18F-labelling of A-ring substituted 16α-fluoro-estradiols as potential radiopharmaceuticals for PET imaging

Article abstract of DOI:10.1016/j.steroids.2008.09.006

The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer agent while the 4-methyl derivative has been shown to interact with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the 16α-¹⁸F-analogs of these estrogens could be suitable radiotracers to evaluate action mechanisms of the parent compounds. In this study we report the synthesis of the 16α-¹⁸F and 16α-¹⁹F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening of the intermediate 16β,17β-O-cyclic sulfones with [¹⁸F]F^- or F^- followed by deprotection.

Full text of DOI:10.1016/j.steroids.2008.09.006

steroids 7 4 ( 2 0 0 9 ) 42–50
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
18F-labelling of A-ring substituted 16␣-fluoro-estradiols as
potential radiopharmaceuticals for PET imaging
Naseem Ahmed1, Guillaume Garcia, Hasrat Ali, Johan E. van Lier∗
Department of Nuclear Medicine and Radiobiology, Faculty of Medicine and Health Sciences,
Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4
a r t i c l e i n f o
a b s t r a c t
Article history:
The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer
agent while the 4-methyl derivative has been shown to interact with cytoplasmic and
nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the
16␣-18F-analogs of these estrogens could be suitable radiotracers to evaluate action mech-
anisms of the parent compounds. In this study we report the synthesis of the 16␣-18F and
16␣-19F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening
of the intermediate 16␤,17␤-O-cyclic sulfones with [18F]F− or F− followed by deprotection.
© 2008 Elsevier Inc. All rights reserved.
Received 8 June 2007
Received in revised form
3 September 2008
Accepted 5 September 2008
Published on line 18 September 2008
Keywords:
2-Methoxyestradiol
4-Methylestradiol
16␣-fluoro[18F]-estradiol
Positron emission tomography (PET)
1.
Introduction
and human leiomyoma cells have been associated with apop-
tosis and collagen production suggesting its potential role in
Many cancers are characterized by the over expression of
selected receptors. Molecular imaging of the distribution and
densities of such receptors facilitates prognosis, the selec-
tion of optimal treatment protocols and the evaluation of
treatment response. Positron emission tomography (PET) is
a particularly attractive modality to visualize in vivo bio-
chemistry since it allows monitoring receptor densities and
a patient’s biochemical response to therapy.
2-Methoxyestradiol (2-MeOES), an endogenous metabolite
of estradiol (ES), has been shown to inhibit the prolifer-
ation of various cancer cell lines including breast cancer
[1–4], myeloma cells [5], osteoclasts [6], inhibit tubulin poly-
merization and possess antimitotic properties [7], inhibit
mitochondrial respiration [8], and affects antiangiogenic
activities [9]. The cytotoxic properties of 2-MeOES against rat
the treatment of uterine fibroids [10]. This derivative has also
been shown to possess potent chemo-preventive and radio-
sensitizing effects [3] and is currently in Phase II clinical trials
as an anticancer agent [11]. The cytotoxicity of 2-MeOES in vivo,
as well as anti-proliferative and pro-apoptotic effects in vitro,
were studied in human prostate cancer cells (PC3) by PET using
[
18F]fluoro-deoxy-d-glucose (FDG), [18F]fluoro-1-(2ꢀ-deoxy-2ꢀ-
fluoro-␤-d-arabinofuranosyl)thymine (FMAU) and 11C-labelled
choline as metabolic tracers [12,13]. Although 2-MeOES is
known to inhibit the growth of PC3 and to induce apoptosis
in a dose-dependent manner [3], these PET studies failed to
confirm a specific action mechanism.
Many physiologic actions are produced by endogenous
hormones such as estrogens. Estrogens have also been impli-
cated in neuroprotective effects with respect to damage from
∗
Corresponding author. Tel.: +1 819 564 5409; fax: +1 819 564 5442.
E-mail address: johan.e.vanlier@usherbrooke.ca (J.E. van Lier).
1
Present address: Department of Chemistry, Indian Institute of Technology Roorkee (IITR), Roorkee 247 667 (U.A.), India.
0039-128X/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2008.09.006

steroids 7 4 ( 2 0 0 9 ) 42–50
43
Alzheimer’s disease or bipolar disorder, major depression and
schizophrenia [14]. Estrogens could have a protector effect
toward schizophrenia by modulating the brain dopaminergic
D2 receptors sensitivity [15]. Estrogenic actions in the brain
are mediated by two major intra nuclear estrogen receptors
(ER-␣ and ER-␤) but there appear to exist a variety of other
ER-like nuclear, cytoplasmic and plasma membrane receptors
in the brain, structurally, functionally and immunochemically
distinct from ER-␣ and ER-␤ [16]. Estrogen replacement ther-
apy (ERT) is often used to alleviate psychological stress that
may occur during menopause; this method has also been pro-
posed for the prevention and treatment of early symptoms
of Alzheimer’s disease. No suitable technique is available to
asses the status of ER in the brain to provide diagnostic and
therapeutic solutions that can correlate different physiologic
and pathologic phenomena.
The 4-MeES has been shown to interact with cytoplasmic
and nuclear estrogen receptors in rat pituitary gland, hypotha-
lamus and uterus [17]. We have previously synthesized steroid
derivatives functionalized at C-2, C-4, C-7, C-11 and C-17 posi-
tions using alkyl, cyano, chloro, fluoro and iodo substituents
and tested their estrogen receptor binding affinity. Several
of these derivatives were labelled with 123I and 18F in order
to establish the effect of such substitutions on ER-mediated
uptake in various animal models. Selected 123I and 18F deriva-
tives were also studied in clinical trials for ER-mediated uptake
in breast cancer [18–22].
HR-MS) were determined with a V9 micromass model, ZAB-1F
apparatus at 70 eV ionization voltage (Chemistry Department,
University of Sherbrooke, Canada). High-pressure liquid chro-
matography (HPLC) was performed with a Waters 600 system,
using a silica gel column (7.8 mm × 250 mm, Waters, Nova-Pak
HR Silica 6 ␮m) with a built-in UV detector operated at 280 nm
and a radioactivity detector (Bioscan). An acetonitrile/water
(MeCN/H2O) solvent system gradient (MeCN/H2O: 8/2 to 2/8)
was used for purification of the radiolabelled products at a
flow of 2 mL/min.
2.1.1. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-17-one
(3)
A solution of 2 (2.7 g, 6.88 mmol) in acetone (80 mL) was cooled
at 0 ◦C in an ice bath and Jones reagent added drop wise with
stirring until a yellow color persisted. The reaction mixture
was further stirred at 0 ◦C for 5 min and then quenched by drop
wise addition of methanol until a green color persisted. The
resulting green suspension was diluted with water (100 mL)
and extracted with CH2Cl2 (3 × 30 mL). The organic fractions
were washed with water (40 mL), saturated aqueous NaHCO3
(15 mL) and brine (15 mL). The organic fraction was filtered
over anhydrous Na2SO4 and the solvent evaporated under
reduced pressure. The residue was crystallized from methanol
to give 3 in 60% yield: mp = 154–156 ◦C; 1H NMR (300 MHz,
CDCl3) 0.92 (3H, s, 18-CH3), 3.86 (3H, s, 2-OCH3), 5.11 (2H, s,
benzyl CH2), 6.64 (1H, s, C4-H), 6.79 (1H, s, C1-H), 7.29–7.46 (5H,
m, benzyl-H); MS (EI) m/z M+: 390.52.
Here we report the synthesis of 2-methoxy- and 4-methyl-
FES and their 16␣-[18F]fluoro analogs as potential tracers for in
vivo imaging of target tissues.
2.1.2. 2-Methoxy-3-benzyloxyestra-1,3,5(10),
16-tetraene-17-enol acetate (4)
Under argon, p-TsOH·H2O (0.2 g, 1.06 mmol) was added to a
mixture of 3 (0.66 g, 1.68 mmol), isopropenyl acetate (10 mL,
90.30 mmol) and acetic anhydride (10 mL, 106 mmol). The mix-
ture was heated to reflux for 5–6 h. Upon completion of
the reaction the mixture (monitored by TLC, 2% acetone in
CH2Cl2) was cooled to room temperature, poured into ice
water (∼150 mL) and stirred for another hour. The mixture
was extracted with CH2Cl2 (3 × 30 mL) and the organic fraction
was washed with water (30 mL), saturated aqueous NaHCO3
(20 mL) and water (30 mL), filtered over anhydrous Na2SO4 and
the solvent evaporated under reduced pressure. The material
was precipitated from methanol to give 4 in 82% yield: 1H NMR
(300 MHz, CDCl3), 0.91 (3H, s, 18-CH3), 2.20 (3H, s, 17-OAc), 3.87
(3H, s, 2-OCH3), 5.11 (2H, s, benzyl-CH2), 5.52 (1H, m, 16-H), 6.63
(1H, s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H, m, benzyl-H); MS
(EI) m/z M+ 432.55.
2.
Experimental
2.1.
General
Melting points (mp) were determined on a Fisher–Johns
melting apparatus and are uncorrected. Starting chemi-
cals and reagents were obtained commercially (analytical
grade) and used without further purification. Solvents were
sure sealed and HPLC grade, used without further purifi-
cation (Aldrich, Oakville, ON, Canada). 19-Nortestosterone
was obtained from Steraloids Inc. (Newport, RI 02840-0600,
USA). The 2-methoxy-3-benzyloxyestra-1,3,5(10)-trien-17␤-ol
(2) was prepared accordingly to a published procedure [23].
4-Bromo-3,17␤-estradiol (12) and 2-bromo-3,17␤-estradiol (13)
were prepared from 17␤-estradiol following a published pro-
cedure [24]. Reaction mixtures were purified by column
chromatography on silica gel (60–200-mesh). Thin layer chro-
matography (TLC) was conducted on silica gel pre-coated
plastic sheets, with fluorescent indicator (UV 254). Com-
pounds were visualized either by their color response upon
spraying with 15% H2SO4 in ethanol and heating at 120 ◦C
or by viewing under short-wave UV light and/or using
KMnO4 solution [KMnO4 (1 g), NaOH (8 g) in water (200 mL)] or
phosphomolybdic acid reagent (20 wt.% solution in ethanol).
Products were characterized by 1H NMR on a Bruker AC-
300 MHz spectrometer in chloroform-d6 using the residual
chloroform proton as an internal standard. Chemical shifts
are given in ı (ppm) and J-values in Hz. Mass spectra (EI and
2.1.3. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-16ˇ-
acetate-17-one (5)
A mixture of 4 (0.433 g, 1.0 mmol), Pb(OAc)4 (0.70 g) and AcOH
(6.0 mL) was stirred for 2.5–3 h. Then another 0.1 g of Pb(OAc)4
was added and the reaction mixture was stirred for 1 h. Fol-
lowing TLC (2% acetone in CH2Cl2) monitoring, the reaction
mixture was diluted with CHCl3 (30 mL), washed (2 × 15 mL,
aqueous 5% sodium thiosulfate; 3 × 50 mL, saturated aqueous
NaHCO3), dried over anhydrous Na2SO4, the solvent evapo-
rated and purified on Florisil. Elution with hexane–EtOAc (10:0
to 9:2) gave 5 in 70% yield: 1H NMR (300 MHz, CDCl3) 1.02 (3H,
s, 18-CH3), 2.17 (3H, s, 16-OAc), 3.87 (3H, s, 2-OCH3), 5.06 (1H,

44
steroids 7 4 ( 2 0 0 9 ) 42–50
t, J = 8.85 Hz, 16-H), 5.11 (2H, s, benzyl-CH2), 6.63 (1H, s, C4-H),
6.85 (1H, s, C1-H), 7.29–7.46 (5H, m, benzyl-H); MS (EI) m/z M+
448.55.
(3H, s, 18-CH3), 3.71 (3H, s, 2-OCH3), 4.08, 4.12 (1H, dd, J = 3.6,
29.5 Hz, 17␣-H), 4.85, 5.03 (1H, dm, J = 53 Hz, 16␤-H), 5.11 (2H, s,
benzyl-CH2), 6.63 (1H, s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H,
m, benzyl-H).
2.1.4. 2-Methoxy-3-benzyloxyestra-1,
3,5(10)-trien-16ˇ-acetate-17ˇ-ol (6)
2.1.8. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-16˛-
fluoro-17ˇ-ol (10)
A
solution of 5 (0.38 g, 0.85 mmol) and lithium tri-tert-
butoxyaluminium hydride (0.72 g) in THF (30 mL) was stirred
for 1 h. The reaction mixture was poured with stirring into
a mixture of ice (40 g), H2O (40 mL), and AcOH (5 mL). After
extraction with CHCl3 and usual work-up the mixture was
purified by flash chromatography in CH2Cl2–EtOAc (10:0 to 8:2)
to give 6 in 86% yield: 1H NMR (300 MHz, CDCl3) 0.88 (3H, s, 18-
CH3), 2.17 (3H, s, 16-OAc), 3.56 (1H, d, J = 3.5 Hz, 17␣-H), 3.86 (3H,
s, 2-OCH3), 5.11 (2H, s, benzyl-CH2), 5.12 (1H, m, 16-H), 6.63 (1H,
s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H, m, benzyl-H); MS (EI)
m/z M+ 450.54.
The tetramethylammonium salt 9 (38 mg) was dissolved in a
mixture of absolute EtOH (10 mL) and concentrated sulfuric
acid (50 ␮L). The solution was heated to 110oC for 5–10 min,
solvent was removed under reduced pressure and the residue
extracted with ether and after usual work-up afforded 10 in
90% yield. An analytical sample was obtained as a white pow-
der after crystallization from ether–EtOH: 1H NMR (300 MHz,
CDCl3) 0.81 (3H, s, 18-CH3), 3.73–3.95 (4H, m, 17␣-H and 2-OMe),
4.90, 5.07 (1H, dm, J = 53 Hz, 16␤-H), 5.11 (2H, s, benzyl-CH2),
6.63 (1H, s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H, m, benzyl-H);
HRMS calcd. for C26H31O3F, 410.5209, found 410.5212.
2.1.5. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-
16ˇ,17ˇ-diol (7)
2.1.9. 2-Methoxy-16˛-fluoro-3,17ˇ-estradiol (11)
Compound 6 (0.66 g, 1.47 mmol) was dissolved in 30 mL of
methanol:water (3:1) containing 5% K2CO3 and stirred at room
temperature for 24 h. Evaporation of the solvent at reduced
pressure afforded a residue, which was partitioned between
water (30 mL) and dichloromethane (30 mL). The organic phase
was separated and the aqueous phase was further extracted
with dichloromethane (50 mL), the combined organic frac-
tion was dried over anhydrous Na2SO4. After usual work up,
the residue was purified by flash chromatography using 2%
Compound 10 (0.50 mmol) was added to 10 mL of a satu-
rated solution of HCOONH4 (prepared by stirring 40–50 mg of
HCOONH4 in MeOH/THF, 2:1, v/v) followed by the addition of
Pd-C (20–30 mg, 10 wt%) and stirred at room temperature for
10–12 h. (Caution: Pd–C reacts violently with MeOH in the pres-
ence of air). Evaporating the solvent at reduced pressure gave
a residue that was dispersed in water and diethyl ether, the
organic fraction was collected, dried over anhydrous Na2SO4,
filtered and evaporated at reduced pressure. Crystallization
from ethanol gave 11 in 85% yield as a white crystalline solid.
Analysis by HPLC on a Waters Nova Pak HR-C18 (7.8 × 250 mm),
in 80:20 to 20:80 H2O/CH3CN at 2.0 mL/min, (UV detector at
280 nm) indicated the material was >99% pure (tR = 24 min);
mp = 154–156 ◦C; 1H NMR (300 MHz, CDCl3) 0.78 (3H, s, 18-CH3),
3.73–3.95 (4H, m, 17␣-H and 2-OMe), 4.85, 5.05 (1H, dm, J = 53 Hz,
16␤-H), 5.42 (1H, bs, 3-OH), 6.63 (1H, s, 4-H), 6.85 (1H, s, 1-H);
HRMS calcd. for C19H25O3F, 320.3984, found 320.3976.
methanol in chloroform as eluent to afford 7 in 90% yield: 1
H
NMR (300 MHz, CDCl3) 0.86 (3H, s, 18-CH3), 3.48 (1H, d, J = 3.2 Hz,
17␣-H), 3.86 (3H, s, 2-OCH3), 4.25 (1H, m, 16-H), 5.11 (2H, s,
benzyl-CH2), 6.63 (1H, s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H,
m, benzyl-H); MS (EI) m/z M+ 408.23.
2.1.6. 2-Methoxy-3-benzyloxyestra-1,
3,5(10)-trien-16ˇ,17ˇ-O-cyclic sulfone (8)
Compound 7 (0.204 g, 0.50 mmol) in anhydrous THF (8 mL)
was added while stirring to NaH (60% suspension in mineral
oil, 2.28 mmol, 90 mg) in THF (5 mL). After 10 min, a solution
of sulfonyldiimidazole (0.65 mmol, 128 mg) in anhydrous THF
(2.5 mL) was added drop wise and the mixture was stirred for
another hour. The reaction mixture was filtered and evapo-
rated. After extraction with ether the material was subjected
to usual work-up to afford product 8. An analytical sample
was purified by silica gel chromatography using 15% EtOAc in
hexane and crystallized from EtOH: 1H NMR (300 MHz, CDCl3)
1.01 (3H, s, 18-CH3), 3.86 (3H, s, 2-OCH3), 4.59 (1H, d, J = 3.8 Hz,
17␣-H), 5.11 (2H, s, benzyl-CH2), 5.16 (1H, m, 16␣-H), 6.63 (1H,
s, C4-H), 6.85 (1H, s, C1-H), 7.29–7.46 (5H, m, benzyl-H); HRMS
calcd. for C26H30O6S, 470.5778, found 470.5779.
2.1.10. 4-Bromo-3-hydroxyestra-1,3,
5(10)-trien-17-one (14)
Compound 14 was prepared from 12 (3.52 g, 10.0 mmol) in ace-
tone (100 mL), following the procedure detailed for compound
3. The material was crystallized from methanol to give 14 in
60.8% yield: 1H NMR (300 MHz, CDCl3) 0.80 (1H, s, 18-CH3), 6.67
(1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d, J = 8.5 Hz, C1-H); MS (EI) m/z
M+ 350.10.
2.1.11. 4-Bromo-3-acetoxyestra-1,3,5(10)-trien-17-one (15)
A
solution of 14 (1.77 g, 5.07 mmol) in dichloromethane
(50 mL) containing acetic anhydride (0.57 mL, 0.62 g,
6.08 mmol), triethylamine (1.41 mL, 1.03 g, 10.1 mmol) and
N,N-dimethylaminopyridine (DMAP) (0.06 g, 0.51 mmol) was
stirred for 18 h. The solution was diluted with 100 mL of
dichloromethane and washed sequentially with water (50 mL)
and saturated aqueous sodium bicarbonate (50 mL), the
organic layer was dried over anhydrous Na2SO4. Filtration
and evaporation at reduced pressure afforded a solid product,
which was purified by flash chromatography (silica gel) using
1% methanol in chloroform as eluent to provide 15 in 96%
yield: 1H NMR (300 MHz, CDCl3) 0.87 (3H, s, 18-CH3), 2.34 (3H,
2.1.7. Tetramethylammonium 2-methoxy-3-benzyl-
oxyestra-1,3,5(10)-trien-16˛-fluoro-17ˇ-ylsulfate (9)
Tetramethylammonium fluoride tetrahydrate (19 mg) was
carefully dried by azeotropic distillation of acetonitrile
(3 × 2.5 mL). A solution of 8 (38 mg) in anhydrous MeCN (4.0 mL)
was added and the mixture was refluxed under dry argon for
15 min. The solvent was removed under reduced pressure to
afford 9 in 85% yield (crude): 1H NMR (300 MHz, CDCl3) 0.81

steroids 7 4 ( 2 0 0 9 ) 42–50
45
s, 3-OAc), 6.68 (1H, d, J = 8.5 Hz, C2-H), 7.45 (1H, d, J = 8.5 Hz,
C1-H); MS (EI) M+ 391.30.
2.1.17. 4-Methyl-3-O-methoxymethylestra-1,3,
5(10)-trien-16,17ˇ-O-cyclic sulfone (21)
Compound 20 (0.17 g, 0.57 mmol) was dissolved in anhydrous
THF (8 mL), to this was added NaH (60% suspension in min-
eral oil, 2.3 mmol, 91 mg) followed by the drop wise addition
of methoxymethyl chloride (68 ␮L, 0.85 mmol) in THF (0.2 mL).
After stirring for 1 h, abs. ethanol (5 mL) was added. The sol-
vent was evaporated and after usual work-up an analytical
samples of 21 was obtained by crystallization from EtOH: 1H
NMR (300 MHz, CDCl3) 0.85 (3H, s, 18-CH3), 2.11 (3H, s, 4-CH3),
3.51 (3H, s, 3-OCH2-OCH3), 4.22 (1H, d, J = 3.15 Hz, 17-H), 4.98
(1H, m, 16-H), 5.17 (2H, s, 3-OCH2–), 6.66 (1H, d, J = 8.5 Hz, C2-H),
7.10 (1H, d, J = 8.5 Hz, C1-H); MS (EI) m/z: M+ 346.46.
2.1.12. 4-Methyl-3-acetoxyestra-1,3,
5(10)-trien-17-one (16)
A solution of 15 (0.83 g, 2.13 mmol) in N, N-dimethylformamide
(40 mL) containing tetramethyltin (1.17 mL, 1.52 g, 8.51 mmol),
bis(triphenylphosphine)palladium
0.32 mmol), triphenylphosphine (0.22 g, 0.85 mmol) and
lithium chloride (0.72 g, 17.0 mmol) was heated at 100 ◦C
under argon for 16 h. Following completion the reaction mix-
ture was cooled to room temperature and poured into 100 mL
of water. After extracted with diethyl ether (2 × 100 mL) and
usual work-up, the residue was purified by flash chromatog-
raphy using 1.5% methanol in chloroform as eluent to provide
21 in 83% yield: 1H NMR (300 MHz, CDCl3) 0.89 (3H, s, 18-CH3),
2.31 (3H, s, 3-OAc), 2.11 (3H, s, 4-CH3), 6.66 (1H, d, J = 8.5 Hz,
C2-H), 7.10 (1H, d, J = 8.5 Hz, C1-H); MS (EI) M+ 326.43.
(II)
chloride
(0.22 g,
2.1.18. 4-Methyl-3-O-methoxymethylestra-1,3,
5(10)-trien-16,17ˇ-O-cyclic sulfone (22)
Compound 22 was prepared from the sulfone 21 (0.17 g,
0.50 mmol) as described for compound 9. Crystallization from
EtOH provided the analytical sample of 21 as a white powder:
1H NMR (300 MHz, CDCl3) 1.01 (3H, s, 18-CH3), 2.11 (3H, s, 4-
CH3), 3.51 (3H, s, 3-OCH2-OCH3), 4.52 (1H, d, J = 3.3 Hz, 17␣-H),
5.17 (1H, m, 16␣-H), 5.17 (2H, s, 3-OCH2–), 6.66 (1H, d, J = 8.5 Hz,
C2-H), 7.10 (1H, d, J = 8.5 Hz, C1-H); HRMS calcd. for C22H30O6S,
422.5350, found 422.5341.
2.1.13. 4-Methyl-3-acetoxyestra-1,3,
5(10)-trien-17-enolacetate (17)
Compound 17 was prepared by reacting 16 (0.7 g, 2.1 mmol)
in isopropenyl acetate (15 mL) with p-TsOH·H2O catalyst, as
detailed for compound 4. The product was purified by Florisil
column chromatography (hexane–EtOAc, 10:0 to 9:1) to afford
17 in 65% yield: 1H NMR (300 MHz, CDCl3) 0.89 (3H, s, 18-CH3),
2.11 (3H, s, 4-CH3), 2.21 (3H, s, 17-OAc), 2.31 (3H, s, 3-OAc),), 5.40
(1H, m, 16-H), 6.66 (1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d, J = 8.5 Hz,
C1-H); MS (EI) m/z M+ 368.46.
2.1.19. 4-Methyl-16˛-fluoro-17ˇ-estradiol (23)
A solution of sulfone 22 (35 mg) in anhydrous MeCN (4.0 mL)
was added to azeotropic dried tetramethylammonium fluo-
ride tetrahydrate (19 mg) and refluxed under dry nitrogen for
15 min whereupon the solvent was removed under reduced
pressure. The reaction mixture was dissolved in EtOH (10 mL)
and concentrated sulfuric acid (50 ␮L). The solution was
heated to 110 ◦C for 5–10 min, solvent was removed under
reduced pressure, the residue extracted with ether, washed
with water, dried over anhydrous Na2SO4, and evaporated to
yield 23 in 88% yield. The analytical sample was obtained as a
white powder after crystallization from ether–EtOH; analysis
by HPLC on a Waters Nova Pak HR-C18 (7.8 × 250 mm), in 80:20
to 20:80 H2O/CH3CN at 2.0 mL/min, (UV detector at 280 nm)
indicated the material was >99% pure (tR = 26 min); 1H NMR
(300 MHz, CDCl3) 0.80 (3H, s, 18-CH3), 2.11 (3H, s, 4-CH3), 3.82
(1H, d, J = 6.5, 28 Hz, 17␣-H), 4.85, 5.07 (1H, dm, J = 52 Hz, 16␤-H),
6.66 (1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d, J = 8.5 Hz, C1-H); HRMS
calcd. for C19H25O2F, 304.3999, found 304.3995.
2.1.14. 4-Methyl-3-acetoxyestra-1,3,5(10)-trien-16ˇ-
acetate-17-one (18)
Reacting 17 (0.46 g, 1.25 mmol) with lead tetraacetate (0.75 g) in
AcOH (7.5 mL), as detailed for compound 5, followed by purifi-
cation over Florisil (hexane–EtOAc 10:0 to 9:1) give 18 in 75%
yield: 1H NMR (300 MHz, CDCl3) 0.90 (3H, s, 18-CH3), 2.17 (3H,
s, 16-OAc), 2.11 (3H, s, 4-CH3), 2.31 (3H, s, 3-OAc), 4.98 (1H,
t, J = 8.85 Hz, 16-H), 6.66 (1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d,
J = 8.5 Hz, C1-H); MS (EI) m/z M+ 384.45.
2.1.15. 4-Methyl-3-acetoxyestra-1,3,
5(10)-trien-16ˇ-acetate-17ˇ-ol (19)
Compound 19 was prepared by reacting 18 (0.31 g, 0.81 mmol)
with lithium tri-tert-butoxyaluminium hydride (0.7 g) in THF
(20 mL), following the procedure detailed for compound 6.
The material was purified by silica gel chromatography
(CH2Cl2–EtOAc, 10:0 to 8:2) to afford 19 in 86% yield: 1H NMR
(300 MHz, CDCl3) 0.88 (3H, s, 18-CH3), 2.17 (3H, s, 16-OAc), 2.11
(3H, s, 4-CH3), 2.31 (3H, s, 3-OAc),), 3.57 (1H, d, J = 3.5 Hz, 17␣-
H), 4.98 (1H, m, 16-H), 6.66 (1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d,
J = 8.5 Hz, C1-H); MS (EI) m/z M+ 386.50.
2.2.
Radiochemistry
2.2.1. Synthesis of 2-methoxy-16˛-[18F]fluoro-3,
17ˇ-estradiol (2-MeO-[18F]FES) ([18F]-11)
The irradiated 18O-enriched water containing carrier-free
[
18F]fluoride was transferred to the module and passed
through the QMA cartridge. The trapped [18F]fluoride on the
cartridge was eluted with 2.5 mL Kryptofix/acetonitrile, trans-
ferred into the reaction vessel and dried twice by adding
1 mL of acetonitrile and evaporation. Precursor 8 (2 mg) in
1 mL acetonitrile was added and the reaction mixture heated
at 110–115 ◦C for 15 min. Solvent was evaporated (helium)
and acid hydrolysis performed with 1.1 mL acidic ethanol
at 110–115 ◦C for 15 min. After cooling to room temperature,
the mixture was dried and 0.8 mL ammonium formate
2.1.16. 4-Methyl-estra-1,3,5(10)-trien-3,16,17ˇ-triol (20)
Compound 20 was prepared from 19 (0.50 g, 1.47 mmol), as
detailed for compound 7, purified by flash chromatography
using 2% methanol in chloroform as eluent to provide 20 in
54% yield: 1H NMR (300 MHz, CDCl3) 0.76 (3H, s, 18-CH3), 2.11
(3H, s, 4-CH3), 4.22 (1H, d, J = 3.2 Hz, 17␣-H), 4.98 (1H, m, 16-H),
6.66 (1H, d, J = 8.5 Hz, C2-H), 7.10 (1H, d, J = 8.5 Hz, C1-H); MS (EI)
m/z M+ 302.40.

46
steroids 7 4 ( 2 0 0 9 ) 42–50
solution in THF/MeOH and Pd/C added and stirred at room
temperature for 15 min. Reaction mixture was diluted with
2 mL acetonitrile/water and passed through a Millex GP nylon
filter (0.22 ␮m). The mixture was removed from the synthesis
module and transferred to the 5-mL sample loop of the HPLC
system for purification. Free fluorine-18 eluted at 5 min with
the solvent front, the radioactive 2-MeO-[18F]FES ([18F]-11)
eluted at 24 min and was collected in a sterile vial. The radio-
chemical purity of the final preparation was verified by re-
injection on the same HPLC system and was >99%. The synthe-
sis time was 100–110 min from the end of bombardment (EOB).
evaporated (helium) and hydrolysis was accomplished by the
addition of 1.1 mL acidic ethanol and heating at 110–115 ◦C
for 15 min. Then the mixture was diluted with 2 mL ace-
tonitrile/water and passed through a Millex GP nylon filter
(0.22 ␮m). The mixture was removed from the synthesis mod-
ule and transferred to the 5-mL sample loop of the HPLC
system for purification. Radioactive 4-Me-[18F]FES ([18F]-23)
eluted between 26 and 27 min and was collected in a sterile
vial. The synthesis time was 80–90 min from EOB.
3.
Results and discussions
2.2.2. Synthesis of 4-methyl-16˛-[18F]fluoro-3,17ˇ-
estradiol (4-Me-[18F]FES) ([18F]-23)
3.1.
Chemistry
Compound 4-Me-[18F]FES ([18F]-23) was prepared as described
above for 2-MeO-[18F]FES ([18F]-11). Precursor 22 (2 mg) in
acetonitrile (1 mL) was added to the vial containing the
18F/Kryptofix and the reaction mixture is heated at 110–115 ◦C
for 15 min to complete the fluorination. The solvent was
3.1.1. Synthesis of 2-methoxy-16˛-fluoro-3,17ˇ-estradiol
(Scheme 1)
A number of procedures to introduce 2-methoxy to estra-
diol have been reported [23,25–29]. The starting material
Scheme 1.

steroids 7 4 ( 2 0 0 9 ) 42–50
47
3-benzyl-2-methoxy-17␤-estradiol (2) was prepared from
estradiol (1) according to a published procedure [29]. Jone’s
16␤-acetate-3-benzylestrone (5) with lead tetraacetate in
acetic acid at room temperature. Reduction of 5 with lithium
tri-tert-butoxy aluminium hydride in tetrahydrofuran at
room temperature gave a mixture of 17␣/␤-distereomers
from which the more polar 2-methoxy-16␤-acetate-3-benzyl-
17␤-estradiol (6) epimer was readily separated by silica gel
flash chromatography. Compound 6 was hydrolyzed with
oxidation of
2
resulted in 2-methoxy-3-benzylestrone
(3), which was converted into 2-methoxy-3-benzyl-
17-enolacetate-estradiol (4) in the presence of isopropenyl
acetate, acetic anhydride and p-TsOH·H2O as catalyst under
reflux [20]. Compound 4 was converted into 2-methoxy-
Scheme 2.

48
steroids 7 4 ( 2 0 0 9 ) 42–50
K2CO3 in methanol-water at room temperature to afford
2-methoxy-3-benzyl-16,17␤-dihydroxyestradiol (7). The cis-
configuration of the 16- and 17-OH groups was confirmed by
the formation of the cyclic sulfone in 8. Attempts to substi-
tute the 3-benzyl in 7 for a methoxymethyl (MOM) protecting
group prior to sulfonation failed likely due to interference
of the 2-OMe group. Thus we retained the 3-benzyl as a
protecting group. Treatment of 7 with sulfonyldiimidazole
and NaH gave 2-methoxy-3-benzyl-16␤,17␤-O-cyclic sulfone
(8), which subsequently was stereoselectively opened via a
nucleophilic fluorination under anhydrous conditions, using
tetramethylammonium fluoride (Me4NF) in acetonitrile at
reflux to yield 2-methoxy-3-benzyl-16␣-fluoro-17␤-yl Me4N+
salt (9). The latter was hydrolyzed in ethanol/sulfuric acid
to give the 3-benzyl protected compound 10. Removal of
the 3-benzyl in 10 gave the 2-methoxy-16␣-fluoestradiol
(11) using previously reported Pd/C catalyzed reduction
method [30]. The stereochemistry of products 10 and 11
was confirmed by their characteristic signals in the 1H
NMR spectra. The 16␣-F,17␤-OH configuration was assigned
from the double doublet at 3.9 ppm (17␣-H) and a double
multiplet at 4.9 ppm (16␤-H). This pattern is readily dis-
tinguishable from that of the 16␣-F,17␤-OH isomer, which
gives a doublet at 3.8 ppm (17␤-H) and a double multiplet
at 5.25 ppm (16␤-H). On the other hand, the 16␤-F,17␤-OH
isomer is characterized by a double doublet at 3.45 ppm
(17␣-H) and a double multiplet at 5.01–5.25 ppm (16␣-H)
[20].
Fig. 1 – HPLC profile of 2-methoxy-16␣-[18F]fluoro-3,
17␤-estradiol ([18F]-11) (lower trace) and co-injected
non-radioactive 2-methoxy-16␣-fluoro-3,17␤-estradiol
(MeOFES; 11) (upper trace) on a normal-phase column
(Waters, Nova-Pak HR Silica 6-␮m) eluted with a linear
gradient of CH3CN-H2O (8:2 to 2:8 over 30 min) at 2 mL/min.
Radioactivity was detected with a flow count detector and
non-radioactive material was monitored at ꢀ = 280 nm.
acetonitrile at reflux to yield the 3-methoxymethyl-4-methyl-
16␣-fluoro-17␤-yl Me4N+ salt. After evaporating the solvent
under reduced pressure, a rapid hydrolysis of the intermediate
salt was accomplished in ethanolic acid solution to afford the
4-methyl-16␣-fluoro-17␤-estradiol (23). The assigned struc-
tures and stereochemistry of all products were confirmed by
their characteristic spectral properties in the 1H NMR spec-
tra.
3.1.2. Synthesis of 4-methyl-16˛-fluoro-3,17ˇ-estradiol
(Scheme 2)
Several reports for the synthesis of 4-Me estradiol have
been published [31,32]. Bromination of estrone at C-2 or
C-4 using NBS or Br2 gave 2,4-dibrominated estrone as a
major product [24]. Selective bromination of estradiol was
accomplished using 2,4,4,6-tetrabromocyclohexa-2,5-dienone
in chloroform/tetrahydrofuran to afford 4-bromoestradiol
(12) and 2-bromoestradiol (13) in 10% and 40% yield,
respectively. Fractional crystallization of the mixture gave
the pure 4-bromoestradiol (12). Jones oxidation of 12
gave the 4-bromoestrone 14 that was converted to the
3-acetoxy-4-bromoestrone (15) in quantitative yield with
acetic anhydride in tertiary ethylamine and DMAP as
catalyst. Compound 15 was methylated by a palladium-
catalyzed Stille coupling reaction [33] using PdCl2(PPh3)2,
tetramethyltin, triphenylphosphine and lithium chloride in
dimethylformamide at 100 ◦C to afford the 3-acetoxy-4-
methylestrone (16). Without protecting the 3-hydroxy group,
the Pd-catalyzed coupling reaction failed. Similarly, 3-acetoxy-
4-iodoestrone was shown to be a poor reactant for the
Pd-catalyzed Stille coupling reaction. Compound 20 was
obtained from 16 in three steps (Scheme 2) similar as
described for compound 7. Protecting the 3-hydroxyl group
of 20 with methoxymethyl (MOM) gave 3-methoxymethyl-
4-methyl-16␤,17␤-dihydroxyestradiol (21), which was reacted
with sulfonyldiimidazole and NaH base in tetrahydrofuran
at room temperature to give the 3-methoxymethyl-4-methyl-
16␤,17␤-O-cyclic sulfone (22) in good yield. The cyclic sulfone
22 was stereoselectively opened via a nucleophilic fluori-
nation reaction under anhydrous conditions with Me4NF in
3.2.
Radiochemistry
3.2.1. Synthesis of
2-methoxy-16˛-[18F]fluoro-3,17ˇ-estradiol (2-MeO-[18F]FES,
[
18F]-11) and 4-methyl-16˛-[18F]fluoro-3,17ˇ-estradiol
(4-Me-[18F]FES, [18F]-23)
The [18F]fluoride was prepared by proton bombardment of
an [18O]H2O target, trapped on an anion exchange resin and
eluted with a solution of K2CO3 and phase transfer catalyst
[2,2,2]Kryptofix in acetonitrile. The 16␤,17␤-O-cyclic sulfones
were stereoselectively opened via a nucleophilic fluorination
with [18F]F− at the 16␣-position using a previously estab-
lished procedures [20,34]. The cyclic sulfone intermediate 8
was stereoselectively opened with [18F]F−, followed by a rapid
acid hydrolysis gave 10, followed by deprotection of the 3-
benzyl with Pd/C and ammonium formate to yield the final
product [18F]-11 (Scheme 1). Similarly cyclic sulfone 22 was
stereoselectively opened with [18F]F−, followed by a rapid
acid hydrolysis of both the protecting methoxymethyl (MOM)
ether and sulfate group to yield the [18F]-23 (Scheme 2).
All final products were purified by normal-phase HPLC
and their assigned identities confirmed by comparing their
HPLC retention times with those of the co-injected non-
radioactive analog (Figs. 1 and 2). Typical synthesis times
were about 90–110 min from the end of bombardment and
radiochemical yields varied between 70 and 84% (decay cor-

steroids 7 4 ( 2 0 0 9 ) 42–50
49
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Two new radiolabelled 16␣-18
F analogs of estradiol, 2-
methoxy-16␣-[18F]fluoro-3,17␤-estradiol (2-MeOFES; [18F]-11)
and 4-methyl-16␣-fluoro[18F]-3,17␤-estradiol (4-MeFES; [18F]-
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