2-Methoxyestradiol and analogs as novel antiproliferative agents: Analysis of three-dimensional quantitative structure-activity relationships for DNA synthesis inhibition and estrogen receptor binding

Article abstract of DOI:10.1124/mol.61.5.1053

2-Methoxyestradiol (2-MEO), a metabolite of estrogen, is an attractive lead compound for the development of novel antitumor and anti-inflammatory agents, because it embodies antiproliferative and antiangiogenic activities in one molecule. However, the aff

Full text of DOI:10.1124/mol.61.5.1053

0026-895X/02/6105-1053–1069$7.00
MOLECULAR PHARMACOLOGY
Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 61:1053–1069, 2002
Vol. 61, No. 5
1545/980502
Printed in U.S.A.
2-Methoxyestradiol and Analogs as Novel Antiproliferative
Agents: Analysis of Three-Dimensional Quantitative Structure-
Activity Relationships for DNA Synthesis Inhibition and
Estrogen Receptor Binding
RICHARD A. HUGHES, TRUDI HARRIS, EMILE ALTMANN, DAVID MCALLISTER, ROSS VLAHOS, ALAN ROBERTSON,
MARK CUSHMAN, ZHIQIANG WANG, AND ALASTAIR G. STEWART
Departments of Pharmacology (R.A.H., T.H., E.A., R.V., A.G.S.) and Chemistry (D.M.), University of Melbourne, Melbourne, Victoria, Australia;
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana (M.C., Z.W.); and Baker Medical
Research Institute, Prahran, Victoria, Australia (A.R.)
Received December 13, 2001; accepted February 11, 2002
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
2-Methoxyestradiol (2-MEO), a metabolite of estrogen, is an ships were developed for these activities using comparative
attractive lead compound for the development of novel antitu- molecular field analysis (CoMFA) techniques. Comparison of
mor and anti-inflammatory agents, because it embodies anti- optimized CoMFA models revealed distinct structural require-
proliferative and antiangiogenic activities in one molecule. ments for DNA synthesis inhibition and ER binding. For exam-
However, the affinity of 2-MEO for the estrogen receptor would ple, DNA synthesis inhibition is enhanced by electropositive
lead to undesirable side effects. As a prelude to the design of substitutions in the 2-position below the plane of the steroid
2-MEO–like compounds with an optimal activity profile, we A-ring, whereas ER binding is favored by electronegative sub-
assayed 2-MEO and a series of analogs for their ability to cause stitution in this position. Similarly, DNA synthesis inhibition
G1 cell-cycle arrest (by measuring inhibition of DNA synthesis in correlates negatively with increased steric bulk in regions clus-
human cultured airway smooth muscle) and to inhibit binding of tered around the A and B rings; changes in steric bulk in these
[3H]estradiol at the estrogen receptor (ER; from rat uterine regions has little correlation with ER binding. These observa-
smooth muscle). One compound, a diacetoxy enediol deriva- tions will guide the design of new analogs with improved po-
tive, was identified with reasonable potency for DNA synthesis tency for desired characteristics (e.g., DNA synthesis inhibition)
(pIC50 ϭ 5.97) but showed negligible affinity for the ER (pIC50 Ͻ with minimal unwanted activities (e.g., ER binding).
5). Three-dimensional quantitative structure-activity relation-
Interest in the metabolites of estrogen has grown in the ER␣ subtype (Brouchet et al., 2001; Hodgin et al., 2001).
last decade, because it has been appreciated that a number of Alternative lower affinity receptors that bind estrogen have
the actions of this sex hormone may be caused by biologically
active metabolites. For example, the glucuronides of estrogen
and its metabolites cause cholestatic jaundice (Zhu et al.,
1996), whereas the catecholestrogens are implicated in the
association between estrogen and increased incidence of cer-
tain tumors (Zhu and Conney, 1998). The cardioprotective
effects of estrogen have been ascribed to an interaction be-
tween estrogen and the high-affinity estrogen receptor (ER);
recent evidence suggests that the cardioprotective effects
of estrogen are mediated largely, if not exclusively, via the
been described, but these do not seem to interact with phys-
iological levels of estrogen, opening up the possibility that a
related ligand binds and activates these receptors under
physiological conditions (Eriksson et al., 1980). 2-Me-
thoxyestradiol (2-MEO, compound 1; Table 1), once regarded
as an inactive metabolite of estrogen, has been identified as
an inhibitor of the proliferation of a variety of cell types and
as an antiangiogenic and antitumor agent (Fotsis et al.,
1994). Inhibition of smooth muscle proliferation by 2-MEO
has given rise to suggestions that this or related compounds
may have therapeutic applications in atherosclerosis (Nishi-
gaki et al., 1995) and asthma (Stewart et al., 1999b), whereas
regulatory effects of 2-MEO on lymphocytes and leukocytes
provide a case for its use to treat rheumatoid arthritis (Jo-
This work was supported in part by AMRAD Operations Ltd, the National
Health and Medical Research Council of Australia, Cryptopharma Pty Ltd,
and contract NO1-CM67260 from the National Cancer Institute.
ABBREVIATIONS: ER, estrogen receptor; 2-MEO, 2-methoxyestradiol; CoMFA, comparative molecular field analysis; 3D-QSAR, three-dimen-
sional quantitative structure-activity relationship; THF, tetrahydrofuran; CIMS, chemical ionization mass spectrometry; DMEM, Dulbecco’s
modified Eagle’s medium; FCS, fetal calf serum; DMF, dimethyl formamide; PBS, phosphate buffered saline; PLS, partial least squares.
1053

1054
Hughes et al.
sefsson and Tarkowski, 1997). Furthermore, cytotoxic and
antiangiogenic actions of 2-MEO on various tumor cell lines
indicate that this agent has potential as an antitumor agent
(Lottering et al., 1992; Fotsis et al., 1994; Cushman et al.,
1995; Hamel et al., 1996; Cushman et al., 1997).
2-MEO has been evaluated for its antitumor potential in
murine models of tumor growth, including immunodeficient
mice bearing human tumor xenografts. Oral doses of 50 to
150 mg/kg have shown efficacy against murine angiosarcoma
(Arbiser et al., 1999), murine Meth A sarcoma, and B16
melanoma (Fotsis et al., 1994). Furthermore, in immunode-
ficient mice, the growth of human pancreatic carcinoma
(Schumacher et al., 1999) and human breast carcinoma
[MDA-MB-435 (Klauber et al., 1997)] are reduced as a result
of treatment with 2-MEO.
The precise mechanisms underlying the various actions of
2-MEO have not been elucidated. 2-MEO inhibits the depo-
lymerization of microtubules and also retards the formation
of polymeric tubulin, giving characteristics that overlap with
those of both paclitaxel (Taxol) and colchicine (D’Amato et al.,
1994; Hamel et al., 1996; Wang et al., 2000). It seems likely
that tubulin binding and consequent inhibition of mitotic
spindle function explains part of the antiproliferative action
of 2-MEO. However, we and others have described an inhib-
itory effect of 2-MEO on cell-cycle progression to S-phase
through G1, as inferred from the inhibition of DNA synthesis
in smooth muscle and in some other cell types (Nishigaki et
al., 1995; Stewart et al., 1999b, 2001). This effect does not
seem to be explained by microtubule actions, because it is not
mimicked by either paclitaxel or colchicine (T. Harris and
A. G. Stewart, unpublished observations). The extent to
which suppression of DNA synthesis rather than microtubule
dysfunction explains the potentially desirable in vivo activi-
ties of 2-MEO is uncertain. However, it is clear that in most
circumstances of the predicted therapeutic use of 2-MEO,
affinity for high-affinity ERs would be undesirable.
In the present study, we have examined the ability of
2-MEO and a series of analogs to inhibit DNA synthesis in
smooth muscle cells and to bind to high affinity ER in rat
uterine cytosol. We then used the technique of comparative
molecular field analysis (CoMFA) to generate models describ-
ing three-dimensional quantitative structure activity rela-
tionships (3D-QSARs) for these two activities. The marked
differences between the respective models suggest that there
are distinct structural requirements for DNA synthesis inhi-
bition and ER binding. The models should be valuable for
guiding the synthesis of compounds showing increased DNA
synthesis inhibitory activity without significant affinity for
ER.
Compounds
Compounds used in this study are shown in Tables 4, 5, and 6. The
syntheses of compounds 1, 2, 4, 9 to 12, 26 (Cushman et al., 1995), 5,
18 (Cushman et al., 1997), 21, 22 (Verdier-Pinard et al., 2000), and
23 to 31 (Wang et al., 2000) have been described previously. Com-
pounds 3, 16, 18, 19, and 34-39 were purchased from Sigma (St.
Louis, MO). ICI 182,780 (17) was purchased from Sapphire Bio-
science (Alexandria, NSW, Australia). The syntheses of additional
new compounds used in this study are described below.
Synthesis of 3,17␤-O-Bis(methoxymethyl)estradiol (40). A
solution of estradiol (16) (10.0 g, 36.7 mmol) in anhydrous tetrahy-
drofuran (THF, 80 ml) containing diisopropylethylamine (70 ml, 401
mmol) was stirred at 0°C under argon for 0.5 h. Methoxymethyl
chloride (25 g, 310 mmol) was introduced and the resulting reaction
mixture stirred at 60°C for 8 h. The reaction mixture was cooled to
room temperature, poured into ice water (300 ml), and extracted
with ethyl acetate (3 ϫ 200 ml). The ethyl acetate layers were
washed with saturated aqueous sodium bicarbonate (150 ml) and
brine (2 ϫ 100 ml), combined, dried over sodium sulfate, and evap-
orated to dryness. Chromatography of the residue (silica gel 230–400
mesh, ethyl acetate/hexane 1:8 by volume) gave compound 40 as a
colorless oil (11.7 g, 88.4%): 1H NMR (CDCl3) ␦ 7.20 (d, J ϭ 8.5 Hz,
1 H), 6.82 (dd, J ϭ 8.5 and 2.6 Hz, 1 H), 6.77 (d, J ϭ 2.6 Hz, 1 H), 5.14
(s, 2 H), 4.65 (d, J ϭ 1.2 Hz, 2 H), 3.61 (t, J ϭ 8.3 Hz, 1 H), 3.47 (s,
3 H), 3.37 (s, 3 H), 2.83 (m, 2 H), 2.15 (m, 5 H), 1.85 (m, 1 H), 1.40 (m,
7 H), 0.81 (s, 3 H).
Synthesis of 2-Formyl-3,17␤-O-bis(methoxymethyl)estra-
diol (41). Sec-BuLi (1.3 M in cyclohexane, 100 ml, 130 mmol) was
added dropwise to a solution of 40 (12.2 g, 33.8 mmol) in anhydrous
THF (200 ml) at Ϫ78°C under argon and the resulting mixture was
stirred at that temperature for 2.5 h. Anhydrous dimethylformamide
(DMF; 20 ml, 255 mmol) was introduced dropwise and the resulting
reaction mixture was stirred from Ϫ78°C to room temperature for
3 h. The reaction mixture was poured into an ice/water mixture (200
ml) and acetic acid (20 ml). The product was extracted with ethyl
acetate (3 ϫ 150 ml) and the ethyl acetate layers were washed with
saturated aqueous sodium bicarbonate (150 ml) and brine (2 ϫ 150
ml), combined, dried over sodium sulfate, and evaporated to dryness.
Chromatography of the residue (silica gel 230–400 mesh, ethyl ace-
tate/hexane 1:6 by volume) gave compound 41 as a colorless oil (11 g,
87%), which solidified after standing at room temperature overnight:
m.p. 89 to 92°C. 1H NMR (CDCl3) ␦ 10.46 (s, 1 H), 7.80 (s, 1 H), 6.94
(s, 1 H), 5.29 (s, 2 H), 4.83 (ABq, J ϭ 7.2 Hz, ⌬␯ ϭ 3.4 Hz, 2 H), 3.64
(t, J ϭ 8.5 Hz, 1 H), 3.54 (s, 3 H), 3.40 (s, 3 H), 2.84 (m, 2 H),
2.42–1.15 (m, 13 H), 0.81 (s, 3 H); CIMS (isobutane) m/z (relative
intensity) 445 (MHϩ, 22), 389 (100). Analysis calculated for
C
23H32O5: C, 71.11; H, 8.19. Found: C, 71.07; H, 8.48.
Synthesis of 2-Formylestradiol (42). A solution of compound 41
(370 mg, 0.95 mmol) and lithium tetrafluoroborate (1.8 g, 20 mmol)
in acetonitrile (15 ml) containing water (1 ml) was heated at 72°C
under nitrogen for 5 h. The reaction mixture was cooled to room
temperature, poured into ice-cooled water (50 ml), and extracted
with ethyl acetate (3 ϫ 30 ml). The ethyl acetate layers were washed
with brine (2 ϫ 30 ml), dried over sodium sulfate, and evaporated to
TABLE 2
Effect of 2-MEO on cell number and viability in the presence and
absence of the mitogen thrombin
Data represent the mean and S.E.M. of the number of cells/well of a six-well plate
from four experiments, each in a culture from a different donor.
TABLE 1
Effect of 2-MEO on [3H]thymidine incorporation in the presence and
absence of the mitogen thrombin
Data represent the mean and S.E.M. of four replicate incubations of one experiment
that is representative of at least 13 different donors.
Cell Number
Incubation
Nonviable Cells
%
(ϫ 10Ϫ4
)
Incubation
[3H]Thymidine Incorporation
Control
1298 Ϯ 97
3959 Ϯ 468*
1082 Ϯ 104
Control
28.5 Ϯ 1.9
31.1 Ϯ 6.4
37.6 Ϯ 4.1
31.6 Ϯ 3.3
3.4 Ϯ 0.5
2.9 Ϯ 0.5
6.5 Ϯ 0.8
6.0 Ϯ 0.6
Thrombin 0.3 U/ml
2-MEO (10 ␮M)
Thrombin ϩ 2MEO (10 ␮M)
Thrombin (0.3 U/ml)
Thrombin ϩ 2-MEO (10 ␮M)
* P Ͻ 0.05, unpaired t test compared with control.

2-Methoxyestradiol QSAR
1055
dryness. Chromatography of the residue with 10% ethyl acetate in
Synthesis of 17␤-Acetoxy-2-ethoxy-3-hydroxy-6-hydroximi-
hexane gave the desired product 42 (200 mg, 70%), which was crys- noestra-1,3,5(10)-triene (8). A solution of compound 43 (Cushman
tallized from ethanol to afford a white crystalline solid: m.p. 236 to
et al., 1997) (800 mg, 1.93 mmol) in pyridine (12 ml) was treated with
237°C literature m.p. 230–232°C (Cushman et al., 1995)]. 1H NMR hydroxylamine hydrochloride (1.07 g, 15.4 mmol). The resulting mix-
(CDCl3) ␦ 9.91 (s, 1 H), 7.58 (s, 1 H), 6.66 (s, 1 H), 3.68 (t, J ϭ 8.4 Hz, ture was stirred and heated at 100°C for 30 min. The mixture was
1 H), 2.88 (m, 2 H), 2.40 (m, 1 H), 2.18 (m, 1 H), 2.10–1.15 (m, 11 H),
0.79 (s, 3 H).
cooled to room temperature and then poured into ice water (150 ml).
The compound was extracted with ethyl acetate (3 ϫ 100 ml). The
organic layers were washed with sodium bicarbonate (100 ml), water
(2 ϫ 100 ml), and brine (2 ϫ 100 ml), dried over sodium sulfate and
evaporated to dryness. Chromatography of the residue on a silica gel
column using 30% ethyl acetate in hexane gave compound 8 (763 mg,
89%), which was crystallized from ethyl acetate-hexane to afford
yellow crystals: m.p. 226 to 227°C. 1H NMR (CDCl3) ␦ 7.47 (s, 1 H),
6.75 (s, 1 H), 4.69 (t, J ϭ 8.5 Hz, 1 H), 4.12 (q, J ϭ 6.9 Hz, 2 H), 3.17
(dd, J ϭ 4.3 and 18 Hz, 1 H), 2.40–1.20 (m, 14 H), 2.05 (s, 3 H), 1.43
(t, J ϭ 6.9 Hz, 3 H), 0.85 (s, 3 H); CIMS (isobutane) m/z (relative
intensity) 388 (MHϩ, 100), 372 (MH-H2O, 10), 328 (75). Analysis
calculated for C22H29O5N: C, 68.20; H, 7.54; N, 3.61. Found: C, 68.04;
H, 7.62; N, 3.54.
Synthesis of 2-Hydroximinomethylestradiol (13). Under ni-
trogen, hydroxylamine hydrochloride (1.1 g, 16 mmol) was added to
a solution of compound 42 (240 mg, 0.8 mmol) in pyridine (10 ml).
The resulting mixture was stirred and heated at 90°C for 1 h. The
pyridine was removed under reduced pressure at 30 to 35°C and the
residue was dissolved in an ethyl acetate (60 ml) and water (50 ml)
mixture. The ethyl acetate solution was washed with water (30 ml)
and brine (30 ml), dried over sodium sulfate, and evaporated to
dryness. Chromatography of the residue on a silica gel column using
15% ethyl acetate in methylene chloride gave compound 13 (185 mg,
73%), which crystallized from ethyl acetate-methylene chloride to
afford a white solid: m.p. 268 to 270°C. 1H NMR (CCl3D) ␦ 8.17 (s, 1
H), 7.13 (s, 1 H), 6.58 (s, 1 H), 4.86 (s, 2 H, OH), 3.66 (t, J ϭ 8.3 Hz,
1 H), 2.81 (m, 2 H), 2.35–1.1 (m, 14 H), 0.77 (s, 3 H); CIMS (isobu-
tane) m/z (relative intensity) 316 (MHϩ, 100). Analysis calculated
for C19H25O3N: C, 72.35; H, 7.99; N, 4.44. Found: C, 72.18; H, 8.22;
N, 4.35.
Synthesis of 2-Methoximinomethylestradiol (14). Methoxy-
lamine hydrochloride (1.1 g, 16 mmol) was added to a solution of
compound 42 (100 mg, 0.8 mmol) in pyridine (5 ml) under nitrogen.
The resulting mixture was stirred and heated at 90°C for 2 h. The
pyridine was removed under reduced pressure at 30 to 35°C and the
residue was dissolved in ethyl acetate (50 ml) and water (50 ml). The
ethyl acetate solution was washed with water (30 ml) and brine (30
ml), dried over sodium sulfate, and evaporated to dryness. Chroma-
tography of the residue on a silica gel column using 15% ethyl acetate
in methylene chloride gave compound 14 (99 mg, 90%), which crys-
tallized from ethyl acetate-methylene chloride to afford a white solid:
m.p. 165 to 166°C; 1H NMR (CDCl3) ␦ 9.61 (s, 0.4 H), 8.11 (s, 0.6 H),
7.03 (s, 1 H), 6.71 (s, 1 H), 3.97 (s, 2 H, OH), 3.72 (t, J ϭ 8.3 Hz, 1 H),
2.82 (m, 2 H), 2.35–1.1 (m, 16 H), 0.79 (s, 3 H); CIMS (isobutane) m/z
(relative intensity) 330 (MHϩ, 100), 312 (MHϩ-H2O, 40). Analysis
calculated for C20H27O6N⅐1/6 H2O: C, 72.26; H, 8.28; N, 4.21. Found:
C, 72.13; H, 8.38; N, 4.19.
Synthesis of B-Homo-6-aza-2-ethoxy-3,17␤-dihydroxyestra-
1,3,5(10)-trien-6-one (44). A solution of compound 8 (700 mg, 1.63
mmol) in pyridine (10 ml) was treated with tosyl chloride (686 mg,
3.6 mmol) at room temperature. The resulting mixture was stirred
for 2 h and the pyridine was removed in vacuo at ambient temper-
ature to afford the crude oxime tosylate as a residue that was used
without further purification. The residue was dissolved in a mini-
mum amount of 40% chloroform in benzene (5 ml) and the material
was applied to the top of a basic alumina column (30 ϫ 3.5 cm). The
column was eluted with chloroform-benzene (40 to 90% chloroform)
and then allowed to stand overnight. The column was washed with
methanol (200 ml) and 80% methanol in water (200 ml). The meth-
anol eluate was collected and then evaporated to dryness. Chroma-
tography of the residue on a silica gel column using ethyl acetate
gave the ring-expanded lactam 44 (267 mg, 47%), which was crys-
tallized from ethyl acetate to afford white crystals: m.p. 222 to 223°C.
IR (KBr) 3336, 2931, 1661, 1514, 1469, 1440, 1371, 1320, 1243, 1119,
1046 cm–1 1H NMR (CDCl3) ␦ 7.31 (brs, 1 H), 6.74 (s, 1 H), 6.58 (s,
;
1 H), 4.12 (q, J ϭ 6.7 Hz, 2 H), 3.75 (t, J ϭ 8.5 Hz, 1 H), 2.41 (m, 2
H), 2.15–1.22 (m, 13 H), 1.44 (t, J ϭ 7 Hz, 3 H), 0.82 (s, 3 H); CIMS
(isobutane) m/z (relative intensity) 346 (MHϩ, 100), 328 (MH-H2O,
20). Analysis calculated for C20H27O4N⅐1/3 H2O: C, 68.35; H, 7.93; N,
3.98. Found: C, 68.25; H, 8.03; N, 3.82.
Synthesis of 2-Formylestradiol Hydrazone (15). Hydrazine
dihydrochloride (594 mg, 16 mmol) was added to a solution of com-
pound 42 (85 mg, 0.8 mmol) in pyridine (5 ml) under nitrogen. The
resulting mixture was stirred and heated at 90°C for 2 h. The
pyridine was removed under reduced pressure at 30 to 35°C and the
residue was suspended in a mixture of ethyl acetate (50 ml) and
saturated aqueous NaHCO3 (50 ml). The yellow solid was collected,
washed with water and ethyl acetate, and dried under vacuum.
Recrystallization from THF-methylene chloride gave compound 15
(62 mg, 70%): m.p. 340°C. 1H NMR (CDCl3) ␦ 10.9 (s, 0.4 H), 8.89 (s,
0.6 H), 7.53 (s, 1 H), 6.65 (s, 1 H), 3.54 (t, J ϭ 8.3 Hz, 1 H), 2.81 (m,
2 H), 2.35–1.1 (m, 17 H), 0.69 (s, 3 H).
Synthesis of B-homo-6-aza-2-ethoxy-3,17␤-dihydroxyestra-
1,3,5(10)-triene (45). A solution of borane in THF (1.0 M, 5.6 ml, 5.6
mmol) was added dropwise by syringe to a solution of compound 44
(97.8 mg, 0.28 mmol) in THF (5 ml) under argon at room tempera-
ture. The resulting solution was stirred for 6 h and then at gentle
reflux for 4 h. The mixture was cooled and 6 N hydrochloric acid (2
ml) was added slowly through a pipette. The solvent was removed
under reduced pressure and the residue was dissolved in ethyl ace-
tate (60 ml). The ethyl acetate solution was washed with sodium
bicarbonate (30 ml) and brine (2 ϫ 20 ml), dried over sodium sulfate
and evaporated to dryness. Chromatography of the residue on a
silica gel column using 50% ethyl acetate in methylene chloride gave
compound 45 (85 mg, 90%), which formed a white stable foam when
it was evaporated from methylene chloride solution under reduced
pressure. 1H NMR (CDCl3) ␦ 6.70 (s, 1 H), 6.28 (s, 1 H), 4.00 (q, J ϭ
6.9 Hz, 2 H), 3.68 (t, J ϭ 8.5 Hz, 1 H), 3.33 (t, J ϭ 6.9 Hz, 1 H), 2.82
(t, J ϭ 6.9 Hz, 1 H), 2.55 (dt, J ϭ 8.5 and 2 Hz, 1 H), 1.20–2.20 (m,
15 H), 1.34 (t, J ϭ 7.0 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z
(relative intensity) 332 (MHϩ, 100), 314 (MH-H2O, 60). Analysis
calculated for C20H29O3N⅐1/4 H2O: C, 71.50; H, 8.85; N, 4.14. Found:
C, 71.64; H, 8.90; N, 4.29.
TABLE 3
Effects of thrombin and 2-MEO on cell-cycle distribution of human
airway smooth muscle cells (n ϭ 13).
Data are presented as mean Ϯ S.E.M. (n ϭ 13).
Cell Cycle Distribution
Incubaton
G0/G1
S
G2/M
%
Control
77 Ϯ 3
69 Ϯ 3*
69 Ϯ 4*
9 Ϯ 2
16 Ϯ 3*
9 Ϯ 2†
14 Ϯ 2
15 Ϯ 1
22 Ϯ 3*†
Thrombin (0.3 U/ml)
Thrombin (0.3 U/ml) ϩ 2-MEO
(10 ␮M)
Synthesis of B-Homo-6-aza-3,17␤,6a-triacetyl-2-ethoxyestra-
1,3,5(10)-triene (46). Under nitrogen, acetic anhydride (1.83 g, 1.7 ml,
17.65 mmol) was added to a solution of compound 45 (146 mg, 0.44
mmol) in pyridine (5 ml). The resulting mixture was stirred at room
temperature for 24 h and then poured into ice water (30 ml). The
* P Ͻ 0.05 by Bonferroni post hoc test compared with corresponding value in
control incubation.
† P Ͻ 0.05 Bonferroni post hoc test thrombin ϩ 2-MEO compared with thrombin.

1056
Hughes et al.
compounds were extracted with ethyl acetate (3 ϫ 50 ml) and the phy of the residue on a silica gel column using 20% ethyl acetate in
organic layers were washed with water (30 ml), saturated aqueous hexane gave compound 48 (1.15 g, 90%), which crystallized from
sodium bicarbonate (30 ml) and brine (30 ml), dried over sodium sulfate, ethyl acetate-hexane to afford white crystals: m.p. 148 to 150°C. 1H
and evaporated to dryness. Chromatography of the residue on a silica NMR (CDCl3) ␦ 7.03 (s, 1 H), 6.88 (s, 1 H), 5.78 (s, 1 H), 4.71 (t, J ϭ
gel column using 33% ethyl acetate in methylene chloride gave com- 8.5 Hz, 1 H), 4.09 (q, J ϭ 6.9 Hz, 2 H), 2.51–1.36 (m, 11 H), 2.29 (s,
pound 46 (169 mg, 84%), which formed a white stable foam when it was 3 H), 2.05 (s, 3 H), 1.35 (t, J ϭ 6.0 Hz, 3 H), 0.82 (s, 3 H); CIMS
evaporated from ethyl acetate/hexane solution under reduced pressure: (isobutane) m/z (relative intensity) 547 (MHϩ, 100). Analysis calcu-
1H NMR (CDCl3) ␦ 6.87 (s, 1 H), 6.78 (s, 1 H), 4.66 (t, J ϭ 8.5 Hz, 1 H), lated for C25H29O8F3S⅐1/2 ethyl acetate: C, 54.94; H, 5.63; F, 9.65.
4.09 (q, J ϭ 6.9 Hz, 2 H), 4.08 (t, J ϭ 6.9 Hz, 1 H), 3.09 (m, 1 H), 2.46
(dt, J ϭ 8.5 and 2 Hz, 1 H), 2.40 (s, 3 H), 2.28 (s, 3 H), 2.20–1.25 (m, 12
Found: C, 55.24; H, 5.39; F, 9.43.
Synthesis of 3,17␤-Diacetoxy-2-ethoxy-7-hydroxy-6-oxoestra-
H), 1.93 (s, 3 H), 1.43 (t, J ϭ 6.8 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) 1,3,5(10)-triene (6). A solution of chromium trioxide (484 mg, 4.84
m/z (relative intensity) 458 (MHϩ, 100), 398 (35). Analysis calculated mmol) on 90% glacial acetic acid (10 ml) was added dropwise at 12 to
for C26H35O6N: C, 68.25; H, 7.71; N, 3.06. Found: C, 68.36; H, 7.90; N,
3.43.
15°C to a solution of compound 43 (500 mg, 1.21 mmol) in glacial acetic
acid (15 ml). The resulting mixture was stirred at 12 to 15°C for 3 h. The
Synthesis of B-homo-6-aza-6-acetyl-2-ethoxy-3,17␤-dihy- mixture was poured into an ice/water mixture (150 ml) and the product
droxyestra-1,3,5(10)-triene (47). A solution of compound 46 (150 mg, was extracted with ethyl acetate (3 ϫ 100 ml). The combined organic
0.33 mmol) in methanol (15 ml) was deoxygenated by bubbling a slow payer was washed with water (2 ϫ 100 ml), sodium bicarbonate (2 ϫ
stream of nitrogen through it for 30 min. A similarly deoxygenated 1 M 100 ml) and brine (2 ϫ 100 ml), dried over sodium sulfate and evapo-
solution of sodium hydroxide in water (3.2 ml, 3.2 mmol) was added and
the mixture was stirred at room temperature for 4.5 h. The reaction
rated to dryness. Chromatography of the residue on a silica gel column
using 30% ethyl acetate in hexane gave compound 6 (185 mg, 36%),
mixture was adjusted to pH 6–7 with acetic acid and the solvents were which was crystallized from ethyl acetate-hexane to afford yellow crys-
removed under reduced pressure. The residue was dissolved in ethyl tals: m.p. 188 to 190°C. 1H NMR (CDCl3) ␦ 7.75 (s, 1 H), 6.95 (s, 1 H),
acetate (60 ml) and the organic layer was washed with water (30 ml), 4.75 (t, J ϭ 8.5 Hz, 1 H), 4.10 (q, J ϭ 6.9 Hz, 2 H), 2.71 (q, J ϭ 4 Hz, 1H),
saturated aqueous sodium bicarbonate (30 ml) and brine (30 ml), dried 1.30–2.41 (m, 19 H), 0.82 (s, 3 H); CIMS (isobutane) m/z (relative
over sodium sulfate, and evaporated to dryness. Chromatography of the intensity) 431 (MHϩ, 100), 413 (MH-H2O, 50). Analysis calculated for
residue on a silica gel column using ethyl acetate gave compound 47
C24H30O7: C, 66.96; H 7.02. Found: C, 66.92; H 6.90.
(105 mg, 86%), which crystallized from ethyl acetate-hexane to afford
Synthesis of 3,17␤-Diacetoxy-2-ethoxyestra-1,3,5(10),6-
white crystals: m.p. 189 to 191°C. 1H NMR (CDCl3) ␦ 6.77 (s, 1 H), 6.67 tetraene (33). Under argon, 96% formic acid (0.31 ml, 8 mmol)
(s, 1 H), 4.09 (q, J ϭ 6.9 Hz, 2 H), 4.08 (t, J ϭ 6.9 Hz, 1 H), 3.71 (t, J ϭ was added to a stirred solution of compound 48 (1.1 g, 2.07 mmol),
8.5 Hz, 1 H), 3.03 (m, 1 H), 2.40 (dt, J ϭ 8.5 and 2.0 Hz, 1 H), 2.12–1.15 palladium acetate (18 mg, 0.08 mmol), triphenylphosphine (42 mg,
(m, 14 H), 1.87 (s, 3 H), 1.43 (t, J ϭ 7.2 Hz, 3 H), 0.78 (s, 3 H); CIMS 0.16 mmol), and triethylamine (1.68 ml, 12 mmol) in anhydrous
(isobutane) m/z (relative intensity) 374 (MHϩ, 100). Analysis calcu- DMF (8 ml). The mixture was stirred for 3 h at 60 to 62°C and then
lated for C22H31O4N⅐1/3 H2O: C, 69.75; H, 8.41; N, 3.69. Found: C, cooled to room temperature, diluted with brine (100 ml), and
69.65; H, 8.25; N, 3.87.
extracted with ethyl acetate (3 ϫ 100 ml). The organic layers were
Synthesis of B-Homo-6-aza-2-ethoxy-6a-ethyl-3,17␤-dihy- washed with brine (3 ϫ 100 ml), dried over sodium sulfate and
droxyestra-1,3,5(10)-triene (32). A solution of borane in THF (1.0 evaporated to dryness. Chromatography of the residue on a silica
M, 4.8 ml, 4.8 mmol) was added dropwise by syringe under argon at gel column using 20% ethyl acetate in hexane provided compound
room temperature to a solution of compound 47 (90 mg, 0.24 mmol) 33 (0.75 g, 89%), which was crystallized from ethyl acetate-hexane
in THF (5 ml), and the resulting solution was stirred for 4 h and then to afford white crystals: m.p. 158 to 159°C. 1H NMR (CDCl3) ␦ 6.87
at gentle reflux for 4 h. The mixture was cooled to room temperature (s, 1 H), 6.71 (s, 1 H), 6.37 (d, J ϭ 9.8 Hz, 1 H), 5.82 (d, J ϭ 9.8 Hz,
and 6 N hydrochloric acid (1 ml) was added slowly through a pipette.
1 H), 4.71 (t, J ϭ 8.5 Hz, 1 H), 4.09 (q, J ϭ 6.9 Hz, 2 H), 2.51–1.36
The solvent was removed under reduced pressure and the residue (m, 11 H), 2.28 (s, 3 H), 2.06 (s, 3 H), 1.37 (t, Jϭ 6.9 Hz, 3 H), 0.82
was neutralized with saturated aqueous sodium bicarbonate solution (s, 3 H); CIMS (isobutane) m/z (relative intensity) 399 (MHϩ,
and then extracted with ethyl acetate (3 ϫ 30 ml). The ethyl acetate 100). Analysis calculated for C24H30O5: C, 72.34; H, 7.59. Found:
layers were washed with brine (2 ϫ 30 ml), dried over sodium sulfate C, 72.45; H, 7.60.
and evaporated to dryness. Chromatography of the residue on a
silica gel column using 20% ethyl acetate in methylene chloride gave
Synthesis of 3,17␤-Diacetoxy-2-ethoxy-7␣-hydroxyestra-
1,3,5(10)-trien-6␣-(m-chlorobenzoate) (49) and 3,17␤-Diace-
compound 32 (70 mg, 81%), which was converted into a white stable toxy-2-ethoxy-7␣-hydroxyestra-1,3,5(10)-trien-6␤-(m-chloro-
foam when it was evaporated from ethyl acetate-hexane solution benzoate) (50). A solution of m-chloroperbenzoic acid (260 mg, 57 to
under reduced pressure: 1H NMR (CDCl3) ␦ 6.68 (s, 1 H), 6.51 (s, 1 81%, 0.86–1.23 mmol) in methylene chloride (10 ml) was added
H), 5.51 (s, 1 H), 4.07 (q, J ϭ 6.9 Hz, 2 H), 3.78 (t, J ϭ 8.5 Hz, 1 H), dropwise to a stirred solution of compound 33 (343 mg, 0.86 mmol) in
3.38 (td, J ϭ 11.5 and 2.2 Hz, 1 H), 3.20 (qd, J ϭ 12.6 and 7.1 Hz, 1 methylene chloride (20 ml) under nitrogen atmosphere at 0°C. The
H), 2.94 (qd, J ϭ 11.7 and 6.8 Hz, 1 H), 2.75 (td, J ϭ 11.5 and 2.2 Hz, mixture was allowed to come to room temperature and stirred for
1 H), 2.55 (dt, J ϭ 8.5 and 2 Hz, 1 H), 2.20–1.20 (m, 16 H), 1.42 (t, Jϭ 20 h. The mixture was poured into ice-cold saturated sodium bicar-
6.4 Hz, 3 H), 0.84 (s, 3 H); CIMS (isobutane) m/z (relative intensity) bonate (100 ml) and the mixture was extracted with methylene
360 (MHϩ, 100), 342 (MH-H2O, 50). Analysis calculated for chloride (3 ϫ 50 ml). The combined organic layer was washed with
C22H33O3N⅐1/6 H2O: C, 72.96; H, 9.27; N, 3.86. Found: C, 72.97; H,
water (2 ϫ 50 ml), sodium bicarbonate (2 ϫ 50 ml) and brine (2 ϫ 50
9.27; N, 3.85.
ml), dried over sodium sulfate, and evaporated to dryness. Chroma-
Synthesis of 3,17␤-Diacetoxy-2-ethoxyestra-1,3,5(10),6-tet- tography of the residue on a silica gel column using 25% ethyl acetate
raene-6-yl Triflate (48). A stirred solution of compound 43 (Cush- in hexane gave compound 49 (153 mg, 31%), which was crystallized
man et al., 1997) (1.0 g, 2.4 mmol) and 2,6-di-tert-butyl-4-methylpyri- from diethyl ether to afford a white solid: m.p. Ͼ 110°C (dec); 1H
dine (743 mg, 3.6 mmol) in dry methylene chloride (48 ml) was NMR (CDCl3) ␦ 8.08 (s, 1 H), 8.00 (d, J ϭ 7.8 Hz, 1 H), 7.57 (d, J ϭ
treated with triflic anhydride (0.52 ml, 3.12 mmol) dropwise under 7.8 Hz, 1 H), 7.40 (t, J ϭ 7.8 Hz, 1 H), 6.91 (s, 1 H), 6.87 (s, 1 H), 6.19
argon at 0°C and stirring was continued for 3 h at 0°C. The mixture (d, J ϭ 3.5 Hz, 1 H), 4.72 (t, J ϭ 8.5 Hz, 1 H), 4.24 (d, J ϭ 3.7 Hz, 1
was poured into ice water (200 ml) and extracted with ethyl acetate
H), 4.01 (q, J ϭ 6.9 Hz, 2 H), 2.88 (bs, 1 H), 2.24 (s, 3 H), 2.04 (s, 3 H).
(3 ϫ 100 ml). The combined organic layer was washed with 2 N HCl 2.04–1.30 (m, 11 H), 1.36 (t, J ϭ 6.9 Hz, 3 H), 0.83 (s, 3 H). Compound
(4 ml) in water (50 ml) and then brine (3 ϫ 100 ml) until neutral, 50 (260 mg, 53%) was crystallized from ethyl acetate-hexane to
dried over sodium sulfate, and evaporated to dryness. Chromatogra- afford a white solid: m.p. 140–145°C. 1H NMR (CDCl3) ␦ 8.0 (s, 1 H),

2-Methoxyestradiol QSAR
1057
7.84 (d, J ϭ 7.8 Hz, 1 H), 7.50 (d, J ϭ 7.8 Hz, 1 H), 7.35 (t, J ϭ 7.8 detail previously (Fernandes et al., 1999; Stewart et al., 1999a).
Hz, 1 H), 7.01(s, 1 H), 6.94 (s, 1 H), 5.90 (d, J ϭ 2.2 Hz), 4.72 (t, J ϭ Approximately 0.1 g of smooth muscle was stripped from the wall
8.5 Hz, 1 H), 4.06 (q, J ϭ 6.9 Hz, 2 H), 4.02 (d, J ϭ 2.2 Hz, 1 H), 2.71 of the bronchus for each cell culture. Dissected tissue was im-
(dt, J ϭ 4.2 and 2.1 Hz, 1 H), 2.24 (s, 3 H), 2.05 (s, 3 H), 2.04–1.30 (m,
11 H), 1.37 (t, J ϭ 6.9 Hz, 3 H), 0.83 (s, 3 H).
mersed in Dulbecco’s modified Eagle’s medium (DMEM; Flow
Laboratories, Irvine, Scotland, UK), supplemented with 100 U/ml
Synthesis of 2-Ethoxy-3,6␣,7␣,17␤-tetrahydroxyestra- penicillin G (CSL Australia, Parkville, Victoria, Australia) and
1,3,5(10)-triene (51). A solution of LiAlH4 in THF (1.0 M, 5 ml, 5 mmol) 100 ␮g/ml streptomycin (CSL Australia). The tissue was rinsed in
was added to a solution of compound 49 (270 mg, 0.49 mmol) in THF phosphate-buffered saline (PBS; Oxoid, Basingstoke, Hampshire,
(10 ml) and the resulting suspension was stirred at gentle reflux England) and the airway smooth muscle was chopped into 2-mm3
overnight (14 h). The reaction mixture was cooled and added cau- pieces and digested for 2 h in DMEM containing elastase (0.5
tiously into ice/water (50 ml), and then acidified to pH 1 with 3 M mg/ml; Worthington Biochemical, Freehold, NJ) followed by a
HCl. The mixture was extracted with ethyl acetate (3 ϫ 60 ml). The 12-h incubation in DMEM containing collagenase (1 mg/ml)
organic layers were washed with water (50 ml), sodium bicarbonate (Worthington Biochemical), at 37°C with agitation. The resulting
(50 ml), and brine (50 ml), dried over sodium sulfate, and evaporated cell suspension was centrifuged and washed three times with
to dryness. Chromatography of the residue on a silica gel column
PBS. After the last centrifugation step, the cells were resuspended
using 30% acetone in methylene chloride gave compound 51 (141 mg, in 25 ml of DMEM supplemented with L-glutamine (2 mM; Sigma),
88%), which was crystallized from ethyl acetate-methylene chloride penicillin G (100 U/ml), streptomycin (100 ␮g/ml), amphotericin B
to afford white crystals: m.p. 231 to 232°C. 1H NMR (CDCl3) ␦ 7.05 (2 ␮g/ml; Wellcome, Stevenage, UK) and heat-inactivated FCS
(s, 1 H), 6.82 (s, 1 H), 4.53 (d, J ϭ 3.7 Hz, 1 H), 4.08 (q, J ϭ 6.9 Hz, (10% v/v; CSL Australia) and seeded into 25-cm2 culture flasks.
2 H), 3.93 (d, J ϭ 3.8 Hz, 1 H), 3.69 (t, J ϭ 8.5 Hz, 1 H), 2.95 (m, 2 The primary isolates were incubated for 7 to 14 days to reach
H), 2.68 (dt, J ϭ 7.6 Hz and 4.0 Hz, 1 H), 2.34 (dd, J ϭ 10.0 and 3.2 confluence. Cells were harvested weekly by 10 min exposure to
Hz, 1 H), 2.14–1.20 (m, 11 H), 1.35 (t, J ϭ 6.8 Hz, 3 H), 0.78 (s, 3 H); trypsin (0.5%; CSL Australia) and EDTA (1 mM in PBS; BDH,
CIMS (isobutane) m/z (relative intensity) 349 (MHϩ, 20), 331 (MH- Kilsyth, Victoria, Australia) and passaged at a 1:3 ratio into
H2O, 100), 313 (MHϩ-2H2O, 50). Analysis calculated for 75-cm2 flasks.
C20H28O5⅐1/3 H2O: C, 67.78; H, 8.15. Found: C, 67.76; H, 8.21.
Synthesis of 2-Ethoxy-3,6␤,7␣,17␤-tetrahydroxyestra-
Inhibition of DNA Synthesis
1,3,5(10)-triene (7). A solution of LiAlH4 in THF (1.0 M, 5 ml, 5
mmol) was added to a solution of compound 50 (120 mg, 0.21 mmol)
in THF (10 ml), and the resulting suspension was stirred at gentle
reflux overnight (14 h). The reaction mixture was cooled and added
cautiously into ice/water (50 ml) and then acidified to pH 1 with 3 M
HCl. The mixture was extracted with ethyl acetate (3 ϫ 60 ml). The
organic layers were washed with water (50 ml), sodium bicarbonate
(50 ml) and brine (50 ml), dried over sodium sulfate, and evaporated
to dryness. Chromatography of the residue on a silica gel column
using 30% acetone in methylene chloride gave compound 7 (48 mg,
66%), which was crystallized from ethyl acetate/diethyl ether to
afford white crystals: m.p. 182 to 184°C. 1H NMR (CDCl3) ␦ 6.84 (s,
1 H), 6.78 (s, 1 H), 4.34 (bs, 1 H), 4.09 (q, J ϭ 6.9 Hz, 2 H), 3.86 (bs,
1 H), 3.71 (t, J ϭ 8.5 Hz, 1 H), 2.95 (bs, 3 H, OH), 2.56 (dt, J ϭ 7.6
and 4.0 Hz, 1 H), 2.34 (dd, J ϭ 9.9 and 3.2 Hz, 1 H), 2.14–1.20 (m, 11
H), 1.38 (t, J ϭ 6.9 Hz, 3 H), 0.78 (s, 3 H); CIMS (isobutane) m/z
(relative intensity) 349 (MHϩ, 18), 331 (MH-H2O, 100), 313 (MHϩ-
2H2O, 50). Analysis calculated for C20H28O5⅐1/3 H2O: C, 67.78; H,
8.15. Found: C, 67.52; H, 8.21.
Cells were subcultured into 24-well plates in a 1:3 split ratio at a
density of approximately 1.5 ϫ 104 cells/cm2 and grown to monolayer
confluence in antimicrobial-supplemented DMEM containing 10%
fetal calf serum, over a 72 to 96 h period (5% CO2 in air, 37°C).
Quiescence was induced 24 h before stimulation by removing the
medium, washing with PBS and replacing the medium with serum-
free DMEM (supplemented as described previously) to synchronize
the cells in G0 of the cell cycle. Cells were stimulated by the addition
of thrombin [bovine plasma, 0.3 U/ml, prepared in PBS containing
bovine serum albumin (0.25% w/v; Sigma). Monomed A (1% v/v; CSL
Australia), a serum-free medium supplement containing insulin,
transferrin, and selenium was added to all wells. 2-MEO and analogs
were dissolved in 100% dimethyl sulfoxide (BDH, Poole, Dorset, UK)
at a concentration of 10 mM and diluted serially in PBS, before
addition of 100 ␮l to 1 ml of medium overlying airway smooth
muscle, up to a maximal final analog concentration of 10 ␮M. This
resulted in a final maximum concentration of 0.1% dimethyl sulfox-
ide, which was added to the corresponding control wells. Concentra-
tion-response relationships were constructed from an evaluation of
the effects of the analogs (0.1 ␮M to 10 ␮M, in half-log increments) on
thrombin-stimulated DNA synthesis. Individual responses were cal-
culated by incubating cells with the analog agents for 24 h (5% CO2
in air, 37°C) then pulsing the cells with [3H]thymidine (1 ␮Ci/ml;
Amersham, Little Chalfont, Buckinhamshire, UK) for 4 h to label
newly synthesized DNA. Before harvest, the medium was removed
and the cells were solubilized by the addition of 100 ␮l sodium
hydroxide (1 M; Selby Biolab, Melbourne, Australia) to each well.
Cellular DNA was immobilized onto glass fiber filters using a Pack-
ard Filtermate 196 Cell Harvester (Packard BioSciences, Melbourne,
Australia). The filters were washed three times with 3 ml distilled
H2O and 1 ml ethanol (100%). Packard Microscint-40 (Packard) was
added to dried filters and radioactivity was measured using a Pack-
ard Top Count Microplate Scintillation Counter.
Materials and Methods
Synthetic Chemistry
Melting points were determined in capillary tubes on a Mel-
Temp apparatus and are uncorrected. Spectra were obtained as
follows: 1H NMR spectra were recorded on a Varian VXR-300S
spectrometer (Palo Alto, CA) using tetramethylsilane as an inter-
nal standard; CIMS spectra were obtained on a Finnigan 4000
spectrometer (Thermo Finnigan, San Jose, CA); microanalyses
were performed at the Purdue Microanalysis Laboratory. Flash
column chromatography was performed on 60-␮m silica gel from
Scientific Adsorbents, Inc. (Atlanta, GA). Most chemicals and
solvents were analytical grade and used without further purifica-
tion. Commercial reagents were purchased from Aldrich Chemical
Company (Milwaukee, WI).
ER Binding Assay
ER affinity of estradiol (16), its metabolites [2-hydroxyestradiol
(19), 2-MEO (1)] and analogs were determined using rat uterine
Culture of Human Airway Smooth Muscle
Human airway smooth muscle was cultured from macroscopi- cytosol as a source of ER (Markaverich and Clark, 1979). Uteri (6–10
cally normal bronchi (0.5- to 2-cm diameter) obtained from lung per batch) were isolated from Brown Norway rats (250–350 g),
resection or heart-lung transplant specimens provided by the washed free from blood in saline, and homogenized in 10 mM Tris
Alfred Hospital (Melbourne) according to methods published in buffer [pH 7.4, 1.5 mM EDTA, 10% (w/v) glycerol, 1 mM phenylm-

1058
Hughes et al.
ethylsulfonylfluoride] using an Ultra Turrax homogenizer for 3 15-s Statistical Analyses
bursts with intervals of 1 min on ice. For additional studies of ER in
All incubations for the [3H]thymidine incorporation assays were
smooth muscle, cytosol from human airway smooth muscle was
prepared from 16- ϫ 175-cm2 flasks of confluent cells grown as
described, and serum-deprived for 24 h before harvest. The cells were
washed 3 times with PBS before the addition of a total of 4 ml of the
10 mM Tris buffer described above. The cells were scraped from the
plates and subjected to homogenization by 30 passes in a glass
homogenizer. The airway smooth muscle and rat uterus prepara-
tions were then processed in the following manner. Nuclear material
and cellular debris were removed by centrifugation at 750g at 4°C for
10 min (Sorvall RT7) and cytosol was prepared by centrifugation at
30,000g for 120 min at 4°C in a Beckman JM1 centrifuge. The cytosol
was diluted to contain approximately 1 mg/ml protein (rat uterine
extract) or 0.015 mg/ml (airway smooth muscle) determined using
the Bradford method (Bio-Rad, Hercules, CA). Binding assays were
carried out by overnight incubation at 4°C in 300 ␮l of the above
described buffer: 200 ␮l of cytosol, 50 ␮l of displacer [10 mM estradiol
(16) or buffer] and 50 ␮L of 0.2 nM [3H]estradiol. Separation of bound
from free radioligand was achieved by the addition of 500 ␮l of
dextran-coated charcoal (400 mg of dextran clinical grade C and 2 g
of charcoal, Norit A in 100 ml of Tris buffer) and centrifugation at
4°C in a Sorval RT7 at 2000g for 10 min. Preliminary saturation
analysis over the range 0.01 to 50 nM [3H]estradiol yielded a Kd and
Bmax values of 0.18 nM and 112 fmol/mg protein, respectively [n ϭ 3,
analysis using Prism (GraphPad Software, San Diego, CA)], indicat-
ing that the 0.2 nM concentration was suitable for displacement
studies. Saturation analysis carried out on airway smooth muscle-
derived cytosol revealed an affinity (KD) of 0.53 nM and a much
higher density of binding sites of 1071 fmol/mg protein. The very low
yield of protein from large amounts of cultured airway smooth mus-
cle made further analyses of ER binding in this cell type impractical.
Analyses of 2-MEO (1) and analogs were initially carried out over the
range 0.1 nM to 10 ␮M in decade increments and, after identification
of the appropriate range, displacement of estradiol was determined
using three to four concentrations per decade. The data were fitted to
the Cheng and Prusoff equation using the Kd value of 0.18 nM
conducted in triplicate or quadruplicate. Experiments were carried
out in at least three cell lines, each derived from at least three
different individuals; results are expressed as the mean of these
estimations. To minimize the influence of variability between tissue
donors on comparisons of data, the response to thrombin in the
presence of the antagonist was expressed as a percentage of the
thrombin control in individual cultures (100%). The potency of the
analogs was determined by fitting log concentration-response data
using nonlinear regression to fit a sigmoidal relationship. The pIC50
values represent the negative log of the concentrations that reduce
the thrombin-stimulated DNA synthesis to 50% of the response in
the absence of inhibitor. The binding data were analyzed by the
Cheng and Prusoff equation (Prism) and are presented as pIC50
values that represent the log of the concentration of analog that
displaces 50% of the specifically bound [3H]estradiol. The standard
errors of these pIC50 values (Tables 4 to 6) did not exceed 5% of the
mean value.
Molecular Modeling Studies
Molecular modeling studies were carried out using SYBYL soft-
ware (version 6.4; Tripos Inc., St Louis, MO) running on a Silicon
Graphics O2 workstation equipped with a 175-MHz R10000 proces-
sor (Silicon Graphics, Mountain View, CA).
Generation of Molecular Models. Molecular models of 2-MEO,
its analogs, and other compounds used in this study were con-
structed from standard SYBYL fragments, and their geometry was
optimized (Powell conjugate gradient minimization, termination at a
gradient of Ͻ0.005 kcal molϪ1
Å
Ϫ1) using the Tripos forcefield and
Gasteiger-Marsili partial atomic charges (Gasteiger and Marsili,
1980). Lowest energy conformations of flexible sidechains were iden-
tified by applying the Systematic Search routine in SYBYL to all
rotatable bonds (10° torsion angle increments of flexible bonds) with
reminimization, as described above, of low-energy candidates. Spec-
troscopic data of compound 6 was consistent with a degree of keto-
enol tautomerism at the 6- and 7-positions; the compound was thus
modeled as the enediol.
(Prism) and the data are presented as pIC50
.
Calculation of CoMFA Fields. 3D-QSAR relationships for the
compounds were generated using the CoMFA routines in the QSAR
Cell Enumeration
Cells were seeded onto six-well plates at a density of 1.5 ϫ 104 cells option in Sybyl (Cramer et al., 1988). Compounds were first aligned
cmϪ2, made quiescent as described previously, and then stimulated (see below) and steric and electrostatic energies calculated for each
for 48 h with thrombin (0.3 U/ml). At the end of the 48 h incubation
period, cells were detached from the culture plate by the addition of
trypsin (0.5% w/v in PBS containing 1 mM EDTA), incubated for 5
min at ambient temperature in PBS containing 0.5% (w/v) trypan
molecule throughout a 2-Å grid, extending 4 Å beyond the van der
Waals’ volume of all molecules, using an sp3 carbon atom probe with
a charge of ϩ1 and a distance-dependent dielectric constant. The
resultant matrices—a row for each compound, a column of biological
blue, washed twice (2% FCS in PBS), isolated by centrifugation activities (either DNA synthesis inhibition or ER binding), and mul-
(12,000g, 5 min) and resuspended in 200 ␮l 2% FCS in PBS for tiple columns for each molecule containing the molecular field
counting in a hemocytometer chamber.
strengths at each grid point—were subjected to partial least-squares
(PLS) regression analysis (see below) to derive 3D-QSAR models.
Three-Dimensional Alignments for CoMFA. 3D-QSAR
models obtained by CoMFA techniques are sensitive to the spatial
alignment of the molecules under study. To examine the effect of
alignment, CoMFA models were generated for the following align-
ments (created by aligning molecules with 2-MEO using the Fit
routine in Sybyl):
Flow Cytometric Analyses of Cell Cycle Status
Cells seeded into 6-well plates at a density of 1.5 ϫ 104 cells cmϪ2
and made quiescent as described previously were then stimulated
with thrombin (0.3 U/ml) for 48 h. Monomed A was added to all cells
(including control cells) at the time of mitogen addition. Cells were
detached from the culture plates by incubation with trypsin for 30
min at 37°C (0.5% w/v). The resulting suspension was washed in PBS
Steroid alignments.
twice before resuspension in 1 ml of 70% ethanol for storage for up to 1. Aligned by overlaying the atoms corresponding to the three
3 weeks at Ϫ20°C. Before staining, cells were washed twice (2% FCS
in PBS) to remove the ethanol. Fixed cells were stained with pro-
pidium iodide (50 ␮g/ml) in Triton X-100 (0.1% v/v) with RNase II
(180 mU/ml). The cell suspension was passed through an 18-gauge
oxygen atoms in 2-MEO. These atoms are common to all ana-
logs and, by analogy to the requirements for ER binding (An-
stead et al., 1997), might therefore correspond to key pharma-
cophoric points.
needle to facilitate the separation of cell clumps. Cells were stored for 2. Aligned as in 1, except that the side chain in position R2 was
24 h before analysis. Cell cycle status was analyzed using a FACScan
Instrument (BD, Franklin Lakes, NJ). Ten thousand events from
each sample were counted and analyzed using a ModFitLT V2.0
analysis package (Verity Software House, Topsham, ME).
oriented to match the conformation of this group in 2-MEO,
except for compounds with an acetate group in this position.
These compounds had their side chains adjusted to the confor-
mation of the side chain in 2-MEO, followed by minimization,

2-Methoxyestradiol QSAR
1059
rather than in their strictly low energy conformations as in
alignment 1. This alignment would be expected to reduce the
could introduce inappropriate structural variation in com-
pounds.
likelihood that alternative side chain comformations (any of 3. Aligned by fitting all common, nonhydrogen atoms. This align-
which might be reasonably accessible in a biological milieu) ment takes weight away from the positions of the oxygen atoms
TABLE 4
Structures of 2-methoxyestradiol (1) and analogs
Biological Activity (pIC50
)
R1
R2
R3
R4
R5
DNA Inhibition
ER Binding
1
CH3O–
CH3O–
CH3O–
HO–
BzlO–
HO–
H–
H–
H–
H–
H–
H–
H–
H–
H–
HO–
HO
H–
H–
H–
H–
H–
H–
H–
H–
H–
HO–
BzlO–
Oϭ
5.21
Ͻ5
7.5
N.D.
4.97
7.37
5.45
Ͻ5
N.D.
N.D.
8.09
N.D.
8.98
8.18
9.16
7.61
N.D.
9.89
8.62
N.D.
9.49
N.D.
2
3
stima
5.04
5.72
5.97
Ͻ5
4
CH3CH2O–
CH3CH2O–
CH3CH2O–
CH3CH2O–
CH3CH2O–
OHC–
HO–
HO–
CH3COO–
HO–
HO–
HO–
CH3COO–
HO–
CH3COO–
HO–
5
Oϭ
Oϭ
HO
HO–Nϭ
H–
6b
7
8
9
HO–
HO–
5.99
Ͻ5
Ͻ5
4.98
5.09
5.95
5.24
Ͻ5
Ͻ5
5.88
Ͻ5
5.03
Ͻ5
10
11
12
13
14
15
16
17
18
19
20
OHC–
BzlO–
HO–
H–
BzlO–
HO–
CH3CH2–
CH3CH2S–
HO–NϭCH–
CH3O–NϭCH–
NH2–NϭCH–
H–
H–
HO–
H–
HO–
HO–
H–
HO–
HO–
H–
HO–
HO–
H–
HO–
HO–
H–
HO–
H–
HO–
H–
C2F5(CH2)3S(0)(CH2)9–
HO–
Br–
BzlO–
HO–
H–
H–
H–
H–
BzlO–
HO–
HO–
H–
HO–
BzlO–
H–
BzlO–
N.D., not determined.
a Caused stimulation of DNA synthesis.
b Modelled as the enediol (see Methods).
TABLE 5
Analogs of 2-ethoxyestradiol (4) with a seven-membered B-ring.
Biological Activity (pIC50
)
R2
R3
R4
R5
X
DNA Inhibition
ER Binding
21
22
23
24
25
26
27
28
29
30
31
32
HO–
CH3COO–
HO–
CH3COO–
HO–
H–
H–
Oϭ
Oϭ
Oϭ
Oϭ
H–
H–
H–
HO–
CH3COO–
HO–
–C–
–C–
–C–
–C–
–C–
–C–
–C–
–C–
–C–
–C–
–C–
–N–
Ͻ5
Ͻ5
Ͻ5
Ͻ5
H–
5.28
5.00
5.32
5.52
5.07
5.6
5.29
5.30
5.51
Ͻ5
Ͻ5
H–
HO–
Ͻ5
H–
CH3COO–
CH3COO–
HO–
Ͻ5
CH3COO–
HO–
H–
Ͻ5
Oϭ
H–
N.D.
Ͻ5
HO–
HONϭ
H–
CH3ONϭ
CH3ONϭ
CH3CH2–
HO–
HO–
HONϭ
H–
HO–
Ͻ5
HO–
HO–
N.D.
Ͻ5
CH3COO–
HO–
H–
H–
CH3COO–
HO–
N.D.
N.D., not determined.

1060
Hughes et al.
TABLE 6
Partial Least Squares (PLS). PLS is a type of regression anal-
ysis particularly suited to situations such as CoMFA, where the
independent variables (i.e., molecular field strengths) greatly out-
number the dependent variables (biological activities) (Wold, 1991).
It uses a means of data reduction to transform the dependent and
independent variables into a set of so-called principal components,
which are mutually orthogonal descriptors from which the QSAR
equations can subsequently be derived. The optimal number of com-
ponents (after which the inclusion of additional components does not
further improve the quality of the model) for the models for ER
binding and DNA synthesis inhibition was determined in so-called
cross-validated runs. In these runs, successive QSAR equations are
generated in which one compound is omitted, and the model derived
in the absence of this compound and used to predict the activity of
the omitted compound. The process was repeated until equations had
been derived with each compound omitted once—the activity of each
omitted compound was predicted—giving a set of n QSAR equations
for n compounds (leave-one-out method). The final CoMFA models
were then derived from non–cross-validated runs using the optimal
number of components determined from the cross-validated runs.
The quality of the QSAR equations generated during the cross-
validated runs was measured by the term q2 (the cross-validated r2),
calculated thus: q2 ϭ (SD Ϫ PRESS) / SD, where SD is the variance
of the biological activities, and PRESS is the sum of the squares of
the differences between predicted and observed biological activities
of each compound as it is left out of the analysis during the cross-
validation process.
Additional compounds used in this study
Biological Activity (pIC50
)
Structure
DNA Inhibition
ER Binding
33
34
35
36
37
38
39
5.24
5.33
Ͻ5
N.D.
9.77
N.D.
5.56
9.14
N.D.
N.D.
stima
Because it involves the omission of data during its calculation, q2
is an inherently conservative estimate of the predictivity of a model.
It is generally accepted that q2 values greater than 0.5—halfway
between no model (q2 ϭ 0.0) and a perfect model (q2 ϭ 1.0)—are
likely to be of useful predictive value.
Ͻ5
To increase the speed of the cross-validated runs, CoMFA columns
with a variance of less than 2.0 kcal/mol (minimum ␴ ϭ 2.0) were
excluded from PLS analyses; a minimum sigma ␴ value of 0.0 kcal/
mol (i.e., no filtering) was used during non–cross-validated analyses,
as recommended in the Tripos manual.
Optimization of CoMFA Models. The CoMFA implementation
in Sybyl allows the user to optimize the model by altering several
parameters.
Ͻ5
Ͻ5
1. Choosing the cut-off value for the energy at a given grid point
(grid points whose computed energy is greater than the cut-off
value are assigned the cut-off value). The effect of cut-off on the
quality and predictive capabilities of the CoMFA models was thus
examined over a range of cut-off values (10–100 kcal/mol).
2. Scaling the field strength data (implementing either CoMFA stan-
dard scaling, the default choice, which attempts to balance the
potential influence of each field and column on the resulting
QSAR by scaling the data to the overall field mean and standard
deviation, or no scaling, which emphasizes variables with a
greater numerical spread, typically those describing the steric
field).
N.D., not determined.
a Caused stimulation of DNA synthesis.
used in alignments 1 and 2 and places more emphasis on the
positions of backbone atoms. Side chains were in their low energy
conformations as in alignment 1. This alignment was thought to
be worthy of examination, given that the estrogens are likely to be
compounds that are well matched to the ER (Andrews et al.,
1984).
3. Dropping electrostatic values at the steric maximum, (i.e., so that
electrostatic field contributions from “inside” a molecule are not
considered).
4. Aligned as in 3, except that side chain conformations were ad-
justed to match those of 2.
Nonsteroid alignments.
The effect of altering combinations of these parameters was ex-
1. 4-OH-tamoxifen (37) was aligned by superimposing the centroids amined systematically. Further optimization of models was carried
of two aromatic rings of 37 with the centroids of the A and D rings out by examining the effects of removing “outlying” compounds. Two
of 2-MEO and the aromatic O-substituents of 37 with the O- methods were used to define outliers: compounds whose residual was
substituents of 2-MEO in positions R2 and R5. This alignment has
greater than three times the S.D. of the residual of the entire dataset
previously been used in developing 3D-QSAR relationships for and compounds which, when removed from the analysis, gave rise to
nonsteroidal ligands for the estrogen receptor (Sadler et al., 1998). CoMFA models with an improved q2 values compared with that
2. Flavones (38 and 39) were aligned by superimposing the carbon obtained with the compound present.
atoms of the aromatic ring of the flavone ␣ to the carbonyl group.
The centroid of the other aromatic ring was fitted to the centroid models were reoptimized with regard to cut-off, scaling, and drop-
of the steroid D-ring. ping of electrostatics as described above, in an iterative fashion.
After the identification and removal of outlying compounds, the

2-Methoxyestradiol QSAR
1061
Default values were used for other settings. The pIC50 of compounds of the oxime tosylate, which underwent the desired Beck-
deemed to be inactive was set to 5, to reduce arbitrary skewing of the
data.
mann rearrangement when passed through a column of basic
alumina and eluted with a mixture of benzene and chloro-
form to afford the lactam 44. Diborane reduction of 44
yielded the expected benzo[f]azepine system 45. Reaction of
45 with acetic anhydride resulted in acetylation of both al-
cohols as well as the amine to give 46, which was hydrolyzed
with sodium hydroxide to provide the diol 47. Diborane re-
duction of the amide present in 47 produced the desired
product 32.
Oxidation of the B ring of 2-ethoxyestradiol has afforded a
number of very potent cytotoxic tubulin polymerization in-
hibitors (Cushman et al., 1997). These included 2-ethoxy-6-
oxoestradiol and the corresponding oxime. Methods have
Presentation of CoMFA Models. The final optimized CoMFA
models for DNA inhibition and ER binding obtained from non–cross-
validated runs are presented as isocontour maps of extreme values of
the product of the S.D. and coefficients of the 3D-QSAR equations
derived by PLS analysis at the grid points, after scaling of this
product from 0 to 100%. (The product of the S.D. and coefficient was
also used to obtain the relative contributions of the steric and elec-
trostatic fields.) Such plots indicate where in space differences in
either the steric or electrostatic fields of the molecules in the data set
correlate most with differences in biological activity (Cramer et al.,
1988). Generally, contours corresponding to Ͼ85% and Ͻ15% of the
signal gave readily interpretable isocontour maps (i.e., regions asso-
ciated with the molecular fields of the datasets that correlated either
positively or negatively with biological activity were represented by
clearly defined, discrete polyhedra), except for the electrostatic field
map for DNA synthesis inhibition, where contours of Ͼ80% and
Ͻ20% were used to give a clearer indication of the position of elec-
trostatically and sterically favorable substituents.
therefore been sought for oxidation of the
B ring of
2-ethoxyestradiol. As shown in Scheme 3, treatment of
2-ethoxy-6-oxoestradiol diacetate (43) (Cushman et al., 1997)
with triflic anhydride under basic conditions afforded the
enol triflate 48, which was reduced in the presence of formic
acid, triethylamine, triphenylphosphine, and palladium ace-
tate in DMF to afford the cyclic alkene 33 (Ciattini et al.,
1990). Instead of the expected 6␣,7␣-epoxide, oxidation of 33
with m-chloroperbenzoic acid gave compounds 49 and 50,
Results
Synthetic Chemistry
Prior work has demonstrated that the cytotoxicities and which are the addition products of m-chloroperbenzoic acid to
tubulin polymerization inhibitory potencies of 2-MEO ana- the 6␣,7␣-epoxide (Burdett et al., 1982). The stereochemical
logs can be modulated by replacement of the 2-methoxyl assignments are based on the larger axial-equatorial cou-
group by other substituents (Cushman et al., 1995, 1997). pling observed between the C-6 and C-7 methine protons in
Replacement of the 2-methoxyl group of 2-MEO by 2-ethoxyl 49 (J ϭ 3.5 Hz) versus the smaller diequatorial coupling
and 2-(E)-1-propenyl substituents resulted in compounds constant (J ϭ 2.2 Hz) seen with 50 (Jackman and Sternhell,
that were more potent than estradiol itself (Cushman et al., 1969). Lithium aluminum hydride reduction of 49 and 50
1995). In general, the optimal substituent in the 2-position cleaved all of the esters, resulting in the tetraols 51 and 7.
proved to be a straight chain containing three atoms from the Oxidation of the starting material 43 with chromium trioxide
second row of the periodic table. To investigate this in more in acetic acid gave the ␣-hydroxyketone 6.
detail, the oxime (13), methoxime (14), and hydrazone (15)
2-MEO Effects on DNA Synthesis, Proliferation and Cell-
Cycle Distribution of Human Airway Smooth Muscle Cells
derivatives were prepared as outlined in Scheme 1. Treat-
ment of estradiol (16) with methoxymethyl chloride in the
presence of diisopropylethylamine afforded the bis(me-
2-MEO (0.3–10 ␮M) concentration-dependently inhibited
thoxymethyl) ether 40. Intermediate 40 was regioselectively 3H-thymidine incorporation in cultures of human airway
lithiated in the 2-position with sec-butyllithium in THF, fol- smooth muscle cells (Table 1). Cell enumeration experiments
lowed by formylation with DMF to yield the aldehyde 41 in revealed that 2-MEO (10 ␮M) prevented 0.3 U/ml thrombin-
high yield as a single regioisomer. Deprotection of the bis- induced increase in cell number but had no effect on the
(methoxymethyl) ether 41 with lithium tetrafluoroborate in number of nonviable cells after incubation for 48 h (Table 2).
acetonitrile provided 2-formylestradiol (42) (Ireland and Var- Flow cytometric analyses of cell-cycle distribution (Fig. 1)
ney, 1986). The oxime oxime (13), methoxime (14), and hy- indicated that 2-MEO (10 ␮M) reduced the proportion of cells
drazone (15) derivatives were then obtained by reaction of 42 in S-phase and increased that in G2/M (Table 3). In a sepa-
with hydroxylamine, methoxylamine, and hydrazine, respec- rate series of experiments, the incubation of cells with throm-
tively.
bin (0.3 U/ml) increased cell number (ϫ10Ϫ4 well of a 6-well
Certain B-ring expanded analogs of 2-ethoxyestradiol re- plate) from 29.8 Ϯ 3.9 to 45.5 Ϯ 8.5; this was significantly
semble paclitaxel in their ability to enhance tubulin polymer- reduced (P Ͻ 0.05, paired t test) to 33.8 Ϯ 6.7 and 32.0 Ϯ 5.0,
ization and stabilize microtubules (Wang et al., 2000). We for 2-MEO (10 ␮M) and compound 5, respectively, without
have therefore synthesized a variety of additional B-ring concomitant increase in the number of trypan blue staining
homologated analogs of 2-ethoxyestradiol, including the ben- (i.e., nonviable) cells.
zo[f]azepine derivative 32 (Scheme 2). Treatment of
Inhibition of Human Airway Smooth Muscle DNA
Synthesis by 2-MEO Analogs
2-ethoxy-3,17␤-diacetoxy-6-oxoestra-1,3,5(10)-triene
(43)
(Cushman et al., 1997) with hydroxylamine resulted in se-
lective deacetylation of the phenolic acetate as well as oxime
The range of data obtained for DNA synthesis inhibition in
formation to afford 8. There are several reports of the use of cultures of human airway smooth muscle (Tables 4–6) was
oxime tosylates as substrates for Beckmann rearrangements narrow, covering only one order of magnitude for active com-
(Tamura et al., 1973; Ciattini et al., 1990). These rearrange- pounds. The most potent inhibitors were the 2-ethoxyestra-
ments are reported to occur when the oxime tosylate is diol derivatives 8 and 6, 2-(hydroximino)estradiol (13) and
passed through a column of acidic or basic alumina. Reaction ICI 182,780 (17) (pIC50 for DNA synthesis inhibition of 5.99,
of 8 with tosyl chloride in pyridine resulted in the formation 5.97, 5.95, and 5.88, respectively), all being considerably

1062
Hughes et al.
more potent than 2-MEO itself (pIC50 5.21). Compound 6 also biological assays (r2 ϭ 0.038 for the 23 compounds for which
displays negligible ER binding potency (see below). Both 8 biological potency was determined in both assays).
and 6 are acetylated at the 17 position (R5), although this
Generation and Optimization of 3D-QSARs
feature is itself not sufficient for potent DNA synthesis inhi-
bition, because other acetylated compounds show reduced
activity (e.g., the diacetylated 33, pIC50 5.24). Two com-
pounds, 3 and 36, stimulated rather than inhibited DNA
synthesis; these compounds were not examined further by
CoMFA.
Selection of Optimal Alignments. Table 7 shows the q2
obtained for the preliminary CoMFA models obtained from
the four different alignments for DNA synthesis inhibition
and ER binding data. For DNA synthesis inhibition data, the
q2 values obtained were low, indicative of models of limited
predictive values. Using the default cut-off (30 kcal/molϪ1),
alignment 4 gave the most predictive model (q2 ϭ 0.144); this
Displacement of [3H]Estradiol from Rat Uterine ER
In contrast to the data obtained for DNA synthesis inhibi- alignment was thus used to generate optimized models. For
tion, ER binding affinities covered a much larger range of ER binding, the highest q2 values were obtained for align-
values (approximately 5 orders of magnitude; Tables 4–6). ments 1 and 3 (q2 ϭ 0.776 and 0.760, respectively). Opti-
The most potent displacement activity was exhibited by 16, mized CoMFA plots were subsequently generated from align-
34, and 19 (pIC50 for ER binding of 9.89, 9.77, and 9.49, ment 3, because initial CoMFA plots obtained from
respectively). All compounds tested with a seven-membered alignment 1 were poorly defined, and difficult to interpret,
B ring (Table 2) were devoid of ER binding, although most of even after preliminary attempts to optimize them (data not
these analogs retained DNA synthesis inhibition (23-31). shown).
Some compounds tested were potent inhibitors of DNA syn-
Optimization of CoMFA Models. Optimized CoMFA
thesis and ER binding (e.g., 13, pIC50 for DNA synthesis models were obtained from the preliminary models by sys-
inhibition of 5.95, pIC50 for ER binding 9.16). Overall, how- tematically altering user-modifiable parameters—cut-off en-
ever, there was no correlation between potency in the two ergy, field-strength scaling, and dropping electrostatic values
Scheme 1. Reagents and conditions: a, CH3OCH2Cl, i-Pr2EtN, THF, 60°C (8 h); b, (1) sec-BuLi, THF, Ϫ78°C (2.5 h), (2) DMF, Ϫ78 to 23°C (3 h); c,
LiBF4, aq CH3CN, 72°C (5 h); d, H2NOH⅐HCl, pyridine, 90°C (1 h); e, H2NOCH3, pyridine, 90°C (2 h); f, H2NNH2⅐2HCl, pyridine, 90°C (2 h).

2-Methoxyestradiol QSAR
1063
at the steric maximum—and examining the effects of remov- of 0.114). However, re-derivation of a model without this
ing outliers. For DNA synthesis inhibition, parameter opti- compound gave only a minor change in q2 (0.799). Thus,
mization alone gave a model with improved predictivity (q2 ϭ compound 1 was not omitted.
0.377, see Table 8). Examination of this model revealed one
Final CoMFA Models. The features of the final CoMFA
compound (the hydroximino derivative 13) for which the models—one for DNA synthesis inhibition, one for ER
residual was greater than three times the S.E.M. residual. binding—are described in Table 9. Apart from differences
Elimination of this compound from the data set and reopti- described above in the parameters used to derive them
mization with respect to user-modifiable parameters further (alignment, use of scaling, cut-offs, etc.), the two models
improved the model (q2 ϭ 0.483). No additional outliers could show other distinct quantitative differences: the CoMFA
be detected on the basis of compound residuals. We therefore model for DNA synthesis inhibition has less predictive
chose to rederive the model in the absence of each compound power than that obtained for ER binding (q2 of 0.566
in turn. The greatest increase in q2 (q2 ϭ 0.566) was obtained versus q2 of 0.773); the model for DNA synthesis (three
when the 7␣-substituted compound, ICI 182,780 (17), was components) is simpler than that for ER binding (eight
removed. The model thus obtained could not be further opti- components); and the relative contributions to the models
mized by changing the user-modifiable parameters. A sum- from steric and electrostatic descriptors is reversed in the
mary of the optimization process for the DNA synthesis two models (72% steric and 28% electrostatic contribution
CoMFA model can be found in Table 8.
for DNA synthesis contribution versus 32% steric and 68%
For ER binding, systematic alterations to user-modifiable electrostatic for ER binding).
parameters produced only a modest change in predictivity (q2
Qualitative differences between the two CoMFA models
ϭ 0.773). One compound (the parent compound 1) could be can be observed in electrostatic and steric CoMFA contour
classified as an outlier with regard to its residual (residual plots (Figs. 2 and 3). For DNA synthesis inhibition, favorable
0.375, compared with the S.D. of the residuals for the dataset electrostatic regions are limited (Fig. 2, a and c): blue poly-
hedra, signifying regions where electropositive substitutions
Scheme 2. Reagents and conditions: a, H2NOH⅐HCl, pyridine, 100°C (30
min); b, (1) TsCl, pyridine, 23°C (2 h), (2) Al2O3, CHCl3-C6H6; c, B2H6,
THF, 23°C (6 h) and reflux (4 h); d, Ac2O, pyridine, 23°C (12 h); e, Aq
NaOH, MeOH, 23°C (4.5 h); f, B2H6, THF, 23°C (4 h) and reflux (4 h).
Scheme 3. Reagents and conditions: a, (TfO)2O, 2,6-(t-Bu)-4-Me-pyri-
dine, CH2Cl2, 0°C (3 h); b, CrO3, AcOH, 12–15°C (3 h); c, HCOOH, Et3N,
Ph3P, Pd(Ac)2, DMF, 60 to 62°C (3 h); d, MCPBA, TsOH, CH2Cl2, 23°C (20
h); e, LiAlH4, THF, reflux (14 h).

1064
Hughes et al.
Fig. 1. Influence of thrombin
(0.3 U/ml) on propidium iodide
DNA profiles in human airway
smooth muscle cells in culture
and its modification by 2-MEO
(10 ␮M). These profiles are rep-
resentative of 12 others (see Ta-
ble 2) and show that thrombin
(B) increases the proportion of
cells in S-phase over that ob-
served in unstimulated cells (A).
The thrombin-induced increase
in cells in S-phase is prevented
by 2-MEO (C), which also in-
creases the proportion of cells in
G2/M.

2-Methoxyestradiol QSAR
1065
favor biological activity, can be found in the 2 position of the vides direct evidence of the synergistic nature of combining
steroid nucleus below the plane of the A ring, and in the these two activities in reducing tumor growth. In contrast,
vicinity of the 17␤ substitutions. An additional small red antagonists of single receptor classes used alone are rarely
polyhedron (favorable position for electronegative substitu- clinically useful in the treatment of chronic inflammatory
tion) can be seen near the B ring. In direct contrast, ER conditions. Moreover, tumor chemotherapy regimens ex-
binding is favored by electronegative properties near the A ploit synergies to achieve maximal tumor shrinkage. 2-Me-
ring, as evidenced by several red polyhedra situated above thoxyestradiol (1) is an attractive lead compound for the
and below the plane of the A ring. (Fig. 2, b and d). The blue development of novel therapeutic agents to treat these
polyhedron near the 17␤ position suggests that electroposi- diseases, because its structure provides for multiple indi-
tive substitutions in this region also favor ER binding, as is vidual activities that may sum or even amplify to provide
the case for DNA synthesis inhibition.
an effective anti-inflammatory and antitumor agent. These
The steric CoMFA field for DNA synthesis inhibition activities include the inhibition of cell cycle progression
(Fig. 3, a and c) defines three regions in which increased through DNA synthesis inhibition (G1 arrest; Stewart et
steric bulk favors activity (green polyhedra): in the plane al., 1999b) and microtubule dysfunction (G2/M arrest;
of the A ring near the 2-position; in the plane of the B ring; D’Amato et al., 1994; Fotsis et al., 1994; Cushman et al.,
and an area near the 17 position, roughly in the plane of 1995; Hamel et al., 1996), in addition to antiangiogenic
the D ring. The steric contributions are further defined by actions via a direct inhibitory effect on endothelial cells
areas in which steric bulk is not favorable to DNA synthe- (Klauber et al., 1997; Reiser et al., 1998; Tsukamoto et al.,
sis inhibition activity (yellow polyhedra), with the major 1998). In contrast, with the possible exception of estrogen-
regions above and below the planes of both the A ring (near responsive tumors, it is unlikely that affinity for ER would
the 2 position) and the B ring. The steric field for ER enhance the potential beneficial actions of 2-MEO. In the
binding is, in comparison, less well defined. The main present study, we have developed 3D-QSAR models based
constraints on steric bulk are found in the vicinity of the 2 on 2-MEO and a series of analogs that may guide the
position—a positive steric influence on activity (green development of novel molecules that possess more or less
polyhedron, located in the plane of the A ring), and a potency as appropriate for the attributes that seem to be of
nearby region of negative steric influence (yellow polyhe- potential therapeutic relevance.
dron, below the plane of the A ring; Fig. 3, b and d).
Inhibition of [3H]thymidine uptake into the airways
Although the most potent compound for ER binding, estra- smooth muscle cells by 2-MEO and analogs has a number
diol (16), has no substituent in this position, several other of potential interpretations including: direct reduction of
compounds that do display good ER binding (e.g., 11 and the activity of enzymes responsible for DNA synthesis;
13) are 2-substituted; presumably, these latter compounds induction of cell cytotoxicity; influence on the transport of
give rise to the positive correlation between bulk in this thymidine into the cell; arrest of mitogen-stimulated cells
position and ER binding. An additional region in which in G0/G1 of the cell-cycle. In the case of 2-MEO, which is
steric bulk positively correlates with improved ER binding likely to have activity indicative of its analogs, there was
can be found near the 17 position.
no evidence of cytotoxicity (loss of membrane integrity or
apoptosis or a reduction in cell number under basal condi-
tions) at the maximum concentration used (10 ␮M). The
reduction in thymidine uptake was paralleled by a reduc-
tion in cells entering S-phase, arguing against the former
observation being explained by direct inhibition of thymi-
dine transport. The reduction in indices of S-phase entry
was accompanied by a reduction in thrombin-induced in-
creases in cell number, indicating an antiproliferative ef-
Discussion
Antitumor and anti-inflammatory therapeutic regimens
are often optimized by the combination of two or more
agents having complementary actions on different aspects
of these complex pathologies. In particular, the recent
description by members of Folkman’s laboratory of synergy
between antiproliferative cytotoxic agents and agents that
specifically target angiogenesis (Nguyen et al., 1994) pro-
TABLE 9
Summary of final CoMFA models for DNA synthesis inhibition and ER
binding.
TABLE 7
q2 values for preliminary CoMFA models for DNA synthesis inhibition
(DNA q2) and ER binding (ER q2) for the four different alignments
CoMFA Model
Parameter
Alignment
DNA Synthesis
ER Binding
Inhibition
1
2
3
4
Alignment Used
Number of Compounds
Outliers Omitted
Steric/Electrostatic Cut-Off
Scaling
#4
35
#3
25
None
DNA q2
ER q2
0.011
0.776
Ϫ0.062
0.127
0.760
0.144
0.636
0.624
13, 17
100 kcal/mol
None
No
80 kcal/mol
CoMFA Standard
Yes
TABLE 8
q2 values obtained for CoMFA models obtained for DNA synthesis
inhibition after removal of statistically outlying compounds and
reoptimization of the CoMFA model
Electrostatics Dropped
q2
0.566
3
0.866
0.107
0.719
0.773
Number of Components
r2
8
0.997
Outlier Optimization Run
S.E.
0.125
Steric Contribution to
Molecular Field
Electrostatic Contribution to
Molecular Field
0.317
1
2
3
Compound Excluded
q2
13
0.483
13,17
0.566
0.281
0.683
0.377

1066
Hughes et al.
fect. Time course studies indicate that when 2-MEO is
The QSAR models were generated using the CoMFA
added more than 6 h after thrombin, there is no inhibition method, developed by Cramer et al. (1988). CoMFA models
of [3H]thymidine incorporation (measured at 24–28 h after are known to be sensitive both to the alignment of the
thrombin addition; T. Harris and A. G. Stewart, unpub- molecules (indicative of the orientation of the molecules
lished observations), in contrast to the expected rapid on- when they bind to their receptor) as well as to the confor-
set of action if 2-MEO were acting by direct inhibition of mation of the individual molecules in the alignment (the
DNA synthesis enzymes. Thus, the effect on [3H]thymidine conformation they adopt when binding). In the absence of
incorporation of 2-MEO, and most probably that of its definitive knowledge of these modes of binding of the com-
analogs, is explained by arrest of cells in G0/G1 phase of pounds, one must examine several alignments and confor-
the cell cycle leading to an antiproliferative effect that is mations and select the combination that gives the CoMFA
contributed to by a further action, identified by fluores- model with the greatest predictive power (i.e., the highest
cence-activated cell-sorting studies, to arrest cells in G2/M q2). In this study, optimal models for DNA synthesis inhi-
phase of the cell-cycle.
bition and ER binding were obtained from different align-
Fig. 2. Orthogonal views of CoMFA
contour plots of electrostatic field con-
tributions to DNA synthesis inhibition
(a and c) and ER binding (b and d). A
potent compound in each of the two
assays is depicted: compound 6 for
DNA synthesis inhibition; estradiol
(16) for ER binding. Blue polyhedra
correspond to regions in which biolog-
ical activity is favored by increased
positive charge, whereas red polyhe-
dra define regions in which more neg-
ative charge is favorable to biological
activity.
Fig. 3. Orthogonal views of CoMFA
contour plots of steric field contribu-
tions to DNA synthesis inhibition (a
and c) and ER binding (b and d). A
potent compound in each of the two
assays is depicted: compound 6 for
DNA synthesis inhibition; estradiol
(16) for ER binding. Green polyhedra
correspond to regions in which biolog-
ical activity is favored by increased
steric bulk, whereas yellow polyhedra
define regions in which reduced steric
bulk is favorable to biological activity.

2-Methoxyestradiol QSAR
1067
ments. For DNA synthesis inhibition, the best alignment airway smooth muscle DNA synthesis (T. Harris and A. G.
was found to occur when the conformation of the substitu- Stewart, unpublished observations), which is not inconsis-
ents in the 2 and 3 positions (R1 and R2) was adjusted to tent with the inhibition of DNA synthesis by the analogs used
match the conformation of the corresponding substituents to generate the QSAR being related to a paclitaxel-like effect.
in 2-MEO (methoxy- and hydroxy-). Conversely, a better
In this study, we have identified compounds with rea-
preliminary CoMFA model derived for ER binding was sonable potency for inhibition of DNA synthesis but that
obtained when these substituents were not constrained to show relatively little ER affinity (e.g., 6 and 5). A similar
2-MEO–like conformation, but rather set to the lowest separation of ER affinity and antitubulin action with an-
energy positions. Differences were also observed in the alogs of 2-MEO has been noted (Cushman et al., 1997).
choice of CoMFA parameters giving the best models as well Nevertheless, airway smooth muscle cells in culture ex-
as in the relative contributions to these models of the steric press high affinity ER binding sites (KD ϭ 0.53 nM; see
and electrostatic fields (Table 9). Together, these data Methods) and antibodies detecting both ER␣ and ER␤ re-
suggest that the receptor for DNA synthesis inhibition vealed immunoreactive ER in the cytoplasm and more
requires quite different conformational and structural fea- prominently in the nucleus of cultured airway smooth
tures of its ligands compared with those required by the muscle (T. Harris and A. G. Stewart, unpublished obser-
ER.
vations). The rat uterine cytosol used to generate the ER
The design of compounds more potent in inhibiting DNA affinity data for the QSAR is known to contain predomi-
synthesis can be assisted by the identification of structural nantly ER␣. It seems unlikely that the compounds used to
features important for this biological activity. The CoMFA generate the QSAR would bind to ER␤ with markedly
studies described here offer a means to discover these different affinities to ER␣ as previous binding studies
features. The two most potent compounds for DNA synthe- show that some of the compounds used in the present
sis inhibition (8 and 6) both have ethoxy substituents at R1 study (compounds 16, 19, 34, and 37) have affinities not
and are acetylated at R5. These structural features are more than 3-fold different between the two receptor iso-
reflected in the steric CoMFA field for DNA synthesis forms (Kuiper et al., 1997) and it has recently been re-
inhibition, which shows favorable regions for steric bulk in ported that 2-MEO has a lower affinity for ER␤ than ER␣
the region of both R1 and R5 (Fig. 3, a and c). The electro- (Pribluda et al., 2001). These observations are not consis-
static CoMFA field for DNA synthesis inhibition indicates tent with a specific role for ER␤ in the effects of 2-MEO
a preference for relatively electropositive substituents in analogs.
these positions. At R1, this is probably a consequence of
The lack of correlation between DNA synthesis inhibi-
weakly active or inactive compounds bearing substituents tion and ER binding, as well as the differences between the
with electronegative atoms (e.g., the formyl substituent of CoMFA models for the two activities suggest that com-
9 and 10; the hydrazone group of 15). At R5, this region is pounds even more selective for DNA synthesis inhibition
centered on the carbonyl carbon atom found in acetylated could be designed. For example, the electrostatic CoMFA
compounds with good DNA synthesis inhibitory potency field for ER binding shows multiple electronegative-favor-
(e.g., 8, 6, 21, and 26). The additional favorable region for ing regions above and below the plane of the A ring, which
steric bulk and negative charge near R3 probably arises are not present in the model for DNA synthesis inhibition.
exclusively from the high biological activity exhibited by 8 These regions resemble the electrostatic CoMFA field ob-
(R3 ϭ hydroximino), 6 (R3 ϭ hydroxyl/keto), and 5 (R3 ϭ tained in an earlier study of the mitogenic effects of estra-
keto). Several compounds with large substituents in the R2 diol analogs (Wiese et al., 1997), despite the fact that the
region (e.g., the benzyl derivatives 2, 10, 18, and 26) are two studies were derived from different data sets. This
inactive; consequently, sterically disfavored regions appear similarity of models across structurally diverse molecules
in the CoMFA contour plots in the region of the A ring. It has suggests that the requirements for ER binding are rela-
been shown previously that size constraints on A ring sub- tively stringent. This latter hypothesis is consistent with
stituents for another biological activity of 2-MEO derivatives, the fact that the data sets include compounds that fit ER
where short, unbranched sidechains have been shown to well (i.e., they have high affinity). Indeed, estrogen has
favor inhibition of tubulin polymerization (Cushman et al., been shown to bind snugly at a site near the region of the
1995). The relationship between antiproliferative activity monomer of ER␣ that is involved with dimerization, as
and microtubule assembly has been questioned by data from determined by X-ray crystallographic studies (Tanenbaum
Attalla et al. (1996) who suggest that the action of 2-MEO is et al., 1998). In our explorations for analogs of 2-MEO
caused by a paclitaxel-like effect on tubulin dynamics induc- with increased potency for DNA synthesis inhibition, we
ing a functional defect in the mitotic spindle. Novel 2-MEO will attempt to design molecules lacking ER affinity, using
analogs substituted to achieve a paclitaxel-like structure the knowledge of these stringent requirements for ER
have increased potency as agents that enhance tubulin poly- binding.
merization without appreciable affinity for the colchicine
Derivation of CoMFA models for DNA synthesis inhibi-
binding site on tubulin (Verdier-Pinard et al., 2000) and are tion from a complete data set (37 compounds) gave models
potent antiproliferative agents [e.g., 2-ethoxyestradiol-17␤- with poor predictive capability, as assessed by the low
acetate (Cushman et al., 1997)]. Whether the similarity be- values obtained for q2 derived from cross-validated analy-
tween the current QSAR model and the SAR observations on ses. Models with significantly greater predictive power
cytotoxicity (Cushman et al., 1997) reflects a similar mecha- could be obtained by excluding outliers, identified in an
nism of action for cytotoxicity and DNA synthesis inhibition iterative manner. Although the act of excluding outliers is
remains unclear. Other inhibitors of microtubule function inherently unsatisfying, it is accepted practice in QSAR
either increase (e.g., colchicine) or decrease (e.g., paclitaxel) studies to omit such compounds in the spirit of developing

1068
Hughes et al.
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DNA synthesis inhibition in airway smooth muscle for a
set of analogs of 2-MEO. The model differs in a number of
characteristics from models derived for ER binding from a
related data set. Application of this molecular modeling
approach to the microtubule action of 2-MEO and analogs
may also be valuable, because it seems likely that micro-
tubule dysfunction would result in dose- and indication-
limiting cytotoxic side effects, such as those seen with
colchicine and paclitaxel. These models would be valuable
tools to assist in the design of compounds with improved
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Acknowledgments
We thank John Wilson and the staff of the Respiratory Medicine
and Pathology Departments at the Alfred Hospital, Prahran, for
providing specimens of human airway tissue.
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Address correspondence to: Dr. Alastair Stewart, Department of Phar-
macology, University of Melbourne, Victoria 3010, Australia. E-mail:
astew@clyde.its.unimelb.edu.au