Synthesis of [18F]fluoroalkyl esters of carfentanil

Article abstract of DOI:10.1002/jlcr.995

With the aim to develop and evaluate new ligands for depicting the μ-opioid receptor with positron emission tomography, the ¹⁸F- fluoroalkyl esters of carfentanil, 3-carboxy-(2-[¹⁸F]fluoroethyl) fentanyl, (2-[¹⁸F]fluoroeth

Full text of DOI:10.1002/jlcr.995

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2005; 48: 771–779.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.995
Research Article
1
Synthesis of [ F]fluoroalkyl esters of carfentanil
8
Gjermund Henriksen, Michael Herz, Markus Schwaiger and
¨
Hans-Jurgen Wester*
Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universita¨t
Mu¨nchen, Ismaninger Strasse 22, 81675 Munich, Germany
Summary
With the aim to develop and evaluate new ligands for depicting the m-opioid receptor
1
8
with positron emission tomography, the
F-fluoroalkyl esters of carfentanil,
1
8
-carboxy-(2-[ F]fluoroethyl)fentanyl, (2-[ F]fluoroethyl-carfentanil) and 3-carboxy-
1
3-[ F]fluoropropyl)fentanyl (3-[ F]fluoropropyl-carfentanil) were prepared by a two-
18
3
8
18
(
step radiosynthesis. Reacting carfentanil carboxylate sodium salt, added 0.96 eqv.
of tetrabutyl ammonium hydroxide (TBAH), with no-carrier-added (n.c.a.) 2-
1
8
[
F]fluoroethyltosylate for 20 min at 1508C in dimethyl formamide (DMF) provided
18
2
-[ F]fluoroethyl carfentanil in an isolated radiochemical yield (RCY) of 36 ꢀ 8%, a
specific activity (S ) of 35 ꢀ 5 TBq/mmol (n=4) within a synthesis time of ꢁ100 min.
A
1
Similarly, 3-[ F]fluoropropyl carfentanil could be obtained by reacting the carfentanil
8
1
8
TBA/Na salt with 3-[ F]fluoropropyl iodide at 1608C in DMF (isolated
RCY=6 ꢀ 2%; ꢁ100 min, S =27 ꢀ 5 TBq/mmol, n=4). The developed methods
A
1
8
allow the production of the two F-labeled carfentanil derivatives in amounts and
specific activities necessary and relevant for a detailed preclinical evaluation of these
new potential m-opioid receptor ligands in vitro and in animal models. Copyright #
2
005 John Wiley & Sons, Ltd.
Key Words: fluorine-18; carfentanil; opioid receptor; fluoroalkyation
Introduction
1
1
11
C-labeled carfentanil ([ C]CAF; t ¼ 20:3 min) is a valuable tracer to
1
=2
assess the m-opiate receptor (mOR) system in physiologic and pathophysiologic
1
11
conditions with positron emission tomography (PET). Although [ C]CAF
has been used in various human PET-studies, its potent agonistic properties
*Corresponding author: Hans-Ju
Technische Universitat Munchen, Ismaninger Strasse 22, 81675 Munich, Germany.
¨
rgen Wester, Department of Nuclear Medicine, Klinikum rechts der Isar,
¨
E-mail: h.j.wester@lrz.tu-muenchen.de
¨
Contract/grant sponsor: Norwegian Research Council; contract/grant number: 151445/432
Contract/grant sponsor: Deutsche Forschungsgemeinschaft; contract/grant number: SFB 391
Copyright # 2005 John Wiley & Sons, Ltd.
Received 12 December 2004
Revised 7 June 2005
Accepted 8 June 2005

7
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G. HENRIKSEN ET AL.
O11CH3
O
O
O(CH2)n18F
O
O
N
N
N
N
n = 2,3
11
18
Figure 1. Structure of [ C]carfentanil and the F-labeled carfentanil derivatives
of study
and pharmacological effects implies administration in amounts of less than
2
11
2
.1 mg/70 kg. For 740 MBq of [ C]CAF administered as a bolus injection a
specific activity at the time of injection >88.8 TBq/mmol is indispensable.
1
1
Consequently, some institutions hesitate to establish [ C]CAF for clinical
research. To overcome this drawback, the development of alternative mOR
ligands with suitable binding characteristics and lower pharmacological
potency is recommended. Furthermore, attempts to measure the central
1
1
release of endogenous opioids by displacement studies in vivo using C-labeled
1
1
ligands are complicated by the relatively short half-life of C and the low
activity at the end of the scanning procedure with a concomitant low
1
8
18
sensitivity for signal changes. A F-labeled tracer ð F : t1 ¼ 109:7 minÞ for
=2
PET could improve the signal intensity and thereby the accuracy of such
protocols.
1
1
The strategy followed for the synthesis of [ C]CAF,
3,4
is based on
alkylation of carfentanil carboxylic acid sodium salt. Thus, a promising route
1
to F-labeled carfentanil analogues may be based on alkylation of the acid by
8
1
8
F-labeled alkylhalides, -tosylates or -triflates. Starting with the evaluation of
the analogue with the smallest structural changes, preclinical experiments with
1
[
8
F]fluoromethyl cafentanil revealed suitable brain uptake in mice but also
5
low in vivo stability. With the aim to stabilize the ester bond we synthesized
18
and evaluated higher F-fluoroalkyl ester homologues. As the ethyl- and
propyl ester of carfentanil (Figure 1) are less potent than carfentanil by a
6
factor of 13 and 133, respectively, they may be used as templates for the
development of radiopharmaceuticals with increased safety profile. Here, the
1
8
18
18
radiosynthesis of 2-[ F]fluoroethyl carfentanil ester ([ F]1) and 3-[ F]fluor-
18
opropyl carfentanil ester ([ F]2) is described.
Results and discussion
18
18
The strategy used for preparation of the F-alkyl esters of carfentanil [ F]1
1
and [ F]2 was based on a two-step radiosynthesis. The two syntheses consist
8
1
of F-fluorination of ethylene glycol-1,2-bistosylate and 1,3-diiodopropane,
8
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

18
F-LABELED CARFENTANIL
773
18
respectively, followed by F-fluoroalkylation of carfentanil carboxylic acid
sodium salt containing 0.96 M equivalents of tetrabutyl ammonium hydroxide
(TBAH). In initial experiments, the effect of temperature of the esterification
was investigated using N,N-dimethyl formamide (DMF) or dimethyl sulfoxide
(
(
DMSO) as solvents. Alkylation of the carboxylate with no-carrier-added
1
8
18
n.c.a.) [ F]fluoroethyltosylate at 1008C in DMF gave [ F]1 in an analytical
radiochemical yield (RCY) of about 7% after 30 min (Figure 2(A)). Although
1
the conditions chosen have been reported to be suitable for the F-
8
7
fluoroethylation of hydroxyl groups and also sterically hindered 28 aliphatic
hydroxyls, the low RCY obtained in this study reflects the comparatively low
8
nucleophilicity of the carboxylate salt. Increasing the reaction temperature to
1
208C increased the yield of the product to about 20% in 30 min. Compared to
the use of DMF, DMSO resulted in lower yields under otherwise identical
conditions, both at 100 and 1208C (Figure 2(A)). At 1408C in DMF, the
product seems to decompose based on the decrease in the RCY observed after
1
0 min (Figure 2(A)). Based on these initial experiments, DMF was chosen for
further studies on reaction optimization. Next, the influence of the addition of
sodium iodide to the reaction mixture on the RCY was investigated. This
1
strategy was chosen since direct preparation of 2-[ F]fluoro-1-iodo ethane,
8
18
another valuable F-alkylation reagent, has been found to result in very low
9
yields. To obtain this intermediate in situ, NaI was added in final
concentrations of 10–30 mM. At 1408C, an increase of the NaI concentration
from 10 to 20 mM increased the yields from 25 to 55% (Figure 2(B)). Highest
Carfentanil carboxylate sodium salt (16.6 mM),
tetrabutyl ammonium hydroxide (15.9 mM),
no-carrier added 2-[18F]fluoroethyltosylate,
7
7
6
6
5
5
5
0
5
0
5
0
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
DMF 120 ˚C
DMF 140 ˚C
DMSO 120 ˚C
DMF 100 ˚C
DMSO 100 ˚C
45
40
35
30
25
20
150 ˚C, 20 mM NaI,
1
1
1
1
40 ˚C, 20 mM NaI,
50 ˚C, 30 mM NaI,
60 ˚C, 20 mM NaI,
40 ˚C, 10 mM NaI,
15
10
5
5
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
(A)
reaction time (min)
(B)
reaction time (min)
18
Figure 2. Analytical RCY of 2-[ F]fluoroethyl carfentanil as function of
temperature and time without (A) or with NaI present (B)
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

7
74
G. HENRIKSEN ET AL.
yields were observed after 10 min at 1508C (20 mM NaI, DMF). However, for
reactions conducted at 150 and 1608C (both with 20 mM NaI, DMF) the
product seems to decompose after 10 min leading to a continuous decrease of
the RCY during the time of observation (Figure 2(B)). Furthermore, no
further improvement was observed when even higher concentrations of NaI
(30 mM) were used (Figure 2(B)).
18
The increase in reactivity of [ F]fluoroethyl bromide towards O- and
N-nucleophiles by addition of sodium iodide has been demonstrated
1
0
18
previously. However, our observation that F-fluroroethylation of a
carboxylate sodium salt, added TBAH, can be improved in the presence of
1
1
sodium iodide seems to be contrary to the observation of Wadsak et al. who
observed a decrease in the formation of flumazenil carboxylic acid ester by the
18
addition of sodium iodide when using 2-[ F]fluoroethyl bromide as alkylation
agent. However, on the basis of the predominantly detected side product, the
11
N-alkylated flumazenil carboxylic acid, it can be speculated that these
differences may be related to sodium iodide predominantly accelerating the
side-reaction of the des-ethyl flumazenil radiolabeling precursor used by
Wadsak et al.
4
11
11
It has been reported that the yield of [ C]CAF, formed via C-
methylation of the carfentanil carboxlic acid sodium salt, added TBAH,
1
8
decreases when excess TBAH is present. As a part of the present study, F-
fluoroethylation of phenyl acetic acid sodium salt was investigated as a simple
model system for the reaction, as this compound possess no other reactive
functional groups than the carboxylate. As can be seen from Table 1, when
using a concentration of NaI of 20 mM, a TBAH/carboxylate sodium salt
ratio of about unity is imperative to provide optimum yields.
Based on the results from the optimization studies, preparative runs of
1
1
8
[
[
F]1 were conducted under the most ‘robust’ conditions, i.e. by reacting 2-
8
F]fluoroethyltosylate with a solution of carfentanil sodium salt (16.6 mM),
TBAH (15.9 mM), NaI (20 mM) in DMF at 1508C for 20 min. For the
preparative runs, 2-[ F]fluoroethyltosylate was trapped on a trace-enrichment
18
1
Table 1. F-fluoroethylation of sodium phenyl acetate
8
a
b
Molar ratio of TBAH to sodium phenyl acetate
RCY (%)
0
0
1
2
3
.5
.95
.2
42 ꢀ 9
70 ꢀ 6
48 ꢀ 5
35 ꢀ 8
17 ꢀ 9
a
18
N.c.a. [ F]fluoroethyltosylate was reacted with sodium phenyl acetate (17 mM) in the presence of various
amounts of TBAH, added as a 1 M solution in methanol, and NaI (20 mM) in DMF at 1508C for 20 min.
b
As determined by analytical HPLC measurements (results are the means ꢀ SD, n=3).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

18
F-LABELED CARFENTANIL
775
cartridge after high-performance liquid chromatography (HPLC). The trace-
enrichment cartridge was directly connected in-line to the HPLC outlet and
subsequently eluted directly from the cartridge with DMF into the reaction
vial. This ensured that the volume of DMF used for the alkylation reaction
could be kept low without additional transfer and evaporation steps. At these
1
8
conditions, [ F]1 could be obtained in an isolated RCY of 36 ꢀ 8%
mean ꢀ SD, n=4) in a radiochemical purity of >97% and a specific activity
SA>35 TBq/mmol.
In comparison to the F-fluoroethylation of carfentanil carboxylic acid,
(
18
18
18
the corresponding F-fluoropropylation to obtain [ F]2 proceeded much
less effective (Figure 3). Even at 1608C using a carfentanil sodium salt
concentration of 16.6 mM, a TBAH of 15.9 mM in DMF, the analytical RCY
1
8
18
of 3-[ F]fluoroproyl iodide into [ F]2 was only about 12% (Figure 3).
18
The apparent lower reactivity of 3-[ F]fluoropropyl iodide as compared
18
to that of the 2-[ F]fluoroethyltosylate/sodium iodide system may reflect the
inferior leaving-group ability of the iodo-substituent of the former compound,
presumably due to less activating effects of fluorine in the 3-position
than is the case for the 2-tosylate/2-iodo substituent of the latter. The
18
preparative RCY of [ F]2 was 6 ꢀ 2%, and the product was obtained in a
radiochemical purity of >97% and a specific activity of S =27 ꢀ 5 TBq/
A
mmol (n=4).
Carfentanil carboxylate sodium salt (16.6 mM),
tetrabutyl ammonium hydroxide (15.9 mM),
no-carrier added 3-[18F]fluoropropyliodide
1
1
1
1
1
4
3
2
1
0
9
8
7
6
5
4
3
2
1
0
1
1
60 ˚C
50 ˚C
140 ˚C
1
1
30 ˚C
20 ˚C
0
10
20
30
40
reaction time (min)
18
Figure 3. Analytical RCY of 3-[ F]fluoropropyl carfentanil at different
temperatures as function of time
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

7
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G. HENRIKSEN ET AL.
18
18
The two new carfentanil analogues [ F]1 and [ F]2 can be prepared in an
overall synthesis time of approximately 100 min (from EOB) consisting of
1
8
18
5 min for the preparation of 2-[ F]fluoroethyltosylate or 3-[ F]fluoropropyl
4
iodide, 20 min for the alkylation reaction and a total of about 35 min for
HPLC separation and product formulation. All radioactive contaminants are
18
eluted before the F-labeled carfentanil analogues on a preparative reverse
phase C18 HPLC column thus ensuring high chemical purity. A specific
18
18
activitiy of 35 TBq/mmol for [ F]1 and 27 TBq/mmol for [ F]2 is less than
11
that required for use of [ C]CAF in the PET imaging of mOR in humans.
However, based on pharmacological data of their parent non-fluorinated
compounds, a lower pharmacological potency than that of carfentanil can be
1
8
18
18
expected for [ F]1 and [ F]2. In addition, the longer half-life of F may
allow a full kinetic analysis of the tracer after administration of a substantially
1
smaller amount of activity than is the case for a C-labeled tracer.
1
Experimental
Materials and equipment
Kryptofix 2.2.2. and acetonitrile were obtained from Merck Eurolab. The
sodium salt of N-[4-(carboxy)-1-(2-phenylethyl)-4-piperidinyl]-N-phenylpro-
panamide sodium salt (carfentanil carboxylate sodium salt) was purchased
from ABX Biochemicals. Other chemicals were purchased from Sigma-Aldrich
and used as received. The preparative and analytical HPLC systems were from
Sykam. For analytical HPLC, in-line detectors included a UV-detector
(254 nm) and a well-type NaI(Tl) detector. Preparative HPLC was performed
using a UV-detector (254 nm) and a Bioscan Flow-count system fitted with a
PIN detector. Mass spectra (MS) were recorded on the liquid chromato-
graphy–mass spectrometry system LCQ from Finnigan using the Hewlett
Packard series 1100 HPLC system.
Synthesis of 2-fluoroethyl carfentanil (1)
Five milligrams of carfentanil carboxylic acid sodium salt (12.4 mmol) was
dissolved in DMF containing 13 ml of a 1 M solution of TBAH in methanol.
1
.7 mg of 1-bromo-2-fluoroethane (13.4 mmol) and 2.0 mg of sodium iodide
(13.4 mmol) was added to the solution and the mixture heated at 1208C for 2 h.
After this the reaction was allowed to cool to room temperature and diluted
with water (10 ml). The resulting solution was extracted with 10 ml
dichloromethane (three times) and dried (MgSO ), filtered and concentrated
4
to an oil which was purified by RP-HPLC (semi-preparative Luna C column,
1
8
1
0 ꢂ 250 mm; Phenomenex) using linear gradient of 15–70% B in 20 min; 0.1%
trifluoro acetic acid (TFA) in water (A), 0.1% TFA in MeCN (B), flow rate of
ml/min. The eluent from the column was continuously monitored for UV
3
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

18
F-LABELED CARFENTANIL
777
absorbance at 254 nm. The fraction containing the product (capacity factor,
0
k ¼ 8:6) was evaporated to yield 1.7 mg of a yellowish oil (31%). Calculated
for C H FN O =426.53; MS: 427.5 ([M+1] ).
+
2
5
31
2
3
3
-fluoropropyl carfentanil (2)
3
-fluoropropyl carfentanil was prepared and purified according to the
synthesis of 1 using 1.9 mg of 1-bromo-3-fluoropropane (13.4 mmol) as
0
alkylation reagent. The fraction containing the product ðk ¼ 8:8Þ was
evaporated to yield 0.6 mg of a yellowish oil (11%). Calculated for
+
C H FN O ¼ 440:56; MS: 441.5 ([m+H] ).
25
31
2
3
Radiosynthesis – general methods
1
1
8
8
18
18
[
[
F]fluoride was produced through the O(p,n) F nuclear reaction. The
F]fluoride was obtained in a 34 mM solution of K CO (0.3 ml) and added
2
3
to a 2 ml conical vial containing 0.5 ml dry MeCN and 15 mg (39.9 mmol)
Kryptofix 2.2.2. The solvent was evaporated with heating under reduced
pressure. Azeotropic drying was repeated three times with 0.5 ml portions of
MeCN.
1
-[ F]fluoroethyltosylate
8
2
1
-[ F]fluoroethyltosylate was prepared according to the method described
8
2
7
previously. Briefly, 5 mg (12 mmol) of ethylene glycol-1,2-ditosylate in 250 ml
+
18 ꢄ
MeCN was added to the dried kryptate ([K ꢃ 2.2.2] / F ), the vial was then
18
sealed and heated at 908C for 5 min. 2-[ F]fluoroethyltosylate was purified by
reversed-phase HPLC (Lichrosorb C18 5 mm; 10 mm ꢂ 250 mm (CS –
chromatographie service) eluted with MeOH/H O (50:50, volume/volume,
2
0
flowrate 4 ml/min; k ¼ 5:7; preparative RCY 65 ꢀ 5%). After on-line fixation
of the product on a Strata X cartridge (33 mm; 30 mg/1 ml; Phenomenex) and
drying of the product by argon-flush, the product was eluted with 0.15 ml
DMF into a reaction vial.
1
-[ F]fluoropropyliodide
8
3
3
.6 mg of 1,3-diiodopropane (12.1 mmol) in 250 ml MeCN was added to the
dried kryptate, the vial was then sealed and heated at 808C for 5 min. 3-
1
[
8
F]fluoropropyliodide was purified by reversed phase HPLC (Lichrosorb C18
5
mm; 10 mm ꢂ 250 mm (CS) eluted with MeOH/H O (60:40, volume/volume,
2
0
flow rate 4 ml/min; k ¼ 6:4; preparative RCY 52 ꢀ 8%). After on-line fixation
of the product on a polystyrene cartridge (LiChrolut EN) and drying of the
product by argon-flush, the product was eluted with 0.15 ml DMF.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2005; 48: 771–779

7
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G. HENRIKSEN ET AL.
1
[
8
F]fluoroalkylesters of carfentanil – general methods
1
The F-fluoroalkylation agent solubilized in DMF and DMSO was added to
8
1
.0 mg of carfentanil carboxylic acid sodium salt (2.5 mmol) and TBAH (2.4 ml
of 1 M solution in methanol) and heated at temperatures of 100–1608C in a
sealed vial for 10–30 min and then cooled to room temperature. For the
determination of the dependency of the RCY on temperature, reaction time
1
8
and solvent as well as the addition of sodium iodide in the case of the F-
fluoroethylated carfentanil analogue, aliquots were drawn for analysis by
radio-HPLC.
For preparative purposes, the reaction mixture was diluted with 4 ml 0.1 M
ammonium formate, loaded into a 7 ml injection loop and transferred onto a
sample enrichment column (PRP-1 -polystyrene-divinylbenzene-, 10 mm;
1
0 mm ꢂ 50 mm, CS). The enrichment column was washed with 0.1 M
ammonium formate at a flow-rate of 2 ml/min for 5 min and subsequently
eluted in the reverse direction onto a semi-preparative column (m-Bondapak
C18 5 mm; 8 mm ꢂ 300 mm, CS) using a mobile phase consisting of MeCN/
0
.1 M ammonium formate (55:45, volume/volume) at a flow rate of 3 ml/min,
continuously monitoring for radioactivity and UV absorbance at 254 nm.
Analytical HPLC was performed on a Nucleosil 100 5 mm CN-column
0
18
18
(
4.6 mm ꢂ 250 mm; CS). The k -value of [ F]1 and [ F]2 under these
1
8
18
conditions was 3.9 and 4.2, respectively, and [ F]1 and [ F]2 were co-eluted
with their respective reference compounds. In addition, a C18 phase was used
to confirm coelution of the labeled compounds and the reference compounds
(
Nucleosil 100 C , 5 mm, 4.6 mm ꢂ 250 mm (CS); acetonitrile/0.1 M ammo-
1
8
0
18
nium formate (65:45, volume/volume)). The k -value of [ F]1 and [ F]2 under
18
these conditions was 5.5 and 5.8, respectively.
Acknowledgements
Financial support was received from the Norwegian Research Council project
No. 151445/432 and from SFB 391 of the Deutsche Forschungsgemeinschaft.
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