Novel derivatives of 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4- tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with improved fluorescent and σ receptors binding properties

Article abstract of DOI:10.1021/jm401874n

Despite the promising potentials of σ₂ receptors in cancer therapy and diagnosis, there are still ambiguities related to the nature and physiological role of the σ₂ protein. With the aim of providing potent and reliable tools to be used in σ₂ receptor research, we developed a novel series of fluorescent σ₂ ligands on the basis of our previous work, where high-affinity σ₂ ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl] piperazine (1, PB28) was used as the pharmacophore. Compared to the previous compounds, these novel ligands displayed improved fluorescence and σ₂ binding properties, were σ₂-specifically taken up by breast tumor cells, and were successfully employed in confocal microscopy. Compound 14, which was the best compromise between pharmacological and fluorescent properties, was successfully employed in flow cytometry, demonstrating its potential to be used as a tool in nonradioactive binding assays for studying the affinity of putative σ₂ receptor ligands.

Full text of DOI:10.1021/jm401874n

Article
pubs.acs.org/jmc
Novel Derivatives of 1‑Cyclohexyl-4-[3-(5-methoxy-1,2,3,4-
tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with Improved
Fluorescent and σ Receptors Binding Properties
Carmen Abate,*,† Mauro Niso,† Roberta Marottoli,† Chiara Riganti,‡ Dario Ghigo,‡ Savina Ferorelli,†
Giulia Ossato,§ Roberto Perrone,† Enza Lacivita,† Don C. Lamb,§ and Francesco Berardi†
†Dipartimento di Farmacia-Scienze del Farmaco, Universita
̀
degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy
‡Department of Oncology, University of Turin, via Santena 5/bis, 10126, Torino, Italy
§Physical Chemistry, Department of Chemistry, Center for Nanoscience (CeNS), and Center for Integrated Protein Science Munich
(CiPSM), Ludwig-Maximilians-Universitat Munchen, Munich D-81377, Germany
̈
̈
S
* Supporting Information
ABSTRACT: Despite the promising potentials of σ2 receptors in cancer therapy and
diagnosis, there are still ambiguities related to the nature and physiological role of the
σ2 protein. With the aim of providing potent and reliable tools to be used in σ2
receptor research, we developed a novel series of fluorescent σ2 ligands on the basis of
our previous work, where high-affinity σ2 ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-
tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1, PB28) was used as the pharma-
cophore. Compared to the previous compounds, these novel ligands displayed
improved fluorescence and σ2 binding properties, were σ2-specifically taken up by
breast tumor cells, and were successfully employed in confocal microscopy.
Compound 14, which was the best compromise between pharmacological and
fluorescent properties, was successfully employed in flow cytometry, demonstrating its
potential to be used as a tool in nonradioactive binding assays for studying the affinity of putative σ2 receptor ligands.
agonists in tumor cells in vitro and supported by data coming
from preclinical tumor models in vivo are very promising and
led to the investigation of σ2 ligands as antitumor agents.12−14
Despite these encouraging data, there are still ambiguities
related to the nature of σ2 receptors, their physiological role,
and their mechanisms of action. This has been mainly due to
the absence of the cloned gene or the purified σ2 receptor
protein, and only recently the σ2 receptor binding site has been
proposed to be located in the progesterone receptor membrane
component 1 (PGRMC1).15 Since interest in σ2 receptor
ligands grows together with the need for alternative anti-cancer
strategies, powerful tools for the study of these still enigmatic
receptors are required.
With the aim of providing more potent and reliable tools to
be used in σ2 receptor research, we synthesized novel
fluorescent derivatives of 1-cyclohexyl-4-[3-(5-methoxy-
1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1,
PB28, Chart 1), one of the highest affinity σ2 ligands
known.16 In previous work, we identified a position in the
structure of 1 where the fluorescent tag could be conveniently
attached without compromising the pharmacological proper-
ties: the 5-position on the tetraline ring, where the methoxy
group could be replaced by a hexamethylenoxy linker.17
Compound 6-{5-[3-(4-cyclohexylpiperazin-1-yl)propyl]-
INTRODUCTION
■
Sigma (σ) proteins were originally characterized as a subtype of
the opiate receptors, but later studies clarified that they are a
distinct class of receptors divided into two subtypes, σ1 and
σ2.1,2 The σ1 protein, which is the better known of the two
subtypes, is distributed throughout the brain, where it has been
shown to modulate a number of central neurotransmitter
systems. Roles in neuroprotection and neuroplasticity have
been suggested for this subtype, and implication in several
central nervous system (CNS) pathologies (e.g., anxiety,
depression, schizophrenia, Parkinson’s and Alzheimer’s disease)
has been shown.3,4 A mutation in the σ1 receptor sequence has
been suggested to cause juvenile amyotrophic lateral sclerosis
(JALS).5 Dimerization and oligomerization of the σ1 receptors
have been recently suggested,6 and a functional form of σ1-D2
heteromer has been proposed to account for the involvement of
D2 receptors in cocaine activity.7 All of these pieces of evidence
keep the interest in σ1 receptors high. As for the σ2 subtypes,
the evidence that they are overexpressed in a wide variety of
peripheral and brain human tumors gave a great impulse to the
σ2 subtype-related research. σ2 Receptors are proposed as
endogenous biomarkers for the proliferative status of tumors,
since they are more overexpressed in proliferative than in the
corresponding quiescent tumor cells.8 Several σ2 radioligands
have been developed for the imaging of tumors, and a phase I
clinical trial for a promising 18F-radiolabeled σ2 ligand has just
been completed.9−11 In addition, the cytotoxic properties of σ2
Received: December 5, 2013
Published: April 3, 2014
© 2014 American Chemical Society
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2,1,3-benzoxadiazol-4-yl). Therefore, ligands emitting in the
green region of the electromagnetic spectrum with higher QY
were obtained. One red-emitting fluorophore was also
connected to the linker to generate σ2 receptor fluorescent
ligands, which fluoresce at longer wavelengths where cellular
autofluorescence is significantly reduced. For all the com-
pounds, the kinetics were studied by confocal microscopy in
human breast tumor cells (MCF7 cells), where the specificity of
the fluorescent tracers for σ2 receptors was also verified. The
best fluorescent σ2 ligand was exploited to perform a σ2
receptor binding assay in MCF7 cells by flow cytometry.
Chart 1
RESULTS AND DISCUSSION
■
Chemistry. The synthetic pathway for 7 is depicted in
Scheme 1. Reaction between potassium phthalimide and 1,10-
dibromodecane gave intermediate 4, which was used to alkylate
the key phenolic intermediate 3,16 affording phthalimide
derivative 5. This phthalimide underwent hydrazinolysis to
afford intermediate primary amine 6, which upon reaction with
NBD-chloride afforded the final compound 7.
5,6,7,8-tetrahydronaphthalen-5-yloxy}-N-(7-nitro-2,1,3-benzox-
adiazol-4-yl)hexanamine (2, PB385, Chart 1) emerged for its
best compromise between pharmacological and fluorescent
properties and was successfully used in microscopy and flow
cytometry studies within pancreatic cancer cells.17,18 However,
both the pharmacological (σ2 receptor affinity and selectivity)
and fluorescence properties (higher quantum yields, QY, and
brightness) of 2 could be improved in order to obtain more
powerful and reliable tools for σ2 receptor research. With this
aim, we first elongated the chain from six to ten methylenes in
the structure of 2. As other authors reported, this elongation
resulted in significantly improved σ2 affinity and selectivity in a
granatane-based pharmacophore series.19 Nevertheless, this was
not the case for our 1-based series, so that subsequently the six-
methylene chain was kept as the optimal linker to connect 1
with fluorophores structurally different from NBD (7-nitro-
The synthetic pathway for 11 is depicted in Scheme 2.
Reaction between the primary amine 817 and 4-nitro-1,8-
naphthalic anhydride (9) afforded the nitro derivative 10.
Hydrogenation of this last compound at atmospheric pressure
in the presence of 10% Pd over carbon afforded the final
compound 11.
The synthetic pathway for 14 is depicted in Scheme 3. 4-
(N,N-Dimethylamino)phthalic acid20 was condensed with 6-
amino-1-hexanol in the presence of carbonyldiimidazole,
affording intermediate alcohol 12. Reaction of 12 with
methanesulfonyl chloride afforded mesyl derivative 13 that
was subsequently used to alkylate the key phenolic intermediate
3, yielding the final fluorescent compound 14.
The synthetic pathway for 17 is depicted in Scheme 4. Acid
15,21 upon activation to the corresponding succinic imide 16,
was reacted with amine 8 to afford the final fluorescent
a
Scheme 1
a
Reagents: (a) NaI, K2CO3; (b) hydrazine hydrate; (c) NBD-chloride.
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a
Scheme 2
a
Reagents: (a) H2, 10% Pd on activated carbon.
a
bearing molecule 7 produced a 3-fold decrease in the affinity for
σ2 receptor binding, compared to the lead compound 2.
However, σ2 receptor selectivity remained unchanged since the
same decrease was also recorded for σ1 receptor binding (Ki =
258 nM at σ1 and Ki = 31.2 nM at σ2 receptors). On the other
hand, the six-methylene chain derivative 11 carrying the green-
emitting fluorophore (4-amino-1,8-naphthylimide) displayed
improved binding properties at σ2 receptors: a slightly higher
affinity at σ2 (Ki = 6.57 nM) and a 2-fold lower affinity at the σ1
subtype (Ki = 156 nM), thus resulting in a convenient increase
in σ2 selectivity. Insertion of 4-(N,N-dimethylamino)-
phthalimide in place of NBD led to the fluorescent derivative
14, with slightly improved binding to the σ2 receptor (Ki = 6.9
nM). However, this last molecule displayed equal high affinity
for the σ1 receptor (Ki = 5.37 nM). Together, these results
suggested that steric hindrance of the fluorophores negatively
affects binding to σ1 receptors while maintaining the high
affinity for the σ2 subtype. As for the red-emitting fluorescent
ligand 17, a significant drop in the affinity for both σ receptors
was recorded (Ki = 235 nM at σ1 and Ki = 161 nM at σ2
receptors), suggesting that either the positive permanent charge
or the more extended structure of the fluorophore, compared to
the other fluorophores, hampers the interaction with both σ
binding sites.
Scheme 3
Fluorescent Ligand Studies. Fluorescent Properties. The
fluorescent properties of final compounds are listed in Table 1.
Excitation and emission spectra were obtained from solutions
of the final compounds in organic solvent (CHCl3) and in
aqueous solution (phosphate-buffered saline, PBS). All of the
compounds showed important differences between maximum
excitation and emission wavelength (λex and λem) (i.e., Stokes
shift), with 14 displaying the highest Stokes shift. QY values
were determined in the above-mentioned solvents to probe the
sensitivity of the final fluorescent ligands to the environment
(i.e., low QY in aqueous solution but high fluorescence in
a
Reagents: (a) MsCl, NEt3; (b) K2CO3.
compound 17. All the final compounds were converted to the
corresponding hydrochloride salts with gaseous HCl except for
17.
Biology: σ Receptor Binding. Results from binding assays
are expressed as inhibition constants (Ki values) in Table 1.
The elongation of the linker to ten methylenes in the NBD-
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a
new fluorescent compounds (11, 14, and 17) is also similar to
what has been observed previously with other σ2 fluorescent
ligands.17,19 At a concentration of 100 nM, the new fluorescent
σ2 receptor ligands localize mostly in the cytoplasm, perinuclear
region of the cell, and vesicles (Figure 1). At higher
concentrations (500 nM), the fluorescence signal increases in
cytosol and vesicles and fluorescence intensity was also
detected in the nuclei (Figure S1 in Supporting Information).
The signal from the nuclei can be clearly visualized in the
intensity profile of pixels in the background and pixels in the
nuclei. The presence of σ2 receptor ligands in the nuclei could
be explained by the hypothesis of Colabufo et al.24 that the σ2
receptor has histone binding proprieties. However, this
behavior needs further characterization since, at this high
concentration (500 nM), unspecific interaction of compounds
with cellular proteins other than σ2 receptors cannot be
excluded. At 500 nM, ligand localization is slightly different
than at 100 nM: 17 tends to accumulate more in vesicles and
11 is in the cytoplasm and concentrates in the perinuclear
region, while 2 and 14 are more homogeneously distributed
(Figure S1 in Supporting Information). No colocalization
experiments were performed with markers for various
organelles, as the high amount of fluorescence of the ligand
observed throughout the cytosol would lead to an apparent
colocalization signal, making the results inconclusive.
Scheme 4
Uptake of σ2 Ligands. Uptake of fluorescent compounds
displaying the highest σ2 affinity (lead compound 2, 11, and
14) was studied in σ2-overexpressing MCF7 cells. Uptake of the
compounds increased in a dose-dependent manner (Figure 2)
at the three concentrations used, reaching a plateau between 50
and 100 nmol/L. Compound 14 showed the highest uptake,
with around 2-fold higher uptake than 2, and 10-fold higher
uptake than 11 (picomoles of fluorescent compound on
milligram of protein). The higher intracellular fluorescence of σ
ligand 14 could be due to its higher QY and/or to its peculiar
chemical structure. Compound 14 displays high affinity for
both σ1 and σ2 receptors, in contrast with the more σ2-receptor-
selective 2 and 11, making it suitable for monitoring dual
nonselective uptake. However, in cell lines such as MCF7
where the density of σ1 receptors is known to be low in
comparison to that of the σ2 subtypes,25 all compounds
including 14 can be used as highly specific σ2 receptor ligands
as demonstrated by competition assays. Pretreatment of cells
with increasing concentration of reference σ2 agonists 113 or cis-
1-cyclohexyl-4-[4-(2,6-difluorophenyl)cyclohexyl]piperazine
(18, Chart 1)17,26 (10, 25, and 50 nmol/L) was followed by
administration of the fluorescent compounds 2, 11, or 14 at 25
nmol/L (Figure 2). Uptake of the three fluorescent compounds
2, 11, and 14 was reduced in a dose-dependent manner by the
two σ2 agonists, confirming that these fluorescent σ2 ligands are
targeting σ2 receptors. Also, in the competition assays, the
absolute values of fluorescence differed for each compounds
and reproduced the differences in fluorescence observed in the
dose-dependence assays, due to the different QY. However, the
general trendthat is, a progressive reduction of the
intracellular fluorescence in the presence of increasing
concentrations of compound 1 or 18strongly suggests that
all the compounds are highly specific for σ2 receptors. The
highest decrease in fluorescence exerted by σ2 agonists 1 or 18
(50 nmol/L) was obtained with 14, indicating that this
compound is by far the most powerful ligand for σ2 receptors.
Flow Cytometry Study. We then set up a σ2 receptor
binding assay, by incubating nonpermeabilized MCF7 cells with
a
Reagents: (a) N,N′-disuccinimidyl carbonate, 4-(dimethylamino)-
pyridine, NEt3; (b) Et3N.
nonpolar solvents or when bound to a hydrophobic sites). As
expected, the highest QY values were those recorded in CHCl3
for all final compounds. On the other hand, all the compounds
exhibited low fluorescence in PBS buffer. Compounds (11, 14,
and 17) with fluorescent tags diverse from NBD displayed
higher QY values and showed improved fluorescent properties
compared to the so far known σ2 fluorescent ligands.17,19,22,23
These ligands were obtained through two methods: by using
fluorescent moieties (β-naphthol and carbazole nuclei) as the
hydrophobic portions of the σ2 ligands22 or by conjugating σ2
receptor pharmacophores with fluorescent moieties (NBD or
dansyl).17,19,23 Among all the compounds tested, the green-
emitting 14 displayed the highest Stokes shift and the highest
QY in CHCl3 and PBS.
Confocal Microscopy and in Vitro Internalization Studies.
All ligands examined (2, 11, 14, and 17) were detectable by
spinning disk confocal microscopy in live and fixed MCF7 cells
starting from a concentration of 50 nM. Compound 7 did not
undergo this study since its binding properties were worse than
2 (its inferior homologue) and the fluorescent properties were
the same. Therefore, we chose to study 2 rather than 7 in order
to obtain more reliable information. We observed the entry of
the new σ2 receptor ligands into MCF7 cells within 40 min.
The fluorescence intensity inside the cells starts increasing 20
min after treatment and reaches a plateau 40 min after
treatment. This uptake kinetics agrees with the observation of
Abate et al.17 for other σ2 ligands. The distribution of all the
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a
Table 1. Receptor Affinities and Fluorescence Properties of Final and Reference Compounds
a
b
Values are the means of n ≥ 2 separate experiments, in duplicate. Fluorescence properties herein reported were evaluated on compounds as free
c
d
bases in CHCl3 and as their corresponding hydrochloride salts in PBS solutions. From ref 17. Compound 17 displayed low solubility in CHCl3.
Therefore, no fluorescence properties were calculated for this compound in this solvent.
increasing concentrations (from 0.1 nmol/L to 10 μmol/L) of
1, followed by 100 nmol/L 14. We detected a progressive
decrease of 14 fluorescence when the concentration of 1 was
increased (Figure 3A). This trend, indicative of the progressive
displacement of 14 exerted by 1, suggests that fluorescent
ligand 14 and compound 1 compete for binding on σ2
receptors. The binding curve (Figure 3A) obtained by this
displacement shows an IC50 = 4.27 nM for 1. This data was in
accordance with what we found by competition binding assay
where [3H]DTG was displaced by 1 (from 0.1 nmol/L to 10
μmol/L) in MCF7 cell membranes (IC50 = 1.2 nM, Figure 3B).
In order to give further support to the specificity of compound
14 for σ2 receptors in intact MCF7 cells, we performed a
binding assay with the σ2 receptor reference compound 1,3-
di(2-tolyl)guanidine (DTG). We incubated MCF7 cells with
increasing concentrations (from 0.1 nmol/L to 10 μmol/L) of
DTG, followed by 100 nmol/L 14. We detected a progressive
decrease of 14 fluorescence when the concentration of DTG
increased (Figure S2 in Supporting Information). The
experiment was performed with and without (+)-pentazocine
to mask the unlikely presence of σ1 receptor, and no difference
was observed, supporting the very low density of σ1 receptors in
this cell line. This is the first assay showing that fluorescent
ligand 14 may be a useful tool in nonradioactive binding assays,
to study the affinity of putative σ2 receptors ligands and to map
σ2 receptors localization and density on cells and tissues.
the compounds accumulate in cells with a distribution in line
with the σ2 receptor distribution previously reported.
Fluorometric experiments showed a clear and σ2-dependent
uptake of the compounds, with ligand 14 (highest QY and
highest σ2 affinity) showing the highest cell uptake. Therefore,
the suitability of 14 as a σ2 fluorescent tracer was tested in flow
cytometry where σ2 binding in MCF7 cells was successfully
verified. Overall, we produced high-affinity σ2 fluorescent
ligands and showed their potential to be used in σ2 receptor
studies within cells. To the best of our knowledge, 14 is the first
σ2 fluorescent ligand successfully employed in binding experi-
ments in living cells by flow cytometry, demonstrating that it is
a useful and safe alternative to the use of radioligands.
EXPERIMENTAL SECTION
■
Chemistry. Both column chromatography and flash column
chromatography were performed with 60 Å pore size silica gel as
the stationary phase (1:30 w/w, 63−200 μm particle size, from ICN,
and 1:15 w/w, 15−40 μm particle size, from Merck, respectively).
Melting points were determined in open capillaries on a Gallenkamp
electrothermal apparatus. Purity of tested compounds was established
by combustion analysis or HPLC, confirming a purity >95%.
Elemental analyses (C, H, N) were performed on an Eurovector
Euro EA 3000 analyzer; analytical results were within 0.4% of
theoretical values. High-performance liquid chromatography (HPLC)
was performed on a Perkin−Elmer series 200 LC instrument with a
Phenomenex Gemini RP-18 column (250 × 4.6 mm, 5 μm particle
size) and equipped with a Perkin−Elmer 785A UV/vis detector set at
λ = 254 nm. 1H NMR spectra were recorded on a Mercury Varian 300
MHz with CDCl3 as solvent. The following data were reported:
chemical shift (δ) in parts per million (ppm), multiplicity (s = singlet,
d = doublet, t = triplet, m = multiplet), integration, and coupling
constant(s) in hertz. Recording of mass spectra was done on an
Agilent 1100 series LCMSD trap system VL mass spectrometer; only
significant m/z peaks, with their percentage of relative intensity in
CONCLUSIONS
■
Novel σ2 fluorescent ligands with different structural and
fluorescence properties were synthesized on the basis of 2 with
the structure of 1 as the pharmacophore. Better results were
obtained when fluorescent ligands carried a green-emitting tag
on a six-methylene linker. Confocal microscopy showed that all
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Figure 1. MCF7 cells treated for 45 min with 100 nM 2 (A), 11 (B), 14 (C), and 17 (D) before fixation with 2% paraformaldehyde (PFA). All
images are background-corrected. All the compounds localize in the cytoplasm and in vesicles. The detected fluorescence from 11 and 17 is higher
compared to the signals from 2 and 14. Scale bar = 25 μm.
parentheses, are reported. Chemicals were from Aldrich and Across
and were used without any further purification.
mL, 0.85 mmol) was added to a solution of phthalimide 5 (179 mg,
0.28 mmol) in methanol (3 mL), and the reaction mixture was stirred
at room temperature for 30 min. HCl (1.5 N, 1.2 mL) was then added
and the mixture was stirred for a further 12 h. Then 3 N HCl was
added until pH < 2 was obtained, and the mixture was heated at reflux
for 30 min. After cooling down to room temperature, the mixture was
filtered, and the solid residue was washed with cold MeOH and with
Et2O and dried under vacuum. The white solid obtained was made free
base with alkaline tratment to afford the target compound as a pale
yellow oil (104 mg, 70% yield). 1H NMR 1.00−1.30 (m, 5H,
cyclohexyl), 1.40−1.85 [m, 29H, 5H cyclohexyl, CH2CH2CH2N,
CHCH2CH2, OCH2(CH2)8CH2N], 1.90−2.10 (br s, 2H, exchanged
D2O), 2.20−2.68 [m, 13H, CH cyclohexyl, CH2CH2CH2N, piperazine
CH2, O(CH2)9CH2N], 2.78−2.90 (m, 3H, benzyl CH2 and CH), 3.92
[t, 2H, J = 6.3 Hz, OCH2(CH2)9N], 6.61 (d, 1H, J = 7.9 Hz,
aromatic), 6.75 (d, 1H, J = 7.7 Hz, aromatic), 7.05 (t, 1H, J = 7.9 Hz,
aromatic). LC-MS (ESI+) m/z 512 [M + H]+.
2-(10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahy-
dronaphthalen-5-yloxy}decyl)-1H-isoindole-1,3(2H)-dione (5). To a
solution of phenol 3 (100 mg, 0.28 mmol) in N,N-dimethylformamide
(DMF) (5 mL) were added a grain of NaI, K2CO3 (52 mg, 0.38
mmol), and 4 (139 mg, 0.38 mmol). The reaction mixture was heated
for 18 h at 100 °C. After cooling, the solvent was removed under
reduced pressure, and then H2O (5 mL) was added to the residue and
the mixture was extracted with AcOEt (3 × 5 mL). The collected
organic layers were dried over Na2SO4 and evaporated to afford a
residue, which was purified by column chromatography with CH2Cl2/
MeOH (95:5) as eluent to give the final compound as a yellow oil
(55.67 mg, 31% yield). 1H NMR δ 1.10−1.48 [m, 10H, (CH2)5
cyclohexyl], 1.55−2.23 [m, 24H, CH2CH2CH2N, CHCH2CH2,
OCH2(CH2)8CH2N], 2.50−2.88 (m, 6H, CH cyclohexyl,
CH2CH2CH2N, benzyl CH2 and CH), 3.05−3.40 (m, 8H, piperazine),
3.67 [t, 2H, J = 7.3 Hz, O(CH2)9CH2N], 3.91 [t, 2H, J = 6.3 Hz,
OCH2(CH2)9N], 6.63 (d, 1H, J = 7.7 Hz, aromatic), 6.71 (d, 1H, J =
7.7 Hz, aromatic), 7.06 (t, 1H, J = 7.98 Hz, aromatic), 7.67−7.74 (m,
2H, aromatic), 7.78−7.86 (m, 2H, aromatic). LC-MS (ESI+) m/z 642
[M + H]+.
10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
naphthalen-5-yloxy}-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-
decylamine (7). 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl, 34
mg, 0.17 mmol) was dissolved in absolute EtOH (4 mL) and added
dropwise to amine 6 (87 mg, 0.17 mmol) dissolved in CH3CN/EtOH
(1:1, 4 mL) and kept under N2. The reaction mixture was stirred for 2
h at room temperature. Then the solvent was removed under reduced
10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
naphthalen-5-yloxy}decylamine (6). Hydrazine hydrate 50% (0.085
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Figure 2. (Left panels) MCF7 cells were incubated with 25, 50, and 100 nmol/L 2, 11, or 14 for 45 min at 37 °C. (Right panels) Cells were
pretreated with 10, 25, and 50 μmol/L 1 or 18 for 45 min at 37 °C followed by 25 nmol/L 2, 11, or 14 for 45 min at 37 °C. The uptake of σ2 ligands
was measured fluorometrically in duplicate as reported in the Experimental Section. Data are presented as mean SE (n = 2).
pressure and a solution of Na2CO3 was added to the residue to obtain
an alkaline pH. The aqueous phase was extracted with CH2Cl2 (3 × 15
mL), and the organic layers were collected and dried with Na2SO4 and
then evaporated under reduced pressure to afford a brown oil. The
crude mixture was purified by column chromatography with AcOEt/
MeOH (7:3) as eluent to give the target compound as a brown oil
(50.4 mg, 44% yield). 1H NMR 1.00−1.89 [m, 34H, (CH2)5
cyclohexyl, CH2CH2CH2N, CHCH2CH2, OCH2(CH2)8CH2N],
2.20−2.78 (m, 14H, CH cyclohexyl, CH2CH2CH2N, piperazine
CH2, benzyl CH2 and CH), 3.40−3.58 [m, 2H, OCH2(CH2)9N],
3.92 [t and br s, 3H, J = 6.3 Hz, O(CH2)9CH2N and NH], 6.17 (d,
1H, J = 8.8 Hz, aromatic), 6.61 (d, 1H, J = 8.0 Hz, aromatic), 6.76 (d,
1H, J = 7.7 Hz, aromatic), 7.06 (t, 1H, J = 7.8 Hz, aromatic), 8.50 (d,
1H, J = 8.5 Hz, aromatic). LC-MS (ESI+) m/z 675 [M + H]+, 697 [M
+ Na]+. Anal. (C39H58N6O4·3HCl·0.5H2O) Calcd: 59.05, 7.88, 10.59.
Found: 59.43, 7.68, 10.24.
2-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
naphthalen-5-yloxy}hexyl)-6-nitro-1H-benzo[de]isoquinoline-
1,3(2H)-dione (10). Under N2 atmosphere, 4-nitro-1,8-naphthalic
anhydride (9) (53.5 mg, 0.22 mmol) was dissolved in absolute EtOH
(5 mL) and added dropwise to hexylamine derivative 8 (102 mg, 0.22
mmol) dissolved in absolute EtOH (10 mL). The reaction mixture was
refluxed for 3 h. The solvent was then removed under reduced
pressure to afford a brown oil. The crude mixture was purified by
column chromatography with CH2Cl2/MeOH (98:2) as eluent to give
1
the target compound as a brown oil (70 mg, 51% yield). H NMR δ
1.05−1.35 [m, 5H, (CHH)5 cyclohexyl], 1.38−2.10 [m, 21H, (CHH)5
cyclohexyl, OCH2(CH2)4CH2N, CH2CH2CH2N, CHCH2CH2], 2.25−
2.90 (m, 14H, piperazine CH2, CH2CH2CH2N, CH cyclohexyl, benzyl
CH2 and CH), 3.92 [t, 2H, J = 6.1 Hz, OCH2(CH2)5N], 4.20 [t, 2H, J
= 7.6 Hz, O(CH2)9CH2N], 6.60 (d, 1H, J = 8.0 Hz, aromatic), 6.74 (d,
1H, J = 7.7 Hz, aromatic), 7.04 (t, 1H, J = 7.8 Hz, aromatic), 7.98 (t,
1H, J = 8.0 Hz, aromatic), 8.40 (d, 1H, J = 8.0 Hz, aromatic), 8.68 (d,
1H, J = 8.0 Hz, aromatic), 8.73 (d, 1H, J = 7.1 Hz, aromatic), 8.84 (d,
1H, J = 8.8 Hz, aromatic).
6-Amino-2-(6-{1-[3-(4-cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-
tetrahydronaphthalen-5-yloxy}hexyl)-1H-benzo[de]isoquinoline-
1,3(2H)-dione (11). Compound 10 (68 mg, 0.10 mmol) was dissolved
in EtOH and hydrogenated with 10% Pd on activated carbon at
atmospheric pressure for 12 h. Then the mixture was filtered off on
Celite and the filtrate was concentrated under reduced pressure to
afford the residue as a yellow oil. The crude mixture was purified by
column chromatography with CH2Cl2/MeOH (9:1) as eluent to give
1
the final compound as a colorless oil (22 mg, 34% yield). H NMR δ
1.05−1.35 [m, 5H, (CHH)5 cyclohexyl], 1.40−2.05 [m, 21H, (CHH)5
cyclohexyl, OCH2(CH2)4CH2N, CH2CH2CH2N, CHCH2CH2], 2.30−
3320
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NCH2), 4.21 (t, 2H, J = 6.5 Hz, CH2O), 6.78 (dd, 1H, J = 8.5 Hz, J′ =
2.5 Hz, aromatic), 7.06 (d 1H, J = 2.4 Hz, aromatic), 7.63 (d, 1H, J =
8.5 Hz, aromatic). LC-MS (ESI+) m/z 391 [M + Na]+.
2-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
naphthalen-5-yloxy}hexyl)-5-dimethylamino-1H-isoindole-1,3(2H)-
dione (14). Intermediate 13 (130 mg, 0.33 mmol), K2CO3 (45.1 mg,
0.33 mmol) and phenol 3 (103 mg. 0.26 mmol) were dissolved in
DMF (10 mL), and the reaction mixture was heated at 150 °C for 24
h. After cooling, the solvent was removed under reduced pressure and
H2O (10 mL) was added to the residue. The mixture was extracted
with CH2Cl2 (3 × 10 mL). The collected organic layers were dried
(Na2SO4) and evaporated to afford a crude residue as a yellow oil,
which was purified by column chromatography with CH2Cl2/MeOH
(98:2) as eluent. The final compound was obtained as a colorless oil
(49 mg, 30% yield). 1H NMR δ 1.15−2.04 [m, 26H, (CH2)5
cyclohexyl, CH2CH2CH2N, CHCH2CH2, OCH2(CH2)4CH2N],
2.35−2.82 [m, 14H, piperazine CH2, CH cyclohexyl,
CH(CH2)2CH2N, benzyl CH2], 3.11 [s, 6H, N(CH3)2], 3.63 [t, 2H,
J = 7.15 Hz, O(CH2)5CH2N], 3.90 [t, 2H, J = 6.3 Hz,
OCH2(CH2)5N], 6.59 (d, 1H, J = 8.0 Hz, aromatic), 6.73−6.79 (m,
2H, aromatic), 7.02−7.07 (m, 2H, aromatic), 7.63 (d, 1H, J = 8.5 Hz,
aromatic). LC-MS (ESI+) m/z 629 [M + H]+, 651 [M + Na]+. Anal.
(C39H56N4O3·2HCl·H2O) Calcd: 65.07, 8.40, 7.78. Found: 64.85,
8.10, 7.71.
(E)-2-[2-(7-Diethylamino-2-oxo-2H-chromen-3-yl)vinyl]-3,3-di-
methyl-1-[6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl]indolium Bro-
mide (16). To acid 15 (200 mg, 0.34 mmol) in anhydrous DMF (2
mL) were added N,N′-disuccinimidyl carbonate (205 mg, 0.80 mmol)
and 4-(dimethylamino)pyridine (79 mg, 0.64 mmol), and the resulting
mixture was stirred for 1.5 h at 70 °C. Upon addition of Et2O (5 mL)
to the solution, crystals were formed, filtered, and dried under vacum
to afford the target compound as a blue-purple solid (156 mg, 68%
yield). The compound was stored at 4 °C. LC-MS (ESI+) m/z 598
[M]+; LC-MS-MS 598:500.
Figure 3. Binding curves of 1: (A) to MCF7 cells by flow cytometry
with fluorescent ligand 14 (IC50 = 4.2 nM) or (B) to MCF7 cell
membranes by radioligand binding assay (IC50 = 1.20 nM). Data
shown represent the mean from two different experiments with
samples in duplicate.
2.85 (m, 14H, piperazine CH2, CH2CH2CH2N, CH cyclohexyl, benzyl
CH2 and CH), 3.91 [t, 2H, J = 6.2 Hz, OCH2(CH2)5N], 4.17 [t, 2H, J
= 7.4 Hz, O(CH2)9CH2N], 4.99 (br s, 2H, D2O exchanged), 6.59 (d,
1H, J = 7.7 Hz, aromatic), 6.72 (d, 1H, J = 7.4 Hz, aromatic), 6.89 (d,
1H, J = 8.3 Hz, aromatic), 7.04 (t, 1H, J = 7.8 Hz, aromatic), 7.65 (t,
1H, J = 7.8 Hz, aromatic), 8.11 (d, 1H, J = 8.5 Hz, aromatic), 8.41 (d,
1H, J = 8.2 Hz, aromatic), 8.60 (d, 1H, J = 8.2 Hz, aromatic). LC-MS
(ESI+) m/z 651 [M + H]+, 673 [M + Na] +. Anal. (C41H54N4O3·
3HCl·0.5H2O) Calcd: 64.01, 7.60, 7.28. Found: 63.93, 7.45, 7.32.
5-Dimethylamino-2-(6-hydroxyhexyl)-1H-isoindole-1,3(2H)-
dione (12). To a solution of 4-dimethylaminophthalic acid (266 mg,
1.28 mmol) in anhydrous DMF (8 mL), kept under Ar, was added 1,1-
carbonyldiimidazole (357 mg, 2.18 mmol). The mixture was stirred for
30 min at room temperature, and then 6-amino-1-hexanol (148 mg,
1.28 mmol) in anhydrous DMF (8 mL) was added. The reaction
mixture was stirred for 12 h at room temperature. The solvent was
then removed under reduced pressure and H2O was added. The
aqueous phase was extracted with AcOEt (3 × 15 mL) and CH2Cl2 (3
× 15 mL). The organic layers were collected, dried over Na2SO4, and
evaporated under reduced pressure to afford the crude mixture as an
orange oil. Purification by column chromatography with CH2Cl2/
MeOH (9:1) as eluent gave the title compound as a yellow oil (160
mg, 43% yield). LC-MS (ESI+) m/z 291 [M + H]+.
(E)-1-[5-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetra-
hydronaphthalen-5-yloxy}hexylaminocarbonyl)pentyl]-2-[2-(7-di-
ethylamino-2-oxo-2H-chromen-3-yl)vinyl]-3,3-dimethyl-3H-indoli-
nium Bromide (17). Compound 8 (68 mg, 0.15 mmol) and NEt3
(0.02 mL, 0.15 mmol) were added to a solution of 16 (90 mg, 0.15
mmol) in anhydrous DMF (3 mL). The mixture was stirred for 24 h at
room temperature. The solvent was then removed under reduced
pressure to afford a brown semisolid, which was purified by column
chromatography with CHCl3/MeOH (95:5) as eluent to give the final
compound 17 as a brown semisolid (42.2 mg, 28% yield). LC-MS
+
(ESI+) m/z 939 [M + H]+, 974.79 [M + Na] . HPLC analysis with
MeOH/H2O/HCOOH (50 mM, pH = 5), 8:2 v/v at a flow rate 1
mL/min indicated that the compound was >98% pure.
Biology. Materials. [3H]DTG (50 Ci/mmol), and (+)-[3H]-
pentazocine (30 Ci/mmol) were purchased from PerkinElmer Life
and Analytical Sciences (Boston, MA). DTG was purchased from
Tocris Cookson Ltd., U.K. (+)-Pentazocine was obtained from
Sigma−Aldrich (Milan, Italy). Male Dunkin guinea pigs and Wistar
Hannover rats (250−300 g) were from Harlan, Italy. Cell culture
reagents were purchased from EuroClone (Milan, Italy).
Competition Binding Assays. All the procedures for the binding
assays were previously described. σ1 and σ2 receptor binding were
carried out according to Matsumoto et al.27 The specific radioligands
and tissue sources were as follows: (a) σ1 receptor, (+)-[3H]-
pentazocine, guinea pig brain membranes without cerebellum; (b) σ2
receptor, [3H]DTG in the presence of 1 μM (+)-pentazocine to mask
σ1 receptors, rat liver membranes. The following compounds were
used to define the specific binding reported in parentheses: (a)
(+)-pentazocine (73−87%), (b) DTG (85−96%). Concentrations
required to inhibit 50% of radioligand specific binding (IC50) were
determined by using 6−9 different concentrations of the drug studied
in two or three experiments with samples in duplicate. Scatchard
parameters (Kd and Bmax) and apparent inhibition constants (Ki)
values were determined by nonlinear curve fitting using Prism
GraphPad software (version 3.0).28
6-[5-Dimethylamino-1,3(2H)-dioxo-1H-isoindol-2-yl]hexyl Meth-
anesulfonate (13). To a solution of 12 (150 mg, 0.50 mmol) in
CH2Cl2 (5 mL) were added methanesulfonyl chloride (0.6 mmol,
0.033 mL) and triethylamine (1.2 mmol, 0.16 mL) at 0 °C, and the
mixture was stirred for 1.5 h. Afterward, H2O was added to the
reaction and the mixture was extracted with CH2Cl2 (3 × 15 mL). The
organic layers were then collected and dried over Na2SO4 and
evaporated under reduced pressure to afford the crude mixture as a
yellow oil. Upon purification by column chromatography with AcOEt/
hexane (8:2) as eluent, the target final compound was obtained as a
1
yellow solid (130 mg, 71% yield), mp 79−81 °C. H NMR δ 1.28−
1.52 (m, 4H, NCH2CH2), 1.58−1.82 (m, 4H, CH2CH2CH2O), 3.0 (s,
3H, SO2CH3), 3.11 [s, 6H, N(CH3)2], 3.63 (t, 2H, J = 7.15 Hz,
3321
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Cell Cultures. Human MCF7 breast adenocarcinoma was purchased
from ICLC (Genoa, Italy). MCF7 cells was grown in Dulbecco’s
modified Eagle’s medium (DMEM) high glucose supplemented with
10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and
100 μg/mL streptomycin, in a humidified incubator at 37 °C with a
5% CO2 atmosphere.
Fluorescence Spectroscopy. Emission and excitation spectra of 7,
11, 14, and 17 were determined in CHCl3 and in PBS buffer solution
as previously reported. Fluorescence quantum yields were calculated
with respect to quinine sulfate as previously reported.17,29
Fluorometric Studies. Uptake of σ2 Ligands. In the dose-
dependence assays, MCF7 cells were incubated for 45 min at 37 °C
with increasing concentrations (25, 50, and 100 nmol/L) of 2, 11, and
14. In the competition assays, cells were pretreated with 10, 25, and 50
μmol/L 1 or 18 for 45 min at 37 °C, followed by 25 nmol/L of 2, 11,
and 14 for 45 min at 37 °C. All the compounds were dissolved in
ethanol and prepared as 1000× stock solutions. At the end of the
incubation periods, cells were washed five times with PBS, detached
with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05/0.02% v/
v), centrifuged at 13000g for 5 min, resuspended in 1 mL of ethanol,
and sonicated (two bursts of 10 s, amplitude 40%, Hielscher UP200S
ultrasound sonicator, Hielscher Ultrasonics GmbH, Teltow, Ger-
many). A 50 μL aliquot was used to measure the cell proteins with the
bicinchoninic acid (BCA) kit (Sigma Chemical Co., St. Louis, MO).
The remaining sample was analyzed for intracellular fluorescence with
a LS-5 spectrofluorometer (PerkinElmer, Waltham, MA), at the
MCF7 cells were seeded onto LabTek slide chamber systems
(eight-well) 24 h before the treatment with σ2 receptor ligands for 45
min. After treatment, cells were fixed with 2% PFA at 37 °C for 30
min, washed with PBS, and imaged.
ASSOCIATED CONTENT
* Supporting Information
■
S
Two figures showing confocal images of MCF7 cells incubated
with novel fluorescent σ2 ligands (500 nM) and binding curve
of reference σ2 ligand DTG by flow cytometry in MCF7 cells.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel +39-080-5442750; fax +39-080-5442231; e-mail carmen.
abate@uniba.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by MIUR with Grant
2009NB3E8M_002. D.C.L. gratefully acknowledges support
of the Deutsche Forschungsgemeinschaft (DFG) through the
SFB 1035 and the excellence cluster Nanosystems Initative
following wavelengths (from spectra in EtOH): λex = 467 nm, λem
520 nm (2); λex = 40 nm, λem = 530 nm (11); and λex = 390 nm, λem
=
=
Munich (NIM), and from the Ludwig-Maximilians-Universitat
̈
520 nm (14). The fluorescence of nontreated cells, considered as a
blank, was subtracted from the fluorescence of each sample.
Fluorescence values were normalized for the protein content and
expressed as picomoles of ligand per milligram of cell proteins,
according to the titration curves performed previously for each
fluorescent ligand.
Flow Cytometry. MCF7 cells were incubated with increasing
concentrations (0.1, 1, 10, and 100 nmol/L and 1 and 10 μmol/L) of
1 followed by 100 nmol/L 14 for 45 min at 37 °C. Analogously, the
same experiment was performed with increasing concentrations of
DTG (0.1, 1, 10, and 100 nmol/L and 1 and 10 μmol/L) with or
without (+)-pentazocine (1 μmol/L) followed by 100 nmol/L 14 for
45 min at 37 °C. At the end of the incubation periods, cells were
washed twice with PBS, detached with 200 μL of cell dissociation
solution (Sigma Chemical Co.) for 10 min at 37 °C, centrifuged at
13000g for 5 min, and resuspended in 500 μL of PBS. The
fluorescence was recorded on a FACSCalibur system (Becton
Dickinson Biosciences, San Jose, CA), with a 530 nm band-pass filter.
For each analysis, 10 000 events were collected and analyzed with Cell
Quest software (Becton Dickinson Biosciences).
Microscopy Studies: σ2 Ligand Localization. Confocal
fluorescence microscopy experiments were performed on a spinning-
disk confocal microscope system (Revolution System; Andor
Technology) that utilized a Nikon microscope base (TE2000E) and
the spinning-disk unit CSU10 from Yokogawa. Measurements were
performed with an oil-immersion fluorescence objective (PlanFluor
40×, NA 1.30, Nikon). The detection path was equipped with a
TuCam system (Andor Technology) for dual-color detection,
corresponding dichroic mirror and filter set for the green and red
detection paths (BS580, HC525/50 and HC617/73; AHF Analy-
sentechnik AG), and two DV-887 Ixon EMCCD cameras (Andor). In
addition, a triple-band dichroic beam splitter was used to separate laser
excitation from fluorescence emission (Di01-T405/488/568/647;
Semrock). The excitation was controlled with an acousto-optic
tunable filter (AOTF). The sample position was controlled with an
xyz piezo stage (ProScan II, NanoScanZ; Prior Scientific).
Compounds 11 and 14 were excited at 405 nm and emission was
observed in the green channel (HC525/50); 2 was excited at 488 nm
and emission was detected in the green channel (HC525/50); and 17
was excited at 561 nm and emission was measured in the red channel
(HC617/73).
Munchen through the LMUInnovativ BioImaging Network and
the Center for NanoScience (CeNS).
̈
ABBREVIATIONS USED
■
CNS, central nervous system; JALS, juvenile amyotrophic
lateral sclerosis; MCF7, human breast adenocarcinoma cells;
NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl; PBS, phosphate-buf-
fered saline; PGRMC1, progesterone receptor membrane
component 1; QY, quantum yield
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