An efficient, practical synthesis of 2-methoxyestradiol

Article abstract of DOI:10.1016/j.steroids.2009.09.013

An efficient and practical scheme to synthesize 2-methoxyestradiol has been developed. The key step was the copper-mediated methoxylation using ethyl acetate as a co-catalyst to introduce a methoxyl group. These synthetic procedures of four steps from 17β-estradiol as starting material gave 2-methoxyestradiol with a 61% overall yield.

Full text of DOI:10.1016/j.steroids.2009.09.013

Steroids 75 (2010) 53–56
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Steroids
journal homepage: www.elsevier.com/locate/steroids
An efficient, practical synthesis of 2-methoxyestradiol
Minhang Xin, Qidong You, Hua Xiang∗
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and practical scheme to synthesize 2-methoxyestradiol has been developed. The key step was
the copper-mediated methoxylation using ethyl acetate as a co-catalyst to introduce a methoxyl group.
These synthetic procedures of four steps from 17␤-estradiol as starting material gave 2-methoxyestradiol
with a 61% overall yield.
Received 17 July 2009
Received in revised form
18 September 2009
Accepted 25 September 2009
Available online 14 October 2009
© 2009 Elsevier Inc. All rights reserved.
Keywords:
2-Methoxyestradiol
Copper-mediated methoxylation
Ethyl acetate
Co-catalyst
Synthesis
1. Introduction
In most cases, these routes suffered from one or more limitations
such as harsh reaction conditions (such as very low temperature,
2-Methoxyestradiol (1), an endogenous metabolite of 17␤-
estradiol (4), was reported to have anti-proliferative and
anti-angiogenic activity [1]. Research on the mechanism of antitu-
mor activity showed that 2-methoxyestradiol could disrupt tubulin
polymerization [2], down-regulate hypoxia inducible factor-1
alpha (HIF-1␣) [3], inhibit superoxide dismutases (SOD) [4] and
so on. These results lead to pharmaceutical scientists’ research
interest in 2-methoxyestradiol and its analogs. Many analogs of
2-methoxyestradiol have been extensively explored to enhance
the inhibitive activity and decrease the metabolic inactivation
[5–8]. Currently, some 2-methoxyestradiol derivatives such as
ENMD-1198 (2) and STX140 (3) have been reported to be ongoing
clinical/preclinical studies (Fig. 1).
Since the first chemical synthesis of 2-methoxyestradiol was
reported by Fishman [9], many synthetic procedures have been
developed. These can be classified mainly into three types. The
first one [10–12] was fulfilled by introducing the bromo (or iodo)
group at the 2-position of 17␤-estradiol and subsequently replac-
ing the 2-bromo (or 2-iodo) atom with sodium methoxide. The
second one mainly involved acylation at the 2-position by Fries
rearrangement [13] or by ortho-lithiation/DMF reaction [14], then
using Baeyer–Villiger oxidation with MCPBA. The last approach
focused on the introduction of 2-hydroxy group via one-pot reac-
tion using LIDAKOR, trimethyl borate and hydrogen peroxide [15].
rigid anhydrous reaction environment, and freshly distilled sol-
vents), low overall yields (some only 28%) and high cost in terms
of large-scale production (mainly due to the catalyst consumed).
Hence, improvements are still needed for the preparation of 2-
methoxyestradiol.
Methoxylation is an important transformation for synthesis of
2-methoxyestradiol. In the past, several efforts have been made
to employ copper-catalyzed methoxylation for the preparation of
2-methoxyestradiol [10–12,16]. Although these results are encour-
aging, there is considerable room for improvement. Capdevielle and
Maumy reported efficient copper/ethyl acetate cooperative cataly-
sis in the assembly of methyl aryl ethers with high yields (98–100%)
[17]. Later some synthetic examples of methoxyaryl-containing
compounds have demonstrated the advantage of this catalytic sys-
tem [18,19]. Encouraged by these results, in connection with our
synthetic work of 2-methoxyestradiol, we considered that this cat-
alytic system could be used to develop a modified procedure to
synthesize 2-methoxyestradiol.
Herein, we report a practical, convenient and efficient method
to prepare 2-methoxyestradiol (Scheme 1). In comparison to cur-
rently existing methods of synthesis of 2-methoxyestradiol, this
approach has some distinguishing characteristics that are worth
mentioning: (1) the key methoxylation step employs an economi-
cally competitive catalytic system that is a combination of readily
available copper-salt and an universal reagent ethyl acetate; (2)
the approach offers experimental simplicity but the overall yield of
pure product is still very high (61%); (3) the cost of this whole route
is low enough in terms of lab-scale production.
∗
Corresponding author. Tel.: +86 25 83271096; fax: +86 25 83271096.
E-mail address: cpuxianghua@sina.com (H. Xiang).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.09.013

54
M. Xin et al. / Steroids 75 (2010) 53–56
Fig. 1. Selected example of clinical/preclinical compounds.
2. Experimental
refluxedfor12 h, andthe end-pointwas detectedby TLC(petroleum
ether/EtOAc, 3:1). After the reaction was completed, the mixture
was filtered and the filtrate was concentrated in vacuo to a small
volume (∼50 ml), then diluted with water (∼200 ml) and extracted
with EtOAc (3 × 100 ml). The organic extracts were combined and
washed with water (50 ml), saturated brine (50 ml), and dried
with anhydrous sodium sulfate. The solvent was removed in vacuo
and the residue was triturated with ethanol below 0 ◦C to give
10.0 g of crude product. The mother liquor was purified via column
chromatography (petroleum ether/EtOAc, 8:1) to give an addi-
tional 3.5 g of product. Recrystallization of combined product above
from ethanol gave compound 5 as white crystals (12.5 g, 94%):
m.p. 99–101 ◦C (lit [20] 99–102 ◦C); 1H NMR (300 MHz, CDCl3), ı
(ppm):0.77 (s, 3H, C18-H), 3.70–3.76 (t, 1H, C17-H, J = 8.3 Hz), 5.02
(s, 2H, C3-OCH2Ph), 6.70–6.71 (d, 1H, C4-H, J = 1.5 Hz), 6.76–6.78
(dd, 1H, C2-H, J1 = 1.6 Hz, J2 = 5.1 Hz), 7.18–7.20 (d, 1H, C1-H,
J = 5.2 Hz), 7.35–7.42 (m, 5H, ArH); EI-MS m/z: 362 (M+).
Melting points of compounds were measured on a RY-1 melting
point apparatus and were uncorrected. Nuclear magnetic res-
onance (1H NMR) spectra were recorded on a Bruker AV-300
(300 MHz) spectrometer as deuterochloroform (CDCl3) solutions
using tetramethylsilane (TMS) as an internal standard (ı = 0) unless
noted otherwise. Electron impact mass spectral (EI-MS) data were
obtained on a SHIMADZU GCMS-QP2010 system. All chemicals
were purchased from commercial sources and were used without
further purification unless otherwise noted. The solvents (such as
MeOH, EtOAc, EtOH, CH2Cl2 and others) were C.P. grade purchased
from Nanjing Chemical Co., Ltd. and used without further purifica-
tion. Anhydrous CH2Cl2 was distilled from continuous reflux with
P2O5. The purity of the commercial 17␤-estradiol used in this study
was at least 95%. Column chromatography (CC) was carried out on
silica gel (200–300 mesh, Qindao Ocean Chemical Company, China).
Thin-layer chromatography (TLC) analyses were carried out on sil-
ica gel GF254 (Qindao Ocean Chemical Company, China) glass plates
(2.5 cm × 10 cm with 250 ␮m layer). Concentration and evapora-
tion of the solvent after reaction or extraction was carried out on a
rotary evaporator operated at reduced pressure.
2.2. 2-Bromo-3-benzyloxyestra-1,3,5(10)-trien-17ˇ-ol (6)
A solution of bromine (0.30 ml, 5.69 mmol) in anhydrous CH2Cl2
(20 ml) was added dropwise to a solution of 5 (2.0 g, 5.52 mmol)
in anhydrous CH2Cl2 (200 ml) while stirring at under 0 ◦C. After
30 min of stirring at −5 to 0 ◦C, the solvent was removed in vacuum
to give the crude product (2.41 g), The crude product above could
be recrystallized from petroleum ether/EtOAc (2:1) to give com-
pound 6 (2.04 g, 84%): m.p. 147–149 ◦C; 1H NMR (300 MHz, CDCl3),
ı (ppm):0.79 (s, 3H, C18-H), 3.70–3.75 (t, 1H, C17-H, J = 8.3 Hz), 5.10
(s, 2H, C3-OCH2Ph), 6.66 (s, 1H, C4-H), 7.24–7.45 (m, 5H, ArH), 7.46
(s, 1H, C1-H); EI-MS m/z: 440 (M+).
2.1. 3-Benzyloxyestra-1,3,5(10)-trien-17ˇ-ol (5)
KOH (2.5 g, 44.2 mmol) was added to a solution of 17␤-estradiol
(4, 10.0 g, 36.8 mmol) in 95% ethanol (250 ml). The reaction mixture
was stirred and heated to reflux under nitrogen atmosphere, then
tetrabutylammonium iodide (1.0 g, 2.7 mmol) and benzyl chlo-
ride (8.5 ml, 73.6 mmol) were added. The reaction mixture was
Scheme 1. Synthetic route to 2-methoxyestradiol (1) from 17␤-estradiol (4).

M. Xin et al. / Steroids 75 (2010) 53–56
55
Table 1
Optimization of the copper catalyst
.
Entry
Cu catalysta
Amount (mol%)
Time (h)
Yield (%)b
1
2
3
4
5
6
7
8
9
10
CuBr
CuBr
CuBr
CuBr
CuI
5
10
20
30
20
20
20
20
20
20
10
10
10
10
10
10
10
10
10
8
52
89
93
92
84
28
91
30
32
28
CuCl
CuBr2
CuCl2
Cu(OAc)2
CuO
a
The amount of Cu catalyst according to the compound 6 (100 mg, 0.23 mmol) in the optimized experiments.
Yield of isolated and purified product.
b
2.3. 2-Methoxy-3-benzyloxyestra-1,3,5(10)-trien-17ˇ-ol (7)
hydrogen atmosphere at room temperature, and the end-point
of reaction was detected by TLC (petroleum ether/EtOAc, 2:1).
After the reaction was complete, the reaction mixture was fil-
trated and the filtrate was concentrated in vacuum. The residue
was crystallized from chloroform to give 0.86 g of crude prod-
uct. The mother liquor was concentrated and purified via column
chromatography (petroleum ether/EtOAc, 5:1) to give an addi-
tional 0.42 g of product. The combined solid was recrystallized
from chloroform to afford compound 1 as white crystals (1.08 g,
83%). Analysis by HPLC (Agilent Eclipse XDB-C18, 55% H2O/45%
CH3CN at 1.0 ml/min, UV = 288 nm) indicated the purity of this
product was 98.6% (Rt = 11.98 min) with no detection of 17␤-
estradiol (Rt = 9.82 min): m.p.186–189 ◦C (lit [22] 188–190 ◦C); 1H
NMR (300 MHz, CDCl3), ı (ppm): 0.79 (s, 3H, C18-H), 3.71–3.76 (t,
1H, C17-H, J = 8.1 Hz), 3.86 (s, 3H, C2-OCH3), 5.43 (s, 1H, C3-OH),
6.64 (s, 1H, C4-H), 6.79 (s, 1H, C1-H); EI-MS m/z: 302 (M+).
Compound 6 (2.04 g, 4.64 mmol) was dissolved in methano-
lic concentrated sodium methoxide (30 ml, 5 mol/L). Then EtOAc
(0.28 ml, 2.78 mmol) and copper (I) bromide (0.14 g, 0.93 mmol)
were added. The reaction mixture was refluxed for 10 h under nitro-
gen and the reaction progress was detected by TLC (petroleum
ether/EtOAc, 3:1). After the reaction was complete, the mixture
was poured into ice water (100 ml), adjusted to a pH ∼7 with 6N
HCl in an ice bath, and extracted with EtOAc (3 × 50 ml). The com-
bined organic fractions were washed with water (50 ml), saturated
brine (50 ml), and dried with anhydrous Na2SO4. The solvent was
concentrated under vacuum to furnish the crude product (2.0 g).
This crude product could be purified by column chromatography
(petroleum ether/EtOAc, 5:1) and crystallized from ether to give
the compound 7 (1.69 g, 93%): m.p. 86–88 ◦C (lit [21] 89–90 ◦C);
1H NMR (300 MHz, CDCl3), ı (ppm):0.78 (s, 3H, C18-H), 3.70–3.75
(t, 1H, C17-H, J = 8.3 Hz), 3.85(s, 3H, C2-OCH3), 5.09 (s, 2H, C3-
OCH2Ph), 6.62 (s, 1H, C4-H), 6.85(s, 1H, C1-H), 7.24–7.45 (m, 5H,
ArH); EI-MS m/z: 392 (M+).
3. Results and discussion
In this synthesis of 2-methoxyestradiol from 17␤-estradiol, the
key step lies in the introduction of 2-methoxy group. The experi-
ments mainly involve region-selective bromination and followed
replacement of the bromo group by a methoxy through cop-
per/ethyl acetate-mediated nucleophilic aromatic substitution.
Typically, the bromination of 17␤-estradiol proceeded simul-
taneously at 2-position, 4-position and 2,4-positions, because the
2.4. 2-Methoxyestradiol (1)
The 2-methoxyestradiol-3-benzyl ether (7, 1.69 g, 4.31 mmol)
and 10% Pd/C (1.0 g) in ethanol (30 ml) was stirred for 8 h under
Table 2
Optimization of the ethyl acetate as a co-catalyst
.
Entry
EtOAc amount (mol%)a
Time (h)
Yield (%)b
1
2
3
4
5
6
0
20
40
50
60
70
10
10
10
10
10
10
55
80
87
89
93
93
a
The amounts of ethyl acetate catalyst according to the compound 6 (100 mg, 0.23 mmol) in the optimized experiments.
Yield of isolated and purified product.
b

56
M. Xin et al. / Steroids 75 (2010) 53–56
A-ring of 17␤-estradiol is very active and the distinction of steric
hindrances between 2-position and 4-position are small. How-
ever, 3-benzyl protecting appeared ideal since it presented some
steric hindrance and lowered the reactivity of the A-ring reason-
ably, which made the bromination reaction predominantly occur
at the 2-position.
2-methoxyestradiol derivatives as potential therapeutic anticancer
agents.
Acknowledgments
This work was supported by the International Science and
Technology Cooperation Program of Jiangsu Province, China (No
BZ2008058). The reviewers’ criticisms and editors’ comments for
improving the quality of our manuscript are greatly appreciated.
Methoxylation was conducted with 2-bromo-3-benzyl estradiol
(6) as the substrate, sodium methoxide as the reagent, and copper
compound/ ethyl acetate as the catalyst. The efficacy of methoxy-
lation was relevant to the types of copper compounds. A brief
comparative study of several readily available copper compounds
as the optimal catalyst was carried out. As shown in Table 1, sev-
eral Cu catalysts such as CuBr, CuI, CuCl, CuBr2, CuCl2, Cu(OAc)2,
and CuO were investigated. Among these, CuCl, CuCl2, CuO and
Cu(OAc)2 did not have obvious impact on the reaction as they
afforded lower yields (28–32%) of product 7 (Table 1, entries 6,
8–10). However, CuI was found to be relatively more active and
its yield (84%) was much better (Table 1, entry 5). CuBr and CuBr2
showed satisfactory results as the alternative copper catalysts
(91–93%, Table 1, entries 3, 4, 7). Interestingly, it was noticed that
some copper compounds sharing same anions such as CuBr and
CuBr2 (Table 1, entries 3, and 7), CuCl and CuCl2 (Table 1, entries
6 and 8), gave almost equivalent yields. Furthermore, the catalyst
loading was also optimized and thus several amounts of a particu-
lar catalyst were investigated such as 5 mol%, 10 mol%, 20 mol% and
30 mol% of CuBr (Table 1, entries 1–4). The highest yield (93%) was
obtained with 20 mol% (Table 1, entry 3). No improvement in the
yield was observed on increasing the catalyst from 20 to 30 mol%
(Table 1, entries 3 and 4). Hence we preferred to utilize CuBr catalyst
in 20 mol% throughout the experiments.
The presence of ethyl acetate is essential for the improvement
of the present copper-mediated methoxylation. We next examined
the optimal amounts of ethyl acetate as co-catalyst and several
amounts were performed. The results are shown in Table 2. 60 mol%
of ethyl acetate gave the best yield (93%, Table 2, entry 5) relative
to other amounts, such as 20 mol%, 40 mol%, and 50 mol% (80–89%,
Table 2, entries 2–4). Moreover, it appeared that further increases
in the amount of ethyl acetate from 60 mol% did not lead to an
improvement of product yield (Table 2, entries 5 and 6). On the
other hand, in the absence of the co-catalyst ethyl acetate only a
moderate yield was obtained (55%, Table 2, entry 1). Consequently,
ethyl acetate plays a very important role as a co-catalyst in this
copper-mediated methoxylation. It seemed that ethyl acetate could
really increase the stabilization and solubility of copper compounds
[17]. Although currently the precise mechanism of ethyl acetate
remains to be well established, its effect is obviously excellent
to improve the methoxylation. Other different reaction parame-
ters such as solvent and the concentration of sodium methoxide
were also performed. A concentrated methoxide solution is nec-
essary to prevent copper salt from precipitating and to provide a
homogeneous reaction system [17]. Finally the methoxylation of
6 proceeded under optimal conditions using CuBr (20 mol%), ethyl
acetate (60 mol%) and MeONa (5 mol/L) in refluxing methanol over
10 h under nitrogen atmosphere.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2009.09.013.
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Under optimized reaction conditions, the 2-methoxyestradiol
was synthesized with a 61% overall yield. The reaction conditions
of the four steps are tolerant and mild.
In conclusion, an efficient method for the synthesis of 2-
methoxyestradiol has been established. The key step is the
copper-mediated methoxylation by using ethyl acetate as a co-
catalyst. This route might provide a new access to the synthesis of