Synthesis and evaluation of tritium labelled 10-methylgalanthamine iodide: A novel compound to examine the mechanism of interaction of galanthamine derivatives with the nicotinic acetylcholine receptors

Article abstract of DOI:10.1002/jlcr.744

A new promising galanthamine derivative, 10-[ ³H]methylgalanthamine iodide, was synthesized for binding studies to nicotinic acetylcholine receptors expressed in Torpedo electric ray electroplaques. Galanthamine was reacted with [³H]methyl iodide to yield 10-[³H]methylgalanthamine iodide with a radiochemical yield of > 70% and a specific activity of 32 Ci/mmol after purification via solid phase extraction. To test the ligand properties of the radioligand, calcium imaging and electrophysiology of the non-radioactive analogue were performed to obtain an EC₅₀ of 270 nM, a Hill coefficient of 1.9 and the induced cell current. Copyright

Full text of DOI:10.1002/jlcr.744

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2003; 46: 1117–1125.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.744
Research Article
Synthesis and evaluation of tritium labelled
10-methylgalanthamine iodide: a novel compound
to examine the mechanism of interaction of
galanthamine derivatives with the nicotinic
acetylcholine receptors
1
,2
1,
1
Andreas Schildan , Ralf Schirrmacher *, Esther Schirrmacher ,
3
Marek Samochocki , Claudia Christner , Alfred Maelicke and
3
3
1
Frank R o. sch
1
Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz,
Fritz Strassmann-Weg 2, Mainz D-55128, Germany
2
Clinic of Nuclear Medicine, University of Leipzig, Liebigstr. 20a,
Leipzig D-04301, Germany
3
Institute of Physiological Chemistry und Pathobiochemistry,
Johannes-Gutenberg-University, Duesbergweg 6, Mainz D-55128, Germany
Summary
3
A new promising galanthamine derivative, 10-[ H]methylgalanthamine iodide,
was synthesized for binding studies to nicotinic acetylcholine receptors
expressed in Torpedo electric ray electroplaques. Galanthamine was reacted
3
3
with [ H]methyl iodide to yield 10-[ H]methylgalanthamine iodide with a
radiochemical yield of >70% and a specific activity of 32 Ci/mmol after
purification via solid phase extraction. To test the ligand properties of the
radioligand, calcium imaging and electrophysiology of the non-radioactive
analogue were performed to obtain an EC50 of 270 nM, a Hill coefficient of 1.9
and the induced cell current. Copyright # 2003 John Wiley & Sons, Ltd.
Key Words: nicotinic acetylcholine receptor (nAChR); acetylcholinesterase
inhibitor (AChE-I); allosterically potentiating ligand (APL); galanthamine;
tritium
*Correspondence to: R. Schirrmacher, Institute of Nuclear Chemistry, Mainz D-55128, Germany.
E-mail: schirrmacher@mail.kernchemie.uni-mainz.de
Copyright # 2003 John Wiley & Sons, Ltd.
Received 12 May 2003
Revised 9 June 2003
Accepted 24 June 2003

1
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A. SCHILDAN ET AL.
Introduction
Galanthamine ð1Þ belongs to a new class of non-competitive nicotinic
acetylcholine receptor agonists which act as allosterically potentiating
1
ligands (APL). As APLs they can enhance the sensitivity of the
receptor towards acetylcholine (ACh), resulting in improved nicotinic
cholinergic neurotransmission. The binding site of APLs was found to
reside within the extracellular part of the a-subunit of nAChR Torpedo
electrocytes (cf. Figure 2) and the mechanism of action of these ligands
2
,3
was clarified. The structure–activity relationship of APLs is not yet
fully understood because most of them interact with additional target
molecules in the mammalian brain. Galanthamine for example is not
only an APL, but also an acetylcholinesterase inhibitor (AChE-I). From
a medical standpoint, APL can intensify the release of different
3
neurotransmitters via a receptor-mediated mechanism. [ H]Tritium
4
labelling of galanthamine has previously been reported. Because of
its higher polarity, 10-methylgalanthamine iodide is less likely to
partition into the membrane than galanthamine itself and it is thus
particularly useful for in vitro applications. The tritiated 10-methylga-
lanthamine iodide ð2Þ was therefore synthesized for a binding assay
using torpedo-fish membrane fragments and the a4 nAChR subtype
5
according to an already established method (Figure 1).
Results and discussion
The non-radioactive standard compound 10-methylgalanthamine io-
dide, which was needed for analytical purposes such as HPLC, was
4
H
H 4
3
H
H
2
O
H
O
H
4a
H
H
1
O
H 12
11
I
H C
3
H
11
9
H
H12
+
9
H
H 7
N
CH3
H
8
CH3
Figure 1. Structure of 10-methylgalanthamine iodide
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1117–1125

SYNTHESIS AND EVALUATION OF TRITIUM LABELLED
1119
1
20
00
1
60
40
EC50 = 0.27 ± 0.48 µM
Hill-Koeffizient = 1.9 ± 0.4
1
8
0
0
0
0
6
1
4
2
0
120
-
20
1E-3
0.01
0.1
log (10-N-Methylgalanthaminium iodid [µM])
1
00
8
0
6
0
0
5
10
0-N-Methylgalanthaminium iodid [µM]
Figure 2. Effect of 10-methylgalanthamine iodide on the dose–response curve for
15
20
25
1
2
+
nicotine-activated Ca
influx into HEK-293 cells expressing the human
a4b2 nAChR subtype
synthesized by a substitution reaction of methyl iodide and galantha-
6
mine in yields of 80%. Several in vitro studies were performed
to evaluate the pharmacological properties of 10-methylgalanthamine
iodide. Galanthamine and 10-methylgalanthamine iodide were
compared according to their APL effect. Under conditions of the
standard calcium imaging assay, galanthamine did not produce any
electric signals in HEK-293 cells expressing the a4b2 subtype of
nAChR, probably because it largely partitioned into the cell membranes
instead of binding to the receptor and activated its integral cation
channel. However, when 10-methylgalanthamine iodide was used
instead, a marked increase of the nicotinic response was observed.
Submicromolar concentrations of 10-methylgalanthamine iodide re-
2+
sulted in a significant increase in Ca influx into the HEK-293-cells. At
2
+
a concentration of 0.5 mM, potentiation of nicotine-induced Ca influx
by 54% was measured. Further increase of 10-methylgalanthamine
2
+
iodide led to inhibition of Ca
the nAChR ion channel (cf. Figure 2). For the potentiating effect of
0-methylgalanthamine on the agonist-induced nAChR response an
influx due to direct blockade of
1
Copyright # 2003 John Wiley & Sons, Ltd.
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A. SCHILDAN ET AL.
co-applicated: 0.5 µM Galanthamine
0.5 µM von 10-methyl-
Galanthaminium iodid
Figure 3. Whole-cell recordings of the effect of galanthamine (1) and 10-
methylgalanthamine iodide on nicotine-induced currents through human
a4b2 nAChR expressed in HEK-293 cells. Nicotine was applied at 50 lM alone
or together with indicated galanthamine derivative. Measurements were done at
holding potential ꢁ70 mV
OH
OH
O
O
3
C H I
O
3
O
H C
H C
3
3
I
toluene, 1 d, RT
+
N
N
H C
3
7
0 %
C H
3
(1)
CH3
(2)
3
3
Figure 4. Radioactive labelling of galanthamine (1) using [ H]methyl iodide
EC50 value of 270 ꢀ 48 nM and a Hill coefficient of 1.9 ꢀ 0.4 were
calculated.
2+
The results of the Ca imaging experiments are supported by similar
findings obtained by electrophysiological measurements (cf. Figure 3).
3
The synthesis of 10-[ H]methylgalanthamine iodide was achieved with
3
0 mCi of [ H]methyl iodide in toluene (10 mCi/ml) at a specific activity
1
of 80 Ci/mmol and 10 mg (34.8 mmol) galanthamine ð1Þ (cf. Figure 4).
After a reaction time of 24 h at room temperature, the synthesized 10-
3
[
H]methylgalanthamine iodide was purified according to a procedure
by Mulholland et al. for the removal of solvent and by-products from
Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1117–1125

SYNTHESIS AND EVALUATION OF TRITIUM LABELLED
1121
11
7
quaternary [ C]methyl-derivatives of amines. After fixation of the
tritiated product on a silica gel cartridge, the product was eluted
with acetic acid (2%). Obviously, the acetic acid interrupts the strong
ionic interactions of the quaternary amine with the material of the
cartridge by protonating anionic binding sites such as silanoate or
other anionic groups in the SiO matrix thus resulting in the elution of
2
the species. The advantage of this method is that the final product
is obtained in a small volume of a solution which can be used for
most biological studies either directly or after adjusting the pH. By
successive elution with ethanol/toluene and pure ethanol the solvent
and the educt were removed. A non-identified polar by-product was
separated by using a small volume of 1% acetic acid before elution
of the product from the cartridge. After work-up, the pure product
could be isolated in radiochemical yields of >70%. By comparison
to the corresponding UV-calibration curve, a specific activity of
3
2 Ci/mmol was determined which is sufficient for the planned binding
assays.
Experimental
3
N.c.a. [ H]methyl iodide was purchased from American Radiolabelled
1
Chemicals and the Silica-Sep-Pak -Plus cartridge from Waters.
Galanthamine hydrobromide ð1Þ was supplied from the Institute of
Physiological Chemistry and Pathological Biochemistry of the Uni-
versity of Mainz. The analysis of the eluted fractions was performed
1
with radio-HPLC. To the eluent Omni-Szintisol by Merck, a cocktail
for scintillation measurements was added [eluent/scintillator 1/2 (v/v)]
prior to the radio-detection. The HPLC system comprised the following
components: UV detector Thermo Separation Products LDC spectro-
monitor 3000; radio detector Berthold Pseudo-LB 506 C-1 with a Z-200-
4
cell; scintillation-adding pump Berthold LB 5035-3; HPLC pump by
Sykam S1121; injection valve Rheodyne 7125. The eluent was methanol/
.05 M Na HPO [60/40 (v/v)] at a flow rate of 1 ml/min. UV detection
0
2
4
was performed at 285 nm. An analytical column by Merck, LiChrospher
1
00 RP 18 – 5 EC (250 ꢂ 4.6 mm) was used. The methanol used as eluent
was especially designed for scintillation measurements, the phosphate
1
buffer was LiChropur -grade and both were purchased from Merck.
For analysing the radiochemical yields, well-defined volumes of the
different eluate fractions were taken with a Hamilton syringe, mixed
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A. SCHILDAN ET AL.
with 4 ml liquid scintillator (Aquasafe 300 by Zinser Analytic) and
measured in a Beckman Coulter LS 6500 scintillation counter.
NMR spectra were recorded on a Bruker 400-MHz-FT-NMR
spectrometer DRX 400. The chemical shifts are quoted in d (ppm),
coupling constants in Hz. IR spectra were recorded on a Mattson-FT-
IR spectrometer Galaxy 2030 with KBr pills. Other instruments used:
CHNS-Analysator Vario EL II by Elementar Analysensysteme for
elemental analyses; Finnigan MAT 90 by Thermo Electron for mass
spectrometry; Perkin–Elmer Lambda 20 for UV/Vis spectrometry and
Perkin–Elmer Polarimeter 241 for determination of optical rotations.
Uncorrected melting points were determined with a Electrothermal IA
Series 9100.
Synthesis of 10-methylgalanthamine iodide
Galanthamine ð1Þ (0.72 g/2.51 mmol) was dissolved in 120 ml dry
diethylether. Two hundred and thirty five microlitres (3.78 mmol,
1
.51 eq.) of methyl iodide was added and the mixture stirred under
protection from light in an argon atmosphere for 15 h at room
temperature. The white precipitate formed was filtered off, washed with
diethylether and dried under vacuum (10ꢁ mbar). Recrystallization
from hot water and methanol yielded 0.88 g of the desired product
3
(
2.05 mmol, 82%) as fine, colourless needles.
1
H-NMR-spectrum (in D O at 298 K, suppression of water signal by
2
0
CW-presaturation): 2.04 (1 H; br s; H12 ); 2.12 (1 H; ddd; 16,4; 5,3; 3,1;
0
00
00
H4 ); 2.32 (1 H; br s; H12 ); 2.46 (1 H; dt; 16,4; 1,5; H4 ); 2.87 (3 H; s;
+
+
N -CH ); 3.31 (3 H; s; N -CH ); 3.58 (1 H; m; H11 ); 3.81 (3 H; s;
3 3
0
|
0
0
-OCH ); 4.04 (1 H; m; H11 ); 4.24 (1 H; br t; 4,9; H3); 4.30 (1 H; br d;
3
0
1
3,9; H9 ); 5.99 (1 H; dd; 9,8; 4,9; H2); 6.12 (1 H; br s; H1); 6.83 (1 H; br
0
0
d; 8,3; H8); 6.91 (1 H; d; 8,3; H7). The signals for H4a and H9 , which
should lie between 4.5 and 5.0 ppm, are missing as expected from the
CW-presaturation; in NMR experiments at higher temperatures these
signals could be detected by corresponding downfield shifts mass
spectrometry (FD) m/z (% rel. int.): 287.7 (6.5%, galanthamine cation);
3
03.7 (100%, 10-methylgalanthamine); 304.7 (18.3%, isotope peak) the
galanthamine elemental analysis: found: C 47.95%; H 5.38%; N 3,28%
(
(
calculated for C H NO I ꢃ H O: C 48.33%; H 5.86%; N 3.13%).
IR (KBr) [n (intensity)]: 3555 (s); 3500–3100 (br m); 3010 (w); 2980
w); 2935 (m); 2900 (w); 1625 (m); 1590 (m); 1480 (s); 1435 (s); 1375 (m);
18
24
3
2
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND EVALUATION OF TRITIUM LABELLED
1123
1
280 (s); 1235 (m); 1215 (m); 1195 (m); 1175 (s); 1065 (s); 1030 (m); 1005
s); 960 (s); 935 (m); 885 (m); 870 (m); 820 (m); 805 (s); 780 (m); 665 (w);
00 (w).
UV (100% EtOH [Uvasol ]): l
(
6
1
¼ 288:41 nm,
max
25
optical rotation: ½aꢄ ¼ ꢁ98:78(c ¼ 0; 55 [g/100 ml]; H O),
D
2
melting point (uncorrected): 280–2838C (decomposition).
3
Synthesis of 10-[ H]methylgalanthamine iodide
3
The synthesis of 10-[ H]methylgalanthamine iodide was achieved by
3
using 10 mCi of [ H]methyl iodide galanthamine ð1Þ (9.9 mg/34.5 mmol)
was dissolved in 1 ml of toluene in a 5 ml reaction vessel flooded with
3
argon. One millilitre of [ H]methyl iodide solution cooled to ꢁ788C was
subsequently transferred via a syringe to the reaction vessel. The
reaction mixture was stirred for 24 h and protected from light. After
dilution with 2 ml ethanol the product was loaded onto a silica cartridge
containing 690 mg of silica gel, which had been conditioned with 20 ml
of an ethanol/toluene mixture [50/50 (v/v)]. The elution of the educt, a
polar by-product presumably formed as a consequence of radiolysis and
the desired product was achieved by successive elution with 20 ml
ethanol/toluene [50/50 (v/v)], 5 ml ethanol, 6 ml 1% acetic acid and
finally several 5 ml fractions of 2% acetic acid. After passing of each
eluent the cartridge was dried with helium. Each fraction was analysed
by HPLC and its radioactivity was measured.
Calcium imaging
With using membrane-permeable fura-2/AM indicator calcium-imaging
measurements were performed to evaluate the effect of the galanthamine
2+
derivatives on nicotine-induced Ca
influx into HEK-293 cells
transfected with a4b2 subtype of nAChR. Cells grown on fibronectin-
coated coverslips were loaded for 1 h at room temperature with 4 mM
2
+
Fura-2/AM (Molecular Probes, USA) and 2 mM Ca
dissolved in
HBSS (10 mM HEPES, 5 mM glucose, 1.3 mM Na HPO , 4 mM
2
4
NaHCO , 137 mM NaCl, 0.4 mM MgSO , 0.5 mM MgCl , 0.4 mM
3
4
2
KH PO , 5.4 mM KCl, pH 7.4). After loading, the ester groups of
2
4
fura-2/AM were immediately cleaved to yield a free, membrane-
inpermeable fura-2 chelator. Measurements were performed in 2 mM
Copyright # 2003 John Wiley & Sons, Ltd.
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A. SCHILDAN ET AL.
2
+
Ca -containing HBBS, pH 7.4 using an imaging system consisting of
an Axiolab 100 microscope (Zeiss, G o. ttingen, Germany), equipped with
an XBO 75W xenon lamp (Osram, M u. nchen, Germany), a TE-1400
CCD camera (Visitron, Puchheim, Germany), a Ludl MAC 2000
controller (Ludl Electronic Products, Hawthorne, USA) and a Physick
LVPS focus device (Visitron, Puchheim, Germany). 10-Methylgalantha-
mine iodide at a concentration range of 0–25 mM, dissolved in 2 mM
2
+
Ca -con-taining HBSS, was added to the cells together with 50 mM
nicotine. Under control condition, 50 mM nicotine was applied alone.
The emission fluorescence intensity at 510 nm was measured at two
excitation wavelengths of 340 and 380 nm in a 5-s interval. Data analysis
was done using a software from Hamamatsu and corresponding EC s
5
0
and Hill coefficients were calculated with Microcal Origin 5.0 software.
Electrophysiology
Whole-cell current recordings were performed using an LM-EPC-7
patch-clamp system (List, Darmstadt, Germany) on nAChR-expressing
HEK-293 cells cultured 3 days on fibronectin-coated cover slips. The
bathing solution was composed of (mM): NaCl 145; KCl 5; MgCl 1;
2
CaCl 2; D-glucose 10; HEPES 10 (pH 7.3; 300 mOsm), and the internal
2
pipette solution contained (mM): CsCl 140 (equilibrated with CsOH);
EGTA 11; HEPES 10; MgCl 1, (pH 7.3; 300 mOsm). The patch
2
microelectrodes were made from borosilicate glass (external diameter
1
.6 mm), and the pipette resistance was measured as 5–7 MO when filled
with the internal solution. After formation of a high-resistance seal with
the cell membrane, capacitance transients were minimized using the
C-Fast facility of the system. No additional capacitance and serial
resistance compensation was applied. All experiments were performed
at room temperature at a holding potential of ꢁ70 mV. Whole-cell
currents were induced by fast application of 10-methylgalanthamine
iodide, dissolved in external solution, using a U-shaped tube positioned
near the investigated cell, at a flow rate of 0.5–1.0 ml/min. To prevent
accumulation of the test compounds in the bath, the cells were
superfused with the bathing solution at the same flow rate. In most
experiments, in order to inhibit intrinsic muscarinic responses, 1 mM
atropine was included in the bathing solutions and in the solutions
applied via the U-tube. Signals were filtered at 3.15 kHz (Bessel),
Copyright # 2003 John Wiley & Sons, Ltd.
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SYNTHESIS AND EVALUATION OF TRITIUM LABELLED
1125
digitized to 10 kHz and analysed on a PC using the pClamp software
package version 6.03 (Axon Instruments, Foster City, USA).
Conclusion
3
We have synthesized 10-[ H]methylgalanthamine iodide in an overall
radiochemical yield of >70% and a specific activity of 32 Ci/mmol in
order to investigate the mechanism of interaction of galanthamine with
nicotinic acetylcholine receptors. In calcium imaging, the non-radio-
active standard compound 10-methylgalanthamine ð2Þ showed a distinct
increase of nicotinic response whereas for the original galanthamine,
performance was not applicable due to its tendency to deposit in cell
membranes. The EC50 value of 270 nM and a Hill coefficient of 1.9 for
ð2Þ were calculated from the potentiating effect. Additionally, electro-
physiological measurements showed a significant potentiation of the
nicotinic effect for ð2Þ compared to galanthamine itself. Therefore,
compound ð2Þ seems to be a valuable new candidate for investigating the
mechanism of interaction of galanthamine derivatives with nicotinic
acetylcholine system.
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Copyright # 2003 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2003; 46: 1117–1125