Novel 4-(4-aryl)cyclohexyl-1-(2-pyridyl)piperazines as Δ8- Δ7 sterol isomerase (emopamil binding protein) selective ligands with antiproliferative activity

Article abstract of DOI:10.1021/jm800965b

To find Δ₈-Δ₇ sterol isomerase (EBP) selective ligands, various arylpiperazines previously studied and structurally related to some σ receptors ligands were preliminarily screened. Consequently, a novel series of 2- or 2,6-disubstitu

Full text of DOI:10.1021/jm800965b

J. Med. Chem. 2008, 51, 7523–7531
7523
Novel 4-(4-Aryl)cyclohexyl-1-(2-pyridyl)piperazines as ∆8-∆7 Sterol Isomerase (Emopamil
Binding Protein) Selective Ligands with Antiproliferative Activity
Francesco Berardi,* Carmen Abate, Savina Ferorelli, Anna F. de Robertis, Marcello Leopoldo, Nicola A. Colabufo,
Mauro Niso, and Roberto Perrone
Dipartimento Farmacochimico, UniVersita` degli Studi di Bari, Via Orabona, 4, I-70125 Bari, Italy
ReceiVed July 30, 2008
To find ∆8-∆7 sterol isomerase (EBP) selective ligands, various arylpiperazines previously studied and
structurally related to some σ receptors ligands were preliminarily screened. Consequently, a novel series
of 2- or 2,6-disubstituted (CH3, CH3O, Cl, F) cis- and trans-4-(4-aryl)cyclohexyl-1-(2-pyridyl)piperazines
was developed. Radioreceptor binding assays evidenced cis-19, cis-30 and cis-33 as new ligands with
nanomolar affinity toward EBP site and a good selectivity relative to EBP-related σ receptors. The most
selective 2,6-dimethoxy derivative (cis-33) demonstrated the highest potency (EC50 ) 12.9 µM) and efficacy
(70%) in inhibiting proliferation of human prostate cancer PC-3 cell line. Among the reference compounds,
σ2 agonist 36 (PB28) reached the maximum efficacy (100%), suggesting the contribution of the σ2 receptor
to the antiproliferative activity. This novel class of EBP inhibitors represents a valuable tool for investigating
the last steps of cholesterol biosynthesis and related pathologies, as well as a starting point for developing
new anticancer drugs.
Chart 1. Structures of Mammalian ∆8-∆7 Sterol Isomerase
(EBP) Ligands
Introduction
Postsqualene sterol biosynthetic pathway is an attractive target
for blood cholesterol lowering. Inhibiting enzymes at the last
steps of cholesterol biosynthesis can reduce the side-effects due
to an early step inhibition such as at 3-hydroxy-3- methylglutaryl
(HMGa) CoA-reductase level by vastatins. On the other hand,
the inborn lack of enzymes acting in the final steps of cholesterol
biosynthesis is known to generate rare but severe disorders. The
discovery of specific inhibitors for such enzymes can help to
better understand the biochemistry of these genetic defects.1,2
CHILD (congenital hemidysplasia with ichthyosiform erythro-
derma and limb defects) syndrome,3 Conradi-Hu¨nermann-
Happle syndrome (or X-linked dominant chondrodysplasia
punctata type 2),4,5 and Smith-Lemli-Opitz syndrome6,7 are
dysmorphogenetic syndromes of variable severity due to muta-
tions to the genes encoding for (i) the C4-sterol dehydrogenase,
(ii) the 3ꢀ-hydroxysteroid ∆8-∆7 isomerase (EBP), and (iii)
the 3ꢀ-hydroxysteroid ∆7-reductase, respectively.8 In the past,
the clinical trials of ∆7-reductase inhibitors such as 20,25-
diazacholesterol and triparanol were discontinued because of
their high toxicity.
EBP (i.e., Emopamil Binding Protein) is a vertebrate sterol
∆8-∆7 isomerase which has demonstrated equal high binding
affinity9 for both enantiomers of emopamil, a Ca2+ channel
blocker. EBP characterization was achieved using the anti-
ischemic drug (-)-(S)-emopamil (1) (Chart 1).10 Functionally,
EBP catalyzes the shift of the double bond from C8-C9 to
C7-C8 position in one of the last steps of cholesterol de novo
biosynthesis. It has been hypothesized that in humans such
isomerizationoccursthroughatranshydrogenaddition-elimination
reaction from 5R-cholesta-8,24-dien-3ꢀ-ol (zymosterol, 9a) or
dihydrozymosterol (9b) to give 5R-cholesta-7,24-dien-3ꢀ-ol
(10a) or lathosterol (10b), respectively (Chart 2).11 cDNA from
human liver encoded EBP as a 230 amino acids, 27-kDa protein9
spanning through four putative transmembrane domains.12 EBP
and its yeast counterpart ERG2p, which catalyzes the same step
in yeast ergosterol biosynthesis, share a strikingly similar
* To whom correspondence should be addressed. Tel.: +39-080-5442751.
Fax: +39-080-5442231. E-mail: berardi@farmchim.uniba.it.
a Abbreviations: EBP, emopamil binding protein; PC-3, prostate cancer
cells; HMG, 3-hydroxy-3-methylglutaryl; CHILD, congenital hemidysplasia
with ichthyosiform erythroderma and limb defects; ERG2p, ∆8-∆7 sterol
isomerase of Saccharomyces cereVisiae; NMDA, N-methyl-D-aspartate;
AEBS, antiestrogen binding site; 5-HT, 5-hydroxytryptamine; TMEDA,
N,N,N,N-tetramethylethylendiamine; LDH, lactate dehydrogenase; ClogD,
calculated logarithm of distribution; SI, sterol isomerase; RPMI, Roswell
park memorial institute; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltet-
razoliumbromide.
10.1021/jm800965b CCC: $40.75
2008 American Chemical Society
Published on Web 11/14/2008

7524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Chart 2. EBP-Catalyzed Isomerization
Berardi et al.
Chart 3. From Tetralin to Arylcyclohexyl Derivatives
In an attempt of finding high-affinity and σ1 and σ2 selective
ligands, we developed several Structure-Affinity Relationship
(SAfiR) studies where the EBP affinity was also taken into
account. Most of the σ1 receptor ligands studied displayed fairly
good to high affinity also toward EBP site, resulting in poor
selectivities.19-22 A good EBP selectivity was found for the
high-affinity ligand 3,3-dimethyl-1-[3-(5-methoxy-1,2,3,4-tet-
rahydronaphthalen-1-yl)n-propyl]piperidine (8).19 The tetralin
or naphthalene moiety in our compounds appeared to be able
to well mime both the benzomorphan template of σ1 ligand (+)-
pentazocine and the steroidal A and B rings of the EBP
substrates 9a,b. On the other hand, the A ring in these steroidal
substrates is less planar than the tetralin nucleus in 11 (Chart
3), and the structures of specific EBP ligand 1 and related
compounds do not present any bicycle nucleus. All things
considered, we thought that such a bicycle might be unessential
for EBP binding and the opening of the tetralin nucleus should
lead to some specific EBP-site affinity. Furthermore, the
intermediate propylenic chain, linked to the amine moiety, had
to be constrained in a cyclohexyl ring in order to reduce the
conformational freedom of the resulting structures 12. We had
followed a similar approach to prepare some arylpiperazine
derivatives as serotonin 5-HT1A receptor ligands.23 Therefore,
we screened a restricted series of these N-arylpiperazines and
related compounds, choosing among the lowest-affinity ligands
for serotonin 5-HT1A, dopamine D2, and adrenergic R1 receptors,
and testing them for EBP, σ1 and σ2 affinity (Chart 4). The
results allowed to highlight the high-affinity and selective EBP
ligand cis-4-[4-(2-methoxyphenyl)cyclohexyl]-1-(2-pyridinyl)pip-
erazine (cis-19) as a lead compound (Table 1). Consequently,
novel 2-pyridinylpiperazine derivatives were designed in order
to investigate the importance of methoxyl substituent on the
phenyl moiety and the role of electronic and steric effects of
other substituents (Me, Cl, F) in determining the binding
affinities. Some 2,6-disubstituted-phenyl derivatives (32-35)
were prepared in order to hinder the free rotation of the phenyl
ring linked to the cyclohexyl ring. The isomers of compound
cis-19 for the methoxyl position and for cis/trans geometry were
tested to complete a SAfiR study. The best EBP ligands were
then tested in antiproliferative assays on human prostate cancer
PC-3 cell line.
pharmacological profile and molecular mass, but their structures
are unrelated. On the other hand, yeast ERG2p and human
sigma-1 (σ1) receptor share a 30% amino acid sequence, so that
it has been proposed that σ receptors belong to the family of
sterol isomerases.12 Nevertheless, no enzymatic activity has been
proven for σ1 receptor, although several structural classes of
ligands present both σ1 and EBP pharmacological behavior. In
addition, EBP ligands exert antiproliferative action in various
tumoral cells, and the expression of the EBP protein together
with the expression of the σ1 receptor has been studied as a
biomarker for prognostic purposes in breast carcinoma.13
The high affinity σ1 receptor ligand N-[(2Z)-3-(3-chloro-4-
cyclohexylphenyl)-2-propen-1-yl]-N-ethyl-cyclohexanamine hy-
drochloride (2, SR 31747A; Chart 1) was known to tightly bind
also EBP and to exert antiproliferative effects.14,15 Compound
2 elicited antitumoral activity partially through the inhibition
of EBP isomerization activity.16 Emopamil and related anti-
ischemic drugs are obviously nonselective EBP ligands, as well
as ifenprodil (3), another prototypic EBP ligand much studied,
but presenting high affinities for NMDA site, σ1 and R1 receptors
too.17 Both emopamil and ifenprodil are noncompetitive inhibi-
tors of EBP. Many other ligands have been proven to bind EBP,
but most of them bind equally σ1 receptor and others are drugs
of various classes used in therapy. An exhaustive list is reported
in a recent work, where different pharmacophores have been
proposed for EBP and σ1 receptor ligands, on the basis of
molecular modeling techniques and virtual screening studies.18
A high EBP affinity with moderate selectivity relative to σ1
receptor was displayed by AEBS ligands, such as antitumor
drugs enclomiphene (4) and tamoxifen (5).18 A good selectivity
was also displayed by estrogen receptor modulators raloxifene
(6) and nafoxidine (7).18 All these compounds inhibit isomer-
ization activity of EBP. Nevertheless, high-affinity and selective
EBP ligands are needed to better understand the role and
importance of EBP function.
Chemistry
The synthesis of final compounds 19-21 has already been
reported.23 The synthesis of all the novel target compounds
29-35 is depicted in the Scheme 1 and the intermediate keys

Arylcyclohexyl-(2-pyridyl)piperazines as EBP Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7525
Scheme 1a
Chart 4. Structures of Preliminarily Screened Compounds
are ketones 26f and 28a-f. Starting from the appropriate
bromobenzene derivatives 22b-d, f, the corresponding Grig-
nard’s reagents were generated with Mg turnings and reacted
with 1,4-cyclohexandione monoethylene ketal (23). The result-
ing alcohols were hydrolyzed and dehydrated with HCl in a
single step to afford the corresponding unsaturated ketones
26b-d, f. Similarly, the intermediate ketone 26e was prepared
starting from the organolithium derivative of 1,3-dimethoxy-
benzene (24). The intermediates 26b-e were not isolated and
were reduced with H2 and 10% Pd on activated carbon to obtain
key-compounds 28b-e.24 Intermediate ketone 28a was com-
mercially available, whereas attempts to prepare compound 28f,
by reduction of corresponding unsaturated ketone 26f, failed.
Intermediate ketone 28f was then obtained through a different
synthetic pathway,25 still starting from Grignard’s reagent of
compound 22f. In the presence of a catalytic amount of FeCl3
and TMEDA (N,N,N,N-tetramethylethylendiamine), 2-bromo-
magnesium-m-xylene was cross-coupled with 8-bromo-1,4-
dioxa-spiro[4,5]-decane (25), which was prepared from the ketal
23 by reduction to the corresponding alcohol with NaBH4 and
subsequent bromination with CBr4.26 The resulting intermediate
27 was deprotected to the corresponding ketone 28f. The
intermediates 28a-f were subsequently condensed with 1-(2-
pyridinyl)piperazine to the corresponding enamines, that were
reduced in situ with NaCNBH3 and ZnCl2 to give the final
compounds cis/trans-29-34 as mixtures of geometric isomers.
Column chromatography purification of the mixture provided
the separation of each cis- from the trans-stereoisomers.
Compound 34 was obtained in a very small amount and only
the cis-isomer was isolated and identified.
a Reagents: (A) Mg turnings; (B) HCl or H2SO4; (C) TMEDA, FeCl3;
(D) n-BuLi; (E) H2, Pd/C 10%; (F) 1-(2-pyridinyl)piperazine, ZnCl2,
NaCNBH3.
corresponding enamine, which was reduced in situ with NaC-
NBH3 to give the desired compound as a racemic mixture. All
final 1-(2-pyridinyl)piperazine derivatives were converted to the
corresponding hydrochloride salts with gaseous HCl in the usual
way. Their physical properties are listed in Table 2, along with
the calculated values of the logarithm of the distribution
coefficient (ClogD) at pH 7.4 for the corresponding free bases.27
Biology
Receptor Binding Studies. The already reported compounds
15, 16, cis-18, cis-19, trans-19, cis-21 and trans-21 were assayed
as free bases, and cis-14 as hydrogen oxalate salt. The
compounds 13, cis-17 and cis-20, trans-20 and the novel target
compounds 29-35 were assayed as hydrochloride salts. All the
compounds were evaluated for in vitro affinity at EBP site and
at σ1 and σ2 receptors by radioreceptor binding assays. The
cyclohexylpiperazine 36 (PB 28, Chart 3, structure 11 with
5-OCH3 and R ) cyclohexyl)28 was tested for radioligand
For the synthesis of compound 35, the intermediate 26f was
isolated and condensed with 1-(2-pyridinyl)piperazine to the
Table 1. Binding Affinity Data for Preliminarily Screened Compounds
Ki ( SEM (nM)
a
a
a
compound
5-HT1A
D2
R1
EBP
σ1
σ2
13
cis-14
15
676 ( 28
373 ( 18
257 ( 17
536 ( 24
74 ( 8
183 ( 22
480 ( 15
815 ( 20
94 ( 8
>850
585 ( 60
>850
32.1 ( 4.2
23.5 ( 2.7
460b
358 ( 7
1250 ( 20
783 ( 32
7.71 ( 1.79
2190 ( 300
1220 ( 120
1310 ( 150
2380 ( 160
2120 ( 130
>850
5160 ( 130
512 ( 16
5540 ( 250
2250 ( 160
712 ( 35
783 ( 32
786 ( 29
16
2640b
220 ( 80
cis-17
cis-18
cis-19
cis-20
cis-21
16.7 ( 2.2
240b
1710b
c
c
c
c
c
c
8.15 ( 1.96
45.4 ( 11.5
32.8 ( 0.2
787 ( 43
a Data already reported (ref 23). b Result from a unique experiment. c See Table 3.

7526 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Table 2. Physical Properties of Novel Compounds
Berardi et al.
and apparent inhibition constants (Ki) values were determined
by nonlinear curve fitting, using the Prism v. 3.0, GraphPad
software.29
compound
formulaa
mp, °Cb
ClogDc
cis-29
trans-29
cis-30
trans-30
cis-31
trans-31
cis-32
trans-32
cis-33
trans-33
cis-34
C21H27N3 ·2HCl·3/4H2O
C21H27N3 ·2HCl
268 (dec)
248 (dec)
285 (dec)
288 (dec)
278 (dec)
272 (dec)
248 (dec)
280 (dec)
194-196
296 (dec)
NDd
3.13
3.13
3.58
3.58
3.73
3.73
3.29
3.29
2.84
2.84
4.04
3.89
Antiproliferative and Cytotoxic Assays. The functional
biochemical assays were carried out on androgen-unsensitive
PC-3 cell line of human prostate epithelial carcinoma, where
the expression of EBP site and σ1 receptor had been previously
reported.15 Among the EBP ligands newly synthesized, five
compounds with different binding profile were selected. The
compounds cis-19, cis-30 and cis-33 were chosen as the highest-
affinity and selective EBP ligands, along with EBP relative to
σ1 selective ligand cis-32 with moderate EBP affinity and trans-
20 as a nonselective one. Furthermore, the following positive
reference compounds were tested: EBP-inhibitor ifenprodil, σ2
agonist 36, σ1 antagonist N,N-dipropyl-2-[4-methoxy-3-(2-
phenylethoxy)phenyl]ethylamine (37, NE 100).30 To complete
the assay panel, the following agents were also tested: σ1 agonist
(+)-pentazocine, σ2 antagonist N-(2-phenylethyl)piperidine (38,
AC 927) and mixed σ1/σ2 agonist DTG. All selected compounds
were tested to evaluate their possible EBP-mediated antipro-
liferative effect at 48 h, and cytotoxic effect at 24 h in PC-3
cell line. The maximum cytotoxic effect was assessed when the
Fluorescence Units from LDH (Lactate dehydrogenase) release
by treated cells in medium were the same as from total LDH
measured from total lysis of cells in untreated control. The EC50
values were obtained from nonlinear iterative curve fitting by
Prism v. 3.0, GraphPad software.
C22H29N3 ·2HCl·H2O
C22H29N3 ·2HCl·1/2H2O
C21H26N3Cl·2HCl·5/4H2O
C21H26N3Cl·2HCl·5/4H2O
C21H25N3F2 ·2HCl·1/2H2O
C21H25N3F2 ·2HCl·1/2H2O
C23H31N3O2 ·2HCl·H2O
C23H31N3O2 ·2HCl·2H2O
C23H31N3 ·2HCl·1/2H2O
C23H29N3 ·2HCl·1/2H2O
35
285 (dec)
a Elemental analyses were within (0.4% of the theoretical values for
the formulas given. b Recrystallized from MeOH/Et2O. c Referred to the
corresponding free bases at pH 7.4; isomeric compounds 19-21 displayed
a ClogD value of 3.04. d Not determined, because it was obtained in a very
small amount.
binding assay at EBP site, before being used as reference
compound in the antiproliferative assay. The specific radioli-
gands and tissue sources were, respectively, (a) EBP site, (()-
[3H]-1, guinea-pig liver membranes; (b) σ1 receptor, (+)-[3H]-
pentazocine ((+)-[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 cerebellum; (c) σ2
receptor, [3H]-DTG (1,3-di-2-tolylguanidine) in the presence of
1 µM (+)-pentazocine to mask σ1 receptors, rat liver mem-
branes. The following compounds were used to define the
specific binding reported in parentheses: (a) (()-ifenprodil
(67-85%), (b) (+)-pentazocine (75-84%), (c) DTG (82-93%).
Concentrations required to inhibit 50% of radioligand specific
binding (IC50) were determined by using six to nine different
concentrations of the drug studied in two or three experiments
Results and Discussion
Radioligand Binding and EBP SAfiR. For a preliminary
screening, the lowest-affinity ligands at serotonin 5-HT1A
,
dopamine D2 and adrenergic R1 receptors were selected among
with samples in duplicate. Scatchard parameters (Kd and Bmax
)
various arylpiperazines previously tested (Chart 4).23 The
Table 3. Binding Affinities and Selectivities
Ki ( SEM (nM)
Ki ratio
compound
cis-19
trans-19
cis-20
trans-20
cis-21
trans-21
cis-29
trans-29
cis-30
trans-30
cis-31
trans-31
cis-32
trans-32
cis-33
R
R1
EBP
σ1
σ2
σ1/EBP
45
0.2
4
1.5
6
4
16
6
200
3.4
13
26
37
1.8
>1210
31
0.2
3.8
1.6
0.07
0.5
<2 · 10-3
0.01
0.12
σ2/EBP
OCH3
OCH3
H
H
H
8.15 ( 1.96
13.5 ( 2.6
45.4 ( 11.5
16.2 ( 2.0
32.8 ( 0.2
71.6 ( 15.1
49.2 ( 6.8
16.8 ( 4.2
5.00 ( 1.50
9.04 ( 1.56
85.1 ( 25.9
7.21 ( 1.71
140 ( 2
369 ( 116
2.86 ( 0.95
178 ( 57
24.8 ( 6.5
194 ( 85
291 ( 3
242 ( 61
30.0 ( 8.8
36.1 ( 9.0
21.9 ( 2.1
63.8 ( 21.9
222 ( 19
62.0 ( 5.3
26.9 ( 7.2
71.0 ( 4.0
29.6 ( 4.4
150 ( 43
29.1 ( 10.4
440 ( 39
138 ( 54
687 ( 66
25.0 ( 5.4
60.2 ( 2.2
113 ( 27
0.34 ( 0.02b
212 ( 24
194 ( 23e
1860 ( 40f
31.1 ( 2.5
2.3h
30
2.2
0.8
1.3
2
3-OCH3
3-OCH3
4-OCH3
4-OCH3
H
H
H
H
H
3
802 ( 43
104 ( 10
1000 ( 20
30.7 ( 7.1
1090 ( 50
190 ( 67
5170 ( 280
190 ( 6
1.3
1.6
14
3.3
1.8
4
H
H
H
H
H
CH3
CH3
Cl
Cl
H
F
F
6-F
6-F
6-OCH3
6-OCH3
6-CH3
6-CH3
3
106 ( 6
1.2
138
3.6
1.8
2.4
0.04
14
0.3
<2
4 · 10-3
0.14
OCH3
OCH3
CH3
CH3
4.95 ( 1.73
7.00 ( 1.04
33.1 ( 2.1
46.7 ( 15.0
8.38 ( 0.81
14.6 ( 4.1
595d
>6000
trans-33
cis-34
215 ( 10
6.32 ( 1.07
177 ( 15
13.6 ( 1.9b
1.03 ( 0.14
309 ( 15e
2.41 ( 0.37
88.5 ( 9.0f
2 ( 0.2g
35a
36
37c
38
(+)-pentazocine
DTG
(()-ifenprodil
>104
7660 ( 790
16.4 ( 1.5
a Racemic cyclohexyl-3-ene derivative (see formula in Scheme 1). b From ref 31. c Binding data from ref 19. d Result from a unique experiment. e From
ref 28. f From ref 30. g From ref 18. h From ref 32.

Arylcyclohexyl-(2-pyridyl)piperazines as EBP Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7527
Table 4. Antiproliferative Effect Measured as Inhibition of PC-3 Cancer
binding data for these compounds are reported in Table 1 and
are expressed as Ki values. As for EBP affinity, the results were
encouraging only for compound cis-19 (Ki ) 8.15 nM). The
compounds 13, cis-14, cis-17, cis-20 and cis-21 displayed fairly
lower EBP affinity, with Ki values ranging from 16.7 to 45.4
nM, whereas compounds 15, 16 and cis-18 displayed poor EBP
affinity and were not further investigated. Some of these
compounds were assayed in σ-receptors binding and compound
cis-19 demonstrated good selectivity relative to either σ1 or σ2
receptors. Even though they could be not selective, trans-
stereoisomers of isomeric compounds cis-19-21 were tested
too, in order to complete SAfiR studies.
Cella Proliferation
compound EC50 ( SEMb(µM)
compound
EC50 ( SEMb(µM)
cis-19
trans-20
cis-30
cis-32
cis-33
36
19.3 ( 8.7
23.0 ( 2.5
38.0 ( 6.0
37.3 ( 3.3
12.9 ( 2.9
37.7 ( 1.3
37
38
47.0 ( 12.8
(30%)c
(()-ifenprodil
(+)-pentazocine
DTG
25.8 ( 3.79
86.9 ( 16.7
(27%)c
a Human prostate carcinoma epithelial cell lines. b Mean of n g 2 separate
experiments. c Percent inhibition at 100 µM.
done for corresponding trans-isomers. When the structure is
closer to planarity, as in the cyclohexenyl compound 35, both
EBP affinity and selectivity dropped. As for electronic effects,
the Ki value of 2-chloro derivative cis-31 (Ki ) 85.1 nM) was
1 order of magnitude greater than that of 2-methoxy derivative
cis-19 (Ki ) 8.15 nM). This was not true for the corresponding
stereoisomers trans-31 (Ki ) 7.21 nM) and trans-19 (Ki ) 13.5
nM). Consistently, in the series of cis-isomers the electron-donor
effect was beneficial as for methyl-substituted cis-30 (Ki ) 5.00
nM). The same effect was indifferent for the stereoisomers trans-
19, trans-31 and trans-30, which displayed similar EBP
affinities. It is possible that the trans-compounds enjoy of a more
conformational freedom, whereas cis-compounds are more
constrained. The highest Ki value (Ki ) 140 nM) for 2,6-difluoro
derivative cis-32 demonstrated the negative influence of small
electron-withdrawing substituents, suggesting that electronic
effects were added to the steric ones. This was confirmed by
compound trans-32, which demonstrated the worst EBP affinity
in the set of trans-compounds. The 2,6-dimethylderivative cis-
34 showed an intermediate value (Ki ) 33.1 nM) between those
of dimethoxy compound cis-33 and difluoro compound cis-32.
The results from binding assays (Ki values) at EBP site, σ1
and σ2 receptors are shown in Table 3 for compounds cis/trans-
19-33, cis-34 and 35. The affinities of compounds 19-35 for
the EBP site were not the highest among the affinities of known
EBP ligands, but they were higher than or comparable to the
corresponding σ-receptor affinities. Only compounds trans-19
and cis-34 displayed low Ki values in σ1 receptor binding. As
for EBP affinity, the Ki values ranged from 4.95 nM (compound
cis-33) to 140 nM (compound cis-32). The highest EBP affinities
were reached by cis-isomers cis-33, cis-30 and cis-19 (Ki )
4.95, 5.00, and 8.15 nM, respectively) and by trans-isomers
trans-33, trans-31 and trans-30 (Ki ) 7.00, 7.21, and 9.04 nM,
respectively). Nevertheless, the 2,6-dimethoxy derivative cis-
33 displayed a more interesting selectivity profile compared to
the corresponding trans-33. The same comment can be done
for 2-methyl derivative cis-30 compared to compound trans-
30 and for 2-methoxycompound cis-19 compared to trans-19.
Conversely, 2-chloroderivative trans-31 was a better EBP ligand
than the corresponding cis-31 but only slightly more selective.
The compound cis-33 was the most selective EBP ligand within
this set of compounds, >1210-fold relative to σ1 receptor and
138-fold relative to σ2 receptor.
As shown in Table 2, the ClogD values were included in a
rather narrow range, covering about one log unit (2.84-4.04).
Apparently, no evident relationship was proven between EBP
affinity and ClogD. High-affinity EBP ligands cis-33, cis-30 and
trans-31 (Ki ) 4.95-7.21 nM) had ClogD ranging from 2.84
to 3.73. The lowest ClogD values were reached by the highest-
affinity 2,6-dimethoxylated EBP ligands cis-33 and trans-33.
Conversely, the isomeric 2-methoxy derivatives 19-21 with the
same ClogD ) 3.04 displayed EBP affinities in a wide range
of Ki values (8.15-71.6 nM).
Generally, not great differences in EBP affinities were noted
between cis-compounds and trans-compounds. Moreover, the
presence and the position of methoxyl groups on the phenyl
ring, moderately affected the EBP affinity. As for monomethoxy
derivatives, a small difference occurred between Ki values of
the 2-methoxy isomer cis-19 (Ki ) 8.15 nM) and 3-methoxy
isomer cis-20 (Ki ) 45.4 nM) or 4-methoxy isomer cis-21 (Ki
) 32.8 nM), or unsubstituted compound cis-29, excluding a
significant influence of the electronic effect of methoxyl group.
The lowest Ki value (4.95 nM) occurred for 2,6-dimethoxy
derivative cis-33, evidencing the main role of steric effects in
the ortho position. Moreover, 2,6-dimethoxy derivative cis-33
presented an EBP affinity only equipotent to 2-monomethoxy
derivative cis-19, but the former displayed lower σ1 and σ2
receptor affinities, resulting in a much better selectivity,
particularly relative to σ1 receptor.
σ SafiR. All compounds were selected as potentially low-
affinity σ ligands; nevertheless, compounds trans-19 (Ki ) 2.86
nM) and cis-34 (Ki ) 6.32 nM) displayed preferentially σ1
receptor affinity and a low selectivity. Several compounds,
mainly in the trans-isomers series displayed moderate σ2 affinity.
The lowest σ1 affinities were recorded for compounds cis-33,
cis-32, cis-31 and cis-30 (Ki g 1000 nM), all belonging to the
cis-isomers series. Within this series, the affinity for the σ1
subtype dramatically dropped when the bulky group was in the
2-position, whereas the affinity for the EBP site was unchanged
leading to higher EBP-site selectivity relative to σ1 receptor.
The highest Ki (>6000 nM) belonged to the best EBP ligand
cis-33. Therefore, compound cis-33 was the most selective high-
affinity EBP ligand. In the trans-isomers series, the 2-substitution
only slightly affected the σ1 receptor affinity, leading to lower
EBP relative to σ1 selectivity. This same trend was also observed
for the affinities toward the σ2 receptor, although at a lower
extent, with a higher decrease in the affinity within the cis-
isomers series rather than in the trans-isomers series. These
findings were in accordance with the virtual pharmacophore
models of EBP and σ1 receptor derived recently, where σ1
receptor model preferentially bound linear ligands, as these new
trans-compounds.18
Functional Assays and SAR. The results expressed as EC50
values were reported in Table 4. In the human prostate cancer
PC-3 cell line, the σ/EBP receptor ligand 2 showed to exert its
antiproliferative activity through the EBP inhibition with a
nanomolar efficacy. σ1 Subtype receptor was demonstrated to
be not responsible for such effect, but interaction of 2 with the
σ2 subtype was hypothesized. In fact, inhibition of sterol
isomerase activity alone could not fully account for the
antiproliferative effect exerted by 2: neither direct correlation
For the cis-compounds the selectivity was high when the
2-position is occupied by a methoxyl or a methyl group; 2-chloro
or 2-fluoro substituent were detrimental for EBP affinity and
consequently for selectivity. Analogous considerations could be

7528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Berardi et al.
were less potent than ifenprodil. As expected, σ1 antagonist 37
had a higher potency and efficacy than (+)-pentazocine. DTG
and compound 38 were practically inactive in this assay. All
tested compounds displayed poor cytotoxic activity (<10% at
100 µM), while only compound 36 had a moderately cytotoxic
activity (EC50 ) 64.2 µM).
The ClogD values for the tested compounds increased from
2.84 for the most active compound cis-33 to 3.58 for the less
active cis-30, displaying a linear relationship between antipro-
liferative activity and ClogD. In this respect, in the most active
compounds cis-33, cis-19 and trans-20, the methoxyl groups
may contribute to the activity also by decreasing ClogD. The
reference compound 36 had a ClogD ) 3.75.
Conclusions
A new class of selective EBP ligands was found by opening
the tetralin moiety and constraining the intermediate chain of
(tetralinalkyl)arylpiperazines in the arylcyclohexyl-1-(2-py-
ridyl)piperazine derivatives. The electronic and steric effects
were investigated in a series of semirigid 2- and 2,6-disubstituted
cis- and trans-phenylcyclohexyl derivatives. Several compounds,
particularly in the cis series, displayed nanomolar affinity and
good selectivities relative to σ1 and σ2 receptors. The 2,6-
dimethoxy derivative cis-33 showed the highest EBP affinity
and the best selectivities. In an antiproliferative assay on PC-3
cell line, cis-33 demonstrated the highest potency but not the
best efficacy that was reached by σ2 agonist 36. Compound cis-
33 appeared as a valuable pharmacological tool for investigating
enzyme activity in the last steps of cholesterol biosynthesis. Its
partial antiproliferative activity has to be further investigated
to define the role of σ2 receptor activation in this assay.
Nevertheless cis-33 may represent a starting point for a new
approach to anticancer treatment.
Figure 1. Antiproliferative effect of compounds tested in androgen-
unsensitive PC-3 cell line of human prostate cancer. Incubation was
carried out for 48 h in the presence (1s100 µM) and absence of tested
compound (ref 15). For compound 36 and (+)-pentazocine, the
concentration ranges were 1s150 and 1s300 µM, respectively.
between cellular EBP content and effect was found, nor the
antiproliferative effect was completely reversed by the addition
of cholesterol to the medium.16 Thus, it appeared important to
define the role of the σ2 receptor in mediating the antiprolif-
erative action in such cell lines. First, we recognized the σ2
receptor content in PC-3 cell line and its density was found
comparable to that of EBP sites. To understand the importance
of the three binding sites in determining the antiproliferative
effect, the following compounds were selected: cis-33 and cis-
19 as selective EBP ligands almost devoid of σ receptors
affinities, the former being the most selective and highest-affinity
EBP ligand; trans-20 for displaying equal and moderate affinity
and cis-32 for displaying low affinity toward the three sites;
cis-30 for being high affinity toward the EBP site and only
slightly selective toward the σ2 subtype. With the purpose to
correlate the antiproliferative effect also with the σ receptor
content, ifenprodil and some σ agents were evaluated. Almost
all the tested compounds displayed a certain antiproliferative
activity. Among the reference compounds, the σ2 agonist 36
was the only one reaching the maximum inhibition of prolifera-
tion. Such result can be explained with the activity of compound
36 both on σ2 receptor and EBP site. Compound cis-33 did not
reach the maximum inhibition (70%), although it was more
potent (EC50 ) 12.9 µM) than 36 (Figure 1), maybe for its lack
of affinity to the σ2 receptor subtype (Ki ) 687 nM). Lower
efficacy and potency were shown by compound cis-19 in
comparison to cis-33, as expected by the slightly lower EBP
affinity of cis-19 along with its low affinity for the σ2 receptor.
Although cis-30 displayed rather higher EBP affinity than cis-
32, this latter surprisingly was more potent and effective than
cis-30. This result may be explained with a possible cis-30
antagonist activity at σ2 receptor. The other tested compounds
did not reach 70% efficacy at 100 µM (Figure 1). Among the
reference compounds, ifenprodil presented the best potency
value (EC50 ) 25.8 µM). Its efficacy was a little over 50% as
for σ1 receptor agents (+)-pentazocine and 37, but these latter
Experimental Section
Chemistry. Column chromatography was performed with 1:30
ICN silica gel 60 Å (63-200 or 15-40 µm, respectively) as the
stationary phase. Melting points were determined in open capillaries
on a Gallenkamp electrothermal apparatus. Elemental analyses (C,
H, N) were performed on a Eurovector Euro EA 3000 analyzer;
the analytical results were within (0.4% of the theoretical values.
1H NMR spectra were recorded at 300 MHz on a Mercury Varian
spectrometer using CDCl3 as solvent unless otherwise reported. The
chemical shift values were reported in ppm (δ). Recording of mass
spectra was done on an Agilent 6890-5973 MSD gas chromato-
graph/mass spectrometer and on an Agilent 1100 series LC-MSD
trap system VL; 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 4-(2-Pyridinyl)pipera-
zine Derivatives cis/trans-29-33, cis-34, and 35. In a typical
reaction, one intermediate (1.0 mmol) among the 4-phenylcyclo-
hexanone derivatives 28a-f and 4-(2,6-dimethylphenyl)-3-cyclo-
hexen-1-one (26f) were reacted with the 1-(2-pyridinyl)piperazine
(1.0 mmol), zinc(II) chloride (0.58 mmol) and NaCNBH3 (1.1
mmol) in 2-propanol (20 mL). The mixture was stirred for 48 h at
room temperature. Then, the reaction mixture was evaporated to
dryness and the residue was diluted with 2 N NaOH (20 mL) and
extracted with AcOEt (3 × 20 mL). The organic layers were washed
with brine (20 mL), dried (Na2SO4) and then concentrated under
reduced pressure to give a crude residue. Purification by column
chromatography afforded the separated cis- and trans-isomers of
final compounds. Consistently, the cis-stereoisomers were eluted
before their trans counterparts. Attribution of cis/trans geometry
1
was done on the basis of H NMR chemical shifts and coupling

Arylcyclohexyl-(2-pyridyl)piperazines as EBP Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23 7529
constants for NCH and CHAr in the cyclohexyl ring, assisted also
by NOESY-NMR.
cis-4-(4-Phenylcyclohexyl)-1-(2-pyridinyl)pipera-
aromatic), 7.03-7.14 (m, 1H, aromatic), 7.44-7.52 (m, 1H, Py),
8.18-8.22 (m, 1H, Py NdCH); GC-MS m/z 358 (M+ + 1, 5), 357
(M+, 15), 238 (58), 107 (100). Anal. (C21H25N3F2 ·2HCl ·1/2H2O) C,
H, N.
zine (cis-29) was obtained as white crystals in 35% yield with
CH2Cl2/AcOEt (4:6) as eluent: 1H NMR δ 1.50-1.72 (m, 4H,
cyclohexylic), 1.94-2.18 (m, 4H, cyclohexylic), 2.27-2.37 (m, 1H,
NCH), 2.56-2.77 [m, 5H, benzyl CH and CHN(CH2)2], 3.48-3.62
[m, 4H, (CH2)2NPy], 6.58-6.68 (m, 2H, Py), 7.14-7.21 (m, 1H,
aromatic), 7.25-7.32 (m, 4H, aromatic), 7.44-7.51 (m, 1H, Py),
8.17-8.22 (m, 1H, Py NdCH); GC-MS m/z 322 (M+ + 1, 6),
321 (M+, 21), 227 (42), 107 (100). Anal. (C21H27N3 ·2HCl·3/4H2O)
C, H, N.
trans-4-(4-Phenylcyclohexyl)-1-(2-pyridinyl)piperazine (trans-
29) was obtained as white crystals in 30% yield with CH2Cl2/AcOEt
(4:6) as eluent: 1H NMR δ 1.40-1.62 (m, 4H, cyclohexylic),
1.95-2.12 (m, 4H, cyclohexylic), 2.40-2.56 (m, 2H, benzyl CH
and NCH), 2.71-2.82 [m, 4H, CHN(CH2)2], 3.52-3.64 [m, 4H,
(CH2)2NPy], 6.58-6.68 (m, 2H, Py), 7.15-7.32 (m, 5H, aromatic),
7.44-7.52 (m, 1H, Py), 8.16-8.22 (m, 1H, Py NdCH); GC-MS
m/z 322 (M+ + 1, 4), 321 (M+, 16), 227 (31), 107 (100). Anal.
(C21H27N3 ·2HCl) C, H, N.
cis-4-[4-(2-Methylphenyl)cyclohexyl]-1-(2-pyridinyl)piperazine
(cis-30) was obtained as white crystals in 42% yield with CH2Cl2/
AcOEt (1:1) as eluent: 1H NMR δ 1.47-1.62 (m, 4H, cyclohexylic),
1.84-2.00 (m, 2H, cyclohexylic), 2.08-2.19 (m, 2H, cyclohexylic),
2.27-2.34 (m, 1H, NCH), 2.35 (s, 3H, CH3), 2.58-2.66 [m, 4H,
CHN(CH2)2], 2.85 (tt, 1H, J ) 11.4 Hz, J′ ) J′′ ) 3.58 Hz, ax benzyl
CH), 3.53-3.60 [m, 4H, (CH2)2NPy], 6.58-6.70 (m, 2H, Py),
7.04-7.22 (m, 3H, aromatic), 7.30 (d, 1H, J ) 6.9 Hz, aromatic),
7.44-7.53 (m, 1H, Py), 8.17-8.24 (m, 1H, Py NdCH); GC-MS
m/z 336 (M+ + 1, 5), 335 (M+, 19), 216 (64), 107 (100). Anal.
(C22H29N3 ·2HCl·H2O) C, H, N.
trans-4-[4-(2-Methylphenyl)cyclohexyl]-1-(2-pyridinyl)pipera-
zine (trans-30) was obtained as white crystals in 36% yield with
CH2Cl2/AcOEt (4:6) as eluent: 1H NMR δ 1.43-1.60 (m, 4H,
cyclohexylic), 1.87-1.98 (m, 2H, cyclohexylic), 2.06-2.17 (m, 2H,
cyclohexylic), 2.33 (s, 3H, CH3), 2.45-2.58 (m, 1H, NCH),
2.64-2.87 [m, 5H, benzyl CH and CHN(CH2)2], 3.53-3.68 [m,
4H, (CH2)2NPy], 6.60-6.69 (m, 2H, Py), 7.05-7.22 (m, 4H,
aromatic), 7.44-7.53 (m, 1H, Py), 8.17-8.23 (m, 1H, Py NdCH);
GC-MS m/z 336 (M+ + 1, 4), 335 (M+, 15), 216 (49), 107 (100).
Anal. (C22H29N3 ·2HCl· 1/2H2O) C, H, N.
cis-4-[4-(2-Chlorophenyl)cyclohexyl]-1-(2-pyridinyl)pipera-
zine (cis-31) was obtained as white crystals in 20% yield with
CH2Cl2/AcOEt (1:1) as eluent: 1H NMR δ 1.52-1.68 (m, 4H,
cyclohexylic), 1.70-1.97 (m, 2H, cyclohexylic), 2.07-2.18 (m, 2H,
cyclohexylic), 2.25-2.36 (m, 1H, NCH), 2.54-2.70 [m, 4H,
CHN(CH2)2], 3.08-3.22 (br t, 1H, J ) 11.4 Hz, benzyl CH),
3.48-3.66 [m, 4H, (CH2)2NPy], 6.60-6.69 (m, 2H, Py), 7.10 (dt,
1H, J ) 7.6 Hz, J′ ) 1.6 Hz, aromatic), 7.19-7.24 (br t, 1H,
aromatic), 7.33 (dd, 2H, J ) 7.7 Hz, J′ ) 1.4 Hz, aromatic),
7.45-7.52 (m, 1H, Py), 8.18-8.21 (m, 1H, Py NdCH); GC-MS
m/z 357 (M+ + 2, 4), 356 (M+ + 1, 3), 355 (M+, 11), 236 (44),
107 (100). Anal. (C21H26N3Cl·2HCl· 5/4H2O) C, H, N.
trans-4-[4-(2-Chlorophenyl)cyclohexyl]-1-(2-pyridinyl)pipera-
zine (trans-31) was obtained as white crystals in 20% yield with
CH2Cl2/AcOEt (1:1) as eluent: 1H NMR δ 1.40-1.70 (m, 4H,
cyclohexylic), 1.98-2.20 (m, 4H, cyclohexylic), 2.53-2.66 (m, 1H,
NCH), 2.75-2.90 [m, 4H, CHN(CH2)2], 2.93-3.07 (br t, 1H, J )
11.6 Hz, benzyl CH), 3.58-3.75 [m, 4H, (CH2)2NPy], 6.61-6.68
(m, 2H, Py), 7.09-7.15 (m, 1H, aromatic), 7.18-7.25 (m, 2H,
aromatic), 7.34 (dd, 1H, J ) 8.3 Hz, aromatic), 7.45-7.53 (m, 1H,
Py), 8.18-8.21 (m, 1H, Py NdCH); GC-MS m/z 357 (M+ + 2,
3), 356 (M+ + 1, 3), 355 (M+, 9), 236 (35), 107 (100). Anal.
(C21H26N3Cl·2HCl·5/4H2O) C, H, N.
trans-4-[4-(2,6-Difluorophenyl)cyclohexyl]-1-(2-pyridinyl)pi-
perazine (trans-32) was obtained as white crystals in 17% yield
with CH2Cl2/AcOEt (4:6) as eluent: 1H NMR δ 1.34-1.52 (m, 2H,
cyclohexylic), 1.80-2.10 (m, 6H, cyclohexylic), 2.48 (tt, 1H, J )
11.6 Hz, J′ ) 3.4 Hz, NCH), 2.68-2.77 [m, 4H, CHN(CH2)2],
2.90-3.02 (m, 1H, benzyl CH), 3.50-3.58 [m, 4H, (CH2)2NPy],
6.58-6.68 (m, 2H, Py), 6.81 (t, 2H, J ) 8.5 Hz, aromatic),
7.05-7.15 (m, 1H, aromatic), 7.43-7.51 (m, 1H, Py), 8.17-8.21
(m, 1H, Py NdCH); GC-MS m/z 358 (M+ + 1, 4), 357 (M+,
15), 238 (43), 107 (100). Anal. (C21H25N3F2 · 2HCl · 1/2H2O) C,
H, N.
cis-4-[4-(2,6-Dimethoxyphenyl)cyclohexyl]-1-(2-pyridinyl)pi-
perazine (cis-33) was obtained as white crystals in 43% yield with
CH2Cl2/AcOEt 1:1 as eluent: 1H NMR δ 1.18-1.31 (m, 1H,
cyclohexylic), 1.42-1.56 (m, 2H, cyclohexylic), 1.58-1.67 (m, 1H,
cyclohexylic), 2.02-2.14 (m, 2H, cyclohexylic), 2.25-2.30 (br m,
1H, NCH), 2.41-2.72 [m, 6H, 2H cyclohexylic and CHN(CH2)2],
3.32 (tt, 1H, J ) 12.2 Hz, J′ ) 4.0 Hz, benzyl CH), 3.50-3.65 [m,
4H, (CH2)2NPy], 3.78 (s, 6H, 2 OCH3), 6.53 (d, 2H, J ) 8.3 Hz,
aromatic), 6.58-6.66 (m, 1H, Py), 6.68 (d, 1H, J ) 8.5 Hz, Py),
7.09 (t, 1H, J ) 8.4 Hz, aromatic), 7.45-7.53 (m, 1H, Py),
8.18-8.23 (m, 1H, Py NdCH); GC-MS m/z 382 (M+ + 1, 5),
381 (M+, 20), 274 (70), 262 (100), 151 (81), 107 (92). Anal.
(C23H31N3O2 ·2HCl·H2O) C, H, N.
trans-4-[4-(2,6-Dimethoxyphenyl)cyclohexyl]-1-(2-pyridinyl)pi-
perazine (trans-33) was obtained as white crystals in 36% yield
with CH2Cl2/AcOEt (4:6) as eluent: 1H NMR δ 1.35-1.54 (m, 2H,
cyclohexylic), 1.61-1.73 (m, 2H, cyclohexylic), 1.98-2.26 (m, 4H,
cyclohexylic), 2.47-2.63 (m, 1H, NCH), 2.72-2.89 [m, 4H,
CHN(CH2)2], 3.20 (tt, 1H, J ) 12.1 Hz, J′ ) 3.5 Hz, benzyl CH),
3.54-3.70 [m, 4H, (CH2)2Py], 3.78 (s, 6H, OCH3), 6.53 (d, 2H, J
) 8.3 Hz, aromatic), 6.58-6.70 (m, 2H, Py), 7.10 (t, 1H, J ) 8.3
Hz, aromatic), 7.44-7.53 (m, 1H, Py), 8.17-8.23 (m, 1H, Py
NdCH); GC-MS m/z 382 (M+ + 1, 5), 381 (M+, 19), 274 (58),
262 (88), 151 (85), 107 (100). Anal. (C23H31N3O2 ·2HCl·2H2O)
C, H, N.
cis-4-[4-(2,6-Dimethylyphenyl)cyclohexyl]-1-(2-pyridinyl)pi-
perazine (cis-34) was afforded by column chromatography in 36%
yield with AcOEt as eluent. Only the cis-isomer was obtained in
enough amount for analytical purposes; 1H NMR δ 1.38-1.45 (m,
4H, cyclohexylic), 1.78-1.82 (m, 2H, cyclohexylic), 1.90-2.18
(m, 2H, cyclohexylic), 2.20-2.52 (m, 7H, NCH and 2 CH3),
2.68-2.80 [m, 4H, CHN(CH2)2], 2.85-3.02 (m, 1H, benzyl CH),
3.50-3.62 [m, 4H, (CH2)2NPy], 6.58-6.70 (m, 2H, Py), 6.82-7.00
(m, 3H, aromatic), 7.42-7.58 (m, 1H, Py), 8.17-8.22 (m, 1H, Py
NdCH); GC-MS m/z 349 (M+, 3), 230 (30), 107 (100). LC-MS
on the hydrochloride salt (ESI+) m/z 350 [M + H+]; LC-MS-MS
350: 149, 121. Anal. C, H, N: not determined, due to the lack of
product.
4-[1-(2,6-Dimethylphenyl)-3-cyclohexen-4-yl]-1-(2-pyridinyl)pi-
perazine (35) was obtained as an oil in 76% yield with CH2Cl2/
AcOEt (1:1) as eluent: 1H NMR δ 1.43-1.78 (m, 2H, CHCH2CH2),
2.10-2.48 (mm, 5H, 2 allylic CH2 and NCH), 2.19 (s, 3H, CH3),
2.23 (s, 3H, CH3), 2.64-2.86 [m, 4H, CHN(CH2)2], 3.48-3.70 [m,
4H, (CH2)2NPy], 5.38-5.47 (m, 1H, vinyl CH), 6.58-6.72 (m, 2H,
Py), 6.98-7.08 (m, 3H, aromatic), 7.44-7.53 (m, 1H, Py),
8.17-8.23 (m, 1H, Py NdCH); GC-MS m/z 338 (M+ + 1, 6),
337 (M+, 21), 240 (31), 107 (100). Anal. (C23H29N3 ·2HCl·1/2H2O)
C, H, N.
Biological Methods: Radioligand Binding Assays. All the
procedures for the binding assays were previously described.
∆8-∆7 SI (EBP) binding was carried out according to Moebius
et al.17 σ1 and σ2 receptor binding were carried out according
to Matsumoto et al.33 The radioligand (()-[3H]-1 (83 Ci/mmol)
was purchased from American Radiolabeled Chemicals, Inc. (St.
Louis, MO). [3H]-DTG (30 Ci/mmol) and (+)-[3H]-pentazocine
(34 Ci/mmol) were purchased from PerkinElmer Life Sciences
cis-4-[4-(2,6-Difluorophenyl)cyclohexyl]-1-(2-pyridinyl)pipera-
zine (cis-32) was obtained as white crystals in 19% yield with CH2Cl2/
AcOEt (4:6) as eluent: 1H NMR δ 1.39-1.63 (m, 4H, cyclohexylic),
2.07-2.38 (m, 5H, cyclohexylic), 2.55-2.68 [m, 4H, CHN(CH2)2],
3.10 (br t, 1H, J ) 12.1 Hz, benzyl CH), 3.48-3.65 [m, 4H,
(CH2)2NPy], 6.58-6.70 (m, 2H, Py), 6.80 (t, 2H, J ) 8.5 Hz,

7530 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 23
Berardi et al.
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(Zavantem, Belgium). (()-Ifenprodil and DTG were purchased
from Tocris Cookson, Ltd., U.K. (+)-Pentazocine 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 prostate cancer PC-3 cell line was
obtained from Interlab Cell Line Collection (ICLC, Genoa). PC-3
line was routinely cultured in RPMI 1640 supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 U/mL penicillin,
and 100 µg/mL streptomycin, 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-diphenyltet-
razoliumbromide (MTT) assay at 48 h.30,34 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 cytotox-
icity. After the established incubation 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 solubi-
lized using 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.
Cytotoxicity Assay. The assay was performed using the
CytoTox-One kit from Promega Corp. (Madison, WI) as reported
by Colabufo et al.30 with minor modifications. Cell death was
determined as release of LDH into the culture medium. The
percentage of cytotoxicity was calculated relative to the LDH
release from total lysis of cells in untreated control. It is assumed
here that the drug-treated wells and the control wells contain
the same total number of cells (dead plus alive cells) at the end
of the treatment period. Therefore, the cytotoxic effect of tested
compounds was unaffected by underestimation of cytotoxicity
that should occur because of decreased total number of cells in
the treated samples compared to the untreated control. Cells were
seeded into 96-well plates for optical performance in the
fluorescent cell-based assay in 100 µL of complete medium in
the presence or absence of different concentrations of test
compounds. The plate was incubated for 24 h in a humidified
atmosphere of 5% CO2 at 37 °C and then 100 µL of substrate
mix in assay buffer was added. Ten microliters of lysis solution
was added to untreated wells in order to estimate total LDH.
Plates were kept protected from light for 10 min at room
temperature, and then 50 µL of stop solution was added to all
wells. The fluorescence was recorded using a LS55 Lumines-
cence Spectrometer PerkinElmer with a 560 nm excitation
wavelength and a 590 nm emission wavelength. Percent cyto-
toxicity was estimated as follows: 100 × (LDH in medium of
treated cells - culture medium background)/(total LDH in
untreated cells - culture medium background).
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Supporting Information Available: Elemental analyses of the
end products, description of the preparation and spectroscopy
data for the intermediate compounds 26f, 27 and 28b-f. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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