Synthesis of 3′-methoxy-E-diethylstilbestrol and its analogs as tumor angiogenesis inhibitors

Article abstract of DOI:10.1016/j.steroids.2011.12.024

3′-Methoxy-E-diethylstilbestrol (2), with the structural and original similarities to 2-methoxyestradiol (2-ME2, 1), was synthesized and screened against HUVEC and a series of human cancer cell lines including RL95-2, SKOV-3, MCF-7 and T-47D in vitro. The

Full text of DOI:10.1016/j.steroids.2011.12.024

Steroids 77 (2012) 419–423
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis of 30-methoxy-E-diethylstilbestrol and its analogs as tumor
angiogenesis inhibitors
Qian Liu a, Weiwei Jin a, Yan Zhu b, Jieyun Zhou b, Min Lu a, Qian Zhang a,
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b Department of Reproductive Pharmacology, NPFPC Key Laboratory of Contraceptives and Devices, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China
a r t i c l e i n f o
a b s t r a c t
30-Methoxy-E-diethylstilbestrol (2), with the structural and original similarities to 2-methoxyestradiol
(2-ME2, 1), was synthesized and screened against HUVEC and a series of human cancer cell lines includ-
ing RL95-2, SKOV-3, MCF-7 and T-47D in vitro. The configuration of the title compound was determined
via the single crystal X-ray diffraction of its benzoyl-ester derivative (10). The fact that 30-methoxy-E-
diethylstilbestrol and its analogues (8 and 11) showed potential antiangiogenesis and anti-tumor activ-
ities at a close level, whereas its ester derivative (10) did not display any cytotoxic activities on all the
screening cell lines indicated that the core scaffold of 30-methoxy-3,4-diphenylhexane and the exposed
hydroxyl-groups in the structures are essential pharmacophores for their anti-tumor activities.
Ó 2011 Elsevier Inc. All rights reserved.
Article history:
Received 28 August 2011
Received in revised form 21 December 2011
Accepted 22 December 2011
Available online 5 January 2012
Keywords:
2-Methoxyestradiol
30-Methoxy-E-diethylstilbestrol
Metabolite
Non-steroidal analog
Angiogenesis inhibitor
1. Introduction
diethylstilbestrol was shown to cause fetal abnormalities during
pregnancy; its metabolites, including 30-methoxy-E-diethylstilbes-
The therapeutic use of 2-methoxyestradiol (2-ME2, 1, Fig. 1), an
endogenous metabolite of 17b-estradiol, against solid tumors has
progressed into Phase II/III clinical trials. This compound has at-
tracted considerable interest owing to its anti-proliferative [1]
and anti-angiogenic activities [2,3] by a mechanism independent
of the estrogen receptor. The transformation of 2-ME2 in humans
involves the 2-hydroxylation of 17b-estradiol by hepatic cyto-
chrome P450 and sequential 2-O-methylation by catechol O-meth-
yltransferase [4–6]. Research on its mechanism of action has
revealed that 2-ME2, as a multitargeted anti-tumor agent, can
disrupt tubulin polymerization [7], downregulate hypoxia induc-
trol, were also presumed to possess fetotoxicity. However, analysis
of the preparation, chemical behavior and bioactivity of 30-meth-
oxy-E-diethylstilbestrol remains limited.
Here, we report the synthesis of 30-methoxy-E-diethylstilbestrol
and its anti-angiogenic and anti-tumor activities, along with its
two
analogs
3-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxy-
phenyl) hex-2-ene (8) and 3-(4-hydroxy-3-methoxyphenyl)-4-(4-
hydroxyphenyl)hexane (30-methoxy-E-diethyldihydrostilbestrol,
11). Compared to that of diethylstilbestrol [14], the synthesis of
30-methoxy-E-diethylstilbestrol was substantially more difficult
due to the elimination of hydroxyl with b-H in 6, the orientation
of the double bond and the separation of these isomers.
ible factor-1a (HIF-1a) [8] and inhibit superoxide dismutases
(SOD) [9], among other functions [10].
Interestingly, 30-methoxy-E-diethylstilbestrol (Fig. 1), with a
similar structure to 2-ME2 [11], was also reported to be an oxida-
tive metabolite of diethylstilbestrol by Gottschlich Regina and co-
workers in 1980. The compound was isolated from the fetus and
placenta of 15-day pregnant Syrian golden hamsters after adminis-
tration of diethylstilbestrol at a dose of 20 mg/kg and was identified
only by mass fragmentography [12,13]. Diethylstilbestrol, a syn-
thetic non-steroidal estrogen, was used clinically as a replacement
for estradiol beginning in the 1940s. It was not until the 1970s that
2. Experimental
2.1. Chemistry
Melting points were measured on a SGW X-4 melting point
apparatus and were uncorrected. Nuclear magnetic resonance (1H
NMR) spectra were recorded on a Bruker-DPX (400 MHz) spec-
trometer, and values are expressed in ppm. ESI-MS spectra were
recorded on an Agilent G1946D mass spectrometer, and EI-MS data
were obtained using an HP5989A mass spectrometer. High-resolu-
tion mass spectra were determined using a Finnigan LTQ-ORBI-
TRAP mass spectrometer with ESI as the ionization source. All
chemicals and solvents were purchased from commercial sources
⇑
Corresponding author. Tel.: +86 21 51980119.
E-mail address: zhangqian511@shmu.edu.cn (Q. Zhang).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.12.024

420
Q. Liu et al. / Steroids 77 (2012) 419–423
5.19 (2H, s, –OCH2–), 5.01 (2H, s, –OCH2–), 4.42 (1H, t,
OH
OH
J = 7.43 Hz, 2-H), 3.89 (3H, s, –OCH3), 2.19–1.75 (2H, m, -CH2CH3),
0.88 (3H, t, J = 7.44 Hz, -CH3); EI-MS m/z (%): 466 (M+, 1.73),
241(59.46), 225(9.83), 91(100).
H3CO
HO
H3CO
HO
2.1.4. 3-(4-Benzyloxy-3-methoxyphenyl)-4-(4-benzyloxyphenyl)
hexan-3-ol (6)
2
2-Methoxyestradiol, 1
To a solution of 5 (560 mg, 1.20 mmol) in dried THF (40 mL) was
added ethylmagnesium bromide (0.6 M in THF, 20 mL) at room
temperature. Following stirring under a nitrogen atmosphere for
3 h at 35 °C, the mixture was poured into 50 mL of ice water. The
aqueous layer was neutralized using 5 N HCl and was extracted
with ethyl acetate (3 ꢀ 30 mL). The combined layer was washed
with saturated brine (50 mL), dried with anhydrous Na2SO4, and
the solvent was removed under vacuum to yield a crude product
(650 mg) that was further purified by column chromatography
(petroleum ether/ethyl acetate, 8:1) and crystallized from ethyl
acetate to yield 6 (600 mg, 82.9%). m.p.: 90–91 °C; 1H NMR (CDCl3,
400 MHz) d: 7.46–7.28 (10H, m, benzyl-H), 6.85 (2H, d, J = 8.22 Hz,
200, 600-H), 6.79 (2H, d, J = 8.22 Hz, 300, 500-H), 6.80–6.76 (1H, d,
J = 8.22 Hz, 50-H), 6.71 (1H, d, J = 1.96 Hz, 20-H), 6.67–6.64 (1H, dd,
J1 = 1.96 Hz, J2 = 8.22 Hz, 60-H), 5.12 (2H, s, –OCH2–), 5.01 (2H, s, –
OCH2–), 3.78 (3H, s, –OCH3), 2.75–2.69 (1H, m, 4-H), 2.18 (1H, s,
3-OH), 1.96–1.77 (2H, m, 5-H), 0.83–0.86 (2H, m, 2-H), 0.74 (3H,
t, J = 7.44 Hz, 1-CH3), 0.64 (3H, t, J = 7.44 Hz, 6-CH3); ESI-MS m/z
(%): 519 (M+23).
Fig. 1. Structures of 2-ME2 and the title compound.
and were used without further purification unless otherwise noted.
Column chromatography (CC) was carried out on silica gel (300–
400 mesh, Qingdao Ocean Chemical Company, China), and thin-
layer chromatography (TLC) analysis was carried out on silica gel
GF254 glass plates (2.5 cm ꢀ 10 cm with a 250
lm silica layer,
Qingdao Ocean Chemical Company, China).
2.1.1. 1-(4-Hydroxy-3-methoxyphenyl)-2-(4-hydroxyphenyl)etha-
none (3)
A mixture of 2-methoxyphenol (8.1 g, 65 mmol) and 2-(4-
hydroxyphenyl)acetic acid (9.93 g, 65 mmol) was dissolved in
30 mL of BF3–Et2O and was stirred continuously at 65 °C for 19 h.
After cooling, the reaction was poured into 300 mL of ice water
and was extracted with ethyl acetate (3 ꢀ 50 mL). The combined
organic layer was washed with saturated brine (50 mL) and dried
with anhydrous Na2SO4. Following removal of the solvent under
vacuum, the crude solid was recrystallized from ethyl acetate,
yielding a white powder as 3 (4.7 g, 27.8%). Melting point [m.p.]:
187–188 °C; 1H NMR (CDCl3, 400 MHz) d: 7.61 (1H, dd,
J1 = 1.96 Hz, J2 = 8.22 Hz, 60-H), 7.55 (1H, d, J = 1.96 Hz, 20-H), 7.13
(2H, d, J = 8.60 Hz, 200, 600-H), 6.93 (1H, d, J = 8.22 Hz, 50-H), 6.78
(2H, d, J = 8.60 Hz, 300, 500-H), 6.04 (1H, s, –OH), 4.52 (1H, s, –OH),
4.17 (2H, s, 2-H), 3.92 (3H, s, –OCH3); EI-MS m/z (%): 258 (M+,
4.61), 123(19.76), 151(100).
2.1.5. 3-(4-Benzyloxy-3-methoxyphenyl)-4-(4-benzyloxyphenyl)hex-
2-ene (7)
H2SO4 (0.67 mL) was added dropwise to a solution of 6 (600 mg,
1.21 mmol) in 15 mL of acetone at room temperature and was stir-
red for 30 min. Following the addition of 20 mL of water, the aque-
ous layer was extracted with ethyl acetate (3 ꢀ 20 mL) and the
combined organic fractions were washed with brine (50 mL) and
dried with anhydrous Na2SO4. A crude product (500 mg) was ob-
tained after removing the solvent under vacuum and was further
purified using column chromatography (petroleum ether/ethyl
acetate, 50:1) and crystallization from ethyl acetate to yield 7
(440 mg, 76%). m.p.: 87–89 °C; 1H NMR (CDCl3, 400 MHz) d:
7.45–7.25 (10H, m, benzyl-H), 7.10–6.99 (2H, d, J = 8.61 Hz, 200,
600-H), 6.87–6.85 (2H, d, J = 8.61 Hz, 300, 500-H), 6.78 (1H, d,
J = 8.22 Hz, 50-H), 6.35 (1H, dd, J1 = 1.96 Hz, J2 = 8.22 Hz, 60-H),
6.19 (1H, d, J = 1.96 Hz, 20-H), 5.60 (1H, q, J = 6.26 Hz, 2-H), 5.11
(2H, s, –OCH2–), 5.03 (2H, s, –OCH2–), 3.63 (3H, s, –OCH3), 3.29
(1H, t, J = 7.43 Hz, 4-H), 2.18–1.74 (2H, m ꢀ 2, 5-H), 1.51 (3H, d,
J = 6.26 Hz, 1-CH3), 0.88 (3H, t, J = 7.43 Hz, 6-CH3); ESI-MS m/z
(%): 501 (M+23).
2.1.2. 1-(4-Benzyloxy-3-methoxyphenyl)-2-(4-benzyloxyphenyl)etha-
none (4)
Benzyl bromide (1.5 g, 8.7 mmol) was added to a mixture of 3
(0.9 g, 3.5 mmol) and K2CO3 (1.0 g, 7.0 mmol) in acetone (20 mL).
Following a 10 h reflux, the reaction was cooled and filtered to
eliminate K2CO3, and the filtrate was concentrated under vacuum
to yield a crude product of 1.53 g. A white solid was obtained via
recrystallization from acetone as 4 (1.4 g, 88.2%). m.p.: 121–
123 °C; 1H NMR (CDCl3, 400 MHz) d: 7.59–7.56 (1H, dd,
J1 = 1.96 Hz, J2 = 7.43 Hz, 60-H), 7.56 (1H, d, J = 1.96 Hz, 20-H),
7.44–7.30 (10H, m, benzyl-H), 7.17 (2H, d, J = 8.61 Hz, 200, 600-H),
6.93 (2H, d, J = 8.61 Hz, 300, 500-H), 6.88 (1H, dd, J = 7.43 Hz, 50-H),
5.21 (2H, s, –OCH2–), 5.02 (2H, s, –OCH2–), 4.15 (2H, s, 2-H), 3.92
(3H, s, –OCH3); EI-MS m/z (%): 438 (M+, 2.58), 241(57.58), 91(100).
2.1.6. 3-(4-Hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)hex-2-
ene (8) and 3-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)
hexane (30-methoxy diethyldihydrostilbestrol, 11)
2.1.3. 1-(4-Benzyloxy-3-methoxyphenyl)-2-(4-benzyloxyphenyl)-
butan-1-one (5)
To a solution of 7 (440 mg, 0.92 mmol) in CH2Cl2 (5 mL) and
MeOH (12 mL), Pd/C (5%, 0.3 g) and ammonium formate (464 mg,
7.38 mmol) were added in turn. The reaction was stirred at room
temperature and was monitored by TLC until the substrate was no
longer visible. The mixture was filtered to recover Pd/C, and the fil-
trate was poured into water (50 mL) and extracted with ethyl ace-
tate (3 ꢀ 20 mL). The combined organic fractions were washed
with saturated brine (50 mL), and dried with anhydrous Na2SO4.
Following removal of the solvent by concentration under vacuum,
a mixture of 8 and 11 (253.2 mg) was obtained that was separated
via column chromatography (petroleum ether/ethyl acetate, 50:1)
and was purified by crystallization from ethyl acetate to yield 8
and 11, respectively. For 8 (111.1 mg, 40.5%), m.p.: 102–104 °C; 1H
NMR (CDCl3, 400 MHz) d: 6.97–6.95 (2H, d, J = 8.60 Hz, 200, 600-H),
6.79–6.77 (1H, d, J = 7.83 Hz, 50-H), 6.72–6.70 (2H, d, J = 8.60 Hz,
A solution of NaH (0.15 g, 6.2 mmol) and 4 (1.4 g, 3.1 mmol) in
toluene (40 mL) was refluxed for 30 min and was cooled to 25 °C,
at which time bromoethane (1.3 g, 12.3 mmol) was added drop-
wise. This mixture was refluxed for 16 h, quenched with ice water,
and extracted with ethyl acetate (3 ꢀ 20 mL). The combined organ-
ic fractions were washed with saturated brine (50 mL), dried with
anhydrous Na2SO4, and filtered and concentrated under vacuum to
yield a crude product (1.3 g) that was further purified by column
chromatography (petroleum ether/ethyl acetate, 10:1) and crystal-
lized from ethyl acetate to yield 5 (1.0 g, 71.0%). m.p.: 106 °C; 1H
NMR (CDCl3, 400 MHz) d: 7.57–7.52 (2H, m, 20, 60-H), 7.42–7.28
(10H, m, benzyl-H), 7.23–7.18 (2H, d, J = 8.61 Hz, 200, 600-H), 6.98–
6.88 (2H, d, J = 8.61 Hz, 300, 500-H), 6.82 (1H, d, J = 8.21 Hz, 50-H),

Q. Liu et al. / Steroids 77 (2012) 419–423
421
300, 500-H), 6.38–6.36 (1H, dd, J1 = 1.56 Hz, J2 = 7.83 Hz, 60-H), 6.13
(1H, d, J = 1.56 Hz, 20-H), 5.60 (1H, q, J = 6.66 Hz, 2-H), 5.46 (1H, s,
–OH); 4.63 (1H, s, –OH), 3.69 (3H, s, –OCH3), 3.30–3.26 (1H, t,
J = 7.44 Hz, 4-H), 1.78–1.64 (2H, m, 5-H), 1.50 (3H, d, J = 6.66 Hz,
1-CH3), 0.89–0.85 (3H, t, J = 7.04 Hz, 6-CH3); ESI-MS m/z (%): 297
(M-1); HR-MS (ESI+), calculated for C19H23O3 [M+H]+: 299.16472,
was found to be 299.16439.
(M-1); HR-MS (ESI+), calculated for C19H23O3 [M+H]+: 299.16472,
was found to be 299.16440.
2.2. X-ray crystal structural determination of 10
Single crystals of 10 were cultured in ethyl acetate, and a color-
less crystal with dimensions of 0.17 mm ꢀ 0.15 mm ꢀ 0.10 mm
was selected and sealed in a capillary. The capillary was mounted
For 11 (130.0 mg, 47.1%), m.p.: 148–150 °C; 1H NMR (CDCl3,
400 MHz) d: 7.03–7.01 (2H, d, J = 8.56 Hz, 200, 600-H), 6.87–6.85
(1H, d, J = 7.95 Hz, 50-H), 6.80–6.77 (2H, d, J = 8.56 Hz, 300, 500-H),
6.68–6.65 (1H, dd, J1 = 1.84 Hz, J2 = 7.95 Hz, 60-H), 6.60 (1H, d,
J = 1.84 Hz, 20-H), 5.48 (1H, s, –OH), 4.65 (1H, s, –OH), 3.88 (3H, s,
–OCH3), 2.46–2.43 (2H, m, 3, 4-H), 1.43–1.23 (4H, m, 2, 5-H),
0.57–0.52 (6H, t, J = 7.33 Hz, 1, 6-CH3); EI-MS m/z (%): 301 (M+,
1.29), 165(100); HR FTMS (ESI+), calculated for C19H25O3 [M+H]+:
301.18037, was found to be 301.17996.
on
a
Bruker SMART APEX CCD area detector diffractometer
radiation
equipped with graphite-monochromatic MoK
a
(k = 0.71073 Å) for the preliminary examination and data collection
at 293(2) K. Out of a total of 14376 reflections collected in the range
of 1.73° 6 h 6 26.01°, 5406 were independent, with Rint = 0.0570.
The structure was solved directly using the SHE-LXL-97 program
and was refined by a full-matrix least-squares procedure with
anisotropic thermal parameters for all non-hydrogen atoms, and
the hydrogen atoms were located geometrically. Structural analysis
demonstrated that 10, C33H30O5 (Mr = 506.57), is of the monoclinic
space group P2(1)/n, with a = 9.722(4) Å, b = 14.962(5) Å,
2.1.7. 3-(4-Hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)hex-3-
ene (9)
To a solution of 8 (240 mg, 0.80 mmol) in 6 mL of acetic acid
was added 48% HBr (13.2 mL). The reaction was stirred at room
temperature for 48 h to reach equilibrium and was poured into
100 mL water and extracted with ethyl acetate (3 ꢀ 20 mL). The
combined organic layer was washed with brine (50 mL) and dried
with anhydrous Na2SO4. The solvent was removed under vacuum
to yield a crude product (235 mg), which was purified via column
chromatography (petroleum ether/ethyl acetate, 7:1) and crystalli-
zation from ethyl acetate to yield 9 (120 mg, 50.0%, 8 was recov-
ered at 120 mg). m.p.: 87–89 °C; 1H NMR (CDCl3, 400 MHz) d:
7.07 (2H, d, J = 8.22 Hz, 200, 600-H), 6.91 (1H, d, J = 8.59 Hz, 50-H),
6.83 (2H, d, J = 8.22 Hz, 300, 500-H), 6.69 (1H, s, 20-H), 6.56 (1H, d,
J = 8.59 Hz, 60-H), 5.55 (1H, s, –OH), 4.76 (1H, s, –OH), 3.91 (3H, s,
–OCH3), 2.13 (4H, q, J = 6.65 Hz, 2, 5-H), 0.78 (6H, t, J = 6.65 Hz, 1,
6-CH3); ESI-MS m/z (%): 297 (Mꢁ1).
c = 19.088(7) Å,
Z = 4, Dc = 1.219 mg/m3,
R = 0.0470 and Rw = 0.1091 (w = 1/[
where P = (Fo2 + 2Fc2)/3), (
max = 0.196, (
max = 0.000 and S = 0.899.
a
= 90°, b = 96.195(5)°,
c
= 90°, V = 2760.3(17) Å3,
l
= 0.081 mmꢁ1, F(000) = 1072, the final
r
2(Fo2) + (0.0614P)2 + 0.0000P]
D
/q
)
D
/q)
min = ꢁ0.172 e/Å3,
(D/r)
2.3. Biological assays
HUVEC (human umbilical vein endothelial cells), RL95-2 (hu-
man uterine epithelial carcinoma cells), SKOV-3 (human ovary car-
cinoma cells), MCF-7 (human breast adenocarcinoma cells) and
T-47D (human breast adenocarcinoma cells) cell lines were cul-
tured in media appropriate for each cell type, supplemented with
10% fetal bovine serum, in a humidified atmosphere of 5% CO2 at
37 °C. Cell suspensions were plated in 96-well plates at a density
of 8 ꢀ 104 cells mLꢁ1. One microliter of each test compound solu-
tion at various concentrations in DMSO was added to each well,
and the culture was incubated for 48 h at 37 °C in a 5% CO2 incuba-
tor. The incubation medium was maintained at a near-physiologi-
cal pH of 7.3–7.4. Plates were incubated 1–4 h at 37 °C in the
2.1.8. 30-Methoxy-E-diethylstilbestrol dibenzoate (10)
Benzoyl chloride (103.6 mg, 0.74 mmol) was added to a mixture
of 9 (100 mg, 0.335 mmol) and K2CO3 (74 mg, 0.536 mmol) in
CH2Cl2 (10 mL) at room temperature. The reaction mixture was
refluxed for 3 h, filtered, and concentrated under vacuum. The
E-configuration isomer was separated by column chromatography
(petroleum ether/ethyl acetate, 40:1) and recrystallized from ethyl
acetate (114 mg, 67.0%). m.p.: 164–165 °C; 1H NMR (CDCl3,
400 MHz) d: 8.22–8.25 and 7.51–7.55 (4H ꢀ 2, m ꢀ 2, benzoyl-2,
3, 5, 6-H), 7.65 (2H, dd, J1 = 8.4 Hz, J2 = 12.0 Hz, benzoyl-4-H),
7.25–7.29 (2H, d, J = 8.4 Hz, 200, 600-H), 7.23–7.25 (2H, d, J = 8.4 Hz,
300, 500-H), 7.16 (1H, dd, J = 7.6 Hz, 50-H), 6.84 (2H, dd, J1 = 1.6 Hz,
J2 = 8.4 Hz, 2, 6-H, 3-ArH), 3.85 (3H, s, –OCH3), 2.20 (4H, m, 2, 5-
CH2), 0.83 (6H, m, 1, 6-CH3); ESI-MS m/z (%): 507 (M + 1); HR-MS
(ESI+), calculated for C33H31O5 [M+H]+: 507.21715, was found to
be 507.21642.
presence of 10 lL of cell counting kit-8 (cck-8) solution per well.
Absorbance was measured at 450 nm. Experiments were per-
formed in triplicate, and the results are presented as the half-max-
imal inhibitory concentration (IC50, shown in Table 2).
3. Results and discussion
3.1. Chemistry
As described in Scheme 1, 2-methoxyphenol was condensed
with 2-(4-hydroxyphenyl)acetic acid in the presence of BF3–Et2O
to provide a skeleton of 1,2-diphenylethanone (3). The phenyl-
hydroxyls of 3 were protected with benzyl groups, and two ethyl
chains were introduced sequentially via substitution of the car-
bonyl a-H with bromoethane and, by the Grignard reaction, of eth-
ylmagnesium bromide with the carbonyl group, with yields of
71.0% and 82.9%, respectively, to produce 6.
Unexpectedly, the dehydration of 6 with H2SO4 in acetone
yielded only the olefinic product with the double bond located at
the 2-position instead of yielding the predicted product following
Sayzteff’s rule, that is 3-(4-benzyloxy-3-methoxyphenyl)-4-(4-
benzyloxyphenyl)hex-3-ene. Despite altering the dehydration re-
agents, solvents, and the reaction temperature, the non-Sayzteff
product remained the predominant product of this elimination
[15]. We attribute this result to steric effects and to the charged
2.1.9. 30-Methoxy-E-diethylstilbestrol (2)
To a solution of 10 (114 mg, 0.225 mmol) in 8 mL of CH2Cl2/
EtOH (1/1) was added 30% NaOH (8 mL). The reaction was refluxed
for 12 h, neutralized with 5 N HCl and extracted with ethyl acetate
(3 ꢀ 30 mL). The combined organic layer was washed with brine
(50 mL) and dried with anhydrous Na2SO4, and the solvent was re-
moved under vacuum, yielding a crude product (650 mg) that was
purified via column chromatography (pure CH2Cl2) and crystallized
from ethyl acetate to yield 2 (42 mg, 62.7%). m.p.: 102–106 °C; 1H
NMR (CDCl3, 400 MHz) d: 7.07 (2H, d, J = 8.61 Hz, 200, 600-H), 6.91
(1H, d, J = 7.83 Hz, 50-H), 6.83 (2H, d, J = 8.61 Hz, 300, 500-H), 6.69
(1H, s, 20-H), 6.56 (1H, d, J = 7.83 Hz, 60-H), 5.53 (1H, s, –OH), 4.76
(1H, s, –OH), 3.94 (3H, s, –OCH3), 2.13 (4H, q, J = 6.65 Hz, 2,
5-CH2), 0.78 (6H, t, J = 6.65 Hz, 1, 6-CH3); ESI-MS m/z (%): 297

422
Q. Liu et al. / Steroids 77 (2012) 419–423
OH
OBn
OH
O
O
MeO
HO
MeO
HO
MeO
BnO
a
b
+
3
4
CH2COOH
OBn
OBn
OBn
O
OH
MeO
BnO
MeO
BnO
MeO
BnO
d
e
c
f
6
5
7
OH
OH
OCOPh
MeO
HO
g
MeO
MeO
h
HO
PhOCO
8
9
10
i
+
OH
OH
MeO
HO
MeO
HO
11
2
Scheme 1. Synthetic route to 30-methoxy-E-diethylstilbestrol. Reagents and conditions: (a) BF3–Et2O, 65 °C, 19 h; (b) BnBr, K2CO3, acetone, reflux, 10 h; (c) EtBr, NaH, toluene,
reflux, 16 h; (d) EtMgBr, THF, 35 °C, 3 h; (e) H2SO4, acetone, room temperature, 30 min; (f) HCO2NH4, 5% Pd/C, CH2Cl2/MeOH, room temperature, 1 h; (g) 48% HBr, acetic acid,
room temperature, 48 h; (h) benzoyl chloride, K2CO3, CH2Cl2, reflux, 3 h; (i) 30% NaOH, CH2Cl2, EtOH, reflux, 12 h.
OHþ2 leaving group that forms under acidic reaction conditions,
which would prefer to follow Hofmann’s rule.
Table 1
Selected bond lengths and angles for compound 10.
The shift of the olefinic bond from the 2-position to the 3-posi-
tion was achieved following the de-protection of 7. The hydrogena-
tion of the benzyl-ether, utilizing ammonium formate as the
hydrogen source and being catalyzed by Pd/C in dichloromethane
and methanol, yielded two products: 3-(4-hydroxy-3-methoxy-
phenyl)-4-(4-hydroxyphenyl)hexane (11), as a thoroughly reduced
product at 47.1%, and 3-(4-hydroxy-3-methoxyphenyl)-4-(4-
hydroxyphenyl)hex-2-ene (8). The latter compound (8) reacted
with HBr (48%) in acetic acid to reach an equilibrium of 2-ene (8)
and 3-ene (9), which could subsequently be separated by column
chromatography to obtain equal amounts of each compound. This
reaction is proposed to occur in a two-step manner: first, the elec-
trophilic addition of HBr to the double bond and, second, the b elim-
ination of bromide. However, separating Z- and E-isomers
(approximately 1:3 by 1H NMR) from the mixture of 9 was problem-
atic. To overcome this difficulty, esterization of 9 was performed by
benzoyl chloride in the presence of K2CO3 to obtain the derivative of
the E-isomer (10), of which the configuration was identified by X-
ray diffraction. The E-isomer was hydrolyzed further to produce
30-methoxy-E-diethylstilbestrol (2) with a yield of 62.7%.
Bond
Dist. (Å)
Bond
Dist. (Å)
C(10)–C(11)
C(11)–C(12)
C(11)–C(16)
C(14)–C(15)
C(15)–C(16)
1.384(2)
1.386(2)
1.510(2)
1.525(3)
1.522(3)
C(16)–C(17)
C(17)–C(18)
C(17)–C(20)
C(18)–C(19)
1.337(2)
1.508(3)
1.508(2)
1.523(3)
Angle
(°)
Angle
(°)
C(11)–C(10)–C(9)
C(11)–C(10)–H(10A)
C(10)–C(11)–C(12)
C(10)–C(11)–C(16)
C(12)–C(11)–C(16)
C(16)–C(15)–C(14)
C(16)–C(15)–H(15A)
C(16)–C(15)–H(15B)
C(14)–C(15)–H(15A)
C(14)–C(15)–H(15B)
121.45(17)
119.3
C(17)–C(16)–C(11)
C(17)–C(16)–C(15)
C(11)–C(16)–C(15)
C(16)–C(17)–C(20)
C(16)–C(17)–C(18)
C(20)–C(17)–C(18)
C(17)–C(18)–C(19)
C(17)–C(18)–H(18A)
C(17)–C(18)–H(18B)
C(19)–C(18)–H(18A)
121.81(15)
125.97(15)
112.22(15)
122.85(16)
123.10(15)
114.04(16)
113.70(15)
108.8
118.09(15)
121.22(15)
120.62(15)
113.22(15)
108.9
108.9
108.9
108.9
108.8
108.8
be concluded that the double bond is oriented between atom
C16 and C17, based on the bond distances of C15–C16, C16–C17
and C17–C18, and on the bond angles of C16 and C17. As shown
in Fig. 2, the C16–C17 double bond adopts a trans-configuration,
and neither the phenyl plane of C8–C13 nor that of C20–C25 is
co-planar with the olefinic bond, indicating that it is not conju-
gated with either of the two attached benzene rings of 10.
However, 2 is likewise unstable in solution and can partially
tautomerize to the 2-ene form. One milligram of 2 was dissolved
in 1 mL of methanol at room temperature for 24 h, and the relative
amount of each isomer was detected by HPLC, with a ratio of
approximately 79:21 (2:8).
The X-ray structure and selected bond lengths and angles for
compound 10 are given in Fig. 2 and Table 1, respectively. It can
3.2. Biological evaluation
Compounds 8, 11, 2 and 10 were evaluated in vitro against HU-
VEC, RL95-2, SKOV-3, MCF-7 and T-47D cell lines using 2-ME2 as a
reference. The 50% inhibition concentration data (IC50, listed in
Table 2) showed that all of the compounds tested except for 10 pos-
sess potential anti-proliferative activity at a concentration approx-
imately 5- to 10-fold lower than that of 2-ME2. Compound 10 was
not cytotoxic to any of the cell lines tested (IC50 > 100 lM), likely
due to the esterization of the two OH-groups by benzoyls, which
is consistent with the SAR findings for 2-ME2 that, in addition to
the 2-methoxy group, modifications at both the 3- and 17-positions
Fig. 2. X-ray crystal structure of 10.

Q. Liu et al. / Steroids 77 (2012) 419–423
423
Table 2
Acknowledgments
Anti-proliferation activities in vitro of 8, 11, 2 and 10.
Compounds
IC50
(
l
M)
This work was supported partially by the National Natural Sci-
ence Foundation of China (No. 30500631) and by the National Drug
Innovative Program (No. 2009ZX09301-011). The authors are
grateful to Mr. Chun-Jun Guo (University of Southern California)
for his advice during manuscript preparation.
HUVEC
RL95-2
SKOV-3
MCF-7
T-47D
8
11
2
10
38.8
38.2
58.3
>100
8.59
25.82
40.75
55.02
>100
4.11
22.98
41.76
73.93
>100
2.48
40.31
52.24
66.44
>100
>100
45.37
47.48
46.57
>100
4.73
2-ME2
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