Synthesis of 7α-substituted derivatives of 17β-estradiol

Article abstract of DOI:10.1016/j.steroids.2005.11.008

Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alternatives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of postmenopausal, receptor-positive human breast cancers. Structurally, these pure

Full text of DOI:10.1016/j.steroids.2005.11.008

steroids 7 1 ( 2 0 0 6 ) 334–342
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Synthesis of 7␣-substituted derivatives of 17␤-estradiol
Xiang-Rong Jiang, J. Walter Sowell, Bao Ting Zhu∗
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of South Carolina, Columbia, SC 29208, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alter-
natives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of post-
menopausal, receptor-positive human breast cancers. Structurally, these pure antagonists
contain the basic core structure of 17␤-estradiol (E2) with a long side chain attached to its C-
7␣ position. We explored and compared in this study various synthetic routes for preparing
a number of C-7␣-substituted derivatives of E2, which are highly useful for the design and
synthesis of high-affinity ER antagonists, ER-based imaging ligands, and other ER-based
multi-functional agents. Using E2 as the starting material and 1-iodo-6-benzyloxyhexane
as a precursor for the C-7␣ side chain, a seven-step synthetic procedure afforded 3,17␤-
bis(acetoxy)-7␣-(6-hydroxyhexanyl)-estra-1,3,5(10)-triene (one of the derivatives prepared)
in an overall yield of ∼45% as compared to other known procedures that afforded sub-
stantially lower overall yield (8–27%). The synthetic steps for this representative compound
include: (1) protection of the C-3 and C-17␤ hydroxyls of E2 using methoxymethyl groups; (2)
hydroxylation of the C-6 position of the bismethoxymethyl ether of E2; (3) Swern oxidation
of the C-6 hydroxy to the ketone group; (4) C-7␣ alkylation of the C-6 ketone derivative of
E2; (5) deprotection of the two methoxymethyl groups; (6) reprotection of the C-3 and C-6
free hydroxyls with acetyl groups; (7) removal of the C-6 ketone and the benzyl group on
the side chain by catalytic hydrogenation in acetic acid. As predicted, two of the represen-
tative C-7␣-substituted derivatives of E2 synthesized in the present study retained strong
binding affinities (close to those of E2 and ICI-182,780) for the human ER␣ and ER␤ subtypes
as determined using the radioligand–receptor binding assays.
Received 16 March 2005
Received in revised form
2 November 2005
Accepted 9 November 2005
Published on line 24 March 2006
Keywords:
Estrogen receptor
Pure antagonist
Chemical synthesis
Abbreviations:
COSY, correlated spectroscopy;
LDA, lithium diisopropylamide;
E2, 17␤-estradiol;
MOM, methoxymethyl;
NMR, nuclear magnetic resonance;
PCC, pyridinium chlorochromate;
t-BuOK, potassium t-butoxide;
TEA, triethylamine;
© 2006 Elsevier Inc. All rights reserved.
TEMPO, tetramethylpyrrolidin-
1-oxyl;
THF, tetrahydrofuran;
TLC, thin-layer chromatography;
TMS, tetramethylsilane
1.
Introduction
weak agonistic activity for the same receptor, is commonly
prescribed nowadays for this purpose [2,3]. Partly because
Breast cancer is a serious health concern worldwide and is one
of the leading causes of cancer-related mortality in women
living in the United States [1]. The use of estrogen receptor
(ER) antagonists has become a common strategy in the treat-
ment of ER-positive human breast cancer [2,3]. Tamoxifen, a
well-known antagonist of the human ER which also retains
of its inherent partial agonistic activity (albeit very weak),
breast cancer patients chronically treated with tamoxifen
often experience relapse of the cancer. The newer ER antago-
nists such as ICI-182,780 (fulvestrant) and ICI-164,384 that are
devoid of any ER agonistic activity have already been devel-
oped as effective alternatives to tamoxifen [4–6]. Studies have
∗
Corresponding author. Tel.: +1 803 777 4802; fax: +1 803 777 8356.
E-mail address: BTZhu@cop.sc.edu (B.T. Zhu).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.11.008

steroids 7 1 ( 2 0 0 6 ) 334–342
335
Scheme 1 – The flow chart for the synthesis of 7␣-substituted derivatives of E2 (compounds 8, 10, 13 and 16) using E2
(compound 1) as the starting material. The reagents and the reaction conditions used are summarized below: (a) MOM
chloride, diisopropylethylamine, THF, reflux, overnight; (b) (1) LDA, t-BuOK, THF, −78 ◦C, (2) B(OMe)3, 0 ◦C and (3) H2O2, H2O,
25 ◦C; (c) Swern oxidation; (d) t-BuOK, THF, 0 ◦C; (e) 6 M HCl in THF, room temperatures, overnight; (f) Ac2O, pyridine; (g) 10%
Pd–C, H2, AcOH, reflux 3 h; (h) Swern oxidation, 98%; (i) CrO3, 3,5-dimethylpyrazole, CH2Cl2 or PCC, CH2Cl2, molecular
sieves, reflux; (j) benzyl bromide, NaOH, 15-crown-5 ether, THF, 10 ◦C; (k) triphenylphosphine, imidazole, I2, CH2Cl2; (l)
BF3·Et2O, Et3SiH, CH2Cl2; (m) NaBH3CN, BF3·Et2O, THF, reflux; (n) 10% Pd–C, H2, ethanol, 60 ◦C, 2 h; (o) H2NNH2, KOH,
(CH2CH2OH)2, 200 ◦C, 3 h; (p) NaBH4, MeOH, 4 h, 98%; (q) 10% Pd(OH)2/C, H2, 40 psi, ethanol, 60 ◦C, 3 h; (r) BF3·Et2O, Et3SiH,
CH2Cl2; (s) MOMCl, diisopropylethylamine, THF, reflux overnight, 93%; (t) 10% Pd–C, H2, ethanol, 40 psi, 2 h.
shown that human breast cancer cells that became resistant
to tamoxifen were still sensitive to the anticancer effect of ful-
vestrant, which was recently approved for use in the United
States for treatment of postmenopausal, ER-positive progres-
sive breast cancers [4,5]. Structurally, these ER pure antago-
nists all contain the E2 core structure with a long side chain
attached to the C-7␣ position. Several of the 7␣-substituted
derivatives of E2 (such as compounds 8, 10, 13 and 16 in
Scheme 1) are highly useful intermediates for the rational
design and synthesis of high-affinity ER antagonistic analogs
as well as ER-based imaging ligands for human breast cancers
or other ER-based multi-functional agents [7–12]. While there
were several published methods describing the syntheses of
this type of intermediates, usually there were also limitations
when they were used for large-scale production of the desired
intermediates, such as the paucity of the expensive starting

336
steroids 7 1 ( 2 0 0 6 ) 334–342
materials required in some of the synthetic procedures, the
highly stringent and difficult conditions for some of the reac-
tion steps involved, and/or the relatively low overall yield for
the final product (ranging from 8 to 27%) [8–10]. In the present
study, we explored and also compared a number of synthetic
procedures for preparing several 7␣-substituted derivatives of
E2 by modifying the reaction steps and also by significantly
improving the overall yield for the desired products.
10% Na2S2O3 (100 ml) was added. After extraction with ethyl
acetate and drying over Na2SO4/Na2CO3, flash column chro-
matography (hexane:ethyl acetate = 3:1) yielded compound 3
(7.70 g, 93%). Data from spectrometric analyses of the afforded
compound are summarized below. 1H NMR (300 Hz, CDCl3):
7.18 (1H, d, J = 2.7 Hz), 7.12 (1H, d, J = 8.4 Hz), 6.83 (1H, dd, J = 8.4,
2.4 Hz), 5.09 (1H, s), 5.08 (1H, s), 4.73 (1H, m), 4.57 (1H, s), 4.56
(1H, s), 3.53 (1H, t, J = 8.7 Hz), 3.39 (3H, s), 3.29 (3H, s), 2.23–1.89
(6H, m), 1.64–1.16 (8H, m) and 0.72 (3H, s). MS (m/z, C22H32O5,
M+ 376): 358 (base peak), 376. HRMS: 376.2250 (calculated) and
376.2255 (observed).
2.
Experimental
2.4.
Synthesis of
2.1.
Chemicals and reagents
3,17ˇ-bis(methoxymethoxy)estra-1,3,5(10)-trien-6-one
(compound 4)
Most of the commercially available reagents were obtained
from the Sigma–Aldrich Chemical Co. (Milwaukee, WI, USA)
or ACROS (through Fisher Scientific, Atlanta, GA, USA). 17␤-
Estradiol (E2) was purchased from Steraloids Inc. (New-
port, RI, USA). Tetrahydrofuran (THF) was distilled over
sodium/benzophenone ketyl and dichloromethane distilled
over calcium hydride in our laboratory prior to use. Most of the
chemicals and solvents used in this study were of ACS grade
and used directly without additional steps of purification.
[2,4,6,7,16,17-3H]E2 (specific activity of 123 Ci/mmol) was
obtained from NEN Life Sciences (Boston, MA, USA), and it was
re-purified using an HPLC method before use [13]. The recom-
binant human ER␣ and ER␤ proteins and bovine serum albu-
min (BSA) were obtained from PanVera Corporation (Madison,
WI, USA). Hydroxylapatite (HAP) and bovine serum albumin
were obtained from Calbiochem (San Diego, CA, USA).
After a solution of CH2Cl2 (70 ml) and oxalyl chloride (3.6 ml,
41.9 mmol) in a 250-ml three-neck round-bottom flask was
cooled to −78 ◦C, Me2SO (5.9 ml, 83 mmol, in 15 ml CH2Cl2)
was added slowly to the stirred oxalyl chloride solution and
the reaction mixture was stirred further for 5 min. Note that
the addition of the Me2SO solution needs to be relatively slow
so that the temperatures of the reaction mixture would not
rise above −50 ◦C (usually controlled between −50 and −60 ◦C),
which is critical for this reaction. After that, compound 3 (13 g,
34.5 mmol, in 30 ml CH2Cl2) was slowly added over 15 min,
and the mixture was stirred for an additional 25 min. Then
TEA (24 ml) was added and the reaction mixture was stirred
for 5 min and allowed to warm to room temperatures. Water
(100 ml) was added and the aqueous phase was extracted with
CH2Cl2 (3 × 50 ml). The organic phase was combined, washed
with a saturated NaCl solution (100 ml) and dried over anhy-
drous Na2SO4. Flash chromatography afforded compound 4
(12.6 g, 98% yield). Data from spectrometric analyses of the title
compound are summarized below. 1H NMR (300 Hz, CDCl3):
7.69 (1H, d, J = 2.7 Hz), 7.35 (1H, d, J = 8.4 Hz), 7.20 (1H, dd, J = 8.4,
2.7 Hz), 5.19 (2H, s), 4.65 (2H, brs), 3.62 (1H, t, J = 8.4 Hz), 3.46
(3H, s), 3.37 (3H, s), 2.73 (1H, dd, J = 16.8, 3.3 Hz), 2.60–1.86 (6H,
m), 1.72–1.52 (3H, m), 1.46–1.30 (3H, m) and 0.80 (3H, s). MS
(m/z, C22H30O5, M+ 374): 374 (base peak). HRMS: 374.2093 (cal-
culated) and 374.2086 (observed).
2.2.
Spectrometric analyses
Mass spectra were recorded with a VG70S analytical mass
spectrometer. An aliquot of the ethanol solution of the test
compound was used for the direct-probe mass analysis. The
nuclear magnetic resonance (NMR) spectra of all test com-
pounds were recorded in deuterochloroform solution using a
Varian Mercury/VX 300 spectrometer with tetramethylsilane
(TMS) as an internal standard (ı = 0), unless noted otherwise.
2.3.
Synthesis of
3,17ˇ-bis(methoxymethoxy)estra-1,3,5(10)-trien-6-ol
2.5.
Synthesis of 3,17ˇ-bis(methoxymethoxy)-7˛-(6-
(compound 3)
benzyloxyhexanyl)-estra-1,3,5(10)-trien-6-one
(compound 5)
Using E2 (compound 1 in Scheme 1) as the starting mate-
rial, the 3,17␤-bisMOM ether of E2 (compound 2) was prepared
according to a previously published method [14] by refluxing
overnight with MOM chloride and diisopropylethylamine in
THF. The yield of this reaction was ∼96%.
A 1.0 M solution of t-BuOK in THF (20 ml, 5.35 mmol) was added
to a cooled solution (at 0 ◦C) of compound 4 (1.0 g, 2.67 mmol) in
THF (20 ml). The reaction mixture was stirred at 0 ◦C for 40 min
and then cooled to −78 ◦C. 6-Benzyl hexane iodide (compound
21, 2.55 g, 8.02 mmol) was added dropwise to the solution. The
reaction mixture was allowed to warm to 0 ◦C and stirred for
2 h, and then to room temperatures and stirred overnight.
The reaction was quenched with water and extracted with
ethyl acetate. The organic phase was dried over Na2SO4 and
solvent was evaporated in vacuo. Flash chromatography (hex-
ane:ethyl acetate = 4:1) afforded compound 5 in 65% yield
(0.98 g). Data from spectrometric analyses of the afforded com-
pound are summarized below. 1H NMR (300 Hz, CDCl3): 7.68
(1H, d, J = 3.0 Hz), 7.29 (6H, m), 7.18 (1H, dd, J = 8.7, 3.0 Hz), 5.18
After a solution of t-BuOK (9.95 g, 88.9 mmol) in dry THF
(100 ml) was cooled to −78 ◦C, LDA (49.4 ml, 1.8 M, 88.9 mmol)
was added and the mixture was stirred for 30 min at −78 ◦C.
Compound 2 (8.0 g, 22.2 mmol) in THF (30 ml) was then added
to the reaction vessel, and the mixture (in a dark red color)
was stirred for 3 h at −78 ◦C. Trimethyl borate (30 ml) was
added, and the reaction was warmed to 0 ◦C, yielding a milky
yellow suspension. After stirring for 2 h, H2O2 (34 ml, 30% in
water) was added, and the mixture was stirred for 1 h at room
temperatures. The reaction mixture was cooled to 0 ◦C and

steroids 7 1 ( 2 0 0 6 ) 334–342
337
(2H, s), 4.64(2H, brs), 4.46 (2H, s), 3.63 (1H, t, J = 8.4 Hz), 3.45
(3H, s), 3.42 (2H, t, J = 6.6 Hz), 3.36 (3H, s), 2.69 (1H, t-d, J = 10.8,
4.2 Hz), 2.47–2.33 (2H, m), 2.16–1.98 (3H, m), 1.64–1.50 (8H, m),
1.41–1.28 (8H, m) and 0.79 (3H, s). MS (m/z, C35H48O6, M+ 564): 91
(base peak), 473, 564. HRMS: 564.3451 (calculated) and 564.3465
(observed).
5.1 Hz), 2.72 (1H, d, J = 16.8 Hz), 2.50 (1H, brs), 2.40–2.10 (3H, m),
2.24 (3H, s), 2.03 (3H, s), 1.86–1.0 (19H, m) and 0.79 (3H, s). MS
(m/z, C28H40O5, M+ 456): 414 (base peak), 456. HRMS: 456.2875
(calculated) and 456.2862 (observed). Compound 9—1H NMR
(300 Hz, CDCl3): 7.24 (1H, d, J = 8.4 Hz), 6.80 (1H, dd, J = 8.4,
2.4 Hz), 6.75 (1H, d, J = 2.4 Hz), 4.68 (1H, t, J = 7.8 Hz), 4.0 (2H, t,
J = 6.7 Hz), 2.88 (1H, dd, J = 16.8, 5.1 Hz), 2.72 (1H, d, J = 16.8 Hz),
2.38–2.16 (3H, m), 2.24 (3H, s), 2.02 (3H, s), 2.0 (3H, s), 1.86–0.94
(19H, m) and 0.79 (3H, s). MS (m/z, C30H42O6, M+ 498): 456
(base peak), 498. HRMS: 498.2981 (calculated) and 498.2983
(observed).
2.6.
Synthesis of 3,17ˇ-bis(hydroxy)-7˛-(6-
benzyloxyhexanyl)-estra-1,3,5(10)-trien-6-one
(compound 6)
A solution containing compound 5 (2.1 g, 3.7 mmol), HCl (25 ml,
6 M) and THF (25 ml) was stirred at room temperatures for
18 h. The reaction mixture was poured into water and then
extracted with ethyl acetate (4 × 50 ml). The combined organ-
ics were dried over Na2SO4, concentrated and then chro-
matographed (hexane:ethyl acetate = 2:1) to afford compound
6 (1.67 g, 94% yield). Data from spectrometric analyses of the
afforded compound are summarized below. 1H NMR (300 Hz,
CDCl3): 7.57 (1H, d, J = 3.0 Hz), 7.33–7.26 (6H, m), 7.05 (1H, dd,
J = 8.4, 2.7 Hz), 6.28 (1H, s), 4.48 (2H, s), 3.77 (1H, brs), 3.43 (2H,
t, J = 6.6 Hz), 2.68 (1H, t-d, J = 11.1, 4.2 Hz), 2.43 (2H, m), 2.11 (3H,
m), 1.77 (1H, s), 1.61–1.18 (16H, m) and 0.78 (3H, s). MS (m/z,
2.9.
Synthesis of 3,17(-bis(acetoxy)-7(-(6-
hexanyladhyde)-estra-1,3,5(10)-triene (compound 10)
A solution of CH2Cl2 (10 ml) and oxalyl chloride (0.095 ml,
1.1 mmol) were placed in a 50-ml three-neck round-botton
flask equipped with a thermometer. Me2SO (0.16 ml, 2.2 mmol)
dissolved in CH2Cl2 (5 ml) was added to the stirred oxalyl chlo-
ride solution at −50 to −60 ◦C. The reaction mixture was stirred
for 5 min and compound 8 (0.5 g, 0.91 mmol, in 30 ml CH2Cl2)
was added over 5 min, and stirring was continued for addi-
tional 25 min. TEA (2 ml) was added, and the reaction mixture
was stirred for 5 min and then allowed to warm to room tem-
peratures. Water (30 ml) was added and then the aqueous layer
was re-extracted with CH2Cl2 (3 × 30 ml). The organic layer was
combined, washed with saturated NaCl solution (50 ml), and
dried over anhydrous Na2SO4. Flash chromatography afforded
compound 10 (0.4 g, 98%). Data from spectrometric analysis
of the afforded compound are summarized below. 1H NMR
(300 Hz, CDCl3): 9.66 (1H, t, J = 1.8Hz), 7.21 (1H, d, J = 8.7 Hz),
6.78 (1H, dd, J = 8.7, 2.7 Hz), 6.72 (1H, d, J = 2.7 Hz), 4.65 (1H, t,
J = 7.5 Hz), 2.85 (1H, dd, J = 16.8, 4.8 Hz), 2.69 (1H, d, J = 16.8 Hz),
2.33 (2H, t-d, J = 7.5, 1.8Hz), 2.24–2.07 (3H, m), 2.21 (3H, s), 1.99
(3H, s), 1.82–0.95 (17H, m) and 0.77 (3H, s). MS (m/z, C28H38O5,
M+ 454): 412 (base peak), 454. HRMS: 454.2719 (calculated) and
454.2725 (observed).
C
31H40O4, M+ 476): 91 (base peak), 385, 476. HRMS: 476.2927
(calculated) and 476.2934 (observed).
2.7.
Synthesis of 3,17ˇ-bis(acetoxy)-7˛-(6-
benzyloxyhexanyl)-estra-1,3,5(10)-trien-6-one
(compound 7)
Under nitrogen, acetic anhydride (8 ml, 85 mmol) was added
to a solution of compound 6 (2.5 g, 5.25 mmol) in dry pyridine
(50 ml). The reaction mixture was stirred at room temper-
atures overnight. The solvents were removed in vacuo and
the residue was purified by column chromatography (hex-
ane:ethyl acetate = 4:1) to yield compound 7 (2.85 g, 97%).
Data from spectrometric analyses of the afforded compound
are summarized below. 1H NMR (300 Hz, CDCl3): 7.73 (1H, d,
J = 2.7 Hz), 7.40 (1H, d, J = 8.7 Hz), 7.31–7.19 (6H, m), 4.71 (1H,
t, J = 8.7 Hz), 4.46 (2H, s), 4.42 (2H, t, J = 6.6 Hz), 2.72 (1H, m),
2.47–1.91 (5H, m), 2.27 (3H, s), 2.04 (3H, s) and 1.87–1.20 (16H,
m), 0.80 (3H, s). MS (m/z, C35H44O6, M+ 560): 91 (base peak), 469,
560. HRMS: 560.3138 (calculated) and 560.3149 (observed).
2.10. Synthesis of 3-methoxy-7˛-(6-benzyloxyhexanyl)-
estra-1,3,5(10)-trien-17ˇ-ol (compound 11)
Triethylsilane (30 ml) was added to a solution of compound 5
(1.46 g, 2.6 mmol) in CH2Cl2 (100 ml). The mixture was cooled
to 0 ◦C and boron trifluoride etherate (100 ml) was added drop-
wise. The yellow-green solution was warmed to room tem-
peratures and stirred overnight. The reaction was hydrolyzed
with the slow addition of 10% K2CO3 (500 ml) and then passed
through a plug of silica. The aqueous layer was extracted
with CH2Cl2, and the organic fractions were combined and
dried over Na2SO4. Following evaporation of the CH2Cl2, the
residue was purified by flash chromatography (hexane:ethyl
acetate = 5:2) to yield compound 11 (0.49 g, 40%) as the major
compound. Data from spectrometric analyses of the afforded
compound are summarized below. 1H NMR (300 Hz, CDCl3):
7.34 (5H, m), 7.14 (1H, d, J = 8.4 Hz), 6.62 (1H, dd, J = 8.4, 2.4 Hz),
6.53 (1H, d, J = 2.7 Hz), 4.51 (2H, s), 3.46 (2H, t, J = 6.6 Hz), 3.40 (3H,
s), 3.36 (1H, t, J = 8.4 Hz), 2.85 (1H, dd, J = 16.8, 5.1 Hz), 2.68 (1H,
d, J = 16.8 Hz), 2.30 (2H, m), 2.05 (2H, m), 1.71–1.08 (19H, m) and
0.81 (3H, s). MS (m/z, C32H44O3, M+ 476): 91 (base peak), 385,
476. HRMS: 476.3290 (calculated) and 476.3285 (observed).
2.8.
Synthesis of 3,17ˇ-bis(acetoxy)-7˛-(6-
hydroxyhexanyl)-estra-1,3,5(10)-triene (compound 8) and
3,17ˇ-bis(acetoxy)-7˛-(6-acetoxyhexanyl)-estra-1,3,5(10)-
triene (compound 9)
Compound 7 (2.5 g, 4.46 mmol) and 10% palladium on carbon
(400 mg) were added to acetic acid (50 ml). The reaction vessel
was purged with nitrogen, and hydrogen was added by contin-
uous bubbling for 3 h at 70 ◦C. The mixture was filtered through
celite and the solvent was removed in vacuo. Flash chromatog-
raphy (hexane:ethyl acetate = 3:1) afforded compound 8 (1.83 g,
90% yield) and compound 9 (67 mg, 3% yield). Data from spec-
trometric analyses of the afforded compounds are summa-
rized below. Compound 8—1H NMR (300 Hz, CDCl3): 7.24 (1H,
d, J = 8.4 Hz), 6.80 (1H, dd, J = 8.4, 2.4 Hz), 6.75 (1H, d, J = 2.4 Hz),
4.67 (1H, t, J = 7.5 Hz), 3.55 (2H, t, J = 6.6 Hz), 2.87 (1H, dd, J = 16.8,

338
steroids 7 1 ( 2 0 0 6 ) 334–342
ane:ethyl acetate = 4:1) to afford compound 15 (0.26 g, 93%).
Data from spectrometric analyses of the afforded compound
are summarized below. 1H NMR (300 Hz, CDCl3): 7.27–7.17 (5H,
m), 7.11 (1H, d, J = 8.4 Hz), 6.75 (1H, dd, J = 8.4, 2.4 Hz), 6.67 (1H,
d, J = 2.4 Hz), 5.07 (2H, s), 4.58(2H, s), 4.42 (2H, s), 3.56 (1H, t,
J = 8.1 Hz), 3.41 (3H, s), 3.37 (2H, t, J = 6.6 Hz), 3.29 (3H, s), 2.79
(1H, dd, J = 16.8, 4.8 Hz), 2.65 (1H, d, J = 16.8 Hz), 2.20 (2H, m),
2.24–1.12 (20H, m) and 0.73 (3H, s). MS (m/z, C35H50O5, M+ 550):
91 (base peak), 550. HRMS: 550.3658 (calculated) and 550.3651
(observed).
2.11. Synthesis of 3,17ˇ-bis(methoxymethoxy)-7˛-(6-
hydroxyhexanyl)-estra-1,3,5(10)-trien-6-ol (compound 12)
and 7˛-(6-hydroxyhexanyl)-17ˇ-estradiol (compound 13)
Compound 5 (0.73 g, 1.3 mmol) and 10% palladium on carbon
(100 mg) were added to ethanol (25 ml). The reaction vessel
was purged with nitrogen, and hydrogen was added by contin-
uous bubbling for 2 h at 60 ◦C. The mixture was filtered through
celite and the solvent was removed in vacuo. Flash chro-
matography (hexane:ethyl acetate = 3:1) afforded compound
12 (0.12 g, 20% yield) and compound 13 (0.20 g, 42%). Data from
spectrometric analyses of the afforded compounds are sum-
marized below. Compound 12—1H NMR (300 Hz, CDCl3): 7.30
(1H, d, J = 2.4 Hz), 7.16 (1H, d, J = 8.4 Hz), 6.88 (1H, dd, J = 8.4,
2.4 Hz), 5.16 (2H, s), 4.87(1H, d, J = 5.1 Hz), 4.64 (2H, s), 3.59 (3H,
m), 3.46 (3H, s), 3.36 (3H, s), 2.22–1.95 (8H, m), 1.63–1.22 (16H,
m) and 0.79 (3H, s). MS (m/z, C28H44O6, M+ 476): 444 (base peak),
458, 476. Compound 13—1H NMR (300 Hz, CDCl3): 7.15 (1H, d,
J = 8.4 Hz), 6.62 (1H, dd, J = 8.4, 2.4 Hz), 6.54 (1H, d, J = 2.7 Hz),
3.74 (1H, t, J = 8.7 Hz), 3.62 (2H, t, J = 6.6 Hz), 2.86 (1H, dd, J = 16.8,
5.1 Hz), 2.69 (1H, d, J = 16.8 Hz), 2.31–1.22 (25H, m) and 0.78 (3H,
s). MS (m/z, C24H36O3, M+ 372): 372 (base peak).
2.14. Synthesis of 3,17ˇ-bis(methoxymethoxy)-7˛-(6-
hydroxyhexanyl)-estra-1,3,5(10)-trien (compound 16)
Compound 15 (98 mg, 0.18 mmol) and 10% palladium hydrox-
ide on carbon (100 mg) were added to 20 ml of ethanol. The
reaction vessel was evacuated of air and filled to a pres-
sure of 40 psi with hydrogen, and then shaken for 2 h at
room temperatures. The mixture was filtered through celite
and the solvent was removed in vacuo. Flash chromatogra-
phy (hexane:ethyl acetate = 3:1) afforded compound 16 (78 mg,
95% yield). Data from spectrometric analyses of the afforded
compound are summarized below. 1H NMR (300 Hz, CDCl3):
7.12 (1H, d, J = 8.4 Hz), 6.75 (1H, dd, J = 8.4, 2.8 Hz), 6.67 (1H, d,
J = 2.8 Hz), 5.06 (2H, s), 4.58 2H, s), 3.55 (1H, t, J = 8.7 Hz), 3.52
(2H, t, J = 6.8 Hz), 3.40 (3H, s), 3.29 (3H, s), 2.81 (1H, dd, J = 16.8,
5.2 Hz), 2.65 (1H, d, J = 16.8 Hz), 2.24 (2H, m), 2.01–1.13 (21H, m)
and 0.73 (3H, s). MS (m/z, C28H44O5, M+ 460): 428 (base peak),
460. HRMS: 460.3189 (calculated) and 460.3184 (observed).
2.12. Synthesis of 3,17ˇ-bis(hydroxy)-7˛-(6-
benzyloxyhexanyl)-estra-1,3,5(10)-trien (compound 14)
Compound 6 (0.34 g, 0.71 mmol) was dissolved in methylene
chloride (20 ml). To this solution was added triethylsilane
(3.5 ml), followed by boron trifluoride etherate (12.5 ml) drop-
wise. The reaction was stirred for 2 days at room temperatures,
cooled to 0 ◦C, and quenched with 10% potassium carbon-
ate (20 ml). The heterogeneous solution was filtered through
a short silica plug, and the organic layer separated. The aque-
ous layer was extracted twice with methylene chloride (20 ml).
The organic fractions were combined, dried over sodium sul-
fate, and evaporated in vacuo to a yellow solid. Purification
by flash chromatography (hexane:ethyl acetate = 2:5) provided
compound 14 (0.23 g, 70%). Data from spectrometric analyses
of the afforded compound are summarized below. 1H NMR
(300 Hz, CDCl3): 7.23–7.15 (5H, m), 7.03 (1H, d, J = 8.4 Hz), 6.54
(1H, dd, J = 8.4, 2.8 Hz), 6.44 (1H, d, J = 2.8 Hz), 4.42 (2H, s), 3.66
(1H, t, J = 8.4 Hz), 3.36 (2H, t, J = 6.4 Hz), 2.75 (1H, dd, J = 16.8,
4.4 Hz), 2.58 (1H, d, J = 16.8 Hz), 2.20 (2H, m), 2.10–1.13 (22H, m)
and 0.71 (3H, s). MS (m/z, C31H42O3, M+ 462): 91 (base peak),
371, 462. HRMS: 462.3134 (calculated) and 462.3141 (observed).
2.15. Synthesis of 3,17ˇ-bis(methoxymethoxy)-7˛-(6-
benzyloxyhexanyl)-estra-1,3,5(10)-trien-6-ol
(compound 17)
Compound 5 (0.5 g, 0.89 mmol) was dissolved in methanol
(20 ml). To this solution was added sodium borohydride (0.68 g,
17.8 mmol). The reaction mixture was stirred at room tem-
peratures for 4 h. The methanol was removed in vacuo, and
the residue was then dissolved in water (20 ml). The aqueous
solution was extracted with methylene chloride (3 × 20 ml).
Organic fractions were combined, dried over sodium sulfate,
and concentrated. The crude product was purified by flash
chromatography (hexane:ethyl acetate = 3:1) to afford com-
pound 17 (0.49 g, 98%). Data from spectrometric analyses of the
afforded compound are summarized below. 1H NMR (300 Hz,
CDCl3): 7.25–7.16 (6H, m), 7.11 (1H, d, J = 8.7 Hz), 6.82 (1H, dd,
J = 8.7, 2.7 Hz), 5.08 (2H, d, J = 5.1 Hz), 4.80 (1H, d, J = 5.1 Hz), 4.58
(2H, s), 4.41 (2H, s), 3.56 (1H, t, J = 8.4 Hz), 3.39 (3H, s), 3.36 (2H, t,
J = 6.6 Hz), 3.29 (3H, s), 2.31 (2H, m), 1.99 (4H, m), 1.59–1.05 (17H,
m) and 0.73 (3H, s). MS (m/z, C35H50O6, M+ 566): 91 (base peak),
548, 566. HRMS: 566.3607 (calculated) and 566.3616 (observed).
2.13. Synthesis of 3,17ˇ-bis(methoxymethoxy)-7˛-(6-
benzyloxyhexanyl)-estra-1,3,5(10)-trien (compound 15)
MOMCl (0.19 ml, 2.5 mmol) was added dropwise to a cold (0 ◦C)
solution of compound 14 (0.23 g, 0.5 mmol) and diisopropy-
lethylamine (0.71 ml, 2.98 mmol) in THF (50 ml). Upon comple-
tion of the addition, the reaction mixture was allowed to warm
up to room temperatures, stirred for 1 h at the same temper-
atures, and then heated at reflux overnight. The mixture was
allowed to cool, and then saturated ammonium chloride solu-
tion (40 ml) was added. After extraction with ether (4 × 50 ml),
the combined organics were washed with saturated brine
(50 ml), dried over sodium sulfate, and concentrated. The
crude product was purified by flash chromatography (hex-
2.16. Synthesis of 3-hydroxy-17ˇ-methoxymethoxy-7˛-
(6-hydroxyhexanyl)-estra-1,3,5(10)-trien (compound 18)
Compound 17 (0.4 g, 0.7 mmol) and 10% palladium hydroxide
on carbon (300 mg) were added to ethanol (50 ml). The reaction
vessel was evacuated of air and filled to a pressure of 40 psi
with hydrogen, and then shaken for 3 h at 60 ◦C. The mix-
ture was filtered through celite and the solvent was removed

steroids 7 1 ( 2 0 0 6 ) 334–342
339
lets were then resuspended in 200 ␮l ethanol, and the content
was transferred to scintillation vials for measurement of the
3H radioactivity in a liquid scintillation counter (Packard Tri-
CARB 2900 TR; Downers Grove, IL, USA). The data obtained
from duplicate measurements were expressed as the percent
specific binding of [3H]E2 versus the log molar concentration
of the competing compound. The IC50 values (calculated using
the SigmaPlot software) represent the concentration of the test
compound required to reduce the [3H]E2 binding by 50%.
in vacuo. Flash chromatography (hexane:ethyl acetate = 3:1)
afforded compound 16 (0.14 g, 43% yield) and compound 18
(0.11 g, 36% yield). Data from spectrometric analyses of the
afforded compound are summarized below. (The data for com-
pound 16 are listed under Section 2.14.) For compound 18—1H
NMR (300 Hz, CDCl3): 7.06 (1H, d, J = 8.4 Hz), 6.55 (1H, dd, J = 8.4,
2.7 Hz), 6.47 (1H, d, J = 2.7 Hz), 4.59 (2H, s), 3.56 (2H, t, J = 6.6 Hz),
3.31 (3H, s), 3.27 (1H, t, J = 8.7 Hz), 2.78 (1H, dd, J = 16.5, 4.8 Hz),
2.56 (1H, d, J = 16.5 Hz), 2.23 (2H, m), 2.02–1.12 (22H, m) and 0.72
(3H, s). MS (m/z, C26H40O4, M+ 416): 386 (base peak), 416. HRMS:
416.2927 (calculated) and 416.2933 (observed).
3.
Results and discussion
2.17. Synthesis of 1-iodo-6-benzyloxyhexane
(compound 21)
With E2 (compound 1) as the starting material, E2 3,17␤-
bisMOM ether (compound 2) was prepared according to a pub-
lished method [14] by refluxing overnight with MOM chloride
and diisopropylethylamine in THF. The reasons for using MOM
chloride to protect the two free hydroxyls of E2 are that the
formation of E2 3,17␤-bisMOM ether gives a very high yield
(almost quantitative) and that this reaction avoids the gen-
eration of diastereomer mixtures which would hamper facile
assignment of the side chain stereochemistry by NMR.
Using 1,6-hexanediol as starting material, its monobenzyla-
tion product (compound 20) was first obtained by using a
known procedure [25] with a yield of 86%. To the dry CH2Cl2
(40 ml) the following reactants were added in a sequential
order: triphenylphosphine (3.14 g, 12 mmol), imidazole (0.82 g,
12 mmol) and iodine (3.05 g, 12 mmol). The mixture was stirred
at room temperatures for 15 min. A solution of the compound
20 (2.1 g, 10 mmol) in dry dichloromethane (10 ml) was added
and the mixture was stirred at room temperatures under N2
for 5 h. The disappearance of compound 20 and the formation
of an alkyl iodide were monitored using TLC. When the reac-
tion was complete, the solvents were removed in vacuo and
the product was purified by passing though a silica gel column
with hexane as the mobile phase and the fractions contain-
ing the desired product were combined (3.1 g, 98% yield). Data
from spectrometric analyses of the afforded compound are
summarized below. 1H NMR (300 Hz, CDCl3): 7.36–7.24 (5H, m),
4.50 (2H, s), 3.62 (2H, t, J = 6.6 Hz), 3.47 (2H, t, J = 6.6 Hz), 1.65–1.54
(4H, m) and 1.42–1.36 (4H, m). MS (m/z, C13H19IO, M+ 318): 91
(base peak), 227, 318. HRMS: 318.0481 (calculated) and 318.0488
(observed).
There are two commonly used strategies to introduce a C-
6 ketone group to compound 2, one is the oxidation of the
6-benzyl position of the O-protected E2 or estrone derivatives
using chromium(VI) compounds [16,17], and the other is the
oxidation of the C-6 organometallic derivatives of E2 obtained
through hydrogen–metal exchange [18,19]. For the first strat-
egy, we tried with two methods, one that used CrO3 and 3,5-
dimethylpyrazole [20,21] and the other that used PCC [22], to
oxidize E2 bisMOM ether (compound 2) to form a C-6 ketone
derivative (compound 4). However, these two methods only
produced a rather low yield (20–25%), and there was a side
product simultaneously formed which was difficult to sepa-
rate from our desired product (compound 4). After a number
of trials with a few other approaches which also gave rela-
tively low yields, the second strategy was then employed [19].
The bisMOM ether of E2 (compound 2) was first deprotonated
under the “superbase” conditions employing a 1:1 ratio of t-
BuOK and LDA. The anion was trapped with trimethyl borate
to yield an intermediate borate ester that was subsequently
oxidized with hydrogen peroxide to an epimeric mixture of
alcohols (compound 3). When we tried to obtain the C-6 ketone
product (compound 4) through oxidation of compound 3 (an
epimeric mixture of alcohols) employing the reaction condi-
tions described in the literature [8,9,18,19], such as PCC [9] or
sodium hypochlorite in the presence of tetramethylpyrrolidin-
1-oxyl (TEMPO) as catalyst [19], we noted that the overall
yield was indeed substantially improved (65–75%). However,
we encountered a similar situation which occurred in the first
strategy, namely, the simultaneous formation of a side prod-
uct (in considerable amounts) which was difficult to separate
from the product of interest (compound 4). This side product
was not further identified. Finally, we found that compound
4 could be obtained quite readily and almost quantitatively
from compound 3 by Swern oxidation [23]. This modified two-
step synthesis procedure employed in this study afforded the
C-6 ketone product with a very high overall yield (∼91%).
Here it should be noted that a number of factors were con-
sidered when we selected the “synthon” as the side chain for
2.18. ER˛ and ERˇ binding assays
The ER␣ and ER␤ competitive binding assays were performed
according to the method described in our recent study [15].
The ER binding buffer used for dilution of the receptor prepa-
rations consisted of 10% glycerol, 2 mM dithiothreitol, 1 mg/ml
BSA and 10 mM Tris–HCl (pH 7.5). The ER␣ washing buffer con-
tained 40 mM Tris–HCl and 100 mM KCl (pH 7.4), but the ER␤
washing buffer contained only 40 mM Tris–HCl (pH 7.5). The
50% hydroxylapatite slurry was adjusted to a final concentra-
tion of 50% (v/v) by using the 50 mM Tris–HCl solution (pH 7.4).
The reaction mixture contained 50 ␮l of varying concentra-
tions of the test compound in the ER binding buffer, and 45 ␮l
of [3H]E2 solution (at 22.22 nM). Then 5 ␮l of ER␣ or ER␤ pro-
tein was added and mixed gently. Nonspecific binding by the
[3H]E2 was determined by addition of a 400-fold concentration
of the nonradioactive E2. The binding mixture was incubated
at room temperatures for 2 h. At the end of the incubation,
100 ␮l of the HAP slurry was added and the tubes were incu-
bated on ice for 15 min with three times of brief vortexing.
An aliquot (1 ml) of the washing buffer was added, mixed and
centrifuged at 10,000 × g for 1 min, and the supernatants were
discarded. This wash step was repeated twice. The HAP pel-

340
steroids 7 1 ( 2 0 0 6 ) 334–342
6 M HCl in THF, which afforded compound 6 almost quanti-
tatively. From compound 6, there are two main approaches
that are of interest to us: One is to remove the 6-keto group
while keeping the benzyl group at the end of the side chain
unchanged, because this method would readily help distin-
guish the 3- and 17␤-hydroxyls from the hydroxyl of the side
chain. The other is to protect the 3- and 17␤-hydroxyls with
protecting groups that are sufficiently stable under the subse-
quent reduction conditions that were designed to remove the
6-keto group as well as the benzyl group on the side chain. For
the first approach, we employed the boron trifluoride ether-
ate and triethylsilane reduction system to remove the 6-keto
group from compound 6. We found that the benzyl group
is stable enough under the conditions we devised. Notably,
there was no published report on whether the benzyl group
would be stable or not under our reaction conditions. Thus,
compound 14 could be readily prepared from compound 6 in
70% yield under the boron trifluoride etherate and triethylsi-
lane reduction conditions. Reprotection of compound 14 with
MOM gave compound 15. Then compound 16 was prepared by
hydrogenolysis of compound 15 under the conditions of Pd–C
and H2 in EtOH. For the second approach, an excellent two-
step reaction route was found: (i) re-protection of the two free
hydroxyls of compound 6 by reaction with acetic anhydride in
dry pyridine to yield the diacetate (compound 7) almost quan-
titatively and (ii) hydrogenolysis of compound 7 under the
reaction conditions of the Pd–C and H2 in AcOH at 70 ◦C for 3 h
to yield compound 8 in 90% yield [28]. It should be noted that
we could almost selectively obtain compound 8 or 9 depending
on the reaction conditions we would use. While a shorter reac-
tion time (3 h) gave mostly compound 8, compound 9 became
the major product when the reaction time was prolonged to 2
days. Compound 8 was easily oxidized to compound 10, which
has a different functional group at the end of the side chain.
Notably, this compound has a very similar structure as com-
pounds that were used earlier for the synthesis of several pure
ER antagonists [29].
Even though we found several excellent reaction condi-
tions to remove the 6-keto group from compound 6, it is still
of considerable interest to have a useful method to remove
the 6-keto group in compound 5 while keeping the 3- and
17␤-protecting groups unchanged. Partly based our experi-
ence gained from the experiments described above, com-
pound 5 was first reduced to compound 17 by sodium borohy-
dride quantitatively, and then hydrogenolysis of compound 17
under the reaction conditions of the Pd(OH)2/C and H2 at 45 psi
in EtOH at 60 ◦C for 3 h yielded two major products, compound
16 (in 43% yield) and compound 18 (in 36% yield, proposed
structure shown in Scheme 1 based on data from NMR, MS and
HRMS). The amount of compound 18 increased as the reaction
was prolonged.
In the present study, we also determined the relative bind-
ing affinities of compounds 13 and 14 for recombinant human
ER␣ and ER␤ proteins, and compared their binding affinities
with those of ICI-182,780 and E2 (data summarized in Fig. 1).
The IC50 values of compound 13 for ER␣ and ER␤ were 41.8
and 22.3 nM, respectively, and the IC50 values of compound 14
were 39.8 and 63.1 nM, respectively. The IC50 values of com-
pounds 13 and 14 are very similar to those of ICI-182,780.
The relative binding affinities of compound 13, 14 or ICI-
attachment to the C-7␣ position of the E2 core structure: (i)
the synthon should be a chain with an appropriate length and
with any polar group(s) away from the C-7 carbon of the E2
core structure after attachment, otherwise the ligand would
not retain high ER binding affinity [24]. (ii) It should have a
functional group at the end of the synthon that can be read-
ily attached to (or transformed to) other functional groups for
extension. Otherwise, after attachment of the side chain to
the C-7␣ position, multi-step modifications of the functional
group will be needed which will lose considerable amounts of
the precious precursor (e.g., compound 5). (iii) The side chain
synthon should be easily synthesized with a high overall yield.
There appeared to be no known procedures that met all of the
aforementioned requirements.
With these considerations in mind, we had designed and
tried a new approach. We used 1-iodo-6-benzyloxyhexane as
a precursor for the side chain. Starting with 1,6-hexanediol,
the monobenzylation product was obtained using a known
procedure [25] which gave a yield of 86%. Then the 1-iodo-
6-benzyloxyhexane was obtained almost quantitatively from
the monobenzylation product under a reaction condition con-
sisting of triphenylphosphine, imidazole and iodine in dry
dichloromethane. The incipient hydroxyl group at the end of
the side chain can be easily transformed into other functional
groups that can be used for various purposes.
Deprotonation of the C-7 position of compound 4 with t-
BuOK generated the corresponding enolate, which was alky-
lated with 1-iodo-6-benzyloxyhexane (the reaction conditions
were similar to those used in the literature) [8,9,26]. This reac-
tion yielded the C-7␣ side chain compound (compound 5) as
a major product in ∼65% yield. The 7␣-configuration of com-
pound 5 was confirmed based on the two-dimensional NMR
analyses (the nuclear Overhauser effect and COSY).
To remove the C-6 ketone group from compound 5, sev-
eral different methods have been explored and compared. One
of the ideal products is compound 16 that has two MOM-
protecting groups on both 3 and 17␤ positions and a free
hydroxyl group at the end of the side chain for extension
of the side chain in future. A well-known reaction condi-
tion consisting of boron trifluoride etherate and triethylsilane
was first employed [9,26]. However, no detectable amount of
compound 16 (the desired product) was formed, but instead
an unexpected product (compound 11, proposed structure
shown in Scheme 1 based on data from NMR, MS and HRMS)
was separated as a major compound. The mechanism for the
formation of this compound is not certain. Our subsequent
attempts to remove the 6-keto group in compound 5 while
keeping the 3- and 17␤-protecting groups unchanged by sev-
eral other methods, such as using a condition consisting of
sodium cyanoborohydride, boron trifluoride etherate, THF and
reflux [27], a condition consisting of Pd–C, H2 and ethanol at
60 ◦C for 2 h [8], or Wolff–Kishner reduction condition [30,31],
were also failed. Under the second experimental conditions,
compounds 12 and 13 were separated as two major products
(see Scheme 1).
The results from the above experiments suggested that
the two protecting MOM groups in compound 5 might readily
interact with the reducing agents or might be unstable under
the employed reduction conditions. We then tried to remove
the 3 and 17␤ MOM groups off compound 5 by treatment with

steroids 7 1 ( 2 0 0 6 ) 334–342
341
Fig. 1 – Comparison of the relative ER binding affinity of compounds 13 and 14 with those of E2 and ICI-182,780. The relative
binding affinity of each chemical was determined by measuring its inhibition of the binding of 10 nM [3H]E2 to the
recombinant human ER␣ and ER␤. Eight concentrations (0.06, 0.24, 0.98, 3.9, 15.6, 62.5, 250 and 1000 nM) of each competing
estrogen were tested. The IC50 values for each competing estrogen was calculated according to the sigmoid inhibition
curves and the relative binding affinity (RBA) for each test compound was calculated against E2 by using the following
equation: RBA = IC50 for E2/IC50 for the test compound. Each point was the mean of duplicate measurements, with average
variations <5%. (᭹) ER␣, (ꢀ) ER␤.
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of E2.
In summary, we have explored and compared in the
present study various synthetic methods for removal of the
6-keto group from C-6 position of 7␣-substituted derivatives
of E2. We described here the improved methods for the chem-
ical synthesis of several C-7␣-substituted derivatives of E2 by
modifying the reaction steps and also by improving the over-
all yield for the desired products. For instance, using E2 as
the starting material and 1-iodo-6-benzyloxyhexane as a pre-
cursor for the C-7␣ side chain, we developed a seven-step
synthetic procedure that produced 3,17␤-bis(acetoxy)-7␣-(6-
hydroxyhexanyl)-estra-1,3,5(10)-triene (compound 8) with an
overall yield of ∼45%. The two C-7␣-substituted E2 deriva-
tives (compounds 13 and 14) retained strong binding affinities
(close to those of E2 and ICI-182,780) for the human ER␣ and
ER␤ subtypes as determined using the radioligand–receptor
binding assays.
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