Synthesis and activity of di- or trisubstituted N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives on the central nervous system

Article abstract of DOI:10.1016/j.bmcl.2018.04.059

Aim of the study was evaluation of anxiolytic, antidepressant, anticonvulsant and analgesic activity in a series of a consistent group of compounds. A series of eleven new N-(phenoxyalkyl)- or N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives has been obtained. Their affinity towards 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, D₂ and α₁ receptors has been assessed, and then functional assays were performed. The compounds were evaluated in mice, i.p. for their antidepressant-like (forced swim test), locomotor, anxiolytic-like (four-plate test) activities as well as – at higher doses – for anticonvulsant potential (MES) and neurotoxicity (rotarod). Two compounds (3, 6) were also evaluated for their analgesic activity in neuropathic pain models (streptozocin test, oxaliplatin test) and they were found active against allodynia in diabetic neuropathic pain at 30 mg/kg. Among the compounds, anxiolytic-like, anticonvulsant or analgesic activity was observed but antidepressant-like activity was not. One of the two most interesting compounds is 1-{2-[2-(2,4,6-trimethylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine dihydrochloride (9), exhibiting anxiolytic and anticonvulsant activity in mice, i.p. 30 min after administration (at 2.5 mg/kg and ED₅₀ = 26.33 mg/kg, respectively), which can be justified by the receptor profile: 5-HT_1A K_i = 5 nM (antagonist), 5-HT₇ K_i = 70 nM, α₁ K_i = 15 nM, D₂ K_i = 189 nM (antagonist). Another interesting compound is 1-[3-(2,4,6-trimethylphenoxy)propyl]-4-(4-methoxyphenyl)piperazine dihydrochloride (3), exhibiting anxiolytic, anticonvulsant and antiallodynic activity in mice, i.p., 30 min after administration (at 10 mg/kg, ED₅₀ = 23.50 mg/kg, at 30 mg/kg, respectively), which can be related with 5-HT_1A weak antagonism (K_i = 146 nM), or other possible mechanism of action, not evaluated within presented study. Additionally, for the most active compound in the four-plate test (7), molecular modeling was performed (docking to receptors 5-HT_1A, 5-HT_2A, 5-HT₇, D₂ and α_1A).

Full text of DOI:10.1016/j.bmcl.2018.04.059

Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and activity of di- or trisubstituted N-(phenoxyalkyl)- or
N-{2-[2-(phenoxy)ethoxy]ethyl}piperazine derivatives on the central
nervous system
a,
b
b
b
b
b
⇑
´
Katarzyna Panczyk , Karolina Pytka , Magdalena Jakubczyk , Anna Rapacz , Kinga Sałat , Anna Furgała ,
c
c
c
d
d
_
´
´
Agata Siwek , Monika Głuch-Lutwin , Anna Grybos , Karolina Słoczynska , Elzbieta Pe˛kala ,
e
e
e
a
a
_
_
Paweł Zmudzki , Adam Bucki , Marcin Kołaczkowski , Dorota Zelaszczyk , Henryk Marona ,
Anna M. Waszkielewicz a
a Department of Bioorganic Chemistry, Chair of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
b Department of Pharmacodynamics, Chair of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
c Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
d Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
e Department of Medicinal Chemistry, Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Aim of the study was evaluation of anxiolytic, antidepressant, anticonvulsant and analgesic activity in a
Received 18 January 2018
Revised 21 April 2018
Accepted 24 April 2018
Available online xxxx
series of a consistent group of compounds. A series of eleven new N-(phenoxyalkyl)- or N-{2-[2-(phe-
noxy)ethoxy]ethyl}piperazine derivatives has been obtained. Their affinity towards 5-HT1A, 5-HT2A
,
5-HT6, 5-HT7, D2 and a1 receptors has been assessed, and then functional assays were performed. The
compounds were evaluated in mice, i.p. for their antidepressant-like (forced swim test), locomotor,
anxiolytic-like (four-plate test) activities as well as – at higher doses – for anticonvulsant potential
(MES) and neurotoxicity (rotarod). Two compounds (3, 6) were also evaluated for their analgesic activity
in neuropathic pain models (streptozocin test, oxaliplatin test) and they were found active against allo-
dynia in diabetic neuropathic pain at 30 mg/kg. Among the compounds, anxiolytic-like, anticonvulsant or
analgesic activity was observed but antidepressant-like activity was not. One of the two most interesting
compounds is 1-{2-[2-(2,4,6-trimethylphenoxy)ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine dihy-
drochloride (9), exhibiting anxiolytic and anticonvulsant activity in mice, i.p. 30 min after administration
(at 2.5 mg/kg and ED50 = 26.33 mg/kg, respectively), which can be justified by the receptor profile: 5-HT1A
Keywords:
5-HT
D2
a1
Allodynia
Docking
Forced swim test
Four-plate test
Hyperalgesia
MES
Ki = 5 nM (antagonist), 5-HT7 Ki = 70 nM, a1 Ki = 15 nM, D2 Ki = 189 nM (antagonist). Another interesting
compound is 1-[3-(2,4,6-trimethylphenoxy)propyl]-4-(4-methoxyphenyl)piperazine dihydrochloride (3),
exhibiting anxiolytic, anticonvulsant and antiallodynic activity in mice, i.p., 30 min after administration
(at 10 mg/kg, ED50 = 23.50 mg/kg, at 30 mg/kg, respectively), which can be related with 5-HT1A weak
antagonism (Ki = 146 nM), or other possible mechanism of action, not evaluated within presented study.
Additionally, for the most active compound in the four-plate test (7), molecular modeling was performed
Piperazine derivatives
(docking to receptors 5-HT1A, 5-HT2A, 5-HT7, D2 and a1A).
Ó 2018 Published by Elsevier Ltd.
Central nervous system disorders, such as depression, anxiety,
epilepsy and neuropathic pain are sometimes comorbid.1 The rea-
son of this phenomenon might be their common etiology, includ-
ing trauma, intoxication, neurodegeneration or genetic factors.2–4
The mentioned disorders also share common treatments, e.g. some
anticonvulsants such as carbamazepine, valproates, or gabapentin
improve mood. On the other hand, tianeptine may cause depres-
sion and seizures can be caused by maprotiline and clomipramine.
Among antidepressants, drugs such as venlafaxine and selective
serotonin reuptake inhibitors are considered relatively safe.5,6 It
has been reported that serotonergic system impairment may be
another common factor for these diseases, which may justify the
above mentioned phenomena.7,8
Taking into account possible multidirectional activity of newly
designed compounds within CNS seems particularly important in
⇑
Corresponding author.
E-mail address: katarzyna.panczyk@uj.edu.pl (K. Pan´ czyk).
https://doi.org/10.1016/j.bmcl.2018.04.059
0960-894X/Ó 2018 Published by Elsevier Ltd.
Please cite this article in press as: Pan´ czyk K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.059

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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
2
terms of drug design. Providing drugs targeting more than one
comorbid disorder would allow to reduce polypragmasy and a risk
of side effects. On the other hand, including research on various
activities within CNS on early stages of drug development would
prevent unexpected off-target activities and adverse effects when
aiming in treatment of one particular disorder.9 Due to involve-
ment of serotonergic system dysregulation in several central ner-
vous system disorders, including broad pharmacological research
is particularly important in case of serotonergic receptors ligands.
ergic receptors and additionally towards receptors D2 and a1.
Based on our former research including structure-activity relation-
ship as well as on literature review, the serotonergic mechanism of
action seems to be the most probable one in case of compounds of
interest. Thus, it was decided to include the multiplicity of seroton-
ergic system influence on major depressive disorder (MDD),22 sei-
zures, neuropathic pain and anticonvulsant activity. Within
current study, in vitro serotonergic (5-HT1A, 5-HT2A, 5-HT6,
5-HT7), D2 and a1 receptors binding assays and in vivo
pharmacological tests were planned. We expected presented
phenylpiperazine derivatives to be multitarget centrally active
compounds with mechanism of action basing mainly on serotoner-
Phenyl
piperazine
(including
particularly
2-
methoxyphenylpiperazine) derivatives constitutes a group of com-
pounds widely investigated in terms of serotonergic activity.10–13
Thus, this group of compounds is of interest regarding search for
multidirectional agents.
gic modulation. Receptors a1 were incorporated due to known fact
that compounds possessing affinity towards 5-HT1A often exhibit
an off-target activity towards receptors a1 and there are many
potent a1 ligands among phenylpiperazine derivatives.23,24 More
than one type of central activity in vivo was expected, basing on
our previous experience with this group of compounds as well as
potential multitarget activity.
Preliminary experiments with the use of serotonergic antago-
nist WAY100635 indicated that mechanism other than serotoner-
gic might be involved in case of some phenylpiperazine
derivatives synthesized before in our laboratory, however particu-
lar targets have been not defined yet.7 For that reason, we decided
to test all compounds in vivo, regardless their receptor profile, in
order to correlate serotonergic receptor profile with in vivo activity.
Potent central activity of close structural analogs served as a
During our ongoing studies in terms of design and synthesis of
new centrally active compounds, derivatives exhibiting anticon-
vulsant, analgesic and/or antidepressant-like activity have been
synthesized. Therefore, inclusion of analysis of all these activities
constitute natural consequence in our research. In order to design
molecules which may possibly be active in treatment of the above
disorders, we based on our former findings in groups of derivatives
of N-(2-methoxyphenyl)piperazine, as well as phenoxyalkyl- or
phenoxyethoxyethyl derivatives of piperazine,14–19 where we
found reference compounds I–IV for this study (Fig. 1), or of
aminoalkanols.20,21
The majority of active compounds which have been obtained in
our laboratory so far, possessed affinities towards various seroton-
Fig. 1. Chemical structures of reference drugs (urapidil14,25 and hydroxyzine) as well as compounds I,14,15 II,16,17 III,18 IV.15,16.
Please cite this article in press as: Pan´ czyk K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.059

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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
premise for such investigations. The results will be indication for
our future studies in terms of rationality of including receptors
other than serotonergic in searching for compounds’ mechanism
of action.
The aim of our study was design, synthesis and examination of
receptor binding, functional assays (for agonism or antagonism)
and evaluation of pharmacological activity in a group of new phe-
noxyalkyl or phenoxyethoxyethyl derivatives of piperazine, where
the other substituent in the piperazine ring may significantly
improve the demanded activity. Another aim was to find possible
correlation between the receptor profiles of the compounds and
their activity on the central nervous system.
This study presents eleven di- or trisubstituted compounds
such as: 2,4-(CH3)2-, 4-Cl-3-CH3-, 2,4,6-(CH3)3-phenoxyalkyl as
well as 2,3-(CH3)2-, 2,3,5-(CH3)3- or 2,4,6-(CH3)3-phe-
noxyethoxyethyl derivatives of piperazine (Table 1).
It was decided to perform all pharmacological testing on the
same species (mice), using the same route of administration
(intraperitoneal i.p.), and at the same time after administration
(30 min), in order to receive comparable results and enable proper
conclusions. However, we needed to take into account different
doses for in vivo testing of activity on anxiety or mood disorders
and seizures, where typically higher doses are used for screening.
The volumes range 326.87–405.85 Å3. None of the compounds
exhibited any violations from the Lipinski rule of five,28 which pre-
sumes that they are potential drug-like agents.
All title compounds (Table 1) were obtained via multistep
chemical synthesis (Scheme 1). The detailed description of reaction
conditions as well as physicochemical properties of obtained prod-
ucts are available in Supplementary materials.
Receptor studies
The synthesized compounds were subject to receptor binding
studies for 5-HT1A, 5-HT2A, 5-HT6, 5-HT7, as well as D2, and a1
receptors. The assay was executed according to previously
described protocol.29 The results are presented in Table 2. The most
advantageous binding properties were observed for compounds 1,
4, 7, and 9, where nanomolar affinities have been achieved. Among
them, compounds 1, 7 and 9 are substituted with 2-methoxy group
in phenylpiperazine moiety. The advantageous impact of such
modification is consistent with literature and our former findings.
Comparing the results with the reference compounds, com-
pound 1 is 2,4-dimethyl analog of the reference compound I and
exhibits comparable receptor binding (e.g. for compound 1 Ki =
2.0 nM towards 5-HT1A and Ki = 15 nM towards 5-HT7, while for
compound I Ki = 0.7 nM towards 5-HT1A and Ki = 26 nM towards
5-HT7). Compound 7 – the 2,3,5-trimethyl analog of III – exhibits
slightly higher binding affinity towards a1 receptors compared to
the reference III (Ki = 14 nM vs Ki = 28.5 nM), but lower toward
5-HT1A (Ki = 32 nM vs Ki = 5 nM). Compound 9 as the 2,4,6-tri-
methyl analog of III exhibits the same affinity toward 5-HT1A
receptors (Ki = 5.0 nM towards 5-HT1A) and slightly better affinity
toward a1 receptors (Ki = 15.0 nM for compound 9 vs Ki = 28.5 for
compound III). It also should be noticed that compounds 6 and 8
as hydroxyethyl analogs of 7 and 9, respectively, do not exhibit
such advantageous receptor profiles.
Chemistry
Eleven derivatives of N-phenoxyalkyl- (1–4) and N-{2-[2-(phe-
noxy)ethoxy]ethyl}piperazine (5–11) in the forms of hydrochlo-
rides have been obtained. Compounds disubstituted in the
phenyl ring with two methyl groups have been extensively studied
within our previous research (e.g. reference compounds I–III). We
also obtained several trisubstituted compounds, among others ref-
erence compound IV, which exhibited favorable receptor profile
and interesting in vivo activity. The design of compounds within
current study based on structural modification of reference com-
pounds I–IV, including change of phenyl ring’s substitution posi-
tions (in case of compound 4 – incorporating chlorine as one of
substituents), manipulation with linker longevity and modification
of arylpiperazine substituent. In case of compounds 2, 6 and 8 an
aryl moiety has been removed from amine component in order
to obtain structural derivatives of hydroxyzine.
Upon completion of affinity assays, functional studies were
performed for relevant compounds (compounds 1, 4, 7, and
9 – 5-HT1A, 5-HT2A, 5-HT7 and D2 receptors, compounds 3 and
11 – 5-HT1A receptor and compound 5 – 5-HT2A receptor).
Antagonistic properties were proved for the majority of performed
evaluations. The exception was weak agonistic properties of
compound 4 towards receptor D2. The data are presented in
Supplementary materials.
Calculation of several physicochemical parameters by means of
Molinspiration online toolkit was an important part in our
research.26 Such calculations are useful at early stage of drug dis-
covery process in terms of predicting essential parameters and
facilitating rational drug design. The parameters include logP (for
lipophilicity), topological polar surface area TPSA, molecular vol-
ume, and violations from the Lipinski rule of five. The structures
and the results are presented in Table 1. The first calculated param-
eter miLogP was found in the range 2.38–5.14, while in the litera-
ture the optimal value for CNS active compounds is about 2–3.27
Our experience with phenylpiperazine derivatives indicates that
in case of this group of compounds deviations from this rule are
possible. We previously obtained centrally active compounds
which were relatively safe in preliminary examinations and exhib-
ited logP values >4, e.g. reference compounds I–IV (Fig. 1). For that
reason, the design of current study did not involve logP value as a
decisive parameter in design of compounds. However, logP values
were calculated for providing information and possible discussion
of relationship between lipophilicity and central activity of pre-
sented compounds. The most advantageous value of TPSA should
be <120 Å2 for orally administered drugs and <60–70 Å2 for com-
pounds designed to penetrate blood-brain barrier. The proposed
structures are consistent with both the rules for oral drugs and
for CNS drugs (calculated values are in the range 15.71–45.17 Å2).
Pharmacology
All compounds were screened for antidepressant-like and anx-
iolytic-like properties in vivo. For this purpose we used the forced
swim test in mice30,31 and the four-plate test in mice,17,32 respec-
tively. The results are presented in Table 3. Influence on locomotor
activity in mice was evaluated in 1-min session22 (Table 4).
Compounds 2–3 and 7–10 exhibited anxiolytic-like properties,
while compounds 1, 4–6, and 11 were inactive in the four-plate
test. Compound 2 (2-(2-{4-[2-(Mesityloxy)ethyl]piperazin-1-yl}
ethoxy)ethanol dihydrochloride) and
9 (1-{2-[2-(mesityloxy)
ethoxy]ethyl}-4-(2-methoxyphenyl)piperazine dihydrochloride)
were active at the doses 2.5 and 5 mg/kg (p < 0.05) and compound
7 (2,3,5-trimethyl substituted analog of 9) at the doses 2.5 (p <
0.0001) and 5 mg/kg (p < 0.01), which was the strongest effect
demonstrated in this group of compounds in this test. Compound
3, 8, and 10 exhibited anxiolytic-like activity at the dose 10 mg/
kg, while the reference clorazepate – at the dose 1.25 mg/kg.
As neither of the compounds affected locomotor activity in mice
at anxiolytic-like doses, the observed effect was specific.
Anticonvulsant evaluation was performed independently from
results of receptor and antidepressant-like or anxiolytic-like activ-
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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
4
Table 1
Structures of the title compounds 1–11.
Compd.
R1
n
3
R2
m
1
miLogPa
4.72
TPSA (Å2)
24.94
Volume (Å3)
355.44
1
2,4-(CH3)2
2
3
2,4,6-(CH3)3
4-Cl-3-CH3
2
3
2
2
2.38
5.14
45.17
24.94
342.46
372.00
4
3
2
4.94
15.71
326.87
Compd
R1
R2
m
2
miLogPa
4.41
TPSA (Å2)
24.94
Volume (Å3)
389.29
5
2,3-(CH3)2
6
7
2,3,5-(CH3)3
2
2
2.38
4.71
45.17
34.18
342.46
397.79
8
9
2,4,6-(CH3)3
2
2
2.38
4.71
45.17
34.18
342.46
397.79
10
11
2
2
4.41
4.81
24.94
24.94
389.05
405.85
a
Calculations performed with use of Molinspiration online toolkit for base forms.26
ity. Typical mode of search for anticonvulsants is performed with
use of in vivo screening. Previously, we performed screening
research related with this group of compounds, on the basis of
some active compounds in depression or anxiety, and in the PTZ
test we did not find them active although we saw some activity
in MES.33 Therefore, this time anticonvulsant activity was screened
only in MES at 30 mg/kg b.w. (mice, i.p.) and in case of positive
results ED50 was derived. Concomitantly, rotarod test was per-
formed for neurotoxicity assessment.
The assays were performed for compounds 1–7, 9, and 11
(Table 5), according to procedure described by Löscher et al.34
together with neurotoxicity evaluation by standard rotarod pro-
cedure.35 The most promising compounds 2–3, 5–6, 9, and 11
were subject to quantitative assays, resulting in ED50 for MES
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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Scheme 1. Synthesis of the title compounds. a: Cl-(CH2)n-OH, K2CO3, acetone/EtOH; b: PBr3; c:
, K2CO3, toluene, or N-methylmorfoline, DMF; d: gaseous
HCl, EtOH; e: Br-(CH2)n-Cl, K2CO3, acetone; f: Cl-(CH2)2-O-(CH2)2-OH, K2CO3, acetone/EtOH; R1, R2, n, m as in Table 1.
Table 2
Binding results for the title compounds 1–11 and reference drugs.
Compound
Ki (nM)
5-HT1A
5-HT2A
5-HT6
5-HT7
D2
a1
[3H] – 8-OH-DPAT
[3H] – ketanserin
[3H] – LSD
[3H] – LSD
[3H] – methylspiperone
[3H] – prazosin
1
2
3
4
5
6
7
8
2.0 0.2
3168.0 112.0
146.0 28.4
20.0 1.2
–
–
527.0 4.0
j
>5000
–
>5000
>5000
200.0 3.5
>5000
1540.0 103.0
>5000
15.0 3.0
–
97.0 3.6
11.0 1.0
–
2117.0 180.0
93.0 3.0
36.0 1.1
–
14.0 0.6
–
j
772.0 45.2
76.0 2.7
220.0 7.3
>5000
3027.0 208.0
90.0 8.0
388.0 7.0
3142.0 218.0
89.0 10.0
–
4963.0 479.0
299.0 9.5
1206.0 13.5
–
162.0 10.3
–
32.0 3.6
–
159.0 12.4
>5000
9
10
11
5.0 0.6
–
552 14.0
4.0 0.4
j
j
j
245.0 5.5
498.0 135
2805.0 249.0
j
2427.0 270.0
>5000
2155.0 129.0
0.9 0.07
j
j
j
70.0 0.5
3160.0 34.5
1050.0 54.0
1.7 0.2
j
189.0 19.3
–
–
15.0 1.5
–
772.0 29.5
Methiothepin
Mianserin
Haloperidol
Phentolamine
j
j
3.2 0.2
j
j
j
j
5.9 0.2
j
j
j
j
12.0 0.6
– No binding at 10ꢀ5 M; j no data.
and TD50 in rotarod. Both ED50 and TD50 values with 95% confi-
dence limits were calculated by probit analysis.36 Further deter-
mination of protection index (PI = TD50/ED50) indicated that the
most favorable neuronal stabilizing properties and safety were
observed for compounds 2, 3, 6, and 11 (PIs 1.88, 2.89, 3.07
and 2.96, respectively).
It was decided that within the tested group of piperazine
derivatives, two compounds with the most favorable PIs and MES
ED50 about 20 mg/kg, were chosen for further evaluation of activity
in neuropathic pain models: 3 and 6 (i.e. 1-[1-(2,4,6-trimethylphe-
respectively.37–39 The results are presented in Figs. 2–5 and they
indicate that both compounds are active in mechanical allodynia
in STZ test at the higher dose 30 mg/kg, but not in thermal hyper-
algesia (hot plate or cold plate tests). Both compounds differ struc-
turally in terms of substitution in the phenyl ring (although they
are both trisubstituted), they have different linker between phe-
noxy group and piperazine (propyl or ethoxyethyl) and the piper-
azine moiety contains different substituent at the distal nitrogen
(4-methoxyphenyl or 2-hydroxyethyl).
noxy)propyl]-4-(4-methoxyphenyl)piperazine
dihydrochloride
and 1-{2-[2-(2,3,5-trimethylphenoxy)ethoxy]ethyl}-4-(2-hydrox-
yethyl)piperazine dihydrochloride, respectively).
Molecular modeling
Evaluation of antiallodynic or antihyperalgesic activity of com-
pounds 3 and 6 in mouse models of neuropathic pain was realized
with use of streptozocin test (STZ) and oxaliplatin test (OXA). The
mechanism of deterioration of nerves in the streptozocin model
resembles the process of human diabetic neuropathy.37 Evaluation
of antiallodynic activity was performed with use of von Frey test.
Moreover, activity against hyperalgesia was also tested in both
STZ and OXA models – with use of hot plate or cold plate test,
The final step of our research was the docking study of com-
pound 7. Despite it was not the one possessing the strongest affini-
ties for the presented receptors, it was the most active anxiolytic
agent among compounds 1–11 tested in vivo. Putative binding
mode of compound 7 was determined by docking to homology
models of the 5-HT1A, 5-HT2A and 5-HT7 serotonin receptors, as
well as for the dopamine D2 and adrenergic a1A receptors, per-
formed according to the published procedures.40–45 The GPCR
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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
6
Table 3
Antidepressant-like and anxiolytic-like activity of 1–11 in mice, i.p.
Compound
Forced swim test (antidepressant-like activity)
Four-plate test (anxiolytic-like activity)
Dose
(mg/kg)
Immobility time
SEM (s)
F statistic (degrees
of freedom)
Dose
(mg/kg)
Number of punished
crossings SEM (s)
F statistic (degrees
of freedom)
Vehicle
1
–
5
10
20
–
164.0 11.7
182.6 17.3
198.1 8.3
197.3 9.9
159.6 19.6
152.5 17.6
135.3 7.0
128.0 15.9
194.5 18.5
179.9 18.5
202.0 13.2
183.9 13.8
201.9 16.0
189.1 14.2
188.9 18.0
172.1 11.6
153.9 11.7
157.6 16.3
161.1 11.7
162.1 10.0
190.9 16.1
171.8 15.7
184.9 15.6
176.4 12.7
163.9 6.7
165.6 19.1
141.9 21.9
156.1 20.4
164.0 11.7
182.6 17.3
198.1 8.3
197.3 9.9
166.9 20.2
138.5 17.9
131.3 14.5
150.6 20.8
194.5 18.5
176.5 9.6
182.4 14.1
157.5 22.8
156.5 21.1
170.5 13.4
173.8 19.5
178.8 10.9
178.4 9.1
152.1 12.9
106.6 9.8***
2.6 0.3
2.1 0.4
1.5 0.5
F(3,28) = 1.674, ns
5
10
F(2,21) = 1.736, ns
Vehicle
2
–
–
3.3 0.4
3.3 0.4
5.5 0.5*
5.0 0.5*
3.9 0.2
3.8 0.4
7.5 0.9***
5
F(3,28) = 1.114, ns
1.25
2.5
5
–
5
F(3,30) = 5.807, p < 0.01
F(2,21) = 14.02, p < 0.001
F(2,21) = 3.190, ns
10
20
–
Vehicle
3
–
5
F (3,28) = 0.388, ns
10
20
–
10
Vehicle
4
–
5
10
3.0 0.3
2.8 0.4
1.9 0.3
5
F(3,28) = 0.6499, ns
F(3,28) = 0.088, ns
F(3,28) = 0.141, ns
F(3,28) = 0.3591, ns
F(3,28) = 1.575, ns
F(3,36) = 0.7080, ns
F(3,28) = 0.828, ns
F(3,28) = 0.324, ns
F(2,27) = 11.65, p < 0.001
10
20
–
Vehicle
5
–
5
10
3.5 0.5
3.8 0.5
2.9 0.4
5
F(2,21) = 0.938, ns
10
20
–
Vehicle
6
–
5
10
2.8 0.4
2.8 0.3
3.0 0.5
5
F(2,21) = 0.769, ns
10
20
–
Vehicle
7
–
3.1 0.5
5
1.25
2.5
5
–
5
3.0 0.4
F(3,32) = 11.89, p < 0.0001
F(2,29) = 15.31, p < 0.0001
F(3,36) = 6.254, p < 0.01
F(2,21) = 12.24, p < 0.001
10
20
–
6.1 0.4****
5.1 0.4**
4.4 0.4
Vehicle
8
5
3.3 0.5
10
20
–
10
7.4 0.7***
Vehicle
9
–
3.1 0.4
3.1 0.3
4.6 0.5*
4.9 0.3*
3.6 0.2
3,6 0.9
8.1 0.9***
5
1.25
2.5
5
–
5
10
20
–
Vehicle
10
5
10
20
–
10
Vehicle
11
–
5
10
3.6 0.4
3.9 0.6
4.0 0.3
5
10
20
–
10
15
F(2,21) = 0.176, ns
Vehicle18
Fluoxetine18
Vehicle
Clorazepate
–
3.0 0.3
3.3 0.3
4.8 0.5*
0.625
1.25
F(2,21) = 5.574, p < 0.0001
All studied compounds and fluoxetine or clorazepate were administered 30 min before the test. Vehicle-treated groups received 0.9% NaCl. The values are expressed as mean
SEM, n = 8–10 mice per group. Statistical analysis: one-way ANOVA (Newman-Keuls post hoc) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. respective vehicle-treated
group, ns – not significant.
structures were represented by several homology models,46,29,47
which simulated conformational flexibility of the proteins. Visual
inspection of the resulting complexes allowed us to capture crucial
ligand-receptor interactions responsible for binding to the biolog-
ical targets.
The predicted binding mode of 7 was considered representative
for other derivatives characterized by high affinity for the main
biological targets in vitro. The molecule posed extended conforma-
tion across the two cavities forming the binding sites in
monoaminergic G protein-coupled receptors. The main anchoring
interactions, important for affinity for the selected receptors,
involved the phenylpiperazine moiety: (i) a charge-reinforced
hydrogen bond between the protonated nitrogen atom and the car-
Phe6.52 (Fig. 6). The compounds lacking the above-mentioned
moiety were unable to bind effectively to the receptors (Table 2).
The substituted N-[(2,3,5-trimethylphenoxy)ethoxyethyl] frag-
ment of the molecule occupied the opposite cavity and found
favorable aromatic/hydrophobic interactions there, which were
distinctive for each receptor type. In the case of serotonin recep-
tors, the 5-HT1A receptor interacted with the phenyl ring of
Tyr2.64 (
with Phe3.28 (CH-
p
–
p
stacking, Fig. 6A), whereas in the 5-HT7 receptor –
interactions, Fig. 6B). In the 5-HT2A receptor
p
site, the latter fragment formed VdW interactions with hydropho-
bic interface of Ile2.65, Trp3.28, Val7.39 and Tyr7.43 (Fig. 6C). The
latter binding mode was similar to the one in the dopamine D2
receptor (Leu2.65, Phe3.28, Val3.29 and Trp71 from extracellular
boxyl group of Asp3.32, as well as (ii) the CH-
p
stacking with
loop 1, Fig. 6D). The compound bounds to the adrenergic a1A recep-
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´
7
K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Table 4
Influence of the studied compounds on locomotor activity of mice in 1-min session.
Compound
Dose
(mg/kg)
Number of crossings
SEM (s)
t statistic (degrees
of freedom)
Vehicle
2
Vehicle
3
Vehicle
7
Vehicle
8
Vehicle
9
–
5
–
10
–
5
–
10
–
5
68.4 12.1
41.4 8.6
69.0 13.7
41.1 10.0
68.4 12.1
51.0 9.7
69.0 13.7
54.4 7.4
72.2 9.7
66.2 8.2
69.0 13.7
68.5 14.2
t(14) = 1.821, ns
t(14) = 1.640, ns
t(14) = 1.122, ns
t(14) = 0.937, ns
t(18) = 0.4732, ns
t(14) = 0.025, ns
Vehicle
10
–
10
All studied compounds were administered i.p. 30 min before the test. Vehicle-
treated groups received 0.9% NaCl. The values are expressed as mean SEM, n = 8–
10 mice per group. Statistical analysis: Student t-test; ns – not significant.
Fig. 2. Evaluation of mechanical nociceptive threshold with use of von Frey test for
compounds 3 and 6 in STZ test. Results are presented as average force against the
plantar surface of the hind paw of the mouse [g] ( SEM), causing pain reaction.
Statistical analysis: one-way ANOVA and Tukey’s post hoc test. Statistical signifi-
cance compared to control group: ###p < 0.001 and compared to measurement
before administration of compound: ***p < 0.001.
tors via
p–p stacking with Tyr2.64 and cation-p interaction with
Lys7.39 (Fig. 6E). The presented simulations provided retrospective
explanation of SAR data and support for future rational design of
multifunctional ligands.
The most important interactions of the tested ligand were in
line with the common binding mode for monoaminergic receptor
ligands determined experimentally,48 and with the results of our
previous studies49 which demonstrated the accuracy of the predic-
tions based on the developed homology models.
and/or antiallodynic activities. Interestingly, in vivo activity was
not always convergent with preferred receptor profile. For that rea-
son it seems reasonable to discuss SAR in terms of receptor profile
and in vivo activity independently.
Within presented series, all compounds possessing phenyl ring
connected directly to piperazine moiety were characterized with
interesting receptor profiles, while other compounds lacked affin-
ity towards tested receptors (apart from compound 5, which binds
Structure-activity relationship
Nine out of eleven synthesized compounds exhibited pharma-
cological activity in vivo, including anxiolytic, anticonvulsant
to receptors a1 and possesses a linker between piperazine and phe-
nyl ring). The results of molecular modeling performed for com-
pound 7 serve for an explanation, showing that phenyl ring takes
part in ligand binding in case of all receptors of interest. Its pres-
ence and proper distance from nitrogen atom of piperazine seem
to be crucial for binding of compound 7 and it might be also impor-
tant when discussing other active compounds.
All compounds binding to receptors 5-HT1A possess also affinity
towards receptors a1, apart from compound 3 – 4-methoxy analog
of reference compound IV. Such a selectivity of compound 3 is
Table 5
Anticonvulsant activity of compounds 1–7, 9 and 11 (mice, i.p.).
Compd. Dose
(mg/kg)
MESa Deaths NTb ED50
(10 (confidence (confidence
TD50
PI
rpm) interval)
(mg/kg)
interval)
(mg/kg)
Control
1
2
–
30
100
60
30
100
45
30
15
30
100
45
30
15
100
30
20
15
30
40
30
20
100
30
20
15
0/6
1/6
6/6
3/6
1/6
6/6
5/6
3/6
2/6
1/6
3/3
6/6
3/6
1/6
6/6
4/6
2/6
1/6
1/6
5/6
3/6
2/6
4/6
0/6
0/6
1/6
2/6
0/6
0/6
0/6
0/6
2/6
0/3
0/6
0/6
2/6
0/6
0/6
1/6
2/6
0/6
0/6
0/6
0/6
1/1
0/6
0/6
0/6
1/6
4/6
45.42
85.26 (75.16– 1.88
96.71)
(37.17–
55.51)
23.50
(13.65–
40.45)
1/6
3/6
2/6
1/6
0/6
3/6
3/3
4/6
2/6
1/6
4/6
1/6
3
67.88 (38.47– 2.89
119.77)
4
5
22.19
(18.36–
26.82)
35.50 (20.77– 1.60
60.67)
6
24.39
(17.66–
33.67)
74.92 (42.22– 3.07
132.94)
7
9
3/6
4/6
3/6
1/6
26.33
31.48 (22.86– 1.19
43.35)
(18.85–
36.78)
18.00
(16.12–
20.11)
11
53.33 (49.47– 2.96
57.48)
6/6
4/6
1/6
0/6
a
Fig. 3. Evaluation of thermal nociceptive threshold in diabetic mice – hot plate test
for compounds 3 and 6 in STZ model. Results are presented as average latency of
pain reaction [s] ( SEM). Statistical analysis: one-way ANOVA and Tukey’s post hoc
test.
The data indicate: number of mice protected against MES seizures/number of
mice tested.
b
The data indicate: number of mice in which motor impairment was observed/
number of mice tested.
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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
8
noteworthy, as search for 5-HT1A ligands devoid off-target
a1 affin-
worth noted that this is the only compound possessing chlorine
as a substituent in the phenyl ring.
In terms of correlation of receptor profile and in vivo activity as
well as SAR regarding pharmacological studies, the following
observations can be made.
ity is an important scientific goal in terms of drug design.50
Compounds 1, 4, 7 and 9 possessed notable affinity towards
receptors 5-HT7, 4 and 7 – towards 5-HT2A, while 1 and 7 – D2.
However, it is difficult to draw extensive conclusions regarding
SAR.
Compound
2
(2-(2-{4-[2-(mesityloxy)ethyl]piperazin-1-yl}
Functional studies showed antagonistic properties of tested
compounds in all performed evaluations, with the exception of
compound 4 being weak agonist. As the affinity of this compound
towards receptors D2 is not very high (Ki = 299 nM), this activity
might not be the crucial one for in vivo activity. Nevertheless, it
ethoxy)ethanol dihydrochloride) exhibits no particular affinity
towards the tested receptors, and still shows anxiolytic-like activ-
ity at 2.5 mg/kg (mice, i.p.) as well as anticonvulsant properties in
MES test with ED50 = 45.42 mg/kg (mice, i.p.) at neurotoxicity in
rotarod test with TD50 = 85.26 mg/kg (mice, i.p.). This compound
Fig. 4. Evaluation of mechanical nociceptive threshold with use of von Frey test for 3 and 6 in OXA test. Results are presented as average force against the plantar surface of
the hind paw of the mouse [g] ( SEM), causing pain reaction. Statistical analysis: ANOVA with repeated measurements and Bonferroni’s post hoc test. Statistical significance
compared to control group: ###p < 0.0001.
Fig. 5. Evaluation of thermal nociceptive threshold in oxaliplatin-treated mice – cold plate test for 3 and 6. Results are presented as average latency of pain reaction [s]
( SEM). Statistical analysis: one-way ANOVA with repeated measurements and Bonferroni’s post hoc test. Statistical significance compared to control group: ####p < 0.0001.
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K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
Fig. 6. Binding modes of compound 7 in the sites of serotonin 5-HT1A (A), 5-HT7 (B) and 5-HT2A (C) receptors, as well as the dopamine D2 and the adrenergic
a1A receptors.
Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are displayed as sticks, whereas those forming H-bonds (dotted yellow lines),
p–p stacking
(dotted cyan lines) and cation- stacking interactions (dotted green lines) are represented as thick sticks. For the sake of clarity, a part of TMH5 and extracellular loop 2
p
residues were undisplayed. TMH – transmembrane helix.
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´
K. Panczyk et al. / Bioorganic & Medicinal Chemistry Letters xxx (2018) xxx–xxx
10
is also a derivative which contains the same elements as com-
pound 8, but in different order. The structure of 2 with unsubsti-
tuted hydroxyl group of N-hydroxyethoxyethylpiperazine as in
hydroxyzine is more advantageous in terms of pharmacological
profile.
propyl]-4-(4-methoxyphenyl)piperazine dihydrochloride) exhib-
ited anxiolytic, anticonvulsant and antiallodynic activity in mice,
i.p., 30 min after administration (at 10 mg/kg, ED50 = 23.50 mg/
kg, at 30 mg/kg, respectively). The observed coexistence of the
three activities is consistent with assumptions described in the
introduction, however, shows some new perspective on search
for new drugs for the central nervous system diseases. Verification
of other possible activities but the searched for in the process of
drug discovery seems to be advantageous, due to possibility of
finding other desirable properties and increase in knowledge of
pharmacological output of a future drug candidate.
It can be concluded, that among presented compounds possess-
ing affinity towards tested receptors, the possible main mechanism
of action is serotonergic antagonism. However, other molecular
targets, including those not evaluated within current study, might
also be involved.
Five compounds (2, 6, 8, 10 and 11) possessed poor pharmaco-
logical profile, but promising in vivo results, including anxiolytic,
anticonvulsant and antiallodynic activities. For that reason, it
seems reasonable to include other than serotonergic, dopaminergic
and adrenergic molecular targets in our future research in this
group of compounds, especially in case of derivatives which do
not possess phenyl ring connected directly to piperazine moiety.
Compound
3
(1-[3-(2,4,6-trimethylphenoxy)propyl]-4-(4-
methoxyphenyl)piperazine dihydrochloride) exhibits binding to
5-HT1A receptors (Ki = 146 nM) and anticonvulsant properties
(MES ED50 = 23.50 mg/kg at TD50 = 67.88 mg/kg). Due to the broad
PI it was subject to evaluation of analgesic activity, where it proved
active in mechanical allodynia at 30 mg/kg (STZ model). The anal-
ogy with reference compound I shows that incorporation of methyl
substituent in position 4 of the phenyl ring and using the methoxy
substituent not in position 2 but 4 in phenyl at piperazine, changes
completely the biological properties of the obtained compound.
Incorporation of phenethylpiperazine as in compound 5 ((2,3-
dimethylphenoxy)ethoxyethyl derivative) produced affinity to a1
receptors, anticonvulsant activity, but not satisfying protection
index (PI = 1.60) in the obtained compound. On the contrary, 11
as also a phenethylpiperazine derivative of (2,4,6-trimethylphe-
noxy)ethoxyethyl exhibits much broader protection index (PI =
2.96) at its anticonvulsant evaluation, disregard of not satisfying
affinity towards the tested receptors.
Compound 6 (1-(2-hydroxyethyl)-4-{2-[2-(2,3,5-trimethylphe-
noxy)ethoxy]ethyl}piperazine dihydrochloride), similarly to com-
pound 3, exhibits anticonvulsant and analgesic activity, disregard
of no particular affinity to any of the tested receptors or no similar-
ity to 3.
Acknowledgements
The authors declare no conflict of interest.
The work was financed by the Polish National Science Centre,
decision on grant no. DEC-2013/11/B/NZ7/04834.
Compound
7
(1-(2-methoxyphenyl)-4-{2-[2-(2,3,5-
trimethylphenoxy)ethoxy]ethyl}piperazine dihydrochloride) exhi-
bits affinity towards 5-HT1A (Ki = 32 nM), 5-HT2A (Ki = 159 nM), 5-
HT7 (Ki = 89 nM), and D2 (Ki = 162 nM). It also exhibits anxiolytic-
like properties at 2.5 mg/kg (mice, i.p.).
Compound 9 (1-{2-[2-(mesityloxy)ethoxy]ethyl}-4-(2-methox-
yphenyl)piperazine dihydrochloride) exhibits affinity towards 5-
HT1A (Ki = 5 nM), 5-HT7 (Ki = 70 nM), D2 (Ki=189 nM), a1 (Ki = 15
nM). Its anticonvulsant activity is observed in MES test with
ED50 = 26.33 mg/kg (mice, i.p.) at neurotoxicity observed by
rotarod test with TD50 = 31.48 mg/kg (mice, i.p.).
It is worth noticing that compound 9 is a 2,4,6-trimethylphe-
noxy analog of compound 7 – a derivative of 2,3,5-trimethylphe-
nol, and change of positions of methyl substituents modified the
pharmacological profiles from anxiolytic-like (both derivatives)
to anticonvulsant at elevated dose (2,4,6-trimethyl derivative 9
only).
Compound 9 is also the 4-methyl derivative of reference com-
pound III. Its receptor profile is comparable, but 9 proved more
neurotoxic in vivo since its TD50 in rotarod is much lower than
TD50 for the reference III.
The experimental protocol was approved by the First Local
Ethics Committee on Animal Testing at the Jagiellonian University
in Krakow (No 108/2014, 232/2015 and 233/2015) and was in
accordance with the European Communities Council Directive of
24 November 1986 (86/609/EEC).
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2018.04.059.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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Please cite this article in press as: Pan´ czyk K., et al. Bioorg. Med. Chem. Lett. (2018), https://doi.org/10.1016/j.bmcl.2018.04.059