Synthesis and evaluation of a 125I-labeled iminodihydroquinoline-derived tracer for imaging of voltage-gated sodium channels

Article abstract of DOI:10.1016/j.bmcl.2013.07.014

In vivo imaging of voltage-gated sodium channels (VGSCs) can potentially provide insights into the activation of neuronal pathways and aid the diagnosis of a number of neurological diseases. The iminodihydroquinoline WIN17317-3 is one of the most potent s

Full text of DOI:10.1016/j.bmcl.2013.07.014

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5170–5173
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of a 125I-labeled iminodihydroquinoline-
derived tracer for imaging of voltage-gated sodium channels q
Carlos Pérez-Medina a, Niral Patel a,b, Mathew Robson c, Mark F. Lythgoe b, Erik Årstad a,
⇑
a Department of Chemistry and Institute of Nuclear Medicine, UCL, 235 Euston Road (T-5), London NW1 2BU, United Kingdom
b Centre for Advanced Biomedical Imaging, UCL, 72 Huntley Street, London WC1E 6BT, United Kingdom
c UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6BT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
In vivo imaging of voltage-gated sodium channels (VGSCs) can potentially provide insights into the acti-
vation of neuronal pathways and aid the diagnosis of a number of neurological diseases. The iminodihy-
droquinoline WIN17317-3 is one of the most potent sodium channel blockers reported to date and binds
with high affinity to VGSCs throughout the rat brain. We have synthesized a 125I-labeled analogue of
WIN17317-3 and evaluated the potential of the tracer for imaging of VGSCs with SPECT. Automated patch
clamp studies with CHO cells expressing the Nav1.2 isoform and displacement studies with [3H]BTX
yielded comparable results for the non-radioactive iodinated iminodihydroquinoline and WIN17317-3.
However, the 125I-labeled tracer was rapidly metabolized in vivo, and suffered from low brain uptake
and high accumulation of radioactivity in the intestines. The results suggest that iminodihydroquinolines
are poorly suited for tracer development.
Received 3 June 2013
Revised 10 July 2013
Accepted 11 July 2013
Available online 18 July 2013
Keywords:
SPECT
Voltage-gated sodium channel
WIN17317-3
Iodine-125
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
Imaging
Voltage-gated sodium channels (VGSCs) are a family of trans-
membrane ion-channels responsible for the rising phase of action
potentials in electrically excitable cells.1 Depolarization results in
lamotrigine (1) and phenytoin (2) (Fig. 1), that can modulate the
gating of sodium channels, are used as antiepileptic drugs (AEDs).5
Demyelinated sclerotic lesions overexpress VGSCs, and the result-
ing excessive influx of sodium ions is believed to cause axonal
injury,12 which underpins the progression and symptoms of multi-
ple sclerosis (MS).13,14 Interestingly, VGSC blockers have shown
efficacy as neuroprotecting agents in animal models of MS,15,16
for treatment of migraine, as well as for psychiatric conditions such
as schizophrenia, post-traumatic stress disorder and bipolar affec-
tive disorder.5,17
Development of radiotracers for noninvasive imaging of VGSCs
with positron emission tomography (PET) and single photon emis-
sion computed tomography (SPECT) can potentially provide tools
to study activation of neurological pathways, to investigate patho-
logical processes, to diagnose disease, and to monitor the response
to treatment.
a
conformational change of the channel from the resting
non-conducting state to the open conducting state, which leads
to selective influx of sodium ions. The ion channel then undergoes
a further conformational change to the inactivated state, before it
reverts back to its resting state.2 VGSCs can rapidly cycle through
the resting, open and inactivated states, allowing neurons to fire
the high-frequency trains of action potentials that are essential
for fast neurotransmission. To date nine distinct alpha subunits
of VGSCs (Nav1.1–Nav1.9) have been reported,3 each with their
own distinct expression pattern in the central (CNS) and peripheral
(PNS) nervous systems.4
VGSCs play a pivotal role in maintaining physiological condi-
tions and higher cognitive functions, but also mediate hyperexcita-
tion and neurodegeneration, and their dysfunction is implicated in
a number of neurological diseases.5–7 Anomalous high-frequency
repetitive spike firing, for instance, is known to take place during
the spread of epileptic seizures,8 and there is an increasing body
of evidence linking VGSC mutations with neuronal hyperexcitabil-
ity in epilepsy.9–11 In fact, state-dependent VGSC blockers such as
O
N
H2N
N
N
NH2
HN
N
NH
O
Cl
Cl
q
This is an open-access article distributed under the terms of the Creative
Cl
Ph
Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are credited.
1
2
WIN17317-3
⇑
Corresponding author. Tel./fax: +44 (0)02076792344.
E-mail address: e.arstad@ucl.ac.uk (E. Årstad).
Figure 1. Structures of the state-dependent VGSC blockers lamotrigine (1),
phenytoin (2) and WIN17317-3.
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.014

C. Pérez-Medina et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5170–5173
5171
WIN17317-3 (Fig. 1) was first reported to be a selective voltage-
gated potassium channel blocker.18,19 However, in a subsequent
study the binding of [3H]WIN17317-3 to rat brain synaptosomal
membranes (Kd 2.2 0.3 nM, Bmax 5.4 0.2 pmol/mg of protein)
was shown to be insensitive to a number of potassium channel
modulators and, on the contrary, could be blocked by several so-
dium channel ligands.20 Moreover, WIN17317-3 was found to inhi-
bit sodium currents in CHO cells transfected with Nav1.2 (Ki 9 nM),
and also blocked muscle sodium channels, and to a lesser degree
sodium channels of the heart. Importantly, autoradiography of
rat brain sections incubated with [3H]WIN17317-3 revealed high
specific binding to sites that correspond with the known distribu-
tion of VGSCs in the CNS.20 The ability to depict VGSC distribution
in vitro and the favorable physiochemical properties make
WIN17317-3 appealing as a lead for tracer development. As part
of our ongoing effort to develop tracers for imaging of VGSCs,21
we herein report the synthesis and biological evaluation of a 125I-
labeled analogue of WIN17317-3.
l
mol (n = 7). The identity of [125I]10 was confirmed by co-elution
with the non-radioactive reference compound 10. The log D7.4 of
[
125I]10 was measured to be 2.98 0.08 (n = 3) using the tradi-
tional n-octanol shake flask method.23
The iodinated reference compound 10 was initially evaluated in
an automated patch clamp assay using CHO cells transfected with
the human Nav1.2 isoform (hNav1.2, SCN2A gene).24 Comparison
with WIN17317-3 (IC50 2.2 1.2
sitive control, suggested that the blocking potency of the iodinated
analogue 10 (IC50 1.5 0.5 M) was fully retained. The discrepancy
between our results and previously reported data for WIN17317-3
(IC50 2.2 1.2 M vs 9 nM) is likely to reflect the proportion of non-
protonated drug in the medium, as a physiological buffered bath
solution was used in this study whereas the higher potency was re-
corded under basic conditions (pH 9.8).20 While the amino acid se-
quence of mammalian VGSCs have been largely preserved across
the species, distinct pharmacological profiles have been reported
for human and rodent isoforms.4 Hence, the potency of the iodin-
ated iminodihydroquinoline 10 was further evaluated by displace-
ment studies with [3H]BTX using rat brain homogenates (Ricerca
Taiwan Ltd, Taiwan).25 The results obtained for WIN17317-3
(IC50 8.1 nM; literature value of 25.6 nM20) and 10 (IC50 21.1 nM)
were comparable and largely in agreement with the patch clamp
studies.
lM), which was included as a po-
l
l
The non-radioactive iodinated compound 10 (Scheme 1) was
synthesized following the pathway described by Lin and Loo22
for the preparation of the key intermediate 4-chloro-7-iodoquino-
line (8). The reaction of 3-iodoaniline (3) with diethyl ethoxymeth-
ylenemalonate (4) gave the acrylate 5, which was converted to
hydroxyquinoline 6 upon heating in diphenyl ether at 250 °C.
Hydrolysis of the ester and subsequent decarboxylation afforded
the intermediate 7 as a poorly soluble solid. Treatment of 7 with
phosphorus oxychloride led to the formation of the chloroquino-
line 8, which was reacted with pentylamine to give the aminoquin-
oline 9. Subsequent alkylation with benzyl bromide provided the
non-radioactive reference compound 10 in 24% overall yield (five
steps). Palladium(0)-catalyzed reaction of the iminodihydroquino-
line 10 with hexamethylditin provided the corresponding labeling
precursor 11 in 37% yield.
To evaluate the suitability of [125I]10 for imaging VGSCs in vivo
we measured the distribution of radioactivity after iv injection in
female BALB/c mice (6–10 weeks old, 15–20 g) over a period of
1 h (5, 15, 30 and 60 min). The distribution of [125I]10 in selected
tissues is depicted in Figure 2. At the early time point a high uptake
was observed in the liver and kidneys with later time points dom-
inated by high uptake in the intestines. The initial brain uptake was
low (0.48 0.04% ID/g at 5 min) and did not show any sign of
retention. The activity in the blood was also low and showed a
moderate clearance, with 1.85 0.17% ID/g at 5 min post-injection
and 0.58 0.04% ID/g at 60 min. Clearance from liver and kidneys
was fast and was paralleled by an increased uptake in the intes-
tines, consistent with hepatobiliary excretion.
Iodo-destannylation of 11 with [125I]NaI in the presence of di-
lute hydrogen peroxide for 30 min at room temperature afforded
the radioligand [125I]10 in 58 9% radiochemical yield (n = 7) with
>99% radiochemical purity and a specific activity of 53.2 7.1 GBq/
In order to assess the metabolic stability of [125I]10 the compo-
sition of radioactive species in plasma and brain tissue was ana-
lyzed by HPLC. In plasma, the fraction of the intact tracer [125I]10
was 22.8% at 5 min, 12.5% at 15 min, and 5.2% at 30 min (n = 2),
with the remaining activity observed as a highly polar metabolite
(Fig. 3). Both the parent compound [125I]10 and its radio-metabo-
lite were also detected in the brain, however, low radioactivity lev-
els made quantification of their relative concentrations difficult.
Finally, whole body SPECT/CT scans were recorded following
injection of [125I]10 in female BALB/c mice (4–10 weeks old). In
agreement with the biodistribution studies, a high initial uptake
of radioactivity was observed in the liver (0–15 min post-injec-
tion). At the later time points (15–35 min post-injection, Fig. 4),
the images were dominated by high levels of activity in the intes-
tines. Unfortunately, the uptake was too low to allow SPECT imag-
ing of the regional distribution of [125I]10 in the brain.
WIN17317-3 was attractive as a lead compound for tracer devel-
opment as the tritiated derivative binds with low nanomolar affin-
ity to VGSCs in rat brain tissue (Kd = 2.2 0.3 nM) and allows
imaging of VGSC expression on cryo-sections with in vitro autora-
diography.20 Furthermore, substitution of the chlorine atom with
radioiodine provided a versatile route to tracer candidates. Io-
dine-125 was chosen as labeling is straightforward and the long
half-life (60 days) simplifies biological evaluation. The blocking
efficiencies observed for the non-radioactive iodinated analogue
10 and WIN17317-3 in patch clamp studies, together with the data
obtained from [3H]BTX displacement studies, suggest that iodine is
well tolerated in the binding site and that 10 largely retained the
biological properties of the parent compound. However, in vivo
O
O
NH2
EtO
EtO
OEt
100 ºC
CO2Et
CO2Et
+
I
N
H
I
3
4
5
OH
O
OH
N
1) 10 % NaOH
2) HCl (c)
3) Ph2O, 260 ºC
Ph2O
250 ºC
POCl3
130 ºC
OEt
I
N
6
I
7
NH
Cl
N
NH
C5H11NH2
120 ºC
BnBr, NaI
Acetone, 60 ºC
I
N
I
I
N
8
9
10
Ph
NH
N
NH
N
[
125I]NaI
Sn2Me6
Pd(PPh3)4
PhMe, 100 ºC
H2O2, HCl
MeOH/H2O
125I
Me3Sn
[
125I]10
11
Ph
Ph
Scheme 1. Synthesis of the iodinated radioligand [125I]10.

5172
C. Pérez-Medina et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5170–5173
Figure 2. Tissue distribution of [125I]10 expressed as % ID/g SD (n P3).
[
125I]10 points to other contributing factors, such as high protein
binding or active extrusion by drug efflux pumps. It should also
be noted that the parent compound is ionized at physiological
pH, which may limit penetration through the blood–brain bar-
rier.27 To rule out the possibility that the distribution pattern
was dominated by specific binding to VGSCs expressed in the
intestines,28 the composition of radioactivity in tissue samples
was analyzed by radio-HPLC. However, the parent tracer [125I]10
constituted only a minor component, with a number of other
metabolites observed.
60
30
15
5
0
In conclusion, we have synthesised a 125I-labeled derivative of
the VGSC blocker WIN17317-3. The non-radioactive reference
compound was found to be a potent blocker of the human VGSC
Nav1.2 isoform, and showed comparable potency to WIN17317-3
in [3H]BTX displacement studies. However, the 125I-labeled tracer
failed to demonstrate specific VGSC binding in vivo, and suffered
from poor metabolic stability and low brain uptake. Taken to-
gether, the results suggest that this class of compounds is poorly
suited for tracer development.
Figure 3. HPLC radioactivity profiles in plasma samples at 0, 5, 15, 30 and 60 min
after injection of [125I]10.
Acknowledgments
This work was undertaken at UCLH/UCL who received a propor-
tion of funding from the Department of Health’s NIHR Biomedical
Research Centres funding scheme (E.Å and C.P.M). We also
acknowledge support from the Medical Research Council (N.P).
Figure 4. SPECT/CT summation image from 15 to 35 min after injection of [125I]10.
evaluation of [125I]10 failed to demonstrate any binding that could
be attributed to VGSC expression. Instead, rapid metabolism was
observed accompanied by accumulation of radioactivity in the
intestines. While iodinated tracers are prone to undergo enzymatic
cleavage of the C–I bond to liberate iodide, the low uptake of radio-
activity in the bladder, stomach and thyroid exclude de-iodination
as a major metabolic route.26 The low metabolic stability of [125I]10
is therefore likely to be related to the iminodihydroquinoline scaf-
fold, and hence to also affect other derivatives of this class of com-
pounds. It is plausible that the imino group undergoes hydrolysis
in vivo; however, the retention time of the radio-metabolite ob-
served is inconsistent with formation of the resulting ketone.
While the rapid metabolism is likely to have impaired the brain
uptake, the low activity level in the brain at 5 min after injection of
Supplementary data
Supplementary data (materials and methods, synthetic proce-
dures, selected 1H and 13C NMR spectra and selected radio-HPLC
chromatograms) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2013.07.014.
References and notes
1. Catterall, W. A. Neuron 2000, 26, 13.
2. Anger, T.; Madge, D. J.; Mulla, M.; Riddall, D. J. Med. Chem. 2001, 44, 115.
3. Yu, F. H.; Catterall, W. A. Genome Biol. 2003, 4, 207.
4. Goldin, A. L. Annu. Rev. Physiol 2001, 63, 871.
5. Mantegazza, M.; Curia, G.; Biagini, G.; Ragsdale, D. S.; Avoli, M. Lancet Neurol.
2010, 9, 413.

C. Pérez-Medina et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5170–5173
5173
6. Dib-Hajj, S. D.; Cummins, T. R.; Black, J. A.; Waxman, S. G. Annu. Rev. Neurosci.
2010, 33, 325.
7. George, A. L., Jr. J. Clin. Invest. 2005, 115, 1990.
8. Kohling, R. Epilepsia 2002, 43, 1278.
9. Scalmani, P.; Rusconi, R.; Armatura, E.; Zara, F.; Avanzini, G.; Franceschetti, S.;
Mantegazza, M. J. Neurosci. 2006, 26, 10100.
19. Nguyen, A.; Kath, J. C.; Hanson, D. C.; Biggers, M. S.; Canniff, P. C.; Donovan, C.
B.; Mather, R. J.; Bruns, M. J.; Rauer, H.; Aiyar, J.; Lepple-Wienhues, A.; Gutman,
G. A.; Grissmer, S.; Cahalan, M. D.; Chandy, K. G. Mol. Pharmacol. 1996, 50, 1672.
20. Wanner, S. G.; Glossmann, H.; Knaus, H.-G.; Baker, R.; Parsons, W.; Rupprecht,
K. M.; Brochu, R.; Cohen, C. J.; Schmalhofer, W.; Smith, M.; Warren, V.; García,
M. L.; Kaczorowski, G. J. Biochemistry 1999, 38, 11137.
10. Lossin, C.; Rhodes, T. H.; Desai, R. R.; Vanoye, C. G.; Wang, D.; Carniciu, S.;
Devinsky, O.; George, A. L., Jr. J. Neurosci. 2003, 23, 11289.
11. Meisler, M. H.; Kearney, J. A. J. Clin. Invest. 2005, 115, 2010.
12. Bechtold, D. A.; Smith, K. J. J. Neurol. Sci. 2005, 233, 27.
13. (a) Moll, C.; Mourre, C.; Lazdunski, M.; Ulrich, J. Brain Res. 1991, 556, 311; (b)
England, J. D.; Gamboni, F.; Levinson, S. R. Brain Res. 1991, 548, 334; (c) Craner,
M. J.; Newcombe, J.; Black, J. A.; Hartle, C.; Cuzner, M. L.; Waxman, S. G. Proc.
Natl. Acad. Sci. 2004, 101, 8168.
21. Pérez-Medina, C.; Patel, N.; Robson, M.; Badar, A.; Lythgoe, M. F.; Årstad, E. Org.
Biomol. Chem. 2012, 10, 9474.
22. Lin, A. J.; Loo, T. L. J. Med. Chem. 1978, 21, 268.
23. Årstad, E.; Platzer, S.; Berthele, A.; Pilowsky, L. S.; Luthra, S. K.; Wester, H. J.;
Henriksen, G. Bioorg. Med. Chem. 2006, 14, 6307.
24. Wible, B. A.; Kuryshev, Y. A.; Smith, S. S.; Liu, Z.; Brown, A. M. Drug Dev. Technol.
2008, 6, 765.
25. Catterall, W. A.; Morrow, C. S.; Daly, J. W.; Brown, G. B. J. Biol. Chem. 1981, 256,
8922.
14. Smith, K. J. Brain Pathol. 2007, 17, 230.
15. Bechtold, D. A.; Miller, S. J.; Dawson, A. C.; Sun, Y.; Kapoor, R.; Berry, D.; Smith,
K. J. J. Neurol. 2006, 253, 1542.
26. Yan, R.; Sander, K.; Galante, E.; Rajkumar, V.; Badar, A.; Robson, M.; El-Emir, E.;
Lythgoe, M.; Pedley, R.; Årstad, E. J. Am. Chem. Soc. 2013, 135, 703.
27. Waterhouse, R. N. Mol. Imaging Biol. 2003, 5, 376.
28. (a) Bartoo, A. C.; Sprunger, L. K.; Schneider, D. A. J. Comp. Neurol. 2005, 486, 117;
(b) Barshack, I.; Levite, M.; Lang, A.; Fudim, E.; Picard, O.; Ben Horin, S.;
Chowers, Y. Digestion 2008, 77, 108; (c) Hao, M. M.; Lomax, A. E.; McKeown, S.
J.; Reid, C. A.; Young, H. M.; Bornstein, J. C. J. Neurosci. 2012, 32, 10949.
16. (a) Lo, A. C.; Black, J. A.; Waxman, S. G. Neuroreport 2002, 13, 1909; (b) Lo, A. C.;
Saab, C. Y.; Black, J. A.; Waxman, S. G. J. Neurophysiol. 2003, 90, 3566.
17. Rogawski, M. A.; Löscher, W. Nature Med. 2004, 10, 685.
18. Michne, W. F.; Guiles, J. W.; Treasurywala, A. M.; Castonguay, L. A.; Weigelt, C.
A.; Oconnor, B.; Volberg, W. A.; Grant, A. M.; Chadwick, C. C.; Krafte, D. S.; Hill,
R. J. J. Med. Chem. 1995, 38, 1877.