Tritium radiolabelling of PB28, a potent sigma-2 receptor ligand: pharmacokinetic and pharmacodynamic characterization

Article abstract of DOI:10.1016/j.bmcl.2007.12.056

A potent sigma-2 receptor ligand, known as PB28, was tritium radiolabelled and biologically evaluated. The results showed that [³H]PB28 and the corresponding unlabelled PB28 had superimposed pharmacodynamic properties. This radioligand appears as a potential candidate for receptor binding and in living cells assays.

Full text of DOI:10.1016/j.bmcl.2007.12.056

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 2183–2187
Tritium radiolabelling of PB28, a potent sigma-2 receptor
ligand: pharmacokinetic and pharmacodynamic characterization
Nicola Antonio Colabufo,* Carmen Abate, Marialessandra Contino, Carmela Inglese,
Savina Ferorelli, Francesco Berardi and Roberto Perrone
`
Dipartimento Farmacochimico, Universita degli Studi di Bari, via Orabona 4, 70125 Bari, Italy
Received 4 December 2007; revised 19 December 2007; accepted 21 December 2007
Available online 25 December 2007
Abstract—A potent sigma-2 receptor ligand, known as PB28, was tritium radiolabelled and biologically evaluated. The results
showed that [3H]PB28 and the corresponding unlabelled PB28 had superimposed pharmacodynamic properties. This radioligand
appears as a potential candidate for receptor binding and in living cells assays.
Ó 2007 Elsevier Ltd. All rights reserved.
Sigma receptors are endoplasmic binding sites clearly
distinct from other known receptors both in pharmacol-
ogy and structure. Two subtypes of sigma receptors
have been identified, sigma-1 and sigma-2, different for
distribution in tissues and for pharmacology and physi-
ology.1 Interest in sigma receptor research is growing as
much evidence shows how sigma proteins, and in partic-
ular sigma-2 subtype, are markedly overexpressed in
many tumour cell lines.2,3 Thus, sigma receptor ligands
are now being developed for the imaging of the two sub-
types with noninvasive techniques (i.e., PET and
SPECT) providing information about the stage of the
tumour.4,5 Besides this diagnostic importance, sigma-2
receptor agonists display a noteworthy therapeutic po-
tential in the oncology field.
N
N
O
Figure 1. PB28 structure.
ary phase.8 The elution of the protein content from
human SK-N-SH neuroblastoma cell line overexpress-
ing sigma-2 receptors led to isolate several histone pro-
teins. Therefore, we concluded that overexpressed
sigma-2 receptors are likely histones or that sigma-2 li-
gands bind histones.
Despite these claimed potentials, sigma-2 receptors have
not been isolated yet. In the last few years our group
developed 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetra-
hydro-naphthalen-1-yl)propyl]piperazine known as
PB28 (Fig. 1).6,7 This compound displayed high sigma-
2 receptor affinity (Ki = 0.34 nM)3,7 and at present, it
is one of the best sigma-2 ligands known in the literature
so that PB28 is commercially available as a pharmaco-
logical tool for sigma-2 receptor research. To contribute
to the isolation and characterization of sigma-2 recep-
tors, our group modified PB28 and linked it to a station-
Several pharmacological investigations have been car-
ried out both in living cells3 and in guinea-pig ileum
and bladder.9 In all biological assays PB28 displayed
sigma-2 receptor agonist properties. In fact, it showed
cytotoxicity by activating apoptotic pathways through
the Ca++ release by inositol 1,4,5-Tris–phosphate
receptor modulation.10 At present, PB28 is the first sig-
ma-2 receptor ligand displaying high affinity and high
agonist activity towards sigma-2 receptors reported in
the literature. On the other hand, SM-21 is reported
as the best sigma-2 ligand displaying antagonist
activity.11,12
Keywords: Sigma-2 receptors; [3H]PB28.
*
Corresponding author. Tel.: +39 080 5442727; fax: +39 080
5442231; e-mail: colabufo@farmchim.uniba.it
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.12.056

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N. A. Colabufo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2183–2187
Moreover, PB28 has been 11C-radiolabelled as a poten-
tial probe for in vivo PET studies. Unfortunately, 11C-
PB28 failed as a PET probe due to non-specific binding
in the CNS.13
[3H]PB28 was tested in biological assays using standard
protocol16 and its Kd value (0.12 nM) was determined in
rat liver, masking sigma-1 receptors with (+)-pentazocine,
as Koff/Kon ratio (Figs. 3 and 4, respectively). These kinetic
constants were obtained by association experiment
(Kon = 0.1836 0.057 minÀ1 nMÀ1 and t1/2 = 3.77 min)
and dissociation experiment (Koff = 0.0220 0.0096
minÀ1 and t1/2 = 45 min). In each kinetic experiment
120 nM [3H]PB28 was employed with the maximal recep-
tor occupancy obtained at this concentration. Moreover,
as depicted in Figure 5, in the rat liver membranes
[3H]PB28 displayed Kd = 0.13 0.03 nM by saturation
analysis. Furthermore, high sigma-2 receptor density
was found (497 34 fmol/mg of protein) in this tissue.
Kd found by saturation analysis matched with the corre-
sponding Kd found in kinetic experiments (0.13 nM and
0.12 nM, respectively).
In MCF-7/Adr cell line overexpressing sigma-2 receptor,
our compound highly modulated P-gp mRNA level and
also inhibited P-gp activity.14 The unsolved question
was to establish if these effects were sigma-2 receptor
mediated or PB28 per se interfered with P-gp activity.
In order to better elucidate the role of sigma-2 receptors
and the involvement of PB28 in P-gp modulating activ-
ity, we planned to H-label PB28. In the present paper
3
we reported the synthesis and the pharmacodynamic
characterization of [3H]PB28.15
The synthesis of [3H]PB28 is depicted in Scheme 1 and
partially differs from the one previously reported for
nonradiolabelled PB28.6
Starting from these results, competition binding assays
were performed using 12 nM [3H]PB28 and evaluating
the receptor affinity of sigma-2 receptor reference
compounds such as DTG, haloperidol and PB28,
(Fig. 6).
The synthetic strategy was aimed to label the compound
in the final step. The already known derivative 16 was
condensed with cyclohexanone providing the corre-
sponding enamine, which was reduced with NaBT4 (spe-
cific activity 15 Ci/mmol) to the final [3H]PB28. The
enamine reduction by NaBT4 was easy and quantitative.
[3H]PB28 was obtained in high yield after being purified
by column chromatography (silica gel, eluent CHCl3/
MeOH 19:1) and the radiochemical purity (>96%) was
determined by TLC. [3H]PB28 UV–vis spectrascopy
was carried out in order to compare the spectroscopic
properties of unlabelled PB28 with those of labelled
PB28 (Fig. 2A and B), respectively). [3H]PB28 was dis-
solved in absolute EtOH and its specific activity was
determined (8 mCi/mmol).
Kd value of radiolabelled PB28 from kinetic experiments
(0.12 nM) fitted the Ki value of the corresponding unla-
belled PB28 (0.34 nM) already reported.7 Moreover, in
binding competition assays, the reference compounds,
DTG, haloperidol and PB28 displayed Ki values
(42.0 nM, 5.85 nM, 0.57 nM, respectively) superimpos-
able on those determined in standard protocol with
[3H]DTG as radioligand (28.2 nM,17 7.38 nM,3
0.34 nM,17 respectively).
Since binding results of labelled (Kd = 0.12 nM) and
unlabelled PB28 (Ki = 0.34 nM) were comparable, it is
NH
N
N
N
a
O
O
1
b
N
*
*
N
O
2
Scheme 1. Radiosynthesis of [3H]PB28. Reagents: (a) cyclohexanone, TFA; (b) NaBT4.

N. A. Colabufo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2183–2187
2185
Figure 2. (A) Unlabelled 10À3 M PB28 UV spectra (EtOH, kmax 277 nm, e = 1396). (B) 10À3 M [3H]PB28 UV spectra (EtOH, kmax 277 nm, e = 1396).
possible to assess that PB28, both in labelled and in
unlabelled form, selectively binds sigma-2 receptors.
known as [3H]RMH-1. This compound, when tested as
unlabelled compound, displayed moderate sigma-2
receptor affinity (Ki = 10.3 nM) and sigma-1/sigma-2
selectivity ratio >400. When this compound was tested
as [3H]RMH-1, it displayed sigma-2 receptor affinity
Recently, Xu et al.18 reported the radiosynthesis and the
biological evaluation of a selective sigma-2 ligand

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2000
1000
0
Ki SEM (nM)
DTG
42.0 1.0
5.85 0.70
0.57 0.05
Haloperidol
PB28
100
75
50
25
0
0
50
100
Time (min)
150
200
Figure 3. Sigma-2 association experiment.
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
Log[drug]
3000
2000
1000
Figure 6. Inhibition curves in sigma-2 competition binding assay.
ilar value (Kd = 0.12 nM and Ki = 0.57 nM, respec-
tively). However, [3H]PB28 displayed lower selectivity
(about 40-fold) towards sigma-1 receptor (Ki = 13.6 nM)
than [3H]RMH-1.
These results evidenced that [3H]PB28 showed a worse
selectivity towards sigma-1 receptor than [3H]RMH-1,
but the receptor binding profile towards sigma-2 recep-
tor of our radiolabelled compound is more reliable.
0
25
50
75 100 125 150
Time (min)
Figure 4. Sigma-2 dissociation experiment.
These preliminary results prompted us to plan future
experiments on living cells in order to identify the local-
ization of sigma-2 subtypes in the different cell
compartments.
600
500
400
300
In conclusion we have developed an efficient method to
radiolabel PB28 starting from the readily obtainable
intermediate 1 through an easy reductive amination of
the cyclohexanone. The biological results given by
[3H]PB28 and its unlabelled counterpart were perfectly
superimposed.
200
100
0
Kd = 0.13 0.03 nM
Bmax = 497 35 fmol/mg of protein
References and notes
0
10
20
30
40
50
[3H]PB28
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Figure 5. Saturation binding analysis with [3H]PB28 towards sigma-2
receptors in rat liver membranes.
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higher than the unlabelled counterpart (Kd = 0.66 nM).
Moreover, several reference compounds have been
tested for determining their sigma-2 receptor affinity
employing [3H]RMH-1 as radioligand in competition
binding assays. These compounds displayed sigma-2
affinity values comparable to the corresponding results
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to suggest that [3H]DTG and [3H]RHM-1 bound the
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by addition of rat liver membranes (500 lg) in a sample
containing incubation buffer (50 mM Tris–HCl, pH 8.0)
and 120 nM [3H]PB28 in a total volume of 500 lL. The
incubation was performed at 25 °C for various periods of
time (0–125 min) before filtration and then the samples
were filtered and washed. The specific binding was
determined in the presence of 10 lM DTG. Moreover,
1 lM (+)-pentazocine was added in the buffer to mask r1
receptors.
The dissociation kinetic experiment was performed by
equilibrating for 120 min at 25 °C the samples containing
120 nM [3H]PB28 and 500 lg rat liver cell membranes in
50 mM Tris–HCl, pH 8.0, in a total volume of 500 lL.
Moreover, 1 lM (+)-pentazocine was added to mask r1
receptors. At equilibrium, an excess of 10 lM DTG was
added for r2 dissociation experiment and the samples were
filtered and washed.
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Perrone, R. Italian Patent RM2006A000517, 2006.
`
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Contino, M.; Perrone, R.; Tortorella, V. J. Med. Chem.
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K. T.; Mach, R. H. Eur. J. Pharmacol. 2005, 525, 8.
1. Briefly: the association kinetic experiment was initiated