Methyl hypofluorite in the synthesis of 16-methoxyestradiol stereoisomers

Article abstract of DOI:10.1016/S0039-128X(98)00051-8

The unusual chemistry of methyl hypofluorite provides a previously unexplored route for functionalizing the 16-position of estradiol. Three isomers of 16-methoxyestradiol were prepared via two synthetic routes, each using methyl hypofluorite. The estrogen

Full text of DOI:10.1016/S0039-128X(98)00051-8

Methyl hypofluorite in the synthesis of
16-methoxyestradiol stereoisomers
Stephanie D. Jonson,*† D. Andre´ d’Avignon,† John A. Katzenellenbogen,‡ and
Michael J. Welch*†
*Mallinckrodt Institute of Radiology, Washington University School of Medicine, Saint Louis,
Missouri, 63110, USA; †Department of Chemistry, Washington University, Saint Louis, Missouri,
63130, USA; and ‡Department of Chemistry, University of Illinois, Urbana, Illinois 61801, USA
The unusual chemistry of methyl hypofluorite provides a previously unexplored route for functionalizing the
16-position of estradiol. Three isomers of 16-methoxyestradiol were prepared via two synthetic routes, each using
methyl hypofluorite. The estrogen receptor binding affinity of these compounds was determined to evaluate their
potential as positron emission tomographic (PET) imaging agents targeting estrogen receptor-positive breast
cancer. Radiolabeled methyl hypofluorite ([11C]CH3OF) would allow the rapid preparation of novel carbon-11
PET imaging agents. The 17-trimethylsilyl enol ethers of 3-benzyloxy and 3-trifloxyestrone were prepared as
substrates to react with methyl hypofluorite. Conditions for the reaction of methyl hypofluorite with simple
substrates were optimized to provide reasonable reaction yields with the steroidal substrates. Following
introduction of the methoxy substituent at the 16-position, reduction and deprotection conditions were manipu-
lated to yield the various methoxyestradiol isomers. Two-dimensional NMR techniques (HMQC and HMQC-
TOCSY) were instrumental in the characterization of the methoxyestradiol isomers. NOESY experiments con-
firmed the stereochemistry of the 16- and 17-positions. 16␣-Methoxyestradiol-17␤ and 16␤-methoxyestradiol-
17␤, each with the preferred ␤ orientation for the 17-alcohol, were determined to have relative binding affinities
of 1.5% and 2.3%, respectively. The stereoisomer with the unfavored ␣ orientation at the 17-position, 16␣-
methoxyestradiol-17␣, exhibited only a 0.5% relative binding affinity for the estrogen receptor. The biological
evaluation of these compounds was not pursued further because of their low binding affinities. (Steroids 63:470–
478, 1998) © 1998 by Elsevier Science Inc.
Keywords: methyl hypofluorite;16-methoxyestradiol; NOESY; HMQC-TOCSY
ligands with decreased in vivo metabolism, higher ER af-
finity, and decreased nonspecific binding.6
Introduction
Positron Emission Tomography (PET) coupled with radio-
labeled estrogens has been used for the diagnostic imaging
of estrogen receptors that are present in estrogen receptor-
positive (ERϩ) breast cancer. Currently, the ER ligand
[18F]-16␣-fluoroestradiol-17␤ ([18F]FES), in conjunction
with 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), is used
clinically for breast cancer imaging.1–3 Studies with
[18F]FES display the ability of PET to effectively stage
breast cancer and monitor therapy response. Several
fluorine-18-labeled estrogens have been prepared and bio-
logically evaluated; however, [18F]FES is the only fluorine-
labeled ER ligand proven clinically useful.4–10 The search
for improved fluorine-18 estrogens continues, focusing on
While fluorine-18 (t1/2 ϭ 110 m) has been the radionu-
clide of choice for whole body PET imaging and for radio-
pharmaceutical syntheses requiring multiple steps, ER li-
gand development has been expanded to the incorporation
of carbon-11 (t1/2 ϭ 20 m).11 With a 20-min half-life,
carbon-11 radiopharmaceuticals can allow for repeat imag-
ing studies in one sitting to follow disease progression and
therapy response of a known tumor site while providing a
lower radiation dose to the patient in comparison to a
fluorine-18 agent. The short half-life of carbon-11, how-
ever, requires that the preparation of carbon-11-labeled ra-
diopharmaceuticals be rapid; in addition, the synthesis of
carbon-11 containing ER ligands is limited by a small set of
precursors commonly available.12
Methyl hypofluorite (CH3OF) has been described as a
new carbon-11 synthon, and its high reactivity provides the
short reaction times appropriate for rapid incorporation of
short-lived isotopes.13 Methyl hypofluorite, reported as the
only source of the novel electrophilic methoxylium ion
species “MeOϩ,” is generated by passing F2 (20% in Ne)
Address reprint requests to Michael J. Welch, Division of Radiological
Sciences, Box 8225, Mallinckrodt Institute of Radiology, Washington
University School of Medicine, 510 S. Kingshighway Blvd., Saint Louis,
MO 63110. E-mail: welch@mirlink.wustl.edu
Received February 23, 1998; accepted April 17, 1998.
Steroids 63:470–478, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00051-8

Synthesis of methoxyestradiol isomers: Jonson et al.
through methanol in acetonitrile at Ϫ40°C.14 The isolation
and characterization of CH3OF and its reactivity toward
various alkenes have been reported.14–16 Enol ethers were
found to react rapidly with CH3OF, forming the correspond-
ing ␣-methoxy ketones. Previously, compounds of this class
were generally prepared by cumbersome multi-step synthe-
ses.17,18 Thus, application of methyl hypofluorite chemistry
to the preparation of novel ER ligands should allow rapid
introduction of a methoxy functionality and thereby provide
a method for the incorporation of carbon-11.
Desiring to use the chemistry of CH3OF to prepare novel
ER ligands, we synthesized various 16-methoxyestradiol
stereoisomers. Four isomers are possible, since substituents
at the 16- and 17-positions can each have the ␣- or
␤-orientation (Figure 1). Since the ER prefers ligands that
have the 17␤-OH orientation, 16␣-methoxyestradiol-17␤
and 16␤-methoxyestradiol-17␤ were considered more de-
sirable than the two isomers having the 17␣-OH configu-
ration.19 Fevig et al.20 synthesized a series of 16␣-
substituted estradiols to ascertain the tolerance of the ER to
polarity and steric interference at this site, and Anstead et
al.21 reviewed the structure–affinity correlations of many
substituted estrogens for ER. These reports concluded that
the receptor can tolerate small, nonpolar substituents at the
16␣-position; however, large substituents displayed poor
receptor affinity, with affinity decreasing further with large
polar substituents. Other binding affinity studies of estro-
gens, substituted at both the 16␣- and 16␤-positions with
fluorine, bromine, and iodine, revealed a clear preference
for 16␣- over 16␤-substitution (Table 1).19,20,22 On the basis
of these literature precedents, a methoxy substituent at the
16-position was predicted to be reasonably well tolerated,19
and the 16␣-methoxyestradiol-17␤ isomer was expected to
have the highest binding affinity.
Table 1 Relative Binding Affinities of Estrogen Receptor
Ligands Substituted at the 16-Position (lamb, 0°C)a
Compound
16␣
16␤
RBA
Estradiol (ES)
—
OH
CH2OH
F
—
—
—
—
F
—
—
Br
—
I
100
20
2.4
76
37
Estriol
16␣-Hydroxymethylestradiolb
16␣-FES
16␤-FES
16␣-Chloroestradiol
16␣-Bromoestradiol
16␤-Bromoestradiol
16␣-Iodoestradiol
16␤-Iodoestradiolc
—
Cl
100
129
5.2
93
Br
—
I
—
57
aReported in Reference 19.
bReported in Reference 20.
cReported in Reference 22.
results were obtained when the methoxy substituent was
introduced by reacting 17-trimethylsilyl enol ether-3-
trifloxy (or benzyloxy) estrone with methyl hypofluorite.
Deprotection and reduction conditions were varied in order
to produce three of the four possible methoxy estradiol
stereoisomers: 16␣-methoxyestradiol-17␤; 16␣-methoxy-
estradiol-17␣; and 16␤-methoxyestradiol-17␤.
1H NMR resonances in steroids are difficult to assign,
due to severe overlap of signals in the aliphatic region, and
the need for 2-D (two-dimensional) NMR methods to make
complete steroid assignments has been recognized.23 To
confirm the isomeric configurations of the methoxyestradiol
compounds, various 2-D correlated NMR techniques were
used: 1H-1H correlated spectroscopy (COSY) methods gen-
1
erally fail for steroids, because the H dispersion is poor,
Conditions that were suitable for the reaction of CH3OF
with simple substrates such as the enol acetate of
1-indanone needed to be modified to obtain satisfactory
results with the more chemically complex steroidal sub-
strates; specifically, alterations were needed to minimize
solubility problems and side product formation.14 Good
whereas the reasonable 13C dispersion found for many ste-
1
roids makes identification techniques like H-13C HMQC
and HMQC-TOCSY useful, because steroids often have
carbons with attached protons on the B, C, and D rings.
HMQC-TOCSY provides information in a 2-D format, in-
dicating the correlations between protons and attached car-
Figure 1
Structures of methoxyestradiol stereoisomers relative to the parent compound estradiol (shown with superimposed
steroidal numbering system).
Steroids, 1998, vol. 63, September 471

Papers
bons belonging to a common spin system.24 Thus, we found
mL, 8.88 mmol). The reaction was refluxed for 21 h, cooled to
room temperature, filtered, and filtrate concentrated under reduced
pressure. Residue was dissolved in CH2Cl2, washed with 1 ϫ 100
mL 1 N HCl, and followed by general work-up. Recrystallization
from MeOH yielded 2 as a white solid (0.978 g, 61%). mp
126–128°C. 1H NMR (CDCl3): ␦ 0.91 (s, 3H, 18-CH3), 1.30–2.60
(m, 13H), 2.90 (m, 2H), 5.04 (s, 2H, PhCH2OAr), 6.74 (d, J ϭ 2.7,
1H), 6.79 (dd, J ϭ 8.6, 2.7, 1H), 7.21 (d, J ϭ 8.7, 1H), 7.32–7.45
(m, 5H). HRMS calcd for C25H28O2 (Mϩ) 360.2089, found
360.2081. Anal. (C25H28O2) C, H.
1
that HMQC experiments correlating H-13C one-bond cou-
pling, combined with the extended coupling identified by
HMQC-TOCSY, allowed us to assign all resonances mak-
ing up the steroid skeleton.
Confirmation of the stereochemistry at the 16- and 17-
position was obtained through a NOESY (nuclear Over-
hauser and exchange spectroscopy) experiment that yielded
information about the relative through-space distances be-
tween proton atoms. The combination of these NMR tech-
niques was essential for assignment of each isomer’s D-ring
stereochemistry. Evaluation of the ER binding affinity
showed that all three isomers are low affinity ER ligands:
Therefore, further biological evaluation was not pursued.
17-Trimethylsilyloxy-3-benzyloxyestra-1,3,5 (10),16-
tetraene (3)
To a solution of 2 (0.770 g, 2.14 mmol) in 15 mL of CH2Cl2 under
N2 was added Et3N (1.55 mL, 11.1 mmol, 5.2 eq). The solution
was stirred for 20 min prior to addition of TMSOTf (1.24 mL, 8.88
mmol, 4 eq), followed by 30 min of stirring. The reaction mixture
was purified directly by pouring onto a basic alumina column that
was eluted (CH2Cl2/hexane/Et3N, 25: 75: 1 v/v), followed by
general work-up. Product, which coeluted with unreacted starting
material under these conditions, was purified by flash column
chromatography (EtOAc/hexane, 20: 80 v/v) on basic alumina to
afford 3 as a white solid (0.92 g, 100%). mp 105–107°C. 1H NMR
(CDCl3): ␦ 0.22 (s, 9H); 0.86 (s, 3H); 1.39–2.4 (m, 11H); 2.85–
2.91 (m, 2H); 4.52 (m, 1H); 5.03 (s, 2H, PhCH2OAr); 6.73 (d, J ϭ
2.7, 1H); 6.78 (dd, J ϭ 8.4, 2.7, 1H); 7.19 (d, J ϭ 8.7, 1H);
7.31–7.45 (m, 5H). HRMS calcd for C28H36O2Si (Mϩ) 432.2485,
found 432.2492.
Experimental
General
All commercial reagents were used as received from the sup-
pliers unless otherwise noted. HPLC solvents were Optima
grade. Fluorine (20% in Ne) was purchased from Acetylene Gas
(St. Louis, MO). Due to the strong oxidizing and corrosive
nature of fluorine, appropriate laboratory safety and person-
nel protective equipment were used.25 2,6-Lutidine was dis-
tilled from barium oxide and stored over molecular sieves.
Methylene chloride (CH2Cl2) and triethylamine (TEA) were
distilled from calcium hydride (CaH2). Column chromatogra-
phy was performed using silica gel (60 Å, 230–400 mesh) or
basic alumina (40 ␮m). Thin-layer chromatography (TLC) was
performed on UV active 250 ␮m silica plates visualized with
phosphomolybdic acid or potassium permanganate. Melting
points are uncorrected. Microanalyses were performed by Gal-
braith Laboratories. 3-Trifluoromethylsulfonyloxyestra-1,3,5
(10)-trien-17-one (6) was prepared according to the literature.26
General work-up of organic solutions included drying over
MgSO4, filtering, and removing solvent under reduced pressure.
17-Trimethylsilyloxy-3-trifluoromethylsulfonyloxyestra-
1,3,5 (10),16-tetraene (7). Procedure A (adapted from
Cazeau)27.
To a flask containing 3-trifluoromethylsulfonyloxyestra-1,3,5
(10)-trien-17-one (6) (0.514 g, 1.28 mmol) under N2, Et3N (221
␮L, 1.59 mmol, 1.24 eq) was added, followed by TMSCl (201 ␮L,
1.59 mmol, 1.24 eq). The resulting white slurry was stirred while
NaI (0.238 g, 1.59 mmol, 1.24 eq) in anhydrous acetonitrile (1.6
mL) was added dropwise. Cold hexane and ice water were added
after the solution had been stirred at rt for ca. 66 h. After decan-
tation, the aqueous layer was washed with hexane, and the com-
bined organic extracts were washed thrice with cold saturated
sodium bicarbonate, followed by general work-up. Purification by
silica flash column chromatography (EtOAc/hexane, 1: 9 v/v)
yielded 7 as a white solid (0.304 g, 50%).
NMR measurements
All NMR data were recorded at 25°C on samples dissolved in
d-chloroform (concentration: 4–10 mg/600 ␮L). Routine H, 19F,
1
and 13C spectra were obtained on a Varian Gemini NMR spec-
trometer at 300, 282, and 75 MHz, respectively, while two-
dimensional HMQC, HMQC-TOCSY, and NOESY experiments
were obtained using a Varian Unity-Plus instrument operating at
500 MHz. Chemical shifts for 1H and 13C were referenced to
internal tetramethylsilane and 19F was referenced to internal
CFCl3. Two-dimensional experiments included H and 13C spec-
1
tral widths of 5,207 and 19,408 Hz, respectively, with 90° pulse
widths of 8 ␮s (1H) and 12 ␮s (13C). In the t2 dimension, 2,048
complex time points were collected and 600 complex time points
in t1 were employed with zero filling to 2,048 ϫ 2,048 with
gaussian weighing in both dimensions prior to Fourier transforma-
tion. The NOESY mixing time was 700 ms. For HMQC-TOCSY
a 15 ms isotropic mixing period was employed and resulted in
Procedure B
To a solution of 6 (2.53 g, 6.29 mmol) in 40 mL CH2Cl2 under N2
was added Et3N (1.76 mL, 12.58 mmol, 2 eq). After the solution
had been stirred for 20 min and then cooled to 0°C, TMSOTf (2.44
mL, 12.58 mmol, 2 eq) was added. The ice bath was removed to
allow the reaction to warm to rt. The reaction was monitored by
TLC (EtOAc/hexane, 23: 77 v/v) and additional Et3N (2.0 mL) and
TMSOTf (1.5 mL) were added to maximize the yield of the enoxy
silane over a reaction time of 3 h. The reaction mixture was
purified directly by passage through a plug of basic alumina
(CH2Cl2/hexane/Et3N, 25: 75: 1 v/v). Solvent was removed under
reduced pressure to afford 7 (2.70 g, 90%). mp 84–88°C. 1H NMR
(CDCl3): ␦ 0.21 (s, 9H); 0.88 (s, 3H); 0.97–2.10 (m, 11H); 2.35–
2.40 (m, 2H); 4.55 (m, 1H); 6.72–6.82 (m, 3H). Anal. Calcd for
C22H29O4F3SSi: C, 55.68; H, 6.16. Found: C, 56.12; H, 6.34.
1
strong 3-bond H-1H correlations, with weak 4-bond interactions
also present as an assignment aide. 13C GARP decoupling was
used for both HMQC and HMQC-TOCSY.
3-Benzyloxyestra-1,3,5 (10)-trien-17-one (2)
A mixture of 50 mL of CHCl3, 25 mL of MeOH, and K2CO3 (1.23
g, 8.88 mmol) was refluxed under N2 for 15 min and then added
to a solution of 1 (1.2 g, 4.44 mmol) and ␣-bromotoluene (1.06
472 Steroids, 1998, vol. 63, September

Synthesis of methoxyestradiol isomers: Jonson et al.
during which time it progressed through a color change from
yellow to clear and colorless. The reaction mixture was diluted
with CH2Cl2, filtered, and concentrated under reduced pressure.
The crude reaction mixture was dissolved in 1 mL EtOAc and
passed through a silica gel plug (EtOAc/hexane, 50: 50 v/v). The
procedure was repeated with two additional aliquots of 4. Analysis
by H NMR showed complete deprotection. H NMR (CDCl3): ␦
0.94 (s, 3H, 18-CH3); 1.95–2.40 (m, 11H); 2.85 (m, 2H); 3.52 (s,
3H, -OCH3); 3.98 (d, J ϭ 7.4, 1H); 5.05 (b, Ͻ 1H, OH); 6.57–6.65
(m, 2H); 7.13 (d, J ϭ 8.1, 1H).
To a solution of an aliquot of deprotected reaction product (13
mg, 0.0433 mmol) in 2 mL of anhydrous MeOH was added PdCl2
(15 mg, 0.087 mmol). While stirring under N2, the reaction was
cooled to 0°C, NaBH4 was added (9.8 mg, 0.260 mmol), and the
reaction mixture was stirred for 4 h. The reaction was filtered into
5% HOAc (8 mL); EtOAc and 1 M NaHCO3 were then added.
Organic and aqueous layers were separated, and the aqueous
fraction was washed with 3 ϫ 15 mL EtOAc; combined organic
fractions were washed with 3 ϫ 30 mL H2O, followed by general
work-up. Reduction was carried out on two additional aliquots of
deprotected reaction product. Crude reaction products were pooled
prior to semi-preparative normal phase HPLC purification (5%
isopropanol in CH2Cl2/hexane, 30: 70 v/v), which yielded 5a as a
white solid (8.1 mg, 20% from 4). mp 95–97°C. 1H NMR
(CDCl3): ␦ 0.81 (s, 3H, 18-CH3); 1.30–2.35 (m, 12H); 2.80–2.85
(m, 2H); 3.39 (s, 3H, -OCH3); 3.64 (d, J ϭ 5.4, 1H, 17-H);
3.68–3.74 (m, 1H, 16-H); 4.50–4.80 (b, Ͻ 1H, OH); 6.58–6.68
(m, 2H); 7.17 (d, J ϭ 8.1, 1H).
General procedure for methyl hypofluorite (CH3OF)
reactions
Anhydrous acetonitrile (48 mL) and anhydrous MeOH (2 mL)
were added to an N2 swept flask and cooled to Ϫ40°C (dry
ice/acetonitrile bath). The nitrogen flow was stopped, and F2 (20%
in Ne) was bubbled through the solution for 35 min. An aliquot
(0.5 mL) of CH3OF⅐ACN was removed and added to a flask
containing 25 mL H2O and KF. The concentration of CH3OF was
determined by titrating the solution with Na2S2O4 (equivalence
point color change: yellow to colorless). The desired substrate was
dissolved in CHCl3 (10 mL) and cooled to 0°C. NaF (30 mg) was
added to the solution of CH3OF and swirled for 30 sec before the
CH3OF was quickly poured into the substrate flask. The reaction
was stirred at 0°C for 5 min and was then allowed to warm to rt
over a 40 min period. The reaction was quenched by the addition
of saturated NaHCO3 (250 mL). Separation of the aqueous phase
was followed by washing the aqueous extract thrice with CHCl3;
combined organic extracts were washed thrice with brine, followed
by general work-up.
1
1
16␣-Methoxy-3-benzyloxyestra-1,3,5 (10)-triene-17-
one (4)
The general procedure was followed to generate 6.90 mmol
CH3OF (0.139 M) that was allowed to react with 3 (570 mg, 1.32
mmol). Purification by silica gel gravity column chromatography
(hexane/CH2Cl2, 30: 70 v/v) afforded 4 as a white solid (52 mg,
1
10%). H NMR (CDCl3): ␦ 0.88 (s, 3H, 18-CH3); 1.26–2.10 (m,
11H); 2.85–2.95 (m, 2H); 3.52 (s, 3H, -OCH3); 3.97 (d, J ϭ 7.5,
1H); 5.03 (s, 2H); 6.70–6.81 (m, 2H); 7.19 (d, J ϭ 8.7, 1H),
7.27–7.44 (m, 5H).
16-Methoxyestra-1,3,5 (10)-triene-3,17-diol (5b, 5c)
3-Trifloxy-16-methoxyestrone (0.0694 mmol, 30 mg 8a or 0.0176
mmol, 7.6 mg 8b) was dissolved in freshly distilled Et2O (0.013
mmol/mL), stirred under N2, and cooled to Ϫ78°C (dry ice/
isopropanol bath). A 1.0 M LiAlH4/Et2O solution (0.350 mmol,
350 ␮L to 8a or 0.087 mmol, 87 ␮L to 8b) was added dropwise
over ca. 2 min. The pale yellow reaction was stirred at Ϫ78°C for
25 min and then warmed to rt over 25 min, giving a cloudy white
appearance. Addition of 6 N HCl (7.8 mmol, 1.3 mL for 8a or
1.044 mmol, 0.174 mL for 8b) quenched the reaction. The aqueous
phase was extracted with 1 ϫ 3 mL Et2O and 2 ϫ 3 mL CH2Cl2/
hexane (50: 50 v/v). Each organic extract was passed through a
MgSO4 plug (2 g) and a 0.22 ␮m filter. Solvent was removed
under reduced pressure. Purification by semi-preparative normal
phase HPLC (5% isopropanol in CH2Cl2/hexane, 40: 60 v/v)
yielded 5b (0.022 mmol, 6.6 mg, 31%) or 5c (0.0175 mmol, 5.3
mg, 83%) as a white solid. 5b: mp 167–171°C. 1H NMR (CDCl3):
␦ 0.71 (s, 3H, 18-CH3); 1.20–2.40 (m, 12H); 2.78–2.85 (m, 2H);
3.40 (s, 3H, -OCH3); 3.76 (d, J ϭ 5.1, 1H, 17-H); 3.99–4.05 (m,
1H, 16-H); 4.68–4.80 (b, Ͻ 1H, OH); 6.55–6.68 (m, 2H); 7.16 (d,
J ϭ 8.4, 1H). HRMS calculated for C19H26O3 (Mϩ) 302.1882,
found 302.1883. 5c: mp 173–175°C. 1H NMR (CDCl3): ␦ 0.79 (s,
3H, 18-CH3); 0.95–2.40 (m, 12H); 2.80–2.85 (m, 2H); 3.37 (s, 3H,
-OCH3); 3.49 (d, J ϭ 7.8, 1H, 17-H); 3.73–3.78 (m, 1H, 16-H);
6.55–6.65 (m, 2H); 7.16 (d, J ϭ 8.7, 1H). HRMS calculated for
C19H26O3 (Mϩ) 302.1882, found 302.1881.
16␣-Methoxy-3-trifluoromethylsulfonyloxyestra-1,3,5
(10)-triene-17-one (8a)
The general procedure was followed to generate 5.11 mmol
CH3OF (0.105 M) that was allowed to react with 7 (330 mg, 0.695
mmol). Purification by silica gel flash column chromatography
(EtOAc/hexane, 15: 85 v/v) followed by semi-preparative normal
phase HPLC (5% isopropanol in CH2Cl2/hexane, 6: 94 v/v) af-
1
forded 8a as a white solid (36 mg, 12%). mp 73–77°C. H NMR
(CDCl3): ␦ 0.96 (s, 3H, 18-CH3); 1.25–2.45 (m, 11H); 2.90–2.98
(m, 2H); 3.53 (s, 3H, -OCH3); 3.98 (d, J ϭ 7.2 Hz, 1H, 16-H);
6.98–7.06 (m, 2H); 7.34 (d, J ϭ 8.5, 1H). HRMS calculated for
C20H23O5F3S (MϩH)ϩ 433.1296, found 433.1300.
16␤-Methoxy-3-trifluoromethylsulfonyloxyestra-1,3,5
(10)-triene-17-one (8b)
The general procedure was followed to generate 5.11 mmol
CH3OF (0.105 M) that was allowed to react with 7 (330 mg, 0.695
mmol). Purification by silica gel flash column chromatography
(EtOAc/hexane, 15: 85 v/v) followed by semi-preparative normal
phase HPLC (5% isopropanol in CH2Cl2/hexane, 6: 94 v/v) af-
1
forded 8b as a white solid (12 mg, 4%). mp 90–94°C. H NMR
(CDCl3): ␦ 1.00 (s, 3H, 18-CH3); 1.22–2.58 (m, 11H); 2.93–2.98
(m, 2H); 3.54 (s, 3H, -OCH3); 3.67 (t, J ϭ 8.2 Hz, 1H, 16-H);
7.00–7.05 (m, 2H); 7.34 (d, J ϭ 8.2, 1H). HRMS calculated for
C20H23O5F3S (Mϩ) 432.1218, found 432.1208.
Results
Synthesis
Our synthetic approach to 16␣-methoxyestradiol-17␤ (5a)
involved reacting the trimethylsilyl enol ether of
3-benzyloxyestrone (3) with CH3OF (Scheme 1). This re-
action yielded the 16␣-methoxy isomer selectively, with
minimal to no formation of a 16␤-methoxy product. The
16␣-Methoxyestra-1,3,5 (10)-triene-3,17␤-diol (5a)
An aliquot of 4 (17.3 mg, 0.045 mmol) was dissolved in 1 mL of
EtOAc, and a suspension of 4 mg PdCl2(CH3CH)2 and 8 ␮L EtOH
was added. The reaction mixture was stirred under H2 for 25 min,
Steroids, 1998, vol. 63, September 473

Papers
Reacting CH3OF with the silyl enol ether of
3-trifloxyestrone (7) yielded an isomeric mixture of 16␣-
and 16␤-methoxy-3-trifloxyestrone (8a and 8b) in a 3:1
ratio, respectively (Scheme 2). The yield of methoxy prod-
ucts from the triflate protected precursor increased to 25–
37%, as ascertained by 1H NMR of the crude reaction
mixture. Isolation of the 16␤-isomer required a two-step
purification (silica gravity column chromatography; HPLC)
to separate the C-16 epimers. This decreased the yield of the
isolated isomers to 16%: 12% and 4% for 16␣- and 16␤-
methoxy-3-trifloxyestrone, respectively.
Reduction and deprotection of 16␣-methoxy-3-
trifloxyestrone (8a) with LiAlH4 resulted in formation of
the low affinity 17␣-OH epimer in 31% yield. Analysis of
the remaining products from this reaction failed to show the
formation of any of the 16␣-methoxyestradiol-17␤ isomer.
Reduction and deprotection of 16␤-methoxy-3-trifloxy-
estrone (8b) under the same reaction conditions yielded
16␤-methoxyestradiol-17␤ in 83% yield.
Scheme 1
2-D NMR
stereoselectivity of this reaction can be readily ascertained
by H NMR: the chemical shift of the 16-H is a doublet at
1
HMQC and HMQC-TOCSY assignments for compounds
5a, 5b, and 5c confirmed their identity as 16-
methoxyestradiols. Representative HMQC and HMQC-
TOCSY spectra are shown for 5a in Figure 2 and the
4.0 ppm in the 16␣-methoxy isomer and a triplet at 3.7 ppm
in the 16␤-methoxy isomer. While the desired methoxy
ketone was shown to be produced in 23% yield by 1H NMR,
the isolated yield after column purification was only 10%
for this reaction.
Deprotection of 16␣-methoxy-3-benzyloxyestrone (4) by
hydrogenation produced the 16␣-methoxyestrone without
adversely affecting other functionality on the steroid. NMR
analysis showed complete deprotection prior to the reduc-
tion. Sodium borohydride reduction in the presence of pal-
ladium resulted in the formation of the desired 16␣-
methoxyestradiol-17␤ (5a) in 20% yield.
1
assignments are given in Table 2. H signals for the 16-H
and 17-H in 5a were differentiated by their splitting pat-
terns: doublet for 17-H; multiplet for 16-H. The one-bond
1H-13C correlations resulting from the HMQC were com-
pared with those from HMQC-TOCSY to identify new
1
cross-peaks arising from three-bond H-1H couplings.
The additional cross-peaks in the HMQC-TOCSY con-
tour plot serve to identify three-bond coupled proton part-
ners and thus to identify adjacently bonded carbons (assum-
Scheme 2
474 Steroids, 1998, vol. 63, September

Synthesis of methoxyestradiol isomers: Jonson et al.
Figure 2 Left, 1H-13C HMQC spectrum of 16␣-methoxyestradiol-17␤ (5a). Vertical lines indicate cross peaks arising from geminal
protons. Numbering refers to the assignment of the carbon position. A high-resolution 1H spectrum is also shown at the far left. Note
that the HMQC spectrum differentiates five protons in the region of 1.3–1.55 ppm that were indistinguishable by 1H NMR. Right, 1H-13
C
HMQC-TOCSY spectrum of 16␣-methoxyestradiol-17␤ (5a). Signals instrumental in the initial assignment of the spectrum are desig-
nated.
ing the carbon atoms are protonated). For example, the
HMQC-TOCSY contour plot (Figure 2) shows additional
cross-peaks labeled 15/16 and 16/15 that arise from three-
bond coupling between protons on carbons 15 and 16. With
the knowledge of the 16-position carbon and proton assign-
ments, cross-peaks 15/16 and 16/15 guide us to the assign-
ment of position 15. Additional cross-peaks along either the
H or C chemical axis for position 15 on the HMQC-TOCSY
plot, when compared to the HMQC plot, allowed for the
assignment of position 14 resonances. In this fashion, all
protonated carbons in the B, C, and D rings were assigned.
HMQC-TOCSY signals between the 16- and 17-positions
were obscured in 5a, due to overlapping HMQC signals.
Comparison of the NOESY information for the 3 isomers
confirmed the stereochemical orientation of the 16- and
17-positions; the results are shown in Table 3. The 18␤-CH3
and 14␣-H have fixed orientations as assigned for estrone
(1), and this facilitated the stereochemical assignment of the
D-ring. The correlation between the magnitude of the
NOESY cross-peak volume integrals and the distance sep-
arating the two interacting proton pairs (as determined from
energy minimized structures) was quite good. For 16␣-
methoxyestradiol-17␤ (5a), larger Overhauser enhancement
between hydrogen atoms at the 14␣- and 17-positions con-
firmed a same-face orientation. The small interaction seen
for the 17␣- and 16-hydrogens was highly suggestive that
they were on opposing faces. Additional evidence came
from the large Overhauser enhancement between the 18␤-
CH3 and the 16␤-H, implying a same-face orientation.
A similar comparison of interactions confirmed the ste-
reochemistry of 16␣-methoxyestradiol-17␣ (5b). Only a
negligible enhancement was seen for the 14␣-H with the
17-H, highly suggestive of a 17␤-H orientation. A large
Overhauser enhancement with the 18␤-CH3 was seen for
both the 16- and 17-hydrogens, suggestive that all three
substituents are on the same (beta) face. Reaffirming evi-
dence for the 16␣- and 17␣-hydrogens was the large en-
hancement between these signals.
Table 2 1H-13C HMQC and HMQC-TOCSY Assignments for 16␣-
methoxyestradiol-17␤ (5a)
16␣-OMe-E2-17␤ HMQC 1H HMQC 13C
HMQC-TOCSY
Assignment
Chemical
Chemical Adjacent Carbon(s)
No.a
Shift (ppm) Shift (ppm)
Shift (ppm)
18
17
16
15
14
12
11
9
0.81
3.64
13.0
88.2
88.0
30.9
48.4
37.0
26.5
44.2
38.8
27.6
29.9
57.9
—
—
3.70
30.9
1.72
1.50
48.4; 88.0
30.9; 38.8
26.5
The orientation of 16␤-methoxyestradiol-17␤ (5c) was con-
firmed by the weak interaction between the 18␤-CH3 and the
16-hydrogen (opposite face), relative to the large interaction
between the 16␣- and 17␣-hydrogens (same face).
1.35; 1.91
1.47; 2.29
2.22
37.0
26.5; 38.8
27.6; 44.2; 48.4
29.9; 38.8
27.6
8
1.43
7
6
1.36; 1.85
2.82
OMe
3.39
—
Relative binding affinities
aCorresponds to the steroidal numbering system shown in Fig-
ure 1.
The relative binding affinities of the 16-methoxyestradiols
for the ER were determined by a competitive radiometric
Steroids, 1998, vol. 63, September 475

Papers
Table 3 NOESY Assignments Represented as the Average Relative Volume to Confirm the Stereochemistry of 16-methoxyestradiol
Isomers
NOESY Volume
NOESY Volume
NOESY Volume
Interacting H
Pairs
16␣-OMe-E2-17␤
Distancea
(Å)
16␣-OMe-E2-17␣
Distancea
(Å)
16␤-OMe-E2-17␤
Distancea
(Å)
(5a)
(5b)
(5c)
H17–H16
0.271
1.852
0.376
1.184
1.160
—
3.05
2.625
4.22
2.35
2.63
3.67
0.825
0.058
0.148
0.828
0.808
0.833
2.35
3.76
4.13
2.36
2.48
2.37
0.271
2.36
2.59
4.41
2.35
3.87
3.67
H17–H14
—
b
H17–OMe
H16–OMe
18-CH3–H16
18-CH3–H17
0.440
0.063
0.112
—A missing value represents that an NOE was not seen for this interaction.
aStructures were built in the modeling program Sybyl with energies minimized. For an interaction involving a methyl or methoxy
group, the distance shown is to the nearest proton.
bRepresents an obscured interaction by either a cross-peak or an artifact.
binding assay using lamb uterine ER.28 The highest RBA
for this methoxyestradiol series was 2.3 for 5c, while the
RBA values for 5a and 5b were 1.5 and 0.5, respectively.
The isomers of 16-methoxyestradiol all displayed low bind-
ing affinity for the ER compared to the natural ligand
estradiol.
NaF acts as a fluoride ion acceptor, decreasing the acidity of
HF through the formation of the HF2Ϫ ion, thereby reducing
its reactivity toward the substrate.29
Reduction and deprotection of 16␣-methoxy-3-
trifloxyestrone with LiAlH4 led to selective formation of
16␣-methoxyestradiol-17␣ (5b). This was unexpected be-
cause LiAlH4 is used to reduce and deprotect 16␣-fluoro-
3-trifloxyestrone, furnishing the deprotected 17␤-OH and
17␣-OH estradiols in a 3:1 ratio.26 Thus, although the
combined reduction-deprotection step with LiAlH4 was ad-
vantageous in these earlier steroid syntheses, with the 16␣-
methoxy isomer it did not furnish the desired 17␤-OH
configuration. Unexpected 17␣- and 17␤-OH ratios have
also been seen in other LiAlH4 reduction/deprotection se-
quences, such as that of 11␤-ethyl-16␤-fluoro-3-
trifloxyestrone. With the ␤-face being blocked by both the
11␤-ethyl, 16␤-fluoro, and 18␤-methyl substituents, hy-
dride attack from the unhindered ␣-face was expected;
however, in this case the attack from the shielded ␤-face
prevailed by 1.6:1.10 By contrast, LiAlH4 reduction of 16␤-
methoxy-3-trifloxyestrone (8b) was not anomalous, giving
16␤-methoxyestradiol-17␤ (5c). This configuration was ex-
pected, because hydride attack from the ␤-face of the steroid
is blocked simultaneously by the 18␤-methyl and 16␤-
methoxy groups.
Choice of reagent and reaction order determined which
stereoisomer was preferentially formed. In order to selec-
tively reduce the protected 16␣-methoxyestrone to the 17␤-
OH, we used sodium borohydride (NaBH4) in the presence
of palladium chloride, as this method is known to reduce
16␣-hydroxyestrone and 16␣-acetoxyestrone selectively to
the corresponding 17␤-estradiols.30 Direct application of
this procedure to 16␣-methoxy-3-trifloxyestrone (8a), how-
ever, proved unsatisfactory, as it led to the formation of
16␣-methoxy-3-deoxyestradiol-17␤. It was clear that the
triflate protecting group had to be removed prior to ketone
reduction, to eliminate deoxygenation at the 3-position. The
triflate could be removed with KOH/methanol at 60°C;
however, these base conditions epimerized the 16␣-
methoxy group, favoring the 16␤-methoxy epimer 2:1. We
avoided these problems by changing the protecting group at
the 3-position. When this position was protected as a benzyl
Discussion
Synthesis
The methyl hypofluorite reagent allowed the facile incor-
poration of a methoxy group at the 16-position of the steroid
skeleton, and by using two related synthetic routes, we were
able to obtain three of the four possible isomers of 16-
methoxyestradiol. This allowed us to evaluate the ability of
these ligands to bind to the ER. This study also prompted us
to expand the chemistry of CH3OF from structurally simple
to more complex molecules, and the methods we have
developed for the synthesis of methoxy substituted estro-
gens will be applied to the preparation of other steroidal
compounds in the future.
In our initial trial reactions with CH3OF, the direct
addition of an enol ether containing substrate dissolved in
CH2Cl2 was made to the methyl hypofluorite-acetonitrile
complex (CH3OF⅐ACN) at Ϫ40°C. This procedure yielded
a crude mixture of ca. 8 products (detected by TLC), with
formation of only minor amounts of the desired product. We
noted that a precipitate formed upon substrate addition to
CH3OF⅐ACN. Further investigation showed that the sub-
strate was insoluble in the ACN/CH2Cl2 solvent combina-
tion at Ϫ40°C, which presumably caused the precipitation.
On the basis of these observations, conditions for substrate
addition to CH3OF⅐ACN were modified to maintain enol
ether solubility, while retaining the reactivity of CH3OF.
Product yields were further increased by changing the sub-
strate solvent to CHCl3, which is more effective in radical
scavenging.
The formation of side products, presumed to result from
the reaction of substrate with HF formed during the gener-
ation of CH3OF, was decreased by the addition of oven-
dried NaF to the CH3OF⅐ACN immediately prior to the
transfer of this solution to the CHCl3 dissolved substrate.
476 Steroids, 1998, vol. 63, September

Synthesis of methoxyestradiol isomers: Jonson et al.
ether, it could be rapidly deprotected by hydrogenolysis;
subsequent reduction with NaBH4 in the presence of palla-
dium yielded 16␣-methoxyestradiol-17␤ (5a).
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1
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Acknowledgments
Support was provided by the Department of Defense
(DAMD17-94-J-4029 to SDJ), the Department of Energy
(DE FG02-84ER60218 to MJW) and the National Institutes
of Health (PHS 5R01 CA25836 to JAK). We thank Kathryn
E. Carlson for determination of relative binding affinities
and Drs. Thomas A. Bonasera, Timothy J. McCarthy, Eyal
Mishani, David Reichert, and Shlomo Rozen for their help-
ful suggestions. Mass spectrometry was provided by the
Washington University Mass Spectrometry Resource and
NIH Research Resource (Grant No. P41RR0954).
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