Synthesis and evaluation of 17alpha-20E-21-(4-substituted phenyl)-19-norpregna-1,3,5(10),20-tetraene-3,17beta-diols as probes for the estrogen receptor alpha hormone binding domain.

Article abstract of DOI:10.1021/jm0205806

As part of our program to develop probes for the hormone binding domain of the estrogen receptor alpha (ERalpha), we prepared a series of 4-para-substituted phenylvinyl estradiol derivatives using a combination of solution and solid-phase Pd(0)-catalyzed

Full text of DOI:10.1021/jm0205806

J . Med. Chem. 2003, 46, 2865-2876
2865
Syn th esis a n d Eva lu a tion of 17r-20E-21-(4-Su bstitu ted
p h en yl)-19-n or p r egn a -1,3,5(10),20-tetr a en e-3,17â-d iols a s P r obes for th e Estr ogen
Recep tor r Hor m on e Bin d in g Dom a in
Robert N. Hanson,*,† Choon Young Lee,† Carolyn J . Friel,† Robert Dilis,† Alun Hughes,‡ and
Eugene R. DeSombre‡
Departments of Chemistry and Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue,
Boston, Massachusetts 02115, and The Ben May Institute for Cancer Research, The University of Chicago,
5841 S. Maryland Avenue, Chicago, Illinois 60637
Received December 27, 2002
As part of our program to develop probes for the hormone binding domain of the estrogen
receptor R (ERR), we prepared a series of 4-para-substituted phenylvinyl estradiol derivatives
using a combination of solution and solid-phase Pd(0)-catalyzed methods. The compounds 5a -j
were evaluated for their binding affinity using the ERR hormone binding domain (HDB) isolated
from transfected BL21 cells. The results indicated that although the new compounds were
somewhat lower in relative binding affinity (RBA at 25 °C is 1-60%) than estradiol (100%),
most had higher affinity than the unsubstituted parent phenylvinyl estradiol (RBA ) 9%).
Because the substituents did not generate a structure-activity relationship directly based on
physicochemical properties, the series was evaluated using molecular modeling and molecular
dynamics to determine key interactions between the ligand, especially the para substituent,
and the protein. The results suggest that the observed relative binding affinities are directly
related to the calculated binding energies and that amino acids juxtaposed to the para position
play a significant but not dominant role in binding. In conclusion, we have identified the 17R-
E-(4-substituted phenyl)vinyl estradiols as a class of ligands that retain significant affinity for
the ERR-HBD. In particular, 4-substitution tends to increase receptor affinity compared to
the unsubstituted analogue, as exemplified by 5e (4-COCH3), which had the highest RBA value
(60%) of the series. Palladium(0)-catalyzed coupling reactions on solid support or in solution
using suitably substituted iodo arenes and 17R-E-tributylstannylvinyl estradiols offer a flexible
approach to their preparation. Molecular modeling studies of the receptor suggest that there
exists additional ligand accessible regions within the ERR-HBD to generate interactions that
may enhance receptor affinity or modify efficacy in developing new therapeutic agents. Studies
to undertake modification in the properties and/or position of the aryl substituents in subsequent
series to further define that role are in progress.
In tr od u ction
agonist response.7-10 Because the orientation of the
helix-12 of the ER-HBD may be affected differently by
various ligands, a variety of approaches can be used to
generate compounds that can bind effectively to the
receptor protein and subsequently produce the desired
pharmacological response. Most strategies have involved
modifications of the nonsteroidal antagonists tamoxifen
and raloxifene11-20 (Figure 1); however, other groups
who have used a heterocyclic moiety to replace the
ethylene bridge have also been successful in preparing
interesting ER ligands.21-24
Breast cancer is the most common cancer diagnosis
among women, with an estimated 181 000 new cases
per year in the U.S.1 Approximately 60% of these newly
diagnosed patients have hormone-responsive breast
cancer, defined as containing estrogen receptor (ER) and
requiring the presence of circulating estrogens for
maintenance of tumor growth.2 This relationship has
generated considerable interest both for understanding
the mechanism of the hormone receptor interactions and
for targeting the ER in therapeutic breast cancer drug
development. Recent publications of the crystal struc-
ture of the liganded ER-HBD have suggested that the
key interaction may involve the N-terminal region
(helix-12) of the receptor.3-6 Antagonists apparently
cause this helical region of the ER-HBD to occupy a
different binding mode compared to that produced by
agonists, thereby disrupting the interaction between the
receptor and the coactivator proteins that initiate the
As part of our program to develop new probes for the
estrogen receptor, we have focused on the preparation
and evaluation of novel steroidal derivatives. Our ap-
proach involved the introduction of substituents at the
17R-position of estradiol as a means to enhance receptor
binding and/or alter receptor response. Our initial
studies described the synthesis and evaluation of several
series of 17R-phenylvinyl estradiols (Figure 2). These
studies, conducted prior to the publication of the first
ER-HBD crystal structures, suggested that there was
significant tolerance for large functional groups at this
site.25-29 Later reviews of the structure-activity rela-
* To whom correspondence should be addressed. Phone: (617) 373-
3313. Fax: (617) 373-8795. E-mail: r.hanson@neu.edu.
† Northeastern University.
‡ The University of Chicago.
10.1021/jm0205806 CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/07/2003

2866 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Hanson et al.
F igu r e 1. Structures of nonsteroidal antiestrogens developed or evaluated as selective estrogen receptor modulators (SERMs).
close to the hinge between helices 11 and 12. The
substituent was also close to Met-421, a residue that is
one of two amino acids that are different from that found
in the binding region of the estrogen receptor â (ERâ)
isoform.9,33-35 Therefore, we proposed that the introduc-
tion of substituents onto the 17R-phenylvinyl group
would provide information regarding the interactions
between ligands and the estrogen receptor isoforms.
However, the model of the interaction between the
ligand and the receptor could not predict either the
F igu r e 2. Structures of 11â-substituted 17R-(Z- and E-)-X-
magnitude of the effects of additional substituents on
the terminal aromatic ring or the effect on the orienta-
tion of the helix 12 and, by extension, the biological
response.
vinyl estradiols prepared and evaluated as estrogen receptor
ligands.
tionships for ER ligands supported these observations
and provided a rationale for the orientation of the 17R-
substituent within the ER-HBD.30-32 To appreciate
these observations, we used molecular modeling to dock
our initial 17R-E-phenylvinyl estradiol with the ER-
HBD and performed energy minimization to identify
potential interactions. This model, in which we have
oriented the steroid nucleus in the same manner as that
found for the estradiol-ER complex, provided two
significant points. The 17R-group was accommodated
within the outer portion of the domain and relatively
The strategy that we utilized in our program involved
the preparation of new 17R-substituted phenylvinyl
estradiol derivatives in which the substituents would
probe the receptor surface. The phenylvinyl group
provides six degrees of variation, i.e., E vs Z stereo-
chemistry around the C-C double bond as well as 2-,
3-, or 4-substitution on the phenyl ring (Figure 3). In
this report, we describe the synthesis, receptor binding,
and computational analysis of a series of 4-substituted
17R-E-phenylvinyl estradiol derivatives. The reasons for

Tetraene-3,17â-diols
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2867
phase Stille coupling approach developed in our labo-
ratories (Scheme 1). The commercially available ethynyl
estradiol 1 was acetylated to give the 3-acetyl interme-
diate 2,41 which was then hydrostannated with tri-n-
butyltin hydride and triethylborane to give predomi-
nantly the E-stannylvinyl estradiol 3. The acetylated
intermediate was then coupled with the 4-substituted
aryl halides (Br/I) using standard Stille coupling condi-
tions to yield the intermediates 4a -f. Hydrolysis with
sodium methoxide in methanol provided the target 17R-
E-(4-substituted phenyl)vinyl estradiols 5a -f, while
saponification of 5f provided the carboxy derivative 5g.
Alternatively, as part of our combinatorial chemistry
approach, ethynyl estradiol 1 was hydrostannated to
give predominately the E-stannylvinyl estradiol 6,
which was coupled to a carboxylated polystyrene resin
to give the intermediate 7. Stille coupling with the
appropriate aryl halide followed by cleavage from the
resin gave the target estradiol derivatives 5h -i (Scheme
2).
A third approach utilized the Suzuki coupling reac-
tion.42,43 This involved first performing iododestanny-
lation of 3 to give the iodovinyl estradiol 8, which
underwent facile Suzuki coupling with 4-fluorophenyl-
boronic acid to give, after hydrolysis, the product 5j
(Scheme 3). The products were purified by column
chromatography, recrystallized, and characterized by
NMR and elemental analysis. Because the objective of
the study was to generate the target compounds and
demonstrate synthetic feasibility, the yields of the
compounds were not optimized (Table 1). Stereochem-
istry of the products was established by the coupling
constant for the vinylic protons, which was J ) 16-18
Hz, consistent with the previously synthesized E-trans
derivatives.27
F igu r e 3. Rationale for selection of 17R-(Z- and E-)-(substi-
tuted phenyl)vinyl estradiols as probes for the hormone
binding site of the estrogen receptor.
this selection included synthetic concerns as well as
conformational issues. Our experience with the Stille
coupling reaction indicated that our synthetic approach
via the vinylstannanes and the commercially available
(or readily accessible) para-substituted aryl halides
would easily generate a series of compounds with a
variety of functional groups.36,37 Ultimately we intended
to extend the solution-phase chemistry to our solid-
phase organic synthesis strategy for combinatorial
chemistry.38 Of equal importance was the recognition
that para substitution would yield products that would
be symmetrical along the aryl axis. This would reduce
the number of potential conformational isomers in
which the compound could exist and simplify modeling
of the interactions between the ligand and the receptor.
Our preliminary NMR studies with the substituted
phenylvinyl estradiols (E and Z isomers) indicated that
the compounds existed in a conformational equilibrium
with a relatively low-energy barrier between them.39,40
Therefore, incorporation of the conformational mobility
of the ligand into the docking interaction with the
receptor would be simplified by the use of the para
substitution. As our results suggest, the presence of the
substituent and its properties had a significant effect
on the binding of the ligand to the ER-HBD.
Biologica l Stu d ies. Rela tive Bin d in g Affin ities.
The new compounds were evaluated for their relative
binding affinities (RBA) at 2 and 25 °C using the ERR-
HBD isolated from the transfected BL21 cells. The RBA
values for the ligands were determined using a competi-
tive radiometric receptor binding assay and were com-
pared to both estradiol and the unsubstituted phenyl-
vinyl estradiol. The results are summarized in Table 2
where the RBA of estradiol is 100%.
Resu lts
Syn th esis of Estr ogen ic Liga n d s. The target com-
pounds in this series were prepared as part of a larger
program to probe ligand-receptor interactions and to
develop potential theraputic agents. As a result, we
utilized several methods to obtain the compounds. The
synthesis of most of the 17R-E-(4-substituted phenyl)-
vinyl estradiols (5a -g) was achieved using the solution-
Although none of the members of this bound as
potently as estradiol, most of the compounds retained
Sch em e 1. Solution-Phase Synthesis of Estrogens

2868 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Sch em e 2. Solid-Phase Synthesis of Estrogens
Hanson et al.
Ta ble 2. Relative Binding to the ERR Ligand Binding Domain
Sch em e 3. Suzuki Synthesis of Estrogen
compd
X
RBAa at 2 °C (%)
RBAa at 25 °C (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
OH
CN
16
21
9
10
53
18
1
5
36
24
9
25
27
18
60
26
1
8
32
22
CH3
COCH3
CO2CH3
CO2H
CF3
OCH3
F
Ta ble 1. Yields of 17R-E-(4-Substituted phenyl)vinyl
Estradiols
5j
a
RBA ) 100 × [E]/[C], where [E] is the concentration of
unlabeled estradiol necessary to reduce the specific binding of
tritiated estradiol to the ERR-LBD by 50% and [C] is the
concentration of competitive ligand necessary to reduce specific
binding by 50%. The RBA of estradiol is 100% at each incubation
temperature. The ERR-LBD was extracted from BL21 cells over-
expressing the 33 kDa pET-23d-ERG vector.58 Curves for ligand
and estradiol had correlation coefficients of >95%.
compd
R
yield of 4 (%)
yield of 5 (%)
methoda
4-fluoro (RBA ) 28%), 4-cyano (RBA ) 27%), 4-meth-
oxycarbonyl (RBA ) 26%), and 4-hydroxy (RBA ) 25%)
phenylvinyl estradiols. The only compound with RBA
values significantly less than that of phenylvinyl estra-
diol at either temperature was the polar 4-carboxy
derivative 5g (RBA ) 1-2%).
Molecu la r Mod elin g Stu d ies. Initially we exam-
ined the structure-activity relationships for this series
using the parameters described by Gao et al.32 for the
17R-substituted estradiols; however, a strong, direct
correlation could not be generated between the sub-
stituent properties and the observed RBA values. There-
fore, molecular modeling of the ligands and the ligand-
ERR-HBD complexes was undertaken to interpret the
relationship between the structure of the compounds
and their receptor binding affinity. Earlier studies with
estrogenic ligands44,45 focused on compounds that were
either substituted directly on the A- or D-rings or were
nonsteroidal analogues of estrogens. As such, the results
were not directly applicable to our work, even though
the approaches were similar.
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
OH
CN
25
21
50
59
89
65
92
89
80
60
83
70
91
49
36
97
I
I
I
I
I
I
I
II
II
III
CH3
COCH3
CO2CH3
CO2H
CF3
OCH3
F
5j
37
a
Method I: two-step solution Stille sequence of 3 f 4 f 5.
Yields are for each step. Method II: solid-phase Stille sequence
of 7 f 5. Yields are for the combined sequence. Method III: Suzuki
reaction sequence of 8 f 4 f 5. Yields are for each step.
significant affinity for the estrogen receptor. The range
of relative binding affinities straddled that of the
unsubstituted phenylvinyl estradiol (RBA ) 16% at 2
°C; RBA ) 9% at 25 °C). At 2 °C, the derivatives with
the highest affinity were the 4-acetyl (RBA ) 53%),
4-methoxy (RBA ) 36%), 4-hydroxy (RBA ) 21%), and
4-fluoro (RBA ) 20%) phenylvinyl estradiols. At 25 °C,
the ligands demostrating the highest RBA values were
the 4-acetyl (RBA ) 60%), 4-methoxy (RBA ) 32%),
The results of our molecular modeling/dynamics study
are depicted in Figures 4 and 5. In this study, we

Tetraene-3,17â-diols
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2869
utilized the X-ray crystal structure of ERR-HBD in
which estradiol was the estrogenic ligand. The ligands
were docked with the complex in a manner in which
the two phenolic A-rings were superimposed. Estradiol
was removed, and the subsequent complex was sub-
jected to molecular dynamics and simulated annealing
manipulations. The docking experiments indicated two
low-energy modes, as previously noted;44 however, only
the complexes similar to the crystal forms, i.e., with the
A-ring orientation toward the Arg-394-Glu-353 site,
were evaluated in this study. Docking with the unsub-
stituted phenylvinyl estradiol gave a complex in which
the 17R-substituent generated new potential interac-
tions with a region of the ER-HBD formed by the
junction of the â-sheet, 2-3 loop, helices 3, 6, 7, and
the 6-7 loop. Critical amino acids that line this pocket
include Met-421, Met-342, Met-343, Leu-410, Leu-346,
Phe-425, and Val-418. It is of interest to note that of
these residues, Met-421 is replaced by Ile in the ERâ-
HBD.
In our proposed binding model, the two edges of the
phenyl ring interact with a different set of residues;
however, conformational mobility around the phenyl-
vinyl axis would allow an ortho or meta substituent to
select its individual low-energy conformation. Para
subsituents, on the other hand, are independent of the
rotation of the phenyl group around the double bond
and would interact with a common set of residues. As
Figure 4A indicates, this set consists of several me-
thionine residues, notably Met-342, -348, and -421, plus
Phe-425. This is consistent with recent evaluations of
ligand-ER-LBD complexes.46 The other amino acids
associated with the ligand-receptor binding have been
identified from earlier studies, i.e., Phe-404, Glu-353,
and Arg- 394, and interact similarly to the other ligands.
The steroidal skeleton interacts with a lipophilic surface,
while the 17R-moiety induces a pocket surrounded by
methionine and phenylalanine residues. The volume of
the induced pocket clearly accommodates the phenyl-
vinyl goup and orients the para substituents toward the
hinge between helices 11 and 12 (Figure 4B). Overall,
the ligands are displaced approximately 1 Å toward the
Glu-Arg interface with a concomitant weakening of the
hydrogen bond interaction between the 17â-hydroxyl
and His-524.
F igu r e 4. (A) Representation of 5d (X ) CH3, RBA(25 °C) )
18%) docked in lowest energy conformation of the ERR-HBD
complex. View is from above the â-face of the steroid looking
toward the junction of helices 11 and 12. Side chains of key
amino acids that are within 5 Å of the ligand are identified.
In particular, the phenylvinyl group is bounded by three
methionines (Met-342, -343, and -421), a phenylalanine (Phe-
425), and the hydrophobic residues (Leu-346, Leu-410, and
Val-418). (B) Representation of 5d and the ERR-HBD from
directly above the C-18 methyl group. The surface of the HBD
is in white (hydrophobic) and shaded to indicate the position
of other functional groups, yellow for sulfur (methionine), red
for oxygen (tyrosine), and blue for nitrogen. The figure clearly
delineates the pocket induced by the binding of the ligand to
the region of the HBD. Also clear are the methionine and
phenylalanine residues that line the binding pocket. The
dimensions easily accommodate the phenyl ring and direct the
para substituent toward Met-342.
The methylthio groups of the Met-342, -343, and -421
form a cage around the phenyl ring with the para
position now oriented toward the junction of Met-342
and Met-421. The introduction of substituents at this
position has relatively little effect on the torsion angle
between the phenyl ring and the C-C double bond
(Figure 5). On the other hand, the conformation of the
substituent is primarily affected by the local environ-
ment of the HBD adjacent to the para position of the
phenyl ring. As the model demonstrates, small groups
such as the 4-F, CN, methyl, trifluoromethyl, and
hydroxy are easily accommodated within the space and
establish few interactions. Larger groups such as the
4-acetyl, methoxycarbonyl, carboxy, and methoxy are
required to undergo torsional motion to establish a low-
energy conformation within the HBD. This equilibration
is reflected not only in the final orientation of the
substituent but also in the translational motion of amino
acid side chains in the vicinity of the ligand. These
movements are ultimately reflected in the calculated
binding energies for the complexes (Figure 6).
Using the parameters described in the Experimental
Section, we calculated the binding energies for each of
the complexes and compared those values to the ob-
served RBA (25 °C) values. Except for two compounds,

2870 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Hanson et al.
the ERR-ligand binding domain. The methods used for
the synthesis of the target compounds were chosen to
demonstrate the feasibility of each approach and did not
represent the optimal conditions. The synthesis of the
target compounds 5a -j, which were obtained in reason-
able yields and in high purity by a combination of
solution- and solid-phase palladium-catalyzed reactions,
illustrated the versatility and flexibility of this strategy.
Given the ability to prepare the requisite stannylvinyl
estradiol precursors, either 3 or 7, and the appropriate
coupling partner, it is possible to prepare any number
of functionalized phenylvinyl estradiol derivatives.
We screened the new compounds with the ERR-HBD
and most of the 4-substituted derivatives displayed high
relative binding affinity (RBA) for the ERR-HBD with
values in the range 25-60%, exceeding that of the
unsubstituted parent. Docking the new ligands in the
ERR-HBD using molecular modeling suggested that the
substituted phenylvinyl group was accommodated by
the outer portion of the ligand binding pocket. The
results of those studies provide the basis for evaluating
the relationship between the 4-substituent and the
binding (RBA) data.
Str u ctu r e-Activity Rela tion sh ip s in th e 4-Su b-
stitu ted P h en ylvin yl Ser ies. Previous studies in our
laboratories have shown that the estrogen receptor
tolerated the introduction of 17R-X-vinyl substituents.
Although the highest affinity was observed for the
halovinyl estradiols, phenyl- and phenylthio/selenovinyl
estradiols also were good ligands. Our topographical
studies of the ER-HBD using the halovinyl estrogens
as probes were limited by the small number of substi-
tuted ligands available. The use of phenylthio/seleno-
vinyl estrogens was hampered as well by the limited
availability of substituted reagents for electrophilic
destannylation and by rotation around the S/Se-vinyl
bond. Introduction of substituents on the phenylvinyl
group via the versatile Pd-catalyzed Stille or Suzuki
reactions made the phenylvinyl estrogens a more prom-
ising method for investigating the ligand-receptor
interactions. The substituents that we have introduced
at the 4-position possess a variety of physicochemical
properties. These included electron-withdrawing as well
as electron-donating, hydrophilic as well as hydrophobic,
and small as well as large groups. Virtually all of the
F igu r e 5. Binding of the 17R-(4-substituted phenyl)vinyl
estradiols 5a -j in the ERR-HBD. Estradiol is represented in
the ball-and-stick format, and the new ligands are represented
as the shaded lines. Compounds 5a -j are displaced ap-
proximately 1 Å toward the Glu-353, Arg-394 binding locus.
The 4-substituents are closely flanked by Met-421 and Phe-
425.
5g and 5h , which have the lowest RBA, a strong
correlation, R2 ) 0.94, was observed. In addition,
binding energies between the docked ligand and selected
amino acids were evaluated using the same method. The
highest contributions to the binding energy were derived
from Phe-404 and Leu-387, which interact respectively
with the R- and â-faces of the steroidal A-ring. The
second highest contribution arises from Leu-346, which
interacts with both the C-ring and the vinyl segment of
the 17R-phenylvinyl moiety. The three methionine
residues that interact with the phenyl group and the
para substituent also provide significantly to the binding
energy. These results suggested the proposed interac-
tions between the ERR-HBD and this homologous set
of ligands represented a reasonable model and a basis
for interpreting the binding of our series of compounds.
Discu ssion
In this study, we prepared a series of 4-substituted
phenylvinyl estradiols and evaluated them as probes for
F igu r e 6. Relationship between the observed RBA (25 °C) values and the calculated binding energy.

Tetraene-3,17â-diols
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2871
new compounds are equal to or better in their ER
affinity relative to the unsubstituted parent compound
except for the 4-trifluoromethyl and 4-carboxy com-
pounds (RBA at 25 °C is 5% and 1%, respectively). The
rest had RBA values that were roughly 20-60% that of
estradiol (RBA ) 100%) and significantly higher than
the unsubstituted compound 5a (RBA ) 9%). This
increase in binding was essentially independent of the
properties of the substituent. For example, the 4-hy-
droxy compound was virtually identical to the 4-cyano
and 4-methoxycarbonyl derivatives (RBA ) 25% vs 27%
vs 26%), and 4- fluoro was similar to 4-methyl (RBA )
22% vs 18%). The highest affinity was observed for the
4-acetyl derivative (RBA ) 60%), although its properties
are similar to those of the methyl ester (RBA ) 26%) or
the methyl ether (RBA ) 32%). The lack of a clear
relationship between physicochemical properties of the
substituents and their RBA values suggested to us that
in the process of binding, both the ligand and receptor
were undergoing structural adjustments to reach a
binding energy minimum for the resultant complex. We
concluded that an analysis of this type of interaction
would best be achieved using molecular modeling and
docking studies.
the highest contribution to binding energy was derived
from Phe-404 and Leu-387 via direct interactions with
the R- and â-faces of the A-ring. The second highest
contribution arose from Leu-346, which interacts di-
rectly with both the steroidal C-ring and the phenylvinyl
group. Met-421 is closest to the 17R-phenyl group, while
Met-342 and Met-343 juxtapose the para and vinyl
groups, respectively. If one includes the consideration
that steric factors could influence translational or
torsional responses on these side chains, then the
interpretation of the individual effects becomes more
complex. As shown in Figure 5, the overlap of the
ligands (deleting the ERR-HBD) shows that the sub-
stituents occupy a reasonably small volume in which
electronegativity is not as critical as the conformation
of the substituent. As a result, the methionines tolerate
a polar substituent (fluoro-, carbonyl-) adjacent to the
phenyl ring as long as the next group is lipophilic
(-methoxy, -methyl). The most significant deviations
were observed for the 4-trifluoromethyl compound 5h ,
which had a greater torsional distortion between the
vinyl group and the phenyl ring, and the 4-carboxy
compound 5g, which may undergo ionization under
binding conditions. These effects may account, at least
in part, for reduction in binding affinity and/or lack of
correlation with the calculated binding energy.
In vestiga tion of th e In ter a ction s of th e 17R-4-
Su bstitu ted P h en ylvin yl Estr a d iols w ith th e ER-
HBD. Molecu la r Mod elin g Stu d ies. We used molec-
ular modeling and molecular dynamics to investigate
the interactions between the phenylvinyl substituent of
our ligands and the amino acid side chains of the ERR-
HBD. We chose the coordinates of the estradiol-ERR-
HBD complex because of the steroidal nature of our
compounds and because preliminary biological data
indicated that the compounds behaved as agonists in
the immature rat uterotrophic assay. Therefore, the
orientation of the critical helix 12, associated with
coactivator binding, was probably the agonist orienta-
tion. Using the modeling program with the Insight II
package,47 we docked the 4-unsubstituted phenylvinyl
estradiol into the estradiol binding site, overlaying the
aromatic rings. Employing molecular mechanics and
energy minimization routines, approximately 20 low-
energy conformers were obtained for each complex. In
each case, the17R-substituent was oriented toward the
external surface of the receptor. The translations of the
internal amino acids associated with the A-B-C ring
interactions were relatively small, consistent with the
crystal structures obtained with the other estrogen
receptor agonists and with the steroidal and nonsteroi-
dal androgens at the androgen receptor.48,49 This effect
has also been observed with the vitamin D analogue-
vitamin D receptor-HBD crystal structures where the
internal structure remains relatively rigid while the side
chain of the analogue tends to undergo the conforma-
tional deformations.50-52 In our model, the phenylvinyl
substituent occupied a region bounded by three me-
thionines (Met-342, -343, -421), a phenylalanine (Phe-
425) as well as two leucines (Leu-346, -410) and a valine
(Val-418). While relatively lipophilic in character, these
residues also can interact through the electron pairs of
the thioether and/or through the π-cloud of the phenyl
ring. Therefore, substituents present at the para posi-
tion of the phenylvinyl group can experience multiple
effects. Analysis of individual amino acids indicated that
There are several conclusions that can be drawn from
this study. First, ERR-HBD can accommodate the
presence of a significant variety of substituents at the
para position of the phenylvinyl estradiols without a
serious reduction in overall receptor binding affinity.
This finding had not been previously observed and leads
to the possibility that other functional groups can be
introduced to impart higher receptor affinity, selectivity,
or altered efficacy. Second, molecular modeling and
molecular dynamics have provided a method for not only
evaluating the interactions between ligands and the
receptor hormone binding domain but, at least within
a homologous (para-substituted) series, possibly predict-
ing the affinity of putative ligands. Last, we have
demonstrated the feasibility of Pd(0) coupling methods
to prepare the diverse members of such a series as may
be required to identify a potential clinical candidate.
Subsequent publications in this project will describe
those efforts to extend these methods to the preparation
of other variations in the phenylvinyl estradiol series.
Exp er im en ta l Section
Gen er a l Meth od s. All reagents and solvents were pur-
chased 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 phosphomolybdic acid
reagent. Column chromatography was performed with 32-63
µm silica gel packing. Melting points were determined using
an Electrotherm capillary melting point apparatus and are
uncorrected. NMR spectral chemical shifts are reported in
parts per million downfield from TMS and referenced either
to TMS internal standard for deuteriochloroform or deuterio-
acetone solvent peak. Coupling constants are reported in hertz.
All compounds gave satisfactory elemental analyses, (0.4%
(Atlantic Microchemical Laboratories, Inc., Norcross, GA),
unless otherwise stated.
Solu tion -P h a se Syn th esis. 17r-E-(Tr i-n -bu tylsta n n yl)-
vin yl Estr a d iol 3-Aceta te (3). To a solution of 3-acetoxy-
17R-ethynyl estradiol 241 (1.5 g, 4.4 mmol) in THF (5 mL) were

2872 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
Hanson et al.
added 1.7 mL (6.3 mmol) of tri-n-butyltin hydride and 3 mL
(26 mmol) of triethylborane. The reaction mixture was stirred
magnetically for 5 h at 60 °C46 and then purified by column
chromatography on silica gel using hexanes-ethyl acetate
(5:1) as the eluent. The reaction afforded 0.5 g (0.79 mmol) of
3-acetoxy-17R-Z-(tri-n-butylstannyl)vinyl estradiol and 1.89 g
(3 mmol) of 3-acetoxy-17R-E-(tri-n-butylstannyl)vinyl estradiol
in a combined yield of 86%. Rf (Z isomer) ) 0.58 (hexanes-
ethyl acetate, 5:1), Rf (E isomer) ) 0.43 (hexanes-ethyl
temperature, the resin was washed three times each with
dichloromethane, methanol, tetrahydrofuran, and warm dim-
ethyl formamide, dried in vacuo, and characterized by FTIR.
The resin was swelled in dichloromethane (10 mL) containing
3 mL of 5 N sodium hydroxide in methanol, and the mixture
was stirred for 1 h. The cleavage step was repeated three
times. The solutions were combined, acidified with dilute acetic
acid, and partitioned between ethyl acetate and water. The
organic phase was washed with 10% sodium bicarbonate and
brine, dried over magnesium sulfate, filtered, and evaporated
to dryness. The crude product was purified by column chro-
matography on silica gel using hexanes-ethyl acetate as the
eluent. The final product was crystallized from hexanes-
acetone/ethyl acetate to obtain analytical samples for the
binding studies.
1
acetate, 5:1), amorphous. H NMR (300 MHz, CDCl3): δ 0.88
(s, 3H, C18-CH3), 1.2-2.4 (steroid envelope), 2.28 (s, 3H,
CH3CdO), 2.7-2.9 (m, 2H, C6R-H and C6â-H), 6.06 (d, 1H, J
) 19.4 Hz, CHdC21H), 6.21 (d, 1H, J ) 19.4 Hz, C20HdCH),
6.79 (d, 1H, J ) 2.4 Hz, C4-H), 6.84 (dd, 1H, J ) 2.6, 8.4 Hz,
C2-H), 7.28 (d, 1H, J ) 8.8 Hz, C1-H). 13C NMR (75.4 MHz,
CDCl3): δ 9.64 (C22, 4C), 13.78 (C24, 4C), 14.18 (C18), 21.13
(CH3CdO), 23.43 (C15), 26.15 (C11), 27.28 (C25, 4C), 27.37 (C7),
29.20 (C23, 4C), 29.59 (C6), 32.35 (C12), 35.87 (C16), 39.05 (C8),
44.06 (C9), 46.61 (C13), 49.06 (C14), 85.47 (C17), 118.54 (C2),
121.48 (C4), 124.68 (C21), 126.39 (C1), 138.05 (C10), 138.27 (C5),
148.38 (C20), 152.40 (C3), 169.89 (CH3CdO).
Meth od I. Gen er a l P r oced u r es for th e Syn th esis of
4a -g. Stille Cou p lin g. To a solution 3-acetoxy-17R-E-(tri-n-
butylstannyl)vinyl estradiol 3 (0.5 mmol) in dry toluene (5 mL)
were added the aryl halide (Br/I) (0.6-0.7 mmol) and a
catalytic amount (5.0 mg) of tetrakis(triphenylphosphine)-
palladium(0) and three crystals of 3,5-di-tert-butyl-4-hydroxy-
toluene. The reaction mixture was stirred for 10 h at 90-100
°C under a nitrogen atmosphere. The reaction mixture was
cooled to ambient temperature and filtered to remove the
catalyst. The filtrate was concentrated by rotary evaporation,
dissolved in ethyl acetate (50 mL), and washed sequentially
with saturated ammonium chloride, saturated potassium
fluoride, and brine. The organic layer was dried over magne-
sium sulfate (anhydrous), filtered, and evaporated to dryness.
The residue was purified by column chromatography on silica
gel using hexanes-ethyl acetate or chloroform-methanol as
the eluent.
Gen er a l P r oced u r e for Dea cet yla t ion . Syn t h esis of
5a -g,j. The purified 3-acetoxy-17R-(4-substituted phenyl)vinyl
estradiols were dissolved in methanol (5 mL) containing 0.4
mL of 10 N sodium hydroxide (or sodium methoxide for 5f).
The solution was stirred for 2 h and then acidified with dilute
acetic acid (4%) and partitioned between ethyl acetate and
water. The organic phase was washed with 10% sodium
bicarbonate, dried over magnesium sulfate, filtered, and
evaporated to dryness. The crude product was purified by
column chromatography on silica gel using hexanes-ethyl
acetate. The final compounds were crystallized from hexanes-
acetone (ethyl acetate) to provide analytical samples for the
binding studies.
Meth od II. Gen er a l P r oced u r e for Solid -P h a se Syn -
th esis of 5h ,i. P r ep a r a tion of th e Resin -Bou n d 17r-Tr i-
n -bu tylsta n n ylvin yl Estr a d iol (7). The 17R-ethynyl estra-
diol 1 (3 g, 10 mmol) was dissolved in THF in a flask and
treated with triethylborane (2 mL, 17 mmol) and tributyltin
hydride (3 g, 11 mmol). The mixture was stirred at 40 °C for
10 h. The reaction mixture was evaporated to dryness, dis-
solved in CH2Cl2, and then transferred to the preswollen
carboxy resin (5 g) in CH2Cl2 (20 mL) in the presence of DCC
(2.3 g, 11 mmol). A catalytic amount of DMAP was added to
the mixture, and the reaction mixture was allowed to stand
for 24 h. The total loading yield for the mixture of E and Z
isomers was 50% (0.59 mmol/g), comprising 47% (0.56 mmol/
g) E isomer and 3% Z isomer (0.03 mmol/g).
17r-20E-21-P h en yl-19-n or p r egn a -1,3,5(10),20-tetr a en e-
3,17â-d iol 3-Aceta te (4a ). 25% yield, Rf ) 0.23 (hexanes-
1
ethyl acetate, 5:1). H NMR (300 MHz, CDCl3): δ 0.88 (s, 3H,
C18-CH3), 1.2-2.4 (m, steroid envelope), 2.28 (s, 3H, CH3Cd
O-), 2.7-2.9 (m, 2H, C6R-H and C6â-H), 6.48 (d, 1H, J ) 16.1
Hz, CHdC21H), 6.60 (d, 1H, J ) 16.1 Hz, C20HdCH), 6.80 (d,
1H, J ) 2.3 Hz, C4-H), 6.83 (dd, 1H, J ) 2.4, 8.4 Hz, C2-H),
7.26 (d, 2H, J ) 7.4 Hz, C1 and C25-H), 7.34 (t, 2H, J ) 7.8
Hz, C24 and C26-H), 7.44 (d, 2H, J ) 7.1 Hz, C23 and C27-H).
13C NMR (75.4 MHz, CDCl3): δ 14.10 (C18), 21.08 (CH3CdO),
23.35 (C15), 26.09 (C11), 27.17 (C7), 29.50(C6), 32.43 (C12), 36.85
(C16), 39.08 (C8), 43.77 (C9), 47.36 (C13), 49.34 (C14), 84.03 (C17),
118.49 (C2), 121.41 (C4), 126.31 (C25), 126.40 (C24, C26), 127.32
(C1), 127.50 (C21), 128.55 (C23, C27), 134.82 (C10), 137.10 (C20),
137.94 (C5), 138.14 (C22), 148.33 (C3), 169.80 (CH3CdO).
17r-20E-21-P h en yl-19-n or p r egn a -1,3,5(10),20-tetr a en e-
3,17â-d iol (5a ). Hydrolysis of the 3-acetate group afforded the
product in 92% yield. Rf ) 0.18 (hexanes-ethyl acetate, 4:1),
mp 176-177 °C, Rf ) 0.17 (hexane-acetone, 4:1), elemental
analysis C26H30O2‚0.5CH3CO2C2H5 . 1H NMR (300MHz, acetone-
d6): δ 1.01 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid envelope),
2.7-2.9 (m, 2H, C6R-H and C6â-H), 3.77 (s, 1H, C17â-OH),
6.52 (d, 1H, J ) 2.6 Hz, C4-H), 6.58 (dd, 1H, J ) 2.7, 8.4 Hz,
C2-H), 6.63 (s, 2H, C20HdC21H), 7.07 (d, 1H, J ) 8.3 Hz, C1-
H), 7.20 (t, 1H, J ) 7.2 Hz, C25-H), 7.31 (t, 2H, J ) 7.7 Hz,
C
24, C26-H), 7.46 (d, 2H, J ) 7.1 Hz, C23, C27-H). 13C NMR
(75.4 MHz, acetone-d6): δ 14.73 (C18), 24.09 (C15), 27.28 (C11),
28.31 (C7), 33.46 (C12), 37.41 (C16), 40.71 (C8), 44.62 (C9), 48.29
(C13), 50.06 (C14), 84.10 (C17), 113.52 (C2), 115.89 (C4), 126.98
(C25), 127.13 (C24and C26), 127.38 (C1), 127.70 (C21), 129.31 (C23
and C27), 132.06 (C10), 137.24 (C20), 138.39 (C5), 138.71(C22),
155.87 (C3).
17r-20E-21-(4-Hyd r oxyp h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol 3-Aceta te (4b). To a solution of
3-acetoxy-17R-E-(tri-n-butylstannyl)vinyl estradiol (0.35 g, 0.56
mmol) in toluene (5 mL) were added 4-iodophenol (0.15 g, 0.68
mmol), three crystals of 3,5-di-tert-butyl-4-hydroxytoluene, and
a catalytic amount (15 mg) of Pd(PPh3)4 to afford 50 mg of the
product in 21% yield as an amorphous solid.
17r-20E-21-(4-Hyd r oxyp h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol (5b). Evaporation followed by silica
gel column chromatography with 2% methanol in chloroform
afforded the amorphous product in 89% yield (0.04 g). Elemen-
tal analysis C26H30O3‚0.5CH3CO2C2H5. 1H NMR (300 MHz,
acetone-d6): δ 0.86 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid
envelope), 2.7-2.9 (m, 2H, C6R-H and C6â-H), 3.67 (s, 1H,
C17â-OH), 6.28 (d, 1H, J ) 16 Hz, CHdC21H), 6.39 (d, 1H, J
) 2.7 Hz, C4-H), 6.40 (d, 1H, J ) 16.1 Hz, C20HdCH), 6.44
(dd, 1H, J ) 2.6, 8.4 Hz, C2-H), 6.66 (d, 2H, J ) 8.7 Hz, C24
,
C26-H), 6.94 (d, 1H, J ) 8.3 Hz, C1-H), 7.17 (d, 1H, J ) 8.6
Hz, C23-H and C27-H), 7.96 (s, 1H, C3-OH), 8.37 (s, 1H, C25
-
The stannylated resin (1.0 g, 0.6 mmol) was placed in the
reaction vessel and swelled with dichloromethane. The solvent
was removed by evacuation, and the resin was treated with
dry toluene (10 mL). To the slurry were added the appropriate
aryl halide (Br/I), two to three crystals of 3,5-di-tert-butyl-4-
hydroxytoluene, and a small amount (5 mg) of the Pd(0)
catalyst. The reaction mixture was heated at 80-90 °C
overnight under nitrogen. The reaction mixture was agitated
to maintain dispersal of the materials. After cooling to ambient
OH). 13C NMR (75.4 MHz, acetone-d6): δ 14.72 (C18), 24.05
(C15), 27.29 (C11), 28.32 (C7), 33.40 (C12), 37.26 (C16), 40.71 (C8),
44.66(C9), 48.16 (C13), 49.94 (C14), 84.02 (C17), 113.43 (C2),
115.80 (C4), 116.07 (C26), 116.15 (C24), 126.99 (C21), 127.21 (C1),
128.33 (C23 and C27), 130.27 (C22), 132.08 (C10), 134.02 (C20),
138.40 (C5), 155.78 (C3), 157.45 (C25).
17r-20E-21-(4-cya n op h en yl)-19-n or p r egn a -1,3,5(10),20-
tetr a en e-3,17â-d iol 3-Aceta te (4c). The purification by silica

Tetraene-3,17â-diols
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2873
gel column chromatography using a hexanes-ethyl acetate
gradient (5/1 f 3/1) afforded the product in 50% yield. Rf )
0.21 (hexanes-ethyl acetate, 3:1). 1H NMR (CDCl3, 300
MHz): δ 0.98 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid envelope),
2.28 (s, 3H, CH3CdO), 2.7-2.9 (m, 2H, C6R-H and C6â-H),
6.63 (t, 2H, J ) 16.6 Hz, C20HdC21H), 6.79 (d, 1H, J ) 2.3 Hz,
C4-H), 6.83 (dd, 1H, J ) 2.5, 8.4 Hz, C2-H), 7.24 (d, 1H, J )
8.3Hz, C1-H), 7.49 (d, 2H, J ) 8.3 Hz, C23-H and C27-H),
7.60 (d, 2H, J ) 8.3 Hz, C24-H and C26-H). 13C NMR (75.4
MHz, acetone-d6): δ 14.05 (C18), 20.99 (CH3CdO), 23.31 (C15),
25.98 (C11), 27.10 (C7), 29.37 (C6), 32.53 (C12), 37.12 (C16), 38.99
(C8), 43.69 (C9), 47.54 (C13), 49.59 (C14), 84.03 (C17), 110.22 (C25),
118.47 (C2), 118.90 (CtN), 121.38 (C4), 125.73 (C1), 126.19
(C21), 126.81 (C23, C27), 132.27 (C24, C26), 137.62 (C10), 137.98
(C5), 138.98 (C20), 141.68 (C22), 148.30 (C3), 169.74 (CH3CdO).
17r-20E-21-(4-Cya n op h en yl)-19-n or p r egn a -1,3,5(10),20-
tetr a en e-3,17â-d iol (5c). The product was purified by silica
gel column chromatography with a hexanes-ethyl acetate
(4:1) eluent. Recrystallization (hexane-acetone) afforded the
pure product (0.11 g, 0.26 mmol) in 80% yield. Rf ) 0.08
(hexanes-ethyl acetate, 4:1), mp 139-140 °C, elemental
analysis C27H29O2N1‚0.5CH3CO2C2H5. 1H NMR (300 MHz,
acetone-d6): δ 1.01 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid
envelope), 2.7-2.9 (m, 2H, C6R-H and C6â-H), 3.92 (s, 1H,
C17â-OH), 6.52 (s, 1H, C4-H), 6.58 (dd, 1H, J ) 2.7, 8.7 Hz,
C2-H), 6.73 (d, 1H, J ) 16 Hz, CHdC21H), 6.90 (d, 1H, J )
16 Hz, C20HdCH), 7.07 (d, 1H, J ) 8.3 Hz, C1-H), 7.67 (d,
2H, J ) 8.9 Hz, C23-H and C27-H), 7.71 (d, 2H, J ) 8.7 Hz,
C24-H and C26-H), 7.97 (s, 1H, C3-OH). 13C NMR (75.4 MHz,
acetone-d6,): δ 14.72 (C18), 24.13 (C15), 27.26 (C11), 28.30 (C7),
(C6), 33.57(C12), 37.65 (C16), 40.69 (C8), 44.55 (C9), 48.54 (C13),
50.18 (C14), 84.29 (C17), 110.76 (C25), 113.53 (C2), 115.90 (C4),
119.51 (CtN), 125.90 (C21), 126.97 (C1), 127.88 (C23, C27),
131.95 (C10), 133.18 (C24, C26), 138.36 (C5), 141.73 (C20), 143.39
(C22), 155.89 (C3).
8.4 Hz, C1-H), 7.50 (d, 2H, J ) 8.4 Hz, C23-H and C27-H),
7.92 (d, 2H, J ) 8.4 Hz, C24-H and C26-H).
17r-20E-21-(4-Acetylp h en yl)-19-n or p r egn a -1,3,5(10),20-
tetr a en e-3, 17â-d iol (5e). The product was purified by
recrystallization (hexanes-ethyl acetate) to afford 0.27 g (0.65
mmol). 83% yield, mp149-150 °C, Rf ) 0.07 (hexanes-ethyl
acetate, 4:1), elemental analysis C28H32O3‚0.5CH3CO2C2H5. 1H
NMR (300 MHz, acetone-d6): δ 1.02 (s, 3H, C18-CH3), 1.2-
2.4 (m, steroid envelope), 2.56 (s, 3H, CdOCH3), 2.7-2.9 (m,
2H, C6R-H and C6â-H), 3.88 (s, 1H, C17â-OH), 6.52 (d, 1H, J
) 2.6 Hz, C4-H), 6.58 (dd, 1H, J ) 2.7, 8.3 Hz, C2-H), 6.72
(d, 1H, J ) 16 Hz, CHdC21H), 6.85 (d, 1H, J ) 16 Hz, C20Hd
CH), 7.07 (d, 1H, J ) 8.3 Hz, C1-H), 7.60 (d, 2H, J ) 8.5 Hz,
C23-H and C27-H), 7.94 (d, 2H, J ) 8.5 Hz, C24-H and C26
-
H). 13C NMR (75.4 MHz, acetone-d6): δ 14.75 (C18), 24.15 (C15),
26.57 (CdOCH3), 27.29 (C11), 28.33 (C7), 33.57 (C12), 37.51 (C16),
40.72 (C8), 44.61 (C9), 48.49 (C13), 50.20 (C14), 84.27 (C17),
113.52 (C2), 115.88 (C4), 126.52 (C1), 126.98 (C21), 127.19 (C24
26), 129.46 (C23, C27), 132.0 (C10), 136.61 (C25), 138.39 (C5),
140.49 (C20), 143.28 (C22), 165.46 (C3), 197.20 (C)OCH3).
,
C
17r-20E-21-(4-Meth oxyca r bon ylp h en yl)-19-n or p r egn a -
1,3,5(10),20-tetr a en e-3,17â-d iol 3-Aceta te (4f). The product
was purified by silica gel column chromatography using a
hexanes-ethyl acetate gradient (4/1 f 3/1) in 65% yield. Rf )
1
0.28 (hexanes-ethyl acetate, 3:1), amorphous. H NMR (300
MHz, CDCl3): δ 0.99 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid
envelope), 2.28 (s, 3H, CH3CdO), 2.7-2.9 (m, 2H, C6R-H and
C6â-H), 3.91 (s, 3H, CdOOCH3), 6.59 (d, 1H, J ) 16.1 Hz,
CHdC21H), 6.66 (d, 1H, J ) 16.1 Hz, C20HdCH), 6.79 (s, 1H,
J ) 2.4 Hz, C4-H), 6.82 (dd, 1H, J ) 2.6, 8.4 Hz, C2-H), 7.24
(d, 1H, J ) 8.7 Hz, C1-H), 7.48 (d, 2H, J ) 8.4 Hz, C23-H
and C27-H), 8.0 (d, 2H, J ) 8.5 Hz, C24-H and C26-H). 13C
NMR (75.4 MHz, CDCl3): δ 14.14 (C18), 21.10 (CH3CdO), 23.41
(C15), 26.10 (C11), 27.20 (C7), 29.51 (C6), 32.58 (C12), 37.14 (C16),
39.12 (C8), 43.80 (C9), 47.56 (C13), 49.58 (C14), 52.03 (CdO-
17r-20E-21-(4-Meth ylp h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol 3-Aceta te (4d ). The product was
purified by silica gel column chromatography using a hex-
anes-ethyl acetate (4:1) eluent. 59% yield, Rf ) 0.26 (hex-
anes-ethyl acetate, 4:1), amorphous. 1H NMR (300 MHz,
CDCl3): δ 0.90 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid envelope),
2.19 (s, 3H, C28-CH3), 2.28 (s, 3H, CH3CdO), 2.7-2.9 (m, 2H,
OCH3), 84.16 (C17), 118.55 (C2), 121.46 (C4), 126.30 (C24, C25
,
C
26), 126.61 (C1), 128.75 (C21), 129.93 (C23, C27), 137.67 (C10),
137.84 (C20), 138.14 (C5), 141.67 (C22), 148.38 (C3), 166.88
(CdOOCH3), 169.83 (CH3CdO).
17r-20E-21-(4-Meth oxycar bon ylyph en yl)-19-n or pr egn a-
1,3,5(10),20-tetr a en e-3,17â-d iol (5f). The product was puri-
fied by silica gel column chromatography using a hexane-
acetone system (3:1). Recrystallization using hexanes-ethyl
acetate afforded the pure product in a 25% yield. Rf ) 0.19
(hexane-acetone, 3:1), mp 144-145 °C, elemental analysis
C
6R-H and C6â-H), 6.34 (d, 1H, J ) 16.1 Hz, CHdC21H), 6.46
(d, 1H, J ) 16 Hz, C20HdCH), 6.71 (s, 1H, C4-H), 6.74 (dd,
1H, J ) 2.6, 8.3 Hz, C2-H), 7.06 (d, 1H, J ) 7.8 Hz, C24-H
and C26-H), 7.16 (d, 1H, J ) 8.5 Hz, C1-H), 7.25 (d, 2H, J )
8.2 Hz, C23-H and C27-H).
C
28H32O4. 1H NMR (300 MHz, acetone-d6): δ 1.01 (s, 3H, C18
-
CH3), 1.2-2.4 (m, steroid envelope), 2.28 (s, 3H, CH3CdO),
2.7-2.9 (m, 2H, C6R-H and C6â-H), 3.87 (s, 3H, CdOOCH3),
6.53 (s, 1H, C4-H), 6.58 (dd, 1H, J ) 2.2, 8.4 Hz, C2-H), 6.72
(d, 1H, J ) 16 Hz, CHdC21H), 6.85 (d, 1H, J ) 15.9 Hz, C20Hd
CH), 7.07 (d, 1H, J ) 8.6 Hz, C1-H), 7.60 (d, 2H, J ) 8.3 Hz,
C23-H and C27-H), 7.93 (s, 1H, C3-OH), 7.95 (d, 2H, J ) 8.3
Hz, C24-H and C26-H). 13C NMR (75.4 MHz, acetone-d6): δ
14.75 (C18), 24.15 (C15), 27.29 (C11), 28.32 (C7), 33.57 (C12), 37.59
(C16), 40.73 (C8), 44.60 (C9), 48.49 (C13), 50.20 (C14), 52.17
(CdOOCH3), 84.28 (C17), 113.55 (C2), 115.92 (C4), 126.48 (C25),
126.99 (C1), 127.16 (C24, C26), 129.33 (C21), 130.51 (C23, C27),
132.03 (C10), 138.40 (C5), 140.50 (C20), 143.41 (C22), 155.80 (C3),
167.01 (C)OOCH3).
17r-20E-21-(4-Ca r boxyp h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol (5g). Compound 5g was prepared by
the same method as compound 5f. 91% yield, mp 157-158 °C,
Rf ) 0.24 (CHCl3-CH3OH, 95:5); elemental analysis C27H32O4.
1H NMR (300 MHz, acetone-d6): δ 1.02 (s, 3H, C18-CH3), 1.2-
2.4 (m, steroid envelope), 2.7-2.9 (m, 2H, C6R-H and C6â-H),
6.54 (d, 1H, J ) 2.5 Hz, C4-H), 6.59 (dd, 1H, J ) 2.6, 8.5 Hz,
C2-H), 6.73 (d, 1H, J ) 16 Hz, CHdC21H), 6.84 (d, 1H, J )
16.1Hz, C20HdCH), 7.06 (d, 1H, J ) 8.8 Hz, C1-H), 7.59 (d,
2H, J ) 8.3 Hz, C23-H and C27-H), 8.0 (d, 2H, J ) 8.3 Hz,
C24-H and C26-H). 13C NMR (75.4 MHz, acetone-d6): δ 14.75
(C18), 24.11 (C15), 27.23 (C11), 28.26 (C7, C6), 33.50 (C12), 37.50
(C16), 40.65 (C8), 44.52 (C9), 48.45 (C13), 50.17 (C14), 84.31 (C17),
17r-20E-21-(4-Meth ylp h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol (5d ). The recrystallization (hexane-
acetone) step afforded the pure product (0.09 g). 60% yield, Rf
) 0.19 (hexanes-ethyl acetate, 4:1), mp 169-170 °C, elemen-
tal analysis C27H32O2‚0.5CH3CO2C2H5. 1H NMR (300 MHz,
acetone-d6): δ 1.00 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid
envelope), 2.30 (s, 3H, C28-CH3), 2.7-2.9 (m, 2H, C6R-H and
C6â-H), 3.72 (s, 1H, C17â-OH), 6.52-6.63 (m, 4H, C2-H, C4-
H, C20HdCH and CHdC21H), 7.07 (d, 1H, J ) 8.8 Hz, C1-H),
7.13 (d, 1H, J ) 7.8 Hz, C24-H and C26-H), 7.35 (d, 2H, J )
8.1 Hz, C23-H and C27-H), 7.95 (s, 1H, C3-OH). 13C NMR
(75.4 MHz, acetone-d6): δ 14.73 (C18), 21.06 (C28), 24.09 (C15),
27.29 (C11), 28.32 (C7, C6), 33.45 (C12), 37.35 (C16), 40.73 (C8),
44.65 (C9), 48.26 (C13), 50.04 (C14), 84.07 (C17), 113.53 (C2),
115.91 (C4), 126.98 (C1), 127.08 (C24, C26), 127.30 (C20), 129.94
(C23, C27), 132.06 (C10), 135.94 (C22), 136.15 (C21), 137.27 (C25),
138.40 (C5), 155.90 (C3).
17r-20E-21-(4-a cetylp h en yl)-19-n or p r egn a -1, 3, 5, (10),
20-tetr a en e-3,17â-d iol 3-Aceta te (4e). The product was
purified by silica gel column chromatography using a hex-
anes-ethyl acetate gradient (8/1 f 1/1). 89% yield, Rf )0.18
1
(hexanes-ethyl acetate, 3:1). H NMR (300 MHz, CDCl3): δ
1.02 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid envelope), 2.28 (s,
3H, C3, CH3-CdO), 2.56 (s, 3H, C29, CdOCH3), 2.7-2.9 (m,
2H, C6R-H and C6â-H), 6.60 (d, 1H, J ) 16.1 Hz, CHdC21H),
6.67 (d, 1H, J ) 16.1 Hz, C20HdCH), 6.79 (d, 1H, J ) 2.3 Hz,
C4-H), 6.82 (dd, 1H, J ) 2.6, 8.4 Hz, C2-H), 7.24 (d, 1H, J )
113.51 (C2), 115.89 (C4), 126.57 (C21), 126.94 (C1), 127.08 (C24
,

2874 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14
26), 129.59 (C25), 130.79 (C23, C27), 132.01 (C10), 138.36 (C5),
Hanson et al.
C
washed with water (5 × 100 mL) and brine (200 mL), dried
over magnesium sulfate, filtered, and concentrated to yield a
yellow powder. The residue was chromatographed on a silica
gel column (50 g) using 98:2 chloroform-methanol as the
eluting solvent to give 4j (0.46 g, 37%). Rf ) 0.2 (hexane-ethyl
140.22 (C20), 143.26 (C22), 155.78 (C3), 167.58 (CdOOH).
17r-20E-21-(4-Tr iflu or om eth ylp h en yl)-19-n or p r egn a -
1,3,5(10),20-tetr aen e-3,17â-diol (5h ). The product was cleaved,
and the resulting mixture was purified by silica gel column
chromatography using chloroform to afford 0.12 g of the E
isomer product and 1 mg of the Z isomer product. 49% yield,
Rf ) 0.15 (hexanes-ethyl acetate, 4:1), mp 215-217 °C,
1
acetate, 4:1). H NMR (CDCl3): δ 0.97 (s, 3H, 18-CH3), 1.2-
2.9 (m, b, 15H, steroid nucleus), 6.37 (d, 1H, J 20-21 ) 15.99
Hz, 20-H), 6.54 (d, 1H, J 21-20 ) 16.11 Hz, 21-H), 6.78 (d, 1H,
J 4-2 ) 2.3 Hz, 4-H), 6.83 (dd, 1H, J 2-4 ) 2.5 Hz, J 2-1 ) 8.4
Hz, 2-H), 6.9 (∼t, 2H, J 24-F and J 26-F ) 8.4 Hz, J 24-23 and J 26-27
) 6.7 Hz, J 24-27 and J 26-23 ) 2 Hz, 24-H and 26-H), 7.24 (d,
1H and CDCl3 peak, J 1-2 ) 8.3 Hz, 1-H), 7.37 (m, 2H, 25-H
and 27-H)
1
elemental analysis C27H29O2F3 . H NMR (300 MHz, acetone-
d6): δ 1.02 (s, 3H, C18-CH3), 1.2-2.4 (m, steroid envelope),
2.7-2.9 (m, 2H, C6R-H and C6â-H), 3.90 (s, 1H, C17â-OH),
6.53 (d, 1H, J ) 2.6 Hz, C4-H), 6.58 (dd, 1H, J ) 2.6, 8.4 Hz,
C2-H), 6.73 (d, 1H, J ) 16 Hz, CHdC21H), 6.85 (d, 1H, J )
16 Hz, C20HdCH), 7.07 (d, 1H, J ) 8.3 Hz, C1-H), 7.64 (d,
2H, J ) 8.7 Hz, C23-H and C27-H), 7.70 (d, 2H, J ) 8.6 Hz,
C24-H and C26-H), 8.0 (s, C3-OH). 13C NMR (75.4 MHz,
acetone-d6): δ 14.73 (C18), 24.13 (C15), 27.26 (C11), 28.31 (C7),
(C6), 33.54 (C12), 37.58 (C16), 40.69 (C8), 44.58 (C9), 48.46 (C13),
50.16 (C14), 84.23 (C17), 113.53 (C2), 115.90 (C4), 125.44 (q, J
) 270.6 Hz, CF3), 125.97(C21), 126.21 (q, J ) 3.5 Hz, C26),
126.22 (q, J ) 3.5 Hz, C24), 126.98 (C1), 127.62 (C23, C27), 128.85
(q, J ) 32 Hz, C25), 131.98 (C10), 138.38 (C5), 140.64 (C20),
142.75 (C22), 155.88 (C3).
17r,20E-21-(4-F lu or op h en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr aen e-3,17â-diol (5j). Our standard deprotection method
of 4j (0.34 g, 0.79 mmol) yielded 5j (0.31 g, 100%). Recrystal-
lization in hexane-acetone, 3:1, produced a fine white powder
(0.31 g, 97%). Rf ) 0.17 (hexane-ethyl acetate, 4:1), mp 189-
191 °C, elemental analysis C26H29FO2‚0.5CH3CO2C2H5. 1H
NMR (acetone-d6): δ 1.0 (s, 3H, 18-CH3), 1.2-2.9 (m, 15H,
steroid nucleus), 6.39 (d, 1H, J 21-20 ) 16.08 Hz, 21-H), 6.55
(d, 1H, J 20-21 ) 16.02, 20-H), 6.56 (d, 1H, J 4-2 ) 2.79 Hz, 4-H),
6.61 (dd, 1H, J 2-1 ) 8.4 Hz, J 2-4 ) 2.82 Hz, 2-H), 7.02 (ddd,
2H, 24-H and 26-H), 7.12 (d, 1H, J 1-2 ) 8.31 Hz, 1-H), 7.38
(dd, 2H, 23-H and 27-H). 13C NMR (acetone-d6): δ 15.07 (C-
18), 24.42 (C-15), 27.60 (C-11), 28.64 (C-7), ∼29 under acetone
peak (C-6), 33.79 (C-12), 37.78 (C-16), 41.02 (C-8), 44.92 (C-
9), 48.61 (C-13), 50.34 (C-14), 84.44 (C-17), 113.86 (C-2), 116.24
(C-4), 116.30 (d, J CCF ) 21 Hz, C-24 and C-26), 126.50 (C-21),
127.30 (C-1), 129.15 (d, J CCCF ) 7.9 Hz, C-23 and C-27), 132.31
(C-10), 135.50 (C-22), 137.52 (C-20), 138.71 (C-5), 156.18 (C-
3), 163.06 (d, J C-F ) 243 Hz, C-25).
Molecu la r Mod elin g a n d Dyn a m ics. We initially evalu-
ated the conformations of our ligands 5a -j using the Builder
module from Insight II. Potentials for each atom were assigned
automatically or manually when necessary. Low-energy con-
formations were generated using the molecular mechanics
method (Discover program, 100 steps, 0.001 final convergence)
and compared to solution conformations determined by NMR.39
The ER-HBD used in our study was obtained from the Protein
Data Bank (PDB code 1QKU, wild-type ERR-HBD cocrystal-
lized with estradiol). Monomer C from the homodimer B/C was
selected for the docking and molecular dynamics studies. All
water molecules were deleted except for the one positioned
near ARG-394 and GLU-353 that is present in all crystal
structures. The monomer C contains all the amino acid
residues between ASN-304 and HIS-550. All manipulations
were performed using the Builder module in Insight II. The
complex of ER-LBD monomer and estradiol bound within the
binding cavity was minimized using the molecular mechanics
method (Discover_3 module, CVFF force field, conjugate
gradient minimization 10 000 steps, 0.001 final convergence).
Docking of the ligands with the ERR-HBD was performed
using the Docking module in InsightII.47 The ligand was
superimposed on the estradiol molecule (A-ring over A-ring),
and the estradiol was then deleted. During the docking
procedure, both the ligand and the protein residues within the
ligand binding cavity (amino acids within 15 Å of the ligand
as well as all amino acids in helix 12, loops 11-12, 1-3, 6-7)
were allowed to flex. In addition, the phenylvinyl side chain
of the ligand was rotated with 30° 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 the rms
distance of more than 0.125 Å were selected and used in
simulated annealing studies. In this procedure, the structures
were subjected to short molecular dynamics runs (100 fs per
stage, total of 50 stages, initial temperature of 500 K, final
temperature of 300 K, 1000 steps). CVFF force field and
default values for all other parameters were used.
17r-20E-21-(4-Meth oxyph en yl)-19-n or p r egn a -1,3,5(10),-
20-tetr a en e-3,17â-d iol (5i). 36% yield, Rf ) 0.23 (CHCl3-
CH3OH, 99:1), elemental analysis C27H32O3‚0.5CH3CO2C2H5.
1H NMR (300 MHz, acetone-d6): δ 0.99 (s, 3H, C18-CH3), 1.2-
2.4 (m, steroid envelope), 2.7-2.9 (m, 2H, C6R-H and C6â-H),
3.68 (s, 1H, C17â-OH), 3.78 (s, 3H, OCH3), 6.46 (d, 1H, J )
16.1 Hz, CHdC21H), 6.51-6.59 (m, 3H, C2-H, C4-H, and C20
-
H), 6.88 (d, 2H, J ) 8.8 Hz, C24-H and C26-H), 7.07 (d, 1H, J
) 8.3 Hz, C1-H), 7.39 (d, 2H, J ) 8.8 Hz, C23-H and C27-H),
7.95 (s, 1H, C3-OH). 13C NMR (75.4 MHz, acetone-d6): δ 14.74
(C18), 24.07 (C15), 27.30 (C11), 28.33 (C7, C6), 33.43 (C12), 37.32
(C16), 40.73 (C8), 44.67 (C9), 48.21 (C13), 49.98 (C14), 55.49
(OCH3), 84.05 (C17), 113.54 (C2), 114.73 (C24, C26), 115.91 (C4),
126.95 (C1), 126.98 (C21), 128.26 (C23, C27), 131.35 (C22), 132.07
(C10), 134.87 (C20), 138.40 (C5), 155.91 (C3), 159.89 (C25).
17r,20E-21-Iod o-19-n or p r egn a -1,3,5(10),20-tetr a en e-3,-
17â-d iol 3-Aceta te (8). To a solution of 3 (2.36 g, 3.75 mmol)
in chloroform-methylene chloride (1:1, 30 mL) was added a
slurry of N-iodosuccinimide (1.0 g, 4.4 mmol) in the same
solvent solution. The reaction mixture was stirred, under
aluminum foil, at 0 °C for 24 h. The reaction was followed by
TLC for the conversion of 3 (Rf ) 0.4, hexane-ethyl acetate,
5:1) to 8 (Rf ) 0.2, same solvent system). The reaction mixture
was washed with saturated sodium bicarbonate/water (50 mL).
Aqueous and organic layers were separated. Aqueous layer was
extracted with chloroform (50 mL × 2). Organic layers were
combined, washed with water (50 mL × 2) and brine (50 mL
× 2), dried over magnesium sulfate, and concentrated. The
yellow oil was separated on a silica gel column (60 g) and
covered with aluminum foil, using chloroform-methanol (98:
2) as the eluting solvent to give 8 as a pure white powder (1.62
g, 93%). Rf ) 0.2 (hexane-ethyl acetate, 5:1). 1H NMR
(CDCl3): δ 0.96 (s, 3H, 18-CH3), 1.2-2.9 (m, 15H, steroid
nucleus), 6.32 (d, 1H, J 21-20 ) 14.34 Hz, 21-H), 6.78 (d, 1H,
J 4-2 ) 2.46 Hz, 4-H), 6.84 (dd, 1H, J 2-4 ) 2.58 Hz, J 2-1 ) 8.04
Hz, 2-H), 6.88 (d, 1H, J 20-21 ) 14.22 Hz, 20-H), 7.29 (d, 1H
and CDCl3 peak, J 1-2 ) 8.28 Hz, 1-H). 13C NMR (CDCl3): δ
14.16 (C-18), 21.17 (-OCOCH3), 22.67 (C-15), 26.08 (C-11),
27.18 (C-7), 29.51 (C-6), 32.47 (C-12), 36.65 (C-16), 39.07 (C-
8), 43.77 (C-9), 47.07 (C-13), 49.35 (C-14), 74.72 (C-21), 87.10
(C-17), 118.62 (C-2), 121.52 (C-4), 126.40 (C-1), 137.80 (C-10),
138.15 (C-5), 150.46 (C-3), 148.43 (C-20), 169.92 (-OCOCH3).
Met h od III. Su zu k i Cou p lin g. Syn t h esis of 17r,20E-
21-(4-F lu or op h en yl)-19-n or p r egn a -1,3,5(10),20-tetr a en e-
3,17â-d iol 3-Aceta te (4j). To a solution of 8 (1.34 g, 2.9 mmol)
in THF (10 mL) was added tris(dibenzylideneacetone)di-
palladium (0.25 g, 0.27 mmol), sodium bicarbonate (1.28 g,
12.08 mmols, 4 equiv, in 5 mL of water), and 4-fluoroben-
zeneboronic acid (0.86 g, 6.1 mmol). The reaction mixture was
protected from light and stirred at room temperature for 12
h. The mixture was extracted with ethyl acetate (4 × 100 mL),
Binding energies were calculated for each of several struc-
tures generated during the docking studies. Values of the
binding energy ∆E
were calculated as the difference
between the potential energy of the complex (Ecomplex) and the
binding

Tetraene-3,17â-diols
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 14 2875
potential energy of the ligand (Eligand) and receptor (Ereceptor).52,53
Binding energy calculations were performed using the Energy
Analysis macro within the Discover_3 module.
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Recep tor Bin d in g Stu d ies. In Vitr o Com p etitive Bin d -
in g Assa y. The compounds were screened for their affinity
for the ERR-LBD isolated from BL 21 cells that overexpressed
the 33 kDa PER-23d ERG vector. The cells were induced with
0.6 mM isopropyl-â-thiogalactopyranoside for 3 h at room
temp, pelleted by centrifugation, frozen, and stored at -75 °C.
The cells were thawed and lysed by sonication (4 × 20 s) in
four volumes of lysis buffer (50 mM Tris, 50 mM NaCl, 1 mM
EDTA, 1 mM dithiothreitol, 1 M urea, pH 7.4) several times.
Clarified fractions, obtained at 30000g for 30 min were pooled,
assayed for receptor binding, and diluted to 50 nM in ER, and
100 µL aliquots were frozen and stored at -75 °C until ready
for use. Then 80 µL of the ERR-LBD-containing extract was
incubated with 10 µL of 10 nM 6,7-[H-3]-estradiol (specific
activity of 51 Ci/mmol) and 10 µL of either buffer, unlabeled
estradiol, or test ligand in 100 µL total volume. The final
concentrations were 1 nM 6,7-[H-3]-estradiol, 2 nM unlabeled
estradiol (using 200 nM estradiol to define specific binding),
and 0.5-5000 nM of the test ligand. In all cases, 10 µL of each
incubation solution was removed for assay of the actual initial
concentration of [H-3]-estradiol and the remainder was incu-
bated at 2 or 25° C for 18 h. After incubation, 100 µL of
dextran-coated charcoal suspension (fines removed) was added
to adsorb the unbound [H-3]-estradiol, the mixture was
incubated for 10 min and centrifuged, and 100 µL samples
were taken from the supernatant fraction for assay of radio-
activity. The results were calculated and plotted as the percent
specific binding as a function of the log of the competitor
concentration using the best fit equation for the binding
inhibition to define the 50% inhibition level. The relative
binding affinity was calculated as 100 × [E]/[C], where [E] was
the concentration of unlabeled estradiol needed to reduce the
specific binding of [H-3]-estradiol by 50% and [C] was the
concentration of test ligand needed to reduce the specific
binding by 50%.
Ack n ow led gm en t. We gratefully acknowledge Dr.
Roger Krautz for his assistance with the 500 MHz NMR
spectrometry. We are grateful for support of this re-
search through grants from the National Institutes of
Health [Grant PHS 1RO1 CA81049 and CA89489
(R.N.H.)], the U.S. Army Breast Cancer Research
Program [Grants DAMD 17-99-1- 9333 and 17-00-1-
00384 (R.N.H.)], and the Boothroyd Foundation (E.R.D.).
Molecular modeling was performed on instruments
supported in part by a grant from the National Science
Foundation [Grant CHE-9974642].
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