Synthesis, biological evaluation and structure-activity relationship of new GABA uptake inhibitors, derivatives of 4-aminobutanamides

Article abstract of DOI:10.1016/j.ejmech.2014.06.024

Six series of 2-substituted 4-aminobutanamide derivatives were synthesized and evaluated for their ability to inhibit GABA transport proteins mGAT1-4 stably expressed in HEK-293 cell lines. The pIC₅₀ values determined were in the range 4.23-5.23. Two compounds (15b and 15c) were selected for further in vitro studies. These compounds were also subjected to preliminary behavioral studies to evaluate their anticonvulsant, antidepressant-like, and antinociceptive activities in mice. Their influence on motor coordination was also assessed. We report that, among a spectrum of in vivo activities, both 15b and 15c displayed significant activity against pentylenetetrazole (PTZ)-induced seizures.

Full text of DOI:10.1016/j.ejmech.2014.06.024

European Journal of Medicinal Chemistry 83 (2014) 256e273
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, biological evaluation and structureeactivity relationship of
new GABA uptake inhibitors, derivatives of 4-aminobutanamides
a
b
c
a
b
€
Paula Kowalczyk , Kinga Sałat , Georg C. Hofner , Marta Mucha , Anna Rapacz ,
Adrian Podkowa b, Barbara Filipek b, Klaus T. Wanner c, Katarzyna Kulig a
a Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,
,
*
ꢀ
Medyczna 9, 30-688 Krakow, Poland
b
ꢀ
Chair of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Krakow, Poland
c
€
Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universitat München, Butenandtstr. 5-13, 81377 Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Six series of 2-substituted 4-aminobutanamide derivatives were synthesized and evaluated for their
ability to inhibit GABA transport proteins mGAT1ꢀ4 stably expressed in HEK-293 cell lines. The pIC50
values determined were in the range 4.23e5.23. Two compounds (15b and 15c) were selected for further
in vitro studies. These compounds were also subjected to preliminary behavioral studies to evaluate their
anticonvulsant, antidepressant-like, and antinociceptive activities in mice. Their influence on motor
coordination was also assessed. We report that, among a spectrum of in vivo activities, both 15b and 15c
displayed significant activity against pentylenetetrazole (PTZ)-induced seizures.
Received 2 April 2014
Received in revised form
4 June 2014
Accepted 12 June 2014
Available online 12 June 2014
Keywords:
GABA uptake inhibitors
mGAT1e4
© 2014 Elsevier Masson SAS. All rights reserved.
4-Aminobutanamides
Seizures
Depression
Pain
1. Introduction
to a minor extent in synaptically apposed astrocytes [7,8], whereas
mGAT4 is predominantly localized on distal astrocytes which are in
4-Aminobutyric acid (GABA) transporters (GATs) have been
extensively investigated as prospective tools for studies on their
key role in the dysfunction of GABAergic neurotransmission. To
date, four different plasma-membrane transport proteins that
mediate the uptake of synaptic GABA into neurons and glial cells
have been identified and characterized. Following the nomencla-
ture used by the Human Genome Organization (HUGO), these
transporters are termed GAT1 (SLC6a1), BGT1 (SLC6a12), GAT2
(SLC6a13) and GAT3 (SLC6a11) which correspond to mouse mGAT1,
mGAT2, mGAT3 and mGAT4, respectively [1e6]. Because the char-
acterization of the test compounds was performed using mouse
GABA transporters, the mouse nomenclature is used in the present
paper.
direct contact with GABAergic neurons [9]. Thus, mGAT1 and
mGAT4 can affect many brain functions including muscle relaxa-
tion, cognition, and memory [10,11]. By contrast, mGAT2 and
mGAT3 are predominantly expressed in the liver and at lower levels
in kidneys, whereas in the brain their significant concentrations are
restricted to the leptomeninges (mGAT3 and mGAT2) and to cere-
bral blood vessels (mGAT3), indicating that these transporters are
unlikely to play an important role in the inactivation of GABA
[12,13].
Following the identification of GAT inhibitors it was demon-
strated that they enhance the GABA tone, and therefore hold
promise the treatment of several diseases in which GABA function
is reduced, including epilepsy, migraine, neuropathic pain, Hun-
tington's chorea, Parkinson's disease [14e18]. However, to explore
both the physiological function of GATs and their individual
structure, further compounds that selectively target and modulate
GATs are needed.
The four transporter subtypes differ in their localization, affinity
for GABA and pharmacological function. mGAT1 and mGAT4 are
almost exclusively located in the central nervous system (CNS).
Moreover, mGAT1 is primarily localized in presynaptic neurons and
Tiagabine (Fig. 1), a derivative of nipecotic acid, has been
developed and marketed as an add-on treatment for partial epi-
lepsy, and this drug is also in clinical trials for new indications
* Corresponding author.
E-mail address: mfkkulig@cyf-kr.edu.pl (K. Kulig).
http://dx.doi.org/10.1016/j.ejmech.2014.06.024
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
257
hydroxybutanamides [31,34,35] and of compounds bearing a
phthalimide group at the 4-position of the butanamides [36].
Following our previous investigations which showed that 4-(1,3-
dioxoisoindolin-2-yl)butanamide derivatives with the 2-(4-
benzhydryl)-piperazin-1-yl residue exhibited only weak GAT inhi-
bition [36], we sought to obtain butanamide derivatives with a
primary amine group at the 4-position. Thus, drug development
commenced from GABA, which shows low selectivity for GATs [37].
Preliminary SAR studies indicated that the benzyl substituent on
the amide group and the aromatic and lipophilic substituent at the
2-position of the 4-hydroxybutanamide moiety are crucial for their
activity. In addition, the presence of a tertiary amino group, for
example N-methyl-4,4-diphenylbut-3-en-1-amine (N-methyl-N-
DPB), in analogues of GABA uptake inhibitors is necessary for
effective interaction with the GABA uptake system [35,38]. To
mimic the biaryl moieties of known GAT inhibitors a different N-
bulky and lipophilic biaryl group was introduced at the 2-position
of 4-aminobutanamide (Fig. 2).
Fig. 1. Structure of lipophilic GABA uptake inhibitors of mGAT1emGAT4.
including diabetic neuropathy and migraine. Tiagabine is a highly
selective blocker of the GAT1 transporter, and this may limit its
activity to regions of the CNS in which GAT1 plays a significant role
(the cortex, cerebellum, and hippocampus) [19]. This drug strongly
inhibits GABA uptake in synaptosomal preparations from rat brain,
as well as in cultured neurons and glial cells in vitro [20]. Tiagabine
generates a dose-dependent increase in extracellular GABA levels
in rat brain in vivo, and specifically suppresses various chemically-
induced seizures evoked by pentylenetetrazole (PTZ) and DMCM
2. Chemistry
To prepare the target compounds 15e20(aee), GABA was used
as a starting material. The reaction routes to all the target com-
pounds are outlined in Scheme 1.
First, the amine group of GABA was protected by phthalimide.
Phthalimide-protected amino acid 1 was easily obtained from the
parent amino acid and phthalic anhydride [39]. In the case of
substituted a-halo amides 3aee, 4-(1,3-dioxoisoindolin-2-yl)buta-
(6,7-dimethoxy-4-ethyl-b-carboline-3-carboxylate), as well as
noic acid (1) was brominated using N-bromosuccinimide. This ionic
bromination procedure proved to be tolerant of phthalimido group
and was superior to the standard Hell-Vollard-Zielinski procedure
[40]. The molecule obtained, 2-bromo-4-(1,3-dioxoisoindolin-2-yl)
butanoic acid (2), was purified by method described by Kolasa et al.
[41]. Further treatment with thionyl chloride generated the acid
chloride which then was reacted with various substituted de-
rivatives of benzylamine at room temperature in dry THF for 12 h,
giving molecules 3aee. The resulting amides (3aee) were a sub-
strate for the N-alkylation of earlier obtained amines (4e8).
The amines necessary for the reactions, namely 4-
diphenylmethylene piperidine (4) and 4-benzhydryl piperidine
(5), were prepared according to a published procedure [42]. N-
Methyl-4,4-diphenylbut-3-en-1-amine (6) and 4,4-diphenylbut-3-
en-1-amine (7) were prepared according to a published proce-
dure using 1,10-(4-bromobut-1-ene-1,1-diyl)dibenzene as a starting
kindled seizures, whereas it only weakly influences maximal
electroshock seizures [19]. Tiagabine demonstrates antinociceptive
effects at moderate doses (7.2 and 24.3 mmol/kg) against thermally
evoked pain in the mouse hot-plate test [21]. Furthermore, it has an
antiallodynic activity and anxiolytic-like properties in rodent
models [21e23]. Unfortunately, the utility of tiagabine is limited in
part because of adverse effects, such as asthenia, diarrhea, dizziness
and tremor [24,25].
Another GAT1 inhibitor which is effective in neuropathic pain is
NO711 (Fig. 1). In the chronic constriction injury (CCI) model in
mice NO711 induced late-onset and long-lasting analgesic effect.
These results indicate that mGAT1 may be involved in the occur-
rence and development of neuropathic pain [26].
mGAT4 inhibitors are represented by moderately potent (S)-
SNAP-5114, the most selective mGAT4 inhibitor up to now (Fig. 1)
[27,28]. Recently, it was found that (S)-SNAP-5114 exerted anti-
nociceptive effects by promoting the activation of GABAA and
GABAB receptors in the spinal cord. Its analgesic efficacy has been
confirmed in the tail flick test (a model of acute thermal noci-
ception), in the late phase of the formalin test (a model of persistent
pain) and in CCI neuropathic pain model. Studies conducted by
Kataoka et al. [29] demonstrated that mGAT4 inhibitors might be
useful in treatment of various painful conditions. However, the
physiological role of mGAT4 in the CNS is not completely under-
stood [27e29].
Our earlier studies focused on the search for novel biologically
active compounds and demonstrated that several 2-substituted 4-
hydroxybutanamides with affinity for GATs display not only anti-
convulsant effects in some rodent models of seizures [30,31] but
also antinociceptive properties in thermally and chemically
induced acute and tonic pain models [30,32,33].
We present herein the synthesis and biological evaluation of six
series of new 4-aminobutanamide derivatives.
The strategy in the development of these new compounds was
motivated by our previous structural characterization and biolog-
ical
evaluation
of
2-substituted
derivatives
of
4-
Fig. 2. Schematic structure of designed 2-substituted 4-aminobutanamides.

258
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
Scheme 1. The synthesis of 2-substituted 4-aminobutanamides 15e20(aee).
material [31,38,43]. N-Methyl-4,4-diphenylbutan-1-amine (8) was
MS binding assay with NO711 as a non-labeled marker [45]. The
compounds were considered as active if GABA uptake or NO711
obtained by the hydrogenation of its derivative
6 [31]. 1-
benzhydrylpiperazine (9) is commercially available.
binding was reduced at least by 50% at a concentration of 100 mM.
The final 2-substituted derivatives of 4-aminobutanamides
15e20 (aee) were obtained by the cleavage of a phthalimide
group with hydrazine hydrate.
Inhibitory activity was expressed as pIC50 (negative log of the 50%
inhibitory concentration; IC50); or pKi values in NO711 MS binding
assay. The results are listed in Table 1.
All the designed compounds were synthesized and subse-
quently tested for their inhibitory potency and selectivity towards
cloned murine GABA transporters mGAT1emGAT4 in uptake as-
says, and their affinity for mGAT1 was evaluated in an MS binding
assay.
4. Results and discussion
4.1. In vitro inhibitory activity
The purpose of this study was to identify GABA uptake inhibitors
with improved pharmacological properties, and a wide range of
new GABA uptake inhibitors has been prepared. The core structure
of the compounds designed is 4-aminobutanamide (15e20(aee)).
Compounds were first assessed for the inhibition of mGAT ac-
tivity in cultured cells stably expressing mGAT1e4.
3. In vitro inhibitory activity
4-Aminobutanoic acid derivatives 15e20 (aee) were evaluated
for their inhibitory potency against the four GABA transporter
subtypes mGAT1emGAT4. The assay system used was based on
[3H] GABA uptake in HEK-293 cells stably expressing the individual
GABA transporters [44]. The affinity for GAT1 was determined by an
Among the amides with the 4,4-diphenylbut-3-en-1-ylamine
moiety 16(aee), compound 16b with a chloro substituent at the

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
259
Table 1
Results of [3H] GABA uptake and NO711 MS-binding assays.
Compound
mGAT1 uptakea
mGAT2 uptakea
mGAT3 uptakea
0.06
mGAT4 uptakea
0.06
mGAT1 NO711 bindingb
15a
15b
15c
15d
15e
16a
16b
16c
16d
16e
17a
17b
17c
17d
17e
18a
18b
18c
18d
18e
19a
19b
19c
19d
19e
20a
20b
20c
20d
20e
4.35
0.05
4.42
4.66
4.68
4.51
4.52
5.10
5.16
5.02
4.92
5.05
5.02
5.11
4.76
4.94
4.85
4.92
5.19
5.01
4.89
5.02
4.81
4.99
4.98
4.99
5.23
4.92
5.23
5.01
5.03
5.10
52%
56%
0.05
4.57
4.98
4.97
4.47
4.74
4.48
4.62
4.75
4.61
4.53
4.53
4.72
4.67
4.57
4.64
4.62
5.00
4.95
4.96
4.92
4.86
4.81
4.89
4.91
4.79
4.69
4.96
5.02
4.67
4.84
64%
4.23
4.82
4.92
4.43
4.63
4.59
4.99
4.93
4.50
4.82
4.59
4.94
4.84
4.87
5.08
4.80
5.04
4.90
4.90
5.01
4.92
4.96
4.92
4.87
4.94
4.70
4.99
4.99
5.07
5.13
73%
100
100
100
m
m
m
M: 83%
M: 55%
M: 53%
4.68
4.59
4.63
4.55
4.51
4.80
4.78
4.56
4.71
4.65
4.90
4.76
4.78
4.91
4.73
4.89
4.89
4.86
5.07
4.96
4.87
4.96
4.96
4.95
4.71
4.86
4.95
4.80
0.10
0.10
0.05
0.10
0.05
0.10
0.10
0.05
0.10
0.09
0.07
0.05
0.04
0.05
0.02
0.10
0.05
0.07
0.08
0.06
0.07
0.07
0.02
0.06
0.09
0.09
0.03
0.10
0.05
0.04
0.07
0.07
0.05
0.02
0.08
0.02
0.05
0.08
0.08
0.04
0.09
0.07
0.02
0.10
0.01
0.06
0.09
0.09
0.07
0.10
0.07
0.11
0.06
0.02
0.10
0.05
0.05
0.06
0.07
0.07
0.07
0.11
0.08
0.09
0.04
0.04
0.06
0.07
0.04
0.04
0.03
0.06
0.08
0.04
0.02
0.05
0.05
0.08
0.04
0.04
0.10
0.05
0.06
0.03
0.06
0.02
0.10
0.07
0.04
0.02
0.05
0.01
0.07
0.10
0.06
0.06
0.10
0.08
0.07
0.10
0.07
0.09
0.06
0.03
0.01
0.02
0.06
0.03
0.04
0.05
0.05
0.01
0.10
0.07
0.06
0.04
4.18
4.20
4.76
100
0.04
0.06
0.11
m
M: 53%
4.79
4.64
4.96
4.51
4.67
4.75
4.67
4.81
4.90
4.96
4.95
4.80
5.04
4.78
4.98
4.62
4.60
4.53
4.49
4.59
4.52
4.47
4.78
0.10
0.11
0.07
0.03
0.02
0.10
0.07
0.06
0.16
0.09
0.09
0.11
0.13
0.14
0.09
0.08
0.03
0.05
0.06
0.04
0.04
0.07
0.05
4.88
Tiagabinec
(S)-SNAP-5114d
6.88 0.12
4.07 0.09
7.41 0.06
4.56 0.02
5.29 0.04
5.71 0.07
a
pIC50 (means SEM; n ꢁ 3.
% of NO711 bound to GAT1 at 100
Data from Ref. [44].
b
c
m
M concentration of tested compound (means; n ¼ 2)or pKi (means SEM; n ¼ 3).
d
Data from Refs. [44,45,53].
ortho-position of the N-benzylamide moiety showed the highest
potency against mGAT2 (pIC50 ¼ 5.16) (Table 1). N-methylation of
the analogs 16(aee) resulted in the increased inhibitory potency of
compounds 18(aee). Increased inhibitory activity against GABA
uptake was observed against mGAT1, mGAT3, and mGAT4,
whereas activity against mGAT2 was unchanged. Moreover, the
compound also bearing a chloro substituent at the ortho-position
of the benzyl fragment of the molecule showed significant potency
against mGAT2 in this series (18b, pIC50 ¼ 5.19) (Fig. 3). Structural
modification involving reduction of the double bond in the amino
moiety of the series discussed above was found to have a negative
effect on the potencies of compounds 17(aee), which were greatly
reduced (Fig. 3).
4.2. Electroconvulsive threshold test
At a dose of 100 mg/kg none of the test compounds elevated the
threshold for induction of electroconvulsive seizures. In control
mice the CS50 value was 6.61 mA. In 15b-treated group the CS50 was
6.22 mA (p > 0.05); in addition, no protection against seizures in-
duction was observed with 15c. For the latter compound, CS50 was
not calculated because an increased mortality rate compared to
control mice was noted in 15c-treated mice (100 mg/kg) after the
animals underwent a single electroconvulsive shock. (S)-SNAP5114
elevated the threshold for induction electroconvulsive seizures to
7.95 mA, but this effect was not statistically significant.
Compound 19e, which contains a methyl substituent at the
para-position of the benzyl fragment of the molecule, showed the
highest potency towards mGAT2 (pIC50 ¼ 5.23). Structural modifi-
cation involving hydrogenation of the double bond in the amino
moiety of 4-(diphenylmethyl)piperidine 20(aee) caused only a
slight decrease in the inhibitory activity against mGAT1, mGAT3,
and mGAT4 transporters compared to 19(aee), whereas this
modification did not affect potency against mGAT2 transporter.
Marked activity against mGAT2 was observed for 20b with a chloro
substituent at the ortho-position of the N-benzylamide moiety
(pIC50 ¼ 5.23) (Fig. 3).
4.3. Maximal electroshock seizure test
None of the test compounds at doses 30 mg/kg and 100 mg/kg
demonstrated protective properties against electrically induced
seizures in the maximal electroshock test. In all 15b-treated mice
tonic hind-limb extension (THLE) was observed after a single
electroshock. 15c at 30 mg/kg was also ineffective in this test. At a
dose of 100 mg/kg it reduced the number of mice with THLE by 44%
(vs. control group) but an increased mortality rate was observed in
this group after the electroshock (65% vs. control, p < 0.01).
Taking into consideration the results of in vitro tests, two com-
pounds (15b and 15c) were selected for further investigation in vivo
in behavioral assays.
4.4. Pentylenetetrazole (PTZ)-induced seizures
In contrast to electrical models of seizures, both compounds
were effective as anticonvulsants in PTZ-induced seizures. At a dose

260
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
Fig. 3. (aef) Inhibition of GABA uptake; the potency of compounds 15e20(aee) for GAT inhibition depends on the nature of the substituents at the benzyl fragment of molecule.
of 100 mg/kg 15b significantly prolonged the latency time to first
seizure episode by 100% (p < 0.05), whereas at 30 mg/kg it had no
effect. Compared to the vehicle-treated group, it also reduced the
Table 2
number of seizure episodes by 53% and 37% at 100 and 30 mg/kg,
Anticonvulsant activity of the test compounds in the PTZ-induced seizure model.
respectively. 15c was even more efficacious than 15b in this test
Compound
Dose [mg/kg] Latency to first
Number of Mortality
because it was able to delay the onset of seizures by 58% and 217%
(vs. control) at doses 15 and 30 mg/kg, respectively. Compared to
the vehicle-treated group, it also reduced the number of seizure
episodes by 58% and 83%, respectively (Table 2). Both compounds:
15b at doses 30 and 100 mg/kg and 15c at 30 mg/kg significantly
reduced the mortality rate of experimental animals as compared to
control mice (p < 0.001; Fig. 4). In this test the anticonvulsant ac-
tivity of (S)-SNAP5114 was not assessed based on literature data
indicating that this GAT inhibitor does not display anticonvulsant
activity in the PTZ seizure model [46]. Ethosuximide (ETX; 100 mg/
kg; i.p.) used as a reference in this test delayed the onset of seizures
by 120% (p < 0.01) and reduced both the number of seizure epi-
sodes by 64% and total mortality rate by 75% (Table 2).
seizure episode [s]a seizure
rate (X/Y)c
episodesb
Vehicle
15b
e
552 48
2.4 0.4
1.5 0.5
1.1 0.5
1.0 0.0*
4/8
30
816 216
2/8***
1/8***
0/8***
100
15
30
e
1104 264*
870 114**
1752 48***
614.9 61.9
1350 187.4**
15c
0.4 0.3*** 0/8***
Vehicle
Ethosuximide 100
1.4 0.3
6/8
0.5 0.2*
0/8***
Each value represents the mean SEM obtained from 7 to 10 mice. The compounds
and the vehicle were administered intraperitoneally 60 min before the assay.
a, b Statistical analysis: one-way analysis of variance (ANOVA), followed by Dunnett's
post hoc comparison (15b, 15c) or Student's t-test (ethosuximide). Latency time to
first seizure episode: F[2,29] ¼ 3.339; p < 0.05 (15b) and F[2,28] ¼ 71.38; p < 0.0001
(15c). Number of seizure episodes: F[2,29]
¼
2.373; p > 0.05 (15b) and F
[2,29] ¼ 9.908; p < 0.001 (15c). Significant difference compared to the control
group: *p < 0.05, **p < 0.01, ***p < 0.001.
4.5. Pilocarpine-induced seizures
c
Mortality rate is expressed as the number of mice that died during the 30 min
observation period (X) as a proportion of the number of mice tested in each group
(Y). The results obtained for the drug-treated group were compared to that of
vehicle-treated group. These differences were statistically evaluated using Fisher's
exact probability test.
At doses tested the investigated compounds 15c and (S)-
SNAP5114 demonstrated
a statistically significant protection
against seizures, whereas 15b had no protective effect (Table 3).

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
261
Table 4
Antidepressant-like activity of the investigated compounds in the forced swim test.
Compound
Dose [mg/kg]
Immobility
time [s] SEM
Reduction
of immobility
time (%)
Vehicle (1% Tween)
15b
e
159.4 10.1
117.9 13.5
98.0 17.10*
153.3 14.8
113.4 13.3*
198.2 4.5
e
15
30
15
30
e
26.0
38.5
3.8
28.9
e
15c
Vehicle
(S)-SNAP-5114
15.0
30.0
174.3 11.4
158.7 9.9**
12.1
19.9
Each value represents the mean SEM obtained from 7 to 8 mice. The compounds
and the vehicle were administered intraperitoneally 60 min before the assay. Sta-
tistical analysis: one-way analysis of variance (ANOVA), followed by Dunnett's post
hoc test: F[2,19] ¼ 4.906, p < 0.05 (15b); F[2,18] ¼ 4.369, p < 0.05 (15c); F
[2,20] ¼ 5.503, p < 0.05 ((S)-SNAP5114). Significant difference versus the vehicle-
treated group: *p < 0.05, **p < 0.01.
Fig. 4. The influence of the test compounds 15b, 15c, and
a reference molecule
(ethosuximide, ETX) on mortality rate in pentylenetetrazole-induced seizures. Results
are shown as the % reduction of mortality rate compared to that observed in vehicle-
treated mice. ***p < 0.001 (vs. control).
Both 15c and (S)-SNAP5114 prolonged the latency time to seizures
by 113% (15c; p < 0.05), and by 158% and 259%, for 100 mg and
200 mg of (S)-SNAP5114, respectively (p < 0.05).
(S)-SNAP5114 demonstrated no antinociceptive activity in the hot
plate test.
Each value represents the mean SEM obtained from 7 to 8
mice. Anticonvulsant activity was assessed by comparison to
vehicle-treated mice. Statistical analysis: Student's t-test. The
compounds and the vehicle were administered i.p. 60 min before
pilocarpine. Significant difference versus the control group:
*p < 0.05. NE: not established.
4.8. Rotarod test
In the rotarod test the investigated compounds at doses 30 and
100 mg/kg did not induce detectable motor impairments in
experimental animals compared to vehicle-treated mice
(Fig. 5aec).
4.6. Forced swim test
4.9. Locomotor activity test
Both 15b and 15c at the dose of 30 mg/kg showed a statistically
significant antidepressant-like effect in the forced swim test
(p < 0.05; Table 4). In addition, a main overall effect of treatment
was observed in the case of (S)-SNAP5114 (F[2,20] ¼ 5.503;
p < 0.05). The dose 30 mg/kg of this compound reduced immobility
time by 20% versus control mice, and this effect was statistically
significant at p < 0.01.
At 30 mg/kg 15b had no influence on locomotor activity (Fig. 6a),
whereas a statistically significant (p < 0.001) decrease in locomotor
activity was observed in 15c-treated mice at each time-point of the
test (Fig. 6b).
5. Conclusion
Each value represents the mean SEM obtained from 7 to 8
mice. The compounds and the vehicle were administered intra-
peritoneally 60 min before the assay. Statistical analysis: one-way
analysis of variance (ANOVA), followed by Dunnett's post hoc test:
F[2,19] ¼ 4.906, p < 0.05 (15b); F[2,18] ¼ 4.369, p < 0.05 (15c); F
[2,20] ¼ 5.503, p < 0.05 ((S)-SNAP5114). Significant difference
versus the vehicle-treated group: *p < 0.05, **p < 0.01.
Six new series of GABA uptake inhibitors have been synthesized
based on the structure of 4-aminobutanamides modified by
changing the N-bulky and lipophilic biaryl side-chain and the po-
sition of substituents in the benzyl fragment of the molecules;
compounds were tested for activity against stably expressed
mGAT1e4 in HEK cells.
The in vitro results demonstrated that an N-bulky and lipophilic
biaryl group in the 2-position of 4-aminobutanamide is significant
for the inhibitory potency towards mGAT1e4. The highest activities
of 4-(diphenylmethylidene)piperidine, 4-(diphenylmethyl)piperi-
dine, and N,N-(methyl)(4,4-diphenyl)but-3-enylamine-substituted
4-aminobutanamides were observed against mGAT2 transporter,
whereas molecules with a 4-benzhydrylpiperazin-1-yl side chain
were the most potent towards mGAT3 transporter.
4.7. Hot plate test
In this test in the vehicle-treated mice the latency time to pain
reaction was 13.9 1.2 s. 15b tested at 30 mg/kg prolonged the
latency time to pain reaction to 18.8 2.3 s (p > 0.05) and 15c at the
same dose prolonged the latency time to 27.5 2.9 s (p < 0.001).
Table 3
Anticonvulsant activity of the investigated compounds in pilocarpine-induced seizure model.
Compound
Dose [mg/kg]
Latency to seizures [s] SEM
Anticonvulsant effect (%)
Latency to death [s] SEM
Anticonvulsant effect (%)
Vehicle
15b
Vehicle
15c
e
392.7 41.10
342.7 49.88
368.3 17.96
785.9 187.6*
950.3 250.1*
1322.0 354.4*
e
519.8 71.58
466.2 26.92
511.1 59.7
874.7 142.3*
1757 544.1*
1547 364.6*
e
100
e
NE
e
NE
e
70
100
200
113.4
158
258.9
71.1
243.8
202.7
(S)-SNAP5114
Each value represents the mean SEM obtained from 7 to 8 mice. Anticonvulsant activity compared to vehicle-treated mice. Statistical analysis: Student's t-test. The com-
pounds and the vehicle were administered i.p. 60 min before pilocarpine. Significant difference compared to the control group: *p < 0.05. NE: not established.

262
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
activity against mGAT1 and mGAT4. Moreover, our results indicate
that the basic character of the amino groups, and the conforma-
tional flexibility of the linker connecting the 4-aminobutanamide
and the aromatic moiety at the second position of this class of
GABA uptake inhibitors, are factors of importance in determining
the activity against GABA uptake.
In the pilocarpine-induced seizure test, a rodent model of status
epilepticus, the molecule 15c, which displayed the most potent
activity against mGAT4, demonstrated a statistically significant
anticonvulsant activity, whereas 15b had no effect.
In the present study we have confirmed that some GAT in-
hibitors exert an antidepressant-like effect in the forced swim test
in mice, in agreement with our recent reports [33] and reports of
other authors [47].
A very significant antinociceptive activity in the hot-plate test
was demonstrated for 15c. On the other hand, this compound
potently decreased experimental animals' locomotor activity at the
dose tested in the hot plate test (30 mg/kg). Taking these sedative
properties of 15c into consideration, the prolongation of the la-
tency time to pain reaction in the hot plate test is likely to be a
false positive effect resulting from sedation caused by this
compound.
To conclude, given the encouraging results obtained with
appropriately 2-substituted GABA derivatives, our study provides a
promising starting point for the search for new and highly potent
GAT inhibitors.
Fig. 5. Influence of the test compounds 15b and 15c (at 30 and 100 mg/kg) on motor
coordination of experimental animals measured in the rotarod test revolving at 6 rpm
(a), 18 rpm (b), or 24 rpm (c). Results are shown as mean time spent on the rotating
rod. Statistical analysis: one-way analysis of variance (ANOVA), followed by Dunnett's
post hoc test: 18 rpm e F[2,21]
[2,21] ¼ 1.000, p > 0.05 (15c). The results were not significant.
¼
1.000, p > 0.05 (15b and 15c); 24 rpm e F
Moreover, the present study indicates that the introduction of
an ortho-chloro or para-methyl substituent on the benzyl fragment
of the molecules is essential for activity against mGAT2.
Of note is the finding that, among the molecules obtained, the
substitution on the N-benzylamide moiety did not produce a dif-
ference in activities against mGAT1 and mGAT4, whereas the
benzhydrylpiperazin-1-yl substituent in 15(aee) was associated
with a reduced activity against mGAT1 and mGAT2.
It is also notable that compounds containing the lipophilic biaryl
(diphenylmethylidene)piperidine and 4-(diphenylmethyl)piperi-
dine moieties were most active against mGAT2, with comparable
Fig. 6. Influence of the test compounds (both at 30 mg/kg) 15b (a) and 15c (b) on
locomotor activity in mice. Data are shown as mean number of light-beam in-
terruptions ( SEM) measured in five periods of 6 min. Statistical analysis of the results
was conducted using repeated-measures analysis of variance (ANOVA), followed by
Bonferroni multiple comparison. 15b: Drug effect: F[1,56] ¼ 0.39; p > 0.05; time effect:
F[4,56] ¼ 0.73; p > 0.05; interaction: F[4,56] ¼ 0.31; p > 0.05. 15c: Drug effect: F
[1,56] ¼ 35.85; p < 0.0001; time effect: F[4,56] ¼ 4.71; p < 0.01; interaction: F
[4,56] ¼ 2.23; p > 0.05. Values were compared to vehicle-treated mice at the same
time-points: ***p < 0.001.

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
263
6. Experimental section
7.70e7.74 (m, 2H, phthalimide), 7.80e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 436.70 [MþH]þ.
6.1. Chemistry
6.2.3. 2-Bromo-N-(4-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)
butanamide (3c)
Reagents and conditions: 8.04 mmol 2 (2.51 g), 20 mL SOCl2,
30 mL dry THF, 24.13 N-(4-chlorobenzyl)-amine (3.42 g); Yield 63%;
white solid; mp 149.0e149.2 ꢂC; Rf: 0.87 (S1); 1H NMR (300 MHz,
Reactions were monitored by thin layer chromatography (TLC)
using silica gel pre-coated 60 F254 plates (0.2 mm; Merck Kieselgel)
and following solvent system: S1 (chloroform:acetone 1:1, v/v), S7
(DCM:acetone 7:3, v/v); S8 (DCM:acetone:MeOH 8:2:0.25 v/v/v);
S9 (DCM:acetone 9:1, v/v); N1:25% NH3/MeOH/DCM/PE
(45:225:600:90); H7:hexane/ethanol/TEA (7:2:1, v/v/v). The re-
actions were carried out using Hydrogen Generators PGX-H2 (Per-
kin Elmer) and reactions under microwave irradiation were carried
out in Discover LabMate (CEM Corporation, USA). The plates were
visualized with the UV light and mixture of ninhydrin (0.3 g
ninhydrin in 100 mL of n-butanol and 3 mL of acetic acid). 1H NMR
and 13C NMR spectra were recorded with VX Mercury Varian
(300 MHz) instrument in CDCl3 at ambient temperature using
solvent signal as an internal standard. Elemental analyses (C, H, N)
were carried out within 0.4% of the theoretical values and were
performed on an ElementarVario EL III (Elementar Analy-
sensysteme, Hanau, Germany). Mass spectra were recorded on
MDX SCILEX API 2000 (Concord, ON, Canada) using the ESI method.
Melting points were determined in open glass capillaries on a
Melting Point Büchi 535 apparatus and are uncorrected.
chloroform-d)
d
ppm 2.36 (dd, J ¼ 14.62, 7.44 Hz, 1H, CH2CHBr),
2.60e2.70 (m, 1H, CH2CHBr), 3.86 (td, J ¼ 6.41, 4.10 Hz, 2H, NCH2),
4.35 (dd, J ¼ 7.44, 6.16 Hz, 1H, CHBr), 4.42 (dd, J ¼ 5.90, 1.80 Hz, 2H,
NHCH2), 6.73 (br. s., 1H, CONH), 7.20e7.26 (m, 2H, arom), 7.27e7.36
(m, 2H, arom), 7.71e7.75 (m, 2H, phthalimide), 7.81e7.87 (m, 2H,
phthalimide); ESI-MS (m/z) 436.70 [MþH]þ.
6.2.4. 2-Bromo-4-(1,3-dioxoisoindolin-2-yl)-N-(4-fluorobenzyl)
butanamide (3d)
Reagents and conditions: 8.04 mmol 2 (2.51 g), 20 mlLSOCl2,
30 mL dry THF, 24.13 N-(4-fluorobenzyl)-amine (3.02 g); Yield 61%;
white solid; mp 151.5e151.7 ꢂC; Rf: 0.82 (S1); 1H NMR (300 MHz,
chloroform-d)
d ppm 2.30e2.43 (m, 1H, CH2CHBr), 2.59e2.72 (m,
1H, CH2CHBr), 3.85 (td, J ¼ 6.41, 3.33 Hz, 2H, NCH2), 4.35 (dd,
J ¼ 7.57, 6.28 Hz,1H, CHBr), 4.41 (d, J ¼ 5.64 Hz, 2H, NHCH2), 6.72 (br.
s., 1H, CONH), 6.98e7.08 (m, 2H, arom), 7.24e7.32 (m, 2H, arom),
7.69e7.76 (m, 2H, phthalimide), 7.80e7.89 (m, 2H, phthalimide); ESI-
MS (m/z) 420.24 [MþH]þ.
6.2. General procedure for the synthesis of N-benzyl-2-bromo-4-
(1,3-dioxoisoindolin-2-yl)butanamide derivatives (3aee)
To a solution of 2-bromo-4-(1,3-dioxoisoindolin-2-yl)butanoic
acid (2) (2.51 g; 8.04 mmol), thionyl chloride (20 mL; 0.27 mol) was
added and the resulting solution was refluxed for three hours. The
excess unreacted thionyl chloride was removed under reduced
pressure and the resulting acid chloride was used in the next step as
such. To the ice-cooled and stirred solution of the obtained above
acid chloride in dry THF (30 mL), the N-benzylamine derivative
(24.13 mmol) was added dropwise and stirring continued for three
hours and left overnight. The reaction mixture was poured into ice.
The organic layer was washed with dil. HCl and evaporated under
vacuum. The aqueous layer was extracted with DCM. The combined
organic layers were dried (NaSO4) and concentrated in vacuo. The
crude product was finally purified by column chromatography
(DCM/acetone 9:1 v/v).
6.2.5. 2-Bromo-4-(1,3-dioxoisoindolin-2-yl)-N-(4-methylbenzyl)
butanamide (3e)
Reagents and conditions: 8.04 mmol 2 (2.51 g), 20 mL SOCl2,
30 mL dry THF, 24.13 N-(4-methylbenzyl)-amine (2.92 g); Yield
60%; white solid; mp 173.7e173.9 ꢂC; Rf: 0.86 (S1); 1H NMR
(300 MHz, chloroform-d) d ppm 2.33 (s, 3H, CH3), 2.35e2.44 (m, 1H,
CH2CHBr), 2.61e2.72 (m, 1H, CH2CHBr), 3.87 (td, J ¼ 6.48, 1.15 Hz,
2H, NCH2), 4.35 (dd, J ¼ 7.82, 5.77 Hz, 1H, CHBr), 4.40 (d, J ¼ 5.13 Hz,
2H, NHCH2), 6.65 (br. s., 1H, CONH), 7.12e7.21 (m, 4H, arom),
7.70e7.74 (m, 2H, phthalimide), 7.81e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 416.28 [MþH]þ.
6.3. General procedure for the synthesis of 2-(4-
benzhydrylpiperazin-1-yl)-N-benzyl-4-(1,3-dioxoisoindolin-2-yl)
butanamide derivatives (9aee)
6.2.1. N-benzyl-2-bromo-4-(1,3-dioxoisoindolin-2-yl)butanamide
(3a)
1.24 mmol 3(aee), 2.14 mmol 1-benzhydrylpiperazine (9;
0.54 g), 29.28 mmol KI (4.86 g) were suspended in acetone (15 mL)
and then the reaction mixture was stirred at 55 ꢂC for 2.4 h using
MV irradiation. The precipitated salt was filtered and the filtrate
was concentrated. The crude product was purified by recrystalli-
zation from methanol.
Reagents and conditions: 8.04 mmol 2 (2.51 g), 20 mL SOCl2,
30 mL dry THF, 24.13 mmol N-benzylamine (2.58 g); Yield 66%;
white solid; mp 152.3e152.5 ꢂC; Rf: 0.87 (S1); 1H NMR (300 MHz,
chloroform-d)
d
ppm 2.37 (dq, J ¼ 14.33, 7.28 Hz, 1H, CH2CHBr),
2.59e2.72 (m, 1H, CH2CHBr), 3.83e3.90 (m, 2H, NCH2), 4.36 (dd,
J ¼ 7.69, 5.90 Hz, 1H, CHBr), 4.44 (d, J ¼ 5.39 Hz, 2H, NHCH2), 6.73
(br. s., 1H, CONH), 7.22e7.42 (m, 5H, arom), 7.72 (m, 2H, phthali-
mide), 7.80e7.88 (m, 2H, phthalimide); ESI-MS (m/z) 401.04
[MþH]þ.
6.3.1. 2-(4-Benzhydrylpiperazin-1-yl)-N-benzyl-4-(1,3-
dioxoisoindolin-2-yl)butanamide (9a)
1.24 mmol 3a (0.50 g), 2.14 mmol 9 (0.54 g), 29.28 mmol KI
(4.86 g) and acetone (15 mL); Yield 57%; white solid; mp
179.7e179.9 ꢂC; Rf: 0.93 (S8); 1H NMR (300 MHz, chloroform-d)
6.2.2. 2-Bromo-N-(2-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)
butanamide (3b)
Reagents and conditions: 8.04 mmol 2 (2.51 g), 20 mL SOCl2,
30 mL dry THF, 24.13 N-(2-chlorobenzyl)-amine (3.42 g); Yield 67%;
white solid; mp 167.1e167.3 ꢂC; Rf: 0.85 (S1); 1H NMR (300 MHz,
d ppm 2.08e2.21 (m, 2H, NCHCH2), 2.27e2.48 (m, 4H, piperazine),
2.59e2.68 (m, 2H, piperazine), 2.71e2.82 (m, 2H, piperazine), 3.28
(dd, J ¼ 8.72, 4.10 Hz, 1H, NCH), 3.72e3.79 (m, 1H, NCHCH2),
3.82e3.91 (m, 1H, CH2N), 4.17 (s, 1H, CHPh2), 4.47 (m, J ¼ 5.39 Hz,
2H, NHCH2), 7.20e7.40 (m, 15H, arom), 7.52 (br. s., 1H, CONH),
7.66e7.71 (m, 2H, phthalimide), 7.77e7.85 (m, 2H, phthalimide); ESI-
MS (m/z) 573.70 [MþH]þ.
chloroform-d)
d ppm 2.30e2.41 (m, 1H, CH2CHBr), 2.58e2.71 (m,
1H, CH2CHBr), 3.86 (td, J ¼ 6.54, 1.28 Hz, 2H, NCH2), 4.36 (dd,
J ¼ 7.95, 5.90 Hz, 1H, CHBr), 4.53 (d, J ¼ 5.90 Hz, 2H, NHCH2), 6.86
(br. s.,1H, CONH), 7.22e7.26 (m, 2H, arom), 7.34e7.41 (m, 2H, arom),

264
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
6.3.2. 2-(4-Benzhydrylpiperazin-1-yl)-N-(2-chlorobenzyl)-4-(1,3-
dioxoisoindolin-2-yl)butanamide (9b)
6.4.1. N-benzyl-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-
3-en-1-yl)amino)butanamide (10a)
1.24 mmol 3b (0.54 g), 2.14 mmol 9 (0.54vg), 29.28 mmol KI
(4.86 g) and acetone (15 mL); Yield 65%; white solid; mp
162.5e162.8 ꢂC; Rf: 0.95 (S8); 1H NMR (300 MHz, chloroform-d)
0.91 mmol 3a (0.37 g), 0.91 mmol 7 (0.20 g), 1.82 mmol KI
(0.30 g), 2.73 mmol K2CO3 (0.38 g) and 15 mL acetonitrile; Yield
35%; light-yellow oil; Rf: 0.84 (S1); 1H NMR (300 MHz, chloroform-
d
ppm 1.96e2.13 (m, 2H, NCHCH2), 2.25e2.45 (m, 4H, piperazine),
d) d ppm 1.88e1.96 (m, 1H, NCHCH2), 2.09e2.15 (m, 1H, NCHCH2),
2.46e2.63 (m, 4H, piperazine), 3.08 (dd, J ¼ 7.82, 4.49 Hz, 1H, NCH),
3.75e3.84 (m, 1H, NCHCH2), 3.86e3.95 (m, 1H, CH2N), 4.17 (s, 1H,
CHPh2), 4.45e4.57 (m, 2H, NHCH2), 7.12e7.30 (m, 9H, arom),
7.34e7.40 (m, 5H, arom), 7.51e7.59 (m, 1H, CONH), 7.66e7.74 (m,
2H, phthalimide), 7.79e7.86 (m, 2H, phthalimide); ESI-MS (m/z)
607.24 [MþH]þ.
2.27 (q, J ¼ 7.27 Hz, 2H, C]CHCH2), 2.58e2.71 (m, 2H, CH2NH), 3.10
(dd, J ¼ 7.31, 5.77 Hz, 1H, NCH), 3.71e3.81 (m, 2H, CH2, phthalimide),
4.34e4.43 (m, 2H, NHCH2), 6.05 (t, J ¼ 7.31 Hz, 1H, C]CH),
7.06e7.44 (m, 15H, arom), 7.60 (t, J ¼ 5.64 Hz, 1H, CONH), 7.68e7.73
(m, 2H, phthalimide), 7.77e7.86 (m, 2H, phthalimide); ESI-MS (m/z)
544.65 [MþH]þ.
6.4.2. N-(2-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbut-3-en-1-yl)amino)butanamide (10b)
0.91 mmol 3b (0.40 g), 0.91 mmol 7 (0.20 g), 1.82 mmol KI
(0.30 g), 2.73 mmol K2CO3 (0.38 g) and 15 mL acetonitrile; Yield
30%; light-yellow oil; Rf: 0.88 (S1); 1H NMR (300 MHz, chloroform-
6.3.3. 2-(4-Benzhydrylpiperazin-1-yl)-N-(4-chlorobenzyl)-4-(1,3-
dioxoisoindolin-2-yl)butanamide (9c)
1.24 mmol 3c (0.54 g), 2.14 mmol 9 (0.54 g), 29.28 mmol KI
(4.86 g) and acetone (15 mL); Yield 60%; white solid; mp
207.2e207.5 ꢂC; Rf: 0.94 (S8); 1H NMR (300 MHz, chloroform-d)
d)
d
ppm 1.89 (dd, J ¼ 14.11, 7.44 Hz, 1H, NCHCH2), 2.12 (dd, J ¼ 14.11,
d
ppm 1.94e2.15 (m, 2H, NCHCH2), 2.21e2.47 (m, 4H, piperazine),
7.18 Hz, 1H, NCHCH2), 2.23e2.33 (m, 2H, C]CHCH2), 2.57e2.69 (m,
2H, CH2NH), 3.08 (dd, J ¼ 7.69, 5.64 Hz, 1H, NCH), 3.68e3.82 (m, 2H,
CH2 phthalimide), 4.47 (d, J ¼ 6.16 Hz, 2H, NHCH2), 6.06 (t,
J ¼ 7.44 Hz, 1H, C]CH), 7.10e7.43 (m, 15H, arom, CONH), 7.67e7.74
(m, 2H, phthalimide), 7.77e7.86 (m, 2H, phthalimide); ESI-MS (m/z)
579.10 [MþH]þ.
2.47e2.76 (m, 4H, piperazine), 3.10 (br. s., 1H, NCH), 3.69e3.80 (m,
1H, NCHCH2), 3.84e3.95 (m, 1H, CH2N), 4.17 (s, 1H, CHPh2),
4.37e4.47 (m, 2H, NHCH2), 7.08e7.46 (m, 15H, arom, CONH),
7.67e7.75 (m, 2H, phthalimide), 7.80e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 607.24 [MþH]þ.
6.4.3. N-(4-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbut-3-en-1-yl)amino)butanamide (10c)
0.91 mmol 3c (0.40 g), 0.91 mmol 7 (0.20 g), 1.82 mmol KI
(0.30 g), 2.73 mmol K2CO3 (0.38 g) and 15 mL acetonitrile; Yield
41%; light-yellow oil; Rf: 0.87 (S1); 1H NMR (300 MHz, chloroform-
6.3.4. 2-(4-Benzhydrylpiperazin-1-yl)-4-(1,3-dioxoisoindolin-2-
yl)-N-(4-fluorobenzyl)butanamide (9d)
1.24 mmol 3d (0.52 g), 2.14 mmol 9 (0.54 g), 29.28 mmol KI
(4.86 g) and acetone (15 mL); Yield 64%; white solid; mp
202.1e202.3 ꢂC; Rf: 0.93 (S8); 1H NMR (300 MHz, chloroform-d)
d)
d ppm 1.85e1.94 (m, 1H, NCHCH2), 2.07e2.15 (m, 1H, NCHCH2),
d
ppm 1.99e2.14 (m, 2H, NCHCH2), 2.24e2.43 (m, 4H, piperazine),
2.26 (q, J ¼ 7.27 Hz, 2H, C]CHCH2), 2.55e2.74 (m, 2H, CH2NH), 3.09
(dd, J ¼ 7.31, 5.77 Hz, 1H, NCH), 3.75 (q, J ¼ 6.50 Hz, 2H, CH2
phthalimide), 4.33 (d, J ¼ 6.16 Hz, 2H, NHCH2), 6.05 (t, J ¼ 7.31 Hz,
1H, C]CH), 7.05e7.39 (m, 14H, arom), 7.63 (t, J ¼ 5.77 Hz, 1H,
CONH), 7.68e7.75 (m, 2H, phthalimide), 7.77e7.87 (m, 2H, phthali-
mide); ESI-MS (m/z) 579.10 [MþH]þ.
2.47e2.64 (m, 4H, piperazine), 3.08 (dd, J ¼ 7.82, 4.23 Hz, 1H, NCH),
3.75e3.84 (m, 1H, NCHCH2), 3.85e3.94 (m, 1H, CH2N), 4.16 (s, 1H,
CHPh2), 4.34e4.48 (m, 2H, NHCH2), 6.95e7.05 (m, 2H, arom),
7.13e7.28 (m, 9H, arom), 7.33e7.39 (m, 4H, arom, CONH), 7.68e7.74
(m, 2H, phthalimide), 7.79e7.87 (m, 2H, phthalimide); ESI-MS (m/z)
591.69 [MþH]þ.
6.4.4. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-yl)
amino)-N-(4-fluorobenzyl)butanamide (10d)
0.91 mmol 3d (0.38 g), 0.91 mmol 7 (0.20 g), 1.82 mmol KI
(0.30 g), 2.73 mmol K2CO3 (0.38 g) and 15 mL acetonitrile; Yield
30%; light-yellow oil; Rf: 0.86 (S1); 1H NMR (300 MHz, chloroform-
6.3.5. 2-(4-Benzhydrylpiperazin-1-yl)-4-(1,3-dioxoisoindolin-2-
yl)-N-(4-methylbenzyl)butanamide (9e)
1.24 mmol 3e (0.51 g), 2.14 mmol 9 (0.54 g), 29.28 mmol KI
(4.86 g) and acetone (15 mL); Yield 66%; white solid; mp
182.8e182.9 ꢂC; Rf: 0.88 (S8); 1H NMR (300 MHz, chloroform-d)
d)
d ppm 1.83e1.96 (m, 1H, NCHCH2), 2.04e2.15 (m, 1H, NCHCH2),
2.21e2.30 (m, 2H, C]CHCH2), 2.54e2.71 (m, 2H, CH2NH), 3.09 (dd,
J ¼ 7.18, 5.90 Hz, 1H, NCH), 3.75 (dq, J ¼ 13.79, 7.20 Hz, 2H, CH2
phthalimide), 4.34 (d, J ¼ 6.16 Hz, 2H, NHCH2), 6.05 (t, J ¼ 7.31 Hz,
1H, C]CH), 6.87e7.04 (m, 2H, arom), 7.08e7.44 (m, 12H, arom),
7.61 (t, J ¼ 5.90 Hz, 1H, CONH), 7.66e7.76 (m, 2H, phthalimide),
7.77e7.88 (m, 2H, phthalimide); ESI-MS (m/z) 562.65 [MþH]þ.
d
ppm 1.99e2.15 (m, 2H, NCHCH2), 2.24e2.44 (m, 7H, CH3, pipera-
zine), 2.49e2.66 (m, 4H, piperazine), 3.08 (dd, J ¼ 8.21, 4.36 Hz, 1H,
NCH), 3.76e3.86 (m, 1H, NCHCH2), 3.86e3.96 (m, 1H, CH2N), 4.15 (s,
1H, CHPh2), 4.34e4.46 (m, 2H, NHCH2), 7.08e7.21 (m, 6H, arom),
7.21e7.28 (m, 5H, arom), 7.33e7.42 (m, 4H, arom; CONH), 7.67e7.74
(m, 2H, phthalimide), 7.79e7.87 (m, 2H, phthalimide); ESI-MS (m/z)
587.62 [MþH]þ.
6.4.5. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-yl)
amino)-N-(4-methylbenzyl)butanamide (10e)
6.4. General procedure for the synthesis of N-benzyl-4-(1,3-
dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-yl)amino)
butanamide derivatives (10aee)
0.91 mmol 3e (0.38 g), 0.91 mmol 7 (0.20 g), 1.82 mmol KI
(0.30 g), 2.73 mmol K2CO3 (0.38 g) and 15 mL acetonitrile; Yield
32%; light-yellow oil; Rf: 0.85 (S1); 1H NMR (300 MHz, chloroform-
d)
d ppm 1.85e1.97 (m, 1H, NCHCH2), 2.07e2.16 (m, 1H, NCHCH2),
0.91 mmol 3(aee), 0.91 mmol 4,4-diphenylbut-3-en-1-amine
(7, 0.20 g), 1.82 mmol KI (0.30 g), 2.73 mmol K2CO3 (0.38 g) and
15 mL acetonitrile. After the addition was complete, the reaction
mixture was heated under reflux for 20 h. The precipitated salt was
filtered and the filtrate was concentrated. The crude oily product
was purified by column chromatography (SiO2:DCM/acetone 9:1).
2.21e2.28 (m, 2H, C]CHCH2), 2.31 (s, 3H, CH3), 2.56e2.70 (m, 2H,
CH2NH), 3.08 (dd, J ¼ 7.44, 5.64 Hz, 1H, NCH), 3.69e3.82 (m, 2H, CH2
phthalimide), 4.34 (d, J ¼ 5.90 Hz, 2H, NHCH2), 6.05 (t, J ¼ 7.44 Hz,
1H, CHPh2), 7.03e7.30 (m,12H, arom), 7.30e7.42 (m, 2H, arom), 7.53
(t, J ¼ 6.03 Hz, 1H, CONH), 7.66e7.76 (m, 2H, phthalimide), 7.76e7.92
(m, 2H, phthalimide); ESI-MS (m/z) 578.65 [MþH]þ.

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
265
6.5. General procedure for the synthesis of N-benzyl-4-(1,3-
6.5.5. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbutyl)(methyl)
dioxoisoindolin-2-yl)-2-((4,4-diphenylbutyl)(methyl)amino)
amino)-N-(4-methylbenzyl)butanamide (11e)
butanamide derivatives (11a-e)
1.50 mmol 3e (0.62 g), 1.50 mmol 8 (0.36 g), 3.07 mmol KI
(0.51 g), 9.11 mmol K2CO3 (1.26 g) and 15 mL acetone; Yield 41%;
light-yellow oil; Rf: 0.90 (S9); 1H NMR (300 MHz, chloroform-d)
1.50 mmol 3(aee), 1.50 mmol N-Methyl-4,4-diphenylbutan-1-
amine (8; 0.36 g), 3.07 mmol KI (0.51 g), 9.11 mmol K2CO3
(1.26 g) and 15 mL acetone. After the addition was complete, the
reaction mixture was heated under reflux for 20 h. The precipitated
salt was filtered and the filtrate was concentrated. The crude oily
product was purified by column chromatography (SiO2:DCM/
acetone 9:1).
d
ppm 1.28e1.37 (m, 2H, CH2CH2N), 1.78e1.99 (m, 4H, Ph2CHCH2;
CH2CH2 phthalimide), 2.11 (s, 3H, CH3), 2.31 (s, 3H, CH3Ph), 2.38 (t,
J ¼ 7.31 Hz, 2H, CH2N), 3.13 (dd, J ¼ 8.59, 3.98 Hz, 1H, NCH),
3.73e3.82 (m, 2H, CH2 phthalimide), 3.89e3.96 (m, 1H, CHPh2),
4.37 (dd, J ¼ 12.31, 5.90 Hz, 2H, NHCH2), 7.06e7.16 (m, 10H, arom),
7.16e7.18 (m, 1H, CONH), 7.23 (d, J ¼ 7.69 Hz, 4H, arom), 7.67e7.71
(m, 2H, phthalimide), 7.80e7.86 (m, 2H, phthalimide); ESI-MS (m/z)
560.70 [MþH]þ.
6.5.1. N-benzyl-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbutyl)(methyl)amino)butanamide (11a)
1.50 mmol 3a (0.60 g), 1.50 mmol 8 (0.36 g), 3.07 mmol KI
(0.51 g), 9.11 mmol K2CO3 (1.26 g) and 15 mL acetone; Yield 31%;
light-yellow oil; Rf: 0.86 (S9); 1H NMR (300 MHz, chloroform-d)
6.6. General procedure for the synthesis of N-benzyl-4-(1,3-
dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-yl)(methyl)
amino)butanamide derivatives (12aee)
d
ppm 1.27e1.39 (m, 2H, CH2CH2N), 1.82e1.96 (m, 4H, ArCHCH2;
1.84 mmol 3(aee), 1.88 mmol N-Methyl-4,4-diphenylbut-3-en-
1-amine (6; 0.45 g), 3.88 mmol KI (0.30 g), 4.60 mmol K2CO3
(0.64 g) and 15 mL acetone. After the addition was complete, the
reaction mixture was heated under reflux for 20 h. The precipitated
salt was filtered and the filtrate was concentrated. The crude oily
product was purified by column chromatography (SiO2:DCM/
acetone 9:1).
CH2CH2 phthalimide), 2.12 (s, 3H, CH3), 2.38 (t, J ¼ 7.05 Hz, 2H,
CH2N), 3.15 (dd, J ¼ 8.59, 3.98 Hz, 1H, NCH), 3.72e3.85 (m, 2H, CH2
phthalimide), 3.89e4.00 (m, 1H, CHPh2), 4.35e4.50 (m, 2H,
NHCH2), 7.07e7.32 (m, 15H, arom), 7.33e7.39 (m, 1H, CONH),
7.64e7.73 (m, 2H, phthalimide), 7.79e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 545.67 [MþH]þ.
6.5.2. N-(2-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbutyl)(methyl)amino)butanamide (11b)
1.50 mmol 3b (0.65 g), 1.50 mmol 8 (0.36 g), 3.07 mmol KI
(0.51 g), 9.11 mmol K2CO3 (1.26 g) and 15 mL acetone; Yield 35%;
light-yellow oil; Rf: 0.88 (S9); 1H NMR (300 MHz, chloroform-d)
6.6.1. N-benzyl-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-
3-en-1-yl)(methyl)amino)butanamide (12a)
1.84 mmol 3a (0.74 g), 1.88 mmol 6 (0.45 g), 3.88 mmol KI
(0.30 g), 4.60 mmol K2CO3 (0.64 g) and 15 mL acetone. Yield 65%;
light-yellow oil; Rf: 0.85 (S1); 1H NMR (300 MHz, chloroform-d)
d
ppm 1.29e1.43 (m, 2H, CH2CH2N), 1.88e1.95 (m, 4H, Ph2CHCH2;
d
ppm 2.13e2.31 (m, 4H, CH2CH2N; C]CHCH2) 2.36 (s, 3H, CH3)
CH2CH2 phthalimide), 2.10 (s, 3H, CH3), 2.38 (t, J ¼ 7.18 Hz, 2H,
CH2N), 3.14 (dd, J ¼ 8.59, 3.98 Hz, 1H, NCH), 3.78 (quin, J ¼ 7.12 Hz,
2H, CH2 phthalimide), 3.88e3.98 (m, 1H, CHPh2), 4.44e4.56 (m, 2H,
NHCH2), 7.10e7.39 (m, 14H, arom), 7.49 (t, J ¼ 6.16 Hz, 1H, CONH),
7.64e7.73 (m, 2H, phthalimide), 7.77e7.86 (m, 2H, phthalimide); ESI-
MS (m/z) 581.12 [MþH]þ.
2.55 (t, J ¼ 6.92 Hz, 2H, CH2N), 3.18 (dd, J ¼ 8.34, 3.98 Hz, 1H, NCH),
3.72e3.82 (m, 1H, CH2 phthalimide), 3.89e3.99 (m, 1H, CH2
phthalimide), 4.32e4.44 (m, 2H, NHCH2), 5.98 (t, J ¼ 7.31 Hz, 1H,
CHPh2), 7.11e7.34 (m, 15H, arom), 7.42 (t, J ¼ 6.80 Hz, 1H, CONH),
7.66e7.72 (m, 2H, phthalimide), 7.80e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 558.68 [MþH]þ.
6.5.3. N-(4-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbutyl)(methyl)amino)butanamide (11c)
1.50 mmol 3c (0.65 g), 1.50 mmol 8 (0.36 g), 3.07 mmol KI
(0.51 g), 9.11 mmol K2CO3 (1.26 g) and 15 mL acetone; Yield 37%;
light-yellow oil; Rf: 0.83 (S9); 1H NMR (300 MHz, chloroform-d)
6.6.2. N-(2-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbut-3-en-1-yl)(methyl)amino)butanamide (12b)
1.84 mmol 3b (0.80 g), 1.88 mmol 6 (0.45 g), 3.88 mmol KI
(0.30 g), 4.60 mmol K2CO3 (0.64 g) and 15 mL acetone. Yield 38%;
light-yellow oil; Rf: 0.89 (S1); 1H NMR (300 MHz, chloroform-d)
d
ppm 1.30e1.39 (m, 2H, CH2CH2N), 1.56e1.66 (m, 2H, Ph2CHCH2),
d
ppm 1.77e1.98 (m, 2H, CH2CH2N), 2.12 (s, 3H, CH3), 2.18e2.26 (m,
1.86e1.95 (m, 2H, CH2CH2 phthalimide), 2.04 (s, 3H, CH3), 2.38 (t,
J ¼ 7.18 Hz, 2H, CH2N), 3.14 (dd, J ¼ 8.72, 3.85 Hz, 1H, NCH),
3.73e3.84 (m, 2H, CH2 phthalimide), 3.87e3.98 (m, 1H, CHPh2),
4.30e4.43 (m, 2H, NHCH2), 7.09e7.20 (m, 6H, arom), 7.20e7.31 (m,
8H, arom), 7.36 (d, J ¼ 4.36 Hz, 1H, CONH), 7.65e7.74 (m, 2H,
phthalimide), 7.78e7.89 (m, 2H, phthalimide); ESI-MS (m/z) 581.12
[MþH]þ.
2H, C]CHCH2), 2.46e2.58 (m, 2H, CH2N), 3.17 (dd, J ¼ 8.34, 4.23 Hz,
1H, NCH), 3.69e3.82 (m, 1H, CH2 phthalimide), 3.88e3.98 (m, 1H,
CH2 phthalimide), 4.38e4.54 (m, 2H, NHCH2), 5.98 (t, J ¼ 7.44 Hz,
1H, CHPh2), 7.04e7.39 (m, 14H, arom), 7.53 (t, J ¼ 6.03 Hz, 1H,
CONH), 7.65e7.74 (m, 2H, phthalimide), 7.78e7.89 (m, 2H, phthali-
mide); ESI-MS (m/z) 593.13 [MþH]þ.
6.6.3. N-(4-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-((4,4-
diphenylbut-3-en-1-yl)(methyl)amino)butanamide (12c)
1.84 mmol 3c (0.80 g), 1.88 mmol 6 (0.45 g), 3.88 mmol KI
(0.30 g), 4.60 mmol K2CO3 (0.64 g) and 15 mL acetone. Yield 45%;
light-yellow oil; Rf: 0.80 (S1); 1H NMR (300 MHz, chloroform-d)
6.5.4. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbutyl)(methyl)
amino)-N-(4-fluorobenzyl)butanamide (11d)
1.50 mmol 3d (0.63 g), 1.50 mmol 8 (0.36 g), 3.07 mmol KI
(0.51 g), 9.11 mmol K2CO3 (1.26 g) and 15 mL acetone; Yield 52%;
light-yellow oil; Rf: 0.87 (S9); 1H NMR (300 MHz, chloroform-d)
d
ppm 1.78e1.99 (m, 1H, CH2CH2N), 2.11 (s, 3H, CH3), 2.17e2.27 (m,
d
ppm 1.26e1.39 (m, 2H, CH2CH2N), 1.85e1.95 (m, 4H, Ph2CHCH2;
3H, CH2CH2N, C]CHCH2), 2.54 (t, J ¼ 6.80 Hz, 2H, CH2N), 3.17 (dd,
J ¼ 8.59, 3.98 Hz, 1H, NCH), 3.78 (dd, J ¼ 14.36, 6.92 Hz, 1H, CH2
phthalimide), 3.88e3.98 (m, 1H, CH2 phthalimide), 4.22e4.39 (m,
2H, NHCH2), 5.98 (t, J ¼ 7.44 Hz, 1H, CHPh2), 7.00e7.10 (m,
4H(arom), 7.10e7.36 (m, 10H, arom), 7.46 (t, J ¼ 6.28 Hz, 1H, CONH),
7.64e7.73 (m, 2H, phthalimide), 7.79e7.90 (m, 2H, phthalimide); ESI-
MS (m/z) 593.13 [MþH]þ.
CH2CH2 phthalimide), 2.12 (s, 3H, CH3), 2.38 (t, J ¼ 7.18 Hz, 2H,
CH2N), 3.14 (dd, J ¼ 8.72, 3.85 Hz, 1, H, NCH), 3.72e3.84 (m, 2H, CH2
phthalimide), 3.87e3.96 (m, 1H, CHPh2), 4.30e4.42 (m, 2H,
NHCH2), 6.90e7.04 (m, 2H, arom), 7.09e7.29 (m, 12H, arom), 7.33 (t,
J ¼ 6.16 Hz, 1H, CONH), 7.66e7.75 (m, 2H, phthalimide), 7.76e7.91
(m, 2H, phthalimide); ESI-MS (m/z) 564.66 [MþH]þ.

266
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
6.6.4. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-
yl)(methyl)amino)-N-(4-fluorobenzyl)butanamide (12d)
1.84 mmol 3d (0.77 g), 1.88 mmol 6 (0.45 g), 3.88 mmol KI
(0.30 g), 4.60 mmol K2CO3 (0.64 g) and 15 mL acetone. Yield 67%;
light-yellow oil; Rf: 0.83 (S1); 1H NMR (300 MHz, chloroform-d)
7.67e7.77 (m, 2H, phthalimide), 7.84 (td, J ¼ 5.00, 3.08 Hz, 2H,
phthalimide); ESI-MS (m/z) 605.14 [MþH]þ.
6.7.3. N-(4-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-(4-
(diphenylmethylene)piperidin-1-yl)butanamide (13c)
d
ppm 1.79e1.99 (m, 2H, CH2CH2N), 2.11 (s, 3H, CH3), 2.17e2.25 (m,
3.58 mmol 3c (1.56 g), 3.58 mmol 4 (0.89 g), 7.16 mmol KI
(1.19 g), 10.74 mmol K2CO3 (1.48 g) and 20 mL acetone. Yield 54%;
light yellow solid; mp 178.3 ꢂC; Rf: 0.71 (S9); 1H NMR (300 MHz,
2H, C]CHCH2), 2.53 (t, J ¼ 6.67 Hz, 2H, CH2N), 3.16 (dd, J ¼ 8.46,
3.85 Hz, 1H, NCH), 3.70e3.82 (m, 1H, CH2 phthalimide), 3.87e3.99
(m, 1H, CH2 phthalimide), 4.22e4.39 (m, 2H, NHCH2), 5.98 (t,
J ¼ 7.31 Hz, 1H, CHPh2), 6.87e6.97 (m, 2H, arom), 7.02e7.18 (m, 5H,
arom), 7.18e7.36 (m, 7H, arom), 7.43 (t, J ¼ 5.90 Hz, 1H, CONH),
7.66e7.74 (m, 2H, phthalimide), 7.78e7.87 (m, 2H, phthalimide); ESI-
MS (m/z) 596.67 [MþH]þ.
chloroform-d)
d ppm 1.93e2.03 (m, 1H, CHCH2CH2), 2.09e2.19 (m,
1H, CHCH2CH2), 2.21e2.40 (m, 4H, piperidine), 2.46e2.67 (m, 4H,
piperidine), 3.16 (dd, J ¼ 8.85, 3.98 Hz, 1H, CHCH2CH2), 3.75e3.85
(m, 1H, CHCH2CH2), 3.95 (ddd, J ¼ 13.72, 7.69, 5.77 Hz, 1H,
CHCH2CH2), 4.37 (dd, J ¼ 14.88, 5.64 Hz, 1H, PhCH2), 4.49 (dd,
J ¼ 15.00, 6.28 Hz, 1H, PhCH2), 7.01e7.11 (m, 4H, arom), 7.13e7.36
(m, 10H, arom), 7.45 (t, J ¼ 6.03 Hz, 1H, NHCO), 7.63e7.76 (m, 2H,
phthalimide), 7.78e7.89 (m, 2H, phthalimide); ESI-MS (m/z) 605.14
[MþH]þ.
6.6.5. 4-(1,3-Dioxoisoindolin-2-yl)-2-((4,4-diphenylbut-3-en-1-
yl)(methyl)amino)-N-(4-methylbenzyl)butanamide (12e)
1.84 mmol 3e (0.76 g), 1.88 mmol 6 (0.45 g), 3.88 mmol KI
(0.30 g), 4.60 mmol K2CO3 (0.64 g) and 15 mL acetone. Yield 86%;
light-yellow oil; Rf: 0.88 (S1); 1H NMR (300 MHz, chloroform-d)
6.7.4. 4-(1,3-Dioxoisoindolin-2-yl)-2-(4-(diphenylmethylene)
piperidin-1-yl)-N-(4-fluorobenzyl)butanamide (13d)
3.58 mmol 3d (1.50 g), 3.58 mmol 4 (0.89 g), 7.16 mmol KI
(1.19 g), 10.74 mmol K2CO3 (1.48 g) and 20 mL acetone. Yield 44%;
light yellow solid; mp 168.2 ꢂC; Rf: 0.75 (S9); 1H NMR (300 MHz,
d
ppm 1.79e2.00 (m, 2H, CH2CH2N), 2.10 (s, 3H, CH3), 2.30 (s, 3H,
CH3Ph), 2.19e2.21 (m, 2H, C]CHCH2), 2.48e2.56 (m, 2H, CH2N),
3.16 (dd, J ¼ 8.46, 4.10 Hz, 1H, NCH), 3.78 (dd, J ¼ 14.23, 7.82 Hz, 1H,
CH2 phthalimide), 3.90e3.98 (m, 1H, CH2 phthalimide), 4.27e4.40
(m, 2H, NHCH2), 5.97 (t, J ¼ 7.44 Hz, 1H, ArCH), 7.01e7.08 (m, 5H,
arom), 7.08e7.25 (m, 6H, arom), 7.29 (s, 3H, arom), 7.32e7.39 (m,
1H, CONH), 7.67e7.72 (m, 2H (phthalimide), 7.80e7.86 (m, 2H,
phthalimide); ESI-MS (m/z) 572.71 [MþH]þ.
chloroform-d)
d ppm 1.92e2.03 (m, 1H, CHCH2CH2), 2.09e2.19 (m,
1H, CHCH2CH2), 2.21e2.38 (m, 4H, piperidine), 2.47e2.64 (m, 4H,
piperidine), 3.15 (dd, J ¼ 8.72, 3.85 Hz, 1H, CHCH2CH2), 3.75e3.86
(m, 1H, CHCH2CH2), 3.90e4.01 (m, 1H, CHCH2CH2), 4.37 (dd,
J ¼ 14.87, 5.64 Hz, 1H, PhCH2), 4.49 (dd, J ¼ 14.75, 6.28 Hz, 1H,
PhCH2), 6.96e7.11 (m, 4H, arom), 7.15e7.33 (m, 10H, arom), 7.43 (t,
J ¼ 6.03 Hz, 1H, NHCO), 7.65e7.76 (m, 2H, phthalimide), 7.77e7.89
(m, 2H, phthalimide); ESI-MS (m/z) 588.68 [MþH]þ.
6.7. General procedure for the synthesis of N-benzyl-4-(1,3-
dioxoisoindolin-2-yl)-2-(4-(diphenylmethylene)piperidin-1-yl)
butanamide derivatives (13aee)
6.7.5. 4-(1,3-Dioxoisoindolin-2-yl)-2-(4-(diphenylmethylene)
piperidin-1-yl)-N-(4-methylbenzyl)butanamide (13e)
3.58 mmol 3e (1.49 g), 3.58 mmol 4 (0.89 g), 7.16 mmol KI
(1.19 g), 10.74 mmol K2CO3 (1.48 g) and 20 mL acetone. Yield 84%;
light yellow solid; mp 149.7 ꢂC; Rf: 0.71 (S9); 1H NMR (300 MHz,
3.58 mmol (3aee), 3.58 mmol 4-diphenylmethylene piperidine
(4; 0.89 g), 7.16 mmol KI (1.19 g), 10.74 mmol K2CO3 (1.48 g) were
suspended in acetone (20 mL). Then the reaction mixture was
stirred and heated at reflux for 24 h. The precipitated salt was
filtered and the filtrate was concentrated. The crude product was
purified by recrystallization from methanol.
chloroform-d)
d
ppm 1.98 (td, J ¼ 10.32, 7.05 Hz, 1H, CHCH2CH2),
2.09e2.19 (m, 1H, CHCH2CH2), 2.19e2.41 (m, 7H, piperidine, PhCH3),
2.45e2.70 (m, 4H, piperidine), 3.16 (dd, J ¼ 8.46, 4.10 Hz, 1H,
CHCH2CH2), 3.76e3.87 (m, 1H, CHCH2CH2), 3.90e4.02 (m, 1H,
CHCH2CH2), 4.37 (dd, J ¼ 14.62, 5.39 Hz, 1H, PhCH2), 4.49 (dd,
J ¼ 14.62, 6.16 Hz, 1H, PhCH2), 7.01e7.31 (m, 14H, arom), 7.32e7.42
(m, 1H, NHCO), 7.66e7.74 (m, 2H, phthalimide), 7.78e7.89 (m, 2H,
phthalimide); ESI-MS (m/z) 584.72 [MþH]þ.
6.7.1. N-benzyl-4-(1,3-dioxoisoindolin-2-yl)-2-(4-
(diphenylmethylene)piperidin-1-yl)butanamide (13a)
3.58 mmol 3a (1.44 g), 3.58 mmol 4 (0.89 g), 7.16 mmol KI
(1.19 g), 10.74 mmol K2CO3 (1.48 g) and 20 mL acetone. Yield 63%;
light yellow solid; mp 154.2 ꢂC; Rf: 0.74 (S9); 1H NMR (300 MHz,
chloroform-d)
d ppm 1.93e2.03 (m, 1H, CHCH2CH2), 2.09e2.19 (m,
1H, CHCH2CH2, 2.21e2.40 (m, 4H, piperidine), 2.46e2.67 (m, 4H,
piperidine), 3.16 (dd, J ¼ 8.85, 3.98 Hz, 1H, CHCH2CH2), 3.75e3.85
(m, 1H, CHCH2CH2), 3.95 (dd, J ¼ 13.72, 7.69, 5.77 Hz, 1H,
CHCH2CH2), 4.37 (dd, J ¼ 14.88, 5.64 Hz, 1H, ArCH2), 4.49 (dd,
J ¼ 15.00, 6.28 Hz, 1H,(ArCH2), 7.01e7.11 (m, 5H, arom), 7.13e7.36
(m, 10H, arom), 7.45 (t, J ¼ 6.03 Hz, 1H, NHCO), 7.63e7.76 (m, 2H,
phthalimide), 7.78e7.89 (m, 2H, phthalimide); ESI-MS (m/z) 570.69
[MþH]þ.
6.8. General procedure for the synthesis of 2-(4-
benzhydrylpiperidin-1-yl)-N-benzyl-4-(1,3-dioxoisoindolin-2-yl)
butanamide derivatives (14aee)
2.2 mmol 3aee, 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone was stirred and
refluxed for 24 h. After the reaction was completed the precipitate
was filtered and the filtrate was concentrated under vacuum. Crude
product was recrystallized from methanol.
6.7.2. N-(2-chlorobenzyl)-4-(1,3-dioxoisoindolin-2-yl)-2-(4-
(diphenylmethylene)piperidin-1-yl)butanamide (13b)
3.58 mmol 3b (1.56 g), 3.58 mmol 4 (0.89 g), 7.16 mmol KI
(1.19 g), 10.74 mmol K2CO3 (1.48 g) and 20 mL acetone. Yield 73%;
light yellow solid; mp 141.9 ꢂC; Rf: 0.69 (S9); 1H NMR (300 MHz,
6.8.1. 2-(4-Benzhydrylpiperidin-1-yl)-N-benzyl-4-(1,3-
dioxoisoindolin-2-yl)butanamide (14a)
2.2 mmol 3a (0.88 g), 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone. Yield 60%; light yellow
solid; mp 184.2 ꢂC; Rf: 0.61 (S9); 1H NMR (300 MHz, chloroform-d)
chloroform-d)
d ppm 1.88e2.08 (m, 2H, CHCH2CH2), 2.23e2.41 (m,
4H, piperidine), 2.44e2.67 (m, 4H, piperidine), 3.12e3.21 (m, 1H,
CHCH2CH2), 3.78e3.87 (m, 1H, CHCH2CH2), 3.93 (dd, J ¼ 8.08,
5.77 Hz, 1H, CHCH2CH2), 4.47e4.60 (m, 2H, PhCH2), 7.02e7.12 (m,
3H, arom), 7.15e7.38 (m, 11H, arom), 7.40e7.46 (m, 1H, NHCO),
d
ppm 0.92e1.18 (m, 2H, CHCH2CH2), 1.47e1.63 (m, 2H, CHCH2CH2),
1.91e2.22 (m, 4H, piperidine), 2.29e2.41 (m, 1H,
piperidine),2.61e2.75 (m, 2H, CHCH2CH2), 3.05e3.12 (m, 1H,
CHCH2CH2), 3.41 (d, J ¼ 10.77 Hz, 1H, Ph2CH), 3.72e3.83 (m, 1H,

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
267
CHCH2CH2), 3.83e3.93 (m, 1H, CHCH2CH2), 4.40 (dd, J ¼ 11.54,
6.9. General procedure for the synthesis of 2-substituted 4-
aminobutanamide derivatives (15e20aee)
5.90 Hz, 2H, PhCH2)) 6.96e7.08 (m, 2H, arom), 7.08e7.29 (m, 11H,
arom), 7.38 (t, J ¼ 6.16 Hz, 1H, NHCO), 7.67e7.75 (m, 2H, phthali-
mide), 7.79e7.87 (m, 2H, phthalimide); ESI-MS (m/z) 572.71
[MþH]þ.
A mixture of hydrazine monohydrate (0.26 g, 0.52 mmol) and
9e14(aee) (0.26 mmol) in ethanol (10 mL) was heated under reflux
for 5 h. After the reaction mixture was cooled to room temperature,
the precipitated was filtered and the filtrate was concentrated. The
aqueous solution was extracted with DCM and the combined
organic phases were washed with saturated aqueous NaCl, dried
over Na2SO4, filtered, and concentrated. The crude product was
6.8.2. 2-(4-Benzhydrylpiperidin-1-yl)-N-(2-chlorobenzyl)-4-(1,3-
dioxoisoindolin-2-yl)butanamide (14b)
2.2 mmol 3b (0.96 g), 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone. Yield 66%; light yellow
solid; mp 156.6 ꢂC; Rf: 0.62 (S9); 1H NMR (300 MHz, chloroform-d)
purified
by
column
chromatography
(SiO2:EtOAc/
MeOH(8:2) / NH3$H2O/MeOH/DCM/PE (45:225:600:90).
d
ppm 1.52 (d, J ¼ 13.34 Hz, 2H, piperidine), 1.62 (br. s., 2H, piperi-
dine), 1.96 (br. s., 1H, piperidine), 2.00e2.21 (m, 3H, piperidine),
2.29e2.39 (m, 1H, piperidine), 2.58e2.74 (m, 2H, CHCH2CH2),
3.01e3.14 (m, 1H, CHCH2CH2), 3.44 (d, J ¼ 10.77 Hz, 1H, Ph2CH),
3.72e3.83 (m, 1H, CHCH2CH2), 3.85e3.97 (m, 1H, CHCH2CH2),
4.48e4.57 (m, 2H, PhCH2), 7.13 (d, J ¼ 5.64 Hz, 2H, arom), 7.18e7.29
(m, 9H, arom), 7.29e7.47 (m, 3H, arom), 7.60 (br. s., 1H, NHCO),
7.68e7.74 (m, 2H, phthalimide), 7.80e7.86 (m, 2H, phthalimide); ESI-
MS (m/z) 607.15 [MþH]þ.
6.9.1. 4-Amino-2-(4-benzhydrylpiperazin-1-yl)-N-
benzylbutanamide (15a)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 9a (0.15 g;
0.26 mmol), ethanol (10 mL) Yield 58%; white solid; mp
112.5e112.7 ꢂC; Rf: 0.15 (N1); Anal. Calcd for C28H34N4O: C, 75.98; H,
7.74; N, 12.66. Found: C, 75.87; H, 7.66; N, 12.64. 1H NMR (300 MHz,
chloroform-d) d ppm 1.82e1.90 (m, 2H, CHCH2CH2), 2.36 (br. s., 4H,
Piperazine), 2.50e2.65 (m, 4H, piperazine), 2.81e2.90 (m, 2H,
CH2NH2), 3.05 (dd, J ¼ 8.21, 5.13 Hz, 1H, NCH), 4.19 (s, 1H, Ph2CH),
4.42 (dd, J ¼ 5.90, 2.82 Hz, 2H, NHCH2), 7.14e7.30 (m, 10H, arom),
7.30e7.43 (m, 6H, arom, CONH); 13C NMR (75 MHz, chloroform-d)
6.8.3. 2-(4-Benzhydrylpiperidin-1-yl)-N-(4-chlorobenzyl)-4-(1,3-
dioxoisoindolin-2-yl)butanamide (14c)
2.2 mmol 3c (0.96 g), 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone. Yield 47%; light yellow
solid; mp 208.4 ꢂC; Rf: 0.60 (S9); 1H NMR (300 MHz, chloroform-d)
d
ppm 29.6 (CH2CH), 36.7 (NH2CH2), 43.6 (CH2NH), 52.5, 54.0
(piperazine), 70.8 (CHCO), 84.5 (CHPh2), 126.3, 126.7, 127.4, 128.2,
128.5, 129.2, 137.9, 142.7 (arom), 171.3 (C]O); ESI-MS (m/z) 443.60
[MþH]þ.
d
ppm 1.42e1.74 (m, 4H, piperidine), 1.90e2.07 (m, 2H, CHCH2CH2),
2.09e2.17 (m, 3H, piperidine), 2.31e2.42 (m, 1H, piperidine),
2.60e2.76 (m, 2H, piperidine), 3.09 (dd, J ¼ 7.69, 3.33 Hz, 1H,
CHCH2CH2), 3.42 (d, J ¼ 10.77 Hz, 1H, Ph2CH), 3.71e3.83 (m, 1H,
CHCH2CH2), 3.84e3.96 (m, 1H, CHCH2CH2), 4.35e4.46 (m, 2H,
PhCH2), 7.11e7.34 (m, 14H, arom), 7.37e7.45 (m, 1H, NHCO),
7.67e7.74 (m, 2H, phthalimide), 7.79e7.86 (m, 2H, phthalimide); ESI-
MS (m/z) 607.15 [MþH]þ.
6.9.2. 4-Amino-2-(4-benzhydrylpiperazin-1-yl)-N-(2-chlorobenzyl)
butanamide (15b)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 9b (0.16 g;
0.26 mmol), ethanol (10 mL) Yield 61%; white solid; mp
172.1e172.3 ꢂC; Rf: 0.18 (N1); Anal. Calcd for C28H33ClN4O: C, 70.50;
H, 6.97; N, 11.74; Found: C, 70.57; H, 7.00; N, 11.68; 1H NMR
(300 MHz, chloroform-d) d ppm 1.71e1.87 (m, 2H, CHCH2CH2), 2.37
(br. s., 4H, piperazine), 2.47e2.62 (m, 4H, piperazine), 2.72e2.88 (m,
2H, CH2NH2), 3.02 (dd, J ¼ 8.08, 5.26 Hz, 1H, NCH), 4.20 (s, 1H,
Ph2CH), 4.48 (dd, J ¼ 6.28, 1.41 Hz, 2H, NHCH2), 7.15e7.41 (m, 14H,
arom), 7.61 (t, J ¼ 6.16 Hz, 1H, CONH); 13C NMR (75 MHz, chloro-
6.8.4. 2-(4-Benzhydrylpiperidin-1-yl)-4-(1,3-dioxoisoindolin-2-yl)-
N-(4-fluorobenzyl)butanamide (11d)
2.2 mmol 3d (0.92 g), 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone. Yield 67%; light yellow
solid; mp 185.4 ꢂC; Rf: 0.56 (S9); 1H NMR (300 MHz, chloroform-d)
form-d)
d ppm 31.3 (CH2CH), 40.1 (NH2CH2), 41.3 (CH2NH), 52.50
(piperazine), 66.51 (CHCO), 76.0 (CHPh2), 127.0, 127.1, 127.9, 128.5,
128.9, 130.3, 133.6, 135.8, 142.5, 142.6 (arom), 173.1 (C]O); ESI-MS
(m/z) 478.04 [MþH]þ.
d
ppm 1.44e1.76 (m, 4H, piperidine), 1.92e2.09 (m, 2H, CHCH2CH2),
2.11e2.19 (m, 3H, piperidine), 2.33e2.44 (m, 1H, piperidine),
2.62e2.78 (m, 2H, piperidine), 3.11 (dd, J ¼ 7.67, 3.31 Hz, 1H,
CHCH2CH2), 3.44 (d, J ¼ 10.78 Hz, 1H, Ph2CH), 3.73e3.85 (m, 1H,
CHCH2CH2), 3.86e3.98 (m, 1H, CHCH2CH2), 4.37e4.48 (m, 2H,
PhCH2), 7.13e7.36 (m, 14H, arom), 7.39e7.47 (m, 1H, NHCO),
7.69e7.76 (m, 2H, phthalimide), 7.81e7.88 (m, 2H, phthalimide); ESI-
MS (m/z) 590.70 [MþH]þ.
6.9.3. 4-Amino-2-(4-benzhydrylpiperazin-1-yl)-N-(4-chlorobenzyl)
butanamide (15c)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 9c (0.16 g,
0.26 mmol), ethanol (10 mL) Yield 65%; white solid; mp
124.3e124.4 ꢂC; Rf: 0.14 (N1); Anal. Calcd for C28H33ClN4O: C, 70.50;
H, 6.97; N, 11.74 Found: C, 70.53; H, 7.03; N, 11.79 1H NMR
(300 MHz, chloroform-d)
d ppm 1.75e1.87 (m, 2H, CHCH2CH2),
6.8.5. 2-(4-Benzhydrylpiperidin-1-yl)-4-(1,3-dioxoisoindolin-2-yl)-
N-(4-methylbenzyl)butanamide (14e)
2.2 mmol 3e (0.91 g), 2.2 mmol 5 (0.55 g), 4.4 mmol KI (0.73 g),
6.6 mmol K2CO3 (0.91 g) and 15 mL acetone. Yield 76%; light yellow
solid; mp 179.3 ꢂC; Rf: 0.67 (S9); 1H NMR (300 MHz, chloroform-d)
2.20e2.47 (m, 4H, piperazine), 2.47e2.63 (m, 4H, piperazine),
2.74e2.87 (m, 2H, CH2NH2), 3.05 (dd, J ¼ 7.95, 5.13 Hz, 1H, NCH),
4.20 (s, 1H, Ph2CH), 4.37 (dd, J ¼ 5.90, 2.82 Hz, 2H, NHCH2),
7.13e7.30 (m, 10H, arom), 7.34e7.41 (m, 4H, arom), 7.44 (t, J ¼ 6.16
Hz, 1H, CONH); 13C NMR (75 MHz, chloroform-d)
d ppm 29.6
d
ppm 0.95e1.17 (m, 2H, CHCH2CH2), 1.45e1.57 (m, 2H, piperidine),
(CH2CH), 37.7 (NH2CH2), 43.6 (CH2NH), 52.5, 54.0 (piperazine), 70.8
(CHCO), 84.5 (CHPh2), 126.3, 126.7, 127.4, 128.2, 128.5, 129.2, 132.3,
142.7 (arom), 171.3 (C]O); ESI-MS (m/z) 478.04 [MþH]þ
1.89e2.23 (m, 4H, piperidine), 2.34 (s, 4H, piperidine, PhCH3),
2.63e2.75 (m, 2H, piperidine), 3.09 (d, J ¼ 4.36 Hz, 1H, CHCH2CH2),
3.41 (d, J ¼ 11.03 Hz, 1H, PhCH), 3.77e3.86 (m, 1H, CHCH2CH2),
3.86e3.96 (m, 1H, CHCH2CH2), 4.32e4.45 (m, 2H, PhCH2), 7.08e7.19
(m, 6H, arom), 7.19e7.29 (m, 9H, arom), 7.31 (t, J ¼ 6.28 Hz, 1H,
NHCO), 7.66e7.76 (m, 2H, phthalimide), 7.77e7.89 (m, 2H, phthali-
mide); ESI-MS (m/z) 443.6 [MþH]þ.
6.9.4. 4-Amino-2-(4-benzhydrylpiperazin-1-yl)-N-(4-fluorobenzyl)
butanamide (15d)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 9d (0.15 g,
0.26 mmol), ethanol (10 mL) Yield 69%; white solid; mp

268
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
100.3e100.5 ꢂC; Rf: 0.12 (N1); Anal. Calcd for C28H33FN4O: C, 73.02;
(m, 2H, NCHCH2), 2.21e2.28 (m, 2H, C]CHCH2), 2.53e2.73 (m, 2H,
CH2NH), 2.77e3.00 (m, 2H, CH2NH2), 3.27e3.41 (m, 1H, NCH),
4.26e4.49 (m, 2H, NHCH2), 5.96e6.09 (m,1H, Ph2CH), 6.85e7.10 (m,
H, 7.22; N, 12.16; found C, 73.07; H, 7.26; N,12.20; 1H NMR
(300 MHz, chloroform-d) d ppm 1.76e1.91 (m, 2H, CHCH2CH2), 2.36
(br. s., 4H, piperazine), 2.49e2.62 (m, 4H, piperazine), 2.77e2.92 (m,
2H, CH2NH2), 3.06 (dd, J ¼ 8.21, 4.87 Hz, 1H, NCH), 4.19 (s, 1H,
(Ph2CH), 4.36 (dd, J ¼ 5.90, 2.56 Hz, 2H, NHCH2), 6.94e7.03 (m, 2H,
arom), 7.16e7.30 (m, 8H, arom), 7.34e7.40 (m, 4H, arom), 7.44 (t,
3H, arom), 7.11e7.42 (m, 11H, arom), 7.52e7.75 (m, 1H, CONH); 13
C
NMR (75 MHz, chloroform-d) d ppm 26.7 (CH2CH]C), 31.8 (CH2CH)
37.4 (NH2CH2), 43.6 (CH2NH), 50.9 (NHCH2CH2), 63.6 (CHCO), 115.3
(CH]CH), 127.9, 128.3, 128.6, 129.2, 134.6, 136.0, 140.0 (arom),133.8
(CHPh2), 171.3 (C]O); ESI-MS (m/z) 414.55 [MþH]þ..
J ¼ 6.16 Hz, 1H, (CONH)); 13C NMR (75 MHz, chloroform-d)
d ppm
38.4 (CH2CH), 39.3 (NH2CH2), 42.4 (CH2NH), 52.1 (piperazine), 66.3
(CHCO), 76.0 (CHPh2), 115.6, 127.8, 128.5, 129.3, 134.3, 142.5, 172.7
(arom), 173.3 (C]O); ESI-MS (m/z) 461.59 [MþH]þ
6.9.9. 4-Amino-2-((4,4-diphenylbut-3-en-1-yl)amino)-N-(4-
fluorobenzyl)butanamide (16d)
Hydrazine monohydrate (0.21 g, 0.42 mmol) and 10d (84 mg,
0.15 mmol), ethanol (5 mL) Yield 46%; yellow oil; Rf: 0.15 (H7); Anal.
Calcd for C27H30FN3O: C, 75.15; H, 7.01; N, 9.74 found: C, 75.02; H,
6.9.5. 4-Amino-2-(4-benzhydrylpiperazin-1-yl)-N-(4-
methylbenzyl)butanamide (15e)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 9e (0.15 g,
0.26 mmol), ethanol (10 mL) Yield 55%; white solid; mp
129.5e129.6 ꢂC; Rf: 0.18 (N1); Anal. Calcd for C29H36N4O: C, 76.28;
H, 7.95; N, 12.27; found C, 76.32; H, 8.03; N, 12.32; 1H NMR
6.94; N, 9.63; 1H NMR (300 MHz, chloroform-d)
d ppm 1.78e2.03
(m, 2H, NCHCH2), 2.20e2.27 (m, 2H, C]CHCH2), 2.52e2.72 (m, 2H,
CH2NH), 2.76e2.99 (m, 2H, CH2NH2), 3.26e3.41 (m, 1H, NCH),
4.25e4.48 (m, 2H, NHCH2), 5.95e6.08 (m, 1H, Ph2CH), 6.84e7.07
(m, 3H, arom) 7.07e7.41 (m, 11H, arom), 7.59e7.74 (m, 1H, CONH);
(300 MHz, chloroform-d) d ppm 1.74e1.87 (m, 2H, CHCH2CH2), 2.32
(s, 3H, CH3), 2.33e2.50 (m, 4H, piperazine), 2.50e2.67 (m, 4H,
piperazine), 2.75e2.89 (m, 2H, CH2NH2), 3.02 (dd, J ¼ 8.21, 4.87 Hz,
1H, NCH), 4.18 (s, 1H, Ph2CH), 4.37 (dd, J ¼ 5.77, 1.92 Hz, 2H, NHCH2),
7.09e7.33 (m, 11H, arom), 7.33e7.42 (m, 4H, arom, CONH); 13C NMR
13C NMR (75 MHz, chloroform-d)
d ppm 26.7 (CH2CH]C), 31.8
(CH2CH) 37.4 (NH2CH2), 43.6 (CH2NH), 50.9 (NHCH2CH2), 63.6
(CHCO),115.3 (CH]CH), 115.6,127.9, 128.5, 128.6,129.2,137.9, 140.7,
160.9 (arom), 133.8 (CHPh2), 171.3 (C]O); ESI-MS (m/z) 432.55
[MþH]þ
(300 MHz, chloroform-d)
d ppm 21.3 (CH3), 29.6 (CH2CH), 37.6
(NH2CH2), 43.4 (CH2NH), 52.5, 54.0 (piperazine), 70.8 (CHCO), 84.5
(CHPh2), 126.3, 126.7, 127.4, 128.1, 128.8, 134.2, 136.9, 142.7 (arom),
171.3 (C]O); ESI-MS (m/z) 457.62 [MþH]þ
6.9.10. 4-Amino-2-((4,4-diphenylbut-3-en-1-yl)amino)-N-(4-
methylbenzyl)butanamide (16e)
Hydrazine monohydrate (0.21 g, 0.42 mmol) and 10e (84 mg,
0.15 mmol), ethanol (5 mL) Yield 59%; yellow oil; Rf: 0.16 (H7); Anal.
Calcd for C28H33N3O: C, 78.65; H, 7.78; N, 9.83; found: C, 78.74; H,
6.9.6. 4-Amino-N-benzyl-2-((4,4-diphenylbut-3-en-1-yl)amino)
butanamide (16a)
Hydrazine monohydrate (0.21 g, 0.42 mmol) and 10a (82 mg,
0.15 mmol), ethanol (5 mL) Yield 55%; yellow oil; Rf: 0.16 (H7); Anal.
Calcd for C27H31N3O: C, 78.42; H, 7.56; N, 10.16; found C, 78.53; H,
7.65; N, 9.79; 1H NMR (300 MHz, chloroform-d)
d ppm 1.79e1.91
(m, 2H, NCHCH2), 2.19e2.29 (m, 2H, C]CHCH2), 2.29e2.33 (m, 3H,
CH3), 2.57e2.68 (m, 2H, CH2NH), 2.72e2.93 (m, 2H, CH2NH2), 3.18
(dd, J ¼ 7.57, 5.51 Hz, 1H, NCH), 4.37 (d, J ¼ 5.90 Hz, 2H, NHCH2),
5.99e6.07 (m, 1H, Ph2CH), 7.09e7.36 (m, 14H, arom) 7.57 (d, J ¼ 7.18
7.60, N, 10.23; 1H NMR (300 MHz, chloroform-d)
d ppm 1.72e1.94
(m, 2H, NCHCH2), 2.25 (q, J ¼ 6.84 Hz, 2H, C]CHCH2), 2.56e2.71 (m,
2H, CH2NH), 2.74e2.86 (m, 2H, CH2NH2), 3.19 (dd, J ¼ 7.57, 5.51 Hz,
1H, NCH), 4.42 (d, J ¼ 5.90 Hz, 2H, NHCH2), 6.02 (t, J ¼ 7.44 Hz, 1H,
Hz, 1H, CONH); 13C NMR (75 MHz, chloroform-d)
d ppm 21.3 (CH3),
26.7 (CH2CH]C), 31.8 (CH2CH) 37.4 (NH2CH2), 43.6 (CH2NH), 50.9
(NHCH2CH2), 63.6 (CHCO),115.3 (CH]CH),128.7,127.4,128.5,129.2,
134.9, 137.9, 140.0 (arom), 133.8 (CHPh2), 171.3 (C]O); ESI-MS (m/z)
428.58 [MþH]þ.
Ph2CH), 7.11e7.34 (m,15H, arom), 7.66 (t, J ¼ 5.64 Hz,1H, CONH); 13
C
NMR (75 MHz, chloroform-d) d ppm 26.7 (CH2CH]C), 31.8 (CH2CH)
37.4 (NH2CH2), 43.6 (CH2NH), 50.9 (NHCH2CH2), 63.6 (CHCO), 115.3
(CH]CH), 126.9, 128.7, 127.4, 128.5, 129.2, 137.9, 140.7 (arom), 133.8
(CHPh2), 171.3 (C]O); ESI-MS (m/z) 414.55 [MþH]þ.
6.9.11. 4-Amino-N-benzyl-2-((4,4-diphenylbutyl)(methyl)amino)
butanamide (17a)
6.9.7. 4-Amino-N-(2-chlorobenzyl)-2-((4,4-diphenylbut-3-en-1-yl)
amino)butanamide (16b)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 11a (95 mg,
0.17 mmol), ethanol (5 mL) Yield 70%; yellow oil; Rf: 0.12 (H7); Anal.
Calcd for C28H35N3O: C, 78.28; H, 8.21; N, 9.78; found: C, 78.32; H,
Hydrazine monohydrate (0.21 g, 0.42 mmol) and 10b (87 mg,
0.15 mmol), ethanol (5 mL) Yield 60%; yellow oil; Rf: 0.15 (H7); Anal.
Calcd for C27H30ClN3O: C, 72.39; H, 6.75; N, 9.38; found: C, 72.45; H,
8.12; N, 9.63; 1H NMR (300 MHz, chloroform-d)
d ppm 1.33e1.41
(m, 2H, CH2CH2N), 1.71e1.87 (m, 2H, ArCHCH2), 1.90e1.99 (m, 2H,
CH2CH2NH2), 2.13 (s, 3H, CH3), 2.35e2.44 (m, 4H, CH2N; NH2),
2.79e2.91 (m, 2H, CH2NH2), 3.10 (dd, J ¼ 8.59, 4.49 Hz, 1H, NCH),
3.81 (t, J ¼ 7.82 Hz, 1H, Ph2CH), 4.39 (dd, J ¼ 6.03, 3.21 Hz, 2H,
NHCH2), 7.12e7.34 (m, 15H, arom), 7.47 (t, J ¼ 5.90 Hz, 1H, CONH);
6.87; N, 9.46; 1H NMR (300 MHz, chloroform-d)
d ppm 1.76e2.01
(m, 2H, NCHCH2), 2.18e2.25 (m, 2H, C]CHCH2), 2.50e2.70 (m, 2H,
CH2NH), 2.74e2.97 (m, 2H, CH2NH2), 3.24e3.39 (m, 1H, NCH),
4.23e4.46 (m, 2H, NHCH2), 5.93e6.06 (m, 1H, Ph2CH), 6.82e7.05
(m, 3H, arom), 7.05e7.39 (m, 11H, arom), 7.49e7.72 (m, 1H, CONH);
13C NMR (75 MHz, chloroform-d)
d ppm 24.6 (CH2CH2CH), 29.3
13C NMR (75 MHz, chloroform-d)
d
ppm 26.7 (CH2CH]C), 31.8
(CH2CH), 36.3 (CH2CHPh2), 37.7 (NH2CH2), 41.6 (CH3N), 43.6
(CH2NH), 50.0 (CH), 56.5 (NCH2CH2), 73.6 (CHCO), 126.9, 128.7,
127.4, 128.5, 129.2, 137.9, 140.7 (arom), 171.3 (C]O); ESI-MS (m/z)
430.60 [MþH]þ.
(CH2CH) 37.4 (NH2CH2), 38.5 (CH2NH), 50.9 (NHCH2CH2), 63.6
(CHCO), 115.3 (CH]CH), 126.9, 128.7, 127.4, 128.5, 129.2, 132.2,
137.9, 140.0, 140.7, 142.4 (arom), 133.8 (CHPh2), 171.3 (C]O); ESI-
MS (m/z) 449.00 [MþH]þ
6.9.12. 4-Amino-N-(2-chlorobenzyl)-2-((4,4-
6.9.8. 4-Amino-N-(4-chlorobenzyl)-2-((4,4-diphenylbut-3-en-1-yl)
amino)butanamide (16c)
diphenylbutyl)(methyl)amino)butanamide (17b)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 11b (0.10 g,
0.17 mmol), ethanol (5 mL) Yield 62%; yellow oil; Rf: 0.15 (H7); Anal.
Calcd for C28H34ClN3O: C, 72.47; H, 7.39; N, 9.06; found: C, 72.36; H,
Hydrazine monohydrate (0.21 g, 0.42 mmol) and 10c (87 mg,
0.15 mmol), ethanol (5 mL) Yield 51%; yellow oil; Rf: 0.14 (H7); Anal.
Calcd for C27H30ClN3O: C, 72.39; H, 6.75; N, 9.38; found: C, 72.54; H,
7.21; N, 8.95; 1H NMR (300 MHz, chloroform-d)
d
ppm 1.34e1.45
6.85; N, 9.43; 1H NMR (300 MHz, chloroform-d)
d ppm 1.79e2.04
(m, 2H, CH2CH2N), 1.67e1.76 (m, 1H, Ph2CHCH2), 1.77e1.88 (m, 1H,

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
269
Ph2CHCH2), 1.92e2.04 (m, 4H, CH2CH2NH2, NH2), 2.11 (s, 3H, CH3),
2.39 (t, J ¼ 7.31 Hz, 2H, CH2N), 2.70e2.80 (m, 1H, CH2NH2),
2.82e2.91 (m, 1H, CH2NH2), 3.07 (dd, J ¼ 8.46, 4.62 Hz, 1H, NCH),
3.83 (t, J ¼ 7.82 Hz, 1H, Ph2CH), 4.41e4.53 (m, 2H, NHCH2),
7.09e7.40 (m, 14H, arom), 7.61 (t, J ¼ 6.16 Hz, 1H, CONH); 13C NMR
Anal. Calcd for C28H33N3O: C, 78.65; H, 7.78; N, 9.83; found: C,
78.54; H, 7.65; N, 8.68; 1H NMR (300 MHz, chloroform-d)
ppm
d
1.70e1.81 (m, 1H, NCHCH2), 1.83e1.94 (m, 1H, NCHCH2), 2.11 (s, 3H,
CH3), 2.18e2.30 (m, 2H, C]CHCH2), 2.49e2.60 (m, 2H, NH2), 2.87 (t,
J ¼ 6.80 Hz, 2H, CH2NH), 3.13 (dd, J ¼ 8.59, 4.23 Hz, 1H, NCH), 3.41
(br. s., 2H, CH2NH2), 4.23e4.43 (m, 2H, NHCH2), 6.01 (t, J ¼ 7.44 Hz,
1H, Ph2CH), 7.01e7.41 (m, 15H, arom), 7.60 (t, J ¼ 6.16 Hz, 1H,
(75 MHz, chloroform-d)
d ppm 24.6 (CH2CH2CH), 29.3 (CH2CH),
36.3 (CH2CHPh2), 37.7 (NH2CH2), 38.5 (CH2NH), 41.6 (CH3N), 50.0
(CH), 56.5 (NCH2CH2), 73.6 (CHCO), 126.9, 128.7, 127.4, 128.5, 129.2,
137.9, 140.7, 145.1 (arom), 171.3 (C]O); ESI-MS (m/z) 465.04
[MþH]þ
CONH); 13C NMR (75 MHz, chloroform-d)
d ppm 24.2 (CH2CH]C),
29.3 (CH2CH), 37.7 (NH2CH2), 41.7 (CH3N), 43.6 (CH2NH), 57.3
(NCH2CH2), 73.7 (CHCO), 126.9, 128.7, 127.4, 128.5, 129.2, 137.9,
140.7 (arom), 133.8 (CPh2), 171.3 (C]O); ESI-MS (m/z) 428.58
[MþH]þ.
6.9.13. 4-Amino-N-(4-chlorobenzyl)-2-((4,4-
diphenylbutyl)(methyl)amino)butanamide (17c)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 11c (0.10 g,
0.17 mmol), ethanol (5 mL) Yield 57%; yellow oil; Rf: 0.10 (H7); Anal.
Calcd for C28H34ClN3O: C, 72.47; H, 7.39; N, 9.06; found C, 72.30; H,
6.9.17. 4-Amino-N-(2-chlorobenzyl)-2-((4,4-diphenylbut-3-en-1-
yl)(methyl)amino)butanamide (18b)
7.29; N, 8.99; 1H NMR (300 MHz, chloroform-d)
d ppm 1.33e1.41
Hydrazine monohydrate (2.00 g, 4 mmol) and 12b (0.69 g,
1.17 mmol), ethanol (10 mL) Yield 66%; yellow oil; Rf: 0.76 (N1);
Anal. Calcd for C28H32ClN3O: C, 72.79; H, 6.98; N, 9.09; found: C,
(m, 2H, CH2CH2N), 1.68e1.86 (m, 2H, Ph2CHCH2), 1.86e2.05 (m, 4H,
CH2CH2NH2, NH2), 2.13 (s, 3H, CH3), 2.36e2.47 (m, 2H, CH2N),
2.82e2.91 (m, 2H, CH2NH2), 3.11 (dd, J ¼ 8.59, 4.23 Hz, 1H, NCH),
3.82 (t, J ¼ 7.95 Hz,1H, Ph2CH) 4.27e4.39 (m, 2H, NHCH2), 7.08e7.32
(m, 14H, arom), 7.49e7.57 (m, 1H, CONH); 13C NMR (75 MHz,
72.65; H, 6.83, N, 8.87; 1H NMR (300 MHz, chloroform-d)
d ppm
1.64e1.78 (m, 1H, NCHCH2), 1.78e1.92 (m, 1H, NCHCH2), 2.12 (s, 3H,
CH3), 2.20e2.31 (m, 2H, C]CHCH2), 2.54 (t, J ¼ 7.18 Hz, 2H, CH2NH),
2.63 (br. s, 2H, NH2), 2.73e2.89 (m, 2H, CH2NH2), 3.11 (dd, J ¼ 8.46,
4.36 Hz, 1H, NCH), 4.35e4.52 (m, 2H, NHCH2), 6.02 (t, J ¼ 7.31 Hz,
1H, Ph2CH), 7.07e7.39 (m, 14H, arom), 7.67 (t, J ¼ 6.16 Hz, 1H,
chloroform-d)
d ppm 24.6 (CH2CH2CH), 29.3 (CH2CH), 36.3
(CH2CHPh2), 37.7 (NH2CH2), 41.6 (CH3N), 43.6 (CH2NH), 50.0 (CH),
56.5 (NCH2CH2), 73.6 (CHCO), 126.2, 128.7, 127.4, 129.2, 137.9, 140.7,
145.1 (arom), 171.3 (C]O); ESI-MS (m/z) 465.04 [MþH]þ.
CONH); 13C NMR (75 MHz, chloroform-d)
d ppm 24.2 (CH2CH]C),
29.3 (CH2CH), 37.7 (NH2CH2), 41.7 (CH3N), 43.6 (CH2NH), 57.3
(NCH2CH2), 73.7 (CHCO), 126.9, 128.7, 127.4, 128.5, 129.2, 132.2,
137.9, 140.7, 142.4 (arom), 133.8 (CPh2), 171.3 (C]O); ESI-MS (m/z)
463.03 [MþH]þ
6.9.14. 4-Amino-2-((4,4-diphenylbutyl)(methyl)amino)-N-(4-
fluorobenzyl)butanamide (17d)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 11d (0.10 g,
0.17 mmol), ethanol (5 mL) Yield 61%; yellow oil; Rf: 0.22 (H7); Anal.
Calcd for C28H34FN3O: C, 75.14; H, 7.66; N, 9.39; found: C, 75.00; H,
6.9.18. 4-Amino-N-(4-chlorobenzyl)-2-((4,4-diphenylbut-3-en-1-
yl)(methyl)amino)butanamide (18c)
7.59; N, 9.25; 1H NMR (300 MHz, chloroform-d)
d ppm 1.32e1.41
(m, 2H, CH2CH2N), 1.67e1.87 (m, 2H, Ph2CHCH2), 1.89e1.99 (m, 2H,
CH2CH2NH2) 2.13 (s, 3H, CH3), 2.36e2.45 (m, 4H, CH2N, NH2),
2.78e2.92 (m, 2H, CH2NH2), 3.10 (dd, J ¼ 8.46, 4.36 Hz, 1H, NCH),
3.78e3.85 (m, 1H, Ph2CH), 4.34 (d, J ¼ 6.16 Hz, 2H, NHCH2),
6.92e7.03 (m, 2H, arom), 7.13e7.30 (m, 12H, arom), 7.49 (t, J ¼ 6.28
Hydrazine monohydrate (2.00 g, 4 mmol) and 12c (0.69 g,
1.17 mmol), ethanol (10 mL) Yield 63%; yellow oil; Rf: 0.73 (N1);
Anal. Calcd for C28H32ClN3O: C, 72.79; H, 6.98; N, 9.09; found: C,
72.68; H, 6.81; N, 9.12; 1H NMR (300 MHz, chloroform-d)
d ppm
1.63e1.77 (m, 1H, NCHCH2), 1.78e1.91 (m, 1H, NCHCH2), 2.09e2.15
(m, 3H, CH3), 2.18e2.31 (m, 4H, C]CHCH2, NH2), 2.50e2.60 (m, 2H,
CH2NH), 2.72e2.89 (m, 2H, CH2NH2), 3.12 (dd, J ¼ 8.46, 4.36 Hz, 1H,
NCH), 4.18e4.36 (m, 2H, NHCH2), 6.02 (t, J ¼ 7.44 Hz, 1H, Ph2CH),
6.97e7.11 (m, 4H, arom), 7.11e7.43 (m, 12H, arom), 7.59 (t,
Hz, 1H, CONH); 13C NMR (75 MHz, chloroform-d)
d ppm 24.6
(CH2CH2CH), 29.3 (CH2CH), 36.3 (CH2CHPh2), 37.7 (NH2CH2), 41.6
(CH3N), 43.6 (CH2NH), 50.0 (CH), 56.5 (NCH2CH2), 73.6 (CHCO),
115.3, 128.7, 127.4, 128.5, 129.2, 145.1, 160.7 (arom), 171.3 (C]O);
ESI-MS (m/z) 448.59 [MþH]þ.
J ¼ 6.03 Hz, 1H, CONH); 13C NMR (75 MHz, chloroform-d)
d ppm
24.2 (CH2CH]C), 29.3 (CH2CH), 37.7 (NH2CH2), 41.7 (CH3N), 43.6
(CH2NH), 57.3 (NCH2CH2), 73.7 (CHCO), 126.9, 128.7, 127.4, 132.3,
134.6, 136.0, 137.9, 140.7 (arom), 133.8 (CPh2), 171.3 (C]O); ESI-MS
(m/z) 463.03 [MþH]þ
6.9.15. 4-Amino-2-((4,4-diphenylbutyl)(methyl)amino)-N-(4-
methylbenzyl)butanamide (17e)
Hydrazine monohydrate (0.26 g, 0.52 mmol) and 11e (98 mg,
0.17 mmol), ethanol (5 mL) Yield 56%; yellow oil; Rf: 0.17 (H7); Anal.
Calcd for C29H37N3O: C, 78.51; H, 8.41; N, 9.47; found: C, 78.41; H,
8.21; N, 9.32; 1H NMR (300 MHz, chloroform-d)
d
ppm 1.29e1.40
6.9.19. 4-Amino-2-((4,4-diphenylbut-3-en-1-yl)(methyl)amino)-N-
(4-fluorobenzyl)butanamide (18d)
(m, 2H, CH2CH2N), 1.71e1.87 (m, 2H, Ph2CHCH2), 1.88e1.98 (m, 2H,
CH2CH2NH2), 2.12 (s, 3H, CH3), 2.28e2.35 (m, 3H, PhCH3), 2.35e2.44
(m, 2H, CH2N), 2.56 (br. s., 2H, NH2), 2.86 (td, J ¼ 6.92, 3.33 Hz, 2H,
CH2NH2), 3.08 (dd, J ¼ 8.46, 4.36 Hz, 1H, NCH), 3.80 (t, J ¼ 7.69 Hz,
1H, Ph2CH), 4.35 (dd, J ¼ 5.77, 3.21 Hz, 2H, NHCH2), 7.05e7.30 (m,
14H, arom), 7.43 (t, J ¼ 6.03 Hz, 1H, CONH); 13C NMR (75 MHz,
Hydrazine monohydrate (2.00 g, 4 mmol) and 12d (0.67 g;
1.17 mmol), ethanol (10 mL) Yield 54%; yellow oil; Rf: 0.79 (N1);
Anal. Calcd for C28H32FN3O: C, 75.48; H, 7.24; N, 9.43; found: C,
75.21; H, 7.17; N, 9.31; 1H NMR (300 MHz, chloroform-d)
d ppm
1.65e1.77 (m, 1H(NCHCH2)) 1.80e1.90 (m, 1H(NCHCH2)) 2.09e2.13
(m, 3H(Me)) 2.18e2.28 (m, 2H(C]CHCH2)) 3.36 (s, 2H(NH2))
2.50e2.59 (m, 2H(CH2NH)) 2.73e2.91 (m, 2H(CH2NH2)) 3.12 (dd,
J ¼ 8.46, 4.36 Hz, 1H(NCH)) 4.20e4.36 (m, 2H(NHCH2)) 6.02 (t,
J ¼ 7.44 Hz, 1H(ArCH)) 6.85e6.98 (m, 2H(Ar)) 7.03e7.09 (m, 3H(Ar))
7.11e7.40 (m, 9H(Ar)) 7.57 (t, J ¼ 5.90 Hz, 1H(CONH)); 13C NMR
chloroform-d)
d ppm 21.3 (CH3), 24.6 (CH2CH2CH), 29.3 (CH2CH),
36.3 (CH2CHPh2), 37.7 (NH2CH2), 41.6 (CH3N), 43.6 (CH2NH), 50.0
(CH), 56.5 (NCH2CH2), 73.6 (CHCO), 126.9, 128.7, 127.4, 128.5, 136.4,
137.9, 145.1 (arom), 171.3 (C]O); ESI-MS (m/z) 444.62 [MþH]þ.
6.9.16. 4-Amino-N-benzyl-2-((4,4-diphenylbut-3-en-1-yl)(methyl)
amino)butanamide (18a)
Hydrazine monohydrate (2.00 g, 4 mmol) and 12a (0.64 g,
1.17 mmol), ethanol (10 mL) Yield 48%; yellow oil; Rf: 0.74 (N1);
(75 MHz, chloroform-d) d ppm 24.2 (CH2CH]C), 29.3 (CH2CH), 37.7
(NH2CH2), 41.7 (CH3N), 43.6 (CH2NH), 57.3 (NCH2CH2), 73.7 (CHCO),
115.3, 128.7, 127.4, 128.5, 129.2, 137.9, 160.9 (arom), 133.8 (CPh2),
171.3 (C]O); ESI-MS (m/z) 446.57 [MþH]þ

270
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
6.9.20. 4-Amino-2-((4,4-diphenylbut-3-en-1-yl)(methyl)amino)-
N-(4-methylbenzyl)butanamide (18e)
137.2 (arom), 142.2 (CPh2), 173.2 (C]O); ESI-MS (m/z) 475.04
[MþH]þ
Hydrazine monohydrate (2.00 g, 4 mmol) and 12e (0.61 g,
1.17 mmol), ethanol (10 mL) Yield 53%; yellow oil; Rf: 0.84 (N1);
Anal. Calcd for C29H35N3O: C, 78.87; H, 7.99; N, 9.52; found: C,
6.9.24. 4-Amino-2-(4-(diphenylmethylene)piperidin-1-yl)-N-(4-
fluorobenzyl)butanamide (19d)
Hydrazine monohydrate (4.21 g, 8.41 mmol) and 13d (1.46 g,
2.48 mmol), ethanol (31 mL). Yield 68%; white solid; mp 137.9 ꢂC;
Rf: 0.77 (N1); Anal. Calcd for C29H32ClN3O: C, 76.12; H, 7.05; N, 9.18;
found: C, 75.94; H, 7.01; N, 9.01; 1H NMR (300 MHz, chloroform-d)
78.78; H, 7.90; N, 9.37; 1H NMR (300 MHz, chloroform-d)
d
ppm
1.72e1.80 (m, 1H, NCHCH2), 1.83e1.91 (m, 1H, NCHCH2), 2.10 (s, 3H,
CH3), 2.18e2.26 (m, 2H, C]CHCH2), 2.30 (s, 3H, PhCH3), 3.68 (s, 2H,
NH2), 2.50e2.57 (m, 2H, CH2NH), 2.88 (dq, J ¼ 12.63, 6.13 Hz, 2H,
CH2NH2), 3.11 (dd, J ¼ 8.72, 4.36 Hz, 1H, NCH), 4.29 (dd, J ¼ 15.39,
5.90 Hz, 2H, NHCH2), 6.01 (t, J ¼ 7.44 Hz, 1H, Ph2CH), 7.01e7.08 (m,
4H, arom), 7.12e7.37 (m, 10H, arom), 7.56 (t, J ¼ 5.90 Hz, 1H, CONH);
d
ppm 1.67 (br. s, 2H, NH2), 1.76e1.90 (m, 2H, CHCH2CH2), 2.23e2.42
(m, 4H, piperidine), 2.48e2.67 (m, 4H, piperidine), 2.73e2.84 (m, 1H,
CHCH2CH2), 2.84e2.96 (m, 1H, CHCH2CH2), 3.12 (dd, J ¼ 7.69, 5.39
Hz, 1H, CHCH2CH2), 4.31e4.49 (m, 2H, PhCH2), 6.89e7.14 (m, 6H,
13C NMR (75 MHz, chloroform-d)
d ppm 21.3 (CH3Ph), 24.2
(CH2CH]C), 29.3 (CH2CH), 37.7 (NH2CH2), 41.7 (CH3N), 43.6
(CH2NH), 57.3 (NCH2CH2), 73.7 (CHCO), 126.9, 128.7, 127.4, 128.5,
129.2, 134.9, 136.4, 140.7 (arom), 133.8 (CPh2), 171.3 (C]O); ESI-MS
(m/z) 442.61 [MþH]þ
arom), 7.14e7.35 (m, 8H, arom), 7.55 (t, J ¼ 5.90 Hz, 1H, NHCO); 13
C
NMR (75 MHz, chloroform-d)
d ppm 30.6 (CH2CH), 32.1, 40.4
(piperidine); 42.5 (NH2CH2), 51.9 (CH2NH), 66.4 (CHCO), 115.4,
115.7, 126.5, 128.0, 129.2, 129.3, 129.7, 134.3, 136.7, (arom), 142.2
(CPh2), 173.1 (C]O); ESI-MS (m/z) 458.58 [MþH]þ.
6.9.21. 4-Amino-N-benzyl-2-(4-(diphenylmethylene)piperidin-1-
yl)butanamide (19a)
Hydrazine monohydrate (4.21 g, 8.41 mmol) and 13a (1.41 g,
2.48 mmol), ethanol (30 mL). Yield 58%; white solid; mp 147.8 ꢂC;
Rf: 0.74 (N1); Anal. Calcd for C29H33N3O: C, 79.23; H, 7.57; N, 9.56;
found: C, 78.96; H, 7.34; N, 9.46; 1H NMR (300 MHz, chloroform-d)
6.9.25. 4-Amino-2-(4-(diphenylmethylene)piperidin-1-yl)-N-(4-
methylbenzyl)butanamide (19e)
Hydrazine monohydrate (4.21 g, 8.41 mmol) and 13e (1.45 g,
2.48 mmol), ethanol (31 mL). Yield 49%; white solid; mp 135.8 ꢂC;
Rf: 0.73 (N1); Anal. Calcd for C30H35N3O: C, 79.43; H, 7.78; N, 9.26;
fond: C, 79.21; H, 7.63; N, 9.03; 1H NMR (300 MHz, chloroform-d)
d
ppm 1.62 (br. s, 2H, NH2), 1.77e1.92 (m, 2H, CHCH2CH2), 2.24e2.41
d
ppm 1.77 (br. ., 2H, NH2), 1.80e1.90 (m, 2H, CHCH2CH2), 2.28e2.37
(m, 4H, piperidine), 2.49e2.69 (m, 4H, piperidine), 2.74e2.85 (m, 1H,
CHCH2CH2), 2.86e2.96 (m, 1H, CHCH2CH2), 3.12 (dd, J ¼ 7.69, 5.39
Hz, 1H, CHCH2CH2), 4.37e4.52 (m, 2H, Ph2CH2), 7.05e7.15 (m, 5H,
(m, 7H, piperidine, PhCH3), 2.49e2.67 (m, 4H, piperidine), 2.76e2.85
(m, 1H, CHCH2CH2), 2.86e2.96 (m, 1H, CHCH2CH2), 3.11 (dd, J ¼ 7.57,
5.51 Hz, 1H, CHCH2CH2), 4.32e4.46 (m, 2H, Ph2CH2), 7.03e7.34 (m,
14H, arom), 7.48 (t, J ¼ 5.64 Hz, 1H, NHCO); 13C NMR (75 MHz,
arom), 7.15e7.40 (m, 10H, arom), 7.54 (t, J ¼ 5.90 Hz, 1H, NHCO); 13
C
NMR (75 MHz, chloroform-d)
d ppm 32.1 (CH2CH), 40.4 (piperi-
chloroform-d)
d ppm 21.1 (CH3), 30.7 (CH2CH), 32.1, 40.4 (piperi-
dine), 43.2 (NH2CH2), 51.9 (CH2NH), 66.5 (CHCO),126.4, 127.4, 127.6,
128.7, 129.7, 134.5, 136.6, 138.6 (arom), 142.2 (CPh2), 173.0 (C]O);
ESI-MS (m/z) 440.59 [MþH]þ
dine); 42.9 (NH2CH2), 51.9 (CH2NH), 66.5 (CHCO), 126.4, 127.6,
128.0, 129.4, 129.7, 134.5, 136.5, 137.1 (arom), 142.2 (CPh2), 173.1
(C]O); ESI-MS (m/z) 454.62 [MþH]þ.
6.9.22. 4-Amino-N-(2-chlorobenzyl)-2-(4-(diphenylmethylene)
piperidin-1-yl)butanamide (19b)
Hydrazine monohydrate (4.21 g, 8.41 mmol) and 13b (1.50 g,
2.48 mmol), ethanol (32 mL). Yield 50%; white solid; mp 161.4 ꢂC;
Rf: 0.78 (N1); Anal. Calcd for C29H32ClN3O: C, 73.48; H, 6.80; N, 8.86;
found: C, 73.05; H, 6.69; N, 8.65; 1H NMR (300 MHz, chloroform-d)
6.9.26. 4-Amino-2-(4-benzhydrylpiperidin-1-yl)-N-
benzylbutanamide (20a)
Hydrazine monohydrate (2.39 g, 4.78 mmol) and 14a (0.80 g,
1.41 mmol), ethanol (19 mL). Yield 76%; white solid; mp 61.1 ꢂC; Rf:
0.92 (N1); Anal. Calcd for C29H35N3O: C, 78.87; H, 7.99; N, 9.52;
found: C, 78.78; H, 8.07; N, 9.46; 1H NMR (300 MHz, chloroform-d)
d
ppm 1.72 (br. s, 2H, NH2), 1.76e1.85 (m, 2H, CHCH2CH2), 2.27e2.42
d
ppm 1.02e1.19 (m, 2H, CHCH2CH2), 1.55 (dt, J ¼ 6.86, 3.11 Hz, 2H,
(m, 4H, piperidine), 2.46e2.63 (m, 4H, piperidine), 2.74e2.83 (m, 1H,
CHCH2CH2), 2.84e2.94 (m, 1H, CHCH2CH2), 3.10 (dd, J ¼ 7.57, 5.51
Hz, 1H, CHCH2CH2), 4.45e4.57 (m, 2H, Ph2CH2), 7.04e7.15 (m, 4H,
piperidine), 1.80 (q, J ¼ 7.18 Hz, 4H, piperidine), 2.06e2.19 (m, 2H,
piperidine), 2.28e2.39 (m, 1H, piperidine), 2.68e2.76 (m, 2H, NH2),
2.77e2.93 (m, 2H, CHCH2CH2), 3.05 (t, J ¼ 6.54 Hz, 1H, CHCH2CH2),
3.45 (d, J ¼ 10.77 Hz, 1H, Ph2CH), 4.42 (dd, J ¼ 5.90, 3.59 Hz, 2H,
arom), 7.15e7.42 (m, 10H, arom), 7.77 (t, J ¼ 6.16 Hz, 1H, NHCO); 13
C
PhCH2), 7.11e7.37 (m,15H, arom), 7.47 (t, J ¼ 5.90 Hz,1H, NHCO); 13
C
NMR (75 MHz, chloroform-d)
d ppm 30.5 (CH2CH), 40.4, 32.1
(piperidine); 41.4 (NH2CH2), 51.9 (CH2NH), 66.4 (CHCO),126.4,127.1,
128.0, 129.0, 129.5, 130.4, 134.5, 135.9 (arom), 142.2 (CPh2), 173.2
(C]O); ESI-MS (m/z) 475.04 [MþH]þ.
NMR (75 MHz, chloroform-d)
d ppm 30.7 (CH2CH), 31.7, 31.9, 39.4
(piperidine); 43.1 (NH2CH2), 48.3 (CH2NH), 58.8 (CPh2), 66.8
(CHCO), 126.2, 127.5, 127.9, 128.5, 128.7, 138.6, 143.6 (arom), 173.3
(C]O); ESI-MS (m/z) 442.61 [MþH]þ.
6.9.23. 4-Amino-N-(4-chlorobenzyl)-2-(4-(diphenylmethylene)
piperidin-1-yl)butanamide (19c)
Hydrazine monohydrate (4.21 g, 8.41 mmol) and 13c (1.50 g,
2.48 mmol), ethanol (32 mL). Yield 34%; white solid; mp 120.3 ꢂC;
Rf: 0.80 (N1); Anal. Calcd for C29H32ClN3O: C, 73.48; H, 6.80; Cl, 7.48;
N, 8.86; found: C, 73.31; H, 6.72; N, 8.59; 1H NMR (300 MHz,
6.9.27. 4-Amino-2-(4-benzhydrylpiperidin-1-yl)-N-(2-
chlorobenzyl)butanamide (20b)
Hydrazine monohydrate (2.39 g, 4.78 mmol) and 14b (0.86 g,
1.41 mmol), ethanol (20 mL). Yield 39%; white solid; mp 152.2 ꢂC;
Rf: 0.79 (N1); Anal. Calcd for C28H34ClN4O: C, 73.17; H, 7.20; N, 8.83;
found: C, 73.00; H, 7.01; N, 8.72; 1H NMR (300 MHz, chloroform-d)
chloroform-d)
d ppm 1.68 (br. s, 2H, NH2), 1.76e1.89 (m, 2H,
CHCH2CH2), 2.24e2.41 (m, 4H, piperidine), 2.49e2.66 (m, 4H,
piperidine), 2.73e2.83 (m, 1H, CHCH2CH2), 2.85e2.95 (m, 1H,
CHCH2CH2), 3.13 (dd, J ¼ 7.69, 5.39 Hz, 1H, CHCH2CH2), 4.33e4.49
(m, 2H, PhCH2), 7.02e7.14 (m, 4H, arom), 7.14e7.35 (m, 10H, arom),
7.58 (t, J ¼ 6.03 Hz, 1H, NHCO); 13C NMR (75 MHz, chloroform-d)
d
ppm 1.07e1.25 (m, 2H, piperidine), 1.54 (d, J ¼ 12.57 Hz, 2H,
piperidine), 1.78 (q, J ¼ 6.92 Hz, 2H, CHCH2CH2), 1.89e2.17 (m, 4H,
piperidine), 2.32 (t, J ¼ 10.64 Hz, 1H, piperidine), 2.68 (t, J ¼ 9.10 Hz,
2H, NH2), 2.74e2.89 (m, 2H, CHCH2CH2), 3.03 (t, J ¼ 6.54 Hz, 1H,
CHCH2CH2), 3.48 (d, J ¼ 10.77 Hz, 1H, Ph2CH), 4.42e4.56 (m, 2H,
PhCH2), 7.12e7.30 (m, 10H, arom), 7.30e7.41 (m, 4H, arom), 7.70 (t,
d
ppm 30.6 (CH2CH), 32.1, 40.1 (piperidine); 42.5 (NH2CH2), 51.9
(CH2NH), 66.4 (CHCO), 126.5, 128.0, 128.8, 128.9, 133.2, 134.3, 136.7,
J ¼ 6.16 Hz,1H, NHCO); 13C NMR (75 MHz, chloroform-d)
d ppm 30.4

P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
271
(CH2CH), 31.7, 31.9, 39.4 (piperidine); 40.3 (NH2CH2), 48.3 (CH2NH),
58.7 (CPh2), 66.7 (CHCO), 126.2, 127.1, 128.0, 128.5, 128.9, 130.1,
133.6, 135.9, 143.6 (arom), 173.4 (C]O); ESI-MS (m/z) 477.05
[MþH]þ
marker were performed as described earlier [45]. NO711 was
analyzed by LC-MS/MS using an API 3200 triple quadrupole mass
spectrometer according to the method described previously [48].
The compounds were considered active if GABA uptake or
NO711 binding was reduced at least by 50% at a concentration of
6.9.28. 4-Amino-2-(4-benzhydrylpiperidin-1-yl)-N-(4-
chlorobenzyl)butanamide (20c)
100 mM. For the active compounds, pIC50 or pKi values were
assessed.
Hydrazine monohydrate (2.39 g, 4.78 mmol) and 14c (0.86 g,
1.41 mmol), ethanol (20 mL). Yield 53%; white solid; mp 130.3 ꢂC;
Rf: 0.77 (N1); Anal. Calcd for C28H34ClN4O: C, 73.17; H, 7.20; N, 8.83;
found: C, 73.00; H, 7.01; N, 8.72; 1H NMR (300 MHz, chloroform-d)
6.11. In vivo activity
6.11.1. Animals
d
ppm 1.00e1.21 (m, 2H, CHCH2CH2), 1.49e1.61 (m, 2H, piperidine),
Adult male Albino Swiss (CD-1) mice obtained from the Animal
Breeding Farm of the Jagiellonian University in Cracow were used
for in vivo tests. The animals weighed between 18 g and 22 g. They
were kept in groups of 10 mice in cages at room temperature of
22 2 ꢂC, under light/dark (12:12) cycle and had free access to food
and water before experiments. The ambient temperature of the
room and humidity were kept consistent throughout all the tests.
For the experiments the animals were selected in a random way.
Each group consisted of 6e10 animals/dose and each mouse was
used only once. The experiments were performed between 8 a.m.
and 3 p.m. Immediately after the assay the animals were killed by
cervical dislocation. All the procedures were approved by the Local
Ethics Committee of the Jagiellonian University in Cracow (ZI/595/
2011).
1.79 (q, J ¼ 6.92 Hz, 2H, piperidine), 1.99 (br.s, 2H, NH2), 2.06e2.19
(m, 2H, CHCH2CH2), 2.34 (td, J ¼ 11.67, 2.05 Hz, 1H, piperidine),
2.64e2.92 (m, 4H, piperidine), 3.05 (t, J ¼ 6.67 Hz, 1H, CHCH2CH2),
3.46 (d, J ¼ 11.03 Hz, 1H, Ph2CH), 4.29e4.44 (m, 2H, PhCH2),
7.09e7.36 (m, 14H, arom) 7.53 (t, J ¼ 5.90 Hz, 1H, NHCO); 13C NMR
(75 MHz, chloroform-d)
d ppm 30.4 (CH2CH), 31.7, 31.9, 39.4
(piperidine); 42.4 (NH2CH2), 48.3 (CH2NH), 58.8 (CPh2), 66.7
(CHCO), 126.2, 127.9, 128.6, 128.8, 128.9, 133.1, 137.2, 143.6 (arom),
173.4 (C]O); ESI-MS (m/z) 442.61 [MþH]þ
6.9.29. 4-Amino-2-(4-benzhydrylpiperidin-1-yl)-N-(4-
fluorobenzyl)butanamide (20d)
Hydrazine monohydrate (2.39 g, 4.78 mmol) and 14d (0.83 g,
1.41 mmol), ethanol (19 mL). Yield 42%; white solid; mp 62.5 ꢂC; Rf:
0.90 (N1); Anal. Calcd for C28H34FN3O: C, 75.79; H, 7.46; N, 9.14;
found: C, 75.63; H, 7.31; N, 9.01; 1H NMR (300 MHz, chloroform-d)
6.11.2. Anticonvulsant activity
6.11.2.1. Electroconvulsive threshold test. The anticonvulsant effi-
cacy of the test compounds at the dose of 100 mg/kg was evaluated
at their previously established time of peak drug effect (60 min
after their i.p. injection) according to the procedure recently
described [33]. Electroconvulsions were produced by an alternating
current (duration of the stimulus: 0.2 s; 50 Hz) delivered via
standard auricular electrodes by an electroshock generator (Hugo
Sachs rodent shocker, Germany). Tonic hind limb extension (i.e., the
hind limbs outstretched 180ꢂ to the plane of the body axis) was an
indicator of seizure episodes. For the evaluation of the electro-
convulsive threshold (ECT) at least four groups of animals per dose
were used. These mice were challenged with electroshocks of
various intensities to yield 10e30%, 30e50%, 50e70% and 70e90%
of animals with seizures. Then, a median current strength value
(CS50 in mA), defined as current intensity required to induce tonic
hind limb extension in 50% of the mice challenged, was estimated
by means of log-probit method [49].
d
ppm 0.97e1.21 (m, 2H, CHCH2CH2), 1.55 (d, J ¼ 13.34 Hz, 2H,
piperidine), 1.81 (q, J ¼ 6.92 Hz, 2H, piperidine), 2.06e2.19 (m, 2H,
piperidine), 2.29e2.44 (m, 3H, piperidine), 2.64e2.78 (m, 2H, NH2),
2.78e2.93 (m, 2H, CHCH2CH2), 3.06 (t, J ¼ 6.54 Hz, 1H, CHCH2CH2),
3.45 (d, J ¼ 11.03 Hz, 1H, Ph2CH), 4.32e4.43 (m, 2H, PhCH2), 7.00 (t,
J ¼ 8.72 Hz, 2H, arom) 7.11e7.30 (m, 12H, arom) 7.51 (t, J ¼ 6.16 Hz,
1H, NHCO); 13C NMR (75 MHz, chloroform-d)
d ppm 30.0 (CH2CH),
31.7, 31.9, 39.4 (piperidine); 40.1 (NH2CH2), 42.4 (CH2NH), 58.8
(CPh2), 66.8 (CHCO), 115.4, 115.7, 126.2, 127.9, 128.5, 129.1, 129.3,
134.4, 143.6, 160.4, 163.7 (arom), 173.3 (C]O); ESI-MS (m/z) 460.60
[MþH]þ.
6.9.30. 4-Amino-2-(4-benzhydrylpiperidin-1-yl)-N-(4-
methylbenzyl)butanamide (20e)
Hydrazine monohydrate (2.39 g, 4.78 mmol) and 14e (0.82 g,
1.41 mmol), ethanol (19 mL). Yield 38%; white solid; mp 110.7 ꢂC;
Rf: 0.92 (N1); Anal. Calcd for C28H34N4O: C, 79.08; H, 8.19; N, 9.22;
found: C, 78.87; H, 7.92; N, 9.26; 1H NMR (300 MHz, chloroform-d)
6.11.2.2. Maximal electroshock seizure test. Maximal electroshock
seizure test was performed as previously described [33]. Briefly, the
vehicle-treated mice and drug-treated mice received a stimulus of
sufficient intensity (25 mA) delivered by an electroshock generator
(Hugo Sachs rodent shocker, Germany) to induce maximal seizures
(tonic extension) of hind limbs. Electroconvulsions were produced
with the use of auricular electrodes and the stimulus duration was
0.2 s. The endpoint was the tonic extension of the hind limbs.
d
ppm 1.00e1.21 (m, 2H, CHCH2CH2), 1.46e1.62 (m, 2H, piperidine),
1.66e1.76 (m, 2H, piperidine), 1.77e1.85 (m, 2H, piperidine),
2.03e2.19 (m, 2H, piperidine), 2.34 (s, 3H, PhCH3), 2.68e2.80 (m, 4H,
NH2, CHCH2CH2), 2.80e2.92 (m, 1H, piperidine), 3.03 (t, J ¼ 6.67 Hz,
1H, CHCH2CH2), 3.46 (d, J ¼ 11.03 Hz, 1H, Ph2CH), 4.38 (dd, J ¼ 5.77,
2.69 Hz, 2H, PhCH2), 7.09e7.21 (m, 6H, arom), 7.21e7.32 (m, 8H,
arom), 7.41 (t, J ¼ 5.77 Hz, 1H, NHCO); 13C NMR (75 MHz, chloro-
form-d) d ppm 21.1 (CH3), 30.8 (CH2CH), 31.7, 31.9, 39.4 (piperidine);
48.4 (NH2CH2), 58.8 (CH2NH), 66.8 (CPh2), 66.8 (CHCO), 126.2,127.5,
128.0, 128.5, 129.3, 135.5, 137.0, 143.6 (arom), 173.2 (C]O); ESI-MS
(m/z) 456.63 [MþH]þ.
6.11.2.3. Pentylenetetrazole-induced seizures. The test was per-
formed according to Łuszczki et al. [50] with some minor modifi-
cation. Clonic convulsions were induced in mice by the
subcutaneous (s.c.) administration of PTZ (Sigma Aldrich, Poland) at
a dose of 100 mg/kg. After the administration of PTZ, the mice were
6.10. [3H] GABA uptake assay
Inhibitory potencies of compounds 15e20(aee) were tested at
four murine GABA transporter subtypes mGAT1emGAT4. The study
was performed as a [3H] GABA uptake assay based on stably
transfected HEK cells, according to the procedure recently
described [44]. Binding assays for mGAT1 based on NO711 as native
placed
separately
into
transparent
Plexiglas
cages
(30 ꢃ 20 ꢃ 15 cm) and were observed during the next 30 min for
the occurrence of clonic seizures. Clonic seizures were defined as
clonus of the whole body lasting more than 3 s, with an accom-
panying loss of the righting reflex. The latency time to first clonus,

272
P. Kowalczyk et al. / European Journal of Medicinal Chemistry 83 (2014) 256e273
the number of seizure episodes and mortality rate were noted and
compared between vehicle-treated and drug-treated groups.
crossings) were counted during the next 30 min in five 6-
min intervals [33].
6.11.2.4. Pilocarpine-induced seizures. The mice were intraperito-
neally treated with the investigated compound or vehicle (1%
Tween 80) and 60 min later the animals received pilocarpine
(400 mg/kg, i.p.; Sigma Aldrich, Poland). These animals were also
pretreated with scopolamine butylbromide (1 mg/kg, i.p.; Sigma
Aldrich, Poland) 45 min before pilocarpine to avoid peripheral
toxicity and diarrhea, masticatory and stereotyped movements.
After the administration of the convulsant, the mice were observed
during 1 h for behavioral changes. The latency time to the onset of
the toniceclonic seizure episode was recorded. The latency time to
death was also noted in the cut-off of 60 min [51].
Acknowledgments
This work was financially supported by the grant from the Na-
tional Science Centre DEC-2012/05/B/NZ7/02705.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.06.
024.
6.11.3. Antidepressant-like activity e the forced swim test
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