New scaffolds for the design of selective estrogen receptor modulators

Article abstract of DOI:10.1039/b806918b

In the present work we report the synthesis of four new ER ligands which can be used as scaffolds for the introduction of the basic side chains necessary for antiestrogenic activity. Affinities and agonist/antagonist characterization of the ligands for both ERα and ERβ have been determined in a competitive radioligand assay, and in an in vitro coactivator recruitment functional assay, respectively. Molecular modelling techniques have been used in order to rationalize the experimental results. Compound 2 is reported as a novel ERβ-agonist/ERα-antagonist. Two compounds show an interesting antitumour profile towards two pancreatic cancer cell lines and have been selected for in vivo assays.

Full text of DOI:10.1039/b806918b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
New scaffolds for the design of selective estrogen receptor modulators
Sonsoles Mart´ın-Santamar´ıa,a Jose´-Juan Rodr´ıguez,a Sonia de Pascual-Teresa,b Sandra Gordon,c
Martin Bengtsson,c Ignacio Garrido-Laguna,d Bele´n Rubio-Viqueira,e Pedro P. Lo´pez-Casas,e
Manuel Hidalgo,d Beatriz de Pascual-Teresa*a and Ana Ramos*a
Received 24th April 2008, Accepted 7th July 2008
First published as an Advance Article on the web 4th August 2008
DOI: 10.1039/b806918b
In the present work we report the synthesis of four new ER ligands which can be used as scaffolds for
the introduction of the basic side chains necessary for antiestrogenic activity. Affinities and
agonist/antagonist characterization of the ligands for both ERa and ERb have been determined in a
competitive radioligand assay, and in an in vitro coactivator recruitment functional assay, respectively.
Molecular modelling techniques have been used in order to rationalize the experimental results.
Compound 2 is reported as a novel ERb-agonist/ERa-antagonist. Two compounds show an interesting
antitumour profile towards two pancreatic cancer cell lines and have been selected for in vivo assays.
Introduction
The estrogen receptor (ER) is a member of the nuclear receptor
gene family binding the steroid hormone estradiol. Two subtypes
of ER (ERa and ERb) which have different tissue distribution,
are known and they are thought to regulate different estrogen
responses.1 While ERais an important receptor in mammary gland
and uterus, and is mainly involved in reproductive events, ERb is
a more generally expressed ER, and its role seems to be relevant
in the central nervous system, bone, lung, cardiovascular system,
ovary, testis, urogenitaltract, kidney, andcolon.2 Selectiveestrogen
Fig. 1 Chemical structures of tamoxifen and raloxifene.
receptor modulators (SERMs) are a therapeutically important
class of ER ligands, which show tissue-dependent agonistic or
antagonistic behavior, and are used as first line treatment for
estrogen-responsive breast cancer and postmenopausal related
disorders.3–5
The SERM tamoxifen has remained the antihormonal therapy
of choice for the treatment of ER positive breast cancer for the last
30 years (Fig. 1).6 For anti-osteoporotic therapy, SERM raloxifene
has a favourable balance of agonist activities in certain tissues
(bone, liver, vasculature)7 and antagonist activities in other tissues,
such as uterus and breast, leading to its use as chemo preventive for
breast cancer.8 A number of ER ligands have been synthesized over
the years.9 They include steroidal and nonsteroidal compounds.
Conformationally restricted analogues of raloxifene are among
the most potent SERMs described, maintaining the biological
structure–activity profile reported previously for raloxifene.10 On
Fig. 2 Structures of some known ERb-selective agonists.
the other hand, since the discovery of ERb in 1996, compounds
that are selective in activating or inhibiting these two ER subtypes
are intensively sought after.1 In particular, ERb agonists (Fig. 2)
constitute potential new drug candidates for diseases such as those
related to inflammation, prostate dysfunction, immune system
aDepartamento de Qu´ımica, Facultad de Farmacia, Universidad San Pablo
disorders, and depression.11
CEU, 28668-Boadilla del Monte, Madrid, Spain. E-mail: aramgon@ceu.es,
The low expression level of ERb in reproductive tissues such
as the uterus, suggests that a selective ERb agonist may maintain
the beneficial effects of estrogen, without the increased risk of
breast and endometrial cancer.12 Genistein is among the first
compounds detected showing a 20-fold greater affinity for ERb
but a number of more selective ligands have since been identified.13
ERb selective agonist ERB-041 (226-fold selective for b) has been
bpaster@ceu.es; Fax: +34 913510496; Tel: +34 913724782
bDepartamento de Metabolismo y Nutricio´n, Instituto del Fr´ıo, C.S.I.C, Jose´
Antonio Novais 10, 28040-Madrid, Spain
cKaro Bio AB, Ha¨lsova¨gen, Huddinge, Sweden
dThe Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins,
Baltimore, MD 21230, USA
eCentro Integral Oncolo´gico Clara Campal (CIOCC), On˜a 10, 28050-
Madrid, Spain
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used to demonstrate that this receptor may be a useful target
for certain inflammatory processes.14 Recently, genistein has been
reported to display strong inhibition in pancreatic cancer cell lines
at doses as low as 20 nM.15 Other estrogenic agents such as 2-
methoxyestradiol16 and estradiol (E2)17 have shown antitumour
benefits against pancreatic cancer in vivo.
Scheme 2. Aldehyde 6 was obtained in three steps from 6-hydroxy-
3-cumarone, by transformation into 6-methoxybenzofuran (14)
and subsequent formylation to give 6. Aldehyde 7 was obtained
by formylation of 6-methoxybenzothiophene, which was obtained
following a previously described method.18
Here we report the design, synthesis, evaluation of their affinity
for both receptors ERa and ERb, and antiproliferative activity of
new scaffolds for the synthesis of potential SERMs (compounds
1–4). These compounds contain a cyano group which allows the
introduction of the basic side chain present in most SERMs, as has
already been demonstrated for compound 24. A computational
study, that has allowed us to rationalize our results, is also
presented.
Results and discussion
Scheme 2 Synthesis of aldehydes 6 and 7. Reagents and conditions: (a) (i)
Me2SO4, 65% (ii) NaBH4, MeOH, 70%; (b) n-BuLi, DMF, THF, −78 ◦C,
61% for 6, 83% for 7.
Chemistry
All target molecules were prepared as depicted in Schemes 1–3.
The key step in all syntheses is a Knoevenagel type condensation
to give an a-cyanostilbene, followed by an efficient photochemical
six electron ring closure which afforded the tetracyclic frame-
work. The synthesis of 1 began with commercially available 1-
benzofuran-2-carbaldehyde (5), which was condensed with (3-
methoxyphenyl)acetonitrile using NaOEt as a base, to give 8.
Irradiation of 8 in the presence of I2 brought about the oxidative
photocyclization of the stilbene type system, to yield 11. The
reaction product precipitated in the photochemical reactor, and
the pure compound was isolated by filtration. Finally, deprotection
was carried out by heating a solution of 11 in DCM with a large
excess of BBr3 in a sealed tube for two days.
Taking into account that the ER has been reported to have some
˚
tolerance for the oxygen–oxygen distance (12.2 A in genistein,
13
˚
10.9 A in estradiol) and that this distance in 2 and 3 is around
˚
˚
10.6 A, compound 4 (12.9 A) was designed in order to increase
and explore the influence of the oxygen–oxygen distance which,
together with the enhanced flexibility of the system, could lead to
better interactions with key amino acids in the ligand binding do-
main of ERb and, therefore, to an improvement of the affinity for
this receptor. Other authors have shown that isoxazole 18 (Fig. 3),
˚
with an oxygen–oxygen distance of 13.4 A has an improved affinity
toward ERb (IC50 = 1.4 nM) compared to the less bulky analogue
19 (oxygen–oxygen distance of 10.5 A, IC50 = 54 nM).19
˚
Fig. 3 Aryl benzisoxazoles as ER b-ligands.
Docking studies for 4 (shown below) suggested that this
compound could present ERbselectivity. Scheme 3shows the route
used to prepare naphthothiophene 4, beginning with commercially
available 5-bromothiophene-2-carbaldehyde. Suzuki coupling led
to 15, and a three-step sequence, analogue to the one used for
the synthesis of compounds 1–3 (Knoevenagel type condensa-
tion, photochemical cyclization and deprotection), provided com-
pound 4.
Our approach to obtaining compounds with potential SERM
activity relied on the introduction of an aromatic nucleus with a
leaving group at the para position, appropriate for a nucleophilic
aromatic substitution (SNAr), as depicted in Scheme 4. This strat-
egy has been used before for the synthesis of raloxifene.20 Thus,
compound 20 was synthesized in 71% yield by reacting 13 with 4-
fluorophenylmagnesium bromide, under standard conditions. At
this stage, a change from methoxy to benzyloxy protective group
became necessary, in order to avoid chain rupture in the last
deprotection step. Treatment of 20 with BBr3 to give 21, followed
by reaction of 21 with benzyl bromide in the presence of K2CO3,
Scheme
1 Synthesis of ligands 1–3. Reagents and conditions: (a)
(3-methoxyphenyl)acetonitrile, NaOEt–EtOH, 62% for 8, 80% for 9, 73%
for 10; b) hm, I2, 65% for 11, 82% for 12, 84% for 13; (c) BBr3, DCM, 99%
for 1, 86% for 2, 60% for 3.
Compounds 2 and 3 were prepared following the same pro-
cedure. The corresponding aldehydes used as starting material
are not commercially available, and were prepared as outlined in
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hydrogenation gave the desired compound 24. This methodology
will allow high yield access to a broad series of SERMs based in
our new scaffolds (1–4), and a SAR study for this type of systems.
Binding affinities and agonist/antagonist characterization towards
ERa and ERb
The ER binding affinity of compounds 1–4 was determined in a
scintillation proximity assay (SPA) using human estrogen receptor
expressed in E. coli (hERa-LBD or hERb-LBD), while their ag-
onist/antagonist character was determined using a commercially
available EnBio estrogen receptor (a and b)/coactivator ligand
assay. Since this assay contains a coactivator peptide (SRC1)
which joins the reaction followed by receptor–ligand binding, both
agonists and antagonists for ERa and ERb can be detected.
Table 1 shows a summary of the results obtained in both assays.
The most interesting result corresponds to benzonaphthofuran
2, which presented reasonably good affinity for ERb (IC50
=
Scheme 3 Synthesis of ligand 4. Reagents and conditions: (a) K2CO3 2 M,
PdCl2(PPh3)2, 51%; (b) (3-methoxyphenyl)acetonitrile, NaOEt–EtOH,
88%; c) I2, hm, EtOH, 57%; (d) BBr3, DCM, 52%.
0.2066 lM), and behaved as an ERb selective agonist in the
ER/coactivator ligand binding assay (EC50 = 0.1 lM). Interest-
ingly, this compound acts through ERa as a weak antagonist
with IC50 = 9.9 lM. Thus, compound 2 appears to be more
potent as an ERb agonist than as an ERa antagonist, which
may be explained by its preferential binding affinity toward ERb.
Although this ligand shows modest affinities for both receptors,
such a ERb-agonist/ERa-antagonist behaviour represents one of
the most wanted profiles sought in SERMs research.21 While b-
agonism has been demonstrated to lead to chemopreventive and
some other beneficial estrogenic effects, the a-antagonism may
avoid undesirable estrogen effects on breast and uterus mediated
by ERa-subtype stimulation.
The affinity toward both receptors decreased in benzonaph-
thothiophene 3, where the furan ring present in 2 is substituted
by a thiophene. Compound 3 clearly showed a reduced ERb
agonism (EC50 = 26 lM) relative to that displayed by 2 in the
scintillation proximity assay. Compound 1 was designed in order
to evaluate the effect that the removal of the benzofuran hydroxyl
group had on the binding affinity. The complete lack of activity
observed for 1, both in the scintillation proximity assay and
in the ER/coactivator ligand binding assay, suggests that the
benzofuran hydroxyl group in 2 makes the major contribution
to ligand binding affinity. Thus, it may be involved in the hydrogen
bonding network between ERb residues Glu305 and Arg346
(ERa residues Glu353 and Arg394), and a highly ordered water
molecule, mimicking the hydroxyl group present in the A-ring of
estradiol. Finally, naphthothiophene 4, where the oxygen–oxygen
distance was increased in order to favour the affinity toward
ERb receptor, did not show the ERb selectivity predicted by
the molecular modelling studies, but it showed a 3-fold selective
affinity toward ERa, and a weak antagonistic activity toward the
same receptor.
Scheme 4 Synthesis of 24. Reagents and conditions: (a) 4-fluorophenyl-
magnesium bromide, THF reflux for 24 h, 71%; (b) BBr3, DCM, 95%;
(c) benzyl bromide, K2CO3, EtOH, 33%; (d) 2-(piperidin-1-yl)ethanol–
NaH, DMF, 57%; (e) H2, Pd/C (10%).
In vitro antiproliferative activity
Recently, it has been shown that in vitro pancreatic cell prolifer-
ation is highly estrogen sensitive.15 ERs are frequently expressed
in them, and ERb expression usually outweighs ERa expression.
Therefore, antiproliferative activity of compounds showing the
highest affinity for ERb (2 and 3) were tested against two
gave 22, which was reacted with 2-dimethylamino-1-ethoxide to
bring about the substitution of the fluoro atom by the nucleophile,
affording 23 (Scheme 4). Finally, deprotection of 23 by catalytic
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Table 1 Agonist/antagonist characterization and binding affinity for compounds 1–4
Coactivator ligand assay (lM)
Scintillation proximity assay (lM)
ERa
ERb
c
c
c
c
Comp.
Agon.a EC50
Antag.b IC50
Agon.a EC50
Antag.b IC50
ERa IC50
ERb IC50
1
2
3
4
Weak
—
—
Weak
9.9
6.5
Weak
0.1
26.1
—
Weak
—
—
Weak
Weak
0.7206
1.0283
0.5443
0.2066
0.2935
1.5606
—
12.5
—
a Agon.: agonist. b Antag.: antagonist. c The effective concentration (EC50) and inhibitory concentration (IC50) for each compound were calculated from
the concentration of sample at which the absorbance was diminished to 50%. Estradiol and bisphenol A were used as controls.
pancreatic cancer cell lines, L36PL and Panc-1. Compounds were
tested at concentrations ranging from 0.1 to 10 lM and the results
are summarized in Fig. 4 and compared with the activity of
gemcitabine. Interestingly, compounds 2 and 3 exhibited a cell
growth inhibition in both cell lines, which was dose-dependent in
the case of L36PL. They showed higher activity than gemcitabine,
which was used as control. Therefore, compounds 2 and 3 have
been selected to carry out in vivo assays and further studies on
their mechanism of action.
the basis of its wide reported use.22 From the Protein Data Bank23
we selected PDB codes 1A52, 1ERR (ERa LBD in complex with
estradiol and the antagonist raloxifene, respectively), and 1L2J
(complex between ERb LBD and the antagonist THC [(R,R)-
5,11-cis-diethyl-5,6,11,12-tetrahydro-chrysene-2,8-diol]). We first
validated AutoDock as an appropriate predictive tool by testing
its ability to predict the binding modes present in the crystal
structures of 1A52, 1ERR and 1L2J, for estradiol, raloxifene and
THC, respectively. Evaluation of the docked structures indicated
that AutoDock was able to predict the crystallographic binding
poses (data not shown), so we considered it as a valid tool to
perform the docking studies.
Estradiol
Predicted binding orientations for estradiol were studied carefully
for comparison purposes. Docking results of estradiol in 1A52
provided different solutions within an energy range of −19.9 to
−16.9 kcal mol−1. AutoDock was able to reproduce the same
key interactions observed in the crystallographic structure: the
hydrogen bonds between OH-3 (A ring) and the Glu353-Arg394-
HOH triad, and between OH-17 (D ring) and His524 (Fig. 5).
With this H-bonding pattern, two poses were predicted: one
with the b-face oriented toward Leu525 (like the crystallographic
orientation), and a second one, with the b-face pointing toward
Met421. We also carried out the docking of estradiol within the
ERaLBD from 1ERR (antagonist-like conformation). AutoDock
did not predict any solution. On the contrary, the docking of
estradiol within the ERb from 1L2J, gave rise to several poses
inside the binding site. Two of them were equivalent to that
predicted for estradiol in the ERa (binding energy of −21.5 to
Fig. 4 Relative growth inhibition of compounds 2 and 3 compared to
gemcitabine against L36PL (above) and Panc-1 (below) pancreatic cancer
cell lines at concentrations of 0.1, 1 and 10 lM.
Molecular modelling
In order to analyze the binding mode of this family of compounds
to the ER receptors, a docking study was undertaken for com-
pounds 1–4, employing the ligand binding domain (LBD) of both
ERaand ERb, and the automated docking program AutoDock on
Fig. 5 Schematic representation of the binding mode of estradiol inside
the ERa LBD.
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−18.0 kcal mol−1), with the b-face oriented toward Leu476 (ERa
Leu525), or with the b-face oriented toward Ile373 (ERaMet421).
both hydroxyl groups, the Glu353-Arg394-HOH triad and His524.
Ring D is placed close to Met421, in a similar way to one of the
orientations obtained from the docking of estradiol.
Compound 1. For the three X-ray structures of ER considered,
several solutions were identified showing a great heterogeneity on
the different orientations obtained from the docking (1A52: −10.5
to −7.7 kcal mol−1; 1ERR: unique solution, −9.1 kcal mol−1; 1L2J:
−13.0 to −10.1 kcal mol−1). These orientations corresponded to
several combinations of interactions, including the interaction
of the OH group with ERa Glu353-Arg394-HOH triad (ERb
Glu305-Arg346-HOH); with ERa His524 (ERb His475) or with
ERa Met343 (ERb Met295). The cyano group is involved in
interactions with ERa His524 (ERb His475) or with ERa Thr347
(ERb Thr299), among others. None of the different binding
orientations seemed to be preferred. These results show that
the benzo[b]naphtho[1,2-d]furan-5-carbonitrile scaffold is capable
of “horizontal and vertical flipping”, as it may bind in two
orientations that differ by 180◦ in each axis. The lack of one of the
two hydroxyl groups, which are well known to be essential for ER
binding, allows a high degree of mobility inside the binding site.
The other non estradiol-like orientations were higher in energy
(−11.0 to −9.4 kcal mol−1) and involved interactions between
the cyano group and Met421, or between the hydroxyl group at
position 3 and Met421. Several poses corresponded to orienta-
tions perpendicular to the estradiol plane, involving interactions
between the OH-9 group and Asp351 (helix-12) and Lys529
(helix-18). This result is in agreement with the weak antagonistic
character detected for this compound, taking into account that
the antiestrogenic character of tamoxifen has been explained by
forcing helix-12 out of position through the interaction of the side
chain with Asp351.24
ERb: Remarkably, predicted docking energies for ERb were
within the narrow range of −12.4 to −12.0 kcal mol−1, and 95 out
of 100 solutions were placed inside the binding site, oriented in four
different poses. Two predicted orientations contained 84 docking
solutions (blue and white, Fig. 6B. and Fig. 7), and corresponded
to orientations equivalent to that for estradiol, in which the
hydroxyl groups interact with Glu305-Arg346-HOH triad and
His475. For the most populated orientation (68 solutions out of
100), an additional interaction is found between the cyano group
and Thr299. It has been reported that hydrogen bonding through
Thr299 (ERa Thr347) rather than His475 (ERa His524) can be an
alternative-binding mode within the ER binding site.25 This may
represent a third anchorage point inside the ER LBD, suggesting
that the cyano group could be modulating the interaction between
the rest of the ligand and the receptor. Additionally, ring D nicely
occupies the cavity between Met336 (ERa Leu384) and Ile373
(ERa Met421), establishing favourable contacts, not observed
for the docking in ERa. Interestingly, it has been proposed that
enhanced selectivity for ERb can be achieved by designing ligands
able to bind differently to ERb Ile373 than to ERa Met421. This
interaction has been reported for some ligands derived from the
2-phenylnaphthalene scaffold.13 Our docking studies show that 2
binds to ERb in a more efficient way than to ERa, as a result of
several subtle interactions that can account for the ERb selectivity
experimentally shown by this compound.
Compound 2. ERa: Predicted docking energies for ERa were
within the range of −12.4 to −9.4 kcal mol−1, and 39 out of 100
solutions were placed inside the binding site, with five different
orientations. One predicted orientation (blue in Fig. 6A ), contains
only three docking solutions with energy of −12.4 kcal mol−1 and
is equivalent to that for estradiol, involving interactions between
Fig. 6 Superimposition of the docking orientations obtained for com-
pound 2 in A) ERa LBD; B) ERb LBD. Estradiol is shown in CPK
colours as reference.
Fig. 7 Compound 2 docked poses into the binding site of ERb. Key
residues of the site are denoted by sticks.
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Compound 3. Energy range for solutions inside ERa LBD
was −12.3 to −11.1 kcal mol−1 (27 solutions out of 100), and
for ERb LBD was −12.3 to −11.9 kcal mol−1 (37 solutions out
of 100). Docking calculations did not predict any orientation
involving similar interactions to those for estradiol, i.e. interac-
tions in which both hydroxyl groups are involved in contacts
with ERa Glu353-Arg394-HOH triad, and with ERa His524,
simultaneously (Fig. 8). Only in ERb, a pattern of interactions
resembling the estradiol-like pattern could be identified (in 17 out
of 37 docking solutions): interactions between OH-3 and His475,
and between OH-9 and Arg346 (but not Glu305). With this pair
of contacts, two “flipped” orientations were identified: with the
cyano group oriented toward Leu394, and with the cyano group
oriented toward Lys315. Nevertheless, the lack of an appropriate
set of interactions could justify the weak ERb agonistic behavior
in comparison to 2.
corresponded to an estradiol-like orientation, with OH-7 pointing
toward His524, and OH-4ꢀ toward Glu353-Arg394-HOH triad.
The sulfur atom is bumping on Leu387 side chain. Docked
solutions within the ERb LBD pointed to a potential ERb
selectivity. The energy range was −11.5 to −9.5 kcal mol−1 (51
solutions out of 100), and four possible orientations could be
identified. One of these orientations (16 solutions) corresponded
to that for estradiol: the hydroxyl group at position 7 is oriented
toward the Glu305-Arg346-HOH triad, and OH-4ꢀ is oriented
toward His475. The cyano group is placed into the cavity delimited
by Leu343 and Leu380. An alternative orientation, and the
most populated one (32 solutions), involves interactions between
the OH-7 group and the sulfur atom of Met340, the OH-4ꢀ
group and the NH of the backbone of His475, and the stacking
interaction between the phenyl ring and the imidazole of His475.
Unexpectedly, naphthothiophene 4 only showed a very weak
antagonistic activity on ERa, in disagreement with the docking
prediction. In order to rationalize this result further computational
work, such as molecular dynamic simulations, becomes necessary.
Experimental
General methods
Melting points (uncorrected) were determined on a Stuart Scien-
tific SMP3 apparatus. Infrared (IR) spectra were recorded with a
Perkin-Elmer 1330 infrared spectrophotometer. 1H and 13C NMR
were recorded on a Bruker 300-AC instrument. Chemical shifts
(d) are expressed in parts per million; coupling constants (J)
are in Hertz. Mass spectra were run on a Bruker Esquire 3000
spectrometer. Elemental analyses (C, H, N) were performed on a
LECO CHNS-932 equipment at the Microanalyses Service of the
University Complutense of Madrid. Thin-layer chromatography
(TLC) was run on Merck silica gel 60 F-254 plates. Unless stated
otherwise, starting materials used were high-grade commercial
products. The photolyses were carried out in a quartz immersion
well apparatus with a Pyrex filter and a 400 W medium-pressure
Hg arc lamp.
6-Methoxy-1-benzofuran 14
To
a solution of 6-hydroxy-1-benzofuran-3(2H)-one (5 g,
33 mmol) in dry acetone (20 cm3) at room temperature were added
K2CO3 (5.9 g, 43 mmol) and Me2SO4 (2.49 g, 20 mmol). After
stirring for 2 h, the mixture was concentrated in vacuo, and diluted
with water. The aqueous layer was extracted with DCM, and the
combined organic layers were washed with brine, dried (MgSO4),
filtered, and concentrated to give 6-methoxy-1-benzofuran-3(2H)-
one (3.5 g, 65%) as a yellow solid, mp 102–103 ◦C; mmax (KBr)/cm−1
2220; dH(300 MHz, CDCl3) 3.85 (3 H, s, OMe), 4.60 (2 H, s, CH2),
6.51 (1 H, d, J 1.8, ArH), 6.62 (1 H, dd, J 8.6 and 1.8, ArH)
and 7.52 (1 H, d, J 9.2, ArH); dC(75.4 MHz, CDCl3) 55.80, 75.44,
96.18, 112.10, 114.20, 124.94, 168.09, 176.43 and 197.49.
To a solution of 6-methoxy-1-benzofuran-3(2H)-one (0.5 g,
3.05 mmol) in MeOH (10 cm3) was added NaBH4 (0.17 g,
4.6 mmol) in four successive portions at room temperature. After
stirring for 4 h, the crude reaction mixture was quenched by
the addition of acetone and treated with an aqueous 3 N HCl
solution for 1 h. The acetone and methanol were evaporated and
Fig. 8 Superimposition of the docking orientations obtained for com-
pound 3 in A) ERa LBD; B) ERb LBD. Estradiol is shown in CPK
colours as reference.
We also observed a repulsive interaction between the sulfur atom
and the carbonyl group of ERa Leu346, due to its proximity (red
in Fig. 8A.). However, in ERb, the sulfur atom is close to the Ca
˚
of Ala302 (3.68 A), and the equivalent carbonyl group (Leu298)
is far away, so there is no repulsive interaction (see Fig. 8B.). In
spite of the slight energy difference between ERa/ERb docking
solutions, this observation could explain the weak ERb selectivity
shown by 3.
Compound 4. Docking studies inside the ERa LBD pre-
dicted only four solutions inside the binding site. Two of them
were grouped into the same cluster (−10.9 kcal mol−1), and
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the aqueous solution was extracted with EtOAc. The combined
organic extracts were dried (MgSO4), filtered, and concentrated to
give 14 (0.305 g, 70%) as an oil; mmax (KBr)/cm−1 1610 and 2810;
dH(300 MHz, CDCl3) 3.87 (3 H, s, OMe), 6.73 (1 H, s, ArH), 6.93
(1 H, d, J 8.5, ArH), 7.09 (1 H, s, ArH), 7.49 (1 H, d, J 8.5, ArH)
and 7.57 (1 H, s, ArH); dC(75.4 MHz, CDCl3) 55.56, 95.76, 106.26,
111.83, 120.54, 121.10, 143.95, 155.84 and 157.88.
3-(1-Benzofuran-2-yl)-2-(3-methoxyphenyl)prop-2-enenitrile 8
To a solution of 3-methoxyphenylacetonitrile (3.02 g, 20 mmol)
and 1-benzofuran-2-carbaldehyde (3 g, 20 mmol) in absolute
EtOH (50 cm3) was added NaOEt (0.68 g, 10 mmol) and the
mixture was stirred at room temperature for 5 h. The precipitate
formed was collected by filtration to give 8 (3.78 g, 62%) as a
solid, mp 88–90 ◦C (Found: C, 78.49; H, 4.81; N, 5.14. C18H13NO2
requires C, 78.53; H, 4.76; N, 5.09%); mmax (KBr)/cm−1 2220;
dH(300 MHz, CDCl3) 3.87 (3 H, s, OMe), 6.95 (1 H, m, ArH), 7.21
6-Methoxy-1-benzofuran-2-carbaldehyde 6
=
(1 H, t, ArH), 7.27–7.50 (6 H, m, ArH, furan-H and HC C), 7.56
To a solution of 14 (2.26 g, 15 mmol) in dry THF (20 cm3), was
(1H, d, J 8.2, ArH) and 7.65 (1 H, d, J 7.7, ArH); dC(75.4 MHz,
CDCl3) 55.4, 110.7, 111.3, 111.6, 115.2, 117.4, 118.2, 122.0, 123.6,
126.8, 128.1, 128.3, 130.2, 134.8, 151.2, 155.2 and 160.1. EIMS
(m/z) 276 [M + H]+.
◦
added dropwise n-BuLi (11.25 cm3, 1.6 M in hexane) at −78 C
under argon. The mixture was stirred for 1 h and then DMF
(2.19 g, 30 mmol) was added. After stirring for 4 h at room
temperature, the mixture was quenched with an aqueous saturated
NH4Cl solution (15 cm3) and stirred at 0 ◦C for 0.5 h. The aqueous
phase was extracted with DCM (3 × 20 mL) and the combined
organic extracts were dried (MgSO4) and concentrated to dryness.
The residue was purified by flash column chromatography using
hexane–EtOAc (9 : 1) as eluent to give 6 (1.6 g, 61%) as brownish
solid, mp 78–80 ◦C; mmax (KBr)/cm−1 1655; dH(300 MHz, CDCl3)
3.69 (3 H, s, OMe), 6.79 (2 H, m, ArH), 7.35 (1 H, s, ArH), 7.42 (1
H, d, J 8.5, ArH) and 9.58 (1 H, s, CHO); dC(75.4 MHz, CDCl3)
55.20, 94.89, 114.32, 118.71, 119.50, 123.57, 151.95, 157.43, 161.29
and 178.28. EIMS (m/z) 199 [M + Na]+.
3-(6-Methoxy-1-benzofuran-2-yl)-2-(3-methoxyphenyl)prop-2-
enenitrile 9
A solution of 3-methoxyphenylacetonitrile (1.25 g, 8.5 mmol),
compound 6 (1.5 g, 8.5 mmol) and NaOEt (0,068 g, 1.0 mmol) in
absolute EtOH (30 cm3) was refluxed for 1 h and cooled to room
temperature to produce a precipitate of yellow crystals. The solid
was removed by filtration and an additional amount of NaOMe
(0.21 g, 3.1 mmol) was added to the filtrate. The mixture was
refluxed for 2 h and cooled to room temperature to precipitate
more crystals. The two crops were washed with a small amount of
EtOH and dried to give 9 (2.1 g, 80%) as a yellow solid, mp
99–101 ◦C (from hexane) (Found: C, 75.24; H, 4.97; N, 4.86.
C19H15NO3 requires C, 74.74; H, 4.95; N, 4.59%); mmax (KBr)/cm−1
2200; dH (300 MHz; CDCl3) 3.89 (3 H, s, OMe), 3.90 (3 H, s,
OMe), 6.91–6.96 (m, 2 H, ArH), 7.08 (1 H, d, J 1.8, ArH), 7.20 (1
H, t, ArH), 7.27–7.30 (1 H, m, ArH), 7.37, (1 H, t, J 7.9, ArH),
6-Methoxy-1-benzothiophene-2-carbaldehyde 7
The procedure described above was used for the synthesis of 7.
From 6-methoxy-1-benzothiophene (3.824 g, 423 mmol), n-BuLi
(14.4 cm3, 1.6 M in hexane) and DMF (3.36 g, 46 mmol), 7 was
obtained (3.69 g, 83%) as a white solid, mp 98–99 ◦C (from EtOH)
(Found: C, 62.71; H, 4.35; S, 16.76. C10H8OS requires C, 62.48;
H, 4.19; N, 16.68%); mmax (KBr)/cm−1 1652; dH(300 MHz, CDCl3)
3.86 (3 H, s, OMe), 7.02 (1 H, dd, J 8.8 and 2.2, ArH), 7.25 (1 H,
d, J 2.2, ArH), 7.76 (1 H, d, J 8.8, ArH), 7.87 (1 H, s, ArH) and
9.97 (1 H, s, CHO); dH(300 MHz, CDCl3) 55.30, 104.23, 116.06,
126.82, 132.20, 134.57, 140.58, 144.68, 160.04 and 183.91; EIMS
(m/z) 193 [M + H]+.
=
7.39 and 7.43 (2 H, 2 s, ArH and CH C) and 7.51 (1 H, d, J 9.2,
ArH). dC (75.4 MHz; CDCl3) 55.42, 55.74, 95.29, 108.58, 111.18,
111.99, 113.86, 114.83, 117.78, 118.12, 121.47, 122.28, 128.16,
130.13, 135.15, 150.52, 156.72, 160.08 and 160.11; EIMS (m/z)
328 [M + Na]+.
3-(6-Methoxy-1-benzothiophen-2-yl)-2-(3-methoxyphenyl)prop-2-
enenitrile 10
5-(4-Methoxyphenyl)thiophene-2-carbaldehyde 15
The procedure described above was used for the synthesis of 10.
From 3-methoxyphenylacetonitrile (2.8 g, 19 mmol), 7 (3.7 g,
19 mmol) and NaOEt (0.32 g, 4.6 mmol◦), 10 (4.427 g, 73%) was
obtained as a yellow solid, mp 149–150 C (Found: C, 70.79; H,
4.76; N, 4.59; S, 9.90. C19H15NO2S requires C, 71.00; H, 4.70;
N, 4.36; S, 9.98%); mmax (KBr)/cm−1 2220; dH(300 MHz, CDCl3)
3.86 (3 H, s, OMe), 3.88 (3 H, s, OMe), 6.91 (1 H, dd, J 8.2,
and 2.2, ArH), 7.00 (1 H, dd, J 8.8 and 2.2, ArH), 7.15 (1 H,
m, ArH), 7.22–7.37 (3 H, m, ArH) and 7.67–7.69 (3 H, m, ArH);
dC(75.4 MHz, CDCl3) 55.35, 55.55, 104.17, 108.47, 111.18, 114.54,
115.69, 118.07, 118.12, 125.36, 130.05, 130.15, 132.47, 135.14,
135.24, 143.14, 159.06 and 159.97; EIMS (m/z) 344 [M + Na]+.
To a solution of 5-bromothiophene-2-carbaldehyde (3.0 g,
16 mmol), 4-methoxyphenylboronic acid (3.65 g, 24 mmol)
and bis(triphenylphosphine)palladium(II) dichloride (5% mol,
0.8 mmol) in THF (50 cm3) was added a 2 M solution of K2CO3
(25 mL, 50 mmol) and the mixture was stirred at 40 ◦C for 3.5 h.
Diethyl ether (30 cm3) was added and the solution was washed
with an aqueous 0.5 N NaOH solution (2 × 20 cm3), water and
brine, and then dried (MgSO4), filtered and evaporated to dryness.
The residue was purified by flash column chromatography using
hexane–EtOAc (9 : 1) as eluent to give 15 (1.78 g, 51%) as an
orange solid, mp 121–122 ◦C (Found: C, 65.97; H, 4.82; S, 14.52.
C2H10O2S requires C, 66.03; H, 4.62; S, 14.69%); mmax (KBr)/cm−1
1650; dH(300 MHz, CDCl3) 3.86 (3 H, s, OMe), 6.95 (2 H, AAꢀXXꢀ,
ArH), 7.30 (2 H, d, J 3.8, thiophene-H), 7.62 (2 H, AAꢀXXꢀ, ArH),
7.71 (1 H, d, J 3.8 Hz, thiophene-H) and 9.86 (1 H, s, CHO);
dC(75.4 MHz, CDCl3) 55.3, 114.4, 122.9, 125.6, 127.7, 137.7, 141.3,
154.4, 160.6 and 182.6; EIMS (m/z) 219 [M + H]+.
2-(3-Methoxyphenyl)-3-[5-(4-methoxyphenyl)thiophen-2-yl]prop-
2-enenitrile 16
To a solution of 3-methoxyphenylacetonitrile (0.5 g, 3.4 mmol) and
5-(4-methoxyphenyl)thiophene-2-carbaldehyde (0.74 g, 3.4 mmol)
3492 | Org. Biomol. Chem., 2008, 6, 3486–3496
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in absolute EtOH (20 cm3) was added NaOEt (0.09 g, 1.7 mmol)
and the mixture was stirred at room temperature for 5 h. The
precipitate formed was collected by filtration to give 16 (1.04 g,
88%) as an orange solid, mp 132–133 ◦C (Found: C, 72.50; H,
5.01; N, 4.11; S, 9.39. C21H17NO2S requires C, 72.60; H, 4.93; N,
4.03; S, 9.23%); mmax (KBr)/cm−1 2220; dH(300 MHz, CDCl3) 3.85
(3 H, s, OMe), 3.87 (3 H, s, OMe), 6.88–6.91 (1 H, m, ArH), 6.94
(2 H, AAꢀXXꢀ, ArH), 7.16 (1 H, t, J 2.2, ArH), 7.22–7.26 (2 H,
m, ArH and thiophene-H), 7.34 (1 H, t, ArH), 7.55 (1 H, d, J 3.8,
orange solid, mp 216–217 ◦C (Found: C, 72.91; H, 4.49; N, 4.24;
S, 9.22. C21H15NO2S requires C, 73.02; H, 4.38; N, 4.06; S, 9.28%);
mmax (KBr)/cm−1 2220; dH (300 MHz, CDCl3) 3.89 (3 H, s, OMe),
4.01 (3 H, s, OMe), 7.01 (2 H, AAꢀXXꢀ, ArH), 7.34 (1 H, dd, J
8.8 and 1.7, ArH), 7.58 (1 H, d, J 1.7, ArH), 7.72 (2 H, AAꢀXXꢀ,
ArH), 7.98 (1 H, s, thiophene-H), 8.21 (1 H, s, ArH) and 8.26 (1
H, d, J 8.8, ArH); dC (75.4 MHz; CDCl3) 55.4, 55.5, 104.7, 114.5,
116.1, 118.6, 119.7, 123.4, 125.7, 126.1, 127.9, 127.9, 128.1, 131.2,
132.4, 140.8, 149.5, 158.8 and 160.4; EIMS (m/z) 346 [M + H]+.
ꢀ
ꢀ
=
thiophene-H), 7.61 (1H, s, HC C) and 7.62 (2 H, AA XX , ArH).
dC(75.4 MHz, CDCl3) 55.3, 106.4, 111.0, 114.2, 114.4, 117.9, 118.3,
122.4, 126.0, 127.4, 130.0, 134.5, 134.5, 135.4, 136.0, 149.2, 160.0
and 160.0. EIMS (m/z) 348 [M + H]+.
3-Hydroxybenzo[b]naphtho[1,2-d]furan-5-carbonitrile 1
To a solution of 11 (0.20 g, 0.73 mmol) in dry DCM (20 cm3),
was added BBr3 (14 cm3, 1 M in DCM, 14 mmol) at 0 ◦C. The
mixture was stirred in a sealed tube at 70 ◦C for 48 h. After cooling
to room temperature, the crude reaction mixture was quenched
carefully with ice, water and 1 N HCl. The aqueous layer was
extracted with AcOEt (3 × 50 cm3) and the combined organic
extracts were washed with saturated aqueous NaHCO3 and brine,
dried (MgSO4) and concentrated to dryness to give 1 (0.11 g, 99%)
as a solid, which was recrystallized from n-butanol, mp > 300 ◦C
(Found: C, 78.22; H, 3.76; N, 5.51. C17H9NO2 requires C, 78.76; H,
3.50; N, 5.40%); mmax (KBr)/cm−1 2220; dH (300 MHz, DMSO) 7.46
(1 H, dd, J 8.5 and 2.4, ArH), 7.53–7.60 (2 H, m, ArH), 7.67 (1 H,
t, ArH), 7.88 (1 H, d, J 7.9, ArH), 8.64 (1 H, s, ArH), 8.67 (1 H, d,
J 7.3, ArH) and 8.79 (1 H, d, J 8.5, ArH); dC (75.4 MHz; DMSO)
105.9, 107.4, 112.3, 118.0, 119.6, 119.7, 121.0, 121.7, 122.2, 122.8,
123.2, 124.1, 126.4, 128.6, 131.0, 149.8, 156.3 and 156.7; EIMS
(m/z) 260 [M + H]+.
3-Methoxybenzo[b]naphtho[1,2-d]furan-5-carbonitrile 11
A solution of 8 (0.6 g, 1.96 mmol) and I2 (0.5 g, 1.96 mmol)
in absolute EtOH (350 mL) was irradiated for 4 h. The solid
formed was isolated by filtration to give 11 (0.39 g, 65%) as a
◦
white solid, mp 202–204 C (Found: C, 79.15; H, 4.20; N, 5.22.
C18H11NO2 requires C, 79.11; H, 4.06; N, 5.13%); mmax (KBr)/cm−1
2220; dH(300 MHz, CDCl3) 4.04 (3 H, s, OMe), 7.46–7.74 (5 H, m,
ArH), 8.15 (1 H, s, ArH), 8.39 (1 H, br d, J 8.8, ArH) and 8.58 (1
H, d, J 9.4, ArH); dC(75.4 MHz, CDCl3) 55.5, 104.7, 107.1, 112.3,
118.7, 118.7, 121.0, 122.6, 122.6, 123.4, 123.7, 125.3, 128.1, 131.0,
150.5, 156.9 and 158.2. EIMS (m/z) 273 [M]+.
3,9-Dimethoxybenzo[b]naphtho[1,2-d]furan-5-carbonitrile 12
A solution of 9 (0.30 g, 0.98 mmol) and I2 (0.25 g, 0.98 mmol)
in absolute EtOH (350 cm3) was irradiated for 4 h in a quartz
immersion well apparatus with a Pyrex filter and a 400 W medium-
pressure Hg arc lamp. The solid formed was isolated by filtration
3,9-Dihydroxybenzo[b]naphtho[1,2-d]furan-5-carbonitrile 2
The procedure described above was used for the synthesis of 2.
From 12 (0.16 g, 0.53 mmol), and BBr3 (10.5 cm3, 1M in DCM,
10.5 mmol), and after work up, the residue was purified by column
chromatography using AcOEt : MeOH (20 : 1) as eluent to afford
2 (0.12 g, 86%) as a solid, mp > 300 ◦C (Found: C, 73.68; H,
3.45; N, 5.28. C17H9NO3 requires C, 74.18; H, 3.30; N, 5.09%);
mmax (KBr)/cm−1 2220; dH(300 MHz, DMSO) 7.01 (1 H, dd, J 8.5
and 2.4, ArH), 7.16 (1 H, d, J 2.4, ArH), 7.41 (1 H, dd, J 9.2 and
2.4 Hz, ArH), 7.56 (1 H, d, J 2.5, ArH), 8.43 (1 H, d, J 8.5 Hz,
ArH), 8.52 (1 H, s, ArH), 8.68 (1 H, d, J 9.2 ArH) and 10.38
(2 H, br s, 2 OH); dC(75.4 MHz, DMSO) 98.29, 103.40, 107.21,
113.55, 114.79, 118.44, 119.25, 120.70, 121.19, 123.14, 123.88,
126.55, 131.34, 149.40, 156.63, 158.34 and 159.04; EIMS (m/z)
274 [M − H]+.
◦
to give 12 (0.247 g, 82%) as a solid, mp 262–263 C (Found: C,
74.89; H, 4.28; N, 4.83. C19H13NO3 requires C, 75.24; H, 4.32; N,
4.62%); mmax (KBr)/cm−1 2200; dH (300 MHz, CDCl3) 3.96 (3 H, s,
OMe), 4.04 (3 H, s, OMe), 7.10 (1 H, dd, J 8.8 and 2.2, ArH), 7.20
(1 H, d, J 2.2, ArH), 7.46 (1 H, dd, J 8.8 and 2.7, ArH), 7.67 (1 H.
d, J 2.7, ArH), 8.12 (1 H, s, ArH), 8.24 (1 H, d, J 8.8, ArH) and
8.52 (1 H, d, J 8.8, ArH); EIMS (m/z) 304 [M + H+].
3,9-Dimethoxybenzo[b]naphtho[1,2-d]thiophene-5-carbonitrile 13
A solution of 10 (0.3 g, 0.94 mmol) and I2 (0.24 g, 0.94 mmol)
in absolute EtOH (350 cm3) was irradiated for 8 h. The solid
formed was isolated by filtration to give 13 (0.2 g, 84%) as a white
solid, mp 226–227 ◦C (Found: C, 71.14; H, 4.27; N, 4.81; S, 10.04.
C19H13NO2S requires C, 71.45; H, 4.10; N, 4.39; S, 10.04%); mmax
(KBr)/cm−1 2200; dH (300 MHz, CDCl3) 3.98 (3 H, s, OMe), 4.05
(3 H, s, OMe), 7.23 (1 H, dd, J 9.2 and 2.4, ArH), 7.43–7.47 (2
H, m, ArH), 7.70 (1 H, d, J 2.4, ArH), 8.28 (1 H, s, ArH), 8.70 (1
H, d, J 9.2, ArH) and 8.90 (1 H, d, J 9.2, ArH); EIMS (m/z) 342
[M + Na]+.
3,9-Dihydroxybenzo[b]naphtho[1,2-d]thiophene-5-carbonitrile 3
The procedure described above was used for the synthesis of 3.
From 13 (0.13 g, 0.41 mmol) and BBr3 (6.12 cm3, 1 M in DCM,
6.12 mmol), and after work-up, the residue was purified by flash
column chromatography using AcOEt : hexane (7 : 3) as eluent
to give 3 (0.071 g, 60%) as a solid, mp > 300 ◦C (Found: C,
69.08; H, 3.52; N, 4.71; S, 10.48. C17H9NO2S·1/3H2O requires C,
68.67; H, 3.28; N, 4.71; S, 10.78%); mmax (KBr)/cm−1 2220, 3250
and 3400; dH(300 MHz, DMSO) 7.12 (1 H, dd, J 9.2 and 2.4,
ArH), 7.40 (1 H, dd, J 9.2 and 2.5, ArH), 7.49 (1 H, d, J 2.4,
ArH), 7.53 (1 H, d, J 2.5, ArH), 8.70 (1 H, s, ArH), 8.80 (1 H,
d, J 9.2, ArH), 8.99 (1 H, d, J 9.2, ArH), 10.30 (1 H, s, OH) and
7-Methoxy-2-(4-methoxyphenyl)naphtho[2,1-b]thiophene-5-
carbonitrile 17
A solution of 16 (0.3 g, 0.867 mmol) and I2 (0.2 g, 0.867 mmol)
in absolute EtOH (350 cm3) was irradiated for 15 h. The solid
formed was isolated by filtration to give 17 (0.17 g, 57%) as an
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10.43 (1 H, s, OH); dC(75.4 MHz; DMSO) 104.49, 107.73, 108.49,
115.56, 118.37, 120.25, 122.85, 126.25, 127.19, 128.99, 129.22,
131.65, 132.10, 133.26, 143.29, 156.62 and 157.26; EIMS (m/z)
290 [M − H]+.
refluxed for 24 h. The reaction was then diluted with AcOEt,
washed with water and brine, dried (MgSO4) and evaporated to
dryness. The residue was purified by flash column chromatography
using hexane : AcOEt (9 : 1) as eluent to give 22 (0.06 g, 33%) as a
yellow oil; dH (300 MHz, CDCl3) 5.11 (2 H, s, CH2), 5.20 (2 H, s,
CH2), 7.14–7.53 (15 H, m, ArH), 7.80 (1 H, s, ArH), 7.94 (3 H, m,
ArH), 8.70 (1 H, d, J 9.2, ArH) and 8.92 (1 H, d, J 9.3 ArH).
7-Hydroxy-2-(4-hydroxyphenyl)naphtho[2,1-b]thiophene-5-
carbonitrile 4
The procedure described above was used for the synthesis of 4.
From 17 (0.145 g, 0.42 mmol) and BBr3 (8.43 cm3, 1 M in DCM,
18.43 mmol), 4 (0.069 g, 52%) was obtained as a solid, mp >
300 ◦C; mmax (KBr)/cm−1 1690 and 3400; dH(300 MHz, acetone-d6)
6.90 (2 H, AAꢀXXꢀ, ArH), 7.30 (1 H, dd, J 9.2 and 2.4, ArH),
7.45 (1 H, d, J 2.4, ArH), 7.74 (2 H, AAꢀXXꢀ, ArH), 8.51 (1 H, s,
thiophene-H), 8.54 (1 H, d, J 9.2, ArH) and 8.63 (1 H, s, ArH),
10.00 (1 H, s OH) and 10.35 (1 H, s OH); dC (75.4 MHz; acetone-
d6) 102.9, 106.5, 115.0, 115.3, 115.4, 117.2, 118.32, 121.9, 124.0,
125.7, 127.0, 127.2, 127.3, 130.6, 130.7, 140.4, 149.1, 156.2 and
157.8; EIMS (m/z) 316 [M − H]+.
(3,9-Dibenzyloxybenzo[b]naphtho[1,2]thien-5-yl)(4-[2-(piperidin-1-
yl)ethoxy]phenyl)methanone 23
To a solution of 2-(piperidin-1-yl)ethanol (0.26 g, 2 mmol) in DMF
(5 ml) was added NaH (0.05 g, 2 mmol) and the mixture was stirred
at RT for 30 minutes. Then 22 (0.06 g, 0.1 mmol) was added and the
stirring was continued for 6 h. To the solution was added water,
and the aqueous layer was extracted with EtOAc, washed with
brine, dried (MgSO4) and evaporated. The residue was purified by
flash column chromatography using AcOEt as eluent to give 23
(0.04 g, 57%) as a yellow oil; dH (300 MHz, CDCl3) 1.47 (2 H,
m, CH2-piperidine), 1.64 (4 H, m, 2CH2-piperidine), 2.52 (4 H,
m, 2CH2-piperidine), 2.82 (2 H, t, J 5.9, CH2N), 4.20 (2 H, t, J
5.9, CH2O), 5.08 (2 H, s, CH2Ph), 5.20 (2H, s, CH2Ph), 6.97 (2
H, d, J 8.8, ArH), 7.37–7.52 (13 H, m, ArH), 7.75 (1 H, d, J 2.4,
ArH), 7.90 (1 H, d, J 8.8, ArH), 7.94 (1 H, s, ArH), 8.71 (1H, d, J
9.3, ArH), 8.92 (1H, d, J 9.8, ArH); dC (75.4 MHz; CDCl3) 24.08,
25.86, 55.03, 57.65, 66.27, 69.89, 70.29, 107.03, 107.45, 114.28,
114.87, 119.39, 123.31, 124.77, 125.68, 125.85, 127.52, 127.72,
127.97, 128.14, 128.50, 128.65, 128.74, 129.87, 131.07, 131.35,
132.77, 133.34, 133.83, 136.44, 136.49, 142.65, 156.30, 157.32,
163.06 and 196.49; EIMS (m/z) 678 [M + H]+.
(3,9-Dimethoxybenzo[b]naphtho[1,2-d]thien-5-yl)(4-
fluorophenyl)methanone 20
To a solution of 13 (0.23 g, 0.72 mmol) in THF (10 cm3) was
added a solution of 4-fluorophenylmagnesium bromide (14.5 ml,
14.5 mmol, 1.0 M in THF) at RT, and the mixture was refluxed for
24 h. After cooling, HCl 3 N (20 mL) was added to the crude,
and a red solid, which was characterized as the imine of 20,
precipitated. The solid was isolated by filtration, and after adding
HCl 3 N (125 cm3), the suspension was refluxed for 48 h. The new
yellow precipitate formed was extracted with DCM (3 × 20 cm3)
and the organic extracts were washed with brine, dried (MgSO4),
and evaporated to give 20 (0.21 g, 71%), mp 179–180 ◦C; mmax
(KBr)/cm−1 1640; dH (300 MHz, CDCl3) 3.87 (3 H, s, OCH3), 3.96
(3 H, s, OCH3), 7.16–7.23 (3 H, m, ArH), 7.40 (1 H, dd, J 9.3 and
2.5, ArH), 7.45 (1 H, d, J 2.5, ArH), 7.71 (1 H, d, J 2.9, ArH),
7.93–7.98 (3 H, m, ArH), 8.70 (1 H, d, J 9.3, ArH) and 8.92 (1
H, d, J 9.8, ArH); dC (75.4 MHz; CDCl3) 55.11, 55.43, 105.65,
106.03, 114.31, 115,45, 115.74, 118.96, 124.33, 124.70, 125.49,
125.81, 129.37, 131.04, 131.97, 132.41, 134.93, 142.88, 157.22,
158.20, 163.99, 167.38 and 196.11.
(3,9-Dihydroxybenzo[b]naphtho[1,2]thien-5-yl)(4-[2-(piperidin-1-
yl)ethoxy]phenyl)methanone 24
To a solution of 23 (40 mg, 0.06 mmol) in EtOH (4 cm3) was
added Pd/C 10% (30 mg) and the mixture was introduced into a
Parr apparatus under an initial pressure of 20 psi. After 2 h the
catalyst was removed and the solvent evaporated. The residue was
purified by flash column chromatography using AcOEt : MeOH
(9 : 1) as eluent to give 24 (13 mg, 46%) as a yellow solid; mmax
(KBr)/cm−1 1640, 3300–3600; dH (300 MHz, CD3OD) 1.65 (2 H,
m, CH2-piperidine), 1.81 (4 H, m, 2CH2-piperidine), 2.84 (4 H, m,
2CH2-piperidine), 3.07 (2 H, t, J 4.8, CH2N), 4.34 (2 H, t, J 4.8,
CH2O), 7.13 (2 H, d, J 7.9, ArH), 7.28 (1 H, d, ArH), 7.46–7.53 (3
H, m, ArH), 7.94–7.96 (3 H, m, ArH), 8.84 (1 H, d, J 9.2, ArH),
9.07 (1H, d, J 9.2, ArH); EIMS (m/z) 498 [M + H]+.
(3,9-Dibenzyloxybenzo[b]naphtho[1,2-d]thien-5-yl)(4-
fluorophenyl)methanone 22
The procedure described above for 1–4 was used for the synthesis
of 21. From 20 (0.13 g, 0.31 mmol), and BBr3 (6.2 cm3, 1 M in
DCM, 6.2 mmol), and after work up, a solid was obtained. Then,
HCl 3 N (125 mL) was added and the mixture was refluxed for 48 h.
After cooling, the mixture was extracted with AcOEt (3 × 50 cm3)
and the combined organic extracts were washed with NaHCO3
and brine, dried (MgSO4) and concentrated to dryness to afford
21 (0.114 g, 95%) as a brown solid, which was used in the next step
without further purification; dH (300 MHz, acetone-d6) 7.22 (1 H,
d, J 8.6, ArH), 7.32 (2 H, m, ArH), 7.42 (1 H, d, J 9.2 Hz, ArH),
7.54 (1 H, s, ArH), 7.58 (1 H, s, ArH), 7.98 (2 H, m, ArH), 8.05
(1 H, s, ArH), 8.83 (1 H, d, J 8.6 Hz, ArH) and 9.05 (1 H, d, J
9.2 Hz, ArH).
Computational methods
Geometries of compounds 1, 2, 3, 4, estradiol, raloxifene, and
THC were first optimized using the ab initio quantum chemistry
program Gaussian 9823 and the B3LYP/3-21G* basis set. Partial
atomic charges were then obtained using the RESP24 methodology
with the 6-31G* basis set. Different conformers of the ligands
were docked using the Lamarckian genetic algorithm (LGA)
implemented in AutoDock, by randomly changing the torsion
angles and overall orientation of the molecule. A volume for
exploration was defined in the shape of a three-dimensional grid
3
3
˚
˚
A solution of 21 (0.12 g, 0.31 mmol), K2CO3 (0.43 g, 3.1 mmol)
(80 × 80 × 90 A for 1A52; 60 × 70 × 95 A for 1ERR; 60 ×
and benzyl bromide (0.4 ml, 3.1 mmol) in EtOH (3 cm3) was
90 × 60 A for 1L2J) with a spacing of 0.375 A that enclosed
3
˚
˚
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the binding site, and included the residues that are known to be
crucial for activity. Missing atoms inside the grids were added
and crystallographic water close to Glu-Arg was kept for the
docking calculations. At each grid point, the receptor’s atomic
affinity potentials for carbon, aromatic carbon, oxygen, nitrogen,
sulfur, and hydrogen atoms were precalculated for rapid intra-
and intermolecular energy evaluation of the docking solutions for
each ligand. The original Lennard–Jones and hydrogen-bonding
potentials provided by the program were used. The parameters
for the docking using the LGA were identical for all docking jobs.
After docking, the 100 solutions were clustered in groups with root
In vitro cytotoxicity assays
Two pancreas tumoural cell lines, Panc-1 and L36PL were used
in this study. In vitro drug sensibility was assessed by the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye
conversion assay. Briefly, cells were trypsinized, seeded at 5 ×
103 per well in 96-well plate, and allowed to grow for 24 h
before treatment with exponential increasing concentrations of
drugs (Gemcitabine, compounds 2 and 3) in the presence 0.5%
fetal bovine serum. After a 96 h period of treatment, 0.02 cm3
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
solution (5 mg cm−3 in PBS; Sigma) were added to each well and the
plates were then incubated for 3 h at 37 ◦C. The medium was then
replaced with 0.1 cm3 DMSO per well. Plates were shaken and the
absorbance was measured at 570 nm using a multiwell plate reader
(Model 550, Bio-Rad, Inc., Hercules, CA). Each experiment was
done in triplicate for each drug concentration and carried out at
least thrice.
˚
mean square deviations less than 1.0 A. The clusters were ranked
by the lowest energy representative of each cluster.
IC50 assessment on hERa-LBD and hERb-LBD with scintillation
proximity assay (SPA)
All dilutions except the compound dilution series were made in
pH 8 assay buffer (1 mM EDTA, 18 mM K2HPO4, 2 mM KH2PO4,
20 mM Na2MoO4, 1mM TCEP). Extract of human estrogen
receptor expressed in E. ◦coli (hERa-LBD or hERb-LBD) was
thawed on ice from −70 C and mixed with streptavidin coated
SPA beads from Perkin Elmer (RPNQ00007). The amount of
receptor used will give an assay signal of approximately 300 ccpm.
Compounds were stored in 10 mM DMSO stock solutions at
−30 ◦C and thawed to room temperature prior to use. The
compounds were diluted in DMSO to 12 concentrations and
0.018 cm3 of each dilution was added in duplicates to a 384 well
assay plate with clear bottom (Corning 3706). The final assay
Conclusion
A set of ER ligands based on novel tetracyclic scaffolds have been
synthesized through an efficient photochemical approach. The
affinity toward both ERa and ERb receptors has been evaluated,
and they have been characterized as agonists or antagonists. The
most interesting result has been found for compound 2, which
behaves as an ERbagonist (EC50 = 0.1 lM) and an ERaantagonist
(IC50 = 9.9 lM), and presents 3.5-fold higher affinity toward ERb.
This result has been rationalized, based on molecular modelling
studies, by an additional interaction between the cyano group
present in 2 and ERb Thr299, which is not present in the complex
formed by 2 with ERa. Further optimization of this scaffold as
ERb-selective agonist is now underway.
All synthesized compounds are candidates to be transformed
into ER antagonists by introduction of an appropriate basic side
chain through the cyano group present in the molecule. In fact, a
basic side chain has been introduced in compound 3 via a five step
synthetic pathway, demonstrating that these systems are scaffolds
suitable for the construction of new potential SERMs.
concentration of tracer was 1.2
0.08 nM and the compound
concentrations ranged from 37 pM to 157 lM in a total volume
of 0.088 cm3. The plates were incubated on a shaker overnight at
room temperature, centrifuged (2000 rpm, 5 min) and measured
with top and bottom detectors on a 12 detector Trilux Microbeta
from Perkin Elmer. A four parameter logistic fit (4PL) was used to
analyze the data with XLfit software from IDBS (Guildford, UK)
in Microsoft Excel.
Agonist/antagonist characterization towards ERa and ERb
Compounds 2 and 3 shown an interesting antitumour profile
towards two pancreatic cancer cell lines and have been selected to
carry out in vivo assays. Interestingly, compound 2 behaves as a
ERb-agonist/ERa-antagonist.
The estrogen receptor a and b competitive binding assays were
conducted using an estrogen receptor (aand b)/coactivator ligand
assay kit (EnBioTec Laboratories, Japan), in accordance with
protocol. In brief, the mixture of recombinant human estrogen
receptor a or b and samples (compounds 1 to 4, estradiol or
bisfenol A) were added to the wells of a plate coated with
biotinylated coactivator peptide, and they were allowed to react
at room temperature for 1 h. After washing the wells with wash
solution, the anti-ER-HRP solution was added to each well. The
absorbance at 450 nm was measured with a microplate reader
(Power Wave XS, Biotek) after incubation with the chromogen.
For antagonist assay the mixture added to the wells of the
plate coated with biotinylated coactivator peptide contained the
samples together with a known concentration of estradiol and
the recombinant human estrogen receptor a or b. The effective
concentration (EC50) and inhibitory concentration (IC50) for each
compound were calculated from the concentration of sample at
which the absorbance was diminished to 50%. Estradiol and
bisphenol A were used as controls.
Acknowledgements
This work has been supported by grants from the Spanish
MEC (SAF2005–02608 and AGL2006–05453) and Fundacio´n
Universitaria San Pablo CEU (USP-PC 15/06 and USP-PC
11/07). S.M.-S. thanks the Spanish MEC for a Ramo´n y Cajal
contract. A grant to J.-J. R from Fundacio´n Universitaria San
Pablo CEU is also acknowledged.
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