Selective and potent agonists for estrogen receptor beta derived from molecular refinements of salicylaldoximes

Article abstract of DOI:10.1016/j.ejmech.2011.03.030

In a continuing effort to improve the subtype selectivity and agonist potency of estrogen receptor β (ERβ) ligands, we have designed and developed a thus far unexplored structural series obtained by molecular refinements of monoaryl-substituted salicylaldoximes (Salaldox B). The most interesting compounds in this series (2c, d) show remarkably high ERβ-binding affinities, with K_i values reaching the sub-nanomolar range (K_i = 0.38 nM for 2c and 0.57 nM for 2d), and have very high levels of ERβ-subtype selectivity. Both compounds show a potent full agonist character on ERβ (EC₅₀ = 0.23 nM for 2c and 1.3 nM for 2d). Furthermore, 2d shows a remarkable functional subtype selectivity, with a β/α transcription potency ratio 50-fold higher than that of estradiol.

Full text of DOI:10.1016/j.ejmech.2011.03.030

European Journal of Medicinal Chemistry 46 (2011) 2453e2462
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original Article
Selective and potent agonists for estrogen receptor beta derived from molecular
refinements of salicylaldoximes
Simone Bertini a, Andrea De Cupertinis a, Carlotta Granchi a, Barbara Bargagli a, Tiziano Tuccinardi a,
Adriano Martinelli a, Marco Macchia a, Jillian R. Gunther b, Kathryn E. Carlson b,
,
John A. Katzenellenbogen b, Filippo Minutolo 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
a r t i c l e i n f o
a b s t r a c t
Article history:
In a continuing effort to improve the subtype selectivity and agonist potency of estrogen receptor
b (ERb)
Received 2 February 2011
Received in revised form
10 March 2011
Accepted 15 March 2011
Available online 23 March 2011
ligands, we have designed and developed a thus far unexplored structural series obtained by molecular
refinements of monoaryl-substituted salicylaldoximes (Salaldox B). The most interesting compounds in
this series (2c, d) show remarkably high ER
molar range (Ki ¼ 0.38 nM for 2c and 0.57 nM for 2d), and have very high levels of ER
tivity. Both compounds show a potent full agonist character on ER
b
-binding affinities, with Ki values reaching the sub-nano-
-subtype selec-
(EC50 ¼ 0.23 nM for 2c and 1.3 nM for
b
b
2d). Furthermore, 2d shows a remarkable functional subtype selectivity, with a b/a transcription potency
Keywords:
Estrogen
Receptor binding
Agonists
ratio 50-fold higher than that of estradiol.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Salicylaldoxime
Docking
1. Introduction
domain (LBD), although the differences within the ligand binding
cavities consist of only two, conservative amino acid substitutions.
In fact, of the 23 amino acid residues that line the ligand binding
cavities of the two ERs (within 4 Å of the ligand), only two are
Since the discovery of estrogen receptor beta (ERb) in 1996 [1],
much effort has been devoted to the search for molecules that are
able to selectively interact with this receptor [2,3]. Such ER -selec-
tive agonists hold promise for the treatment of diverse diseases,
such as rheumatoid arthritis and inflammatory diseases [4], certain
cancers [5,6], endometriosis [7], as well as cardiovascular [8] and
b
different: Leu384 and Met421 of ER
Ile373, respectively, in ER [1]. These slight modifications and other
minor alterations of tertiary structure make the volume of the ER
binding pocket smaller than the one in ER and somewhat different
a are replaced by Met336 and
b
b
-
a
CNS pathologies [9]. The therapeutic potential of ER
compounds appears to be particularly favourable because the
beneficial effects of stimulation through ER would be expected to
be free from undesired proliferative effects on breast and uterus,
b
-selective
in shape. Due to the lack of a pronounced difference between the
two receptor subtypes, the design and development of molecules
that selectively bind to and activate ERb is not a trivial task,
although there are now several pharmacophore models for the kind
of molecular frameworks more likely to engender ER subtype
selectivity [3].
b
which are mediated largely by the other receptor subtype, ER
[10,11].
a
ER
b
, as well as ER
a
, belong to the superfamily of nuclear
We recently reported on two restricted series of monoaryl-
substituted salicylaldoximes bearing a para-hydroxylated aryl
substituent at either position 4 (Salaldox A) [13,14] or 5 (Salaldox B)
[14] of the central ring. These were inspired by a consolidated
pharmacophore model originally developed for indazole deriva-
tives [15] (Fig. 1). These compounds derive from our original
observation that one of the two phenol groups typically present in
non-steroidal ER-ligands could be isosterically replaced by a six-
membered pseudocycle, formed by an intramolecular H-bond
involving the phenol and the oxime nitrogen atom, which had led
receptors that act as ligand-regulated transcription factors [12].
Their amino acid sequence shows 59% identity in the ligand binding
Abbreviations: ER, estrogen receptor; ER
b, estrogen receptor subtype beta; LBD, ligand binding domain; RBA, relative
binding affinity; RTP, relative transcriptional potency; TLC, thin-layer chromatog-
raphy; CG, conjugated gradient.
a, estrogen receptor subtype alpha;
ER
* Corresponding author. Tel.: þ39 050 2219557; fax: þ39 050 2219605.
E-mail address: filippo.minutolo@farm.unipi.it (F. Minutolo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.030

2454
S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
We later identified the simplest member of the Salaldox B class
(2a) as a promising beta-selective ligand and, notably, also as the
first ER full agonist among our oxime derivatives [14]. Nonethe-
less, despite its high ER -binding preference, compound 2a also
b
b
experienced a loss of beta-selectivity in transcriptional assays, most
likely for the same reasons reported above for compounds 1c and
1d.
To overcome these limitations, we have continued our efforts to
obtain compounds that can both bind and activate ERb in a highly
efficient and selective fashion. To this purpose, we herein report
a logical extension of our structural optimization process to the
Salaldox B class, using some of the same molecular interventions
that proved successful in the Salaldox A series of derivatives studied
previously, combined with new insights from molecular modeling.
In the process, we prepared a focused series of analogs of 2a
through which we have investigated the effect of introducing
a 6-chloro atom in the central ring (2c) and an additional 30-F-
group in the aryl substituent (2d). We also introduced relatively
small substituents (such as methyl and chlorine) in position 3 of the
central ring, a place for molecular variations that has so far been
unexplored within these salicylaldoxime derivatives. We first
introduced a 3-methyl group (2e) and then inverted the respective
3/6-positions of the methyl and chlorine groups, such as in 2f and
in its 30-fluoro-substituted analog 2g. Finally, we obtained
compound 2b as a Salaldox B analog of compound 1b, to verify
whether the same structural restrictions in the Salaldox A series
Fig. 1. Structural derivation of the two series of salicylaldoximes, Salaldox A (1) and
Salaldox B (2), from the ERb pharmacophore model.
us to initially develop several diaryl-substituted salicyl- [16] and
anthranyl-aldoximes [17] as non-subtype-selective ER-ligands.
We initially obtained highly selective ERb-ligands belonging to
the Salaldox A series (Fig. 2) by progressively refining the substi-
tution pattern (compounds 1a, c, and d) [13,14], and we confirmed
would also apply to this new series of ERb-ligands.
the detrimental effect of a second aryl substituent on ER
(1b) [16]. However, even the best Salaldox A members proved to be
only partial agonists for ER , since their maximal activation values,
compared to estradiol, were of 60% for 1c and 85% for 1d [13,14].
Moreover, the beta-selectivities shown by these two compounds in
b binding
2. Chemistry
b
The synthesis of compounds 2bed started from 3-bromo-
2-chloro-6-methoxybenzaldehyde (3) [18], as shown in Scheme 1.
When compound 3 was submitted to a single cycle of Pd-catalyzed
cross-coupling reaction under classical Suzuki conditions [19]
(specifically, in situ formation of Pd(PPh3)4 by reaction of palla-
dium acetate with a 5-fold excess of triphenylphosphine with an
aqueous base and conventional heating at 100 ꢀC overnight) in the
presence of 1.2 equivalent of the boronic acid, 4-methox-
yphenylboronic or 3-fluoro-4-methoxyphenylboronic acid, it
selectively formed the corresponding monoaryl-substituted
adducts 4c, d, respectively. In fact, these conditions resulted in the
chemo-selective replacement of only the bromine atom of 3, with
the chloro group remaining intact. On the other hand, the repeti-
tion of two reaction cycles under the same Suzuki conditions, using
a total of 3.2 equivalents of boronic acid, afforded mostly the diaryl-
substituted intermediate 4b. The resulting adducts were treated
with boron tribromide to obtain O-demethylated salicylaldehydes
5bed, which were then condensed with hydroxylamine hydro-
chloride, thus yielding final salicylaldoximes 2bed.
functional assays were considerably less than their ER
ities in binding assays, probably because of differences in the
manner in which the ER - and ER -ligand complexes interact with
b-selectiv-
a
b
various cellular coregulators, which can act as modulators of ligand
potency.
The synthetic route to methyl/chlorine-substituted oximes
2eeg was slightly longer, as shown in Scheme 2. Commercially
available phenols 6e, f underwent O-allylation upon treatment
with allyl bromide. Claisen rearrangement of the resulting ethers
7e, f at 210 ꢀC yielded o-allyl-phenols 8e, f. The terminal double
bond of 8e, f was shifted to the internal position by alkaline iso-
merisation with potassium tert-butoxide in DMSO at 55 ꢀC, to give
b-methylstyrene derivatives 9e, f as E-diastereomers. Oxidative
cleavage of the styrene-type double bond with sodium periodate in
the presence of catalytic amounts of osmium tetroxide yielded
aldehydes 10e, f, which were then treated with bromine in glacial
acetic acid, affording mono-brominated salicylaldehyde derivatives
11e, f.
At this point, we initially tried to carry out a cross-coupling
reaction of 11e, f with the appropriate boronic acid, to effect the
replacement of the bromine atom with a suitable aryl substituent.
Fig. 2. Reference salicylaldoximes (1aed, 2a) and new derivatives (2beg) designed as
ERb-selective agonists.

S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
2455
Scheme 1. Synthesis of salicylaldoximes 2bed. Reagents and conditions: (a) 4-MeO-
C6H4B(OH)2 or 3-F-4-MeO-C6H4B(OH)2 (1.2 eq), Pd(OAc)2, PPh3, aqueous 2 M Na2CO3,
1:1 toluene/EtOH, 100 ꢀC, 16 h; (b) 2 times: 4-MeO-C6H4B(OH)2 (1.6 eq), Pd(OAc)2,
PPh3, aqueous 2 M Na2CO3, 1:1 toluene/EtOH, 100 ꢀC, 16 h; (c) BBr3, CH2Cl2, ꢂ78 to
0 ꢀC, 1 h; (d) NH2OH$HCl, EtOHeH2O, 50 ꢀC, 5 h.
Unfortunately, we were not able to perform this reaction efficiently,
probably because of the bidentate chelating effect that the free
vicinal OH/CHO groups of the salicylaldehyde portion have on both
the boron atom of the boronic acid and on the palladium catalyst of
the cross-coupling reaction, thus diverting the reactants from the
correct reaction pathway during the Suzuki step. Therefore, we first
protected the phenol hydroxyl with a methyl group, by reaction
with iodomethane and potassium carbonate in acetone, and then
submitted the resulting anisole derivatives 12e, f to the Suzuki
coupling reaction. This time the reaction worked nicely, and
monoaryl adducts 4eeg were so obtained. Final steps included
O-demethylation with BBr3, to give intermediates 5eeg, and
subsequent condensation with hydroxylamine hydrochloride,
which afforded final oximes 2eeg.
All the oximes (2beg) were obtained as single E-diastereoiso-
meric forms, presumably because the intramolecular hydrogen
bond, which can only form in the E-isomer, contributes to the
oxime stability. This selectivity had already been demonstrated for
other oxime analogs previously reported [13,14,16,17], and it was
confirmed here by the chemical shift values of the oxime protons of
all the new products, which were always found downfield from
Scheme 2. Synthesis of salicylaldoximes 2eeg. Reagents and conditions: (a) allyl
bromide, K2CO3, acetonitrile, 80 ꢀC; (b) neat, 180 ꢀC; (c) t-BuOK, DMSO, 55 ꢀC; (d) OsO4,
NaIO4, dioxane-H2O; (e) Br2, AcOH, RT; (f) MeI, K2CO3, acetone, RT; (g) 4-MeO-C6H4B
(OH)2 or 3-F-4-MeO-C6H4B(OH)2, Pd(OAc)2, PPh3, aqueous 2 M Na2CO3, 1:1 toluene/
EtOH, 100 ꢀC, 16 h; (h) BBr3, CH2Cl2, ꢂ78 to 0 ꢀC, 1 h; (i) NH2OH$HCl, EtOHeH2O, 50 ꢀC,
5 h.
methods that have been previously described [21,22]. The relative
binding affinity (RBA) values for the newly reported compounds,
together with those previously obtained for compounds 1aed and
2a [13,14,16b], are summarized in Table 1. RBA values are reported
as percentages (%) of that of estradiol, which is set at 100% (Entry 1).
We first analyzed some relevant results obtained previously
with the Salaldox A series (Table 1, Entries 2e5): It turns out that the
8 ppm (
d
ꢁ 8, see Section 7) [20].
3. Estrogen receptor binding assays
ERa- and ER
b
-binding affinities of new oximes 2beg were
simplest member of this class (1a) is already a very ER
b-selective
determined by a radiometric competitive binding assay, using
ligand (RBA
b/a
ratio ¼ 79), although its binding affinity for the

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S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
Table 1
series [14]. The binding affinity of 2d for ERb is also remarkably
Relative binding affinitiesa of compounds of the Salaldox A (1aed) and Salaldox B
high, as shown by its 87% RBA value (corresponding to a Ki of
0.57 nM). By contrast, the addition of a methyl group to the central
ring of compound 2c results in a compound (2e) that has a dramatic
(2aeg) series for the estrogen receptors
a and b.
Entry
1
Ligand
hER
a
(%)
hER
b
(%)
b
/a ratio
Estradiol
(100)
(100)
1
drop in affinity for both receptor subtypes (RBA ¼ 0.074% for ER
a
and 0.639% for ER ). A marked recovery of binding properties is
b
Salaldox A Series
2
3
4
5
1ab
1bc
1cb
1dd
0.007 ꢃ 0.001
0.92 ꢃ 0.04
0.55 ꢃ 0.11
0.35 ꢃ 0.01
4.21 ꢃ 0.66
7.01 ꢃ 1.00
79
0.38
65
64
obtained, however, when the relative positions of the methyl and
chlorine substituents of 2e are reversed, as shown by the good
0.065 ꢃ 0.016
0.11 ꢃ 0.03
affinity values associated with compound 2f (RBA ¼ 1.47% for ER
and 15.8% for ER ), although the beta-selectivity is not as high as
desired (RBA
ratio ¼ 11). Here again, introduction of a meta-
fluorine atom into the 4-hydroxyphenyl substituent of 2f, leading
to 2g (RBA ¼ 0.392% for ER and 7.90% for ER ), effects a 2-fold
increase of ER -selectivity (RBA
a
b
b/a
Salaldox B Series
6
7
8
2ad
2b
2c
2d
2e
2f
0.064 ꢃ 0.016
88.4 ꢃ 18.1
4.46 ꢃ 0.60
1.88 ꢃ 0.30
0.074 ꢃ 0.006
1.47 ꢃ 0.04
0.39 ꢃ 0.04
2.64 ꢃ 0.62
101 ꢃ 2
41
1.1
29
46
8.6
a
b
130 ꢃ 25
b
b
/
a
ratio ¼ 20). Finally, the single
9
87.1 ꢃ 15.0
0.64 ꢃ 0.09
15.8 ꢃ 3.5
7.90 ꢃ 0.40
10
11
12
Salaldox B member bearing two para-hydroxyphenyl substituents
(2b) is the only one not showing any appreciable preference for the
11
20
2 g
beta-subtype (RBA
receptors (RBA ¼ 88.4% for ER
b
/
a
ratio ¼ 1.1), although its affinities for both
a
Determined by a competitive radiometric binding assay with [3H]estradiol;
a
and 101% for ER ) are remarkably
b
preparations of purified, full-length human ER
used; see Section 7. Values are reported as the mean ꢃ the range or SD of 2 or more
independent experiments; the Kd for estradiol for ER is 0.2 nM and for ER is
a and ERb (Invitrogen, PanVera) were
higher than those of its Salaldox A analog 1b, reaching values that
are surprisingly close to those of estradiol. This last result further
supports our original hypothesis that the monoaryl-substitution
a
b
0.5 nM. Ki values for the new compounds can be readily calculated by using the
formula: Ki ¼ (Kd[estradiol]/RBA) ꢄ 100.
motif is a strict prerequisite for obtaining good ERb-selectivity in
b
See Ref. [13].
See Ref. [16b].
See Ref. [14].
this type of salicylaldoxime derivative [13,14].
c
d
4. Molecular modeling
An automated computational analysis of the newly synthesized
compounds was performed to try to rationalize their binding
beta-receptor was rather modest (0.55%). Insertion of a chlorine
into the 6 position of the central scaffold, as in compound 1c,
properties. Docking of the ligands into ERa and ERb (PDB codes 2I0J
preserved the high selectivity level (RBA
markedly increased the ER -binding affinity (4.21%) [13]. By
contrast, insertion of a second para-hydroxyphenyl substituent in
this scaffold produces a compound (1b) whose affinity for ER was
b
/
a
ratio ¼ 65) and
and 2I0G, respectively) was carried out using AUTODOCK 4.0 soft-
ware [25]. Fig. 3A and B displays the docking results for diaryl-
substituted compound 2b into both ERs. In agreement with its
similar affinity for both receptor subtypes, this compound shows
b
b
dramatically reduced (0.35%) [16b], thus confirming the impor-
tance of the monoaryl-substitution motif within this class. Being
inspired by a few very successful examples reported in the litera-
ture, such as biphenylcarbaldehyde oxime derivatives [23] and
benzoxazole ERB-041 [24], we introduced a fluorine atom in the 30-
position of the para-hydroxyphenyl group of 1c, and the compound
the same interactions in the ER
The para-hydroxyl group on the aryl substituent distal to the oxime
function is involved in an H-bond network, which includes (ER
residues in parentheses) E305 (E353), R346 (R394), and a water
molecule. The para-hydroxyl of the aryl substituent proximal to the
oxime function group forms an H-bond with T299 (T347). Finally,
the pseudocycle/oxime system forms an H-bond with H475 (H524).
All of these supposedly strong interactions confirm the very high
a and ERb ligand binding pockets.
a
thus obtained (1d) possessed a higher affinity for ER
its non-fluorinated counterpart, together with a similar subtype
selectivity (RBA
b (7.01%) than
b/
a
ratio ¼ 64).
binding affinities that 2b has for both ER
The docking of monoaryl-substituted compound 2c (Fig. S1B,
Supplementary data) and 2d (Fig. 3D) into ER produces results
a and ERb.
We then turned to analysis of the binding affinity of members of
the Salaldox B series. Although this series was designed by
a completely different structural modification of 1a, which involved
an inversion of the respective positions of the hydroxy and oxime
groups of the salicylaldoxime scaffold, we were surprised to find
that the simplest member of Salaldox B series (2a) had a 5-fold
b
substantially similar to those previously obtained with their
non-halogenated analog 2a [14], and highlights that: (1) the
pseudocycle/oxime system is engaged in the H-bond network of
the E305-R346-water system; and (2) the OH of the p-hydrox-
yphenyl ring forms a H-bond with T299. In both compounds, the
chlorine atom is inserted into a pocket delimited by A302, W335,
M336, and L339. It should be noted that this orientation is
completely different from that previously found in the docking
higher ER
2 and 6). Most importantly, the ER
quite low (0.064%), so that this compound retained a notable beta-
selectivity (RBA
b
-binding affinity (2.64%) compared to 1a (Table 1, Entries
a
binding affinity of 2a remained
b
/
a
ratio ¼ 41) [14]. This promising behavior of the
“progenitor” member of the Salaldox B series (2a) indicated that
this series merited further exploration.
analysis of Salaldox A derivatives such as 1c and 1d into ERb-LBD,
which instead place their p-hydroxyphenyl substituent in the
H-bond network of the E305-R346-water system and the pseudo-
cycle/oxime portion forming a H-bond with H475 [14]. It is inter-
esting to note that the strong interaction between the 40-hydroxyl
of the ligands and T299 is possible only in the ERb-LBD cavity,
because only in this ER subtype is there enough space for the
phenol group to reach the OH of T299 by occupying an area close to
Among the newly synthesized Salaldox B derivatives (Table 1,
Entries 7e12), compound 2c, possessing a chlorine atom in the
central scaffold, displays an outstanding affinity for ER
value of 130% (corresponding to a Ki of 0.38 nM), and a robust beta-
selectivity (RBA
ratio ¼ 29). It should be noted that the affinity
of 2c for ER is significantly higher than that of estradiol itself.
Compound 2d, derived from an addition of a meta-fluorine in the
para-hydroxylated aryl substituent, has a considerably higher ER
selectivity (RBA
b, with a RBA
b
/a
b
M336. The same does not happen in ER
(M336 in ER ) is replaced by a bulkier and less flexible leucine
(L384 in ER ) [26], causing a completely different disposition of the
two compounds in this receptor subtype. In fact, compared to what
happens in ER , in ER 2c (Fig. S1A, Supplementary data) and 2d
a, where the methionine
b
-
b
b
/
a
ratio ¼ 46) than 2c, thus confirming that this
a
kind of structural modification often leads to an improved prefer-
ence for the beta-subtype, as was seen before in the Salaldox A
b
a

S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
2457
Fig. 3. Docking analysis into ER
ER ; (E) docking of 2e (light blue) into ER
version of this article.)
a
and ER
b
: (A) docking of 2b (green) into ER
a
; (B) docking of 2b (green) into ER
b
; (C) docking of 2d (yellow) into ER
a
; (D) docking of 2d (yellow) into
b
b; (F) docking of 2f (orange) into ERb. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
(Fig. 3C) are turned upside down such that their phenol 40-OH
group is involved in the H-bond network with E353, R394, and
a water molecule, and their pseudocycle/oxime system forms
a H-bond with H524.
The addition of a methyl group in the position para to the
chloro-substituent of the central ring, as in 2e (Fig. 3E), is not
tolerated in the same low energy conformation assumed by 2c and
using Gaussian09 to verify the reliability of our docking results [27].
The ER e2d complex obtained from the docking studies was
b
energy-minimized and then subjected to QM/MM, which has been
so far used with good success by many authors in the field of drug
design to find correct interactions within biological systems [28]. In
these calculations, the zone of highest interest is treated quantum
mechanically, while the rest of the system is treated by classical
mechanics, thus reducing computational expenses. We used the
ONIOM module of Gaussian09, using the B3LYP chemical model for
the quantum mechanics (QM) system [29], with a 6-31Gþþ** basis
set and the Amber force field (parm96) for the molecular
mechanics (MM) system. The QM system consisted of the ligand,
the structural water molecule, and the side chains of T299, E305
and R346. Fig. 4 shows the superimposition between the starting
2d in ER
not large enough to tolerate the presence of a CH3. This causes the
orientation of 2e to become rotated by 180ꢀ in the ER
-binding
pocket, resulting in a complex that is less stable than those with 2c
or 2d. Thus, 2e has a dramatically reduced binding affinity for ER
b, because the binding region in the proximity of M340 is
b
b
.
On the other hand, if the position of the two CH3/Cl central
substituents is interchanged, the resulting compounds, 2f (Fig. 3F)
and 2g (Fig. S2, Supplementary data), have a preferred conforma-
ERbe2d complex and the QM/MM-optimized one. The ligand
tion in ER
b
that is the same as the one found for compounds 2c and
maintained the interactions with T299 and the E305-R346-water
system, showing a Root Mean Square Deviation (RMSD) from the
starting structure of only 0.3 Å. Also, the binding site residues
showed only small movements, with an RMSD of 0.7 Å.
2d, because the methyl substituent of 2f and 2g can now be
accommodated in the pocket delimited by A302, W335, M336, and
L339, while the chlorine atom, being smaller than the methyl group
present in an analogous position of 2e, now fits nicely in the pocket
close to M340. These factors result in the good binding affinity that
both 2f and 2g have for ERb, although their values are not better
5. Transcription assays
than those found for 2c and 2d.
It should be noted that in our modeling studies, the analyzed
ligands were simply docked into a single, rigid version of the
receptor, and we did not computationally evaluate the possible fit-
induced effects. This kind of approach requires the use of a flexible
receptor and, hence, is much more computationally intensive than
rigid receptor docking. At present, the main docking software
programs are able to take into account the flexibility of only a small
number of residues, making the possibility of evaluating the flexi-
bility of a binding site very difficult. For these reasons, we used the
ER
b
-selective ligands 2bed, displaying the highest levels of
, as well as previously
binding affinity and selectivity for ER
b
reported reference compounds 1c, 1d and 2a, were submitted to
further biological testing to assess their pharmacological character.
They were assayed for transcriptional activity through both
receptor subtypes, together with estradiol for reference. Reporter
gene transfection assays were conducted in human endometrial
(HEC-1) cells, using expression plasmids for either full-length
human ER
a or ERb and an estrogen-responsive luciferase reporter
ERbe2d complex as a test set for a two-layer QM/MM calculation
gene system [30].

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S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
Table 2
Transcription potencies of ER
series through human estrogen receptors
b-ligands of the Salaldox A (1c, d) and Salaldox B (2aed)
a
and b a
.
Entry Ligand
hERa
activation
hER
b
activation
b/a EC50-
selectivity ratio
EC50 (nM)b EMAX (%)c EC50 (nM)b EMAX (%)c
Salaldox A Series
1
2
1cd
1de
26
19
80
95
11
4.8
60
85
2.4
4.0
Salaldox B Series
3
4
5
6
7
2ae
2b
2c
17
0.30
0.58
8.4
100
90
100
100
100
10
0.50
0.23
1.3
100
80
100
100
100
1.7
0.6
2.5
6.5
0.12
2d
Estradiol 0.09
0.72
a
Human endometrial cancer (HEC-1) cells, transfected with expression vectors
or ER and an (ERE)2-pS2-luc reporter gene, were treated with the indicated
for ER
a
b
compound and resulting luciferase activity was measured as expression of tran-
scription (see Section 7).
b
Half-maximal effective concentration.
Maximal effect normalized to the activity with 100 nM estradiol, which was set
c
at 100%.
d
See Ref. [13].
See Ref. [14].
e
Overall, as we observed in the past [13,14], there is a general
reduced ERb-selectivity in terms of transcriptional potency vs.
binding affinity. This apparent discrepancy may be attributed to the
fact that the receptoreligand complex is present by itself in the
binding assays, whereas in the cellular transcription assays it is
engaged in various interactions with the many coregulators present
in the cell; these receptorecoregulator binding interactions can act
as additional modulators of ligand potency [31]. It should be noted
that both 2c and 2d are by far more beta-selective full agonists than
Fig. 4. QM/MM simulation of the ERbe2d complex. Superimposition between the QM/
MM results (binding site and ligand coloured light blue and pink, respectively) and the
structure resulting from the docking calculations (binding site and ligand coloured
white and yellow, respectively). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
is estradiol, which has EC50 values of 0.72 nM on ER
ER (Table 2, Entry 7); one of them (2c) is even more potent on ER
than estradiol itself.
b vs. 0.09 nM on
a
b
The first representative examples of the previously reported
Salaldox A series (Table 2, Entries 1e2), compound 1c, reached only
In order to make a better comparisons of the ER subtype tran-
scriptional potencies of our derivatives with their ER subtype
binding affinities, we converted the EC50 values from the functional
assays to relative transcriptional potency (RTP) values, which were
partial activation of ER
b
(EMAX ¼ 60%) [13], whereas its “fluori-
nated” analog 1d displayed a general improvement in all functional
properties, such as potency (EC50 ¼ 4.8 nM), maximal effect
(EMAX ¼ 85%), and subtype selectivity
(b
/a
EC50-selectivity
calculated
as
RTP ¼ EC50(estradiol)/EC50(ligand) ꢄ 100
(RTP,
ratio ¼ 4.0) [14]. In any case, these Salaldox A members could only
be classified as partial agonists, because none of them reached
maximal activation of transcription (normalized to that resulting
from 100 nM estradiol). By contrast, the first example of the Sal-
aldox B series, compound 2a, behaved as a full agonist on both
receptor subtypes, but its functional beta-selectivity was not
satisfactory (Entry 3).
estradiol ¼ 100). The RTPs give
a
measure of transcriptional
potency relative to that of estradiol and, therefore, are suitable
factors to be used in comparisons with their binding affinities,
which are also measured relative to estradiol. In our present assays,
estradiol has a 2.5-fold preference in favour of ER
binding (Kd [ER ] ¼ 0.5 nM) and a 8-fold pref-
] ¼ 0.2 nM vs. [ER
erence in terms of transcriptional potency (EC50 [ER
] ¼ 0.09 nM vs.
[ER
] ¼ 0.72 nM). This seems to be caused by the fact that, when
stimulated by estradiol, ER is a more potent transcription activator
than ER in inducing cellular responses [32]. We report herein the
RTP values of our most potent ER -agonists 2bed and that of
a in terms of
a
b
a
Among the newly synthesized Saladox B ligands (Table 2, Entries
4e6), diaryl-substituted derivative 2b, which proved to be non-
subtype-selective inbindingassays, shows a similarlack of selectivity
in these transcriptional assays, where it shows only a slight prefer-
b
a
b
b
ence for ER
EMAX ¼ 80%). Monoaryl-substituted derivatives 2c and 2d show
preferential activation of ER rather than ER , consistent with their
ER -binding selectivities, and, similar to their “progenitor” 2a, both
cause full activation of transcription. Inparticular, the single insertion
of a chlorine atomin the central ringof 2a generates a full agonist (2c)
a
(EC50 ¼ 0.30 nM, EMAX ¼ 90%) vs. ER
b
(EC50 ¼ 0.50 nM,
estradiol, together with their doseeresponse curves for transcrip-
tional activation (Fig. 5). According to these metrics, compound 2c
b
a
has an RBA(
(Fig. 5, Panel C), and compound 2d has an RBA(
(Table 1) and an RTP( ) ratio of 52 (Fig. 5, Panel D). Hence,
measured relative to estradiol, the ER affinity preference of these
b/
a) ratio of 29 (Table 1) and an RTP(
b
/a) ratio of 20
b
b/a) ratio of 46
b/a
b
having a >40-fold increased potency on ER
b
and reaching a sub-
compounds is, indeed, preserved in their ER
potency preference.
b transcriptional
nanomolar EC50 value (0.23 nM) and a slightly better beta-selectivity
than 2a. The combined addition of the central chlorine group and the
30-fluorine atom in the pendant aryl substituent, as in compound 2d,
Although there are several examples of previously reported
-agonists showing higher subtype selectivities in functional
assays (i.e., DPN, SERBA-1, ERB-041) [3], compounds 2c and d are
definitely the most ER -selective agonists of the whole salicy-
laldoxime class so far synthesized.
ERb
results in a jump in the selectivity of ERb-activation (b/a EC50-
selectivity ratio ¼ 6.5), together witha substantial preservationoffull
b
agonist potency (EC50 ¼ 1.3 nM).

S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
2459
Fig. 5. Doseeresponse curves for transcriptional activation by estradiol (Panel A), compound 2b (Panel B), compound 2c (Panel C) and compound 2d (Panel D) through ER
b
(dashed
line) and ER
a
(solid line). Human endometrial cancer (HEC-1) cells were transfected with expression vectors for ER
a
b
or ER
-galactosidase activity from an internal control plasmid. The
/ER relative transcriptional potency
b and an (ERE)2-pS2-luc reporter gene and were treated
with estradiol or compound 2bed at the concentrations indicated. Luciferase activity was expressed relative to
maximal activity with 100 nM E2 was set at 100%. Values are the mean of duplicate determinations. EC50 values give absolute potencies. The ER
b
a
(RTP( )) ratios are calculated as explained in the text.
b/a
6. Conclusions
ER
b-binding cavity [33]. Efforts to obtain X-ray structures of
complexes of ER
b
with these salicylaldoxime derivatives are
Our present studies confirm that, in contrast to our previously
developed Salaldox A analogs, which never reached full activation
currently underway and, if successful, should shed further light on
the way these compounds interact and activate this intriguing and
therapeutically exploitable nuclear receptor.
of ER
b (partial agonists), the monoaryl-substituted Salaldox B
derivatives herein reported display full agonist character on ER
b
and have much higher binding affinity than their earlier counter-
parts. In fact, representative compounds 2c and 2d have affinities
7. Experimental section
for ER
sub-nanomolar range (Ki ¼ 0.38 nM for 2c and 0.57 nM for 2d),
together with notable levels of selectivity for ER over ER . Most
b comparable to that of estradiol itself, with Ki values in the
7.1. Chemistry
b
a
7.1.1. General
importantly, one of them (2d) has a remarkably improved beta-
selectivity even in functional assays, which is unprecedented for
any of the salicylaldoxime derivatives so far developed. This is
Commercially available chemicals were purchased from Sig-
maeAldrich or Alfa Aesar and used without further purification,
with the exception of 3, which was prepared as previously reported
[18]. NMR spectra were obtained with a Varian Gemini 200 MHz
demonstrated by ER
times higher than that of estradiol, together with a comparable
ER
-agonist potency (EC50 ¼ 1.3 nM vs. 0.73 nM of estradiol).
Curiously, the very same structural modifications that were previ-
ously shown to successfully improve selective ER -binding affinity
b/ERa selective activation by 2d, which is >50
spectrometer. Chemical shifts (d) are reported in parts per million
b
downfield from tetramethylsilane and referenced from solvent
references. Electron impact (EI, 70 eV) mass spectra were obtained
on a ThermoQuest Finningan (TRACE GCQ plus MARCA) mass
spectrometer. Melting points were measured with a Kofler appa-
ratus. Purity was routinely measured by HPLC on a Waters SunFire
b
and transcriptional activation in the Salaldox A series, such as the
insertion of a Cl in the central phenyl ring and a meta-fluorine in the
phenol substituent (compounds 1c and 1d), also proved to be
beneficial when made at the corresponding positions of this new
Salaldox B class, as shown by the behavior of compounds 2c and 2d.
This could have not been easily predicted, because our docking
studies indicate that the Salaldox B derivatives assume a completely
RP 18 (3.0 ꢄ 150 mm, 5
mm) column (Waters, Milford, MA, www.
waters.com) using a Beckmann SystemGold instrument consisting
of a chromatography 125 Solvent Module and a 166 UV Detector.
Mobile phases: 10 mM ammonium acetate in Millipore purified
water (A) and HPLC grade acetonitrile (B). A gradient was formed
from 5% to 80% of B in 10 min and held at 80% for 10 min; flow rate
different orientation in ERb-binding cavity, compared to that found
for the other series. As a matter of fact, in these studies, compounds
2c and 2d have their oxime portion participating in the H-bond
network with residues R346 and E305, and place their peripheral
phenol OH in a position where it can establish a strong interaction
with a threonine residue (T299). This last interaction is quite
was 0.7 mL/min and injection volume was 30 mL; retention times
(HPLC, tR) are given in minutes. HPLC purity of final compounds (2c,
d) was determined by monitoring at 254 and 300 nm and was
found in the range 96e99%. Chromatographic separations were
performed on silica gel columns by flash (Kieselgel 40,
0.040e0.063 mm; Merck) or gravity column (Kieselgel 60,
0.063e0.200 mm; Merck) chromatography. Reactions were
unusual for ER
b-agonists, since it has only been reported so far to
occur when ligands having antagonist properties interact with the

2460
S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
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 or Biotage microwave synthesizer.
2.02 mmol) in water (3.5 mL), and the mixture was heated to 50 ꢀC
for 5 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 chromatog-
raphy over silica gel. Elution with n-hexane/EtOAc 6:4 (Rf ¼ 0.48)
afforded pure 2c (99% yield) as a white solid; 1H NMR (CD3OD)
7.1.2. 2-Chloro-4,40-dimethoxybiphenyl-3-carbaldehyde (4c)
0
0
A solution of Pd(OAc)2 (2.9 mg, 0.013 mmol) and triphenyl-
phosphine (16.9 mg, 0.064 mmol) in ethanol (1.0 mL) and toluene
(1.0 mL) was stirred at RT under nitrogen for 10 min. After that
period, the bromo-aryl precursor 3 [18] (0.43 mmol), an aqueous
solution of Na2CO3 (1.0 mL, 2 M), and 4-methoxyphenylboronic
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 were dried and
concentrated. The crude product was purified by flash chroma-
tography over silica gel. Elution with n-hexane/EtOAc 8:2 (Rf ¼ 0.16)
d
(ppm): 6.81 (AA0XX0, 2H, JAX ¼ 8.6 Hz, JAA /XX ¼ 2.5 Hz, H3 , H5 ),
0
0
6.89 (d, 1H, J ¼ 8.6 Hz, H5), 7.17 (AA0XX0, 2H, JAX ¼ 8.6 Hz, JAA /
0
¼ 2.5 Hz, H20, H60), 7.20 (d, 1H, J ¼ 8.6 Hz, H6), 8.79 (s, 1H, eCH]
XX0
Ne). 13C NMR (CD3OD)
d (ppm): 115.73, 115.86, 116.22, 131.50,
131.72, 131.98, 132.83, 133.87, 150.27, 157.83, 158.85. MS m/z 263
(Mþ, 15), 155 (M þ Hþ eOH eC6H4O, 100). Mp: 144e146 ꢀC. HPLC, tR
10.3 min.
7.1.7. (E)-2-chloro-30-fluoro-4,40-dihydroxybiphenyl-3-
carbaldehyde oxime (2d)
Compound 2d was prepared from aldehyde 5d by following the
same procedure described above for the preparation of 2c. The
crude product was purified by flash chromatography over silica gel.
Elution with n-hexane/EtOAc 7:3 (Rf ¼ 0.35) afforded pure 2d (93%
afforded 4c as a yellow solid (70% yield). 1H NMR (CDCl3)
d (ppm):
3.86 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.95e6.99 (m, 3H, H5, H30,
0
0
H50), 7.31 (AA0XX0, 2H, JAX ¼ 8.8 Hz, JAA /XX ¼ 2.2 Hz, H2 , H6 ), 7.45
0
0
(d, 1H, J ¼ 8.6 Hz, H6), 10.56 (s, 1H, CHO). Mp: 57e58 ꢀC.
yield) as a white solid; 1H NMR (acetone-d6)
d (ppm): 6.96 (d, 1H,
J ¼ 8.6 Hz, H5), 7.04e7.19 (m, 3H, H20, H50, H60), 7.30 (d, 1H,
J ¼ 8.4 Hz, H6), 8.82 (exchangeable bd, 1H, J ¼ 1.3 Hz, 40-OH), 8.84 (s,
1H, eCH]Ne),10.91 (exchangeable s,1H, OH),11.14 (exchangeable s,
7.1.3. 2-Chloro-30-fluoro-4,40-dimethoxybiphenyl-3-carbaldehyde
(4d)
Compound 4d was prepared by a cross-coupling reaction of 3
with 3-fluoro-4-methoxyphenylboronic acid (1.2 eq), following the
same procedure described above for the preparation of 4c. The
crude product was purified by flash chromatography over silica gel.
Elution with n-hexane/EtOAc 8:2 (Rf ¼ 0.16) afforded 4d (80% yield)
1H, OH). 13C NMR (acetone-d6)
d (ppm): 115.55, 116.38, 118.12 (d,
J ¼ 22.8 Hz), 118.28, 126.78 (d, J ¼ 3.7 Hz), 132.20 (d, J ¼ 6.4 Hz),
132.35, 132.61, 133.92, 145.17 (d, J ¼ 12.8 Hz), 150.36, 151.73 (d,
J ¼ 238.6 Hz), 159.06. MS m/z 281 (Mþ, 100), 263 (Mþ ꢂ H2O, 64).
Mp: 125e127 ꢀC. HPLC, tR 10.7 min.
as a yellow solid; 1H NMR (CDCl3)
d (ppm): 3.94 (s, 3H, OCH3), 3.95
(s, 3H, OCH3), 6.97 (d,1H, J ¼ 8.8 Hz, H5), 7.03 (d,1H, J ¼ 7.7 Hz, H50),
7.06e7.17 (m, 2H, H20, H60), 7.43 (d, 1H, J ¼ 8.8 Hz, H6), 10.55 (s, 1H,
CHO). Mp: 73e74 ꢀC.
7.2. Biological methods
7.2.1. General
Full-length human ER
Invitrogen (Carlsbad, CA). [3H]Estradiol ([3H]E2) ([2,4,6,7-3H]estra-
1,3,5(10)-triene-3,17 -diol) was obtained from PerkinElmer, Inc.
(Waltham, MA) and had a specific activity of 70e120 Ci/mmol. 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
a and ERb were obtained from PanVera/
7.1.4. 2-Chloro-4,40-dihydroxybiphenyl-3-carbaldehyde (5c)
A solution of methoxy-substituted aldehyde 4c (0.12 mmol) in
anhydrous dichloromethane (1.5 mL) was cooled to ꢂ78 ꢀC and
treated dropwise with a solution of BBr3 in dichloromethane
(0.7 mL, 1 M), and the resulting solution was stirred at the same
temperature for 5 min and 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 7:3 (Rf ¼ 0.35) afforded pure 5c (42% yield) as a yellow solid;
b
(Atlanta, GA). The expression vectors for human ER
a (pCMV5-
hER ) and human ER (pCMV5-ER ) were as described previously
a
b
b
[34,35]. 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
1H NMR (CDCl3) d0 (ppm): 6.90 (AA0XX0, 2H, JAX ¼ 8.8 Hz, JAA /
0
¼ 2.4 Hz, H30, H5 ), 6.95 (d, 1H, J ¼08.9 Hz, H5), 7.27 (AA0XX0, 2H,
10.53 (s, 1H, CHO), 12.11 (exchangeable s, 1H, OH). Mp: 58e59 ꢀC.
XX0
0
0
0
JAX ¼ 8.5 Hz, JAA /XX ¼ 2.6 Hz, H2 , H6 ), 7.46 (d, 1H, J ¼ 8.8 Hz, H6),
(Madison, WI). The plasmid pCMVb-gal (Clontech, Palo Alto, CA),
which contains the -galactosidase gene, was used as an internal
b
control for transfection efficiency.
7.1.5. 2-Chloro-30-fluoro-4,40-dihydroxybiphenyl-3-carbaldehyde
(5d)
7.2.2. Estrogen receptor binding assays
Compound 5d was prepared from methoxy-substituted alde-
hyde 4d by following the same procedure described above for the
preparation of 5c. The crude product was purified by flash chro-
matography over silica gel. Elution with n-hexane/EtOAc 7:3
(Rf ¼ 0.39) afforded pure 5d (81% yield) as a yellow solid; 1H NMR
Relative binding affinities were determined by competitive
radiometric binding assays with 2 nM [3H]E2 as tracer, as a modi-
fication of methods previously described [21,22]. The source of ER
was purified full-length human ERa and ERb purchased from Pan
Vera/Invitrogen (Carlsbad, CA). Incubations were done at 0 ꢀC for
18e24 h, and hydroxyapatite was used to absorb the purified
receptoreligand complexes (human ERs) [22]. The binding affini-
ties are expressed as relative binding affinity (RBA) values, where
the RBA of estradiol is 100%; under these conditions, the Kd of
(CDCl3)
d
(ppm): 6.95 (d, 1H, J ¼ 8.8 Hz, H5), 7.04e7.16 (m, 3H, H20,
H50, H60), 7.44 (d, 1H, J ¼ 8.8 Hz, H6), 10.52 (s, 1H, CHO), 12.12
(exchangeable s, 1H, OH). Mp: 63e64 ꢀC.
7.1.6. (E)-2-chloro-4,40-dihydroxybiphenyl-3-carbaldehyde oxime
(2c)
A solution of aldehyde 5c (1.0 mmol) in ethanol (15 mL) was
treated with a solution of hydroxylamine hydrochloride (140 mg,
estradiol for ERa is ca. 0.2 nM, and for ERb 0.5 nM. The determi-
nation of these RBA values is reproducible in separate experiments
with a CV of 0.3, and the values shown represent the aver-
age ꢃ range or SD of 2 or more separate determinations.

S. Bertini et al. / European Journal of Medicinal Chemistry 46 (2011) 2453e2462
2461
7.2.3. Cell culture and transient transfections
into a rectangular parallelepiped water box; an explicit solvent
model for water, TIP3P, was used, and the complex was solvated
with a 10 Å water cap. Sodium ions were added as counter ions to
neutralize the system. Two steps of minimization were then carried
out. In the first stage, we kept the complex fixed with a position
restraint of 500 kcal/(mol Å2) and we solely minimized the posi-
tions of the water molecules. In the second stage, we minimized the
entire system through 20,000 steps of steepest descent followed by
conjugate gradient until a convergence of 0.05 kcal/(mol Å) was
Human endometrial cancer (HEC-1) cells were maintained in
culture as described [30]. 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 containing 5
ogies, Rockville, MD), 20 L of transferrin (Sigma, St. Louis, MO),
0.2 g of pCMV -galactosidase as internal control, 0.5 g of the
mL of lipofectin (Life Technol-
m
m
b
m
reporter gene plasmid, 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% charcoaledextran 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
attained. All the
a carbons of the protein were blocked with
a harmonic force constant of 10 kcal/(mol Å2). The minimized
structure was used as starting structure for the QM/MM calcula-
tions. The QM region was described by the B3LYP chemical model
with a 6-31Gþþ** basis set and contained the ligand, the structural
water molecule and the side chains of T299, E305 and R346. The
B3LYP model is a combination of the Becke three-parameter hybrid
functional [41] with the LeeeYangeParr correlation functional
(which also includes density gradient terms) [42]. On the MM
region, the parm96 force field was applied using no constraints.
internal control
cribed [30].
b-galactosidase activity, was assayed as des-
7.3. Docking methods
The crystal structure of ERa (pdb code 2I0J) [36] and ERb (pdb
code 2I0G) [36] was taken from the Protein Data Bank. After adding
hydrogen atoms the two proteins complexed with their reference
inhibitor were minimized using Amber 9 software [37] 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 counter ions 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 mole-
cules. 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.
Acknowledgment
One of the authors (CG) is grateful to the Italian Ministry for
University and Research (MIUR), Rome, Italy, for a triennial
(2008e2010) PhD fellowship (“grandi progetti strategici”). Dr.
Giorgio Placanica and Dr. Caterina Orlando are gratefully
acknowledged for technical assistance in the analysis of the
chemical products. Molecular graphic images were produced using
the UCSF Chimera package from the Resource for Biocomputing,
Visualization, and Informatics at the University of California, San
Francisco (supported by NIH P41 RR001081). Support from the
National Institutes of Health is gratefully acknowledged (PHS R37
DK015556, JAK; NRSA F30 ES016484-01 and T32 GM070421, JRG).
The ligands were built using Maestro [38] and were minimized
by means of Macromodel [38] 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.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2011.03.030.
Automated docking was carried out by means of the AUTODOCK
4.0 program [25]; Autodock Tools 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 intra-
molecular bond. The regions of interest used by Autodock were
defined by considering SERBA-1 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.
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