Novel estrogen receptor ligands based on an anthranylaldoxime structure: Role of the phenol-type pseudocycle in the binding process

Article abstract of DOI:10.1021/jm0308390

The 3,4-diphenylsalicylaldoxime system 1 is an estrogen receptor (ER) ligand of unusual structure, having a hydrogen-bonded pseudocyclic A′-ring in place of the paradigmatic phenolic A-ring that is characteristic of most estrogens. We have investigated th

Full text of DOI:10.1021/jm0308390

4032
J . Med. Chem. 2003, 46, 4032-4042
Novel Estr ogen Recep tor Liga n d s Ba sed on a n An th r a n yla ld oxim e Str u ctu r e:
Role of th e P h en ol-Typ e P seu d ocycle in th e Bin d in g P r ocess
Filippo Minutolo,† Michela Antonello,† Simone Bertini,† Gabriella Ortore,† Giorgio Placanica,†
Simona Rapposelli,† Shubin Sheng,‡ Kathryn E. Carlson,§ Benita S. Katzenellenbogen,‡
J ohn A. Katzenellenbogen,§ and Marco Macchia*,†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, Via Bonanno 6, 56126 Pisa, Italy, Department of Molecular and
Integrative Physiology, University of Illinois, 407 S. Goodwin Avenue, Urbana, Illinois 61801, and Department of Chemistry,
University of Illinois, 600 S. Mathews Avenue, Urbana, Illinois 61801
Received March 13, 2003
The 3,4-diphenylsalicylaldoxime system 1 is an estrogen receptor (ER) ligand of unusual
structure, having a hydrogen-bonded pseudocyclic A′-ring in place of the paradigmatic phenolic
A-ring that is characteristic of most estrogens. We have investigated the role played by the
pseudocycle A′ in binding to the ER by preparing 3,4-diphenylbenzaldoxime (4), a compound
that completely lacks this ring but still preserves all of the other features of the original molecule
1, as well as a series of 3,4-diphenylanthranylaldoximes (5a -c) in which the nature of the
heteroatom participating in the formation of pseudoring A′ has been changed from an oxygen
(1) to a nitrogen that is either unsubstituted (5a ) or substituted with small alkyl groups (a
methyl in 5b, or an ethyl in 5c). The importance of hydrogen-bonded pseudocycle A′ in the
binding process was confirmed by the fact that benzaldoxime 4 showed a greatly reduced binding
affinity compared to salicylaldoxime 1. Moreover, the binding affinity improved considerably
when the A′-ring contained either an unsubstituted nitrogen (5a ) or an N-Me group (5b). On
the other hand, the N-Et-substituted anthranyl derivative 5c showed a marked drop in binding
affinity. Molecular modeling docking studies on ERR confirmed that compounds 5a and 5b fit
nicely in the ligand binding pocket, with an especially comfortable fit for the N-Me group of 5b
in a small hydrophobic pocket surrounded by nonpolar residues. The limited size of this pocket
does not allow accommodation of N-substituents larger than a methyl group, which is consistent
with the low binding affinity of the N-Et compound 5c.
In tr od u ction
where stimulation would be undesirable, such as the
breast and uterus, but at the same time stimulate
estrogen actions in other tissues where they are desired,
such as the bone and liver.11,15 A great deal of effort has
been devoted to the task of understanding the processes
by which SERMs are able to exert tissue-specific
estrogen agonist and antagonist effects and to improve
upon their already rather favorable profile of selectivity.
More recently, a second subtype of the estrogen
receptor, named ERâ, was discovered.16,17 The two ER
subtypes (ERR and ERâ) show a very high amino acid
sequence homology in their DNA-binding domains
(DBDs) and considerable homology in their ligand-
binding domains (LBDs).15 The tissue distribution pat-
terns of ERR and ERâ, however, are rather different,15,18
as are their biological functions, some of which have
been revealed by in vivo studies on receptor subtype-
specific knockout mice.19-21 The tissue-selective phar-
macology of SERMs is thought to result from their
different action on each ER subtype and/or by the
different interactions that the ER-ligand complex
might have with the cellular coregulatory proteins or
effector components that vary from tissue to tissue.22,23
The estrogen receptor (ER) is a member of the nuclear
receptor superfamily that functions as a ligand-regu-
lated transcription factor.1 Many physiological processes
can be regulated by selectively activating or inhibiting
the ER with appropriate agonist or antagonist ligands.
Positive effects on the maintenance of bone mineral
density,2 on blood lipid levels, and on vasomotor and
central nervous system functions3 are generally consid-
ered to justify the use of estrogen agonists as agents in
the treatment of postmenopausal osteoporosis,4 athero-
sclerosis,5 hot flush responses, and Alzheimer’s disease.6
Unfortunately, the activation of the ER also results in
an increase in breast and uterine cancer.7-9 Therefore,
molecules, such as tamoxifen and raloxifene, that block
the tumor-promoting effects of endogenous estrogens are
currently used in the therapy and prevention of breast
cancer.10-12 These drugs, however, are not pure estrogen
antagonists, because they promote estrogen-like effects
in certain tissues; rather, they are termed selective
estrogen receptor modulators (SERMs).13,14 SERMs are
particularly attractive as therapeutic agents because
they are able to block estrogen action at those sites
Nonsteroidal ER ligands known so far encompass a
large variety of molecular structures. However, the
striking chemical feature common to nearly all synthetic
ligands possessing a good ER binding affinity is the
presence of a phenolic ring (A, ER pharmacophore,
Figure 1) that seems to directly mimic the steroid A-ring
* Author for correspondence and reprints. (Tel: +39-050-500209.
Fax: +39-050-40517. E-mail: mmacchia@farm.unipi.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Department of Molecular and Integrative Physiology, University
of Illinois.
§ Department of Chemistry, University of Illinois.
10.1021/jm0308390 CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/16/2003

Novel Estrogen Receptor Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4033
Sch em e 1
a
Key: (a) 2 times: Pd2(dba)3, Cy3P, PhB(OH)2, Cs2CO3, dioxane,
80 °C; (b) NH2OH‚HCl, MeOH-H2O, 50 °C.
cle A′, to see whether we could improve the ER binding
affinity by modifying the stereoelectronic nature of this
ring. Therefore, we first synthesized benzaldoxime 4,
F igu r e 1. Schematic representation of the H-bond network
occurring between the phenolic portions (A-rings) of estradiol
or the synthetic ER pharmacophore with the protein residues
Glu353(305) and Arg394(346) of ERR(ERâ).
in which the pseudocycle was absent, and then anthra-
nylaldoxime 5a , in which the oxygen atom of the original
compound 1 was replaced by an aniline-type nitrogen.
We also prepared anthranyl derivatives possessing
small alkyl groups on the nitrogen atom (a methyl in
5b and an ethyl in 5c) to see whether these groups
would affect the binding properties of this class of
molecules.
F igu r e 2. Modifications to salicylaldoxime 1 that caused a
decrease in ER binding affinity: OH-removal (2) and O-
methylation (3).
present in natural estrogens (e.g., estradiol, Figure 1).24
This phenolic group was found to participate in a
hydrogen-bond network that includes two specific resi-
dues of the ER ligand binding domain, Glu353(305) and
Arg394(346) of ERR(ERâ), as well as a bound molecule
of water.25,26 This interaction is thought to be respon-
sible for much of the binding affinity between the ligand
and the ERs.
Resu lts
Syn th etic Ch em istr y. Benzaldoxime 4 was synthe-
sized as shown in Scheme 1. Commercially available 3,4-
dichlorobenzaldehyde 6 underwent a double cross-
coupling reaction with phenylboronic acid, using a
catalytic system containing Pd2(dba)3 and tricyclohexyl-
phosphine,29 which efficiently promotes cross-coupling
reactions on aryl-chloride bonds (otherwise unreactive
under more classical Suzuki conditions30). Diphenyl-
substituted aldehyde 7 was then treated with hydroxyl-
amine hydrochloride in a 10:1 methanol-water mixture
at 50 °C, yielding oxime 4, which was obtained as the
pure (E)-diastereomer after column chromatography.
The (E)-configuration of the oxime moiety of compound
4 was confirmed by the chemical shift value of the oxime
proton, which is well below 8 ppm (8.22 ppm). Such a
downfield shift is typical for aromatic oximes possessing
an (E)-configuration, whereas the same group with a
(Z)-configuration is usually found between 7.3 and 7.6
ppm. This downfield shift occurs when the oxime proton
is on the same side and in a close spatial contact with
an electronegative heteroatom such as an oxygen atom
[(E)-configuration of the oxime]; through hydrogen
bonding, it is deshielded to a greater extent than when
it is positioned on the other side [(Z)-configuration].31
The synthesis of compounds 5a -c was accomplished
as shown in Scheme 2. 2,3-Dichloroaniline (10a ) was
commercially available. N-Methylated aniline 10b was
prepared from trifluoroacetamide 832 by an initial
methylation with methyl iodide in the presence of
sodium carbonate, to give intermediate 9, which was
then submitted to alkaline hydrolysis, yielding 10b.
N-Ethylated aniline 10c was obtained by reductive
amination of acetaldehyde with 10a in the presence of
In an investigation of new molecular entities that
might bind to the ER and thereby increase the chemical
diversity of estrogen ligands, we recently reported that
3,4-diphenylsalicylaldoxime (1, Figure 2), a compound
that possesses an unprecedented bioisosteric replace-
ment of the phenolic A group, proved to have interesting
binding properties for both estrogen receptor subtypes
[RBA ) 1.13% with ERR and 1.71% with ERâ; RBA
(estradiol) ) 100%].27 The good interaction of compound
1 with ERR and ERâ was rationalized by the fact that
the six-membered pseudocycle A′, formed by intramo-
lecular hydrogen bond between the phenolic OH and the
oxime nitrogen atom, might be functioning as an effec-
tive mimic of the phenolic A-ring that is found in most
ER ligands.28 To verify the importance of the OH oxime
moiety, we also investigated the binding of aldehyde 2
and O-methyloxime 3, in which the oxime hydroxyl
group was either deleted altogether or methylated,
respectively. Large reductionssof a 100-fold or greaters
were found in the binding affinity of both 2 and 3 for
ERR and ERâ.27 These observations confirmed the
crucial role being played by the oxime OH in the binding
of the structurally novel salicylaldoxime estrogens to the
ERs.
In this report, we have investigated further the
structural basis for the phenol mimicry of the pseudocy-

4034 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Minutolo et al.
Ta ble 1. Relative Binding Affinitiesa of Compounds 1-5 for
the Estrogen Receptors R and â
Sch em e 2
ligand
hERR
(100)
hERâ
estradiol
(100)
1b
1.1 ( 0.2b
0.15 ( 0.05
2.2 ( 0.2
1.7 ( 0.4b
0.27 ( 0.01
2.8 ( 0.2
5.2 ( 1.2
0.048 ( 0.013
4
5a
5b
5c
3.7 ( 1.1
0.47 ( 0.10
a
Determined by a competitive radiometric binding assay with
[3H]estradiol; preparations of purified, full-length human ERR and
ERâ (PanVera) were used (see the Experimental Section). Values
are reported as the mean ( SD of three independent experiments;
the Kd for estradiol for ERR is 0.2 nM and for ERâ is 0.5 nM. See
ref 27.
b
binding affinity (RBA) values determined with purified
full-length human alpha (hERR) and beta (hERâ) recep-
tor subtypes, together with those previously obtained
for 1.27 Binding affinity (RBA) values are reported
relative to estradiol (E2), which is set at 100%.
From the results shown in Table 1, it is evident that
benzaldoxime 4 experiences a significant reduction in
its binding affinity with respect to the reference com-
pound 1.27 In particular, when compared to 1, the RBA
values for compound 4 are 8 times lower with ERR and
6 times lower with ERâ.
A different trend is found with anthranylaldoxime
derivatives 5a -c. Here, the N-unsubstituted derivative
5a proved to possess better binding properties than its
salicylic analogue 1,27 showing a 2-fold improvement in
both receptor subtypes. Similar binding affinities are
observed with the N-methylated analogue 5b, which
shows RBA values of 3.7 with ERR and 5.2 with ERâ.
These values are three times higher than reference
compound 1 with both receptor subtypes, and they
correspond to Ki values of 5 and 10 nM with ERR and
ERâ, respectively. On the other hand, the N-ethylated
derivative 5c showed a very significant drop in binding
properties when compared to its N-methylated analogue
5b, with a nearly 10-fold reduction on ERR and a
remarkable >100-fold reduction on ERâ.
Tr a n scr ip tion Assa ys. Benzaldoxime 4 and anthra-
nylaldoximes 5a -c were assayed for transcriptional
activity through both receptor subtypes. These cotrans-
fection assays were conducted in human endometrial
(HEC-1) cells, using expression plasmids for either full-
length human ERR or ERâ and an estrogen-responsive
luciferase reporter gene system.38 In this initial screen,
agonist activity was determined at two concentrations,
10-8 and 10-6 M, and antagonist activity was assayed
at 10-6 M, in the presence of 10-9 M E2. In all cases,
transcriptional activity is normalized relative to that
obtained with 10-9 M estradiol, which is set at 100%.
These data are summarized in Table 2, where the
percent efficacy of the compounds tested as agonists (at
10-8 and 10-6 M) and as antagonists (at 10-6 M in the
presence of 10-9 M E2) is given.39
Salicylaldoxime 1 was previously reported to be a
partial agonist on ERR and a partial antagonist on
ERâ.39 “Low-affinity” benzaldoxime derivative 4 showed
a marked agonist character on ERR and a weak partial
agonist character on ERâ. The simplest anthranylal-
doxime derivative 5a is a partial agonist on ERR and a
partial antagonist on ERâ. N-Methyl-substituted an-
thranylaldoxime 5b proved to be a weak partial agonist
a
Key: (a) MeI, K2CO3, DMF; (b) K2CO3, H2O-MeOH; (c)
CH3CHO, NaBH3CN, MeOH; (d) allyl bromide, K2CO3, acetoni-
trile, 80 °C; (e) BF3‚Et2O, sulfolane, 120 °C; (f) t-BuOK, DMSO,
55 °C; (g) 2 times: Pd2(dba)3, Cy3P, PhB(OH)2, Cs2CO3, dioxane,
80 °C; (h) OsO4, NaIO4, dioxane-H2O (1:1); (i) NH2OH‚HCl,
MeOH-H2O, 50 °C.
sodium cyanoborohydride. Treatment of 10a -c with
allyl bromide and potassium carbonate in acetonitrile
afforded N-allyl derivatives 11a -c. Subsequent aza-
Claisen transposition of 11a -c with boron trifluoride
etherate at 120 °C33 selectively gave the ortho-allylated
anilines 12a -c. Alkaline rearrangement of the terminal
double bond of 12a -c to the internal position afforded
the styrene derivatives 13a -c as E/ Z diastereomeric
mixtures.34 The two chloro-aryl groups of 13a -c were
then submitted to two identical, sequential Pd-catalyzed
cross-coupling steps with phenylboronic acid (1.5 equiv
each step), using Pd2(dba)3 as the catalyst, tricyclohexyl-
phosphine as the ligand, Cs2CO3 as the base, and
dioxane as the solvent.29 Under these conditions, diphen-
yl-substituted products 14a -c were obtained. Anthra-
nylaldehydes 15a -c were then obtained by oxidative
cleavage of the double bond of 14a -c, using sodium
periodate in the presence of catalytic amounts of os-
mium tetroxide.35 Final condensation of aldehydes
15a -c with hydroxylamine hydrochloride in refluxing
ethanol yielded anthranylaldoximes 5a -c.
In all cases (5a -c), the (E)-form of the oxime was the
only diastereoisomer formed, presumably because the
intramolecular hydrogen bond, which can only form in
the (E)-isomer, contributes to the oxime stability. As
seen before for oxime 4, in these cases the chemical shift
value of the oxime proton, which is always found below
8 ppm (8.34 for 5a , 8.37 for 5b, and 8.54 for 5c),
confirmed the (E)-configuration of oximes 5a -c.31
Estr ogen Recep tor Bin d in g Assa ys. The binding
affinity of oximes 4 and 5a -c for both ERR and ERâ
was determined by a radiometric competitive binding
assay, using methods that have been described else-
where in detail.36,37 In Table 1 are reported the relative

Novel Estrogen Receptor Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4035
Ta ble 2. Transcriptional Efficacy of Selected Salicylaldoxime Analogues on ERR and ERâa
% efficacy ERRb
agonistc (M)
10-8
% efficacy ERâb
agonistc (M)
10-8
antag.c (M)
10-6
antag.c (M)
10-6
compd
10-6
pharmacol. characterd
10-6
pharmacol. characterd
1
4
5a
5b
5c
12 ( 0.1
17 ( 2
74 ( 8
85 ( 2
71 ( 4
58 ( 1
70 ( 4
98 ( 0.4
85 ( 5
85 ( 1
part. agon.
full agon.
part. agon.
wk part. agon.
wk part. agon.
2.6 ( 0.2
1.3 ( 0.1
6.0 ( 0.1
1.1 ( 0.1
0.94 ( 0.03
24 ( 0.3
20 ( 1
53 ( 8
wk part. antag.
wk part. antag.
part. antag.
wk part. antag.
weak
69 ( 13
31 ( 0.3
71 ( 14
85 ( 13
38 ( 7
23 ( 1
1.6 ( 0.2
0.58 ( 0.03
22 ( 1
4.9 ( 1.3
59 ( 1 106 ( 15
a
Transcriptional efficacy determined in cotransfection assay in HEC-1 cells using ERR or ERâ expression plasmids and an estrogen
regulated reporter gene (see the Experimental Section for details). Values are percent of the transcriptional response of estradiol (E2) at
10-9 M. Abbreviations: “antag.” stands for antagonist, “agon.” stands for agonist, part. stands for partial, and “wk” stands for weak
b
throughout Table 2. Values are percent of the transcriptional response of estradiol at 10-9 M, and they represent the average of triplicate
determinations (CV < 0.15). c Agonist assays are done with compound alone; antagonist assays are done with compound together with
d
10-9 M estradiol. For a definition of these terms, see ref 39.
on both receptor subtypes. Similarly, its N-ethyl ana-
logue 5c proved to be an even weaker partial agonist
on ERR and ERâ.To confirm their pharmacological
character in the transcription assays, complete dose-
response curves were obtained for compounds 5a and
5b (Figure 3), the two compounds that had the highest
ERR binding affinities. The dose-response curves ob-
tained with estradiol alone (Figure 3a) serve as a
reference point and confirm that this compound is a full
agonist on both ERs, with only a slightly higher potency
on ERR. The full dose-response curves show that
anthranylaldoxime derivative 5a is a nearly full agonist
on ERR and partial antagonist on ERâ, having moderate
potency in both cases, although the antagonist effect of
5a on ERâ is clearly evident (Figure 3b). The N-methyl
substituted analogue 5b has a similar pharmacological
profile (Figure 3c), being a stronger agonist on ERR than
on ERâ. Surprisingly, even though the N-methyl ana-
logue (5b) has higher ER binding affinity than its
unmethylated congener (5a ), in these transcription
assays, it appeared less potent on both ERs (Figure 3c).
Molecu la r Mod elin g of Oxim es in th e Liga n d
Bin d in g P ock ets of th e ERs. To better understand
the possible binding modes of compounds 1 and 5a -c,
we performed molecular docking experiments on ERR.
The protein backbone was taken from the X-ray struc-
ture of ERR bound to estradiol25,40 (Figure 4a), where
it is clear that the A-ring hydroxyl group makes
hydrogen bonds with Arg394 and Glu353. Docking
calculations were carried out by replacing the estradiol
molecule with salicylaldoxime 1 or anthranylaldoximes
5a -c. We found that the pseudoring A′ of salicylal-
doxime 1 could be positioned where estradiol had its
phenolic A-ring (Figure 4b), allowing for an efficient
participation of the oxime OH in the same hydrogen-
bonding network with Arg394 and Glu353. The aromatic
central core of 1 replaces the B-ring of estradiol, and
the two phenyl substituents protrude toward the rela-
tively hydrophobic empty spaces in the receptor ligand
binding pocket. Although 1 binds to ERR in a fashion
similar to estradiol, the overall complexes may not be
perfectly identical, since 1 exhibits only a partial ago-
nism on this receptor subtype.
the case of 1. N-methyl derivative 5b places its N-
methyl group in a small hydrophobic pocket defined by
Leu346, Leu349, and Ala350.
By contrast, the higher homologue 5c, which has an
N-ethyl substituent, was not able to assume a similar
binding mode within the ligand binding pocket as
defined in the ERR-E2 X-ray structure when the pocket
was rigidly fixed during the modeling. To examine
whether the rigidity of the receptor was responsible for
this behavior, we performed molecular dynamics on the
ERR structure, fixing 5c in a binding mode that was
similar to the one found for its lower homologues (5a ,b).
Under this molecular dynamics simulation, some amino
acid residues underwent spatial shifts to accommodate
5c (Figure 5), in particular expanding the small hydro-
phobic pocket that hosts the N-alkyl substituent.
The most evident difference between the minimized
protein (green, Figure 5) and the X-ray structure
(purple, Figure 5) lies in a 0.5 Å shift of the Leu349 side
chain, which is the residue closest to the N-ethyl
substituent of 5c. We verified that the ERR-5c complex
obtained after molecular dynamics (green, Figure 5) was
of lower energy that the one obtained previously by
automated docking in the rigid ERR structure, by
minimizing both ERR-5c complexes and performing
energy calculations after minimization. The structure
obtained after molecular dynamics was of lower energy
by 5.3 kcal/mol. Because of this increase in ligand
stabilization energy, we also submitted the ERR-1,
ERR-5a , and ERR-5b complexes to the same minimi-
zation procedure, so that we could evaluate the relative
stability of all of the modeled ER-ligand complexes on
a common basis.
In this manner, we found that the interaction energy
between the ligand and the receptor was less favorable
with 5c (4.4 kcal/mol higher) than with 5a and 5b, both
of which showed practically identical interaction ener-
gies. Salicylaldoxime derivative 1 formed a complex with
2.5 kcal/mol higher energy than those with 5a and 5b.
The van der Waals contribution in the case of 5c is
particularly unfavorable, because the N-ethyl group is
forced to push against the residue of Leu349.A view of
the final docked conformation obtained with the highest
affinity compound 5b (Figure 6) shows the presence of
a hydrophobic pocket of limited size fitting tightly
around the N-Me substituent of 5b. Both the front view
(Figure 6a) and the top view (Figure 6b) show that this
pocket is formed by the aliphatic side chains of Leu349
and Ala350, and by the peptide carbonyl portion of
Both anthranylaldoximes 5a ,b (Figure 4c) could be
positioned in a binding mode which is very similar to
1, placing their oxime OH and pseudorings A′ in
analogous sites. The only significant difference is in the
torsional angles of the two phenyl substituents, which
are close to 90° in 5a and 5b, but only around 70° in

4036 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Minutolo et al.
F igu r e 3. Dose-response curve for transcriptional activation
by (a) estradiol (E2) through ERR (solid circles) and ERâ (solid
triangles); (b) 5a in the agonist mode through ERR (solid
circles) and ERâ (solid triangles), or in the antagonist mode,
i.e., in the presence of 1 nM estradiol (E2) through ERR (open
circles) and ERâ (open triangles); (c) 5b in the agonist mode
through ERR (solid circles) and ERâ (solid triangles), or in the
antagonist mode, i.e., in the presence of 1 nM estradiol (E2)
through ERR (open circles) and ERâ (open triangles). Human
endometrial cancer (HEC-1) cells were transfected with ex-
pression vectors for ERR or ERâ and an (ERE)2-pS2-luc
reporter gene and were treated with compounds E2, 5a , or 5b
alone, or with compounds 5a or 5b plus 1 nM estradiol (E2)
for 24 h, at the concentrations indicated. Luciferase activity
was expressed relative to â-galactosidase activity from an
internal control plasmid. The maximal activity with 1 nM E2
was set at 100 (a). Values are the mean ( SD from three or
more separate experiments.
Leu346. As a result, there is no room for the extra
carbon unit that is present in 5c, unless the receptor is
forced to expand this hydrophobic cavity.
Figu r e 4. (a) X-ray structure of the complex ERR estradiol.25,40
(b) Docking of compound 1 into ERR. (c) Docking of compounds
5a and 5b into ERR. Most H’s omitted for clarity.
Discu ssion a n d Con clu sion s
Our prior work on the salicylaldoxime estrogens
established the fundamental role of the free oxime OH
group of compound 1 in determining the high binding
affinity of this ligand to the estrogen receptor.27 In this
study, we have shown that it is not just the oxime but
the full pseudoring A′ in 1 (formed by intramolecular
hydrogen bond between the phenolic OH and the oxime
nitrogen atom) that is important in ensuring a high
affinity interaction with the ERs. This is evident from
that fact that the affinity of benzaldoxime 4 for the ERs

Novel Estrogen Receptor Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4037
F igu r e 5. Docking of 5c into ERR after molecular dynamics
and minimization: comparison of the relative spatial position
of representative amino acid residues between the receptor
X-ray structure25,40 (purple) and the “relaxed” structure after
molecular dynamics (green). Most H’s omitted for clarity.
is 6-8 times lower than that of the salicylaldoxime
compound 1.
The difference in binding affinity of the salicylal-
doxime (1) and the benzaldoxime (4) might be due to
the fact that in the latter compound [4, of (E)-configu-
ration], rotation around the single bond between the
oxime carbon atom and the aromatic ring is constrained
only by resonance overlap with the benzene ring and
not by the intramolecular hydrogen bond, so that two
low-energy conformations are possible, (s-cisoid and
s-transoid; Figure 7). This conformational freedom
might enable the oxime OH to populate a “nonpharma-
cophoric” (i.e., s-transoid) conformation that would be
expected to have lower ER binding affinity. This rotation
is impeded when the pseudoring A′ is present (as in 1),
because the position of the oximic OH is locked into the
s-cisoid conformation by the intramolecular hydrogen
bond (Figure 7). Alternatively, the pseudoring A′ might
be acting as a real mimic of the phenyl A-ring of
estradiol by means of its rather extended π-conjugation,
which might engender a greater degree of attractive van
der Waals or π-π interactions between these ligands
and the A-ring binding pocket of the receptor.
Once we verified the importance of the pseudoring A′
by comparing the binding affinity of the congeneric
benzaldoxime (4) and salicylaldoxime (1) systems, we
improved the binding affinity of the lead compound 1
by replacing the oxygen atom contained in A′ with a
nitrogen atom, as in anthranylaldoxime 5a . The nitro-
gen atom of 5a is of the aniline type, and therefore, its
hybridization state has a greater sp2 character than its
oxygen counterpart in 1.41 We believe that this results
in a greater degree of delocalization of the nitrogen lone
pair through the aromatic central core to the oxime C-N
double bond, giving the pseudocycle a greater degree of
π-conjugation. Thus, the A′-rings of the anthranylic
derivatives would have greater aromatic character,
which would make them better mimics for the phenolic
A-ring of estradiol and other classical estrogen ligands.
F igu r e 6. (a) Front view and (b) top view of van der Waals
volumes of 5b (green), docked into ERR-LBD, together with
selected amino acid residues that define a small hydrophobic
pocket occupied by the N-methyl group (purple). Most H’s
omitted for clarity.
Another factor that might be responsible for the
higher affinity of anthranyl derivative 5a is the consid-
erably higher torsional angles of the phenyl substitu-
ents, which were observed to be around 90° in the
docking experiments (Figure 4c), as compared to the
salicylic derivative 1, where the same torsionals mea-
sure about 70° (Figure 4b). These torsional angle

4038 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Minutolo et al.
by the peptide carbonyl portion of Leu346 has limited
dimensions; it is able to comfortably host the N-Me
group of 5b (Figure 6), but it needs some rearranging
to be able to do the same with even the slightly bulkier
ethyl moiety, as in 5c. Thus, the reduced affinity of 5c
effectively probes the spatial dimension of this hydro-
phobic pocket, which, as visualized in Figure 6, only has
space enough to efficiently embrace a methyl group.
The transcriptional efficacy of these compounds con-
firmed what had been previously found for their sali-
cylaldoxime analogues,39 that is, a greater level of
agonism on ERR than on ERâ (Table 2). A curiosity is
that the N-methyl anthranyloxime (5b), which binds to
ER with higher affinity than the unsubstituted analogue
(5a ), has lower potency in the transcription assay. It is
not uncommon to find such discrepancies between the
rank order of receptor binding and transcriptional
potency with ER ligands, and this has been ascribed to
differences in the interaction with cellular coregulators,
which can act as modulators of ligand potency.22,23
In conclusion, we have found in the anthranylal-
doximes a new class of estrogen receptor ligands en-
dowed of better binding affinity properties when com-
pared with their oxygenated predecessor salicylaldoximes.
Among the anthranyl derivatives, we have recognized
in the N-Me analogue 5b the highest affinity member
of this class. Docking experiments confirmed the binding
affinity data and explained, through consideration of the
properties of a small hydrophobic pocket, the highest
affinity of the N-Me derivative 5b and the lowest affinity
of the N-Et derivative 5c. It will be now interesting to
decorate the external aromatic substituents, as we have
previously done with related compounds,39 to explore
whether it is possible to find other members of this class
of molecules that possess improved biological properties.
F igu r e 7. Free rotation of the oxime group in compound 4
and effect of intramolecular H-bond in blocking the s-cis
conformation.
differences arise from the presence of the exocyclic N-H
bond in 5a , which sterically hinders the adjacent phenyl
substituent in a way not found in the salicylic analogue
1, where the corresponding atom (oxygen) has only
exocyclic lone pairs. To minimize this hindrance, the
phenyl ring in position 3 adopts a conformation that is
approximately orthogonal to the central aromatic core,
a change that also forces the second phenyl substituent
to assume a similar, more perpendicular conformation.
The greater torsion angles result in an expansion of the
molecular volume of these portions of the ligand, so that
they more fully fill the empty spaces that surround the
ligand in the ligand binding pocket of the receptor
(Figure 4c). A similar effect of ligand bulk normal to
the plane of the steroid system as a factor that enhances
binding affinity has been noted in the tetrahydrochry-
sene system,42 in 7R- and 11â-substituted steroids,24,43
and in the stilbene series, where X-ray crystallography
shows that the two ethyl groups of DES are, in fact,
filling hydrophobic voids above and below the plane
adopted by steroidal ligands, rather than within this
plane.44
The binding affinity of the anthranylic oxime system
turned out to be preserved when a methyl group was
placed on the aniline nitrogen atom of the anthranylic
system, as in compound 5b, which showed the best RBA
values of this series (Table 1). The docking experiments
showed that, as expected, the structure of 5b in the
ERR-LBD perfectly superimposes that found for 5a
(Figure 4c), with the torsional angles of the two phenyl
substituents of about 90°. The only structural difference
between 5a and 5b is the N-methyl group, which is
found to occupy a small hydrophobic pocket whose
boundaries are formed by three residues, Leu346,
Leu349, and Ala350 (Figures 4c and 6).
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot-stage apparatus and are uncorrected. NMR spectra were
obtained with
a Varian Gemini 200 MHz spectrometer.
Chemical shifts (δ) are reported in parts per million downfield
from tetramethylsilane and from solvent references. Electron
impact (EI) mass spectra were obtained on a HP-5988A mass
spectrometer. The elemental compositions of the compounds
agreed to within (0.4% of the calculated value. Chromato-
graphic separations were performed on silica gel columns by
flash (Kieselgel 40, 0.040-0.063 mm; Merck) or gravity column
(Kieselgel 60, 0.063-0.200 mm; Merck) chromatography.
Reactions were followed by thin-layer chromatography (TLC)
on Merck aluminum silica gel (60 F254) sheets that were
visualized under a UV lamp. Evaporation was performed in
vacuo (rotating evaporator). Sodium sulfate was always used
as the drying agent. 3,4-Dichlorobenzaldehyde (6) and 2,3-
dichloroaniline (10a ) were purchased from Sigma-Aldrich (St.
Louis, MO). 2,3-Dichloro-N-(trifluoroacetyl)aniline (8) was
prepared as previously described.32
3,4-Dip h en ylsa licyla ld oxim e (1). Preparation of 1 and
most characterization data have already been reported.27 13C
NMR (CDCl3) δ 115.58, 121.86, 126.80, 126.91, 127.76, 127.87,
129.75, 129.80, 131.09, 136.14, 140.82, 144.46, 152.69, 154.71.
3,4-Dip h en ylben za ld eh yd e (7). A solution of 3,4-dichlo-
robenzaldehyde (6) (0.300 g, 1.71 mmol) in dioxane (2 mL) was
treated with 0.957 g (2.94 mmol) of cesium carbonate, 0.314 g
(2.57 mmol) of phenylboronic acid, 0.050 g (0.055 mmol) of tris-
(dibenzylideneacetone)dipalladium(0), and 0.21 mL of a 20%
solution of tricyclohexylphosphine (0.13 mmol) in toluene. The
resulting suspension was heated at 80 °C for 16 h under an
argon atmosphere. The reaction mixture was then cooled to
room temperature, diluted with diethyl ether, and filtered
This trend undergoes an early break, however, when
an N-ethyl substituent is introduced, as in compound
5c, where an unexpected drop in binding affinity is
found. However, our docking experiments showed that
5c is able to effectively interact with ERR only after the
receptor LBD has undergone a certain degree of defor-
mation (Figure 5) and that the resulting complex is less
stable than the ERR-5b one, basically because of
repulsive van der Waals interactions. In fact, the small
hydrophobic pocket defined by Leu349 and Ala350 and

Novel Estrogen Receptor Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4039
through a Celite pad. The solvent was removed under vacuum,
and the resulting crude mixture was submitted again to the
same treatment described above. The crude product deriving
from the second step was purified by column chromatography,
eluting with a 9:1 f 7:3 n-hexane/ethyl acetate mixture, to
obtain 0.352 g (1.36 mmol, 79% yield, two steps) of aldehyde
7 as an oil: 1H NMR (CDCl3) δ 7.12-7.25 (m, 10H), 7.60 (d,
1H, J ) 8.4 Hz), 7.90-7.94 (m, 2H), 10.10 (s, 1H). Anal.
diethyl ether, and the filtrate was concentrated under vacuum.
The crude product was purified by flash chromatography (n-
hexane/ethyl acetate 9:1) to yield pure 11a (1.39 g, 6.87 mmol,
68% yield) as an oil: 1H NMR (CDCl3) δ 3.81-3.88 (m, 2H),
4.64 (br, 1H, NH), 5.20 (dq, 1H, J ) 10.3, 1.5 Hz), 5.29 (dq,
1H, J ) 17.4, 1.5 Hz), 5.94 (ddt, 1H, J ) 17.2, 10.3, 5.1 Hz),
6.54 (dd, 1H, J ) 8.2, 1.1 Hz), 6.79 (dd, 1H, J ) 7.9, 1.2 Hz),
7.04 (t, 1H, J ) 8.1 Hz); MS m/z 201 (M+, 59), 174 (M+ - CHd
CH2, 100), 166 (M+ - Cl, 51), 130 (M+ - 2Cl, 55).
C
19H14O (C, H).
N-Allyl-N-m eth yl-2,3-d ich lor oa n ilin e (11b). Compound
11b was prepared from 10b (0.357 g, 2.02 mmol) by following
the same procedure described above for 11a . The crude product
was purified by flash chromatography (n-hexane/ethyl acetate
9:1) to yield pure 11b (0.345 g, 1.60 mmol, 79% yield) as an
(E)-3,4-Dip h en ylben za ld oxim e (4). A solution of 3,4-
diphenylbenzaldehyde (7) (0.329 g, 1.28 mmol) in methanol
(20 mL) was treated with a solution of hydroxylamine hydro-
chloride (0.177 g, 2.55 mmol) in water (3 mL), and the resulting
mixture was heated to 50 °C for 1 h. After being cooled to room
temperature, the solvent was partially removed under vacuum
and the residue was extracted with ethyl acetate. The com-
bined organic phase were dried and concentrated under
vacuum and the crude product was purified by flash chroma-
tography (n-hexane/ethyl acetate 8:2) to yield pure 1b (0.267
g, 0.980 mmol, 74% yield) as an off-white solid: mp 118-120
1
oil; H NMR (CDCl3) δ 2.76 (s, 3H), 3.64 (d, 2H, J ) 6.3 Hz),
5.20 (ddt, 1H, J ) 10.1, 1.8, 1.3 Hz), 5.25 (dq, 1H, J ) 17.2,
1.6 Hz), 5.91 (ddt, 1H, J ) 17.2, 10.2, 6.1 Hz), 6.98-7.01 (m,
1H), 7.12-7.15 (m, 2H); MS m/z 215 (M+).
N-Allyl-N-eth yl-2,3-dich lor oan ilin e (11c). Compound 11c
was prepared from 10c (1.79 g, 9.47 mmol) by following the
same procedure described above for 11a . The crude product
was purified by flash chromatography (n-hexane/ethyl acetate
9:1) to yield pure 11c (1.68 g, 7.32 mmol, 77% yield) as an oil:
1H NMR (CDCl3) δ 1.02 (t, 3H, J ) 7.1 Hz), 3.14 (q, 2H, J )
7.0 Hz), 3.67 (d, 2H, J ) 6.0 Hz), 5.13 (dq, 1H, J ) 10.3, 1.4
Hz), 5.20 (dq, 1H, J ) 17.4, 1.5 Hz), 5.83 (ddt, 1H, J ) 17.2,
10.1, 6.1 Hz), 6.96 (dd, 1H, J ) 7.0, 2.7 Hz), 7.06-7.16 (m,
2H); MS m/z 229 (M+).
6-Allyl-2,3-d ich lor oa n ilin e (12a ). Compound 11a (2.73 g,
13.7 mmol) was dissolved in sulfolane (5 mL) and treated
under argon with BF3‚Et2O (5.2 mL, 41.0 mmol). The resulting
mixture was heated to 120 °C overnight. After cooling to room
temperature, the reaction mixture was poured into water. The
resulting aqueous suspension was neutralized with aqueous
NaOH (1 N) and extracted with diethyl ether. The organic
phase was washed with brine, dried, and concentrated under
vacuum. The crude product was purified by flash chromatog-
raphy (n-hexane/dichloromethane 9:1 f 6:4) to yield pure 12a
(1.66 g, 8.22 mmol, 60% yield) as an oil: 1H NMR (CDCl3) δ
3.29 (d, 2H, J ) 6.0 Hz), 5.14-5.20 (m, 2H), 5.90 (ddt, 1H, J
) 16.5, 10.4, 6.0 Hz), 6.82 (d, 1H, J ) 8.2 Hz), 6.89 (d, 1H, J
) 8.1 Hz); MS m/z 201 (M+).
6-Allyl-N-m eth yl-2,3-d ich lor oa n ilin e (12b). Compound
12b was prepared from 11b (0.260 g, 1.21 mmol) by following
the same procedure described above for 12a . The crude product
was purified by flash chromatography (n-hexane/dichlo-
romethane 9:1 f 8:2) to yield pure 12b (0.160 g, 0.738 mmol,
61% yield) as an oil: 1H NMR (CDCl3) δ 2.85 (s, 3H), 3.45 (dt,
2H, J ) 6.0, 1.6 Hz), 5.07 (dq, 1H, J ) 16.7, 1.6 Hz), 5.14 (dq,
1H, J ) 10.1, 1.6 Hz), 5.96 (ddt, 1H, J ) 16.8, 10.3, 6.1 Hz),
6.98 (d, 1H, J ) 8.4 Hz), 7.06 (d, 1H, J ) 8.3 Hz); MS m/z 215
(M+).
1
°C; H NMR (CDCl3) δ 7.11-7.24 (m, 10H), 7.46 (d, 1H, J )
8.4 Hz), 7.63-7.67 (m, 2H), 8.22 (s, 1H); 13C NMR (CDCl3) δ
125.92, 126.89, 128.05, 129.53, 129.82, 129.87, 131.22, 140.82,
140.85, 141.16, 142.35, 150.08; MS m/z 273 (M+, 100), 256 (M+
- OH, 16), 255 (M+ - H2O, 20), 229 (M+ - CHdNOH, 59).
Anal. C19H15NO (C, H, N).
2,3-Dich lor o-N-m eth yl-N-(tr iflu or oa cetyl)a n ilin e (9). A
solution of 2,3-dichloro-N-(trifluoroacetyl)aniline (8)32 (0.472
g, 1.82 mmol) in anhydrous DMF was treated under nitrogen
with 0.377 g of potassium carbonate (2.73 mmol) and 0.33 mL
of methyl iodide (2.2 mmol) at room temperature for 30 min.
The reaction mixture was then quenched with water and
extracted with ethyl acetate. The combined organic phase was
washed with water, dried, and concentrated under vacuum.
The crude residue was purified by flash chromatography (n-
hexane/ethyl acetate 9:1) to yield pure 9 (0.435 g, 1.60 mmol,
88% yield) as an oil: 1H NMR (CDCl3) δ 3.32 (s, 3H), 7.22-
7.34 (m, 2H), 7.56 (dd, 1H, J ) 7.2, 2.5 Hz).
2,3-Dich lor o-N-m et h yla n ilin e (10b ). A solution of 2,3-
dichloro-N-methyl-N-(trifluoroacetyl)aniline (9) (0.311 g, 1.14
mmol) in methanol (50 mL) and water (20 mL) was treated
with potassium carbonate (1.05 g, 7.60 mmol) and the resulting
mixture was stirred at room temperature for 1 h. The solvent
was partially removed under vacuum and the residue ex-
tracted with ethyl acetate. The combined organic phase was
washed with water, dried, and concentrated under vacuum.
The crude product was purified by flash chromatography (n-
hexane/ethyl acetate 9:1) to yield pure 10b (0.162 g, 0.923
mmol, 81% yield) as an oil: 1H NMR (CDCl3) δ 2.91 (s, 3H),
4.29 (br, 1H), 6.53 (dd, 1H, J ) 8.1, 1.5 Hz), 6.79 (dd, 1H, J )
8.1, 1.5 Hz), 7.08 (t, 1H, J ) 8.1 Hz); MS m/z 174 (M+, 100),
139 (M+ - Cl, 10).
2,3-Dich lor o-N-eth yla n ilin e (10c). A solution of 2,3-
dichloroaniline (10a ) (3.00 g, 18.5 mmol) in methanol (20 mL)
was treated with 1.0 mL of acetaldehyde (19 mmol) and 1.4 g
of sodium cyanoborohydride (22 mmol). The resulting mixture
was stirred at room temperature for 4 h and then diluted with
chloroform, washed with brine, dried, and concentrated under
vacuum. The crude product was purified by flash chromatog-
raphy (n-hexane/ethyl acetate 9:1) to yield pure 10c (2.16 g,
11.4 mmol, 62% yield) as an oil: 1H NMR (CDCl3) δ 1.31 (t,
3H, J ) 7.1 Hz), 3.20 (q, 2H, J ) 7.1 Hz), 4.35 (br, 1H), 6.54
(dd, 1H, J ) 8.2, 1.5 Hz), 6.77 (dd, 1H, J ) 8.2, 1.5 Hz), 7.06
(t, 1H, J ) 8.2 Hz); MS m/z 189 (M+, 27), 174 (M+ - CH3,
100).
N-Allyl-2,3-d ich lor oa n ilin e (11a ). A solution of 2,3-
dichloroaniline (10a ) (1.62 g, 10.1 mmol) in acetonitrile (10
mL) was treated with 2.5 g of potassium carbonate (18 mmol).
The mixture was then heated to 80 °C and treated, dropwise,
with a solution of allyl bromide (1.5 g, 12 mmol) in acetonitrile
(6 mL). Heating was continued for 3 h and then the mixture
was left under stirring at the same temperature overnight.
After cooling to room temperature, the reaction mixture was
filtered by suction, with repeated washing of the filter with
6-Allyl-N-eth yl-2,3-d ich lor oa n ilin e (12c). Compound 12c
was prepared from 11c (1.65 g, 7.23 mmol) by following the
same procedure described above for 12a . The crude product
was purified by flash chromatography (n-hexane/dichlo-
romethane 9:1 f 8:2) to yield pure 12c (0.993 g, 4.34 mmol,
60% yield) as an oil: 1H NMR (CDCl3) δ 1.20 (t, 3H, J ) 7.1
Hz), 3.10 (q, 2H, J ) 7.1 Hz), 3.41 (dt, 2H, J ) 6.2, 1.5 Hz),
5.07 (dq, 1H, J ) 16.8, 1.7 Hz), 5.14 (dq, 1H, J ) 10.1, 1.6
Hz), 5.94 (ddt, 1H, J ) 16.8, 10.3, 6.2 Hz), 6.97 (d, 1H, J ) 8.4
Hz), 7.04 (d, 1H, J ) 8.4 Hz); MS m/z 229 (M+).
(E,Z)-2,3-Dich lor o-6-(1-p r op en yl)a n ilin e (13a ). Com-
pound 12a (1.00 g, 4.95 mmol) was dissolved in dimethyl
sulfoxide (6 mL) and treated with potassium tert-butoxide (1.38
g, 12.3 mmol). The resulting suspension was heated to 55 °C
for 4 h. After cooling to room temperature, the mixture was
neutralized with a saturated aqueous ammonium chloride
solution and extracted with diethyl ether. The organic phase
was washed with brine, dried, and concentrated under vacuum.
The crude product was purified by flash chromatography (n-
hexane/dichloromethane 6:4) to yield 13a as a 9:1 E/ Z
diastereomeric mixture (0.690 g, 3.42 mmol, 69% yield) as an
oil: 1H NMR (CDCl3, 9:1 E/ Z mixture, asterisk denotes minor

4040 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Minutolo et al.
isomer peaks) δ 1.71* (dd, 3H, J ) 6.9, 1.3 Hz), 1.90 (dd, 3H,
J ) 6.4, 1.3 Hz), 6.08 (dq, 1H, J ) 15.6, 6.4 Hz), 6.32 (dd, 1H,
J ) 15.6, 1.2 Hz), 6.81 (d, 1H, J ) 8.4 Hz), 7.02 (d, 1H, J )
8.3 Hz); MS m/z 201 (M+, 100), 186 (M+ - CH3, 43), 174 (M+
- CHdCH2, 42), 165 (M+ - Cl, 53), 130 (M+ - 2Cl, 84).
denotes minor isomer peaks) δ 0.89* (t, 3H, J ) 7.1 Hz), 0.91
(t, 3H, J ) 7.1 Hz), 1.93* (dd, 3H, J ) 6.8, 1.5 Hz), 1.95 (dd,
3H, J ) 6.6, 1.6 Hz), 2.95 (q, 2H, J ) 7.1 Hz), 3.10 (br, 1H),
5.85* (dq, 1H, J ) 11.4, 7.0 Hz), 6.21 (dq, 1H, J ) 15.7, 6.6
Hz), 6.53* (dq, 1H, J ) 11.6, 1.4 Hz), 6.69 (dq, 1H, J ) 15.6,
1.5 Hz), 6.99-7.25 (m, 11H), 7.44 (d, 1H, J ) 8.1 Hz); MS m/z
313 (M+).
(E,Z)-2,3-Dich lor o-N-m eth yl-6-(1-pr open yl)an ilin e (13b).
Compound 13b was prepared from 12b (0.402 g, 1.86 mmol)
by following the same procedure described above for 13a . The
crude product was purified by flash chromatography (n-
hexane/diethyl ether 9:1) to yield 13b as a 9:1 E/ Z diastere-
omeric mixture (0.309 g, 1.43 mmol, 77% yield) as an oil: 1H
NMR (CDCl3, 9:1 E/ Z mixture, asterisk denotes minor isomer
peaks) δ 1.76* (dd, 3H, J ) 7.0, 1.8 Hz), 1.91 (dd, 3H, J ) 6.6,
1.5 Hz), 2.83 (s, 3H), 2.87* (s, 3H), 3.09 (bs, 1H), 5.84* (dq,
1H, J ) 11.4, 7.0 Hz), 6.10 (dq, 1H, J ) 15.6, 6.6 Hz), 6.38*
(dq, 1H, J ) 11.4, 1.8 Hz), 6.52 (dq, 1H, J ) 15.6, 1.5 Hz),
6.98 (d, 1H, J ) 8.6 Hz), 7.15 (d, 1H, J ) 8.4 Hz); MS m/z 215
(M+).
(E,Z)-2,3-Dich lor o-N-eth yl-6-(1-p r op en yl)a n ilin e (13c).
Compound 13c was prepared from 12c (2.62 g, 11.4 mmol) by
following the same procedure described above for 13a . The
crude product was purified by flash chromatography (n-
hexane/dichloromethane 8:2) to yield 13c as a 9:1 E/ Z dia-
stereomeric mixture (1.94 g, 8.44 mmol, 74% yield) as an oil:
1H NMR (CDCl3, 9:1 E/ Z mixture, asterisk denotes minor
isomer peaks) δ 0.99* (t, 3H, J ) 7.1 Hz), 1.16 (t, 3H, J ) 7.1
Hz), 1.90 (dd, 3H, J ) 6.6, 1.6 Hz), 3.13 (q, 2H, J ) 7.1 Hz),
3.20* (q, 2H, J ) 7.1 Hz), 3.83 (br, 1H), 4.19* (br, 1H), 5.83*
(dq, 1H, J ) 11.2, 6.9 Hz), 6.10 (dq, 1H, J ) 15.7, 6.6 Hz),
6.36* (dq, 1H, J ) 11.3, 1.8 Hz), 6.50 (dq, 1H, J ) 15.7, 1.6
Hz), 6.75* (d, 1H, J ) 8.2 Hz), 6.88* (d, 1H, J ) 8.4 Hz), 6.98
(d, 1H, J ) 8.2 Hz), 7.15 (d, 1H, J ) 8.4 Hz); MS m/z 229 (M+).
(E,Z)-2,3-Dip h en yl-6-(1-p r op en yl)a n ilin e (14a ). A solu-
tion of 13a (0.300 g, 1.49 mmol) in dioxane (1.5 mL) was
treated with 0.834 g (2.56 mmol) of cesium carbonate, 0.273 g
(2.24 mmol) of phenylboronic acid, 0.044 g (0.048 mmol) of tris-
(dibenzylideneacetone)dipalladium(0), and 0.18 mL of a 20%
solution of tricyclohexylphosphine (0.11 mmol) in toluene. The
resulting suspension was heated at 80 °C for 16 h under an
argon atmosphere. The reaction mixture was then cooled to
room temperature, diluted with diethyl ether, and filtered
through a Celite pad. The solvent was removed under vacuum,
and the resulting crude mixture was submitted again to the
same treatment described above. The crude product deriving
from the second step was purified by column chromatography,
eluting with a 9:1 n-hexane/ethyl acetate mixture, to obtain
14a as a 9:1 E/ Z diastereomeric mixture (0.371 g, 1.30 mmol,
87% yield, two steps) as an oil: 1H NMR (CDCl3, 9:1 E/ Z
mixture, asterisk denotes minor isomer peaks) δ 1.86* (dd, 3H,
J ) 6.9, 1.7 Hz), 1.94 (dd, 3H, J ) 6.5, 1.6 Hz), 3.71 (br, 1H),
6.17 (dq, 1H, J ) 15.6, 6.5 Hz), 6.48 (dq, 1H, J ) 15.6, 1.6
Hz), 6.84 (d, 1H, J ) 7.9 Hz), 7.03-7.31 (m, 11H); MS m/z
285 (M+).
(E,Z)-2,3-Diph en yl-N-m eth yl-6-(1-pr open yl)an ilin e (14b).
Compound 14b was prepared from 13b (0.129 g, 0.597 mmol)
by following the same procedure described above for 14a . The
crude product was purified by flash chromatography (n-
hexane/ethyl acetate 9:1) to yield 14b as a 9:1 E/ Z diastere-
omeric mixture (0.131 g, 0.436 mmol, 73% yield, two steps) as
an oil: 1H NMR (CDCl3, 9:1 E/ Z mixture, asterisk denotes
minor isomer peaks) δ 1.92* (dd, 3H, J ) 6.8, 1.6 Hz), 1.97
(dd, 3H, J ) 6.6, 1.6 Hz), 2.65 (d, 3H, J ) 5.7 Hz), 2.95* (d,
3H, J ) 5.2 Hz), 3.30 (br q, 1H, J ) 5.5 Hz), 3.42* (br, 1H),
5.87* (dq, 1H, J ) 11.4, 7.1 Hz), 6.23 (dq, 1H, J ) 15.7, 6.6
Hz), 6.54* (dq, 1H, J ) 11.5, 1.8 Hz), 6.70 (dq, 1H, J ) 15.6,
1.5 Hz), 7.02 (d, 1H, J ) 8.0 Hz), 7.06-7.14 (m, 6H), 7.21-
7.39 (m, 4H), 7.45 (d, 1H, J ) 7.9 Hz); MS m/z 299 (M+).
3,4-Dip h en yla n th r a n yla ld eh yd e (15a ). A solution of 14a
(0.370 g, 1.30 mmol) in dioxane (10 mL) was treated with 5
mL of water, 0.639 g of sodium periodate (2.99 mmol), and
0.05 mL of a 2.5% solution of osmium tetroxide in tert-butyl
alcohol (0.05 mmol), and the mixture was stirred at room
temperature for 4 h. The mixture was then diluted with water
and extracted with chloroform. The organic phase was washed
with aqueous sodium thiosulfate, dried, and concentrated to
afford a crude residue that was purified by flash chromatog-
raphy (n-hexane/ethyl acetate 9:1) to yield 15a (0.167 g, 0.611
mmol, 47% yield) as a yellow solid: mp 125 °C; 1H NMR
(CDCl3) δ 6.84 (d, 1H, J ) 8.1 Hz), 7.05-7.17 (m, 8H), 7.23-
7.31 (m, 2H), 7.56 (d, 1H, J ) 8.2 Hz), 9.95 (s, 1H); MS m/z
273 (M+, 100), 245 (M+ - CO, 52), 244 (M+ - CHO, 67). Anal.
C
19H15NO (C, H, N).
3,4-Dip h en yl-N-m eth yla n th r a n yla ld eh yd e (15b). Com-
pound 15b was prepared from 14b (0.288 g, 0.963 mmol) by
following the same procedure described above for 15a . The
crude product was purified by flash chromatography (n-
hexane/dichloromethane 1:1) to yield 15b (0.122 g, 0.424 mmol,
44% yield) as a yellow solid: mp 118-120 °C; 1H NMR (CDCl3)
δ 2.28 (s, 3H), 6.77 (d, 1H, J ) 8.1 Hz), 6.94-6.99 (m, 2H),
7.06-7.19 (m, 8H), 7.51 (d, 1H, J ) 7.9 Hz), 8.30 (br, 1H), 9.90
(s, 1H); MS m/z 287 (M+). Anal. C20H17NO (C, H, N).
3,4-Dip h en yl-N-eth yl-a n th r a n yla ld eh yd e (15c). Com-
pound 15c was prepared from 14c (0.337 g, 1.08 mmol) by
following the same procedure described above for 15a . The
crude product was purified by flash chromatography (n-
hexane/dichloromethane 1:1) to yield 15c (0.149 g, 0.497 mmol,
1
46% yield) as a yellow solid: mp 75-77 °C; H NMR (CDCl3)
δ 0.96 (t, 3H, J ) 7.1 Hz), 2.52 (q, 2H, J ) 7.1 Hz), 6.89 (d,
1H, J ) 8.0 Hz), 6.94-6.99 (m, 2H), 7.09-7.23 (m, 8H), 7.56
(d, 1H, J ) 7.9 Hz), 9.94 (s, 1H); MS m/z 301 (M+). Anal. C21H19
NO (C, H, N).
-
3,4-Dip h en yla n th r a n yla ld oxim e (5a ). A solution of 15a
(0.030 g, 0.11 mmol) in methanol (2 mL) was treated with a
solution of hydroxylamine hydrochloride (0.016 g, 0.22 mmol)
in water (0.5 mL), and the resulting mixture was heated to
50 °C for 3 h. After being cooled to room temperature, the
solvent was partially removed under vacuum and the residue
was extracted with ethyl acetate. The combined organic phase
were dried and concentrated under vacuum, and the crude
product was purified by flash chromatography (n-hexane/ethyl
acetate 8:2) to yield 5a (0.023 g, 0.079 mmol, 72% yield) as a
1
solid: mp 135-137 °C; H NMR (CDCl3) δ 6.81 (d, 1H, J )
7.9 Hz), 7.02-7.32 (m, 11H), 8.34 (s, 1H);13C NMR (CDCl3) δ
113.47, 118.71, 126.47, 127.25, 127.58, 128.84, 129.60, 131.09,
131.57, 137.10, 141.49, 143.38, 153.80; MS m/z 288 (M+, 100),
271 (M+ - OH, 45), 254 (M+ - H2O - NH2, 13), 244 (M+
CHdNOH, 82). Anal. C19H16N2O (C, H, N).
-
3,4-Dip h en yl-N-m eth yla n th r a n yla ld oxim e (5b). Com-
pound 5b was prepared from 15b (0.033 g, 0.11 mmol) by
following the same procedure described above for 5a . The crude
product was purified by flash chromatography (n-hexane/ethyl
acetate 8:2) to yield 5b (0.026 g, 0.087 mmol, 79% yield) as a
solid: mp 130 °C; 1H NMR (CDCl3) δ 2.40 (s, 3H), 6.92 (d, 1H,
J ) 7.9 Hz), 6.97-7.01 (m, 2H), 7.09-7.22 (m, 8H), 7.39 (d,
1H, J ) 8.0 Hz), 8.37 (s, 1H); 13C NMR (CDCl3) δ 36.14, 115.49,
119.98, 122.84, 126.52, 127.11, 127.58, 128.27, 129.60, 130.20,
131.20, 137.78, 141.31, 144.51, 151.81; MS m/z 302 (M+, 13),
285 (M+ - OH, 100). Anal. C20H18N2O (C, H, N).
3,4-Dip h en yl-N-et h yla n t h r a n yla ld oxim e (5c). Com-
pound 5c was prepared from 15c (0.116 g, 0.385 mmol) by
following the same procedure described above for 5a . The crude
product was purified by flash chromatography (n-hexane/ethyl
acetate 8:2) to yield 5c (0.093 g, 0.293 mmol, 76% yield) as a
solid: mp 103 °C; 1H NMR (CDCl3) δ 1.06 (t, 3H, J ) 7.1 Hz),
(E,Z)-2,3-Dip h en yl-N-eth yl-6-(1-p r op en yl)a n ilin e (14c).
Compound 14c was prepared from 13c (1.63 g, 7.10 mmol) by
following the same procedure described above for 14a . The
crude product was purified by flash chromatography (n-
hexane/dichloromethane 8:2 f 6:4) to yield 14c as a 9:1 E/ Z
diastereomeric mixture (1.44 g, 4.62 mmol, 65% yield, two
steps) as an oil: 1H NMR (CDCl3, 9:1 E/ Z mixture, asterisk

Novel Estrogen Receptor Ligands
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 4041
2.86 (q, 2H, J ) 7.1 Hz), 7.13 (d, 1H, J ) 7.9 Hz), 7.15-7.18
(m, 2H), 7.24-7.42 (m, 8H), 7.61 (d, 1H, J ) 8.0 Hz), 8.54 (s,
1H); 13C NMR (CDCl3) δ 15.45, 44.79, 115.50, 121.02, 123.61,
124.27, 126.54, 127.21, 127.60, 127.94, 128.36, 129.60, 131.04,
137.63, 141.24, 144.22, 151.29; MS m/z 316 (M+, 11), 299 (M+
- OH, 100). Anal. C21H20N2O (C, H, N).
Biologica l Meth od s. Purified human full-length ERR and
ERâ were obtained from PanVera (Madison, WI). Cell culture
media were purchased from Gibco BRL (Grand Island, NY).
Calf serum was obtained from Hyclone Laboratories, Inc.
(Logan, UT), and fetal calf serum was purchased from Atlanta
Biologicals (Atlanta, GA). The luciferase assay system was
from Promega (Madison, WI). The expression vectors for
human ERR (pCMV5-hERR) and human ERâ (pCMV5-ERâ)
were constructed previously as described.45,46 The estrogen
responsive reporter plasmid was (ERE)2-pS2-Luc, was con-
structed by inserting the (ERE)2-pS2 fragment from (ERE)2-
pS2-CAT into the MluI/BglII sites of pGL3-Basic vector
(Promega, Madison, WI). The plasmid pCMVâ (Clontech, Palo
Alto, CA), which contains the â-galactosidase gene, was used
as an internal control for transfection efficiency.
A manual docking of the N-Et derivative (5c) in ERR was
performed, imposing the orientation of the ligand in a binding
mode that overlapped with the previously found orientation
of the other analogues 1, 5a ,b. A molecular dynamic simulation
of 100 ps, T ) 300 K, time step ) 1 fs (Amber force field) was
performed on the complex so obtained by the MacroModel
program. During the simulation, all the protein backbone
atoms were fixed, as were the ligand pseudocycle atoms and
the hydrogen bond of the oxime OH with Glu353 and Arg394.
All the constraints were relaxed during minimization after the
molecular dynamic, performed through the conjugate gradient
method, using the derivative convergence criterion at 0.05 kJ /
mol. The interaction energy between ligand and receptor was
obtained through a MacroModel complete current energy
calculation, performed considering the steric and electrostatic
terms of all the atoms of the ligand with respect to the protein.
Ack n ow led gm en t. This work was partly supported
by CNR, Rome. We are grateful for support of this
research through grants from the National Institutes
of Health [PHS 5R37 DK15556 (J .A.K.) and 5R01
CA19118 (B.S.K.)].
Estr ogen Recep tor Bin d in g Assa ys. Relative binding
affinities were determined by competitive radiometric binding
assays using 10 nM [3H]E2 as tracer, using methods previously
described.36,37 The source of ER was purified full-length human
ERR and ERâ purchased from Pan Vera (Madison, WI).
Incubations were done at 0 °C for 18-24 h, and hydroxyl-
apatite was used to absorb the purified receptor-ligand
complexes (human ERs).37 The binding affinities are expressed
as relative binding affinity (RBA) values, where the RBA of
estradiol is 100%; under these conditions, the Kd of estradiol
for ERR is ca. 0.2 nM and for ERâ is 0.5 nM. The determination
of these RBA values is reproducible in separate experiments
with a CV of 0.3, and the values shown represent the average
( range or SD of two or three or more separate determinations,
respectively.
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