*Chem. Res. Toxicol.* **2001,** *14,* 280-294

**Structure-Activity Relationships for a Large Diverse Set\
of Natural, Synthetic, and Environmental Estrogens**

Hong Fang,^†^ Weida Tong,\*^,†^ Leming M. Shi,^†,‡^ Robert Blair,^§^
Roger Perkins,^†^\
William Branham,^§^ Bruce S. Hass,^§^ Qian Xie,^†^ Stacy L. Dial,^§^\
Carrie L. Moland,^§^ and Daniel M. Sheehan^§^

*R.O.W. Sciences, Inc., 3900 NCTR Road, MC 910, Jeferson, Arkansas
72079, and\
Division of Genetic and Reproductive Toxicology, National Center for
Toxicological Research,\
Jeferson, Arkansas 72079*

*Received October 3, 2000*

Understanding structural requirements for a chemical to exhibit estrogen
receptor (ER) binding has been important in various fields. This
knowledge has been directly and indirectly applied to design drugs for
human estrogen replacement therapy, and to identify estrogenic endocrine
disruptors. This paper reports structure-activity relationships (SARs)
based on a total of 230 chemicals, including both natural and
xenoestrogens. Activities were generated using a validated ER
competitive binding assay, which covers a 10^6^-fold range. This study
is focused on identification of structural commonalities among diverse
ER ligands. It provides an overall picture of how xenoestrogens
structurally resemble endogenous 17β-estradiol (E2) and the synthetic
estrogen diethylstilbestrol (DES). On the basis of SAR analysis, five
distinguishing criteria were found to be essential for xenoestrogen
activity, using E2 as a template: (1) H-bonding ability of the phenolic
ring mimicking the 3-OH, (2) H-bond donor mimicking the17β-OH and O-O
distance between 3- and 17β-OH, (3) precise steric hydrophobic centers
mimicking steric 7α- and 11β-substituents, (4) hydrophobicity, and (5) a
ring structure. The 3-position H-bonding ability of phenols is a
significant requirement for ER binding. This contributes as both a
H-bond donor and acceptor, although predominantly as a donor. However,
the 17β-OH contributes as a H-bond donor only. The precise space (the
size and orientation) of steric hydrophobic bulk groups is as important
as a 17β-OH. Where a direct comparison can be made, strong estrogens
tend to be more hydrophobic. A rigid ring structure favors ER binding.
The knowledge derived from this study is rationalized into a set of
hierarchical rules that will be useful in guidance for identification of
potential estrogens.

**Introduction**

There is a growing body of evidence that some man­made chemicals, now
called endocrine-disrupting chemi­cals (EDCs),^1^ have the potential to
disrupt the endocrine system by mimicking endogenous hormones such as
estrogens and androgens *(1).* Recent legislation mandates that the
Environmental Protection Agency develop a screening and testing program
for potential EDCs, of which xenoestrogens figure predominately *(2).*
Xenoestro­gens contain a number of chemical classes that display a broad
range of structural diversity *(3).* For example, DES, DDTs,
polychlorinated biphenyls (PCBs), alkylphe­nols, phthalates, and parabens
have been found to be estrogenic. It has long been an enigma why such
struc‑

\* To whom correspondence should be addressed: R.O.W. Sciences, Inc.,
3900 NCTR Road, MC 910, J efferson, AR 72079. Telephone: (870) 543-7142.
Fax: (870) 543-7382. [[E-mail:
wtong@nctr.fda.gov.]{.underline}](mailto:wtong@nctr.fda.gov.)

> † R.O.W. Sciences, Inc.
>
> ‡ Current address: BASF Corp., P.O. Box 400, Princeton, NJ 08543. §
> National Center for Toxicological Research.

1 Abbreviations: SAR, structure-activity relationship; QSAR,
quan­titative structure-activity relationship; ER, estrogen receptor;
hERR, human estrogen receptor R subtype; EDCs, endocrine disrupting
chemicals; RBA, relative binding affinity; NA, not active; NCTR,
National Center for Toxicological Research; 2D, two-dimensional; 3D,
three-dimensional; E2, 17*â*-estradiol; E1, estrone; E3, estriol; EE,
ethynylestradiol; DES, diethylstilbestrol; DMS, dimethylstilbestrol;
DDTs, 1,1,1-trichloro-2,2-bischlorophenylethane derivatives; PCBs,
polychlorinated biphenyls; log *P,* hydrophobicity.

turally diverse chemicals have estrogenic activity like the endogenous
hormone estradiol.

SARs for estrogens date back more than six decades to the early work of
Dodds et al. *(4, 5).* The succeeding two decades saw the discovery of
nonsteroidal estrogens, such as DES, based on understanding of the
important structural features governing potency for steroidal es­trogens.
Recently, a number of SAR studies have been reported for steroidal
estrogens *(6)* and nonsteroidal estrogens *(7).* These are generally
focused on identifica­tion of structural characteristics for chemicals
within similar two-dimensional (2D) structural frameworks, including E2
derivatives *(6),* DES derivatives *(8),* PCBs *(9),* phytoestrogens
*(10),* alkylphenols *(11),* raloxifenes *(12),* and others. Modern
computer-based tools have enabled the development of quantitative
structure­activity relationship (QSAR) models for identifying steric and
electrostatic features of a molecule in three-dimen­sional (3D) space for
estrogenic activity *(8, 13*-*19).* Recent crystallographic structures
of the human ER R subtype (hERα) with a number of ligands, including E2,
DES, raloxifene, and 4-OH-tamoxifene, have also been reported *(20,
21).* By aligning these four ligands on the basis of the superposition
of their ER binding sites, we have been able to demonstrate the common
binding characteristics among these ligands *(22).*

10.1021/tx000208y CCC: \$20.00 © 2001 American Chemical Society\
Published on Web 02/10/2001

In principle, chemicals with similar biological activity share common
structural features. This implies that structurally diverse estrogens
possess a certain degree of structural commonality essential to
eliciting estrogenic activity. Although a number of chemical classes are
known to be estrogenic, little thorough structural evalu­ation has been
presented for their structural resemblance to the E~2~ or the strong
synthetic estrogen DES. Moreover, only limited efforts have explored the
structural similari­ties between different chemical classes of
xenoestrogens. To better understand the structural requirements for ER
binding, it is important to have a reliable data set, obtained with
consistent assay design, covering a broad range of chemical classes.
Recently, we reported ER binding activity data for a large number of
chemicals, including natural, synthetic, and environmental estro­gens,
using a validated rat ER competitive binding assay *(23, 24).* This data
set, called the NCTR data set, currently contains 230 chemicals. It was
designed a priori to cover broad structural diversity and a wide range
of binding activities for elucidating the structural charac­teristics of
xenoestrogens and natural estrogens.

The rat uterine cytosol ER competitive binding assay is the gold
standard for in vitro ER assays. When our results are compared to
results from other ER binding assays, there is a general consistency
between relative ER activities across different assay methods and
species *(25).* For example, we found a high linear correlation for ER
binding affinities among a diverse group of chemicals assayed with the
ER from rat uterine cytosol and hERα.^2^ Further, we also found that ER
assay results correlated very well with those from a yeast-based
reporter gene assay and a MCF-7 cell proliferation assay. These find­ings
demonstrate that ER binding is the major determi­nant across three levels
of biological complexity (receptor binding, a yeast reporter gene
response, and cell prolif­eration) of estrogen action. Moreover,
chemicals positive in uterotrophic responses (in vivo estrogenic
activity) are also positive in the ER binding assay, indicating that
binding affinity is a good predictor of in vivo activity with few false
negatives observed *(26).* Therefore, understand­ing the structural
requirements for ER binding provides strong guidance in identifying
potential in vivo estrogenic EDCs.

The NCTR data set covers most known estrogenic chemical classes as well
as some new estrogenic chemi­cals. A careful SAR examination of the NCTR
data set in conjunction with knowledge of the recently reported
ligand-ER crystal structures should allow a better understanding of the
general structural requirements of a chemical binding to the ER in a
quantitative manner. In turn, the knowledge can be used to develop a
predic­tive toxicology model for rapidly identifying potential estrogenic
EDCs.

**Materials and Methods**

**ER Binding Activities.** The ER binding affinities of chemi­cals were
determined by using a competitive receptor binding assay described
previously *(23).* Briefly, a chemical's binding activity was determined
by competing with radiolabeled \[^3^H\]-E2 for the ER in rat uterine
cytosol. The IC~50~ (50% inhibition of \[^3^H\]E~2~ binding) for each
competitor was determined. The relative binding affinity (RBA) for each
competitor was calcu­lated by dividing the IC~50~ of E~2~ by the IC~50~
of the competitor

> ^2^ H. Fang, W. Tong, and D. Sheehan, unpublished results.

and multiplying by 100 (E~2~ RBA ) 100). The validated assay incubation
conditions were 20 h at 4°C using 17 mg of uterine

tissue/mL (*B*~max~) 0.22 nM) with 1 nM \[^3^H\]E~2~. The competing

chemical concentrations ranged from 1 nM to 1 mM. Chemicals that failed
to compete for \[^3^H\]E~2~ binding to the ER were designated as "not
active" (NA). Chemicals that exhibited binding, but did not reach 50%
inhibition in the designed concentration range, were designated as
"slight binders". All assays were repeated at least twice; the IC~50~
values of positive chemicals are the means of the replicate values. The
standard deviation of IC~50~ for each chemical was reported *(23),* and
only the mean RBA value was used for this study. The purity of chemicals
as well as their effect on RBAs was also studied by Blair et al. *(23).*
The largest fold difference (\~10-fold) was found for nonylphenol from
different commercial sources due to the impurity of the sample.

**Molecular Modeling.** The crystal structures of E~2~, DES, raloxifene,
and 4-OH-tamoxifen bound to the ER were obtained from the Protein Data
Bank (PDB) as entries 1A52, 3ERD, 1ERR, and 3ERT, respectively. The
alignment of these four ligands, based on root-mean-square (RMS) fitting
of their receptor coordinates, was performed using the InsightII
software package (Molecular Simulations, Inc., San Diego, CA). Log *P*
was calculated using the atom/fragment contribution method *(27).*
Pharmacophore searching was performed with the CATA­LYST package
(Molecular Simulations, Inc.). The energy dif­ferences for E2 and DES
between the conformation in their binding modes and that in the minimum
conformation of the free ligands were calculated using the AM1 model
Hamiltonian ofthe AMPAC/MOPAC module in InsightII (Molecular
Simula­tions, Inc.). The atom-atom distance was also measured using
InsightII (Molecular Simulations, Inc.).

**Results**

We determined the ER RBA of 230 chemicals, of which 130 were active and
100 inactive. To the best of our knowledge, this is the largest
published ER competitive binding data set. This NCTR data set has been
exten­sively used to build and validate a series of computational models
proposed for priority setting of potential estro­genic EDCs *(22).*

For the convenience of analysis and presentation, the NCTR data set is
divided into seven major categories according to the chemical structural
characteristics. The chemical names and structures, as well as RBA
values, are shown in Figures 1-7 for 130 active chemicals and a few of
the selected inactive chemicals. Steroids are chemicals with a steroidal
backbone. E~2~ derivatives have a phenolic A ring (Figure 1A), whereas
the others lack a phenolic A ring (Figure 1B). The common structural
feature of DES-like chemicals is two benzene rings sepa­rated by two
carbons that are connected by a double bond in DES derivatives (Figure
2A) and by a single bond in hexestrol derivatives (Figure 2B). In this
group of chemi­cals, triphenylethylene derivatives (Figure 2C) are
struc­turally similar to DES derivatives, but have an additional phenyl
group attached to the ethylene bridge group. Most synthetic
antiestrogens contain this structural feature. Phytoestrogens contain
four major structurally distinct chemical classes: flavonoids,
coumestans, chalconoids, and mycoestrogens. Flavonoids are the largest
class, containing flavones (Figure 3A), flavanones (Figure 3B), and
isoflavones (Figure 3C). They share a benzene ring directly connected to
a chromone or 4-chromanone. Coumestans (Figure 3D) contain the smallest
number of chemicals, of which coumestrol is the most rigid and flat
molecule of all the phytoestrogens used. In contrast, chalconoids
(Figure 3E) are the most structurally flexible

chemicals of the phytoestrogens. The generic structure of chalconoids is
two benzene rings separated by three carbons. Mycoestrogens (Figure 3F)
have a large ring fused with a benzene ring that contains two OH groups
at positions 14 and 16. Chemicals with twobenzene rings connected by one
carbon atom are classified as diphe­nylmethanes, including
diphenolalkanes (Figure 4A), benzophenones (Figure 4B), and DDTs (Figure
4C). Biphenyls are chemicals with two phenyl rings directly connected to
each other. They can be chlorinated as PCBs (Figure 5A) or
nonchlorinated (Figure 5B). Alkylphenols (Figure 6A), parabens (Figure
6B), and alkyloxyphenols (Figure 6C) are categorized as phenols. They
all contain a single phenolic ring. Most chemicals in this group have a
long alkyl chain substituted at the para position. The chemicals that do
not belong to any of these groups are found in miscellaneous (Figure
7).![](media/image1.png){width="6.970138888888889in"
height="5.4631944444444445in"}

Chemicals that exhibit ER binding have a broad range of structural
diversity and RBAs. A common structural feature for steroids, DES-like
chemicals, and most phyto­estrogens is the presence of two rings (one of
them usually a phenolic ring) separated by two carbons. Chemicals either
with two rings separated by one carbon atom (diphenylmethanes),
connected directly (PCBs), or possessing only one ring (alkylphenols,
phthalates, and kepone) typically have relatively lower binding
affinities than chemicals with two rings separated by two atoms *(25),*
although there is overlap in RBAs among some

chemical classes. Table 1 shows the mean RBA values for each chemical
class. Steroids and DES-like chemicals have the strongest binding
activities. The RBAs of the rest of the chemical classes follow this
order: phyto­estrogens \> diphenylmethanes \> biphenyls \> phenols. There
are only a few inactive chemicals in the DES-like chemical and phenol
classes. The mean RBA ofDES-like chemicals is 2.63, which is the highest
ofthe groups that were examined, while phenols (mean RBA ) 0.0015) has
the lowest mean RBA. These observations suggest that although both
classes are likely to bind to the ER, their binding specificity is
different due to the nature of their structures.

**Steroids.** E2, with an RBA of 100, is one of the most active
estrogens. It has a hydrophobic backbone with OH groups at each end of
the molecule. Such a molecular configuration was considered as the most
important structural features for ER binding *(6).* The recently
reported crystal structure ofthe ER-E2 complex reveals that the 3- and
17*β*-OH groups primarily serve as H-bond donors and acceptors in
interacting with the receptor binding site *(20).* The elimination or
modification of either of these two OH groups significantly reduces a
chemical's binding affinity for the receptor, as shown in Table 2. This
impact is more dramatic at the 3-position than at the 17*β*-position.
For example, the loss ofbinding activity of3-deoxy-E~2~ is 28-fold
greater than the loss for 17-deoxy-E~2~, indicating that the 3-OH is
more important than the

**Table 1. General Information for the ER Ligand Categories, Including
the Number of Chemicals with the Ratio of\
Active and Inactive Ligands, Mean RBA Values,*^a^* and Representative
Chemicals with Key Structural
Features*^b^***![](media/image2.png){width="6.778472222222222in"
height="3.439583333333333in"}

*^a^* Mean RBA values are calculated for active ligands only. *^b^* The
presence and absence of a structural feature are represented as 1 and 0,
respectively. *^c^* A ring mimics one of the B, C, and D rings of E2.

**Table 2. Effects of Elimination and Modification of the\
3- and 17*â***-OH on RBAs

+--------------------+------------+-----------------------+-----------+
| > elimination or   | > from     | > to                  | fold      |
+--------------------+------------+-----------------------+-----------+
| > modification     | > (RBA)    | (RBA)                 | d         |
|                    |            |                       | ifference |
+--------------------+------------+-----------------------+-----------+
| > 3-OH             | E~2~ (100) | 3-deoxy-E2 (0.5)      | 200       |
+--------------------+------------+-----------------------+-----------+
|                    | E~1~       | 3-deoxy-E1(0.006)     | 1218      |
|                    | (7.31)     |                       |           |
+--------------------+------------+-----------------------+-----------+
|                    | EE (190)   | mestranol (2.26)      | 84        |
+--------------------+------------+-----------------------+-----------+
|                    | E~3~       | 3-methylestriol       | 442       |
|                    | (9.72)     |                       |           |
+--------------------+------------+-----------------------+-----------+
| > 17*β*-OH         | E~2~ (100) | 17-deoxy-E2 (14.1)    | 7         |
+--------------------+------------+-----------------------+-----------+
|                    | E~2~ (100) | E1 (7.31)             | 14        |
+--------------------+------------+-----------------------+-----------+

17*β*-OH in ER binding. This is consistent with the findings from the
ER-E2 crystal structure that the 3-OH has H-bonding interactions with
Glu 353, Arg 394, and a water molecule, whereas 17*â*-OH only forms one
H-bond with His 524.

The elimination of 3-OH caused a greater reduction in the activity for
estrone (E1) than for E2 (Table 2). A similar result was also observed
for 3-OH methylation of estriol (E3) and ethynylestradiol (EE). In both
cases, the fold reduction in activity for stronger estrogens (E2 and EE)
was about 6 times lower than the fold reduction for relatively weak
estrogens (E1 and E3). This indicates that the phenolic ring is more
critical for weak estrogens than strong ones.

The lack of a phenolic ring is a likely explanation for why most other
steroids such as androgens, progester­ones, and cholesterols were
inactive in ER binding, or exhibited very low activity, such as
dihydrotestosterone (Figure 1B). Clearly, the precise distance
~(*d*O-O)~ between the 3- and 17*β*-OH groups of steroids, as well as
their orientation, governs binding affinity. For examples, because the
two OH groups had the same distance and orientation as those ofE2
(*d*~O-O~ ) 11.0 Å), 3*β*-androstane­diol (RBA ) 0.12, *d*O-O ) 11.3 Å)
was one of the most active steroids that does not contain a phenolic
ring. Also, 17R-E2 (RBA ) 3.07, *d*O-O ) 10.4 Å) has a RBA 33-fold lower
than that of E2, and the RBA of 3α-androstanediol

(RBA ) 0.002, *d*O-O ) 9.95 Å) is 60-fold lower than that of its
*β*-isomer.

The RBA of 17-deoxy-E2 (RBA = 14.1) was slightly larger than that of
estrone (RBA = 7.31), indicating that the 17-ketone group might not
contribute significantly to the binding as a H-bond acceptor. If the
ketone group of E1 was moved from the 17-position to the 16-position to
form estra-1,3,5(10)-trien-16-one-3-ol, the RBA was decreased 16-fold. A
similar activity reduction was also observed with introduction of an OH
group at the 16R-position of E2, resulting in a relatively weak
estrogen, E3. This demonstrates that a small polar group at the
16-position reduces a chemical's activity.

Systematic studies on the influences of substituents at various
positions of E2 revealed that for most positions the introduction of
substituents results in a loss of binding affinity *(17, 28).* For
example, the E2 metabolites 2-OH-E2 (RBA = 22.4) and 4-OH-E2 (RBA =
52.9), 6α­OH-E2 (RBA = 0.71), and E3 (RBA = 9.72) were less active than
E2 with 2-140-fold differences. The only positions where introduction of
a substituent does not interfere with binding are 7R, 11*β*, and 17α
*(6).* The degree of increase or decrease in activity is strongly
dependent on the substituents in those positions. The 7R-and
11*â*-positions are structurally equivalent for steroids and can bear
large substituents. Small steric substituents introduced at these
positions generally increase activity *(29).* Large substituents reduce
the RBA and give rise to antiestrogenic activity, such as ICI 182,780
(RBA = 37.5) and ICI 164,384 (RBA = 14.5). A 17α-ethynyl substituent is
favorable for ER binding. EE is a stronger estrogen (RBA = 190) than E2
(RBA = 100). Norethynodrel (RBA = 0.22) is a progesterone derivative
with a 17α-ethynyl substitution, and is a popular oral contraceptive
drug. It is a moderately strong binder (454-fold weaker than E2).

**DES-like Chemicals.** DES (RBA = 400) is one of the highest-affinity
synthetic estrogens. Its activity is 4 times

![](media/image3.png){width="2.8430555555555554in" height="7.35in"}

**Figure 2.** (A) DES, (B) hexestrol, and (C) triphenylethylene
derivatives.

higher than that of E2. The symmetry of the molecule, the distance
between the two OH groups, and the two ethyl side chains provide
precisely correct spacing for hydrophobic and H-bond interactions
*(21).*

To efficiently bind to a receptor, the ligand tends to adopt a
conformation that generally is not the one with the lowest energy. The
free ligand which is in a stable conformation adjusts to a less stable
conformation to achieve optimal binding. Nevertheless, ligand binding to
the ER will release energy in general. The energy difference of a
molecule, between the conformation in its binding mode and that in the
minimum conformation of the free ligand, contributes to the binding
affinity in a

negative way. The less energy a molecule sacrifices during the binding,
the stronger the binding affinity will be. E2 has a much larger energy
difference *(∆H*f *=* 40.5 kcal/mol) than DES *(∆H*f *=* 17.8 kcal/mol),
which might help explain why DES is a better binder.

The O-O distance is 1 Å larger in DES (*d*O-O ) =12.1 Å) than in E2 and
functionally resembles the 3- and 17*β*-OH's of E2. The loss of the
H-bonding capability of one OH group reduces the RBA. For example, if
one of the OH groups is converted to its methyl ether, DES mono­methyl
ether (RBA = 20.43), the RBA decreases 20-fold (Figure 2A). A similar
activity reduction was also observed for hexestrol (RBA = 300) (Figure
2B); replacing one of the OH groups with a methoxy group resulted in a
32-fold less active derivative, hexestrol monomethyl ether (RBA = 9.37).
The reduction in activity is much more significant when both OH groups
of DES are methylated; the RBA decreases more than 7000-fold for DES
dimethyl ether (RBA = 0.056). This demonstrates that while the loss of
one H-bond donor will reduce the binding affinity by 20-fold, the loss
of the second H-bond donors diminishes the activity by a further
350-fold.

4-Methyl-substituted hexestrol \[meso -*p*-(α,*β*-diethyl -*p*­
methylphenethyl)phenol, RBA = 4.00\] and hexestrol monomethyl ether (RBA
= 9.37) have comparable binding affinities. This is consistent with the
observation that 17-deoxy-E2 has an RBA close to that of E1. In both
cases, the chemicals with or without a H-bond acceptor exhibit similar
RBAs. It suggests that with a phenolic ring at one end, the contribution
of the OH group at the other end is mainly through H-bond donor
interaction with the receptor.

The degree of contributions to the binding activity of the two ethyl
groups of DES is as significant as its OH groups. DES (RBA = 400),
dimethylstilbestrol (DMS, RBA = 14.50), and 4,4-dihydroxystilbene (RBA )
0.28) are in sequence one carbon atom shorter at the two ethylene side
chains. The RBAs of these latter two chemicals were 28- and 1423-fold
lower than that of DES. The ethyl groups may contribute to ER binding in
three distinct ways. First, they increase the molecule's hydro­phobicity.
The hydrophobicity of DES (log *P=* 5.64) is larger than that ofE2 (log
*P=* 3.94). The two ethyl groups contribute more than one-third of the
hydrophobicity of the overall structure. Second, they maintain the rigid
binding conformation of DES. The decrease in the length of the side
chains introduces more flexibility for both phenolic rings. Third,
probably the most important contribution of the ethyl groups to binding
is to occupy the precise space that interacts with the receptor binding
pocket site, which resembles the 11*β*- and 7α-substituents of E2, and
therefore increase its RBA. It is evident that the attachment of small
alkyl substituents to the 11*β*­position can lead to a considerable
increase in binding affinity for E2, such as 11*β*-chloromethyl-,
ethyl-, and vinyl-E2 *(28)* with RBA values in the range of 120-230. On
the basis of the recent crystal structure of the E2-and DES-ER
complexes, both ligands could be aligned together by overlaying their
receptor coordinates. By modifying the ethyl group to the 11*β*-position
of the E2 binding conformation, we have been able to compare the
relative position of the ethyl groups from 11*β*-ethyl-E2 with that of
DES. As shown in Figure 8, the ethyl groups from both molecules are
positioned extremely close to each other, and fit well in the
hydrophobic binding pocket. This observation also suggests that the
specific

**Figure 3.** (A) Flavones, (B) flavanones, (C) isoflavones, (D)
coumestans, (E) chalconoids, and (F)
mycoestrogens.![](media/image4.png){width="6.876388888888889in"
height="7.9534722222222225in"}

orientation of the two ethyl groups of DES is critical to its function.
That may explain why meso-hexestrol (RBA = 300) was 80-fold stronger
than DL-hexestrol (RBA = 3.60).

In general, DES derivatives were stronger binders than hexestrol
derivatives, because the former has a rigid double bond connecting the
two benzene rings. This is evident from the RBA comparison of three
pairs of chemicals between these two chemical classes: DES versus
hexestrol, DES monoether versus hexestrol mono-

ether, and 4,4-dihydroxystilbene versus 4,4'-ethylene­diphenol. However,
4-phenylethylphenol (RBA = 0.002) with single phenol group was more
active than 4-stilbenol (slight binder). These observations indicate
that when both OH groups contribute to the binding, a rigid structure is
critical for a better fit to the ER. However, when a chemical contains
only one phenolic ring, binding is dependent on how well the rest of the
structure fits into the binding pocket and the binding activity is more
favorable to a chemical with certain flexibility.

![](media/image5.png){width="3.283333333333333in"
height="2.1868055555555554in"}

![](media/image6.png){width="0.5868055555555556in"
height="6.666666666666667e-2in"}

![](media/image7.png){width="3.1666666666666665in"
height="4.097222222222222in"}

**Figure 4.** (A) Diphenolalkanes, (B) benzophenones, and (C) DDTs.

Triphenylethylenes (Figure 2C), which act as anti­estrogens, are
structurally similar to DES, but have an additional phenyl group
attached at the ethylene moiety. The RBA value, presented here, cannot
distinguish agonist from antagonist activity. 4-OH-tamoxifen (RBA )
175.24) binds to the ER 100 times stronger than tamoxifen (RBA ) 1.62)
and 10 times stronger than droloxifene (RBA ) 15.24) because its
phenolic ring resembles the A ring of E2. If its OH group is eliminated
(tamoxifen, RBA ) 1.62) or its position is changed (droloxifene, RBA )
15.24), the binding affinity is re­duced. For clomiphene, a chloro
substituent at the ethylene and, for toremifene, a chloro substituent at
the ethyl side chain both produce binding affinities similar to that of
tamoxifen.

**Phytoestrogens.** There are four major structurally distinct chemical
classes of phytoestrogens *(10):* fla‑

![](media/image8.png){width="0.3229166666666667in"
height="7.708333333333334e-2in"}

![](media/image9.png){width="3.1770833333333335in"
height="2.783333333333333in"}

**Figure 5.** (A) PCBs and (B) nonchlorinated biphenyls.

vonoids, coumestans, chalconoids, and mycoestrogens. The binding
activity of these four classes follows this order: mycoestrogens \>
coumestans \> flavonoids \> chalconoids. The representative chemicals
for each class are R-zearalenol (RBA = 43), coumestrol (RBA = 0.9),
genistein (RBA = 0.45), and phloretin (RBA = 0.07).

Flavonoids are the largest class of phytoestrogens, which include
flavones (Figure 3A), flavanones (Figure 3B), and isoflavone (Figure
3C). Their basic construction consists of a benzene ring directly
connected to C2 or C3 of chromone or 4-chromanone. The positions of the
A and C benzene rings of flavonoids resemble those in DES, where they
are separated by two atoms in a trans conformation. Thus, the binding
affinity of flavonoids is largely dependent on the relative positions of
the OH groups at the A and C rings, respectively, to mimic the 4,4'-OH
of DES. It is generally considered that isofla­vones are more active than
flavones *(30).* That is because most isoflavones, such as genistein,
equol, and daidzein, have two OH groups at the 7- and 4'-positions that
match the 4- and 4-positions of DES, respectively (Figure 3C). Although
most flavones such as apigenin, kaempferol, fisetin, and morin also have
the 7- and 4'-positions occupied by OH groups, these two positions
correspond to the 3- and 4-positions of DES (Figure 3A). Actually, the
6- and 4-positions of flavones match the 4- and 4'-positions ofDES,
respectively. This was evident because 3,6,4'-trihydroxyflavone (RBA =
0.45) and 6,4'-dihydoxy­flavone (RBA = 0.15) have RBAs comparable to
those of isoflavones.

Like the 4- and 4'-OH groups of DES, the 6- and 4'-OH groups of
flavonoids (7- and 4'-OH in isoflavones) play a similar role in binding.
Although a small difference (\~2.5-fold) in RBA values was observed for
the biochanin A(RBA = 0.0043) and prunetin (RBA ) 0.0018) pair and the
6-hydroxyflavanone (RBA ) 0.0009) and 4-hydroxy­flavanone (RBA ) 0.0023)
pair, it is difficult to eliminate the possibility that both phenolic
rings (A and C) of flavonoids can functionally mimic the phenolic A ring
of E2. This suggests the possibility that flavonoids may bind to the ER
in either of two orientations that differ by 180°. The recent
description of the crystal structure for the genistein-ER*β* complex
revealed that its phenolic C ring

![](media/image10.png){width="3.0965277777777778in"
height="5.9430555555555555in"}

**Figure 6.** (A) Alkylphenols, (B) parabens, and (C) alkyloxy­phenols.

served the same function as the A ring of E2 to form the H-bond
interaction with the receptor *(31).*

Like DES derivatives that have larger RBAs than hexestrol derivatives,
flavones are slightlybetter binders than flavanones because their
benzene rings are attached to the double bond of the B ring of chromone,
which leads to a more rigid and flat structure. Within the same
structural frame, the flavone apigenin (RBA = 0.028) has an RBA 4 times
higher than that of the flavanone naringenin (RBA = 0.0075). Similarly,
genistein was more potent than 2,3-dihydroxygenistein *(32).* However,
the result is only applicable for flavonoids containing both OH groups
at the A and C rings. The relative activity is reversed between
6-hydroxyflavanone (RBA = 0.0009) and 6-hydroxyflavone (RBA = 0.0004),
which are consis­tent with observations on DES-like chemicals.

Generally, a large steric hindrance will reduce activity. Genistein and
naringin are inactive compared to genistein (RBA = 0.45) and naringenin
(RBA = 0.0075). However, compared to quercetin (NA), rutin exhibited a
very low activity. This indicates that the 3-position of flavones is
equivalent to the 7α-position of E2 where a large group can be fitted in
the binding pocket.

![](media/image11.png){width="2.870138888888889in"
height="3.4131944444444446in"}

**Figure 7.** Miscellaneous chemicals.

![](media/image12.png){width="3.3069444444444445in"
height="1.8138888888888889in"}

**Figure 8.** Superimposed structures of 11*β*-ethyl-E2 (green) and DES
(yellow). E2 and DES are first aligned on the basis of the superposition
of the receptor coordinates of the crystal struc­tures between the E2-
and DES-ER complexes. The 11*â*­position of the E2 is then modified by
adding an ethyl group to form 11*â*-ethylestradiol. The ethyl groups of
11*β*-ethyl-E2 and DES are positioned closely in a hydrophobic binding
pocket.

> Converting one of the OH groups to a methyl ether

significantly reduced the RBA of isoflavones. Prunetin (RBA = 0.0018)
and biochanin A(RBA = 0.0043) have

250- and 100-fold lower RBAs, respectively, than their parent compound,
genistein (RBA = 0.45), while the RBA of formononetin (RBA = 0.0013) is
17-fold lower than that of daizein.

Unlike the fact that 1-OH-E2 is less potent than E2 *(33),* an
additional OH group in position 5 of isoflavones, which

resembles the 1-position of E2, increases estrogenic activity *(10,
34).* Specifically, genistein is 20-fold more

active than daizein, and biochanin A is 4-fold more active than
formononetin. This may be explained by the forma­tion of an
intramolecular H-bond between the 5-OH and the carbonyl groups which
enhances the electron with­drawal of the carbonyl group and lead to a
better 7-OH H-bond donor.

> Two coumestans were assayed, and both had RBAs

\~100-fold lower than that of E2 (Figure 3D). While coumestrol has a
relatively rigid and flat structure that is similar to E2,
4-ethyl-7-hydroxy-3-(*p*-methoxyphenyl)-

**Table 3. Relationship between the O-**O Distance and\
the RBA

  ----------------- -------- ----------- ----------------- ------- ---------
  mycoestrogen      RBA      *d*O-O (Å)  mycoestrogen RBA          *d*O-O
                                                                   (Å)

  α-zearalanol      30       11.00       *β*-zearalenol    0.20    9.85

  α -zearalenol     43       11.28       zearalanone       2.1     9.77

  *β*-zearalanol    0.64     9.23                                  
  ----------------- -------- ----------- ----------------- ------- ---------

2*H*-1-benzopyran-2-one has an ethyl group that is func­tionally similar
to that of DES. The latter had the same RBA as coumestrol because its
ethyl group favors binding which compensates for the reduced RBA form
converting one of the OH groups to a methyl ether.

The generic structure of chalconoids is two benzene rings connected by
three carbons (Figure 3E). The O-O distance between the two OH groups in
the B ring and the one in the A ring is \~11 and \~12 Å, respectively,
which is comparable to those ofDES and E2. Since their flexible
molecular structures do not favor binding, chal­conoids have RBAs that
are 1000-fold lower than that of E2. The elimination of one of the OH
groups on one side, such as 4-hydroxychalcone and 4'-hydroxychalcone,
caused a loss in activity of \~20-fold. The results further demon­strate
that two OH groups within a precise distance play an important role in
ER binding.

Mycoestrogens are the most active chemicals in phyto­estrogens (Figure
3F). As shown in Table 3, α-zearalenol and β-zearalanol had RBAs 2
orders of magnitude higher than those of their β-isomers. The activity
of zearalanone was between those of its α- and β -isomers. The order of
their RBAs was consistent with that of their O-O distances. In
β-zearalenol and β-zearalanol, these were about 11.0 Å, the same
distance found between the 3-and 17 β-OH groups of E2; β-zearalanol and
β-zeralenol are 1 Å shorter (Table 3). This indicates that their RBAs
are dependent on one of the critical structural features, the O-O
distance, when chemicals have the same structural frameworks.

**Diphenylmethanes.** The generic structure for this chemical class is
two benzene rings separated by one carbon (or other atoms) (Figure 4).
The 4-OH substituent is critical for binding, which is supported by a
number of observations:

All chemicals with a 4-OH substituent exhibit binding activity, except
4,4-methylenebis(2,6-di-*tert*-butylphenol) that contains four steric
hindered *tert*-butyl groups ortho to the 4- and 4'-OH groups. These
prevent H-bond interactions between the phenolic rings and the receptor.

Two benzophenones (Figure 4B), 2,4'-dihydroxy­benzophenone and
4,4'-dihydroxybenzophenone, both containing a 4-OH group, exhibit weak
binding activity. However, 2,2'-dihydroxybenzophenone is inactive due to
the absence of a 4-OH group. Similarly,
2,2'-dihydroxy-4-methoxybenzophenone and 2-OH-4-methoxybenzo­phenone are
inactive because of the methylation of the 4-OH.

Among the 12 DDTs (Figure 4C), the four with 4-OH substitution show much
stronger binding than the others. Methoxychlor and methoxychlor olefin
are inactive be­cause of methylation of their OH groups.

Bisphenol A(RBA = 0.008) and cumyl phenol (RBA = 0.005) have comparable
binding affinities, as do mono­hydroxymethoxychlor (RBA = 0.13) and HPTE
(RBA= 0.25). It suggests that only one phenolic ring is critical in
binding for chemicals with the sp^3^ hybrid bridged carbon. However,
this is not the case for chemicals with the sp^2^ hybrid bridged carbon.
Dihydroxymethoxylchlor

**Table 4. Hydrophobicity (log *P) ***of DDTs

+---------------+--------+--------------+--------+--------------+------+
| > DDT         | log    | > DDT        | log    | > DDT        | log  |
|               | *P*    |              | *P*    |              | *P*  |
+---------------+--------+--------------+--------+--------------+------+
| *o*,*p'*-DDT  | 6.79   | *o*,*p'*-DDD | 5.87   | *o*,*p'*-DDE | 6.00 |
+---------------+--------+--------------+--------+--------------+------+
| *p*,*p'*-DDT  | 6.91   | *p*,*p'*-DDD | 6.02   | *p*,*p'*-DDE | 6.51 |
+---------------+--------+--------------+--------+--------------+------+

olefin (RBA = 2.47) binds 10 times stronger than mono­hydroxymethoxychlor
olefin (RBA = 0.23). This suggests that the second phenolic ring of
methoxychlor olefin derivatives contributes to the binding, which may be
analogous to the 17 β-OH of E2. The crystal structures of four ligands
with the ER reveal that a considerable flexibility of the His 524
residue of the receptor permits H-bond interactions with ligands in a
wide range of relative positions. Because of the sp^2^ hybridization,
the angle between the two phenyls in olefins (120° with sp^2^) are
larger than that in the non-olefin methoxychlors (109° with sp^3^). The
distance between the two O atoms for olefin derivatives is 9.7 Å, and
that for the non-olefins is \~9.3 Å. Therefore, the O-O distance for
olefin methoxy­chlors is closer to that in E2 and more favorable for
binding.

For diphenolalkanes, changes in chain length at the bridging carbon lead
to the RBA changes: bisphenol B (RBA = 0.086) \> bisphenol A(RBA -
0.008) \> bisphenol F(RBA = 0.0009). The longer the side chain, the
greater the binding affinity for the ER *(35).* Common pharmaco­phore
identification indicated that the optimal structural superposition
between bisphenol A and E2 is one in which the two benzene rings of
bisphenol A are positioned over the A and C rings ofE2, with the bridge
carbon overlaying the B ring (Figure 9). Thus, the effect of steric
substit­uents on the bridge atom of diphenylmethanes on ER binding is
analogous to the effect of 7α-substituents on E2 binding.

DDT, DDD, and DDE isomers have structural frame­works similar to that
ofbisphenol A(Figure 4C). The *o,p'*­isomers are active in binding, while
*p*,*p'*-isomers are not. The orthochlorine of *o*,\'*p'*-isomers mimics
the steric 11 β-substituent of E2 and increases structural rigidity,
which favors binding. DDT isomers have greater hydrophobicity (log *P)*
than either DDD or DDE isomers (Table 4), which might explain why
*o*,*p'*-DDT is the strongest binder among the six DDT derivatives that
were evaluated.

**Biphenyls.** 4-OH-PCBs tend to be good binders (Figure 5A), which is
consistent with observations in other chemical classes. As the number of
chloro substitu­tions at the nonphenolic ring increases, more electron
withdrawal is found in the phenolic ring, which results in higher p*K*a
values *(36)* and a better H-bond donor.
2',3',4',5'-Tetrachloro-4-biphenylol and 2',5'-dichloro-4-biphenylol are
the strongest binders in the group. Both chemicals have an orthochlorine
substitution at the nonphenolic ring. Korach et al. *(9)* reported that
PCB compounds with the strongest affinities possess either single- or
multi-orthochlorine substitution. The ortho substitution restricts the
conformational flexibility of PCBs, which favors binding. However, our
data show that a considerable improvement of binding is only associated
with the orthochlorine substitution at the nonphenolic ring. For
example, 2-chloro-4-biphenylol (RBA = 0.002) has binding affinity
similar to that of 4'-chloro-4-biphenylol (RBA = 0.007), but 28 times
lower than that of 2',5'-dichloro-4-biphenylol (RBA = 0.036). Analyzing
common pharmacophores between E2 and 2,3,4,5-tetrachloro-4-biphenylol
(Figure 9), we found

![](media/image13.png){width="3.1069444444444443in"
height="1.8229166666666667in"}

> ![](media/image14.png){width="2.796527777777778in"
> height="3.3368055555555554in"}
>
> ![](media/image15.png){width="2.310416666666667in"
> height="1.7694444444444444in"}

**Figure 9.** Four-point pharmacophore ofE2, the 3-OH and the centers of
rings A, B, and C, identified three structurally diverse xenoestrogens,
bisphenol A, 2,3,4,5-tetrachloro-4-biphenylol, and ketone.

that the two benzene rings of PCBs match the A and C ring of E2 very
well while the orthochlorine occupies the B ring position. For PCBs
having two orthochlorine substitutions at the nonphenolic ring, the
second ortho­chlorine is most likely to overlay the 11 β-position of E2
where the introduction of small steric substituents

normally improves binding. Thus, these types of PCBs are better binders.
It is evident that 2\',4',6'-trichloro-4‑biphenylol is more active than
2','3,4',5'-tetrachloro-4-biphenylol *(9).* It is safe to conclude that
the contributions of orthochlorine substitution to binding are
associated not only with the restriction of conformational flexibility
but

> *Chem. Res. Toxicol., Vol. 14, No. 3, 2001* **289**

![](media/image16.png){width="2.8131944444444446in"
height="2.783333333333333in"}

**Figure 10.** Correlation of log RBA with log *P* for phenols.

also with possibly acting as a hydrophobic group resem­bling the B ring
and/or 11 β-substituent of E2.

The phenylphenols (Figure 5B) are relatively lower affinity binders than
the 4-OH-PCBs. With the change of OH position from 4 to 3 and to 2, the
RBA decreases in the following order due to the decrease in the
accessi­bility of the OH group for H-bond interaction: 4-phenyl­phenol \>
3-phenylphenol \> 2-phenylphenol.

**Phenols.** Alkylphenols (Figure 6A), parabens (Figure 6B), and
alkyloxyphenols (Figure 6C) are contained in this class; nonylphenol has
the highest RBA (0.031). The activities of the phenols are largely
dependent on the alkyl chain length at the para position.
2-Ethylhexyl­paraben (RBA = 0.018) has an RBA close to that of
4-*tert*-octylphenol (RBA = 0.015), and a chain length with the same
number of carbons (eight). The activity of parabens follows this order:
2-ethylhexyl \> heptyl \> benzyl \> butyl \> propyl = ethyl \> methyl. A
similar trend is also observed in alkylphenols *(11, 23).* The
relationship of ER binding activity (log RBA) with hydrophobicity (log
*P)* is linearly correlated for this class *(r*^2^*=* 0.82, log RBA )
0.58 log *P-* 5.20; Figure 10). Each unit of log *P* change will cause
0.58 unit of logRBA change, demonstrating the importance
ofhydrophobicity for binding. However, dodecylphenol is an outlier,
indi­cating that this linear correlation is only valid within a certain
range.

**Miscellaneous.** Anumber ofmiscellaneous chemicals in the NCTR data
set were identified to be active in ER binding (Figure 7). Some have
similar structural frame­works as previously discussed structural
classes, while others do not.

Doisynolic and allenolic acids are nonsteroidal estro­gens. Their binding
affinities for the ER are normally 100-fold lower than that of E2, but
their in vivo potencies are high because of a long duration of action
*(3 7).* Two of their derivatives, α,α-dimethyl- β -ethylallenolic acid
and doisynoestrol, were tested; both exhibited an ability to compete E2
for ER binding. Their OH and acid groups as well as their naphthalene
substructures match the 3-and 17 β -OH and A and B rings of E2,
respectively, very well in 3D space.^3^ Because of the methylation of
its OH

~3~ X. Qian, H. Fang, H. Hong, W. Tong, R. Perkins, and D. M. Sheehan,
unpublished results.

group, doisynoestrol has activity \~100-fold lower than that of
doisynolic acid, which is consistent with the observations for steroids.

4,4'-Methylenedianiline and 4-aminophenyl ether struc­turally resemble
bisphenol F, where the OH groups are replaced with the amino groups.
Both chemicals are inactive in binding. A similar result is also
observed for butyl-2-aminobenzoate, which is structurally similar to
butylparaben. This evidence reinforces the importance of the phenolic
ring in binding, particularly its essential role for weak estrogens.

Benzil is a selective inducer and a potent in vitro activator of
microsomal epoxide hydrolase *(38).* It is also used as a photoinitiator
for the visible light polymerizing resin system that is widely applied
in modern dentistry *(39).* Benzil has a structural resemblance to
stilbene and has been studied as a new type of inducer of
drug­metabolizing enzyme along with stilbene oxide *(40).* The
4,4'-hydroxylated benzils share a similar structural construction with
the 4,4'-hydroxylated stilbenes: two OH groups separated by two benzene
rings with two sp^2^ (carbonyl) carbons. Their close structural
similarity leads to similar binding affinities found for
2,2',4,4'-tetrahydroxybenzil (RBA = 0.209) (Figure 7) and
4,4'-dihydroxystilbene (RBA = 0.281) (Figure 2A).

Nordihydroguariaretic acid is an antioxidant for fats and oils in foods.
It is a flexible molecule with two catechols separated by four carbons.
According to our recent study on common pharmacophore identification of
estrogens,^3^ nordihydroguariatic acid shares structural commonalities
with E2: its two catechol groups mimic E2's A and D rings, while its
bridged carbons fold in such a way to occupy the space recognized by
7α-substituents of E2. Normally, hydroxylation of phenol to create
cate­chols reduces estrogenic activity *(34),* and the flexible structure
is not favorable for binding. Therefore, even though nordihydroguariatic
acid shares the key phar­macophores of E2, its RBA is much lower.

Siloxanes are widely used for industrial and consumer product
applications *(41).* A single subcutaneous injection in mice using
samples from breast implant or poly-(dimethylsiloxane) results in the
wide distribution of the low-molecular weight siloxanes throughout the
body *(42).* Studies by Bennett et al. *(43)* have shown that a series
of low-molecular weight siloxanes alter male reproductive function in a
number of mammalian species. An estro­genic effect on the immature rat
uterus has also been demonstrated for several organosiloxane compounds
*(44).* Two linear disiloxanes were included for this study. Neither
chemical possesses H-bonding capability at either end of the structure
which is normally con­sidered to be essential for a chemical to bind to
the ER. However, 1,3-diphenyltetramethyldisiloxane exhibits marginal
activity. Chalcone (RBA = 0.0015), 1,3-diphenyl­tetramethyldisiloxane
(RBA = 0.0007), and butylbenzyl­phthalate (slight binder) share similar
structural frame­works: two benzene rings (hydrophobic centers) sepa­rated
by three atoms. Their activities are proportional to the rigidity of
their backbone, suggesting that the precise distance of two hydrophobic
centers with a rigid linkage favors binding.

Of the variety of estrogens whose structures have little resemblance to
E2, kepone is of particular interest for many reasons. Not only does it
produce a variety of "estrogen-like" effects on the female reproductive
system, but it also causes other toxic effects, including in the

neuromuscular system, liver, etc. *(45).* Kepone (RBA = 0.013) was found
to be 10 000 times weaker than E2 in its affinity for the ER. How its
structural features relate to E2 in binding is still puzzling. Kepone
contains four cyclopentanes in the left, right, top, and bottom sides
fused together to form a cagelike structure (Figure 7). It appears that
the carbonyl group at the C2-position is essential because mirex, its
analogue that has the car­bonyl group replaced with two chlorine atoms,
is inactive in binding. Common pharmacophore identification indi­cates
that kepone is similar to E2 in various structural features: the
carbonyl group along with the hydrophobic center of the
2,3,3a,5b,1a-cyclopentane ring, C4-Cl, and the hydrophobic center of the
two Cl atoms at the C5-position mimics the function of the phenolic A,
B, and C rings in E2 (Figure 9). It is important to point out that
kepone can rapidly pick up moisture to form kepone hydrate that should
be more active for the ER *(46).*

**Discussion**

In principle, the biological activity of a chemical is determined by its
structure. The chemical structure can be represented in three different
general ways: 2D substructures, 3D pharmacophores, and physicochemical
properties. A 2D substructure is a structural fragment of a molecule,
which often can be used as a strong indicator of a particular activity,
such as the phenolic ring for the ER. A 3D pharmacophore is a portion of
a chemical's 3D structure that is considered essential in eliciting the
biological activity of interest, such as the precise O-O distance, the
orientation of the OH group, and the location of the hydrophobic center.
A physico­chemical property of a molecule is a measure of one property of
a whole molecule. For example, log *P* mea­sures a chemical's
hydrophobicity. ER binding activity relates to all these structural
features. The SAR studies for ER binding by individual chemical classes
indicate that some features may well represent binding depend­encies for
one structural class, while other features may better represent binding
dependencies for a different structural class. These structural features
are inherently related, suggesting that structural commonality exists
among structurally diverse estrogens.

It is well-accepted that the precise spacing of two OH groups at either
end of an essential planar and primarily hydrophobic molecule is
considered the structural basis for ER binding *(47).* The E2-ER
H-bonding network (Figure 11) demonstrates the critical role of
H-bonding ability for a chemical to bind to the ER *(20).* Although the
"anchor-like" H-bonding network at the A ring of E2 imposes an absolute
requirement that an effective ligand must contain a phenolic ring, the
remainder of the binding pocket can accept a number of different
hydro­phobic groups. By comparing various estrogen classes through SAR
studies, we can summarize the general structural requirements relevant
to the template E2 structure: (1) H-bonding ability of the phenolic ring
mimicking the 3-OH, (2) H-bond donor mimicking the 17 β-OH and O-O
distance between 3- and 17 β-OH, (3) precise steric hydrophobic centers
mimicking steric 7R-and 11 β-substituents, (4) hydrophobicity, and (5) a
ring structure. These important features provide the struc­tural basis
for a xenoestrogen to exhibit binding activity.

**H-Bonding Ability of the Phenolic Ring Mimick­ing the 3-OH.** The
importance of H-bonding has been

![](media/image17.png){width="3.2in" height="2.373611111111111in"}

**Figure 11.** ER-E2 hydrogen-bonding network *(52).*

Table 5. H-Bond Distances between Two OH Groups of\
Four Ligands and the Receptor

+-------------------+------------+------------+------------+----------+
|                   |            | > 3-OH     |            | 17-OH    |
+-------------------+------------+------------+------------+----------+
|                   | Glu 353    | Arg 394    | > water    | His 524  |
+-------------------+------------+------------+------------+----------+
| ER ligand         | \[*d*~O-O~ | \[*d*~O-N~ | \[*d*~O-O~ | \[       |
|                   | (Å)\]      | (Å)\]      | (Å)\]      | *d*~O-N~ |
|                   |            |            |            | (Å)\]    |
+-------------------+------------+------------+------------+----------+
| E2                | 2.3 7      | 3.08       | 2.82       | 2.77     |
+-------------------+------------+------------+------------+----------+
| DES               | 2.51       | 3.33       | 3.19       | 2.67     |
+-------------------+------------+------------+------------+----------+
| raloxifene        | 2.3 8      | 2.93       | 2.97       | 2.62     |
+-------------------+------------+------------+------------+----------+
| 4-OH-tamoxifen    | 2.45       | 3.04       | 3.07       |          |
+-------------------+------------+------------+------------+----------+

recognized in most biological systems. The O atom is one of the most
important heteroatoms directly associated with H-bonding. Of the 130
active chemicals in the NCTR data set, 125 chemicals contain an O atom
and 3 chemicals contain a Cl atom that may serve as a weak H-bond
acceptor. Even though the degree of specificity is reduced because of
two active chemicals without H-bonding ability that produce specific
steric effects, it is difficult to avoid the conclusion that the
requirement ofH-bonding ability is the most important characteristic for
estrogen.

It has been long understood that the phenolic ring is normally
associated with estrogenic activity. One hundred eight of 130 active
chemicals (83%) in the NCTR data set contain a phenolic ring. The
contribution of the phenolic ring in binding is much more significant
than any other structural feature. The current crystal structures of
four ligand-ER complexes reveal that the phenolic rings of all four
ligands are closely positioned at the same location to allow H-bond
interactions with Glu 353, Arg 394 of the receptor, and a water
molecule. The distance ofthe phenolic O atom ofthese four ligands to Glu
353, Arg 394, and the water molecule is sum­marized in Table 5. It
appears that the H-bonding ofthe phenolic OH is much stronger with Glu
353 than Arg 394 and the water molecule because of the shorter distance.
Thus, the phenolic OH contributes to binding largely through its H-bond
donor ability because of the H-bond acceptor properties of Glu 353. The
phenolic OH is a better H-bond donor than an acceptor. Replacing OH with
the weak H-bond donor NH2 reduces activity for E2 *(17),* and diminishes
activity for bisphenol F and butylparaben (4-aminophenyl ether and
butyl-4-aminobenzoate, Figure 7). It is important to point out that the
H-bond donor ability of the phenolic OH is dependent on a number of
factors, particularly the nature of ortho substituents that affect the
OH accessibility. The H-bond donor ability for several
ortho-substituented phenols follows this trend:

phenol \> 2-methylphenol = 2-*tert*-butylphenol \> 2,6-dimethylphenol \>
2,6-di-*tert*-butylphenol *(48),* in which 2,6-di-*tert*-butylphenol is
not a H-bond donor. This is consistent with the lack of binding activity
observed for 4,4'-methylenebis(2,6-di-*tert*-butylphenol) (Figure 4A).
In conclusion, a good H-bond donor mimicking the 3-OH of E2 may be more
important than a H-bond acceptor for xenoestrogen.

**H-Bond Donor Mimicking the 17** β**-OH and O**-**O Distance between 3-
and 17** β**-OH.** Chemicals could be strong estrogens if they possess a
phenolic ring and an additional OH group within a certain distance
range. Chemicals containing only one phenolic group are most likely to
be weak to medium ER ligands. The 17 β-OH of E2 makes a single H-bond as
a donor with His 524, because the two N atoms of imidazole act as an
H-bond acceptor in most cases. This is consistent with the observation
of similar RBAs for the E1 and 17-deoxy-E2 (Figure 1A) pair and the
meso-*p*-(α, β-diethyl-*p*-methyl­phenethyl)phenol and hexestrol
monomethyl ether pair (Figure 2B). Therefore, a stronger H-bond donor,
such as the phenolic OH, favors binding at the position that resembles
the 17 β-OH of E2.

The imidazole of His 524 is not only able to rotate (because of the
alkyl chain), but its two equivalent imidazole N atoms could compensate
for the change in the oxygen position of 17 β-OH and maintain a
favorable H-binding position. This was recognized in the E2-and
raloxifene-ER crystal structure. The phenolic A rings of both molecules
overlap quite well, whereas the 17 β-OH group of E2 and the OH group of
raloxifene are 5 Å apart. However, these two OH groups have a similar
distance to the N atom of the imidazole (Table 5). It suggests that the
flexible His 524 is more tolerant of the second OH groups of a chemical
in forming an effective H-bond interaction. Although the H-bonding
strength is depend­ent on the relative positions (angle and distance)
between the imidazole of His 524 and the OH group, the O-O distance
could provide a fair estimation of the H-bonding ability of the second
OH group analogous to the 17 β-OH

of E2.

For purposes of analysis, we have divided the NCTR data set into four
activity categories: strong (RBA \> 1), medium (1 \> RBA \> 0.01), weak
(0.01 \> RBA \> detectable activity), and inactive. As shown in Figure
12, most strong to medium ER ligands contain two OH groups with an O-O
distance ranging from 9.7 to 12.3 Å. The chemicals with an O-O distance
not in this range are highly likely to be weak estrogens.

**Precise Steric Hydrophobic Centers Mimicking Steric 7**r- **and
11** β**-Substituents.** The volume of the ER binding pocket (450 Å^3^)
is about twice that of E2 (245 Å^3^) *(20).* The length and breadth of
the E2 skeleton are well matched by the receptor, but there are large
unoccupied cavities at the 7α- and 11 β-positions of E2. The positions
of these cavities allow steric groups of certain sizes to fit, and are
of great importance for xenoestrogens, including DES-like chemicals,
diphenyl­methanes, and biphenyls. For example, the binding affinities
ofDES, DMS, and 4,4'-dihydroxystilbene change dramatically with changes
in the side chain length on the C-C double bond. This trend was also
observed *(4)* in in vivo activity; 4,4'-dihydroxystilbene had 250-fold
lower potency than DMS and 16700-fold lower potency than DES. Even
triphenylethylene (RBA = 0.002) (Figure 2C) that has no H-bonding
ability still exhibits weak

![](media/image18.png){width="3.2104166666666667in"
height="2.053472222222222in"}

**Figure 12.** Effect of the range of the O-O distance on RBA. The
activity categories were arbitrarily defined as follows. For strong
estrogens, RBA \> 1. For medium estrogens, the RBA is between 0.01
and 1. For the weak estrogens, RBA is between 0.01 and a detectable
activity. Inactive chemicals show no detectable activity in the assay.
The RBA for E2 is set to 100.

![](media/image19.png){width="2.55in" height="1.4833333333333334in"}

**Figure 13.** Relationship of the mean log *P* values with binding
activity. The activity categories are defined in the legend of Figure
12.

binding. Clearly, the precise location of steric bulk in the region of
the 7α- and/or 11 β-positions of E2 is critical for binding. It is
evident that DL-hexestrol's RBA activity is about 100-fold lower than
that of mesohexestrol. The two isomers only differ in the direction of
the two ethyl substitutes. Similarly, indenestrol A was found to have
higher affinity for the ER *(49)* than the enantiomers of indenestrol B
*(50).*

**Hydrophobicity.** The ER ligand-binding pocket has a 3D "cross"-like
shape; the center and vertical ends are mainly hydrophobic, whereas the
polar functions are located at opposite ends of the horizontal cavity.
An effect of hydrophobicity in binding was observed for phthalates
*(51).* Hydrophobicity (log *P),* expressed as a ratio of solubility
between octanol and water, is important for many biological end points.
Log *P* values too high or too low can be associated with poor transport
characteristics. Unlike the aforementioned three estrogen-related
struc­tural features that are critical in binding only by inter­acting
with specific amino acids of the receptor, hydro­phobicity is a
physicochemical property.

Although the mean log *P* values for strong, medium, and weak estrogens
exhibited a positive trend (Figure 13), the inactive chemicals also have
a wide range of log *P* values. This suggests that better binders tend
to have larger log *P* values but not vice versa. We plotted log RBA
versus log *P* for individual chemical classes; the only linear
correlation was for phenols (Figure 10). Phenols consist of only a
phenolic ring serving as a H-bond anchor and an alkyl chain for
hydrophobicity. The linear correlation between log RBA and log *P* for
phenols

suggests that log *P* is only important in binding when the key
pharmacophores are in place. This can be further demonstrated by
plotting mean log *P* values versus activity categories for chemicals
containing two OH groups with distances between 9.7 and 12.3 Å(mimicking
the 3- and 17*β*-OH of E2) and only one 3-OH group. As shown in Figure
13, the positive effect of hydrophobicity on binding is much more
apparent for chemicals contain­ing the 3- and 17* β*-OH, and a 3-OH.
Interestingly, for each activity category, the mean log *P* value of
chemicals with one OH group is larger by almost the same amount compared
to that of chemicals containing two OH groups mimicking the 3- and
17* β*-OH of E2. This indicates that chemicals lacking the effective O-O
distance require greater hydrophobicity to reach binding activities
similar to those of chemicals with the 3- and 17* β*-OH groups.

**Ring Structure.** Effective ER ligands must possess a ring structure.
A literature survey of more than 2000 unique chemicals that were tested
by various in vitro and/or in vivo assays for estrogenic activity
reveals that no estrogenic chemical has been found that does not possess
a ring structure. The ring construction increases the rigidity of both
the structure and the steric center, which favors ER binding. It appears
that the flat aro­matic ring, which fits better to the narrow
"tunnel-like" arrangement in the receptor for the phenolic A ring, is
more important than other ring structures. The majority of xenoestrogens
contain at least one aromatic ring. Only five chemicals without aromatic
rings were found to be active, ofwhich four are steroids (Figure 1B) in
addition to kepone. All five chemicals possess H-bond capability with a
rigid hydrophobic backbone that matches the A, B, and C rings of E2 very
well. It suggests that nonaro­matic estrogens require several key
structural features to exhibit binding activity, at least including a
strong electronegative atom (O, S, N, etc.) for H-bond interaction and a
rigid hydrophobic backbone.

There are several substructures inherently associated with ER binding
activity. The common structural frame­work for most estrogens contains an
aromatic ring, normally a phenolic ring, separated from a hydrophobic
center that mimics one of the centers of E2's B, C, or D ring. PCBs have
two benzene rings connected directly, which resemble the A and C rings.
The ortho substituents of PCBs match the B ring and/or
11* β*-substituents of E2 and increase binding affinity. Bisphenol A and
DDT-like chemicals have two benzene rings separated by one atom, whereas
two rings mimic the A and C ring and the substituents on the bridge atom
match the B ring. The two benzene rings of DE S derivatives resemble the
A and D rings. The binding of DES derivatives is mainly governed by the
presence or absence of two OH groups at the 4,4'-position and two ethyl
groups to mimic the 7R- and 11*β*-substituents ofE2. It seems that
twobenzene rings separated by three atoms provide an optimal steric
interaction with the receptor, which is demonstrated by three weak
estrogens, chalcone, 1,3-diphenyltetramethyl­disiloxane, and
butylbenzylphthalate, which contain no H-bonding capability mimicking
either the 3- or 17*β*-OH

of E2.

**Summary.** The SAR studies on 230 diverse chemicals reveal five
structural features that are most important for chemical binding to ER.
The representative chemical for each structural category listed in Table
1 demon­strates that the more key structural features a chemical
contains, the more active it is. These findings can be

![](media/image20.png){width="3.2666666666666666in"
height="3.4402777777777778in"}

**Figure 14.** Flowchart for identification of ER ligands.

generalized as a set of "if-then" rules for guidance in identifying
potential ER ligands, which is depicted in Figure 14:

(1) If a chemical contains no ring structure, then it is unlikely to be
    an ER ligand.

(2) If a chemical has a nonaromatic ring structure, then it is unlikely
    to be an ER ligand if it does not contain an O, S, N, or other
    heteroatom for H-bonding. Otherwise, its binding potential is
    dependent on the existence ofthe key structural features. Kepone,
    dihydrotestosterone, norethynodrel, and 3α- and 3*β*-androstanediol
    are active ER ligands that fall into this category.

(3) If a chemical has a non-OH aromatic structure, then its binding
    potential is dependent on the existence ofthe key structural
    features. A total of 16 chemicals in the NCTR data set, including
    *o*,*p'*-DDT, 1,3-diphenyltetra­methyldisiloxane, 3-deoxyl-E2,
    mestranol, and others, fall into this category.

(4) If a chemical contains a phenolic ring, then it tends to be an ER
    ligand if it contains any additional key structural features. For
    the chemicals containing a phenolic ring separated from another
    benzene ring with the number of bridge atoms ranging from none to
    three, it will most likely be an ER ligand.

**Conclusion**

The goal of the study presented here was to explore the change in ER
binding activity with the change of structure based on the NCTR data
set, which to the best of our knowledge is so far the largest published
structurally diverse data set with a consistent quantita­tive binding
activity measurement. The data set conveys information far beyond the
scope of this paper. The knowledge derived from this study enhances our
under­standing of the important ER binding-related structural features,
which in turn should be important in guiding the development of
predictive toxicology models for rapid identification of xenoestrogens
using computational ap­proaches. No matter what computational approaches
areused, the primary step is to identify ER-related structural
characteristics. The knowledge then could be used to design alignment
for 3D QSAR, to select ER-related structural descriptors for QSAR, and
to identify key structural features for pharmacophore models. All this
tells us that structure-activity relationship analyses of xenoestrogens
are essential.

**Acknowledgment.** We gratefully acknowledge the U.S. Department of
Energy and the U.S. Food and Drug Administration (FDA) for postdoctoral
support through the Oak Ridge Institute for Science and Education. This
work was partially supported by the FDA's Office of Women's Health and
the Chemical Manufacturer's Association.

**References**

\(1\) Kavlock, R. J., Daston, G. P., DeRosa, C., Fenner-Crisp, P., Gray,
L. E., Kaattari, S., Lucier, G., Luster, M., Mac, M. J., Maczka,

> C., Miller, R., Moore, J., Rolland, R., Scott, G., Sheehan, D. M.,
> Sinks, T., and Tilson, H. A. (1996) Research needed for the risk
> assessment of health and environmental effects of endocrine
> disruptors: a report of the U.S. EPA-sponsored workshop. *Envi­ron.
> Health Perspect.* **104** (Suppl. 4), 715-740.

\(2\) Patlak, M. (1996) A testing deadline for endocrine disrupters.
*Environ. Sci. Technol.* **30,** 540A-544A.

\(3\) Katzenellenbogen, J. A. (1995) The structural pervasiveness of
estrogenic activity. *Environ. Health Perspect.* **103** (Suppl. 7),
99-101.

\(4\) Dodds, E., Goldberg, L., and Lawson, W. (1938) Oestrogenic
activity of alkylated stilboestrols. *Nature* **142,** 34.

\(5\) Dodds, E., and Lawson, W. (1938) Molecular structure in relation
to oestrogenic activity. Compounds without a phenanthrene nucleus.
*Proc. R. Soc.* **125,** 222-232.

\(6\) Anstead, G. M., Carlson, K. E., and Katzenellenbogen, J. A. (1997)
The estradiol pharmacophore: ligand structure-estrogen receptor binding
affinity relationships and a model for the receptor binding site.
*Steroids* **62,** 268-303.

\(7\) Dodge, J., and Jones, C. (1999) Non-steroidal estrogens. In
*Estrogens and antiestrogens I: Physiology and Mechanisms of Avtion of
estrogens and antiestrogens* (Oettel, M., and Schillinger,

> E., Eds.) pp 43-53, Springer-Verlag, Berlin.

\(8\) Sadler, B. R., Cho, S. J., Ishaq, K. S., Chae, K., and Korach, K.
S. (1998) Three-dimensional quantitative structure-activity rela­tionship
study ofnonsteroidal estrogen receptor ligands using the comparative
molecular field analysis/cross-validated r2-guided region selection
approach. *J. Med. Chem.* **41,** 2261-2267.

\(9\) Korach, K., Sarver, P., Chae, K., McLachlan, J., and McKinney, J.
(1988) Estrogen receptor-binding activity of polychlorinated
hydroxybiphenyls: conformationally restricted structural probes. *Mol.
Pharmacol.* **33,** 120-126.

\(10\) Miksicek, R. J. (1994) Interaction of naturally occurring
non­steroidal estrogens with expressed recombinant human estrogen
receptor. *J. Steroid Biochem. Mol. Biol.* **49,** 153-160.

\(11\) Routledge, E. J., and Sumpter, J. P. (1997) Structural features
of alkylphenolic chemicals associated with estrogenic activity. *J.
Biol. Chem.* **272,** 3280-3288.

\(12\) Grese, T. A., Cho, S., Finley, D. R., Godfrey, A. G., Jones, C.
D., Lugar, C. W., III, Martin, M. J., Matsumoto, K., Pennington, L.

> D., Winter, M. A., Adrian, M. D., Cole, H. W., Magee, D. E., Phillips,
> D. L., Rowley, E. R., Short, L. L., Glasebrook, A. L., and Bryant, H.
> U. (1997) Structure-activity relationships of selective estrogen
> receptor modulators: modifications to the 2-arylbenzo­thiophene core
> ofraloxifene. *J. Med. Chem.* **40,** 146-167.

\(13\) Tong, W., Perkins, R., Strelitz, R., Collantes, E. R., Keenan,
S., Welsh, W. J., Branham, W. S., and Sheehan, D. M. (1997) Quantitative
structure-activity relationships (QSARs) for estro­gen binding to the
estrogen receptor: predictions across species. *Environ. Health
Perspect.* **105,** 1116-1124.

\(14\) Waller, C. L., Oprea, T. I., Chae, K., Park, H. K., Korach, K.
S., Laws, S. C., Wiese, T. E., Kelce, W. R., and Gray, L. E., Jr. (1996)
Ligand-based identification of environmental estrogens. *Chem. Res.
Toxicol.* **9,** 1240-1248.

\(15\) Tong, W., Perkins, R., Xing, L., Welsh, W. J., and Sheehan, D. M.
(1997) QSAR models for binding of estrogenic compounds to estrogen
receptor α and *β *subtypes. *Endocrinology* **138,** 4022-4025.

\(16\) Tong, W., Lowis, D. R., Perkins, R., Chen, Y., Welsh, W. J.,
Goddette, D. W., Heritage, T. W., and Sheehan, D. M. (1998) Evaluation
of quantitative structure-activity relationship meth­ods for large-scale
prediction of chemicals binding to the estrogen receptor. *J. Chem. Inf.
Comput. Sci.* **38,** 669-677.

\(17\) Wiese, T. E., Polin, L. A., Palomino, E., and Brooks, S. C.
(1997) Induction of the estrogen specific mitogenic response of MCF-7
cells by selected analogues of estradiol-17 beta: a 3D QSAR study. *J.
Med. Chem.* **40,** 3659-3669.

\(18\) Gantchev, T. G., Ali, H., and van Lier, J. E. (1994) Quantitative
structure-activity relationships/comparative molecular field analy­sis
(QSAR/CoMFA) for receptor-binding properties ofhalogenated estradiol
derivatives. *J. Med. Chem.* **37,** 4164-4176.

\(19\) Waller, C. L., Minor, D. L., and McKinney, J. D. (1995) Using
three-dimensional quantitative structure-activity relationships to
examine estrogen receptor binding affinities ofpolychlorinated
hydroxybiphenyls. *Environ. Health Perspect.* **103,** 702-707.

\(20\) Brzozowski, A. M., Pike, A. C., Dauter, Z., Hubbard, R. E., Bonn,
T., Engstrom, O., Ohman, L., Greene, G. L., Gustafsson, J. A., and
Carlquist, M. (1997) Molecular basis of agonism and antago­nism in the
oestrogen receptor. *Nature* **389,** 753-758.

\(21\) Shiau, A. K., Barstad, D., Loria, P. M., Cheng, L., Kushner, P.

> J., Agard, D. A., and Greene, G. L. (1998) The structural basis of
> estrogen receptor/coactivator recognition and the antagonism of this
> interaction by tamoxifen. *Cell* **95,** 927-937.

\(22\) Shi, L. M., Fang, H., Tong, W., Wu, J., Perkins, R., Blair, R.,
Branham, W., and Sheehan, D. (2001) QSAR models using a large diverse
set of estrogens. *J. Chem. Inf. Comput. Sci.* **41 (1),** 186-195.

\(23\) Blair, R., Fang, H., Branham, W. S., Hass, B., Dial, S. L.,
Moland, C. L., Tong, W., Shi, L., Perkins, R., and Sheehan, D. M. (2000)
Estrogen Receptor Relative Binding Affinities of 188 Natural and
Xenochemicals: Structural Diversity ofLigands. *Toxicol. Sci.* **54,**
138-153.

\(24\) Branham, W. S., Dial, S. L., Moland, C. L., Hass, B., Blair, R.,
Fang, H., Shi, L., Tong, W., Perkins, R., and Sheehan, D. M. (2001)
Phytoestrogen and Mycoestrogen Binding to Rat Uterine Estrogen Receptor.
*Am. J. Nutr.* (in press).

\(25\) Fang, H., Tong, W., Perkins, R., Soto, A., Prechtl, N., and
Sheehan, D. M. (2000) Quantitative comparison of *in vitro* assays for
estrogenic activity. *Environ. Health Perspect.* **108,** 723-729.

\(26\) Zacharewski, T. (1998) Identification and assessment of endocrine
disruptors: limitations of in vivo and in vitro assays. *Environ. Health
Perspect.* **106** (Suppl. 2), 577-582.

\(27\) Meylan, W., and Howard, P. (1995) Atom/Fragment Contribution
Method for Estimating Octanol-Water Partition Coefficients. *J. Pharm.
Sci.* **84,** 83-92.

\(28\) Von Angerer, E. (1995) in *The Estrogen Receptor As Target For
Rational Drug Design* (von Angerer, E., Ed.) p 36, R. G. Landes Co.,
Austin, TX.

\(29\) Bucourt, R., Vignau, M., and Torelli, V. (1978) New biospecific
adsorbents for the purification of estradiol receptor. *J. Biol. Chem.*
**253,** 8221-8228.

\(30\) Jordan, V. C., Mittal, S., Gosden, B., Koch, R., and Lieberman,
M. E. (1985) Structure-activity relationships of estrogens. *En­viron.
Health Perspect.* **61,** 97-110.

\(31\) Pike, A., Brzozowski, A., Hubbard, R., Bonn, T., Thorsell, A.,
Engstrom, O., Ljunggren, J., Gustafsson, J., and Carlquist, M. (1999)
Structure of the ligand-binding domain of oestrogen receptor *â* in the
presence of a partial agonist and a full antagonist. *EMBO J.* **18,**
4608-4618.

\(32\) Nishirara, T., Nishikawa, J., Kanayama, T., Dakeyama, F., Saito,

> K., Imagawa, S., Takatori, S., Kitagawa, Y., Hori, S., and Utsumi, H.
> (2000) Estrogenic Activities of 517 Chemicals by Yeast Two-Hybrid
> Assay. *J. Health Sci.* **46,** 282-298.

\(33\) Anstead, G. M., Wilson, S. R., and Katzenellenbogen, J. A. (1989)
2-Arylindenes and 2-arylindenones: Molecular structures and
considerations in binding orientation of unsymmetrical nonsteroidal
ligands to the estrogen receptor. *J. Med. Chem.* **32,** 2163-2172.

\(34\) Miksicek, R. J. (1995) Estrogenic flavonoids: structural
require­ments for biological activity. *Proc. Soc. Exp. Biol. Med.*
**208,** 44-50.

(35) Perez, P., Pulgar, R., Olea-Serrano, F., Villalobos, M., Rivas, A.,
     Metzler, M., Pedraza, V., and Olea, N. (1998) The estrogenicity
     ofbisphenol A-related diphenylalkanes with various substituents at
     the central carbon and the hydroxy groups. *Environ. Health
     Perspect.* **106,** 167-174.

(36) Ebner, K., and Braselton, W. (1985) Structural and chemical
     requirements ofhydroxylated polychlorinated biphenyls (PCBOH) for
     inhibition ofenergy-dependent swelling ofrat liver mitochon­dria.
     *Toxicologist* **5,** 260.

(37) Scribner, A. W., Jonson, S. D., Welch, M. J., and
     Katzenellen­bogen, J. A. (1997) Synthesis, estrogen receptor
     binding, and tissue distribution of \[^18^F\]fluorodoisynolic
     acids. *Nucl. Med. Biol.* **24,** 209-224.

(38) Seidegard, J., and DePierre, J. W. (1981) Benzil, a selective
     inducer and a potent in vitro activator of microsomal epoxide
     hydrolase. *Adv. Exp. Med. Biol.* **136,** 717-728.

(39) Fujisawa, S., Kadoma, Y., and Masuhara, E. (1986) Effects of
     photoinitiators for the visible-light resin system on hemolysis of
     dog erythrocytes and lipid peroxidation of their components. *J.
     Dent. Res.* **65,** 1186-1190.

(40) Seidegard, J., DePierre, J. W., Morgenstern, R., Pilotti, A., and
     Ernster, L. (1981) Induction of drug-metabolizing systems and
     related enzymes with metabolites and structural analogues of
     stilbene. *Biochim. Biophys. Acta* **672,** 65-78.

(41) McKim, J. M., Jr., Choudhuri, S., Wilga, P. C., Madan, A.,
     Burns-Naas, L. A., Gallavan, R. H., Mast, R. W., Naas, D. J.,
     Parkinson, A., and Meeks, R. G. (1999) Induction of hepatic
     xenobiotic metabolizing enzymes in female Fischer-344 rats
     following re­peated inhalation exposure to
     decamethylcyclopentasiloxane (D5). *Toxicol. Sci.* **50,** 10-19.

(42) Kala, S. V., Lykissa, E. D., Neely, M. W., and
     Lieberman, M. W. (1998) Low molecular weight silicones are widely
     distributed after a single subcutaneous injection in mice. *Am. J.
     Pathol.* **152,** 645-649.

(43) Bennett, D. R., Gorzinski, S. J., and LeBeau, J. E. (1972)
     Structure-activity relationships of oral organosiloxanes on the
     male reproductive system. *Toxicol. Appl. Pharmacol.* **21,**
     55-67.

(44) Hayden, J. F., and Barlow, S. A. (1972) Structure-activity
     relationships of organosiloxanes and the female reproductive
     system. *Toxicol. Appl. Pharmacol.* **21,** 68-79.

(45) Guzelian, P. S. (1982) Comparative toxicology of chlordecone
     (kepone) in humans and experimental animals. *Annu. Rev. Pharmacol.
     Toxicol.* **22,** 89-113.

(46) Wilson, N. K., and Zehr, R. D. (1979) Structures of some kepone
     photoproducts and related chlorinated pentacyclodecanes by
     carbon-13 and proton nuclear magnetic resonance. *J. Org. Chem.*
     **44,** 1278-1282.

(47) Keasling, H., and Schueler, F. (1950) The relationship between
     estrogenic action and chemical constitution in a group of
     azo­methine derivatives. *J. Am. Pharm. Assoc.* **39,** 87-90.

(48) Abraham, M., Duce, P., and Prior, D. (1989) Hydrogen Bonding.
     Part 9. Solute Proton Donor and Proton Acceptor Scales for Use in
     Drug Design. *J. Chem. Soc., Perkin Trans. 2,* 1355-1375.

(49) Korach, K. S., Chae, K., Levy, L. A., Duax, W. L., and
     Sarver, P. J. (1989) Diethylstilbestrol metabolites and analogs.
     Stereochem­ical probes for the estrogen receptor binding site. *J.
     Biol. Chem.* **264,** 5642-5647.

(50) Duax, W. L., Griffin, J. F., Weeks, C. M., and Korach, K. S. (1985)
     Molecular conformation, receptor binding, and hormone action of
     natural and synthetic estrogens and antiestrogens. *Environ. Health
     Perspect.* **61,** 111-121.

(51) Nakai, M., Tabira, Y., Asai, D., Yakabe, Y., Shimyozu, T., Noguchi,
     M., Takatsuki, M., and Shimohigashi, Y. (1999) Binding
     charac­teristics of dialkyl phthalates for the estrogen receptor.
     *Biochem. Biophys. Res. Commun.* **254,** 311-314.

(52) Tanenbaum, D. M., Wang, Y., Williams, S. P., and
     Sigler, P. B. (1998) Crystallographic comparison of the estrogen
     and proges­terone receptor's ligand binding domains. *Proc. Natl.
     Acad. Sci. U.S.A.* **95,** 5998-6003.

> TX000208Y