The high specific activity tritium labeling of the ganglion-blocking nicotinic antagonist chlorisondamine

Article abstract of DOI:10.1002/jlcr.1069

Chlorisondamine is a bisquaternary ganglion-blocking nicotinic antagonist that accumulates in dopaminergic, serotonergic, and noradrenergic cell bodies; the mechanism of uptake and time-course of retrograde transport are not known. Chlorisondamine could possibly be taken into monoaminergic neurons when blocked nicotinic receptors on those cells are internalized. In order to more easily study the mechanism of the capture and recycling of chlorisondamine, a ligand is needed that has high specific activity. For that purpose, we now report the preparation of ³H-labeled chlorisondamine (4,5,6,7-tetrachloro-2- [³H]methyl-2-(2-trimethylammoniumethyl)-isoindolinium diiodide) of high specific activity (94 Ci/mmol). The compound was synthesized by quarternization of 4,5,6,7-tetrachloro-2-(2-trimethylammoniumethyl-isoindolinium iodide with pertritiated methyl iodide in dimethylformamide at high temperature in a sealed vessel. Copyright

Full text of DOI:10.1002/jlcr.1069

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 471–478.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1069
Research Article
The high specific activity tritium labeling of the
ganglion-blocking nicotinic antagonist
chlorisondamine
Josef Zezula1, Hay-Yan J. Wang2, Amina S. Woods2, Roy A. Wise2,
Arthur E. Jacobson1 and Kenner C. Rice1,*
1 Laboratory of Medicinal Chemistry, Building 8, Room B1-23, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS,
8 Center Drive, MSC 0815, Bethesda, MD 20892, USA
2 Behavioral Neuroscience Branch, National Institute for Drug Abuse, Intramural
Research Program, National Institutes of Health, DHHS, 5500 Nathan Shock Dr,
Baltimore, MD 21224, USA
Summary
Chlorisondamine is a bisquaternary ganglion-blocking nicotinic antagonist that
accumulates in dopaminergic, serotonergic, and noradrenergic cell bodies; the
mechanism of uptake and time-course of retrograde transport are not known.
Chlorisondamine could possibly be taken into monoaminergic neurons when blocked
nicotinic receptors on those cells are internalized. In order to more easily study the
mechanism of the capture and recycling of chlorisondamine, a ligand is needed
that has high specific activity. For that purpose, we now report the preparation of
3H-labeled chlorisondamine (4,5,6,7-tetrachloro-2-[3H]methyl-2-(2-trimethylammoniu-
methyl)-isoindolinium diiodide) of high specific activity (94 Ci/mmol). The compound
was synthesized by quarternization of 4,5,6,7-tetrachloro-2-(2-trimethylammoniumethyl-
isoindolinium iodide with pertritiated methyl iodide in dimethylformamide at high
temperature in a sealed vessel. Copyright # 2006 John Wiley & Sons, Ltd.
Key Words: chlorisondamine; nicotinic antagonist; tritiation
*Correspondence to: Kenner C. Rice, Laboratory of Medicinal Chemistry, Building 8, Room B1-23,
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, DHHS, 8
Center Drive, MSC 0815, Bethesda, MD 20892, USA. E-mail: kr21f@nih.gov
Contract/grant sponsor: National Institute of Diabetes and Digestive and Kidney Diseases and the National
Institute on Drug Abuse
Contract/grant sponsor: National Institute on Drug Abuse, NIH, DHHS
Copyright # 2006 John Wiley & Sons, Ltd.
Received 31 January 2006
Revised 8 March 2006
Accepted 8 March 2006

472
J. ZEZULA ET AL.
Introduction
Chlorisondamine (6, Scheme 1) is a bisquaternary ganglion-blocking nicotinic
antagonist with negligible effects at the skeletal muscle endplate.1,2 While it
penetrates the brain poorly when given systemically, a high dose blocks the
locomotor stimulating effects3 and reward-enhancing effects4 of nicotine. Both
the locomotor-stimulating effects3 and the rewarding effects5 of nicotine are
blocked when the drug is given intracerebro-ventricularly. While effects of
chlorisondamine at the autonomic ganglion last only hours or days, blockade
of the locomotor-stimulating, rewarding, or reward-enhancing effects of
nicotine, effects thought to be mediated by actions of nicotine on the
mesocorticolimbic dopamine system, lasts for many weeks.3–5 The long-lasting
central effects of chlorisondamine on nicotine-induced locomotion and reward
appear to be due to the intraneuronal accumulation and protection of
chlorisondamine by dopaminergic neurons; indeed, radiolabeled chlorisonda-
mine has been found in synaptosome and tissue samples 3 weeks after
intracerebroventricular injection.6,7 Moreover, radiolabelled chlorisondamine
injected into the caudate putamen was found, 1 week after injection, not only
to label the injection site but also to be concentrated in the zona compacta of
the substantia nigra and the dorsal raphe nuclei, nuclei that innervate the
caudate with dopaminergic and serotonergic terminals, respectively.6 Thus
chlorisondamine appears to be taken up by monoaminergic nerve terminals
expressing the receptors that it blocks, and transported from those terminals
to their parent cell bodies.
Cl
Cl
Cl
O
O
N
Cl
Cl
Cl
Cl
Cl
Cl
N
N
+
a
b
O
N
N
NH2
O
O
Cl
Cl
Cl
1
2
3
4
c
Cl
Cl
Cl
Cl
Cl
Cl
N
d
e
N
N
I
I
4
I
N
Cl
Cl
5
6
Scheme 1. Synthesis of starting materials and standards. Reaction conditions:
(a) Toluene/D (93%), (b) LiAlH4/THF/D (43%), (c) MeI (2.1–3.0 eq.)/90%
EtOH or DMF/1008C (65–68%), (d) MeI (1.1 eq.)/absolute EtOH/208C
(58–71%), (e) MeI (1.05 eq.)/DMF/1008C (58%)
Copyright # 2006 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 471–478

3H-LABELED CHLORISONDAMINE
473
While it is known to accumulate in dopaminergic, serotonergic, and
noradrenergic8 cell bodies, the mechanism of uptake and time-course of
retrograde transport are not known. One possibility is that chlorisondamine is
taken into monoaminergic neurons when blocked nicotinic receptors on those
cells are internalized. The mechanism of this capture and recycling of
chlorisondamine is of considerable interest and could be more easily studied
with a ligand that has high specific activity.
The tritiated chlorisondamine used for these studies6 was prepared, as
previously reported, by tritium exchange9 between chlorisondamine and
tritium gas; a method that provided the material with a specific activity of
3.0 Ci/mmol and 98% purity. We thought that it would be more convenient to
use tritiated methyl iodide as a source of radioactivity since the direct
precursor of chlorisondamine has two nucleophilic sites that can be alkylated
stepwise with methyl halides. This strategy would yield labeled chlorisonda-
mine with very high specific activity and in good yield.
Results and discussion
The starting material, 4,5,6,7-tetrachloro-2-(2-dimethylaminoethyl)-isoindo-
line (4, Scheme 1), was prepared from commercially available tetrachlor-
ophthalanhydride (1) by a condensation10 with N,N-dimethylethylenediamine
(2), followed by reduction11 of the resulting phthalimide (3) with lithium
aluminum hydride in two steps (40% overall yield) utilizing published
procedures (Scheme 1). Two previously reported procedures for bisquaterni-
12
zation to obtain 6 with methyl iodide used refluxing in 90% ethanol
or
dimethylformamide.11 Both procedures call for an excess of methyl iodide
(ca. 5–6 eq.). With the goal of minimizing the amount of methyl iodide used,
the procedures above were modified using sealed vessels (heated to 90–1008C).
Heating of diamine 4 in 90% ethanol in the presence of 3.0 eq. of methyl
iodide for 5 days gave a 68% yield of chlorisondamine (6) after recrystalliza-
tion. Lowering the amount of methyl iodide to 2.1 eq. and shortening the
reaction time (1 day) also gave a satisfactory yield (65%) of 6. However, in
that case the presence of a trace amount of the mono salt 5 was observed in the
1H-NMR spectrum. When DMF was used as a solvent11 a similar results were
obtained.
In 1957, Rosen et al.,11 reported that the nitrogen atoms of 4,5,6,7-
tetrachloro-2-(2-dimethylaminoethyl)-isoindoline (4) displayed remarkably
different reactivity towards various alkylating reagents. The terminal nitrogen
was easily alkylated under mild conditions, while the alkylation of the
isoindoline nitrogen required increased temperatures and higher pressures.
Rosen et al.,11 rationalized this in terms of the negative inductive effect of the
four chlorine substituents on the aromatic ring significantly decreasing the
basicity of the isoindoline nitrogen. According to Rosen et al.,11 this basicity is
Copyright # 2006 John Wiley & Sons, Ltd.
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474
J. ZEZULA ET AL.
further decreased in the presence of the b-ammonio cation-bearing side-chain
group in 5. When both of those influences are present, the basicity of
isoindoline nitrogen was said to be decreased by a factor of about
50 000–65 000 (pKa from 7.7 to 2.9–3.0). They further reported the
monomethochloride, monomethobromide, and monomethiodide salts of 4 in
1957.11,13 We used this differential reactivity to selectively prepare the
monomethiodide 5 by the reaction of 4 with 1.1 eq. of methyl iodide in
absolute ethanol at ambient temperature. The desired product 5 precipitated
out of the reaction mixture and was obtained in good yield (58%) after two
recrystallizations. A second quarternization, using a slight excess of methyl
iodide (1.05 eq., in DMF in a sealed tube) proceeded smoothly to give the
bismethiodide (chlorisondamine, 6) in good yield (58%, recrystallized). A
similar procedure using 90% ethanol gave a lower yield (35%) of
bismethiodide, and it was contaminated with starting material.
Monomethiodide salt 5 was easily prepared and it seemed likely that the
second alkylation with labeled methyl iodide could be accomplished using the
above-mentioned procedure (sealed tube/CT3I/DMF). The labeling was
successfully carried out by Amersham Biosciences using an excess of CT3I
in DMF in a sealed vessel at 1008C to give crude product in >60%
radiochemical purity (Scheme 2). Purification on a reverse phase support gave
pure product (7) with the desired high specific activity (94 Ci/mmol by MS).
Since the addition of three tritium atoms would give a maximum specific
activity of 82 Ci/mmol, there was probably some nonspecific exchange as well.
Mass spectroscopic analysis (MALDI-TOF) of unlabeled material showed
weak molecular peaks (355.12, 357.13, 359.12) and strong base peaks (296.09,
298.09, 299.09) corresponding to the loss of the (CH3)3N moiety. The
spectrum of labeled material showed an analogous pattern, with the
corresponding peaks having a larger mass of 6 amu, confirming the successful
labeling. The labeling on the much less reactive site was very important since
the strongest peak in the MS spectra corresponded to the loss of the alkylated
side-chain nitrogen. The labeled moiety was preserved for easier detection. In
summary, we prepared labeled chlorisondamine 7 with very high specific
Cl
Cl
Cl
Cl
Cl
Cl
CT3
N
a
N
N
I
I
I
N
Cl
Cl
5
7
Scheme 2. Quaternization with labeled methyl iodide. Reaction conditions: (a)
CT3I/DMF/sealed tube/1008C
Copyright # 2006 John Wiley & Sons, Ltd.
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3H-LABELED CHLORISONDAMINE
475
activity and in good yield, and this material should be suitable for the
contemplated biological experiments.
Experimental
Tetrachlorophthalic anhydride (96%), N,N-dimethylethylenediamine (95%),
and methyl iodide (CH3I, 99.5%, stabilized over copper) were purchased
from Sigma Aldrich and used without further purification. Diethyl ether
(Et2O) was freshly distilled from LiAlH4 before use. The melting points were
taken on a Thomas Hoover apparatus and are corrected. The Ace Glass
threaded thick-walled tubes (of ca. 24 ml volume) were used for the
reactions at elevated temperatures, with proper safety shielding. The actual
labeling was performed by Tritium Custom Preparations Group, Amersham
Biosciences, The Maynard Centre, Whitchurch, Cardiff, CF147YT. The
NMR spectra were recorded on a Varian Gemini 300 spectrometer in
d6-DMSO (2.50 ppm). The mass spectra were measured at NIDDK/NIH, at
Amersham Biosciences facilities, and at NIDA/IRP (MALDI-TOF), the latter
using a PE-Biosystems DE-PRO mass spectrometer (Farmingham, MA),
equipped with a nitrogen laser (337 nm) firing at 3 Hz using an a-cyano-4-
hydroxycinnamic acid matrix, prepared as a saturated solution in 50% v/v
EtOH/H2O).
10
4,5,6,7 -Tetrachloro-2-(2-dimethylamino-ethyl)-isoindole-1,3-dione (3)
A solution of N,N-dimethylethylenediamine (2, 13.2 mL, 0.12 mol, 1.20 eq.) in
dry toluene (100 ml) was added dropwise to a solution of tetrachlorophthalic
anhydride (1, 28.6 g, 0.10 mol) in toluene (500 ml) at ambient temperature. The
resulting white suspension was stirred at room temperature for 1 h. A Dean-
Stark trap was attached and the reaction mixture was heated to reflux for 3 h,
by which time the theoretical amount of H2O was collected. After cooling to
room temperature, the reaction mixture was washed with H2O (2 Â 100 ml),
5% NaHCO3 (2 Â 100 ml) and H2O again (2 Â 100 ml). The organic layer was
dried over anhydrous Na2SO4, filtered, evaporated and recrystallized from
CHCl3 (ca. 150 ml) to give the first crop of 3 as pale-yellow crystals (20.96 g,
59%), m.p. 180–1838C (Lit.11 m.p. 183–1858C). Evaporation of mother liquors
and trituration of the residue with toluene (200 ml) gave an additional amount
of 3 as yellow crystals (11.75 g, 33%). HRMS [M-H]+ calculated for
C12H11N2O2Cl4: 354.9575, found 354.9579.
4,5,6,7-Tetrachloro-2-(2-dimethylaminoethyl)-isoindoline (4)11
Tetrachlorophthalimide derivative 3 (10.68 g, 0.03 mol) was added portion-
wise to a stirred suspension of LiAlH4 (4.50 g, 0.12 mol, 4.0 eq.) in dry Et2O
(600 ml) over ca. 20 min. The resulting yellow–green suspension was heated to
Copyright # 2006 John Wiley & Sons, Ltd.
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J. ZEZULA ET AL.
reflux under an argon atmosphere for 3 h, after which the heating was turned
off and the reaction mixture was allowed to stir overnight at ambient
temperature. After cooling in an ice bath to 08C the excess hydride was
carefully quenched with 10% aqueous Na2SO4 (24 ml) and the resulting slurry
was stirred 1 h at room temperature. A fine granular precipitate was filtered
and washed with Et2O (5 Â 75 ml). The pale-yellow filtrate was dried over
anhydrous MgSO4 (ca. 2 h), filtered and evaporated to give crude 4.
Recrystallization from acetone (ca. 20 ml) gave 4 as fine needles (4.24 g,
43%), m.p. 68–69.58C (Lit.11 m.p. 71.5–738C). HRMS [M-H]+ calculated for
C12H15N2Cl4: 326.9989, found 326.9986.
4,5,6,7-Tetrachloro-2-methyl-2-(2-trimethylammoniumethyl)-isoindolinium diiodide
(6): procedure A12
In a thick-walled tube, CH3I (114 mL, 1.83 mmol, 3.0 eq.) was added to a
stirred solution of 4,5,6,7-tetrachloro-2-(2-dimethylaminoethyl)-isoindoline
(4, 0.200 g, 0.61 mmol) dissolved in 90% EtOH (1 ml) The monomethiodide
salt 5 rapidly precipitated. The tube was briefly purged with argon, tightly
capped and heated in an oil bath at 908C for 5 days. After cooling to room
temperature, the reaction mixture was cooled in an ice bath and the precipitate
filtered and washed with absolute EtOH (2 Â 3 ml) to give 0.355 g of crude 6,
which was recrystallized from 95% EtOH (50 ml + ca. 7 ml of H2O) to
furnish 0.255 g of chlorisondamine (6) as white needle-like crystals (68%, m.p.
239–2418C, Lit.11 m.p. 239–2418C). 1H-NMR (d6-DMSO)
d
5.23
(ABq, J=15.0 Hz, 4 H), 4.25 (m, 2 H), 4.01 (m, 2 H), 3.40 (s, 1 H), 3.19
(s, 9 H). 13C-NMR (d6-DMSO) d 132.8, 132.1, 126.7, 70.0, 57.4, 56.7, 53.2
(3 Me), 52.0 (Me). Analytically calculated for C14H20Cl4I2N2: C 27.48%,
H 3.29%, N 4.58%; found: C 27.71%, H 3.34%, N 4.58%.
Procedure B for 4,5,6,7-Tetrachloro-2-methyl-2-(2-trimethylammoniumethyl)-
isoindolinium diiodide (6)
In a thick-walled tube (ca. 24 ml volume), CH3I (78 mL, 1.28 mmol, 2.1 eq.) was
added to a stirred solution of 4,5,6,7-tetrachloro-2-(2-dimethylaminoethyl)-
isoindoline (4, 0.200 g, 0.00061 mol) dissolved in 90% EtOH (1 ml) The
monomethiodide salt 5 quickly precipitated. The tube was briefly purged
with argon, tightly capped and heated in an oil bath at 948C for 1 day.
After cooling to room temperature, the reaction mixture was cooled further in
an ice bath and the precipitate was filtered off and washed with abs. EtOH
1
(2 Â 3 ml) to give 0.318 g of crude product. The H-NMR spectrum (d6-
DMSO) revealed the presence of a small amount of monomethiodide 5 (ca.
1
6% by integration of H-NMR spectra). Recrystallization from 90% EtOH
(ca. 15 ml) gave 0.242 g of chlorisondamine (6) as white crystals (65%, m.p.
239–2418C).
Copyright # 2006 John Wiley & Sons, Ltd.
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3H-LABELED CHLORISONDAMINE
477
Stepwise monoquarternizations
4,5,6,7-Tetrachloro-2-(2-trimethylammoniumethyl)-isoindolinium iodide (5).
CH3I (190 ml, 0.433 g, 0.00305 mol, 1.1 eq.) was added to a stirred solution
of 4,5,6,7-tetrachloro-2-(2-dimethylaminoethyl)-isoindoline
4
(0.910 g,
0.00277 mol) dissolved in absolute EtOH (10 mL). The reaction mixture was
stirred at ambient temperature under an argon atmosphere for 2 days. After
cooling in an ice bath, the precipitate was filtered off and washed with ice-cold
absolute EtOH (2 Â 5 ml), dried and recrystallized from 95% EtOH (ca. 20 ml)
to give 0.990 g (76%) of 5 as a pink/yellow solid (m.p. 241–2438C). The
compound was dissolved in 95% EtOH, boiled with charcoal, filtered hot,
and cooled to give 0.756 g (58%) of 5 as off-white crystals (m.p. 241.5–2438C).
1H-NMR (d6-DMSO) d 4.17 (s, 4 H), 3.52 (t, J=5.4 Hz, 2 H), 3.18 (m, 2 H),
3.14 (s, 9 H), 13C-NMR (d6-DMSO) d 139.6, 129.8, 125.9, 62.4, 58.9, 52.8,
48.5. Analytically calculated for C13H17Cl4IN2: C 33.22%, H 3.65%,
N 5.96%; found: C 32.97%, H 3.64%, N 5.80%. HRMS: [M-H]+ calculated
for C13H17Cl4N2: 341.0146, found 341.0144.
4,5,6,7-Tetrachloro-2-methyl-2-(2-trimethylammoniumethyl)-isoindolinium diio-
dide (6)11. In a thick-walled tube (inner volume ca. 25ml), CH3I was added
(7mL, 0.112 mmol, 1.05eq.) to 4,5,6,7-tetrachloro-2-(2-trimethylammoniu-
methyl)-isoindolinium iodide 2 (0.050g, 0.106 mmol) dissolved in dry N,N-
dimethylformamide (1 ml). The tube was briefly purged with argon, tightly
capped and heated in an oil bath at 1008C for 1 day. After cooling to room
temperature, the reaction mixture (dark-brown solution) was diluted with
acetone (3ml) and cooled to 08C. The precipitate was filtered and recrystallized
from 95% EtOH (7 ml plus ca. five drops of H2O) to give 6 as white crystals
(0.038 g, 58%), m.p. 241–242.58C). Analytically calculated for C14H20Cl4I2N2:
C 27.48%, H 3.29%, N 4.58%; found: C 27.63%, H 3.25%, N 4.37%.
4,5,6,7-Tetrachloro-2-CT3-2-(2-trimethylammoniumethyl)-isoindolinium diiodide
(7). [3H]chlorisondamine was prepared by combining monomethiodide 5
(10.1 mg, 0.02 mmol) in a borosilicate glass tube with N,N-dimethylformamide
(1 ml) and [3H]methyl iodide (10 Ci, total activity) and heating the mixture at
1008C for 16 h. The volatile materials were pumped off at high vacuum, labile
tritium removed by repeated evaporation of EtOH (3 Â 1 ml), and the orange
residue was dissolved in EtOH: H2O mixture (3:1; 40 ml). Yield: 839 mCi.
Crude radiochemical purity: >60%, specific activity: 94 Ci/mmol. Purified
(ꢀ100 mCi) by column chromatography (SepPak C18; 3 cm3; 500 mg) eluting
with MeOH: H2O: 55% hydriodic acid (80:20:0.2). Desired fractions were
collected and analyzed. Yield: ꢀ40 mCi. Radiochemical purity was ascer-
tained by thin layer chromatography on Whatman LKC18F in: (i) MeOH:
H2O: trifluoroacetic acid (90:10:0.2): 97%, (ii) MeOH: H2O: 55% hydriodic
Copyright # 2006 John Wiley & Sons, Ltd.
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J. ZEZULA ET AL.
acid (80:20:0.2): 96% and also on cellulose (Merck 5718) in 1-butanol
saturated with 0.1 M ammonia to be 97%. Labeled material co-eluted with the
non-labeled material and the mass spectrum was consistent with the proposed
structure and non-labeled reference. TOF MS ES+ (Amersham):
C14H20Cl4N22+: m/z: 178.0285, 179.0253, 180.0248. Labeled material:
C14H17T3Cl4N22+: 181.0381, 182.0377, 183.0345.
Acknowledgements
This research was supported by the NIH Intramural Research Program of the
National Institute of Diabetes and Digestive and Kidney Diseases and the
National Institute on Drug Abuse. We (LMC, NIDDK) thank the National
Institute on Drug Abuse, NIH, DHHS for partial support of this work. We
also thank John D. Lloyd, NIDDK/NIH for some of the mass spectra and
assistance with interpretation of the results.
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