Chlorophenoxy aminoalkyl derivatives as histamine H3R ligands and antiseizure agents

Article abstract of DOI:10.1016/j.bmc.2015.11.021

A series of twenty new chlorophenoxyalkylamine derivatives (9-28) was synthesized and evaluated on their binding properties at the human histamine H₃ receptor (hH₃R). The spacer alkyl chain contained five to seven carbon atoms. The h

Full text of DOI:10.1016/j.bmc.2015.11.021

Bioorganic & Medicinal Chemistry 24 (2016) 53–72
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Chlorophenoxy aminoalkyl derivatives as histamine H3R ligands and
antiseizure agents
Kamil Kuder a,y, Dorota Łazewska a,y, Gniewomir Latacz a, Johannes Stephan Schwed b, Tadeusz Karcz a,
_
b
c
a,
⇑
´
Holger Stark , Janina Karolak-Wojciechowska , Katarzyna Kiec-Kononowicz
a Jagiellonian University Medical College, Faculty of Pharmacy, Department of Technology and Biotechnology of Drugs, Medyczna 9, 30-688 Kraków, Poland
b Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Universitaetsstr. 1, 40225 Duesseldorf, Germany
c
_
´
´
Institute of General and Ecological Chemistry, Technical University of Łódz, Zeromskiego 116 Str., 90-924 Łódz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of twenty new chlorophenoxyalkylamine derivatives (9–28) was synthesized and evaluated on
their binding properties at the human histamine H3 receptor (hH3R). The spacer alkyl chain contained five
to seven carbon atoms. The highest affinities have shown the 4-chloro substituted derivatives 10 and 25
(Ki = 133 and 128 nM, respectively) classified as antagonists in cAMP accumulation assay (EC50 = 72 and
75 nM, respectively). Synthesized compounds were also evaluated for anticonvulsant activity in
Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA).
Two compounds (4-chloro substituted derivatives: 20 and 26) were the most promising and showed
in the MES seizure model in rats (after ip administration) ED50 values of 14 mg/kg and 13.18 mg/kg,
respectively. Protective indexes (PI = TD50/ED50) were 3.2 for 20 and 3.8 for 26. Moreover, molecular mod-
eling and docking studies were undertaken to explain affinity at hH3R of target compounds, and the
experimentally and in silico estimation of properties like lipophilicity and metabolism was performed.
Antiproliferative effects have been also investigated in vitro for selected compounds (10 and 25).
These compounds neither possessed significant antiproliferative and antitumor activity, nor modulated
Received 12 August 2015
Revised 12 November 2015
Accepted 19 November 2015
Available online 2 December 2015
Keywords:
Histamine H3 receptor
Histamine H3R ligands
Anticonvulsant
Ethers
Piperidine derivatives
Drug-like properties
CYP3A4 activity up to concentration of 10 lM.
Ó 2015 Published by Elsevier Ltd.
1. Introduction
as experimental findings showed the effectiveness of histamine
H3R antagonists/inverse agonists in various experimental models
of epilepsy.13 Epilepsy, as one of the major neurological disorders,
is characterized by unprovoked or reflex seizures.14 The mecha-
nism of seizures is still not fully understood15 and available
antiepileptic drugs (AEDs) could not help all epileptic patients as
almost 30% of them are resistant to current therapy. Moreover,
as long life medication is required, caused side effects should be
minimized, and the search for new and more efficacious AEDs with
good safety profile is important. Conducted research studies show
an association between histaminergic central nervous system and
seizure pathophysiology. Histamine itself is considered as an
endogenous anticonvulsant,16 whereas histamine H1 receptor
antagonists induce convulsions.17,18 Recent studies in a lamotrig-
ine-resistant kindled model of epilepsy in mice showed decreased
level of brain histamine and suggested a role of this neurotransmit-
ter in resistance development.19 Last two years have brought more
information of anticonvulsant activities of histamine H3R antago-
nists/inverse agonists tested in animal models of epilepsy.20,21
Both imidazole20 as well as nonimidazole histamine H3R21 ligands
Histamine receptors belong to the class A super family of G-pro-
tein coupled receptors (GPCR).1 Histamine H3 receptors (H3R) are
involved in the central and peripheral regulation of histamine
levels2,3 as well as other neurotransmitters such as acetylcholine
(Ach), serotonin (5-HT), noradrenalin (NA), and neuropeptide Y,
by postsynaptic modulation.4,5 Therefore, the blockade of these
receptors by inhibiting post synaptic H3R might be a useful phar-
macological target in the treatment of many CNS-based diseases.
Intensive pharmacological studies suggested the utility of his-
tamine H3R antagonists/inverse agonists in treatment of various
diseases: schizophrenia, Alzheimer (AD) and Parkinson’s disease,
obesity, narcolepsy and attention-deficit hyperactivity disorder
(ADHD),6–8 also as dual acting ligands with additionally different
pharmacological activity.9–12 Epilepsy could be another indication
⇑
Corresponding author. Tel.: +48 12 6205580.
E-mail address: mfkonono@cyf-kr.edu.pl (K. Kiec´-Kononowicz).
These authors contributed equally to this manuscript.
y
http://dx.doi.org/10.1016/j.bmc.2015.11.021
0968-0896/Ó 2015 Published by Elsevier Ltd.

54
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
showed some kind of protection in the maximal electroshock
(MES)-induced seizure in Wistar rats. Activity mostly depended
on the chemical structure of tested compounds. Hybrid
molecules,21 combining H3R pharmacophore motif (3-piperido-
propyloxy moiety) with main structural elements of known AEDs
(e.g., the hydantoin element of phenytoin; PHT), unfortunately
did not lead to high protection against seizures induced in rats
by MES or pentylenetetrazole (PTZ)-kindled epilepsy (up to a dose
of 10 mg/kg ip of tested compounds). One PHT derivative showed
only moderate protection whereas the another one even induced
epileptogenic effect.21 In mice model of temporal lobe epilepsy
(TLE), where seizures are induced by injection of kainic acid (KA),
the known histamine H3R ligand—ABT-239 (3 mg/kg ip) signifi-
cantly attenuated seizures elicited by KA.22 This research showed
also that coadministration of ABT-239 with sodium valproate
(150 mg/kg ip) enhanced anticonvulsive effect. Moreover, this
study confirmed the neuroprotective ability of ABT-239 observed
previously in mice model of Alzheimer disease.23
Amongst a number of active compounds obtained in both aca-
demia and industry, so far only one histamine H3R antagonist, pito-
lisant (PIT; Bioprojet Biotech; Fig. 1) completed phase III of clinical
trials with positive results in narcolepsy and daytime sleepiness in
Parkinson’s disease,14,24,25 and is under evaluation as an orphan
drug (Wakix; pitolisant hydrochloride) by Bioprojet Pharma.26
Recently, clinical utility of PIT was also checked in early phase II
studies in the human photosensitivity model.27 PIT tested in doses
of 20, 40 and 60 mg/kg caused statistically significant suppressive
effect in 64% of patients.
two the most potent compounds and evaluated their antiprolifera-
tive effects and metabolic stability. Lipophilicity using planar RP-
TLC method and molecular modelling studies were also included
for better understanding behaviour and properties of the
compounds.
2. Results and discussion
2.1. Chemistry
The synthesis of desired ether derivatives 9–28 was achieved
through the efficient synthetic route outlined in Scheme 1.
Chlorophenoxyalkyl bromides (4–8) were obtained in simple
one-step alkylation of suitable chlorophenols with
a,x-dibro-
moalkanes refluxed in sodium propanolate. Obtained bromides
were then reacted with corresponding piperidines and a homopi-
peridine in the mixture of ethanol/water with powdered potassium
carbonate and a catalytic amount of potassium iodide. 3-Methyl-
piperidine was used as a racemic mixture. The desired products
were obtained as free bases and crystallized as salt of oxalic acid.
The structures of compounds were confirmed by elemental and
spectral analyses (1H NMR, 13C NMR, MS, UV, IR). All compounds
except 9 are new ones.33,34 Compound 9 (free base) was prepared
some time ago (1958) as analgesic agent but proved only weak
activity.33
2.2. Pharmacology
2.2.1. Histamine H3 receptor affinity
Since years our research group is engaged in the search for
potent histamine H3R compounds.28–31 Some time ago we obtained
and described a series of diether histamine H3R ligands, that
showed very good histamine H3R affinities (hH3R Ki = 3.2–62 nM;
selected compounds 1–3 are shown in Fig. 1).30,31
All compounds in their hydrogen oxalate forms were evaluated
in radioligand displacement binding assays for affinity at human
recombinant histamine H3 receptor (hH3R) stably expressed in
human embryonic kidney (HEK-239) cells as described by Kottke
a
et al.35 The radioligand [3H]N methylhistamine was used as
Encouraged by the aforementioned results in experimental and
clinical studies we decided to test selected of our compounds in
MES-induced and PTZ-kindled seizure models in rats.32 A few of
these compounds showed promising anticonvulsant activity,
among which was compound 1 (Fig. 1). In the MES-induced seizure
1 (10 mg/kg ip; rats) showed protective effects significantly greater
than those of PIT. Moreover, derivative 1 (5 mg/kg ip; rats; the low-
est effective dose) concomitant with PHT (5 mg/kg ip; rats; ineffec-
tive dose) reduced the duration of tonic hind limb extension.
Taking into account these results we decided to obtain N-alkyl
(substituted)(homo)piperidine ether derivatives. As a lead struc-
tures for the introduced modifications, a drug candidate pitolisant
(WakixÒ) and compound 1 have been chosen. Modifications
included: introduction only one ether group, placing ether group
straightaway to the phenyl ring, different cycloalkylamine moi-
eties in the basic part of compounds, ascending alkyl chain length
(5–7 methylene groups) as well as changeable position of chlorine
in the benzene ring (3-Cl or 4-Cl). General structures of final com-
pounds 9–28 are presented in Scheme 1. Compound 9 was prelim-
inary tested in the-MES-induced and PTZ-kindled seizure models
in rats at a dose of 10 mg/kg (ip) as described by Sadek et al.32
The results showed some kind of anticonvulsant protection32 but
lower than for 1. Considering the above, we examined the whole
series of compounds (9–28) for their anticonvulsant activity and
neurotoxicity within the Anticonvulsant Screening Program (ASP)
carried out at National Institute of Neurological Disorders and
Stroke, National Institutes of Health (Rockville, USA). In vitro phar-
macological evaluation of compounds (9–28) included histamine
hH3R affinity in a binding assay (HEK 239 cells) and in cAMP accu-
mulation assay at HEK293 cells. Moreover, as absorption, distribu-
tion, metabolism, elimination and toxicity (ADME-Tox) are very
important properties of compounds, in vitro studies are often
applied at the earlier stages of drug discovery. We have chosen
competitor.
Pharmacological results are assembled in the Table 1. All of the
compounds, except compound 24 (due to its very low solubility)
showed moderate affinities for hH3R in nanomolar concentration
range (Ki: 128–805 nM). Moreover, most of the compounds
showed affinities in Ki range of 100–500 nM (with exception of
compounds 11, 23 and 24).
For the tested group of novel ligands, amongst four different
used cycloalkylamine moieties (piperidine, 3-methylpiperidine,
4-methylpiperidine, homopiperidine), it could be clearly seen, that
3-methylpiperidine is the most preferable basic moiety for his-
tamine H3R binding pocket. Surprisingly, in the group of heptyl
derivatives (e.g., compounds 22 and 26) 3-methylpiperidine
showed slightly lower affinity in comparison to ‘bare’ piperidine
derivatives (e.g., 21 vs 22; hH3R Ki = 333 45 vs Ki = 466 146 nM,
respectively). On the other hand, for 3-Cl series of compounds,
elongation of the alkyl chain resulted in the decrement of his-
tamine H3R affinity (14 vs 22—hH3R Ki = 174 46 vs
Ki = 446 146 nM, respectively), whereas in 4-Cl series elongation
of the alkyl spacer resulted in high increment of the affinity (e.g., 9
vs 17 vs 25; hH3R Ki = 427 83 vs Ki = 265 128 vs Ki = 128 49
nM, respectively). Compound 25 showed in fact the highest affinity
towards hH3R in the tested group of compounds. However, com-
pound 17, being direct structural homologue of pitolisant, showed
radical loss of affinity when compared to the reference ligand
(hH3R Ki = 265 128 nM vs Ki = 0.3—1 nM,24 respectively). This sit-
uation clearly shows that the position of ether moiety plays a cru-
cial role in receptor–ligand interactions for this group of
derivatives. Also, the exchange of the one ether moiety in com-
pound 1 for methylene group as in compound 25 had not positive
impact on histamine H3R affinity (hH3R Ki = 3.20 nM30 for 1 vs
Ki = 128 49 nM for 25). In summary, compounds with chloro

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
55
Figure 1. In vitro activity of selected previously described H3R ligands.30 In vitro and in vivo activity of pitolisant (WakixÒ).24
R2
R2
R1-H
ii
R2
+
O
Br
Br
Br
O
R1
n
n
n
HO
R2: 3-Cl, 4-Cl
i
9-28
n = 3-5
4-8
Compounds
R1
n
R2
R1
n
R2
4-Cl
11
19
27
4-Cl
9
17
25
3-Cl
---
13
3-Cl
---
15
3
4
5
3
4
5
21
23
3
4
5
10
18
26
---
14
22
3
4
5
12
20
28
---
16
24
Scheme 1. Synthetic pathway of described compounds 9–28. Reagents and conditions: (i) sodium propanolate (0.05 mol Na in 50 mL), room temperature, 150; 60 °C 3 h;
reflux 3 h; 32–74% (ii) K2CO3, KI, EtOH, H2O, reflux 20 h, 17–62%.
substituent in the para-position of the phenyl ring seem to confer
higher affinities than those of meta-substituted (being seven of
them 10, 18, 19, 20, 25, 26 and 28 with hH3R Ki < 250 nM). It can
be then concluded, that chloro substitution in the 4-position of
phenyl moiety is the most favorable for the binding pocket of his-
tamine H3R among the examples in this small series.
of 100 mg/kg (13–15, 18, and 22–28) showed also protection at
the both tested times (30 min and 4 h). In the rotarod test
compounds revealed toxicity especially in the higher doses
(100 mg/kg and 300 mg/kg). In the most cases they caused inability
to grasp rotarod or/and death.
Two compounds 20 and 26 were also tested in the MES test in
rats after ip administration. Data are collected in Table 3. Both of
them were active but compound 26 showed 100% protection
lasting from 0.5 h to 4 h.
According to ASP procedures for most promising compounds in
the MES test in mice (9, 10, 13–15, 18–20, and 25–28; Table 2)
showing anticonvulsant activity at the dose of 30 mg/kg and no
neurotoxicity at the same dose, additional MES tests in rats after
oral administration were performed. Results are presented in
Table 4 (Supplemental materials). Only three compounds (9, 14
and 27) were devoid of anticonvulsant protection, whereas the
other showed some kind of activity protecting in most cases 25%
of animals in two time points. All compounds in the tested dose
30 mg/kg did not show any toxicity.
2.2.2. Functional properties at histamine H3 receptor
Selected structures (10 and 25) were additionally tested in
cAMP accumulation assay at HEK293 cells expressing recombinant
hH3R. Addition of considered compounds resulted in a blockade
of histamine H3R agonist activity, leading to dose-dependent
increase of cAMP level in cells co-treated with H3R agonist
((R)(ꢀ)-
a-methylhistamine) and forskolin (H3R is a Gi-coupled
receptor). Basing on the obtained results 10 and 25 were classified
as histamine H3R antagonists. Antagonist potencies (EC50) deter-
mined in functional assays were as follows: 10—EC50 = 72 8 nM;
25—EC50 = 75 17 nM (Fig. 2).
2.2.3. Anticonvulsant activity
Selected compounds were screened to block psychomotor sei-
zures induced by low-frequency (6 Hz) and longer duration of
stimuli (3 s) delivered through corneal electrodes (test 6 Hz;
Table 5). Compounds effective in the 6 Hz test can be useful in
the treatment of pharmacoresistant epilepsy.38 Only two com-
pounds 15 and 26 showed some protection (from 25% to 100%)
in all the tested time at the dose 50 mg/kg and 60 mg/kg respec-
tively. Three compounds (17, 19 and 26) were tested at two doses,
but no protection was observed at the lowest one. All compounds
showed high toxicity in the active dose. For compound 20 even
TOX ED50 value was calculated: TOX ED50 = 36.2 mg/kg (t = 0.25 h).
Four the most promising compounds (18, 19, 20 and 26) were
selected for quantitative evaluation in mice or/and rats via ip
administration. The tests provided an evaluation of the ED50 and
TD50 values and the data are shown in Table 6. The ED50 values
are from 13.04 mg/kg for 19 (mice) to 16.80 mg/kg for 18 (mice).
Calculated protection index PI (PI = TD50/ED50) values for 18, 19,
Antiepileptic tests were carried out at National Institute of Neu-
rological Disorders and Stroke, National Institutes of Health (Rock-
ville, USA) within Anticonvulsant Screening Program (ASP)
according to known procedures.36,37 In vivo screening tests in mice
included: the maximal electroshock (MES), subcutaneous pente-
trazole (scMET) and neurotoxicity test (TOX, rotarod). In the MES
and scMET tests activity against generalized tonic–clonic seizures
and nonconvulsive (absence or myoclonic) seizures might be
predicted, respectively.38
Results of the anticonvulsant screening are presented in Table 2.
In the scMET test none of the tested compounds showed any pro-
tection. In the MES test compounds were administered in mice
intraperitoneally (ip) in the doses from 3 to 300 mg/kg, mostly in
the dose of 30 mg/kg. All of them (except 21–24) showed some
kind of protection at this dose (30 mg/kg) at 30 min after
administration. A few compounds which were active at the dose

56
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Table 1
Six compounds (17–21 and 26) were assessed for their ability to
halt pilocarpine-induced convulsive status epilepticus in rats
(ip).38 Tested compounds (17–21) at a dose 65 mg/kg and 26 at a
dose 50 mg/kg did not show any protection but some animals died
during the testing (Table 7; Supplemental materials). Compound
20 was also tested in a mouse model of temporal lobe epilepsy
(the corneal kindled mouse model).38,39 However, in the dose of
30 mg/kg 20 did not show any protection.
Selected compounds (17, 18, 19, 20) were also tested in neuro-
protection studies. Severe and repetitive seizures (e.g., status
epilepticus) could lead to neuronal damage and death.40 Tested
compounds did not show any cytoprotection against cells death
Human histamine H3 receptor in vitro affinities of compounds 9–28
No.
R1
n
R2
hH3Ra Ki (nM)
N
9
3
4-Cl
427 83b
N
N
10
11
3
3
4-Cl
4-Cl
133 39
caused by either KA (kainic acid) or NMDA (N-methyl-D-aspartate).
800 237
As certain antiepileptic drugs possess analgesic activity41 (e.g.,
carbamazepine, lamotrigine), therefore, many drug candidates for
epilepsy, still in preclinical stage, are evaluated for their analgesic
potency. The formalin test is usually performed to study whether
the compound possesses any analgesic activity.42 Compounds 19
(13 mg/kg; 0.25 h) and 26 (16 mg/kg; 0.25 h) did not cause scien-
tifically significant decrease of animals’ pain response in either
acute and inflammatory phase.
N
12
13
14
15
3
4
4
4
4-Cl
3-Cl
3-Cl
3-Cl
371 91
255 10
174 46
412 10
N
N
N
Analyzing the anticonvulsant activities of the synthesized com-
pounds it was noticeable that most compounds showing anticon-
vulsant activity contain chloro substituent in the para position of
the benzene ring. Moreover, more active were compounds with a
chain of six carbon atoms as more of them reached to further
ASP phases. However, in the whole series of compounds, 20 (hexyl
derivative) and 26 (heptyl derivative) are considered as the most
active. Unfortunately no straight correlation between hH3R affinity
and anticonvulsant activity of compounds can be seen. The most
active in ASP compounds 20 and 26 are not the most potent
hH3R ligands (Ki values about 200 nM vs Ki values about 130 nM
for 10 and 25; Table 1). Discrepancies could be connected with spe-
cies differences in affinities.43 In vivo tests were conducted in
rodents whereas in vitro binding assay on HEK-293 cell stably
expressing hH3R.
N
16
17
18
19
4
4
4
4
3-Cl
4-Cl
4-Cl
4-Cl
293 78
265 128
205 49
189 110
N
N
N
N
20
21
22
23
4
5
5
5
4-Cl
3-Cl
3-Cl
3-Cl
237 138
333 35
466 146
N
N
N
2.3. X-ray studies and in silico molecular and docking studies
Monocrystal of compound 19 (in the hydrogen oxalate form)
was selected as suitable for X-ray structure analysis. Molecule
adopts extended conformation and proton is located at N1
nitrogen. In the structure hydrogen oxalate ion was also located
(charge on O2B atom). Both charged species are joined via three
H-bonds: N1–H1. . .O2B = 2.843(4) Ǻ, N1–H1. . .O1B = 2.932(4) Ǻ
and O1A–H1A. . .O2B = 2.522(3) Ǻ. Finally in the crystal protonated
main species are fastened to oxalate ions chain (for details see
Fig. 3).
805
2
N
24
25
26
27
5
5
5
5
3-Cl
4-Cl
4-Cl
4-Cl
>150,000c
128 49
180 75
467 41
N
N
N
Computational approaches using Schrödinger Maestro 9.3.544
allowed the in silico visualization of the investigated compounds
binding to histamine H3R model obtained from GPCRM Structure
Modeling Server.45 The model was built on the crystal structure
of M3 muscarinic acetylcholine receptor, due to its’ highest
sequence homology to histamine H3R aa sequence (PDB code:
4DAJ; resolution: 3.4 Å). Validation with reference ligand pitolisant
according to the procedure described by Levoin et al. was also per-
formed.45 Docking studies results were interpreted by the values of
Docking Score function that resulted in the range of ꢀ7.95 to
ꢀ4.12. Previously described salt bridge formation between the pro-
tonated cycloalkylamine nitrogen and GLU20631 could be observed
for most of the compounds despite their in vitro activity. As it has
N
28
5
4-Cl
203 48
a
a
[3H]N -methylhistamine binding assay performed with cell membrane prepa-
ration of HEK-hH3R cells stably expressing human H3 receptor, mean value SEM,
n = 3; measurement as previously described.35
b
Ref. 32.
Difficulties with solubility.
c
20 and 26 are about 3 (PI: 3 for 18 and 26 in mice; 3.1 for 19 in mice,
and PI: 3.2 for 20 and 3.8 for 26 in rats) and are comparable to PI
values for phenobarbital (ED50 = 21.8 mg/kg, TD50 = 69.0 mg/kg;
PI = 3.2; data from Ref. 36) and lamotrigine (ED50 = 7.47 mg/kg,
TD50 = 30 mg/kg; PI = 4.0; data from Ref. 36). PI values ꢁ3 are not
high and suggest that the tested compounds (18, 19, 20 and 26)
can exert antiepileptic effects but with minor motor disturbances.
been previously stated, this might be
antagonists binding at the histamine H3R.45,46 Moreover, for all of
the ligands stacking could be observed between TYR189
a crucial residue for
p–p
and chlorinated phenyl ring. Interestingly for methylated
piperidine derivatives which have higher in vitro affinity than

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
57
Figure 2. cAMP accumulation studies in HEK293 cells expressing the hH3R, co-treated with forskolin, (R)(ꢀ)-
a-methylhistamine and tested compounds: 10 (left) and 25
(right).
non-methylated ones, nonadditional interactions between the
methyl group and side-chains amino acids were found. A binding
modes of compounds 10 and 19 with histamine H3R homology
model are presented in Figure 4. In order to test the possible influ-
ence of methyl group and its position on ligands affinity, possible
Van der Waal’s interactions between the ligand and binding site
aminoacids in the distance of 12 Å from ligand were studied. It
appeared that the interaction strength were higher for 3-methyl
derivatives than for 4-methyl derivatives (e.g., THR375 10 vs 11:
ꢀ1.4 vs ꢀ0.94), as well as methylated versus non-methylated
piperidine derivatives.
For all of the docked ligands after superimposition it could be
observed, that regardless of the alkyl chain length chloro substi-
tuted phenyl rings are situated in close proximity of TRP 174,
LEU 177, ALA 190, GLU 191, TYR 194, TRP 196. The reason for this
is probably the result of the interaction between lipophilic part of
the ligands and the functional groups of five partly polar and partly
lipophilic amino acids constituting the pocket at receptor binding
site.
forms. Such obtained theoretical values seem to be more realistic,
bearing in mind that all of the tested compounds are hydrogen
oxalates. Most similar theoretical-to-practical values were
obtained using Marvin software (R = 0.91598, R2 = 0.83902,
SD = 0.38036).
2.5. Antiproliferative effect study
The most active H3R ligands described in this study 10 and 25
were chosen to examine the selected ADME-TOX parameters—tox-
icity and metabolic stability. The drug candidates which have
acceptable ADME-TOX parameters are defined as a drug-like and
are suitable to the human Phase I clinical trials.50 The formazan
dye based EZ4U was applied for determination of the effect of both
tested compounds on proliferation of two eukaryotic cell lines—
human embryonic kidney HEK-293 cell line and neuroblastoma
IMR-32 cell line. The results were next compared to the reference
compound doxorubicin (DX). As was shown below, the incubation
of HEK-293 cells in the different concentrations of H3R ligands for
48 h revealed the dose dependent antiproliferative effect (Fig. 5).
The significant inhibition of cell viability was observed above
2.4. Lipophilicity evaluation by RP-TLC
10
the IC50 values of the reference compound DX (IC50 = 0.46
with the calculated IC50 value for 25 (IC50 = 33.62 M), the similar
effect of H3R ligand 25 was observed at more than 70-fold higher
concentration.
The observed antiproliferative effect of the examined
compounds against neuroblastoma cell line IMR-32 was slightly
higher than against HEK-293 (Fig. 6). The calculated IC50 values
l
M for 25 whereas for 10 at only 250
l
M. However comparing
As there is a strong aim to determine physicochemical prop-
erties of novel compounds at the early stage of preclinical
research, their lipophilicity was experimentally evaluated
(expressed by RM0 values) using planar RP-TLC method.47 As a
mobile phase, ternary solvent mixture—methanol/acetic acid/
water—was used, with constant, 10% acetic acid concentration
and varying methanol concentration in the range of 50–80%.
Tested set of compounds showed RM0 values in the range
between 0.914 and 1.505. Lipophilicity studies data are collected
in Table 8.
lM)
l
were 15.5
l
M for 25 and 89.0
l
M for 10. However, relative to
M) the examined com-
the calculated DX IC50 value (IC50 = 0.0051
l
pounds did not possess significant antiproliferative and antitumor
As it was assumed, lipophilicity rises with the elongation of
the alkyl chain. Moreover, for structural analogues (3-Cl and 4-
Cl derivatives) values resulted in the similar range, for example,
compounds 13–16 and 17–20. Similar dependence could be
observed for 3- and 4-methylpiperidine structural isomers, for
example, compounds 10 and 11. Slightly lower values could be
observed for homopiperidine derivatives in comparison with 3
(4)-methylpiperidine analogues. No correlation between
lipophilicity and hH3R affinity was observed for the tested series
(data not shown) as well as for lipophilicity and anticonvulsant
activity.
activity.
2.6. In silico metabolism study
The metabolic stability of 10 and 25 was first examined in silico
by using MetaSite51,52 computational method in two models of
metabolism: liver and CYP3A4 exclusively, and compared to the
results obtained for pitolisant. For compound 10 the highest prob-
ability of metabolism calculated by liver model occurred at the
3-substituted position of piperidine moiety. However, for com-
pound 25 MetaSite predicted similar to pitolisant highest possibil-
ity of metabolism at the aliphatic linker. The use of CYP3A4 model
showed for both examined compounds (10 and 25) and for pitoli-
sant as the most reactive position of metabolism, the methylene
bridge straightly connected to nitrogen in the piperidine moiety.
In order to compare the theoretical partition coefficient param-
eters (clogP) with practical ones, calculations using computer var-
ious programs: Chem3D, Marvin and QikProp for Schrödinger were
also performed.44,48,49 All of the compounds were used in ionic

58
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Table 2
Preliminary anticonvulsant evaluation of compounds 9–28 in the MES, the scMET and TOX test (mice, ip)*
Compd
Dose (mg/kg)
MES1
scMET2
TOX3
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
9
3
0/4
0/4
1/1
2/2
—
—
—
0/1
—
—
—
—
0/1
0/1
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
—
—
10
30
100
300
0/4
8/8a,b
4/4b
10
11
12
13
14
15
16
17
18
19
20
3
0/4
1/4
1/1
—
—
—
0/1
—
—
—
—
0/1
0/1
—
—
—
0/1
0/1
—
0/4
0/4
—
—
0/2
—
—
10
30
100
300
0/4
8/8a,b
4/4b
—
3
0/4
0/4
1/1
—
—
—
1/1
—
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
—
—
0/2
—
—
10
30
100
300
0/4
1/4f
8/8b
4/4b
—
3
0/4
0/4
1/1
—
—
—
0/1
1/1
—
—
—
0/1
0/1
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
0/1
—
10
30
100
300
0/4
8/8a,b,d
4/4b
—
3
0/4
0/4
1/1
2/2
—
—
—
0/1
3/3
—
—
—
0/1
0/1
—
—
—
0/1
0/1
—
0/4
0/4
—
—
0/2
0/4
—
10
30
100
300
0/4
8/8a,b
4/4b
3
0/4
0/4
1/1
3/3
—
—
—
0/1
3/3
—
—
—
0/1
0/1
—
0/4
0/4
0/4
8/8a
4/4b
—
—
0/2
0/4
0/0
10
30
100
300
0/1
0/1
—
3
0/4
0/4
1/1
1/1
—
—
—
0/1
1/1
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
0/4
0/4
0/4
0/2
0/1
—
10
30
100
300
0/4
8/8a,b
4/4b
3
0/4
0/4
1/1
1/1
—
—
—
0/1
—
—
—
—
0/1
0/1
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
—
—
10
30
100
300
1/4
8/8a,b
4/4b
3
0/4
1/4
1/1
—
0/4
0/4
1/1
—
-
-
0/1
—
—
-
-
0/1
—
—
0/4
0/4
1/4
0/4
0/4
0/2
—
10
30
100
300
8/8b
4/4b
—
—
—
3
0/4
0/4
1/1
3/3
—
—
—
0/1
3/3
1/1
—
—
0/1
0/1
—
—
—
0/1
0/1
—
0/4
0/4
0/4
—
—
0/2
0/4
0/1
10
30
100
300
0/8
4/4b,d
3
0/4
4/4
1/1
—
—
—
1/1
—
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
—
—
10
30
100
300
0/4
8/8b
4/4b
—
3
0/4
0/4
1/1
1/1
—
—
—
0/1
—
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
—
—
10
30
100
300
0/4
8/8b,g,h
4/4b
21
22
23
30
100
300
0/1
2/3
—
0/1
0/3
—
0/1
0/1
—
0/1
0/1
—
0/4
0/8
4/4b
0/2
0/4
1/1b
30
100
300
0/1
3/3
—
0/1
2/2
—
0/1
0/1
—
0/1
0/1
—
0/4
0/2
0/3
—
8/8a,b
4/4b
30
0/1
0/1
0/1
0/1
0/4
0/2

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
59
Table 2 (continued)
Compd
Dose (mg/kg)
MES1
scMET2
TOX3
0.5 h
4.0 h
0.5 h
4.0 h
0.5 h
4.0 h
100
300
1/2
1/1
3/3
—
0/1
—
0/1
—
6/8a,b
4/4a–c
2/4
—
24
25
30
100
300
0/1
2/2
—
0/1
2/3
—
0/1
—
—
0/1
0/1
—
0/4
7/8
4/4
0/2
0/4
—
3
0/4
0/4
1/1
1/1
—
—
—
0/1
1/1
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
2/3b
—
10
30
100
300
0/4
8/8a,b
4/4b
26
27
28
3
0/4
1/4
1/1
3/3
—
0/4
0/4
1/1
2/2
—
—
—
0/1
0/1
0/1
—
—
0/1
—
—
0/4
0/4
0/4
0/4
0/2
0/2
—
10
30
100
300
0/4
7/8a–e
4/4b,c
3
0/4
0/4
1/1
3/3
—
—
—
0/1
3/3
—
—
—
0/1
0/1
—
—
—
0/1
0/1
—
0/4
0/4
—
—
0/2
1/4
—
10
30
100
300
0/4
6/8a,c,e
4/4b
3
0/4
0/4
1/1
1/1
—
—
—
0/1
2/2
—
—
—
0/1
—
—
—
—
0/1
—
—
0/4
0/4
—
—
0/2
3/3b
—
10
30
100
300
0/4
8/8b,d
4/4
— The compound was not tested in the particular case.
a–g Kind of toxicity: a unable to grasp rotarod; b death; c diarrhea; d loss of righting reflex; e clonic seizures; f vocalization; g sedated; h myoclonic jerks.
*
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
or 32 mass units in compare to the substrates, suggesting the addi-
tion of one or two oxygen atoms and in consequence one or two
hydroxyl groups. To locate the site(s) of hydroxylation, the precise
analysis of the fragment ions produced by substrates and metabo-
lites under ion fragment analysis conditions were undertaken and
supported by in silico data. The obtained results showed the simi-
lar metabolism pathway of pitolisant (metabolite M1 with
m/z = 328 corresponding to the quasimolecular ion [M+H]+; Fig. 9B)
and compound 25 (metabolite M1 with m/z = 342 corresponding
to the quasimolecular ion [M+H]+; Fig. 10B). Based on the compar-
ison of ion fragment analyses of pitolisant and 25 to the analysis of
their aforementioned metabolites, the lack of characteristic peak
coming from N-methyl-piperidine with m/z = 98 produced by sub-
strates and the appearance of the new ion fragment with m/z = 101
produced by metabolites were observed. Additionally, further
analysis of the another ion fragments produced by both
metabolites excluded the double hydroxylation of benzene ring
or at aliphatic linker of pitolisant and 25 suggesting rather
the degradation of piperidine moiety followed by oxidation
(Figs. 9A–C and 10A–C). The metabolite M2 of pitolisant
(m/z = 312; Fig. 9C) was observed as a hydroxyl derivative with
hydroxyl group located at the methylene bridge situated probably
next to the ether bound. The site of hydroxylation was determined
due to the fragment ions with m/z = 142 and m/z = 125 produced
by M2, which excluded the presence of the hydroxyl group at the
piperidine and benzene ring as well as at the part of aliphatic linker
situated closer to the piperidine moiety (Fig. 9C). Moreover, the in
silico pitolisant metabolism prediction by liver model showed that
the methylene bridges which are nearby to the ether bound, are
susceptible for the modifications (Fig. 7). The molecular mass of
the another metabolite of 25 (M2) indicated the presence of one
Table 3
Anticonvulsant activity in the MES test in rats (ip)*
Compd
Test
Dose (mg/kg)
Pretreatment time
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
20
26
MES
TOX
30
30
2/4
0/4
2/4
0/4
2/4
1/4
0/4
0/4
0/4
0/4
MES
TOX
30
30
3/4
0/4
4/4
0/4
4/4
4/4
1/4
0/4
0/4
0/4
MES—maximum electroshock seizure: number of animals protected/number of
animals tested.
TOX—neurotoxicity assay (the rotarod test): number of animals exhibiting toxicity/
number of animals tested.
*
Ratios where at least one animal was protected have been highlighted in bold
for easier data interpretation.
The places of metabolism are shown in Figures 7 and 8. The circle
marked the site of H3R ligand involved in metabolism with the
highest probability calculated by MetaSite method. The darker
color of the marked functional group indicates its higher likelihood
to be involved in the metabolic pathway.
2.7. In vitro metabolism study
The metabolic stability of compounds 10 and 25 was also deter-
mined in vitro. The examined H3R ligands were incubated for 2 h
with human liver microsomes (HLMs). A full scan chromatograms
of the reaction mixtures obtained by UPLC–MS/MS detected the
presence of unreacted substrates and the two metabolites of pito-
lisant, 10 and 25 each (Figs. 9–11). The mass spectra gave the accu-
rate molecular masses of all metabolites which were up either 16

60
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Table 5
position of the benzene moiety (Fig. 10C). It was observed, that
metabolite M2 of 25 produced two fragment ions with m/z = 98
and m/z = 182, which excluded hydroxylation at the piperidine
moiety or at the aliphatic linker, and the fragment ion with
m/z = 144 which confirmed the presence of hydroxyl group at the
benzene moiety (Fig. 10C). Moreover, the results of 25 metabolism
prediction in silico by liver model showed the higher probability of
hydroxylation rather at the ortho than at the meta position of the
benzene moiety (Figs. 7 and 10C).
Metabolic pathway of compound 7 included the addition of one
(M1) or two (M2) hydroxyl groups (Fig. 11). According to 10 in
silico metabolism prediction, the highest probability to be involved
in the metabolic pathway was shown at the 3-position of piperi-
dine moiety, probably due to the presence of the methyl sub-
stituent (Fig. 7). The ion fragment analysis of M1 showed
hydroxylation of the parent ion (m/z = 312) and its major fragment
(m/z = 294) corresponding to ion where the water elimination
occurred (Fig. 11B). The precise study of the fragmentation routes
of the parent ion as well as the major dehydrated fragment did not
confirm the in silico data, which indicated the presence of the
Results of anticonvulsant activity of tested compounds in the 6-Hz test in mice (ip)*
Com
Test
Dose (mg/kg)
Pretreatment time
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
13
14
15
16
17
6 Hz
TOX
50
50
1/4
3/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
6 Hz
TOX
50
50
0/4
1/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
6 Hz
TOX
50
50
4/4
2/4
2/4
1/4
2/4
0/4
1/4
0/4
1/4
0/4
6 Hz
TOX
30
30
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
6 Hz
TOX
6 Hz
TOX
30
30
60
60
0/4
0/4
3/4
3/4
0/4
0/4
3/4
2/4
0/4
0/4
2/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
18
19
6 Hz
TOX
100
100
2/3
4/4
0/4
4/4
0/2
4/4
2/2
4/4
1/3
1/4
6 Hz
TOX
6 Hz
TOX
25
25
50
50
0/4
0/4
2/4
3/4
0/4
0/4
3/4
4/4
0/4
0/4
4/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
hydroxyl group at the
a position to piperidine nitrogen
(Figs. 7 and 11B). The molecular mass of metabolite M2 of 10 indi-
cated the presence of two additional hydroxyl groups (m/z = 328;
Fig. 11C). The sites of di-hydroxylation were determined due to
the fragment ions with m/z = 98, m/z = 126 and m/z = 168 produced
by M2, which excluded the presence of the hydroxyl group at the
piperidine moiety or aliphatic linker (Fig. 11C). Moreover, as dis-
cussed before for 25, the results of metabolism prediction in silico
by liver model, showed the higher probability of hydroxylation of 7
at the ortho than at the meta position of the benzene moiety
(Figs. 7 and 11C).
20
6 Hz
TOX
TOX
TOX
50
35
45
50
0/4
4/8
8/8
4/4
1/4
1/8
8/8
4/4
0/4
—
5/8
4/4
0/4
—
1/8
0/4
0/4
—
0/8
0/4
25
26
6 Hz
TOX
50
50
0/4
0/4
0/4
1/4
2/4
0/4
1/4
0/4
0/4
0/4
6 Hz
TOX
6 Hz
TOX
30
30
60
60
0/4
0/4
2/4
2/4
0/4
0/4
3/4
3/4
0/4
0/4
2/4
0/4
0/4
0/4
2/4
0/4
0/4
0/4
1/4
0/4
28
6 Hz
TOX
50
50
0/4
4/4
1/4
3/4
2/4
3/4
0/4
0/4
0/4
0/4
The luminescent CYP3A4 P450-GloTM assay was also used for
testing the effects compounds 10 and 25 on CYP3A4 activities. As
it is shown in Figure 12, compounds 10, 25 and pitolisant did not
— The compound was not tested in the particular case.
modulate cytochrome activity up to 10
compound ketoconazole inhibited completely the CYP3A4 at
10 M.
lM, whereas the reference
6 Hz—psychomotor 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.
l
*
Ratios where at least one animal was protected have been highlighted in bold
for easier data interpretation.
3. Conclusions
In this series of compounds, structures with promising submi-
cromolar affinity were obtained. Although, changing of place of
the ether function in investigated group of chlorophenoxyalkyl
additional hydroxyl group (m/z = 326; Fig. 10C). However, in com-
parison to the metabolite M2 of pitolisant the hydroxylation of 25
occurred not at the aliphatic linker but probably at the ortho
Table 6
Quantitative anticonvulsant data in mice and/or rats (ip)
Compd
Animals
Mice
MES ED50 (mg/kg) (95% confidence interval)
scMET ED50 (mg/kg)
>50a
TOX TD50 (mg/kg) (95% confidence interval)
PI TD50/ED50
3
18
16.80a
50.40a
(13.3–19.3)
(45.4–57.4)
19
20
26
Mice
Rats
13.04b
>30b
>44a
>47a
>50b
>40
40.77a
3.1
3.2
3
(11.31–15.16)
14.00a
(39.4–41.8)
44.10a
(8.4–18.7)
(38.3–48.5)
Mice
Rats
16.14a
48.28a
(14.89–17.3)
13.18c
(8.06–18.78)
7.47
(45.6–52.1)
50.35b
(45.7–55.8)
30
3.8
4.0
Lamotrigined
Phenobarbitald
Valproic acidd
Mice
Mice
Mice
(6.13–9.11)
21.8
(15.0–25.5)
263
(24.8–36.2)
69.0
(62.8–72.9)
398
13.2
(5.87–15.9)
220
3.2MES
5.2scMET
1.5MES
(237–282)
(177–268)
(356–445)
1.8scMET
a
b
c
Results observed after 0.25 h of administration of compound.
Results observed after 0.5 h of administration of compound.
Results observed after 1 h of administration of compound.
Data from Ref. 37.
d

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
4. Experimental
61
4.1. Chemistry
All reagents were purchased from commercial suppliers and
were used without further purification. Pitolisant was synthesized
by us in the Department of Technology and Biotechnology of Drugs
(Kraków, Poland) as described by Meier et al.53 Melting points (mp)
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 spec-
trometer. 1H NMR spectra were recorded on a Varian Mercury
300 MHz PFG spectrometer in DMSO-d6 or a Bruker AMX 300
(300 MHz) spectrometer (Bruker, Germany) in DMSO-d6. Chemical
shifts were expressed in parts per million (ppm) using the solvent
signal as an internal standard. Data are reported in the following
order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; qu,
quintet, m, multiplet; br, broad; cPropPh, phenyl; Pip, piperidine;
Figure 3. ORTHEP drawing of 19 hydrogen oxalate structure (light grey color for
carbons, red for oxygen, blue for nitrogen and green for chlorine).
hPip, homopiperidine), approximate coupling constants
J
expressed in Hertz (Hz), number of protons. 13C NMR spectra were
recorded on Varian-Mercury-VX 300 MHz PFG or 400 MHz
spectrometer 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
Analyser Vario El III (Hanau, Germany) and agreed with theoretical
values 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
with Merck Silica gel 60 RP-18 F254S glass plates, using planar chro-
matographic CHROMDES chambers. Compounds solutions were
derivatives (as compared to pitolisant) significantly influence the
affinity of the examined compounds toward hH3R. The highest
affinities were observed for 4-chloro substituted derivatives: 10
and 25 (Ki = 133 and 128 nM, respectively) with five and seven car-
bons alkyl chain. Also, the character of cyclic amine had a huge
influence on the affinity of the evaluated compounds—generally,
for this group the most potent were 3-methylpiperidine deriva-
tives. The results of anticonvulsant screening revealed that all of
the synthesized compounds showed activity in the MES screen
(mice; ip). Two compounds 20 and 26 were especially promising
and were also tested in different models of epilepsy (e.g., 6 Hz test,
corneal kindled in mice, pilocarpine in rats). Quantitative assays for
20 and 26 in mice (ip) and/or rats (ip) provided an evaluation of the
ED50 values. The highest activity was observed for 20 with ED50 of
14 mg/kg (MES; mice) and 26 with ED50 of 16.14 mg/kg (MES;
mice) and with ED50 of 13.18 mg/kg (MES; rats). Moreover, the
examined compounds show satisfying, selected ADME-TOX
parameters.
applied using Hamilton 25 lL syringes. Detection of compounds
was obtained by the staining of TLC plates with iodine
vapors. 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).
Figure 4. Position of compounds 10 and 19 in the binding pocket of H3R (ligand: white color for hydrogens, dark green for carbons, blue for nitrogen, light green for chlorine.
Green lines represent salt bridges).

62
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Table 8
Practical (RM0
compounds
4.1.3. 1-((6-Bromohexyl)oxy)-3-chlorobenzene (5)
) and theoretical lipophilicity values for the series of described
Synthesis from 3-chlorophenol (6.4 g, 0.05 mol), 1,6-dibromo-
hexane (12.2 g, 0.05 mol) in sodium propanolate (50 mL propan-
1-ol, 1.15 g, 0.05 mol Na). Obtained 4.7 g after purification by FC.
Yield 32%.
No.
RM0
QikPropa
Chem3Db
Marvinc
9
0.914
0.994
0.993
0.921
0.99
1.202
1.203
1.115
0.99
1.203
1.202
1.115
1.059
1.504
1.503
1.473
1.059
1.505
1.504
1.479
4.513
4.925
4.914
4.848
4.910
4.870
5.295
5.074
4.962
5.297
5.292
5.025
5.359
5.548
5.714
5.711
5.293
5.674
5.722
5.609
5.187
5.537
5.537
5.746
5.716
6.065
6.065
6.275
5.716
6.065
6.065
6.275
6.245
6.594
6.594
6.804
6.245
6.594
6.594
6.804
0.81
1.17
1.17
1.25
1.25
1.62
1.62
1.70
1.25
1.62
1.62
1.70
1.70
2.06
2.06
2.14
1.70
2.06
2.06
2.14
1H NMR (DMSO-d6) d: 7.27 (t, J = 8.0 Hz, 1H, Ph-5-H), 6.91–7.01
(m, 2H, Ph-2,4-H), 6.87 (dd, J = 1.7, 8.3 Hz, 1H, Ph-6-H), 3.95 (t,
J = 6.4 Hz, 2H, CH2-O), 3.51 (t, J = 6.7 Hz, 2H, CH2-Br), 1.79 (quin,
J = 6.80 Hz, 2H, AO-CH2-CH2A), 1.68 (quin, J = 6.5 Hz, 2H, ACH2-
CH2-Br), 1.31–1.50 (m, 4H, 2 ꢂ ACH2A); 13C NMR (300 MHz,
DMSO-d6) d: 160.06, 134.16, 131.24, 120.76, 114.84, 113.97,
68.16, 35.51, 32.62, 28.81, 27.70, 25.05.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
4.1.4. 1-((6-Bromohexyl)oxy)-4-chlorobenzene (6)56
Synthesis from 4-chlorophenol (3.2 g, 0.025 mol), 1,6-dibromo-
hexane (6.1 g, 0.025 mol) in sodium propanolate (25 mL propan-1-
ol, 0.575 g, 0.025 mol Na). Obtained 5.4 g of crude product. Yield
74%.
4.1.5. 1-((7-Bromoheptyl)oxy)-3-chlorobenzene (7)
Synthesis from 3-chlorophenol (3.2 g, 0.025 mol), 1,7-dibromo-
heptane (6.45 g, 0.025 mol) in sodium propanolate (25 mL propan-
1-ol, 0.575 g, 0.025 mol Na). Obtained 2.6 g after purification by FC.
Yield 34%.
a
Ref. 44.
Ref. 48.
Ref. 49.
b
c
1H NMR (300 MHz, DMSO-d6) d: 7.26 (t, J = 8.1 Hz, 1H, Ph-5-H),
6.91–7.01 (m, 2H, Ph-2,4-H), 6.87 (dd, J = 1.5, 8.5 Hz, 1H, Ph-6-H),
3.95 (t, J = 6.4 Hz, 2H, CH2-O), 3.50 (t, J = 6.7 Hz, 2H, CH2-Br), 1.78
(quin, J = 6.8 Hz, 2H, AO-CH2-CH2A), 1.67 (def quin, 2H, ACH2-
CH2-Br), 1.26–1.45 (m, 6H, 3 ꢂ CH2A); 13C NMR (300 MHz,
DMSO-d6) d: 160.08, 134.16, 131.24, 120.74, 114.82, 113.97,
68.21, 35.57, 32.63, 28.23, 27.90, 25.72.
4.1.1. General procedure for compounds 4–8*
⁄Compounds 4,54–56 56 and 857 were obtained previously but in
the different conditions.
To a solution of freshly prepared sodium propanolate (50 mL;
1.15 g, 0.05 mol Na), proper substituted phenols were added and
6
stirred in room temperature (RT) for 15 min.
a,x-dibromoalkanes
were then added dropwisely in the time of one hour. The reaction
mixture was stirred in 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 crude product, 100 mL of 10% NaOH was added
and left overnight in cold. To a resulting white oil, CH2Cl2 was
added, mixed and layers were then separated. Organic layer was
dried over sodium sulphate filtered and evaporated. Used without
further purification (4, 6, 8) or purified by Flash Chromatography
(FC; 5, 7) (petroleum ether/CH2Cl2 [95:5]).
4.1.6. 1-((7-Bromoheptyl)oxy)-4-chlorobenzene (8)57
Synthesis from 4-chlorophenol (6.4 g, 0.05 mol), 1,7-dibromo-
heptane (12.9 g, 0.05 mol) in sodium propanolate (50 mL propan-
1-ol, 1.15 g, 0.05 mol Na). Obtained 11.3 g of crude product. Yield
74%.
4.1.7. General procedure for compounds 9–28
To a suspension of potassium carbonate and catalytic amount of
potassium iodide in water, a proper cyclic amine and compounds
4–8 in ethanol were added. Mixture was then refluxed for 20 h
(TLC controlled). After cooling down to RT, the solid was filtrated
and the residue evaporated under the reduced pressure and puri-
fied by one of procedures:
4.1.2. 1-((5-Bromopentyl)oxy)-4-chlorobenzene (4)54–56
Synthesis from 4-chlorophenol (6.4 g, 0.05 mol), 1,5-dibro-
mopentane (11.5 g, 0.05 mol) in sodium propanolate (50 mL pro-
pan-1-ol, 1.15 g, 0.05 mol Na). Obtained 8.8 g of crude product.
Yield 63%.
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.
Figure 5. The antiproliferative effect of 10, 25 and doxorubicin (reference) against
HEK-293 cell line. Values represent the mean of n = 4 experiments.
Figure 6. The antiproliferative effect of 10, 25 and doxorubicin against IMR-32 cell
line. Values represent the mean of n = 4 experiments.

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
63
procedure A. Yield 50%. Raw product was transformed into oxalic
acid salt yielding 0.82 g of final compound. Mp: 134–136 °C.
[1-(5-(4-Chlorophenoxy)pentyl)piperidine hydrochloride, mp.
178–180 °C).34
]
C16H24NOCl ꢂ C2H2O4 (MW = 371.19). 1H NMR
(DMSO-d6) d: 7.31 (d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz,
2H, Ph-2,6-H), 3.94 (t, J = 6.3 Hz, 2H, CH2-O), 3.27–2.91 (m, 6H,
Pip-2,6-H2 + Pip-CH2), 1.75–1.61 (m, 8H, Pip-3,5-H2, 2CH2), 1.50–
1.34 (m, 4H, Pip-4-H2, ACH2). 13C NMR (400 MHz, DMSO-d6) d:
165.20, 157.91, 129.66, 124.54, 116.64, 67.92, 56.21, 52.24, 28.49,
23.35, 23.22, 22.97, 21.99. UV–VIS, kmax (loge): 217 (4.24), 224
(4.26), 227 (4.28), 281 (4.16). LC–MS: purity 100% tR = 4.94, (ESI)
m/z [M+H]+ 282.23. IR (cmꢀ1): 3422.06 (N(CH2A)3), 3030.59 (aro-
matic CH@), 2944.77 (het. CH2A), 2864.74, 2677.68, 2546.54,
1718.26, 1646.91, 1491.67, 1249.79 (O-CH aromatic), 1091.51,
1015.34, 946.88, 857.20, 720.28 (alif. CH2A). Anal. calcd for
C
16H24NOCl ꢂ C2H2O4: C, 58.24; H, 7.06; N, 3.77. Found: C, 58.05;
H, 7.22; N, 3.75.
4.1.9. 1-(5-(4-Chlorophenoxy)pentyl)-3-methylpiperidine
hydrogen oxalate (10)
Synthesis from 4 (1.39 g, 0.005 mol) and 3-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.8 g of oil. Puri-
fied by procedure A. Yield 54%. Raw product was transformed into
oxalic acid salt yielding 0.93 g of final compound. Mp: 119–121 °C.
Figure 7. Plots of MetaSite predictions for sites of metabolism for 10 (A), 25 (B) and
pitolisant (C) by liver. The darker color of the marked functional group indicates its
higher probability to be involved in the metabolic pathway. The blue circle marked
the site of H3R ligand involved in metabolism with the highest probability
calculated by MetaSite method.
C
17H26NOCl ꢂ C2H2O4 (MW = 385.90) 1H NMR (DMSO-d6) d: 7.29
(d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 8.7 Hz, 2H, Ph-2,6-H),
3.94 (t, J = 6.2 Hz, 2H, CH2-O), 3.31 (t, J = 6.2 Hz, 2H, Pip-2,6-
CH2a), 2.95 (t, J = 6.1 Hz, 2H, Pip-CH2), 2.64–2.71 (m, 1H, Pip-2-
He), 2.49–2.38 (m, 1H, Pip-6-He), 1.84–1.66 (m, 8H, 3 ꢂ CH2, Pip-
5-H2), 1.44–1.34 (m, 2H, Pip-4-H2), 1.08–0.99 (m, 1H, Pip-3-H),
0.96(d, J = 6.7 Hz, 3H, CH3). 13C NMR (400 MHz, DMSO-d6) d:
165.27, 157.91, 129.66, 124.54, 116.64, 67.92, 57.83, 56.22, 51.86,
Organic layer was evaporated and dissolved in 3% HCl solution, fol-
lowed by washing with diethyl ether and neutralization with 10%
NaOH solution. Final product was extracted with CH2Cl2 from the
basic solution, dried over Na2SO4, filtered and evaporated.
Procedure B: Column chromatography (MeOH/CH2Cl2; 9:1).
Oily products (from procedure A or B) were transformed into
oxalic acid salts using 10% excess of oxalic acid solution in absolute
ethanol in RT, and then precipitated by addition of ethyl ether.
30.56, 28.86, 28.49, 23.35, 23.23, 22.60, 19.05. UV–VIS kmax (loge):
211 (4.29), 214 (4.31), 217 (4.32), 220 (4.33), 223 (4.33), 229
(4.32), 282 (4.19). IR (cmꢀ1): 3422.06 (N(CH2A)3), 3009.37 (aro-
matic CH@), 2939.95 (het. CH2A), 2874.38 (CH3A), 2673.68,
2556.18, 1720.19, 1637.27, 1492.63, 1474. 31, 1402.96, 1280.50,
1242.90 (O-CH aromatic), 1092.48, 1028.84, 824.42, 720.28 (alif.
CH2A). LC–MS: purity 100% tR = 5.26, (ESI) m/z [M+H]+ 296.32.
Anal. calcd for C17H26NOCl ꢂ C2H2O4: C, 59.14; H, 7.31; N, 3.63.
Found: C, 59.10; H, 7.53; N, 3.60.
4.1.8. 1-(5-(4-Chlorophenoxy)pentyl)piperidine hydrogen
oxalate (9)
Synthesis from 4 (1.39 g, 0.005 mol) and piperidine (0.85 g,
0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount) in etha-
nol (105 mL) and water (20 mL). Obtained 0.7 g of oil. Purified by
4.1.10. 1-(5-(4-Chlorophenoxy)pentyl)-4-methylpiperidine
hydrogen oxalate (11)
Synthesis from 4 (1.39 g, 0.005 mol) and 4-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.8 g of oil. Puri-
fied by procedure A. Yield 54%. Raw product was transformed into
oxalic acid salt yielding 0.88 g of final compound. Mp: 125–127 °C.
C
17H26NOCl ꢂ C2H2O4 (MW = 385.90). 1H NMR (DMSO-d6) d: 7.30
(d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 8.7 Hz, 2H, Ph-2,6-H),
3.94 (t, J = 6.2 Hz, 2H, CH2-O), 3.34 (br d, J = 12 Hz,
2H, Pip-2,6-H2a), 2.95 (t, J = 6.1 Hz, 2H, Pip-CH2), 2.83–2.76 (m,
2H, Pip-2,6-He), 1.75–1.60 (m, 7H, 3 ꢂ CH2, Pip-4-H), 1.44–1.27
(m, 4H, Pip-3,5-H2), 0.90 (d, J = 6.4 Hz, 3H, CH3). 13C NMR
(300 MHz, DMSO-d6) d: 165.24, 157.88, 129.65, 124.51, 116.60,
67.88, 28.46, 23.45, 23.20, 21.29. UV–VIS, kmax (loge): 215 (4.31),
218 (4.32), 222 (4.32), 225 (4.33), 281 (4.19). IR (cmꢀ1): 3430.74
(N(CH2A)3), 3019.01 (aromatic CH@), 2956.34 (het. CH2A),
2930.31, 2866.67 (CH3A), 2752.90, 2703.71, 2544.61, 1716.34,
1655.59, 1596.77, 1577.49, 1475.28, 1455.99, 1394.28, 1282.43,
1248.68 (O-CH aromatic), 1092.48, 1024.98, 824.42, 755.96 (alif.
CH2A). LC–MS: purity 100% tR = 5.25, (ESI) m/z [M+H]+ 296.32.
Anal. calcd for C17H26NOCl ꢂ C2H2O4: C, 59.14; H, 7.31; N, 3.63.
Found: C, 59.31; H, 7.45; N, 3.62.
Figure 8. Plots of MetaSite predictions for sites of metabolism for 10 (A), 25 (B) and
pitolisant (C) by CYP3A4. The darker color of the marked functional group indicates
its higher probability to be involved in the metabolism pathway. The blue circle
marked the site of H3R ligand involved in metabolism with the highest probability
calculated by MetaSite method.

64
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Figure 9A. MS/MS spectra and ion fragments analysis of pitolisant.
Figure 9B. MS/MS spectra and ion fragments analysis of metabolite M1 of pitolisant in the total ion chromatogram.
Figure 9C. MS/MS spectra and ion fragments analysis of metabolite M2 of pitolisant in the total ion chromatogram.
Figure 10A. MS/MS spectra and ion fragments analysis of 25 and its metabolites in the total ion chromatogram.
Figure 10B. MS/MS spectra and ion fragments analysis of metabolite M1 of 25 in the total ion chromatogram.
4.1.11. 1-(5-(4-Chlorophenoxy)pentyl)homopiperidine
hydrogen oxalate (12)
into oxalic acid salt yielding 0.93 g of final compound. Mp: 114–
116 °C. C16H24NOCl ꢂ C2H2O4 (MW = 385.90). 1H NMR (DMSO-d6)
d: 7.30 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 8.7 Hz, 2H, Ph-2,6-
H), 3.94 (t, J = 6.2 Hz, 2H, CH2-O), 3.17 (br s, 2H, hPip-2,6-H2),
3.02 (def t, 2H, hPip-CH2), 1.75–1.57 (m, 8H, hPip-(3–6)-H2),
1.64–1.54 (m, 4H, 2 ꢂ CH2), 1.40 (def qu, 2H, CH2). 13C NMR
Synthesis from
4 (1.39 g, 0.005 mol) and homopiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.88 g of oil.
Purified by procedure A. Yield 59%. Raw product was transformed

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
65
Figure 10C. MS/MS spectra and ion fragments analysis of metabolite M2 of 25 in the total ion chromatogram.
Figure 11A. MS/MS spectra and ion fragments analysis of 10 in the total ion chromatogram.
Figure 11B. MS/MS spectra and ion fragments analysis of M1 metabolite of 10 in the total ion chromatogram.
Figure 11C. MS/MS spectra and ion fragments analysis of M2 metabolite of 10 in the total ion chromatogram.
(400 MHz, DMSO-d6) d: 165.30, 157.91, 129.66, 124.54, 116.65,
67.95, 56.57, 53.91, 28.50, 26.55, 23.77, 23.42, 23.23. UV–VIS: kmax
(log
): 225 (4.29), 228 (4.29), 281 (4.14), 398 (2.62). IR (cmꢀ1):
3445.21 (N(CH2A)3), 3036.37 (aromatic CH@), 2939.95 (het.
CH2A), 2866.67, 2699.85, 2632.36, 2570.65, 2533.04, 1718.26,
1701.87, 1597.73, 1578.45, 1491.67, 1402.96, 1242.90 (O-CH aro-
matic), 1169.62, 1104.48, 1004.73, 825.38, 720.38 (alif. CH2A),
668.21. LC–MS: purity 98.46% tR = 5.23, (ESI) m/z [M+H]+ 296.26.
Anal. calcd for C16H24NOCl ꢂ C2H2O4: C, 59.14; H, 7.31; N, 3.63.
Found: C, 59.36; H, 7.67 N, 3.55.
4.1.12. 1-(6-(3-Chlorophenoxy)hexyl)piperidine hydrogen
oxalate (13)
e
Synthesis from 5 (1.46 g, 0.005 mol) and piperidine (0.85 g,
0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount) in etha-
nol (105 mL) and water (20 mL). Obtained 0.6 g of oil. Purified by
procedure A. Yield 41%. Raw product was transformed into oxalic
acid salt yielding 0.72 g of final compound. Mp: 109–111 °C.
C
17H26NOCl ꢂ C2H2O4 (MW = 385.89). 1H NMR (DMSO-d6) d: 7.30
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.99–6.95 (m, 2H, Ph-2,4-H), 6.92–6.88
(m, 1H, Ph-6-H), 3.98 (t, J = 6.2 Hz, 2H, CH2-O), 3.07 (br s, 4H,

66
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
Pip-2,6-He), 1.77–1.59 (m, 7H, CH2, Pip-3,4,5-CH), 1.47–1.30 (m,
6H, 3 ꢂ CH2), 0.91 (d, J = 6.4 Hz, 3H, CH3). 13C NMR (400 MHz,
DMSO-d6) d: 165.19, 160.08, 134.18, 131.31, 120.81, 114.86,
114.02, 68.16, 28.73, 28.54, 26.34, 25.47, 23.81. UV–VIS kmax
(loge
): 221 (4.25), 275 (4.19), 282 (4.14). IR (cmꢀ1): 3432.67
(N(CH2A)3), 3028.66 (aromatic CH@), 2944.77 (het. CH2A),
2869.56 (CH3A), 2636.14, 2615.00, 2538.83, 1718.26, 1595.81,
1578.45, 1470.46, 1418.39, 1397.17, 1340.28, 1310.39, 1281.47,
1241.93 (O-CH aromatic), 1101.15, 1070.30, 1022.09, 945.91,
885.17, 742.46 (alif. CH2A), 684.61. LC–MS: purity 99.50%
tR = 5.61, (ESI) m/z [M+H]+ 310.28. Anal. calcd for C18H28NOCl ꢂ C2-
H2O4: C, 60.07; H, 7.56; N, 3.50. Found: C, 60.14; H, 7.76 N, 3.51.
Figure 12. The effect of 10, 25, pitolisant and ketoconazole on CYP3A4 activity.
Values represent the mean of n = 3 experiments.
4.1.15. 1-(6-(3-Chlorophenoxy)hexyl)homopiperidine hydrogen
oxalate (16)
Synthesis from
5 (1.46 g, 0.005 mol) and homopiperidine
Pip-2,6-CH2), 2.94 (t, J = 6.1 Hz, 2H, N-CH2), 1.75–1.59 (m, 8H,
2 ꢂ CH2, Pip-3,5-H2), 1.52–1.30 (m, 6H, 2 ꢂ CH2, Pip-4-H2). 13C
NMR (300 MHz, DMSO-d6) d: 165.24, 160.05, 134.15, 131.29,
120.78, 114.81, 113.99, 68.12, 59.26, 52.34, 28.70, 26.29, 25.44,
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.7 g of oil. Puri-
fied by procedure A. Yield 45%. Raw product was transformed into
oxalic acid salt yielding 0.74 g of final compound. Mp: 127–129 °C.
23.52, 22.94, 21.96. UV–VIS kmax (loge): 212 (4.25), 215 (4.27),
C
18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.29
219 (4.28), 226 (4.27), 275 (4.19), 282 (4.13). IR (cmꢀ1): 3431.71
(N(CH2A)3), 3025.76 (aromatic CH@), 2944.77 (het. CH2A),
2862.81, 2643.93, 2541.72, 1718.26, 1701.87, 1596.77, 1482.99,
1469.49, 1397.17 1334.50, 1281.47, 1243.86 (O-CH aromatic),
1097.30, 1066.48, 1032.69, 948.98, 883.24, 720.28 (alif. CH2A),
684.61. LC–MS: purity 100% tR = 5.36, (ESI) m/z [M+H]+ 296.26.
Anal. calcd for C17H26NOCl ꢂ C2H2O4: C, 59.14; H, 7.31; N, 3.63.
Found: C, 59.40; H, 7.46 N, 3.62.
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.99–6.95 (m, 2H, Ph-2,4-H), 6.92–6.88
(m, 1H, Ph-6-H), 3.98 (t, J = 6.2 Hz, 2H, CH2-O), 3.20–3.17 (m, 4H,
hPip-2,6-CH2), 3.04–2.99 (m, 2H, hPip-CH2), 1.77–1.59 (m, 12H,
hPip-3,4,5,6-CH2, 2 ꢂ CH2), 1.48–1.28 (m, 4H, 2 ꢂ CH2). 13C NMR
(400 MHz, DMSO-d6) d: 165.24, 160.07, 134.18, 131.31, 120.81,
114.86, 114.02, 68.15, 56.67, 53.91, 40.61, 28.73, 26.53, 26.28,
25.45, 23.97, 23.42. UV–VIS kmax (loge): 221 (4.25), 229 (4.24),
275 (4.18), 282 (4.12). IR (cmꢀ1): 3437.49 (N(CH2A)3), 3030.59
(aromatic CH@), 2941.88 (het. CH2A), 2860.87, 2709.50, 2648.75,
2536.90, 2362.37, 1701.87, 1635.34, 1596.77, 1470.46, 1398.14,
1310.39, 1249.65 (O-CH aromatic), 1155.15, 1073.19, 1034.62,
884.20, 721.25 (alif. CH2A), 683.64. LC–MS: purity 100% tR = 5.60,
(ESI) m/z [M+H]+ 310.28. Anal. calcd for C18H28NOCl ꢂ C2H2O4: C,
60.07; H, 7.56; N, 3.50. Found: C, 60.05; H, 7.69 N, 3.52.
4.1.13. 1-(6-(3-Chlorophenoxy)hexyl)-3-methylpiperidine
hydrogen oxalate (14)
Synthesis from 5 (1.46 g, 0.005 mol) and 3-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.5 g of oil. Purified
by procedure A. Yield 32%. Raw product was transformed into oxalic
acid salt yielding 0.54 g of final compound. Mp: 110–113 °C. C18H28
-
4.1.16. 1-(6-(4-Chlorophenoxy)hexyl)piperidine hydrogen
oxalate (17)
NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.31
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.99–6.95 (m, 2H, Ph-2,4-H), 6.92–6.88
(m, 1H, Ph-6-H), 3.98 (t, J = 6.2 Hz, 2H, CH2-O), 3.37–3.27 (m, 2H,
Pip-2,6-CH2a), 2.95 (t, J = 6.1 Hz, 2H, Pip-CH2), 2.73–2.66 (m, 1H,
Pip-2-He), 2.51–2.57 (m, 1H, Pip-6-He), 1.75–1.50 (m, 8H, 2 ꢂ CH2,
Pip-4,5-CH2), 1.47–1.30 (m, 4H, 2 ꢂ CH2), 1.11–0.97 (m, 1H, Pip-3-
H), 0.88 (d, J = 6.6 Hz, 3H, CH3). 13C NMR (300 MHz, DMSO-d6) d:
165.26, 160.05, 134.15, 131.29, 120.78, 114.82, 113.99, 68.12,
57.80, 56.30, 51.84, 30.56, 28.87, 28.70, 26.30, 25.44, 23.56, 22.61,
Synthesis from 6 (1.46 g, 0.005 mol) and piperidine (0.85 g,
0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount) in etha-
nol (105 mL) and water (20 mL). Obtained 0.25 g of oil. Purified by
procedure B. Yield 17%. Raw product was transformed into oxalic
acid salt yielding 0.22 g of final compound. Mp: 135–137 °C.
C
17H26NOCl ꢂ C2H2O4 (MW = 385.89). 1H NMR (DMSO-d6) d: 7.31
(d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.95 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.94
(t, J = 6.3 Hz, 2H, CH2-O), 3.36–3.26 (m, 4H, Pip-2,6-CH2), 2.93 (t,
J = 6.1 Hz, 2H, Pip-CH2), 1.82–1.61 (m, 8H, 2 ꢂ CH2, Pip-3,5-CH2),
1.57–1.47 (m, 2H, Pip-4-H2CH2), 1.08–0.99 (m, 2H, CH2), 0.97–
0.80 (m, 2H, CH2). 13C NMR (400 MHz, DMSO-d6) d: 165.21,
157.95, 129.66, 124.50, 116.63, 68.11, 56.30, 52.38, 28.76, 26.33,
19.05. UV–VIS kmax (loge): 213 (4.26), 216 (4.26), 223 (4.28), 275
(4.19), 282 (4.13). IR (cmꢀ1): 3421.10 (N(CH2A)3), 3024.80 (aromatic
CH@), 2940.91 (het. CH2A), 2866.67 (CH3A), 2674.48, 2554.25,
2348.87, 1720.19, 1596.77, 1471.42, 1428.99, 1395.25, 1334.50,
1310.39, 1283.39, 1249.65 (O-CH aromatic), 1231.11, 1100.19,
1024.02, 883.24, 683.64 (alif. CH2A). LC–MS: purity 100% tR = 5.73,
(ESI) m/z [M+H]+ 310.28. Anal. calcd for C18H28NOCl ꢂ C2H2O4:
C, 60.07; H, 7.56; N, 3.50. Found: C, 59.94; H, 7.93 N, 3.70.
25.48, 23.57, 22.97, 21.99. UV–VIS kmax (loge): 209 (4.09), 215
(4.16), 225 (4.24), 282 (3.66), 398 (2.68). IR (cmꢀ1): 3428.38 (N
(CH2A)3), 3020.52 (aromatic CH@), 2948.86 (het. CH2A), 2868.54,
2690.77, 2538.19, 2362.37, 1703.63, 1656.40, 1595.56, 1471.54,
1394.95, 1332.21, 1244.27 (O-CH aromatic), 1169.39, 1069.24,
1055.30, 939.71, 836.56, 752.26 (alif. CH2A), 669.35. LC–MS:
purity 100% tR = 5.35, (ESI) m/z [M+H]+ 296.28. Anal. calcd for
4.1.14. 1-(6-(3-Chlorophenoxy)hexyl)-4-methylpiperidine
hydrogen oxalate (15)
Synthesis from 5 (1.46 g, 0.005 mol) and 4-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.47 g of oil. Puri-
fied by procedure A. Yield 30%. Raw product was transformed into
oxalic acid salt yielding 0.54 g of final compound. Mp: 144–146 °C.
C
17H26NOCl ꢂ C2H2O4: C, 59.14; H, 7.31; N, 3.63. Found: C, 59.08;
H, 7.04 N, 3.64.
4.1.17. 1-(6-(4-Chlorophenoxy)hexyl)-3-methylpiperidine
hydrogen oxalate (18)
Synthesis from 6 (1.46 g, 0.005 mol) and 3-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.55 g of oil.
Purified by procedure B. Yield 38%. Raw product was transformed
C
18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.29
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.99–6.95 (m, 2H, Ph-2,4-H), 6.92–6.88
(m, 1H, Ph-6-H), 3.98 (t, J = 6.2 Hz, 2H, CH2-O), 3.37–3.33 (m, 2H,
Pip-2,6-CH2a), 2.97–2.92 (m, 2H, Pip-CH2), 2.86–2.78 (m, 2H,

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
67
into oxalic acid salt yielding 0.37 g of final compound. Mp: 132–
135 °C. C18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6)
d: 7.30 (d, J = 9.0 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-
H), 3.93 (t, J = 6.3 Hz, 2H, CH2-O), 3.37–3.27 (m, 2H, Pip-2,6-
CH2a), 2.93 (t, J = 6.1 Hz, 2H, Pip-CH2), 2.73–2.66 (m, 1H, Pip-2-
He), 2.51–2.57 (m, 1H, Pip-6-He), 1.75–1.50 (m, 8H, 2 ꢂ CH2, Pip-
4,5-CH), 1.47–1.30 (m, 4H, 2 ꢂ CH2), 1.02 (def q, 1H, Pip-3-H),
0.86 (d, J = 6.7 Hz, 3H, CH3). 13C NMR (400 MHz, DMSO-d6) d:
165.29, 157.93, 129.66, 124.49, 116.63, 68.11, 57.84, 51.90, 30.50,
28.73, 26.30, 25.45, 23.57, 19.01. IR (cmꢀ1): 3420.79 (N(CH2A)3),
3030.02 (aromatic CH@), 2942.24 (het. CH2A), 2871.79, 2871.79,
2729.70, 2686.52, 2539.54, 1722.85, 1597.58, 1491.53, 1392.88,
1284.25, 1247.05 (O-CH aromatic), 1168.28, 1090.58, 1013.03,
983.35, 830.93, 702.30 (alif. CH2A), 666.87, 476.89. UV–VIS kmax
ethanol (105 mL) and water (20 mL). Obtained 0.56 g of oil. Puri-
fied by procedure A. Yield 55%. Raw product was transformed into
oxalic acid salt yielding 0.63 g of final compound. Mp: 115–117 °C.
C
18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.27
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.97–6.94 (m, 2H, Ph-2,4-H), 6.90–6.85
(m, 1H, Ph-6-H), 3.96 (t, J = 6.2 Hz, 2H, CH2-O), 3.05 (br s, 4H,
Pip-2,6-CH2), 2.92 (t, J = 6.1 Hz, 2H, Pip-CH2), 1.70–1.26 (br m,
16H, CH2, Pip-3,4,5-CH2, 5 ꢂ CH2). 13C NMR (400 MHz, DMSO-d6)
d: 165.20, 160.09, 134.17, 131.29, 120.78, 114.85, 114.01, 68.24,
56.34, 52.37, 28.86, 28.65, 26.55, 25.66, 23.54, 22.96, 21.98. UV–
VIS kmax (loge): 213 (4.28), 221 (4.28), 275 (4.20), 282 (4.15). IR
(cmꢀ1): 3431.71 (N(CH2A)3), 3020.94 (aromatic CH@), 2938.98
(het. CH2A), 2866.67, 2684.43, 2548.37, 1721.16, 1627.63,
1593.88, 1482.03, 1470.26, 1392.35, 1349.93, 1284.36, 1230.36
(O-CH aromatic), 1198.54, 1167.69, 1073.19, 1042.34, 1022.09,
868.77, 850.45, 775.24 (alif. CH2A), 699.07, 680.75. LC–MS: purity
(log
e): 212 (4.20), 222 (4.26), 224 (4.26), 282 (3.78). LC–MS: purity
98% tR = 5.59, (ESI) m/z [M+H]+ 310.28. Anal. calcd for C18H28
-
NOCl ꢂ C2H2O4: C, 60.07; H, 7.56; N, 3.50. Found: C, 59.94; H,
100% tR = 5.75, (ESI) m/z [M+H]+ 310.35. Anal. calcd for C18H28
-
7.52 N, 3.46.
NOCl ꢂ C2H2O4: C, 60.07; H, 7.56; N, 3.50. Found: C, 59.91; H,
7.50; N, 3.59.
4.1.18. 1-(6-(4-Chlorophenoxy)hexyl)-4-methylpiperidine
hydrogen oxalate (19)
4.1.21. 1-(7-(3-Chlorophenoxy)heptyl)-3-methylpiperidine
hydrogen oxalate (22)
Synthesis from 6 (1.46 g, 0.005 mol) and 4-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.35 g of oil. Puri-
fied by procedure A followed by B. Yield 24%. Raw product was
transformed into oxalic acid salt yielding 0.3 g of final compound.
Mp: 129–131 °C. C18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR
(DMSO-d6) d: 7.30 (d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz,
2H, Ph-2,6-H), 3.93 (t, J = 6.2 Hz, 2H, CH2-O), 3.37–3.33 (m, 2H,
Pip-2,6-CH2a), 2.97–2.92 (m, 2H, Pip-CH2), 2.86–2.78 (m, 2H, Pip-
2,6-He), 1.77–1.59 (m, 7H, CH2, Pip-3,5-CH2, Pip-4-H), 1.47–1.30
(m, 6H, 4 ꢂ CH2), 0.90 (d, J = 6.4 Hz, 3H, CH3). IR (cmꢀ1): 3431.94
(N(CH2A)3), 3023.99 (aromatic CH@), 2940.34 (het. CH2A),
2927.34, 2869.32, 2646.85, 2548.52, 1718.50, 1628.17, 1492.99,
1473.80, 1404.70, 1284.51, 1245.61 (O-CH aromatic), 1229.73,
1172.01, 1090.47, 1017.93, 945.17, 831.85, 700.23 (alif. CH2A),
Synthesis from 7 (1.53 g, 0.005 mol) and 3-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.52 g of oil. Puri-
fied by procedure A. Yield 38%. Raw product was transformed into
oxalic acid salt yielding 0.63 g of final compound. Mp: 112–115 °C.
C
19H30NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.28
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.97–6.94 (m, 2H, Ph-2,4-H), 6.90–6.85
(m, 1H, Ph-6-H), 3.96 (t, J = 6.3 Hz, 2H, CH2-O), 3.38–3.21 (m, 2H,
Pip-2,6-CH2a), 2.93 (def t, 2H, Pip-CH2), 2.68 (br s, 1H, Pip-2-He),
2.43–2.41 (m, 1H, Pip-6-He), 1.93 (br s, 2H, CH2), 1.80–1.66 (m,
7H, Pip-3,4,5-CH, CH2), 1.48–1.27 (m, 6H, 3 ꢂ CH2), 0.86 (d,
J = 6.7, 3H, CH3). 13C NMR (300 MHz, DMSO-d6) d: 165.20, 160.06,
134.15, 131.28, 120.77, 114.82, 113.99, 68.21, 57.83, 56.34, 51.81,
30.56, 28.83, 28.64, 26.54, 25.64, 23.56, 19.04. UV–VIS kmax (loge):
512.08. UV–VIS kmax (log
e
): 223 (4.25), 225 (4.24), 282 (3.76).
213 (4.25), 220 (4.28), 225 (4.28), 231 (4.26), 275 (4.19), 282
(4.14). IR (cmꢀ1): 3437.79 (N(CH2A)3), 2937.06 (aromatic CH@),
2852.34 (het. CH2A), 2692.20 (CH3A), 2502.19, 2413.48, 1801.19,
1771.30, 1712.48, 1594.84, 1467.56, 1455.99, 1394.28, 1282.43,
1248.68 (O-CH aromatic), 1092.48, 1024.98, 824.42, 755.96 (alif.
CH2A). LC–MS: purity 98.47% tR = 6.06, (ESI) m/z [M+H]+ 324.31.
Anal. calcd for C19H30NOCl ꢂ C2H2O4 ꢂ 0.5 H2O: C, 59.63; H, 7.86;
N, 3.31. Found: C, 59.25; H, 8.20; N, 3.42.
LC–MS: purity 100% tR = 5.69, (ESI) m/z [M+H]+ 310.28. Anal. calcd
for C18H28NOCl ꢂ C2H2O4: C, 60.07; H, 7.56; N, 3.50. Found: C,
59.93; H, 7.38; N, 3.52.
4.1.19. 1-(6-(4-Chlorophenoxy)hexyl)homopiperidine hydrogen
oxalate (20)
Synthesis from
6 (1.46 g, 0.005 mol) and homopiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.45 g of oil. Puri-
fied by procedure B. Yield 31%. Raw product was transformed into
oxalic acid salt yielding 0.53 g of final compound. Mp: 127–129 °C.
4.1.22. 1-(7-(3-Chlorophenoxy)heptyl)-4-methylpiperidine
hydrogen oxalate (23)
Synthesis from 7 (1.53 g, 0.005 mol) and 4-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.65 g of oil. Puri-
fied by procedure A. Yield 58%. Raw product was transformed into
oxalic acid salt yielding 0.4 g of final compound. Mp: 110–112 °C.
C
18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.30
(d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.93
(t, J = 6.2 Hz, 2H, CH2-O), 3.3–3.10 (m, 4H, hPip-2,6-CH2), 3.02–
2.97 (m, 2H, hPip-CH2), 1.75–1.43 (m, 12H, hPip-3,4,5,6-CH2,
2 ꢂ CH2), 1.41–1.30 (m, 4H, 2 ꢂ CH2). IR (cmꢀ1): 3428.37 (N
(CH2A)3), 3016.69 (aromatic CH@), 2942.39 (het. CH2A), 2863.90,
2638.12, 2535.47, 2361.48, 1717.47, 1697.76, 1598.54, 1494.04,
1452.26, 1405.09, 1341.13, 1289.48, 1250.90 (O-CH aromatic),
1089.58, 1027.49, 1006.54, 925.13, 828.78, 794.61, 705.29 (alif.
C
19H30NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.28
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.98–6.94 (m, 2H, Ph-2,4-H), 6.90–6.85
(m, 1H, Ph-6-H), 3.96 (t, J = 6.2 Hz, 2H, CH2-O), 3.35–3.31 (m, 2H,
Pip-2,6-CH2a), 2.95–2.90 (m, 2H, Pip-CH2), 2.80 (br s, 2H, Pip-2,6-
H2e), 1.75–1.48 (m, 7H, CH2, Pip-3,5-CH2, Pip-4-H), 1.44–1.30 (m,
8H, 4 ꢂ CH2), 0.90 (d, J = 6.2 Hz, 3H, CH3). 13C NMR (400 MHz,
DMSO-d6) d: 165.23, 160.10, 134.17, 131.29, 120.78, 114.85,
114.01, 68.24, 30.91, 28.86, 28.65, 28.47, 26.55, 25.66, 23.67,
CH2A), 669.64, 474.55. UV–VIS kmax (loge): 226 (4.26), 282
(3.70). LC–MS: purity 100% tR = 5.61, (ESI) m/z [M+H]+ 310.35. Anal.
calcd for C18H28NOCl ꢂ C2H2O4: C, 60.07; H, 7.56; N, 3.50. Found: C,
60.08; H, 7.07 N, 3.52.
21.30. UV–VIS kmax (loge): 207 (5.01), 219 (4.95), 276 (4.44), 283
(4.40), 399 (3.62), 563 (3.05). IR (cmꢀ1): 3421.10 (N(CH2A)3),
3066.26, 3028.66 (aromatic CH@), 2941.88 (het. CH2A), 2870.52,
2857.99 (CH3A), 2611.14, 2510.86, 1717.30, 1591.95, 1579.41,
1469.49, 1393.32, 1282.43, 1247.72 (O-CH aromatic), 1247.72,
1177.33, 1158.04, 1090.50, 1072.23, 1030.70, 944.95, 883.24,
4.1.20. 1-(7-(3-Chlorophenoxy)heptyl)piperidine hydrogen
oxalate (21)
Synthesis from 7 (1.53 g, 0.005 mol) and piperidine (0.85 g,
0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount) in

68
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
766.57 (alif. CH2A), 682.68. LC–MS: purity 100% tR = 6.05, (ESI) m/z
[M+H]+ 324.31. Anal. calcd for C19H30NOCl ꢂ C2H2O4: C, 60.93; H,
7.79; N, 3.38. Found: C, 61.75; H, 8.35; N, 3.57.
1718.26, 1701.87, 1625.70, 1597.73, 1492.63, 1473.35, 1402.96,
1280.50, 1245.79 (O-CH aromatic), 1168.65, 1092.48, 823.46,
721.25 (alif. CH2A), 667.25. LC–MS: purity 100% tR = 5.98, (ESI)
m/z [M+H]+ 324.31. Anal. calcd for C19H30NOCl ꢂ C2H2O4: C,
60.93; H, 7.79; N, 3.38. Found: C, 60.86; H, 8.10; N, 3.47.
4.1.23. 1-(7-(3-Chlorophenoxy)heptyl)homopiperidine
hydrogen oxalate (24)
Synthesis from
7
(1.53 g, 0.005 mol) and homopiperidine
4.1.26. 1-(7-(4-Chlorophenoxy)heptyl)-4-methylpiperidine
hydrogen oxalate (27)
Synthesis from 8 (1.53 g, 0.005 mol) and 4-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
inethanol(105 mL) and water(20 mL). Obtained0.75 g ofoil. Purified
by procedure A. Yield 46%. Raw product was transformed into oxalic
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.72 g of oil. Puri-
fied by procedure B. Yield 62%. Raw product was transformed into
oxalic acid salt yielding 0.7 g of final compound. Mp: 85–87 °C.
C
19H30NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.29
(t, J = 8.7 Hz, 1H, Ph-5-H), 6.99–6.94 (m, 2H, Ph-2,4-H), 6.90–6.86
(m, 1H, Ph-6-H), 3.97 (t, J = 6.2 Hz, 2H, CH2-O), 3.27–3.16 (m, 4H,
hPip-2,7-CH2), 3.00–2.97 (m, 2H, hPip-CH2), 1.76–1.51 (m, 12H,
2 ꢂ CH2, hPip-3,4,5,6-CH2), 1.48–1.30 (m, 6H, 3 ꢂ CH2). 13C NMR
(300 MHz, DMSO-d6) d: 165.30, 160.06, 134.14, 131.28, 120.76,
114.81, 113.99, 68.21, 56.66, 53.84, 28.83, 28.64, 26.54, 25.66,
acid salt yielding 0.78 g of final compound. Mp: 139–141 °C. C19H30-
NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.30 (d,
J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.93 (t,
J = 6,3 Hz, 2H, CH2-O), 3.35–3.31 (m, 2H, Pip-2,6-CH2a), 2.95–2.89
(m, 2H, Pip-CH2), 2.83–2.75 (m, 2H, Pip-2,6-H2e), 1.76–1.48 (m, 7H,
CH2, Pip-3,5-CH2, Pip-4-H), 1.44–1.31 (m, 8H, 4 ꢂ CH2), 0.90 (d,
J = 6.4 Hz, 3H, CH3). 13C NMR (300 MHz, DMSO-d6) d: 165.16,
157.93, 129.64, 124.44, 116.59, 68.16, 28.87, 28.64, 26.52, 25.67,
23.95, 23.38. UV–VIS kmax (loge): 218 (4.29), 220 (4.29), 275
(4.20), 282 (4.14). IR (cmꢀ1): 3442.31 (N(CH2A)3), 3015.36 (aro-
matic CH@), 2938.98 (het. CH2A), 2856.06, 2649.71, 1720.19,
1698.02, 1595.81, 1471.42, 1439.60, 1402.96, 1283.39 (O-CH aro-
matic), 1231.33, 1197.58, 1074.16, 1042.34, 855.28, 721.25 (alif.
CH2A), 704.85, 680.75. LC–MS: purity 98.59% tR = 5.99, (ESI) m/z
[M+H]+ 324.31. Anal. calcd for C19H30NOCl ꢂ C2H2O4: C, 60.93; H,
7.79; N, 3.38. Found: C, 61.12; H, 7.91; N, 3.40.
23.67. UV–VIS kmax (loge): 215 (4.26), 225 (4.30), 230 (4.29), 282
(4.23), 289 (4.19). IR (cmꢀ1): 3438.46 (N(CH2A)3), 3032.51 (aromatic
CH@), 2930.31 (het. CH2A), 2855.10 (CH3A), 2713.35, 2570.65,
2553.29, 1719.23, 1638.23, 1596.77, 1491.67, 1474.31, 1391.39,
1281.47, 1242.90 (O-CH aromatic), 1170. 58, 1092.48, 1024.98,
823.46, 720.28 (alif. CH2A), 655.32. LC–MS: purity 100% tR = 6.05,
(ESI) m/z [M+H]+ 324.31. Anal. calcd for C19H30NOCl ꢂ C2H2O4: C,
60.93; H, 7.79; N, 3.38. Found: C, 60.91; H, 8.08; N, 3.50.
4.1.24. 1-(7-(4-Chlorophenoxy)heptyl)piperidine hydrogen
oxalate (25)
Synthesis from 8 (1.53 g, 0.005 mol) and piperidine (0.85 g,
0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount) in etha-
nol (105 mL) and water (20 mL). Obtained 0.5 g of oil. Purified by
procedure A. Yield 48%. Raw product was transformed into oxalic
acid salt yielding 0.55 g of final compound. Mp: 150–152 °C.
4.1.27. 1-(7-(4-Chlorophenoxy)heptyl)homopiperidine
hydrogen oxalate (28)
Synthesis from
8 (1.53 g, 0.005 mol) and homopiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.7 g of oil. Puri-
fied by procedure A. Yield 43%. Raw product was transformed into
oxalic acid salt yielding 0.67 g of final compound. Mp: 128–131 °C.
C
18H28NOCl ꢂ C2H2O4 (MW = 399.92). 1H NMR (DMSO-d6) d: 7.29
(d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.93
(t, J = 6.3 Hz, 2H, CH2-O), 3.29–3.05 (m, 4H, Pip-2,6-CH2), 2.91 (t,
J = 6.1 Hz, 2H, Pip-CH2), 1.70–1.27 (br m, 16H, Pip-3,4,5-CH2,
C
19H30NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.30
(d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.93
(t, J = 6.3 Hz, 2H, CH2-O), 3.27–3.16 (m, 4H, hPip-2,7-CH2), 3.02–
2.98 (m, 2H, hPip-CH2), 1.75–1.50 (m, 12H, 2 ꢂ CH2, hPip-3,4,5,6-
CH2), 1.48–1.29 (m, 6H, 3 ꢂ CH2). 13C NMR (300 MHz, DMSO-d6)
d: 165.26, 157.93, 129.64, 124.44, 116.59, 68.16, 56.66, 53.85,
5 ꢂ CH2). UV–VIS kmax (log
e): 215 (4.27), 219 (4.29), 224 (4.30),
228 (4.30), 235 (4.27), 282 (4.15). IR (cmꢀ1): 3429.78 (N(CH2A)3),
3031.55 (aromatic CH@), 2940.91 (het. CH2A), 2859.92, 2691.18,
2598.61, 2483.21, 1719.23, 1644.02, 1596.77, 1579.41, 1491.67,
1474.231, 1394.28, 1285.32, 1243.86 (O-CH aromatic), 1169.62,
1104.05, 1091.51, 1002.68, 961.43, 945.91, 823.46, 720.28 (alif.
CH2A), 667.25, 523.58. LC–MS: purity 100% tR = 5.71, (ESI) m/z
[M+H]+ 310.28. Anal. calcd for C18H28NOCl ꢂ C2H2O4: C, 60.07; H,
7.56; N, 3.50. Found: C, 60.33; H, 8.01; N, 3.55.
28.86, 28.66, 26.53, 25.68, 23.96, 23.40. UV–VIS kmax (loge): 215
(4.26), 225 (4.30), 230 (4.30), 282 (4.23), 289 (4.19). IR (cmꢀ1):
3432.67 (N(CH2A)3), 3035.41 (aromatic CH@), 2937.06 (het.
CH2A), 2858.95, 2717.21, 2651.64, 2361.41, 1718.26, 1701.87,
1636.30, 1596.77, 1579.41, 1491.67, 1474.31, 1396.21, 1280.50,
1244.83 (O-CH aromatic), 1168.65, 955.56, 823.46, 721.24 (alif.
CH2A), 692.32, 666.29. LC–MS: purity 100% tR = 5.96, (ESI) m/z
[M+H]+ 324.31. Anal. calcd for C19H30NOCl ꢂ C2H2O4: C, 60.93; H,
7.79; N, 3.38. Found: C, 61.08; H, 8.04; N, 3.46.
4.1.25. 1-(7-(4-Chlorophenoxy)heptyl)-3-methylpiperidine
hydrogen oxalate (26)
Synthesis from 8 (1.53 g, 0.005 mol) and 3-methylpiperidine
(0.99 g, 0.01 mol), K2CO3 (2.07 g, 0.015 mol), KI (catalytic amount)
in ethanol (105 mL) and water (20 mL). Obtained 0.73 g of oil. Puri-
fied by procedure A. Yield 45%. Raw product was transformed into
oxalic acid salt yielding 0.65 g of final compound. Mp: 130–132 °C.
4.2. X-ray structure analysis
4.2.1. 1-(6-(4-Chlorophenoxy)hexyl)-4-methylpiperidine
hydrogen oxalate (19)
C
19H30NOCl ꢂ C2H2O4 (MW = 413.93). 1H NMR (DMSO-d6) d: 7.30
(d, J = 9.2 Hz, 2H, Ph-3,5-H), 6.93 (d, J = 9.0 Hz, 2H, Ph-2,6-H), 3.93
(t, J = 6.3 Hz, 2H, CH2-O), 3.35–3.25 (m, 2H, Pip-2,6-CH2a), 2.92 (t,
J = 6.1 Hz, 2H, Pip-CH2), 2.67–2.63 (m, 1H, Pip-2-He), 2.47–2.41
(m, 1H, Pip-6-He), 1.89–1.27 (br m, 14H, Pip-4,5-CH2, 5 ꢂ CH2),
1.04 (def q, 1H, Pip-3-CH), 0.87 (d, J = 6.7 Hz, 3H, CH3). 13C NMR
(300 MHz, DMSO-d6) d: 165.19, 157.94, 129.64, 124.44, 116.59,
68.16, 57.81, 51.84, 30.54, 28.88, 28.66, 26.53, 25.66, 23.55,
Crystal data for 19: C18H29ClNO, C2HO4, M = 399.90, monoclinic,
space group P21/c, a = 8.2950(15) Å, b = 25.609(5) Å, c = 11.0624
(19) Å, b = 111.584(3) (°), V = 2185.2(7) Ǻ3, Z = 4, Dx = 1.216 g cmꢀ3
,
T = 296 K,
l
= 0.203 mmꢀ1
,
k = 0.71073 Ǻ, data/parameters =
3731/253; final R1 = 0.07.
The structures were solved by direct methods and refined with
SHELXTL.58 Crystallographic data (excluding structural factors) for
the structure reported in this paper have been deposited at the
Cambridge Crystallographic Data Centre and allocated with the
deposition number CCDC 1022232.
19.04. UV–VIS kmax (loge): 206 (3.81), 228 (4.08), 282 (3.37), 538
(2.14). IR (cmꢀ1): 3433.64 (N(CH2A)3), 2935.13 (aromatic CH@),
2857.99 (het. CH2A), 2671.89, 2550.40 (CH3A), 2361.41, 2342.12,

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
69
4.3. Docking studies
4.5.2. cAMP accumulation assay in cells expressing hH3R
Intracellular cAMP accumulation was measured with homoge-
nous TR-FRET immunoassay, using LANCE Ultra cAMP kit (Perki-
nElmer) and HEK293 cells, stably expressing hH3R. An antagonist
dose–response experiments were performed in a total assay vol-
In silico visualization of binding to histamine H3R model
obtained from GPCRM Modelling Server,45 was held using
Schrödinger MacroModel 9.7.44 All of the tested compounds were
used in their N-protonated form and generated using Schrödinger
suite. For the tested set of compounds, energetically optimal con-
formers were found using ConfGen (MMFFs forcefield, PRCG, con-
vergence threshold = 0.05, 20 steps per rotatable bond, max. ring
conformations = 16). For all of the structures 5 energetically best
conformations were used for docking.
ume of 20
(ꢀ)- -Methylhistamine (15 nM), forskolin (10
nists in appropriate concentrations (in range 0.1 nM–10
l
L in white 384-well plates, using 300 cells/well. (R)
M) and antago-
M) were
a
l
l
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
Receptor grid was generated by GlideGrid module and validated
with previously described pitolisant (WakixÒ). Docking studies
were performed via GlideDock module (OPLS 2001 force field, sol-
vent = water, standard precision, post-docking minimization).
Results were interpreted by the means of Docking Score functions.
Receptor–ligand interactions were visualized using UCSF
Chimera.59,60
and 5 lL of ULight-labeled anti-cAMP mAb working solutions were
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 presented
as % of maximal response. Sigmoidal dose–response curve fitting
was performed with use of GraphPad PrismTM software (version
5.01, San Diego, CA, USA). Showed results represent the mean of
three separate experiments, each performed in triplicates.
4.4. Lipophilicity
4.5.3. Anticonvulsant Screening Program
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% for each step. Mobile phase compositions as well as concentra-
Anticonvulsant evaluation of compounds was performed and
sponsored by National Institute of Neurological Disorders and
Stroke, National Institutes of Health (Rockville, USA) for the Anti-
convulsant Screening Program (ASP). Compounds were injected
as suspensions in 0.5% methylcellulose at the selected doses (3,
10, 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
(sometimes additionally 0.25, 1 and 2 h) after administration.
Groups of eight mice or four rats were employed. Phase 1 of
ASP includes three tests performed in mice (ip): maximal
electroshock (MES), subcutaneous pentylenetetrazole (scMET),
and neurotoxicity (rotarod test). Compounds interested for ASP
program, underwent additional tests including: MES and TOX tests
in rats after po; MES and TOX tests in rats after ip; 6-Hz test in
mice; quantitative MES, scMET and TOX in mice or/and rats ip;
pilocarpine induced status epilepticus (rats, ip); corneal kindled
mice model (ip); formalin test (mice, ip), and hippocampal slice
culture neuroprotection assay in vitro.
tions were chosen experimentally. For each concentration, 10 lL of
compounds methanol solution in 1 mg/mL concentration was
used. Planar chromatographic chambers were saturated with
proper mixture for 45 min. followed by 15 min. saturation together
with the plate. Glass plates were then evaluated on the distance of
90 mm, dried and spots were visualized using iodide fumes. For
each of the compounds, on the base of Rf values, RM values were
determined. RM0 values were read from RM/methanol concentra-
tion charts, after extrapolation to zero methanol concentration.
4.5. Pharmacology
General remarks Competition binding data were analyzed by
the software GraphPad PrismTM (200, version 3.02, San Diego, CA,
USA), using non-linear least squares fit. Affinity values (Ki) were
expressed as mean from at least two experiments in triplicates.
Ki values were calculated from IC50 values according to Cheng–Pru-
soff equation.61
4.5.3.1. Maximal electroshock seizure (MES) test.
Seizures
are elicited by 60 Hz alternating current (50 mA in mice and
150 mA in rats) 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 electrodes. Protection is defined as abolition
of the hindlimb tonic extension component of the seizure.62,63
a
4.5.1. [3H]N -methylhistamine hH3R displacement assay
The displacement binding assay was carried out as described by
Kottke et al.35 In summary, frozen crude membrane preparations of
HEK-293 cells stably expressing the full-length recombinant hH3R
were thawed, homogenized, incubated for 90 min at 25 °C and
4.5.3.2.
Subcutaneous
pentetrazole
induced
seizures
(scMET).
A dose of pentetrazole which induce convulsions
a
shook at 250 rpm with [3H]N -methylhistamine (2 nM) and differ-
in 97% of animals (CD97: 85 mg/kg mice or 56.4 mg/kg rats) 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.37
ent concentrations of the test compounds (seven appropriate con-
centrations between 0.01 nM and 10
volume of 200 l per well. Non-specific binding was determined in
the presence of pitolisant (10 M). In saturation binding experi-
lM were used) in a final assay
l
l
ments Bmax was found to be 0.89 pmol/mg and the Kd value of
a
[3H]N -methylhistamine was 2.98 nM. The bound radioligand
was separated from free radioligand by filtration through GF/B fil-
ters (pre-treated with 0.3% (m/v) polyethyleneimine) using an Ino-
tech cell harvester (Dottikin, Switzerland). Unbound radioligand
was removed by three washing steps with 0.3 mL/well of ice-cold
50 mM Tris–HCl buffer (pH 7.4) containing 120 mM NaCl. Scintilla-
tion cocktail was added and the liquid scintillation counting was
performed with a Perkin Elmer Trilux Betacounter (Perkin Elmer,
Germany).
4.5.3.3. Neurotoxicity (TOX) test.
The rotarod test was used
to evaluate neurotoxicity in rodents (mice, rats). was measured
by. Animal was placed on a 1 inch diameter 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. In rats, neurologi-
cal deficit was indicated by ataxia, loss of placing response and
muscle tone.64

70
K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
4.5.3.4. 6 Hz psychomotor seizure test.
Compounds are
Dr. Christa Müller (Pharmaceutical Institute, Pharmaceutical
Chemistry I, University of Bonn).
preadministered to mice via ip injection. Corneal stimulation
(0.2 ms monopolar rectangular pulses at 6 Hz and 32 mA for 3 s)
was delivered by a constant-current device. Untreated animals dis-
play seizures after such stimulation described as minimal clonic
phase, whereas mice not displaying this behavioral are considered
protected.65
4.5.4.2. Cell culture.
HEK-293 and IMR-32 cell lines were cul-
tured in Dulbecco’s Modified Eagle’s Medium—DMEM (Gibco) con-
tained 10% fetal bovine serum (FBS), 100 mgꢃmLꢀ1 streptomycin
and 100 UꢃmLꢀ1 penicillin. Cells were cultured in an atmosphere
containing 5% of carbon dioxide at 37 °C.
4.5.3.5. Pilocarpine induced status epilepticus (SE)66
.
Com-
4.5.4.3. In vitro antiproliferative assay.
The cells were
pounds were administered via ip to male Sprague Dawley rats
(150–180 gm). Then pilocarpine (50 mg/kg; ip) is administered
and treatment-effects were observed. In the initial convulsive-SE
behavioral observation studies, adult rats received a dose of the
investigated compound, which was found in (Quantitative Rat
(ip) Study) to produce minimal motor impairment. Rats were
dosed either immediately upon the first behavioral Stage 3 seizure
(e.g., 0 min) or 30 min following the first behavioral Stage 3
seizure. The number of animals which protected from Stage 3 or
greater Racine67 scale seizures is determinated.
seeded in 96-well plates at a concentration of 2 ꢂ 104 cells/well
(IMR-32) or 1.5 ꢂ 104 cells/well (HEK-293) in 200
lL culture med-
ium. Next, the cell lines were cultured for 24 h to reach ꢁ60% con-
fluence. The stock solutions of examined H3R ligands in DMSO
were diluted into fresh growth medium. The maximal DMSO con-
centration did not exceed 1%. Next the H3R ligands were added into
the microtiter plates at the final concentrations 0.01–250
lM. After
48 h of incubation 20 L of EZ4U labeling mixture (EZ4U Non-
l
radioactive cell proliferation and cytotoxicity assay, Biomedica)
was added to the each well according to the manufacturer proto-
col. The cells were next incubated for 5 h at 37 °C, 5% CO2. The
absorbance of the samples was measured using a PerkinElmer
EnSpire microplate reader at 492 nm. GraphPad Prism 5.01 soft-
ware was used to calculate the IC50 values. The activity of the stan-
dard drug Doxorubicin was estimated as we described
previously.73
4.5.3.6. Corneal kindled mice model.
Adult male CF1 mice
were kindled to a criterion of 5 consecutive secondarily general-
ized seizures (stage 4 or 5, as described by Racine67) according to
the corneal kindling protocol previously described.39,68 Tested
compound was administered to 4 fully kindled mice and tested
at 0.5 h after dosing. Mice displaying a seizure score < 3 are consid-
ered protected.
4.6. Physicochemical studies
4.5.3.7. Formalin test (mice, ip).
The formalin test was per-
4.6.1. In silico metabolism study
formed according to the method of Tjolsen et al.69 A sub-dermal
injection of 0.5% formalin was made into the plantar region of
the right hindpaw of a mouse. The response to the formalin injec-
tion is characterized by the mouse licking the affected paw. The
investigational compounds or vehicle were administered ip at a
dose 13 mg/kg (20) and 16 mg/kg (26). After 0.25 h formalin was
sub-dermally injected into the plantar surface of the right hind-
paw. Following the formalin injection, each animal was then
observed for the first 2 min of each 5-min epoch until a total of
40 min has elapsed since the administration of the test compound
or vehicle. The cumulative duration of licking (in seconds) during
each 2-min recording period was measured for analysis across
compound- and vehicle-pretreated groups.
The computational procedure MetaSite 4.1.152 was used for pre-
diction of H3R ligands metabolic biotransformations in silico.
MetaSite identifies the most likely sites of metabolism in examined
compound and predicts the structures of the metabolites by con-
sidering two factors: thermodynamic factor (enzyme-substrate
recognition) and kinetic factor describing the chemical transforma-
tions catalyzed by the enzymes.74,75 The highest metabolism prob-
ability sites were analyzed during this study using either liver or
cytochrome CYP3A4 model exclusively.
4.6.2. In vitro metabolism study
4.6.2.1. Reaction with recombinant human liver microsomes
(HLMs).
Commercial, pooled, human (adult male and female)
liver microsomes (HLMs) from Sigma–Aldrich were used for all
biotransformations. The reaction was carried out using 1 mg/mL
4.5.3.8. In vitro hippocampal slice culture—neuroprotection
assay.
Test in vitro qualitative assessment of the ability of
HLMs in 200
7.4), NADPH Regeneration System (Promega) and histamine H3R
ligand with final volume of 50 M. The reaction was initiated by
adding 50 L of Regeneration System after 5 min. of preincubation
at 37 °C. The 200 L of cold methanol was added to terminate the
ll of reaction buffer containing 0.1 M Tris–HCl (pH
compound to prevent excitotoxic cell death. Organotypic hip-
pocampal slice cultures, prepared as described by Stoppini et al.,70
l
are treated with N-methyl-D-aspartate (NMDA) or kainic acid (KA)
l
to induce neuronal death. Propidium iodide (PI), a membrane-
impermeant compound is included in all wells of the culture plate.
PI binds to the DNA of dead and dying cells and fluoresces. The
intensity of the PI fluorescence is proportional to the amount of
the cell death in the individual slices. Hippocampal slice cultures
are treated with the excitotoxin alone, or with the excitotoxin
and either one or two investigational compounds at the indicated
concentrations. If neuroprotection occurs as a consequence of the
added compound, slice cultures will have visibly reduced fluores-
cent intensity when compared to the slice cultures that have been
treated with the excitotoxin alone.
l
reaction after 2 h of incubation at 37 °C. The mixture was cen-
trifuged at 13,000 rpm for 15 min. and next the LC/MS analysis of
the supernatant was performed. Mass spectra were recorded on
LC/MS system consisted of a Waters Acquity UPLC, coupled to a
Waters TQD mass spectrometer (electrospray ionization mode
ESI-tandem quadrupole).
4.6.2.2. CYP3A4 P450-GloTM assay.
The CYP3A4 P450-GloTM
assays (Promega) were performed according to the manufacturer’s
procedure. The enzymatic reactions were conducted in white poly-
styrene, flat-bottom NuncTM MicroWellTM 96-Well Microplates
(Thermo Scientific). The mixture consisting 0.5 pmol of CYP3A4
membranes and 4ꢂ Luciferin-PPXE in 100 mM Tris–HCl was
titrated in 96-well plate. Next, an equal volume of the histamine
H3R ligand or Luciferin-Free Water was added to give one-half of
the final reaction volume. After the 10-min pre-incubation
4.5.4. Antiproliferative assay
4.5.4.1. Cell lines.
Neuroblastoma IMR-32 cell line was
provided by Department of Oncogenomics, Academisch Medisch
Centrum, Amsterdam, Holland.71,72 Human embryonic kidney
HEK-293 cell line (ATCC CRL-1573) was kindly donated by Prof.

K. Kuder et al. / Bioorg. Med. Chem. 24 (2016) 53–72
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one-half of the final volume of 2ꢂ NADPH Regeneration System
(Promega) was added to initiate CYP3A4 reaction. The reaction
mixture was next stirred for 1 min and then incubated at 37 °C
for 30 min. The final concentrations of H3R ligands were in the
range of 0.025–10 lM and the total volume of DMSO did not
exceed of recommended by manufacturer 0.2%. The reaction mix-
ture with inactive control membranes (control) was also prepared
as described above. Afterwards, to initiate the luminescence the
reconstituted Luciferin Detection Reagent containing firefly lucifer-
ase was added. The luminescence was measured with a PerkinEl-
mer EnSpire microplate reader in luminescence mode after 20
min incubation of the reaction mixture at room temperature. For
calculation the total luminescence, the average luminescence of
the control reaction containing inactive membranes was sub-
tracted from the luminescence of CYP3A4 containing reactions.
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Acknowledgements
_
´
28. Łazewska, D.; Ligneau, X.; Schwartz, J.-C.; Schunack, W.; Stark, H.; Kiec-
Kononowicz, K. Bioorg. Med. Chem. 2006, 14, 3522.
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We would like to thank prof James P. Sables, 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 grateful
thank Senior scientist Mohamed Hamdi from Department of
Oncogenomics Academisch Medisch Centrum Amsterdam, Holland
for providing neuroblastoma IMR-32 cell line. We sincerely
acknowledge Nicolas Levoin from Bioprojet Biotech for providing
the histamine H3R homology model, and Bioprojet for providing
HEK H3R cell lines. The authors are grateful to Maria Kaleta
(Krakow, Poland) for excellent technical assistance in chemical
synthesis part of the project and to Tim Kottke and Lilia Weizel
(Frankfurt, Germany) and Agnieszka Olejarz (Krakow, Poland) for
excellent support in pharmacological screening and to Natalia
Groth (Krakow, Poland) for technical assistance in ADME-Tox
screening. The authors acknowledge the partial support of National
Science Center granted on the basis of decision Nos. DEC-2011/02/
A/NZ4/00031 and K/ZDS/004689, of the European GLISTEN—
CM1207 as well as of the DFG INST 208/664-1 FUGG.
_
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