Synthesis of a novel fluorescent probe for estrogen receptor

Article abstract of DOI:10.1016/S0960-894X(02)00144-0

A novel estradiol-mimetic fluorescent probe 5 was synthesized from diethylstilbestrol (DES, 1), which is useful for probing estrogen receptor (ERα), a prognostic indicator of estrogen-dependent cancers, and for developing a homogeneous fluorescence polarization (FP) assay to identify the ligands of estrogen receptor.

Full text of DOI:10.1016/S0960-894X(02)00144-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1283–1285
Synthesis of a Novel Fluorescent Probe for Estrogen Receptor
Maciej Adamczyk,* Rajarathnam E. Reddy and Zhiguang Yu
Department of Chemistry (9NM, Bldg AP20), Diagnostics Division, Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, IL 60064-6016, USA
Received 17 December 2001; accepted 20 February 2002
Abstract—A novel estradiol-mimetic fluorescent probe 5 was synthesized from diethylstilbestrol (DES, 1), which is useful for
probing estrogen receptor (ERa), a prognostic indicator of estrogen-dependent cancers, and for developing a homogeneous fluor-
escence polarization (FP) assay to identify the ligands of estrogen receptor. # 2002 Elsevier Science Ltd. All rights reserved.
Estrogen receptors (ER) regulate the expression of
genes,1 which control the differentiation, growth and
function of reproductive system in females.2 They also
play an important role in the growth of estrogen-
dependent tumors such as breast cancer and other
endocrinological disorders.3 Breast cancer is the second
leading cause of death in women in United States and
the levels of estrogen receptors (ER) on tumor cells
(estimated range: 5000–50,000 molecules/cell) have been
shown to be a prognostic indicator of the disease.4 As
with most cancers, the early diagnosis of breast cancer is
key for long-term survival, and the choice of treatment
is aided by the determination of estrogen receptor levels
on a tumor cell. Additionally, development of screening
assays for estrogen receptor ligands facilitate the search
for new and improved agonists and antagonists of
estrogens,5 which are potentially useful for treatment of
cancers, Alzheimer’s disease, cardiovascular diseases,
diabetes, osteoporosis, and urinary incontinence.6 Gen-
erally, the estrogen receptor content in a breast tumor is
determined either by imaging methods, for example
positron emission tomography (PET), single photon
emission computerized tomography (SPECT) using
radiolabeled tracers or in vitro analysis of tumor biopsy
sample.7 Limitations of these methods, coupled with the
increasing need for high-throughput screening (HTS),8
have stimulated interest in the development of new
homogeneous assay technologies for determination of
estrogen receptors as well as its ligand screening.
Fluorescence polarization (FP)9 method is useful to
study molecular interactions, which is based on the
principle that a fluorescent molecule when excited with
polarized light will emit fluorescence having a degree of
polarization inversely proportional to its rate of rota-
tion. Small fluorescent molecules (e.g., probe) rotate
faster in solution than the larger molecules, (e.g., ER–
probe complex) and hence, when the probe bound to
ER, its rotation is slowed and the polarization value
(mP) is increased. Thus, homogeneous FP technique is
widely used to investigate molecular binding events in
solution.9 The probes for ER, from chemistry point of
view, to be able to serve effectively in a homogeneous
FP format would be those derived from its native
ligand, 17b-estradiol. We10 and others11 have reported a
variety of fluorescent probes needed for development of
immunoassays for 17b-estradiol. However, in our
hands, the 17b-estradiol-derived fluorescent probes,9
which were labeled at the 6-, 7- and 17-positions of 17b-
estradiol, exhibited poor binding to estrogen receptor
(ERa) and were not suitable for development of homo-
geneous FP assay. A possible explanation for the lack of
binding of these 17b-estradiol-derived probes to ER
might be that the critical binding determinants were
blocked when the 17b-estradiol was conjugated with a
fluorescent label and thus rendering them to be less than
perfect mimics of 17b-estradiol to the native protein,
ER.10 Recently, Parker et al.12 reported an FP-based
assay for estrogen receptor, which is now commercially
available from PanVera Corporation (Madison, WI,
USA), with undisclosed chemistry of the probe. In con-
tinuation of our interest in the development of fluores-
cence and chemiluminescence assays for the measurement
of 17b-estradiol9 and other steroidal hormones,13 we
decided to design a new fluorescent probe for studies of
*Corresponding author. Tel.: +1-847-937-0225; fax: +1-847-938-
8927; e-mail: maciej.adamczyk@abbott.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00144-0

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M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1283–1285
estrogen receptor (ERa) in homogeneous fluorescence
polarization assay format.
from 34.6 to 0.017 nM. As a control, bovine serum
albumin (BSA) solution was prepared similarly to the
ERa and mixed with 2.0mL of probe 5 solution (1.0
nM) in individual tubes. All samples were then incu-
bated at room temperature in dark for 2 h and fluores-
cence polarization value (mP) was measured in
triplicates (Fig. 1). The titration curve showed that the
probe 5 bound tightly to ERa (KD, 5.8Æ1.2 nM). The
dynamic range of fluorescence polarization for 5 was
more than 350mP. The control experiment using BSA
showed only 50mP increase FP value even at a
concentration of about 40nM of ER a.
We envisioned that a fluorescent conjugate derived from
non-steroidal substrates, which are known to bind to
estrogen receptor (ER), could be more efficient for
probing the estrogen receptor. Although, the overall
molecular topology of a non-steroidal ER ligand, di-
ethylstilbestrol (DES, 1) is different from the native ER
ligand, 17b-estradiol, it has been reported that the oxy-
gen–oxygen distance in DES (1, 12.1 A) is close to the
O–O distance between the 3- and 17-positions of 17b-
estradiol (10.9 A).14 The difference of O–O distance (1.2
A) between DES (1) and 17b-estradiol is well within the
mobility (7 A) of ER binding pocket,14 and thus ren-
dering DES (1) to be a promising candidate for estrogen
receptor probe development. Accordingly, DES (1) was
selectively alkylated (Scheme 1) with ethyl 4-bromo-
butyrate15 using 1.1 equiv of NaH in DMF to afford the
mono O-alkylated product 2 in 42% yield. The ester
group in 2 was then hydrolyzed with LiOH in THF–
water medium to afford the corresponding acid 3 in
95% yield. The acid 3 was reacted with 6-amino-
methylfluorescein hydrobromide (6-Fln-CH2NH2 HBr,
4) in the presence of HOBt, EDAC and triethylamine in
DMF at room temperature for 16 h. The crude product
was purified by preparative reversed-phase HPLC16 and
lyophilized to afford the fluorescent probe 5 in 20%
yield as an orange powder in >99% purity.17
Competitive displacement studies were then conducted
with three ligands (17b-estradiol, diethylstilbestrol and
tamoxifen) for their ability to displace the probe 5 from
estrogen receptor (ERa) complex. Thus, solution of
estrogen receptor (ERa, 36 mL, 5800 nM) was added to
30mL of 0.2 nM probe 5 in 100 mM potassium phos-
phate buffer containing 0.5 mg/mL b-cyclodextrin and
the mixture was aliquoted into individual tubes (2.0mL/
tube). The stock solution of 17b-estradiol (10mM in
DMSO) was diluted with the same buffer (100 mM
potassium phosphate buffer containing 0.5 mg/mL
b-cyclodextrin) and different amounts were added to the
tubes containing ERa-probe 5 complex to achieve a
final concentration of 0.1, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 12,
and 22 nM of 17b-estradiol, respectively. The measured
fluorescence polarization value (mP) was plotted against
the concentration (nM) of 17b-estradiol. A four-para-
meter logistic fitting in Grafit was used to obtain an
inhibition constant (IC50) of 3.40( Æ1.0) nM for 17b-
estradiol (Fig. 2). A similar displacement assay was
carried out with diethylstilbestrol and tamoxifen as the
inhibitors and the IC50 values were determined to be
0.33 (Æ0.1) and 8.40 (Æ1.5) nM, respectively.
We then proceeded to assess the suitability of 5 as an
estradiol-mimetic probe for ER in a homogeneous fluor-
escence polarization assay format using ISS PC1 photon
counting spectrofluorometer. Thus, a 1.0mM stock
solution of fluorescent probe 5 in DMSO was prepared,
which was diluted with 100 mM potassium phosphate
buffer (pH 7.5) to obtain 1.0nM solution, and aliquoted
into individual tubes (2.0mL/tube). Different amounts
of estrogen receptor (ERa, 5800 nM) was added to the
each tube to achieve final concentration of ERa ranging
In summary, a novel estradiol-mimetic probe 5 was
synthesized and its application in the development of
homogeneous fluorescence polarization (FP) assay for
Figure 1. A titration curve for binding of estrogen receptor (ERa) and
fluorescent probe 5, with BSA as a control. Excitation wavelength in
the ISS PC1 photon counting spectrofluorometer was set to 480nm
via a monochromator (1.0mm slit). Polarized fluorescence was passed
through a Crion 383U XM-530-F filter (530 nm, bandpass 25 nm) and
the intensity was monitored by an internal PMT.
Scheme 1. Synthesis of fluorescent probe 5.

M. Adamczyk et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1283–1285
1285
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