2-Substituted 4-hydroxybutanamides as potential inhibitors of γ-aminobutyric acid transporters mGAT1-mGAT4: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2013.06.038

A series of 2-substituted 4-hydroxybutanamide derivatives has been synthesized by the aminolysis of appropriate 2-substituted dihydrofuran-2(3H)- one derivatives with various substituted benzylamines. The final compounds have been evaluated for their capability of inhibiting the GABA transport proteins GAT1-4 stably expressed in HEK-239 cell lines. The pIC₅₀ values determined were in the range 4.21-5.14. Two compounds (16a and 16d), which displayed the most interesting profiles in in vitro tests, have also been subjected to further preliminary behavioral studies, evaluating their antinociceptive activity in hot-plate, writhing, and formalin tests. Their influence on motor coordination has also been assessed.

Full text of DOI:10.1016/j.bmc.2013.06.038

Bioorganic & Medicinal Chemistry 21 (2013) 5154–5167
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Substituted 4-hydroxybutanamides as potential inhibitors
of c-aminobutyric acid transporters mGAT1–mGAT4: Synthesis
and biological evaluation
Paula Kowalczyk a, Kinga Sałat b, Georg C. Höfner c, Natalia Guzior a, 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 Kraków, Poland
b Chair of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
c Department für Pharmazie—Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandstr. 5-13, 81377 München, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-substituted 4-hydroxybutanamide derivatives has been synthesized by the aminolysis of
appropriate 2-substituted dihydrofuran-2(3H)-one derivatives with various substituted benzylamines.
The final compounds have been evaluated for their capability of inhibiting the GABA transport proteins
GAT1-4 stably expressed in HEK-239 cell lines. The pIC50 values determined were in the range 4.21–5.14.
Two compounds (16a and 16d), which displayed the most interesting profiles in in vitro tests, have also
been subjected to further preliminary behavioral studies, evaluating their antinociceptive activity in hot-
plate, writhing, and formalin tests. Their influence on motor coordination has also been assessed.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 5 April 2013
Revised 12 June 2013
Accepted 16 June 2013
Available online 26 June 2013
Keywords:
GABA uptake inhibitors
GAT1-4
Butanamides
Paw licking response
Stretching behavior
Motor deficits
1. Introduction
mans and rats, a different nomenclature, also adopted by the Hu-
man Genome Organization (HUGO), is used. According to this,
c
-Aminobutyric acid (GABA) plays a significant role in the
these transporters are named as GAT1, GAT2, GAT3, and BGT1,
respectively.
inhibitory function within the mammalian central nervous system
(CNS). As 20–50% of all central synapses use GABA as their neuro-
transmitter, it controls the activity of a large percentage of neu-
rons.1 A decrease of GABA-ergic neurotransmission appears to be
involved in the development and outbreak of several neurological
and psychiatric disorders, such as epilepsy, anxiety, neuropathic
pain, Parkinson’s disease, and Huntington Chorea.2,3
Currently, a number of strategies are available for increasing the
GABAergic tone, which include agonism for GABAergic receptors,
inhibition of GABA catabolism, or its reuptake. Since the identifica-
tion of GABA transporter proteins (GATs) and the realization of
their importance in the regulation of GABA concentration in the
brain, these have become interesting targets as new drugs for the
treatment of epilepsy or neuropathic pain syndromes.4,5 To date,
five GABA transporters are known, of which one is the vesicular
GABA transporter (VGAT) and the other four (GAT1, 2, 3, and 4)
are membrane proteins. GAT1-4 belong to the SLC6 superfamily
of Na+-dependent transporters.6,7 For other species, including hu-
The GABA transporters display different physiological activities
and distributions in the CNS. mGAT1 and mGAT4 are located in the
CNS, while mGAT2 and mGAT3 are poorly expressed in the CNS
and can also be found in peripheral tissues.8,9 In view of the fact
that the biological characterization of the test compounds was per-
formed using murine GABA transporters, the ‘mouse’ nomencla-
ture is used in the present paper.
Since the identificationof the GABAtransport systemasa pharma-
cological target, a number of acyclic and cyclic structures have been
tested as potential GAT inhibitors10–13 (Fig. 1). However, the thera-
peutic potential of GAT inhibition has been confirmed with the suc-
cessful development of the GAT1-selective drug tiagabine, which is
used in the treatment of partial seizures and neuropathic pain.14
The therapeutic potential of other GAT1–GAT4 inhibitors is still un-
clear. However, anticonvulsant activity has been demonstrated for
(S)-SNAP-5114, which is slightly selective for mGAT44,15 (Fig. 1).
In recent years, much attention has been paid to the function of
mGAT1 and mGAT3 in nociception. It was shown that the overex-
pression of mGAT1 is responsible for hyperalgesic effects in mice,
whereas in mice lacking GAT1 hypoalgesia was observed.16
⇑
Corresponding author. Tel.: +48 12 6205 452; fax: +48 12 6205 403.
E-mail address: mfkkulig@cyf-kr.edu.pl (K. Kulig).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.038

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5155
MeO
S
OMe
S
O
N
N
N
MeO
COOH
COOH
COOH
Tiagabine
SKF89976A
(S)-SNAP-5114
S
N
S
N
O
HO
N
N
N
N
O
OMe
COOH
HO
EF1502
NO-711
NNC 05-2090
S
N
N
N
H
COOH
N
S
N
N
I
II
Figure 1. Selected structure of lipophilic GABA uptake inhibitors.10,11
Based on this knowledge, numerous mGAT1 inhibitors, includ-
ing tiagabine, have been tested for their antinociceptive activity
in hot-plate, tail flick, formalin, and writhing tests in mice.16–18
GAT1-selective inhibitors, such as ethyl nipecotate and NO-711,
exhibited significant antinociceptive effects in these nociceptive
assays in naïve and GAT1-overexpressing transgenic mice. The re-
sults indicated that GAT1 is involved in the regulation of pain pro-
cesses, and pointed to the possibility of developing analgesic drugs
that target GAT1.
The nociceptive responses of GAT1-knockout mice (GAT1(ꢀ/ꢀ))
were compared with those of heterozygous (GAT(+/ꢀ)) and wild-
type (GAT(+/+)) mice in some of these pain screening models.
These data demonstrated that GAT1 deficiency (due to genetic
knockout or acute blockade by selective inhibitors) leads to hypo-
algesia in mice. These results confirmed the crucial role of GAT1 in
the regulation of nociceptive threshold and indicated that two
GAT1-selective inhibitors, NO-711 and tiagabine, have potential
for clinical use in pain therapy.17
The present work is part of an extensive search for new GABA
uptake inhibitors among derivatives of 4-hydroxybutanamide,
and is based on the results of our earlier studies.21–23
Previously, we reported the synthesis and biological evaluation
of the series of 4-hydroxybutanamide derivatives III and IV (Fig. 2)
as potential GABA uptake inhibitors.21–23 Preliminary SAR studies
on this group of compounds indicated that the benzyl substituent
on the amide group and an aromatic substituent at the 2-position
of the 4-hydroxybutanamide moiety are crucial for their activity.
In order to verify the above hypothesis, we present herein the
synthesis and biological evaluation of new 4-hydroxybutanamide
derivatives with further structural modifications. An N-bulky and
lipophilic biaryl group was introduced at the 2-position of
4-hydroxybutanamide in order to mimic the biaryl moieties of
known GAT inhibitors.24 Other modifications included the intro-
duction of substituents on the benzyl fragment of the molecules.
Schematic structures of the designed and synthesized compounds
are presented in Fig. 3. All of the designed compounds were
synthesized and subsequently tested for their inhibitory potency
and selectivity towards cloned murine GABA transporters GAT1–
In addition, it was demonstrated that a peripherally located
mGAT3 transporter might play a regulatory role in peripheral GAB-
Aergic mechanisms controlling numerous pathological processes,
including pain.19
Our earlier studies, which were focused on the search for novel
biologically active compounds, demonstrated that several 2-
substituted 4-hydroxybutanamides with affinity for GAT display
not only anticonvulsant but also antinociceptive properties in
some rodent models of seizures,20 as well as thermally- and chem-
ically-induced acute and tonic pain models.20–21 Promising results
obtained by our research team confirmed that the pharmacological
activity of some derivatives from this group extends far beyond the
well-known therapeutic targets, which suggests that there are
more opportunities for their use.
H
N
H
N
O
O
N
R
R
N
H
N
OH
OH
IV
III
R = H, 2-Cl, 4-Cl, 4-F, 4-Me
Figure 2. The structure of the 4-hydroxybutanamide derivatives (III and IV).21–23

5156
P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
R2
R2:
O
R3
NH
N
N
N
N
R1
R3: H, 2-Cl, 4-Cl, 4-F, 4-Me
OH
OH
O
Me
Me
R3
NH
HN
N
N
HN
R1
R1: H, Me
Figure 3. Schematic structure of designed 2-substituted 4-hydroxybutan- or 4-hydroxypentanamides.
GAT4 in uptake assays, and their affinity for GAT1 in an MS binding
assay was evaluated. As the next step, the most interesting com-
pounds were further investigated in vivo in some behavioral assays
designed to evaluate their antinociceptive properties, namely hot-
plate, writhing, and formalin tests. For a preliminary evaluation of
the safety profiles of these compounds, the rotarod test was used.
2. Chemistry
All of the derivatives of 4-hydroxybutanamides or 4-hydroxy-
pentanamides presented herein were synthesized by aminolysis
of the appropriate substituted butyrolactones (10–15). The lac-
tones were prepared by N-alkylation of the corresponding amines
1. Potassium phthalimide,
ether 18-crown-6;
toluene; reflux; 20h
Br
10% Pd/C
NH2
NH2
2. NH2NH2 in H2O
EtOH; reflux; 3h
EtOH; r.t.; 8h
6
7
3
33 % r-r CH3NH2 in EtOH;
r.t.; 72h
10% Pd/C
N
H
N
H
EtOH, r.t.; 9h
4
5
O
benzylamine
derivatives
O
O
R2
R3
R2
1,2,4,5,7
Br
N
H
O
O
R1
THF
K2CO3; TBAB
R1
R1
OH
8, 9
10 - 15
16 - 22 (a-e)
O
R2
R3
N
H
R1
OH
N
N
R2
N
N
N
H
R1
H
CH3
H
H
H
H
Lactones
Amides
10
cis-11 trans-11
12
13
14
15
R3
16a
16b
18a
18b
19a
19b
17a
17b
20a
20b
21a
21b
22a
22b
Cl
Cl
F
16c
16d
16e
18c
18d
18e
19c
19d
19e
17c
17d
17e
20c
20d
20e
21c
21d
21e
22c
22d
22e
Scheme 1. The synthesis of 2-substituted 4-hydroxybutanamides 16a–e to 22a–e.

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5157
Table 1
Results of [3H] GABA uptake and NO711 MS-binding assays
Compound
mGAT1 uptakea
mGAT2 uptakea
mGAT3 uptakea
mGAT4 uptakea
mGAT1 NO-711 bindingb
5.04 0.13
100 M: 68%
4.12 0.05
4.94 0.13
4.36 0.08
16a
16b
16c
16d
16e
17a
17b
17c
4.83 0.05
4.33 0.05
4.12 0.05
4.71 0.11
4.78 0.12
4.51 0.11
4.43 0.05
4.25 0.08
4.49 0.10
4.55 0.05
4.48 0.06
4.38 0.11
4.44 0.05
4.49 0.13
4.46 0.13
4.37 0.08
4.51 0.05
4.54 0.13
4.37 0.12
4.26 0.14
4.48 0.09
4.66 0.07
4.48 0.01
4.17 0.08
4.75 0.05
4.58 0.03
4.47 0.05
4.23 0.09
4.17 0.05
4.16 0.10
4.09 0.07
4.06 0.10
4.14 0.11
100
l
M: 58%
l
4.47 0.07
5.01 0.09
4.42 0.18
4.88 0.11
4.71 0.07
4.62 0.07
4.74 0.05
4.52 0.11
4.62 0.10
100
lM: 51%
100
100
100
100
100
100
100
100
100
100
100
100
lM: 55%
lM: 89%
lM: 68%
lM: 49%
lM: 55%
lM: 51%
lM: 95%
lM: 69%
lM: 52%
lM: 57%
lM: 50%
lM: 99%
4.31 0.09
4.22 0.10
4.26 0.06
4.42 0.10
4.21 0.10
17d
17e
18a (3RS,5RS)
18b (3RS,5RS)
18c (3RS,5RS)
18d (3RS,5RS)
18e (3RS,5RS)
19a (3RS, 5SR)
19b (3RS, 5SR)
19d (3RS, 5SR)
19e (3RS, 5SR)
20a
20b
20c
20d
20e
21a
21b
21c
21d
100
l
M: 47%
100
lM: 73%
100
4.16 0.10
100 M: 65%
lM: 71%
4.18 0.08
4.46 0.09
4.27 0.04
5.11 0.05
4.14 0.04
4.00 0.07
4.30 0.02
4.31 0.10
l
4.46 0.08
4.05 0.07
100
4.79 0.04
100 M: 46%
l
M: 95%
100
lM: 69%
100
lM: 79%
100
4.38 0.07
100 M: 616%
lM: 99%
4.19 0.10
4.62 0.09
4.57 0.10
4.90 0.13
4.88 0.11
4.67 0.12
4.84 0.13
4.40 0.02
4.54 0.05
4.64 0.04
4.38 0.11
4.54 0.09
4.54 0.07
4.66 0.06
4.96 0.01
4.57 0.06
4.77 0.09
52%
4.57 0.11
4.24 0.06
4.87 0.14
5.14 0.10
5.02 0.07
4.95 0.12
4.93 0.09
4.49 0.06
4.79 0.04
4.88 0.08
4.41 0.10
4.70 0.10
4.45 0.06
4.74 0.02
4.83 0.05
4.64 0.08
4.63 0.10
64%
4.37 0.09
l
l
100
100
100
100
100
100
100
100
100
100
lM: 58%
lM: 50%
lM: 54%
lM: 61%
lM: 73%
lM: 50%
lM: 79%
lM: 85%
lM: 67%
lM: 74%
4.98 0.10
4.72 0.07
4.63 0.03
4.66 0.10
4.65 0.09
4.23 0.06
4.43 0.10
4.41 0.03
4.01 0.09
4.43 0.07
4.21 0.11
4.48 0.04
4.63 0.03
4.41 0.05
4.26 0.08
6.88 0.12
4.07 0.09
4.64 0.04
5.00 0.04
4.95 0.08
4.70 0.10
4.78 0.08
4.44 0.07
4.59 0.07
4.64 0.11
4.23 0.11
4.69 0.02
4.41 0.05
4.68 0.10
4.78 0.09
4.60 0.04
4.63 0.11
73%
21e
22a
22b
22c
22d
22e
4.09 0.07
100 M: 62%
l
4.33 0.02
4.25 0.10
100 lM: 82%
4.29 0.04
7.41 0.06
4.56 0.02
Tiagabinec
(S)-SNAP-5114d
56%
5.29 0.04
5.71 0.07
a
b
c
% of remaining GABA uptake at 100
% of NO-711 binded to GAT1 at 100
Data from.18
l
l
M concentration of tested compound (means; n = 2) or pIC50 (means SEM; n P3 if not specified otherwise).
M concentration of tested compound or pKi (means SEM; n = 3).
d
Data from.41
(1, 2, 4, 5, and 7) with 3-bromodihydrofuran-2(3H)-one (8) or
3-bromo-5-methyldihydrofuran-2(3H)-one (9). The reactions were
carried out in acetonitrile in the presence of anhydrous K2CO3 and
tetrabutylammonium bromide (TBAB) at room temperature for
12–48 h. In the case of lactone 11, two geometric isomers cis
(11-cis) and trans (11-trans) were obtained.
293 cells stably expressing the individual GABA transporters.28
The affinity for GAT1 was determined by MS binding assay with
NO711 as a non-labeled marker.29 The compounds were consid-
ered as active if GABA uptake or NO711 binding was reduced by
at least 50% at a concentration of 100 lm. For the active com-
pounds, pIC50 or pKi values were assessed. The results are listed
The compounds needed for the reactions, namely 4-diphenylm-
ethylene piperidine (1) and 4-benzhydryl piperidine (2), were pre-
pared according to a literature procedure.25
in Table 1.
The compounds (16a and 16d) that displayed the most interest-
ing profile in in vitro tests were then subjected to further behav-
ioral studies in vivo. The performed investigations included pain
(hot plate, writhing, formalin) and rotarod tests.
N-Methyl-4,4-diphenylbut-3-en-1-amine (4) was prepared
according to
1-ene-1,1-diyl)dibenzene (3) as
a
literature procedure using 1,10-(4-bromobut-
a
starting material.23,26,27 N-
Methyl-4,4-diphenylbutan-1-amine (5) and 4,4-diphenylbutan-
1-amine (7) were obtained by the hydrogenation of their deriva-
tives 4 and 6.
Aminolyses of the obtained lactones 10–15 were performed un-
der different conditions depending on the reactivity and solubility
of both the lactone and the amine, using conventional methods of
heating or microwave irradiation. The course of the performed
reactions is presented in Scheme 1.
4. Results and discussion
With the aim of identifying GABA uptake inhibitors with im-
proved pharmacological properties, a wide range of new GABA up-
take inhibitors has been prepared. The core structure of the
designed compounds is 4-hydroxybutanamide (16a–e, 17a–e,
20a–e to 22a–e) or 4-hydroxypentanamide (18a–e, 19a–e).
The biological evaluation revealed that among the obtained
amides with the 4,4-diphenylbutan-1-amine moiety (22a–e), the
highest potency towards transporters mGAT1-4 was shown by
3. Biological evaluation
22c with
a chloro substituent at the para-position of the
4-Hydroxybutanoic acid and 4-hydroxypentanoic acid deriva-
tives 16a–e to 22a–e were evaluated for their inhibitory potency
towards the four GABA transporter subtypes mGAT1–mGAT4.
The assay system used was based on [3H]GABA uptake in HEK-
N-benzylamide moiety (pIC50 = 4.63–4.96) (Table 1). The introduc-
tion of a double bond in the amino moiety at the 2-position of the
acidic fragment of the amides obtained and a tertiary amine led to
an increase in the inhibitory potency of compounds 20a–e.

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P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
The introduction of an additional methyl group at the 4-position
of 4-hydroxybutanamide enabled the synthesis of two series of geo-
metric isomers 18a–e (from 11-cis) and 19a, b, d, e (from 11-trans).
Among the obtained compounds containing a 4-(diphenylmethylid-
ene)piperidine moiety, unsubstituted amide 19a showed the highest
inhibitory activity towards mGAT1 (pIC50 = 5.11); however, this
compound was not active in NO711 binding studies.
Taking into consideration the results of in vitro tests, two com-
pounds 16a (the most active in NO711 binding and active in uptake
studies) and 16d (active in both uptake and binding studies), which
displayed the most interesting uptake profiles, were subjected to
further in vivo tests.
Behavioral studies concerning the antinociceptive properties of
compounds 16a and 16d proved their activity in some tests in
mice. In the hot-plate assay, both 16a and 16d at 30 mg/kg pro-
longed the latency time to nocifensive response in mice, but this
effect did not reach statistical significance (F(2,25) = 1.332; p
>0.05). The reference (S)-SNAP-5114 showed no antinociceptive
activity in this test.
A statistically significant and dose-dependent analgesic activity
of the test compound 16d was observed in the acetic acid-induced
writhing test (Table 2). ED50 values of 18.1 mg/kg for 16d and
7.1 mg/kg for (S)-SNAP-5114 were determined. The analgesic po-
tency of 16d in the writhing test (ED50 = 18.1 mg/kg) (Table 2) indi-
cates that it has the ability to attenuate the symptoms of
inflammatory pain. This observation was confirmed using another
model of chemogenic pain, namely the formalin test (Table 3,
Fig. 5). The latter is a tonic pain model, which in rodents reflects
persistent pain dependent on both sensory C-fiber activation as
well as sensitization within the spinal cord dorsal horn and the
brain; however, peripheral inflammation is also an important con-
tributor 30–32. In the formalin test, both 16a and 16d demon-
strated significant analgesic properties. 16a was antinociceptive
in both phases of the test, but its activity was particularly pro-
nounced in the second (inflammatory) phase of this assay (Table 3;
Fig. 5). This can be ascribed to the formalin-evoked combination of
inflammatory reactions in the peripheral tissues and functional
changes in the dorsal horn of the spinal cord (central sensitization
of pain and neuroplasticity of the CNS).30,31,33 16d did not influence
the duration of the licking response in the first phase of the test,
but it had significant antinociceptive properties in the inflamma-
tory phase (Fig. 5). (S)-SNAP-5114 at 30 mg/kg (ip) displayed weak
antinociceptive activity in the second phase of the formalin test
(40% vs vehicle-treated mice).
Several lines of evidence indicate that there is a strong concen-
tration dependence of nociceptive responses induced by formalin
in both mice and rats. There is also evidence that distinct nocicep-
tive mechanisms might underlie animals’ pain reaction in response
to low or high concentrations of formalin 32. In our study, we used
5% formalin solution. At this concentration of formalin, in the late
phase of the test, a significant dependence of nociceptive responses
on peripheral inflammatory changes and to a lesser degree on cen-
tral sensitization is observed 33,34. In our research, this fact was
confirmed by the observed significant plasma extravasation after
formalin injection into the hind paws of control animals (data
not shown). This observation is consistent with results reported
by other authors 32, who demonstrated that 5% but not 1% forma-
lin produced significant inflammation in peripheral tissues and
plasma extravasation, which were attenuated by high doses of
anti-inflammatory drugs 32,34.
Figure 4. Potencies of compound series 20, 21 and 22.
Increased inhibitory activity of GABA uptake was observed towards
mGAT3 and mGAT4 transporters. Moreover, the compound bearing
a chloro substituent at the ortho-position of the benzyl fragment of
the molecule showed the highest potency towards mGAT3 (20b,
pIC50 = 5.14). Structural modification involving hydrogenation of
the double bond in the amino moiety of the series discussed above
was found to have a negative effect as the potencies of compounds
21a–e were greatly reduced. The rigid amine group of 4-(diph-
enylmethylidene)piperidine (16a–e) in the 2-position of the acidic
fragment of the amides obtained provided optimal results only to-
wards mGAT1. Compound 16d, which contains a fluoro substituent
at the para-position of the benzyl fragment of the molecule,
showed significant potency towards mGAT1 (pIC50 = 5.01). Struc-
tural modification involving hydrogenation of the double bond in
the amino moiety of 4-(diphenylmethyl)piperidine 17a–e caused
only a slight increase in the inhibitory activity towards mGAT1
compared with 16a–e. Unfortunately, this modification proved to
be unfavorable for the other transporters. A marked decrease in
activity was observed towards GAT4.
Based on the obtained results, it is worth noting that the antin-
ociceptive effect is of a rather peripheral origin, as indicated by the
lack of activity in the hot-plate test (this test reveals compounds
with central analgesic properties), yet statistically significant activ-
ities in two chemogenic pain models, i.e. the writhing test and in
the second phase of the formalin test.
The dependences of the potency towards inhibition of GAT for
the compound series 20, 21, and 22 on the nature of the substitu-
ents on the benzyl amide function are presented in Fig. 4.

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5159
Table 2
Antinociceptive activity of 16a, 16d and (S)-SNAP-5114 in the acetic acid-induced writhing test
Compound
Dose (mg/kg)
Number of writhes (mean SEM)
Analgesic effect (%)
ED50 (mg/kg)
Vehicle (0.5% MC)
16a
16d
F(3,44) = 13.51;
p <0.0001
(S)-SNAP-5114
F(3,37) = 13.12;
p <0.0001
—
55.3 3.9
38.8 8.3
39.3 6.6
—
—
—
30.0
15.0
20.0
30.0
3.75
7.5
29.8
28.9
69.9
79.4
23.3
62.7
70.5
18.1 (14.1–23.4)
16.6 8.1⁄⁄⁄
11.4 7.6⁄⁄⁄
42.4 8.5⁄⁄⁄
20.6 3.9
7.1 (4.2–12.2)
15.0
16.3 4.9⁄⁄⁄
Each value represents the mean SEM. The test compounds and the vehicle were administered ip 30 min before the assay. ED50 value: the dose that reduced the number of
stretches (i.e., the dose that diminished the pain reaction) for 50% as compared to vehicle-treated animals. MC: methylcellulose (vehicle).
Statistical analysis: Student’s t-test (16a) and one-way ANOVA, followed by post hoc Dunnett’s comparison (16d) and (S)-SNAP-5114). Significant difference compared to MC-
treated mice: ⁄⁄⁄p <0.0001.
The lack of motor coordination impairments in animals pre-
treated with 16a or 16d, together with their antinociceptive activ-
ity in two chemogenic pain models, indicate that these molecules
are interesting lead structures for the further development of novel
analgesic drugs (Table 4).
unsubstituted amide 19a showed the highest inhibitory activity to-
wards mGAT1 (19a; pIC50 = 5.11).
The presence of a chloro substituent at the para-position of the
N-benzylamide moiety had a beneficial effect on the activity
against mGAT2. Slightly lower values were obtained by switching
the chloro substituent to the ortho position. Nevertheless, this sub-
stitution afforded compound 22c, the most active agent against
mGAT2 (22c; pIC50 = 4.96). However, replacement of the chloro
substituent in the para-position by a fluoro substituent or reverting
to an unsubstituted N-benzylamide moiety caused a decrease in
activity. As in the case of mGAT1, a para-methyl group showed
no significant effect on the activity towards mGAT2.
Among the obtained series, substitution on the N-benzylamide
moiety did not produce a significant effect on the activity towards
mGAT3. However, compound 18b, bearing a chloro substituent at
the ortho-position, exhibited a slightly improved subtype selectiv-
ity with respect to mGAT3 (18b; pIC50 = 4.54). Among the obtained
compounds, compound 20b, which contains a chloro substituent in
the ortho position, was the most active toward mGAT3 (20b;
pIC50 = 5.14). It is worthy of note that the N,N-(methyl)(4,4-diphe-
nyl)but-3-enylamine moiety is the most preferred lipophilic biaryl
group toward mGAT3, regardless of the substituent on the benzyl
fragment of the molecule.
5. Conclusion
Seven series of new compounds have been produced. An N-
bulky and lipophilic biaryl group in the 2-position of 4-hydroxybu-
tanamide proved to be significant for inhibitory potency towards
GAT1-4. The highest activities against the mGAT1 transporter were
observed for 4-(diphenylmethylidene)piperidine- and 4-(diphenyl-
methyl)piperidine-substituted 4-hydroxybutanamides (16 and 17)
and
the
4-(diphenylmethylidene)piperidine-substituted
4-
hydroxypentanamide (18). Modification that involved opening of
the piperidine ring was well-tolerated against mGAT2 (22). Also,
the introduction of an additional methyl group on the amino group
in the 2-position of 4-hydroxybutanamide improved the activity
with respect to mGAT3 and mGAT4 (20, 21).
Moreover, the introduction of substituents on the benzyl frag-
ment of molecules is essential for activity against mGAT1. The
introduction of an ortho- or para-chloro substituent led to a de-
crease in activity towards mGAT1. However, replacement of the
chloro substituent in the para-position by a fluoro substituent
yielded better results, but a methyl group in the para-position
did not significantly affect the activity. Among the obtained com-
pounds containing a 4-(diphenylmethylidene)piperidine moiety,
The effect on the activity toward mGAT4 was comparable to
that toward mGAT3. Increased activity toward mGAT4 was also ob-
served for compound 20b (20b; pIC50 = 5.00).
The results of in vivo studies indicated that the test compounds
displayed antinociceptive properties in these pain models, in
which pain is induced by chemical stimuli acting within peripheral
Table 3
Antinociceptive activity of 16a, 16d and (S)-SNAP-5114 in the formalin test
Compound
Dose
(mg/kg)
Duration of licking response (s)
SEM (phase 1)
Analgesic
activity (%)
ED50 (mg/kg)
(phase 1)
Duration of licking response (s)
SEM (phase 2)
Analgesic
activity (%)
ED50 (mg/kg)
(phase 2)
Vehicle
(0.5% MC)
16a
—
55.4 6.6
—
111.6 17.1
—
7.5
39.0 6.1
31.0 4.9⁄
27.7 5.0⁄⁄
59.9 7.4
50.4 8.4
58.1 5.6
34.8 9.4
29.6
44.0
50.0
NA
NA
NA
27.1 (7.8–94.4)
—
75.1 6.5
32.7
64.5
85.4
72.3
84.1
78.5
—
11.1 (7.1–17.3)
—
15.0
30.0
7.5
15.0
30.0
—
39.6 7.2⁄⁄⁄
16.3 8.1⁄⁄⁄
30.9 14.4⁄⁄⁄
17.8 7.3⁄⁄⁄
24.0 3.3⁄⁄⁄
78.8 15.9
16d
Vehicle
(0.5% MC)
(S)-SNAP-
5114
—
—
—
—
—
30.0
35.6 5.9
NA
47.7 11.6
39.5
Each value represents the mean SEM. The test compounds and the vehicle were administered ip 30 min before the assay. ED50 value: the dose that reduced the duration of
licking response (i.e., the dose that diminished the pain reaction) for 50% as compared to vehicle-treated animals. MC: methylcellulose (vehicle). NA: not active.
Statistical analysis: Student’s t-test ((S)-SNAP-5114) and one-way ANOVA, followed by post hoc Dunnett’s comparison (16a and 16d). 16a: F(3,29) = 4.777; p <0.01 (phase 1)
and F(3,32) = 11.38; p <0.0001 (phase 2).
16d: F(3,29) = 0.3149; p >0.05 (phase 1) and F(3,31) = 12.39; p <0.0001 (phase 2). Significant difference compared to MC-treated mice: ⁄p <0.05; ⁄⁄p <0.01; ⁄⁄⁄p <0.001.

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P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
ada) using the ESI method. Melting points were determined in
open glass capillaries on a Melting Point Büchi 535 apparatus
and are uncorrected. Reactions under microwave irradiation were
carried out in Discover LabMate (CEM Corporation, USA).
The synthesis of the 4-(diphenylmethylidene)piperidine (1) and
4-benzhydryl piperidine (2) was performed based on method de-
scribed in literature.25 The synthesis of the 1,10-(4-bromobut-1-
ene-1,1-diyl)dibenzene (3) was performed according to methods
described in literature.26 Synthesis of N-methyl-4,4-diphenylbut-
3-en-1-amine (4) and 4,4-diphenylbut-3-en-1-amine (6) was per-
formed as previously described.23,27
6.1.1. General procedure for the synthesis of amines (5) and (7)
To the solution of 1 mmol (4 or 6) in ethanol (6 ml) 70 mg Pd/C
(10%) was added. After hydrogenation for 8 h at room temperature,
the catalyst was removed through celite and the filtrate was con-
centrated in vacuo to afford (5) or (7) as oils, which were used
for the next step without further purification.
6.1.1.1. N-Methyl-4,4-diphenylbutan-1-amine (5).
Thick
yellow oil; yield: 80%; Rf: 0.40 (S2); 1H NMR (300 MHz, chloro-
form-d) d ppm 1.43–1.53 (m, 2H(CH2CH2NH)) 2.05–2.14 (m,
2H(CHCH2)) 2.39 (s, 3H(Me)) 2.60 (t, J = 7.18 Hz, 2H(CH2NH)) 3.91
(t, J = 7.82 Hz, 1H(CHCH2)) 7.13–7.36 (m, 10H(Ar)), ESI-MS (m/z)
239.1 [M+H]+.
6.1.1.2. 4,4-Diphenylbutan-1-amine (7).
Thick yellow oil;
yield: 92%; Rf: 0,27 (S2); 1H NMR (300 MHz, chloroform-d) d ppm
1.37–1.44 (m, 2H(CH2CH2NH2)) 2.03–2.14 (m, 2H(CHCH2)) 2.71 (t,
J = 7.05 Hz, 2H(CH2NH2)) 3.90 (t, J = 7.82 Hz, 1H(CHCH2)) 7.13–
7.40 (m, 10H(Ar)), ESI-MS (m/z) 226.1 [M+H]+.
6.1.2. General procedure for the synthesis of 3-subsituted
dihydrofuran-2(3H)-one derivatives and 3-subsituted 5-methy-
ldihydrofuran-2(3H)-one derivatives (10–15)
Anhydrous K2CO3 (5 equiv) was added to the solution of rele-
vant amine (1 equiv) and tetrabutylammonium bromide (TBAB)
(0.01 equiv) in the acetonitrile and the mixture was stirred at at
0 °C for 1.5 h. Then a solution of 3-bromodihydrofuran-2(3H)-one
(8) or 3-bromo-5-methyldihydrofuran-2(3H)-one (9) (1 equiv)
was added dropwise and stirring was continued for 12–48 h at
room temperature. After the reaction was completed, the precipi-
tate was filtered off and the filtrate was concentrated under vac-
uum. Obtained crude products were purified by column
chromatography.
Figure 5. Analgesic activity of the test compounds in the neurogenic (0–5 min)
phase (Fig. 5A) and in the second—inflammatory (15–30 min) phase (Fig. 5B) of the
formalin test. Significant difference compared to MC-treated mice: ⁄p <0.05; ⁄⁄p
<0.01; ⁄⁄⁄p <0.001.
tissues. The effect of GAT inhibition as the mechanism underlying
antinociception requires further study. Nevertheless, our research
indicates a potential role of GAT subtypes in peripheral pain and
inflammation.
6.1.2.1. 3-[4-(Diphenylmethylidene)piperidin-1-yl]dihydrofu-
ran-2(3H)-one (10).
Reagents and conditions: 21.25 mmol 1
6. Experimental
6.1. Chemistry
(5.30 g), 85 mmol K2CO3 (11.75 g), 0.65 mmol TBAB (0.21 g),
21.25 mmol 8 (3.50 g), 50 ml MeCN, 24 h; purification by column
chromatography (S7); Yield 98%; yellow solid; mp 164.1–
165.3 °C; Rf: 0.89 (S3); 1H NMR (300 MHz, chloroform-d) d ppm
2.30–2.39 (m, 2H(NCHCH2CH2)) 2.41–2.52 (m, 4H(piperidine))
2.60–2.69 (m, 2H(piperidine)) 2.89 (dt, J = 10.64, 5.45 Hz, 2H(piper-
idine)) 3.66 (t, J = 9.62 Hz, 1H(NCH)) 4.16–4.25 (m, 1H(CH2CH2O))
4.33–4.40 (m, 1H(CH2CH2O)) 7.09–7.31 (m, 10H(Ar)), ESI-MS (m/
z) 334.1 [M+H]+.
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 (PE/EtOAc 7:3, v/v); S2 (n-hexan/
ethanol/triethylamine 7:2:1, v/v/v), S3 (chloroform/acetone 1:1, v/
v), S4 (PE/EtOAc 1:1, v/v), S5 (DCM/acetone 9:1, v/v), S6 (chloro-
form/acetone 9:1, v/v), S7 (DCM/acetone 7:3, v/v). The plates were
visualized with the UV light and mixture of ninhydrin (0.3 g ninhy-
drin in 100 mL of n-butanol and 3 mL of acetic acid). NMR spectra
were recorded with VX Merkury 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 Elementa-
rVario EL III (Elementar Analysensysteme, Hanau, Germany). Mass
spectra were recorded on MDX SCILEX API 2000 (Concord, ON, Can-
6.1.2.2.
3-[4-(Diphenylmethylidene)piperidin-1-yl]-5-meth-
Reagents
yldihydrofuran-2(3H)-one (cis-11 or trans-11).
and conditions: 16 mmol 1 (3.98 g), 16 mmol K2CO3 (2.21 g),
0.16 mmol TBAB (0.05 g), 16 mmol 9 (2.85 g), 20 ml MeCN, 20 h;
purification by column chromatography (S6) and recrystallization
from n-hexane/EtOAc 1:1; Yield 71%; white solid; cis-11: 0.83;
trans-11 0.78 (S7); 1H NMR (300 MHz, chloroform-d) d ppm
(cis-18): 1.43 (d, J = 6.16 Hz, 3H(Me)) 2.20–2.54 (m, 6H(piperidine))

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5161
Table 4
Influence of 16a, 16d and (S)-SNAP-5114 on motor coordination in the rotarod test
Compound
Dose Rotations per minute
(rpm)
Mean time spent on the rotarod ( SEM) Number of animals with motor impairments/number of animals tested
(s)
(X/Y)
Vehicle
(0.5% MC)
—
6
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
60.0 0.0
59.6 0.4
56.3 3.8
59.0 1.0
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
0/8
1/8
1/8
1/8
18
24
6
18
24
6
18
24
6
18
24
16a
16d
30.0
30.0
30.0
(S)-SNAP-
5114
Motor coordination measured on the rotarod apparatus rotating at 6, 18 and 24 rpm 30 min after the ip administration of each compound. Data are expressed as: mean time
spent on the rotarod ( SEM) or X/Y: the number of animals falling off the rod (X) per the number of mice tested (Y). Animals per group were: 8.
Statistical analysis of data: average time spent on the rotarod: one-way ANOVA followed by Dunnett’s test, F(3,28) = 1.000; p >0.05 (6, 18, 24 rpm). Qualitative variables (i.e.,
the number of mice showing motor impairments vs number of mice that performed the test) were statistically evaluated by means of the Fisher’s exact probability test
(differences not significant).
2.61 (dt, J = 10.45, 5.42 Hz, 2H(piperidine)) 2.82–2.92 (m, 2H(piper-
idine)) 3.71 (dd, J = 12.18, 8.34 Hz, 1H(NCH)) 4.38–4.51 (m,
1H(CHMe)) 7.08–7.14 (m, 4H(Ar)) 7.18–7.31 (m, 6H(Ar)); (trans-
11): 1.43 (d, J = 6.16 Hz, 3H(Me)) 2.20–2.54 (m, 6H(piperidine))
2.61 (dt, J = 10.45, 5.42 Hz, 2H(piperidine)) 2.82–2.92 (m, 2H(piper-
idine)) 4.09–4.16 (q, 1H(NCH)) 4.64–4.73 (m, 1H(CHMe)) 7.08–7.14
(m, 4H(Ar)) 7.18–7.31 (m, 6H(Ar)), ESI-MS (m/z) 338.1 [M+H]+.
40.37 mmol K2CO3 (5.58 g), 0.08 mmol TBAB (0.03 g), 8.18 mmol
8 (0.80 g), 10 ml MeCN, 48 h; purification by column chromatogra-
phy (S5); Yield 60%; yellow oil; Rf: 0.74 (S3); 1H NMR (300 MHz,
chloroform-d) d ppm 1.42–1.54 (m, 2H(CH2CH2NH)) 2.03–2.17
(m, 3H(NHCHCH2); (CHCH2CH2)) 2.37–2.50 (m, 1H(CHCH2CH2))
2.64 (dt, J = 11.22, 7.21 Hz, 1H(CH2NH)) 2.76 (dt, J = 11.16,
6.99 Hz, 1H(CH2NH)) 3.49 (dd, J = 10.39, 8.08 Hz, 1H(NHCH)) 3.89
(t, J = 7.82 Hz, 1H(ArCHCH2)) 4.17 (td, J = 9.81, 6.28 Hz, 1H(CH2CH2-
O)) 4.37 (td, J = 8.91, 2.18 Hz, 1H(CH2CH2O)) 7.09–7.40 (m,
10H(Ar)), ESI-MS (m/z) 310.2 [M+H]+.
6.1.2.3.
3-[4-(Diphenylmethyl)piperidin-1-yl]dihydrofuran-
Reagents and conditions: 4.85 mmol
2(3H)-one (12).
2
(1.22 g), 24.25 mmol K2CO3 (3.35 g), 0.05 mmol TBAB (0.02 g),
4.85 mmol 8 (0.80 g), 20 ml MeCN, 20 h; purification by column
chromatography (S5); Yield 72%; yellow oil; Rf: 0.76 (S7); 1H NMR
(300 MHz, chloroform-d) d ppm 1.27–1.39 (m, 2H(piperidine))
1.54–1.65 (m, 2H(piperidine)) 2.15 (dt, J = 11.41, 3.53 Hz, 1H(piper-
idine)) 2.22–2.35 (m, 3H (piperidine)) 2.60 (td, J = 11.41, 2.56 Hz,
1H(CHCH)) 2.71–2.80 (m, 1H(NCHCH2CH2)) 2.95–3.02 (m,
1H(NCHCH2CH2)) 3.47–3.52 (d, 1H (CHCH)) 3.54–3.61 (t, 1H
(NCH)) 4.17 (td, J = 8.91, 7.82 Hz, 1H(CH2CH2O)) 4.27–4.38 (m, 1H
(CH2CH2O)) 7.07–7.41 (m, 10H(Ar)), ESI-MS (m/z) 336.2 [M+H]+.
6.1.3. 2-Substituted 4-hydroxybutanamides (16a–e to 22a–e)
General procedure 1 (GP1): 2-Substituted dihydrofuran-2(3H)-
one derivative (1 equiv) was heated with relevant amine (2.5 or
1.2 equiv) in THF at reflux for 48–72 h. Then the solvent was evap-
orated under vacuum and crude product was purified by column
chromatography and recrystallized from n-hexane/EtOAc 1:1.
General procedure 2 (GP2): The process vial was charged with 2-
substituted dihydrofuran-2(3H)-one derivative (1 equiv) and rele-
vant benzylamine derivative (3 equiv). The vial was then closed
and its content was magnetically stirred and microwave heated
at 160 °C for 1 h. After the reaction was finished, the crude product
was purified by column chromatography (S3) and recrystallized
from n-hexane/EtOAc 1:1.
6.1.2.4. 3-[(4,4-Diphenylbut-3-en-1-yl)(methyl)amino]dihydro-
furan-2(3H)-one (13).
Reagents and conditions: 6.03 mmol 4
(1.43 g), 30.19 mmol K2CO3 (4.17 g), 0.06 mmol TBAB (0.02 g),
6.03 mmol 8 (1.00 g), 15 ml MeCN, 48 h; purification by column
chromatography (S6); Yield 78%; yellow oil; Rf: 0.70 (S6); 1H NMR
(CDCl3) d [ppm]: 2.17–2.20 ppm (m, 2H(CH2CH2N)), 2.23–2.30
(m, 2H(NCHCH2)) 2.31 (s, 3H (Me)), 2.68–2.74 ppm (t, 2H
(CH2N)), 3.62–3.68 ppm (t, 1H(NCH)), 4.13–4.37 ppm (m, 2H(CH2-
CH2O), 6.13 (t, 1H(C@CH)), 7.17–7.40 ppm (m, 10H (Ar)), ESI-MS
(m/z) 322.1 [M+H]+.
6.1.3.1. N-Benzyl-4-hydroxy-2-(4-(diphenylmethylidene)piperi-
din-1-yl)butanamide (16a).
white solid; mp 157–160 °C; Rf: 0.62 (S3). Anal. Calcd for
29H32N2O2: C, 79.06; H, 7.32; N, 6.36. Found: C. 79.15; H, 7.46;
Procedure GP2. Yield: 34%;
C
N, 6.42. 1H NMR (300 MHz, chloroform-d) d ppm 1.81–1.99 (m,
2H(CH2CH2OH)) 2.25–2.42 (m, 4H(piperidine)) 2.49–2.67 (m,
4H(piperidine)) 3.26 (dd, J = 9.49, 3.08 Hz, 1H(NCH)) 3.59–3.68
(m, 1H(CH2OH)) 3.84–3.91 (m, 1H(CH2OH)) 4.39–4.55 (m,
2H(NHCH2)) 7.07–7.14 (m, 4H(Ar)) 7.14–7.36 (m, 11H(Ar)) 7.84
(br s, 1H(CONH)); 13C NMR (300 MHz, chloroform-d) d ppm 33.5,
56.9 (piperidine) 34.6 (CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH)
70.1 (CHCH2) 129.3 (C@CPh2) 137.8 (CPh2) 126.7, 126.9, 127.9,
128.3, 128.6, 137.9, 142.0 (arom) 171.3 (carbonyl); ESI-MS (m/z)
441.2 [M+H]+.
6.1.2.5.
2(3H)-one (14).
3-[(4,4-Diphenylbutyl)(methyl)amino]dihydrofuran-
Reagents and conditions: 1.78 mmol
5
(0.40 g), 8.90 mmol K2CO3 (1.23 g), 0.02 mmol TBAB (0.01 g),
1.78 mmol 8 (0.29 g), 7 ml MeCN, 24 h; purification by column
chromatography (S7); Yield 75%; yellow oil; Rf: 0.76 (S7); 1H NMR
(300 MHz, chloroform-d) d ppm 1.41–1.53 (m, 2H(CH2CH2N))
2.03–2.13 (m, 2H(CHCH2CH2)) 2.16–2.25 (m, 2H(NCHCH2)) 2.29
(s, 3H(Me)) 2.59 (t, J = 7.44 Hz, 2H(CH2N)) 3.59 (t, J = 9.62 Hz,
1H(NCH)) 3.90 (t, J = 7.82 Hz, 1H(ArCHCH2)) 4.09–4.19 (m, 1H(CH2-
CH2O)) 4.29–4.37 (m, 1H(CH2CH2O)) 7.05–7.42 (m, 10H(Ar)), ESI-
MS (m/z) 324.2 [M+H]+.
6.1.3.2. N-(2-Chlorobenzyl)-4-hydroxy-2-(4-(diphenylmethylid-
ene)piperidin-1-yl)butanamide (16b).
Procedure GP2. Yield:
36%; white solid; mp 174–175 °C; Rf: 0.72 (S3). Anal. Calcd for C29-
H31ClN2O2: C, 73.33; H, 6.58; N, 5.90. Found: C, 73.25; H, 6.66; N,
5.82. 1H NMR (300 MHz, chloroform-d) d ppm 1.76–1.87 (m,
1H(CH2CH2OH)) 1.89–1.98 (m, 1H(CH2CH2OH)) 2.29–2.41 (m,
6.1.2.6. 3-[(4,4-Diphenylbutyl)amino]dihydrofuran-2(3H)-one
(15).
Reagents and conditions: 8.17 mmol
7
(1.84 g),

5162
P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
4H(piperidine)) 2.48–2.60 (m, 4H(piperidine)) 3.23 (dd, J = 9.49,
3.08 Hz, 1H(NCH)) 3.62 (ddd, J = 11.28, 8.34, 3.46 Hz, 1H(CH2OH))
3.80–3.88 (m, 1H(CH2OH)) 4.49–4.62 (m, 2H(NHCH2)) 7.05–7.14
(m, 4H(Ar)) 7.15–7.33 (m, 10H(Ar)) 8.00–8.10 (m, 1H(CONH));
13C NMR (300 MHz, chloroform-d) d ppm 33.5, 56.9 (piperidine)
34.6 (CHCH2CH2) 38.5 (CH2Ph) 58.3 (CH2OH) 70.1 (CHCH2) 129.3
(C@CPh2) 137.8 (CPh2) 126.6, 127.9, 128.3, 128.6, 132.2, 142.0,
142.4 (arom) 171.3 (carbonyl); ESI-MS (m/z) 375.5 [M+H]+.
(300 MHz, chloroform-d) d ppm 26.7, 38.0, 44.2 (piperidine) 34.6
(CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH) 53.7 (CPh2) 70.0 (CHCH2)
126.2, 126.7 126.9, 128.2, 128.5, 137.9, 143.0 (arom) 171.3 (car-
bonyl); ESI-MS (m/z) 443.1 [M+H]+
6.1.3.7. N-(2-Chlorobenzyl)-4-hydroxy-2-(4-(diphenylmethyl)-
piperidin-1-yl)butanamide (17b).
Procedure GP1. Reagents
and conditions: 12 (2.50 mmol, 0.84 g), 2-chlorobenzylamine
(3 mmol, 0.42 g), THF (15 mL), 72 h; purification by column chroma-
tography (S7); recrystallization from n-hexane/EtOAc 1:1; yield: 60%;
whitesolid;mp173.6 °C;Rf: 0.53(S3). Anal. CalcdforC29H33ClN2O2:C,
73.02; H, 6.97; N, 5.87. Found: C, 73.17: H, 7.01: N, 5.99. 1H NMR
(300 MHz, chloroform-d) dppm 1.08–1.30 (m, 2H(piperidine)) 1.52–
1.61 (m, 2H(piperidine)) 1.73–1,97 (m, 2H(piperidine)) 2.02–2.21 (m,
2H(piperidine)) 2.34–2.47 (m, 1H(NCHCO)) 2.52–2.62 (m,
1H(NCHCH2CH2)) 2.64–2.75 (d, 1H (NCHCH2CH2)) 3.12–3.21 (m,1H
(ArCHCH))3.41–3.52 (d, J = 11.03 Hz, 1H(ArCH)) 3.54–3.65 (m,
1H(CH2OH)) 3.77–3.87 (m, 1H(CH2OH)) 4.41–4.62 (m, 2H (NHCH2))
7.09–7.40 (m, 14H(Ar)) 7.96 (br s, 1H(CONH)), 13C NMR (300 MHz,
chloroform-d) d ppm 26.7, 38.0, 44.2 (piperidine) 34.6 (CHCH2CH2)
38.5 (CH2Ph) 58.3 (CH2OH) 53.7 (CPh2) 70.0 (CHCH2) 126.2, 126.6,
128.1, 128.2, 128.3, 129.2, 132.2, 143.0 (arom) 171.3 (carbonyl);
ESI-MS (m/z) 477,5 [M+H]+
6.1.3.3. N-(4-Chlorobenzyl)-4-hydroxy-2-(4-(diphenylmethylid-
ene)piperidin-1-yl)butanamide (16c).
Procedure GP2. Yield:
34%; white solid; mp 130-132 °C; Rf: 0.64 (S3). Anal. Calcd for C29-
H31ClN2O2: C, 73.33; H, 6.58; N, 5.90. Found: C, 73.47; H, 6.56; N,
5.94. 1H NMR (300 MHz, chloroform-d) d ppm 1.79–1.92 (m,
1H(CH2CH2OH)) 1.93–2.02 (m, 1H(CH2CH2OH)) 2.23–2.45 (m,
4H(piperidine)) 2.47–2.66 (m, 4H(piperidine)) 3.25 (dd, J = 9.49,
3.33 Hz, 1H(NCH)) 3.60–3.69 (m, 1H(CH2OH)) 3.81–3.91 (m,
1H(CH2OH)) 4.31–4.55 (m, 2H(NHCH2)) 7.09 (d, J = 9.49 Hz,
4H(Ar)) 7.14–7.41 (m, 10H(Ar)) 7.84 (s, 1H(CONH)), 13C NMR
(300 MHz, chloroform-d)
d ppm 33.5, 56.9 (piperidine) 34.6
(CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH) 70.1 (CHCH2) 129.3
(C@CPh2) 137.8 (CPh2) 127.9, 128.6, 127.9, 132.2, 134.6, 136.0,
142.0 (arom) 171.3 (carbonyl); ESI-MS (m/z) 375.5 [M+H]+.
6.1.3.4. N-(4-Fluorobenzyl)-4-hydroxy-2-(4-(diphenylmethylid-
6.1.3.8.
N-(4-Chlorobenzyl)-4-hydroxy-2-(4-(diphenyl-
Procedure GP1.
ene)piperidin-1-yl)butanamide (16d).
Procedure GP2. Yield:
methyl)piperidin-1-yl)butanamide (17c).
29%; white solid; mp 68–69 °C; Rf: 0.71 (S3). Anal. Calcd for C29H31-
FN2O2; C, 75.96; H, 6.81; N, 6.11. Found: C, 75.89; H, 6.96; N, 6.28
1H NMR (300 MHz, chloroform-d) d ppm 1.79–2.01 (m, 2H(CH2CH2-
OH)) 2.23–2.44 (m, 4H(piperidine)) 2.49–2.64 (m, 4H(piperidine))
3.25 (dd, J = 9.62, 3.21 Hz, 1H(NCH)) 3.58–3.69 (m, 1H(CH2OH))
3.81–3.93 (m, 1H(CH2OH)) 4.32–4.55 (m, 2H(NHCH2)) 6.94–7.14
(m, 5H(Ar)) 7.14–7.34 (m, 9H(Ar)) 7.84 (br s, 1H(CONH)), 13C
NMR (300 MHz, chloroform-d) d ppm 33.5, 56.9 (piperidine) 34.6
(CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH) 70.1 (CHCH2) 129.3
(C@CPh2) 137.8 (CPh2) 115.3, 127.9, 128.3, 128.5, 128.6, 133.5,
142.0, 160.9 (arom) 171.3 (carbonyl);ESI-MS (m/z) 459.3 [M+H]+.
Reagents and conditions: 12 (2.50 mmol, 0.84 g), 4-chlorobenzyl-
amine (3 mmol, 0.42 g), THF (15 mL), 72 h; purification by column
chromatography (S7); recrystallization from n-hexane/EtOAc 1:1;
yield: 69%; white solid; mp 141.4 °C; Rf: 0.47 (S3). Anal. Calcd for
C29H33ClN2O2: C, 73.02; H, 6.97: N, 5.87. Found: C, 73.20; H,
7.16; N, 6.01. 1H NMR (300 MHz, chloroform-d) d ppm 1.06–1.24
(m, 2H(piperidine)) 1.49–1.66 (m, 2H(piperidine)) 1.74–1,98 (m,
2H(piperidine)) 2.02–2.21 (m, 2H(piperidine)) 2.35–2.48 (t,
1H(NCHCO)) 2.51–2.64 (m, 1H(NCHCH2CH2)) 2.65–2.76 (d, 1H
(NCHCH2CH2)) 3.11–3.23 (m,1H (ArCHCH)) 3.42–3.50 (d,
1H(ArCH)) 3.55–3.68 (m, 1H(CH2OH)) 3.77–3.90 (m, 1H(CH2OH))
4.33–4.50 (m, 2H (NHCH2)) 7.06–7.40 (m, 14H(Ar)) 7.76 (br s,
1H(CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 26.7, 38.0,
44.2 (piperidine) 34.6 (CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH)
53.7 (CPh2) 70.0 (CHCH2) 126.2, 128.2, 128.6, 129.2, 132.3, 134.6,
136.0, 143.0 (arom) 171.3 (carbonyl); ESI-MS (m/z) 477,5 [M+H]+.
6.1.3.5. N-(4-Methylbenzyl)-4-hydroxy-2-(4-(diphenylmethylid-
ene)piperidin-1-yl)butanamide (16e).
30%; white solid; mp 140–142 °C; Rf: 0.64 (S3). Anal. Calcd for
30H34N2O2: C, 79.26; H, 7.54; N, 6.16. Found: C, 79.34; H, 7.69;
N, 6.28. 1H NMR (300 MHz, chloroform-d)
ppm 1.85 (d,
Procedure GP2. Yield:
C
d
J = 18.72 Hz, 1H(CH2CH2OH)) 1.94–2.03 (m, 1H(CH2CH2OH)) 2.28–
2.43 (m, 7H(piperidine; Me)) 2.47–2.65 (m, 4H(piperidine)) 3.26
(d, J = 3.08 Hz, 1H(NCH)) 3.62 (d, J = 7.95 Hz, 1H(CH2OH)) 3.81–
3.91 (m, 1H(CH2OH)) 4.38–4.52 (m, 2H(NHCH2)) 7.04–7.32 (m,
14H(Ar)) 7.77 (br s, 1H(CONH)), 13C NMR (300 MHz, chloroform-
d) d ppm 21.3 (CH3) 33.5, 56.9 (piperidine) 34.6 (CHCH2CH2) 43.6
(CH2Ph) 58.3 (CH2OH) 70.1 (CHCH2) 129.3 (C@CPh2) 137.8 (CPh2)
127.9, 128.1, 128.3, 128.6, 128.8, 134.9, 136.4, 142.0 (arom)
171.3 (carbonyl); ESI-MS (m/z) 455.1 [M+H]+.
6.1.3.9. N-(4-Fluorobenzyl)-4-hydroxy-2-(4-(diphenylmethyl)-
piperidin-1-yl)butanamide (17d).
Procedure GP1. Reagents
and conditions: 12 (2.33 mmol, 0.78 g), 4-fluorobenzylamine
(2.80 mmol, 0.35 g), THF (15 mL), 72 h; purification by column
chromatography (S7); recrystallization from n-hexane/EtOAc 1:1;
yield: 70%; white solid; mp 131.1 °C; Rf: 0.51 (S3). Anal. Calcd for
C29H33FN2O2: C, 75.62; H, 7.22; N, 6.08. Found: C, 75.91; H, 7.43;
N, 6.22. 1H NMR (300 MHz, chloroform-d) d ppm 0.96–1.31 (m,
2H(piperidine)) 1.50–1.64 (dd, J = 13.08, 2.31 Hz, 2H(piperidine))
1.86–1,99 (m, 2H(piperidine)) 2.01–2.24 (m, 2H(piperidine)) 2.37–
2.48 (m, 1H(NCHCO)) 2.55–2.64 (m, 1H(NCHCH2CH2)) 2.65–2.76
(m, 1H (NCHCH2CH2)) 3.15–3.23 (dd, J = 9.62, 3.21 Hz, 1H
(ArCHCH)) 3.43–3.50 (d, J = 11.03 Hz, 1H(ArCH)) 3.56–3.68 (m,
1H(CH2OH)) 3.80–3.89 (m, 1H(CH2OH)) 4.33–4.49 (m, 2H (NHCH2))
6.96–7.09 (m, 2H(Ar)) 7.09–7.40 (m, 12H(Ar)) 7.76 (br s,
1H(CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 26.7, 38.0,
44.2 (piperidine) 34.6 (CHCH2CH2) 43.6 (CH2Ph) 58.3 (CH2OH)
53.7 (CPh2) 70.0 (CHCH2) 115.3, 126.2, 128.5, 129.2, 143.0, 160.9
(arom) 171.3 (carbonyl); ESI-MS (m/z) 461,5 [M+H]+.
6.1.3.6. N-Benzyl-4-hydroxy-2-(4-(diphenylmethyl)piperidin-1-
yl)butanamide (17a).
Procedure GP1. Reagents and condi-
tions: 12 (2.33 mmol, 0.78 g), benzylamine (2.80 mmol, 0.30 g),
THF (15 mL), 72 h; purification by column chromatography (S7);
recrystallization from n-hexane/EtOAc 1:1; yield: 76%; white solid;
mp 112.1 °C; Rf: 0.62 (S3). Anal. calcd for C29H34N2O2: C, 78.70; H,
7.74; N, 6.33. Found: C, 79.19; H, 7.56; N, 6.42. 1H NMR (300 MHz,
chloroform-d) d ppm 1.06–1.21 (m, 2H(piperidine)) 1.51–1.67 (m,
2H(piperidine)) 1.77–2.24 (m, 4H(piperidine)) 2.38–2.48 (m,
1H(NCHCO)) 2.57–2.81 (m, 2H(NCHCH2CH2))) 3.19 (dd, J = 9.49,
3.08 Hz, 1H(ArCHCH)) 3.46 (d, J = 11.03 Hz, 1H(ArCH)) 3.56–3.67
(m, 1H(CH2OH)) 3.80–3.90 (m, 1H(CH2OH)) 4.43–4.50 (m, 2H
(NHCH2)) 7.05–7.41 (m, 15H(Ar)) 7.76 (br s, 1H(CONH)), 13C NMR
6.1.3.10. N-(4-Methylbenzyl)-4-hydroxy-2-(4-(diphenylmethyl)-
piperidin-1-yl)butanamide (17e).
Procedure GP1. Reagents
and conditions: 12 (2.60 mmol, 0.87 g), 4-methylbenzylamine

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5163
(3.12 mmol, 0.38 g), THF (15 mL), 72 h; purification by column
chromatography (S7); recrystallization from n-hexane/EtOAc 1:1;
yield: 64%; white solid; mp 147.4 °C; Rf: 0.69 (S3). Anal. Calcd for
6.1.3.13. N-(4-Chlorobenzyl)-4-hydroxy-2-(4-(diphenylmethy-
lidene)piperidin-1-yl)pentanamide (18c). Procedure GP1.
Reagents and conditions: 11 (1.50 mmol, 0.52 g), 4-chlorobenzyl-
amine (3.32 mmol, 0.47 g), THF (10 mL), 72 h. Crude product which
was a mixture of diastereomers were separated by column chro-
matography (S5) and recrystallized from n-hexane/EtOAc 1:1;
yield: 47%; white solid; mp 150.1–156.6 °C; Rf: 0.72 (cis); (S3). Anal.
Calcd for C30H33ClN2O2: C, 73.68; H, 6.80; N, 5.73. Found: C, 73.64;
H, 6.83; N, 5.77. 1H NMR (300 MHz, chloroform-d) d ppm: 1.26 (d,
J = 6.16 Hz, 3H(Me)) 1.71–1.82 (m, 2H(CH2CH2OH)) 2.28–2.42 (m,
4H(piperidine)) 2.46–2.63 (m, 4H(piperidine)) 3.31 (d, J = 11.54 Hz,
1H(NCH)) 3.72–3.85 (m, 1H(CHOH)) 4.35–4.46 (m, 1H(NHCH2))
4.46–4.54 (m, 1H(NHCH2)) 7.07 (m, 3H(Ar)) 7.20 (m, 2H(Ar)) 7.14
(m, 3H(Ar)) 7.28 (m, 6H(Ar)) 8.00 (br s, 1H(CONH)), 13C NMR
(300 MHz, chloroform-d) d ppm 22.8 (CHCH3) 33.5, 56.9 (piperi-
dine) 43.8 (CHCH2CH) 43.6 (CH2Ph) 62.6 (CH2OH) 67.6 (CHCH2)
129.3 (C@CPh2) 137.8 (CPh2) 127.9, 128.3, 128.6, 132.3, 134.6,
136.0, 142.0 (arom) 171.3 (carbonyl); ESI-MS (m/z) 489,5 [M+H]+.
C30H36N2O2: C, 78.91; H, 7.95; N, 6.13. Found: C, 79.04; H, 7.83;
N, 6.17;1H NMR (300 MHz, chloroform-d) d ppm 1.48–1.67 (m,
3H(piperidine)) 1.77–1.86 (m, 1H(piperidine)) 1.89–1,97 (m,
1H(piperidine)) 1.97–2.25 (m, 3H(piperidine)) 2.33 -2.36 (s, 3H
(Me)) 2.38–2.47 (m, 1H(NCHCO)) 2.58–2.74 (m, 2H(NCHCH2CH2))
3.14–3.21 (m,1H (ArCHCH)) 3.42–3.49 (d, J = 10.77, 1H(ArCH))
3.57–3.65 (m, 1H(CH2OH)) 3.81–3.88 (m, 1H(CH2OH)) 4.38–4.44
(dd, J = 5.77, 2.44 Hz, 2H (NHCH2)) 7.13–7.27 (m, 14H(Ar)) 7.71
(br s, 1H(CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 21.3
(CH3) 26.7, 38.0, 44.2 (piperidine) 34.6 (CHCH2CH2) 43.6 (CH2Ph)
58.3 (CH2OH) 53.7 (CPh2) 70.0 (CHCH2) 126.2, 128.1, 128.2,
128.5, 128.8, 134.9, 136.4, 143.0 (arom) 171.3 (carbonyl); ESI-MS
(m/z) 456,1 [M+H]+.
6.1.3.11. N-benzyl-4-hydroxy-2-(4-(diphenylmethylidene)piper-
idin-1-yl)pentanamide (18a; 19a).
Procedure GP1. Reagents
and conditions: 11 (1.50 mmol, 0.52 g), benzylamine (1.80 mmol,
0.19 g), THF (8 mL), 72 h. Crude product was a mixture of diaste-
reomers which were separated by column chromatography (S5)
and recrystallized from n-hexane/EtOAc 1:1; yield: 60% (18a);
40% (19a); white solid; mp 136.4-139 °C (18a);136.8-140.5 °C
(19a); Rf: 0.74 (18a); 0.70 (19a) (S3). Anal. Calcd for C30H34N2O2:
C, 79.26; H, 7.54; N, 6.16. Found: (18a) C, 79.28; H, 7.69; N, 6.17.
Found: (19a) C, 79.31; H, 7.70; N, 6.24. 1H NMR (300 MHz, chloro-
form-d) d ppm (18a); 1.26 (s, 3H(Me)) 1.70–1.85 (m, 2H(CH2CH2-
OH)) 2.22–2.43 (m, 4H(piperidine)) 2.43–2.69 (m, 4H(piperidine))
3.32 (dd, J = 9.10, 2.44 Hz, 1H(NCH)) 3.73–3.83 (m, 1H(CHOH))
4.48–4.59 (m, 2H(NHCH2)) 7.06–7.15 (m, 5H(Ar)) 7.15–7.39 (m,
10H(Ar)) 7.99 (br s, 1H(CONH)); (19a) 1.19 (d, J = 6.16 Hz,
3H(Me)) 1.74–1.83 (m, 2H(CH2CH2OH)) 2.23–2.41 (m, 4H(piperi-
dine)) 2.46–2.74 (m, 4H(piperidine)) 3.37 (dd, J = 6.80, 1H(NCH))
3.70–3.83 (m, 1H(CHOH)) 4.40–4.54 (m, 2H(NHCH2)) 7.10–7.49
(m, 15H(Ar)) 7.73 (br s, 1H(CONH)), 13C NMR (300 MHz, chloro-
form-d) d ppm 22.8 (CHCH3) 33.5, 56.9 (piperidine) 43.8 (CHCH2-
CH) 43.6 (CH2Ph) 62.6 (CH2OH) 67.6 (CHCH2) 129.3 (C@CPh2)
137.8 (CPh2) 126.7, 126.9, 127.9, 128.3, 128.5, 128.6, 137.9, 142.0
(arom) 171.3 (carbonyl); ESI-MS (m/z) 455,1 [M+H]+.
6.1.3.14. N-(4-Fluorobenzyl)-4-hydroxy-2-(4-(diphenylmethy-
lidene)piperidin-1-yl)pentanamide (18d; 19d).
Procedure
GP1. Reagents and conditions: 11 (1.50 mmol, 0.52 g),4-fluoroben-
zylamine (1.80 mmol, 0.22 g), THF (10 mL), 72 h. Crude product
which was a mixture of diastereomers were separated by column
chromatography (S5) and recrystallized from n-hexane/EtOAc
1:1; yield: 60% (18d); 44% (19d); white solid; mp 152.2–162.4 °C
(18d);153.4–163.6 °C (19d); Rf: 0.77 (18d); 0.76 (19d) (S3). Anal.
Calcd for C30H33FN2O2: C, 76.24; H, 7.04; N, 5.93. Found: (18d) C,
76.28; H, 7.19; N, 5.97. Found: (19d) C, 76.31; H, 7.10; N, 5.84.
1H NMR (300 MHz, chloroform-d) d ppm (18d) 1.28 (s, 3H(Me))
1.68–1.89 (m, 2H(CH2CH2OH)) 2.06–2.42 (m, 4H(piperidine))
2.42–2.76 (m, 4H(piperidine)) 3.31 (dd, J = 9.23, 2.31 Hz,1H(NCH))
3.72–3.84 (m, 1H(CHOH)), 4.31–4.42 (m, 1H(NHCH2)) 4.45–4.57
(m, 1H(NHCH2)) 6.80–7.15 (m, 5H(Ar)) 7.15–7.39 (m, 9H(Ar))
7.98 (br s, 1H(CONH)); (19d)1.19 (d, J = 6.16, 3H(Me)) 1.72–1.83
(m, 2H(CH2CH2OH)) 2.34 (q, J = 5.13 Hz, 4H(piperidine)) 2.57 (t,
J = 5.51 Hz, 4H(piperidine)) 3.32–3.39 (m, 1H(NCH)) 3.97–4.12 (m,
1H(CHOH)), 4.32–4.54 (m, 2H(NHCH2)) 6.98–7.15 (m, 5H(Ar))
7.15–7.32 (m, 9H(Ar)) 7.40 (br s, 1H(CHOH)), 13C NMR (300 MHz,
chloroform-d) d ppm 22.8 (CHCH3) 33.5, 56.9 (piperidine) 43.8
(CHCH2CH) 43.6 (CH2Ph) 62.6 (CH2OH) 67.6 (CHCH2) 129.3
(C@CPh2) 137.8 (CPh2) 115.3, 127.9, 128.3, 128.5, 128.6, 133.5,
142.0, 160.9 (arom) 171.3 (carbonyl); ESI-MS (m/z) 473,1 [M+H]+.
6.1.3.12. N-(2-Chlorobenzyl)-4-hydroxy-2-(4-(diphenylmethy-
lidene)piperidin-1-yl)pentanamide (18b; 19b).
Procedure
GP1. Reagents and conditions: 11 (1.50 mmol, 0.52 g), 2-chloro-
benzylamine (3.67 mmol, 0.52 g), THF (10 mL), 72 h. Crude product
which was a mixture of diastereomers were separated by column
chromatography (S5) and recrystallized from n-hexane/EtOAc
1:1; yield: 50% (18b); 30% (19b); white solid; mp 190.1–192.6 °C
(18b);198.2–207.3 °C (19b); Rf: 0.74 (18b); 0.72 (19b) (S3). Anal.
Calcd for C30H33ClN2O2: C, 73.68; H, 6.80: N, 5.73. Found: (18b)
C, 73.74; H, 7.99; N, 5.81. Found: (19b) C, 73.61; H, 6.70; N, 5.64.
1H NMR (300 MHz, chloroform-d) d ppm(18b) 1.25 (d, J = 6.16 Hz,
3H(Me)) 1.66–1.85 (m, 2H(CH2CH2OH)) 2.20–2.43 (m, 4H(piperi-
dine)) 2.43–2.66 (m, 4H(piperidine)) 3.29 (dd, J = 9.36, 2.18 Hz,
1H(NCH)) 3.71–3.82 (m, 1H(CHOH)) 4.43–4.64 (m, 2H(NHCH2))
6.76–7.16 (m, 4H(Ar)) 7.16–7.32 (m, 8H(Ar)) 7.37 (d, J = 5.64 Hz,
2H(Ar)) 8.21 (s, 1H(CONH)); (19b) 1.17 (d, J = 6.16 Hz, 3H(Me))
1.71–1.80 (m, 2H(CH2CH2OH)) 2.26–2.44 (m, 4H(piperidine))
2.46–2.66 (m, 4H(piperidine)) 3.29–3.36 (m, 1H(NCH)) 4.03 (dd,
J = 12.18, 5.00 Hz, 1H(CHOH)) 4.46–4.61 (m, 2H(NHCH2)) 6.99–
7.14 (m, 4H(Ar)) 7.14–7.47 (m, 10H(Ar)) 7.61 (s, 1H(CONH)), 13C
NMR (300 MHz, chloroform-d) d ppm 22.8 (CHCH3) 33.5, 56.9
(piperidine) 43.8 (CHCH2CH) 38.5 (CH2Ph) 62.6 (CH2OH) 67.6
(CHCH2) 129.3 (C@CPh2) 137.8 (CPh2) 126.6, 127.9, 128.1, 128.3,
128.6, 132.2, 142.0. 142.4 (arom) 171.3 (carbonyl); ESI-MS (m/z)
489,5 [M+H]+.
6.1.3.15. N-(4-Methylbenzyl)-4-hydroxy-2-(4-(diphenylmethy-
lidene)piperidin-1-yl)pentanamide (18e; 19e).
Procedure
GP1. Reagents and conditions: 11 (1.01 mmol, 0.35 g), 4-methylob-
enzylamine (2.31 mmol, 0.29 g), THF (10 mL), 48 h. Crude product
which was a mixture of diastereomers were separated by column
chromatography (S5) and recrystallized from n-hexane/EtOAc
1:1; yield: 64% (18e); 31% (19e); white solid; mp 165.8–167.7 °C
(18e);151.3–154.5 °C (19e); Rf: 0.73 (18e); 0.71 (19e) (S3). Anal.
Calcd for C31H36N2O2: C, 79.45; H, 7.74; N, 5.98. Found: (18e) C,
79.58; H, 7.79; N, 6.07. Found: (19e) C, 79.51; H, 7.70; N, 5.94.
1H NMR (300 MHz, chloroform-d)
d ppm (18e) 1.26 (d,
J = 6.16 Hz, 3H(Me)) 1.69–1.86 (m, 2H(CH2CH2OH)) 2.21–2.31 (m,
2H(piperidine)) 2.33 (s, 3H(ArMe)) 2.34–2.45 (m, 2H(piperidine))
2.45–2.68 (m, 4H(piperidine)) 3.30 (dd, J = 8.98, 2.56 Hz,
1H(NCH)) 3.73–3.83 (m, 1H(CHOH)) 4.34–4.43 (m, 1H(NHCH2))
4.43–4.55 (m, 1H(NHCH2)) 6.98–7.36 (m, 14H(Ar)) 7.93 (br s,
1H(CHOH)); (19e) 1.18 (d, J = 6.41 Hz, 3H(Me)) 1.75–1.82 (m,
2H(CH2CH2OH)) 2.33 (s, 3H(ArMe)) 2.6 (m, J = 7.44 Hz, 4H(piperi-
dine)) 2.63 (m, J = 5.00, 4H(piperidine)) 3.41 (dd, J = 9.49 Hz,
1H(NCH)) 3.99–4.07 (m, 1H(CHOH)) 4.35–4.48 (m, 2H(NHCH2))
7.05–7.31 (m, 14H(Ar)) 7.43 (br s, 1H(CHOH)), 13C NMR
(300 MHz, chloroform-d) d ppm 21.3 (CH3) 22.8 (CHCH3) 33.5,

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P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
56.9 (piperidine) 43.8 (CHCH2CH) 43.6 (CH2Ph) 62.6 (CH2OH) 67.6
(CHCH2) 129.3 (C@CPh2) 137.8 (CPh2) 127.9, 128.3, 128.6, 128.8,
134.9, 136.4, 142.0 (arom) 171.3 (carbonyl); ESI-MS (m/z) 469,1
[M+H]+.
(CHCH2CH2) 34.3 (CHCH2CH) 41.7 (CH3) 43.6 (CH2Ph) 57.3 (CH2-
CH2N) 58.3 (CH2OH) 72.6 (CHCH2) 115.3 (C@CPh2) 133.8 (CPh2)
115.3, 127.9, 128.3, 128.5, 128.6, 133.5, 140.0, 160.9 (arom)
171.3 (carbonyl); ESI-MS (m/z) 445,3 [M+H]+.
6.1.3.16. N-Benzyl-4-hydroxy-2-(N-methyl-4,4-diphenylbut-3-
6.1.3.20.
N-(4-methylbenzyl)-4-hydroxy-2-(N-methyl-4,4-
Procedure
en-1-amino)butanamide (20a).
Procedure GP1. Reagents
diphenylbut-3-en-1-amino)butanamide (20e).
and conditions: 13 (2.12 mmol, 0.68 g), benzylamine (2.54 mmol,
0.27 g), THF (7 mL), 48 h; purification by column chromatography
(S3); yield: 63%; yellow oil; Rf: 0.77 (S3). Anal. Calcd for
GP1. Reagents and conditions: 13 (1.56 mmol, 0.50 g), 4-methyl-
benzylamine (1.88 mmol, 0.23 g), THF (5 mL), 48 h; purification
by column chromatography (S3); yield: 74%; yellow oil; Rf: 0.78
(S3). Anal. Calcd for C29H34N2O2: C, 78.70; H, 7.74; N, 6.33. Found:
C, 78.72; H, 7.78; N, 6.47. 1H NMR (CDCl3) d [ppm]: 1.36 (s, 3H
(Me)) 1.81–1.84 (m, 2H (CH2CH2N)) 2.11 (s, 3H (Me)) 2.53–2.57
(m, 2H (CH2CH2OH)) 3.23–3.28 (m, 1H (CH2N)) 3.54–3.58 (m, 1H
(CH2N)) 3.82–3.87 (m, 1H (NCH)) 4.13–4.19 (q, 2H (CH2OH))
4.31–4.35 (d, 2H (NHCH2)) 6.03–6.09(t, 1H (C@CH)) 6.97–6.60
(m,14H (Ar)), 7.84-7.87 (s, 1H (CONH)), 13C NMR (300 MHz, chloro-
form-d) d ppm 21.3 (CH3) 24.2 (CHCH2CH2) 34.3 (CHCH2CH) 41.7
(CH3) 43.6 (CH2Ph) 57.3 (CH2CH2N) 58.3 (CH2OH) 72.6 (CHCH2)
115.3 (C@CPh2) 133.8 (CPh2) 127.9, 128.1, 128.3, 128.6, 128.8,
134.9, 136.4, 140.0 (arom) 171.3 (carbonyl); ESI-MS (m/z) 443,1
[M+H]+.
C
28H32N2O2: C, 78.47; H, 7.53; N, 6.54. Found: C, 78.54; H, 7.43;
N, 6.67. 1H NMR (CDCl3) d [ppm]: 1.82–1.85 (m, 2H (CH2CH2N)
2.21 (s, 3H (Me)) 2.54–2.58 (m, 2H (CH2CH2OH)) 3.20–3.29 (m,
1H (CH2N)) 3.50–3.56 (m, 1H (CH2N)) 3.79–3.81 (m, 1H (NCH))
4.10–4.15 (m, 2H (CH2OH)) 4.29–4.32 (m, 2H (NHCH2)) 6.03 (t,
1H (C@CH)) 7.14–7.37 (m, 15H (Ar)) 7.82 (s, 1H (CONH)), 13C
NMR (300 MHz, chloroform-d) d ppm 24.2 (CHCH2CH2) 34.3
(CHCH2CH) 41.7 (CH3) 43.6 (CH2Ph) 57.3 (CH2CH2N) 58.3 (CH2OH)
72.6 (CHCH2) 115.3 (C@CPh2) 133.8 (CPh2) 126.7, 126.9, 127.9,
128.5, 128.6, 137.9, 140.0 (arom) 171.3 (carbonyl); ESI-MS (m/z)
429,3 [M+H]+.
6.1.3.17. N-(2-Chlorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diphe-
nylbut-3-en-1-amino)butanamide (20b).
Procedure GP1.
6.1.3.21. N-Benzyl-4-hydroxy-2-(N-methyl-4,4-diphenylbutyla-
Reagents and conditions: 13 (1.56 mmol, 0.50 g), 2-chlorobenzyl-
amine (1.88 mmol, 0.27 g), THF (5 mL), 48 h; purification by col-
umn chromatography (S3); yield: 60%; yellow oil; Rf: 0.79 (S3).
Anal. Calcd for C28H31ClN2O2: C, 72.63; H, 6.75; N, 6.05. Found: C,
72.64; H, 6.73; N, 6.07. 1H NMR (CDCl3) d [ppm]: 1.81 (m, 2H (CH2-
CH2N)) 2.15 (s, 3H (Me)) 2.52 (m, 2H (CH2CH2OH)) 3.23 (m, 1H
(CH2N)) 3.53 (m, 1H (CH2N)) 3.81 (m, 1H (NCH)) 4.15 (q, 2H (CH2-
OH)) 4.29 (d, 2H (NHCH2)) 6.01 (t, 1H (C@CH)) 7.04–7.41 (m, 14H
(Ar)) 7.71 (s, 1H (CONH)), 13C NMR (300 MHz, chloroform-d) d
ppm 24.2 (CHCH2CH2) 34.3 (CHCH2CH) 41.7 (CH3) 38.5 (CH2Ph)
57.3 (CH2CH2N) 58.3 (CH2OH) 72.6 (CHCH2) 115.3 (C@CPh2)
133.8 (CPh2) 126.6, 127.9, 128.1, 128.3, 128.6, 140.0, 142.4 (arom)
171.3 (carbonyl); ESI-MS (m/z) 463,5 [M+H]+.
mino)butanamide (21a).
Procedure GP1. Reagents and con-
ditions: 14 (1.5 mmol, 0.50 g), benzylamine (3.75 mmol, 0.40 g),
THF (10 mL), 72 h; purification by column chromatography (S7);
yield: 78%; yellow oil; Rf: 0.59 (S3). Anal. Calcd for C28H33N2O2: C,
78.10; H, 7.96; N, 6.51. Found: C, 78.12; H, 7.98; N, 6.57. 1H NMR
(300 MHz, chloroform-d) d ppm 1.32–1.44 (m, 2H(CH2CH2N))
1.81–1.89 (m, 2H(CH2CH2OH)) 1.90–2.01 (m, 2H(CHCH2)) 2.13 (s,
3H(Me)) 2.39 (t, J = 7.18 Hz, 2H(CH2N)) 3.18–3.25 (m, 1H(NCH))
3.52–3.61 (m, 1H(CHCH2)) 3.78–3.88 (m, 2H(CH2OH)) 4.39–4.49
(m, 2H(NHCH2)) 7.13–7.35 (m, 15H(Ar)) 7.76 (br s, 1H(CONH)),
13C NMR (300 MHz, chloroform-d) d ppm 24.6 (CHCH2CH2) 34.3
(CHCH2CH) 36.3 (CH2CHPh2) 41.6 (CH3) 43.6 (CH2Ph) 50.0 (CHPh2)
56.5 (CH2CH2N) 58.3 (CH2OH) 72.5 (CHCH2) 126.2, 126.7, 126.9,
128.2, 128.5, 128.7, 137.9, 145.1 (arom) 171.3 (carbonyl); ESI-MS
(m/z) 430,1 [M+H]+.
6.1.3.18. N-(4-Chlorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diphe-
nylbut-3-en-1-amino)butanamide (20c).
Procedure GP1.
Reagents and conditions: 13 (1.56 mmol, 0.50 g), 4-chlorobenzyl-
amine (1.88 mmol, 0.27 g), THF (5 mL), 48 h; purification by col-
umn chromatography (S3); yield: 58%; yellow oil; Rf: 0.76 (S3).
Anal. Calcd for C28H31ClN2O2: C, 72.63; H, 6.75; N, 6.05. Found: C,
72.69; H, 6.77; N, 6.11. 1H NMR (CDCl3) d [ppm]: 1.83 (m, 2H (CH2-
CH2N)) 2.11 (s, 3H (Me)) 2.53 (m, 2H (CH2CH2OH)) 3.21 (m, 1H
(CH2N)) 3.53 (m, 1H (CH2N)) 3.82 (m, 1H (NCH)) 4.13 (q, 2H (CH2-
OH)) 4.31 (d, 2H (NHCH2)) 6.03 (t, 1H (C@CH)) 6.97–6.60 (m, 14H
(Ar)) 7.84–7.87 (s, 1H (CONH)), 13C NMR (300 MHz, chloroform-
d) d ppm 24.2 (CHCH2CH2) 34.3 (CHCH2CH) 41.7 (CH3) 43.6 (CH2Ph)
57.3 (CH2CH2N) 58.3 (CH2OH) 72.6 (CHCH2) 115.3 (C@CPh2) 133.8
(CPh2) 126.6, 127.9, 128.5, 128.6,132.3, 134.6, 136.0, 140.0 (arom)
171.3 (carbonyl); ESI-MS (m/z) 463,5 [M+H]+.
6.1.3.22. N-(2-Chlorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diph-
enylbutylamino)butanamide (21b).
Procedure GP1. Re-
agents and conditions: 14 (1 mmol, 0.33 g), 2-chlorobenzylamine
(2.50 mmol, 0.35 g), THF (10 mL), 72 h; purification by column
chromatography (S7); yield: 75%; yellow oil; Rf: 0.63 (S3). Anal.
Calcd for C28H33ClN2O2: C, 72.32; H, 7.15; N, 6.02. Found: C,
72.42; H, 7.18; N, 6.07. 1H NMR (300 MHz, chloroform-d) d ppm
1.35–1.44 (m, 2H(CH2CH2N)) 1.78–1.86 (m, 2H(CH2CH2OH))
1.95–2.04 (m, 2H(CHCH2)) 2.11 (s, 3H(Me)) 2.39 (t, J = 7.18 Hz,
2H(CH2N) 3.16–3.21 (m, 1H(NCH)) 3.50–3.59 (m, 1H(CHCH2))
3.79–3.87 (m, 2H(CH2OH)) 4.52 (dd, J = 6.16, 2.82 Hz, 2H(NHCH2))
7.08–7.41 (m, 14H(Ar)) 7.91 (br s, 1H(CONH)), 13C NMR
(300 MHz, chloroform-d) d ppm 24.6 (CHCH2CH2) 34.3 (CHCH2CH)
36.3 (CH2CHPh2) 41.6 (CH3) 43.6 (CH2Ph) 50.0 (CHPh2) 56.5 (CH2-
CH2N) 58.3 (CH2OH) 72.5 (CHCH2) 126.2, 126.6, 128.1, 128.2,
128.6, 129.2, 132.2, 142.4, 145.1 (arom) 171.3 (carbonyl); ESI-MS
(m/z) 465,5 [M+H]+.
6.1.3.19. N-(4-Fluorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diphe-
nylbut-3-en-1-amino)butanamide (20d).
Procedure GP1.
Reagents and conditions: 13 (1.68 mmol, 0.54 g), 4-fluorobenzyl-
amine (2.02 mmol, 0.25 g), THF (5 mL), 48 h; purification by col-
umn chromatography (S3); yield: 66%; yellow oil; Rf: 0.76 (S3).
Anal. Calcd for C28H31FN2O2: C, 75.31; H, 7.00; N, 6.27. Found: C,
75.34; H, 7.03; N, 6.27. 1H NMR (CDCl3) d [ppm]: 1.84-1.87 (m,
2H (CH2CH2N)) 2.25 (s, 3H (Me)) 2.53-2.56 (m, 2H (CH2CH2OH))
3.22-3.27 (m, 1H (CH2N)) 3.53- 3.58 (m, 1H (CH2N)) 3.82- 3.89
(m, 1H (NCH)) 4.11–4.15 (q, 2H (CH2OH)) 4.30-4.34 (d, 2H
(NHCH2)) 6.03-6.07 (t, 1H (C@CH)) 7.04–7.37 (m, 14H (Ar)) 7.83
(s, 1H (CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 24.2
6.1.3.23. N-(4-Chlorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diph-
enylbutylamino)butanamide (21c).
Procedure GP1. Re-
agents and conditions: 14 (1 mmol, 0.33 g), 4-chlorobenzylamine
(2.50 mmol, 0.35 g), THF (10 mL), 72 h; purification by column
chromatography (S7); yield: 80%; yellow oil; Rf: 0.66 (S3). Anal.
Calcd for C28H33ClN2O2: C, 72.32; H, 7.15; N, 6.02. Found: C,
72.39; H, 7.14; N, 6.11. 1H NMR (300 MHz, chloroform-d) d ppm
1.32–1.46 (m, 2H(CH2CH2N)) 1.79–1.88 (m, 2H(CH2CH2OH))

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5165
1.92–2.04 (m, 2H(CHCH2)) 2.13 (s, 3H(Me)) 2.39 (t, J = 7.31 Hz,
2H(CH2N)) 3.17–3.23 (m, 1H(NCH)) 3.52–3.61 (m, 1H(CHCH2))
3.79–3.87 (m, 2H(CH2OH)) 4.31–4.43 (m, 2H(NHCH2)) 7.09–7.35
(m, 14H(Ar)) 7.78 (br s, 1H(CONH)), 13C NMR (300 MHz, chloro-
form-d) d ppm 24.6 (CHCH2CH2) 34.3 (CHCH2CH) 36.3 (CH2CHPh2)
41.6 (CH3) 43.6 (CH2Ph) 50.0 (CHPh2) 56.5 (CH2CH2N) 58.3 (CH2OH)
72.5 (CHCH2) 126.2, 128.2, 128.6, 129.2, 132.3, 134.6, 136.0, 145.1
(arom) 171.3 (carbonyl); ESI-MS (m/z) 465,5 [M+H]+.
0.34 g), THF (5 mL), 72 h; purification by column chromatography
(S5); recrystallization from n-hexane/EtOAc 1:1; yield: 60%; white
solid; mp 93.5 °C; Rf: 0.47 (S3). Anal. Calcd for C27H31ClN2O2: C,
71.90; H, 6.93; N, 6.21. Found: C, 71.98; H, 6.98; N, 6.27. 1H NMR
(300 MHz, chloroform-d) d ppm 1.36–1.47 (m, 2H(CH2CH2NH))
1.75–1.85 (m, 2H(CH2CH2OH)) 1.95–2.06 (m, 2H(CHCH2)) 2.46–
2.60 (m, 2H(CH2NH)) 3.19 (t, J = 6.80 Hz, 1H(NHCH)) 3.68–3.78
(m, 2H(CH2OH)) 3.83 (t, J = 7.82 Hz, 1H(CHCH2)) 4.47–4.58 (m,
2H(NHCH2)) 7.13–7.38 (m, 14H(Ar)) 7.54 (d, J = 11.54 Hz,
1H(CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 27.1 (CHCH2-
CH2) 36.8 (CHCH2CH) 36.0 (CH2CHPh2) 38.6 (CH2Ph) 50.0 (CHPh2)
48.2 (CH2CH2N) 58.0 (CH2OH) 64.2 (CHCH2) 126.2, 126.6, 128.1,
128.2, 128.6, 129.2, 142.4, 145.1 (arom) 171.3 (carbonyl); ESI-MS
(m/z) 451,5 [M+H]+.
6.1.3.24. N-(4-Fluorobenzyl)-4-hydroxy-2-(N-methyl-4,4-diph-
enylbutylamino)butanamide (21d).
agents and conditions: 14
Procedure GP1. Re-
(2,3 mmol, 0.77 g), 4-
fluorobenzylamine (5,75 mmol, 0.72 g), THF (10 mL), 72 h; purifi-
cation by column chromatography (S7); yield: 71%; yellow oil; Rf:
0.63 (S3). Anal. Calcd for C28H33FN2O2: C, 74.97; H, 7.42; N, 6.25.
Found: C, 74.92; H, 7.48; N, 6.27; 1H NMR (300 MHz, chloroform-
d) d ppm 1.32–1.46 (m, 2H(CH2CH2N)) 1.78–1.88 (m, 2H(CH2CH2-
OH)) 1.91–2.01 (m, 2H(CHCH2)) 2.12 (s, 3H(Me)) 2.39 (t,
J = 7.18 Hz, 2H(CH2N)) 3.16–3.23 (m, 1H(NCH)) 3.51–3.60 (m,
1H(CHCH2)) 3.78–3.87 (m, 2H(CH2OH)) 4.37 (dd, J = 6.16, 3.59 Hz,
2H(NHCH2)) 6.95–7.02 (m, 2H(Ar)) 7.14–7.29 (m, 12H(Ar)) 7.77
(br s, 1H(CONH)), 13C NMR (300 MHz, chloroform-d) d ppm 24.6
(CHCH2CH2) 34.3 (CHCH2CH) 36.3 (CH2CHPh2) 41.6 (CH3) 43.6
(CH2Ph) 50.0 (CHPh2) 56.5 (CH2CH2N) 58.3 (CH2OH) 72.5 (CHCH2)
115.3, 126.2, 128.2, 128.5, 129.2, 133.5, 145.1 (arom) 171.3 (car-
bonyl); ESI-MS (m/z) 449,1 [M+H]+.
6.1.3.28. N-(4-Chlorobenzyl)-4-hydroxy-2-(4,4-diphenylbutyla-
mino)butanamide (22c).
Procedure GP1. Reagents and con-
ditions: 15 (1.5 mmol, 0.46 g), 4-chlorobenzylamine (3.75 mmol,
0.53 g), THF (10 mL), 72 h; purification by column chromatography
(S5); recrystallization from n-hexane/EtOAc 1:1; yield: 57%; white
solid; mp 105.9 °C; Rf: 0.38 (S3). Anal. Calcd for C27H31ClN2O2: C,
71.90; H, 6.93; N, 6.21. Found: C, 71.87; H, 7.02; N, 6.17. 1H NMR
(300 MHz, chloroform-d) d ppm 1.35–1.45 (m, 2H(CH2CH2NH))
1.78–1.86 (m, 2H(CH2CH2OH)) 1.97–2.08 (m, 2H(CHCH2)) 2.48–
2.62 (m, 2H(CH2NH)) 3.20 (t, J = 6.80 Hz, 1H(NHCH)) 3.71–3.79
(m, 2H(CH2OH)) 3.80–3.86 (m, 1H(CHCH2)) 4.39 (t, J = 5.51 Hz,
2H(NHCH2)) 7.14–7.31 (m, 14H(Ar)) 7.46 (br s, 1H(CONH)), 13C
NMR (300 MHz, chloroform-d) d ppm 27.1 (CHCH2CH2) 36.8
(CHCH2CH) 36.0 (CH2CHPh2) 43.6 (CH2Ph) 50.0 (CHPh2) 48.2 (CH2-
CH2N) 58.0 (CH2OH) 64.2 (CHCH2) 126.2, 128.2, 128.6, 129.2, 132.3,
136.0, 145.1 (arom) 171.3 (carbonyl); ESI-MS (m/z) 451,5 [M+H]+.
6.1.3.25. N-(4-Methylbenzyl)-4-hydroxy-2-(N-methyl-4,4-diph-
enylbutylamino)butanamide (21e).
agents and conditions: 14
Procedure GP1. Re-
(1 mmol, 0.33 g), 4-
methylbenzylamine (2.50 mmol, 0.30 g), THF (10 mL), 72 h; purifi-
cation by column chromatography (S7); yield: 86%; yellow oil; Rf:
0.67 (S3). Anal. Calcd for C29H38N2O2: C, 77.99; H, 8.58; N, 6.27.
Found: C, 77.96; H, 8.58; N, 6.26; 1H NMR (300 MHz, chloroform-
d) d ppm 1.31–1.44 (m, 2H(CH2CH2N)) 1.81–1.89 (m, 2H(CH2CH2-
OH)) 1.90–2.02 (m, 2H(CHCH2)) 2.12 (s, 3H(NMe)) 2.33 (s,
3H(ArMe)) 2.38 (t, J = 7.18 Hz, 2H(CH2N)) 3.16–3.23 (m, 1H(NCH))
3.52–3.61 (m, 1H(CHCH2)) 3.77–3.89 (m, 2H(CH2OH)) 4.33–4.44
(m, 2H(NHCH2)) 7.06–7.38 (m, 14H(Ar)) 7.72 (br s, 1H (CONH)),
13C NMR (300 MHz, chloroform-d) d ppm 21.3 (CH3) 24.6 (CHCH2-
CH2) 34.3 (CHCH2CH) 36.3 (CH2CHPh2) 41.6 (CH3) 43.6 (CH2Ph)
50.0 (CHPh2) 56.5 (CH2CH2N) 58.3 (CH2OH) 72.5 (CHCH2) 126.2,
128.1, 128.2, 128.8, 129.2, 134.9, 136.4, 145.1 (arom) 171.3 (car-
bonyl); ESI-MS (m/z) 442,2 [M+H]+.
6.1.3.29. N-(4-Fluorobenzyl)-4-hydroxy-2-(4,4-diphenylbutyla-
mino)butanamide (22d).
Procedure GP1. Reagents and con-
ditions: 15 (1.5 mmol, 0.46 g), 4-fluorobenzylamine (3.75 mmol,
0.50 g), THF (10 mL), 72 h; purification by column chromatography
(S5); recrystallization from n-hexane/EtOAc 1:1; yield: 65%; white
solid; mp 87.0 °C; Rf: 0.37 (S3). Anal. Calcd for C27H31FN2O2: C,
74.63; H, 7.19; N, 6.45. Found: C, 74.67; H, 7.23; N, 6.58; 1H
NMR (300 MHz, chloroform-d) d ppm 1.34–1.44 (m, 2H(CH2CH2-
NH)) 1.77–1.85 (m, 2H(CH2CH2OH)) 1.98–2.08 (m, 2H(CHCH2))
2.47–2.61 (m, 2H(CH2NH)) 3.20 (t, J = 6.80 Hz, 1H(NHCH)) 3.70–
3.78 (m, 2H(CH2OH)) 3.83 (t, J = 7.82 Hz, 1H(CHCH2)) 4.39 (dd,
J = 5.90, 3.33 Hz, 2H(NHCH2)) 6.92–7.04 (m, 2H(Ar)) 7.09–7.35
(m, 12H(Ar)) 7.43 (br s, 1H(CONH)), 13C NMR (300 MHz, chloro-
form-d) d ppm 27.1 (CHCH2CH2) 36.8 (CHCH2CH) 36.0 (CH2CHPh2)
43.6 (CH2Ph) 50.0 (CHPh2) 48.2 (CH2CH2N) 58.0 (CH2OH) 64.2
(CHCH2) 115.3, 126.2, 128.2, 129.2, 133.5, 145.1 (arom) 171.3 (car-
bonyl); ESI-MS (m/z) 435,1 [M+H]+.
6.1.3.26.
butanamide (22a).
N-Benzyl-4-hydroxy-2-(4,4-diphenylbutylamino)
Procedure GP1. Reagents and conditions:
15 (0.97 mmol, 0.30 g), benzylamine (2.42 mmol, 0.26 g), THF
(5 mL), 72 h; purification by column chromatography (S5); recrys-
tallization from n-hexane/EtOAc 1:1; yield: 54%; white solid; mp
97.3 °C; Rf: 0.36 (S3). Anal. Calcd for C27H32N2O2: C, 77.85; H,
7.74; N, 6.73. Found: C, 77.92; H, 7.79; N, 6.78. 1H NMR
(300 MHz, chloroform-d) d ppm 1.37–1.47 (m, 2H(CH2CH2NH))
1.77–1.84 (m, 2H(CH2CH2OH)) 2.51–2.63 (m, 2H(CHCH2)) 3.18–
3.30 (m, 2H(CH2NH)) 3.69–3.75 (m, 1H(NHCH)) 3.77–3.94 (m,
2H(CH2OH)) 4.12 (q, J = 7.18 Hz, 1H(CHCH2)) 4.36–4.51 (m,
2H(NHCH2)) 7.19–7.33 (m, 15H(Ar)) 7.39–7.49 (m, 1H(CONH)),
13C NMR (300 MHz, chloroform-d) d ppm 27.1 (CHCH2CH2) 36.8
(CHCH2CH) 36.0 (CH2CHPh2) 43.6 (CH2Ph) 50.0 (CHPh2) 48.2 (CH2-
CH2N) 58.0 (CH2OH) 64.2 (CHCH2) 126.2, 126.9, 126.7, 128.2, 128.5,
129.2, 137.9, 145.1 (arom) 171.3 (carbonyl); ESI-MS (m/z) 417,1
[M+H]+.
6.1.3.30. N-(4-Methylbenzyl)-4-hydroxy-2-(4,4-diphenylbutyla-
mino)butanamide (22e).
Procedure GP1. Reagents and con-
ditions: 15 (0.5 mmol, 0.15 g), 4-methylbenzylamine (1.25 mmol,
0.15 g), THF (5 mL), 72 h; purification by column chromatography
(S5); recrystallization from n-hexane/EtOAc 1:1; yield: 65%; white
solid; mp 107.3 °C; Rf: 0.53 (S3). Anal. Calcd for C28H34N2O2: C,
78.10; H, 7.96; N, 6.51. Found: C, 78.18; H, 6.99; N, 6.57. 1H NMR
(300 MHz, chloroform-d) d ppm 1.36–1.43 (m, 2H(CH2CH2NH))
1.82 (td, J = 6.92, 4.87 Hz, 2H(CH2CH2OH)) 1.97–2.06 (m,
2H(CHCH2)) 2.33 (s, 3H(Me)) 2.50–2.59 (m, 2H(CH2NH)) 3.20 (t,
J = 6.80 Hz, 1H(NHCH)) 3.72–3.79 (m, 2H(CH2OH)) 3.79–3.86 (m,
1H(CHCH2)) 4.36–4.43 (m, 2H(NHCH2)) 7.12–7.21 (m, 10H(Ar))
7.23–7.28 (m, 4H(Ar)) 7.29 (t, J = 1.41 Hz, 1H(CONH)), 13C NMR
(300 MHz, chloroform-d) d ppm 21.3 (CH3) 27.1 (CHCH2CH2) 36.8
(CHCH2CH) 36.0 (CH2CHPh2) 43.6 (CH2Ph) 50.0 (CHPh2) 48.2 (CH2-
CH2N) 58.0 (CH2OH) 64.2 (CHCH2) 126.2, 126.7, 128.1, 128.5, 128.8,
6.1.3.27. N-(2-Chlorobenzyl)-4-hydroxy-2-(4,4-diphenylbutyla-
mino)butanamide (22b).
Procedure GP1. Reagents and con-
ditions: 15 (0.97 mmol, 0.30 g), 2-chlorobenzylamine (2.42 mmol,

5166
P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
129.2, 134.9136.4, 145.1 (arom) 171.3 (carbonyl); ESI-MS (m/z)
6.2.2.3.2. Writhing test. In this test mice were placed individu-
ally into glass beakers and 30 min before the experimentation
were allowed to habituate. Then, each mouse was weighed, in-
jected with the test compound or vehicle (10 ml/kg) and then
placed back into the cylinder. 30 min later, 0.9% acetic acid (in sal-
ine) was injected ip (10 ml/kg). Mice were placed in the beakers
once again and observed continuously for 30 min. Stereotypical
writhes (lengthwise constrictions of the torso with a concomitant
concave arching of the back) were counted over this period.37
6.2.2.3.3. Formalin test. In mice the injection of diluted forma-
lin produces a biphasic nocifensive behavioral response (i.e., lick-
ing or biting the injected paw). The acute nociceptive phase lasts
for the first 5 min, whereas the second (inflammatory) phase oc-
curs between 15 and 30 min after formalin injection. The analgesic
activity is indicated by the reduction of time spent on licking or
biting the injected paw in drug-treated groups compared to control
animals.
431,1 [M+H]+.
6.2. Pharmacology
6.2.1. In vitro activity
6.2.1.1. [3H]GABA uptake assay.
Inhibitory potency of com-
pounds (16a–e to 22a–e) were tested at four murine GABA transporter
subtypes mGAT1–mGAT4. The study was performed as a [3H] GABA up-
take assay based on stably transfected HEK cells, according to the pro-
cedure recently described.29 Briefly, the affinity for mGAT1 was
determined by MS-binding assay with NO 711 as a non-labeled
marker.35 Binding assays for mGAT1 based on NO 711 as native marker
were performed as described earlier.29 NO 711 was analyzed by LC–MS/
MS using an API 3200 triple quadrupole mass spectrometer according
to the method described previously.35
The compounds were considered active if GABA uptake or NO
711 binding was reduced at least by 50% at a concentration of
100 lM. For the active compounds, pIC50 values were assessed.
The formalin test in mice was performed according to.38 Briefly,
20 ll of a 5% formalin solution was injected intraplantarly (ip) into
the right hind paw using a 26-gauge needle. Immediately after for-
malin injection, the animals were placed individually into glass
beakers and were observed for the next 30 min. The time (in sec-
onds) spent on licking the injected paw during periods of 0–
5 min, and then 15–30 min was measured and was an indicator
of nociceptive behavior.
6.2.2. In vivo activity
6.2.2.1. Animals.
The behavioral experiments were carried out
at the Department of Pharmacodynamics, Faculty of Pharmacy, Jagiel-
lonian University in Kraków. Adult male Albino Swiss (CD-1) mice
weighing 18–25 g were used in the experiments. The animals were
kept in groups of 15 mice in cages at a room temperature of
22 2 °C, under light/dark (12:12) cycle and had free access to food
and water. The ambient temperature of the room and the humidity
were kept consistent throughout all the tests. For the experiments
the animals were selected in a random way and killed by cervical dis-
location immediately after the assay. Experimental groups consisted
of 6–12 animals/dose and all the animals were used only once. The
number of animals was kept at minimum to obtain definite results
with the test utilized. Prior to the test, the mice were allowed to accli-
mate to the holding cages for a minimum of 2 h. The experiments
were performed between 8 a.m. and 3 p.m. All the procedures were
approved by the Local Ethics Committee of the Jagiellonian University
in Kraków (ZI/595/2011).
6.2.2.3.4. Rotarod test. The test was performed according to the
method described by Talarek et al.39 with some minor modifica-
tions. The mice were trained daily for 3 days on the rotarod appa-
ratus (Rotarod apparatus, May Commat RR0711, Turkey; rod
diameter: 2 cm) rotating at a constant speed of 18 rotations per
minute (rpm). During each training session, the animals were
placed on a rotating rod for 3 min with an unlimited number of tri-
als. The proper experimentation was conducted at least 24 h after
the final training trial. On the test day, 30 min before the rotarod
test the mice were intraperitoneally pretreated with the test com-
pound. Then the animals were tested on the rotarod revolving at 6,
18, 24 rpm. Motor impairments, defined as the inability to remain
on the rod for 1 min, were measured at each speed and were ex-
pressed as the number of animals falling from the roller per the
number of mice tested. The mean time to fall off the rotating rod
was also counted for each dose.
6.2.2.2. Chemicals used in pharmacological assays.
For the
6.2.2.3.5. Data analysis. The data obtained in behavioral exper-
iments are expressed as mean SEM (standard error of the mean).
To compare the results between three or more groups of animals
(the investigated compound groups vs. the vehicle-treated group)
one-way ANOVA, followed by Dunnett’s post hoc statistics were
used. To compare the results between only two groups (drug-trea-
ted group vs control group) Student’s t-test was used. Qualitative
variables from the rotarod test were compared by the use of the
Fisher’s exact probability test. In each assay the difference of
means was statistically significant if p <0.05.
pharmacological studies the test compounds were suspended in
0.5% methylcellulose solution (Loba Chemie, Germany) and admin-
istered intraperitoneally (ip) 30 min before the test. Control mice
were given an appropriate amount of vehicle (i.e., methylcellulose
solution).
Acetic acid was purchased from Polskie Odczynniki Chemiczne
(Poland). It was diluted to obtain a 0.9% solution which was used in
the subsequent behavioral test. Morphine hydrochloride was pro-
vided by Polfa Kutno (Poland). 37% formaldehyde was provided
by Polskie Odczynniki Chemiczne (Poland). It was diluted to obtain
a 5% solution which was used in the subsequent behavioral test.
In the writhing test and in the formalin test ED50 values were
calculated using log-probit method.40
6.2.2.3. Behavioral tests.
The behavioral measures were
Acknowledgments
scored by trained observers blind to the experimental conditions.
6.2.2.3.1. Hot plate test. In the hot plate test mice were treated
ip either with the test compound or the vehicle 30 min before plac-
ing the animal on the hot plate apparatus (Hot Plate 2A Type Ome-
ga, Poland). This apparatus has an electrically heated surface and is
supplied with a temperature-controller that maintains the temper-
ature at 55–56 °C. The latency time until the animal licked its back
paws or jumped was recorded by means of the stop-watch. In this
assay the cut-off time was established (30 s) to avoid tissue dam-
age and mice not responding within 30 s were removed from the
apparatus and assigned a score of 30 s.36
The study was supported by the Jagiellonian University Medical
College Grant no. KDZ/001919, and the Grant from the National
Science Centre DEC-2012/05/B/NZ7/02705.
References and notes
1. Gether, U.; Andersen, P. H.; Larsson, O. M.; Schousboe, A. Trends Pharmacol. Sci.
2006, 27, 375.
2. Cherlyn, S. Y.; Woon, P. S.; Liu, J. J.; Ong, W. Y.; Tsai, G. C.; Sim, K. Neurosci.
Biobehav. Rev. 2010, 34, 958.
3. Luscher, B.; Shen, Q.; Sahir, N. Mol. Psychiatry 2011, 16, 383.

P. Kowalczyk et al. / Bioorg. Med. Chem. 21 (2013) 5154–5167
5167
4. Schousboe, A.; Sarup, A.; Larsson, O. M.; White, H. S. Biochem. Pharmacol. 2004,
68, 1557.
5. Madsen, K. K.; Clausen, R. P.; Larsson, O. M.; Krogsgaard-Larsen, P.; Schousboe,
A.; White, H. S. J. Neurochem. 2009, 109, 139144.
22. Kulig, K.; Wie˛ckowski, K.; Wie˛ckowska, A.; Gajda, J.; Pochwat, B.; Hoefner, G.
C.; Wanner, K. T.; Malawska, B. Eur. J. Med. Chem. 2011, 46, 183190.
23. Kowalczyk, P.; Hoefner, G. C.; Wanner, K. T.; Kulig, K. Acta Pol. Pharm. 2012, 69,
157160.
6. Borden, L. A. Neurochem. Int. 1996, 29, 335356.
7. Dalby, N. O. Eur. J. Pharmacol. 2003, 479, 127137.
24. Sałat, K.; Kulig, K. Future Med. Chem. 2011, 3, 211222.
25. Lee, J.; Kang, S. U.; Lim, J. O.; Choi, H. K.; Jin, M. K.; Toth, A.; Pearce, L. V.; Tran,
R.; Wang, Y.; Szabo, T.; Blumberg, P. M. Bioorg. Med. Chem. 2004, 12, 371385.
26. Chen, G.; Xia, H.; Cai, Y.; Ma, D.; Yuan, J.; Yuan, C. Bioorg. Med. Chem. Lett. 2011,
21, 234.
27. Falch, E.; Korgsgaard, P. Eur. J. Med. Chem. 1991, 26, 69.
28. Kragler, A.; Hoefner, G.; Wanner, K. T. Eur. J. Med. Chem. 2008, 43, 24042411.
29. Zepperitz, C.; Höfner, G.; Wanner, K. T. ChemMedChem 2006, 1, 208217.
30. Hunskaar, S.; Hole, K. Pain 1987, 30, 103.
8. Madsen, K. K.; White, H. S.; Schousboe, A. Pharmacol. Ther. 2010, 125, 394.
9. Palacín, M.; Estévez, R.; Bertran, J.; Zorzano, A. Physiol. Rev. 1998, 78, 9691054.
10. Vogensen, S. B.; Jørgensen, L.; Madsen, K. K.; Borkar; Wellendorph, M.;
Skovgaard-Petersen, J.; Schousboe, A.; White, H. S.; Krogsgaard-Larsen, P.;
Clausen, R. P J. Med. Chem. 2013, 56, 2160.
11. Schaffert, E. S.; Höfner, G.; Wanner, K. T. Bioorg. Med. Chem. 2011, 19,
64926504.
12. Soudijn, W.; van Wijngaarden, I. Curr. Med. Chem. 2000, 7, 106379.
13. Høg, S.; Greenwood, J. R.; Madsen, K. B.; Larsson, O. M.; Frølund, B.; Schousboe,
A.; Krogsgaard-Larsen, P.; Clausen, R. P. Curr. Top. Med. Chem. 2006, 6,
18611882.
31. Tjølsen, A.; Berge, O. G.; Hunskaar, S.; Rosland, J. H.; Hole, K. Pain 1992, 51, 517.
32. Yashpal, K.; Coderre, T. J. Eur. J. Pain 1998, 2, 6368.
33. Wang, Z. Q.; Porreca, F.; Cuzzocrea, S.; Galen, K.; Lightfoot, R.; Masini, E.;
Muscoli, C.; Mollace, V.; Ndengele, M. H.; Salvemini, D. J. Pharmacol. Exp. Ther.
2004, 309, 869878.
14. Froestl, W. Future Med. Chem. 2011, 3, 163.
15. Dalby, N. O. Neuropharmacology 2000, 39, 2399407.
16. Hu, J. H.; Yang, N.; Ma, Y. H.; Zhou, X. G.; Jiang, J.; Duan, S. H.; Mei, Z. T.; Fei, J.;
Guo, L. H. J. Neurosci. Res. 2003, 73, 565572.
34. Munro, G. Eur. J. Pharmacol. 2009, 605, 95102.
35. Höfner, G.; Wanner, K. T. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 2010,
878, 1356.
17. Xu, Y. F.; Cai, Y. Q.; Cai, G. Q.; Jiang, J.; Sheng, Z. J.; Wang, Z. G.; Fei, J. J. Neurosci.
Res. 2008, 86, 465470.
18. Ipponi, A.; Lamberti, C.; Medica, A.; Bartolini, A. Eur. J. Pharmacol. 1999, 368,
205211.
19. Schlessinger, A.; Wittwer, M. B.; Dahlin, A.; Khuri, N.; Bonomi, M.; Fan, H.;
Giacomini, K. M.; Sali, A. J. Biol. Chem. 2012, 287, 3774537756.
20. Sałat, K.; Kulig, K.; Sałat, R.; Filipek, B.; Malawska, B. Pharmacol. Rep. 2012, 64,
102112.
36. Eddy, N.; Leimbach, D. J. Pharmacol. Exp. Ther. 1953, 107, 385393.
37. Wilson, S. G.; Bryant, C. D.; Lariviere, W. R.; Olsen, M. S.; Giles, B. E.; Chesler, E.
J.; Mogil, J. S. J. Pharmacol. Exp. Ther. 2003, 305, 755764.
38. Laughlin, T. M.; Tram, K. V.; Wilcox, G. L.; Birnbaum, A. K. J. Pharmacol. Exp.
Ther. 2002, 302, 11681175.
39. Talarek, S.; Orzelska, J.; Listos, J.; Fidecka, S. Pharmacol. Rep. 2010, 62, 627634.
40. Litchfield, J. T.; Wilcoxon, E. J. Pharmacol. Exp. Ther. 1949, 96, 99113.
41. Pabel, J.; Faust, M.; Prehn, C.; Wörlein, B.; Allmendinger, L.; Höfner, G.; Wanner,
K. T. ChemMedChem 2012, 7, 12451255.
21. Sałat, K.; Wie˛ckowska, A.; Wie˛ckowski, K.; Höfner, G. C.; Kamin´ ski, J.; Wanner,
K. T.; Malawska, B.; Filipek, B.; Kulig, K. Pharmacol. Rep. 2012, 64, 81733.