Synthesis and functional analysis of novel bivalent estrogens

Article abstract of DOI:10.1016/j.steroids.2010.05.019

The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen mediates its effects through binding and activation of specific estrogen receptors alpha (ERα) and beta (ERβ), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been the target of large therapeutic development efforts. The binding of estrogen to its respective receptors initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for manipulating steroid receptor gene expression activity through the development of bivalent steroid hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can activate estrogen receptor alpha (ERα)-mediated transcription events with efficacy and potency equal to or greater than that of ERα's cognate ligand, 17β-estradiol. These bivalent estrogen structures open the door to the development of a variety of steroid therapeutics that could dramatically impact future drug development in this area.

Full text of DOI:10.1016/j.steroids.2010.05.019

Steroids 75 (2010) 825–833
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and functional analysis of novel bivalent estrogens
Alison E. Wendlandt, Sharon M. Yelton, Dingyuan Lou, David S. Watt, Daniel J. Noonan∗
Department of Molecular and Cellular Biochemistry, University of Kentucky, 741 South Limestone Street, Lexington, KY 40536-0509, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The steroid hormone estrogen plays a critical role in female development and homeostasis. Estrogen
mediates its effects through binding and activation of specific estrogen receptors alpha (ER␣) and beta
(ER␤), members of the steroid/nuclear receptor family of ligand-induced transcription factors. Due to
their intimate roles in genomic and nongenomic signaling pathways, these hormones and their receptors
have been also implicated in the pathologies of a variety of cancers and metabolic disorders, and have been
the target of large therapeutic development efforts. The binding of estrogen to its respective receptors
initiates a cascade of events that include receptor dimerization, nuclear localization, DNA binding and
recruitment of co-regulatory protein complexes. In this manuscript, we investigate the potential for
manipulating steroid receptor gene expression activity through the development of bivalent steroid
hormones that are predicted to facilitate hormone receptor dimerization events. Data are presented
for the development and testing of novel estrogen dimers, linked through their C-17 moiety, that can
activate estrogen receptor alpha (ER␣)-mediated transcription events with efficacy and potency equal to
or greater than that of ER␣’s cognate ligand, 17␤-estradiol. These bivalent estrogen structures open the
door to the development of a variety of steroid therapeutics that could dramatically impact future drug
development in this area.
Received 28 December 2009
Received in revised form 26 April 2010
Accepted 24 May 2010
Available online 2 June 2010
Keywords:
ER␣
Nuclear hormone
Girard reagent
Dimerization
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
and mediate genomic and non-genomic signaling events [11,12].
These data suggest that the specific targeting and manipulation of
Estrogens regulate gene expression events through their abil-
ity to bind either of two intracellular receptors, estrogen receptor
alpha (ER␣) or estrogen receptor beta (ER␤) [1,2]. This binding
event facilitates dimerization of the receptor and modulation of
both nongenomic and genomic signaling pathways [3–5]. Nonge-
nomic signaling pathways are mediated through a subpopulation
of estrogen-bound ER dimers that bind peripheral membranes
and activate G protein and extracellular signal-regulated kinase
(ERK)-mediated events. The classical genomic signaling pathway
is mediated by estrogen-ER binding events that result in recep-
tor nuclear localization, binding to specific DNA response elements
motifs (EREs) in gene promoters, and regulation of transcription of
these genes [6]. Furthermore, as might be predicted, recent stud-
ies suggest the convergence of these two signaling pathways in the
mediation of their signaling events [6,7].
the dimeric state of ERs might serve as a mechanism for regulat-
ing their activity if suitable agents were available to modulate this
dimerization in either a positive or negative sense.
The above mechanisms of activity permit estrogens to coordi-
nate the expression of a large number of genes in a variety of tissues.
This is a positive facet of estrogen activity in that it orchestrates the
large pleiotropic effects that are needed, but it can also have serious
negative impacts on an organism in situations where the hormone
or its receptors are improperly expressed, improperly regulated,
or expressed at the wrong time. Accordingly, estrogen, its receptor
and its coregulatory proteins, has been implicated in the patholo-
gies of a variety of diseases and cancers [13–16]. Furthermore, these
large pleiotropic effects are a serious complication in using hor-
mone or hormone derivatives as potential therapeutics [17–19].
Technologies that increase specificity for estrogen activity or possi-
bly target it to cells, tissues or independent genes, have tremendous
applicability in therapeutics. In this manuscript, we investigate
the potential for developing steroid-based molecules that would
directly impact the critical steroid receptor dimerization event.
Using estrogen as a model system, evidence is presented for the
development of novel estrogen dimers (bivalent estrogens), linked
through their C-17 moiety, that can function as potent agonists
of transcriptional activation events mediated by human ER␣. It is
further shown that these bivalent estrogens retain both their speci-
Estrogen binding activates its intracellular receptors by altering
the conformation of its ligand-binding domain [8–10]. Unliganded
estrogen receptor adopts a mobile and partially disordered globu-
lar state that rearranges upon estrogen binding and goes through
multiple conformational changes that finally allow it to dimerize
∗
Corresponding author. Tel.: +1 859 257 7498; fax: +1 859 323 1037.
E-mail address: dnoonan@uky.edu (D.J. Noonan).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.05.019

826
A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
ficity and stability throughout the in vitro analyses employed. These
data foster a host of subsequent studies in which the linker moiety
might be manipulated to create targeted agonists and antagonists
of ER␣-mediated events.
a product that was chromatographed in 2:1 ethyl acetate:hexane
and recrystallized from 1:10 dichloromethane:methanol to afford
45 mg (29%) of 5a: mp 182–183 ◦C; 1H NMR (400 MHz, DMSO-d6,
ppm) ı 7.1–7.2 (2H, d, J = 8.8 Hz), 6.55–6.68 (4H, m) 4.6–4.7 (2H, s,
OH), 2.8–2.9 (4H, m), 2.5–2.72 (4H, m), 2.35–2.42 (6H, m), 2.2–2.3
(4H, m), 1.9–1.98 (4H, m), 1.6–1.8 (6H, m), 1.4–1.6 (10H, m, over-
laps with H2O peak), 1.26–1.4 (12H, m) 1.1–1.4 (6H, s); 13C NMR
(400 MHz, DMSO-d6, ppm) ı 178.73, 171.81, 153.68, 138.17, 132.35,
126.77, 115.45, 113.04, 52.96, 45.60, 44.02, 38.36, 33.91, 33.31,
29.66, 29.59, 29.43, 29.38, 27.36, 27.37, 26.31, 25.19, 23.03, 17.26;
m/z (MALDI-TOF, DHB matrix) m/z (intensity): 787.4 (M+Na+, 600),
803.4 (M+K+, 240). Anal. Calcd. for C48H64N2O6·3H2O: C, 70.39; H,
8.61. Found: C, 70.78; H, 8.14.
2. Experimental
2.1. Chemicals and reagents
Estrone (1), estrone 3-methyl ether (2), and equilenin (3)
were purchased from Sigma–Aldrich. 3-Hydroxyestra-1,3-5(10)-
triene-17-oxime (4a) [20] and 3-methoxyestra-1,3-5(10)-triene-
17-oxime (4b) [21] were synthesized according to literature
procedures. Solvents were used from commercial vendors with-
out further purification unless otherwise noted. Infrared spectra
were determined on an Avatar 360 FT. Nuclear magnetic res-
onance spectra were determined on a Varian 200 or 400 MHz
instrument. LRMS electron-impact (EI) ionization mass spectra
were recorded at 70 eV on a ThermoFinnigan PolarisQ (ion trap
mass spectrometer). Samples were introduced via a heatable direct
probe inlet. MALDI mass spectra were obtained on a Bruker
Autoflex time-of-flight mass spectrometer (Billerica, MA), using
various matrices (i.e., CHCA: alpha-cyano-4-hydroxycinnamic acid;
SA: sinapinic acid; DHB: 2,5-dihydroxybenzoic acid), as noted
in the following experimentals. HRMS electron impact (EI) ion-
ization mass measurements were recorded at 25 eV on a JEOL
JMS-700T MStation (magnetic sector instrument) at a resolution of
greater than 10,000. Samples were introduced via heatable direct
probe inlet. Perfluorokerosene (PFK) was used to produce ref-
erence masses. Elemental analyses were determined by Atlantic
Microlabs, Inc., Norcross, GA. Compounds were chromatographed
on preparative layer Merck silica gel F254 unless otherwise indi-
cated.
2.4. Synthesis of
bis(3-hydroxyestra-1,3,5(10)-triene-17-oximinyl)
1,22-docosanedioate (5b)
The procedure described for 5c was repeated using 138 mg
(0.486 mmol, 1.2 equiv.) of oxime 4b, 75 mg (0.202 mmol) of
1,22-docosanedioic acid, 66 mg (0.486 mmol, 1.2 equiv.) of 1-
hydroxybenzotriazole, and 93 mg (0.486 mmol, 1.2 equiv.) of
N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochloride
in 1 mL of 1:1 acetonitrile:tetrahydrofuran to afford, after stir-
ring at reflux for 24 h, a product that was chromatographed using
2:1 ethyl acetate/hexane, to afford 131 mg (72%) of 5b: 1H NMR
(400 MHz, CDCl3, ppm) ı 7.13–7.15 (2H, d, J = 8.4 Hz), 6.57–6.66
(4H, m), 5.22 (2H, s, OH), 2.8–2.87 (4H, m), 2.5–2.72 (4H, m),
2.35–2.42 (6H, m), 2.18–2.3 (4H, m), 1.88–1.96 (4H, m) 1.64–1.78
(9H, m), 1.4–1.58, (9H, m), 1.2–1.4 (34H, m), 1.01 (6H, s); 13C
NMR (400 MHz, CDCl3, ppm) ı 178.92, 172.03, 153.93, 138.00,
132.03, 126.65, 115.50, 113.12, 52.92, 45.62, 43.98, 38.34, 33.83,
33.30, 29.92, 29.90, 29.86, 29.79, 29.64, 29.42, 29.34, 27.37, 26.28,
25.17, 23.00, 17.22; LRMS m/z: 816 (M+Na, 3000), 534 (M+Na−258,
1200).
2.2. Synthesis of 3-hydroxyestra-1,3-5(10)-triene-17-oximinyl
17-laurate (4c)
To 50 mg (0.175 mmol) of oxime 4a in 0.9 mL anhydrous pyri-
dine was added 67 mg (0.175 mmol) of lauric anhydride. The
mixture was stirred for 72 h at 25 ◦C and quenched with ice to give
a white precipitate. The mixture was diluted with ethyl acetate,
washed successively with saturated copper sulfate solution, and
brine, and dried over anhydrous MgSO4. The product was chro-
matographed in 1:2 ethyl acetate:hexane and recrystallized from
ethyl acetate to give 18 mg (23%) of 4c: mp, 72–74 ◦C; 1H NMR
(400 MHz, DMSO, ppm) ı 7.28–7.31 (1H, d, J = 8.8), 7.82–7.84 (1H,
dd, J1,4 = 2.4, J1,2 = 8), 6.70–6.80 (1H, s), 2.8–2.85 (2H, m), 2.25–2.55
(4H, m, overlaps with DMSO peak), 1.8–2.0 (3H, m), 1.2–1.65 (26H,
m), 0.8–0.87 (6H, m); 13C NMR (200 MHz, CDCl3, ppm) ı 172.88,
171.32, 148.80, 138.21, 137.71, 126.53, 121.76, 118.91, 53.14, 44.39,
38.01, 34.65, 34.21, 32.13, 29.82, 29.68, 29.56, 29.48, 29.34, 27.25,
26.22, 25.37, 25.22, 23.15, 22.91, 17.40, 14.34; LRMS m/z (inten-
sity): 467 (M+, 40), 468 (MH+, 17), 268 (M+−199, 83); HRMS Calcd.
for C30H45O3N 467.3399, found 467.3392 (mean of five determina-
tions, SD 2.1 ppm; error −0.7 ppm).
2.5. Synthesis of
bis-(3-methoxyestra-1,3-5(10)-triene-17-oximinyl)
1,12-dodecanedioate (5c)
To 123 mg (0.411 mmol, 0.8 equiv.) of oxime 4b and 57 mg
(0.247 mmol, 1 equiv.) of 1,12-dodecanedioic acid in 1 mL of
1:2 methanol:dichloromethane was added 67 mg (0.494 mmol, 1
equiv.) of 1-hydroxybenzotriazole hydrate followed immediately
by 158 mg (0.823 mmol, 1.7 equiv.) of N-(3-dimethylaminopropyl)-
Nꢀ-ethylcarbodiimide hydrochloride. The mixture was stirred
at 25 ◦C for 48 h, evaporated to dryness, and diluted with
dichloromethane. The solution was washed with water; the result-
ing emulsion was broken with brine; and the product was dried
over anhydrous MgSO4. The crude product was chromatographed
using 1:1 ethyl acetate/hexane and recrystallized twice from ethyl
acetate/hexane to give 15.4 mg (8%) of 5c: mp 119–121 ◦C; IR (KBr)
1766 (C O) cm−1 1H NMR (400 MHz, CDCl3, ppm) ı 7.18–7.24 (2H,
;
d, J = 8.4 Hz), 6.7–6.73 (2H, dd, J1,4 = 2.4 Hz J1,2 = 8.4 Hz), 6.64 (2H,
s), 3.8 (6H, s), 2.84–2.9 (4H, m), 2.5–2.8 (4H, m), 2.2–2.5 (12–14H,
m), 1.8–2.0 (4H, m), 1.4–1.8 (25H, m), 1.2–1.4 (12H, m), 1.03 (6H,
s); 13C NMR (400 MHz, CDCl3, ppm) ı 178.707, 171.769, 157.778,
137.867, 132.273, 126.557, 114.10, 114.06, 111.754, 55.421, 52.977,
45.591, 44.027, 38.372, 33.923, 33.308, 29.854, 29.589, 29.437,
29.39, 27.349, 26.309, 25.193, 23.30, 17.268; LRMS m/z (inten-
sity): 793 (M+H+, 87), 815 (M+Na−H+, 100), 816 (M+Na+, 57),
831 (M+K−H+, 12), 832 (M+K+, <1). Anal. Calcd. for C50H68N2O6
hydrate: C, 74.04; H, 8.70; N, 3.45. Found: C, 74.24; H, 8.68; N,
3.44.
2.3. Synthesis of
bis-(3-hydroxyestra-1,3,5(10)-triene-17-oximinyl)
1,12-dodecanedioate (5a)
The procedure described for the preparation of 5c was repeated
using 137 mg (0.48 mmol, 1.2 equiv.) of oxime 4a, 46 mg (0.2 mmol)
of 1,12-dodecanedioic diacid, 65 mg (0.48 mmol, 1.2 equiv.) of 1-
hydroxybenzotriazole hydrate, and 92 mg (0.48 mmol, 1.2 equiv.)
of N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochlo-
ride in 1.2 mL anhydrous THF to afford, after stirring at 25 ◦C for 24 h,

A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
827
2.6. Synthesis of bis(3-methoxyestra-1,3-5(10)-triene-17-oxime)
2.9. Synthesis of Girard P adduct (7)
1,22-docosanedioate (5d)
The procedure described for the preparation of 6b was repeated
using 25 mg (9.4 ␮mol) of equilenin (3), 18 mg (9.4 ␮mol, 1 equiv.)
of Girard’s reagent P, and 10 mg Dowex 50 W × 2 acid resin in
940 ␮L of methanol to afford, after refluxing for 16 h and filter-
ing, a crude solid. The product was recrystallized from methanol
to yield 27 mg (66%) of 7: mp 210–212 ◦C (with decomposi-
tion); 1H NMR (400 MHz, DMSO-d6) ı 10.99 (1H, s), 9.67 (1H, s),
9.05–9.08 (2H, t, J = 5.6 Hz), 8.67–8.71 (1H, t, J = 8 Hz), 8.20–8.24
(2H, t, J = 7.2 Hz), 7.82–7.87 (1H, t, J = 9.2 Hz), 7.54–7.56 (1H, d,
J = 4.8 Hz), 7.19–7.21 (1H, d, J = 8.8), 7.10–7.12 (1H, m), 5.85–5.97
(m, 2H), 3.12–3.47 (m, overlaps with H2O), 2.95–2.99 (1H, m),
2.4–2.8 (4H, m), 2.25–2.29 (1H, m), 1.81–2.00 (2H, m), 0.74–0.78
(3H, m); 13C NMR (200 MHz, DMSO-d6) ı 173.30, 172.24, 159.981,
151.92, 151.72, 151.53, 138.97, 136.81, 132.91, 131.55, 130.12,
129.98, 124.01, 114.92, 67.197, 53.68, 49.79, 36.78, 33.194, 29.112,
28.55, 21.45; m/z (MALDI-TOF, DHB matrix) 400 (M−Cl−, 5000);
Anal. Calcd. for C25H26N3O2Cl·2H2O: C, 63.62%; H, 6.41%. Found: C,
63.52%; H, 6.05%.
The procedure described for 5c was repeated using 185 mg
(0.617 mmol, 0.9 equiv.) of oxime 4b, 137 mg (0.371 mmol, 1 equiv.)
of 1,22-docosanedioic acid, 101 mg (0.741 mmol, 1 equiv.) of 1-
hydroxybenzotriazole hydrate, and 237 mg (1.23 mmol, 1.6 equiv.)
of N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochlo-
ride to afford, after stirring at 25 ◦C for 72 h, a product that was
chromatographed using 1:1 ethyl acetate:hexane and recrystal-
lized from ethyl acetate:hexane to afford 71 mg (21%) of 5d:
mp 63–66 ◦C; 1H NMR (400 MHz, CDCl3, ppm) ı 7.20–7.26 (2H,
d, J = 8.4 Hz), 6.7–6.73 (2H, dd, J1,4 = 2 Hz, J1,2 = 8.4 Hz), 6.63–6.64
(2H, d, J = 2.4 Hz), 3.77 (6H, s), 2.87–2.91 (4H, m), 2.5–2.7 (4H,
m), 2.36–2.42 (5H, m), 2.20–2.32 (4H, m), 1.9–1.98 (4H, m),
1.2–1.8 (60H, m), 1.02 (6H, s); 13C NMR (CDCl3) ı 178.669,
171.761, 157.763, 137.844, 132.257, 126.557, 114.069, 111.724,
55.406, 52.946, 45.568, 44.019, 38.364, 33.908, 33.316, 29.908,
29.839, 29.665, 29.460, 29.391, 27.425, 27.327, 26.287, 25.201,
23.007, 17.246; m/z (MALDI-TOF, DHB matrix) 955.8 (M+Na,
2400), 971.8 (M+K, 1400). Anal. Calcd. for
C60H88N2O6: C,
77.21%; H, 9.50%; N, 3.00%. Found: C, 77.10%; H, 9.54%; N,
2.97%.
2.10. Synthesis of bis-Girard adduct (8a)
Suberoyl chloride (0.586 g, 2.776 mmol) was added dropwise
to a solution of 4-aminopyridine (0.627 g, 6.66 mmol), triethy-
lamine (0.773 mL, 5.55 mmol), and 10 mL MeCN. After 60 min
of stirring at room temperature, TLC of the reaction indicated
completion; the reaction was refluxed for an additional 30 min
to ensure full conversion of the starting materials. The reaction
mixture was then dumped into an excess (75 mL) of saturated
NaHCO3, and allowed to stir for 30 min. The aqueous solution
was then filtered, and the solids rinsed with additional saturated
NaHCO3. Solids were dried overnight under vacuum, and then
recrystallized from boiling methanol to give 0.835 g (92%) of bis-
2.7. Synthesis of Girard P adduct (6a)
The procedure described for the preparation of 6b was repeated
using 25 mg (0.092 mmol) of estrone (1), 17 mg (0.092 mmol, 1
equiv.) of Girard’s reagent P, and 10 mg of Dowex 50 W × 2 acid
resin to afford, after refluxing for 16 h and filtering, a crude solid.
The product was recrystallized from abs EtOH to give 33 mg (81%)
of 6a: mp 199–201 ◦C (with decomposition); 1H NMR (400 MHz,
DMSO-d6, ppm) ı 10.92 (0.5H, s), 9.05–9.06 (2H, d, J = 5.6 Hz),
8.65–8.69 (1H, t, J = 7.6 Hz), 8.19–8.23 (2H, t, J = 7.6 Hz), 7.04–7.09
(1H, t, J = 8.4 Hz), 6.47–6.54 (2H, m), 5.79–5.91 (2H, q, J = 16.8 Hz),
1.8–2.8 (10H, m, overlaps DMSO peak), 1.3–1.6 (6H, m), 0.82–0.92
(3H, m); 13C NMR (400 MHz, DMSO-d6, ppm) ı 169.18, 166.75,
137.10, 129.97, 127.37, 126.06, 114.97, 112.79, 61.74, 55.99, 51.98,
44.75, 43.65, 37.93, 34.05, 29.08, 26.98, 26.80, 25.86, 22.76, 18.57,
16.92; IR (KBr) 3199 (phenol); m/z 404 (M−Cl−, 100); Anal. Calcd for
N-(4-pyridyl) 1,8-octanediamide as a white solid: mp 195–196; 1
H
NMR (400 MHz, DMSO-d6, ppm) ı 10.20 (2H, s, NH), 8.33–8.35 (4H,
dd, Ja = 1.6 Hz, Jb = 4.8 Hz), 7.49–7.51 (4H, dd, Ja = 1.6 Hz, Jb = 4.8 Hz),
2.27–2.31 (4H, t, J = 7.2 Hz), 1.52–1.56 (4H, m), 1.26–1.28 (4H, m);
13C NMR (400 MHz, DMSO-d6, ppm): ı 172.47, 150.29, 145.75,
113.02, 36.44, 28.37, 24.65; HRMS Calcd. for C18H22N4O4 326.1742,
found 326.1744 (mean of 5 determinations, SD 1.0 ppm; error
0.6 ppm).
The recrystallized bis-N-(4-pyridyl) 1,8-octanediamide (0.350,
1.07 mmol) was added slowly to a solution of ethyl chloroacetate
(0.228, 2.14 mmol) in 1 mL absolute ethanol at room temperature.
The mixture was heated to reflux, at which point the solids fully
dissolved. After refluxing for 4 h, the reaction was removed from
heat and allowed to cool to room temperature, and then cooled to
0 ◦C with an ice/water bath. Hydrazine (0.067 mL, 2.14 mmol) was
added dropwise, resulting in a very thick suspension. The solids
were obtained by filtration, and then recrystallized in 75 mL of
boiling methanol.
This recrystallized bis-Girard reagent (0.200 g, 0.368 mmol)
was then suspended in 5 mL absolute ethanol. Estrone (0.199 g,
0.736 mmol), and catalytic Dowex 50 W × 2 resin (50 mg) were
added and the reaction was brought to reflux. After 24 h the reac-
tion was filtered, solids were washed with hot methanol, and
the liquids were concentrated to give a crude solid. The result-
ing solid was brought up in a minimum of methanol, and dropped
slowly into 100 mL of cold diethyl ether. The fine white precipitate
was collected from the ether by gravity filtration through a sin-
tered glass filter, giving bis-Girard adduct 8a: mp >230 ◦C; 1H NMR
(400 MHz, MeOD-d4, ppm): ı 7.19–7.21 (4H, d, J = 8 Hz), 6.28–6.31
(2H, d, J = 8.8 Hz), 6.04–6.06 (4H, d, J = 7.2 Hz), 5.73–5.76 (2H, d,
J = 8.8 Hz), 5.69–5.70 (2H, d, J = 2.8), 4.5–4.6 (4H, q, AB), 4.25 (2H,
s), 1.95–2.10 (4H, m), 1.1–1.9 (20H, m), 0.6–0.9 (18H, m), 0.17 (6H,
C
25H30N3O2Cl·H2O: C, 65.56%; H, 7.04%; N, 9.17%. Found: C, 65.21%;
H, 7.27%; N, 8.77%.
2.8. Synthesis of Girard P adduct (6b)
To 50 mg (0.176 mmol) of estrone 3-methyl ether (2) in 1.8 mL
abs EtOH was added 33 mg (0.176 mmol, 1 equiv.) of Girard’s
reagent P followed immediately by 20 mg of Dowex 50 W × 2
acid resin. The mixture was refluxed for 16 h, cooled, and fil-
tered through Celite. The crude product was recrystallized from
hexane/ethanol to give 38 mg (48%) of 6b: mp 218–220 ◦C (with
decomposition); 1H NMR (400 MHz, DMSO-d6, ppm) ı 10.92 (0.5H,
s), 9.04–9.05 (2H, d, J = 6 Hz), 8.65–8.69 (1H, t, J = 7.6 Hz), 8.19–8.22
(2H, t, J = 7.6 Hz), 7.18–7.20 (1H, d, J = 7.6 Hz), 6.63–6.70 (2H, m),
5.78–5.90 (2H, q, J = 17.2 Hz), 3.69 (3H, s), 2.79–2.85 (2H, m),
2.16–2.6 (8H, m, overlaps with DMSO peak), 1.86–2.04 (4H, m),
1.2–1.6 (8H, m), 0.9–0.91 (3H, m); 13C NMR (400 MHz, DMSO-d6,
ppm) ı 169.11; 166.72; 157.12; 146.45, 146.06, 137.37, 131.72,
127.43, 126.18, 113.46, 111.54, 61.73, 54.89, 51.95, 44.72, 43.61,
37.80, 34.01, 29.16, 26.96, 26.69, 25.79, 22.74, 16.89; m/z (MALDI-
TOF, DHB Matrix) 418 (M−Cl−, 2700), 137 (MNa+−114, 275); Anal.
Calcd. for C26H32N3O2Cl·H2O: C, 65.27%; H, 7.45%. Found: C, 65.66%;
H, 7.39%.

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A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
s); 13C NMR (400 MHz, MeOD-d4, ppm): ı 168.28, 166.68, 158.32,
153.41, 142.83, 142.59, 136.03, 129.49, 124.48, 113.41, 111.15,
107.56, 56.67, 51.11, 43.77, 42.82, 37.21, 32.83, 27.92, 25.73, 24.79,
21.51, 14.76; m/z (MALDI, SA matrix): 975 (M, 220), 841 (M−134,
400), 643 (M−332, 50).
was removed, the solid was added slowly to a flask containing
1.12 g (11.92 mmol) 4-aminopyridine, 1.66 mL (11.92 mmol) tri-
ethylamine, and 25 mL MeCN. The mixture was stirred at room
temperature for 30 min, then refluxed for 2 h, then cooled to room
temperature. The reaction mixture was dumped into an excess
(100 mL) of saturated NaHCO3, and allowed to stir for 30 min. The
aqueous solution was then filtered, and the solids rinsed with
additional saturated NaHCO3. Solids were dried overnight under
vacuum, and then recrystallized from boiling methanol to give an
off-white solid. One recrystallization gave 1.89 g (4.05 mmol, 85%)
of bis-N-(4-pyridyl) 1,18-octadecanediamide: mp 141–143 ◦C; 1H
NMR (400 MHz, DMSO-d6, ppm): ı 10.22 (2H, s, NH), 8.38–8.40
(4H, d, J = 6.4 Hz), 7.53–7.55 (4H, d, J = 6.8 Hz), 2.31–2.35 (4H,
t, J = 7.2 Hz), 1.55–1.59 (4H, m), 1.15–1.30 (24H, m); 13C NMR
(400 MHz, DMSO-d6, ppm): ı 172.53, 150.30, 145.75, 113.00,
36.48, 28.98 (several), 28.88, 28.74, 28.57, 24.76; HRMS: Calcd. for
2.11. Synthesis of bis-Girard adduct (8b)
To 2.26 g (24 mmol, 2.4 equiv.) of 4-aminopyridine and 2.02 g
(20 mmol, 2 equiv.) of triethylamine in 40 mL of acetonitrile at
0 ◦C was added 2.67 g (10 mmol) of 1,12-dodecanedioyl dichloride
dropwise. The reaction was stirred at 25 ◦C for 30 min, refluxed for
16 h, and quenched with saturated NaHCO3. The resulting white
solid was collected and recyrstallized from acetone to yield 2.8 g
(72%) of bis-N-(4-pyridyl) 1,12-dodecanediamide: mp 138–139 ◦C;
IR (KBr) 3312 (NH), 1697 (C O) cm−1 1H NMR (DMSO-d6) ı
;
C28H42N4O2 466.3307, found 466.3307 (mean of 5 determinations,
10.3 (2H, s), 8.35–8.45 (4H, d, J = 6.3 Hz), 7.5–7.6 (4H, d, J = 6.3 Hz),
2.3–2.38 (4H, t, J = 7.48), 1.5–1.6 (4H, m), 1.2–1.35 (12H, m); 13C
NMR (DMSO-d6) ı 172.55, 150.30, 145.77, 113.00, 36.47, 28.85,
28.74, 28.58, 24.77; m/z (EI) 383 (MH+, 100), (ESI-MS–MS) 95
(MH+−289, 100), 289 (MH+−94, 100); HRMS calcd for C22H40N4O2:
382.2363, found 382.2377 (mean of five determinations, error
0.0014 ppm). Anal. Calcd. for C22H30N4O2–CH3OH: C, 66.64%; H,
8.27%; N, 13.52%. Found: C, 66.35%; H, 8.10%; N, 13.77%.
To 1 g (8.16 mmol, 1 equiv.) of ethyl chloroacetate in 3.5 mL
of absolute EtOH was added 1.56 g (4.08 mmol) of bis-N-(4-
pyridyl) 1,12-dodecanediamide. The reaction was refluxed for 2.5 h
and cooled to 25 ◦C. To this solution at 0 ◦C was added 262 mg
(8.16 mmol, 1 equiv.) of hydrazine. The solid product was col-
lected, dried under high vacuum, recrystallized from methanol
to afford 699 mg (29%) of the bis-Girard Reagent: mp 233–250 ◦C
with decomposition; 1H NMR (200 MHz, D2O) ı 8.5–8.6 (d, 4H,
J = 7.4 Hz), 8.05–8.15 (d, 4H, J = 7.4 Hz), 5.2–5.3 (s, 4H), 2.45–2.6
(t, 4H, J = 7.2 Hz), 1.5–1.8 (m, 4H), 1.1–1.4 (br m, 12H); 13C NMR
(D2O) ı 179.99, 155.42, 148.60, 118.20, 99.80, 39.87, 31.12, 31.02,
30.85, 27.13; m/z (ESI) 264.1 (MH+−Cl2/2, 100). Anal. Calcd. for
C26H40N8O4Cl2–CH3OH: C, 51.35%; H, 7.02%; N, 17.74%. Found: C,
51.69%; H, 6.79%; N, 18.04%.
To 75 mg (0.13 mmol) of bis-Girard reagent in 0.65 mL methanol
was added 81 mg (0.3 mmol, 1.2 equiv.) of estrone (1), followed by
10 mg of Dowex 50 W × 2 acid resin. The reaction was refluxed for
24 h and filtered through a warm sintered-glass funnel. The fun-
nel was rinsed several times with warm methanol. The methanolic
solutions were concentrated to give a solid product. The product
was recrystallized from 1:1 acetonitrile:methanol to give 14 mg
(28%) of bis-Girard adduct 8b: mp >240 ◦C; 1H NMR (200 MHz,
DMSO-d6) ı 11.81 (s, 2H), 10.83 (s, 2H), 9.04 (s, 2H), 8.70–8.74 (d,
4H, J = 7); 8.12–8.16 (d, 4H, J = 7.4), 7.05–7.09 (m, 2H), 6.45–6.54
(m, 4H), 5.62–5.65 (m, 4H), 3.8–4.2 (br s, 4H); 2.70–2.80 (m, 4H),
1.65–2.60 (m, 21H, overlaps with DMSO peak); 1.28–1.62 (m, 29H);
0.82–0.90 (m, 6H); 13C NMR (200 MHz, DMSO-d6) ı 179.54, 174.41,
172.56, 160.49, 157.69, 152.17, 142.52, 135.40, 131.47, 120.41,
119.44, 118.24, 65.40, 57.42, 50.15, 49.09, 48.98, 42.15, 39.45, 34.48,
34.27, 34.14, 33.90, 32.30, 32.22, 31.34, 29.81, 28.20, 22.31; m/z
(MALDI-TOF, SA matrix) 1032 (M−2Cl−, 2500); 707 (M−325, 1500).
SD 1.8 ppm, error 0.0 ppm).
Bis-Girard reagent, 8c, was prepared as described above
for bis-Girard adduct, 8a, using bis-N-(4-pyridyl) 1,18-
octadecanediamide: 1H NMR (400 MHz, CD3OD, ppm): 7.00–8.01
(4H, d, J = 7.2 Hz), 7.08–7.10 (2H, d, J = 8.4 Hz), 6.84–6.86 (4H, d,
J = 6.8 Hz), 6.54–6.56 (2H, d, J = 8.4 Hz), 6.49–6.50 (2H, d, J = 2.4 Hz),
5.3–5.41 (4H, q, AB), 5.06 (2H, s, OH), 2.7–2.9 (4H, m), 1.90–2.7
(20H, m), 1.3–1.75 (38H, m), 0.97 (6H, s); 13C NMR (400 MHz,
CD3OD, ppm): d 168.25, 166.68, 158.31, 153.42, 142.69, 136.03,
129.48, 124.49, 113.29, 111.05, 107.49, 56.68, 51.09, 43.76, 42.71,
37.19, 35.52, 32.55, 27.52–28.06 (many), 25.81, 25.65, 24.82,
21.62, 14.70; m/z (MALDI-TOF, CHCA matrix): 1117 (M, 100), 982
(M−135, 400).
2.13. Plasmids
The development of the human estrogen (pRSV-ER␣), proges-
terone (pRSV-PR), androgen (pRSV-AR), glucocorticoid (pRSV-GR)
and beta galactosidase (pRSV-␤Gal) mammalian expression plas-
mids used in these studies were as described elsewhere [22–24].
The development and use of the estrogen (pBL-ERE-tk-Luc),
androgen (pBL-ARE-tk-Luc), progesterone pBL-PRE-tk-Luc, and
glucocorticoid (pBL-MMTV-tk-Luc) reporter plasmids have like-
wise been described elsewhere.
2.14. Transcription assays
The cell line used throughout these studies was the human
embryonic kidney 293 (HEK) cell line obtained from ATCC (CRL-
1573). These cells were grown and maintained at 37 ◦C/5% CO2 in
DMEM media plus 5% FBS. Mammalian transient co-transfections
were performed in the HEK293 cell line as previously described
[25]. To determine the efficiency of transfection and to standard-
ize the expression of activity, a pRSV-␤Gal expression plasmid
was included in all co-transfections and analyzed as previously
described [25]. Each transfection was run in sextuplet and the
average luciferase response for the 6 independent transfections
was normalized to the average ␤Gal rate [average luciferase
response/(average ␤Gal response/minute)]. Statistical evaluations
of these data were expressed as standard error of their means (SEM)
for the sextuplet wells of each treatment dose. Data were analyzed
using the graphical and statistical program Prism (GraphPad, San
Diego, CA).
2.12. Synthesis of bis-Girard reagent (8c)
A
flask containing 1,18-octadecanedioic acid (1.50 g,
4.77 mmol), thionyl chloride (10 mL), and DMF (1 drop) was
stirred at room temperature for 30 min. The suspension was then
brought to reflux, at which point the solids dissolved. Reaction
was refluxed for 2 h. The solvents were removed under reduced
pressure, and the remaining white solid was re-suspended in
MeCN and evaporated several times. When all the thionyl chloride
2.15. Stability assays
To evaluate the chemical stability of the bis(3-hydroxyestra-
1,3,5(10)-triene-17-oximinyl) 1,22-docosanedioate, the bivalent
estrogen 5b, HEK cells, aliquoted at 5 × 105 cells per well of a 96-

A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
829
afforded good yields of the corresponding “monovalent” oximes
(Fig. 1: compounds 4a and 4b). Coupling of these oximes to either
1,12-dodecanedioic diacid or 1,22-docosanedioic diacid using
N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochloride
(EDCI) and 1-hydroxybenzotriazole hydrate (HOBT) [32] afforded
the tethered bivalent oxime esters compounds 5a–d in modest
yields after chromatography and recrystallization. No effort was
made to optimize yields. The “monovalent” oxime ester compound
4c was also prepared from a regioselective reaction of oxime com-
pound 4b and lauric anhydride.
At the turn of the last century, Girard developed water-soluble
semicarbazone reagents P and T for the purpose of extracting
and purifying ketone-containing steroids [33]. Reaction of Girard
reagent P with estrone (Fig. 1: compound 1), estrone 3-methyl
ether (Fig. 1: compound 2), and equilenin (Fig. 1: compound 3)
afforded good yields of the corresponding “monovalent” Girard
reagents (Fig. 2: compounds 6a, 6b, and 7). We anticipated that bis-
Girard reagents would offer not only suitable tethers but also better
water solubility than seen with the oxime esters. Preparation of bis-
Girard reagents paralleled the published synthesis of Girard reagent
P in so far as: (1) reaction of 4-aminopyridine and 1,12-dodecanoyl
dichloride provided a bispyridylamide, and (2) treatment of the
bispyridylamide with ethyl chloroacetate and hydrazine furnished
the desired bis-Girard reagent [34]. Coupling of this bis-Girard
reagent with estrone led to the bis-Girard derivative bivalent estro-
gen (Fig. 2: compound 8b). Comparable procedures were used to
synthesize compounds 8a and 8c.
Fig. 1. Monovalent and bivalent 17-oxime esters. Presented are the 6 monovalent (1,
2, 3, 4a, 4b and 4c) and 4 bivalent (5a–d) 17-oxime esters evaluated for their stability,
specificity and ability to modulate ER␣-mediated gene expression parameters.
well plate, were treated for 36 h in the presence and absence of
10 ␮M bivalent estrogen. The liquid in the wells was aspirated; the
wells were treated with methanol to lyse the cells; and the con-
tents of 1 well, 5 wells, 10 wells and 25 wells were collected and
concentrated to dryness. Each of these samples was diluted with
500 ␮L of methanol. The samples were applied to a Merck silica
gel F254 analytical plate, developed in 1:2 ethyl acetate–hexanes
(a solvent system that readily separates the estrone oxime 4a from
the bivalent estrogen 5b), sprayed with 10% phosphomolybdic acid
in ethanol, and heated to detect compounds as purple spots. The
bivalent estrogen 5b and none of the estrone oxime 4a was detected
in the samples from 1 well, 5 wells, 10 wells and 25 wells.
3.2. Defining optimal linkage points for estrogens
In preliminary investigations of the impact of modifying estro-
gen at either C-3 or C-17, we created and evaluated the modified
estrogens 4a–7 shown in Fig. 1. The “monovalent” and “bivalent”
estrogens were analyzed for their ability to modulate estrogen
genomic signaling as measured using an in vitro transcription
assay. Estrogen genomic signaling was reconstituted in a human
embryonic kidney (HEK) cell line by co-transfecting this cell line
with a mammalian expression construct for human ER␣ along
with an estrogen response element (ERE)-driven luciferase reporter
plasmid. All transfections also included a mammalian expression
construct for ␤-galactosidase (␤-Gal), which was used to normal-
ize for transfection efficiency. All compounds were analyzed at a
concentration of 100 nM. Fig. 3 presents the results for “monova-
lent” C-17 oximes of estrone that possess either a hydroxyl group
or a methoxy group at C-3. As seen here, the compounds 4a, 4c, 5a,
5b, 6a and 7, containing a C-3 hydroxyl, activated ER␣-mediated
reporter gene expression. Compounds 4b, 5c, 5d and 6b with the
methoxy group at this position were inactive. This is not totally
unexpected in that structural studies looking at estrogen bind-
ing of the ER␣ ligand binding domain demonstrated that the C-3
hydroxyl participates in crucial hydrogen-bonding with an Arg,
Glu and water molecule within the binding site [8,34,35]. More
recently, it has also been shown that the ER␣ ligand binding domain
displays structural plasticity with respect to C-17 modifications
[36]. These data led us quickly to the C-17 carbonyl group of estrone
as a logical site on which to construct estrogen dimers.
3. Results and discussion
3.1. Synthesis of estrogens
A prerequisite to steroid hormone genomic signaling is the
ligand-induced dimerization of its intracellular receptor, and the
concept of creating bivalent estrogens dates back to 1994 [26],
when Bergmann et al. published the synthesis and characterization
of bivalent hexestrols linked through polymethylene and polyethy-
lene glycol spacers. One of these displayed partial agonism for
the estrogen receptor while the remainder were characterized as
antagonists. Since then several other groups have published biva-
lent or dimeric steroid syntheses using a variety of linkers [27–30],
but these papers focused on design, synthesis and/or binding, with
little to no characterization of relative efficacy, potency or speci-
ficity. More recently, the Katzenellenbogen laboratory, publishers
of the original manuscript on the synthesis of bivalent hexestrols,
extended these studies with bivalent estrogen designs that dis-
played variable estrogen receptor binding affinities that correlated
with linker length [31]. These estrogen dimers were evaluated com-
prehensively with respect to linker composition, tether length and
receptor binding, but did not evaluate biological activity.
We began our investigation of bivalent steroid synthesis using
estrogen as a template for these studies. The estrogen molecule
has any number of chemically accessible positions, but two stood
out as logical targets for our initial efforts: the hydroxyl group at
the C-3 position of ␤-estradiol or the carbonyl group at the C-17
position of estrone. We elected to modify the C-17 keto group of
estrone (Fig. 1: compound 1), estrone 3-methyl ether (Fig. 1: com-
pound 2), or equilenin (Fig. 1: compound 3) as either oximes or
semicarbazones in order to provide functionality to which a suit-
able tether could be attached. Reaction of hydroxylamine with
estrone (compound 1) or estrone 3-methyl ether (compound 2)
3.3. Dose response analyses of “bivalent” estrogens
We further evaluated the bivalent oxime esters, 5a and 5b in
Fig. 1, with either a (CH2)10 or a (CH2)20 spacer respectively, along
with the monovalent estrogen 4c containing a simple laurate ester,
for their ability to activate ER␣-mediated transcription in a dose-
dependent manner (Fig. 4). As seen here, ER␣ was agonized by
all three modified estrogens in a dose-dependent manner with an
order of efficacy of estrogen > 5b > 5a > 4c. These data support the

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A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
Fig. 2. Monovalent and bivalent Girard P-based oxime derivatives. Presented are the 2 monovalent (6 and 7) and 3 bivalent (8a, 8b, 8c) estrogens evaluated for their ability
to modulate ER␣-mediated gene expression events.
Fig. 3. Monovalent and bivalent estrogens modified at their C-3 and C17 moi-
eties analyzed for their ER␣ agonist activities. Compound number identifiers are
as designated in Fig. 1. ER␣-mediated transcription was reconstituted in an HEK cell
line through the co-transfection of an ER␣ mammalian expression plasmid and an
ERE-driven luciferase reporter plasmid. As controls, parallel analyses were run to
evaluate solvent impact as well as background attributable to the ERE reporter vec-
tor (ERE). ER␣’s cognate ligand 17␤-estradiol (E2) along with a series of monovalent
and bivalent estrogens modified at their C-3 and C-17 positions (A) were analyzed
(B) in a 96-well plate format. For normalization of data, included in all transfections
was a mammalian expression construct for ␤-galactosidase (␤-Gal). All compounds
were analyzed at 100 nM, and data from 6 wells for each ligand was averaged and
divided by the average ␤-Gal rate for these wells. Error bars represent the SEM for
the 6 wells.
Fig. 5. Efficacy comparison of the bivalent estrogen 5b to its monovalent oxime
derivative 4c. Utilizing the ER␣-specific HEK co-transfection analysis described in
Fig. 3, the bivalent estrogen 5b and its monovalent oxime derivative 4c were ana-
lyzed in molar equivalents of estrogen for their ability to stimulate ER␣-specific
reporter gene expression. Data are expressed as averaged reporter gene luciferase
values for sextuplet wells divided by the average ␤-Gal rate for these wells.
hypothesis that estrogen dimers are able to enter the cell effectively
and modulate ER␣ genomic signaling.
3.4. Stability of bivalent estrogens in the oxime ester family
We evaluated three concerns in the initial phases of devel-
oping these estrogen dimers: (1) potential hydrolytic liability of
the oxime ester tether between the two estrone moieties; (2) the
hydrophobicity of the aliphatic linker; and (3) the specificity of
the modified steroid structure. To evaluate the issue of stability,
transcriptional activation mediated by bivalent estrogen 5a was
compared to a monovalent estrogen, 4c, containing a simple lau-
rate ester (Fig. 5). A dose–response curve was analyzed for both
compounds, and monovalent 4c was assayed at a molar equivalent
of estrogen concentration (2×) to that of the bivalent estrogen 5a.
The monovalent estrogen 4c displayed substantially lower efficacy
(∼75% at the highest concentration tested) to that of bivalent estro-
gen 5a. Although these results may involve differences in cellular
uptake, differences in receptor binding, or differences in dimer-
ization efficiency, we interpret these findings in support of the
chemical stability of these compounds during the course of the 24-
hour assay time-frame in which they were analyzed, and in support
of the increase in dimerization efficiency incumbent in the bivalent
estrogen 5b structure.
Fig. 4. Dose Response analysis of the bivalent estrogens 5a and 5b. Utilizing the
ER␣-specific HEK co-transfection analysis described in Fig. 3, the bivalent estrogens
5a and 5b, along with the monovalent estrogen 4c and 17␤-estradiol (E2), were
analyzed at concentrations varying from 0.1 nM to 100 nM. Data are expressed as
averaged reporter gene luciferase values for sextuplet wells divided by the average
␤-Gal rate for these wells.

A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
831
Fig. 7. Analysis of the Girard-based bivalent estrogens 8a, 8b, and 8c for their ER␣
agonist activity. Utilizing the ER␣-specific HEK co-transfection analysis described in
Fig. 3, the Girard-based bivalent estrogens 8a, 8b, and 8c along with 17␤-estradiol
(E2), were analyzed at concentrations varying from 0.1 nM to 100 nM. Data are
expressed as averaged reporter gene luciferase values for sextuplet wells divided
by the average ␤-Gal rate for these wells.
Fig. 6. Stability evaluation of the bivalent estrogen 5b. HEK cells, aliquoted at 5 × 105
cells per well of a 96-well plate, were treated for 36 h in the presence and absence of
100 nM bivalent estrogen 5b and analyzed by thin layer chromatography using 1:2
ethyl acetate–hexanes with 10% phosphomolybdic acid spray for detection. Lane 1:
bivalent estrogen 5b + oxime 4a; lane 2: bivalent estrogen 5b; lane 3: oxime 4a; lane
4: extract from 25 wells; lane 5: extract from 10 wells; lane 6: extract from 5 wells;
lane 7: extract from 1 well; lane 8: bivalent estrogen 5b; lane 9: bivalent estrogen
5b + oxime 4a.
3.5. Analysis of an estrogen dimer with increased hydrophilicity
These promising preliminary studies clearly suggested that
estrone dimers linked through their C-17 position function as
specific agonists of ER␣, but with a slightly lower efficacy rela-
tive to that of estrogen. These results propelled our interest in
a second generation of C-17 linked estrogen dimers in which
we manipulated the composition of the linker and its attach-
ment to the estrone moieties. These modifications also provided
us with an opportunity to augment the solubility of the dimer
and evaluate its impact on efficacy. To achieve this goal, we
explored the development of “Girard-based” estrogen dimers, as
described above (Fig. 2). Girard-based estrogen dimers 8a, 8b, and
8c along with 17␤-estradiol, were analyzed in the ER␣ in vitro
co-transfection assay (Fig. 7). As seen in Fig. 7, the Girard-based
estrogen dimer 8b displayed a potency equivalent to estrogen
and an efficacy that was approximately 125% that of estrogen at
its optimal 100 nM concentration. Dimers 8a and 8b had reduced
efficacy in these assays, potentially reflecting an inability to span
the estrogen binding sites in the case of 8a, or too much linker-
An analysis was performed that directly examined the chem-
ical stability of the bivalent estrogen 5b. HEK cells, aliquoted at
5 × 105 cells per well of a 96-well plate, were treated for 36 h in
the presence and absence of 100 nM bivalent estrogen 5b. The lipid
components for cell populations from 1, 5, 10 and 25 wells were
extracted into methanol and analyzed by thin layer chromatog-
raphy for the presence of monovalent 4a and bivalent estrogen 5b
components (Fig. 6). As seen here, only the bivalent estrogen 5b was
detected even in the sample where the contents of 25 cells were
combined and analyzed. These results support the stability of the
oxime ester bonds to the conditions of the assay, and exclude the
possibility that adventitious esterases cleave the bivalent estrogen
5b to produce 4a. The stability of the bivalent Girard P deriva-
tives was not an issue since these derivatives possess robust amide
linkages.
Fig. 8. Specificity analysis of the Girard-based bivalent estrogen 8b. HEK cells were reconstituted with receptor/reporter systems for ER␣, human progesterone receptor
beta (PR), human androgen receptor (AR), and human glucocorticoid receptor (GR) through co-transfection of specific receptor and reporter plasmids as described in Fig. 3.
Assays were performed in the presence of solvent, 8b and receptor-specific ligands [ER: 17␤-estradiol (E2); AR: dihydrotestosterone (Test.); GR: dexamethasone (Dex); PR:
progesterone (Prog.)]. For normalization of data, included in all transfections was a mammalian expression construct for ␤-Gal. All compounds were analyzed at 100 nM, and
data from 6 wells for each ligand was averaged and divided by the average ␤-Gal rate for these wells. Error bars represent the SEM for the 6 wells.

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A.E. Wendlandt et al. / Steroids 75 (2010) 825–833
associated flexibility leading to inefficient dimerization in the
case of 8c. It is anticipated that futures studies investigating the
receptor binding specificities of these compounds may help define
the mechanism by which linker length is influencing receptor
dimerization and activity. Cumulatively, these data suggest that
manipulation of the dimer length, linkage, and hydrophobicity may
indeed serve as a mechanism for optimizing dimer efficacy and
potency.
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To evaluate the specificity of the bivalent estrogens, the bivalent
estrogen 8b was analyzed for its ability to modulate transcription
events mediated by three other homodimerizing steroid hor-
mone receptors; the progesterone receptor (PR), the androgen
receptor (AR), and the glucocorticoid receptor (GR) (Fig. 8). As
described above for ER␣, a receptor-specific in vitro transcrip-
tion system was reconstituted in HEK cells for each receptor
through the co-transfection of human AR, GR or PR␤ recep-
tor mammalian expression plasmids, along with a corresponding
receptor-specific response element-driven luciferase reporter plas-
mid. Transfected cells were analyzed in the presence of ligand
solvent, in the presence of 100 nM cognate ligand, and in the pres-
ence of 100 nM of the bivalent estrogen 8b. As seen in Fig. 8,
the bivalent estrogen 8b was able to stimulate reporter gene
expression only with the ER␣ reconstituted transcription sys-
tem.
In summary, we evaluated the hypothesis that bivalent steroid
estrogens can be created capable of facilitating ER dimerization
and consequently ER efficacy. This is an initial attempt to mimic
the ER dimerization event using a synthetic bivalent ligand. As dis-
cussed above, this is not a novel idea, but separating itself from
previous publications are both the synthetic approach and the
functional characterizations employed. The chemistry required to
achieve these results harkens back to the early days of the steroid
literature, although this is the first time that bis-Girard reagents
have been used to create bivalent steroids. More to the point, the
chemistry developed here is straightforward and provides access
to an array of other modified tethers or even unsymmetrical dimers
between different steroids. The identification of C-17 as a readily
modified position on the estrogen backbone that does not grossly
impact its activity opens the door to a variety of alternative struc-
tures that might be developed. This might include bivalent steroid
linkers capable of targeting bivalent hormone agonists or antag-
onists to any number of extracellular or intracellular molecules,
such as: bone hydroxylapatite, cell surface proteins, proteaso-
mal complexes, lysosomes, transcription coregulatory complexes,
cell-specific proteases or perhaps even in the creation of novel tran-
scription factor heterodimers that target specific gene promoters.
Two issues not addressed in this study, but currently under investi-
gation, are the estrogen receptor binding affinity for these steroids
and the impact they have on estrogen non-genomic signaling path-
ways. Finally, the basic concept applied here to estrogen dimers
would conceptually appear feasible for any of these steroid/nuclear
receptor ligands.
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One of us (DSW) thanks the Vice President for Research for
research funding. We also thank the University of Kentucky, Center
for Structural Biology, Organic Chemistry Core Facility supported
in part by funds from NIH National Center for Research Resources
(NCRR) grant P20 RR020171. Mass spectra were acquired at the
University of Kentucky Mass Spectrometry Facility.
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