Exploring the importance of piperazine N-atoms for σ2 receptor affinity and activity in a series of analogs of 1-cyclohexyl-4-[3-(5- methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-propyl]piperazine (PB28)

Article abstract of DOI:10.1021/jm9007505

σ2-Agonist 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen- 1-yl)propyl]piperazine (7, PB 28), which proved to revert doxorubicin resistance in breast cancer cells,was taken as a template to prepare new analogs. One of the two basic N-atoms was

Full text of DOI:10.1021/jm9007505

J. Med. Chem. 2009, 52, 7817–7828 7817
DOI: 10.1021/jm9007505
Exploring the Importance of Piperazine N-Atoms for σ2 Receptor Affinity and Activity in a
Series of Analogs of 1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-
propyl]piperazine (PB28)†
Francesco Berardi,* Carmen Abate, Savina Ferorelli, Vincenzo Uricchio, Nicola A. Colabufo, Mauro Niso, and
Roberto Perrone
ꢀ
Dipartimento Farmacochimico, Universita degli Studi di Bari, Via Orabona, 4, I-70125 Bari, Italy
Received May 29, 2009
σ2-Agonist 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (7, PB 28),
which proved to revert doxorubicin resistance in breast cancer cells, was taken as a template to prepare new
analogs. One of the two basic N-atoms was alternatively replaced by a methine or converted into an amide
or ammonium function, with the aim of finding out which of them was essential for σ2 receptor affinity and
activity. Some simply 4-substituted 1-cyclohexylpiperazineswere also investigated. None of the compounds
was as high-affinity as 7 (σ2Ki = 0.68, σ1Ki = 0.38 nM), proving that both basic N-atoms ensure better σ2
receptor binding. Amide 36 emerged as high-affinity (Ki = 0.11 nM) and noteworthy selective (1627-fold)
σ1 ligand. Small N-cyclohexylpiperazine 59 displayed the highest σ2 affinity (Ki = 4.70 nM). The σ2/σ1
selectivities were generally low. Antiproliferative assay in SK-N-SH cells revealed piperidines 24 and 15 as
putative σ2 agonists (EC50s 1.40 and 3.64 μM respectively) more potent than 7.
Introduction
to induce cell death by both caspase-independent and cas-
pase-dependent apoptoses.11,12
Initially considered as belonging to the opioid receptor
family,1 at present, sigma (σ) receptors are known as a distinct
class consisting of two subtypes, σ1 and σ2 receptors.2 They
are present with high density in the central nervous system
(CNS)a and inperipheral organs.3,4 There areseveralpieces of
evidence that show both σ receptors to be overexpressed in
many tumor cell lines, where σ2 subtype overexpression is
prevalent.5 These findings prompted the development of
σ radioligands as potential tumor imaging agents by single
photon emission computed tomography (SPECT) and posi-
tron emission tomography (PET) techniques.6,7 However,
only σ1 receptor has been identified and cloned, showing a
sequence different from any other mammalian protein.8
Recently, the σ2 receptor has been likely isolated and char-
acterized, and it seems to belong to the histone protein
family.9 The biochemical mechanism through which both
the subtypes act is still under investigation. Different experi-
ments suggest that σ receptors are endocellular proteins and
play a role in signal transduction through the mobilization of
Ca2þ from intracellular stores.10 σ2 Receptor agonists proved
Several selective σ1 receptor ligands have been proposed to
develop new classes of CNS agents for neuroprotection, anxi-
ety, depression, psychosis, and learning and memory improve-
ment.13 Conversely, only few σ2 agents are now under develop-
ment: putative agonists have been proposed as anticancer
drugs, whereas antagonists have been suggested to be useful
in inhibiting extrapyramidal symptoms of neuroleptics.10 Most
of the σ2 receptor ligands show a low selectivity relative to the σ1
subtype. Poorly selective 1,3-di-2-tolylguanidine (1, DTG) is
the most used radioligand in binding affinity assays (Chart 1).
Excellent σ2 receptor ligands were found within the series of
tetrahydroisoquinoline derivatives, where the highest selectivity
was displayed by N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoqui-
nolin-2-yl)butyl]-2-(2-fluoro-ethoxy)-3-methoxy-5-iodo-benza-
mide (2). The corresponding radiolabeled [18F]-derivative
has been developed to be used in PET analysis.14 The σ2
receptor agonist (þ)-(1R,5R)-(E)-8-3,4-dichlorobenzylidene-
5-(3-hydroxyphenyl)-2-methyl-2-azabicyclo[3.3.1]nonan-7-one
(3, CB 184) widely used to characterize σ2 receptors, also binds
the μ opioid receptor.15 A promising agent for tumor therapy is
the σ2 receptor ligand 10-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]-
1-butyl]spiro-[isobenzofuran-1(3H),40-piperidine] (4, sirame-
sine)16 that causes lysosome leakage leading the tumor cells to
death.17 Very few are the compounds claimed to be σ2 receptor
antagonists such as the moderate-affinity 1-(2-phenylethyl)-
4-(2-pyridyl)piperazine (5, UMB 24),18 which displays only a
poor σ2 over σ1 selectivity. (()-3R-Tropanyl 2-(4-chlorophe-
noxy)butyrate [6, (()-SM 21] is a σ2 selective but hydrolyzable
antagonist.19
†Contribution to celebrate the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Tel.: þ39-080-
5442751. Fax: þ39-080-5442231. E-mail: berardi@farmchim.uniba.it.
a Abbreviations: CNS, central nervous system; SPECT, single photon
emission computed tomography; PET, positron emission tomography;
SAfiR, structure-affinity relationship; SK-N-SH, neuroblastoma cells;
MCF7, breast cancer cells; PC-3, prostate cancer cells; MDR, multidrug
resistance; MCF7 ADR, breast cancer cells resistant to Adriamycin (or
doxorubicin); EBP, emopamil binding protein; SAR, structure-activity
relationship; ClogD, calculated logarithm of distribution; CoMFA,
comparative molecular field analysis; DCC, dicyclohexylcarbodiimide;
RPMI, Roswell park memorial institute; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide.
In our previous works, the high-affinity σ2 receptor ligand
1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphtha-
len-1-yl)propyl]piperazine (7),20 later named PB 28, emerged
r
2009 American Chemical Society
Published on Web 10/20/2009
pubs.acs.org/jmc

7818 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Chart 1. Structures of the Most Known σ2 Ligands
Berardi et al.
piperidine derivatives were prepared with an N-atom or an
O-atom in the intermediate chain to balance the increase
in hydrophobicity. Moreover, one or both the piperazine
N-atoms were quaternized to evaluate the effect of positive
permanent charges on the ligand binding. Derivatives bearing
an opened piperazinone ring or a symmetric (5-methoxy)-
tetralinpropyl substituent were prepared, as well as some
analogs devoid of N-cyclohexyl substituent, and with an
isosteric ring replacing the piperazine ring. A series of known
and rather more simply substituted N-cyclohexylpiperazines
were also investigated. To further define their selectivity, most
of these compounds also underwent explorative binding
assays at emopamil binding protein (EBP), a sterol isomerase
thought to belong to the σ receptor family.27,32 The most
significant ligands, amongthosehereinreported, wereselected
to be assayed for their ability in inhibiting cell proliferation.
Thus, a limited structure-activity relationship (SAR) was
generated to define the structural features for σ2 agonist or
antagonist activity.
Chemistry
The synthetic pathway for final compounds 11, 15, 16 and
24 is depicted in the Scheme 1. PtO2-catalyzed hydrogenation
of 4-(3-cyclohexen-1-yl)pyridine (8) yielded 4-cyclohexylpi-
peridine (9). Alkylation of intermediate 9 with (5-chloropen-
tyl)tetralin 1021 afforded final compound 11. 4-Cyclohexyl-4-
hydroxy-piperidine (13) was given by Grignard’s reaction
between 1-benzyl-4-piperidone (12) and cyclohexylmagne-
sium bromide, followed by debenzylation with H2 and 10%
palladium on activated carbon. Compounds 9 and 13 were
alkylated by (3-bromopropyl)tetralin 14,20 affording final
compounds 15 and 16. The synthesis of the final piperidine
24 was achieved in several steps. Bromopropyl intermediate
14 was reacted with diethyl malonate in the presence of NaH
to afford diethyl ester derivative 17. Reduction of this latter
compound by LiAlH4 yielded the diol 18, which was subse-
quently mesylated to the compound 19 with methanesulfonyl
chloride. Nucleophilic substitution of compound 19 with
NaCN provided dinitrile 20, which was subsequently hydro-
lyzed to the corresponding dicarboxylic acid monoamide21 in
alkaline medium (NaOH). Cyclization of such amide to the
imide 22 was provided in the presence of carbonyl diimida-
zole. Imide 22 was reduced by LiAlH4 to 4-substituted
piperidine derivative 23, which was condensed to the corre-
sponding enamine with cyclohexanone in the presence of
trifluoroacetic acid. Reduction in situ with NaBH4 gave the
final compound 24.
The synthesis of final piperidines 29 and 33 is depicted in the
Scheme 2. Carboxylic acid intermediate 26 was obtained by
alkylation of 5-methoxy-1,2,3,4-tetrahydro-1-naphthalenol
(25)21 with chloroacetic acid, through previous activation
with NaH,33 and then was reduced with LiAlH4 to the
corresponding alcohol 27, that generated compound 28 upon
mesylation. This last compound was reacted with 4-cyclohex-
ylpiperidine (9) to achieve final compound 29. Acylation of
5-methoxy-1,2,3,4-tetrahydro-1-naphthalenamine (30) with
bromoacetyl chloride gave bromoacetamide 31 according to
a previously reported procedure.34 The subsequent substitu-
tion with 4-cyclohexylpiperidine (9) led to the amide deriva-
tive 32, which was then reduced with borane dimethyl sulfide
complex to the final diamine compound 33.
from a class of N-cyclohexylpiperazine derivatives.21 On
the basis of earlier binding studies, it proved to be selective
relative to σ1 receptor (40-fold) and a number of other CNS
receptors.22,23 A summary of structure-affinity relationship
(SAfiR) studies on compound7 and itsanalogs is reported in a
recent review.24 Compound 7 was widely studied in functional
assays and demonstrated an agonist σ2 activity because it
inhibited electrically evoked twitch in guinea pig ileum,23 and
proliferation of SK-N-SH,25 MCF7,26 and PC3 tumor cell
lines.27 Compound 7 also proved to down regulate P-gp pump
expression and torevertmultidrugresistance (MDR) in breast
cancer tumor cells treated with doxorubicin,26 two effects
consistent with agonist σ2 receptor activity. Contradictory
binding results for compound 7 and its enantiomers,21
prompted us to conduct a reinvestigation with an updated
protocol. Although selectivity was quite lost from new assays
in animal tissues,24 the σ2 over σ1 receptor selectivity was
confirmed in MCF7 and MCF7 ADR cell lines (46-fold and
59-fold respectively) for racemic compound 7.26 Since piper-
idine derivatives provided a good pharmacophore for σ1
affinity,28,29 we assumed that the poor σ2 over σ1 selectivity
found in the N-cyclohexylpiperazines series was possibly
caused by the piperazine overlapping the piperidine ring.
Moreover, the two N-atoms in the piperazine ring should
allow reverse binding modes to both the σ receptor subtypes,
as already suggested.30,31 To find out which N-atom is
essential for σ2 binding, we followed an approach similar to
theone conducted for investigating theσ1 pharmacophorein a
series of phenylalkyl-piperidine and -piperazine analogs.30
In the present work, we synthesized a series of analogs of
compound 7 to explore the effect of the lack of one of the two
basic piperazine N-atoms in σ receptor binding. Alternatively,
proximal and distal N-atoms were replaced by an isosteric
methine group or were transformed in amidic functions. Two
The final compound 36 was prepared by hydrolysis of
carboxylic acid ethyl ester 3435 in alkaline medium and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7819
Scheme 1. Synthesis of 7-Related Cyclohexylpiperidine Derivatives 11, 15, 16, and 24a
a Reagents: (a) H2, PtO2; (b) cyclohexylmagnesium bromide; (c) H2, Pd-C; (d) CH2(COOC2H5)2, NaH; (e) LiAlH4; (f) CH3SO2Cl; (g) NaCN; (h)
NaOH; (i) 1,10-carbonyldiimidazole; (j) cyclohexanone, CF3COOH; (k) NaBH4.
subsequent condensation of the corresponding carboxylic
acid36 with commercial 1-cyclohexylpiperazine (35) by activa-
tion with dicyclohexylcarbodiimide (DCC) (Scheme 3).
The synthesis of final compounds 40, 41, 43, 44, 49, and 50 is
reported in the Scheme 4. All these compounds were prepared
starting from the common key intermediate 14. The synthesis
of final compound 40 required the monomethylammonium
intermediate 39. Commercially available 1-acetyl-4-cyclohexy-
lpiperazine (37) was prepared by acetylation of 1-cyclohexylpi-
perazine (35) and then underwent methylation affording the
corresponding methylpiperazinium iodide salt 38. Subsequent
deacetylation with HCl afforded the intermediate methylpiper-
azinium iodide salt 39 that was subjected to alkylation with the
key compound 14 to give the final compound 40. Final bis-1,4-
dimethylpiperazinium iodide 41 was obtained by methylation
of known compound 7 with an excess of CH3I. Intermediate 42
was synthesized as already reported37 to provide the precursor
for final compounds 43, 44 and 49. Deacetylation of acetylpi-
perazine 42 furnished known compound 4320 that was reacted
with cyclohexanecarbonyl chloride to afford amide 44. The
compound 14 was the key intermediate to prepare also
known37 derivatives 45 and 46 (Table 2) with piperidine and
morpholine, respectively. Methylation of the intermediate 42 to
the corresponding ammonium salt 47 with CH3I, followed by
deacetylation, gave the methylpiperazinium iodide salt 48.
Alkylation of this last compound with bromocyclohexane
afforded the final piperazinium salt 49. Symmetrical com-
pound 50 was afforded by reaction of an excess of interme-
diate 14 with piperazine.
Furthermore, compound 14 was used as starting material
for the synthesis of final compound 54 (Scheme 5). Through
standard reaction between 14 and potassium phthalimide,
derivative 51 was synthesized. Then, the latter compound
underwent hydrazinolysis to intermediate amine 52. Final
amine 54 was obtained by derivatization of amine 52 with
amide 53 which is also commercially available.38
Small piperazine derivatives 37 and 55-59 (Table 3) were
prepared through standard procedures39 starting from com-
mercial N-acetylpiperazine or N-cyclohexylpiperazine (35).
Compounds 37 and 56-58 were also commercially available.
To be assayed, all target compounds obtained as free bases
were converted into hydrochloride salts with gaseous HCl in

7820 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Berardi et al.
Scheme 2. Synthesis of 7-Related 4-Cyclohexylpiperidines with Hetero Spacer 29 and 33a
a Reagents: (a) NaH, ClCH2COOH; (b) LiAlH4; (c) CH3SO2Cl; (d) BrCH2COCl; (e) BH3 S(CH3)2.
3
Scheme 3. Synthesis of Amide Compound 36a
ligands, strategic compounds 15, 16, 24, 40, 49, 57, and 59
were selected on the basis of their high σ2 receptor affinity
(Kis < 30 nM) and different binding profile. Compounds 15
and 40 were σ2 receptor preferring ligands; 16, 24, 49, and
small piperazines 57 and 59 were both σ receptors almost
equally high-affinity ligands; 41 was a poor σ ligand. Fur-
thermore, we prepared the σ2 agonist 7 and σ2 antagonist 5,
and included them in the assay as positive and negative
reference compounds, respectively. All selected compounds
were tested for evaluating their possible σ2-mediated anti-
proliferative effect at 48 h in SK-N-SH cell line. The EC50
values were obtained from nonlinear iterative curve fitting by
Prism, version 3.0, GraphPad software.41
a Reagents: (a) 2N NaOH; (b) DCC.
Et2O, and then recrystallized from MeOH/Et2O. Calculated
values of the logarithm of the distribution coefficient (ClogD)
at pH 7.440 for all target compounds are inserted in
Tables 1-3 along with the binding results.
Results and Discussion
Biology
Radioligand Binding and σ2 SAfiR. Several N-cyclohexyl-
piperazines which are analogs of σ agent 7 were found to be
high-affinity σ2 ligands in our previous works,21,24 and this
suggested an important role for piperazine ring N-atoms in
optimal σ2 receptor binding. Are both N-atoms important
for σ2 receptor affinity? Is the proximal N-atom (the one
linked to the intermediate chain) to be important? Or the
distal one (the one bearing the cyclohexyl group)? To answer
to these questions, compounds 15 and 24 were first prepared.
In these derivatives, the distal N-atom and the proximal
N-atom, respectively, were replaced by an isosteric methine
group in the piperazine ring (Table 1). The binding data at
σ2 receptor for both these piperidine derivatives revealed
quite similar affinities (Kis 18.8 and 26.6 nM) although lower
than the subnanomolar affinity displayed by compound 7
(Ki=0.68 nM). Conversely, the affinity toward σ1 receptor
increased for the 1-cyclohexylpiperidine derivative 24 (Ki=
1.10 nM), showing a better fitting at the σ1 receptor for distal
N-atom. On the other hand, the 4-cyclohexylpiperidine
derivative 15 resulted to be a σ2 receptor ligand slightly
selective relative to σ1 receptor (∼8-fold). In summary,
though compounds 15 and 24 were not subnanomolar σ2
ligands, the decrease in their affinity was not dramatic.
Elongation of the intermediate chain to five methylene
groups (compound 11) led to a slightly decreased σ2 affinity
(Ki=72.3 nM), compared to the three-methylene compound
15, but σ1 affinity still remained low. Besides, the loss of an
Receptor Binding Studies. All the compounds listed in
Tables 1-3 were evaluated as hydrochloride salts except
for methylammonium iodide salts 40, 41, and 49, by radio-
receptor binding assays for in vitro affinity at σ1 and σ2
receptors and almost all of them were evaluated, at least
once, at EBP site. The specific radioligands and tissue
sources were respectively: (a) σ1 receptor, (þ)-[3H]-penta-
zocine ((þ)-[2S-(2R,6R,11R)]-1,2,3,4,5,6-hexahydro-6,11-di-
methyl-3-(3-methyl-2-butenyl)-2,6-methano-3-benzazocine-
8-ol), guinea-pig brain membranes without cerebellum; (b)
σ2 receptor, [3H]-1 in the presence of 1 μM (þ)-pentazocine
to mask σ1 receptors, rat liver membranes; (c) EBP site,
(()-[3H]-emopamil, guinea-pig liver membranes. The fol-
lowing compounds were used to define the specific binding
reported in parentheses: (a) (þ)-pentazocine (73-87%), (b)
compound 1 (85-96%), (c) (()-ifenprodil (70-85%). Con-
centrations 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 experi-
ments 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.41
Antiproliferative Assays. The functional biochemical as-
says were performed on human SK-N-SH neuroblastoma
cell line, where the expression of σ2 receptor had been pre-
viously reported.25 Among the newly synthesized σ2 receptor

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7821
Scheme 4. Synthesis of Methylpiperazinium Derivatives 40, 41, and 49 and Compounds 43, 44, and 50a
a Reagents: (a) CH3I; (b) HCl; (c) N-acetylpiperazine; (d) cyclohexanecarbonyl chloride; (e) bromocyclohexane; (f) piperazine.
N-atom resulted in an increase of hydrophobic parameter
ClogD, therefore compound 16 was prepared as a 4-hydro-
xy-piperidine derivative of 15. This modification not only led
to a small improvement of σ2 affinity but also to a significant
enhancement of σ1 affinity, so that 16 turned out to be a
nonselective ligand. Preparation of 1-cyclohexyl-4-hydroxy-
piperidine derivative, isomer of 16, failed because of syn-
thetic difficulties. As an alternative way, the hydrophobicity
was lowered by introduction of an O-atom or a NH-group in
the intermediate chain adjacently to the tetralin nucleus. In
the case of compound 29 both σ1 and σ2 affinities were
moderate resulting in an absence of selectivity; in the case
of compound 33 a high σ1 affinity appeared, whereas a
moderate σ2 affinity was retained, comparable to the affinity
of longer-chain compound 11. One can deduce that the
presence of a further basic N-atom could increase the
possibility of more binding modes at the σ1 receptor.
derivatives 36, 44, and 54 were prepared. Compound 36
showed lower σ2 affinity (Ki = 179 nM), suggesting a certain
role of proximal N-atom basicity in σ2 receptor binding.
Moreover, its σ1 affinity reached the subnanomolar range
(Ki =0.11 nM) revealing the compound 36 as the highest-
affinity and selective σ1 receptor ligand among this class. The
insertion of a carbonyl group in the R-position of the distal
piperazine N-atom led to the amide 44 (Table 2). This
compound displayed low σ2 affinity (Ki=304 nM) and very
low σ1 affinity (Ki > 5000 nM). However, it should be noted
that in compound 44 the cyclohexyl substituent was spaced
out by a carbon atom from the piperazine N-atom. These
results taken together with those for 36 confirmed the
importance of the basicity of distal piperazine N-atom for
σ1 receptor binding and of both N-atoms for excellent σ2
receptor binding within this series. Similar results were
obtained when the amide 54 was assayed as opened piper-
azinone ring derivative (Ki = 798 nM): neither σ2 nor σ1
receptor affinity was significant (Table 2). This same beha-
vior was evident in simpler 1,4-disubstituted piperazines
Another way to demonstrate the importance of N-atom
basicity was to render unavailable one electron lone pair at a
time for each N-atom of the piperazine ring. Thus, amide

7822 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Berardi et al.
Table 1. ClogD Values and Binding Data for 7-Related σ Ligands with Cyclohexylpiperazine or Cyclohexylpiperidine Moiety
Ki ( SEM (nM)
Ki ratio
compound
A
B
X
Y
ClogDa
σ1
σ2
EBP
σ1/σ2
σ2/σ1
7b
CH2
(CH2)3
CH2
CH2
CH2
O
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CO
N
N
N
N
N
CH
3.75
6.60
4.81
3.69
5.43
4.01
2.95
4.96
1.76
1.55
1.83
0.38 ( 0.10
236 ( 85
143 ( 18
5.56 ( 1.92
1.10 ( 0.71
91.0 ( 30.7
0.67 ( 0.11
0.11 ( 0.01
338 ( 66
0.68 ( 0.20
72.3 ( 7.3
18.8 ( 5.9
9.27 ( 1.18
26.6 ( 9.6
161 ( 12
8.38 ( 0.82
49
7.4
2
11
15
16
24
29
33
36
40
41
49
3.3
7.6
CH
COH
N
CH
10.4 ( 2.4
1.46 ( 0.18
NTc
NTc
1.6
24
CH
N
1.8
NH
N
CH
74.7 ( 6.1
179 ( 56
111
1627
CH2
CH2
CH2
CH2
N
N
7.4
23
CH2
CH2
CH2
N
þNCH3
þNCH3
N
13.0 ( 2.9
1710 ( 400
6.78 ( 0.38
1860 ( 40d
32.0 ( 1.7
26
>3
9.4
þNCH3
þNCH3
>5000
150
32
63.7 ( 17.2
3.31 ( 0.39
88.5 ( 9.0d
(þ)-pentazocine
1
(()-ifenprodil
10.2 ( 1.6
a Referred to pH 7.4. b σ1 And σ2 data are from ref 24. c Not tested. d From ref 25.
Table 2. ClogD Values and Binding Data for 7-Related σ Ligands with Modified Cyclohexylpiperazine Moiety
Ki ( SEM (nM)
compound
Α
X
Clog Da
σ1
σ2
EBP
σ1/σ2
σ2/σ1
43b
44
45
46
50
54
R1
R1
R1
R1
R2
R3
NH
NCO-c-Hexc
0.98
4.33
2.26
2.46
4.50
3.86
218 ( 74
>5000
3.38 ( 0.20d
9.10 ( 2.60
>5000
590 ( 86
304 ( 7
41.2 ( 9.6
442 ( 135
120 ( 30
798 ( 186
35.4 ( 9.4
550
2.7
>16
CH2
O
11.1 ( 1.4
NTe
470
12
49
>42
5
3910 ( 610
1230
a Referred to pH 7.4. b Previously assayed in σ total binding: Ki > 877 nM, [3H]-1, guinea-pigbrain (ref 20). c c-Hex stands for cyclohexyl. d Previous σ1
binding: Ki = 18.2 nM, (þ)-[3H]-pentazocine, whole rat brain (ref 37). e Not tested.
when the affinities of compounds 58 and 57 were compared
(Table 3). The importance of a hydrophobic group such as
the cyclohexyl substituent was better stressed in the high-
affinity mixed σ1/σ2 ligand 59, where a three-methylene
spacer supplies the appropriate distance from N-atoms to
fit the known putative σ1 receptor model.42
To investigate if N-atoms bind the receptor in the proto-
nated form, one or two positively charged N-atoms were
inserted, by quaternization to ammonium salts 40, 41, and
49. The results for these three compounds were surprisingly
interesting (Table 1). The presence of two charged methy-
lammonium functions in compound 41 was quite detrimen-
tal for both σ1 and σ2 binding, lowering the σ2 affinity at
a micromolar range (Ki=1710 nM). This could be either due
to the low hydrophobicity provided by the doubly charged
piperazine or to its conformation inferred by the two methyl
groups. When only a methylammonium function was pre-
sent, the σ2 binding was not hampered, the σ2 affinities
being rather comparable for 40 and 49 (Ki values 13.0 and
6.78 nM, respectively). A greater difference was observed

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7823
Table 3. ClogD Values and Binding Data for Simple N-Cyclohexylpiperazine-Related σ Ligands
Ki ( SEM (nM)
Ki ratio
σ2/σ1
compound
Α
Β
ClogDa
σ1
σ2
EBP
>1000
>1000
64.4 ( 4.1
>1000
17
230
35
37
55
56
57
58
59
H
CH3CO
c-Hex
c-Hex
c-Hex
CH3
-0.38
1.28
0.95
>5000
>5000
>5000
>5000
CH3CH2
CH3CO
c-Hex
62.4 ( 11.3
>5000
349 ( 27
>10000
11.5 ( 1.8
5.6
- 0.58
2.27
3.36
c-Hex
c-Hex
c-Hex
2.05 ( 0.63
3.59 ( 0.98
0.25 ( 0.07
5.6
56
c-HexCO
c-Hex(CH2)3
201 ( 33
4.09
4.70 ( 0.58
4.7
19
a Referred to pH 7.4.
Scheme 5. Synthesis of Opened Piperazinone Analog 54a
piperazines were tested, where the N-cyclohexyl was retained
and the 5-methoxytetralin nucleus removed and replaced by a
small group (Table 3). The sole removal of the proximal
N-atom substituent led to the 1-cyclohexylpiperazine (35),
with loss of any affinity (Kis>5000). In this case as well, a high
hydrophilicity and/or an intramolecular H-bond can be
claimed. The 4-ethyl substitution on the 1-cyclohexylpipera-
zine (compound 55) was sufficient to raise a certain affi-
nity, which was more pronounced toward σ1 receptor (Ki =
62.4 nM). When both piperazine N-atoms were substituted
with a cyclohexyl group (compound 57), better affinities were
reached either for σ2 (Ki = 11.5 nM) or σ1 receptor (Ki =
2.05 nM). These affinities became even higher (σ2, Ki = 4.70;
σ1, Ki=0.25) when one of the two cyclohexyl was spaced from
the N-atom by a three-methylene chain (compound 59). As
already said, the inclusion of the N-atoms into an amide
function results in a worse σ2 (amide 58, Ki =201 nM) but
not σ1 affinity (Ki=3.59 nM). These results suggest that both
basic N-atoms of piperazine ring are needed for an optimal σ2
binding, whereas an amidic function still allows a good
interaction with σ1 receptor likely through a H-bond interac-
tion. Nevertheless, the hydrophobic contribution in the amide
function (compounds 36, 44, 58) was beneficial since the
acetamide 37 displayed no affinity at both σ receptors. The
importance of more hydrophobic substituents was evidenced
by the moderate affinities of ethyl-derivative 55, which were
higher than 37, but lower than 57 affinities. When no cyclo-
hexyl group was present and only one N-atom was basic
(acetylderivative 56), both σ affinities were lost, as for 37.
As for affinities toward EBP site (i.e., mammalian Δ8;Δ7
sterol isomerase), we already demonstrated that the presence
of a tetralinalkyl moiety brought EBP affinity.27,29 All the
EBP binding results were rather comparable to those at σ1 or
σ2 receptor. Binding data have only an indicative value,
being mainly from a unique experiment. Among the com-
pounds tested in this work, 24 reached the highest EBP
affinity (Ki=1.46 nM), rather similar to its σ1 receptor affi-
nity, whereas small piperazine derivative 59 demonstrated
the same high affinity toward EBP site and σ2 receptor (Ki=
4.7 nM).
a Reagents: (a) Potassium phthalimide; (b) NH2NH2, HCl.
between the σ1 affinity values of the same compounds
(Ki values 338 and 63.7 nM, respectively). Again, a more pro-
nounced decrease in σ1 affinity was caused by distal N-atom
quaternization (compound 40), suggesting the need of an
available electron lone pair of the piperazine distal N-atom
for optimal σ1 receptor binding. On the other hand, when this
N-atom was secondary (compound 43) by removal of the
cyclohexyl group, both σ1 and σ2 affinities became moderate
(Table 2). These results could be explained by either a lower
hydrophobicity of 43 or a piperazine ring boat conforma-
tional arrangement, which weakens N-atoms basicity through
an intramolecular H-bond between them. As a confirmation
of this, piperidine 45 and morpholine 46, which are isosteric
derivatives of 43, regained high σ1 affinity (Ki values 3.38 and
9.10 nM, respectively), whereas σ2 affinity increased only for
45. Likely, a reduced interaction with the corresponding
hydrophobic pocket in the σ2 receptor might be hypothesized
for the morpholine moiety of 46, because of the loss of the
cyclohexyl group together with the presence of a hydrophilic
O-atom. Furthermore, from these results it derives that a
certain additional role is played by the 5-methoxytetralin
nucleus. Still, testing the symmetrically substituted piperazine
50, σ2 receptor affinity was poor, suggesting the need of a
relatively small substituent on one of the piperazine N-atoms.
To investigate more deeply these aspects, a molecular
simplification was conducted. Some simpler-substituted
The ClogD values of the six compounds with the highest
σ2 receptor affinities (Ki < 20 nM) ranged between 1.76
(compound 40) and 4.81 (compound 15). In particular, the
ClogD of 59 (4.09), the best σ2 ligand of the series, was very
close to that of reference compound 7 (3.75). Linear relation-
ships were evidenced for small piperazines 56, 55, 57, and 59
series and piperidines 16, 15, 24, and 11 series where the rise

7824 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Berardi et al.
Table 4. Antiproliferative Effect Measured as Inhibition of SK-N-SH
Cella Proliferation
compound EC50 ( SEMb (μM) compound EC50 ( SEMb (μM)
and 24. Despite the unambiguous action of σ2 receptors in
mediating the antiproliferative effect in SK-N-SH cells, a
linear correlation between the σ2 receptor affinity and the
activity could not be found, so that other receptor systems
should be taken into account.
5
(38%)c
40
41
49
57
59
(22%)c
(30%)c
7
9.97 ( 0.55
3.64 ( 0.68
15.9 ( 0.6
1.40 ( 0.38
15
16
24
(26%)c
(4%)c
Conclusions
52.1 ( 0.8
None of the newly tested compounds herein presented
displayed σ2 receptor affinity comparable to that of com-
pound 7. Therefore, one can deduce that both N-atoms in the
piperazine ring are important for a subnanomolar σ2 receptor
binding in this class. The loss of only one of the basic N-atoms
did not lead to a dramatic drop in affinity, indicating just a
weakened binding. The same conclusions were in part
achieved in a recent molecular modeling work on some series
of our cyclohexylpiperazines studied with a comparative
molecular field analysis (CoMFA) method.39 Among the
changes made to repress N-atom basicity, introduction of
the amide function was the most detrimental for σ2 receptor
binding (compounds 36 and 44). On the basis of this find-
ing, compound 36, which was found to possess the highest
σ1 affinity (Ki =0.11 nM), reached a noteworthy selectivity
(1627-fold). Small N-cyclohexylpiperazine 59 emerged as the
highest-affinity σ2 ligand (Ki=4.70 nM). Activity results from
antiproliferative assay did not allow to define a clear SAR
consistently with binding results. Indeed, isomeric piperidines
24 and 15 elicited putative σ2 agonist activity higher than
reference compound 7, although their σ2 receptor affinities
were lower. Conversely, compound 57 was found to be a
possible σ2 antagonist.
a
Human neuroblastoma cell line. b Mean of n g 2 separate experi-
ments. c Percent inhibition at 100 μM.
Figure 1. Antiproliferative effect of compounds tested in human
SK-N-SH neuroblastoma cell line. Incubation was carried out for
48 h in the presence (1;100 μM) and absence of tested compound
(ref 25 and 26).
in ClogD values was associated with an increase or decrease
of σ2 receptor affinity respectively. This confirmed an opti-
mal ClogD range for σ2 receptor affinity.
Functional Assays and SAR. The results expressed as EC50
values are reported in Table 4. As previously reported, SK-
N-SH human neuroblastoma cell line expressed σ1 receptor
in a low affinity state.25 Investigation by Scatchard analysis
proved the absence of EBP binding sites in the same cell line.
Thus, we speculated that the antiproliferative effect was
mediated by σ2 receptor rather than the other two investi-
gated binding sites. When tested in SK-N-SH human neuro-
blastoma cell line, compound 5 displayed only 38% of
antiproliferative activity at 100 μM, confirming its σ2 an-
tagonist activity, which had been previously claimed by in
vivo assay.18 The compounds selected to be tested in the
antiproliferative assay as the highest-affinity σ2 ligands were
also representative of the different structural changes made.
The ammonium salts 40, 41, and 49 showed a comparable
low percentage of inhibition of the proliferation, despite
their different σ2 affinities. This may be ascribed to the
permanent charges that likely prevent the compounds to
penetrate the cell and exert the effect. The symmetric ligand
57 may be claimed as σ2 receptor antagonist because it
showed an antiproliferative activity as low as 4% at
100 μM. The other five tested compounds displayed anti-
proliferative activity in the micromolar range (EC50s ranging
from 1.40 μM to 52.1 μM), and thus they can be claimed as σ2
receptor agonists (Figure 1). The piperidine compounds
15 and 24 displayed antiproliferative activity (EC50s 3.64
and 1.40 μM respectively) higher than 7, although their σ2
receptor affinity was moderate. Furthermore, compounds 15
and 24 presented the highest ClogD values among the
compounds tested (4.81 and 5.43 respectively). In the
1.40-15.9 μM range, a linear relationship was observed
between the antiproliferative activity and the ClogD rise
within the sequence of the most active compounds 16, 7, 15,
Experimental Section
Chemistry. Both column chromatography and flash column
˚
chromatography were performed with 60 A pore size silica gel as
the stationary phase (1:30 w/w, 63-200 μm particle size, from
ICN and 1:15 w/w, 15-40 μm particle size, from Merck
respectively). Melting points were determined in open capillaries
on a Gallenkamp electrothermal apparatus. Purity of tested
compounds was established by combustion analysis, confirming
a purity g95%. 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, unless otherwise
indicated. HPLC analyses were performed on a 1525 Micro
Binary pump instrument (Waters) equipped with a 996 Photo
Diode Array (PDA) detector and on a Perkin-Elmer series 200
LC instrument equipped with a Perkin-Elmer 785A UV/vis
detector. 1H NMR spectra were recorded on a Mercury Varian
300 MHz using CDCl3 as solvent, unless otherwise reported.
The following data were reported: chemical shift (δ) in ppm,
multiplicity (s=singlet, d=doublet, t=triplet, q=quadruplet,
m = multiplet), integration and coupling constants 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 parentheses, are reported. Chemicals were from Aldrich and
Across and were used without any further purification.
General Procedure to Obtain Final Amine Derivatives 11, 15,
16, and 46. In a typical reaction, the haloalkyl intermediate, 1-(5-
chloropentyl)- (10) or 1-(3-bromopropyl)-5-methoxy-1,2,3,4-
tetrahydronaphthalene (14) (1.0 mmol), was stirred and refluxed
overnight with the appropriate amine (9 or 13 or morpholine)
(1.2 mmol) and Na2CO3 (1.0 mmol, 0.11 g) in CH3CN. The
work up for final amine compounds was carried out as pre-
viously reported for similar derivatives.21 Purification of the
crude residue was achieved by column chromatography with

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7825
CH2Cl2/MeOH (9:1) as eluent affording the title amine deriva-
tives (11, 15, 16, and 46) as colorless oils in 70% yield, unless
otherwise stated.
(m, 5H, benzyl CH and CH2 and 2 OH, D2O exchanged),
3.58-3.72 [m, 2H, (CHHOH)2], 3.78-3.85 (sþm, 5H, OCH3
and (CHHOH)2], 6.60-7.10 (m, 3H, aromatic); GC-MS m/z 278
(Mþ, 15), 161 (100).
4-Cyclohexyl-1-[5-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl-pentyl)]piperidine (11). Compound 11 was obtained as
colorless oil in 40% yield (0.16 g) with CH2Cl2/MeOH (95:5)
as eluent: 1H NMR δ 0.85-1.98 [m, 28H, cyclohexyl CH and 5
CH2, piperidine CH(CH2)2, and (CH2)2CH(CH2)4], 2.28-2.35
[m, 6H, CH2N(CH2)2], 2.55-3.05 (m, 3H, benzyl CH and CH2),
3.80 (s, 3H, OCH3), 6.62-7.05 (m, 3H, aromatic); signal attri-
bution was supplementary assisted by NOESY-NMR; GC-MS
m/z 398 (Mþ þ1, 6), 397 (Mþ, 22), 236 (43), 180 (100); Anal.
(C27H43NO HCl 5/2H2O) C, H, N, H calcd 10.31, found 9.73.
2-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1,
3-propanediol Dimethanesulfonate (19). Mesylchloride (15.5
mmol, 1.20 mL) was added to a solution of 18 (4.14 mmol,
1.15 g) and Et3N (16.1 mmol, 2.26 mL) in anhydrous CH2Cl2 at
0 °C. The mixture was kept under stirring at the same tempera-
ture for 1 h. Then it was poured into H2O and extracted with
CH2Cl2 (3 ꢀ 15 mL). The organic layers were collected, dried
(Na2SO4), and concentrated under reduced pressure to give a
crude mixture which was purified by column chromatography
with CH2Cl2/ethyl acetate (8:2) as eluent. The brown oil ob-
tained underwent further purification by column chromatogra-
phy with CH2Cl2/ethyl acetate (7:3) as eluent affording pure 19
(1.53 g) as a colorless oil in 85% yield: 1H NMR δ 1.40-2.25 [m,
11H, (CH2)2CH(CH2)3CH], 2.55-2.80 (m, 3H, benzyl CH and
CH2), 3.05 (s, 6H, 2 SO2CH3), 3.80 (s, 3H, OCH3), 4.18-4.35
(m, 4H, 2 CH2O), 6.60-7.15 (m, 3H, aromatic); LC-MS (ESIþ)
m/z 457 [M þ Na]þ; LC-MS-MS 457: 361, 243.
3
3
A purity >95% was assessed by HPLC analysis for the free base
and the corresponding hydrochloride salt.
4-Cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl-propyl)]piperidine (15): 1H NMR δ 0.85-2.00 [m, 26H,
cyclohexyl CH and 5 CH2, N(CHH)2CH2CHCH2 and (CH2)2-
CH(CH2)2], 2.25-2.80 [m, 4H, CH2N(CHH)2], 2.95-3.05 (m,
3H, benzyl CH and CH2), 3.80 (s, 3H, OCH3), 6.62-7.05 (m,
3H, aromatic); GC-MS m/z 370 (Mþ þ 1, 6), 369 (Mþ, 20), 180
(100); Anal. (C25H39NO HCl 3/2 H2O) C, H, N.
3-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
pentane-1,5-dinitrile (20). A solution of 19 (3.06 mmol, 1.33 g)
and NaCN (12.25 mmol, 0.600 g) in CH3CN (15 mL) in the
presence of 1 μL of 15-Crown-5 ether was refluxed under stirring
overnight. After the mixture was cooled to room temperature,
the solvent was evaporated under reduced pressure, and the
residue was taken up with H2O and extracted with CH2Cl2 (3 ꢀ
15 mL). The organic layers were collected, dried (Na2SO4), and
concentrated under reduced pressure to give a crude mixture,
which was purified by column chromatography with CH2Cl2
affording a yellow oil (0.635 g) in a 70% yield: 1H NMR δ
1.38-1.85 [m, 11H, (CH2)2CH(CH2)3CH], 2.15-2.20 [m, 2H,
(CHH)2CN], 2.45-2.58 [m, 2H, (CHH)2CN], 2.60-2.82 (m,
3H, benzyl CH and CH2), 3.80 (s, 3H, OCH3), 6.62-7.15 (m,
3H, aromatic); GC-MS m/z 297 (Mþ þ 1, 7), 296 (Mþ, 34), 161
(100).
3-(Carbamoylmethyl)-6-(5-methoxy-1,2,3,4-tetrahydronaph-
thalen-1-yl)hexanoic Acid (21). A solution of intermediate 20
(2.18 mmol, 0.97 g), NaOH (6.05 mmol, 0.24 g) and 15-Crown-5
ether (1 μL) in EtOH (20 mL, 90%) was refluxed for 40 h under
stirring. The solvent was then removed under reduced pressure,
and the residue was taken up with H2O and added with 6 N HCl
until the solution became acidic. The acidic solution was ex-
tracted with AcOEt (3 ꢀ 15 mL), the organic layers collected
were dried (Na2SO4) and concentrated under reduced pressure
to give the title compound (0.65 g) as a white solid in 90% yield:
1H NMR δ 1.35-1.90 [m, 11H, (CH2)2CH(CH2)3CH], 2.10-
2.30 [m, 2H, (CHH)2CO], 2.45-2.80 [m, 5H, (CHH)2CO, benzyl
CH and CH2], 3.80 (s, 3H, OCH3), 6.60-7.15 (m, 3H, aromatic),
7.90 (br s, 0.8 H, NH2, D2O exchanged); LC-MS (ESIþ) m/z 356
[M þ Na]þ.
3
3
4-Cyclohexyl-4-hydroxy-1-[3-(5-methoxy-1,2,3,4-tetrahydro-
1
naphthalen-1-yl-propyl)]piperidine (16): H NMR δ 0.98-1.25
(m, 7H, 3 cyclohexyl CH2 and OH, D2O exchanged), 1.35-1.98
[m, 17H, piperidine CH2CHCH2, cyclohexyl CH2CHCH2, and
(CH2)2CH(CH2)2], 2.18-2.38 [m, 4H, CH2N(CHH)2], 2.52-
2.78 [m, 5H, N(CHH)2, benzyl CH and CH2], 3.80 (s, 3H,
OCH3), 6.62-7.05 (m, 3H, aromatic); GC-MS m/z 386 (Mþ
þ1, 7), 385 (Mþ, 26), 196 (100), 178 (28); Anal. (C25H39NO2
3
HCl) C, H, N.
4-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
morpholine (46). Compound 46 was obtained as a colorless oil in
1
98% yield (0.28 g): H NMR δ 1.45-1.95 [m, 8H, (CH2)2CH-
(CH2)2], 2.40-2.82 [m, 9H, benzyl CH and CH2, and CH2N-
(CH2)2], 3.58-3.82 (m, 4H, CH2OCH2), 3.80 (s, 3H, OCH3),
6.62-7.07 (m, 3H, aromatic); GC-MS m/z 290 (Mþ þ1, 6), 289
(Mþ, 30), 100 (100); Anal. (C18H27NO2 HCl 1/4H2O) C, H, N.
3
3
2-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
malonic Acid Diethyl Ester (17). Diethyl malonate (8.8 mmol,
1.34 mL) was added in a dropwise manner to a suspension of
NaH (8.8 mmol, 0.21 g), in dry THF (20 mL). The mixture was
stirred for 30 min under N2 at room temperature. Then, a
solution of intermediate 14 (8.8 mmol, 2.5 g) in dry THF
(10 mL) was added and the mixture was refluxed for 18 h. After
it was cooled to room temperature, the mixture was added with
H2O (10 mL) and extracted with Et2O (3 ꢀ 20 mL). The
combined organic phases were dried (Na2SO4) and concentrated
under reduced pressure to give a crude mixture, which was
purified by flash column chromatography with petroleum
ether/CH2Cl2 (1:1) as eluent affording title compound (2.01 g)
1
as a colorless oil in 63% yield: H NMR δ 1.20-1.30 [m, 6H,
4-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
piperidin-2,6-dione (22). A mixture of the intermediate 21 (2.04
mmol, 0.680 g) and 1,10-carbonyl diimidazole (0.330 g, 2.04
mmol) in THF is refluxed under stirring overnight. The solvent
was evaporated under reduced pressure and the crude material
obtained was purified by column chromatography with
CH2Cl2/MeOH (95:5) as eluent affording 22 (0.257 g) as a white
solid in 40% yield; 1H NMR δ 1.38-1.85 [m, 11H, (CH2)2-
CH(CH2)3CH], 2.08-2.30 (m, 3H, benzyl CH and CH2),
2.55-2.82 (m, 4H, 2 CH2CO), 3.80 (s, 3H, OCH3), 6.60-7.15
(m, 3H, aromatic), 8.00 (br s, 1H, NH, D2O exchanged); GC-
MS m/z 316 (Mþ þ 1, 8), 315 (Mþ, 38), 161 (100).
(OCH2CH3)2], 1.35-2.03 [m, 10H, (CH2)2CH(CH2)3], 2.45-
2.80 (m, 3H, benzyl CH and CH2), 3.33 (t, 1H, J = 7.4 Hz,
CHCO), 3.80 (s, 3H, OCH3), 4.17-4.25 [m, 4H, (OCH2CH3)2],
6.61-7.17 (m, 3H, aromatic); GC-MS m/z 363 (Mþ þ 1, 12), 362
(Mþ, 51), 161 (100).
2-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]pro-
pane-1,3-diol (18). A solution of 17 (5.52 mmol, 2.00 g) in
anhydrous Et2O (10 mL) was added in a dropwise manner to
a suspension of LiAlH4 (11.4 mmol, 0.422 g) in the same solvent
kept under N2 and at 0 °C. The mixture was then refluxed for 4 h,
and after it was cooled to room temperature, few drops of H2O
were carefully added into the reaction pot. The obtained mixture
was extracted with Et2O (3 ꢀ 15 mL) and the collected organic
phases were dried (Na2SO4) and concentrated under reduced
pressure to give a crude mixture. This latter was purified by
column chromatography with CH2Cl2/ethyl acetate (8:2) as
4-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
piperidine (23). A solution of the imide 22 (1.52 mmol, 0.480 g) in
dry THF (5 mL) was added in a dropwise manner to a suspen-
sion of LiAlH4 (3.04 mmol, 0.115 g) in the same solvent (8 mL)
cooled at 0 °C under N2. The mixture was then refluxed over-
night, and after it was cooled to room temperature, a few drops
1
eluent affording 18 (1.17 g) as a colorless oil in 76% yield; H
NMR δ 1.32-1.85 [m, 11H, (CH2)2CH(CH2)3CH], 2.55-2.80

7826 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Berardi et al.
of H2O were carefully added. The resulting mixture was ex-
tracted with Et2O (3 ꢀ 10 mL), and the organic layers were
collected and dried (Na2SO4). Concentration under reduced
pressure gave a crude residue, which was purified by column
chromatography with CH2Cl2/MeOH (9:1) as eluent to afford
23 (0.306 g) as a yellow oil in 70% yield: 1H NMR δ 1.02-1.82
[m, 16H, (CH2)2CH(CH2)3, piperidine CH(CH2)2, and NH D2O
exchanged], 2.45-2.80 [m, 5H, (CHH)2NH, benzyl CH and
CH2], 3.02-3.18 [m, 2H, (CHH)2N], 3.80 (s, 3H, OCH3),
6.60-7.15 (m, 3H, aromatic); GC-MS m/z 288 (Mþ þ 1, 8),
287 (Mþ, 11), 126 (100).
collected, dried and evaporated under reduced pressure to
afford a crude residue, which was purified by column chroma-
tography with CH2Cl2/MeOH (95:5) as eluent affording the
final amine 33 (0.30 g) as a colorless oil in 50% yield: 1H NMR δ
0.80-1.95 [m, 23H, cyclohexyl CH and 5 CH2, piperidine CH-
(CH2)2(CHH)2N, CH(CH2)2, and NH, D2O exchanged], 2.40-
2.98 [m, 8H, benzyl CH2, NCH2CH2N(CHH)2], 3.75-3.80 (m,
1H, NCH), 3.82 (s, 3H, OCH3), 6.62-7.05 (m, 3H, aromatic);
GC-MS m/z 370 (Mþ, 2), 180 (100), 161 (15); Anal. (C24H38-
N2O 2HCl 5/4 H2O) C, H, N.
3
3
4-Cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)propionyl]piperazine (36). 3-(5-Methoxy-1,2,3,4-tetrahy-
dronaphthalen-1-yl)propanoic acid ethyl ester (34) obtained
as already reported was hydrolyzed to the corresponding
carboxylic acid, as for its lower homologue.35 A solution of
3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propanoic acid
(1.28 mmol, 0.300 g) and dicyclohexylcarbodiimide (DCC)
(1.69 mmol, 0.348 g) in CH2Cl2 (15 mL) was added in a dropwise
manner to a solution of N-cyclohexylpiperazine (35) (1.28
mmol, 0.215 g) in the same solvent (10 mL). The mixture was
stirred at room temperature overnight; then it was added with
glacial acetic acid, filtered, and washed with 2N NaOH. The
organic phase was separated from the alkaline phase, was dried
(Na2SO4) and evaporated under reduced pressure. The crude
residue was purified by column chromatography with CH2Cl2/
MeOH (95:5) as eluent to afford the final compound 36 (0.345 g)
as a pale yellow oil in 70% yield; 1H NMR δ 1.20-2.18 [m, 16H,
cyclohexyl 5 CH2, (CH2)2CHCH2], 2.38-2.95 (m, 10H, CHN-
(CH2)2, CH2CO, benzyl CH and CH2), 3.42-3.80 [m, 4H,
(CH2)2NCO], 3.80 (s, 3H, OCH3), 6.62-7.05 (m, 3H, aromatic);
GC-MS m/z 385 (Mþ þ 1, 13), 384 (Mþ, 50), 138 (78), 125 (100);
Anal. (C24H36N2O2 HCl H2O) C, H, N.
1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)propyl]piperidine (24). A solution of 23 (0.52 mmol, 0.150 g),
cyclohexanone (0.96 mmol, 0.10 mL), and CF3COOH (2 drops)
in toluene (20 mL) was refluxed overnight with azeotropic
removal of H2O. After cooling, the reaction mixture was added
to a solution of NaBH4 (1.06 mmol, 0.040) in EtOH (10 mL),
and the resulting mixture was stirred at room temperature for
2 h. Then few drops of H2O were added; the solvent was
removed under reduced pressure, and the residue was taken
up with H2O (10 mL) and extracted with CH2Cl2 (3 ꢀ 15 mL).
The collected organic layers were dried (Na2SO4) and concen-
trated under reduced pressure to give a crude material, which
was purified by column chromatography with CH2Cl2/MeOH
(95:5) as eluent, affording the title compound (0.144 g) as a
yellow oil in 75% yield; 1H NMR δ 1.02-1.92 [m, 25H, (CH2)2-
CH(CH2)3, piperidine CH(CH2)2 and cyclohexyl 5 CH2],
2.05-2.40 [m, 3H, (CHH)2NCH], 2.45-2.80 [m, 3H benzyl
CH and CH2], 2.85-3.02 [m, 2H, (CHH)2N], 3.80 (s, 3H,
OCH3), 6.60-7.15 (m, 3H, aromatic); GC-MS m/z 370 (Mþ þ
1, 5), 369 (Mþ, 20), 326 (100), 208 (26); LC-MS (ESIþ) m/z 370
[M þ H]þ, LC-MS-MS 370: 288; Anal. (C25H39NO HCl 1/
3
3
3
3
2H2O) C, H, N.
1-Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)propyl]-1-methylpiperazinium Iodide (40). A mixture of 1-(3-
bromopropyl)-5-methoxy-1,2,3,4-tetrahydronaphthalene (14)
(3.2 mmol, 0.91 g) with intermediate 39 (3.2 mmol, 1.00 g) and
Na2CO3 (3.7 mmol, 0.39 g) in CH3CN (25 mL) and EtOH
(2 mL) was refluxed and stirred overnight. After the mixture was
cooled, the solvent was removed under reduced pressure, and
the solid residue was dissolved in MeOH and warmed under
stirring for 20 min. The methanolic solution, while still warm,
was filtered and the filtrates were evaporated under reduced
pressure to afford a white solid, which was passed over silica gel
(CH2Cl2/MeOH 8:2 as eluent). The resulting gummy solid was
recrystallized from EtOH to give the final compound 40 (1.15 g)
as pale yellow crystals in 70% yield: mp 173-175 °C; 1H NMR
(CD3OD) δ 1.18-1.70 [m, 14H, cyclohexyl (CH2)3, and (CH2)2-
CH(CH2)2], 1.78-1.85 [m, 2H, cyclohexyl CH(CHH)2],
2.05-2.10 [m, 2H, cyclohexyl CH(CHH)2], 2.15-2.62 [m, 9H,
benzyl CH and CH2, and CH2N(CH2)2], 2.77 (s, 3H, NCH3),
3.20-3.40 [m, 5H, (CH2)2NCH], 3.80 (s, 3H, OCH3), 6.45-
6.95 (m, 3H, aromatic); LC-MS (ESIþ) m/z 385 [Mþ - I]; Anal.
(C25H41IN2O) C, H, N.
4-Cyclohexyl-1-[2-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yloxy)ethyl]piperidine (29). Mesyl chloride (1.1 mmol, 0.085
mL) was added to a solution of the alcohol 27 (0.90 mmol, 0.20
g) and Et3N (1.8 mmol, 0.25 mL) in CH2Cl2 (10 mL) at 0 °C. The
mixture was left under stirring at 0 °C for 1 h, then poured into
H2O (10 mL), and extracted with CH2Cl2 (3 ꢀ 10 mL). The
organic phases were collected, dried (Na2SO4) and evaporated
under reduced pressure to afford the sulfonate derivative 28
(0.24 g) as a yellow oil (85% yield) used for the next step without
any further purification: GC-MS m/z 300 (Mþ, 2), 160 (100). A
mixture of 28 (∼0.7 mmol, 0.22 g), N-cyclohexylpiperazine (9)
(0.95 mmol, 0.16 g), and Na2CO3 (0.7 mmol, 0.074 g) in CH3CN
(10 mL) was stirred under reflux overnight. After it was cooled
to room temperature, the mixture was concentrated under
reduced pressure. The resulting residue was taken up with
H2O (10 mL) and extracted with CH2Cl2 (3 ꢀ 10 mL). The
organic layers were collected, dried (Na2SO4) and evaporated
under reduced pressure to afford a brown oil, which was
purified by column chromatography with CH2Cl2/MeOH
(9:1) as eluent. The final compound 29 (0.203 g) was obtained
as a yellowish oil in 78% yield: 1H NMR δ 0.85-2.05 [m, 22H,
cyclohexyl CH and 5 CH2, piperidine CH(CH2)2(CHH)2N,
OCH(CH2)2], 2.42-2.78 [m, 4H, benzyl CH2, and CH2N],
2.90-3.05 [m, 2H, CH2N(CHH)2], 3.58-3.70 (m, 2H, OCH2),
3.80 (s, 3H, OCH3), 4.38-4.42 (m, 1H, CHO), 6.75-7.15 (m,
3H, aromatic); GC-MS m/z 371 (Mþ, 0.2), 180 (100), 167 (24);
Anal. (C24H37NO2 HCl 1/2H2O) C, H, N.
1-Cyclohexyl-1,4-dimethyl-4-[3-(5-methoxy-1,2,3,4-tetrahy-
dronaphthalen-1-yl)propyl]piperazinium Diiodide (41). To a solu-
tion of 7 (0.75 mmol, 0.28 g) in CH3CN (20 mL) and DMF
(0.5 mL), CH3I (3.0 mmol, 0.2 mL) was added, and the mixture
was stirred under reflux overnight. After it was cooled to room
temperature, more CH3I (6.0 mmol, 0.40 mL) was added to the
mixture, which was stirred under reflux for further 80 h. After
the mixture was allowed to cool to room temperature, the
solvent was evaporated under reduced pressure. The residue
was taken up with MeOH, and the mixture was refluxed for 30
min. The warm methanolic suspension was filtered, affording
the target compound 41 (0.17 g) as white crystals in 35% yield:
3
3
4-Cyclohexyl-1-[N-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)-2-aminoethyl]piperidine (33). A 10 M borane dimethyl
sulfide complex (6.0 mmol, 0.60 mL) was diluted in dry THF
(10 mL) and added in a dropwise manner to a solution of 32 (1.6
mmol, 0.61 g) in the same solvent (10 mL) kept at 0 °C and under
N2. The mixture was refluxed for 4 h, and after cooling, the
excess of reagent was quenched with MeOH. Then 2N HCl
(20 mL) was added and the mixture was refluxed for 1 h. After
cooling, 2 N NaOH (30 mL) was added, and the mixture was
extracted with AcOEt (3 ꢀ 15 mL). The organic phases were
1
mp 240-241 °C; H NMR (D2O) δ 0.95-1.98 [m, 18H,
cyclohexyl 5 CH2, and (CH2)2CH(CH2)2], 2.38-2.55 (m, 2H,
benzyl CH2), 2.70-2.80 (m, 1H, benzyl CH), 2.98 (s, 3H,
NCH3), 3.10 (s, 3H, NCH3), 3.35-3.58 (m, 3H, NCH and
CH2N), 3.65 (s, 3H, OCH3), 3.75-3.85 (m, 8H, piperazinium

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7827
4 CH2), 6.65-7.05 (m, 3H, aromatic); LC-MS (ESIþ) m/z
527 [Mþ - I]; LC-MS-MS 527: 317, 260, 203; Anal. (C26H44I2-
N2O) C, H, N.
σ1 And σ2 receptor binding were carried out according to
Matsumoto et al.43 EBP binding was carried out according to
Moebius et al.44 [3H]-1 (30 Ci/mmol) and (þ)-[3H]-pentazocine
(34 Ci/mmol) were purchased from PerkinElmer Life Sciences
(Zavantem, Belgium). The radioligand (()-[3H]-emopamil
(83 Ci/mmol) was purchased from American Radiolabeled
Chemicals, Inc. (St. Louis, MO). Ligand 1 and (()-ifenprodil
were purchased from Tocris Cookson Ltd., U.K. (þ)-Pentazo-
cine was obtained from Sigma-Aldrich-RBI srl (Milan, Italy).
Male Dunkin guinea-pigs and Wistar Hannover rats (250-
300 g) were from Harlan, Italy.
Cell Culture. The human SK-N-SH neuroblastoma cell line
was obtained from Interlab Cell Line Collection (ICLC, Gen-
oa). SK-N-SH cell line was routinely cultured in RPMI 1640
supplemented with 10% fetal bovine serum, 2 mM glutamine,
100 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium
pyruvate, and 1% non essential amino acids in a humidified
incubator at 37 °C with a 5% CO2 atmosphere.
Antiproliferative Assay. Determination of cell growth was
performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) assay at 48 h.25,26 On day 1, 10 000
cells/well were plated in 96-well plates in a volume of 200 μL
and on day 2, the various drugs alone or in combination were
added. In all the experiments, the various drug-solvents
(ethanol, DMSO) were added in each control to evaluate
a possible solvent cytotoxicity. After the established incuba-
tion time with drugs, 0.5 mg/mL MTT was added to each
well and, after 3 h incubation at 37 °C, the supernatant was
removed. The formazan crystals were solubilized by 100 μL of
DMSO and the absorbance values at 570 and 630 nm were
determined on the microplate reader Victor 3 from PerkinElmer
Life Sciences.
1-Cyclohexanecarbonyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-
naphthalen-1-yl)propyl]piperazine (44). Cyclohexanecarbonyl
chloride (2.57 mmol, 0.34 mL) was added in a dropwise manner
to a solution of 43 (2.14 mmol, 0.615 g) and triethylamine
(0.4 mL) in toluene (8 mL) kept at 0 °C. The mixture was then
heated at reflux for 6 h. The reaction mixture was allowed to cool
to room temperature, and the solvent was evaporated under
reduced pressure. The residue was taken up with H2O (10 mL)
and extracted with CH2Cl2 (3 ꢀ 10 mL). The combined organic
layers were dried (Na2SO4) and concentrated under reduced
pressure. The crude residue was eluted by column chromatog-
raphy with CH2Cl2/MeOH (95:5) affording the title compound
(0.60 g) as a pale yellow oil in 70% yield: 1H NMR δ 1.20-1.85
[m, 18H, cyclohexyl 5 CH2, and (CH2)2CH(CH2)2], 2.38-2.55
[m, 7H, COCH, CH2N(CH2)2], 2.58-2.80 (m, 3H, benzyl CH
and CH2), 3.52-3.90 [m, 4H, (CH2)2NCO], 3.80 (s, 3H, OCH3),
6.62-7.05 (m, 3H, aromatic); GC-MS m/z 399 (Mþ þ 1, 10), 398
(Mþ, 38), 258 (50), 209 (100); Anal. (C25H38N2O2 HCl 1/
3
3
4H2O) C, H, N.
4-Cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
1-yl)propyl]-1-methylpiperazinium Iodide (49). A mixture of 48
(1.1 mmol, 0.42 g), bromocyclohexane (1.3 mmol, 0.21 g), and
Na2CO3 (1.4 mmol, 0.15 g) in CH3CN (20 mL) was refluxed
under stirring overnight. After it was cooled, the solvent was
removed under reduced pressure, and the resulting solid residue
was dissolved in MeOH and warmed under stirring for 20 min.
The methanolic solution, while still warm, was filtered and the
filtrates were evaporated under reduced pressure to afford a
white solid, which was purified through silica gel with a mixture
of CH2Cl2/MeOH (8:2) as eluent. The solid obtained was
recrystallized from EtOH affording the final compound 49
(0.37 g) as pale yellow crystals in 65% yield: mp 196-198 °C;
1H NMR (CD3OD) δ 1.13-1.40 (m, 5H, cyclohexyl 5 CHH),
1.60-1.98 [m, 13H, (CH2)2CH(CH2)2 and cyclohexyl 5 CHH],
2.35-2.98 [m, 8H, (CH2)2NCH, and benzyl CH and CH2], 3.08
(s, 3H, NCH3), 3.35-3.45 [m, 6H, CH2N(CH2)2], 3.77 (s, 3H,
OCH3), 6.64-7.05 (m, 3H, aromatic); LC-MS (ESIþ) m/z 385
[Mþ - I], LC-MS-MS m/z 385: 203, 161; Anal. (C25H41IN2O)
C, H, N.
Supporting Information Available: Formulas, appearances,
and melting points of hydrochloride salts of the novel final
compounds 11, 15, 16, 24, 29, 33, 36, 44, 46, 50, and 54,
elemental analyses of the novel end products, description of
the preparation and spectroscopy data for the intermediate
compounds 9, 13, N-benzyl-13, 26, 27, 32, 38, 39, 47, 48, 51
and 52, and purity HPLC analysis for compound 11. This
material is available free of charge via the Internet at http://
pubs.acs.org.
1,4-Bis[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)pro-
pyl]piperazine (50). A mixture of 1-(3-bromopropyl)-5-meth-
oxy-1,2,3,4-tetrahydronaphthalene (14) (2.0 mmol, 0.56 g)
with piperazine (1.0 mmol, 0.086 g) and Na2CO3 (2.0 mmol,
0.21 g) in CH3CN (20 mL) was refluxed and stirred overnight.
Work up and purification were conducted as in general proce-
dure followed for 16, affording the target compound as an oil in
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1
92% yield (0.90 g): H NMR δ 1.52-1.85 [m, 16H, 2 (CH2)2-
CH(CH2)2)], 2.35-2.85 (m, 18H, piperazine 4 CH2, 2 NCH2,
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1
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MS m/z 359 (Mþ þ 1, 9), 358 (Mþ, 34), 232 (100), 201 (64); Anal.
(C22H34N2O2 HCl H2O) C, H, N.
3
3
Biological Methods: Radioligand Binding Assays. All the
procedures for the binding assays were previously described.

7828 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
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