Synthesis, structure, and estrogenic activity of 2- and 3-substituted 2,3-dihydro-4H-1-benzopyran-4-ones

Article abstract of DOI:10.1007/s00044-012-0058-2

Molecules with potent estrogenic activity are ~270 A³ hydrophobic structures that encompass two hydroxyls among which is at least one phenol. However, compounds with only one phenol or devoid of such a ring have been shown to enhance ERα-mediat

Full text of DOI:10.1007/s00044-012-0058-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:681–691
DOI 10.1007/s00044-012-0058-2
ORIGINAL RESEARCH
Synthesis, structure, and estrogenic activity of 2- and 3-substituted
2,3-dihydro-4H-1-benzopyran-4-ones
•
•
•
Yves Jacquot Cillian Byrne Alain Xicluna
Guy Leclercq
Received: 26 December 2011 / Accepted: 4 April 2012 / Published online: 25 April 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Molecules with potent estrogenic activity are
3
Introduction
˚
*270 A hydrophobic structures that encompass two
hydroxyls among which is at least one phenol. However,
compounds with only one phenol or devoid of such a ring
have been shown to enhance ERa-mediated transcription at
concentrations much larger than those measured with E2. In
this context, we show here that benzopyrans sharing one
hydroxyl/methoxyl and containing an additional benzy-
lidenyl or a spirocyclohexyl motif are able to induce ERE-
dependent transcription in breast carcinoma cells.
Most non-steroidal compounds with potent estrogenic
3
˚
activity are *270 A hydrophobic molecules flanked by
two hydroxyls (of which at least one is phenol) playing the
role of corresponding functions in 17b-estradiol (E2).
These hydroxyls are commonly considered as a part of the
pharmacophore for the onset of an estrogenic activity
(Wurtz et al., 1998). However, recent studies have revealed
that they are not absolutely required: chlorinated pesticides
(such as DDT, chlordecone, and methoxychlor), organo-
phosphorus compounds (such as parathion), or pyrethroids
Keywords Benzylidenylbenzopyranones ꢀ
Spirocyclohexylbenzopyranones ꢀ Estrogenic activity ꢀ
ERa binding affinity
´ ´
(Lorand et al., 2010) also give estrogenic responses.
In an attempt to produce new heterocyclic estrogens
(Jacquot et al., 2001; Gust et al., 2001; Gust et al., 2002;
Jacquot et al., 2002; von Rauch et al., 2005) and to improve
reported structure/activity relationships (SAR), we synthe-
sized two series of benzopyrans sharing structural and phys-
icochemical similarities with apigenin (flavonoid) and
genistein (isoflavonoid), two phytoestrogens (Fig. 1), i.e.,
3-benzylidenyl-2,3-dihydro-4H-1-benzopyran-4-ones (homoi-
Y. Jacquot ꢀ A. Xicluna
´
Laboratoire de Chimie Organique et Therapeutique, Faculte de
Medecine et de Pharmacie, Universite de Franche-Comte, Place
´
´
´
´
´
soflavonoid derivatives) (Farkas et al., 1971; Levai and
Saint-Jacques, 25030 Besanc¸on cedex, France
´
´
Schag, 1979; Levai et al., 1981; Al Nakib et al., 1990; Fisera
et al., 1997; Hoshino et al., 1998; Subramaniyan et al., 2002;
Koorbanally et al., 2006; Biju et al., 2010) and 2-spi-
rocyclohexyl-2,3-dihydro-4H-1-benzopyran-4-ones (Dike
et al., 1991). The ability of some of these compounds to
enhance ERa-mediated transcription confirmed the impor-
tance not only of one hydroxy- but also of methoxy-con-
taining phenyl groups. Molecular structures established by
Fourier-transformed infrared (FTIR) and homo/heteronuclear
magnetic resonance (NMR) spectroscopy, X-ray crystallog-
raphy, and high-resolution mass spectrometry (HRMS), as
well as theoretical calculations, suggest that these active
compounds may localize within the ligand-binding pocket of
Y. Jacquot (&) ꢀ C. Byrne
´
Departement de Chimie, Laboratoire des BioMolecules, CNRS-
UMR 7203, Ecole Normale Superieure/Universite Pierre et
´
´
´
´
Marie Curie, Ecole Normale Superieure, 24 rue Lhomond, 75231
Paris Cedex 05, France
e-mail: yves.jacquot@upmc.fr
Y. Jacquot ꢀ C. Byrne ꢀ G. Leclercq
Fondation Pierre-Gilles de Gennes pour la Recherche,
29, rue d’Ulm, 75005 Paris, France
G. Leclercq
Laboratoire J.-C. Heuson de Cancerologie Mammaire,
´
Universite Libre de Bruxelles, Institut Jules Bordet,
´
1 rue Heger-Bordet, 1000 Brussels, Belgium
´
123

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Med Chem Res (2013) 22:681–691
OH
O
OH
O
OH
C
D
C
B
B
C
O
OH
A
B
A
A
O
OH
HO
HO
HO
Apigenin
Genistein
E2
Fig. 1 Chemical structure of 17b-estradiol (E2), genistein (isoflavone), and apigenin (flavone)
the receptor even though no interaction could be detected by
classical [3H]E2 competitive binding assay.
being for all compounds of the same intensity as that
corresponding to the C=O (frequency between
m = 1651 cm-1 and m = 1668 cm-1) (inset of Scheme 1a)
(Hassner and Mead, 1964). Even if NMR and FTIR data
suggest strongly that the synthesized 3-benzylidenyl-2,3-
dihydro-4H-1-benzopyran-4-ones were isolated under the
cisoid form I, some doubts may subsist between the latter
and the thermodynamic-driven transoid form III. To dis-
criminate between these two conformers, the structure of
3e has been resolved by X-ray diffraction. Unambiguously,
the X-ray structure reveals that the cisoid form I was the
only isolated form (Fig. 2), confirming definitively the
NMR and FTIR results as expected.
Chemistry
3-benzylidenyl-2,3-dihydro-4H-1-benzopyran-4-ones
A series of planar 3-benzylidenyl-2,3-dihydro-4H-1-ben-
zopyran-4-ones (molecules 3a–f) mimicking non-steroidal
estrogens (such as genistein and apigenin) were synthe-
sized following the reaction of Knoevenagel by condensing
the substituted benzaldehydes 2a–f on the chroman-4-one 1
in solvent-free medium and in the presence of a few drops
Spiro[cylohexyl-2,40-(2,3-dihydro-4H-1-benzopyran-4-
ones)]
´
´
of piperidine (Scheme 1a) (Levai and Schag, 1979). The
pure expected products were obtained from direct crystal-
lization on cooling at room temperature. During the course
of the reaction, the temperature was carefully kept at
110 °C to avoid exocyclic (cisoid forms I or/and II) to
endocyclic (transoid form III) isomerization (Scheme 1b),
which occurs at *150 °C and which can be accompanied
by a rearrangement into 3-methylflavone (Mulvagh et al.,
Substituted spiro[cylohexyl-2,40-(2,3-dihydro-4H-1-benzo-
pyran-4-ones)] 6a–d were obtained in one-step following
the Kabbe’s method (Kabbe, 1978; Bergmann and Gericke,
1990). Briefly, the cyclohexanones 5a–c were condensed
on the substituted acetophenones 4a or 4b (Bergmann and
Gericke, 1990; Panteleon et al., 2008) in refluxing toluene
and in the presence of pyrrolidine (Scheme 2). The
assembly was equipped with a Dean–Stark apparatus to
eliminate the molecules of water resulting from the con-
densation reaction. The products were purified by flash
column chromatography with the appropriate eluant. It
should be noted that the cyclohexyl motif was structurally
defined under the chair conformer, allowing therefore the
substituent group in the position 13 of the crystal structure
to orientate equatorially, as shown by the X-ray of com-
pound 6c (Fig. 3).
´
1979; Levai et al., 1981). In this regard, it is noteworthy
that the absence of electron acceptor substituents on the
benzaldehyde motif would favor the formation of the exo
(cisoid) forms I or II (Subramaniyan et al., 2002). The
cisoid isomer II resulting principally from a photoisomer-
ization process of the former (Hassner and Mead, 1964;
´
Bennett et al. 1972; Mulvagh et al., 1979; Levai et al.,
´
1981; Levai et al., 1999), the expected form I, would be
exclusively observed.
To confirm the presence of the cisoid form I, we carried
out a complete structural study on 3e. The ROESY NMR
spectrum revealed a correlation between the CH2 motif in
position 2 and the aromatic proton in the ortho position of
the benzylidenyl phenyl ring (inset of Scheme 1a). Like-
wise, no coupling was recorded between this CH2 and the
alkenic CH in position 9, excluding therefore the cisoid
form II. FTIR data were also in accordance with the cisoid
form I, the band corresponding to the C=C double bond
Results and discussion
Transcription assays
The effect of 3-benzylidenylchroman-4-ones 3a–f on ERa-
mediated transcription was assayed on MVLN breast
(frequency between m = 1590 cm-1 and m = 1610 cm-1
)
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Med Chem Res (2013) 22:681–691
683
(a)
O
R1
O
O
R1
R2
R3
R2
R3
piperidine
110°C, 1h
O
+
O
R5
R5
R4
R4
1
2a : R1 = H, R2 = H, R3 = H, R4 = H, R5 = H
3a : R1 = H, R2 = H, R3 = H, R4 = H, R5 = H
3b : R1 = H, R2 = H, R3 = OH, R4 = H, R5 = H
3c : R1 = H, R2 = H, R3 = CH3, R4 = H, R5 = H
3d : R1 = H, R2 = H, R3= N(CH3)2, R4 = H, R5 = H
3e : R1 = H, R2 = OCH3, R3 = H, R4 = H, R5 = H
2b : R1 = H, R2 = H, R3 = OH, R4 = H, R5 = H
2c : R1 = H, R2 = H, R3 = CH3, R4 = H, R5 = H
2d : R1 = H, R2 = H, R3= N(CH3)2, R4 = H, R5 = H
2e : R1 = H, R2 = OCH3, R3 = H, R4 = H, R5 = H
2f : R1 = OCH3, R2 = H, R3 = OCH3, R4 = H, R5 = OCH3
3f : R1 = OCH3, R2 = H, R3 = OCH3, R4 = H, R5 = OCH3
(b)
R3
R3
R2
R1
R4
R5
R2
R4
R5
R1
O
O
R1
O
R2
R3
O
R5
O
O
Form I
Form II
R4
Transoid (Form III)
Cisoid
Scheme 1 a Synthesis of 3-benzylidenyl-2,3-dihydro-4H-1-benzo-
pyran-4-ones 3a–f. Inset infrared spectrum (left) and NMR coupling
observed by using the sequence ROESY (right) of the compound 3e.
Inset a coupling between the protons in position 2 and the proton in
the ortho position of the benzylidene ring is relevant to the cisoid
form I (or eventually the transoid form III). The intensity of the two
bands at m = 1,662 cm-1 and m = 1,664 cm-1 recorded using
infrared being identical, the cisoid form I is likely (Hassner and
Mead, 1964). b Structure of the two cisoid (isomers I and II) and the
transoid forms
cancer cells (MCF-7 cells stably transfected with a plasmid
containing an estrogen-response element (ERE) triggering
the luciferase gene (Pons et al., 1990)). At 10-6 M, a
concentration usually tested for endocrine disrupting
chemicals (see for example (Miksicek, 1994)), 3b and 3e
increased luciferase activity with an almost equivalent
potency (236 and 243 %, respectively; control: 100 %,
Fig. 4a) while 3a was totally ineffective. The acceptor
character of the OCH3 and OH oxygen carried by the
benzylidenyl motif of these molecules seemed important
for transcription suggesting some SAR connection with the
role of the 17b and 3 OH action of E2 (or corresponding
position in non-steroidal estrogens) in terms of interaction
with ERa (Jacquot et al., 2003) even the efficiency of the
hormone is at least 1,000 times higher (E2 operates at
10-10 M). Thus, the presence of three methoxy groups on
the benzylidenyl motif (compound 3f: 134 %) appears
deleterious probably due to steric and electronic reasons. In
the same context, a low activity was observed with the
N,N-dimethylamino group (3d: 145 %).
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Med Chem Res (2013) 22:681–691
Fig. 2 ORTEP drawing of the
3-(30-methoxybenzylidenyl-2,3-
dihydro-4H-1-benzopyran-4-
ones 3e
O
O
4
CH3
pyrrolidine
O
R2
+
7
2
toluene, reflux, 3hours
R2
R1
OH
R1
O
1
11
6a R1=OCH3, R2 = H
6b R1=OH, R2 = H
6c R1=OCH3, R2 = -C(CH3)3
5a R2 = H
5b R2 = -C(CH3)3
5c R2 = C6H5
4a : R1 = OCH3
4b : R1 = OH
6d R1 OCH , R = C H
=
3
2
6 5
Scheme 2 Synthesis of the 2-spirocyclohexylbenzopyran-4-ones 6a–d
With regard to the 2-spirocyclohexylchromanones 6a–d,
6b at 10-6 M solely increased luciferase activity (279 %,
Fig. 4b). Hence, the donor character of the hydroxyl in
position 7 of the benzopyran core structure seemed
essential as the corresponding methoxylated analogue 6a
was devoid of activity.
2004), or synthetic peptides corresponding to a regulatory
motif of ERa (Gallo et al., 2007).
Binding assays
All investigated compounds failed to compete with [3H]E2
for ERa binding (assays carried out with a highly purified
recombinant human ERa (Maaroufi and Leclercq, 1994))
raising the question regarding the mechanism by which 3b,
3e, and 6b may enhance transcription. The implication of
ERb may be excluded because MVLN cells, as MCF7 cells
from which they derive do not express significant amounts
of this receptor (Kuiper et al., 1998). Although an indirect
activation of ERa through an interaction with a target
contributing to a cross-talk mechanism could not be ruled
out, a participation in living cells of factors that may
enhance the potency of these compounds to associate with
the receptor (coregulators) (Gee et al., 1999) could be
advocated, as proposed for other non-steroidal estrogens
similarly unable to displace [3H]E2 in binding assays (Gust
et al., 2001; Gust et al., 2002; Jacquot et al., 2002; von
Transcriptional activities of the most active compounds
3b, 3e, and 6b were assayed at a tenfold higher concen-
tration (10-5 M) to optimize their efficiency (Fig. 5).
Under such conditions, luciferase expression reached val-
ues slightly lower than those measured with E2 at 10-10 M.
Interestingly, the antiestrogen fulvestrant (ICI) totally
abrogated the capacity of these three compounds to
enhance transcription, confirming unambiguously that they
contribute to the acquisition by ERa of an active confor-
mation. This property does not necessarily imply an
insertion procedure within the ligand-binding pocket of
ERa since a similar antiestrogen-induced suppression of
transcription enhancement occurs with activating mole-
cules operating at other receptor sites or associated proteins
(coregulators), such as cyclic nucleotides (Aronica and
Katzenellenbogen, 1991), bisphosphonates (Journe et al.,
¨
Rauch et al., 2004; Jacquot et al., 2007; Laıos et al., 2007).
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Med Chem Res (2013) 22:681–691
685
Fig. 3 ORTEP drawing of
spiro[tert-butylcyclohexyl-2,40-
(2,3-dihydro-7-methoxy-4H-1-
benzopyran-4-one)] (6c)
In fact, transcription induced by a compound usually
occurs at lower concentrations than expected from its
efficiency to compete with [3HE2] (RBA value), indicating
that biological tests performed on living cells surpass
conventional ligand binding assays in terms of sensitivity.
Potential insertion within the E2-binding pocket
Interactions between residues of the ligand-binding pocket
of ERa and hydroxyls/methoxyls of weak non-steroidal
estrogens without significant binding affinity for the
receptor have been reported (Gust et al., 2001; Jacquot
et al., 2001; Jacquot et al., 2002; Gust et al., 2002; von
Rauch et al., 2005). Hence, the question of whether the
ability of 3b, 3e, and 6b to enhance transcription is or not
related to such a capacity is of interest.
Similarities between our compounds and genistein as well
as apigenin guided our analysis. Indeed, we previously
reported that these two phytoestrogens enhance ERa-medi-
ated transcription with an efficiency closely related to their
binding affinity for ERa (RBA: genistein = 0.5–1.0, api-
genin = 0.1–0.2; E2 = 100) (Seo et al., 2006). According to
X-Ray crystallographic data, this property results from an
ability of these molecules to dock within the E2-stabilized
conformation of the ligand-binding pocket. Our active
compounds being unable to compete with E2 for ERa
binding, we failed to use such a docking approach because
we felt that results might be subjected to controversy. We
preferred to restrict our analysis to a comparison of the
structural and physicochemical properties of our compounds
Fig. 4 Effect of compounds on ERa-mediated transcription. Assays
were carried out in MVLN breast cancer cells in the presence of E2 at
10-10 M, ICI (fulvestrant) at 10-7 M or investigated compounds at
10-6 M (24 h incubation). Values refer to the increase of basal
luciferase expression (reporter gene) (n = 4, SD)
123

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Med Chem Res (2013) 22:681–691
3
of active compounds is especially low, i.e., 224.84 A for
˚
350
300
250
200
150
100
50
3
3b, 242.34 A for 3e, and 215.16 A for 6b (references: E2:
3
˚
˚
3
3
˚
˚
268.74 A , genistein, and apigenin: 270.24 A ). The pla-
narity of 3b and 3e conjugated with some structural anal-
ogies with genistein and apigenin suggests similar
interactions with residues of the binding pocket (Manas
et al., 2004). Indeed, the exocyclic localization of the
double bond may restrain the rotation of the benzylidenyl
aromatic ring in the cisoid form I (Scheme 1b), maintain-
ing its orientation in an appropriate direction for such
interactions.
0
control
E2
ICI
3b
3e
6b 3b + ICI 3e + ICI 6b + ICI
Fig. 5 Effect of active compounds on ERa-mediated transcription in
the presence of ICI (fulvestrant). Assays were carried out in MVLN
breast cancer cells in the presence of E2 at 10-10 M, ICI (fulvestrant)
at 10-7 M or investigated compounds at 10-5 M in the presence or
absence of ICI (fulvestrant) at 10-7 M (24 h incubation). Values refer
to the increase of basal luciferase expression (reporter gene) (n = 3,
SD)
LogP values (3.14 for 3b, 3.65 for 3e, and 3.07 for 6b),
which reflect lipophilicity, are in the same range as E2
(3.43). Thus, not only molecular volumes but also lipo-
philicity of active compounds are relevant to a possible
insertion within the ligand-binding pocket.
Despite the absence of polar groups at both extremities,
3e shares a polar surface within the range calculated for our
reference molecules. Important polar surface area was also
recorded for 6b. Hence, the presence of polar groups, and
more specifically of donor/acceptor OCH3 or OH groups
might be crucial for an interaction with either Arg-353/
Glu-394 or His-524, known to associate with the 3 and 17b
hydroxyls of E2, respectively. Hence, even if purely
hypothetical, the methoxylated/hydroxylated benzylidenyl
in position 3 of the 3-benzylidenylchroman-4ones (3b and
3e) or in the position 7 of the benzopyran moiety of the
2-spirocyclohexylbenzopyran-4-ones (6b) may mimic, at
least partially, the phenolic hydroxyls of genistein and
apigenin to interact with these important anchoring deter-
minants of the E2-binding pocket.
Table 1 Physicochemical parameter values (Molecular volumes,
logP, and polar surface area) of the synthesized derivatives 3a–f and
6a–d (references: E2, genistein, and apigenin)
3
Volume (A )
2
Polar surface area (A )
˚
˚
Compounds
LogP
E2
268.74
270.24
270.24
216.80
224.84
233.36
262.70
242.34
293.43
233.19
215.16
299.40
304.62
3.43
2.27
2.46
3.62
3.14
4.07
3.72
3.65
3.47
3.61
3.07
4.71
4.70
40.46
90.90
90.90
26.31
46.53
26.30
29.54
35.54
54.01
35.54
46.53
35.54
35.54
Genistein
Apigenin
3a
3b
3c
3d
3e
3f
Although our structural analysis provides pertinent data
to explain the biological activity of 3b, 3e, and 6b, the lack
of competition between these compounds and [3H]E2 for
recombinant ERa binding still raises questions about their
mechanism of action. Even though an activating metabo-
lism giving rise to a hydroxylation/O-demethylation of 3b,
3e, and 6b suitable for significant ERa binding may occur
in cell culture, association with another receptor site such
as its coactivator-binding groove (Moore et al., 2010;
Leclercq et al., 2011) could not be excluded. Likewise, the
specific conformational modifications induced by specific
ERa partners, the activity of which could be modulated by
these compounds, is another alternative hypothesis.
6a
6b
6c
6d
Molecular volumes and Polar surface area (PSA) and are expressed in
3
A and A , respectively
2
˚
˚
with those of genistein, apigenin, and E2, taken as reference
ligands.
Remarkably, the calculated physicochemical properties
of compounds able to enhance transcription (3b, 3e, and
6b) were found to be similar to those observed for our
reference ligands (Table 1). Thus, their possible insertion
within the E2-binding pocket appears conceivable.
Indeed, all tested compounds are characterized by rea-
sonable smaller molecular volumes than E2, genistein, and
apigenin, suggesting that potency to enhance transcription
may result, in terms of steric hindrance, from possible
interactions within the ligand-binding pocket, except for 3f,
6c, and 6d, which were inactive, and for which a higher
value was calculated (Table 1). In this regard, the volume
Conclusion
In summary, we have synthesized phenol- or methoxy-
phenyl-containing benzopyrans which activate ERa-medi-
ated transcription. While interactions of these compounds
with residues of the ligand-binding pocket of the receptor
directly implicated in the onset of estrogenic responses
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Med Chem Res (2013) 22:681–691
687
seems likely, their lack of competition with [3H]E2 for ERa
binding raises questions upon the mechanism by which
they operate. Thus, and as already shown for other non-
steroidal estrogens with similar ERa-binding properties,
the capacity to interact with the receptor and to increase its
ability to enhance transcription do not seem to be strictly
relevant to identical chemical features. While the presence
of one phenol or one methoxyphenyl may fail to confer a
significant binding affinity for the receptor, such a function
may be largely sufficient to induce a weak ERE-dependent
transcription giving rise to an estrogenic response.
Hence, investigations are obviously required to decrypt
the mechanism of action of non-steroidal estrogens unable
to compete with [3H]E2 for ERa binding, such as the ones
described here. In this regard, extensive structural (NMR
and X-ray crystallography) or FRET studies may be fruitful
in opening new avenues for pharmacomodulation since the
estrogen pharmacophore appears more complex than usu-
ally thought.
1 eq.) were heated to 110 °C for 1 h in solvent-free med-
ium in the presence of 10 drops of piperidine. On cooling
to room temperature, the product crystallized and was fil-
tered under vacuum, and rinsed with diethylether.
3-Benzylidenyl-2,3-dihydro-4H-1-benzopyran-4-one (3a)
Prepared by condensing the benzaldehyde 2a on the
chroman-4-one 1. White powder. 53 % yield.
mp = 111 °C. FTIR (KBr) (m cm-1): 3057, 2856, 1668,
1601, 1493, 1475, 1450, 1016; 1H-NMR (CDCl3) d (ppm) :
5.36 (2H, d, 4J = 2.2 Hz, CH2); 6.94 (1H, d, 3J =
3
10.0 Hz, Ar–H); 7.08 (1H, m, J = 8.9 Hz, Ar–H); 7.32
3
(2H, m, J = 9.2 Hz, Ar–H); 7.41–7.53 (4H, m, Ar–H);
7.89 (1H, s, CH); 8.01–8.05 (1H, dd, 3J = 9.5 Hz,
4J = 2.2 Hz, Ar–H). 13C-NMR (DMSO-d6) d (ppm):
67.33, 117.91, 121.99, 127.25, 128.79, 129.69, 130.28,
130.77, 133.76, 136.25, 136.54, 160.63, 181.18. MS (m/z):
259 [MNa?] (calc: [MH?] = 236.08); Anal calcd for
C16H12O2: C, 81.36, H, 5.08. Found: C, 80.87, H, 5.15.
3-(40-Hydroxybenzylidenyl)-2,3-dihydro-4H-1-benzopyran-
4-one (3b) Prepared by condensing the 4-hydroxybenz-
aldehyde 2b on the chroman-4-one 1. Gray powder. 83 %
yield. mp = 213 °C. FTIR (KBr) (m cm-1): 3163, 3063,
2947, 2866, 1651, 1593, 1508, 1450, 1018; 1H-NMR
(DMSO-d6) d (ppm) : 5.41 (2H, s, CH2); 6.83–6.89 (3H, m,
Ar–H); 7.04-7.16 (2H, m, 3J = 10.0 Hz, Ar–H); 7.27–7.33
(1H, t, 3J = 9.2 Hz, Ar–H); 7.56–7.67 (2H, m, CH, Ar–H);
7.87–7.91 (1H, dd, 3J = 9.6 Hz, 4J = 2.2 Hz, Ar–H); 9.72
(1H, s, OH). 13C-NMR (DMSO-d6) d (ppm): 67.36, 116.70,
117.88, 121.08, 121.47, 121.92, 127.24, 129.83, 130.58,
134.96, 136.15, 157.51, 160.61, 181.16. MS (m/z): 275
[MNa?] (calc: [MH?] = 252.08); Anal calcd for
C16H12O3: C, 76.19, H, 4.76. Found: C, 76.34, H, 4.69.
Experimental protocols
Chemistry
All chemical products were purchased from Aldrich Inc.
(Saint-Quentin Fallavier, France). The synthesized com-
pounds were characterized by elemental analyses, FTIR
spectroscopy, 1H- and 13C-NMR spectroscopy, and HRMS.
Infrared spectra were recorded from potassium bromide
pellets on a Shimadzu FTIR-8201 PC spectrometer (m in
cm-1). 1H-NMR and 13C-NMR spectra were recorded on a
Bruker AC 300 spectrometer. Samples were dissolved in
DMSO-d6 or CDCl3. All measurements were performed at
293 K, and chemical shift values were referenced to tetra-
methylsilane (CEA Saclay, France) as internal standard. 2D
NMR experiments heteronuclear multiple bond correlation,
HMQC (Heteronuclear Multiple Quantum Coherence),
HSQC (Heteronuclear Single Quantum Coherence), and
ROESY (Rotating frame Overhause Effect SpectroscopY)
were recorded from a Bruker AC 500 apparatus equipped
with a Cryo Platform (Bruker). High-resolution mass spectra
were acquired using a hybrid linear ion trap LTQ-Orbitrap
(Thermo Fisher Scientific, Les Ulis, France). Melting points
were determined on a Bu¨chi N°510 apparatus. Microanal-
yses were carried out by the Service de Microanalyses of the
University Pierre et Marie Curie in Paris.
3-(40-Methylbenzylidenyl)-2,3-dihydro-4H-1-benzopyran-
4-one (3c) Prepared by condensing the 4-methylbenzal-
dehyde 2b on the chroman-4-one 1. White powder. 40 %
yield. mp = 115 °C. FTIR (KBr) (m cm-1): 3067, 2934,
1
2843, 1665, 1607, 1512, 1462, 1011; H-NMR (CDCl3) d
(ppm) : 2.42 (3H, s, CH3); 5.38 (2H, d, 4J = 1.7 Hz, CH2);
6.98 (1H, m, 3J = 8.3 Hz, Ar–H); 7.08–7.11 (1H, m,
3J = 7.29 Hz, Ar–H); 7.24 (4H, m, Ar–H); 7.50 (1H, m,
3
Ar–H); 7.88 (1H, s, CH); 8.03–8.06 (1H, dd, J = 7.7 Hz,
4J = 1.6 Hz, Ar–H). 13C-NMR (DMSO-d6) d (ppm): 20.99,
67.41, 117.87, 121.49, 121.94, 127.22, 129.42, 129.94,
130.42, 130.99, 136.15, 136.59, 139.76, 160.58, 181.13. MS
(m/z): 273 [MNa?] (calc: [MH?] = 250.10); Anal calcd for
C17H14O2: C, 81.60, H, 5.60. Found: C, 81.35, H, 5.72.
Substituted 3-benzylidenyl-2,3-dihydro-4H-1-benzopyran-
4-ones (3a–f)
3-(40-N,N-Dimethylaminobenzylidenyl)-2,3-dihydro-4H-1-
benzopyran-4-one (3d) Prepared by condensing the 4-
N,N-dimethylaminobenzaldehyde 2d on the chroman-4-
2,3-dihydro-4H-1-benzopyran-4-one (chroman-4-one 1,
5 mmol) and substituted benzaldehyde 2a–f (5 mmol,
123

688
Med Chem Res (2013) 22:681–691
one 1. White powder. 26 % yield. mp = 154 °C. FTIR
the presence of 5 mL pyrrolidine and in refluxing toluene
during 3 h. The assembly was equipped with a Dean–Stark
trap to remove water resulting from the reaction. Toluene
was evaporated under vacuum. Then, the crude product
was purified by column chromatography (ethyl acetate/
cyclohexane: 3/7) to afford the final product which crys-
tallized in \48 h.
1
(KBr) (m cm-1): 3000, 2914, 1659, 1607, 1477, 1066; H-
NMR (CDCl3) d (ppm) : 3.08 (6H, s, CH3); 5.45 (2H, s,
CH2); 6.74 (2H, d, 3J = 8.8 Hz, Ar–H); 6.97 (1H, d,
3J = 8.3 Hz, Ar–H); 7.07 (1H, t, 3J = 7.2 Hz, Ar–H); 7.27
(2H, m, Ar–H); 7.47 (1H, m, Ar–H), 7.85 (1H, s, CH);
8.02–8.05 (1H, dd, 3J = 7.9 Hz, 4J = 1.7 Hz, Ar–H). 13C-
NMR (DMSO-d6) d (ppm): 39.54, 67.82, 111.71, 117.63,
121.08, 121.69, 121.87, 125.30, 127.06, 132.69, 132.94,
135.52, 137.41, 151.16, 160.27, 180.62. MS (m/z): 302.11
[MNa?] (calc: [MH?] = 279.13); Anal calcd for
C18H17NO2: C, 77.42, H, 6.09, N, 5.02. Found: C, 76.84,
H, 6.23, N, 4.99.
Spiro[cylohexyl-2,40-(2,3-dihydro-7-methoxy-4H-1-benzo-
pyran-4-one)] (6a) Prepared by condensing the cyclo-
hexanone 5a on the 4-methoxy-6-hydroxyacetophenone
4a. Yellow crystals. 45 % yield. mp = 68 °C. FTIR
(KBr) (m cm-1): 3080, 2928, 2861, 1672, 1574, 1437,
1
1443, 1198, 1059; H-NMR (CDCl3) d (ppm): 1.31–1.68
3-(30Methoxybenzylidenyl)-2,3-dihydro-4H-1-benzopyran-
4-one (3e) Prepared by condensing the 3-methoxybenz-
aldehyde 2e on the chroman-4-one 1. White powder. 25 %
yield. mp = 83 °C. FTIR (KBr) (m cm-1): 3026, 2966,
2837, 1663, 1605, 1512, 1462, 1215, 1024; 1H-NMR
(CDCl3) d (ppm) : 3.86 (3H, s, OCH3); 5.37 (2H, s, CH2);
6.85–6.91 (2H, m, 3J = 7.8 Hz, Ar–H); 6.95–7.06 (2H, m,
(8H, m, cyclohexyl); 1.72–1.80 (2H, m, cyclohexyl);
2.65 (2H, s, CH2); 3.84 (3H, s, OCH3); 6.42 (1H, d,
4J = 3.0 Hz, Ar–H); 6.53 (1H, dd, 3J = 10.6 Hz,
3
4J = 2.9 Hz, Ar–H); 7.78 (1H, d, J = 10.7 Hz, Ar–H).
13C-NMR (DMSO-d6) d (ppm): 20.98, 24.62, 33.93,
47.04, 55.66, 80.13, 101.28, 109.05, 114.17, 127.45,
160.99, 165.75, 190.17. MS (m/z): 269.03 [MNa?] (calc:
[MH?] = 246.13); Anal calcd for C15H18O3: C, 73.17,
H, 7.32. Found: C, 73.15, H, 7.48.
3
Ar–H), 7.06–7.11 (1H, m, J = 8.0 Hz, Ar–H), 7.35–7.40
(1H, t, 3J = 8.0 Hz, Ar–H); 7.48–7.50 (1H, t, 3J = 6.9 Hz,
4J = 1.3 Hz, Ar–H), 7.86 (1H, s, CH); 8.00–8,04 (1H, dd,
3J = 9.2 Hz, 4J = 1.9 Hz, Ar–H). 13C-NMR (DMSO-d6) d
(ppm): 55.68, 79.30, 101.39, 108.89, 114.30, 127.52,
160.89, 165.73, 190.27. MS (m/z): 289.08 [MNa?] (calc:
[MH?] = 266.09); Anal calcd for C17H14O3: C, 76.69, H,
5.26. Found: C, 75.59, H, 5.42.
Spiro[cylohexyl-2,40-(2,3-dihydro-7-hydroxy-4H-1-benzo-
pyran-4-one)] (6b) Prepared by condensing the cyclo-
hexanone 5a on the 4,6-dihydroxyacetophenone 4b.
Yellow crystals. 68 % yield. mp = 175 °C. FTIR (KBr) (m
cm-1): 3105, 3044, 2941, 2847, 1651, 1495, 1447; 1184,
1119; 1H-NMR (CDCl3) d (ppm) : 1.24–1.56 (8H, m,
cyclohexyl); 1.81–1.85 (2H, m, cyclohexyl); 2.62 (2H, s,
3-(20,40,60-Trimethoxybenzylidenyl)-2,3-dihydro-4H-1-ben-
zopyran-4-one (3f) Prepared by condensing the 2,4,6-
trimethoxybenzaldehyde 2f on the chroman-4-one 1. Yel-
low powder. 42 % yield. mp = 130 °C. FTIR (KBr) (m
cm-1): 3100, 2897, 1663, 1605, 1466, 1416, 1207, 1057;
1H-NMR (CDCl3) d (ppm) : 3.82 (6H, s, OCH3); 3.88 (3H,
s, OCH3); 4.87 (2H, s, CH2); 6.17 (2H, s, Ar–H); 6.94 (1H,
3
CH2); 6.28 (1H, s, Ar–H); 6.43 (1H, dd, J = 10.6 Hz,
3
4J = 3.0 Hz, Ar–H); 7.57 (1H, d, J = 10.4 Hz, Ar–H);
10.49 (1H, s, OH). 13C-NMR (DMSO-d6) d (ppm): 21.03,
24.66, 33.99, 47.10, 79.77, 102.93, 109.79, 113.30, 127.81,
160.93, 164.90, 189.88. MS (m/z): 255.09 [MNa?] (calc:
[MH?] = 232.11); Anal calcd for C14H16O3: C, 72.41, H,
6.90. Found: C, 72.61, H, 6.91.
3
3
d, J = 8.3 Hz, Ar–H); 7.04 (1H, t, J = 7.7 Hz, Ar–H);
3
7.46 (1H, t, J = 7.8 Hz, Ar–H); 7.76 (1H, s, CH); 8.05
(1H, d, 3J = 7.6 Hz, Ar–H). 13C-NMR (DMSO-d6) d
(ppm): 55.28, 55.53, 55.74, 69.10, 72.95, 90.71, 90.88,
104.12, 117.77, 121.21, 121.75, 127.18, 129.49, 129.99,
135.77, 159.12, 161.13, 163.09, 181.35. MS (m/z): 349.00
[MNa?] (calc: [MH?] = 326.12); Anal calcd for
C19H18O5: C, 69.94, H, 5.52. Found: C, 70.16, H, 5.79.
Spiro[ter-butylcyclohexyl-2,40-(2,3-dihydro-7-methoxy-4H-
1-benzopyran-4-one)] (6c) Prepared by condensing the
tert-butylcyclohexanone 5b on the 4-methoxy-6-hydro-
xyacetophenone 4a. Yellow crystals. 71 % yield.
mp = 146 °C. FTIR (KBr) (m cm-1): 3026, 2937, 2862,
1680, 1580, 1445, 1205, 1070; 1H-NMR (CDCl3) d (ppm) :
0.91 (9H, s, CH3); 1.10 (1H, m, cyclohexyl); 1.31–1.60
3
Spiro[cylohexyl-2,40-(2,3-dihydro-4H-1-benzopyran-4-
one)] (6a–d)
(6H, m, cyclohexyl); 2.20 (2H, m, J = 11.9 Hz, cyclo-
hexyl); 2.62 (2H, s, CH2); 3.87 (3H, s, OCH3); 6.44 (1H, d,
4J = 2.2 Hz, Ar–H); 6.56 (1H, dd, 3J = 8.8 Hz,
4J = 2.4 Hz, Ar–H); 7.82 (1H, d, 3J = 8.8 Hz, Ar–H).
13C-NMR (DMSO-d6) d (ppm): 21.57, 23.03, 27.36, 32.08,
34.02, 35.18, 46.69, 47.90, 55.69, 79.34, 81.63, 101.40,
109.03, 114.30, 127.50, 160.90, 165.74, 189,82. MS (m/z):
The synthesis of the spiro[cylohexyl-2,40-(2,3-dihydro-4H-
1-benzopyran-4-ones)] 6a–6d was carried out by con-
densing the appropriate cyclohexanones 5a–5c (1.1 eq.)
with the 20-hydroxyacetophenones 4a or 4b (3 mmol) in
123

Med Chem Res (2013) 22:681–691
689
325.18 [MNa?] (calc: [MH?] = 302.19); Anal calcd for
Cells were plated in six-well plates at a density of 104 cells/
cm2 in 10 % DCC-treated fetal calf serum, cultured for
3 days, and then maintained in the absence of any com-
pound (control) or exposed to one of the investigated
C19H26O3: C, 75.49, H, 8.61. Found: C, 75.78, H, 8.36.
Spiro[4-phenylcyclohexyl-2,40-(2,3-dihydro-7-methoxy-4H-
1-benzopyran-4-one)] (6d) Prepared by condensing 4-
phenylcyclohexanone 5c on 4-methoxy-6-hydroxyaceto-
phenone 4a. Yellow crystals. 65 % yield. mp = 153 °C.
FTIR (KBr) (m cm-1): 3028, 2939, 2907, 2866, 1709, 1497,
benzopyran derivatives (10-6 M). 10-10 M E2 or 10-7
M
fulvestrant (ICI) were used as complementary positive and
negative luciferase-inducing agents. At the end of treatment,
the medium was removed, and the cell monolayers were
rinsed twice with PBS. A fivefold diluted lysis solution
(250 ll, Promega E153A) was then added to the plates,
which were subsequently maintained under mild agitation
for 20 min to extract luciferase. Lysed cells were detached
with a scraper, and the resulting suspensions clarified by
centrifugation (5 s, 10,0009g). Finally, 20 ll of such clar-
ified extracts were mixed at room temperature with 100 ll
of luciferase reagent mixture (Promega E151A/E152A),
prepared according to the manufacturer’s protocol. Lumi-
nescence was measured in a Lumat LB 9507 luminometer
(Berthold Technologies, Bad Wildbad, Germany). Lucifer-
ase induction, expressed in arbitrary units with regard to a
blank (relative luciferase units) was normalized according to
the protein content of the extract (coomassie assay) and the
data given as percentages of the mean value ( SD) of
control untreated cells.
1
1447, 1250, 1167; H-NMR (CDCl3) d (ppm) : 1.59–1.97
(6H, m, cyclohexyl); 2.17–2.22 (2H, m, cyclohexyl); 2.63
(1H, m, cyclohexyl); 2.92 (2H, s, CH2); 3.84 (3H, s,
4
OCH3); 6.41 (1H, d, J = 2.9 Hz, Ar–H); 6.54 (1H, m,
3J = 10.7 Hz, 4J = 2.9 Hz, Ar–H); 7.21–7.34 (5H, m,
Ar–H); 7.79 (1H, d, 3J = 10.7 Hz, Ar–H). 13C-NMR
(DMSO-d6) d (ppm): 33.33, 40.74, 41.64, 126.19, 126.67,
128.36, 145.32, 210.12. MS (m/z): 345.15 [MNa?] (calc:
[MH?] = 322.16); Anal calcd for C21H22O3: C, 78.26, H,
6.83. Found: C, 77.63, H, 6.47.
X-ray crystallography
Compounds 3e and 6c were recrystallized from a mixture
of n-pentane/methanol (80/20) and n-pentane alone, res-
pectively. Selected single crystals were mounted onto a
glass fiber and set-up on a diffractometer Nonius Kappa-
CCD. Data were recorded at room temperature under Mo
ERa binding assay
˚
Ka radiation (k = 0.71073 A). Data collection, integration,
and unit cell parameters were carried out using the Nonius
EVAL-14 program. The data were corrected from absorp-
tion by means of SHELXS-86 (Sheldrick, 1986) and
refined by full least-squares on F2, and completed with
SHELXL-97 (Sheldrick, 1997). Graphics were carried out
with DIAMOND (Brandenburg and Berndt 1999). All non-
H atoms were refined with anisotropic displacement
parameters, and H atoms were included in calculated
positions. Crystallographic data: 3e: C17H14O3, mono-
‘‘Hydroxylapatite (HAP) assay’’ (Maaroufi and Leclercq,
1994) was performed using highly purified recombinant
hERa (Calbiochem Novabiochem, San Diego, CA) diluted
in a bovine serum albumin solution (1 mg/ml). Before-
hand, recombinant ERa was adsorbed onto HAP. After
removal of unbound material by centrifugation, HAP-
bound ERa was incubated overnight at 0–4 °C with
10-9 M [3H]E2 (Amersham Biosciences, Roosendaal, The
Netherlands) in the presence of increasing amounts of
either unlabeled E2 (10-10 to 10-6 M) or one of the
investigated compounds. Radioactivity adsorbed onto HAP
was then extracted with ethanol and measured by liquid scin-
tillation counting. Relative concentration of E2 and compound
required to reduce [3H]E2 binding by 50 % gave the rela-
˚
˚
clinic, P21/c, a = 12.545(9) A, b = 6.789(8) A, c =
3
˚
˚
16.290(11) A, V = 1371.2(2) A , Z = 4, 4742 observed
data, with I [ 2r (I). Crystallographic data: 6c: C19H26O3,
˚
monoclinic, P21/a, a = 7.572(5) A, b = 23.507(38) A,
˚
3
˚
˚
c = 9.624(11) A, V = 1712.4(3) A , Z = 4, 2739
observed data, with I [ 2r (I).
tive binding affinity (RBA), i.e., RBA = (IC50compound
/
IC50E2) 9 100.
Biological tests
Theoretical physicochemical properties calculations
ERa-mediated transcription
The physicochemical properties of each compound were
theoretically calculated by using the free online service
ERa-mediated transcription was assayed in MCF-7 cells
stably transfected with a pVit-tk-Luc reporter plasmid
(MVLN cells (Pons et al., 1990)). Expression of this lucif-
erase reporter gene was measured according to a procedure
already described (Demirpence et al., 1993) using the
Luciferase Assay System from Promega (Madison, WI).
Molinspiration
(http://www.molinspiration.com/cgi-bin/
properties). The hydrophobicity (LogP), the polar surface
area, and the volume of the different molecules were cal-
culated and compared with the references E2, genistein,
and apigenin.
123

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Med Chem Res (2013) 22:681–691
Acknowledgments This study was supported in part by grants from
Hassner A, Mead TC (1964) The stereochemistry of 2-benzalcyclo-
hexanones and 2-benzalcyclopentanones. Tetrahedron 20:
2201–2210
´
La Ligue Nationale contre le Cancer (comite de Montbeliard) and the
Fonds J.C. Heuson de Cancerologie Mammaire. We are very grateful
´
´
´
to Denis Lesage (UMR 7201, the Universite Pierre et Marie Curie,
Hoshino Y, Tanaka H, Takeno N (1998) Regioselective reduction of
the homoisoflavone system with dialkylboranes in the presence
of palladium catalysts. Bull Chem Soc Jpn 71:2923–2928
Jacquot Y, Bermont L, Giorgi H, Refouvelet B, Adessi GL,
Daubrosse E, Xicluna A (2001) Substituted benzopyranoben-
zothiazinones. Synthesis and estrogenic activity on MCF-7
breast carcinoma cells. Eur J Med Chem 36:127–136
Paris6) for mass spectrometry experiments. We are also grateful to
Kamal Boubekeur of the Laboratoire de Chimie Inorganique et
´
´
´
Materiaux Moleculaires, CNRS UMR 7071 (Universite Pierre et
Marie Curie, Paris6) for X-ray experiments; and to Olivier Lequin for
2D NMR experiments. Further, we are grateful to Dominique Gallo,
Ioanna Laios, and Anny Cleeren for their precious participation in the
biological assays.
Jacquot Y, Cleeren A, La¨ıos I, Ma Y, Boulahdour A, Bermont L,
A (2002)
Refouvelet B, Adessi G, Leclercq G, Xicluna
Pharmacological profile of 6,12-dihydro-3-methoxy-1-benzopyr-
ano[3,4-b][1,4]benzothiazin-6-ones, a novel estrogen receptor
agonist. Biol Pharm Bull 25:335–341
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