Synthesis of 11C-labelled metomidate analogues as adrenocortical imaging agents

Article abstract of DOI:10.1002/jlcr.1517

Clinical findings using [¹¹C]methyl 1-[(1R)-1-phenylethyl]-1H- imidazole-5-carboxylate ([¹¹C]MTO, 1) show high uptake in lesions of adrenocortical origin, including adenomas, but low uptake in lesions of non-adrenocortical origin. In this paper the synthesis and preclinical evaluation of two new ¹¹C-labelled analogues of MTO, [ ¹¹C]methyl 1-[(1R)-1-(4-chlorophenyl)ethyl]-1H-imidazole-5- carboxylate ([¹¹C]CLM, 2) and [¹¹C]methyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carboxylate ([¹¹C]BRM, 3), using frozen-section autoradiography, organ distribution and a metabolic study are presented. Copyright

Full text of DOI:10.1002/jlcr.1517

Research Article
Received 18 December 2007,
Revised 28 February 2008,
Accepted 4 March 2008
Published online in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1517
11
Synthesis of C-labelled metomidate
analogues as adrenocortical imaging agents
a
Farhad Karimi, Maria Erlandsson, Orjan Lindhe, and Bengt
b
a
¨
a,b
Ã
˚
Langstrom
¨
Clinical findings using [11C]methyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylate ([11C]MTO, 1) show high uptake in
lesions of adrenocortical origin, including adenomas, but low uptake in lesions of non-adrenocortical origin. In this paper
the synthesis and preclinical evaluation of two new 11C-labelled analogues of MTO,
[
11C]methyl 1-[(1R)-1-(4-
chlorophenyl)ethyl]-1H-imidazole-5-carboxylate ([11C]CLM, 2) and [11C]methyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imida-
zole-5-carboxylate ([11C]BRM, 3), using frozen-section autoradiography, organ distribution and a metabolic study are
presented.
Keywords: PET; [11C]methylation; MTO; metomidate; adrenocortical tumours
incidentally around the adrenals during CT of the abdomen.
This tracer could differentiate between malign and benign
Introduction
lesions found in incidentalomas.2,3 In this report, two potential
The 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carboxylic acid esters
are a class of compounds that interact selectively with the
11C-analogues of MTO were synthesized and a biological assay
mitochondrial cytochrome P-450 species in the adrenal cortex.1
with MTO was used to decrease liver uptake compared with
Compounds labelled with b1-emitting radionuclides such as 11
C
the lead. The calculated log P-values for the MTO analogues
using ACD-labs5 were 2.7370.34 (1), 3.3270.35 (2) and
3.5070.43 (3).
1-[(1R)-1-Phenylethyl]-1H-imidazole-5-carboxylate analogues
were synthesized from the corresponding enantiopure pheny-
lethylamine as described in the literature,6 and labelled as
shown in Scheme 1.
(t1/2 = 20.3 min) and 18F (t1/2 = 109.7 min) might serve as radio-
tracers for the diagnosis of adrenal cortical masses such as
incidentalomas, adenomas and primary and metastatic adreno-
cortical carcinoma. Clinical findings with the radio-
tracer [11C]methyl 1-[(1R)-1-phenylethyl]-1H-imidazole-5-carbox-
ylate [11C]MTO (1) have indicated high uptake in lesions of
adrenocortical origin, including adenomas, but very low uptake
in lesions of non-adrenocortical origin.2,3 A study of the value of
[11C]MTO 1 in the diagnosis of gliomas, and in differentiation
between malignant and benign ones, showed that tumours
of all types had remarkable uptake of the positron emission
tomography (PET)-tracer.2 A retrospective study demonstrated
that MTO-PET is very useful in the imaging work up of adrenal
incidentalomas and may be beneficial for the examination
of patients with primary aldosteronism or adrenocortical
cancer.4 Although [11C]MTO 1 has good biological properties
for visualization of the adrenals and their tumours, there remains
interest in improved tracers with improved properties especi-
ally in regard to the adrenal-to-liver ratio. The assessment
of the right adrenal is hampered by the large uptake in the
liver.
Frozen-section autoradiography
As the first validation step, frozen-section autoradiography was
used to show binding properties.2,7–9 The incubation of frozen
sections with [11C]MTO and its analogues was used to show
specific binding to the adrenal cortex and few other tissues
(Figure 1). The degree of specific binding in rat adrenal was 83%
(1), 62% (2) and 21% (3). In rhesus adrenal the specific binding
was 76, 65 and 35%, respectively.
Organ distribution
Organ distribution was used to access the adrenal uptake as well
as the adrenal-to-organ ratio for the different analogues. The
The halogenated analogues of 1, [11C]CLM (2) and [11C]BRM
(3) were synthesized and some of their biological properties
were explored.
aUppsala Imanet, GE Healthcare, Box 967, S-751 09 Uppsala, Sweden
bDepartment of Biochemistry and Organic Chemistry, Uppsala University, Box
576, Husargatan 3, BMC, S-751 23 Uppsala, Sweden
Results and discussion
˚
¨
*Correspondence to: Bengt Langstrom, Department of Biochemistry and Organic
Chemistry, Uppsala University, Box 576, Husargatan 3, BMC, S-751 23 Uppsala,
Sweden.
[11C]MTO (1), which is a tracer binding to 11b-hydroxylase,2 was
developed in response to the clinical need for a PET imaging
agent that could visualize and characterize lesions found
E-mail: Bengt.Langstrom@biorg.uu.se
J. Label Compd. Radiopharm 2008, 51 273–276
Copyright r 2008 John Wiley & Sons, Ltd.

F. Karimi et al.
11C
O
Table 1. Organ distribution of (1), (2) and (3) after 15, 30
and 60 min in rat (n = 2, except for à where n = 1)
H3
O
O
OH
1
2
3
1) TBAH
2) 11CH3I
N
N
Mean
SD
Mean
SD
Mean
SD
N
N
X
X
15 min
Blood
Heart
Lung
Liver
Pancreas
Adrenal
Kidney
Testis
Brain
30 min
0.80
0.74
11.53
2.97
2.80
12.02
8.13
0.64
0.77
0.27
0.19
1.88
0.58
0.35
0.33
2.19
0.54
0.18
0.15
0.22
2.81
0.55
0.89
2.71
0.43
0.21
0.20
0.02
0.04
1.82
0.02
0.19
0.38
0.06
0.05
0.03
0.28 0.00
0.47 0.04
8.35 0.43
1.39 0.20
2.06 0.24
8.30 2.57
0.94 0.08
0.53 0.08
0.40 0.04
(1) X = H
(2) X = Cl
(3) X = Br
(4) X = Cl
(5) X = Br
Scheme 1. 11C-labelled ester analogues.
Ã
Ã
Ã
Ã
Ã
Ã
Ã
Ã
Ã
Blood
Heart
Lung
0.85
0.78
5.20
3.27
1.82
12.38
4.14
0.79
0.73
0.13
0.15
3.39
0.61
0.56
2.71
0.29
0.18
0.12
0.02
0.01
2.08
0.06
0.03
0.37 0.22
0.45 0.25
6.51 7.64
1.78 0.70
2.21 1.11
Liver
Pancreas
Adrenal
Kidney
Testis
Brain
0.19 12.87 3.54
0.02
0.01
0.01
0.86 0.46
0.53 0.21
0.34 0.19
60 min
Blood
Heart
Lung
0.79
0.62
3.31
3.12
1.48
13.12
3.18
0.55
0.56
0.02
0.00
0.71
0.13
0.36
0.62
0.35
0.04
0.02
0.09
0.09
1.14
0.35
0.32
2.47
0.17
0.07
0.06
0.01
0.02
0.15
0.04
0.04
0.29 0.05
0.24 0.07
3.46 2.07
1.47 0.45
1.15 0.20
Liver
Pancreas
Adrenal
Kidney
Testis
Brain
0.55 11.87 1.88
0.05
0.02
0.01
0.48 0.11
0.21 0.05
0.18 0.08
Organ values are shown as SUV (mean7SD).
recovery was 69710%. One hydrophilic metabolite was
observed. The metabolite analysis revealed that tracer 3 was
52 and 37% intact after 5 and 30 min of injection, respectively.
The recovery was 499%. One hydrophilic metabolite was
observed. The results from the metabolite study indicate that
analogue 3 is more stable over time in rat, than analogue 2.
Figure 1. Frozen-section autoradiography of [11C]MTO (1), [11C]BRM (3), and
11C]CLM (2). Total binding and non-specific binding (after blocking with 1 mM
etomidate) are shown. Organs, from top: (a) rhesus monkey kidney, (b) rhesus
monkey liver, (c) rhesus monkey adrenal, (d) pig kidney, (e) pig liver, (f) pig adrenal,
(g) rat kidney, (h) rat liver and (i) rat adrenal.
[
standardized uptake values (SUVs) for all organs were lower for
the analogues having a halogen in the para position on
the phenyl group than for MTO (Table 1). However, the
adrenal-to-organ ratios increased more rapidly for these
analogues than for MTO itself (Figure 2). In liver the adrenal-
to-liver and blood ratio remains unchanged at the time
points studied, whereas those for the halogen analogues
increases with time.
Materials and methods
General
[
11C]Carbon dioxide was produced at Uppsala Imanet by the
14N(p,a)11C reaction in a gas target containing nitrogen (AGA,
Nitrogen 6.0) and 0.1% oxygen (AGA, Oxygen 4.8), using
17 MeV protons produced from a Scanditronix MC-17 cyclo-
tron. [11C]Carbon dioxide was converted in two steps to
[
11C]iodomethane via reduction with lithium aluminium
Analysis of radiolabelled metabolites in plasma
hydride and subsequent treatment with hydroiodic acid.10
Liquid chromatographic analysis was performed with a Beck-
A metabolite study was performed on 2 and 3. Blood samples
were collected 5 and 30 min after injection to measure the
amounts of unchanged tracer and radioactive metabolites in
plasma. The metabolite analysis revealed that tracer 2 was 62
and 16% intact after 5 and 30 min of injection, respectively. The
man 126 gradient pump and
a Beckman 166 variable
wavelength UV-detector (Fullerton, CA, USA) in series with a b
1-flow detector. The following mobile phases were used:
50 mM ammonium formate pH 3.5 (A) and acetonitrile/water
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J. Label Compd. Radiopharm 2008, 51 273–276

F. Karimi et al.
Adrenal to organ ratios
45
40
35
30
25
20
15
10
5
1, 15 min
2, 15 min
3, 15 min
1, 30 min
2, 30 min
3, 30 min
1, 60 min
2, 60 min
3, 60 min
0
Blood
Heart
Lung
Liver
Pancreas
Kidney
Testis
Brain
Figure 2. Adrenal-to-organ ratios with compound (1), (2) and (3) after 15, 30 and 60 min in rat (calculated from Table 1). This figure is available in colour online at
www.interscience.wiley.com/journal/jlcr.
(50:7) (B). For analytical LC, a Jones chromatography genesis respectively), flow 5 ml/min, mobile phase A:B (40:60). The
C18, 4 mm ꢀ 250 mm ꢀ 4.6 mm column was used at a flow of organic solvent was evaporated from the collected fractions,
1.5 ml/min. For semi-preparative LC, a Jones chromatography and the residue was dissolved in phosphate buffer (pH = 7.5,
genesis C18 column, 4 mm ꢀ 250 mm ꢀ 10 mm, was used at a 4 ml) containing ethanol (99.5%, 0.4 ml). Sterile filtration of the
flow of 5 ml/min. Synthias, an automated synthesis system,11 final product solution through a filter (Dynagard ME, 0.22 mm
was used for LC injection and fraction collection. Data pore size) into a sterile vial was performed. Radiochemical purity
collection and LC control were performed with the use of a (497%) and analytical radiochemical yield 60–80% were
Beckman System Gold chromatography software package determined by analytical LC. Analytical LC (1, 2 and 3;
(USA). Radioactivity was measured in an ion chamber (Veenstra tR = 5.50, 6.71 and 7.52 min, respectively), mobile phase A:B
Instrumenten bv, VDC-202, Holland). For coarse estimations of (40:60).
radioactivity during synthesis, a portable dose-rate meter was
˚
used (Langenas elektriska AB, Sweden). In the analysis of Precursors and references
labelled substances, reference substances were used for
¨
Methyl 1-[(1R)-1-(4-chlorophenyl)ethyl]-1H-imidazole-5-carboxy-
comparison in LC runs. All syntheses were conducted under a
nitrogen atmosphere using dried glassware and magnetic
stirring. All chemicals and anhydrous solvents were bought
from commercial suppliers. 1H and 13C NMR spectra were
recorded in CDCl3 (7.26 ppm 1H, 77.0 ppm 13C) on a Varian
late (2): 1H NMR (CDCl3): d 7.66 (s, 1H), 7.60 (s, 1H), 7.08 (d,
2H), 6.92 (d, 2H), 6.14 (q, 1H), 3.58 (s, 3H), 1.64 (d, 3H). 13C NMR
(CDCl3): d 160.0, 139.5, 139.2, 137,6, 133.2, 128.2, 127.3, 121.8,
54.4, 51.0, 21.6. LC-MS (ESI1), m/z 265 (75.8%), 267 (24.2%)
[M1H]1.
1
Unity 400 spectrometer (400 MHz for H and 100.6 MHz for 13C
Methyl 1-[(1R)-1-(4-bromophenyl)ethyl]-1H-imidazole-5-carbox-
ylate (3): 1H NMR (CDCl3): d 9.17 (s, 1H), 7.95 (s, 1H), 7.55 (d, 2H),
7.22 (d, 2H), 6.49 (q, 1H), 3.93 (s, 3H), 2.01 (d, 3H). 13C NMR
(CDCl3): d 158.4, 150.4, 137.4, 132.8, 128.8, 127.4, 123.7, 111.9,
58.4, 53.2, 22.2. LC-MS (ESI1), m/z 309 (50.7%), 311 (49.3%)
[M1H]1.
1-[(1R)-1-(4-Chlorophenyl)ethyl]-1H-imidazole-5-carboxylic acid
(4): 1H NMR (CDCl3): d 8.06 (s, 1H), 7.85 (s, 1H), 7.29 (d, 2H), 7.16
(d, 2H), 6.58 (q, 1H), 1.85 (d, 3H). 13C NMR (CDCl3): d 176.1, 163.0,
138.9, 137.6, 134.0, 133.2, 129.0, 127.8, 55.0, 21.0. LC-MS (ESI1),
m/z 251 (75.8%), 253 (24.2%) [M1H]1.
nuclei). Liquid chromatography mass spectrometry (LC-MS)
analyses were performed using a Gilson HPLC and Finnigan
AQA mass spectrometer, in ESI mode.
General method for 11C-labelling: [11C]methyl 1-[(1R)-1-
phenylethyl]-1H-imidazole-5-carboxylate analogues (1), (2)
and (3)
The corresponding carboxylic acid (1.470.1 mg) in dichloro-
methane (300 ml) was treated with tetrabutylammonium hydro-
xide (TBAH, 20%, 371 ml). The solvent was then evaporated and
co-evaporated with dichloromethane (2 ꢀ 300 ml). The activated
carboxylate was dissolved in dimethyl formamide (300 ml).
[1-11C]Iodomethane10 was trapped in the solution and heated
at 1301C for 7 min. The crude product was injected on semi-
preparative LC (1, 2 and 3; tR = 5.54, 6.80 and 7.47 min,
1-[(1R)-1-(4-Bromophenyl)ethyl]-1H-imidazole-5-carboxylic acid
1
(5): H NMR (CDCl3): d 12.97 (bs, OH), 8.11 (s, 1H), 7.87 (s, 1H),
7.45 (d, 2H), 7.10 (d, 2H), 6.53 (q, 1H), 1.85 (d, 3H). 13C NMR
(CDCl3): d 162.7, 138.9, 136.8, 131.9, 130.4, 128.1, 126.7, 122.2,
55.4, 21.5 LC-MS (ESI1), m/z 295 (50.7%), 297 (49.3%) [M1H]1.
J. Label Compd. Radiopharm 2008, 51 273–276
Copyright r 2008 John Wiley & Sons, Ltd.
www.jlcr.org

F. Karimi et al.
Frozen-section autoradiography12–14
eluted in the second fraction. The radioactivity of different
fractions was measured in a g-counter. Even if no detectable
radiopeak co-eluted with the unlabelled standard, the fraction
was collected and its radioactivity was measured.
Frozen sections (20 mm) were prepared in a cryomicrotome and
put on superfrost glass slides. Kidney, liver and adrenal from
rhesus monkey, pig and rat were used. The slides were kept in a
freezer (-201C) before use. At the start of the experiment, the
slides were pre-incubated for 10 min in TRIS (50 mM, pH = 7.4)
buffer. The slides were then transferred to containers containing
approximately 1 nM of compound (1), (2) or (3) in TRIS buffer. In
a duplicate set of containers, 1 mM of etomidate was added to
block specific binding. After incubation for 30 min, the slides
were washed for 3 ꢀ 3 min in buffer. The slides were dried and
then 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).
Conclusion
MTO-PET is a specific and sensitive method for diagnosing
adrenal incidentalomas and for the examination of patients with
adrenocortical cancer. The addition of a halogen in compound 2
and 3, two new 11C-labelled analogues of 1, showed increased
adrenal-to-liver ratios in rats and primates compared with 1. This
may be advantageous in the further characterization of
incidentalomas since assessment of the right adrenal can be
made more readily. The possibility to detect adrenal metastases
in the liver is also improved with an increased ratio. The adrenal-
to-organ ratios indicate that analogue 3 has better properties
with regard to ratio to liver and stability in blood over time.
Organ distribution12–14
The in vivo studies were carried out in adult male Sprague
Acknowledgement
˚
¨
Dawley rats (300–700 g) (Mollegard, Denmark). All animals were
handled according to the guidelines of the Swedish Animal
Welfare Agency, and the experiments were approved by the
local Ethics Committee for Animal Research, permit no: C46/3. In
order to evaluate uptake in normal tissues, a biodistribution
study was performed. Six rats were injected intravenously with
20 MBq/kg (1), (2) or (3) solution. At 15, 30 and 60-min post-
injection, two animals/time point were sacrificed and their
organs were dissected out, weighed and measured. Organ
values were calculated as SUVs as follows:
This work was conducted in collaboration with Uppsala Imanet,
¨
GE Healthcare. We are grateful to Elisabeth Bergstrom-Petter-
mann for her assistance in the biological study.
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¨
¨
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Copyright r 2008 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2008, 51 273–276