1-Cyclohexyl-4-(4-arylcyclohexyl)piperazines: Mixed σ and Human Δ8-Δ7 Sterol Isomerase Ligands with Antiproliferative and P-Glycoprotein Inhibitory Activity

Article abstract of DOI:10.1002/cmdc.201000371

Many new chemotherapeutic agents are under preclinical investigation and, despite efforts to more selectively target cancer cells, limitations such as toxicity and inherent resistance are often encountered. Therefore, alternative strategies are needed to treat cancer and overcome such limitations. We describe novel cyclohexylpiperazine derivatives, designed as mixed affinity ligands for sigma (σ) receptors and human Δ₈-Δ₇ sterol isomerase (HSI) ligands, which also exhibit P-glycoprotein (P-gp) inhibitory activity, with the aim of exploiting the antiproliferative effects mediated by σ and HSI sites while overcoming P-gp-mediated resistance. All of the compounds displayed high affinities for σ receptors and HSI sites, P-gp inhibitory activity, and σ₂ receptor agonist antiproliferative activity. Antiproliferative activity was also tested in PC-3 cells to establish σ₁ and HSI contribution. Compound cis-11, which displayed the best antiproliferative and P-gp inhibitory activities, was co-administered with 0.1 μM doxorubicin in MDCK-MDR1 cells. Compound cis-11 caused 70 % and 90 % cell death when co-administered at 30 μM and 50 μm, respectively. When administered alone, cis-11 resulted in 50 % cell death, demonstrating its single agent antitumor properties in a tumor cell line overexpressing P-gp.Synergistic mixed activity: A series of cyclohexylpiperazines with mixed σ and HSI affinities and P-gp inhibitory activity were synthesized. Their antiproliferative activity, combined with P-gp inhibitory activity, shows the potential of these compounds to be used as antitumor agents devoid of P-gp-mediated resistance, or in association with classic antitumor agents susceptible to P-gp activity.

Full text of DOI:10.1002/cmdc.201000371

DOI: 10.1002/cmdc.201000371
1-Cyclohexyl-4-(4-arylcyclohexyl)piperazines: Mixed s and
Human D8–D7 Sterol Isomerase Ligands with
Antiproliferative and P-Glycoprotein Inhibitory Activity
Carmen Abate,* Mauro Niso, Marialessandra Contino, Nicola Antonio Colabufo,
Savina Ferorelli, Roberto Perrone, and Francesco Berardi[a]
Many new chemotherapeutic agents are under preclinical in-
vestigation and, despite efforts to more selectively target
cancer cells, limitations such as toxicity and inherent resistance
are often encountered. Therefore, alternative strategies are
needed to treat cancer and overcome such limitations. We de-
scribe novel cyclohexylpiperazine derivatives, designed as
mixed affinity ligands for sigma (s) receptors and human D8–
D7 sterol isomerase (HSI) ligands, which also exhibit P-glyco-
protein (P-gp) inhibitory activity, with the aim of exploiting the
antiproliferative effects mediated by s and HSI sites while over-
coming P-gp-mediated resistance. All of the compounds dis-
played high affinities for s receptors and HSI sites, P-gp inhibi-
tory activity, and s2 receptor agonist antiproliferative activity.
Antiproliferative activity was also tested in PC-3 cells to estab-
lish s1 and HSI contribution. Compound cis-11, which displayed
the best antiproliferative and P-gp inhibitory activities, was co-
administered with 0.1 mm doxorubicin in MDCK-MDR1 cells.
Compound cis-11 caused 70% and 90% cell death when co-
administered at 30 mm and 50 mm, respectively. When adminis-
tered alone, cis-11 resulted in 50% cell death, demonstrating
its single agent antitumor properties in a tumor cell line over-
expressing P-gp.
Introduction
Despite considerable efforts in cancer research, tumors remain
one of the main causes of human illness and death. There are
several chemotherapeutic agents that, through different mech-
anisms, are used to treat cancer, but they are frequently ac-
companied by serious side effects mainly due to lack of tumor
specificity.[1] Although physical targeting techniques, such as
directed radiotherapy or intratumoral injections, enable precise
delivery to the tumor, metastases are often difficult to reach.
The newer molecular targeted therapies aim to treat cancer
cells more selectively by exploiting tumor-specific characteris-
tics. Monoclonal antibodies such as trastuzumab,[2] or tyrosine
kinase inhibitors such imatinib[3] or sunitinib,[4] belong to this
class. Many different types of targeted therapies have been de-
veloped to treat cancer, and a number of potential drugs are
under investigation in clinical trials.[3,5] However, many of these
strategies show limitations such as tissue toxicity and resist-
ance.[6] Some tumors are intrinsically resistant, others may
become resistant to pharmacological therapy, and drug efflux
is one of the main reasons for resistance.[7] Drug efflux is medi-
ated by plasma membrane transporters such as P-glycoprotein
(P-gp), breast cancer resistant protein (BCRP), and multidrug
resistant associated proteins (MRPs), all of which belong to the
ATP binding cassette (ABC) protein superfamily.[8–10]
of P-gp inhibitors have been developed to date in an attempt
to decrease the toxicity associated with original inhibitors (“L-
type” calcium channel blocker verapamil and immunosuppres-
sant cyclosporin A). Less toxic compounds from the most
recent generation (e.g., tariquidar, laniquidar, and elacridar)
have entered clinical trials but have not exhibited suitable re-
sults;[12] therefore, clinical applications remain distant. Conse-
quently, alternative strategies are needed to counteract trans-
porter-mediated resistance.
The two subtypes of sigma (s) receptors, s1 and s2, repre-
sent novel targets for potential tumor treatment.[13] Both sub-
types are overexpressed in a variety of tumors and activate
apoptotic pathways. Several studies have been undertaken
with labeled s receptor ligands for diagnostic PET analysis.[14,15]
The mechanism through which s receptors induce apoptosis is
not yet understood, because contradictory results were ob-
tained for different cells and/or different ligands.[16–20] Studies
[a] Dr. C. Abate, Dr. M. Niso, Dr. M. Contino, Prof. Dr. N. A. Colabufo,
Prof. S. Ferorelli, Prof. Dr. R. Perrone, Prof. Dr. F. Berardi
Dipartimento Farmacochimico, Universitꢀ degli Studi di Bari ALDO MORO
Via Orabona 4, 70125 Bari (Italy)
Fax: (+39)080-5442231
E-mail: abate@farmchim.uniba.it
These transporters are overexpressed in chemoresistant
tumors and decrease or abolish the effects of several antitumor
agents (e.g., doxorubicin) that are their substrates. Therefore,
inhibition of such proteins represents a strategy to circumvent
cellular resistance to chemotherapeutic agents. Upon co-ad-
ministration, pump inhibitors allow antitumor agents to enter
cells and exert their tumoricidal action.[10,11] Three generations
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000371: Elemental analyses of the
novel end products, formulas, melting points of hydrochloride salts, and
clogP values of final compounds cis- and trans-7–14 are listed in the
table of physical properties. Information is also provided for chromatog-
raphy eluents for purification, melting points, yields, and spectroscopic
characterizations for compounds cis- and trans-8 and cis- and trans-10–
14.
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with (R,S)-1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaph-
thalen-1-yl)-n-propyl]piperazine (1, (R,S)-PB28; Figure 1) gener-
ated the hypothesis that the s2 receptor could be a histone
protein,[21,22] while other s2 receptor ligands demonstrated that
From this series, 6,7-dimethoxy-2-{[5-methoxy-3,4-dihydro-
(1E)(2H)-naphthalenylidene]propyl}-1,2,3,4-tetrahydroisoquino-
line (4, MC18)[34] and 4’-[(6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinolin-2-yl)methyl]biphenyl-4-ol (5, MC70)[35] (Figure 2)
Figure 2. Structures of P-gp inhibitors.
Figure 1. Structures of ligands with s receptor or mixed s/HSI activity.
emerged as the most potent P-gp inhibitors. We observed that
a decrease in conformational freedom, relative to 1, led to
better P-gp inhibitory properties. This evidence prompted us
to develop a series of conformationally constrained analogues
of 1 in which the propylene linker was locked into a cyclohexyl
ring, while a basic 4-cyclohexylpiperazine moiety was able to
provide excellent s affinity[36–38] and appreciable P-gp inhibitory
activity.[39]
this subtype is not localized within the nucleus.[23] Despite
these contradictory results, existing evidence shows that s1 an-
tagonists and s2 agonists exert antiproliferative activity in sev-
eral tumor cell lines.[24,25] Recently, promising preliminary re-
sults were reported for two tumors types that generally have
the poorest prognosis: pancreatic adenocarcinoma[26,27] and
glioblastoma.[28] The s2 agonist N-(9-(4-aminohexyl)-9-
azabicyclo[3.3.1]nonan-3a-yl)-N-(2-methoxy-5-methylphenyl)-
carbamate (2, SV119; Figure 1) augmented tumoricidal activity
of paclitaxel and gemcitabine in pancreatic adenocarcinoma
cells,[26,27] and various s1 ligands exhibited antiproliferative ef-
fects in glioma cells.[28] Human D8–D7 sterol isomerase (HSI) is
pharmacologically related to s receptors, and was originally
proposed to be a member of the s receptor family.[29] There-
fore, s receptor ligands were also tested against HSI, the inhib-
ition of which was shown to induce antiproliferative effects in
a series of human tumor cell lines.[30] Ligands such as N-[(2Z)-3-
(3-chloro-4-cyclohexylphenyl)-2-propen-1-yl]-N-ethylcyclohex-
anamine hydrochloride (3, SR 31747A; Figure 1) which has a
mixed s1, s2, and HSI affinity profile, have also been widely
studied in phase IIb clinical trials in androgen-independent
non-metastatic prostate cancer (ODYSSEY) cells.[31]
We designed our new molecules keeping in mind a series of
(2-pyridyl)piperazines that were synthesized as HSI inhibitors.[33]
Our rationale was that substitution of the pyridyl group with a
cyclohexyl ring could extend affinity to the s subtypes.[33] De-
velopment of this novel series may lead to the identification of
a new compound with potent antitumoral activity (resulting
from combined s receptor affinity and HSI inhibition), along
with potent P-gp inhibitory activity. Changes were made to
evaluate the role of steric and electronic effects of different
phenyl ring substituents (OCH3, CH3, F, Cl) on s receptor affini-
ty, as well as HSI and P-gp activities. For each substituent, the
corresponding cis and trans isomers were obtained.
Chemistry
Procedures for the synthesis of final compounds cis/trans 7–14
were previously reported for similar derivatives[33,40] and are de-
picted in Scheme 1. Intermediate ketones 6b–d, although
commercially available, were synthesized according to the pro-
cedure previously reported for the other known intermediate
ketones 6a, e–h.[33] Starting from the appropriate bromoben-
zene derivatives, corresponding Grignard reagents were gener-
ated and combined with 1,4-cyclohexandione monoethylene
ketal. The resulting alcohols were hydrolyzed and dehydrated
to the unsaturated ketones, which were reduced to known in-
termediate ketones 6a–h. All ketones were subsequently con-
densed with cyclohexylpiperazine to the corresponding enam-
ines. Reduction with NaCNBH3 gave final compounds cis/trans
7–14 as mixtures of geometric isomers, which were separated
by column chromatography to afford the cis- and trans-stereo-
isomers.
Gathering our inspiration from compound 3, the aim of this
work was to design new ligands that retain a mixed affinity
profile while incorporating P-gp inhibitory activity, thus identify
antitumor agents that can exploit these new molecular targets,
combined with an intrinsic ability to overcome transporter-
mediated resistance. Previously, we developed compound 1,[32]
one of the highest affinity s receptor ligands to date that dis-
plays potent agonist activity in several tumor cell lines, as an
important pharmacological tool for the investigation of s re-
ceptor activation pathways.[19,33] According to studies of s2 re-
ceptor ligands from other research groups, compound 1 both
decrease P-gp expression and directly inhibited pump activi-
ty.[20] Structural modifications to 1 led to a series of compounds
displaying potent P-gp inhibitory activity that were devoid
both of s receptor affinity and P-gp expression modulation.
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Cyclohexylpiperazines with Antiproliferative and P-gp Inhibitory Activity
reference compound 1, were tested for possible s2-mediated an-
tiproliferative effects after 48 h in SK-N-SH cells, where expres-
sion of the s2 receptor was previously reported.[19] Compounds
with the best compromise between s1 and HSI affinity and ref-
erence compound cis-4-[4-(2,6-dimethoxyphenyl)cyclohexyl]-1-
(2-pyridinyl)piperazine33 (15) were selected for the evaluation of
the s1/HSI-mediated antiproliferative effect at 48 h in PC3 cell
line.[33] The EC50 values were obtained from nonlinear iterative
curve fitting by GraphPad Prism v. 3.0 software.[41]
Calcein-AM experiment
P-gp inhibitory activities of the newly synthesized cis/trans 7–
14, along with reference compound 4, were determined by
fluorescence measurement using calcein-AM fluorescent
probes in MDCK-MDR1 cells according to the published proce-
dure.[42] These transfected canine kidney epithelial cells overex-
press only P-gp of all the transporters, so the measured biolog-
ical effect can be attributed to inhibition of this pump. EC50
values were obtained from nonlinear iterative curve fitting by
GraphPad Prism v. 3.0 software.[41]
Scheme 1. Reagents and conditions: A) Mg turnings, THF, RT, 2–4 h; B) HCl,
RT, 2 h; C) H2, Pd/C 10%, EtOAc, RT, 24 h; D) 1-cyclohexylpiperazine, p-tolue-
nesulfonic acid, toluene, reflux, overnight and NaCNBH3, CH3OH, 40 min, or
cyclohexylpiperazine, ZnCl2 and NaCNBH3, 2-propanol, RT, 48 h.
All final cyclohexylpiperazine derivatives were converted,
using gaseous HCl, to the corresponding hydrochloride salts,
which were isolated as white crystals. Their physical properties
are listed in the table of physical properties reported in the
Supporting Information.
Results and Discussion
On the basis of previous structure–affinity relationship (SAfiR)
studies, new cyclohexylpiperazine derivatives were designed in
order to obtain ligands with s/HSI site mixed affinity and P-gp
modulation activity.[33,36,39]
Biology
Receptor binding studies
s1 and s2 Receptors and HSI affinities
Novel target compounds cis/trans 7–14 were biologically eval-
uated as hydrochloride salts. All compounds were evaluated
for in vitro affinity at the s1 and s2 receptors and at the HSI
site by radioreceptor binding assays. Respective radioligands
and tissue sources used were as follows: 1) s1 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; 2) s2 re-
ceptor, [3H]DTG (1,3-di-2-tolylguanidine) in the presence of
1 mm (+)-pentazocine to mask s1 receptors, rat liver mem-
branes; 3) HSI site, (Æ)-[3H]emopamil, guinea pig liver mem-
branes. The following compounds were used to define the
specific binding reported in parentheses: 1) (+)-pentazocine
(75–84%), 2) DTG (82–93%), 3) (Æ)-ifenprodil (67–85%). Con-
centrations required to inhibit 50% of radioligand specific
binding (IC50) were determined by using six to nine different
drug concentrations in two or three experiments in duplicate.
Values for Scatchard parameters (Kd and Bmax) and apparent in-
hibition constants (Ki) were determined by nonlinear curve fit-
ting by GraphPad Prism v. 3.0 software.[41]
Results are expressed in Ki and reported in Table 1. As expect-
ed, all newly synthesized compounds displayed high affinities
for both s receptors, in accordance with known pharmaco-
phoric models.[43,44] Sub-nanomolar values were recorded for s1
receptor binding, with the best results obtained when the
phenyl ring was unsubstituted (Ki =0.042 nm and 0.045 nm for
cis- and trans-7, respectively). No significant differences in the
affinity between cis and trans isomers were detected, in con-
trast with the cis isomers of 2-pyridylpiperazine analogues,
which displayed low affinities for the s1 receptor.[33] These re-
sults suggested that, for this series of ligands, the two isomers
were accommodated in a similar manner within the s1 recep-
tor. The only exception was compound trans-10 (Ki =4.08 nm
at the s1 receptor), which displayed 13-fold lower affinity than
cis-10 (Ki =0.30 nm). Ki values could not be determined for
compounds trans-12, cis/trans-13, and cis/trans-14, as these li-
gands did not compete with (+)-[3H]pentazocine for binding
to the s1 receptor. Percentages of binding inhibition of (+)-
[3H]pentazocine at drug concentration of 10À12 m are reported
for each of these compounds in Table 1. This is likely due to
the higher hydrophobicity of the 2-chloro, 2,6-difluoro, and 2-
methyl substituents (clogP values: 5.00, 5.46, 5.32 for trans-12,
cis/trans-13, and cis/trans-14, respectively; table of physical
properties, Supporting Information).[45]
Antiproliferative assays
Functional biochemical assays were performed with human SK-
N-SH neuroblastoma and human PC-3 prostate cancer cell
lines.[19] All of the s receptor ligands reported herein, along with
For the s2 receptor, differences in affinities between cis and
trans isomers were generally detectable, with trans isomers dis-
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Table 1. Binding affinities of novel cyclohexylpiperazine derivatives.
Table 2. Biological activity of novel cyclohexylpiperazine derivatives.
EC50 ÆSEM [mm]
Compd
P-gp[a]
SK-N-SH[b]
PC-3[c]
cis-7
trans-7
cis-8
trans-8
cis-9
trans-9
cis-10
trans-10
cis-11
trans-11
cis-12
trans-12
cis-13
trans-13
cis-14
trans-14
1
20Æ4
49.3Æ0.9
88.7Æ7.7
32.7Æ5.1
55.0Æ0.7
59.7Æ7.6
33.1Æ3.3
40.2Æ5.5
45.7Æ12.7
32.5Æ1.9
36.4Æ6.3
12.1Æ0.2
43.1Æ2.9
13.8Æ0.7
35.5Æ3.3
18.7Æ3.0
35.8Æ3.3
9.97[e]
>100
7.6Æ1.5
6.4Æ1.2
6.9Æ2.0
4.1Æ1.0
2.2Æ0.3
1.1Æ0.2
4.6Æ0.6
0.8Æ0.1
2.8Æ0.5
7.3Æ2.0
2.4Æ0.2
1.8Æ0.4
1.4Æ0.3
6.8Æ1.8
4.4Æ0.6
3.0[d]
Ki ÆSEM [nm]
Compd
R
R1
s1
s2
HSI
>100
cis-7
trans-7
cis-8
trans-8
cis-9
trans-9
cis-10
trans-10
cis-11
trans-11
cis-12
trans-12
cis-13
trans-13
cis-14
trans-14
H
H
H
H
H
H
H
H
H
0.042Æ0.005 3.66Æ0.71
0.045Æ0.015 4.77Æ1.04
12.3Æ3.1
14.2Æ1.6
44.4Æ7.0
4-OCH3
4-OCH3
3-OCH3
3-OCH3
OCH3
OCH3
OCH3
OCH3
F
F
Cl
Cl
CH3
0.81Æ0.07
0.23Æ0.12
0.50Æ0.15
0.10Æ0.04
0.30Æ0.14
4.08Æ0.09
0.26Æ0.05
0.32Æ0.08
0.63Æ0.06
79%[c]
1.59Æ0.69 6.51Æ0.86
63.4Æ12.5
1.21Æ0.52
2.05Æ0.74
62%[a]
13.6Æ2.4
12.6Æ4.3
0.4[b]
4.1[b]
31.3Æ8.4
40.6Æ10.2
22.6Æ4.6
59.8Æ12.0
37.7[f]
6.89Æ3.30
H
6-OCH3
6-OCH3
6-F
6-F
H
H
H
H
61%[a]
10.7Æ1.5
7.92Æ0.11
0.21Æ0.07
14.1Æ5.1
1.93Æ0.07
21.1Æ5.4
4.95Æ1.98
7.6[b]
11[b]
16[b]
18[b]
69%[c]
16.9Æ3.4
20.6Æ1.8
71%[c]
4
15
1.3Æ0.3
67%[c]
1.66Æ0.36 9.00Æ2.80
1.65Æ0.39 9.73Æ0.03
0.68Æ0.20
22.4Æ5.0
CH3
70%[c]
1[d]
0.38Æ0.10
>6000
2.83Æ0.35
[a] Transport inhibition in MDCK-MDR1 cells using calcein-AM (2.5 mm) as
a probe. [b] Antiproliferative effects in human SK-N-SH neuroblastoma
cells. [c] Antiproliferative effects in human PC-3 prostate cancer cells.
[d] From ref. [39]. [e] From ref. [46]. [f] From ref. [33]. Values represent the
mean of nꢀ2 separate experiments.
15[e]
687
4.95
(+)-pentazocine
DTG
25.7Æ1.41
(Æ)-ifenprodil
14.7Æ2.0
[a] Percentage of [3H]-(+)-DTG binding inhibition at 10À12 m compound.
[b] Data are from a single experiment. [c] Percentage of [3H]-(+)-pentazo-
cine binding inhibition at 10À12
m compound. [d] From ref. 36[36].
ferative activity in the micromolar range (EC50 range: 12.1–
88.7 mm); thus, they can be described as s2 receptor ago-
nists.[19] In general, cis isomers were more potent than their
trans counterparts, a trend opposite that for s2 receptor affini-
ty, and better results were obtained for substituents other than
a methoxy group (cis-12, cis-13, and cis-14). Compounds cis-12
and cis-13 exhibited antiproliferative activity similar to that of
1 in the same cell line. However, despite the unambiguous role
of s2 receptors in mediating antiproliferative activity in SK-N-
SH cells, a linear correlation between s2 receptor affinity and
antiproliferative activity could not be determined. The pres-
ence of receptor systems in the cells, other than the s2 recep-
tor, should also be taken into account.[46]
[e] From ref. [33]. Values are the mean of nꢀ3 separate experiments.
playing better binding affinities than cis isomers when the sub-
stituent was in the ortho position, in accordance with what
was observed for the 2-pyridylpiperazine analogue series.[33]
These differences were greater for the 2,6-dimethoxy substitu-
ent: trans-11 (Ki =0.21 nm) displayed a 40-fold higher affinity
than its cis counterpart (Ki =7.92 nm). Compound trans-11 was
the only compound with sub-nanomolar affinity at the s2 re-
ceptor. The Ki values for compounds trans-9 and trans-10
could not be determined, because these ligands did not com-
pete with [3H]DTG for binding to the s2 receptor. Percentages
of binding inhibition at compound concentrations of 10À12
m
The SH-SY5Y human neuroblastoma cell line was initially se-
lected for estimation of activity at s1 receptors, as it had been
reported to express only the s1 receptor subtype.[47] Presence
of the s1 subtype was measured within this cell line through
saturation analysis with (+)-[3H]pentazocine, resulting in values
of Kd =7.48 nm and Bmax =207 fmolmgÀ1 protein, suggesting
that the s1 receptor was present, but only at low density. For
this reason, this cell line was not considered a reliable model
for evaluation of s1 receptor-mediated activity. In the absence
of a model for the evaluation of s1 receptor-mediated antipro-
liferative activity, we selected the human PC-3 prostate cancer
cell line, because high expression levels of HSI sites and s1 re-
ceptors[33] within these cells could allow evaluation of com-
pounds binding at both sites and contributing to overall anti-
proliferative effects. We also previously verified expression of
s2 receptors within the same cell line.[33] Because s2 receptor-
mediated antiproliferative activity was evaluated in SK-N-SH
cells, compounds displaying the best s1/HSI affinity profile (cis-
are reported in Table 1. The other compounds had similar
nanomolar affinities, independent of steric or electronic effects.
As with the 2-pyridylpiperazine series, no significant differen-
ces between the cis and trans isomers of these compounds
were detected at the HSI site, and nanomolar HSI affinities,
similar to those of our best HSI ligand compound, 15 (Ki =
4.95 nm),[33] were obtained for all the compounds, independent
of the phenyl ring substituent.
Antiproliferative activity in tumor cell lines
The SK-N-SH human neuroblastoma cell line was selected to
test compound-mediated s2 receptor activity. This cell line has
been reported to express the s1 receptor only in a low affinity
state;[19] HSI binding sites are also reported to be absent.[46] Re-
sults, expressed as EC50 values, are reported in Table 2. All com-
pounds within the series were tested and displayed antiproli-
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Cyclohexylpiperazines with Antiproliferative and P-gp Inhibitory Activity
7, 8, and 10–14 and trans-9) were evaluated for their s1-/HSI-
mediated antiproliferative activity. All of these compounds ex-
hibited moderate antiproliferative activity (EC50: from 22.6 to
63.4 mm), with cis-7 and 8 displaying lower effects (EC50 >
100 mm), possibly due to s1 receptor agonist activity lowering
the overall antiproliferative effect. The best antiproliferative ac-
tivities were exhibited by compounds cis-11 and cis-13 (EC50:
31.3 and 22.6 mm, respectively). These values were similar to
that of the reference compound, HSI-selective agent 15 (EC50 =
22.4 mm). However, while the latter did not reach maximum in-
hibition (60%), compound cis-11 and cis-13 exerted 100% effi-
cacy at the highest concentration (100 mm, data not shown),
likely due to the involvement of both s1 and s2 receptors. Both
cis-11 and cis-13 displayed a behavior similar to that of refer-
ence compound 1, which reached maximum inhibition but
with a lower potency (EC50 =37.7 mm).[33]
Figure 3. Co-administration of doxorubicin with compound cis-11. Antiproli-
ferative effect of 0.1 mm doxorubicin (white bar) at 48 h in the MDCK-MDR1
cell line. In comparison, cis-11 (30 mm and 50 mm) was administered for 24 h
(black bars). After washing, cis-11 was co-administered with doxorubicin
(0.1 mm) at the same concentration for 24 h (grey bars).
Calcein-AM experiment, SAR, and co-administration
experiment
compounds as antiproliferative agents, a neuroblastoma cell
line (SK-N-SH) overexpressing the s2 receptor and a prostate
cancer cell line (PC-3) expressing the three subtypes were se-
lected. Results from SK-N-SH cells demonstrated that the novel
compounds were s2 receptor agonists, and three of these (cis-
12, cis-13, and cis-14) displayed antiproliferative activities simi-
lar to that of compound 1. Compounds cis-11 and cis-13 also
exhibited the best results in PC-3 cells, which was hypothe-
sized to be the result of binding to all three receptor subtypes.
In addition, the therapeutic usefulness of our best P-gp inhibi-
tor (cis-11) was tested in a co-administration experiment with
doxorubicin. The lack of antiproliferative activity of 0.1 mm dox-
orubicin in MDCK-MDR1 cell line was reverted in the presence
of 30 mm cis-11, increasing cell death to 70%. Higher concen-
trations of cis-11 (50 mm) led to a 90% antiproliferative effect
in the presence of 0.1 mm doxorubicin and a 50% antiprolifera-
tive effect in the absence of doxorubicin, demonstrating anti-
tumor properties in P-gp overexpressing tumor cell lines as
well.
Activity at the P-gp pump was determined for all compounds
to determine their ability to overcome P-gp-mediated drug re-
sistance. Inhibition of the P-gp pump, expressed as EC50 values,
was in the micromolar range for all compounds in the series.
Better results were obtained when a substituent, such as a me-
thoxy group or a chlorine atom, was present, particularly at
position 2 (cis-10, cis-11, cis- and trans-13, with EC50 values of
1.1, 0.8, 1.8, and 1.4 mm, respectively). Cis-11 displayed one of
the highest levels of P-gp inhibitory activity, relative to com-
pound 4 (EC50 =1.30 mm). Because compound cis-11 has mod-
erate s- and HSI-mediated antiproliferative activity, it was se-
lected as a promising candidate for co-administration with
doxorubicin, a widely used chemotherapeutic and P-gp sub-
strate, in the P-gp overexpressing cell line MDCK-MDR1. Ad-
ministered alone, doxorubicin did not show any antiprolifera-
tive effect (Figure 3). However, administration of 50 mm cis-11
alone decreased cell viability by 50%, demonstrating its effica-
cy as a single agent in an MDR cell line. Administration of
30 mm cis-11 exhibited no effect (Figure 3). Only co-administra-
tion of doxorubicin with 30 mm cis-11 resulted in an antiproli-
ferative effect, decreasing cell viability by 70%. The antiproli-
ferative effect of 50 mm cis-11, combined with 0.1 mm doxorubi-
cin, decreased cell viability by 90%. Therefore, compound cis-
11 appeared to be an antiproliferative agent that can intrinsi-
cally eluding P-gp activity (Figure 3).
Although more data are needed from different cell lines to
test the contribution to antiproliferative activity of each recep-
tor bound by these novel ligands, these preliminary results are
promising for this class of compounds as potential antitumor
agents that can act simultaneously at different molecular tar-
gets. One or all of the targets may be present in the tumor,
leading to the potential for synergistic activity. Such antiproli-
ferative effects, combined with the P-gp inhibitory activity ex-
hibited by these compounds, gives them the potential to be
used as either antitumor agents devoid of P-gp-mediated re-
sistance, or in association with classic antitumor agents suscep-
tible to P-gp activity. In vivo studies will follow to test the effi-
cacy of these novel compounds.
Conclusions
We have described herein the synthesis of novel cyclohexylpi-
perazine derivatives, designed as s receptor and HSI ligands,
with high P-gp inhibitory activity. All of the compounds dis-
played considerable (nanomolar range) affinities for s recep-
tors, some presenting sub-nanomolar binding values at both
receptor subtypes. All of the compounds also exhibited nano-
molar affinities at the HSI site and micromolar P-gp inhibitory
activity, with values similar to those of both HSI and P-gp in-
hibitor lead compounds. To test the activity of these mixed
Experimental Section
Chemistry
Both column chromatography and flash column chromatography
were performed with 60 ꢁ pore size silica gel as the stationary
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77

C. Abate et al.
MED
phase (1:30 w/w, 63–200 mm particle size and 1:15 w/w, 15–40 mm
particle size, from ICN and Merck, respectively). Melting points
were determined in open capillaries on a Gallenkamp electrother-
mal apparatus. Purity of tested compounds was determined by
combustion analysis and confirmed as ꢀ95%. Elemental analyses
(C, H, N) were performed on a Eurovector Euro EA 3000 analyzer;
action mixture was then evaporated to dryness and the residue
was diluted with 2 N NaOH (20 mL) and extracted with EtOAc (3ꢂ
20 mL). The organic layers were washed with brine (20 mL), dried
(Na2SO4), and concentrated under reduced pressure to give a
crude residue, which was purified by column chromatography to
provide the separated cis and trans isomers of final compounds.
Consistently, the cis stereoisomers eluted before their trans coun-
terparts, and attribution of cis/trans geometry was determined as
for cis/trans-7 and cis/trans-8.
1
analytical results were within Æ0.4% of theoretical values. H NMR
spectra were recorded on a Mercury Varian 300 MHz using CDCl3
as solvent. The following data were reported: chemical shift (d) in
ppm, multiplicity (s=singlet, d=doublet, t=triplet, m=multiplet),
integration, and coupling constant(s) in Hertz. Mass spectra were
recorded 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 per-
centage of relative intensity in parentheses, are reported. Chemi-
cals were from Sigma–Aldrich and Acros and were used without
any further purification.
cis-1-Cyclohexyl-4-[(3-methoxyphenyl)cyclohexyl]piperazine (cis-
9): was obtained as a colorless oil (0.12 g, 34%), using EtOAc/
1
CH3OH/NH3 (95:5:0.01) as an eluent: H NMR: d=1.07–1.37 (m, 5H,
cyclohexyl), 1.43–1.65 (m, 5H, cyclohexyl), 1.68–2.00 [m, 8H, PhCH-
(CH2)4], 2.18–2.25 [m, 2H, benzyl CH and NCH(CH2)5], 2.45–2.75 (m,
9H, piperazine, and NCH), 3.80 (s, 3H, OCH3), 6.68–6.90 (m, 3H, aro-
matic), 7.18–7.27 (m, 1H, aromatic); GC–MS m/z 357 (M++1, 18),
356 (M+, 68), 313 (24), 207 (100). Anal. (C23H36N2O·2HCl) C, H, N.
General procedure to obtain final cyclohexylpiperazine deriva-
tives cis/trans-7 and cis/trans-8: In a typical reaction, 4-phenylcy-
clohexanone 6a or its derivative 6d (1.0 mmol) and cyclohexylpi-
perazine (2.0 mmol, 0.33 g) were dissolved in toluene (20 mL) and
condensed at reflux in the presence of a catalytic amount of p-tol-
uenesulfonic acid (PTSA). After 18 h, the mixture was cooled and
the solvent removed under reduced pressure to afford a crude resi-
due which was dissolved in dry THF. HCl(g) was introduced to the
solution, and the solid salt that formed was filtered, dissolved in
anhydrous CH3OH (20 mL), and cooled to 08C. NaCNBH3 (1.8 mmol,
0.11 g) was added to the cooled solution, and the mixture stirred
at room temperature for 40 min. The mixture was then evaporated
to dryness and the residue was treated with 0.1 N NaOH (50 mL)
and extracted with CH2Cl2 (3ꢂ25 mL). The organic layers were
washed with brine (30 mL), dried (Na2SO4), and concentrated under
reduced pressure to afford a crude residue, which was purified by
column chromatography to yield the separated cis and trans iso-
mers of final compounds. Consistently, the cis stereoisomers were
eluted before their trans counterparts, and cis and trans geometry
was determined as previously reported for analogous deriva-
tives.[33]
trans-1-Cyclohexyl-4-[(3-methoxyphenyl)cyclohexyl]piperazine
(trans-9): was obtained as a white crystalline solid (0.11 g, 30%),
using EtOAc/CH3OH/NH3 (95:5:0.01) as an eluent: mp=129–1318C;
1H NMR: d=1.05–1.30 (m, 5H, cyclohexyl), 1.34–1.56 (m, 5H, cyclo-
hexyl), 1.62–2.05 [m, 8H, PhCH(CH2)4], 2.16–2.45 [m, 3H, benzyl CH,
NCH, and NCH(CH2)5], 2.55–2.75 (m, 8H, piperazine), 3.80 (s, 3H,
OCH3), 6.68–6.82 (m, 3H, aromatic), 7.16–7.23 (m, 1H, aromatic);
GC–MS m/z 357 (M++1, 15), 356 (M+, 57), 313 (21), 207 (100).
Anal. (C23H36N2O·2HCl·1= H2O) C, H, N.
3
Biological methods and materials
Radioligand binding assays. All procedures for binding assays
were previously described. Receptor binding studies for s1 and s2
were carried out according to Matsumoto et al.[48] HSI binding was
evaluated according to Moebius et al.[49] [3H]DTG (30 CimmolÀ1
)
and (+)-[3H]pentazocine (34 CimmolÀ1) were purchased from Perki-
nElmer Life Sciences (Zavantem, Belgium). The radioligand (Æ)-
[3H]emopamil (83 CimmolÀ1) was purchased from American Radio-
labeled Chemicals, Inc. (St. Louis, MO). DTG and (Æ)-ifenprodil
were purchased from Tocris Bioscience (Bristol, UK). (+)-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.
cis-1-Cyclohexyl-4-(4-phenylcyclohexyl)piperazine (cis-7): Com-
pound cis-7 was obtained as a white crystalline solid (0.065 g,
20%), using CH2Cl2/EtOAc/NH3 (1:1:0.01) as an eluent: mp=78–
1
808C; H NMR: d=1.04–1.37 (m, 5H, cyclohexyl), 1.49–1.68 (m, 5H,
cyclohexyl), 1.73–2.04 [m, 8H, PhCH(CH2)4], 2.17–2.23 [m, 2H,
benzyl CH and NCH(CH2)5], 2.38–2.78 (m, 9H, piperazine and NCH),
7.13–7.35 (m, 5H, aromatic); GC–MS m/z 327 (M++1, 22), 326 (M+,
77), 207 (100). Anal. (C22H34N2·2HCl) C, H, N.
Cell culture. The MDCK-MDR1 cell line was a gift from Prof. P.
Borst, NKI-AVL Institute (Amsterdam, the Netherlands). MDCK-
MDR1 cells were grown in DMEM high glucose supplemented with
10% fetal bovine serum, 2 mm glutamine, 100 U/mL penicillin, and
100 mgmLÀ1 streptomycin in a humidified incubator at 378C with a
5% CO2 atmosphere. Human SK-N-SH neuroblastoma and PC-3
human prostate cell lines were obtained from Interlab Cell Line
Collection (ICLC, Genoa). The SH-SY5Y cell line was a gift from Prof.
M. Buttiglione (Dipartimento di Farmacologia e Fisiologia umana,
Universitꢃ degli Studi di Bari Aldo Moro). SK-N-SH and SH-SY5Y cell
lines were routinely cultured in RPMI 1640 supplemented with
10% fetal bovine serum, 2 mm glutamine, 100 U/mL penicillin,
100 mgmLÀ1 streptomycin, 1 mm sodium pyruvate, and 1% non es-
sential amino acids in a humidified incubator at 378C with a 5%
CO2 atmosphere. PC-3 cell lines were routinely cultured in
RPMI 1640 supplemented with 10% fetal bovine serum, 2 mm glu-
tamine, 100 U/mL penicillin, and 100 mgmLÀ1 streptomycin in a hu-
midified incubator at 378C with a 5% CO2 atmosphere. Cell culture
reagents were purchased from Celbio srl (Milano, Italy). Culture-
trans-1-Cyclohexyl-4-(4-phenylcyclohexyl)piperazine
(trans-7):
Compound trans-7 was obtained as a white crystalline solid
(0.033 g, 10%), using CH2Cl2/EtOAc/NH3 (1:1:0.01) as an eluent:
1
mp=2108C (dec); H NMR: d=1.07–1.34 (m, 5H, cyclohexyl), 1.38–
1.73 (m, 5H, cyclohexyl), 1.78–2.14 [m, 8H, PhCH(CH2)4], 2.19–2.50
[m, 3H, benzyl CH, NCH, and NCH(CH2)5], 2.63–2.70 (m, 8H, pipera-
zine), 7.13–7.32 (m, 5H, aromatic); GC–MS m/z 327 (M++1, 14),
326 (M+, 58), 207 (100). Anal. (C22H34N2·2HCl·1= H2O) C, H, N.
2
General procedure to obtain final cyclohexylpiperazine deriva-
tives cis/trans- 9–14: Typically, an intermediate (1.0 mmol) of 4-
phenylcyclohexanone derivative 6b, c, or e–k was combined with
cyclohexylpiperazine
(1.0 mmol,
0.17 g),
zinc(II)
chloride
(0.58 mmol, 0.11 g), and NaCNBH3 (1.1 mmol, 0.07 g) in 2-propanol
(20 mL). The mixture stirred for 48 h at room temperature. The re-
78
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Keywords: cyclohexylpiperazines
modulators · sigma receptors · antitumor agents
·
HSI ligands
·
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