Development of 3,4-dihydroisoquinolin-1(2H)-one derivatives for the Positron Emission Tomography (PET) imaging of σ2 receptors

Article abstract of DOI:10.1016/j.ejmech.2013.09.018

σ₂ Receptors are promising biomarkers for cancer diagnosis given the relationship between the proliferative status of tumors and their density. With the aim of contributing to the research of σ₂ receptor Positron Emission Tomography (PET) probes, we developed 2-[3-[6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-3, 4-dihydroisoquinolin-1(2H)-one (3), with optimal σ₂ pharmacological properties and appropriate lipophilicity. Hence, 3 served as the lead compound for the development of a series of dihydroisoquinolinones amenable to radiolabeling. Radiosynthesis for compound 26, which displayed the most appropriate σ₂ profile, was developed and σ₂ specific binding for the corresponding [¹⁸F]-26 was confirmed by in vitro autoradiography on rat brain slices. Despite the excellent in vitro properties, [¹⁸F]-26 could not successfully image σ₂ receptors in the rat brain in vivo, maybe because of its interaction with P-gp. Nevertheless, [¹⁸F]-26 may still be worthy of further investigation for the imaging of σ₂ receptors in peripheral tumors devoid of P-gp overexpression.

Full text of DOI:10.1016/j.ejmech.2013.09.018

European Journal of Medicinal Chemistry 69 (2013) 920e930
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of 3,4-dihydroisoquinolin-1(2H)-one derivatives for the
Positron Emission Tomography (PET) imaging of s2 receptors
,
b
b
b
Carmen Abate a , Svetlana V. Selivanova , Adrienne Müller , Stefanie D. Krämer ,
Roger Schibli b, Roberta Marottoli a, Roberto Perrone a, Francesco Berardi a, Mauro Niso a,
Simon M. Ametamey b
*
a Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy
b Center for Radiopharmaceutical Sciences, ETH Zurich, Zurich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
s2 Receptors are promising biomarkers for cancer diagnosis given the relationship between the prolif-
erative status of tumors and their density. With the aim of contributing to the research of s2 receptor
Received 16 May 2013
Received in revised form
6 September 2013
Positron
Emission Tomography
(PET)
probes,
we
developed
2-[3-[6,7-dimethoxy-3,4-
dihydroisoquinolin-2(1H)-yl]propyl]-3,4-dihydroisoquinolin-1(2H)-one (3), with optimal s2 pharmaco-
logical properties and appropriate lipophilicity. Hence, 3 served as the lead compound for the devel-
opment of a series of dihydroisoquinolinones amenable to radiolabeling. Radiosynthesis for compound
26, which displayed the most appropriate s2 profile, was developed and s2 specific binding for the
corresponding [18F]-26 was confirmed by in vitro autoradiography on rat brain slices. Despite the
excellent in vitro properties, [18F]-26 could not successfully image s2 receptors in the rat brain in vivo,
maybe because of its interaction with P-gp. Nevertheless, [18F]-26 may still be worthy of further inves-
tigation for the imaging of s2 receptors in peripheral tumors devoid of P-gp overexpression.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Accepted 7 September 2013
Available online 20 September 2013
Keywords:
s
receptors
s2 receptors PET probe
6,7-Dimethoxytetrahydroisoquinoline
Dihydroisoquinolinone
PET
1. Introduction
addiction, Parkinson’s and Alzheimer’s diseases so that s1 receptor
appears as an interesting therapeutic target [3].
Sigma receptors are a unique pharmacologically defined family
of proteins, which has been thus far divided into s1 and s2 sub-
types. The s1 receptors, which were purified and cloned in 1996 [1],
are the most well characterized. A chaperone function has been
assigned to this protein and increasing lines of evidence demon-
strate that s1 proteins are involved in the intracellular signaling
through the modulation of intracellular Ca2þ levels via inositol
1,4,5-trisphosphate (IP3) receptors [2]. A role in neuroprotection
and neuroplasticity has also been shown for this subtype which
seems to be involved in a series of Central Nervous System (CNS)
pathologies such as anxiety, depression, schizophrenia, drug
The s2 receptor gene has yet to be cloned. s2 receptor structure,
intracellular localization and pathways activated are still unclear.
Diverse attempts to isolate this receptor led to contradictory results
[4], and only recently s2 receptors have been proposed as the
progesterone receptor membrane component 1 (PGRMC1) protein
[5,6]. Nevertheless, interest in s2 receptors is on the increase
because of the important therapeutic potentials that are foreseen
for this subtype [7e9]. In fact, the s2 subtype in particular, is
overexpressed in several tumor cell lines and its activation with s2
agonists leads cancer cells to apoptotic pathways [10]. Diverse s2
agonists have been demonstrated to be powerful anticancer agents
in vitro as well as in tumor xenografts in vivo [11e13].
In addition, s2 subtype has been validated as an endogenous
biomarker for cancer diagnosis as a relationship between the pro-
liferative status of tumors and the density of s2 receptors has been
demonstrated [14]. A clinical study (NCT00968656A) has just been
completed with fluorine-18 radiolabeled (N-[4-(6,7-dimethoxy-
3,4-dihydro-1H-isoquinolin-2-yl)butyl]-2-(2-fluoroethoxy)-5-
iodo-3-methoxybenzamide (ISO-1) for the assessment of cellular
proliferation in tumors by Positron Emission Tomography (PET)
[15]. ISO-1 belongs to a series of flexible benzamides which were
Abbreviations: Calcein-AM, acetoxymethylester of calcein; BBB, bloodebrain
barrier; CNS, central nervous system; DMEM, Dulbecco’s modified eagle medium;
DTG, 1,3-di-o-tolyl-guanidine; IP3, inositol 1,4,5-trisphosphate; MDCK-MDR1,
Madin Darby canine kidney cells transfected with the human MDR1 gene; PET,
Positron Emission Tomography; P-gp, P-glycoprotein; Papp, apparent permeability;
PGRMC1, progesterone receptor membrane component 1.
* Corresponding author. Tel.: þ39 080 5442750; fax: þ39 080 5442231.
E-mail address: carmen.abate@uniba.it (C. Abate).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.09.018

C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
921
OCH3
N
OCH3 O
N
N
N
H
OCH3
OCH3
1, RHM-1
2, PB28
O
OCH3
OCH3
N
N
3
Fig. 1. s2 receptors reference compounds.
generated using the s2 receptor high-affinity ligand 5-methyl-2-
methoxy-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)
butyl]benzamide (1, RHM-1; Fig. 1) as the lead compound. A few
high s2 affinity and selectivity radioligands belonging to this series
provided a clear image of diverse solid tumors with optimal tumor
uptake which depended upon the log P values of the radioligands
[16,17].
With the aim of contributing to the research of s2 receptor PET
probes, we developed a series of compounds as hybrids of one of the
highest affinity s2 ligands (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-
10e15 were obtained from the corresponding indanones except for
intermediate 3,4-dihydro-6-methoxy-isoquinolin-1(2H)-one 16
(Scheme 1) which was obtained, by cyclization of the ethyl-3-
methoxyphenethylcarbamate as previously reported [21].
7-Methoxy-indan-1-one 4 was obtained starting from 3-(2-bromo-
5-methoxyphenyl)propanoic acid [22]. Cyclization of this latter com-
pound in polyphosphoric acid provided 4-Br-5-methoxy-indan-1-one,
and its debromination led to indanone 4 with a little change of the
procedure already reported [23]. Indanon-1-ones 8 and 9 were ob-
tained by alkylation of the 5-hydroxy-indan-1-one 5 with CH3I or with
2-fluoroethyl-4-methylbenzenesulfonate [24] respectively, although
the former compound was commercially available. Indan-1-ones 4e9
treated with NaN3 in HCl or in Cl3CCOOH led to key 3,4-dihydro-iso-
quinolinones 10e15 [25]. 5-Hydroxy derivative 10, obtained by the
corresponding indanone 5 according to a previously reported proce-
dure [25], was benzylated by the use of benzylbromide and K2CO3 to
afford the already known 5-benzyl-3,4-dihydro-isoquinolinone 17
[26], through a different procedure. Alkylation of 3,4-dihydro-iso-
quinolinones 11e17 with 1-Bromo-3-chloropropane using NaH as a
base afforded 2-(3-chloropropyl)-3,4-dihydroisoquinolin-1(2H)-one
derivatives (18e24) whose reaction with 6,7-dimethoxy-3,4-dihydro-
1H-isoquinoline led to final compounds 25, 26, 28e31 and 5-benzyl
intermediate 27 (Scheme 1).
Debenzylation of this last compound in the presence of H2 and
Pd on activated carbon 10% as the catalyst furnished intermediate
32 which was subsequently reacted with 2-bromoethylacetate to
afford intermediate 33. Hydrolysis of the acetyl group in basic
medium led to the 2-ethoxy-ethanol intermediate 34. Reaction of
this last compound with p-toluensulfonyl chloride gave the pre-
cursor 35 which was then used for the radiolabeling to afford [18F]-
26. All of the final amine compounds were converted into their
hydrochloride salts with gaseous HCl, in anhydrous diethyl ether
except for compound 29 which could not be recrystallized and
therefore it was analyzed as free base. Physical properties of the
hydrochloride salts are listed in the Table of Physical Properties of
Novel Compounds in the Supporting information.
tetrahydronaphthalen-1-yl)-n-propyl]piperazine
[18,19], and one of the best flexible benzamides (1, Fig. 1) [20].
Dihydroisoquinolinone derivative 2-[3-[6,7-dimethoxy-3,4-
2 PB28; Fig. 1)
dihydroisoquinolin-2(1H)-yl]propyl]-3,4-dihydroisoquinolin-1(2H)-
one (3, Fig. 1) displayed an optimal s2 pharmacological profile
together with an appropriate lipophilicity value. In addition (and in
contrast to flexible benzamides), a rather weak interaction of 3 with
the P-glycoprotein (P-gp) [21], suggested that this class of com-
pounds might be well suitable to selectively visualize s2 receptors in
P-gp-overexpressing tumors. Therefore, 3,4-dihydroisoquinolin-
1(2H)-one 3 served as a lead compound for the development of a
series of dihydroisoquinolinone derivatives whose functionalization
with methoxy, fluoroethoxy or fluorine could easily lead to the
development of corresponding carbon-11 or fluorine-18 radiolabeled
ligands for PET imaging. Radiosynthesis for compound [18F]-26,
which displayed the best s2 pharmacological profile was developed
and prior to using s2-positive tumor-expressing animals, a pre-
liminary study involving in vitro autoradiography on rat brain slices
and biodistribution in rats were performed to evaluate the specificity
of [18F]-26 binding to s2 receptors.
2. Chemistry
The synthesis of final compounds herein reported is depicted in
Scheme 1. The synthetic pathway leading to [18F]-26 is depicted in
Schemes 2 and 3. All the substituted 3,4-dihydro-isoquinolinones

922
C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
Scheme 1. Reagents and conditions: a) Polyphosphoric acid, 60 ꢀC, 1 h (synthesis of intermediate 4); b) H2, Pd/C 10%, NaOAc, EtOH, RT, 4 h (synthesis of intermediate 4); c) MeI or 2-
Fluoroethyl-4-methylbenzenesulfonate, K2CO3, Acetone, reflux, 6 h or 36 h respectively; d) NaN3, HCl 37%, RT, 12 h; e) Benzylbromide, K2CO3, Acetone, reflux, 18 h; f) 1-Bromo-3-
chloropropane, NaH, DMF, RT, 30 min (Procedure A); 1-Bromo-3-chloropropane, NaH, THF, reflux, 18 h (Procedure B); g) 6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinoline hydro-
chloride, K2CO3, reflux, 18 h.
3. Results and discussion
appropriate in conferring remarkable s2 receptor affinity and
selectivity. The presence of either an electron-donating (methoxy)
or withdrawing substituent (fluorine) did not alter the affinity at
the s2 receptor compared to the un-substituted lead-compound 3
as long as the group was introduced in 5- or 6-position of the
benzene ring (compound 25, 28 and 31, s2 Ki values ¼ 4.24 nM,
6.16 nM and 3.56 nM respectively). The presence of the methoxy
group in 7- or 8- position (30 and 29) of the 3,4-
dihydroisoquinolinone system led to a slight reduction (around 4-
fold) of the affinity at the s2 receptor. The presence of both the
electron-donating (methoxy) and electron-withdrawing (fluorine)
substituents was only explored for the 6-position of the benzene
ring, where no electronic effect was detected compared to the
unsubstituted 3. 5-Methoxy-substituted 25 emerged as the most
s2-selective ligand (574-fold) with a selectivity 4-fold higher
compared to the lead compound 3. Therefore, we identified the 5-
methoxy-substitution of the 3,4-dihydroisoquionolinone system as
a key feature to preserve high s2 selectivity. Introduction of 11C into
the 5-methoxy group would be one of the possible routes for the
radiolabeling. However, the half-life of the 11C radioisotope
(20 min), prompted us to develop the corresponding 2-
fluoroethoxy derivative 26 which displayed a lower but yet
3.1. s1 and s2 receptor affinities
Results from binding assays are expressed as inhibition con-
stants (Ki values) in Table 1. The Ki values at the s1 subtype for the
present series of compounds are in the micromolar range generally
presenting a 3- to 4-fold reduction in the affinity compared to the
unsubstituted isoquinolinone derivative 3 (Ki ¼ 709 nM). Excep-
tions to this were 6-substituted compounds: 6-methoxy- and 6-
fluoro- derivatives 28 and 31 exhibited a 2-fold improvement in
the affinity (Ki values ¼ 402 nM and 311 nM respectively),
compared to 3. These results suggested that independently of their
electronic effects, substituents at the isoquinolinone 6-position are
less detrimental for the interaction with the s1 receptor. On the
other hand, the presence of an electron-donating methoxy group in
5- or 7- or 8- position of the isoquinolinone benzene ring exerted a
similar decrease in the s1 receptor binding (compounds 25, 26, 29
and 30). The Ki values at the s2 subtype for the newly synthesized
compounds ranged from 3.56 nM to 20.1 nM (compounds 31 and
30 respectively) confirming the template of the 6,7-
dimethoxytetrahydroisoquinoline linked to benzamides as

C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
923
Scheme 2. Reagents and conditions: a) H2 10 atm, Pd/C 10%, EtOH, RT, 12 h; b) 2-Bromoethylacetate, K2CO3, Acetone, reflux, 24 h; c) NaOH, MeOH/H2O, RT, 18 h; d) TsCl, NEt3,
CH2Cl2, RT, 1 h.
O
O
N
N
N
N
a
O
O
OCH3
OCH3
OCH3
OCH3
O
18F
O2S
18F]-26
35
[
Scheme 3. Radiolabeling with 18F. Reagents and conditions: a) [18F]-KF, K2CO3, CH3CN, 100 ꢀC, 10 min.

924
C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
Table 1
Since the results from Calcein-AM experiment and from the
Binding data of novel compounds at
s receptors.
apparent permeability (Papp) determination suggested could not
allow to undoubtedly predict whether the compound may over-
come BBB to bind s2 receptors, we developed the corresponding
[
18F]-26 to test its suitability as PET radiotracer, given the encour-
aging s2-binding profile of the compound.
3.3. Radiosynthesis
One step radiolabeling with 18F was achieved via aliphatic
nucleophilic substitution of tosyl leaving group in the precursor
molecule (35). The reaction between precursor (2.4 mg, 4 mmol)
Compd
R
Ki ꢁ SEM (nM)a
and dry 18F-fluoride was accomplished in anhydrous acetonitrile at
100 ꢀC for 10 min. The product was purified by semi-preparative
HPLC and formulated for biological experiments. Decay corrected
radiochemical yield was 34.3 ꢁ 1.5% and the total synthesis time
was w90 min. Radiochemical purity exceeded 98% and specific
s1
s2
s1//s2
3b
H
709 ꢁ 133
2435 ꢁ 995
3095 ꢁ 750
402 ꢁ 7
4.74 ꢁ 0.74
4.24 ꢁ 0.84
9.24 ꢁ 2.55
5.74 ꢁ 0.42
16.3 ꢁ 0.8
20.1 ꢁ 2.5
3.56 ꢁ 0.4
149
574
330
70
101
127
87
25
26
28
29
30
31
5-OCH3
5-OCH2CH2F
6-OCH3
8-OCH3
6,7-OCH3
6-F
activity was in a range of 75e88 GBq/mmol. Typically, w8 GBq of the
1628 ꢁ 61
2567 ꢁ 162
311 ꢁ 8
formulated ready for in vivo application product could be produced
starting with w55 GBq 18F-fluoride. No attempts were made to
optimize the reaction yield since produced amount of [18F]-26 was
sufficient for our studies.
(þ)-Pentazocine
3.0 ꢁ 0.21
DTG
25.7 ꢁ 1.41
a
Values are the means of n ꢂ 3 separate experiments, in duplicate.
b
From Ref. [20].
3.4. In vitro autoradiography
appreciable s2 receptor affinity (Ki ¼ 9.24 nM) compared to 25, and
a high s2-selectivity (330-fold). This promising result encouraged
us to further study this compound as PET radiotracer. For this
purpose, we extended the binding of compound 26 to more re-
ceptors, as well as interaction at the P-gp was studied, so that
possible and misleading interaction with other targets might be
predicted.
To evaluate binding of [18F]-26 to s2 receptors in vitro, we per-
formed autoradiography using rat brain slices. [18F]-26 showed
heterogeneous accumulation clearly delineating various brain re-
gions (Fig. 2). Maximal binding was detected in cerebral cortex and
hippocampus. High radioactivity accumulation was also observed
in cerebellum. Overall, the binding pattern was consistent with
binding of a known s2 radioligand, [3H]-Lu28-179 [27]. Binding of
[
[
18F]-26 was blocked when rat brain slices were incubated with
3.2. Compound 26: affinity at a1, 5HT7 and 5HT1a receptors, activity
18F]-26 in the presence of 90
m
M haloperidol. The binding was also
at P-gp and apparent permeability (Papp
)
diminished in a dose-dependent manner by co-incubation with
non-radioactive analog 26: at 0.9
partially, while blocking with 90
m
M, binding was disrupted only
Binding of 26 towards other CNS receptors such as a1, 5HT7 and
5HT1a was studied, and results are expressed as Ki values in Table 2.
Affinity at both the serotonin receptors was low (Ki
mM of 26 had a similar effect as
blocking with 90 mM haloperidol. Our data thus confirm the spec-
ificity of [18F]-26 binding to s2 receptors in rat brain in vitro.
values > 1000 nM), as well as at the
a-adrenergic receptor
(Ki ¼ 764 nM), supporting the s2-selectivity of this compound. Also,
interaction with P-gp was measured through the Calcein-AM
experiment in the P-gp overexpressing cell line MDCK-MDR1.
3.5. PET imaging in the rat brain
PET imaging showed a fast washout of [18F]-26 from rat brain.
Virtually, no radioactivity accumulation was visible during 0e
90 min scan after tracer injection (Fig. 3). Derived timeeactivity
curves (TACs) showed elevated radioactivity levels in cerebral cor-
tex, olfactory, and cerebellum, which is in agreement with the
autoradiography results. However, radioactivity levels in hippo-
campus were not higher than in the remaining brain regions, in
contrast to the autoradiography data. A PET scan under blocking
conditions with 1 mg/kg haloperidol revealed similar TACs (data
not shown). The increased radioactivity in cortex, olfactory and
cerebellum under baseline and blocking conditions may result from
spill over from the periphery rather than s2 receptor-related
accumulation. The TACs may represent blood radioactivity,
without any transfer of [18F]-26 across the BBB, despite its relatively
high estimated lipophilicity (clogP 3.5 [28]). As data standing, the
ambiguous interaction with P-gp that we evaluated through
Calcein-AM and Papp experiments, resulted in the absence of BBB
passage, suggesting that 26 may be a P-gp substrate, strongly
effluxed out of the CNS. Another speculation on possible reasons for
the discrepancy between the in vitro and in vivo data and for the
low brain SUV in general, is that [18F]-26 could undergo rapid
biotransformation to radiometabolite(s) with low BBB permeation.
The slight steady but unspecific increase in brain radioactivity could
Compound 26 displayed
(EC50 ¼ 5 M, Table 2), but stronger than the interaction of the lead
compound 3 (EC50 ¼ 12 M). The Apparent Permeability (Papp) in
a moderate interaction with P-gp
m
m
Caco-2 cell monolayer was determined in order to predict the Blood
Brain Barrier (BBB) permeation of 26, but the result obtained was
borderline. The flux of the compound from basolateral to apical
(BA) represents the passive transport, whereas the flux from apical
to basolateral (BA) represents the active transport, and in agree-
ment to the current classification, compound is a P-gp inhibitor for
BA/AB < 2; compound is a P-gp substrate for BA/AB > 2. Compound
26 displayed BA/AB ¼ 2.58 (Table 2).
Table 2
Compound 26: binding data at 5HT1a, 5HT7 and a1 receptors, P-gp activity and
apparent permeability (Papp).
Compd
Ki ꢁ SEM (nM)a
EC50
P-gp
(m
M)a (BA/AB)a
5HT1a
5HT7
a1
Papp
26
5-HT
5-CT
Phentolamine
MC18
>1000
9.5 ꢁ 1.1
>1000
764 ꢁ 98
5.0 ꢁ 0.8
2.58
0.4 ꢁ 0.05
15.2 ꢁ 1.2
1.1 ꢁ 0.2
a
Values are the means of n ꢂ 3 separate experiments, in duplicate.

C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
925
Fig. 2. In vitro autoradiography using rat brain slices under baseline (A) and blocking (BeD) conditions. A: 9 nM [18F]-26 solution; B: co-incubation of 9 nM [18F]-26 and 0.9
mM
unlabeled 26; C: co-incubation of 9 nM [18F]-26 and 90 M unlabeled 26; D: co-incubation of 9 nM [18F]-26 plus 90
m mM haloperidol.
Fig. 3. Time series of a rat’s head PET scan (left) and timeeactivity curves (right) from 0 to 90 min after injection of [18F]-26.
result from radiometabolite(s). In many cases, defluorination of PET
tracers precludes successful imaging. In this study, we did not
observe any appreciable radioactivity accumulation in the bones,
which confirmed that no free 18F-fluoride was released by [18F]-26.
Extensive binding at the blood macromolecule, that is another
possible reason for low BBB permeation, was excluded since the
TRANSILXL assay predicted only a low fraction (84%) of 26 bound to
plasma proteins (data not shown) [29].
1:15 w/w, 15e40 mm particle size, from Merck respectively).
Melting points were determined in open capillaries on a Gallen-
kamp electrothermal apparatus. Purity of tested compounds was
established by combustion analysis, confirming a purity ꢂ 95%.
Elemental analyses (C, H, N) were performed on an Eurovector Euro
EA 3000 analyzer; the analytical results were within ꢁ0.4% of the
theoretical values. 1H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were recorded on a Mercury Varian: CDCl3 was used as
solvent to record 1H NMR on intermediate and final compounds as
4. Conclusion
free basis and the following data were reported: chemical shift (d)
in ppm, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
m ¼ multiplet), integration and coupling constant(s) in Hertz.
CD3OD was used as solvent to record 13C NMR on hydrochloride
We synthesized a series of dihydroisoquinolinone derivatives
and evaluated their binding properties to
s receptors. Selected
compound, 26, showed high affinity and selectivity towards s2, but
ambiguous interaction with the efflux pump P-gp. After radio-
labeling with 18F, 26 was evaluated as a potential PET tracer. Despite
encouraging results from in vitro autoradiography, [18F]-26 was not
successful in imaging s2 receptors in the rat brain in vivo, maybe
because its BBB passage is hampered by interaction with P-gp,
although other reasons may not be excluded. Nevertheless, the
tracer may still be worthy of further investigation for the imaging of
s2 receptors in peripheral tumors devoid of P-gp overexpression.
salts of final compounds where reported and chemical shift (d) in
ppm were reported. Recording of mass spectra was done on an
Agilent 6890e5973 MSD gas chromatograph/mass spectrometer
and on an Agilent 1100 series LC-MSD trap system VL mass spec-
trometer; only significant m/z peaks, with their percentage of
relative intensity in parentheses, are reported. Infrared spectra
were recorded on a PerkineElmer, Spectrum one, FTIR. Chemicals
were from Aldrich and Acros, and were used without any further
purification. Radiolabeling reactions were monitored by ultra-
performance liquid chromatography (UPLC). The system was Acq-
uity UPLC from Waters, equipped with a built-in PDA detector and
an additional FlowStar LB 513 radiodetector (Berthold Technolo-
gies) and a reversed-phase Acquity BEH C18 column (particle size
5. Chemistry: materials and methods
5.1. Chemistry
1.7
m
m, 100 ꢃ 2.1 mm). The mobile phase consisted of a gradient of
Both column chromatography and flash column chromatog-
acetonitrile in aqueous 50 mM NH4COOH buffer (pH 4.45) from 5 to
65% over 2 min at a flow rate 0.7 mL/min. Semi-preparative HPLC
for radiolabeled product purification was carried out on an HPLC
ꢀ
raphy were performed with 60 A pore size silica gel as the sta-
tionary phase (1:30 w/w, 63e200 mm particle size, from ICN and

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C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
system equipped with a Merck-Hitachi L-6200A Intelligent pump, a
5 mL injection loop, a Knauer Variable Wavelength Monitor UV-
detector, and an Eberline RM-14 radiodetector on a reversed-
5.2.2.1. 2-(3-Chloropropyl)-6-methoxy-3,4-dihydroisoquinolin-
1(2H)-one (21). Compound 21 was obtained as yellow oil (0.12 g,
39%); 1H NMR
d
¼ 2.09e2.18 (m, 2H, CH2CH2Cl), 2.97 (t, 2H,
ꢀ
phase Waters
mBondapack C18, 10
m
m, 125 A, 300 ꢃ 7.8 mm col-
J ¼ 6.5 Hz, ArCH2), 3.57e3.70 (m, 6H, 2 CH2N, and CH2Cl), 3.84 (s,
3H, OCH3), 6.66 (d,1H, J ¼ 2.7 Hz, aromatic), 6.84 (dd,1H, J’ ¼ 8.8 Hz,
J00 ¼ 2.7 Hz, aromatic), 8.00 (d, 1H, J ¼ 8.8 Hz, aromatic); GCeMS m/z
253 (Mþ, 10), 218 (100), 190 (50).
umn. The mobile phase consisted of acetonitrile and 50 mM
NH4COOH buffer (pH 4.45). The gradient was: 0e10 min, isocratic
5% acetonitrile; 10e40 min, 5% / 65% acetonitrile and eluted at
4 mL/min flow rate. UV-absorbance was detected at 235 nm.
Analytical HPLC was an Agilent 1100 series system equipped with a
Raytest Gabi Star radiodetector. Analytical reversed-phase column
5.2.2.2. 2-(3-Chloropropyl)-8-methoxy-3,4-dihydroisoquinolin-
1(2H)-one (22). Compound 22 was obtained as yellow oil (0.09 g,
was Agilent Eclipse XDB-C18, particle size 5
m
m, 50 ꢃ 4.6 mm. A
30%); 1H NMR
d
¼ 2.10e2.19 (m, 2H, CH2CH2Cl), 2.92 (t, 2H,
gradient of acetonitrile in aqueous 50 mM NH4COOH buffer (pH
4.45) from 10% to 65% over 13 min at a flow rate of 1 mL/min was
used.
J ¼ 6.0 Hz, ArCH2), 3.53 (t, 2H, J ¼ 6.3 Hz, CH2Cl), 3.61e3.70 (m, 4H, 2
CH2N), 3.91 (s, 3H, OCH3), 6.76 (d, 1H, J ¼ 7.4 Hz, aromatic), 6.88 (d,
1H, J ¼ 8.2 Hz, aromatic), 7.33 (t, 1H, J ¼ 8.0 Hz, aromatic); GCeMS
m/z 253 (Mþ, 78), 218 (100), 204 (44), 190 (58).
5.2. General procedures for the synthesis of alkyl chloride
derivatives
5.2.2.3. 2-(3-Chloropropyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-
1(2H)-one (23). Compound 23 was obtained as yellow semi-solid
(0.16 g, 46%); mp ¼ 94e96 ꢀC (Litt. mp ¼ 92e95ꢀ C) [30]; 1H
5.2.1. General procedure A
To a suspension of NaH (0.11 g, 4.5 mmol) in DMF (10 mL), a
solution in DMF (5 mL) of the appropriate 3,4-dihydroisoquinolin-
1-one (1.8 mmol), was added in a dropwise manner, at 0 ꢀC under a
stream of N2. After 15 min, 1-bromo-3-chloropropane was added in
a dropwise manner and the mixture was allowed to warm to room
temperature and was kept under stirring for 30 min. After cooling
to 0 ꢀC H2O was added and the solvent was removed under reduced
pressure. The residue was taken up with water and extracted with
AcOEt (3 ꢃ 10 mL). The organic layers were collected, dried over
Na2SO4 and evaporated under reduced pressure. The crude residue
was purified by column chromatography with CH2Cl2/AcOEt (1:1)
as eluent.
NMR
d
¼ 2.11e2.15 (m, 2H, CH2CH2Cl), 2.93 (t, 2H, J ¼ 6.3 Hz,
ArCH2), 3.48e3.70 (m, 6H, 2 CH2N, and CH2Cl), 3.91 (s, 6H, OCH3),
6.63 (s, 1H, aromatic), 7.57 (s, 1H, aromatic); GCeMS m/z 283 (Mþ,
36), 248 (100), 220 (54).
5.2.2.4. 2-(3-Chloropropyl)-6-fluoro-3,4-dihydroisoquinolin-1(2H)-
one (24). Compound 24 was obtained as colorless oil (0.11 g, 38%);
1H NMR
d
¼ 2.09e2.18 (m, 2H, CH2CH2Cl), 3.00 (t, 2H, J ¼ 6.6 Hz,
ArCH2), 3.59e3.69 (m, 6H, 2 CH2N, and CH2Cl), 6.87 (dd, 1H,
JHF ¼ 8.8 Hz, JHH ¼ 3 Hz, aromatic), 7.02 (td, 1H, JHF ¼ 8 Hz,
JHH ¼ 8.7 Hz, JHH ¼ 3 Hz, aromatic), 8.07 (dd, 1H, JHH ¼ 8.7 Hz,
JHF ¼ 5.8 Hz, aromatic); GCeMS m/z 241 (Mþ, 10), 206 (98),178 (100).
5.2.1.1. 2-(3-Chloropropyl)-5-methoxy-3,4-dihydroisoquinolin-
5.3. General procedure for the synthesis of final compounds 25, 26,
28e31 and intermediate 27
1(2H)-one (18). Compound 18 was obtained as yellow oil (0.26 g,
58%); 1H NMR
d
¼ 2.10e2.19 (m, 2H, CH2CH2Cl), 2.97 (t, 2H,
J ¼ 6.6 Hz, ArCH2), 3.46e3.75 (m, 6H, 2 CH2N, and CH2Cl), 3.85 (s,
3H, OCH3), 6.98 (d, 1H, J ¼ 8 Hz, aromatic), 7.26e7.32 (m, 1H, aro-
matic), 7.68 (d, 1H, J ¼ 7.7 Hz, aromatic); LCeMS (ESIþ) m/z: 276
[M þ H]þ; LCeMSeMS 276; 218.
To a solution of the appropriate alkyl chloride derivative
(1.0 mmol) in CH3CN, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline
hydrochloride (0.23 g, 1.0 mmol) and K2CO3 (0.21 g, 1.5 mmol)
were added. The mixture was heated to reflux overnight. After
cooling to room temperature, the solvent was evaporated to dryness.
The residue was treated with H2O and extracted with CH2Cl2
(3 ꢃ 10 mL) and the collected organic layers, were dried over Na2SO4
and concentrated under reduced pressure. The crude residue was
purified by column chromatography with CH2Cl2/MeOH (98:2) as
eluent.
5 . 2 .1. 2 . 2 - ( 3 - C h l o r o p r o p yl ) - 5 - ( 2 - fl u o r o e t h o x y ) - 3 , 4 -
dihydroisoquinolin-1(2H)-one (19). Compound 19 was obtained as
1
yellow oil (0.25 g, 55%); H NMR
d
¼ 2.10e2.19 (m, 2H, CH2CH2Cl),
3.02 (t, 2H, J ¼ 6.6 Hz, ArCH2), 3.47e3.71 (m, 6H, 2 CH2N, and
CH2Cl), 4.18e4.38 (m, 2H, CH2O), 4.65e4.86 (m, 2H, CH2F), 6.97 (d,
1H, J ¼ 8.2 Hz, aromatic), 7.26e7.31 (m, 1H, aromatic), 7.72 (d, 1H,
J ¼ 7.8 Hz, aromatic); GCeMS m/z 285 (Mþ, 12), 250 (100), 222
(57).
5.3.1. 2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-methoxy-3,4-dihydroisoquinolin-1(2H)-one (25)
Compound 25 was obtained as yellow oil (0.11 g, 28%); 1H NMR
5.2.1.3. 2-(3-Chloropropyl)-5-benzyloxy-3,4-dihydroisoquinolin-
1(2H)-one (20). Compound 20 was obtained as yellow oil (0.34 g,
58%); GCeMS m/z 329 (Mþ, 12), 91 (100); LCeMS (ESIþ) m/z 330
[M þ H]þ; m/z 352 [M þ Na]þ.
d
¼ 1.85e2.05 (m, 2H, CH2CH2CH2), 2.50e3.00 (m, 8H, ArCH2
-
CH2NCO, CH2NCH2CH2Ar), 3.55e3.70 (m, 6H, NCH2Ar, NCO(CH2)2),
3.80 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 6.51 (s, 1H,
aromatic), 6.58 (s, 1H, aromatic), 7.00 (d, 1H, J ¼ 8.2 Hz, aromatic),
7.22e7.31 (m, 1H, aromatic), 7.70 (d, 1H, J ¼ 8.0 Hz, aromatic); LCe
5.2.2. General procedure B
MS (ESIþ) m/z: 411 [M
þ
H]þ, 433 [M
þ
Na]þ. Anal.
To a solution of the appropriate 3,4-dihydroisoquinolin-1-one
(1.2 mmol) in THF, NaH (0.07 g, 3 mmol) was added at 0 ꢀC under
a stream of N2. After 10 min, the mixture was allowed to warm to
room temperature and 1-bromo-3-chloropropane was added in a
dropwise manner. The reaction mixture was heated to reflux
overnight. After cooling H2O was added and the mixture was
extracted with Et2O (3 ꢃ 5 mL), the organic layers were collected,
dried over Na2SO4 and evaporated under reduced pressure. The
crude residue was purified by column chromatography with
CH2Cl2/AcOEt (9:1) as eluent.
(C24H30N2O4$HCl$H2O) C, H, N.
5.3.2. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-(2-fluoroethoxy)-3,4-dihydroisoquinolin-1(2H)-one (26)
Compound 26 was obtained as yellow oil (0.11 g, 25%); 1H NMR
d
¼ 1.90e2.00 (m, 2H, CH2CH2CH2), 2.59 (t, 2H, J ¼ 7.5 Hz,
CH2CH2CH2N), 2.70e3.05 (m, 6H, NCH2CH2Ar and ArCH2CH2NCO),
3.54e3.66 (m, 6H, NCH2Ar, NCO(CH2)2), 3.82 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 4.19 (t, 1H, J ¼ 4.2 Hz, CHHO), 4.28 (t, 1H, J ¼ 4.0 Hz,
CHHO), 4.67e4.70 (m, 1H, CHHF), 4.83e4.86 (m, 1H, CHHF), 6.51 (s,

C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
927
1H, aromatic), 6.58 (s, 1H, aromatic), 6.95 (d, 1H, J ¼ 8 Hz, aromatic),
7.26e7.30 (m, 1H, aromatic), 7.73 (d, 1H, J ¼ 8 Hz, aromatic); 13C
NMR: 20.90; 22.58; 24.74; 44.20; 45.89; 50.11; 52.51; 53.27; 55.15;
55.21; 68.00 (d, 2J C,F ¼ 20 Hz), 81.50 (d, 1J C,F ¼ 168 Hz); 109.51;
111.35; 115.24; 119.37; 119.54; 119.91; 123.04; 127.31; 127.71;
129.52; 148.60; 149.43; 154.79; 166.07. LC-eMS (ESIþ) m/z 443
3.56e3.66 (m, 6H, NCH2Ar, NCO(CH2)2), 3.82 (s, 3H, OCH3), 3.83 (s,
3H, OCH3), 6.58 (s, 1H, aromatic), 6.63 (s, 1H, aromatic), 6.86 (dd,
1H, JHF ¼ 8.8 Hz, JHH ¼ 2.2 Hz, aromatic), 7.00 (td, 1H, JHF ¼ 8.0 Hz,
JHH ¼ 8.7 Hz, JHH ¼ 2.5 Hz, aromatic), 8.00e8.10 (m, 1H, aromatic);
GCeMS m/z 398 (Mþ, 2),192 (100); Anal. (C23H27FN2O3$HCl$H2O) C,
H, N.
[M
þ
H]þ;
LCeMSeMS
443:
250,
222;
Anal.
(C25H31FN2O4$HCl$1.5H2O) C, H, N.
5.3.8. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-hydroxy-3,4-dihydroisoquinolin-1(2H)-one (32)
5.3.3. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
A solution of benzyl derivative 27 (0.4 mmol, 0.19 g) in CH3OH
(10 mL) was added with Pd on 10% activated carbon and hydro-
genated for 6 h. After filtration of the catalyst on Celite pad and the
filtrate was concentrated under reduced pressure to afford the
target compound as yellow oil (0.16 g, 100%); LCeMS (ESIꢄ) m/z:
395 [M ꢄ H]ꢄ; IR cmꢄ1: 3182, 3055, 2936, 1667, 1644.
propyl)-5-benzyloxy-3,4-dihydroisoquinolin-1(2H)-one (27)
Compound 27 was obtained as brown oil (0.19 g, 40%); 1H NMR
d
¼ 1.90e2.05 (m, 2H, CH2CH2CH2N), 2.57e3.05 (m, 8H, ArCH2
-
CH2NCOCH2CH2CH2NCH2CH2Ar), 3.48e3.70 (m, 6H, CON(CH2)2,
ArCH2N), 3.82 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 5.05 (s, 2H,
PhCH2O), 6.48 (s, 1H, aromatic), 6.58 (s, 1H, aromatic), 7.02 (d, 1H,
J ¼ 8.0 Hz, aromatic), 7.30e7.45 (m, 6H, aromatic), 7.72 (d, 1H,
J ¼ 7.7 Hz, aromatic); GC/MS m/z 486 (Mþ., 0.5), 192 (100); LCeMS
(ESIþ) m/z: 487 [M þ H]þ, 509 [M þ Na]þ; LCeMSeMS: 403.
5.3.9. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-(2-acetoxy-ethoxy)-3,4-dihydroisoquinolin-1(2H)-one
(33)
A solution of 32 (0.54 mmol, 0.22 g) in acetone (30 mL) (which
was warmed to improve the solubility of the compound) was added
with 2-bromoethylacetate (3.56 mmol, 0.4 mL) and K2CO3
(3.56 mol, 0.50 g) and refluxed overnight. The mixture was evap-
orated under reduced pressure and the residue taken up with H2O
and extracted with ethyl acetate (3 ꢃ 20 mL). The collected organic
layers were dried over Na2SO4 and evaporated under reduced
pressure. The crude residue was purified by column chromatog-
raphy with CH2Cl2/CH3OH (95:5) as eluent to give the target
compound as yellow oil (0.12 g, 46%); LCeMS (ESIþ) m/z: 483
[M þ H]þ; 505 [M þ Na]þ.
5.3.4. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-6-methoxy-3,4-dihydroisoquinolin-1(2H)-one (28)
Compound 28 was obtained as colorless oil (0.12 g, 29%); 1H
NMR
d
¼ 1.76e1.98 (m, 2H, CH2CH2CH2), 2.58e3.00 (m, 8H,
ArCH2CH2NCO, CH2NCH2CH2Ar), 3.55e3.65 (m, 6H, NCH2Ar,
NCO(CH2)2), 3.82 (s, 3H, OCH3), 3.83 (s, 6H, 2 OCH3), 6.51 (s, 1H,
aromatic), 6.58 (s, 1H, aromatic), 6.65 (d, 1H, J ¼ 2.2 Hz, aromatic),
6.84 (dd, 1H, J00 ¼ 8.8 Hz, J0 ¼ 2.5 Hz, aromatic), 8.01 (d, 1H,
J ¼ 8.8 Hz, aromatic); 13C NMR: 22.76; 24.95; 27.89; 44.11; 46.46;
50.23; 52.63; 53.37; 54.82; 55.26; 55.31; 109.55; 111.43; 111.92;
112.70; 119.54; 121.02; 123.19; 129.77; 141.33; 148.70; 149.51;
163.37; 166.48. GC/MS m/z 410 (Mþ., 1.2), 192 (100); LCeMS (ESIþ)
m/z: 411 [M þ H]þ, 433 [M þ Na]þ. Anal. (C24H30N2O4$HCl$H2O) C,
H, N.
5.3.10. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-(2-hydroxy-ethoxy)-3,4-dihydroisoquinolin-1(2H)-one
(34)
NaOH (0.52 mmol, 0.02 g) was added to a solution of interme-
diate 33 (0.26 mmol, 0.12 g) in CH3OH (12 mL) and H2O (6.0 mL) and
the mixture was stirred overnight. HCl 2 N was added to neutralize
the solution and the solvent was evaporated under reduced pres-
sure to afford a residue that was taken up with water and then
extracted with ethyl acetate (3 ꢃ 10 mL). The collected organic
layers were dried over Na2SO4 and evaporated under reduced
pressure. The crude residue was purified by column chromatog-
raphy with CH2Cl2/CH3OH (9:1) as eluent to give the target com-
pound as yellow oil (0.08 g, 70%); LCeMS (ESIþ) m/z: 441 [M þ H]þ;
463 [M þ Na]þ, LCeMSeMS 441: 248.
5.3.5. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-8-methoxy-3,4-dihydroisoquinolin-1(2H)-one (29)
Compound 29 was obtained as yellow oil (0.041 g, 10%); 1H NMR
d
¼ 1.80e2.00 (m, 2H, CH2CH2CH2N), 2.55e2.95 (m, 8H, ArCH2,
CH2NCH2CH2Ar), 3.45e3.65 (m, 6H, NCH2Ar, NCO(CH2)2), 3.82 (s,
3H, OCH3), 3.85 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.51 (s, 1H, aro-
matic), 6.58 (s, 1H, aromatic), 6.74 (d, 1H, J ¼ 7.4 Hz, aromatic), 6.87
(d, 1H, J ¼ 8.2 Hz, aromatic), 7.32 (t, 1H, J ¼ 8.2 Hz, aromatic); GC/MS
m/z 410 (Mþ., 1), 192 (100); LCeMS (ESIþ) m/z: 433 [M þ Na]þ. HPLC
analysis using acetonitrile/20 mM NH4COOH (65:35 v/v), at a flow
rate 1 mL/min indicated the compound was >98% pure.
5.3.11. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-5-(2-ethyltosylate)-3,4-dihydroisoquinolin-1(2H)-one (35)
A solution of 34 (0.18 mmol, 0.08 g) in CH2Cl2 kept at 0 ꢀC was
added with Tosyl chloride (0.21 mmol, 0.040 g) and NEt3
(0.21 mmol, 0.0027 mL). The mixture was kept under stirring at
room temperature for 1 h, then added with NH4Cl and extracted
with CH2Cl2 (3 ꢃ 10 mL). The collected organic layers were dried
over Na2SO4 and evaporated under reduced pressure. The crude
residue was purified by column chromatography with CH2Cl2/
CH3OH (95:5) as eluent to give the target compound as pale yellow
oil (0.069 g, 65%); LCeMS (ESIþ) m/z: 595 [M þ H]þ; 617 [M þ Na]þ,
LCeMSeMS 595: 402.
5.3.6. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-1(2H)-one (30)
Compound 30 was obtained as yellow oil (0.10 g, 25%); 1H NMR
d
¼
1.91e2.00 (m, 2H, CH2CH2CH2), 2.59e2.95 (m, 8H,
CH2NCH2CH2Ar and ArCH2CH2NCO), 3.54e3.58 (m, 6H, NCH2Ar,
NCO(CH2)2), 3.82 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.91 (s, 6H,
OCH3), 6.51 (s, 1H, aromatic), 6,60 (s, 1H, aromatic), 6.62 (s, 1H,
aromatic), 7.59 (s, 1H, aromatic); 13C NMR: 22.81; 24.90; 27.15;
44.25; 46.67; 50.23; 52.60; 53.42; 55.26; 55.32; 55.38; 109.57;
109.90; 110.33; 111.41; 119.49; 120.710; 123.17; 133.29; 148.42;
148.68; 149.51; 152.99; 166.37. LCeMS (ESIþ) m/z: 441 [M þ H]þ,
463 [M þ Na]þ. Anal. (C25H32N2O5$HCl$½H2O) C, H, N.
6. Biological materials and methods
5.3.7. 2-(3-(6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)
propyl)-6-fluoro-3,4-dihydroisoquinolin-1(2H)-one (31)
Compound 31 was obtained as yellow oil (0.11 g, 27%); 1H NMR
6.1. Materials
d
¼ 1.90e2.00 (m, 2H, CH2CH2CH2), 2.59 (t, 2H, J ¼ 7.3 Hz,
Human recombinant serotonin 5-HT7 receptors expressed in
CHOeK1 cells, human recombinant serotonin 5-HT1A receptors
CH2CH2CH2N), 2.70e3.00 (m, 6H, NCH2CH2Ar and ArCH2CH2NCO),

928
C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
expressed in HEK293-EBNA cells, [3H]-DTG, (þ)-[3H]-pentazocine,
[3H]-5-CT, [3H]-8-OH-DPAT and [3H]-Prazosin were obtained from
PerkinElmer Life and Analytical Sciences (Boston, MA, USA). DTG
and 5-CT were purchased from Tocris Bioscience (Bristol, UK).
(þ)-Pentazocine, 8-OH-DPAT hydrobromide, phentolamine hydro-
chloride, and Calcein-AM were from SigmaeAldrich (Milan, Italy).
Male Dunkin guinea-pigs and Wistar Hannover rats (250e300 g)
were from Harlan, Italy. Cell culture reagents were purchased from
EuroClone (Milan, Italy).
studied were incubated. Nonspecific binding was defined using
10
M phentolamine. Samples were incubated at 25 ꢀC for 50 min
and then filtered on Whatman GF/C glass microfiber filters pre-
soaked in polyethylenimine 0.5% for 50 min. The filters were
washed twice with 2 mL of ice-cold buffer (50 mM TriseHCl, pH
7.4).
m
7. Radiochemistry
No-carrier-added (n.c.a) 18F-fluoride was produced via the
18O(p,n)18F nuclear reaction in a fixed-energy Cyclone 18/9 cyclo-
tron (IBA, Belgium) by irradiating >98% isotopically enriched 18O-
water (Nukem GmbH, Germany) by 18 MeV proton beam. Produced
18F-fluoride/18O-water solution was transferred using a helium
stream from the target to a shielded hot cell equipped with a
manipulator, where radiosynthesis was performed. 18F-fluoride
was trapped on an anion exchange Sep-Pak Light Accell Plus QMA
cartridge (Waters) preconditioned with 5 mL 0.5 M potassium
carbonate solution, 10 mL water and flushed with 10 mL air. It was
eluted with 1 mL solution of potassium carbonate (1 mg) and
Kryptofix K2.2.2 (5 mg) in acetonitrile/water (4:1 v/v). Effluent was
collected to a tightly closed 5 mL Reacti-Vial (Thermo Fisher Sci-
entific) and solvent was evaporated under a stream of nitrogen and
reduced pressure (50e80 mbar) at 95 ꢀC. The residue was azeo-
tropically dried by addition of 3 ꢃ 0.8 mL anhydrous acetonitrile.
6.2. s1 and s2 radioligand binding assays
All the procedures for the binding assays were previously
described. s1 And s2 receptor binding were carried out according to
Matsumoto et al. [31]. The specific radioligands and tissue sources
were respectively: (a) s1 receptor, (þ)-[3H]-pentazocine, guinea-
pig brain membranes without cerebellum; (b) s2 receptor, [3H]-
DTG in the presence of 1
mM (þ)-pentazocine to mask s1 receptors,
rat liver membranes. The following compounds were used to define
the specific binding reported in parentheses: (a) (þ)-pentazocine
(73e87%), (b) DTG (85e96%). Concentrations required to inhibit
50% of radioligand specific binding (IC50) were determined by using
six to nine different concentrations of the drug studied in two or
three experiments with samples in duplicate. Scatchard parameters
(Kd and Bmax) and apparent inhibition constants (Ki) values were
determined by nonlinear curve fitting, using the Prism, version 3.0,
GraphPad software [32].
Precursor 35 (2.4 mg, 4 mmol) predissolved in 300 mL anhydrous
acetonitrile was added to the dry 18F-fluoride and the reaction
mixture was heated at 100 ꢀC for 10 min. After cooling for 5 min at
room temperature the reaction mixture was diluted with 2 mL
acetonitrile/50 mM NH4COOH (1:1 v/v) and loaded into semi-
preparative HPLC. The radioactive fraction containing the product
was collected into 30 mL water and passed through a Sep-Pak Light
C18 cartridge (preconditioned with 5 mL ethanol followed by 10 mL
water). The cartridge was washed with additional 5 mL water for
injection. The product was eluted with 0.5 mL ethanol to a peni-
cillin vial and diluted with 9.5 mL 0.9% sodium chloride solution. An
aliquot of known volume and radioactivity of the final formulated
solution was injected into analytical HPLC for the quality control.
The specific radioactivity was determined matching the area of the
UV absorbance peak at 235 nm, which co-eluted with the radio-
labeled product, to a standard calibration curve calculated using
6.3. 5-HT7 radioligand binding assay
Binding of [3H]-5-CT at human cloned 5-HT7 receptor was per-
formed according to Jasper et al. [33] with minor modifications. In
0.5 mL of incubation buffer (50 mM TriseHCl, 10 mM MgSO4 and
0.5 mM EDTA, pH 7.4) were suspended 34 mg of membranes, 1.5 nM
[3H]-5-CT, the drugs or reference compound (six to nine concen-
trations). The samples were incubated for 120 min at 27 ꢀC. The
incubation was stopped by rapid filtration on Whatman GF/C glass
microfiber filters (pre-soaked in 0.3% polyethylenimine for 30 min).
The filters were washed with 3 ꢃ 1 mL of ice-cold buffer (50 mM
TriseHCl, pH 7.4). Nonspecific binding was determined in the
presence of 10 mM 5-CT. Approximately 90% of specific binding was
determined under these conditions.
known concentrations (0.5e20
reference compound 26.
mg/ml) of the non-radioactive
6.4. 5-HT1A radioligand binding assay
8. Animal care
Human 5-HT1A serotonin receptors stably expressed in HEK293-
EBNA cells were radiolabelled with 1.0 nM [3H]-8-OH-DPAT [34].
Animal care and all experimental procedures were according to
Swiss Animal Protection legislation and approved by the Veterinary
Office of the Canton Zurich. Male Wistar rats were purchased from
(Charles River, Sulzfeld, Germany) and were allowed free access to
food and water.
Samples containing 32
trations of each compound ranging from 0.1 nM to 10
incubated in a final volume of 500 L of 50 mM TriseHCl pH 7.4,
mg of membrane protein, different concen-
mM were
m
5 mM MgSO4 for 120 min at 37 ꢀC. After this incubation time,
samples were filtered through Whatman GF/C glass microfiber fil-
ters pre-soaked in polyethylenimine 0.5% for at least 30 min prior to
use. The filters were washed twice with 1 mL of ice-cold buffer
(50 mM TriseHCl, pH 7.4). Nonspecific binding was determined in
9. In vitro autoradiography
A male Wistar rat was sacrificed by decapitation under iso-
flurane anesthesia. The brain was removed, frozen, and cut in
the presence of 10 mM 5-HT.
horizontal slices (20
m
m) at ꢄ20 ꢀC using a MICROM microtome
6.5. a1 adrenoceptors radioligand binding assay
cryostat HM 505 N. The brain slices were mounted on SuperFrost
Plus slides (Menzel, Thermo Fisher Scientific) and stored at ꢄ80 ꢀC.
The slices were thawed at room temperature (rt) for 30 min before
the experiment and incubated in 50 mM TRIS-HCl buffer (pH 7.4) at
4 ꢀC for 10 min. A solution of [18F]-26 (9 nM in TRIS-HCl containing
1% bovine serum albumin, BSA) was pipetted on the brain slices
(n ¼ 4). Another three sets of brain slices (n ¼ 4) were incubated
Binding experiments were performed according to Glossman
and Hornung with minor modifications [35]. Each tube received, in
a final volume of 1 mL, incubation buffer (50 mM TriseHCl pH 7.4,
120 mM NaCl, 5 mM KCl, 5 mM CaCl2, 1 mM MgCl2), 500 mg rat
cerebral cortex membranes and 1 nM [3H]-Prazosin. For competi-
tive inhibition experiments various concentrations of the drugs
with the [18F]-26 solution containing either, 0.9
mM unlabeled 26,

C. Abate et al. / European Journal of Medicinal Chemistry 69 (2013) 920e930
929
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at rt, the slides were washed with TRIS-HCl/BSA and twice with
TRIS-HCl for 3 min each and then rinsed twice with distilled water.
The sections were air-dried and exposed to a BAS-TR 2025 phos-
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imaging plates were scanned in a BAS-5000 bio-imaging analyzer
(Fujifilm). Data were analyzed and processed with the AIDA 4.5
software (Raytest Fuji).
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Male Wistar rats were anesthetized by isoflurane inhalation
using an oxygen/air mixture as carrier gas. One rat (302 g) was
injected with 47 MBq (0.8 nmol) [18F]-26 into a tail vein and the
head was scanned in a GE eXplore VISTA PET/CT tomograph (axial
field of view 4.8 cm) in PET list mode from 0 to 90 min after in-
jection, followed by a CT scan for anatomical orientation. A second
rat (291 g) was injected into a tail vein 1 mg/kg haloperidol
immediately before tracer injection and scanned as above (34 MBq,
1.3 nmol). Raw data were reconstructed in user-defined time
frames (5 ꢃ 2, 6 ꢃ 5, 2 ꢃ 10 min) with a voxel size of
0.3875 ꢃ 0.3875 ꢃ 0.775 mm3 by 2D-ordered subsets expectation-
maximization (2D-OSEM). Random and singlet but no attenuation
correction was applied. Image files were evaluated by region of
interest (ROI) analysis using PMOD software (PMOD Technologies,
Switzerland) and standardized uptake values (SUV) were calculated
from the image-derived radioactivity per cm3 tissue (Bq/cm3)
normalized by the injected radioactivity dose per g body weight
(Bq/g).
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Cyclohexylpiperazine and 3,3-dimethylpiperidine derivatives as sigma-1
Acknowledgments
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We thank Claudia Keller and Martin Hungerbühler for the
excellent technical help in conducting in vitro and in vivo experi-
ments, and Dr. Marialessandra Contino and Dr. Maria Grazia Per-
rone for performing Calcein-AM and permeability assays.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.09.018.
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