Synthesis and evaluation of 11β-(4-Substituted phenyl) estradiol analogs: Transition from estrogen receptor agonists to antagonists

Article abstract of DOI:10.1016/j.bmc.2012.04.041

Introduction: As part of our program to develop estrogen receptor (ER) targeted imaging and therapeutic agents we chose to evaluate 11β-substituted estradiol analogs as a representative scaffold. Previous synthetic studies provided an entry into this class of compounds and other work indicated that 11β-(substituted aryl) estradiol analogs were potent antagonists of the ER. Little information existed about the specific structural features involved in the transition from agonism to antagonism for the 11β-aryl estradiol analogs or their potential as scaffolds for drug conjugation. Methods: We prepared and characterized a series of 11β-(4-Substituted phenyl) estradiol analogs using modifications of existing synthetic methods. The new compounds, as well as standard steroidal agonists and antagonists, were evaluated as competitive ligands for the ERβ-LBD. Functional assays used the induction of alkaline phosphatase in Ishikawa cells to determine potency of the compounds as ER agonists or antagonists. Results: The synthetic strategy successfully generated a series of compounds in which the 4-substituent was sequentially modified from hydroxyl to methoxy to azidoethoxy/N,N-dimethylaminoethoxy and eventually to a prototypical 1,4-naphthoquinone-containing moiety. The new compounds all retained high relative binding affinity (RBA) for the ERα-LBD, ranging from 13-83% that of estradiol. No subtype selectivity was observed. More importantly, the transition from agonist to antagonist activity occurs at the 4-methoxy stage where the compound is a mixed antagonist. More notably, antagonism appeared to be more dependent upon the size of the 11β-substituent than upon the nature of the terminal group Conclusions: We have developed a synthetic strategy that provides facile access to potent 11β-(4-substituted phenyl) estradiol analogs. The resultant compounds retain high affinity for the ERα-LBD and, more importantly, demonstrate potent antagonist activity in cells. Large functionalities distal to the 11β-phenyl ring had little additional effect on either affinity or efficacy, suggesting the incorporation of diverse imaging or biologically active groups can be attached without significantly compromising the ER-binding capacity. Future studies are in progress to exploit the 11β-aryl estradiol analogs as potential drug delivery systems and imaging agents.

Full text of DOI:10.1016/j.bmc.2012.04.041

Bioorganic & Medicinal Chemistry 20 (2012) 3768–3780
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 11b-(4-Substituted phenyl) estradiol analogs:
Transition from estrogen receptor agonists to antagonists
Robert N. Hanson a, , Edward Hua a, J. Adam Hendricks a, David Labaree b, Richard B. Hochberg b
⇑
a Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115-5000, USA
b Department of Obstetrics/Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction: As part of our program to develop estrogen receptor (ER) targeted imaging and therapeutic
agents we chose to evaluate 11b-substituted estradiol analogs as a representative scaffold. Previous syn-
thetic studies provided an entry into this class of compounds and other work indicated that 11b-(substi-
tuted aryl) estradiol analogs were potent antagonists of the ER. Little information existed about the
specific structural features involved in the transition from agonism to antagonism for the 11b-aryl estra-
diol analogs or their potential as scaffolds for drug conjugation.
Methods: We prepared and characterized a series of 11b-(4-Substituted phenyl) estradiol analogs using
modifications of existing synthetic methods. The new compounds, as well as standard steroidal agonists
and antagonists, were evaluated as competitive ligands for the ERb-LBD. Functional assays used the
induction of alkaline phosphatase in Ishikawa cells to determine potency of the compounds as ER ago-
nists or antagonists.
Received 14 February 2012
Revised 11 April 2012
Accepted 21 April 2012
Available online 7 May 2012
Keywords:
Steroidal anti-estrogens
Estrogen receptor
Synthesis
Agonist
Antagonist
Conformational analysis
Results: The synthetic strategy successfully generated a series of compounds in which the 4-substituent
was sequentially modified from hydroxyl to methoxy to azidoethoxy/N,N-dimethylaminoethoxy and
eventually to a prototypical 1,4-naphthoquinone-containing moiety. The new compounds all retained
high relative binding affinity (RBA) for the ERa-LBD, ranging from 13–83% that of estradiol. No subtype
selectivity was observed. More importantly, the transition from agonist to antagonist activity occurs at
the 4-methoxy stage where the compound is a mixed antagonist. More notably, antagonism appeared
to be more dependent upon the size of the 11b-substituent than upon the nature of the terminal group
Conclusions: We have developed a synthetic strategy that provides facile access to potent 11b-(4-substi-
tuted phenyl) estradiol analogs. The resultant compounds retain high affinity for the ERa-LBD and, more
importantly, demonstrate potent antagonist activity in cells. Large functionalities distal to the 11b-phe-
nyl ring had little additional effect on either affinity or efficacy, suggesting the incorporation of diverse
imaging or biologically active groups can be attached without significantly compromising the ER-binding
capacity. Future studies are in progress to exploit the 11b-aryl estradiol analogs as potential drug delivery
systems and imaging agents.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
transcription (agonist responses).6–11 The process leading to ER
antagonism, although less well defined, has been described with
The estrogen receptor (ER) is a member of the nuclear receptor
(NR) superfamily of transcription factors, a group of proteins that
mediate a wide variety of physiological and developmental pro-
cesses.1,2 Because inappropriate or over-expression of ER is associ-
ated with a number of endocrine disorders, such as breast,
endometrial and ovarian cancer, and osteoporosis, chemical mod-
ulation of the ER-regulated pathways is a critical clinical objec-
tive.3–5 A number of reviews have described the structure of the
ER, including its subtypes, and the general mechanism by which
binding of the endogenous ligand initiates the events leading to
increasing detail.12–14 While many individual steps are involved
in the overall process, the initial binding of the ligand to the
(apo)receptor to generate a stable complex constitutes the key step
that defines the subsequent events. Because virtually all subse-
quent biological responses, including antagonist responses, are
influenced by the conformation of the receptor–ligand complex,
studies that enhance our understanding of this initial interaction
and its therapeutic implications are valuable.
Our research has primarily focused on the systematic prepara-
tion of modified derivatives of estradiol that evaluate the influence
of structural properties on receptor affinity, selectivity and efficacy.
Although most of our efforts focused on the 17
a-position, through
⇑
Corresponding author. Tel.: +1 617 373 3313; fax: +1 617 373 8795.
E-mail address: r.hanson@neu.edu (R.N. Hanson).
the use of the phenyl vinyl moiety,15–22 we also conducted studies
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.041

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3769
evaluating the effect of small 11b-substituents on estrogenic func-
tion.23–28 In particular, we observed, as have others,29–35 the po-
tency-enhancing influence of small substituents, such as
methoxy, ethyl and vinyl, at the 11b position of estradiol. Notably,
all of these small 11b-alkyl/alkoxy substituted estradiol derivatives
were ER agonists.
In our current research program we were interested in develop-
ing new drug/imaging-estradiol conjugates for the treatment or
diagnosis of diseases that over-express ER, such as hormone
responsive breast cancer (Fig. 1). Recent reviews reported that
et al. subsequently used the 11b-aryl estradiol scaffold as the basis
for developing electrophilic agents for alkylating the estrogen
receptor.47–49 In those studies, simple 11b-aryl (phenyl/thienyl)
estradiol analogs were agonists but that introduction of tamoxi-
fen-like side chains (RU-39411) or substituted alkyoxy phenyl side
chains (RU-58668) yielded antagonism.35,40,50–52 Hochberg et al.
also reported that within a homologous series of 11b-alkyl estra-
diol analogs, the transition from agonism to antagonism occurred
within a 1–2methylene group transition.53,54 These observations
suggested that small structural modifications produced significant
alterations in the conformational preferences for helix-12 of the
the most common sites of conjugation were the 3,6,16a, 17a and
17b-positions of estradiol, however, in almost all examples, intro-
duction of the therapeutic or imaging groups led to significant loss
of ER-binding affinity and therefore target cell selectivity.36,37 Of
equal concern to us was the observation that the conjugates were
based on agonist structures which would tend to lead to cell pro-
liferation rather than cytotoxicity. The only structural variations
that addressed the issues of affinity and efficacy were the 7a-
substituted estradiol-conjugates. Upon binding to ER, the steroid
scaffold in these analogs apparently rotates around the 3–17 axis
and the 7a-group occupies the 11b-binding pocket of the ER. Those
few studies that used this approach reported retention of signifi-
cant ER binding affinity, antagonist properties, and modest cyto-
toxic activity in breast cancer cell lines.38,39 Although 11b-
substituted estradiols have been described as potent ER antago-
nists, no studies have been reported to date in which the 11b-posi-
tion was successfully exploited for drug conjugation. Therefore this
study was initiated with the aims of developing the chemistry
needed to efficiently introduce 11b-substituents, to demonstrate
that these 11b-substituted estradiol analogs would retain high ER
binding affinity and generate antagonist responses, and that the
11b-substituent could be ultimately modified with terminal
groups similar to those found in therapeutic drugs or imaging
moieties.
ERa-LBD. Notably, no studies specifically addressed the transition
from agonist to antagonist conformations with aryl groups within
the 11b-binding pocket or the extent to which binding affinity was
affected. Although previous studies suggested that terminal groups
beyond the amine of tamoxifen and raloxifene-like antagonists oc-
cupy solvent accessible space, none had looked at the influence of
the alkoxy group or the heteroatom on the interactions with com-
plementary receptor residues. In this study we describe the syn-
thesis a series of 11b-(4-substituted phenyl) estradiol analogs,
their evaluation as probes (affinity and efficacy) for the ER-LBD,
and their assessment as scaffolds for diagnostic/therapeutic drug-
estradiol conjugates.
2. Experimental
2.1. General information
All reagents and solvents were purchased from Aldrich or Fisher
Scientific. THF and toluene were distilled from sodium/benzophe-
none. Reactions were monitored by TLC, performed on 0.2 mm sil-
ica gel plastic backed sheets containing F-254 indicator.
Visualization on TLC was achieved using UV light, iodine vapor
and/or phosphomolybdic acid reagent. Column chromatography
The strategy in this study addresses each of these aims. As part
of earlier studies directed toward high affinity estrogens, we had
established synthetic methods for the facile introduction of sub-
stituents at the 11b-position using Cu(I)-catalyzed Grignard reac-
was performed with 32–63 lm silica gel packing. Melting points
were determined using an Electrotherm capillary melting point
apparatus and are uncorrected. 1H NMR spectra were recorded
with a Varian Mercury 300 MHz, a Varian 500 MHz or a Bruker
700 MHz spectrometer. DEPT and 13C experiments were performed
on a Varian Mercury instrument at 75 MHz. NMR spectra chemical
shifts are reported in parts per million downfield from TMS and
referenced either to TMS, or internal standard for chloroform-d1,
tions.23,27
A
significant body of work published by the
pharmaceutical industry (Roussel-UCLAF and more recently Aven-
tis)40–46 demonstrated that estradiol, when substituted with
appropriate 11b-aryl groups, were potent anti-estrogens. Borgna
OH
Drug/Imaging
linker
HO
group
Concept of drug-estradiol conjugate
17β
17α
OH
11β
OH
16α
3
HO
HO
7α
6
"Antagonist" sites of conjugation
"Agonist" sites of conjugation
Figure 1. Drug-estradiol conjugates for targeted drug delivery and primary sites of conjugation.

3770
R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
acetone-d6, methanol-d4, and THF-d8 solvent peak. Coupling con-
stants are reported in hertz. High-resolution mass spectra were ob-
tained by electron impact (EI) or fast atom bombardment (FAB) on
MStation JMS700 (JEOL) by University of Massachusetts Amherst,
Mass Spectrometry Center using sodium iodide as an internal stan-
dard. NMR spectra for the intermediates and final compounds are
provided in the Supplementary data.
temperature between 18 and 20 °C. The solution was stirred for
5 h at room temperature (TLC monitoring: ethyl acetate/hexanes,
3:7). The mixture was carefully poured into aqueous sodium bicar-
bonate (10 mL, 1.34 g, 10 equiv). The mixture was stirred for 18 h
at room temperature. The organic layer was separated, washed
with 1N sodium hydroxide solution (25 mL ꢀ 2), water
(30 mL ꢀ 2) adjusted to pH 5–6, dried over magnesium sulfate,
and concentrated under reduced pressure.
2.2. 3,3,17,17-Diethylenedioxy-5,10-
and 3,3,17,17-Diethylenedioxy-5,10-b-epoxy-estr-9(11)-ene (2)
a
-epoxy-estr-9(11)-ene 3
The crude product (0.78 g) was dissolved in a mixture of meth-
anol (10 mL) and methylene chloride (5 mL). A solution of potas-
sium hydroxide (0.147 g, 2.63 mmol, 1.5 equiv) in methanol
(10 mL) was added dropwise over 5 min at 0–5 °C. The mixture
was stirred at 0–5 °C for 2 h (TLC monitory: ethyl acetate/hexanes,
3:7). The organic layer was separated, washed with water
(30 mL ꢀ 3) to pH ꢂ6, and brine solution (30 mL), dried over mag-
nesium sulfate, and concentrated under vacuum. The crude prod-
uct was purified using a silica gel column (25 g, ethyl acetate/
hexanes, 2:3). The fractions containing the product were combined
and concentrated under reduced pressure to give an oily product 4.
Yield = 0.21 g, 26%. Rf = 0.4 (ethyl acetate/hexanes, 3:7). 1H
Estra-5(10),9(11)-diene 3,17 diethylene ketal 127 (1 g,
2.79 mmol), hexafluoroacetone trihydrate (0.04 mL, 0.279 mmol),
pyridine (0.005 mL), 50% hydrogen peroxide (0.3 mL, 4.74 mmol,
ca. 18 M) and dichloromethane (10 mL) were charged into a round
bottom flask at room temperature under argon atmosphere. The
mixture was stirred for 20 h at room temperature (TLC monitoring:
ethyl acetate/hexanes, 3:7). After reductive workup (aqueous so-
dium thiosulfate solution, 2 g in 50 mL of water), the organic layer
was washed with water (25 mL ꢀ 2), extracted with dichlorometh-
ane (30 mL ꢀ 2). The organic layer was dried over magnesium sul-
fate and concentrated under reduced pressure to give 2 as mixture
NMR(300 MHz, CDCl3)
d 6.97 (d, J = 8.7 Hz, 2H), 6.82 (d,
J = 8.1 Hz, 1H, C2-H), 6.63 (d, J = 8.7 Hz, 2H), 6.61 (s, 1H, C4-H),
6.42 (dd, J = 8.4 Hz, J = 6.4 Hz, 1H, C1-H), 3.85 (t, J = 4.2 Hz, 1H,
of isomers (ratio of
purified from other components by chromatographic separation on
a
:b ꢁ3:1, 1H NMR). The crude mixture could be
C11-Ha
),3.69 (s, 3H, OCH3), 0.44 (s, 3H, CH3). 13C NMR (75 MHz,
a silica gel column (25 g, ethyl acetate/hexanes, 1:4). The combined
CDCl3, d 219.9, 156.9, 153.3, 150.7, 137.7, 135.4, 130.7, 130.4,
127.9, 115.1, 113.7, 113.3, 55.2, 52.4, 48.4, 47.7, 40.2, 38.3, 35.6,
35.3, 30.2, 27.5, 21.6, 15.4.
fractions containing the individual
under reduced pressure.
a/b products were concentrated
Yield = 0.806 g, 76% Rf = 0.4 (ethyl acetate/hexanes 5:1). 1H
NMR (300 MHz, CDCl3, d 6.05 (m, 1H, 2
a
), 5.86 (m, 1H, 2b), 0.88
2.5. 11b-(4-Methoxyphenyl)estra-1,3,5(10)-trien-3,17b-diol (5)
(s, 3H, 2b), 0.89 (s, 3H, 2
a
). 13C NMR (75 MHz, CDCl3) d 191.1,
164.9, 132.2, 1145, 55.8.
To a solution (0–5 °C) of compound 4 (55 mg, 0.147 mmol,
1 equiv) in methanol (10 mL), sodium borohydride (6 mg,
0.153 mmol, 1.05 equiv) was added portion wise. The mixture
was stirred at 0–5 °C for 1.5 h (TLC monitoring: ethyl acetate/hex-
anes, 1:1). The excess borohydride was consumed by addition of
acetone (5 mL). The suspension was stirred at 0–5 °C for 0.5 h,
and poured onto a mixture of cold water (25 mL) and methylene
chloride (25 mL). The organic layer was separated, washed with
water (30 mL ꢀ 2), brine (30 mL), dried over magnesium sulfate,
and concentrated to dryness under reduced pressure. The crude
product was dissolved in isopropanol (1 mL) at room temperature.
The product crystallized as needles upon standing and was col-
lected by filtration. The crystals were rinsed with small amount
of cold water and dried under reduced pressure overnight to give
pure 5.
2.3. 11b-(4-Methoxyphenyl)estra-4,9-diene-3,17 dione (3)
Copper (I) chloride (0.24 g, 2.41 mol, 0.15 equiv) was added at
20 °C (water bath) to a 1 M solution of 4-methoxyphenyl magne-
sium bromide in THF (5 mL, anhydrous, 2.0 equiv). A solution of
the mixture 2 (a- and b-isomers, 0.8 g, 2.14 mmol, 1.0 equiv) in
THF (10 mL, anhydrous) was added dropwise over 30 min at
20 °C. The reaction mixture was then stirred for 1 h at 20 °C. Upon
completion (TLC monitoring: ethylacetate:hexanes, 3:7), the mix-
ture was poured into a mixture of aqueous ammonium chloride
(8 mL, 15 equiv) and methylene chloride (8 mL) at 10–15 °C. The
organic layer was separated, washed with water (20 mL ꢀ 2), con-
centrated under reduced pressure to ꢂ5 mL, and diluted with
methylene chloride (5 mL). Aqueous hydrochloric acid (6 equiv,
0.47 g in 2.6 mL of water) was added at 0–5 °C. The mixture was
stirred for 2 h at 0–5 °C and then diluted with water (20 mL). The
pH of the mixture was <1 (pH paper). The organic phase was
washed with water (20 mL ꢀ 2), neutralized with aqueous 10% so-
dium bicarbonate solution to pH 8–9, washed with water
(30 mL ꢀ 3), dried over magnesium sulfate, and concentrated to
dryness under vacuum. This crude product (0.66 g) was purified
by column chromatography (silica gel, 25 g; ethyl acetate/hexanes,
3:7). The fractions containing the product were combined and con-
centrated under reduced pressure to compound 3.
Yield = 36 mg, 65% mp: 209–211 °C. Rf = 0.4 (ethyl acetate/hex-
anes, 3:7). 1H MNR (300 MHz, CDCl3): d 6.92 (d, J = 8.7 Hz, 2H), 6.75
(d, J = 8.1 Hz, 1H, C2-H), 6.56 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H, C4-H),
6.35 (dd, J = 8.4 Hz, J = 6.4 Hz, 1H, C1-H), 3.88 (t, J = 4.2 Hz, 1H,
C11-Ha) 3.64 (s, 3H, OCH3), 0.44 (s, 3H, CH3).
2.6. 11b-(4-Hydroxyphenyl)-estra-1,3,5(10)-triene-3,17b-diol (6)
To a solution of compound 4 (90 mg, 0.25 mmol) in methylene
chloride (5 mL, anhydrous), 1 M boron tribromide (0.3 mL in meth-
ylene chloride) was added dropwise over 10 min under argon
atmosphere at ꢃ78 °C (acetone/dry ice bath). The mixture was stir-
red at ꢃ78 °C for 1 h, and then at room temperature for 2 h. The
mixture was poured into a beaker containing cold water and so-
dium bicarbonate (10 mg), and stirred for 10 min. The mixture
was extracted with methylene chloride (30 mL ꢀ 3). The combined
organic layers were washed with water (30 mL), brine (30 mL),
dried over magnesium sulfate, and concentrated under reduced
pressure. The crude product was purified by column chromatogra-
phy (silica gel, 25 g; ethyl acetate/hexanes, 1:1). The fractions con-
taining the product were combined and concentrated under
Yield = 0.23 g, 25%. 1H NMR (300 MHz, CDCl3): d 6.72 and 7.01
(AA’BB’, 4H), 5.78 (s, 1H), 4.35 (d, J = 6.6 Hz, 1H), 3.75 (s, 3H),
0.55 (s, 3H).
2.4. 11b-(4-Methoxyphenyl)estra-1,3,5-trien-3-ol-17-one (4)
To a solution of 3 (0.66 g, 1.75 mmol) in methylene chloride
(10 mL), acetic anhydride (0.17 mL, d = 1.08 g/mL, 1.75 mmol,
1 equiv) was added over 5 min. Acetyl bromide (0.32 mL,
4.38 mmol, 2.5 equiv) was added dropwise over 5 min, and at a

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3771
vacuum to give pure intermediate 11b-(4-hydroxyphenyl)-estra-
1,3,5(10)-trien-17-one-3-ol.
hexanes, 1:4). The organic solvents were removed under reduced
pressure. The residue was dissolved in methylene chloride
(20 mL), washed with sodium bicarbonate (20 mL, saturated),
water (20 mL ꢀ 2), brine (30 mL), dried over magnesium sulfate
and concentrated under vacuum. Colorless crystals of triethylene
glycol ditosylate were obtained from ethyl acetate.
Yield = 92 mg, 99% Rf = 0.3 (ethyl acetate/hexanes = 1:1). 1H
NMR(300 MHz, DCCl3,):
d 6.90 (d, J = 8.1 Hz, 2H), 6.80 (d,
J = 8.7 Hz, 1H, C2-H), 6.56 (d, J = 3 Hz,1H, C4-H), 6.53 (d, J = 8.4 Hz,
2H), 6.39 (dd, J = 6 Hz, J = 3 Hz, 1H, C1-H), 4.48 (d, J = 11.1 Hz, 1H,
C
17-H
a
), 3.90 (t, J = 9 Hz, 1H, C11-Hb), 0.38 (s, 3H).
Yield = 5.8 g, 64% mp: 75–77 °C. Rf = 0.5 (ethyl acetate/hexanes,
1:4). 1H NMR (300 MHz, CDCl3): d 7.78 and 7.34 (AA’BB’, 8H), 4.12
(d, 4H), 3.65 (t, 4H), 3.53 (s, 4H), 2.45 (s, 6H). 13C NMR (75 MHz,
CDCl3, d 145.1, 133.3, 130.1, 128.2, 70.9, 69.4, 69.0, 21.8.
To a solution (0–5 °C) of the intermediate 11b-(4-hydroxy-
phenyl)-estra-1,3,5(10)-trien-17-one-3-ol (37 mg, 0.098 mmol) in
methanol (10 mL), sodium borohydride (4 mg, 0.103 mmol,
1.05 equiv) was added and the reaction mixture was stirred for
1.5 h (TLC monitoring: ethyl acetate/hexanes, 2:3). The excess hy-
dride was consumed by addition of acetone (5 mL). The suspension
was stirred for 0.5 h at 0–5 °C and then poured onto a mixture of
cold water (25 mL) and methylene chloride (25 mL). The organic
layer was washed with water (25 mL ꢀ 2), brine (25 mL), dried
over magnesium sulfate, and concentrated under the reduced pres-
sure. The crude product was purified by column chromatography
(silica gel, 25 g: ethyl acetate/hexanes, 2:3). The fractions contain-
ing the product were combined and concentrated under reduced
pressure to give product 6.
To a solution of the triethylene glycol ditosylate (2 g, 4.4 mmol)
in ethanol (50 mL), sodium azide (142 mg, 2.18 mmol) was added
at room temperature. The mixture was heated at reflux for 2 h
(TLC monitoring: ethyl acetate/hexanes, 1:1). The mixture was al-
lowed to cool to room temperature. The colorless solid that formed
upon cooling was collected by filtration and washed with small
amount of solution of ethyl acetate/hexanes = 1:1. The filtrate
was evaporated to dryness under reduced pressure. The oily resi-
due was dissolved in diethyl either (100 mL), washed with water
(20 mL ꢀ 2), and brine (20 mL). The organic phase was dried over
magnesium sulfate, filtered and evaporated to dryness. Column
chromatography on silica gel (25 g; ethyl acetate/hexanes, 2:3),
Yield = 8 mg, 22%. Rf = 0.2 (ethyl acetate/hexanes, 2:3). 1H
51
NMR(300 MHz, CDCl3):
J = 8.7 Hz, 1H, C2-H), 6.56 (d, J = 5.5 Hz, 2H), 6.52 (s, 1H, C4-H),
6.39 (dd, J = 8.7 Hz, J = 6.4 Hz, 1H, C1-H), 4.48 (d, J = 11.1 Hz, 1H),
d
6.90 (d, J = 8.1 Hz, 2H), 6.80 (d,
gave a colorless oil X.
Yield = 0.26 g, 37%. Rf = 0.5 (ethyl acetate/hexanes, 1:1). 1H NMR
(300 MHz, CDCl3): d 7.78 and 7.34 (AA’BB’, 4H), 4.12 (t, 2H), 3.70 (t,
2H), 3.64 (t, 2H), 3.60 (s, 4H), 3.36 (t, 2H), 2.44 (s, 3H). 13C NMR
(75 MHz, CDCl3, d 145.0, 133.3, 130.0, 128.2, 70.0, 70.8, 70.3,
3.91 (t, J = 8.1 Hz, 1H, C11-Ha), 0.31 (s, 3H, CH3).
2.7. 11b-(4-hydroxyphenyl)-estra-4,9-diene-3,17-dione (7)
69.5, 69.0, 50.9, 21.8. IR (thin film) 2105 cmꢃ1
.
Copper (I) chloride (35 mg, 0.35 mmol) was added at room tem-
perature to a ca. 1 M solution of 4-trimethylsilyloxyphenyl magne-
sium bromide in THF (10 mL) under argon atmosphere. A solution
2.9. 11b-4-(2-Azidoethoxy)phenyl-estra-4,9-diene-3,17-dione (9)
11b-(4-Hydroxyphenyl)estra-4,9-diene-3,17-dione 7 (65.0 mg,
0.179 mmol) was dissolved in acetonitrile (5 mL). Cesium carbon-
ate (234 mg, 0.717 mmol) was added to the solution and the
homogeneous mixture was stirred for 15 min after which ethylene
glycol ditosylate (100 mg, 0.269 mmol) was added. After 13 h, the
reaction mixture was poured into a biphasic mixture of ethyl ace-
tate (20 mL) and water (20 mL). The organic layer was washed
with water (2 ꢀ 25 mL). All of the aqueous layers were combined;
sodium chloride was added and then back extracted with ethyl
acetate (30 mL). The organic layer was dried over magnesium sul-
fate, filtered and the solvent removed under reduced pressure.
Flash column chromatography with hexane–ethyl acetate afforded
(60 mg, 0.107 mmol, 60% yield) of the product 11b-[4-(2-tosyloxy-
ethoxy)-phenyl]-estra-4,9-diene-3,17-dione. 1H NMR (300 MHz,
CDCl3): d 0.53 (s, 3H), 2.44 (s, 3H), 4.10 (t, 2H), 4.33 (t, 2H) 4.38
(d, J = 6.9, 1H), 5.78 (s, 1H), 6.71 (d, J = 8.4, 2H), 7.04 (d, J = 8.5,
2H), 7.33 (d, J = 8.5, 2H), 7.80 (d, J = 8.3, 2H) 13C NMR (500 MHz,
acetone-d6): d 14.23, 20.92, 21.79, 26.03, 27.09, 30.71, 35.06,
36.88, 38.17, 38.38, 39.74, 47.55, 50.74, 65.80, 69.17, 114.77,
122.95, 128.30, 130.14, 133.56, 137.53, 145.29, 155.92, 156.61,
197.54, 217.44.
of the mixture of 2
a
/b (ratio ꢁ3:1, 1H NMR) (760 mg, 2.03 mmol)
in THF (10 mL) was added during ꢂ30 min at room temperature
(exothermic). The mixture was then stirred for 1 h at room temper-
ature (TLC monitoring: ethyl acetate/hexanes = 3:7). When the
reaction went to completion, the resulting solution was poured
into a biphasic mixture of aqueous ammonium chloride (15 equiv,
6 mL) and methylene chloride (8 mL) at 10–15 °C. The organic
layer was separated, washed with water (20 mL ꢀ 2), concentrated
the total volume to ꢂ5 mL, and diluted with methylene chloride
(5 mL). Aqueous hydrochloric acid (6 equiv, 0.47 g in 2.6 mL of
water) was added at 0–5 °C. This biphasic mixture was stirred for
2 h at 0–5 °C (pH <1, pH paper) and then diluted with water
(20 mL). The organic phase was separated, washed with water
(20 mL ꢀ 2) and carefully neutralized to pH ꢁ8 (10% sodium bicar-
bonate, ꢂ1.5 mL, pH ꢂ7–8). The neutralized solution washed with
water (30 mL ꢀ 2). The combined organic layer was dried over
magnesium sulfate. Compound 7 (0.66 g, 90.4%) was isolated from
a silica gel flash column chromatography (ethyl acetate/hexanes,
3:7).
Yield = 0.66 g, 90% mp 248 °C, 1H NMR (300 MHz, CDCl3: d 6.75
and 7.07 (AA’BB’, 4H), 5.81 (s, 1H), 4.35 (d, J = 7.2 Hz, 1H), 4.01 (t,
J = 6 Hz, 2H), 2.85 (t, J = 6 Hz, 2H), 2.63 (q, J = 7 Hz, 4H), 0.56 (s,
3H). 13C NMR (75 MHz, CDCl3, d 219.3, 200.0, 156.6, 154.3, 145.6,
135.8, 130.2, 128.2, 123.5, 115.9, 50.8, 47.9, 39.8, 38.2, 38.0, 36.9,
35.6, 31.1, 26.9, 26.0, 22.1, 14.6.
Rf (dichloromethane/ethyl acetate = 7:3): 0.60.
Intermediate
11b-[4-(2-tosyloxyethoxy)-phenyl]-estra-4,9-
diene-3,17-dione (80 mg, 0.143 mmol) was charged to a reaction
tube and dissolved in ethanol (5 mL). Sodium azide (37.1 mg,
0.571 mmol) was added to the solution and the reaction was heated
to 80 °C. After 4 h the reaction was poured into ice cold ethyl acetate
(20 mL) and washed with water (3 ꢀ 20 mL). Sodium chloride was
added to the combined aqueous layers and then back extracted with
ethyl acetate (20 mL). The organic fractions were combined and
dried over magnesium sulfate. The magnesium sulfate was filtered
and the solvent removed under reduced pressure. Column chroma-
tography using hexane/ethyl acetate afforded 11-[4-(2-azidoeth-
oxy)-phenyl]-estra-4,9-diene-3,17-dione 9 (60 mg, 0.139 mmol,
97% yield). 1H NMR (300 MHz, CDCl3): d 0.54 (s, 3H), 3.56 (t, 2H)
2.8. 2-[2-(2-Azidoethoxy)ethoxy)ethoxy]-4-methylbenzenesulf
onate (8)
To a solution of triethylene glycol (3 g, 0.02 mol) in diethyl
ether (40 mL), triethylamine (7.8 g, 0.04 mol) was added at room
temperature, followed by addition of p-toluenesulfonyl chloride
(7.8 g, 0.04 mol) under argon atmosphere. The mixture was stirred
at room temperature for 18 h (TLC monitoring, ethyl acetate/

3772
R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
4.01 (t, 2H), 4.65 (d, J = 6.9, 1H) 5.77 (s, 1H), 6.82 (d, J = 8.8, 2H), 7.07
(d, J = 8.8, 2H).
hexane; ethyl acetate afforded 11b-[4-(2-azidoethoxy)-phenyl]-es-
tra-1,3,5(10)-triene-3,17b-diol 11 (26 mg, 0.06 mmol, 98% yield).
1H NMR (300 MHz, CDCl3): d 0.33 (s, 3H), 3.59 (t, 2H), 3.95 (m,
1H 11-H)), 4.09 (t, 2H), 6.37 (dd, 1H (2-H)), 6.55 (d, J = 2.4, 2H),
6.67 (d, J = 8.8, 2H (4-H)), 6.78 (d, J = 8.8, 1H (1-H)), 7.06 (d,
J = 8.6, 1H), 13C NMR (500 MHz, acetone-d6): d 12.98, 23.21,
28.29, 29.75, 30.12, 35.85, 38.70, 43.96, 45.99, 47.68, 50.34,
52.09, 67.02, 81.77, 113.25, 113.54, 115.27, 127.65, 128.12,
129.72, 130.21, 131.03, 137.25, 137.67, 154.00, 155.67 IR:
2086.03, 3351.50 cmꢃ1 HRMS calcd for C26H31N3O3 m/e 433.2365,
found m/e 433.2325.
13C NMR (300 MHz, acetone-d6): d 14.26, 21.80, 26.02, 27.07,
30.72, 35.07, 36.89, 38.14, 38.35, 39.74, 47.55, 50.32, 50.77,
67.25, 114.77, 122.96, 128.47, 130.00, 137.44, 145.32, 155.94,
156.79, 197.54, 217.44 IR: 2108.98 cmꢃ1 (N3–).
2.10. 11b-(4-((2-(2-(2-Azidoethoxy)ethoxy)ethoxy)phenyl)-
estra-4,9-diene-3,17-dione (10)
11b-(4-Hydroxyphenyl)-estra-4,9-dien-3,17-dione
7 (14 mg,
0.04 mmol), potassium carbonate (44 mg, 0.32 mmol) and acetoni-
trile (2.4 mL, anhydrous) were added to a round bottom flask at
room temperature and stirred for 5 min, then azido-triethylene
glycol tosylate 8 (26 mg, 0.08 mmol) was added at room tempera-
ture. The mixture was stirred at 80 °C for 10 h (TLC monitoring:
ethyl acetate/hexanes, 1:1). The reaction mixture was poured onto
a mixture of cold water (10 mL) and methylene chloride (20 mL),
stirred for 10 min, extracted with methylene chloride
(25 mL ꢀ 2), washed with water (20 mL), brine (20 mL), dried over
magnesium sulfate, and concentrated under reduced pressure.
Without further purification, a crude material was obtained con-
taining mixture of product and starting materials.
2.12. 11b-(4-((2-(2-(2-Azidoethoxy)ethoxy)ethoxy)phenyl)
estra-1,3,5(10)-trien-3,17b-diol (12)
To a round bottom flask, the mixture containing 10 (24 mg,
0.046 mmol) and methylene chloride (5 mL, anhydrous) were
added under argon atmosphere at room temperature. Then acetic
anhydride (4
over ꢂ5 min at room temperature, followed by acetyl bromide
(9 L, 14 mg, d = 1.663 g/mL, 0.115 mmol) for 5 min at room tem-
lL, 4.7 mg, d = 1.080 g/mL, 0.046 mmol) was added
l
perature under argon atmosphere. The light yellow mixture was
stirred at room temperature for 10 h (TLC monitoring: ethyl ace-
tate/hexanes, 1:1). The mixture was then carefully poured into
aqueous sodium carbonate (40 mg, in 5 mL of water, 10 equiv),
and stirred for 14 h at room temperature. The organic layer was
separated, washed with 1 N sodium hydroxide solution
(25 mL ꢀ 2), water (25 ꢀ 2), dried over magnesium sulfate and
concentrated under reduced pressure to give the crude 3-acetoxy
estra-trien-17-one intermediate (69 mg).
1H NMR (300 MHz, CDCl3,): d 7.08 and 6.84 (AA’BB’, J = 8.7 Hz,
J = 9.0 Hz, 4H), 5.80 (s, 1H, C4-H), 4.38 (d, J = 6.6 Hz, 2H, C11 -H),
a
4.10 (t, J = 4.8 Hz, 2H), 3.86 (t, J = 5.4 Hz, 2H), 3.74 (m, 2H), 3.68
(m, 2H), 3.39 (t, J = 5.1 Hz, 2H), 0.55 (s, 3H, C18-CH3). 13C NMR
(75 MHz, CDCl3,): d 219.0, 199.4, 157.2, 156.1, 145.1, 136.3,
130.3, 128.5, 123.6, 114.9, 71.1, 70.9, 70.3, 67.6, 50.9, 50.9, 47.9,
39.8, 38.2, 38.0, 37.0, 35.6, 31.1, 27.0, 26.1, 22.1, 14.6.
To a solution (0–5 °C) of crude intermediate (69 mg) in methy-
lene chloride (2 mL), a solution of potassium hydroxide (4 mg) in
methanol (5 mL) was added at 0–5 °C. The mixture was stirred at
0–5 °C for 4 h (TLC monitoring: ethyl acetate/hexanes, 1:1). The
mixture was diluted with methylene chloride (30 mL), washed
with water (20 mL ꢀ 2), brine (20 mL), dried over magnesium sul-
fate, and concentrated under reduced pressure. The crude product
was purified by column chromatography on silica gel (10 g, ethyl
acetate/hexanes, 2:3). The fractions containing the product were
combined and evaporated to dryness, yielding the estra-trien-17-
one-3-ol intermediate.
2.11. 11b-(4-(2-azidoethoxy)phenyl)-estra-1,3,5(10)-trien-
3,17b-diol (11)
11b-[4-(2-Azidoethoxy)-phenyl)-estra-4,9-dien-3,17-dione
(19.0 mg, 0.044 mmol) was charged to a round bottom flask and
dissolved in methylene chloride (3 mL). To this solution was added
8
acetic anhydride (4.16 lL, 0.044 mmol) and acetyl bromide (8.2 lL,
0.110 mmol). After 4 h the reaction mixture was poured into a
solution of sodium bicarbonate (37.0 mg, 0.44 mmol) in water
(15 mL). The product was extracted with ethyl acetate (10 mL)
and washed with water (3 ꢀ 15 mL). The aqueous washes were
combined and back extracted with ethyl acetate (30 mL). The or-
ganic fractions were combined and dried over magnesium sulfate,
which was removed by filtration and the solvent removed under
reduced pressure leaving (20 mg, 0.042 mmol, 96% yield) of 3-acet-
oxy-11b-(2-azidoethoxyphenyl)-estra-1,3,5(10)-triene-17-one. 1H
NMR (300 MHz, acetone-d6): d 0.41 (s, 3H), 2.17 (s, 3H), 2.99 (m,
1H (11-H)), 3.59 (t, 2H) 4.10 (t, 2H), 6.63 (dd, 1H (2-H)), 6.70 (d,
J = 8.8, 2H), 6.87 (d, J = 2.4, 1H (4-H)), 7.01 (d, J = 8.5, 1H (1-H)),
7.09 (d, J = 8.3, 2H) 13C NMR (500 MHz, acetone-d6): d 15.00,
20.35, 21.27, 27.26, 29.96, 34.89, 35.18, 38.39, 40.46, 47.7, 48.00,
50.32, 52.14, 67.05, 113.87, 119.29, 121.90, 127.58, 130.93,
135.82, 136.31, 137.88, 148.75, 156.00, 168.96, 217.27.
Yield = 15 mg, 63% Rf = 0.4 (ethyl acetate/hexanes, 1:1). 1H NMR
(300 MHz, CDCl3) 13C NMR (75 MHz, CDCl3) [Spectra in Supple-
mentary data].
To a 0–5 °C solution of the estra-trien-17-one-3-ol intermediate
(15 mg, 0.029 mmol) in methanol (10 mL), sodium borohydride
(2 mg, 0.03 mmol) was added as one portion. The mixture was stir-
red for 1 h at 0–5 °C (TLC monitoring: ethyl acetate/hexanes, 1:1).
An additional equivalent of sodium borohydride (2 mg, 0.03 mmol)
was added and the reaction mixture was stirred for an additional
1.5 h. The excess borohydride was consumed by addition of ace-
tone (5 mL). The suspension was stirred for 0.5 h at 0–5 °C, and
then poured into a mixture of ice-water (10 mL) and methylene
chloride (30 mL). The organic layer was extracted with methylene
chloride (20 mL x 2), washed with water (25 mL ꢀ 2) to pH ꢁ6–7,
brine (30 mL), dried over magnesium sulfate, and concentrated un-
der reduced pressure. After a silica gel column chromatography
purification (ethyl acetate/hexanes, 1:1), 11b-(4-((2-(2-(2-azido-
ethoxy)ethoxy)ethoxy)phenyl) estra-1,3,5(10)-trien-3,17b-diol 12
was isolated.
Intermediate
3-acetoxy-11b-(2-azidoethoxyphenyl)-estra-
1,3,5(10)-triene-17-one (29.0 mg, 0.06 mmol) was dissolved in
methanol (5 mL). To this solution was added sodium borohydride
(3.0 mg, 0.079 mmol). After 1 h 10 N sodium hydroxide
(0.024 mL, 0.245 mmol) was added and the reaction continued
for 16 h. The reaction was poured into an ice cold biphasic mixture
of ethyl acetate (20 mL) and water (20 mL), after which the organic
layer was washed with water (2 ꢀ 25 mL). The aqueous washes
were combined, sodium chloride was added and then back ex-
tracted with ethyl acetate (30 mL). The organic layer was dried
over magnesium sulfate. After filtration the solvent was adsorbed
onto Florisil under reduced pressure. Flash chromatography with
Yield = 12 mg, 80% Rf = 0.5 (ethyl acetate/hexanes, 1:1). 1H NMR
(300 MHz, CDCl3, d 6.63 (d, J = 4.8 Hz, 2H), 6.48 (d, J = 5.4 Hz, 1H),
6.29 (d, J = 5.1 Hz, 2H), 6.25 (d, J = 1.5 Hz, 1H), 6.08 (dd, J = 5 Hz,
J = 1. 5 Hz, 1H), 4.00 (t, J = 2.7 Hz, 2H), 3.92 (t, 1H, H-11a), 3.79 (t,
J = 3.3 Hz, 1H), 3.70 (m, 3H), 3.65 (m, 4H), 1.77 (t, J = 3 Hz, 1H),
2.96 (t, J = 9 Hz, 1H), 2.84 (s, 1H), 2.80 (d, J = 3 Hz, 1H), 2.49 (dd,

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3773
J = 1.2 Hz, J = 7.7 Hz, 1H), 1.00–2.30 (m, 10H), 0.32 (s, 3H, C18-CH3)
13C NMR (75 MHz, CDCl3, d 155.9, 153.3, 137.9, 136.2, 130.8, 127.9,
115.5, 113.8, 113.5, 82.8, 71.0, 70.9, 70.3, 70.1, 67.3, 52.0, 50.9,
47.6, 45.7, 43.8, 38.5, 35.6, 30.7, 30.4, 28.2, 23.4, 13.1. HRMS calcd
for C30H39N3O5 m/e 521.2890, found m/e 521.2840.
the ratio of the EC50 of E2 to that of the steroid analog ꢀ 100 using
the curve fitting program Prism to determine the EC50. Selected
estradiol analogs were evaluated as ligands for the ERb-LBD using
a similar protocol.
2.15.2. Estrogenic potency in Ishikawa cells
2.13. N-Propargyl-2-amino-1,4-naphthoquinone (13)
The estrogenic potency of the E2-analogs was determined in an
estrogen bioassay, the induction of AlkP in human endometrial
adenocarcinoma cells (Ishikawa) grown in 96-well microtiter
plates as we have previously described.59 The cells are grown in
phenol red free medium with estrogen depleted (charcoal
stripped) bovine serum in the presence or absence of varying
amounts of the steroids, across a dose range of at least 6 orders
of magnitude. After 3 days, the cells are washed, frozen and
thawed, and then incubated with 5 mM p-nitrophenyl phosphate,
a chromogenic substrate for the AlkP enzyme, at pH 9.8. To ensure
linear enzymatic analysis, the plates are monitored kinetically for
the production of p-nitrophenol at 405 nm. For antagonists, the ef-
fect (Ki) of each compound tested at a range of 10ꢃ6 to 10ꢃ12 M was
measured for the inhibition of the action of 10ꢃ9 M E2 (EC50
ꢂ0.2 nM). Each compound was analyzed in at least 3 separate
experiments performed in duplicate. The Ki and RSA (RSA = ratio
of 1/EC50 of the steroid analog to that of E2 ꢀ 100) were determined
using the curve fitting program Prism.
To
a solution of 2-methoxyl-1,4-naphthoquinone (2.28 g,
18 mmol) in methylene chloride (30 mL), propargylamine (1 g,
12 mmol) was added at room temperature under argon atmo-
sphere. The mixture was heated at reflux (ꢂ45 °C) and stirred for
18 h (TLC monitoring: methylene chloride, 100% and checked by
1H NMR in CDCl3). The solvents were removed under reduced pres-
sure and the crude product was purified column chromatography
on silica gel (70 g, dichloromethane, 100%). The fractions contain-
ing the product were combined and concentrated under vacuum
to give the pure material 13.56
Yield = 0.24 g, 6%. Rf = 0.3 (methylene chloride, 100%). 1H NMR
(300 MHz, CDCl3): d 8.12 (d, 1H), 8.10 (d, 1H), 7.75 (t, 1H), 7.64
(t, 1H), 5.85 (s, 1H), 4.00 (d, 2H), 2.4 (t, 1H). 13C NMR (75 MHz,
CDCl3): d 183.4, 181.7, 150.7, 147.3, 135.0, 133.5, 132.5, 130.7,
126.6, 126.5, 102.8, 73.6, 32.5.
2.14. 2-(((1-(2(2(2-(4–3,17b-Dihydroxy-estra-1,3,5(10)-trien-
11b-yl)phenoxy)ethoxy)ethoxy)ethyl-1H-1,2,3-triazol-4-
yl)methyl)amino)naphthalenen-1,4-dione (14)
3. Results and discussion
3.1. Synthesis of estrogen receptor ligands
N-Propargyl-2-amino-1,4-naphthoquinone
13
(12 mg,
0.023 mmol) and compound 12 (7.3 mg, 0.035 mmol) were sus-
pended in a mixture of water and t-butyl alcohol (5 mL, water/t-
butyl alcohol = 1:1). A freshly prepared solution of sodium ascor-
bate (23 mg, 0.115 mmol, 5 mol %) in water (0.5 mL) was added,
followed by a freshly prepared solution of copper (II) sulfate penta-
hydrate (6 mg, 0.023 mmol, 1 mol %) in water (0.5 mL) at room
temperature. The heterogeneous mixture was stirred at the same
temperature for 18 h (TLC monitoring: ethyl acetate, 100%). The
mixture was diluted with ice-water (15 mL), extracted with meth-
ylene chloride (30 mL ꢀ 3). Combined organic layers were washed
with water (20 ml ꢀ 2), brine (30 mL), dried over magnesium sul-
fate, and concentrated to dryness under reduced pressure. The
crude product was purified by column chromatography on silica
gel (5 g; ethyl acetate/hexanes, 9:1) to give pure 14.
The synthetic targets in this study were the 11b-(4-substituted
phenyl) estradiol analogs in which the 4-substituent varies from
hydroxy to methoxy to longer substituted azido-alkoxy deriva-
tives. Because the quinone moiety is a key structural feature in
anticancer antibiotics, such as, mitomycin and geldanamycin,60,61
,
and comparable in size to many imaging groups, introduction of
the terminal naphthoquinone would provide preliminary informa-
tion regarding the ability of the ER to tolerate such substituents.
Our synthetic strategy [Schemes 1 and 2] was based on our pre-
vious work and incorporated more recent contributions from the lit-
erature.44–46 In this approach, we began with diketal intermediate 1,
available from estrone 3-methyl ether as described in our previous
study.27 Epoxidation of 1 gave a mixture of
a- and b-epoxides 2
Yield = 8 mg, 48%. 1H NMR (300 MHz, CDCl3: d 8.08 (q, 2H), 7.90
(s, 1H), 7.73 (t, J = 6.7 Hz, 1H), 7.63 (t, J = 7.0 Hz, 1H), 6.94 (d,
J = 8.7 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.61 (d,s, 3H), 6.46 (d,
J = 9 Hz, 1H), 6.39 (s, 1H, NH), 5.74 (s, 1H), 4.52 (t, J = 4.2 Hz, 2H),
4.22 (m, 2H), 4.02 (q, 2H), 3.91 (br, 1H), 3.83 (d, J = 4.5 Hz, 2H),
3.78 (t, J = 4.2 Hz, 2H), 3.68–3.60 (br, 4H), 0.32 (s, 3H).
(3:1), isolated in a yield of 76% and used directly in the next step.
Cu(I)-catalyzed 1,4-Grignard addition (4-methoxyphenyl magne-
sium bromide) followed by dehydration, deprotection and chro-
matographic separation gave the 11b-(4-methoxyphenyl)-estra-
4,9-diene-3,17-dione 3, in a 25% yield. Aromatization, deprotection
and reduction gave 11b-(4-methoxyphenyl) estradiol 5 (18% for 3
steps).62 O-Demethylation of the intermediate estrone 4 with bor-
on tribromide followed by reduction afforded the corresponding
11b-(4-hydroxyphenyl) analog 6 in 21% yield (2 steps). The synthe-
sis of the remaining estradiol analogs is shown in Scheme 2. Be-
cause alkylation of 6 could proceed at either phenolic position,
we prepared the 11b-(4-hydroxyphenyl)-estra-4,9-diene-3,17-
dione 7 via 1,4-Grignard addition of 4-trimethylsilyloxyphenyl
2.15. Biological assays
2.15.1. Competitive binding to ERa-LBD
Binding affinities of the 11b-substituted estradiol derivatives
relative to E2 were performed in incubations with the LBD of
ERa. in lysates of Escherichia coli in which the LBD of human ERa
(M250–V595)] is expressed as described57,58 The assay was per-
formed overnight in phosphate buffered saline + 1 mM EDTA at
room temperature. The competition for binding of [3H]E2 to the
LBD of the E2-derivatives in comparison to E2, relative binding
affinity (RBA) was determined over a range of concentrations from
10ꢃ12 to 10ꢃ6 M. After incubation, the media is aspirated, the plates
are washed 3 times and the receptor bound radioactivity absorbed
to the plates are extracted with methanol and counted. The results,
as RBAs compared to E2, of all receptor studies shown in Table 1,
are from 3 experiments performed in duplicate. RBAs represent
magnesium bromide to 2a/b. The resulting phenol 7 was elabo-
rated using Williamson ether synthesis with ethylene glycol
ditosylate to give the crude alkylation product which was con-
verted with sodium azide in ethanol to 9. In a similar fashion, 7
was converted to the corresponding 11b-(4-((2-(2-(2-azidoeth-
oxy)ethoxy)ethoxy)phenyl)-estra-4,9-diene-3,17-dione 10. Aroma
tization, reduction, and hydrolysis gave 11b-(2-azidoethoxyphe-
nyl) estradiol analog 1163 and 11b-(4-((2-(2-(2-azidoethoxy)eth-
oxy)ethoxy)phenyl) estradiol analog 12. In anticipation of future
studies in which we would introduce a second bioactive or imaging

3774
R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
Table 1
Relative binding affinity (RBA) and relative stimulatory activity/Ki of 11b-substituted estadiol analogs at ERa-LBD
R
OH
OH
R
HO
HO
5
6
11
R = CH3O-
R = HO-
R = N3CH2CH2O-
15
R = CH3O-
16 R = CH2=CH-
12 R = N3(CH2CH2O)3-
N
N
O
H
(CH2CH2O)-
N
14
R =
N
O
17
R = (CH3)2NCH2CH2O-
18 R = C2F5SO2(CH2)5O-
Compound
RBAa E2 = 100%
RSAb E2 = 100
Ki(nM)c
n.d.
Efficacyd
5
6
11
12
14
15
16
12.7 1.5
Max stim = 35–45%
17.0 8.5
Mixed Agonist-Antagonist
Agonist
Antagonist
Antagonist
Antagonist
Agonist
67
39
26
8
9
9
n.d.
2.4 0.6
1.1 0.1
51 13
19.0 4.6
22.5 0.7
76 15
70
5
Agonist
17 RU 39411
18 RU 58668
38
83 13
9
41.5 9.2
5.3 9.2
Antagonist
Antagonist
a
Relative binding affinity (RBA) values were determined by a competitive binding assay using [3H] estradiol and ER
a-LBD, as described in the Experimental Section. The
RBA of estradiol (E2) is defined as 100, and the measured RBA values represent the mean of two or more independent determinations.
b
The relative stimulatory activity (RSA) values for ER agonists were determined using the induction of alkaline phosphastase (AlkP) in human endometrial adenocarcinoma
(Ishikawa) cells as described in the Experimental section. The RSA of estradiol (E2) is defined as 100.
c
The inhibitory constants Ki of each antagonist compound tested at a range of 10ꢃ6 to 10ꢃ12 M and measured for the inhibition of the action of 10ꢃ9 M E2 (EC50 ꢂ0.2 nM).
Each compound was analyzed in at least 3 separate experiments performed in duplicate. D Agonists generated full stimulatory response in Ishikawa assay in the absence of
estradiol. Antagonists generated no stimulatory response in the absence of estradiol but elicited full block of 0.1 nM estradiol effect with increasing concentration of test
compound. n.d. = not determined.
O
O
O
O
H3C
H3C
O
O
O
O
O
b,c
a
d,e
O
O
O
HO
O
O
O
4
2α/2β [3:1]
1
3
g.f
f
HO
H3C
OH
OH
HO
HO
6
5
Scheme 1. Synthesis of 11b-(4-methoxy/hydroxyphenyl)-estradiol analogs 5 and 6 Reagents and conditions. (a) H2O2-50%, CF3COCF3ꢄH2O, pyridine, CH2Cl2 (b) Cu(I)Cl, 4-
CH3OC6H4MgBr, THF (c) 6N HCl–CH2Cl2 (d) Ac2O, AcBr, CH2Cl2 (e) KOH, CH3OH (f) NaBH4, CH3OH (g) BBr3, CH2Cl2.

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3775
HO
O
O
O
R
R
c,d/e
OH
O
O
a,b
f,g,h
2α/2β [3:1]
HO
O
7
11 R = CH2CH2N3
9
10
R = CH2CH2N3
R = CH2CH2(OCH2CH2)2N3
12
R = CH2CH2(OCH2CH2)2N3
O
O
O
N
N
O
OH
NH
i
N
11
O
HO
14
Scheme 2. Synthesis of 11b-(4-(substituted alkoxy)phenyl)-estradiol analogs 11,12,14. Reagents and conditions. (a) Cu(I)Cl, 4-(CH3)3SiOC6H4MgBr, THF (b) 6N HCl–CH2Cl2 (c)
TsOCH2CH2OTs, K2CO3, CH3CN (d) NaN3, EtOH (e) 8, Cs2CO3, CH3CN (f) Ac2O, AcBr, CH2Cl2 (g) NaBH4, CH3OH (h) KOH, CH3OH (i) 13, CuSO4ꢄ5 H2O, H2O–t-BuOH.
moiety, we elaborated this intermediate with N-propargyl-2-
aminonaphthoquinone 13, using the [3+2] Huisgen cycloaddition
(‘click’) reaction, to give the terminally functionalized 11b-substi-
tuted estradiol analog 14.64–66
Although the synthesis of the target compounds involved a
multi-step process, it should be noted that the individual steps
proceeded in satisfactory, although unoptimized, yields. Among
the key features of this synthetic scheme was the observation that
the affinities were essentially the same (RBA = 26–39%). This find-
ing is of interest as the dimethylamino group of RU39411, compa-
rable to the dialkylamino groups of tamoxifen and raloxifene, is
believed to interact with Asp-351 though ionic or hydrogen bond-
ing.72 No significant differences in RBA values were observed for
these compounds, suggesting that other factors may be more
important. Clearly the region of the ligand binding domain that is
complementary to the 11b-position of the steroid scaffold is capa-
ble of accommodating significant structural diversity at that posi-
tion. It should be noted that the affinity was also not significantly
affected by whether the ligand binds to the agonist or antagonist
conformation of the receptor.
separation of the two epoxide isomers, 2a and 2b, while possible,
was unnecessary. As noted in the literature,40–42 work up of the
subsequent Grignard reaction under acidic conditions generated
the more stable 11b-substituted product. Depending upon the ulti-
mate target, introduction of the substituent at the 4-oxyphenyl po-
sition could either precede aromatization or follow it. In either
case, reproducible introduction of phenyl (aromatic) substituents
with the b-orientation was critical for generating the desired bio-
logical response and high affinity. This route is a more efficient
and versatile method than the alternative approach which involves
Grignard addition to an 11-oxo derivative followed by eventual
deoxygenation.33,34,67
We were also interested in determining whether the 11b-aryl
estradiol derivatives were ER-substype selective. A few studies
suggested that simple 11b-substituted estradiols, such as the
11b-methoxyestradiol, are relatively nonselective ligands for the
ERa
/ERb subtypes.54,73, Therefore, we evaluated three 11b-(4-
substituted phenyl)estradiol analogs (6,17, 18) as ERb ligands.
The observed ERb-LBD RBA values were 41%, 17% and 27% respec-
tively, vs 67%, 38%, and 83% for the ERa-LBD, indicating that the
analogs retained high affinity for the ERb-subtype but were rela-
tively nonselective. This is consistent with other studies that relate
the lack of subtype selectivity to the presence of a similar receptor
topography in the 11-beta pocket, with the major difference being
3.2. Biological evaluation of estradiol analogs
The compounds were initially evaluated as competitive inhibi-
tors of estradiol binding using ER
a-LBD, and subsequently as ago-
Leu384 (ERa
) and Met 336 (ERb).74,75
nists or antagonists using the induction of alkaline phosphatase
activity in Ishikawa cells.55–58 11b-Methoxy estradiol 15, 11b-vinyl
estradiol 16, two agonists which we had previously pre-
pared,23,68,69 and two steroidal antagonists, RU 39411 17 and RU
58668 18,70,71 were used for comparison purposes. The results of
the binding (RBA) and stimulatory (RSA) assays are shown in Table
1. The binding results indicate that all of the steroidal derivatives
Of particular interest to us was the effect of 11b-substitution on
efficacy, especially for the 4-substituted phenyl derivatives. The
11b-methoxy and 11b-vinyl estradiols 15 and 16 are well estab-
lished as potent estrogens, and this effect was observed in the cur-
rent assay. Previous studies had shown that simple 11b-aryl
estradiol analogs also were agonists, but that more highly substi-
tuted derivatives expressed anti-estrogenic properties. The specific
effects on efficacy of functional groups emanating from a 11b-phe-
nyl(aryl) substituent were not well described as most studies tend
to focus on variations of the dialkylaminoalkoxy moiety. Very few
studies, even with the triphenylethylenes (TPEs) have examined
the structural transition from hydroxyphenyl to methoxyphenyl.
In their study of triphenyl acrylonitriles (Fig. 2), Dore, et al.,
possessed high relative binding affinity (RBA) for the ERa-LBD.
The binding affinity was essentially independent of the efficacy
of the estradiol analogs as agonists with small 11b-substituents,
such as 15 and 16, had RBA values comparable to the antagonists
with much large substituents, such as 12, 14, or 18.
Binding for the antagonists was also not significantly affected
by the nature of the atom at the 2-position of the alkoxy moiety.
Whether the substituent was azido (11), amino (17) or oxy (12),
observed that the
a,a’,b-tris(4-hydroxyphenyl) compound was a
full agonist with high binding affinity (RBA = 166%), whereas the

3776
R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
O
OH
R
O
O
R
CN
OH
S
HO
Benzothiophene Derivatives
OH
R = H
Agonist
?
R = CH
3
Triphenyl acrylonitriles
R = H
-
CH CH
R =
2
2
Agonist
Partial Agonist
N
R = CH
3
R = (C2 H5 )NCH2 CH2 - Antagonist
Raloxifene Antagonist
Figure 2. Evaluation of methoxy/hydroxyl analogs of estrogens/anti-estrogens.
a
,b-bis(4-hydroxyphenyl)-
a
’-(4-methoxyphenyl) analog had re-
recruitment of coregulatory peptides.81 Weatherman and co-work-
duced binding (RBA = 17%) and ‘partial’ agonist responses.76,77
A
ers, noted in their study of GW7604 derivatives, in which a termi-
nal carboxy group of a nonsteroidal antiestrogen was replaced by
an amido or methyl ketone moiety, that affinity but not efficacy
was affected.82 Further extension of the 4-substituent, as seen in
larger alkoxy group, such as isopropoxy had even lower RBA and
agonist effects. In other studies that examined the benzothiophene
class of antiestrogens, that is, structurally related to raloxifene
(Fig. 2), one can find similar effects. Griffin and co-workers used
hydrogen-deuterium exchange mass spectrometry (HDX-MS) to
evaluate the stability of ligand–ER-LBD complexes.78,79 In their
work which included raloxifene, the de-alkylated 4-hydrox-
ybenzoyl derivative, and other analogs of raloxifene, they
determined that the 4-hydroxybenzoyl compound behaved as an
agonist, had high binding affinity (RBA ꢂ25%), and gave an
HDX-MS profile similar to that for estradiol. The corresponding
4-methoxybenzoyl analog was not evaluated. What was particu-
larly notable in this study, which evaluated a number of raloxifene
analogs and other antiestrogens, was that the antagonist com-
plexes appeared to be more stable than the agonist complexes,
based upon the slower exchange rates within the ligand binding
core of the receptor.
In our study, we examined the influence of the substituents on
phenolic group, as well as the contribution of the more distal com-
ponents. As the results show, the 11b-(4-hydroxyphenyl) estradiol
analog exhibited high affinity (RBA = 67%) but was a slightly less
potent agonist (RSA = 17%) than estradiol. Capping the terminal
phenolic hydroxyl with a methyl group reduced binding affinity
somewhat (RBA = 13%), but the compound now displayed mixed
agonist/antagonist activity (See Supplementary data for binding
curves). This effect is similar to that observed in the triphenylacr-
ylonitrile and benzothiophene studies. What our series provides,
and which was not evaluated in the other studies, is the effect of
progressive modification of the alkoxy moiety. Extension of the
4-substituent from a methoxy to a 2-(azidoethoxy)-moiety trans-
formed the ligand into essentially a full antagonist with high affin-
ity (RBA = 39%). The distal azido group is a nonbasic, weak
hydrogen bonding group which would not interact strongly with
the complementary Asp-351 moiety usually associated with antag-
onist effects. Substitution of the azido-group by the N,N-dimethyl-
amino group yields the known antagonist RU39411 17
(RBA = 38%), which has much higher affinity than tamoxifen
(ꢂ1%). 80 This substitution provides essentially no change in either
binding affinity or antagonist potency, suggesting that neither
nitrogen basicity nor hydrogen bonding are the dominating effects
at this site. It has been observed previously that replacement of
Asp-351 by neutral amino acids did not significantly affect the
binding affinity of antagonists but did influence the subsequent
RU58668 18, or the
x-substituted triethyleneglycol derivatives
12 and 14, in which the beta atom is carbon or oxygen gave equally
potent ER ligands with roughly comparable anti-estrogenic proper-
ties. Introduction of a terminal 1,4-naphthoquinone moiety to give
derivative 14 or the fluoroethylsulfonyl group in 18 produced,
slight improvements in ERa-LBD binding and maintenance of full
antagonist properties. Therefore the key feature that contributes
to the antiestrogenic response appears to be the loss of the pheno-
lic group and addition of at least a two carbon moiety.
The results from the current study demonstrated that the tran-
sition from agonist to antagonist properties occurs within a very
narrow structural range for this class of compounds. One can con-
sider the initial interaction of the steroidal ligand with the
(apo)receptor as proceeding via an interaction with a receptor con-
formation in which the helix-12 component is loosely bound. This
initial complex can then undergo transformations to generate
either an agonist conformation or an antagonist conformation,
depending upon the nature of the 11b-subsituent. Recruitment of
coactivator proteins for the agonist conformation or corepressor
proteins for the antagonist conformation then leads to the subse-
quent biological response. [Fig. 3.] For small functional groups at
the 11b-position, represented by 11b-methoxy- or vinyl estradiol
15 or 16, the preferred conformation is clearly the agonist confor-
mation. Folding of helix-12 to give a complex similar to that ob-
served for estradiol is favored and yields a stabilized agonist
ligand–receptor complex. As observed in this study, 11b-(4-
hydroxyphenyl)estradiol 6 also prefers the agonist conformation,
presumably by interactions between the 4-hydroxy moiety and
the complementary residues, such as Thr-347, in the receptor bind-
ing pocket.[Fig. 4A] Such interactions have been used to explain the
binding affinities observed with 1,1-bis(4-hydroxyphenyl)ethylene
derivatives, such as the cyclofenil analogs.83,84 Replacement of the
terminal hydrogen with a methyl group as in 11b-(4-methoxy-
phenyl) estradiol 5 apparently disrupts those interactions suffi-
ciently, either by loss of hydrogen bonding or by enhanced steric
interactions, such that the alternate complex with the antagonist
helix-12 conformation is more competitive.[Fig. 4B] The difference
in binding energy may be small as the compound displays a signif-
icant estrogenic component (30%), even at high concentrations. In
addition, HDX-MS experiments suggest that the antagonist

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3777
ER-LBD
CH3
O
LBP
5
H12
Coactivator
proteins
Corepressor
proteins
ER-LBD
ER-LBD
ER-LBD
CH3
O
CH3
O
H12
CH3
O
H12
H12
Agonist
Intermediate open
Antagonist
Figure 3. Proposed binding of 11b-(4-methoxyphenyl)estradiol analog with ERa-LBD. The initial interaction of the ligand with the receptor is reversible as are the
conformational states. The equilibrium is influenced by the contributions of interactions of the terminal methoxy group with complementary residues in the ligand binding
pocket (LBP) in the different conformations of the receptor.
Thr-347
Thr-347
Leu-525
His-524
His-524
Leu-540
Glu-353
H2O
Glu-353
H2O
Arg-394
Arg-394
6
5
2B
2A
Figure 4. (A, B) Representation of major interactions between the 11b-(4-substituted phenyl)estradiol analogs and the agonist conformation (A) and antagonist conformation
(2B) of ER -LBD. In (A), compound 6 would have strong interactions with the HO-group of Thr-347, in addition to hydrogen bonding with Glu-353 and Arg-394 (3-phenolic –
a
OH) and His-524 (17b-OH), stabilize the agonist conformation. In (B), compound 5 retains the strong hydrogen bonding interactions with Glu-353 and Arg-394 (3-phenolic–
OH) and His-524 (17b-OH), but the methoxy-group would have weaker bonding to the HO-of Thr-347. In addition, the steric interactions would disfavor the agonist
conformation, while hydrophobic interactions with Leu-525,540 present in helix-12 of the antagonist conformation would be favorable.
conformations are more stable than agonist conformations which
may also influence the observed balance.78,79 Further modification
of estradiol analogs with longer/larger substituents on the 4-oxy-
phenyl group results in a clear preference for the antagonist con-
formation, similar to that formed with tamoxifen, raloxifene and
other nonsteroidal antagonists. The substituents in this study that
extend beyond the methoxy-group appear to have little additional
effect on the binding affinity.
One potential consequence of the identification of the agonist-
antagonist transition point relates to the assessment of the metab-
olism of anti-estrogens. With conventional nonsteroidal antiestro-
gens, such as tamoxifen, primary attention has focused on
metabolism of the aromatic ring (4-hydroxytamoxifen) or the
terminal amine group (endoxifene).85,86 [Fig. 5] In these cases,
metabolism yields pharmacologically active compounds that re-
tain antiestrogenic properties. Further metabolism of the terminal
amine to a carboxylic acid, or conjugation with glucoronic acid
generates inactive compounds. However, a minor but detectable
pathway involves the oxidative cleavage of the dialkylaminoeth-
oxy side chain to give the corresponding phenolic derivative. As
we and others have shown, these phenolic compounds (metabo-
lites) are potent estrogenic substances whose levels would need
to be suppressed in disorders such as hormone responsive breast
cancer. It would be of interest to determine the extent to which
these metabolites contribute to the Selective Estrogen Receptor
Modulation (SERM) effect.
In considering the design and synthesis of drug-estradiol conju-
gates for the targeted therapy of ER-expressing breast cancer, most

3778
R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
O
O
O
N
H
HO
Endoxifene
Inactive acid
O
HO
N
Potent TPE agonist
OH
Tamoxifen
O
O
N
N
OGluc
Inactive conjugate
OH
Hydroxytamoxifen
Figure 5. Metabolic pathways for tamoxifen. Cleavage of basic side chain yields compound with estrogenic properties.
O
N
HN
NH
Linker
Linker
H3C
O
O
O
H2N
O
N3
O
Linker
Mitomycin C Derivative
OH
O
NH
O
N
HO
H
O
HO
OCH3
Steroidal Antiestrogen Derivative
O
O
H2N
Geldanamycin Derivative
Figure 6. Putative alkynylated therapeutic coupling partners for azido-linker substituted steroidal antiestrogen.
studies of the interaction of anti-estrogens with ER
nonsteroidal agents as the ligands. Crystal structures of complexes
between ER -LBD and tamoxifen or raloxifene have shown that
the N,N-dialkylaminoethoxyphenyl moiety extends almost perpen-
dicularly from the nonsteroidal scaffold, into a pocket that has an
interaction between Asp-351 and the terminal amino.86–88 In these
and other complexes, the position of the terminal amine is
essentially at the receptor–solvent interface. For the new com-
pounds that display antagonist properties, one can imagine that
the 11b-(4-alkoxyphenyl) group occupiesessentially thesame space
a
-LBD have used
as the N,N-dialkylaminoethoxyphenyl moiety of the nonsteroidal
antagonists. In that scenario, virtually all of the atoms that extend
beyond the equivalent of the N,N-dialkylamino group would be
external to the receptor surface and therefore extend into the sol-
vent space. Because the interactions between more distal parts of
the ligand and the receptor surface are likely to be weaker, receptor
affinities should be relatively similar. Although our series does not
have sufficient representatives to confirm that hypothesis, the re-
sults tend support this proposal. The RBA values for the azidoethoxy
and N,N-dialkylaminoethoxy derivatives, as well as the more highly
a

R. N. Hanson et al. / Bioorg. Med. Chem. 20 (2012) 3768–3780
3779
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Support of this research was provided by grants from the Na-
tional Institutes of Health [PHS 1R01 CA81049 (R.N.H.), PHS 1R01
CA 37799 and R21 MH082252 (R.B.H.)], Department of Energy
(DE-SC0001781)[RNH], the Susan
G
Komen Foundation
(BCTR0600659)[RNH] and NSF-IGERT Award # 0504331 (J.A.H.).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.041.
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