Prostate cancer PET bioprobes: Synthesis of [18F]-radiolabeled hydroxyflutamide derivatives

Article abstract of DOI:10.1016/j.bmc.2005.06.033

Approximately 80-90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl- propanamide [¹⁸F]-1 and N-(4-fluoro-3-(trifluoromethyl)phenyl)-2- hydroxy-2-methylpropanamide [¹⁸F]-2, derivatives of hydroxyflutamide, were synthesized as a fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were successfully labeled with a 10 ± 3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of 1500 ± 200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molecular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of prostate cancer.

Full text of DOI:10.1016/j.bmc.2005.06.033

Bioorganic & Medicinal Chemistry 13 (2005) 6195–6205
Prostate cancer PET bioprobes: Synthesis
of [18F]-radiolabeled hydroxyflutamide derivatives
Orit Jacobson,a Yossi Bechor,b Avi Icar,b Nurit Novak,b Atalia Birman,b Hanit Marom,b
Ludmila Fadeeva,b Elizabeth Golan,b Ilan Leibovitch,c Mordechai Gutman,d
Einat Even-Sapir,e Roland Chisin,a Michael Gozinb,* and Eyal Mishania,*
aDepartment of Medical Biophysics and Nuclear Medicine, The Hebrew University of Jerusalem,
Hadassah Hospital, Jerusalem 91120, Israel
bSchool of Chemistry, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv 69978, Israel
cDepartment of Urology, Meir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Saba 44281, Israel
dDepartment of Surgery A, Sapir Medical Center, Sackler School of Medicine, Tel Aviv University, Kfar Sava 44281, Israel
eDepartment of Nuclear Medicine, Tel Aviv Sourasky Medical Center, Sackler School of Medicine, Tel Aviv University,
Tel Aviv 64239, Israel
Received 1 June 2005; revised 16 June 2005; accepted 17 June 2005
Available online 27 July 2005
Abstract—Approximately 80–90% of prostate cancers are androgen dependent at initial diagnosis. The androgen receptor (AR) is
present in most advanced prostate cancer specimens and is believed to have a critical role in its development. Today, treatment of
prostate cancer is done by inhibition of AR using antiandrogens such as flutamide (pro-drug of hydroxyflutamide), nilutamide, and
bicalutamide. However, there is currently no noninvasive imaging modalities to detect, guide, and monitor specific treatment of AR-
positive prostate cancer. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-propanamide [18F]-1 and N-(4-
fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-2, derivatives of hydroxyflutamide, were synthesized as a
fluorine-containing imaging agent candidates. A three-step fluorine-18 radiosynthesis route was developed, and the compounds were
successfully labeled with a 10 3% decay corrected radiochemical yield, 95% radiochemical purity, and a specific activity of
1500 200 Ci/mmol end of bombardment (n = 10). These labeled biprobes not only may enable for the future quantitative molec-
ular imaging of AR-positive prostate cancer using positron emission tomography but may also allow for image-guided treatment of
prostate cancer.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
advanced prostate cancer specimens.5,6 Males who are
castrated at a young age do not develop prostate cancer,
implying that androgens are risk factors for prostate
cancer development.6 In addition, prostate-specific
expression of an androgen receptor transgene in a trans-
genic mouse induces prostate intraepithelial neoplasia.6
The androgen receptor (AR) is a member of the steroid/
thyroid hormone receptor superfamily and plays a crit-
ical role in the development and maintenance of male
secondary sexual phenotypes such as muscle, hair and
bone mass, prostate growth, and spermatogenesis.1–4
The AR is a cellular regulatory protein that upon andro-
gen binding migrates into the nucleus, binds to specific
DNA sequences called androgen response elements,
and modulates the transcription of target genes.1
Prostate cancer is estimated to represent 30% of new can-
cer cases in US men.6 Approximately 80–90% of prostate
cancers are androgen dependent upon initial diagnosis
and endocrine therapy of prostate cancer is directed to-
ward the reduction of serum androgens and inhibition
of AR.7 On the other hand, some very aggressive forms
of prostate cancer were shown to have lost the expression
of AR and are insensitive to inhibition of the AR.6,7 Tes-
tosterone and 5a-dihydrotestosterone (5a-DHT) are nat-
ural steroids that serve as the natural ligands for the AR
and can be used as replacement therapy for androgen
The AR is also believed to be involved in prostate carci-
nogenesis, and amplification of AR is present in most
Keywords: Antiandrogen; PET; Fluorine-18; Prostate cancer.
*
Corresponding authors. Tel.: +972 2 677 7931; fax: +972 2 642
1203; e-mail: mishani@md2.huji.ac.il
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.06.033

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O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
deficiency.8,9 Different ligands of the AR were reported
and can be divided into two main chemical structures—
steroidal and nonsteroidal—with two different function-
alities—androgenic and antiandrogenic classes.1 Antian-
drogens are used to counteract the undesirable actions of
excessive androgens. Nonsteroidal antiandrogens, such
as flutamide, nilutamide, and bicalutamide (Fig. 1), were
shown to bind exclusively to the AR and, therefore, have
few side effects.1,4 These agents are advantageous over
steroidal antiandrogens (e.g., megestrol acetate and
cyproterone acetate) in terms of specificity, selectivity,
and pharmacokinetic properties and are successfully
used in the clinic for the treatment of AR-dependent
prostate cancer.1,10 Since AR is a specific target for pros-
tate cancer treatment and loss of expression of AR has
been observed in several aggressive tumors, it is critical
to determine its role in individual patients to guide and
monitor treatment. To date, imaging tools, including
positron emission tomography (PET) for diagnosing lo-
cal recurrence and metastatic sites of prostate cancer, are
suboptimal.11–13 PET is a nuclear medicine imaging
modality, which allows for the three-dimensional, quan-
titative determination of the distribution of radioactivity
within the human body.14 PET allows accurate measure-
ment of radioactivity concentration in small volume ele-
ments in vivo as well as the ability to follow tracer
kinetics. PET requires the administration to the subject
of a molecule labeled with a positron-emitting nuclide
such as 15O, 13N, 11C, and 18F, which have half-lives of
2.037, 9.965, 20.39, and 109.8 min, respectively. The
major effort to visualize AR in prostate cancer using
PET has been focused on the labeling of steroids, such
as testosterone and dihydrotestosterone, or synthetic
steroid, mibolerone and metribolone (R1881) deriva-
tives.15 The most advanced PET agent for AR is a
derivative of dihydrotestosterone (Ki = 0.28 nM)4—
[18F]fluoro-dihydrotestosterone.15i,j,k Although the met-
abolic rate of this compound was rapid, fast tumor up-
take and prolonged retention of radioactivity were
observed in human studies.15i,j,k While androgenic ste-
roid radiopharmaceuticals have high affinity to the
AR, they often have inadequate AR selectivity and like
other steroids, tend to bind with other steroid recep-
tors.16 Anilide analogs such as flutamide, nilutamide,
and bicalutamide (Fig. 1) were the first group of nonste-
roidal androgen antagonists to be used as drugs, and
their chemical structures are natural candidates for
PET imaging agent development.
Figure 2. 18F-radiolabeled nonsteroidal antiandrogen derivatives.
report our successful radiosynthesis of (R)-3-bromo-N-
(4-fluoro-3-(trifluoromethyl)phenyl)-2-hydroxy-2-meth-
ylpropanamide [18F]-1 and N-(4-fluoro-3-(trifluoro-
methyl)phenyl)-2-hydroxy-2-methylpropanamide [18F]-
2 hydroxyflutamide derivatives (Fig. 2). These novel
radiopharmaceuticals may not only enable future nonin-
vasive-specific molecular imaging of AR-dependent
prostate cancer using PET but also for image-guided
treatment of prostate cancer.
2. Results and discussion
Hydroxyflutamide is a symmetric nonchiral molecule
(Ki = 175 nM);17 however, substitution of a single
hydrogen atom by halogen on one of the methyl groups
of the hydroxyflutamide molecule, introduces an asym-
metry, which results in the formation of a chiral center.
Several reports have indicated the large differences in
biological activity (AR-binding constants) of different
stereoisomers of hydroxyflutamide derivatives such as
bromine, fluorine, and iodine relative to flutamide and
bicalutamide. It was found that the R-enantiomers of
these derivatives would typically have much higher
AR-binding constants than their S counterparts [(R)-3-
bromo hydroxyflutamide Ki = 0.3 nM; (S)-3-bromo
hydroxyflutamide, Ki = 16.5 nM] flutamide and bicalu-
tamide (11 nM).2 According to the present working
hypothesis, it was assumed that radiolabeled ligands
with higher AR-binding constants would produce better
images, due to higher target affinity. On the basis of
this assumption, a radiochemical transformation was
designed to synthesize the (R)-enantiomer of 3-[18F]fluo-
ro-2-hydroxy-2-methyl-N-(4-nitro-3-(trifluoromethyl)-
phenyl)propanamide [18F]-6 in a one-step radiosynthesis
(Fig. 4). Since the nonlabeled fluorine derivative only
served as the chromatographic reference standard for
the identification of the labeled analog by HPLC, it
was prepared as a racemic mixture by a three-step syn-
thesis (Fig. 3), starting with biphasic reaction of fluoro-
acetone solution in diethyl ether with aqueous NaCN to
form the corresponding cyanohydrin 318 with a 50%
yield (Fig. 3). Refluxing of 3 in concentrated HCl pro-
duced ( )-3-fluoro-2-hydroxy-2-methylpropanoic acid
We report our efforts on the design, synthesis and radio-
synthesis of novel prostate cancer PET imaging agent
candidates labeled with fluorine-18. We will begin by
first discussing our unsuccessful approach to aliphatical-
ly incorporate the radiolabeled isotopes, and finally
Figure 1. Nonsteroidal antiandrogens.

O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
6197
Figure 3. Synthesis of compound 6. Reagents and conditions: (i) NH4Cl(q), NaCN(aq), diethyl ether, 10 ꢁC; (ii) concd HCl(aq), reflux, 3 h; (iii) thionyl
chloride, dimethylacetamide; 4-nitro-3-(trifluoro-methyl)benzenamine, ꢀ12 ꢁC.
Figure 4. First attempt to synthesize [18F]-radiolabeled hydroxyflutamide derivative [18F]-6. Reagents and conditions: (i) methacryloyl chloride,
NaOH(aq), acetone, 0 ꢁC; (ii) N-bromosuccinimide, CCl4, DMF, 0 ꢁC; (iii) 24% HBr(aq), 105 ꢁC; (iv) thionyl chloride, dimethylacetamide; 4-nitro-3-
(trifluoro-methyl)benzenamine, ꢀ12 ꢁC; (v) K[18F]FÆKryptofix complex, DMSO, 110 ꢁC; (vi) acetic anhydride, dimethylaminopyridine, pyridine,
35 ꢁC.
418 with a 32% yield similar to the reported procedure.
Attempts to improve the yield of this latter step by using
different acids such as H2SO4 or H3PO4, or under other
temperature conditions, resulted in even lower yields. In
the third step, the coupling process was performed via in
situ generation of acyl chloride 5 that was obtained by
the reaction of hydroxy acid 4 with thionyl chloride in
dry dimethylacetamide at ꢀ12 ꢁC. Formation of com-
pound 6 was found to be sensitive to the reaction tem-
perature. Optimization of this parameter showed that
the highest yield of the product of 45% was obtained
when the reaction temperature was kept within
ꢀ12 5 ꢁC range.
Compound 1119 and its derivatives served as precursors in
attempts to radiolabel the (R)-enantiomer of 3-[18F]fluo-
ro-2-hydroxy-2-methyl-N-(4-nitro-3-(trifluoro-meth-
yl)phenyl)propanamide [18F]-6 (Fig. 4). On the basis of
the published procedures, pure (R)-enantiomer of com-
pound 11 was prepared in four steps (Fig. 4). The required
chirality was defined by utilizing D-Proline as a chiral aux-
iliary.19 The synthesis was started by coupling of methac-
ryloyl chloride with D-Proline in 2.0 M NaOH aqueous
solution in acetone at 0 ꢁC.20 It was found that the use
of freshly distilled methacryloyl chloride and careful con-
trol of pH conditions within a 10.3 0.3 range are prereq-
uisites for this reaction and the desired (R)-1-(2-
methylacryloyl)pyrrolidine-2-carboxylic acid 719 was iso-
lated with a 81% yield. In the following cyclization step,
substantial improvement in the yield of (3R,8aR)-3-(bro-
momethyl)-3-methyl-tetrahydro-3H-pyrrolo[2,1-c][1,4]oxa-
zine-1,4-dione 819 (from 64% to 96%) was achieved by using
a light-protected reaction vessel and a mixture of dry CCl4
and DMF (4:5) as the solvent. Compound 8 was then
hydrolyzed in aqueous HBr at 105 ꢁC to the correspond-
ing hydroxy acid 919 with a 84% yield. Conversion
of 9 into (R)-3-bromo-2-hydroxy-2-methyl-N-(4-nitro-
3-(trifluoro-methyl)phenyl)-propanamide was
achieved by reacting in situ generated acyl chloride 10
with 4-nitro-3-(trifluoromethyl)-benzenamine in dry
1119

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O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
dimethylacetamide at ꢀ12 ꢁC. Compound 11 was ob-
tained with a 45% yield after silica gel chromatography
followed by selective precipitation from a diethyl ether/
hexanes solution.
that reaction of 12 with silver triflate furnished the epox-
ide 13;20 reaction with silver nosylate in dry THF at 60 ꢁC
furnished the (S)-3-hydroxy-2-methyl-1-(4-nitro-3-(tri-
fluoro-methyl)phenylamino)-1-oxopropan-2-yl acetate
14. The only desired derivative that could be obtained
with a 39% yield, following RP-C18 column chromatogra-
phy, was the acetyl-protected tosylate derivative 15, by
the reaction of 12 and silver tosylate (Fig. 5).
Attempts to substitute the bromine of compound 11 with
[18F]fluoride ion were unsuccessful and only H[18F]F gas
was detected. It was proposed that under fluorination
conditions, the hydroxyl group of compound 11 would
undergo deprotonation. This would interfere with the
labeling reaction since it would capture most of the
fluoride ions. Thus, the acetyl protecting group was
introduced by reacting 11 with acetic anhydride and dim-
ethylaminopyridine in dry pyridine. Similarly, the pro-
tected precursor, compound 12, did not produce any
fluorination products and therefore, it was concluded that
the bromide atom should be replaced by a much better
leaving group. Several good leaving groups, based on sul-
fonyl moiety, are frequently used in 18F-radiopharmaceu-
tical precursors, among them being the nosyl, tosyl, and
triflate groups. Compound 12 was subjected to a series
of reactions where silver salts of triflate, nosylate, and tos-
ylate in dry acetonitrile or THF were used, under light-
protecting conditions (Fig. 5). Due to the expected reac-
tivity of sulfonyl derivatives, a preliminary analysis of
crude reaction mixtures was performed by 1H, 13C
NMR, and MS, immediately after silver bromide filtra-
tion and evaporation of the reaction solvent. It was found
Attempts to convert precursor 15 to the target com-
pound [18F]-6 included a two-step reaction, in which
15 was reacted with K[18F]FÆKryptofix complex in dry
DMSO at 110 ꢁC, followed by treatment with 1.0 M
aqueous HCl at 105 ꢁC (Fig. 6). The radio-HPLC anal-
ysis of the products at the end of the reaction with
[18F]fluoride indicated formation of low quantities of
compound [18F]-16 probably because of (a) the tosyl
group is not a sufficient leaving group (b) The amide
NAH is also acidic because the electronegativity associ-
ated with the aromatic ring also undergoes deprotona-
tion that interferes with the labeling reaction by
capturing some of the fluoride ions. However, acid
hydrolysis did not produce the desired product [18F]-6.
Increasing the temperature of the hydrolysis step above
110 ꢁC resulted in the formation of side products, most
probably due to the hydrolysis of the amide bond. It was
then suggested that a better leaving and labile protecting
groups may produce better results.
Figure 5. Synthesis of compound 15. Reagents and conditions: (i) silver triflate, acetonitrile, 60 ꢁC; (ii) silver nosylate, THF, 60 ꢁC; (iii) silver
tosylate, acetonitrile, 60 ꢁC.
Figure 6. Attempt to convert potential precursor 15 to compound [18F]-6. Reagents and conditions: (i) K[18F]FÆKryptofix complex, DMSO, 110 ꢁC;
(ii) 1.0 M HCl(aq), 105 ꢁC.

O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
6199
Following a search for a better protecting group that
could be easily removed, the O-BOC derivative 18 was
selected.21 Compound 11 was treated with (BOC)2O
under various conditions. Figure 7 and Table 1 present
the results obtained during the development of suitable
conditions for the synthesis of the target BOC-derived
intermediate 18.
tion could have an important influence on the ability of
the OH group to act as a hydrogen bond donor. More-
over, these proposed mechanisms are also supported by
1H NMR measurements of compound 11 in CD3CN
and C6D6. Substantial changes in chemical shifts, from
4.69 ppm (in CD3CN) to 2.30 ppm (in C6D6) of the
OH proton in compound 11, are indicative that with
acetonitrile, intramolecular hydrogen bonds are disrupt-
ed by the solvent, making the proton of the OH group to
be more acidic (Table 2).
Reaction of 11 with (BOC)2O, dimethylaminopyridine,
and pyridine in acetonitrile at 40 ꢁC produced exclusive-
ly epoxide 13.3 Replacing the media to a less polar
solvent such as toluene resulted in the formation of
lactam 17, as a major product. The changes in products
obtained under the different conditions could be de-
scribed with two different reaction mechanisms (Fig.
8). With acetonitrile, the formation of epoxide 13 was
derived from a base-assisted intramolecular attack of
the hydroxyl group on the methylenebromide moiety.
On the other hand, with toluene, due to intramolecular
hydrogen bonds,19,22 the preferred conformation of 11
was placing the anilide nitrogen toward the methylene
group and enabling intramolecular ring closure to form
lactam 17. These suggested mechanisms are supported
by the work of Morris and co-workers23 who performed
an infrared spectroscopic study of hydroxyflutamide
derivatives and related compounds. By examining the
intramolecular hydrogen bonding in these types of mol-
ecules, they concluded that a single dominant conforma-
Performing the reaction in the absence of dimethylami-
nopyridine produced a mixture of products from which
the desired BOC derivative 18 was isolated with a 25%
yield (Fig. 7). Upon increasing the temperature of the
latter process to 60 ꢁC, a substantial reduction in the
yield of the desired product was observed. Attempts to
obtain the nosylate derivative of 18 by reaction with sil-
ver nosylate in dry acetonitrile at room temperature and
up to 40 ꢁC resulted in the recovery of the starting mate-
rial, while increased temperatures led to the decomposi-
tion of 18. It was suggested that since the BOC group
was too bulky, it may have blocked the SN2 attack on
the methylenebromide moiety.
At that point, the strategy was reevaluated and another
position for incorporating the fluorine atom was
selected. Specifically, the labeling was performed on
Figure 7. Reaction of compound 11 with (BOC)2O under various conditions. Reagents, specific conditions and obtained product yields are presented
in Table 1.
Table 1. Changes in product distribution due to variations in reaction conditions of 11 with (BOC)2O. Indicated yields of products correspond to the
actual isolated yields of the products
Solvent
Amount of base
Amount of
(BOC)2O (equiv)
Temperature
(ꢁC)
Yield of
13 (%)
Yield of
17 (%)
Yield of
18 (%)
i.
ii.
Acetonitrile
Toluene
Toluene
0.25 equiv DMAP + 1.50 equiv pyridine
0.25 equiv DMAP + 1.50 equiv pyridine
1.50 equiv pyridine
2.0
5.0
5.0
40
40
40
98
<2
37
0
90
37
0
<2
25
iii.

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O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
Figure 8. Proposed reaction mechanisms of the conversion of 11 to: (i) 13 in acetonitrile and (ii) 17 in toluene.
anhydride.25 Both of these methods resulted with very
1
Table 2. Chemical shifts in H NMR spectra of NH and OH protons
of compound 11 and of flutamide in CD3CN and C6D6
low conversions (20%). The final method used for oxida-
tion was with the HOFÆCH3CN complex in aqueous ace-
tonitrile26 which furnished 19 with a 95% yield and a
synthesis time of 5 min. Compound 19 was reacted with
the K[18F]FÆKryptofix complex in DMSO, using a con-
ventional microwave oven heating. [18F]fluoride SN2
nucleophilic attack took place on the most reactive
elecrophilic aromatic carbon attached to the nitro group
and a to the trifluoromethyl group27 as analyzed by cold
standards coinjection to the HPLC. The corresponding
fluorine-18-labeled derivative [18F]-20 was obtained
exclusively with a 60% radiochemical yield. The second
step of the radiosynthesis was the reduction of the nitro
residue to the corresponding amino [18F]-21, with a 50%
radiochemical yield, using Raney nickel and hydrazine
hydrate at 55 ꢁC.27 The third and final step was coupling
of compound 9 and 2-hydroxy-2-methylpropanoic acid
chlorides28 with the labeled amino moiety (30% radio-
chemical yield) to give the labeled hydroxyflutamide
derivatives [18F]-1 and [18F]-2 with an overall decay cor-
rected yield of 10 3% (end of synthesis), a total synthe-
sis time of 4 h and SA of 1500 200 Ci/mmol EOB
(determined by HPLC and calibration curve with non-
labeled standard; n = 10).
Solvent
OH (11) d
NH (11) d
NH (flutamide) d
CD3CN
C6D6
4.69
2.30
9.87
8.36
10.16
10.12
the aromatic ring replacing the nitro group to form new
labeled derivatives of hydroxyflutamide [18F]-1 and
[18F]-2 (Fig. 2). On the basis of the published studies,
these derivatives should also have high affinity to the
AR.1 Nonlabeled chromatographic standards 1 and 2
were synthesized. Compound 9 and 2-hydroxy-2-meth-
ylpropanoic acids were used as starting materials for
the syntheses of compounds 1 and 2, under similar
conditions described above, and were obtained with
56% (1) and 61% (2) isolated yields.
Compound 19 served as the key starting material for the
radiosynthesis of compounds [18F]-1 and [18F]-2 (Fig. 9).
It could be obtained by oxidation of the commercial
4-nitro-3-(trifluoromethyl)-aniline, using various oxida-
tion methods, such as 3-chloro-peroxybenzoic acid24 or
combination of hydrogen peroxide and trifluoroacetic
Figure 9. Radiosynthesis of [18F]-radiolabeleld hydroxyflutamide derivatives [18F]-1 and [18F]-2. Reagents and conditions: (i) HOFÆCH3CN(aq)
,
acetonitrile, dichloromethane, 0 ꢁC; (ii) K[18F]FÆKryptofix complex, DMSO, microwave; (iii) Raney nickel, hydrazine hydrate; (iv) compound 9,
oxalyl chloride, dimethylformamide, 0 ꢁC; (v) 2-hydroxy-2-methylpropanoic acid, oxalyl chloride, dimethylformamide, 0 ꢁC.

O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
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3. Conclusions
4.1.1. ( )-3-Fluoro-2-hydroxy-2-methylpropanenitrile (3).
To a stirred solution of NH4Cl (16.0 g, 299 mmol) in
water (45 mL), a solution of fluoroacetone (17 mL,
236 mmol) in diethyl ether (50 mL) at 10 ꢁC was added.
Then, to the resulting emulsion, a solution of NaCN
(13.0 g, 265 mmol) in water (30 mL) was added and
the reaction mixture was stirred at room temperature
for 10 h. After that time, organic and aqueous phases
were separated, the aquoeus phase was extracted with
diethyl ether (3· 200 mL), and ethereal extracts were
combined with organic phase and dried over MgSO4.
Evaporation of organic solution gave 3 as a yellow oil
(12.13 g, 50%) that was used further without purifica-
The objective of the described work was to develop
radiosynthetic methodologies for the preparation of
novel 18F-containing hydroxyflutamide derivatives, with
potential affinity for the androgen receptor. These com-
pounds may present a better alternative to radiolabeled
steroid-based androgen ligands for noninvasive molecu-
lar imaging of AR-dependent prostate cancer using
PET. This developed methodology for nonsteroidal ani-
lide-type 18F-labelled radiopharmaceuticals can serve as
a platform for the preparation of a wide spectrum of
specific prostate cancer PET imaging agents and may
lead to image-guided treatment of prostate cancer.
1
tion. H NMR (400 MHz, DMSO-d6): d 1.47 (d, 3H,
J = 2 Hz), 4.35, 4.46 (ABq, 2H, J = 2.5 Hz). 13C NMR
(100 MHz, DMSO-d6): d 23.22 (d, J = 9 Hz), 66.75 (d,
J = 19 Hz), 86.44 (d, J = 178 Hz), 121.06 (d,
J = 3.5 Hz). 19F NMR (376 MHz, CDCl3): d ꢀ129.53
(dd, JHAF = 54 .41 Hz). IR (CH2Cl2): 3562, 2980, 1601,
1045, 885/cm.
4. Experimental
4.1. General methods
All operations with air- and moisture-sensitive com-
pounds were performed by the Schlenk techniques un-
der argon atmosphere. All solvents were of analytical
grade or better. Toluene and THF were distilled over
sodium/benzophenone; other solvents were purchased
4.1.2. ( )-3-Fluoro-2-hydroxy-2-methylpropanoic acid
(4). A stirred suspension of 3 (12.0 g, 116 mmol) in con-
centrated aqueous HCl (30 mL) was refluxed for 3 h.
After cooling to room temperature, a white precipitate
was filtered out and washed with cold ether to give 4
as a white solid (4.59 g, 32%). Mp 97.0–98.6 ꢁC (lit.18
99–100 ꢁC). 1H NMR (400 MHz, DMSO-d6): d 1.23
(d, 3H, J = 2 Hz), 4.38 (ddd, 2H, J = 57, 48, 9 Hz). 13C
NMR (100 MHz, DMSO-d6): d 21.51 (d, J = 18 Hz),
73.20 (d, J = 18 Hz), 87.72 (d, J = 173 Hz), 174.02 (d,
1
as anhydrous. H and 13C NMR spectra were recorded
on 200 or 400 MHz spectrometers in CDCl3 or DMSO-
1
1
d6. H and 13C NMR signals are reported in ppm. H
NMR signals are referenced to the residual proton
(7.26 ppm for CDCl3 or 2.50 ppm for DMSO-d6) of a
deuterated solvent and for 13C NMR spectra, the signal
of CDCl3 (77.16 ppm) or DMSO-d6 (39.52 ppm) was
used as a reference. 13C NMR spectra interpretations
were supported by DEPT experiments. Mass spectra
were obtained on a spectrometer equipped with CI,
EI, and FAB probes and on a spectrometer equipped
with ESI probe. HRMS results were obtained on MAL-
DI-TOF and ESI mass spectrometers. IR spectra were
recorded on FTIR spectrometer. Optical activity of
the chiral molecules was measured in a polarimeter
equipped with optical rotation 1 dc cell in CH2Cl2 or
DMSO solutions. The progress of reactions was moni-
tored by TLC (SiO2) and visualized by UV light or
developed with vanillin spray (1.0 g in 95.0 mL ethanol,
5.0 mL water, and 1.0 mL concentrated H2SO4) or with
iodine chamber. Flash chromatography was carried out
on SiO2 (0.04–0.063 mm). Microwave heating was per-
formed in a conventional oven operating at 500 W (full
power). HPLC was performed on system with variable
wavelength detector operating at 254 nm, unless other-
wise noted, and with a radioactivity detector with a
NaI crystal. Two systems were used: a reverse phase sys-
tem employing C-18 column (5 lm, 250· 10 mm) and
35% CH3CN/65% H2O as eluent, at flow rate of
3.5 mL/min. Fractions of 4.0 mL were collected. Analy-
sis of formulated radiotracer was performed on a re-
versed phase system using a C-18 column (10 lm,
300· 3.9 mm) and 40% CH3CN/60% H2O as solvents
at a flow rate of 1 mL/min. The selected eluent fractions
were diluted with water, loaded onto an activated
(EtOH) and equilibrated (water) C-18 Sep-Pak (classic,
short body). The cartridge was washed with 10% EtOH
and 90% saline.
J = 4.5 Hz). 19F NMR (376 MHz, DMSO-d6):
d
ꢀ131.98 (ddd, J = 722, 278, 55 Hz). HR-MS (CI): m/z
123.0 (MH+, 100); m/z calcd for C4H8FO3 (MH+):
123.0457. Found: 123.0452. IR (KBr): 3109, 2933,
1753, 1157, 815/cm.
4.1.3. ( )-3-Fluoro-2-hydroxy-2-methyl-N-(4-nitro-3-(tri-
fluoromethyl)phenyl)propanamide (6). To a solution of 4
(4.59 g, 37.62 mmol) in dry dimethylacetamide (44 mL),
thionyl chloride (3.45 mL, 48 mmol) was added drop-
wise at ꢀ12 ꢁC under Ar. At the same temperature,
the resulting mixture was stirred for 3 h and then a
solution of 4-nitro-3-(trifluoromethyl)benzenamine
(7.75 g, 38 mmol) in dry dimethylacetamide (51 mL)
was added dropwise. The reaction mixture was allowed
to warm up to room temperature and stirred for addi-
tional 6 h. After dimethylacetamide evaporation, the
resulting orange oil was purified by flash chromatogra-
phy (SiO2; CH2Cl2; Rf = 0.3) to give crude 6 as a yel-
low solid that was further purified by selective
precipitation from diethyl ether/hexanes to yield pure
6 as a light-yellow solid (5.25 g, 45%). Mp 99.8–
1
101.1 ꢁC. H NMR (400 MHz, CDCl3): d 1.71 (d, 3H,
J = 2 Hz), 4.70 (dd, 1H, J = 48, 10.5 Hz), 4.81 (dd,
1H, J = 48, 10.5 Hz), 8.03 (dd, 1H, J = 8.5, 12 Hz),
7.07 (d, 1H, J = 8.5 Hz), 8.14 (d, 1H, J = 2 Hz). 13C
NMR (100 MHz, CDCl3): d 17.21 (d, J = 4.5 Hz),
83.12 (d, J = 180 Hz), 85.18 (d, J = 18.5 Hz), 121.44
(q, J = 272 Hz), 124.32, 126.41, 1344.77, 1447.05,
151.63, 170.71 (d, J = 3.5 Hz). HRMS (MALDI-
TOF): m/z 133.03 (MNa+, 100); m/z calcd for
C11H11N2O4F4 (MH+): 311.0649. Found: 311.0650.

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O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
IR (KBr): 3447, 1735, 1032, 519/cm. UV–vis (MeCN):
kmax 201, 253 nm.
tate (4· 200 mL). The combined ethyl acetate fractions
were extracted with saturated NaHCO3 aqueous solu-
tion (4· 200 mL), the resulting aqueous solution was
acidified to pH 1, and then was extracted with ethyl ace-
tate (4· 200 mL). The combined ethyl acetate extracts
were dried over MgSO4 and evaporated to yield 9 as a
white solid (5.86 g, 84%). Mp 111.4–113.1 ꢁC (lit.19
106.5–109.0 ꢁC). 1H NMR (200 MHz, DMSO-d6): d
1.47 (s, 3H), 3.62, 3.75 (ABq, 2H, J = 10.0 Hz). 13C
NMR (50 MHz, DMSO-d6): d 24.55, 41.02, 73.19,
174.60. MS (CI): m/z 182.9 (M+, 25), 103.0 (M+ꢀBr,
100).
4.1.4. (R)-1-(2-Methylacryloyl)pyrrolidine-2-carboxylic
acid (7). To a solution of D-proline (50.0 g, 434 mmol)
in 2.0 M aqueous NaOH (240 mL) and acetone
(240 mL), a solution of methacryloyl chloride (32 mL,
443 mmol) in acetone (240 mL) was added dropwise at
0 ꢁC. During the addition, the reaction pH was moni-
tored and kept within 10.3 0.3 range via simultaneous
dropwise addition of 2.0 M aqueous NaOH. After the
end of addition, the reaction mixture was allowed to
warm up to room temperature and stirred for additional
4 h. Following the evaporation of acetone, the mixture
was acidified to pH 2 with concentrated HCl and NaCl
was added till saturation. The resulting solution was
extracted with ethyl acetate (3· 250 mL) and combined
organic extracts were dried over MgSO4. After solvent
evaporation, the crude product was purified by crystalli-
zation (ethyl acetate/hexanes) to yield 7 as a white solid
(64.05 g, 81%). Mp 103.5–104.5 ꢁC (lit.19 102.5–
4.1.7. (R)-3-Bromo-2-hydroxy-2-methylpropanoyl chlo-
ride (10). A round bottom flask with a nitrogen flow
connector was charged with compound 9 (40 mg,
0.22 mmol) in 2.0 mL of dry dichloromethane. To this
solution, oxalyl chloride (38.5 lL, 0.44 mmol) in dry
DMF (100 lL) was added under nitrogen at ꢀ5 ꢁC to
ꢀ10 ꢁC. The resulting mixture was stirred for 3 h under
the same conditions. The excess of oxalyl chloride and
the dichloromethane were evaporated under vacuum.
Compound 10 was used for the radiosynthesis without
further purification.
1
103.5 ꢁC). H NMR (200 MHz, DMSO-d6) [Major rot-
amer]: d 1.94 (s, 3H), 1.90–2.10 (m, 2H), 2.25–2.40 (m,
2H), 3.35–3.55 (m, 2H), 4.32–4.35 (m, 1H), 5.20, 5.33
(ABq, 2H, J = 12.0 Hz); [Minor rotamer]: d 1.88 (s,
3H), 1.90–2.10 (m, 2H), 2.25–2.40 (m, 2H), 3.60–3.65
(m, 2H), 4.52–4.55 (m, 1H), 5.20–5.33 (ABq, 2H,
J = 24 Hz). 13C NMR (50 MHz, DMSO-d6) [Major rot-
amer]: d 19.53, 24.72, 28.91, 48.77, 58.25, 116.44, 140.83,
169.13, 173.27; [Minor rotamer]: d 19.66, 22.35, 30.97,
40.67, 60.25, 115.28, 141.60, 170.01, 174.04. MS (CI):
m/z 184.1 (MH+, 100). IR (KBr): 3509, 2960, 1734,
1587, 1458, 1174/cm.
4.1.8. (R)-3-Bromo-2-hydroxy-2-methyl-N-(4-nitro-3-(tri-
fluoromethyl)phenyl)propanamide (11). To a solution of 9
(5.0 g, 27.32 mmol) in dry dimethylacetamide (32 mL),
thionyl chloride (2.5 mL, 31.90 mmol) was added drop-
wise at ꢀ12 ꢁC under Ar. At the same temperature, the
resulted mixture was stirred for 3 h and then a solution
of
4-nitro-3-(trifluoromethyl)benzenamine
(5.63 g,
27.32 mmol) in dry dimethylacetamide (37 mL) was
added dropwise. The reaction mixture was allowed to
warm up to room temperature and stirred for additional
6 h. After dimethylacetamide evaporation, the crude
orange oil was purified by flash chromatography
(SiO2; CH2Cl2; Rf = 0.3) to give a yellow wax that was
further purified by selective precipitation from diethyl
ether/hexanes solution to give 11 as a light-yellow solid
(4.56 g, 45%). Mp 103.0–103.8 ꢁC (lit.19 100.0–
4.1.5. (3R,8aR)-3-(Bromomethyl)-3-methyl-tetrahydro-
3H-pyrrolo[2,1-c][1,4]oxazine-1,4-dione (8). To a solu-
tion of 7 (112.59 g, 615 mmol) in dry CCl4 (320 mL)
and dry DMF (400 mL), a solution of NBS (142.20 g,
800 mmol) in dry DMF (500 mL) was added dropwise
in light-protected environment at 0 ꢁC. After the end
of addition, the reaction mixture was stirred at 0 ꢁC
for 2 h, allowed to warm up to room temperature and
stirred for additional 48 h. Following the evaporation
of CCl4, saturated NaCl aqueous solution (700 mL)
was added and the mixture was extracted with ethyl ace-
tate (3· 400 mL). The combined organic extracts were
washed with water (3· 200 mL) and dried over MgSO4.
After ethyl acetate evaporation, the crude product was
purified by crystallization (CH2Cl2/hexanes) to yield 8
as a white solid (154.64 g, 96%). Mp 158.1–159.9 ꢁC
1
101.5 ꢁC). H NMR (200 MHz, DMSO-d6): d 1.50 (s,
3H), 3.60, 3.85 (ABq, 2H, J = 10.5 Hz), 6.43 (s, 1H),
8.21 (d, 1H, J = 9.0 Hz), 8.37 (dd, 1H, J = 2.0, 9.0 Hz),
8.57 (d, 1H, J = 2.0 Hz), 10.60 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): d 24.79, 41.28, 74.35, 118.21 (q,
J = 6 Hz), 122.37, 122.98 (q, J = 33 Hz), 123.02,
127.15, 141.60, 142.89, 173.55. MS (CI): m/z 373.0
(MH+, 7), 291.1 (MH+ꢀHBr, 100).
1
(lit.19 152–154 ꢁC). H NMR (200 MHz, DMSO-d6): d
4.1.9. (R)-3-Bromo-2-methyl-1-(4-nitro-3-(trifluorometh-
yl)phenylamino)-1-oxopropan-2-yl acetate (12). To a
solution of 11 (400 mg, 1.078 mmol) and DMAP
(13 mg, 0.106 mmol) in dry pyridine (5.0 mL) acetic
anhydride (310 lL, 3.279 mmol) was added by syringe.
The mixture was heated at 35 ꢁC under Ar for 10 h.
After solvent evaporation, the crude yellow oil was puri-
fied by flash chromatography (SiO2; ethyl acetate/hex-
anes, 2:3) to give 15 as a light-yellow oil (270 mg,
1.67 (s, 3H), 1.85–2.10 (m, 2H), 2.31–2.43 (m, 2H),
3.48–3.69 (m, 2H), 3.96, 4.13 (ABq, 2H, J = 12.0 Hz),
4.81 (dd, 1H, J = 7.5, 9.5 Hz). 13C NMR (50 MHz,
DMSO-d6): d 21.63, 22.95, 29.01, 37.86, 45.47, 57.265,
83.92, 163.11, 167.33. MS (CI): m/z 262.0 (MH+, 100).
IR (KBr): 3861, 1744, 1686, 1449, 1061, 649/cm.
4.1.6. (R)-3-Bromo-2-hydroxy-2-methylpropanoic acid
(9). A solution of 8 (10.0 g, 38.15 mmol) in 24% aqueous
HBr (170 mL) was heated to 105 ꢁC for 90 min. After
cooling to room temperature, solid NaCl was added till
saturation and the mixture was extracted with ethyl ace-
20
D
1
61%). ½aꢁ ꢀ17 (c 1.5, CH2Cl2). H NMR (400 MHz,
CDCl3): d 1.90 (s, 3H), 2.29 (s, 3H), 3.99, 4.31 (ABq,
2H, J = 11 Hz), 7.94 (d, 1H, J = 10 Hz), 8.02–8.03 (m,
2H), 8.43 (s, 1H). 13C NMR (100 MHz, CDCl3): d

O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
6203
21.84, 21.97, 34.84, 82.85, 119.01 (q, J = 6 Hz), 120.40,
123.17, 125.19 (q, J = 34 Hz), 127.06, 141.18, 143.51,
168.62, 169.44. HR-MS (CI): m/z 413.0 (MH+, 100);
m/z calcd for C13H12BrF3N2O5 (MH+): 412.99241.
Found: 412.99296. IR (CH2Cl2): 3411, 1753, 1532,
1170, 912/cm. UV–vis (MeCN): kmax 224, 286 nm.
(s, 3H), 4.31, 4.83 (ABq, 2H, J = 11 Hz), 7.31 (d, 2H,
J = 8 Hz), 7.71 (d, 2H, J = 8 Hz), 7.94–7.99 (m, 3H),
8.42 (s, 1H). 13C NMR (50 MHz, CDCl3): d 14.77,
19.98, 22.44, 70.67, 82.41, 119.45, 123.39, 127.51,
128.45, 130.62, 132.75, 141.51, 146.16, 169.03, 169.41.
MS (CI): m/z 505.10 (MH+, 15). UV–vis (MeCN): kmax
290 nm.
4.1.10.
(S)-N-(3-(Trifluoromethyl)-4-nitrophenyl)-2-
methyloxirane-2-carboxamide (13). To a solution of 11
(300 mg, 0.808 mmol) and DMAP (25 mg, 0.205 mmol)
and dry pyridine (100 lL, 1.236 mmol) in dry acetoni-
trile (32 mL) heated to 40 ꢁC under Ar, di-tert-butyl
dicarbonate (372 lL) was added by syringe in one por-
tion. The resulting yellow solution was stirred at 40 ꢁC
for 10 h. After solvent evaporation, the crude product
was purified by chromatography (SiO2; CH2Cl2/ethyl
4.1.13. tert-Butyl-(S)-1-(3-(trifluoromethyl)-4-nitrophen-
yl)-3-methyl-2-oxoazetidin-3-yl carbonate (17). To a solu-
tion of 11 (300 mg, 0.808 mmol) and DMAP (25 mg,
0.205 mmol) and dry pyridine (100 lL, 1.236 mmol) in
dry toluene (32 mL) heated to 40 ꢁC under Ar, di-tert-bu-
tyl dicarbonate (930 lL) was added by syringe in one por-
tion. The resulting yellow solution was stirred at 40 ꢁC for
10 h. After solvent evaporation, the crude product was
purified by chromatography (SiO2; CH2Cl2/ethyl ace-
tate/hexanes, 18%:18%:64%; Rf = 0.54) to give 17 as a yel-
acetate/ hexanes, 18%:18%:64%; Rf = 0.38) to give
20
D
13 as a yellow oil (230 mg, 98%). ½aꢁ ꢀ36 (c 1.6,
CH2Cl2). H NMR (400 MHz, CDCl3): d 1.85 (s, 3H),
3.73, 3.85 (ABq, 2H, J = 12 Hz), 8.02 (dd, 1H, J = 9,
2 Hz), 8.07 (d, 1H, J = 9 Hz), 8.12 (d, 1H, J = 2 Hz).
13C NMR (100 MHz, CDCl3): d 21.21, 33.60, 84.31,
123.96, 125.24 (q, J = 34 Hz), 126.46, 129.13, 134.64,
147.15, 151.50, 171.20. MS (ESI): m/z 289.20 (MꢀH+,
100), 306.90 (M+H2O, 100). IR (CH2Cl2): 3023, 2932,
1758, 1540, 1250, 418/cm. UV–vis (MeCN): kmax
253 nm. Compound 13 was also obtained by reaction
of 12 with silver triflate in acetonitrile at 60 ꢁC.
low solid (284 mg, 90%). Mp 142–145 ꢁC. ½aꢁ20 +24 (c 2.0,
1
D
1
CH2Cl2). H NMR (400 MHz, CDCl3): d 1.51 (s, 9H),
1.79 (s, 3H), 3.84, 4.20 (ABq, 2H, J = 3 Hz), 7.69 (dd,
1H, J = 1, 4.5 Hz), 7.75 (d, 1H, J = 1 Hz), 8.00 (d, 1H,
J = 4.5 Hz). 13C NMR (100 MHz, CDCl3): d 19.70,
27.68, 52.97, 84.46, 115.59 (q, J = 5.5 Hz), 119.69,
125.81 (q, J = 34 Hz), 127.50, 141.49, 143.05, 151.448,
165.27. HRMS (MALDI-TOF): m/z 413.08 (MNa+,
80); m/z calcd for C16H17N2O6F3Na (MNa+): 413.0936.
Found: 413.0812. IR (KBr): 2971, 1766, 1576, 1136,
826/cm. UV–vis (MeCN): kmax 225, 307 nm.
4.1.11. (S)-3-Hydroxy-2-methyl-1-(4-nitro-3-(trifluoro-
methyl)phenylamino)-1-oxopropan-2-yl acetate (14). To
a solution of 12 (77 mg, 0.187 mmol) in dry THF
(15 mL), solid silver p-nitrobenzenesulfonate (64 mg,
0.208 mmol) was added in a light-protected environment
at room temperature under Ar, the vessel was sealed and
stirred at room temperature for 10 h. After filtration of
silver bromide and solvent evaporation, the crude brown
wax was purified by flash chromatography (SiO2; ethyl
4.1.14.
(R)-2-(3-(Trifluoromethyl)-4-nitrophenylcarba-
moyl)-1-bromopropan-2-yl-tert-butyl carbonate (18). To
a solution of 11 (300 mg, 0.808 mmol) in dry pyridine
(100 ll, 1.236 mmol) and dry toluene (32 mL) heated
to 42 ꢁC under Ar, di-tert-butyl dicarbonate (930 lL)
was added by syringe in one portion. The resulting yel-
low solution was stirred at 42 ꢁC for 10 h. After solvent
evaporation, the crude product was purified by chroma-
acetate/hexanes, 2:3) to give 14 as a yellow solid
tography
(SiO2;
CH2Cl2/ethyl
18%:18%:64%; Rf = 0.58. Under these chromatographic
acetate/hexanes,
20
D
(62 mg, 95%). Mp 116.5–117.1 ꢁC. ½aꢁ ꢀ15 (c 2.3,
1
CH2Cl2). H NMR (400 MHz, CDCl3): d 1.56 (s, 3H),
2.16 (s, 3H), 4.43, 4.51 (ABq, 2H, J = 12 Hz), 8.03 (d,
1H, J = 9 Hz), 8.07 (dd, 1H, J = 9, 2 Hz), 8.13 (d, 1H,
J = 2 Hz), 9.22 (s, 1H). 13C NMR (100 MHz, CDCl3):
d 21.96, 23.55, 29.88, 69.86, 118.38 (q, J = 6 Hz),
122.18, 127.26, 141.53, 172.20, 172.93. MS (CI): m/z
351.1 (MH+, 100). HR-MS (CI): m/z calcd for
conditions Rf of 11 = 0.17, Rf of 13 = 0.38 and Rf of
20
D
17 = 0.54) to give 18 as a yellow oil (95 mg, 25%). ½aꢁ
ꢀ4 (c 2.5, CH2Cl2). 1H NMR (400 MHz, CDCl3): d
1.55 (s, 9H), 1.87 (s, 3H), 3.99, 4.33 (ABq, 2H,
J = 11 Hz), 7.99 (d, 1H, J = 9 Hz), 8.04 (dd, 1H, J = 9,
2 Hz), 8.07 (d, 1H, J = 2 Hz), 8.61 (s, 1H). 13C NMR
(100 MHz, CDCl3): d 23.80, 27.26, 57.99, 79.98, 84.65,
118.73, 122.76, 124.10, 126.48, 129.15, 140.60, 143.31,
14.74, 149.49, 150.57, 168.71. HRMS (MALDI-TOF):
m/z 493.02 (MNa+, 100); m/z calcd for C16H18N2O6F3-
NaBr (MNa+): 493.0192. Found: 493.0151. IR
(CH2Cl2): 3401, 2929, 1754, 1533, 1153, 811/cm. UV–
vis (MeCN): kmax 290 nm.
C13H14F3N2O6
(MH+):
351.080396.
Found:
351.080649. IR (KBr): 3487, 3316, 2928, 1526, 1142,
807/cm. UV–vis (MeCN): kmax 198, 227, 294 nm.
4.1.12.
(S)-2-Methyl-1-(4-nitro-3-(trifluoromethyl)phe-
nylamino)-1-oxo-3-(tosyloxy)propan-2-yl acetate (15).
To a solution of 12 (77 mg, 0.187 mmol) in dry THF
(15 mL) solid silver p-toluenesulfonate (78 mg,
0.280 mmol) was added in a light-protected environment
at room temperature under Ar, the vessel was sealed and
heated at 65 ꢁC for 10 h. After filtration of silver bro-
mide and solvent evaporation, the crude brown wax
was purified by flash chromatography (RP-C18;
4.1.15. (R)-3-Bromo-N-(4-fluoro-3-(trifluoromethyl)phen-
yl)-2-hydroxy-2-methylpropanamide (1). To a solution of
9 (1.0 g, 5.464 mmol) in dry dimethylacetamide (10 mL),
thionyl chloride (0.5 mL, 6.892 mmol) was added drop-
wise at ꢀ12 ꢁC under Ar. At the same temperature, the
resulted mixture was stirred for 3 h and then 4-fluoro-
3-(trifluoromethyl)benzenamine (0.8 mL, 6.222 mmol)
was added dropwise. The reaction mixture was allowed
to warm up to room temperature and stirred for
CH3CN) to give 15 as a yellow solid (37 mg, 39%).
20
Mp 144–146 ꢁC. ½aꢁ ꢀ4 (c 0.6, MeOH). 1H NMR
D
(200 MHz, CDCl3): d 1.73 (s, 3H), 2.23 (s, 3H), 2.44

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O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
additional 16 h. After dimethylacetamide evaporation,
the crude orange oil was purified by flash chromatogra-
phy (SiO2; CH2Cl2; Rf = 0.2). Compound 1 was obtained
target at the Hadassah-Hebrew University IBA 18/9
cyclotron and then transferred into a fluorination mod-
ule by a flow of argon. After trapping, it was loaded on
an anion exchange column, dried, eluted with 1 mL of
K2CO3 solution (2.76 mg/mL), and transferred to the
reactor and finally to the collecting vial. Reactive organ-
ic [18F]fluoride ion was prepared by adding 50–100 lL of
the K[18F]F solution to Kryptofix 2.2.2 (1.0 mg,
2.7 lmol) in acetonitrile. Azeotropic removal of water
and acetonitrile was achieved by heating under a stream
of nitrogen. The dried K[18F]FÆKryptofix 2.2.2 complex
was then dissolved in 300 lL anhydrous DMSO for use
in the radiolabeling.
20
as a white solid (1.04 g, 56%). Mp 103.0–104.5 ꢁC. ½aꢁ
D
1
ꢀ28 (c 0.5, CH2Cl2). H NMR (400 MHz, CDCl3): d
1.66 (s, 3H), 3.39 (s, 1H), 3.63, 4.04 (ABq, 2H,
J = 10.5 Hz), 7.21 (t, 1H, J = 9.0 Hz), 7.77 (dt, 1H,
J = 9.0, 4.0 Hz), 7.92 (dd, 1H, J = 6.0, 4.0 Hz), 8.85 (s,
1H). 13C NMR (100 MHz, CDCl3): d 24.91, 41.41,
75.44, 117.49, 117.65 (d, J = 22 Hz), 118.84 (q,
J = 5.0 Hz), 122.35 (q, J = 122.0 Hz), 125.27 (d,
J = 8.0 Hz), 133.28, 156.40 (d, J = 253.0 Hz), 171.48.
19F NMR (376 MHz, CDCl3): d ꢀ119.34, ꢀ61.93. MS
(CI): m/z 344.1 (MH+, 100), 343.1 (M+, 20). HRMS
(MALDI-TOF): m/z calcd for C11H10NO2F4NaBr
(MNa+): 365.9723. Found: 365.9692. IR (KBr): 3333,
2928, 1674, 1513, 1133, 609/cm. UV–vis (MeCN): kmax
283, 241 nm.
4.1.19.
yl)phenyl)-2-hydroxy-2-methylpropanamide
(R)-3-Bromo-N-(4-[18F]fluoro-3-(trifluorometh-
([18F]-1).
The K[18F]FÆKryptofix 2.2.2 complex in DMSO
(300 lL) was added to 19 (2–3 mg, 8–13 lmol) in a
screw-cap test tube (8 mL). The tube was capped, shak-
en and heated in the microwave for 3.5 min. After cool-
ing to ambient temperature in a water bath, the vial
content was diluted with 10 mL of water and loaded
onto an activated (EtOH) and equilibrated (water) C18
Sep-Pak cartridge (classic, short body). The cartridge
was washed with water (10 mL) and [18F]-20 was eluted
with EtOH (2 mL) into a small glass test tube. [18F]-20
was analyzed using HPLC and compared to cold stan-
dards (Sigma) by coinjection; this analysis indicated that
only the nitro group at the ortho to the trifluoromethyl
group was substituted exclusively. The reduction vessel
was prepared by adding to a V-vial (5 mL), sequentially,
a few borosilicate glass beads, EtOH–water (100 lL,
4:1), Raney nickel (50% slurry in water, 250 lL), and
hydrazine monohydrate (60 lL, 1.2 lmol). After cap-
ping with a septum-equipped screw cap (vented with a
large diameter needle), the vial was shaken and placed
in a 55 ꢁC heating block for 5 min. The solution of
[18F]-20 in ethanol was diluted with water (0.5 mL)
and was added slowly to the reduction vessel. After
5 min, an additional portion of hydrazine hydrate
(40 lL, 0.8 lmol) was added to the reaction vessel.
The reaction was stirred for additional 10 min and then
the vessel was cooled to ambient temperature in a water
bath and the vial content was filtered through a 0.45 mm
filter (polypropylene) into another glass test tube. [18F]-
21 was analyzed using HPLC and compared to cold
standard (Sigma) by coinjection. To the filtered solution
of [18F]-21 water (10 mL) was added and loaded onto an
activated (EtOH) and equilibrated (water) C18 Sep-Pak
cartridge (classic, short body). The cartridge was washed
with dry dimethylacetamide (0.7 mL) into a 10 mL flask,
which contained 10 under nitrogen at 0 ꢁC. The reaction
solution was stirred for 0.5 h in an ice bath and addi-
tional 1 h at room temperature to give 30% conversion
to the product [18F]-1. The solution was filtered through
a 0.2 mm filter (nylon) and injected onto the reverse
phase HPLC (semipreparative column). [18F]-1 was elut-
ed with a retention time of 28 min with a decay corrected
radiochemical yield of 10% (SOS). The product was then
analyzed by analytical reversed phase HPLC [retention
time = 15.2 min; chemical purity 90%; radiochemical
purity 95%, SA of 1500 Ci/mmol (EOB)] and compared
with nonlabeled standard by coinjection.
4.1.16.
N-(4-Fluoro-3-(trifluoromethyl)phenyl)-2-hy-
droxy-2-methylpropanamide (2). To a solution of 2-hy-
droxy-2-methylpropanoic acid (1.5 g, 14.408 mmol) in
dry dimethylacetamide (35 mL), thionyl chloride
(1.3 mL, 17.920 mmol)was addeddropwise at ꢀ12 ꢁC un-
der Ar. At the same temperature, the resulted mixture was
stirred for 3 h and then 4-fluoro-3-(trifluoromethyl)ben-
zenamine (1.90 mL, 14.76 mmol) was added dropwise.
The reaction mixture was allowed to warm up to room
temperature and stirred for additional 16 h. After dime-
thylacetamide evaporation, the crude orange oil was puri-
fied by flash chromatography (SiO2; CH2Cl2; Rf = 0.1).
Compound 2 was obtained as a light yellow solid
1
(2.327 g, 61%). Mp 93.3–94.9 ꢁC. H NMR (400 MHz,
CDCl3): d 1.58 (s, 6H), 2.79 (s, 1H), 7.19 (t, 1H,
J = 9.5 Hz), 7.75 (dt, 1H, J = 4.0, 9.0 Hz), 7.93 (dd, 1H,
J = 3.0, 6.0 Hz), 8.91 (s, 1H). 13C NMR (100 MHz,
CDCl3): d 28.89, 75.40, 118.41 (d, J = 22 Hz), 119.44 (q,
J = 6 Hz), 123.33 (q, J = 271 Hz), 125.87 (d, J = 8 Hz),
134.73 (d, J = 3.5 Hz), 157.10 (d, J = 250 Hz), 175.77.
19F NMR (376 MHz, CDCl3): d ꢀ120.08, ꢀ61.94. MS
(CI): m/z 266.2 (MH+, 100). HRMS (CI): m/z calcd for
C11H12NO2F4 (MH+): 266.080417. Found: 266.080291.
IR (KBr): 3291, 2926, 1668, 1134, 671/cm. UV–vis
(MeCN): kmax 284, 241 nm.
4.1.17. 1,4-Dinitro-2-(trifluoromethyl)benzene (19). A
solution of 3-(trifluoromethyl)-4-nitrobenzenamine
(1.0 g, 4.854 mmol) in CH2Cl2 (50 ml) was added to a
solution of HOF26 in aqueous CH3CN (0.4 M, 24 mL)
at 0 ꢁC. The reaction mixture was stirred for 5 min.
Evaporation gave 19 as a yellow solid (1.145 g, 95%).
1
Mp 35.0–36.0 ꢁC. H NMR (400 MHz, DMSO-d6): d
8.46 (d, 1H, J = 9 Hz), 8.69 (d, 1H, J = 2.5 Hz), 8.77
(dd, 1H, J = 9, 2.5 Hz). 13C NMR (100 MHz, DMSO-
d6): d 119.80, 122.37–122.71 (m), 123.80 (q, J = 5 Hz),
127.36, 129.95, 149.18, 150.17. 19F NMR (376 MHz,
DMSO-d6): d ꢀ59.99. MS (CI): m/z 237.1 (MH+, 25).
IR (KBr): 3102, 1550, 1294, 1172, 839/cm. UV–vis
(MeCN): kmax 248 nm.
4.1.18. K[18F]FÆKryptofix 2.2.2 complex. [18F]Fluoride
ion was produced by the 18O(p,n) 18F nuclear reaction
on 2 mL enriched [18O]water (95% isotopic purity) as a

O. Jacobson et al. / Bioorg. Med. Chem. 13 (2005) 6195–6205
6205
4.1.20. N-(4-[18F]Fluoro-3-(trifluoromethyl)phenyl)-2-hy-
droxy-2-methylpropanamide ([18F]-2). Compound [18F]-2
was prepared in the same manner as described above
and obtained with a decay corrected radiochemical yield
of 10% (SOS). [18F]-2 was analyzed by analytical
reversed phase HPLC with a retention time of 10 min,
chemical purity of 95%; radiochemical purity of 95%,
SA of 1500 Ci/mmol (EOB) and compared with nonla-
beled standard by coinjection.
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Acknowledgments
The authors thank the Israel Cancer Association, Van
Bitz Foundation and Tel Aviv University for their gen-
erous financial support (to M.G.); the Maiman Institute
for Proteome Research of Tel-Aviv University, for
their important contribution in performing MALDI-
and ESI-TOF HRMS experiments.
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