Synthesis and evaluation of 17α-(dimethylphenyl)vinyl estradiols as probes of the estrogen receptor-α ligand binding domain

Article abstract of DOI:10.1016/j.steroids.2012.01.003

As part of our program to explore the influence of small structural modifications on the biological response of the estrogen receptor-α (ERα), we prepared and evaluated a series of mono-and di-substituted phenyl vinyl estradiols. The target compounds were

Full text of DOI:10.1016/j.steroids.2012.01.003

Steroids 77 (2012) 471–476
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and evaluation of 17
a-(dimethylphenyl)vinyl estradiols as probes
of the estrogen receptor-
a
ligand binding domain
a
a
a
b
Robert N. Hanson a, , Emmett McCaskill , Pakamas Tongcharoensirikul , Robert Dilis , David Labaree ,
⇑
Richard B. Hochberg b
a Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States
b Department of Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of our program to explore the influence of small structural modifications on the biological response
Received 5 December 2011
Received in revised form 22 December 2011
Accepted 6 January 2012
of the estrogen receptor-
vinyl estradiols. The target compounds were prepared in 45–80% yields using the Stille coupling reaction
and evaluated using competitive binding analysis with the ER -ligand binding domain (hER -LBD) and
a (ERa), we prepared and evaluated a series of mono-and di-substituted phenyl
a
a
Available online 17 January 2012
estrogenic activity (induction of alkaline phosphatase in Ishikawa cells). Results indicated that the
2,4- and 2,5-dimethyl derivatives, 5b and 5c, had the highest relative binding affinity (RBA = 20.5 and
37.3%) and relative stimulatory activity (RSA = 101.0% and 12.3%) of the di-methyl series.
Ó 2012 Elsevier Inc. All rights reserved.
Keywords:
Steroidal estrogens
Estrogen receptor
Ligand binding domain
Synthesis
Molecular modeling
Binding assay
1. Introduction
[8]. In parallel with biological assays, we used computational and
crystallographic methods to evaluate the results. Molecular model-
Our research program has focused on the preparation of specif-
ically modified derivatives of estradiol as probes for the ligand
binding domain (LBD) of the estrogen receptor (ER). These struc-
tural probes would permit enhanced insight into the physicochem-
ical factors that influence receptor affinity, subtype selectivity and
efficacy resulting from cofactor recruitment. Our early studies sug-
gested the presence of a region within the LBD that could accom-
ing studies suggested, and crystallographic studies confirmed [9],
that the phenyl vinyl group induced the formation of a new pocket
on the alpha face of the ligand binding pocket (LBP), similar to one
observed for thyroid (TR), glucocorticoid (GR) and progesterone
(PgR) receptors [10–12]. The position of functional groups on the
phenyl ring and their physicochemical properties produce interac-
tions with specific amino acid side chains, leading to significant
differences in affinity (RBA values) and in vivo potency profiles.
Notably, all of our derivatives evaluated to date exhibited full ago-
nist responses, indicating that the compounds induced ER agonist,
not antagonist, conformations. Because of the close interactions
between the ligand and the LBP, we hypothesized that the intro-
duction of additional substituents on the phenyl group would im-
part further changes in the biological responses. For example, small
changes at the 11b-position, such as the extension of an alkyl chain
by a single methylene group, converted a potent ER agonist to an
ER antagonist [13,14]. We initially considered evaluating the series
of bis(trifluoromethyl)phenyl vinyl isomers, however, the entire
set of requisite isomeric bis(trifluoromethyl)phenyl starting mate-
rials was neither commercially available nor readily accessible via
synthesis. However, the corresponding isomeric di-methylphenyl
iodides (iodoxylenes) were available and, because the differences
in RBA values between the methyl and trifluoromethyl substituted
phenylvinyl estradiols were relatively small, we undertook this
modate phenyl (-X-) vinyl groups at the 17a-position of estradiol
[1–4]. Analysis of the E-(4-substituted phenyl)vinyl estradiols indi-
cated that there existed within the LBD significant steric tolerance
toward 4-substituent and that some polar influences were present
[5]. Comparison with the corresponding Z-(4-substituted phenyl)-
vinyl isomers suggested that the two phenyl vinyl moieties
accessed different regions of the LBD, leading to different struc-
ture–activity relationships [6]. Subsequent examination of the E-
(2-,3-, and 4-trifluoromethylphenyl)vinyl estradiols demonstrated
significant dependence on substitution patterns, as the 2-isomer
was more potent in vitro as well as in vivo [7]. Introduction of an
11b-methoxy group into the E-(2-,3-, and 4-trifluoromethylphenyl)
vinyl estradiols had little effect on the affinity for theER-LBD,
however, it provided a significant enhancement of in vivo potency
⇑
Corresponding author. Tel.: +1 617 373 3313; fax: +1 617 373 8795.
E-mail address: r.hanson@neu.edu (R.N. Hanson).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.01.003

472
R.N. Hanson et al. / Steroids 77 (2012) 471–476
study investigating the effect that di-methyl substitution on the
phenyl ring would have on ER binding and estrogenic activity. In
this paper we report the preparation and evaluation of a series of
(mono- and di-substituted phenyl)vinyl estradiols as ligands for
the estrogen receptor ligand binding domain.
and concentrated to give the crude estradiol derivative. The substi-
tuted phenyl vinyl estradiol derivative was purified using flash
chromatography and characterized by 1H NMR and 13C NMR, and
elemental analysis.
2.2.4. (17
3,17-diol 3
Method A was used and the reaction conditions were performed
0.68 mmol scale. Yield = 176 mg, 69%. Mp 163–165 °C,
26H30O2 2ÁH2O, Anal: C, 76.06; H, 8.35, Found: C, 75.96; H, 8.28.
1H NMR (d6-acetone); 13C NMR (d6-acetone).
a,20E)-21-(phenyl)-19-norpregna-1,3,5(10),20-tetraene-
2. Experimental
on
C
a
2.1. General methods
All reagents and solvents were purchased from Aldrich or Fisher
Scientific. THF and toluene were distilled from sodium/benzophenone.
Reactions were monitored by TLC, performed on 0.2 mm silica gel
plastic backed sheets containing F-254 indicator. Visualization on
TLC was achieved using UV light, iodine vapor and/or phosphomo-
lybdic acid reagent. Column chromatography was performed on an
Argonaut Flashmaster using prepacked Isolute silica gel columns
(Biotage). Melting points were determined using an Electrotherm
capillary melting point apparatus and are uncorrected. NMR spec-
tra chemical shifts are reported in parts per million downfield
from TMS and referenced either to TMS internal standard for
deuterochloroform or deuteroacetone solvent peak. All compounds
gave satisfactory elemental analyses, 0.4%, (Desert Analytics,
Tucson, AZ) unless otherwise stated. 1H spectra and 13C spectra
and elemental analyses are provided in the Supporting
Information.
2.2.5. (17a,20E)-21-[2-methylphenyl]-19-norpregna-1,3,5(10),20-
tetraene-3,17-diol 4a
Method B was used and the reaction conditions were performed
on a 0.626 mmol scale. Yield = 205 mg, 74%, of the acetate. Hydro-
lysis at the 0.247 mM scale gave the product. Yield 92 mg, 96%
yield. Mp 193–195 °C, C27H32O2 0.25ÁH2O, Anal: C, 82.51; H, 8.33,
Found: C, 82.32; H, 8.46 1H NMR (d6-acetone); 13C NMR (d6-
acetone).
2.2.6. (17a,20E)-21-[3-methylphenyl]-19-norpregna-1,3,5(10),
20-tetraene-3,17-diol 4b
Method B was used and the reaction conditions were performed
on a 0.50 mmol scale. Yield = 132 mg, 64.4%. Mp 177–178 °C.
C
27H32O2 0.75ÁH2O, Anal: C, 80.66; H, 8.40, Found: C, 80.93; H,
8.26 1H NMR (d6-acetone); 13C NMR (d6-acetone).
2.2. Synthesis of substituted phenylvinyl estradiols
2.2.7. (17a,20E)-21-[4-methylphenyl]-19-norpregna-1,3,5(10),
20-tetraene-3,17-diol 4c
2.2.1. General procedure for the Stille coupling with 17a-E-tri-n-
butylstannylvinyl estradiol and the substituted phenyl/xylyl iodides.
Method A
Method A was used and the reaction conditions were performed
on
27H32O2 1.5ÁH2O, Anal: C, 78.04; H, 8.49, Found: C, 78.23; H,
8.56. 1H NMR (d6-acetone); 13C NMR (d6-acetone).
a 0.34 mmol scale. Yield = 104 mg, 79%. Mp 195–197 °C.
To a reaction tube containing (17
19-norpregna-1,3,5(10)20-tetraene-3,17b-diol, 2a, were added
few crystals of 2,6 di-tert-butyl-4-methylphenol and the
a-20E)-21-(tri-n-butylstannyl)-
C
a
substituted phenyl/xylyl iodide. The tube was dried under
vacuum for 24 h, then exchanged with argon at least four times.
Tetrakis(triphenylphosphine) palladium (0) (0.024 g, 0.02 mmol)
and dried, degassed toluene (5 mL) were added and the reaction
was heated at 110 °C for 6–18 h. After cooling to room temperature,
the reactuib mixture was transferred to a flask with ethyl acetate
(50 mL), activated charcoal was added, the mixture heated to
boiling, and then filtered through a Celite pad. To the filtrate
containing the substituted phenyl vinyl estradiol derivative, fluorsil
(4–8 g) was added and then mixture was evaporated to dryness.
Hexane was then added to the slurry and the mixture was again
evaporated to dryness. The substituted phenyl vinyl estradiol was
isolated using flash chromatography and characterized by 1H and
13C NMR, elemental analysis.
2.2.8. (17a,20E)-21-[2,3-dimethylphenyl]-19-norpregna-1,3,5(10),
20-tetraene-3,17-diol 5a
Method A was used and the reaction conditions were performed
on
28H34O2 2ÁH2O, Anal: C, 76.68; H, 8.73, Found: C, 76.04; H, 8.28
1H NMR (d6-acetone); 13C NMR (d6-acetone).
a 0.34 mmol scale. Yield = 75 mg, 54%. Mp 185–187 °C.
C
2.2.9. (17a,20E)-21-[2,4-dimethylphenyl]-19-norpregna-1,3,5(10),
20-tetraene-3,17b-diol 5b
Method B was used and the reaction conditions were performed
on a 0.64 mmol scale. Yield = 129 mg, yield 45%, of the acetate.
Hydrolysis of acetate (0.176 mM) gave 66 mg, Yield 94%, Mp
183–184 °C. C28H34O2 2ÁH2O, Anal: C, 81.71; H, 8.57, Found: C,
81.54; H, 8.76, 1H NMR (d6-acetone); 13C NMR (d6-acetone).
2.2.2. General procedure for the Stille coupling with 17-E-tri-n-
butylstannylvinyl estradiol 3 acetate and the selected substituted
phenyl iodide. Method B
2.2.10. (17a,20E)-21-[2,5-dimethylphenyl]-19-norpregna-
1,3,5(10),20-tetraene-3,17b-diol 5c
Method A was used and the reaction conditions were performed
The procedure for coupling 17a-E-tri-n-butylstannylvinyl
on
28H34O2 2ÁH2O, Anal: C, 76.68; H, 8.76, Found: C, 75.85; H, 8.06
1H NMR (d6-acetone); 13C NMR (d6-acetone).
a 0.34 mmol scale. Yield = 98 mg, 72%. Mp 193–194 °C.
estradiol-3-acetate 2b was the same as given above. The substi-
tuted phenyl vinyl estradiol-3-acetate was isolated, characterized
and hydrolyzed using the method given below.
C
2.2.3. Hydrolysis of the acetate
2.2.11. (17a,20E)-21-[2,6-dimethylphenyl]-19-norpregna-1,3,5(10),
To a methanolic solution of 17a-(substituted phenyl)vinyl
20-tetraene-3,17b-diol 5d
estradiol acetate, 10 N sodium hydroxide (0.1 mL) was added and
the reaction solution was stirred for 5–10 min at room tempera-
ture. The reaction solution was neutralized with 50 mL of
ammonium acetate (10%), extracted with ethyl acetate
(3 Â 100 mL), dried over anhydrous magnesium sulfate, filtered
Method B was used and the reaction conditions were performed
a 0.64 mmol scale. Yield = 135 mg, 52%, of the acetate.
Hydrolysis of the acetate (0.265 mM) gave 56 mg, 52% yield. Mp
163–165 °C. C28H34O2 0.75ÁH2O, Anal: C, 80.83; H, 8.60, Found: C,
80.93; H, 8.30 1H NMR (d6-acetone)
on

R.N. Hanson et al. / Steroids 77 (2012) 471–476
473
2.2.12. (17a,20E)-21-[3,4-dimethylphenyl]-19-norpregna-
Builder module in Insight II. The complex of ERa-HBD monomer
1,3,5(10),20-tetraene-3,17b-diol 5e
and estradiol bound within the binding cavity was minimized
using the molecular mechanics method with restraints applied to
the backbone atoms of the protein (Discover_3 module, CVFF force
field, dielectric constant 2.0, conjugate gradient minimization
10,000 steps or until 0.001 final convergence). All ligands used in
this study were constructed using the Builder module from Insight
II. Potentials for each atom were assigned automatically or manu-
ally when necessary. Each ligand was optimized using the molecu-
lar mechanics method as done with the receptor. Partial charges
for each atom were calculated using the Mopac program from
the Ampac/Mopac module in the Insight II package. In addition, li-
gands were further optimized using semi-empirical method (calcu-
lation method:PM3; calculation type:optimization; optimizer
type: native).
Method B was used and the reaction conditions were performed
on
28H34O2 2ÁH2O, Anal: C, 81.71; H, 8.57, Found: C, 81.54; H, 8.76
1H NMR (d6-acetone); 13C NMR (d6-acetone).
a 0.55 mmol scale. Yield = 125 mg, 50%. Mp 135–138 °C.
C
2.2.13. (17a,20E)-21-[3,5-dimethylphenyl]-19-norpregna-
1,3,5(10),20-tetraene-3,17b-diol 5f
Method B was used and the reaction conditions were performed
on
28H34O2Á 0.5ÁH2O; Anal: C, 81.71; H, 8.57; Found: C, 81.49; H,
8.84, 1H NMR (d6-acetone); 13C NMR (d6-acetone).
a 0.55 mmol scale. Yield = 185 mg, 80%. Mp 180–182 °C.
C
2.3. Competitive binding to human LBD-ER
a
The Affinity program within the Docking module in InsightII
was used to perform the docking studies of the ligands with the
Binding affinities of the mono- and di-methylphenyl vinyl
estradiol derivatives relative to E2 were performed in incubations
ERa-HBD. This module includes elements from Monte Carlo, simu-
with the LBD of ER
a
in lysates of Escherichia coli in which the
lated annealing and minimization for automatically docking and
finding the best structures of the ligand complexed to the receptor
based on the energy of the ligand–receptor complex. The ligand
was superimposed on the estradiol molecule (A-ring over A-ring)
and the estradiol was then deleted. The complex was subjected
to energy minimization to obtain a starting structure in which
bad steric contacts are removed and internal energies are relieved.
During the docking procedure both the ligand and the protein res-
idues within the ligand binding cavity (amino acids within 15 ang-
stroms of the ligand as well as all amino acids in helix-12, loops
11–12, 1–3, 6–7) were allowed to flex while the backbone atoms
and the rest of the protein were restrained in their original posi-
tions. In addition, the phenylvinyl side chain of the ligand was ro-
tated with maximum of 180° increments in order to more fully
explore the potential binding modes of the conformational choices
of the ligand. After each docking procedure, structures within
10 kcal/mol of the lowest energy structure and RMS distance of
more than 0.125 Å were selected and used in simulated annealing
studies. At the beginning of each run, the ligand–receptor complex
was minimized over 5000 steps or until 0.001 final convergence.
Then each structure was heated from 300 to 500 K over 5000 fs
and allowed to equilibrate for and additional 5000 fs. Each struc-
ture was allowed to cool to 300 K in 20 stages with 10 K decre-
ments for each stage and 100 fs long equilibration periods for
each stage. The structure at the end of the final stage was recorded
in an archive file and further minimized 200 steps. Each of the
dynamics and simulated annealing cycles was repeated 10 times.
During these calculations additional restraints were applied to
amino acids facing the outer surface of the protein. All calculations
involving docking and refinement of generated structures were
performed with a dielectric constant = 2.0.
LBD of human ER
a
(M250–V595) [16] is expressed as described
[17] The assay was performed overnight in phosphate buffered sal-
ine + 1 mM EDTA at room temperature. The competition for bind-
ing of [3H] E2 to the LBD of the E2-derivatives in comparison to
E2, relative binding affinity (RBA) was determined over a range of
concentrations from 10À12 to 10À6 M. After incubation, the media
is aspirated, the plates are washed three times and the receptor
bound radioactivity absorbed to the plates is extracted with meth-
anol and counted. The results, as RBAs compared to E2, of all recep-
tor studies are from three experiments performed in duplicate.
RBAs represent the ratio of the EC50 of E2 to that of the steroid ana-
log  100 using the curve fitting program Prism to determine the
EC50
.
2.4. Estrogenic potency in Ishikawa cells
The estrogenic potency of the E2-analogs was determined in an
estrogen bioassay, the induction of AlkP in human endometrial
adenocarcinoma cells (Ishikawa) grown in 96-well microtiter
plates as we have previously described [18]. The cells are grown
in phenol red free medium with estrogen depleted (charcoal
stripped) bovine serum in the presence or absence of varying
amounts of the steroids, across a dose range of at least six orders
of magnitude. After 3 days, the cells are washed, frozen and
thawed, and then incubated with 5 mM p-nitrophenyl phosphate,
a chromogenic substrate for the AlkP enzyme, at pH 9.8. To ensure
linear enzymatic analysis, the plates are monitored kinetically for
the production of p-nitrophenol at 405 nm. Each compound was
analyzed in at least three separate experiments performed in
duplicate. The RSA (RSA = ratio of 1/EC50 of the steroid analog to
that of E2 Â 100) was determined using the curve fitting program
Prism.
Results of the docking studies were analyzed using a combina-
tion of modules: analysis, Discover_3, Docking and Viewer. Each
structure generated during the docking, simulated annealing and
dynamics runs was analyzed in terms of binding energy, ligand en-
2.5. Molecular modeling and dynamics
ergy and protein energy. Values of the binding energy
DEbinding
We initially evaluated the conformations of our ligands using
the Builder module from Insight II [19]. Potentials for each atom
were assigned automatically or manually, when necessary. Low
energy conformations were generated using the molecular
mechanics method (Discover program, 100 steps, 0.001 final
convergence) and compared to solution conformations determined
were calculated as the difference between the potential energy of
the complex (Ecomplex) and the potential energy of the ligand
(Eligand) and receptor (Ereceptor) [20,21]. Binding energy calculations
were performed using the Energy Analysis macro within the
Discover_3 module.
by NMR. The ERa-LBD used in our study was obtained from our
crystal structure of E-(2-trifluoromethylphenyl)vinyl estradiol [9]
was selected for the docking and molecular dynamics studies. All
water molecules present in the crystal structure were deleted.
The monomer contains all the amino acid residues between ASN
304 and HIS 550. All manipulations were performed using the
3. Results and discussion
The target compounds selected for this study were three
E-mono- and six di-methylphenyl vinyl estradiol derivatives
4b–d and 5a–f, as well as one mono-trifluoromethyl and two

474
R.N. Hanson et al. / Steroids 77 (2012) 471–476
Table 1
bis(trifluoromethyl)phenyl estradiol derivatives 4d and 5g,h. The
Stille coupling procedure, developed in our previous studies, was
used (Scheme 1). Hydrostannation of ethynyl estradiol or ethynyl
estradiol 3-acetate gave the E-tri-n-butylstannyl intermediates
2a,b as the major products (70% isolated yield), readily separable
from the Z-isomers (20% isolated yield), with separation easier
with the acetate 2b derivative than with the free phenol 2a. Cou-
pling with the appropriate iodotoluene or iodoxylene isomer
(Method A or B), followed by flash chromatography on silica gel,
gave the products in good (45–80%) overall yields. The coupling
reactions were generally complete within 2 h, except with the
2,6-dimethyl derivative 5d for which 16 h was necessary. A change
from triphenylphosphine to tri-tert-butylphosphine as the
ligand improved the coupling reaction [15]. Synthesis of the
trifluoromethyl analogs 4d, 5g and 5h proceeded in a similar man-
ner. The E-stereochemistry was established by 1H NMR where the
coupling constant for the vinylic protons was J = 16–18 Hz.
Relative binding and stimulatory activity for estradiol and derivatives 3, 4a–d, 5a–h.
Compound Substitution
ER
a
-LBD (RBA)a Ishikawa AlkP (RSA)b
Estradiol
3
4a
4b
4c
4d
5a
5b
5c
5d
5e
5f
5g
None
X = H, Y = H
X = 2-CH3, Y = H
X = 3-CH3, Y = H
X = 4-CH3, Y = H
X = 2-CF3, Y = H
100
100
9.4 2.5
10.3 2.9
14.0 0.8
6.3 1.5
7.3 0.5
80.2 16.5
31.8
7
5.0 1.4
5.5 0.7
The compounds were evaluated for their relative binding affin-
ity (RBA) using the human ERa-LBD [16,17]. Estrogenic potency
was determined using relative stimulatory activity (RSA) of an
estrogen responsive gene, alkaline phosphatase, in the human
endometrial adenocarcinoma (Ishikawa) cell line [18]. The
100
8.4
9
4
X = 2-CH3, Y = 3-CH3 10.5 1.7
X = 2-CH3, Y = 4-CH3 20.5 1.9
X = 2-CH3, Y = 5-CH3 37.3 4.3
11.0 3.7
12.3 4.3
2.5 0.7
X = 2-CH3, Y = 6-CH3
X = 3-CH3, Y = 4-CH3
X = 3-CH3, Y = 5-CH3
X = 2-CF3, Y = 5CF3
X = 3-CF3, Y = 5CF3
9.3 0.5
6.0 0.8
5.0 0.8
26.3 1.5
9.0 1.8
1.5
2
ERa-LBD binding reflects the initial interaction with the ER while
3.5 0.7
29.7 3.4
3.5 0.7
the alkaline phosphatase assay permits us to compare the relation-
ship between binding and efficacy (ER responsiveness). The results
for the binding and stimulation assays are shown in Table 1. All of
5h
a
RBA = 100 Â [E]/[C] where [E] is the concentration of unlabeled estradiol nec-
essary to reduce the specific binding of tritiated estradiol to the ER -HBD by
a
the new compounds demonstrated significant binding to the ERa-
50% and [C] is the concentration of the competitive ligand necessary to reduce
specific binding by 50%. The RBA of estradiol is 100% at 25 °C. Curves for ligand
and estradiol had correlation coefficients >95%.
RSA, relative stimulatory activity compared to E2 = 100%, in stimulation of
alkaline phosphatase (AlkP) in the Ishikawa cell line. EC50 for E2 = 0.9 0.2 nM.
LBD and were agonists in the AlkP assay system. Introduction of a
single methyl group onto the phenylvinyl moiety generated
derivatives with higher (4a, RBA = 14.0%) as well as lower (4b,
RBA = 6.3%; 4c, RBA = 7.3%) affinity than the parent compound 3
(RBA = 10.3%). This trend is similar to that previously observed
for the mono-trifluoromethylated series [7]. Introduction of a sec-
ond methyl group onto the phenylvinyl moiety expanded the range
of RBA values for the series and identified the influence of the
second substituent. For example, 2-(ortho)-substitution was
still optimal but the highest affinity was observed for those com-
pounds in which the second methyl group was either in the
5-(meta)- (RBA = 37) or 4-(para)- position (RBA = 20.5). 2,3- and
2,6-Di-methyl substitution gave RBA values approximately equal
to that of the unsubstituted parent compound (RBAs = 10.5 and
9.3 vs 10.3) while 3,4- and 3,5- di-methyl substitution gave prod-
ucts that were comparable to the 3- and 4-mono-methyl
derivatives.
b
Ishikawa cell line. The parent unsubstituted phenylvinyl estradiol
3 possessed an RSA value essentially identical to its RBA value
(9.4% vs 10.3%), suggesting that the relationship between receptor
binding and biological potency of the ligand–receptor complex is
parallel to that formed with estradiol. This relationship between
RBA and RSA values held with some but not all of the compounds
evaluated in this study. In particular, the 2-methylphenylvinyl
estradiol 4a demonstrated
a more potent stimulatory effect
(RSA = 31.8) than binding affinity (RBA = 14.0). This relationship
was reversed for the 2,4-, the 2,5- and 2,6-dimethyl derivatives
for which the RSA values were 1/3 to 1/2 those of the RBA values.
Although a complete series of trifluoromethylphenyl vinyl
estradiols was not prepared, we synthesized and evaluated three
The second component of the study involved the ability of the
ligands to stimulate the induction of alkaline phosphatase in the
X
SnBu3
X
Y
OH
OH
OH
Y
i
I
ii
HO
RO
RO
a. R = H; b.R = CH3CO
1a,b
2a,b
3
X = Y = H 4a X = 2-CH3, Y = H 5a X = 2-CH3, Y = 3-CH3
4b X = 3-CH3, Y = H 5b X = 2-CH3, Y = 4-CH3
4c X = 4-CH3, Y = H 5c X = 2-CH3, Y = 5-CH3
4d X = 2-CF3, Y = H 5d X = 2-CH3, Y = 6-CH3
5e X = 3-CH3, Y = 4-CH3
Reagents and Conditions.
i. Bu3SnH; Et3B; THF; 60oC
5f
X = 3-CH3, Y = 5-CH3
5g X = 2-CF3, Y = 5-CF3
5h X = 3-CF3, Y = 5-CF3
ii. [(C6H5)3P]4Pd(0); I-Ar-X,Y; Toluene, 100oC
Scheme 1. Synthesis of mono- and di-substituted phenylvinyl estradiols.

R.N. Hanson et al. / Steroids 77 (2012) 471–476
475
analogs, the 2-trifluoromethyl, 2,5- and 3,5-bis(trifluoromethyl)
phenyl vinyl estradiols 4d,5g,5h for comparison purposes. In the
binding assays, the mono-trifluoromethylated derivative 4d
had a higher RBA value than 4a (80.2 vs 14.0) while the 2,5-
bis(trifluoromethyl) derivative 5g had a lower RBA values than the
corresponding analog 5c (26.3 vs 37.3). The 3,5-bis(trifluoromethyl)
derivative 5h also had a slightly higher affinity than the correspond-
ing analog 5f (9.0 vs 5.0). Disparities were also observed in the
Ishikawa assay. The 2-trifluoromethyl derivative was very potent
(RSA = 100) and more active than the methyl analog (RSA = 31.8)
while the 2,5-bis(trifluoromethyl) derivative 5g was somewhat
weaker (RSA = 29.7), but still more potent than the 2,5-di-methyl
analog (RSA = 12.3). There was no significant difference between
the 3,5-di-methyl and 3,5-bis(trifluoromethyl) analogs (RSA = 3.5
for each).
Although there may be alternate explanations for the observed
differences between ER binding and biological activity, for exam-
ple, cell penetration or intracellular metabolism, it appears more
likely that the presence of the second substituent affects the ability
of the ligand–receptor complex to readily assume the agonist
conformation, thereby leading to altered in vitro potency. The
presence of the second substituent clearly does not prevent
the complex from forming an agonist conformation as evidenced
by the observation that all of the compounds were full agonists
in this assay, however, it may influence how easily this
derivative 4a is likely due to enhanced dipole–dipole interactions
between the fluoro-group and the complementary peptide
backbone [9].
Based upon predictions that ligand–protein remodeling may be
involved with the biological response, we undertook to correlate
measured RBA and RSA values with calculated binding energies,
including total, ligand and protein energy components (see Sup-
porting Information). Given the small number of compounds in-
volved in this study it may not be possible to generate definitive
conclusions, however, distinct trends could be noted. When RBA
values were initially plotted against the total binding energy for
the complexes, there was a poor correlation for the di-methyl
substituted ligands, however, inclusion of a term for protein energy
(0.9 protein energy), reflecting the energy price paid for remodel-
ing of the receptor, yielded a plot with a clear correlation
(R2 = 0.804). Plotting the RSA values, which reflect the generation
of a physiologically competent complex, against the protein energy
also gave very clear correlation (R2 = 0.811).
4. Conclusions
The results of this study suggest that relatively small changes in
structure of the steroidal estrogens produce significant variations
in the response of the target ERa-LBD. The introduction of the sec-
ond methyl group provides a generally consistent change in phys-
icochemical properties, which may affect pharmacokinetic
parameters such as rates of metabolism and clearance. However,
at the molecular level, where pharmacodynamic properties are
generated, small changes in protein architecture can influence
the orientation of functional groups involved with recruitment of
activation factors or conformational equilibria. In order to accom-
modate the second methyl substituent on the phenyl ring, the ami-
conformation is obtained. Using the crystal structure of 17
E-(2-trifluoromethylphenyl)vinyl estradiol 4d complexed with
ER -LBD [9] we evaluated the low energy conformations of the
a-
a
corresponding di-methylphenyl vinyl estradiol complexes. The
binding modes for the compounds, superimposed on each other,
are shown in Fig. 1.
As can be seen, the general conformation of the ligand within the
binding pocket is the essentially the same for each compound, with
minor torsional differences around the C20–C21 bond resulting from
the need to accommodate the additional methyl group within the
receptor. Subtle movements of the surrounding peptide side chains
are required to provide adequate space for the methyl groups,
resulting in perturbations of the conformations of the amino
acids associated with ligand binding. Enhanced affinity for the
2-trifluoromethyl compound 4d compared to the 2-methyl
no acids within the 17a-region of the ER ligand binding pocket
must undergo differential remolding [9]. This process can lead to
conformations which possess higher affinity for the ligand, but
not necessarily more favorable orientations for subsequent coreg-
ulatory peptide recruitment (efficacy). Our results suggest that
substituents at the 2 and/or 5-positions of the phenylvinyl group
lead to compounds that retain high binding affinity yet do not
stimulate the receptor as effectively. Such compounds may func-
tion as ‘‘impeded estrogens’’ and studies to explore that possibility
are in progress.
Acknowledgments
We gratefully acknowledge Dr. Roger Krautz for his assistance
with the 500 MHz NMR spectrometry. We are grateful for support
of this research through grants from the National Institutes of
Health [PHS 1R01 CA81049 (R.N.H.) and PHS 1R01 CA 37799 and
R21MH082252 (R.B.H.)], and the US Army Breast Cancer Research
Program [DAMD 17-99-1-9333 and 17-00-1-00384 (R.N.H.)].
Molecular modeling was performed on instruments supported in
part by a grant from the National Science Foundation [CHE-
9974642].
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.steroids.2012.01.003.
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Fig. 1. Superimposition of 17
a-(di-methylphenyl)vinyl estradiol isomers 5a–f
within ligand binding pocket (LBP) of ER
a
. As with the mono-substituted phenyl
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