4-Nitro-2,1,3-benzoxadiazole derivatives as potential fluorescent sigma receptor probes

Article abstract of DOI:10.1039/c5ra08639f

New fluorescent derivatives for σ receptors were designed and synthesized. To achieve this purpose, a 4-nitro-2,1,3-benzoxadiazole fluorescent tag was connected through a piperazine linker to a modified skeleton derived from selected σ receptor agonists o

Full text of DOI:10.1039/c5ra08639f

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Martorana, A. N. Colabufo, M. Contino, M. Niso, M. G. Perrone, G. de Guidi, A. Catalfo, G. Rappazzo, E.
Zuccarello, O. Prezzavento, E. Amata, A. Rescifina and A. Marrazzo, RSC Adv., 2015, DOI:
10.1039/C5RA08639F.
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4-Nitro-2,1,3-benzoxadiazole derivatives as potential
fluorescent sigma receptor probes
Cite this: DOI: 10.1039/x0xx00000x
Barbara Schininà,a† Andrea Martorana,a† Nicola Antonio Colabufo,b
Marialessandra Contino,b Mauro Niso,b Maria Grazia Perrone,b Guido De Guidi,c
Alfio Catalfo,c Giancarlo Rappazzo,d Elisa Zuccarello,a Orazio Prezzavento,a
Emanuele Amata,a Antonio Rescifina,a Agostino Marrazzo*a
Received 00th January 2015,
Accepted 00th January 2015
DOI: 10.1039/x0xx00000x
New fluorescent derivatives for
purpose, 4ꢀnitroꢀ2,1,3ꢀbenzoxadiazole fluorescent tag was connected through a piperazine
linker to a modified skeletons derived from selected receptor agonists or antagonists.
Compounds 5g 7b 7e and 7g displayed high σ1 affinity and low σ1 σ2 selectivity (Kiσ1
ranging from 31.6 nM to 48.5 nM, Kiσ1 σ2 = 5–18), while compound 5d exhibited high σ2
affinity and selectivity (Kiσ2 = 56.8 nM, Kiσ1 >5,000 nM). Binding affinity studies revealed
that compounds 5d 5g 7b 7e and 7g showed no affinity towards several receptors including
opioid, dopaminergic, serotoninergic, adrenergic, muscarinic, histaminergic, ꢀmethylꢀ
σ receptors were designed and synthesized. To achieve this
www.rsc.org/
σ
,
,
/
/
,
,
,
N
Dꢀ
aspartate (NMDA), NMDA receptor channel, or dopamine and serotonine transporters. The
fluorescent properties, cellular uptake and confocal microscopy studies on 5d suggest a
potential use of this probe to further clarify the molecular role of σ2 receptor subtypes in the
normal and cancer cells.
(PGMRC1) represents a possible σ2 binding site.23 However,
1. Introduction
even though PGRMC1 was highly expressed in proliferating
Sigma (σ) receptor was initially classified as a subtype of the
tumour cells, its molecular weight (25 kDa)23 is different from
opiate receptors. Subsequent studies revealed that sigmaꢀ
binding sites are a distinct class of receptors that are located in
the central nervous system (CNS), in endocrine tissues, in the
liver and kidneys, and in immune system cells.1ꢀ4 Early studies
using radioligand binding methods and biochemical analyses
demonstrated that there are two subtypes of sigma receptors,
named σ1 and σ2. They differ each other in function,
pharmacology, and molecular size.5, 6
σ1 Receptor is involved in the release of neurotrasmitters, in
particular in the NMDA, serotonergic, dopaminergic and
muscarinic neurotransmission, suggesting its potential
applications in the treatment of a number of neurological
disorders and cognitive disease7 including depression,8
schizophrenia,9 Alzheimer’s disease,10ꢀ13 frontotemporal lobar
degenerationꢀmotor neuron disease14 and drug abuse.15
In recent studies σ1 receptor has been classified as a protein
chaperone at the endoplasmatic reticulum (ER)ꢀmitochondrion
interface that regulates Ca2+ signaling and cell survival.16
To date, σ2 subtype has not been completely characterized.
Although its structure is still unknown, σ2 receives relevant
interest related to its overexpression in a wide variety of cancer
tissues.17 Therefore, several σ2 ligands are designed for cancer
treatment and diagnosis.18ꢀ22 Moreover, it was recently
proposed that progesterone receptor membrane component 1
the σ
2 receptor binding sites observed with [3H]ꢀazidoꢀ1,3ꢀdiꢀoꢀ
tolylguanidine ([3H]ꢀAzꢀDTG) photoaffinity labeling (21.5
and/or 18 kDa).6, 24
In spite of several studies, there are still major shortcomings
related to
σ receptors: i) although σ1 receptor has been cloned
and displays a 30% sequence homology with the yeast C8–C7
sterol isomerase,25 it lacks C8–C7 isomerase activity; ii)
moreover, the σ2 receptor has not been cloned, and most of
what is known regarding it has been obtained through the use of
in vitro receptor binding studies aimed at its pharmacological
characterization;26 iii) finally, the absence of the cloned gene or
the purified σ2 receptor protein has also prevented the
generation of antibodies to study the subcellular localization of
this receptor using the immunohistochemical techniques.27 For
these reasons, new fluorescent probes have been synthesised as
useful tools to understand and better characterize the
biomolecular role of the
Generally, considering the low molecular weight of selective
σ1 and σ
2 receptors.28ꢀ30
σ
ligands, the introduction of bulky fluorescent probes could
provide a loss in receptor affinity. In order to avoid this, new
potential
σ fluorescent probes, having as typical fluorophore the
4ꢀnitrobenzoꢀ2ꢀoxaꢀ1,3ꢀdiazole (NBD), were designed and
synthesized.31 This moiety showed reduced steric hindrance and
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DOI: 10.1039/C5RA08639F
a low impact on the ligand affinity. Moreover, the NBD 5a
fluorophore generally showed low emission quantum yield in yl)ethanol (
aqueous solution, but it became highly fluorescent in nonpolar
–
f
were obtained in three steps by reaction of 2ꢀ(piperazinꢀ1ꢀ
) with the commercially available 4ꢀchloroꢀ7ꢀ
nitrobenzoꢀ2ꢀoxaꢀ1,3ꢀdiazole (NBDꢀCl). Conversion of alcohol
into alkyl chloride with SOCl2, followed by condensation
1
2
3
Fig. 1 Structures of selected sigma ligands.
solvents, when it bound to the cell membrane or inserted in a
hydrophobic pocket of target protein.28, 29
Several
N,N´ꢀdisubstituted piperazines, like SAꢀ4503 (Fig. 1),
and morpholine esters, like PREꢀ084 (Fig. 1), have been
reported as potent and selective σ1 receptor agonist ligands
inducing antiamnesic and antidepressive activity,32ꢀ34 or able to
reduce cocaineꢀinduced toxic effects.15, 35ꢀ39 Others piperazine
(PB28, a σ2 receptors agonist),40, 41 piperidine (Haloperidol, σ1
antagonist and σ2 agonist),42 and pyrrolidine (AC915, σ1
antagonist)42 derivatives (Fig. 1), showed an antiproliferative
activity towards different cancer cell lines.
Scheme 1 Synthetic strategy for the preparation of compounds 5a
and conditions: (a) THF, 0 °C, 5 min; (b) SOCl2, CH2Cl2, reflux, 2 h; (c) only for 4a
–g. Reagents
–
f
, K2CO3/H2O, reflux, 12 h; (d) i-PrOH, reflux, 48 h.
with the potassium salts of the corresponding acids 4a–f
(Scheme 1) under refluxing
i
ꢀPrOH, gave the desired products
On the other hand, 4ꢀ(4ꢀChlorophenyl)ꢀ4ꢀ
hydroxypiperidine (4g) reacted directly with to give the
desired compound 5g
44
5a– .
f 43,
Thus, starting from the framework of typical sigma ligands,
including the above mentioned ones (Fig. 1), we designed and
3
.
synthesized a new series of piperazine derivatives 5a
–g and 7a–
Target compounds 7a
–
g
, reported in Scheme 2, were obtained
g
carrying the NBD fluorophore (Schemes 1 and 2), as new
by reaction of suitable
N
ꢀfunctionalized piperazines (6a ) with
–g
potential fluorescent probes for sigma receptors.
NBDꢀCl according to the literature.45
2. Results and discussion
2.2. Radioligand binding and σ1 and σ2 receptor affinities
The pharmacological results of binding assays for receptors
were expressed in terms of inhibition constant (Ki) and are
reported in Table 1. The ester derivatives 5a and 5e displayed
irrelevant σ1 σ2 affinity, while compound 5b showed moderate
2.1. Chemistry
σ
The 4ꢀnitroꢀ2,1,3ꢀbenzoxadiazole derivatives (5a–g and 7a–g)
were synthesized as outlined in Schemes 1 and 2. Compounds
–
f
/
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affinity at both
negligible σ1 and moderate σ2 affinities, while compound 5d (B
exhibited good 2 affinity and selectivity (Ki 2 = 56.8 nM, Kiσ1 were determined. The B
> 5,000 nM) (Table 1). This result suggests that the permeation of tested compound, whereas the A
σ
receptor subtypes. Compound 5c showed In this assay, both the fluxes of ligand from basolateral to apical
A) and from apical to basolateral (A B) compartment
A flux describes the passive
B flux is
→
→
σ
σ
→
→
replacement of the morpholine ring present in the σ1 selective representative for active transport Pꢀglycoprotein (Pꢀgp)
ligand PREꢀ084 with the 4ꢀnitroꢀ7ꢀ(piperazinꢀ1ꢀyl)ꢀ2,1,3ꢀ mediated.
benzoxadiazole moiety, shifted the affinity and selectivity
Table 1 Radioligand Binding Assays
towards the σ2 receptor subtype.
Compound
Binding constant Ki (nM)±SEMa
σ1
σ2
5a
5b
2172 ±410
355 ±50
>10000 (36%)b
278 ±15
5c
5d
5e
5f
>5000 (23%)
>5000 (42%)
>5000 (24%)
>5000 (13%)
41 ±8
393 ±20
56.8 ±2
>10000 (39%)
>10000 (28%)
211 ±8
5g
7a
264 ±40
358 ±15
7b
48.5 ±9
313 ±25
7c
127 ±25
381 ±10
7d
7e
944 ±150
32.3 ±5
>10000 (44%)
577 ±30
7f
7g
526 ±80
31.6 ±3
4690 ±150
225 ±25
PREꢀ084
PB 28
DTG
33.5 ±2
18.5 ±2
61 ±5
>10000 (29%)
1.0 ±0.2
42 ±4
a
The value is the mean of three independent experiments, sample in
b
duplicate. Ki has been not obtained and the percentage of radioligand
Scheme 2. Synthetic strategy for the preparation of compounds 7a–g. Reagents
and conditions: (a) THF, 0 °C, 5 min.
binding inhibition at 10–5 M of tested compound is reported in parentheses.
Table 2 Cell permeability
The piperazine derivatives 5g and 7a
moderate affinity for both receptor subtypes. Among these,
the greater σ1 affinity is showed by the ligand with a
phenethylic substituent 7g Ki = 31.6 nM). It should be noted
that the corresponding benzyl derivative 7f was about 16 fold
less potent (Ki = 526 nM). On the other hand, the derivative
with cyclohexylmethyl substituent at piperazine moiety showed
a reduced σ1 affinity respect to the cyclohexyl substituted one
–g, revealed high or
Comp.
εb
λ (nm)
Papp A→B
(nm/sec)
±SEMa
Papp B→A
(nm/sec)
B→A/A→B
σ
±SEMa
(
5d
5g
7b
7e
7g
575 ±78
280 ±21
363 ±57
356 ±18
558 ±30
2059 ±198
2182 ±355
1581 ±291
2083 ±153
2028 ±117
3.6
7.8
4.4
5.8
3.6
26220
28290
15170
26110
25030
230
498
484
496
230
a
The value is the mean of three independent determinations, sample in
(
7c
with 4ꢀphenylpiperidine and
respectively, display a σ1 affinity of the same order of 7g
Ki = 41 nM; 7e Ki = 32.3 nM). However, among these This biological assay permit to establish if a compound is a
,
Ki = 127 nM; 7b
,
Ki = 48.5 nM). Compounds 5g and 7e
ꢀmethylpiperidine moiety,
5g
,
duplicate. b ε = molar extinction coefficient measured at 10ꢀ5 M at listed λmax
N
for each tested compound.
(
,
,
derivatives, compound 7e displays the better selectivity for σ1 substrate of Pꢀgp and therefore unable to permeate into CNS or
receptors. In order to assess the binding profile of compounds Pꢀgp not transported and consequently brain penetrant. This
5d
different receptors and transporters (Tables S1 and S2). All both fluxes contributions, (B
tested compounds showed negligible affinity for opioid, displaying B
A/A B >2 are considered Pꢀgp substrates and
dopaminergic (D1, D2, D3, D4.2), serotoninergic (5ꢀHT2A, 5ꢀ thus unable to cross BBB, whereas ligands showing
HT2C, 5ꢀHT3, 5ꢀHT4, 5ꢀHT6), adrenergic (α1 2), muscarinic,
A/A B <2 are defined Pꢀgp not transported and therefore
histaminergic H1, NMDA, NMDA receptor channel, such as could permeate BBB and meet specific areas of CNS. The five
,
5g
,
7b
,
7e and 7g, we investigated their affinities towards pharmacokinetic parameter is estimated taking into account
→
A/A B). Normally, compounds
→
→
→
,
α
B
→
→
dopamine and serotonine transporters confirming an excellent
selectivity.
σ
compounds analysed showed a ratio B
thus they can be estimated Pꢀgp substrates.
→A/A→B >2 (Table 2),
In light of these finding, the aforementioned compounds could
be useful as diagnostic tools in detecting Pꢀgp mediated
2.3. Permeability experiments
Among the overall synthesized compounds, 5d, 5g, 7b, 7e and chemoresistant tumours, in peripheral organs where a high
7g, have been tested for their membrane permeability. Apparent concentration of
permeability (Papp) through a monolayer of Cacoꢀ2 cells was
assessed in order to define the pharmacokinetic profile.
σ
receptors has been reported.22
2.4. Spectroscopic properties
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The absorbance and emission properties of some selected 2.5. Subcellular localization by confocal microscopy
compounds are listed in Table 3. The excitation and emission
Both isolated and confluent MCF7 breast cancer cell patches
spectra were registered in organic solvents (EtOH and CHCl3)
were observed. In both conditions, fluorescence was evenly
and in phosphate buffered saline (PBS) at pH 7.4 solutions. The
distributed as punctuated signals throughout the cytoplasm (Fig.
NBDꢀbearing compounds 5d, 5g, 7b, 7e and 7g displayed
2A,B). At 100 nM, the least dilution tested, the signal was still
well distributed, suggesting that compound 5d could be further
diluted while keeping its ability to bind to σ2 receptor in vivo
(Fig. 3).
absorbance maxima at two different wavelengths (at about 335
and at about 470 nm). After excitation in both these maxima
regions, the corresponding emission maximum (λem) was in the
range 530–550 nm. Thus, we selected the excitation wavelength
(
λexc) of 450 nm to avoid autofluorescence phenomena. This
was selected in light of potential future studies employing
living cells. In the corresponding excitation spectra, the
compounds displayed λexc maxima centred in the UVA region
(about 330 nm), as expected considering the relative
absorbance spectra. All compounds showed an important
difference between λexc and λem (Stokes shift). Quantum yields
(
Φ
)
were
determined
in
the
Table 3 Spectroscopic propertiesa
Fig. 2 (A) Microscopic field showing both isolated and confluent MCF-7 cells labelled
with compound 5d (300 nM). Arrows point to fluorescence spots at cell periphery. The
cells in the red square are further enlarged in (B), which shows orthogonal projections
of cells at the top and right sides.
Comp.
5d
ΦFL
0.142
Solvent
λmax1
323
332
ndb
336
334
351
318
328
347
nd
335
353
336
334
nd
ε1
λmax2
ε2
CHCl3
EtOH
PBS
CHCl3
EtOH
PBS
CHCl3
EtOH
PBS
CHCl3
EtOH
PBS
5930
7325
nd
8323
8256
1918
5850
6048
1412
nd
5026
7393
7229
8381
nd
454
472
nd
472
472
486
446
463
487
nd
471
496
472
473
nd
18476
20808
nd
29982
24098
6048
19520
17708
4925
nd
13188
30459
25599
23670
nd
4.47×10–3
5.93×10–4
7.46×10–3
2.26×10–3
1.00×10ꢀ4
4.05×10–3
3.13×10–3
1.05×10–3
6.01×10–3
3.24×10–3
1.34×10–3
0.100
5g
7b
7e
7g
CHCl3
EtOH
PBS
4.05×10–3
1.63×10–3
a
Fluorescence properties herein reported were evaluated on compounds as
Fig. 3 (A) A clump of MCF-7 cells at the 100 nM dilution. (B) Negative control.
free bases, but they were also evaluated on their corresponding hydrochloride
salts in EtOH and PBS solutions. b nd = not detected.
The fluorescence signals were sometimes clustered in a
paranuclear environment, suggestive of accumulation within
membraneꢀdelimited subcellular components, such as ER or
Lysosomes. This is in agreement with similar observations
obtained with living and fixed fluorescent microscopy on the
same cells46 as well as on different cell lines, both murine and
human.28ꢀ30 Moreover, fluorescent aggregates were seen at
specific sites in some cell’s periphery (Fig. 2A, arrows),
selected solvents to probe the environment affecting the
sensitivity of the final fluorescent ligand. Indeed, the selected
fluorophore (NBD) is endowed with environment sensitivity
properties, (i.e., low quantum yield in aqueous solution, but
high fluorescence in nonꢀpolar solvents or when bound to a
hydrophobic site). In this view, 5d exhibited about two order
lower fluorescence in PBS buffer with respect to that measured
in the nonꢀpolar organic solvent. For the vast majority of
compounds, the highest quantum yields were those recorded in
suggesting
involvement
of
σ2
receptor
with
filopodia/lamellipodia system in the course of cell spreading.
As expected, nuclei were thoroughly devoid of fluorescent
signals, as seen in orthogonal projections of labelled cells (Fig.
2B).
More interestingly, nucleoli were found positive, a finding so
far not reported for other σ2ꢀspecific ligands. We do not know
CHCl3:
and severalꢀfold higher than in PBS buffer. The relative polar
compound 7b showed a less pronounced increase in values
from EtOH to CHCl3 solutions. Molar extinction coefficients
) were determined for most of the compounds, with the lowest
Φ values were generally higher in CHCl3 than in EtOH
Φ
(
ε
whether our observation could be due to the high resolution of
our pictures or to a different effects of drug uptake by the cells.
Vertical sectioning of cells showed no sign of fluorescence
within nuclei. On the other hand, being nucleoli, at least
partially, membraneꢀdelimited organelles, our observation does
not contradict the assumption of coꢀlocalization of σ2 receptor
values often displayed in PBS; Low solubility of 5d and 7g in
PBS, as well as that of relatively polar 7e in CHCl3, did not
allow a reliable evaluation of molar extinction coefficient. On
the other hand, solubility limits were sufficient to obtain the
emission data.
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with cytomembranes, but further extends the actual knowledge 102.52, 59.08, 52.42, 49.23, 40.77. Anal. Calcd for
at this regard.
C12H14ClN5O3: C, 46.24; H, 4.53; N, 22.47. Found: C, 46.08;
H, 4.55; N, 22.41.
3. Experimental section
3.2. Preparation of potassium salt of acids 4a–f
3.1. Chemistry
To an aqueous solution of K2CO3 at 120 °C was added an
aqueous solution of the corresponding acid 4a–f (ratio 1:1) and
All starting materials were obtained commercially from
Aldrich, Inc, and were used without further purification. Flash
column chromatography were performed with 60 Å pore size
silica gel as the stationary phase (0.040–0.063 mm, 230–400
mesh ASTM, Merck). Melting points were determined in open
capillaries on a Büchi 530 apparatus and are uncorrected.
Elemental analyses (C, H, N) were performed on a Carlo Erba
CHNSꢀ0 EA1106 analyser; the analytical results were within
the reaction mixture was refluxed for 12 h until the solution
become clear. The water was evaporated in vacuum and the
crude was washed with ether and MeOH several times. The
product was stored under argon at room temperature.
3.3. General procedure for the synthesis of compounds 5a–g
An isopropanolic solution of the potassium salt of acid 4a
compound 4g (3.2 mmol) was added to an heated solution of
(200 mg, 0.642 mmol) in
–
f
or
3
1
±0.4% of the theoretical value assuring a purity ≥95%. H and
13C NMR spectra were recorded on a Varian Inova 200 MHz
using CDCl3 as solvent. The following data were reported:
i
ꢀPrOH (100 mL). The mixture
changed colours from orange to red and was thus refluxed for
48 h. The solvent was evaporated in vacuum and the crude was
dissolved in CH2Cl2 (20 mL). The solution was extracted with
an 1M aqueous solution of NaHCO3, dried over Na2SO4, the
solvent evaporated under reduced pressure and the residue was
purified by flash chromatography using CHCl3 as eluent. The
chemical shift (δ) in ppm respect to TMS, multiplicity, and
coupling constants in hertz. PBS consisted of sodium phosphate
10 mM at pH 7.4, NaCl 138 mM and KCl 2.7 mM. Chemicals
were from SigmaꢀAldrich (Milan, Italy) and were used without
any further purification.
purified compounds 5a
corresponding chlorohydrates by treatment with an anhydrous
solution of hydrogen chloride in diethyl ether.
–
g
were converted into the
3.1.1. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL)-PIPERAZIN
1-YL]-ETHANOL (2)
A solution of 4ꢀchloroꢀ7ꢀnitroꢀ2,1,3ꢀbenzoxadiazole (NBDꢀCl)
(2.2 g, 11 mmol) in anhydrous THF (80 mL) was slowly
-
3.3.1. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL
)PIPERAZIN-
1-YL ETHYL 2-METHYL-2-PHENYLPROPANOATE (5A)
]
dropped into a solution of 2ꢀpiperazinꢀ1ꢀylꢀethanol (1) (5.0 g,
1
246 mg, 72% yield, orange solid: mp 214–217 °C. H NMR
(200 MHz, CDCl3) 8.41 (d, 1H, =7.4 Hz), 7.41–7.23 (m,
5H), 6.24 (d, 1H, =9.0 Hz), 4.24 (t, 2H, =5.2 Hz), 3.95 (t, 4H,
=5.2 Hz), 2.62 (t, 2H, =5.2 Hz), 2.54 (t, 4H, =5.0 Hz), 1.60
(s, 6H). 13C NMR (50 MHz, CDCl3)
170.50, 141.04, 138.22,
38.4 mmol) in anhydrous THF at 0 °C and the reaction mixture
was stirred for 5 min. The solution colour change from orange
to red. The solvent was then evaporated in vacuum, the crude
was dissolved in the minimum amount of methanol/acetone 1:1
and dripped in chloroform (500 mL). The resulting mixture was
then washed with water, dried over Na2SO4, the solvent was
then evaporated in vacuum, and the residue was purified by
δ
J
J
J
J
J
J
δ
132.04, 128.96, 128.06, 127.50, 126.74, 125.57, 120.08, 85.16,
63.50, 59.06, 56.73, 50.34, 45.34, 27.77. Anal. Calcd for
C22H25N5O5•HCl: C, 55.52; H, 5.51; N, 14.72. Found: C,
55.66; H, 5.52; N, 14.67.
3.3.2. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL)PIPERAZIN-
flash chromatography (chloroform/ethanol 9:1) to give
2
as a
red powder (3.0 g, 92% yield). H NMR (200 MHz, CDCl3)
8.44 (d, 1H, =8.8 Hz), 6.32 (d, 1H, =9.0 Hz), 4.14 (t, 4H,
=5.0 Hz), 3.72 (t, 2H, =5.4 Hz), 2.8 (t, 2H, =5.0 Hz), 2.67 (t,
=5.2 Hz). 13C NMR (50 MHz, CDCl3)
138.22, 132.04,
1
δ
J
J
1-YL
]
ETHYL
2-(4-CHLOROPHENYL)-2-METHYLPROPANOATE
J
J
J
(5
B
)
4H,
J
δ
1
320 mg, 80% yield, orange solid: mp 211–215 °C. H NMR
125.69, 125.57, 120.08, 102.67, 59.20, 57.95, 52.48; 49.34.
Anal. Calcd for C12H15N5O4: C, 49.14; H, 5.16; N, 23.88.
Found: C, 49.37; H, 5.12; N, 23.79.
(200 MHz, CDCl3)
4H), 6.27 (d, 1H, =9.0 Hz), 4.24 (t, 2H,
(m, 4H), 2.64 (t, 2H, =5.4Hz), 2.60–2.55 (m, 4H), 1.59, (s,
6H). 13C NMR (50 MHz, CDCl3)
174.97, 144.78, 144.69,
δ
8.39 (d, 1H,
J
=9.0 Hz), 7.45–7.18 (m,
J
J=5.4 Hz), 3.99–3.95
J
3.1.2. 4-[4-(2-CHLOROETHYL)PIPERAZIN-1-YL]-7-NITRO-2,1,3-
δ
BENZOXADIAZOLE (3)
144.58, 143.02, 136.22, 131.52, 128.29, 127.91, 123.00,
104.83, 59.73, 53.90, 50.49, 46.00, 45.76, 25.98. Anal. Calcd
SOCl2 (0.2 mL, 309 mg, 2.6 mmol) was added to a solution of
compound (200 mg, 0.682 mmol) in CH2Cl2 (10 mL) at 0 °C
2
for C22H24ClN5O5•HCl: C, 51.77; H, 4.94; N, 13.72. Found: C,
51.56; H, 4.92; N, 13.78.
under magnetic stirring and, after 15 min, the mixture was
refluxed for 2 h and successively stirred overnight at room
temperature. The solvent was evaporated in vacuum and the
crude was washed several times with ether to remove the excess
3.3.3. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL
)
PIPERAZIN-
1-YL]ETHYL (3,4-DICHLOROPHENYL)ACETATE (5C)
294 mg, 79% yield, orange solid: mp 215–218 °C. H NMR
1
of SOCl2 to give
1H NMR (200 MHz, CDCl3)
1H, =9.0 Hz), 4.07 (t, 4H,
2.78 (t, 2H, =6.8 Hz), 2.74 (t, 4H,
MHz, CDCl3) 144.96, 144.58, 135.83, 135.11, 123.02,
3
as an orange powder (200 mg, 94% yield).
8.57 (d, 1H, =9.0 Hz), 6.22 (d,
=4.8 Hz), 3.58 (t, 2H, = 6.6 Hz),
=5.2 Hz). 13C NMR (50
(200 MHz, CDCl3)
=1.2 =6.4 Hz), 7.26 (d, 1H,
2.4 Hz), 6.29 (d, 1H, =9.0 Hz), 4.29 (t, 2H,
4H,
=5.0 Hz), 3.62 (s, 2H), 2.70 (m, 6H). 13C NMR (50 MHz,
CDCl3)
δ
8.43 (d, 1H,
=0.5 Hz), 7.14 (dd, 1H,
=5.4 Hz), 4.03 (t,
J
=9.0 Hz), 7.42 (dd, 1H,
δ
J
J
J
J
J=1.2,
J
J
J
J
J
J
J
J
δ
δ
167.29, 138.22, 136.22, 133.29, 132.54, 132.53,
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DOI: 10.1039/C5RA08639F
131.26, 129.66, 125.69, 125.57, 120.08, 85.16, 62.49, 59.06, and, after the evaporation of the solvent under reduced pressure
56.73, 45.34, 39.76 Anal. Calcd for C20H19Cl2N5O5
46.48; H, 3.90; N, 13.55. Found: C, 46.36; H, 3.92; 13.50.
3.3.4. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL
1-YL ETHYL 2-PHENYLCYCLOESANCARBOSSILATE (5
•
HCl: C, the residue was purified by flash chromatography
(CHCl3/EtOH 9:1).
)
PIPERAZIN
-
3.4.1.
4-(4-BUTYLPIPERAZIN-1-YL)-7-NITRO-2,1,3-
]
D
)
1
BENZOXADIAZOLE (7
A)
1
251 mg, 68% yield, orange solid: mp 225–228 °C. H NMR 468 mg, 83% yield, orange solid: mp 250–255 °C. H NMR
(200 MHz, CDCl3) 8.38 (d, 1H, =7.6 Hz), 7.45–7.18 (m, (200 MHz, CDCl3) 8.41 (d, 1H, =9.0 Hz), 6.30 (d, 1H, =9.0
5H), 6.23 (d, 1H, =9.0 Hz), 4.24 (t, 2H, =5.4 Hz), 3.94 (t, 4H, Hz), 4.13 (t, 4H, =5.2 Hz), 2.69 (t, 4H, =5.2 Hz), 2.43 (t, 2H,
=5 Hz), 2.62 (t, 2H, =5.0 Hz), 2.53 (t, 4H, =4.8 Hz), 1.80– =7.8 Hz), 1.60–1.25 (m, 4H), 0.95 (t, 3H,
=7.2 Hz). 13C NMR
1.25 (m, 10H). 13C NMR (50 MHz, CDCl3)
174.91, 145.03, (50 MHz, CDCl3) 166.41, 145.16, 144.81, 135.15, 129.97,
δ
J
δ
J
J
J
J
J
J
J
J
J
J
J
δ
δ
144.72, 144.70, 143.59, 135.12, 128.36, 126.74, 125.98, 102.39, 57.69, 52.74, 49.45, 28.87, 20.56, 13.98. Anal. Calcd
123.20, 102.36, 61.79, 56.11, 52.42, 50.70, 49.32, 34.47, 25.50, for C14H19N5O3: C, 55.07; H, 6.27; N, 22.94. Found: C, 55.31;
23.52. Anal. Calcd for C25H29N5O5•HCl: C, 58.19; H, 5.86; N, H, 6.29; N, 22.87.
13.57. Found: C, 57.97; H, 5.84; N, 13.57.
3.4.2.
4-(4-CYCLOHEXYLPIPERAZIN-1-YL)-7-NITRO-2,1,3-
3.3.5. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL
)
PIPERAZIN
-
BENZOXADIAZOLE (7
B)
1
1-YL
]
ETHYL NAPHTHALEN-1-YLACETATE (5
E)
415 mg, 72% yield, red solid: mp 240–246 °C. H NMR (200
8.35 (d, 1H, =9.0 Hz), 6.24 (d, 1H, =9.0 Hz),
=5.0 Hz), 2.78 (t, 4H,
=9.0 Hz), 10H). 13C NMR (50 MHz, CDCl3)
1
294 mg, 82% yield, orange solid: mp 196–202 °C. H NMR MHz, CDCl3)
δ
J
J
(200 MHz, CDCl3)
δ
8.42 (d, 1H,
J=9.0 Hz), 8.07 (m, 1H), 7.82 4.07 (t, 4H,
J
J
=5.0 Hz), 1.84–1.03 (m,
δ 145.14, 144.81, 144.76,
(m, 1H), 7.48 (m, 4H), 7.25 (s, 1H), 6.15 (d, 1H,
J
J
4.25 (t, 2H,
2.57 (t, 2H,
MHz, CDCl3)
J
J
=5.0 Hz), 4.10 (s, 2H), 3.73 (t, 4H,
=4.8 Hz), 2.36 (t, 4H,
174.07, 171.21, 144.73, 138.00, 137.23, Found: C, 57.76; H, 6.37; N, 21.18.
135.06, 133.75, 132.08, 130.54, 128.73, 128.17, 128.06, 3.4.3. 4-[4-(CYCLOHEXYLMETHYL
126.36, 125.84, 125.53, 123.88, 102.32, 62.19, 56.07, 52.36, 2,1,3-BENZOXADIAZOLE (7
49.09, 39.47. Anal. Calcd for C24H23N5O5
=4.5 Hz), 135.21, 102.24, 96.99, 63.32, 49.96, 48.68, 28.86, 26.12, 25.66.
J
=4.8 Hz). 13C NMR (50 Anal. Calcd for C16H21N5O3: C, 57.99; H, 6.39; N, 21.13.
δ
)
PIPERAZIN-1-YL]-7-NITRO-
C
)
1
•
HCl: C, 57.89; H, 412 mg, 77% yield, orange solid: mp 230–237 °C. H NMR
4.86; N, 14.06. Found: C, 57.64; H, 4.85; N, 14.12.
3.3.6. 2-[4-(7-NITRO-2,1,3-BENZOXADIAZOL-4-YL
(200 MHz, CDCl3)
Hz), 4.12 (t, 4H, =5.0 Hz), 2.65 (t, 4H,
=7.2 Hz), 1.83–0.82 (m, 11H). 13C NMR (50 MHz, CDCl3)
δ
δ
8.42 (d, 1H,
J
=9.0 Hz), 6.29 (d, 1H,
J=9.0
)
PIPERAZIN
-
J
J
=5.0 Hz), 2.21 (d, 2H,
1-YL
]
ETHYL NAPHTHALEN-2-YLACETATE (5
F
)
J
1
305 mg, 85% yield, orange solid: mp 210–214 °C. H NMR 145.18, 144.79, 135.80, 135.21, 123.13, 102.30, 65.09, 53.12,
(200 MHz, CDCl3)
(m, 3H), 6.03 (d, 1H,
Hz), 3.78 (m, 6H), 2.66 (t, 2H,
Hz). 13C NMR (50 MHz, CDCl3)
δ
8.38 (d, 1H,
=9.0 Hz), 5.30 (s, 2H), 4.29 (t, 2H,
=5.2 Hz), 2.52 (t, 4H,
J=8.0 Hz), 7.79 (m, 3H), 7.45 49.48, 34.94, 31.69, 26.68, 26.02. Anal. Calcd for C17H23N5O3:
J
J
J
=5.4 C, 59.12; H, 6.71; N, 20.28. Found: C, 58.93; H, 6.72; N, 20.53.
=5.2 3.4.4.
J
4-NITRO-7-[4-(TETRAHYDROFURAN-2-
δ
171.24, 169.11, 144.71, YLMETHYL
)
PIPERAZIN-1-YL]-2,1,3-BENZOXADIAZOLE (7
D
)
1
141.10, 139.22, 135.02, 133.33, 132.37, 131.50, 128.21, 490 mg, 81% yield, orange solid: mp 223–227 °C. H NMR
128.02, 127.64, 127.55, 127.43, 126.31, 125.93, 102.38, 62.23, (200 MHz, CDCl3)
56.08, 52.54, 49.06, 41.78. Anal. Calcd for C24H23N5O5 HCl: Hz), 4.16–3.73 (m, 7H), 2.80 (t, 4H,
C, 57.89; H, 4.86; N, 14.06. Found: C, 57.75; H, 4.85; N, 14.11. 2H), 2.11–1.47 (m, 4H). 13C NMR (50 MHz, CDCl3)
3.3.7. 4-(4-CHLOROPHENYL)-1-{2-[4-(7-NITRO-2,1,3- 145.14, 144.78, 137.20, 135.18, 102.37, 76.47, 68.31, 62.81,
δ
8.42 (d, 1H,
J
=8.8 Hz), 6.30 (d, 1H,
=5.2 Hz), 2.58–2.47 (m,
171.41,
J=9.2
•
J
δ
BENZOXADIAZOL-4-YL
)
PIPERAZIN-1-YL
]
ETHYL}-PIPERIDIN-4-
53.23, 49.35, 30.18, 25.36. Anal. Calcd for C15H19N5O4: C,
54.05; H, 5.75; N, 21.01. Found: C, 53.81; H, 5.74; N, 21.11.
OL (5
G)
Directly starting from 4g; 233 mg, 75% yield, orange solid: mp 3.4.5.
4-[4-(1-METHYLPIPERIDIN-4-YL
)
PIPERAZIN-1-YL]-7-
1
190–194 °C. H NMR (200 MHz, CDCl3)
Hz), 7.47–7.27 (m, 4H), 6.31 (d, 1H,
J
δ
8.41 (d, 1H,
J
=9.0 NITRO-2,1,3-BENZOXADIAZOLE (7
E)
1
J
=9.0 Hz), 4.13 (t, 4H, 400 mg, 70% yield, orange solid: mp 280–284 °C. H NMR
=4.2 Hz), 2.86–2.55 (m, 12H), 2.14–1.70 (m, 5H). 13C NMR (200 MHz, CDCl3)
δ
8.42 (d, 1H,
=4.8 Hz), 2.98–2.79 (m, 7H), 2.30 (s, 3H),
175.00,
•HCl: 162.20, 149.70, 145.98, 135.41, 102.56, 61.49, 55.39, 50.03,
J=9.0 Hz), 6.30 (d, 1H, J=9.0
(50 MHz, CDCl3)
δ 149.05, 145.25, 144.72, 136.25, 130.74, Hz), 4.12 (t, 4H, J
128.30, 127.69, 126.80, 121.00, 103.47, 69.34, 58.77, 55.48, 2.05–1.60 (m, 6H). 13C NMR (50 MHz, CDCl3)
δ
53.33, 50.09, 49.58, 38.48. Anal. Calcd for C23H27ClN6O4
C, 52.78; H, 5.39; N, 16.06. Found: C, 52.97; H, 5.40; N, 15.99. 49.01, 46.28, 28.29. Anal. Calcd for C16H22N6O3: C, 55.48; H,
6.40; N, 24.26. Found: C, 55.75; H, 6.42; N, 24.21.
3.4. General procedure for the synthesis of compounds 7a–g
3.4.6.
4-(4-BENZYLPIPERAZIN-1-YL)-7-NITRO-2,1,3-
A solution of NBDꢀCl (0.4136 g, 2.07 mmol) in anhydrous BENZOXADIAZOLE (7
THF was added to a solution of substituted piperazine 6a 430 mg, 73% yield, red solid: mp 176–180 °C. H NMR (200
(1.727 mmol) in anhydrous THF at 0 °C and the reaction MHz, CDCl3) 8.41 (d, 1H, =8.8 Hz), 7.36ꢀ7.23 (m, 5H), 6.28
mixture was stirred for 5 min. The solution colour change from (d, 1H, =9.2 Hz), 4.12 (t, 4H, =5.0 Hz), 3.61 (s, 2H), 2.71 (t,
orange to red. After the solvent evaporation the crude was 4H, 145.00, 144.12,
=5.0 Hz). 13C NMR (50 MHz, CDCl3)
dissolved in CHCl3, extracted with water, dried over Na2SO4 137.15, 130.84, 128.10, 127.91, 125.69, 125.57, 120.08,
F)
1
–
g
δ
J
J
J
J
δ
6 | RSC Adv., 2015, 00, 1-3
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DOI: 10.1039/C5RA08639F
102.08, 62.70, 59.20, 46.69. Anal. Calcd for C17H17N5O3: C, Emission spectra of compounds were determined in EtOH,
60.17; H, 5.05; N, 20.64. Found: C, 59.94; H, 5.03; N, 20.69. CHCl3 and in PBS solutions. In all experiments, the excitation
3.4.7.
4-NITRO-7-[4-(2-PHENETHYL)PIPERAZIN-1-YL]-2,1,3- and the emission bandpass was set at 5 nm (S/R mode; response
BENZOXADIAZOLE (7
G
)
time 0.5 s). The emission spectra were obtained from about 300
1
395 mg, 71% yield, red solid: mp 193–197 °C. H NMR (200 to 700 nm, with excitation set at the appropriate excitation
MHz, CDCl3)
6.31 (d, 1H,
8H). 13C NMR (50 MHz, CDCl3)
δ
8.42 (d, 1H,
J
=9.0 Hz), 7.35–7.22 (m, 5H), wavelength. Fluorescence quantum yields were calculated with
=5.0 Hz), 2.91–2.68 (m, respect to quinine sulphate in 0.5 M H2SO4 as a standard (ΦFL
145.22, 144.01, 136.80, 0.546).49 Solutions of both the sample and the reference were
J
=9.0 Hz), 4.15 (t, 4H,
J
δ
=
129.07, 128.63, 126.05, 125.69, 125.57, 120.08, 102.08, 59.05, prepared from original solutions diluted with the appropriate
58.20, 45.69, 34.16 . Anal. Calcd for C18H19N5O3: C, 61.18; H, solvent so that absorbance was below 0.2 at the same excitation
5.42; N, 19.82. Found: 61.43; H, 5.44; N, 19.78.
wavelength (356 nm). Fluorescence measurements were carried
out for each solution with the same instrument parameters, and
the fluorescence spectra were corrected for instrumental
response before integration. The quantum yield for each sample
3.5. Cell permeability experiments
3.5.1. PREPARATION OF CACO-2 MONOLAYER
This procedure has been previously reported.47 Briefly, Cacoꢀ2 was calculated according the following equation:
cells were harvested with trypsin–EDTA and seeded onto
MultiScreen Cacoꢀ2 assay system at a density of 10,000 where
cells/well. The culture medium was replaced every 48 h for the the excitation wavelength,
first 6 days and every 24 h thereafter, and after 21 days in emission curve,
2
ΦS
is the emission quantum yield,
is the area under the corrected
is the refractive index of the solvent (1.34,
= ΦQ(AQ/AS)(FS/FQ)(ηS/ηQ)
Φ
A is the absorbance at
F
η
culture, the Cacoꢀ2 monolayer was utilized for the permeability 1.36 and 1.44 for 0.5 M H2SO4, EtOH and CHCl3, respectively)
experiments. The transepitelial electrical resistance (TEER) of for the sample (S) and the standard (Q). Absorption spectra
the monolayers was measured daily before and after the were recorded with a Beckman DU 650 spectrophotometer, and
experiment using a epithelial voltꢀohm meter (MillicellꢀERS; fluorescence spectra were obtained with a Yvon Jobin Spex
Millipore, Billerica, MA). Generally, TEER values obtained are Fluorologꢀ2 (model Fꢀ111) spectrofluorimeter. Molar extinction
greater than 1000X for a 21ꢀday culture.
3.5.2. DRUG TRANSPORT EXPERIMENT
coefficients (
solvents were determined.
εCl, where (
) is the molar concentration of the sample, and (l) was the
ε
) of soluble sample dissolved in the various
values were determined by fitting
) is the absorbance at the λexc
ε
Papp
A
→
B and Papp
B
→
A of drugs were measured at 120 min the Beer’s law:
A
=
A
;
and at various drugs concentrations (1–100 ꢁM).48 Drugs were
(
C
dissolved in Hank’s balanced salt solution (HBSS, pH 7.4) and optical path length (l = 1 cm) of standard quartz cuvettes.
sterile filtered. After 21 days of cell growth, the medium was Triplicates of measurements were always made.
removed from filter wells and from the receiver plate. The filter
3.7. Radioligand Binding Assays
wells were filled with 75 ꢁL of fresh HBSS buffer and the
receiver plate with 250 ꢁL per well of the same buffer. This σ1 and σ2 receptor binding experiments were carried out with
procedure was repeated twice, and the plates were incubated at [3H]ꢀ1,3ꢀdiꢀoꢀtolylguanidine ([3H]ꢀDTG) (30 Ci/mmol) and (+)ꢀ
37 °C for 30 min. After incubation time, the HBSS buffer was [3H]ꢀpentazocine (34 Ci/mmol). These were purchased from
removed and drug solutions added to the filter well (75 ꢁL). PerkinElmer Life Sciences (Zavantem, Belgium). [3H]ꢀDTG
HBSS without the drug was added to the receiver plate (250 and unlabeled DTG were purchased from Tocris Cookson Ltd.,
ꢁL). The plates were incubated at 37 °C for 120 min. After UK. (+)ꢀPentazocine was obtained from SigmaꢀAldrich (Milan,
incubation time, samples were removed from the apical (filter Italy). Male Dunkin guinea pigs and Wistar Hannover rats
well) and basolateral (receiver plate) side of the monolayer and (250–300 g) were from Harlan, Italy. The specific radioligands
then were stored in a freezer (–20 °C) for pending analysis.
The concentration of compounds was analysed using UV–Vis (a) σ1 receptor, (+)ꢀ[3H]ꢀpentazocine, (2
spectroscopy. dimethylꢀ3ꢀ(3ꢀmethylbutꢀ2ꢀenꢀ1ꢀyl)ꢀ1,2,3,4,5,6ꢀhexahydroꢀ2,6ꢀ
The Papp, in units of nm/s, was calculated using the following methanoꢀ3ꢀbenzazocinꢀ8ꢀol, guinea pig brain membranes
equation: without cerebellum; Nonspecific binding was determined in the
presence of 1 M (+)ꢀpentazocine.
and tissue sources were respectively:
S,6R,11R)ꢀ6,11ꢀ
ꢎ
ꢒ
= ꢃꢆꢇꢈꢉ×ꢊꢋꢌꢈꢍ × ꢃ ꢏꢇꢐꢑ ꢓꢔꢔꢕꢖꢗꢘꢙꢍ
ꢄ
ꢅ
ꢀ
ꢀ
ꢁꢂꢂ
ꢎ
ꢒ
ꢏꢇꢐꢑ
(b) σ2 receptor, [3H]ꢀDTG in the presence of 1 ꢁM (+)ꢀ
pentazocine to mask σ1 receptors, rat liver membranes.
ꢚꢛꢚꢗꢚꢓꢜ
where VA is the volume (in mL) in the acceptor well; area is the
surface area of the membrane (0.11 cm2 of the well); time is the
total transport time in seconds (7200 s); [Drug]acceptor is the
concentration of the drug measured by UV spectroscopy;
[Drug]initial is the initial drug concentration (1×10–4 M) in the
apical or basolateral wells.
Nonspecific binding was determined in the presence of 10
DTG
Concentrations required to inhibit 50% of radioligand specific
binding (IC50) were determined by using sixꢀnine different
concentrations of the drug studied in two or three experiments
ꢀM
with samples in duplicate. Scatchard parameters (Kd and Bmax
)
and apparent inhibition constants (Ki) values were determined
3.6. Absorption and Emission Spectroscopy
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DOI: 10.1039/C5RA08639F
by nonlinear curve fitting by using the Prism, version 3.0, magnifications; optical sections (0.25
ꢀ
m) were taken when
GraphPad software.
appropriate.
Binding affinities towards the target receptors and transporters
listed in Table S2 were achieved as previously reported.50
Briefly, different rat brain regions were dissected to assess the
following binding assays; rat striatum for D1 and D2 receptors
and DAT; rat olfactory tubercle for D3 receptors; rat cortex for
4. Conclusions
A new series of 4ꢀnitroꢀ2,1,3ꢀbenzoxadiazole derivatives, as
potential fluorescent receptor ligands, were synthesized.
Binding results displayed that compounds 5d 7e and 7g
showed high sigma receptor affinity as well as a different
selectivity feature. Cell permeability evaluation led to address
these compounds as useful fluorescent in vitro probes for
detecting peripheral chemoresistant tumors where sigma
receptors are overexpressed.
Indeed, the fluorescent properties of σ2 selective ligand 5d and
preliminary confocal microscopy analysis on MCF7 breast
cancer cells, provide evidence that this new ligand represent a
useful probe to characterize the function and molecular
mechanisms in which this enigmatic receptors are involved.
σ
,
muscarinic, 5ꢀHT2A, 5ꢀHT3, α1, α2, H1, and NMDA receptors
and SERT; guinea pig cortex for 5ꢀHT4 receptors. 5ꢀHT2C
binding assays were carried out on pig brains (frontal cerebral
cortex) obtained from a local slaughterhouse and promptly
placed on ice until use. Binding experiments on 5ꢀHT6
receptors were performed using a ratꢀcloned serotonin receptor
subtype 6 in HEKꢀ293 cells, as reported by Boess et al.51
Tissue preparation steps and binding assays were performed
according to Gobbi et al.52 and Mennini et al.53 After incubation
the samples were filtered through Whatman GF/B or GF/C
glass fiber filters using a Brandel apparatus (model Mꢀ48R) or
Millipore filter apparatus. The filters were washed with iceꢀcold
buffer (3 × 4 mL). Radioactivity was counted in 4 mL of
“Ultima Gold MV” or Filter Count Cocktail (Packard) in a
DSA 1409 (Wallac) or 1414 Winspectral PerkinꢀElmer Wallac
liquid scintillation counter. For inhibition experiments, the
drugs were added to the binding mixture at seven to nine
different concentrations. Total opioid receptor binding assays
were performed using [3H]ꢀnaloxone (55.5 Ci/mM; Kd = 6.6
Acknowledgements
A free academic license to OpenEye Scientific Software and
ChemAxon for their suites of programs is gratefully
acknowledged.
Notes and references
±0.7 nM;
n = 3), and rat brain membranes were prepared as
a
Department of Drug Sciences, University of Catania, Viale A. Doria 6,
previously reported.54 Nonspecific binding was assessed in the
presence of 10 ꢁM of unlabeled naloxone. Inhibition constants
95125 Catania, Italy. Eꢀmail: marrazzo@unict.it.
b Department of Pharmacy, University of Bari “A. Moro”, Via Orabona 4,
70125 Bari, Italy.
(
Ki values) were calculated using the “Allfit” program or the
EBDA/LIGAND program purchased from Elsevier/Biosoft.
c Department of Chemical Sciences, University of Catania, Viale A. Doria
6, 95125 Catania, Italy.
3.8. Confocal Microscopy
d
Department of Biological, Geological and Environmental Sciences –
3.8.1. CELL CULTURE AND ANALYSIS
Division of Animal Biology, University of Catania, Via Androne 81,
95124 Catania, Italy.
MCF7 cells were grown in Dulbecco’s MEM supplemented
with 10% fetal calf serum, 2 mM glutamine, 100 units/mL
penicillin, and 100 ꢁg/mL streptomycin, in a humidified
incubator at 37 °C in 5% CO2. About 3000 cells were seeded in
each well of a 16ꢀwells glass Chamberslide (LabꢀTek, Thermo
Scientific Nunc). After 24 hours, the medium was replaced with
fresh complete medium containing serial dilutions of 5d from a
20 mM stock solution in DMSO, added in duplicate to the wells
from 1 mM to 100 nM. Some wells were kept aside for
negative control. The incubation time was limited to one hours
in order to keep cells alive, since it is well known σ2 receptor
agonists may lead to cell death by apoptosis.30 Medium was
then withdrawn, wells washed twice with prewarmed PBS and
fixed with 4% formaldehyde in PBS for 2 hour, then washed in
PBS two more times. Before microscopic examination, the well
strips, with their silicone gasket, were teared from the glass
slide; fixed cells were again PBS washed, briefly air dried and
mounted in DAPIꢀcontaining antifade (Vectashield Mounting
Medium, Vector Labs, Peterborough, UK). Several fields,
including both isolated and confluent cells, were observed in a
Zeiss LSM 700 confocal microscope at 40× and 60×
†
These authors contributed equally.
Electronic Supplementary Information (ESI) available: radioligand
binding profile of compounds 5d 5g 7b 7e and 7g against different
,
,
,
receptors and transporters, and tabulation of radioligand binding assay
conditions; NMR spectra. See DOI: 10.1039/b000000x/
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