Oxa-azaspiro derivatives: A novel class of triple re-uptake inhibitors

Article abstract of DOI:10.1002/cmdc.200900482

(Figure Presented) Oxa-aza spiro compounds: In silico techniques were successfully employed to predict the rank order of derivatives based on their binding affinities to the monoamine transporters, SERT, NET and DAT. The proposed scaffolds derived from the pharmacophore model were experimentally evaluated in vitro and in vivo. A new class of selective triple reuptake inhibitors was discovered, that may be developed further to give novel antidepressant agents.

Full text of DOI:10.1002/cmdc.200900482

DOI: 10.1002/cmdc.200900482
Oxa-azaspiro Derivatives: a Novel Class of Triple Re-uptake Inhibitors
Michela Bettati,[a] Paolo Cavanni,[a] Romano Di Fabio,[a] Beatrice Oliosi,[b] Ornella Perini,[b] Gunther Scheid,[a]
Giovanna Tedesco,[b] Laura Zonzini,[a] and Fabrizio Micheli*[a]
Drugs able to interfere with either the uptake or the metabo-
lism of endogenous monoamines have been used for many
years to treat depression. The first drugs of this type, such as
the monoamine oxidase (MAO) inhibitors and the tricyclic anti-
depressants, achieved wide diffusion in the market, but are un-
fortunately associated with side effects that may influence pa-
tient compliance.[1] Over the last few years, compounds acting
through the selective blockade of neurotransmitter re-uptake
have demonstrated their efficacy as successful antidepressant
agents. This blockade can take place in either serotoninergic
neurons (SSRI; e.g., paroxetine) or noradrenergic neurons
(SNRI; e.g., reboxetine). Moreover, drugs known as “dual
uptake” inhibitors, which act on both serotoninergic and nora-
drenergic transporters (e.g., venlafaxine) or on both noradre-
nergic and dopaminergic transporters (e.g., bupropion), have
also demonstrated good efficacy.[1]
More precisely, three specific pharmacophore models for the
monoamine transporters, SERT (serotonin transporter), NET
(norepinephrine transporter) and DAT (dopamine transporter),
were built using structurally rigid and selective derivatives.
These compounds were derived both from the GSK proprietary
collection and from the public domain (e.g., derivatives de-
scribed in Reference [3]). The structures were modeled within
the Maestro modeling environment.[4] Conformational analyses
were carried out for the three structures with BatchMin[5] using
the following parameters: 1000 steps/rotatable bond, OPLS_
2005 FF, implicit water model of solvation, 5000 minimization
steps with PRCG. Conformations lying within a 3 kcalmolꢀ1
energy window were kept.
A single-ligand pharmacophore was then built over these
structures using the Phase[6] module within Maestro, and se-
lecting the most relevant pharmacophoric features. The three
pharmacophores were finally merged together to create the
triple re-uptake inhibitor pharmacophore. Average coordinates
for the common pharmacophoric features were taken to deter-
mine their three-dimensional arrangement, as illustrated in
Figure 1.
More recently, compounds acting as “triple re-uptake” inhibi-
tors (TRUI),[2] such as indatraline and DOV 21,947, have been
disclosed. Given the extensive experience of investigators at
GlaxoSmithKline (GSK) with selective, dual and triple re-uptake
inhibitors, new TRUI scaffolds were rationally planned exploit-
ing a pharmacophore model developed in house
Figure 1. The triple re-uptake inhibitor pharmacophore. Coding of features:
P6 sphere, positive ionizable; A2 sphere, H-bond acceptor; H3, H4, and H5
spheres, hydrophobic; R7 sphere, aromatic ring. Distances between the
pharmacophoric points are given in ꢀ.
[a] Dr. M. Bettati, Dr. P. Cavanni, Dr. R. Di Fabio, Dr. G. Scheid, Dr. L. Zonzini,
Dr. F. Micheli
The aim of the work described herein was twofold: the first
objective was to set up the chemistry to prepare a new series
of oxa-spiro derivatives; the second was to validate the capa-
bility of the pharmacophore model to predict the rank order
of affinity of the new derivatives.
Neurosciences Centre of Excellence for Drug Discovery
GlaxoSmithKline
Via Fleming 4, 37135 Verona (Italy)
Fax: (+39)045-8218196
E-mail: Fabrizio.E.Micheli@gsk.com
[b] Dr. B. Oliosi, Dr. O. Perini, Dr. G. Tedesco
Molecular Discovery Research
GlaxoSmithKline
It was hypothesized that the introduction of an appropriate-
ly located tetrahydrofuran (1) or a tetrahydropyran (2) moiety
on the 4-phenyl piperidine scaffold should have satisfied at
Via Fleming 4, 37135 Verona (Italy)
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least five out of the six pharmacophoric points in the model as
depicted in Figure 2, suggesting that the proposed derivatives
should be endowed with good affinity at the three mono-
amine transporters, namely SERT, NET and DAT.
Scheme 1. Reagents and conditions: a) NaOEt, AcOEt/THF, RT, methyl 3-oxo-
3-[(phenylmethyl)amino]propanoate, multistep; b) Ethyl bromoacetate,
K2CO3, 858C, 1.5 h; c) BH3 ·THF, 658C; d) PPh3, DEAD, CH2Cl2; e) ACE-Cl, 1,2-di-
chloroethane, reflux, 5 h.
proaching the reacting centre from the opposite side to the
bulky aromatic group.
The subsequent exhaustive reduction using borane·THF
complex led to the isolation of derivative 8 in 87% yield. The
final cyclization was performed under Mitsunobu’s conditions[8]
and the target derivative 1 was isolated after deprotection of
the benzyl group with 1-chloroethyl chloroformate[9] in 43%
overall yield. Derivative 1 was tested in biological assays as the
racemate.
Figure 2. The proposed scaffolds a) 1 and b) 2 fitted in the TRUI pharmaco-
phore model.
As described in Scheme 2, intermediate 6 was reacted under
Michael reaction conditions with ethyl acrylate to obtain, as
previously, a single diastereoisomer (9) in 71% yield. Subse-
quently, this intermediate was reacted with borane·THF com-
Such model compounds were meant to provide a rapid
reply to the working hypothesis before embarking on a more
complex synthesis of final scaffolds designed to fully fit all six
of the pharmacophoric points.
From an in silico point of view, both derivatives 1 and 2 pro-
vided excellent fitting in the TRUI pharmacophore, with 2
being slightly superior in its ability to fit the desired features
(Figure 2). However, it is evident that the sixth feature (i.e., the
H5 hydrophobic region) is not satisfied by either derivative 1
or 2, but that the introduction of an appropriate substituent
able to reach this area might further increase the potential af-
finity of the molecules.
Given these promising theoretical results, the necessary syn-
thetic steps to prepare compounds 1 and 2 were put in place
and their preparation is reported in Scheme 1 and 2, respec-
tively (n.b., relative stereochemistry shown).
3,4-Dichlorobenzaldehyde was reacted with methyl 3-oxo-3-
[(phenylmethyl)amino]propanoate under basic conditions[7] to
provide the versatile intermediate 6 in 55% yield as a race-
mate. This derivative was alkylated with ethyl bromoacetate
under thermodynamic conditions leading to the isolation of a
single diastereoisomer 7 in 58% yield. The stereochemistry of
the reaction is probably controlled by the electrophile ap-
Scheme 2. Reagents and conditions: a) Ethyl acrylate, K2CO3, acetone, over-
night, RT; b) BH3 ·THF, 608C, 8 h; c) PPh3, DEAD, CH2Cl2; d) ACE-Cl, 1,2-di-
chloroethane, reflux, 5 h.
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ChemMedChem 2010, 5, 361 – 366

plex to obtain the diol 10 in 29% yield. Cyclization under Mit-
sunobu’s conditions[8] led to the N-benzylated derivative 11 in
21% yield. Deprotection with 1-chloroethyl chloroformate led
to the desired product 2 in 65% yield. As for derivative 1, com-
pound 2 was tested in biological assays as the racemate.
Both compounds were evaluated for their ability to bind to
the three monoamine transporters (SERT, NET and DAT) in scin-
tillation proximity assay (SPA)-binding assays on membranes
from cells transiently transduced with BacMam virus. Commer-
cially available indatraline was used as a reference compound.
Results are reported in Table 1.
pothesis, derivatives 3 and 4 were also prepared. Figure 3
shows that these two model systems were endowed with an
inferior “theoretical” ability to fit the pharmacophore. Despite
the poor two-dimensional depiction of a three-dimensional re-
ality, it can be appreciated that the overlap of the oxetane ring
of derivative 3 with the A2 region of the pharmacophore is
poor and inferior to the 5- and 6- membered spiro systems of
compounds 1 and 2, respectively. In derivative 4, the oxygen
atom is behind the A2 sphere and only a minimal overlap is
present with this region.
The synthesis of compound 3 from the versatile intermedi-
ate 6 is described in Scheme 3. The electrophile used, in this
case, was an aqueous solution of formaldehyde, which allowed
Table 1. Binding at the three transporters (SERT, NET, DAT).
Entry
pKi
hNET SPA[a]
hSERT SPA[a]
hDAT SPA[a]
DOV 21,947
Indatraline
1
2
3
4
20
21[b]
22[c]
7.8
9.7
8.3
9.2
7.2
7.5
4.3
8.9
9.5
7.2
8.8
7.1
8.0
6.8
7.3
4.5
7.2
8.4
7.1
9.1
7.4
7.7
6.8
7.4
5.6
7.5
7.9
[a] SEM for hSERT/NET/DAT data sets is ꢁ0.1. [b] 21 is the enantiomer 1
of derivative 2. [c] 22 is the enantiomer 2 of derivative 2
As can be clearly appreciated, and in agreement with the
suggestions provided by the pharmacophore model, derivative
2 showed better affinity than 1 towards SERT and NET, while
the affinity of the two molecules towards DAT was comparable.
Interestingly, both derivatives were endowed with similar or
superior affinity for these transporters to those reported in lit-
erature for the clinical candidate DOV 21,947.[2b]
Scheme 3. Reagents and conditions: a) Formaline 37%, Et3N, dioxane, 1.5 h,
RT; b) BH3·THF, 608C, 8 h; c) MsCl/Py, 08C; d) THF, NaOH, reflux, 6 h; e) ACE-
Cl, 1,2-dichloroethane, reflux, 5 h.
To further increase the confidence in the ability of the phar-
macophore model to rank order the affinity of the derivatives
in this specific series, and therefore to reinforce the original hy-
the isolation of intermediate 12 in almost quantitative yield.
The subsequent reaction with borane·THF complex led to de-
rivative 13 in 91% yield. A two-step cyclization, through the
formation of a mesylate intermediate, allowed the isolation of
14 in 30% yield. The final deprotection to derivative 3 was in-
efficient due to formation of a series of by-products, leading to
an isolated yield of just 10%.
Concerning derivative 4, the synthetic route to the desired
fused bicyclic system had to be carefully evaluated so as to
obtain the appropriate stereochemistry to fit the pharmaco-
phore model. Actually, in this novel, substituted octahydro-2H-
pyrano[4,3-b]pyridine template, a cis junction of the two six-
membered rings led to a better fit in the pharmacophore
model than the trans junction. For this specific reason, a hydro-
genation step was considered as the key, last step in the syn-
thesis.
The desired compound 4 was prepared from ethyl 4-(3,4-di-
chlorophenyl)-3-pyridinecarboxylate 15 (Scheme 4) by treating
it with 3-chloro perbenzoic acid at room temperature to give
intermediate 16 in quantitative yield. Reaction with phospho-
rus oxychloride allowed the preparation of the intermediate 17
Figure 3. The proposed model scaffolds a) 3 and b) 4 fitted in the TRUI phar-
macophore model. The overlap of the oxetane ring with the H-bond accept-
or region A2 is the least pronounced in the series of oxa-spiro derivatives.
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Figure 4. The scaffold a) 4 and b) intermediate 20 fitted in the TRUI pharma-
cophore model.
isolated. Accordingly, compounds 21 (2, enantiomer 1) and 22
(2, enantiomer 2) were obtained through chiral chromatogra-
phy and tested for their binding affinity. Results are reported
in Table 1.
Derivative 22 showed high affinity for the three transporters.
For this reason, the ability of 22 to block [3H]5-HT, [3H]NE and
[3H]DA uptake was then tested in functional uptake SPA assays
using LLCPK cells stably transfected with human SERT, NET or
DAT. The results of these experiments are reported in Table 2.
Scheme 4. Reagents and conditions: a) mCPBA, CH2Cl2, overnight, RT;
b) POCl3, overnight, 808C; c) allyl triethyl stannane, Pd(PPh3)4, toluene, reflux,
5 h; d) O3, EtOH, ꢀ788C followed by NaBH4 from ꢀ788C to RT, overnight;
e) P2O5, toluene, dioxane, 1308C, 5 h; f) PtO2, H2, EtOH, RT, 2 h.
Table 2. Ability of 22 to inhibit the uptake of all of the three mono-
in 45% yield, which was then subjected to a Stille coupling[10]
to give the desired allyl derivative 18 in 40% yield. Ozonolysis
of this compound at low temperature, followed by reduction
with excess sodium borohydride, allowed the isolation of the
diol 19 in quantitative yield. The subsequent cyclization step
proved difficult; neither of the previously employed cyclization
procedures worked for this system. Mitsunobu’s conditions
stopped at the activated intermediate stage, while an attempt
to pass through a mesylate intermediate only led to degrada-
tion products. In the end, cyclization to the desired intermedi-
ate 20 was achieved in low yield (10%) through dehydrating
conditions (P2O5 at high temperature). Despite the potential in-
terference of the chlorine atoms, the planned hydrogenation
step with platinum dioxide worked very well, giving the de-
sired product 4 in 90% yield, with just 10% formation of deha-
logenated by-products. The desired cis junction of the two
rings and the reported stereochemistry was confirmed by NMR
experiments.
amines.
Entry[a]
pIC50 ꢁSEM
hSERT SPA[b]
hNET SPA[b]
hDAT SPA[b]
DOV 21,947
Indatraline
22
7.2
8.5
8.6
7.6
8.4
8.9
7.1
8.8
8.0
[a] DOV 21,947 and indatraline were included as reference compounds.
[b] SEM for hSERT/NET/DAT data sets is ꢁ0.2
The functional pIC50 value observed in the DAT assay was in
line with the affinity of compound 22 determined in the bind-
ing assay, while the functional pIC50 value observed at NET was
slightly higher. Conversely, the functional potency of 22 at
SERT was lower than its binding affinity for this transporter. In
the latter case, it is important to note that a difference was ex-
pected since the SERT uptake-SPA assay is known to be partic-
ularly sensitive to test conditions (e.g., cell numbers) and, from
previous experience in house, most SERT blockers tested in
uptake assays showed pIC50 values lower than the correspond-
ing affinity in the binding assay.
Derivatives 3 and 4 were biologically evaluated along with
intermediate 20. Results for these derivatives are reported in
Table 1. As predicted by the pharmacophore model, their affini-
ty was inferior to derivatives 1 and 2. The almost complete in-
activity of intermediate 20 was also expected. In fact, this com-
pound only fitted the lipophilic regions of the pharmacophore
marginally and it was not endowed with the basic sp3 positive-
ly ionizable feature identified by the P6 sphere (Figure 4).
Given that derivative 2 showed a good binding profile at
the three monoamine transporters, the two enantiomers were
Given its excellent in vitro profile, both in the binding and
uptake assays, derivative 22 was submitted to CYPEX bacto-
some P450 inhibition assays and rat and human liver micro-
somes in vitro clearance assays. This further step was per-
formed to validate the potential for development of such a
scaffold. The compound showed an excellent CYP450 inhibi-
tion profile demonstrating IC50 values greater than 10 mm
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borane–THF complex in THF (23.25 mmol) and stirred at 608C for
8 h. The reaction mixture was cooled to RT and then MeOH (5 mL)
was added, followed by 1.0m HCl in Et2O (21 mL); the resulting
mixture was stirred at RT overnight. The solvent was removed in
vacuo and the residue purified by SCX cartridge (100% MeOH then
2.0n NH3 in MeOH) to give intermediate 10 as a colorless oil. Com-
pound 10 (0.6 mmol) was dissolved in CH2Cl2 and PPh3 (0.70 mmol)
was added, followed by dropwise addition of diethyl azodicarboxy-
late (40% in 0.86 mmol). The reaction mixture was stirred at RT
overnight. The solvent was removed in vacuo and the residue puri-
fied by SCX cartridge (100% MeOH then 2.0n NH3 in MeOH) to
give compound 11 as colorless oil. This intermediate (0.13 mmol)
was dissolved in 1,2-dichloroethane (10 mL), 1-chloroethyl chloro-
formate (1.3 mmol) was added and the mixture was refluxed for
5 h. It was then cooled to RT and MeOH (5 mL) added. The mixture
refluxed for 2 h, cooled to RT and the residue purified by SCX car-
tridge (100% MeOH then 2.0n NH3 in MeOH) to give derivative 2.
1H NMR (500 MHz, CDCl3): d=7.41–7.33 (d, 1H, J=8.3 Hz), 7.21–
7.18 (d, 1H, J=2.0 Hz), 6.97–6.92 (dd, 1H, J=8.3, 2.0 Hz), 4.36–4.29
(dd, 1H, J=11.0, 2.4 Hz), 3.87–3.80 (dd, 1H, J=11.0, 4.6 Hz), 3.74–
3.67 (d, 1H, J=12.2 Hz), 3.27–3.17 (m, 1H), 3.11–3.05 (d, 1H, J=
11.0 Hz), 3.07–2.95 (m, 1H), 2.78–2.67 (m, 1H), 2.49–2.40 (dd, 1H,
J=13.2, 3.7 Hz), 2.31–2.24 (d, 1H, J=12.4 Hz), 2.05–1.92 (m, 1H),
1.85–1.72 (m, 1H), 1.54–1.47 (m, 1H), 1.47–1.36 (m, 1H), 1.35–
1.22 ppm (m, 2H); Relative stereochemistry confirmed on the basis
of nOe experiments; MS (ESI): m/z (%): 301 (100) [M+H]+.
against all isoforms, with the exception of CYP2D6 (IC50 =
4 mm). The rat and human intrinsic clearance values proved to
be relatively low (0.6 and 1.5 mLminꢀ1 gꢀ1 of protein, respec-
tively), further confirming the tractability of this template. On
the basis of these promising in vitro results, the in vivo phar-
macokinetic profile of compound 22 was determined in rats.
The overall blood clearance of compound 22 was estimated
to be moderate (34 mLminꢀ1 kgꢀ1), while its distribution
volume was in the moderate to high range (4.9 Lkgꢀ1). The re-
sulting half-life was relatively long (2.2 h) and, importantly, the
compound was demonstrated to readily cross the blood–brain
barrier (Brain/Blood AUC ratio=9.6). Notwithstanding its
modest clearance, derivative 22 was prone to efficient metabo-
lism during the absorption process. After oral administration,
despite a good absorption (fraction absorbed=91%), high
hepatic extraction (EH=90%) led to an overall bioavailability
of 9%.
However, this potential drawback might be appropriately
fixed when a rational decoration will be used for the scaffolds
designed to fully fit the six features of the pharmacophore
model as stated at the beginning of the manuscript.
Considering the declared objective of the work, the experi-
mental results described here can be considered to be very
positive. The pharmacophore model proved to be proficient in
rank ordering the potency of the scaffolds designed in this
novel series of spiro derivatives. The synthetic routes designed
were successful in preparing the new oxa-spiro scaffolds. The
oxa-spiro template 2 proved to be a potent TRUI scaffold, and
the potential exists to further increase its affinity and develop
its druglike characteristics by appropriately decorated final de-
rivatives.
The enantiomeric purity of each enantiomer of compound 2, ob-
tained after preparative chromatography on a chiral column (Chir-
alpak AD-H, 25ꢂ2.0 cm; UV: 225 nm; 23 mg in EtOH per injection;
mobile phase: n-hexane/EtOH+0.1% iPrNH2, 70:30; flow rate=
13 mLminꢀ1), was verified on an analytical column (Chiralpak AD-
H; DAD=210–340 nm; CD=230 nm; mobile phase: n-hexane/
EtOH+0.1% iPrNH2, 70:30; flow rate=0.8 mLminꢀ1
;
RT=
16.746 min). Enantiomeric excess (ee) was determined for the sepa-
rated enantiomers by means of SFC analytical techniques using a
Chiralpak AD-H column (25ꢂ0.46 cm) with EtOH (+0.1% iPrNH2)
15% as modifier, flow rate=2.5 mLminꢀ1, P=180 bar at 358C with
detection at 220 nm. ee=97%.
Experimental Section
Chemistry
9-(3,4-Dichlorophenyl)-2-oxa-6-azaspiro[3.5]nonane (3): 1H NMR
(400 MHz, CDCl3): d=7.48–7.46 (d, 1H, J=8.0 Hz), 7.37–7.36 (d, 1H,
J=2.0 Hz), 7.12–7.09 (dd, 1H, J=8.0, 2.0 Hz), 4.63–4.62 (dd, 1H, J=
6.4, 1.6 Hz), 4.46–4.44 (d, 1H, J=6.4 Hz), 4.38–4.36 (d, 1H, J=
6.0 Hz), 4.11–4.09 (d, 1H, J=6.0 Hz), 3.63–3.60 (d, 1H, J=12.8 Hz),
3.13–2.91 (m, 1H), 2.87–2.84 (m, 1H), 2.84–2.83 (m, 1H), 2.75–2.72
(m, 1H), 2.72–2.60 (m, 1H), 1.70–1.68 ppm (m, 2H); MS (ESI): m/z
(%): 273 (100) [M+H]+.
DAD chromatographic traces, mass chromatograms and mass spec-
trums were taken on a on a UPLC/MS Acquity system coupled with
a Micromass ZQ mass spectrometer operating in ESI positive or
negative. Mobile phases: A) H2O/MeCN (95:5) +0.1% TFA; B) H2O/
MeCN (5:95) +0.1% TFA. Gradient: t=0 min) 95% A, 5% B; t=
0.25 min) 95% A, 5% B; t=3.30 min) 100% B; t=4.0 min) 100% B;
followed by 1 min of reconditioning.
1
10-(3,4-Dichlorophenyl)-2-oxa-7-azaspiro[4.5]decane (1): H NMR
4-(3,4-Dichlorophenyl)octahydro-2H-pyrano[4,3-b]pyridine
(4):
1H NMR (500 MHz, CDCl3): d=7.40–7.35 (d, 1H, J=8.3 Hz), 7.26–
7.23 (d, 1H, J=2.0 Hz), 7.03–6.96 (dd, 1H, J=8.3, 2.0 Hz), 3.84–3.76
(m, 1H), 3.76–3.68 (m, 1H), 3.68–3.60 (m, 1H), 3.39–3.26 (m, 1H),
3.19–3.13 (m, 1H), 3.13–3.06 (dd, 1H, J=11.0, 4.6 Hz), 2.91–2.77 (m,
2H), 2.20–2.09 (m, 1H), 2.06–1.97 (m, 1H), 1.96–1.85 (m, 1H), 1.58–
1.50 (m, 1H), 1.49–1.38 (m, 1H); MS (ESI): m/z (%): 287 (100) [M+
H]+.
(500 MHz, CDCl3): d=7.41–7.37 (d, 1H, J=8.3 Hz), 7.31–7.28 (d, 1H,
J=2.0 Hz), 7.08–7.03 (dd, 1H, J=8.0, 2.0 Hz), 4.00–3.94 (d, 1H, J=
9.0 Hz), 3.77–3.70 (d, 1H, J=9.0 Hz), 3.70–3.60 (m, 1H), 3.27–3.20
(m, 1H), 3.22–3.15 (m, 1H), 3.11–3.05 (d, 1H, J=12.0 Hz), 2.81–2.70
(m, 2H), 2.69–2.63 (d, 1H, J=12.0 Hz), 1.92–1.78 (m, 2H), 1.74–1.67
(m, 1H), 1.52–1.42 ppm (m, 1H); Relative stereochemistry con-
firmed on the basis of nOe experiments; MS (ESI): m/z (%): 287
(100) [M+H]+.
11-(3,4-Dichlorophenyl)-2-oxa-8-azaspiro[5.5]undecane
(2):
EtOAc (3.1 mmol) was added to a solution of 6 (2.4 mmol) in ace-
tone (5 mL) containing K2CO3 (2.4 mmol). The reaction was stirred
overnight at RT. The solvent was removed in vacuo and compound
9 was purified by chromatography (silica cartridge, 10!30%
EtOAc/cyclohexane). Only one diastereoisomer was isolated (71%).
Compound 9 (2.114 mmol) was then treated with 1.0m solution of
Biology
The research described here was conducted in compliance with
company policy, and with national and European legislation on the
care and use of animals in scientific experiments, and related
codes of practice.
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SPA-binding assay: The affinity of the compounds towards the
human transporters has been assessed with radioligand displace-
ment binding of [3H]citalopram, [3H]nisoxetine and [3H]WIN-35,428
for SERT, NET, and DAT in BacMam-recombinant human SERT, NET
and DAT membranes with SPA technology. The SPA beads (product
number: RPNQ0260) were obtained from GE HealthCare (www.ge
healthcare.com). Briefly, 0.3 mL of test compound in DMSO were
added to 30 mL of the SPA mixture containing: 1 mgmLꢀ1 SPA
beads (SERT) or 2 mgmLꢀ1 SPA beads (NET and DAT); 6, 40 or
20 mgmLꢀ1 of SERT, NET or DAT BacMam membranes; 0.02% Plur-
onic F-127; 3 nm [3H]citalopram, 10 nm [3H]nisoxetine or 10 nm
[3H]WIN-35,428 for SERT/NET/DAT-binding SPA in the assay buffer
(20 mm HEPES, 145 mm NaCl, 5 mm KCl, pH 7.4). Incubation was
performed overnight at room temperature. Bound radioactivity
was measured with a ViewLuxꢃ instrument. Data analysis was per-
formed using a four-parameter logistic equation with ActivityBase
software (version 5.4; ID Business Solutions Ltd., UK), and pKi
values were calculated from the pIC50 values using the Cheng–
Prusoff equation.[11]
Acknowledgements
We would like to thank Dr. Daniele Donati, Dr. Stefano Fontana,
Dr. Simone Braggio, Dr. Sylvie Gehanne and Dr. Filippo Visentini
for their support in the preparation of the manuscript. We would
also like to thank Dr. Colin P. Leslie for careful proofreading of
the manuscript.
Keywords: dopamine · inhibitors · noradrenaline · serotonin
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initiated by the addition of substrate: 10 mL of 35 nm [3H]serotonin,
45 nm [3H]noradrenalin or 75 nm [3H]dopamine for SERT, NET or
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Received: November 23, 2009
Revised: January 7, 2010
Published online on January 28, 2010
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