Fluorescent derivatives of σ Receptor ligand 1-cyclohexyl-4-[3-(5- methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) as a tool for uptake and cellular localization studies in pancreatic tumor cells

Article abstract of DOI:10.1021/jm200591t

Fluorescent derivatives of σ₂ high affinity ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl] piperazine 1 (PB28) were synthesized. NBD or dansyl fluorescent tags were connected through a 5- or 6-atom linker in two d

Full text of DOI:10.1021/jm200591t

ARTICLE
pubs.acs.org/jmc
Fluorescent Derivatives of σ Receptor Ligand 1-Cyclohexyl-4-[3-(5-
methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazine
(PB28) as a Tool for Uptake and Cellular Localization Studies in
Pancreatic Tumor Cells
Carmen Abate,*,† John R. Hornick,‡ Dirk Spitzer,‡ William G. Hawkins,‡ Mauro Niso,† Roberto Perrone,†
and Francesco Berardi†
†Dipartimento Farmacochimico, Universitꢀa degli Studi di Bari “Aldo Moro”, Via Orabona 4, I-70125 Bari, Italy
‡Division of Hepatobiliary, Pancreatic, and Gastrointestinal Surgery, Department of Surgery, Washington University School of Medicine,
St. Louis, Missouri, United States
S Supporting Information
b
ABSTRACT: Fluorescent derivatives of σ2 high affinity ligand
1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-
yl)propyl]piperazine 1 (PB28) were synthesized. NBD or
dansyl fluorescent tags were connected through a 5- or 6-atom
linker in two diverse positions of 1 structure. Good σ2 affinities
were obtained when the fluorescent tag was linked to 5-meth-
oxytetralin nucleus replacing the methyl function. NBD-bearing
compound 16 displayed high σ2 affinity (Ki = 10.8 nM) and
optimal fluorescent properties. Its uptake in pancreatic tumor
cells was evaluated by flow cytometry, showing that it partially occurs through endocytosis. In proliferating cells, the uptake was
higher supporting that σ2 receptors are markers of cell proliferation and that the higher the proliferation is, the stronger the
antiproliferative effect of σ2 agonists is. Colocalization of 16 with subcellular organelles was studied by confocal microscopy: the
greatest was in endoplasmic reticulum and lysosomes. Fluorescent σ2 ligands show their potential in clarifying the mechanisms of
action of σ2 receptors.
’ INTRODUCTION
selector used was a derivative of 1-cyclohexyl-4-[3-(5-meth-
oxy-1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1,
PB28), one of the highest affinity σ2 receptor ligands known,18,19
concluding that either σ2 receptors may be histones or histone
binding proteins or compound 1 binds such proteins as well as σ2
receptors, with modeling studies conducted to rationalize these
hypotheses.20 Such results were in disagreement with findings
from fluorescence microscopy, which localizes σ2 subtypes in
several organelles except the nucleus through the use of fluor-
escent σ2 ligands.21,22 Besides the intracellular localization, there
are other ambiguities related to the σ2 receptors: evidence shows
that σ2 ligands activate different apoptotic pathways in diverse
tumor cells.11ꢀ13,23 With the aim of helping to clarify some of
these ambiguities, we synthesized a small series of fluorescent
derivatives of compound 1 to be used in microscopy studies
for the purpose of localizing σ2 receptors subcellularly within
cancer cells.
After their first discovery in 1976, sigma (σ) receptor research
met a renovated enthusiasm in the early 1990s when the two
subtypes, σ1 and σ2, were identified.1 The σ1 subtype was soon
thereafter isolated and cloned from different sources,2 and it has
been recently classified as a receptor chaperone at the endoplas-
mic reticulum (ER) membrane that regulates ER-mitochondrial
Ca2+ signaling and cell survival.3 Although their mechanism of
action is still unclear, σ proteins receive much interest because of
their potential applications as drug targets for a wide range of
diseases. σ1 receptor ligands display neuroprotective and neuror-
egulative functions and are under evaluation for the treatment of
a number of neurological disorders4 such as depression,5
schizophrenia,6 Alzheimer’s and Parkinson’s diseases,7ꢀ9 and
for drug abuse (e.g., cocaine).10 The high therapeutic potential
of σ2 receptors comes from the evidence that this subtype is
overexpressed in a wide variety of cancer tissues, and activation of
σ2 receptors lead tumor cells to death through different apoptotic
pathways.11ꢀ13 Therefore, a number of σ2 receptor ligands are
under investigation for cancer treatment and diagnosis.14ꢀ16
Nevertheless, the σ2 subtype is not as well as characterized as
the σ1. It has not yet been cloned, and attempted characterization
from homogenate of σ2-overexpressing tumor cells led to isola-
tion of histone proteins by affinity chromatography.17 The σ2
Intrinsically fluorescent compound 1 analogues have been
synthesized in the past, with appreciable σ receptor affinity but
with maximum excitation and emission wavelength (λexc and
λem) inappropriate for use in living cells for fluorescence
Received: May 11, 2011
Published: July 11, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of Fluorescent Compound 1 Analogues: Fluorescent Tag at the Piperazinea
a Reagents: (a) 6-Br(CH2)5CN; (b) LiAlH4; (c) CH3COCl; (d) HCl; (e) ZnCl2, NaCNBH3; (f) dansyl chloride; (g) NBD-chloride.
microscopy.24 Therefore, we followed a common approach to
overcome this limitation: dansyl (λexc ∼315 nm) and 7-nitro-
2,1,3-benzoxadiazole (NBD) (λexc ∼420 nm) moieties were
alternatively inserted in two different positions on compound 1
structure through a 5- or 6-atom linker. Such separation between
the pharmacophore and the fluorescent tag should prevent the
loss of affinity, leaving the fluorescent properties of the fluor-
escent moieties almost unchanged. Compound 16, with the best
fluorescence/pharmacological properties, was used for prelimi-
nary fluorescence microscopy analyses in murine and human
pancreatic tumor cells which have been previously shown to
overexpress σ2 receptors.11 Human pancreatic tumor cells
(BxPC3) were selected for more extensive studies of compound
16, whose internalization and colocalization by confocal micro-
scopy with subcellular organelles were evaluated.
with 6-bromohexanenitrile, affording intermediate 2. Upon re-
duction with LiAlH4 to the corresponding amine and subsequent
amine protection through acetylation, compound 2 provided
derivative 3. Acidic deprotection of the ethyleneꢀacetal function
with HCl led to intermediate 4, which underwent reductive
amination with piperazine 5,25 providing the acetyl derivative of
6, which was deacetylated, affording key amine 6. Reaction of this
latter alternatively with NBD-chloride or dansyl chloride af-
forded the fluorescent compounds 7 and 8, respectively
(Scheme 1).
The final compounds 16ꢀ19 were synthesized as outlined in
Scheme 2. The reaction between potassium phthalimide and 1,6-
dibromohexane gave intermediate 9,26 which was used to alkylate
the key phenolic intermediate 10,19 affording phthalimide 11.
Alkylation of the phenolic intermediate 9 with 2-(2-chloroethoxy)-
ethanol afforded the corresponding alcohol 12 that underwent
Mitsunobu condensation with phthalimide in the presence of
triphenylphosphine and diisopropylazodicarboxylate (DIAD) to
yield compound 13. Phthalimide derivatives 11 and 13 under-
went hydrazinolysis to afford intermediate primary amines 14
and 15, respectively. Reaction of 14 with NBD-chloride or
dansyl chloride afforded the fluorescent compounds 16 and 18,
’ RESULTS AND DISCUSSION
Chemistry. The synthetic pathways for final compounds 7, 8,
and 16ꢀ19 are depicted in Schemes 1 and 2. Key intermediate 6
was prepared starting from commercial piperidin-4-one ethylene
ketal (1,4-dioxa-8-aza-spiro[4.5]decane), which was alkylated
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Scheme 2. Synthesis of Fluorescent Compound 1 Analogues: Fluorescent Tag at the Tetralin Nucleusa
a Reagents: (a) K2CO3; (b) ClCH2CH2OCH2CH2OH, K2CO3; (c) Ph3P, phthalimide, DIAD; (d) hydrazine hydrate; (e) NBD-chloride; (f) dansyl
chloride.
respectively. Reaction of 15 with NBD-chloride or dansyl
chloride afforded respectively the fluorescent final compounds
17 and 19. All the final compounds were converted to the
corresponding hydrochloride salts with gaseous HCl.
alkyl fluorescent tag on the 5-methoxy-tetralin ring in place of the
methyl group displayed nanomolar affinities at both σ receptors
(compounds 16ꢀ19). Previous SAfiR studies demonstrated how
the methoxy substituent was unessential for σ2 receptor binding
indeed.28 NBD or dansyl moieties were tolerated at the σ2 receptor,
with the highest affinity displayed by the NBD-bearing compound
16 (Ki = 10.8 nM). The presence of the NBD, but not of the dansyl
moiety, appeared to be detrimental (Ki = 78.8 nM for 16 and Ki =
96.2 nM for 17) for σ1 receptor binding. With σ1 and σ2 affinities in
the same range, dansyl-bearing ligands 18 and 19 did not show
any σ2 versus σ1 selectivity, whereas compounds bearing NBD (16
and 17) displayed a moderate σ2 selectivity (8-fold and 2.5-fold,
respectively). Selectivity was missing in lead compound 1 when
binding assays were performed on animal tissues according to
literature protocols.18 Results obtained with this small series of
compounds demonstrated that a fluorescent tag spaced out from
the tetralinoxy moiety by a 5- or 6-atom linker leads to molecules
with good σ receptor affinities useful for in living cells visualization
of σ2 receptors, with compound 16 displaying the best pharmaco-
logical properties for further investigation.
Radioligand Binding and σ1 and σ2 Receptor Affinities.
Results from binding assays are expressed as inhibition constants
(Ki values) in Table 1. The introduction of the fluorescent tag
and the linker produced a decrease in the affinity at both σ
receptors with respect to lead compound 1. The most dramatic
drop in the affinity at both σ receptors was observed with
compounds 7 (Ki = 2570 nM for σ1 and Ki = 1720 nM for σ2
receptor) and 8 (Ki > 5000 nM for σ1 and Ki = 5020 nM for σ2
receptor), which reached micromolar values. In such com-
pounds, the piperidine ring replacing the cyclohexyl ring was
functionalized with the fluorescent tag through a six-methylene
chain. The drop in the affinity was independent from the nature
of the fluorescent tag, indicating that functionalization (at least
with a six-atom linker) in that position of the pharmacophore was
not tolerated by the σ2 receptors, in accordance with a previous
study in which substitution of the cyclohexyl with more hindered
substituents led to reduced σ affinities.18,27 On the other hand,
fluorescent final compounds obtained through insertion of the
Fluorescent Ligand Studies. The fluorescent properties of
final compounds are listed in Table 1. The excitation and emission
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Table 1. Receptor Affinities and Fluorescence Properties of Final Compounds
Ki nMa
CHCl3
EtOHb
PBSb,c
λem
λexc
λem
λexc
λem
λexc
compd
X
Y
Z
σ1
σ2
(nm)
(nm)
Φ
(nm)
(nm)
Φ
(nm)
(nm)
εd
1e
7
CH3
CH3
CH3
A
CH2
A
0.38 ( 0.10
2570f
>5000 f
0.68 ( 0.20
1720 ( 160
5020 ( 180
10.8 ( 3.0
39.3 ( 11.8
20.8 ( 1.5
25.7 ( 4.7
CH2CH2
CH2CH2
CH2CH2
O
450
346
451
450
340
345
515
490
514
512
490
490
0.17
0.32
0.20
0.18
0.30
0.48
476
335
467
465
335
335
520
507
520
520
507
507
0.08
0.29
0.05
0.04
0.20
0.23
480
340
460
460
345
343
535
510
520
520
485
510
14391
2600
11300
6544
4000
2741
8
B
16
17
18
19
CH2
CH2
CH2
CH2
78.7 ( 18.2
96.2 f
A
B
CH2CH2
O
9.08 ( 1.32
19.8 ( 8.7
2.62 ( 0.25
B
(+)-pentazocine
DTG
24.6 ( 2.2
a Values are the means of n g 2 separate experiments. b Fluorescence properties herein reported were evaluated on compounds as free bases, but they
were also evaluated on their corresponding hydrochloride salts in EtOH and PBS solutions. A maximum of 5 nm shift was observed in the excitation and
emission wavelengths when compared to the excitation and emission wavelengths from the corresponding free bases. c All compounds solubilized in PBS
gave Φ value very close to 0 and therefore they are not reported. d From EtOH solutions of compounds in EtOH. e From ref 18, where results from
binding on human cells, in which compound 1 displays about 40-fold σ2 versus σ1 selectivity, are also reported. f From a unique experiment.
spectra were obtained from solution of the final compounds in
organic solvents (EtOH and CHCl3) and in aqueous solution
(PBS buffer). The NBD-bearing compounds (8, 16, 17) displayed
excitation peaks at two different wavelengths (∼335 and
∼450 nm), and for both the wavelengths, the corresponding
λem was ∼520 nm. The λexc selected to perform the assays in living
cells was 450 nm to avoid cells autofluorescence phenomena.
Dansyl-bearing compounds (7, 18, 19) displayed a λexc more
shifted toward the UV region (∼340 nm). All of the compounds
showed an important difference between λexc and λem (Stokes
shift). Quantum yields (Φ) were determined in the above-
mentioned solvents to probe the environment affecting the
sensitivity of the final fluorescent ligands because the fluorophores
selected (Dansyl and NBD) are endowed with environment
sensitivity properties, (i.e., low quantum yield in aqueous solution
but high fluorescence in nonpolar solvents or when bound to a
hydrophobic sites). All tested compounds exhibited very low
fluorescence in PBS buffer but became fluorescent in the organic
solvents. The highest quantum yields were those recorded in
CHCl3 for all the final compounds: Φ values were 2- or 4-fold
higher in CHCl3 than in EtOH for NBD-bearing compounds
(8, 16, 17) and several-fold higher than in PBS buffer. Dansyl-
bearing compounds(7, 18, 19) showeda lesspronouncedincrease
in Φ values from EtOH to CHCl3 solutions, although the highest
Φ was shown by compound 19 (Φ = 0.48). Molar extinction
coefficients (ε) were determined for all final compounds (in
EtOH), with the lowest values displayed by the dansyl-bearing com-
were conducted with compound 16, which displayed the best
combination between pharmacological (σ2 receptor affinity and
selectivity) and fluorescence properties (convenient excitation
and emission wavelengths and high ε value), and promising
results were obtained in different tumor pancreatic cells which
were previously shown to overexpress σ2 receptors.11 Therefore,
compound 16 was evaluated in more details in in vitro inter-
nalization studies and cellular colocalization by confocal micro-
scopy in human pancreatic tumor cells (BxPC3).
In Vitro Internalization Studies. Uptake of compound 16 in
BxPC3 pancreatic cancer cells was analyzed immediately follow-
ing treatment (25 nM) and the mean fluorescence recorded over
time (Figure 1A). The T1/2 of maximum fluorescence at 60 min
was 7.8 ( 1.5 min (mean ( SEM). We further studied uptake for
the purpose of better understanding the involvement of endo-
cytosis of these compounds and the receptor. Caveolin-mediated
(lipid rafts-mediated) endocytotis can be inhibited by Filipin
III29 and clathrin-mediated endocytosis by phenylarsine oxide
(PAO).30 BxPC3 cells were pretreated with Filipin III (5 μg/mL)
or PAO (10 mM) for 30 min prior to treatment with compound
16 (25 nM) and mean fluorescence was collected over 60 min.
The rate of uptake was decreased from 7.8 ( 1.5 min to 6.8 ( 0.8
min for Filipin III and 4.9 ( 2.1 min for PAO. As well, the overall
uptake at 60 min was decreased to 69% and 50% for Filipin III
and PAO, respectively. Taken together, the T1/2 in the range of
minutes, and the decreased uptake in the presence of endocytosis
inhibitors suggest that the internalization of 16 occurs, in part,
through the caveolin- and clathrin-mediated endocytotic path-
ways in addition to simple membrane diffusion. Uptake by
the clathrin-dependent pathway has been described previously
with other σ2 receptor ligands,21,22 but uptake by the caveolin-
pounds (2600ꢀ4000 L/mol cm) and the highest values displayed
3
by the NBD-bearing compounds (6544ꢀ14391 L/mol cm),
3
indicating that the fluorescence intensity of the latter compounds
is stronger. Preliminary fluorescence microscopy experiments
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Figure 1. Cellular internalization of the fluorescent σ2 receptor ligand 16. (A) Kinetic uptake of compound 16: BxPC3 pancreatic cancer cells were
treated for 1 h with the endocytosis inhibitors Filipin III (5 μg/mL) or phenylarsine oxide (PAO, 10 μM) or DMSO vehicle for 60 min prior to kinetic
uptake analysis of 16 (25 nM) by flow cytometry. Compound 16 uptake represents the percentage of the mean fluorescence of maximum signal intensity.
(B) Competition of compound 16 by σ2 agonist structural analogues. BxPC3 cells were treated with increasing doses of compounds 1 or 20 for 45 min
prior to replacement with compound 16 (25 nM) for 45 min and fluorescence intensity quantified by flow cytometry. (C) Cell density dependence σ2
agonist uptake and cell death. BxPC3 cells were seeded at increasing densities to achieve a range of dividing, subconfluent and quiescent, confluent
cultures. The following day, cells were treated with compound 16 (25 nM) and fluorescence intensity quantitated by flow cytometry. Alternatively, cell
were treated with compound 1 or 20 (100 μM) for 24 h and viability compared to DMSO control.
dependent/lipid raft pathway has not been previously reported.
Interaction and endocytosis of compound 16 through lipid rafts
is of note considering that σ2 receptor agonists were initially
found to bind to protein constituents of the lipid rafts,31,32
cholesterol-rich domains in the cell membrane. They form
flask-shaped invaginations called caveolae, for the caveolin
protein that coats them, and act as platforms for glycopho-
sphatidylinositol-linked protein mediated signaling pathways
and internalization of cholesterol.33 Interest has grown in
targeting this pathway in cancer cells, which may have disrupted
lipid rafts, contributing to aberrances in pathways implicated in
chemoresistance such as the epidermal growth factor receptor
and tumor necrosis factor R receptor.34
Therefore, implication of lipid raft disruption in oncology
signaling and response to chemotherapy together with the
presence of σ2 proteins in lipid rafts suggest that σ2 receptor
mediated toxicity and overcame chemoresistance (which has
been shown with different σ2 receptor agonists),23,35 likely
involve lipid rafts.
compound functionally competes for localization in the same
plane as the parent and analogue compound.
The correlation between the proliferative status of tumors and
the expression of the σ2 receptors has been widely demonstrated
in different cancer cell lines,22,37,38 and we have further detailed
the expression and apoptosis response in pancreatic cancer
cells.11,39 In this study, we further evaluated the impact of
proliferation on σ2 agonist uptake and sensitivity (Figure 1C).
To maintain proliferating versus quiescent cell cultures, subcul-
tured cells were seeded at increasing densities in order to achieve
subconfluent and confluent cultures, respectively. Compound 16
mean fluorescence at 30 min decreased as cell density increased,
in accordance with earlier findings that σ2 receptors are markers
of cell proliferation.40 In addition, the decreased uptake was
associated with decreased cell death as the density increased so
that the reduction of the antiproliferative activity of σ2 agonists 1
and 20 was likely due to the σ2 reduced presence in nonprolifer-
ating cells. Together, these findings show that compound 16
performs biologically as expected for a σ2 ligand and that
increased uptake of σ2 agonists by proliferating cells is a critical
step for mediating cell death.
In vitro competition was performed to quantify interaction of
compound 16 with σ2 agonists in the live cell. Preloading with 1
or the recently produced σ2 agonist cis-1-cyclohexyl-4-[4-(2,6-
difluorophenyl)cyclohexyl]piperazine36 (20) for 45 min prior to
addition of compound 16 for 45 min decreased the mean
fluorescence intensity of 16 with increasing concentrations of
σ2 agonist (Figure 1B). This indicates that the fluorescent
Cellular colocalization studies of these fluorescent analogues
of 1 were initially screened by epifluorescent microscopy
(Supporting Information), and confocal microscopy results were
in accordance with those findings (Figure 2). BxPC3 cells were
incubated with fluorescent ligand 16 and MitoTracker Red,
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16 displayed high σ2 receptor affinity and moderate σ1/σ2
selectivity. Therefore, compound 16 internalization was studied
by flow cytometry and by confocal microscopy for colocalization
with subcellular organelles in BxPC3 pancreatic tumor cells. The
uptake of fluorescent ligand 16 decreased in the presence of
nonfluorescent σ2 ligands, showing that fluorescent and non-
fluorescent compounds compete for localization in the same
plane. Endocytosis inhibitors decreased the uptake of compound
16, showing that internalization occurs, in part, through endo-
cytotic pathways besides simple membrane diffusion, and that
interaction with lipid rafts, which may be able to influence the
membrane composition and downstream signaling, takes place
so that σ2 receptor mediated toxicity and chemoresistance over-
come likely involve lipid rafts. The influence of proliferation, with
cell density as a surrogate, on σ2 agonist uptake and sensitivity,
was studied and showed that the uptake is higher in proliferating
cells, supporting that σ2 receptors are markers of cell
proliferation.37,38 Furthermore, the sensitivity of BxPC3 cells
for σ2 agonists decreased, together with the uptake, as the density
(i.e., quiescent cells) increased, suggesting that uptake is a critical
step for mediating σ2 agonists-dependent cell death. Compound
16 colocalized to the greatest extent in the endoplasmic reticu-
lum and lysosomes, with moderate colocalization in the mito-
chondria and the plasma membrane but no colocalization in the
nucleus was observed, in disagreement with the histone hypoth-
esis which will have to be further analyzed.
All in all, it was demonstrated that the use of fluorescent σ2
ligands may help in clarifying the mechanisms of action of the still
enigmatic σ2 receptors. The use of such compounds in different
cell lines may contribute to understand the different cell type
apoptotic pathways activated by σ2 ligands. Furthermore, a
scaffold which appears optimal for inferring high σ receptor
affinities was herein produced, and it can be further exploited for
obtaining fluorescent molecules with λexc and λem more shifted
toward the near-infrared (NIR) region of the spectrum for a
wider application of σ receptor fluorescent ligands in optical
molecular imaging techniques.
Figure 2. Cellular colocalization of 16 with subcellular organelles.
BxPC3 pancreatic cancer cells were incubated with 16 and subcellular
markers, as described in the Materials and Methods, and imaged by
confocal microscopy. 16 is presented as green, organelle markers in red,
and overlays in yellow.
’ MATERIALS AND METHODS
Chemistry. Both column chromatography and flash column chro-
matography were performed with 60 Å pore size silica gel as the
stationary phase (1:30 w/w, 63ꢀ200 μm particle size, from ICN, and
1:15 w/w, 15ꢀ40 μm particle size, from Merck, respectively). Melting
points were determined in open capillaries on a Gallenkamp electro-
thermal apparatus. Purity of tested compounds was established by
combustion analysis, confirming a purity g95%. Elemental analyses
(C, H, N) were performed on an Eurovector Euro EA 3000 analyzer; the
analytical results were within (0.4% of the theoretical values unless
ERTracker Red, LysoTracker Red, or Vybrant cholera-toxin B
subunit (CT-B), at 37 °C for 30 min prior to fixation and nuclear
staining with TO-PRO-3. Compound 16 is found in the mem-
brane fractions of the cell and colocalizes greatest in the
endoplasmic reticulum and lysosomes, with moderate colocaliza-
tion in the mitochondria and the plasma membrane (CT-B).
Colocalization in the nucleus was not observed with TO-PRO-3.
1
otherwise indicated. H NMR spectra were recorded on a Mercury
Varian 300 MHz using CDCl3 as solvent. The following data were
reported: chemical shift (δ) in 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 6890ꢀ5973 MSD gas
chromatograph/mass spectrometer and on an Agilent 1100 series LC-
MSD trap system VL mass spectrometer; only significant m/z peaks,
with their percentage of relative intensity in parentheses, are reported.
Chemicals were from Aldrich and Across and were used without any
further purification.
6-(1,4-Dioxa-8-azaspiro[4.5]dec-8-yl)hexanenitrile (2). A
mixture of 1,4-dioxa-8-azaspiro[4.5]decane (0.72 mL, 5.6 mmol),
triethylamine (0.78 mL, 5.6 mmol), and 6-bromohexanenitrile
(0.74 mL, 5.6 mmol) in CH2Cl2 was stirred at room temperature.
’ CONCLUSIONS
Fluorescent σ2 ligands were obtained linking dansyl or NBD
moieties in two different positions of compound 1 structure.
High affinity σ2 ligands were obtained when the fluorescent tag
was attached on the tetralin ring through an alkyl linker replacing
the methyl in the methoxy function. On the other hand, the
approach of attaching a fluorescent tag at the compound 1
cyclohexyl moiety (replaced by a piperidine ring) was unsuccess-
ful, and a dramatic drop in the affinity was recorded. NBD-
bearing compounds displayed better fluorescent properties
(more convenient λexc and λem and high fluorescence intensity)
than dansyl-bearing compounds, and among them, compound
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The reaction mixture was washed with H2O, and the separated organic
layer was concentrated under reduced pressure to give a crude mixture,
which was purified by column chromatography with CH2Cl2/MeOH
(95:5) as eluent, to afford the target compound as a pale-yellow oil
2-(6-[5-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-
tetrahydronaphthalen-1-yloxy]hexyl)isoindole-1,3-dione
(11). A grain of NaI, K2CO3 (0.14 g, 1.0 mmol), and phthalimide 9
(0.29 g, 1.0 mmol) were added to a solution of phenol 10 (0.26 g, 0.74
mmol) in DMF (5 mL), and the reaction mixture was heated at 100 °C
for 18 h. 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 ꢁ 10 mL). The crude was purified by flash
chromatography with ethyl acetate/CH2Cl2 (6:4) as eluent to give
compound 11 as a yellow oil (0.094 g, 16% yield). 1H NMR δ 1.21ꢀ1.97
(m, 26H), 2.10ꢀ2.95 (m, 14H), 3.69 (t, 2H, J = 7.2 Hz), 3.90 (m, 2H),
6.57ꢀ7.07 (m, 3H), 7.60ꢀ7.86 (m, 4H). LC-MS (ESI+) m/z 586 [M +
H]+, 608 [M + Na]+.
2-[2-[5-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-te-
trahydronaphthalen-1-yloxy]ethoxy]ethanol (12). To a solu-
tion of phenol 10 (0.14 g, 0.4 mmol) in DMF (5 mL), a grain of NaI,
K2CO3 (0.06 g, 0.5 mmol), and 2-(2-chloroethoxy)ethanol (0.05 mL,
0.5 mmol) were added, and the reaction mixture was heated at 120 °C
for 18 h. After cooling, the solvent was evaporated under reduced
pressure, and then water was added to the residue. The mixture was
extracted with AcOEt (3 ꢁ 5 mL), and the collected organic layers
collected were dried (Na2SO4) and evaporated to afford a crude, which
was purified by flash chromathography with AcOEt/CH2Cl2 (7:3) as
eluent to give the target compound 12 as a yellow oil (0.11 g, 62% yield).
1H NMR δ 1.00ꢀ1.40 (m, 6H), 1.40ꢀ2.00 (m, 12H), 2.40ꢀ3.20 (m,
15H), 3.65ꢀ3.80 (m, 4H), 3.86ꢀ3.90 (m, 2H), 4.06ꢀ4.12 (m, 2H),
6.65ꢀ7.05 (m, 3H). GC-MS m/z 445 (M+ + 1, 5), 444 (M+, 22), 181
(100). LC-MS (ESI+) m/z 445 [M + H]+, 467 [M + Na]+.
2-[2-[2-[5-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-
tetrahydronaphthalen-1-yloxy]ethoxy)]ethyl]isoindole-1,3-
dione (13). To a stirred solution of alcohol 12 (0.12 g, 0.27 mmol) in
dry THF (10 mL) kept under N2, triphenylphosphine (0.12 g, 0.46
mmol), phthalimide (0.069 g, 0.47 mmol), and DIAD (0.12 mL, 0.60
mmol) were added, and the mixture was stirred at room temperature for
18 h. Then the solvent was evaporated under reduced pressure, and the
resulting residue was treated with H2O and the aqueous layer was
extracted with AcOEt (3 ꢁ 20 mL). The combined organic layers were
dried (Na2SO4) and concentrated under reduced pressure to give a
crude residue, which was purified by column chromatography using
CH2Cl2/MeOH (95:5) as eluent, to afford the target compound as a
yellow oil (0.10 g, 65% yield). LC-MS (ESI+) m/z 574 [M + H]+, 596
[M + Na]+.
General Procedure for the Synthesis of 6-[1-[3-(4-Cyclo-
hexylpiperazin-1-yl)-propyl]-1,2,3,4-tetrahydronaphthale-
n-5-yloxy]hexylamine (14) and 2-[2-[1-[3-(4-Cyclohexylpi-
perazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-ylox-
y]ethoxy]ethylamine (15). Hydrazine hydrate 50% (0.085 mL,
0.85 mmol) was added to a solution of either 11 or 13 (0.29 mmol)
in methanol (3 mL), and the reaction mixture was stirred at room
temperature for 30 min. Then 1.5 N HCl (1.2 mL) was then added, and
the mixture was stirred for further 12 h. Then 3 N HCl was added until a
pH < 2 was obtained, and the mixture was heated at reflux for 30 min
After cooling down to room temperature, the mixture was filtered, 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 treatment to afford the target compound as a pale-yellow oil
(70% yield).
1
(1.16 g, 87% yield). H NMR δ 1.42ꢀ1.56 (m, 2H), 1.64ꢀ1.84 (m,
4H), 1.86ꢀ2.10 (m, 4H), 2.36 (t, 2H, J = 7.2 Hz), 2.68 (t, 2H, J = 8 Hz),
2.80ꢀ3.00 (m, 4H), 4.00 (s, 4H). GC-MS m/z 239 (M+ + 1, 1), 238
(M+, 3), 156 (100).
N-[6-(1,4-Dioxa-8-aza-spiro[4.5]dec-8-yl)hexyl]acetamide
(3). A solution of the nitrile 2 (1.0 g, 4.2 mmol) in anhydrous Et2O
(25 mL) was added in a dropwise manner to a suspension of LiAlH4
(0.32 g, 8.4 mmol) in the same solvent kept under N2 at 0 °C. The
mixture was stirred at 0 °C for 45 min and then at room temperature
overnight. H2O was carefully added into the reaction pot, and the
obtained mixture was filtered on Celite pad and the filtrate evaporated
under reduced pressure to give the corresponding amine as a pale-yellow
gummy solid (1.0 g, 99% yield). GC-MS m/z 242 (M+, 1), 156 (100).
Such amine (1.0 g, 4.2 mmol) was dissolved in anhydrous CH2Cl2
(40 mL) and added with triethylamine (1.2 mL, 8.6 mmol). Acetyl
chloride (0.45 mL, 6.4 mmol) was then dropped at 0 °C, under N2. The
mixture was stirred at room temperature for 3 h and then treated with
NaHCO3 (satd solution 20 mL), and extracted with CH2Cl2 (3 ꢁ
20 mL). The organic layers collected were dried (Na2SO4) and
concentrated under reduced pressure to give a crude mixture, which
was purified by column chromatography with CH2Cl2/MeOH (9:1) as
eluent to yield title compound as a pale-yellow oil (0.78 g, 66% yield).
GC-MS m/z 284 (M+, 1), 156 (100).
N-[6-(4-Oxopiperidino)hexyl]acetamide (4). To a solution of
amide 3 (0.32 g, 1.13 mmol) in acetone, 2 N HCl (37 mL) was added
and the mixture was heated at reflux for 1 h followed by 1 h at room
temperature. The solvent was removed under reduced pressure, and
conc NaOH was added to obtain an alkaline pH. The aqueous phase was
extracted with AcOEt (3 ꢁ 15 mL), and the organic layers collected and
dried (Na2SO4) and evaporated under reduced pressure to afford the
target compound as a yellow oil (0.17 g, 63% yield), which was used for
1
the next step without further purification. H NMR δ 1.42ꢀ1.84 (m,
8H), 2.10ꢀ2.68 (s+m, 13H), 2.90ꢀ3.10 (m, 2H), 5.10 (broad s, 1H).
GC-MS m/z 240 (M+, 1), 112 (100).
6-[4-[4-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-
yl)propyl]piperazin-1-yl]piperidino]hexylamine (6). The pi-
peridine 4 (0.16 g, 0.67 mmol) and piperazine 5 (0.19 g, 0.68 mmol)
were reacted with ZnCl2 (0.05 g, 0.39 mmol) and NaCNBH3 (0.044 g,
0.70 mmol) in 2-propanol (20 mL). The mixture was stirred for 48 h at
room temperature. Then, the reaction mixture was evaporated to
dryness, and the residue was diluted with 2 N NaOH and extracted
with AcOEt. The organic layers were washed with brine, dried
(Na2SO4), and then concentrated under reduced pressure to give a
crude residue, which was purified by column chromatography with
CH2Cl2/MeOH (8:2) as eluent to afford the intermediate N-acetyl
derivative of compound 6 as a white solid (0.13 g, 39% yield). 1H NMR δ
1.20ꢀ2.00 (m, 23H), 2.20ꢀ2.40 (m, 6H), 2.45ꢀ3.00 (m, 14H), 3.20
(m, 2H), 3.80 (s, 3H), 5.45 (broad s, 1H), 6.65 (d, 1H, J = 7.7 Hz), 6.80
(d, 1H, J = 7.7 Hz), 7.08 (t, 1H, J = 7.9 Hz). LC-MS (ESI+) m/z 513
[M + H]+, 535 [M + Na]+. Such intermediate acetamide (0.22 g,
0.44 mmol) was refluxed in 3N HCl (6.5 mL) for 4 h. After cooling, the
mixture was made alkaline with K2CO3 (satd solution, 10 mL) and
extracted with CH2Cl2 (3 ꢁ 10 mL). The collected organic layers were
dried (Na2SO4), and the solvent was evaporated to produce a brown
semisolid (0.20 g, 99% yield), which was used for the next step without
6-[1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
naphthalen-5-yloxy]hexylamine (14). 1H NMR δ 1.00ꢀ1.30 (m, 5H),
1.40ꢀ2.00 (m, 23H), 2.20ꢀ2.80 (m, 16H), 3.90 (t, 2H, J = 6.0 Hz), 6.60
(d, 1H, J = 7.9 Hz), 6.75 (d, 1H, J = 7.7 Hz), 7.05 (t, 1H, J = 7.9 Hz). LC-
MS (ESI+) m/z 456 [M + H]+.
1
any further purification. H NMR δ 1.20ꢀ2.00 (m, 20H), 2.20ꢀ2.80
(m, 19H), 2.85ꢀ3.05 (m, 3H), 3.80 (s, 3H), 5.45 (broad s, 2H, D2O
exchanged), 6.65 (d, 1H, J = 7.7 Hz), 6.80 (d, 1H, J = 7.7 Hz), 7.08
(t, 1H, J = 7.9 Hz). LC-MS (ESI+) m/z 471 [M + H]+.
2-[2-[1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydro-
1
naphthalen-5-yloxy]ethoxy]ethylamine (15). H NMR δ 1.05ꢀ1.50
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Journal of Medicinal Chemistry
ARTICLE
(m, 6H), 1.60ꢀ2.10 (m, 12H), 2.40ꢀ3.20 (m, 18H), 3.65ꢀ3.90 (m,
6H), 6.65ꢀ7.05 (m, 3H). LC-MS (ESI+) m/z 444 [M + H]+, 466 [M +
Na]+.
General Procedure for the Synthesis of Final Compounds
7, 16, 17. 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl, 1.0 mmol)
was dissolved in absolute EtOH (15 mL) and added in a dropwise
manner to one among amines 6, 14, or 15 (1.0 mmol) dissolved in the
same solvent (15 mL). The mixture was stirred for 1.5 h at room
temperature. Then the reaction mixture was filtered and the filtrate
evaporated under reduced pressure.
using CH2Cl2/MeOH (97:3) as eluent to give the target compound 18 as
1
a pale-green oil (0.33 g, 48% yield). H NMR δ 1.00ꢀ1.90 (m, 24H),
2.00ꢀ2.20 (m, 2H), 2.40ꢀ3.10 (m, 22H), 3.80 (t, 2H, J = 6 Hz),
4.70ꢀ4.80 (m, 1H, D2O exchanged), 6.55 (d, 1H, J = 7.9 Hz), 6.73 (d,
1H, J = 7.7 Hz), 7.00ꢀ7.05 (m, 1H), 7.20 (d, 1H, J = 7.9 Hz), 7.50ꢀ7.60
(m, 2H), 8.20ꢀ8.40 (m, 2H), 8.51 (d, 1H, J = 8.5 Hz). LC-MS (ESI+)
m/z 689 [M + H]+. Anal. (C41H60N4O3S 3HCl 3/2H2O) C, H, N.
3
3
5-Dimethylaonaphthalene-1-sulfonic acid 2-[2-[5-[3-(4-cyclohex-
ylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronaphthalen-5-yloxy]etho-
xy]ethanamide (19). The crude semisolid was purified by column
chromatography using CH2Cl2/MeOH (98:2) as eluent to give the
6-[4-[4-[3-(5-Methoxy-1,2,3,4-tetrahydronaphthalen-1-yl]propyl]-
piperazin-1-yl]piperidino]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexa-
namine (7). The crude semisolid was purified by column chromatog-
raphy with AcOEt/MeOH (7:3) as eluent to give the final compound 7
1
target compound as a pale-green oil (0.47 g, 70% yield). H NMR δ
1.00ꢀ2.30 (m, 18H), 2.50ꢀ3.40 (m, 22H), 3.45 (t, 2H, J = 5 Hz), 3.55
(t, 2H, J = 6 Hz), 3.90 (t, 2H, J = 6 Hz), 5.20ꢀ5.30 (m, 1H, D2O
exchanged), 6.55 (d, 1H, J = 7.9 Hz), 6.76 (d, 1H, J = 7.7 Hz), 7.00ꢀ7.10
(m, 1H), 7.14 (d, 1H, J = 7.9 Hz), 7.40ꢀ7.54 (m, 2H), 8.20ꢀ8.26 (m,
2H), 8.51 (d, 1H, J = 8.5 Hz). LC-MS (ESI+) m/z 677 [M + H]+. Anal.
1
as an orange semisolid (0.26 g, 42% yield). H NMR δ 1.20ꢀ2.20
(m, 21H), 2.25ꢀ2.35 (m, 8H), 2.45ꢀ2.80 (m, 11H), 3.00ꢀ3.10
(m, 2H), 3.20ꢀ3.40 (broad s, 1H), 3.80 (s, 3H), 6.18 (d, 1H, J = 8.5
Hz), 6.60 (d, 1H, J = 7.7 Hz), 6.80 (d, 1H, J = 7.7 Hz), 7.05 (t, 1H, J = 7.9
Hz), 8.50 (d, 1H, J = 8.5 Hz). LC-MS (ESI+) m/z 634 [M + H]+, 656 [M +
(C39H56N4O4S 3HCl) C, H, N.
3
Fluorescence Spectroscopy and Molar Extinction Coeffi-
cient. Emission spectra of compounds 7, 8, and 16ꢀ19 were deter-
mined in EtOH, CHCl3, and in PBS buffer solution. In all experiments,
the excitation and the emission bandpass was set at 10 nm. The emission
spectra were obtained from 300 to 700 nm, with excitation set at the
appropriate excitation wavelength. The excitation spectra of compounds
8, 18, and 19 were obtained from 250 to 450 nm, with the emission being
recorded at the appropriate wavelength. The excitation spectra of
compounds 7, 16, and 17 were obtained from 300 to 550 nm, with
the emission being recorded at the appropriate wavelength. Fluores-
cence quantum yields were calculated with respect to quinine sulfate
(Fluka) in 0.5 M H2SO4 as a standard (Φ = 0.546).41 Solutions of both
the sample and the reference were prepared from original solutions
diluted with the appropriate solvent so that absorbance was below 0.2 at
the same excitation wavelength (347 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 was calculated
according the following equation:42
Na]+. Anal. (C35H51N7O4 3.8HCl) C, H, N.
3
6-[5-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydronap-
hthalen-5-yloxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine
(16). The crude semisolid was purified by column chromatography
using CH2Cl2/MeOH (99:1) as eluent to give the target compound 16
1
as a brown oil (0.37 g, 60% yield). H NMR δ 1.00ꢀ1.40 (m, 10H),
1.50ꢀ2.00 (m, 16H), 2.18ꢀ2.80 (m, 13H), 3.45ꢀ3.55 (m, 3H), 3.94
(t, 2H, J = 6.0 Hz), 6.17 (d, 1H, J = 8.5 Hz), 6.20ꢀ6.30 (broad s, 1H,
D2O exchanged), 6.60 (d, 1H, J = 7.7 Hz), 6.78 (d, 1H, J = 7.7 Hz), 7.05
(t, 1H, J = 7.9 Hz), 8.50 (d, 1H, J = 8.5 Hz). LC-MS (ESIꢀ) m/z 617
[M ꢀ H]ꢀ. Anal. (C35H50N6O4 3HCl 5/4H2O) C, H, N.
3
3
2-[2-[5-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-5,6,7,8-tetrahydro-
naphthalen-1-yloxy]ethoxy]-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)eth-
anamine (17). The crude semisolid was purified by column chroma-
tography using CH2Cl2/MeOH (99:1) as eluent to give the final
1
compound 17 as a brown oil (0.37 g, 62% yield). H NMR δ 1.40ꢀ
2.20 (m, 18H), 2.25ꢀ2.80 (m, 14H), 3.50ꢀ3.75 (m, 3H), 3.85ꢀ4.00
(m, 4H), 4.08ꢀ4.15 (m, 2H), 6.17 (d, 1H, J = 8.5 Hz), 6.60 (d, 1H, J =
7.7 Hz), 6.80 (d, 1H, J = 7.7 Hz), 7.05 (t, 1H, J = 7.9 Hz), 8.45 (d, 1H, J =
Φx ¼ ΦSðAS=AXÞðFX=FSÞðnX=nSÞ2
8.5 Hz). LC-MS (ESI+) m/z 607 [M + H]+. Anal. (C33H46N6O5 3HCl)
3
C, H, N.
where Φ is the emission quantum yield, A is the absorbance at the
excitation wavelength, F is the area under the corrected emission curve, n
is the refractive index of the solvent for the sample (X) and the standard
(S). Absorption spectra were recorded with a PerkinElmer UVꢀvis-NIR
spectrophotometer, and fluorescence spectra were obtained with a
PerkinElmer LS55 spectrofluorometer. Molar extinction coefficients
(ε) were determined for each final compound (7, 8, 16ꢀ19) dissolved
in EtOH, with concentration ranging from 1 to 100 μM and absorbance
spectra recorded from 200 to 600 nm in standard quartz cuvettes. ε
values were determined by fitting the Beer’s law: A = ε ꢁ c ꢁ d where (A)
is the absorbance at the λexc; (c) is the molar concentration of the
solution, and (d) was the optical path length (d = 1 cm). Measurements
were repeated twice.
Biological Methods and Materials. Radioligand Binding As-
says. All the procedures for the binding assays were previously described.
σ1 And σ2 receptor binding were carried out according to Matsumoto
et al.43 [3H]-DTG (30 Ci/mmol) and (+)-[3H]-pentazocine (34 Ci/
mmol) were purchased from PerkinElmer Life Sciences (Zavantem,
Belgium). DTG was purchased from Tocris Cookson Ltd., UK. (+)-
Pentazocine was obtained from Sigma-Aldrich-RBI srl (Milan, Italy).
Male Dunkin guinea pigs and Wistar Hannover rats (250ꢀ300 g) were
from Harlan, Italy. The specific radioligands and tissue sources were
respectively: (a) σ1 receptor, (+)-[3H]-pentazocine (+)-[2S-(2R,6R,11-
R)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2,6-metha-
no-3-benzazocine-8-ol), guinea pig brain membranes without cerebellum;
General Procedure for the Synthesis of Final Compounds
8, 18, 19. A solution of 5-(dimethylamino)naphthalene-1-sulfonyl chlo-
ride (dansyl chloride) (0.21 g, 0.8 mmol) in anhydrous CH2Cl2 (15 mL)
was added in a dropwise manner to one among amines 6, 14, or 15 (1.0
mmol) dissolved in the same solvent (15 mL). The mixture was stirred at
room temperature for 18 h. Then, the reaction mixture was washed with
H2O (2ꢁ 30 mL) andthe organicphaseswerecollected, dried (Na2SO4),
and evaporated under reduced pressure to afford a crude residue, which
was purified as described below.
5-Dimethylaonaphthalene-1-sulfonic acid 6-[4-[4-[3-(5-methoxy-
1,2,3,4-tetrahydronaphthalen-1-yl)propyl]piperazin-1-yl]piperidino]-
hexanamide (8). The crude semisolid was purified by column chroma-
tography using CHCl3/MeOH (9:1) as eluent to give the target
1
compound as a green oil (0.29 g, 42% yield). H NMR δ 1.04ꢀ1.45
(m, 8H), 1.50ꢀ2.10 (m, 12H), 2.20ꢀ2.40 (m, 6H), 2.45ꢀ2.80
(m, 11H), 2.92ꢀ3.02 (m, 11H), 3.80 (s, 3H), 4.90ꢀ5.00 (m, 1H
D2O exchanged), 6.63 (d, 1H, J = 7.7 Hz), 6.78 (d, 1H, J = 7.7 Hz), 7.08
(t, 1H, J = 7.7 Hz), 7.18 (d, 1H, J = 7.4 Hz), 7.50ꢀ7.60 (m, 2H), 8.22
(d, 1H, J = 7.4 Hz), 8.30 (d, 1H, J = 7.4 Hz), 8.55 (d, 1H, J = 7.4 Hz). LC-
MS (ESI+) m/z 704 [M+H]+. Anal. (C41H61N5O3S 4HCl 2H2O)
3
3
C, H, N.
5-Dimethylaonaphthalene-1-sulfonic acid 6-[5-[3-(4-cyclohexylpi-
perazin-1-yl)propyl]-5,6,7,8- tetrahydronaphthalen-1-yloxy]hexana-
mide (18). The crude semisolid was purified by column chromatography
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Journal of Medicinal Chemistry
ARTICLE
(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 (73ꢀ87%), (b) DTG (85ꢀ96%). 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
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +39-080-5442748. Fax: +39-080-5442231. E-mail: abate@
farmchim.uniba.it.
experiments with samples in duplicate. Scatchard parameters (Kd and Bmax
)
’ ACKNOWLEDGMENT
and apparent inhibition constants (Ki) values were determined by nonlinear
curve fitting by using the Prism, version 3.0, GraphPad software.44
Cell Culture. BxPC3 pancreatic cancer cells were maintained in
Roswell Park Memorial Institute (RPMI) media (GIBCO) supplemen-
ted with L-glutamine (2 mM), 4-(2-hydroxyethyl)-1-piperazineethane-
sulfonic acid (HEPES) (1 mM), pyruvate (1 mM), sodium bicarbonate
(0.075% w/v), penicillin, and streptomycin (100 IU/mL), amphotericin
(0.25 μg/mL), and 10% fetal bovine serum (Atlanta Biologicals, Law-
renceville, GA). Cells were seeded at a density of 2 ꢁ 105/mL unless
otherwise stated and maintained in a humidified atmosphere of 5% CO2
at 37 °C.
This project was partially financed by Veterans Administration
Merit Award (project 1136919) (W.G.H.), American Cancer
Society (project MRSG08019-01CDD) (W.G.H.), and National
Institutes of Health T32 Training Grant (project 5T32CA0-
09621-22) (J.R.H.).
’ ABBREVIATIONS USED
NBD, 7-nitro-1,2,3-benzoxadiazole; ER, endoplasmic reticulum;
DIAD, diisopropylazodicarboxylate; SAfiR, structureꢀaffinity
relationship; PBS, phosphate buffered saline; PAO, pheny-
larsine oxide; UV, ultraviolet; NIR, near-infrared; DMSO, di-
methyl sulfoxide
Confocal Microscopy. For subcellular compartmentalization, cells
grown on glass coverslips were incubated with compound 16 (100 nM)
and either ERTracker Red (1 μM), MitoTracker Red (100 nM), or
LysoTracker Red (50 nM) for 30 min at 37 °C. The plasma membrane
was visualized using the Vybrant Alexa Fluor 594 lipid raft labeling kit as
directed by the manufacturer. All reagents were obtained from Molec-
ular Probes. Cells were washed with PBS and fixed in 2% paraformalde-
hyde for 30 min at 37 °C prior to additional washing and mounting to a
slide with ProLong Gold antifade reagent. Imaging was performed on a
Carl Zeiss Axiovert 100 inverted microscope, fitted with LSM 510 laser
scanning microscope camera and software. Images were collected with
filter bandwidths corresponding to 505ꢀ530 nm for green,
560ꢀ615 nm for red, and >650 nm for far red, with 4 scans over 11.8 s.
Internalization of Compound 16 by Flow Cytometry. To quantify
internalization of compound 16, cells were pretreated with the endocy-
tosis inhibitors phenylarsine oxide (10 μM) or Filipin III (5 μg/mL) for
60 min at 37 °C prior to washing and resuspension in cell media.
Compound 16 (25 nM) was added and the mean fluorescence (FL1)
quantified with a FACSCalibur flow cytometer (BD Biosciences, San
Jose, CA) with kinetic readings over a time period of 60 min. To detect
competition of compound 16 with parent and analogous compounds,
BxPC3 cells were treated with compounds 1 or 20 at increasing
concentrations for 45 min prior to replacement with compound 16
(25 nM) for 45 min at 37 °C and fluorescence intensity quantified by
flow cytometry. To deter the influence of cell proliferation by increasing
the cell seeding density, BxPC3 cells were seeded at increasing con-
centrations, and the following day, cells were treated with compound 16
(25 nM) for 30 min at 37 °C and fluorescence intensity quantified by
flow cytometry.
’ REFERENCES
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Cell Viability. Subcultured BxPC3 cells were seeded at increasing
densities from 1 ꢁ 105 to 9 ꢁ 105 into 96-well clear-bottom plates 24 h
prior to treatment with compounds 1 or 20 (100 μM). Then 18 h later,
cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min
at 37 °C, and stained with crystal violet for 15 min at 37 °C. Cells were
then washed with PBS and cell density detected with a Bio-Rad
Laboratories ChemiDoc XRS+ Imager and quantified with Quantity
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’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analyses of the no-
b
vel end products; formulas, melting points of hydrochloride salts;
fluorescence microscopy images taken with compound 16 and
subcellular organelles trackers. This material is available free of
charge via the Internet at http://pubs.acs.org.
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