Monoaryl-substituted salicylaldoximes as ligands for estrogen receptor β

Article abstract of DOI:10.1021/jm701396g

Salicylaldoximes possess a hydrogen-bonded pseudocyclic A′ ring in place of the typical phenolic A ring that is characteristic of most estrogen receptor (ER) ligands. Monoaryl-substituted salicylaldoximes were obtained by replacing the phenol moiety (ring

Full text of DOI:10.1021/jm701396g

1344
J. Med. Chem. 2008, 51, 1344–1351
Monoaryl-Substituted Salicylaldoximes as Ligands for Estrogen Receptor ꢀ
Filippo Minutolo,*,‡ Rosalba Bellini,‡ Simone Bertini,‡ Isabella Carboni,‡ Annalina Lapucci,‡ Letizia Pistolesi,‡ Giovanni Prota,‡
Simona Rapposelli,‡ Francesca Solati,‡ Tiziano Tuccinardi,‡ Adriano Martinelli,‡ Fabio Stossi,# Kathryn E. Carlson,§
Benita S. Katzenellenbogen,# John A. Katzenellenbogen,§ and Marco Macchia‡
Dipartimento di Scienze Farmaceutiche, UniVersità di Pisa, Via Bonanno 6, 56126 Pisa, Italy, and Department of Molecular and IntegratiVe
Physiology and Department of Chemistry, UniVersity of Illinois, 600 S. Goodwin AVenue, Urbana, Illinois 61801
ReceiVed NoVember 7, 2007
Salicylaldoximes possess a hydrogen-bonded pseudocyclic A′ ring in place of the typical phenolic A ring
that is characteristic of most estrogen receptor (ER) ligands. Monoaryl-substituted salicylaldoximes were
obtained by replacing the phenol moiety (ring A) of the ERꢀ pharmacophore with the pseudocycle A′ ring,
which has previously been shown to behave as a bioequivalent of phenols in nonselective ER ligands. In
this series, small substituents (CH3, CN, Cl) were introduced into the central phenyl scaffold. An efficient
sequential halogen-selective double cross-coupling reaction was developed for the synthesis of the methyl-
substituted ER ligand. The measured ERꢀ affinity proved to be very sensitive to the effect of central core
substituents. The binding affinities of the compounds herein reported were in good agreement with the
results of computational docking analysis. The chloro-substituted derivative showed the highest ꢀ affinity
and selectivity, and it also proved to be an ERꢀ partial agonist with an EC50 of 11 nM.
Introduction
The estrogen receptor (ER)a exists in two isoforms, R (ERR)
and ꢀ (ERꢀ), that have different distributions in various estrogen
target tissues and also have different functions, some of which
have not yet been clarified. In fact, although a clear role has
been established for ERR in the classical effects of estrogen
activity, the physiological role of ERꢀ is less well understood.1
Figure 1. Structural derivation of monoaryl-substituted salicylal-
doximes from the ERꢀ pharmacophore.
In several situations ERꢀ seems to counteract the actions of
the other estrogen receptor subtype (ERR), the form that
cavities caused by variations at amino acid residues positioned
promotes cancer development in some tissues (uterus, breast
in proximity around the outer side of the cavity borders. This
glands, ovary). Moreover, the cancer-controlling properties of
ERꢀ seem to extend to colorectal and prostate cancer.2
Therefore, there is growing evidence that stimulation of ERꢀ
by a ꢀ-selective agonist may lead to a chemopreventive or even
high similarity in the binding pocket of the two ERs constitutes
a serious impediment to the development of highly ꢀ-selective
ligands.
In addition to an ERꢀ-selective binding affinity, compounds
with potent transcriptional activity are also sought, since ERꢀ-
selective stimulation is considered to be therapeutically relevant1,2
and, therefore, would be obtained only with selective ERꢀ
agonists (SERBAs).6 The silencing/activation process of the ERs
is also strongly affected, not only by the ligand structure but
also 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.7,8 Thus, obtaining
selective ERꢀ agonists with a desirable pharmacological profile
is a very challenging goal in this quite complicated framework.
The structural analysis of some examples of either natural
(genistein)9,10 or synthetic nonsteroidal compounds (i.e., DNP,11
ERB-041,12 indazoles13), showing specific ERꢀ agonist activity,
shows several common features that were collected in a ERꢀ
pharmacophoric model (Figure 1).13
therapeutic effect in these tumors. Furthermore, highly selective
ERꢀ agonists may present some of the “most wanted” features
sought in SERMs (selective estrogen receptor modulators)3–5
by providing beneficial estrogenic effects in some targets (for
example, bone, CNS, liver, adipocytes, and cardiovascular
system) while at the same time avoiding estrogen agonist effects
on breast and uterus mediated by the ERR-subtype.
Unfortunately, there are only minor structural differences in
the ligand binding cavities of ERR and ERꢀ that can be
exploited to obtain highly ꢀ-selective ligands. These consist of
slightly different pocket volumes, with the ERꢀ binding cavity
being somewhat smaller than that of ERR and with just two
amino acid differences: ERꢀ has a methionine (M336) in the
place of the leucine at position 384 of ERR and has an isoleucine
(I373) instead of the methionine at position 421 of ERR. There
are also some slight differences in the shape of the binding
In an attempt to enlarge the chemical diversity of estrogen
receptor–ligands, we had previously shown that some oxime
derivatives, comprising a six-membered pseudocycle formed by
an intramolecular H-bond, could isosterically replace one phenol
ring in ER ligands. Thus, we have developed diaryl-substituted
salicylaldoximes14,15 and anthranylaldoximes16,17 that could
efficiently bind the ERs. Other scientists have also independently
reported that some aryl-carbaldehyde oximes can efficiently
replace one phenol ring in ERꢀ-selective ligands.18
* To whom correspondence should be addressed. Telephone: +39 050
2219557. Fax: +39 050 2219605. E-mail: minutolo@farm.unipi.it.
‡
Università di Pisa.
#
Department of Molecular and Integrative Physiology, University of
Illinois.
§
Department of Chemistry, University of Illinois.
a Abbreviations: ER, estrogen receptor; SERM, selective estrogen
receptor modulator; SERBA, selective estrogen receptor ꢀ agonist; SPhos,
2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; RBA, relative binding
affinity.
10.1021/jm701396g CCC: $40.75
2008 American Chemical Society
Published on Web 02/13/2008

Salicylaldoximes as SelectiVe ERꢀ Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1345
Chart 1. Monoaryl-Substituted Salicylaldoximes Designed as
ERꢀ-Selective Ligands
Scheme 1. Synthesis of 3-Unsubstituted Salicylaldoximes 1-3a
On the basis of this evidence we envisaged the possibility of
adapting our molecular approach to the newly developed ERꢀ
pharmacophoric model by replacing the phenol A-ring with an
A′-pseudocycle formed by an intramolecular H-bond between
the oxime N-atom and the o-OH group. The design was
completed by the insertion of an additional p-phenol substituent
in position 4 of the central ring (Figure 1). Moreover, we were
intrigued by the possibility of exploring the effect that could
be caused by the introduction of several small substituents in
position 3 of the central ring (gray sphere, Figure 1).
In fact, we were particularly attracted by the effect that was
previously observed in an indazole series of ERꢀ ligands, where
the introduction into the central scaffold of a halogen atom (Cl,
Br, I), a small alkyl (Et), or a CN group, dramatically increased
the ERꢀ affinities of the resulting molecules and also remarkably
enhanced their ꢀ selectivities.13
a Reagents and conditions: (a) allyl bromide, K2CO3, acetonitrile, 80 °C;
(b) N-methylaniline, 180 °C; (c) t-BuOK, DMSO, 55 °C; (d) 4-R-
C6H4B(OH)2, Pd(PPh3)4, Na2CO3, toluene, EtOH, ∆; (e) OsO4, NaIO4,
dioxane-H2O; (f) NH2OH·HCl, MeOH-H2O, 50 °C; (g) BBr3, CH2Cl2,
-78 to 0 °C.
We therefore designed a series of new monoaryl-substituted
salicylaldoximes (1-7, Chart 1) that could potentially show
selective binding for ERꢀ and a satisfactory potency as ERꢀ
agonists. Initially, we considered the 3-unsubstituted monoar-
ylsalicylaldoxime 1. Then, among the possible substituents to
be introduced into the central ring, the choice of the methyl
(4), the chloro (6), or the cyano (7) group was based on the
pharmacophoric considerations reported above and on the
synthetic accessibilities of the target molecules. For some of
them (1 and 4), the importance of the additional phenolic p-OH
group (R2 ) OH) was assessed by preparing their analogues
either with a p-methoxy moiety (R2 ) OCH3, 2 and 5) or
completely unsubstituted (R2 ) H, 3).
Figure 2. Sequential halogen-selective double cross-coupling reaction.
3-Methyl-substituted oximes 4 and 5 required a slightly
different synthetic approach. To this aim we had to introduce a
“sequential halogen-selectiVe double cross-coupling reaction”
into the synthetic plan, as shown in Figure 2.
We had previously exploited a similar approach for the
preparation of 3,4-heterodiaryl-substituted salicylaldoximes15
and N-methylanthranylaldoximes.17 In detail, our idea was to
perform this reaction sequence on an appropriate bromochloro
disubstituted precursor, where the more reactive Br group could
be initially replaced by an aryl under classical Suzuki conditions,
using Pd(PPh3)4 as the catalyst. As a matter of fact, under these
“mild” conditions, the chloroaryl group is typically unreactive.19
Later, the methyl could be introduced by submitting the Cl group
to a highly active catalytic system, although this second Pd-
catalyzed cross-coupling between an alkylboronic acid and an
aryl chloride is generally considered to be quite challenging.19
Scheme 2 shows how we applied this strategy. We first
verified the suitability of our double cross-coupling sequence
by model reactions on 3-bromo-2-chlorophenol (17), prepared
as previously reported by a remarkably regioselective ortho
lithiation, occurring exclusively at the 2-position, of N,N-
diethylcarbamate of 3-bromophenol, followed by quenching with
hexachloroethane and hydrolysis.20 The first step with 4-meth-
oxyphenylboronic acid was carried out in the presence of
Pd(PPh3)4 (Suzuki conditions19) and afforded intermediate 18.
The second step was conducted with methylboronic acid in the
presence of Pd(OAc)2 as the catalyst and 2-dicyclohexylphos-
phino-2′,6′-dimethoxybiphenyl (Buchwald Ligand, SPhos) be-
cause this catalytic system was known to efficiently promote
the cross-coupling reaction even between alkylboronic acids and
aryl chlorides.21 We found that compound 19 could be obtained
quite efficiently this way. So these model reactions confirmed
that the halogen-selective double cross-coupling was achievable
Results and Discussion
Synthetic Chemistry. Different synthetic strategies were
applied, depending on the kind of substituent to be introduced
into the 3-position of the central ring (R1). The 3-unsubstituted
molecules (1-3) were synthesized as reported in Scheme 1.
Commercially available 3-bromophenol (8) was treated with
allyl bromide to give allyl ether 9, which was submitted to a
Claisen rearrangement at 180 °C in N-methylaniline, yielding
2-allyl-5-bromophenol (10) together with the other o-allyl-
substituted derivative (2-allyl-3-bromophenol), from which it
was separated by column chromatography. Alkaline isomeriza-
tion of the terminal double bond of 10 afforded an E/Z
diastereoisomeric mixture of 11. Palladium-catalyzed cross-
coupling reaction of 11 with phenylboronic or 4-methoxyphe-
nylboronic acid afforded biphenyl derivatives 12 or 13, respec-
tively. Oxidative cleavage of the double bond of 12 and 13,
carried out with sodium periodate in the presence of catalytic
amounts of osmium tetroxide, yielded salicylaldehydes 14 and
15, and their condensation with hydroxylamine hydrochloride
respectively afforded oximes 3 and 2. Treatment of 15 with
BBr3 caused the removal of the O-methyl group, and subsequent
condensation of the resulting aldehyde (16) with hydroxylamine
hydrochloride produced 4-hydroxyphenyl-substituted oxime 1.

1346 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Minutolo et al.
Scheme 2. Synthesis of 3-Methyl-Substituted Salicylaldoximes
4 and 5a
Scheme 3. Synthesis of 3-Chloro-Substituted Salicylaldoxime 6a
a Reagents and conditions: (a) OsO4, NaIO4, dioxane-H2O; (b) BBr3,
CH2Cl2, -78 to 0 °C; (c) NH2OH·HCl, MeOH-H2O, 50 °C.
Scheme 4. Synthesis of 3-Cyano-Substituted Salicylaldoxime 7a
a Reagents and conditions: (a) 4-MeOC6H4B(OH)2, Pd(OAc)2, PPh3,
aqueous 2M Na2CO3, 1:1 toluene/EtOH, 100 °C, 16 h; (b) MeB(OH)2,
Pd(OAc)2, 2-dicyclohexylphosphine-2′,6′-dimethoxybiphenyl (SPhos),
K3PO4, toluene, 100 °C; (c) allyl bromide, K2CO3, acetonitrile, 80 °C; (d)
N-methylaniline, 180 °C; (e) t-BuOK, DMSO, 55 °C; (f) OsO4, NaIO4,
dioxane-H2O; (g) BBr3, CH2Cl2, -78 to 0 °C; (h) NH2OH · HCl,
MeOH-H2O, 50 °C.
with this kind of compound, and therefore, it was applied to
the real synthesis of oximes 4 and 5 (Scheme 2).
3-Bromo-2-chlorophenol (17)20 was submitted to O-allylation
(20), Claisen rearrangement (21), and double bond isomerization
(22), similar to the synthesis reported above (Scheme 1). At
this point, the bromochloro-substituted intermediate 22 was
submitted to the halogen-selective double cross-coupling se-
quence, first introducing the p-methoxyphenyl group in the place
of the Br atom (23) under mild conditions and then replacing
the less reactive Cl atom by a methyl group, under harsher
Buchwald conditions.21 Compound 24 so obtained was treated
with sodium periodate in the presence of catalytic amounts of
osmium tetroxide, yielding salicylaldehyde 25. Subsequent
condensation of 25 with hydroxylamine hydrochloride afforded
oxime 5. Demethylation of 25 by BBr3 afforded aldehyde 26
which, after reaction with hydroxylamine hydrochloride, eventu-
ally produced oxime 4.
The synthesis of chloro-substituted oxime 6 is shown in
Scheme 3 and started from intermediate 23, which was prepared
as reported above (Scheme 2). Oxidative cleavage of the styrene-
type double bond of 23 was achieved with the catalytic OsO4/
NaIO4 system, producing aldehyde 27, which was then sub-
mitted to O-demethylation in the presence of boron tribromide.
The final step involved a condensation of 28 with NH2OH·HCl,
which yielded salicylaldoxime 6.
a Reagents and conditions: (a) (1) AcOK, 18-crown-6, CH3CN, ∆, (2)
aqueous NaOH (10 M); (b) allyl bromide, K2CO3, acetonitrile, 80 °C; (c)
microwave, basic Al2O3, 200 °C; (d) t-BuOK, DMSO, 55 °C; (e)
4-MeOC6H4B(OH)2, Pd2(dba)3, Cy3P, Cs2CO3, dioxane, microwave, 80 W,
30 min; (f) OsO4, NaIO4, dioxane-H2O; (g) BBr3, CH2Cl2, -78 to 0 °C;
(h) NH2OH·HCl, MeOH-H2O, 50 °C.
bromide to give O-allylphenol 31. At this point we observed
an unexpected reactivity of 31 in the Claisen rearrangement step.
When 31 was submitted to the same reaction conditions reported
for Claisen rearrangements in Schemes 1 and 2, we found that
the main product obtained was dihydrobenzofuran 33 rather than
the ortho-allylated phenol 32. Since the classical conditions used
in Schemes 1 and 2 (180 °C in N-methylaniline, 24–48 h) were
not suitable for a fast optimization, we tried to run the reaction
with neat compound 31 in a microwave synthesizer. In this case,
in spite of the very fast conversion (>95% after 20 min), the
reaction still produced the cyclized product 33 as the major or
exclusive product, regardless of the microwave power used.
In considering why this unexpected side reaction was
encountered in this system, we thought that it might be a result
of the stronger acidity of cyano-substituted phenol 32 when
compared to unsubstituted (10, Scheme 1) and chloro-substituted
(21, Scheme 2) phenols. In fact, the phenol OH group might
promote a cyclization reaction by protonating the terminal
carbon of the allylic substituent, with the resulting phenoxide
then forming a bond with the cation that forms on the internal
carbon atom of the allyl double bond (Figure 3). This “acid-
The synthesis of the cyano-substituted oxime followed a
slightly different approach in the first steps, which started from
commercially available 2-chloro-6-fluorobenzonitrile (29), as
shown in Scheme 4. Aryl fluoride 29 was submitted to
nucleophilic aromatic substitution with potassium acetate, and
the resulting aryl acetate was submitted in situ to an alkaline
hydrolysis.22 The resulting phenol 30 was then treated with allyl

Salicylaldoximes as SelectiVe ERꢀ Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1347
Table 1. Relative Binding Affinitiesa of Compounds 1–7 for the
Estrogen Receptors R and ꢀ
relative binding affinity
(% relative to that of estradiol)
Figure 3. Acid-promoted cyclization process affected by the acid
strength of the phenol.
ligand
hERR
(100)
hERꢀ
(100)
ꢀ/R ratio
estradiol
1
1
2
3
4
5
6
7
0.007 ( 0.001
0.010 ( 0.003
<0.004
0.249 ( 0.062
0.005 ( 0.001
0.065 ( 0.016
0.004 ( 0.001
0.553 ( 0.110
79
<0.004
<0.004
4.10 ( 1.00
0.007 ( 0.002
4.21 ( 0.66
0.078 ( 0.016
16
65
20
Figure 4. Base-promoted opening reaction of dihydrobenzofuran 33.
a Determined by a competitive radiometric binding assay with [3H]es-
tradiol. Preparations of purified, full-length human ERR and ERꢀ (PanVera/
Invitrogen) were used (see 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 ERꢀ it is 0.5 nM. Ki values for the new compounds
can be readily calculated by using the formula Ki ) (Kd[estradiol]/RBA) ×
100.
promoted” reaction would then be favored by an increased
acidity of the phenol OH group, as in cyano-substituted phenol
32, whereas it would be less likely when the phenol is less
acidic, as in 10 and 21.
In light of these considerations, we decided to repeat the
microwave experiments in the presence of solid supports
characterized by different levels of acidity/basicity, such as
silica, Celite, and neutral and basic alumina. In particular, we
thought that more alkaline conditions due to basic alumina could
“quench” the excessive acidity of the CN-substituted phenol,
and therefore, the cyclization reaction would not occur signifi-
cantly. In fact, when the reaction was run in the most acidic
media (silica), only the cyclized derivative 33 was recovered,
whereas we were pleased to find that basic alumina caused a
significant improvement of the reaction outcome, affording a
75:25 ratio of the desired o-allylphenol 32 and the benzofuran
33. Neutral solid supports (Celite and neutral alumina) caused
a simple loss of the allyl group with no Claisen rearrangement,
producing exclusively phenol 30.
Fortuitously, while optimizing this step, we found to our
surprise that desired compound 34 could be formed not only
from o-allyl phenol 32 but also from benzofuran 33 upon
treatment with potassium tert-butoxide in DMSO. We think that
the base-promoted ring opening of 33 starts with an initial
deprotonation of the benzylic methylene group, followed by
formation of the internal double bond and simultaneous ring
opening (Figure 4). Therefore, we could use the 75:25 mix-
ture of 32 and 33obtained in the previous step for the preparation
of 34, which was obtained as a 90:10 mixture of E/Z diastere-
oisomers. The final steps follow the same synthetic pathway
reported above and involve a Pd-catalyzed cross-coupling with
p-methoxyphenylboronic acid (35), an oxidative cleavage with
OsO4/NaIO4 (36), an O-demethylation with BBr3 (37), and a
condensation with NH2OH·HCl to produce oxime 7.
All the oximes (1-7) were obtained as single E-diastereoi-
someric forms. This is probably due to the fact that only these
isomers can form intramolecular hydrogen bonds that contribute
to the energetic stabilization of the products. This selectivity
had already been verified for other oxime analogues previously
reported,14–17 and it was confirmed by the chemical shift value
of the oxime proton, which is always found downfield from
the 8 ppm value (δ > 8 ppm; see Experimental Section).23
Estrogen Receptor Binding Assays. ERR and ERꢀ binding
affinities of oximes 1–7 were determined by a radiometric
competitive binding assay, using methods that have been
previously described.24,25 In Table 1 are reported the RBA
values for the newly reported compounds, determined with
purified full-length human R (hERR) and ꢀ (hERꢀ) receptor
subtypes. Binding affinity (RBA) values are reported as percent-
ages (%) relative to that of estradiol, which is set at 100%
(absolute affinities for estradiol: Kd ) 0.2 nM on ERR and 0.5
nM on ERꢀ).
As would be expected by an analysis of the ERꢀ pharma-
cophoric model (Figure 1), only p-hydroxyphenyl-substituted
compounds (1, 4, 6, and 7; R2 ) OH, Chart 1) show significant
affinities for the ERs. Among this series, compound 1 is
unsubstituted in the central core and shows a remarkable ERꢀ
selectivity, although the affinity for this receptor subtype is quite
modest (RBA ) 0.553%). The introduction of substituents (R1,
Chart 1) in the central ring have significant effects on binding.
The methyl-substituted 4 (R1 ) CH3) shows a very high ERꢀ
affinity (RBA ) 4.10%), but its subtype selectivity is not very
satisfactory. Better results are obtained with the introduction
of a chlorine, as in 6 (R1 ) Cl), where high affinity for ERꢀ is
observed, while at the same time, remarkable ERꢀ selectivity
(ERꢀ/R RBA ratio of 65) is maintained. On the other hand,
introduction of a cyano group, as in derivative 7, causes a loss
of affinity for both receptor subtypes. These results establish
that in this series chloro-substituted compound 6 is the best ERꢀ-
selective ligand, with an ERꢀ RBA of 4.21% relative to
estradiol, corresponding to an absolute binding (Ki) of 12 nM,
whereas its binding on ERR is much weaker (RBA ) 0.065%,
Ki ) 0.31 µM).
Transcription Assays. Compound 6, showing the highest
affinity and binding selectivity for ERꢀ, was assayed for
transcriptional activity through both receptor subtypes to
determine its pharmacological character. Reporter gene trans-
fection assays were conducted in human endometrial (HEC-1)
cells, using expression plasmids for either full-length human
ERR or ERꢀ and an estrogen-responsive luciferase reporter gene
system.26 In these assays, compound 6 proved to be an ERꢀ
partial agonist, reaching a maximum activation of 60% when
compared to estradiol, and had an EC50 value of 11 nM (Figure
5, solid line). This value is very close to the absolute affinity
Ki (12 nM) found in the binding assay (section above).
However, this compound also proved able to stimulate ERR
with an EC50 of 26 nM (Figure 5, dashed line), thus showing a
reduced level of ERꢀ selectivity in transcriptional potency
compared to that of its ERꢀ and ERR binding. This may be
ascribed to differences in the interaction of these receptor–ligand
complexes with the various coregulators present in these cellular
assays, which can act as modulators of ligand potency.
Molecular Modeling. An automated computational analysis
of all the compounds reported herein (1-7) docking into ERR
and ERꢀ (PDB codes 2I0J and 2I0G, respectively) was
performed, using the AUTODOCK 4.0 software.27 Figure 6A

1348 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Minutolo et al.
forming H-bonds with the E353-R394-water system (see
Figure 6E). This overturned disposition of compounds 4 and 6
in ERR, when compared to ERꢀ, seems to result from different
interactions of the two ERs with the R1 substituents. In fact, in
ERꢀ this group is able to interact with the lipophilic pocket
delimited by A302, W335, M336, and L339, whereas in ERR,
M336 is replaced by a leucine residue (L384), which restricts
the size of this cavity, thus reducing the ability of this pocket
to host a substituent and resulting in the different binding
disposition of the ligands in ERR. These observations are in
agreement with previously reported results,28,29 which indicate
that the leucine/methionine replacement results in differences
in the receptor binding cavities that can be exploited for the
development of selective ligands.
Compound 4, which shows the best ERR-binding affinity of
the series (and therefore a low ERꢀ selectivity), differs a bit
from the others by arranging its central-ring substituent (CH3)
in the lipophilic pocket delimited by M388, L391, F404, and
L428 (Figure 6F), thus benefiting from favorable hydrophobic
interactions. This is likely the main cause of its lower ERꢀ
selectivity when compared to the other ERꢀ ligands of this
series.
Figure 5. Dose–response curves for transcriptional activation by 6
through ERꢀ (solid line) and ERR (dashed line). Human endometrial
cancer (HEC-1) cells were transfected with expression vectors for ERR
or ERꢀ and an (ERE)2-pS2-luc reporter gene and were treated with
compound 6 at the concentrations indicated. Luciferase activity was
expressed relative to ꢀ-galactosidase activity from an internal control
plasmid. The maximal activity with 1 nM E2 was set at 100%. Values
are the mean of duplicate determinations.
Conclusions
Monoaryl-substituted salicylaldoximes were designed in
agreement with a recently developed ERꢀ pharmacophoric
model and were efficiently synthesized by means of Pd-
catalyzed cross-coupling reactions that, in some cases, required
an optimization of the catalytic system to have a halogen-
selective single reaction or sequential double reactions. This
class of compounds generally provided good ERꢀ ligands. A
particularly relevant effect was found with the introduction into
the 3-position of a methyl or a chloro substituent, which
considerably increased the ERꢀ binding affinities of the resulting
oximes 4 and 6, respectively, when compared to their 3-unsub-
stituted analogue 1. In particular, the 3-Cl substituent gives high
ERꢀ selectivity to 6, not by increasing its ERꢀ binding affinity
but by decreasing its binding ability for the ERR-subtype, when
compared to 4. Computational docking studies could explain
the high ERꢀ affinity observed with 4 and 6 and the low
ꢀ-selectivity of 4, by showing that ERꢀ has a small side pocket
(delimited by A302, W335, M336, and L339) that is able to
accommodate both the CH3 (4) and the Cl (6) substituents but
not a larger one such as CN (7). On the other hand, ERR can
only efficiently accommodate the methyl group (4) in its
corresponding lipophilic cavity delimited by M388, L391, F404,
and L428. The most interesting ERꢀ-selective ligand 6 also
proved to be an efficient ERꢀ partial agonist in functional assays,
although these tests revealed that the functional activity of 6 is
much less ERꢀ-selective than its binding affinity. Studies are
underway in an attempt to produce new monoaryl-subsituted
salicylaldoximes that would retain the ERꢀ binding affinity and
selectivity of 6 but simultaneously give improved ERꢀ-selective
agonist character in transcriptional activity assays.
displays the docking results for compounds 4 and 6 into ERꢀ.
In agreement with their very similar ERꢀ affinity levels, they
show comparable binding modes. The phenol OH group is
involved in a H-bond network, which includes E305, R346, and
a water molecule. The pseudocycle/oxime system forms H-
bonds with G472 and H475, whereas the methyl substituent of
4 and the chlorine atom of 6 are inserted into the lipophilic
pocket delimited by A302, W335, M336, and L339. Compound
1 has an 8-fold lower ERꢀ affinity than 4 and 6, and it is
characterized by the absence of the substituent on the central
ring (R1 ) H). It was found (Figure 6B) in an overturned
disposition with the phenol OH group interacting with G472
and H475 and the pseudocycle/oxime system engaged in the
H-bond network of the E305-R346-water system. The sub-
stitution of the phenolhydroxyl with a methoxy group, as in
compounds 2 and 5, results in a much greater decrease of affinity
for both receptors. As shown in Figure 6C, the docking studies
confirm that the presence of the methoxy group results in the
loss of the H-bond interactions with G472 and H475, thus
explaining the dramatic decrease of binding ability. Finally,
docking into ERꢀ of cyano-substituted compound 7 (R1 ) CN),
whose ERꢀ affinity is about 54-fold lower than that of
compounds 4 and 6, shows that the steric hindrance of the CN
group prevents its insertion in the lipophilic pocket delimited
by A302, W335, M336, and L339 (Figure 6D). This results in
a rotation of about 180° along the long molecular axis of 7
docked into ERꢀ when compared to 4 and 6, thus resulting in
a higher energy situation that explains its lower ERꢀ affinity.
The inability of the lipophilic small pocket delimited by A302,
W335, M336, and L339 to host a relatively long and linear
substituent, such as the CN sticking out of the 3-position of the
central phenyl ring in compound 7, is also displayed in Figure
7, where the hydrophobic cavity can barely fit the methyl (4)
or chloro (6) substituents (cyan circle).
Experimental Section
Chemistry. 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 referenced from solvent references. Electron impact (EI, 70
eV) mass spectra were obtained on a HP-5988A mass spectrometer.
Where indicated, the elemental compositions of the compounds
agreed to within (0.4% of the calculated value. Chromatographic
separations were performed on silica gel columns by flash (Kieselgel
All the analyzed compounds proved to be weaker ligands
with ERR. Docking shows that none of the compounds are able
to assume a disposition similar to those assumed in ERꢀ by the
most active ligands 4 and 6. On the contrary, they assume a
disposition similar to that of 1 in ERꢀ, that is, phenol OH
interacting with H524 and the pseudocycle/oxime system

Salicylaldoximes as SelectiVe ERꢀ Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1349
Figure 6. Docking analysis into ERꢀ and ERR: (A) docking of 4 (orange) and 6 (magenta) into ERꢀ; (B) docking of 1 (cyan) into ERꢀ; (C)
docking of 2 (green) and 5 (violet) into ERꢀ; (D) docking of 7 (yellow) into ERꢀ; (E) docking of 1, 6, and 7 into ERR; (F) docking of 4 into ERR.
procedures and characterization data of intermediate structures have
been included in the Supporting Information.
4-Phenylsalicylaldoxime (3). A solution of 14 (200 mg, 1.01
mmol) in ethanol (17 mL) was treated with a solution of hydroxyl-
amine hydrochloride (140 mg, 2.02 mmol) in water (3.5 mL), and
the mixture was heated to 50 °C for 1 h. After the mixture was
cooled to room temperature, part of the solvent was removed under
vacuum, and the mixture was diluted with water and extracted with
EtOAc. The organic phase was dried and evaporated to afford a
crude residue that was purified by column chromatography (n-
hexane/ethyl acetate, 90:10) to yield pure 3 (212 mg, 98% yield)
as a white solid: mp 130 °C; 1H NMR (CDCl3) δ (ppm) 7.18–7.27
(m, 3H), 7.37–7.49 (m, 3H), 7.59–7.64 (m, 2H), 8.27 (s, 1H), 9.84
(bs, 1H). MS m/z 213 (M+). Anal. (C13H11NO2) C, H, N.
4-(4-Methoxyphenyl)salicylaldoxime (2). Compound 14 (40
mg, 0.18 mmol) was submitted to the same procedure described
above for the preparation of 3. The crude product was purified by
flash chromatography (n-hexane/EtOAc, 70:30) to yield 2 as a white
solid (38 mg, 87% yield): mp 153 °C; 1H NMR (acetone-d6) δ
(ppm) 3.85 (s, 3H), 7.03 (AA′XX′, 2H, JAX ) 9.0 Hz, JAA′/XX′
)
2.5 Hz), 7.14 (d, 1H, J ) 1.6 Hz), 7.20 (dd, 1H, J ) 7.9, 1.6 Hz),
7.41 (d, 1H, J ) 8.1 Hz), 7.64 (AA′XX′, 2H, JAX ) 9.2 Hz, JAA′/
XX′ ) 2.6 Hz), 8.39 (s, 1H), 10.71 (bs, 1H). MS m/z 243 (M+).
Anal. (C14H13NO3) C, H, N.
Figure 7. Free space (red polyhedra) around 4 (orange) and 6 (magenta)
docked into ERꢀ. The cyan circle highlights the R1 substituents (CH3 and
Cl, respectively) and the absence of free extra space around these groups.
4-(4-Hydroxyphenyl)salicylaldoxime (1). Compound 16 (53
mg, 0.25 mmol) was submitted to the same procedure described
above for the preparation of 3. The crude product was purified by
flash chromatography (n-hexane/EtOAc, 6:4) to yield 1 as a white
solid (39 mg, 69% yield): mp 138 °C; 1H NMR (acetone-d6) δ
(ppm) 6.93 (AA′XX′, 2H, JAX ) 8.4 Hz, JAA′/XX′ ) 2.5 Hz), 7.12
(d, 1H, J ) 1.5 Hz), 7.18 (dd, 1H, J ) 8.1, 1.8 Hz), 7.39 (d, 1H,
J ) 8.2 Hz), 7.55 (AA′XX′, 2H, JAX ) 8.6 Hz, JAA′/XX′ ) 2.6 Hz),
8.38 (s, 1H), 8.56 (bs, 1H), 10.11 (bs, 1H), 10.65 (bs, 1H). MS
m/z 229 (M+, 100), 212 (M+ - OH, 43). Anal. (C13H11NO3) H,
N. C: calcd, 68.11; found, 67.49.
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. Microwave assisted
reaction were run in a CEM microwave synthesizer. The preparation

1350 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Minutolo et al.
4-(4-Methoxyphenyl)-3-methylsalicylaldoxime (5). Compound
25 (49 mg, 0.20 mmol) was submitted to the same procedure
described above for the preparation of 3. The crude product was
purified by flash chromatography (CH2Cl2) to yield 5 as a white
solid (36 mg, 71% yield): mp 145 °C; 1H NMR (CDCl3) δ (ppm)
2.20 (s, 3H), 3.86 (s, 3H), 6.83 (d, 1H, J ) 7.9 Hz), 6.90 (AA′XX′,
2H, JAX ) 8.8 Hz, JAA′/XX′ ) 2.2 Hz), 7.05 (d, 1H, J ) 7.9 Hz),
7.10 (bs, 1H), 7.25 (AA′XX′, 2H, JAX ) 8.6 Hz, JAA′/XX′ ) 1.8
Hz), 8.25 (s,1H), 10.00 (bs, 1H). MS m/z: 257 (M+). Anal.
(C15H15NO3) H, N. C: calcd, 70.02; found, 69.25.
containing 5 µL of lipofectin (Life Technologies, Rockville, MD),
20 µL of transferrin (Sigma, St. Louis, MO), 0.2 µg of pCMVꢀ-
galactosidase as internal control, 0.5 µg of the reporter gene plasmid,
and 50 ng of ER expression vector. The cells were incubated at 37
°C in a 5% CO2 containing incubator for 4 h. The medium was
then replaced with fresh medium containing 5% charcoal-dextran
treated calf serum and the desired concentrations of ligands.
Reporter gene activity was assayed at 24 h after ligand addition.
Luciferase activity, normalized for the internal control ꢀ-galactosi-
dase activity, was assayed as described.26
4-(4-Hydroxyphenyl)-3-methylsalicylaldoxime (4). Compound
26 (51 mg, 0.22 mmol) was submitted to the same procedure
described above for the preparation of 3. The crude product was
purified by flash chromatography (CH2Cl2) to yield 4 as a white
solid (51 mg, 95% yield): mp 125 °C; 1H NMR (CDCl3) δ (ppm)
2.19 (s, 3H), 4.80 (bs, 1H), 6.82 (d, 1H, J ) 8.0 Hz), 6.88 (AA′XX′,
2H, JAX ) 8.8 Hz, JAA′/XX′ ) 2.4 Hz), 7.05 (d, 1H, J ) 7.8 Hz),
7.20 (AA′XX′, 2H, JAX ) 8.8 Hz, JAA′/XX′ ) 2.4 Hz), 8.25 (s, 1H),
10.01 (bs, 1H). MS m/z: 243 (M+). Anal. (C14H13NO3) C, H, N.
3-Chloro-4-(4-hydroxyphenyl)salicylaldoxime (6). Compound
28 (39 mg, 0.16 mmol) was submitted to the same procedure
described above for the preparation of 3. The crude product was
purified by flash chromatography (n-hexane/AcOEt 6:4) to yield
Docking Methods. The crystal structures of ERR (PDB code
2I0J32) and ERꢀ (PDB code 2I0G32) were taken from the Protein
Data Bank.33 After adding hydrogen atoms and a water molecule
able to interact with E353-R394 in ERR and E305, R346 in ERꢀ,17
the two proteins complexed with their reference inhibitor (i.e.,
benzopyran selective estrogen receptor ꢀ agonist-1, SERBA-132)
were minimized using Amber 9 software34 and parm03 force field
at 300 K. The complexes were placed in a rectangular parallelepiped
water box. An explicit solvent model for water, TIP3P, was used,
and the complexes were solvated with a 10 Å water cap. Sodium
ions were added as counterions to neutralize the system. Two steps
of minimization were then carried out. In the first stage, we kept
the protein fixed with a position restraint of 500 (kcal/mol)/Å2 and
we solely minimized the positions of the water molecules. In the
second stage, we minimized the entire system through 5000 steps
of steepest descent followed by conjugate gradient (CG) until a
convergence of 0.05 kcal/(Å ·mol).
1
27 as a yellow solid (33 mg, 77% yield): mp 163 °C; H NMR
(acetone-d6) δ (ppm): 6.92 (AA′XX′, 2H, JAX ) 8.6 Hz, JAA′/XX′
)
2.5 Hz), 6.95 (d, 1H, J ) 7.9 Hz), 7.32 (AA′XX′, 2H, JAX ) 8.6
Hz, JAA′/XX′ ) 2.5 Hz), 7.37 (d, 1H, J ) 7.9 Hz), 8.43 (s, 1H), 8.56
(bs, 1H), 10.86 (bs, 1H), 10.91 (bs, 1H). MS m/z 263 (M+). Anal.
(C13H10ClNO3) C, H, N.
The ligands were built by means of Maestro35 and were then
minimized in a water environment (using the generalized Born/
surface area model) by means of Macromodel.36 They were
minimized using the CG method until a convergence value of 0.05
kcal/(Å·mol) was attained, using the MMFFs force field and a
distance-dependent dielectric constant of 1.0.
3-Cyano-4-(4-hydroxyphenyl)salicylaldoxime (7). Compound
37 (42 mg, 0.18 mmol) was submitted to the same procedure
described above for the preparation of 3. The crude product was
purified by flash chromatography (CHCl3/MeOH, 95:5) to produce
pure 7 (43 mg, 94% yield) as a white solid: mp 245 °C; 1H NMR
Automated docking was carried out by means of AUTODOCK
4.0 software.27 AUTODOCK TOOLS37 was used to identify the
torsion angles in the ligands, add the solvent model, and assign
the Kollman and the Gasteiger partial atomic charges to the protein
and the ligands, respectively. In order to prevent the loss of the
intramolecular H-bond of the pseudocycle/oxime system, during
the docking we blocked the torsions involved in this intramolecular
bond.17
The regions of interest used by AUTODOCK were defined by
considering SERBA-1 in both receptors as the central group. In
particular, a grid of 50, 40, and 46 points in the x, y, and z directions
was constructed centered on the center of mass of this compound.
A grid spacing of 0.375 Å and a distance-dependent function of
the dielectric constant were used for the energetic map calculations.
By use of the Lamarckian genetic algorithm, all docked
compounds were subjected to 100 runs of the AUTODOCK search,
in which the default values of the other parameters were used.
Cluster analysis was performed on the docked results using an rms
tolerance of 1.0 Å, and the best docked conformation was used for
the analysis.
(CD3OD) δ (ppm) 6.89 (AA′XX′, 2H, JAX ) 8.8 Hz, JAA′/XX′
)
2.4 Hz), 7.04 (d, 1H, J ) 8.1 Hz), 7.43 (AA′XX′, 2H, JAX ) 8.8
Hz, JAA′/XX′ ) 2.5 Hz), 7.55 (d, 1H, J ) 8.2 Hz), 8.32 (s, 1H). MS
m/z 254 (M+). Anal. (C14H10N2O3) C, H. N: calcd, 11.02; found,
10.50.
Biological Methods. Full-length human ERR and ERꢀ were
obtained from PanVera/Invitrogen (Carlsbad, CA). 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 expression vectors for human ERR (pCMV5-
hERR) and human ERꢀ (pCMV5-ERꢀ) were as described
previously.30,31 The estrogen responsive reporter plasmid, (ERE)2-
pS2-Luc, was constructed 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 luciferase assay system was
from Promega (Madison, WI). The plasmid pCMVꢀ-gal (Clontech,
Palo Alto, CA), which contains the ꢀ-galactosidase gene, was used
as an internal control for transfection efficiency.
Estrogen Receptor Binding Assays. Relative binding affinities
were determined by competitive radiometric binding assays with 2
nM [3H]E2 as tracer, as a modification of methods previously
described.24,25 The source of ER was purified full-length human
ERR and ERꢀ purchased from Pan Vera/Invitrogen (Carlsbad, CA).
Incubations were done at 0 °C for 18–24 h, and hydroxylapatite
was used to absorb the purified receptor–ligand complexes (human
ERs).25 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 about 0.2 nM, and
for ERꢀ it 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 more
separate determinations, respectively.
All graphic manipulations and visualizations were performed by
means of Chimera.38
Acknowledgment. We thank Karen J. Kieser for technical
assistance. Support of this research from the National Institutes
of Health is gratefully acknowledged (Grants R37DK15556,
R01CA18119, and P01AG024387 to J.A.K. and B.S.K.).
Molecular graphics images were produced using the UCSF
Chimera package from the Resource for Biocomputing, Visu-
alization, and Informatics at the University of California, San
Francisco (supported by NIH Grant P41 RR-01081).
Supporting Information Available: Experimental procedures
for the intermediate compounds and a table reporting the combus-
tion analysis data of the final products. This material is available
free of charge via the Internet at http://pubs.acs.org.
Cell Culture and Transient Transfections. Human endometrial
cancer (HEC-1) cells were maintained in culture as described.26
Transfection of HEC-1 cells in 24-well plates used a mixture of
0.35 mL of serum-free MEM medium and 0.15 mL of HBSS

Salicylaldoximes as SelectiVe ERꢀ Ligands
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