Synthesis and characterization of 4,6-dichloroindole-based radioligands for imaging the glycine site of the NMDA ion channel

Article abstract of DOI:10.1002/jlcr.528

To provide effective PET or SPECT ligands for the glycine binding site of the NMDA ion channel, we have synthesized and characterized in vitro four substituted derivatives of the potent glycine site antagonist 3-[2-[(phenylamino)carbonyl]ethenyl]-4,6-dich

Full text of DOI:10.1002/jlcr.528

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 91–102.
DOI: 10.1002/jlcr.528
Research Article
Synthesis and characterization of
4,6-dichloroindole-based radioligands
for imaging the glycine site of the NMDA
ion channel
R. N. Waterhouse1,*, A. Sultana1, N. Guo1, Bora Lee1, N. Simpson2,
Lee Collier2,3 and M. Laruelle1,2
1 Department of Psychiatry, College of Physicians and Surgeons, Columbia
University, New York, NY 10032, USA
2 Department of Radiology, Columbia University College of Physicians and
Surgeons, New York, NY 10032, USA
3 Department of Medicine, Division of Cardiology, Columbia University College
of Physicians and Surgeons, New York, NY 10032, USA
Summary
To provide effective PET or SPECT ligands for the glycine binding site of the
NMDA ion channel, we have synthesized and characterized in vitro four
substituted derivatives of the potent glycine site antagonist 3-[2-[(phenylami-
no)carbonyl]ethenyl]-4,6-dichloroindole-2-carboxylic acid (Ki=3.0 nM). These
new ligands contain groups amenable to labeling with C-11 for PET, or I-123
for SPECT. In vitro analysis of these compounds revealed that placement
of a methoxy group at either the ortho or para position of the phenylamino-
carbonyl group significantly reduced receptor affinity (Ki=74.0 ꢀ 8.1 and
26.5 ꢀ 4.9 nM, respectively), as did placement of an iodine at the para position
(Ki=60.4 ꢀ 8.2 nM). However, the meta-methoxy derivative (4b) maintained
high affinity (Ki=4.8 ꢀ 0.9 nM) for the glycine site and was therefore labeled
with carbon-11 by reacting the corresponding desmethyl derivative with
[11C]methyl iodide. Radiochemical yields of 14 ꢀ 10% (EOS), and high specific
activity (1.2 ꢀ 0.5Ci/ mmol (EOS, n=7)) were realized, and the product was
*Correspondence to: R. N. Waterhouse, New York State Psychiatric Institute, 1051 Riverside
Drive, Box # 31, New York, NY 10032, USA. E-mail: rnw7@columbia.edu
Copyright # 2002 John Wiley & Sons, Ltd.
Received 12 July 2001
Revised 25 July 2001
Accepted 6 September 2001

92
R. N. WATERHOUSE ET AL.
prepared in a sterile saline solution suitable for in vivo use. Copyright # 2002
John Wiley & Sons, Ltd.
Key Words: NMDA; glutamate; glycine; PET; radioligand; carbon-11
Introduction
The N-methyl-d-aspartate (NMDA) ion channel, a major site of action
for glutamate, is known to play a central role in many biological
functions including neuroprotection, neurodegeneration, long-term
potentiation, and cognition.1–6 Alterations in normal NMDA receptor
composition, densities and function have been implicated in the
pathophysiology of certain neurological and neuropsychiatric disorders
such as Parkinson’s Disease, Huntington’s Chorea, schizophrenia,
alcoholism and stroke.6–11 Stimulation of the different binding sites
located on or within the NMDA ion channel has been shown to
modulate the expression of behavior in different ways.12–17 In addition
to a site of binding for l-glutamate, there exists a second site that binds
Mg2+ and phencyclidine (PCP) that mediates ion flux through the
strychnine-insensitive glycine binding site, a polyamine binding site and
a Zn2+ binding site. The glycine, glutamate and polyamine sites are
located outside the ion channel, while the Zn2+ and PCP sites are found
within. Glycine is a known co-agonist acting of the NMDA receptor as
its presence is required to allow channel activation by glutamate and
NMDA. Antagonists of the glycine site are reported to be neuropro-
tective against focal ischemia and trauma, anxiolytic, anti-epileptic,
anti-convulsant, anti-psychomimetic, and able to effect improvements in
cognition and memory processes. 6, 8, 18, 19 In line with this, radiotracers
suitable for imaging the glycine site of the NMDA ion channel would
facilitate a means for assessing the status of this site to provide
important biochemical information relevant to the study of these
disorders.
To date the development of effective PET ligands for imaging the
glycine site has proven difficult. Past efforts include the high affinity
3-substituted-4-hydroxyquinolin-2-ones such as [11C]L703,71720,21
,
[
123I]7-Bromo-5-iodo-4-oxo-1,4-dihidroquinoline-2-carboxylic acid and
123I]7-chloro-5-iodo-4-oxo-1,4-dihidroquinoline-2-carboxylic acid.22 How-
[
ever, to date these tracers have not been proven effective for
in vivo imaging, and the development of radioligands for the NMDA/
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4,6-DICHLOROINDOLE-BASED RADIOLIGANDS
93
glycine site remains problematic. High lipophilicity, excessive acidic
character, and binding to the warfarin binding site on human serum
albumin are reportedly the major factors contributing to high plasma
protein binding and low brain uptake.23 This has been reported for
radiotracers, as well as for glycine site antagonists developed as
potential therapeutic agents. A detailed study has been undertaken to
probe the relationships between lipophilicity, plasma protein binding
and in vivo activity of 76 different glycine site antagonists.23 A parabolic
relationship was found between lipophilicity and in vivo activity, and
suitable log P values for optimum in vivo potency occurred between 2.0
and 3.0, with a maximum at 2.39. Also, compounds which possess an
excess number of acidic functional groups exhibited higher degrees of
binding to plasma proteins.
In an effort to provide effective ligands for imaging the glycine site of
the NMDA receptor, we considered the 5,7-dichloroindole-2-carbox-
ylate class of compounds. Of the ligands reported thus far, 3-(2-
((phenylamino)carbonyl)ethenyl)-4,6-dichloroindol-2-carboxylic acid,
GV150526A, is one of most potent glycine site antagonists when
administered intravenously (i.v.).24–26 This compound inhibits NMDA
induced convulsions in mice with an ED50 of 0.06 mg/kg (i.v.). The high
in vivo potency seemed a reasonable indication that this ligand may
adequately cross the blood/brain barrier. Other advantages are its high
affinity (Ki=1–3 nM) and reasonable lipophilicity (log P=2.24).23
However, there is no direct method to quickly label GV150526A with
PET isotopes using standard procedures. Published structure/affinity
relationships for glycine site ligands reveal that a general region exists
around the phenyl ring where there is bulk tolerance.25 We hypothesized
that substituents amenable to direct labeling with PET or SPECT
isotopes could best be placed in this region to hopefully provide ligands
with preserved high affinity for the glycine site.
Results and discussion
Four compounds were synthesized and evaluated in vitro to identify
candidate radiotracers that can be labeled with either C-11 or I-123
using established methods (Scheme 1). (E)-3-(2’-carboxyethenyl)-4,6-
dichloroindole-2-carboxylic acid 7 was prepared in 3 steps from the
known indole 6 according to the methods of Di Fabio et al.24
Amidation of 7 was accomplished by a modification of the method
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R. N. WATERHOUSE ET AL.
Scheme 1. Synthesis of the target compounds 1b–5b
described by Di Fabio et al.24 Included was activation of the carboxylic
acid group through the formation of the corresponding 2-pyridyl
thioester intermediate, followed by the addition of the appropriately
substituted aniline to provide the ethyl esters (1a–5a). Hydrolysis of the
ester with sodium hydroxide in isopropanol afforded the target
compounds 1b–4b and the meta phenol precursor 5b in good yield
(62–89%). By established methods, in vitro assays were subsequently
performed to determine the inhibition constants (Ki values) of the new
ligands.27,28 It was found that placement of a methoxy group at either
the ortho or para position of the phenyl ring significantly reduced
receptor affinity (Ki=74.0 ꢀ 8.1 and 26.5 ꢀ 4.9 nM, respectively), as
did placement of an iodine at the para position (Ki=60.4 ꢀ 8.2 nM)
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(Table 1). However, the meta-methoxy derivative (4b) maintained high
affinity (Ki=4.8 ꢀ 0.9 nM) for the glycine site and was therefore
considered a good candidate for labeling with C-11 for further in vitro
and in vivo testing.
The desired tracer [11C]-4b was synthesized by methylation of the
corresponding 3-hydroxyphenyl derivative using [11C]methyl iodide in
dimethylformamide in the presence of sodium hydroxide. This mixture
was heated at 65–708C for 5min, and purified by a reversed phase semi-
preparative HPLC method as described in the experimental section.
Radiochemical yields of 14–20% (EOS) and high specific activity
(2.2 ꢀ 0.5Ci/ mmol EOS) were realized (Scheme 2). The radiochemical
purity was>95% and up to 69 mCi of product was obtained after
Table 1. In vitro inhibition constants for 2-carboxyindole derivatives
Scheme 2. Radiosynthesis of [11C]-4b
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R. N. WATERHOUSE ET AL.
HPLC purification. The fractions containing [11C]-4b were diluted with
de-ionized water (100 ml) and this solution was passed through a C-18
Sep-Pak. This procedure is performed to remove the acetonitrile and
buffer from the product, and to facilitate its concentration. The
radioactivity was well retained, and the radiotracer was eluted from
the Sep-Pak with 1–1.5ml absolute ethanol. The ethanol solution was
diluted with sterile saline to provide approximately 200 mCi/100 ml, and
the preparation was passed through a sterile 0.22 mm filter into a sterile
vial. This formulation is suitable for in vivo use.
Materials and methods
General
Proton NMR spectra were recorded on a Brucker 400 MHz FT-NMR
spectrometer (Department of Chemistry, Columbia University). Che-
mical shifts were recorded in ppm (d) from an internal tetramethylsilane
standard in either dichloromethane-d2 or methylsulfoxide-d6 and
coupling constants (J) are reported in Hz. Chromatographic purifica-
tion of unlabeled compounds was performed using silica gel (Aldrich,
70-230 mesh, ASTM) using the solvent systems indicated in the text. For
mixed solvent systems, the ratios are given with respect to volumes. All
reagents were purchased from commercial sources and were used
without further purification. (E)-3-(2’-Carboxyethenyl)-4,6-dichloroin-
dole-2-carboxylic acid 6 was prepared using reported methods.[27,28]
[11C]Methyl iodide is synthesized routinely at Columbia University
Radioligand Laboratory using established methods. HPLC analysis of
the radioligand was performed using a Waters.
515 HPLC pump, a Waters PDA UV detector, and a Bicron Flow-
Scint radiation detector. The column used was a reverse-phase base-
deactivated column (Phemomenex, ODS, analytical: 4 ꢁ 250 mm2, 5 mm
particle size; semipreparative: 10 ꢁ 250 mm2, 10 mm particle size) and the
mobile phases and flow rates used are indicated in the text below.
Synthesis
General synthesis of Aryl Amides. To a solution of (E)-3-(20-carbox-
yethenyl)-4,6-dichloroindole-2-carboxylic acid in DMF were added 2,20-
dipyridyl disulfide and triphenylphosphine at room temperature under
an N2 atmosphere. The solution was stirred for 2 h, then the
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appropriately substituted aniline was added. The temperature was
increased to 858C, and stirring continued for another 20 h. Water
was added to effect precipitation, and the resulting precipitate was
collected and dried to obtain the desired ethyl esters 1a–5a in good yield
(62–89%).
3-[2-[(4-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
1
carboxylic acid ethyl ester, 1a. mp=242–2438C; H-NMR: 1.23. (t,
3 H), 4.1 (q, 2 H), 6.33 (d, 1 H, J=15.0), 7.2–7.7 (m, 7 H), 10.18 (s, 1 H),
12.3 (br s, 1 H).
3-[2-[(2-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
carboxylic acid ethyl ester, 2a. mp=250–2538C; 1H-NMR: 1.3. (t, 3 H),
3.85(s, 3 H), 4.2 (q, 2 H), 6.8–7.7 (m, 7 H), 8.55(d, 1 H, J=15.0), 10.2
(s, 1 H), 12.3 (br s, 1 H).
3-[2-[(4-iodophenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-car-
1
boxylic acid ethyl ester, 3a. mp=231–2358C; H-NMR: 1.3. (t, 3 H),
3.85(s, 3 H), 4.2 (q, 2 H), 6.8–7.7 (m, 7 H), 8.55(d, 1 H, J=15.0), 10.2
(s, 1 H), 12.3 (br s, 1 H).
3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
carboxylic acid ethyl ester, 4a. mp=257–2608C; 1H-NMR: 1.3. (t, 3 H),
3.85(s, 3 H), 4.2 (q, 2 H), 6.8–7.7 (m, 7 H), 8.55(d, 1 H, J =15.0), 10.2
(s, 1 H), 12.3 (br s, 1 H).
3-[2-[(3-hydroxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
carboxylic acid ethyl ester, 5a. mp=279–2818C; 1H-NMR: 1.3. (t, 3 H),
4.2 (q, 2 H), 6.5–7.6 (m, 8 H), 8.55 (d, 1 H, J=15.0), 10.2 (s, 1 H), 12.3
(br s, 1 H).
General Procedure for the basic hydrolysis of ethyl esters. The 2-indole
carboxylic acid ethyl esters were dissolved in isopropanol and heated at
reflux for 6 h in the presence of sodium hydroxide (1 equivalent). The
solution was cooled, and water was added to effect precipitation. The
alcohol was evaporated in vacuo, and the precipitate filtered and
washed with water to provide the target compounds 1b–5b in high yield
(91–98%).
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3-[2-[(4-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
1
carboxylic acid, 1b. mp=220–2238C; H-NMR: 3.85. (s, 3 H), 6.8–7.5
(m, 7 H), 8.75 (d, 1H, J=15.0); MS m/z: 403.
3-[2-[(2-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
carboxylic acid, 2b. mp=>2308C; 1H-NMR: 3.85. (s, 3 H), 6.8–7.5 (m,
7 H), 8.75 (d, 1H, J=15.0); MS m/z: 403.
3-[2-[(4-iodophenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-car-
boxylic acid, 3b. mp=>2508C; 1H-NMR: 6.12. (d, 1 H, J=16 Hz), 7.0–
7.6 (m, 6 H), 8.6 (d, 1H, J=16.0); MS m/z: 499.
3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
1
carboxylic acid, 4b. mp=227–2308C; H-NMR: 3.9. (s, 3 H), 6.8-7.5
(m, 7 H), 8.75 (d, 1H, J=15.0); MS m/z: 403.
3-[2-[(3-hydroxyphenylamino)carbonyl]ethenyl]-4,6-dichloroindole-2-
carboxylic acid, 5b. mp=>2308C; 1H-NMR: 6.5–7. 6 (m, 8 H), 8.55 (d,
1 H, J=15.0); MS m/z: 391.
Radiolabeling
[11C]3-[2-[(3-methoxyphenylamino)carbonyl]ethenyl]-4,6-dichloroin-
dole-2-carboxylic acid, [11C]-4b.
Into a 1 ml reaction vial was added 2 mg 5b, 5 ml aqueous sodium
hydroxide (5N) and dimethylformamide (400 ml). The vessel was
sealed and [11C]methyl iodide (820–1,430 mCi) was transferred into
the solution via a stream of argon. The vessel activity was monitored,
and once maximized, the mixture was heated at 65–708C for 5min.
The crude product was diluted with 1 ml mobile phase, and then
injected onto an HPLC column (ammonium acetate (0.1 M, pH=5)/
acetonitrile 35:65 (v/v); flow rate=12 ml/min). The desired product
eluted between 11-13 min, and the eluent was diluted with 100 ml de-
ionized water, passed through a C-18 Sep-Pak. [11C]-4b was eluted from
the Sep-Pak with ethanol (1.0–1.5ml) and this solution was subjected to
QC analysis to determine overall radiochemical purity, specific activity,
and product yield. From this analysis, the desired radiotracer was
produced in yields of 14–20% (EOS), specific activities of 2.2 ꢀ 0.5Ci/
mmol (EOS, n=4) and a radiochemical purity>95%. Synthesis, HPLC
purification and initial QC of the ethanol was typically completed
within 40–45min.
To obtain preparations of the radiotracer suitable for use in vivo,
the ethanol solution was subsequently diluted with saline (0.9% NaCl,
sterile) to provide the desired concentration of [11C]-4b while maintain-
ing a maximum of 10% ethanol by volume. This final saline solution
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was passed through a sterile filter (22 mM) into an evacuated sterile to
provide approximately 10 mCi [11C]-4b per ml solution. The formula-
tion step typically took 5–7 min. The range in the amount of product
formulated was 12.3–68 mCi when prepared in this manner. Aliquots
were further diluted with sterile saline to provide concentrations of
200 mCi of [11C]-4b per 100 ml solution, which is suitable for use in
rodent studies.
Ligand binding assay
Once prepared, compounds 1b–4b were evaluated for selective
binding to the glycine site through receptor binding assays established
using highly purified rat cortex membrane preparations and
established in vitro assay conditions.27,28 The radioligand used was
the well characterized glycine site antagonist [3H]MDL 105,519
(KD=3.7 nM). Briefly, assays were performed in triplicate, and the
ability of various concentrations of unlabeled 1b–4b to inhibit
specific binding of [3H]MDL 105,519 (3 nM) was determined. Unlabeled
ligands were dissolved as 1 mM stock solutions in 10% DMSO in
buffer and diluted with binding buffer as appropriate. The presence
of 1% DMSO has been reported to not alter the binding of [3 H]
MDL 105,519.29 Samples varying in concentration (10–12 to 10–5 M)
of 1b–4b were incubated for 1 h at room temperature in a total
volume of 1 ml binding buffer (tris acetate 0.05M, pH=7.4) in the
presence of membrane (2 mg) and tracer. Samples were filtered
using a 48-well cell harvestor (Brandel) and unbound tracer was
removed by washing three times (approx. 4 ml per wash) with ice
cold buffer. The radioactivity was determined by scintillation
counting (Packard TriCarb 1500) after drying the membrane filters
at room temperature for 2 h, followed by the addition of scintilla-
tion fluid (4 ml, Packard Ultima Gold). The effect of different
concentrations of test ligand 1b–4b was examined using non-
linear regression analysis (Prism), and the IC50 and Ki values
determined.
Conclusion
Four new glycine site ligands have been synthesized and their inhibition
constants evaluated in vitro. Of these, one ligand, [11C]-4b, exhibited
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high affinity for the glycine site. This compound was radiolabeled with
the positron emitting isotope carbon-11 in good yield and high specific
activity, and subsequently prepared as a formulation suitable for in vivo
studies.
Acknowledgements
The authors thank Eric Pringle for his assistance with purification of
chemical intermediates, and Julie Montoya for the contributing to the
specific activity analysis of the target radiotracer.
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