Tritium labeling of a γ-secretase inhibitor and two modulators as in vitro imaging agents

Article abstract of DOI:10.1002/jlcr.1955

The γ-secretase inhibitor dibenzazepine (DBZ) and the γ-secretase modulators 1 and AZ8349 were prepared as tritium-labeled compounds with high specific activity and radiochemical purity. [ ³H]DBZ was labeled via an iodinated precursor, [^3<

Full text of DOI:10.1002/jlcr.1955

Research Article
Received 8 September 2011,
Revised 1 November 2011,
Accepted 18 November 2011
Published online 23 January 2012 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1955
Tritium labeling of a g-secretase inhibitor and
two modulators as in vitro imaging agents
a
b
a
*
Jonas Malmquist, Alexandra Bernlind, Johan Sandell,
Peter Ström,a and Magnus Waldmanb
The g-secretase inhibitor dibenzazepine (DBZ) and the g-secretase modulators 1 and AZ8349 were prepared as tritium-labeled
compounds with high specific activity and radiochemical purity. [3H]DBZ was labeled via an iodinated precursor, [3H]1 was
labeled by [3H]methylation of an O-desmethyl precursor, and [2-3H]AZ8349 was labeled via a tribromoacetyl precursor by
catalytic hydrogenation. [3H]DBZ, [3H]1, and [2-3H]AZ8349 are promising in vitro imaging radioligands and have the potential
to provide key information with regard to g-secretase expression, function, stoichiometry, and pharmacology.
3
Keywords: isotopically labeled synthesis; H-3; H; DBZ; AZ8349; GSI; GSM
of detection of the radioligand, which enables the radioligand to
be used at a correspondingly lower concentration. This is of
importance when imaging a protein that is expressed in a low
density, or expressed only in very discrete brain areas. Accordingly,
Introduction
g-Secretase is one of the enzymes that catalyze the cleavage of the
amyloid precursor protein into shorter amyloid beta peptide
fragments. Amyloid beta peptides aggregate into senile plaques
in the brains of patients with Alzheimer’s disease and are one of
the labeling strategies of 1 and AZ8349 took advantage of the
presence of methyl groups in the compound structures.
the neuropathological hallmarks of the disease. Two pharmacolo-
Borontribromide was used for the demethylation of 1. The
gical classes of small molecules targeting g-secretase have been
O-desmethyl precursor 2 (Scheme 1) was prepared in high yield
described to date: g-secretase inhibitors (GSIs) and g-secretase
and purity after purification by flash chromatography. Methylation
modulators (GSMs).1 GSIs and GSMs have attracted much attention
as potential novel disease-modifying therapeutics for Alzheimer’s
was accomplished by deprotonation of the phenol with sodium
hydroxide in demethyl sulfoxide and subsequent addition of high
specific activity, 2.8 TBq/mmol, [3H]iodomethane at ambient
disease, and several g-secretase-based drug discovery programs
are ongoing.2,3
temperature. The reaction was completed after 10 min. The high
specific activity was retained, and [3H]1 was isolated in 20%-
Isotope-labeled g-secretase binding molecules have been
shown to be useful pharmacological tools that have provided
radiochemical yield with no detectable impurities. The substance
valuable insights into the biology of g-secretase and g-secretase-
was characterized by co-elution on HPLC with 1 and by mass
based drug discovery efforts.4,5,6 In this paper, we describe
spectroscopy.
the radiolabeling of the two GSMs, 1 ((E)-1-(1-(4-fluorophenyl)
Treatment of DBZ with N-iodosuccinimide in trifluoroacetic acid
ethyl)-3-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)benzylidene)
afforded the iodinated precursor 3 (Scheme 2). The position of
piperidin-2-one7 ) and AZ8349 (1-(4-benzyl-2-(3-methoxy-4-(4-
methyl-1H-imidazol-1-yl)phenylamino)-7,8-dihydropyrido[4,3-
iodination was confirmed with NMR spectroscopy. Catalytic
deiodohydrogenation was performed in the presence of palladium
d]pyrimidin-6(5H)-yl)ethanone8 ), and a GSI, dibenzazepine (DBZ)
(II) oxide with an excess of tritium at 803 mbar. A specific activity of
0.6 TBq/mmol was obtained for [3H]DBZ in 32% yield and a
((2S)-2-(2-(3,5-difluorophenyl)acetamido)-N-(5-methyl-6-oxo-6,7-
dihydro-5H-dibenzo[b,d]azepin-7-yl)propanamide,9,10 (Figure 1).
radiochemical purity of 99.7%. The tritium position was confirmed
A preliminary pharmacological characterization of [3H]1, [3H]
DBZ, and [3H]AZ8349 including imaging with in vitro autoradio-
graphy has previously been reported by Borgegård et al.11 By
cross-competition and radioligand displacement studies, it
was shown that the GSM and GSI radioligands have different
g-secretase binding sites and, thus, are valuable imaging tools in
the study of target expression and drug–target interactions.
by 3H NMR, and because proton atoms were decoupled in the 3H
NMR experiment, 3-bond as well as 5-bond J-couplings between
3H and 19 F could be measured in the spectrum (Figure 2).
aIsotope Chemistry, Screening and Profiling Global DMPK IM, AstraZeneca
Research and Development Innovative Medicines, Södertälje SE-151 85, Sweden
bMedicinal Chemistry, CNSP iMed Science Södertälje, AstraZeneca Research and
Development Innovative Medicines, Södertälje SE-151 85, Sweden
Results and discussions
*Corresponndence to: Jonas Malmquist, Isotope Chemistry, Screening and
Profiling Global DMPK IM, AstraZeneca Research and Development Innovative
Medicines, Södertälje SE-151 85, Sweden.
Methyl groups provide an entry to a radioligand with high specific
activity because of the opportunity to simultaneously introduce
three tritium atoms. High specific activity increases the sensitivity E-mail: jonas.malmquist@astrazeneca.com
J. Label Compd. Radiopharm 2012, 55 80–83
Copyright © 2012 John Wiley & Sons, Ltd.

J. Malmquist et al.
O
A
O
N
N
F
N
1
ppm
7.02
7.01
7.00
6.99
6.98
6.97
F
F
O
NH
O
B
NH
N
O
DBZ
ppm
7.02
7.01
7.00
6.99
6.98
6.97
Figure 2. 3H spectra of [3H]DBZ, expanded to show the 3H signal. A: 3H spectrum.
B: 3H spectrum with 1H decoupling, which allows the 3-bond and 5-bond 3H, 19F
couplings to be identified.
N
achieved by reacting tribromoacetyl chloride with 48 to provide
5 in moderate yield (Scheme 3). Tritium was introduced at
621 mbar with palladium catalysis to perform a debromohydro-
genation of 5. The reaction was run overnight in ethanol. The
unoptimized yield of [3H]AZ8349 was 1.3%, with a radiochemi-
cal purity of 97% and a specific activity of 1.2TBq/mmol. The
substance was characterized by co-elution on HPLC with
O
N
N
N
O
N
H
N
AZ8349
Figure 1. Structure of substances labeled.
3
AZ8349 and by mass spectroscopy. H NMR was not performed
because of the small amount isolated. The yield and specific
activity would possibly improve with a shorter exposure time
to tritium gas.
Several routes were investigated for tritium incorporation into
AZ8349 and found unsuccessful:
• H/3H exhange with an iridium (I) complex for catalytic hydrogen
isotope exchange:12 No reaction was observed.
• Methylation with iodomethane of an O-desmethyl precursor:
Only N-alkylation was observed.
Experimental
• Methylation with iodomethane of an O-desmethyl precursor General methods
with N-BOC or N-SEM protection: The desired product could
All solvents used were of analytical grade and commercially
not be identified.
• Acylation of 4 with [3H]acetic anhydride:13 Unfruitful, although
acylation worked on a larger scale with acetic anhydride.8
available. Anhydrous solvents were routinely used for reactions.
Reactions were typically run under an inert atmosphere of nitrogen
or argon. Tritium gas was handled in a tritium gas manifold system
To circumvent this unexpected problem, another substrate was (RC TRITEC AG, Teufen). [3H]Iodomethane was purchased from
prepared by the introduction of a tribromoacetyl moiety. The Larodan Fine Chemicals AB.
objective was to have a fair option of a decent specific activity.
3H and 1H spectra were recorded on a Bruker DRX600 NMR
To our knowledge, this method of labeling an acetyl group has Spectrometer, operating at 640 MHz for tritium and at 600MHz
not been reported in the literature before. The N-acylation was for proton, equipped with a 5-mm 3H/1H SEX probehead with
3H
O
O
C3H3I
NaOH
3H 3H
O
O
BBr3
HO
N
O
N
N
N
N
F
F
CH Cl
2
DMSO
20%
2
F
N
N
N
N
72%
[3H]1
1
2
Scheme 1.
3H2
PdO
O
F
F
O
F
F
NIS
O
HN
O
HN
DBZ
NH
NH
MeOH
32%
TFA
I
3H
[3H]DBZ
N
N
O
O
70%
3
Scheme 2.
J. Label Compd. Radiopharm 2012, 55 80–83
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org

J. Malmquist et al.
O
Br
Br
3H2
N
N
N
O
O
Cl
3H
3H
Br
Br
N
O
N
O
N
O
Br
Pd/C
N
N
N
NH
N
N
Br
3H
Et3N
THF
6.5%
EtOH
1.3%
N
H
N
N
N
N
N
H
H
4
5
[3H]AZ8349
Scheme 3.
Z-gradients. H decoupled H spectra were recorded on samples [3-3H3]-(E)-1-(1-(4-Fluorophenyl)ethyl)-3-(3-methoxy-4-(4-
dissolved in CD3OD. For 3H NMR spectra referencing, a ghost methyl-1H-imidazol-1-yl)benzylidene)piperidin-2-one ([3H]1)
reference frequency was used, as calculated by multiplying the
1
3
1
(E)-1-(1-(4-Fluorophenyl)ethyl)-3-(3-hydroxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)piperidin-2-one, 2, (1.2 mg, 3.0mmol)
was dissolved in dimethyl sulfoxide (0.5ml). Sodium hydroxide
(10%, 5 ml) was added. The mixture was stirred for 5 min at ambient
temperature. [3H]Iodomethane (2.8 TBq/mmol, 1850 MBq in 0.05 ml
toluene, 0.66 mmol) was added to this mixture. After 10 min of stir-
ring at ambient temperature, the mixture was filtered through silica
(1cm in a Pasteur pipette) and was eluted with dichloromethane.
The concentrated filtrate was purified on HPLC using the Kromasil
column and 60% acetonitrile. Pooled fractions were concentrated
and dissolved in ethanol (abs. 12 ml) to give [3H]1 (371 MBq,
2.8 TBq/mmol) in a 20% yield and >99.9% radiochemical purity.
LC-MS m/z 424, 426 ([M + H]+).
frequency of internal tetramethylsilane in an H spectrum with
the Larmor frequency ratio between 3H and 1H (1.06663975),
according to the description by Al-Rawi et al.14 1H spectra were
referenced to external tetramethylsilane, which was set to 0 ppm.
Mass spectra were recorded on a Waters liquid chromatogra-
phy coupled mass spectroscopy (LC-MS) consisting of a Waters
1525 micro (LC), Waters photodiode array 2996, and evaporative
light scattering detector (Sedex 75) and a z-spray mass detector
(ZMD) single quadrupole mass spectrometer.
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 scin-
tillator (SolarScint) cell with a volume of 33 ml. A Gemini column
(C18, 3 Â 100 mm, 5 mm) with the column temperature set to
40ꢀC use a gradient of 5%–95% acetonitrile in 10 mmol/L ammo-
nium acetate in MilliQ water at 1 ml/min over 13.5 min. Purities
were reported as UV area% or radioactive area% by HPLC.
Preparative chromatography was run on an XBridgeTM column
(C8, 10 mm 19 Â 250 mm) using a gradient of 55%–90% metha-
nol/ 0.2% ammonia in MilliQ Water at 20 ml/min or a Kromasil
column (C8, 5 mm, 10 Â 250 mm) using acetonitrile/ 50 mmol/L
ammonium acetate in MilliQ Water at 2 ml/min.
(2S)-2-(2-(3,5-Difluoro-2-iodophenyl)acetamido)-N-
(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)
propanamide (3)
To (2S)-2-(2-(3,5-difluorophenyl)acetamido)-N-(5-methyl-6-oxo-6,7-
dihydro-5H-dibenzo[b,d]azepin-7-yl) propanamide, DBZ (10 mg,
0.02 mmol) in trifluoroacetic acid (1 ml) was added N-iodosuccinimide
(5 mg, 0.02 mmol), and the reaction mixture was stirred over-
night at ambient temperature. The mixture was concentrated.
The residue was purified on an HPLC with the Kromasil column
and 60% acetonitrile. Pooled fractions were concentrated to
give 3 (8mg, 0.014 mmol) in a 63% yield. 1H NMR (500 MHz, CDCl3)
d ppm 1.46 (d, J = 6.94 Hz, 3 H), 3.37 (s, 3 H), 3.77 (s, 2 H), 4.74
(quin, J = 7.01 Hz, 1 H), 5.25 (d, J = 6.62 Hz, 1 H), 6.32 (br. s., 1 H),
6.79 (td, J = 8.12, 2.68 Hz, 1 H), 7.00 (d, J = 8.83 Hz, 1 H), 7.27–7.31
(m, 1H), 7.34–7.39 (m, 2 H), 7.40–7.46 (m, 2 H), 7.46–7.51 (m, 1 H),
7.53 (d, J= 5.36 Hz, 1 H), 7.55–7.58 (m, 1 H), 7.61 (d, J=7.57Hz, 1H).
LC-MS m/z 590 ([M + H]+).
Liquid scintillation analysis was performed on a PACKARD TRI-
CARB 2900TR. Specific activities were determined with mass spec-
trometry and confirmed with 3H NMR. Thin layer chromatography
was performed on Merck TLC-plates (Silica gel 60 F254), and UV
light (254 nm) visualized the spots. Flash column chromatography
™
was performed on Redisep prepacked columns.
(E)-1-(1-(4-Fluorophenyl)ethyl)-3-(3-hydroxy-4-(4-methyl-
1H-imidazol-1-yl)benzylidene)piperidin-2-one (2)
(E)-1-(1-(4-Fluorophenyl)ethyl)-3-(3-methoxy-4-(4-methyl-1H-
imidazol-1-yl)benzylidene)piperidin-2-one, 1,7 (20 mg, 0.05 mmol)
was dissolved in dichloromethane (1ml) under an argon atmo-
sphere and was cooled to approximately À78ꢀC. Borontribromide
[2-3H]-(2S)-2-(2-(3,5-Difluorophenyl)acetamido)-N-
(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)
propanamide ([3H]DBZ)
(1mol/L in dichloromethane, 100 ml, 0.10 mmol) was added, and (2S)-2-(2-(3,5-Difluoro-2-iodophenyl)acetamido)-N-(5-methyl-6-
the mixture was warmed to ambient temperature over a couple oxo-6,7-dihydro-5Hdibenzo[b,d]azepin-7-yl)propanamide, 3, (1.6mg,
of hours and was stirred overnight. The reaction was neutralized 2.7 mmol) and palladium (II) oxide (1.4 mg, 0.01 mmol) were
with sat. sodium bicarbonate and was extracted with ethyl acetate dissolved in methanol (0.3ml). The mixture was stirred for 1 h in
(4Â 1 ml). The organic phase was dried over sodium sulfate, 803 mbar tritium atmosphere at ambient temperature. The
filtered, and concentrated. The residue was purified by chromato- mixture was filtered, and the residue was lyophilized with
graphy on silica gel (4g) using a gradient of dichloromethane to methanol (1ml) twice. The product was isolated by purification
dichloromethane/methanol 25:1 to give 2 (14 mg, 0.03 mmol) in on HPLC using the Kromasil column and 60% acetonitrile. Pooled
1
72% yield and 98% UV purity. H NMR (500 MHz, CDCl3) d ppm fractions were concentrated to give [3H]DBZ, (540 MBq,
1.48 (d, J = 6.94 Hz, 3 H), 1.55–1.65 (m, 2 H), 1.68–1.80 (m, 1 H), 0.60 TBq/mmol) in 32% yield and a radiochemical purity of
2.69 (dq, J = 20.77, 10.36 Hz, 2 H), 2.82–2.91 (m, 1 H), 3.17 (ddd, 99.7%. 1H NMR (600 MHz, CD3OD) d ppm 1.43 (d, J= 7.12Hz, 3 H),
J = 12.30, 8.67, 3.47 Hz, 2 H), 6.07 (q, J = 6.99 Hz, 2 H), 6.91 (d, 1.92 (s, 3 H), 3.62 (d, J= 1.57 Hz, 2 H), 4.59 (q, J= 7.12 Hz, 1 H), 5.20
J = 8.35 Hz, 1 H), 6.94–7.00 (m, 4 H), 7.08 (s, 2 H), 7.64 (s, 2 H), 8.38 (s, 1 H), 6.78–6.84 (m, 1 H), 6.95 (d, J = 8.26 Hz, 1 H), 7.33–7.36
(br s, 1 H). LC-MS m/z 406.0 ([M + H]+).
(m, 1 H), 7.37–7.40 (m, 1H), 7.41–7.46 (m, 2H), 7.50–7.57 (m, 2 H),
www.jlcr.org
Copyright © 2012 John Wiley & Sons, Ltd. J. Label Compd. Radiopharm 2012, 55 80–83

J. Malmquist et al.
3
7.62 (d, J = 7.54 Hz, 1 H), 7.67 (dd, J = 7.76, 1.21 Hz, 1 H). H NMR
Conclusions
3
(640MHz, CD3OD) d ppm 6.99 (dd, J = 9.8, 0.7 Hz). The H signal
is split into a double doublet by JTF and JTF. LC-MS m/z 464,
Methods for the preparation of [3H]1, [3H]DBZ, and [3H]AZ8349
have been developed providing a specific activity sufficient for
in vitro imaging of brain tissue g-secretase. To overcome the
unexpected problems with N-acylation of 4, the tribromoacyl
motif was introduced as a precursor for tritium-labeled acetyl
groups and derivatives thereof.
3
5
466 ([M + H]+).
1-(4-Benzyl-2-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)
phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-
2,2,2-tribromoethanone (5)
Conflict of interest
4-Benzyl-N-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)phenyl)-5,6,7,8-
tetrahydropyrido[4,3-d]pyrimidin-2-amine, 4, (56 mg, 0.13 mmol)
was mixed with triethylamine (27 ml, 0.20 mmol) and 2,2,2-
tribromoacetyl chloride (25ml, 0.13 mmol) in tetrahydrofuran
(2ml).The mixture was stirred at ambient temperature for 2½ h.
The mixture was then concentrated, and the residue was dissolved
in dimethyl sulfoxide (1ml) and was purified on a preparative
HPLC twice using the XBridge column. Pooled fractions were
The authors did not report any conflict of interest.
References
[1] G. Tian, S. V. Ghanekar, D. Aharony, A. B. Shenvi, R. T. Jacobs, X. Liu,
B. D. Greenberg, J. Biol. Chem. 2003, 278, 28968–28975.
[2] Y.-M. Li, M. Xu, M.-T. Lai, Q. Huang, J. L. Castro, J. DiMuzio-Mower,
T. Harrison, C. Lellis, A. Nadin, J. G. Neduvelil, R. B. Register, M. K.
Sardana, M. S. Shearman, A. L. Smith, X.-P. Shi, K.-C. Yin, J. A. Shafer,
S. J. Gardell, Nature 2000, 405, 689–694.
1
concentrated to give 5 (6mg, 8.5 mmol) in 6.5% yield. H NMR
(600MHz, DMSO-d6) d ppm 1.09 (br. s., 3 H), 2.14 (s, 3 H), 2.94
(br. s., 1 H), 3.73 (s, 2 H), 4.03 (s, 2 H), 4.14 (s, 1 H), 4.81 (br. s., 1 H),
7.01 (s, 1 H), 7.16 (d, J = 8.40 Hz, 1 H), 7.21–7.27 (m, 1 H), 7.28–7.38
(m, 6 H), 7.64 (s, 1 H), 7.89 (s, 1 H), 9.81 (br. s., 1 H). LC-MS m/z 703,
705, 707, 709 ([M + H]+).
[3] D. Beher, E. E. Clarke, J. D. J. Wrigley, A. C. L. Martin, A. Nadin,
I. Churcher, M. S. Shearman, J. Biol. Chem. 2004, 279, 43419–43426.
[4] J. Malmquist, A. Bernlind, P. Ström, J. Label. Compd. Radiopharm.
2010, 53, 44–46.
[5] M. E. Goldstein, Y. Cao, T. Fiedler, J. Toyn, L. Iben, D. M. Barten,
M. Pierdomenico, J. Corsa, C. V. C. Prasad, R. E. Olson, Y.-W. Li, R. Zaczek,
C. F. Albright, J. Pharm. Exp. Therap. 2007, 323, 102–108.
[6] E. E. Clarke, I. Churcher, S. Ellis, J. D. J. Wrigley, H. D. Lewis, T. Harrison,
M. S. Shearman, D. Beher, J. Biol. Chem. 2006, 281, 31279–31289.
[7] I. Kushida, E. Doi, K. Ito, T. Nakamura (August 6, 2008) European
Patent Application 1953151 (1: 24).
[8] R. Forsblom, K. Paulsen, D. Rotticci, E. Santangelo, M. Waldman
(May 14, 2010) WO 2010/53438 (2: 175–176; 4: 174–175).
[9] H. Fuwa, Y. Okamura, Y. Morohashi, T. Tomita, T. Iwatsubo, T. Kan,
T. Fukuyama, H. Natsugari, Tet. Lett. 2004, 45, 2323–2326.
[10] J. Wu, J. S. Tung, E. D. Thorsett, M. A. Pleiss, J. S. Nissen, J. Neitz, L. H.
Latimer, V. John, S. Freedman, T. C. Britton, J. E. Audia, J. K. Reel, T. E.
Mabry, B. A. Dressman, C. L. Cwi, (January 21, 2009) EP 0951466 B1
(Page/column: 62/79).
[11] T. Borgegård, F Olsson, A. Juréus, R. Klintenberg, A. Sabirsh, D. Rotticci,
M. Waldman, J. Johansson, J. Malmquist, J. Sandell, A. Regnér,
E. Hagström, P. Wollberg, D. Malinowsky, M. Berg, S. Parpal, K. Strömberg,
H. Eriksson, P. Arvidsson, S. Rosqvist, J. Lundkvist, Poster presentation,
ADPD2011 (March 8–9, 2011), Barcelona.
[12] Complex 5c in: J. A. Brown, S. Irvine, A. R. Kennedy, W. J. Kerr,
S. Andersson, G. N. Nilsson, Chem. Commun. 2008, 1115–1117.
[13] American Research Chemicals, ART 0203 C, lot# 101001.
[14] 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.
[2-3H]1-(4-Benzyl-2-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)
phenylamino)-7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)
ethanone ([3H]AZ8349)
1-(4-Benzyl-2-(3-methoxy-4-(4-methyl-1H-imidazol-1-yl)phenylamino)-
7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)-2,2,2-tribromoethanone, 5,
(0.89 mg, 1.26 mmol) and palladium (10% on carbon) (1.3mg) were
mixed in ethanol (0.4ml). The mixture was connected to the
tritium manifold and placed under tritium atmosphere (excess) at
621mbar. The reaction mixture was stirred at ambient tempera-
ture overnight. The reaction mixture was filtered and lyophilized
with ethanol (1ml) thrice. The residue was dissolved in acetonitrile
(0.4ml) and purified by preparative HPLC with the Kromasil
column and 47% acetonitrile. Pooled fractions were concentrated
and dissolved in ethanol (2.00 ml) to give [3H]AZ8349 (20 MBq,
1.2TBq/mmol) in 1.3% yield and a radiochemical purity of 97%.
LC-MS m/z 469, 471, 473, 475, 477 ([M + H]+).
J. Label Compd. Radiopharm 2012, 55 80–83
Copyright © 2012 John Wiley & Sons, Ltd.
www.jlcr.org