Arylamides hybrids of two high-affinity σ2 receptor ligands as tools for the development of PET radiotracers

Article abstract of DOI:10.1016/j.ejmech.2011.05.057

1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl] piperazine 1 (PB28) and 2-Methoxy-5-methyl-N-[4-(6,7-dimethoxy-3,4-dihydro-1H- isoquinolin-2-yl)butyl]benzamide 2 (RHM-1) represent leads for tumor diagnosis, given their high affinity

Full text of DOI:10.1016/j.ejmech.2011.05.057

European Journal of Medicinal Chemistry 46 (2011) 4733e4741
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Arylamides hybrids of two high-affinity s2 receptor ligands as tools for the
development of PET radiotracers
*
Carmen Abate , Savina Ferorelli, Marialessandra Contino, Roberta Marottoli, Nicola Antonio Colabufo,
Roberto Perrone, Francesco Berardi
Dipartimento Farmacochimico, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine
1 (PB28) and 2-
Received 28 February 2011
Received in revised form
19 May 2011
Accepted 23 May 2011
Available online 30 May 2011
Methoxy-5-methyl-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)butyl]benzamide 2 (RHM-1)
represent leads for tumor diagnosis, given their high affinity at s2 receptors. With the purpose of obtaining
good candidates for s2 PET tracers development, hybrid structures between 1 and 2 were designed.
Excellent s1
methoxy substituted arylamide (11a, 12a, 15a), and for these benzamides an intramolecular H-bond in the
active conformation at the sites, was hypothesized. However these excellent s2 ligands were accom-
/s2 selectivities were reached when 6,7-dimethoxytetrahydroisoquinoline was linked to an o-
s
Keywords:
s2 Receptor
Cyclohexylpiperazine
6,7-Dimethoxytetrahydroisoquinoline
Dihydroisoquinolinone
P-Glycoprotein
panied by interaction with P-gp, which may limit their use as s2 receptor PET agents when tumors over-
express P-gp. Compound 15a whose P-gp interaction was just moderate represents an interesting tool for
the development of s2 PET tracers useful in tumors overexpressing P-gp.
Ó 2011 Elsevier Masson SAS. All rights reserved.
PET
1. Introduction
subtype has been classified as a chaperone of inositol(1,4,5)
triphosphate receptor, ensuring proper ER-mitochondrial Ca2þ
After the discovery of sigma (
s
) receptors in 1976 [1], initial
signaling and cell survival [5]. s1 Receptor ligands have been
reported to modulate the release of a number of neurotrasmitters
and are under evaluation for the treatment of several Central
Nervous System (CNS) disorders [6e9] despite their mechanism of
action is still unclear.
interest was generated by the evidence that a number of antipsy-
chotic agents displayed affinity for this protein and selective
ligands might have served as novel antipsychotic drugs. However,
the lack of selective agents and the concurrent use of different
protocols led to contradictory findings for over a decade [2].
Therefore, interest in receptor research waned until the early
1990’s, when the two subtypes of receptors, s1 and s2, were
s
s
The s2 subtype is even more elusive. It has yet to be cloned and
a recent attempt to characterize it using an affinity chromatography
technique led to the isolation of possible histone proteins [10,11].
Such result was in disagreement with findings from fluorescence
microscopy, which localizes s2 subtypes in several organelles
except the nucleus [12]. Regardless of these ambiguities, the
development of many high-affinity ligands [13,14] is improving
the understanding of s2 receptor subtypes, and interest is on the
increase since s2 receptors are overexpressed in a wide variety of
human tumor cell lines [15]. Therefore, s2 receptors represent
endogenous biomarkers for the diagnosis of tumors with non-
invasive techniques such as Positron Emission Tomography (PET)
or Single Photon Emission Computed Tomography (SPECT) [15]. In
addition, s2 receptor density and the proliferative status of tumors
proved to be highly correlated in a number of cell lines (e.g. s2
receptors density is about 10-fold higher in proliferative than
quiescent cells), so that s2 receptor radiotracers may serve for the
s
s
identified, renovating enthusiasm in these proteins [3]. Soon
thereafter the s1 subtype was isolated and cloned showing its
difference from any other known mammalian protein [4]. Located
primarily at endoplasmic reticulum (ER) mitochondrion contact, s1
Abbreviations: Calcein-AM, acetoxymethylester of Calcein; CNS, central nervous
system; DMEM, dulbecco’s modified eagle medium; DTG, 1,3-di-o-tolyl-guanidine;
EMT-6, mouse mammary carcinoma; ER, endoplasmic reticulum; MDCK-MDR1,
Madin Darby canine kidney cells transfected with the human MDR1 gene; MDR,
multidrug resistance; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium-
bromide; PET, positron emission tomography; P-gp, P-glycoprotein; SAfiReaffinity
relationship, structure; SPECT, single photon emission computed tomography.
* Corresponding author. Tel.: þ39 080 5442748; fax: þ39 080 5442231.
E-mail address: abate@farmchim.uniba.it (C. Abate).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.057

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C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
imaging of the proliferative status of tumors helping to select the
tumor treatment [16]. Several are the s2 receptor radioligands
which have been developed as PET or SPECT agents [15,17] with
diverse results. 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahy-
dronaphthalen-1-yl)-n-propyl]piperazine (1, PB28; Fig. 1), one of
the highest affinity s2 receptor ligands known [13,18], was [11C]-
radiolabeled at the methoxy group, but its in vivo evaluation in
mice failed for a high degree of non-specific binding in the brain,
maybe related to a not appropriate lipophilicity of the compound
[19]. On the other hand, the s2 receptor high-affinity ligand 5-
methyl-2-[11C]-methoxy-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-
isoquinolin-2-yl)butyl]benzamide (2, RHM-1; Fig. 1) [20] provided
a clear image of murine breast tumors. Studies followed with the
corresponding fluoroethoxy derivative [18F]-radiolabeled which is
reported to be currently under clinical study for imaging solid
tumors with PET [16]. A correlation between the logP values of the
s2-receptor ligands flexible benzamides and their tumor up-take
was found out indicating that, in addition to receptor affinity, lip-
ophilicity is an important property that must be considered when
evaluate their interaction at the P-glycoprotein (P-gp). Such protein,
responsible of the multidrug resistance (MDR), is known to be
overexpressed in several tumors [28,29], so that a tracer interacting
with both s2 receptor and P-gp would lead to an ambiguous infor-
mation about the tumor, as we recently speculated [24]. The data
about the P-gp interaction appears particularly important if one
considers that lead compound 1 interacts with P-gp as well as
several other s2 receptor ligands do [24,25,30]. As for compound 2,
its promising results as PET agent were obtained from mice
implanted with EMT-6 cells, where P-gp is not overexpressed [31].
The evaluation of the P-gp activity for the novel hybrid compounds
add important pieces of information for the development of s2
receptor imaging agents reliable also in tumors overexpressing P-gp.
2. Chemistry
The synthesis of target compounds (11a,be15a,b) reported
herein is depicted in Schemes 1 and 2. The synthesis of the inter-
mediate propylamines (5a,b) started from the alkylation of either
6,7-dimethoxytetrahydroisoquinoline (Scheme 1) or 1-cyclohexyl-
piperazine (Scheme 2) with 3-bromopropanenitrile to afford nitrile
derivatives 4a,b which were reduced to amine 5a,b with borane
dimethyl sulfide complex. Substituted benzoic acid (6) or 1-
tetraline carboxylic [32] acids 7 were transformed into their cor-
responding acyl chloride, through the use of oxalyl chloride, and
then reacted with propylamine intermediates (5a,b) to afford final
amides (11a,be14a,b, Scheme 1 and 2). The synthesis of interme-
diate mesylates 9a,b was obtained through initial alkylation of
either 6,7-dimethoxytetrahydroisoquinoline (Scheme 1) or 1-
cyclohexypiperazine (Scheme 2) with 3-bromopropanol to alco-
hols 8a,b followed by reaction with methanesulphonyl chloride.
Alkylation of the already known 3,4-dihydroisoquinolin-1(2H)-one
[33,34] with mesylate intermediates (9a,b) in the presence of NaH
afforded final compounds 15a,b (Scheme 1 and 2).
receptor-based tumor imaging agents are planned. Amide
2
showed the highest tumor up-take and its logD value (2.85)
appeared as optimal for tumor imaging. With the aim of contrib-
uting to the research of s2 receptor PET probes we designed a series
of hybrid structures combining the moieties of compound 1 (which
displays subnanomolar s2 receptor affinity) and compound 2
(which displays a very high s2 selectivity and in vivo activity). The
first hybrid was obtained through the replacement with 6,7-
dimethoxytetrahydroisoquinoline nucleus (A) of 1-cyclohexy-
lpiperazine basic moiety (B) present in compound 1, generating
compound 3 (Table 1) [21]. Such compound showed a noncom-
petitive binding at s2 receptor so that its structural template was no
longer considered useful for our purposes. Therefore, moiety B, that
was demonstrated to infer excellent
s affinities [13,22e27], was
connected by a three-methylene linker to substituted arylamides
which are moieties proper of compound 2 analogous structures.
The three-methylene linker was selected for a better overlap of
these new compounds with ligand 1, also knowing from previous
Structure Affinity Relationship (SAfiR) studies that high s2 receptor
affinity was kept in shorter chain length analogous benzamides
[16]. For each of these newly synthesized cyclohexylpiperazine
derivatives, the corresponding 6,7-dimethoxytetrahydroiso-
quinoline derivatives were prepared in order to compare the
3. Biology
3.1. Receptor binding studies
The target compounds 2, 3 and 11a,b-15a,b were assayed as
hydrochloride salts. All the compounds were evaluated for in vitro
affinity at s1 and s2 receptors by radioligand binding assays. The
specific radioligands and tissue sources were, respectively: (a) s1
receptor, (þ)-[3H]-pentazocine ((þ)-[2S-(2R,6R,11R)]-1,2,3,4,5,6-
hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2,6-methano-3-
benzazocine-8-ol), guinea-pig brain membranes without cere-
bellum; (b) s2 receptor, [3H]-DTG (1,3-di-2-tolyl-guanidine) in the
impact of each of the two basic moieties in
s receptors binding.
Preliminary binding results suggested that in the active confor-
mation of these benzamides at the binding sites an hydrogen bond
occurs between the amidic N-atom and the O-atom in the o-
methoxy substituted compounds, and such hypothesis was verified
through the synthesis of dihydroisoquinolinone derivatives (Fig. 2).
Since all of these novel compounds served to strengthen the
development of s2 receptor imaging agents, we considered useful to
presence of 1
m
M (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
Fig. 1. Structures of High-Affinity s2 Receptors Ligands.

Table 1
Receptors Affinities, P-gp Activities and Lipophilicity.
s
OCH
OCH
3
3
N
X- (CH ) -Y
2 n
Y =
N
N
A
B
a
Ki nM
Ki ratio s1/s2
EC50 m
M
logD7.4
Comp.
X
n
Y
s1
s2
P-gpb
1
3
B
0.38 ꢀ 0.10c
0.68 ꢀ 0.20c
8.68 ꢀ 0.4e
96%f
3.0 ꢀ 0.2c
3.75d
OCH3
O
CH3O
NH
2
3
4
3
A
A
2150 ꢀ 50e
247
1.6 ꢀ 0.3
1.1 ꢀ 0.2
2.83
4.33
CH3
151 ꢀ 20
OCH3
O
CH3O
11a
11b
3
3
A
B
534 ꢀ 104
45.5 ꢀ 5.1
2.63 ꢀ 0.20
26.1 ꢀ 0.7
203
1.7
5.3 ꢀ 1.3
8.6 ꢀ 1.5
2.7
1.94
NH
CH3
O
CH3O
H3CO
H3CO
12a
12b
3
3
A
B
4400 ꢀ 80
25.8 ꢀ 1.7
12.9 ꢀ 1.7
21.2 ꢀ 2.0
341
1.2
2.5 ꢀ 0.4
2.7 ꢀ 0.8
3.22
2.43
NH
NH
Br
CH3O
13a
13b
3
3
A
B
2570 ꢀ 1470
268 ꢀ 9
50.9 ꢀ 14.8
467 ꢀ 3
50
0.6
45 ꢀ 4
45 ꢀ 8
2.89
2.11
Br
O
O
NH
14a
14b
3
3
A
B
146 ꢀ 12
21.1 ꢀ 6.9
16.5 ꢀ 7.9
7
2
2.7 ꢀ 0.6
8.6 ꢀ 1.7
3.21
2.46
35.1 ꢀ 7.8
OCH3
O
15a
15b
3
3
A
B
709 ꢀ 133
24.7 ꢀ 1.2
3.09 ꢀ 0.26
4.84 ꢀ 0.74
26.6 ꢀ 4.1
147
1
12 ꢀ 3
20 ꢀ 4
2.25
1.49
N
(þ)-pentazocine
DTG
26.3 ꢀ 0.7
16
1.1 ꢀ 0.3
a
Calculated for compounds at pH 7.4 [37].
b
c
d
e
f
Transport Inhibition in MDCK-MDR1 cells with Calcein-AM (2.5
From Ref. [13].
mM) as probe Ref. [43].
LogD7.4 experimental value was 3.99; Ref. [24].
Ki ¼ 3078 ꢀ 87 nM for s1; Ki ¼ 10.3 ꢀ 1.5 nM for s2, Ref. [20].
[3H]-(þ)-DTG binding inhibition percentage at 10ꢁ11 M of the compound.

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C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
H3C
H
Linker
O
Basic Moiety
obtained from non-linear iterative curve fitting by Prism v. 3.0,
GraphPad software [35].
Basic Moiety
Linker
N
O
N
O
4. Results and discussion
R
4.1. Structureeactivity relationships studies and lipophilicity
Fig. 2. Intramolecular Hydrogen Bond Formation in 2-Methoxybenzamide compounds
in a sort of byciclic structure.
s1 and s2 receptors affinities. Results from binding assays are
expressed as inhibition constants (Ki values) in the Table 1. The Ki
values at the s1 receptor subtype for the newly synthesized
compounds ranged from 24.7 nM to 4400 nM. The highest Ki values
were displayed by arylamides 11a, 12a, 13a and 15a bearing the 6,7-
dimethoxytetrahydroisoquinoline moiety (A) (Kis from 534 nM to
4400 nM) in accordance to the result shown by the lead compound
2. The replacement of the aryl ring with the 5-methoxytetralin one
slightly improved s1 subtype affinity (compound 3 and 14a
Ki ¼ 151 nM and Ki ¼ 146 nM respectively). The presence of the
cyclohexylpiperazine moiety (B) always improved s1 receptor
affinity compared to moiety A (from 4- to 400-fold). The lowest
affinity at the s1 receptor in the new cyclohexylpiperazine deriv-
atives was displayed by 13b (Ki ¼ 268 nM), whereas for all the other
cyclohexylpiperazine derivatives Kis were in a narrow lower range
(from 24.7 nM to 45.5 nM, 15b and 11b respectively).
(76e85%), (b) DTG (85e93%). Concentrations required to inhibit
50% of radioligand specific binding (IC50) were determined using
six to nine different concentrations of the drug studied in two or
three experiments with samples in duplicate and inhibition
constants (Ki) values were determined by non-linear curve fitting,
using the Prism v. 3.0, GraphPad software [35].
3.2. Calcein-AM experiment
P-gp inhibiting activity of the compounds 2 and newly synthe-
sized 11a,b-15a,b and the reference compound 6,7-Dimethoxy-2-
{3-[4-methoxy-3,4-dihydro-2H-naphthalen-(1E)-ylidene]propyl}-
1,2,3,4-tetrahydroisoquinoline [21] (16) was determined by fluo-
rescence measurement using Calcein-AM probe in MDCK-MDR1
cell line according to the published procedure [36]. These trans-
fected canine kidney epithelial cells overexpress only P-gp among
MDR transporters, so that the measured biological effect is ascribed
essentially to the inhibition of this ATP-pump. The EC50 values were
As for the s2 receptor affinity, differently from compound 1,
none of the compounds reached subnanomolar values, but most of
them displayed results comparable to the s2 receptor affinity that
we found for compound 2. The replacement of moiety B with
moiety A in the structure of lead compound 1, surprisingly led to
a noncompetitive binding at the s2 receptor (compound 3) so that,
Scheme 1. Reagents: (a): Br(CH2)2CN, triethylamine; (b): BH3$S(CH3)2 (10M); (c): (COCl)2; (d): triethylamine; (e) Br(CH2)3OH, K2CO3; (f): CH3SO2Cl, triethylamine; (g): NaH.

C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
4737
Scheme 2. Reagents: (a): Br(CH2)2CN, triethylamine; (b): BH3$S(CH3)2 (10M); (c): (COCl)2; (d): triethylamine; (e) Br(CH2)3OH, K2CO3; (f): CH3SO2Cl, triethylamine; (g): NaH.
this scaffold appeared to be not appropriate for a specific s2
receptor interaction. Compound 2 inferior homolog 11a displayed
the highest s2 receptor affinity with a 4-fold improved Ki values at
both s1 and s2 receptors (Ki ¼ 534 nM for s1 and Ki ¼ 2.63 nM for
s2), so that the selectivity remained almost unchanged (about 200-
fold). The highest s2 receptor selectivity was displayed by 12a
(about 340-fold) (Ki ¼ 4400 nM at s1 and Ki ¼ 12.9 nM at s2
receptors). The lowest affinity was shown by 2-bromobenzamide
derivative 13b (Ki ¼ 467 nM), whereas its corresponding isomeric
3-bromobenzamide 12b displayed a 20-fold higher affinity value
(Ki ¼ 21.2 nM). Such a difference suggested that an intramolecular
hydrogen bond may occur in this kind of compounds between the
amidic NH group and the O-atom in o-methoxy substituted deriv-
atives, mimicking a byciclic ring. This suggestion is further sup-
ported by the difference in affinity between the two isomers 13a
(Ki ¼ 50.9 nM) and 12a (Ki ¼ 12.9 nM) as well as by the high affinity
shown by all of the amides where this intramolecular hydrogen
bond is possible. Tetralin derivatives, which naturally contain
a bicyclic structure, are endowed with a high affinity too. Results
from final compounds 15a,b (Kis ¼ 4.84 nM and 26.6 nM at s2
receptor respectively), in which the presence of the above
mentioned intramolecular H-bond was frozen in the dihy-
droisoquinolinone ring, strengthened the hypothesis of such an
active conformation for these benzamides at the binding site
(Fig. 2). Compound 15a bearing moiety A, and mimicking the
intramolecular H-bond resulted as a high affinity, highly selective
s2 ligand (147-fold s2 relative to s1 receptor), showing a s2
receptor profile comparable to lead compound 2. All of these results
taken together confirmed that the presence of the basic moiety A is
strongly detrimental for s1 receptor affinity when compared to
basic moiety B, so that 6,7-dimethoxytetrahydroisoquinoline
moiety is more appropriate than cyclohexylpiperazine to infer
high selectivity for s2 receptors. As demonstrated in a previous
work, the presence of the second N-atom is responsible of the high
affinity for both the
s subtypes [23]. Finally, when the A moiety is
linked to an arylamide o-methoxy substituted, as in lead compound
2, the best compromise between affinity and high selectivity for
excellent s2 receptor ligands is reached. In addition, the calculated
logD7.4 (Table 1, logD range 1.49e3.22) displayed by most of the
novel amides (logD w 2.5 for compounds 11a, 12b, 13a, 13b, 14b,
15a) was close to the optimal value of the in vivo active compound
2 (logD 2.83) and therefore consistent with entry into tumor cells
[37]. It has been shown that radiotracers with similar affinities at s2
receptors have different tumor up-take depending on their lip-
ophilicity, suggesting that high receptor affinity and lipophilicity
are both important for the design of receptor-based tumor imaging
agents [16].
4.2. Calcein-AM experiment
Activity at the P-gp efflux pump, expressed as EC50 in Table 1,
was determined for compound 2 and each of the target compounds.

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C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
The MDCK-MDR1 cells used in the assay overexpress the P-gp
transporter, so that the measured biological effect can be ascribed
to the interaction with this pump. All the compounds herein
reported displayed activity at P-gp with EC50 values in a range from
EA 3000 analyzer; the analytical results were within ꢀ0.4% of the
theoretical values unless otherwise indicated. 1H NMR (300 MHz)
and 13C NMR (75 MHz)spectra were recorded on a Mercury Varian
using CDCl3 as solvent. The following data were reported: chemical
1.1
m
M to 45
m
M. Apparently, P-gp interaction was independent
shift (
d
) in ppm, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
from the nature of the basic moiety since A and their corresponding
B counterparts displayed the same degree of activity (1 and 3, 11a
and 11b, 12a and 12b, 13a and 13b, 14a and 14b and 15a and 15b).
m ¼ multiplet), integration and coupling constant(s) in Hertz.
Recording of mass spectra was done on an Agilent 6890-5973 MSD
gas chromatograph/mass spectrometer and on an Agilent 1100
series LC-MSD trap system VL mass spectrometer; only significant
m/z peaks, with their percentage of relative intensity in paren-
theses, are reported. Infrared spectra were recorded on a Per-
kineElmer, Spectrum one, FTIR. Chemicals were from Aldrich and
Across and were used without any further purification.
Compound 2 proved to be a P-gp modulator (EC50 ¼ 1.6
together with compound 3 which showed the strongest P-gp
activity (EC50 ¼ 1.1 M). In fact, in a previous work [38], compound
11C]-3 and [11C]-16 were successfully used to image P-gp in rats,
therefore we reasonably hypothesize that compounds with P-gp
M), will image this efflux
mM)
m
[
activities similar to 3 and 16 (EC50 ¼ 1.1
m
pump as well. Furthermore, radiolabeled 3 and 16 proved an up-
take in several periphery organs, so that an interference with the
s2 receptor imaging of peripheral resistant tumors may occur with
radiotracers displaying such P-gp activities [38]. Unfortunately
compounds 11a and 12a which displayed the best s2 selectivity
profiles, displayed also significant P-gp interactions (EC50
5.2. General procedure for the synthesis of nitrile derivatives (4a,b)
3-Bromopropionitrile (0.43 mL, 5.18 mmol) and triethylamine
(1.4 mL, 10.4 mmol) were added to a solution of either 6,7-
dimethoxytetrahydroisoquinoline or 1-cyclohexylpiperazine (5.2
mmol) in CH2Cl2. The solution was stirred at room temperature for
6 h and then the reaction mixture was washed with H2O and dried
over Na2SO4. The solvent was removed under reduced pressure and
the crude product was purified by column chromatography using
CH2Cl2/MeOH (95:5) as eluent.
values ¼ 5.3
compound 2. On the other hand, compound 15a which displayed
M)
mM and 2.5 mM respectively), only slightly lower than
important s2 selectivity, showed a P-gp interaction (EC50 ¼ 12
m
8-fold lower than lead 2, appearing as a tool worthy of further
investigation for the development of s2 receptor tracers.
N-[6,7-Dimethoxy-3,4-dihydro-isoquinolin-2(1H)-yl]propane-
nitrile (4a) was obtained as white solid (0.70 g, 55% yield);
4.3. Conclusions
mp ¼ 84e86 ꢃC; 1H NMR
2.64 (t, 2H, J ¼ 6.9 Hz, CH2CN), 2.82e2.92
d
[m, 6H, NCCH2CH2N, N(CH2)2Ar], 3.66 (s, 2H, NCH2Ar), 3.82 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 6.51 (s, 1H, aromatic), 6.59 (s, 1H,
aromatic); GCeMS m/z 246 (Mþ, 50), 245 (52), 206 (100), 164 (80);
IR cmꢁ1: 2248 (CN).
All the results from
s receptors binding taken together show
that the combination of 6,7-dimethoxytetrahydroisoquinoline
linked to an o-methoxy substituted arylamide is responsible of
excellent s1 relative to s2 selectivity, and an intramolecular
hydrogen bond formation is suggested for the active conformation
of such compounds. As shown by other authors, this class of s2
receptor ligands may be exploited for the imaging of s2 over-
expressing tumors by PET analysis [15,16,39] through [11C]- or [18F]-
3-(4-cyclohexylpiperazin-1-yl)propanenitrile
(4b)
was
obtained as a white solid (0.97 g, 85% yield); mp ¼ 32e34 ꢃC; 1H
NMR
d 1.05e1.28 [m, 5H, cyclohexyl (CHH)5], 1.61e1.87 [m, 5H,
cyclohexyl (CHH)5], 2.18e2.25 (m, 1H, cyclohexyl CHN), 2.47e2.59
(m, 10H piperazine, and CH2CN), 2.69 (t, 2H, J ¼ 6.9 Hz,
NCH2CH2CN); GCeMS m/z 222 (Mþþ1, 5), 221 (Mþ, 33), 178 (100);
IR cmꢁ1: 2250 (CN).
radiolabeling. However, novel compounds with the best s1/s2
selectivity profile (11a and 12a), as well as lead compound 2, are
also accompanied by an important interaction with P-gp which is
overexpressed in several resistant tumors. Such interaction, which
is comparable to the activity shown by P-gp reference compounds 3
and 16, suggests that interference with the imaging of s2 receptors
may occur, leading to misinterpretation, as already demonstrated
for other classes of ligands which are characterized by P-gp activity
besides a high affinity for their targets [40,41]. An interesting bio-
logical profile worthy of further investigation is shown by
5.3. General procedure for the synthesis of amine derivatives (5a,b)
At the solution of 4a or 4b (1.00 mmol) in dry THF (10 mL), kept
at 0 ꢃC and under a stream of N2, borane dimethyl sulfide 10 M
(0.8 mL, 8.55 mmol) was added, in a dropwise manner. The reaction
mixture was stirred under reflux for 4 h. Methanol and 30 mL of HCl
6 N were then added to the reaction mixture and the solution was
refluxed for 1 h. After cooling the solvent was evaporated under
reduced pressure. The acid aqueous layer was made basic with
NaOH and extracted with CH2Cl2 (3 ꢄ 10 mL). The organic layers
were dried over Na2SO4 and concentrated under reduced pressure.
The crude residue was purified by flash column chromatography
with CHCl3/MeOH (95:5).
compound 15a: s2 affinity and s1/s2 selectivity comparable to the
in vivo active lead compound 2, but a 8-fold lower interaction with
P-gp. Therefore, compound 15a which can potentially be developed
as s2 radioligand, provides, above all, the template to drive future
synthesis of optimal s2 receptor tumors tracers devoid of P-gp
interaction.
3-[3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl]propan-1-
amine (5a) was obtained as a white semi-solid (0.68 g, 96% yield);
5. Experimental
1H NMR
d 1.58e1.88 (m, 4H, CH2CH2NH2, and NH2, D2O exchanged),
5.1. Chemistry
2.56 (t, 2H, CH2CH2CH2NH2), 2.62e2.96 (m, 6H, ArCH2CH2N,
CH2NH2), 3.52e3.57 (br s, 2H, ArCH2N), 3.78e3.88 (2s, 6H, 2 OCH3),
6.51 (s, 1H, aromatic), 6.57 (s, 1H, aromatic); GCeMS m/z 250 (Mþ,
1.6), 206 (20), 192 (100); IR cmꢁ1: 3303 (NH2), 3020, 1613.
Both column chromatography and flash column chromatog-
raphy were performed with 60 Å pore size silica gel as the
stationary phase (1:30 w/w, 63e200
m
m particle size, from ICN and
3-(4-cyclohexylpiperazin-1-yl)propan-1-amine
obtained as a yellow semi-solid (0.42 g, 66% yield); 1H NMR
d 1.02e1.22 [m, 5H, cyclohexyl, (CHH)5], 1.55e1.86 [m, 7H, cyclo-
hexyl, (CHH)5, and CH2CH2NH2], 2.13e2.21 (m, 1H, CHN),
2.34e2.65 (m, 10H, piperazine, and CH2CH2CH2NH2), 2.68e2.85
(m, 2H, CH2NH2), 3.30 (br s, 2H, NH2, D2O exchanged); GCeMS m/z
(5b)
was
1:15 w/w, 15e40 m particle size, from Merck respectively).
m
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

C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
4739
225 (Mþ, 3), 195 (32), 181 (94), 71 (100); IR cmꢁ1: 3281 (NH2),
2930, 1646.
in CH2Cl2 (10 mL), at 0 ꢃC. The resulting mixture was heated under
stirring to reflux for 1 h. Then, the solution was cooled and
extracted with 3 N HCl. The aqueous layer was made basic with
a solution of NaOH and extracted with CH2Cl2 (3 ꢄ 10 mL). The
combined organic extracts were dried over Na2SO4 and concen-
trated under reduced pressure to give a crude mixture which was
purified by column chromatography to achieve the final product.
N-[3-[6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-
2-methoxy-5-methylbenzamide (11a) was obtained as a yellow oil
using AcOEt/CH2Cl2 (7:3) as eluent (0.095 g, 13% yield); 1H NMR
5.4. General procedure for the synthesis of alcohol 8a,b
3-Bromo-1-propanol (0.22 mL, 2.5 mmol) and K2CO3 (0.41 g,
3.0 mmol) were added to a solution of either 6,7-dimethoxy-
tetrahydroisoquinoline or 1-cyclohexylpiperazine (3.0 mmol) in
CH3CN (20 mL). The reaction mixture was stirred under reflux
overnight. Then the solvent was removed under reduced pressure
and, H2O was added to the crude residue. The aqueous mixture was
extracted with CH2Cl2 (3 ꢄ 10 mL) and the organic layers were dried
over Na2SO4 and concentrated under reduced pressure to afford
a crude residue which was purified by column chromatography
with CH2Cl2/MeOH (95:5) as eluent.
d
1.86e1.90 (m, 2H, NHCH2CH2), 2.31 (s, 3H, CH3), 2.58e2.75 (m, 6H,
ArCH2CH2NCH2), 3.51e3.62 (m, 4H, NHCH2, ArCH2N), 3.74e3.84 (m,
9 H, 3 OCH3), 6.47 (s, 1H, aromatic), 6.57 (s, 1H, aromatic), 6.78 (d,
1H, J ¼ 8.5 Hz, aromatic), 7.18e7.24 (m, 1H, aromatic), 7.98 (s, 1H,
aromatic), 8.08e8.18 (br s, 1H, NH); 13C NMR: 20.55; 27.08; 28.85;
38.65; 51.32; 56.01; 56.05; 56.10; 56.14; 56.55; 109.60; 111.39;
111.51; 121.58; 126.38; 126.79; 130.71; 132.65; 133.11; 147.41;
147.71; 155.58; 165.69; GCeMS m/z 398 (Mþ, 0.1), 192 (100); Anal.
C23H30N2O4$HCl$0.5H2O (C, H, N).
3-[6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propan-1-
ol (8a) was obtained as a yellow solid (0.43 g, 70% yield);
mp ¼ 70e72 ꢃC; 1H NMR
d 1.84e1.98 (m, 2H, CH2CH2OH),
2.88e2.97 (m, 6H, ArCH2CH2N and CH2CH2CH2OH), 3.73e3.85 (m,
10H, 2 OCH3, ArCH2N and CH2OH), 4.81 (br s, 1H, OH, D2O
exchanged), 6.52 (s, 1H, aromatic), 6.59 (s, 1H, aromatic); GCeMS
m/z 251 (Mþ, 18), 206 (100), 164 (48).
N-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-2-methoxy-5-
methylbenzamide (11b) was obtained as a yellow oil using CH2Cl2/
MeOH (8:2) as eluent (0.20 g, 20% yield); 1H NMR
d 1.09e1.37 [m,
3-(4-cyclohexylpiperazin-1-yl)propan-1-ol (8b) was obtained
5H, cyclohexyl (CHH)5], 1.63e1.67 (m, 1H, cycloexyl), 1.78e1.85 [m,
4H, cyclohexyl (CHH)4], 1.94e2.08 (m, 2H, NHCH2CH2), 2.31 (s, 3H,
CH3), 2.44e2.58 (m, 3H, CHN and NHCH2CH2CH2), 2.75e2.84 (m,
8H, piperazine), 3.48e3.58 (m, 2H, CH2NH), 3.92 (s, 3H, OCH3), 6.86
(d, 1H, J ¼ 8.3 Hz, aromatic), 7.22 (dd, 1H, J ¼ 8.53 and J0 ¼ 2.5 Hz,
aromatic), 7.95 (d, 1H, J ¼ 2.2 Hz, aromatic), 7.96e7.97 (br s,
1H, NH); GCeMS m/z 373 (Mþ, 1.0), 235 (79), 149 (100); LCeMS
as a brown semi-solid (0.48 g, 86% yield); 1H NMR
d 1.04e1.28 [m,
5H, cyclohexyl, (CHH)5], 1.59e1.87 [m, 7H, cyclohexyl, (CHH)5, and
CH2CH2OH], 2.19e2.27 (m, 1H, CHN), 2.58e2.62 (m, 10H, pipera-
zine, and CH2CH2CH2OH), 2.89 (br s, 1H, OH, D2O exchanged),
3.76e3.80 (m, 2H, CH2OH); GCeMS m/z 226 (Mþ, 60),183 (100),181
(75), 111 (78).
(ESIþ) 374 [M
þ
H]þ; LCeMSeMS 374: 206, 149. Anal.
5.5. General procedure for the synthesis of methanesulphonate
C22H35N3O2$2HCl$0.5H2O (C, H, N).
(9a,b)
N-[3-[6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-
5-bromo-2,3-dimethoxybenzamide (12a) was obtained as colorless
oil (0.13 g, 17%) with CHCl3/MeOH (99:1) as eluent; 1H NMR
To a solution of the appropriate alcohol 8 (2.8 mmol) in dry
CH2Cl2 (5 mL) kept at 0 ꢃC, methanesulphonyl chloride (0.2 mL,
2.6 mmol) and triethylamine (1.0 mL, 7.3 mmol) were added. The
mixture was stirred for 1 h. Then, H2O was added to the mixture
reaction and the aqueous solution was extracted with CHCl3
(3 ꢄ 10 mL). The combined organic extracts were dried over Na2SO4
and concentrated under reduced pressure. The crude residue was
purified by column chromatography with AcOEt/MeOH (9:1) as
eluent.
d
1.85e1.89 (m, 2H, NHCH2CH2), 2.67e2.74 [m, 6H,
Ar(CH2)2NCH2(CH2)2], 3.53e3.60 (m, 4H, NHCH2 and ArCH2N),
3.80e3.85 (4s, 12H, 4 OCH3), 6.45 (s, 1H aromatic), 6.47 (s, 1H
aromatic), 7.11 (d, J ¼ 2.5 Hz, 1H, aromatic), 7.72 (d, 1H, J ¼ 2.5 Hz,
aromatic), 8.38e8.58 (br s, 1H, NH); GCeMS m/z 492 (Mþ, 0.2), 192
(100); LCeMS (ESIþ) m/z 493 [M þ H]þ; LCeMSeMS 493: 302, 300,
245. Anal. C23H29BrN2O5$(COOH)2 (C, H, N).
N-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5-bromo-2,3-
3-[6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propan-1-
methanesulphonate (9a) was obtained as a yellow oil (0.21 g; 25%
dimethoxybenzamide (12b) was obtained as colorless oil using
CH2Cl2/MeOH (9:1) as eluent (0.57 g, 78% yield); 1H NMR
yield); 1H NMR
d
2.00e2.07 (m, 2H, CH2CH2O), 2.61e2.81 (m, 6H,
d 1.02e1.28 [m, 5H, cyclohexyl (CHH)5], 1.55e1.68 (m, 1H, cyclo-
ArCH2CH2N and CH2CH2CH2O), 3.00 (s, 3H, CH3SO2), 3.83e3.86 (m,
8H, 2 OCH3 and ArCH2N), 4.35 (t, 2H, J ¼ 6.3 Hz, CH2O), 6.51 (s, 1H,
aromatic), 6.59 (s, 1H, aromatic).
hexyl), 1.68e1.92 (m, 6H, cyclohexyl (CHH)4 and NHCH2CH2],
2.18e2.28 (m, 1H, CHN), 2.35e2.68 (m, 10H, piperazine and
NHCH2CH2CH2), 3.41e3.58 (m, 2H, NHCH2), 3.85 (s, 3H, OCH3), 3.87
(s, 3H, OCH3), 7.11 (d, J ¼ 2.5 Hz,1H, aromatic), 7.72 (d,1H, J ¼ 2.5 Hz,
aromatic), 8.38e8.58 (br s, 1H, NH); 13C NMR: 26.14; 26.36; 26.54;
29.10; 39.07; 48.97; 53.98; 56.49; 56.90; 61.60; 63.68; 117.23;
118.22; 125.31; 129.28; 146.61; 153.56; 164.17; GCeMS m/z 469
(Mþþ2, 2), 468 (Mþþ1, 2), 467 (Mþ, 4), 329 (72), 245 (100), 243
(93). Anal. C22H34BrN3O3$2HCl$0.5H2O (C, H, N).
3-(4-cyclohexylpiperazin-1-yl)propan-1-methanesulphonate
(9b) was obtained as a yellow semi-solid (0.35 g, 46% yield); 1H
NMR
d 1.05e1.28 [m, 5H, cyclohexyl, (CHH)5], 1.59e1.94 [m, 7H,
cyclohexyl, (CHH)5, and CH2CH2O], 2.19e2.33 (m, 1H, CHN),
2.42e2.58 (m, 10H, piperazine CH2, and CH2CH2CH2O), 3.01 (s, 3H,
CH3SO2), 4.28 (t, 2H, J ¼ 6.3 Hz, CH2O); LCeMS (ESIþ) m/z 305
[M þ H]þ; LCeMSeMS 305: 209, 127.
N-[3-[6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-
2-bromo-4,5-dimethoxybenzamide (13a) was obtained as a yellow
oil (0.14 g, 16% yield) with CH2Cl2/MeOH (98:2) as eluent; 1H NMR
5.6. General procedure for the synthesis of amides 11a,be14a,b
d
1.86e1.90 (m, 2H, NHCH2CH2), 2.66e2.73 [m, 6H,
Oxalyl chloride (0.24 mL, 2.7 mmol) was added in a dropwise
manner to a solution of the carboxylic acid 7 (0.30 g, 1.81 mmol) in
dry CH2Cl2 (10 mL) added with dry DMF (0.2 mL). The mixture was
stirred at room temperature for 1 h and then it was refluxed for
30 min. The solvent was removed under reduced pressure, and the
crude residue was dissolved in dry CH2Cl2 and added to a solution
of the appropriate amine (2.7 mmol) and triethylamine (3.6 mmol)
Ar(CH2)2NCH2(CH2)2], 3.55e3.62 (m, 4H, NHCH2 and ArCH2N),
3.79e3.84 (4s, 12H, 4 OCH3), 6.45 (s, 1H aromatic), 6.47 (s, 1H
aromatic), 6.79 (s, 1H aromatic), 7.03 (s, 1H, aromatic), 7.78 (br s, 1H,
NH); GCeMS m/z 492 (Mþ, 0.8), 192 (100); LCeMS (ESIþ) m/z 493
[M þ H]þ. Anal. C23H29BrN2O5$HCl (C, H, N).
N-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-2-bromo-4,5-
dimethoxybenzamide (13b) was obtained as a colorless oil using

4740
C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
CH2Cl2/MeOH (9:1) as eluent (1.24 g, 49% yield); 1H NMR
1.02e1.32 [m, 5H, cyclohexyl (CHH)5], 1.58e1.68 (m, 1H, cyclo-
127.02; 127.23; 128.39; 129.83; 131.70; 138.17; 164.58; GCeMS m/z
355 (Mþ, 2), 230 (39), 217 (100), 195 (47), 188 (62). Anal.
C22H33N3O$2HCl$0.5H2O (C, H, N).
d
hexyl), 1.69e1.85 (m, 6H, cyclohexyl (CHH)4 and NHCH2CH2),
2.12e2.28 (m, 1H, CHN), 2.34e2.68 (m, 10H, piperazine and
NHCH2CH2CH2), 3.51e3.60 (m, 2H, NHCH2), 3.87 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 6.98 (s, 1H, aromatic), 7.09 (s, 1H, aromatic),
7.95e8.02 (br s, 1H, NH); 13C NMR: 24.83; 26.12; 26.51; 28.87;
40.62; 48.89; 53.72; 56.37; 57.90; 63.61; 109.86; 112.51; 115.62;
130.93; 148.56; 150.61; 167.32; GCeMS m/z 469 (Mþþ2, 8), 468
(Mþþ1, 5), 467 (Mþ, 12), 331 (86), 329 (85), 245 (100), 243 (99); IR
cmꢁ1: 3263 (NH), 1645 (CO). Anal. C22H34BrN3O3$2HCl (C, H, N).
1,2,3,4-Tetrahydro-N-[3-[6,7-dimethoxy-3,4-
5.8. Biological methods and materials: radioligand binding assays
All the procedures for the binding assays were previously
described. s1 And s2 receptor binding experiments were carried
out according to Matsumoto et al. [42]. [3H]-DTG (30 Ci/mmol) and
(þ)-[3H]-pentazocine (34 Ci/mmol) were purchased from Per-
kineElmer Life Sciences (Zavantem, Belgium). DTG was purchased
from Tocris Cookson Ltd., U.K. (þ)-Pentazocine was obtained from
SigmaeAldrich-RBI s.r.l. (Milan, Italy). Male Dunkin guinea-pigs
and Wistar Hannover rats (250e300 g) were from Harlan, Italy.
dihydroisoquinolin-2(1H)-yl]propyl]-5-methoxynaphthalene-1-
carboxamide (14a) was obtained as a colorless semi-solid (1.0 g,
75%) with CH2Cl2/MeOH (9:1) as eluent; 1H NMR
d 1.61e1.92 (m,
6H, NHCH2CH2 and CHCH2CH2), 2.42e2.79 (m, 8H, ArCH2CH2NCH2
and CHCH2CH2CH2), 3.28e3.38 (m, 2H, ArCH2N), 3.48e3.65 (m, 2H,
NHCH2), 3.75e3.88 (m, 10H, 3 OCH3 and CHCO), 6.47 (s, 1H
aromatic, tetrahydroisoquinoline), 6.55 (s, 1H aromatic, tetrahy-
droisoquinoline), 6.68e6.78 (m, 2H, aromatic), 7.08 (t, 1H,
J ¼ 7.8 Hz, aromatic), 7.90 (br s, 1H, NH); GCeMS m/z 438 (Mþ, 2),
206 (91), 192 (100); Anal. C26H34N2O4$HCl$0.25H2O (C, H, N).
N-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-5-
methoxynaphthalene-1-carboxamide (14b) was obtained as
a colorless semi-solid (0.20 g, 27% yield) using CH2Cl2/MeOH (98:2)
5.9. Cell culture
MDCK-MDR1 cell line was a gift from Prof. P. Borst, NKI-AVL
Institute, Amsterdam, Nederland. MDCK-MDR1 cells were grown
in DMEM high glucose supplemented with 10% fetal bovine serum,
2 mM glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, in
a humidified incubator at 37 ꢃC with a 5% CO2 atmosphere. Cell
culture reagents were purchased from Celbio s.r.l. (Milano, Italy).
CulturePlate 96/wells plates were purchased from PerkineElmer
Life Science; Calcein-AM, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazoliumbromide) were obtained from SigmaeAldrich
(Milan, Italy).
as eluent; 1H NMR
d 1.10e1.30 [m, 5H, cyclohexyl (CHH)5],
1.56e1.98 [m, 11H, cyclohexyl (CHH)5, CHCH2CH2, NHCH2CH2],
2.11e2.23 (m, 11H, piperazine CH2, CH2N, CHN), 2.51e2.78 (m, 2H,
benzyl CH2), 3.24e3.34 (m, 2H, NHCH2), 3.64 (t, 1H, J ¼ 5.5 Hz,
CHCO), 3.82 (s, 3H, OCH3), 6.20 (br s, 1H, NH), 6.70e7.12 (m, 3H,
aromatic); 13C NMR: 20.26; 23.28; 25.86; 26.04; 26.50; 27.35;
29.27; 29.30; 39.04; 47.84; 49.14; 53.94; 55.46; 56.95; 63.57;
108.37; 122.01; 126.59; 126.96; 135.76; 157.71; 175.05; GCeMS m/z
413 (Mþ, 7), 289 (57), 275 (97), 181 (66), 161 (100); Anal.
C25H39N3O2$2HCl (C, H, N).
5.10. Calcein-AM experiment
The experiments were carried out as described by Feng et al.
with minor modifications [36]. Each cell line (50 000 cells per well)
was seeded into black CulturePlate 96-well plates with 100
medium and allowed to become confluent overnight. Test
compounds were dissolved in 100 L culture medium and were
added to the cell monolayers. The plates were then incubated at
37 ꢃC for 30 min. Calcein-AM was added in 100
L phosphate-
buffered saline (PBS) to yield a final concentration of 2.5 M, and
plate incubation was continued for 30 min. Each well was washed
three times with ice-cold PBS. Saline buffer was added to each well,
and the plates were read with a Victor3 fluorimeter (PerkineElmer)
at excitation and emission wavelengths of 485 nm and 535 nm,
respectively. Under these experimental conditions, calcein cell
accumulation in the absence and presence of tested compounds
was evaluated, and basal-level fluorescence was estimated by
untreated cell fluorescence. In treated wells, the increase in fluo-
rescence was measured relative to the basal level. EC50 values were
determined by fitting the percent fluorescence increase percentage
versus log[dose] [35].
mL
5.7. General procedure for the synthesis of amide (15a,b)
m
To a solution of 10 (0.1g, 0.72 mmol) in dry toluene (5 mL), NaH
60% dispersion in mineral oil (0.07 g, 1.8 mmol) was added at 0 ꢃC
cautiously. Then, a solution of appropriate methansulphonate
derivative 9ab (1.15 mmol) in dry toluene (5 mL) was added in
a dropwise manner. The resulting mixture was heated under stir-
ring to reflux overnight. Then water was added to the reaction
mixture which was extracted with AcOEt (3 ꢄ 5 mL). The collected
organic layers were dried over Na2SO4 and evaporated under
reduced pressure. The crude residue was purified by column
chromatography with CH2Cl2/MeOH (95:5) as eluent.
m
m
2-[3-[6,7-Dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl]propyl]-
3,4-dihydroisoquinolin-1(2H)-one (15a) was obtained as a yellow
solid (0.18 g, 65% yield); mp ¼ 70e72 ꢃC; 1H NMR
d 1.90e2.00 (m,
2H, CH2CH2CH2NCO), 2.56e3.00 [m, 8H, ArCH2CH2NCO(CH2)2CH2,
ArCH2CH2], 3.56e3.67 (m, 6H, ArCH2CH2NCOCH2, ArCH2N), 3.82 (s,
3H, OCH3), 3.84 (s, 3H, OCH3), 6.50 (s, 1H, aromatic), 6.58 (s, 1H,
aromatic), 7.17e8.07 (m, 4H, aromatic); GCeMS m/z 380 (Mþ, 1),
192 (100); LCeMS (ESIþ) m/z 381 [M þ H]þ; LCeMSeMS 381: 188,
160. Anal. C23H28N2O3$HCl$0.75H2O (C, H, N).
Contributors
CA and FB conceived the project and wrote the paper. MC and
NAC conducted all the biological assays that provided the activity
data. SF and RM synthesized all the compounds that were used in
this study. R P planned and supervised the research.
2-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-3,4-
dihydroisoquinolin-1(2H)-one (15b) was obtained as a yellow oil
(0.11 g; 45% yield); 1H NMR
d 1.11e1.31 [m, 5H, cyclohexyl, (CHH)5],
1.58e2.04 [m, 7H, cyclohexyl (CHH)5 and NCH2CH2CH2NCO],
2.28e2.49 (m, 3H, NCH2CH2CH2NCO and CHN), 2.52e2.82 (m, 8H,
piperazine), 2.98 (t, 2H, J ¼ 6.6 Hz, ArCH2), 3.54e3.62 [m, 4H,
(CH2)2NCO], 7.16e8.05 (m, 4H, aromatic); 13C NMR: 25.47; 26.09;
26.51; 28.44; 29.14; 45.98; 46.45; 49.10; 53.88; 56.07; 63.71;
Appendix. Supplementary material
The supplementary data associated with this article can be
found in the on-line version at doi:10.1016/j.ejmech.2011.05.057.

C. Abate et al. / European Journal of Medicinal Chemistry 46 (2011) 4733e4741
4741
activity in series of analogs of 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-
tetrahydronaphthalen-1-yl)propyl]piperazine (PB28), J. Med. Chem. 52
(2009) 7817e7828.
a
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