Synthesis and antimitotic activity of novel 2-methoxyestradiol analogs.

Article abstract of DOI:10.1016/S0039-128X(02)00066-1

The syntheses and antimitotic activity of several novel 2-methoxyestradiol analogs are described. Structural modifications investigated include introduction of additional unsaturation in rings B and D; inversion at C-13; and substitution at the C-2, C-15, C-16, and C-7 alpha positions. Of 15 analogs synthesized, 2 have demonstrated superior biological activities compared to 2-methoxyestradiol.

Full text of DOI:10.1016/S0039-128X(02)00066-1

Steroids 67 (2002) 1079–1089
Synthesis and antimitotic activity of novel 2-methoxyestradiol analogsଝ
Pemmaraju N. Raoa,∗, James W. Cessaca, Tina L. Tinleyb, Susan L. Mooberryb
Department of Organic Chemistry, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, TX 78245-0549, USA
a
b
Department of Physiology and Medicine, Southwest Foundation for Biomedical Research, P.O. Box 760549, San Antonio, TX 78245-0549, USA
Received 10 April 2002; received in revised form 24 May 2002; accepted 7 June 2002
Abstract
The syntheses and antimitotic activity of several novel 2-methoxyestradiol analogs are described. Structural modifications investigated
include introduction of additional unsaturation in rings B and D; inversion at C-13; and substitution at the C-2, C-15, C-16, and C-7␣
positions. Of 15 analogs synthesized, 2 have demonstrated superior biological activities compared to 2-methoxyestradiol.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Steroids; Estrogens; 2-Methoxyestradiol; Antiangiogenesis; Antimitotic activity
1. Introduction
The biological activities of 2-ME2 are generating con-
siderable excitement because of its efficacy without toxic-
ity. Because 2-ME2 is a promising drug for cancer therapy,
work has begun on second-generation derivatives with su-
perior properties, including better oral availability and dif-
ferent chemosensitivity profiles.
The sequential biochemical hydroxylation and methyla-
tion of the natural hormone estradiol (E2, 1, Fig. 1) gives
rise to the endogenous mammalian metabolite 2-methoxy-
estradiol (2-ME2, 2, Fig. 1) [1]. 2-Methoxyestradiol is a
natural metabolite of estrogen devoid of uterotropic or es-
trogenic activity in vivo. Recent studies [2] have shown that
2-ME2 inhibits the cellular machinery involved in repli-
cating 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 advantages over current anticancer thera-
pies. The mechanism of action of 2-ME2 involves disruption
of cellular microtubules leading to mitotic arrest and initia-
tion of apoptosis. Preclinical studies with 2-ME2 reveal that
it is orally active, inexpensive to produce and, in contrast to
most antitumor agents, exhibits no overall toxicity at thera-
peutically effective doses. Both NCI and private sector spon-
sored Phase I clinical trials have been initiated to determine
if 2-ME2 is safe for use in humans. Preliminary results indi-
cate no occurrence of life-threatening toxicities and no max-
imal tolerated dose level was achieved. Subsequently, Phase
II clinical trials have been initiated to determine if 2-ME2
is effective against multiple myeloma and prostate cancer.
Recently [4–8], several synthetic analogs of 2-ME2 have
been developed, some of which exhibit increased antitumor
activity compared to the parent 2-methoxyestradiol. All of
the new compounds synthesized involve structural modifica-
tions in ring A or ring B of 2-ME2. Our continuing interest
in the field of estrogen metabolites [9–15] prompted us to
devise a new efficient approach to synthesize 2-ME2 based
upon the regioselective zirconium tetrachloride-mediated
Fries rearrangement carried out on estradiol diacetate [16].
Subsequently, we have conceived and carried out the syn-
thesis and biological testing of a series of novel 2-ME2
analogs. Structural modifications investigated include in-
troduction of additional unsaturation in rings B and D;
inversion at C-13; and substitution at the C-2, C-15, C-16,
and C-7␣ positions. Of 15 analogs synthesized and tested,
2 have demonstrated potentially superior biological effects
compared to 2-ME2.
2. Experimental
2.1. Chemistry
ଝ
The compounds described in this communication are the subject of a
Melting points were determined on a Thomas–Hoover
apparatus and are uncorrected. Nuclear magnetic reso-
nance spectra were recorded on a General Electric GE-300
pending U.S. patent.
∗
Corresponding author. Tel.: +1-210-258-9414; fax: +1-210-670-3321.
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)00066-1

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P.N. Rao et al. / Steroids 67 (2002) 1079–1089
Fig. 1. Estradiol and 2-methoxyestradiol.
(300 MHz) spectrometer as deuterochloroform (CDCl3)
solutions using tetramethylsilane (TMS) as an internal stan-
dard (δ = 0) unless noted otherwise. Infrared 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). ‘Flash column’ chro-
matography was performed on 32–64 ␮M silica gel obtained
from EM Science, Gibbstown, New Jersey. ‘Dry column’
chromatography was performed on 70–230 mesh silica gel,
also obtained from EM Science. Thin-layer chromatography
(TLC) analyses were carried out on silica gel GF (Anal-
tech) glass plates (2.5 cm × 10 cm with 250 ␮M layer and
prescored). Most chemicals and solvents were analytical
grade and used without further purification. Commercial
reagents were purchased from Aldrich Chemical Company
(Milwaukee, WI). 2-Methoxyestrone was purchased some
time ago from Organon Inc., W Orange, New Jersey.
2.3. 17,17-Ethylenedioxy-2-methoxyestra-1,3,5(10)-
trien-3-ol 3-benzyl ether (5)
Under nitrogen, triethylorthoformate (3.8 ml, 22.8 mmol),
ethylene glycol (2.5 ml, 44.8 mmol) and toluenesulfonic acid
monohydrate (0.1 g, 0.53 mmol) were added to a solution of
the 17-keto steroid (3, 3.5 g, 6.96 mmol) in methylene chlo-
ride (35 ml). The reaction mixture was stirred at room tem-
perature for 16 h. After that time, analysis by TLC (CH2Cl2)
indicated a complete reaction. The reaction mixture was di-
luted with methylene chloride (100 ml) and washed with sat-
urated sodium bicarbonate solution (1×), water (1×), and
brine (1×). The organic fractions were filtered through anhy-
drous sodium sulfate, combined and concentrated in vacuo
to give 3.8 g of a clear oil. This material was purified by flash
chromatography (CH2Cl2) followed by crystallization from
methanol to give the pure 17-ketal (5, 3.28 g, 84.2%): mp =
87–88 ◦C; FT-IR (KBr, diffuse reflectance) νmax: 2940 and
1606 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.89 (s,
18-CH3), 3.86 (s, 2-OCH3), 3.89–3.98 (m, ketal CH2’s),
5.10 (s, benzyl CH2), 6.62 (s, 4-H), 6.85 (s, 1-H), 7.29–7.46
(m, benzyl aromatic). Analysis calculated for C28H34O4: C,
77.39; H, 7.89. Found: C, 77.10; H, 7.86.
2.2. 3-(Benzyloxy)-2-methoxyestra-1,3,5(10)-
trien-17-one (4)
A solution of 3-(benzyloxy)-2-methoxyestra-1,3,5(10)-
trien-17␤-ol [16] (3, 8.1 g, 20.64 mmol) in acetone (300 ml)
was cooled to 0 ◦C in an ice bath and treated dropwise
with Jones reagent with stirring until a yellow color per-
sisted. At that point, the reaction mixture was stirred at
0 ◦C for another 5 min and then was treated dropwise with
isopropanol until a green color persisted. The resulting
green suspension was diluted with water (500 ml) and ex-
tracted with methylene chloride (3×). The organic fractions
were washed with water (1×), saturated sodium bicarbon-
ate solution (1×), and brine (1×). The organic fractions
were filtered through anhydrous sodium sulfate, combined
and concentrated in vacuo. The residue was crystallized
from methanol to give the pure product (4, 4.9 g, 60.8%):
2.4. 17,17-Ethylenedioxy-2-methoxyestra-1,3,5(10)-
trien-3-ol (6)
Under nitrogen, ammonium formate (0.5 g, 7.9 mmol) and
palladium on charcoal (10%, 0.5 g) were added to a solu-
tion of the 3-benzyl ether (5, 0.5 g, 1.15 mmol) in methanol
(10 ml) and THF (5 ml). The reaction mixture was then
stirred overnight at room temperature. After that time, anal-
ysis by TLC (CH2Cl2) indicated a complete reaction. The
mixture was diluted with methylene chloride (50 ml), filtered
through Celite, and concentrated in vacuo. The residue was
taken up in methylene chloride, washed with water (2×)
and brine (1×), filtered through anhydrous sodium sulfate,
combined and concentrated in vacuo. The residue was crys-
tallized from methanol to give the pure product (6, 0.33 g,
83.3%): mp = 150–151 ◦C; FT-IR (KBr, diffuse reflectance)
νmax: 3441, 2932, and 1619 cm−1; NMR (300 MHz, CDCl3),
δ (ppm): 0.89 (s, 18-CH3), 3.85 (s, 2-OCH3), 3.88–3.98 (m,
mp = 154–156 ◦C; FT-IR (KBr, diffuse reflectance) νmax
:
2929, 1732, and 1605 cm−1; NMR (300 MHz, CDCl3), δ
(ppm): 0.92 (s, 18-CH3), 3.86 (s, 2-OCH3), 5.11 (s, benzyl
CH2), 6.638 (s, 4-H), 6.84 (s, 1-H), 7.29–7.46 (m, benzyl
aromatic). Analysis calculated for C26H30O3·1/5H2O: C,
79.24; H, 7.77. Found: C, 79.13; H, 7.76.

P.N. Rao et al. / Steroids 67 (2002) 1079–1089
1081
ketal CH2’s), 5.53 (br.s, OH), 6.63 (s, 4-H), 6.79 (s, 1-H).
Analysis calculated for C21H28O4·1/10MeOH: C, 72.90; H,
8.23. Found: C, 72.77; H, 8.09.
xylene (70 ml). A solution of the 16-bromo compound (8,
1.07 g, 2.3 mmol) in dry xylene (100 ml) was concentrated
to 50 ml to remove any moisture. After cooling to room
temperature, the steroid solution was added to the potas-
sium t-butoxide solution and the mixture heated to reflux
for 17 h. The reaction mixture was cooled to room temper-
ature, poured into water, and extracted with ethyl acetate
(3×). The organic fractions were washed with water (3×),
combined, dried over sodium sulfate, filtered, and concen-
trated in vacuo to give 1.01 g residue. This material was
dissolved in benzene and filtered through a small Florisil
column. The elute was concentrated in vacuo to give 0.73 g
residue. This material was crystallized from methanol to
give the pure tetraene (9, 0.64 g, 87%): mp = 161–162 ◦C;
FT-IR (KBr, diffuse reflectance) νmax: 3432, 2935, 2900,
and 1619 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.97
(s, 18-CH3), 3.87 (s, 2-OCH3), 3.96–4.03 (m, ketal CH2’s),
5.43 (s, OH), 5.75 (dd, J1 = 6.2 Hz, J2 = 3.4 Hz, 15-H),
6.26 (dd, J1 = 6.2 Hz, J2 = 1.2 Hz, 16-H), 6.65 (s, 4-H),
6.79 (s, 1-H). Analysis calculated for C21H26O4·1/2MeOH:
C, 72.04; H, 7.87. Found: C, 72.16; H, 7.78.
2.5. 17,17-Ethylenedioxy-2-methoxyestra-1,3,5(10)-
trien-3-ol acetate (7)
Under nitrogen, acetic anhydride (25 ml, 265 mmol) was
added to a solution of the ketal (6, 4.5 g, 13.06 mmol) in
dry pyridine (25 ml, 310 mmol). The reaction mixture was
stirred overnight at room temperature in the dark. After that
time, methanol (50 ml) was added and solvents removed
in vacuo. The treatment with methanol was repeated and
the residue obtained crystallized from methanol to give
the pure 3-acetate (7, 4.85 g, 96%): mp = 166–167 ◦C;
FT-IR (KBr, diffuse reflectance) νmax: 2933, 1765, and
1615 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.885 (s,
18-CH3), 2.30 (s, OAc), 3.801 (s, 2-OCH3), 3.88–3.98 (m,
ketal CH2’s), 6.73 (s, 4-H), 6.90 (s, 1-H). Analysis calcu-
lated for C23H30O5·1/6MeOH: C, 71.01; H, 7.89. Found:
C, 71.04; H, 7.81.
2.8. 2-Methoxyestra-1,3,5(10),15-tetraen-3-ol-
17-one (10)
2.6. 17,17-Ethylenedioxy-16α-bromo-2-methoxyestra-
1,3,5(10)-trien-3-ol acetate (8)
Under nitrogen toluenesulfonic acid monohydrate (0.1 g,
0.53 mmol) was added to a solution of the 17-ketal (9, 1.88 g,
5.5 mmol) in acetone (120 ml) and water (20 ml). The re-
action mixture was stirred at room temperature for 1.5 h,
diluted with cold water (150 ml), and extracted with ben-
zene (3×). The organic fractions were washed with satu-
rated sodium bicarbonate solution (2×) and brine (3×), dried
over anhydrous sodium sulfate, filtered and concentrated
in vacuo to give 1.78 g residue. This material was purified
by chromatography followed by crystallization from methy-
lene chloride/hexanes to give the pure 17-ketone (10, 1.05 g,
64%): mp = 212–214 ◦C; FT-IR (KBr, diffuse reflectance)
νmax: 3332, 2930, and 1696 cm−1; NMR (300 MHz, CDCl3),
δ (ppm): 1.12 (s, 18-CH3), 3.87 (s, 2-OCH3), 5.46 (s, OH),
6.09 (dd, J1 = 6.0 Hz, J2 = 3.2 Hz, 16-H), 6.678 (s, 4-H),
6.79 (s, 1-H), 7.64 (dd, J1 = 6.0 Hz, J2 = 1.1 Hz, 15-H).
Analysis calculated for C19H22O3: C, 76.48; H, 7.43. Found:
C, 76.42; H, 7.27.
Under nitrogen, solid phenyltrimethylammonium tri-
bromide (4.92 g, 13 mmol) was added to a solution of
the 3-acetate (7, 4.6 g, 11.9 mmol) in dry THF (100 ml)
cooled to −5 ◦C in an ice-salt bath. The reaction mixture
was stirred at 2 ◦C overnight. After that time, saturated
sodium bicarbonate solution (50 ml) was added and the
mixture was extracted with ethyl acetate (3×). The organic
fractions were washed with saturated sodium bicarbonate
solution (2×), sodium thiosulfate solution (10%, 1×), and
ice-cold water (3×). The organic fractions were dried over
sodium sulfate, filtered and concentrated in vacuo to give
6.5 g residue. This material was crystallized from methanol
to give the pure 16-bromo compound (8, 2.97 g, 54%):
mp = 210–212 ◦C; FT-IR (KBr, diffuse reflectance) νmax
:
2918, 1766, and 1616 cm−1; NMR (300 MHz, CDCl3), δ
(ppm): 0.92 (s, 18-CH3), 2.30 (s, OAc), 3.80 (s, 2-OCH3),
3.92–4.31 (m, ketal CH2’s), 4.55 (dd, J1 = 10.4 Hz, J2 =
4.4 Hz, 16-H), 6.73 (s, 4-H), 6.87 (s, 1-H). Analysis calcu-
lated for C23H29BrO5: C, 59.36; H, 6.28. Found: C, 59.51;
H, 6.30.
2.9. 2-Methoxyestra-1,3,5(10),15-tetraen-3,17β-
diol (11)
2.7. 17,17-Ethylenedioxy-2-methoxyestra-1,3,5(10),
15-tetraen-3-ol (9)
Under nitrogen, solid lithium aluminum hydride (0.15 g,
3.95 mmol) was added to a solution of the 17-ketone (10,
0.06 g, 0.2 mmol) in dry ether (60 ml) cooled to −5 ◦C in
an ice-salt bath. The reaction mixture was stirred at −5 ◦C
for 1 h then quenched cautiously with dropwise addition of
water. The mixture was neutralized with 5% H2SO4, diluted
with water and extracted with ethyl acetate (3×). The or-
ganic fractions were washed with saturated sodium bicar-
bonate solution (2×) and water (3×), combined, dried over
Under argon, freshly cut potassium metal (0.77 g,
19.7 mmol) was added to tert-butanol (33 ml) and the mix-
ture heated to reflux until all of the metal had reacted. The
mixture was cooled slightly and the excess tert-butanol was
removed under a slight vacuum. Xylene (70 ml) was added
and distilled off three times followed by a fourth addition of

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P.N. Rao et al. / Steroids 67 (2002) 1079–1089
sodium sulfate, filtered and concentrated in vacuo to give
0.073 g residue. This material was triturated with ether to
give the pure 17-ol (11, 0.055 g, 91%): mp = 191–193 ◦C;
FT-IR (KBr, diffuse reflectance) νmax: 3506, 3241, 2938,
and 1607 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.89 (s,
18-CH3), 3.86 (s, 2-OCH3), 4.40 (br.s, 17-H), 5.45 (s, OH),
5.72 (ddd, J1 = 5.9 Hz, J2 = 3.3 Hz, J3 = 1.1 Hz, 16-H),
6.03 (m, 15-H), 6.66 (s, 4-H), 6.79 (s, 1-H). Analysis calcu-
lated for C19H24O3·1/3H2O: C, 74.49; H, 8.11. Found: C,
74.35; H, 8.05.
(ppm): 1.02 (s, 18-CH3), 3.86 (s, 2-OCH3), 4.10 (t, J =
8.4 Hz), 5.21 (m, 15-H), 6.66 (s, 4-H), 6.81 (s, 1-H). Anal-
ysis calculated for C19H24O3·2/5H2O: C, 74.19; H, 8.13.
Found: C, 74.04; H, 8.01.
2.12. 2-Methoxyestra-1,3,5(10),15-tetraen-3,17β-diol
3,17-diacetate (14)
Under nitrogen, acetic anhydride (30 ml, 318 mmol)
was added to a solution of 2-methoxyestra-1,3,5(10),15-
tetraene-3,17␤-diol (11, 1.15 g, 3.83 mmol) in dry pyri-
dine (35 ml, 434 mmol). The reaction mixture was stirred
overnight at room temperature. Solvents were azeotropi-
cally removed in vacuo using benzene (2×). The residue
was crystallized from methanol to give the pure diacetate
(14, 1.07 g, 72.4%): mp = 164–165 ◦C; FT-IR (KBr, dif-
2.10. 2-Methoxy-3,17-diacetoxyestra-1,3,5(10),14,16-
pentaene (12)
Under nitrogen, toluenesulfonic acid monohydrate (0.1 g,
0.53 mmol) was added to a solution of 2-methoxyestra-1,3,
5(10),15-tetraen-3-ol-17-one (10, 0.25 g, 0.84 mmol), iso-
propenyl acetate (5 ml, 45.15 mmol) and acetic anhydride
(5 ml, 53 mmol) and the mixture was heated to reflux for
6 h. At the end of that time, analysis by TLC (2% acetone in
CH2Cl2) indicated a complete reaction. The reaction mix-
ture was cooled to room temperature, poured into ice water
(∼100 ml) and stirred for 1 h. The mixture was extracted
with methylene chloride (3×). The organic fractions were
washed with water (1×), saturated sodium bicarbonate so-
lution (1×), and water (1×), filtered through anhydrous
sodium sulfate, combined and concentrated in vacuo. The
residue was crystallized from methanol to give the pure
diacetate (12, 0.259 g, 80.8%): mp = 191–194 ◦C; FT-IR
fuse reflectance) νmax: 2934, 1757, 1735, and 1613 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 0.88 (s, 18-CH3), 2.11
(s, 17-OAc), 2.30 (s, 3-OAc), 3.80 (s, 2-OCH3), 5.38 (t,
J = 3.5 Hz, 17-H), 5.70 (ddd, J1 = 6 Hz, J2 = 3.2 Hz,
J3 = 1.4 Hz, 16-H), 6.09 (m, 15-H), 6.75 (s, 4-H), 6.88
(s, 1-H). Analysis calculated for C23H28O5·1/10MeOH: C,
71.57; H, 7.38. Found: C, 71.52; H, 7.46.
2.13. 2-Methoxyestra-1,3,5(10)-trien-3,15ξ,16ξ,
17β-tetrol 3,17-diacetate (15)
Under nitrogen, a solution of the ꢀ15-steroid (14, 27.84 g,
72.4 mmol) in dry benzene (315 ml) was added to a solution
of osmium tetroxide (20.5 g, 80.6 mmol) in benzene (520 ml)
and pyridine (56 ml). The reaction mixture was stirred me-
chanically at room temperature for 4 h, and then left without
stirring at room temperature for 64 h. The reaction mixture
was diluted with benzene (510 ml) and methanol (940 ml).
Aqueous solutions of potassium bicarbonate (123 g/720 ml)
and sodium sulfite (123 g/720 ml) were added and the mix-
ture was stirred at room temperature for 4 h. The reac-
tion mixture was filtered and extracted with ethyl acetate
(3×). The organic fractions were washed with cold brine
(4×), combined, and concentrated in vacuo. Water was re-
moved azeotropically in vacuo using benzene to give 32.3 g
residue as crude product. This material was not charac-
terized or further purified and was carried on to the next
step.
(KBr, diffuse reflectance) νmax: 2931, 1763, and 1615 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 1.11 (s, 18-CH3),
2.23 (s, OAc), 2.31 (s, OAc), 3.81 (s, 2-OCH3), 5.87 (dd,
J1 = 2.6 Hz, J2 = 1.5 Hz, 15-H), 6.16 (d, J = 2.6 Hz,
16-H), 6.79 (s, 4-H), 6.90 (s, 1-H). Analysis calculated for
C23H26O5·1/20MeOH: C, 72.09; H, 6.88. Found: C, 72.02;
H, 6.81.
2.11. 2-Methoxyestra-1,3,5(10),14-tetraen-3,17β-diol (13)
A solution of the diacetate (12, 0.08 g, 0.21 mmol) in
ethanol (5 ml) and THF (3 ml) was cooled to 0 ◦C in an ice
bath. A solution of sodium borohydride (0.05 g, 1.32 mmol)
in ethanol/water (10:3, 5 ml) was cooled to 0 ◦C in an ice
bath and added to the steroid solution. The reaction mixture
was stirred at 0 ◦C for 1 h, then allowed to warm to room
temperature and stirred overnight. After that time, the reac-
tion mixture was diluted with water (20 ml) and neutralized
with glacial acetic acid. The organic solvents were removed
in vacuo under a stream of nitrogen, the residue was di-
luted with water (50 ml) and extracted with methylene chlo-
ride (3×). The organic fractions were washed with water
(2×), brine (1×), filtered through sodium sulfate, combined
and concentrated in vacuo. The residue was triturated with
ether to give the pure product (13, 0.035 g, 56%): mp =
169–171 ◦C; FT-IR (KBr, diffuse reflectance) νmax: 3499,
3185, 2920, and 1606 cm−1; NMR (300 MHz, CDCl3), δ
2.14. 2-Methoxyestra-1,3,5(10)-trien-3,15α,16α,
17β-tetrol tetraacetate (16a) and 2-methoxyestra-1,3,
5(10)-trien-3,15β,16β,17β-tetrol tetraacetate (16b)
Acetic anhydride (100 ml, 1.06 mol) was added to a solu-
tion of the crude mixture (15, 32.3 g, assume 72.4 mmol) in
dry pyridine (100 ml, 1.24 mol) and the mixture was stirred
overnight at room temperature. Solvents were removed in
vacuo under a stream of nitrogen with solvent residues be-
ing removed azeotropically using methanol (2×) to give

P.N. Rao et al. / Steroids 67 (2002) 1079–1089
1083
37 g crude product. This material was combined with 39 g
2.17. 2-Methoxyestra-1,3,5(10)-trien-3,15α,16α,
17β-tetrol 15,16-acetonide (18a)
of crude product obtained from a separate batch to give a to-
tal amount of 76 g crude product as an isomer mixture. This
material was resolved by stepwise crystallization (methanol)
and dry column chromatography (ether) to give the pure
15␣,16␣-isomer (16a, 43.08 g, 59.2%): mp = 168–171 ◦C;
FT-IR (KBr, diffuse reflectance) νmax: 2930, 1758, 1738,
and 1613 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.95
(s, 18-CH3), 2.04 (s, OAc), 2.08 (s, OAc), 2.09 (s, OAc),
2.30 (s, 3-OAc), 3.80 (s, 2-OCH3), 5.01 (d, J = 6.6 Hz,
17-H), 5.16 (dd, J1 = 14.7 Hz, J2 = 8.4 Hz, 15-H), 5.40
(dd, J1 = 8.4 Hz, J2 = 6.6 Hz, 16-H), 6.75 (s, 4-H), 6.88
(s, 1-H). Analysis calculated for C27H34O9: C, 64.53; H,
6.82. Found: C, 64.63; H, 6.84. And the 15␤,16␤-isomer
(16b, 11.78 g, 16.2%): mp = 169–171 ◦C; FT-IR (KBr, dif-
fuse reflectance) νmax: 2939, 1743, and 1612 cm−1; NMR
(300 MHz, CDCl3), δ (ppm): 1.09 (s, 18-CH3), 2.03 (s,
OAc), 2.06 (s, OAc), 2.06 (s, OAc), 2.31 (s, 3-OAc), 3.81 (s,
2-OCH3), 4.72 (d, J = 7.4 Hz, 17-H), 5.39 (t, J = 6.3 Hz,
15-H), 5.50 (t, J = 7.4 Hz, 16-H), 6.750 (s, 4-H), 6.88 (s,
1-H). Analysis calculated for C27H34O9: C, 64.53; H, 6.82.
Found: C, 64.73; H, 6.80.
Under nitrogen, a solution of the tetrol (17a, 0.15 g,
0.45 mmol) in acetone (10 ml) was treated with perchloric
acid (70%, one drop). The reaction mixture was stirred at
room temperature overnight. Analysis by TLC (5% acetone
in CH2Cl2) indicated a complete reaction. The mixture
was quenched with saturated sodium bicarbonate solu-
tion (1 ml) and solvent removed in vacuo under a stream
of nitrogen. The residue was extracted with ethyl acetate
(3×). The organic fractions were washed with saturated
sodium bicarbonate solution (1×), water (1×), and brine
(1×), combined, dried over sodium sulfate, filtered and
concentrated in vacuo. The residue was crystallized from
ether/hexanes to give the purified acetonide (18a, 0.16 g,
95%): mp = 155–157 ◦C; FT-IR (KBr, diffuse reflectance)
νmax: 3534, 3208, 2934, and 1594 cm−1; NMR (300 MHz,
CDCl3), δ (ppm): 0.89 (s, 18-CH3), 1.32 (s, acetonide
CH3), 1.52 (s, acetonide CH3), 3.78 (br.s, 17-H), 3.86 (s,
2-OCH3), 4.43 (t, J = 8 Hz, 16-H), 4.51 (dd, J1 = 8 Hz,
J2 = 4.4 Hz, 15-H), 5.45 (s, OH), 6.65 (s, 4-H), 6.78
(s, 1-H). Analysis calculated for C22H30O5·1/4hexane: C,
71.27; H, 8.53. Found: C, 71.35; H, 8.70.
2.15. 2-Methoxyestra-1,3,5(10)-trien-3,15α,16α,17β-
tetrol (17a)
2.18. 2-Methoxyestra-1,3,5(10)-trien-3,15β,16β,
17β-tetrol 15,16-acetonide (18b)
Under nitrogen, a solution of potassium carbonate (6.0 g,
43.4 mmol) in water (330 ml) was added to a solution of the
tetraacetate (16a, 10.0 g, 19.9 mmol) in methanol (1800 ml).
The reaction mixture was stirred overnight at room temper-
ature. Glacial acetic acid (5.2 ml, 90.48 mmol) was added
and the solvents removed in vacuo. The residue was crys-
tallized from methanol/water to give the purified tetrol
(17a, 6.1 g, 91.7%): mp = 228–230 ◦C; FT-IR (KBr, dif-
fuse reflectance) νmax: 3342, 2930, and 1589 cm−1; NMR
(300 MHz, CDCl3 + D6DMSO + D2O), δ (ppm): 0.81 (s,
18-CH3), 3.52 (d, J = 5.7 Hz, 17-H), 3.85 (s, 2-OCH3),
3.94 (m, 15- and 16-H), 6.64 (s, 4-H), 6.78 (s, 1-H). Anal-
ysis calculated for C19H26O5: C, 68.24; H, 7.84. Found: C,
68.29; H, 8.00.
Following the same procedure given for the preparation
of 18a, the tetrol (17b, 0.1 g, 0.299 mmol) was reacted
with perchloric acid (70%, one drop) in acetone (10 ml)
overnight at room temperature. Identical workup gave after
crystallization from ether/hexanes the pure acetonide (18b,
0.064 g, 57.2%): mp = 188–191 ◦C; FT-IR (KBr, diffuse
reflectance) νmax: 3574, 3516, 2927, 1621, and 1594 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 1.04 (s, 18-CH3), 1.34
(s, acetonide CH3), 1.52 (s, acetonide CH3), 3.44 (m, 17-H),
3.86 (s, 2-OCH3), 4.53 (t, J = 6.1 Hz, 16-H), 4.64 (dd,
J1 = 6.1 Hz, J2 = 4.5 Hz, 15-H), 5.44 (OH), 6.65 (s, 4-H),
6.78 (s, 1-H). Analysis calculated for C22H30O5·1/5H2O:
C, 69.89; H, 8.10. Found: C, 69.88; H, 8.15.
2.16. 2-Methoxyestra-1,3,5(10)-trien-3,15β,16β,17β-
tetrol (17b)
2.19. 2-Methoxyestra-1,3,5(10),7-tetraen-3,17β-diol (20)
Following the same procedure given for the preparation
of 17a, the tetraacetate (16b, 10.0 g, 19.9 mmol) in methanol
(1800 ml) was hydrolyzed with potassium carbonate (6.0 g,
43.4 mmol) in water (330 ml) to give the pure tetrol (17b,
3.87 g, 58.2%): mp = 224–225 ◦C; FT-IR (KBr, diffuse re-
Under nitrogen, a solution of l-selectride in THF (1 M,
0.081 ml, 0.081 mmol) was added dropwise to a solution of
2-methoxy-3-hydroxyestra-1,3,5(10),7-tetraen-17-one [15]
(19, 0.008 g, 0.027 mmol) in dry THF (1.0 ml) cooled to
0 ◦C in an ice bath. The reaction mixture was allowed to
warm to room temperature and stirred for 45 min. Methanol
(three drops) followed by methanolic KOH (3%, three
drops) were added and the mixture was cooled to 0 ◦C in
an ice bath. Hydrogen peroxide solution (30%, five drops)
was added and the mixture was allowed to warm to room
temperature. The reaction mixture was diluted with water,
acidified with HCl, and extracted with methylene chloride
flectance) νmax: 3526, 3398, 3266, 2938, and 1621 cm−1
;
NMR (300 MHz, CDCl3 +D6DMSO+D2O), δ (ppm): 0.92
(s, 18-CH3), 3.42 (d, J = 7.2 Hz, 17-H), 3.85 (s, 2-OCH3),
4.12 (t, J = 6.9 Hz, 16-H), 4.26 (dd, J1 = 6.9 Hz, J2 =
5.1 Hz, 15-H), 6.63 (s, 4-H), 6.79 (s, 1-H). Analysis calcu-
lated for C19H26O5·1/10MeOH: C, 67.95; H, 7.88. Found:
C, 67.93; H, 7.79.

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P.N. Rao et al. / Steroids 67 (2002) 1079–1089
(3×). The organic fractions were washed with water (1×)
and brine (1×), filtered through sodium sulfate, combined
and concentrated in vacuo to give 0.006 g residue. This
material was crystallized from ether/hexanes to give the
purified product (20, 0.0037 g, 45.9%): NMR (300 MHz,
CDCl3), δ (ppm): 0.66 (s, 18-CH3), 3.38 (m, 17-H), 3.87
(s, 2-OCH3), 5.40 (t, J = 1.65 Hz, 7-H), 6.66 (s, 4-H), 6.69
(s, 1-H).
white solid indicated by NMR to contain none of the 13␤-
isomer.
For purposes of characterization and biological testing,
100 mg of this material was crystallized from methanol/
water to give the pure 13␣-product (24, 0.065 g): mp =
179–180.5 ◦C; FT-IR (KBr, diffuse reflectance) νmax: 3442,
2922, 2874, 2860, 1724, 1709, 1611, and 1587 cm−1. (Note:
an unusual occurrence of 17-ketone absorbance showing up
as two peaks.) NMR (300 MHz, CDCl3), δ (ppm): 1.06 (s,
18-CH3), 3.84 (s, 2-OCH3), 5.46 (s, OH), 6.62 (s, 4-H),
6.75 (s, 1-H). Analysis calculated for C19H24O3·1/10H2O:
C, 75.52; H, 8.07. Found: C, 75.43; H, 8.06.
2.20. 2-Methoxyestra-1,3,5,7,9-pentaen-3,17β-diol (22)
Under nitrogen, a solution of l-selectride in THF (1 M,
0.09 ml, 0.09 mmol) was added dropwise to a solution of
2-methoxy-3-hydroxyestra-1,3,5,7,9-pentaen-17-one [15]
(21, 0.009 g, 0.03 mmol) in dry THF (1.0 ml) cooled to 0 ◦C
in an ice bath. The reaction mixture was allowed to warm
to room temperature and stirred for 45 min. Methanol (four
drops) followed by methanolic KOH (3%, four drops) were
added and the mixture was cooled to 0 ◦C in an ice bath.
Hydrogen peroxide solution (30%, five drops) was added
and the mixture was allowed to warm to room tempera-
ture. The reaction mixture was diluted with water, acidified
with HCl, and extracted with methylene chloride (3×). The
organic fractions were washed with water (1×) and brine
(1×), filtered through sodium sulfate, combined and con-
centrated in vacuo to give 0.011 g residue. This material
was crystallized from ether/hexanes to give the purified
product (22, 0.0066 g, 73%): NMR (300 MHz, CDCl3), δ
(ppm): 0.71 (s, 18-CH3), 3.96 (m, 17-H), 4.03 (s, 2-OCH3),
7.06 (d, J = 8.25 Hz, 6-H), 7.18 (s, 4-H), 7.26 (s, 1-H),
7.50 (d, J = 8.25 Hz, 7-H).
2.22. 2-Methoxy-13α-estra-1,3,5(10)-trien-3,17β-diol (25)
and 2-methoxy-13α-estra-1,3,5(10)-trien-3,17α-diol (26)
A solution of the 13␣-isomer (24, 1.0 g, 3.33 mmol) in
methanol (30 ml) and THF (25 ml) was cooled to 0 ◦C in an
ice bath and treated portionwise with solid NaBH4 (0.25 g,
6.6 mmol). The reaction mixture was allowed to warm to
room temperature and stirred for 3 h. Acetone (∼2 ml) was
added followed by ice-water (∼10 ml). The mixture was
poured into ice-water (100 ml) and the resulting precipi-
tate collected by filtration, washed with water and air-dried.
The crude product mixture was separated via flash chro-
matography (10% acetone in toluene) to give the less po-
lar 17␤-isomer (25, 0.53 g, 52.6%) as a white solid: mp =
164–166 ◦C (MeOH/H2O); FT-IR (KBr, diffuse reflectance)
νmax: 3373, 3166, 2944, 2901, 1614, 1598, and 1516 cm−1
;
NMR (300 MHz, CDCl3), δ (ppm): 0.95 (s, 18-CH3), 3.81
(t, J = 3 Hz, 17-H), 3.84 (s, 2-OCH3), 5.46 (s, OH), 6.61
(s, 4-H), 6.75 (s, 1-H). Analysis calculated for C19H26O3:
C, 79.68; H, 9.15. Found: C, 79.49; H, 9.25. And the more
polar 17␣-isomer (26, 0.31 g, 30.8%) as a white solid: mp =
150–152 ◦C (MeOH/H2O); FT-IR (KBr, diffuse reflectance)
2.21. 2-Methoxy-13α-estra-1,3,5(10)-trien-2-ol-
17-one (24)
νmax: 3533, 3316, 2915, 2874, 1623, 1593, and 1506 cm−1
;
Under nitrogen, a mixture of 2-methoxyestrone (23, 1.0 g,
3.33 mmol) and 1,2-phenylenediamine (0.6 g, 5.55 mmol)
in glacial acetic acid (10 ml) was heated to reflux for 4 h.
The mixture was allowed to cool to room temperature and
poured into ice-water (∼400 ml). The resulting precipitate
was collected by filtration, washed with water and air dried
to give 0.9 g crude product as an off-white solid. This ma-
terial was combined with that obtained from a second 1 g
batch, and the total crude material (1.8 g) was purified via
flash chromatography (3% acetone in CH2Cl2) to give 1.4 g
of a white solid indicated by NMR to consist of the ex-
pected 13␣-product (24) plus 5–10% starting material (23).
This material was suspended in ethanol (50 ml). Girard’s
Reagent P (1.5 g, 8.0 mmol) and glacial acetic acid (1 ml,
17.4 mmol) were added and the mixture was heated to re-
flux under nitrogen for 1.5 h. The reaction mixture was al-
lowed to cool to room temperature, poured into half saturated
sodium bicarbonate solution (350 ml) and extracted with
methylene chloride (3×). The organic fractions were washed
with water (2×), filtered through Na2SO4, combined and
concentrated in vacuo to give 1.18 g crude product (24) as a
NMR (300 MHz, CDCl3), δ (ppm): 0.93 (s, 18-CH3), 3.84 (s,
2-OCH3), 4.18 (t, J = 8.4 Hz, 17-H), 5.60 (br.s, OH), 6.61
(s, 4-H), 6.79 (s, 1-H). Analysis calculated for C19H26O3:
C, 79.68; H, 9.15. Found: C, 79.73; H, 9.02.
2.23. 2-Acetylestra-1,3,5(10)-trien-3,17β-diol (28)
Under nitrogen, a suspension of 2-acetylestra-1,3,5(10)-
trien-3,17␤-diol 17-acetate [16] (27, 0.5 g, 1.4 mmol)
in methanol (100 ml) was treated with 1N KOH (4 ml,
4 mmol). Water (10 ml) was added and the reaction mixture
was stirred overnight at room temperature. After that time,
analysis by TLC (5% acetone in CH2Cl2) of a small aliquot
made acidic indicated ∼90% reaction. The reaction mixture
was heated to reflux for 30 min, after which time analysis
by TLC indicated a complete reaction. The solvents were
removed in vacuo, the residue diluted with water (100 ml)
and glacial acetic acid (0.6 ml, 7 mmol) was added. The
resulting suspension was extracted with methylene chloride
(3×). The organic fractions were washed with water (3×),

P.N. Rao et al. / Steroids 67 (2002) 1079–1089
1085
filtered through Na2SO4, combined and concentrated in
vacuo to give 0.46 g yellow foam as residue. This mate-
rial was crystallized from ether to give the pure diol (28,
0.406 g, 92%): mp = 192–195 ◦C; FT-IR (KBr, diffuse re-
flectance) νmax: 3477, 2972, 2937, 2913, 2867, 1634, 1607,
and 1570 cm−1; NMR (300 MHz, CDCl3), δ (ppm): 0.80 (s,
18-CH3), 2.60 (s, acetyl), 3.75 (t, J = 8.4 Hz, 17-H), 6.69
(s, 4-H), 7.61 (s, 1-H), 12.04 (s, 3-OH). Analysis calculated
for C20H26O3·1/5Et2O: C, 75.88; H, 8.57. Found: C, 75.92;
H, 8.42.
by acid catalyzed ketal exchange of (9) in acetone gave the
ꢀ15-17-one derivative (10) in 56% overall yield. Lithium
aluminum hydride reduction of (10) to the ꢀ15-17␤-ol
derivative (11) was successfully carried out in 91% yield
using ether as solvent at −5 ◦C.
Following the procedure of Rasmusson and Arth [21],
the ꢀ15-17-ketone (10) was converted to the dienyl acetate
(12) in 80.8% yield by reaction with isopropenyl acetate
and toluenesulfonic acid monohydrate in acetic anhydride
at reflux. The subsequent sodium borohydride reduction of
this material gave the ꢀ14-17␤-ol derivative (13) in 55.7%
yield.
2.24. Biological assays
Starting from the ꢀ15-3,17␤-diol (11), the syntheses of the
3,15␣,16␣,17␤-tetrol (17a) and the 3,15␤,16␤,17␤-tetrol
(17b) were carried out following the procedure of Nam-
bara et al. [22]. Diacetylation of (11) was carried out in
72.4% yield followed by reaction with osmium tetroxide in
pyridine/benzene to give a mixture of the 15,16-diols (15).
This mixture was converted to the corresponding tetraac-
etates which could be separated by chromatography and
crystallization to give the 3,15␣,16␣,17␤-tetraacetate (16a)
in 59.2% yield and the 3,15␤,16␤,17␤-tetraacetate (16b) in
16.2% yield. The assignment of the 15,16-configuration was
based upon proton chemical shift data for the C-18 methyl
group and the 17␣-proton. Compound 16b has greater
deshielding of the 18-methyl (δ = 1.093) compared to 16a
(δ = 0.945) and compound 16a has increased deshielding
of the 17␣-H (δ = 5.012) compared to 16b (δ = 4.724).
Subsequent mild base hydrolysis of the tetraacetates gave
the corresponding 3,15␣,16␣,17␤-tetrol (17a) and the
3,15␤,16␤,17␤-tetrol (17b) in 91.7 and 58.2% yields, re-
spectively. Conversions of the tetrols (17a and 17b) to the
corresponding acetonides (18a and 18b) were carried out
by reaction with acetone and catalytic perchloric acid in 95
and 57.2% yields, respectively.
The equine estrogen derivatives 2-methoxyestra-1,3,5(10),
7-tetraen-3,17␤-diol (20) and 2-methoxyestra-1,3,5,7,9-
pentaene-3,17␤-diol (22) were obtained from the previously
synthesized [15] 17-ketones (19 and 21) by reduction with
l-selectride in THF at 0 ◦C in 45.9 and 73% yields, respec-
tively, as shown in Fig. 3. Having only small quantities of
starting materials, these products were only characterized
by NMR.
The syntheses of the 13␣-compounds (24, 25, and 26)
were carried out following the procedure of Schönecker et al.
[24] and is outlined in Fig. 4. Reaction of 2-methoxyestrone
(23) with 1,2-phenylenediamine in glacial acetic acid at re-
flux gave a 70% yield of the 13␣-derivative (24) contami-
nated with 5–10% starting material (23). This material was
purified by reaction with Girard’s Reagent P which selec-
tively reacts with the 13␤-starting material. The overall yield
of the purified 13␣-ketone (24) was 59%. Sodium borohy-
dride reduction of the 13␣-ketone (24) gave a chromato-
graphically separable mixture of the 17␤- and 17␣-alcohols
(25 and 26) in 52.6 and 30.8%, respectively. The assign-
ment of configuration at C-17 was based upon comparison
2.24.1. Inhibition of proliferation
The sulforhodamine B (SRB) assay was used to evaluate
the antiproliferative activity of 2-ME2 and 2-ME2 deriva-
tives in the MDA-MB-435 and SK-OV-3 cell lines [17,18].
Cells were plated into 96-well plates and allowed to grow
and attach for 24 h followed by addition of the test sub-
stances or vehicle controls. The cells were incubated with
drugs for 48 h and then the cellular protein was fixed, stained,
and concentration determined by absorbance at 560 nm. Log
dose–response curves were constructed for each experiment
and the IC50 for inhibition of proliferation determined.
2.24.2. Microtubule depolymerizing activity
The effects of the parental compound and derivatives on
cellular microtubule depolymerization were determined by
indirect immunofluorescence techniques in rat aortic smooth
muscle A-10 cells. Microtubules were visualized using a
␤-tubulin antibody as previously described [19]. Three view-
ers determined the percent microtubule loss for each treat-
ment concentration. The data were averaged and plotted as
percent microtubule loss versus drug concentration and the
EC50s for microtubule depolymerization calculated from the
log dose–response curves.
3. Results and discussion
3.1. Chemistry
The syntheses of compounds 2, 3, 19, 21, 27, and 29 were
described previously [15,16]. The syntheses of the ꢀ15- and
ꢀ14-derivatives (11 and 13), the tetrols (17a and 17b), and
the acetonides (18a and 18b) are outlined in Fig. 2 and are
based upon well known synthetic methodologies [20–22].
Jones oxidation of the previously synthesized [16] benzyl
ether (3) gave the 17-ketone (4) in 60.8% yield. Ketalization
of (4) followed by catalytic transfer hydrogenation using
ammonium formate [23] to remove the benzyl ether gave the
3-ol-17 ketal compound (6) in 70.1% overall yield. Acety-
lation of (6) followed by bromination using phenyltrimethy-
lammonium tribromide gave the 16␣-bromo-3-acetate (8) in
51.8% overall yield. Subsequent dehydrobromination of (8)
using potassium tert-butoxide in refluxing xylene followed

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P.N. Rao et al. / Steroids 67 (2002) 1079–1089
Fig. 2. Syntheses of 2-methoxyestradiol analogs.
Fig. 3. Syntheses of equine estrogen derivatives.

P.N. Rao et al. / Steroids 67 (2002) 1079–1089
1087
Fig. 4. Syntheses of 13␣-analogs.
Fig. 5. Synthesis of 2-acetyl analog.
of the chemical shifts of the 17-proton with those obtained
by Schönecker et al. [24] for the 17␣- and 17␤-isomers of
13␣-estradiol 3-methyl ether.
The synthesis of the 2-acetyl-3,17␤-diol derivative (28) is
shown in Fig. 5 and was carried out by base hydrolysis of
the previously synthesized [16] 3-acetate (27).
rived from a breast carcinoma and SK-OV-3 is an ovarian
carcinoma cell line. The data are presented in Table 1. The
most potent analog is the ꢀ14 analog (13) and in these cell
lines it is 23 and 12 times more potent than the parental
compound, 2-ME2 (2). The ꢀ15-compound (11) is approx-
imately 1.3–3.3-fold more potent than the parental com-
pound. In the MDA-MB-435 cell line, the ꢀ7-compound
(20) is slightly less potent than the reference compound
and almost 3-fold less potent towards SK-OV-3 cells. The
15␣,16␣-acetonide derivative (18a) is 0.34–0.38 as potent
as the reference compound.
3.2. Biological activity
The compounds tested for biological activity are shown
in Fig. 6.
The IC50s for inhibition of proliferation were generated
in two cancer cell lines. The MDA-MB-435 cells are de-
The effects of the derivatives on cellular microtubule de-
polymerization were determined using rat aortic smooth
Table 1
IC50 for inhibition of proliferation
Compound
Inhibition of proliferation IC50 (␮M)
MDA-MB-435 SK-OV-3
EC50 for microtubule depolymerization in
A-10 cells (␮M)
2
1.38
0.86
0.06
47.4
265
3.66
136
7.85
1.97
105
10.5
36.3
60.4
16.9
22.6
26.3
6.03
0.10
0.11
0
1.79
1.37
0.15
85.8
>200
5.16
N.D.
18.1
4.49
>200
28.3
32.1
114
0.24
0.26
0.07
16.0
7.5
6.5
0.3
11
13
17a
17b
18a
18b
19
20
21
22
23
24
25
26
28
29
4.45
71.4
1.17
24.1
5.73
0.40
36.3
2.80
30.4
28.2
3.87
1.70
1.61
1.38
>150
>150
17.4
N.D.
N.D.
N.D.
150
>150
>150
>150
>150
>150
>150
45.0
0.99
3.49
0.18
1.86
3.96
27.8
6.15
8.13
5.15
0.40
56.3
65.3
29.7
6.27
Three or four experiments were conducted and the IC50 for each experiment was determined. The values presented are the means S.D. EC50 for
microtubule depolymerization is the concentration of compound that causes 50% loss of cellular microtubules in A-10 cells. N.D.: experiment not carried
out.

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P.N. Rao et al. / Steroids 67 (2002) 1079–1089
Fig. 6. Compounds tested for biological activity.
muscle A-10 cells. This data is also shown in Table 1. Con-
sistent with the inhibition of cellular proliferation data, both
(11) and (13) were more potent than the reference compound
for causing microtubule depolymerization. Compound (18a)
was 0.43 as potent as 2-ME2, consistent with its antiprolif-
erative effects. The results suggest that the new derivatives
and 2-ME2 share the same intracellular target, tubulin.
(2, 20, 22, 25, and 26) are more active than their 17-oxo
counterparts (23, 19, 21, and 24), thus supporting the gen-
eralization for the free 17-OH group being required for
potent activity. However, an exception to this generalization
is 2-methoxyestrone-3-O-sulfamate which was shown by
Purohit et al. [7] to be 10 times as active as 2-ME2 against
human breast cancer cells.
Other structural modifications on 2-ME2 carried out by
Cushman et al. [4,5] have focused primarily on variations
at the 2- and 6-positions of the parent compound. The au-
thors conclude that the optimum 2-substituent for cytotoxic
activity appears to be an unbranched chain containing three
atoms chosen from the second row of the periodic table, with
activity increasing with increased electron density adjacent
to the aromatic ring. This generalization is supported by the
decreased cytotoxicity we observed for compound 28 with
the decreased electron density of the carbonyl carbon be-
ing adjacent to the aromatic ring. Concerning the 6-position,
3.3. Structure–activity relationships
Prior testing of 2-ME2 variants for inhibition of tubu-
lin polymerization [25] and inhibition of endothelial cell
proliferation [3] would seem to indicate that free hy-
droxyl groups at the 3- and 17-positions are required
for potent biological activity. The compounds previously
tested include 2-methoxyestrone, 2,3-dimethoxyestradiol,
and 2-hydroxyestradiol 3-methyl ether. From the data we
present in Table 1 it can be seen that the 17-hydroxy analogs

P.N. Rao et al. / Steroids 67 (2002) 1079–1089
1089
most modifications carried out by Cushman decreased cy-
totoxic activity by varying degrees with only the 6-oximino
derivatives being more active than the parent compound.
The introduction of a double bond at the 6-position for the
2-ethoxy analog led to a 90-fold decrease in cytotoxic activ-
ity. This is in contrast to the equipotent activity we observed
for compound 20 with the double bond at the 7-position.
The total aromatization of the ring B in compound 22 led
to decreased cytotoxic activity, but not as dramatic as that
seen by Cushman for the ꢀ6 analog.
enhanced inhibitory effects on tubulin polymerization and cancer cell
growth. J Med Chem 1997;40:2323–34.
[6] (a) Sachdeva Y, Ram S (inventors, Pharm-Eco Laboratories, Inc.,
USA). Synthesis of 2-alkoxyestradiols. World Patent No. 9840398
(1998);
(b) Ram S, Varma R, Sachdeva
Y (inventors, United States
Department of Health and Human Services, USA; Pharm-Eco
Laboratories, Inc.). Preparation of 2-alkoxyestradiols as antitumor
agents. US Patent No. 6136992 (2000).
[7] Purohit A, Hejaz HAM, Walden L, Maccarthy-Morrogh L, Packham
G, Potter BVL, et al. The effect of 2-methoxyoestrone-3-O-sulfamate
on the growth of breast cancer cells and induced mammary tumors.
Int J Cancer 2000;85:584–9.
Outside of 2-methoxyestrone, the only other ring D mod-
ified 2-ME2 analogs previously tested for antimitotic ac-
tivity are 2-methoxyestriol and 2-methoxyethynylestradiol
both of which proved to be inactive with regard to inhibi-
tion of tubulin polymerization [25]. The data for compounds
17a, 17b, 18a, and 18b seem to indicate cis-substitution
at the 15,16-positions leads to decreased activity with the
␤-substituted analogs being much less active than the ␣.
The inversion of configuration at the C-13 position in com-
pounds 24, 25, and 26 also leads to decreased cytotoxic ac-
tivity. The introduction of additional unsaturation in ring D
for compounds 11 and 13 gives the most interesting results
and leads to a modest to dramatic increase in cytotoxicities
relative to 2-ME2. Whether these structural modifications
lead to an increased binding affinity towards tubulin or per-
haps a decreased rate of metabolic inactivation remains to
be investigated.
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Detailed biological testing of the most promising analogs
will continue. Currently, compounds 11, 13, and 18a are
being evaluated in murine models for toxicity and antitumor
activity in head-to-head tests with 2-ME2.
[14] Rao PN, Burdett Jr JE.
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Financial support from Southwest Foundation for Bio-
medical Research and The San Antonio Area Foundation is
gratefully acknowledged.
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