Synthesis of benzoylbenzamide derivatives of 17α-E-vinyl estradiol and evaluation as ligands for the estrogen receptor-α ligand binding domain

Article abstract of DOI:10.1016/j.steroids.2019.02.003

A series consisting of substituted benzoylbenzamide derivatives of 17α-E-vinyl estradiol 6a-i and 7a-d was prepared in good overall yields from the corresponding novel iodinated benzoylbenzamide precursors using Pd(0)-catalyzed Stille coupling. Biological

Full text of DOI:10.1016/j.steroids.2019.02.003

Accepted Manuscript
Synthesis of Benzoylbenzamide Derivatives of 17α-E-Vinyl Estradiol and Eval-
uation as Ligands for the Estrogen Receptor-α Ligand Binding Domain
Robert N. Hanson, Emmett McCaskill, Edward Hua, Pakamas
Tongcharoensirikul, Robert Dilis, Jessa L. Silver, Timothy A. Coulther, Mary
Jo Ondrechen, David Labaree, Richard B. Hochberg
PII:
S0039-128X(19)30020-0
https://doi.org/10.1016/j.steroids.2019.02.003
STE 8370
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
29 October 2018
10 January 2019
4 February 2019
Please cite this article as: Hanson, R.N., McCaskill, E., Hua, E., Tongcharoensirikul, P., Dilis, R., Silver, J.L.,
Coulther, T.A., Ondrechen, M.J., Labaree, D., Hochberg, R.B., Synthesis of Benzoylbenzamide Derivatives of
17α-E-Vinyl Estradiol and Evaluation as Ligands for the Estrogen Receptor-α Ligand Binding Domain, Steroids
(2019), doi: https://doi.org/10.1016/j.steroids.2019.02.003
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Synthesis of Benzoylbenzamide Derivatives of 17α-E-Vinyl Estradiol and Evaluation as
Ligands for the Estrogen Receptor-α Ligand Binding Domain
Robert N. Hansona*, Emmett McCaskilla, Edward Huaa, Pakamas Tongcharoensirikula, Robert Dilisa,Jessa L.
Silvera, Timothy A. Coulthera, Mary Jo Ondrechena, David Labareeb and Richard B. Hochbergb
aDepartment of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
bDepartment of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520
Author to whom correspondence should be addressed:
Robert N Hanson, PhD
Matthews Distinguished University Professor
Department of Chemistry and Chemical Biology
Northeastern University
360 Huntington Avenue
Boston, MA 02115-5000
Tel. 617-373-3313
Fax. 617-373-8795
e-mail. r.hanson@neu.edu

Abstract:
A series consisting of substituted benzoylbenzamide derivatives of 17α-E-vinyl estradiol 6a-i and 7a-d was
prepared in good overall yields from the corresponding novel iodinated benzoylbenzamide precursors using
Pd(0)-catalyzed Stille coupling. Biological evaluation using competitive binding assays indicated that all
compounds were effective ligands for the ERα- and ERβ-LBD (RBA = 0.5-10.0% of estradiol). Most of the
compounds expressed lower stimulatory (agonist) potency (RSA <0.2-0.5%) compared to their binding affinity,
however, the meta-substituted isomer 6h demonstrated a level of efficacy (RSA = 5.7%) comparable to its
affinity (RBA = 9.5%). Docking studies of 6b,6h, and 6i with the 2YAT crystal structure suggested that higher
affinity and efficacy of 6h are due to an effective set of interactions with exposed receptor sidechains not
observed with the ortho- and para- isomers. In this binding model, the terminal ring of the ligand is exposed to
the solvent space, which would explain both the small variation in RBA values and the narrow SAR for the
diverse structural features.
Keywords:
Estradiol derivatives, Stille coupling, relative binding affinity, relative stimulatory activity, docking studies

1. Introduction
The estrogen receptor (ER) is a member of the nuclear receptor (NR) superfamily, a group of receptor
transcription factors that mediate a wide variety of physiological and developmental responses.[1,2] Because
inappropriate or over-expression of ER is associated with endocrine disorders, such as breast and endometrial
cancer, and osteoporosis, modulation of these ER-regulated responses is a critical clinical objective.[3-5] A
number of reviews have highlighted the structure of the ER, including its subtypes, and the general mechanism
by which binding of the endogenous ligand initiates the events leading to transcription.[6-10] While many
individual steps are involved in the overall estrogenic process, the initial binding of ligand to the unliganded
(apo)receptor to form a stable complex remains a key step. Because subsequent biological responses are
influenced by the receptor conformation induced by this interaction, research efforts to characterize that initial
step remain important for understanding how the subsequent biological effects are generated.
As part of our program to develop steroidal probes of the ligand binding pocket (LBP) of the estrogen
receptor (ER), we previously prepared and evaluated several series of E-17α-( ortho-, meta- and para-mono-
substituted phenyl) vinyl estradiols.[11-14] More recently we have examined the binding pocket further using
17α-heteroaryl vinyl estradiols to examine the effects of electronic character independent of steric factors and
17α-(disubstituted phenyl) vinyl estradiols to probe multiple small steric interactions.[15,16]
[Figure 1]
Figure 1. Representative 17α-E-(Substituted Phenyl)vinyl Estradiols [11-16]
Those series of compounds, readily accessible via Stille coupling of the requisite aryl iodide/bromide
and the E-stannylvinyl estradiol, displayed a range of relative binding affinities (RBAs) compared to estradiol,
ranging from <1% to >200%. Structure activity relationship (SAR) studies indicated a clear preference for
ortho- substitution with meta- and para-substituents inducing poorer receptor binding properties. Electron
withdrawing groups, such as fluoro- and trifluoromethyl- appeared to provide better RBA values than electron
donating groups. The phenyl derivative possessing the ortho-trifluoromethyl substituent was of particular
interest because it displayed the highest binding affinity and greatest in vivo estrogenic activity. Molecular
modeling studies, coupled with x-ray crystallography, indicated that the plasticity of the ER-ligand binding
domain protein allowed the residues within the ligand binding pocket (LBP) to accommodate the modest steric
demands imposed by the 17α-(substituted-phenyl)vinyl substituent.[17] However, replacement of the phenyl
ring by either electron rich (thienyl) or electron deficient (pyridyl/pyrimidinyl) heterocycles was generally
detrimental to ER-LBD binding.

Because the initially evaluated compounds used relatively small functional groups, little information was
available to evaluate more significant steric effects. Recent studies using a 17α-(substituted-pyridyl)ethynyl
estradiol suggested that this region of the receptor may exhibit significant steric tolerance.[18] An X-ray crystal
structure of the Eu-chelated complex with ERα-LBD indicated that the protein can accommodate the sterically
demanding substituent, however, binding and functional data for this compound as an ER ligand were not
provided. The objective of this current study was to further explore the limits to which the ER-LBD could
undergo adaptation by using electron donating and electron withdrawing groups both at the inner phenyl
substitutent and on the terminal group. Herein we describe the synthesis and evaluation of a series of
(substituted)benzyl and benzoyl derivatives of E-phenylvinyl estradiol as ligands for the estrogen receptor
ligand binding domain (ER-LBD).
2. Experimental
General Methods. All solvents were purchased from Fisher Scientific. Toluene was distilled from
sodium/benzophenone. All reagents, including substituted benzylamines 1a-e and substituted benzoyl chlorides
2a-i were purchased from Sigma-Aldrich. 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 phosphomolybdic acid reagent Separations were performed using automated flash chromatography
(Argonaut FlashMaster) or packed column chromatography with Sorbent Technologies silica gel particle size
32-63 μm and 60 Å pore size. Liquid chromatography-mass spectroscopy (LC-MS) was performed using
Alliance HT -LCT Premier 2489, Waters® instrument equipped with time-of-flight (TOF) MS module. All
compounds were >95% by UV analysis. Melting points were determined using an Electrotherm capillary
melting point apparatus and are uncorrected. NMR spectra chemical shifts are reported in parts per million
downfield from TMS and referenced either to TMS internal standard for deuterochloroform or
hexadeuteroacetone solvent peak. All compounds gave satisfactory elemental analyses,  0.4%, (Desert
Analytics, Tucson, AZ) unless otherwise stated. Mass spectra were determined with LC-TOF-MS-ES+.
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13
Elemental analyses and mass spectral data for all compounds as well as representative H-, and C-spectra for
precursors and final products are provided in the Appendix (Supporting Information).
2.1.
General procedure for preparation of iodinated N-benzylbenzamide derivatives 3a-i and
4a-d.
2-/3-/4-Iodobenzyl chloride (1a-c) was dissolved in chloroform containing 1.1 equivalents of triethylamine.
A solution of substituted benzoyl chloride (2a-g) in chloroform was added dropwise with stirring over 15
min. The precipitate that formed was removed by filtration and the resulting filtrate was washed sequentially
with water, 1N hydrochloric acid, 5% sodium carbonate and brine. The organic phase was dried over
magnesium sulfate (anhyd.), filtered, evaporated to dryness and the resultant 2-/3/-4-substituted- N-(2/3/4-
iodo)- benzylbenzamide 3a-i was recrystallized from alcohol.
To obtain the corresponding 2-/4-iodo- N-substituted benzyl benzamide derivatives, the same procedure was
followed, except that the 2-/4-iodobenzoyl chlorides 2h,2i and isomeric 2-/4-methoxybenzylamines 1d,1e
were used.
1
All new compounds 3a-i and 4a-d were characterized by H, 13C-NMR spectrometry, and elemental
analysis or LC-MS.
2.2.
General procedure for the Stille coupling with 17-E-Tri-n-butylstannylvinyl estradiol and
the N-(iodobenzyl) substituted benzamides (3a-i) and N-(substituted benzyl) iodobenzamides (4a-

d). Synthesis of N-[ 17α-(E)-vinyl estradiol – benzyl] benzamides 6a-i and N-benzyl-[-[ 17α-(E)-
vinyl estradiol] benzamides 7a-d
To a reaction tube containing (17α -20E)- 21-(tri-n-butylstannyl)-19-norpregna-1,3,5(10)20-tetraene-3,17-
diol, 5 [11], a few crystals of 2,6 di-tert-butyl-4-methylphenol and the iodobenzoyl/iodobenzyl derivatives 3
or 4 were dried under vacuum for 24 hours 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
heated at 110˚C for 6 – 18 hours. On cooling to ambient temperature, the mixture was transferred to a flask
with ethyl acetate (50mL), activated charcoal added, heated to boiling, and filtered through a celite pad. To
the filtrate containing the (substituted phenyl) vinyl estradiol derivative, fluorosil (4 – 8 g.) was added and
evaporated to dryness. Hexane was then added to the slurry and again evaporated to dryness. The mixture
containing the substituted phenyl vinyl estradiol was purified using automated flash chromatography,
1
13
isolated and characterized by H-and C- NMR spectrometry, and LC- mass spectrometry and elemental
analysis.
2.3.
Competitive Binding to Human ERα- and ERβ-LBD.
Binding affinities of the substituted benzyl and benzoyl vinyl estradiol derivatives relative to E2 were
performed in incubations with the LBD of ERα in lysates of Escherichia coli in which the LBD of human
ERα (M250–V595) is expressed as described.[19,20] The assay was performed overnight in phosphate
buffered saline + 1 mM EDTA at room temperature. The competition for binding 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 10-12 to 10-6 M. After incubation, the media is aspirated, the plates are washed 3 times
and the receptor bound radioactivity absorbed to the plates is extracted with methanol and counted. The
results, as RBAs compared to E2, of all receptor studies are from 3 experiments performed in duplicate.
RBAs represent the ratio of the EC50 of E2 to that of the steroid analog x 100 using the curve fitting program
Prism to determine the EC50.
2.4.
Molecular Modeling and Docking Studies
Structure files for the compounds were created in ChemDraw Professional 16.0. Low energy conformations
for compounds 6b, 6h, and 6i are shown in Supporting Information. [21] Energy minimization was
performed on the compound structures using the OPLS3 force field. [22] The structure of the ER was
downloaded from the Protein Data Bank (PDB), [23] using the structure of ERα complexed with an
estradiol derivative, estradiol-pyridine tetra acetate europium (III), PDB code 2YAT.[18] The structure was
prepared for docking with the Protein Preparation Wizard in the Schrödinger suite of programs. Protonation
states were set at a pH= 7.5 and unresolved residues from the crystal structures were modeled using Prime,
comprising residues 331-340, 414-422, and 462-463. This ER structure is a triple mutant,
C381S/C417S/C530S, constructed for ease of expression. Therefore the mutations were reverted back to the
wild-type sequence followed by energy minimization inside the Schrödinger Suite. Docking was performed
using the Schrödinger Induced Fit Docking (IFD) protocol, consisting of GLIDE docking with the rigid
protein followed by energy minimization of the side chain orientations with Prime of those residues within
7.5 Å of the ligand. In the first Glide docking round, the ring containing carbons 1,2,3,4,5, and 10, and the
hydroxyl at C3 of the estradiol core were restricted to an RMSD of 1.0 Å from the crystal pose. A final
round of GLIDE docking was then performed towards the optimized receptor structures, from which final
Induced Fit Docking scores for each ligand-receptor complex were calculated.

3. Results and Discussion
Our initial studies in the 17α-(E)-phenyl vinyl estradiol series, as well as other studies with 17α-substituted
phenyl ethynyl estradiols suggested steric tolerance within that region of the ER ligand binding domain. [11-16]
Target compounds in this series were derivatives of E-phenylvinyl estradiol in which the phenyl ring was
substituted at the ortho,-meta or para-position with an additional aromatic substituent. The linker between the
two aromatic groups would be either electron donating (aminomethyl) or electron withdrawing
(aminocarbonyl), and the terminal aromatic group would also be substituted with either electron donating
(methoxy) or electron withdrawing (trifluoromethyl) groups. Such substitution patterns would provide an initial
assessment of steric and/or electronic factors in receptor binding.
Our synthetic strategy (Scheme 1) was adapted from our previous studies. [11-14] Stannylvinyl estradiol
5 was readily prepared via hydrostannation of ethynyl estradiol using triethyl borane as the radical initiator. The
iodoarene coupling partners 3a-i and 4a-d were prepared in excellent yields by either (methoxy/trifluoromethyl)
benzoylation
of
the
4-/3-/2-iodobenzylamines
1a-c
or
4-/2-iodobenzoylation
of
the
(methoxy/trifluoromethyl)benzylamines. Notably, most of these precursors had generally not been previously
prepared and characterized. [24, 25] Both procedures gave products that were readily purified by
chromatography or recrystallization and characterized. Coupling of stannylvinyl estradiol 5 and the iodinated N-
(substituted benzyl)benzamides 3a-i and 4a-d using the Stille coupling method gave the desired products 6a-i
and 7a-d in modest (13-62%) isolated yields, after flash chromatographic purification. Although yields were
poorer than observed with the simpler aryl halides, sufficient material was obtained to undertake full
1
13
characterization and evaluation. H- and C-NMR spectra were consistent with the proposed structures, but it
was difficult to assign specific signals due to the complexity of the aromatic regions of the spectra.
.
Reagents and conditions. (i) triethylamine, CH2Cl2, r.t.; (ii) [(C6H5)3P]4Pd(0), BHT, toluene, 110°C,6-16h
Scheme 1. Preparation of N-Benzylcarboxamido/-Aminomethylbenzoyl conjugated phenyl vinyl
estradiols 6a-i, 7a-d.

The new compounds 6a-i and 7a-d were evaluated as ligands for the ERα-and ERβ-LBD using a
competitive binding assay and as agonists using the ER-induced alkaline phosphatase assay in Ishikawa cells.
[16, 17] Results for the assays are shown in Table 1. The relative binding affinity (RBA) values were compared
to both estradiol (RBA at both receptor subtypes = 100%) and the parent 17α-E-phenylvinyl estradiol (RBA
ERα= 7%, RBA ERß = 7.8%). As the results show, all compounds tested demonstrated significant ERα/ß-LBD
binding with RBA values ranging from 0.5-10%. The highest RBA value was found for the meta-benzylamine
Table 1. ER-alpha and ER-beta Relative Binding Affinity (RBA) of Conjugated Phenylvinyl Estradiols 6a-i and
7a-d
Compound
Phenylvinyl
Inner Substitution
Outer Substitution
-
RBA ERαa
7.0
RBA ERßa
7.8
RSAb
6.7
-
6a
6b
6c
6d
6e
6f
6g
6h
6i
7a
7b
7c
7d
4-
4-
4-
4-
4-
4-
4-
3-
2-
4-
4-
2-
H-
4.2±1.9
2.4±0.9
3.6±1.0
4.5±1.4
1.9±1.0
2.4±0.5
2.3±0.9
9.5±1.7
2.6±1.6
7.9±1.8
2.6±1.6
2.1±1.6
1.4±1.1
1.1±0.2
3.7±0.6
3.0±1.0
5.3±0.6
1.0±0.1
2.7±1.5
3.3±1.5
7.3±2.1
2.6±1.6
4.7±1.5
2.7±1.5
1.0±0.7
0.2±0.1
<0.1
<0.1
4-OCH3
3-OCH3
2-OCH3
4-CF3
3-CF3
2-CF3
4-OCH3
4-OCH3
4-OCH3
2-OCH3
4-OCH3
2-OCH3
0.2±0.1
0.2±0.1
<0.1
0.2±0.1
<0.1
5.7±1.5
<0.1
0.6±0.3
<0.17
<0.1
2-
<0.1
(a) RBA = 100 X [E]/[C] where [E] is the concentration of unlabeled estradiol necessary to reduce the
specific binding of tritiated estradiol to the ERβ-HBD by 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%. (b) 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
derivative 6h, functionalized with the terminal 4-methoxybenzoyl group, while the lowest RBA value was
obtained for its ortho-benzylamine isomer 6i. Virtually all other compounds had values in the 1.5-7.0% range.
There appeared to be no selectivity for ERα-LBD versus ERβ-LBD within this set of compounds as ERα/ERß
binding ratios were in the range of 0.7-3.7:1. Therefore, this series of compounds demonstrated retention of
competitive binding to the ER-LBD comparable to the parent phenyl vinyl estradiol and to previously reported
(substituted phenyl) vinyl estradiol derivatives.
Efficacy studies were undertaken using the induction of alkaline phosphatase in the Ishikawa cell line.
Stimulation of the enzyme compared to estradiol (RSA = 100) provides a measure of agonism whereas the

ability to reduce the effect of estradiol would suggest antagonist activity. All compounds demonstrated agonist
responses in the Ishikawa cells as indicated by a stimulatory response, however, maximal stimulation was not
reached for most compounds at concentrations below 1 μM (RSA < 0.1-0.5%) and actual RSA values could not
be determined except for 6h and 7a. Because none of the compounds tested blocked or reduced the effect of
estradiol, even at the highest concentrations tested (1 µM), this series appeared to be ER agonists. It was
apparent that for all of the compounds tested, except for 6h, that stimulatory activity (RSA) of the ligand at the
estrogen receptor was almost an order of magnitude weaker than the corresponding binding affinity (RBA).
Generating SAR from the data was complicated, given the molecular dimensions, physicochemical
properties of the additional functional groups appended to the phenylvinyl substituent and the limited number of
compounds in the series. It is noteworthy that the maximal loss of relative binding affinity was less than one
order of magnitude compared to the parent unsubstituted phenylvinyl estradiol. The introduction of an
additional functionalized benzoyl/benzylamino group into a binding pocket that is as closely bounded as the
17α-position of estradiol was expected to have a greater impact on binding based upon the crystal structure of
the ortho-trifluoromethylphenylvinyl estradiol-ERαLBD complex.[19] Within the previous mono-substituted
phenylvinyl estradiol series, shifting a trifluoromethyl group from the ortho to meta to para position reduced
binding by an order of magnitude.[13] Similar effects were recently observed with the di-subsituted phenylvinyl
estradiols.[20] In this present case, introduction of an additional aromatic ring, along with the amide linkage and
a peripheral methoxy/trifluoromethyl moiety, had relatively little effect on the ability of the new ligand to
compete with the binding of estradiol at the ERα/ß-LBD. This effect was comparable to that observed in studies
with a variety of (hetero)arylethylnyl estradiols in which introduction of large substituents to the terminus gave
significantly reduced but similar RBA values (0.1-1.0%). [26-30]
Of equal significance in this study was the observation that the RBA range was narrow and that the
values were essentially unaffected by the variation either with the amide linkage or the substitution pattern on
either inner or terminal aryl ring (6a-i or 7a-d). In general, the range of RBA values was less than one order of
magnitude for the variety of substitution patterns, which is significant when compared to our previous series.
[11-16] Preliminary docking studies of ligand-ER-LBD complexes, based upon the crystal structure obtained
with the ortho-trifluoromethylphenyl derivative suggested that major conformational adaptions of the protein
would be necessary to accommodate the ligand within the “standard” steroidal binding mode. [31,32] In order
to accommodate the extended derivatives prepared in this study, the protein would have to undergo even greater
conformational adaptations. Apparently other conformations were involved with these compounds.
As previously noted, the crystal structure of a europium chelate of 17α-pyridine ethynyl estradiol
tetraacetate revealed the extent of protein remodeling of the ER-LBD needed to accommodate a sterically
demanding terminal group. [18] In this complex, the metallated chelating moiety is exposed to the solvent and
oriented toward the H7-H8 interface, suggesting that further modifications would have little additional effect on
the binding affinity. In a recent study directed toward the preparation of moxestrol conjugated o-iodobenzyl
alcohol, [33] docking studies with the final ligand and all three published complexes suggested that significant
remodeling of the H7 region of the receptor was required to accommodate the extended 17α-substitutent.
Structure 2YAT most successfully accommodated the ligand and rationalized its binding affinity. It is highly
probable that the derivatives described in this present study adopted a similar orientation and that this complex
could be used to evaluate the binding properties of the new derivatives.
The results generated by our evaluation of the interactions between the three isomeric ligands 6b, 6h,
and 6i, and the ER derived from ER-LBD 2YAT [Figure 2] ]indicated a stronger ∆Gbinding for the meta isomer
compared to the ortho- or para-isomers. This calculation is in agreement with the observed binding data. Of
greater interest was the network of ligand-receptor interactions [Figure 3] present in the meta-isomer pose.
While all three poses share some common interactions, such as a hydrogen bond between the C3 hydroxyl and

E353, the meta-isomer has more favorable interactions, including its exclusive π-π stacking interaction between
the terminal aromatic ring and F425 and a hydrogen bond between the oxygen in the amide bond and C417.
6b
6h
6i
Figure 2 Comparative docking of ligands 6b, 6h, and 6i with ER-ligand binding domain derived from
2YAT. Steroidal scaffold of each ligand occupies common internal binding pocket while divergent 17α-
substituents access different components of the H7, H8 and loop segments.
Although the three poses share multiple interactions, neither the ortho- nor the para-isomer have the
same extent of interactions across the ligand, suggesting their looser accommodation within the receptor. It was
observed in the para-isomer that the terminal aromatic ring, especially its functional group, is exposed to the
solvent and may have significant conformational flexibility. As such, this would account for the low variability
in RBA values for the para-derivatives. The ortho-isomer did not produce such solvent exposure, suggesting
that the protein may adapt to enclose the terminal group at an energy cost, as those ligands expressed the lowest
RBA values.
This alternative binding mode may also explain the observed reduced efficacy of most of the compounds as
agonists. Assuming that the compounds generate the alternative binding mode in which the terminal ring is
exposed to the solvent, the resulting complex may not be stable enough to efficiently recruit coregulatory

proteins needed for downstream events. While complexes with the coregulatory proteins may occur, their
capacity to stimulate downstream events may be impeded. In the previously reported series of arylethynyl
estradiols, the compounds either demonstrate low estrogenicity compared to their ligand binding capacity, or
not data are provided. [33]. Almost all compounds in this study also appear to be impeded estrogens. The
capacity to generate an agonist response is approximately an order of magnitude weaker than its binding
affinity. Compound 7a, which has an RBA value comparable to that of the parent phenylvinyl estradiol (7.9%
vs 7.0%), has an RSA value one-tenth (0.6% vs 6.7%).
Figure 3. Key interactions between 6h and the residues within the ligand binding domain derived from 2YAT.
Major contributors within the steroid binding site are maintained while additional interactions with Met-343 and
Asp-411 are generated.

The most significant finding from this study, however, was that the inner meta-substituted derivative had RBA
and RSA values comparable to the parent phenylvinyl estradiol, in distinction from all other derivatives. The
key factor appears to be the meta-substitution which allows the terminal ring to project toward a region of the
estrogen receptor that is more accommodating. [Figure 3] The sp3 benzylic carbon also allows the terminal ring
more conformational mobility to interact with key residues of the receptor, e.g., D411, C417 and F425. Not
only does this change in orientation appear to enhance (modestly) binding affinity, it significantly restores the
capacity to recruit the coactivator proteins required to generate the agonist response (enhanced RSA). These
subtle modifications now provide a basis for continuing to explore the link between the binding interaction and
activation processes for the estrogen receptor, and for generating novel imaging probes.
This study is of interest to us as we have shown that steroidal antagonists, functionalized through an 11β-
alkoxyphenyl group, exhibit high substituent tolerance. [34-36] Terminal groups on these compounds are also
solvent-exposed. This property allows us to append a variety of functional groups, including fluorescent dyes
and other imaging moieties, with the objective of imaging estrogen receptors in their antagonist conformation.
The observation that we have identified a steroidal agonist that can also tolerate significant substitution at a
synthetically accessible site without significant loss of affinity or efficacy raises the possibility of preparing
comparable fluorescent derivatives. The ability to visualize estrogen receptors in both conformations using
fluorescent steroidal derivatives should enhance our capacity to study the structure and function of the receptors
in a cellular context.
4. Conclusion
In summary, we have demonstrated the facile preparation of a novel class of steroidal ER ligands and
their binding to the ERα- and ERβ-LBD. The compounds demonstrate significant affinity for both ER subtypes
with RBA values in the 2-10% range, but do not demonstrate subtype selectivity. Most of the new derivatives
appear to be impeded agonists, with RSA values approximately an order of magnitude lower than the RBA
values. In general, the binding and stimulatory effects can be rationalized on the basis of a model in which the
large 17α-substituent accesses the solvent space and also modulates coactivator protein recruitment. One
derivative, 6h, however, which has an inner-meta substitution pattern not only exhibits the highest binding
affinity (RBA = 9.5%), it also has a stimulatory capacity comparable to its receptor binding (RSA = 5.7%).
Docking studies with this compound and the ortho-/para-isomers provided a rationale for the higher affinity and
stimulatory activity. These interesting findings provide the basis for further studies to explore the link between
estrogen receptor binding and agonist responses using 17α-substituted estradiol derivatives. Further studies
investigation the preparation of additional analogs and fluorescent derivatives are now in progress.
5. Acknowledgments:
This work was supported by grants from the PHS [5RO1-CA81049 (RNH), CA 37799 (RBH), HL-61432
(RBH)] and DoD [DAMD-17-99-1-9333 (RNH), DAMD –17-1-00-1-0384 and W81XWH-04-1-0544(RNH)
and NSF MCB-1517290 (MJO) and a National Institute of Justice Pre-doctoral Fellowship (TAC)]. We also
than Jenifer N. Winters for her assistance with the ligand-receptor modeling studies.

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Graphical Abstract.
Synthesis of Benzoylbenzamide Derivatives of 17α-E-Vinyl Estradiol and Evaluation as
Ligands for the Estrogen Receptor-α Ligand Binding Domain
Robert N. Hansona*, Emmett McCaskilla, Edward Hua1, Pakamas Tongcharoensirikula, Robert Dilisa,Jessa L.
Silvera, Timothy A. Coulthera, Mary Jo Ondrechena, David Labareeb and Richard B. Hochbergb
aDepartment of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115
bDepartment of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520

Highlights:
Efficient synthesis of novel series of benzoylbenzamide derivatives of 17α-vinyl estradiols
All compounds demonstrate high relative binding affinity (1-9%) at both estrogen receptor subtypes
One derivative, the meta -substituted compound demonstrates high affinity and full efficacy whereas all others
appear to be impeded estrogens
Molecular modeling studies of the ortho-, meta- and para isomers suggest a binding mode for 6h that accounts
for its biological activity
Results suggest approach to new subclass of 17α-substituted estradiol derivatives as molecular probes