Sigma-2 receptor agonists as possible antitumor agents in resistant tumors: Hints for collateral sensitivity

Article abstract of DOI:10.1002/cmdc.201300291

With the aim of contributing to the development of novel antitumor agents, high-affinity σ₂ receptor agonists were developed, with 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-1,2,3, 4-tetrahydroisoquinoline (15) and 9-[4-(6,7-di

Full text of DOI:10.1002/cmdc.201300291

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DOI: 10.1002/cmdc.201300291
Sigma-2 Receptor Agonists as Possible Antitumor Agents
in Resistant Tumors: Hints for Collateral Sensitivity
Mauro Niso,[a] Carmen Abate,*[a] Marialessandra Contino,[a] Savina Ferorelli,[a]
Amalia Azzariti,[b] Roberto Perrone,[a] Nicola Antonio Colabufo,[a] and Francesco Berardi[a]
With the aim of contributing to the development of novel anti-
tumor agents, high-affinity s2 receptor agonists were devel-
oped, with 6,7-dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-
yl]butyl]-1,2,3,4-tetrahydroisoquinoline (15) and 9-[4-(6,7-di-
methoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-9H-carbazole
(25) showing exceptional selectivity for the s2 subtype. Most
of the compounds displayed notable antiproliferative activity
in human MCF7 breast adenocarcinoma cells, with similar ac-
tivity in the corresponding doxorubicin-resistant MCF7adr cell
line. Surprisingly, a few compounds, including 25, displayed
enhanced activity in MCF7adr cells over parent cells, recalling
the phenomenon of collateral sensitivity, which is under study
for the treatment of drug-resistant tumors. All of the com-
pounds showed interaction with P-glycoprotein (P-gp), and 15
and 25, with the greatest activity, were able to revert P-gp-
mediated resistance and reestablish the antitumor effect of
doxorubicin in MCF7adr cells. We therefore identified a series
of s2 receptor agonists endowed with intriguing antitumor
properties; these compounds deserve further investigation for
the development of alternate strategies against multidrug-
resistant cancers.
Introduction
In the early 1990s, two distinct sigma (s) receptor subtypes,
namely s1 and s2, were identified after the existence of s pro-
teins was first proposed in 1976.[1,2] Initial research was focused
on s receptors within the central nervous system (CNS), where
the most studied s1 receptors modulate the release of
a number of neurotransmitters. Involvement in pathologies
such as anxiety, depression, schizophrenia, drug addiction, and
Parkinson’s and Alzheimer’s diseases has been demonstrated
for this subtype, which seems to play a role in neuroprotection
and neuroplasticity.[3–5] s1 Proteins have also been shown to
take part in intracellular signaling through modulation of intra-
cellular Ca2+ levels via inositol 1,4,5-triphosphate (IP3) recep-
tors.[6] A role in lipid compartmentalization and modulation of
K+ channels has also been suggested for this subtype, the sig-
naling mechanism for which has yet to be fully understood.[6,7]
Less is known about s2 receptors, which have yet to be
cloned. The various attempts[8] to characterize this subtype re-
cently led to the identification of the s2 protein as the proges-
terone receptor membrane component 1 (PGRMC1).[9] A signifi-
cant boost in s2 receptor research has been given by evidence
that s proteins—mostly the s2 subtype—are overexpressed in
a variety of human peripheral and brain tumors. In particular,
s2 receptor density has been shown to be higher in proliferat-
ing than in quiescent tumor cells; therefore, this subtype has
been proposed as an endogenous biomarker for the prolifera-
tive status of tumors.[10] Moreover, activation of s2 receptors
with s2 agonists exerts antiproliferative and cytotoxic effects in
tumor cells in vitro as well as in tumor xenografts in vivo.[11]
Therefore, s2 receptors are an intriguing target for tumor diag-
nosis and treatment. The apoptotic mechanisms activated by
s2 proteins are currently under investigation. There are multi-
ple pathways activated by s2 receptor agonists to induce cell
death; these include caspase-dependent and -independent
mechanisms,[12] generation of reactive oxygen species (ROS),
and autophagy.[13] Apparently, activation of the pathways de-
pends on the tumor cell type and on the structure of the s2
ligand used. 1’-[4-[1-(4-Fluorophenyl)-1H-indol-3-yl]butan-1-yl]-
spiro[isobenzofuran-1(3H)],4’-piperidine, (1, siramesine)[11] and
1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-
yl)propyl]piperazine (2, PB28)[11] are among the most potent s2
agonists known (Figure 1), with the former inducing cell death
by lysosomal leakage and oxidative stress,[14] and the latter
modulating Ca2+ release from intracellular stores.[15] These two
s2 agonists are important reference compounds in the study of
s2 receptor ligands, and have often been used as lead com-
pounds for the development of new generations of s2 receptor
ligands.[11,16–21] The corresponding 4-(4-fluorophenyl)piperidine
derivative of siramesine (1), compound 3 (Figure 1), has
emerged for its sub-nanomolar s2 receptor affinity and appre-
ciable selectivity.[22] Among the ligands developed from PB28
(2), 4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-
[a] Dr. M. Niso, Dr. C. Abate, Dr. M. Contino, Prof. S. Ferorelli,
Prof. Dr. R. Perrone, Prof. Dr. N. A. Colabufo, Prof. Dr. F. Berardi
Dipartimento di Farmacia-Scienze del Farmaco
Universitꢀ degli Studi di Bari ALDO MORO
Via Orabona 4, 70125 Bari (Italy)
E-mail: carmen.abate@uniba.it
[b] Dr. A. Azzariti
Clinical Experimental Oncology Laboratory
National Cancer Institute, “Giovanni Paolo II”
Via O. Flacco 65, 70124 Bari (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300291.
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the co-administration approach (administration of a P-gp in-
hibitor with a classic antineoplastic drug), which is one of the
strategies to overcome multidrug resistance (MDR),[26–31] but
which has shown little success so far.
Results and Discussion
Chemistry
The synthesis of intermediate and final compounds reported
herein is depicted in Schemes 1 and 2. [(4-Fluorophenyl)-1H-
indol-3-yl]butanoic acid (7) was obtained from 4-(1H-indol-3-
yl)butanoic acid by addition of 1-fluoro-4-iodobenzene, upon
microwave-assisted reaction in 2-ethoxyethanol, in the pres-
ence of copper(I) iodide and potassium carbonate (Scheme 1).
Reduction of 7 with lithium aluminum hydride provided the
butanol derivative 8,[22] which underwent reaction with metha-
nesulfonyl chloride to afford the known key intermediate 4-[1-
Figure 1. Reference s2 receptor ligands 1–6.
1-yl-propyl)]piperidine (4)[19] and 1-cyclohexyl-4-[3-(9H-carbazol-
9-yl)propyl]piperazine (5, F281)[23,24] are the most promising in
terms of antiproliferative activity and appreciable s2 receptor
affinity. 6,7-Dimethoxy-2-[3-(5-methoxy-1,2,3,4-tetrahydronaph-
thalen-1-yl)propyl]-1,2,3,4-tetrahydroisoquinoline
(6,
Figure 1)[16] has emerged for its s2 receptor selectivity and in-
spired the synthesis of novel classes of 6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline derivatives.[16] In our continued efforts
to produce high-affinity s2 receptor ligands with agonist (anti-
proliferative) activity, we gathered our inspiration from the
aforementioned compounds 1–6.
Scheme 1. Synthesis of final compounds 13–15. Reagents and conditions:
a) CuI, K2CO3, 1-fluoro-4-iodobenzene, ethoxyethanol, hn, 2008C, 40 min;
b) LiAlH4, Et2O, reflux, 2 h, then RT overnight; c) MsCl, Et3N, CH2Cl2, 08C, 1 h;
d) CH3CN or DMF and K2CO3 and either 1-cyclohexylpiperazine, 4-cyclohexyl-
piperidine, or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.
The basic moieties (a–e, Table 1 below) contained in these
compounds were alternatively connected to the hydrophobic
portions of the same compounds (R, Table 1) in order to com-
bine the structural features that are likely responsible for high
s2 receptor affinity and/or activity. Binding at s receptors and
antiproliferative effects of novel and reference s2 receptor li-
gands were studied in human MCF7 breast adenocarcinoma
cells and in corresponding adriamycin- (or doxorubicin)-resist-
ant cells (MCF7adr). Data from assays with these cell lines pro-
vide an indication of the efficacy of the s2 receptor ligands in
resistant tumors when resistance is due to overexpression of
the P-glycoprotein efflux pump (P-gp, also known as multi-
drug-resistance protein 1, MDR1), which has been found in
50% of human cancers.[25] With this same purpose, we evaluat-
ed the interaction of our s2 compounds with P-gp, also based
on previous results showing that s2 ligands are often accompa-
nied by P-gp activity.[16,18] As we have already showed, inter-
action with P-gp by our s2 agonists may be exploited in two
directions in resistant tumors: by co-administration with a clas-
sic antineoplastic drug whose activity is hampered by P-gp
overexpression, or as single antitumor agents that are able to
overcome P-gp-mediated resistance. This second hypothesis
would also solve the pharmacokinetic problems generated by
Scheme 2. Synthesis of final compounds 16–25. Reagents and conditions:
a) CH3CN or DMF and K2CO3 and one amine among spiro[isobenzofuran-1-
(3H),4’piperidine], 4-(4-fluorophenyl)piperidine, 1-cyclohexylpiperazine,
4-cyclohexylpiperidine, or 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline.
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(4-fluorophenyl)-1H-indol-3-yl)butyl methanesulfonate (9).[22]
Nucleophilic substitution of 9 with 1-cyclohexylpiperazine, 4-
cyclohexylpiperidine,[19] or 6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline (basic moieties c, d, or e, respectively, Table 1), in the
presence of potassium carbonate afforded final compounds
13–15, respectively. Similarly, reaction of 4-(1H-indol-3-yl)butyl
methanesulfonate (10) with the same basic moieties gave final
compounds 16–18. Reaction of (3-bromopropyl)tetralin 11[32]
with either spiro[isobenzofuran-1-(3H),4’piperidine][22] (a) or 4-
(4-fluorophenyl)piperidine[22] (b), in the same way, afforded
final compounds 19 and 20, respectively. Each of the basic
moieties a–e was allowed to react with 9-(4-chlorobutyl)carba-
zole 12[33] to afford final compounds 21–25. Siramesine was
prepared according to published methods[22] in order to com-
pare our results with this lead compound. All final amine com-
pounds were converted into their hydrochloride or oxalate
salts, and their physical properties are listed in table 1 of the
Supporting Information.
venient as in the tetralin series for s2 receptor affinity, with N-
substituted (13) and N-unsubstituted (16) indoles showing Ki
values of 28.9 and 6.50 nm respectively, in agreement with car-
bazole 5 (Ki =12.6 nm). Notable is the s2 receptor affinity we
observed with our protocols for siramesine (1, Ki =12.6 nm),
which is 100-fold lower than the reported value (IC50 =
0.12 nm).[22] In accordance, all siramesine N-(4-fluorophenyl)in-
dole analogues displayed similar Ki values, with the four basic
moieties (a, c–e) conferring similar affinity (compounds 1, 13,
14, and 15). All carbazole derivatives displayed s2 affinities
generally similar to that of 5 (compounds 22 and 24), with
compounds 21 and 25 showing the highest s2 affinities (Ki
values of 3.24 and 0.04 nm, respectively) among the novel
compounds. The exceptional Ki value shown by 25 was coun-
teracted by a low Hill slope (nH =0.46). Nevertheless, com-
pound 25 emerged for its exceptional s2 selectivity (54750-
fold) due to very low affinity for the s1 receptor. N-Unsubstitut-
ed indoles 17 and 18, together with carbazole 23, displayed
non-competitive binding with [3H]DTG, with the radioligand
being displaced by very low test compound concentrations
Biology
s Receptor binding
Table 1. s Receptor affinities of reference compounds 1–6 and new compounds 13–
25.
Affinity values at the s receptor subtypes for refer-
ence compounds 1–6 and novel compounds 13–25
are listed in Table 1 and expressed as Ki values. As for
affinity at the s1 receptor, 6,7-dimethoxy-1,2,3,4-tetra-
hydroisoquinoline (e) was confirmed as a detrimental
basic moiety, with 25 displaying the lowest affinity
(Ki =2190 nm). In the indole series, the presence of
the 1-(4-fluorophenyl) moiety at the indole nitrogen
atom led to a dramatic decrease in affinity for the s1
receptor, in accordance with a previous report.[22] Cu-
riously, cyclohexylpiperidine (d) and 6,7-dimethoxy-
1,2,3,4-tetrahydroisoquinoline (e) moieties provided
appreciable s1 affinity in the N-unsubstituted indoles
(Ki =1.07 nm for 17, and Ki =46.9 nm for 18), in con-
trast to the low s1 receptor binding observed for the
tetralin-, carbazole-, and 1-(4-fluorophenyl)-substitut-
ed indole series. These differences between the N-un-
substituted and N-substituted indoles suggest that
a hydrogen bond may occur between the indole NH
group and the s1 receptor (compare 17 with 4, 14,
24, and 18 with 6, 15, and 25). A few compounds
Ki [nm][a]
Compound
R
n
Y
s1
s2
siramesine (1)[22]
3[22]
13
14
15
4
4
4
4
4
a
b
c
d
e
10.5Æ2.6
16[b]
25.7Æ7.3
98.9Æ7.1
1390Æ20
12.6Æ0.1
0.27[b]
28.9Æ6.6
7.28Æ1.11
5.34Æ1.22
16
17
18
4
4
4
c
d
e
70%
6.50Æ1.45
90%
63%
1.07Æ0.28
46.9Æ3.8
PB28 (2)[34]
3
3
3
3
3
c
0.38Æ0.10
143Æ18
151Æ20
76%
0.68Æ0.20
18.8Æ5.9
96%
40.6Æ6.4
22.6Æ3.9
4[19]
6[16]
19
d
e
a
b
20
80%
displayed
non-competitive
binding
with
(+)-[3H]pentazocine: two were from the tetralin series
(19 and 20), one from the N-unsubstituted indoles
(16), and one from the carbazole series (23). As for s2
receptor binding, in the tetralin series replacement of
the cyclohexylpiperazine moiety (which is present in
compound 2) with a piperidine-type group (19 and
20) led to a 30–60-fold decrease in s2 affinity, as pre-
viously shown with compound 4.[19] The only excep-
tion was 6, which displaced [3H]-1,3-di-O-tolylguani-
dine ([3H]DTG) in a very strong and non-competitive
manner. Nevertheless, the presence of a cyclohexylpi-
perazine group in the indole series was not as con-
F281 (5)[23]
3
4
4
4
4
4
c
3450Æ1660
27.3Æ5.7
29.0Æ1.2
91%
139Æ18
2190Æ230
12.6Æ3.2
3.24Æ1.34
11.7Æ1.8
60%
21
22
23
24
25
a
b
c
d
e
7.65Æ2.5
0.04Æ0.01[c]
DTG
(+)-pentazocine
31.5Æ3.3
3.38Æ0.31
[a] Values represent the mean ÆSEM of nꢀ2 separate experiments carried out in du-
plicate; percent displacement at a concentration of 10À11 m is reported if a complete
displacement curve was not obtained. [b] IC50 values as reported in Ref. [22]. [c] Hill
slope nH =0.46.
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(10À11 m). Therefore, the importance of the basic moiety types
for conferring s2 selectivity was confirmed. As previously
shown with a diverse panel of tetralin analogues,[19,17] the cy-
clohexylpiperazine ring is not optimal for conferring s2 selec-
tivity, likely for the presence of the two N-atoms. By contrast,
and as already shown in the tetralin series, 4-cyclohexylpiperi-
dine,[19,34] and in particular 6,7-dimethoxy-1,2,3,4-tetrahydroiso-
quinoline,[16] were found to have high s2 selectivity. Indoles
and carbazoles bearing these two moieties were accompanied
by a significant decrease in s1 affinity (14, 15, 24 and 25).
Compounds 15 and 25 emerged for their notable s2 selectivity
(260- and 54750-fold, respectively).
ence of the 4-fluorophenyl substituent at the indole nitrogen
atom led to a 10-fold increase in P-gp activity, only with 6,7-di-
methoxy-1,2,3,4-tetrahydroisoquinoline as the basic moiety
(compare 15 with 18). Therefore, the combination of the 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety and N-substi-
tuted indoles appeared as the optimal approach to confer ex-
cellent interaction with P-gp, with 15 and 25 emerging as
novel P-gp modulators worthy of further investigation.
Antiproliferative activity in MCF7 and MCF7adr cell lines
With the exception of compound 3, the antiproliferative activi-
ties of novel and reference compounds were evaluated in
MCF7 and MCF7adr cells, expressed as EC50 values (Table 2).
Compounds such as the N-unsubstituted indoles 16 and 18
displayed antiproliferative activity in neither of the two cell
lines (EC50 >100 mm). In contrast, the corresponding N-substi-
tuted indoles 13 and 15 displayed notable and similar antipro-
liferative activities in both cell lines (EC50 values: 12.7 and
13.6 mm for 13, and 17.8 and 21.8 mm for 15, in MCF7 and
MCF7adr cells, respectively). This same difference in the anti-
proliferative activity between N-unsubstituted and N-substitut-
ed indoles was not shown by the couple 17/14, with both
compounds exerting notable antiproliferative effects in both
cell lines (EC50 values: 8.86 and 16.4 mm for 17, and 14.2 and
12.9 mm for 14, in MCF7 and MCF7adr, respectively). Further-
more, N-unsubstituted indole 17 displayed the most potent ac-
tivity of all the compounds studied in MCF7 cells. All the novel
carbazole derivatives (21–25) displayed antiproliferative activity
in both cell lines (EC50 from 12.6 to 28.2 mm), with spiro-isoben-
zofurane derivative 21 giving the best results. As for the tetra-
lin series, all of the derivatives displayed antiproliferative activi-
ty except for the reference compound 6. This compound, bear-
ing a 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety, curi-
ously displayed an EC50 value in MCF7adr (EC50 =83.9 mm)
lower than that in the parental MCF7 line (EC50 >100 mm).
Basic moieties such as spiro-isobenzofurane- (a) and cyclohex-
yl- (d) piperidines led to the highest activity among tetralins in
both cell lines (EC50: 14.4 and 17.1 mm for 19, and 16.0 and
17.9 mm for 4, in MCF7 and MCF7adr cells, respectively). Moder-
ate activity was displayed by 4-fluorophenyl derivative 20
(EC50: 31.4 and 28.1 mm in MCF7 and MCF7adr, respectively),
and unexpectedly disappointing activity was shown by cyclo-
hexylpiperazine derivative 2 (EC50: 28.4 and 77.5 mm in MCF7
and MCF7adr, respectively).
Calcein-AM experiments
Activity at P-gp was determined for all of the compounds
except 3 by using the P-gp-overexpressing cell line MDCK-
MDR1, and data are expressed as EC50 values (Table 2). Activity
was in the micromolar range for all compounds (0.21 mm–
5.38 mm), with 1 showing a considerable interaction with the
Table 2. Activities of reference compounds 1–6 and new compounds
13–25.
EC50 [mm][a]
MCF7[c]
Compound
P-gp[b]
MCF7adr[d]
siramesine (1)
PB28 (2)
4
F281 (5)
6
13
14
15
16
17
18
19
20
21
22
23
24
25
1.41Æ0.31
3.0Æ0.2[35]
1.92Æ0.32
2.90Æ0.21
1.15Æ0.2[16]
3.44Æ0.21
3.70Æ0.23
0.21Æ0.02
5.03Æ0.61
4.60Æ0.43
2.80Æ0.41
2.68Æ0.43
1.98Æ0.32
1.58Æ0.11
3.40Æ0.61
1.52Æ0.23
5.38Æ0.50
0.42Æ0.02
12.3Æ0.6
28.4Æ6.1
16.0Æ2.0
24.0Æ3.1
5.90Æ1.21
77.5Æ10.1
17.9Æ1.5
35.2Æ4.1
83.9Æ13.2
13.6Æ1.1
12.9Æ0.8
21.8Æ1.5
>100
12.7Æ0.1
14.2Æ2.3
17.8Æ0.4
>100
>100
16.4Æ2.1
>100
8.86Æ1.81
>100
14.4Æ0.6
31.4Æ3.3
12.6Æ2.5
25.0Æ0.2
23.9Æ0.9
19.9Æ3.1
28.2Æ5.0
17.1Æ1.2
28.1Æ2.4
17.8Æ2.1
19.0Æ0.9
18.6Æ1.1
20.1Æ2.5
17.1Æ1.2
[a] Values represent the mean ÆSEM of nꢀ2 separate experiments car-
ried out in duplicate. [b] Transport inhibition in MDCK-MDR1 cells using
calcein-AM (2.5 mm) as a probe. [c] Antiproliferative effect in the MCF7
cell line. [d] Antiproliferative effect in MCF7adr cells.
Collateral sensitivity
efflux pump (EC50 =1.41 mm). All tetralins (2, 4, 6, 19, and 20)
displayed similar activity at P-gp, independent of the basic
moiety type (EC50 values from 1.15 to 2.68 mm). Similar behav-
ior was shown by carbazole derivatives 5 and 21–24 (EC50
values ranged from 1.52 to 5.38 mm), with the exception of 6,7-
dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative 25, which
displayed a very potent interaction with P-gp (EC50 =0.42 mm).
The strongest interaction with P-gp was recorded by another
6,7-dimethoxytetrahydroisoquinoline derivative: the N-substi-
tuted indole derivative 15 (EC50 =0.21 mm). Curiously, the pres-
Surprisingly, some of the tested compounds displayed higher
antiproliferative activity in resistant cells than in parent cells,
recalling a phenomenon that has was recently termed collater-
al sensitivity (CS).[36,37] According to this phenomenon, activity
is potentiated rather than decreased by the overexpression of
P-gp; therefore, the MDR1-selective ratio (SR)—the com-
pound’s EC50 value in parental cells divided by its EC50 value in
P-gp-overexpressing cells—should be >1. There are different
hypotheses that explain CS, and based on the evidence that
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several CS agents are P-gp substrates, one of the suggested
mechanisms is the activation of a futile ATP cycle, followed by
increased production of ROS. In accordance with this hypothe-
sis, 1, which is known to activate ROS production and which
was shown in this work to interact with P-gp, displayed an in-
teresting SR, with an EC50 value in MCF7adr cells 2.1-fold lower
than in parental cells. Lower values, but still >1, were the SR
ratios observed for 22, 23, and 25, all of which belong to the
carbazole series. On the other hand, the differences in EC50
values were too small between parental and resistant cells for
compounds 6, 14, and 20 to recall the CS phenomenon. Be-
cause the increase in ROS production generated by a futile
ATP cycle may be responsible for CS, ATP consumption and
ROS generation were studied for compounds 1 and 25. For all
of the compounds with antiproliferative activity in MCF7
(EC50 <100 mm), involvement of ROS was indirectly demonstrat-
ed by the administration of the lipid antioxidant a-tocopherol:
100 mm a-tocopherol administered before the drug was able
to completely rescue MCF7 cells from death (Table 3). To deter-
mine whether more ROS generation in MCF7adr than in MCF7
cells was responsible for CS, both cell lines were pretreated
with increasing concentrations of a-tocopherol (1–50 mm)
before the administration of 1 (25 mm) or 25 (25 mm), i.e., com-
pounds with higher CS (Figure 2). At 1 mm, a-tocopherol was
able to rescue neither MCF7 nor MCF7adr cells treated with 1,
whereas a small rescue of viability (13%) was observed in
MCF7 cells treated with 25. The differences in viability between
MCF7 and MCF7adr pretreated with 10 mm a-tocopherol
before administration of 1 or 25 were more significant, with vi-
ability reaching 70% only in MCF7 cells. On the other hand,
MCF7adr viability was only slightly increased by a-tocopherol
at 10 mm, with 40% of cells surviving after treatment with 25,
and 20% of cells surviving after administration of 1. Pretreat-
Figure 2. a-Tocopherol effect on MCF7 and MCF7adr cell viability. Antiproli-
ferative effect of compounds A) 1 and B) 25 at 24 h in MCF7 and MCF7adr
cells as indicated. Compounds 1 or 25 (25 mm) were administered alone or
in combination with a-tocopherol at the indicated concentrations.
ment with a-tocopherol at 50 mm led to an almost complete
rescue of viability in MCF7 cells treated with 1 or 25, whereas
a lower percentage of MCF7adr cells (~80%) survived. a-Toco-
pherol at 100 mm rescued the viability of MCF7adr as for MCF7
cells, treated with 1 or 25. These results are in agreement with
the hypothesis that the enhanced antiproliferative activity
shown in resistant cells (CS) may be due to higher ROS produc-
tion, likely induced by the futile ATP cycle activated by P-gp
substrates. Therefore, consumption of ATP induced by com-
pounds endowed with CS properties was evaluated, and
higher ATP hydrolysis in MCF7adr than in MCF7 cells was dem-
onstrated (Figure 3). In accordance with such a hypothesis,
compound 15 did not cause enhanced ATP hydrolysis, and did
not display CS, despite its very potent activity toward P-gp.
Therefore, it appeared as a P-gp inhibitor that does not acti-
vate the futile ATP cycle as substrates do (Figure 3).
Table 3. Antiproliferative effect of 100 mm a-tocopherol on MCF7 cell via-
bility.
Compound[b]
Cell viability [%][a]
a-tocopherol[c]
siramesine (1)
PB28 (2)
4
F281 (5)
6
À
+
26Æ2.4
80Æ5.2
60Æ4.3
60Æ3.6
–
100
100
100
100
–
13
14
15
16
20Æ0.9
20Æ1.6
31Æ3.2
–
100
100
85Æ6.2
–
17
18
15Æ0.8
–
80Æ7.3
–
19
20
21
22
23
24
25
43Æ2.4
70Æ6.1
20Æ1.2
25Æ1.1
43Æ3.2
20Æ2.1
60Æ7.1
90Æ8.9
100
100
100
100
100
100
[a] Compounds tested at 25 mm. [b] Values represent the mean ÆSEM of
nꢀ2 separate experiments carried out in duplicate. [c] a-Tocopherol was
used at 100 mm.
Figure 3. ATP consumption in MCF7 and MCF7adr cells as affected by com-
pounds 1, 15, and 25 at a final concentration of 25 mm. Control: untreated
cells.
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Co-administration experiments
Compounds showing the stron-
gest interaction with P-gp, 15
and 25, were selected for co-ad-
ministration experiments in
MCF7adr cells with doxorubicin,
which is a widely used chemo-
therapeutic drug and a P-gp
substrate. As expected from
a cell line in which overexpres-
sion of P-gp had been previous-
ly induced by treatment with
doxorubicin, doxorubicin admin-
istered alone (up to 100 mm) did
not exert any antiproliferative
effect. Administration of 15
alone at 1, 10, and 25 mm left re-
spectively 96, 89, and 12% of
the cells surviving. When the
compound was co-administered
with 1 mm doxorubicin at the
same concentrations above,
a small synergistic effect was
observed (cell survival rates of
76, 67, and 9%). This synergistic
effect became much more pro-
nounced with co-administration
of doxorubicin at 10 mm (cell
survival rates of 55, 15, and 9%).
These data show that 15 inhibits
P-gp, allowing doxorubicin to
Figure 4. Co-administration of doxorubicin with s2/P-gp-active compounds A) 1, B) 15, and C) 25. Antiproliferative
effect of doxorubicin (white bars) at 1 mm (left side) and 10 mm (right side) at 24 h in the MCF7adr cell line. In
comparison, s2/P-gp-active compounds (1, 10, and 25 mm) were administered for 24 h (black bars). After washing,
compounds were co-administered with doxorubicin (1 or 10 mm) at the same concentration for 24 h (grey bars).
enter cells and to exert its cytotoxic effects. Better synergistic
results were obtained when both 15 and doxorubicin were ad-
ministered at 10 mm, although 15 at 1 mm also led to moderate
doxorubicin activity. At 25 mm, 15 was administered above its
EC50 value (21.8 mm, Table 2), and demonstrated its efficacy as
a single agent in an MDR cell line (Figure 4). Similar behavior
was shown by 25, with better results obtained with co-admin-
istration of doxorubicin and 25 both at 10 mm (28% cell surviv-
al). Compound 25 at 25 mm also displayed its single-agent anti-
tumor property with 15% cell survival, as already proven by its
EC50 value (Table 2). Because compound 1 also appeared to un-
dergo interaction with P-gp, we evaluated the properties of
1 in co-administration with doxorubicin. At 10 mm, 1 deter-
mined an 80% cell mortality as a single agent because of its
EC50 value (5.9 mm in MCF7adr), and co-administration with
doxorubicin at either 1 or 10 mm only slightly decreased viabili-
ty. Compound 1 co-administered at 1 mm with 10 mm doxorubi-
cin led to a lower synergistic effect (64% cell survival) than
compound 15 (55% survival).
6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline was connected to
an N-substituted indole ring, with 15 displaying a 260-fold se-
lectivity for s2 over s1. Despite the low Hill slope from the s2
receptor binding assay, carbazole 25 displayed exceptional s2
versus s1 selectivity (54750-fold). Therefore, novel scaffolds for
high-affinity and highly s2-selective ligands were identified. Al-
though no linear correlation between s2 receptor affinity and
activity was found, most of the novel compounds exerted ap-
preciable antiproliferative activity in breast tumor cell lines,
and generally no decrease in activity was shown in MCF7adr
cells, demonstrating the efficacy of these compounds in cells
with P-gp-induced resistance. All compounds showed a certain
degree of interaction with P-gp, with 15 and 25 displaying an
unexpectedly potent activity. In co-administration with doxoru-
bicin, both of these compounds at 10 mm were able to revert
P-gp-mediated resistance, and to re-establish the antitumor ac-
tivity of 10 mm doxorubicin, with some improvement also ob-
served with doxorubicin at 1 mm. Therefore, 15 and 25 appear
to be promising agents in MDR tumors, for use as single
agents able to elude P-gp activity and kill tumor cells, or in co-
administration with classic chemotherapeutic substrates of P-
gp. However, the most surprising results given by some of the
studied compounds (1, 22, 23 and 25) were the enhanced ac-
tivities recorded in resistant MCF7adr cells over those observed
in parent (non-resistant cells), recalling a phenomenon known
Conclusions
Most of the compounds synthesized displayed high s2 affinity,
and a few displayed excellent s2 over s1 receptor selectivity.
The best results in terms of s2 selectivity were obtained when
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3-yl)butanoic acid (7) (0.47 mmol, 0.14 g) in the same solvent
(5 mL) was added in a dropwise manner. After holding at reflux for
2 h, the mixture was stirred at room temperature overnight. The
reaction mixture was then cooled, quenched with H2O (10 mL),
and the aqueous layer was extracted with Et2O (3ꢂ10 mL). The
combined organic extracts were dried over Na2SO4 and concentrat-
ed under reduced pressure to give a crude residue, which was pu-
rified by column chromatography with CH2Cl2/EtOAc (8:2) as
eluent to afford the title compound as a yellow semisolid (0.130 g,
98%); GC–MS m/z 284 [M+1]+ (7), 283 [M+1]+ (34), 224 (100);
1H NMR (300 MHz, CDCl3): d=1.60–1.90 (m, 5H, CH2CH2CH2OH),
2.78–2.83 (m, 2H, ArCH2), 3.78 (t, 2H, CH2O), 7.09–7.70 ppm (m,
9H, aromatic).
as collateral sensitivity. Exploitation of this phenomenon is
under study as a potential alternative approach to treating
drug-resistant tumors. We examined some of the possible
mechanisms suggested for CS and found that 1 and 25 gener-
ate more ROS in the MCF7adr than in the MCF7 cell line, likely
through a higher consumption of ATP due to interaction with
P-gp. Overall, with this work we developed a few interesting
and potent s2 receptor ligands endowed with unexpected an-
titumor properties that are surely worthy of further investiga-
tion for the development of alternate strategies against multi-
drug-resistant cancers.
Experimental Section
General procedure for the synthesis of amines 13–15, 21, 22,
and 25
Chemistry
One intermediate between 9 or 12 (0.95 mmol, 0.24 g) with one
among the appropriate amines a–e (1.1 mmol) and K2CO3
(1.16 mmol, 0.16 g) in DMF (5 mL) was stirred at 1008C for 20 min.
The solvent was then removed under reduced pressure, and H2O
(10 mL) was added to the crude residue, and the mixture was ex-
tracted with CH2Cl2 (3ꢂ10 mL). The organic layers collected were
dried (Na2SO4) and concentrated under reduced pressure to afford
a crude residue, which was purified by column chromatography
with CH2Cl2/MeOH (95:5) as eluent.
Microwave reactions were conducted in a Biotage Initiator Micro-
wave Synthesizer. Both column chromatography and flash column
chromatography were performed with 60 ꢁ pore size silica gel as
the stationary phase (1:30 w/w, 63–200 mm particle size, ICN; 1:15
w/w, 15–40 mm particle size, Merck, respectively). Melting points
were determined in open capillaries on a Gallenkamp electrother-
mal apparatus. The purity of tested compounds (in all cases
ꢀ95%) was established by combustion analysis. Elemental analy-
ses (C, H, N) were performed on a Eurovector Euro EA 3000 ana-
lyzer, and results were within Æ0.4% of the theoretical values
unless otherwise indicated. 1H NMR (300 MHz) and 13C NMR
(75 MHz) spectra were recorded on a Mercury Varian instrument;
3-[4-(4-Cyclohexyl-1-piperazinyl)butyl]-1-(4-fluorophenyl)-1H-
indole (13) was obtained as a yellow oil (0.27 g, 66% yield).
1H NMR (300 MHz, CDCl3): d=1.06–1.30 [m, 5H, cyclohexyl,
(CHH)5], 1.58–1.97 [m, 9H, cyclohexyl (CHH)5 and ArCH2CH2CH2],
2.17–2.31 (m, 1H, CHN), 2.40 (t, 2H, J=7.6 Hz, CH2N), 2.51–2.74 (m,
8H, piperazine), 2.81 (t, 2H, J=7.3 Hz, ArCH2), 7.13 (s, 1H, aromat-
ic), 7.13–7.25 (m, 4H, aromatic), 7.40–7.46 (m, 3H, aromatic),
7.64 ppm (dd, 1H, JHH =7 Hz, JHF =1.5 Hz, aromatic); LC–MS (ESI+)
m/z 434 [M+H]+; LC–MS–MS 434: 352, 266, 224; Anal.
1
CDCl3 was used as solvent to record H NMR on intermediate and
final compounds as free basis, and the following data are reported:
chemical shifts (d) in ppm, multiplicity (s=singlet, d=doublet, t=
triplet, m=multiplet), integration and coupling constant(s) in
Hertz. CD3OD was used as solvent to record 13C NMR on hydrochlo-
ride salts of final compounds where reported, and chemical shifts
(d) in ppm were reported. Recording of mass spectra was done on
an Agilent 6890-5973 MSD GC/MS instrument and on an Agilent
1100 series LC–MSD trap system VL mass spectrometer; only signif-
icant m/z peaks, with percent relative intensities in parentheses,
are reported. Chemicals were obtained from Aldrich and Alfa Aesar
and were used without further purification.
(C28H36FN3·2C2H2O4·1= H2O) C, H, N.
4
3-[4-(4-Cyclohexyl-1-piperidino)butyl]-1-(4-fluorophenyl)-1H-
indole (14) was obtained as a yellow oil (0.29 g, 70%). 1H NMR
(300 MHz, CDCl3): d=0.85–1.86 [m, 22H, cyclohexyl, piperidine
CH2CHCH2, and ArCH2CH2CH2CH2N], 2.32–2.37 (m, 2H, piperidine
CHHNCHH), 2.81 (t, 2H, J=7.1 Hz, ArCH2), 2.93–2.97 (m, 2H, piperi-
dine CHHNCHH), 7.07 (s, 1H, aromatic), 7.13–7.26 (m, 4H, aromatic),
4-[1-(4-Fluorophenyl)-1H-indol-3-yl)butanoic acid (7). A mixture
of 4-(1H-indol-3-yl)butanoic acid (0.98 mmol, 0.20 g), K2CO3
(1.30 mmol, 0.18 g), CuI (0.25 mmol, 0.047 g), and 1-fluoro-4-iodo-
benzene (1.49 mmol, 0.33 g) in ethoxyethanol (3.5 mL) was
warmed at 2008C under stirring in a microwave oven for 40 min.
After cooling to room temperature, H2O (5 mL) was added to the
reaction mixture, and the solvent was removed under reduced
pressure. The residue was taken up with water, acidified with 3n
HCl (5 mL) and extracted with Et2O (3ꢂ10 mL). The collected or-
ganic layers were dried (Na2SO4) and evaporated under reduced
pressure to afford a crude residue as a brown oil, which was puri-
fied by crystallization from CHCl3/n-hexane to provide the target
compound as light-brown crystals (0.145 g, 50%): mp: 123–1258C;
1H NMR (300 MHz, CDCl3): d=2.00–2.15 (t, 2H, J=7.4 Hz,
ArCH2CH2), 2.47 (t, 2H, J=7.4 Hz, CH2COOH), 2.87 (t, 2H, J=7.4 Hz,
ArCH2), 3.80 (brs, 1H, COOH, D2O exchanged), 7.10–7.66 ppm (m,
9H, aromatic); LC–MS (ESIÀ) m/z: 296 [MÀH]À; LC–MS–MS 296:
252.
7.41–7.46 (m, 3H, aromatic), 7.64 ppm (dd, 1H, JHH =7 Hz, JHF
=
1.5 Hz, aromatic); LC–MS (ESI+) m/z 433 [M+H]+; LC–MS–MS 433:
266, 224; Anal. (C29H37FN2·HCl·H2O) C, H, N.
6,7-Dimethoxy-2-[4-[1-(4-fluorophenyl)-1H-indol-3-yl]butyl]-
1,2,3,4-tetrahydroisoquinoline (15) was obtained as a yellow oil
(0.28 g, 65%). 1H NMR (300 MHz, CDCl3): d=1.70–1.88 (m, 4H,
ArCH2CH2CH2), 2.56 (t, 2H, J=7.1 Hz, CH2N), 2.7 (t, 2H, J=5.8 Hz,
CH2N), 2.80–2.87 (m, 4H, 2ArCH2), 3.55 (s, 2H, ArCH2N), 3.83 (s, 6H,
2 OCH3), 6.50 (s, 1H, aromatic), 6.58 (s, 1H, aromatic), 7.16 (s, 1H,
aromatic), 7.17–7.25 (m, 3H, aromatic), 7.41–7.47 ppm (m, 3H, aro-
matic), 7.65 (d, 1H, J=8.2 Hz, aromatic); 13C NMR (75 MHz, CD3OD):
d=22.55, 23.27, 25.34, 48.53, 51.18, 53.70, 53.80, 54.50, 107.98,
108.45, 109.85, 114.53, 114.84, 115.10, 117.40, 118.00 (d, J2-CF
=
23 Hz), 120.93, 121.58, 123.95, 124.17, 124.20 (d, J3-CF =8 Hz),
127.44, 134.73, 135.00, 147.14, 147.95, 159.52 ppm (d, J1-CF
=
243 Hz); GC–MS m/z: 458 [M]+ (17), 246 (42), 206 (60), 192 (100);
Anal. (C29H31FN2O2·HCl·1= H2O) C, H, N.
4
4-[1-(4-Fluorophenyl)-1H-indol-3-yl)-1-butanol (8). To a suspension
of LiAlH4 (0.66 mmol, 0.025 g) in dry THF (5 mL) kept at 08C and
under a stream of N2, a solution of 4-[1-(4-fluorophenyl)-1H-indol-
1’-[4-(9H-Carbazol-9-yl)-butyl]spiro[isobenzofuran-1(3H),4’-piper-
idine] (21) was obtained as a light-yellow oil (0.22 g, 50%);
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1H NMR (300 MHz, CDCl3): d=1.60–2.00 (m, 8H, CH2CCH2,
NCH2CH2CH2), 2.25–2.50 [m, 4H, CH2N(CHH)2], 2.78–2.90 [s, 2H,
N(CHH)2], 4.38 (t, 2H, J=7.2 Hz, carbazole NCH2), 5.05 (s, 2H,
CH2O), 7.10–8.15 (m, 12H, aromatic); GC–MS m/z: 410 [M]+ (8), 202
6,7-Dimethoxy-2-[4-(1H-indol-3-yl)butyl]-1,2,3,4-tetrahydroiso-
quinoline (18) was obtained as yellow oil (0.35 g, 50% yield).
1H NMR (300 MHz, CDCl3): d=1.67–1.82 (m, 4H, ArCH2CH2CH2), 2.54
(t, 2H, J=7.3 Hz, CH2N), 2.70 (t, 2H, J=5.6 Hz, CH2N), 2.78–2.95 (m,
4H, 2ArCH2), 3.54 (s, 2H, ArCH2N), 3.83 (s, 6H, 2OCH3), 6.51 (s, 1H,
aromatic), 6.58 (s, 1H, aromatic), 6.97 (s, 1H, aromatic), 7.10 (t, 1H,
J=7.4 Hz, aromatic), 7.18 (t, 1H, J=7.7 Hz, aromatic), 7.34 (d, 1H,
J=7.1 Hz, aromatic), 7.61 (d, 1H, J=7.7 Hz, aromatic), 7.97 ppm
(brs, 1H, NH D2O exchanged); GC–MS m/z: 365 [M+1]+ (12), 364
(100); Anal. (C28H30N2O·HCl·1= H2O) C, H, N.
4
9-[4-(4-(4-Fluorophenyl)piperidin-1-yl)butyl]-9H-carbazole
(22)
was obtained as a light-brown solid, which was recrystallized from
EtOH to provide an ivory solid (0.17 g, 40%): mp: 121–1238C;
1H NMR (300 MHz, CDCl3): d=1.70–2.05 [m, 10H, CH(CH2)2,
NCH2CH2CH2CH2N(CHH)2], 2.30–2.50 (m, 3H, ArCH, NCH2), 2.98–3.02
[m, 2H, N(CHH)2], 4.38 (t, 2H, J=7.2 Hz, carbazole NCH2), 6.90–
8.10 ppm (m, 12H, aromatic); GC–MS m/z: 400 [M]+ (12), 192
(100); LC–MS (ESI+) m/z 401 [M+H]+; LC–MS–MS 401: 222, 180;
Anal. (C27H29N2F·H2O) C, H, N (H calcd: 7.47, found: 7.01).
[M]+
(C23H28N2O2·HCl·11= H2O) C, H, N.
(54),
246
(46),
206
(100),
192
(96);
Anal.
4
1’-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]spir-
o[isobenzofuran-1(3H),4’-piperidine] (19) was obtained as
1
a yellow oil (0.58 g, 78%). H NMR (300 MHz, CDCl3): d=1.48–2.20
[m, 12H, CH(CH2CH2)2 and piperidine C(CH2)2], 2.40–2.82 (m, 7H,
N(CH2)3 and benzyl CH), 2.90–3.05 (m, 2H, benzyl CH2), 3.80 (s, 3H,
OCH3), 5.04 (s, 2H, CH2O), 6.65 (d, 1H, J=7.7 Hz, aromatic), 6.80 (d,
1H, J=7.7 Hz, aromatic), 7.10 (t, 1H, J=7.7 Hz, aromatic), 7.15–
7.30 ppm (m, 4H, aromatic); GC–MS m/z: 391 [M]+ (26), 202 (100);
LC–MS (ESI+) m/z: 392 [M+H]+; LC–MS–MS 392: 203, 161; Anal.
9-[4-(6,7-Dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-9H-
carbazole (25) was obtained as a white solid (0.32 g, 83%): mp:
104–1068C; 1H NMR (300 MHz, CDCl3): d=1.60–2.05 (m, 4H,
NCH2CH2CH2), 2.45–2.85 (m, 6H, ArCH2CH2NCH2), 3.45 (s, 2H,
ArCH2N), 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 4.38 (t, 2H, J=
7.2 Hz, carbazole NCH2), 6.45 (s, 1H, aromatic), 6.60 (s, 1H, aromat-
ic), 7.18–8.15 ppm (m, 8H, aromatic); 13C NMR (75 MHz, CD3OD):
d=20.33, 23.10, 24.31, 40.19, 48.45, 51.03, 53.70, 53.80, 54.11,
107.28, 107.91, 109.82, 117.41, 117.67, 118.52, 121.42, 124.19,
138.96, 147.11, 147.92 ppm; GC–MS m/z: 414 [M]+ (66), 206 (44),
192 (100); LC–MS (ESI+) m/z: 415 [M+H]+; LC–MS–MS 390: 222,
(C26H33NO2·HCl·3= H2O) C, H, N.
4
4-(4-Fluorophenyl)-1-[3-(5-methoxy-1,2,3,4-tetrahydronaphtha-
len-1-yl)propyl]piperidine (20) was obtained as a white semisolid
(0.43 g, 60% yield). 1H NMR (300 MHz, CDCl3): d=1.45–1.85 [m,
12H, CH(CH2CH2)2 and piperidine CH(CH2)2], 1.98–2.08 (m, 1H, pi-
peridine CH), 2.35–2.85 (m, 7H, N(CH2)3 and benzyl CH), 3.02–3.08
(m, 2H, benzyl CH2), 3.80 (s, 3H, OCH3), 6.65 (d, 1H, J=7.7 Hz, aro-
matic), 6.80 (d, 1H, J=7.7 Hz, aromatic), 6.92–7.05 (m, 2H, aromat-
ic), 7.10 (t, 1H, J=7.7 Hz, aromatic), 7.16–7.30 ppm (m, 2H, aromat-
180; Anal. (C27H30N2O2·HCl·11= H2O) C, H, N.
2
ic); GC–MS m/z 381 [M]+
(26), 202 (100); Anal.
(C25H32NOF·HCl·1= H2O) C, H, N.
2
General procedure for the synthesis of amines 16–20, 23, and
24
9-[4-(4-Cyclohexylpiperazin-1-yl)butyl]-9H-carbazole (23) was ob-
tained as a white solid (0.44 g, 60%): mp: 84–868C; 1H NMR
(300 MHz, CDCl3): d=1.00–1.98 [m, 14H, cyclohexyl (CH2)5 and
NCH2CH2CH2], 2.05–2.78 (m, 11H, CHN, and piperazine), 4.32 (t, 2H,
J=7.2 Hz, carbazole NCH2), 7.15–8.10 ppm (m, 8H, aromatic); GC–
MS m/z: 390 [M+1]+ (17), 389 [M]+ (66), 346 (27), 181 (100); LC–
MS (ESI+) m/z: 390 [M+H]+; LC–MS–MS 390: 222, 180; Anal.
To a solution of one among intermediates 10–12 (1.9 mmol) in
CH3CN (25 mL), one among the appropriate amines a–e (2.3 mmol)
and K2CO3 (2.3 mmol, 0.32 g) were added. The reaction mixture
was held at reflux under stirring overnight. The solvent was then
removed under reduced pressure, and H2O (15 mL) was added to
the crude residue. The aqueous mixture was extracted with CH2Cl2
(3ꢂ10 mL), and the organic layers were dried over Na2SO4 and
concentrated under reduced pressure to afford a crude residue,
which was purified by column chromatography with CH2Cl2/MeOH
(95:5) as eluent.
(C26H35N3·2HCl·3= H2O) C, H, N.
4
9-[4-(4-Cyclohexyl-1-piperidino)butyl]-9H-carbazole (24) was ob-
tained as a white solid (0.46 g, 62%): mp: 87–898C; 1H NMR
(300 MHz, CDCl3): d=0.82–1.38 (m, 8H, cyclohexyl and piperidine
CH and CH2), 1.50–1.90 (m, 12H, cyclohexyl and piperidine CH,
CH2, and NCH2CH2CH2), 1.95–2.00 (m, 2H, CH2N), 2.25–2.40 (m, 2H,
CHHN piperidine), 2.82–2.95 (m, 2H, CHHN piperidine), 4.32 (t, 2H,
J=7.2 Hz, carbazole NCH2), 7.15–8.20 ppm (m, 8H, aromatic); GC–
MS m/z: 388 [M]+ (8), 346 (27), 180 (100); Anal.
3-[4-(4-Cyclohexyl-1-piperazinyl)butyl]-1H-indole (16) was ob-
tained as a yellow semisolid (0.58 g, 90%). 1H NMR (300 MHz,
CDCl3): d=1.07–1.25 [m, 5H, cyclohexyl (CHH)5], 1.55–1.92 [m, 9H,
cyclohexyl (CHH)5 and ArCH2CH2CH2], 2.11–2.29 (m, 1H, CHN), 2.40
(t, 2H, J=7.6 Hz, CH2N), 2.54–2.66 (m, 8H, piperazine), 2.77 (t, 2H,
J=7.3 Hz, ArCH2), 6.97 (s, 1H, aromatic), 7.07–7.20 (m, 2H, aromat-
ic), 7.31–7.36 (m, 1H, aromatic), 7.58–7.61 (m, 1H, aromatic),
7.97 ppm (brs, 1H, NH D2O exchanged); GC–MS m/z: 340 [M+1]+
(C27H36N2·HCl·1= H2O) C, H, N.
4
(25), 339 [M]+ (100), 181 (95), 130 (48); Anal. (C22H33N3·2HCl·1= H2O)
2
Biology
C, H, N.
3-[4-(4-Cyclohexyl-1-piperidino)butyl]-1H-indole (17) was ob-
tained as yellow oil (0.47 g, 73%). 1H NMR (300 MHz, CDCl3): d=
1.07–1.75 [m, 20H, cyclohexyl, piperidine CH2CHCH2, and
ArCH2CH2CH2], 1.78–1.86 (m, 2H, CH2CH2CH2N), 2.31–2.36 (m, 2H,
piperidine CHHNCHH), 2.77 (t, 2H, J=7.3 Hz, ArCH2), 2.94–3.00 (m,
2H, piperidine CHHNCHH), 6.97 (s, 1H, aromatic), 7.07–7.20 (m, 2H,
aromatic), 7.35 (d, 1H, J=7.7 Hz, aromatic), 7.60 (d, 1H, J=7.5 Hz,
aromatic), 7.95 (brs, 1H, NH D2O exchanged); GC–MS m/z: 338
[M]+ (11), 180 (100), 130 (66); Anal. (C23H34N2·HCl) C, H, N.
Materials:
[3H]DTG
(50 CimmolÀ1),
(+)-[3H]pentazocine
(30 CimmolÀ1) and ATPlite 1-step Kit were purchased from Perkin
Elmer Life and Analytical Sciences (Boston, MA, USA). DTG was pur-
chased from Tocris Cookson Ltd., (UK). (+)-Pentazocine was ob-
tained from Sigma–Aldrich-RBI s.r.l. (Milan, Italy). Male Dunkin
guinea pigs and Wistar Hannover rats (250–300 g) were obtained
from Harlan, Italy. Cell culture reagents were purchased from Euro-
Clone (Milan, Italy). CulturePlate 96-well plates were purchased
from PerkinElmer Life Science. Calcein-AM, 3-(4,5-dimethylthiazol-2-
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yl)-2,5-diphenyltetrazoliumbromide (MTT), a-tocopherol, and dox-
orubicin were obtained from Sigma–Aldrich (Milan, Italy).
at 378C, the supernatant was removed. The formazan crystals were
solubilized with 100 mL DMSO/EtOH (1:1), and the absorbance
values at l 570 and 630 nm were determined on a microplate
reader Victor 3 (PerkinElmer Life Sciences).
Competition binding assays: All procedures for binding assays were
described previously; s1 and s2 receptor binding assays were car-
ried out according to Matsumoto et al.[38] The specific radioligands
and tissue sources were: a) s1 receptor, (+)-[3H]pentazocine,
guinea pig brain membranes without cerebellum; b) s2 receptor,
[3H]DTG in the presence of 1 mm (+)-pentazocine to mask s1 recep-
tors, rat liver membranes. The following compounds were used to
define the specific binding reported in parentheses: a) (+)-penta-
zocine (73–87%), b) DTG (85–96%). Concentrations required to in-
hibit 50% of radioligand specific binding (IC50) were determined by
using six to nine different concentrations of the drug studied in
two or three experiments with samples in duplicate. Scatchard pa-
rameters (Kd and Bmax) and apparent inhibition constant (Ki) values
were determined by nonlinear curve fitting by using Prism v. 3.0
(GraphPad software).[39]
Calcein-AM experiments: These experiments were carried out as de-
scribed by Feng et al. with minor modifications.[42] Calcein-AM is
a pro-fluorescent probe and a P-gp substrate. In cells overexpress-
ing P-gp, calcein-AM is unable to permeate the cell membrane,
whereas when the efflux pump is not present or is inhibited, the
probe enters living cells and is converted into fluorescent calcein
by intracellular esterases. Calcein is unable to diffuse through the
membrane, as it is hydrophilic and is not a P-gp substrate; there-
fore, calcein accumulates in cells when the pump is blocked.
Therefore, the fluorescent signal is directly correlated to the
amount of P-gp inhibition. The MDCK-MDR1 cell line (50000 cells
per well) was seeded into black CulturePlate 96-well plates with
100 mL medium and allowed to reach confluency overnight. Test
compounds at various concentrations (0.1–100 mm) were solubi-
lized in culture medium (100 mL) and added to monolayers; 96-well
plates were incubated at 378C for 30 min. Calcein-AM was added
in 100 mL phosphate-buffered saline (PBS) to yield a final concen-
tration of 2.5 mm, and plates were incubated for 30 min. Each well
was washed with ice-cold PBS (3ꢂ100 mL). Saline buffer (100 mL)
was added to each well, and the plate was read in a Victor 3 instru-
ment (PerkinElmer) at excitation and emission wavelengths of 485
and 535 nm, respectively. Under these experimental conditions, cal-
cein cell accumulation in the absence and presence of test com-
pounds was evaluated, and basal-level fluorescence was estimated
by untreated cells. In treated wells, the increase in fluorescence
with respect to basal levels was measured. EC50 values were deter-
mined by fitting the percent increase in fluorescence versus
log[dose].
Cell cultures: Human MCF7 breast adenocarcinoma was purchased
from ICLC (Genoa, Italy), human MCF7adr breast adenocarcinoma
(resistant to doxorubicin), were kindly provided by Prof. G. Zupi
(IRE, Rome, Italy), MDCK-MDR1 cells were a gift of Prof. P. Borst,
NKI-AVL Institute, Amsterdam (Netherlands). MCF7 and MDCK-
MDR1 cells were grown in high-glucose DMEM supplemented with
10% fetal bovine serum, 2 mm glutamine, 100 UmLÀ1 penicillin,
100 mgmLÀ1 streptomycin, in a humidified incubator at 378C under
a 5% CO2 atmosphere. MCF7adr cells were grown in RPMI 1640
supplemented with 10% fetal bovine serum, 2 mm glutamine, 100
UmLÀ1 penicillin, 100 mgmLÀ1 streptomycin, in a humidified incu-
bator at 378C under a 5% CO2 atmosphere.
Cell viability: Determination of cell growth was performed by MTT
assay at 48 h.[40,41] On day 1, 25000 cells per well were seeded into
96-well plates at a volume of 100 mL. On day 2, the various drug
concentrations (0.1–100 mm) were added. In all the experiments,
the various drug solvents (EtOH, DMSO) were added in each con-
trol to evaluate possible cytotoxicity by solvent. After the estab-
lished incubation time with drugs, MTT (0.5 mgmLÀ1) was added
to each well, and after 3–4 h incubation at 378C, the supernatant
was removed. The formazan crystals were solubilized using 100 mL
DMSO/EtOH (1:1), and the absorbance values at l 570 and 630 nm
were determined on a microplate reader Victor 3 (PerkinElmer Life
Sciences).
Bioluminescence ATP assays: These experiments were performed as
reported in the technical sheet of the ATPlite 1-step Kit for lumi-
nescence ATP detection based on firefly (Photinus pyralis) luciferase
(PerkinElmer Life Sciences).[43,44] The ATPlite assay is based on the
production of light caused by the reaction of ATP with added luci-
ferase and d-luciferin (substrate solution), and the amount of light
emitted is proportional to the ATP concentration. MCF7 and
MCF7adr cells were seeded into black CulturePlate 96-well plates
in 100 mL of complete medium at a density of 2ꢂ104 cells per well.
Plates were incubated overnight in a humidified atmosphere con-
taining 5% CO2 at 378C. The medium was removed, and 100 mL of
complete medium in the presence or absence of various test com-
pound concentrations (0.1–100 mm) was added. Plates were incu-
bated for 2 h under a humidified atmosphere 5% CO2 at 378C.
Mammalian cell lysis solution (50 mL) was then added to all wells,
and the plates were agitated for 5 min on an orbital shaker. Sub-
strate solution (50 mL) was added to all wells, and the plates were
stirred for another 5 min on an orbital shaker. Plates were dark-
adapted for 10 min, and luminescence was measured on a micro-
plate reader Victor 3 (PerkinElmer Life Sciences).
Effect of a-tocopherol on cell viability: The interference of ROS in
cell viability was indirectly determined by MTT assay at 24 h. On
day 1, 25000 cells per well were seeded into 96-well plates in the
presence or absence of various concentrations of a-tocopherol (1–
100 mm). On day 2, the drugs (25 mm) were added alone and in
combination with various concentrations of a-tocopherol (1–
100 mm). After incubation (24 h) with drugs, MTT (0.5 mgmLÀ1) was
added to each well, and after 3–4 h incubation at 378C, the super-
natant was removed. The formazan crystals were solubilized with
100 mL DMSO/EtOH (1:1), and the absorbance values at l 570 and
630 nm were determined on a microplate reader Victor 3 (Perki-
nElmer Life Sciences).
Keywords: antitumor agents
· breast cancer · collateral
Co-administration assays: The co-administration assay with doxoru-
bicin was performed at 72 h.[41] On day 1, 25000 cells per well were
seeded into 96-well plates at a volume of 100 mL. On day 2, three
drug concentrations (1, 10, and 25 mm) were added. On day 3, the
medium was removed, and the three drug concentration were
added alone and in combination with doxorubicin (1 or 10 mm).
After the established incubation time with drugs, MTT
(0.5 mgmLÀ1) was added to each well, and after 3–4 h incubation
sensitivity · multidrug resistance · sigma receptors
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Received: July 1, 2013
Revised: September 6, 2013
Published online on && &&, 0000
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Collateral damage: We developed
promising s2 ligands for alternative
strategies against multidrug-resistant
cancer. New high-affinity s2 agonists dis-
play antiproliferative activity in breast
tumor cells; their interaction with P-gp
generates higher activity in resistant
than in parent cells (collateral sensitivi-
ty). Compounds co-administered with
doxorubicin revert P-gp-mediated
resistance.
M. Niso, C. Abate,* M. Contino,
S. Ferorelli, A. Azzariti, R. Perrone,
N. A. Colabufo, F. Berardi
&& – &&
Sigma-2 Receptor Agonists as Possible
Antitumor Agents in Resistant
Tumors: Hints for Collateral Sensitivity
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