UNIVERSITEIT GENT
FACULTEIT FARMACEUTISCHE WETENSCHAPPEN
VAKGROEP FARMACEUTISCHE ANALYSE
LABORATORIUM VOOR RADIOFARMACIE
ACADEMIEJAAR 2010-2011
SYNTHESIS OF [18F]FLUORO-N,N-DIMETHYLAMINOETHANOL
AS RADIOTRACER IN THE DETECTION OF PROSTATE CANCER
VIA POSITRON EMISSION TOMOGRAPHY
Gwendoline TROISPONT
Eerste Master in de Geneesmiddelenontwikkeling
Promotor
Prof. Dr. Apr. Filip De Vos
Commissarissen
Dr. K. Kersemans
Dr. A. Heyerick
UNIVERSITEIT GENT
FACULTEIT FARMACEUTISCHE WETENSCHAPPEN
VAKGROEP FARMACEUTISCHE ANALYSE
LABORATORIUM VOOR RADIOFARMACIE
ACADEMIEJAAR 2010-2011
SYNTHESIS OF [18F]FLUORO-N,N-DIMETHYLAMINOETHANOL
AS RADIOTRACER IN THE DETECTION OF PROSTATE CANCER
VIA POSITRON EMISSION TOMOGRAPHY
Gwendoline TROISPONT
Eerste Master in de Geneesmiddelenontwikkeling
Promotor
Prof. Dr. Apr. Filip De Vos
Commissarissen
Dr. K. Kersemans
Dr. A. Heyerick
First of all, I would like to thank my promotor Prof. Dr. Filip De Vos for his
supervision and for his valuable input in the completion of this thesis
A very special thanks in particular goes out to my mentor Dominique Slaets. Her continued guidance and insights during the development of my thesis have been a very good support for me. I will never forget the wisdom and
expertise she shared with me during the past four months.
Finally, I would like to thank my family for always believing in me, for encouraging me and for supporting my ambitions.
They are the guiding light throughout my life.
Table of Contents
1. INTRODUCTION ................................................................................................................... 1
1.1. MOLECULAR IMAGING IN CANCER DIAGNOSIS AND MANAGEMENT ......................... 1
1.2. PROSTATE CANCER ....................................................................................................... 2
1.2.1. Pathogenesis of prostate cancer ............................................................................. 2
1.2.2. Screening methods for the detection of prostate cancer ...................................... 3
1.3. POSIRON EMISSION TOMOGRAPHY ............................................................................. 4
1.4. PET TRACERS IN PROSTATE CANCER DETECTION ......................................................... 5
1.4.1. [18F]Fluoro-2-deoxyglucose ..................................................................................... 6
1.4.2. 18F- or 11C-labeled acetate ....................................................................................... 7
1.4.3. 18F- or 11C-labeled choline ....................................................................................... 8
1.4.3.1. Physiological role and metabolism .................................................................... 8
1.4.3.2. Choline uptake in tumors ................................................................................ 10
1.4.3.3. Role in prostate cancer screening ................................................................... 10
1.5. RADIOCHEMISTRY WITH 18FLUOR .............................................................................. 12
1.5.1. Physical characteristics ......................................................................................... 12
1.5.2. Radiofluorination .................................................................................................. 13
1.5.2.1. Nucleophilic fluorination ................................................................................. 14
1.5.2.2. Electrophilic fluorination ................................................................................. 14
2. OBJECTIVES ....................................................................................................................... 15
3. MATERIALS ........................................................................................................................ 16
4. METHODS .......................................................................................................................... 17
4.1. TRITYLMETHYLAMINOETHANOL ................................................................................. 17
4.1.1. Synthesis and Purification ..................................................................................... 17
4.1.2. HPLC Identification ................................................................................................ 18
4.1.3. Radiosynthesis of [18F]FCH2 -TrMAE+ .................................................................... 19
4.1.3.1. Radiosynthesis on a SPE Cartridge ................................................................... 19
4.1.3.2. Radiosynthesis in a Heated Reaction Vial ........................................................ 19
4.2. BENZYLMETHYLAMINOETHANOL ............................................................................... 21
4.2.1. Radiosynthesis of [18F]FCH2 -BzMAE+ .................................................................... 21
4.2.1.1. Synthesis of the Cold Ligand (CH3-BzMAE+) ..................................................... 21
4.2.1.2. HPLC Identification .......................................................................................... 21
4.2.1.3. Radiosynthesis on a SPE Cartridge ................................................................... 21
4.2.1.4. Radiosynthesis in a Heated Reaction Vial ........................................................ 22
4.2.2. Purification of [18F]FCH2 -BzMAE+ ......................................................................... 22
4.2.3. Deprotection of [18F]FCH2 -BzMAE+ ....................................................................... 22
4.2.3.1. Catalytic Transfer Hydrogenolysis of CH3-BzMAE+I- ......................................... 22
4.2.3.2. Catalytic Transfer Hydrogenolysis of [18F]FCH2-BzMAE+ .................................. 23
4.2.4. Identification and Purification of [18F]FDMAE ...................................................... 23
4.3. 2-[(N-BENZYLOXYCARBONYL)METHYLAMINO]ETHANOL ........................................... 24
4.3.1. Assessment of the Reactivity of a Carbamate ...................................................... 24
4.3.2. Synthesis and Purification of 2-[(N-Benzyloxycarbonyl)methylamino]ethanol ... 24
4.3.3. Radiosynthesis of [18F]FCH2 -CbzMAE+ .................................................................. 25
4.3.3.1. Radiosynthesis on a SPE Cartridge ................................................................... 25
4.3.3.2. Radiosynthesis in a Heated Reaction Vial ......................................................... 26
4.4. METHYLAMINOETHANOL ........................................................................................... 26
4.4.1. HPLC Identification ................................................................................................ 26
4.4.2. Radiosynthesis of [18F]FDMAE .............................................................................. 26
4.4.2.1. Radiosynthesis on a SPE Cartridge ................................................................... 26
4.4.2.2. Radiosynthesis in a Heated Reaction Vial ......................................................... 26
5. RESULTS ............................................................................................................................. 27
5.1. TRITYLMETHYLAMINOETHANOL ................................................................................. 27
5.1.1. Synthesis and Purification ..................................................................................... 27
5.1.2. HPLC Identification ................................................................................................ 31
5.1.3. Radiosynthesis of [18F]FCH2 -TrMAE+ .................................................................... 31
5.1.3.1. Radiosynthesis on a SPE Cartridge ................................................................... 31
5.1.3.2. Radiosynthesis in a Heated Reaction Vial ......................................................... 32
5.2. BENZYLMETHYLAMINOETHANOL ............................................................................... 33
5.2.1. Radiosynthesis of [18F]FCH2 -BzMAE+ .................................................................... 33
5.2.1.1. Synthesis of the Cold Ligand (CH3-BzMAE+)...................................................... 33
5.2.1.2. HPLC Identification ........................................................................................... 33
5.2.1.3. Radiosynthesis on a SPE Cartridge and in a Heated Reaction Vial ................... 33
5.2.2. Purification of [18F]FCH2 -BzMAE+ ......................................................................... 34
5.2.3. Deprotection of [18F]FCH2 -BzMAE+ ....................................................................... 35
5.2.3.1. Catalytic Transfer Hydrogenolysis of CH3-BzMAE+I- ......................................... 35
5.2.3.2. Catalytic Transfer Hydrogenolysis of [18F]FCH2-BzMAE+ .................................. 35
5.2.4. Identification and Purification of [18F]FDMAE ...................................................... 36
5.3. 2-[(N-BENZYLOXYCARBONYL)METHYLAMINO]ETHANOL ........................................... 37
5.3.1. Assessment of the Reactivity of a Carbamate ...................................................... 37
5.3.2. Purification and Identification of CbzMAE ............................................................ 38
5.3.3. Radiosynthesis of [18F]FCH2 -CbzMAE+ .................................................................. 40
5.4. METHYLAMINOETHANOL ........................................................................................... 40
5.4.1. HPLC Identification ................................................................................................ 40
5.4.2. Radiosynthesis of [18F]FDMAE .............................................................................. 41
6. DISCUSSION ....................................................................................................................... 42
7. CONCLUSION ..................................................................................................................... 46
8. REFERENCES ...................................................................................................................... 47
LIST OF ABBREVIATIONS
amu Atomic mass unit
ACN Acetonitrile
ARA Androgen receptor coactivators
AR Androgen receptor
β+ Positron
BzMAE N-benzyl,N-methylaminoethanol
CbzCl Benzylchloroformate
CbzMAE 2-[(N-benzyloxycarbonyl)methylamino]ethanol
CDCl3 Deuterated chloroform
CH2Cl Dichloromethane
CH2Br2 Dibromomethane
CH3I Methyliodide
CHT1 High-affinity choline transporter
CK Choline kinase
CT Computer Tomography
CTH Catalytic transfer hydrogenolysis
CTL Choline transporter-like protein
CoA Coenzym A
DHT α-dihydrotestosterone
DMAE N,N-dimethylaminoethanol
DMAP Dimethylaminopyridine
DMSO Dimethylsulfoxide
DRE Digital rectal examination
E.C. Electron capture
EDTA Ethylenediaminetetraacetic acid
EK Kinetic energy
ESI Electrospray ionization
EtOH Ethanol
EtOAc Ethylacetate
[18F]F2 [18F]fluorine
FAS Fatty acid synthase
[18F]FCH [18F]fluorocholine
[18F]FCH2Br [18F]fluorobromomethane
[18F]FDG [18F]fluorodeoxyglucose
[18F]FDMAE [18F]fluoro-N,N-dimethylaminoethanol
GLUT Glucose transporter
HCl Hydrochloric acid
HCOOH Formic acid
HNO3 Nitric acid
HPLC High performance liquid chromatography
H2SO4 Sulfuric acid
IGF-1 Insulin-like growth factor 1
IL-6 Interleukine-6
J Coupling constant
K222 Kryptofix 222
K2CO3 Potassium carbonate
keV kiloelectron Volt
KHCO3 Potassium hydrogen carbonate
MAE N-methylaminoethanol
MeOH Methanol
MeV megaelectron Volt
MR Magnetic resonance
MRglu Metabolc rate of glucose
MRI Magnetic Resonance Imaging
mRNA Messenger ribonuclein acid
MRSI Magnetic Resonance Spectroscopy Imaging
MS Mass spectroscopy
m/z Mass-to-charge ratio
NH4OH Ammoniumhydroxide
NMR Nuclear magnetic resonance
OCT Organic cation transporter
PC Phosphatidylcholine
Pca Prostate cancer
Pcho Phosphocholine
Pd/C Palladium on carbon
PE Phosphatidylethanolamine
PET Positron Emission Tomography
PG-MAE Nitrogen-protected
ppm parts per million
PSA Prostate-specific antigen
Qβ+ Positron maximum energy
R2 Coefficient of determination
RAS Rat Sarcoma
RI Refractive index
Rf Ratio-to-front value
Rpm Rounds per minute
SAM S-adenosylmethionine
SD Standard deviation
δH Chemical shift
SN2 Nucleophilic substitution
SPE Solid phase extraction
SPECT Single Photon Emission Computed Tomography
t1/2 Half-life
TEA Triethylamine
TLC Thin layer chromatography
TGFβ Transforming growth factor β
TMS tetramethylsilane
tR Retention time
TrCl Tritylchloride
TrMAE Tritylmethylaminoethanol
TRUS Transrectal ultrasound
US Ultrasound
UV Ultraviolet
WCX Weak cation exchange
1. Introduction
1
1. INTRODUCTION
1.1. MOLECULAR IMAGING IN CANCER DIAGNOSIS AND MANAGEMENT
Molecular imaging techniques are an essential and vital part in the diagnosis, staging
and follow-up of cancer. They have the capacity to visualize fundamental cellular and
metabolic processes within living organisms in a noninvasive way. This allows us to gain a
greater understanding of the key underlying processes involved in cancer pathogenesis. Over
the past 30 years, there has been a massive increase in the variety of imaging techniques
available to investigate patients with cancer. Many different imaging modalities such as
ultrasound (US), computerized tomography (CT), magnetic resonance imaging (MRI),
magnetic resonance spectroscopic imaging (MRSI) and radionuclide imaging exist now for
physiological and functional imaging in order to detect abnormal cell growth. Each of the
previously listed imaging techniques have their own advantages and shortcomings and will
therefore be used in a corresponding way in specific applications (Kim E. E. & Yang D. Y.
(2001). Targeted Molecular Imaging in Oncology. Springer-Verlag, New York, USA, Chapter
2).
Radionuclide imaging or scintigraphy is currently the most commonly used modality in
cancer detection because of its high sensitivity and target specificity. Single photon emission
computed tomography (SPECT) and positron emission tomography (PET) are the main tools
in scintigraphy and they both require radio-labeled pharmaceuticals in order to translate
disease processes into a signal perceptible through imaging contrast. Radioisotope tracers
are in this modality incorporated into natural biomolecules that, once injected in the body,
will accumulate in areas of disease where they are preferentially metabolized or where they
can specifically interact with tumor biomarkers or even specific receptors. Measurement of
these short-lived radioisotopes can differentiate cancer tissue from normal tissue in an early
phase before symptoms become apparent, providing the patient this way with an early
diagnosis, accurate follow-up and a higher chance of recovery (Michalski & Chen, 2011).
Each imaging advance has in addition been accompanied by a diversity of new
developments in radiopharmaceuticals. Typical examples are metabolic tracers such as
[18F]fluorodeoxyglucose ([18F]FDG), [11C]methionine, [11C]thymidine, [18F]choline,.. Each of
1. Introduction
2
these radiopharmaceuticals focuses on a specific target of a biochemical process. In the case
of [18F]FDG, the target will be the enzyme hexokinase in order to specifically assess glucose
metabolism in vivo. The most recently developed radiopharmaceuticals include radiolabeled
oligonucleotides (nucleic acid aptamers) and multimodal imaging probes (MR particles
containing organic dyes) and led to a broadening of the range of application of molecular
imaging. (Yun-Sang Lee et al., 2010)
1.2. PROSTATE CANCER
1.2.1. Pathogenesis of prostate cancer
Prostate cancer (PCa) is the most common diagnosed cancer in men. It is a
heterogeneous disease characterized by an over-activity of the nuclear androgen receptor
(AR). Androgens are required for the development, growth and normal function of the
prostate. The effects of androgens, testosterone and α-dihydrotestosterone (DHT) in
particular, are exerted via binding to the ligand-dependent AR and through inducing of the
AR transcriptional activity (Figure 1.1).
Figure 1.1 : Signal Transduction Pathways in the Prostate (Heinlein et al., 2004)
1. Introduction
3
The transcriptional activity of AR leads to cell proliferation and differentiation and is
modulated by the association of AR with AR coactivators (ARAs) in response to testosterone
and DHT and by the phosphorylation of AR or ARAs in response to growth factors like
transforming growth factor β (TGFβ), intereukine-6 (IL-6) and insulin-like growth factor 1
(IGF-I) (Heinlein et al., 2004).
At initial diagnosis, approximately 80-90% of all patients responds favorably to an
androgen ablation by reduction of the serum androgens and inhibition of AR. However, all
patients eventually relapse to a hormone-refractory state where androgen ablation therapy
no longer helps. This state is believed to occur through dysregulation of the AR pathway,
through amplification in the expression of AR coactivators or through AR mutations that
enable the receptor to be active in a ligand-independent manner.
1.2.2. Screening methods for the detection of prostate cancer
Standard screening tests for prostate cancer are performed via digital rectal
examination (DRE) and measurement of the prostate-specific antigen (PSA) level in the
serum. PSA is a serine protease enzyme that liquefies the semen via cleavage of seminogelin
in the seminal fluid and its production is stimulated by the androgen receptor. PSA reflects
thus AR activity and can therefore be measured as a prostate tumor marker in the serum.
Frequently used PSA parameters include PSA density, free to total PSA ratio, PSA velocity
and PSA half life. A PSA level higher than 4.0 ng/ml has predictive value for prostate cancer.
However, the effectiveness of DRE and PSA measurements has often been questioned. Since
only an inexact indication of the local extent of the disease can be obtained, newer, non-
invasive screening methods need to be developed. In addition, prostatitis, irritation and
benign prostatic hyperplasia may cause elevated PSA levels, hereby rendering a false positive
result in the screening test. A complete absence of elevated PSA levels can also occur in
prostate cancer (Freedland, S. et al., 2002). There are hence limitations to these two
screening tests, neither DRE nor the PSA test are 100% reliable.
Transrectal ultrasound (TRUS) is frequently used to perform prostate biopsies on
patients where disease is suspected based on abnormal findings of DRE or high PSA levels in
the serum. Despite its usefulness during biopsy and treatment of PCa, TRUS is no longer
recommended as first-line screening test for prostate cancer. Other conventional imaging
1. Introduction
4
techniques such as CT and MRI are not sufficiently accurate in the diagnosis and
characterization of prostate cancer, but their role is still evolving. In recent years, positron
emission tomography has proven to be a useful and valuable diagnostic tool in the
assessment of several types of cancer and metastatic spread. A major limitation of PET is its
lack of anatomical details. However, combination of a PET camera with a CT scanner (PET-
CT) presented interesting possibilities. Two sets of complementary data can be obtained,
therefore the resulting fusion images display both anatomic and functional details in the
body, permitting an improvement in the diagnostic performance of PET. Much depends still
on the chosen radiotracer used in the PET screening method. In urologic tract tumors and
prostate cancer, radiotracers like for instance [18F]FDG tends to accumulate significantly in
the kidney and the urinary bladder due to urinary excretion, consequently impeding
adequate visualization of tumors in the pelvis. Therefore, development of new
radiopharmaceuticals that interfere with other metabolic pathways than those in the urinary
tract are desirable and have an important advantage compared to [18F]FDG (Delgado Bolton
R. C. et al., 2009).
1.3. POSITRON EMISSION TOMOGRAPHY
Positron emission tomography is one of the most powerful and fastest growing imaging
modalities worldwide and its clinical applications in cancer diagnosis are increasing. It is a
tracer technique which provides unique information about the metabolic activities of
tumors. The technique utilizes tracers labeled with short lived positron-emitting
radioisotopes, typically 11C, 13N, 15O and 18F. The isotope shows radioactive decay, emitting a
positron that annihilates with an electron in the neighboring tissue. This results in the
simultaneous emission of two back-to-back gamma rays of equal energy (511keV) that are
detected by a ring of detectors surrounding the patient (Figure 1.2). No collimators are
required since the PET-camera is able to detect the simultaneous arrival of each pair of
gamma rays. This increases the detection efficiency significantly. The location of annihilation
can be found by drawing a line that connects the two opposing activated detectors. 3D
volume images are finally obtained after retroprojection of the detected coincidence lines
resulting from the annihilation events (Le Bars et al., 2006).
1. Introduction
5
Figure 1.2 : Mechanism of positron emission tomography
(http://www.heartandmetabolism.org/issues/hm34/hm34refresherc.asp (30-03-2011)) The high specificity of the tracers and high sensitivity of the tomographs make PET one of
the most specific and sensitive means for imaging specific molecular pathways and
interactions in the body. As a result, PET is able to detect picomolar concentrations of
chemical compounds. This sensitivity is of paramount importance when the radiotracer
targets certain receptors, enzymes or transporters which occur in the sub-micromolar
concentration range (Jones, T. (1996). Startegy fot creating accurate functional imaging with
PET and its relevance to. In: Tomography in Nuclear Medicine, Proceedings of an
International Symposium, IAEA (Ed.), IAEA, Vienna, Austria, pp. 81-88).
1.4. PET TRACERS IN PROSTATE CANCER DETECTION
Most radionuclides used in PET are produced by means of nuclear reactions in a
cyclotron using a high energetic proton or deuteron beam. Production of a proton or
deuteron beam is achieved by accelerating these charged particles in the presence of an
alternating electric field. Perpendicular to the electric field, a magnetic field is applied that
causes the moving particles to bend between the accelerations into a semi-circular path.
Eventually, the high energetic protons/deuterons are smashed against the target material,
enabling the formation of unstable, radioactive isotopes.
The 3 most studied PET radiotracers used in prostate cancer are [18F]FDG, 18F- or 11C-
labeled acetate, and 18F- or 11C-labeled choline (Jadvar et al., 2011).
1. Introduction
6
1.4.1. [18F]Fluoro-2-deoxyglucose
[18F]FDG is the most common used radiopharmaceutical in PET to identify and localize
tumors within normal tissue. The ability of this glucose analogue to accumulate in malignant
tissue can be explained by the fact that tumor cells show accelerated rates of glycolysis
compared to normal tissue. This is the result of an increased expression and translocation of
glucose transporters, primarily GLUT-1 and GLUT-3, and an elevated hexokinase II activity in
tumor cells (Jadvar et al., 2009). GLUT proteins make the facilitative transport of glucose
across the cell membrane, down its concentration gradient, possible while hexokinase II
catalyzes the phosphorylation of glucose to glucose-6-phosphate. Unlike glucose, [18F]FDG
cannot enter the glycolysis pathway due to the presence of fluorine instead of a hydroxyl
group in position 2 and so, it becomes trapped in the cell under the form of [18F]FDG-6-
phosphate (see Figure 1.3). The radioactive accumulation of [18F]FDG is proportionally to the
metabolic rate of glucose (MRglu). Since cancer cells are characterized by an increased
glucose consumption for energy production, [18F]FDG can be used to distinguish malignant
from normal tissue and to provide valuable imaging information about the tumor grade or
stage. (Price et al., 2010)
Figure 3.1 : Metabolic trapping of 18FDG in the cell
(http://movies-tatecalebzane.blogspot.com/2011/03/glycolysis-and-gluconeogenesis-concept.html (20-04-2011))
Nevertheless, the clinical use of 18F-FDG in prostate cancer screening is limited for
several reasons. First, in contrast to other primary tumors, prostate carcinoma is a low-grade
tumor and shows thus low metabolic activities including glucose turnover. This results in a
reduced uptake of [18F]FDG in most primary prostate tumors and so, less effective
delineation of the tumor from surrounding tissue is established. In addition, [18F]FDG also
accumulates in other tissues with high levels of glucose metabolism. An increased uptake
can therefore be observed in sites of active inflammation (prostatitis), tissue repair and
1. Introduction
7
benign prostatic hyperplasia. This makes the differentiation between benign hyperplasia and
malignant prostatic disease impossible. Also, [18F]FDG is rapidly excreted in urine, hence
causing an overwhelming abundant radioactivity in the bladder and ureters. Because of the
close proximity of the prostate and the urinary bladder, levels of radioactivity in the bladder
may interfere with [18F]FDG accumulation in the prostate area, possibly masking small
tumors or lesions in the vicinity and deteriorating the image quality. Bladder catherization,
forced diuresis as well as iterative image reconstruction could not solve this problem
completely (Hofer et al., 1999). Therefore, [18F]FDG is not appropriate for prostate cancer
detection and more specific and sensitive radiotracers are desired.
1.4.2. 18F- or 11C-labeled acetate
Acetate is a principal source of carbon that participates as a substrate in the Krebb’s
cycle and in the cytoplasmic lipid synthesis. The biologic basis for radiolabeled acetate
uptake in malignant tumors is an increased lipid synthesis through overexpression of fatty
acid synthase (FAS) at protein and mRNA levels (Vavere et al., 2007).
FAS is an anabolic, multifunctional enzyme complex that plays an essential role in the
conversion of carbohydrates (acetyl-CoA or malonyl-CoA) to fatty acids. In most normal
tissues, low FAS levels are expressed because of the high availability of dietary fatty acids
that are responsible for downregulation of “de novo” fatty acid synthesis (Pflug et al., 2003).
In cancer tissue however, the expression of FAS is increased to allow prostate cancer growth
and survival. This explains why acetate is found to be an effective and sensitive imaging
biomarker for delineation of prostate cancer since it is actively incorporated into
phospholipids like phosphatidylcholine (PC) and other lipids of malignant cells (Liu Y et al.,
2006).
Also, the metabolic fate of radiolabeled acetate in tumor cells is different from that in
normal tissue. The exact pathway of acetate metabolism has not yet been fully clarified.
Soloviev et al. (2007) reported that [11C]acetate in tumor tissue is rather incorporated into
[11C]palmitate via activation of the fatty acid synthesis, while benign cells oxidize acetate in
the Krebb’s cycle to [11C]CO2 for energy production. Eventually [11C]CO2 leaves the body via
exhalation by the lungs after 15-20 minutes. Although prostate cancer is a relative slowly
proliferating tumor compared to other cancers, fuel nutrients are still required to meet the
1. Introduction
8
cellular energy demands for rapid cell growth and proliferation. ATP as well as acetyl-CoA
are essential energy sources that are provided by an increased fatty acid oxidation and β-
oxidation pathway in prostate cancer rather than glycolysis (Liu Y et al., 2006).
The radiotracer shows very little urinary excretion, which is favourable in primary
tumor evaluation because no bladder activity can profoundly interfere with the visualisation
of pelvic structures. Acetate is thus considered superior to [18F]FDG as a tracer for prostate
cancer imaging because of its higher sensitivity and specificity (Jadvar et al., 2011). In
addition, 18F-labeled acetate presents the possibility of delayed imaging due to the longer
half-life of 18F (109,8 min) compared to 11C (20 min). This allows further increasement of the
tumour-to-background ratios and thus a better way to visually distinguish normal from
tumor tissue (Matthies et al., 2004).
1.4.3. 18F- or 11C-labeled choline
1.4.3.1. Physiological role and metabolism
Choline (N,N,N-trimethylaminoethanol) is a an essential nutrient for animals and
humans. It can be acquired from the diet (mainly from liver, eggs and wheat germ) or via the
methylation of phosphatidylethanolamine (PE) to phosphatidylcholine, followed by
phospholipase degradation to choline (Figure 1.4). Nevertheless, de novo synthesis of
choline alone does not meet the human requirements (Li Z. and Vance D., 2008).
Figure 1.4 : Metabolic fate of choline (Michel et al., 2006)
1. Introduction
9
Choline is involved in three important biochemical processes: the biosynthesis of
acethylcholine for cholinergic neurotransmission, the synthesis of S-adenosylmethionine
(SAM) as methyl group donor for numerous methyltransferase reactions and most
importantly, the synthesis of essential lipid components such as phosphatidylcholine,
lysophosphatidylcholine, choline plasmalogen and sphingomyelin for the structural integrity
of cell membranes, but also for lipid transport as well as cell membrane signaling (Penry J,
2008). The majority of the total choline pool in our body is used for the conversion to PC, an
essential phospholipid in cellular membranes and an important precursor of signaling
molecules (Zeisel & Da Costa, 2009).
Choline is a quaternary ammonium base and needs therefore specific transporters to
pass the lipophilic cell membrane. Three different choline transporter families have been
characterised in a variety of species: the Na+-dependent, high-affinity choline transporters
(CHT1), the polyspecificic organic cation transporters (OCTs) with low affinity for choline and
the Na+-independent choline transporter–like proteins (CTLs) with intermediate affinity for
choline (N.-Y. Lee et al., 2009). CHT1 is abundant in presynaptic cholinergic nerve terminals,
mainly to provides choline for the biosynthesis of acetylcholine, whereas CTLs are
ubiquitously located and supply choline for the synthesis of PC and other membrane
phospholipids (Michel et al., 2006; Sebastian A. Müller et al., 2009).
Once choline enters the cell, it is immediately phosphorylated by choline kinase (CK)
to phosphocholine which in turn will be converted to PC (see Figure 1.4). In some cell types
such as hepatocytes and nephrocytes, choline is oxidized to betaine by the enzyme complex
choline oxidase (Zeisel & Da Costa, 2009). Betaine (trimethylglycine) is an important
osmolyte in the cell and participates also in the one carbon cycle as a methyl group donor to
produce methionine out of homocysteine and to generate eventually the methylation agent
S-adenosylmethionine. A small amount of choline is acetylated by choline acyltransferase to
acetylcholine, a neurotransmitter that plays an important role in cognitive processes like
learning and memory (Roivainen et al., 2000). The oxidation of choline to betaine is
irreversible whereas conversion to phosphatidyl- or acetylcholine is not.
Since choline is involved in a wide range of metabolic pathways, it is essential for the
normal functioning of all cells throughout the body. Adequate intake levels for man and
1. Introduction
10
women are respectively 550 mg/day and 425 mg/day. A deficiency in choline often results in
fatty liver disease, hemorrhagic kidney necrosis, atherosclerosis and possibly neurological
disorders (Zeisel & Da Costa, 2009).
1.4.3.2. Choline uptake in tumors
In many malignant tumors, including prostate cancer, increased phosphocholine
(PCho) levels are observed. This is largely explained by an over expression and increased CK
activity, which leads to the integration of choline in the tumor cell membrane and eventually
to PCho trapping (J. Leyton et al., 2009). Growth factors, chemical carcinogens and ras
oncogene transfection are all responsible for the induction of CK activity (Kouji et al., 2008).
An increased expression of choline transporters and an up regulated transport rate have
been reported as well in prostate tumors, this most likely to provide the tumor with enough
phospholipids for the increased cell proliferation and concomitant membrane forming.
However, elevated PCho levels did not correlate well with proliferation rates of tumor cells
(Plathow & Weber, 2008).
An additional explanation for the alteration of choline metabolites is reported by
Ackerstaff et al. (2001) and Glunde et al. (2004). Phospholipase C, an enzyme that accounts
for the breakdown of many phospholipids, appears also to be upregulated in cancer cells.
This contributes to the accumulation of PCho in malignant cells. Since PCho, both a
precursor and a breakdown product of PC, also acts as a second messenger in cell growth
signaling, it is essential for PC to be metabolised by phospholipase C to enable mitogenic
signal transduction in tumor cells (Kouji et al., 2008).
1.4.3.3. Role in prostate cancer screening
The aberrant choline phospholipid metabolism observed in tumor tissues is strongly
associated with their malignant progression which makes radiolabeled choline a prominent
diagnostic marker for detection of prostate tumors and metastases. Many PET studies have
been published, using 11C- or 18F-labeled choline as potential radiotracer in the detection of
prostate, breast and colon carcinomas.
[11C]choline uptake in prostate carcinoma is significantly higher than the discrete
uptake of 18F-FDG. As a consequence, [11C]choline shows a higher sensitivity for malignant
1. Introduction
11
prostate tissue when compared to [18F]FDG. Another advantage of [11C]choline is its rather
low urinary excretion. Minimal accumulation of radioactivity may appear in the urine due to
incomplete tubular reabsorbtion of [11C]choline or to enhanced excretion of the labeled
oxidative metabolite betaine. Nevertheless, bladder activity is much lower than that of
[18F]FDG and mostly too low to profoundly interfere with pelvic image interpretation (Scher
et al., 2008). Tracer uptake of acetate and choline shows a very similar pattern, which
suggests that both radiotracers are about equally useful in imaging prostate cancer lesions
(Kotzerke et al., 2002).
However, the short half-life of the radioisotope 11C (t½ = 20,4 min) limits the
widespread application of [11C]choline, since only PET centers with an on-site cyclotron are
able to use it. For this reason, the longer-lived radioisotope 18F (t½ = 109,8 min) is generally
favored over 11C. Encouraging results were found in numerous articles using 18F-labeled
choline as radiotracer in the evaluation of prostate cancer. Fluorocholine ([18F]FCH) shows
very similar biodistribution to [11C]choline and moreover, no significant differences in tracer
uptake of malignant lesions were found (Sher et al., 2008; McCarthy et al., 2010).
Nonetheless, [18F]FCH has certain limitations, having a higher urinary excretion
pattern and radiation dose to kidneys when compared to [11C]choline. The extremely rapid
renal clearance of [18F]FCH suggests a species difference in the tubular reabsorption of
[18F]FCH relative to [11C]choline (Bansal et al., 2008). Bladder activity is observed within the
first 20 min. after administration and has consequently the potential to complicate image
interpretation in the pelvis. To reduce the impact of this problem, delayed scanning can be
performed. This results in lower urinary activity in the bladder and in higher tumor-to-
background contrast ratios due to the rapid circulatory and urinary clearance and little
washout of [18F]FCH from malignant tumors. The use of image acquisition protocols is
another way to overcome the problem of bladder activity. This is possible by performing
dynamic imaging of the pelvic region for the first 10 min. after injection. The frames taken
before radioactivity appeared in the ureter or bladder, will show a clear delineation of
[18F]FCH tumor uptake, those that show significant urinary interference are retrospectively
excluded (DeGrado et al., 2007; Kotzerke et al., 2003).
1. Introduction
12
1.5. RADIOCHEMISTRY WITH 18FLUOR
1.5.1. Physical characteristics
Fluorine-18 has a half life of 109,8 minutes and is the most stable radioisotope of the
naturally occurring fluorine-19 isotope. It decays via positron (β+) emission to the ground
state of oxygen-18 with a probability of 96,86% (Figure 1.6). In a much lesser extent, 18F
desintegrates also via electron capture (probability 3,14%) to 18O (Bé M.-M. et al., 2004). In
β+ decay, a proton is converted into a neutron, while a positron and a neutrino are emitted
from the nucleus (Figure 1.5). PET imaging is based on this principle (Figure 1.2).
Figure 1.5 : β+ decay reaction of Fluorine 18
(http://www.learner.org/courses/physics/unit/pdfs/unit2.pdf (11-05-2011))
The decay scheme for β+ decay of 18F is shown in Figure 1.6. The emitted β+ particles are
not monoenergetic, but show a continuous kinetic energy distribution with an average of
0,250 MeV. The positron maximum energy Qβ+ contains 0,638 MeV and is calculated from
the following equation :
Qβ+ = { M(18F) – [M(18O) + 2me] } * c2
= (18,000937u – 17,999160u)c2 – 2me c2
= 0,001777u * 931,5 MeV/u – 2 * 0,511 MeV
= 1,660 MeV – 1,022 MeV
= 0,638 MeV
with M : nuclear mass of the isotope (u)
me : mass of the positron (u)
c2 : squared speed of light (931,5 MeV/u)
The energy corresponding to 2mec2 (= 1,022 MeV) is required to enable the conversion
of a proton to a neutron (Podgorsak, 2010).
1. Introduction
13
Figure 1.6 : Decay scheme of 18F (Podgorsak, 2010)
1.5.2. Radiofluorination
Fluorine-18 is currently the radioisotope of choice for PET because of its relatively long
half life (109,8 min), its positron decay (96,86%), the low tissue range (2,4 mm) and the low
β+ energy (0,638 MeV). These properties are of major importance in terms of resolution and
dosimetry (Le Bars et al., 2006). Fluorine-18 can be produced in a cyclotron via the following
two nuclear reactions :
(1) The carrier added; 20Ne(d,α)18F reaction yields electrophilic fluorine [18F]F2 using
neon-20 as target gas. Inactive fluorine-19 carrier was added to the target to prevent
adsorption of 18F to the target wall. As a consequence, a lower specific activity was obtained
(Elsinga et al., 2002).
(2) The non carrier added; 18O(p,n)18F reaction produces nucleophilic [18F]fluoride in
large amounts by proton irradiation of an 18O-enriched water target. This reaction has many
advantages, including higher specific activity, easy separation of 18F- and [18O]H2O on a anion
exchange resin and eventually a higher yield. When the same reaction is performed on a 18O-
gas target, carrier added [18F]F2 is produced (Le Bars et al., 2006). A common sideproduct of
the 18O(p,n)18F reaction is 13N (t1/2 = 9,97 min) due to (p,α) reaction with little amounts of
16O present in the enriched water target.
1. Introduction
14
1.5.2.1. Nucleophilic fluorination
Nucleophilic fluorine is obtained in the form of the [18F]fluoride. Because the latter is
strongly solvated in aqueous solutions, its nucleophilic activity is rather poor. For this reason,
nucleophilic fluorinations are performed in polar aprotic solvents and in the presence of the
crown ether Kryptofix 222 (K222), after consecutive azeotropc evaporations (Le Bars et al.,
2006).
Nucleophilic fluorination is highly favored nowadays because of the easy preparation
and use of [18F]fluoride and because of the high specific activities and radiochemical yields
obtained from the radiochemical reactions. A variety of methods exist for this type of
fluorination, ranging from aliphatic and aromatic substitution to the use of 18F-labeled
synthons. In nucleophilic substitutions with aliphatic compounds, halogens or sulphonates
(mesylate, tosylate, or triflate) are generally used as leaving groups. Afterwards, hydrolysis
of protective groups is often performed. The nucleophilic aromatic substitution is the most
efficient method for radionuclide incorporation into an aryl position. This reaction requires a
leaving group (nitro-, cyano- or acylgroup), activated by an electron-withdrawing substituent
placed in the ortho or para position (Erik M. van Oosten, 2009). In the cases where direct
fluorination is not possible, a [18F]fluorinated intermediate is prepared via a nucleophilic
substitution reaction (SN2), followed by an alkylation of the latter with the target molecule
(Elsinga et al., 2002).
1.5.2.2. Electrophilic fluorination
Electrophilic fluorine is obtained in the form of the highly reactive [18F]F2, but it can
also be converted into [18F]acetylhypofluorite, which is a more mild and regioselective agent.
Electrophilic fluorinations are preferably performed on vinyl or aromatic derivatives.
However, a mixture of 18F-labeled products with low specific activities are obtained, which
makes this type of reaction less favorable (Le Bars et al., 2006).
2. Objectives
15
2. OBJECTIVES
Recently, several 11C- and 18F-labeled choline analogues have been developed as PET
tracers for the evaluation and staging of patients with prostate cancer. [18F]FCH in particular
has shown potential usefulness in the detection of many cancers, including prostate tumors.
However, [18F]FCH is disadvantaged because of the radioactivity accumulation observed in
the urinary bladder.
N,N-dimethylaminoethanol (DMAE), a choline precursor and analog, seemes to have the
required characteristics of a potentially better radiotracer than [18F]FCH. Geldenhuys et al.
(2010) reported based on structure-activity relationship experiments, that DMAE acts as a
substrate for the CHT1 transporter. This is favourale since choline transport in prostate
cancer cells is mediated by CHT1 and CLT (Sebastian A. Müller et al., 2009). Moreover,
[14C]DMAE showed two to seven times higher uptake in tumour cells than [14C]choline
(Mintz et al., 2008). The enzym choline oxidase shows additionally a relative activity of only
5,2% of the total activity seen with choline, when DMAE was used as substrate (Ikuta et al.,
1977; Gadda et al., 2004). This means that DMAE will be less susceptible to metabolisation.
The aim of this study was to synthesize [18F]fluoro-N,N-dimethylaminoethanol
([18F]FDMAE) as a promising PET probe for prostate cancer. Different synthetic approaches
were evaluated. The direct nucleophilic fluoromethylation of a nitrogen-protected N-
methylaminoethanol (PG-MAE) with [18F]fluorobromomethane ([18F]FCH2Br) is assessed. The
effect of three different protecting groups on the fluoromethylation is also evaluated.
Furthermore, nucleophilic fluoromethylation of MAE is performed.
3. Materials
16
3. MATERIALS
MAE, DMAE, dibromomethane (CH2Br2), potassium hydrogen carbonate (KHCO3),
potassium carbonate (K2CO3), dimethylsulfoxide (DMSO), benzylchloroformate, ethanol
(EtOH), ethylenediaminetetraacetic acid (EDTA), nitric acid (HNO3), dichloromethane,
triethylamine, calciumchloride, methyliodide, phenyl-N,N-dimethylcarbamate, citric acid,
palladium on carbon extent of labeling (10 wt. % loading, matrix activated carbon support),
aluminium oxide cards (with fluorescent indicator 254 nm) and Duran® sintered disc filter
funnels were purchased from Sigma Aldrich (St Louis, Missouri, USA). Dry acetonitrile (ACN),
N-benzyl,N-methylaminoethanol (BzMAE) and ammonium hydroxide (NH4OH) were
obtained from Acros Organics (Geel, Belgium). Triethylamine (TEA) and tritylchloride (TrCl)
were purchased from Fluka Chemicals (Buchs, Switzerland). HPLC-grade solvents
(acetonitrile and methanol) were obtained from Lab-Scan Analytical Sciences (Sowinskiego,
Poland). Hexane and ethylacetate (EtOAc) were purchased from Chem-Lab (Zedelgem,
Belgium). MilliQWater was obtained from a Nanopure Ultrapure purification system
(Barnstead, Dubuque, Iowa USA) and formic acid was obtained from Janssen Chimica (Geel,
Belgium). Polygram® SIL G/UV254 was purchased from Machery-Nagel (Oensingen,
Switzerland) and the Nylon Acrodisc® syringe filter was obtained from Schleicher & Schuell
(Brunswick way, London UK). The magnetic stirrer, heating plate and temperature sensor
were purchased from Heidolph (Schwabach, Germany) and balances from Mettler-Toledo
(Tielen, Nederland). All three solid-phase cartridges (Sep-Pak Light Acell plus QMA, HLB Oasis
plus, Oasis WCX plus) were obtained from Waters (Milford, Massachusetts, USA).
4. Methods
17
4. METHODS
4.1. TRITYLMETHYLAMINOETHANOL
4.1.1. Synthesis and Purification
Tritylmethylaminoethanol (TrMAE) is synthesized by a nucleophilic substitution reaction
of methylaminoethanol (MAE) with tritylchloride (TrCl) in ethylacetate (EtOAc) (Figure 4.1).
TrCl (1394 mg, 5 mmol) was dissolved in 20 ml EtOAc (dried over molecular sieves) in a
round bottom flask and placed on a magnetic stirrer. At room temperature and under
atmospherical pressure, MAE (400 µl, 5 mmol) was dropwise added to the TrCl-solution.
Furthermore, the synthesis was carried out as described previously, but with TrCl (1394 mg,
5 mmol) and MAE (364 μl, 4,5 mmol). The latter reaction was also once performed at 60°C.
Homogeneous heat distribution was achieved by placing the flask in an oil bath on a heating
plate and using a temperature sensor to set and maintain the temperature at 60°C. The
course of the reaction was followed with thin layer chromatography (TLC) using aluminium
oxide plates and EtOAc/hexane 1:4 v/v as mobile phase. Spots of the reactionmixture (10
min and 1h) were evaluated under an ultraviolet (UV) lamp (254 nm) and compared to a
TrCl-standard solution.
Figure 4.1 : Reaction Scheme for the synthesis of TrMAE-Cl+
After one hour, 30 ml MilliQWater was added to the reactionmixture to end the
nucleophilic reaction. The mixture was transferred into a separatory funnel whereupon it
was shaken gently by inverting the funnel multiple times. After the extraction, the aqueous
phase was discarded while the organic phase was transferred in a round-bottom flask. An
amount of EtOAc was added to the organic phase and the flask was placed in the refrigerator
overnight for recrystallization.
Purification was carried out by filtration of the formed TrMAE.HCl crystals followed by base
extraction with triethylamine (TEA). A Duran® sintered disc filter funnel was used for
4. Methods
18
vacuum filtration (35mm, 10-16 microns). To ensure removal of trace quantities of
tritylchloride from the product, the filter and precipitate was washed twice with an
additional 200 ml icecold hexane. When TLC indicated the removal of TrCl from the
reactionproduct, the resulting amount TrMAE.HCl was weighed and transferred into a
separation funnel. A three equivalent amount of TEA, 50 ml EtOAc and 20 ml MilliQWater
were added to the separation funnel. The solution was shaken vigorously and the aqueous
phase was disposed. The extraction was repeated several times until the pH of the washing
phase equaled 7. Then, the solution was washed one last time with 20 ml MilliQWater and
finally the organic phase was collected into a previously tared flask. TrMAE was hereafter
concentrated at 45-50°C under reduced pressure with a rotary evaporator (Büchi, Flawil,
Switzerland) until constant weight.
TrMAE identification was accomplished with massa spectroscopy (MS) and nuclear
magnetic resonance (NMR) spectroscopy. The MS sample was diluted in MeOH and
experiments were performed in positive mode by electrospray ionisation (ESI) with a LCT
Premier XE orthogonal acceleration time of flight mass spectrometer (Waters, Milford, MA,
USA). Afterwards, TrMAE was dissolved in deuterated chloroform (CDCl3) for 1H NMR
experiments with tetramethylsilane (TMS) as internal standard. These were conducted on a
Varian 300 MHz FT-NMR (Palo Alto, California, USA).
4.1.2. HPLC Identification
The high performance liquid chromatography (HPLC) system consisted of a 1525 Binary
HPLC pump subsequently coupled with a 2487 UV-detector (Waters, Milford, Massachusetts,
USA) and a radioactivity detector (Ludlum Measurements inc., Sweetwater, Texas, USA).
Separation of the compounds took place on a reversed-phase Alltima C18 column (4,6 x 250
mm, 5 µm; Alltech, Deerfield, IL, USA) at a flow rate of 1ml/min. The isocratic mobile phase
contained acetonitrile (ACN) and MilliQWater (85:15, v/v) + 10mM TEA and was degassed by
sonication in a ultrasonic bath (Branson, Danbury, Connecticut, USA) before use.
Retention times of TrMAE, TrCl and [18F]FCH2Br were determined. Stock solutions were
prepared : 10 mg TrCl dissolved in 10 ml ACN and 10 mg TrMAE dissolved in 10 ml ACN. The
stock solutions were 1 to 4 diluted in MilliQWater and 100 µl of the latter was injected into
4. Methods
19
the HPLC. To determine the retention time of [18F]FCH2Br, 300 µl dimethyl sulfoxide (DMSO)
was loaded on an ®Oasis HLB plus cartridge. Gaseous [18F]FCH2Br, produced on an
automated [18F]FCH Scintomics module (Fürstenfeldbruck, Germany), was then leaded over
the cartridge and elution took place with 2 ml ACN. Finally 100 µl of the elute was injected
into the HPLC.
4.1.3. Radiosynthesis of [18F]FCH2-TrMAE+
4.1.3.1. Radiosynthesis on a SPE Cartridge
Radiosynthesis was performed on the automated Scintomics module as follows (Figure
4.2): after irradiation of 18O enriched water at 16 µA for 10 min, [18F]fluoride was isolated
from the latter using an anion exchange QMA cartridge previously conditioned with
potassium hydrogen carbonate (KHCO3) and H2O. Then, 18F- was eluted from the anion
exchange cartridge with a solution containing 3.5 mg potassium carbonate (K2CO3), 18,8 mg
K222, 1,92 ml ACN and 80 µl H2O. Subsequent azeotropic evaporations with 2 x 1 ml ACN
were performed to remove all traces of water (Slaets D. et al., 2010).
Dibromomethane (300 µl) in 2 ml acetonitrile was added to the dried
[18F]fluoride/K222/K+ complex to yield [18F]FCH2Br. The heated reaction mixture was then
purged with a N2 stream (30ml/min) through a combination of 4 silica cartridges where
chromatographic separation could take place. After the [18F]FCH2Br gas eluted from the silica
cartidges, radioactive labeling was carried out. The nucleophilic substitution took place on an
®Oasis HLB plus cartridge previously loaded with 300 µl TrMAE. The cartridge was eluted
with 2 ml ACN. The elute (100 µl) was then diluted with 900 µl ACN and finally 100 µl of the
diluted solution was injected into the HPLC for identification of the elute.
4.1.3.2. Radiosynthesis in a Heated Reactionvial
Radiosynthesis of [18F]FCH2-TrMAE was also performed in a reactionvial. To this end, the
automated Scintomics module was modified: the SPE cartridge was replaced by an alltech
vial so that later on, the vial could be removed from the module to place it in a heated oil
bath for the actual radiolabeling.
4. Methods
20
Dry acetonitrile (300 µl) was added to 300 µl TrMAE in the reactionvial. [18F]FCH2Br was
bubbled into the solution and thereafter, the vial was place in an oil bath and heated to 80°C
with a magnetic stirrer. After 15, 30, 45 and 60 min, 15µl of the reactionmixture was diluted
with 200 µl ACN in an eppendorf tube. The solution (100 µl) was then injected into the HPLC
system to identify the radioactive compounds.
After one hour, 60 µl DMAE was added to the reactionvial. Twelve minutes later, 15 µl of
the solution in the vial was diluted with 1,2 ml MilliQWater. The mixture was then
centrifuged at 13 000 rpm for a few minutes and finally 100 µl of the supernatant was
injected in the HPLC using an IC PAK Cation M/D column (3,9 x 150 mm, 5 µm; Waters,
Milford, Massachusetts, USA) and 0,1 mM ethylenediaminetetraacetic acid (EDTA) + 4 mM
nitric acid (HNO3) as isocratic mobile phase. Separation took place at a flow rate of 1 ml/min
and the 2414 Refractive Index (RI)-detector (Waters, Milford, Massachusetts, USA) coupled
with the radioactive detector was used for detection.
Figure 4.2 : Schematic diagram of the automated [18F]FCH Scintomics module
4. Methods
21
4.2. BENZYLMETHYLAMINOETHANOL 4.2.1. Radiosynthesis of [18F]FCH2-BzMAE+
4.2.1.1. Synthesis of the cold ligand (CH3-BzMAE+)
Benzylmethylaminoethanol (BzMAE) (8180 µl, 5 mmol) was treated with methyliodide
(CH3I) (311 ml, 5 mmol) in a round-bottom flask filled with 25 ml EtOAc. The reaction mixture
was stirred at room temperature and under atmospherical pressure for one hour and then
placed under a rotary vapor until the solvent and the unreacted CH3I had evaporated. An
amount of EtOAc was added to the precipitate before placing the flask in the refrigerator.
After one hour, the precipitate was filtered under vacuum with a Duran® sintered disc filter
funnel (60 mm, 16-40 microns). An additional wash step was carried out with 100 ml EtOAc
and finally the resulting amount CH3-BzMAE+I- was weighed.
4.2.1.2. HPLC Identification
Retention times of BzMAE, CH3BzMAE+ and [18F]FCH2Br were determined on an Alltech
C18 column (specifications described in 4.1.2) with 50:50 ACN/H2O + 10mM TEA as mobile
phase and 1ml/min flow. Here fore, a stock solution of BzMAE was prepared (10 µl in 10 ml
ACN), which was then 1 to 4 diluted with MilliQWater before injecting 100 µl in the
previously described HPLC system. [18F]FCH2Br was obtained by leading [18F]FCH2Br gas over
an ®Oasis HLB cartridge, previously loaded with 300 µl DMSO, followed by elution with 2 ml
ACN. Finally, 100 µl of the elute was injected into the HPLC system. The retention time of
CH3-BzMAE+ was determined by injecting 100 µl of a 100 ppm solution into the HPLC system
described in 4.2.1.1.
4.2.1.3. Radiosynthesis on a SPE Cartridge
The Scintomics module described in 4.1.3. was applied for synthesis of [18F]FCH2-
BzMAE+. An ®Oasis HLB plus cartridge was loaded with 300 µl BzMAE and eluted with 2 ml
ACN after reaction with [18F]FCH2Br. The elute (100 µl) was diluted with 900 µl H2O and
finally 100 µl of the diluted solution was injected into the HPLC system described in 4.2.1.1.
for identification of the reaction products.
4. Methods
22
4.2.1.4. Radiosynthesis in a Heated Reaction vial
Radiosynthesis in a heated reaction vial was carried out as described in 4.1.3.2. Dry
acetonitrile (300µl) was added to 300 µl BzMAE in an Alltech reaction vial. [18F]FCH2Br was
bubbled into the solution and thereafter, the vial was placed in an oil bath and heated to
80°C. After 15, 30 and 60 min, the reaction mixture (10 µl) was 1 to 6000 diluted in an
eppendorf tube with 50:50 ACN/H2O + 10 mM TEA. Finally, 100 µl was injected into the
previously described HPLC system to identify the radioactive compounds.
4.2.2. Purification of [18F]FCH2-BzMAE+
After 30 minutes and one hour, an ammonium hydroxide (NH4OH) solution (6%, 12
ml) was added to the reaction mixture and the radioactivity in the vial was measured with a
dose calibrator (Comecer, Ravenna, Italy). The mixture was hereafter applied to an ®Oasis
weak cation-exchange (WCX) cartridge, successively preconditioned with 10 ml H2O and 10
ml ethanol (EtOH). Afterwards, 18F-radioactivity was measured on the WCX cartridge, in the
elute and in the reaction vial. The WCX cartridge was additionally washed with 12 ml 50:50
6% NH4OH/EtOH and 12 ml EtOH while the elute was collected in a Falcon tube.
Radioactivity on the cartridge, in the elute and in the empty reaction vial was measured with
the dose calibrator.
A solution of 10 µl formic acid (HCOOH) in 1 ml H2O and 4 ml EtOH was used for elution
of the remaining compound(s) from the WCX cartridge. Afterwards, the elute and the
cartridge were measured for radioactivity.
4.2.3. Deprotection of [18F]FCH2-BzMAE+
4.2.3.1. Catalytic transfer hydrogenolysis of CH3-BzMAE+I-
A suspension of CH3-BzMAE+I- (307,2 mg, 1 mmol), formic acid (115,05 µl, 3 mmol) in
5 ml 1:4 H2O/EtOH was stirred and treated with palladium on carbon (Pd/C) (50 mg, 15 µm)
at reflux temperature for 1,5h. The course of the reaction was followed by HPLC. After 15,
30, 60 and 90 min, the reaction mixture (100 µl) was filtered through a Nylon Acrodisc®
syringe filter (0,2 µm) and the filtrate was collected in an eppendorf tube, 1 to 10 diluted
with H2O and injected into the HPLC system described in 4.2.1.1.
4. Methods
23
4.2.3.2. Catalytic transfer hydrogenolysis of [18F]FCH2 - BzMAE+
To determine the optimum reaction time, the elute, obtained after purification and
elution (described in 4.2.2), was collected in a new Alltech vial containing 50 mg Pd/C as
catalyst for the subsequent hydrogenolysis. The suspension was stirred at 80°C in an oil bath
and every 15 minutes, 500 µl of the mixture in the vial was diluted with 100 µl H2O in an
eppendorf tube. The dilution was filtered through a Nylon Acrodisc® syringe filter (0,2 µm)
and the filtrate was applied to a newly preconditioned WCX cartridge. The latter was then
washed with 10 ml 6% NH4OH and eventually the radioactivity in the elute, on the cartridge
and on the membrane filter was measured.
Figure 4.3 : Reaction Scheme for the hydrogenolysis of [18F]FCH2-BzMAE+ using Pd/C as
a catalyst and HCOO- as H-donor
4.2.4. Identification and Purification of [18F]F-DMAE+
After hydrogenolysis (20 min), the suspension was filtered through a Nylon Acrodisc®
syringe filter (0,2 µm) and the filter was additionally washed with 4 ml H2O. The elute was
collected in a Falcon tube and measured for radioactivity. The pH of the elute was adjusted
to 7 by adding a small amount of a 5,2% hydrochloric acid (HCl) solution and 100 µl of the
solution was injected into the HPLC system, using the IC PAK Cation M/D column and 0,1
mM EDTA + 4 mM HNO3 as mobile phase. The 2414 refractive index detector in series with
the radioactivity detector was used for identification of compounds in the solution.
Thereupon, the solution was applied on a WCX cartridge, preconditioned with H2O (15 ml).
The cartridge and the resulting elute were measured for radioactivity. The remaining
product on the cartridge was eluted with 2 ml 6% NH4OH and again, radioactivity of the
elute and WCX was measured. Afterwards, 100 µl of the elute (adjusted to pH 7) was
injected into the HPLC system for identification of the radioactive compounds.
The same procedure was one more time repeated using 20 mg Pd/C. After membrane
filtration of the reaction mixture, the filtrate (300 µl) was collected in an eppendorf tube and
adjusted to pH 7 by adding 30 µl of a 5,2% HCl solution. The filtrate was diluted with mobile
4. Methods
24
phase and injected into the previously described HPLC system. Finally, 10 ml 6% NH4OH was
added to the remaining filtrate, the resulting solution was applied on a preconditioned WCX
cartridge and radioactivity was measured in the elute and on the cartridge.
4.3. 2-[(N-BENZYLOXYCARBONYL)METHYLAMINO]ETHANOL 4.3.1. Assessment of the reactivity of a carbamate
The same procedure described in 4.2.1.3. was adopted for the radiosynthesis of
[18F]fluoromethylated phenyl-N,N-dimethylcarbamate, using phenyl-N,N-dimethylcarbamate
as nucleophilic agent. After 15, 30, 45 and 60 min., samples of the reactionmixture were
taken, diluted with mobile phase and injected into the HPLC system (Alltima RP C18 column;
ACN/H2O (50:50, v/v) + 10mM TEA). The identity of the 18F-labeled carbamate was
confirmed by comparison of the observed retention time to those obtained after injection of
the cold referenceproduct (Phenyl-N,N,N-trimethylcarbamate). The latter was synthesized
by treating phenyl-N,N-dimethylcarbamate with CH3I in equimolar concentrations. Isolation
and purification of the cold ligand was carried out as described in 4.2.1.4.
4.3.2. Synthesis and Purification of 2-[(N-Benzyloxycarbonyl)methylamino]ethanol
2-[(N-benzyloxycarbonyl)methylamino]ethanol (CbzMAE) was synthesized according to
a previously reported method (Mohler & Shen, 2006), in which MAE (4.4 ml, 54.0 mmol),
benzylchloroformate (CbzCl) (8.4 ml, 55.0 mmol) and TEA (9.6 ml, 68.8 mmol) were added to
a solution of CH2Cl2 (180 ml) at 0°C (Figure 4.4). After removal of the ice bath, the reaction
mixture was left stirring under nitrogen for 24h. Three consecutive washing steps were
performed using 10% citric acid (120 ml) and H2O (2 x 120 ml) before the organic phase was
dried with calcium chloride (CaCl2) and concentrated under the rotavapor to give the yellow
oil CbzMAE.
Figure 4.4 : Reaction Scheme for the synthesis of CbzMAE (Mohler & Shen, 2006)
4. Methods
25
The reaction product was isolated from CbzCl by flash chromatography using a glass
column (Büchi, Flawil, Germany) connected to a Shimadzu LC-8A solvent delivery pump
(Kyoto, Japan). The column was therefore packed with 400 ml silica gel using 40% EtOAc-
hexane and afterwards loaded with 10 g reactionmixture dissolved in dichloromethane
(CH2Cl2). During separation with 40% EtOAc-hexane at a flow rate of 20 ml/min, the column’s
progress was monitored by TLC. Fractions of 50 ml were collected in test tubes for a period
of 80 minutes. Spots of the reaction mixture, CbzCl and each flash fraction were applied on a
SIL G/UV254 plate and the latter was developed in 40% EtOAc-hexane. When TLC indicated
the removal of CbzCl, the reaction product was eluted from the column with 100%
methanol. Fractions of 50 ml were again collected and submitted to TLC (eluent: 40% EtOAc-
hexane) for evaluation of the elution process. The test tubes containing fractions of similar
purity were pooled in round-bottomed flasks and concentrated under the rotary vapor.
A second purification was performed with flash chromatography to dispose a residual
impurity. Preparation of the flash column was carried out as mentioned above, using 100 ml
silica gel. During elution with 40% EtOAc-hexane (flow rate: 50 ml/min.), fractions of 50 ml
were collected and spotted on a SIL G/UV254 plate (mobile phase: 40% EtOAc-hexane) to
monitor the elution of CbzMAE. The fractions that only contained the pure CbzMAE were
pooled and concentrated.
Identification of the reactionproduct was verified with MS in positive ion mode and the
retention time was determined under the same HPLC conditions as specified in 4.4.1.
4.3.3. Radiosynthesis of [18F]FCH2-CbzMAE+
4.3.3.1. Radiosynthesis on a SPE cartridge
Radiosynthesis was performed on the Scintomics module described in 4.1.3. The
Oasis® HLB C18 cartridge was instead loaded with 300 μl CbzMAE. After the cartridge was
eluted with 2 ml ACN, 100 μl elute was 1 to 200 diluted with mobile phase (50:50 ACN/H2O +
10 mM TEA) and eventually 100 μl was injected into the HPLC system described in 4.4.1.
4. Methods
26
4.3.3.2. Radiosynthesis in a Heated Reactionvial
Radiosynthesis in a heated reactionvial was carried out as described in 4.1.3.2. After 15,
30, 45 and 60 min, 10 µl of the reactionmixture was 1 to 200 diluted with mobile phase and
100 µl of this solution was injected into the same HPLC system for identification of the
radioactive compounds.
4.4. METHYLAMINOETHANOL
4.4.1. HPLC Identification
Retention times of MAE, DMAE, choline and [18F]FCH2Br were determined under the
same HPLC conditions as those specified in 4.2.4. Here fore, standard solutions of DMAE (10
ppm), MAE (50 ppm) and choline (50 ppm) were prepared. [18F]FCH2Br was obtained as
described in 4.1.2 and subsequently injected in the HPLC system. The elution process was
monitored using the 2414 refractive index detector in series with a radioactivity detector.
4.4.2. Radiosynthesis of [18F]FDMAE
4.4.2.1. Radiosynthesis on a SPE Cartridge
Radiosynthesis was performed on the Scintomics module in a manner similar to 4.1.3.
The Oasis® HLB C18 cartridge was instead loaded with 300 μl MAE. After the cartridge was
eluted with 2 ml ACN, 100 μl elute was diluted with 1900 μl H2O and eventually 100 μl was
injected into the HPLC system.
4.4.2.2. Radiosynthesis in a Heated Reactionvial
Radiosynthesis with MAE (300 µl) was carried out as described in 4.1.3.2. After 15, 30,
45 and 60 min, 15 µl of the reactionmixture was 1 to 400 diluted with MilliQWater and 100
µl of this solution was injected in the HPLC system. One minute fractions were collected in
test tubes during the HPLC program and radioactivity of these samples was measured with
the Packard Cobra® gamma (γ)-counter (Packard Instrument Company, Meriden CT, USA).
5. Results
27
5. RESULTS
5.1. TRITYLMETHYLAMINOETHANOL
5.1.1. Synthesis and Purification
After 5 minutes, the reaction mixture turned opaque which suggested the formation of
an insoluble compound, most likely TrMAE+Cl-. Figure 5.1. shows the TLC plates after 10 min
and 1 hour of reaction: TrCl appeared as a quenched spot with Rf = 0,64. The two other spots
(respectively Rf = 0,86 and Rf = 0,97) were probably due to apolar impurities in the TrCl
standard solution. Comparison of the standard solution with the reaction mixture after 1
hour indicates the presence of TrCl in the latter. This means that the SN2-reaction had not
proceeded completely. The new quenched spot with a Rf = 0,28 will very likely represent
TrMAE+Cl- since this compound is more polar than TrCl and therefore less far migrates than
TrCl. The third quenched spot (Rf = 0,95) is probably the same apolar impurity observed in
the standard solution (Rf = 0,97).
Figure 5.1 : TLC plates after 10 min (left) and after 1h (right) under UV light (gray) and after treatment with H2SO4 (yellow)
Additional visualisation of TrCl and TrMAE+Cl- could be achieved by a more sensitive
method. Acidic treatment with 50% sulfuric acid (H2SO4) and heating of the TLC plates will
turn the spots containing a trityl cation yellow. This was the case for the spots with Rf = 0,28
5. Results
28
and 0,64 (and also 0,86 and 0,97) in the standard solution. The appearance of a new yellow
spot with Rf = 0,28 confirms the suspicion of TrMAE+Cl- migration with Rf = 0,28. Purified
TrMAE+Cl- was obtained after successive washing steps with hexane since the developed TLC
plate after the washing steps showed no spots of TrCl or other impurities.
Figure 5.2 : Full-scan mass spectra of TrMAE at positive mode
Additional confirmation of the identification of the new spot (Rf : 0,28) requires MS
experiments. Figure 5.2 shows a full-scan mass spectrum with an intensive peak at m/z
317,87. This indicates the presence of the protonated molecular ion TrMAE+ (318,43 amu).
The ESI-MS/MS spectrum of m/z 317,87 displays fragment ions at m/z 243,08 and 165,31
(Figure 5.3a). The ion at m/z 243,08 was formed by neutral loss of MAE (75,13 amu) from the
protonated molecular ion, yielding a trityl cation (243,3 amu) in the process. The other ion at
m/z 165,31 reflects possibly futher fragmentation of the trityl cation into a phenyl group (77
amu) and a positive diphenylmethyl compound (166,23 amu). Additional confirmation of this
fragmentation mechanism was obtained by the MS/MS spectrum of m/z 243,08 (Figure
5.3b), which showed an abundant peak at m/z 165,13. The ion (m/z 317,8) and his fragment
ions (m/z 243,08 and 165,31) are indicative of the presence of TrMAE+ as parent compound.
5. Results
29
(a) (b)
Figure 5.3 : ESI-MS/MS spectra at positive mode of (a) the [M + H]+ ion with m/z 318,12 and (b) the fragment ion with m/z 243,13
TrMAE was futher confirmed via 1H NMR spectroscopy. Figure 5.5a is a close-up of
the obtained 1H NMR spectrum (Figure 5.4) and shows one methyl singlet at δH 2.209 ppm,
two methylene triplets at δH 2.256 and 3.862 ppm (respectively N- and OH-substituted) and
one hydroxyl proton at δH 2.934 ppm as characteristic signals. Spin-spin coupling is
responsible for peak splitting of both the methylene groups (J = 6.0 Hz). The structure of
MAE can be reconciled with the observed chemical shifts in Figure 5.5a.
Figure 5.5b shows 3 multiplets between 7.1-7.7 that correspond most likely to the
aromatic protons of the trityl group. The most deshielded protons (H-2/H-6; Figure 5.4) of
the three phenyl rings were displayed as a doublet at δ = 7.629-7.653 ppm (J = 7.2 Hz)
because of spin-spin coupling with H-3/H-5. Likewise, vicinal coupling with the ortho and
para protons yields a triplet at δ = 7.346 ppm (J = 7.5 Hz) for H-3/H-5. Finally, the signal of H-
4 is observed as a triplet of a triplet at δ = 7.225 ppm (J = 1.2 and 7.2 Hz) due to vicinal
coupling with H-3/H-5 and also longe range coupling with H-2/H-6. Since both MS and NMR
data interpretation confirm the suggested structure of TrMAE, our synthesized compound is
identified as TrMAE.
5. Results
30
Figure 5.4 : Typical 1H NMR spectrum of TrMAE at 300 MHz in CDCl3
(a)
(b)
Figure 5.5 : Expansion of the 1H NMR spectrum (a) between 2.2 and 4.0 ppm and (b)
between 7.1 and 7.7 ppm
5. Results
31
The following two reaction parameters (concentration and temperature) with their
corresponding yields are listed in table 5.1. The reaction with the highest yield of TrMAE+Cl-
synthesis was obtained at roomtemperature with an excess reaction. Heating of the reaction
did not promote synthesis of TrMAE+Cl-, on the contrary, yield values decreased significantly.
Table 5.1 : Optimalisation of the synthesis of TrMAE
TrCl (mmol) MAE (mmol) Reactiontemp. (°C) n Yield (%)
5 5 20°C 2 18 ± 2,5
5 4,545 20°C 1 25
5 4,545 60°C 3 1 ± 1,4
5.1.2. HPLC Identification
Retention times of [18F]FCH2Br, TrCl and TrMAE are shown in Table 5.2. The TrMAE
working solution showed a small peak at 4,59 min, so it still contained little amounts of TrCl
although a purity of more than 95% was achieved (data not shown).
Table 5.2 : Retention times of standard compounds
Components Retention Time (min)
[18F]FCH2Br 3,18
TrCl 4,59
TrMAE 5,43
5.1.3. Radiosynthesis of [18F]FCH2-TrMAE+
5.1.3.1. Radiosynthesis on a SPE Cartridge
The chromatogram obtained after radioactive labeling on the Oasis HLB plus cartridge
(Figure 5.7) showed one radioactive peak at 3,46 min. Comparison of the retention time with
that of the standard compounds in Table
5.2 reveals that the observed peak in
Figure 5.7 corresponds with [18F]FCH2Br.
As a result, no [18F]FCH2-TrMAE+ (Figure
5.6) was formed. Figure 5.6 : [18F]FCH2-TrMAE+
5. Results
32
5.1.3.2. Radiosynthesis in a Heated Reactionvial
Figure 5.7 illustrates the HPLC chromatograms after 15, 30, 45, and 60 min of reaction in
a heated alltech vial. As in 5.1.3.1., only one radioactive peak (tR = 3,46 min.) is observed.
This peak matches the retention time of [18F]FCH2Br, which indicates that also here, no
[18F]FCH2-TrMAE+ was formed.
Figure 5.7 : Overlayed HPLC Radiochromatogram of the reaction mixture after SPE
synthesis and after 15, 30, 45 and 60 min radiosynthesis in a heated reactionvial
To test the reactivity of [18F]FCH2Br, DMAE was added to the reactionvial. After injection
into the HPLC system, a high peak was observed at 11,3 min (Figure 5.8). This indicates the
formation of [18F]FCH, which implies that the reaction conditions for the nucleophilic
substitution are favored. Accordingly, TrMAE cannot be radiolabeled with fluorine-18 via SN2
reaction of TrMAE with [18F]FCH2Br.
Figure 5.8 : HPLC Radiochromatogram for the assessment of [18F]FCH2Br reactivity
5. Results
33
5.2. BENZYLMETHYLAMINOETHANOL
5.2.1. Radiosynthesis of [18F]FCH2-BzMAE+
5.2.1.1. Synthesis of the cold ligand (CH3-BzMAE+)
The drop wise addition of BzMAE to the CH3I-EtOAc solution generated an opaque
solution. After recrystallization and washing of the precipitate, the reaction produced 188
mg CH3BzMAE+I-, which corresponds to a reaction yield of 12%.
5.2.1.2. HPLC Identification
Retention times for [18F]FCH2Br, BzMAE and CH3-BzMAE+ are given in Table 5.3.
Table 5.3. Retention times of standard compounds
Components Retention Time (min)
[18F]FCH2Br 5.60 – 6.20 BzMAE 4.78
CH3-BzMAE+ 1.69
5.2.1.3. Radiosynthesis on a SPE Cartridge and in a Heated Reaction vial
The radiochromatogram obtained after radioactive labeling of BzMAE on a ®Oasis HLB
plus cartridge (Figure 5.9) showed no indication of [18F]FCH2-BzMAE+ formation. Only one
radioactive compound (tR = 6,07) was observed and by comparing its retention time to that
of the standard compounds in Table 5.3, the peak could be identified as [18F]FCH2Br.
Figure 5.9 : Overlaid HPLC radiochromatograms of the reaction mixture after SPE
synthesis and after 15, 30 and 60 min radiosynthesis in a heated reaction vial.
5. Results
34
Table 5.4 : % peak area of the radiolabeled compounds in the reaction mixture
Peak at 1.98 min Peak at 6.0 min Peak at 14.49 min
SPE synthesis / 100% / 15 min. 80°C 59% 41% / 30 min. 80°C 89% 11% / 60 min. 80°C 18% 2% 80%
However, when radiosynthesis was performed in a heated reaction vial, more
promising results were obtained (Figure 5.9). After 15, 30 and 60 minutes of reaction, the
peak height of [18F]FCH2Br declined significantly while a new radiolabeled compound was
formed with tR = 1,98 min. Because the peak corresponds to the retention time of
CH3BzMAE+ (Table 5.3), formation of [18F]FCH2-BzMAE+ is confirmed during radiosynthesis.
The unidentified peak at 14,5 min., which arises after 60 min radiosynthesis, is probably a
radiolabeled side product.
5.2.2. Purification of [18F]FCH2-BzMAE+
After one hour of radiosynthesis, a 6% NH4OH solution was added to the reaction
mixture. The solution was thereafter applied to a conditioned Oasis WCX cartridge which
captured 14% (not corrected for decay) of the reaction mixture on the WCX column. This
amount corresponds to the percentage of [18F]FCH2-BzMAE+ formed after 60 min. of
radiosynthesis (Table 5.4). Therefore we can conclude that [18F]FCH2-BzMAE+ can be isolated
on a WCX cartridge. Since a radiolabeled side product is formed after 60 min. of
radiosynthesis and the highest yield of [18F]FCH2-BzMAE+ is achieved after 30 min., further
synthesis will be carried out with 30 min. of radiosynthesis.
The calculated yield values (calculated with decay corrected data) of the three
performed purifications are listed in Table 5.5. An acceptable yield of 69 ± 15% (means ± SD)
was achieved for [18F]FCH2-BzMAE+.
Table 5.5 : Yield determination of the reaction by WCX isolation of [18F]FCH2-BzMAE+
WCX isolation (%) Elute (%) VialEMPTY (%)
54 43 3,2 83 3,3 0,89 71 25 4,2
Mean ± SD 69% ± 15%
5. Results
35
Table 5.6 shows the corresponding yield values of each elution of [18F]FCH2-BzMAE+. A
yield of up to 93 ± 6% (means ± SD) was observed when a solution of 10 µl HCOOH in 1 ml
H2O and 4 ml EtOH was used. This is a good result if the intention is to isolate [18F]FCH2-
BzMAE+ for hydrogenolysis in the presence of HCOO- as a H-donor.
Table 5.6 : Yield determination of the elution of [18F]FCH2-BzMAE+ from the WCX using a 10 µl HCOOH in 1 ml H2O and 4 ml EtOH solution.
AWCX (mCi) AELUTED WCX (mCi) AELUTE (mCi) Yield (%)
5,8 0,8 5,1 87 20,2 2,0 19,0 92
17,1 0,7 17,1 100
5.2.3. Deprotection of [18F]FCH2-BzMAE+
5.2.3.1. Catalytic transfer hydrogenolysis of CH3-BzMAE+I-
The time-course plot of the catalytic transfer hydrogenolysis (CTH) of CH3-BzMAE+
(Figure 5.10) is an exponentially-declining curve (R2 0,9942). This demonstrates that CTH of
CH3-BzMAE+ proceeds with HCOO- as a H-donor and Pd/C as a catalyst.
Figure 5.10 : Kinetic plot of the catalytic transfer hydrogenolysis of CH3-BzMAE+ in
which peak areas are expressed in relation to the reaction time (min)
5.2.3.2. Catalytic transfer hydrogenolysis of [18F]FCH2-BzMAE+
The progress in time of the catalytic transfer hydrogenolysis of [18F]FCH2 BzMAE+ is
shown in Table 5.7. Highest CTH yield values were obtained after 15 and 30 min of reaction
(almost 90% of deprotection), which suggested that the optimum reaction time for
5. Results
36
hydrogenolysis ranges from 15 to 30 min., since a 60 min. CTH does not allow complete
deprotection of [18F]FCH2-BzMAE+.
Table 5.7 : Catalytic transfer hydrogenolysis yield values at different reaction times
Reaction Time (min) AWCX (%) AELUTE (%)
15 11 89 30 11 89 45 27 73 60 23 77
5.2.4. Identification and Purification of [18F]FDMAE+
The radiochromatogram obtained after CTH of [18F]FCH2-BzMAE+ (Figure 5.11)
revealed three peaks. The first peak (tR = 4,66 min) stands for a radioactive compound
formed during hydrogenolysis with very little column interaction. The compound with tR =
9,62 could represent [18F]FDMAE and the third compound (tR = 12,3 min) could correspond
to a ternairy amine, probably [18F]FCH2-BzMAE+, or to [18F]FCH, since a match in retention
time is observed for the latter. Figure 5.12 shows the radiochromatogram acquired after
elution of the WCX cartridge with 6% NH4OH. In this elute, [18F]FDMAE was expected but
only the compound with tR = 3,89 min. was observed.
Figure 5.11 : HPLC radiochromatogram obtained after hydrogenolysis
5. Results
37
Figure 5.12 : HPLC radiochromatogram obtained after CTH and elution from the
WCX cartridge with a 6% NH4OH solution
When the test was performed for a second time, with 20 mg of Pd/C, the absence of
[18F]FDMAE was proved (Figure 5.13) since only a compound with retention time 3.86 min. is
observed in the filtrate after CTH. Furthermore, the refractive index detector gives a signal at
5.06 min., which corresponds to the retention time of MAE.
Figure 5.13 : HPLC chromatogram (left) and radiochromatogram (right) obtained
after catalytic transfer hydrogenolysis
5.3. 2-[(N-BENZYLOXYCARBONYL)METHYLAMINO]ETHANOL
5.3.1. Assessment of the reactivity of a carbamate
The radiochromatogram shown in Figure 5.14 is acquired after radioactive labeling of
phenyl-N,N-dimethylcarbamate in a heated reactionvial. After 15 min, two peaks are
observed with respectively tR = 2,32 and 6,54 min. In the course of time, the peak area of the
latter decreases whereas the first peak increases in intensity (Table 5.8). By comparing their
retention times with that of the standard compounds (Table 5.3), the two peaks could be
5. Results
38
identified as [18F]FCH2Br (tR = 6,54 min) and [18F]fluoromethylated phenyl-N,N-
dimethylcarbamate (tR = 2,32 min). Thus, radiofluorination of the carbamate had occured.
Figure 5.14 : Overlayed HPLC radiochromatogram of the reactionmixture after 15, 30,
45 and 60 min radiosynthesis in a heated reactionvial
Table 5.8 : % peak area of the radiolabeled compounds in the reaction mixture
Peak at 2,3 min Peak at 6.3 min
15 min. 80°C 12,08% 87,92% 30 min. 80°C 16,07% 83,93% 45 min. 80°C 49,67% 50,33% 60 min. 80°C 47,74% 52,26%
5.3.2. Purification and identification of CbzMAE
Flash chromatography was performed to remove and dispose the remaining CbzCl and
possible impurities in the reactionmixture. The elution of CbzCl from the flash column was
monitored by TLC. As can be seen from the TLC plate (Figure 5.15), CbzCl has an RF = 0.847,
0.719 and an RF = 0. The fractions after 400, 420 and 440 ml elution showed a quenched spot
with a similar RF value (= 0,858), which indicates that CbzCl is eluted from the column after
an amount of 440 ml mobile phase (40% EtOAc-hexane).
5. Results
39
Figure 5.15 : TLC plate developed in 40% EtOAc-hexane after elution of the column with 40% EtOAc-hexane (left) and later on 100% MeOH (right)
When the column was eluted with 100% MeOH (Figure 5.15), the reaction product
(CbzMAE) with RF = 0,110 was collected in the fractions of 400, 450, 500, 550 and 600 ml
elution. However, another impurity (RF = 0), that also appeared in CbzCl, was observed in the
reaction product and in the fractions of 450 to 600 ml elution. Therefore, a second
purification was performed using 40% EtOAc-hexane as mobile phase, which eventually
resulted in the purified CbzMAE.
Figure 5.16 : Close-up of the MS spectrum of CbzMAE in positive ionisation mode
The retention time of CbzMAE, obtained after HPLC analysis, is 4,458 min (data not
shown). The MS spectrum of CbzMAE (Figure 5.16) shows two abundant adduct molecular
ions. The most intensive peak at m/z 232.0943 corresponds to the sodium adduct molecular
ion [M + Na]+ and the peak at m/z 248.0691 to the potassium adduct ion [M + K]+. These two
5. Results
40
characteristic ions enable a reliable identification of CbzMAE (209 amu) since the two MS
peak mass values (232 and 248 amu) match the molecular mass of respectively [CbzMAE +
Na]+ and [CbzMAE + K]+.
5.3.3. Radiosynthesis of [18F]FCH2-CbzMAE+
The radiochromatogram obtained after radiosynthesis on an ®Oasis HLB plus cartridge
(data not shown) revealed only one peak at 6,13 min due to [18F]FCH2Br. This indicates that
no [18F]FCH2-CbzMAE+ (Figure 5.17) was formed.
Figure 5.17: Reaction scheme of the radiosynthesis of [18F]FCH2-CbzMAE+
When radiofluorination was carried out in a heated reactionvial, the same result was
observed. Only the [18F]FCH2Br peak appeared (tR = 6,238 min) (Figure 5.18a). The HPLC
chromatogram after 60 min reaction (Figure 5.18b) showed one peak corresponding to
CbzMAE (tR = 4,452), indicating that degradation of CbzMAE did not occur.
(a) (b)
Figure 5.18 : HPLC radiochromatogram (a) and HPLC chromatogram (b) of the reactionmixture after 60 min. radiosynthesis in a heated reactionvial
5.4. METHYLAMINOETHANOL
5.4.1. HPLC Identification
Retention times of the standard compounds obtained from the HPLC analysis are
presented in Table 5.90. MAE, DMAE and choline were detected with the RI detector
whereas [18F]FCH2Br and [18F]FCH were detected with the radioactivity detector.
5. Results
41
Table 5.9 : Retention times of standard compounds
Components Retention Time (min)
[18F]FCH2Br 3,88
MAE 4,53
DMAE 7,85 Choline 11,3
[18F]FCH 13,2
5.4.2. Radiosynthesis of [18F]FDMAE
When radiolabeling was performed on the SPE cartridge, no peak was revealed at 9,9
min., which is the expected retention time for [18F]FDMAE. On the other hand, two other
compounds with retention times of approximately 6,8 min and 15 min. were detected. After
15 min radiosynthesis in a reaction vial, a new compound was detected (tR = 9,9 min.) in the
presence of the two same compounds observed after SPE synthesis (tR = 6,8 and 15 min.).
This suggested the presence of [18F]FDMAE when compared to the tR of DMAE (Table 5.5).
During further reaction (60 min.), the proportion of [18F]FDMAE had declined. Additionnaly,
the amount activity of the collected fractions did not correspond tot the activity injected on
the column. Investigation with the Geiger counter (Fidgeon Limited, Peterlee, Durham UK)
indicated that a great amount activity stayed behind on the HPLC column.
Figure 5.20 : Overlaid HPLC chromatograms of the reaction mixture after SPE synthesis and after 15 and 60 min radiosynthesis in a heated reactionvial
6. Discussion
42
6. DISCUSSION
Choline radiotracers are widely used as PET imaging agents for the clinical diagnosis of
cancer. In previous studies, various choline analogs, including [18F]FCH, have been tested on
their sensitivity and specificity to distinguish cancer from normal tissue. Althought [18F]FCH
has shown to be an effective tracer for prostate cancer screening, it exhibits still a few
limitations, mainly urinary excretion. In this study, [18F]fluorodimethylaminoethanol was
introduced as a choline analog with potentially better properties than [18F]FCH.
Several approaches were explored in order to develop this radiotracer. Initially,
fluoromethylation of MAE with the alkylating agent [18F]FCH2Br seemed the easiest method
for [18F]FDMAE synthesis. However, difficulties arise during purification of [18F]FDMAE. In the
case of [18F]FCH, Slaets et al. reported that a high purity is achieved using a WCX cartridge
and an eluting solution with pH 12. Because of the small pKa difference (ΔpKa = 0,1 – 1)
between MAE and [18F]FDMAE, separation based on pH between the radiolabeled
compound and its cold precursor is very unlikely to occur. As a result, the WCX cartridge
could not be used for purification of [18F]FDMAE. In addition, since the degree of alkylation
with amines is difficult to control, a certain amount of [18F]FCH could be formed besides
[18F]FDMAE (Figure 6.1). This impedes the purification process even more.
Figure 6.1 : Formation of [18F]FDMAE and [18F]FCH
Therefore, other purification methods need to be developed. Derivatisation after
fluorlabeling is a way to separate [18F]FDMAE from the other compounds in the mixture.
A first approach is the radiosynthesis of [18F]FDMAE, based on the use of N-protecting
groups introduced on MAE. This solves the problems of purification that occur with MAE.
After nucleophilic substitution with [18F]FCH2Br, a quaternary amine is formed. Due to the
positive charge, the latter is trapped on the WCX during purification, whereas [18F]FDMAE (a
6. Discussion
43
tertiar amine) is not. Additionally, only one alkylation step can proceed, which results in the
formation of one product instead of a mixture of reactionproducts in the case of MAE.
However, to end up with [18F]FDMAE, an additional deprotection and purification step are
required after radioactive labeling.
The selected N-protection groups (a trityl-, a benzyl- and a benzylcarboxycarbonyl group)
must meet a number of conditions including a high stability at pH 12 and an easy way to
deprotect. The reactivity of the resulting compounds (TrMAE, BzMAE and CbzMAE) with
[18F]FCH2Br were tested in the present study. BzMAE is commercially available, whereas
TrMAE and CbzMAE are not. Those two need to be synthesized and purified before
radiosynthesis is started. TrMAE synthesis at roomtemperature, with an excess reaction gave
the highest yield.
Our results indicated however that TrMAE was unable to react with [18F]FCH2Br, nor on
the cartridge, nor in the heated reactionvial. A possible explanation for this, is the steric
hinder caused by the trityl group. To ensure that the lack of reaction was not the
consequence of bad reaction conditions, the reactivity of [18F]FCH2Br with DMAE was also
tested. Since [18F]FCH was generated from the latter reaction, the assumption that TrMAE
cannot be methylated is confirmed.
BzMAE on the other hand showed initially more promising results than TrMAE.
Radiofluorination did occur in a reactionvial at 80°C and the resulting [18F]FCH2-BzMAE+ was
isolated on the WCX and eluted with 10 µl HCOOH in 1:4 H2O/EtOH (5 ml). The optimum
reaction time of the hydrogenolysis varies from around 15 to 30 min. Based on the obtained
data from our experiment, it can be concluded that exceeding of the optimum reaction time
leads to a decrease in yield. Possible reasons for this deterioration are the low stability
exhibited by the reaction product against CTH.
After hydrogenolysis, the presence of MAE was confirmed whereas [18F]FDMAE was not
detected. This is an unsatisfiable result since the main goal of this study was to synthesize
[18F]FDMAE. The generation of MAE during hydrogenolysis was not unexpected and can be
explained as follows. The selectivity of CTH to remove N-benzyl protecting groups is strongly
influenced by the electronic properties of the benzyl ring and its affinity to the metal surface
6. Discussion
44
(Papageorgiou et al., 2000). Since [18F]fluorine is a very electronegative element, the carbon
to which it is bonded will be activated. As a consequence, hydrogenolysis will also proceed
on the activated carbon, yielding MAE and [18F]FCH3 in the process (Figure 6.2). The
recurrent unidentified peak with tR = 3,8 – 4,6 min. will most likely represent [18F]FCH3.
Figure 6.2 : Possible reactionmechanism of the CTH of [18F]FCH2-BzDMAE+
All together, it can be concluded that the amount [18F]FDMAE generated from [18F]FCH2-
BzDMAE+, is immediately hydrogenated to MAE (Figure 6.2), which means that this method
did not allow synthesis of [18F]FDMAE.
Besides TrMAE and BzMAE, the carbamate CbzMAE was eventually tested on its ability to
be fluoromethylated. Firstly, to determine whether a carbamate (-NH(CO)O-) is able to form
a quaternary amide in the presence of the carboxyl group, the reactivity of a commercially
available carbamate (phenyl-N,N-dimethylcarbamate) with [18F]FCH2Br was investigated.
Since our results implied that the reaction between the two compounds occured, synthesis
of CbzMAE was started.
However, as in the case of TrMAE, radiofluorination of CbzMAE did not take place. The
most plausible explanation for this observation is the low tendency of CbzMAE to be
methylated. This problem might be solved by using a catalyst, dimethylaminopyridine
(DMAP), in the alkylation reaction of CbzMAE or another carbamate. Herein, DMAP would
be methylated by [18F]FCH2Br, yielding [18F]FCH2-DMAP+. The latter will in turn be
demethylated in the next step, catalysing this way the fluoromethylation of the carbamate.
Since 18F is very electronegative, the [18F]FCH2-group has a higher tendency to be detached
from [18F]FCH2-DMAP+ than the other two methyl groups on the nitrogen atom. Neverteless,
the chance exists that the carbamate is methylated instead of fluoromethylated.
In the current work, reaction of MAE with [18F]FCH2Br was also assessed on a HLB plus
cartridge and in a heated reactionvial. The latter requires a slight modification of the
6. Discussion
45
Scintomics module, wherein the SPE cartridge is replaced by a reactionvial. Once the formed
[18F]FCH2Br was collected in the vial, the latter is removed from the module and placed in a
heated oil bath where the actual radiosynthesis could take place. In contrast to the SPE
cartridge, higher temperatures and a longer reaction time are achieved in the reactionvial,
which is beneficial for the radiosynthesis. However, since the SN2-reaction is performed
outside the hot-cell, radiofluorination in a reactionvial is related to a higher radiation
exposure.
Our experiments with MAE showed that instead of [18F]FDMAE, two other unidentified
compounds were formed on the SPE cartridge. When radioactive labeling was carried out in
a reactionvial heated to 80°C, the same two radioactive components appeared. These
probably came to pass via adduct formation. However, after 15 min, an additional peak that
could correspond to [18F]FDMAE was detected, but after 60 min the intensity of the latter
had decreased significantly. This could be explained by a possible effect of the temperature
on the yield of the [18F]FDMAE formation. Datta E. Ponde et al. (2009) reported earlier that
the yield of the radiosynthesis of [11C]DMAE decreased at higher temperatures. The sample
at 60 min is much longer subjected to a temperature of 80°C, thus a lower yield is obtained.
However, the data obtained from our experiments were not consequent enough to be
able to conclude that [18F]FDMAE was formed. Several unexpected events, like the high
amount activity that stayed on the HPLC column, the shift in retention time of [18F]FDMAE
and the adductformation are still unclear. Further experiments are needed in order to gain a
better insight in the radiosynthesis of [18F]FDMAE based on reaction of MAE with
[18F]FCH2Br.
7. Conclusion
46
7. CONCLUSION
The synthesis of the choline analog, [18F]fluoro-N,N-dimethylaminoethanol, was
attempted by two related routes involving [18F]fluoromethylation. Both approaches were
unsuccessful. Initially, the reaction of a nitrogen-protected N-methylaminoethanol with
[18F]FCH2Br was assessed. It was found that MAE protected with either a trityl- or benzyl
carbamate group was unable to be fluoromethylated and form respectively [18F]FCH2-
TrMAE+ or [18F]FCH2-CbzMAE+. The benzyl protecting group on MAE did not impede
fluoromethylation, allowing the formation of [18F]FCH2-BzMAE+ with an acceptable yield of
69% ± 15%. However, deprotection of the benzyl group via hydrogenolysis was accompanied
by side reactions, causing the formation of MAE instead of [18F]FDMAE.
Direct nucleophilic fluoromethylation of MAE with [18F]FCH2Br on a SPE cartridge
generated two unidentified compounds, but still no [18F]FDMAE. The same reaction
performed in a reaction vial heated to 80°C resulted possibly in the formation of [18F]FDMAE
after 15 min. However, when the reaction progressed in time, the amount of [18F]FDMAE
declined in our experiment. The two same unidentified compounds were also present here.
In order to fully understand what occurs during fluoromethylation with MAE in a heated
reaction vial, further analysis should be carried out. The obtained data from the performed
analysis are unexpected and not reliable enough for us to be able to come up with a correct
explanation or conclusion.
Overall can be concluded that no suitable and efficient synthesis method has yet been
found for the synthesis of [18F]FDMAE as potential radiotracer in prostate cancer imaging.
8. References
47
8. REFERENCES
Ackerstaff, E.; Pflug, B. R.; Nelson, J. B.; Bhujwalla, Z. M. (2001). Detection of Increased
Choline Compounds with Proton Nuclear Magnetic Resonance Spectroscopy Subsequent to
Malignant Travsformation of Human Prostatic Epithelial Cells. Cancer res, 61, 3599-3603.
Bé, M.-M.; Chisté, V.; Dulieu, C.; Browne, E.; Chechev, V.; Kuzmenko, N.; Helmer, R.; Nichols,
A.; Schönfeld, A.; Dersch, R. (2004). Table of Radionuclides. Bureau International des Poids et
Mesures, Pavillon de Breteuils, Sèvres, France.
Bansal, A; Shuyan, W.; Hara, T.; Harris, R. A.; DeGrado, T. R. (2008). Biodisposition and
metabolism of [18F]fluorocholine in 9L glioma cells and 9L glioma-bearing fisher rats. Uer J
Nucl Med Mol Imaging, 35, 1192-1203.
Degrado, T. R.; Kwee, S. A.; Coel, M. N.; Coleman, R. E. (2007). The Impact of Urinary
Excretion of 18F-Labeled Choline Analogs. J Nucl Med, 48, 1225-1225.
Delgado-Bolton, R. C.; Carreras Delgado, J. L. (2010). Évaluation de la réponse au traitement
du lymphome avec la TEP 18F-FDG: existe-t-il un consensus dans l’évaluation de la réponse?.
Médecine Nucléaire, 35, 29-37.
Elsinga, P. H. (2002). Radiopharmaceutical chemistry for positron emission tomography.
Methods, 27, 208-217.
Freedland, S. J.; Wieder, J. A.; Jack, G.S.; Dorey, F.; dekernion, J. B.; Aronson, W. J. (2002).
Improved risk stratification for biochemical recurrence after radical prostatectomy using a
novel risk group system based on prostate specific antigen density and biopsy Gleason score.
The Journal of Urology, 168, 110-115.
Gadda, G.; Powell, N. L. N.; Menon, P. (2004). The trimethylammonium headgroup of choline
in a major determinant for substrate binding and specificity in choline oxidase. Arch Biochem
Biophys, 430, 264-273.
Geldenhuys, W. J.; Allen, D. D.; Lockman, P. R. (2010). 3-D-QSAR and docking studies on the
neuronal choline transporter. Bioorg Med Chem Lett, 20, 4870-4877.
8. References
48
Glunde, K.; Jie, C.; Bhujwalla, Z. M. (2004). Molecular Causes of the Aberrant Choline
Phospholipid Metabolism in Breast Cancer. Cancer Research, 64, 4270-4276.
Heinlein, C. A.; Chang, C. (2004). Androgen Receptor in Prostate Cancer. Endocr Rev, 25, 276-
308.
Hofer, C.; Laubenbacher, C.; Block, T; Breul, J.; Hartung, R.; Schwaiger, M. (1999). Fluorine-
18-Fluorodeoxyglucose Positron Emission Tomography Is Useless for the Detection of Local
Recurrence after Radical Prostatectomy. Eur Urol, 36, 31-35.
http://movies-tatecalebzane.blogspot.com/2011/03/glycolysis-and-gluconeogenesis-
concept.html (20-04-2011)
http://www.heartandmetabolism.org/issues/hm34/hm34refresherc.asp (30-03-2011)
http://www.learner.org/courses/physics/unit/pdfs/unit2.pdf (11-05-2011)
Ikuta, S.; Imamura, S.; Misaki, H.; Horiuti, Y. (1977). Purification and Characterization of
Choline-oxidase from Arthrobacter-Globiformis. J Biochem, 82, 1741-1749.
Jadvar, H. (2009). Molecular imaging of prostate cancer with 18F-fluorodeoxyglucose PET.
Nat Rev Urol, 6, 317-323.
Jadvar, H. (2011). Prostate Cancer: PET with 18F-FDG, 18F-FDG, 18F- or 11C-Acetate, and 18F- or
11C-Choline. J Nucl Med, 52, 81-89.
Jones, T. (1996). Startegy fot creating accurate functional imaging with PET and its relevance
to SPECT. In: Tomography in Nuclear Medicine, Proceedings of an International Symposium,
International Atomic Energy Agency (Ed.), Vienna, Austria, pp. 81-88.
Kim E. E.; Yang D. Y. (2001). Targeted Molecular Imaging in Oncology. Springer-Verlag, New
York, USA, Chapter 2.
Kotzerke, J.; Gschwend, J. E.; Neumaier, B. (2002). PET for Prostate Cancer Imaging: Still a
Quandary or the Ultimate Solution? J Nucl Med, 43, 200-202.
8. References
49
Kouji, H.; Inazu, M.; Yamada, T.; Tajima, H.; Aoki, T.; Matsumiya, T. (2009). Molecular and
functional characterization of choline transporter in human colon carcinoma HT-29 cells.
Arch Biochem Biophys, 483, 90-98.
Le Bars, D. (2006). Fluorine-18 and medical imaging: Radiopharmaceuticals for positron
emission tomography. Journal of Fluorine Chemistry, 127, 1488-1493.
Lee, N.-Y.; Choi, H.-M.; Kang, Y.-S. (2009). Choline Transport via Choline Transporter-like
Protein 1 in Conditionally Immortalized Rat Syncytiotrophoblast Cell Lines TR-TBT. Placenta,
30, 368-374.
Lee, Y.-S. (2010). Radiopharmaceuticals for Molecular Imaging. The Open Nuclear Medicine
Journal, 2, 178-185.
Leyton, J.; Smith, G.; Zhao, Y.; Perumal, M.; Nguyen, Q.; Robins, E.; Arstad, E.; Alboagye, E. O.
(2009). [18F]Fluoromethyl-[1,2-2H4]-Choline: A Novel Radiotracer for Imaging Choline
Metabolism in Tumors by Positron Emission Tomograpgy. Cancer Res, 69, 7721-7728.
Li, Z.; Vance, D. E. (2008). Phosphatidylcholine and choline homeostasis. J Lipid res, 49, 1187-
1194.
Liu, Y. (2006). Fatty acid oxidation is a dominant bioenergetics pathway in prostate cancer.
Prostate Cancer Prostatic Diseases, 9, 230-234.
Matthies, A.; Ezziddin, S.; Ulrich, E.-M.; Palmedo, H.; Biersack, H.-J.; Bender, H.; Guhlke, S.
(2004). Imaging of prostate cancer metastases with 18F-fluoroacetate using PET/CT. Eur J
Nucl Med Mol Imaging, 31, 797.
McCarthy, M.; Siew, T.; Campbell, A.; Lenzo, N.; Spry, N.; Vivian, J.; Morandeau, L. (2011).
18F-Fluoromethylcholine (FCH) PET imaging in patients with castration-resistant prostate
cancer: prospective comparison with standard imaging. Eur J Nucl Med Mol Imaging, 38, 14-
22.
Michalski, M. H.; Chen, X. (2011). Molecular imaging in cancer treatment. Eur J Nucl Med Mol
Imaging, 38, 358-377.
8. References
50
Michel, V.; Yuan, Z.; Ramsubir, S.; Bakovic, M. (2006). Choline Transport for Phospholipid
Synthesis, Exp Biol Med, 231, 490–504.
Mintz, A.; Wang, L.; Ponde, D.E. (2008). Comparison of radiolabeled choline and
ethanolamine as probe for cancer detection. Cancer Biology & Therapy, 7, 742-747.
Mohler, D. L.; Shen, G. (2006). The synthesis of tethered ligand dimers for PPARc–RXR
protein heterodimers. Org Biomol Chem, 4, 2082-2087.
Müller, S. A.; Holzapfel, K.; Seidi, C.; Treiber, U.; Krause, B. J.; Senekowitsch-Schmidtke, K.
(2009). Characterization of choline uptake in prostate cancer cells following bicalutamide
and docetaxel treatment. Eur J Nucl Med Mol Imaging, 36, 1434-1442.
Papageorgiou, E. A.; Gaunt, M. J.; Yu, J.-Q.; Spencer, J. B. (2000). Selective Hydrogenolysis of
Novel Benzyl Carbamate Protecting Groups. Organic Letters, 2, 1049-1051.
Penry, J. T.; Manore, M. M. (2008). Choline: An important micronutrient for maximal
endurance-exercise performance? Int J Sport Nutr Exe, 18, 191-203.
Plathow, C.; Weber, W. A. (2008). Tumor Cell Metabolism Imaging. J Nucl Med, 49, 43S-63S.
Pflug, B. R.; Pecher, S. M.; Brink, A. W.; Nelson, J. B.; Foster, B. A. (2003). Increased Fatty Acid
Synthase Expressionand Activity During Progression of Prostate Cancer in the TRAMP Model.
Prostate, 57, 245-254.
Podgorsak, E. B. (2000). Radiation Physics for Medical Physicists. Springer-Verlag, Berlin
Heidelberg, Germany, Chapter 11.
Price, P. M.; Green, M. M. (2011). Positron emission tomography (PET) imaging approaches
for external beam radiation therapies: current status and future developments. The British
Journal of Radiology, published ahead-of-print as doi: 10.1259/bjr/21263014.
Roivainen, A.; Forsback, S.; Grönroos, T.; Lehikoinen, P.; Kähkönen, M.; Sutinen, E.; Minn, H.
(2000). Blood metabolism of [methyl-11C]choline; implications for in vivo imaging with
positron emission tomography. Eur J Nucl Med, 27, 25-32.
8. References
51
Scher, B.; Seitz, M.; Albinger, W.; Reiser, M.; Schlenker, B.; Stief, Ch.; Mueller-Lisse, U.;
Dresel, S. (2008). Value of PET and PET/CT in the Diagnosis of Prostate and Penile Cancer. In:
PET in Oncology, Dresel, S. (Ed.), Springer-Verlag, Berlin Heidelberg, Germany, pp. 159-179.
Slaets, D.; De Bruyne, S.; Dumolyn, C.; Moerman, L.; Mertens, K.; De Vos, F. (2010). Reduced
dimethylaminoethanol in [18F]fluoromethylcholine: an important step towards enhanced
tumour visualization. Eur J Nucl Med Mol Imaging, 37, 2136-2145.
Soloviev, D.; Fini, A.; Chierichetti, F.; Al-Nahhas, A.; Rubello, D. (2008). PET imaging with 11C-
acetate in prostate cancer: a biochemical, radiochemical and clinical perspective. Eur J Med
Mol Imaging, 35, 942-949.
Van Oosten, E. M. (2009) Synthesis of fluorine-18 labelled radiotracers for positron emission
tomography. Master thesis at the University of Toronto, Canada.
Vavere, A. L.; Kridel, S. J.; Wheeler, F. B.; Lewis, J. S. (2008). 1-11C-Acetate as a PET
Radiopharmaceutical for Imaging Fatty Acid Synthase Expression in Prostate Cancer. J Nucl
Med, 49, 327-334.
Wang, L. M.; Ponde, D. E. (2009). Radiosynthesis of [11C] N-Methyl and N,N'-Dimethyl
Ethanolamine for Measuring Phospholipid Metabolism Using PET Imaging. Synthetic
Commun, 39, 2804-2814.
Zeisel, S. H.; Da Costa, K.-A. (2009). Choline: An Essential Nutrient for Public Health. Nutr Rev,
67, 615-623.
Samenvatting
In deze thesis werd gepoogd de synthese van een nieuwe radiotracer te ontwikkelen ter
detectie van prostaatkanker. Het gaat hier om het choline-analoog, [18F]fluoro-N,N-
dimethylaminoethanol ([18F]FDMAE). Choline is een essentiële precursor van belangrijke
celmembraancomponenten zoals fosfatidylcholine en sfyngomyeline. Door een upregulatie
van choline kinase en choline transporters, zal choline accumuleren in tumorcellen onder de
vorm van fosfocholine. Er bestaan al diverse PET-tracers voor prostaatkankerdetectie,
waaronder [11C]choline en [18F]fluorocholine. Door de korte halfwaardetijd van 11C (20 min.)
krijgt 18F-gelabeld choline de voorkeur boven [11C]choline. De hogere urine-excretie die
voorkomt bij [18F]fluorocholine ten gevolge van een onvolledige tubulaire reabsorptie of een
verhoogde excretie van geoxideerde metabolieten (betaïne), bemoeilijkt echter visualisatie
in de regio van de prostaat. Aangezien DMAE van hetzelfde transportsysteem gebruik maakt
als choline, maar minder onderhevig is aan oxidatie door het enzym choline oxidase, lijkt
DMAE dan ook een radiotracer met meer gunstige eigenschappen te zijn.
Methoden: De synthese van [18F]FDMAE werd op 2 manieren benaderd. Eerst werd de
reactiviteit geëvalueerd van N-beschermd methylaminoethanol met [18F]FCH2Br op een SPE
cartridge en in een verwarmde reactievial (80°C). De beschermgroepen die hier werden
aangewend waren een trityl-, benzyl-, en carboxybenzylgroep. Daaropvolgende opzuivering
over een WCX en deprotectie via een katalytische transfer hydrogenolyse zouden uiteindelijk
moeten leiden tot het gewenste [18F]FDMAE. In een tweede, minder efficiënte methode
werd enkel methylaminoethanol gereageerd met [18F]FCH2Br op een SPE cartridge en in de
verwarmde reactievial.
Resultaten: TrMAE en CbzMAE waren noch op de SPE cartridge, noch in de reactievial in
staat om te reageren met [18F]FCH2Br. BzMAE daarentegen gaf enkel na radiosynthese in de
reactievial bij 80°C aanleiding tot [18F]FCH2-BzMAE+. Opzuivering over een WCX leverde het
zuiver radioactief eindproduct op met een rendement van 69% ± 15%. Na deprotectie van de
benzylgroep via hydrogenolyse werd MAE bekomen in plaats van [18F]FDMAE. In de tweede
methode wordt [18F]FDMAE mogelijks gevormd in de reactievial na 15 min., maar bij verdere
reactie daalt het aandeel [18F]FDMAE terug.
Conclusie: Verder onderzoek is vereist om een efficiënte synthesemethode voor [18F]FDMAE
te ontwikkelen.
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