Synthesis and biological evaluation of [carboxyl-11C]eprosartan

Article abstract of DOI:10.1002/jlcr.1598

Essential hypertension occurs in approximately 25% of the adult population and one cause of hypertension is primary aldosteronism. Targeting the angiotensin II AT₁ receptor using PET and an appropriate tracer may offer a diagnostic method for a

Full text of DOI:10.1002/jlcr.1598

Research Article
Received 21 January 2009,
Revised 9 February 2009,
Accepted 16 February 2009
Published online 1 June 2009 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1598
Synthesis and biological evaluation
of [carboxyl-11C]eprosartan
a
b
c
d
b
Ola Aberg, Orjan Lindhe, Hakan Hall, Per Hellman, Tor Kihlberg, and
˚
Bengt Langstrom
¨
˚
a,bÃ
˚
¨
Essential hypertension occurs in approximately 25% of the adult population and one cause of hypertension is primary
aldosteronism. Targeting the angiotensin II AT1 receptor using PET and an appropriate tracer may offer a diagnostic
method for adrenocortical tissue. This report describes the synthesis of the selective AT1 receptor antagonist
[carboxyl-11C]eprosartan 10, 4-[2-butyl-5-((E)-2-carboxy-3-thiophen-2-yl-propenyl)-imidazol-1-ylmethyl]-[carboxyl-11C]ben-
zoic acid, and its precursor (E)-3-[2-butyl-3-(4-iodo-benzyl)-3H-imidazol-4-yl]-2-thiophen-2-ylmethyl-acrylic acid 9.
11C-carboxylation of the iodobenzyl moiety was performed using a palladium-mediated reaction with [11C]carbon
monoxide in the presence of tetra-n-butyl-ammonium hydroxide in a micro-autoclave using a temperature gradient from
25 to 1401C over 5 min. After purification by semipreparative HPLC, [carboxyl-11C]eprosartan 10 was obtained in
37–54% decay-corrected radiochemical yield (from [11C]carbon monoxide) with a radiochemical purity 495% within
35 min of the end of bombardment (EOB). A 5-lAh bombardment gave 2.04 GBq of 10 (50% rcy from [11C]carbon monoxide)
with a specific activity of 160 GBq lmolꢀ1 at 34 min after EOB. Frozen-section autoradiography shows specific binding
in kidney, lung and adrenal cortex. In vivo experiments in rats demonstrate a high accumulation in kidney, liver and
intestinal wall.
Keywords: angiotensin II; AT1; eprosartan; [11C]carbon monoxide; carboxylation
observed. Human levels of AT1 mRNA were altered in
hearts with dilated cardiomyopathy as well as old myocardial
infarctions.13–17
Introduction
Hypertension occurs in approximately 25% of the adult
population and predispose for cardiac diseases and arterio-
sclerosis. Hypertension may be caused by a chronic excessive
secretion of aldosterone from the glomerulosa cells of the
adrenal cortex; this is known as primary aldosteronism.1 Primary
aldosteronism is the cause of hypertension in 10–15% of all
patients with essential hypertension.2,3 When caused by an
aldosterone producing adenoma; primary aldosteronism can be
treated by surgical removal of the hypersecreting gland. Since
nodular hyperplasia of the adrenal glands may mimic an
adenoma; it may be a challenge to determine which of the
adrenals to remove.4
Another clinical problem is adrenal incidentalomas that are
frequently found (1–5%) in CT examinations.5–7 Though the
majority of incidentalomas are benign masses, characterization
is difficult and time-consuming since sensitive methods to
exclude benign masses, which do not need further attention, are
lacking. For this purpose, PET scanning with 11C-metomidate
and 11C-hydroxyephederine have been studied.8,9 As a potential
complementary method we are investigating the use of the
angiotensin II type 1 receptor (AT1-R) as target for PET.
Pathological adrenal cortex may express different amounts of
AT1-R, adenoma classically been angiotensin-unresponsive while
the tissue in idiopathic hyperplasia is angiotensin-responsive.
This receptor is present mainly in the zona glomeruosa in
human,10 and is known to also be present in colon,
kidney, aorta, CNS and the heart.10–14 AT1 up-regulation in the
atria of patients with end-stage heart failure has also been
One class of compounds that is targeting the AT1-receptor is
the sartans. Eprosartan is a drug that selectively inhibits the
action of angiotensin II on the AT1 receptor and is prescribed for
hypertension. The structure has two carboxylic acid moieties
that potentially could be labelled with 11C by carboxylation of
the corresponding aryl-halide or vinyl-halide using [11C]carbon
monoxide. [11C]Carbon monoxide is a versatile labelled pre-
cursor that has been used to synthesize a whole array of
different carbonyl compounds by transition metal mediated
reactions with high specific activity.18 Aromatic carboxylations
can be performed using [11C]carbon dioxide and a correspond-
ing Grignard reagent, but the reaction is moisture sensitive and
is not compatible with acidic functional groups. Another
drawback is the laborious preparation and handling of the
Grignard reagent to avoid isotopic dilution with atmospheric
carbon dioxide. Carbon monoxide on the other hand is less
aDepartment of Biochemistry and Organic Chemistry, Box 599, Husargatan 3,
BMC, S-751 24 Uppsala, Sweden
bUppsala Imanet, GE Healthcare, Box 967, S-751 09 Uppsala, Sweden
cUppsala ASL, GE Healthcare, Box 967, S-751 09 Uppsala, Sweden
dDepartment of Surgery, University Hospital, SE-75185 Uppsala, Sweden
˚
*Correspondence to: Bengt Langstro¨m, Department of Biochemistry and Organic
Chemistry, Box 599, Husargatan 3, BMC, S-751 24 Uppsala, Sweden.
E-mail: bengt.langstrom@biorg.uu.se
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Copyright r 2009 John Wiley & Sons, Ltd.

˚
O. Aberg et al.
abundant in the atmosphere, thus [11C]carbon monoxide is less
prone to isotopic dilution. Transition-metal mediated/catalyzed
carboxylation is a very general reaction and tolerates a wide
variety of functional groups.
Results and discussion
Precursor synthesis
The precursor was synthesized as outlined by Keenan et al.
(Schemes Knoevenagel reaction between
and 2).30
The metabolic profile of a PET-tracer may influence the
decision where to incorporate the radiolabel. However, eprosar-
tan is reported to be very metabolically stable in human and no
active metabolites are reported, thus the radiolabel may in this
case be incorporated in any of the two carboxylic acid moieties.
Following an intravenous administration of [14C]eprosartan, about
61% of the radioactivity was recovered in the feces and about
37% in the urine, indicating that both the biliary and renal routes
are important. Elimination occurs via the liver and kidneys,
primarily as unchanged drug, with 7% of an intravenous dose
excreted in urine as the corresponding acyl glucuronide.
Eprosartan is not metabolized by cytochrome P450 enzymes.19,20
Previously, the following compounds targeting the AT1
receptor have been labelled with 11C; [11C-tetrazoyl]irbesartan,21
[11C]MK-996,22 [11C]L-159,88423–27 and [11C]KR31173.28,29
Here we report the synthesis of [carboxyl-11C]eprosartan 10
(Scheme 3). Eprosartan was selected as a tracer candidate
as it (a) is extensively investigated in the human, including
toxicity studies, (b) is metabolically stable in the human, and (c)
can be obtained in high specific activity using a one-step
synthesis.
1
A
2-thiophenecarboxaldehyde and diethyl malonate formed the
condensation product 1, which was subsequently reduced using
sodium borohydride in ethanol to give the diester 2. The diester
was selectively hydrolyzed using potassium hydroxide to give
the ester/carboxylic acid 3 in good yields.
2-Butyl-4(5)-(hydroxymethyl)-imidazole was protected by stir-
ring with acetic anhydride to give 4 in good yield. In the next
step
4 was combined with trifluoromethanesulfonic acid
4-iodobenzyl ester (prepared from (4-iodophenyl)methanol,
DIPEA and triflic anhydride at ꢀ781C), in a one-pot reaction to
give the crude acetate 5. The crude acetate was hydrolyzed
using potassium carbonate to give the alcohol 6. The position of
the 4-iodobenzyl group was confirmed by NOE NMR by selective
irradiation of the CH2 groups. Oxidation of the alcohol using
technically activated manganese oxide gave the desired
aldehyde 7.
A Knoevenagel-like condensation between the acid 3 and the
aldehyde 7 and subsequent elimination of CO2 formed the
intermediate ester 8.31 The acid 3 was added in excess due to a
competing decarboxylation of the acid. The E-geometry of the
Scheme 1. (a) Diethyl malonate, piperidine, benzoic acid in cyclohexane, reflux 20 h. (b) NaBH4 in EtOH, 40 min. (c) KOH 1.0 eq in EtOH, r.t. 48 h.
f
Scheme 2. (a) Acetic anhydride. (b) (4-iodophenyl)methanol, triflic anhydride, DIPEA in dichloromethane. (c) K2CO3 in MeOH. (d) Activated MnO2 in dichloromethane.
(e) 3, piperidine and benzoic acid in toluene. (f) NaOH, EtOH.
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O. Aberg et al.
3
double bond E-form was confirmed by measuring the JH-C
The conversion to [11C]carbonate was determined by acidify-
coupling constants of the vinyl proton by means of an HMBC ing the purged crude sample with ammonium formate buffer
experiment. The cis coupling to the carbonyl carbon was 7.4 Hz (pH 3.5) and then purging once more to remove [11C]O2. Both
and the trans coupling to the CH2-carbon was 11.2 Hz. The [11C]O conversion and the amount of [11C]carbonate formed in
precursor 9 was finally obtained by basic hydrolysis of the ester. the reaction rapidly dropped at elevated temperatures. Above
1501C, [11C]carbonate could no longer be detected; instead
unidentified lipophilic radioproducts were formed (entries 1–9).
The temperature range 65–1601C gave low product yields
(22711%) and no clear correlation between radiochemical yield
Labelling reaction
The 11C-carboxylation of 9 was performed according to a
reported method using a palladium-mediated carboxylation of
the aryl iodide with [11C]carbon monoxide in the presence of
tetra-n-butyl-ammonium hydroxide in a micro-autoclave for
5 min (Scheme 3 and Table 1).32 Different temperatures were
tested using constant reagent concentrations. Temperatures
below 1201C gave high conversion of [11C]carbon monoxide,
but 61–77% resulted in [11C]carbonate and only 11–31%
radiochemical yield of 10 (entries 1–4). The side reaction that
trapped the radioactivity as [11C]carbonate occurs probably via
the water–gas shift reaction (Scheme 4) and subsequent
reaction of [11C]carbon dioxide with OHꢀ.
and temperature was found. When the amounts of base, water
and DMSO were decreased and the temperature was raised
from r.t. to around 1401C over 5 min, the reaction was cleaner
and formed less [11C]carbonate (entry 10). However, 51%
conversion of [11C]carbon monoxide was not satisfactory, so
the amount of Pd complex was increased in order to favor the
[11C]O insertion that forms the Pd-[11C]acyl complex. This gave
90% [11C]O conversion in a very clean reaction that produced
radiochemically pure product (entry 11). The presence of
DMSO as a co-solvent had no effect on the radiochemical
yields but helped to prevent precipitation while diluting the
Scheme 3. (a) [11C]carbon monoxide, Pd(PPh3)4, tetra-n-butylammonium hydroxide 30 hydrate, water, DMSO, THF, 5 min.
Table 1. Optimization of the 11C-carboxylation reaction
Pd(PPh3)4
(mM)
Q-OHa
(mM)
H2Ob
(M)
DMSO
(M)
Temp
(1C)
Conversion of
[11C]O (%)c,d
Analytical
RCY (%)d,e
Entry
1
2
3
4
5
6
7
8
5
5
5
5
5
5
5
5
5
25
25
25
25
25
25
25
25
25
25
6.8
6.8
6.8
–
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
3.5
0.75
0.90
0.20
0.20
–
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
–
65
90
110
120
130
140
150
160
100
99
98
83
57
36
40
24
23
31
19
11
28
20
33
16
o6
4172(2)
91
82
79
9
180
30
10
11
12
13
14
5
25–140
25–140
25–140
25–140
25–140
5176(2)
91
86
89
81
14
14
14
14
0.35
0.35
–
–
33
Reaction conditions were [11C]carbon monoxide (10–100 nmol), and 200 mL solution of 9 (17 mM), Pd(PPh3)4, tetra-n-
butylammonium hydroxide 30 hydrate. Additives were H2O and DMSO.
aQ-OH: tetra-n-butylammonium hydroxide 30 hydrate.
bThe total amount of added water including crystal water from QꢀOH.
cDecay corrected conversion yield of [11C]carbon monoxide to non-volatile products remaining in the reaction mixture after
evaporation of solvent by purging with nitrogen.
dThe numbers in brackets indicate the number of experiments.
eAnalytical radiochemical yield as the sum of the decay corrected analytical radiochromatogram and the conversion yield.
J. Label Compd. Radiopharm 2009, 52 295–303
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˚
O. Aberg et al.
Scheme 4. The water–gas shift reaction.
nitrogen-purged crude product for HPLC injection (entry 12 and
13). When base, water and DMSO were omitted the reaction
resulted in an unidentified compound (47%) along with the
product (33%, entry 14).
For biological deliveries of 10, the conditions were similar as
for entry 13 except that the amount of Pd complex was
decreased to 10 mM for practical reasons to avoid precipitation
upon dilution of the crude product. Those conditions gave a
slightly less clean reaction than entry 11 (as estimated from the
preparative UV chromatogram), but at the same time better
chromatography, since DMSO was no longer needed to keep
crude product mixture in solution. The crude product was
purified using semipreparative HPLC and formulated in a
phosphate buffer solution with a pH of 7.2. In this way the
decay-corrected radiochemical yield of the 11C-labelled and
formulated [carboxyl-11C]eprosartan 10 was 37–54% (n = 5),
calculated from [11C]carbon monoxide, within 35 min from
EOB and with a radiochemical purity 495%.
{
Figure 1. Frozen section autoradiography of [carboxyl-11C]eprosartan left, in the
right column binding was blocked with 10 mM unlabelled eprosartan.
Specific activity
High specific activity is important to avoid saturating or
perturbating the biological system under study, especially when
the binding site density (Bmax) is low. The concept of using
[11C]carbon monoxide as the labelled precursor allows very easy
handling and preparation of the reagent solutions because the
aryl halide and palladium complex used are not particularly
reactive toward carbon monoxide. Additionally, carbon mon-
oxide has low solubility in most organic solvents at normal
pressures and temperatures and has low abundance in the
atmosphere.33 This is an advantage over the laborious handling
of reactive Grignard reagents used for carboxylation with
[11C]carbon dioxide, due to the more abundant carbon dioxide
that is prone to cause isotopic dilution and lower specific
activity.34 It should be noted that the basic quaternary
ammonium hydroxide salt used in this report is prone to react
with carbon dioxide to form carbonate and should be stored
under inert atmosphere. Addition of cold carbonate could, at
least in theory, lead to isotopic dilution of [11C]carbon monoxide
via the water–gas shift reaction. However, in this work we have
not noticed tendencies of isotopic dilution from the base. The
specific activity of [carboxyl-11C]eprosartan 10 was determined
following a 5-mAh bombardment giving 10.3 GBq [11C]carbon
monoxide at 7.5 min from EOB. At 34 min, 2.04 GBq of 10 (50%
from [11C]carbon monoxide) was isolated with a specific activity
of 160 GBq mmolꢀ1 (520 GBq mmolꢀ1 at EOB). A 12-mAh bom-
bardment gave a specific activity of 360 GBq mmolꢀ1 at 38 min
from EOB (1250 GBq mmolꢀ1 at EOB) and is in the same order as
previously reported.
the adrenal cortex (presumably zona glomerulosa) and was also
affected by the addition of excess unlabelled eprosartan
(Figure 2). However, the binding density (Bmax) was very low.
Quantitation of the binding in whole pig adrenals followed by
saturation analysis showed that the binding was saturable, with
a Kd-value of approximately 10 nM (Figure 2). The specific
binding in pig adrenals was approximately 60% at Kd (10 nM)
and 70% at half Kd (5 nM). Specific [carboxyl-11C]eprosartan
binding was also found in human adrenals, and especially with
those affected with aldosterone producing adenomas (Figure 3).
It should be noted that no binding at all was found in one
case of cortisol producing adenoma and in two cases of
adrenocortical hyperplasia (images not shown).
Several research groups have studied the receptor expression
of AT1 at the mRNA or receptor level in diseased adrenal glands.
While the reported levels of AT1 compared to controls some-
times are contradictory, aldosteron producing adenoma seems
in most cases to have a higher degree of variability.35–46 No
studies have yet clearly described differences in AT1-R expres-
sion between adenomas and hyperplasia.
[Carboxyl-11C]eprosartan in vivo organ distribution
A high accumulation in male rat kidney, liver and intestinal wall
was observed as well as retention of uptake after 40 min in lung,
testis and fat (Table 2). In an earlier distribution study using
pharmacological doses of [14C]eprosartan (3 and 10 mg kgꢀ1
)
the highest concentrations were found in the intestines, kidney
and liver.47 In the present investigation using tracer amounts of
[11C]eprosartan (o0.1 mg kgꢀ1), the highest concentrations were
found in the corresponding organs.
[Carboxyl-11C]eprosartan in vitro autoradiography
Using in vitro autoradiography on frozen organ section, specific
[carboxyl-11C]eprosartan binding (affected by excess of unla-
belled eprosartan) was observed in rat lung and kidney as well
as pig adrenal, and to some degree also in rat spleen and liver.
No specific binding was seen in striatum and cerebellum of rat
Experimental
General
brain (Figure 1). Studies of [carboxyl-11C]eprosartan binding in All chemicals were purchased from Aldrich/Fluka and used
pig adrenal showed most intense binding in the outer layer of without further purification. Dry tetrahydrofuran was distilled
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˚
O. Aberg et al.
Figure 2. The left panel shows the total and non-specific binding of [carboxyl-11C]eprosartan to pig adrenal glands at four different concentrations. Quantitative analysis
of the binding was used for a saturation analysis, revealing saturable binding with an estimated of Kd 10 nM and Bmax of 8.3 pixels (corresponding to approximately
1.1 fmol mgꢀ1 tissue). Lower panel: (ꢁ) Total binding, ( & ) non-specific binding, (m) specifically bound tracer.
Table 2. Organ distribution of [carboxyl-11C]eprosartan 10
in adult male Sprague Dawley rats 15 and 40 min post
injection (SUV, average7S.E.M.)
Organ distribution of [carboxyl-11C]eprosartan 10 in rats
SUVa in rat
15 min (n = 9)
40 min (n = 8)
Blood
Heart
Lung
Liver
0.26970.057
0.11670.022
0.49170.094
5.2870.583
0.19470.025
0.26370.046
0.87570.113
7.0071.01
15.4172.55
0.41770.065
0.04870.004
0.10070.018
0.05270.007
0.01070.002
0.13070.032
0.05070.008
0.43470.127
2.5270.387
0.06470.017
0.09370.017
0.31870.054
2.3070.358
3.5870.570
0.71470.283
0.05470.009
0.04970.009
0.05970.016
0.00570.001
Pancreas
Spleen
Adrenal
Kidney
Intestines
Urinary bladder
Testis
Muscle
Fat
Brain
aSee experimental section for details.
spectrometer equipped with non-polar column SE-54. LC-MS
analyses were performed on a Gilson reverse phase HPLC
equipped with a Finnigan mass spectrometer (MeCN/H2O and
0.1% formic acid). [11C]Carbon was produced by a Scandtronix
MC-17 cyclotron at the Uppsala Imanet, GE Healthcare by the
14N(p, a)11C nuclear reaction using a gas target containing
nitrogen (AGA, Nitrogen 6.0) and 0.05% oxygen (AGA, Oxygen
4.8) that was bombarded with 17 MeV protons. [11C]Carbon was
obtained as [11C]carbon dioxide in the target and was reduced
to [11C]carbon monoxide in a zinc furnace at 4001C, and
transferred to the micro-autoclave using a remote-controlled
workstation.48,49 Analytical HPLC was performed on a Beckman
system, equipped with a Beckman 126 pump, a Beckman 166 UV
Figure 3. 1 and 2: Parts of two adrenal glands extirpated for non-cortical reasons,
but expressing normal cortex 3: from a patient with unspecified adrenocortical
cancer (ACC), 4 and 5 from patients with aldosterone producing adenomas (APA).
over sodium and benzophenone. Thin-layer chromatography
analyses were performed on pre-coated Merck silica gel plates
1
(60F254) and visualized with UV light. H and 13C NMR spectra
were recorded on a Varian 300, 400 or 500 MHz spectrometer
and chemical shifts are given in ppm (d) using CHCl3 (7.26 ppm
1
for H, and 77.16 for 13C) as internal standard. GC-MS analyses
were performed on a Finnigan MAT Thermoquest GCQ mass
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O. Aberg et al.
detector in series with a Bioscan b1-flow count detector and (t, J = 7.1 Hz, 3H, CH3), 1.30 (t, J = 7.1 Hz, 3H, CH3) ppm. GC-MS for
a Beckman Ultrasphere ODS dp 5 m column (250 ꢂ 4.6 mm). C12H14O4S: m/z (rel. intensity) 254 (M1, 44), 209 (85), 164 (86),
A Gilson 231 XL was used as auto injector. Mobile phase: 136 (79), 108 (100).
(A) 50 mM ammonium formate pH 3.5, (B) acetonitrile; 30% B for
Diethyl (thien-2-ylmethyl)malonate (2): NaBH4 (2.94 g,
10 min then 95% B for 10 min, 1 mL minꢀ1. Purification with 77.7 mmol) was added portion wise to a solution of 1 (39.54 g,
semipreparative HPLC was performed on a similar Beckman 155.5 mmol) in ethanol (250 mL) at 01C. The temperature was
system equipped with a Genesis C18 120 4 m (250 ꢂ 10 mm). allowed to rise to r.t. and stirred for 40 min. Glacial acetic acid
Mobile phase: 65%A, 35% B, 5 mL minꢀ1
.
(ꢃ4 mL) was added to pH = 6 (indicated by Merck pH paper),
whereupon a white precipitate was formed and removed by
filtration. The solvent was removed under reduced pressure to
give a brown residue, which was taken up between water
(75 mL) and diethyl ether (175 mL). The organic layer was
washed with water (50 mL) and brine (40 mL) and dried over
MgSO4. The solvent was removed under reduced pressure to
11C-labelling of eprosartan in the carboxyl position
4-[2-Butyl-5-((E)-2-carboxy-3-thiophen-2-yl-propenyl)-imidazol-1-
ylmethyl]-[carboxyl-11C]benzoic acid (10): The following text
describes the procedure used for biological deliveries. To a
(0.8 mL) vial was added tetrakis(triphenylphosphine)-palla-
dium(0) (4.8 mg, 4.1 mmol) and precursor 9 (3.5 mg, 6.8 mmol).
The mixture was purged with argon before dissolving in THF
(400 mL). The resulting solution was added to an argon-purged
vial containing tetra-n-butylammonium hydroxide 30 hydrate
(2.2 mg, 2.8 mmol) and then injected into the 200 mL-injection
loop of the synthesis system. [11C]Carbon monoxide was
transferred to the micro-autoclave (200 mL), which was then
pressurized to 35 MPa (5 kpsi) with the reaction mixture from the
injection loop. The micro-autoclave was immersed and heated in
a heating block set at 1501C for 5 min. The crude product was
transferred to an evacuated vial (3 mL) and the radioactivity was
measured. The vial was heated in a heating block (701C) and
purged with nitrogen gas to remove unreacted [11C]carbon
monoxide and solvent, and the radioactivity was measured
again to determine the conversion yield of [11C]carbon
monoxide. The crude product was diluted first with MeCN
(500 mL) and then water (200 mL), and was purified using
semipreparative HPLC (Rt = 5.5–7.5 min). The product fraction
was concentrated under reduced pressure or by purging with
nitrogen at 1001C and then diluted with phosphate buffer
(pH = 7.2) before the radioactivity was measured (dc rcy 37–54%,
n = 5). The purity of the final product was analyzed with
analytical UV-radio-HPLC (Rt = 6.9 min), and characterization was
made by co-eluting with an isotopically unmodified sample. LC-
MS, ESI1 for C23H24N2O4S m/z [M1H]: 425. Specific activity was
determined by measuring the concentration, volume and
radioactivity of the purified product solution at a specified time
point from EOB. An aliquot was withdrawn and 50 mL injections
were analyzed by analytical UV-radio-HPLC, l = 254 nm, (three
replicates) and compared to a calibration curve made from three
standard solutions (1, 5 and 10 mM) of eprosartan.
1
give 2 (35.47 g, 89%) as a brown oil. H NMR (300 MHz, CDCl3,
251C) d: 7.12 (dd, J = 1.2, 5.1 Hz, 1H, Ar-H), 6.88 (dd, J = 3.5, 5.1 Hz,
1H, Ar-H), 6.83 (m, 1H, Ar-H), 4.17 (dq, J = 0.9, 7.2 Hz, 4H,
2 ꢂ CH2), 3.65 (dd, J = 7.2, 8.1 Hz, 1H, CH), 3.42 (m, 2H, CH2), 1.22
(t, J = 7.2, 6H, 2 ꢂ CH3). GC-MS for C12H16O4S: m/z (rel. intensity)
256 (M1, 84), 182 (64), 154 (43), 137 (99), 110 (100), 97 (55).
3-Ethoxy-3-oxo-2-(thien-3-ylmethyl)propanoic acid (3): A solu-
tion of KOH (5.10 g, 77.25 mmol) dissolved in ethanol (150 mL)
was added drop wise to a solution of 2 (20.0 g, 78.0 mmol) in
ethanol (75 mL). The reaction mixture was stirred at r.t. for 48 h
and then concentrated. The residue was diluted with water and
washed with ether. The aqueous phase was acidified with 2 M
HCl to pH = 1 and extracted with ether (3 ꢂ 60 mL). The
combined extracts were washed with water and brine and then
dried over MgSO4. The solvent was removed under reduced
pressure to give 3 (15.6 g, 89%) as a yellow oil. 1H NMR
(300 MHz, CDCl3, 251C) d: 10.66 (brs, 1H, COOH), 7.16 (dd, J = 1.3,
5.1 Hz, 1H, Ar-H), 6.91 (dd, J = 3.5, 5.1 Hz, 1H, Ar-H), 6.86 (dm,
J = 3.5 Hz, 1H, Ar-H), 4.22 (q, J = 7.1 Hz, 2H, CH2CH3), 3.73 (t,
J = 7.4 Hz, 1H, CH), 3.46 (m, 2H, CH2), 1.25 (t, J = 7.1 Hz, 3H, CH3)
ppm. The compound decompose upon vaporization in GC-MS
to the decarboxylated product, 3-thiophen-2-yl-propionic
acid ethyl ester, GC-MS for C12H16O4S: m/z (rel. intensity)
184 (M1,100), 120 (88), 97 (66).
Acetic acid 1-acetyl-2-butyl-1H-imidazol-4-ylmethyl ester (4):
2-Butyl-4(5)-(hydroxymethyl)imidazole (5.00 g, 32.4 mmol) was
added portion wise to a flask containing stirred ice cold acetic
anhydride (15.1 mL, 162 mmol). When the reaction mixture
occasionally solidified it was warmed a little to dissolve again.
The reaction mixture was allowed to slowly rise to r.t. and then
stirred for 36 h. The excess acetic anhydride was removed under
reduced pressure and the residue was dissolved in dichlor-
omethane and washed with sat. aqueous NaHCO3 (3 ꢂ 25 mL)
and brine and dried over MgSO4. The solvent was removed
under reduced pressure to give 4 (6.99 g, 90%) as a yellow oil. 1H
NMR (400 MHz, CDCl3, 251C) d: 7.19 (m, 1H, Ar-H), 4.96 (d, J = 0.8
Hz, 2H), 2.98 (m, 2H), 2.52 (s, 3H, CH3), 2.05 (s, 3H, CH3), 1.67 (m,
2H, CH2), 1.39 (m, 2H, CH2), 0.90 (t, J = 7.4 Hz, 3H, CH3). 13C NMR
(100 MHz, CDCl3, 251C) d: 170.8, 167.6, 152.6, 136.2, 116.8, 59.7,
30.4, 29.4, 24.5, 22.5, 20.9, 13.7 ppm.
Eprosartan precursor synthesis
Diethyl (thien-2-ylmethylene)malonate (1):
A mixture of 2-
thiophenealdehyde (20.15 g, 180 mmol), diethylmalonate
(29.63 g, 185 mmol), piperidine (2.4 mL, 24 mmol) and benzoic
acid (50 mg, 41 mmol) in cyclohexane (120 mL) was heated to
reflux for 22 h under a Dean-Stark water trap. Removal of the
solvent gave a brown oil, which was dissolved in diethyl ether
(150 mL) and washed with 2 M HCl (3 ꢂ 30 mL), sat. aqueous
NaHCO3 (3 ꢂ 30 mL) and brine (30 mL) then dried over MgSO4.
The solvent was removed under reduced pressure to give 1
Acetic acid 2-butyl-3-(4-iodo-benzyl)-3H-imidazol-4-ylmethyl es-
ter (5): To a solution of triflic anhydride (4.91 g, 17.4 mmol) in dry
dichloromethane (30 mL) was added drop wise a solution of
diisopropylethylamine (2.30 g, 17.8 mmol) and (4-iodophenyl)-
methanol in dichloromethane (20 mL) over 15 min at ꢀ781C. The
reaction mixture was stirred at the same temperature for 30 min.
A solution of 4 (4.00 g, 16.8 mmol) in dichloromethane was
then added drop wise over 15 min at the same temperature.
1
(39.76 g, 87%) as a brown oil. H NMR (300 MHz, CDCl3, 251C) d:
7.84 (unresolved dd, J = 0.6 Hz, 1H), 7.51 (ddd, J = 0.9, 1.2, 5.1 Hz,
1H), 7.36 (ddd, J = 0.6, 1.2, 3.7 Hz, 1H), 7.07 (dd, J = 3.7, 5.1 Hz,
1H), 4.39 (q, J = 7.1 Hz, 2H, CH2), 4.28 (q, J = 7.1 Hz, 2H, CH2), 1.35
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2009, 52 295–303

˚
O. Aberg et al.
The reaction mixture was allowed to slowly warm to r.t. and eluted ahead of the desired product 8 during chromatography.
then stirred for 36 h. The solvent was removed under reduced 1H NMR (400 MHz, CDCl3, 251C) d: 7.22 (dd, J = 1.2, 5.1 Hz, 1H),
pressure and the residue was taken up in ethyl acetate, washed 6.91 (dd, J = 3.4, 5.1 Hz, 1H), 6.82 (dd, J = 1.2, 3.4 Hz, 1H), 4.15 (q,
with sat. aqueous NaHCO3, 2 M HCl and brine and dried over J = 7.1 Hz, 2H), 3.17 (m, 2H), 2.68 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H)
MgSO4. The solvent was removed under reduced pressure to ppm. 13C NMR (100 MHz, CDCl3, 251C) d: 172.4, 143.2, 126.9,
give the crude acetate 5 as a pale solid that was used in the next 124.7, 123.6, 60.7, 36.3, 25.3, 14.3 ppm.
step without further purification.
(E)-3-[2-Butyl-3-(4-iodo-benzyl)-3H-imidazol-4-yl]-2-thiophen-2-
[2-Butyl-3-(4-iodo-benzyl)-3H-imidazol-4-yl]-methanol (6): The ylmethyl-acrylic acid (9): The ester 8 (0.883 g, 1.65 mmol) was
crude acetate 5 was dissolved in methanol (100 mL) and water hydrolyzed in 10 wt% NaOH in EtOH at r.t. overnight. The
(10 mL) and treated with K2CO3 (4.60 g, 33.0 mmol). The reaction reaction was concentrated under reduced pressure and the
mixture was stirred at r.t. for 90 min. The reaction mixture was residue was dissolved in water. The solution was acidified with
filtered and concentrated under reduced pressure and the 2 M HCl and extracted with dichloromethane to give
a
residue was taken up in ethyl acetate and washed with water semicrystalline material that was further purified using flash
and brine and dried over MgSO4. The solvent was removed chromatography, eluting with 0–5% MeOH in dichloromethane
under reduced pressure to give a white crystalline material, to give the acid 9 (0.678 g, 81%) as beige solid. 1H NMR
which was purified by flash chromatography ( | = 7 cm, h = 7 cm) (400 MHz, CDCl3, 251C) d: 7.64 (AA0BB0, 2H, Ar-H), 7.51 (s, 1H),
using 10% methanol in dichloromethane as the eluent to give 7.47 (s, 1H), 7.11 (dd, J = 1.3, 5.3 Hz, 1H), 6.88 (dd, J = 3.5, 5.2 Hz,
1
the alcohol 6 (4.24 g, 68%) as a white solid. H NMR (500 MHz, 1H), 6.79 (dm, J = 3.5 Hz, 1H), 6.69 (AA0BB0, 2H, Ar-H), 5.12 (s, 2H,
CDCl3, 251C) d: 7.62 (AA0BB0, 2H, Ar-H, ortho to the iodo CH2), 4.06 (s, 2H, CH2), 2.69 (m, 2H), 1.62 (m, 2H, CH2), 1.31 (m,
substituent), 6.82 (s, 1H, Ar-H on imidazole), 6.70 (AA0BB0, 2H, Ar- 2H, CH2), 0.86 (t, J = 7.4 Hz, 3H) ppm. LC-MS, ESI1 for
H, meta to the iodo subsituent), 5.17 (s, 2H, -CH2-N), 4.44 (m, 2H, C22H23IN2O2S m/z [M1H]: 507.
-CH2-O), 3.94 (brs, 1H, -OH), 2.51 (m, 2H, -CH2-imidazole), 1.60
(m, 2H, CH2), 1.30 (m, 2H, CH2), 0.85 (t, J = 7.3 Hz, 3H, CH3) ppm. Preparation of eprosartan reference
13C NMR (100 MHz, CDCl3, 251C) d: 150.0, 137.9, 136.4, 131.5,
127.9, 126.0, 93.0, 54.3, 46.4, 29.7, 26.6, 22.4, 13.7 ppm. LC-MS,
4-[2-Butyl-5-((E)-2-carboxy-3-thiophen-2-yl-propenyl)-imidazol-1-
ylmethyl]-benzoic acid: A sample of the commercially available
ESI1 for C15H19IN2O m/z [M1H]: 371.
2-Butyl-1-(4-iodobenzyl)-1H-imidazole-5-carbaldehyde (7): To a
solution of the alcohol 6 (4.02 g, 10.9 mmol) in dichloromethane
(100 mL) was added technically activated MnO2 (9.44 g,
109 mmol). The heterogeneous mixture was stirred for 90 min
at r.t. then filtered through a thin layer of Celite, which was then
Tevetens, manufactured by Solvay Pharmaceuticals, was finely
powdered in a mortar. The powder (1.99 g) was slurried in water
(20 mL) and the pH was adjusted to 412 with 6 M NaOH before
the solution was filtered. 6 M HCl was added drop wise to the
filtrate until pH was o2. The mixture was filtered and dried to
obtain the practically pure organic compound as an off-white
1
rinsed with several portions of dichloromethane. The solvent
was removed under reduced pressure to give the aldehyde 7
crystalline material. H NMR (400 MHz, DMSO-d6, 251C) d: 12.90
(br s, 1H, COOH), 7.91 (AA0BB0, 2H, Ar-H), 7.68 (s, 1H, Ar-H), 7.37
(3.73 g, 93%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3, 251C)
(s, 1H, Ar-H), 7.30 (dd, J = 1.2, 5.1 Hz, 1H, Ar-H), 7.17 (AA0BB0, 2H,
Ar-H), 6.90 (dd, J = 3.5, 5.1 Hz, 1H), 6.71 (dm, J = 3.4 Hz, 1H), 5.60
(s, 2H, CH2), 4.00 (s, 2H, CH2), 2.92 (m, 2H, CH2), 1.58 (m, 2H, CH2),
d: 9.64 (s, 1H, COH), 7.77 (s, 1H, Ar-H), 7.61 (AA0BB0, 2H, Ar-H),
6.74 (AA0BB0, 2H, Ar-H), 5.49 (s, 2H, CH2), 2.63 (m, 2H, CH2), 1.67
(m, 2H, CH2), 1.34 (m, 2H, CH2), 0.88 (t, J = 7.3 Hz, 3H, CH3) ppm.
1.28 (m, 2H, CH2), 0.80 (t, J = 7.3 Hz, 3H, CH3) ppm. LC-MS, ESI1
13C NMR (75 MHz, CDCl3, 251C) d: 178.6, 156.6, 143.5, 137.9,
135.8, 131.2, 128.1, 93.2, 47.6, 29.3, 26.4, 22.4, 13.6 ppm.
for C23H24N2O4S m/z [M1H]: 425.
(E)-3-[2-Butyl-3-(4-iodo-benzyl)-3H-imidazol-4-yl]-2-thiophen-2-
ylmethyl-acrylic acid ethyl ester (8): A solution of aldehyde 7
Frozen section autoradiography50,51
(1.46 g, 3.97 mmol), piperidine (0.270 g, 3.18 mmol) and acid 3
Frozen sections (20 mm) were prepared in a cryomicrotome and
(0.905 g, 3.97 mmol), in cyclohexane (25 mL) and 2 mL of toluene
was heated in an oil bath at 1001C under a gentle stream of
nitrogen. A Dean-Stark trap was used to remove water.
Additional portions of acid 3 (in total 0.453 g, 1.99 mmol) in
cyclohexane were added at at 2, 5 and 7 h. The reaction mixture
was heated for a total of 8 h. The reaction mixture was
concentrated under reduced pressure and the residue purified
by flash chromatography eluting with 0–3% MeOH in dichlor-
omethane, to give the product (1.392 g, 73%) as pale yellow oil.
1H NMR (400 MHz, CDCl3, 251C) d: 7.61 (AA0BB0, 2H, Ar-H) 7.43 (d,
J = 0.8 Hz, 2H), 7.41 (s, J = 0.8 Hz, 1H) 7.06 (dd, J = 1.2, 5.2 Hz, 1H),
6.85 (dd, J = 3.5, 5.2 Hz, 1H), 6.77 (dm, J = 3.5 Hz, 1H), 6.69
(AA0BB0, 2H, Ar-H), 5.08 (s, 2H, CH2), 4.15 (q, J = 7.2 Hz, 2H, CH2),
4.06 (m, 2H, CH2), 2.62 (m, 2H, CH2), 1.66 (m, 2H, CH2), 1.35 (m,
2H, CH2), 1.21 (t, J = 7.2 Hz, 3H, CH3), 0.87 (t, J = 7.2 Hz, 3H) ppm.
13C NMR (100 MHz, CDCl3, 251C) d: 167.1, 151.3, 141.0, 138.0,
135.5, 131.7, 128.4, 127.6, 127.0, 126.6, 125.2, 124.4, 123.4, 93.3,
60.9, 46.2, 29.5, 28.7, 26.9, 22.3, 14.1, 13.6. LC-MS, ESI1 for
put on superfrost glass slides. Organs used were: liver, lung,
spleen, heart, kidney, striatum and cerebellum from rat as well
as pig adrenal. The slides were kept in a freezer (ꢀ201C) until
used. At the start of the experiment the slides were pre-
incubated for 10 min in TRIS1 (50 mM TRIS1120 mM NaCl1
5 mM KCl11 mM MgCl212.5 mM CaCl2, pH 7.4) buffer. The slides
were then transferred to containers containing [carboxyl-11C]-
eprosartan in TRIS1buffer. In a duplicate set of containers 10 mM
of unlabelled eprosartan was added to block-specific binding.
After incubation for 30 min the slides were washed 3 ꢂ 3 min in
buffer. The slides were dried in a heated (371C) oven and the
exposed to phosphor imaging plates (Molecular Dynamics, USA)
for 40 min and scanned in a Phosphor Imager Model 400S
(Molecular Dynamics, USA, purchased from Amersham
Bioscience, Uppsala, Sweden).
Organ distribution52,53
C24H27IN2O2S m/z [M1H]: 535. Decarboxylation of 3 resulted in The in vivo studies were carried out in adult male Sprague
3-thiophen-2-yl-propionic acid ethyl ester, a yellow oil that Dawley rats (315–550 g) (Scanbur B&K, Sweden). All animals
J. Label Compd. Radiopharm 2009, 52 295–303
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www.jlcr.org

˚
O. Aberg et al.
were handled according to the guidelines by the Swedish
Animal Welfare Agency, and the experiments were approved by
the local Ethics Committee for Animal Research, permit no:
C234/5.
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In order to evaluate uptake in normal tissues, a biodistribution
study was performed. Rats were injected intravenously in the tail
vein with 10 MBq/kg [carboxyl-11C]eprosartan solution. At 15 and
40 min post injection, nine or eight animals/time point were
sacrificed and their organ were dissected out, weighed and
radioactivity was measured in a well counter. Organ values were
calculated as standardized uptake value (SUV), calculated as follows:
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ACTðBq=gÞ
SUV ¼
DOSEðBqÞ=BWðgÞ
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where ACT is the measured concentration of radioactivity corrected
for physical decay, DOSE is the administered amount of radioactivity
(Bq) and BW is the body weight of the animal (g).
Conclusions
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In an effort to find a new tracer for the imaging of aldosterone
producing adenomas we have synthesized [carboxyl-11C]epro-
sartan 10, specifically labelled with 11C in the carboxyl position
using
a palladium-mediated carboxylation reaction with
[11C]carbon monoxide. The labelling reaction gave sufficient
radiochemical yields to be used for biological experiments, and
with high specific activity.
Biological screening revealed expected binding/accumulation
in tissues reported to express the AT1 receptor, such as kidney,
lung and adrenal. The adrenal binding warrants further
examination of human adrenal tissues, especially those of
pathological origin. 11C-labelled eprosartan is thus a possible
tracer for imaging AT1 receptors in vivo.
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Acknowledgement
This work was conducted in collaboration with Uppsala Imanet,
GE Healthcare.49 We acknowledge Dr Tamara Church for
commenting on the text, Dr Adolf Gogoll for expertise in 2D-
¨
NMR-analysis and Elisabeth Bergstrom-Pettermann for technical
assistance.
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