Synthesis and antimitotic activity of novel 2-methoxyestradiol analogs. Part III

Article abstract of DOI:10.1016/j.steroids.2007.10.016

The syntheses and antimitotic activity of several novel analogs of 2-methoxyestradiol are described. Structural modifications include ring-D homologation, aromatization of the six-membered ring-D to a chrysine type molecule, and introduction of unsaturation in five-membered ring-D along with substitution of alkyl and ethynyl groups for the 17β-hydroxy function. Of nine analogs synthesized, five have demonstrated superior antiproliferative activities compared to 2-methoxyestradiol.

Full text of DOI:10.1016/j.steroids.2007.10.016

steroids 7 3 ( 2 0 0 8 ) 171–183
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antimitotic activity of novel
2-methoxyestradiol analogs. Part III
Pemmaraju N. Raoa,∗, James W. Cessaca, James W. Boyda,
Arthur D. Hansonb, Jamshed Shahb
a
Department of Organic Chemistry, Southwest Foundation for Biomedical Research, P.O. Box 760549, 7620 NW Loop 410, San Antonio,
TX 78245-0549, USA
b
EntreMed, Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses and antimitotic activity of several novel analogs of 2-methoxyestradiol are
described. Structural modifications include ring-D homologation, aromatization of the six-
membered ring-D to a chrysine type molecule, and introduction of unsaturation in five-
membered ring-D along with substitution of alkyl and ethynyl groups for the 17␤-hydroxy
function. Of nine analogs synthesized, five have demonstrated superior antiproliferative
activities compared to 2-methoxyestradiol.
Received 20 June 2007
Received in revised form
4 October 2007
Accepted 11 October 2007
Published on line 17 November 2007
© 2007 Elsevier Inc. All rights reserved.
Keywords:
Estrogens
2-Methoxyestradiol
Antiangiogenesis
Antimitotic activity
Antiproliferative activity
1.
Introduction
compounds with increased activity [5]. It is speculated that
the 17␤-hydroxyl protected analogs of 2ME2 are not readily
2-Methoxyestradiol (2ME2), a natural metabolite of estra-
diol which has no estrogenic activity, was found to be a
potent antitumor and antiangiogenic compound [1,2]. It is
currently in clinical trials for treatment of a variety of can-
cers. Several structural modifications of 2ME2 were reported
in the literature to obtain biologically more potent analogs.
We have previously shown [3,4] that modification of ring-D
by incorporation of additional unsaturation or introduction
of an ␣-substituted functional group such as a 15␣,16␣ ace-
tonide significantly increases the biological activity in vitro.
We also observed that in some cases protection of the
17␤-hydroxyl function as a methoxy derivative resulted in
amenable to metabolic oxidation to the less active 17-oxo-
analogs.
In continuation of our studies on the structure–activity
relationship in 2ME2 series, we synthesized several novel ring-
D-modified compounds. Structural modifications include the
expansion of the five-membered ring-D to a six-membered
structure (D-homo), aromatization of the six-membered
ring-D to chrysine type molecules, and substituting the
17␤-hydroxyl function in five-membered ring-D with alkyl
and ethynyl functions. We now present their synthesis and
evaluation of their cytotoxic activity in a variety of cell
types.
∗
Corresponding author. Tel.: +1 210 258 9414; fax: +1 210 670 3321.
E-mail address: pnrao@sfbr.org (P.N. Rao).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.10.016

172
steroids 7 3 ( 2 0 0 8 ) 171–183
1508 cm−1.NMR (300 MHz, CDCl3), ı (ppm): 0.92 (s, 18-CH3), 2.31
2.
Experimental
(s. 3-OAc), 3.81 (s, 2-OCH3), 6.77 (s, 4-H), 6.90 (s, 1-H).
Anal. Calcd. for C21H26O4: C, 73.66; H, 7.65. Found: C, 73.53;
H, 7.67.
2.1.
Chemistry
Melting points were determined on a Thomas–Hoover appa-
ratus and are uncorrected. Nuclear magnetic resonance
spectra were recorded on a General Electric GE-300 (300 MHz)
spectrometer as deuterochloroform (CDCl3) solutions using
tetramethylsilane (TMS) as an internal standard (ı = 0) unless
noted otherwise. Infrared spectra were recorded on Thermo-
Nicolet model 370 FT-IR instrument equipped with an
attenuated reflectance (ATR) accessory. Combustion analyses
were performed by Midwest Microlabs Ltd. (Indianapolis, IN).
‘Flash column’ chromatography 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 chro-
matography (TLC) analyses were carried out on silica gel GF
(Analtech) 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-Methoxyestradiol was provided as a gift by EntreMed,
Inc., 9640 Medical Center Drive, Rockville, MD.
2.1.3. 2-Methoxy-3-hydroxyestra-1,3,5(10)-trien-17-one
(4)
Under nitrogen, a solution of the 3-acetate derivative (3, 11.3 g,
33 mmol) in 1:1 THF/H2O (100 ml) was treated with NaOH (2 M,
75 ml, 150 mmol) at room temperature for 1 h. Analysis by TLC
(5% acetone/CH2Cl2) indicated a complete reaction. The reac-
tion was cooled to 0 ◦C, quenched with 3 M HCl, concentrated
in vacuo, and extracted with ethyl acetate (3×). The organic
fractions were washed with water, brine, combined, dried over
Na2SO4, and concentrated in vacuo to give 2-methoxyestrone
(4, 10 g, 100%) as a white solid. mp = 189–190 ◦C (Lit. [6],
mp = 189–191 ◦C); FT-IR (ATR) ꢀmax: 3359, 2928, 1720, 1588,
and 1503 cm−1. NMR (300 MHz, CDCl3) ı (ppm): 0.93 (s, 18-
CH3), 3.87 (s, 2-OCH3), 5.51 (s, 3-OH), 6.67 (s, 4-H), 6.80 (s,
1-H).
2.1.4. 2-Methoxy-3-methoxymethoxyestra-1,3,5(10)-
trien-17-one
(5)
Under nitrogen, 2-methoxyestrone (4, 9.2 g, 30 mmol) was
dissolved in 60 ml of THF. N,N-Diisopropylethylamine (35 ml,
200 mmol) and chloromethyl methyl ether (12.5 ml, 160 mmol)
were added and the reaction mixture stirred at 65 ◦C overnight.
The reaction was cooled to room temperature, quenched
with 20% NH4Cl solution, and extracted with ethyl acetate
(3×). The organic phase washed with water (3×), brine,
dried over Na2SO4, and concentrated in vacuo to give the
pure methoxymethyl ether (5, 9.39 g, 89%) as a white solid.
mp = 117 ◦C; FT-IR (ATR) ꢀmax: 2928, 2854, 1731, 1606 and
1509 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.93 (s, 18-CH3),
3.53 (s, 3-OCH2OCH3) 3.87 (s, 2-OCH3), 5.21 (s, 3-OCH2OCH3),
6.85 (s, 4-H), 6.90 (s, 1-H). Anal. Calcd for C21H28O4: C, 73.23; H,
8.19. Found: C, 73.09; H, 8.16.
2.1.1. 2-Methoxy-3-acetoxy-17ˇ-hydroxyestra-1,3,5(10)-
triene
(2)
To a solution of 2-methoxyestradiol (1, 20 g, 58 mmol) in iso-
propanol (350 ml) was added sodium hydroxide solution (2 M,
100 ml, 400 mmol) and acetic anhydride (16 ml, 169 mmol).
The reaction mixture was stirred at room temperature and
monitored by TLC (5% acetone/CH2Cl2) which indicated a com-
plete reaction after 2 h. The reaction was slowly quenched
with methanol, diluted with water, and concentrated in vacuo.
The residue was acidified with HCl (3 M) and extracted with
ethyl acetate (3×). The organic fractions were washed with
water, brine, dried over Na2SO4, and concentrated in vacuo
to give the 3-acetate derivative (2, 24.4 g) as a white solid.
mp = 148–149 ◦C; FT-IR (ATR) ꢀmax: 3459, 2926, 2859, 1758, and
2.1.5. 2-Methoxy-3-methoxymethoxy-17˛-cyano-17ˇ-
trimethylsilyloxyestra-1,3,5(10)triene
(6)
1505 cm−1
.
NMR (300 MHz, CDCl3), ı (ppm): 0.76 (s, 18-CH3), 2.30 (s. 3-
To
a solution of the 3-methoxymethyl ether (5, 4.9 g,
OAc), 3.80 (s, 2-OCH3), 3.71 (t, J = 8.5 Hz, 17-H), 6.73 (s, 4-H), 6.90
(s, 1-H).
Anal. Calcd for C21H28O4: C, 73.26; H, 8.19. Found: C, 73.13;
H, 8.04.
14.3 mmol) in chloroform (40 ml) were added zinc iodide
(20 mg, 0.063 mmol) and trimethylsilylcyanide (3.0 ml,
23.5 mmol) and the reaction mixture stirred at room tem-
perature overnight. The reaction mixture was quenched
with water, concentrated in vacuo, and extracted with ethyl
acetate (3×). The organic phase was washed with water
(3×), brine, dried over Na2SO4, and concentrated in vacuo
to give 7.0 g of residue. Analysis by 1H NMR showed an
incomplete reaction. The residue was re-reacted for another
4 h, quenched and extracted as above and purified by flash
column (2% acetone/CH2Cl2) to give 2.51 g of material. Anal-
ysis by 1H NMR showed the loss of the 3-methoxymethyl
ether.
2.1.2. 2-Methoxy-3-acetoxyestra-1,3,5(10)-trien-17-one
(3)
Under nitrogen, the 17-hydroxy compound (2, 10 g, 29 mmol)
was dissolved in 20 ml of acetone and chilled to 0 ◦C. Jones
reagent was slowly added with stirring until the yellow–orange
color persisted (18 ml). The reaction was stirred an additional
5 min then slowly quenched with isopropanol. The solution
was concentrated in vacuo, diluted with water, and extracted
with ethyl acetate (3×). The organic fractions were washed
with water, brine, combined, dried over Na2SO4, and concen-
trated in vacuo to give the 17-ketone (3, 9.07 g, 91%) as a white
solid. mp = 152 ◦C; FT-IR (ATR) ꢀmax: 2939, 1761, 1733, 1616, and
In this material, the 3-hydroxyl function was re-protected
by reaction with chloromethyl methyl ether and N,N-
diisoproplyethylamine in THF at 65 ◦C overnight. The reaction
was cooled to room temperature, quenched with 20% NH4Cl,

steroids 7 3 ( 2 0 0 8 ) 171–183
173
and extracted with ethyl acetate (3×). The organic fractions
2.1.8. 2-Methoxy-D-homoestra-1,3,5(10)-trien-3,17aˇ-
diol (10) and
2-methoxy-D-homoestra-1,3,5(10)-trien-3,17a˛-diol (11)
were washed with water (3×), brine, dried over Na2SO4, and
concentrated in vacuo to give the pure 3-methoxymethoxy-
17␣-cyano product (6, 2.78 g, 40%) as a yellow foam. mp = 63 ◦C;
FT-IR (ATR) ꢀmax: 2929, 1608, and 1507 cm−1. NMR (300 MHz,
CDCl3), ı (ppm): 0.20 (s, 17B-OSiMe3), 0.79 (s, 18-CH3), 3.45 (s,
3-OCH2OCH3) 3.8 (s, 2-OCH3), 5.12 (s, 3-OCH2OCH3), 6.74 (s, 4-
H), 6.76 (s, 1-H). Anal. Calcd for C25H37NO4Si·(2/5)H2O: C, 66.60;
H, 8.45; N, 3.11. Found: C, 66.56; H, 8.17; N, 3.24.
Under nitrogen, the 17a-ketone (8, 1.0 g, 2.79 mmol) was
dissolved in 20 ml of THF and cooled to 0 ◦C. Lithium tri-
tert-butoxyaluminum hydride solution (1 M in THF, 5.6 ml,
5.6 mmol) was added dropwise and the solution stirred at
room temperature for 2.5 . The reaction was quenched with
ethyl acetate, concentrated in vacuo, diluted with water, acid-
ified with 1 M HCl, and extracted with ethyl acetate (3×). The
organic phase was washed with water (3×), brine, dried over
Na2SO4, and concentrated in vacuo to give a residue that was
a mixture of the 17a␤ and 17a␣ alcohols. The residue was
separated by flash chromatography (4:1 ether/hexanes) and
subsequently hydrolyzed with 6 M HCl at 60 ◦C. Trituration
from ether/hexanes yielded the 17a␤-OH and 17a␣-OH com-
pounds, (10, 300 mg, 32%) and (11, 100 mg, 10%) as white solids.
mp (10) = 138 ◦C; FT-IR (ATR) ꢀmax: 3503, 3384, 2927, 2860, 1589,
and 1505 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.85 (s, 18-CH3),
3.28 (dd, J1 = 10.95 Hz, J2 = 4.65 Hz, 17-H), 3.87 (s, 2-OCH3), 6.64 (s,
4-H), 6.80 (s, 1-H). Anal. Calcd for C20H28O3·(1/4)H2O: C, 74.85;
H, 8.95. Found: C, 74.73; H, 8.88.
2.1.6. 2-Methoxy-3-methoxymethoxy-17˛-
aminomethylestra-1,3,5(10)-trien-17ˇ-ol
(7)
Under nitrogen, the 17␣-cyano compound (6, 2.7 g, 6.26 mmol)
in THF (40 ml) was slowly added to an ice cold solution
of LiAlH4 (1 M in THF, 12.5 ml, 12.5 mmol). After the addi-
tion was complete, the reaction was allowed to come to
room temperature and stirred overnight. The reaction mix-
ture was chilled to 0 ◦C, quenched with saturated sodium
sulfate, then solid sodium sulfate. The resulting mixture was
filtered through Celite and washed (3×) with THF. The solu-
tion was concentrated in vacuo and the residue extracted
with ethyl acetate (3×). The organic phase washed with
water (3×), brine, dried over Na2SO4, and concentrated in
vacuo. The residue was purified by flash column to give
the pure 17␣-aminomethyl (7, 1.72 g, 75%) as a yellow solid.
mp = 114 - 115 ◦C; FT-IR (ATR) ꢀmax: 3350, 2918, 1607, and
1513 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.8 (s, 18-CH3),
1.274 (d, J = 7.5 Hz, 1H), 3.40 (s, 3-OCH2OCH3) 3.75 (s, 2-
OCH3), 5.08 (s, 3-OCH2OCH3), 6.73 (s, 4-H), 6.75 (s, 1-H). Anal.
mp (11) = 146 ◦C; FT-IR (ATR) ꢀmax: 3561, 3499, 3271, 2937,
2863, 1608, and 1524 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.87
(s, 18-CH3), 3.43 (m, 17-H), 3.87 (s, 2-OCH3), 6.64 (s, 4-H), 6.81
(s, 1-H). Anal. Calcd for C20H28O3: C, 75.91; H, 8.92. Found: C,
75.49; H, 8.82.
2.1.9. 2-Methoxy-D-homoestra-1,3,5(10)-trien-3,17ˇ-diol
(12)
Calcd for C22
8.82.
H
33NO4: C, 70.37; H, 8.86. Found: C, 70.22; H,
The 17-ketone (9, 90 mg, 0.251 mmol) in 10 ml of THF
was reacted similarly with lithium tri-tert-butoxyaluminum
hydride solution (1 M in THF, 0.5 ml, 0.5 mmol). Purification by
flash chromatography (4:1 ether/hexanes), subsequent hydrol-
ysis with 6 M HCl at 60 ◦C and trituration from ether/hexanes
yielded the pure17␤-OH compound (12, 36 mg, 43%) as a white
solid. mp = 207 ◦C; FT-IR (ATR) ꢀmax: 3544, 3466, 3272, 2925, 1595,
1513 and 1501 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 1.10 (s, 18-
CH3), 3.86 (s, 2-OCH3), 4.15 (m, 17-H), 6.64 (s, 4-H), 6.80 (s, 1-H).
Anal. Calcd for C20H28O3·(2/5)H2O: C, 74.22; H, 8.97. Found: C,
74.02; H, 8.66.
2.1.7. 2-Methoxy-3-methoxymethoxy-D-homoestra-
1,3,5(10)-trien-17a-one (8) and
2-methoxy-3-methoxymethoxy-D-homoestra-1,3,5(10)-
trien-17-one
(9)
Under nitrogen, the 17␣-aminomethyl compound (7, 1.72 g,
4.58 mmol) was dissolved in 48 ml of 5:1 HOAc/H2O and chilled
to 0 ◦C. Sodium nitrite (1.67 g, 24.2 mmol) in 7 ml of H2O was
added dropwise over 1/2 h, the solution was then stirred an
additional 3 h at 0 ◦C, then warmed to room temperature and
stirred overnight. Analysis by TLC (5% i-prOH/CH2Cl2) indi-
cated a complete reaction. The solvent was removed and
the residue extracted with ethyl acetate (3×). The organic
phase was washed with water (3×), brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash
column (4:1 ether/hexanes) to give the pure 17a and 17-
ketone (8, 1.0 g, 61% and 9, 90 mg, 5.5%, respectively) as white
solids. mp (8) = 115 ◦C; FT-IR (ATR) ꢀmax: 2936, 2866, 1697,
1606 and 1505 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 1.116
(s, 18-CH3), 3.502 (s, 3-OCH2OCH3) 3.850 (s, 2-OCH3), 5.183 (s,
3-OCH2OCH3), 6.843 (s, 4-H), 6.855 (s, 1-H). Anal. Calcd for
C22H30O4: C, 73.71; H, 8.44. Found: C, 73.06; H, 8.29.
mp (9) = 104–105 ◦C; FT-IR (ATR) ꢀmax: 2935, 1707, 1608 and
1507 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.85 (s, 18-CH3), 3.53
(s, 3-OCH2OCH3) 3.86 (s, 2-OCH3), 5.21 (s, 3-OCH2OCH3), 6.86 (s,
4-H), 6.89 (s, 1-H). Anal. Calcd for C22H30O4: C, 73.71; H, 8.44.
Found: C, 73.31; H, 8.28.
2.1.10. 2-Methoxy-3-methoxymethoxyestra-1,3,5(10)-
trien-17ˇ-ol
(13)
Under nitrogen, sodium borohydride (1.3 g, 34.35 mmol) in 1:1
EtOH:H2O (65 ml) was added dropwise to a solution of the 17-
ketone (5, 4.76 g, 13.74 mmol) in 1:1 THF:EtOH (250 ml) and
stirred at room temperature for 1/2 h. Analysis by TLC (5%
acetone/CH2Cl2) indicated a complete reaction. The reaction
was chilled in an ice bath, quenched with acetic acid until
acidic, then concentrated in vacuo. The residue was diluted
with water, acidified with 6 M HCl, and extracted with ethyl
acetate (3×). The organic phase was washed with water, brine,
dried over Na2SO4 and concentrated in vacuo. The residue
was purified by flash chromatography (5% acetone/CH2Cl2) to
give the 17-hydroxy compound (13, 4.74 g, 99%) as a yellow
semisolid that still contained some solvent. FT-IR (ATR) ꢀmax
:
3422, 2923, 2866, 1608 and 1507 cm−1. NMR (300 MHz, CDCl3), ı
(ppm): 0.79 (s, 18-CH3), 3.51 (s, 3-OCH2OCH3) 3.73 (t, J = 8.4 Hz,

174
steroids 7 3 ( 2 0 0 8 ) 171–183
then allowed to warm to room temperature. After stirring
at room temperature for 2 h, TLC (sat. NH4Cl aliquot, 2%
acetone/CH2Cl2) indicated complete formation of the lithium
intermediate. Methanesulfonyl chloride (1 ml, 4.77 mmol) was
added followed 1/2 h later by pyridine (2 ml, 1.8 eq). The reac-
tion mixture was then stirred overnight at room temperature.
The reaction mixture was quenched with saturated NaHCO3
solution and extracted with ethyl acetate (3×). The organic
phase was washed with water, 1 M HCl (2×), water, brine,
dried over Na2SO4, and concentrated in vacuo to give 1.6 g
of a dark violet residue. The material was passed through
silica gel (2% acetone/CH2Cl2) to give a crude mixture com-
posed mainly of the 3-methoxymethyl (16a) and 3-hydroxy
(16b) derivatives. This mixture was used directly for the next
reaction.
17-H), 3.85 (s, 2-OCH3), 5.19 (s, 3-OCH2OCH3), 6.84 (s, 4-H), 6.87
(s, 1-H). Anal. Calcd for C21H30O4·(1/6)H2O: C, 72.18; H, 8.75.
Found: C, 72.09; H, 8.52.
2.1.11. 2-Methoxy-3-methoxymethoxyestra-1,3,5(10)-
trien-17ˇ-yl-tosylate
(14)
Under nitrogen, p-toluenesulfonic anhydride (97%, 5.5 g,
16.3 mmol) was added to a solution of the 17-hydroxy com-
pound (13, 4.74 g, 13.7 mmol) in anhydrous pyridine (80 ml) at
0 ◦C and stirred for 2 h. Analysis by TLC (5% acetone/CH2Cl2)
indicated a complete reaction. The reaction mixture was
quenched with water and extracted with ethyl acetate (3×).
The organic phase was washed with cold 2 M HCl, water,
cold saturated NaHCO3 solution, brine, dried over Na2SO4
and concentrated in vacuo. Purification by flash column (2%
acetone/CH2Cl2) and crystallization from acetone/hexanes
afforded the 17-tosylate (14, 6.7 g, 95%) as a white solid.
mp = 115 ◦C; FT-IR (ATR) ꢀmax: 2969, 2922, 2868, 1598, and
1515 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.83 (s, 18-CH3),
2.46 (s, OTs-CH2), 3.50 (s, 3-OCH2OCH3), 3.84 (s, 2-OCH3), 4.36
(t, J = 8.1 Hz, 17-H), 5.18 (s, 3-OCH2OCH3), 6.79 (s, 4-H), 6.85 (s,
1-H), 7.34 (d, J = 3.9 Hz, OTs-Ar-H), 7.80 (d, J = 4.0 Hz, OTs-Ar-H).
Anal. Calcd for C28H36O6S: C, 67.17; H, 7.25; S, 6.40. Found: C,
67.02; H, 6.98; S, 6.35.
2.1.14. 2-Methoxy-17-ethynylestra-1,3,5(10),16-tetraen-3-
ol
(17)
Under nitrogen, the crude 21-trimethylsilyl mixture (16, 0.85 g,
∼2 mmol) was dissolved in 15 ml of THF. Tetrabutylammo-
nium fluoride (1 M/THF, 3.7 ml, 3.7 mmol) was added and
the reaction stirred at room temperature for 2 h. The reac-
tion was quenched with water and extracted with ethyl
acetate (3×). The organic phase was washed with water,
brine, dried over Na2SO4, and concentrated in vacuo to give
0.65 g of a brown foam. The residue was twice purified by
flash column (1% acetone/CH2Cl2), then (7:3 CH2Cl2/hexanes)
to give 120 mg of material. The material was twice crystallized
from acetone/hexanes to give the pure product (17, 40 mg,
6.4%). mp = 202 ◦C; FT-IR (ATR) ꢀmax:3490, 3293, 2932, 1591, and
1504 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 0.895 (s, 18-CH3),
1.549 (s, 17-CCH), 3.874 (s, 2-OCH3), 6.158 (t, J = 2.55 Hz, 16-H),
6.658 (s, 4-H), 6.794 (s, 1-H). Anal. Calcd for C21H24O2: C, 81.78;
H, 7.84. Found: C, 81.73; H, 7.97.
2.1.12. 2-Methoxy3-hydroxy-17-methylgona-
1,3,5(10),13(17)-tetraene
(15)
Under nitrogen, the 17-tosylate derivative (14, 6.23 g,
12.5 mmol) in benzene (125 ml) was added dropwise to a
solution of ethylmagnesium bromide (3M in ether, 53 ml,
159 mmol). Once the addition was complete, the ether
was distilled off, the solution refluxed for 3 h, then stirred
overnight at room temperature. The reaction was cooled to
0 ◦C, quenched with the dropwise addition of cold 2 M H2SO4,
and extracted with ethyl acetate (3×). The organic phase was
washed with cold saturated NaHCO3 solution, water, brine,
dried over Na2SO4 and concentrated in vacuo. Purification by
flash column (2% acetone/CH2Cl2) and crystallization from
acetone/hexanes afforded the17-methyl product (15, 2.86 g,
79%) as a white solid. mp = 108 ◦C; FT-IR (ATR) ꢀmax: 3444,
2911, 2855, 2832, 1591, and 1505 cm−1. NMR (300 MHz, CDCl3),
ı (ppm): 1.65 (s, 17-CH3), 3.87 (s, 2-OCH3), 5.42 (s, 3-OH), 6.64 (s,
4-H), 6.83 (s, 1-H). Anal. Calcd for C19H24O2: C, 80.24; H, 8.51.
Found: C, 79.86; H, 8.14.
2.1.15. 17,17a-Dimethyl-3-methoxy-D-homogona-
1,3,5(10),13,15,17(17a)-hexaene
(19)
Compound (19) was prepared from mestranol (18) by reacting
with formic acid as reported in the literature [11].
2.1.16. 17,17a-Dimethyl-3-hydroxy-D-homogona-
1,3,5(10),13,15,17(17a)-hexaene
(20)
Under nitrogen, the 3-methoxy derivative (19, 1.0 g, 3.41 mmol)
was dissolved in toluene (10 ml) and treated with DIBAL
(1 M/toluene, 17 ml, 17 mmol) at 95 ◦C overnight. Analysis by
TLC (5% acetone/CH2Cl2) showed a complete reaction. The
reaction mixture was quenched with 1:1 ethanol/water, the
resulting precipitate was dissolved with 5% HCl and extracted
with ethyl acetate (3×). The organic phase was washed
with water, brine, dried over Na2SO4, and concentrated
in vacuo. Purification by flash column (2% acetone/CH2Cl2)
gave the 3-hydroxy compound (20, 0.73 g, 76%) as a white
solid. mp = 177 ◦C; FT-IR (ATR) ꢀmax: 3372, 2918, 2828, and
1607 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 2.186 (s, 17-H),
2.30 (s, 17␣-H), 4.82, (s, 3-OH), 6.62 (d, J = 2.7 Hz, 4-H), 6.68
(dd, J1 = 8.1 Hz, J2 = 3.0 Hz, 2-H), 7.05 (d, J = 9.6 Hz, 1-H), 7.20
(d, J = 8.1 Hz, 15-CH3), 7.28 (d, J = 8.4 Hz, 16-H). Anal. Calcd for
2.1.13. 2-Methoxy-3-methoxymethoxy-21-trimethylsilyl-
19-norpregna-1,3,5(10),16-tetraene-20-yne (16a) and
2-methoxy-3-hydroxy-21-trimethylsilyl-19-norpregna-
1,3,5(10),16-tetraene-20-yne
(16b)
Under nitrogen, a solution of trimethylsilylacetylene (1 ml,
7 mmol) in THF (10 ml) was cooled to −78 ◦C. Butyl lithium
(1.6 M in hexanes, 4.4 ml, 7 mmol) was slowly added and the
mixture stirred at −78 ◦C for 10 min, and then allowed to warm
to 0 ◦C in an ice bath. After stirring at 0 ◦C for 1/2 h, the mix-
ture was cooled again to −78 ◦C and transferred to a solution
of the ketone (5, 1 g, 2.9 mmol) in 12 ml of 2:1 THF/benzene,
also at −78 ◦C. The reaction mixture was stirred for 15 min

steroids 7 3 ( 2 0 0 8 ) 171–183
175
C20H22O·(1/10)CH2Cl2: C, 84.15; H, 7.80. Found: C, 84.08; H, 7.86.
(7:3 ether/hexanes) showed a complete reaction. The reaction
mixture was quenched with water and extracted with ethyl
acetate (3×). The organic phase was washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. Purification by
flash column (7:3 ether/hexanes) gave the 2-methoxy deriva-
tive (23, 0.49 g, >100%) as a white foam. mp = 82 ◦C; FT-IR (ATR)
ꢀmax: 2917, 2857, 1608 and 1514 cm−1. NMR (300 MHz, CDCl3), ı
(ppm): 2.17 (s, 17-H), 2.28 (s, 17␣-H), 3.52 (s, 3-OCH2OCH3), 3.89
(s, 2-OCH3), 5.21 (m, 3-OCH2OCH3), 6.94 (s, 4-H), 6.95 (s, 1-H),
7.03 (d, J = 8.7 Hz, 15-CH3), 7.20 (d, J = 8.1 Hz, 16-H). Anal. Calcd
for C23H28O3: C, 78.38; H, 8.01. Found: C, 78.07; H, 8.08.
2.1.17. 17,17a-Dimethyl-3-methoxymethoxy-D-homogona-
1,3,5(10),13,15,17(17a)-hexaene
(21)
Under nitrogen, the 3-hydroxy compound (20, 0.9 g,
3.23 mmol) was dissolved in 15 ml of dry THF. Chloromethyl
methyl ether (1.25 ml, 16.5 mmol) and diisopropylethy-
lamine (3.4 ml, 19.5 mmol) were added and the reaction
mixture heated to 65 ◦C overnight. Analysis by TLC (2%
acetone/CH2Cl2) showed a complete reaction. The reaction
mixture was cooled to room temperature, quenched with
20% NH4Cl solution, and extracted with ethyl acetate (3×).
The organic phase was washed with water (3×), brine, dried
over Na2SO4, and concentrated in vacuo. Purification by flash
column (1.5% acetone/CH2Cl2) gave the 3-methoxymethoxy
ether product (21, 0.8 g, 77%) as a white solid. mp = 102 ◦C;
2.1.20. 17,17a-Dimethyl-2-methoxy-3-hydroxy-D-
homogona-1,3,5(10),13,15,17(17a)-hexaene
(24)
Under nitrogen, the 3-methoxymethoxy ether (23, 0.46 g,
1.3 mmol) was hydrolyzed with 6 M HCl (15 ml) in THF (20 ml)
at room temperature over the weekend. Analysis by TLC
(7:3 ether/hexanes) indicated a complete reaction. The reac-
tion mixture was quenched with water and extracted with
ethyl aceate (3×). The organic phase was washed with water,
brine, dried over Na2SO4, and concentrated in vacuo. Purifi-
cation by flash column (1% acetone/CH2Cl2) and subsequent
crystallization from ether gave the 3-hydroxy derivative (24,
FT-IR (ATR) ꢀmax
: . NMR
2953, 2826, 1613 and 1575 cm−1
(300 MHz, CDCl3), ı (ppm): 2.16 (s, 17-H), 2.27 (s, 17␣-H), 3.47 (s,
3-OCH2OCH3), 5.18 (m, 3-OCH2OCH3), 6.84 (d, J = 2.4 Hz, 4-H),
6.88 (dd, J1 = 8.7 Hz, J2 = 3.0 Hz, 2-H), 7.02 (d, J = 8.1 Hz, 1-H), 7.18
(d, J = 8.4 Hz, 15-CH3), 7.30 (d, J = 8.4 Hz, 16-H). Anal. Calcd for
C22H26O2·(1/4)H2O: C, 80.82; H, 8.17. Found: C, 80.96; H, 7.97.
2.1.18. 17,17a-Dimethyl-2-hydroxy-3-methoxymethoxy-D-
homogona-1,3,5(10),13,15,17(17a)-hexaene
(22)
0.35 g, 87%) as a white solid. mp = 177 ◦C; FT-IR (ATR) ꢀmax
:
3511, 2920, 2841, 1592, and 1514 cm−1. NMR (300 MHz, CDCl3),
ı (ppm): 2.19 (s, 17-H), 2.30 (s, 17␣-H), 3.91 (s, 2-OCH3), 5.48 (s,
3-OH), 6.72 (s, 4-H), 6.91 (s, 1-H), 7.05 (d, J = 8.1 Hz, 15-CH3), 7.22
(d, J = 7.8 Hz, 16-H). Anal. Calcd for C21H24O2: C, 81.78; H, 7.84.
Found: C, 81.05; H, 7.75.
Under nitrogen, the 3-methoxymethoxy ether (21, 0.8 g,
2.48 mmol) was dissolved in 20 ml of dry THF and chilled
to -78 ◦C. To this was added sec-BuLi (1.4 M/cyclohexane,
3.5 ml, 4.9 mmol) such that the temperature did not exceed
−65 ◦C and the reaction mixture was stirred at −78 ◦C for
3 h. Trimethyl borate (1.1 ml, 9.9 mmol) was then added such
that the temperature did not exceed −65 ◦C and the mixture
stirred for 1 h at −78 ◦C. The reaction mixture was quenched
with 15 ml of 20% NH4Cl solution, allowed to come to room
temperature, and stirred for 1 h. Sodium perborate tetrahy-
drate (1.5 g, 9.7 mmol) was then added at such a rate that
the temperature did not exceed 35 ◦C and the reaction mix-
ture was stirred at room temperature overnight. The reaction
mixture was quenched with water and extracted with ethyl
acetate (3×). The organic phase was washed with water, brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by flash column (8:2 CH2Cl2/hexanes) to give the 2-
hydroxy compound (22, 0.26 g, 31%) as a white solid. mp = 145
2.1.21. 2-Methoxy-3-acetoxy-17-methylgona-
1,3,5(10),13(17)-tetraene
(25)
Under nitrogen, the 3-hydroxy compound (15, 14.2 g, 50 mmol)
was acetylated in pyridine (250 ml) with acetic anhydride
(60 ml, 634.8 mmol) at room temperature overnight. Analysis
by TLC (1% acetone/CH2Cl2) indicated a complete reaction. The
reaction mixture was quenched with methanol and concen-
trated in vacuo to give the 3-acetate derivative (25, 17.5 g, 100%)
as a yellow solid. This material was used in the subsequent
reaction without further purification. mp = 91 ◦C; FT-IR (ATR)
ꢀmax: 2924, 2837, 1762, 1615, and 1508 cm−1. NMR (300 MHz,
CDCl3), ıH (ppm): 1.6 (s, 17-CH3), 2.30 (s, 3-OAc), 3.80 (s, 2-
OCH3), 6.72 (s, 4-H), 6.92 (s, 1-H). Anal. Calcd for C21H26O3: C,
77.27; H, 8.03. Found: C, 77.20; H, 7.96.
- 146 ◦C; FT-IR (ATR) ꢀmax: 3354, 2949, 1618, and 1508 cm−1
.
NMR (300 MHz, CDCl3), ı (ppm): 2.16 (s, 17-H), 2.27 (s, 17␣-H),
3.50 (s, 3-OCH2OCH3), 5.15 (m, 3-OCH2OCH3), 6.85 (s, 1-H), 7.02
(d, J = 7.8 Hz, 15-CH3), 7.17 (d, J = 8.4 Hz, 16-H). Anal. Calcd for
C22H26O3·(1/3)H2O: C, 76.73; H, 7.80. Found: C, 76.76; H, 7.59.
2.1.22. (8s,4bS,8aR)-3-Methoxy-7-oxo-8-(3-oxobutyl)-
5,6,8,9,10,4b,8a-heptahydrophenanthra-2-yl acetate
(26)
Under nitrogen, the 3-acetate derivative (25, 2.0 g, 6.1 mmol) in
3:1 dioxane/water (10 ml) was treated with a solution of OsO4
(6.2 mg, 0.024 mmol) in t-BuOH (2 ml). Sodium periodate (0.52 g,
2.43 mmol) and pyridine (0.1 ml, 1.24 mmol) were added and
the mixture was heated at 65–70 ◦C for 3 h and then stirred at
room temperature overnight. Analysis by TLC (CH2Cl2) indi-
cated an incomplete reaction. Additional OsO4 in t-BuOH (3 mg
in 1 ml, 0.012 mmol) was added and the reaction heated at
65–70 ◦C for 4 h. Analysis by TLC showed disappearance of
the starting material. The reaction mixture was cooled to
2.1.19. 17,17a-Dimethyl-2-methoxy-3-methoxymethoxy-D-
homogona-1,3,5(10),13,15,17(17a)-hexaene
(23)
To a solution of the 2-hydroxy (22, 0.46 g, 1.36 mmol) in
DMF (20 ml) was added potassium carbonate (1.8 g, 13 mmol)
and tetrabutylammonium iodide (30 mg, .08 mmol) and the
reaction mixture stirred for 15 min. Methyl iodide (1.9 ml,
30.5 mmol) was added and the mixture stirred at 45 ◦C for 4 h
then stirred overnight at room temperature. Analysis by TLC

176
steroids 7 3 ( 2 0 0 8 ) 171–183
room temperature, quenched with water, and extracted with
EtOAc (3×). The organic fractions were washed with water (3×)
and brine, combined, dried over Na2SO4, filtered, and con-
centrated in vacuo. Purification by flash chromatography (5%
acetone/CH2Cl2) gave compound (26, 0.9 g, 41%) as a white
solid. mp = 111–112 ◦C; FT-IR (ATR) ꢀmax: 2950, 2863, 1760, 1704,
1615 and 1509 cm−1. NMR (300 MHz, CDCl3), ıH (ppm): 2.14 (s,
3-oxobutyl CH3), 2.30 (s, 2-OAc), 3.80 (s, 3-OCH3), 6.76 (s, 1-H),
6.89 (s, 4-H). Anal. Calcd for C21H26O5: C, 70.37; H, 7.31. Found:
C, 70.44; H, 7.38.
OAc), 3.84 (s, 3-OCH3), 5.13 (s, 8-OH), 6.59 (d, J = 2.7 Hz, 7-H),
6.62 (dd, J1 = 8.25 Hz, J2 = 3.0 Hz, 9-H), 6.80 (s, 1-H), 6.96 (s, 4-H),
7.20 (d, J = 8.4 Hz, 10-H). Anal. Calcd for C21H22O4: C, 74.54; H,
6.55. Found: C, 74.11; H, 6.58.
2.1.26. (4bS,10bR)-3-Methoxy-5,6,11,12,10b,4b-
hexahydrochrysene-2,8-diol
(30)
Under nitrogen, the 3-acetate compound (29, 0.17 g, 0.5 mmol)
was hydrolyzed in MeOH/H2O (60 ml, 5:1) with potassium car-
bonate (1.9 g, 13.7 mmol) at room temperature for 2 h. Analysis
by TLC (5% acetone/CH2Cl2) indicated a complete reaction.
The reaction mixture was quenched with water and extracted
with EtOAc (3×). The organic fractions were washed with
water (3×) and brine, combined, dried over Na2SO4, filtered,
and concentrated in vacuo. Purification by flash chromatogra-
phy (5% acetone/CH2Cl2) and subsequent crystallization from
ether/hexanes gave compound (30, 130 mg, 88%) as a white
solid. mp = 225 ◦C; FT-IR (ATR) ꢀmax: 3458, 3312, 2922, 2829, 1614,
1584, 1497, and 1463 cm−1. NMR (300 MHz, CDCl3), ıH (ppm):
3.89 (s, 3-OCH3), 4.74 (s, 2-OH), 5.49 (8-OH), 6.63 (d, J = 3.0 Hz, 7-
H), 6.68 (dd, J1 = 8.4 Hz, J2 = 3.0 Hz, 9-H), 6.71 (s, 1-H), 6.87 (s, 4-H),
7.25 (d, J = 8.4 Hz, 10-H). Anal. Calcd for C19H20O3·(1/2)H2O: C,
74.73; H, 6.93. Found: C, 74.82; H, 6.70.
2.1.23. (10bS,4aS,4bS)-8-Hydroxy-9-methoxy-
3,4,5,6,11,12,10b,4a,4b-nonahydrochrysen-2-one
(27)
Under nitrogen, the diketone compound (26, 3 g, 8.4 mmol)
was cyclized in methanol (100 ml) with 10% KOH solution
(40 ml) at reflux for 2.5 h. Analysis by TLC (5% acetone/CH2Cl2)
indicated a complete reaction. The reaction mixture was
quenched with water, acidified with 10% HCl, concentrated
in vacuo, and extracted with EtOAc (3×). The organic fractions
were washed with water (3×) and brine, combined, dried over
Na2SO4, filtered, and concentrated in vacuo to give the cyclized
compound (27, 2.4 g, 99%) as a yellow solid. mp = 211–213 ◦C;
FT-IR (ATR) ꢀmax: 3267, 2923, 2863, 1658, and 1505 cm−1. NMR
(300 MHz, CDCl3), ıH (ppm): 3.86 (s, 9-OCH3), 5.91 (s, 1-H) 6.64 (s,
7-H), 6.78 (s, 10-H). Anal. Calcd for C19H22O3: C, 76.48; H, 7.43.
Found: C, 76.48; H, 7.48.
2.1.27. (4bS,10bR)-2,8-bis(Methoxymethoxy)-3-methoxy-
5,6,11,12,10b,4b-hexahydrochrysene
(31)
2.1.24. (10aS,10bS,4bS)-3-Methoxy-8-oxo-
5,6,9,10,11,12,10a,10b,4b-nonahydrochrysen-2-yl acetate
(28)
Under nitrogen, the 2,8-diol derivative (30, 1 g, 3.4 mmol)
in THF (100 ml) was treated with N,N-diisopropylethylamine
(2.1 ml, 5.75 mmol) and chloromethyl methyl ether (6 ml, 79
mmol) and the reaction mixture stirred at 65–70 ◦C overnight.
Analysis by TLC (5% acetone/CH2Cl2) indicated a complete
reaction. The reaction mixture was cooled to room temper-
ature, quenched with 20% NH4Cl solution, concentrated in
vacuo, and extracted with EtOAc (3×). The organic fractions
were washed with water (3×) and brine, combined, dried
over Na2SO4, filtered, and concentrated in vacuo. Purifica-
tion by flash chromatography (5% acetone/CH2Cl2) gave the
dimethoxymethyl ether derivative (31, 1.1 g, 88%) as a low
melting solid. mp = 83–84 ◦C; FT-IR (ATR) ꢀmax: 2908, 2834, 1606,
and 1498 cm−1. NMR (300 MHz, CDCl3), ıH (ppm): 3.48 (s, 8-
OCH2OCH3), 3.52 (s, 2-OCH2OCH3) 3.88 (s, 3-OCH3), 5.16 (s,
8-OCH2OCH3), 5.21 (s, 2-OCH2OCH3), 6.84 (d, J = 2.4 Hz, 7-H),
6.88 (dd, J1 = 8.4 Hz, J2 = 2.7 Hz, 9-H), 6.92 (s, 1-H), 6.93 (s, 4-H).
7.30 (d, J = 8.4 Hz, 10-H). Anal. Calcd for C23H28O5: C, 71.85; H,
7.34. Found: C, 71.68; H, 7.32.
Under nitrogen, the 3-hydroxy compound (27, 0.62 g,
2.0 mmol) was acetylated in pyridine (50 ml) with acetic anhy-
dride (10 ml, 105.8 mmol) at room temperature overnight.
Analysis by TLC (3% acetone/CH2Cl2) indicated a complete
reaction. The reaction mixture was quenched with methanol
and concentrated in vacuo. Purification by flash chromatogra-
phy (3% acetone/CH2Cl2) and subsequent crystallization from
ether gave the 2-acetate derivative (28, 0.36 g, 51%) as a white
solid. mp = 139 ◦C; FT-IR (ATR) ꢀmax: 2916, 2860, 1759, 1665,
1615 and 1508 cm−1. NMR (300 MHz, CDCl3), ıH (ppm): 2.30 (s,
2-OAc), 3.81 (s, 3-OCH3), 5.90 (s, 7-H) 6.76 (s, 1-H), 6.89 (s, 4-H).
Anal. Calcd for C21H24O4·(1/5)H2O: C, 73.32; H, 7.15. Found: C,
73.37; H, 7.01.
2.1.25. (4bS,10bR)-8-Hydroxy-3-methoxy-
5,6,11,12,10b,4b-hexahydrochrysene-2-yl acetate
(29)
Under nitrogen, compound (28, 2.3 g, 5.3 mmol) in CH3CN
(150 ml) was aromatized with CuBr2 (1.4 g, 6.3 mmol) at room
temperature for 24 h. Analysis by TLC (5% acetone/CH2Cl2)
indicated a complete reaction. The reaction mixture was
quenched with water and extracted with EtOAc (3×). The
organic fractions were washed with water (3×) and brine, com-
bined, dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by flash chromatography (5% acetone/CH2Cl2)
and subsequent crystallization from ether gave the hex-
ahydrochrysene derivative (29, 1.6 g, 70%) as a white solid.
mp = 199–200 ◦C; FT-IR (ATR) ꢀmax: 3455, 2928, 2839, 1745, 1610,
and 1503 cm−1. NMR (300 MHz, CDCl3), ıH (ppm): 2.33 (s, 2-
2.1.28. (10bS,4bR)-2,8-bis(Methoxymethoxy)-9-methoxy-
5,6,11,12,10b,4b-hexahydrochrysen-3-ol
(32)
Under nitrogen, the dimethoxymethyl ether derivative (31,
0.5 g, 1.3 mmol) in anhydrous THF (10 ml) at −78 ◦C was treated
with the dropwise addition of sec-BuLi (1.4 M/cyclohexane,
1.8 ml, 2.52 mmol) and the reaction stirred for 3 h. Trimethyl
borate (0.6 ml, 5.4 mmol) was added dropwise and the reac-
tion mixture stirred for 20 min. The reaction mixture was
warmed to 0 ◦C and quenched with 20% NH4Cl solution (10 ml)
and stirred at room temperature for 1 h. Sodium perborate
tetrahydrate (0.8 g, 5.2 mmol) was added and the reaction

steroids 7 3 ( 2 0 0 8 ) 171–183
177
mixture stirred at room temperature overnight. The mixture
was extracted with EtOAc (3×), the organic fractions were
washed with water (3×) and brine, combined, dried over
Na2SO4, filtered and concentrated in vacuo. Purification by
flash chromatography (5% acetone/CH2Cl2) gave a mixture of
the starting material (0.14 g) and the 16-hydroxy derivative
(32, 0.31 g, 84%), which crystallized from ether/hexanes as a
white solid. mp = 117–119 ◦C; FT-IR (ATR) ꢀmax: 3440, 2909, 2838,
1608, and 1502 cm−1. NMR (300 MHz, CDCl3), ıH (ppm): 3.50 (s,
2-OCH2OCH3), 3.52 (s, 8-OCH2OCH3) 3.87 (s, 9-OCH3), 5.15 (s,
2-OCH2OCH3), 5.21 (s, 8-OCH2OCH3), 5.99 (3-OH), 6.86 (s, 1-H),
6.91 (d, J = 8.4 Hz, 4-H), 6.92 (s, 7-H), 6.98 (s, 10-H). Anal. Calcd
for C23H28O6: C, 69.98; H, 7.05. Found: C, 69.83; H, 6.99.
tion mixture stirred at room temperature for 2 h. Analysis by
TLC (5% acetone/CH2Cl2) indicated a complete reaction. The
reaction was slowly quenched with methanol, diluted with
water, concentrated in vacuo and extracted with ethyl acetate
(3×). The organic phase was washed with water, brine, dried
over Na2SO4, and concentrated in vacuo to give the 3-acetate
derivative (36, 0.82 g, 96%) as a white solid. mp = 84 ◦C; FT-IR
(ATR) ꢀmax: 3425, 2927, 2843,1757, 1615, and 1508 cm−1. NMR
(300 MHz, CDCl3), ı (ppm): 1.03 (s, 18-CH3), 2.31 (s, 3-OAc), 3.81
(s, 2-OMe), 4.11 (t, J = 8.2 Hz, 17-H), 5.22 (s, 15-H), 6.77 (s, 4-H),
6.93 (s, 1-H). Anal. Calcd for C21H26O4: C, 73.66; H, 7.65. Found:
C, 73.77; H, 7.59.
2.1.32. 2-Methoxy-3-acetoxyestra-1,3,5(10),14-tetraen-17-
one
2.1.29. (4bS,10bR)-2,8-bis(Methoxymethoxy)-3,9-
dimethoxy-5,6,11,12,10b,4b-hexahydrochrysene
(37)
(33)
Under nitrogen, the 17-hydroxy compound (36, 0.82 g,
2.4 mmol) was dissolved in 20 ml of acetone and chilled to
0 ◦C. Jones reagent was slowly added with stirring until the
yellow–orange color persisted (∼3 ml). The reaction was stirred
an additional five minutes then slowly quenched with iso-
propanol. The solution was concentrated in vacuo, diluted with
water, and extracted with ethyl acetate (3×). The organic phase
was washed with water, brine, dried over Na2SO4, and con-
centrated in vacuo to give the 17-ketone (37, 0.82 g, 100%) as
a yellow solid. mp = 133 ◦C; FT-IR (ATR) ꢀmax: 2966, 2938, 1762,
1734, 1615 and 1508 cm−1. NMR (300 MHz, CDCl3), ı (ppm): 1.18
(s, 18-CH3), 2.31 (s, 3-OAc), 3.81 (s, 2-OMe), 5.63 (s, 15-H), 6.79 (s,
4-H), 6.89 (s, 1-H). Anal. Calcd for C21H24O4·(1/10)H2O: C, 73.70;
H, 7.13. Found: C, 73.81; H, 7.06.
Under nitrogen, the 16-hydroxy derivative (32, 0.5 g,
1.24 mmol) in DMF (20 ml) was methylated with methyl
iodide (5 ml, 80.3 mmol) in the presence of potassium car-
bonate (2 g, 14.5 mmol) and tetrabutyl ammonium iodide
(20 mg) at room temperature overnight. Analysis by TLC (3%
acetone/CH2Cl2) indicated an incomplete reaction. Additional
methyl iodide (5 ml) was added and the reaction mixture
stirred another 2 h. Analysis by TLC indicated no further
progress. The reaction mixture was quenched with brine
and extracted with EtOAc (3×). The organic fractions were
washed with brine, combined, dried over Na2SO4, filtered, and
concentrated in vacuo. Purification by flash chromatography
(3% acetone/CH2Cl2) gave the dimethoxy compound (33,
0.38 g, 73%) as a yellow solid. mp = 151–153 ◦C; FT-IR (ATR)
ꢀmax: 2944, 2913, 2827, 1607, and 1510 cm−1. NMR (300 MHz,
CDCl3), ıH (ppm): 3.53 (s, 2,8-OCH2OCH3), 3.89 (s, 3,9-OCH3),
5.22 (s, 2,8-OCH2OCH3), 6.94 (s, 1,4,7,10-H). Anal. Calcd for
2.1.33. 2-Methoxy-3-acetoxyestra-1,3,5(10),14-tetraene-
17,17-ethylenethioketal
(38)
C
24H30O6: C, 69.55; H, 7.30. Found: C, 69.56; H, 7.23.
To a solution of the 17-ketone compound (37, 0.8 g, 2.4 mmol)
in acetic acid (35 ml) was added ethanedithiol (1.9 ml,
22.6 mmol) and boron trifluoride diethyl etherate (1.9 ml,
15.4 mmol) and the reaction mixture stirred at room tempera-
ture for ninety minutes. Analysis by TLC (5% acetone/CH2Cl2)
indicated a complete reaction. The reaction was quenched
with water and extracted with ether (3×). The organic phase
was washed with saturated NaHCO3 solution (2×), water,
brine, dried over Na2SO4, and concentrated in vacuo. Purifi-
cation by flash chromatography (CH2Cl2) gave the thioketal
derivative (38, 0.7 g, 71%) as a white foam. mp = 123 ◦C; FT-
2.1.30. (4bS,10bR)-3,9-Dimethoxy-5,6,11,12,10b,4b-
hexahydrochrysene-2,8-diol
(34)
Under nitrogen, the dimethoxymethyl ether derivative (33,
0.5 g, 1.2 mmol) in THF (30 ml) was hydrolyzed with 6M HCl
(10 ml) at room temperature overnight. Analysis by TLC (3%
acetone/CH2Cl2) indicated a complete reaction. The reaction
mixture was quenched with water and extracted with EtOAc
(3×). The organic fractions were washed with water (3×) and
brine, dried over Na2SO4, filtered, and concentrated in vacuo.
Purification by flash chromatography (5% acetone/CH2Cl2) and
subsequent crystallization from MeOH/EtOAc gave compound
(34, 0.16 g, 40%) as a white solid. mp = 295 ◦C (dec); FT-IR (ATR)
ꢀmax: 3511, 3346, 2958, 2909, 2841, 1622, and 1500 cm−1. NMR
(300 MHz, (CD3)2SO), ıH (ppm): 3.73 (s, 3,9-OCH3), 6.50 (s, 1,7-H),
6.88 (s, 4,10-H). Anal. Calcd for C20H22O4·(1/2)H2O: C, 72.60; H,
6.85. Found: C, 72.65; H, 6.67.
IR (ATR) ꢀmax: 2926, 2857, 2835, 1762, 1614, and 1508 cm−1
.
NMR (300 MHz, CDCl3), ı (ppm): 1.12 (s, 18-CH3), 2.31 (s,
3-OAc), 3.15-3.37 (m, 17-thioketal CH2’s) 3.82 (s, 2-OMe),
5.47 (s, 15-H), 6.77 (s, 4-H), 6.94 (s, 1-H). Anal. Calcd for
C23H28O3S2: C, 66.31; H, 6.77; S, 15.39. Found: C, 66.33; H, 6.83;
S, 15.62.
2.1.34. 2-Methoxyestra-1,3,5(10),14,16-pentaen-3-yl-
acetate
2.1.31. 2-Methoxy-3-acetoxyestra-1,3,5(10),14-tetraen-
17ˇ-ol
(36)
To a solution of compound (35, 0.75 g, 2.5 mmol) [3] in iso-
propanol (20 ml) was added 2 M sodium hydroxide (15 ml,
30 mmol) and acetic anhydride (0.7 ml, 7.4 mmol) and the reac-
(39)
The thioketal compound (38, 0.68 g, 1.63 mmol) in acetone
(20 ml) was added to a mixture of deactivated Raney nickel
(5 g) in acetone (50 ml) and the mixture refluxed for 8 h.
Analysis by TLC (3% acetone/CH2Cl2) indicated a complete
reaction. The reaction mixture was filtered through Celite,

178
steroids 7 3 ( 2 0 0 8 ) 171–183
and concentrated in vacuo. The residue was purified by flash
chromatography (1:1 ether/hexanes) to give the ꢁ14,16 deriva-
tive (39, 60 mg, 9.3%) as a white solid. mp = 88 ◦C. FT-IR (ATR)
ꢀmax: 2924, 2856, 1762, 1614 and 1509 cm−1. NMR (300 MHz,
CDCl3), ı (ppm): 1.08 (s, 18-CH3), 2.31 (s, 3-OAc), 3.81 (s, 2-
OMe), 5.92 (s, 15-H), 6.33 (dd, J1 = 4.8 Hz, J2 = 1.8 Hz, 16-H),
6.38 (d, J = 2.4 Hz, 17-H), 6.79 (s, 4-H), 6.92 (s, 1-H). Anal.
Calcd for C23H28O3·(1/2)H2O: C, 75.65; H, 7.56. Found: C, 75.60;
H, 7.58.
H), 6.82 (s, 1-H). Anal. Calcd for C19H22O2·(1/4)H2O: C, 79.55; H,
7.91. Found: C, 79.51; H, 7.92.
2.2.
Biological assays
Biological assays were carried out by EntreMed Inc., Rockville
MD.
2.3.
Cell cultures
Human umbilical vein endothelial cells (HUVEC) were
obtained from Clonetics (San Diego, CA), MDA-MB-231, and
U87-MG cells were obtained from ATCC (American Type
Culture Collection, Manassas, VA). HUVEC cultures were main-
tained for up to five passages in EGM (Endothelial Growth
Medium) containing bovine brain extract (Clonetics) and 1×
antibiotic-animycotic (BioWhittaker, Walkersville, MD). MDA-
MB-231, and U87-MG cells were maintained in DMEM/F12 (1:1)
containing 10% (v/v) fetal bovine serum (Hyclone Laboratories,
Logan, UT) and 1× antibiotic–antimycotic.
2.1.35. 2-Methoxy-3-hydroxyestra-1,3,5(10),14,16-
pentaene
(40)
To a solution of the 3-acetate compound (39, 50 mg, 0.15 mmol)
in 90% methanol/water (20 ml) was added potassium carbon-
ate (0.2 g, 1.45 mmol) and the reaction mixture stirred at room
temperature for 2 h. Analysis by TLC (1:1 ether/hexanes) indi-
cated a complete reaction. The reaction mixture was acidified
with 0.5 M HCl and extracted with ether (3×). The organic
phase was washed with, water, brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by flash
chromatography (1:1 ether/hexanes) to give the 3-hydroxy
2.4.
In vitro inhibition of proliferation
compound (40, 44 mg, 100%) as a clear oil. FT-IR (ATR) ꢀmax
:
3549, 2923, 2854, 1592, and 1505 cm−1. NMR (300 MHz, CDCl3),
ı (ppm): 1.08 (s, 18-CH3), 3.87 (s, 2-OMe), 5.92 (s, 15-H), 6.33 (dd,
J1 = 5.1 Hz, J2 = 1.8 Hz, 16-H), 6.37 (d, J = 2.4 Hz, 17-H), 6.70 (s, 4-
Proliferation assays were performed by evaluating detection
of DNA synthesis by the use of the 5-bromo-2ꢀ-deoxyuridine
(BrdU) cell proliferation colorimetric ELISA kit from Roche
Fig. 1 – Synthesis of 2-methoxy-D-homoestrogens.

steroids 7 3 ( 2 0 0 8 ) 171–183
179
(Indianapolis, IN) according to the manufacturer’s instruc-
Table 1 – Selected proton chemical shifts for 10, 11, and
tions. The cells were seeded at 1,000 cells/well (MDA-MB-231
and U87-MG cells, anti-tumor activity) or 3,000 cells/well
(HUVEC, anti-angiogenic activity) in a 96 well plate, allowed
to attach for 24 h and then exposed to the compound to be
tested for 48 h. IC50 values and standard deviation (S.D.) were
calculated for each assay using Sigma Plot (Systat Software
Inc., San Jose, CA). Reported IC50 values are the mean and S.D.
of a minimum of three separate assays.
12
Compound
18-CH3
H–C–OH
10
11
12
0.85
0.87
1.10
3.28
3.43
4.15
Jones reagent in acetone at 0 ◦C afforded the 17-ketone
derivative (3) in 91% yield. Hydrolysis of this material with
sodium hydroxide in THF/methanol gave 2-methoxyestrone
(4) in quantitative yield. Treatment of compound (4) with
chloromethyl methyl ether and diisopropylethylamine in THF
at 65 ◦C overnight afforded the 3-methoxymethoxy ether (5)
in 89% yield. This material was then reacted with zinc iodide
and trimethylsilylcyanide in chloroform at room temperature
overnight. Analysis by 1H NMR indicated an incomplete reac-
tion. The residue was re-reacted for 4 h and purified by flash
column. Analysis of the recovered material by 1H NMR indi-
cated the loss of the 3-methoxymethyl ether. The residue was
re-protected by reaction with chloromethyl methyl ether and
diisopropylethylamine at 65 ◦C overnight to give (6) in 40%
overall yield. Reduction of the 17␣-cyano group of (6) was
carried out with LiAlH4 in THF to give the 17␣-aminomethyl-
derivative (7) in 75% yield. Reaction of compound (7) with
sodium nitrite in aqueous acetic acid gave compounds (8 and
9) in 61% and 5.5% respectively. Treatment of compound (8)
with lithium tri-tert-butoxyaluminum hydride solution in THF
at 0 ◦C followed by hydrolysis with HCl gave products (10
and 11) in 32% and 10%, respectively. Finally compound (12)
was produced from (9) in 43% yield using the same condi-
tions.
2.5.
Computational analysis
All calculations were carried out using the MM3 forcefield
[×] as implemented by PCMODEL (Version 9.1, Serena Soft-
ware, Bloominton, IN). After initial input of basic starting
geometries, the conformational space of each compound was
explored by means of the GMMX routine (Global Energy Mini-
mization) in PCMODEL using the MM3 forcefield, and an energy
window of 20 kcal/mol. In determining the global minima, all
degrees of conformational freedom were considered includ-
ing ring conformations and rotatable bond orientations. The
overlap of energy minimized structures depicted in Fig. 6 was
obtained by deleting the hydrogen atoms of the minimized
structures and superimposing the A-ring carbon atoms of each
analog with those of 2ME2 using the RMS Fit algorithm as
implemented in Alchemy III (Tripos Inc., St. Louis, MO).
3.
Results and discussion
3.1.
Chemistry
The syntheses of the D-homo analogs of 2ME2 are out-
lined in Fig. 1 and are based on the Tiffeneau–Demjanov
rearrangement as carried out by Avery et al. [7]. Reac-
tion of 2-methoxyestradiol (1) with sodium hydroxide and
acetic anhydride in isopropanol selectively gave the 3-acetate
compound (2) in quantitative yield. Oxidation of (2) using
Stereochemistry for compounds (10), (11) and (12) were
assigned based on the NMR chemical shifts for the 18-methyls
and 17a-protons or 17-protons. This data is summarized in
Table 1. According to Bhacca and Williams [8], axial pro-
tons attached to hydroxyl-substituted carbons resonate at
Fig. 2 – Syntheses of 2-methoxy-17-methyl and 17-ethynyl steroids with unsaturated ring-D.

180
steroids 7 3 ( 2 0 0 8 ) 171–183
Fig. 3 – Synthesis of dimethylhexahydrochrysine analog.
higher field than the corresponding equatorial protons in the
epimeric alcohol. As can be seen from Table 1, this observation
is consistent with the assignment of 17a␤-OH for compound
(10) and 17a␣-OH for compound (11). These assignments agree
with those made by Hillisch et al. [9] for the corresponding
compounds as the 3-sulfamate derivatives. The assignment
of 17␤-OH for compound (12) is based on the strong downfield
shift of the 18-methyl group due to 1,3-diaxial interaction with
the 17␤-axial OH.
The syntheses of D-ring unsaturated 2ME2 analogs (13–17)
are outlined in Fig. 2. The 17-ketone (5) was converted to the
17␤-hydroxy derivative (13) by reduction with sodium boro-
hydride in 99% yield. Compound (13) was then treated with
p-toluenesulfonic anhydride in pyridine at 0o C to give the
17␤-tosylate product (14) in 95% yield. This material was then
subjected to a Miescher–Ka¨gi rearrangement following the
procedure of Engel et al. [10] by refluxing with ethyl mag-
nesium bromide in benzene to give the 18-nor-13(17)-ene
derivative (15) in 79% yield.
Compound (5) was treated with trimethylsilylacetylene
and butyl lithium at −78 ◦C in THF/benzene then stirred at
room temperature for 2 h. Once TLC confirmed the forma-
tion of the lithium intermediate, methanesulfonyl chloride
was added followed 0.5 h later with pyridine and the mixture
stirred at room temperature overnight. Subsequent extraction
and purification gave a crude mixture indicated by NMR to be
composed mainly of the 3-methoxymethyl ether (16a) and the
3-hydroxy compound (16b) which were used directly for the
next reaction. This material was then treated with tetrabuty-
lammonium fluoride at room temperature for 2 h. Multiple
purifications by flash column and subsequent crystallization
from acetone/hexanes gave the product (17) in 6.4% yield.
Apparently, the tetrabutylammonium fluoride reagent reacts
with the free phenol of compound (16b) to generate HF which
can potentially react with the acid sensitive ene-yne function
to give byproducts. In retrospect, a better preparation of com-
pound (17) could be achieved by isolation and purification of
(16a) prior to removal of the silyl group.
The synthesis of the dimethylhexahydrochrysine ana-
log (24) is outlined in Fig. 3. Compound (19) was prepared
as described by Vincze et al. [11] by refluxing mestranol
(18) in formic acid. This material was converted to the
free hydroxy derivative (20) in 76% yield by reaction with
DIBAL in toluene at 95 ◦C overnight. Treatment of (20) with
chloromethyl methyl ether and N,N-diisopropylethylamine at
65 ◦C overnight afforded the 3-methoxymethoxy ether (21)
in 77% yield. Introduction of the 2-methoxy-group was car-
ried out following the procedure of Paaren et al. [12] whereby
metallation of compound (21) was achieved by reaction with n-
BuLi in THF at −65 ◦C. Subsequent addition of trimethyl borate
and sodium perborate tetrahydrate gave the 2-hydroxy com-
pound (22) in 31% yield. Methylation of compound (22) with
methyl iodide in DMF in the presence of potassium carbonate
and tetra-n-butyl ammonium iodide at 45 ◦C for 4 h afforded
the 2-methoxy derivative (23) in quantitative yield. Hydrolysis
of compound (23) in THF with 6M HCl at room temperature
for 48 h followed by crystallization from ether gave compound
(24) in 87% yield.
The syntheses of the 2,8-dihydroxyhexahydrochrysene
derivatives (30 and 34) are outlined in Fig. 4. The 18-nor-
13(17)-ene derivative (15) was treated with acetic anhydride in
pyridine at room temperature overnight to give the 3-acetate
derivative (25) in quantitative yield. Oxidative cleavage of the
13–17 double bond was carried out following the procedure
of Wenshang et al. [13] using a catalytic amount of OsO4 in
dioxane/water/t-BuOH in the presence of excess sodium peri-
odate and pyridine to give the diketone derivative (26) in 41%
yield. Robinson annulation of diketone (26) following the pro-
cedure of Engle et al. [14] was accomplished by refluxing in
methanol with 10% KOH to give compound (27) in quanti-
tative yield. Compound (27) was converted to the 3-acetate
derivative (28) in 51% yield by reaction with acetic anhydride in

steroids 7 3 ( 2 0 0 8 ) 171–183
181
Fig. 4 – Syntheses of 2,8-dihydroxyhexahydrochrysene derivatives.
gave the 3,9-dimethoxyhexahydrochrysene-2,8-diol deriva-
tive (34) in 40% yield.
pyridine. Aromatization of compound (28) to give the chrysene
derivative (29) was carried out in 70% yield, following the pro-
cedure of Rao et al. [15] using copper II bromide in acetonitrile.
Hydrolysis of compound (29) in methanol/water with potas-
sium carbonate gave the 2,8-diol derivative (30) in 88% yield.
The diol (30) was converted to the dimethoxymethyl ether (31)
in 88% yield, by reaction with chloromethyl methyl ether and
N,N-diisopropylethylamine in THF at 65 ◦C. Introduction of a
methoxy-group at the C-9 position was carried out following
the procedure of Paaren et al. [12] as outlined above for com-
pound (22) to give the 8-hydroxy compound (32) in 84% yield.
O-Methylation of compound (32) was carried out following the
procedure used for the preparation of compound (23) to afford
the 3,9-dimethoxychrysine derivative (33) in 73% yield. Finally,
hydrolysis of compound (33) in THF with 6M HCl followed by
subsequent chromatographic purification and crystallization
The syntheses of compounds (35–40) are outlined in
Fig. 5. The 3,17␤-dihydroxy-ꢁ14 derivative (35) was prepared
as previously described [3]. This material was selectively
acetylated with acetic anhydride in isopropanol in the
presence of 2 M NaOH to give the 3-acetate compound
(36) in 96% yield. Treatment of (36) with Jones reagent
in acetone at 0 ◦C afforded the 17-ketone derivative (37)
in quantitative yield. The 17-ketone (37) was converted
to the 17-thioketal (38) in 71% yield by reaction with
ethanedithiol and boron trifluoride diethyl etherate in acetic
acid at room temperature for ninety minutes. This mate-
rial was then refluxed with deactivated Raney nickel in
acetone to give the ꢁ14,16 derivative (39) in 9.3% yield. Sim-
ple base hydrolysis of (39) with potassium carbonate in
Fig. 5 – Synthesis of 2-methoxyestra-1,3,5(10),14,15-pentaen-3-ol.

182
steroids 7 3 ( 2 0 0 8 ) 171–183
Table 2 – IC50 values for inhibition of proliferation
Compound
Structure
Inhibition of cell proliferation IC50 (␮M) S.D.
No.
HUVEC
MDA-MB-231
U87-MG
1
0.68 0.15
0.69 0.14
1.48 0.62
10
11
2.04 0.51
7.25 0.78
>100
31.90 28.98
24.75 1.91
>100
>100
12
15
0.39 0.13
5.53 1.00
0.26 0.05
19.11 4.16
14.33 10.26
17
24
0.20 0.10
0.66 0.01
0.24 0.01
1.14 0.52
0.19 0.01
0.22 0.00
0.94 0.04
0.10 0.01
30
34
40
0.18 0.11
>10
n.d.a
>10
0.31 0.04
0.49 0.40
0.59 0.24
a
Not done.
methanol/water gave the 3-hydroxy compound (40) in quan-
titative yield.
cells (HUVEC), human breast carcinoma cells (MDA-MB-231)
and human gliomablastoma cells (U87-MG). The data are pre-
sented in Table 2. The IC50 value is the concentration at which
cell proliferation is inhibited by 50%.
3.2.
Biological activity
Simple D-homologation to give compounds (10), (11) and
(12) resulted in total loss of activity in the U87-MG cell line for
all of these compounds. Compounds (10) and (11), with the
The IC50 values for inhibition of proliferation were gener-
ated in three cell types: human umbilical vein endothelial

steroids 7 3 ( 2 0 0 8 ) 171–183
183
to this model is compound (34) which has no biological activ-
ity and whose D-ring projects to the ␣-side similar to the other
two chrysene analogs (24) and (30). The reasons for the inactiv-
ity of compound (34) are unknown, although increased steric
bulk of the additional methoxy group could be an explanation.
Further investigations in this area are needed to clarify this.
r e f e r e n c e s
Fig. 6 – A-ring superimposed MM3 minimized structures
for compounds 2ME2 (red), 15 (orange), 17 (green), 24 (blue),
30 (cyan), 34 (purple), and 40 (black). Hydrogen atoms are
omitted for clarity.
[1] Seegers JC, Aveling M-L, van Aswgan CH, Cross M, Koch F,
Joubert WS. The cytotoxic effects of estradiol-17␤,
catecholestradiols and methoxyestradiols on dividing MCF-7
and HeLa cells. J Steroid Biochem 1989;32:
797–809.
D-ring hydroxyl function most resembling that of the parent
2ME2 show much less activity in the HUVEC and MDA-MB-231
cell lines compared to 2ME2 with the 17a␤-OH (10) showing
more activity than the 17a␣-OH (11). Interestingly, when the
D-ring hydroxyl group is at the 17␤-position in compound (12),
the activity is about twice that observed for 2ME2 in both the
HUVEC and the MDA-MB-231 cell lines.
[2] Fotsis T, Zhang Y, Pepper MS, Aldercreutz H, Montesano R,
Nawroth PP, et al. The endogenous oestrogen metabolite
2-methoxyestradiol inhibits angiogenesis and suppresses
tumor growth. Nature 1994;368:237–9.
[3] Rao PN, Cessac JW, Tinley TL, Mooberry SL. Synthesis and
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[4] Tinley TL, Leal RM, Randall-Hlubek DA, Cessac JW, Wilkins
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[6] Leese MP, Hejaz HAM, Mahon MF, Newman SP, Purohit A,
Reed MJ, et al. A-ring-substituted estrogen-3-O-sulfamates:
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2005;48:5243–56.
The chrysene analogs (24), (30), and (34) vary in biological
activity depending on the type and position of substituents on
ring-D. The dimethylhexahydrochrysine analog (24) is 0.6, 3.1,
and 1.6 times as active as 2ME2 in the HUVEC, MDA-MB-231,
and U87-MG cell lines respectively. It is notable that compound
(24) is comparable to 2ME2 in biological activity despite the
lack of a hydroxyl or polar group on ring-D. The chrysene ana-
log (30) is the most active compound in this series, exhibiting
3.6, 3.8, and 14.8 times the antiproliferative activity of 2ME2.
However, introduction of an additional methoxy-group at the
nine-position to give the symmetrical chrysene derivative (34)
results in a complete loss of biological activity.
The remaining new analogs (15, 17, and 40) share three
common characteristics. The D-ring of all three of these com-
pounds are five-membered, lack oxygen functionality, and
have additional unsaturation. As can be seen from Table 2,
compounds (17) and (40) are slightly more active than 2ME2 in
all three cell lines whereas compound (15) is much less active,
being 0.123, 0.036, and 0.103 times the activity of 2ME2 in the
HUVEC, MDA-MB-231, and U87-MG cell lines, respectively.
While the various ring-D modifications carried out here can
increase activity compared to 2ME2, clearly other factors play
a role. In particular, the loss of activity for compound (15) was
at first somewhat puzzling. A comparison of the MM3 mini-
mized molecular structures of compounds 2ME2, 15, 17, 24,
30, 34, and 40 is shown in Fig. 6 whereby the A-ring of com-
pounds 15, 17, 24, 30, 34, and 40 are superimposed on the
A-ring of 2ME2 via the best root mean square fit of the A-ring
carbon atoms. As can be seen from this comparison, the active
compounds (17, 24, 30 and 40) have a D-ring that is either in
line with that of 2ME2 or projects towards the ␣-side of the
molecule. In contrast, the less active compound (15) has a D-
ring that projects above that of 2ME2, towards the ␤-side of the
molecule. Prior investigation has suggested that substituents
on the ␤-side of the D-ring decrease antiproliferative activity
[5]. In the case of compound (15) the ꢁ13,17 bond projects the
entire D-ring towards the ␤-face of the molecule which could
explain its decreased antiproliferative activity. The exception
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Y. Favorskii rearrangements of ␣-halogenated
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Steroids 1993;58:220–4.
[12] Paaren HE, Duff SR. Synthesis of 2-hydroxyestradiol
derivatives. US Patent No. 6488419B1; 2002.
[13] Wensheng Y, Yan M, Ying K, Zhengmao H, Zhendong J.
Improved procedure for the oxidative cleavage of olefins by
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