[11C] Methyl-losartan as a potential ligand for PET imaging angiotensin II AT1 receptors

Article abstract of DOI:10.1002/jlcr.1917

The renin-angiotensin system regulates blood pressure via activation of the angiotensin II type 1 receptor (AT₁R). The AT₁R is involved in the pathology of cardiac and renal diseases such as heart failure and diabetic nephropathy. The aim of this study was to synthesize and characterize the O-[¹¹ C]methylated derivative of the clinically used AT₁ receptor blocker losartan as a novel AT₁R PET imaging radioligand. [¹¹ C]Methyl-losartan was reliably synthesized (n ≥ 40) via methylation of tetrazole-protected losartan followed by deprotection using HCl in an overall yield of 30%-60% (decay-corrected from [¹¹ C]MeI). Radiochemical purity was >99% and specific activity 700-3600 mCi/μmol. Copyright 2011 John Wiley & Sons, Ltd. [¹¹ C]Methyl-losartan has been synthesized in three steps: 1) protection of the tetrazole group, 2) [¹¹ C]methylation of hydroxyl group, and 3) deprotection in acidic conditions. Copyright

Full text of DOI:10.1002/jlcr.1917

Research Article
Received 6 May 2011,
Revised 5 July 2011,
Accepted 6 July 2011
Published online 8 September 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.1917
[11C]Methyl-losartan as a potential ligand for
PET imaging angiotensin II AT1 receptors
Tayebeh Hadizad,a Jeffrey Collins,a Rawad E. Antoun,a,b
Rob S. Beanlands,a,b and Jean N. DaSilvaa,b
*
The renin-angiotensin system regulates blood pressure via activation of the angiotensin II type 1 receptor (AT1R). The AT1R is
involved in the pathology of cardiac and renal diseases such as heart failure and diabetic nephropathy. The aim of this study
was to synthesize and characterize the O-[11C]methylated derivative of the clinically used AT1 receptor blocker losartan as
a novel AT1R PET imaging radioligand. [11C]Methyl-losartan was reliably synthesized (n≥40) via methylation of tetrazole-
protected losartan followed by deprotection using HCl in an overall yield of 30%–60% (decay-corrected from [11C]MeI).
Radiochemical purity was >99% and specific activity 700–3600mCi/mmol.
Keywords: tetrazol-protected losartan; [11C]methylation; HCl hydrolysis; cardiovascular disorder
on structure-activity relationship studies, losartan and its methy-
lated analog, methyl-losartan (Scheme 1) exhibit similar binding
Introduction
The renin-angiotensin system is a key hormonal system involved
affinities for AT1Rs: IC50=19 nM, IC50=32 nM, respectively, and
in the regulation of blood pressure.1 Angiotensin II (Ang II) is the
main effector hormone of this system and acts principally by sti-
mulating the Ang II Type 1 receptors (AT1R) which are expressed
in the kidneys, adrenal glands, heart, brain, and vasculature.2,3
Several lines of evidence suggest that there are alterations in
the expression levels of the AT1R associated with cardiovascular
diseases. In a streptozotocin-induced diabetic rat model, signifi-
cant increases in the AT1R receptors and mRNA expression levels
were observed in the heart 14days post-streptozotocin injec-
tion.4 Secondly, in a rat model of myocardial infarction, an
increase in AT1R was observed in the heart and brain, and a
decrease in the kidneys at 4 and 8weeks post-myocardial infarc-
tion.5 Thirdly, increases in renal AT1R levels were observed in a
swine model of renal artery stenosis, suggesting involvement
with renovascular hypertension, the most common form of sec-
ondary hypertension.6
thus good potential.18 The aim of this work is to describe the
synthesis of the O-[11C]methylated derivative of the clinically
used losartan.
Experimental
General methods
All reagents and solvents were purchased from Sigma-Aldrich
Chemical and were used as received without further purification.
Losartan was purchased from Toronto Research Chemicals Inc.
North York, ON, Canada. HPLC analysis was performed with a
Waters pump (Waters, Milford, MA, USA) with inline radiation
detector and Waters 486 UV detector (Meadows Instrumentation
Company, Zion, IL, USA) at 254nm. The analytical HPLC column
was Luna C18 (10m, 250Â4.6mm). Melting point of tetrazol-pro-
tected losartan was measured by differential scanning calorime-
try (DSC) on DSC Q100 Instrument at 10ꢀC/minute heating rate
under nitrogen. 1H and 13C NMR were recorded on Varian Inova
500MHz spectrophotometer. The high resolution mass spectrum
was obtained on a Micromass Q-TOF mass spectrometer.
Several groups have developed radioligands aimed at imaging
the AT1R noninvasively in vivo using positron emission tomogra-
phy (PET) and single photon emission computed tomography
(SPECT). Verjans and colleagues have developed 99mTc-losartan
as a potential SPECT tracer for imaging the AT1R, which displayed
increased uptake in the infarcted area post-myocardial infarction
in mice.7 The Johns Hopkins group reported three 11C-labeled
tracers: MK-996, L-159,884, and most recently KR31173 for ima-
ging renal and cardiac AT1Rs in vivo.6,8–14 Finally, we have
recently reported that [11C]methyl-candesartan is a suitable
and promising agent for PET imaging of renal AT1 receptors at
aCardiac PET Centre, University of Ottawa Heart Institute, Ottawa, Ontario,
Canada
specific activity below 1500Ci/mmol.15 Despite these efforts, bUniversity of Ottawa, Department of Cellular and Molecular Medicine Ottawa,
Ontario, Canada
work is still underway to develop a PET radioligand that binds
to brain AT1 receptors and is suitable for PET imaging in humans.
*Correspondence to: Jean N. DaSilva, Cardiac PET Centre, University of Ottawa
Heart Institute.
Losartan is a selective AT1R antagonist widely used clinically
in the management of hypertension and heart failure.16,17 Based Email: jdasilva@ottawaheart.ca
J. Label Compd. Radiopharm 2011, 54 754–757
Copyright © 2011 John Wiley & Sons, Ltd.

T. Hadizad et al.
Cl
Cl
Cl
Cl
N
N
N
N
N
N
N
N
C4H9
C4H9
C4H9
C4H9
CH2O11CH3
CH2O11CH3
CH2OH
CH2OH
a
b
c
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
H
CPh3
H
Ph3C
1
[
11C]2
[
11C]3
Scheme 1. Reagents and conditions: a) Triethylamine, Trityl chloride, DMF; b) [11C]MeI, NaH, DMF, 80ꢀC, 2minutes; c) HCl (4N), DMF, 80ꢀC, 3minutes.
Chemistry
chromatography. Pure tetrazole-protected methyl-losartan, 2 was
eluted with 3% methanol in dichloromethane (20mg, 39%).
1H NMR (d6-DMSO, 500MHz) dH 7.79 (d, 1H, J=7.4Hz), 7.60–7.20
(m, 12H), 7.05 (d, 2H, J=7.5Hz), 6.83–6.89 (m, 8H), 5.11 (s, 2H),
4.11 (s, 2H), 3.08 (s, 3H), 2.38 (t, 2H, J=7.5Hz), 1.40 (quint, 2H,
J=7.5Hz), 1.15 (sext, 2H, J=7.4Hz), 0.72 (t, 3H, J=7.4Hz).
2-Butyl-4-chloro-5-(hydroxymethyl)-1-[[2'-[(triphenylmethyl)tetra-
zole-5-yl]biphenyl-4-yl]methyl]imidazole (tetrazole-protected
losartan, 1)
Trityl chloride (0.275g, 1mmol) was added to an ice-cooled solu-
tion of losartan (0.300g, 0.7mmol) and triethylamine (0.100g, 1
mmol) in dimethylformamide (DMF) (1.8mL). The resulting solu-
tion (yellow in color) was stirred at 0–4ꢀC for onehour and then
at room temperature overnight. The progress of the reaction was
monitored by thin layer chromatography (silica gel, hexane/ethyl
acetate, 30/70, Rf=0.8) as well as analytical HPLC (acetonitrile/0.1
M ammonium formate solution, 35/65, 2mL/minute) with HPLC
confirming the disappearance of losartan (Rt=3.2minute). The
reaction mixture was quenched and the product was extracted
by ethyl acetate, dried over sodium sulfate (Na2SO4), and the sol-
vent was evaporated. The tetrazole-protected losartan, 1 was
purified by flash column chromatography (silica gel, hexane/
ethyl acetate, 30/70, v/v) to provide 0.453g (96%) as white pow-
der. MP: 162ꢀC (measured by DSC, heating rate: 10ꢀC/minute).
The compound was decomposed immediately after melting
and did not exhibit any crystallization while cooling. 1H NMR
(d6-DMSO, 500MHz) dH 7.79 (d, 1H, J=7.7Hz), 7.61 (t, 1H,
J=7.5Hz), 7.54 (t, 1H, J=7.5Hz), 7.44–7.30 (m, 10H), 7.05 (d, 2H,
J=8.1Hz), 6.90–6.85 (m, 8H), 5.23 (t, 1H, J=5.2Hz), 5.18 (s, 2H), 4.20
(d, 2H, J=5.2Hz), 2.37 (t, 2H, J=7.6Hz), 1.40 (quint, 2H, J=7.5Hz),
1.14 (sext, 2H, J=7.4Hz), 0.73 (t, 3H, J=7.4Hz). Two-dimensional
H/H COSY (COrrelation SpectroscopY) spectrum confirmed the
coupling of the hydroxy proton at 5.23ppm to the methylene
protons at 4.20ppm, whereas in 1H/13C HSQC (Heteronuclear
Single Quantum Coherence, acquired at 400MHz) spectrum no
2-Butyl-4-chloro-5-(methoxymethyl)-1-[[2'-tetrazole-5-yl]biphenyl-
4-yl]methyl] imidazole (methyl-losartan, 3)
To the tetrazole-protected methyl-losartan, 2 (0.015g, 0.022
mmol) dissolved in 1,4-dioxane (1.2ml) was added HCl (1N in
anhydrous methanol, 1.6mL). The reaction mixture was stirred
at room temperature overnight and progress was monitored
by HPLC (acetonitrile/0.1M ammonium formate, 35/65, 2mL/
minute, Rt=6.0minutes). The mixture was diluted with water
and extracted with ethyl acetate, dried over anhydrous sodium
sulfate, filtered, and concentrated. The crude product was puri-
fied by column chromatography (hexane/ethyl acetate/acetic
acid, 50/50/0.5v/v). Recrystallization from ethyl acetate provided
8.5mg (88%) of methyl-losartan. 1H NMR (d6-DMSO, 500MHz) dH
7.65–7.48 (m, 4H), 7.07 (d, 2H, J=8.2Hz), 6.97 (d, 2H, J=8.2Hz),
5.17 (s, 2H), 4.25 (s, 2H), 3.14 (s, 3H), 2.50 (t, 2H, J=7.6Hz), 1.46
(quint, 2H, J=7.7Hz), 1.23 (sext, 2H, J=7.6Hz), 0.80 (t, 3H, J=7.5
Hz). 13C NMR (CDCl3, 500MHz) dC 171.21, 148.67, 140.70,
139.55, 135.03, 131.06, 130.99, 130.44, 129.72, 128.25, 125.52,
123.60, 122.35, 60.41, 57.56, 47.10, 29.90, 26.31, 22.31, 14.18.
High resolution mass spectrum calcd. for C23H25ON6Cl:
436.1795, found: 436.1796.
Radiochemistry
signal was observed for the proton of hydroxy group confirm- 2-Butyl-4-chloro-5-([11C]methoxymethyl)-1-[[2'-tetrazole-5-yl]
ing that this proton is not attached to carbon.
biphenyl-4-yl]methyl] imidazole ([11C]methyl-losartan, [11C]3)
About 15minutes before end of beam, a solution of tetrazole-
protected losartan, 1 (1.0mg, 1.5Â10-3mmol) in 0.2mL of dry
DMF was transferred to a 1mLV-vial containing approximately
2mg (8.3Â10-2mmol) of NaH. The mixture was vortexed, soni-
2-Butyl-4-chloro-5-(methoxymethyl)-1-[[2'-[(triphenylmethyl)tetrazole-
5-yl]biphenyl-4-yl]methyl]imidazole (tetrazole-protected methyl-
losartan, 2)
Tetrazole-protected losartan, 1 (0.050g, 0.075mmol) in THF (2.0 cated (5minutes each) and purged with argon. [11C]CO2 pro-
mL) was added to the flask containing sodium hydride (NaH, duced from a CTI/Siemens RDS-111 cyclotron was converted to
0.200g, 8.3mmol), and the reaction mixture was stirred at room
[
11C]methyl iodide using the wet method (lithium aluminum
temperature under argon for 1.5hour, while the resulting hydro- hydride reduction/hydroiodic acid).15
[
11C]Methyl iodide was dis-
gen gas was vented. To this milky mixture, methyl iodide (1.36g, tilled through P2O5 trap to the reaction vial via helium flow (30
9.6mmol) was added dropwise while stirring in ice bath. The ice mL/minute). Once maximum activity is trapped, the reaction vial
bath was removed, and the reaction mixture was allowed to was sealed, heated at 80ꢀC for 2minutes. The reaction mixture
stir at room temperature overnight. Water was added to the was cooled to approximately À24ꢀC, and 4N HCl in DMF (0.2mL)
light yellow reaction mixture and the product was extracted was added. The temperature was then raised to 80ꢀC for 3minutes.
with ethyl acetate. Organic solvents were evaporated and the The reaction mixture was cooled down to À40ꢀC, quenched
yellow crude product was loaded on a silica gel column with water (0.3mL) and was injected onto the semipreparative
J. Label Compd. Radiopharm 2011, 54 754–757
Copyright © 2011 John Wiley & Sons, Ltd.
www.jlcr.org

T. Hadizad et al.
HPLC column (Luna C18, 10m, 250Â10mm, acetonitrile/0.1% and solvents such as DMSO, DMF or acetonitrile.19 To overcome
ammonium formate, 35/65, 8.0mL/minute). [11C]Methyl-losartan, these side reactions, tetrabutylammonium hydroxide was used
[
11C]3 was eluted at 7.4minutes (Figure 1). The fraction contain- as a base; however, results were unsuccessful because the base
ing the product was evaporated to dryness and the final product was not strong enough to generate the desired intermediate.
was dissolved in a saline/sterile water/8.4% sodium bicarbonate Methylation also failed when a mixture of potassium carbonate
(5/4.4/0.66v/v/v) mixture. [11C]Methyl-losartan was produced in and kryptofix (4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-
30%–60% radiochemical yield (decay-corrected, n≥40) with hexacosane) was used. The use of 15-crown-15 along with NaH
>99% radiochemical purity as determined by injection onto ana- did not improve the yield.
lytical HPLC (acetonitrile/0.1% ammonium formate, 40/60, 2mL/
Compound 2 was characterized by 1H NMR spectrometry. The
minute, Rt: 3.5minutes). Similar results were obtained with the singlet peak at d=3.08ppm is a clear indication of incorporation
Prodigy 10m and Partisil SCX 10m columns using the same sol- of the methyl group (Ha). Furthermore, the triplet at d=5.23ppm
vent system.
corresponding to the proton of the hydroxyl group (Hb) disap-
peared (Figure 2). The doublet at d=4.20ppm corresponding to
Hc of tetrazole-protected losartan is converted to a singlet and
it is upfielded (d=4.11ppm) because of the presence of an elec-
Results and discussion
The synthetic route used for preparation of methyl-losartan is tron-donating group (methyl). No change was observed in the
illustrated in Scheme 1. Losartan has three possible positions aliphatic and aromatic areas.
for methylation (the hydroxyl group and two positions on the
The N-trityl protective group was removed by the addition of
tetrazole ring). Because the anion charged tetrazole ring of losar- HCl (1N in methanol) to a solution of compound 2 in 1,4-dioxane
tan mimics the anion charges in the NH2-terminal of Ang II, and stirred at room temperature overnight. Acid-deprotection of the
because of the higher tendency of the tetrazole group for tetrazole group at high temperature (HCl, 65–70ꢀC, 12–13min-
1
methylation, the protection of this group was required. The tet- utes) produced losartan. The H NMR spectrum of compound 3
razole-protected losartan, 1 was prepared following a literature demonstrated disappearance of the trityl peaks in the aromatic
procedure15 in high yields. Trityl chloride solution was added region, whereas a singlet corresponding to the methyl group
to an ice cooled solution of losartan in DMF in the presence was observed at d=3.14ppm.
of triethylamine. The product was characterized by 1H NMR
[
11C]Methyl-losartan was synthesized from tetrazole-protected
(Figure 2). Further analysis by NMR spectroscopy (two-dimensional losartan, 1 in DMF in high yields (30%–60% from [11C]MeI,
H-H COSY and C-H HSQC) revealed that the 2H doublet at d=4.20 decay-corrected) with a total synthesis and purification time
ppm was a methylene coupled to the 1H triplet at d=5.23ppm of approximately 30minutes+5minutes for quality control
1
(OH). The H NMR spectrum of compound 1 demonstrated dis- (from end-of-beam). The tracer was obtained in high chemical
appearance of the tetrazole proton.
(>95%) and radiochemical (>99%) purities, whereas the resulting
Synthesis of tetrazole-protected methyl losartan, 2 was per- specific activity was 700–3600mCi/mmol. The methylation reaction
formed with a modification to the Williamson reaction by using did not proceed in THF at 50ꢀC (3minutes). The optimal conditions
sodium hydride as a base in THF. Although the methylation reac- for the deprotection step were heating at 80ꢀC for 3minutes in
tion was successful in DMF, more undesired by-products were DMF. The deprotection reaction was also successful with trifluoroa-
formed and the violent exothermic reaction could not be con- cetic acid in DMF. The identity of this novel radiotracer as [11C]
trolled. Sodium hydride plays a dual role as a base and a source methyl-losartan was confirmed by co-injection with an authentic
of hydride in competing reactions with the intended substrate standard onto analytical HPLC using three different columns.
[11C]Methyl-Losartan
0
2
4
6
8
10
12
14
16
18
20
Methanol
min
Figure 1. HPLC profile for the synthesis of [11C]methyl-losartan. Dotted line is the radioactivity chromatogram and solid line presents the ultraviolet (UV) absorption.
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Copyright © 2011 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2011, 54 754–757

T. Hadizad et al.
Cl
Hc
OR
N
N
C4H9
Hd
Hd
Hc
A) R=CH3 (Ha)
B) R=H (Hb)
N
N
N
N
CPh3
Ha
Hc
Hd
Hd
A
B
Hc
Hb
EOAc
Figure 2. 1H NMR spectrum of tetrazole-protected methyl-losartan (A) compared with tetrazole-protected losartan (B).
A. Cuthbertson, M. A. Vannan, C. P. M. Reutelingsperger, JACC
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Conclusion
[
11C]Methyl-losartan was produced reliably by O-[11C]methyla-
tion followed by acid removal of the trityl protecting group.
Synthesis of the methylated analog of the clinically used drug
losartan is expected to facilitate translational work on imaging
AT1 receptors to humans.
Acknowledgement
We thank the radiochemistry and biotesting staff, and the [13] T. G. Zober, W. B. Mathews, E. Seckin, S. E. Yoo, J. Hilton, J. Xia,
K. Sandberg, H. T. Ravert, R. F. Dannals, Z. Szabo, Nucl. Med. Biol.
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Canadian Institutes of Health Research MOP-80203 for funding
this work.
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