Stereoselective radiosynthesis of L- and D-3-[18F]fluoro-α-methyltyrosine

Article abstract of DOI:10.1016/j.jfluchem.2015.07.015

A three-step radiosynthesis is described which allows for the first time the preparation of L- and D-3-[¹⁸F]fluoro-α-methyltyrosine starting from [¹⁸F]fluoride. Corresponding 3-fluoro-4-formyl-benzylated Sch?llkopf derivatives were <

Full text of DOI:10.1016/j.jfluchem.2015.07.015

Accepted Manuscript
Title: Stereoselective radiosynthesis of L- and
D-3-[18F]fluoro-␣-methyltyrosine
´
Author: Johnny Castillo Melean Sven Humpert Johannes
Ermert Heinz H. Coenen
PII:
S0022-1139(15)00215-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jfluchem.2015.07.015
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9-7-2015
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Stereoselective radiosynthesis of L- and D-3-[18F]fluoro-α-methyltyrosine
Johnny Castillo Meleán, Sven Humpert, Johannes Ermert, Heinz H. Coenen
Institut für Neurowissenschaften und Medizin, INM-5: Nuklearchemie, Forschungszentrum
Jülich, 52425 Jülich, Germany
Keywords: radiofluorination, fluoroaromatic amino acids, Bayer-Villiger oxidation, isotopic
exchange, fluorine-18
Abstract
A three-step radiosynthesis is described which allows for the first time the preparation of L-
and D-3-[18F]fluoro-α-methyltyrosine starting from [18F]fluoride. Corresponding 3-fluoro-4-
formyl-benzylated Schöllkopf derivatives were 18F-fluorinated as precursors by isotopic
exchange, followed by Baeyer-Villiger oxidation and subsequent hydrolysis of protecting
groups in acidic medium. The three-step, two-pot radiosynthesis provided each enantiomer of
3-[18F]fluoro-α-methyltyrosine with an overall radiochemical yield of 32 ± 8 % and an
enantiomeric excess ≥ 95 % within 140 min in carrier-added form with a molar activity of 20
GBq/mmol.
Highlights
A nucleophilic synthesis of L‐ and D‐3‐[18F]fluoro‐α‐methyltyrosine is described for the first time.
This was achieved by a three‐step, two‐pot procedure.
Each enantiomer is obtained in an overall RCY of 32 ± 8 % and an ee of ≥ 95 %.
1
Page 1 of 22

Graphical Abstract
O
O
OH
NH2.HCl
N
N
HO
18F
L-[18F]FAMT
O
F
O
Graphical Abstract
A three‐step radiosynthesis of 3‐[18F]fluoro‐α‐methyltyrosine provided both enantiomers with 32 ±8
% radiochemical yield and an enantiomeric purity of ≥ 95 % within 140 min.
Introduction
L-3-[18F]Fluoro-α-methyltyrosine (L-[18F]FAMT) is a very promising radiotracer for tumor
detection with positron emission tomography (PET), a non-invasive imaging technique. L-
[18F]FAMT accumulates in areas with high metabolic activity but shows low accumulation in
tissues with high glucose metabolism, allowing an accurate diagnosis of malignancy in the
brain [1]. In contrast to other labeled amino acids, L-[18F]FAMT has also been discussed as a
potentially useful tracer for depiction of peripheral tumors and even of metastatic lesions in
the cardiac region [2]. Distinct from other amino acid PET tracers, it is selective to the L-type
2
Page 2 of 22

amino acid transporter 1 (LAT1) due to its α-methyl group. This feature is proposed to
contribute to its highly specific tumor accumulation [3]. In human lung cancer, L-[18F]FAMT
uptake was closely correlated with LAT1, glycoprotein CD98, cell proliferation and
angiogenesis [4]. The usefulness of L-[18F]FAMT in PET imaging combined with computed
tomography (CT) for the detection of bone metastases, regardless of the bone lesion
phenotype, has also been demonstrated [5]. Moreover, it has been suggested that this tracer
may also be a suitable replacement for [18F]FDG in assessing malignant lymph nodes of oral
cancer, especially non-tongue cancer [6].
The enantiomeric tracer, D-[18F]FAMT, has also been pharmacologically evaluated. It is
stable both in vitro and in vivo, and it is also transported into the tumor via LAT1 [7]. In PET-
studies on mouse models it showed rapid clearance from the blood and reduced radioactivity
in non-target organs. Compared to L-[18F]FAMT, D-[18F]FAMT thus provides a higher
tumor-to-background contrast and lower exposure dose to patients, suggesting that it could
potentially serve as an even better tracer for imaging of malignant tumors [7].
So far, [18F]FAMT has only been synthesized by electrophilic radiofluorination of α-
methyltyrosine employing either [18F]AcOF in trifluoroacetic acid [8] or [18F]F2 in anhydrous
HF [9] which limits a broader use of this tracer. Nucleophilic radiofluorination has some
principal advantages over electrophilic methods which are especially restricted by limited
amounts of activity [10, 11]. Nucleophilic preparation of 18F-labelled aromatic amino acids, as
mainly demonstrated for the synthesis of 6-[18F]fluoro-L-DOPA, can be performed by build-
up synthesis, using a multi-step phase-transfer catalyst strategy [12] which was recently
modified and adapted to an automatable process [13]. However, this modification necessitates
a remote-controlled synthesizer with two reactors. Very recently, several new strategies for
the nucleophilic synthesis of 6-[18F]fluoro-L-DOPA have been developed [14]. Also, during
18
the progress of our study, methods using a copper-catalyzed nucleophilic F-fluorination of
mesityl diaryliodonium precursors [15] or of boronic esters derivatives [16] opened new,
3
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more direct ways for the syntheses of 18F-labelled aromatic amino acids. However, an
automated synthesis procedure of those methods still requires further optimization [17, 18]. A
method known for longer, which has the advantage of being easier to automate, is based on
nucleophilic substitution with [18F]fluoride on carbonyl-activated precursors, producing
aromatic amino acids in a three-step, two-pot procedure. For this, following reaction sequence
was optimized: isotopic exchange, Baeyer-Villiger oxidation or decarbonylation reaction, and
hydrolysis, as exemplified with 6-[18F]fluoro-L-DOPA [19], 2-[18F]fluoro-L-phenylalanine, 2-
[18F]fluoro-L-tyrosine [20] and 6-[18F]fluoro-meta-L-tyrosine [21]. The aim of this work was
to adapt this three step radiosynthetic approach, which particularly has the advantage of easy
automation, to the preparation of L- and D-[18F]FAMT, starting from [18F]fluoride.
Results and discussion
Synthesis of the standards
In earlier studies non-radioactive standards of FAMT were prepared through direct
electrophilic fluorination of α-methyl tyrosine leading to a mixture of position isomers that
are difficult to separate [8]. Here, L- and D-FAMT were prepared by a five-step linear
synthesis according to Scheme 1. Benzylation of commercially available 3-fluoro-4-
hydroxybenzoic acid 1 under basic conditions produced compound 2 in 91 % yield. Reduction
of the ester led to benzyl alcohol 3 in 93 % yield. The benzyl bromide 4 was prepared by
means of an Appel reaction [22] with 89 % yield. Then, the benzyl bromide derivative 4 was
reacted with [(5R)-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-2,5-dihydropyrazin-2-yl]lithium
(Li-Me-(R)-Schöllkopf) [23], furnishing the adduct 5a with 60 % yield. Finally, 5a was
hydrolyzed with HCl at high temperature using a pressure tube. The crude product was
purified by high-performance-liquid-chromatography (HPLC) giving L-FAMT with 75 %
yield, rendering an overall synthesis yield of 34 %. The D-FAMT standard was prepared
4
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using the same synthetic pathway, but with the Li-Me-(S)-Schöllkopf auxiliary instead with
an overall yield of 32 %. An enantiomeric excess of ≥ 99 % was confirmed by chiral HPLC.
O
O
OH
OH
OBn
a
b
c
BnO
HO
BnO
F
F
F
2
3
1
O
O
O
Br
d
N
OH
NH3Cl
e
N
BnO
BnO
HO
F
F
F
4
L-FAMT (6a)
5a
Scheme 1. Synthesis of L-3-fluoro-α-methyltyrosine (L-FAMT).
a) BnBr, Na2CO3, acetone, reflux; b) LiAlH4, THF; c) CBr4, PPh3, CH2Cl2, 0 °C; d) Li-Me-
Schöllkopf, THF, -78 °C – room temperature (r.t.).; e) HCl, 160 °C.
Synthesis of the labelling precursor
The diastereoselective derivatives of 4-([-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-2,5-
dihydropyrazin-2-yl]methyl)-2-fluorobenzaldehyde were synthesized in a 3-step synthesis
starting from 3-fluoro-4-iodotoluene as delineated in Scheme 2. The latter was at first
converted to the benzyl bromide derivative 8 using a Wohl-Ziegler reaction [24] with 86 %
yield. The next step was the substitution of the benzyl bromide 8 with the lithium enolate of
the chiral auxiliary Me-(R)Schöllkopf. This reaction furnished compound 9a with 60 % yield.
Finally, compound 9a was formylated using a halogen-metal exchange, yielding the
enantiomerically pure labeling precursor 10a with 58 % yield. The overall yield for the
synthesis of the precursor was 31 %. Following an analogous procedure, but using the Me-
5
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(S)-Schöllkopf derivative instead, the enantiomeric precursor 10b was obtained with an
overall yield of 30 %.
Br
a
b
I
I
F
F
7
8
O
O
O
O
N
N
c
N
N
I
O
F
F
9a
10a
Scheme 2. Synthesis of the precursor for L-[18F]FAMT. Conditions: a) N-bromosuccinimide,
benzoylperoxide, CCl4, reflux; b) Li-Me-(R)-Schöllkopf, THF, -78 °C – r.t.; c) 1. i-PrMgBr,
THF, 0 °C; 2. DMF, r.t.
Radiosynthesis
As mentioned in the introduction, the corresponding precursors 10a and 10b were 18F-
fluorinated by nucleophilic isotopic exchange, followed by the conversion of the activating
aldehyde into a formic acid arylester ([18F]11a) by Baeyer-Villiger oxidation (BVO). This
resulting formate was then cleaved simultaneously with the Schöllkopf auxiliary by
hydrolysis in concentrated HCl (Scheme 3).
O
O
O
O
O
O
O
a)
b)
c)
N
N
N
OH
NH2.HCl
N
N
N
O
HO
18F
18F
O
F
18F
L-[18F]FAMT
O
O
Scheme 3. Radiosynthesis of L-[18F]FAMT.
6
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a) TBA18F, DMF, 120 °C, 7 min, RCY: 64 ± 6 %; b) peracetic acid, CF3CH2OH, H2SO4, 80
°C, 30 min, RCY: 63 ± 2 %; c) HCl, 160 °C, 30 min, RCY: quantitative; overall RCY: 32 ±
8 %.
In the following, a given radiochemical yield (RCY) is related to each single reaction step
18
including its work-up procedure. The purity of the reaction product upon F-exchange was
confirmed by TLC and of that after oxidation and quantitative hydrolysis by radio-HPLC. The
RCY over all three reaction steps is denoted as ‘overall RCY’.
Previous studies have shown that DMF is the solvent of choice for the isotopic exchange
reaction for this class of precursors. The results of kinetic examinations showed that the
maximum RCY was reached after 5 minutes and it remained almost constant until about 10
minutes. Thereafter, the RCY decreased and several side products were detected by radio-
TLC. The reaction time for further experiments was therefore set at 7 min. Two anion
activator systems were compared: The traditional Kryptofix®2.2.2./K2CO3 and
tetrabutylammonium bicarbonate. The results obtained with the two systems are depicted in
Figure 1.
7
Page 7 of 22

Figure 1. Influence of anion activator and reaction temperature on isotopic exchange;
conditions: 15 µmol 10, TBAHCO3 (16.5 µmol), or Kryptofix©2.2.2 (26 μmol) and 1 M
potassium carbonate solution (13 μL), 1 mL DMF, 7 min reaction time (n = 3).
The best radiochemical yield of 64 ± 6 % was achieved with TBAHCO3 at 120 °C. It was also
observed that the RCY decreased at lower as well as at higher temperatures: the RCY
detriment at higher temperatures being a consequence of the formation of multiple unknown
side products. The α-methyl group of [18F]FAMT prevented an epimerization of the precursor
during the labeling process. Thus, in contrast to the synthesis of 2-[18F]fluoro-L-tyrosine [20]
and 6-[18F]fluoro-meta-L-tyrosine [21] by isotopic exchange, harsher conditions are
applicable for the isotopic exchange reaction.
The subsequent Baeyer-Villiger reaction was carried out using 2,2,2-trifluoroethanol and
sulfuric acid as adequate solvent and additive, respectively, for the oxidation of non-activated
[18F]fluorobenzaldehydes as recently reported [25]. A good RCY of 63 ± 2 % was achieved
using m-chloroperoxybenzoic acid (m-CPBA) as oxidant. However, the reactions employing
m-CPBA gave a solid residue after hydrolysis, which can be a problem for the foreseen
automation process. Further experiments proved peracetic acid as good replacement since it
did not form solid by-products and delivered still a good RCY of 57 ± 6 %. Oxone® was also
explored as oxidant; however, this proved to be a non-reliable alternative since the RCY of
the reaction varied between 27 and 70 %, with an average value of 43 ± 16 %.
The cleavage of the auxiliary was performed using aqueous concentrated HCl. A high
temperature of 160 °C was needed in order to complete the reaction within a reasonable time
of 30 min quantitatively. Reaction times and temperatures lower than 30 min and 160 °C,
respectively, led to significantly lower yields.
8
Page 8 of 22

After hydrolysis, the crude product was purified by semi-preparative HPLC on a C-18 column
with embedded polar groups allowing the use of sodium phosphate buffer with 2 % ethanol as
eluent. [18F]FAMT was obtained in an about 8 mL volume and can directly be used for
injection after filtering through a 0.2 µm Millipore filter.
First analyses of the purity revealed only an enantiomeric excess of > 90 % of the final
products. It is known from literature that coupling of the benzyl bromide with the
Schöllkopf reagent (cf. Scheme 2) generally leads to a diastereomeric excess of 95% or
more [26, 27]. A further analysis by HPLC was therefore done and it revealed an impurity
which turned out to be the corresponding diastereomer. This was obviously not separated
under the chromatographic conditions chosen before and also not found in the NMR
spectra. Therefore, a preparative HPLC purification of precursor 10 was performed to
improve the enantiomeric excess of [18F]FAMT. In fact, after this purification an
enantiomeric excess ≥ 95 % of FAMT was achieved which is in the acceptable range for
clinical application.
The synthesis was completed within 140 min and the overall radiochemical yield obtained for
both L-[18F]FAMT or D-[18F]FAMT was 32 ± 8 % (n = 3), each with an enantiomeric excess
≥ 95 %. The whole synthetic sequence was performed and optimized manually with starting
activities of ≤ 300 MBq, yielding a molar activity of ≤ 20 GBq/mmol.
The specific activity obtained by isotopic exchange reactions is a function of the degree of
substitution and of both the amount of radioactivity of [18F]fluoride and that of the precursor
[20]. When starting with high amounts of [18F]fluoride activity of > 20 GBq, which are easily
produced, a much higher specific activity can be expected than that obtainable with
electrophilic 18F-fluorination.
Summary
9
Page 9 of 22

The procedure developed here constitutes an efficient and amenable alternative to previous
syntheses of L-[18F]FAMT and D-[18F]FAMT carried out by electrophilic 18F-fluorination
which are limited to low amounts of activity. The method presented here is based on a
nucleophilic exchange reaction using the advantage of the large scale production of
[18F]fluoride and offers an easy automation. The best radiochemical yield of 64 ± 6 % of the
isotopic exchange was achieved using TBAHCO3 at 120 °C. The combined RCY of the BVO
and hydrolysis reactions was 57 ± 6 %. Thus, the overall RCY for the three-step synthesis
amounted to 32 ± 8 % which was achieved within 140 minutes of total synthesis time with a
>95 % e.e. of the respective enantiomer.
Experimental
General
Dry solvents and chemicals were purchased from Aldrich and Merck, Germany. All were
used without further purification.
1H, 13C and 19F NMR spectra were recorded on a Varian Inova 600 MHz spectrometer
using CDCl3 or d6-DMSO as solvents. All chemical shifts given below are in δ ppm using
the signals of the appropriate solvent as a reference. HRMS spectra were obtained on an
FTICR ‘LTQ FT Ultra’ device (Thermo Fisher Scientific, Germany). Melting points are
uncorrected and were determined on a Mettler FP-61 apparatus in open capillaries.
Elemental analyses were performed on a Leco CHNS-932 CHNS analyser (Central
Institute for Engineering, Electronics and Analytics (ZEA), Forschungszentrum Jülich).
Flash chromatography was performed either on Macherey-Nagel (Germany) 60 M (0.04-
0.06 mm) silica gel or using a Grace Reveleris® flash chromatography system with ELSD
and UV detection on 40 µm flash-cartridges (Grace, Belgium). Thin layer chromatography
(TLC) was performed on precoated plates of silica gel 60 F254 (Merck, Darmstadt,
Germany) and the compounds were detected at 254 nm. Radioactivity on radio-TLC was
10
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measured by a Raytest minigita device (Raytest, Germany). High-performance liquid
chromatography (HPLC) separations were performed with a Knauer 1000 pump (Knauer,
Germany), a Knauer Smartline 2520 UV/VIS detector, a manual Rheodyne injector (50-
µL or 5.0-mL loop), and a NaI(Tl) well-type scintillation detector (EG&G Ortec (Ortek
AMETEK, Germany); model 276 Photomultiplier Base) with an ACE Mate Amplifier and
BIAS supply (Ortec AMETEK, Germany) for radioactivity detection. Data acquisition and
interpretation were performed with Gina software (Raytest, Germany).
Chromatographic systems
System A: Analytical HPLC of [18F]FAMT was performed with an analytical reverse-
phase Synergi 4µm Hydro-RP 80 Å column (250 x 4.6 mm; Phenomenex, Germany). The
mobile phase was water with 2 % ethanol and 0.1 % acetic acid, used at a flow rate of 1
mL/min.
System B: Preparative HPLC of [18F]FAMT was carried out with a Synergi 4µm Hydro-
RP 80 Å column (250 x 10 mm; Phenomenex, Germany). The mobile phase was prepared
by adding 20 mL of sodium phophate buffer (B.Braun, Germany) and 2 % ethanol into 1 L
of sterile water, and the flow rate was 4 mL/min.
System C: The enantiomeric purity of [18F]FAMT was determined by HPLC using a
Chirex 3126 (D)-penicillamine column (150 x 4.6 mm; Phenomenex, Germany) and 3 mM
CuSO4 in water and 2-propanol (95:5 v/v) as eluent at a flow rate of 1 mL/min.
System D: Preparative HPLC of 6a and 6b were carried out with a Synergi Hydro RP,
(250 x 10 mm, Phenomenex, Germany) with 3 % EtOH + 0.1% AcOH in H2O as eluent at
a flow rate of 4.7 mL/min.
System E: Preparative HPLC of 10a and 10b were carried out with a Gemini 5µ 18C 110
Å (250 x 10 mm, Phenomenex, Germany) and water and acetonitrile (40:60 v/v) as eluent
at a flow rate of 4.7 mL/min.
11
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Table 1.
k'-values of the final compounds analyzed by radio-HPLC.
Compound
HPLC System
k’
[18F]6
[18F]6a
[18F]6b
A
C
C
3.4
3.1
4.5
Syntheses of standards
Benzyl-4-(benzyloxy)-3-fluorobenzoate (2)
Benzyl bromide (2.24 g, 13.12 mmol) and potassium carbonate (3.30 g) were added to a
solution of 3-fluoro-4-hydroxybenzoic acid (0.93 g, 5.96 mmol) in 50 mL acetone. The
mixture was refluxed overnight. Thereafter, the reaction mixture was cooled to room
temperature (r.t.), the potassium carbonate filtered and the solid residue washed with acetone
(3 x 15 mL). The solvent was removed in vacuo and the crude product purified by flash
chromatography (AcOEt/petroleum ether (PE) 5:95) giving 1.34 g (67%) of 2 as a white solid.
1
Rf = 0.24 (AcOEt/PE 5:95). mp = 75 °C. H NMR (600 MHz, CDCl3): δ 7.81 (m, 2H), 7.44
(d, J= 7.3 Hz, 4H), 7.36 (m, 2H), 7.02 (t, J= 8.1 Hz, 1H), 5.34 (s, 2H), 5.20 (s, 2H); 13C NMR
(150 MHz, CDCl3): δ 165.3, 152.9 (J= 247 Hz), 150.9 (J= 10.1 Hz), 136.0, 135.8, 128.9,
128.7, 128.5 (J= 9.8 Hz), 127.9 (J= 120.5 Hz), 126.7 (J= 3.3 Hz), 123.4, 117.7 (J= 19.8 Hz),
114.3, 71.2, 66.9; 19F (564 MHz, CDCl3): δ -133.1 (t, J=9.5 Hz). Anal. calcd for C21H17FO3
+
C, 74.99; H, 5.09; found: C, 74.77; H, 5.04; HRMS (ESI), m/z: calcd for C21H18FO3
337.1234, [M+H]+ found 337.1235.
4-Benzyloxy-3-fluorobenzyl alcohol (3)
Lithium aluminum hydride (1.0 M in THF, 4.0 mL, 4 mmol) was added dropwise to a solution
of compound 2 (1.3 g, 3.98 mmol) in 60 mL of THF at r.t. and the mixture stirred during 60
min. The mixture was quenched with 60 mL of water and acidified with H2SO4 until the solid
12
Page 12 of 22

was dissolved. The mixture was extracted with ether (3 x 25 mL), the organic phase washed
with brine (2 x 30 mL), separated and dried over Na2SO4. The solvent was removed and the
crude product purified by flash chromatography (AcOEt/PE 25:75) yielding product 3 (0.76 g,
1
82%) as a white solid. Rf = 0.21 (AcOEt/PE 20:80). mp = 56 °C. H NMR (600 MHz,
CDCl3): δ 7.44 (d, J= 7.4 Hz, 2H), 7.38 (t, J= 7.3 Hz, 2H), 7.32 (t, J= 7.3 Hz, 1H), 7.12 (d, J=
13
11.8 Hz, 1H), 6.98 (m, 2H), 5.14 (s, 2H), 4.58 (s, 2H), 1.79 (s, 1H); C NMR (150 MHz,
CDCl3): δ 153.0 (J= 246 Hz), 146.2 (J= 10.7 Hz), 136.6, 134.7 (J= 6.0 Hz), 128.7, 128.2,
19
127.5, 122.8 (J= 3.6), 115.8, 115.3 (J= 19.0), 71.6, 64.5; F (564 MHz, CDCl3): δ -133.3 (t,
J= 9.5 Hz). Anal. calcd for C14H13FO2 C, 72.40; H, 5.64; found C, 72.78; H, 5.70; HRMS
(ESI), m/z: calcd for C14H13FO2Na+ 255.0797 [M+Na]+, found 255.0793.
4-Benzyloxy-3-fluorobenzylbromide (4)
A stirred solution of alcohol 3 (0.73 g, 3.14 mmol) and 1.15 g (3.46 mmol) of
carbontetrabromide in 10 mL of dichloromethane was cooled to 0 °C and triphenylphosphine
(1.23 g, 4.71 mmol) was added. Then the mixture was stirred for further 10 min, whereupon
5 g of silica gel were added and the solvent was removed in vacuo at room temperature. The
adsorbed compound was purified by flash chromatography (AcOEt/PE 5:95) giving 0.89 g
(96%) of the bromide as a white solid.
1
Rf = 0.50 (AcOEt/PE 5:95). mp = 82 °C. H NMR (600 MHz, CDCl3) δ 7.44 (d, J= 7.6 Hz,
2H), 7.39 (t, J= 7.3 Hz, 2H), 7.34 (t, J= 7.3 Hz, 1H), 7.15 (d, J= 11.3 Hz, 1H), 7.06 (d, J= 8.3
13
Hz, 1H), 6.94 (t, J= 8.3 Hz, 1H), 5.15 (s, 2H), 4.44 (s, 2H); C NMR (150 MHz, CDCl3) δ
152.6 (J= 247.4 Hz), 147.0 (J= 10.8 Hz), 136.4, 131.2 (J= 6.5 Hz), 128.7, 128.3, 127.5, 125.0
(J= 3.3 Hz), 117.2 (J= 19.0 Hz), 115.6, 71.4, 32.9; 19F (564 MHz, CDCl3) δ – 132.7 (t, J= 9.8
Hz). Anal. calcd for C14H12BrFO C, 56.97; H, 4.10; found C, 57.06; H: 4.09.
13
Page 13 of 22

(2S,5R)-2-{[4-(Benzyloxy)-3-fluorophenyl]methyl}-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-
2,5-dihydropyrazine (5a)
A stirred solution of methyl-(R)-Schöllkopf (0.49 g, 2.47 mmol) in dry THF (2 mL) was
cooled down to -78 °C, then n-BuLi (2.5 M/hexane, 1.09 mL, 2.72 mmol) was added
dropwise. The resulting solution was stirred for 30 min. Benzyl bromide 4 (0.73 g, 2.47
mmol) in THF (2 mL) was added, the reaction mixture was allowed to warm to room
temperature and stirring was continued for 3 h. The reaction was quenched with saturated
aqueous ammonium chloride (10 mL) and 30 mL of water were added. Then the reaction
mixture was extracted with ether (3 x 25 mL), the organic layer was dried over Na2SO4 and
evaporated, giving the crude product which was purified by flash chromatography (AcOEt/PE
2:98) (0.63 g, 62%, yellowish oil).
Rf = 0.41 (AcOEt/PE 5:95). 1H NMR (600 MHz, CDCl3) δ 7.42 (d, J= 7.3 Hz, 2H), 7.37 (t, J=
7.3 Hz, 2H), 7.31 (t, J= 6.9 Hz, 1H), 6.79 (m, 2H), 6.67 (d, J= 8.1 Hz, 1H), 5.08 (s, 2H), 3.68
(s, 3H), 3.67 (s, 3H), 3.32 (d, J= 3.1 Hz, 1H), 3.05 (d, J= 13.0 Hz, 1H), 2.68 (d, J= 13.0 Hz,
1H), 2.13 (m, 1H), 1.44 (s, 3H), 0.96 (d, J= 6.9 Hz, 3H), 0.61 (d, 6.7Hz, 3H); 13C NMR (150
MHz, CDCl3) δ 164.1, 162.3, 152.3 (J= 244 Hz), 145.3 (J= 10.9 Hz), 137.0, 131.7 (J= 6.0
Hz), 128.8, 128.3, 127.8, 125.6 (J=2.9 Hz), 118.1 (J= 18.3 Hz), 115.3, 71.7, 60.9, 59.9, 52.4,
52.2, 46.6, 30.9, 28.7, 19.5, 16.9; 19F (564 MHz, CDCl3) δ -134.9 (t, J= 10.4 Hz); HRMS
(ESI), m/z: calcd for C24H30FN2O3+ 413.2235 [M+H]+; found 413.2234.
(2R,5S)-2-{[4-(Benzyloxy)-3-fluorophenyl]methyl}-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-
2,5-dihydropyrazine (5b)
Compound 5b was prepared following the procedure described for compound 5a, but using
Me-(S)-Schöllkopf instead. Yield was 80 %.
1H NMR (600 MHz, CDCl3) δ 7.41 (d, J= 7.3 Hz, 2H), 7.36 (t, J= 7.3 Hz, 2H), 7.31 (t, J= 7,1
Hz, 1H), 6.79 (m, 2H), 6.67 (d, J= 8.3 Hz, 1H), 5.07 (s, 2H), 3.68 (s, 3H), 3.67 (s, 3H), 3.31
14
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(d, J= 3.1 Hz, 1H), 3.05 (d, J= 12.8 Hz, 1H), 2.69 (d, J= 12.8 Hz, 1H), 2.13 (m, 1H), 1.44 (s,
13
3H), 0.96 (d, J= 6.8 Hz, 3H), 0.60 (d, 6.8 Hz, 3H); C NMR (150 MHz, CDCl3) δ 164.1,
162.3, 152.3 (J= 244 Hz), 145.3 (J= 10.9 Hz), 137.0, 131.7 (J= 6.0 Hz), 128.8, 128.3, 127.8,
125.6 (J= 2.9 Hz), 118.1 (J= 18.3 Hz), 115.3, 71.7, 60.9, 59.9, 52.4, 52.2, 46.6, 30.9, 28.7,
19
19.5, 16.9; F (564 MHz, CDCl3) δ -134.9 (t, J= 10.4 Hz); Anal. calcd for C24H29FN2O C,
69.88; H, 7.09; N, 6.79; found: C, 69.88; H, 7.09; N, 6.52; HRMS (ESI), m/z: calcd for
C24H30FN2O3+ 413.2235 [M+H]+; found 413.2235.
(2S)-2-Amino-3-(3-fluoro-4-hydroxyphenyl)-2-methylpropanoic acid (L-3-fluoro-α-methyl-
tyrosine (6a)
Compound 5a (0.22 g) was reacted with 2 mL HCl (32 %) at 160 °C during 30 min in a
pressure tube. The mixture was then cooled to room temperature and the solid residues
filtered through a PTFE-frit. The acid solution was diluted with 3 mL EtOH:H2O (1:1) and
purified by HPLC (System D), giving 0.10 g (75%) of 6a as a white solid. The 1H NMR data
were in agreement with the previously reported ones [7].
(2R)-2-Amino-3-(3-fluoro-4-hydroxyphenyl)-2-methylpropanoic acid (D-3-fluoro-α-methyl-
tyrosine (6b)
1
Compound 6b was prepared following the procedure described for compound 6a. The H
NMR data were in agreement with the previously reported ones [7].
Syntheses of the precursors
3-Fluoro-4-iodobenzylbromide (8)
N-Bromosuccinimide (2.63 g, 14.78 mmol) and benzoylperoxide (0.20 g, 0.62 mmol) were
added to a solution of 3-fluoro-4-iodotoluene (2.90 g, 12.29 mmol) in 7 mL of
tetrachloromethane. The resulting solution was refluxed for 20 h. Thereafter, the reaction was
allowed to cool to room temperature and was then diluted with 30 mL of CH2Cl2. The solid
15
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phase was filtered off and the filtrate was washed with 30 mL of a saturated solution of
sodium thiosulphate. The organic layer was dried over Na2SO4 and evaporated. The crude
product was purified by flash chromatography (AcOEt/PE 2:98), giving 3.45 g (86 %) of 8 as
a white solid.
1
Rf = 0.76 (AcOEt/PE 5:95); mp = 46-48 °C. H NMR (600 MHz, CDCl3) δ 7.71 (m, 1H),
13
7.11 (m, 1H), 6.94 (m, 1H), 4.41 (s, 2H); C NMR (150 MHz, CDCl3) δ 161.0 (J= 246.5
Hz), 140.6 (J= 7.6 Hz), 139.9 (J= 1.3 Hz), 126.4 (J= 3.2 Hz), 116.4 (J= 24.5 Hz), 81.3 (J=
19
25.6 Hz), 31.5; F (564 MHz, CDCl3) δ -92.9 (t, J= 7.3 Hz). Anal. calcd for C7H5BrFI: C,
26.70; H, 1.60; found: C, 26.70; H, 1.58.
(2S,5R)-2-[(3-Fluoro-4-iodophenyl)methyl]-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-2,5-
dihydropyrazine (9a)
Compound 9a was prepared following the procedure described for compound 5a and isolated
by flash chromatography (AcOEt/PE 3:97). Yield was 62%; the product was a yellowish oil.
1
Rf = 0.29 (AcOEt/PE 1:99); H NMR (600 MHz, CDCl3) δ 7.53 (dd, J= 8.0 Hz, J= 6.7 Hz,
1H), 6.76 (dd, J= 9.2 Hz, J= 1.9 Hz, 1H) 6.58 (dd, J= 8.0 Hz, J= 1.9 Hz, 1H), 3.68 (s, 3H),
3.67 (s, 3H), 3.37 (d, J= 3.3 Hz, 1H), 3.08 (d, J= 12.8 Hz, 1H), 2.73 (d, J= 12.8 Hz, 1H), 2.13
13
(m, 1H), 1.45 (s, 3H), 0.96 (d, J= 7.3 Hz, 3H), 0.60 (d, 7.3 Hz, 3H); C NMR (150 MHz,
CDCl3) δ 163.7, 162.4, 161.2 (J= 245.2 Hz), 140.9 (d, J= 6.9 Hz), 138.4, 127.6 (J= 3.2 Hz),
117.4 (J= 23.7 Hz), 78.6 (J= 25.4 Hz), 60.8, 59.6, 52.3, 52.1, 46.7, 30.8, 28.8, 19.3, 16.8; 19F
(564 MHz, CDCl3) δ -95.4 (t, J= 7.9 Hz); HRMS (ESI), m/z: calcd. for C17H22FIN2O2H+
433.0783 [M+H]+; found 433.0785.
(2R,5S)-2-[(3-Fluoro-4-iodophenyl)methyl]-3,6-dimethoxy-2-methyl-5-(propan-2-yl)-2,5-
dihydropyrazine (9b)
16
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Compound 9b was prepared following the procedure described for compound 5a but using
Me-(S)-Schöllkopf instead. The isolation was performed by flash chromatography (AcOEt/PE
3:97). Yield was 56 %, the product a yellowish oil.
Rf = 0.29 (AcOEt/PE 1:99); 1H NMR (600 MHz, CDCl3) δ 7.53 (dd, J= 8.3 Hz, J= 6.9 Hz,
1H), 6.76 (dd, J= 9.5 Hz, J=2.1 Hz, 1H) 6.58 (dd, J= 8.0 Hz, J= 2.0 Hz, 1H), 3.68 (s, 3H),
3.67 (s, 3H), 3.37 (d, J= 3.2 Hz, 1H), 3.09 (d, J= 12.8 Hz, 1H), 2.73 (d, J= 12.8 Hz, 1H), 2.13
(m, 1H), 1.45 (s, 3H), 0.96 (d, J= 7.3 Hz, 3H), 0.60 (d, 6.8 Hz, 3H); 13C NMR (150 MHz,
CDCl3) δ 163.6, 162.3, 161.0 (J= 244.5 Hz), 140.8 (J= 7.0 Hz), 138.3, 127.5 (J= 3.0 Hz),
117.3 (J= 24.2 Hz), 78.4 (J= 25.3 Hz), 60.7, 59.5, 52.2, 52.0, 46.6, 30.7, 28.7, 19.2, 16.7; 19F
(564 MHz, CDCl3) δ -95.4 (t, J= 8.1 Hz); HRMS (ESI), m/z: calcd. for C17H23FIN2O2+
433.0783 [M+H]+; found 433.0784.
4-{[(2S,5R)-3,6-Dimethoxy-2-methyl-5-(propan-2-yl)-2,5-dihydropyrazin-2-yl]methyl}-2-
fluorobenzaldehyde (10a)
A solution of compound 9a (0.65 g, 1.50 mmol) in THF (4 mL) was cooled to 0 °C.
Isopropylmagnesiumbromide (2.9 M in 2-methyl-THF, 1.05 mL, 3.05 mmol) was added
dropwise and the mixture was allowed to react for 1 h. Thereafter, N,N-dimethylformamide
(0.46 mL, 5.95 mmol) was added and the mixture was warmed to r.t. and stirred overnight.
The reaction was quenched with saturated aqueous NH4Cl, and extraction was done with ether
(3 x 25 mL). The organic phase was dried over Na2SO4 and evaporated under reduced
atmosphere. After purification by flash chromatography (AcOEt/PE 5:95) the desired product
10a was obtained as colorless oil (0.28 g, 56 %). To obtain higher diastereomeric purity the
product was finally purified by semi-preparative HPLC (System E).
Rf = 0.33 (AcOEt/PE 5:95); 1H NMR (600 MHz, CDCl3) δ 10.28 (s, 1H), 7.68 (t, J= 7.6 Hz,
1H), 6.94 (d, J= 8.0 Hz, 1H), 6.86 (d, J= 11.5 Hz, 1H), 3.68 (s, 6H), 3.36 (d, J= 2.7 Hz, 1H),
3.19 (d, J= 12.5 Hz, 1H), 2.84 (d, J= 12.5, 1H), 2.13 (m, 1H), 1.47 (s, 3H), 0.95 (d, J= 6.7 Hz,
17
Page 17 of 22

13
3H), 0.60 (d, J= 6.7 Hz, 3H); C NMR (150 MHz, CDCl3) δ 187.3 (J= 6.3 Hz) 164.2 (J=
258.1 Hz), 163.5, 162.6, 148.2 (J= 8.7 Hz), 128.0, 126.5 (J= 3.0 Hz), 122.5 (J= 8.1 Hz), 117.9
(J= 20.4 Hz), 60.8, 59.7, 52.4, 52.3, 47.4, 30.9, 29.0, 19.3, 16.8; 19F (564 MHz, CDCl3) δ -
123.0 (t, J=8.8 Hz); HRMS (ESI), m/z: calcd. for C18H24FN2O3+ 335.1765 [M+H]+; found
335.1765.
4-{[(2R,5S)-3,6-Dimethoxy-2-methyl-5-(propan-2-yl)-2,5-dihydropyrazin-2-yl]methyl}-2-
fluorobenzaldehyde (10b)
Compound 10b was prepared following the procedure described for compound 10a with a
yield of 59 % as colorless oil.
Rf = 0.33 (AcOEt/PE 5:95); 1H NMR (600 MHz, CDCl3) δ 10.28 (s, 1H), 7.68 (t, J= 7.6 Hz,
1H), 6.94 (d, J= 7.8 Hz, 1H), 6.86 (d, J= 11.6 Hz, 1H), 3.68 (s, 6H), 3.36 (d, J= 2.3 Hz, 1H),
3.19 (d, J= 12.6 Hz, 1H), 2.84 (d, J= 12.6, 1H), 2.13 (m, 1H), 1.47 (s, 3H), 0.95 (d, J= 6.7 Hz,
13
3H), 0.60 (d, J= 6.7 Hz, 3H); C NMR (150 MHz, CDCl3) δ 187.3 (J= 6.0 Hz) 164.2 (J=
257.9 Hz), 163.5, 162.6, 148.2 (J= 8.7 Hz), 127.9, 126.5 (J= 2.7 Hz), 122.5 (J= 8.0 Hz), 117.9
19
(J= 20.7 Hz), 60.8, 59.7, 52.4, 52.2, 47.4, 30.9, 29.0, 19.3, 16.8; F (564 MHz, CDCl3) δ -
123.0 (t, J= 8.8 Hz); Anal. calcd for C18H23FN2O3: C, 64.65, H, 6.93, N, 8.38, found C,
64.92, H, 7.12, N, 7.99; HRMS (ESI), m/z: calcd. for C18H24FN2O3+ 335.1765 [M+H]+;
found 335.1766.
Radiosynthesis
Preparation of [18F]fluoride
18
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18
N.c.a. [18F]fluoride was produced by the O(p,n)18F nuclear reaction by bombardment of an
isotopically enriched [18O]water target with 17 MeV protons at the JSW cyclotron BC 1710
(Forschungszentrum Jülich) [28]. The [18F]fluoride produced was diluted with 2 mL H2O. An
aliquot of the [18F]fluoride solution (200 – 300 MBq) was added to 150 µL (19.5 µmol) of an
aqueous 0.13 M tetrabutylammonium bicarbonate solution (TBAHCO3) [29]. The mixture
was diluted with 1.0 mL of dry acetonitrile and transferred by syringe into a reaction flask.
The solvent was evaporated under a stream of argon at 80 °C and 650 mbar. The azeotropic
evaporation was repeated twice with 1.0 mL of acetonitrile and afterwards the vial was
evacuated for 5 min at 20-30 mbar.
Radiosynthesis of L- and D-[18F]FAMT
A solution of 15 µmol of the corresponding precursor (10a or 10b) in 1.0 mL of DMF was
added to the dried residue of TBA18F. The mixture was heated at 120 ºC for 7 min. After
labeling, the DMF solution was diluted with 10 mL of water, and passed through a pre-
conditioned Waters C-18 cartridge. Then, the product was eluted from the cartridge with 6.0
mL of ether and the eluate was dried using a column packed with Na2SO4. Subsequently, the
solvent was evaporated in vacuum and a solution of the oxidant (6 equivalents, e.g. 90 µmol
of m-CPBA) in 1 mL of 2,2,2-trifluoroethanol and 10 µL of H2SO4(conc.) were added and the
mixture heated to 80 ºC. After 30 min the reaction was quenched with a solution of ascorbic
acid (10 mg in 50 µL H2O and 300 µL acetonitrile) and the mixture evaporated to dryness at
the same temperature. 1 mL of hydrochloric acid (32 %) was added to the residue and the
solution was heated at 160 ºC for 30 min. The mixture was then cooled to r.t., diluted with
water and the product purified by HLPC (System B).
Acknowledgements
19
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The authors wish to thank Drs. Dirk Bier and Marcus Holschbach (INM-5) and Dr. Sabine
Willbold (ZEA-3), all Forschungszentrum Jülich, for recording the spectroscopic data.
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