No carrier added synthesis of O-(2′-[18F]fluoroethyl)-l-tyrosine via a novel type of chiral enantiomerically pure precursor, NiII complex of a (S)-tyrosine Schiff base

Article abstract of DOI:10.1016/j.bmc.2008.03.040

O-(2′-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) has gained much attention as a promising amino acid radiotracer for tumor imaging with positron emission tomography (PET) due to favorable imaging characteristics and relatively long half-life of ¹⁸F (110 min) allowing remote-site application. Here we present a novel type of chiral enantiomerically pure labeling precursor for [¹⁸F]FET, based on NiII complex of a Schiff's base of (S)-[N-2-(N′-benzylprolyl)amino]benzophenone (BPB) with alkylated (S)-tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH2CH2X (X = OTs (3a), OMs (3b) and OTf (3c)). A series of compounds 3a-c was synthesized in three steps from commercially available reagents. Non-radioactive FET as a reference was prepared from 3a in a form of (S)-isomer and (R,S) racemic mixture. Radiosynthesis comprised two steps: (1) n.c.a. nucleophilic fluorination of 3a-c (4.5-5.0 mg) in the presence of either Kryptofix 2.2.2.or tetrabutylammonium carbonate (TBAC) in MeCN at 80 °C for 5 min, followed by (2) removal of protective groups by treating with 0.5 M HCl (120 °C, 5 min). The major advantages of this procedure are retention of enantiomeric purity during the ¹⁸F-introduction step and easy simultaneous deprotection of amino and carboxy moieties in 3a-c. Radiochemically pure [¹⁸F]FET was isolated by semi-preparative HPLC (C18 μ-Bondapak, Waters) eluent aq 0.01 M CH₃COONH₄, pH 4/C₂H₅OH 90/10 (v/v). Overall synthesis time operated by Anatech RB 86 laboratory robot was 55 min. In a series of compounds 3a-c, tosyl derivative 3a provided highest radiochemical yield (40-45%, corrected for radioactive decay). Enantiomeric purity was 94-95% and 96-97%, correspondingly, for Kryptofix and TBAC assisted fluorinations. The suggested procedure involved minimal number of synthesis steps and suits perfectly for automation in the modern synthesis modules for PET radiopharmaceuticals. Preliminary biodistribution study in experimental model of turpentine-induced aseptic abscess and Glioma35 rat's tumor (homografts) in Wistar rats has demonstrated the enhanced uptake of radiotracer in the tumor area with minimal accumulation in the inflamed tissues.

Full text of DOI:10.1016/j.bmc.2008.03.040

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4994–5003
0
18
No carrier added synthesis of O-(2 -[ F]fluoroethyl)-L-tyrosine
via a novel type of chiral enantiomerically pure precursor,
Ni complex of a (S)-tyrosine Schiff base
II
a,
a
a
b
*
Raisa N. Krasikova, Olga F. Kuznetsova, Olga S. Fedorova, Victor I. Maleev,
b
b
Tatyana F. Saveleva and Yuri N. Belokon
a
Institute of Human Brain Russian Academy of Science, Laboratory of Radiochemistry, Acad. Pavlova Str. 9,
St. Petersburg 197376, Russia
b
A.N. Nesmeyanov Institute of Organoelement Compounds Russian Academy of Science, B-334, Vavilova Str. 28,
Moscow 119991, Russia
Received 3 October 2007; revised 5 March 2008; accepted 14 March 2008
Available online 17 March 2008
0
18
Abstract—O-(2 -[ F]fluoroethyl)-L-tyrosine ([ F]FET) has gained much attention as a promising amino acid radiotracer for tumor
18
1
8
imaging with positron emission tomography (PET) due to favorable imaging characteristics and relatively long half-life of
(
[
F
110 min) allowing remote-site application. Here we present a novel type of chiral enantiomerically pure labeling precursor for
1
8
0
F]FET, based on NiII complex of a Schiff’s base of (S)-[N-2-(N -benzylprolyl)amino]benzophenone (BPB) with alkylated (S)-
tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH2CH2X (X = OTs (3a), OMs (3b) and OTf (3c)). A series of compounds 3a–c was synthesized
in three steps from commercially available reagents. Non-radioactive FET as a reference was prepared from 3a in a form of (S)-
isomer and (R,S) racemic mixture. Radiosynthesis comprised two steps: (1) n.c.a. nucleophilic fluorination of 3a–c (4.5–5.0 mg)
in the presence of either Kryptofix 2.2.2.or tetrabutylammonium carbonate (TBAC) in MeCN at 80 ꢀC for 5 min, followed by
(
2) removal of protective groups by treating with 0.5 M HCl (120 ꢀC, 5 min). The major advantages of this procedure are retention
18
of enantiomeric purity during the F-introduction step and easy simultaneous deprotection of amino and carboxy moieties in 3a–c.
1
8
Radiochemically pure [ F]FET was isolated by semi-preparative HPLC (C18 l-Bondapak, Waters) eluent aq 0.01 M
CH COONH , pH 4/C H OH 90/10 (v/v). Overall synthesis time operated by Anatech RB 86 laboratory robot was 55 min. In a
3
4
2
5
series of compounds 3a–c, tosyl derivative 3a provided highest radiochemical yield (40–45%, corrected for radioactive decay). Enan-
tiomeric purity was 94–95% and 96–97%, correspondingly, for Kryptofix and TBAC assisted fluorinations. The suggested procedure
involved minimal number of synthesis steps and suits perfectly for automation in the modern synthesis modules for PET radiophar-
maceuticals. Preliminary biodistribution study in experimental model of turpentine-induced aseptic abscess and Glioma35 rat’s
tumor (homografts) in Wistar rats has demonstrated the enhanced uptake of radiotracer in the tumor area with minimal accumu-
lation in the inflamed tissues.
ꢁ
2008 Elsevier Ltd. All rights reserved.
1
,2
1
. Introduction
ing, which exploits the abnormal glucose metabolism
3
18
of malignant cells. Although [ F]FDG has been effec-
tive radiotracer for a variety of malignancies, it has
limited application in brain tumors imaging, because
of high glucose uptake in a grey matter. [ F]FDG accu-
mulation in inflamed tissues and granulation cells makes
it difficult to differentiate malignant tumors from benign
lesions as the main source of false-positive PET find-
ings. Labeled amino acids constitute an alternate class
of PET tracers for indicating tumor activity by measur-
ing trans-membrane transport rate, which is accelerated
1
,2
Positron emission tomography (PET), based on the
imaging of pharmaceuticals labeled with short-lived pos-
itron-emitting radionuclides, is a rapidly growing
modality for the diagnosis and management of cancer.
At present 2-[ F]-fluoro-2-deoxy-D-glucose ([ F]FDG)
is the most widely used PET radiotracer for tumor imag-
1
8
1
1
8
18
4
,5
0
18
Keywords: Fluorine-18; O-(2 -[ F]fluoroethyl)-L-tyrosine; Labeling
6
–8
in malignant cells. They demonstrated a minimal up-
take in the normal brain parenchyma and relatively low
accumulation in inflamed tissues. Among amino acids
precursor; Asymmetric synthesis.
*
Corresponding author. Tel.: +7 812 234 2359; fax: +7 812 234
247; e-mail: raisa@ihb.spb.ru
3
0
968-0896/$ - see front matter ꢁ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.040

R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
4995
1
1
11
radiotracers, L-[ C-methyl]methionine (L-[ C]MET)
remains the most popular one, mainly due to the ease
of its synthesis. However the short half-life of carbon-
alkylating intermediate from starting tosyloxyethane
by semi-preparative HPLC. This laborious procedure
with double preparative HPLC steps was not easy
adaptable to automation. Later the purification of
1
1 (t1/2 = 20.4 min) has a potential disadvantage of being
1
8
used only in PET centers with an expensive in-house
cyclotron. Fluorinated compounds labeled with longer-
lived fluorine-18 (t1/2 = 109.8 min) have practical bene-
fits, they can be transported to centers remote from pro-
duction/cyclotron site.
[ F]fluoroethyltosylate on disposable SPE cartridges
2
6
was reported, however this method was not ideal for
automated productions due to multiplicity of purifica-
tion steps using various solvents.
1
8
The synthesis of [ F]FET was substantially improved
by the implementation of direct nucleophilic radiofluori-
nation process on protected alkyl tyrosine derivative, O-
(2-tosyloxyethyl)-N-trityl-L-tyrosine tert.-butylester (1,
1
8
Despite
(
a
[ F]FAAs) have being suggested as tumor seeking
variety of
F-labeled amino acids
1
8
7
,8
agents for PET, their clinical application has been
hampered by difficult synthesis. Particularly difficult is
nucleophilic synthesis of the [ F]FAAs with label intro-
duced into the aromatic moiety. Such procedures in-
volve several synthetic steps and are difficult to adapt
2
7
Fig. 1). Deprotection of amino and carboxy functions
was carried out in solution of trifluoroacetic acid in
dichloromethane. Enantiomerically pure [ F]FET was
1
8
1
8
obtained in a radiochemical yield of 60% using re-
28
9
–11
for routine productions.
Alternatively the introduc-
mote-controlled synthesis module.
However, the
1
8
tion of F-label into the alkyl chain can be achieved
via aliphatic nucleophilic substitution and allows to pro-
duce radiotracers in clinically relevant amounts. Of sev-
eral structurally similar o-fluoroalkyl substituted
necessity to remove the solvent and an aggressive TFA
by SPE technique preceding the final HPLC purification
step resulted in a relatively long and complex radio-
chemistry sequence.
0
18
18
12
tyrosines, O-(2 -[ F]fluoroethyl)-L-tyrosine ([ F]FET),
1
8
13
0
18
O-(3-[ F]fluoropropyl)-L-tyrosine, and O-(2 -[ F]flu-
In the further studies similar precursor structure with
different protecting groups, O-(2-tosyloxyethyl)-N-tert.-
butyloxycarbonyl-L-tyrosine benzyl ester (2, Fig. 1),
1
4
18
oromethyl)-L-tyrosine, [ F]FET has gained particular
attention. An initial study demonstrated that an uptake
of [ F]FET in brain tumors is similar to that of
1
8
29
was suggested. It should be noted that the change-
over of the protecting groups may lead to racemiza-
1
1
15 18
L-[ C]MET. [ F]FET is not metabolized and not
incorporated into proteins, it is actively transported into
tumor cells by specific amino acid transport system
1
tion of amino acid moiety within the course of F-
8
fluorination reaction, performing under basic condi-
27
+
0+ 16,17
L and in part by Na -dependent system B .
reports have shown high uptake of [ F]FET in cere-
Further
tions.
[ F]FET obtained via
of 2 was not reported.
Unfortunately, the enantiomeric purity of
F-nucleophilic fluorination
1
8
18
18
1
8–21
22
29
bral
L-[ C]MET and [ F]FDG, [ F]FET exhibits negligible
and peripheral tumors.
In contrast to
1
1
18
18
23,24
uptake in inflammatory tissues in animals model.
the recent studies in humans the usefulness of [ F]FET
as a molecular probe for the differentiation of tumor and
In
Based on our experience in asymmetric synthesis of ami-
30–33
1
8
no acids,
[ F]FET by elaborating a new leading structure of
we pursued a different approach to
1
8
2
5
34
inflammation has been confirmed.
labeling precursor. The novel type of the precursor ar-
II
0
ose from Ni complex of a Schiff’s base of (S)-[N-2-(N -
benzylprolyl)amino]benzophenone (BPB) with alkylated
(S)-tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH CH X (3, Fig. 1);
1
8
Growing interest to clinical application of [ F]FET has
stimulated developments of new synthesis strategies that
could be suitable for routine use. In the first published
2
2
X = OTs (3a), OMs (3b), OTf (3c). The precursor has
some advantages which include (a) simple preparation
from commercial reagents; (b) stability under reaction
basic conditions and retention of desired (S)-configura-
1
2
18
synthesis [ F]FET was prepared via alkylation of
the di-potassium salt of L-tyrosine with [ F]fluoroethyl-
1
8
1
tosylate. The process involved separation of the F-
8
1
8
Figure 1. The structure of labeling precursors for [ F]FET, including Ni-(S)-BPB-(S)-Tyr-OCH
2 2
CH X (3) of the present work; X = OTs (3a), OMs
(
3b), OTf (3c).

4996
R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
3
0,31
tion of amino acid;
(c) one stage removal of the
and tosylation of the O-hydroxyethyl-group under basic
chiral auxiliary and simultaneous deprotection of amino
and carboxy functions under mild conditions.
conditions. Using this synthesis route a series of precur-
sors carrying mesylate and triflate leaving groups in an
alkyl moiety, 3b and 3c, respectively, were prepared
(Fig. 1). All intermediate compounds and the final prod-
Herein, we describe the preparation of this novel type of
precursor 3 and authentic FET. We also report one-pot
1
13
ucts were characterized by
spectroscopy.
H
and
C NMR
1
8
two-steps radiosynthesis of [ F]FET via direct nucleo-
philic fluorination of 3 with no carrier added (n.c.a.)
[
1
8
F]fluoride in the presence of two phase transfer cata-
lysts, tetra butylammonium carbonate (TBAC) and
Kryptofix 2.2.2. (K2.2.2.)/K CO complex. Biological
It should be emphasized that it is most beneficial to use
in PET studies single-enantiomer tracers. The distribu-
tion and kinetics of (S)-isomers of amino acids are
known to be different from those for (R)-isomers, and
the (S)-configuration is generally preferred for brain
2
3
1
8
evaluation of [ F]FET obtained via new synthesis ap-
proach has been performed in experimental rat’s model
of tumor and inflammation.
1
7
tumors imaging. Finally, for precursors 1 and 2 the
reactions in the basic solutions may be a cause of
possible amino acid moiety racemization either during
1
8
2
. Results and discussion
F-introduction or in the process of cold synthesis of
3
0
the standard. The reason for that is relatively high
CH-acidity of their amino acid moieties. Fortunately,
in the case of 3 it is no longer racemization but epimer-
ization of the amino acid moiety that would take place
under the reaction conditions, leading to an equilibrium
ratio of (R,S)- and (S,S)-diastereoisomers, namely
Ni-(S)-BPB-(S)-Tyr-[18(19)F]OCH CH F and Ni-(S)-
2
.1. Chemistry
The synthesis of a novel chiral diastereoisomerically
pure precursor for [ F]FET, a Ni complex of a Schiff’s
base of (S)-[N-2-(N -benzylprolyl)amino]benzophenone
(BPB) with (S)-tyrosine, in which the OH-group of the
tyrosine moiety was converted into OCH CH OTs
1
8
II
0
2
2
BPB-(R)-Tyr-[18(19)F]OCH CH F. Due to the chiral
2
2
2
2
derivative Ni-(S)-BPB-(S)-Tyr-OCH CH OTs (3a), is
2
structure of diastereoisomerically pure complex 3 and
greater thermodynamic stability of its (S,S)-form, rela-
2
depicted in Scheme 1. At the first synthesis stage the
Ni-(S)-BPB-(S)-Tyr complex (4) was easily prepared
from a nickel nitrate salt, (S)-BPB and racemic tyrosine
in the presence of base. Under the reaction basic condi-
tions the epimerization of the unstable intermediate
((R,S)-diastereoisomer) leads to the formation of a sta-
ble (S,S)-diastereoisomer as the only red colored reac-
tion product. The next two stages include O-alkylation
1
8
tive to (S,R)-isomer, F-fluorination should proceed
with the retention of desired (S)-configuration of the
tyrosine moiety. In other words even if the a-proton of
the (S)-tyrosine moiety leaves under the effect of a base,
it should come back preferentially from the same side
and thus the danger of side reaction of racemization
becomes insignificant.
Scheme 1. Synthesis of labeling precursor, Ni-(S)-BPB-(S)-Tyr-OCH
2
CH
2
OTs (3a).

R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
4997
The reference compound, (S)-FET, was obtained via
reaction of 3a with KF in the presence of tertabutylam-
moinim fluoride, followed by the decomposition of the
resulting complex and purification of product by column
chromatography. The compound was characterized by
added in succession and each evaporated in turn, to give
the corresponding F-reactive complex as a slightly
brown residue.
1
8
The course of radiofluorination reactions was monitored
by radio TLC. Initially, the aliquot of the reaction mix-
ture was quenched with water. A radio TLC chromato-
1
13
19
H, C, and F NMR spectroscopy. Racemic FET
5
was prepared according to earlier described procedure.
3
1
8
gram showed only two peaks of unbounded [ F]fluoride
and F-fluorinated precursor 3d. Radiochemical yields
1
8
2
.2. Radiochemistry
1
8
of F-incorporation (incorporation rates) were evalu-
ated from the TLC data and defined as the ratio of
F-labeled intermediate 3d over total fluorine-18 radio-
The two-step reaction sequence for the preparation of
[
1
8
18
F]FET using new labeling precursors 3a–c is outlined
in Scheme 2. The three compounds were carefully inves-
tigated for their applicability in routine production pro-
cedures, including radiofluorination effectiveness,
chemical stability, and other related characteristics.
activity (Table 1).
Concerning the influence of the leaving group, precur-
sors 3a and 3b gave similar and reasonably good radio-
fluorination yields of 50–60%, when using TBAC as a
phase transfer catalyst (PTC). Although not systemati-
cally investigated, increasing the amount of precursors
up to 9–10 mg did not further increase the radiochemical
yields. Noteworthy, radiochemical yields for 3c were
much lower, and no further attempts were made to im-
prove the yield. As for a choice of PTC, similar incorpo-
ration rates were obtained for precursor 3a for both
complexing agents. However, from the current data,
the fluorination yields for 3b were substantially lower
in cryptate-assisted reactions (Table 1). Also, we ob-
served reduction in fluorination efficiency of 3b in
TBAC mediated process from 50% to 60% to 15% with-
in 6 months of storage period at 4 ꢀC. Nevertheless, no
chemical instability of 3a was observed within 2.5 years.
1
.2.1. F nucleophilic substitution reactions on labeling
precursors 3a–c. One-pot radiosynthesis of [ F]FET was
accomplished in two steps: (1) n.c.a. nucleophilic fluori-
nation of 3a–c in the presence of phase transfer catalyst
8
2
1
8
(
PTC) followed by (2) removal of protective groups by
treating with aqueous solution of HCl. The first stage
was carried out under routine conditions for nucleo-
philic displacement reactions on the aliphatic substrates
1
8
using n.c.a. [ F]fluoride. Typically, 4.5–5.0 mg of 3a–c
was used, radiofluorinations were conducted in acetoni-
2
7,29
trile at 80 ꢀC for 5 min. In previous reports
was chosen as a PTC for this type of reaction because
TBAC
1
8
it showed higher F-incorporation rate comparing to
the commonly employed Kryptofix 2.2.2/K CO com-
2
3
+
18
ꢀ
plex ([K/K2.2.2] [ F] ). Because the Kryptofix system
is a generally preferred one for the most of n.c.a. fluori-
1
8
2.2.2. Hydrolysis/deprotection of [ F]-3d. In terms of
automation of the synthesis procedure, the hydrolysis/
deprotection step warrants particular attention. In the
previous communication the cleavage of protective
groups in the labeled intermediate, N-trityl-O-(2-
1
8
nations, we compared the performance of F-nucleo-
philic displacement reactions on the 3a–c in the
presence of both complexing agents. Detailed procedure
2
7
+
18
ꢀ
for the preparation of the activated [K/K2.2.2] [ F]
1
1
complex has been described elsewhere.
[
Briefly,
F]fluoride generated in water cyclotron target via
1
8
1
8
18
O(p,n) F nuclear reaction was collected on the pre-
18
Table 1. F-incorporation rates into 3a–c
conditioned QMA cartridge. It was eluted from the
QMA resin by passing 2 mL of stock solution of either
TBAC (pH 8) or K2.2.2/K CO in MeCN/H O. The elu-
ate was collected into 5 mL conic vial, the solvents were
removed under stream of nitrogen gas and heated until
dryness. Two portions of acetonitrile (0.5 mL) were
1
8
18
F-incorporation
rate in %, Kryptofix
Precursor
F-incorporation
rate in %, TBAC
2
3
2
3a
3b
3c
56.7 ± 9.8 (n = 5)
51.3 ± 9.6 (n = 5)
<10
57.3 ± 8.5 (n = 3)
28.0 ± 14.0 (n = 4)
<5
18
Scheme 2. Radiosynthesis of [ F]FET via direct nucleophilic fluorination of 3a–c.

4998
R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
1
8
[
F]fluoroethyl)-L-tyrosine tert.-butylester, was carried
and 96–97%, correspondingly, for Kryptofix and TBAC
assisted fluorinations.
out in a non-aqueous solution consisting of trifluoroace-
tic acid (TFA) in dichloromethane. In order to remove
the corrosive TFA and to separate the radiotracer from
the water insoluble triphenylcarbinol, the crude
To ensure that the final dose is devoid of any nickel
impurities, the nickel content of a few batches of
[ F]FET was determined by the inductively coupled
1
8
18
[
F]FET was preliminarily isolated on Silica Sep-Pak.
In our view this represents a difficult procedure to per-
form if automated synthesis has to be accomplished.
In our workup a removal of the chiral auxiliary and
simultaneous deprotection of amino and carboxy func-
tions of tyrosine was easily achieved under moderate
aqueous conditions (0.5 M HCl, 5 min, 120 ꢀC). It
should be noted that aqueous acid solution has to be
added into the reactive vial which contained acetonitrile
used in the previous radiofluorination step. Our studies
depicted that the presence of acetonitrile on that stage of
the synthesis was a critical factor for an exhaustive
cleavage of protective groups. The completeness of
plasma mass spectroscopy. The nickel content was neg-
ligible, ranging between 0.2 and 0.5 parts per million.
The radiochemical yield of [ F]FET using 3a as an opti-
1
8
mal labeling precursor was 40–45% (corrected for radio-
1
8
active decay). The specific radioactivity of the [ F]FET
was >14 GBq/lmol at the end of the synthesis. Synthesis
time operated by laboratory robot Anatech RB 86 was
55 min including HPLC purification.
2.3. Preliminary in vivo evaluation in rats
1
8
The biodistribution of [ F]FET was investigated in
Wistar rats using experimental model of turpentine-in-
duced aseptic abscess and Glioma35 rats tumor
(homografts). To compare the radiotracer uptake in
tumor and inflamed areas, the corresponding tumor-
to-muscle and abscess-to-muscle ratios were calculated
(Tables 2 and 3).
1
8
decomposition of labeling intermediate [ F]-3d and re-
moval of the protective groups was confirmed by radio-
TLC analysis of the aliquot of the crude reaction
mixture.
2
.2.3. The HPLC purification and the formulation steps.
Crude product obtained after hydrolysis/deprotection
step was partly neutralized and directly injected into
semi-preparative HPLC (C18 l-Bondapak column,
Waters) 300 · 7.8 mm, 10 lm; eluent aq 0.01 M
CH COONH , pH 4/C H OH 90/10 (v/v). An interme-
2
3,24
As expected from the literature data,
the uptake
1
8
of [ F]FET was not significantly different in the in-
flamed muscle than in the contralateral healthy tissues.
The abscess-to-muscle ratios were 1.30, 1.32, 1.39, and
1.33 at 20, 40, 60, and 120 min of post injection, cor-
respondingly. At the same time points the radiotracer
uptake in Glioma35 rat’s tumor was almost double:
the tumor-to-muscle ratios reached the values of
3.17, 2.95, 2.54 and 2.93, correspondingly. The results
obtained in our experimental models confirmed that
3
4
2
5
2
7
diary cartridge purification suggested earlier was elim-
inated and thus radiochemistry sequence was simplified.
HPLC analysis of the product fraction revealed high
radiochemical (>99%) and chemical purities of
1
8
[ F]FET. The enantiomeric purity was analyzed by chi-
ral HPLC (Fig. 2) and (R,S)-FET was used as a refer-
ence. The enantiomeric purity was in range of 94–95%
1
8
[ F]FET may serve as a suitable radiotracer for the
differentiation of tumor and inflammatory tissues. This
observation is in accordance with previously reported
2
3
1
4 mV
1
findings.
D-FET
2
L-FET
3. Conclusions
In conclusion, the nucleophilic substitution of the leav-
ing group in the novel labeling precursor, Ni complex
II
0
of a Schiff’s base of (S)-[N-2-(N -benzylprolyl)amino]-
benzophenone ((S)-BPB) with alkylated (S)-tyrosine,
Ni-(S)-BPB-(S)-Tyr-OCH CH X (3a–c), is a feasible
ch1
2
4
6
8
10
12
min
2
2
1
8
way to a fully automated production of [ F]FET, an
important amino acid tracer for tumor diagnosis with
PET. Based on the comparison within a series of label-
ing precursors 3a–c, the tosylate derivative 3a has ap-
peared to be the best for routine preparation of
619 mV
2
1
8
L-[ F]FET
1
8
1
8
[ F]FET. According to the current results, TBAC seems
to be more suitable phase transfer catalyst for the nucle-
D-[ F]FET
1
1
8
ophilic fluorination of 3a with n.c.a. [ F]fluoride pro-
viding high radiochemical and enantiomeric purity.
The simple procedure with minimal intermediate purifi-
cations steps, the mild conditions for the removal of
ch2
2
4
6
8
10
12
min
Figure 2. A typical chiral HPLC radiochromatogram of purified
1
8
1
8
18
protective groups in [ F]-3d offer a better opportunity
to automate the overall synthesis process. In terms of
large scale productions, using Curie amounts of cyclo-
[
F]FET obtained by n.c.a. [ F]fluorination of 3a (down) and UV
chromatogram of racemic D,L-tyrosine (up); Crownpack CR (+),
eluent HClO pH 2/CH OH 90/10 (v/v), flow rate 0.8 mL/min; k
54 nm.
4
3
1
8
2
tron-produced [ F]fluoride, the amenability of the syn-

R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
Table 2. Tissue distribution of radioactivity in five Wistar rats bearing Glioma35 rat tumor at 40 min post-injection of [ F]FET, in % ID/g
4999
1
8
Blood
Kidney
Heart
Liver
Urinary bladder
Brain
Muscle
Tumor
Tumor-to-muscle ratio
1
1
1
1
1
.24
.05
.20
.16
.12
0.73
0.73
0.86
0.70
0.66
0.89
0.77
0.88
0.98
0.73
1.03
0.85
0.82
0.71
0.56
2.51
1.59
1.42
7.02
1.12
0.54
0.38
0.45
0.41
0.46
0.75
0.62
0.84
0.74
0.61
2.53
2.35
2.44
2.00
1.25
3.36
3.77
2.89
2.69
2.06
thetic approach to automation becomes increasingly
important. Taking into account a high price of both pre-
cursor 1 and authentic reference of FET from commer-
MeOH were placed in a three neck round-bottom flask
equipped with a mechanical stirrer, thermometer, and
reflux condenser. The mixture was heated up to 45 ꢀC,
the thermometer was replaced with a funnel and then
cial vendor, the availability of
alternative might be another important aspect slashing
a non-expensive
ꢀ
2
6.5 mL of 4 N MeONa (2.6 · 10 mol) was quickly
added under stirring. The mixture was refluxed with stir-
ring for 70 min. The reaction was monitored by TLC
(SiO , CHCl /Me CO, 7:1), following the disappearance
1
8
the costs of single clinical dose of [ F]FET. Preliminary
biodistribution study in rats has demonstrated the suit-
1
8
ability of [ F]FET as a radiotracer for the differentia-
tion of tumor and inflammatory tissues.
2
3
2
of the parent BPB spot on the chromatogram. Then the
reaction mixture was neutralized with 1.5 mL of AcOH
and diluted with 25 mL of water and the precipitate (the
target product Ni-(S)-BPB-(S)-Tyr admixed with some
residual initial tyrosine impurity) was filtered off. The
4
. Materials and methods
4
4
.1. Chemistry
.1.1. Materials and general procedures. All NMR spec-
precipitate was treated with CHCl (the target product
3
was dissolved) and the residual tyrosine was removed
by filtration. The solution was evaporated to give
1.16 g (96%) of the target product Ni-(S)-BPB-(S)-Tyr.
The final complex was used without additional purifica-
tion. To get high-quality analytical data the portion of
the product was additionally purified by gel-filtration
chromatography (Sephadex LH-20, elution with C H /
tra were recorded on a Bruker Avance 400 instrument
(
1
400.13 MHz for H, 100.16 MHz for
13
C
61 MHz for F) using CDCl as a solvent (unless indi-
and
1
9
1
3
cated otherwise). Chemical shifts of peaks are presented
in ppm (d-scale with 0.00 ppm for TMS) with, using
residual deuterated solvent signal as an internal stan-
dard. Optical rotations were measured with a Perkin-El-
mer 241 polarimeter in a thermostated cell at 25 ꢀC. All
solvents were distilled prior to use. Melting points were
determined on Electrothermal Melting Point Apparatus
and are uncorrected. Elemental analysis was carried out
in the laboratory of Microanalysis of INEOS RAS using
Carlo-Erba 1106. Enantiomeric analysis of FET was
performed on a Bischoff HPLC instrument (detector:
UV at 254 nm) using the ‘Chirobiotic T’ column (250·
6
6
1
EtOH (3:1)). H NMR: 1.65 (m, 1H, Pro), 1.94 (m,
1H, Pro), 2.34 (m, 3H, Pro), 2.72 and 2.92 (AB part of
ABX, JAB = 13.7 Hz, JAX = 4.6 Hz, JBX = 2.1 Hz, 2H,
–CH -Tyr), 3.07 (m, 1H, d-Pro), 3.33 (m, 1H, a-Tyr),
2
4.24 (m, 1H, a-Pro), 3.32 and 4.21 (AB, JAB = 12 Hz,
2H, –CH -Bn), 6.64 (m, 2H, Ar), 6.83 (d, 1H, Ar),
2
6.91 and 7.00 (AB, JAB = 7.7 Hz, 4H, Tyr), 7.13 (m,
2H, Ar), 7.27 (m, 3H, Ar), 7.41 (m, 1H, Ar), 7.52 (m,
2H, Ar), 8.05 (d, 2H, Ar), 8.14 (d, 1H, Ar), 8.98 (s,
1H, OH).
4
.0 mm), eluent MeOH/H O (1:1), flow rate 1 mL/min,
2
1
3
temperature 25 ꢀC. Retention time: 5.3 min for (R)-
enantiomer and 5.8 min for (S)-enantiomer. For TLC
analysis Alugram SIL G/UV from Macherey-Nagel
was used. Chiral auxiliary BPB was available from Ac-
ros. (R,S)-tyrosine, 2-chloroethanol and other reagents
were available from Aldrich and were used without
additional purification.
C NMR: 23.13 c-C [Pro], 30.98 b-C [Pro], 38.54 b-C
[Tyr], 57.85 d-C [Pro], 63.81 CH2 [Bn], 70.81 a-C
[Tyr], 71.83 a-C [Pro], 116.01 m-Ar [Tyr], 121.13 3-Ar
[2-ABPh], 123.64 5-Ar [2-ABPh], 126.21 i-Ar [Tyr],
126.49 1-Ar [2-ABPh], 127.24 4-Ph [Bn], 127.86 6-Ar
ꢂ
0
[2-ABPh], 128.94 3,3 -Ph [Bn], 129.00 4-Ph [2-ABPh],
0
129.24 2-Ar [Tyr], 129.98 4-Ar [2-ABPh], 131.53 3.3 -
0
Ph [2-ABPh], 131.67 2,2 -Ph [Bn], 132.42 2-Ph [2-ABPh],
0
4
1
.1.2. Ni-(S)-BPB-(S)-Tyr (4). Racemic tyrosine (1.8 g,
133.55 i-Ph [Bn], 133.63 2 -Ph [2-ABPh], 134.19 i-Ph [2-
ABPh], 142.28 2-Ar [2-ABPh], 157.14 4-Ar [Tyr], 171.24
ꢀ
2
ꢀ3
· 10 mol), (S)-BPB · HCl (0.84 g, 2 · 10 mol),
ꢀ3
Ni(NO ) · 6H O (1.16 g, 4 · 10 mol), and 10 mL of
–CONH–, 179.33 >C@N–, 180.79 –COO–.
3
2
2
1
Table 3. Tissue distribution of radioactivity in five Wistar rats with turpentine-induced aseptic abscess at 40 min post-injection of [ F]FET, in % ID/g
8
Blood
Kidney
Heart
Liver
Urinary bladder
Brain
Muscle
Abscess
Abscess-to-muscle ratio
1
1
1
1
1
.45
.15
.19
.29
.13
1.58
0.89
1.13
0.85
0.64
1.47
1.28
1.00
1.16
1.08
1.24
0.88
1.04
1.17
1.03
2.84
2.30
0.19
3.94
2.50
1.00
0.66
0.87
0.96
0.85
1.32
1.16
0.85
0.92
0.97
1.75
1.41
1.17
1.52
0.98
1.33
1.22
1.37
1.66
1.02

5000
R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
Found, %: C 68.18; H 5.24; N 6.71. Calculated for
C H N NiO , %: C, 67.57; H, 5.17; N, 6.95. Mp
vent was removed in vacuo and Ni-(S)-BPB-(S)-Tyr-
CH CH OTs was separated by preparative TLC (SiO ,
elution with CHCl /Me CO (7:1)). The complex was
3 2
3
4
31
3
4
2
2
2
D
1
80–182 ꢀC. ½aꢁ25 +2154 (c 0.031, MeOH).
additionally purified by gel-filtration chromatography
(Sephadex LH-20, elution with C H /EtOH (3:1)), dried
4
.1.3. Ni-(S)-BPB-(S)-Tyr-CH CH OH (5). Ni-(S)-
2
2
6
6
ꢀ3
1
BPB-(S)-Tyr (4) (1 g, 1.65 · 10 mol) was placed in a
round-bottom flask, 2-chloroethanol (4 g, 4.96 ·
on air. Yield 67% (0.08 g). H NMR: 1.65 (m, 1H, Pro),
1.95 (m, 1H, Pro), 2.30 (m, 3H, Pro), 2.42 (s, 3H,
CH C H SO –), 2.77 and 2.99 (AB part of ABX,
ꢀ
2
1
a magnetic stirrer until the substance was dissolved.
0
mol) was added, and the mixture was stirred with
3
6
4
3
JAB = 13.9 Hz, JAX = 5.7 Hz, JBX = 4.3 Hz, 2H, –CH2-
Tyr), 3.07 (m, 1H, d-Pro), 3.29 (m, 1H, a-Tyr), 4.20 (m,
1H, a-Pro), 3.42 and 4.24 (AB, JAB = 12.5 Hz, 2H, –
CH -Bn), 4.12 (m, 2H, OCH CH OTs), 4.34 (m, 2H,
+
ꢀ
Then benzene (15 mL) and Bu N I (0.6 g, 1.65 ·
4
ꢀ
4
1
0
mol) were added to the flask, followed by 12 mL
of aqueous KOH (50%) solution in portions of 1 mL
over a period of 3 h under vigorous stirring. The reac-
tion mixture was additionally stirred for 14 h at room
temperature, neutralized with 1 mL of AcOH, and trea-
2
2
2
OCH CH OTs), 6.64 (d, 2H, Ar), 6.84 (d, 1H, Ar), 6.80
2
2
and 7.04 (AB, JAB = 9 Hz, 4H, Tyr), 7.12 (m, 2H, Ar),
7.27 (m, 3H, Ar), 7.41 (m, 1H, Ar), 7.51 (m, 2H, Ar),
7.33 and 7.79 (AB, JAB = 8.2 Hz, 4H, Ts), 8.00 (d, 2H,
Ar), 8.19 (d, 1H, Ar).
ted with 3 mL of CHCl . The chloroform extract was
3
separated, washed with water (3· 5 mL), dried over
Na SO and evaporated in vacuo. The residue was twice
2
4
1
3
treated with 5 mL of acetone and each time evaporated
to remove the remaining 2-chloroethanol, 10 mL of ace-
tone was added to the solid residue, and the suspension
filtered off and washed with acetone (2· 5 mL) to give
C NMR: 21.70 CH -[Ts], 23.10 c-C [Pro], 30.70 b-C
3
[Pro], 38.92 b-C [Tyr], 57.35 d-C [Pro], 63.42 CH2
[Bn], 65.58 –OCH CH OTs, 68.15 a-C [Tyr], 70.35 a-C
2
2
[Pro], 71.56 –OCH CH OH, 114.75 m-Ar [Tyr], 120.59
3-Ar [2-ABPh], 123.37 5-Ar [2-ABPh], 126.11 1-Ar [2-
ABPh], 127.19 4-Ph [Bn], 127.82 6-Ar [2-ABPh],
2
2
0
.754 g (70.5%) of Ni-(S)-BPB-(S)-Tyr-CH CH OH.
2
2
The product was additionally purified by gel-filtration
chromatography (Sephadex LH-20, mixture C H /
0
0
128.01 2,2 -Ar [Ts], 128.58 i-Ar [Tyr], 128.78 3,3 -Ph
[Bn], 2-Ar [Tyr], 128.90 4-Ph [ABPh], 129.12 4-Ph [2-
ABPh], 129.80 4-Ar [2-ABPh], 129.79 4-Ar [Ts], 129.92
6
6
1
EtOH (3:1)). H NMR: 1.66 (m, 1H, Pro), 1.96 (m,
H, Pro), 2.34 (m, 3H, Pro), 2.78 and 3.01 (AB part of
ABX, JAB = 14 Hz, JAX = 5.7 Hz, JBX = 4.3 Hz, 2H, –
1
0
0
0
3,3 -Ar [Ts], 131.52 3.3 -Ph [2-ABPh], 131.61 2,2 -Ph
[Bn], 132.34 2-Ph [2-ABPh], 133.33 i-Ph [Bn], 133.51
2 -Ph [2-ABPh], 134.20 i-Ph [2-ABPh], 142.85 2-Ar [2-
ABPh], 145.03 i-Ar [Ts],157.72 4-Ar [Tyr], 171.05 –
CONH–, 178.55 >C@N–, 180.34 –COO–.
CH -Tyr), 3.09 (m, 1H, d-Pro), 3.29 (m, 1H, a-Tyr),
2
0
3
.41 (s, 1H, OH), 4.21 (m, 1H, a-Pro), 3.42 and 4.25
(
CH OH), 4.07 (m, 2H, OCH CH OH), 6.64 (d, 2H, Ar),
AB, JAB = 12.5 Hz, 2H, –CH -Bn), 3.94 (m, 2H, OCH2-
2
2
2
2
6
.86 (d, 1H, Ar), 6.93 and 7.07 (AB, JAB = 8.4 Hz, 4H,
Tyr), 7.13 (m, 2H, Ar), 7.28 (m, 3H, Ar), 7.42 (m, 1H,
Ar), 7.51 (m, 2H, Ar), 8.00 (d, 2H, Ar), 8.19 (d, 1H, Ar).
Found, %: C, 62.54; H, 4.90; N, 4.90. Calculated for
C H N NiO S · 1.25H O, %: C, 62.6; H, 5.31; N,
1
3
43 41
3
7
2
C NMR: 23.13 c-C [Pro], 30.66 b-C [Pro], 38.88 b-C
Tyr], 57.35 d-C [Pro], 61.35 CH [Bn], 63.46 –OCH2-
D
5
.09. Mp 220–223 ꢀC. ½aꢁ +1922 (c 0.03, MeOH).
2
5
[
2
CH OH, 69.33 a-C [Tyr], 70.41 a-C [Pro], 71.61 –OCH
2
2-
4
.1.5. Ni-(S)-BPB-(S)-Tyr-CH CH OMs (3b). To the
2 2
CH OH, 114.82 m-Ar [Tyr], 120.66 3-Ar [2-ABPh],
123.38 5-Ar [2-ABPh], 126.17 1-Ar [2-ABPh], 127.19 4-
Ph [Bn], 127.85 6-Ar [2-ABPh], 128.19 i-Ar [Tyr],
2
ꢀ
4
stirred solution of (5) (0.087 g, 1.34 · 10 mol) in 2 mL
of dry CH Cl at 0 ꢀC was added Et N (0.02 mL,
2
2
3
ꢀ
ꢀ
4
0
1.42 · 10 mol) followed by MeSO Cl (0.01 mL,
1
ABPh], 129.80 4-Ar [2-ABPh], 131.51 3.3 -Ph [2-ABPh],
28.79 3,3 -Ph [Bn], 128.90 2-Ar [Tyr], 129.12 4-Ph [2-
0
2
4
1.34 · 10 mol). After the addition was complete, the
cooling was stopped and the reaction mixture was stirred
for another 18 h at ambient temperature. Then addition-
0
1
[
1
31.64 2,2 -Ph [Bn], 132.36 2-Ph [2-ABPh], 133.31 i-Ph
0
Bn], 133.53 2 -Ph [2-ABPh], 134.20 i-Ph [2-ABPh],
42.82 2-Ar [2-ABPh], 154.39 4-Ar [Tyr], 171.07 –
ꢀ
4
ally Et N (0.02 mL, 1.42 · 10 mol) and MeSO Cl
3
2
ꢀ
4
(
0.01 mL, 1.34 · 10 mol) were added sequentially, and
CONH–, 178.66 >C@N–, 180.33 –COO–.
the mixture was stirred extra 6 h at room temperature.
The reaction was monitored by TLC (SiO , CHCl :Me
2-
Found, %: C, 65.37; H, 5.40; N, 5.91. Calculated for
C H N NiO · EtOH, %: C, 65.72; H, 5.95; N, 6.05.
2
3
CO (3:1)) by the disappearance of the spot of the initial
Ni-(S)-BPB-(S)-Tyr-CH CH OH complex. After com-
pletion of the reaction, the solvent was removed in vacuo
and Ni-(S)-BPB-(S)-Tyr-CH CH OMs was separated by
3
6
35
3
5
D
5
Mp 250–252 ꢀC. ½aꢁ2 +1206 (c 0.03, MeOH).
2
2
4
.1.4. Ni-(S)-BPB-(S)-Tyr-CH CH OTs (3a). To the
2
2
2
2
ꢀ
4
preparative TLC (SiO , elution with CHCl /Me CO
2 3 2
stirred solution of (5) (0.1 g, 1.54 · 10 mol) in 2 mL of
(3:1)). The complex was additionally purified by gel-filtra-
dry CH Cl at 0 ꢀC was added Et N (0.07 mL,
2
2
3
ꢀ
4
tion chromatography (Sephadex LH-20, elution with
C H /EtOH (3:1)), dried on air. Yield, 68% (0.066 g).
H NMR: 1.71 (m, 1H, Pro), 1.83 (m, 1H, Pro), 1.96 (m,
4
4
.78 · 10 mol) followed by CH C H SO Cl (0.09 g,
3
6
4
2
ꢀ
4
.72 · 10 mol). After the addition was complete, the
6
6
1
cooling was stopped; the reaction mixture was allowed
to warm up to an ambient temperature and the mixture
was additionally stirred for 20 h. The reaction was moni-
tored by TLC (SiO , elution with CHCl /Me CO (3:1)) by
1
3
H, Pro), 2.34 (m, 2H, Pro), 2.42 (m, 1H, Pro), 2.81 and
.00 (AB part of ABX, JAB = 14 Hz, JAX = 6 Hz,
JBX = 4.2 Hz, 2H, –CH -Tyr), 3.07 (s, 3H, CH SO –),
2
3
3
3
2
3
2
.11 (m, 1H, d-Pro), 3.31 (m, 1H, a-Tyr), 4.20 (m, 1H,
the disappearance of the initial Ni-(S)-BPB-(S)-Tyr-
CH CH OH spot. After the reaction was complete, sol-
a-Pro), 3.44 and 4.26 (AB, JAB = 12.5 Hz, 2H, –CH2-
2
2

R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
5001
Bn), 4.22 (m, 2H, OCH CH OMs), 4.55 (m, 2H, OCH
2-
CH OMs), 6.67 (d, 2H, Ar), 6.87 (d, 1H, Ar), 6.92 and
2
Found, %: C, 63.115; H, 4.98; N, 5.81. Calculated for
C H F N NiO S, %: C, 66.94; H, 4.39; N, 5.38. Mp
2
2
37 34
3
3
7
D
7
(
.08 (AB, JAB = 8.4 Hz, 4H, Tyr), 7.14 (m, 2H, Ar), 7.28
m, 3H, Ar), 7.43 (m, 1H, Ar), 7.53 (m, 2H, Ar), 8.01 (d,
193–195 ꢀC with decomposition. ½aꢁ +2226 (c 0.03,
25
MeOH).
2
H, Ar), 8.19 (d, 1H, Ar).
0
19
.1.7. O-(2 -[ F]fluoroethyl)-(S)-tyrosine (FET): (S)-FET
4
and (R,S)-FET. To the solution of Ni-(S)-BPB-(S)-Tyr-
1
3
C NMR: 23.14 c-C [Pro], 30.67 b-C [Pro], 37.78
ꢀ
3
MeSO –, 38.99 b-C [Tyr], 57.25 d-C [Pro], 63.42 CH
OCH CH OTs, (1.29 g 1.57 · 10 mol) in 15 mL of dry
2
2
2
2
ꢀ
2
[
Bn], 65.92 –OCH CH OMs, 68.03 a-C [Tyr], 70.33 a-C
MeCN were added dry KF, 2.33 g (4.02 · 10 mol) and
2
2
[
Pro], 71.54 –OCH CH OMs, 114.78 m-Ar [Tyr], 120.66
1.6 mL of Bu NF (1 N solution in THF). The reaction
2
2
4
3
ABPh], 127.22 4-Ph [Bn], 127.82 6-Ar [2-ABPh], 128.34
-Ar [2-ABPh], 123.38 5-Ar [2-ABPh], 126.15 1-Ar [2-
mixture was stirred for 10 h at 80 ꢀC, then the mixture
was cooled to ambient temperature. The inorganic salts
formed were filtered and the filtrate was washed with
dry MeCN. The combined filtrates were evaporated and
the target product Ni-(S)-BPB-(S)-Tyr-OCH CH F was
0
i-Ar [Tyr], 128.82 2-Ar [Tyr], 3,3 -Ph [Bn], 129.16 4-Ph
0
[
ABPh], 131.73 2,2 -Ph [Bn], 132.40 2-Ph [2-ABPh],
2-ABPh], 129.84 4-Ar [2-ABPh], 131.52 3.3 -Ph [2-
0
2
2
0
1
33.27 i-Ph [Bn], 133.53 2 -Ph [2-ABPh], 134.18 i-Ph [2-
separated by column chromatography on SiO column
2
ABPh], 142.80 2-Ar [2-ABPh], 157.64 4-Ar [Tyr], 171.10
CONH–, 178.56 > C@N–, 180.29 –COO–.
(300 · 35 mm, CHCl /Me CO,7:1). The yield was 0.53 g
3
2
ꢀ
4
1
–
(8.14 · 10 mol), 52%. H NMR: 1.68 (m, 1H, Pro),
1.96 (m, 1H, Pro), 2.35 (m, 3H, Pro), 2.78 and 3.02 (AB
part of ABX, JAB = 14 Hz, JAX = 5.6 Hz, JBX = 4.2 Hz,
Found, %: C, 60.45; H, 4.970; N, 5.38. Calculated for
C H N NiO S · 0.1CHCl , %: C, 60.35; H, 5.06; N,
2H, –CH -Tyr), 3.11 (m, 1H, d-Pro), 3.31 (m, 1H, a-
3
7
37
3
7
3
2
D
5
5
.69. Mp 125–127 ꢀC. ½aꢁ2 +1975 (c 0.032, MeOH).
Pro), 4.16 (dd, 1H, a-Tyr), 3.43 and 4.26 (AB,
JAB = 12.6 Hz, 2H, –CH -Bn), 4.19 and (m, 2H,
OCH CH F, J
2
4
.1.6. Ni-(S)-BPB-(S)-Tyr-CH CH OTf (3c). To the
2
= 20.1 Hz), 4.74 (m, 2H, OCH CH F,
H–F 2 2
2
2
2
ꢀ
4
stirred solution of (5) (0.2 g, 3.08 · 10 mol) in
3
JH–F = 47.1 Hz), 6.65 (d, 2H, Ar), 6.86 (d, 1H, Ar), 6.95
and 7.10 (AB, JAB = 9 Hz, 4H, Tyr), 7.14 (m, 2H, Ar),
7.29 (m, 3H, Ar), 7.42 (m, 1H, Ar), 7.51 (m, 2H, Ar),
.5 mL of dry CH Cl at 0 ꢀC was added Et N
2
2
3
ꢀ
4
(0.14 mL, 9.59 · 10 mol) followed by CF SO Cl
(0.15 mL, 1.41 · 10 mol). After the addition was com-
3
2
ꢀ
3
19
8.00 (d, 2H, Ar), 8.19 (d, 1H, Ar). F NMR: ꢀ226, (–
D
plete, the cooling was stopped; the reaction mixture was
allowed to warm up to an ambient temperature and the
mixture was stirred for another 20 h. The reaction was
monitored by TLC (SiO , eluent CHCl /Me CO (7:1))
CH F). Mp 130–132 ꢀC. ½aꢁ +2300 (c 0.038, MeOH).
2
25
Found, %: C, 66.49; H, 5.31; N, 6.38. Calculated for
C H FN NiO , %: C, 66.48; H, 5.27; N, 6.46.
3
6
34
3
4
2
3
2
following the disappearance of the initial Ni-(S)-BPB-
S)-Tyr-CH CH OH. After the reaction was complete,
the solvent was removed in vacuo and Ni-(S)-BPB-(S)-
Enantiomerically pure (S)-FET (ee > 99.8%) was pre-
pared after the decomposition of the complex Ni-(S)-
BPB-(S)-Tyr-OCH CH F under standard procedure
(
2
2
2
2
3
0
1
Tyr-CH CH OTf was separated by preparative TLC
2
conditions.
H NMR: 3.15 and 3.26 (AB part of
2
(
SiO , elution with CHCl /Me CO (7:1)). The complex
2
ABX, JAB = 15 Hz, JAX = 7.56 Hz, JBX = 5.4 Hz, 2H, –
CH Ar), 4.26 (m, 2H, –OCH CH F), 4.34 (m, 1H, a-
2
3
was additionally purified by gel-filtration chromatogra-
phy (Sephadex LH-20, elution with C H /EtOH (3:1)),
2
2
2
H), 4.73 (m, 2H, –OCH CH F J = 47 Hz), 6.99
and 7.25 (AB, JAB = 8.7 Hz, 4H, Ar).
6
6
2
2
H–F
1
dried on air. Yield 33% (0.08 g). H NMR: 1.67 (m,
H, Pro), 1.96 (m, 1H, Pro), 2.33 (m, 3H, Pro), 2.77
and 3.00 (AB part of ABX, JAB = 13.8 Hz, JAX = 5.7 Hz,
1
1
3
C NMR: 34.75 (–CH Ar), 54.25 (a-C), 67.57 (–
2
JBX = 4.4 Hz, 2H, –CH -Tyr), 3.10 (m, 1H, d-Pro), 3.29
OCH CH F, J
= 18 Hz), 82.71 (–OCH CH F, J
2
2
2
C–F 2 2 C–
(
–
m, 1H, a-Tyr), 3.43 and 4.25 (AB, JAB = 12.5 Hz, 2H,
CH -Bn), 3.80 (m, 2H, OCH CH OTf), 4.21 (m, 3H,
= 164 Hz), 115.35 (C4, Ar), 126.99 (C1, Ar), 130.75
F
1
9
(C2, Ar), 157.37 (C4, Ar), 171.59 (COOH). F NMR:
2
2
2
D
25
OCH CH OTs and a-Pro), 6.64 (d, 2H, Ar), 6.87 (d,
ꢀ223 (–CH F). ½aꢁ ꢀ3.94 (c 0.5, 6 N HCl).
2
2
2
1H, Ar), 6.93 and 7.09 (AB, JAB = 8.6 Hz, 4H, Tyr),
7.13 (m, 2H, Ar), 7.28 (m, 3H, Ar), 7.42 (m, 1H, Ar),
7.51 (m, 2H, Ar), 8.00 (d, 2H, Ar), 8.20 (d, 1H, Ar).
Found, %: C, 58.19; H, 6.19; N, 6.11. Calculated for
C H FNO , %: C, 58.14; H, 6.21; N, 6.16.
1
1
14
3
1
3
C NMR: 23.12 c-C [Pro], 30.647 b-C [Pro], 38.85 b-C
To prepare the racemic FET, part of the (S)-FET was
3
racemized, using earlier described procedure.
5
[
[
Tyr], 42.08 –OCH CH OTf, 57.32 d-C [Pro], 63.39 CH
Bn], 68.13 a-C [Tyr], 70.34 a-C [Pro], 71.57 –OCH2-
2
2
2
CH OMs, 72.52 CF SO -, 114.91 m-Ar [Tyr], 120.58
3-Ar [2-ABPh], 123.38 5-Ar [2-ABPh], 126.12 1-Ar [2-
ABPh], 127.19 4-Ph [Bn], 127.84 6-Ar [2-ABPh],
4.2. Radiochemistry
2
3
2
4.2.1. Materials and reagents. The commercially avail-
able reagents and solvents were used without further puri-
0
1
1
3
28.57 i-Ar [Tyr], 128.78 2-Ar [Tyr], 3,3 -Ph [Bn],
29.11 4-Ph [2-ABPh], 129.79 4-Ar [2-ABPh], 131.52
.3 -Ph [2-ABPh], 131.69 2,2 -Ph [Bn], 132.35 2-Ph [2-
0
1
8
fication unless stated otherwise. O-enriched water (97%)
was purchased from the Global Scientific Technologies,
Sosnovy Bor, Russia; anhydrous potassium carbonate,
4,7,13,16,20,24-hexaoxa-1,10-diazabicyclo-[8.8.8]hexac-
osane (Kryptofix, K2.2.2) were obtained from Aldrich;
anhydrous acetonitrile (DNA-quality) was bought from
0
0
ABPh], 133.29 i-Ph [Bn], 133.50 2 -Ph [2-ABPh],
1
34.25 i-Ph [2-ABPh], 142.89 2-Ar [2-ABPh], 157.90 4-
Ar [Tyr], 171.06 –CONH–, 178.54 >C@N–, 180.33 –
1
9
COO–. F NMR spectra: ꢀ72.92, s, CF SO –.
3
2

5002
R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
TM
Merck; Sep-Pak Light Waters Accell Plus QMA car-
tridges were purchased from Waters. O-(2-fluoroethyl)-
L-tyrosine (trifluoroacetate salt) was delivered by ABX,
Germany. Tetrabutylammonium hydrogen carbonate
was obtained by bubbling carbon dioxide through the
solution of commercially available 20% aqueous tetrabu-
tylammonium hydroxide (Merck) to the pH 8; the stock
solution obtained was kept in a sealed vial at 4 ꢀC.
v). The resulting solution was collected into 5 mL conic
vial, the solvents were gently removed under a stream of
nitrogen gas at 130 ꢀC until dryness. Two portions of
acetonitrile (0.5 mL) were added in succession and each
was evaporated in turn, to give the corresponding F-
reactive complex as a slightly brown residue.
1
8
1
8
4.2.5. General F-labeling procedure. The appropriate
precursor 3a–c (4.5–5.0 mg) was dissolved in 0.5 mL of
anhydrous acetonitrile. This solution was added through
the Teflon rubber septa into the vial containing the dry
4
.2.2. HPLC, TLC and other equipment and conditions.
The HPLC purification system consisted of a Gilson
Pump 305; an automatic sample injector (type VICI Val-
co) equipped with 2 mL loop; Gilson 116 UV absor-
1
8
[ F]fluorinating agent. Reaction mixture was heated at
80 ꢀC for 5 min without stirring. The aliquot of the reac-
tion mixture was taken by syringe, quenched with water,
and then analyzed by radioTLC (SiO2 plate, eluent:
bance detector in series with
a
Beckman 170
radiodetector to monitor the effluent radioactivity;
1
8
semi-preparative C18 l-Bondapak column, Waters,
EtOAc/CHCl /CH COOH (4:1:1)); (R [ F]fluoride:
3 3 f
1
0.1; R [ F]4: 0.53). Immediately, 0.5 mL of aqueous
8
3
00 · 7.8 mm; eluent aq 0.01 M CH COONH , pH 4/
3
4
f
C H OH 90/10 (v/v); flow rate 4.0 mL/min; k 254 nm.
0.5 M HCl was added following heating at 120 ꢀC for
5 min. An aliquot of the crude reaction mixture was ana-
lyzed by radioTLC (SiO2 plate, eluent: n-BuOH/
2
5
Analytical HPLC system included a Gilson Pump 305,
a Rheodyne type injector fitted with 20 lL loop, Gilson
16 UV absorbance detector in series with a Beckman
70 radiodetector; Chrom & Spec data acquisition soft-
1
8
1
1
CH COOH/H O/EtOH (4:1:1.6:0.5)); (R [ F]fluoride:
3 2 f
1
0.1; R [ F]FET: 0.63).
8
f
ware, Ampersand, Russia; system A: column Nucleosil
C18, Supelco, 250 · 4.6 mm, 5 lm; eluent aq 0.05 M
CH COONH , pH 3/C H OH 90/10 (v/v), flow
4.2.6. Semi-preparative HPLC purification. Crude reac-
tion mixture was cooled down, partly neutralized until
pH 3, and diluted three times with HPLC eluent until to-
tal volume of 2 mL. The resultant solution was intro-
duced onto HPLC loop with the help of a motor-
driven 5 mL disposable plastic syringe. The radioactive
3
4
2
5
1
.0 mL/min, k 254 nm; system B: column Crownpak
CR (+), Daicel Chemical Industries, Ltd, 150 · 4 mm;
eluent HClO pH 2/CH OH 90/10 (v/v), flow rate
.8 mL/min; k 254 nm. TLC analyses were run on pre-
4
3
0
1
8
coated plates of silica gel F254, Sorbfil, Lenchrom, Rus-
sia; radioactivity spots were detected using an automatic
radioTLC scanner, raytest GmBH, Germany. Authentic
FET was visualized by spraying with 0.2% ninhydrin
solution and heating. The nickel content of a few
batches of the final [ F]FET was determined by the
inductively coupled plasma mass spectroscopy (ICP-
MS PQ-3; Plasma Quad, VG, UK).
fraction containing [ F]FET, eluted between 8.0 and
9.5 min, was collected into the sterile 10 mL sealed vial.
Finally, the solution was passed through a sterile filter
(0.22 lm pore size, Millipore) and mixed with PBS buf-
fer, to adjust pH, isotonic formula, and ethanol concen-
tration in injectable solution (Table 3).
1
8
4.2.7. Quality control. The radiochemical and chemical
purities of the final preparation were determined by re-
versed phase HPLC (system A, Section 4.2.2). The iden-
1
8
.2.3. Radioisotope production. N.c.a. [ F]fluoride was
4
produced via the O(p,n) F nuclear reaction by the
1
8
18
18
tity of [ F]FET was confirmed under the same
1
8
proton bombardment (17 MeV, 15 lA) of [ O]H O
conditions by co-injection of the authentic sample avail-
able from commercial vendor (ABX, Germany) or syn-
thesized within the present study. t FET: 4.54 min; tR
2
(
97% isotopic enrichment) in a small volume low pres-
sure target of the MC-17 cyclotron, Scanditronix, Swe-
den. Radioactivity was measured with dose
R
1
8
a
[ F]FET: 4.99 min (radiodetector is situated after UV
1
8
calibrator PTW Curiementor-2, Germany. The entire
radiochemical synthesis was operated with the use of
computer-assisted Anatech RB 86 laboratory robot
detector). Enantiomeric purity of [ F]FET was deter-
mined by chiral HPLC (system B, Section 4.2.2) using
optically pure (S)-FET and a racemic mixture of the
(
ware. The system has been described elsewhere.
Anatech, Uppsala, Sweden) and associated ARC soft-
3
(R,S)-isomers, as the reference standards. t (R)-
R
6
18 18
[ F]FET: 7.21 min; t (S)-[ F]FET: 8.85 min. Specific
R
radioactivity was determined by HPLC (system A),
using reference (S)-FET of known concentration.
+ 18
.2.4. Preparation of the [Bu N] [ F]
ꢀ
4 and [K/
K2.2.2] [ F] Fluorinating agents. [ F]fluoride gener-
4
+
18
ꢀ
18
ated in water cyclotron target was collected on the
TM
Sep-Pak Light Waters Accell Plus QMA cartridge
4.3. Preliminary in vivo evaluation in rats
pre-conditioned by passing 10 mL of 0.5 M K CO
2
The biodistribution study was carried out in Wistar rats
(males, weighing 150–200 g) bearing Glioma35-derived
rats tumors (homografts) and turpentine-induced
inflammatory foci (aseptic abscess). For the induction
3
and 15 mL of water. Cartridge was flushed with helium
for at least 5 min to maximally remove the last traces of
1
8
18
O]water. The [ F]fluoride was than eluted from the
[
1
1
QMA resin by passing 2 mL of stock solution A or B
of the following composition: A: 2 mL MeCN,
of the tumor we used previously reported procedure.
Briefly, 10% suspension of Glioma35 tumor cells in 5%
dimethylsulfoxide solution in saline, stored in liquid
nitrogen, was thawed quickly at 37 ꢀC and injected sub-
cutaneously into the experimental animal (0.5 mL per
0
9
.075 mL of 20% aq solution of TBAC, pH 8; B:
.5 ± 0.4 mg (0.025 mmol) of K2.2.2, 1.7 ± 0.2 mg
(
0.012 mmol) of K CO , 2 mL of MeCN/H O (96/4 v/
2 3 2

R. N. Krasikova et al. / Bioorg. Med. Chem. 16 (2008) 4994–5003
5003
rat). When the tumor size reached the weight of about
0 g, the rats were killed by cervical dislocation, tumor
12. Wester, H.; Herz, M.; Weber, W.; Heiss, P.; Senekovitsch-
Schmidtke, R.; Schwaiger, M.; Stocklin, G. J. Nucl. Med.
5
1
999, 40, 205.
tissues were taken, pulverized in saline (1:10 w/v) by
passing through the sieve with 1 mm pore size and
pumping through the injection needle with 1 mm inner
diameter. This suspension (0.5 mL per rat) was injected
subcutaneously or into the muscle of the right hind limb
of rats. The tumors were allowed to grow for 14 days.
To induce aseptic inflammation, 10 lL of turpentine
oil was inoculated subcutaneously into the muscle of
the right hind limb for 24 h before the experiment.
1
1
1
3. Tang, G. H.; Wang, M. F.; Tang, X. L.; Luo, L.; Gan, M.
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1
8
of [ F]FET in 0.3 mL of the isotonic solution via jugu-
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gamma counter. The biodistribution of radioactivity
was accessed in 20, 40, 60, and 120 min after injection
2
004, 31, 1464.
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(
five rats for each time point) and the percentage of in-
jected dose per gram (% ID/g) was calculated. The tu-
mor-to-muscle or abscess-to-muscle ratios were then
calculated using right hind limb as a contralateral re-
gion. The animal distributions experiments were per-
formed in the laboratory of the Institute of
Experimental Medicine RAMS, St. Petersburg, under
Institutional Animal Care and National regulations gov-
erning the safe and humane use of laboratory animals in
research.
20. Rachinger, W.; Goetz, C.; Popperl, G.; Gildehaus, F. J.;
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007, 48, 519.
2
2
2
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Acknowledgments
The authors thank the cyclotron engineers Mr. Alexandr
Demianov and Mr. Vladimir Obolencev for performing
the irradiations of the fluorine-18 target. This work was
supported by Grant 2780 from the ISTC and a Grant
06-0308089-ofi from Russian Foundation of Basic
Researches.
25. Pauleit, D.; Zimmermann, A.; Stoffels, G.; Bauer, D.;
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