dziubina, A.; Bednarski, M.; Müller, C.E.; Sapa; Kie c´ -Kononowicz, K. PSB 603—A known
selective adenosine A2B receptor antagonist–has anti-inflammatory activity in mice. Biomed. Pharmacother. 2021, 135, 111164.
CrossRef]
8. Benzie, I.F.F.; Strain, J.J. Ferric reducing/antioxidant power assay: Direct measure of total antioxidant activity of biological
fluids and modified version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods
Enzymol. 1999, 299, 15–27. [CrossRef]
[
3
4
4
9. Schrödinger Release, 2019-1; Schrödinger, LLC: New York, NY, USA, 2019.
0. Schrödinger Release, 2019-1: ConfGen; Schrödinger, LLC: New York, NY, USA, 2019.
1. Watts, K.S.; Dalal, P.; Murphy, R.B.; Sherman, W.; Friesner, R.A.; Shelley, J.C. ConfGen: A Conformational Search Method for
Efficient Generation of Bioactive Conformers. J. Chem. Inf. Model. 2010, 50, 534–546. [CrossRef]
2. Schrödinger Release, 2019-1; Schrödinger Suite 2019-1 Induced Fit Docking Protocol; Glide, Schrödinger, LLC; Prime, Schrödinger,
LLC: New York, NY, USA, 2019.
4
4
4
3. Schrödinger Release, 2019-1: Glide; Schrödinger, LLC: New York, NY, USA, 2019.
4. Halgren, T.A.; Murphy, R.B.; Friesner, R.A.; Beard, H.S.; Frye, L.L.; Pollard, W.T.; Banks, J.L. Glide: A New Approach for Rapid,
Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. J. Med. Chem. 2004, 47, 1750–1759. [CrossRef]
5. Schrödinger Release, 2019-1: Jaguar; Schrödinger, LLC: New York, NY, USA, 2019.
6. Bochevarov, A.D.; Harder, E.; Hughes, T.F.; Greenwood, J.R.; Braden, D.A.; Philipp, D.M.; Rinaldo, D.; Halls, M.D.; Zhang, J.;
Friesner, R.A. Jaguar: A high-performance quantum chemistry software program with strengths in life and materials sciences. Int.
J. Quantum Chem. 2013, 113, 2110–2142. [CrossRef]
4
4
4
7. Molecular Operating Environment (MOE), 2019.01; Chemical Computing Group: Montreal, QC, Canada, 2019.