Dual target ligands with 4-tert-butylphenoxy scaffold as histamine H3 receptor antagonists and monoamine oxidase B inhibitors

Article abstract of DOI:10.3390/ijms21103411

Dual target ligands are a promising concept for the treatment of Parkinson’s disease (PD). A combination of monoamine oxidase B (MAO B) inhibition with histamine H₃ receptor (H₃R) antagonism could have positive effects on dopamine re

Full text of DOI:10.3390/ijms21103411

Article
Dual Target Ligands with 4-tert-Butylphenoxy
Scaffold as Histamine H Receptor Antagonists and
3
Monoamine Oxidase B Inhibitors
Dorota Łażewska 1,*, Agnieszka Olejarz-Maciej 1, David Reiner 2, Maria Kaleta 1,
Gniewomir Latacz 1, Małgorzata Zygmunt 3, Agata Doroz-Płonka 1, Tadeusz Karcz 1,
Annika Frank 2, Holger Stark 2 and Katarzyna Kieć-Kononowicz 1,*
1
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, 9
Medyczna Str, 30-688 Kraków, Poland; agnieszka.olejarz@uj.edu.pl (A.O.-M.);
maria.kaleta@uj.edu.pl (M.K.); glatacz@cm-uj.krakow.pl (G.L.); a.doroz-plonka@uj.edu.pl (A.D.-P.);
t.karcz@uj.edu.pl (T.K.)
2
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf,
Universitaetsstr. 1, 40225 Duesseldorf, Germany; david.reiner@hhu.de (D.R.); a.frank@hhu.de (A.F.);
stark@hhu.de (H.S.)
3
Department of Pharmacodynamics, Jagiellonian University Medical College, 9 MedycznaStr,
30-688 Kraków, Poland; malgorzata.zygmunt@uj.edu.pl
* Correspondence: dlazewska@cm-uj.krakow.pl (D.Ł.); mfkonono@cyf-kr.edu.pl (K.K.-K.)
Received: 18 April 2020; Accepted: 9 May 2020; Published: 12 May 2020
Abstract: Dual target ligands are a promising concept for the treatment of Parkinson’s disease (PD).
A combination of monoamine oxidase B (MAO B) inhibition with histamine H
3
receptor (H R)
3
antagonism could have positive effects on dopamine regulation. Thus, a series of twenty-seven 4-
tert-butylphenoxyalkoxyamines were designed as potential dual-target ligands for PD based on the
structure of 1-(3-(4-tert-butylphenoxy)propyl)piperidine (DL76). Probed modifications included the
introduction of different cyclic amines and elongation of the alkyl chain. Synthesized compounds
were investigated for human H
Most compounds showed good hH
showed potent inhibitory activity for hMAO B with IC50 values below 50 nM. However, the most
balanced activity against both biological targets showed DL76 (hH R: K = 38 nM and hMAO B: IC50
3
R (hH
3
R) affinity and human MAO B (hMAO B) inhibitory activity.
3
R affinities with K
i
values below 400 nM, and some of them
3
i
= 48 nM). Thus, DL76 was chosen for further studies, revealing the nontoxic nature of DL76 in
HEK293 and neuroblastoma SH-SY5Ycells. However, no neuroprotective effect was observed for
DL76 in hydrogen peroxide-treated neuroblastoma SH-SY5Y cells. Furthermore, in vivo studies
showed antiparkinsonian activity of DL76 in haloperidol-induced catalepsy (Cross Leg Position
Test) at a dose of 50 mg/kg body weight.
Keywords: dual-target ligands; histamine H receptor; monoamine oxidase B; 4-tert-butylphenyl
3
derivatives; antagonists; inhibitors; neurodegenerative disease; Parkinson’s disease
1. Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor
problems. Although the entire pathology of PD is still unknown, several factors have been proposed
to contribute to PD development, such as environmental toxins, neuroinflammation, genetic
mutations, oxidative stress, or mitochondrial dysfunction [1]. Generally, PD is characterized by a
severe lack of dopamine (DA) (80%–90%) in striatum due to a progressive loss of dopaminergic
neurons in the substantia nigra [1]. Current therapy for PD can only mitigate symptoms and slow the
progress. However, there is no cure for the disease to date. Commonly administered drugs include
Int. J. Mol. Sci. 2020, 21, 3411; doi:10.3390/ijms21103411
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levodopa (as a DA precursor), DA agonists (e.g., pramipexole and rotigotine), and monoamine
oxidase (MAO) B inhibitors (e.g., selegiline, rasagiline, or safinamide; Figure 1).
selegiline
rasagiline
safinamide
Figure 1. Structures of antiparkinsonian drugs—monoamine oxidase (MAO) B inhibitors.
MAO B plays a crucial role in the pathogenesis of PD. This enzyme belongs to the family of
MAOs that catalyze the deamination of neurotransmitters (e.g., DA) and release reactive oxygen
species as by-products. MAO B dominates in the human brain and deaminates β-phenylethylamine
(PEA). PEA increases the synaptic levels of DA and blocks its reuptake into neurons. An increase in
the activity of MAO B with age and some diseases as PD was observed in humans. Inhibitors of MAO
B stop the activity of this enzyme and block the breakdown of DA. Moreover, MAO B inhibitors show
neuroprotection and reduce oxidative stress [2]. Thus, MAO B inhibition is an important factor in the
search for effective drugs in the treatment of PD. However, due to the multifactorial etiology of PD,
it is thought that ligands acting on several targets at the same time (so-called Multi-Target-Directed
Ligands (MTDL)) will be more effective in treatment than a one-target compound [3]. Thus, for
improving the pharmacotherapy of PD, it is important to find MAO B inhibitors with combined
activity at other targets.
Histamine H
3
receptors (H
3Rs) are widely distributed in the human brain and dominantly in
areas connected with cognition (such as the striatum, cortex, or hippocampus). H
3
Rs influence the
release not only of histamine itself but also of other neurotransmitters, such as DA or acetylcholine
[4], and increase the level of mentioned neurotransmitters in the synaptic cleft. Numerous
pharmacological studies show that blocking H Rs provides beneficial effects in the treatment of
3
various neurological diseases such as narcolepsy, neurodegenerative diseases (e.g., Alzheimer’s
disease and PD), attention deficit hyperactivity disorder, epilepsy, obesity, or neuropathic pain [5].
For years, many scientific centers and pharmaceutical companies have been involved in the search
for active ligands of these receptors. Intensive synthetic work has led to a large number of structurally
diverse compounds. Some of them have reached clinical trials, but so far, only one (pitolisant
(Wakix®); Figure 2) has entered into the market as an orphan drug for narcolepsy [6].
One strategy to obtain MTDL is to combine two or more pharmacophores into a single molecule.
Pharmacophores can be connected by linkers, attached directly (fused), or merged [7]. A
propargylamine moiety is known to be important for MAO B inhibition [8], and it is present in the
marketed drugs selegiline and rasagiline. The piperidinepropoxy motif is a part of many potent H
ligands, e.g., pitolisant (Figure 2). The idea to combine MAO B inhibition with H R antagonism is
quite new. In 2017, the first of such compounds, contilisant (Figure 2), was described by Bautista-
Aguilera et al. [9]. Contilisant not only proved to be a H R antagonist (K = 11 nM) and human MAO
3
R
3
3
i
B (hMAO B) inhibitor (IC50 = 78 nM) but also showed moderate inhibition of cholinesterases (AChE
IC50 = 530 nM; BuChE IC50 = 1690 nM). Further, this idea was continued by Lutsenko et al. [10] with
the fused rasagiline derivative 1 as a dual-target ligand (DTL) with high hH
and good hMAO B inhibitory activity (IC50 = 256 nM) (Figure 2). Moreover, Affini et al. [11] described
indanone derivatives as DTL for PD (compound 2; hH R K = 6.5 nM; hMAO B IC50 = 276 nM; Figure
2). Recently, we have described a new group of DTL hMAO B inhibitors, tert-amylphenoxy
derivatives [12]. These compounds showed also affinity for hH R (e.g., compound 3; Figure 2). In
contrast to the previously described DTL (contilisant, 1 and 2), some of them showed an inhibitory
3
R affinity (K
i
= 6.7 nM)
3
i
3
activity for hMAO B that was higher than their affinity for hH
3
R (3: hH
3
R K = 63 nM; hMAO B IC50 =
i
4.5 nM).

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pitolisant
contilisant
1
2
3
DL76
Figure 2. Structures of pitolisant and histamine H
3
receptor ligands with MAO B inhibitory activity.
To continue this work, we synthesized a series of 4-tert-butylphenoxy derivatives as analogues
of histamine H R ligand DL76 (hH R K = 22 nM in CHO K1 cells [13]; Figure 2) which showed also
3
3
i
good inhibitory activity for hMAO B with an IC50 of 48 nM. Encouraged by these results, we designed
a series of novel 4-tert-butylphenoxy derivatives. Designed structural modifications included the
following:
-
-
exchange of piperidine moiety for other cyclic amines (pyrrolidine, substituted piperidine,
or azepane)
elongation of alkyl chain from three up to six atoms.
All compounds were evaluated for their affinity towards hH R and inhibition of hMAO B.
3
Furthermore, we selected one of compounds for antiparkinsonian activity tests in vivo (in
haloperidol-induced catalepsy) and neuroprotection studies in vitro (in neuroblastoma SH-SY5Y
cells). Moreover, the toxicity of this compound in HEK293 cells and neuroblastoma SH-SY5Y cells
was evaluated.
2. Results and Discussion
2.1. Synthesis of Compounds
Compounds were synthesized as shown in Scheme 1. Briefly, proper 4-tert-butylphenoxyalkyl
bromides (4a–4d) were obtained by nucleophilic substitution of 4-tert-butylphenol with α,ω-
dibromoalkanes in freshly prepared sodium propanolate as described previously [14]. Then, the
bromides 4a–4d were refluxed with corresponding amines in the mixture of ethanol–water (21:4) with
powdered potassium carbonate and a catalytic amount of potassium iodide. The purified free bases
were converted into hydrogen oxalates. Structures and purity of novel compounds 5–31 were
confirmed by spectral analyses (1H and 13C NMR spectra; see Supplementary Materials S1), mass
spectrometry (MS), and elemental analysis.

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Scheme 1. General synthetic pathway of synthesized compounds 5–31. Reagents and conditions: (a)
sodium propanolate (0.05 mol Na in 50 mL), room temperature, 15′; 60 °C 3 h; reflux 3 h; 69%–78%;
(b) (i) amine, K
2
CO
3
, KI, EtOH:H O (21:4), and reflux 20 h and (ii) oxalic acid, EtOH, room
2
temperature, 1 h; (Et)
2O; crystallization from EtOH, 5%–53%.
2.2. Human Histamine H
3
Receptor Affinity
The affinity of compounds (5–31) for H
HEK293 cells stably expressing hH
R [14]. [3H]Nα-methylhistamine was used as a radioligand.
Results are presented as K values in Table 1. The compounds showed variable affinities for hH
ranging from good (K < 100 nM) to weak (K > 1500 nM), depending on the kind of the introduced
cyclic amine moiety and alkyl chain length. Analyzing the influence of a cyclic amine moiety on the
affinity for hH R, it was noticed that derivatives of piperidine (DL76, 6–8), 3-methylpiperidine (12–
15), and azepane (28–31) were the most active. On the other hand, 3,3-dimethylpiperidine derivatives
(16–19) showed weaker affinities (K > 1600 nM). Unfortunately, none of the synthesized compounds
(5–31) showed higher hH R affinity than the lead structure DL76 (K = 38 nM). The most potent was
compound 9 with a K of 69 nM.
3
R was evaluated in a radioligand-binding assay in
3
i
3
R
i
i
3
i
3
i
i
Table 1. Human histamine H
3
receptor affinity and human MAO B inhibition of tested compounds
5–31 and DL76.
H
3
R a
(nM)
MAO B b
IC50 (nM)
(%Inh.) c
MAO A b
IC50 (nM)
(%Inh.) d
Compounds
R
n
1
1
Ki
(95%CI)
371
5
2.7 ± 0.4
nt e
(136, 1009)
>10,000
(9%)
38 f
DL76
48 ± 15
(8, 181)
309
(166, 574)
252
>10,000
(10%)
6
7
2
3
4
1
2
3
1
2
290 ± 7
(28%)
nt e
nt e
(64, 990)
225
8
(13%)
(98, 519)
69
>10,000
(5%)
9
11 ± 1
(49, 96)
153
>10,000
(2%)
10
11
12
13
475 ± 38
(36%)
(46, 505)
1556
nt e
(349, 6941)
98
>10,000
(10%)
117 ±12
1405 ± 494
(43, 226)
102
nt e
(18, 571)

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114
14
3
4
(33%)
(13%)
nt e
nt
(33, 397)
351
15
(223, 552)
1624
(1075, 2453)
3437
>10,000
(6%)
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
476 ± 38
(38%)
nt e
(2701, 4374)
3535
>10,000
(19%)
2777 ± 66
1953 ± 45
(37%)
(2528, 4942)
2575
>10,000
(24%)
(542, 12227)
341
nt e
nt e
nt e
nt e
nt e
nt e
nt e
nt e
(49, 2388)
1381
(35%)
(923, 2066)
2235
(41%)
(1136, 4397)
2083
(34%)
(936, 4637)
316
(37%)
(123, 808)
400
(33%)
(152, 1050)
531
(39%)
(344, 822)
1350
(10%)
(651, 2798)
111
>10,000
(10%)
45 ± 4
(68, 180)
299
>10,000
(23%)
1627 ± 78
(18%)
(105, 855)
324
nt e
nt e
(121, 870)
829 g
(23%)
(313, 2194)
nt e
rasagiline
pargiline
15 ± 1
360 ± 138
7.7 ± 1.2
nt e
nt e
nt e
nt e
nt e
nt e
nt e
safinamide
clorgiline
1.76 ± 0.5 nM
a
[3H]Nα-Methylhistamine-binding assay in HEK293 cells stably expressing the human H
R; mean
3
value within the 95% confidence interval (CI); b fluorometricAmplex™ Red MAO assay [15]; mean ±
c
SEM of 2–5 independent experiments; the percent of inhibition at 1 μM, mean values of two
d
independent experiments; the percent of inhibition at 10 μM, mean values of two independent
experiments; e nt = not tested; f K
i
(± SEM) = 22 ± 3 nM in [125I]Iodoproxyfan binding assay in CHO K1
cells, data from Reference [13]; g data from Reference [16].

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2.3. Human Monoamine Oxidase B Inhibitory Activity
2.3.1. Screening and Determination of IC50
The inhibitory activity of the compounds against hMAO B was evaluated using Amplex Red®
Monoamine Oxidase kits [15]. Rasagiline, pargyline, and safinamide were used as reference
inhibitors. Following the initial screening of compounds at 1 μM concentration, IC50 values were
determined for those which exhibited inhibitory activity greater than 50% (Table 1). Sixteen
compounds out of the thirty-one showed a percentage of inhibition between 10% and 41%. Results
of IC50 determinations indicated the influence of both an amine moiety and a length of alkyl chain on
hMAO B inhibition. Except the series of 3,5-dimethylpiperidine and 4-methylpiperidine derivatives,
the inhibitory activity of the compounds was more pronounced with a shorter (three or four) alkyl
linker. Thus, an elongation of alkyl chain caused a drop of activity. The highest activity was observed
for the compounds with the propylene linker (DL76, 9, 12, 16, 20, 24, and 28). This observation is
simillar to our previous finding [12] concerning 4-tert-amylphenoxy derivatives as DTL ligands.
Exchange of a cyclic amine group (piperidine in DL76) for other moieties (pyrrolidine, substituted
piperidine, or azepane) caused variable influences: increased (pyrrolidine-5 or 2-methylpiperidine-
9), maintained (azepane-28), or decreased activity (3-methylpiperidine-12, 3,3-dimethylpiperidine-
16, 3,5-dimethylpiepridine-20, or 4-methylpiperidine-24). The most potent inhibitor 5 (IC50 = 2.7 nM)
showed higher activity than the reference compounds rasagiline (IC50 = 15 nM) and safinamide (IC50
= 7.7 nM).
2.3.2. Reversibility Studies
To investigate the type of inhibition (reversible or irreversible) of hMAO B by 4-tert-
butylphenoxy derivatives, experiments were performed with selected compounds (DL76, 5, and 9)
and conducted as described previously [12]. Rasagiline (irreversible inhibitor) and safinamide
(reversible inhibitor) were used as standards. Results from the experiment are shown in Figure 3A–
C. All tested compounds presented a signal similar to the reversible reference inhibitor safinamide
so they were considered as reversible. However, the signal for rasagiline was slightly higher than
expected for the IC80 concentration. High signal for rasagiline can be explained by the fact that
irreversible inhibitors need more time to create the covalent bond with the enzyme. The used protocol
for the reversibility testing did not contain the preincubation of enzyme with inhibitors. Lack of
preincubation could also change the observed reversibility curves for reversible inhibitors because
they could present different affinity towards the free enzyme and the enzyme that was bound to the
substrate [17]. Thus, the experiment was modified and the reversibiblity of investigated inhibitors
with and without the preincubation with the enzyme was performed (see Materials and Methods).
Results from the experiments after the modification are shown in Figure 3E,F. Safinamide, 5, and 9
did not show differences between experiments with and without the preincubation, suggesting that
the inhibitors did not bind covalently to the enzyme. On the other hand, rasagiline (irreversible
inhibitor) showed higher inhibition when preincubated with hMAOB.

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Figure 3. Reversibility of DL76 (A), 5 (B), 9 (C), and reference inhibitors without the preincubation
and comparison of reversibility of 5 (D), 9 (E), and reference inhibitors that were (rings) or were not
(lines) preincubated with hMAOB. Concentrations of compounds correspond to their IC80 values.
2.3.3. Kinetic Studies
In order to determine the mode of hMAO B inhibition, compounds 9 and DL76 were chosen and
used in three concentrations corresponding to their IC20, IC50, and IC80 values (0.2 nM, 10 nM, and 40
nM for 9 and 7 nM, 48 nM, and 164 nM for DL76). Results from the kinetic experiment were used for
determination of Michaelis–Menten curves (Figure 4A,C). Later, data were transformed using the
Lineweaver–Burk equation to double reciprocal plot (lines from different concentrations of both
compound 9 and DL76 converged to the left of y-axis and above x-axis; Figure 4B,D). From Michaelis–
Menten curves, Vmax and K
M
values were fitted (Table 2). For both compounds, Vmax values decreased
curvilinearly and K values increased curvilinearly with the increased inhibitor concentrations. The
M
behavior of these parameters suggested the mixed mode inhibition of 9 and DL76 [17,18]. This
observation was further confirmed by a value of calculated constant α (α = 1.6 for 9 and α = 4.6 for
DL76) by GraphPad Prism software from the nonlinear regression (see Supplementary Materials S2).
According to Copeland [18,19], α > 1 is characteristic for the mixed mode of inhibition where an
inhibitor is able to bind to both the free enzyme and the enzyme–substrate complex unequally and
its affinity for binding to the free enzyme is higher (see Table S1, Supplementary Materials S2).

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Figure 4. Michaelis–Menten curves for compound 9 (A) and DL76 (C): Inhibitors were tested in three
concentrations (0.2 nM, 10 nM, and 40 nM for 9 and 7 nM, 48 nM, and 164 nM for DL76) representing
their IC20, IC50, and IC80, respectively. Substrate (p-tyramine) was used in six concentrations: 0.05 mM,
0.1 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM. (B,D) Double-reciprocal (Lineweaver–Burk) plots from
enzyme kinetic studies.
Table 2. Vmax and K
Parameters
m
calculated from Michaelis–Menten curves of compound 9 and DL76.
Compound 9 DL76
Concentration
(nM)
0
0.2
10
40
0
7
48
164
V
max (RFU/min)
(mM)
1713
0.24
1652
0.27
1544
0.27
1238
0.28
2046
0.11
1628
0.14
1165
0.21
726.3
0.27
K
M
2.4. Human Monoamine Oxidase A Inhibitory Activity
Selected compounds (DL76, 6, 9, 10, 12, 16, and 28) were screened for hMAO A inhibition using
Amplex Red® Monoamine Oxidase kits [15]. Tested compounds showed a percentage of inihibition
<25% at a concentration of 10 μM. Results indicated very weak or no inhibition of hMAO A by these
compounds.
2.5. Toxicity and Neuroprotection Studies In Vitro
For toxicity and neuroprotection studies, DL76 was chosen as the most promising DTL. The
human embryonic kidney (HEK-293) cell line was used for the estimation of safety of DL76. As shown
in Figure 5, the statistically significant decrease of HEK-293 cell viability in the presence of DL76 was
observed only at the highest doses of 125 μM and 250 μM. The used reference cytostatic drug
doxorubicin (DX) decreased cell viability <50% under the same conditions at very low concentrations
of 0.2 and 0.05 μM. The neuroprotection effect of DL76 was investigated in vitro in the model of
neuronal damage. Oxidative damage was induced by very high toxic levels of hydrogen peroxide
(H2
O2; 300 μM), the popular cell model for PD research, in neuroblastoma SH-SY5Y cell line.
Compound DL76 was tested at two doses, 10 μM and 50 μM, within two independent experiments.
Salsolinol (SAL) was used at a concentration of 50 μM as the reference compound with the proven

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neuroprotection activity [20]. DL76 displayed no increase of cell survival either at 10 μM (Figure 6A)
or 50 μM (Figure 6B), whereas SAL statistically significant increased cells viability in both
experiments. However, the safety of DL76 was confirmed here, as no toxic effect against SH-SY5Y
cells was observed at both used concentrations 10 and 50 μM (Figure 6A,B).
Figure 5. The effect of DL76 and doxorubicin (DX) on HEK-293 viability: Statistical significance (****
p < 0.0001) was analyzed by GraphPadPrism™ 8 software using one-way ANOVA and Bonferroni’s
multiple comparison posttest in comparison with control (1% DMSO in cell culture medium).
Figure 6. The effect of salsolinol (SAL) at 50 μM and DL76 at 10 μM (A) or at 50 μM (B) on SH-SY5Y
neuroblastoma cells viability damaged by 300 μM of H
2O
2
after 24 h of incubation: Statistical
significance was set at *** p < 0.001 and ** p < 0.01 by GraphPadPrism™ 8 software using one-way
ANOVA and Bonferroni’s multiple comparison posttest in comparison with the positive control H
2O2
(300 μM).
2.6. Antiparkinsonian Activity in Haloperidol-Induced Catalepsy in Wistar Rats
Haloperidol (dopamine D antagonist)-induced catalepsy in rodents is a popular model for
2
screening of antiparkinsonian activity of compounds [21]. Caused symptoms in animals are similar
to the inability of PD patients to initiate movements. We used this test to preliminary evaluate
antiparkinsonian activity of DL76. Haloperidol (0.63 mg/kg; s.c.) was administered to induce
catalepsy in Wistar rats; 5 min later, DL76 was added at doses of 25 or 50 mg/kg (i.p.). The reversal
of catalepsy was tested for 6 min (3 times in 3 min intervals) after 1 h of haloperidol administration.
As the reference compound was used MSX-3, the adenosine A2A receptor antagonist with confirmed

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ability to reverse catalepsy mediated by D
1
and D
2
receptor antagonists [22]. To characterize the
antiparkinsonian activity of DL76, two tests were performed: bar test and crossed-leg position test.
2.6.1. Bar Test
Haloperidol-induced catalepsy was not reduced by DL-76 administrated at a dose of 25 mg/kg
body weight (Table 3). After administration of DL-76 at a dose of 50 mg/kg body weight, low
antiparkinsonian activity in the bar test was observed. The compound reduced the duration of
catalepsy by 36.4% compared to the control group, which was obtained after administration of
haloperidol (16.4 versus 25.8). This effect was weaker than the anti-Parkisonian activity of MSX-3.
Table 3. Antiparkinsonian activity of DL76 in the bar test.
Dose
Times of Observation of Catalepsy (s)1
Compound
(mg/kg; i.p.)
0 min
3 min
30.0 ± 0
6 min
30.0 ± 0
30.0 ± 0
22.0 ± 5.0
0 ± 0 ***
0 ± 0 ***
control
DL-76
-
17.5 ± 5.3
19.5± 4.2
13.8 ± 5.9
0 ± 0 ***
0 ± 0 ***
25
50
25
50
26.5 ± 3.5
13.5 ± 4.2 *
0 ± 0 ***
0 ± 0 ***
MSX-3
1 mean ± SEM (n = 6); * p < 0.05; *** p < 0.001.
2.6.2. Crossed-Leg Position Test
The strongest, statistically significant antiparkinsonian activity was demonstrated by DL-76 at a
dose of 50 mg/kg body weight, which, compared to the control group, reduced the duration of
haloperidol-induced catalepsy by 99.7% (0.053 versus 19.25) (Table 4). This effect was comparable to
the antiparkinsonian activity of MSX-3.
The weaker, antiparkinsonian activity was demonstrated by DL-76 administered at a dose of 25
mg/kg body weight. DL76, in comparison to the control group, reduced the duration of haloperidol-
induced catalepsy by 54.5% (8.75 versus 19.25) (Table 4), but the effect was not statistically significant.
Table 4. Antiparkisonian activity of DL76 in the crossed-leg position test.
Times of observation of catalepsy (s)1
Compound Dose (mg/kg)
0 min
3 min
6 min
30.0 ± 0
control
DL-76
-
4.2 ± 1.6
23.5 ± 6.5
25
50
25
50
0.66 ± 0.68 10.6 ± 6.1
15.0 ± 6.7
0.16 ± 0.4 ***
0 ± 0 ***
0 ± 0 ***
0 ± 0 ***
0 ± 0 ***
0 ± 0 ***
0 ± 0 ***
0 ± 0 ***
MSX-3
0 ± 0 ***
1 mean ± SEM (n = 6); *** p < 0.001.
3. Materials and Methods
3.1. Chemistry
All reagents were purchased from commercial suppliers Alfa Aesar (Karlsruhe, Germany) or
Sigma Aldrich (Darmstadt, Germany) and were used without further purification. TLC data were
obtained with Merck silica gel 60F254 aluminum sheets with UV light detection and evaluation with
Dragendorff’s reagent (solvent systems: methylene chloride: methanol 9:1 or methylene chloride).
Melting points (m.p.) were determined on a MEL-TEMP II (LD Inc., Long Beach, CA, USA) melting
point apparatus and are uncorrected. Mass spectra (LC/MS) were performed on Waters TQ Detector
(Water Corporation., Milford, CT, USA) mass spectrometer. Retention times (t ) are given in minutes.
R
The UPLC/MS purity of all final compounds was determined (%). All compounds (except 14, 19, and
1
26) showed purity above 96%. H NMR and 13C NMR spectra were recorded in DMSO-d
6
on a

Int. J. Mol. Sci. 2020, 21, 3411
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Mercury 300 MHz PFG spectrometer (Varian, Palo Alto, CA, USA), Avance III HD 400 MHz
spectrometer (Bruker, Billerica, MA, USA), or FT-NMR 500 MHz spectrometer (Jeol Ltd., Akishima,
Tokyo, Japan). 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 (br, broad; d, dublet; m,
multiplet; quin, quintet; s, singlet; and t, triplet), approximate coupling constants J expressed in Hertz
(Hz), and number of protons. Elemental analyses were measured on analyzer Vario EL III 2
(Elementar, Langenselbold, Germany) and are within ±0.5% of the theoretical values (except carbon
for 7 (±0.6%) and 28 (±0.7%).
4-tert-Butylphenoxyalkyl bromides (4a–4d) were synthesized according to the method described
previously [14]. All of them are known and reported in Chemical Abstract Database:
1-(3-bromopropoxy)-4-tert-butylbenzene(4a):CAS3245-63-4; 1-(4-bromobutoxy)-4-tert-butylbenzene
(4b): CAS53669-73-1;
1-(5-bromopentyloxy)-4-tert-butylbenzene (4c): CAS53669-74-2;
1-(6-bromohexyloxy)-4-tert-butylbenzene (4d): CAS53669-73-3.
Designed compounds 5–31 were synthesized according to the procedure described previously
[12]. Briefly, to a proper 4-tert-butylphenylalkoxy bromide (2.5 mmol) in the mixture of C
mL) and H O (10 mL) and in the presence of K CO (7.5 mmol) with the catalytic amount of KI was
added a proper amine (5 mmol), and the solution was refluxed from 10 to 48 h. Then, C OH was
evaporated, and to the residue were added 50 mL of CH Cl and 40 mL of water. The organic solution
was washed with 50 mL of 1% HCl, evaporated to dryness, dissolved in 3% HCl followed by washing
with (C O, and neutralized with 5% NaOH. The final product was extracted with CH Cl , dried
over Na , and evaporated. The oily residue was transformed into oxalic acid salt in absolute
OH and precipitated (C O. The solid was crystallized from C OH.
2H
5OH (52.5
2
2
3
2
H
5
2
2
2
H5
)
2
2
2
2
SO
4
C2
H
5
2H
5
)
2
2H
5
1-(3-(4-tert-Butylphenoxy)propyl)pyrrolidine hydrogen oxalate (5)
1
White solid, yield 27%, m.p. 147–149 °C, C17
DMSO-d ) δ: 7.29 (d, J = 8.61 Hz, 2H), 6.86 (d, J = 8.61 Hz, 2H), 4.01 (t, J = 6.06 Hz, 2H), 3.20–3.31 (m,
6H), 2.05–2.14 (m, 2H), 1.93 (br s, 4H), 1.25 (s, 9H). 13C NMR (126 MHz, DMSO-d
) δ: 165.3, 156.5,
143.4, 126.5, 114.4, 65.3, 53.5, 51.9, 34.2, 31.8, 25.9, 23.2.LC-MS: purity 100% t
= 5.27, (ESI) m/z [M+H]+
262.24. Anal. calcd. for C19 29NO : C, 64.93; H, 8.32; N, 3.99%. Found: C, 64.90; H, 8.35; N, 3.93%.
H
27NO x C
2
H
2O4
(MW = 351.43). H NMR (400 MHz,
6
6
R
H
5
1-(4-(4-tert-Butylphenoxy)butyl)piperidine hydrogen oxalate (6)
1
White solid, yield 52%, m.p. 150–152 °C, C19
DMSO-d ) δ: 7.28 (d, J = 9.00 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 3.95 (t, J = 5.87 Hz, 2H), 2.95–3.31 (m,
6H), 1.64–1.88 (m, 8H), 1.52 (br. s., 2H), 1.25 (s, 9H). 13C NMR (101 MHz, DMSO-d
) δ: 165.2, 156.7,
143.2, 126.5, 114.4, 67.2, 56.1, 52.4, 40.9, 34.2, 31.8, 26.5, 23.0, 22.0, 20.8.LC-MS: purity 100% t = 5.69,
: C, 66.46; H, 8.76; N, 3.69%. Found: C, 66.45; H,
H
31NO x C
2
H
2O4
(MW = 379.48). H NMR (400 MHz,
6
6
R
(ESI) m/z [M+H]+ 290.22. Anal. calcd. for C21
H
33NO
5
8.48; N, 3.62%.
1-(5-(4-tert-Butylphenoxy)pentyl)piperidine hydrogen oxalate (7)
White solid, yield 42%, m.p. 170–172 °C, C20 33NO x C
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 8.61 Hz, 2H), 3.93 (t, J = 6.06 Hz, 1H), 2.89–3.39 (m,
6H), 1.72 (br. s., 8H), 1.52 (br. s., 2H), 1.37–1.48 (m, 2H), 1.25 (s, 9H). 13C NMR (101 MHz, DMSO-d
δ: 165.1, 156.8, 143.0, 126.5, 114.4, 67.5, 56.3, 52.4, 40.9, 34.2, 31.8, 28.7, 23.4, 23.3, 23.0, 22.0. LC-MS:
1
H
2
H
2
O (MW = 393.51). H NMR (400 MHz,
4
6
6)
purity 98.98% t
R
= 5.98, (ESI) m/z [M+H]+ 304.18. Anal. calcd. for C22
H
35NO5: C, 67.14; H, 8.96; N, 3.56%.
Found: C, 66.50; H, 8.71; N, 3.42%.
1-(6-(4-tert-Butylphenoxy)hexyl)piperidine hydrogen oxalate (8)
1
White solid, yield 16%, m.p. 156–158 °C, C21
H
35NO x C
2
H
2O4
(MW = 407.54). H NMR (400 MHz,
DMSO-d
6
) δ: 7.27 (d, J = 9.00 Hz, 2H), 6.83 (d, J = 8.61 Hz, 2H), 3.92 (t, J = 6.26 Hz, 2H), 2.86–3.21 (m,

Int. J. Mol. Sci. 2020, 21, 3411
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6H), 1.59–1.87 (m, 8H), 1.38–1.56 (m, 4H), 1.34 (d, J = 7.04 Hz, 2H), 1.25 (s, 9H). 13C NMR (101 MHz,
DMSO-d ) δ: 165.3, 156.9, 143.0, 126.5, 114.3, 67.6, 56.3, 52.4, 40.9, 34.2, 31.8, 29.0, 26.4, 25.6, 23.6, 22.9,
22.0.LC-MS: purity 96.38% t 37NO : C, 67.78; H,
= 6.39, (ESI) m/z [M+H]+ 318.20. Anal. calcd. for C23
6
R
H
5
9.15; N, 3.44%. Found: C, 67.26; H, 9.13; N, 3.42%.
1-(3-(4-tert-Butylphenoxy)propyl)-2-methylpiperidine hydrogen oxalate (9)
1
White solid, yield 19%, m.p. 114–116 °C, C19
DMSO-d ) δ: 7.30 (d, J = 8.61 Hz, 2H), 6.86 (d, J = 9.00 Hz, 2H), 3.93–4.12 (m, 2H), 3.18–3.44 (m, 3H),
3.06–3.17 (m, 1H), 2.92–3.05 (m, 1H), 2.01–2.20 (m, 2H), 1.78–1.91 (m, 1H), 1.53–1.76 (m, 4H), 1.38–1.52
(m, 1H), 1.28 (d, J = 6.65 Hz, 3H), 1.25 (s, 9H).13C NMR (101 MHz, DMSO-d
) δ: 165.2, 156.5, 143.4,
126.5, 114.5, 65.4, 57.3, 49.3, 40.9, 34.2, 31.8, 23.4, 22.8. LC-MS: purity 100% t
= 5.68, (ESI) m/z [M+H]+
290.22. Anal. calcd. for C21 33NO : C, 66.46; H, 8.76; N, 3.69%. Found: C, 66.29; H, 8.80; N, 3.64%.
H
31NO x C
2
H
2O4
(MW = 379.48). H NMR (400 MHz,
6
6
R
H
5
1-(4-(4-tert-Butylphenoxy)butyl)-2-methylpiperidine hydrogen oxalate (10)
White solid, yield 34%, m.p. 107–110 °C, C20 33NO x C (MW = 393.51). H NMR (400 MHz,
DMSO-d ) δ: 7.29 (d, J = 9.00 Hz, 2H), 6.85 (d, J = 9.00 Hz, 2H), 3.95 (m, 2H), 3.27 (br.s., 2H), 3.08–3.19
(m, 1H), 2.91–3.08 (m, 2H), 1.68–1.87 (m, 7H), 1.39–1.67 (m, 3H), 1.20 – 1.33 (m, 12H).13C NMR (101
MHz, DMSO-d ) δ: 165.2, 156.7, 143.2, 126.5, 114.4, 67.3, 51.6, 34.2, 31.8, 26.5, 22.7, 20.2.LC-MS: purity
100% t 35NO : C, 67.14; H, 8.96; N, 3.56%. Found:
= 5.94, (ESI) m/z [M+H]+ 304.24. Anal. calcd. for C22
C, 67.00; H, 8.95; N, 3.52%.
1
H
2
H
2O4
6
6
R
H
5
1-(5-(4-tert-Butylphenoxy)pentyl)-2-methylpiperidine hydrogen oxalate (11)
White solid, yield 11%, m.p. 96–99 °C, C21 35NO x C
(MW = 407.54). 1H NMR (400 MHz, DMSO-
) δ: 7.28 (d, J = 6.81 Hz, 2H), 6.84 (d, J = 9.00 Hz, 2H), 3.94 (t, J = 6.26 Hz, 2H), 3.26 (br.s., 2H), 3.03–
3.13 (m, 1H), 2.91–3.02 (m, 2H), 1.56–1.86 (m, 9H), 1.40–1.50 (m, 3H), 1.23–1.30 (m, 12H).13C NMR (101
MHz, DMSO-d ) δ: 165.2, 156.8, 143.0, 126.5, 114.4, 67.5, 51.7, 34.2, 31.8, 28.7, 23.4, 22.9.LC-MS: purity
H
2
H
2
O4
d
6
6
96.49% tR = 6.23, (ESI) m/z [M+H]+318.27. Anal. calcd. for C23
H
37NO : C, 67.78; H, 9.15; N, 3.44%. Found:
5
C, 67.50; H, 8.97; N, 3.41%.
1-(3-(4-tert-Butylphenoxy)propyl)-3-methylpiperidine hydrogen oxalate (12)
1
White solid, yield 29%, m.p. 141–143 °C, C19
H
31NO x C
2
H
2O4
(MW = 379.48). H NMR (500 MHz,
DMSO-d ) δ: 7.24 (d, J = 8.88 Hz, 2H), 6.80 (d, J = 8.59 Hz, 2H), 3.95 (t, J = 5.87 Hz, 2H), 3.24–3.43 (m,
6
2H), 3.07 (t, J = 7.59 Hz, 2H), 2.63–2.77 (m, 1H), 2.37–2.45 (m, 1H), 2.00–2.16 (m, 2H), 1.78–1.90 (m, 1H),
1.61–1.77 (m, 3H), 1.20 (s, 9H), 0.94–1.10 (m, 1H), 0.85 (d, J = 6.59 Hz, 3H). 13C NMR (126 MHz, DMSO-
d ) δ: 165.4, 156.6, 143.4, 126.6, 114.5, 65.5, 58.0, 54.0, 52.1, 34.3, 31.9, 30.6, 29.0, 24.1, 22.8, 19.1. LC-MS:
6
purity 98.04% t
R
= 5.75, (ESI) m/z [M+H]+ 290.22. Anal. calcd. for C21
H
33NO
5
: C, 66.46; H, 8.76; N, 3.69%.
Found: C, 66.44; H, 8.42; N, 3.65%.
1-(4-(4-tert-Butylphenoxy)butyl)-3-methylpiperidine hydrogen oxalate (13)
1
White solid, yield 53%, m.p. 148–150 °C, C20
DMSO-d
2H), 2.94–3.11 (m, 2H), 2.67–2.78 (m, 2H), 1.62–1.97 (m, 8H), 1.25 (s, 9H), 1.06 (m, 1H), 0.89 (d, J = 6.65
Hz, 3H).13C NMR (101 MHz, DMSO-d
) δ: 165.2, 156.7, 143.1, 126.5, 114.4, 67.2, 57.9, 56.1, 51.9, 40.9,
34.2, 31.8, 30.6, 28.9, 26.5, 22.6, 20.8, 19.1.LC-MS: purity 99.46% t
= 6.13, (ESI) m/z [M+H]+ 304.24. Anal.
calcd. for C22 35NO : C, 67.14; H, 8.96; N, 3.56%. Found: C, 66.86; H, 8.62; N, 3.51%.
H
33NO x C
2
H
2O4
(MW = 393.51). H NMR (400 MHz,
6
) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 3.95 (t, J = 6.06 Hz, 2H), 3.24–3.45 (m,
6
R
H
5
1-(5-(4-tert-Butylphenoxy)pentyl)-3-methylpiperidine hydrogen oxalate (14)
1
White solid, yield 8%, m.p. 179–181 °C, C21
DMSO-d ) δ: 7.28 (d, J = 8.22 Hz, 2H), 6.84 (d, J = 7.83 Hz, 2H), 3.94 (br. s., 2H), 3.19–3.48 (m, 2H), 2.97
(br. s., 4H), 1.57–1.99 (m, 8H), 1.35–1.54 (m, 2H), 1.26 (br. s., 9H), 1.06 (d, J = 9.39 Hz, 1H), 0.69–0.97
H
35NO x C
2
H
2
O (MW = 407.54). H NMR (400 MHz,
4
6

Int. J. Mol. Sci. 2020, 21, 3411
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(m, 3H). 13C NMR (101 MHz, DMSO-d
6
) δ: 165.0, 156.8, 143.1, 126.5, 114.4, 67.5, 58.0, 56.4, 52.0, 41.0,
34.2, 31.8, 30.6, 28.7, 23.5, 23.3, 19.1.LC-MS: purity 93.40% t
R
= 6.40, (ESI) m/z [M+H]+ 318.20. Anal.
calcd. for C23 37NO : C, 67.78; H, 9.15; N, 3.44%. Found: C, 67.44; H, 8.90; N, 3.43%.
H
5
1-(6-(4-tert-Butylphenoxy)hexyl)-3-methylpiperidine hydrogen oxalate (15)
White solid, yield 7%, m.p. 140–142 °C, C22 37NO x C (MW = 421.56). H NMR (400 MHz,
DMSO-d ) δ: 7.27 (d, J = 8.61 Hz, 2H), 6.83 (d, J = 9.00 Hz, 2H), 3.92 (t, J = 6.46 Hz, 2H), 3.23–3.43 (m,
1
H
2
H
O
2 4
6
2H), 2.88–3.04 (m, 2H), 2.68–2.76 (m, 2H), 1.57–1.95 (m, 8H), 1.43 (quin, J = 7.34 Hz, 2H), 1.33 (quin, J
= 7.04 Hz, 2H), 1.25 (s, 9H), 0.97–1.14 (m, 1H), 0.89 (d, J = 6.65 Hz, 3H). 13C NMR (101 MHz, DMSO-
d
6
) δ: 165.2, 156.9, 143.0, 126.5, 114.3, 67.6, 57.8, 56.3, 51.9, 40.9, 34.2, 31.8, 30.6, 29.0, 28.9, 26.4, 25.6,
23.6, 22.6, 19.1. LC-MS: purity 100% t 39NO : C,
R
= 6.75, (ESI) m/z [M+H]+ 332.22. Anal. calcd. for C24
H
5
68.37; H, 9.32; N, 3.32%. Found: C, 68.14; H, 9.20; N, 3.28%.
1-(3-(4-tert-Butylphenoxy)propyl)-3,3-dimethylpiperidine hydrogen oxalate (16)
1
White solid, yield 44%, m.p. 183–186 °C, C20
DMSO-d ) δ: 7.30 (d, J = 8.61 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 3.99 (t, J = 5.67 Hz, 2H), 2.79–3.30 (m,
6H), 2.10 (br. s., 2H), 1.74 (br. s., 2H), 1.36 (br. s., 2H), 1.25 (s, 9H), 1.00 (s, 6H).13C NMR (101 MHz,
DMSO-d ) δ: 164.7, 156.5, 143.4, 126.5, 114.4, 65.6, 62.1, 55.0, 52.8, 40.9, 35.3, 34.2, 31.8, 30.9, 24.2, 20.2.
LC-MS: purity 100% t 35NO : C, 67.14; H, 8.96;
= 6.02, (ESI) m/z [M+H]+ 304.24. Anal. calcd. for C22
H
33NO x C
2
H
2O4
(MW = 393.51). H NMR (400 MHz,
6
6
R
H
5
N, 3.56%. Found: C, 67.19; H, 8.50; N, 3.51%.
1-(4-(4-tert-Butylphenoxy)butyl)-3,3-dimethylpiperidine hydrogen oxalate (17)
White solid, yield 23%, m.p. 140–142 °C, C21
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 3.95 (t, J = 5.87 Hz, 2H), 2.91–3.20 (m,
4H), 2.80 (br. s., 2H), 1.63–1.90 (m, 6H), 1.37 (d, J = 4.70 Hz, 2H), 1.25 (s, 9H), 0.99 (s, 6H).13C NMR (101
MHz, DMSO-d ) δ: 165.0, 156.7, 143.1, 126.5, 114.4, 67.2, 61.8, 57.1, 52.5, 40.9, 35.2, 34.2, 31.8, 30.9, 26.6,
20.8, 20.1.LC-MS: purity 100% t 37NO : C, 67.78;
= 6.29, (ESI) m/z [M+H]+318.27. Anal. calcd. for C23
H
35NO x C
2
H
2O4
(MW = 407.54).1H NMR (400 MHz,
6
6
R
H
5
H, 9.15; N, 3.44%. Found: C, 67.57; H, 9.03; N, 3.39%.
1-(5-(4-tert-Butylphenoxy)pentyl)-3,3-dimethylpiperidine hydrogen oxalate (18)
1
White solid, yield 40%, m.p. 150–153 °C, C22
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 9.00 Hz, 2H), 3.93 (t, J = 6.26 Hz, 2H), 2.88–3.24 (m,
4H), 2.81 (br. s., 2H), 1.61–1.83 (m, 6H), 1.30–1.45 (m, 4H), 1.25 (s, 9H), 0.99 (s, 6H). 13C NMR (101
MHz, DMSO-d ) δ: 165.0, 156.8, 143.0, 126.5, 114.4, 67.4, 61.8, 57.3, 52.5, 40.9, 35.2, 34.2, 31.8, 30.9, 28.7,
23.4, 23.3, 20.0.LC-MS: purity 96.27% t 39NO : C,
= 6.58, (ESI) m/z [M+H]+ 332.29. Anal. calcd. for C24
H
37NO x C
2
H
2O4
(MW = 421.56). H NMR (400 MHz,
6
6
R
H
5
68.37; H, 9.32; N, 3.32%. Found: C, 67.94; H, 8.99; N, 3.24%.
1-(6-(4-tert-Butylphenoxy)hexyl)-3,3-dimethylpiperidine hydrogen oxalate (19)
1
White solid, yield 21%, m.p. 122–125 °C, C23
DMSO-d ) δ: 7.27 (d, J = 8.61 Hz, 2H), 6.83 (d, J = 8.61 Hz, 2H), 3.83–4.01 (m, J = 6.46, 6.46 Hz, 2H),
2.75–3.13 (m, 6H), 1.57–1.83 (m, 6H), 1.29–1.54 (m, 6H), 1.25 (s, 9H), 0.99 (s, 6H). 13C NMR (101 MHz,
DMSO-d ) δ: 165.1, 156.9, 143.0, 126.5, 114.3, 67.6, 61.7, 57.3, 52.4, 40.9, 35.2, 34.2, 31.8, 30.9, 29.0, 26.4,
25.6, 23.5, 20.0. LC-MS: purity 94.18% t 41NO
= 6.97, (ESI) m/z [M+H]+ 346.25. Anal. calcd. for C25
H
39NO x C
2
H
2O4
(MW = 435.59). H NMR (400 MHz,
6
6
R
H
5
:
C, 68.93; H, 9.46; N, 3.22%. Found: C, 68.80; H, 9.26; N, 3.22%.
1-(3-(4-tert-Butylphenoxy)propyl)-3,5-dimethylpiperidine hydrogen oxalate (20)
1
White solid, yield 43%, m.p. 143–145 °C, C20
H
33NO x C
2
H
2
O (MW = 393.51). H NMR (400 MHz,
4
DMSO-d ) δ: 7.29 (d, J = 9.00 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 4.00 (t, J = 5.67 Hz, 2H), 3.34 (d, J = 10.17
6
Hz, 2H), 2.97–3.23 (m, 2H), 2.40 (t, J = 11.93 Hz, 2H), 2.02–2.23 (m, 2H), 1.91 (d, J = 3.52 Hz, 2H), 1.73
(d, J = 12.91 Hz, 1H), 1.33–1.49 (m, 1H), 1.25 (s, 9H), 0.99 (d, J = 6.65 Hz, 6H). 13C NMR (101 MHz,

Int. J. Mol. Sci. 2020, 21, 3411
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DMSO-d
15.62% t
calcd. for C22
6
) δ: 165.0, 156.5, 143.4, 126.5, 114.4, 65.5, 57.6, 53.9, 34.2, 31.8, 28.8, 24.1, 18.9.LC-MS: purity
= 6.08, (ESI) m/z [M+H]+ 304.24 and purity 84.38% t = 6.15, (ESI) m/z [M+H]+ 304.24. Anal.
35NO : C, 67.14; H, 8.96; N, 3.56%. Found: C, 67.03; H, 9.14; N, 3.52%.
R
R
H
5
1-(4-(4-tert-Butylphenoxy)butyl)-3,5-dimethylpiperidine hydrogen oxalate (21)
White solid, yield 39%, m.p. 170–172 °C, C21 35NO x C (MW = 407.54). H NMR (400 MHz,
DMSO-d ) δ: 7.29 (d, J = 8.61 Hz, 2H), 6.85 (d, J = 8.61 Hz, 2H), 3.96 (t, J = 6.06 Hz, 2H), 3.31 (d, J = 9.78
Hz, 2H), 2.90–3.09 (m, 2H), 2.37 (t, J = 11.93 Hz, 2H), 1.61–2.02 (m, 7H), 1.13–1.44 (s, 10H), 0.87–1.04
(m, 6H). 13C NMR (101 MHz, DMSO-d
) δ:165.0, 156.7, 143.2, 126.5, 114.4, 67.2, 57.4, 56.1, 34.2, 31.8,
28.7, 26.5, 20.8, 18.9. LC-MS: purity 11.21% t
= 6.31, (ESI) m/z [M+H]+ 318.46 and purity 88.79% t
37NO : C, 67.78; H, 9.15; N, 3.44%. Found: C, 68.14;
1
H
2
H
O
2 4
6
6
R
R
=
6.37, (ESI) m/z [M+H]+ 318.46. Anal. calcd. for C23
H
5
H, 8.87; N, 3.40%.
1-(5-(4-tert-Butylphenoxy)pentyl)-3,5- dimethylpiperidine hydrogen oxalate (22)
White solid, yield 31%, m.p. 175–178 °C, C22 37NO x C (MW = 421.56). H NMR (400 MHz,
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 8.61 Hz, 2H), 3.94 (t, J = 6.06 Hz, 2H), 3.31 (d, J = 10.56
Hz, 2H), 2.80–3.06 (m, 2H), 2.36 (t, J = 11.74 Hz, 2H), 1.59–2.12 (m, 7H), 1.15–1.52 (m, 12H), 0.83–0.99
(m, 6H). 13C NMR (101 MHz, DMSO-d
) δ: 164.9, 156.8, 143.1, 126.5, 114.4, 67.4, 57.5, 34.2, 31.8, 28.7,
23.5, 23.3, 18.9.LC-MS: purity 6.64% t = 6.78,
= 6.73, (ESI) m/z [M+H]+ 332.29 and purity 93.36% t
: C, 68.37; H, 9.32; N, 3.32%. Found: C, 68.25; H,
1
H
2
H
2O4
6
6
R
R
(ESI) m/z [M+H]+ 332.29. Anal. calcd. for C24
H
39NO
5
9.06; N, 3.30%.
1-(6-(4-tert-Butylphenoxy)hexyl)-3,5- dimethylpiperidine hydrogen oxalate (23)
1
White solid, yield 43%, m.p. 143–145 °C, C23
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 8.61 Hz, 2H), 3.93 (t, J = 6.26 Hz, 2H), 3.30 (d, J = 10.17
Hz, 2H), 2.81–3.04 (m, 2H), 2.36 (t, J = 11.93 Hz, 2H), 1.88 (br. s., 2H), 1.60–1.78 (m, 5H), 1.20–1.53 (m,
14H), 0.89 (d, J = 6.65 Hz, 6H). 13C NMR (101 MHz, DMSO-d
) δ: 164.9, 156.9, 143.0, 126.5, 114.4, 67.6,
57.5, 34.2, 31.8, 28.9, 26.4, 25.6, 23.7, 18.9.LC-MS: purity 5.30% t
= 6.96, (ESI) m/z [M+H]+ 346.31 and
purity 94.70% t
= 7.00, (ESI) m/z [M+H]+ 346.31. Anal. calcd. for C25
Found: C, 68.74; H, 9.64; N, 3.19%.
H
39NO x C
2
H
2O4
(MW = 345.56). H NMR (400 MHz,
6
6
R
R
H
41NO5: C, 68.93; H, 9.49; N, 3.22%.
1-(3-(4-tert-Butylphenoxy)propyl)-4-methylpiperidine hydrogen oxalate (24)
White solid, yield 29%, m.p. 158–160 °C, C19 31NO x C
DMSO-d
1
H
2
H
2O4
(MW = 379.48). H NMR (500 MHz,
6
) δ: 7.25 (d, J = 8.88 Hz, 2H), 6.81 (d, J = 8.59 Hz, 2H), 3.95 (t, J = 6.01 Hz, 8H), 3.35 (d, J = 11.74
Hz, 2H), 3.02–3.15 (m, 2H), 2.82 (t, J = 11.60 Hz, 2H), 2.00–2.11 (m, 2H), 1.72 (d, J = 13.17 Hz, 2H), 1.57
(d, J = 6.01 Hz, 1H), 1.29–1.43 (m, 2H), 1.20 (s, 9H), 0.88 (d, J = 6.59 Hz, 3H). 13C NMR (126 MHz,
DMSO-d
31.2, 31.1, 31.1, 31.1, 31.1, 31.0, 28.5, 24.2, 21.4, 21.4.LC-MS: purity 98.17% t
290.22. Anal. calcd. for C21 33NO : C, 66.46; H, 8.76; N, 3.69%. Found: C, 65.97; H, 8.46; N, 3.63%.
6
) δ: 165.2, 156.5, 143.4, 126.6, 114.5, 65.5, 53.8, 53.7, 52.2, 52.1, 52.1, 52.1, 52.1, 34.3, 31.9, 31.2,
R
= 5.84, (ESI) m/z [M+H]+
H
5
1-(4-(4-tert-Butylphenoxy)butyl)-4-methylpiperidine hydrogen oxalate (25)
1
White solid, yield 47%, m.p. 155–157 °C, C20
DMSO-d ) δ: 7.28 (d, J = 9.00 Hz, 2H), 6.85 (d, J = 9.00 Hz, 2H), 3.95 (t, J = 5.87 Hz, 2H), 3.37 (d, J = 11.74
Hz, 2H), 2.97–3.13 (m, 2H), 2.84 (t, J = 11.54 Hz, 2H), 1.67–1.90 (m, 6H), 1.61 (d, J = 5.87 Hz, 1H), 1.33–
1.50 (m, 2H), 1.25 (s, 9H), 0.91 (d, J = 6.65 Hz, 3H). 13C NMR (101 MHz, DMSO-d
) δ: 165.2, 156.7, 143.1,
126.5, 114.4, 67.2, 55.8, 51.8, 40.9, 34.2, 31.8, 30.9, 28.5, 26.5, 21.3, 20.9. LC-MS: purity 99.02% t = 6.07,
: C, 67.14; H, 8.96; N, 3.56%. Found: C, 67.41; H,
H
33NO x C
2
H
2O4
(MW = 393.51). H NMR (400 MHz,
6
6
R
(ESI) m/z [M+H]+ 304.24. Anal. calcd. for C22
H
35NO
5
8.48; N, 3.56%.

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1-(5-(4-tert-Butylphenoxy)pentyl)-4-methylpiperidine hydrogen oxalate (26)
1
White solid, yield 8%, m.p. 167–170 °C, C21
DMSO-d ) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 8.61 Hz, 2H), 3.94 (t, J = 6.26 Hz, 2H), 3.31–3.41 (m,
2H), 2.93–3.03 (m, 2H), 2.77–2.87 (m, 2H), 1.64–1.82 (m, 6H), 1.54–1.64 (m, 1H), 1.32–1.48 (m, 4H), 1.20–
1.31 (m, 9H), 0.92 (d, J = 6.26 Hz, 3H). 13C NMR (101 MHz, DMSO-d
) δ: 165.0, 156.8, 143.0, 126.5, 114.4,
67.4, 40.9, 34.2, 31.8, 28.7, 23.6, 23.3.LC-MS: purity 92.34% t
= 6.34, (ESI) m/z [M+H]+ 318.20. Anal.
calcd. for C23 37NO : C, 67.78; H, 9.15; N, 3.44%. Found: C, 67.65; H, 8.81; N, 3.42.
H
35NO x C
2
H
2
O (MW = 407.54). H NMR (400 MHz,
4
6
6
R
H
5
1-(6-(4-tert-Butylphenoxy)hexyl)-4-methylpiperidine hydrogen oxalate (27)
1
White solid, yield 12%, m.p. 136–139 °C, C22
DMSO-d ) δ: 7.27 (d, J = 8.61 Hz, 2H), 6.83 (d, J = 9.00 Hz, 2H), 3.92 (t, J = 6.46 Hz, 2H), 3.36 (d, J = 11.74
Hz, 2H), 2.89–3.03 (m, 2H), 2.76–2.87 (m, 2H), 1.54–1.82 (m, 7H), 1.29–1.48 (m, 6H), 1.25 (s, 9H), 0.91
(d, J = 6.26 Hz, 3H). 13C NMR (101 MHz, DMSO-d
) δ: 165.3, 156.9, 143.0, 126.5, 114.3, 67.6, 56.0, 51.8,
40.9, 34.2, 31.8, 30.9, 29.0, 28.5, 26.4, 25.6, 23.7, 21.3. LC-MS: purity 98.49% t
= 6.77, (ESI) m/z [M+H]+
332.22. Anal. calcd. for C24 39NO : C, 68.37; H, 9.32; N, 3.32%. Found: C, 68.51; H, 9.40; N, 3.30%.
H
37NO x C
2
H
2
O
4
(MW = 421.56). H NMR (400 MHz,
6
6
R
H
5
1-(3-(4-tert-Butylphenoxy)propyl)-azepane hydrogen oxalate (28)
1
White solid, yield 32%, m.p. 138–140 °C, C19
DMSO-d ) δ: 7.24 (d, J = 3.15 Hz, 2H), 6.81 (d, J = 2.86 Hz, 2H), 3.95 (br. s., 2H), 2.88–3.47 (m, 6H), 2.08
(br. s., 2H), 1.76 (br. s., 4H), 1.56 (br. s., 4H), 1.20 (br. s., 9H). 13C NMR (126 MHz, DMSO-d
) δ: 165.5,
156.6, 143.4, 126.6, 114.5, 65.5, 54.3, 54.1, 34.3, 31.9, 26.6, 24.4, 23.6. LC-MS: purity 99.17% t = 5.82,
: C, 66.46; H, 8.76; N, 3.69%. Found: C, 65.74; H,
H
31NO x C
2
H
2O4
(MW = 379.48). H NMR (500 MHz,
6
6
R
(ESI) m/z [M+H]+ 290.22. Anal. calcd. for C21
H
33NO
5
8.41; N, 3.65%.
1-(4-(4-tert-Butylphenoxy)butyl)-azepane hydrogen oxalate (29)
1
White solid, yield 49%, m.p. 149–151 °C, C20
DMSO-d ) δ: 7.28 (d, J = 8.22 Hz, 2H), 6.85 (d, J = 7.82 Hz, 2H), 3.95 (t, J = 5.67 Hz, 2H), 3.16–3.32 (m,
4H), 3.11 (d, J = 7.04 Hz, 2H), 1.68–1.89 (m, 8H), 1.60 (br. s., 4H), 1.25 (s, 9H). 13C NMR (101 MHz,
DMSO-d ) δ: 165.3, 156.7, 143.1, 126.5, 114.4, 67.2, 56.4, 53.9, 40.9, 34.2, 31.8, 26.6, 26.5, 23.5, 21.2. LC-
MS: purity 99.24% t
H
33NO x C
2
H
2O4
(MW = 393.51). H NMR (400 MHz,
6
6
R
= 6.01, (ESI) m/z [M+H]+ 304.24. Anal. calcd. for C22
H
35NO
5
: C, 67.14; H, 8.96; N,
3.56%. Found: C, 67.40; H, 8.51; N, 3.55%.
1-(5-(4-tert-Butylphenoxy)pentyl)-azepane hydrogen oxalate (30)
1
White solid, yield 38%, m.p. 151–153 °C, C21
DMSO-d
4H), 2.94–3.08 (m, 2H), 1.79 (br. s., 4H), 1.66–1.75 (m, 4H), 1.54–1.64 (m, 4H), 1.43 (d, J = 7.43 Hz, 2H),
1.25 (s, 9H). 13C NMR (101 MHz, DMSO-d
) δ: 165.3, 156.8, 143.0, 126.5, 114.4, 67.5, 56.6, 53.9, 40.9,
34.2, 31.8, 28.7, 26.6, 23.8, 23.4, 23.3. LC-MS: purity 100% t
for C23 37NO
H
35NO x C
2
H
2O4
(MW = 407.53). H NMR (400 MHz,
6
) δ: 7.28 (d, J = 8.61 Hz, 2H), 6.84 (d, J = 9.00 Hz, 2H), 3.93 (t, J = 6.26 Hz, 2H), 3.13–3.32 (m,
6
R
= 6.27, (ESI) m/z [M+H]+ 366.25. Anal. calcd.
H
5
: C, 67.78; H, 9.15; N, 3.44%. Found: C, 67.47; H, 8.75; N, 3.39%.
1-(6-(4-tert-Butylphenoxy)hexyl)-azepane hydrogen oxalate (31)
1
White solid, yield 5%, m.p. 130–132 °C, C22
DMSO-d
4H), 2.98–3.04 (m, 2H), 1.58–1.82 (m, 12H), 1.28–1.47 (m, 4H), 1.25 (s, 9H). 13C NMR (101 MHz, DMSO-
) δ: 165.3, 156.8, 143.0, 126.5, 114.3, 67.6, 56.7, 53.9, 40.9, 34.2, 31.8, 29.0, 26.6, 26.4, 25.6, 24.0, 23.4. LC-
MS: purity 97.93% t 39NO : C, 68.37; H, 9.32; N,
= 6.79, (ESI) m/z [M+H]+ 332.22. Anal. calcd. for C24
3.32%. Found: C, 68.46; H, 8.98; N, 3.32%.
H
37NO x C
2
H
2O4
(MW = 421.56). H NMR (400 MHz,
6
) δ: 7.27 (d, J = 8.61 Hz, 2H), 6.83 (d, J = 8.61 Hz, 2H), 3.92 (t, J = 6.46 Hz, 2H), 3.16–3.23 (m,
d
6
R
H
5

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3.2. Biological Studies In Vitro
3.2.1. Affinity for Human Histamine H
3
Receptor
The radioligand displacement binding assay was carried out in HEK-293 cells stably expressing
the recombinant hH
R as described by Kottke et al. [23] with slight modifications [14]. [3H]Nα-
methylhistamine was used as radiolabeled tracer (2 nM, K = 3.08). Obtained data from at least three
experiments (in at least duplicates) were analyzed with GraphPad Prism 6.1 (San Diego, CA, USA)
using nonlinear regression (one-site competition on logarithmic scale), and K values were
3
D
i
transformed from IC50 according to Cheng-Prusoff [24]. Statistical analysis was performed on −log K
values. Mean values and 95% confidence intervals were converted to nanomolar concentrations.
i
3.2.2. Human Monoamine Oxidase Inhibitory Activity
General Method for Determining Activity Against MAO Isoforms
Inhibitory potency of compounds on MAO isoenzymes were carried out by a fluorometric
method with a commercial Amplex™ Red Monoamine Oxidase Assay Kit (ThermoFisher Scientific
A12214, Waltham, MA, USA) as described previously [12,25]. For all tests, recombinant human MAO
B and MAO A (from Sigma AldrichM7441 and M7316, Darmstadt, Germany) were used. Inhibitors’
activity was measured in the presence of p-tyramine (200 μM). In all experiments, reference inhibitors
in the concentrations that fully inhibited the MAO isoform were included. Reference inhibitors for
MAO-B were pargyline 10 μM, rasagiline 1 μM, and safinamide 1 μM and, for MAO-A, was
clorgyline 1 μM.
Screening and Determination of IC50
Firstly, inhibitors’ activity was measured in a concentration of 1 μM. The results were
normalized (no inhibition = 0% and fully inhibited enzyme 100%). For compounds that inhibited the
enzyme by more than 50%, further studies were conducted to obtain IC50 from concentration–
response curves. All calculations were made in Microsoft Excel and GraphPadPrism software. All
experiments were performed in duplicate, and data are expressed as mean ± SEM of 2–5 independent
experiments.
Reversibility Studies
Reversibility of MAO B inhibitors was tested as described previously [12] with slight
modification. Two experiments were performed simultaneously. In the first experiment, inhibitors
(in concentrations corresponding to their IC80 values) were incubated with the enzyme and p-
tyramine (10 μM) for 22 min (measuring fluorescence every two minutes). Next, the concentration of
the substrate was increased to 1 mM and the fluorescence was measured every 5 min for 5 h. In the
second experiment, inhibitors and enzyme were preincubated for 30 min in room temperature before
addition of 10 μM p-tyramine; then, continuation of the experiment was carried out identically as the
first.
Kinetic Studies
The mode of the inhibition was tested according to the standard procedure described in
Reversibility Studies using different concentrations of the substrate [12,26]. Inhibitors (compound 9
and DL76) were used in three concentrations corresponding to their IC20, IC50, and IC80 values.
Substrate was used in six concentrations: 0.05 nM, 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM. After
the experiment, velocities were calculated and put on the graph (y-axis) against the concentration of
the substrate (x-axis). From the Michaelis–Menten plot Vmax, K , and α values were calculated for
M
different concentrations of the inhibitor. Double-reciprocal plot (Lineweaver–Burk plot) were
prepared to display the data.

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3.2.3. Toxicity and Neuroprotection Evaluation In Vitro
Cell Lines
SH-SY5Y CRL-2266™ neuroblastoma cell line was purchased directly from American Type
Culture Collection (Manassas, VG, USA) and were cultured as described previously [20]. Human
embryonic kidney HEK-293 cell line (ATCC CRL-1573) was kindly donated by Prof. Dr Christa
Müller (Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Germany). The
cells were cultured as described previously [27].
Toxicity Studies
The HEK-293 cells were seeded at a concentration of 1.5 × 104 cells/well in 200 μL culture medium
and incubated for 24 h at 37 °C and 5% CO
dissolved in DMSO were added at various concentrations into microplate (total concentration of
DMSO in media was 1%) and incubated for 48 h at 37 °C and 5% CO . Then, 25 μL EZ4U labelling
2. Next, the cytostatic reference doxorubicin (DX) or DL76
2
mixture (Biomedica, Vienna, Austria) was added and the cells were incubated for 2 h. The
spectrophotometric absorbance of the samples was measured using a microplate reader (EnSpire,
PerkinElmer, Waltham, MA, USA) at 492 nm. All measurements were performed in triplicate, and
results are shown as mean ± SD.
Neuroprotection Studies
SH-SY5Y cells were seeded in microplate at a concentration of 2.5 × 104 cells/well in 100 μL
culture medium and cultured for 24 h at 37 C and 5% CO to reach 70% confluence. The cells were
2
preincubated first for 1 h with DL76 (10 and 50 μM) or with the reference neuroprotectant salsolinol.
(R,S)-salsolinol (purity ≥ 99%) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Next,
H2O2
was added at final concentration 300 μM and the cells were placed into the incubator. After 24
h of compounds co-incubation with H
2O2
, the CellTiter 96®AQueous Non-Radioactive Cell
Proliferation Assay (MTS) labeling mixture was added to each well, and the microplates were
incubated under the same conditions for 5 h. The absorbance was measured using a microplate reader
EnSpire (PerkinElmer, Waltham, MA USA) at 490 nm. All measurements were performed in
triplicate, and results are shown as mean ± SD.
3.3. Antiparkinsonian Activity in Haloperidol-Induced Catalepsy In Vivo
3.3.1. Animals
The experiments were carried out on male rats Wistar (180–220 g). Animals were housed in
plastic cages in room at a constant temperature of 20 ± 2 °C with natural light–dark cycles. The
animals had free access to standard pellet diet and water and were used after a minimum of 3 days
of acclimatization to the housing conditions. Control and experimental groups consisted of 6 animals
each. All experimental procedures were performed according to the European Union Directive of 22
September 2010 (2010/63/EU) and Polish legislation concerning animal care and use and was
approved by the Local Ethics Committee for Experiments on Animals in Kraków, Poland (Resolution
No. 70/2014, approval date: 20 May 2014). The examined compound was administered as the
suspension in 0.5% methylcellulose in constant volume of 10 mL/kg.
3.3.2. Drugs
Haloperidol was purchased from Sigma Aldrich (Darmstadt, Germany). MSX-3 ((E)-phosphoric
acid mono-[3-[8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydro-
purin-3-yl]propyl] ester disodium salt) was synthesized in the laboratory of Pharmaceutical Institute,
Pharmaceutical Chemistry I, University of Bonn, Germany and donated by Prof. Dr Christa Müller.

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3.3.3. Statistical Analysis
The data are expressed as the means  SEM. All statistical calculations were carried out with the
GraphPad Prism 5 program. The data were evaluated by one-way analysis of variance (ANOVA)
followed by Duncan test; p < 0.05 was considered significant.
3.3.4. Determination of Antiparkinsonian Activity in Catalepsy Tests
Antiparkinsonian activity in catalepsy tests was performed as described in [28]. Haloperidol was
administered s.c. at a dose of 0.63 mg/kg, which in 100% of control animals caused catalepsy. Tested
compounds were administered i.p. at the doses 50 and 25 mg/kg body weight, 5 min after haloperidol
injection. After 60 min from the injection of haloperidol, to assess the occurrence of catalepsy, the
animals were placed in a forced position and the time they remained in this position was measured.
Two tests were performed to determine the antiparkinsonian activity: crossed leg position test and
bar test. In the first test, the animals were put on the hind paws behind the front (forced position); in
the second, the animals were supported on a wooden block (the front paws were placed on a block
suspended 10 cm above the ground). Then, the time that the animals remain is measured in a forced
position (time was measured to a maximum of 60 s). Observation was carried out 3 times in 3-min
intervals. The shortening of the time of catalepsy to the control group was adopted for the
antiparkinsonian activity.
4. Conclusions
In summary, in this study, new potential DTL for PD have been designed and synthesized. As
the lead structure, DL76 was chosen, the compound with proven high hH
in CHO K1 cells and K = 38 nM in HEK 293 cells) and hMAO B inhibitory activity in vitro (IC50 = 48
nM). The introduced modifications were aimed at assessing the influence of cyclic amines and the
length of the alkyl chain on hH R affinity and hMAO B inhibition. Most compounds showed
nanomolar range affinities for hH R. The significant increase in the inhibitory effect for hMAO B
3
R affinity (K = 22 nM [13]
i
i
3
3
occurred for pyrrolidine (5) and 2-methylpiperidine (9) derivatives. These results confirmed our
previous observations concerning 4-tert-amylphenoxy derivatives [12] where such compounds were
among the most potent hMAO B inhibitors.
In vitro toxicity studies with DL76 in the HEK293 cells and neuroblastoma SH-SY5Y cells did
not show risk of toxicity at the dose of 50 μM of this compound. However, no neuroprotection effect
of DL76 against very high toxic levels of hydrogen peroxide (300 μM) in neuroblastoma SH-SY5Y
cells was observed.
Conducted in vivo studies showed that tested DL76 caused just statistically significant
antiparkinsonian activity in the crossed-leg position test. The tested compound, at the dose of 50
mg/kg body weight, practically completely reduced the duration of catalepsy, whereas in the bar test
at this dose, a low positive effect was observed. Moreover, DL76 did not show any cataleptic effects.
Considering the dual functional profile, the most valuable compound proved to be DL76 with
balanced activity against both biological targets (hH
3
R K = 38 nM and hMAO B IC50 = 48 nM).
i
Structural modification in DTL is not easy as it requires optimization towards both targets. By direct
exchange of a cyclic amine, it was possible to obtain a very potent hMAO B inhibitor, compound 5
with the IC50 of 2.7 nM. However, this compound proved to be only a moderate hH
nM). Generally, very potent hMAO B inhibitors (5, 9, and 28) showed higher strength to inhibit
hMAO B than to block hH R. Thus, it seems that, in this class of compounds, a cyclic amine moiety
3
R ligand (K = 371
i
3
in the western part of a molecule plays the very important role in the interaction with hMAO B, but
further studies should be performed to confirm it. However, the presented compounds are promising
starting materials in further search for new active DTL for PD.
Supplementary Materials: Supplementary Materials can be found at www.mdpi.com/1422-0067/21/10/3411/s1.
Author Contributions: Conceptualization, D.Ł. and K.K.-K.; synthesis of compounds: M.K.; in vitro hMAO B
and A studies: A.O.-M., A.D.-P., and T.K.; in vitro hMAO B kinetic and reversibility studies: A.O.-M.; in vitro
histamine H
3
R affinity: D.R., A.F., and H.S.; supervision of in vitro H R studies: H.S.; in vivo studies: M.Z.; in
3

Int. J. Mol. Sci. 2020, 21, 3411
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vitro toxicity and neuroprotection studies: G.L.; writing—original draft preparation: D.Ł.; writing—review and
editing: D.Ł and K.K.-K.; project administration, D.Ł. All authors have read and agreed to the published version
of the manuscript.
Funding: This research was funded by National Science Centre, Poland grant no. UMO-2016/23/B/NZ7/02327
(D.Ł.), UMO-2019/03/X/NZ7/00180 (G.L.), and DFG INST 208/664-1 FUGG (H.S.). Further, the authors
acknowledge the contribution of EU-COST action CA18133 ERNEST (“European Research Network on Signal
Transduction”).
Acknowledgments: The generous gift of MSX-3 and human embryonic kidney HEK-293 cell line (ATCC CRL-
1573) by Christa Müller (Univeristy of Bonn, Germany) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AChE
BuChE
DA
Acetylcholinesterase
Butyrylcholinesterase
Dopamine
DMSO
DTL
Dimethyl sulfoxide
Dual Target Ligands
Doxorubicin
DX
H3
R
Histamine H receptor
3
HEK293
i.p.
Human embryonic kidney
Intraperitoneal
MAO B
MTDL
PD
Monoamine oxidase B
Multi-Target-Directed Ligands
Parkinson’s disease
β-phenylethylamine
Salsolinol
PEA
SAL
s.c.
Subcutaneous
TLC
Thin-layer Chromatography
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