Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium channel blocker for imaging with SPECT

Article abstract of DOI:10.1039/c2ob26695d

Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions. Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one derived VGSC blockers for imaging, we have prepared a ¹²⁵I-labelled analogue of BNZA and evaluated the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive compound blocked the Na_v1.2 and Na_v1.7 VGSC isoforms with IC₅₀ values of 4.1 ± 1.5 μM and 0.25 ± 0.07 μM, respectively. [³H]BTX displacement studies revealed a three-fold difference in affinity between the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging revealed highly localized uptake in the interscapular region, an area typically associated with brown adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the potential of 1-benzazepin-2-ones for in vivo imaging.

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PAPER
Evaluation of a 125I-labelled benzazepinone derived voltage-gated sodium
channel blocker for imaging with SPECT†
Carlos Pérez-Medina,a Niral Patel,a,b Mathew Robson,c Adam Badar,b Mark F. Lythgoeb and Erik Årstad*a
Received 30th April 2012, Accepted 16th October 2012
DOI: 10.1039/c2ob26695d
Voltage-gated sodium channels (VGSCs) are a family of transmembrane proteins that mediate fast
neurotransmission, and are integral to sustain physiological conditions and higher cognitive functions.
Imaging of VGSCs in vivo holds promise as a tool to elucidate operational functions in the brain and to
aid the treatment of a wide range of neurological diseases. To assess the suitability of 1-benzazepin-2-one
derived VGSC blockers for imaging, we have prepared a 125I-labelled analogue of BNZA and evaluated
the tracer in vivo. In an automated patch-clamp assay, a diastereomeric mixture of the non-radioactive
compound blocked the Nav1.2 and Nav1.7 VGSC isoforms with IC50 values of 4.1 1.5 μM and 0.25
0.07 μM, respectively. [3H]BTX displacement studies revealed a three-fold difference in affinity between
the two diastereomers. Iodo-destannylation of a tin precursor with iodine-125 afforded the two
diastereomerically pure tracers, which were used to assess binding to VGSCs in vivo by comparing their
tissue distributions in mice. Whilst the results point to a lack of VGSC binding in vivo, SPECT imaging
revealed highly localized uptake in the interscapular region, an area typically associated with brown
adipose tissue, which in addition to high metabolic stability of the iodinated tracer, demonstrate the
potential of 1-benzazepin-2-ones for in vivo imaging.
conformational change to the inactivated state before reverting
Introduction
back to their resting state.4
Voltage-gated sodium channels (VGSCs) are a family of trans-
membrane proteins that initiate and propagate action potentials
in electrically excitable cells.1 Nine VGSC isoforms (Nav1.1–
Nav1.9) have been reported to date, each characterized by their
distinct pharmacological and electrophysiological properties, as
well as their unique expression patterns. Nav1.1, Nav1.2, Nav1.3
and Nav1.6 are mainly found in the central nervous system,
whereas Nav1.4 and Nav1.5 are expressed in cardiac and skeletal
muscle, and Nav1.7, Nav1.8 and Nav1.9 are principally found in
the peripheral nervous system.2,3 VGSCs are activated by mem-
brane depolarisation which triggers a conformational change
from the resting state to the open state, thereby allowing selective
influx of sodium ions. The channels then undergo rapid
In addition to their role in mediating fast neurotransmission,
VGSCs have been implicated in a number of pathological con-
ditions including neuropathic pain, migraine, epilepsy, multiple
sclerosis and neurodegeneration.5,6 Interestingly, up-regulation of
VGSC expression has also been reported for several types of
cancer,7,8 and is believed to enhance cellular migration and inva-
sion.9 Radiotracers for imaging of VGSCs with positron emis-
sion tomography (PET) and single photon emission computed
tomography (SPECT) therefore hold significant potential as tools
to study activation of neuronal pathways, as well as for diagnos-
tic imaging and treatment monitoring. Despite this potential, the
field of VGSC imaging still remains largely unexplored.10
There are currently no established tracers for imaging of exci-
tatory ion-channels in vivo. However, a number of tracers have
been evaluated for imaging of N-methyl-D-aspartate receptors
(NMDARs), and depending on their binding site, tracer distri-
bution either reflects the activity or the expression pattern of the
ion-channel.11–15 For VGSC tracers it is likely that both ion-
channel expression and function can influence the binding
pattern, at least for state-dependent ligands.4 In addition, the
subtype selectivity of a tracer will dictate its potential appli-
cations. Yet, the high degree of homology between VGSC iso-
forms makes development of subtype selective ligands
challenging, and at least for small molecules, the potency is
aDepartment of Chemistry and Institute of Nuclear Medicine, UCL, 235
Euston Road (T-5), NW1 2BU London, United Kingdom.
E-mail: e.arstad@ucl.ac.uk; Fax: +44 (0)02076792344;
Tel: +44 (0)02076792344
bCentre for Advanced Biomedical Imaging, Department of Medicine and
UCL Institute of Child Health, 72 Huntley Street, WC1E 6DD London,
United Kingdom
cTumour Biology Section, UCL Cancer Institute, 72 Huntley Street,
WC1E 6DD UCL London, United Kingdom
†Electronic supplementary information (ESI) available: Synthetic pro-
cedures, selected 1H and 13C NMR spectra and selected radio-HPLC
chromatograms. See DOI: 10.1039/c2ob26695d
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Cove Road, Billerica, MA 01862, USA) as a non-carrier-added
solution in reductant free 10−5 M aqueous sodium hydroxide
solution (pH 8–11).
Radio-HPLC
HPLC purification and analytical runs were carried out on a C18
Agilent Eclipse Plus column (4.6 × 150 mm, 5 μm) and moni-
tored with a 254 UV detector and a Raytest Gabi NaI detector.
The solvent systems used were water (0.1% TFA, solvent A) and
Fig. 1 Structure of BNZA.
often similar for many, if not all, of the Nav isoforms.4 The
binding of VGSC tracers in vivo is therefore likely to broadly
reflect VGSC expression in the periphery as well as in the brain,
with state-dependent ligands showing increased uptake in tissues
with high electrical activity.
methanol (0.1% TFA, solvent B) with a flow rate of 1 mL min−1
.
Metabolite analysis was carried out on a C18 Agilent XDB
column (4.6 × 150 mm, 5 μm) using water (0.1% formic acid,
solvent A) and methanol (0.1% formic acid, solvent B) as
The 1-benzazepin-2-one derivative [3H]BNZA (Fig. 1) was
recently reported to be a highly potent and state-dependent
VGSC blocker that binds with low nanomolar affinity to rat
solvent systems with a flow rate of 1 mL min−1
.
brain synaptosomal membranes (Kd = 1.53 0.46 nM, Bmax
=
Log D determination
3.4 1.2 pmol mg−1 of protein) as well as to transfected human
hNav1.5 (Kd 0.97 nM) and hNav1.7 (Kd 1.6 nM) VGSC iso-
forms.16 Furthermore, analogues of BNZA have been shown to
have good metabolic stability, and their anticonvulsant efficacy
in vivo implies that derivatives of this compound class can pene-
trate the blood-brain barrier.17 Taken together, the results suggest
that 1-benzazepin-2-ones meet many of the key criteria for tracer
development, and that suitably radiolabelled derivatives of
BNZA may enable imaging of VGSCs. To assess the suitability
of 1-benzazepin-2-one derived VGSC blockers for imaging, we
have prepared a 125I-labelled analogue of BNZA and evaluated
the tracer in vivo.
Log D7.4 of [125I]h-9 was determined using the n-octanol shake
flask method.18 Briefly, a solution of radioligand in n-octanol
(500 μL, presaturated with PBS 7.4) was mixed with an equal
volume of PBS 7.4 (presaturated with n-octanol) and shaken for
15 minutes at 900 rpm, after which time the mixture was centri-
fuged at 10 000 rpm for 5 minutes. The radioactivity present in
both layers was measured in a gamma counter, and the
n-octanol/PBS partition coefficient determined by dividing the
radioactivity found in the n-octanol layer by that found in the
PBS layer. The log value of this coefficient was then calculated.
The process was repeated until a consistent value was obtained.
The experiments were carried out in quadruplicate.
Materials and methods
Animals
Chemistry
Female Balb/C mice were obtained from Charles River, UK.
After arrival, the mice were allowed to acclimatise for at least 5
days in a room with constant temperature (21 °C) and humidity
(30%). All efforts were made to reduce the number of animals
used, and all experiments were conducted in accordance with the
UK Animals (Scientific Procedures) Act, 1986 and the European
Community Council Directive, 1986.
All reagents were purchased from Sigma-Aldrich, except for
3-bromo-1,3,4,5-tetrahydro-benzo[b]azepin-2-one (1) which was
purchased from Fluorochem, and were used without further
purification. The synthetic procedures and analytical data for the
compounds described herein can be found in the ESI.† Column
chromatography was performed on silica-gel (VWR BDH-Pro-
labo 40–63 μm). 1H and 13C NMR spectra were recorded at
room temperature on Bruker Avance 300 or 500 instruments
1
operating at the frequency of 300 or 500 MHz for H, and 75 or
Metabolite analysis
125 MHz for 13C. All were internally referenced to the residual
solvent peaks, CDCl3 (7.26 ppm), DMSO-d6 (2.49 ppm) or
CD3OD (3.31 ppm) for 1H, CDCl3 (δ 77.0 ppm), DMSO-d6
(39.5 ppm) or CD3OD (49.1 ppm) for 13C. High resolution mass
data were recorded on a thermo Finnigan MAT900xp (CI/EI), a
Waters LCT Premier XE (ES) or a VG70-SE (FAB) mass spec-
trometers. HPLC analysis was performed with an Agilent 1200
HPLC system equipped with a 1200 Series Diode Array Detector
and Fluorescence Detector. The specific rotation ([α]2D0, c = 1.0,
MeOH) of amine 3 was measured using a Perkin Elmer 241
polarimeter.
Female Balb/C mice (6–10 weeks old and 15–20 g of weight)
were injected with 2.4–3.4 MBq [125I]h-9 in 120–300 μL saline
solution (5% ethanol) via their lateral tail vein. The mice were
anesthetized with isoflurane (5% mixed with medical air at a
flow of 2 mL min−1). At 5, 15, 30 and 60 min post injection
blood was drawn out by cardiac puncture and, immediately after-
wards, animals were sacrificed by cervical dislocation and brains
removed. Blood was collected into heparised tubes and centri-
fuged at 13 000 rpm for 1 min to separate plasma; 300 μL of
plasma were mixed with 1200 μL of cold acetonitrile, vortexed
and centrifuged at 13 000 rpm for 1 min. The supernatant was
then transferred to a glass vial and mixed with 900 μL of water.
Brain tissues were mixed with 1.5 mL acetonitrile–water 2 : 1
and homogenized, vortexed and centrifuged at 13 000 rpm for
1 min. The resulting supernatant was then mixed with 500 μL of
Radiochemistry
[
125I]NaI (>12.95 GBq mL−1, 629 GBq mg−1) was purchased
from Perkin Elmer Life and Analytical Sciences (331 Treble
This journal is © The Royal Society of Chemistry 2012
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water and the resulting solution was centrifuged again as
described above. Pellets and supernatants were separated and
counted for radioactivity to determine recovery efficiency. An
aliquot (1000 μL) of the supernatant obtained from the plasma
and brain extracts was subjected to reversed-phase high-perform-
ance liquid chromatography (HPLC) analysis (Agilent C18 XDB
column, 4.6 × 150 mm, 5 μm) using gradient elution from 50 to
90% solvent B over 15 minutes at a flow rate of 1 mL min−1
.
The recovery from plasma was measured to be 93.4 1.0% (n =
3) and from brain 80.8 0.1% (n = 3). Sample workup was iden-
tical as described above. Results are expressed as percentages of
the total activity S.D.
Biodistribution studies
Biodistribution experiments were conducted on female Balb/C
mice (6–10 weeks old and 15–20 g of weight). The radiotracer
(0.2–0.4 MBq in 150–300 μL saline solution (5% ethanol)) was
administered via the lateral tail vein. The mice were anesthetized
with isoflurane (5%) mixed with medical air at a flow rate of
2 mL min−1 at predetermined time points after administration (5,
15, 30 and 60 min for [125I]h-9 and 5 and 30 min for [125I]l-9),
blood was sampled through cardiac puncture, and the animals
were then sacrificed by cervical dislocation. The radioactivity
content in tissues of interest, (large and small intestine, stomach,
kidneys, brain, bone (femur), liver, lungs, heart, skin, spleen,
bladder, tail) was measured using a Wizard2 2470 Automatic
Gamma Counter (Perkin Elmer). For each time point, 4 mice
were used and the radioactivity uptake was calculated as the
mean percentage injected dose per gram tissue S.D.
Scheme 1 Synthesis of BNZA derivatives 7–9.
The racemic mixture of 3 was resolved by selective precipi-
tation of the chiral D-pyroglutamate salt of the R enantiomer.20
The recovery was 68% and the enantiomeric excess of the free
amine 3 > 96%, as determined by polarimetry.21 Once isolated,
the enantiomerically enriched amine 3 was tritylated to give 4.
Alkylation with isopropyl iodide in DMF provided the N-isopro-
pyl lactam 5 in 61% yield. After deprotection of the trityl group
with p-toluenesulfonic acid in methanol, the amine 6 was reacted
with racemic N-Boc-DL-3-fluorophenylalanine using BOP as
coupling agent to afford amide 7 in 88% yield. Removal of the
Boc group with trifluoroacetic acid in dichloromethane provided
the amine 8 in near quantitative yield. Finally, coupling of 8
with 2-iodobenzoic acid in the presence of BOP provided the
non-radioactive reference compound 9 in 83% yield. Since
racemic N-Boc-DL-3-fluorophenylalanine was used, compounds
7, 8 and 9 were obtained as a mixture of diasteroisomers.
Enriched fractions of both diastereomers of 9 were obtained by
column chromatography, and excellent diastereomerical purity
was achieved after separation by semi-preparative HPLC. The
two diastereomers of 9 were termed high-9 (h-9), and low-9 (l-9)
according to their relative affinities (see below). The absolute
configuration around the α-carbon of the 3-fluorophenylalanine
moiety was not determined for this study.
SPECT imaging
Female Balb/C mice (4–10 weeks old) were injected with 20–
35 MBq [125I]h-9 or [125I]l-9 in 200–300 μL saline solution
(5–7% ethanol, 0.1% TWEEN-80) via the lateral tail vein and
then anesthetized with isofluorane mixed with medical air (5%
for induction, 2% for maintenance). Animals were immediately
placed in prone position on a heated bed (37 °C) under isofluor-
ane anesthesia and scans were then performed using a Nano-
SPECT/CT small animal in vivo scanner (Bioscan Inc., 4590
MacArthur Blvd., NW, Washington D.C. 20007, USA). Whole
body scans consisted of sixteen projections of varying duration
and CT were recorded once the SPECT scans were finished.
SPECT Images were reconstructed using HiSPECT software and
in vivo Scope software was used for CT image reconstruction.
Results
The tin precursor for radiolabelling with iodine-125 was pre-
pared from amine 8 by reaction with the active ester 12, which
in turn was obtained from acid 10 after a two-step synthesis.
2-iodobenzoic acid (10) was treated with TSTU in DMF to
produce the succinimidyl ester 11, which upon reaction with
hexamethylditin in the presence of palladium (0) furnished the
stannated ester 12 in 70% yield. Subsequent reaction of 12 with
amine 8 afforded the desired tin precursor 13 in a moderate 46%
yield (Scheme 2).
Chemistry
A nine step synthesis based on modified literature procedures19
was followed leading to the preparation of compound 9 in 9%
overall yield (Scheme 1). Racemic 3-bromo-1,3,4,5-tetrahydro-
benzo[b]azepin-2-one (1) was reacted with sodium azide in the
presence of sodium iodide in DMF to afford azide 2, which was
subsequently converted via a Staudinger reaction to provide
amine 3.
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was measured to be 3.93 0.01 (n = 4) using the n-octanol
shake flask method.18
Biodistribution studies
The distribution of radioactivity was measured after i.v. injection
of both diastereoisomers of [125I]9 in female Balb/C mice at pre-
determined time points (5, 15, 30 and 60 minutes for [125I]h-9,
and 5 and 30 min for [125I]l-9). Table 1 shows the uptake in
selected tissues. Initially, a high uptake in the liver and kidneys
was observed for [125I]h-9 with later time points dominated by
high uptake in the intestines. The brain uptake was moderate
(0.92% ID/g for [125I]h-9 at 5 min post-injection) and gradually
decreased over time. Blood clearance was slow, with 7.17
1.10% ID/g at 5 min post-injection and 3.25 0.45% ID/g at
60 min, resulting in poor brain-to-blood ratios values at all time
points. The tissue distribution of [125I]l-9 was similar to that of
Scheme 2 Synthesis of precursor 13 and radiolabelling of [125I]9.
In vitro evaluation
[
125I]h-9, with high uptake in liver and kidneys at 5 min post-
injection, and high uptake in the intestines at 30 min. The brain
uptake was lower than that of [125I]h-9 at both time points inves-
tigated (0.68 vs. 0.92% ID/g at 5 min), and blood clearance was
slightly faster, resulting in similar brain-to-blood ratios for both
diastereomers.
The diastereomeric mixtures of compounds 7 and 9 were evalu-
ated as VGSC blockers against the cloned human Nav1.2
(hNav1.2, SCN2A gene) and Nav1.7 (hNav1.7, SCN9A gene) iso-
forms expressed in CHO cells by means of automated patch-
clamp as described elsewhere (ChanTest Corp., USA).22 The
IC50 values for compound 9 were 4.1 1.5 μM and 0.25
0.07 μM for Nav1.2 and Nav1.7, respectively. For comparison,
the IC50 values for compound 7 were 0.52 0.2 μM (Nav1.2)
and 0.13 0.02 μM (Nav1.7). As an indirect way to measure the
relative affinities of both diastereoisomers of 9, a receptor [3H]
BTX binding assay (Ricerca Taiwan Ltd, Taiwan) was carried
out.23 Compound h-9 displaced [3H]BTX with an IC50 compar-
able to that of 7 (0.151 vs. 0.101 μM), whereas l-9 had three
times lower affinity than its diastereoisomer (IC50 = 0.484 μM).
Metabolite analysis
To assess the metabolic stability of [125I]h-9, the composition of
radioactive species in plasma and brain tissue was analyzed by
radio-HPLC over a period of one hour after injection. [125I]h-9
was found to have excellent stability in both plasma and brain,
and was the predominant radioactive species at all time points.
In plasma, the fraction of the intact tracer [125I]h-9 was 93% at
5 min post-injection, 70% at 30 min, and 58% at 60 min (n = 2)
(Fig. 2). Three metabolites were identified, all more polar than
the parent compound. In the brain, the intact tracer [125I]h-9 was
the only radioactive species found at the earlier times points
(5, 10 and 15 min), with some minor metabolites (20%, n = 2)
occurring 60 min after administration.
Radiochemistry
Treatment of the trimethyltin precursor 13 with [125I]NaI in the
presence of 0.1 M HCl and 1.4% H2O2 for 30 min at 60 °C pro-
vided [125I]9 in 24
4% (n = 11) radiochemical yield with
specific activity of 37.0 10.4 GBq μmol−1 (n = 8) (Scheme 2).
The two diastereisomeric products of [125I]9, h-9 and l-9, were
separated by radio-HPLC with excellent radiochemical (>99%)
and diastereomeric (>99%) purities. The log D7.4 of [125I]h-9
Imaging
Whole body SPECT/CT scans were recorded for both diastereo-
isomers of [125I]9 in female Balb/C mice. Overall, the SPECT
Table 1 Tissue distribution of [125I]h-9 and [125I]l-9 expressed as % ID/g SD (n ≥ 3)
125I]h-9
[
[
125I]l-9
5 min
15 min
30 min
60 min
5 min
30 min
Blood
Heart
Liver
Kidneys
Brain
7.17 1.10
7.84 1.20
36.10 2.1
24.40 1.0
0.92 0.08
7.98 0.85
3.47 0.18
10.10 1.7
2.11 0.56
9.89 1.03
4.42 0.77
4.66 0.23
4.27 0.70
27.50 2.8
12.80 0.9
0.49 0.11
6.89 0.93
2.53 0.58
5.78 0.61
1.27 0.36
10.60 1.8
4.60 1.0
5.60 0.21
6.01 0.81
43.40 3.4
17.80 1.7
0.59 0.07
6.28 0.46
4.30 1.3
6.94 0.58
1.76 0.81
39.50 2.5
10.20 1.3
3.25 0.45
2.96 0.38
18.80 1.3
7.34 0.77
0.26 0.03
4.51 0.68
1.71 0.26
2.71 0.23
1.97 0.76
25.60 2.7
12.6 2.4
6.89 0.81
8.78 1.84
40.60 3.5
32.70 2.9
0.68 0.10
9.10 1.52
4.05 0.56
10.10 1.7
5.09 3.48
9.63 1.02
6.39 1.68
4.05 0.55
4.27 0.70
31.70 3.9
20.30 3.2
0.34 0.05
4.86 0.69
2.41 0.36
6.33 0.83
3.41 1.33
33.80 8.2
7.38 1.7
Lungs
Muscle
Spleen
Bone
Small Int.
Stom./L. Int.
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Discussion
We have prepared the novel radioiodinated 1-benzazepin-2-one
125I]9 with the aim to evaluate the suitability of this compound
[
class for imaging of VGSCs. Previously reported binding studies
with [3H]BNZA using rat brain synaptosomal membranes
suggest that radiolabelled 1-benzazepin-2-ones can be developed
with sufficient affinity and binding potential for in vivo
imaging.16 Our primary goal was therefore to assess the organ
distribution and metabolic stability of [125I]9 over a time frame
relevant for imaging with PET and SPECT.
Iodine-125 was chosen for this study as it enables straightfor-
ward and reliable labelling by iodo-destannylation, and the long
half-life (60 days) facilitates rapid biological evaluation. In
addition, previously reported SAR studies with 1-benzazepin-2-
ones revealed that benzamide derivatives bearing bulky lipophi-
lic substituents in the 2-position, such as tert-butyl and trifluoro-
methyl, are well tolerated in the binding site.24 As iodine is
comparable in size and polarity, we envisaged that incorporation
of a 2-[125I]iodobenzamide moiety as in [125I]9 would allow lab-
elling whilst retaining the biological activity of the lead com-
pound 7. However, the use of radioiodine for evaluation of small
molecule tracers can be problematic as it results in high lipophili-
city, a significant increase in molecular weight, and often poor
metabolic stability. Despite these disadvantages, radioiodine has
proven useful for evaluation of scaffolds as putative PET tracers,
for instance to identify ligands with the required binding proper-
ties in biological tissues, and to assess biodistribution and meta-
bolic stability.25,26
Fig. 2 HPLC radioactivity profiles of plasma samples at 0, 15, 30 and
60 min after injection of [125I]h-9.
Fig. 3 SPECT/CT images showing the distribution of [125I]h-9 (top)
and [125I]l-9 (bottom) in mice.
The non-radioactive target compound 9 was prepared in 9%
overall yield following a nine step synthesis. Coupling of the
enantiomerically enriched amine 6 with racemic N-Boc-DL-3-
fluorophenylalanine yielded a diastereomeric mixture of the pre-
viously reported VGSC blocker 7. Deprotection of the BOC
group and coupling of the resulting amine 8 with 2-iodobenzoic
acid (2) provided the iodinated target compound 9. Purification
with semi-preparative HPLC allowed isolation of both diastereo-
mers of 9 with excellent diastereomerical purity.
To assess the potency of 9 as a VGSC blocker the diastereo-
meric mixtures of 7 and 9 were evaluated in an automated patch-
clamp assay (IWQ) against the human Nav1.2 (hNav1.2, SCN2A
gene) and Nav1.7 (hNav1.7, SCN9A gene) isoforms expressed in
CHO cells.22 Whereas the potency of 9 was largely retained
against the Nav1.7 isoform (0.25 0.07 μM vs. 0.13 0.02 μM
for 7), the structural modifications were less well tolerated for
Nav1.2 (4.1 1.5 μM vs. 0.52 0.2 for 7). It is interesting to
note that incorporation of the 2-iodobenzamide moiety increased
the selectivity for hNav1.7 over Nav1.2 from 4-fold for com-
pound 7 to 16-fold for compound 9. BNZA and the analogue 7
have previously been reported to be equipotent in a FRET assay
with an IC50 of 0.03 0.02 μM against hNav1.7.16 The discre-
pancy between our results and the previously reported blocking
efficiency for compound 7 is most likely due to the use of auto-
mated patch-clamp (IWQ) instead of FRET as the read-out, as
well as the use of a diastereomeric mixture of 7 in this study.
However, it should be noted that data obtained with IWQ can
differ significantly from manual patch-clamp readings when
using test compounds with high lipophilicity, and this may have
affected the IC50 values obtained for 7 and 9.22
Fig. 4 Lateral (left) and anterior/posterior (right) SPECT/CT images of
the uptake of [125I]h-9 in the interscapular region at 55–65 min post-
injection.
images (Fig. 3) were in good agreement with the biodistribution
data, showing a high initial uptake of radioactivity in the liver
(15–25 min post-injection). At the later time points (35–55 min
post-injection) the images were dominated by high levels of
activity in the intestines. Interestingly, in three out of a total of
seven mice imaged with SPECT, a high uptake was observed in
the interscapular region (Fig. 4). The uptake in this region
appeared to be age related, as it was observed in adult mice
(8–10 weeks old, [125I]h-9 (n = 2) and [125I]l-9 (n = 1)), but not
in a group of younger animals (4–6 weeks, [125I]h-9 (n = 2) and
[
125I]l-9 (n = 2)). However, other factors may be involved, and
further studies are required to determine the reason why the
signal in the interscapular region was only observed in some,
and not all, of the animals investigated. Unfortunately, the
activity levels were too low to allow SPECT imaging of the
regional distribution of [125I]9 in the brain.
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Since the VGSC blocker WIN17317-3 has been reported to
displace both batrachotoxin ([3H]BTX)27 and [3H]BNZA16 from
their respective binding sites, we performed a [3H]BTX displace-
ment study as an indirect way to measure the relative affinities of
the two diastereoisomers of 9. The diastereomer with the shortest
retention on HPLC displaced [3H]BTX with an IC50 of
0.151 μM, whereas the IC50 of the slow eluting diastereomer
was 0.484 μM. In comparison, the diastereomeric mixture of 7
was found to have an IC50 of 0.101 μM. The diastereomers of 9
were then termed high affinity 9, h-9, and low affinity 9, l-9,
according to their relative affinities. Whilst the IC50 values for 7
and 9 are well above the low nanomolar affinity range typically
required for in vivo imaging, the data cannot be directly related
to binding affinities as the relationship with blocking potency
and [3H]BTX displacement are complex.
administration in mice. The distribution of both diastereomers
was similar, and pointed to a lack of VGSC binding in vivo.
However, a highly localized uptake in the interscapular region,
and high metabolic stability of the iodinated tracer, demonstrate
the potential of 1-benzazepin-2-ones for in vivo imaging. Further
development of this compound class as VGSC tracers is there-
fore warranted, and we are currently investigating strategies for
labelling 1-benzazepin-2-ones with fluorine-18.
Acknowledgements
This work was undertaken at UCLH/UCL who received a pro-
portion of funding from the Department of Health’s NIHR Bio-
medical Research Centres funding scheme (E. Årstad and
C. Pérez-Medina). We also acknowledge support from the
Medical Research Council (N. Patel) and King’s College
London and UCL Comprehensive Cancer Imaging Centre
CR-UK & EPSRC, in association with the MRC and DoH
(England) (M.F. Lythgoe).
The two radiolabelled diastereomers [125I]h-9 and [125I]l-9
were obtained in moderate radiochemical yield by treatment of
the tin precursor 13 with [125I]NaI in the presence of hydrogen
peroxide under acidic conditions. As lipophilicity affects brain
uptake, metabolism and protein binding, we determined the log
D7.4 of [125I]h-9 using the n-octanol shake flask method. The
high lipophilicity of [125I]h-9 (log D7.4 of 3.93 0.01) is a limi-
tation with this study as it is likely to impair brain uptake, and
result in low specific binding and high plasma protein binding
in vivo.
Notes and references
1 W. A. Catterall, Neuron, 2000, 26, 13–25.
2 F. H. Yu and W. A. Catterall, Genome Biology, 2003, 4, 207.
3 A. L. Goldin, Annu. Rev. Physiol., 2001, 63, 871–894.
4 T. Anger, D. J. Madge, M. Mulla and D. Riddall, J. Med. Chem., 2001,
44, 115–137.
5 M. Mantegazza, G. Curia, G. Biagini, D. S. Ragsdale and M. Avoli,
Lancet Neurol., 2010, 9, 413–424.
6 S. D. Dib-Hajj, T. R. Cummins, J. A. Black and S. G. Waxman, Annu.
Rev. Neurosci., 2010, 33, 325–347.
7 J. K. Diss, S. N. Archer, J. Hirano, S. P. Fraser and M. B. Djamgoz, Pros-
tate, 2001, 48, 165–178.
8 D. Diaz, D. M. Delgadillo, E. Hernandez-Gallegos, M. E. Ramirez-Dom-
inguez, L. M. Hinojosa, C. S. Ortiz, J. Berumen, J. Camacho and
J. C. Gomora, J. Cell. Physiol., 2007, 210, 469–478.
9 W. J. Brackenbury, M. B. Djamgoz and L. L. Isom, Neuroscientist, 2008,
14, 571–583.
10 No papers have yet been published on tracers for imaging of VGSCs,
however, a patent has been filed (Arstad, E. US2010247435 (A1), 2007)
and we and others have published conference abstracts: C. Pérez-Medina,
et al., J. Label. Compd. Radiopharm., 2011, 54(S1), S287–S288;
A. Hoehne, et al., J. Labelled Compd. Radiopharm., 2011, 54, S1–S2.
11 J. Owens, A. A. Tebbutt, A. L. McGregor, K. Kodama, S. S. Magar,
M. E. Perlman, D. J. Robins, G. J. Durant and J. McCulloch, Nucl. Med.
Biol., 2000, 27, 557–564.
In biodistribution studies, [125I]h-9 and [125I]l-9 behaved simi-
larly, with the early time points dominated by uptake by the liver
and kidneys. The brain uptake was moderate (0.92% ID/g 5 min
post-injection) and gradually decreased over time. At later time
points the majority of the activity was found in the intestines,
consistent with hepatic clearance. There were no noteworthy
differences in the tissue distributions of the two diastereomers of
[
125I]9, and the lack of retention in the brain implies that no
specific binding to VGSC channels was achieved. Surprisingly,
whilst the SPECT images overall confirmed the results from the
biodistribution studies, in the older animals (8–10 weeks) a high
uptake was observed in the interscapular region in what appears
to be a brown adipose tissue (BAT) depot. BAT has no known
expression of VGSCs,28 and the high uptake of [125I]9 in the
interscapular region is therefore most likely caused by a physio-
logical effect, or potentially, cross activity with an unknown
protein. Nevertheless, the highly localized uptake of [125I]9 in
the interscapular region demonstrates the potential of 1-benzaze-
pin-2-ones for in vivo imaging, provided that sufficient VGSC
binding affinity can be achieved. Whilst the brain uptake of
12 J. M. Stone, K. Erlandsson, E. Årstad, R. A. Bressan, L. Squassante,
V. Teneggi, P. J. Ell and L. S. Pilowsky, Nucl. Med. Biol., 2006, 33, 239–
243.
13 R. J. J. Knol, K. De Bruin, B. L. F. Van Eck-smit, S. Pimlott, D. J. Wyper
and J. Booij, Synapse, 2009, 63, 557.
14 E. G. Robins, Y. J. Zhao, I. Khan, A. Wilson, S. K. Luthra and E. Årstad,
Bioorg. Med. Chem. Lett., 2010, 20(5), 1749–1751.
15 F. Sobrio, G. Gilbert, C. Perrio, L. Barré and D. Debruyne, Mini Rev.
Med. Chem., 2010, 10(9), 870–886.
[
125I]9 is moderate, it is likely that this at least in part is due to
the high lipophilicity (log D7.4 3.93 0.01) and high molecular
weight (MW 612.45) of 9.
16 B. S. Williams, J. P. Felix, B. T. Priest, R. M. Brochu, K. Dai, S. B. Hoyt,
C. London, Y. S. Tang, J. L. Duffy, W. H. Parsons, G. J. Kaczorowski and
M. L. Garcia, Biochemistry, 2007, 46, 14693–14703.
17 S. B. Hoyt, C. London, M. J. Wyvratt, M. H. Fisher, D. E. Cashen,
J. P. Felix, M. L. Garcia, X. Li, K. A. Lyons, M. D. Euan, W. J. Martin,
B. T. Priest, M. M. Smith, V. A. Warren, B. S. Williams,
G. J. Kaczorowski and W. H. Parsons, Bioorg. Med. Chem. Lett., 2008,
18, 1963–1966.
18 E. Arstad, S. Platzer, A. Berthele, L. S. Pilowsky, S. K. Luthra,
H. J. Wester and G. Henriksen, Bioorg. Med. Chem., 2006, 14, 6307–
6313.
19 S. B. Hoyt, C. London, D. Gorin, M. J. Wyvratt, M. H. Fisher,
C. Abbadie, J. P. Felix, M. L. Garcia, X. Li, K. A. Lyons, E. McGowan,
Conclusions
We have synthesized and radiolabelled a novel iodinated 1-ben-
zazepin-2-one in order to evaluate the suitability of this com-
pound class for imaging of VGSCs. The non-radioactive target
compound was obtained as a pair of diastereomers. Iodo-destan-
nylation of a tin precursor with iodine-125 afforded the diaster-
eomerically pure tracers, which allowed us to assess binding to
VGSCs in vivo by comparing their tissue distributions after
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 9474–9480 | 9479

View Article Online
D. E. Macintyre, W. J. Martin, B. T. Priest, A. Ritter, M. M. Smith,
V. A. Warren, B. S. Williams, G. J. Kaczorowski and W. H. Parsons,
Bioorg. Med. Chem. Lett., 2007, 17, 4630–4634.
24 S. B. Hoyt, C. London, H. Ok, E. Gonzalez, J. L. Duffy, C. Abbadie,
B. Dean, J. P. Felix and M. L. Garcia, et al., Bioorg. Med. Chem. Lett.,
2007, 17, 6172–6177.
20 J. D. Armstrong III, K. K. Eng, J. L. Keller, R. M. Purick, F. W. Hartner
Jr., W. Choi, D. Askin and R. P. Volante, Tetrahedron Lett., 1994, 35,
3239–3242.
25 Z. P. Zhuang, M. P. Kung, C. Hou, D. M. Skovronsky, T. L. Gur,
K. Plössl, J. Q. Trojanowski, V. M. Y. Lee and H. F. Kung, J. Med.
Chem., 2001, 44, 1905–1914.
21 W. R. Schoen, J. M. Pisano, K. Prendergast, M. J. Wyvratt Jr.,
M. H. Fisher, K. Cheng, W. W. Chan, B. Butler, R. G. Smith and
R. G. Ball, J. Med. Chem., 1994, 37, 897–906.
22 B. A. Wible, Y. A. Kuryshev, S. S. Smith, Z. Liu and A. M. Brown,
Assay Drug Dev. Technol., 2008, 6, 765–780.
26 G. Smith, M. Glaser, M. Perumal, Q. D. Nguyen, B. Shan, E. Årstad and
E. O. Aboagye, J. Med. Chem., 2008, 51(24), 8057–8067.
27 S. G. Wanner, H. Glossmann, H. G. Knaus, R. Baker, W. Parsons,
K. M. Rupprecht, R. Brochu, C. J. Cohen, W. Schmalhofer, M. Smith,
V. Warren, M. L. Garcia and G. J. Kaczorowski, Biochemistry, 1999, 38,
11137–11146.
23 W. A. Catterall, C. S. Morrow, J. W. Daly and G. B. Brown, J. Biol.
Chem., 1981, 256, 8922–8927.
28 B. Cannon and J. Nedergaard, Physiol. Rev., 2004, 84, 277–359.
9480 | Org. Biomol. Chem., 2012, 10, 9474–9480
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