Synthesis and biological evaluation of new derivatives of 2-substituted 4-hydroxybutanamides as GABA uptake inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.11.001

This study presents the synthesis of novel substituted 4-hydroxybutanamides and their influence on the activity of murine GABA transport proteins GAT1-GAT4. The active compounds, derivatives of N-arylalkyl-2-(4- diphenylmethylpiperazin-1-yl)-4-hydroxybuty

Full text of DOI:10.1016/j.ejmech.2010.11.001

European Journal of Medicinal Chemistry 46 (2011) 183e190
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new derivatives of 2-substituted
4-hydroxybutanamides as GABA uptake inhibitors
Katarzyna Kulig a, Krzysztof Wie˛ckowski a, Anna Wie˛ckowska a, Justyna Gajda a, Bart1omiej Pochwat a,
,
Georg C. Höfner b, Klaus T. Wanner b, Barbara Malawska a
*
a Department of Physicochemical Drug Analysis, Chair of Pharmaceutical Chemistry, Jagiellonian University Medical College, Medyczna 9, 30-688 Kraków, Poland
b Department für Pharmazie-Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 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:
This study presents the synthesis of novel substituted 4-hydroxybutanamides and their influence on the
activity of murine GABA transport proteins GAT1eGAT4. The active compounds, derivatives of N-aryl-
alkyl-2-(4-diphenylmethylpiperazin-1-yl)-4-hydroxybutyramide, are characterized by pIC50 values in
range of 3.92e5.06 and by slight subtype-selectivity. Among them N-4-chlorobenzylamide was the most
potent GAT inhibitor (mGAT3), while N-benzylamide was the most active in GAT1-binding assay (pKi =
4.96). The results pointed out that benzhydryl and benzylamide moieties are crucial for the activity of this
class of compounds as murine GAT inhibitors.
Received 6 May 2010
Received in revised form
19 October 2010
Accepted 1 November 2010
Available online 4 November 2010
Keywords:
GABA-uptake inhibitors
GAT1e4
Ó 2010 Elsevier Masson SAS. All rights reserved.
Butanamides
1. Introduction
treatment of epilepsy [9,10]. Although the extensively investigated
GAT1 seems to be the most promising one, the three latter still
4-Aminobutanoic acid (
g
-aminobutyric acid, GABA) is the
remain in the area of interest of current medicinal chemistry.
Potent and selective inhibitors of GAT2eGAT4 are sought-after not
only as potential drugs, but mainly as research tools for investiga-
tion of the physiological role and therapeutic potential of each
subtype [11e17]. Among GAT inhibitors, the GABA analogs nipe-
cotic acid (2), guvacine (3), and compounds SKF-89976-A (4), Cl-
966 (5), NO711 (6) (Fig. 1) represent a significant and large group.
The present work is a part of an extended search for new anti-
convulsant agents and potential antiepileptics in the group of
derivatives of 4-hydroxybutanamide [18,19]. In this paper, we present
the synthesis of substituted 4-hydroxybutanamides and biological
evaluation of their influence on murine GABA uptake proteins
essential inhibitory neurotransmitter in mammalian brain which
influences all neurological and psychological processes in central
nervous system (CNS) [1]. Since the activity of GABAergic system is
so extensive, its dysfunctions can lead to various neurological
disorders such as epilepsy, insomnia, spasticity, neuropathic pain,
as well as anxiety and other mental disorders [2e5]. The majority of
drugs effective in the treatment of disturbances mentioned above
exert the pharmacological activity by direct influence on GABA
receptors or increasing GABA accessibility in synaptic cleft through
inhibition of its metabolism or inhibition of specific transporters.
The identification of GABA transporter proteins (GATs) and their
crucial role in regulation of GABA concentration in the brain
opened up new possibilities in the search for GABAergic-active
compounds and thereby potential CNS drugs [6]. To date, four
subtypes of membrane-bound proteins transporting GABA have
been identified and cloned: GAT1, GAT2, GAT3, and GAT4 [7]. The
various types of GAT display different physiological activities and
distribution in CNS [8]. Tiagabine (1) is an example of a selective
GAT1 inhibitor and one of the most potent drugs used in the
GAT1eGAT4. 4-Hydroxybutyric acid (
g-hydroxybutyric acid, GHB),
which was chosen as the core-structure of the new compounds, is an
endogenous substance, a metabolite of GABA, which acts as an
inhibitory neurotransmitter in mammalian CNS [20,21]. Based on
structures of GAT inhibitors and derivatives of N-benzylamides of
GHB with anticonvulsant activity, a group of new structures was
designed. The carboxylic acid function of GHB was transformed into
more lipophilic primary amides or arylalkylamides. In position 2 of
GHB, a N-diphenylmethylpiperazine moiety was introduced as a part
mimicking the biaryl moieties of known GAT inhibitors and a frag-
ment corresponding to the anticonvulsant-active arylpiperazine
derivatives of GHB [22]. Other modifications in position 2, including
* Corresponding author. Tel.: þ48 12 62 05 464; fax: þ48 12 657 02 62.
E-mail address: mfmalaws@cyf-kr.edu.pl (B. Malawska).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.001

184
K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
CH3
O
O
O
S
N
OH
HN
OH
HN
OH
CH3
S
Guvacine (3)
Tiagabine (1)
Nipecotic acid (2)
CF3
O
O
O
N
OH
N
OH
O
O
N
N
OH
CF3
SKF 89976-A (4)
Cl-966 (5)
NO 711 (6)
Fig. 1. Structures of selected GABA uptake inhibitors.
introduction of analogous arylalkyl- and alkylamine residues were
also applied. Schematic structures of the designed and synthesized
compounds are presented in Fig. 2. all the designed compounds were
synthesized and tested for their inhibitory potency and selectivity
towards cloned murine GABA transporters GAT1eGAT4 in uptake
assays and their affinity to GAT1 in an MS-binding assay.
assay with NO711 as a non-labeled marker [23]. The compounds
were considered as active if GABA uptake or NO711 binding was
reduced at least by 50% at a concentration of 100
compounds, pIC50- or pKi-values were assessed.
mM. For the active
4. Results and discussion
2. Chemistry
The majority of the herein described compounds share the
same 2-piperazin-1-yl-4-hydroxybutanamide scaffold that
corresponds to arylpiperazine derivatives of the anticonvulsant-
active N-benzylamides of GHB and GABA [18,19]. These
compounds can be classified by the type of substituent on the
piperazine ring and on nitrogen atom of the amide functionality.
The present study focuses on two series of arylalkylamides
bearing on the piperazine-part benzhydryl (compounds 13e21) or
4,40-difluorobenzhydryl moieties (compounds 22e30) (series A
and B respectively) mimicking the lipophilic groups present in the
known GAT inhibitors (Fig. 1).
To verify the influence of the substituents on the inhibitory
potency of the GHB derivatives, several analogs of structures
mentioned above were synthesized. The modifications included the
exchange of benzhydrylpiperazine for 4-benzylpiperidine (38),
unsubstituted piperazine (34e36), or its oxo-analog e morpholine
(37). In the amide-functionality, apart from differently substituted
benzyl- and phenethyl- substituents, primary amide was also
obtained (21, 30, 36).
The herein presented derivatives of 4-hydroxybutanamide were
obtained by the aminolysis of the appropriate substituted butyro-
lactones (Fig. 3). The lactones (8e12) were prepared by N-alkylation
of the substituted piperazines, morpholine, or 4-benzylpiperidine by
3-bromodihydrofuran-2(3H)-one (7). The reactions were carried out
in the different solvents in the presence of an anhydrous K2CO3 at
room temperature for 3e20 h. Aminolysis of the obtained lactones
by ammonia or primary arylalkylamines resulted in primary or
secondary amides of 2-substituted-4-hydroxybutanoic acid (13e33,
37, 38). The aminolysis was performed in the different conditions
depending on reactivity and solubility of both lactones and amines,
including reactions in the solution or fusion in ambient or increased
temperature, using conventional methods of heating or microwave
irradiation. Compounds 34e36 were obtained by deprotection of
carbamates 31e33 by trifluoroacetic acid (TFA) in DCM (Fig. 3). These
methods led to 23 final compounds.
3. Biological evaluation
Biological evaluation revealed that among the tested
compounds only the benzhydryl derivatives series A (13e21) and
B (22e30) were active (Table 1). The derivatives were characterized
by pIC50 values in range of 3.92e5.06 and slight subtype-selectivity.
Among them, (R, S) 2-(4-benzhydrylpiperazin-1-yl)-N-(4-chlor-
obenzyl)-4-hydroxybutanamide (15) was found to be the most
Inhibitory potency of the amides 13e30 and 34e38 was tested
at four murine GABA transporter subtypes GAT1eGAT4. The study
was performed as a [3H] GABA uptake assay based on stably
transfected HEK cells, according to the procedure recently
described [17]. The affinity to GAT1 was determined by MS-binding
potent with an IC50 value at GAT3 of about 9
mM. Two amides of
H or F
R
A
N
HN
O
O
R"
H
N
R'
H or F
n
N
H
n = 1, 2
OH
Fig. 2. Schematic structure of designed derivatives of 2-substituted-4-hydroxybutanamides as GAT inhibitors.

K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
185
O
O
R
A
NH
R
A
Br
N
O
O
K2CO3
rt, 3 - 20 h
7
8 - 12
R
A
O
R'
N
R'
H2N
N
H
20 - 140 °C
0.5 - 72 h
OH
13 - 33, 37, 38
31 - 33
TFA, DCM
1h
34 - 36
F
R
A
O
N
N
O
N
HN
O
F
Lactones
10
Amides
8
9
-
11
37
12
38
R'
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
34
35
Cl
32
Cl
F
F
O
H
33
36
Fig. 3. Synthesis of 3-substituted dihydrofuran-2(3H)-one derivatives (8e12) and 2-substituted 4-hydroxybutanamides (13e38).
series A, N-benzylamide 13 and N-(4-fluorobenzylamide) 17 were
found to display a moderate potency at GAT1 with pIC50 values of
transport proteins are among the most promising biological targets
in the search for new antiepileptic drugs. In this study, a new group
of 2-substituted 4-hydroxybutanamides was synthesized and
evaluated for their affinity to GABA transporters GAT1eGAT4. The
active compounds were found in the group of N-substituted 4-
hydroxybutanamides with 4-(diphenylmethyl)piperazin-1-yl and
4-bis(4-fluorophenylmethyl)piperazin-1-yl substituents. Among
them, compound 15 showed the highest inhibitory potency against
GATs, however was not selective towards the individual subtypes.
Preliminary SAR studies for this group of compounds point out that
the benzyl substituent in the amide group and benzhydrylpiper-
azinyl are crucial for the activity. Structural optimization of the
active compounds which increases the selectivity and the potency
is planned.
4.66 and 4.84, i.e. IC50 values of 22 and 14
mM, respectively.
Comparison of the corresponding amides from series A (13e21)
and B (22e30) shows that the introduction of the fluorine atoms
led to decrease in activity and loss of the binding affinity to GAT1
(22e30). Among substituents in the amide part, 4-methoxy ary-
lalkylamides were not tolerated (19, 28). The influence of the
halogen atom in ortho position on the activity can vary. All further
modifications, like degradation, exchange of the piperazine part, or
removal of substituent of the amide nitrogen, led to loss of the
activity. Since none of the applied modifications gave positive
results, we conclude that this kind of bulky and lipophilic biaryl
group and substituted amides are crucial for the inhibition of GAT
activity in this group of compounds.
6. Experimental
5. Conclusions
6.1. Chemistry
Due to a great number of drug-resistant cases of epilepsy and
severe side effects of the available treatment, there is an urgent
need for new, more efficient, and safer antiepileptics. GABA
Melting points were determined in open glass capillaries on the
Electrothermal 9300 apparatus and are uncorrected. Reactions

186
K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
Table 1
Results of [3H] GABA uptake and NO711 MS-binding assays.
Compound
GAT1 uptakea
GAT2 uptakea
GAT3 uptakea
GAT4 uptakea
GAT1 NO711 bindingb
13
14
15
16
17
18
19
4.66 ꢁ 0.08
83%
4.47(n¼1)
72%
46%
73%
54%
76%
4.96 ꢁ 0.07
e
4.88 ꢁ 0.10(n¼9)
4.48 ꢁ 0.11(n¼7)
4.84 ꢁ 0.08
52%
4.56 ꢁ 0.13(n¼7)
4.33 ꢁ 0.12(n¼7)
52%
5.06 ꢁ 0.11(n¼5)
4.26 ꢁ 0.04(n¼4)
4.34 ꢁ 0.09
4.36 ꢁ 0.10
61%
4.91 ꢁ 0.12
4.33 ꢁ 0.03
55%
4.82 ꢁ 0.12
61%
e
82%
51%
52%
59%
4.54
78%
55%
20
68%
93%
80%
80%
e
21
22
23
24
25
26
27
28
29
30
34
35
103%
49%
83%
66%
98%
63%
93%
102%
92%
75%
92%
101%
98%
106%
76%
e
4.19 ꢁ 0.11
4.20 ꢁ 0.13
4.14 ꢁ 0.09
97%
4.03 ꢁ 0.07
4.23 ꢁ 0.10
93%
69%
66%
63%
79%
4.33 ꢁ 0.02
4.17 ꢁ 0.17
4.35 ꢁ 0.08
65%
4.13 ꢁ 0.05
4.44 ꢁ 0.11
65%
4.15 ꢁ 0.06
4.72 ꢁ 0.04
4.54 ꢁ 0.05
75%
4.22 ꢁ 0.14
4.52 ꢁ 0.10
75%
4.10 ꢁ 0.06
4.01 ꢁ 0.08
3.92 ꢁ 0.06
84%
4.12 ꢁ 0.14
4.20 ꢁ 0.08
84%
4.43 ꢁ 0.08
4.00 ꢁ 0.02
4.15 ꢁ 0.90
84%
86%
91%
98%
98%
62%
80%
75%
80%
36
84%
110%
110%
98%
e
37
38
113%
75%
98%
49%
52%
3.31 ꢁ 0.03
100%
65%
64%
4.59 ꢁ 0.05
100%
88%
73%
4.59 ꢁ 0.05
95%
86%
e
Tiagabine (2)c
Guvacine (4)c
6.88 ꢁ 0.12
4.87 ꢁ 0.07
e
a
% of remaining GABA uptake at 100
% of NO711 binded to GAT1 at 100
Data from Ref. [17].
m
m
M concentration of tested compound (means; n ¼ 2) or pIC50 (means ꢁ SEM; n ¼ 3 if not specified otherwise).
b
c
M concentration of tested compound or pKi (means ꢁ SEM; n ¼ 3).
were monitored by thin layer chromatography (TLC) using silica gel
plates silica gel 60 F254 (Merck) and following solvent systems: S1
(Me2CO/CHCl3, 1/1) and S2 (CHCl3/MeOH/CH3COOH, 12/2/1). The
spots were visualized under UV lamp and by iodine solution
(0.05 M in 10% HCl). 1H NMR and 13C NMR spectra were recorded
with Varian Mercury 300 spectrometer (300 MHz) in CDCl3 and
DMSO-d6, with signal of solvent as an internal standard. Elemental
analyses were carried out on a Vario EL III Model Elemental
Analyzer. Reactions under microwave irradiation were carried out
in Discover LabMate (CEM Corporation).
(OCH2CH2), 78.1 (CHCH2), 84.5 (CHPh2), 126.2, 128.2, 129.2, 142.7
(arom), 175.0 (carbonyl); TLC: Rf (S1) ¼ 0.77, Rf (S2) ¼ 0.91.
6.1.1.2. (R, S) 3-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]dihy-
drofuran-2(3H)-one (9). Reagents and conditions: 3-bromodihy-
drofuran-2(3H)-one (1.65 g, 10 mmol), 1-[bis(4-fluorophenyl)
methyl]piperazine (2.88 g, 10 mmol), K2CO3 (1.38 g, 10 mmol),
MeCN, 20 h; yield 60%; purification by column chromatography
(S1); anal. calcd for C21H22N2O2F2: C% 67.73, H% 5.95, N% 7.52 found:
C% 67.83, H% 5.89, N% 7.49; 1H NMR (CDCl3)
d (ppm): 2.28e3.42 (m,
6H, CHCH2, piperazine), 2.55e2.61 (m, 2H, piperazine), 2.83e2.93
(m, 2H, piperazine), 3.53 (t, J ¼ 9.4 Hz, 1H, CH), 4.31 (s, 1H, CHPh2),
4.31e4.40 (m, 2H, CH2O), 7.15e7.40 (m, 8H, Ar-H); 13C NMR (CDCl3)
6.1.1. Synthesis of 3-substituted dihydrofuran-2(3H)-one
derivatives 8e12 (general procedure)
Anhydrous K2CO3 (1 equiv.) was added to the solution of rele-
vant amine (1 equiv.) in 20 mL of solvent (acetonitrile, DCM or
Me2CO) and the mixture was stirred at room temperature for 0.5 h.
Then a solution of 3-bromodihydrofuran-2(3H)-one (1 equiv.) in
5 mL of appropriate solvent was added dropwise and stirring was
continued for 3e20 h. In the synthesis of compounds 11 and 12
tetrabutylammonium bromide (TBAB) (0.1 equiv.) was added. After
the reaction was completed, the precipitate was filtered off and the
filtrate was concentrated under vacuum. Obtained crude products
were recrystallized from suitable solvent (solid) or purified by
column chromatography (oil). Lactone 11 was isolated as a hydro-
chloride salt and recrystallized from DCM. Synthesis of compound
13 was described elsewhere [24].
d (ppm): 28.8 (CHCH2), 53.1, 54.7 (piperazine), 66.2 (OCH2CH2), 78.6
(CHCH2), 84.5 (CHPh2), 116.2, 129.8, 138.2, 160.7 (arom), 175.0
(carbonyl); TLC: Rf (S1) ¼ 0.57, Rf (S2) ¼ 0.83.
6.1.1.3. (R, S) Ethyl 4-(2-oxotetrahydrofuran-3-yl)piperazine-1-carbox-
ylate (10). Reagents and conditions: 3-bromodihydrofuran-2(3H)-
one (1.65 g, 10 mmol), ethyl 1-piperazinecarboxylate (1.58 g,
10 mmol), K2CO3 (1.38 g, 10 mmol), Me2CO, 20 h; yield 66%; puri-
fication by column chromatography (S1); anal. calcd for
C11H18N2O4: C% 54.53, H% 7.49, N% 11.56 found: C% 54.40, H% 7.52, N
% 11.60; 1H NMR (CDCl3)
d
(ppm): 1.22 (t, J ¼ 1.6 Hz, 3H, CH2CH3),
2.21e2.31 (m, 2H, CH2CH), 2.42e2.50 (m, 4H, piperazine),
2.72e2.84 (m, 4H, piperazine), 3.48 (t, 1H, J ¼ 5.2 Hz, CH2CHCO),
4.05e4.35 (m, 4H, CH2O, CH2CH3); 13C NMR (CDCl3)
d (ppm): 13.8
6.1.1.1. (R, S) 3-(4-Benzhydrylpiperazin-1-yl)dihydrofuran-2(3H)-one
(8). Reagents and conditions: 3-bromodihydrofuran-2(3H)-one
(1.65 g, 10 mmol), 1-benzhydrylpiperazine (2.52 g, 10 mmol), K2CO3
(1.38 g, 10 mmol), MeCN, 3 h; yield 65%; mp 161e162 ꢀC (EtOAc);
anal. calcd for C21H24N2O2: C% 74.97, H% 7.19, N% 8.33 found: C%
(CH3), 28.6 (CHCH2), 46.2, 52.0 (piperazine), 62.0 (OCH2CH3), 66.2
(OCH2CH2), 78.1 (CHCH2),156.3, 175.0 (carbonyl); TLC: Rf (S1) ¼ 0.65
6.1.1.4. (R, S) 3-Morpholinodihydrofuran-2(3H)-one hydrochloride
(11). Reagents and conditions: 3-bromodihydrofuran-2(3H)-one
(1.65 g, 10 mmol), morpholine (0.87 g, 10 mmol), K2CO3 (1.38 g,
10 mmol), DCM, 3 h; yield 50%; mp 202e204 ꢀC (MeOH); anal. calcd
for C8H13NO3$HCl: C% 46.27, H% 6.80, N% 6.75 found: C% 45.65, H%
74.36, H% 7.30, N% 8.22; 1H NMR (CDCl3)
d (ppm): 2.30e3.46 (m, 2H,
CHCH2), 2.46e2.60 (m, 4H, piperazine), 2.78e2.94 (m, 4H, pipera-
zine), 3.54 (t, J ¼ 9.4 Hz, 1H, CH), 4.16e4.20 (m, 1H, CHPh2),
4.32e4.47 (m, 2H, CH2O), 7.15e7.40 (m, 10H, Ar-H); 13C NMR
7.16, N% 6.67; 1H NMR (DMSO-d6)
d
(ppm): 2.52 (m, 2H, CHCH2),
(CDCl3)
d (ppm): 28.6 (CHCH2), 52.9, 54.4 (piperazine), 66.3
3.05e3.26 (m, 4H, morpholine), 3.26e3.87 (m, 4H, morpholine),

K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
187
4.23 (m, 1H, CH), 4.47 (m, 2H, CH2O); 13C NMR (DMSO-d6)
d
(ppm):
7 mmol), 120 ꢀC; reaction time 3 h; yield 88%; mp 195e197 ꢀC
28.4 (CHCH2), 53.3, 66.4 (piperazine), 66.8 (OCH2CH2), 78.4
(CHCH2), 175.0 (carbonyl); TLC: Rf (S1) ¼ 0.39, Rf (S2) ¼ 0.61.
(toluene); anal. calcd for C28H32N3O2Cl: C% 70.35, H% 6.75, N% 8.79
found: C% 70.25, H% 6.90, N% 8.82; 1H NMR (CDCl3)
d (ppm): 1.80 (m,
2H, CHCH2), 2.45e2.54 (m, 8H, piperazine), 3.17 (dd, J ¼ 3.2, 9.7 Hz,
1H, CH), 3.62e3.83 (m, 2H, CH2OH), 4.21 (s, 1H, CHPh2), 4.43e4.59
(m, 3H, CH2Ph, OH), 7.06e7.49 (m, 14H, Ar-H), 7.94 (t, J ¼ 6.1 Hz, 1H,
6.1.1.5. (R, S) 3-(4-Benzylpiperidin-1-yl)dihydrofuran-2(3H)-one
(12). Reagents and conditions: 3-bromodihydrofuran-2(3H)-one
(1.65 g, 10 mmol), 4-benzylpiperidine (1.75 g, 10 mmol), K2CO3
(1.38 g, 10 mmol), Me2CO, 5 h; yield 60%; mp 65e66 ꢀC (iPrOH);
anal. calcd for C16H21NO2: C% 74.1, H% 8.16, N% 5.40 found: C% 73.85,
amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 43.6 (NHCH2Ph),
52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.6 (CHCH2), 84.5
(CHPh2), 126.2, 128.2, 129.2, 142.7 (arom), 171.3 (carbonyl); 13C NMR
H% 8.19, N% 5.37; 1H NMR (CDCl3)
d
(ppm): 1.24e1.45 (m, 2H,
(CDCl3) d (ppm): 34.8 (CHCH2), 38.5 (NHCH2Ph), 52.5, 54.0 (pipera-
piperidine), 1.47e1.73 (m, 3H, CH2CH, piperidine), 2.18e2.34 (m,
3H, CH2CH, piperidine), 2.45e2.60 (m, 3H, CH2Ph, piperidine), 2.83
(d, J ¼ 10.3, 1H, piperidine), 2.96 (d, J ¼ 10.5, 1H, piperidine), 3.63 (t,
J ¼ 9.5,1H, CH), 4.24 (dd, J ¼ 8.8,16.8,1H, CH2O), 4.42 (dt, J ¼ 5.9, 9.1,
zine), 58.3 (HOCH2CH2), 69.7 (CHCH2), 84.5 (CHPh2), 126.2, 126.6,
128.1, 128.2, 128.6, 129.2, 132.2, 142.4, 142.7 (arom), 171.3 (carbonyl);
TLC: Rf (S1) ¼ 0.43, Rf (S2) ¼ 0.76.
1H, CH2O), 7.08e7.36 (m, 5H, Ar-H); 13C NMR (CDCl3)
d
(ppm): 28.8
6.1.2.7. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-N-(4-chlorobenzyl)-4-
hydroxybutanamide (15). Procedure GP4; reagents: compound 8
(1.68 g, 5 mmol), 4-chlorobenzylamine (0.99 g, 7 mmol); reaction
time 12 h; yield 20%; mp 146e147 ꢀC (EtOAc); anal. calcd for
C28H32N3O2Cl:. C% 70.35, H% 6.75, N% 8.79 found: C% 70.25, H% 6.75,
(CHCH2), 41.7 (CH2Ph), 32.6, 37.2, 44.3 (piperidine), 66.2 (OCH2CH2),
78.6 (CHCH2), 125.9, 128.1, 128.5, 139.7 (arom), 175.0 (carbonyl);
TLC: Rf (S1) ¼ 0.66, Rf (S2) ¼ 0.64.
6.1.2. Synthesis of 2-substituted 4-hydroxybutanamides 13e33,
37e38
N% 8.34; 1H NMR (CDCl3)
d (ppm): 1.84e2.00 (m, 2H, CHCH2),
2.39e2.56 (m, 8H, piperazine), 3.17e3.20 (m, 1H, CH), 3.64e3.85
(m, 2H, CH2OH), 4.21 (s, 1H, CHPh2), 4.35e4.53 (m, 3H, CH2Ph, OH),
7.00e7.39 (m, 14H, Ar-H), 7.72 (t, 1H, amide); 13C NMR (CDCl3)
6.1.2.1. General procedure 1 (GP1). 2-Substituted dihydrofuran-2
(3H)-one derivative (1 equiv.) was heated with relevant amine
(1.15e2.5 equiv.) at 100e120 ꢀC. The oily residue was then dissolved
in 5 mL of EtOAc, 2e5 mL of n-hexane was added, and the mixture
was refrigerated for about 24 h. The obtained solid was filtered off
and purified by recrystallization.
d
(ppm): 34.9 (CHCH2), 43.4 (NHCH2Ph), 52.6, 54.3 (piperazine),
58.0 (HOCH2CH2), 69.76 (CHCH2), 84.2 (CHPh2), 126.2, 128.2, 128.6,
129.2, 132.3, 134.6, 136.0, 142.7 (arom), 171.8 (carbonyl); TLC: Rf
(S1) ¼ 0.39, Rf (S2) ¼ 0.75.
6.1.2.2. General procedure 2 (GP2). The process vial was charged
with 2-substituted dihydrofuran-2(3H)-one derivative (1.0 equiv.),
amine (1.4e2 equiv.), and relevant solvent. The vial was then closed
and its content was magnetically stirred and microwave heated at
120e140 ꢀC. After the reaction was finished, the mixture was
refrigerated, then, the obtained solid was filtered and washed with
EtOH and Et2O.
6.1.2.8. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-N-(2-fluorobenzyl)-4-
hydroxybutanamide (16). Procedure GP1; reagents and conditions:
compound 8 (1.68 g, 5 mmol), 2-fluorobenzylamine (1.25 g,
10 mmol), 120 ꢀC; reaction time 4 h; yield 36%; mp 148e149 ꢀC
(EtOAc); anal. calcd for C28H32N3O2F: C% 72.86, H% 6.99, N% 9.10
found: C% 72.59, H% 6.74, N% 9.14; 1H NMR (CDCl3)
d (ppm):
1.81e1.99 (m, 2H, CHCH2), 2.40e2.51 (m, 8H, piperazine), 3.16e3.18
(m, 1H, CH), 3.62e3.83 (m, 2H, CH2OH), 4.21 (s, 1H, CHPh2),
4.41e4.63 (m, 2H, CHPh, OH), 4.49 (m, 1H, CHPh), 7.00e7.40 (m,
6.1.2.3. General procedure
3 (GP3). The mixture of relevant
2-substituted dihydrofuran-2(3H)-one derivative and 25% NH3 aq.
was stirred in closed vessel at room temperature for 48e72 h. The
obtained crude products were purified according to procedures
described in the detailed part.
14H, Ar-H), 7.81 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.5
(CHCH2), 36.6 (NHCH2Ph), 52.5, 54.0 (piperazine), 58.3
(HOCH2CH2), 69.9 (CHCH2), 84.5 (CHPh2), 115.3, 126.2, 128.2, 128.3,
129.2, 129.6, 131.5, 142.7, 159.4 (arom), 171.3 (carbonyl); TLC: Rf
(S1) ¼ 0.54, Rf (S2) ¼ 0.76.
6.1.2.4. General procedure 4 (GP4). 2-Substituted dihydrofuran-2
(3H)-one derivative (1 equiv.) was refluxed in toluene (10 mL) with
relevant amine (1.4e2 equiv.) for 5e20 h. Then the solvent was
evaporated under vacuum and crude product was refrigerated. The
obtained solid was recrystallized from EtOAc.
6.1.2.9. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-N-(4-fluorobenzyl)-4-
hydroxybutanamide (17). Procedure GP1; reagents and conditions:
compound 8 (1.68 g, 5 mmol), 4-fluorobenzylamine (1.25 g,
10 mmol), 120 ꢀC; reaction time 4 h; yield 42%; mp 145e147 ꢀC (n-
hexane/EtOAc, 1/4); anal. calcd for C28H32N3O2F: C% 72.86, H% 6.99,
6.1.2.5. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-N-benzyl-4-hydroxybuta-
namide (13). Procedure GP2; reagents and conditions: compound 8
(0.84 g, 2.5 mmol), benzylamine (0.54 g, 5 mmol), toluene (3 mL),
140 ꢀC; reaction time 25 min; yield 60%; mp 144e145 ꢀC; anal. calcd
for C28H33N3O2: C% 75.82, H% 7.50, N% 9.47 found: C% 75.80, H%
N% 9.10 found: C% 72.75, H% 6.84, N% 8.93; 1H NMR (CDCl3)
d (ppm):
1.85e2.01 (m, 2H, CHCH2), 2.39e2.57 (m, 8H, piperazine), 3.18e3.20
(m, 1H, CH), 3.64e3.86 (m, 2H, CH2OH), 4.19 (s, 1H, CHPh2),
4.40e4.57 (m, 3H, CH2Ph, OH), 7.15e7.40 (m, 14H, Ar-H), 7.71 (t, 1H,
amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 43.6 (NHCH2Ph),
7.70, N% 9.66; 1H NMR (CDCl3)
d
(ppm): 1.76e2.03 (m, 2H, CHCH2),
52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2), 84.5
(CHPh2), 115.3, 126.2, 128.2, 128.5, 133.5, 142.7, 160.9 (arom), 171.3
(carbonyl); TLC: Rf (S1) ¼ 0.38, Rf (S2) ¼ 0.70.
2.21e2.70 (m, 8H, piperazine), 3.19 (dd, J ¼ 3.3, 9.7 Hz, 1H, CH),
3.64e3.86 (m, 2H, CH2OH), 4.19 (s, 1H, CHPh2), 4.43 (s, 1H, OH), 4.52
(t, J ¼ 6.2 Hz, 2H, CH2Ph), 7.12e7.41 (m,15H, Ar-H), 7.71 (t, J ¼ 5.5 Hz,
1H, amide); 13C NMR (CDCl3)
d
(ppm): 34.6 (CHCH2), 43.6
6.1.2.10. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-4-hydroxy-N-(4-methyl-
benzyl)butanamide (18). Procedure GP4; reagents: compound 8
(1.68 g, 5 mmol), 4-methylbenzylamine (0.85 g, 7 mmol); reaction
time 5 h; yield 22%; mp 158e160 ꢀC (EtOAc); anal. calcd for
C29H35N3O2: C% 76.12, H% 7.71, N% 9.18 found: C% 76.59, H% 7.74, N%
(NHCH2Ph), 52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.6
(CHCH2), 84.5 (CHPh2), 126.2, 126.7, 126.9, 128.2, 128.5, 129.2, 137.9,
142.7 (arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.57, Rf (S2) ¼ 0.73.
6.1.2.6. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-N-(2-chlorobenzyl)-4-
hydroxybutanamide (14). Procedure GP1; reagents and conditions:
compound 8 (1.68 g, 5 mmol), 2-chlorobenzylamine (0.99 g,
9.08; 1H NMR (CDCl3)
d (ppm): 1.84e2.00 (m, 2H, CHCH2), 2.33 (s,
3H, CH3), 2.40e2.54 (m, 8H, piperazine), 3.17e3.19 (m, 1H, CH),
3.64e3.86 (m, 2H, CH2OH), 4.19 (s, 1H, CHPh2), 4.35e4.53 (m, 3H,

188
K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
CH2Ph, OH), 7.13e7.40 (m, 14H, Ar-H), 7.65 (t, 1H, amide); 13C NMR
(CDCl3) (ppm): 21.3 (CH3), 34.6 (CHCH2), 43.6 (NHCH2Ph), 52.5,
(EtOAc); anal. calcd for C28H30N3O2F2Cl: C% 65.43, H% 5.88, N% 8.18
found: C% 65.40, H% 5.91, N% 8.12; 1H NMR (CDCl3)
(ppm):
d
d
54.0 (piperazine), 58.3 (HOCH2CH2), 69.5 (CHCH2), 84.05 (CHPh2),
126.2, 128.1, 128.2, 128.8, 129.2, 134.9, 136.4, 142.9 (arom), 171.9
(carbonyl); TLC: Rf (S1) ¼ 0.39, Rf (S2) ¼ 0.78.
1.82e2.03 (m, 2H, CHCH2), 2.32e2.62 (m, 8H, piperazine),
3.18e3.22 (m, 1H, CH), 3.67e3.87 (m, 2H, CH2OH), 4.17 (s, 1H,
CHPh2), 4.56e4.61 (m, 3H, CH2Ph, OH), 6.96e7.35 (m, 12H, Ar-H),
7.83 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 38.6
6.1.2.11. (R,
S)
2-(4-Benzhydrylpiperazin-1-yl)-4-hydroxy-N-(4-
(NHCH2Ph), 52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.7
(CHCH2), 84.5 (CHPh2), 116.3, 126.6, 128.1, 128.6, 129.8, 132.2, 138.3,
142.4 (arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.51.
methoxybenzyl)butanamide (19). Procedure GP1; reagents and
conditions: compound 8 (1.68 g, 5 mmol), 4-methoxybenzylamine
(0.96 g, 7 mmol), 100 ꢀC; reaction time 3 h; yield 37%; mp
138e139 ꢀC (EtOAc); anal. calcd for C29H35N3O3: C% 73.54, H% 7.45,
N% 8.87 found: C% 73.44, H% 7.73, N% 8.87; 1H NMR (CDCl3)
6.1.2.16. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-N-(4-
chlorobenzyl)-4-hydroxybutanamide (24). Procedure GP1; re-
agents: compound 9 (1.86 g, 5 mmol), 4-chlorobenzylamine
(1.42 g, 10 mmol), 120 ꢀC; reaction time 3 h; yield 24%; mp
137e138 ꢀC (EtOAc); anal. calcd for C28H30N3O2F2Cl: C% 65.43, H%
5.88, N% 8.18 found: C% 65.39, H% 5.62, N% 8.24; 1H NMR (CDCl3)
d
(ppm): 1.82e2.01 (m, 2H, CHCH2), 2.14e2.67 (m, 8H, piperazine),
3.17 (dd, J ¼ 3.3, 9.7 Hz, 1H, CH), 3.58e3.69 (m, 1H, CH2OH), 3.80 (s,
3H, CH3O), 3.81e3.91 (m, 1H, CH2OH), 4.19 (s, 1H, CHPh2), 4.37 (t,
J ¼ 6.0 Hz, 2H, CH2Ph), 4.57 (s, 1H, OH), 6.75e6.92 (m, 2H, Ar-H),
7.12e7.41 (m, 12H, Ar-H), 7.63 (t, J ¼ 6.4 Hz, 1H, amide); 13C NMR
d
(ppm): 1.82e2.03 (m, 2H, CHCH2), 2.36e2.71 (m, 8H, piperazine),
(CDCl3)
d
(ppm): 34.6 (CHCH2), 43.6 (NHCH2Ph), 52.5, 54.0 (piper-
3.18e3.22 (m, 1H, CH), 3.67e3.87 (m, 2H, CH2OH), 4.20 (s, 1H,
CHPh2), 4.40e4.47 (m, 2H, CH2Ph), 4.56 (s, 1H, OH), 6.96e7.35 (m,
azine), 55.8 (OCH3), 58.3 (HOCH2CH2), 69.7 (CHCH2), 84.5 (CHPh2),
114.3, 126.2, 128.2, 130.2, 130.5, 142.7, 159.9 (arom), 171.3
(carbonyl); TLC: Rf (S1) ¼ 0.49, Rf (S2) ¼ 0.70.
12H, Ar-H), 7.54 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6
(CHCH2), 43.6 (NHCH2Ph), 52.5, 54.0 (piperazine), 58.3
(HOCH2CH2), 69.7 (CHCH2), 84.5 (CHPh2), 116.0, 128.6, 128.8, 132.3,
134.6, 136.0, 138.3, 160.4 (arom), 171.3 (carbonyl); TLC: Rf
(S1) ¼ 0.48.
6.1.2.12. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-4-hydroxy-N-phene-
thylbutanamide (20). Procedure GP2; reagents: compound
8
(1.68 g, 5 mmol), 2-phenethylamine (0.85 g, 7 mmol), toluene
(3 mL), 120 ꢀC; reaction time 40 min; yield 33%; mp 133e134 ꢀC;
anal. calcd for C29H35N3O2: C% 76.12, H% 7.71, N% 9.18 found: C%
6.1.2.17. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-N-(2-
fluorobenzyl)-4-hydroxybutanamide (25). Procedure GP1; reagents
and conditions: compound 9 (1.86 g, 5 mmol), 2-fluorobenzylamine
(1.25 g, 10 mmol), 120 ꢀC; reaction time 3 h; yield 35%; mp
154e155 ꢀC (EtOAc); anal. calcd for C28H30N3O2F3: C% 67.59, H% 6.08,
76.32, H% 8.01, N% 9.19; 1H NMR (CDCl3)
d (ppm): 1.66e1.99 (m, 2H,
CHCH2), 2.00e2.55 (m, 8H, piperazine), 2.85 (t, J ¼ 6.8 Hz, 2H,
CH2CH2Ph), 3.06 (m, 1H, CH), 3.41e3.81 (m, 4H, CH2OH, CH2Ph),
4.15 (s, 1H, CHPh2), 4.64 (s, 1H, OH), 7.07e7.55 (m, 16H, Ar-H,
N% 8.45 found: C% 67.69, H% 6.04, N% 8.44; 1H NMR (CDCl3)
d (ppm):
amide); 13C NMR (CDCl3)
d
(ppm): 34.6 (CHCH2), 35.1 (CH2Ph), 40.6
1.83e2.05 (m, 2H, CHCH2), 2.37e2.46 (m, 8H, piperazine), 3.16e3.21
(m, 1H, CH), 3.65e3.88 (m, 2H, CH2OH), 4.21 (s, 1H, CHPh2),
4.42e4.53 (m, 2H, CH2Ph), 4.64 (s, 1H, OH), 7.00e7.40 (m, 12H, Ar-H),
(NHCH2), 52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2),
84.5 (CHPh2), 125.9, 126.2, 127.7, 128.2, 128.6, 129.2, 139.5, 142.7,
(arom), 171.9 (carbonyl); TLC: Rf (S1) ¼ 0.53, Rf (S2) ¼ 0.73.
7.81 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 36.8
(NHCH2Ph), 52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2),
84.5 (CHPh2), 115.3, 116.0, 124.1, 128.3, 129.5, 131.5, 159.7, 160.4
(arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.57, Rf (S2) ¼ 0.78.
6.1.2.13. (R, S) 2-(4-Benzhydrylpiperazin-1-yl)-4-hydroxybutanamide
(21). Procedure GP3; reagents: compound 8 (1.34 g, 4 mmol), 25%
NH3 aq. (5 g); reaction time 72 h; purification: washed with water,
EtOH, Et2O; yield 37%; mp 167e168 ꢀC; anal. calcd for C21H27N3O2:
C% 71.36, H% 7.70, N% 11.89 found: C% 71.69, H% 7.41, N% 11.67; 1H
NMR (CDCl3)
piperazine), 3.20e3.36 (m, 1H, CH), 3.64e3.82 (m, 2H, CH2OH), 4.22
(s,1H, CHPh2), 4.67 (s,1H, OH), 6.99e7.48 (m,12H, Ar-H, amide); 13
NMR (CDCl3) (ppm): 34.3 (CHCH2), 52.5, 54.0 (piperazine), 58.3
6.1.2.18. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-N-(4-
fluorobenzyl)-4-hydroxybutanamide (26). Procedure GP1; reagents:
compound 9 (1.86 g, 5 mmol), 4-fluorobenzylamine (1.25 g,
10 mmol), 120 ꢀC; reaction time 3 h; yield 19%; mp 87e88 ꢀC
(EtOAc); anal. calcd for C28H30N3O2F3: C% 67.59, H% 6.08, N% 8.45
d (ppm): 1.99e2.34 (m, 2H, CHCH2), 2.43e2.73 (m, 8H,
C
d
found: C% 67.42, H% 6.17, N% 8.39; 1H NMR (CDCl3)
d (ppm):
(HOCH2CH2), 71.7 (CHCH2), 84.5 (CHPh2), 126.2, 128.2, 129.5, 142.7
(arom), 176.3 (carbonyl); TLC: Rf (S1) ¼ 0.68.
1.85e2.07 (m, 2H, CHCH2), 2.42e2.78 (m, 8H, piperazine), 3.18e3.22
(m, 1H, CH), 3.67e3.87 (m, 2H, CH2OH), 4.18 (s, 1H, CHPh2),
4.40e4.47 (m, 2H, CH2Ph), 4.56 (s,1H, OH), 6.96e7.35 (m,12H, Ar-H),
6.1.2.14. (R, S) N-Benzyl-2-[4-(bis(4-fluorophenyl)methyl)piperazin-
7.58 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 43.6
1-yl]-4-hydroxybutanamide
(22). Procedure
GP1;
reagents:
(NHCH2Ph), 52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2),
84.5 (CHPh2), 115.3, 116.0, 128.5, 129.8, 133.5, 138.5, 160.4, 160.9
(arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.52.
compound 9 (1.86 g, 5 mmol), benzylamine (1.07 g,10 mmol),120 ꢀC;
reaction time 3 h; yield 20%; mp 108e109 ꢀC (EtOAc); anal. calcd for
C28H31N3O2F2: C% 70.13, H% 6.52, N% 8.76 found: C% 70.27, H% 6.31, N
% 8.82; 1H NMR (CDCl3)
d
(ppm): 1.76e1.98 (m, 2H, CHCH2),
6.1.2.19. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-4-
2.14e2.65 (m, 8H, piperazine), 3.17e3.19 (m, 1H, CH), 3.51e3.79 (m,
2H, CH2OH), 4.15 (s, 1H, CHPh2), 4.33e4.47 (m, 3H, CH2Ph, OH),
7.27e7.55 (m, 13H, Ar-H), 7.84 (t, 1H, amide); 13C NMR (CDCl3)
hydroxy-N-(4-methylbenzyl)butanamide
(27). Procedure
GP1;
reagents: compound 9 (1.86 g, 5 mmol), 4-methylbenzylamine
(1.21 g, 10 mmol), 120 ꢀC; reaction time 3 h; yield 21%; mp
129e130 ꢀC (EtOAc); anal. calcd for C29H33N3O2F2: C% 70.57, H%
6.74, N% 8.51 found: C% 70.27, H% 6.62, N% 8.45; 1H NMR (CDCl3)
d
(ppm): 34.6 (CHCH2), 43.6 (NHCH2Ph), 52.5, 54.0 (piperazine), 58.3
(HOCH2CH2), 43.6 (CHCH2), 84.5 (CHPh2), 116.3, 126.7, 128.5, 129.8,
137.5, 138.7, 160.4 (arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.54.
d
(ppm): 1.80e2.14 (m, 2H, CHCH2), 2.30 (s, 3H, CH3), 2.36e2.67 (m,
8H, piperazine), 3.21e3.26 (m, 1H, CH), 3.63e3.82 (m, 2H, CH2OH),
4.20 (s, 1H, CHPh2), 4.40e4.47 (m, 2H, CH2Ph), 4.59 (s, 1H, OH),
6.96e7.35 (m, 12H, Ar-H), 7.84 (t, 1H, amide); 13C NMR (CDCl3)
6.1.2.15. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-N-(2-
chlorobenzyl)-4-hydroxybutanamide (23). Procedure GP1; reagents:
compound 9 (1.86 g, 5 mmol), 2-chlorobenzylamine (1.42 g,
10 mmol), 120 ꢀC; reaction time 3 h; yield 22%; mp 138e139 ꢀC
d
(ppm): 21.3 (CH3), 34.6 (CHCH2), 43.6 (NHCH2Ph), 52.5, 54.0
(piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2), 84.5 (CHPh2), 116.0,

K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
189
125.9, 127.7, 128.6, 129.8, 138.3, 139.4, 160.4 (arom), 173.9
56.20, H% 6.71 N% 10.86; 1H NMR (CDCl3)
d
(ppm): 1.24 (d,
(carbonyl); TLC: Rf (S1) ¼ 0.55.
J ¼ 6.2 Hz, 3H, CH3), 1.70e1.96 (m, 2H, CHCH2), 2.60e2.78 (m, 4H,
piperazine), 3.05e3.24 (m, 5H, piperazine, CH), 3.67e3.87 (m, 2H,
CH2OH), 4.13 (q, J ¼ 6.2 Hz, 2H, CH2CH3), 4.53 (s, 1H, OH), 4.67e4.85
(m, 2H, CH2Ph); 6.96e7.35 (m, 4H, Ar-H), 7.83 (t, 1H, amide); 13C
6.1.2.20. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-4-
hydroxy-N-(4-methoxylbenzyl)butanamide (28). Procedure GP1;
reagents and conditions: compound 9 (1.86 g, 5 mmol), 4-
methoxybenzylamine (1.37 g, 10 mmol), 100 ꢀC; reaction time 3 h;
yield 43%; mp 135e136 ꢀC (EtOAc); anal. calcd for C29H33N3O3F2: C%
68.35, H% 6.53, N% 8.25 found: C% 68.44, H% 6.73, N% 8.37; 1H NMR
NMR (CDCl3)
d (ppm): 13.8 (CH3CH2), 34.6 (CHCH2), 43.6
(NHCH2Ph), 45.5, 51.8 (piperazine), 58.3 (HOCH2CH2), 62.0
(CH3CH2), 69.7 (CHCH2), 128.6, 132.3, 134.6, 136.0 (arom), 171.3
(carbonyl); TLC: Rf (S1) ¼ 0.50, Rf (S2) ¼ 0.64.
(CDCl3)
d (ppm): 1.85e2.07 (m, 2H, CHCH2), 2.23e2.67 (m, 8H,
piperazine), 3.19 (dd, J ¼ 3.3, 9.7 Hz, 1H, CH), 3.60e3.74 (m, 1H,
CH2OH), 3.80 (s, 3H, CH3O), 3.84e3.98 (m, 1H, CH2OH), 4.19 (s, 1H,
CHPh2), 4.37 (t, J ¼ 6.0 Hz, 2H, CH2Ph), 4.59 (s, 1H, OH), 6.75e6.92
(m, 2H, Ar-H), 7.12e7.41 (m, 10H, Ar-H), 7.63 (t, J ¼ 6.4 Hz, 1H,
6.1.2.25. (R, S) Ethyl 4-(1-amino-4-hydroxy-1-oxobutan-2-yl)piper-
azine-1-carboxylate (33). Procedure GP3; reagents: compound 10
(0.97 g, 4 mmol), 25% NH3 aq. (4 g); reaction time 72 h; purification:
extraction of reaction mixture with EtOAc; yield 42%; mp
120e122 ꢀC (EtOAc); anal. calcd for C11H21N3O4: C% 50.95, H% 8.16,
N% 16.21 found: C% 50.86, H% 8.12 N% 16.26; 1H NMR (CDCl3)
amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 43.6 (NHCH2Ph),
52.5, 54.0 (piperazine), 58.3 (HOCH2CH2), 55.8 (CH3), 69.7 (CHCH2),
84.5 (CHPh2), 114.1, 116.3, 129.8, 130.2, 130.5, 158.6, 160.4 (arom),
171.3 (carbonyl); TLC: Rf (S1) ¼ 0.54, Rf (S2) ¼ 0.73.
d
(ppm): 1.24 (d, J ¼ 6.2 Hz, 3H, CH3), 1.80e2.11 (m, 2H, CHCH2),
2.60e2.78 (m, 4H, piperazine), 3.05e3.24 (m, 5H, piperazine, CH),
3.76e3.91 (m, 2H, CH2OH), 4.17 (q, 2H, CH2CH3),5.24e5.48 (s wide,
6.1.2.21. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]- 4-
hydroxy-N-phenethylbutanamide (29). Procedure GP1; reagents:
compound 9 (1.86 g, 5 mmol), 2-phenethylamine (1.21 g, 10 mmol),
120 ꢀC; reaction time 4 h; yield 33%; mp 142e143 ꢀC; anal. calcd for
C29H33N3O2F2: C% 70.57, H% 6.74, N% 8.51 found: C% 70.32, H% 6.83,
1H, OH), 7.58 (s, 2H, amide); 13C NMR (CDCl3)
d (ppm): 13.8
(CH3CH2), 34.6 (CHCH2), 45.8, 51.6 (piperazine), 58.3 (HOCH2CH2),
62.0 (CH3CH2), 71.9 (CHCH2), 156.3 (carbonyl); TLC: Rf (S2) ¼ 0.74.
6.1.2.26. (R, S) N-Benzyl-4-hydroxy-2-morpholinobutanamide
(37). Procedure GP1; reagents: compound 11 (1.28 g, 7.5 mmol),
benzylamine (1.07 g, 10 mmol), 115 ꢀC; reaction time 2 h; yield 50%;
mp 93e94 ꢀC (EtOAc); anal. calcd for C15H22N2O3: C% 64.73, H% 7.97,
N% 10.06 found: C% 64.60, H% 8.36, N% 10.13; 1H NMR (CDCl3)
N% 8.59; 1H NMR (CDCl3)
d (ppm): 1.66e1.99 (m, 2H, CHCH2),
2.00e2.55 (m, 8H, piperazine), 2.85 (t, J ¼ 6.8 Hz, 2H, CH2CH2Ph),
3.06 (m, 1H, CH), 3.41e3.81 (m, 4H, CH2OH, CH2Ph), 4.15 (s, 1H,
CHPh2), 4.64 (s, 1H, OH), 7.07e7.55 (m, 14H, Ar-H, amide); 13C NMR
(CDCl3)
d
(ppm): 34.6 (CHCH2), 35.1 (CH2Ph), 40.6 (NHCH2), 52.5,
d (ppm): 1.83e2.05 (m, 2H, CHCH2), 2.45e2.63 (m, 4H, morpho-
54.0 (piperazine), 58.3 (HOCH2CH2), 69.7 (CHCH2), 84.5 (CHPh2),
115.3, 126.2, 128.2, 128.5, 133.5, 142.7, 160.9 (arom), 171.3
(carbonyl); TLC: Rf (S1) ¼ 0.55, Rf (S2) ¼ 0.79.
line), 3.22 (dd, J ¼ 3.5, 9.7 Hz, 1H, CH), 3.58e3.87 (m, 6H, CH2OH,
morpholine), 4.18 (s, 1H, OH), 4.40e4.55 (m, 2H, CH2Ph), 7.19e7.43
(m, 5H, Ar-H), 7.62 (s, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6
(CHCH2), 43.6 (NHCH2Ph), 52.5, 66.8 (morpholine), 58.3
(HOCH2CH2), 70.0 (CHCH2), 126.7, 126.9, 128.5, 137.9 (arom), 171.3
(carbonyl); TLC: Rf (S1) ¼ 0.23, Rf (S2) ¼ 0.60.
6.1.2.22. (R, S) 2-[4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl]-4-
hydroxybutanamide (30). Procedure GP3; reagents: compound 9
(1.86 g, 5 mmol), 25% NH3 aq. (5 g); reaction time 72 h; purification:
extraction of reaction mixture with EtOAc; yield 12%; mp
148e149 ꢀC (EtOAc); anal. calcd for C21H25N3O2F2: C% 64.77, H%
6.47, N% 10.79 found: C% 64.69, H% 6.41, N% 10.67; 1H NMR (CDCl3)
6.1.2.27. (R, S) N-Benzyl-2-(4-benzylpiperidin-1-yl)-4-hydroxybuta-
namide (38). Procedure GP1; reagents: compound 12 (1.56 g,
6 mmol), benzylamine (1.61 g, 15 mmol), 100 ꢀC; reaction time 3 h;
yield 86%; mp 96e97 ꢀC (iPrOH); anal. calcd for C23H30N2O2: C%
75.38, H% 8.25, N% 7.64 found: C% 75.08, H% 8.34 N% 7.58; 1H NMR
d
(ppm): 1.92e2.17 (m, 2H, CHCH2), 2.38e2.73 (m, 8H, piperazine),
3.18e3.22 (m, 1H, CH), 3.64e3.82 (m, 2H, CH2OH), 4.22 (s, 1H,
CHPh2), 4.63 (s, 1H, OH), 6.99e7.48 (m, 10H, Ar-H, amide); 13C NMR
(CDCl3)
d (ppm): 1.01e1.36 (m, 2H, piperidine), 1.42e1.57 (m, 1H,
(CDCl3)
d
(ppm): 34.6 (CHCH2), 52.5, 54.0 (piperazine), 58.3
CH, piperidine), 1.58e1.72 (m, 2H, CH2, piperidine), 1.73e2.00 (m,
2H, CHCH2), 2.13e2.38 (m, 2H, piperidine), 2.50 (d, J ¼ 7.1 Hz, 2H,
CH2Ph), 2.63e2.73 (m, 2H, CH2, piperidine), 3.19 (dd, J ¼ 3.2, 9.5 Hz,
1H, CH), 3.56e3.68 (m, 1H, CH2OH), 3.79e3.91 (m, 1H, CH2OH),
4.39e4.56 (m, 2H, CH2Ph), 4.75 (s, 1H, OH), 7.05e7.41 (m, 10H, Ar-
(HOCH2CH2), 71.9 (CHCH2), 84.5 (CHPh2), 116.0, 129.8, 138.3, 160.4
(arom), 176.7 (carbonyl); TLC: Rf (S1) ¼ 0.71.
6.1.2.23. (R, S) Ethyl 4-(1-benzylamino-4-hydroxy-1-oxobutan-2-yl)
piperazine-1-carboxylate (31). Procedure GP4; reagents: compound
10 (0.97 g, 4 mmol), benzylamine (1.13 g, 8 mmol); reaction time
6 h; yield 53%; mp 132e133 ꢀC (EtOAc); anal. calcd for C18H27N3O4:
C% 61.87, H% 7.79, N% 12.03 found: C% 61.68, H% 7.71 N% 12.16; 1H
H), 7.72e7.86 (s, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6
(CHCH2), 41.6 (CHCH2Ph), 32.2, 37.2, 43.9 (piperidine), 58.3
(HOCH2CH2), 70.7 (CHCH2), 126.7, 126.9, 128.1, 128.5, 125.9, 137.9,
139.7 (arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.36, Rf (S2) ¼ 0.35.
NMR (CDCl3)
d
(ppm): 1.28 (d, J ¼ 6.3 Hz, 3H, CH3),1.76e1.96 (m, 2H,
CHCH2), 2.60e2.78 (m, 4H, piperazine), 3.05e3.24 (m, 5H, pipera-
zine, CH), 3.67e3.87 (m, 2H, CH2OH), 4.20 (q, J ¼ 6.3 Hz, 2H,
CH2CH3), 4.61 (s, 1H, OH), 4.69e4.86 (m, 2H, CH2Ph); 6.96e7.35 (m,
6.1.3. Synthesis of 4-Hydroxy-2-(piperazin-1-yl)butanamides
34e36 (general procedure)
To solution of 31, 32 or 33 (2 mmol) in 5 mL of anhydrous DCM,
5 mL TFA was added and stirred for 1 h at room temperature. Then
excess of reagent and solvent was removed under vacuum. The
resulting oil was neutralized by 2 M KOH, extracted with DCM
(4 ꢂ 10 mL), dried over Na2SO4 and evaporated. The crude product
was purified by column chromatography (Me2CO/CHCl3, 1/1).
5H, Ar-H), 7.83 (t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 13.8
(CH3CH2), 34.6 (CHCH2), 43.6 (NHCH2Ph), 45.5, 51.8 (piperazine),
58.3 (HOCH2CH2), 62.0 (CH3CH2), 69.7 (CHCH2), 126.7, 126.9, 128.5,
137.9 (arom), 171.3 (carbonyl); TLC: Rf (S1) ¼ 0.56, Rf (S2) ¼ 0.65.
6.1.2.24. (R, S) Ethyl 4-[1-(4-chlorobenzylamino)-4-hydroxy-1-oxo-
butan-2-yl]piperazine-1-carboxylate (32). Procedure GP4; reagents:
compound 10 (0.97 g, 4 mmol), 4-chlorobenzylamine (1.13 g,
8 mmol); reaction time 6 h; yield 57%; mp 129e130 ꢀC (EtOAc);
anal. calcd for C18H26N3O4Cl: C% 56.32, H% 6.83, N% 10.95 found: C%
6.1.3.1. (R, S) N-Benzyl-4-hydroxy-2-(piperazin-1-yl)butanamide
(34). Reagents: compound 31 (0.97 g); yield 34%; mp 121e122 ꢀC;
anal. calcd for C15H22N3O2: C% 64.95, H% 8.36, N% 15.15 found: C%
64.89, H% 8.24 N% 15.21; 1H NMR (CDCl3)
d (ppm): 1.68e1.84 (m,

190
K. Kulig et al. / European Journal of Medicinal Chemistry 46 (2011) 183e190
3H, CHCH2, NH piperazine), 2.64e2.80 (m, 4H, piperazine),
3.09e3.26 (m, 5H, piperazine, CH), 3.69e3.93 (m, 2H, CH2OH), 4.24
(s, 1H, OH), 4.48e4.59 (m, 2H, CH2Ph), 6.96e7.49 (m, 5H, Ar-H), 7.83
MS using an API 3200 triple quadrupole mass spectrometer
according to the method described previously [25].
(t, 1H, amide); 13C NMR (CDCl3)
d (ppm): 34.6 (CHCH2), 43.6
Acknowledgements
(NHCH2Ph), 45.8, 54.8 (piperazine), 58.3 (HOCH2CH2), 69.7
(CHCH2), 126.7, 126.9, 128.5, 137.9 (arom), 171.3 (carbonyl); TLC: Rf
(S1) ¼ 0.49, Rf (S2) ¼ 0.68.
Financial support of this work by the DAAD/54/2006 grants is
gratefully acknowledged.
6.1.3.2. (R, S) N-(4-Chlorobenzyl)-4-hydroxy-2-(piperazin-1-yl)buta-
namide (35). Reagents: compound 32 (0.77 g); yield 27%; mp
119e120 ꢀC; anal. calcd for C15H22N3O2Cl: C% 57.78, H% 7.11, N%
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(CHCH2), 43.6 (NHCH2Ph), 45.8, 54.8 (piperazine), 58.3
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6.2.2. MS-binding assays
Binding assays for mGAT1 based on NO711 as native marker
were performed as described [23]. NO711 was analyzed by LC-MS/