Steroidal bivalent ligands for the estrogen receptor: Design, synthesis, characterization and binding affinities

Article abstract of DOI:10.1016/j.bmc.2009.04.016

Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ERα dimers as a template. The syntheses of several 17α-ethynylestradiol-based bivalent ligands with varying linker compositions and lengths are described. The binding affinities of these bivalent ligands for ERα and ERβ were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation between linker length and binding affinity, both of which reach a maximum at the same tether length. Further studies are underway to explore aspects of bivalent ligand and control compound binding to the ERs and their effects on ER dimer formation; these results will be reported in a subsequent publication.

Full text of DOI:10.1016/j.bmc.2009.04.016

Bioorganic & Medicinal Chemistry 17 (2009) 3528–3535
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Steroidal bivalent ligands for the estrogen receptor: Design, synthesis,
characterization and binding affinities
*
Andrew L. LaFrate, Kathryn E. Carlson, John A. Katzenellenbogen
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 February 2009
Revised 1 April 2009
Accepted 9 April 2009
Available online 12 April 2009
Steroidal bivalent ligands for the estrogen receptor (ER) were designed using crystal structures of ER
a
dimers as a template. The syntheses of several 17
linker compositions and lengths are described. The binding affinities of these bivalent ligands for ER
a
-ethynylestradiol-based bivalent ligands with varying
and
a
ERb were determined. In the two series of bivalent ligands that we synthesized, there is a clear correlation
between linker length and binding affinity, both of which reach a maximum at the same tether length.
Further studies are underway to explore aspects of bivalent ligand and control compound binding to
the ERs and their effects on ER dimer formation; these results will be reported in a subsequent
publication.
Keywords:
Estrogen receptor
Bivalent ligand
Multivalent ligand
Dimer
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
er affinity ligands with varying tether lengths and compositions
which could offer insight into ER dimer formation and stability.
A multivalent ligand is a single molecule comprised of multiple
individual ligands linked by a tether. Such ligands have often been
used to increase binding affinity, to probe dimer or cluster forma-
tion or to explore subsite binding on a receptor.1–7 In medicinal
chemistry, multivalent ligands have been used as inhibitors of sev-
eral biological targets, including, among others, HIV protease, gly-
cosidase and the opioid and muscarinic receptors.8
2. Results and discussion
2.1. Steroidal bivalent ligand design
Our bivalent ER ligands were designed based on the crystal
structure of the ERa ligand binding domain bound to estradiol
The estrogen receptor has a brief history involving multivalent,
specifically bivalent, ligands (Fig. 1). In 1994, our research group
was the first to investigate the effects of bivalent ligands on ER
by tethering two hexestrol (a non-steroidal ER agonist) ligands
with varying lengths of polymethylene and polyethylene glycol
(PEG) spacers (1).9 Another early bivalent ER ligand involved dimer
formation through an iron carbonyl system built from ethynylest-
radiol (2); this molecule gave poor results in binding assays, pre-
sumably because its tether length was too short.10 In 1999,
Berube and co-workers11 reported the synthesis of tethered tri-
phenylethylene molecules (3); ER binding affinities were not re-
ported, and these compounds showed low potencies in cell-based
assays, with no evidence that effects were ER mediated. The most
recent reports of bivalent ligands for ER involved the synthesis of
(E2) and literature reports of estrogen ligand conjugates. The biva-
lent ligands prepared by Bergmann et al.9 and Osella et al.10 were
published before the crystal structure of the ER ligand binding do-
main was released,14 so it could not have been used as a guide for
their ligand design. Although Berube and co-workers reported their
first bivalent ligands in 1999,11 two years after the publication of
the first ER crystal structure, they do not mention using it as a basis
for bivalent ligand design in this work,11 or in their later pa-
pers.12,13 We hoped to gain a better understanding of bivalent li-
gand binding to ER by considering not only the structure of the
receptor but also by carefully selecting how the ligands are teth-
ered, relying as well on published reports of functionalized
estradiol.15
The crystal structure of the ER
a
dimer with E2 bound16 was
two estradiol molecules linked at the 17a position by varying
modelled using the Sybyl modelling software package (Fig. 2). It
is worth noting that the ER dimer possesses C2 symmetry, which
simplifies bivalent ligand design, because bivalent ligands built
from the same enantiomer of E2 are also C2 symmetric. The closest
distance between the E2 ligands is between the carbons at the 17-
position of the steroids and was found to be 26 Å, seemingly close
enough to be linked by a molecular tether. It is fortunate that the
lengths of PEG chains (4), but again the binding affinities for these
compounds were not reported.12,13 By using the crystal structure of
ER to rationally design bivalent ligands, we hoped to prepare high-
* Corresponding author. Tel.: +1 217 333 6310; fax: +1 217 333 7325.
E-mail address: jkatzene@uiuc.edu (J.A. Katzenellenbogen).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.016

A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
3529
OH
(CO)6
H
OH
HO
Fe
H
O
O
O
H
OH
HO
O
HO
H
OH
HO
2, RBA = 0.5%
1, RBA = 6.9%
Osella et al, 1997
Bergman et al, 1994
H/OH
H/OH
HO
OH
O
O
O
HO/H
H/OH
n
OH
HO
4, RBA not reported
Rabouin et al, 2003
Berube et al, 2006
3, RBA not reported
H/OH
H/OH
Groleau et al, 1999
Figure 1. Some previously reported ER bivalent ligands and their relative binding affinity (RBA) values, estradiol = 100%.
and provides access to the exterior of the protein.25 This suggests
that ER will respond in the same way to the phenylethynyl group,
which is nearly isostructural with the phenylvinyl group.
Due to the synthetic accessibility of 17a-phenylethynylestradi-
ols and the well established and structurally confirmed tolerance
of ER for this substitution, we chose to prepare bivalent ligands
with varying tether lengths and composition based on 17a-pheny-
lethynylestradiol (5, Scheme 1). The synthesis of these estrogens is
relatively straightforward and involves palladium-catalyzed Sono-
gashira coupling of EE2 with an aryl halide.26
2.2. Synthesis of bivalent ligands
Initially, bivalent ligands with hexaethylene glycol tethers were
prepared because the linker is commercially available and seemed
long enough to bridge the distance between the ligand binding
pockets in the ER dimer complex (26 Å). With this commercial
tether, the distance between the 17-carbons in the hexaethylene
glycol bivalent ligand in a fully extended conformation was found
to be 35.7 Å using Chem3D Pro 7.0 (see below). Because it is possi-
ble that the hexaethylene glycol linker could adopt a non-stag-
gered, coiled conformation (see below),27–30 we felt that the
additional 10 Å in the tether length might help the two steroids
Figure 2. The distance between the 17
filling: grey = carbon, red = oxygen) within the ER
Figures were generated with Sybyl 7.3 from the corresponding research collabo-
ratory for structural bioinformatics protein data bank (RCSB-PDB file names: 1ERE
for E2).
a
carbons of two estradiol molecules (space
homodimer (teal ribbon).
to span the 17a–17a distance of 26 Å.
a
The synthesis of a hexaethylene glycol tethered bivalent ligand
with amine linkages (10) is shown in Scheme 2. Hexaethylene gly-
col was refluxed with p-toluenesulfonyl chloride in CH2Cl2 in the
presence of excess triethylamine and DMAP to afford di-tosylated
hexaethylene glycol (6) in good yield (Scheme 2). Diamino hexa-
ethylene glycol (8) was prepared from the ditosylate 6 by forming
the diazide (7), followed by reduction in the presence of palladium
on carbon in methanol under a hydrogen atmosphere. Bivalent li-
gand 10 was prepared by stirring the benzaldehyde derivative of
point of closest distance between ligands in the crystal structure,
the 17a
-position, is known to tolerate substitution very well;15
in fact, 17a-ethynylestradiol (EE2) binds with a nearly three-fold
higher affinity to ER than E2 itself.17
Our design was also supported by the numerous reports of 17
a-
17a
-ethynylestradiol (9)18 and diamine 8 in CH2Cl2 in the presence
phenylethynylestradiol derivatives tethered to other molecules
such as dendrimers,18 fluorophores,19 radioactive and luminescent
organometallic complexes,20–22 Pt(II) fragments23 and photo affin-
ity tags,24 all of which show reasonably high binding affinity for ER.
While modelling of a phenyl group onto the end of the ethynyl sub-
of excess anhydrous K2CO3 for 24–48 h. Formation of the imine
was confirmed by proton NMR by the disappearance of the alde-
hyde peak around 10 ppm and the appearance of the imine peaks
(cis and trans) near 8 ppm.18 The crude imine was dissolved in
MeOH and excess NaBH4 was added and stirred at room tempera-
ture for 5–10 min to give the bivalent estrogen 10 in good yield
over two steps.
A second hexaethylene glycol bivalent ligand, with phenyl ether
linkages, was also synthesized (12, Scheme 3). Bis-aryl iodide (11)
was prepared by stirring the PEG ditosylate 6 with 4-iodophenol
stituent causes severe interactions with the ER
structure, X-ray analysis of closely related 17 -phenylvinyl estro-
gens with ER shows that when presented with this larger substi-
tuent at 17 , the ER unwinds a short helix (helix 8), thereby
generating a large pocket that accommodates the phenyl group
a-estradiol protein
a
a
a

3530
A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
OH
OH
Me
Me
HO
Me
HO
Ethynylestradiol (EE2)
Estradiol (E2)
OH
HO
Me
Linker
5
HO
OH
Scheme 1.
TsCl, DMAP,
Et3N, CH2Cl2
NaN3, DMF
87%
Ts
OTs
6
N3
N3
5
H
OH
6
O
O
O
72%
7
6
E2
CHO
H2, Pd/C
CH3OH
H2N
NH2
5
O
9
8
100%
1. K2CO3, CH2Cl2
2. NaBH4, CH3OH
79%
OH
HO
O
5
N
N
H
H
10
HO
OH
Scheme 2.
p
-iodophenol,
EE2, PdCl2(Ph3P)2, CuI,
CH3CH, Et3N
K2CO3, acetone
I
O
Ts
OTs
6
O
I
O
6
92%
30%
6
11
OH
HO
O
O
6
12
HO
OH
Scheme 3.
and excess K2CO3 in 2-butanone for 24 h. Palladium-catalyzed
Sonogashira coupling31,32 was used to make phenylethynyl deriva-
tives of 17a-ethynylestradiol (EE2) from aryl iodide 11. By stirring
of the ethylene glycol unit should be relaxed. Polyethylene glycols
are commercially available in discrete lengths, but polypropylene
and polybutylene glycols are not. Therefore, we prepared discrete
polypropylene and polybutylene glycols using modifications of
previously described syntheses,34 the details of which can be found
in the Supplementary data. Bivalent ligands with these polypropyl-
ene and polybutylene linkers were synthesized as shown in
Scheme 4. The phenol linkage was chosen over the amine because
it can be prepared in fewer synthetic steps. The linker diol was con-
verted to the methanesulfonate, which was found to be more reac-
tive and easier to prepare than the corresponding tosylate. The
mesylates were then alkylated with p-iodophenol and subjected
to Sonogashira coupling to give the bivalent ligands.
11 with EE2 in the presence of CuI and Pd catalyst in Et3N and
CH3CN at room temperature, bivalent ligand 12 was isolated in
low yield. The low yield for the Sonogashira coupling can be attrib-
uted to homo coupling of the EE2, giving a diacetylene dimer, and
incomplete reaction to give the mono-coupled product, both of
which were isolated as side products. Homo coupling of alkynes
is a common side reaction in Sonogashira couplings.33
Because ethylene glycol polymers are reported to form coiled
conformations27–30 and are relatively polar, we chose to explore
tethers constituted of less polar glycol ethers, namely the polypro-
pylene and polybutylene glycol ethers. In these systems, the helical
or coiled conformation enforced by the inherent gauche preference
Bis-mesylates were prepared from the polypropylene and poly-
butylene glycol linkers in good to excellent yield by stirring with

A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
3531
I
OH
MsCl
O
O
n
O
O
n
I
H
H
Ms
Ms
O
O
I
x
x
K2CO3
Et3N, CH2Cl2
76-100%
n
x
2-Butanone
63-84%
HO
OH
EE2, Pd(Ph3P)4,
CuI, piperidine
O
O
n
x
THF
20-53%
13- 21
OH
HO
Scheme 4.
Table 1
Structures, ER
estrogens
methanesulfonyl chloride in Et3N and CH2Cl2 at 0 °C for 2 h
(Scheme 4). The yields for this reaction ranged from 76% to 100%,
with the differences being attributed to different workup and puri-
fication procedures. Some isolated products were quite pure by 1H
NMR and could be used in subsequent reactions without further
purification (see below), while others needed to be purified by col-
umn chromatography. In these latter cases, the yields were lower
because these ether methanesulfonates lack a chromophore; so,
it was difficult to determine by TLC when they were eluting from
the column. The mesylates were then converted to the correspond-
ing bis-aryliodides in good yield by stirring with p-iodophenol and
K2CO3 in 2-butanone at reflux. Once chromophores were present,
purification of the alkylated products using silica gel flash chroma-
tography was straightforward, and any impurities remaining in the
mesylates could be easily removed in this step.
Bivalent ligands were prepared in moderate yields by coupling
the bis-aryliodides with EE2 under improved Sonogashira condi-
tions. By changing solvents from CH3CN to THF, using piperidine
in place of Et3N as the base, and palladium tetrakis(triphenylphos-
phine) as the catalyst, yields were improved substantially. Gener-
ally, purification on multiple (2–3) silica gel columns using
different solvent systems was necessary to obtain the products in
pure form, usually as white foamy solids. All of the bivalent ligands
were completely characterized by 1H and 13C NMR and high reso-
lution mass spectrometry. Compound purity was determined using
1H NMR and HPLC, with all bivalent ligands being at least 95% pure.
The presence of even a small amount of residual EE2 in the bivalent
ligand product would result in higher than actual RBA values. The
difference in HPLC retention times of EE2 and the bivalent ligands
is substantial, making HPLC an excellent tool for the detection of
residual EE2. All bivalent ligands were free of EE2 by this analysis
method.
a and ERb binding affinities and maximum tether lengths for bivalent
E2
O
O
E2
n
x
Bivalent ligand
n
x
C17–C17 distancea
(Å)
ER
(%)
a
RBAb
ERb RBAb
(%)
EE2-(eg)6-EE2 (Amine,
10)
EE2-(eg)6-EE2 (Phenol,
12)
2
2
6
6
39.8
35.7
6.8 1.0
1.2 0.4
3.6 0.3
0.68 0.22
EE2-(pg)2-EE2 (13)
EE2-(pg)3-EE2 (14)
EE2-(pg)4-EE2 (15)
EE2-(pg)5-EE2 (16)
EE2-(pg)6-EE2 (17)
3
3
3
3
3
2
3
4
5
6
24.2
29.4
34.1
39.1
43.9
0.9 0.04
2.1 0.5
6.9 1.8
2.3 0.6
1.5 0.5
0.38 0.21
0.53 0.23
0.39 0.003
0.69 0.56
0.38 0.34
EE2-(bg)2-EE2 (18)
EE2-(bg)3-EE2 (19)
EE2-(bg)4-EE2 (20)
EE2-(bg)5-EE2 (21)
4
4
4
4
2
3
4
5
26.4
33.5
38.9
45.3
1.8 0.4
4.5 0.4
2.6 0.7
1.6 0.3
0.82 0.25
1.5 0.6
0.76 0.28
0.22 0.04
a
The maximum C17–C17 distance was determined using Chem 3D Pro to gen-
erate structures with fully extended chains (all staggered conformations), and then
minimizing the energy to 0.05 RMS gradient and measuring the distance between
the 17-carbons of the steroids.
b
RBA is relative binding affinity, that is, binding relative to estradiol (E2); the Kd
of E2 for ERa is 0.2 nM and for ERb is 0.5 nM.
at the 17 position (C17) of the steroid were then measured; this
gives what are considered maximum tether lengths, which are re-
corded in Table 1. By plotting the C17–C17 distance against the
RBA, clear affinity–length relationships can be seen (Fig. 3).
Figure 3 is a plot of the RBA versus the maximum tether length
in Angstroms (Å). For both the propylene and butylene glycol
2.3. Biological results for steroidal bivalent ligands
ether-linked bivalent ligands (black lines), an optimum ERa bind-
ing affinity is observed for ligands with C17–C17 maximum tether
lengths of 34–35 Å. Notably, this maximum tether length of ꢀ35 Å
is considerably greater than the 26 Å C17–C17 distance measured
The 11 bivalent ligands were tested in the competitive radiomet-
ric relative binding affinity (RBA) assay for each subtype of ER, using
[3H]estradiol as a tracer and estradiol as a standard.35,36 Binding
affinities expressed as RBA values (i.e., relative to the binding affinity
of estradiol = 100%) are shown in Table 1. All of the bivalent ligands
had lower affinity for ER than estradiol, with the best compounds(10
and 15) having RBA values just below 7%. In all cases, the RBAs were
in the estradiol-liganded ER
a crystal structure (Fig. 2). The
straight-line distance measured directly between the ligands in
the crystal structure, however, actually passes through some por-
tions of the ER. Realistically, when the ligands bind with their
phenylethynyl linkages projecting outward from the monomer
units of the receptor dimer, the tether would need to find a mini-
mum energy path—probably more tortuous than direct—to connect
to the ends of the two phenyl groups. It is also possible that the
tether chain adopts a conformation different from the all-extended
one used to estimate the maximum tether length; conformations
other than a fully staggered one would result in contraction of
the tether, giving a somewhat shorter distance between the two
steroids. Thus, a combination of conformational effects shortening
the tether end-to-end distance and a non-linear low-energy route
higher for ER
athan for ERb, which may be attributed to the generally
larger ligand binding pocket in ERa 37
.
We examined the relationship between the tether length and
binding affinity in the propylene glycol ethers and butylene glycol
ethers—two series in which bivalent ligands linked with multiple
tether lengths were prepared. To estimate tether lengths, the biva-
lent ligands were drawn using ChemDraw 7.0 and then transferred
to Chem3D Pro 7.0 and minimized to a RMS gradient of 0.05 with
staggered chain conformations. The distance between the carbons

3532
A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
metric competitive binding assay. Members of the propylene glycol
and butylene glycol ether bivalent ligand series show a pro-
nounced peak in ER binding affinity with tether lengths of
ꢀ35 Å. Affinities for ERb are lower, but a similar maximum binding
with the butylene glycol-linked ligands was evident. Further bio-
logical studies of these bivalent ligands and control compounds
are underway to examine the effect of bivalent ligand tether length
and composition on the binding affinity, agonist/antagonist func-
tion and stability and conformation of ER dimers. The results from
these studies will be the subject of a separate publication.
ERα
propylene glycol
a
7.5
5.0
2.5
0.0
butylene glycol
ERβ
propylene glycol
butylene glycol
4. Experimental
All reagents were purchased from commercial suppliers and
were used without further purification. Anhydrous solvents (with
the exception of DMF) were obtained from an anhydrous solvent
dispensing system, and anhydrous DMF was obtained by distilla-
tion over molecular sieves. For all reactions employing anhydrous
solvents, glassware was oven-dried overnight and cooled under
vacuum, then purged with argon; all such reactions were con-
ducted under argon.
20
30
40
50
Maximum 17-17 distance (Å)
Figure 3. A plot of relative binding affinity versus maximum tether length between
the C17 carbons for propylene glycol and butylenes glycol ether-tethered bivalent
ligands. The ethyleneglycol ether-tethered (12) and amine-tethered (10) ligands are
shown as single points at 35.7 Å and 39.8 Å, respectively.
1H NMR spectra were recorded on either a 400 or 500 MHz Var-
ian Oxford instrument and 13C NMR were recorded at either 100 or
125 MHz on the same instruments. NMR spectra are reported in
ppm and were referenced to the solvent peak and processed using
ACD Labs 5.0 software. EI mass spectra were recorded at 70 eV
using the 70-VSE mass spectrometer, and ESI mass spectra were re-
corded using the Quattro mass spectrometer. Melting points are
uncorrected and were obtained using a Thomas Hoover Uni-Melt
capillary melting point apparatus.
connecting the two steroids result in bivalent ligands with the
ꢀ35-Å maximum tether length giving the highest ER
a binding
affinities.
The binding affinities of the two ethylene glycol-linked bivalent
ligands appear relevant to this point. Compound 12 has the same
ether linkage to the phenylethynyl group as do the bivalent ligands
in the other two series, and its maximum tether length (35.7 Å) is
close to that which gives the peak of binding affinity shown by the
other two series. Its RBA for ERa, however, is considerably lower
(1.2%) than that of the comparable tether-length members of the
other two series (6.9% and 4.5%). In water, polyethylene glycols
are known to adopt a helical or coiled-like conformation, due to
the preferred gauche conformation around the C–C bond bearing
two electronegative substituents.29,30 Because of this tendency to-
wards helicity, ethylene glycol ether tethers are more likely to span
a considerably shorter distance than do the propylene glycol and
butylene glycol ether tether chains, even though their maximum,
fully extended (fully staggered) lengths might be the same. It
was this conformational uncertainty that led us to focus on ligands
linked by the butylene and propylene glycol ether tethers in which
this preferred gauche effect-enforced helicity no longer applies. We
believe as well that the higher lipophilicity of the three and four-
carbon ether monomer units might be contributing to their higher
binding affinities. It is notable that the other bivalent ligand having
4.1. Hexaethylene glycol 1,20-ditosylate (6)
Hexaethylene glycol (1.41 g, 5 mmol) and DMAP (120 mg,
1 mmol) were dissolved in Et3N (7 mL, 50 mmol) and CH2Cl2
(100 mL) and cooled to 0 °C. Tosyl chloride (3.21 g, 20 mmol) was
added and the reaction warmed to room temperature and stirred
for 24 h until complete by TLC (EtOAc). The mixture was diluted
with CH2Cl2 (200 mL), washed with saturated NaHCO3
(3 Â 60 mL), dried over MgSO4, filtered and concentrated. Purifica-
tion by silica gel column (2:1 EtOAc/hexanes) gave the product as a
clear oil (2.1 g, 72%). 1H NMR (500 MHz, chloroform-d) d: 2.42 (s,
6H), 3.55 (s, 8H), 3.56–3.62 (m, 8H), 3.63–3.68 (m, 4H), 4.08–
4.16 (m, 4H), 7.28–7.36 (m, 4H), 7.73–7.81 (m, 4H). 13C NMR
(125 MHz, chloroform-d) d: 21.56, 68.58, 69.19, 70.42, 70.47,
70.52, 70.65, 127.91, 129.78, 132.89, 144.77. HRMS: Calcd for
C26H38O11S2 [M]+: 590.1856. Found: 590.1871.
an ethylene glycol tether (10) has an ERa binding affinity compa-
rable to the best of those in the other two series. However, it has
a tether that is 4 Å longer than that in compound 12, which might
compensate for the greater tendency of the polyethylene glycol
chain to coil. Also, the tether in this compound is attached to the
phenylethynyl group via a benzyl amine rather than a phenyl
ether. Thus, the structure of the tether-to-steroid linkage, as well
as the tether length and composition likely all contribute to the
binding affinity of these bivalent ligands.
All of these compounds have lower binding affinities for ERb.
Although the linear scale for RBA values compresses the curve, it
is clear that the butylene glycol bivalent ligands also reach maxi-
mum ERb binding affinity at 35 Å. The ERb binding of the propylene
glycol linked bivalent ligands, however, does not show a clear rela-
tionship to tether length.
4.2. Hexaethylene glycol 1,20-diazide (7)
Hexaethylene glycol ditosylate (6, 1 g, 1.7 mmol) was dissolved
in DMF (10 mL) and sodium azide (663 mg, 10.2 mmol) was added
and the mixture was heated to 80 °C and stirred for 24 h. The reac-
tion mixture was cooled to room temperature and added to water
(50 mL) and extracted with EtOAc (2 Â 75 mL). The combined or-
ganic layers were washed with saturated LiCl (2 Â 30 mL), dried
over MgSO4, filtered and concentrated. The product was isolated
as a clear oil (493 mg, 87%). 1H NMR (500 MHz, chloroform-d) d:
3.61 (m, 20H), 3.34 (m, 3H). 13C NMR (125 MHz, chloroform-d) d:
70.6. 70.5, 70.4, 70.3, 69.1, 50.1. ESI MS: [M+H]+ = 333.0.
4.3. Hexaethylene glycol 1,20-diamine (8)
3. Conclusions
Hexaethylene glycol 1,20-diazide (7, 480 mg, 1.44 mmol) was
dissolved in CH3OH (20 mL) and 10% Pd/C (100 mg) was added
and the mixture was stirred at room temperature for 24 h under
Bivalent ligands for the estrogen receptor (ER) were prepared
with glycol ether tethers of different lengths and compositions,
and their binding affinities for ERa and ERb were tested in a radio-

A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
3533
a H2 atmosphere. The mixture was filtered through Celite and con-
centrated, affording the product as a yellow oil (445 mg, 100%). 1H
NMR (500 MHz, chloroform-d) d: 3.59 (m, 10H), 3.47 (t, J = 5.35 Hz,
4H), 2.82 (t, J = 5.25 Hz, 4H), 1.47 (s, 4H). 13C NMR (125 MHz, chlo-
roform-d) d: 73.4, 70.53, 70.52, 70.49, 70.2, 41.73. HRMS: Calcd for
C12H28N2O5 [M]+: 281.2076. Found: 281.2064.
91.36, 112.74, 114.53, 115.19, 115.27, 126.46, 132.30, 133.04,
138.15, 153.56, 158.72. ESI MS [M+H2O]+ = 1040.5.
4.7. General procedure for formation of linker bis-mesylate
Linker diol (1 equiv) was added to an oven-dried flask under ar-
gon, dissolved in Et3N (4 equiv) and CH2Cl2 (10 mL/mmol linker
diol) and cooled to À78 °C. Mesyl chloride (3 equiv) was added
and the reaction was warmed to 0 °C and stirred for 2 h until start-
ing material was consumed by TLC (10% CH3OH/EtOAc). The mix-
ture was diluted with CHCl3, washed with saturated NaHCO3 and
brine, dried over MgSO4, filtered, concentrated and purified by sil-
ica gel column chromatography. Details for individual products
including yield, NMR and mass spectral data can be found in the
Supplementary data.
4.4. Hexaethylene glyocol bivalent EE2 ligand with amine
linker (10)
The benzaldehyde derivative of EE2 (9, 31.0 mg, 0.078 mmol)
was dissolved in CH2Cl2 (5 mL) and anhydrous K2CO3 (54 mg,
0.39 mmol) and hexaethyleneglycol diamine (8, 10.8 mg,
0.039 mmol) were added. The mixture was stirred at room temper-
ature for 48 h and concentrated. 1H NMR of the crude product indi-
cated complete conversion to the imine. The product was
redissolved in CH3OH (5 mL) and NaBH4 (43 mg, 0.12 mmol) was
added and stirred for 5 min at which point no imine was observed
by TLC (EtOAc, silica gel). The mixture was concentrated and redis-
solved in EtOAc (50 mL) and washed with water (2 Â 15 mL), dried
over MgSO4, filtered and concentrated. The product was a clear oil
(27 mg, 79%). 1H NMR (500 MHz, chloroform-d) d: 1H NMR
(500 MHz, chloroform-d) d: 3.50–3.67 (m, 24H), 3.76–3.85 (m,
4H), 6.42–6.69 (m, 4H), 7.06–7.17 (m, 2H), 7.23–7.32 (m, 4H),
7.37 (t, J = 8.15 Hz, 4H). 13C NMR (125 MHz, chloroform-d) d:
154.3, 154.0, 138.1, 128.3, 121.8, 115.4, 113.2, 112.9, 80.2, 70.5,
70.4, 70.2, 70.1, 69.9, 53.3, 49.7, 48.4, 43.7, 39.5, 33.1, 29.7, 27.2,
26.5, 22.9, 12.9, 1.0. HRMS: Calcd for C66H85N2O9 [M]+:
1049.6255. Found: 1049.6296.
4.8. General procedure for formation of linker bis-aryl iodide
Linker bis-mesylate (1 equiv), p-iodophenol (4 equiv) and
K2CO3 (8 equiv) were dissolved in 2-butanone (15 mL) and re-
fluxed for 20 h. The reaction mixture was diluted with EtOAc
(100 mL), washed with 3 M KOH (4 Â 25 mL) and brine (25 mL),
dried over MgSO4, filtered, concentrated and purified by silica gel
column chromatography. Details for individual products including
yield, NMR and mass spec data can be found in the Supplementary
data.
4.9. General procedure for Sonogashira coupling for bivalent
ligands
4.5. Hexaethylene glycol 1,20-di(4-iodophenyl)ether (11)
17a-Ethynylestradiol (2.5 equiv), Pd(Ph3P)4 (10 mol %), CuI
(20 mol %) and piperidine (20 equiv) were added to an oven-dried
flask under argon. A solution of the linker bis-aryl iodide (1 equiv)
in anhydrous THF (15 mL) was added, and the reaction was re-
fluxed for 18 h until complete by TLC (7:1 CHCl3/acetone). The
mixture was diluted with EtOAc (100 mL), washed with water
(3 Â 35 mL) and brine (2 Â 25 mL), dried over MgSO4, filtered, con-
centrated and purified by silica gel chromatography.
Hexaethylene glycol ditosylate (6, 300 mg, 0.5 mmol) was dis-
solved in acetone (20 mL) and K2CO3 (474 mg, 3 mmol) and p-iodo-
phenol (330 mg, 1.5 mmol) were added and the mixture was
refluxed for 24 h. The reaction mixture was concentrated and
redissolved in CHCl3 (100 mL) and washed with 1 M NaOH
(2 Â 25 mL) and dried over MgSO4, filtered and concentrated. The
product was a clear oil (630 mg, 92%). 1H NMR (500 MHz, chloro-
form-d) d: 7.51 (m, 4H), 6.66 (m, 4H), 4.05 (dd, J = 5.25, 4.27 Hz,
4H), 3.80 (m, 4H), 3.68 (m, 4H), 3.63 (m, 12H). 13C NMR
(125 MHz, chloroform-d) d: 158.5, 138.0, 118.0, 116.9, 82.8, 70.7,
70.5, 70.4, 69.4, 67.3. HRMS: Calcd for C24H32I2O7 [M]+: 686.0237.
Found: 686.0230.
4.10. Dipropylene glycol bivalent ligand (13)
Following the general Sonogashira coupling procedure, diprop-
ylene glycol bis-aryl iodide (89 mg, 0.17 mmol) was coupled with
17a-ethynylestradiol to give the product as a white solid (26 mg,
20%), after purification by silica gel column chromatography (col-
umn 1: 1:1 hexanes/EtOAc, column 2: 10% acetone/CHCl3, column
3: 3:2 hexanes/acetone). Mp 110–112 °C. 1H NMR (400 MHz, chlo-
roform-d) d: 0.90–0.98 (m, 6H), 1.18–1.92 (m, 18H), 1.91–2.28 (m,
12H), 2.29–2.50 (m, 4H), 2.72–2.94 (m, 4H), 4.01 (t, J = 6.23 Hz,
4H), 4.88 (br s, 2H), 6.57 (d, J = 2.69 Hz, 2H), 6.64 (dd, J = 8.55,
2.69 Hz, 2H), 6.72–6.82 (m, 4H), 7.17 (d, J = 8.30 Hz, 2H), 7.31–
7.40 (m, 4H). 13C NMR (125 MHz, chloroform-d) d: 12.94, 22.92,
26.49, 27.17, 29.43, 29.65, 33.06, 39.05, 39.44, 43.62, 47.57,
49.67, 64.76, 67.09, 77.79, 80.36, 85.85, 91.30, 112.65, 114.35,
115.22, 126.55, 133.06, 138.27, 153.26, 158.95, 177.21. HRMS:
Calcd for C58H66O7Na [M+Na]+: 897.4706. Found: 897.4714.
4.6. Hexaethylene glycol bivalent EE2 ligand with phenol linker
(12)
Hexaethylene glycol 1,20-di(4-iodophenyl)ether (11, 172 mg,
0.25 mmol),
17a
-ethynylestradiol
(133 mg,
0.45 mmol),
PdCl2(Ph3P)2 (7 mg, 0.01 mmol) and CuI (1 mg, 0.005 mmol) were
added to an oven-dried flask under argon and dissolved in anhy-
drous, degassed CH3CN (15 mL) and Et3N (5 mL). The reaction
was stirred for 24 h at room temperature, until complete by TLC
(EtOAc). The mixture was concentrated and purified by silica gel
column chromatography (column 1: 5% CH3OH/CHCl3, column 2:
3:1 CHCl3/acetone) to give the product as a white solid (54 mg,
21%). Mp 83–84 °C. 1H NMR (500 MHz, chloroform-d) d: 0.90 (s,
6H), 1.21–1.55 (m, 10H), 1.66–1.90 (m, 8H), 1.91–2.00 (m, 2H),
2.02–2.12 (m, 2H), 2.14–2.24 (m, 4H), 2.27–2.42 (m, 4H), 2.68–
2.86 (m, 4H), 3.58–3.66 (m, 12H), 3.66–3.72 (m, 4H), 3.76–3.84
(m, 4H), 4.04–4.12 (m, 4H), 6.54 (d, J = 2.69 Hz, 2H), 6.62 (dd,
J = 8.48, 2.75 Hz, 2H), 6.78–6.87 (m, 4H), 7.11 (d, J = 8.42 Hz, 2H),
7.31–7.37 (m, 4H). 13C NMR (125 MHz, chloroform-d) d: 12.89,
22.88, 26.45, 27.16, 29.62, 33.02, 38.99, 39.44, 43.58, 47.56,
49.65, 67.36, 69.57, 70.48, 70.52, 70.54, 70.74, 80.36, 85.77,
4.11. Tripropylene glycol bivalent ligand (14)
Following the general Sonogashira coupling procedure, tripro-
pylene glycol bis-aryl iodide (106 mg, 0.18 mmol) was coupled
with 17a-ethynylestradiol to give the product as a white solid
(63 mg, 38%), after purification by silica gel column chromatogra-
phy (column 1: 3:1 CHCl3/acetone, column 2: 3:2 hexanes/ace-
tone). Mp 110 °C. 1H NMR (400 MHz, chloroform-d) d: 0.93 (s,
6H), 1.24–1.59 (m, 8H), 1.63–1.92 (m, 10H), 1.92–2.15 (m, 8H),

3534
A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
2.16–2.30 (m, 4H), 2.29–2.49 (m, 4H), 2.75–2.91 (m, 4H), 3.50 (t,
J = 6.35 Hz, 4H), 3.56 (t, J = 6.10 Hz, 4H), 4.03 (t, J = 6.23 Hz, 4H),
5.25 (br s, 2H), 6.57 (d, J = 2.44 Hz, 2H), 6.64 (dd, J = 8.30,
2.69 Hz, 2H), 6.78–6.86 (m, 4H), 7.10–7.19 (m, 2H), 7.32–7.42 (m,
4H). 13C NMR (125 MHz, chloroform-d) d: 12.92, 22.89, 26.46,
27.16, 29.46, 29.63, 29.88, 33.02, 39.01, 39.42, 43.60, 47.57,
49.65, 64.82, 67.13, 67.75, 80.40, 85.85, 91.20, 112.67, 114.38,
114.85, 115.24, 126.51, 132.43, 133.04, 138.21, 153.36, 158.94.
HRMS: Calcd for C61H72O8Na [M+Na]+: 955.5125. Found: 955.5106.
47.89, 49.97, 65.19, 67.43, 68.04, 68.12, 68.18, 68.22, 78.10,
80.64, 86.12, 91.57, 112.97, 114.72, 115.20, 115.54, 126.84,
132.76, 133.36, 138.55, 153.70, 159.29. HRMS: Calcd for
C70H90O11Na [M+Na]+: 1129.6381. Found: 1129.6376.
4.15. Dibutylene glycol bivalent ligand (18)
Following the general Sonogashira coupling procedure, dibut-
ylene glycol bis-aryl iodide (43 mg, 0.08 mmol) was coupled with
17a-ethynylestradiol to give the product as a white solid (37 mg,
4.12. Tetrapropylene glycol bivalent ligand (15)
54%), after purification by silica gel column chromatography (col-
umn 1: 7:1 CHCl3/acetone, column 2: 3:2 hexanes/acetone). Mp
84–86 °C. 1H NMR (400 MHz, chloroform-d) d: 0.90–1.00 (m,
6H), 1.22–1.58 (m, 12H), 1.69–1.92 (m, 12H), 1.91–2.27 (m, 8H),
2.30–2.49 (m, 4H), 2.75–2.91 (m, 4H), 3.50 (t, J = 6.23 Hz, 4H),
3.97 (t, J = 6.23 Hz, 4H), 5.37 (br s, 2H), 6.57 (d, J = 2.69 Hz, 2H),
6.64 (dd, J = 8.30, 2.69 Hz, 2H), 6.76–6.87 (m, 4H), 7.16 (d,
J = 8.55 Hz, 2H), 7.32–7.42 (m, 4H). 13C NMR (125 MHz, chloro-
form-d) d: 26.03, 26.20, 26.47, 27.17, 29.17, 29.63, 31.71, 33.02,
39.00, 39.43, 43.59, 47.56, 49.64, 53.64, 67.67, 70.39, 80.38,
85.84, 112.67, 114.38, 115.23, 126.49, 132.38, 133.03, 138.19,
153.42, 158.96. HRMS: Calcd for C60H70O7Na [M+Na]+: 925.5109.
Found: 925.5034.
Following the general Sonogashira coupling procedure, tetra-
propylene glycol bis-aryl iodide (115 mg, 0.18 mmol) was coupled
with 17a-ethynylestradiol to give the product as a white solid
(79 mg, 45%), after purification by silica gel column chromatogra-
phy (column 1: 4:1 CHCl3/acetone, column 2: 3:2 hexanes/ace-
tone). Mp 100–102 °C. 1H NMR (400 MHz, chloroform-d) d: 0.93
(s, 6H), 1.23–1.60 (m, 8H), 1.68–1.92 (m, 12H), 1.91–2.15 (m,
8H), 2.15–2.30 (m, 4H), 2.30–2.50 (m, 4H), 2.75–2.89 (m, 4H),
3.45 (t, J = 6.35 Hz, 4H), 3.50 (t, J = 6.47 Hz, 4H), 3.58 (t,
J = 6.10 Hz, 4H), 4.04 (t, J = 6.23 Hz, 4H), 5.43 (br. s., 2H), 6.57 (d,
J = 2.69 Hz, 2H), 6.64 (dd, J = 8.55, 2.69 Hz, 2H), 6.77–6.87 (m,
4H), 7.15 (d, J = 8.30 Hz, 2H), 7.32–7.42 (m, 4H). 13C NMR
(125 MHz, chloroform-d) d: 12.92, 22.89, 26.46, 27.17, 29.49,
29.64, 29.89, 33.03, 39.01, 39.43, 43.59, 47.56, 49.65, 64.86,
67.11, 67.72, 67.86, 80.37, 85.83, 91.22, 112.68, 114.39, 114.86,
115.24, 126.49, 132.36, 133.04, 138.19, 153.44, 158.95. HRMS:
Calcd for C64H78O9Na [M+Na]+: 1013.5544. Found: 1013.5530.
4.16. Tributylene glycol bivalent ligand (19)
Following the general Sonogashira coupling procedure, tributyl-
ene glycol bis-aryl iodide (49 mg, 0.08 mmol) was coupled with
17a-ethynylestradiol to give the product as a white solid (39 mg,
52%), after purification by silica gel column chromatography (1:
7:1 CHCl3/acetone, 2: 3:2 hexanes/acetone). Mp 64–66 °C. 1H
NMR (400 MHz, chloroform-d) d: 0.91–0.97 (m, 6H), 1.14–2.28
(m, 36H), 2.29–2.50 (m, 6H), 2.75–2.89 (m, 4H), 3.38–3.53 (m,
8H), 3.97 (t, J = 6.35 Hz, 4H), 6.57 (d, J = 2.44 Hz, 2H), 6.64 (dd,
J = 8.30, 2.44 Hz, 2H), 6.77–6.87 (m, 4H), 7.16 (d, J = 8.30 Hz, 2H),
7.33–7.43 (m, 4H). 13C NMR (125 MHz, chloroform-d) d: 12.91,
22.91, 26.03, 26.22, 26.44, 27.17, 29.20, 29.64, 33.03, 39.01,
39.45, 43.60, 47.57, 49.65, 53.66, 67.70, 70.31, 70.65, 80.37,
85.86, 91.17, 112.66, 114.39, 115.22, 126.52, 132.45, 133.03,
138.22, 153.38, 158.99. HRMS: Calcd for C64H78O8Na [M+Na]+:
997.5594. Found: 997.5565.
4.13. Pentapropylene glycol bivalent ligand (16)
Following the general Sonogashira coupling procedure, pent-
apropylene glycol bis-aryl iodide (65 mg, 0.09 mmol) was cou-
pled with 17a-ethynylestradiol to give the product as a white
solid (48 mg, 50%), after purification by silica gel column chro-
matography (column 1: 3:1 CHCl3/acetone, column 2: 3:2 hex-
anes/acetone). Mp 80–82 °C. 1H NMR (500 MHz, chloroform-d)
d: 0.93 (s, 6H), 1.31–1.58 (m, 8H), 1.69–1.93 (m, 14H), 1.93–
2.15 (m, 8H), 2.16–2.27 (m, 4H), 2.32–2.47 (m, 4H), 2.75–2.91
(m, 4H), 3.41–3.48 (m, 8H), 3.51 (t, J = 6.43 Hz, 4H), 3.58 (t,
J = 6.11 Hz, 4H), 4.05 (t, J = 6.32 Hz, 4H), 5.14 (br s, 2H), 6.57
(d, J = 2.57 Hz, 2H), 6.64 (dd, J = 8.36, 2.79 Hz, 2H), 6.80–6.87
(m, 4H), 7.16 (d, J = 8.36 Hz, 2H), 7.33–7.41 (m, 4H). 13C NMR
(125 MHz, chloroform-d) d: 12.90, 22.88, 26.46, 27.15, 29.50,
29.63, 29.91, 29.93, 33.02, 39.01, 39.42, 43.59, 47.56, 49.64,
64.87, 67.11, 67.71, 67.82, 67.90, 80.34, 85.83, 89.34, 91.25,
112.67, 114.42, 114.89, 115.24, 126.54, 132.48, 133.07, 138.25,
159.00. HRMS: Calcd for C67H84O10Na [M+Na]+: 1071.5692.
Found: 1071.5966.
4.17. Tetrabutylene glycol bivalent ligand (20)
Following the general Sonogashira coupling procedure, tetr-
abutylene glycol bis-aryl iodide (55 mg, 0.08 mmol) was coupled
with 17a-ethynylestradiol to give the product as a white solid
(43 mg, 53%), after purification by silica gel column chromatogra-
phy (column 1: 15% acetone/CHCl3, column 2: 3:2 hexanes/ace-
tone). Mp 80 °C. 1H NMR (400 MHz, chloroform-d) d: 1.23–1.31
(m, 6H), 1.31–1.54 (m, 8H), 1.56–1.68 (m, 6H), 1.68–1.92 (m,
12H), 1.91–2.26 (m, 16H), 2.38 (d, J = 9.28 Hz, 4H), 2.81 (d,
J = 4.88 Hz, 4H), 3.38–3.52 (m, 10H), 3.97 (t, J = 6.35 Hz, 2H), 5.35
(br s, 4H), 6.57 (d, J = 2.69 Hz, 2H), 6.64 (dd, J = 8.42, 2.81 Hz, 2H),
6.78–6.86 (m, 4H), 7.16 (d, J = 8.06 Hz, 2H), 7.33–7.41 (m, 4H).
13C NMR (125 MHz, chloroform-d) d: 12.89, 22.86, 25.98, 26.17,
26.37, 26.44, 27.15, 29.15, 29.61, 33.00, 38.98, 39.42, 43.57,
47.55, 49.62, 67.68, 70.30, 70.57, 70.64, 80.36, 85.85, 91.20,
112.69, 114.39, 114.80, 115.25, 126.49, 132.32, 133.04, 138.18,
153.53, 159.00. HRMS: Calcd for C68H86O9Na [M+Na]+:
1069.6170. Found: 1069.6149.
4.14. Hexapropylene glycol bivalent ligand (17)
Following the general Sonogashira coupling procedure, hexa-
propylene glycol bis-aryl iodide (69 mg, 0.09 mmol) was coupled
with 17a-ethynylestradiol to give the product as a white solid
(42 mg, 42%), after purification by silica gel column chromatogra-
phy (column 1: 3:1 CHCl3/acetone, column 2: 3:2 hexanes/ace-
tone). Mp 76 °C. 1H NMR (400 MHz, chloroform-d) d: 0.93 (s, 6H),
1.29–1.60 (m, 8H), 1.72–1.92 (m, 16H), 1.93–2.15 (m, 8H), 2.16–
2.30 (m, 4H), 2.32–2.51 (m, 4H), 2.81 (d, J = 4.88 Hz, 4H), 3.41–
3.54 (m, 16H), 3.59 (t, J = 6.10 Hz, 4H), 4.05 (t, J = 6.23 Hz, 4H),
5.17 (br s, 2H), 6.57 (d, J = 2.69 Hz, 2H), 6.64 (dd, J = 8.30,
2.69 Hz, 2H), 6.79–6.88 (m, 4H), 7.16 (d, J = 8.55 Hz, 2H), 7.33–
7.42 (m, 4H). 13C NMR (125 MHz, chloroform-d) d: 13.24, 23.22,
26.80, 27.49, 29.84, 29.98, 30.27, 33.35, 39.34, 39.76, 43.93,
4.18. Pentabutylene glycol bivalent ligand (21)
Following the general Sonogashira coupling procedure, pent-
abutylene glycol bis-aryl iodide (61 mg, 0.08 mmol) was coupled

A. L. LaFrate et al. / Bioorg. Med. Chem. 17 (2009) 3528–3535
3535
with 17a-ethynylestradiol to give the product as a white solid
References and notes
(42 mg, 49%), after purification by silica gel column chromatogra-
phy (column 1: 15% acetone/CHCl3, column 2: 3:2 hexanes/ace-
tone). Mp 60–62 °C. 1H NMR (400 MHz, chloroform-d) d: 1.22–
2.28 (m, 48H), 2.29–2.52 (m, 4H), 2.73–2.89 (m, 4H), 3.36–3.52
(m, 16H), 3.97 (t, J = 6.35 Hz, 4H), 5.66 (br s, 4H), 6.56 (d,
J = 2.69 Hz, 2H), 6.63 (dd, J = 8.42, 2.81 Hz, 2H), 6.78–6.87 (m,
4H), 7.15 (d, J = 8.30 Hz, 2H), 7.32–7.41 (m, 4H). 13C NMR
(125 MHz, chloroform-d) d: 12.89, 22.87, 26.01, 26.19, 26.39,
26.41, 27.16, 29.63, 33.01, 39.00, 39.44, 43.59, 47.56, 49.64,
67.03, 67.23, 67.69, 70.31, 70.56, 70.59, 70.66, 80.33, 85.83,
91.22, 112.68, 114.41, 114.82, 115.24, 126.51, 133.05, 137.10,
138.21, 153.53, 160.99. HRMS: Calcd for C72H44O10Na [M+Na]+:
1141.6745. Found: 1141.6732.
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We are grateful for support of this research from the National
Institutes of Health (PHS R37 DK15556). Mass spectra were ob-
tained on a 70-VSE mass spectrometer, purchased in part with a
grant from the Division of Research Resources, National Institutes
of Health (RR 04648)on a Quattro mass spectrometer, purchased
in part with a grant from the Division of Research Resources, Na-
tional Institutes of Health (RR 07141).
Supplementary data
Supplementary data (procedural details for the synthesis of the
propylene and butylenes glycol tethers, with spectroscopic charac-
terization data) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2009.04.016.