Diaryl-substituted salicyl- and anthranyl-ketoximes as potential estrogen receptor ligands

Article abstract of DOI:10.1016/j.farmac.2004.01.007

3,4-Diphenylsalicylaldoxime and 3,4-diphenylanthranylaldoxime derivatives, containing small groups (methyl or ethyl) on the imine carbon atom, were synthesized and submitted to biological assays. Binding tests performed on uterine cytosol estrogen recepto

Full text of DOI:10.1016/j.farmac.2004.01.007

IL FARMACO 59 (2004) 601–607
www.elsevier.com/locate/farmac
Diaryl-substituted salicyl- and anthranyl-ketoximes
as potential estrogen receptor ligands
Filippo Minutolo a, Michela Antonello a, Simone Bertini a, Giorgio Placanica a,
Simona Rapposelli a, Kathryn E. Carlson, John A. Katzenellenbogen b, Marco Macchia a,*
a
Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
b
Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, USA
Received 20 November 2003; accepted 24 January 2004
Available online 20 April 2004
Abstract
3,4-Diphenylsalicylaldoxime and 3,4-diphenylanthranylaldoxime derivatives, containing small groups (methyl or ethyl) on the imine
carbon atom, were synthesized and submitted to biological assays. Binding tests performed on uterine cytosol estrogen receptor (ER)
preparation and on purified full-length human ERa and ERb, showed that the newly synthesised compounds exhibit considerably lower
binding affinities with respect to reference non-substituted 3,4-salicylaldoxime.
© 2004 Elsevier SAS. All rights reserved.
1. Introduction
gens. In particular, non-steroidal molecules such as tamox-
ifen and raloxifene, which possess partial antagonist
properties, are currently used in the therapy and prevention of
these diseases.[11-13]
Estrogens (see for example estradiol in Figure 1) are
steroidal hormones that play a crucial role in the develop-
ment and function of female reproductive tissues. Besides,
they have positive effects on the maintenance of bone min-
eral density,[1] on the liver, and on the cardiovascular and
central nervous systems.[2]
An interesting aspect of the pharmacology of natural and
synthetic ER-ligands is that they demonstrate remarkable
patterns of selectivity. Some ER-ligands, particularly those
of the newly designated class of ″selective estrogen receptor
modulators″ (SERMs)[14,15] promote estrogenic effects in
those tissues where they are desired, such as bone, liver and
the cardiovascular system, but block estrogen action at other
sites where stimulation might be undesirable, such as breast
and uterus.[6,12]
Estrogens exert their physiological effects by activating a
specific receptor (ER) which functions as a ligand-inducible
nuclear transcription factor.[3]
Two subtypes of the estrogen receptor, ERa and ERb,
have been described.[4,5] They show a very high aminoacid
sequence homology in their DNA-binding domains (DBDs)
and a considerable homology in their ligand-binding do-
mains (LBDs).[6] However, these two ER subtypes have
different tissue distributions and, in some cases, show differ-
ences in their responses to certain receptor ligands.[6, 7]
It has been proposed that the tissue-selective pharmacol-
ogy of SERMs is a result of their different action on each ER
subtype and/or differential interactions that the ER-ligand
complex might have with the cellular coregulatory proteins
or effector components, which vary from tissue to tissue.[16]
Alterations of the activation/inhibition balance of ER are
also involved in several diseases, such as breast and uterine
cancer,[8-10] prostate hypertrophy and osteoporosis;[9]
therefore, particular therapeutic relevance resides in mol-
ecules able to modulate the negative effects of natural estro-
* Corresponding author.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
Fig. 1.
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.01.007

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F. Minutolo et al. / IL FARMACO 59 (2004) 601–607
Fig. 3.
In order to obtain further information on the steric require-
ments of type 1 and 2 compounds for the ER binding affinity
and ERa/b selectivity, starting from the observation that the
introduction of small alkyl groups in the C4-position of
estradiol resulted in the maintenance of receptor affinity
(Figure 3),[26-28] we decided to affect the same type of
chemical manipulation on the spatially corresponding imine
carbon of type 1 and 2 compounds.
Fig. 2.
Non-steroidal ER-ligands known so far include a large
variety of molecular structures.[17-19] The typical estrogen
ligand pharmacophore model[12] (Figure 1) contains a ge-
neric and quite variable core structure bearing one phenolic
ring (A) and a second aromatic substituent which can be
differently substituted (R’). In addition, this generalized
ligand structure typically tolerates (or, in fact, may prefer)
the presence of one or two additional substituents, one of
which may be another aromatic group. Therefore, the strik-
ing chemical feature common to nearly all synthetic ligands
possessing a good ER binding affinity is the presence of a
phenolic ring A, (see ER pharmacophore of Figure 1), which
seems to mimic the steroid ″A-ring″ present in natural estro-
gens (see estradiol, Figure 1).[20] This phenolic group has
been found to participate in a hydrogen-bond network that
includes two specific residues of the ER ligand-binding do-
main, Glu353(305) and Arg394(346) of ERa(ERb), together
with a bound molecule of water.[21,22] This interaction is
thought to be responsible for much of the attraction between
the ligand and ER receptors.
In this paper we describe the synthesis and biological
properties of salicylketoximes 3a and 3b (Figure 3), contain-
ing small alkyl groups like a methyl or an ethyl (for 3a and
3b, respectively) on the imine carbon atom and the anthranyl
derivative 4, bearing a methyl group on the same imine
carbon.
2. Chemistry
The synthesis of compound 3a was accomplished as
shown in Scheme 1. 2,3-Dichloro-6-acetyl-phenol (5), pre-
pared from commercially available 2,3-dichlorophenol as
previously reported,[29] was submitted to two identical, sub-
sequential Pd-catalyzed cross-coupling steps with phenylbo-
ronic acid, under typical Fu conditions[31] to yield meth-
ylketone 6. Treatment of compound 6 with hydroxylamine
hydrochloride in ethanol[32] gave the desired salicylke-
toxime 3a.
In a prior work carried out in order to obtain new molecu-
lar entities that possess good estrogen receptor-binding affin-
ity with potential SERM activity,[7,23] we reported that
3,4-diphenylsalicylaldoxime (1, Figure 2), designed as ana-
logue of a typical non-steroidal diarylnaphthalenic estrogen
in which the phenolic A group is replaced by an unprec-
edented bioequivalent pseudocycle A’, proved to have inter-
esting binding properties for both estrogen receptor subtypes
(relative binding affinities of 1.13% with ERa and 1.71%
with ERb).[24] The good interaction of compound 1 with
ERa and ERb was rationalized by the fact that the six-
membered pseudocycle A’, formed by an intramolecular hy-
drogen bond between the phenolic OH and the oxime nitro-
gen atom, might be an effective mimic of the phenolicA-ring
that is found in most ER ligands.[19]
Compound 3b was synthesized as shown in Scheme 2.
2,3-Dichlorophenol (7) was treated with propionyl chloride
in the presence of tetrabutylammonium hydrogen sulfate in
dioxane[29] to produce the related aryl-propionate ester (8).
Fries rearrangement[30] in the ortho position of the propio-
nyl group of 8 achieved with alluminium chloride at 120-130
°C afforded the propionylphenol 9 which was submitted to
two identical, subsequential Pd-catalyzed cross-coupling
steps with phenylboronic acid, under typical Fu condi-
tions[31] to yield the diphenyl-substituted propionylphenol
Furthermore, in a subsequent work we reported that an-
thranylaldoxime 2 (Figure 2), in which the oxygen atom of
the original salicylaldoxime derivative 1 is replaced by an
aniline-type nitrogen, showed better binding affinity proper-
ties when compared with its “oxygenated” model compound
1.[25]
Scheme 1. a Key: (a) Cs2CO3, phenylboronic acid, Pd2(dba)3, PCy3, 80 °C
(two steps); (b) NH2OH.HCl, EtOH, 100-110 °C.

F. Minutolo et al. / IL FARMACO 59 (2004) 601–607
603
Table 1
Relative BindingAffinitiesa of Compounds 3a,b and Reference Compounds
Salicylaldoxime 1 and Anthranylaldoxime 2 for uterine cytosol and the
Estrogen Receptors a and b
Ligand
uterine ER
(100)
hERa
hERb
estradiol
(100)
(100)
1b
2c
3a
3b
4
0.85 0.21
1.71. .0.42
<0.005
1.13 0.18
2.21 0.18
0.020 0.001
0.004 0.001
0.137 0.042
1.71 0.42
2.79 0.23
0.005 0.001
0.009 0.001
0.119 0.002
0.014
0
0.030 0.007
a Determined by a competitive radiometric binding assay with [3H]estra-
diol; cytosol preparations of lamb uterus or full-length human ERa and ERb
(Pan Vera) were used; see Experimental. [23, 36]
Values are reported as the mean range or SD of 2-3 independent experi-
ments; the Kd for estradiol for uterine ER and ERa is 0.2 nM, and for ERb is
0.5 nM.
Scheme 2. a Key: (a) NaOH, tetrabutylammonium hydrogen sulfate,
CH3CH2COCl, dioxane, t. a.; (b)AlCl3, 120-130 °C; (c) Cs2CO3, phenylbo-
ronic acid, Pd2(dba)3, PCy3, 80 °C (two steps); (d) NH2OH.HCl, EtOH,
100-110 °C.
b See ref. 24.
10. Treatment of 10 with hydroxylamine hydrochloride in
ethanol[32] afforded salicylketoxime 3b.
c See ref. 25.
The synthesis of compound 4 was accomplished as shown
in Scheme 3. 2,3-Dichloroaniline 11 was treated with aceto-
nitrile in the presence of boron tribromide in dichlo-
romethane[33] to yield the dichloro-substituted acetylaniline
12, which was then submitted to two identical cross-coupling
reactions under Fu conditions[31] to afford the diphenyl-
substituted acetylaniline 13. Condensation of 13 with hy-
droxylamine hydrochloride in ethanol[32] gave the desired
diaryl-substituted antranylketoxime 4.
three receptor preparations. The anthranylic derivative 4,
which possesses a methyl group on the imine carbon, exhib-
its slightly higher RBA values than compounds 3a,b; never-
theless, its affinity is still 8–30 fold lower with respect to
reference compound 1, and 15-20 fold lower with respect to
anthranylaldoxime 2 on ERa and ERb, respectively.
The dramatic reduction in binding affinity observed with
the new derivatives 3a,b and 4 might be due to the increased
steric tension caused by a possible interaction between the
methyl or ethyl group on their imine carbon and the spatially
adjacent aromatic hydrogen. This steric repulsion could force
a rotation around the Cimine-Caryl bond of 3a,b and 4 Figure 4
thus determining a distortion of the pseudocycle A’. In this
“distorted” form, the salicyl- or the anthranylketoxime por-
tions may lose some of the features (planarity, hexagonal
geometry, p-delocalization) which make it an effective
bioequivalent replacement for the aromatic A-ring of typical
estrogen ligands. A further hypothesis is that, because of the
distortion of the pseudocycle, the alkyl group (methyl or
ethyl) linked to the imine carbon may assume a spatial
orientation that prevents the best fit with the ER site(s).
3. Results and Discussion
The binding affinity of compounds 3a,b and 4 was deter-
mined in a radiometric competitive binding assay, using
methods described elsewhere in detail.[34, 35]
Table 1 reports the results obtained for these compounds
in an uterine cytosol ER preparation, as well as with purified
full-length human ERa and ERb. These data are expressed as
the relative binding affinity (RBA) values relative to estra-
diol, which is set at 100%. In Table 1 the results obtained in
the same type of tests for the reference compounds salicyl
and anthranylaldoximes (1 and 2, respectively) are also re-
ported.
4. Conclusions
The binding values shown in Table 1 indicate that com-
pounds 3a and 3b, bearing a methyl and an ethyl group on the
imine carbon, respectively, show considerably lower binding
affinities than the non-substituited salicylaldoxime 1, in all
This study was carried out with the aim of determining the
effects induced by the introduction of small alkyl groups
(methyl and ethyl) on the imine carbon of the structures of
Scheme 3. a Key: (a) 1) CH3CN, BBr3, CH2Cl2, 55 °C; 2) HCl 2 N, 70 °C;
(b) Cs2CO3, phenylboronic acid, Pd2(dba)3, PCy3, 80 °C (two steps); (c)
NH2OH.HCl, EtOH, 95 °C.
Fig. 4.

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F. Minutolo et al. / IL FARMACO 59 (2004) 601–607
two previously reported estrogen ligands (1 and 2). The
results show that this structural modification causes a signifi-
cant reduction in binding affinity on both receptor subtypes,
probably due to the loss of planarity of the pseudocycleA’ or
to the position of the alkyl group on the imine carbon which
may not be tolerated by the receptor.
solution of 6 (0.120 g, 0.417 mmol) in ethanol (6.4 mL)
previously heated at 50 °C. The resulting mixture was stirred
at the same temperature for 12 h, then refluxed for 7 h. After
cooling at r. t., the solvent was evaporated under vacuum, and
the residue was diluted with water and extracted with ethyl
acetate. The organic layer was dried and concentrated to
obtain a crude residue which was purified by flash chroma-
tography (n-hexane/ethyl acetate 9:1) to yield 3a (0.0561 g,
0.185 mmol, 44% yield) that was crystallized from 95%
ethanol; m. p.: 203-204 °C; 1H NMR (CDCl3) d: 2.44 (s, 3H),
7.02 (d, 1H, J = 8.2 Hz), 7.06-7.24 (m, 10H), 7.52 (d, 1H,
J = 8.2 Hz); MS m/z: 303 (M+, 87), 287 (M+ –CH4, 24), 286
(M+ –OH, 78), 285 (M+ –H2O, 60), 284 (M+ –H2O –H, 100),
269 (M+ –OH –OH, 38), 226 (M+ –C6H5, 34).
5. Experimental
5.1. Chemistry
5.1.1. General
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 (d) are reported in parts per million downfield from
tetramethylsilane and referenced from solvent references.
Electron impact (EI) mass spectra were obtained on a HP-
5988A mass spectrometer. The elemental compositions of
the compounds agreed to within 0.4% of the calculated
value. Chromatographic separation was 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, which were visualized under a UV lamp
(254 nm). Evaporation was performed in vacuo (rotating
evaporator). Sodium sulfate was always used as the drying
agent. 2,3-dichlorophenol and 2,3-dichloroaniline was pur-
chased from Sigma-Aldrich (St. Louis, MO).
5.1.4. Synthesis of O-propionyl-2,3-dichlorophenol 8
NaOH (3.00 g, 75.0 mmol) and tetrabutylammonium hy-
drogensulfate (36.8 mg, 0.108 mmol) were added to a solu-
tion of 2,3-dichlorophenol 7 (5.00 g, 30.7 mmol) in dioxane
(45 mL). A solution of propionyl chloride (3.22 mL,
36.8 mmol) in dioxane (30 ml) was then added dropwise
(over 30 min.). The resulting suspension was filtered under
vacuum, and the filtrate was concentrated to give a crude
residue that was purified by flash chromatography (n-
hexane/ethyl acetate 9:1) to yield pure 8 (2.00 g 9.17 mmol,
1
30% yield) as an oil; H NMR (CDCl3) d: 1.30 (t, 3H,
J = 7.6 Hz), 2.66 (q, 2H, J = 7.5 Hz), 7.06 (dd, 1H, J = 8.1,
1.5 Hz), 7.21 (t, 1H, J = 8.1 Hz), 7.36 (dd, 1H, J = 8.1,
1.6 Hz).
5.1.5. Synthesis of 2,3-dichloro-6-propionyl-phenol 9
5.1.2. Synthesis of 2,3-diphenyl-6-acetyl-phenol 6
AlCl3 (1.59 g, 11.9 mmol) was added to compound 8
(2.00 g 9.17 mmol); the resulting mixture was heated at
120-130 °C in a sealed vial for 2 h. After being cooled to r.t.
the mixture was diluted with diethyl ether and extracted with
HCl 2 N. The organic layer was dried and concentrated under
vacuum. The crude residue was purified by flash chromatog-
raphy (chloroform/acetone 8:2) to yield pure 9 (0.750 g,
3.44 mmol, 38% yield) as a yellow crystalline solid; m. p.:
97-98 °C; 1H NMR (CDCl3) d: 1.25 (t, 3H, J = 7.2 Hz), 3.04
(q, 2H, J = 7.2 Hz), 7.03 (d, 1H, J = 8.8 Hz), 7.64 (d, 1H,
J = 8.8 Hz).
A solution of 5 (1.00g, 4.90 mmol) in dioxane (5.0 ml)
was treated with cesium carbonate (2.74 g, 8.41 mmol),
phenylboronic acid (0.897 g, 7.35 mmol), tris(dibenzylide-
neacetone)dipalladium(0) (0.144 g, 0.158 mmol) and 0.6 mL
of a 20% solution of tricyclohexylphosphine in toluene
(0.38 mmol). The resulting mixture was heated at 80 °C in a
sealed vial for 24 h.After being cooled to r.t., the mixture was
diluted with diethyl ether, filtered through a Celite pad and
concentrated to dryness. The residue was dissolved in diox-
ane (4.9 mL), and the resulting solution was submitted again
to the same treatment described above. The crude product
deriving from the second step was purified by column chro-
matography eluting with a 9:1 n-hexane/ethyl acetate mix-
ture, to obtain 0.120 g (0.417 mmol, 85% yield, 2 steps) of
pure 6 as a crystalline solid; m. p.:138 °C; 1H NMR (CDCl3)
d: 2.71 (s, 3H), 7.02 (d, 1H, J = 8.4 Hz), 7.06-7.22 (m, 10H),
7.82 (d, 1H, J = 8.2 Hz).
5.1.6. Synthesis of 2,3-diphenyl-6-propionyl-phenol 10
A solution of 9 (0.70 g, 3.21 mmol) in dioxane (3 mL) was
treated with cesium carbonate (1.79 g, 5.05 mmol), phenyl-
boronic acid (0.587 g, 4.82 mmol), tris(dibenzylideneac-
etone)dipalladium(0) (0.0941 g, 0.103 mmol) and 0.39 mL
of a 20% solution of tricyclohexylphosphine in toluene
(0.25 mmol). The resulting mixture was heated at 80 °C in a
sealed vial for 24 h.After being cooled to r.t., the mixture was
diluted with diethyl ether, filtered through a Celite pad and
concentrated to dryness. The residue was dissolved in diox-
ane (3.2 mL) and the resulting solution was submitted again
5.1.3. Synthesis of 2-hydroxy-3,4-diphenyl-acetophenone-
oxime 3a
A solution of hydroxylamine hydrochloride (0.058 g,
0.834 mmol) in water (1.5 mL) was added dropwise to a

F. Minutolo et al. / IL FARMACO 59 (2004) 601–607
605
to the same treatment described above. The crude product
deriving from the second step was purified by column chro-
matography eluting with a 9:1 n-hexane/ethyl acetate mix-
ture, to obtain 0.470 g (1.55 mmol, 48% yield, 2 steps) of
lphosphine in toluene (0.06 mmol). The resulting mixture
was heated at 80 °C in a sealed vial for 24 h. After being
cooled to r.t., the mixture was diluted with diethyl ether,
filtered through a Celite pad and concentrated to dryness. The
residue was dissolved in dioxane (3.55 mL) and the resulting
solution was submitted again to the same treatment described
above. The crude product deriving from the second step was
purified by column chromatography eluting with a 8:2
1
pure 10 as yellow crystals; m. p.:129-130 °C; H NMR
(CDCl3) d: 1.29 (t, 3H, J = 7.2 Hz), 3.13 (q, 2H, J = 7.3 Hz),
7.01 (d, 1H, J = 8.2 Hz), 7.06-7.22 (m, 10H), 7.85 (d, 1H,
J = 8.3 Hz).
n-hexane/ethyl acetate mixture, to obtain 0.198
g
1
(0.688 mmol, 93% yield, 2 steps) of pure 13. H NMR
(CDCl3) d: 2.66 (s, 3H), 6.53 (br, 2H), 6.74 (d, 1H,
J = 8.3 Hz), 7.02-7.18 (m, 5H), 7.20-7.34 (m, 5H), 7.80 (d,
1H, J= 8.4 Hz).
5.1.7. Synthesis of 2-hydroxy-3,4-diphenyl-propiophenone-
oxime 3b
A solution of hydroxylamine hydrochloride (0.203 g,
2.91 mmol) in water (5.5 mL) was added dropwise to a
solution of 10 (0.440 g, 1.46 mmol) in ethanol (35 mL)
previously heated at 70 °C. The resulting mixture was re-
fluxed for 5 h. After cooling at r. t., the resulting precipitate
was collected by filtration, dried under vacuum and crystal-
lized from 95% ethanol, to obtain pure 3b (0.0374 g,
0.118 mmol). The filtrate was concentrated, diluted with
water and extracted with ethyl acetate. The organic layer was
dried and concentrated to obtain a crude residue that was
purified by flash chromatography (n-hexane/ethyl acetate
9:1) to yield pure 3b (0.0363 g, 0.114 mmol), (total amount
of 3b: 0.0747g, 0.232 mmol, 16% yield); m. p.:220-221 °C;
1H NMR (CDCl3) d: 1.30 (t, 3H, J = 7.7 Hz), 2.96 (q, 2H,
J = 7.7 Hz), 7.02 (d, 1H, J = 8.2 Hz), 7.06-7.21 (m, 10H),
7.52 (d, 1H, J = 8.2 Hz). MS m/z: 317 (M+, 69), 300 (M+ –
OH, 30), 299 (M+ – H2O, 27), 270 (M+ – H2O – CH2CH3,
100).
5.1.10. Synthesis of 2-amino-3,4-diphenyl-acetophenone-
oxime 4
A solution of hydroxylamine hydrochloride (0.047 g,
0.67 mmol) in water (0.4 mL) was added dropwise to a
solution of 13 (0.097 g, 0.338 mmol) in ethanol (4.0 mL)
previously heated at 50 °C. The resulting mixture was re-
fluxed for 3 h. After cooling at r. t., the solvent was evapo-
rated under vacuum, and the residue was diluted with water
and extracted with ethyl acetate. The organic layer was dried
and concentrated to obtain a crude residue, which was puri-
fied by flash chromatography (n-hexane/diethyl ether 6:4) to
yield pure 4 (0.053 g, 0.175 mmol, 51% yield) as a yellow
powder; m. p.: 149-150 °C; 1H NMR (CDCl3) d: 2.40 (s, 3H),
6.81 (d, 1H, J = 8.1 Hz), 7.02-7.18 (m, 5H), 7.18-7.33 (m,
5H), 7.45 (d, 1H, J= 8.2 Hz).
5.1.8. Synthesis of 2,3-dichloro-6-acetyl-aniline 12
5.2. Biological Assays
5.2.1. Materials
A solution of 2,3-dichloroaniline 11 (1.00 g, 6.17 mmol)
in dichloromethane (5.0 mL) was treated, under a nitrogen
atmosphere, with a solution of acetonitrile (0.915 mL,
9.15 mmol) in dichloromethane (2.5 mL). A 1 M solution of
BBr3 in dichloromethane (15.0 mL, 14.8 mmol) was then
added with a syringe through a silicon septum and the result-
ing mixture was heated at 55 °C for 71 h. After this time, HCl
2 N (7 mL) was added and the mixture was heated at 70 °C
for 30 min.. After being cooled to r. t., the mixture was
Purified human ERa and ERb were obtained from Pan-
Vera (Madison, WI). Cell culture media were purchased from
Gibco BRL (Grand Island, NY). Calf serum was obtained
from Hyclone Laboratories, Inc. (Logan, UT), and fetal calf
serum was purchased from Atlanta Biologicals (Atlanta,
GA). The luciferase assay system was from Promega (Madi-
son, WI). The expression vectors for human ERa (pCMV5-
hERa) and human ERb (pCMV5-ERb) were constructed as
previously described.[37,38] The estrogen responsive re-
porter plasmid was (ERE)2-pS2-Luc, which 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 plasmid pCMVb (Clontech,
PaloAlto, CA), which contains the b-galactosidase gene, was
used as an internal control for transfection efficiency.
neutralized (pH
7) with NaHCO3 and extracted with
dichloromethane. The organic layer was dried and concen-
trated to obtain a crude residue, which was purified by flash
chromatography (n-hexane/diethyl ether 8:2) to yield pure 12
(0.336 g, 1.64 mmol, 27% yield); 1H NMR (CDCl3) d: 2.58
(s, 3H), 6.75 (d, 1H, J = 8.8 Hz), 7.04 (br, 2H), 7.60 (d, 1H,
J= 8.6 Hz).
5.1.9. Synthesis of 2,3-diphenyl-6-acetyl-aniline 13
A solution of 12 (0.150 g, 0.735 mmol) in dioxane
(0.5 mL) was treated with cesium carbonate (0.410 g,
1.26 mmol), phenylboronic acid (0.134 g, 1.10 mmol), tris-
5.2.2. Estrogen receptor binding assays
Relative binding affinities were determined by competi-
tive radiometric binding assays using 10 nM [3H]E2 as tracer,
using methods previously described.[34, 35] The source of
(dibenzylideneacetone)dipalladium(0)
(0.022
g,
0.023 mmol) and 0.09 mL of a 20% solution of tricyclohexy-

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F. Minutolo et al. / IL FARMACO 59 (2004) 601–607
[15] B.S. Katzenellenbogen, J.K. Katzenellenbogen, Defining the «S» in
SERMs, Science 295 (2002) 2380–2381.
ER was purified full-length human ERa and ERb purchased
from Pan Vera (Madison, WI). Incubations were performed
at 0 °C for 18-24 h, and hydroxylapatite was used to absorb
the purified receptor-ligand complexes (human ERs).[35]
The binding affinities are expressed as relative binding affin-
ity (RBA) values, where the RBA of estradiol is 100%; under
these conditions, the Kd of estradiol for uterine ER and ERa
is ca. 0.2 nM, and for ERb 0.5 nM. The determination of
these RBA values is reproducible in separate experiments
with a CV of 0.3 nM, and the values shown represent the
average range or SD of 2 or 3 or more separate determina-
tions, respectively.
[16] J.A. Katzenellenbogen, B.W. O’Malley, B.S. Katzenellenbogen, Tri-
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of these hormones, Mol. Endocrinol. 10 (1996) 119–131.
[17] R.A. Magarian, L.B. Overacre, S. Singh, K.L. Meyer, The medicinal
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[18] H. Gao, J.A. Katzenellenbogen, R. Garg, C. Hansch, Comparative
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