Novel anilide and benzylamide derivatives of arylpiperazinylalkanoic acids as 5-HT1A/5-HT7 receptor antagonists and phosphodiesterase 4/7 inhibitors with procognitive and antidepressant activity

Article abstract of DOI:10.1016/j.ejmech.2020.112437

A library of novel anilide and benzylamide derivatives of ω-(4-(2-methoxyphenyl)piperazin-1-yl)alkanoic acids as combined 5-HT_1A/5-HT₇ receptor ligands and phosphodiesterase PDE4B/PDE7A inhibitors was designed using a structure-based drug design approach. The in vitro studies of 33 newly synthesized compounds (7-39) allowed us to identify 22 as the most promising multifunctional 5-HT_1A/5-HT₇ receptor antagonist (5-HT_1A K_i = 8 nM, K_b = 0.04 nM; 5-HT₇ K_i = 451 nM, K_b = 460 nM) with PDE4B/PDE7A inhibitory activity (PDE4B IC₅₀ = 80.4 μM; PDE7A IC₅₀ = 151.3 μM). Compound 22 exerted a very good ability to passively penetrate through biological membranes and a high metabolic stability in vitro. Moreover, the pharmacological evaluation of 22 showed its procognitive and antidepressant properties in rat behavioral tests. Compound 22 at a dose of 3 mg/kg (i.p.) significantly reversed MK-801-induced episodic memory deficits in the novel object recognition test, while at a dose of 10 mg/kg (i.p.) reduced the immobility time of animals (by about 34percent) in the forced swimming test. The antidepressant-like effect produced by compound 22 was stronger than that of escitalopram used as a reference drug. This study opens a new perspective in the search for efficacious drugs for the treatment of cognitive and depressive disorders.

Full text of DOI:10.1016/j.ejmech.2020.112437

Journal Pre-proof
Novel anilide and benzylamide derivatives of arylpiperazinylalkanoic acids as
5-HT /5-HT receptor antagonists and phosphodiesterase 4/7 inhibitors with
1A
7
procognitive and antidepressant activity
Agnieszka Jankowska, Grzegorz Satała, Marcin Kołaczkowski, Adam Bucki, Monika
Głuch-Lutwin, Artur Świerczek, Krzysztof Pociecha, Anna Partyka, Magdalena
Jastrzębska-Więsek, Annamaria Lubelska, Gniewomir Latacz, Alicja Gawalska,
Andrzej J. Bojarski, Elżbieta Wyska, Grażyna Chłoń-Rzepa
PII:
S0223-5234(20)30408-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112437
EJMECH 112437
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 March 2020
Revised Date: 5 May 2020
Accepted Date: 6 May 2020
Please cite this article as: A. Jankowska, G. Satała, M. Kołaczkowski, A. Bucki, M. Głuch-Lutwin, A.
Świerczek, K. Pociecha, A. Partyka, M. Jastrzębska-Więsek, A. Lubelska, G. Latacz, A. Gawalska,
A.J. Bojarski, Elż. Wyska, Graż. Chłoń-Rzepa, Novel anilide and benzylamide derivatives of
arylpiperazinylalkanoic acids as 5-HT /5-HT receptor antagonists and phosphodiesterase 4/7
1A
7
inhibitors with procognitive and antidepressant activity, European Journal of Medicinal Chemistry (2020),
doi: https://doi.org/10.1016/j.ejmech.2020.112437.
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5-HTRs
affinity
Functional
activity
PDE4B/7A
inhibition
ADME
properties
Behavioral
activity
Design
Synthesis
5-HT1A/5-HT7 receptor antagonist
PDE4B/PDE7A inhibitor
n = 3, 4 or 5
Procognitive-like activity
Antidepressant-like activity
m = 0 or 1
Hybrid compd 7
Compd 8-39
Compd 22
• 2-OH, 5-C(CH3)3
• 3-C(CH3)3
• 3-CH(CH3)2
• 4-C(CH3)3
• 4-CH(CH3)CH2CH3
• 4-COOC(CH3)3
• 2-OH
• 2-CONH2
• 2-OCH3
Suitable ADME properties
• 4-CH(CH3)2

Novel Anilide and Benzylamide Derivatives of Arylpiperazinylalkanoic Acids as 5-HT1A/5-HT7
Receptor Antagonists and Phosphodiesterase 4/7 Inhibitors with Procognitive and
Antidepressant Activity
Agnieszka Jankowskaa, Grzegorz Satałab, Marcin Kołaczkowskia, Adam Buckia, Monika Głuch-
Lutwinc, Artur Świerczekd, Krzysztof Pociechad, Anna Partykae, Magdalena Jastrzębska-Więseke,
Annamaria Lubelskaf, Gniewomir Lataczf, Alicja Gawalskaa, Andrzej J. Bojarskib, Elżbieta Wyskad,
and Grażyna Chłoń-Rzepaa,*
aJagiellonian University Medical College, Faculty of Pharmacy, Department of Medicinal Chemistry,
9 Medyczna Street, 30-688 Kraków, Poland
bPolish Academy of Sciences, Maj Institute of Pharmacology, Department of Medicinal Chemistry, 12
Smętna Street, 31-343 Kraków, Poland
cJagiellonian University Medical College, Faculty of Pharmacy, Department of Pharmacobiology, 9
Medyczna Street, 30-688 Kraków, Poland
dJagiellonian University Medical College, Faculty of Pharmacy, Department of Pharmacokinetics and
Physical Pharmacy, 9 Medyczna Street, 30-688 Kraków, Poland
eJagiellonian University Medical College, Faculty of Pharmacy, Department of Clinical Pharmacy, 9
Medyczna Street, 30-688 Kraków, Poland
fJagiellonian University Medical College, Faculty of Pharmacy, Department of Technology and
Biotechnology of Drugs, Medyczna 9, 30-688 Kraków, Poland
Corresponding author*:
Grażyna Chłoń-Rzepa,
Jagiellonian University Medical College,
Faculty of Pharmacy, Department of Medicinal Chemistry,
9 Medyczna Street, 30-688 Kraków, Poland,
Phone: +48(12) 6205450,
Fax: +48(12) 6205458,
E-mail: grazyna.chlon-rzepa@uj.edu.pl
1

Abstract:
A
library of novel anilide and benzylamide derivatives of ω-(4-(2-
methoxyphenyl)piperazin-1-yl)alkanoic acids as combined 5-HT1A/5-HT7 receptor ligands and
phosphodiesterase PDE4B/PDE7A inhibitors was designed using a structure-based drug design
approach. The in vitro studies of 33 newly synthesized compounds (7-39) allowed us to identify 22 as
the most promising multifunctional 5-HT1A/5-HT7 receptor antagonist (5-HT1A Ki = 8 nM, Kb = 0.04
nM; 5-HT7 Ki = 451 nM, Kb = 460 nM) with PDE4B/PDE7A inhibitory activity (PDE4B IC50 = 80.4
ꢀM; PDE7A IC50 = 151.3 ꢀM). Compound 22 exerted a very good ability to passively penetrate
through biological membranes and a high metabolic stability in vitro. Moreover, the pharmacological
evaluation of 22 showed its procognitive and antidepressant properties in rat behavioral tests.
Compound 22 at a dose of 3 mg/kg (i.p.) significantly reversed MK-801-induced episodic memory
deficits in the novel object recognition test, while at a dose of 10 mg/kg (i.p.) reduced the immobility
time of animals (by about 34 %) in the forced swimming test. The antidepressant-like effect produced
by compound 22 was stronger than that of escitalopram used as a reference drug. This study opens a
new perspective in the search for efficacious drugs for the treatment of cognitive and depressive
disorders.
Keywords: anilide; benzylamide; 1-(2-methoxyphenyl)piperazine derivative; PDE4/PDE7 inhibitor;
5-HT1A/5-HT7 receptor antagonist; procognitive and antidepressant activity.
Abbreviations: PDE, phosphodiesterase; 5-HT receptor, serotonin receptor; cAMP, cyclic-3′,5′-
adenosine monophosphate; LCAP, long-chain arylpiperazine; BBB, blood-brain barrier; RLMs, rat
liver microsomes; IBMX, 3-isobutyl-1-methylxanthine; NOR test, novel object recognition test; FST,
forced swimming test.
2

1. Introduction
Due to the growing number of patients suffering from memory and depressive disorders [1,2], the
search for new drugs with procognitive and antidepressant properties is one of the leading research
directions around the world [3–5]. Cognitive functioning is moderately to severely impaired in patients
with depression [6], schizophrenia [7], and various forms of dementia including this caused by
Alzheimer’s disease [8]. The impairment of cognition is the prime driver of the significant disabilities
in occupational, social, and economic functioning in patients. Unfortunately, we have limited
treatment options for cognitive disorders, which are often accompanied by deterioration of mood, a
factor that worsens patients’ quality of life and leads to their premature death [9,10].
According to latest literature data, drugs with multidirectional activity combining procognitive and
antidepressant effects may be achieved by interaction with serotonin 5-HT1A and 5-HT7 receptors [10]
and inhibition of cyclic-3′,5′-adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE)
type 4 and 7 [11–13]. Therapeutic paradox that both agonists and antagonists of 5-HT1A and 5-HT7
receptors may exert procognitive activity is still unclear [14]. In turn, the action of PDE4/PDE7
inhibitors probably results from the regulation of cAMP signaling pathway and their anti-
inflammatory [15,16] and neuroprotective properties [17–19].
Fig. 1. Representative 5-HT1A/5-HT7 receptor ligands with antidepressant activity from previously
described studies [20–23].
For several years, we have been developing 5-HT1A/5-HT7 receptor ligands with a diverse
functional ago/antagonist profile in a long-chain arylpiperazines (LCAPs) class containing purine-2,6-
dione scaffold as the terminal fragment. Some compounds from the evaluated series showed
antidepressant activity in an animal behavioral test, e.g., I-V (Fig. 1) [20–23]. Recently, novel LCAP
3

derivatives of 8-alkoxypurine-2,6-dione and imidazo[2,1-f]purine-2,4-dione combining 5-HT1A/5-HT7
receptor affinity and PDE4B/PDE10A inhibitory activity have been designed, synthesized, and
evaluated as potential antidepressant drugs, e.g., VI and VII (Fig. 1) [24–27]. However, the
pharmacological studies aimed to assess the effects of above mentioned LCAP derivatives of purine-
2,4-dione on cognitive functions have not been performed yet.
In this work we made an attempt to obtain innovative multifunctional ligands with ability to
improve cognition and mood. Our research hypothesis is that simultaneous interaction with 5-HT1A/5-
HT7 receptors and inhibition of PDE4B/PDE7A isoenzymes may provide both procognitive and
antidepressant activities by their synergistic effect on the intracellular cAMP level.
The aim of this study was to design, synthesize, and evaluate procognitive and antidepressant
activity of novel anilide and benzylamide derivatives of ω-(4-(2-methoxyphenyl)piperazin-1-
yl)alkanoic acids as 5-HT1A/5-HT7 receptor ligands and PDE4B/PDE7A inhibitors. The innovative
group of hybrid compounds that linked the 1-(2-methoxyphenyl)piperazine scaffold with a differently
substituted anilide or benzylamide moiety via 3, 4, or 5-carbon aliphatic chain was designed using a
structure-based drug design approach. For the newly synthesized compounds, their 5-HT1A/5-HT7
receptor affinity and PDE4B/PDE7A inhibitory activity were determined in vitro. For the most potent
ligands, the evaluation of functional activity toward targeted receptors as well as membrane
permeability and metabolic stability was performed. Finally, for the most promising compound its
procognitive and antidepressant-like activity was tested in vivo using well-established experimental
paradigms.
2. Results and discussion
2.1.Computer-aided molecule design
In order to obtain innovative multifunctional ligands that combine 5-HT1A and 5-HT7 receptor
affinity and ability to inhibit PDE4B and PDE7A isoforms, a ligand-based drug design approach was
applied that relies on the knowledge of molecules that bind to the biological targets of interest. As a
result of combining LCAP moiety being one of the most universal templates used for designing 5-
HT1A and 5-HT7 receptors ligands (Fig. 2) [28–31] and substituted anilide group found to be crucial
for inhibitory activity of the 1,3-dimethylpurine-2,6-diones toward PDE4B and PDE7A (Fig. 3)
[32,33], a new hybrid compound 7, anilide derivative of arylpiperazinylalkanoic acid as a potential
multi-target-directed ligand was designed (Fig. 4).
4

Fig. 2. The structures of representative 5-HT1A/5-HT7 receptor ligands based on the LCAP moiety [28–
31].
Fig. 3. The structures of representative PDE4B/PDE7A inhibitors based on the 1,3-dimethylpurine-
2,6-dione scaffold [32,33].
To confirm the advisability of the proposed hybrid hypothesis, molecular modeling studies
were performed. The results showed that compound 7 was able to interact as both a 5-HT1A and 5-HT7
receptor ligand as well as a PDE4B and PDE7A inhibitor.
In 5-HT1A and 5-HT7 receptors, hybrid compound 7 was docked in the orthosteric binding sites
(the phenylpiperazine moiety), whereas the (5-tert-butyl-2-hydroxyphenyl)carbamoylpropyl
fragment bound in the additional (allosteric) binding sites. The key anchoring interactions of
phenylpiperazine fragment, common for both the receptors, are salt bridges (charge reinforced
hydrogen bonds) to Asp3.32, and aromatic π-π stackings with Phe6.52. In the 5-HT1A receptor,
additional stabilizing interactions are as follows: π-π stacking with Tyr2.64, hydrogen bonds to
Asn7.39 and Thr188 (Fig. 5A). In the case of the 5-HT7 receptor, the additional interactions comprise
π-π stacking with Phe3.28 and hydrogen bonds to Arg7.36 and Tyr7.43 (Fig. 5B). Favorable
5

complementarity of ligand-receptor interactions suggests a high affinity for the target proteins and
justifies further research within this chemotype.
Fig. 4. Design of anilide and benzylamide derivatives of arylpiperazinylalkanoic acids as potential
multifunctional ligands combining 5-HT1A/5-HT7 receptor affinity and PDE4B/PDE7A inhibitory
activity.
In the case of PDE4B, compound 7 was docked in the center of the catalytic site, placing the
phenylpiperazine moiety (responsible for dual 5-HT1A and 5-HT7 receptor activity) in the hydrophobic
pocket (H pocket) between Phe446 and Phe414 residues. Its phenyl ring formed π-π stacking
interactions with Phe446. Another fragment of the structure
-
(5-tert-butyl-2-
hydroxyphenyl)carbamoylpropyl was situated in the area of the metal-binding site (M site) and formed
two hydrogen bonds with His234 (carbonyl group) and Asp275 (hydroxy group) amino acid residues.
Additionally, there were several weak interactions found – aromatic hydrogen bonds with Ser442,
Phe414 and Glu304 (Fig. 5C).
In the active site of PDE7A, compound 7 was placed analogously. The phenyl ring of the
phenylpiperazine moiety formed π-π stacking interactions with Phe416. The protonated nitrogen atom
of piperazine ring formed an H-bond network with a water molecule, which in turn interacted
6

simultaneously with Tyr211 (hydroxy group) and Asp362 (carbonyl group). (5-tert-butyl-2-
hydroxyphenyl)carbamoylpropyl fragment also stabilized the proper compound conformation through
a hydrogen bond with Glu282 (a hydroxy group at position 2 of phenyl ring) and aromatic hydrogen
bond with Gln261 (Fig. 5D).
Fig. 5. The predicted binding modes of hybrid compound 7 in the models of 5-HT1A (A) and 5-HT7
(B) receptors, as well as PDE4B (C) and PDE7A (D) catalytic sites. Interactions with the key amino
acids are displayed as follows: hydrogen bonds (yellow), aromatic hydrogen bonds (green), and
aromatic π-π stacking (blue).
It was proved that PDE4B and PDE7A inhibitory activity depends on the occurrence of 3
interactions: hydrogen bond with Gln443/Gln413, and π-π stacking interactions with Phe446/Phe416,
and Phe414/Phe384 [34]. The lack of these important hydrogen bonds and one of the aromatic
interactions could pose an explanation of compound 7 weak PDE4B and PDE7A inhibitory activity.
For the hybrid compound 7 its molecular parameters, physicochemical, pharmacological, and
toxicological properties were determined using appropriate software (Table 1). Based on the
Lipinski’s rule of five [35], it was estimated using the Instant JChem software that compound 7 has
physical and chemical properties that would make it a likely orally active drug in humans. Using the
Van de Watembeerd [36] and Kelder [37] rules, and the brain or intestinal estimated permeation
(BOILED-Egg) method [38], it was found that compound 7 is likely to cross the blood-brain barrier
(BBB) and act within CNS. The risk of mutagenicity, tumorigenicity, irritating, and reproductive
7

effects of compound 7 were assessed as low using the OSIRIS Property Explorer software [39].
Finally, using the SwissADME program [40], no structural fragments known as PAINS (Pan-assay
interference compounds) were found in the designed hybrid compound 7 that could give false positive
results in in vitro pharmacological tests.
Table 1. Prediction of ability to cross the BBB and toxicity of compound 7 using Instant JChema,
SwissADMEb, or OSIRIS Property Explorerc.
Molecular and
CNS penetrationa,b
Risk of toxicityc
physicochemical propertiesa
MW
HBD
425.56
Lipinski’s rule
Van de Waterbeemd’s rule
Kelder’s rule
Yes
Yes
Yes
Mutagenicity
Tumorigenicity
Irritating effect
No
No
No
2
5
HBA
CLogP
TPSA
3.53
65.04 Å2
BOILED-Egg
Yes Reproductive effect Low
Taking into account the results of in silico studies, a library of new hybrid compounds was
designed as potential 5-HT1A/5-HT7 receptor ligands and PDE4B/PDE7A inhibitors based on the
structure of the pilot compound 7 (Fig. 4). 2-Methoxyphenylpiperazine was left as a terminal amine
fragment. The benzene ring at the anilide group was substituted with a branched alkyl, ester, hydroxy,
methoxy, or carboxamide group. Moreover, instead of the anilide group, the benzylamide fragment
was introduced. Both terminal fragments were connected by a 3, 4, or 5-carbon aliphatic chain. The
modifications were aimed at checking how the length of aliphatic linker and changing the substitution
mode of the benzene ring at the amide fragment affect the receptor and enzymatic affinities of the
obtained compounds.
2.2.Synthesis
Synthesis of the designed compounds 7-39 was performed according to the multistep procedure
presented in Scheme 1. In the first step, commercially available 1-(2-methoxyphenyl)piperazine was
alkylated using an appropriate ethyl bromoalkanoate in the presence of anhydrous K2CO3 in refluxing
acetonitrile, yielding ethyl esters 1-3. Intermediate esters were subsequently hydrolyzed with KOH
solution in a water-acetone medium, and the resulting acids 4-6 were isolated after acidification of the
reaction mixture. In the next step, the obtained acids were coupled with amine (aniline or benzylamine
derivative) in the presence of di(1H-imidazol-1-yl)methanone (CDI) in DMF. The synthesized amides
were treated with 1 N HCl in methanol to give the final products 7-39 as hydrochloride salts.
8

Scheme 1. The synthesis of the anilide and benzylamide derivatives of ω-(4-(2-
methoxyphenyl)piperazin-1-yl)alkanoic acids 7-39. Reagents and conditions: (i) ethyl 4-
bromobutanoate, ethyl 5-bromopentanoate, or ethyl 6-bromohexanoate, K2CO3, acetonitrile, reflux, 15
h; (ii) KOH, acetone/water, reflux, 8 h; (iii) conc. HCl; (iv) aniline or benzylamine derivative, CDI,
DMF, room temp., 72 h; (v) HCl in methanol.
2.3.Preliminary in vitro pharmacology
2.3.1. Radioligand binding assays
All the newly synthesized compounds 7-39 were tested for their 5-HT1A and 5-HT7 receptor
affinity in the radioligand binding assays according to the previously described protocols [41]. The
inhibition constants (Ki) were calculated from the Cheng-Prusoff equation and presented in Table 2.
The experiments were carried out using [3H]-8-OH-DPAT (135.2 Ci/ mmol) and [3H]-5-CT (39.2
Ci/mmol) for the human 5-HT1A and 5-HT7 receptors, respectively, which were all stably expressed in
human embryonic kidney 293 (HEK293) cells. Buspirone and clozapine were used as reference 5-
HT1A and 5-HT7 receptor ligands, respectively.
In general, the synthesized 2-methoxyphenylpiperazine derivatives 7-39 containing 3, 4, or 5-
carbon aliphatic linker and differently substituted benzene ring at the amide fragment exerted higher
affinity for the 5-HT1A receptor than for the 5-HT7 receptor (Table 2). The compounds showed 5-HT1A
receptor affinity in the range of 8-590 nM. The most potent 5-HT1A receptor ligands were compounds
12, 18-28, 30-35, and 37-39 exerting Ki < 100 nM. Among them, compounds 19-24 provided a similar
or higher affinity (Ki = 8-29 nM) comparing with buspirone (Ki = 20 nM). In turn, compounds 8, 9, 11,
13, 14, 16, 17, 29, and 36 behaved as moderate 5-HT1A receptor ligands (100 nM < Ki < 500 nM). The
lowest 5-HT1A receptor affinity (Ki > 500 nM) was found for compounds 7, 10, and 15.
As presented, all the structural modifications of the prototype hybrid molecule (pilot compound 7)
resulted in an improved affinity for the 5-HT1A receptor. Considering the aliphatic linker length
between 2-methoxyphenylpiperazine and amide fragments it can be concluded that the 4-carbon linker
was more preferred for the enhanced 5-HT1A receptor affinity than 3- or 5-carbon ones (compounds
18-29 vs. 7-17 and 30-39) what is in line with the previously reported SAR analysis results in the
group of purine-based 5-HT1A receptor ligands [20,23,24]. Moreover, the analysis of the benzene ring
substitution at the amide group revealed that branched alkyl groups were more favorable than
hydroxy, ester, and amide groups (e.g., compounds 19-23 vs. 18 and 24-26).
9

Table 2. Structures and 5-HT1A and 5-HT7 receptor affinities of compounds 7-39.
Compd
n
m
R
Ki [nM]
5-HT1A
Ratio Ki
5-HT7/5-HT1A
5-HT7
328
130
157
1196
954
354
3282
471
1143
319
869
159
75
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
1
1
1
-
-
-
-
-
-
-
-
1
1
2-OH, 5-C(CH3)3
3-C(CH3)3
539
224
187
590
150
37
287
405
526
339
292
61
<1
<1
<1
2
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
3-CH(CH3)2
4-C(CH3)3
4-CH(CH3)2
4-CH(CH3)CH2CH3
4-COOC(CH3)3
2-OH
2-CONH2
2-OH
2-OCH3
2-OH, 5-C(CH3)3
3-C(CH3)3
3-CH(CH3)2
4-C(CH3)3
4-CH(CH3)2
4-CH(CH3)CH2CH3
4-COOC(CH3)3
2-OH
6
10
11
1
2
1
3
3
5
7
24
56
23
32
10
23
8
4
3
2
4
16
21
21
8
14
29
31
82
43
82
138
82
53
34
40
43
69
125
95
151
496
451
323
927
300
1874
343
366
391
154
190
141
514
434
260
799
254
289
515
-
2-CONH2
2-OH
2-OCH3
4-C(CH3)3
2-OH, 5-C(CH3)3
3-C(CH3)3
3-CH(CH3)2
4-C(CH3)3
4-CH(CH3)2
4-CH(CH3)CH2CH3
4-COOC(CH3)3
2-OH
4
13
10
4
6
3
2-OH
2-OCH3
70
64
20
4
8
Buspirone
Clozapine
-
18
The replacement of the anilide fragment by benzylamide moiety resulted in a slight increase in 5-
HT1A receptor affinity in the case of 2-hydroxy phenyl derivatives (14 vs. 16 and 37 vs. 38). On the
other hand, in the 4-tert-butyl analogs this modification decreased (about 7-fold) 5-HT1A receptor
affinity (21 vs. 29).
10

The evaluated compounds 7-39 exerted 5-HT7 receptor affinity in the range of 75-3282 nM.
Compound 19 showed the highest 5-HT7 receptor affinity (Ki = 75 nM) in this group of derivatives but
its activity was 4-fold lower than that for clozapine (Ki = 18 nM). Compounds 7-9, 12, 14, 16, 18, 20-
23, 25, 27-32, 34, 35, 37, and 38 were characterized as moderate 5-HT7 receptor ligands (100 nM < Ki
< 500 nM), while other studied compounds possessed a low affinity toward the 5-HT7 receptor (Ki >
500 nM).
Similarly to the 5-HT1A receptor, the 5-HT7 receptor affinity of the tested compounds was highest
for the derivatives with 4-carbon chain than for the compounds with 3- and 5-carbon linker.
Considering the benzene ring substitution at the amide moiety it can also be concluded that branched
alkyl groups were preferred, especially, at the meta position (e.g., compounds 8, 9, 19, 20, 31, and 32).
It was also found that only the combination of 2-methoxyphenylpiperazine, a 4-carbon aliphatic
linker, and a 3-(isopropyl)phenyl group provided a potent and dual 5-HT1A and 5-HT7 receptor ligand
(compound 19) with Ki value of 16 and 75 nM, respectively. The shortening of the alkyl linker from 4-
carbon to 3-carbon one maintained the dual nature of interaction of compound 8 but its 5-HT receptor
affinity was reduced.
Finally, the SAR study around amide group showed that the replacement of the anilide by
benzylamide (compounds 14 vs. 16, 21 vs. 29, 25 vs. 27, 38 vs. 39) did not significantly change the
pharmacological activity of the tested compounds toward the 5-HT7 receptor.
2.3.2. Functional activity evaluation
On the basis of the binding affinity results, a series of the high affinity 5-HT1A and 5-HT7 receptor
ligands was selected for functional profile analysis according to previously described protocols using
in vitro cellular assays [42]. For the 5-HT1A receptor ligands, a cellular aequorin-based functional
assay was performed with recombinant Chinese hamster ovary (CHO)-K1 cells expressing
mitochondrially targeted aequorin, the human 5-HT1A receptor, and the promiscuous G protein α16 for
the 5-HT1A receptor. For the 5-HT7 receptor ligands, adenylyl cyclase activity was monitored using
cryopreserved CHO-K1 cells with expression of the human 5-HT7 receptor. NAN-190 and SB-269970
were used as reference 5-HT1A and 5-HT7 receptor antagonists, respectively, and serotonin as a 5-
HT1A/5-HT7 receptor agonist.
All the tested compounds showed antagonistic properties toward 5-HT1A and 5-HT7 receptors
(Table 3), while no activation of 5-HT1A and 5-HT7 receptors was observed in the cell-based assays.
The evaluated compounds were characterized as very potent 5-HT1A receptor antagonists and weak to
moderate 5-HT7 receptor antagonists. The most potent 5-HT1A receptor antagonists (compounds 19,
20, 22, and 24) affected the receptor with a similar or higher potency than that of NAN-190 (Kb = 0.07
nM) used as a reference. Other 5-HT1A receptor antagonists (compounds 7, 10, 16, 27, 30, and 33)
showed the Kb value in the range of 0.89 to 4.20 nM. The antagonistic activity of all tested compounds
toward 5-HT7 receptor was much more diverse and provided the Kb value in the range of 54 nM
11

(compound 19) to 9900 nM (compound 24) compared to Kb value of 0.2 nM for the reference 5-HT7
receptor antagonist SB-269970. However, most of the tested 5-HT7 receptor antagonists 16, 20, 22, 27,
30, and 33 gave the Kb value at submicromolar level in the range of 120-870 nM.
Table 3. Antagonistic activity of the selected 5-HT1A and 5-HT7 receptor ligands.
Compd
Kb [nM]
5-HT1A
5-HT7
2300
N.C.
120
54
100
460
9900
240
480
870
-
3.40
4.20
4.20
0.21
0.13
0.04
0.11
0.89
1.10
1.40
0.07
-
7
10
16
19
20
22
24
27
30
33
NAN-190
SB-269970
0.2
N.C. – not calculable
The results obtained in this selected set of compounds showed the importance of the aliphatic
linker length on the functional profile toward 5-HT1A receptor as the potency of the antagonistic
activity was observed in the following order: 4-carbon (compounds 19, 20, 22, 24, and 27) > 5-carbon
(compounds 30 and 33) > 3-carbon aliphatic chain (compounds 7, 10, and 16). No similar relationship
was found for the 5-HT7 receptor.
2.3.3. PDE4B and PDE7A inhibitory activity
All the synthesized compounds 7-39 were tested in vitro for their PDE4B and PDE7A inhibitory
activity using PDE-GloTM Phosphodiesterase Assay and human recombinant PDE4B and PDE7A
expressed in Spodoptera frugiperda 9 (Sf9) cells according to previously described protocols [43]. A
non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) was used as a reference compound
in this study. The data obtained were analyzed using nonlinear regression (ADAPT 5 (BMSR, Los
Angeles, CA, USA). PDE4B and PDE7A inhibitory activities were expressed as IC50 values.
Compounds 7-39 exerted various PDE4B and PDE7A inhibitory activity in vitro. The IC50 values
for active compounds 7, 10, and 18-23 were summarized in Table 4. Other tested compounds 8, 9, 11-
17, and 24-39 showed PDE4B and PDE7A IC50 values > 200 µM.
12

Table 4. The PDE4B and PDE7A inhibitory activities of compounds 7, 10, and 18-23.
Compd
IC50 [µM]
PDE4B
69.0
96.0
PDE7A
57.0
27.0
7
10
36.5
130.0
88.1
138.5
80.4
168.9
28.2
> 200
> 200
> 200
> 200
151.3
125.5
85.6
18
19
20
21
22
23
IBMX
The hybrid compound 7 showed double-digit micromolar IC50 values of 69.0 and 57.0 ꢀM for
PDE4B and PDE7A, respectively, and its inhibitory activity was similar to that obtained for IBMX
(Table 4). The structural modification of compound 7 which comprised the elongation of the aliphatic
linker between 2-methoxyphenylpiperazine and amide fragments from 3- to 4-carbon chain gave
compound 18 with increased activity toward PDE4B (IC50 = 36.5 ꢀM). However, this structural
change decreased PDE7A inhibitory activity (IC50 > 200 ꢀM). In turn, the removal of the hydroxy
group from the amide fragment and introduction of the tert-butyl group at para position of the benzene
ring provided compound 10 with increased activity toward PDE7A (IC50 = 27.0 ꢀM). On the other
hand, this modification decreased activity toward PDE4B (IC50 = 96.0 ꢀM). For several other
compounds 19-23, their PDE4B IC50 values were determined in the range of 80.4-168.9 µM. For
compounds 22 and 23, their PDE7A inhibitory activity was estimated as 151.3 and 125.5 µM,
respectively. Compounds 7, 10, 22, and 23 were found to be weak dual PDE4B/PDE7A inhibitors.
The results obtained in this set of compounds confirmed the influence of the aliphatic linker length
and the mode of the benzene ring substitution at the amide fragment on PDE4B and PDE7A
inhibition. Some compounds (7, 10, 22, and 23) with 3- or 4-carbon aliphatic chain were characterized
as dual PDE4B/PDE7A inhibitors, while the 5-carbon homologs were inactive. Furthermore, the
replacement of the anilide by benzylamide moiety (compound 21 vs. 29) decreased the inhibitory
activity toward both PDE4B and PDE7A. Finally, analysis of the benzene ring substitution at the
amide group revealed that branched alkyl groups were more favorable than hydroxy, ester, and amide
groups for the activity toward PDE4B/PDE7A (e.g., compounds 18-23 vs. 24-26). For the alkyl
substituents, the activity was established in the following order: isopropyl > tert-butyl > sec-butyl
group. It is worth noting that according to the results of molecular modeling studies, the combination
of 2-hydroxy and 5-tert-butyl substituents at the benzene ring leads to the strongest inhibitory activity
toward PDE4B (compounds 7 and 18) but has a different effect on PDE7A.
13

2.4.In vitro ADME studies
2.4.1. Parallel artificial membrane permeability assay
The penetration through biological membranes is the crucial aspect determining in vivo activity of
CNS-targeted compounds. Thus, compounds 19, 20, and 22 characterized as potent 5-HT1A receptor
antagonists, weak or moderate 5-HT7 receptor antagonists with additional inhibitory activity toward
PDE4B or PDE7A were selected to extended in vitro ADME studies in order to determine their
passive transport and predict oral absorption.
The membrane permeability of compounds 19, 20, and 22 was estimated by the commercially
available Pre-coated PAMPA Plate System Gentest™ according to the previously described protocols
and formulas [44,45]. Norfloxacin and caffeine were used as a low- and high-permeable reference
drug, respectively. The probes were analyzed by LC/MS Waters ACQUITY™ TQD system with the
TQ Detector.
Table 5. Membrane permeability of the selected compounds 19, 20, 22, and reference substances.
Compd
19
Pea [10-6 cm/s] ± SD
4.8 ± 1.0
8.6 ± 1.0
20
10.5 ± 3.1
22
0.6 ± 0.1
15.1 ± 0.4
Norfloxacin
Caffeine
aPAMPA plate’s manufacturer breakpoint for permeable compounds: Pe ≥ 1.5 x 10-6 cm/s [46]
The calculated permeability coefficients (Pe) for compounds 19, 20, and 22 were much higher than
that for a low-permeable reference drug, norfloxacin and lower than that for a high-permeable
compound, caffeine (Table 5). Moreover, the obtained Pe values for compounds 19, 20, and 22 were 3-
, 6-, or 7-fold higher, respectively, than that indicated as the breakpoint for permeable compounds (Pe
≥ 1.5 x 10-6 cm/s) [46].
In this study, the passive transport of compounds 19, 20, and 22 across the cell membranes as well
as an opportunity for their oral absorption were estimated as high. The highest ability to passively
penetrate through biological membranes was found for the 4-isopropylanilide derivative 22.
2.4.2. Metabolic stability study
Determination of metabolic stability and metabolites of new chemical entities is a key step in the
process of drug discovery, since it influences pharmacological, pharmacokinetic, and toxicological
properties of the tested compounds. The preliminary metabolic stability study of compounds 19, 20,
and 22 was performed in vitro using rat liver microsomes (RLMs) according to the previously
described protocols [47]. The supernatants were analyzed by UPLC/MS using LC/MS Waters
ACQUITY™ TQD system with the TQ Detector. The results of biotransformation study and
characteristics of metabolites were presented in Table 6. Additionally, the in silico study was
14

performed by MetaSite 6.0.1 and the sites of biotransformation of compound 22 were predicted (Fig.
6).
UPLC/MS analyses of the reaction mixtures after 2 hours of incubation with RLMs indicated the
rapid and almost complete decomposition of compounds 19 and 20, possibly due to the hydrolysis of
anilide (amide) bond to alkylcarboxylic acid (19-M1, 20-M1) and an appropriate aniline derivative
(19-M2, 20-M2) (Table 6).
Table 6. Biotransformation study results of compounds 19, 20, and 22 and their metabolite
characteristics.
Parent compounds
and their metabolites
[M+H]+
Metabolic pathway % content after incubation
with RLMs
424.34
293.21
150.14
410.38
293.21
136.11
410.38
426.33
426.33
hydrolysis
23.8
66.2
10.0
6.5
80.9
12.6
90.1
5.9
19
-
-
19-M1
19-M2
20
20-M1
20-M2
22
hydrolysis
-
-
hydroxylation
-
-
22-M1
22-M2
4.0
According to the UPLC/MS spectra, compound 22 was characterized by a high metabolic stability
using RLMs. After 2 hours of incubation with RLMs, only around 10 % of compound 22 (tR = 5.19
min, [M+H]+ = 410.38) was metabolized into two metabolites: 22-M1 (tR = 4.64 min, [M+H]+ =
426.33) and 22-M2 (tR = 3.72 min, [M+H]+ = 426.33) (Supp. Fig. S1-4). The MetaSite in silico
prediction (Fig. 6), the ion fragmentation analyses performed for compound 22 and its metabolites as
well as increased [M+H]+ values of both metabolites 22-M1 and 22-M2 by 16 mass units relative to
the initial [M+H]+ value of compound 22 indicated the hydroxylation at the benzene ring of the
phenylpiperazine scaffold (22-M1) or the hydroxylation at the isopropyl group of the anilide fragment
(22-M2) as the most probable biotransformation sites of compound 22.
Analysis of the structure-metabolic stability relationship in the studied group of anilide derivatives
enabled the statement that branched alkyl substituent at the para position of the benzene ring at the
amide fragment increased the metabolic stability (compound 22), while substitution at the meta
position decreased the metabolic stability in the RLMs (compound 19 and 20). The results obtained
make compound 22 a good candidate for further studies, including behavioral tests in animals.
15

Fig. 6. The plot of MetaSite 6.0.1 prediction for sites of biotransformation of compound 22. The
darker red color of the marked functional group indicates its higher probability to be involved in the
metabolism pathway. The blue circle marked the site of compound 22 which may be involved in
metabolism with the highest probability.
2.5.In vivo behavioral evaluation
The in vitro pharmacological profile and suitable ADME properties of the presented compounds
prompted us to evaluate their procognitive and antidepressant activity in vivo. We selected for
behavioral studies compound 22 which was characterized as a potent 5-HT1A receptor antagonist with
weak 5-HT7 receptor antagonistic properties and an inhibitory activity toward both PDE4B and
PDE7A isoenzymes. The choice was dictated by the results of functional studies suggesting the
insignificance of antagonistic properties of compound 22 toward the 5-HT7 receptor and its negligible
effect on the reduction of cAMP level. Moreover, several studies provided evidence that 5-HT7
receptor blockade may be also useful in treating cognitive and antidepressant disorders [5,48].
The procognitive and antidepressant activity of compound 22 was tested in Wistar rats at doses of
1, 3, and 10 mg/kg (i.p.) using well-established experimental paradigms, i.e., novel object recognition
(NOR) and forced swimming (FST) tests, respectively. Moreover, in order to exclude the possibility of
competing behaviors, such as general locomotor activity, the open field (OF) test was carried out and
the influence of effective doses of 22 was studied.
2.5.1. Ability of compound 22 to reverse memory impairment
Following a single administration, compound 22 at doses of 1 and 3 mg/kg reversed MK-801-
induced memory impairment in the NOR test in rats (Fig. 7) but the effect was statistically significant
only at the dose of 3 mg/kg. Moreover, effective dose of 3 mg/kg obtained in the NOR test had no
influence on the spontaneous locomotor activity of the animals in the OF test (Table 7).
16

Fig. 7. Effect of compound 22 on MK-801-induced memory deficits in rat NOR test. MK-801 was
administered i.p. 30 min and the tested compound 60 min before the T1 phase. Data represent mean ±
SEM. Rats per group N = 6–8. *p < 0.05 vs. vehicle-treated group, #p < 0.05 vs. MK-801-treated
group (one-way ANOVA followed by Bonferroni’s posthoc test).
Table 7. Effect of compound 22 and escitalopram on the rat locomotor activity measured in the OF
test.
Treatment
Dose
(mg/kg)
Total distance
(cm)
Rearings
Ambulations
X
Ambulations
Y
0
3
10
0
2334.7 ± 201.4
2257.2 ± 229.6
1156.8 ± 105.0**
1897.3 ± 98.6
2058.5 ± 113.1
48.0 ± 6.5
44.0 ± 4.1
18.5 ± 1.3***
42.6 ± 4.8
223.5 ± 23.5
116.8 ± 32.1
103.0 ± 14.6*
194.5 ± 21.4
205.1 ± 24.0
203.3 ± 31.1
219.8 ± 35.4
82.7 ± 6.7**
201.1 ± 26.3
192.5 ± 15.2
Vehicle
Cmpd 22
Vehicle
S-cit
20
39.9 ± 6.3
The compound 22 was injected i.p. 60 min, while escitalopram (S-cit) was given i.p. 30 min before the
test. Values represent the mean ± SEM during 5-min test session compared to the respective vehicle-
treated group. Rats per group N = 4–6. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA
followed by the Bonferroni’s post hoc test).
2.5.1. Antidepressant-like effect of compound 22
Following a single administration, compound 22 reduced the immobility time of animals in the
FST by about 34 % at dose of 10 mg/kg, while the reference drug, escitalopram, revealed
antidepressant-like effect at 2-fold higher dose of 20 mg/kg, shortening the immobility time by about
49 % (Fig. 8). The antidepressant-like effect produced by compound 22 as well as escitalopram seems
to be specific, since both compounds given at their effective doses did not increase the spontaneous
locomotor activity of rats in the OF test (Table 7). However, compound 22 significantly reduced the
mobility of animals which indicates its sedative properties.
17

Fig. 8. Effect of compound 22 and escitalopram in the FST in rats. The compound 22 was injected i.p.
60 min, while escitalopram 30 min before the test. Values represent mean ± SEM and are expressed as
percentage of control. Rats per group N = 6–8. ***p < 0.001 vs. vehicle-treated group (one-way
ANOVA followed by the Bonferroni’s post hoc test).
Animal tests showed both procognitive and antidepressant effects of compound 22 being a
representative of a new class of compounds with unique mechanism of action. However, the in vivo
activity of compound 22 seems to be related to its antagonistic properties toward the 5-HT1A receptor.
Further in vivo studies, including pharmacokinetic evaluation, are needed to provide evidence for the
participation of individual targets in the in vivo activity. In addition, the in vitro studies to specify the
affinity of obtained compounds for other receptors which are recognized therapeutic targets in the
treatment of CNS disorders or are associated with adverse effects should be performed. The obtained
compound 22 represents the new lead structure for further optimization to find more active and
metabolically stable derivatives showing this desired multi-target mechanism of action. An improved
multifunctional ligand with a balanced activity profile toward 5-HT1A/5-HT7 receptors and
PDE4B/PDE7A will be eventually tested for its efficacy in an animal model of Alzheimer’s disease.
3. Conclusions
In this paper, the computer-aided design and synthesis of 33 innovative anilide and benzylamide
derivatives of ω-(4-(2-methoxyphenyl)piperazin-1-yl)alkanoic acids were described. The synthesized
compounds were found to be potent to moderate 5-HT1A receptor antagonists with moderate to weak
5-HT7 receptor antagonistic properties and additional PDE4B and PDE7A inhibitory activities.
Binding mode predictions shed light on structural requirements for multifunctional 5-HT1A/5-HT7
receptor ligands and PDE4B/PDE7A inhibitors. SAR analysis showed that the 4-carbon aliphatic
chain between cyclic amine and amide fragments was the most favorable for obtaining multi-target-
directed ligands of 5-HT1A/5-HT7 receptors and PDE4B/PDE7A inhibitors. Moreover, the benzene
ring substitution site at the amide fragment was found as a possible modification site of the metabolic
stability of the tested compounds. The selected compound 22 characterized as a potent 5-HT1A
receptor antagonist, a weak 5-HT7 receptor antagonist with an additional PDE4B/PDE7A inhibitory
activity and suitable in vitro ADME properties showed in vivo procognitive-like and antidepressant-
18

like activity in well-established experimental tests in rats. The in vitro studies suggested that the
behavioral effects of compound 22 were most likely related to its antagonistic properties toward 5-
HT1A receptor; however, the detailed selectivity and pharmacokinetic studies are needed to establish
the participation of individual targets in in vivo activity. The obtained compound 22 represents a new
lead structure for further optimization to find more active and metabolically stable derivatives with
procognitive and antidepressant activity.
4. Materials and methods
4.1.In silico studies
The design of compounds was supported by calculation of molecular properties and prediction of
physicochemical properties, BBB permeation, and risk of toxicity using appropriate software. The
calculation of molecular properties and prediction of physicochemical characteristics were performed
using Instant JChem software [49]. PAINS alerts and the ability of compound to cross the BBB was
checked using SwissADME server [50]. OSIRIS Property Explorer was used to estimate the risk of
toxicity [39]. The prediction of metabolic biotransformation was performed using MetaSite 6.0.1
(Molecular Discovery Ltd, UK) [51].
The studies on the binding mode were performed using Small-Molecule Drug Discovery Suite
(Schrӧdinger Ltd.) [52]. Ligand structures were optimized using LigPrep tool and OPLS-3 force field.
The structural models of serotonin receptors 5-HT1A and 5-HT7 and PDEs 4 and 7 have been described
previously [4,43]. The docking procedure was carried out by Glide XP tool using default settings.
Complexes were additionally optimized by Prime tool (Refine Protein-Ligand Complex) in order to
minimize their energy and correct ligand arrangement in the active site. The results were analyzed for
both Glide gscore and the presence of proper interactions.
4.2.Synthesis
All the reagents and solvents were purchased from Sigma-Aldrich, TCI Europe, or Fluorochem.
Thin layer chromatography was performed on Merck silica gel 60 F254 aluminium sheets (Merck,
Darmstadt, Germany) with the following solvent: (A) dichloromethane/methanol (9:1). Spots were
detected by their absorption under UV light (λ = 254 nm). Column chromatography was performed on
Merck silica gel 60 (63-200 mm) with the solvent A. 1H NMR and 13C NMR spectra were recorded on
Varian Mercury 300 at 300 MHz and 75 MHz, respectively, using TMS (0.00 ppm) as an internal
standard and DMSO-d6 as solvent. Chemical shifts were expressed in δ (ppm) and the coupling
constants J in Hertz (Hz) and splitting patterns were designated as follows: s (singlet), b.s. (broad
singlet), d (doublet), t (triplet), q (quartet), and m (multiplet). LC/MS analyses were performed on
Waters Acquity TQD apparatus with eλ DAD detector. For mass spectrometry, ESI+ (electrospray
positive) ionization mode was used. UV spectra were taken in the range of 200-700 nm. For
establishing the purity of compounds, UV chromatograms were used. The UPLC/MS purity of all the
19

synthesized compounds was determined to be over 98 %. Melting points (mp) were determined with a
Büchi apparatus and are uncorrected. Elemental analyses were taken with Elementar Vario EL III
apparatus. Analyses indicated by the symbols of the elements were within ± 0.4 % of the theoretical
values.
4.2.1. General procedure for the synthesis of ethyl ω-(4-(2-methoxyphenyl)piperazin-1-yl)alkanoates
(1-3) and ω-(4-(2-methoxyphenyl)piperazin-1-yl)alkanoic acids (4-6)
The synthesis of the intermediates 1-3 from 1-(2-methoxyphenyl)piperazine and 4-6 from ethyl ω-(4-
(2-methoxyphenyl)piperazin-1-yl)alkanoates 1-3 has been reported previously [53]:
•
•
•
•
•
•
Ethyl 4-(4-(2-methoxyphenyl)piperazin-1-yl)butanoate (1),
Ethyl 5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanoate (2),
Ethyl 6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanoate (3),
4-(4-(2-Methoxyphenyl)piperazin-1-yl)butanoic acid (4),
5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentanoic acid (5),
6-(4-(2-Methoxyphenyl)piperazin-1-yl)hexanoic acid (6).
4.2.2. General procedure for the synthesis of hydrochlorides of the ω-(4-(2-
methoxyphenyl)piperazin-1-yl)alkanamides (7-39)
A mixture of 1 mmol of alkanoic acid 4, 5, or 6 with 1.5 mmol of CDI in DMF (5 mL) was stirred at
room temperature for 30 minutes. Then, 1 mmol of appropriate aniline or benzylamine was added and
stirring was continued for 72 hours. Afterwards, 2-3 drops of water were added and the mixture was
concentrated under reduced pressure. The obtained crude product was purified by silica gel
chromatography with solvent A as eluent. The hydrochlorides of compounds 7-39 were prepared by
dissolving the compounds in a minimum quantity of methanol. Then, the solution was treated with 1 N
solution of HCl(g) in methanol, evaporated under reduced pressure, washed, and dried.
4.2.2.1. N-(5-(tert-butyl)-2-hydroxyphenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide
hydrochloride (7)
The title compound was obtained from 4 and 2-amino-4-(tert-butyl)phenol as a white solid; yield 59
%; mp 176-178 °C; Rf = 0.42 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.21 (s, 9H, C(CH3)3), 1.89 -
2.13 (m, 2H, CH2CH2CH2), 2.53 (t, J = 7.00 Hz, 2H, CH2CH2CH2), 2.94 - 3.23 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2), 3.42 - 3.60 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.78 (d, J
= 8.79 Hz, 1H, 3-Ph’), 6.84 - 7.07 (m, 5H, 3,4,5,6-Ph, 4-Ph’), 7.74 (d, J = 2.34 Hz, 1H, 6-Ph’), 9.44 (s,
1H, CONH), 9.55 (s, 1H, OH), 10.65 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.8,
31.8, 33.2, 34.2, 47.3, 51.6, 55.6, 55.8, 112.3, 116.0, 118.6, 120.0, 121.3, 121.9, 123.9, 126.0, 139.9,
20

141.6, 146.1, 152.2, 171.0; LC/MS: m/z calc. 426.28, found 426.27; MW 462.02; Anal. C25H36ClN3O3
(C, H, N).
4.2.2.2. N-(3-(tert-butyl)phenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (8)
The title compound was obtained from 4 and 3-(tert-butyl)aniline as a white solid; yield 69 %; mp
201-203 °C; Rf = 0.39 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.24 (s, 9H, C(CH3)3), 1.97 - 2.11
(m, 2H, CH2CH2CH2), 2.43 (t, J = 7.03 Hz, 2H, CH2CH2CH2), 3.00 - 3.22 (m, 6H, N(CH2CH2)2NH+,
CH2CH2CH2), 3.40 - 3.61 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.85 - 7.08 (m, 5H, 3,4,5,6-
Ph, 4-Ph’), 7.19 (t, J = 7.91 Hz, 1H, 5-Ph’), 7.48 (d, J = 7.60 Hz, 1H, 6-Ph’), 7.61 (s, 1H, 2-Ph’), 10.11
(s, 1H, CONH), 10.92 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ 19.7, 31.6, 33.6, 34.8, 47.3,
51.6, 55.5, 55.8, 112.3, 116.6, 116.8, 118.6, 120.5, 121.3, 123.8, 128.7, 139.4, 139.9, 151.6, 152.2,
170.3; LC/MS: m/z calc. 410.28, found 410.25; MW 446.03; Anal. C25H36ClN3O2 (C, H, N).
4.2.2.3. N-(3-isopropylphenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (9)
The title compound was obtained from 4 and 3-isopropylaniline as a white solid; yield 57 %; mp 197-
199 °C; Rf = 0.41 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.16 (d, J = 7.03 Hz, 6H, CH(CH3)2),
1.95 - 2.11 (m, 2H, CH2CH2CH2), 2.43 (t, J = 7.03 Hz, 2H, CH2CH2CH2), 2.72 - 2.89 (m, 1H,
CH(CH3)2), 2.99 - 3.19 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2), 3.42 - 3.61 (m, 4H,
N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.84 - 7.05 (m, 5H, 3,4,5,6-Ph, 4-Ph’), 7.18 (t, J = 7.91 Hz,
1H, 5-Ph’), 7.43 (d, J = 8.21 Hz, 1H, 6-Ph’), 7.48 (s, 1H, 2-Ph’), 10.07 (s, 1H, CONH), 10.75 (br.s.,
1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.7, 24.3, 33.6, 33.9, 47.2, 51.5, 55.5, 55.8, 112.3,
117.2, 117.5, 118.6, 121.3, 121.6, 123.8, 129.0, 139.7, 139.9, 149.3, 152.2, 170.3; LC/MS: m/z calc.
396.26, found 396.29; MW 432.00; Anal. C24H34ClN3O2 (C, H, N).
4.2.2.4. N-(4-(tert-butyl)phenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (10)
The title compound was obtained from 4 and 4-(tert-butyl)aniline as a white solid; yield 58 %; mp
196-198 °C; Rf = 0.44 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.23 (s, 9H, C(CH3)3), 1.76 (m, J =
7.03 Hz, 2H, CH2CH2CH2), 2.21 - 2.42 (m, J = 7.03 Hz, 4H, CH2CH2CH2), 2.51 (m, 2H,
N(CH2CH2)2NH+), 2.93 (m, 4H, N(CH2CH2)2NH+), 3.74 (s, 3H, OCH3), 6.76 - 6.99 (m, J = 2.34 Hz,
4H, 3,4,5,6-Ph), 7.27 (m, J = 8.79 Hz, 2H, 3,5-Ph’), 7.49 (m, J = 8.79 Hz, 2H, 2,6-Ph’), 9.77 (s, 1H,
CONH), 10.78 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.7, 31.6, 33.6, 34.4, 47.3,
51.6, 55.5, 55.8, 112.4, 118.6, 119.4, 121.3, 123.9, 125.7, 137.1, 139.9, 145.8, 152.2, 170.2; LC/MS:
m/z calc. 410.28, found 410.32; MW 446.03; Anal. C25H36ClN3O2 (C, H, N).
4.2.2.5. N-(4-isopropylphenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (11)
The title compound was obtained from 4 and 4-isopropylaniline as a white solid; yield 59 %; mp 200-
1
202 °C; Rf = 0.40 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.15 (d, J = 7.03 Hz, 6H, CH(CH3)2),
21

1.83 - 2.05 (m, 2H, CH2CH2CH2), 2.39 (t, J = 7.03 Hz, 2H, CH2CH2CH2), 2.72 - 2.85 (m, 1H,
CH(CH3)2), 2.85 - 3.47 (m, 10H, N(CH2CH2)2NH+, CH2CH2CH2), 3.77 (s, 3H, OCH3), 6.84 - 7.03 (m,
4H, 3,4,5,6-Ph), 7.14 (d, J = 8.79 Hz, 2H, 3,5-Ph’), 7.50 (d, J = 8.21 Hz, 2H, 2,6-Ph’), 9.97 (s, 1H,
CONH), 10.73 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 20.3, 24.4, 33.3, 33.8, 47.9,
52.0, 55.8, 56.0, 112.3, 118.6, 119.7, 121.3, 123.6, 126.8, 137.5, 140.2, 143.5, 152.3, 170.4; LC/MS:
m/z calc. 396.26, found 396.22; MW 432.00; Anal. C24H34ClN3O2 (C, H, N).
4.2.2.6. N-(4-(sec-butyl)phenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (12)
The title compound was obtained from 4 and 4-(sec-butyl)aniline as a white solid; yield 55 %; mp
1
168-170 °C; Rf = 0.42 (A); H NMR (300 MHz, DMSO-d6) δ ppm 0.73 (t, J = 7.60 Hz, 3H,
CH(CH3)CH2CH3), 1.14 (d, J = 6.45 Hz, 3H, CH(CH3)CH2CH3), 1.38 - 1.60 (m, 2H,
CH(CH3)CH2CH3), 1.93 - 2.12 (m, 2H, CH2CH2CH2), 2.34 - 2.46 (t, J = 7.03 Hz, 2H, CH2CH2CH2),
2.93 - 3.24 (m, 7H, N(CH2CH2)2NH+, CH2CH2CH2, CH(CH3)CH2CH3), 3.38 - 3.61 (m, 4H,
N(CH2CH2)2NH+,), 3.77 (s, 3H, OCH3), 6.81 - 7.04 (m, 4H, 3,4,5,6-Ph), 7.10 (d, J = 8.80 Hz, 2H, 3,5-
13
Ph’), 7.50 (d, J = 8.20 Hz, 2H, 2,6-Ph’), 10.05 (s, 1H, CONH), 10.74 (br.s., 1H, NH+); C NMR (75
MHz, DMSO-d6) δ ppm 12.5, 19.7, 22.3, 31.0, 33.6, 40.8, 47.2, 51.6, 55.5, 55.8, 112.4, 118.6, 119.7,
121.3, 123.8, 127.4, 137.5, 139.9, 142.3, 152.2, 170.2; LC/MS: m/z calc. 410.28, found 410.25; MW
445.25; Anal. C25H36ClN3O2 (C, H, N).
4.2.2.7. Tert-butyl 4-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamido)benzoate hydrochloride (13)
The title compound was obtained from 4 and tert-butyl 4-aminobenzoate as a white solid; yield 79 %;
1
mp 180-182 °C; Rf = 0.42 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.51 (s, 9H, C(CH3)3), 1.96 -
2.13 (m, 2H, CH2CH2CH2), 2.53 (t, J = 7.00 Hz, 2H, CH2CH2CH2), 3.01 - 3.22 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2), 3.38 - 3.53 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.75 -
7.09 (m, 4H, 3,4,5,6-Ph), 7.73 (d, J = 8.80 Hz, 2H, 3,5-Ph’), 7.83 (d, J = 8.80 Hz, 2H, 2,6-Ph’), 10.55
(s, 1H, CONH), 10.90 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.5, 28.3, 33.7, 47.3,
51.6, 55.5, 55.8, 80.7, 112.3, 118.6, 118.7, 121.3, 123.9, 126.0, 130.5, 139.9, 143.7, 152.2, 165.0,
171.0; LC/MS: m/z calc. 454.27, found 454.18; MW 490.03; Anal. C26H36ClN3O4 (C, H, N).
4.2.2.8. N-(2-hydroxyphenyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride (14)
The title compound was obtained from 4 and 2-aminophenol as a white solid; yield 55 %; mp 229-231
1
°C; Rf = 0.38 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.91 - 2.13 (m, 2H, CH2CH2CH2), 2.52 -
2.56 (t, J = 7.03 Hz, 2H, CH2CH2CH2), 2.93 - 3.25 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2), 3.37 -
3.61 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.67 - 6.79 (m, 1H, 3-Ph’), 6.82 - 7.06 (m, 6H,
3,4,5,6-Ph, 4,5-Ph’), 7.74 (d, J = 7.03 Hz, 1H, 6-Ph’), 9.38 (s, 1H, CONH), 9.80 (s, 1H, OH), 10.67
(br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.8, 33.3, 47.3, 51.6, 55.6, 55.8, 112.4,
22

116.3, 118.6, 119.3, 121.3, 123.0, 123.9, 125.1, 126.6, 139.9, 148.5, 152.2, 170.8; LC/MS: m/z calc.
370.21, found 370.17; MW 405.92; Anal. C21H28ClN3O3 (C, H, N).
4.2.2.9. 2-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butanamido)benzamide hydrochloride (15)
The title compound was obtained from 4 and 2-aminobenzamide as a white solid; yield 60 %; mp 209-
211 °C; Rf = 0.33 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.99 - 2.12 (m, 2H, CH2CH2CH2), 2.41
(t, J = 7.03 Hz, 2H, CH2CH2CH2), 2.96 - 3.24 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2), 3.39 - 3.61 (m,
4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.83 - 7.04 (m, 4H, 3,4,5,6-Ph), 7.10 (t, J = 7.00 Hz, 1H,
5-Ph’), 7.47 (t, J = 7.60 Hz, 1H, 4-Ph’), 7.79 (dd, J = 7.91, 1.47 Hz, 1H, 3-Ph’), 8.28 (br.s., 2H,
CONH2), 8.42 (d, J = 7.62 Hz, 1H, 6-Ph’), 10.70 (br.s., 1H, CONH), 11.68 (s, 1H, NH+); 13C NMR (75
MHz, DMSO-d6) δ ppm 18.9, 34.0, 46.7, 51.1, 54.9, 55.3, 111.8, 118.1, 119.9, 120.3, 120.8, 122.4,
123.3, 128.6, 132.0, 139.3, 139.4, 151.7, 169.7, 170.6; LC/MS: m/z calc. 397.22, found 397.22; MW
432.94; Anal. C22H29ClN4O3 (C, H, N).
4.2.2.10.
(16)
N-(2-hydroxybenzyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide hydrochloride
The title compound was obtained from 4 and 2-(aminomethyl)phenol as a white solid; yield 63 %; mp
1
144-146 °C; Rf = 0.40 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.86 - 2.04 (m, 2, CH2CH2CH2),
2.20 - 2.32 (m, 2H, CH2CH2CH2), 2.93 - 3.20 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2), 3.43 - 3.56 (m,
4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 4.19 (d, J = 5.86 Hz, 2H, CONHCH2), 6.70 - 7.11 (m, 8H,
3,4,5,6-Ph, 3,4,5,6-Ph’), 8.39 (t, J = 5.86 Hz, 1H, CONH), 9.59 (s, 1H, OH), 10.65 (br.s., 1H, NH+);
13C NMR (75 MHz, DMSO-d6) δ ppm 19.9, 32.5, 38.0, 47.3, 51.5, 55.6, 55.8, 112.4, 115.5, 118.6,
119.2, 121.3, 123.9, 125.5, 128.3, 129.0, 139.8, 152.2, 155.4, 171.8; LC/MS: m/z calc. 384.23, found
384.26; MW 419.94; Anal. C22H30ClN3O3 (C, H, N).
4.2.2.11.
N-(2-methoxybenzyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)butanamide
hydrochloride (17)
The title compound was obtained from 4 and (2-methoxyphenyl)methanamine as a white solid; yield
1
82 %; mp 97-99 °C; Rf = 0.40 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.90 - 2.05 (m, 2H,
CH2CH2CH2), 2.23 - 2.32 (m, 2H, CH2CH2CH2), 2.93 - 3.21 (m, 6H, (CH2CH2)2NH+, CH2CH2CH2),
3.39 - 3.57 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, CH3OPh), 3.78 (s, 2H, CH3OPh’), 4.22 (d, J = 5.86
Hz, 2H, CONHCH2), 6.79 - 7.07 (m, 6H, 3,4,5,6-Ph, 3,5-Ph’), 7.08 - 7.28 (m, 2H, 4,6-Ph’), 8.33 (t, J =
5.86 Hz, 1H, CONH), 10.83 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 19.9, 32.6, 37.6,
47.2, 51.5, 55.6, 55.7, 55.8, 110.9, 112.3, 118.6, 120.6, 121.3, 123.9, 127.1, 128.2, 128.5, 139.9,
152.2, 157.0, 171.5; LC/MS: m/z calc. 398.24, found 398.22; MW 433.97; Anal. C23H32ClN3O3 (C, H,
N).
23

4.2.2.12.
N-(5-(tert-butyl)-2-hydroxyphenyl)-5-(4-(2-methoxyphenyl)piperazin-1-
yl)pentanamide hydrochloride (18)
The title compound was obtained from 5 and 2-amino-4-(tert-butyl)phenol as a white solid; yield 63
1
%; mp 86-88 °C; Rf = 0.49 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.20 (s, 9H, C(CH3)3), 1.54 -
1.69 (m, 2H, CH2CH2CH2CH2), 1.69 - 1.85 (m, 2H, CH2CH2CH2CH2), 2.91 - 3.23 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.47 - 3.59 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.77
(d, J = 8.21 Hz, 1H, 3-Ph’), 6.83 - 7.07 (m, 5H, 3,4,5,6-Ph, 4-Ph’), 7.69 (d, J = 2.34 Hz, 1H, 6-Ph’),
9.45 (s, 1H, CONH), 9.57 (s, 1H, OH), 10.55 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm
22.9, 23.1, 31.8, 34.2, 35.6, 47.3, 51.5, 55.6, 55.8, 112.3, 116.3, 118.6, 119.9, 121.3, 122.0, 123.9,
126.1, 139.8, 141.6, 146.1, 152.2, 171.9; LC/MS: m/z calc. 440.29, found 440.16; MW 476.05; Anal.
C26H38ClN3O3 (C, H, N).
4.2.2.13. N-(3-(tert-butyl)phenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide hydrochloride
(19)
The title compound was obtained from 5 and 3-(tert-butyl)aniline as a white solid; yield 78 %; mp
1
216-218 °C; Rf = 0.45 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.23 (s, 9H, C(CH3)3), 1.55 - 1.67
(m, 2H, CH2CH2CH2CH2), 1.69 - 1.84 (m, 2H, CH2CH2CH2CH2), 2.36 (t, J = 7.33 Hz, 2H,
CH2CH2CH2CH2), 3.03 - 3.21 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.38 - 3.57 (m, 4H,
N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.85 - 7.07 (m, 5H, 3,4,5,6-Ph, 4-Ph’), 7.18 (t, J = 7.91 Hz,
1H, 5-Ph’), 7.45 - 7.53 (m, 1H, 6-Ph’), 7.61 (m, 1H, 2-Ph’), 10.04 (s, 1H, CONH), 10.95 (br.s., 1H,
NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 22.7, 23.2, 31.6, 34.8, 36.1, 47.3, 51.4, 55.6, 55.8, 112.4,
116.5, 116.8, 118.7, 120.4, 121.3, 124.1, 128.7, 139.5, 139.6, 151.6, 152.2, 171.1; LC/MS: m/z calc.
424.30, found 424.34; MW 460.05; Anal. C26H38ClN3O2 (C, H, N).
4.2.2.14.
N-(3-isopropylphenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (20)
The title compound was obtained from 5 and 3-isopropylaniline as a white solid; yield 64 %; mp 227-
1
229 °C; Rf = 0.47 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.16 (d, J = 7.03 Hz, 6H, CH(CH3)2),
1.53 - 1.68 (m, 2H, CH2CH2CH2CH2), 1.69 - 1.84 (m, 2H, CH2CH2CH2CH2), 2.36 (t, J = 7.03 Hz, 2H,
CH2CH2CH2CH2), 2.80 (m, 1H, CH(CH3)2), 2.98 - 3.18 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2),
3.39 - 3.58 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.79 - 7.04 (m, 5H, 3,4,5,6-Ph, 4-Ph’),
7.17 (t, J = 7.62 Hz, 1H, 5-Ph’), 7.37 - 7.52 (m, 2H, 2,6-Ph’), 10.00 (s, 1H, CONH), 10.69 (br.s., 1H,
NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 22.8, 23.1, 24.3, 33.9, 36.1, 47.2, 51.5, 55.6, 55.8, 112.3,
117.1, 117.4, 118.6, 121.3, 121.5, 123.9, 128.9, 139.8, 139.9, 149.3, 152.2, 171.1; LC/MS: m/z calc.
410.28, found 410.32; MW 446.03; Anal. C25H36ClN3O2 (C, H, N).
24

4.2.2.15.
N-(4-(tert-butyl)phenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (21)
The title compound was obtained from 5 and 4-(tert-butyl)aniline as a white solid; yield 62 %; mp
1
222-224 °C; Rf = 0.45 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.23 (s, 9H, C(CH3)3), 1.53 - 1.84
(m, 4H, CH2CH2CH2CH2), 2.35 (t, J = 6.74 Hz, 2H, CH2CH2CH2CH2), 2.94 - 3.22 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.46 (t, J = 14.07 Hz, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3),
6.83 - 7.05 (m, 4H, 3,4,5,6-Ph), 7.28 (d, J = 8.21 Hz, 2H, 3,5-Ph’), 7.51 (d, J = 8.79 Hz, 2H, 2,6-Ph’),
13
9.97 (s, 1H, CONH), 10.65 (br.s., 1H, NH+); C NMR (75 MHz, DMSO-d6) δ ppm 22.8, 23.1, 31.7,
34.4, 36.0, 47.2, 51.5, 55.6, 55.8, 112.3, 118.6, 119.3, 121.3, 123.9, 125.6, 137.2, 139.8, 145.7, 152.2,
171.0; LC/MS: m/z calc. 424.30, found 424.21; MW 460.05; Anal. C26H38ClN3O2 (C, H, N).
4.2.2.16.
N-(4-isopropylphenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (22)
The title compound was obtained from 5 and 4-isopropylaniline as a white solid; yield 64 %; mp 204-
1
206 °C; Rf = 0.45 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.15 (d, J = 6.45 Hz, 6H, CH(CH3)2),
1.53 - 1.84 (m, 4H, CH2CH2CH2CH2), 2.35 (t, J = 7.03 Hz, 2H, CH2CH2CH2CH2), 2.80 (m, 1H,
CH(CH3)2), 2.94 - 3.20 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.48 (t, J = 11.00 Hz, 4H,
N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.83 - 7.05 (m, 4H, 3,4,5,6-Ph), 7.13 (d, J = 8.20 Hz, 2H, 3,5-
13
Ph’), 7.50 (d, J = 8.20 Hz, 2H, 2,6-Ph’), 9.98 (s, 1H, CONH), 10.70 (br.s., 1H, NH+); C NMR (75
MHz, DMSO-d6) δ ppm 22.8, 23.1, 24.4, 33.3, 36.0, 47.2, 51.4, 55.6, 55.8, 112.3, 118.6, 119.6, 121.3,
123.9, 126.7, 137.6, 139.8, 143.4, 152.2, 170.9; LC/MS: m/z calc. 410.28, found 410.25; MW 446.03;
Anal. C25H36ClN3O2 (C, H, N).
4.2.2.17.
N-(4-(sec-butyl)phenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (23)
The title compound was obtained from 5 and 4-(sec-butyl)aniline as a white solid; yield 67 %; mp
1
198-200 °C; Rf = 0.47 (A); H NMR (300 MHz, DMSO-d6) δ ppm 0.73 (t, J = 7.33 Hz, 3H,
CH(CH3)CH2CH3), 1.13 (d, J = 7.03 Hz, 3H, CH(CH3)CH2CH3), 1.42 - 1.55 (m, 2H,
CH(CH3)CH2CH3), 1.56 - 1.68 (m, 2H, CH2CH2CH2CH2), 1.69 - 1.84 (m, 2H, CH2CH2CH2CH2), 2.35
(t, J = 7.03 Hz, 2H, CH2CH2CH2CH2), 2.94 - 3.21 (m, 7H, N(CH2CH2)2NH+, CH2CH2CH2CH2,
CH(CH3)CH2CH3), 3.38 - 3.56 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.83 - 7.04 (m, 4H,
3,4,5,6-Ph), 7.08 (d, J = 8.80 Hz, 2H, 3,5-Ph’), 7.51 (d, J = 8.20 Hz, 2H, 2,6-Ph’), 9.97 (s, 1H,
CONH), 10.69 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 12.5, 22.3, 22.9, 23.1, 31.1,
36.1, 40.8, 47.2, 51.5, 55.6, 55.8, 112.3, 118.6, 119.6, 121.3, 123.8, 127.3, 137.6, 139.9, 142.1, 152.2,
170.9; LC/MS: m/z calc. 424.30, found 424.27; MW 460.05; Anal. C26H38ClN3O2 (C, H, N).
25

4.2.2.18.
Tert-butyl
4-(5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamido)benzoate
hydrochloride (24)
The title compound was obtained from 5 and tert-butyl 4-aminobenzoate as a white solid; yield 57 %;
1
mp 242-244 °C; Rf = 0.47 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.50 (s, 9H, C(CH3)3), 1.55 -
1.68 (m, 2H, CH2CH2CH2CH2), 1.68 - 1.79 (m, 2H, CH2CH2CH2CH2), 2.42 (t, J = 6.74 Hz, 2H,
CH2CH2CH2CH2), 2.93 - 3.37 (m, 10H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.76 (s, 3H, OCH3), 6.84
- 7.02 (m, 4H, 3,4,5,6-Ph), 7.73 (d, J = 9.40 Hz, 2H, 3,5-Ph’), 7.82 (d, J = 8.80 Hz, 2H, 2,6-Ph’), 10.44
(s, 1H, CONH), 10.80 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 22.6, 23.3, 28.3, 36.2,
47.5, 51.7, 55.8, 80.6, 110.0, 112.3, 118.6, 118.7, 121.3, 123.8, 125.9, 130.4, 140.0, 143.8, 152.2,
165.1, 171.9; LC/MS: m/z calc. 468.34, found 468.29; MW 504.06; Anal. C27H38ClN3O4 (C, H, N).
4.2.2.19.
N-(2-hydroxyphenyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (25)
The title compound was obtained from 5 and 2-aminophenol as a white solid; yield 52 %; mp 218-220
°C; Rf = 0.43 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.57 - 1.70 (m, 2H, CH2CH2CH2CH2), 1.70 -
1.79 (m, 2H, CH2CH2CH2CH2), 2.43 (t, J = 6.50 Hz, 2H, CH2CH2CH2CH2), 2.96 - 3.18 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.46 (t, J = 12.30 Hz, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3),
6.67 - 6.80 (m, 1H, 3-Ph’), 6.84 - 7.04 (m, 6H, 3,4,5,6-Ph, 4,5-Ph’), 7.71 (d, J = 7.03 Hz, 1H, 6-Ph’),
9.34 (s, 1H, CONH), 9.79 (s, 1H, OH), 10.50 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm
22.9, 23.1, 35.6, 47.2, 51.5, 55.6, 55.8, 112.3, 116.4, 118.6, 119.3, 121.3, 122.9, 123.9, 125.1, 126.8,
139.9, 148.4, 152.2, 171.7; LC/MS: m/z calc. 384.23, found 384.13; MW 419.94; Anal. C22H30ClN3O3
(C, H, N).
4.2.2.20.
2-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentanamido)benzamide hydrochloride (26)
The title compound was obtained from 5 and 2-aminobenzamide as a white solid; yield 58 %; mp 199-
1
201 °C; Rf = 0.48 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.54 - 1.70 (m, 2H, CH2CH2CH2CH2),
1.70 - 1.91 (m, 2H, CH2CH2CH2CH2), 2.41 (t, J = 7.03 Hz, 2H, CH2CH2CH2CH2), 2.93 - 3.09 (m, 6H,
N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.37 - 3.61 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.81
- 7.05 (m, 4H, 3,4,5,6-Ph), 7.10 (t, J = 7.60 Hz, 1H, 5-Ph’), 7.47 (t, J = 7.00 Hz, 1H, 4-Ph’), 7.79 (dd,
J = 7.91, 1.47 Hz, 1H, 3-Ph’), 8.28 (br.s., 2H, CONH2), 8.44 (d, J = 7.60 Hz, 1H, 6-Ph’), 10.61 (br.s.,
13
1H, CONH), 11.67 (s, 1H, NH+); C NMR (75 MHz, DMSO-d6) δ ppm 22.5, 23.0, 37.1, 47.2, 51.5,
55.6, 55.8, 112.3, 118.6, 120.3, 120.7, 121.3, 122.8, 123.9, 129.1, 132.6, 139.9, 140.0, 152.2, 170.9,
171.2; LC/MS: m/z calc. 411.24, found 411.38; MW 446.97; Anal. C23H31ClN4O3 (C, H, N).
4.2.2.21.
N-(2-hydroxybenzyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (27)
26

The title compound was obtained from 5 and 2-(aminomethyl)phenol as a white solid; yield 69 %; mp
207-209 °C; Rf = 0.39 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.52 - 1.63 (m, 2H,
CH2CH2CH2CH2), 1.63 - 1.74 (m, 2H, CH2CH2CH2CH2), 2.20 (t, J = 7.03 Hz, 2H, CH2CH2CH2CH2),
2.98 - 3.14 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.45 (t, J = 7.60 Hz, 4H, N(CH2CH2)2NH+),
3.77 (s, 3H, OCH3), 4.18 (d, J = 5.27 Hz, 2H, CONHCH2), 6.70 - 7.11 (m, 8H, 3,4,5,6-Ph, 3,4,5,6-
Ph’), 8.34 (t, J = 5.86 Hz, 1H, CONH), 9.61 (s, 1H, OH), 10.61 (br.s., 1H, NH+); 13C NMR (75 MHz,
DMSO-d6) δ ppm 22.9, 23.1, 34.9, 37.9, 47.3, 51.5, 55.6, 55.8, 112.3, 115.6, 118.6, 119.2, 121.3,
123.9, 125.7, 128.3, 129.1, 139.8, 152.2, 155.4, 172.6; LC/MS: m/z calc. 398.24, found 398.29; MW
433.97; Anal. C23H32ClN3O3 (C, H, N).
4.2.2.22.
N-(2-methoxybenzyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (28)
The title compound was obtained from 5 and (2-methoxyphenyl)methanamine as a white solid; yield
1
57 %; mp 213-215 °C; Rf = 0.43 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.43 - 1.62 (m, 2H,
CH2CH2CH2CH2), 1.62 - 1.79 (m, 2H, CH2CH2CH2CH2), 2.20 (t, J = 7.03 Hz, 2H, CH2CH2CH2CH2),
2.90 - 3.19 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.36 - 3.57 (m, 4H, N(CH2CH2)2NH+), 3.77
(s, 3H, CH3OPh), 3.78 (s, 3H, CH3OPh’), 4.21 (d, J = 5.86 Hz, 2H, CONHCH2), 6.80 - 7.05 (m, 6H,
3,4,5,6-Ph, 3,5-Ph’), 7.10 - 7.27 (m, 2H, 4,6-Ph’), 8.22 (t, J = 5.57 Hz, 1H, CONH), 10.69 (br.s., 1H,
NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 23.0, 23.2, 35.0, 37.4, 47.3, 51.5, 55.7, 55.7, 55.8, 110.9,
112.3, 118.6, 120.6, 121.3, 123.9, 127.3, 128.1, 128.4, 139.9, 152.2, 157.0, 172.2; LC/MS: m/z calc.
412.26, found 412.24; MW 448.00; Anal. C24H34ClN3O3 (C, H, N).
4.2.2.23.
N-(4-(tert-butyl)benzyl)-5-(4-(2-methoxyphenyl)piperazin-1-yl)pentanamide
hydrochloride (29)
The title compound was obtained from 5 and (4-(tert-butyl)phenyl)methanamine as a white solid;
1
yield 78 %; mp 119-121 °C; Rf = 0.49 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.24 (s, 9H,
C(CH3)3), 1.56 (d, J = 7.03 Hz, 2H, CH2CH2CH2CH2), 1.61 - 1.83 (m, 2H, CH2CH2CH2CH2), 2.18 (t, J
= 7.03 Hz, 2H, CH2CH2CH2CH2), 3.00 - 3.16 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2), 3.39 - 3.57
(m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 4.20 (d, J = 5.86 Hz, 2H, CONHCH2), 6.84 - 7.05 (m,
4H, 3,4,5,6-Ph), 7.13 - 7.19 (m, J = 8.21 Hz, 2H, 3,5-Ph’), 7.28 - 7.35 (m, 2H, 2,6-Ph’), 8.36 (s, 1H,
CONH), 10.64 (br.s., 1H, NH+); 13C NMR (75 MHz, DMSO-d6) δ ppm 23.0, 23.1, 31.6, 34.6, 35.0,
42.2, 47.2, 51.4, 55.6, 55.8, 112.3, 118.6, 121.3, 123.9, 125.4, 127.5, 137.1, 139.9, 149.5, 152.2,
172.0; LC/MS: m/z calc. 438.31, found 438.23; MW 474.08; Anal. C27H40ClN3O2 (C, H, N).
4.2.2.24.
N-(5-(tert-butyl)-2-hydroxyphenyl)-6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanamide
hydrochloride (30)
27

The title compound was obtained from 6 and 2-amino-4-(tert-butyl)phenol as a white solid; yield 73
1
%; mp 63-65 °C; Rf = 0.48 (A); H NMR (300 MHz, DMSO-d6) δ ppm 1.20 (s, 9H, C(CH3)3), 1.26 -
1.42 (m, 2H, CH2CH2CH2CH2CH2), 1.52 - 1.92 (m, 4H, CH2CH2CH2CH2CH2), 2.42 (t, J = 7.03 Hz,
2H, CH2CH2CH2CH2CH2), 2.88 - 3.24 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2CH2), 3.36 - 3.51
(m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.77 (d, J = 8.21 Hz, 1H, 3-Ph’), 6.82 - 7.11 (m, 5H,
3,4,5,6-Ph, 4-Ph’), 7.67 (d, J = 2.34 Hz, 1H, 6-Ph’), 9.46 (s, 1H, CONH), 9.59 (br.s., 1H, OH), 10.72
13
(br.s., 1H, NH+); C NMR (75 MHz, DMSO-d6) δ ppm 23.2, 25.2, 26.1, 31.8, 34.2, 35.9, 47.3, 51.4,
55.7, 55.8, 112.3, 116.4, 118.6, 119.8, 121.3, 122.0, 124.0, 126.2, 139.7, 141.7, 146.1, 152.2, 172.4;
LC/MS: m/z calc. 453.62, found 454.31; MW 490.08; Anal. C27H40ClN3O3 (C, H, N).
4.2.2.25.
N-(3-(tert-butyl)phenyl)-6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanamide
hydrochloride (31)
The title compound was obtained from 6 and 3-(tert-butyl)aniline as a white solid; yield 73 %; mp
143-145 °C; Rf = 0.46 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.23 (s, 9H, C(CH3)3), 1.28 - 1.38
(m, 2H, CH2CH2CH2CH2CH2), 1.58 - 1.78 (m, 4H, CH2CH2CH2CH2CH2), 2.32 (t, J = 7.33 Hz, 2H,
CH2CH2CH2CH2CH2), 2.99 - 3.18 (m, 6H, N(CH2CH2)2NH+, CH2CH2CH2CH2CH2), 3.40 - 3.58 (m,
4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.84 - 7.07 (m, 5H, 3,4,5,6-Ph, 4-Ph’), 7.18 (t, J = 7.91
Hz, 1H, 5-Ph’), 7.48 (d, J = 8.79 Hz, 1H, 6-Ph’), 7.59 (s, 1H, 2-Ph’), 9.93 (s, 1H, CONH), 10.62 (br.s.,
13
1H, NH+); C NMR (75 MHz, DMSO-d6) δ ppm 23.2, 25.1, 26.2, 31.6, 34.8, 36.5, 47.2, 51.5, 55.7,
55.8, 112.3, 116.4, 116.7, 118.6, 120.3, 121.3, 123.9, 128.7, 139.6, 139.9, 151.5, 152.2, 171.5;
LC/MS: m/z calc. 438.31, found 438.30; MW 474.08; Anal. C27H40ClN3O2 (C, H, N).
4.2.2.26.
N-(3-isopropylphenyl)-6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanamide
hydrochloride (32)
The title compound was obtained from 6 and 3-isopropylaniline as a white solid; yield 72 %; mp 166-
168 °C; Rf = 0.48 (A); 1H NMR (300 MHz, DMSO-d6) δ ppm 1.16 (d, J = 6.45 Hz, 6H, CH(CH3)2),
1.24 - 1.38 (m, 2H, CH2CH2CH2CH2CH2), 1.54 - 1.82 (m, 4H, CH2CH2CH2CH2CH2), 2.32 (t, J = 7.33
Hz, 2H, CH2CH2CH2CH2CH2), 2.71 - 2.85 (m, 1H, CH(CH3)2), 2.92 - 3.20 (m, 6H, N(CH2CH2)2NH+,
CH2CH2CH2CH2CH2), 3.38 - 3.57 (m, 4H, N(CH2CH2)2NH+), 3.77 (s, 3H, OCH3), 6.83 - 7.05 (m, 5H,
3,4,5,6-Ph, 4-Ph’), 7.17 (t, J = 7.91 Hz, 1H, 5-Ph’), 7.41 (d, J = 8.21 Hz, 1H, 6-Ph’), 7.48 (s, 1H, 2-
13
Ph’), 9.92 (s, 1H, CONH), 10.62 (br.s., 1H, NH+); C NMR (75 MHz, DMSO-d6) δ ppm 23.2, 24.3,
25.1, 26.1, 33.9, 36.5, 47.2, 51.4, 55.7, 55.8, 112.3, 117.1, 117.4, 118.6, 121.3, 121.4, 123.9, 128.9,
139.8, 139.9, 149.2, 152.2, 171.5; LC/MS: m/z calc. 424.30, found 424.21; MW 460.05; Anal.
C26H38ClN3O2 (C, H, N).
4.2.2.27.
N-(4-(tert-butyl)phenyl)-6-(4-(2-methoxyphenyl)piperazin-1-yl)hexanamide
hydrochloride (33)
28