Synthesis of a spin-labeled anti-estrogen as a dynamic motion probe for the estrogen receptor ligand binding domain

Article abstract of DOI:10.1016/j.bmcl.2011.12.091

The preparation and characterization of a novel nitroxide spin probe based on a steroidal anti-estrogen is described. The probe 5 demonstrated very high binding affinity for both the alpha and beta isoforms of the estrogen receptor-ligand binding domain. EPR spectrometric studies demonstrate conformational constraints for the ligand, consistent with the nitroxyl moiety occupying a position just beyond the receptor-solvent interface.

Full text of DOI:10.1016/j.bmcl.2011.12.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1743–1746
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Bioorganic & Medicinal Chemistry Letters
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Synthesis of a spin-labeled anti-estrogen as a dynamic motion probe
for the estrogen receptor ligand binding domain
⇑
J. Adam Hendricks, Stefano V. Gullà, David E. Budil, Robert N. Hanson
Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115-5000, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparation and characterization of a novel nitroxide spin probe based on a steroidal anti-estrogen is
described. The probe 5 demonstrated very high binding affinity for both the alpha and beta isoforms of
the estrogen receptor–ligand binding domain. EPR spectrometric studies demonstrate conformational
constraints for the ligand, consistent with the nitroxyl moiety occupying a position just beyond the
receptor-solvent interface.
Received 18 November 2011
Revised 14 December 2011
Accepted 19 December 2011
Available online 8 January 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Nitroxide spin label
Estrogen receptor-ligand binding domain
Selective estrogen receptor modulator
Anti-estrogen
Dynamic motion probe
Estrogen receptor-alpha (ER
a
) is a member of the family of
X-ray crystallography4,7 and nuclear magnetic resonance spec-
troscopy8 (NMR) are the primary techniques from which most
conformational data regarding ligand–ERa–LBD complexes have
nuclear hormone dependent transcription factors responsible for
mediating the effect of endogenous estrogens.1 Because of its
important role in hormone responsive breast cancer,2,3 significant
efforts have been directed toward elucidating conformational
changes elicited by complexes between this protein and its natural
and synthetic ligands. The folding of helix 12, located at the C-ter-
been drawn. However, crystallography requires crystallization of
the complex under nonphysiological conditions and more impor-
tantly, only provides a conformational ‘snapshot’ of the complex,
providing little or no information about the dynamic nature of
the interactions. These interactions can be simulated using molec-
ular modeling,9 thereby providing an approximation of the process
and surrounding environment. NMR spectroscopy complements in
silico studies by providing additional data reflecting the dynamics
of the ligand–receptor complex, but is limited by the size of the
biomolecule.
Electron paramagnetic resonance (EPR) spectroscopy is a sensi-
tive technology for studying protein dynamics. EPR typically
employs nitroxide radicals as the paramagnetic species, which,
due to their biological stability, makes them ideal for studying
ligand–receptor interactions at a molecular level under physiolog-
ical conditions. Nitroxide radicals also possess T1 contrast proper-
ties extending their application into magnetic resonance imaging
(MRI).10 This particular property allows for the potential use
of spin labeled ligands in vitro and in vivo molecular imaging
studies.
minus of ERa–ligand binding domain (ERa–LBD), after ligand
binding, is a critical factor in determining an agonist or antagonist
response4–6 through recruitment of either coactivators or corepres-
sors, respectively. In the agonist conformation, helix 12 folds into a
compact structure, and the ‘LxxLL’ binding motif of activation func-
tion 2 (AF2) is exposed on the surface where coactivator proteins
bind, promoting transcriptional activity, including cell prolifera-
tion. However, in the antagonist conformation, the folding of helix
12 is disrupted, causing AF2 to be buried within the receptor,
thereby preventing coactivator recruitment and inhibiting cell
proliferation. This is the mechanism by which selective estrogen
receptor modulators (SERMs), such as Tamoxifen or Raloxifene,
exhibit their beneficial pharmacological effects.
Abbreviations: ERa, estrogen receptor alpha; ERa-LBD, estrogen receptor alpha-
ligand binding domain; AF2, activation function 2; SERM, selective estrogen
receptor modulator; NMR, nuclear magnetic resonance; EPR, electron paramagnetic
resonance; SDSL, site directed spin labeling; DEER, double electron-electron
resonance; RBA, relative binding affinity; ERb, estrogen receptor beta.
To date most research on ERa–LBD, and other receptors, using
EPR has used site directed spin labeling (SDSL) where the nitroxide
label is attached to a specific amino acid on the receptor,11 rather
than using a labeled ligand. When both ERa–LBD and ligand are
⇑
Corresponding author. Tel.: +1 617 373 3313; fax: +1 617 373 8795.
E-mail address: r.hanson@neu.edu (R.N. Hanson).
labeled, interspin distances can be determined through double
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.091

1744
J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1743–1746
triazole. The spin label 7 was prepared in 91% yield via reductive
amination of commercially available 4-oxo-TEMPO with propargyl
amine in the presence of acetic acid in 1,2-dichloroethane.16 The
complementary azido-estradiol derivative was synthesized, as out-
lined in Scheme 1. Starting from estradiene dione-3-ketal, 1, regio-
selective epoxidation17 of the 5,10 olefin using hexafluoroacetone
and 50% hydrogen peroxide afforded a 3:1 mixture of
in 56% overall yield. Separation of the isomers was critical as only
the isomer gave the desired 11b intermediate. The Grignard
a:b isomers
a
Figure 1. Target compound 5 designed from RU 39411.
reagent of 4-bromo(phenoxy) trimethylsilane was prepared and
used immediately undergoing a copper (I) mediated 1,4-addition
electron–electron resonance (DEER) spectroscopy. This approach
provides not only spatial information but also insight regarding
the biological microenvironment, such as polarity and proticity.12
Previous work has demonstrated that 11b-functionalized estradi-
to the pure
(4-hydroxyphenyl)estra-4,9-diene-3,17-dione
11b-(4-hydroxyphenyl)estra-4,9-diene-3,17-dione
a-epoxide which, after acid hydrolysis, afforded 11b-
2
in 87% yield.13
underwent
alkylation with ethylene glycol ditosylate,18 followed by the dis-
placement of the terminal tosylate by sodium azide. Aromatization
of the A ring using acetic anhydride and acetyl bromide in dichlo-
romethane furnished 11b-(2-azidoethoxyphenyl-3-acetoxyl-estra-
1,3,5(10)-trien-17-one 3. Huisgen 3+2 cyclization of 3 with 4-prop-
argylamino-2,2,6,6-piperidine-N-oxide 7 followed by reduction
and saponification gave the spin labeled antiestrogen 5 in a 71%
yield (3 steps). Overall yield for 11 steps was 12%. Reduction and
saponification of 3 afforded 11b-(4-azidoethoxy)phenyl estradiol
6 as the reference anti-estrogen.
ols generate ER
a
antagonists13,14 while maintaining high affinity
for the receptor.15 Judicious modifications of the synthesis would
permit facile entry into anti-estrogens bearing the requisite spin
label. In this study, we describe the preparation and characteriza-
tion of a molecular EPR probe for ER
a designed to address this
‘gray area’ left by modeling, NMR and crystallography.
The target for our study, compound 5, shown in Figure 1, com-
bines the high affinity antiestrogenic component associated with
RU 3941116 and the TEMPO spin label, linked together via a
O
HO
N3
O
O
O
h,i
d,e,f
a,b,c
O
6
O
O
O
O
3
2
1
HN
O
N
O
O
N
N
N
N
OH
N
O
N
O
h,i
7
g
3
NH
NH
O
HO
N
O
N
O
5
4
Scheme 1. Synthesis of spin labeled 11b-estradiol derivative 5 and parent steroid 6. Reagents and conditions: (a) H2O2 (50%, 2.3 equiv), CF3C(O)CF3 (0.11 equiv),
pyridine(0.09 equiv), DCM, 0?23 °C, 12 h; (b) (i) Mg (1.7 equiv), I2 (catalytic), 4-bromo(phenoxy) trimethylsilane (1 equiv), THF, 65 °C, 5 h (ii) Cu(I)Cl (0.1 equiv), THF,
À10 °C?23 °C, 12 h; (c) 70% AcOH 30% H2O, 60 °C, 1.5 h; (d) ethylene glycol ditosylate (1.5 equiv), K2CO3 (4.0 equiv), 80 °C, 12 h.; (e) NaN3 (4.0 equiv), EtOH, 80 °C, 6 h; (f)
Ac2O (1.0 equiv), AcBr (2.5 equiv), DCM, 23 °C, 6 h.; (g) Cu2SO4 (0.01 equiv), NaC6H7O6 (0.05 equiv), 1:1 t-butanol–water, 12 h; (h) NaBH4 (1.2 equiv), MeOH (i) NaOH
(4.0 equiv), MeOH.
Figure 2. Relative binding affinities (RBA) of spin probe 5 and its parent steroid 6.

J. Adam Hendricks et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1743–1746
1745
as N = R||/R\ from 1 (isotropic motion) for the free label to 16 in
the presence of ER –LBD, with the axis of fastest rotation lying
a
along the N–O bond of the nitroxide. The complete set of parame-
ters obtained from the least-squares lineshape fitting of the data is
given in the Supplementary data.
free label
The EPR results are consistent with the binding mode predicted
for the label 5 based on molecular docking studies, as shown in Fig-
ure 4. The steroidal(estradiol) moiety of the spin-labeled compound
(shown in yellow) occupies the same position as tamoxifen bound
with ER
3380
α
in the antagonist conformation of ERa–LBD (shown in green). One
should note the position of the triazole ring which occupies the
same region as the basic dimethylamino group of tamoxifen. This
binding mode places the triazole at the receptor-solvent interface,
with the TEMPO group extended beyond the binding pocket, allow-
ing relatively rapid motion of the nitroxide around the axis parallel
to the N–O bond.
3300
3320
3340
Field (Gauss)
3360
3400
Figure 3. EPR spectra of free 5 (top) and 5 in the presence of ERa–LBD (bottom).
Lines show least-squares fits to the experimental data as detailed in the Supple-
In summary, we have synthesized, characterized and evaluated
a high affinity spin labeled anti-estrogenic ligand designed to study
mentary data.
Figure 4. Binding poses of tamoxifen (green) and 5 (yellow) after molecular docking with ERa–LBD monomer, illustrating the disruption of Helix 12 (red).
Relative binding affinities (RBA) of spin probe 5 and azido-anti-
estrogen 6 as ligands for ER –LBD and ERb–LBD were determined
using a competitive binding assay, the results of which are given in
Figure 2. The RBA values for the azidoethoxy derivative 6 (39% and
34%) are comparable to those reported for RU39411,15,19 the
dimethylaminoethoxy analog. Incorporation of the spin label re-
the real time response of ERa–LBD upon ligand binding. These re-
a
sults provide the basis for the design and preparation of second
generation spin probes for the steroid hormone receptors as com-
plements to current spectroscopic methods.
Acknowledgments
duces affinity of
(RBA = 4.5%) toward ER
5
by less than an order of magnitude
–LBD, indicating that extension of the sub-
a
This work was supported by Army CDMRP Contract W81XWH06-
1-0551 (D.E.B. and R.H.) and NSF-IGERT Award # 0504331 (J.A.H.) We
gratefully acknowledge Professor John A. Katzenellenbogen, whose
research group carried out the ER binding assays for the spin labeled
anti-estrogen and parent steroid.
stituent does not completely abolish binding within the ligand
binding pocket. The affinity at the ERb–LBD was lower
(RBA = 1.9%) indicating that binding of the larger substituent is
somewhat less favored at this subtype. While the terminal azido
group was well tolerated, introduction of the substituted triazole
moiety did affect the affinity somewhat, but overall antagonist
activity was maintained. Based on these results, we proceeded to
Supplementary data
evaluate 5 as a spin labeled molecular probe for ER
An initial characterization of the binding of 5 to the ER
was made by standard continuous wave EPR at 9.5 GHz. Figure 3
compares spectra of free 5 in solution (top) and 5 in the presence
of ERa–LBD (bottom). Although both spectra exhibit the three lines
a–LBD.
Supplementary data (includes detailed experimental proce-
dures, spectra and characterization data, and lineshape fitting re-
sults) associated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2011.12.091.
a
–LBD
characteristic of fast probe motion, the major component exhibits
lines that are considerably broadened and have different relative
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