Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)piperidine derivatives as histamine H3R ligands

Article abstract of DOI:10.1016/j.bmc.2017.03.031

As a continuation of our search for novel histamine H₃ receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2–21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their bi

Full text of DOI:10.1016/j.bmc.2017.03.031

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)
piperidine derivatives as histamine H3R ligands
a
a
a
a
b
a
_
Kamil J. Kuder , Dorota Łazewska , Maria Kaleta , Gniewomir Latacz , Tim Kottke , Agnieszka Olejarz ,
a
c
a
c
´
´
Tadeusz Karcz , Andrzej Fruzinski , Katarzyna Szczepanska , Janina Karolak-Wojciechowska ,
d
a,
⇑
´
Holger Stark , Katarzyna Kiec-Kononowicz
a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland
b Institute of Pharmaceutical Chemistry, Biozentrun, ZAFES, Frankfurt/Main 60438, Germany
c
_
´
´
Institute of General and Ecological Chemistry, Technical University of Łódz, Zeromskiego 116 str., Łódz 90-924, Poland
d Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Universitaetsstr. 1, Düsseldorf 40225, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
As a continuation of our search for novel histamine H3 receptor ligands a series of twenty new tert-amyl
phenoxyalkylamine derivatives (2–21) was synthesized. Compounds of four to eight carbon atoms spacer
alkyl chain were evaluated on their binding properties at human histamine H3 receptor (hH3R). The high-
est affinities were observed for pentyl derivatives 6–8 (Ki = 8.8–23.4 nM range) and among them piper-
idine derivative 6 with Ki = 8.8 nM. Structures 6, 7 were also classified as antagonists in cAMP
accumulation assay (with EC50 = 157 and 164 nM, respectively). Moreover, new compounds were also
evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of
Neurological Disorders and Stroke (USA). Seven compounds (2–4, 9, 11, 12 and 20) showed anticonvul-
sant activity at maximal electroshock (MES) test in the dose of 30 mg/kg at 0.5 h. In the subcutaneous
pentetrazole (scMET) test compound 4 showed protection at 100 and 300 mg/kg dose at mice, however
compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies
were undertaken, to explain affinity of compounds at hH3R (taking into the consideration X-ray analysis
of compound 18). In order to estimate ‘‘drug-likeness” of selected compounds in silico and experimental
evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.
Received 15 December 2016
Revised 13 March 2017
Accepted 15 March 2017
Available online xxxx
Keywords:
Histamine H3 receptor
Histamine H3 receptor ligands
Non-imidazole histamine H3R ligands
Piperidine derivatives
Anticonvulsants
Molecular docking
Drug-like properties
Ó 2017 Published by Elsevier Ltd.
1. Introduction
in the treatment of many CNS-based diseases. This assumption has
been proved by intensive pharmacological studies confirming the
Histamine mediates its action through four histamine receptors,
that belong to the G-protein coupled receptors superfamily
(GPCRs).1 While histamine H1 and H4 receptors are mainly
involved in inflammatory processes and response, histamine H2
receptors mediate the gastric acid secretion, histamine H3 recep-
tors (H3R) are involved in the central and peripheral regulation of
histamine levels.2,3 Also, by postsynaptic modulation (as heterore-
ceptors) they regulate the levels of other neurotransmitters such as
utility of histamine H3R antagonists/inverse agonists in the
treatment of Alzheimer and Parkinson’s diseases, schizophrenia,
narcolepsy, obesity and attention-deficit hyperactivity disorder
(ADHD).6,7 Dual- and multi target acting ligands addressing, apart
of H3R, various therapeutic targets have also been described.8–10
Among a number of active compounds obtained in both academia
and industry,11,12 only one histamine H3R antagonist, Pitolisant
(Bioprojet Biotech) completed phase III of clinical trials with posi-
tive results in narcolepsy and daytime sleepiness in Parkinson’s
disease,13 and is accepted by European Medicines Agency as an
orphan drug under the name of Wakix (Pitolisant hydrochloride)
by Bioprojet Pharma.14 However, safety pharmacology studies
highlighted the ability of Pitolisant to affect the QT interval in
humans, as well as its pro-convulsant potential, The European
Medicines Agency’s Committee for Medicinal Products for Human
acetylcholine, serotonin, noradrenaline, glutamate, c-aminobutyric
acid and neuropeptide Y.4,5 Therefore, blockade of these ‘‘multi-
functional” receptors might provide useful pharmacological target
⇑
Corresponding author.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
http://dx.doi.org/10.1016/j.bmc.2017.03.031
0968-0896/Ó 2017 Published by Elsevier Ltd.
Please cite this article in press as: Kuder K.J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.031

2
K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Use decided that Wakix’s benefits are greater than its risks and
recommended that it be approved for use in the EU.
2.2. Pharmacology
Despite the variety of the structures of active H3R ligands, all of
them fit to the proposed blueprint: basic, mostly cyclic, amine con-
nected by aliphatic (rigidified) linker with polar moiety, connected
with arbitrary region, that may contain polar, acidic, basic, lipophi-
lic and even metal-containing substituents.15 While amine-alipha-
tic part is mostly responsible for ligand’s affinity, the arbitrary
region might influence activity and pharmacokinetic proper-
ties.16,17 One must also pay attention to the fact, that at least six
H3R isoforms in the human and more than 20 in other species have
been found. A wide variety of such isoforms might result in differ-
ent brain expression and signaling. Thus binding heterogeneity of
ligands might be the result of different splicing variants of the
same protein.18
Basing on our previous results, as well as on those described in
literature, novel structures were designed according to proposed
pharmacophore pattern for histamine H3R ligands. The main aim
of this work was to obtain N-alkyl(substituted)piperidine ether
derivatives with expected histamine H3R affinity. As a lead struc-
ture for further modifications, previously described and obtained
in our group compound DL7719,20 has been chosen. Modifications
included: ascending alkyl chain length (4–8 methylene groups) as
2.2.1. Histamine H3 receptor affinity
All compounds in their oxalate forms were then presented for
histamine H3R in vitro binding studies, as described by Ligneau
et al.23 and Kottke et al.24
[
125I]Iodoproxyfan or [3H]N -methylhis-
a
tamine were used as the radioligands in binding assays to test the
affinity at human recombinant histamine H3R stably expressed in
CHO-K123 cells or human embryonic kidney (HEK-293) cells.24
Pharmacological results are assembled in Table 1. All of the
compounds showed good to very good affinities for human H3R
(hH3R) in Ki range of 8–325 nM.
In 1992 Lipp and co-workers25 proposed the general pharma-
cophore for histamine H3R antagonists, where nitrogen containing
ring is connected via aliphatic linker with polar moiety. Customar-
ily, the length of such linker is 3 methylene groups. One of the first
to describe active ligands with longer linker consisted of 3–6
methylene groups was Ganellin et al. in 1998,26 where the
in vitro affinity of the compounds was of high values (hH3R
Ki = 0.019–0.46 lM) independently of the linker length (3–5
methylene groups alkyl spacer).
In this work we focused on compounds with elongated alkyl
chain (4–8 methylene groups), derived from previously described,
well as modifications of piperidine moiety
piperidines, hexamethyleneimine (homopiperidine), in the basic
part of compounds (Fig. 1).
–
un/substituted
high potent compound DL77 (hH3R Ki = 8.4 nM; ED50 = 2.1 mg/kg)
19
_
described by Łazewska et al. For the series of presented ligands,
one can observe that the in vitro affinity to H3R decreases with
the elongation of the linker. However, compounds of 4 methylene
linker group whose activity is placed in-between the group of 7
and 8 methylene linker groups, are still on high level (Ki = 46–
238 nM), they seem to be an exception to this rule. Compounds
with 5 methylene linker (6–9) were the most active group (Ki
8.8–23.4 nM). For each of the presented subgroups, 3-methylpiper-
idine derivatives show the highest affinity to H3R (e.g. compounds:
3, 11, 15, 19), with, again, the exception of compound 7, that has
slightly lower affinity than its piperidine homologue 6 – the most
active compound of herein described series (hH3R Ki = 8.8 nM). In
fact compound 6 retained the affinity of reference compound –
DL77. Oppositely, the lowest affinities in the subgroups show the
4-methylpiperidine derivatives, with the exception of previously
mentioned subgroups of 7 and 8 methylene long linker, where
homopiperidine derivatives were of lowest hH3R affinity (eg. com-
pound 16 vs. 17 and 20 vs. 21).
2. Results and discussion
2.1. Chemistry
The synthesis of desired ether derivatives 2–21 was achieved
through the efficient synthetic route outlined in Scheme 1.
Para tert-amylphenoxyalkyl bromides (1a–1e, synthesis accord-
ing,21 modified by authors) were obtained in one-step alkylation of
para tert-amylphenol with
a,x-dibromoalkanes refluxed in pro-
pan-1-ol.22 Obtained precursor bromides, after the separation from
bis products, that was created during synthesis, were then coupled
with corresponding cycloalkylamines in the mixture of ethanol/
water with powdered potassium carbonate and a catalytic amount
of potassium iodide. The desired products were obtained as free
bases and isolated as oxalic acid salts.
Paying attention to the overall high affinity of the obtained
compounds, it can be assumed, that the linker length, and various
piperidine derivatives determines their affinity for histamine H3R.
2.2.2. Functional properties at histamine H3 receptor
Selected, most potent structures (6 and 7) were further investi-
gated in functional tests in order to determine their intrinsic activ-
ity. For that purpose cAMP accumulation assay at HEK293 cells
expressing recombinant human histamine H3R was employed.
Investigated compounds caused a blockade of cAMP level reduc-
tion by receptor agonist, in cells co-treated with forskolin (H3R is
a Gi-coupled receptor) and were therefore classified as histamine
H3R antagonists. Antagonist potency (EC50) was evaluated by per-
forming a dose-response curve experiment in presence of H3R ago-
nist – (R)(ꢀ)-
a-methylhistamine (15 nM, corresponding to its EC80)
and forskolin (10 mM) (Fig. 2). Obtained EC50 values were as fol-
lows: 6 – EC50 = 157 16 nM; 7 – EC50 = 164 37 nM (Fig. 2).
2.2.3. Anticonvulsant activity
Given H3R localization and their ability to influence multiple
neurotransmitter systems (eg. glutamate, GABA), targeting central
H3R’s might find its use in epilepsy treatment. Therefore, selected
(16) compounds were screened in vivo for anticonvulsant activity.
Fig. 1. Pitolisant (WakixÒ – top left),13 DL77(top right)18 and general structure of
described ligands 2–21 (bottom); hH3R – human histamine H3 receptor; rH3R – rat
histamine H3R.
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Scheme 1. General synthetic pathway for compounds 2–21.
Table 1
Structures of compounds 2–21 and their pharmacological in vitro properties.
No.
n
R
hH3R Ki [nM]
No.
n
R
hH3R Ki [nM]
DL77
2
3
4
5
6
7
8
9
3
4
4
4
4
5
5
5
5
6
6
–
–
8.4 1.3a
140 47b,c
46 22b
238 95b
68 20b
8.8c,d
12
13
14
15
16
17
18
19
20
21
6
6
7
7
7
7
8
8
8
8
4-CH3
–CH2–
–
3-CH3
4-CH3
–CH2–
–
3-CH3
4-CH3
–CH2–
60.5d
41.2d
3-CH3
4-CH3
–CH2–
–
3-CH3
4-CH3
–CH2–
–
55c,d
36.6d
64.4d
128.3d
325.9d
120.7d
308.1d
182.5d
13.6d
23.4d
21.4c,d
10
11
46.5c,d
3-CH3
33.8d
a
b
c
Data from Ref. 18.
a
[3H] N Methylhistamine as radioligand, mean values SD, as described by Kottke et al.24
.
Histamine hH3R affinity has been previously shown by Sadek et al.20
.
d
[
125I] Iodoproxyfan binding to membranes of CHO-K1 cells expressing human H3R as described by Ligneau et al.,23 data from a single experiment with each concentrations
tested at least in triplicate.
Fig. 2. cAMP accumulation studies in HEK293 cells expressing the human histamine H3 receptor, co-treated with forskolin, (R)(ꢀ)-
6 (left) and 7 (right).
a-methylhistamine and tested compounds:
All tests were carried out at National Institute of Neurological
Disorders and Stroke, National Institutes of Health (Rockville,
USA) within Anticonvulsant Screening Program (ASP) according
to known, standardized procedures.27,28 Sixteen compounds (2–7,
9–16, 19, 20) were subjected to standard protocols including three
tests: the maximal electroshock (MES), subcutaneous pentetrazole
for anticonvulsant activity (scMET) and rotarod test (TOX) for neu-
rotoxicity. Tests were performed in mice at 0.5 h and 4 h after
intraperitoneal (i.p.) administration of the studied compounds.
Results of the anticonvulsant screening are presented in Table 2.
Seven compounds (2–4, 9, 11, 12, and 20) showed anticonvulsant
activity tested in the dose of 30 mg/kg at 0.5 h. All tested com-
pounds were active at the dose of 100 mg/kg at 0.5 h after admin-
istration, while twelve of them (2, 3, 5, 7, 9, 10–12, 15, 16, 19 and
20) were also active up to 4 h after administration. One compound
(4-methyl piperidinyl hexyl derivative 12) proved to be active in
the MES at the dose of 30 and 100 mg/kg up to 4 h of tested time.
All compounds showed high toxicity in the active dose, and caused
the death of all animals. In the scMET study only 4-methyl piperi-
dine derivative 4 with butyl chain showed protection at 100 and
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4
K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 2
Preliminary anticonvulsant evaluation of compounds 2–20 in the MES, scMET and TOX test (mice, i.p.).*
Com
Dose [mg/kg]
MES1
0.5 h
scMET2
0.5 h
TOX3
0.5 h
4.0 h
4.0 h
4.0 h
2
3
4
5
6
7
30
1/1
3/3
–
1/1
3/3
–
1/1
3/3
–
0/1
2/2
–
0/1
3/3
–
0/1
2/3
–
0/1
2/3
–
0/1
1/1
–
0/1
0/3
–
0/1
1/1
–
0/1
0/3
–
0/1
3/3
–
0/1
0/1
–
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
–
–
–
–
–
0/1
0/1
–
0/1
0/1
–
2/4
0/2
0/4
–
0/2
4/4b
–
0/2
–
–
0/2
1/2b
–
0/2
0/4
–
0/2
1/4
–
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30–300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30
100
300
30–300
30–300
30
100
300
30
100
300
30–300
8/8a
4/4b
3/4
8/8a
4/4b
1/4
–
0/1
1/1
1/1
–
–
–
0/1
0/1
–
0/1
0/1
–
8/8b
4/4b,c,d
1/4b
8/8b,c
4/4b,c
0/4
6/8a
4/4b
0/4
7/8a
4/4b
nt
8
9
nt
nt
nt
nt
nt
1/1
3/3
–
0/1
3/3
–
0/1
1/2
–
0/1
3/3
–
0/1
3/3
–
1/1
3/3
–
0/1
0/3
–
0/1
0/3
–
0/1
3/3
0/1
0/1
3/3
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
0/1
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/1
0/1
–
0/4
0/2
3/4b
–
0/2
0/4
–
0/2
3/4
–
0/2
3/4
–
6/8a,e,f
4/4b
0/4
10
11
12
13
14
15
16
7/8
4/4a,b
0/4
1/1
3/3
1/1
1/1
3/3
1/1
0/1
3/3
–
0/1
3/3
1/1
0/1
2/3
1/1
0/1
3/3
–
8/8g
4/4b,d
0/4
6/8a
4/4b
0/4
0/2
3/4
–
8/8a
4/4b
0/4
0/2
0/4
0/1
0/2
2/4
1/2b
0/2
3/4a
–
7/8a
4/4a,b
0/4
0/1
0/1
6/8
3/4
0/1
0/1
–
nt
nt
0/1
0/1
0/1
0/1
0/1
0/1
nt
0/4
7/8a
4/4b
nt
17
18
19
nt
nt
nt
nt
nt
nt
nt
nt
nt
0/4
1/8
2/4
0/1
1/3
1/1
1/1
1/3
1/1
nt
0/1
3/3
–
0/1
2/2
–
0/1
0/1
0/1
0/1
0/1
0/1
nt
0/2
0/4
2/2b
0/2
4/4b
1/1b
nt
20
21
1/4
8/8
4/4a,b
nt
nt
nt – not tested – the compound was not tested in the particular case.
a–gKind of toxicity: aunable to grasp rotorod; bdeath; cdiarrhea; dcontinuous seizure activity; eloss of righting reflex; fmyoclonic jerks; gtremors.
*
Ratios where at least one animal was protected have been highlighted in bold for easier data interpretation.
MES – maximum electroshock seizure: number of animals protected/number of animals tested.
scMet – subcutaneous pentetrazole evoked seizure test: number of animals protected/number of animals tested.
TOX – neurotoxicity assay (the rotarod test): number of animals exhibiting toxicity/number of animals tested.
1
2
3
300 mg/kg, but these doses caused also severe toxicity. Three com-
pounds (2, 3, and 19) were also tested in MES after oral administra-
tion to rats (30 mg/kg) but they did not show any activity (data not
shown).
values.29 Ternary solvent mixture – methanol:acetic acid:water –
as the mobile phase was used, with constant, 10% concentration
of acetic acid and varying methanol concentration in the range of
50–80%. Due to high similarity of 3- and 4-methylpiperidine iso-
mers, lipophilicity was estimated only for 3-methylpiperidine
derivatives. Tested set of compounds showed RM0 values in range
between 1.883 and 3.562. All of the values are over 2 (except com-
pound 2), which may suggest good biological barriers permeability.
Lipophilicity studies data are collected in Table 3. Sample graph,
showing the relationship between RM0 values and methanol con-
centration for compound 18 is shown in Fig. 3. As it was expected,
2.3. Physicochemical properties
2.3.1. Lipophilicity evaluation
As physicochemical properties might determine the fate of
novel compounds, at the early stage of research, their lipophilicity
was evaluated using planar RP-TLC method and expressed by RM0
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
lipophilicity of the tested set of compounds rises with the elonga-
tion of the alkyl chain length. Moreover, for structural analogues
(3-methylpiperidine and azepane derivatives) values resulted in
the similar range, e.g. compounds 11 and 13, 15 and 17. Although,
no correlation between lipophilicity and hH3R affinity was
observed for the tested series.
In order to compare the theoretical partition coefficient
parameters (logP) with ones obtained practically, calculations
using computer programs: Chem3D,30 Marvin31 and QikProp for
Schrödinger32 were also performed. All of the compounds were
used in both: base and ionic forms. The latter obtained theoretical
values seem to be more realistic, due to the fact, that all of the
tested compounds are hydrogen oxalates. The values vary between
programs, due to various algorithms used by software to deter-
mine lipophilicity values. The highest theoretical to practical val-
ues ratio was obtained using Marvin software (R = 0.92158,
R2 = 0.84931). Correlation coefficient (R), regression line slope (a),
standard deviation (SD) and R2 values are collected in Table 3.
Fig. 3. Relationship between RM0 values and methanol concentration for compound
18.
Computational approaches, using Schrödinger Maestro
10.5.014,32 allowed the in silico binding visualization of the inves-
tigated compounds to histamine H3R homology model obtained
from GPCRM Structure Modeling Server.33 The model was built
on the crystal structure of M3 muscarinic acetylcholine receptor,
due to its’ highest sequence homology to histamine H3R aminoacid
sequence (PDB code: 4DAJ; resolution: 3.4 Å). Validation with ref-
erence ligand Pitolisant, according to the procedure described by
Levoin et al. was also performed.34 Docking studies results were
interpreted by the means of Docking Score function values, that
resulted in the range of ꢀ9.001 (17) to ꢀ6.398 (21). Previously
described salt bridge formation between the protonated cycloalky-
2.3.2. In silico solubility estimation
As low aqueous solubility of compounds might be the major
problem encountered with development of new chemical entities,
by affecting e.g. absorption and distribution, thus, activity towards
desired biological target, conformation independent (CI) solubility
factor, by means of logS [mol/dm3] was calculated for the series of
compounds, using QikProp 3.2 for Schrödinger32 (Table 3). For all of
the compounds the predicted solubility is moderate (with lowest
value for compound 9), although still in the characteristic range
for market drugs. Also, no correlation between logS values and
hH3R affinity was observed for the tested series.
lamine nitrogen and GLU2065.461 35
antagonist/inverse agonist binding,
,
described as crucial for H3R
could be observed for all of
36
the compounds regardless of their in vitro affinity (Fig. 5, Table 1).
Moreover, for all of the ligands
p–p stacking could be observed
2.4. X-ray studies and in silico molecular and Docking studies
between PHE1985.39 and/or TYR189(ECL2) and substituted ben-
zene ring of docked compounds. Moreover, for compounds of
Monocrystal of compound 18 (in the hydrogen oxalate form)
was selected as suitable for X-ray structure analysis. The structure
was solved in non-centrosymmetric triclinic space group P1 with
two independent molecules. Both molecules adopt extended con-
formation with proton located at nitrogen (Fig. 4). Independent
molecules differ in C7-C8-C9-C10-C11-C12-C13-C14 chain confor-
mation details and tert-pentyl rotation. In the crystals main proto-
nated molecules are twisted around the oxalate ions chain. For
detailed information see Appendix A. Supplementary Materials.
longer (6–8 methylene groups) alkyl chain length, additional
p–p
stacking with TYR1153.33 was observed. This phenomenon was
likewise observed for the reference compound DL77.
None of the compounds showed previously described interac-
tion between ether moiety oxygen and TYR3746.51, although
p–p
stacking with substituted phenyl ring was observed. What was
interesting, for methylated piperidine derivatives, which are of
higher in vitro affinity than non-methylated ones, none additional
Table 3
Theoretical and practical lipophilicity and solubility (CI logS) values for compounds 2–21, as well as R, R2, a and SD values for each of the used computational programs.
No
RM0
Marvin 5.4.1.1
Ionic
ChemBio3D Ultra 11.0
Ionic
QikProp 3.2
Ionic
CI logS
Base
Base
2
3
5
6
7
9
10
11
13
14
15
17
18
19
21
1.883
2.294
2.292
2.172
2.342
2.389
2.456
2.521
2.567
2.624
2.983
2.926
3.075
3.562
3.384
1.75
2.12
2.19
2.19
2.56
2.64
2.64
3.00
3.08
3.08
3.45
3.53
3.53
3.89
3.97
5.25
5.62
5.70
5.70
6.06
6.14
6.14
6.51
6.59
6.59
6.95
7.03
7.03
7.39
7.47
6.581
7.100
7.140
7.110
7.629
7.669
7.639
8.158
8.198
8.168
8.687
8.727
8.697
9.216
9.256
6.161
6.680
6.720
6.690
7.209
7.249
7.219
7.738
7.778
7.748
8.267
8.307
8.277
8.796
8.836
5.128
5.627
5.016
5.451
5.813
5.750
5.853
6.200
5.596
6.240
6.596
6.615
6.570
6.884
7.057
ꢀ3.590
ꢀ3.888
ꢀ3.888
ꢀ3.463
ꢀ3.888
ꢀ6.099
ꢀ4.185
ꢀ3.761
ꢀ4.185
ꢀ4.483
ꢀ4.483
ꢀ4.059
ꢀ4.483
ꢀ4.781
ꢀ4.781
R
0.92158
0.84931
0.72523
1.30901
0.92141
0.84898
0.72483
2.19606
0.93709
0.87815
0.77804
2.92496
0.93692
0.87783
0.78491
2.73370
0.91834
0.84335
1.11644
2.11186
R2
a
SD
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6
K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 4. ORTHEP drawing of 18 hydrogen oxalate structure (light grey color for carbons, red for oxygen, blue for nitrogen).
Fig. 5. Compound 6, and 7, 11, 15 in the binging pocket of histamine H3 receptor homology model (ligands: green color for carbons, white – hydrogens, blue – nitrogen, red –
oxygen; receptor – cyan for carbons, white – hydrogens, blue – nitrogen, red – oxygen).
interactions between methyl group and side-chains amino acids
were found. Although, nevertheless the spacer length, methylene
groups are placed in one position, surrounded by small pocket
formed by CYS1183.37, THR1193.38 and ALA1223.40 (Figs. 5 and 6).
This interaction might condition higher affinity to H3R of 3-
methylpiperidine derivatives.
its way to be useful tools in drug discovery and development of
new anticancer therapeutics.39 Therefore, the additional tests were
performed with neuroblastoma IMR-32 cell line, where com-
pounds 7, 8, 9 showed similar activity against IMR-32. The highest
activity in these tests showed compound 9, the derivative with
azepane moiety, with IC50 = 12.31 mM. However, significant effect
of 9 against IMR-32 was observed under more than 1000-fold
higher concentration in comparison to IC50 value of the reference
compound DX, which was found to be only 5.1 nM. (Fig. 8).
2.5. Drug-like properties
The acceptable ADME-Tox parameters of the biologically
active compound describe its ‘‘drug-likeness” – namely, the ability
to become an ideal clinical candidate.37 Presented in this study,
most active H3R ligands 6, 7, 8 and 9 were chosen for examination
of two important ADME-Tox parameters: toxicity and metabolic
stability.
2.5.2. Metabolic stability
In first instance, compounds 6, 7, 8 and 9 were examined in sil-
ico using MetaSite 4.1.1software,40 to predict the most likely sites
of metabolic transformations. The highest probability of metabo-
lism calculated by liver model for compounds 7, 8 occurred at 3-
and 4-substituted position of piperidine moiety, whereas for com-
pounds 6 and 9 MetaSite predicted the highest possibility of meta-
bolism at the aliphatic linker. For all of the compounds the
possibility of hydroxylation of tert-amyl moiety was also consid-
ered. The locations of metabolic modifications are shown in Fig. 9.
In the next step the examined H3R ligands were incubated for
2 h with human liver microsomes (HLMs). A full scan chro-
matogram of the reaction mixtures showed the presence of 3
metabolites for compounds 6, 7, 8 and the presence of 5 metabo-
lites for compound 9. The LC/MS analysis allowed to indentify
the molecular masses of obtained metabolites, as well as to
confirm the structures of HLMs obtained metabolites, ion
fragments analysis was performed. The obtained data showed the
2.5.1. Toxicity
Toxicity of novel compounds was determined in vitro after incu-
bation of HEK-293 cell line in the presence of H3R ligands for 48 h.
Obtained data showed the decreasing viability of examined cells
above the 10 mM concentration with IC50 values from 25.1 mM to
41.19 mM (Fig. 7). The highest activity against HEK-293 cell line
was shown for compound 7, the derivative with the methyl sub-
stituent at 2-position of piperidine moiety (Fig. 7). However,
observed antiproliferative effects of examined compounds could
be described as weak, being 50–90-fold lower in comparison to
IC50 value of the reference compound doxorubicin (DX)
(0.46 mM).38 On the other hand, antiproliferative assays have found
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Fig. 6. Ligand – interaction diagrams (generated using Schrödinger Suite) for compounds 7, 11, 15.
Fig. 7. Activity of DX (positive standard) and H3 receptor ligands (6–9) against HEK-293 cell line.
similar metabolism pathway of compounds 6 ([M+H]+ = 318 m/z), 7
([M+H]+ = 332 m/z), and 8 ([M+H]+ = 332 m/z), where the hydroxy-
lation occurred in three different ways at the tert-amyl moiety
(metabolites: M1, M2 and M3;see Appendix A). At first, it was
observed that the molecular masses of metabolites increased to
334 m/z compared to substrate 6 or to 348 m/z compared to sub-
strates 7, 8. Moreover, the characteristic for all substrates tert-amyl
ion fragment (71 m/z) was absent in the all metabolites spectra and
the precise analysis of the another ion fragments excluded the
hydroxylation in piperidine moiety or in the aliphatic linker. The
metabolic pathway of compound 9 ([M+H]+ = 332 m/z) included
similar to compounds 6, 7 and 8 reactions of hydroxylation at
tert-amyl moiety (metabolites M2, M3 and M4) and additional
two metabolites M1 and M5. The additional metabolite M1
([M+H]+ = 320 m/z) is probably the product of demethylation
followed by hydroxylation. The molecular mass of second addi-
tional metabolite M5 ([M+H]+ = 364 m/z) suggested the double
hydroxylation of the substrate. The presence of characteristic
tert-amyl ion fragment 71 m/z excluded the hydroxylation of
tert-amyl moiety. The precise study of ion fragments of M5 showed
that the hydroxylation occurred at the azepane moiety and at the
methylene bridge connected to nitrogen in the azepane moiety.
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 8. Activity of DX (positive standard) and H3 receptor ligands (6–9) against IMR-32 cell line.
CYP3A4 activity at 10 mM with calculated IC50 = 0.14 l
M.42
Depending on the structure of the H3R ligand, weak or very strong
activations of CYP3A4 were observed. In the presence of 25 mM of
compound 8 with the methyl substituent at the 4-position of
piperidine moiety, the CYP3A4 activity increased surprisingly by
325%. However, compound 7 with the same substituent at the 3-
position increased the cytochrome activity only by 52% at 25 mM,
similar to compound 9 with azepane moiety (76%). The weakest
influence on CYP3A4 showed compound 6 with unsubstituted
piperidine moiety, which increased the cytochrome activity only
by 21% at 25 mM (Fig. 10).
3. Conclusions
A novel series of tert-amyl phenoxy alkylamine derivatives
investigated in the present study proved to be potent histamine
H3R ligands with the H3R affinities in the nanomolar range. The
highest affinities were observed for pentyl derivatives 6 and 7
(Ki = 8.8 and 13.6 nM, respectively) with piperidine and 3-methyl
piperidine cycloalkylamines. The length of alkyl chain and charac-
ter of amine have pronounced effect on the activity – generally, for
this group, 3-methyl piperidine derivative with five carbon atoms
chain was the most potent. Further elongation of the alkyl linker
resulted in decrement of H3R affinity. Selected, representative
structures were described as antagonists in cAMP test. Performed
in vivo studies proved anticonvulsant activity in MES test for all
of the examined compounds. On the other hand, only one com-
pound has shown the activity in scMET test. Docking studies to his-
tamine H3R homology model built on the crystal structure of M3
muscarinic acetylcholine receptor allowed the observation of pre-
viously proposed ligand-receptor interactions. Experimentally esti-
mated lipophilic properties (by means of RM0) may suggest good
tissues barriers permeability. Moreover, the examined compounds
show satisfying, selected (cytotoxicity, metabolic stability, influ-
Fig. 9. Plots of MetaSite predictions for sites of metabolism for H3 receptor ligands
by liver model. Higher color intensity of the marked functional group indicates
higher probability of its involvement in the metabolism pathway. The blue circle
indicates the highest probability to be involved in the metabolism pathway.
2.5.3. Influence on cytochrome CYP3A4 activity
The luminescence CYP3A4 P450-GloTM Assay based on the con-
version of the luciferin-PPXE into
D-luciferin by recombinant
human CYP3A4 was next used to determine the influence of exam-
ined H3R ligands on cytochrome CYP3A4 activity. After addition of
the firefly luciferase, the measured amount of light produced was
proportional to
D-luciferin concentration and allowed to assess
the effect of examined compound on CYP3A4.41 In that assay, the
reference compound ketoconazole inhibited completely the
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
Fig. 10. The effect of 6, 7, 8, 9 (0.025 lM–25 lM) and ketoconazole (0.010 lM–10 lM) on CYP3A4 activity.
ence on cytochrome CYP3A4 activity) drug-like properties. Paying
attention to described herein ligands’ biological activity and dock-
ing results, further directions of research include modification of
the ‘‘eastern part” of the molecule (e.g. introduction of bulky aro-
matic groups: naphthyl, biphenyl) in order to test, whether larger
substituents are also tolerated.
with Merck Silica gel 60 RP-18 F254S glass plates, using planar chro-
matographic CHROMDES chambers. Compounds solutions were
applied using Hamilton 25 ml syringes. The following detection
was obtained by evaluation in iod-saturated glass chambers. For
CC (column chromatography using silica gel 60 (0.063–0.20 mm;
Merck) solvent systems were used: I-CH2Cl2; II: CH2Cl2:MeOH
(9:1).
4. Experimental
4.1.1. General procedure (a) for compounds 1a–1e
Starting compounds were obtained according the procedure
described in Ref. 22. Analytical data of compounds 1a–1e could
be found in Ref. 20. Detailed synthetic procedure for compounds
1b–1e could be found in Appendix A: Supplementary Data.
A solution of freshly prepared sodium propylate, proper substi-
tuted phenols were added and stirred in room temperature for
4.1. Chemistry
All reagents were purchased from commercial suppliers and
were used without further purification. 3-methyl piperidine was
used as racemate, and the stereochemistry of its’ final derivatives
(3, 7, 11, 15, 19) were therefore not determined. Melting points
(m.p.) were determined on a MEL-TEMP II (LD Inc., USA) melting
point apparatus and are uncorrected. IR spectra were measured
as KBr pellets on FT Jasco IR spectrometer. UV spectra were
recorded on a Jasco UV/Vis V-530 apparatus in 10ꢀ5 mol/L in
methanol. Mass spectra (LC/MS) were performed on Waters TQ
Detector mass spectrometer. 1H NMR spectra were recorded on a
Varian Mercury 300 MHz PFG spectrometer in DMSO-d6 or in
CDCl3. Chemical shifts were expressed in parts per million (ppm)
using the solvent signal as an internal standard. Data are reported
in the following order: multiplicity (s, singlet; d, dublet; t, triplet;
qi quintet, m, multiplet; br, broad; t-p, tert-pentyl; Ph, phenyl; Pip,
15 min.
a,x-dibromoalkanes were then added drop wisely in the
time of 1 h. The reaction mixture was stirred in 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%
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 evapo-
rated. Rough product was used for further reactions without
purification.
4.1.1.1. 1-((4-Bromobutyl)oxy)-4-(tert-pentyl)benzene (1a). Synthe-
sis from tert-pentylphenol (16.42 g, 0.1 mol), 1,4-dibromobutane
(g, 0.2 mol) in sodium propylate (0.1 mol, 100 ml). Obtained 27 g
of raw product, purified by CC (II). Yield 78%.
piperidine, Azp, azepane), 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.
The UPLC/MS purity of all final compounds was determined (%).
Elemental analyses (C, H, N) were performed on an Elemental Anal-
yserVario El III (Hanau, Germany) and agreed with theoretical val-
ues within 0.4%. TLC data were obtained with Merck silica gel
60F254 aluminum sheets with the following detection with UV light
and evaluation with Dragendorff’s reagent (solvent system:
methylene chloride: methanol 9:1). RP-TLC data were obtained
4.1.2. General synthetic procedure for compounds 2–21
To a suspension of potassium carbonate and catalytic amount of
potassium iodide in water, a mixture of proper cyclic amine and
compound 1a–1e in ethanol was added. Mixture was then refluxed
for 8–20 h (TLC controlled). After cooling down to room tempera-
ture, reaction mixture was filtrated, evaporated and purified by
one of the procedures:
Procedure A: To a resulting oil, 100 ml of CH2Cl2 was added and
washed with: 0.5% HCl solution, 0.5% NaOH solution and water.
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10
K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
After drying over anhydrous Na2SO4 and evaporation of organic
layer product was further purified using CC (CH2Cl2:MeOH).
Procedure B: To a resulting oil 100 ml of CH2Cl2 was added and
washed with: 0.5% HCl solution. After drying over anhydrous
Na2SO4 and evaporation of organic layer, product was suspended
in 50 ml of 3% HCl solution and washed with diethyl ether. Organic
layer was then alkalized and extracted to CH2Cl2.
the deposition number CCDC 1485473. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road,
CambridgeCB2 1EW, UK (Fax: Int code +(1223)336-033; E-mail:
deposit@ccdc.cam.ac.uk).
4.4. Docking studies
Resulting oils were transformed into hydrogen oxalates, using
10% excess of oxalic acid ethanol solution in room temperature,
and then precipitated by addition of ethyl ether.
Detailed synthetic procedure and analytical data for compounds
2–21 could be found in Appendix A: Supplementary Data.
In silico visualization of binding to histamine H3R model
described by Levoin et al., was held using Glide module of
Schrödinger MacroModel 10.5.43–46 All of the tested compounds
were used in their N-protonated form, and generated using
Schrödinger suite, despite compound 18 where crystallographic
structure was used for docking. For the tested compounds energet-
ically optimal conformers were found using ConfGen (MMFFs
forcefield, PRCG, convergence threshold = 0.05, 20 steps per rotat-
able bond, max. ring conformations = 16). For all of the structures
5 energetically best conformations were used for docking.
Receptor grid was generated by GlideGrid module and validated
with previously described BP.294 (Pitolisant). Docking studies
were performed via GlideDock module (OPLS 2001 forcefield, sol-
vent = water, standard precision, post-docking minimization).
Results were interpreted by the means of Docking Score functions.
Receptor-ligand interactions were visualized using PyMOLver
1.4.1.47
4.1.2.1. 1-(4-(4-(tert-Pentyl)phenoxy)butyl)piperidine hydrogen oxa-
late (2). Synthesis from piperidine (0.85 g, 10 mmol), and com-
pound 1a (1.5 g, 5 mmol) in ethanol (105 ml) in the presence of
K2CO3 (2.07 g, 15 mmol) and catalytic amount KI in water
(20 ml). Refluxed for 20 h. Purified by Procedure A, CC eluent:
CH2Cl2:MeOH (9:1). Obtained 0.75 g of oil. Yield 35%. Raw product
was transformed into oxalic acid salt yielding 0.63 g of final
compound. Mp: 133–135 °C. Rf = 0.28. 1H NMR (DMSO-d6) d:
7.22–7.17 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.85–6.80(d, J = 8.7 Hz, 2H,
Ph-2,6-H), 3.95–3.92 (t, J = 6.2 Hz, 2H, CH2-O), 3.03–2.98(m, 6H,
Pip-2,6-CH2 + N-CH2), 1.80–1.60 (m, 8H, 3ꢁ CH2 + t-p-CH2), 1.59–
1.51(m, 4H, 2ꢁ CH2), 1,18 (s, 6H, 2ꢁ CH3), 0.61–0.56(t, J = 7.3 Hz,
3H, CH2-CH3). 13C NMR (DMSO-d6)TM: 165.11, 156.61, 141.27,
127.13, 114.35, 67.14, 56.11, 52.43, 37.36, 36.72, 28.92, 26.52,
4.5. Pharmacology
23.00, 21.98, 20.78, 9.51;UV–VIS k [nm] (lg
e
): 216 (3.89), 223
4.5.1. General remarks
(3.89), 276 (3.78), 282 (3.71). IR (cmꢀ1): 3429.78 (N(CH2–)4),
3022.87 (aromatic CH@), 2961.16 (het. CH2–), 2875.34, 2679.60,
2537.86, 2365.26, 1719.23, 1700.91, 1608.34, 1579.41, 1491.67,
1475.28, 1384.64, 1362.46, 1294.97 (O–CH aromatic), 1186.01,
1115.62, 1034.62, 993.16, 953.63, 831.17, 779.10, 704.85 (alif.
CH2–), 662.43, 521.65. LC–MS: purity 100% tR = 5.90, (ESI) m/z [M
+H]+ 304.24. Anal. Calcd for C20H33NO ꢁ C2H2O4: C, 67.14, N, 3.56,
H, 8.96%. Found: C, 67.22, N, 3.61, H, 9.25.
Competition binding data were analyzed by the GraphPad-
PrismTM (200, version 3.02, San Diego, CA, USA) software, using
non-linear least squares fit. Affinity values (Ki) were expressed as
mean from at least two experiments in triplicates with SEM. Ki val-
ues were calculated from IC50 values according to Cheng-Prusoff
equation.48
4.5.2. [125I]Iodoproxyfan hH3R displacement assay
The displacement binding assay was carried out as described by
Ligneau et al.23 In brief, membrane preparations of CHO-K1 cells
expressing the recombinant hH3R gene were homogenized, incu-
bated for 90 min at 25 °C and shook at 250 rpm with [125I]
Iodoproxyfan (16–50 pM) and different concentrations of the test
compound. Non-specific binding was determined in the presence
of imetit (1 mM). In saturation binding experiments Bmax was found
to be 0.6 pmol/mg and the Kd value was 0.044 pM. The bound radi-
oligand was separated from free radioligand by filtration through
GF/B filters (pre-treated with 0.3% (m/v) polyethyleneimine using
an Inotech cell harvester Dottikin, Switzerland). Unbound radioli-
gand was removed by four washing steps with 5 ml/well of binding
buffer.
4.2. Lipophilicity
Lipophilicity, by means of RM0 values, was estimated using RP-
TLC planar method. Methanol:acetic acid:water (with constant,
10% acid concentration) solvent mixture was used as a mobile
phase. Organic solvent concentration varied from 85% to 55%, by
5% each step. Mobile phase composition as well as concentrations
were chosen experimentally. For each concentration, 10 ml of
compounds methanol solution in 1 mg/ml concentration was used.
Planar chromatographic chambers (Chromdes) were saturated
with proper mixture for 45 min. followed by 15 min. saturation
together with the plate. Glass plates (Merck RP-18, FP254s) were
then evaluated on the distance of 90 mm, dried and spots
were visualized using iod fumes. For each of the compounds, on
the base of Rf values, RM values were determined. RM0 values were
read from RM/methanol concentration charts, after extrapolation to
zero methanol concentration using in-house script.
a
4.5.3. [3H]N -Methylhistamine hH3R displacement assay
The displacement binding assay was carried out as described by
Kottke et al.24 Frozen crude membrane preparations of HEK-293
cells stably expressing the recombinant hH3R in full length were
thawed, homogenized, incubated for 90 min at 25 °C and shook
a
4.3. X-ray structure analysis
at 250 rpm with [3H]N -methylhistamine (2 nM) and different
concentrations of the test compounds (seven appropriate concen-
trations between 0.01 nM and 10 mM were used) in a final assay
volume of 200 ml per well. Non-specific binding was determined
in the presence of Pitolisant (10 mM). In saturation binding experi-
ments Bmax was found to be 0.89 pmol/mg and the Kd value of [3H]
Crystal data for 18: C24 H42 N O, C2 H2 O4, M = 449.61, triclinic,
space group P1, a = 8.4543(2) Å, b = 11.1330(3) Å, c = 14.7601
(11) Å, h = 89.363(1) (°), Ò = 84.8700(9) (°), Ó = 73.511(6) (°),
V = 1326.63(12) Ǻ3,
Z = 2,
Dx = 1.126 g cmꢀ3
,
T = 296 K,
m = 0.612 mmꢀ1, k = 0.71073 Ǻ, data/parameters = 3703/559; final
R1 = 0.06.
Crystallographic data (excluding structural factors) for the
structure reported in this paper has been deposited at
the Cambridge Crystallographic Data Centre and allocated with
N -methylhistamine was 2.98 nM. The bound radioligand was sep-
a
arated from free radioligand by filtration through GF/B filters (pre-
treated 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
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K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
50 mM Tris-HCl buffer (pH 7.4) containing 120 mM NaCl. Scintilla-
4.7. Antiproliferative assay
4.7.1. Cell lines
Neuroblastoma IMR-32 cell line was provided by Department
of Oncogenomics, AcademischMedisch Centrum, Amsterdam,
Holland.52,53 Human embryonic kidney HEK-293 cell line (ATCC
CRL-1573) was kindly donated by Prof. Dr. Christa Müller
(Pharmaceutical Institute, Pharmaceutical Chemistry I, University
of Bonn).
tion cocktail was added and the liquid scintillation counting was
performed with a Perkin Elmer TriluxBetacounter (Perkin Elmer,
Germany).
4.5.4. cAMP accumulation assay in cells expressing hH3R
Intracellular cAMP accumulation was measured with homoge-
nous TR-FRET immunoassay, using LANCE UltracAMP kit (PerkinEl-
mer) and HEK293 cells, stably expressing hH3R. An antagonist
dose-response experiments were performed in a total assay vol-
ume of 20
(ꢀ)- -Methylhistamine (15 nM), forskolin (10
nists in appropriate concentrations (in range 0.1 nM–10
added simultaneously to cell suspension. Cells stimulation was
performed for 30 min at room temperature. After incubation per-
iod, five microliters of europium (Eu) chelate-labeled cAMP tracer
l
l in white 384-well plates, using 300 cells/well. (R)
M) and antago-
M) were
4.7.2. In vitro antiproliferative assay
a
l
DMEM cell culture medium (Gibco) with 10% fetal bovine
serum (FBS), 100 mg mLꢀ1 streptomycin and 100 U mLꢀ1 penicillin
was used for both cell lines. The cells were seeded in 96-well plates
at a concentration of 2 ꢁ 104 cells/well (IMR-32) or 1.5 ꢁ 104 cells/
l
well (HEK-293) in 200 ll culture medium and incubated for 24 h at
and 5
ll of ULight-labeled anti-cAMPmAb working solutions were
37 °C and 5% CO2to reach 60% confluence. The stock solutions of H3
added, mixed and incubated for 1 h. TR-FRET signal was read on
an EnSpire microplate reader (PerkinElmer). Measured TR-FRET
signal was translated into actual quantities of produced cAMP on
the basis of cAMP standard curve and obtained results were pre-
sented as% of maximal response. Sigmoidal dose-response curve
fitting was performed with use of GraphPad PrismTM software (ver-
sion 5.01, San Diego, CA, USA). Showed results represent the mean
of three separate experiments, each performed in triplicates.
receptor ligands in DMSO (25 mM) were diluted into fresh growth
medium to the final concentrations 0.01 lM–250 lM and added to
the culture. The DMSO concentration did not exceed 1%. After 48 h
of incubation, the 20 ml ofEZ4U (EZ4U Non-radioactive cell prolifer-
ation and cytotoxicity assay, Biomedica) labeling mixture was
added to each well and the cells were next incubated for 5 h. The
absorbance of the samples was measured using a microplate
reader (PerkinElmer) at 492 nm. The activity of the standard drug
doxorubicin (DX) was estimated as we described previously.36
4.6. In vivo studies
4.8. Metabolic stability
4.6.1. Anticonvulsant screening program
Anticonvulsant evaluation of compounds was performed and
sponsored by National Institute of Neurological Disorders and
Stroke, National Institutes of HealthCountry name of ‘‘National
Institutes of Health” is ‘‘United States”. (Rockville, USA) for the
Anticonvulsant Screening Program (ASP). Compounds were
injected as suspensions in 0.5% methylcellulose at the selected
doses (30, 100 or/and 300 mg/kg). As experimental animals male
albino mice (CF-1 strain) and male albino rats (Sprague-Dawley)
were used. Observations were carried out after 0.5 h and 4 h after
administration. Groups of eight mice or four rats were employed.
Phase 1 of ASP includes three tests performed in mice (i.p.): max-
imal electroshock (MES), subcutaneous pentylenetetrazole
(scMET), and neurotoxicity (rotarod test). Selected compounds
were administered orally into rats using four animals at a fixed
dose of 30 mg/kg (MES test) (Phase VIa).
4.8.1. Reaction with recombinant human liver microsomes (HLM)
Commercial, pooled, human (adult male & female) liver micro-
somes (HLMs) from Sigma-Aldrich were used for all biotransfor-
mations. The reaction was carried out using 1 mg/ml HLMs in
200 ll of reaction buffer containing 0.1 M Tris-HCl (pH 7.4),
NADPH Regeneration System (Promega) and H3 receptor ligand
with final volume of 50 mM at 37° for 2 h. The reaction was initi-
ated by adding 50 ml of Regeneration System after 5 min. preincu-
bation at 37 °C. The 200 ml of cold methanol was added to
terminate the reaction. The mixture was centrifuged at
13,000 rpm for 15 min. and then LC/MS analysis of the supernatant
was performed. Mass spectra were recorded on LC/MS system con-
sisted of a Waters Acquity UPLC, coupled to a Waters TQD mass
spectrometer (electrospray ionization mode ESI-tandem
quadrupole).
4.6.1.1. Maximal electroshock seizure test (MES) test. Seizures were
elicited by 60 Hz alternating current (50 mA in mice) delivered
for 2 s by corneal electrodes. A drop of 0.5% tetracaine HCl in
0.9% NaCl solution was placed into each eye prior to applying elec-
trodes. Protection was defined as abolition of the hindlimb tonic
extension component of the seizure.49,50
4.8.2. CYP3A4 P450-GloTM assay
The CYP3A4 inhibitor ketoconazole was purchased from Sigma-
Aldrich. The enzymatic reactions were performed in white poly-
styrene, flat-bottom NuncTM MicroWellTM 96-Well Microplates
(Thermo Scientific). The luminescence signal was measured with
a microplate reader in luminescence mode (PerkinElmer). The
CYP3A4 P450-GloTM assay and protocol were provided by
Promega.41 The IC50 value of the reference compound ketoconazole
was determinate and calculated as we described previously.42 The
4.6.1.2. Subcutaneous pentetrazole induced seizures. A dose of pente-
trazole which induce convulsions in 97% of animals (CD97: 85 mg/
kg mice) was injected into a loose fold of skin in the midline of the
neck. Animals were observed for 30 min for the presence or
absence of a seizure. Failure of observing even a threshold seizure
(a single episode of clonic spasm which remains at least 5 s) was
classified as protection.27
final concentrations of H3R ligands were 0.025
CYP3A4 inhibitor ketoconazole 0.010
l
M–25
lM and the
lM–10 mM.
4.8.3. In silico study
The metabolic biotransformation of H3R ligands was studied in
silico by using computational software MetaSite 4.1.1 provided by
Molecular Discovery Ltd (2013)54,55 using liver model.
4.6.1.3. Neurotoxicity test. The rotarod test was used to evaluate
neurotoxicity in rodents (mice). Animal was placed on a 1 in. diam-
eter knurled plastic rod rotating at 6 rpm. The animal can maintain
its equilibrium for long periods of time. Neurotoxicity was
indicated if animal falls off this rotating rod three times during a
1-min period.51
Acknowledgements
We grateful thank Senior scientist Mohamed Hamdi from
Department of Oncogenomics Academisch Medisch Centrum
Please cite this article in press as: Kuder K.J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.031

12
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Amsterdam, Holland for providing neuroblastoma IMR-32 cell line.
We would like to thank prof. James P. Stables, prof. Jeff Jiang and
Tracy Chen, Ph.D. for providing the results of anticonvulsant assays
through ASP at National Institute of Neurological Disorders and
Stroke, National Institutes of Health (Rockville, USA).We are
pleased to acknowledge the generous support of National Science
Center, Poland granted on the basis of decision No. DEC-2011/02/
A/NZ4/00031 and K/ZDS/004689 and of the European COST Action
GLISTEN-CM1207.
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Please cite this article in press as: Kuder K.J., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2017.03.031