Synthesis of [11C] N-(2-chloro-5-thiomethylphenyl)-N′-(3-methoxyphenyl)- N′-methylguanidine ([11C]GMOM): A candidate PET tracer for imaging the PCP site of the NMDA ion channel

Article abstract of DOI:10.1002/jlcr.622

The N-methyl-D-aspartate (NMDA) ion channel plays an important role in a number of neurodegenerative disorders including stroke, Parkinson's disease, Huntington's Chorea, Alzheimer's disease, schizophrenia and epilepsy. To provide effective radioligands for imaging the PCP binding site of the NMDA ion channel, we synthesized and characterized in vitro the candidate PCP site ligand N-(2-chloro-5-thiomethylphenyl)-N′-(3-methoxyphenyl)- N′-methylguanidine (GMOM: K_i = 5.2 ± 0.3 nM, log P = 2.34). The corresponding PET radiotracer [¹¹C]GMOM was synthesized with a radiochemical yield of 8.4 ± 3.2% EOS and with a specific activity of 1.23 ± 0.25 Ci/μmol EOS (n = 5). The average time required for synthesis, purification and formulation was 52 ± 5 min. The final product was prepared in a sterile saline solution suitable for in vivo use. Copyright

Full text of DOI:10.1002/jlcr.622

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 955–964.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.622
Research Article
Synthesis of [11C] N-(2-chloro-
5-thiomethylphenyl)-N0-(3-methoxyphenyl)-
N0-methylguanidine ([11C]GMOM):
a candidate PET tracer for imaging
the PCP site of the NMDA ion channel
Rikki N. Waterhouse1,*, Filip Dumont1, Abida Sultana1,
Norman Simpson2 and Marc Laruelle1,2
1 Department of Psychiatry, Columbia University College of Physicians and
Surgeons and New York State Psychiatric Institute, New York, NY 10032, USA
2 Department of Radiology, Columbia University College of Physicians and
Surgeons and New York State Psychiatric Institute, New York, NY 10032, USA
Summary
The N-methyl-d-aspartate (NMDA) ion channel plays an important role in a
number of neurodegenerative disorders including stroke, Parkinson’s disease,
Huntington’s Chorea, Alzheimer’s disease, schizophrenia and epilepsy. To
provide effective radioligands for imaging the PCP binding site of the NMDA
ion channel, we synthesized and characterized in vitro the candidate PCP site
ligand N-(2-chloro-5-thiomethylphenyl)-N0-(3-methoxyphenyl)-N0-methylgua-
nidine (GMOM: Ki ¼ 5:2 ꢀ 0:3 nM, log P ¼ 2:34). The corresponding PET
radiotracer [11C]GMOM was synthesized with a radiochemical yield of
8.4 ꢀ 3.2% EOS and with a specific activity of 1.23 ꢀ 0.25 Ci/mmol EOS
(n ¼ 5). The average time required for synthesis, purification and formulation
was 52 ꢀ 5 min. The final product was prepared in a sterile saline solution
suitable for in vivo use. Copyright # 2002 John Wiley & Sons, Ltd.
Key Words: NMDA; glutamate; phencyclidine; PET; radiotracer
*Correspondence to: Rikki N. Waterhouse, Department of Psychiatry, 1051 Riverside Drive, Unit
31, New York, NY 10032, USA. E-mail: rnw7@columbia.edu
Contract/grant sponsor: National Institutes of Health; contract/grant numbers: NIAAA IP50AA-
12870-01, NIMH MH59342-01
Copyright # 2002 John Wiley & Sons, Ltd.
Received 29 November 2001
Revised 12 December 2001
Accepted 14 May 2002

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R. N. WATERHOUSE ET AL.
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, memory, and cognition.1–5 Alterations in normal NMDA
channel 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.6ꢁ12 This ion channel is complex in respect to
both its structure and function and is comprised of at least one NR-1
subunit along with one or more NR-2 subunits (NR-2A, -2B, -2C, or
-2D).13–15 In addition to the site of binding for the agonist l-glutamate,
receptors for the known co-agonist glycine also exist. Several NMDA
receptor modulatory sites have also been well characterized, including
those for polyamines, Zn2ꢁ, Mg2+ and phencyclidine (PCP). It has been
established that the specific subunit composition of a particular NMDA
ion channel, and the nature of modulation by NMDA receptor ligands,
dictate subsequent biological responses.
Non-invasive methods for imaging the PCP site in vivo would allow
for quantification of these sites in living subjects and, in addition, would
make possible drug occupancy studies to evaluate new NMDA receptor
targeted drugs that might be tested clinically as neuroprotective or anti-
stroke agents. In addition, it is known that the in vitro and in vivo
binding of [3H]MK801 is affected by the state of activation of NMDA
receptors.16ꢁ18 Agonists and antagonists at the glutamate or glycine
sites increase and decrease, respectively, [3H]MK801 binding to the PCP
site which is located inside the ion channel. Therefore, in vivo binding of
a PET PCP site ligand might report on the state of activation of NMDA
receptors in vivo.
To date, several candidate PCP site radioligands have been reported.
Examples include [11C]ketamine,19,20 123I, and 125I-labeled dizocilpine
(MK 801),21ꢁ23 123I, 18F and 11C-labeled derivatives of phencyclidine
(PCP), such as [18F]fluorothienylcyclohexylpiperidine ([18F]FETCP),24
and adamantane derivatives including [18F]1-amino-3-fluoromethyl-5-
methyl-adamantane ([18F]AFA).25 Although many of these compounds
show promise in vitro for labeling the PCP site, all suffer in vivo from
either fast metabolism and subsequent fast brain clearance, as is the case
for [11C]ketamine, or a lack of demonstrable specific binding. A low
target-to-background ratio is possibly a result of non-selectivity, as with
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF [11C] GMOM
957
[18F]AFA and other ligands that also binds sigma receptors, or
high lipophilicity as exemplified by [123I]MK801. We have chosen
as a starting point a group of selective non-competitive antagonists
consisting of N-(2,5-disubstitutedphenyl)-N0-(3-substitutedphenyl)-
N0-methylguanidines.26,27 Compounds of this series comprise some of
the highest affinity PCP site ligands reported to date, and these also
exhibit specificity for the PCP site vs sigma receptors. Herein, we report
the synthesis, in vitro characterization, lipophilicity estimation and
radiolabeling of the first novel PET tracer of this series, [11C]N-(2-
chloro-5-thiomethylphenyl)-N0-(3-methoxyphenyl)-N0-methylguanidine
([11C]GMOM).
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 chloroform-d3 or methylsulfoxide-d6 and coupling
constants (J) are reported in Hz. Chromatographic purification 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 and solvents were purchased from commercial sources and
were used without further purification. The 3-substituted anilines
purchased from Aldrich Chemical Company (Milwaukee Wisconsin).
[11C]Methyl iodide is synthesized routinely at Columbia University
Radioligand Laboratory. This radioactive synthon is prepared by the
lithium aluminum hydride reduction of [11C]carbon dioxide to form
[11C]methanol, followed by treatment of the [11C]methanol with
hydrogen iodide and distillation of newly formed [11C]methyl iodide.
HPLC analysis of the target radioligand [11C]GMOM 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 (Phenomenex, ODS, analytical:
4 ꢂ 250 mm, 5 mm particle size; semi-preparative: 10 ꢂ 250 mm, 10 mm
particle size) and the mobile phases and flow rates used are indicated in
the text below.
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R. N. WATERHOUSE ET AL.
Synthesis
The synthesis of N-(2-chloro-5-thiomethylphenyl)-N0-(3-ethylphenyl)-
N0-methylguanidine (1), N-(2-chloro-5-thiomethylphenyl)-N0-(3-thio-
methylphenyl)-N0-methylguanidine were synthesized according to
established methods (2). The novel compounds 7 and 8 were prepared
using the general method of Hu et al. with modifications as required.26
The N-methylated 3-substituted cyanamides were prepared using the
general methods described in Hu et al. and the crude products obtained
were subjected to column chromatography (hexanes/ethyl acetate
2:1 v/v) to provide the desired purified compounds.
N-methyl-3-hydroxyphenylcyanamide: mp=92–948C; 1H-NMR:
7.22 (t, 1H, Ar), 6.66 (t, 1H, Ar), 6.59 (m, 2H, Ar), 3.32 (s, 3H, N-CH3).
1
N-methyl-3-methoxyphenylcyanamide: oil; H-NMR: 7.28 (t, 1H,
Ar), 6.62–6.71 (t, 3H, Ar), 3.83 (s, 3H, O-CH3), 3.33 (s, 3H, N-CH3).
Synthesis of 7 and 8 (GMOM)
A mixture of the appropriate N-methyl-3-substituted phenylcyanamide
and 2-chloro-5-thiomethylaniline hydrochloride (1.1 equivalents) in
chlorobenzene was heated at 1408C under a nitrogen atmosphere for
4 h. The mixture was diluted with aqueous HCl solution (0.1 M) and the
product was extracted into dichloromethane. The organic layer was
separated, dried over sodium sulfate, and the solvent was evaporated
under reduced pressure. Column chromatography (silica; dichloro-
methane/methanol 95:5 v/v) provided the desired substituted guanidines
in good yield (40–68%).
N-(2-chloro-5-thiomethylphenyl)-N0-(3-hydroxyphenyl)-N0-methyl-
guanidine, 7: mp=160–1648C; 1H–NMR: 9.72 (br s, 1H) 8.33 (br s, 2H),
6.65–7.20 (m, 7H, Ar), 3.65 (s, 3H, N-CH3), 2.42 (s, 3H, S-CH3); MS
m=z: 322.
N-(2-chloro-5-thiomethylphenyl)-N0-(3-methoxyphenyl)-N0-methyl-
1
guanidine (GMOM) 8: mp=149–1518C; H-NMR: 9.80 (br s, 1H) 8.4
(br s, 2H), 6.85–7.30 (7H), 3.82 (s, 3H, O-CH3), 3.65 (s, 3H, N-CH3),
2.45 (s, 3H, S-CH3); MS m=z found (and theory), 336.0953 (336.0937).
Ligand binding assay
Compounds 1, 2 and 8 were evaluated for binding to the PCP site
through receptor binding assays using established methods.16,8 Male
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SYNTHESIS OF [11C] GMOM
959
Sprague–Dawley rats were decapitated under CO2 anesthesia, and the
frontal cortex was removed and homogenized with a PowerGen 125
tissue homogenizer (Fisher Scientific; 15 s, half-maximum speed) in a 30-
fold (w/v) excess of ice-cold Tris–HCl buffer (50 nM, pH=7.4). The
resulting homogenate was centrifuged at 10000g for 10 min at 48C using
an Allegra 64R ultracentrifuge (Beckman Coulter). The pellet was
collected and resuspended in 30 volumes (w/v) of the same buffer, and
was centrifuged a second time. The process was repeated once more.
The final pellet was resuspended in ice-cold Tris–HCl buffer (50 mm,
pH=7.4) containing glutamate (10 mM) and glycine (10 mM) to
provide a final concentration of 30 mg membrane per 1 ml solution.
[3H]MK801 (10 mM) was incubated for 1.5 h in a total volume of 1.0 ml
Tris acetate buffer (pH=7.4, 228C) including glutamate (10 mM) and
glycine (10 mM) in the presence of rat cortical membranes (3 mg/tube).
The effect of different concentrations of test ligand (10ꢁ12–10ꢁ4 M)
GMOM was examined, and all assays were performed in triplicate.
Experiments were terminated by the addition of 3 ml ice-cold buffer, and
the unbound tracer was removed by washing twice more (approx. 4 ml/
wash) cold buffer using a 48 well cell harvester (Brandel). The membranes
were incubated in 4 ml scintillation fluid (Packard Ultrascint) and the
radioactivity measured with a Tricarb 1500 scintillation counter (Packard).
Lipophilicity estimations
The lipophilicity of 1, 2 and 8 was examined by determination of the
log P value using a HPLC method previously described.28 Briefly,
samples were analyzed using an analytical C18 column (Phenominex
Protegy 10 mm, 4.6 ꢂ 250 mm) and a mobile phase of methanol and
phosphate buffer (85:15 v/v, pH=7.5) at 1.0 ml/min. Lipophilicity was
determined by comparison of the retention time of the compound to
that of standards having known log P values. Relative retention times
(RRT) were calculated, and a calibration curve of log P vs log RRT was
generated. The calibration equations were polynomial with r2 of 0.986.
All standard and sample injections were done in triplicate and the
results averaged to provide the final values.
Radiolabeling
[11C]N-(2-chloro-5-thiomethylphenyl)-N0-(3-methoxyphenyl)-N0-methyl-
guanidine, [11C]GMOM. Into a 1 ml reaction vial was added 1 mg 7,
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R. N. WATERHOUSE ET AL.
5 mL aqueous sodium hydroxide (5 N) and dimethylformamide (400 mL).
The vessel was sealed and [11C]methyl iodide was transferred into the
solution via a stream of argon. The vessel radioactivity was monitored,
and once maximized, the mixture was heated at 708C for 5 min. The
crude product was diluted with 1 ml mobile phase, and then injected
onto a C-18 HPLC column (mobile phase: ammonium acetate (0.1 M,
pH=5)/acetonitrile 30:70 (v/v); flow rate=12 ml/min). The product
eluted with a retention time between 12 and 14 min.
To obtain preparations of the radiotracer suitable for use in vivo, the
eluted radioactive peak corresponding to [11C]GMOM was collected
and diluted with 100 ml de-ionized water, passed through a solid phase
extraction cartridges (Water C-18 Sep-Pak) and the product was eluted
with 1 ml ethanol. A 10–50 ml sample of the ethanol solution was
analyzed to determine radiotracer purity and specific activity.
Results and discussion
The synthesis of the 3-methoxy derivative 8 (GMOM) and the precursor
7 was carried out using reported methods with modifications as
required,26 and the general method is outlined in Scheme 1. For our
initial studies, we chose to prepare and characterize the 3-methoxy
analog GMOM as a PET radiotracer instead of the 3–thiomethyl
derivative 2 due to the ease of synthesis and expected stability of the
precursor 7 required to prepare [11C]GMOM. In vitro characterization
was carried out in rat forebrain membranes using a standard PCP site
NCH3CN
NH2
NHCN
1. NaH
2. CH3I
CNBr
Et2O
X
X
X
DMF
3
4
5
NH3Cl
Cl
NCH3CN
H
N
CH3
N
Cl
X
+
NH
chlorobenzene
140˚C
SCH3
X
4 hrs
SCH3
6
5
7
8
X = OH
X = OCH (GMOM)
3
Scheme 1. Synthesis of the target compounds 7 and 8 (GMOM)
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SYNTHESIS OF [11C] GMOM
961
Table 1. In vitro inhibition constants and experimental log P values for selected
substituted N; N0-diphenyl-N0-methylguanidines
Cl
H
N
CH3
N
X
NH
SCH3
x
Ki (PCP site)a
Ki (PCP site)b
log P
1
2
8
CH2CH3
SCH3
OCH3
5.1 ꢀ 0.3
1.87 ꢀ 0.61
6.28 ꢀ 0.28
1.87 ꢀ 0.25
5.2 ꢀ 0.3
3.00
2.68
2.34
a See References 26.
b Performed in house by methods outlined in Reference 16.
assay with [3H]MK801 as the radioligand.16,18 Analysis of GMOM
provided a Ki value of 5.2 ꢀ 0.3 nM (n ¼ 3), indicating that the 3-
methoxy group provided an affinity similar to the 3-ethyl derivative 1
but somewhat lower than the 3-thiomethyl compound 2 (Table 1).
Compounds 1 and 2 were also analyzed to ensure our assay provided Ki
values close to those already reported in the literature. The Ki values of
6.28 ꢀ 0.28 and 1.87 ꢀ 0.25 nM for 1 and 2, respectively, compared
very well with those reported by Hu et al.26 In addition, an HPLC
method was used to determine the log P value of these ligands at
physiological pH (pH=7.5). GMOM was found to have the lowest
lipophilicity compared to the 3-ethyl and 3-thiomethyl derivatives
(Table 1). Its log P value of 2.34 is sufficient to allow passage across the
blood/brain barrier, but not so high as to suggest that a large degree of
non-specific binding would occur, as is often associated with radi-
oligands such as 3-Iodo-MK801 and phencyclidine-based tracers with
log P values above 3.0.
The corresponding PET tracer [11C]GMOM was synthesized by
methylation of the 3-hydroxyphenyl derivative 7 with [11C]methyl iodide
in dimethylformamide in the presence of aqueous sodium hydroxide
(Scheme 2). This mixture was heated briefly at 708C, and [11C]GMOM
was purified by reversed phase semi-preparative HPLC. After complete
work-up as described in detail in the Experimental section, [11C]GMOM
was provided in radiochemical yields of 5.8–13.0% (8.4 ꢀ 3.2%, n ¼ 5,
EOS) and high specific activity (1.25 ꢀ 0.25 Ci/mmol, n ¼ 5, EOS). The
radiochemical purity was 96.7 ꢀ 1.5% and up to 39 mCi of product was
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Cl
H
NaOH
11
Cl
H
CH3
CH3
11
N
CH I
3
N
N
N
CH
O
3
OH
NH
NH
DMF
70˚C
5 min
SCH3
SCH3
11
[
C]GMOM
Scheme 2. Radiosynthesis of [11C]GMOM
obtained. The chemical purity was demonstrated by HPLC analysis to
be >95%, as no UV associated peaks other than those attributed to
GMOM and ethanol were noted. Diluted solution of the tracer was
made with sterile saline to provide approximately 200 mCi/100 ml
solution, and the final preparation was passed through a sterile
0.22 mM filter into a sterile vial. The radiochemical and chemical purity
of the final formulation was >95% as determined by the analytical
HPLC method described in the experimental section. The average time
required for synthesis, purification and formulation was 52 ꢀ 5 min.
The final formulation was deemed suitable for in vivo use.
Conclusions
The novel high-affinity guanidine-based NMDA/PCP site radioligand
[11C]GMOM has been synthesized. This compound exhibits high
affinity (Ki ¼ 5:2 ꢀ 0:3 nM) for the PCP site as well as a log P value
(2.34) suitable for good brain penetration and low non-specific binding.
The corresponding desmethyl precursor was labeled with the positron
emitting isotope carbon-11 in good yield and high specific activity, and
[11C]GMOM was subsequently prepared as a formulation suitable for
use in in vivo studies.
Acknowledgements
The authors sincerely thank Julie Montoya for her excellent technical
assistance regarding the tracer characterization. Funding for this work
was provided by grants from the National Institutes of Health (NIAAA
IP50AA-12870-01 and NIMH MH59342-01).
Copyright # 2002 John Wiley & Sons, Ltd.
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SYNTHESIS OF [11C] GMOM
963
References
1. Curran H, Morgan C. Addiction 2000; 95(4): 575–590.
2. Lees KR. Neurology 1997; 49(5 Suppl 4): S66–S69.
3. Li H, Matsumoto K, Tohda M, Yamamoto M, Watanabe H. Behav Brain
Res 1997; 83(1–2): 225–228.
4. Mohr E, Knott V, Sampson M, Wesnes K, Herting R, Mendis T. Clin
Neuropharmacol 1995; 18(1): 28–38.
5. Newcomer J, Farber N, Jevtovic-Todorovic V, et al. Neuropsychopharma-
cology 1999; 20(2): 106–118.
6. Becker J, Li Z, Noe CR. Eur J Biochem 1998; 256(2): 427–435.
7. Grimwood S, Slater P, Deakin JF, Hutson PH. Neuroreport 1999; 10(3):
461–465.
8. Krupp JJ, Vissel B, Heinemann SF, Westbrook GL, Neuron 1998; 20(2):
317–327.
9. Laube B, Hirai H, Sturgess M, Betz H, Kuhse J. Neuron 1997; 18(3):
493–503.
10. Yamakura T, Shimoji K. Prog Neurobiol 1999; 59(3): 279–298.
11. Christie JM, Jane DE, Monaghan DT. J Pharmacol Exp Ther 2000; 292(3):
1169–1174.
12. Magnusson KR. J Neurosci 2000; 20(5): 1666–1674.
13. Quirk K, Foster AC, McKernan RM. Mol Membr Biol 1994; 11(1):
17–21.
14. Snell LD, Nunley KR, Lickteig RL, Browning MD, Tabakoff B, Hoffman
PL. Brain Res Mol Brain Res 1996; 40(1): 71–78.
15. Dingledine R, Myers SJ, Nicholas RA. FASEB J 1990; 4(9): 2636–2645.
16. Javitt DC, Zukin SR. Mol Pharmacol 1989; 35(4): 387–393.
17. Murray F, Kennedy J, Hutson PH, et al. Eur J Parmacol 2000; 398:
263–270.
18. Javitt DC, Zukin SR, Proc Natl Acad Sci USA 1989; 86(2): 740–744.
19. Hartvig P, Valtysson J, Antoni G, et al. Nucl Med Biol 1994; 21(7):
927–934.
20. Shiue CY, Vallabhahosula S, Wolf AP et al. Nucl Med Biol 1997; 24(2):
145–150.
21. Ransom RW, Eng WS, Burns HD, Gibson RE, Solomon HF. Life Sci
1990; 46(15): 1103–1110.
22. Brown DR, Wyper DJ, Owens J, Patterson J, Kelly RC, Hunter R,
McCulloch J. J Psychiatr Res 1997; 31(6): 605–619.
23. Gibson EG, Burns HD, Thorpe HH, Eng WS, Ransom R, Solomon H, Int
J Rad Appl Instrum B 1992; 19(3): 319–326.
24. Ferrarese C, Guidotti A, Costa E, et al. Neuropharmacology 1991; 30(8):
899–905.
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 955–964

964
R. N. WATERHOUSE ET AL.
25. Ametamey SM, Samnick S, Leenders KL, Vontobel P, Quack G, Parsons
CG, Schubiger PA, J Recept Signal Transduct Res 1999; 19(1–4): 129–141.
26. Hu LY, Guo J, Magar SS, Fischer JB, Burke-Howie KJ, Durant GJ. J
Med Chem 1997; 40(26): 4281–4289.
27. Keana JF, McBurney RN, Scherz MW, et al. Proc Natl Acad Sci USA
1989; 86(14): 5631–5635.
28. Waterhouse RN, Mardon K, Giles KM, Collier TL, O’Brien JC. J Med
Chem 1997; 40(11): 1657–1667.
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