Highly selective salicylketoxime-based estrogen receptor β agonists display antiproliferative activities in a glioma model

Article abstract of DOI:10.1021/jm501829f

Estrogen receptor β (ERβ) selective agonists are considered potential therapeutic agents for a variety of pathological conditions, including several types of cancer. Their development is particularly challenging, since differences in the ligand binding cavities of the two ER subtypes α and β are minimal. We have carried out a rational design of new salicylketoxime derivatives which display unprecedentedly high levels of ERβ selectivity for this class of compounds, both in binding affinity and in cell-based functional assays. An endogenous gene expression assay was used to further characterize the pharmacological action of these compounds. Finally, these ERβ-selective agonists were found to inhibit proliferation of a glioma cell line in vitro. Most importantly, one of these compounds also proved to be active in an in vivo xenograft model of human glioma, thus demonstrating the high potential of this type of compounds against this devastating disease.

Full text of DOI:10.1021/jm501829f

Article
pubs.acs.org/jmc
Highly Selective Salicylketoxime-Based Estrogen Receptor β Agonists
Display Antiproliferative Activities in a Glioma Model
Ilaria Paterni,† Simone Bertini,† Carlotta Granchi,† Tiziano Tuccinardi,† Marco Macchia,†
Adriano Martinelli,† Isabella Caligiuri,‡ Giuseppe Toffoli,‡ Flavio Rizzolio,‡ Kathryn E. Carlson,§
Benita S. Katzenellenbogen,∥ John A. Katzenellenbogen,§ and Filippo Minutolo*,†
†Dipartimento di Farmacia, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡Division of Experimental and Clinical Pharmacology, Department of Molecular Biology and Translational Research, CRO National
Cancer Institute and Center for Molecular Biomedicine, IRCCS, 33081 Aviano, Pordenone, Italy
§Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, Illinois 61801, United States
∥Department of Molecular and Integrative Physiology, University of Illinois, 407 S. Goodwin Avenue, Urbana, Illinois 61801, United
States
S
* Supporting Information
ABSTRACT: Estrogen receptor β (ERβ) selective agonists are
considered potential therapeutic agents for a variety of pathological
conditions, including several types of cancer. Their development is
particularly challenging, since differences in the ligand binding
cavities of the two ER subtypes α and β are minimal. We have
carried out a rational design of new salicylketoxime derivatives
which display unprecedentedly high levels of ERβ selectivity for this
class of compounds, both in binding affinity and in cell-based
functional assays. An endogenous gene expression assay was used to
further characterize the pharmacological action of these com-
pounds. Finally, these ERβ-selective agonists were found to inhibit
proliferation of a glioma cell line in vitro. Most importantly, one of
these compounds also proved to be active in an in vivo xenograft
model of human glioma, thus demonstrating the high potential of
this type of compounds against this devastating disease.
subtype generally counteracts the ERα-promoted cell hyper-
proliferation in tissues such as breast and uterus and is generally
considered a tumor suppressor in these organs. This
antiproliferative effect exerted by ERβ was also observed in
several cancer tissues, such as, for example, breast,3 prostate,4
colon,5 renal,6 pleural mesothelioma,7 and glioma.8 In
particular, the protective role of ERβ in gliomas is also
supported by the fact that the incidence of developing this type
of cancer is smaller in women than in men,9 and the use of
exogenous estrogens further reduces this incidence.10 All this
evidence suggests that selective activation of this receptor
subtype may be exploited in order to obtain an antitumor effect.
Several efforts have been dedicated so far to the development
of ERα- or ERβ-selective ligands.11 In particular, a great deal of
attention has been focused on ERβ-selective agonists,12 which
have the potential to be used as antitumor agents because they
predominantly activate the β-subtype, thus being free from the
undesired ERα-promoted proliferative effects on breast and
uterus. However, this endeavor is particularly difficult since, in
INTRODUCTION
■
Estrogen receptors (ERs) are nuclear transcription factors that
mediate the physiological functions of estrogenic compounds.
These receptors exert many of their actions in the nucleus,
where they bind to associated DNA regulatory sequences and
modulate the transcription of specific target genes. Two ER
subtypes, α (ERα) and β (ERβ), are known,1 and subsequent
studies have indicated the presence of up to five different ERβ
isoforms (ERβ1−5) that arise from alternative splicing of the
last exon coding for ERβ.2 Nevertheless, the only fully
functional ERβ isoform appears to be the originally cloned 59
kDa ERβ1 isoform; hence, this is the isoform referred to simply
as ERβ.
Both ERα and ERβ are widely distributed throughout the
human body, where they modulate biological functions in
several organ systems. In addition to their obvious control of
the female reproductive system, they also play key roles in
skeletal, cardiovascular, and central nervous systems. ERα plays
a more prominent role in the mammary gland and uterus, on
the preservation of bone homeostasis, and on the regulation of
metabolism. ERβ has more pronounced effects on the central
nervous system (CNS) and immune system. Moreover, the β-
Received: September 3, 2014
Published: January 5, 2015
© 2015 American Chemical Society
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spite of a limited overall sequence identity (59%) in the ligand
binding domains (LBD) of the two subtypes, the differences
within the ligand binding cavities are at only two amino acid
positions and consist of minor changes between hydrophobic
residues. Thus, Leu384 and Met421 in ERα are replaced by
Met336 and Ile373, respectively, in ERβ. A more important
difference arises from the smaller volume of ERβ binding
pocket when compared to that of ERα, which may be exploited
in the design of ERβ-selective ligands.
We have been involved in the optimization of selective ERβ
agonists that were developed by structural refinements of a
monoaryl-substituted salicylaldoxime scaffold.13 In this article
we describe how molecular modeling has indicated a simple
way to introduce molecular variations that produced some
salicylketoxime derivatives displaying significant improvements
in binding affinity, transactivation activity, and subtype
selectivity over their aldoxime counterparts. Furthermore, for
the first time further pharmacological evaluations were
conducted on our oxime-based ERβ-agonists, both in vitro,
on a glioma U87 cell line, and in vivo on a murine xenograft
model of the same tumor.
that might enhance ERβ binding affinity or selectivity. From
this modeling analysis we realized that there is an empty
hydrophobic cavity that abuts the aldoxime hydrogen atom and
is delimited by Phe356 and Leu301 (Figure 1 and Figure 2A).
Therefore, we decided to fill this cavity with a suitable small
lipophilic group, such as a methyl, an ethyl, or a trifluoromethyl
substituent so that the binding affinity of the resulting
compounds could be improved. A similar docking analysis of
a methyl-ketoxime analogue of 1, such as compound 2a (Figure
1), showed that this compound nicely fits into the receptor
binding site and neatly places its methyl group in the lipophilic
cavity (Figure 2B). It should be noted that the presence of the
ketoxime portion slightly distorts the pseudocycle, which is
formed because of the intramolecular H-bond of these oxime
derivatives. This is due to the larger steric interaction of the
methyl group compared to a hydrogen atom with the adjacent
arene C−H bond. Nevertheless, the most important
interactions that are typical of these types of ERβ ligands13c
are maintained: in detail, both 1 and 2a have their oxime OH
group participating in a highly energetic H-bond network with
residues Glu305 and Arg346, and their antipodal phenolic OH
group forms another H-bond with Thr299 (Figure 2A and
Figure 2B).
In light of these theoretical results, we planned the synthesis
of a selected series of methyl-, ethyl-, and trifluoromethyl-
ketoximes (2a−m, Chart 1), where we could also investigate
the effect due to the variation of the substitution pattern in the
aryl substituent (R2, Chart 1). In particular, we wanted to verify
whether the addition of an extra substituent (F, Cl, or CH3) in
the 3-position or the replacement of the phenol 4-OH with a
group that is exclusively able to act as a H-bond acceptor
(OCH3, F, Cl) could lead to any further improvement of ERβ
affinity and selectivity. In fact, that portion of the molecules
binds to the OH group of a threonine residue (Thr299) of the
receptor, which may act as a H-bond donor or acceptor. In fact,
compounds possessing a p-OH group (2a−d, 2i, 2k, 2m, R2 =
OH; Chart 1) are likely to mainly donate a H-bond to Thr299,
whereas the others (2e−h, 2j, 2l, R2 = OCH3, F, Cl; Chart 1)
can only function as H-bond acceptors in their interaction with
the same residue. It should be noted that oximes 2a−l were
obtained as single (E)-diastereoisomers, whereas trifluorometh-
yl-substituted oxime 2m could only be obtained as a (E/Z)-
diastereoisomeric mixture (see discussion below).
RESULTS AND DISCUSSION
■
Molecular Modeling and Design. Some of the most
potent and selective salicylaldoxime-based ERβ-selective
agonists were obtained by interchanging the respective
positions of the hydroxyl and oxime groups of the Salaldox A
class, to produce compounds belonging to the Salaldox B class
(Figure 1).13c We then decided to further analyze the complex
derived by a docking procedure of the simplest member of the
Salaldox B class, compound 1 (Figure 1), into ERβ-binding
cavity, in order to search for additional productive interactions
Chemistry. The synthesis of methyl- and ethyl-ketoximes
2a−l followed a common, straightforward reaction sequence,
starting from commercially available 5-bromo-2-hydroxyaceto-
phenone (3) or 5-bromo-2-hydroxypropiophenone (4),
respectively (Scheme 1). The first step involved a Pd-catalyzed
cross-coupling reaction under classical Suzuki conditions.14 In
detail, compounds 3 and 4 were treated with 1.2 equiv of the
appropriate arylboronic acid in the presence of aqueous sodium
carbonate and a solvent mixture composed of toluene/ethanol
(1:1). Catalyst Pd(PPh3)4 was formed in situ by reaction of
palladium acetate with a 5-fold excess of triphenylphosphine.
Conventional heating at 100 °C overnight produced the desired
aryl-substituted acetophenone (5e−h, 5j, 5l) and propiophe-
none (6, 7) derivatives in good yields. Ketoxime containing
methoxy- or halogen-substituted aryl rings (2e−h, 2j, 2l) were
then obtained by a direct condensation with hydroxylamine
hydrochloride. On the other hand, an intermediary BBr3-
promoted deprotection of compounds 5e, 5f, 5j, 5l, 6, and 7
was needed to produce hydroxyaryl-substituted ketoximes (2a−
d, 2i, 2k), via the initial formation of ketones (8a−d, 8i, 8k)
Figure 1. General structures of Salaldox A and Salaldox B compounds;
position of the hydrophobic cavity in the complex of aldoxime 1 into
ERβ; design of new ketoxime derivatives containing a methyl (2a), an
ethyl (2b), or a trifluoromethyl (2m) group.
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Figure 2. Docking analysis of salicylaldoxime 1 (A) and salicylketoxime 2a (B) into ERβ binding site. The volume of the small hydrophobic cavity is
represented in yellow (PDB code of the starting ERβ crystal structure is 2I0G).
As previously observed for structurally related salicylaldoxime
derivatives,13 all methyl- and ethyl-ketoximes obtained in this
manner possess the E-configuration in their oxime portion,
which is also consistent with the high degree of stabilization
induced by the energetic intramolecular H-bond between the
oxime nitrogen atom and the adjacent OH group. These
configurations were demonstrated by 1H and 13C NMR analysis
of the final compounds 2a−l. In the case of the methyl-
Chart 1. Structures of the Newly Synthesized
Salicylketoximes 2a−m
1
ketoximes (2a, 2c, 2e−l), the H NMR chemical shift values
(δ) of the methyl protons (2.44 ≤ δ ≤ 2.46 ppm) closely
correspond to the values reported in the literature for the E-
isomer of analogous aromatic methyl-ketoximes.15 Moreover,
the most significant results are given by the 13C NMR chemical
shift values of the methyl carbon atom (10.80 ≤ δ ≤ 10.87
ppm), which nicely overlaps with the values observed for the E-
isomer of previously reported aromatic methyl-ketoximes (11.5
≤ δ ≤ 12.4 ppm) and which differs substantially from the values
reported for the Z-isomers (21 ≤ δ ≤ 21.4 ppm).16 By analogy,
an E-configuration was assigned to ethyl-ketoximes 2b,d.
The synthesis of trifluoromethyl-ketoxime 2m required a
different reaction sequence starting from commercially available
5-bromo-2-methoxybenzaldehyde (9), as shown in Scheme 2.
We followed a trifluoromethylation/oxidation protocol,17
which had already been applied to the synthesis of
trifluoromethylketone 11, via the isolation of intermediate
trifluoromethylcarbinol 10.18 Subsequent Pd-catalyzed cross-
coupling reaction of bromoaryl 11 with 4-methoxyphenylbor-
onic acid produced biphenyl derivative 12, which was
deprotected with BBr3. The resulting dihydroxylated trifluor-
omethylketone 13 was then condensed with hydroxylamine
hydrochloride, thus affording the final product 2m.
a
Scheme 1. Synthesis of Salicylketoximes 2a−l
In distinction with the ketoximes 2a−l, which were obtained
as single (E)-diastereoisomers, the trifluoromethylketoxime 2m
was obtained as an 8:2 E/Z-diastereoisomeric mixture (note
that in 2m there is a nominal inversion of the E/Z-
diastereoisomers). Assignment of the (E)-geometry to the
most abundant isomer is based on a comparison of the NMR
signals with a 2-hydroxyaryl-substituted trifluoromethylketox-
ime, which was characterized by crystallographic X-ray
analysis.19 In fact, 13C NMR spectra of 2m display peaks of
a
Reagents and conditions: (a) ArB(OH)2 (1.2 equiv), Pd(OAc)2 (0.04
equiv), PPh3 (0.2 equiv), aqueous 2 M Na2CO3, 1:1 toluene/EtOH,
100 °C, 16 h; (b) NH2OH·HCl, EtOH−H2O, 50 °C, 16 h; (c) BBr3,
CH2Cl2, −78 to 0 °C, 1 h.
followed by final condensation with hydroxylamine hydro-
chloride.
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a
a
Scheme 2. Synthesis of Trifluoromethylketoxime 2m
Table 1. Relative Binding Affinities of Aldoxime 1 and
Ketoximes 2a−l for the Estrogen Receptors α and β
ligand
hERα (%)
(100)
hERβ (%)
(100)
β/α ratio
estradiol
1
b
1
0.064 0.016
0.54 0.03
3.0 0.6
0.16 0.00
1.9 0.5
<0.005
2.6 0.6
46 14
41
85
15
75
26
>2
2a
2b
2c
2d
2e
2f
46
12
49
9
3
5
0.011 0.000
<0.005
<0.005
2g
2h
2i
<0.005
0.015 0.003
0.099 0.030
1.6 0.1
>3
a
<0.005
>20
16
Reagents and conditions: (a) TMS−CF3, TBAF, THF, rt, 10 h; then
HCl (4.4 M), rt, 1 h; (b) TEMPO, PhI(OAc)2 CH2Cl2, rt, 13 h; (c) 4-
methoxyphenylboronic acid (1.2 equiv), Pd(OAc)2 (0.04 equiv), PPh3
(0.2 equiv), aqueous 2 M Na2CO3, 1:1 toluene/EtOH, 100 °C, 16 h;
(d) BBr3, CH2Cl2, −78 to 0 °C, 1 h; (e) NH2OH·HCl, EtOH−H2O,
50 °C, 16 h.
0.10 0.02
<0.005
2j
<0.005
2k
2l
0.033 0.008
<0.005
1.1 0.1
33
21
<0.005
2m
0.036 0.006
0.76 0.10
a
Determined by a competitive radiometric binding assay with
[3H]estradiol. Preparations of purified, full-length human ERα and
ERβ (PanVera) were used; see Experimental Section. Values are
reported as the mean the range or SD of two or more independent
experiments. The Kd of estradiol for ERα is 0.2 nM and for ERβ is 0.5
nM. Ki values for the new compounds can be readily calculated by
the major isomer corresponding to the CF3 at 122.23 (quartet,
1JC−F = 273.0 Hz) and to the oxime carbon atom at 146.23
2
(quartet, JC−F = 33.0 Hz), which compare favorably to the
corresponding peaks reported in the literature for the (E)-
isomer of 1-(2,4-dihydroxy-3,5-dipropylphenyl)-2,2,2-trifluor-
b
1
using the formula Ki = (Kd[estradiol]/RBA) × 100. See ref 13b.
oethanone oxime: 123.1 (quartet, JC−F = 274.5 Hz), 148.3
(quartet, JC−F = 27.9 Hz).19 This peculiar outcome of the
2
condensation reaction of trifluoromethyl-ketone 13 with
hydroxylamine is probably dictated by the substantial stereo-
electronic repulsion that takes place when the OH and CF3
groups are placed on the same side, as in the (Z)-isomer
(Figure 3).
fold improvement in ERβ-selectivity over its nonmethylated
counterpart 1. It should be noted that the ERβ-RBA value of
45.7% observed for 2a corresponds to a Ki of 1.1 nM, thus
confirming the remarkably high affinity of this compound for
the β-subtype. An enlargement of the oxime alkyl substituent,
from a methyl to an ethyl group, produces a compound (2b),
which preserves an excellent affinity for ERβ but also gains
some affinity for ERα, thus resulting in an ERβ selectivity that is
less than that of 2a. The introduction of a m-fluorine atom into
the 4-hydroxyphenyl group of compounds 2a and 2b,
respectively, produced compounds 2c and 2d, which did not
display any significant improvements over their nonfluorinated
counterparts. Rather, a marked loss of affinity for ERβ was
observed for methyl-ketoxime 2c. Furthermore, when a m-
methyl group (2i) or a m-chlorine atom (2k) was analogously
inserted in the structure of 2a, an even more dramatic reduction
of the binding to ERβ was observed. As previously observed for
other oxime derivatives,13 the presence of a p-methoxy group
into the aryl substituent of these ketoxime derivatives always
compromises the receptor binding affinities of the resulting
compounds (2e, 2f, 2j, and 2l). On the other hand, the
replacement of the p-hydroxy group with a fluorine (2g) or
chlorine atom (2h) restores a certain, though minimal, affinity
for ERβ. Overall, these binding assays confirm that the ERβ
ligand cavity may profitably host methyl- (2a) and an ethyl-
(2b) ketoxime portion, although the highest β-selectivity is
obtained with the former, and that the 4-hydroxyphenyl
substituent still constituted an ideal moiety for an efficient
binding to ERβ.
Figure 3. Stereoelectronic repulsion occurring in the (Z)-diaster-
eoisomer of 2m, which favors the formation of (E)-2m.
Unfortunately, we were not able to separate the two isomers,
and therefore, compound 2m was submitted as a 8:2 (E/Z)-
diastereoisomeric mixture to the receptor binding assays
reported below.
Biological Evaluation. The binding affinity of ketoximes
2a−l for ERα and ERβ was measured by a radiometric
competitive binding assay by using previously reported
methods.20,21 The relative binding affinity (RBA) values for
the newly reported compounds, together with that previously
obtained for reference aldoxime 1,13b are summarized in Table
1. RBA values are reported as percentage (%) of that of
estradiol, which is set at 100%.
The most important finding from these binding measure-
ments is the confirmation of our initial hypothesis derived from
the computer-aided drug design. In fact, when a methyl group
is inserted onto the oxime portion of aldoxime 1, a general
improvement of the binding affinity of the resulting ketoxime
2a is observed, which is particularly evident for ERβ. In fact, 2a
displays a 17-fold improvement in ERβ-binding affinity and a 2-
In order to evaluate the binding disposition of these
derivatives in ERα, the most interesting compounds 2a and
2b were also analyzed for their interaction with this receptor
subtype (Figure 4). As already reported,13b the interaction of
Thr299 with the 4′-hydroxyl of 2a and 2b is only possible in
ERβ because only in this subtype is there enough space for the
phenol group to reach Thr299, because of its proximity to
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Figure 4. Docking analysis of 2a (A) and 2b (B) into ERα (PDB code of the starting ERα crystal structure is 2I0J).
Figure 5. Dose−response curves for transcriptional activation by estradiol (E2), 2a, and 2c through ERα (red line) and ERβ (green line) in the
reporter gene transfection assay. Human endometrial cancer (HEC-1) cells were transfected with expression vectors for ERα or ERβ and an (ERE)2-
pS2-luc reporter gene and were treated for 24 h with estradiol, 2a, or 2c at the concentrations indicated. Luciferase activity was expressed relative to
β-galactosidase activity from an internal control plasmid. The maximal activity with 1 nM E2 was set at 100%. Values are the mean and SD of
triplicate determinations.
Table 2. Reporter Gene Transfection Assay in Human Endometrial (HEC-1) Cells: Transcriptional Activities of Estradiol and
a
Compounds 2a and 2c
hERα
hERβ
ligand
EC50 (nM)
RTP (%)
EC50 (nM)
RTP (%)
EC50(β/α) ratio
RTP(β/α) ratio
estradiol
2a
0.16 0.03
29.3 7.6
194 98.6
100
0.38 0.09
3.97 0.76
14.7 5.2
100
9.62
2.59
0.42
7.4
13
1
0.546
0.083
18
31
2c
a
EC50 values give absolute potencies. The ERβ/ERα relative transcriptional potencies (RTP) and ratios (RTP(β/α)) are calculated as explained in
the text. Cells were exposed to the compounds for 24 h prior to measurements. See refs 22 and 23.
Met336, one of the two nonconserved residues that in ERα is
replaced by bulkier Leu384. As shown in Figure 3, the docking
results showed that for both compounds 2a and 2b the
phenolic OH interacts in the ERα with the Glu305−Arg346−
water hydrogen bonding system, the pseudocycle/oxime
system does not show any important interactions, and the
methyl (2a)/ethyl (2b) substituents show lipophilic inter-
actions with Phe404, Phe425, and Leu428. This binding
disposition is very similar to that hypothesized for compound
1,13b and the lipophilic interactions of the methyl/ethyl group
may explain the increase in ERα affinity associated with
ketoximes 2a and 2b, when compared to that of aldoxime 1.
Nevertheless, the ERβ-binding affinities of 2a increase to a
larger extent (17-fold) when compared to its improvement in
binding to ERα (8-fold), and therefore, the newly synthesized
ketoxime 2a display a substantial enhancement of both affinity
and selectivity for ERβ.
The compounds showing the highest levels of β-selectivity in
the receptor binding assays (2a and 2c) were assayed for
transcriptional activity through ERα and ERβ by two different
methods, and estradiol was always used as the reference
receptor activator. The first method was a reporter gene
transfection assay, which was conducted in human endometrial
(HEC-1) cells, using expression plasmids for either full-length
human ERα or ERβ and an estrogen-responsive luciferase
reporter gene system.22,23 These assays (Figure 5, Table 2)
showed that estradiol (E2) has a 2.4-fold preference in favor of
ERα in terms of transcriptional potency (EC50[ERα] = 0.16
nM vs EC50[ERβ] = 0.38 nM), as has been widely noted. We
had previously reported13b that aldoxime 1 is also a potent ERβ
full agonist, with an EC50 of 10 nM, but it also stimulated ERα
with an EC50 of 17 nM. New ketoxime 2a displayed significant
improvements over aldoxime 1, in terms of both potency on
ERβ (EC50[ERβ] = 3.97 nM) and subtype-selectivity
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Table 3. Activation of Endogenous Genes in Human Breast Cancer (MCF-7) Cells Containing Only ERα or Both ERα and
a
ERβ: Transcriptional Activities of Estradiol and Compounds 2a and 2c
hERα
hERα+β
EC50 (nM)
ligand
EC50 (nM)
RTP (%)
RTP (%)
EC50((α + β)/α) ratio
RTP((α + β)/α) ratio
estradiol
2a
0.0084 0.0060
83.8 17.0
100
0.30 0.20
12.7 5.0
35.0 20.0
100
0.028
6.58
59.7
1
0.0095
0.0004
2.35
0.857
247
2140
2c
2090 1781
a
EC50 values give absolute potencies. The ERβ/ERα relative transcriptional potencies (RTP) and ratios (RTP (β/α)) are calculated, as explained in
the text. Cells were exposed to the compounds for 24 h prior to measurements. See refs 24−26.
Figure 6. Dose−response curves for transcriptional activation of endogenous genes by estradiol (E2), 2a, and 2c. The response of progesterone
receptor (PR, red curves), a gene activated predominantly through ERα, and otubain 2 (OTUB2, green curves), a gene activated predominantly
through ERβ, was measured by qPCR at 24 h after exposure of human breast cancer (MCF-7) cells containing only ERα (solid curves) or both ERα
and ERβ (dashed curves) to compounds at the indicated concentrations. Values are the mean SD of triplicate determinations. For details, see ref
24.
(EC50[ERα] = 29.3 nM). Ketoxime 2c proved to suffer from a
∼4-fold reduction in its potency on ERβ (EC50[ERβ] = 14.7
nM) when compared to 2a, although an even more
pronounced loss of activity on ERα (EC50[ERα] = 194 nM)
elevates its β-selectivity.
only ERα (solid red curve), with this stimulation being
somewhat less in cells containing ERα and ERβ (dashed red
curve). By contrast, the OBUT2 gene (green curves) requires
the presence of ERβ (ERα plus ERβ cells, dashed green curve)
to be stimulated effectively, the response being very minimal in
cells with only ERα (solid green curve). The OTUB2 gene is
also stimulated in ERα + ERβ cells with the two ERβ-selective
ketoximes (2a and 2c; green dashed curves), though at a
somewhat lowered potency, and as expected, no stimulation of
OTUB2 with these compounds was seen in cells with only ERα
(solid green curves). While there was some stimulation of PR
by these compounds, this required high concentrations (the red
curves were right shifted compared to that for E2; see also
Table 3).
Again, as was the case with the reporter gene assays (Figure
5, Table 2), comparisons of potencies in transcription assays
with binding affinities require that the EC50 values from the
transcription assay be referenced to the values of E2, expressed
as RTP values (Table 3). Clearly, the RTP values of
compounds 2a and 2c are far greater for the ERβ-mediated
response (OTUB2 activation in ERα + ERβ cells) than the
ERα-mediated response (PR activation in ERα only cells). The
RTP (α + β)/α ratios, in particular, highlight the very high ERβ
selectivity of these compounds.
We then wanted to evaluate a possible application of our
most promising ERβ agonists as antitumor agents. Among the
beneficial effects of estrogens, a possible protective role in the
progression of gliomas has been reported.27 In fact, this deadly
disease has a significantly lower incidence in reproductive-aged
females than in males. Recently, a naturally occurring ERβ
agonist, liquiritigenin, was shown to be active in vitro against
U87 glioma cells and also in a murine model of the same
disease.8 Therefore, we decided to test the ability of our best
ketoximes 2a and 2c, together with aldoxime 1 and
To compare the ER subtype transcriptional potencies of
these compounds with their subtype binding affinities in a more
proper way, we calculated their relative transcriptional potency
(RTP) values from their EC50 values according to the formula
RTP = (EC50(estradiol)/EC50(ligand)) × 100 (RTP of estradiol =
100). These RTP values provide an estimate of transcriptional
potency relative to that of estradiol and, therefore, are more
appropriate to compare with their binding affinities, which are
also measured relative to estradiol as RBA. By these metrics
(Tables 1 and 2), compound 2a has an RBA(β/α) ratio of 84
and an RTP(β/α) ratio of 18, and compound 2c has an
RBA(β/α) ratio of 72 and an RTP(β/α) ratio of 31. It is worth
noting that differences in ERβ-selectivity in terms of transcrip-
tional potency vs binding affinity may be ascribed to changes in
the manner in which the ERα- and ERβ-ligand complexes
interact with numerous cellular coregulators, which act as
modulators of ligand potency. Therefore, when measured
relative to estradiol, most of the ERβ affinity preference of these
compounds is actually maintained in their ERβ transcriptional
potency preference.
In addition to the reporter gene assays, we also examined the
regulation of two endogenous genes, the progesterone receptor
(PR), which is mostly activated through ERα, and otubain 2
(OTUB2), which is stimulated largely through ERβ.24−26 We
studied the activation of these genes in MCF-7 breast cancer
cells containing either ERα only, or ERα and ERβ (Figure 5
and Table 3).
As we have observed before,24,25 the PR gene (Figure 6, red
curves) is effectively stimulated by estradiol (E2) in cells with
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Table 4. Effects on Cell Growth (IC50, μM) of Human Glioma (U87), Colon (LoVo, HCT), and Breast (MDA-MB-231, MCF7,
a
SkBr3) Cancer Cells by Compounds 1, 2a, and 2c and Liquiritigenin
IC50 (μM)
compd
U87
LoVo
HCT
MDA231
167
MCF7
>250
SkBr3
liquiritigenin
88.3 7.8
45.8 4.2
35.0 1.7
68.8 6.5
77.7 2.9
36.7 2.7
38.1 1.6
27.1 2.8
51.4 2.9
27.4 4.1
17.2 2.9
15.2 2.0
9
175.6 8.9
96.2 3.3
86.3 5.6
92.4 4.8
1
12.4 1.4
9.3 1.2
10.0 3.1
16.4 2.6
8.7 1.4
2.7 0.7
2a
2c
a
IC50: inhibitory concentration causing a 50% reduction in cell growth, in μM. Mean values SD calculated from at least two triplicate cytotoxicity
experiments (see Experimental Section).
liquiritigenin, a major component in licorice root extracts, to
block proliferation of glioma (U87), colon (LoVo, HCT), and
breast (MDA-MB-231, MCF7, SKBR3) cancer cells. The
results are reported in Table 4.
As for the U87 glioma cells, in our hands, liquiritigenin
displayed an IC50 value of 88.3 μM in this assay, whereas all our
oxime-based ERβ-agonists proved to be more potent than this.
In particular, ketoxime 2a, which was also found to be the most
potent ERβ agonist in both the reporter (Table 2) and the
endogenous (Table 3) gene assays, demonstrated the highest
potency as an antiproliferative agent from this series, with an
IC50 value of 35.0 μM. In addition, since activation of ERβ is
also known to exert an antiproliferative effect in colorectal and
breast tumors,10 we extended our in vitro screening to colon
(LoVo, HCT) and breast (MDA231, MCT7) cancer cell lines.
In all these cell lines, oxime derivatives 1, 2a, and 2c still
displayed the most potent inhibition of proliferation when
compared to liquiritigenin. Finally, we included in our study the
ERβ-negative breast cancer cell line SkBr328 in order to verify
the contribution of ERβ activation to the antiproliferative effect
of these compounds. Notably, the activities of the oxime-based
ERβ agonists 1, 2a, and 2c against SkBr3 cells were significantly
lower than their activities against all the other ERβ-positive
cancer cells. For a more significant evaluation of the
involvement of ERβ in the antiproliferative effect of these
compounds, we can restrict our comparison to only breast
cancer cells. Then the differences in the IC50 values found in
ERβ-positive MDA231 and MCF7 cells (2.7−16.4 μM range)
and those in ERβ-negative SkBr3 cells (86.3−96.2 μM) are
even more evident, with 6- to 34-fold reductions of
antiproliferative activities in the ERβ-negative cancer cells. Of
course, we cannot exclude the involvement of other
mechanisms in the inhibition of proliferation by compounds
1, 2a, and 2c, since some activity is also noted in SkBr3 cells.
Nevertheless, these results confirm a highly significant
involvement of the activation of ERβ in the antiproliferative
effects of these compounds.
To complete the evaluation of our compounds 1, 2a, and 2c
against glioma, we carried out an in vivo study using U87
glioma cells grown as xenografts in nude mice, an in vivo model
that has already been used to evaluate the antiglioma effect of
liquiritigenin.8 After tumors reached a measurable size, the
indicated compounds (10 mg/kg/mouse/day) were adminis-
tered for 14 days, and the tumor volume was measured with a
caliper. After 14 days of treatment no signs of animal weight
loss were observed (see Table S1, Supporting Information). As
shown in Figure 7, ketoxime 2a produced a statistically
significant reduction of the tumor volume when compared to
control vehicle, whereas the effects of 1 and 2c were negligible.
This is not surprising since compound 2a, when compared to
the other two compounds, displayed the highest ERβ-binding
Figure 7. Tumor volume (normalized to control, which was set at 1)
of subcutaneous implanted U87 cells in nude mice after 14 days of
treatment with 10 mg/kg compounds. The values are derived from ≥5
mice, and bars show SE.
affinity and ERβ-transcriptional activation, as well as the most
potent antiproliferative activity against U87 cells. Therefore,
these in vivo results indicate that our most potent ERβ-agonist,
2a, is effective in reducing the progression of tumor growth in
both an in vitro and in vivo model of this glioma.
CONCLUSION
■
In summary, molecular modeling studies drove us to the
rational design of some new ketoxime derivatives by
introducing small alkyl groups on the oxime carbon atom of
previously developed salicylaldoxime-based ERβ agonists. Some
of the newly synthesized compounds displayed remarkably high
subtype-selective binding affinities for ERβ, which is
unprecedented for this chemical class of ligands. In particular,
these compounds proved to behave as full agonists on ERβ and
to activate transcription of reporter genes and endogenous
genes, highlighting their very high ERβ potency selectivity.
Finally, we could then demonstrate that one of these ketoxime
derivatives efficiently inhibited tumor progression of ERβ-
expressing human glioma cells, both in vitro and in vivo. These
results further extend the therapeutic potential of ERβ-selective
agonists.
EXPERIMENTAL SECTION
■
General Procedures and Materials. All solvents and chemicals
were used as purchased without further purification. Chromatographic
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. Proton (1H) and carbon (13C) NMR spectra
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were obtained with a Varian Gemini 200 MHz spectrometer using the
indicated deuterated solvents. Chemical shifts are given in parts per
Hz), 6.87−6.96 (m, 3H), 7.43−7.50 (m, 3H), 7.69 (d, 1H, J = 2.2 Hz),
11.52 (exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 11.60,
18.39, 116.42 [2C], 118.28, 118.48, 126.00, 128.36 [2C], 129.20,
132.82, 132.90, 157.41, 158.24, 164.03. MS m/z 257 (M+, 100), 239
(M+ − H2O, 39). HPLC analysis: retention time = 0.983 min; peak
area, 99.01% (254 nm), >99.95% (300 nm); eluent A, NH4OAc
solution (10 mM); eluent B, CH3CN; a gradient was formed from
70% to 95% of B in 10 min and held at 95% for 10 min; flow rate was
1.0 mL/min. HRMS (ESI): m/z calculated for C15H15NO3 + H+ [M +
H+]: 258.1130. Found: 258.1129.
1
million (ppm) (δ relative to residual solvent peak for H and 13C).
Electron impact (EI, 70 eV) mass spectra were obtained on a HP-
5988A mass spectrometer. High-resolution mass spectrometry
(HRMS) analysis was performed using a Waters Quattro II
quadrupole−hexapole−quadrupole liquid chromatography/mass spec-
trometry apparatus (Waters, Milford, MA) equipped with an
electrospray ionization source. Purity of the final compounds 2a−m
was determined by high performance liquid chromatography (HPLC)
on a Waters SunFire RP 18 (3.0 mm × 150 mm, 5 μm) column
(Waters, Milford, MA, www.waters.com) using a Beckmann System-
Gold instrument consisting of chromatography 125 solvent module
and a 166 UV detector set at 254 and 300 nm, and injection volume
was 30 μL (see below for details about mobile phase and flow rate.The
purity was always ≥95%, unless otherwise specified. Yields refer to
isolated and purified products derived from nonoptimized procedures.
Preparation of Ketones 5e−h, 5j, 5l, 6, 7, and 12. General
Procedure. A solution of Pd(OAc)2 (50 mg, 0.22 mmol) and
triphenylphosphine (294 mg, 1.12 mmol) in ethanol (12 mL) and
toluene (12 mL) was stirred at rt under nitrogen for 10 min. After that
period, 5.8 mmol of commercially available 5-bromo-2-hydroxyaceto-
phenone (3) or 5-bromo-2-hydroxypropiophenone (4), a 2 M
aqueous solution of Na2CO3 (13 mL), and the appropriate arylboronic
acid (1.2 equiv) were sequentially added. The resulting mixture was
heated at 100 °C in a sealed vial under nitrogen overnight. After being
cooled to rt, the mixture was diluted with water and extracted with
EtOAc. The combined organic phase was dried and concentrated. The
crude product was purified by flash chromatography over silica gel.
Elution with n-hexane/EtOAc (95:5 to 8:2) afforded the desired
ketone intermediates. The same procedure was applied to 1-(5-bromo-
2-methoxyphenyl)-2,2,2-trifluoroethanone (11), which was synthe-
sized as previously reported.18
(E)-1-(6-Hydroxy-3-(3-fluoro-4-hydroxyphenyl)phenyl)-
ethanone Oxime (2c). White solid; yield 91% from 8c; mp 193 °C.
1H NMR (acetone-d6) δ (ppm): 2.46 (s, 3H), 6.94 (d, 1H, J = 8.6 Hz),
7.05 (dd, 1H, J = 9.0, 8.6 Hz 1H), 7.30 (ddd, 1H, J = 8.4, 2.2, 0.9 Hz),
7.40 (dd, 1H, J = 12.6, 2.2 Hz), 7.50 (dd, 1H, J = 8.4, 2.4 Hz), 7.74 (d,
1H, J = 2.4 Hz), 11.52 (exchangeable s, 1H). 13C NMR (acetone-d6) δ
(ppm): 10.87, 114.74 (d, J = 19.2 Hz), 118.13, 118.93, 120.01, 123.26
(d, J = 2.7 Hz), 126.56, 129.18, 131.51, 133.89 (d, J = 6.4 Hz), 144.61
(d, J = 12.8 Hz), 152.54 (d, J = 239.0 Hz), 158.71, 159.26. MS m/z
261 (M+, 100), 243 (M+ − H2O, 51), 228 (M+ − H2O − CH3, 57).
HPLC analysis: retention time = 10.100 min; peak area, >99.95% (254
nm), 99.91% (300 nm); eluent A, NH4OAc solution (10 mM); eluent
B, CH3CN; a gradient was formed from 5% to 80% of B in 10 min and
held at 95% for 10 min; flow rate was 0.7 mL/min. HRMS (ESI): m/z
calculated for C14H12FNO3 + H+ [M + H+]: 262.0879. Found:
262.0880.
(E)-1-(6-Hydroxy-3-(3-fluoro-4-hydroxyphenyl)phenyl)-
propan-1-one Oxime (2d). White solid; yield 45% from 8d; mp 172
°C. 1H NMR (acetone-d6) δ (ppm): 1.24 (t, 3H, J = 7.6 Hz), 3.04 (q,
2H, J = 7.6 Hz), 6.95 (d, 1H, J = 8.4 Hz), 7.05 (dd, 1H, J = 9.0, 8.4 Hz
1H), 7.30 (ddd, 1H, J = 8.4, 2.2, 0.9 Hz), 7.39 (dd, 1H, J = 12.5, 2.2
Hz), 7.50 (dd, 1H, J = 8.4, 2.2 Hz), 7.73 (d, 1H, J = 2.2 Hz), 8.70
(exchangeable bs, 1H), 10.61 (exchangeable bs, 1H), 11.58
(exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 11.60,
18.41, 114.76 (d, J = 18.3 Hz), 118.42, 118.62, 118.97, 123.30 (J = 2.7
Hz), 126.22, 129.31, 131.60, 133.96 (d, J = 6.4 Hz), 144.62 (d, J = 12.8
Hz), 152.50 (d, J = 239.9 Hz), 158.74, 164.11. MS m/z 275 (M+, 100),
259 (M+ − O, 18), 257 (M+ − H2O, 12). HPLC analysis: retention
time = 10.533 min; peak area, 98.25% (254 nm), 98.99% (300 nm);
eluent A, NH4OAc solution (10 mM); eluent B, CH3CN; a gradient
was formed from 5% to 80% of B in 10 min and held at 95% for 10
min; flow rate was 0.7 mL/min. HRMS (ESI): m/z calculated for
C15H14FNO3 + H+ [M + H+]: 276.1036. Found: 276.1034.
Preparation of O-Deprotected Ketones 8a−d, 8i, 8k, and 13.
General Procedure. A solution of pure ketones 5e, 5f, 5j, 5l, 6, 7,
and 12 (0.90 mmol) in anhydrous CH2Cl2 (11 mL) was cooled to
−78 °C and treated dropwise with a 1.0 M solution of BBr3 CH2Cl2 (3
mL) under nitrogen. The mixture was left under stirring at the same
temperature for 5 min and then at 0 °C for 1 h. The mixture was then
diluted with water and extracted with ethyl acetate. The organic phase
was dried and concentrated. The crude product was purified by flash
chromatography over silica gel. Elution with n-hexane/EtOAc (8:2 to
7:3) afforded the desired O-deprotected ketones.
(E)-1-(6-Hydroxy-3-(4-methoxyphenyl)phenyl)ethanone
Preparation of Final Products 2a−m. General Procedure. A
solution of pure ketones 5e−h, 5j, 5l, 8a−d, 8i, 8k, 13 (1.2 mmol) in
ethanol (20 mL) was treated with a solution of hydroxylamine
hydrochloride (257 mg, 3.72 mmol) in water (4 mL), and the mixture
was heated to 50 °C for 16 h. After being cooled to rt, 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 7:3) to afford the desired
ketoxime derivatives.
1
Oxime (2e). White solid; yield 74% from 5e; mp 164 °C. H NMR
(acetone-d6) δ (ppm): 2.45 (s, 3H), 3.83 (s, 3H), 6.94 (d, 1H, J = 8.6
Hz), 6.99 (AA′XX′, 2H, JAX = 8.8 Hz, JAA′/XX′ = 2.6 Hz), 7.49 (dd, 1H,
J = 8.4, 2.4 Hz), 7.57 (AA′XX′, 2H, JAX = 8.8 Hz, JAA′/XX′ = 2.6 Hz),
7.73 (d, 1H, J = 2.4 Hz), 10.71 (exchangeable s, 1H), 11.48
(exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 10.83, 55.59,
115.00 [2C], 118.06, 119.95, 126.45, 128.27 [2C], 129.18, 132.39,
133.94, 157.88, 159.15, 159.74. MS m/z 257 (M+, 90), 239 (M+ −
H2O, 34), 224 (M+ − H2O − CH3, 100). HPLC analysis: retention
time = 1.583 min; peak area, >99.95% (254 nm), 99.00% (300 nm);
eluent A, NH4OAc solution (10 mM); eluent B, CH3CN; a gradient
was formed from 70% to 95% of B in 10 min and held at 95% for 10
min; flow rate was 1.0 mL/min.
(E)-1-(6-Hydroxy-3-(4-hydroxyphenyl)phenyl)ethanone
1
Oxime (2a). White solid; yield 50% from 8a; mp 188 °C. H NMR
(acetone-d6) δ (ppm): 2.44 (s, 3H), 6.88−6.94 (m, 3H), 7.44−7.52
(m, 3H), 7.70 (d, 1H, J = 2.2 Hz), 8.36 (exchangeable s, 1H), 10.70
(exchangeable s, 1H), 11.45 (exchangeable s, 1H). 13C NMR
(acetone-d6) δ (ppm): 10.80, 116.43 (2C), 117.98, 119.91, 126.32,
128.32 [2C], 129.06, 132.74, 132.83, 157.45, 157.69, 159.18. MS m/z
243 (M+, 71), 242 (M+ − H, 100), 227 (M+ − H2O, 31). HPLC
analysis: retention time = 1.550 min; peak area, 98.97% (254 nm),
99.17% (300 nm); eluent A, NH4OAc solution (10 mM); eluent B,
CH3CN; a gradient was formed from 50% to 95% of B in 10 min and
held at 95% for 15 min; flow rate was 1.0 mL/min. HRMS (ESI): m/z
calculated for C14H13NO3 + H+ [M + H+]: 244.0974. Found:
244.0971.
(E)-1-(6-Hydroxy-3-(3-fluoro-4-methoxyphenyl)phenyl)-
ethanone Oxime (2f). White solid; yield 68% from 5f; mp 171 °C.
1H NMR (acetone-d6) δ (ppm): 2.46 (s, 3H), 3.91 (s, 3H), 6.95 (d,
1H, J = 8.4 Hz), 7.18 (t, 1H, J = 8.9 Hz), 7.37−7.48 (m, 2H), 7.52 (dd,
1H, J = 8.6, 2.4 Hz), 7.76 (d, 1H, J = 2.2 Hz), 10.76 (exchangeable s,
1H), 11.60 (exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm):
10.87, 56.61, 114.74 (d, J = 19.3 Hz), 114.87, 118.19, 120.03, 123.05
(d, J = 3.7 Hz), 126.69, 129.29, 131.19, 134.71 (d, J = 6.4 Hz), 147.49
(d, J = 10.1 Hz), 153.29 (d, J = 243.6 Hz), 158.33, 159.32. MS m/z
275 (M+, 100), 257 (M+ − H2O, 53). HPLC analysis: retention time =
1.517 min; peak area, >99.95% (254 nm), >99.95% (300 nm); eluent
A, NH4OAc solution (10 mM); eluent B, CH3CN; a gradient was
formed from 70% to 95% of B in 10 min and held at 95% for 10 min;
(E)-1-(6-Hydroxy-3-(4-hydroxyphenyl)phenyl)propan-1-one
1
Oxime (2b). White solid; yield 37% from 8b; mp 171 °C. H NMR
(acetone-d6) δ (ppm): 1.24 (t, 3H, J = 7.6 Hz), 3.02 (q, 2H, J = 7.6
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flow rate was 1.0 mL/min. HRMS (ESI): m/z calculated for
C15H14FNO3 + H+ [M + H+]: 276.1036. Found: 276.1037.
H2O, 100). HPLC analysis: retention time = 1.050 min; peak area,
>99.95% (254 nm), >99.95% (300 nm); eluent A, NH4OAc solution
(10 mM); eluent B, CH3CN; a gradient was formed from 70% to 95%
of B in 10 min and held at 95% for 10 min; flow rate was 1.0 mL/min.
HRMS (ESI): m/z calculated for C14H12ClNO3 + H+ [M + H+]:
278.0584. Found: 278.0582.
(E)-1-(6-Hydroxy-3-(3-chloro-4-methoxyphenyl)phenyl)-
ethanone Oxime (2l). White solid; yield 59% from 5l; mp 178 °C.
1H NMR (acetone-d6) δ (ppm): 2.46 (s, 3H), 3.94 (s, 3H), 6.95 (d,
(E)-1-(6-Hydroxy-3-(4-fluorophenyl)phenyl)ethanone Oxime
(2g). White solid; yield 53% from 5g; mp 203 °C. 1H NMR (acetone-
d6) δ (ppm): 2.45 (s, 3H), 6.96 (d, 1H, J = 8.4 Hz), 7.19 (double
AA′XX′, 2H, 3JHF‑o = 9.0 Hz, JAX = 8.8 Hz, JAA′/XX′ = 2.6 Hz), 7.52 (dd,
1H, J = 8.4, 2.2 Hz), 7.67 (double AA′XX′, 2H, 4JHF‑m = 5.3 Hz, JAX
=
8.8 Hz, JAA′/XX′ = 2.6 Hz), 7.77 (d, 1H, J = 2.2 Hz), 10.76
(exchangeable s, 1H), 11.57 (exchangeable s, 1H). 13C NMR
(acetone-d6) δ (ppm): 10.85, 116.18 (d, 2C, J = 22.0 Hz), 118.20,
120.03, 126.98, 129.11 (d, 2C, J = 7.3 Hz), 129.57, 131.58, 137.92,
158.43, 159.28, 162.83 (d, J = 243.5 Hz). MS m/z 245 (M+, 100), 227
(M+ − H2O, 40). HPLC analysis: retention time = 1.817 min; peak
area, 99.50% (254 nm), 97.90% (300 nm); eluent A, NH4OAc
solution (10 mM); eluent B, CH3CN; a gradient was formed from
70% to 95% of B in 10 min and held at 95% for 10 min; flow rate was
1.0 mL/min. HRMS (ESI): m/z calculated for C14H12FNO2 + H+ [M
+ H+]: 246.0930. Found: 246.0931.
1H, J = 8.6 Hz), 7.18 (d, 1H, J = 8.4 Hz), 7.49−7.59 (m, 2H), 7.67 (d,
1H, J = 1.8 Hz), 7.77 (d, 1H, J = 2.0 Hz), 10.73 (exchangeable s, 1H),
11.55 (exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 10.87,
56.61, 113.67, 118.20, 120.10, 123.14, 126.69, 126.84, 128.69, 129.29,
131.02, 135.05, 154.93, 158.35, 159.23. MS m/z 291 (M+, 100), 273
(M+ − H2O, 36), 258 (M+ − H2O − CH3, 91). HPLC analysis:
retention time = 1.883 min; peak area, >99.95% (254 nm), 99.83%
(300 nm); eluent A, NH4OAc solution (10 mM); eluent B, CH3CN; a
gradient was formed from 70% to 95% of B in 10 min and held at 95%
for 10 min; flow rate was 1.0 mL/min. HRMS (ESI): m/z calculated
for C15H14ClNO3 + H+ [M + H+]: 292.0740. Found: 292.0737.
(E/Z)-1-(6-Hydroxy-3-(4-hydroxyphenyl)phenyl)-2,2,2-tri-
fluoroethanone Oxime (2m). White solid; yield 68% (unresolved
(E)-1-(6-Hydroxy-3-(4-chlorophenyl)phenyl)ethanone
1
Oxime (2h). White solid; yield 60% from 5h; mp 207 °C. H NMR
(acetone-d6) δ (ppm): 2.46 (s, 3H), 6.98 (d, 1H, J = 8.4 Hz), 7.45
(AA′XX′, 2H, JAX = 8.6 Hz, JAA′/XX′ = 2.3 Hz), 7.55 (dd, 1H, J = 8.6,
2.3 Hz), 7.69 (AA′XX′, 2H, JAX = 8.6 Hz, JAA′/XX′ = 2.3 Hz), 7.80 (d,
1H, J = 2.2 Hz), 10.76 (exchangeable s, 1H), 11.61 (exchangeable s,
1H). 13C NMR (acetone-d6) δ (ppm): 10.87, 118.33 [2C], 120.15,
127.04, 128.89 [2C], 129.58 [2C], 131.24, 132.99, 140.27, 158.772,
159.28. MS m/z 261 (M+, 100), 243 (M+ − H2O, 45). HPLC analysis:
retention time = 2.483 min; peak area, 98.79% (254 nm), 98.05% (300
nm); eluent A, NH4OAc solution (10 mM); eluent B, CH3CN; a
gradient was formed from 70% to 95% of B in 10 min and held at 95%
for 10 min; flow rate was 1.0 mL/min. HRMS (ESI): m/z calculated
for C14H12ClNO2 + H+ [M + H+]: 262.0635. Found: 262.0634.
(E)-1-(6-Hydroxy-3-(4-hydroxy-3-methylphenyl)phenyl)-
ethanone Oxime (2i). White solid; yield 60% from 8i; mp 209 °C.
1H NMR (acetone-d6) δ (ppm): 2.26 (s, 3H), 2.44 (s, 3H), 6.88 (d,
1H, J = 8.1 Hz), 6.91 (d, 1H, J = 8.4 Hz), 7.28 (dd, 1H, J = 8.2, 2.4
Hz), 7.38 (d, 1H, J = 2.4 Hz), 7.46 (dd, 1H, J = 8.4, 2.2 Hz), 7.69 (d,
1H, J = 2.2 Hz), 8.19 (exchangeable s, 1H), 10.67 (exchangeable s,
1H), 11.42 (exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm):
10.85, 16.35, 115.82, 117.95, 119.84, 125.30, 125.58, 126.31, 129.13,
129.73, 132.88, 132.95, 155.42, 157.62, 159.28. MS m/z 257 (M+,
100), 239 (M+ − H2O, 33). HPLC analysis: retention time = 1.000
min; peak area, >99.95% (254 nm), >99.95% (300 nm); eluent A,
NH4OAc solution (10 mM); eluent B, CH3CN; a gradient was formed
from 70% to 95% of B in 10 min and held at 95% for 10 min; flow rate
was 1.0 mL/min. HRMS (ESI): m/z calculated for C15H15NO3 + H+
[M + H+]: 258.1130. Found: 258.1133.
1
8:2 E/Z-mixture) from 13; mp 187 °C. H NMR (acetone-d6; E/Z
mixture, asterisk denotes minor isomer peaks) δ (ppm): 6.91
(AA′XX′, 2H, JAX = 8.6 Hz, JAA′/XX′ = 2.5 Hz), 7.02* (d, 1H, J =
8.4 Hz), 7.05 (d, 1H, J = 8.5 Hz), 7.40 (d, 1H, J = 2.1 Hz), 7.44
(AA′XX′, 2H, JAX = 8.6 Hz, JAA′/XX′ = 2.6 Hz), 7.55 (dd, 1H, J = 8.6,
2.4 Hz), 8.36 (exchangeable bs, 1H). 1H NMR (CD3OD; E/Z
mixture, asterisk denotes minor isomer peaks) δ (ppm): 6.83
(AA′XX′, 2H, JAX = 8.7 Hz, JAA′/XX′ = 2.5 Hz), 6.88* (d,1H, J = 8.5
Hz), 6.93 (d,1H, J = 8.5 Hz), 7.21 (d,1H, J = 2.2 Hz), 7.30* (d,1H, J =
2.4 Hz), 7.35 (AA′XX′, 2H, JAX = 8.7 Hz, JAA′/XX′ = 2.5 Hz), 7.46 (dd,
1H, J = 8.5, 2.4 Hz). 13C NMR (acetone-d6; E/Z mixture, asterisk
denotes minor isomer peaks) δ (ppm): 116.55, 116.58 [2C], 117.29,
122.23 (q, 1JC−F = 273.0 Hz), 128.08, 128.40 [2C], 129.55*, 130.10*,
130.16, 132.33*, 132.39*, 133.59, 133.60, 146.23 (q, 2JC−F = 33.0 Hz),
154.95, 157.65. HRMS (ESI): m/z calculated for C14H10F3NO3 + H+
[M + H+]: 298.0691. Found: 298.0686.
Modeling. The crystal structure of ERα (PDB code 2I0J) and ERβ
(PDB code 2I0G)29 was taken from the Protein Data Bank.30 After
addition of hydrogen atoms, the two proteins complexed with their
reference inhibitor were minimized using AMBER 9 software31 and
parm03 force field at 300 K. The two 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). The two ligands were
built using Maestro32 and were minimized by means of Macromodel33
in a water environment using the CG method until a convergence
value of 0.05 kcal/(Å·mol), using the MMFFs force field and a
distance-dependent dielectric constant of 1.0. Automated docking was
carried out by means of the AUTODOCK 4.0 program;34 Autodock
Tools35 was used in order to identify the torsion angles in the ligands,
add the solvent model, and assign the Kollman atomic charges to the
protein. The ligand charge was calculated using the Gasteiger method.
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. The regions of interest
used by Autodock were defined by considering SERBA-129 into 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 the 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, the docked compounds were subjected to 100 runs of the
(E)-1-(6-Hydroxy-3-(4-methoxy-3-methylphenyl)phenyl)-
ethanone Oxime (2j). White solid; yield 50% from 5j; mp 154 °C.
1H NMR (acetone-d6) δ (ppm): 2.23 (s, 3H), 2.44 (s, 3H), 3.86 (s,
3H), 6.90−6.99 (m, 2H), 7.39−7.43 (m, 2H), 7.48 (dd, 1H, J = 8.4,
2.4 Hz), 7.72 (d, 1H, J = 2.4 Hz), 10.69 (exchangeable s, 1H), 11.46
(exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 10.87, 16.46,
55.77, 111.21, 118.02, 119.92, 125.67, 126.47, 127.20, 129.26, 129.49,
132.66, 133.63, 157.83, 157.86, 159.28. MS m/z 271 (M+, 100), 253
(M+ − H2O, 21), 238 (M+ − H2O − CH3, 54). HPLC analysis:
retention time = 2.133 min; peak area, 99.17% (254 nm), 98.93% (300
nm); eluent A, NH4OAc solution (10 mM); eluent B, CH3CN; a
gradient was formed from 70% to 95% of B in 10 min and held at 95%
for 10 min; flow rate was 1.0 mL/min. HRMS (ESI): m/z calculated
for C16H17NO3 + H+ [M + H+]: 272.1287. Found: 272.1284.
(E)-1-(6-Hydroxy-3-(3-chloro-4-hydroxyphenyl)phenyl)-
ethanone Oxime (2k). White solid; yield 50% from 8k; mp 208 °C.
1H NMR (acetone-d6) δ (ppm): 2.46 (s, 3H), 6.94 (d, 1H, J = 8.6 Hz),
7.08 (d, 1H, J = 8.4 Hz), 7.44 (dd, 1H, J = 8.6, 2.2 Hz), 7.49 (dd, 1H, J
= 8.4, 2.2 Hz), 7.62 (d, 1H, J = 2.2 Hz), 7.74 (d, 1H, J = 2.2 Hz), 8.80
(exchangeable bs, 1H), 10.60 (exchangeable bs, 1H), 11.52
(exchangeable s, 1H). 13C NMR (acetone-d6) δ (ppm): 10.87,
117.88, 118.17, 120.03, 121.48, 126.58, 126.93, 128.49, 129.24, 131.30,
134.45, 152.76, 158.23, 159.30. MS m/z 277 (M+, 90), 259 (M+ −
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Autodock search, using 500 000 steps of energy evaluation and the
default values of the other parameters. Cluster analysis was performed
on the results using an rms tolerance of 1.0 Å, and the best docked
conformation was used for the analysis. For the docking of compound
2b into ERβ, F356 was considered as a flexible residue. The reported
docking procedure has recently been indirectly validated by the
deposition of the crystal structure of ERα complexed with 2-chloro-3′-
fluoro-3-[(E)-(hydroxyimino)methyl]biphenyl-4,4′-diol (PDB code
4IWF).36 Before the deposition of this structure, in 2011 we reported
a docking analysis of this compound into ERα;13c superimposing the
docking with the experimental results, the proposed binding mode was
correctly predicted, as the two compounds showed a root-mean-square
deviation of their disposition of 1.0 Å.
Relative Binding Affinity Assay. Relative binding affinities were
determined by competitive radiometric binding assays with 2 nM
[3H]E2 as tracer, as a modification of methods previously
described.20,21 The source of ER was purified full-length human
ERα and ERβ purchased from Pan Vera/Invitrogen (Carlsbad, CA).
Incubations were done at 0 °C for 18−24 h, and hydroxyapatite was
used to absorb the purified receptor−ligand complexes (human
ERs).21 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 ERα is ∼0.2 nM, and for ERβ 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.
Reporter Gene Assays. The procedures used for the ERα and
ERβ-responsive reporter gene assays in HEC-1 cells have been fully
described in prior publications.22,23
ACKNOWLEDGMENTS
■
Intramural funding support from the University of Pisa (to
F.M.) and support from the National Institutes of Health
(Grants PHS R01CA025836 and R01DK015556 to J.A.K.) are
gratefully acknowledged. We thank Dr. Giorgio Placanica of the
University of Pisa and Dr. Sung Hoon Kim of the University of
Illinois for technical assistance in the analysis of the products
and Dr. Yan Jiang of the University of Illinois for conducting
some of the cell-based assays.
ABBREVIATIONS USED
■
ER, estrogen receptor; CNS, central nervous system; LBD,
ligand binding domain; E2, estradiol; RBA, relative binding
affinity; RTP, relative transcriptional potency; ERE, estrogen
response element; PR, progesterone receptor; OTUB2, otubain
2; qPCR, quantitative real-time polymerase chain reaction; SD,
standard deviation; ppm, parts per million; NMR, nuclear
magnetic resonance; HPLC, high performance liquid chroma-
tography; TLC, thin layer chromatography; rt, room temper-
ature; MS, mass spectrometry; EI, electron impact; HRMS,
high-resolution mass spectrometry; s, singlet; d, doublet; dd,
double doublet; ddd, double double doublet; t, triplet; q,
quartet; m, multiplet; bs, broad signal; CG, conjugate gradient;
MMFF, Merck molecular force field
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■
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ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC chromatograms, mass spectra, analytical data of
intermediate compounds, docking of 2b into ERβ binding
site, time-based body weight analysis of treated animals. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: filippo.minutolo@farm.unipi.it. Telephone: (+39) 050
2219557. Fax: (+39) 050 2219605.
■
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The authors declare no competing financial interest.
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