2-(hydroxyalkyl)estradiols: Synthesis and biological evaluation

Article abstract of DOI:10.1021/jm9508245

Synthetic estrogens possessing hydroxyalkyl side chains at the C-2 position of the A-ring were designed in order to further elucidate the structural and electronic requirements of the estrogen receptor to A-ring modifications. Furthermore, these compounds were envisaged as being stable analogs of the estradiol metabolite 2-hydroxyestradiol. The homologous series of 2-(hydroxy-alkyl)estradiols 1-3 has been prepared by chain extension of 2- formylestradiol 6, which, in turn, was prepared via ortholithiation of estradiol. The substituted estradiols 1-3 were assayed for their abilities to bind to the estrogen receptor in MCF-7 cells and induce estrogen-responsive gene expression. The estradiol homologs exhibited significantly weaker affinity than estradiol for the MCF-7 cell estrogen receptor, with relative binding affinities (estradiol = 100) ranging from 1.11 for 2- (hydroxymethyl)estradiol (1) to 0.073 for 2-(hydroxypropyl)estradiol (3). The relative activities for mRNA induction of the pS2 gene by the estradiol homologs closely parallel the relative binding affinities for the estrogen receptor in MCF-7 cells. 2-(Hydroxymethyl)estradiol (1) exhibited similar estrogen receptor affinity and pS2 gene induction to the catechol estrogen 2- hydroxyestradiol and may prove useful in examination of the further biological effects of 2-hydroxyestrogen homologs.

Full text of DOI:10.1021/jm9508245

J . Med. Chem. 1996, 39, 1917-1923
1917
Notes
2
-(Hyd r oxya lk yl)estr a d iols: Syn th esis a n d Biologica l Eva lu a tion 1,2
Carl J . Lovely, Nancy E. Gilbert, Muriel M. Liberto, Damon W. Sharp, Young C. Lin, and
Robert W. Brueggemeier*
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Department of Veterinary Biosciences, College of
Veterinary Medicine, and The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
X
Received November 8, 1995
Synthetic estrogens possessing hydroxyalkyl side chains at the C-2 position of the A-ring were
designed in order to further elucidate the structural and electronic requirements of the estrogen
receptor to A-ring modifications. Furthermore, these compounds were envisaged as being stable
analogs of the estradiol metabolite 2-hydroxyestradiol. The homologous series of 2-(hydroxy-
alkyl)estradiols 1-3 has been prepared by chain extension of 2-formylestradiol 6, which, in
turn, was prepared via ortholithiation of estradiol. The substituted estradiols 1-3 were assayed
for their abilities to bind to the estrogen receptor in MCF-7 cells and induce estrogen-responsive
gene expression. The estradiol homologs exhibited significantly weaker affinity than estradiol
for the MCF-7 cell estrogen receptor, with relative binding affinities (estradiol ) 100) ranging
from 1.11 for 2-(hydroxymethyl)estradiol (1) to 0.073 for 2-(hydroxypropyl)estradiol (3). The
relative activities for mRNA induction of the pS2 gene by the estradiol homologs closely parallel
the relative binding affinities for the estrogen receptor in MCF-7 cells. 2-(Hydroxymethyl)-
estradiol (1) exhibited similar estrogen receptor affinity and pS2 gene induction to the catechol
estrogen 2-hydroxyestradiol and may prove useful in examination of the further biological effects
of 2-hydroxyestrogen homologs.
In tr od u ction
The structure-activity relationships of estrogens
have received extensive investigations over the past 3
decades, with studies focusing on the evaluation of
uterotrophic activity, estrogen receptor affinities, and
stimulation of cell growth.3 Estradiol is the most potent
endogenous estrogen, and the estrogen metabolites
F igu r e 1. Structure of 2-(hydroxyalkyl)estradiols.
estrone and estriol exhibit limited estrogenic activity
compared to estradiol. Two other metabolites, 2-hy-
electronic requirements of the estrogen receptor to
droxyestradiol and 4-hydroxyestradiol, also have sig-
A-ring modifications, estradiols 1-3 possessing a hy-
4
nificantly lower estrogenic activity than estradiol.
droxyalkyl side chain were designed. Furthermore,
More recently, the ability of estrogenic substances to
these previously unprepared compounds were envisaged
induce gene transcription at the mRNA level of estrogen-
as being stable analogs of the estradiol metabolite
5
responsive sequences has been investigated. The
results of these studies have shed light on some of the
structural and electronic requirements of the estrogen
receptor and the mechanisms through which estrogens
elicit a biological response.3
1
2
-hydroxyestradiol. Estradiol is metabolized in vivo by
oxidation by a cytochrome P450 enzyme complex termed
/4-hydroxylase. The catechol estradiols are then rap-
2
idly methylated by catechol O-methyltransferase, which
renders the products much less estrogenic.
It has long been believed that only minor modifica-
tions in the A-ring can be made without losing signifi-
cant binding affinity.6 However, a recent report has
shown that larger substituents can be tolerated and that
electronic considerations may be important.7 In addi-
tion, estrogen analogs containing the hydroxyl group at
different positions of the A-ring (at C-2, C-3, or C-4) have
demonstrated subtle differences in estrogen receptor
affinity but significant differences in abilities to induce
The catechol estradiols have been implicated as a
possible causitive agent in estrogen-induced tumorigen-
esis. It has been postulated that the mechanism of
carcinogenesis stems from the formation of further
oxidized species, such as semiquinones or orthoquinones
9
which form protein or possibly DNA adducts leading to
10,11
changes in function.
A further postulate is that
these quinone species undergo redox cycling producing
12
hydroxyl radicals, leading to DNA damage. We were
8
the expression of various estrogen-responsive genes. In
order to further the understanding of the structural and
interested in examining the potential of oxidative dam-
age of the catechol estradiols in tumorigenesis; however,
as these compounds are both chemically and biochemi-
cally short-lived, the use of them in vitro and in vivo is
difficult. The synthetic compounds 1-3 (Figure 1)
should not suffer from chemical instability and would
not be susceptible to quinone/semiquinone formation.
*
Address all correspondence to this author at College of Pharmacy,
5
00 W. 12th Ave., The Ohio State University, Columbus, OH 43210.
Phone: (614) 292-5231. Fax: (614) 292-2435. E-mail: Brueggemeier.1@
osu.edu.
X
Abstract published in Advance ACS Abstracts, March 15, 1996.
0
022-2623/96/1839-1917$12.00/0 © 1996 American Chemical Society

1
918 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Notes
Sch em e 1a
a
Reagents and conditions: (a) MOMCl, i-Pr2NEt, THF, ∆, 96%; (b) (i) s-BuLi, THF, -78 °C, (ii) DMF, -78 °C f room temperature,
6%; (c) HCl, THF, H2O, room temperature, quantitative; (d) LiAlH4, THF, 0 °C f room temperature, 7 f 1, 71%, 11 f 12, quantitative;
8
(
e) Ph3PdCH2, THF, room temperature, 90%; (f) (i) BH3‚THF, THF, 0 °C, (ii) NaOH, H2O2, ∆, 8 f 9, 80%, 13 f 12, 77%; (g) PPTS,
MeOH, ∆, 9 f 2, quantitative, 12 f 3, 72%; (h) EtO2CCH2P(O)(OEt)2, KHMDS, THF, RT, 81%; (i) PhI(OAc)2, NH2NH2, CH2Cl2, room
temperature, 93%; (j) (i) s-BuLi, THF, -78 °C, (ii) CuI, (iii) allyl bromide, 66%.
If redox cycling of catechol estrogens is important for
tumor formation, then these homologs would be ex-
pected to have greatly diminished activity. On the other
hand, compounds 1-3 do contain hydroxyl groups at
positions 2 and 3 available for hydrogen bonding in
protein interactions (receptors or enzymes) and may
exhibit classical estrogenic activity. Therefore, these
homologs may prove useful as chemical probes for
differentiating receptor-mediated vs redox-mediated
events which operate in estrogen-induced tumorigen-
esis. This report describes the synthesis of an initial
series of 2-(hydroxyalkyl)estradiols 1-3, their relative
estrogen receptor affinities, and their ability to induce
estrogen-responsive gene expression.
s-BuLi at low temperature, and the resulting anion was
quenched with dimethylformamide to give the 2-formy-
lated estradiol 6 in 86% yield as a single regioisomer.
For this reaction to be successful, it was essential to
distill the dimethylformamide from calcium hydride just
prior to use, otherwise the reaction would fail. It is
noteworthy that the formylated product was obtained
as a single regioisomer, as evidenced by one aldehyde
1
proton signal observed in the H NMR spectrum of the
crude product. It had previously demonstrated that 6
could be deprotected with aqueous acid to give 7.14 After
removal of the MOM ethers, 7 was reduced with lithium
aluminum hydride to give the first estratriol 1 in 71%
yield (Scheme 1).
It had originally been our intention to prepare the
hydroxyethyl and hydroxypropyl analogs by lithiation
of 5 and subsequent alkylation with ethylene oxide or
allyl bromide, respectively. Several experiments were
performed to probe the workability of this approach;
however, they were uniformally unsuccessful. Although
we were later able to prepare the propyl derivative in
this way (vide infra), it was decided to synthesize the
higher analogs via homologation of 6.
Resu lts a n d Discu ssion
Ch em istr y. The synthesis of the catechol analogs
began by converting estradiol 4 to the known bisMOM
ether 5 in 96% yield by treatment with methoxymethyl
chloride and diisopropylethylamine in THF at reflux.
The protected estradiol 5 was formylated in the 2-posi-
1
3
1
4
tion according to the procedure of Pert and Ridley.
Thus, the 2-position was lithiated by treatment of 5 with

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1919
Ta ble 1. Estrogen Receptor Affinities of Estradiol Homologs
compd
EC50 (M)
RBAa
Estrogen Receptor Preparations
-
-
10
7
estradiol
1
9.96 × 10
100.00
0.68
1.46 × 10
Whole Cell Estrogen Receptor Assays
-
-
-
-
-
-
9
7
6
6
7
6
estradiol
3.80 × 10
3.42 × 10
4.89 × 10
5.22 × 10
7.82 × 10
1.66 × 10
100.00
1.11
0.078
0.073
0.486
0.229
1
2
3
7
2
-hydroxyestradiol
a
The relative binding affinity (RBA) for each synthetic homolog
was determined using the following equation: RBA ) (EC50 for
estradiol)/(EC50 for homolog) × 100.
F igu r e 2. Estrogen receptor binding for estradiol (O), 2-(hy-
droxymethyl)estradiol (9), and 2-(hydroxyethyl)estradiol (0).
Although several homologation strategies were ex-
plored, simple Wittig methylenation of 6 with Ph3PdCH2
was the most successful, giving the vinylestradiol 8 in
9
0% yield (Scheme 1). Hydroboration of the double bond
followed by oxidative workup gave the (hydroxyethyl)-
estradiol 9 in 80% yield. The triol 2 could then be
readily obtained in good yield by exposure of 9 to PPTS
in refluxing methanol.15
The hydroxypropyl compound 3 was obtained in a
somewhat similar fashion. Thus, treatment of 6 with
the anion generated from triethyl phosphonacetate and
potassium hexamethyldisilazide gave the cinnamate 10
in 81% yield as a 20:1 mixture of E/ Z isomers. At-
tempted reduction of the double bond with NaBH4 or
by catalytic hydrogenation was unsuccessful; however,
use of diimide, generated in situ from hydrazine hydrate
and iodobenzene diacetate, gave the dihydrocinnamate
1
6
1
1 in 93% yield. The ester was reduced in quantita-
F igu r e 3. Northern analysis of mRNA levels for estrogen-
responsive pS2 gene and control 36B4 gene expressed by
2-(hydroxymethyl)estradiol, 2-(hydroxyethyl)estradiol, and 2-(hy-
droxypropyl)estradiol.
tive yield with lithium aluminum hydride to the propyl
alcohol 12; removal of the MOM groups was then
achieved as before by refluxing a methanol solution of
1
2 with PPTS.
After preparing the triol 3 by homologation of 6, it
Ta ble 2. Induction of pS2 Gene Expression by Estradiol
Homologs
was subsequently found that the anion generated by
ortholithiation of 5 could be alkylated efficiently with
allyl bromide provided certain modifications of the
reaction conditions were carried out. Thus, if the anion
generated from 5 and s-BuLi was transmetalated with
copper iodide to form the cuprate, followed by treatment
with allyl bromide, then the 2-allylated estradiol 13
could be obtained in 65% (89% based on recovered
starting material) yield.17 Hydroboration of 13 followed
by oxidative workup gave 12 in 77% yield (Scheme 1).
Biologica l Eva lu a tion s. The affinities of the syn-
thetic estradiol homologs for the estrogen receptor were
compd
estradiol
1
2
EC50 (M)
relative activitya
-
10
0.996 × 10
100.00
0.967
0.0367
0.0201
0.0198
-
8
1.03 × 10
-7
2.72 × 10
4.95 × 10-7
3
-
7
2-hydroxyestradiol
5.04 × 10
a
The relative activity for each synthetic homolog was deter-
mined using the following equation: relative activity ) (EC50 for
estradiol)/(EC for homolog) × 100.
50
genes. The induction of transcription of the pS2 gene
in human MCF-7 mammary carcinoma cells is a pri-
mary response to estrogen.
6
19
assessed in isolated estrogen receptor preparations and
The induction of pS2
whole cell estrogen receptor binding assays using MCF-7
human mammary cancer cells.18 The EC50 value for
estradiol binding to the estrogen receptor preparations
using 2.0 nM [ H]estradiol was found to be 0.996 nM,
while the affinity for compound 1 was 146 nM (Table
mRNA expression by estradiol and estrogen homologs
was determined by Northern analysis (Figure 3). The
EC50 value for estradiol induction of pS2 mRNA was
found to be 0.292 nM. The estradiol homologs exhibited
significantly weaker activity than estradiol for pS2
mRNA induction, with relative activities (estradiol )
100) ranging from 2.79 for compound 1 to 0.004 for
compound 3 (Table 2, Figure 4).
Thus, the 2-(hydroxyalkyl)estradiols 1-3 exhibited
significantly weaker affinity for the estrogen receptor
than estradiol. These results are congruous with the
established structure-activity relationships of estrogens
and the limitations for A-ring substitutions. Further-
more, the relative activities for pS2 mRNA induction
of the estradiol homologs closely parallel the relative
3
1
). The EC50 value for estradiol binding to the estrogen
3
receptor in these whole cell assays using 3.0 nM [ H]-
estradiol was found to be 3.80 nM. The estradiol
homologs exhibited significantly weaker affinity for the
estrogen receptor than estradiol, with relative binding
affinities (RBA; estradiol ) 100) ranging from 1.11 for
compound 1 to 0.073 for compound 3 (Table 1, Figure
2
).
Estradiol acts through the estrogen receptor to induce
the transcription of a variety of hormone-responsive

1
920 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Notes
2
1
925-2787, 1610, 1576, 1498, 1471, 1446, 1403, 1248, 1205,
1
151, 1134, 1116, 1105, 1076, 962, 922, 873; H NMR 7.19 (1H,
d, J ) 8.5 Hz), 6.82 (1H, dd, J ) 8.5, 2.7 Hz), 6.76 (1H, d, J )
3
3
(
.0 Hz), 5.14 (2H, s), 4.65 (2H, ABq, J ) 6.6 Hz, ∆v ) 3.4 Hz),
.61 (1H, t, J ) 8.4 Hz), 3.46 (3H, s), 3.37 (3H, s), 2.87-2.81
2H, m), 0.80 (3H, s); 13C NMR 155.2, 138.1, 134.1, 126.3, 116.4,
1
2
13.8, 96.1, 94.6, 86.7, 55.8, 55.1, 50.2, 44.1, 43.1, 38.7, 37.4,
9.1, 28.1, 27.2, 26.3, 23.1, 11.7; MS m/ z (M ) calcd 360.2301,
+
obsd 360.2306.
-F or m ylest r a -1,3,5(10)-t r ien e-3,17â-d iol 3,17â-Bis-
m eth oxym eth oxy) Eth er (6). s-BuLi (50 mL, 65.0 mmol)
was added dropwise to a cold (-78 °C) solution of 5 (6.00 g,
6.7 mmol) in THF (100 mL). The resulting brown solution
2
(
1
was stirred at -78 °C for 2 h, at which time freshly distilled
DMF was added and the reaction mixture was allowed to
warm up to room temperature overnight. The reaction
mixture was cooled to 0 °C; then 10% HCl (40 mL) was added
cautiously. After separation of the organic layer, the aqueous
F igu r e 4. Induction of pS2 gene expression by estradiol (O),
2
2
-(hydroxymethyl)estradiol (9), 2-(hydroxyethyl)estradiol (0),
-(hydroxypropyl)estradiol (]), and 2-hydroxyestradiol (b).
layer was extracted with Et
organic extracts were washed with water (2 × 100 mL) and
saturated brine (100 mL), dried (MgSO ), and concentrated.
The residue was purified by chromatography (3:1) to afford
.56 g (86%) of the desired product as a colorless oil: IR (neat,
2
O (4 × 100 mL). The combined
4
binding affinities for the estrogen receptor in MCF-7
cells. Interestingly, 2-(hydroxymethyl)estradiol (1) ex-
hibited similar estrogen receptor affinity and pS2 gene
induction to the catechol estrogen 2-hydroxyestradiol.
This catechol estradiol has been implicated as a possible
causitive agent in estrogen-induced tumorigenesis; how-
ever, in vitro and in vivo investigations with 2-hydroxy-
estradiol are difficult due to its chemical and biochemi-
cal instability. Thus, 2-(hydroxymethyl)estradiol (1)
may be viewed as a chemically stable catechol estrogen
homolog and may therefore prove useful in examination
of the role of catechol estrogens in normal physiology
and pathological states, such as estrogen-induced
tumorigenesis.
5
-1
cm ) 2929-2871, 1684, 1608, 1491, 1471, 1446, 1423, 1392,
1
1
261, 1209, 1151, 1105, 1054, 1025, 997, 919; H NMR 10.41
(1H, s), 7.75 (1H, s), 6.90 (1H, s), 5.25 (2H, s), 4.65 (2H, ABq,
J ) 6.7 Hz, ∆v ) 3.1 Hz), 3.60 (1H, t, J ) 8.4 Hz), 3.50 (3H,
1
3
s), 3.36 (3H, s), 2.91-2.87 (2H, m), 0.79 (3H, s); C NMR 189.4,
1
5
1
57.5, 146.2, 134.6, 125.3, 123.5, 115.1, 96.0, 94.7, 86.5, 56.5,
5.1, 50.1, 43.7, 43.0, 38.4, 37.1, 30.3, 28.1, 26.8, 26.1, 23.0,
1.6; MS m/ z (M ) calcd 388.2250, obsd 388.2273.
+
2
-F or m ylestr a -1,3,5(10)-tr ien e-3,17â-d iol (7). A solution
of 6 (2.00 g, 5.15 mmol), 6 M aqueous HCl (25 mL), and THF
(25 mL) was stirred at room temperature for 3 h. The reaction
mixture was poured into water and then extracted with EtOAc
(
4 × 50 mL). The combined organics were dried (MgSO
4
),
concentrated, and then chromatographed (1:3 increasing to 1:2)
to afford the title compound (1.52 g, 98%) as a pale yellow,
crystalline solid: mp 225-227 °C (lit. mp 231-233 °C); IR
(KBr, cm ) 3361, 3116, 3023, 2964, 2927, 2865, 1658, 1612,
1442, 1384, 1352, 1342, 1290, 1267, 1242, 1117, 1051, 1020,
Ma ter ia ls a n d Meth od s
1
4
-
1
Syn th esis. Gen er a l In for m a tion . Steroids were pur-
chased from Steraloids (Wilton, NH); all other chemicals were
purchased from Aldrich Chemical Co. (Milwaukee, WI) and
used as received unless otherwise indicated. Anhydrous
solvents were dried by standard procedures, and amines were
1
870; H NMR (DMSO) 10.41 (1H, brs), 10.12 (1H, s), 7.55 (1H,
s), 6.66 (1H, s), 4.50 (1H, d, J ) 4.7 Hz), 3.52 (1H, q, J ) 8.2
Hz), 2.49 (2H, brm), 0.65 (3H, s); 13C NMR (DMSO) 192.2,
158.2, 146.6, 131.9, 126.5, 120.0, 116.4, 79.9, 49.4, 42.9, 42.6,
2
stirred over CaH , distilled, and stored over KOH pellets.
+
Silica gel TLC plates (60 F254) were purchased from Analtech
Inc. (Newark, NE) and visualized with a UV lamp and/or 5%
ethanolic phosphomolybdic acid followed by charring. All
intermediates were purified by flash column chromatography
on silica gel (Merck Kieselgel 60) using the indicated mixtures
of hexanes and ethyl acetate. Melting points were determined
in open capillaries on a Thomas Hoover capillary melting point
apparatus and are uncorrected. IR spectra were recorded on
a Laser Precision Analytical RFX-40 FTIR spectrometer in the
38.1, 36.3, 29.8, 29.3, 26.3, 25.8, 22.6, 11.0; MS m/ z (M ) calcd
300.1725, obsd 300.1732.
2-(Hyd r oxym eth yl)estr a -1,3,5(10)-tr ien e-3,17â-d iol (1).
4
LiAlH (0.20 g, 5.20 mmol) was added portionwise to a solution
of the aldehyde 7 (0.21 g, 0.70 mmol) in cold (0 °C) THF (10
mL). The resulting mixture was stirred for 2 h while warming
to room temperature. The reaction was quenched by the
addition of water (0.2 mL), 15% NaOH (0.2 mL), and water
(0.6 mL). The granular precipitate was removed by filtration
1
13
phase indicated. H and C NMR were recorded on an IBM
AF/250 spectrometer at 250 and 67.5 MHz, respectively, in
4
through a pad of Celite and MgSO (1:1). After concentration,
the residue was recrystallized from water and methanol to give
CDCl
3
solutions unless otherwise indicated using the residual
the desired triol (150 mg, 71%) as a colorless solid: mp > 270
-
1
protiosolvent signal as internal reference. NMR data are
reported in δ units. Mass spectra were obtained at The Ohio
State University Chemical Instrumentation Center on either
a VG 70-2505, a Nicolet FTMS-200, or a Finnigan MAT-900
mass spectrometer. Elemental Analyses were performed by
Oneida Research Services, Inc. (Whitesboro, NY).
E st r a -1,3,5(10)-t r ien e-3,17â-d iol 3,17â-Bis(m et h oxy-
m eth oxy) Eth er (5). MOMCl (9.1 mL, 120 mmol) was added
dropwise to a cold (0 °C) solution of estradiol (6.50 g, 23.9
mmol) and diisopropylethylamine (34 mL, 143 mmol) in THF
°C; IR (KBr, cm ) 3514, 3319, 3147, 2954, 2922, 2866, 1614,
1508, 1431, 1381, 1354, 1340, 1319, 1286, 1252, 1213, 1186,
1
1130, 1115, 1095, 1070, 1052, 1034, 1016, 972, 866; H NMR
(DMSO) 8.92 (1H, s), 7.15 (1H, s), 6.41 (1H, s), 4.80 (1H, t, J
) 5.6 Hz), 4.49 (1H, d, J ) 4.8 Hz), 4.40 (2H, d, J ) 5.5 Hz),
2.48-2.66 (2H, m), 0.65 (3H, s); 13C NMR (DMSO) 151.8, 135.0,
130.0, 125.4, 124.4, 114.4, 80.0, 58.4, 49.5, 43.5, 42.7, 38.5, 36.5,
+
29.8, 28.7, 26.9, 26.1, 22.7, 11.1; MS m/ z (M ) calcd 302.1875,
obsd 302.1882. Anal. Calcd for C19
H, 8.70. Found: C, 74.25; H, 8.46.
H
26
O
3
2
‚0.25H O: C, 74.36;
(
200 mL). On completion of the addition, the reaction mixture
2-E t h en ylest r a -1,3,5(10)-t r ien e-3,17â-d iol 3,17â-Bis-
(m eth oxym eth oxy) Eth er (8). A solution of KHMDS (0.64
g, 3.22 mmol) in THF (10 mL) was added by cannula to a
suspension of methyltriphenylphosphonium iodide (1.30 g, 3.22
mmol) in THF (10 mL) at room temperature followed by
stirring for 1 h at the same temperature. 6 (0.50 g, 1.29 mmol)
was added to the ylide solution and then stirred for an
was allowed to warm up to room temperature, stirred for 1 h
at the same temperature, and then heated at reflux overnight.
The mixture was allowed to cool; then saturated NH
solution (100 mL) was added. After extraction with Et O (4
100 mL), the combined organics were washed with saturated
4
Cl
2
×
brine (100 mL), dried (MgSO
4
), and concentrated. The crude
product was purified by chromatography (4:1) to afford 8.30 g
additional 2 h at room temperature. Saturated NH
was added; then the aqueous solution was extracted with
4
Cl (50 mL)
-
1
(97%) of the title compound as a colorless oil: IR (neat, cm )

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1921
EtOAc (3 × 100 mL). The combined organic extracts were
calcd 458.2668, obsd 458.2671. Anal. Calcd for C27
70.72; H, 8.35. Found: C, 70.81; H, 8.34.
38 6
H O : C,
4
washed with brine (100 mL), dried (MgSO ), and concentrated.
The residue was purified by chromatography (4:1) to afford
Eth yl 3,17â-Bis(m eth oxym eth oxy)estr a -1,3,5(10)tr ien e-
-1
0
1
7
)
4
.48 g (96%) of 8 as a colorless oil: IR (neat, cm ) 2927, 1496,
149, 1105, 1065, 1043, 1022, 999, 918; H NMR 7.40 (1H, s),
2-p r op ion oa te (11). Iodobenzene diacetate (0.57 g, 1.77
1
mmol) in CH
solution of 10 (0.54 g, 1.18 mmol) and hydrazine hydrate (0.24
g, 4.72 mmol) in CH Cl (5 mL) at room temperature. The
2 2
Cl (15 mL) was added dropwise to a stirred
.01 (1H, dd, J ) 11.2, 17.8 Hz), 6.80 (1H, s), 5.68 (1H, dd, J
1.6, 17.7 Hz), 5.20 (1H, dd, J ) 1.6, 11.2 Hz), 5.17 (2H, s),
2
2
.66 (2H, ABq, J ) 6.6 Hz, ∆v ) 3.5 Hz), 3.61 (1H, t, J ) 8.4
reaction mixture was stirred overnight; then the same amount
of reagents was added neat and followed by overnight stirring.
This procedure was repeated until the reaction was complete
Hz), 3.45 (3H, s), 3.37 (3H, s), 2.85-2.81 (2H, m), 0.80 (3H, s);
1
3
C NMR 152.4, 137.8, 134.0, 132.0, 125.1, 123.5, 115.2, 113.5,
6.1, 95.0, 86.7, 56.1, 55.1, 50.2, 44.1, 43.1, 38.7, 29.7, 28.2,
9
2
3
(TLC), requiring a total of 4 days. Aqueous NaHCO
(50 mL) was added to the reaction mixture, which was
extracted with CH Cl
(3 × 50 mL). The combined extracts
were dried (MgSO ), concentrated, and chromatographed (6:
3
solution
+
7.3, 26.3, 23.1, 11.7; MS m/ z (M ) calcd 386.2457, obsd
86.2462. Anal. Calcd for C24
H
34
O
4
:
C, 74.58; H, 8.87.
2
2
Found: C, 74.64; H, 8.89.
-(2′-Hyd r oxyeth yl)estr a -1,3,5(10)-tr ien e-3,17â-d iol 3,-
7â-Bis(m eth oxym eth oxy) Eth er (9). BH ‚THF complex in
THF (5.0 mL, 5.00 mmol) was added dropwise to a solution of
(0.48 g, 1.24 mol) in THF (10 mL) at 0 °C. The cooling bath
was removed, and stirring was continued for 1 h; 1 N NaOH
10 mL) was cautiously added followed by 30% H (10 mL).
4
-
1
1
2
1
) to yield 0.50 g (93%) of 13 as a colorless oil: IR (neat, cm )
2
929-2825, 1736, 1502, 1468, 1446, 1371, 1286, 1252, 1178,
1
3
1
105; H NMR 7.07 (1H, s), 6.78 (1H, s), 5.16 (2H, s), 4.65 (2H,
s), 4.12 (2H, q, J ) 7.1 Hz), 3.60 (1H, t, J ) 8.4 Hz), 3.47 (3H,
s), 3.36 (3H, s), 2.91 (2H, t, J ) 8.0 Hz), 2.81-2.78 (2H, m),
8
2
.58 (2H, t, J ) 8.0 Hz), 1.24 (3H, t, J ) 7.1 Hz). 0.79 (3H, s);
C NMR 173.3, 153.1, 136.0, 133.6, 127.0, 126.8, 114.2, 96.1,
4.5, 86.7, 60.2, 56.0, 55.1, 50.2, 44.1, 43.1, 38.7, 37.4, 34.8,
(
2 2
O
1
3
The resulting mixture was heated to a gentle reflux for 1 h.
After cooling, the reaction mixture was extracted with EtOAc
9
2
4
+
9.6, 28.2, 27.3, 26.4, 26.1, 23.1, 14.2, 11.2; MS m/ z (M ) calcd
(
3 × 50 mL), washed with brine (2 × 50 mL), dried (MgSO
4
),
60.2825, obsd 460.2822. Anal. Calcd for C27
40 6
H O : C, 70.41;
and concentrated. The residue was chromatographed (1:1) to
give 0.40 g (80%) of 9 as a colorless oil: IR (neat, cm ) 3344,
-
1
H, 8.50. Found: C, 70.41; H, 8.75.
-(3′-Hyd r oxypr op yl)estr a -1,3,5(10)-tr ien e-3,17â-diol 3,-
7â-Bis(m eth oxym eth oxy) Eth er (12). 11 (0.48 g, 1.04
mmol) in THF (25 mL) was added dropwise to a cold (0 °C)
suspension of LiAlH (198 mg, 5.20 mmol). On completion of
2
2
1
9
931, 1502, 1471, 1444, 1417, 1400, 1382, 1382, 1369, 1356,
340, 1317, 1284, 1259, 1207, 1182, 1149, 1117, 1105, 1061,
20; H NMR 7.08 (1H, s), 6.80 (1H, s), 5.16 (2H, s), 4.65 (2H,
1
1
4
s), 3.87-3.79 (3H, m), 3.60 (1H, t, J ) 8.4 Hz), 3.46 (3H, s),
.37 (3H, s), 2.89-2.79 (4H, m), 0.79 (3H, s); 1 C NMR 153.3,
3
the addition, the mixture was allowed to warm to room
temperature and then stirred for 9 h. The reaction was
quenched by the addition of water (0.2 mL), 15% NaOH (0.2
mL), and water (0.6 mL), and the resulting granular precipi-
tate was removed by filtration through a 1:1 mixture of Celite
3
1
5
2
36.4, 133.9, 128.0, 124.8, 114.4, 96.1, 94.7, 86.7, 63.1, 56.1,
5.1, 50.1, 44.0, 43.1, 38.7, 37.4, 34.1, 29.7, 28.2, 27.3, 26.4,
+
3.1, 11.8; MS m/ z (M ) calcd 404.2563, obsd 404.2563. Anal.
Calcd for C24
.87.
-(2′-Hyd r oxyeth yl)estr a -1,3,5(10)-tr ien e-3,17â-d iol (2).
36 5
H O : C, 71.26; H, 8.97. Found: C, 70.93; H,
4
and MgSO . The filtrate was concentrated and purified by
8
chromatography (1:1) to give 0.43 g (quantitative) of the alcohol
2
-1
as a colorless oil: IR (neat, cm ) 3855-3435, 3028-2927,
A solution containing 9 (120 mg, 0.30 mmol) and pyridinium
p-toluenesulfonate (0.34 g, 1.35 mmol) in methanol (5 mL) was
heated at reflux overnight. After cooling to room temperature,
the reaction mixture was poured into water (50 mL) which
was then extracted with EtOAc (3 × 50 mL). The combined
1
1
614, 1576, 1502, 1473, 1448, 1417, 1400, 1382, 1355, 1149,
1
117, 1105, 920; H NMR 7.25 (1H, s), 7.07 (1H, s), 5.18 (2H,
s), 4.65 (2H, s), 3.85-3.50 (3H, m), 3.48 (3H, s), 3.37 (3H, s),
2
(
9
2
4
.83-2.81 (2H, m), 2.69 (2H, t, J ) 7.5 Hz), 1.84 (2H, m), 0.80
13
3H, s); C NMR 153.1, 135.7, 133.8, 127.9, 127.2, 114.4, 96.1,
4
organics were washed with brine (50 mL), dried (MgSO ), and
4.9, 86.8, 62.2, 56.1, 55.1, 50.2, 44.1, 43.1, 38.7, 37.4, 33.4,
concentrated. The residue was recrystallized from methanol
and water to give 94 mg (quantitative) of a colorless crystalline
+
9.6, 28.2, 27.3, 26.4, 26.2, 23.1, 11.8; MS m/ z (M ) calcd
18.2719, obsd 418.2717. Anal. Calcd for C25
38 5
H O : C, 71.74;
-1
solid: mp 201-202 °C; IR (KBr, cm ) 3400, 2919, 2870, 2856,
H, 9.15. Found: C, 71.46; H, 8.88.
2
1
840, 1512, 1469, 1437, 1421, 1383, 1354, 1340, 1282, 1267,
253, 1213, 1209, 1186, 1117, 1087, 1053, 1009, 989; H NMR
2
-(3′-Hydr oxypr opyl)estr a-1,3,5(10)-tr ien e-3,17â-diol (3).
1
A solution containing 12 (0.56 g, 1.33 mmol) and pyridium
p-toluenesulfonate (2.96 g, 13.30 mmol) in methanol (25 mL)
was heated at reflux overnight. After cooling, water (50 mL)
and EtOAc (50 mL) were added. The separated aqueous layer
was extracted with EtOAc (2 × 50 mL); the combined organics
(
CDCl
3 3
/CD OD) 7.00 (1H, s), 6.51 (1H, s), 3.77 (2H, t, J ) 6.8
Hz), 3.67 (1H, t, J ) 8.5 Hz), 2.84-2.67 (2H, m), 2.82 (2H, t,
J ) 7.1 Hz), 0.78 (3H, s); C NMR 153.7, 136.7, 132.5, 128.5,
1
2
3
8
1
3
23.9, 116.3, 82.2, 63.6, 51.0, 44.9, 44.0, 40.0, 37.7, 35.0, 30.0,
8.2, 27.3, 23.8, 11.5; MS m/ z (M ) calcd 330.2195, obsd
30.2194. Anal. Calcd for C20
.94. Found: C, 74.79; H, 8.94.
+
4
were washed with brine (50 mL), dried (MgSO ), and concen-
H
28
O
3
2
‚0.25H O: C, 74.85; H,
trated. The crude product was chromatographed (1:1) to give
the desired product, which was recrystallized from methanol
and water to afford 315 mg (72%) of 3 as a colorless crystalline
Eth yl 3,17â-Bis(m eth oxym eth oxy)estr a-1,3,5(10)-tr ien e-
-p r op en oa te (10). Triethyl phosphonacetate (0.52 mL, 3.34
-1
2
solid: mp 203-204 °C; IR (KBr, cm ) 3411, 2924, 2866, 1509,
1
mmol) was added dropwise to a solution of KHMDS (0.67 g,
.34 mmol) in THF (10 mL) at room temperature and stirred
1471, 1423, 1371, 1284, 1263, 1205, 1055, 1010; H NMR
3
(CDCl /CD OD) 7.00 (1H, s), 6.49 (1H, s), 3.67 (1H, t, J ) 8.5
3
3
for 1 h. The resulting solution was added by cannula to a
solution of 6 (0.65 g, 1.68 mmol) in THF (10 mL) followed by
Hz), 3.59 (2H, t, J ) 6.5 Hz), 2.80-2.75 (2H, m), 2.63 (2H, t,
13
J ) 7.5 Hz), 1.82 (2H, t, J ) 7.3 Hz), 0.78 (3H, s); C NMR
(CDCl /CD OD) 153.3, 136.0, 132.5, 127.7, 126.3, 115.9, 82.2,
4
h of stirring at room temperature. The reaction was
3
3
quenched with water (50 mL), and then the aqueous mixture
62.2, 51.0, 44.9, 44.0, 40.0, 37.7, 33.7, 30.5, 30.0, 28.2, 27.3,
26.8, 23.7, 11.5; MS m/ z (M ) calcd 330.2195, obsd 330.2193.
+
was extracted with EtOAc (4 × 50 mL). The combined organic
solutions were dried (MgSO
4
), concentrated, and chromato-
Anal. Calcd for
Found: C, 75.71; H, 9.12.
C
21
H
30
O
3
2
‚0.25H O: C, 75.30; H, 9.17.
graphed (4:1) giving 0.62 g (81%) of 10 as a colorless oil (20:1,
E/ Z): IR (neat, cm- ) 2873, 2931, 1739, 1712, 1629, 1608,
1
2-(2′-P r op en yl)estr a -1,3,5(10)-tr ien e-3,17â-d iol 3,17â-
Bis(m eth oxym eth oxy) Eth er (13). s-BuLi (1.3 M, 9.7 mL,
12.6 mmol) was added dropwise to a cold (-78 °C) solution of
5 (0.97 g, 2.69 mmol) in THF (30 mL). After stirring for 2 h
at -78 °C, the reaction mixture was transferred by cannula
to a suspension of flame-dried CuI (2.81 g, 14.7 mmol) in THF
(5 mL) and maintained at -78 °C for 1 h. Allyl bromide (2.4
mL, 15.4 mmol) was added, and then the reaction mixture was
allowed to warm to room temperature overnight. Any pre-
cipitated salts were removed by filtration through Celite, and
1
1
496, 1467, 1446, 1421, 1398, 1367, 1315, 1278, 1252, 1213,
151, 1117, 1105, 1047, 989, 920, 867; H NMR 7.98 (1H, d, J
1
)
16.2 Hz), 7.44 (1H, s), 6.85 (1H, s), 6.46 (1H, d, J ) 16.2
Hz), 5.21 (2H, s), 4.65 (2H, ABq, J ) 6.6 Hz, ∆v ) 3.4 Hz),
4
3
.24 (2H, q, J ) 7.1 Hz), 3.61 (1H, t, J ) 8.3 Hz), 3.48 (3H, s),
.38 (3H, s), 2.87-2.82 (2H, m), 1.32 (3H, t, J ) 7.1 Hz), 0.80
1
3
(3H, s); C NMR 167.5, 154.0, 141.1, 140.3, 134.2, 125.6, 121.8,
1
3
17.8, 115.1, 96.1, 94.7, 86.6, 60.2, 56.2, 55.1, 50.1, 43.8, 43.0,
8.5, 37.2, 29.9, 28.1, 27.1, 26.3, 23.1, 14.4, 11.7; MS m/ z (M )
+

1
922 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9
Notes
5
NH Cl (50 mL) was added to the filtrate. The organic layer
was separated, and then the aqueous solutions were extracted
4
divided into 9.4 cm2 wells on a six-well plate at 1.5-2 × 10
cells/well in modified MEM (2-3 mL) containing 10% steroid
free fetal calf serum and gentamycin (20 mg/mL). After 12-
24 h at 37 °C, the culture media were replaced, and the culture
was continued for a further 48 h. The media were removed,
and then serum free MEM media (888 µL) containing insulin
(5.0 mg/L), transferrin (5.0 mg/L), glutamine (2 mM), albumin
(2.0 mg/mL), and the synthetic estrogens 1-3 at various
with ethyl acetate (3 × 50 mL). The combined organic extracts
4
were washed with brine (100 mL), dried (MgSO ), and con-
centrated. The residue was purified by chromatography (4:1)
to afford 696 mg (65%) of the desired alkene as a colorless oil
and 260 mg (27%) of unreacted starting material: IR (neat,
-
1
cm ) 3050-2775, 1620, 1600, 1500, 1175, 1150, 1100, 1050,
1
-5
9
5
8
2
1
4
75; H NMR 7.07 (1H, s), 6.79 (1H, s), 6.04-5.91 (1H, m),
.16 (2H, s), 5.10-4.99 (2H, m), 4.66 (2H, s), 3.62 (1H, t, J )
.3 Hz), 3.48 (3H, s), 3.38 (3H, s), 3.46-3.36 (2H, m), 2.86-
.87 (2H, m), 0.81 (3H, s); 13C NMR 152.9, 137.4, 135.7, 133.7,
27.0, 126.6, 115.0, 114.5, 96.1, 94.7, 86.8, 55.9, 55.1, 50.2, 44.2,
concentrations ((3-1) × 10 M, 100 µL) were added and
incubated for 10 min at 37 °C. To determine total binding,
3
[
H]estradiol (3.0 nM, 1.0 µCi) was added and the plates were
then incubated for 1 h at 37 °C. The cells were washed twice
with PBS at 4 °C; then 95% ethanol (1 mL) was added followed
by standing for 30 min at room temperature. An aliquot (500
µL) of the ethanol solution was added to Formula 963 and
counted on a liquid scintillation counter. The blank samples
with no cells and the nonspecific binding samples, containing
3.1, 38.8, 37.4, 34.4, 29.6, 28.2, 27.3, 26.4, 23.1, 11.8; MS m/ z
M ) calcd 400.2618, obsd 400.2616.
+
(
2
-(3′-Hydr oxypr opyl)estr a -1,3,5(10)-tr ien e-3,17â-d iol 3,-
7â-Bis(m eth oxym eth oxy) Eth er (12). BH ‚THF (5.2 mL,
.2 mmol) was added dropwise to a solution of the alkene 13
1
5
3
6
µM unlabeled estradiol, were performed in a comparable
(560 mg, 1.40 mmol) in THF (10 mL) at 0 °C. On completion
3
manner. Specific binding of [ H]estradiol was calculated by
subtracting the nonspecific binding data from total binding
data. The EC50 value for each synthetic estrogen homolog
represents the concentration of homolog to produce a half-
of the addition, the mixture was allowed to warm to room
temperature and stirred for 1 h. NaOH (1 M, 10 mL) was
added cautiously followed by 30% H O (10 mL), and the
2 2
resulting mixture was heated to a gentle reflux for 1 h. After
cooling, the reaction mixture was extracted with ethyl acetate
3
maximal displacement of specific [ H]estradiol binding and was
calculated by a nonlinear regression analysis using the Mar-
quardt method (SAS Institute, Cary, NC).
(
3 × 50 mL). The combined organic solutions were washed
with brine (100 mL), dried (MgSO ), and concentrated. The
residue was purified by chromatography (4:1) and then MPLC
3:1) to give the desired alcohol (445 mg, 76%) as a colorless
oil.
Biologica l Eva lu a tion s. Gen er a l In for m a tion . [2,4,6,7-
4
p S2 In d u ction . MCF-7 cells were maintained in a similar
fashion as described above. Cells were placed on defined
media for 3 days. Twelve hours prior to RNA isolation, the
(
-3
-6
cells were treated with 1-3 (10 -10 M), estradiol, or carrier
(95% ethanol). The final concentration of ethanol was 0.1%
in all flasks. Total RNA was isolated by an adaptation of the
method of Chomczynski and Sacchi.20 The cells were lysed
with a 4 M guanidine isothiocyanate solution and transferred
to Phase-Lock Gel tubes (5Prime-3Prime, Inc.). After acidi-
fication with 3 M NaOAc, pH 5.2 (1:10 vol), RNA was extracted
with phenol:chloroform:isoamyl alcohol (60:24:1), phenol:chlo-
roform:isoamyl alcohol (25:24:1), and then chloroform:isoamyl
alcohol (24:1). RNA was collected by ethanol precipitation. The
pellets were washed with 70% ethanol (3×) and then resus-
pended in RNase-DNase free water for spectral analysis.
Aliquots of RNA equal to 10 µg were loaded onto a 1.5%
agarose: 0.66 M formaldehyde gel and electrophoresed for 2
h. Northern transfer was accomplished by downward capillary
action under neutral conditions using a turboblotter (Schle-
icher and Scheull, Keene, NH). Nylon membranes were
prehybridized at 42 °C for 1 h in a buffer of 50% formamide,
2.5× SSPE, 2.5× Denhardt’s solution, and 2% SDS. Mem-
branes were hybridized simultaneously with pS2 and 36B4
probes (10 cpm/mL each; specific activity of ca. 1 × 10 cpm/
µg each) for 12-20 h in a similar solution lacking SDS. The
membranes were washed in 5× SSPE, 15′, 55 °C; 2× SSPE,
15′, 60 °C; and 0.1× SSPE, 20′, 65 °C; all wash solutions
contained 1% SDS. The extent of pS2 induction was normal-
ized to the 36B4 signal in each lane and corrected for base-
line pS2 expression on an Ambis scanning proportional
counter. The EC50 value for each synthetic estrogen homolog
represents the concentration of homolog to produce a half-
maximal induction of pS2 and was calculated by a nonlinear
regression analysis using the Marquardt method (SAS Insti-
tute, Cary, NC).
3
3
2
H]Estradiol (98.4 Ci/mmol; H-E ) was purchased from Du-
Pont/NEN (Boston, MA) and used as received. MCF-7 human
breast adenocarcinoma cells were obtained from ATCC, and
cells were incubated in a humidified CO
2
incubator (Forma
model 3052) with 5% CO atmosphere. A modified Eagle’s
2
minimum essential medium (MEM) supplemented with es-
sential amino acids (1.5×), vitamins (1.5×), nonessential amino
acids (2×), and l-glutamine (1×) was obtained from Gibco BRL
(Long Island, NY) and used for maintaining the cells. The
sterilized liquid medium was prepared by The OSU Compre-
hensive Cancer Center by dissolving the powder in water
containing sodium chloride (0.487 g/L), pyruvic acid (0.11 g/L),
and sodium bicarbonate (1.5 g/L), and the pH was adjusted to
6
.8. Fetal calf serum was obtained from Gibco BRL. Steroids
were removed from the fetal calf serum by two treatments with
dextran-coated charcoal. Tissue culture flasks and supplies
were obtained from Corning Glass Works (Corning, NY).
Biochemicals were purchased from Sigma Chemical Co. (St.
Louis, MO). Radioactive samples were detected with a Beck-
man LS 6800 scintillation counter using Formula 963 (DuPont/
NEN) as the counting solution. Probes for Northern analysis
6
9
(pS2:ATCC 57137, 36B4:ATCC 65917) were obtained as puri-
fied plasmids from the American Type Culture Collection and
amplified by PCR for use in hybridization. Primers used were
synthesized by OLIGOS, ETC (Wilsonville, OR) and were for
pS2:sense, 5′-ATC CCT GAC TCG GGG TCG CCT TTG-3′;
antisense, 5′-CAA TCT GTG TTG TGA GCC GAG GCA CAG-
3
′; for 36B4:sense, 5′-AAA CTG CTG CCT CAT ATC CG-3′;
antisense, 5′-TTT CAG CAA GTG GGA AGG TG-3′.
Probes were labeled by random priming with Klenow
fragment. Analysis of the Northern blots was carried out with
2
an Ambis 100 instrument to obtain values for the cpm/mm .
Permanent visual records were made by exposing the mem-
Ack n ow led gm en t. This research was supported in
part by NIH grants R01 CA58003 and R21 CA66193.
branes to Kodak XOMAT film at -80 °C for 1-4 days.
6
Estr ogen Recep tor P r ep a r a tion s. MCF-7 mammary
2
cells were grown in 75 cm plastic flasks at 37 °C in a modified
Eagle’s MEM culture medium containing 10% fetal calf serum
and gentamycin (20 mg/L). Estrogen receptors were isolated
from homogenized cells by differential centrifugation, and
binding affinity was determined by competitive ligand binding
Refer en ces
(
1) A preliminary communication has appeared on the synthetic
aspects of this work; see: Lovely, C. J .; Brueggemeier, R. W.
Synthesis of 2-substituted hydroxyalkyl and aminoalkylestra-
diols. Tetrahedron Lett. 1994, 35, 8735-8738.
3
assays using 2 nM [ H]estradiol, as previously described by
6
(2) Aspects of the biological data have been presented at the
following meetings. Brueggemeier, R. W.; Lovely, C. J .; Gilbert,
N. E.; Hu, Y.; Lin, Y. C. Catechol Estrogens As Probes of
Estrogen Carcinogenesis. Poster presentation at the 2nd Inter-
national Symposium on Hormonal Carcinogenesis, J uly 4-9,
1994, Stockholm, Sweden. Brueggemeier, R. W.; Lovely, C. J .;
Brueggemeier et al.
Wh ole Cell Estr ogen Recep tor Stu d ies.18 MCF-7 cells
were maintained in a similar fashion as described above. Cells
from 90-100% confluent cultures were harvested by treatment
with 0.01% trypsin solution, and the washed cell pellet was

Notes
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1923
Gilbert, N. E.; Liberto, M. M.; Burak, W. E. Synthesis of Catechol
Estrogen Analogs and Evaluation in Estrogen-Responsive Hu-
man Breast Cells. Presented at the IXth International Congress
on Hormonal Steroids, Sept. 24-29, 1994, Dallas, TX.
(11) Zhu, B. T.; Roy, D.; Liehr, J . G. The carcinogenic activity of
ethinyl estrogens is determined by both their hormonal char-
acteristics and their conversion to catechol estrogens. Endocri-
nology 1993, 132, 577-583.
(
3) Duax, W. L.; Griffin, J . F.; Rhorer, D. C.; Swenson, C.; Weeks,
C. M. Molecular details of receptor binding and hormonal action
of steroids derived from x-ray crystallographic investigations.
J . Steroid Biochem. 1981, 15, 41-47.
4) Merriam, G. R., Lipsett, M. F., Eds. Catechol Estrogens; Raven
Press: New York, 1983.
(12) Liehr, J . G.; Roy, D. Free radical generation by redox cycling of
estrogens, Free Radical Biol. Med. 1990, 8, 415-423.
(
(
(
13) Pert, D. J .; Ridley, D. D. Formylation of oestrogens. Aust. J .
Chem. 1989, 42, 405-419.
(
14) Pert, D. J .; Ridley, D. D. An alternative route to 2-bromo-and
2
-iodoestradiol. Aust. J . Chem. 1987, 40, 4303-4309.
(
5) Sathyamoorthy, N.; Wang, T. T. Y.; Phang, J . M. Stimulation of
pS2 expression by diet derived compounds. Cancer Res. 1994,
15) Monti, H.; Leandri, G.; Klos-Ringuet, M.; Corriol, C. An efficient
deprotective method for allylic alcohols protected as methoxy-
ethoxymethyl (MEM) and methoxymethoxy (MOM) ethers.
Synth. Commun. 1983, 12, 1021-1026.
5
4, 957-961.
(
6) Brueggemeier, R. W.; Katlic, N. E.; Palmer, C. E., J r.; Stevens,
J . M. Catechol estrogen formation in MCF-7 cell culture and
effects of bromoestrogen inhibitors. Mol. Cell. Endocrinol. 1989,
(
(
(
16) Moriarty, R. M.; Vaid, R. K.; Duncan, M. P. Hypervalent iodine
oxidation of hydrazine hydrate in dichloromethane. Synth.
Commun. 1987, 17, 703-708.
6
4, 101-107.
(
7) Omar, A.; Ahmed, I. C.; Aboul Wafa, O. M.; Hassan, A. M.;
Ismail, K. A.; El-Din, M. M. M.; Mansour, N. A. Synthesis,
binding affinities and uterotrophic activity of some 2-substituted
estradiol ring-A fused pyrone derivatives. Eur. J . Med. Chem.
17) Ziegler, F. E.; Fowler, K. W. Substitution reaction of specifically-
ortho-metallated piperonal cyclohexylimine. J . Org. Chem. 1978,
4
1, 1564-1566.
18) Taylor, C.; Blanchard, B.; Zava, D. A simple method to determine
whole cell uptake of radiolabelled oestrogen and progesterone
and their subcellular localiztion in breast cancer cell lines in
monolayer culture. J . Steroid Biochem. 1984, 20, 1083-1088.
1
994, 29, 25-32.
(
8) VanderKuur, J . A.; Wiese, T.; Brooks, S. C. Influence of estrogen
structure on nuclear binding and progesterone receptor induction
by the receptor complex. Biochemistry 1993, 32, 7002-7008.
Pilat, M. J .; Hafner, M. S.; Kral, L. G.; Brooks, S. C. Differential
induciton of pS2 and cathepsin D mRNAs by structurally altered
estrogens. Biochemistry 1993, 32, 7009-7015.
(19) Brown, A. M. C.; J eltsch, J .-M.; Roberts, M.; Chambon, P.
Activation of pS2 gene transcription is a primary response to
estrogen in the human breast cancer cell line MCF-7. Proc. Natl.
Acad. Sci. U.S.A. 1984, 81, 6344-6348.
(20) Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem. 1987, 162, 156-159.
(
9) Li, J . J .; Li, S. A. Estrogen carcinogenesis in hamster tissue: A
critical review. Endocrin. Rev. 1990, 11, 524-531.
(
10) Li, J . J .; Li, S. A.; Klicka, J . K.; Parsons, J . A.; Lam, L. K.
Relative carcinogenic activity of various synthetic and natural
estrogens in the Syrian hamster. Cancer Res. 1983, 51, 5200-
5
204.
J M9508245