18f]fluoro-n,n-dimethylaminoethanol as...

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


4.2.1.4. 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


4.3.3.2. Radiosynthesis in a Heated Reaction Vial ......................................................... 26

4.4. METHYLAMINOETHANOL ........................................................................................... 26


4.4.2. Radiosynthesis of [18F]FDMAE .............................................................................. 26



5. RESULTS ............................................................................................................................. 27

5.1. TRITYLMETHYLAMINOETHANOL ................................................................................. 27

5.1.1. Synthesis and Purification ..................................................................................... 27


5.1.3. Radiosynthesis of [18F]FCH2 -TrMAE+ .................................................................... 31



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


5.4.2. 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).

http://www.heartandmetabolism.org/issues/hm34/hm34refresherc.asp

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

http://movies-tatecalebzane.blogspot.com/2011/03/glycolysis-and-gluconeogenesis-concept.html


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).

http://en.wikipedia.org/wiki/Positron_emission

http://www.learner.org/courses/physics/unit/pdfs/unit2.pdf

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.


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


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


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


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.


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


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+


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


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


[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


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


[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

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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.

18f]fluoro-n,n-dimethylaminoethanol as...

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