Synthesis and affinity for serotonin and dopamine transporters of some benzophenone oxime ether derivatives

Article abstract of DOI:10.1016/S0014-827X(03)00102-2

In a previous study, we reported the synthesis of several 3-(methylenaminoxymethyl)-substituted piperidine derivatives and their ability to interfere with the transmission mediated by biogenic amines. The present study describes the preparation of some ne

Full text of DOI:10.1016/S0014-827X(03)00102-2

Il Farmaco 58 (2003) 977Á981
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www.elsevier.com/locate/farmac
Synthesis and affinity for serotonin and dopamine transporters of
some benzophenone oxime ether derivatives
Annalina Lapucci a,*, Susanna Nencetti a, Gian Carlo Demontis b
a Dipartimento di Scienze Farmaceutiche, Facolta` di Farmacia, Universita` di Pisa, Via Bonanno 6, 56100 Pisa, Italy
b Facolta` di Farmacia, Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, Via Bonanno 6, 56100 Pisa, Italy
Received 4 September 2002; accepted 12 December 2002
Abstract
In a previous study, we reported the synthesis of several 3-(methylenaminoxymethyl)-substituted piperidine derivatives and their
ability to interfere with the transmission mediated by biogenic amines. The present study describes the preparation of some new
oxime derivatives and their capacity to inhibit serotonin (SERT) and dopamine (DAT) transporters expressed at the level of the
rabbit cortex and the striatal membranes, respectively. All the compounds showed Ki values in the micromolar range on both
transporters.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Dopamine; Serotonin; Oxime derivatives
1. Introduction
In a previous work [4], we evaluated the inhibitory
activity of some 3-(methylenaminoxy)methyl piperidines
of type 1 (Fig. 1) on biogenic amine reuptake (NA, DA,
5-HT) in rabbit brain synaptosomal fractions. This
study showed that all compounds possess an appreciable
activity, and the one in which R and R1 are two phenyls
(1a) is the most active in this series of compounds, with
Ki values in the micromolar range. On the basis of these
data, with the aim to gain further knowledge about the
influence of certain molecular portions of 1a on its
biological properties, we synthesised compounds 2a, 2b,
3a and 3b, in which the benzophenone oximic portion of
1a is still present, while the aminic side-chain, ethylpi-
peridinic (2a and 2b) or ethyldimethylaminic (3a and
3b), is different.
According to the monoamine theory, depression is a
disease of the central nervous system associated with a
dysfunction of the metabolism and/or of the synaptic
transmission mediated by the biogenic amines dopamine
(DA), serotonin (5-HT), noradrenaline (NA) [1,2]. The
therapeutic treatment of this pathology is linked with
the use of drugs that enhance the synaptic levels of these
amines such as biogenic amines reuptake inhibitors. In
this class of antidepressant agents, a particular impor-
tance is attached to selective serotonin reuptake inhibi-
tors (SSRIs), such as fluoxetine, sertraline, paroxetine,
citalopran and fluvoxamine, which have been used with
success in depressed patients in view of their favourable
side-effect profile with respect to classic tricyclic anti-
depressants (TCAs) [1,3].
2. Chemistry
The oxime ethers 2a, 2b, 3a and 3b were synthesised as
indicated in Scheme 1. Reaction of benzophenone or 4-
* Corresponding author.
E-mail address: alapucci@farm.unipi.it (A. Lapucci).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00102-2

978
A. Lapucci et al. / Il Farmaco 58 (2003) 977Á
/981
Fig. 1.
fluorobenzophenone with hydroxylamine hydrochloride
afforded the oximes 4a and 4b [5,6]. The reaction of 4a
and 4b with the appropriate alkyl chloride in DMSO
and sodium hydroxide, in the presence of 18-Crown-6,
yielded the crude oxime ethers 2a, 2b, 3a and 3b, which
were purified by column chromatography and then
transformed into the corresponding hydrochloride salts.
The structure of compounds 2 and 3 was confirmed
by their spectral data. The E configuration around the
double bond of the methylenaminoxy group of com-
Scheme 1.

A. Lapucci et al. / Il Farmaco 58 (2003) 977Á
/981
979
pounds 2b and 3b was assumed on the basis of the
configuration of the starting oxime 4b [6] which have
been proved to be configurationally stable in the
reaction conditions employed.
change in their affinity for DAT, with a ratio of 4.1 for
compound 3b.
In conclusion, it may be said that the changes made in
the structure of compound 1a lead to a slight increase in
the affinity for SERT, in particular in compounds in
which RꢀF (2b and 3b). Overall results indicate that
/
3. Pharmacology
compounds 2a, 2b, 3a and 3b exhibit a certain selectivity
for DAT compared with SERT, but this tendency is
attenuated by the insertion of a fluorine atom on one of
the aromatic rings.
Compounds 2a, 2b, 3a and 3b were tested for their
ability to interfere with the systems of transmission of
biogenic amines, by directly evaluating the degree to
which they bind to the serotonin (SERT) and dopamine
(DAT) transporters expressed at the level of the rabbit
cerebral cortex and the striatal membranes, respectively.
In order to allow a more homogeneous comparison of
all biological data, the same type of test was also used
for compound 1a, which was previously tested [4] by
means of the direct uptake method. [3H]-paroxetine was
used as the specific tritiated ligand for SERT, while
[3H]WIN 35,428 was used to label DAT. The binding
affinity indices of oxime ethers 2a, 2b, 3a and 3b are
reported in Table 1, together with those obtained in the
same tests with the previously studied compound 1a and
fluoxetine.
5. Experimental
5.1. Chemistry
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. IR spectra for compar-
ison between compounds were recorded with a PerkinÁ
/
Elmer model 1310, as nujol mulls in the case of solid
1
substances, or as liquid film in the case of liquids. H
NMR spectra were routinely recorded with a Varian
CFT 20 (80 MHz) instrument in ca. 5% solution of
CDCl3 (for neutral compounds or the free bases) or D2O
(for the salts), using Me4Si or Me3Si(CH2)3SO3Na as the
internal standard, respectively. The oximes 4a and (E)-
4b were prepared by the method described in literature.
[5,6]. Analytical TLC were carried out on 0.25 mm layer
silica gel plates containing a fluorescent indicator; spots
were detected under UV light (254 nm). Column
4. Results and discussion
As regard affinity for SERT, the data in Table 1 show
that all the synthesised compounds 2a, 2b, 3a and 3b
exhibit very low affinity values with Ki in the micro-
molar range, in the same way as the previously studied
compound 1a whereas the affinity value of the reference
compound, fluoxetine is in the nanomolar range.
chromatography was performed using 70Á230 mesh
/
silica gel. MgSO4 was always used as the drying agent.
Evaporation was carried out in vacuo (rotating eva-
porator). Elemental analyses were performed by our
analytical laboratory and agreed with theoretical values
Also, the affinity for DAT of the compounds 2a and
3a, in which Rꢀ/H, is low with Ki values in the
to within 90.4%.
/
micromolar range, so the DAT/SERT affinity ratio is
very low, whereas for the fluoxetine is 689.
5.1.1. General procedure for the synthesis of oximethers
2a, 2b, 3a and 3b
Furthermore, it may be seen from Table 1 that the
compounds in which one of the aromatic rings is
substituted in the para position with a fluorine atom
(2b and 3b) exhibit a SERT affinity index slightly lower
than that of the corresponding compounds unsubsti-
tuted on the aromatic ring, without any appreciable
N-(2-Chloroethyl) piperidine or 2-(dimethylamino)-
ethyl chloride hydrochloride (23 mmol), NaOH (46
mmol) and 18-Crown-6 (23 mmol) were added to a
stirred solution of the appropriate oxime 4a and 4b [5,6]
(23 mmol) in anhydrous DMSO (20 ml). After comple-
Table 1
Affinity for SERT and DAT of compounds 1a, 2a, 2b, 3a and 3b
Comp.
R
R1
5-HT-T Ki (mM)
11.492.7
DAT Ki (mM)
Ratio
2a
2b
H
F
bdcÃ
bdcÃ
N(Me)2
N(Me)2
/
C5H10
N
N
/
3.69
3.99
7.29
4.99
n.t.
4.09
/
1.2
1.1
2.6
1.4
0.3
0.9
0.8
4.1
/C5H10
4.29
9.09
1.29
/
1.3
3.1
0.3
/
3a
3b
H
F
/
/
/
/
1a
Fluoxetine
25.09
/
6.1
10ꢂ39
5.8ꢁ
/
/2.9ꢁ
/
10ꢂ3
/
1.7
689

980
A. Lapucci et al. / Il Farmaco 58 (2003) 977Á981
/
tion of the addition the reaction mixture was stirred at
40 8C for 48 h, diluted with water and extracted with
CHCl3. The evaporation of the washed (H2O) and dried
CHCl3 extracts give an oily residue that was purified by
column chromatography (CHCl3/5% MeOH) to yield
Competition assays were performed in duplicate glass
tubes in a total volume of 2000 ml, with a concentration
of [3H]-paroxetine of 100Á
200 pM, using an incubation
/
time of 2 h at room temperature. In the experimental
conditions used, specific binding (see below) reached
equilibrium after 90 min. Separation of ligand bound to
the transporter from free ligand was performed by
filtration using a 30 well manyfold (Brandel), with glass
fiber filters (GF/C) pre-soaked for 2 h at room
temperature with 0.6% (w:v) polyethylenimine. Filters
pure 2a, 2b, 3a and 3b.
1
2a: (39%) H NMR d 1.25Á
/
2.40 (m, 10H), 2.58Á
4.37 (m, 2H), 7.06Á7.89 (m, 10H). 2b:
(96%) H NMR d 1.43Á2.34 (m, 10H), 2.52Á2.65 (m,
2H), 4.18Á4.40 (m, 2H), 7.01Á7.43 (m, 9H). 3a: (59%)
1H NMR d 2.24 (m, 6H), 2.57Á
2.72 (m, 2H), 4.20Á4.36
(m, 2H), 7.29Á
7.39 (m, 10H). 3b: (37%) 1H NMR d 2.25
(m, 6H), 2.48Á2.72 (m, 2H), 4.19Á4.36 (m, 2H), 6.96Á
7.37 (m, 9H).
/
2.73
(m, 2H), 4.22Á
/
/
1
/
/
/
/
/
/
were quickly washed four times with cold (4 8C) TrisÁ
/
/
HCl 50 mM. Filters were removed, put into scintillation
vials and let to stay overnight in 3 ml of Cytoscint ES
(ICN). The following day, samples were read by
scintillation spectroscopy in a b-counter (Packard).
Unspecific binding, defined in the presence of 2 mM
serotonin, was subtracted from total binding (in the
absence of competitors) to obtain specific binding. In a
typical assay, total binding was 900 d.p.m., unspecific
was 200 d.p.m., and specific binding was 700 d.p.m.
/
/
/
Compounds 2a, 2b, 3a and 3b were converted to the
corresponding hydrochloride salts by dissolving the
bases in Et2O and subsequent treatment with an excess
of Et2O×
crystallised from MeOH/Et2O to yield pure 2a×
HCl, 3a×HCl, 3b×HCl. Melting points are reported in
Table 2.
/HCl. The solid precipitate was filtered and
/
HCl, 2b×
/
/
/
5.2.2. Affinity for DA transporter (DAT)
5.2. Binding studies
The affinity of compounds (2a, 2b, 3a, and 3b) for the
DAT was assayed in rabbit striatal membranes, labelled
with the cocaine analogue [3H]-WIN 35,428, whose
specific activity was 84.5 Ci/mmol.
5.2.1. Affinity for 5-HT transporter (SERT)
The affinity of compounds (2a, 2b, 3a, and 3b) for the
serotonin transporter was assayed in rabbit cortex
membranes, labelled with [3H]-paroxetine, whose speci-
fic activity ranged from 15 to 20 Ci/mmol.
Tissue from the anterior pole of the rabbit frontal
cortex was collected and stored at ꢂ35 8C until used.
Tissue was collected and stored at ꢂ35 8C until used.
/
Membranes were prepared from frozen tissue using an
ultraturrax homogeniser, in a saline solution whose
composition was (mM): NaCl (48); sucrose (320), pH
7.7 at 4 8C, by mixing Na2HPO4/NaH2PO4 (25). The
homogenate was centrifuged for 10 min at 48,000
/
Membranes were prepared from frozen tissue using an
ultraturrax homogeniser, in a saline solution whose
relative centrifugal force of 46,000ꢁg, keeping the
/
composition was (mM): NaCl (120); KCl (5), TrisÁ
HCl (50), pH 7.4 at 4 8C, using a dilution factor of
1:30 (w:v). The homogenate was centrifuged for 10 min
/
temperature at 4 8C. The supernatant was discarded
and the pellet resuspended by diluting 1:100 (w:v) in the
homogenisation buffer. The pellet was recentrifuged at
at a relative centrifugal force of 46,000ꢁ
/
g, keeping the
46,000ꢁ
1:100 in buffer without sucrose, splitted in 3.5 ml
aliquots and stored frozen at ꢂ35 8C. On the day of
the experiment, one 3.5 ml aliquot was quickly thawed
at 37 8C, centrifuged for 10 min at 46,000ꢁg at 4 8C
/
g for 10 min and the pellet was resuspended
temperature at 4 8C. The supernatant was discarded and
the pellet was resuspended by diluting 1:30 (w:v) in the
homogenisation buffer. The pellet was recentrifuged at
/
46,000ꢁ
buffer without sucrose, splitted in 1 ml aliquots and
stored frozen at ꢂ35 8C. On the day of the experiment,
one aliquot was quickly thawed at 37 8C, centrifuged for
10 min at 46,000ꢁg at 4 8C and the pellet resuspended
/g for 10 min and the pellet resuspended 1:30 in
/
and the pellet was resuspended in buffer without sucrose
at a dilution of 1:200 (w:v).
/
Competition assays were done in duplicate glass tubes
in a total volume of 500 ml, with a concentration of [3H]-
/
in homogenisation buffer at a dilution of 1:30 (w:v).
WIN 35,428 of 0.5Á/2 nM, using an incubation time of 2
Table 2
Affinity for SERT and DAT of compounds 1a, 2a, 2b, 3a and 3b
Comp.
R
R1
m.p. (8C)
Formula
Analysis
2a×
2b×
3a×
3b×
/
HCl
HCl
HCl
HCl
H
F
bdcÃ
bdcÃ
N(Me)2
N(Me)2
/
C5H10
N
N
182Á
165Á
185Á
154Á
/
184
166
186
155
C20H25ClN2O
C20H24ClFN2O
C17H21ClN2O
C17H20ClFN2O
C, H, N
C, H, N
C, H, N
C, H, N
/
/C5H10
/
/
H
F
/
/
/

A. Lapucci et al. / Il Farmaco 58 (2003) 977Á
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981
h at 4 8C. In the experimental conditions used, specific
binding (see below) reached equilibrium after 60 min.
Separation of ligand bound to the transporter from free
ligand was by filtration using a 30 well manyfold
(Brandel), with glass fiber filters (GF/C) pre-soaked
for 2 h at room temperature with 0.6% (w:v) poly-
ethylenimine. Filters were quickly washed three times
ments, and gave an average dissociation constant (Kd) of
6.1 nM.
References
with cold (4 8C) TrisÁHCl 50 mM. Filters were
/
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removed, put in scintillation vials and let to stay
overnight in 3 ml of Cytoscint ES (ICN). The following
day, samples were read by scintillation spectroscopy in a
b-counter (Packard). Unspecific binding, defined in the
presence of 10 mM dopamine, was subtracted from total
binding (in the absence of competitors) to obtain specific
binding. In a typical assay, total binding was 600 d.p.m.,
unspecific was 150 d.p.m., and specific binding was 450
d.p.m.
[2] C.L.E. Broekkamp, D. Leysen, B.W.M.M. Peeters, R.M. Pinder, J.
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transporter was assessed in separate saturation experi-
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/