A new, practical synthesis of 2-methoxyestradiols.

Article abstract of DOI:10.1016/S0039-128X(02)00065-X

An efficient and practical approach to synthesize moderate to large amounts of 2-methoxyestradiol (2-ME2) is described. The key step in the synthesis is the regioselective introduction of an acetyl group at the C-2 position of estradiol using a zirconium tetrachloride mediated Fries rearrangement carried out on estradiol diacetate. The seven step synthetic procedure readily gave 2-ME2 in 49% overall yield. Application of this method to the synthesis of 2-methoxy-7 alpha-methylestradiol is also described.

Full text of DOI:10.1016/S0039-128X(02)00065-X

Steroids 67 (2002) 1065–1070
A new, practical synthesis of 2-methoxyestradiolsଝ
Pemmaraju N. Rao∗, James W. Cessac
Department of Organic Chemistry, Southwest Foundation for Biomedical Research, P.O. Box 760549,
7620 N.W. Loop 410 (at Military Drive), San Antonio, TX 78227-5301, USA
Received 10 April 2002; received in revised form 24 May 2002; accepted 6 June 2002
Abstract
An efficient and practical approach to synthesize moderate to large amounts of 2-methoxyestradiol (2-ME2) is described. The key
step in the synthesis is the regioselective introduction of an acetyl group at the C-2 position of estradiol using a zirconium tetrachloride
mediated Fries rearrangement carried out on estradiol diacetate. The seven step synthetic procedure readily gave 2-ME2 in 49% overall
yield. Application of this method to the synthesis of 2-methoxy-7␣-methylestradiol is also described.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Steroids; 2-Methoxyestradiol; Zirconium tetrachloride; Fries rearrangement; Antiangiogenesis
1. Introduction
2. Experimental
The sequential biochemical hydroxylation and methy-
lation of the natural hormone estradiol (E2) gives rise to
the endogenous mammalian metabolite 2-methoxyestradiol
(2-ME2) [1]. 2-ME2 is a natural metabolite of estrogen
devoid of uterotropic or estrogenic activity in vivo. Recent
studies [2] have shown that 2-ME2 inhibits the cellular
machinery involved in replicating cancer cells, specifically
microtubules. In addition, 2-ME2 has been demonstrated to
act as an antiangiogenic agent that prevents the growth of
new blood vessels required to nourish tumors [3]. Initiation
of either of these events will cause tumors to shrink but
the combination of effects may provide significant advan-
tages over current anticancer therapies. These results have
increased the demand for 2-ME2 for preclinical and clini-
cal testing as anticancer agents in humans. Prior syntheses
of 2-ME2 [4–11] suffer from low overall yields or require
chromatographic purification of intermediates and as such,
are not applicable to moderate to large-scale synthesis. The
present communication describes a new, practical prepara-
tion of 2-ME2 from estradiol in 49% overall yield and its
successful application to the synthesis of 2-methoxy-7␣-
methylestradiol.
Melting points were determined on a Thomas–Hoover
apparatus and are uncorrected. Nuclear magnetic resonance
(NMR) spectra were recorded on a General Electric GE-300
(300 MHz) spectrometer as deuterochloroform (CDCl3)
solutions using tetramethylsilane (TMS) as an internal stan-
dard (δ = 0) unless noted otherwise. Infared spectra were
recorded on a Perkin-Elmer model 1600 FT-IR instrument
equipped with a diffuse reflectance accessory using a KBr
matrix. Combustion analyses were performed by Midwest
Microlabs Ltd. (Indianapolis, IN). Thin-layer chromatog-
raphy (TLC) analyses were carried out on silica gel GF
(Analtech) glass plates (2.5 cm × 10 cm with 250 ␮M layer
and prescored). HPLC analyses were performed using a
system composed of the following components: Waters
Associates model 6000A pump and model 481 UV–VIS
detector, Hewlett-Packard model 3396A integrator and
Rheodyne model 7125 injector.
Most chemicals and solvents were analytical grade and
used without further purification. Commercial reagents
were purchased from Aldrich Chemical Company (Mil-
waukee, WI). Estradiol was purchased from Schering AG
(Berlin, Germany). 7␣-Methylestrone was obtained from
Ciba-Geigy, now known as Novartis International AG.
2.1. Estra-1,3,5(10)-trien-3,17β-diol diacetate (2a)
ଝ
The method presented here is the subject of a pending US patent.
Corresponding author. Tel.: +1-210-258-9414; fax: +1-210-670-3321.
∗
Under nitrogen, acetic anhydride (50 ml, 264.5 mmol) was
added to a solution of estradiol (1a, 20.0 g, 73.43 mmol) in
E-mail address: pnrao@icarus.sfbr.org (P.N. Rao).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(02)00065-X

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P.N. Rao, J.W. Cessac / Steroids 67 (2002) 1065–1070
dry pyridine (200 ml). The reaction mixture was stirred at
room temperature overnight (16 h). The next morning, anal-
ysis by TLC (5% acetone/CH2Cl2) indicated a complete
reaction. The mixture was cooled in an ice bath and the
excess acetic anhydride quenched by addition of methanol
(25 ml). The mixture was stirred at 0 ◦C for 1 h and then
allowed to warm to room temperature. The solvents were
removed in vacuo and the solid residue crystallized from
hot methanol to give the pure diacetate (2a, 24.87 g, 95%):
mp = 127–128 ◦C (lit. [12] 125–126 ◦C); FT-IR (KBr, dif-
1-H), 12.041 (s, 3-OH). Analysis calculated for C22H28O4:
C, 74.13; H, 7.92. Found: C, 74.02; H, 7.92.
2.4. 2-Acetyl-7α-methylestra-1,3,5(10)-triene-3,17β-diol
17-acetate (3b)
Following the procedure outlined for the synthesis of 3a,
zirconium tetrachloride (33 g, 141.6 mmol) was added to
a solution of the diacetate (2b, 11.0 g, 29.7 mmol) in dry
dichloromethane (1 l). The suspension was stirred at room
temperature for 48 h. At the end of that time, NMR analysis
of a small aliquot taken to dryness indicated only ∼33% re-
action. Additional ZrCl4 (33 g, 141.6 mmol) was added and
the reaction was stirred at room temperature for an additional
3 days. At the end of that time, NMR analysis indicated a
complete reaction. The reaction mixture was cooled in an ice
bath, diluted with water (∼200 ml) and stirred at 0 ◦C for 1 h.
The mixture was then extracted with dichloromethane (3×).
The organic fractions were washed with water (2×), filtered
through Na2SO4, combined and concentrated in vacuo to
give 11.3 g crude product as a yellow foam. Crystallization
of this material from hot methanol gave 7.0 g of a light yel-
low solid. The mother liquors were concentrated and purified
via Flash column chromatography (methylene chloride) to
give an additional 0.6 g product. Total yield (3b, 7.6 g, 69%):
fuse reflectance) νmax: 2924, 2874, 1765, and 1734 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 0.823 (s, 18-CH3), 2.056
(s, 17-OAc), 2.277 (s, 3-OAc), 4.688 (dd, J1 = 9.3 Hz,
J2 = 7.5 Hz, 17-H), 6.786 (d, J = 2.7 Hz, 4-H), 6.834 (dd,
J1 = 8.55 Hz, J2 = 2.7 Hz, 2-H), 7.275 (d, J = 8.55 Hz,
1-H). Analysis calculated for C22H28O4: C, 74.13; H, 7.92.
Found: C, 73.98; H, 7.99.
2.2. 7α-Methylestra-1,3,5(10)-triene-3,17β diacetate (2b)
Following the procedure outlined for the synthesis of 2a,
7␣-methylestradiol (1b, 10.0 g, 34.91 mmol) was reacted
with acetic anhydride (25 ml, 264.5 mmol) in dry pyridine
(100 ml) to give the pure diacetate (2b, 11.57 g, 89.4%):
mp = 143–144 ◦C; FT-IR (KBr, diffuse reflectance) νmax
:
2973, 2917, 2883, 1766, and 1734 cm−1; NMR (300 MHz,
CDCl3), δ (ppm): 0.829 (s, 18-CH3), 0.841 (d, J = 7.2 Hz,
7-CH3), 2.060 (s, 17-OAc), 2.276 (s, 3-OAc), 4.699 (dd,
J1 = 8.6 Hz, J2 = 7.6 Hz, 17-H), 6.779 (d, J = 2.4 Hz,
4-H), 6.842 (dd, J1 = 8.33 Hz, J2 = 2.4 Hz, 2-H), 7.287 (d,
J = 8.33 Hz, 1-H). Analysis calculated for C23H30O4: C,
74.56; H, 8.16. Found: C, 74.40; H, 8.13.
mp = 145–147 ◦C; FT-IR (KBr, diffuse reflectance) νmax
:
3230, 2948, 1726, 1640, and 1619 cm−1; NMR (300 MHz,
CDCl3), δ (ppm): 0.840 (d, J = 7.2 Hz, 7-CH3), 0.852 (s,
18-CH3), 2.070 (s, 17-OAc), 2.604 (s, 2-Ac), 4.712 (dd,
J1 = 9.45 Hz, J2 = 7.65 Hz, 17-H), 6.684 (s, 4-H), 7.628 (s,
1-H), 12.003 (s, 3-OH). Analysis calculated for C23H30O4:
C, 74.56; H, 8.16. Found: C, 74.45; H, 8.10.
2.3. 2-Acetylestra-1,3,5(10)-triene-3,17β-diol 17-acetate
(3a)
2.5. 2-Acetyl-3-benzyloxyestra-1,3,5(10)-trien-17β-ol
17-acetate (4a)
Under nitrogen, solid zirconium tetrachloride (60 g,
257.5 mmol) was added to a solution of the diacetate (2a,
20.0 g, 56.11 mmol) in dry dichloromethane (1.5 l). The
suspension was stirred at room temperature for 48 h. At the
end of that time, analysis by TLC (1% acetone in CH2Cl2)
indicated a complete reaction. The brown-yellow suspen-
sion was cooled in an ice bath and quenched by the slow
addition of water (250 ml) with stirring. The yellow mix-
ture was stirred at 0 ◦C for 1 h, diluted further with water
(500 ml) and extracted with methylene chloride (3×). The
organic extracts were washed with water (2×), filtered
through anhydrous sodium sulfate, combined, and concen-
trated in vacuo to give 19.8 g crude product as a yellow
solid. Crystallization of this material from hot methanol
gave the pure product (3a, 16.8 g, 84%) as a light yellow
solid: mp = 198–200 ◦C (lit. [5] 202–204 ◦C); FT-IR (KBr,
diffuse reflectance) νmax: 3231, 2924, 1727, 1642, and
1619 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.844 (s,
18-CH3), 2.069 (s, 17-OAc), 2.604 (s, 2-Ac), 4.703 (dd,
J1 = 9.3 Hz, J2 = 7.5 Hz, 17-H), 6.693 (s, 4-H), 7.597 (s,
Under nitrogen, benzyl bromide (16 ml, 134.5 mmol) was
added to a mixture of the 2-acetyl compound (3, 16.0 g,
43.19 mmol) and anhydrous potassium carbonate (25 g,
180.9 mmol) in dry dimethylformamide (500 ml). The mix-
ture was heated to 60 ◦C over the weekend (62 h). At the
end of that time, analysis by TLC (CH2Cl2) indicated
about a 75% completion of reaction. Additional benzyl bro-
mide (16 ml, 134.5 mmol) and potassium carbonate (25 g,
180.9 mmol) were added and the reaction heated at 60 ◦C
for a further 4 h. At the end of that time, analysis by TLC
indicated a complete reaction. The reaction mixture was
cooled to room temperature, poured into ice water (∼1.5 l)
and stirred until the ice melted. The resulting precipitate
was collected by filtration and washed well with water
until the filtrate was neutral. The light yellow solid crude
product was crystallized from methylene chloride/methanol
to give the pure product (4a, 17.62 g, 91.4%) as a white
solid: mp = 172–174 ◦C (lit. [5] 170–172 ◦C); FT-IR (KBr,
diffuse reflectance) νmax: 2936, 2920, 1730, 1663, and
1604 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.823 (s,

P.N. Rao, J.W. Cessac / Steroids 67 (2002) 1065–1070
1067
18-CH3), 2.059 (s, 17-OAc), 2.576 (s, 2-Ac), 4.680 (dd,
J1 = 8.85 Hz, J2 = 7.35 Hz, 17-H), 5.122 (s, benzyl CH2),
6.735 (s, 4-H), 7.343–7.456 (m, benzyl aromatic), 7.708 (s,
1-H). Analysis calculated for C29H34O4: C, 78.00; H, 7.67.
Found: C, 77.80; H, 7.68.
6.713 (s, 4-H), 6.957 (s, 1-H), 7.301–7.381 (m, benzyl aro-
matic). Analysis calculated for C29H34O5: C, 75.30; H, 7.41.
Found: C, 75.54; H, 7.49.
2.8. 3-Benzyloxy-7α-methylestra-1,3,5(10)-triene-
2,17β-ol diacetate (5b)
2.6. 2-Acetyl-3-benzyloxy-7α-methylestra-1,3,5(10)-
trien-17β-ol 17-acetate (4b)
Following the procedure outlined for the synthesis of
5a, meta-chloroperbenzoic acid (77%, 6.5 g, 29 mmol) was
added to a mixture of the 2-acetyl compound (4b, 6.5 g,
14.1 mmol) and disodium phosphate (4.2 g, 29.6 mmol)
in dry dichloromethane (300 ml). The mixture was stirred
overnight at room temperature, after which time, analysis
by TLC (2% acetone in CH2Cl2) indicated a complete
reaction. The reaction mixture was transferred to a separa-
tory funnel and washed with 10% sodium sulfite solution
(1×), water (1×), and 50% saturated sodium bicarbonate
solution (1×). The organic fractions were filtered through
sodium sulfate, combined and concentrated in vacuo to give
7.1 g of yellow foam. Crystallization of this material from
methanol containing 1% water gave 5.47 g of product as a
white solid. The mother liquors were concentrated in vacuo
and the residue purified via flash chromatography (1% ace-
tone in CH2Cl2) to give an additional 0.59 g of product.
Total yield (5b, 6.06 g, 90%): mp = 127–128 ◦C; FT-IR
(KBr, diffuse reflectance) νmax: 2943, 1763, 1722, and
1615 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.819 (s,
18-CH3), 0.841 (d, J = 7.2 Hz, 7-CH3), 2.054 (s, 17-OAc),
2.253 (s, 2-OAc), 4.695 (dd, J1 = 8.7 Hz, J2 = 7.8 Hz,
17-H), 5.029 (dd, J1 = 13.81 Hz, J2 = 11.71 Hz, benzyl
CH2), 6.703 (s, 4-H), 6.965 (s, 1-H), 7.304–7.385 (m, ben-
zyl aromatic). Analysis calculated for C30H36O5: C, 75.60;
H, 7.61. Found: C, 75.42; H, 7.58.
Following the procedure outlined for the synthesis of
4a, benzyl bromide (7 ml, 58.85 mmol) was added to a
mixture of the 2-acetyl compound (3b, 7.0 g, 18.89 mmol)
and anhydrous potassium carbonate (11 g, 79.6 mmol) in
dry dimethylformamide (250 ml). The reaction mixture was
then heated to 60 ◦C overnight. Analysis by TLC (CH2Cl2)
indicated an incomplete reaction. Additional benzyl bro-
mide (10 ml, 84.1 mmol) and potassium carbonate (11 g,
79.6 mmol) were added and the reaction continued for
another 24 h. Analysis by TLC at that time indicated a com-
plete reaction. The reaction mixture was cooled to room
temperature, filtered, diluted with water (∼1 l) and extracted
with dichloromethane (3×). The organic fractions were
washed with water (2×) saturated sodium bicarbonate so-
lution (1×), filtered through sodium sulfate, combined and
concentrated in vacuo to give 15 g of a yellow oily solid.
Crystallization of this material from hot methanol gave
the pure benzyl ether (4b, 6.7 g, 77%): mp = 179–181 ◦C;
FT-IR (KBr, diffuse reflectance) νmax: 2927, 1734, 1663,
and 1603 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.832 (s,
18-CH3), 0.842 (d, J = 5.7 Hz, 7-CH3), 2.060 (s, 17-OAc),
2.572 (s, 2-Ac), 4.694 (dd, J1 = 9.16 Hz, J2 = 7.95 Hz,
17-H), 5.117 (s, benzyl CH2), 6.731 (s, 4-H), 7.335–7.459
(m, benzyl aromatic), 7.723 (s, 1-H). Analysis calculated
for C30H36O4: C, 78.23; H, 7.88. Found: C, 78.09; H, 7.90.
2.9. 3-Benzyloxyestra-1,3,5(10)-triene-2,17β-diol (6a)
2.7. 3-Benzyloxyestra-1,3,5(10)-triene-2,17β-diol
diacetate (5a)
Under nitrogen, a solution of sodium hydroxide (1N,
100 ml, 100 mmol) was added to a solution of the diacetate
(5a, 14.6 g solid + residue from mother liquors, assume
38.5 mmol). The mixture was heated to 60 ◦C for 2 h. At that
time, analysis by TLC (5% acetone in CH2Cl2) indicated an
incomplete reaction. Additional sodium hydroxide solution
(100 ml, 100 mmol) was added and the reaction continued
for an additional hour. Analysis by TLC at that time indi-
cated a complete reaction. The mixture was cooled to room
temperature and acetic acid (glacial, 8 ml, 139.2 mmol)
was added. The mixture was diluted with water (1.5 l) and
the resulting precipitate collected by filtration. After air
drying, the solid was dissolved in methylene chloride, fil-
tered through Na2SO4 and concentrated in vacuo to give
13.1 g crude product. Trituration of this material with ether
gave the pure product (6a, 12.56 g, 86.2% from 4a): mp =
218–220 ◦C (lit. [5] 227–228 ◦C); FT-IR (KBr, diffuse re-
flectance) νmax: 3524, 3280, 2919, and 1605 cm−1; NMR
(300 MHz, CDCl3), δ (ppm): 0.776 (s, 18-CH3), 3.728 (t,
J = 9.15 Hz, 17-H), 5.061 (s, benzyl CH2), 5.464 (s, OH),
Under nitrogen, meta-chloroperbenzoic acid (77%, 17.0 g,
75 mmol) was added to a mixture of the 2-acetyl benzyl
ether (4a, 17.4 g, 39 mmol) and disodium phosphate (14 g,
98 mmol) in methylene chloride (800 ml). The reaction mix-
ture was stirred overnight at room temperature. After that
time, analysis by TLC (2% acetone in CH2Cl2) indicated a
complete reaction. The mixture was diluted with water (1 l)
and extracted with methylene chloride (3×). The organic
fractions were washed with water (1×), 10% Na2SO3 so-
lution (1×) and half saturated sodium bicarbonate solution
(1×), filtered through Na2SO4, combined and concentrated
in vacuo. The residue was crystallized from methanol to give
the pure 2-acetoxy derivative (5a, 14.85 g, 82.4%) as a white
crystalline solid: mp = 151–153 ◦C (lit. [5] 157–159 ◦C);
FT-IR (KBr, diffuse reflectance) νmax: 929, 1758, 1733, and
1615 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.818 (s,
18-CH3), 2.054 (s, 17-OAc), 2.259 (s, 2-OAc), 4.682 (dd,
J1 = 8.85 Hz, J2 = 7.65 Hz, 17-H), 5.039 (s, benzyl CH2),

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P.N. Rao, J.W. Cessac / Steroids 67 (2002) 1065–1070
6.649 (s, 4-H), 6.905 (s, 1-H), 7.359–7.422 (m, benzyl
aromatic). Analysis calculated for C25H30O3·1/10H2O: C,
78.95; H, 8.00. Found: C, 79.09; H, 7.97.
2.12. 2-Methoxy-3-benzyloxy-7α-methylestra-
1,3,5(10)-trien-17β-ol (7b)
Following the procedure outlined for the synthesis of 7a,
reaction of the diol (6b, 4.37 g, 11.13 mmol) with lithium
hydroxide monohydrate (0.6 g, 14.01 mmol) and dimethyl
sulfate (1.1 ml, 11.6 mmol) in dry THF (60 ml) gave the pure
2-methoxy product (7b, 3.5 g, 77.3%) after crystallization
from methanol/water: mp = 68–70 ◦C; FT-IR (KBr, dif-
fuse reflectance) νmax: 3415, 2953, and 1607 cm−1; NMR
(300 MHz, CDCl3), δ (ppm): 0.783 (s, 18-CH3), 0.818 (d,
J = 6.9 Hz, 7-CH3), 3.745 (t, J = 8.25 Hz, 17-H), 3.854
(s, OCH3), 5.095 (s, benzyl CH2), 6.607 (s, 4-H), 6.842
(s, 1-H), 7.292–7.464 (m, benzyl aromatic). Analysis calcu-
lated for C27H34O3·1/10H2O: C, 79.41; H, 8.44. Found: C,
79.44; H, 8.52.
2.10. 3-Benzyloxy-7α-methylestra-1,3,5(10)-
triene-2,17β-diol (6b)
Under nitrogen, a solution of sodium hydroxide (1N,
30 ml, 30 mmol) was added to a solution of the diacetate
5b in methanol (300 ml). The reaction mixture was stirred
at room temperature and monitored by TLC (2% acetone in
CH2Cl2) which indicated an incomplete reaction after 2 h.
Additional sodium hydroxide solution (30 ml) was added
and the reaction was heated to 60 ◦C. Analysis by TLC
indicated a complete reaction after 1 h. The mixture was
allowed to cool to room temperature and then concentrated
in vacuo. The residue was diluted with water and the so-
lution was adjusted to a pH ∼ 5 (pH paper) with glacial
acetic acid. The resulting precipitate was collected by fil-
tration, washed well with water and air-dried to give 5.1 g
light purple solid. This material was dissolved in ethyl ac-
etate (∼300 ml), dried over sodium sulfate, filtered through
Celite and concentrated in vacuo. The residue was crystal-
lized from ether/heptane to give the pure diol (6b, 4.5 g,
92%) as a light purple solid: mp = 146.5–147.5 ◦C; FT-IR
(KBr, diffuse reflectance) νmax: 3514, 2955, 2902, and
1607 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.777 (s,
18-CH3), 0.831 (d, J = 7.5 Hz, 7-CH3), 3.739 (m, 17-H),
5.053 (dd, J1 = 13.1 Hz, J2 = 10.96 Hz, benzyl CH2),
6.636 (s, 4-H), 6.904 (s, 1-H), 7.351–7.425 (m, benzyl
aromatic). Analysis calculated for C26H32O3·1/10H2O: C,
79.19; H, 8.23. Found: C, 79.05; H, 8.13.
2.13. 2-Methoxyestradiol (8a)
A mixture of the 3-benzyl ether (7a, 1.0 g, 2.55 mmol)
and 5% palladium on charcoal (1.0 g) in ethanol (50 ml)
was hydrogenated in a Parr shaker apparatus at 38 psi hy-
drogen pressure for 16 h. The mixture was filtered through
Celite and concentrated in vacuo to give 0.72 g residue.
Crystallization of this material from CH2Cl2/hexanes gave
the pure product (8a, 0.65 g, 84.4%) as a white crystalline
solid. Analysis by HPLC (Waters Associates NovaPak C18,
66% H2O/34% CH3CN at 1.0 ml/min, UV = 288 nm) in-
dicated this material to be greater than 99% pure (RT
=
10.73 min) with no detection of 4-methoxyestradiol (RT =
9.35 min) or estradiol (RT = 8.51 min): mp = 183–185 ◦C
(lit. [10] 188–190 ◦C); FT-IR (KBr, diffuse reflectance)
νmax: 3424, 3202, 2905, 2863, and 1607 cm−1; NMR
(300 MHz, CDCl3), δ (ppm): 0.787 (s, 18-CH3), 3.734
(t, J = 8.1 Hz, 17-H), 3.858 (s, OCH3), 5.433 (s, OH),
6.641 (s, 4-H), 6.795 (s, 1-H). Analysis calculated for
C26H32O3·2/7CH2Cl2: C, 70.99; H, 8.21. Found: C, 70.98;
H, 8.22.
2.11. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-17β-ol
(7a)
Under nitrogen, solid lithium hydroxide monohydrate
(1.4 g, 32.7 mmol) and dimethyl sulfate (2.8 ml, 29.59 mmol)
were added to a solution of the diol (6a, 10.0 g, 26.42 mmol)
in dry THF (150 ml). The reaction mixture was heated to
reflux and monitored by TLC (3% acetone in CH2Cl2)
which indicated a complete reaction after 3 h. The mixture
was cooled to room temperature and solvents removed in
vacuo. The residue was taken up in methylene chloride
and washed with water (2×) and brine (1×). The organic
fractions were filtered through Na2SO4, combined and con-
centrated in vacuo to give 10.2 g of a foam. Crystallization
of this material from benzene/ether gave the pure product
(7a, 9.61 g, 92.7%): mp = 113–114 ◦C (lit. [5] 89–90 ◦C);
FT-IR (KBr, diffuse reflectance) νmax: 3318, 2925, 2862,
and 1606 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.784
(s, 18-CH3), 3.733 (t, J = 8.7 Hz, 17-H), 3.861 (s,
OCH3), 5.104 (s, benzyl CH2), 6.623 (s, 4-H), 6.851 (s,
1-H), 7.29–7.46 (m, benzyl aromatic). Analysis calcu-
lated for C26H32O3: C, 79.56; H, 8.22. Found: C, 79.37;
H, 8.21.
2.14. 2-Methoxy-7α-methylestradiol (8b)
Following the procedure outlined for the synthesis of 8a, a
mixture of the benzyl ether (7b, 3.4 g, 8.36 mmol) in ethanol
(100 ml) was hydrogenated over 5% Pd/C (3.4 g) in a Parr
shaker apparatus at a hydrogen pressure of 38 psi for 16 h.
Filtration followed by concentration in vacuo gave 2.8 g solid
residue. Crystallization of this material from methanol gave
the pure 2-methoxy compound (8b, 2.25 g, 85%) as a white
crystalline solid: mp = 159–161 ◦C; FT-IR (KBr, diffuse
reflectance) νmax: 3414, 3296, 2955, 2888, and 1608 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 0.789 (s, 18-CH3), 0.820
(d, J = 6.9 Hz, 7-CH3), 3.737 (t, J = 8.25 Hz, 17-H),
3.855 (s, OCH3), 4.769 (br.s, OH), 6.623 (s, 4-H), 6.789 (s,
1-H). Analysis calculated for C20H28O3: C, 75.91; H, 8.92.
Found: C, 76.05; H, 9.04.

P.N. Rao, J.W. Cessac / Steroids 67 (2002) 1065–1070
1069
3. Results and discussion
tion with lithium hydroxide-hydrate and dimethyl sulfate
in tetrahydrofuran following the procedure of Basak et al.
[14]. Removal of the benzyl ether of 7a was accomplished
in 84.4% yield by hydrogenation over Pd/C at 38 psi. The
overall yield of 2-ME2 from estradiol was 49%. All the inter-
mediates 2a–7a are solids purified by simple crystallization.
In a similar fashion, 7␣-methylestradiol (1b) was con-
verted to 2-methoxy-7␣-methylestradiol (8b) in 25.8%
yield. The lower overall yield for the preparation of 8b was
due to decreased yields in the Fries rearrangement (2b–3b),
benzylation (3b–4b), and methylation (6b–7b) steps. All
intermediates 2b–8b were also solids, purifiable by crystal-
lization. However, in some steps, column chromatography
was carried out on the mother liquors in order to increase
the overall yield.
The key step in this scheme is the zirconium tetrachloride-
mediated Fries rearrangement [13] of diacetate 2a to give
the 2-acetyl derivative 3a in 84% yield after crystallization.
This reaction was carried out at room temperature regiose-
lectively with no indication of acyl migration to the C-4 po-
sition. As a method of C-2 functionalization, this method is
superior to halogenation, which gives mixtures of the 2- and
4-monosubstituted halides as well as the 2,4-disubstituted
dihalides as crude reaction mixtures [6,7,9,11].
For the projected syntheses of a series of 2-ME2 deriva-
tives, a method for the preparation of a large amount of 2-ME
was required. While reviewing the prior syntheses, we were
attracted in particular to the procedure of Nambara et al. [5]
whereby estradiol 3-methyl ether 17-acetate is converted to
2-ME2 utilizing Friedel–Crafts and Baeyer–Villiger reac-
tions as key steps. Nambara reports a 50% overall yield for
this process, however, the experimental data he gives indi-
cates a much lower yield (22%). We felt we could improve
this procedure by incorporating the recently discovered zir-
conium tetrachloride-mediated Fries rearrangement [13] in
place of the Friedel–Crafts acylation. Nambara reported that
the aluminum chloride mediated Fries rearrangement carried
out on estrone 3-acetate gave an unsatisfactory yield (54%).
2-ME2 and 2-methoxy-7␣-methylestradiol were synthe-
sized as outlined in Fig. 1. Estradiol was converted to the
3,17␤-diacetate (2a) in 95% yield by reaction with acetic an-
hydride in pyridine. Reaction of diacetate 2a with zirconium
tetrachloride in methylene chloride at room temperature
following the procedure of Harroven [13] gave the 2-acetyl
derivative 3a in 84% yield. Formation of the 3-benzyl ether
derivative 4a was carried out in 91.4% yield by reaction of 3a
with benzyl bromide and potassium carbonate in dimethyl-
formamide. Subsequent Baeyer–Villiger oxidation followed
by base hydrolysis gave the 2,17␤-dihydroxy derivative 6a
in 86.2% overall yield. The phenolic hydroxyl group of 6a
was then selectively methylated in 92.7% yield by reac-
Pert and Ridley [15] have reported the regioselective
2-formylation of the 3,17␤-bis(methoxymethyl) ether of
estradiol by reaction with sec-butyl lithium at low tem-
perature followed by quenching with anhydrous dimethyl-
formamide. This procedure was adopted by Wang and
Fig. 1. Synthesis of 2-ME2 and its 7␣-methyl derivative.

1070
P.N. Rao, J.W. Cessac / Steroids 67 (2002) 1065–1070
Cushman [10] into a five-step synthesis of 2-ME2 with an
overall yield of 63%. However, the first three intermediates
in this synthesis are oils that require chromatographic pu-
rification [10,16], making this approach unattractive for a
moderate to large-scale synthesis.
estradiol inhibits angiogenesis and suppresses tumor growth. Nature
1994;368:237–9.
[4] Fishman J. Synthesis of 2-methoxyestrogens. J Am Chem Soc
1958;80:1213–6.
[5] Nambara T, Honma S, Akiyama S. Studies on steroid conjugates. III.
New syntheses of 2-methoxyestrogens. Chem Pharm Bull 1970;18:
474–80.
Agoston et al. [17] have reported that all commercially
available preparations of 2-ME2 are either less than 98%
pure or contain undesirable steroid contaminants that are
of concern for pharmaceutical uses. Using HPLC, the au-
thors identified estradiol and 4-methoxyestradiol as the
major contaminants. The 4-methoxyestradiol contaminant
arises from small amounts of 4-brominated intermediate
that is carried through the reaction sequence. The estradiol
contaminant arises upon the reaction of the 2-brominated
intermediate with sodium methoxide in the presence of a
copper catalyst. A small amount (1–2%) of the reactive
copper complex is quenched by a hydride rather than a
methoxide. Since the procedure we presented here does
not rely on the halogenation method, no indication of these
contaminants is detected in the final product. Analysis by
HPLC indicated a purity of >99%.
[6] Rao PN, Burdett JE. A novel two-step synthesis of 2-methoxy-
estradiol. Synthesis 1977;168–9.
[7] Chen S-H, Luo G-R, Wu X-S, Chen M, Zhao H-M. A new synthetic
route to 2- and 4-methoxyestradiols by nucleophilic substitution.
Steroids 1986;47:63–6.
[8] Cushman M, He H-M, Katzenellenbogen JA, Lin CM, Hamel E.
Synthesis, antitubulin and antimitotic activity, and cytotoxicity of
analogs of 2-methoxyestradiol, an endogenous mammalian metabolite
of estradiol that inhibits tubulin polymerization by binding to the
colchicine site. J Med Chem 1995;38:2041–9.
[9] Cushman M, He H-M, Katzenellenbogen JA, Varma RK, Hamel
E, Lin CM, et al. Synthesis of analogs of 2-methoxyestradiol with
enhanced inhibitory effects on tubulin polymerization and cancer cell
growth. J Med Chem 1997;40:2323–34.
[10] Wang Z, Cushman M. An optimized synthesis of 2-methoxyestradiol,
a naturally occurring human metabolite with anticancer activity. Synth
Commun 1998;28:4431–7.
[11] Stewart AG, Mcallister DJ, Robertson AD (inventors). Estradiol
conjugates and uses thereof. International Patent Application WO
01/27132 (2001).
[12] Nagao Y, Fujita E, Kohno T, Yagi M. An efficient method for
selective acetylation of alcoholic hydroxyl groups. Chem Pharm Bull
1981;29:3202–7.
[13] Harroven DC, Dainty RF. Zirconium tetrachloride as a mediator for
ambient temperature ortho-Fries rearrangements. Tetrahedron Lett
1996;37:7659–60.
Acknowledgments
Financial support from Southwest Foundation for
Biomedical Research is gratefully acknowledged.
[14] Basak A, Nayak MK, Chakraborti AK. Chemoselective O-methyla-
tion of phenols under non-aqueous condition. Tetrahedron Lett
1998;39:4883–6.
References
[1] Breuer H, Knuppen R. Biosynthesis of 2-methoxyestradiol in human
liver. Naturwissenschaften 1960;12:280–1.
[15] Pert DJ, Ridley DD. Formylation of oestrogens. Aust J Chem 1989;
42:405–19.
[2] Seegers JC, Aveling M-L, van Aswegan CH, Cross M, Koch F,
Joubert WS. The cytotoxic effects of estradiol-17␤, catecholestradiols
and methoxyestradiols on dividing MCF-7 and HeLa cells. J Steroid
Biochem 1989;32:797–809.
[3] Fotsis T, Zhang Y, Pepper MS, Adlercreutz H, Montesano R,
Nawroth PP, et al. The endogenous oestrogen metabolite 2-methoxy-
[16] Lovey CJ, Gilbert NE, Liberto MM, Sharp DW, Lin YC, Bruegge-
meier RW. 2-(Hydroxyalkyl)estradiols: synthesis and biological
evaluation. J Med Chem 1996;39:1917–23.
[17] Agoston GE, Treston AM, Shah JH (inventors, Entrmed Inc., USA
assignees). Methods of obtaining 2-methoxyestradiol of high purity.
International Patent Application WO 01/14405 (2001).