Imaging agents for myeloperoxidase

Article abstract of DOI:10.1002/jlcr.2961

The myeloperoxidase (MPO) inhibitors 1 and 2 were prepared as their isotopologues with carbon-14, carbon-13, and nitrogen-15 or tritium with high specific activity and purity. Starting from potassium [¹⁴C]cyanide or [¹⁴C]formate prov

Full text of DOI:10.1002/jlcr.2961

Research Article
Received 15 May 2012,
Revised 9 July 2012,
Accepted 29 July 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.2961
Imaging agents for myeloperoxidase
a
c
b
*
Jonas Malmquist, Alexandra Bernlind, Maria Johansson,
Anders Juréus,d and Maria Nilssond
The myeloperoxidase (MPO) inhibitors 1 and 2 were prepared as their isotopologues with carbon-14, carbon-13, and
nitrogen-15 or tritium with high specific activity and purity. Starting from potassium [14C]cyanide or [14C]formate provided
metabolically stable 14C-labels on [14C]-1 and [14C]-2. Catalytic hydrogenation was used for the preparation of [3H]-2, giving
multiple enriched positions as shown by 3H NMR. 1 and 2 are promising in vitro and in vivo imaging radioligands and have
the potential to provide key information with regard to MPO expression, function, stoichiometry, and pharmacology.
3
Keywords: isotopically labeled synthesis; H; 13C; 14C; 15N; MPO; SIL; xanthine; imaging; NMR; QWBA; autoradiography
Further binding and safety experiments were needed in sup-
port of the development of the enantiomerically pure xanthine
1d. The label in the 6-position was possible to use for these
Introduction
Myeloperoxidase (MPO) is a prime candidate for promoting
oxidative stress during inflammation. This abundant enzyme of
neutrophils uses hydrogen peroxide to oxidize chloride to highly
experiments, as this synthetic route is faster and higher yielding.
Using lithium [14C]formate,6 prepared by treating [14C]-carbon
reactive and toxic chlorine bleach. 2-Thioxanthines1 and pyrrolo
[3,2-d]pyrimidin-4-ones2 have been identified as potent mechan-
ism-based inactivators of MPO, as these inhibitors become cova-
lently attached to the heme prosthetic groups of the enzyme.3
This report describes the isotopologue syntheses of some thiox-
anthines 1 and pyrrolo-pyrimidone 2 (Figure 1), used for imaging
in in vitro and in vivo binding experiments.
dioxide with superhydride or sodium [14C]formate, the label was
introduced in the 6-position of the xanthine (Scheme 2). Com-
pound [14C]-1d was isolated in 12–20% yield with a specific
activity of 2.1 GBq/mmol at a very high radiochemical, UV, and
enantiomeric purity. The compound was then administered to
rats for a quantitative whole body autoradiography experiment.
The tissue distribution of the MPO ligand [14C]-1d showed a high
radioactive concentration in the thyroid gland. The finding is a
result of the MPO ligand’s cross-reactivity of the thyroid peroxi-
dase. The results of that study are shown in Figure 3.
Results and discussions
14C-label in the thiocarbonyl position was needed to investi-
Pyrrolo[3,2-d]pyrimidin-4-one labeled with C-14 was required
also. Compound [14C]-2 (Scheme 3) was prepared by reacting
benzoyl [14C]isothiocyanate5 5 with the amine 13 in acetone at
elevated temperature. This was followed by a base catalyzed
cyclization to obtain the pyrimidone [14C]-2. The product was
isolated by preparative HPLC to obtain [14C]-2 in good specific
activity and high purity. The stability pattern of this compound
reflects that previously observed with [14C]-1a and [14C]-1b; it
was stable when stored long term as a solid but rapidly decom-
posed in solution.
A
gate the binding of 1 and 2 to MPO. Use of [14C]potassium thio-
cyanate4 3 to prepare benzoyl [14C]isothiocyanate5 5 provided a
good starting point for the preparation of xanthine derivatives
with the label in the 2-position. The syntheses of [2-14C]xanthine
derivatives of 1a and 1b were initiated by coupling the corre-
spondingly substituted 1ꢀ amine with 5 to give benzamides 6a
and 6b, respectively (Scheme 1). The benzamides were then
hydrated under basic conditions to afford the corresponding
ureas 7a and 7b. The ureas were condensed with ethyl 2-cyanoa-
cetate in sodium ethoxide to give the dihydropyrimidones 8a
and 8b, and a nitroso group was introduced using sodium nitrite
in acetic acid to give 9a and 9b. Portionwise addition of sodium
dithionite to 9a or 9b at elevated temperature produced 10a or
10b, respectively, and a condensation with formic acid provided
the substituted xanthines [14C]-1a and [14C]-1b in good specific
activity and ~100% purity. The substances decomposed (e.g.,
dimerization) to 95% over 2 weeks when stored in ethanol solu-
tion at ꢁ20 ꢀC. In contrast, the solid stability was almost 2 years,
showing <1% radiolysis impurity traces on HPLC at that time.
The same route was used to prepare stable isotope-labeled
derivatives [13C,15N]-1a, [13C,15N]-1b, and [13C,15N]-1c using
potassium [13C,15N]thiocyanate, sodium [15N]nitrite, and [13C]
formic acid in the corresponding transformations (Figure 2).
aIsotope Chemistry, Screening and Profiling Global DMPK IM, AstraZeneca,
Research and Development | Innovative Medicines, SE-151 85 Södertälje, Sweden
bGlobal Distribution Imaging, Screening and Profiling Global DMPK IM, AstraZeneca,
Research and Development | Innovative Medicines, SE-151 85 Södertälje, Sweden
cMedicinal Chemistry, CNSP iMed Science Södertälje, AstraZeneca, Research and
Development | Innovative Medicines, SE-151 85Södertälje, Sweden
dNeuroscience, CNSP iMed Science Södertälje, AstraZeneca, Research and
Development | Innovative Medicines, SE-151 85Södertälje, Sweden
*Correspondence to: Jonas Malmquist, Isotope Chemistry, Screening and Profiling
Global DMPK IM, AstraZeneca, Research and Development | Innovative Medicines,
SE-151 85 Södertälje, Sweden.
E-mail: jonas.malmquist66@gmail.com
J. Label Compd. Radiopharm 2012
Copyright © 2012 John Wiley & Sons, Ltd.

J. Malmquist et al.
O
N
O
N
O
N
O
N
H
N
H
N
15NH
13CH
N
H
N
H15
13C
N
HN
HN
O
HN
N
S
N
S
N
S
S
O
F
[
13C,15N]-1a
1a
1b
1c
O
N
O
H
H
N
N
K13C15
N
HN
HN
Na15NO2
H13CO2H
O
N
S
S
N
O
N
15NH
13CH
N
H
15N
13C
15NH
13CH
N
H15
13C
N
O
S
N
N
S
1d
2
Figure 1. Thioxanthines 1 and pyrrolo-pyrimidone 2 of which isotopologues have
been developed.
F
13C,15N]-1b
[
13C,15N]-1c
[
To perform high resolution imaging, a tritiated isotopologue
was needed. No success was found reacting 10 or 11 with
tritiated formate to give tritium-labeled 1, presumably because
Figure 2. Summary of stable label isotopologues of 1 prepared.
of the acidity of the position giving rapid tritium/proton dehalogenation: tritium was incorporated on the pyrrole, the
exchange. Iodination of 1a and 1b was unsuccessful using pyridine, and the benzylic positions of [3H]-2 (Figure 4a, b and
N-iodo succinimide and trifluoroacetic acid at ambient tempera- Table 1). The tritium contained in the CH2 was a metabolically
ture and at 100 ꢀC. Bromination of 2 using bromine in acetic acid stability concern. Tritium located in benzylic positions has been
was successful, but tritio-dehalogenation failed to provide [3H]-2, observed to exchange with water in biological systems, which
probably because of the sulfur poisoning the catalysts. To could lead to an elevated background in the autoradiography
circumvent this, pyrrole 13 was brominated (Scheme 4) with image. Therefore, the tritiated substance was treated with palla-
bromine in acetic acid, and 15 was isolated in >50% yield. The dium on carbon in absolute ethanol overnight. However, the
dibromide was targeted so that a higher specific activity com- benzylic tritons of [3H]-2 were tightly bound and were not
pound would be prepared by tritiodehalogenation. Tritiodehalo- exchanged by this method. Therefore, [3H]-2 was used in the
genation of 15 was performed at subatmospheric pressure with binding experiments, and no elevated background problem
tritium gas and palladium oxide. The thiourea function was then was observed. The tritiated sample was stable for several weeks
introduced with benzoyl isothiocyanate, and a base catalyzed in ethanol solution before it started dropping drastically in
ring closure at elevated temperature gave the target pyridinyl purity, for similar reasons as for [14C]-2. Additives that are often
[3H]-2 in 7% yield from di-bromide 15. Preparative HPLC purified used to slow radiochemical decomposition—such as ascorbic
[3H]-2 had a specific activity of 1.6 TBq/mmol and a radio-HPLC acid—resulted in accelerated decomposition instead.
3
purity of 97%. H NMR analysis of the product showed tritium
An example of in vitro binding of [3H]-2 in spinal cord is shown
incorporation in several positions7 besides those expected from in Figure 5. The MPO ligand [3H]-2 could specifically label
O
H
14C
H
R-NH2
KS14CN
N
N
N
+
Cl
14C
R
acetone
acetone
S
O
O
S
O
3
4
5
6a/b
O
O
O
N
H
N
NH2
O
KOH
HN
14C
HN
14C
N
NaNO2
AcOH
14C
R
EtOH/H2O
EtONa
EtOH
NH2
8a/b
NH2
S
S
N
R
S
N
R
7a/b
9a/b
O
O
O
H
N
H
N
O
OH
HN
14C
HN
14C
NH2
NH2
Na2S2O4
NH3(aq)
HN
14C
H
N
N
S
N
S
N
S
N
R
ii) NaOH
14C]-1a
[
14C]-1b
10a/b
[
O
F
Scheme 1. Synthesis of carbon-14-labeled thioxanthines 1 in the thio-carbonyl position. R = tetrahydrofurfuryl for compounds a and 4-fluorobenzyl for compounds b.
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J. Malmquist et al.
O
14C
O
Superhydride
THF
O
N
O
N
H
N
NH2
NH2
HN
O
HN
O
14CH
N
O
14C
i) EDAC/DMF
ii) NaOH
+
S
S
H
O-
11
12
[
14C]-1d
Scheme 2. Synthesis of xanthine 1d having the carbon-14 label in position 6.
brain
muscle
kidney
thyroid gland heart
liver
caecum
testis
O
Figure 3. Autoradiogram showing tissue distribution at 4 h after oral administration of [14C]-1d to a male rat.
O
O
H
N
H
N
H
O
N
HN
14C
O
EtONa
N
+
5
N
S
HN
14C
N
acetone,
reflux
EtOH
65°C
NH
S
O
N
14C]-14
[
14C]-2
N
Tot.: 6 %
13
[
Scheme 3. Synthesis of pyrrolo-pyrimidone 2 14C-labeled in the thio-carbonyl position.
O
H
N
HN
3H
O
O
O
O
i) SCNCOBn
CH Cl
3H
3
2
2
H (807 mbar)
S
N
2
Br
2
H
H
N
N
3H
3H
PdO
13
NH
Br
NH
3H
3H
ii) 2% NaOH
EtOH, 55-65°C
o.n.
HOAc
N
N
3H
Et N
3
DMF
3H
3H
3H
[3H]-13
3H
Br
N
[3H]-2
15
3H
Scheme 4. Three-step synthesis of tritiated pyrrolo-pyrimidone 2.
gas was handled in a tritium gas manifold system from RC TRITEC AG,
Teufen. [14C]-Carbon dioxide was handled in a [14C]-carbon dioxide gas
manifold system from RC TRITEC AG, Teufen.
inflammatory lesions in the spinal cord that also was positive for
CD68,a marker for phagocytic microglia/macrophages that are
abundant in experimental autoimmune encephalomyelitis
(EAE) lesions. MPO is expected to be expressed in the same cell
population, suggesting [3H]-2 to be an in vitro imaging ligand
for MPO in the rat EAE spinal cord.
3H and 1H spectra were recorded on a Bruker DRX600 NMR Spectro-
meter, operating at 640 MHz for tritium and at 600 MHz for proton,
equipped with a 5 mm 3H/1H SEX probehead with Z-gradients. 1H
decoupled 3H spectra were recorded on samples dissolved in CD3OD.
3
For H NMR spectra referencing, a ghost reference frequency was used,
as calculated by multiplying the frequency of internal tetramethylsilane
in a 1H spectrum with the Larmor frequency ratio between 3H and 1H
(1.06663975), according to the description by Al-Rawi et al.8 1H spectra
were referenced to tetramethylsilane that was set to 0 ppm. Mass spectra
Experimental
General methods
All solvents used were analytical grade and commercially available. were recorded on a Waters liquid chromatography–mass spectrometry
Anhydrous solvents were routinely used for reactions. Reactions were (LCMS) consisting of a Waters 1525 micro (LC), Waters PDA 2996, an ELS
typically run under an inert atmosphere of nitrogen or argon. Tritium detector (Sedex 75), and a ZMD single quadrupole mass spectrometer.
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J. Malmquist et al.
Figure 5. Images of adjacent cryo-cut tissue sections from experimental autoim-
mune encephalomyelitis (EAE) rat spinal cord comparing the binding distribution
of [3H]-2 to the microglia marker ED-1. (A) Low magnification autoradiogram of
10 nM [3H]-2 bound to rat EAE spinal cord. (B) Magnification of outlined area with
high level of [3H]-2 binding (C) ED1 immunostaining on consecutive sections to
(A). (D) Higher magnification of outlined area shown in (C).
HPLC. Enantiomerical purity was performed on a ReproSil AM (Chiral,
4.6 ꢂ 250 mm, 5 mm) using a mobile phase of 0.01% trifluoroacetic acid
and acetic acid in methanol at 0.5 mL/min and 40 ꢀC.
Preparative chromatography was run on a Kromasil column (C8, 5 mm,
10 ꢂ 250 mm or 20 ꢂ 250 mm) using acetonitrile/50-mM ammonium
acetate in MilliQ Water at 2–15 mL/min.
Liquid scintillation analysis was performed on a PACKARD TRI-CARB
2900TR. Thin layer chromatography (TLC) was performed on Merck
TLC-plates (Silica gel 60 F254) and UV light (254 nm) was used to visualize
the spots. Flash column chromatography was performed on a Redisep™
prepacked column.
Figure 4. (a) NMR of methylene region of [3H]-2 and (b) NMR of aromatic region
of [3H]-2.
Benzoyl [14C]isothiocyanate5 (5)
Table 1. Distribution of tritium in [3H]-2 and isotopic distri-
bution in the benzylic position
[
14C]Potassium thiocyanate,9 3, (1850 MBq, 0.98 mmol) was mixed with
benzoyl chloride, 4, (120 mL, 1.04 mmol) in acetone (4 mL). The mixture
was heated to reflux for 10 min, and 5 was then used as is in the follow-
ing step.
Position
Incorporation (%)
6
7
50
8
5
~4
27
300
600
20
[2-14C]N-((Tetrahydrofuran-2-yl)methylcarbamothioyl)
benzamide ([14C]-6a)
Tetrahydrofurfurylamine (110 mL, 1.1 mmol) was added to a solution of
benzoyl [14C]isothiocyanate,5 5, (~1.85 GBq, ~0.98 mmol) in acetone
(4 mL) at 0 ꢀC. The mixture was heated to reflux for 1 h. Water (1 mL)
was added, and the mixture was concentrated to dryness. The crude
Thereof:
CH2
CHT
60
25
15
[
14C]-6a (257 mg) was used as is in the next step.
CT2
[2-14C]-1-((Tetrahydrofuran-2-yl)methyl)thiourea ([14C]-7a)
HPLC analyses were performed on an Agilent 1100 HPLC-system with
a binary pump, auto-injector, diode array detector (DAD), and column
oven, coupled in series with a Packard Radiomatic Flow Scintillator
525TR, equipped with a solid scintillator (SolarScint) cell with a volume
of 33 mL. A Gemini column (C18, 3 ꢂ 100 mm, 5 mm) with the column tem-
perature set to 40 ꢀC using a gradient of 5–95% acetonitrile in 10-mM
ammonium acetate (MilliQ water) over 13.5 min at a flow rate of 1 mL/
min. Purities were reported as UV area% or radioactive area% (RCP) by
[2-14C]N-((Tetrahydrofuran-2-yl)methylcarbamothioyl)benzamide, [14C]-6a,
(~1.85 GBq) was dissolved in ethanol (1.5 mL). Freshly ground potas-
sium hydroxide (0.38 mg, 6.8 mmol) in water (0.5 mL) was added. The
mixture was heated to reflux for 75 min. The mixture was concentrated
to dryness and filtered through silica (1 cm) with dichloromethane and
ethanol to give [14C]-7a (253 mg, ~1.85 MBq). LCMS m/z 204.0 (5%),
206.0 (100%) ([M + H]+).
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J. Malmquist et al.
[2-14C]-6-Amino-1-((tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-
[2-14C]-6-Amino-1-(4-fluorobenzyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one ([14C]-8b)
dihydropyrimidin-4(1H)-one ([14C]-8a)
Small pieces of sodium (41 mg, 1.8mol) were added to a solution of [2-14C]- [2-14C]-1-(4-Fluorobenzyl)thiourea, [14C]-7a, (344 mg) was treated in a
1-((tetrahydrofuran-2-yl)methyl)thiourea, [14C]-7a, (~1.85 GBq, ~1mmol) in similar way as for the preparation of [14C]-8a to give the title compound
ethanol (1mL). Ethyl 2-cyanoacetate (110mL, 1.03mmol) was added, and
the mixture was heated to reflux for 4 h under a nitrogen atmosphere.
The reaction was concentrated to dryness. The residue was dissolved in
water (4mL), and the solution was neutralized with hydrochloric acid
(2M, 3mL). The water phase was extracted with dichloromethane
(4ꢂ 1 mL). The combined organic phase was dried over sodium sulfate,
filtered, and concentrated. The residue was purified by chromatography
on silica with dichloromethane/methanol/acetic acid (100/10/1) to give
[
14C]-8b (344 mg). LCMS m/z 252 (10%), 254 (100%) ([M + H]+).
[2-14C]-6-Amino-1-(4-fluorobenzyl)-5-nitroso-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one ([14C]-9b)
[2-14C]-6-Amino-1-(4-fluorobenzyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-
one, [14C]-8b, (344 mg) was treated in a similar way as for the preparation
of [14C]-9a to give the title compound [14C]-9b (500 mg). LCMS m/z 281
(10%), 283 (100%) ([M + H]+).
[
14C]-8a (27mg, 0.12 mmol). LCMS m/z 228.1 (5%), 230.1 (100%) ([M+H]+).
[2-14C]-5,6-Diamino-1-(4-fluorobenzyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one ([14C]-10b)
[2-14C]-6-Amino-5-nitroso-1-((tetrahydrofuran-2-yl)methyl)-2-
thioxo-2,3-dihydropyrimidin-4(1H)-one ([14C]-9a)
[2-14C]-6-Amino-1-(4-fluorobenzyl)-5-nitroso-2-thioxo-2,3-dihydropyrimidin-
4(1H)-one, [14C]-9b, (500mg) was treated in a similar way as for the prepara-
tion of [14C]-10a to give a precipitate of the title compound [14C]-10b
(28mg). LCMS m/z 267 (10%), 269 (100%) ([M + H]+).
[2-14C]-6-Amino-1-((tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-dihydropyr-
imidin-4(1H)-one, [14C]-8a, (27 mg, 0.12mmol) was mixed with sodium
nitrite (13 mg, 0.19 mmol) in acetic acid (2 M, 1 mL), and the mixture was
heated to 75 ꢀC. The mixture was extracted with dichloromethane (4 ꢂ 1
mL) to give [14C]-9a (17 mg, 0.07 mmol). LCMS m/z 257.0 (5%), 259.0
(100%) ([M + H]+).
[2-14C]-3-(4-Fluorobenzyl)-2-thioxo-2,3-dihydro-1H-purin-6
(7H)-one ([14C]-1b)
[2-14C]-5,6-Diamino-1-(4-fluorobenzyl)-2-thioxo-2,3-dihydropyrimidin-4
(1H)-one, [14C]-10b, (28 mg) was treated in a similar way as for the
preparation of [14C]-1a. The crude product was recrystallized from N,
N-dimethylformamide (0.2 mL) and ethanol (0.4 mL). The precipitate
was dissolved in sodium hydroxide (10%) and precipitated with acetic
acid (2 M). The precipitate was washed with water and dried to give
the title compound [14C]-1b (10 mg, 72 MBq) at 2.0 GBq/mmol and a
100% RCP (in a 2% yield from potassium [14C]cyanide). LCMS m/z 277
(10%), 279 (100%) ([M + H]+).
[2-14C]-5,6-Diamino-1-((tetrahydrofuran-2-yl)methyl)-2-thioxo-
2,3-dihydropyrimidin-4(1H)-one ([14C]-10a)
[2-14C]-6-Amino-5-nitroso-1-((tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one, [14C]-9a, (17 mg, 66 mmol) was mixed with
water (0.2 mL) and ammonia (32%, 0.2 mL). The mixture was heated to
70 ꢀC, and sodium dithionite (29 mg, 0.17 mmol) was added in small
portions. Stirring was continued for 15 min at 70 ꢀC and then 1 h at ambi-
ent temperature. The mixture was extracted with dichloromethane
(4 ꢂ 1 mL) to give [14C]-10a (17 mg, 0.07 mmol). LCMS m/z 243 (5%),
245 (100%) ([M + H]+).
[2,8-13C2, 3,7-15N2]-3-((Tetrahydrofuran-2-yl)methyl)-2-thioxo-
2,3-dihydro-1H-purin-6(7H)-one ([13C,15N]-1a)
Tetrahydrofurfurylamine (110mL, 1.1mmol) was treated in a similar way as
for the preparation of [14C]-1a using potassium [13C,15N]cyanide, sodium
[
[2-14C]-3-((Tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-dihydro-
1H-purin-6(7H)-one ([14C]-1a)
15N]nitrite, and [13C]formic acid. The crude product was recrystallized
from N,N-dimethylformamide (0.2mL) and ethanol (0.4 mL). The precipitate
was dissolved in sodium hydroxide (10%) and precipitated with acetic
acid (2M). The precipitate was washed with water and dried to give the title
compound [13C,15N]-1a (15 mg) in 6% yield and 99% purity. LCMS m/z 253
(<1%), 254 (<1%), 255 (<1%), 256 (6%), 257 (100%) ([M + H]+).
[2-14C]-5,6-Diamino-1-((tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-dihydro-
pyrimidin-4(1H)-one, [14C]-10a, (17mg, 0.07 mmol) was mixed with formic
acid (0.3mL) and heated to 70 ꢀC for half an hour, and then the mixture was
concentrated. Sodium hydroxide (10% aq., 0.4mL) was added, and the
mixture was heated to 70 ꢀC for 40 min. Hydrochloric acid (2M, 0.8mL)
was added, and the precipitate was extracted with dichloromethane and
acetonitrile 9:1 (4ꢂ 1 mL). The concentrated residue of organic phase was
purified by chromatography on silica using dichloromethane/methanol/
acetic acid (100/10/1) as eluent. The residue was triturated with methanol
(0.3mL) to give [14C]-1a (2.3mg, 9 mmol) at 2.1GBq/mmol and a radioche-
mical purity of 98.2% (UV 98.5%) (in a yield of 0.9% from potassium [14C]
cyanide). LCMS m/z 253 (5%), 255 (100%) ([M + H]+).
[2,8-13C2, 3,7-15N2]-3-(4-Fluorobenzyl)-2-thioxo-2,3-dihydro-1H-
purin-6(7H)-one ([13C,15N]-1b)
4-Fluorobenzyl amine (115 mL, 1.0 mmol) was treated in a similar way as
for the preparation of [13C,15N]-1a with similar yields and purity. LCMS
m/z 277 (<1%), 278 (<1%), 279 (<1%), 280 (6%), 281 (100%) ([M + H]+).
[2,8-13C2, 3,7-15N2]-3-Isobutyl-2-thioxo-2,3-dihydro-1H-purin-
6(7H)-one ([13C,15N]-1c)
[2-14C]N-(4-Fluorobenzylcarbamothioyl)benzamide ([14C]-6b)
Iso-butylamine (99 mL, 1 mmol) was treated in a similar way as for the pre-
paration of [13C,15N]-1a with similar yields and purity. LCMS m/z 225
(<1%), 226 (<1%), 227 (<1%), 228 (6%), 229 (100%) ([M + H]+).
4-Fluorobenzyl amine (460 mL, 4.0 mmol) was treated in a similar way as
for the preparation of [14C]-6a to give the title compound (510 mg). LCMS
m/z 166.1 (10%), 168.1 (100%) ([M ꢁ H]ꢁ).
[6-14C]-(R)-3-((Tetrahydrofuran-2-yl)methyl)-2-thioxo-2,3-
dihydro-1H-purin-6(7H)-one ([14C]-1d)
[2-14C]-1-(4-Fluorobenzyl)thiourea ([14C]-7b)
[2-14C]N-(4-Fluorobenzylcarbamothioyl)benzamide, [14C]-6b, (510mg) was Lithium triethylborohydride (Super-HydrideW) (1mL, 1 M in tetrahydro-
treated in a similar way as for the preparation of [14C]-7a to give the title furan) and [14C]-carbon dioxide (0.069 g, 1.51 mmol, 2.12 GBq/mmol) were
compound (344 mg). LCMS m/z 183.3 (10%), 185.3 (100%) ([M ꢁ H]ꢁ).
mixed under an argon atmosphere at ꢁ78 ꢀC and allowed to attain
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J. Malmquist et al.
ambient temperature over 1 h to give a colorless solution. The mixture (5 mL, 4 mmol) in dichloromethane (1 mL). The mixture was stirred at
was concentrated with methanol (3ꢂ 2 mL). The residue was dissolved in ambient temperature for 2 h, and then concentrated to give [3H]-14
N,N-dimethylformamide (4mL). (R)-5,6-Diamino-1-((tetrahydrofuran-2-yl) (~1.5 mg). LCMS (ES+) m/z 409 (20%), 411 (80%), 413 (100%), 415
methyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one, 11, (0.242g, 1.00 mmol) (40%), 417 (6%) [M-EtOH + H]+.
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EDAC,
(0.383 g, 2.00 mmol) were added. The mixture was stirred at ambient
[6,7,20,300,600-3H]-1-(Pyridin-2-ylmethyl)-2-thioxo-2,3-dihydro-
temperature for 18h. The presence of formylated product was checked
by LCMS. Sodium hydroxide (2M, 2 mL) was added, and the mixture was
heated at 100 ꢀC for 1 h. The solution was neutralized with hydrochloric acid
(2M, 2 mL, 4mmol). The mixture was concentrated to ~1/5 its volume and
was dissolved in water (1mL) and dimethyl sulfoxide (4mL), and then
1H-pyrrolo[3,2-d]pyrimidin-4(5H)-one ([3H]-2)
[4,5,20,300,600-3H]Ethyl 3-(3-benzyl-1-(pyridin-2-ylmethyl)thioureido)-1H-
pyrrole-2-carboxylate, [3H]-14, (~1.5 mg, ~3.7 mmol) was mixed with
purified by HPLC (50 ꢂ 250 mm) using 20% acetonitrile. The fractions con-
sodium hydroxide (2% aq., 400 mL, 0.40 mmol) in ethanol (200 mL). The
mixture was heated at 65 ꢀC for 1.5 h, and then at 50 ꢀC overnight. The
mixture was neutralized with hydrochloric acid (2 M, 200 mL, 0.40 mmol)
and was extracted with ethyl acetate (4 ꢂ 1 mL). The combined organic
layers were dried over sodium sulfate, filtered, and concentrated. The
residue was purified by chromatography on silica gel (4 cm in a Pasteur
pipette) using chloroform and methanol (25:1). The fractions containing
product were combined and concentrated, and the resulting residue
was purified by HPLC using 22% acetonitrile at 3 mL/min. The pooled
fractions were concentrated and the resulting residue was dissolved in
ethanol to give [3H]-2 (503 MBq) in 8.5% yield with a specific activity
of 1.6 TBq/mmol at 78 MBq/mL and a radiochemical purity of 97%. 1H
NMR (185 MBq, 600 MHz, CD3OD) d ppm 5.80–5.89 (m, 2H), 6.05
(d, J = 2.28 Hz, 1H), 7.22 (d, J = 2.85 Hz, 1H), 7.25–7.34 (m, 3H), 7.76
(t, J = 7.69 Hz, 1H), 8.49 (d, J = 4.55 Hz, 2H). 3H NMR (185 MBq, 640 MHz,
CD3OD) d ppm 5.73–5.88 (m), 6.10 (s), 7.26 (s), 7.36 (d, J = 3.03 Hz), 8.53
(br. s.). LCMS (ES+) m/z 259 (33%), 261 (100%), 263 (88%), 265 (32%),
267 (6%) [M + H]+.
taining product were pooled and concentrated to dryness, and the residue
was lyophilized four times with acetonitrile and water (10:1) and to give
[
14C]-1d (30mg, 0.12 mmol) in 12% yield at a specific activity of 2.1GBq/
mmol and >99.9% purity (UV, RCP, and enantiomerical purity). LCMS m/z
253 (5%), 255 (100%) ([M + H]+).
[2-14C]-1-(Pyridin-2-ylmethyl)-2-thioxo-2,3-dihydro-1H-pyrrolo
[3,2-d]pyrimidin-4(5H)-one ([14C]-2)
Ethyl 3-(pyridin-2-ylmethylamino)-1H-pyrrole-2-carboxylate, 13, (253 mg,
1.03 mmol) was mixed with benzoyl [14C]isothiocyanate,5 5, (1850 MBq,
0.98 mmol) in acetone (5 mL). The mixture was heated to reflux for
1.5 h, and then concentrated to dryness. The ethyl [2-14C]-3-(3-benzyl-1-
(pyridin-2-ylmethyl)thioureido)-1H-pyrrole-2-carboxylate ([14C]-14, crude)
was mixed with sodium hydroxide (2%, 4 mL) in ethanol (2 mL), and the
mixture was heated at 65 ꢀC overnight. The mixture was neutralized with
hydrochloric acid (2 M), and then extracted with ethyl acetate (4 ꢂ 2 mL).
The combined organic phase was dried over sodium sulfate, and then
filtered and concentrated. The residue was purified by chromatography
on silica gel (12 g) using chloroform and methanol (25:1). The material
was then purified by HPLC using 25% acetonitrile to give [14C]-2
(15 mg, 58 mmol) at 1.9 GBq/mmol in 5.9% yield and a radiochemical
purity of 98.6%. LCMS m/z 259 (15%), 261 (100%) ([M + H]+).
Quantitative whole body autoradiography
Animals
Male Long Evans black-hooded rats (5–7 weeks old; ~200 g) were obtained
from Taconic M & B A/S (Denmark). All animals were acclimatized for 1 week
to a 12-h light/dark cycle in temperature controlled environment (20–21 ꢀC)
with a humidity similar to ambient air. The animals were allowed free
access to a standard pellet diet (Brogaarden, Denmark) and tap water ad
libitum. All animal experimental procedures were performed in accordance
with relevant guidelines and regulations provided by the Swedish Board of
Agriculture.
Ethyl 4,5-dibromo-3-(pyridin-2-ylmethylamino)-1H-pyrrole-2-
carboxylate (15)
To a solution of ethyl 3-(pyridin-2-ylmethylamino)-1H-pyrrole-2-carboxy-
late,2 13, (21mg, 86 mmol) in acetic acid (0.5mL) was added bromine
(206mL, 100mg/mL acetic acid, 130 mmol) dropwise at 0 ꢀC. The reaction
was quenched with sodium bisulfite (1mL, 1 M) and was extracted with
ethyl acetate (1mL) thrice. The combined organic phase was dried over
sodium sulfate, and then filtered, and concentrated. The crude product
was purified by HPLC using 30–70% acetonitrile to give 15 (18mg) in
52% yield and a UV purity of 99%. 1H NMR (400 MHz, DMSO-d6) d ppm
1.28 (t, J = 7.03Hz, 3H), 4.22 (q, J = 7.11 Hz, 2H), 4.67 (d, J = 6.53 Hz, 2H),
6.24 (t, J= 6.40 Hz, 1H), 7.26 (dd, J = 6.90, 5.14 Hz, 1H), 7.33 (d, J= 7.78 Hz,
1H), 7.67–7.86 (m, 1H), 8.52 (d, J = 4.77Hz, 1H), 12.29 (br. s., 1H). LCMS
(AP+) m/z 401.7, 403.7 (100%), 405.8 [M + H]+.
Experimental design
Compound [14C]-1d (10 mmol/kg; 10 MBq/kg; 5 mL/kg) was formulated as
a solution in 0.3 M meglumine (pH 10). The animals were administered a
single oral dose and sacrificed at 15 min, 1 h, 4 h, 24 h, 2 days, and 7 days
after administration (n = 1 animal per time-point). The animals were
sacrificed with enflurane (EfraneW, Abbott Laboratories, USA) and frozen
in acetone, cooled to ꢁ70 ꢀC with solid CO2, and thereafter sectioned
according to the procedure of Ullberg.10 Briefly, the carcass was
embedded in 2.5% aqueous solution of carboxymethyl cellulose and
frozen for at least 10 min in acetone and cooled to ꢁ70 ꢀC with solid
CO2. At sectioning, each block was mounted in a Leica CM3600 Cryostat
(Leica Microsystems AB, Sweden) maintained at approximately ꢁ20 ꢀC.
Whole-body sections (20 mm) were mounted on type 6890 tape
(3 M, USA) and lyophilized.
[4,5,20,300,600-3H]Ethyl 3-(pyridin-2-ylmethylamino)-1H-pyrrole-
2-carboxylate ([3H]-13)
Ethyl 4,5-dibromo-3-(pyridin-2-ylmethylamino)-1H-pyrrole-2-carboxylate,
15, (1.74 mg, 4.32 mmol) and palladium(II) oxide (2.8 mg, 0.02 mmol) were
mixed with triethylamine (5 mL, 0.04 mmol) and N,N-dimethylformamide
(400 mL). The mixture was stirred in 807 mbar tritium atmosphere at
ambient temperature overnight. The reaction was filtered and lyophilized
with ethanol (1 mL) thrice to give [3H]-13 (~1 mg). LCMS (ES+) m/z 246
(30%), 248 (80%), 250 (100%), 252 (60%), 254 (30%) [M + H]+.
The whole-body sections, together with a set of carbon-14 nuclide cali-
bration standards made in an aqueous solution of gelatine (100mg/mL),
were exposed to phosphor imaging plates (FujiFilm Sverige AB, Sweden)
for 3 days (+8 ꢀC). The imaging plates were enclosed in light tight cassettes
in a lead shielding box to protect from environmental radiation. After
exposure, the imaging plates were scanned at a pixel size of 50 mm using
BAS2500 (FujiFilm Sverige AB, Sweden), and tissue concentrations were
[4,5,20,300,600-3H]Ethyl 3-(3-benzyl-1-(pyridin-2-ylmethyl)
thioureido)-1H-pyrrole-2-carboxylate ([3H]-14)
[4,5,20,300,600-3H]Ethyl 3-(pyridin-2-ylmethylamino)-1H-pyrrole-2-carboxy- quantified using AIDA (Raytest, Germany). The quantification limit was
late, [3H]-13, (~1 mg, ~4 mmol) was mixed with benzoyl isothiocyanate defined as twice the background value of the imaging plate.
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J. Label Compd. Radiopharm 2012

J. Malmquist et al.
In vitro imaging
stable isotope-labeled compounds were prepared for use as
internal standards in bioanalyses.
Animals (experimental autoimmune encephalomyelitis induction)
All animal experimental procedures were performed in accordance with
relevant guidelines and regulations provided by the Swedish Board of
Agriculture. Female (8–12 weeks) Dark Agouti rats (Scanbur, Sollentuna,
Sweden) were injected intradermally/subcutaneously at the base of the
tail with 0.2 mL inoculum, containing 85–100 mg recombinant rat
MOG1–125 in Incomplete Freund’s adjuvant.
Acknowledgement
Peter Ström is kindly acknowledged for valuable discussions on
practical issues.
Autoradiography
Conflict of interest
In vitro binding on tissue sections was adapted for MPO ligands from
previously described protocols.11 Briefly, frozen spinal cords from rats
were sectioned (10 mm) with a cryostat through the horizontal plain, air
dried, and stored at ꢁ80 ꢀC until used. Sections were warmed to room
temperature, preincubated for 10 min at room temperature in 50 mM
tris-buffer (pH 7.4), and then transferred to the same buffer containing
10 nM of [3H]-2, and incubated for 30 min at room temperature. The sec-
tions were subsequently washed several times at 1 ꢀC, dipped in cold
H2O, and finally, air dried. Samples and plastic tritium standards
(Amersham, Piscatway, NJ, USA) were exposed to imaging plates (Fuji
BAS-TR2040) for 5 days and then processed with a FLA7000 Imaging
Reader (Fujifilm).
The authors did not report any conflict of interest.
References
[1] S. Hanson, G. Nordvall, A. Tidén. WO 03/089430 A1, October 30, 2003.
[2] A. Bögevig, Y. Lo-Alfredsson, D. Pivonka, A. Tidén. WO 2006/062465
A1, June 15, 2006.
[3] A.-K. Tidén, T. Sjögren, M. Svensson, A. Bernlind, R. Senthilmohan,
F. Auchère, H. Norman, P.-O. Markgren, S. Gustavsson, S. Schmidt,
S. Lundquist, L. V. Forbes, N. J. Magon, L. N. Paton, G. N. L. Jameson,
H. Eriksson, A. J. Kettle, J. Biol. Chem. 2011, 286, 37578–37589.
[4] P. Ström, J. Malmquist, J. Label. Compd. Radiopharm. 2008, 51, 419–421.
[5] G. Zòlyomi, L. Toldy, D. Bánfi, J. Label. Compd. Radiopharm. 1973, 9,
243–251.
[6] a) J. G. Burr, H. E. Heller, J. Am. Chem. Soc. 1950, 72, 2560–2562; b)
C. S. Elmore, D. C. Dean, D. G. Melillo, J. Label. Compd. Radiopharm.
2000, 43, 1135–1144. c) J. Z. Ho, C. S. Elmore, M. A. Wallace, D. Yao,
M. P. Braun, D. C. Dean, D. G. Melillo, C.-Y. Chen, Helv. Chim. Acta
2005, 88, 1040–1047
[7] A. Bernlind, J. Malmquist, J. Sandell, P. Ström, Poster presentation:
The use of 3H NMR to verify structure and labelling position(s) of
3H labelled compounds. International Isotope Society - Central
European Division, 18th Workshop, Bad Soden, September 22–23, 2011.
[8] J. M. A. Al-Rawi, J. P. Bloxsidge, C. O’Brien, D. E. Caddy, J. A. Elvidge,
J. R. Jones, E. A. Evans, J. Chem. Soc. Perkin Trans. II 1974, 1635–1638.
[9] Ström P., Malmquist J., J. Label. Compd. Radiopharm. 2008, 51, 419–421.
[10] S. Ullberg, B. Larsson, H. Tjälve, Autoradiography. In Biologic applica-
tion of radiotracers (Ed.: J. Glenn), CRC Press: Boca Raton, FL, 1982,
pp. 55–108.
Immunohistochemistry
To detect areas with inflammation, immunohistochemistry was performed
on a Ventana DiscoveryW XT staining module (Ventana, Illkirch, France).
Acetone-fixed tissue slides were run through a no pretreatment protocol.
ED-1, a monoclonal antibody detecting CD 68 (Serotec, Hamar, Norway),
was manually applied at a 1:500 dilution. A detection 1, Donkey anti-mouse,
biotinylated secondary antibody was used together with Ventana DAB
detection kit to visualize ED-1 immunostained cells.
Conclusions
Methods for the preparation of isotopologues of 1 and 2 have
been developed providing material with specific activity sufficient
for in vitro imaging of MPO in brain and spinal tissue and for in vivo
imaging (e.g., quantitative whole body autoradiography). Several
[11] A. Juréus, B. M. Swahn, J. Sandell, F. Jeppsson, A. E. Johnson,
P. Johnström, J. A. Neelissen, D. Sunnemark, L. Farde, S. P. Svensson,
J. Neurochem. 2010, 114, 784–794.
J. Label Compd. Radiopharm 2012
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