Synthesis, pharmacological evaluation, and structure-activity relationships of benzopyran derivatives with potent SERM activity

Article abstract of DOI:10.1016/j.bmc.2004.05.015

The synthesis, binding affinity for estrogen receptor subtypes (ERα and ERβ) and pharmacological activity on rat uterus of a new class of potent ligands, characterized by a 3-phenylbenzopyran scaffold with a basic side chain in position 4, are reported. S

Full text of DOI:10.1016/j.bmc.2004.05.015

Bioorganic & Medicinal Chemistry 12 (2004) 3763–3782
Synthesis, pharmacological evaluation, and structure–activity
relationships of benzopyran derivatives with potent SERM activity
Gabriele Amari,a,* Elisabetta Armani,a Silvia Ghirardi,a Maurizio Delcanale,a
Maurizio Civelli,b Paola Lorenza Caruso,b Elisabetta Galbiati,b Milco Lipreri,c
Silvia Rivara,d Alessio Lodolad and Marco Mord
aDepartment of Medicinal Chemistry, Chiesi Farmaceutici S.p.A., via Palermo 26/A, I-43100 Parma, Italy
bDipartimento Farmaceutico, Universitꢀa degli Studi di Parma, Parco Area delle Scienze 27/A, I-43100 Parma, Italy
cDepartment of Pharmacology, Chiesi Farmaceutici S.p.A., via Palermo 26/A, I-43100 Parma, Italy
dDepartment of Analytical Chemistry, Chiesi Farmaceutici S.p.A., via Palermo 26/A, I-43100 Parma, Italy
Received 19March 2004; revised 6 May 2004; accepted 11 May 2004
Abstract—The synthesis, binding affinity for estrogen receptor subtypes (ERa and ERb) and pharmacological activity on rat uterus
of a new class of potent ligands, characterized by a 3-phenylbenzopyran scaffold with a basic side chain in position 4, are reported.
Some of these compounds, endowed with very high receptor affinity, showed potent inhibition of agonist-stimulated uterine growth,
with no or limited proliferative effect. Binding affinity mostly depended on the nature and position of substituents at the 3-phenyl
ring, while the uterine activity seems to be affected by basic chain length. Compound 9c (CHF4227) showed excellent binding affinity
and antagonist activity on the uterus. The docking of benzopyran derivatives explained the structure–affinity relationships observed
for 3-phenyl substitution: a small, hydrophobic 40-substituent could interact with a small accessory binding cavity, while
di-substitution at 40 and 30 led to some ERa selectivity. This selectivity can be ascribed to differences in amino acid composition and
side chain conformation in the region accommodating the 3-phenyl ring at human ERa and ERb ligand-binding domain.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
of these pathologies, are characterized by important side
effects, greatly reducing the compliance of such treat-
ments.5–7 This has led to an increased interest in the
search for new pharmacological approaches, such as the
tissue-selective modulation of estrogen receptors.
Estrogens are known to play an important role in the
regulation of the development and maintenance of the
female reproductive system, in particular of the uterus,
ovaries and breast, through interaction with two differ-
ent receptor subtypes, named ERa and ERb,1 which are
members of the nuclear receptor superfamily of ligand-
inducible transcription factors. Moreover, estrogens are
involved in the growth and/or function of several other
tissues such as the bone, liver, brain, and the cardio-
vascular system.2 In fact, the decreased production of
ovarian steroids, occurring after the climacteric, has
been linked to typical menopausal disturbances, such as
osteoporosis and coronary artery diseases.3;4 Estrogen
replacement therapy (ERT) and hormone replacement
therapy (HRT), used for the prevention and treatment
In this light, at first tamoxifen,8 and later a second gen-
eration of compounds with an agonist/antagonist
behavior9 were developed: raloxifene10 (Fig. 1) was the
first approved selective estrogen receptor modulator
(SERM), marketed for the treatment and prevention of
osteoporosis. The efforts to obtain compounds with a
better activity profile led to a third generation of SERMs
characterized by improved potency and absence of ad-
verse effects, such as those on breast and the cardiovas-
cular system.11 These new compounds, briefly
summarized in Figure 1, belong to different chemical
classes, such as benzothiophenes (arzoxifene), indoles
(pipindoxifene, bazedoxifene), tetrahydronaphthalenes
(lasofoxifene), and benzopyrans (EM-800), albeit sharing
with raloxifene the known pharmacophoric elements for
SERM activity. These are a core scaffold, mimicking the
Keywords: SERM; Estrogen receptor; Benzopyran; Docking.
* Corresponding author. Tel.: +39-0521-279428; fax: +39-0521-2798-
80; e-mail: g.amari@chiesigroup.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.05.015

3764
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
N(CH3)2
O
O
O
N
O
N
N
(CH2)n
O
O
N
OH
O
OH
HO
S
S
HO
HO
tamoxifen
raloxifene
n=1 pipindoxifene
n=2 bazedoxifene
arzoxifene
N
N
O
O
O
O
C(CH3)3
O
O
O
(CH3)3C
O
N
O
HO
O
lasofoxifene
Figure 1. Structure of SERMs.
levormeloxifene
EM-800
17b-estradiol structure (often in a stilbene-like arrange-
ment)9 with at least one hydroxyl group, known to confer
high binding affinity for estrogen receptors (ERs).12;13
Additionally, the feature most characteristic of many
SERMs is a basic chain, necessary to achieve an anta-
gonist action in some tissues, projecting from the middle
of the core, generally through a phenolic spacer. In fact,
SERMs appear to achieve their antiestrogen effect by
inducing a conformational change on helix-12 of both the
estrogen receptor subtypes, a and b, because of the
necessity to accommodate the basic chain in the binding
site.14;15 Chemical modulations at the polar side chain of
SERMs widely affect their activity, as has been shown by
SAR exploration.16 In particular, a coplanar arrange-
ment between the basic side chain and the stilbene moi-
ety, as in tamoxifen, produces uterine proliferating
effects, while an orthogonal orientation between the two
substructures, like that conferred by the carbonyl hinge
of raloxifene, leads to an antagonist effect on the uterus.
O
O
RO
A: R = H
B: R = isopropyl
Figure 2. Isoflavone derivatives.
nolic hydroxyl of 17b-estradiol and raloxifene, which is
known to be strongly involved in electrostatic interac-
tions with a carboxyl group at both the receptor sub-
types. Moreover, the carbonyl group at position 4 of the
benzopyranone nucleus could be easily modified
through a C–C forming reaction, and this gave us the
possibility to attach a piperidinyl-alkoxy-phenyl side
chain at that position. We decided to make the con-
nection through a methylene hinge, thus allowing for an
orthogonal disposition of the side chain with respect to
the plane of the stilbene scaffold, in analogy with the
carbonyl group of raloxifene. As stated above, it had
been speculated that this specific arrangement could lead
to a reduction of the uterotrophic effect.16 As expected,
the resulting compound (CHF4056, 9g in Table 1) be-
haved like a new nonsteroidal estrogen agonist/antago-
nist, binding with high affinity to human ERa and ERb,
and displaying a very interesting pharmacological pro-
file, because of its ability to prevent bone loss and its
positive action on cholesterol levels, while maintaining
antagonist effects on the uterus.20 Moreover, 9g and its
derivatives presented the additional advantage over
other benzopyran derivatives such as EM800 (Fig. 1) of
being devoid of chiral centers, thus avoiding the prob-
lems related to racemization and optical purity.
In this light, we undertook a project aimed at the dis-
covery of a new class of third generation SERMs, en-
dowed with good binding affinity for the a and b
estrogen receptors and with an improved pharmaco-
logical profile compared to that of raloxifene, for the
treatment of postmenopausal pathologies such as oste-
oporosis, and of estrogen-dependent cancer. Our
approach started from the hypothesis that 7-hydroxyi-
soflavone (7-hydroxy-3-phenyl-4H-1-benzopyran-4-one,
A in Fig. 2), a metabolite of ipriflavone (OsteofixTM, B in
Fig. 2),17 marketed for the treatment of osteoporosis,
could be a good starting point for the preparation of
compounds able to bind to estrogen receptors, even
though the precise mechanism of ipriflavone action was
not clear.18;19 This hypothesis was supported by the
presence of a stilbene-like substructure, typical of clas-
sical ER ligands, within the isoflavone structure, while
the hydroxyl group at position 7 may mimic the phe-
The present work reports the synthesis, pharmacological
characterization, and structure–activity relationship
(SAR) studies of a series of compounds belonging to the

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3765
Table 1. Binding affinity (pKi) for ERa and ERb, uterotrophic (% increase in uterine weight, IUW), and antiuterotrophic (% inhibition of EE2-
induced uterine weight increase) activity of the tested compounds
pKiaa
pKiba
Kib/Kia
IUWb;c
23
112
Inhib.b;d
e
raloxifene
10.15 0.05
9.57 0.04
10.70 0.02
9.29 0.04
8.79 0.09
8.74 0.12
9.97 0.19
9.20 0.03
22.9
6.8
5.4
1.2
1
80
4
41 5f
n.a.g
50h
revormeloxifene
4-hydroxytamoxifen
tamoxifen
9
n.a.g
153h
O
(CH2)n
N
(CH2)m
R1
R2
O
R1
R2
n
m
i
9g
10a
9b
9c
9d
9e
9f
H
OH
OCH3
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
2
2
2
2
2
2
2
2
2
2
2
3
3
1
1
1
1
1
1
1
1
1
0
2
1
1
10.39 0.12
9.59 0.09
9.68 0.03
10.77 0.05
10.49 0.03
9.95 0.03
10.15 0.03
10.35 0.03
9.87 0.03
10.48 0.03
10.40 0.03
10.12 0.03
10.34 0.03
9.80 0.08
8.17 0.09
9.28 0.07
10.00 0.02
10.15 0.03
8.96 0.04
8.80 0.03
10.08 0.03
9.52 0.03
10.04 0.03
9.82 0.03
9.74 0.03
9.53 0.03
3.9
26.0
2.5
––
11
35
25
6
4
1
H
4-OH
––i
11
1
4-OCH3
4-CH3
5.95
2.2
1
86
20
1
1
71
59
21
62
10
54
42
20
30
4
4-O-n-C4H9
3-OCH3, 4-OCH3
2-OCH3
3-OCH3
4-OCH3
4-OCH3
H
9.8
8
1
3
1
3
1
2
2
1
1
22.4
1.9––
2.2
––j
j
9a
9h
9i
2
1f
1
2.8
3.8
26
––j
24
85
9l
9j
9k
2.4
6.5
2
3
4-OCH3
a Average of three determinations made in duplicate.
b n ¼ 8.
c % Increase in uterine weight at 1 mg/kg, unless differently indicated.
d % Inhibition of 0.05 mg/kg day EE2-induced uterine weight increase at 0.1 mg/kg, unless differently indicated.
e At 1 mg/kg.
f At 10 mg/kg.
g Data not available.
h Ref. 8. ED50 ¼ 0:1 mg/kg.
i Inactive at 1 mg/kg.
j Inactive at 10 mg/kg.
benzopyran class, developed to explore the possibility to
obtain new derivatives with an improved SERM profile,
lacking stimulating effects on the reproductive system.
Starting from the reference compound 9g, we investi-
gated the importance of some chemical modulations on
the binding affinity at the two estrogen receptor sub-
types, and on in vivo uterotrophic and antiuterotrophic
activity. The hydroxyl group at position 7 on the
benzopyran nucleus was masked by an ether to evaluate
its importance for receptor binding, and some substit-
uents were added to the phenyl ring at position 3 (also
called the ‘distal ring’), to look for additional interac-
tions at the binding site, in analogy with what had been
observed for the 40-OH group of raloxifene. Minor
modifications were applied to the basic side chain, pre-
paring longer derivatives and modulating the basic ring
hindrance, in order to test the influence on binding
affinity and pharmacological profile. Moreover, in
order to rationalize the binding affinity data obtained,
we built 3D-models of the ligand-binding domains of
human ERa and ERb, employing the crystallographic
coordinates present in the Protein Data Bank
(PDB)21 and we performed docking studies with our
compounds.
2. Chemistry
The benzopyran derivatives 9a–l were obtained follow-
ing a common synthetic route, which involves seven
steps (Schemes 1 and 2), starting from the commercially
available 7-hydroxy-isoflavone derivatives (compounds
1a–h). Of all the possible protecting groups of the phe-
nol hydroxyl in position 7, we chose the pivaloyl group
since it was simple to introduce and remove, and it was
not reactive in the Grignard addition.22
In detail, protection of the phenolic group of 7-hydroxy-
isoflavones (1a–h) as a pivaloyl ester (step a in Scheme 1)
gave compounds 2a–h; the subsequent catalytic selective
reduction of the carbon–carbon double bond gave
compounds 3a–h, which yielded compounds 5a–h by
reaction with the Grignard reagent obtained from
compound 4. After catalytic deprotection of the benzyl
group, the free phenol hydroxyl of compounds 6a–h was
alkylated by reaction with the desired tertiary chloro-
alkyl amine in the presence of a base, to obtain com-
pounds 7a–l. For the synthesis of the benzopyran
products 9a–l, compounds 7a–l were first deprotected
from the pivaloyl group, furnishing compounds 8a–l,

3766
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
O
O
O
m(H2C)
N
(CH2)n
O
a
a
R1
6a-h
OH
R1
O
O
R1
HO
O
O
O
1a-h
2a-h
O
7a-l
O
c
b
O
m(H2C)
N (CH2)n
R1
Cl
b
O
O
O
OH
O
R1
4
3a-h
HO
O
O
HO
O
8a-l
O
m(H2C)
N (CH2)n
OH
c
d
OH
R1
R1
O
R1
O
O
HO
O
O
O
9a-l
5a-h
6a-h
R1
R1= 2-OCH3
Compounds
m
n
7a, 8a, 9a
7b
8b, 9b
1
1
1
1
1
1
1
1
1
0
1
1
2
Compounds
1a, 2a, 3a, 5a, 6a
1b
2b, 3b, 5b, 6b
1c, 2c, 3c, 5c, 6c
1d, 2d, 3d, 5d, 6d
1e, 2e, 3e, 5e, 6e
1f, 2f, 3f, 5f, 6f
1g, 2g, 3g, 5g, 6g
1h, 2h, 3h, 5h, 6h
R1
2
R1= 4-OCOC(CH3)3
R1= 4-OH
2
2
2
2
2
2
2
2
2
3
3
2
R1= 2-OCH3
R1= 4-OH
R1= 4-OCH3
R1= 4-CH3
R1= 4-O-nC4H9
R1= 3-OCH3, 4-OCH3
R1= H
R1= 3-OCH3
R1= 4-OCH3
R1= H
R1= 4-OCH3
R1= 4-OCH3
7c, 8c, 9c
7d, 8d, 9d
7e, 8e, 9e
7f, 8f, 9f
7g, 8g, 9g
7h, 8h, 9h
7i, 8i, 9i
7j, 8j, 9j
7k, 8k, 9k
7l, 8l, 9l
R1= 4-OCOC(CH3)3
R1= 4-OCH3
R1= 4-CH3
R1= 4-O-nC4H9
R1= 3-OCH3, 4-OCH3
R1= H
R1= 3-OCH3
Scheme 1. Reagents and conditions: (a) K2CO3, pivaloyl chloride,
acetonitrile, room temperature; (b) H2, Pd/C, dioxane, room temper-
ature; (c) Mg, THF dry, 70 ꢁC then )20 ꢁC to room temperature; (d)
H2, Pd/C, ethyl acetate, room temperature.
Scheme 2. Reagents and conditions: (a) N-chloroalkyl-cycloalkyl-
amine, K2CO3, acetone, reflux; (b) CH3OH/H2O, K2CO3 room tem-
perature; (c) acetonitrile or ethanol, HCl, reflux.
which were dehydrated to give the desired products 9a–l
(Scheme 2). Compound 9b was obtained by protecting
(step a of Scheme 1) and deprotecting (step b of Scheme
2) both the phenolic hydroxyls as pivaloyl esters.
Compound 10a was obtained by methylation of com-
pound 9g with methyl iodide.
4. Results and discussion
Table 1 reports the pKi values for the benzopyran deriv-
atives at ERa and ERb and their in vivo effects. The
uterine pharmacological profile was investigated because
the uterotrophic effects represent one of the most
important drawbacks for SERMs. Agonist activity is
expressed as percent of uterine weight gain at 1 mg/kg of
tested compounds and the antagonist effect as percent
inhibition of 17a-ethynylestradiol (EE2) induced weight
gain on immature female rat, at 0.1 mg/kg. For compar-
ison, pKi values and uterine profile activity were also
evaluated for two reference SERMs: the marketed
raloxifene, and levormeloxifene. In addition, we mea-
sured the binding affinity of tamoxifen and of its metab-
olite 4-hydroxytamoxifen (OHT). All the benzopyran
derivatives 9a–l and 10a showed good binding affinity for
both receptor subtypes and inhibited estrogen-stimulated
uterus growth at doses lower than 1 mg/kg (po). Inter-
estingly, some derivatives (e.g., 9c and 9a) resulted as
being significantly more potent in vivo than the unsub-
stituted compound 9g, despite the small differences in
their pKi values; this could be due to differing metabolic
stability or access to the target. Most of the tested com-
3. Pharmacology
Binding affinity to human ERa and ERb was deter-
mined by the inhibition of [3H]-17b-estradiol binding, in
accordance with the reported procedure for estrogen
receptors.23 pKi values were calculated from concentra-
tion response curves of test compounds.
Estrogen agonist and antagonist activity was tested in
vivo using an immature female rat model24 since in these
animals, in the absence of endogenous estrogen, the uterus
is fully responsive to exogenous estrogen and allows for
the ready measurement of an agonist and antagonist ac-
tion. We used the increase in uterine wet weight as a
measure of estrogenic activity, while antiestrogenic
activity was assessed by evaluating the ability of a com-
pound to block estrogen-stimulated uterine weight gain.

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3767
pounds showed uterotrophicity similar to raloxifene,
giving no or limited weight increase; the only exception
was compound 9k, characterized by a longer side chain.
The role of the basic side chain in the modulation of
receptor intrinsic activity has been widely recognized. As
a result of the side chain modulation, we found a good
agreement with the SARs previously reported for ra-
loxifene derivatives. In fact, an optimal pharmacological
profile was achieved when the chain attached to the
methylene hinge was a 4-(piperidinoethoxy)phenyl one.16
Compounds 9j and 9k, having a propoxy chain, showed a
limited decrease in binding affinity and in vivo antiute-
rotrophic activity, although, as stated before, compound
9k led to an increase in uterus weight. At the same time we
observed that, although cyclic amine modifications are
tolerated by the receptor binding site, ring contraction or
enlargement, as in compounds 9i and 9l, led to a decrease
in antiuterotrophic activity compared to compound 9c.
The 7-OH group, mimicking the 3-OH one of the A-ring
of estradiol, was silenced by a methyl ether in order to
evaluate its importance on binding affinity. In analogy
with SAR studies on estradiol and 2-arylbenzothioph-
ene12 derivatives, a loss in binding affinity at ERa and
ERb was observed, with a 10-fold reduction of potency
between 10a and 9g.
Figure 3. Superposition of 4-hydroxytamoxifen (cyan carbons),
raloxifene (green carbons), and 9g (magenta carbons) into ERa ligand-
binding site deriving from the crystal structure 3ERT, displaying the
side chains of the principal amino acids involved in ligand recognition.
bene substructure in the 3-phenylbenzopyran scaffold can
be superimposed to the unconstrained stilbene fragment
of OHT with a better fitting than to that of the 2-phenyl-
benzothiophene in raloxifene. This binding mode,
which is illustrated in Figure 3, may explain the affinity
decrease observed for 9b, having a 40-OH group, com-
pared to the parent compound 9g, owing to the place-
ment of a polar group in a hydrophobic environment. We
thus explored the possibility of increasing affinity and in
vivo activity by introducing a small hydrophobic group
at the 40-position of the distal ring. Compounds 9c and
9d, having a methoxy and a methyl group, respectively,
gained a significant increase of affinity compared to 9b,
resulting as being more potent than the parent 9g. A
butoxy group was introduced at the 40-position to test the
steric tolerance of the binding pocket; the significant
decrease in affinity of compound 9e indicated that there is
not enough room to accommodate bulky groups. The
methoxy group was also moved to position 20 (9a) or 30
(9h), and a 30,40-disubstituted derivative (9f) was pre-
pared. As a result of this limited exploration, we observed
that while the substitution in 20 was tolerated by both
receptors, the methoxy group in 30 caused a 3-fold
reduction in binding affinity on ERa and a 2-fold one on
ERb, compared to compound 9g. Although compound 9f
had an intermediate affinity between compounds 9c and
9h on ERa, it showed an unexpected drop of affinity on
ERb. The pharmacological data in Table 1 show that, by
introducing a small lipophilic group in 20 and 40, the
antagonist effect on uterine growth is improved: in fact,
9a and 9c gave higher inhibition of EE2-induced uterine
weight increase. Moreover, these compounds showed low
(9c) or no (9a) uterotrophic activity at the highest doses
tested.
Observing the crystal structures of the complexes ERs-
raloxifene, it is possible to note that the 2-phenyl ring is
accommodated in a hydrophobic region, and that the 40-
OH group undertakes a hydrogen bond interaction with
a flexible histidine. Grese et al.12 have underlined the
importance of this interaction, in particular observing
that the replacement of the raloxifene 40-hydroxyl group
with a hydrogen atom caused a 5-fold affinity decrease
versus ERa. In contrast, compound 9b in our benzo-
pyran series, having a 40-hydroxyl group, exhibited
lower affinity, on both ERa and ERb, than the unsub-
stituted compound 9g. We thought that SAR differences
between 2-arylbenzothiophenes and 3-arylbenzopyrans
could arise from different binding modes. This was in
accordance with the observations of Egner et al.25 aris-
ing from the superposition of the ligand-binding domain
of all known human ERa structures, co-crystallized with
different ligands. In fact, this superposition has evi-
denced that the steroidal A-ring was tightly fixed, in
contrast to the D-ring region, where substituents of
differing size and shape could be accommodated, sug-
gesting greater flexibility than in other regions of the
ligand-binding site. In fact, comparing the crystal
structures of the complexes ERa-OHT and ERa-ra-
loxifene, it can be observed that His524, the last amino
acids of helix 11 and a b-turn, linking helices 6 and 7,
show different conformations. In particular, the absence
of the hydroxyl group in the ‘estradiol D-ring region’
induces His524 to move backward, allowing for a deeper
penetration of the OHT phenyl ring into the hydro-
phobic pocket at the ligand-binding domain site.
In this context, we supposed that our benzopyran class
may arrange the distal phenyl ring in the ligand-binding
domain like OHT rather than like raloxifene, as the stil-
In order to rationalize the SAR data obtained for the benz-
opyran compounds, the models of the ligand-binding

3768
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
domains of human ERa and ERb were built, starting
from the crystallographic coordinates of ER-ligand
complexes, and used for docking studies. The observa-
tion that the introduction of a 40-OH decreases the
binding affinity, while, at same position, small lipo-
philic groups are generally tolerated, suggested that the
binding of benzopyran derivatives does not involve
His524 as a H-bond acceptor, similarly to what was
observed for the ERa-OHT complex. Based on this
hypothesis, we used the high resolution crystal structure
of the complex ERa-OHT (PDB code 3ERT) to build a
refined model of human ERa, while the building of
the human ERb model started from the coordinates of
the rat ERb-raloxifene complex (PDB code 1QKN), the
crystallographic coordinates of the human ERb in
complex with OHT being unavailable.
backward shift of His524/475 not involved in H-bonding
with the ligand, opposite to what was observed in the
complexes with raloxifene. This supposed binding mode,
resembling that of OHT, may explain the small increase
in affinity observed for the methoxy and methyl deriva-
tives, 9c and 9d, and the significant decrease in affinity of
the hydroxy derivative 9b. Moreover, the conformation
of the distal phenyl ring was a little different for 9c (green
structure in Fig. 4) in the two binding sites, owing to the
differing shapes of the hydrophobic pockets. In both
cases, however, the limited size of the accessory binding
pocket led to bumping interactions when the
40-butoxy derivative 9e was docked into the ligand-
binding domain. The docking of compound 9h (orange
structure in Fig. 4) showed that the 30-methoxy group can
be accommodated at the ERa binding site close to
Met343, and steric clashes could account for the slight
decrease in affinity; this also happened at the ERb
binding site, the 30-methoxy group being positioned close
to Met295, owing to a slight rotation of the distal phenyl
ring of 9h. On ERa, the 30,40-dimethoxy derivative 9f had
an intermediate affinity, between those of compounds 9c
and 9h, and it assumed the same binding orientation as
the other derivatives represented in Figure 4A; in con-
trast, on ERb the two methoxy groups could not be
accommodated in the same way, mainly because of the
different orientation of Phe377, and they were forced into
a crowded region of the hydrophobic binding pocket
(yellow structure in Fig. 4), leading to a decrease in
affinity. The differing interactions led to a 22-fold selec-
tivity of 9f for ERa, and this result suggests that addi-
tional steric hindrance in 30 and 40 regions may be useful
to further increase subtype selectivity, as also suggested
by the small selectivity observed for the 40-butoxy
derivative 9e. The differences in steric tolerance at ERa
and ERb may also explain the different selectivity ob-
served for some recently reported compounds of the
tetrahydroisoquinoline series:27 in fact, those derivatives
having a bulky substituent at the 2-phenyl ring, such as a
p-iPr or a m-NMe2, are characterized by the greatest a
selectivity, mainly due to a drop in ERb affinity. The
similarity between the binding modes of this class and
ours is evidenced by the comparison of the crystal
structure of 2-phenyl-1-[4-(2-piperidin-1-yl-ethoxy)-
phenyl]-1,2,3,4-tetrahydroisoquinolin-6-ol in complex
with ERa ligand-binding domain (PDB code 1UOM)
with that of OHT and raloxifene (PDB codes 3ERT and
1ERR,14 respectively). In fact, the 2-phenyl ring of the
tetrahydroisoquinoline derivative occupies the same
region of space as that of OHT, slightly apart from the
2-phenyl ring of raloxifene, as proposed for the benzo-
pyran derivatives by our docking models.
Compound 9g and its derivatives were docked into the
ER binding sites superimposing their benzopyran scaf-
fold to the styrene fragment of OHT. While this scaffold
was rather rigid, the basic side chain was much more
flexible; the conformation that provided the best fit into
the binding channel, allowing for interaction of the basic
nitrogen with Asp351 (in ERa, corresponding to Asp303
in ERb), was chosen. In particular, the methylene hinge
allowed for an orthogonal arrangement of the side chain
with respect to the benzopyran scaffold, and the ethyloxy
chain adopted a gauche conformation. After ligand ori-
entation, a minimization of the ligand-receptor interac-
tion energy was performed. The result obtained for 9g
docked into ERa is illustrated in Figure 3, where the
minimized structures of OHT and raloxifene are also
represented for a comparison. Although the side chain
of 9g better fitted that of raloxifene, the distal phenyl ring
of 9g resulted as being strictly superimposed to that of
OHT. The distal ring was surrounded by several hydro-
phobic amino acids, in particular Met343 and Leu346,
lining the so-called beta face14 of the ligand-binding do-
main pocket, Leu384, Met388, and Ile424, delimiting the
alpha one; at the same time, Met421 defined the bottom
wall, while His524 and Leu525 formed the side border.
The docking of 9g into the ERb binding site evidenced an
analog network of interactions, where most amino acids
were of the same type, with the exception of Met336 in
helix-5 of ERb, corresponding to Leu384 in ERa, and
Ile373 in helix-7 of ERb, corresponding to Met421 in
ERa. In the ERb ligand-binding domain, Phe377 and
Ile376 adopted conformations different from those as-
sumed in ERa by the corresponding Phe425 and Ile424;
their spatial disposition is that observed in the crystal
structures of the beta ligand-binding domain in complex
with raloxifene, genisteine, and the pure antagonist
ICI164,384;15;26 This could be due to the different steric
properties of Met421 (in ERa) and Ile373 (in ERb), and
probably reflects a conformational equilibrium, which
can be affected by ligand binding. This difference resulted
in a projection of the phenyl ring of Phe377 into the
hydrophobic binding pocket of ERb, whose volume is
further reduced by the side chain conformation of Ile376.
The ortho-substituted derivative 9a (not shown) placed
the methoxy group in a small cavity, corresponding to
that occupied by the ethyl tail emerging from the stil-
bene scaffold of OHT.
5. Conclusions
Docking simulations for compounds having a sub-
stituent in position 40 showed that it can be accommo-
dated in an accessory binding pocket created by the
3-Phenyl-benzopyran derivatives having a basic side
chain, connected to position 4 through a methylene

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3769
Figure 4. Compounds 9c (green), 9f (yellow), and 9h (orange) docked into the binding site of ERa (A) and ERb (B). Residues interacting with the
distal ring of the ligands are shown. A small lipophilic cavity between His524/475 and Ile424/376 can accommodate a substituent of limited size.
€
hinge, represent a new class of potent ERa and b
ligands, with high antiuterotrophic activity and no or
reduced stimulation of uterine proliferation. The
40-methoxy derivative 9c is endowed with a receptor
affinity higher than that of known SERMs, and there-
fore warrants further investigation for its therapeutic
potential.28;29 This series of compounds probably binds
to ERs in a manner similar to the traditional classes, like
those of OHT and raloxifene. On the other hand, some
differences were observed in their SAR profile: a 40-hy-
droxy group seems not to interact with His524/475,
which interacts with the phenol hydroxyl of raloxifene,
and a 40-methoxy or methyl group is optimal for
receptor binding. Moreover, the 30,40-dimethoxy deriv-
ative evidenced some selectivity for the ERa subtype,
and the docking into receptor models suggested a pos-
sible way to design selective compounds. In fact, it was
possible to observe a reduced steric tolerance of the so-
called D-ring region at the ERb ligand-binding domain,
compared to the ERa one, which may be ascribed to
different amino acids and side chain conformations.
Therefore, steric modulation of fragments occupying
this region can be critical for subtype selectivity, as
observed in other series of ER ligands.
on a Buchi 535 instrument. Purity of compounds 9a–l,
and 10a were analyzed by two different HPLC methods.
Method 1: on a Rainin-Dinamax HPLC instrument;
Column: Lichrochart C18 RP Select B (4.6 · 250 mm;
5 lm); isocratic elution A/B ¼ 40/60 (where A was a
pH ¼ 3 phosphate buffer and B was a mixture CH3CN/
CH3OH ¼ 8/2); flow 1 mL/min; k ¼ 230 nm. Method 2:
on a Waters 2695 HPLC system equipped with a DAD
detector and a ZQ 2000 single quadrupole MS spec-
trometer (used in ES+mode); column: Waters C18
XTerra MS (4.6 · 50 mm; 2.5 lm); gradient elution: at
0 min 100% A, at 5 min 100% B, at 6 min 100% A, at
10 min 100% A (where A ¼ water/CH3CN/formic acid ¼
95/5/0.05 and B ¼ CH3CN/water/formic acid ¼ 95/5/
0.05); flow ¼ 0.5 mL/min. All samples were >98% pure
with both methods. The ESþ/MS spectra were obtained
on a QUATTRO LC Micromass and on a ZQ Waters
instrument; the high resolution mass spectra (HRMS)
were obtained on a Q-TOF micro Micromass instru-
ment (ESþ mode). NMR spectra were recorded on a
Bruker AC200 spectrometer. The abbreviations THF
and DMSO refer to tetrahydrofuran and dimethyl
sulfoxide.
6.2. 2,2-Dimethyl-propanoic acid 3-(2-methoxyphenyl)-4-
oxo-4H-1-benzopyran-7-yl ester (2a)
6. Experimental
6.1. Chemistry
K2CO3 (7.8 g; 56.4 mmol) was added to a suspension of
compound 1a (10.2 g; 38 mmol) in acetonitrile (170 mL).
After some min at rt, when basic pH was achieved,
pivaloyl chloride (6.8 g, 56.4 mmol) was added dropwise.
After 1 h at rt, water was added (350 mL) to obtain a
precipitate, which was filtered. The solid was washed
with water, then dissolved in methylene chloride
(100 mL); residual water was separated and the organic
fraction was dried on Na2SO4 and evaporated under
vacuum, obtaining a yellow solid (12.44 g; yield ¼ 93%).
All solvents and reagents were used as obtained from
commercial sources, unless otherwise indicated. Dry
tetrahydrofuran was purchased from Fluka and used
without further distillations. Flash chromatography was
carried out on Isolute columns SI (silica cartridges).
TLC analyses were performed on Merck Silica gel 60
F254 UV sensitive plates. Melting points were measured

3770
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
The product was used for the subsequent steps without
any purification.
J ¼ 2:15 Hz, J ¼ 8:61 Hz, 1H, Ar-H), 7.48–7.56 (m, 3H,
Ar-H), 8.13–8.21 (d, J ¼ 8:61 Hz, 1H, Ar-H), 8.48 (s,
1H, Ar–O–CH@C); MS (ESI) m=e 394 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.34 (s, 9H, 3CH3),
3.72 (s, 3H, Ar–O–CH3), 6.93–7.15 (m, 2H, Ar-H), 7.20–
7.32 (m, 2H, Ar-H), 7.33–7.45 (m, 1H, Ar-H), 7.52–7.56
(d, J ¼ 2:26 Hz, 1H, Ar-H), 8.10–8.18 (d, J ¼ 8:76 Hz,
1H, Ar-H), 8.34 (s, 1H, Ar–O–CH@C); MS (ESI) m=e
352 (Mþ).
6.7. 2,2-Dimethyl-propanoic acid 3-(3,4-dimethoxyphen-
yl)-4-oxo-4H-1-benzopyran-7-yl ester (2f)
The title compound was prepared in quantitative yield
from 1f by a method similar to that described for 2a.
6.3. 2,2-Dimethyl-propanoic acid 4-[7-(2,2-dimethyl-1-
oxopropoxy)-4-oxo-4H-1-benzopyran-3-yl]-phenyl ester
(2b)
1H NMR (200 MHz, DMSO-d6) 1.34 (s, 9H, 3CH3),
3.79(s, 6H, Ar–[O–C H3]2), 6.98–7.05 (d, J ¼ 8:38 Hz,
1H, Ar-H), 7.13–7.23 (m, 2H, Ar-H), 7.24–7.32 (dd,
J ¼ 2:15 Hz, J ¼ 8:61 Hz, 1H, Ar-H), 7.53–7.56 (d,
J ¼ 1:92 Hz, 1H, Ar-H), 8.15–8.22 (d, J ¼ 8:86 Hz, 1H,
Ar-H), 8.53 (s, 1H, Ar–O–CH–C@C); MS (ESI) m=e 382
(Mþ).
The title compound was prepared in quantitative yield
from 1b by a method similar to that described for 2a.
1H NMR (200 MHz, DMSO-d6) 1.32–1.34 (2s, 18H,
6CH3), 7.13–7.22 (d, J ¼ 2:07 Hz, 2H, Ar-H), 7.26–7.34
(dd, J ¼ 2:07 Hz, J ¼ 8:62 Hz, 1H, Ar-H), 7.56–7.59(d,
J ¼ 2:07 Hz, 1H, Ar-H), 7.61–7.69(d, J ¼ 8:96 Hz, 2H,
Ar-H), 8.15–8.23 (d, J ¼ 8:62 Hz, 1H, Ar-H), 8.59(s,
1H, Ar-H); MS (ESI) m=e 422 (Mþ).
6.8. 2,2-Dimethyl-propanoic acid 3-phenyl-4-oxo-4H-1-
benzopyran-7-yl ester (2g)
The title compound was prepared in quantitative yield
from 1g by a method similar to that described for 2a.
6.4. 2,2-Dimethyl-propanoic acid 3-(4-methoxyphenyl)-4-
oxo-4H-1-benzopyran-7-yl ester (2c)
1H NMR (200 MHz, DMSO-d6) 1.34 (s, 9H, 3CH3),
7.25–7.33 (dd, J ¼ 2:09Hz, J ¼ 8:64 Hz, 1H, Ar-H),
7.37–7.50 (m, 3H, Ar-H), 7.52–7.64 (m, 3H, Ar-H),
8.13–8.24 (d, J ¼ 8:64 Hz, 1H, Ar-H), 8.54 (s, 1H, Ar–
O–CH–C@C); MS (ESI) m=e 322 (Mþ).
The title compound was prepared in quantitative yield
from 1c by a method similar to that described for 2a.
1H NMR (200 MHz, DMSO-d6) 1.34 (s, 9H, 3CH3),
3.79(s, 3H, Ar–O–C H3), 6.95–7.05 (d, J ¼ 8:97 Hz, 2H,
Ar-H), 7.23–7.31 (dd, J ¼ 2:07 Hz, J ¼ 8:96 Hz, 1H, Ar-
H), 7.48–7.58 (m, 3H, Ar-H), 8.13–8.21 (d, J ¼ 8:96 Hz,
1H, Ar-H), 8.49(s, 1H, Ar–C @CH); MS (ESI) m=e 352
(Mþ).
6.9. 2,2-Dimethyl-propanoic acid 3-(3-methoxyphenyl)-4-
oxo-4H-1-benzopyran-7-yl ester (2h)
The title compound was prepared in quantitative yield
from 1h by a method similar to that described for 2a.
1H NMR (200 MHz, DMSO-d6) 1.34 (s, 9H, 3CH3),
3.80 (s, 3H, Ar–O–CH3), 6.90–7.00 (m, 1H Ar-H), 7.12–
7.20 (m, 2H, Ar-H), 7.25–7.40 (m, 2H, Ar-H), 7.55–7.60
(d, J ¼ 2:00 Hz, 1H, Ar-H), 8.15–8.22 (d, J ¼ 8:75 Hz,
1H, Ar-H), 8.55 (s, 1H, Ar–O–CH@C); MS (ESI) m=e
352 (Mþ).
6.5. 2,2-Dimethyl-propanoic acid 3-(4-methylphenyl)-4-
oxo-4H-1-benzopyran-7-yl ester (2d)
The title compound was prepared in quantitative yield
from 1d by a method similar to that described for 2a.
1H NMR (200 MHz, DMSO-d6) 1.35 (s, 9H, 3CH3),
2.35 (s, 3H, Ar–CH3), 7.20–7.31 (m, 3H, Ar-H), 7.44–
7.55 (m, 3H, Ar-H), 8.14–8.21 (d, J ¼ 8:86 Hz, 1H, Ar-
H), 8.48 (s, 1H, Ar–O–CH@C); MS (ESI) m=e 336 (Mþ).
6.10. 2,2-Dimethyl-propanoic acid 3,4-dihydro-3-(2-
methoxyphenyl)-4-oxo-2H-1-benzopyran-7-yl ester (3a)
Compound 2a (4.2 g; 11.9mmol) was dissolved in ace-
tone (120 mL), Pd 5%/C (50% water) (3 g) was added
and the mixture was hydrogenated (30 psi, rt) for 9h.
After filtering, the solution was evaporated under vac-
uum and purified by flash chromatography (CH2Cl2/
petroleum ether ¼ from 90/10 to 70/30). After evapora-
tion of the solvent, ꢀ2.5 g of 3a were obtained
(yield ¼ 60%).
1H NMR (200 MHz, DMSO-d6) 1.25–1.90 (2m, 6H,
3CH2), 2.80–3.10 (m, 2H, Nþ–CH2), 3.32–3.55 (m, 4H,
Nþ–[CH2]2), 3.72 (s, 2H, Ar–CH2–C@C), 4.24–4.42 (t,
J ¼ 5:18 Hz, 2H, Ar–O–CH2–CH2–Nþ), 4.91 (s, 2H,
6.6. 2,2-Dimethyl-propanoic acid 3-(4-butoxyphenyl)-4-
oxo-4H-1-benzopyran-7-yl ester (2e)
The title compound was prepared in quantitative yield
from 1e by a method similar to that described for 2a.
1H NMR (200 MHz, DMSO-d6) 0.88–1.00 (t,
J ¼ 7:19Hz, 3H, C H3), 1.34 (s, 9H, C–[CH3]3), 1.38–
1.56 (m, 2H, CH3–CH2–CH2), 1.62–1.80 (m, 2H, CH3–
CH2–CH2), 3.95–4.05 (t, J ¼ 6:46 Hz, Ar–O–CH2),
6.95–7.03 (d, J ¼ 8:85 Hz, 2H, Ar-H), 7.23–7.31 (d, d,

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3771
Ar–O–CH2–C@C), 6.17–6.31 (m, 2H, Ar-H), 6.81–6.96
(m, 3H, Ar-H), 7.04–7.17 (d, J ¼ 8:63 Hz, 2H, Ar-H),
7.23–7.47 (m, 5H, Ar-H), 9.58 (s, 1H, Ar–OH), 10.18–
10.48 (br, 1H, Nþ–H); MS (ESI) m=e 354 (Mþ).
7.88 (d, J ¼ 8:57 Hz, 1H, Ar-H); MS (ESI) m=e 396
(Mþ).
6.15. 2,2-Dimethyl-propanoic acid 3-(3,4-dimethoxyphen-
yl)-3,4-dihydro-4-oxo-2H-1-benzopyran-7-yl ester (3f)
6.11. 2,2-Dimethyl-propanoic acid 4-[7-(2,2-dimethyl-1-
oxopropoxy)-3,4-dihydro-4-oxo-2H-1-benzopyran-3-yl]-
phenyl ester (3b)
The title compound was prepared from 2f by a method
similar to that described for 3a (yield ¼ 65%).
The title compound was prepared in 70% yield from 2b
by a method similar to that described for 3a.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
3.71 and 3.73 (2s, 6H, Ar–[O–CH3]2), 4.08–4.20 (dd,
J ¼ 4:94 Hz, J ¼ 9:88 Hz, 1H, Ar–O–CH2–CH–CO),
4.58–4.88 (m, 2H, Ar–O–CH2–CH–CO), 6.74–6.81 (dd,
J ¼ 1:98 Hz, J ¼ 8:40 Hz, 1H, Ar-H), 6.82–6.94 (m, 4H,
Ar-H), 7.82–7.89(dd, J ¼ 0:50 Hz, J ¼ 8:40 Hz, 1H, Ar-
H); MS (ESI) m=e 384 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.29–1.31 (2s, 18H,
6CH3), 4.21–4.36 (dd, J ¼ 5:21 Hz, J ¼ 9:46 Hz, 1H,
CO–CH–CH2), 4.62–4.88 (m, 2H, Ar–O–CH2–CH),
6.82–6.92 (d+m, J ¼ 2:13 Hz, 2H, Ar-H), 7.04–7.11 (d,
J ¼ 9:31 Hz, 2H, Ar-H), 7.29–7.36 (d, J ¼ 9:31 Hz, 2H,
Ar-H), 7.83–7.90 (dd, J ¼ 8:51 Hz, J ¼ 0:47 Hz, 1H, Ar-
H); MS (ESI) m=e 424 (Mþ).
6.16. 2,2-Dimethyl-propanoic acid 3,4-dihydro-3-phenyl-
4-oxo-2H-1-benzopyran-7-yl ester (3g)
6.12. 2,2-Dimethyl-propanoic acid 3,4-dihydro-3-(4-
methoxyphenyl)-4-oxo-2H-1-benzopyran-7-yl ester (3c)
The title compound was prepared in 60% yield from 2g
by a method similar to that described for 3a.
The title compound was prepared in 85% yield from 2c
by a method similar to that described for 3a.
1H NMR (200 MHz, DMSO-d6) 1.31 (s, 9H, 3CH3),
4.15–4.30 (dd, J ¼ 5:20 Hz, J ¼ 9:61 Hz, 1H, Ar–CH–
C@O), 4.62–4.88 (m, 2H, Ar–O–CH2–CH), 6.81–6.93
(m, 2H, Ar-H), 7.21–7.42 (m, 5H, Ar-H), 7.80–7.92 (dd,
J ¼ 0:52 Hz, J ¼ 8:31 Hz, 1H, Ar-H); MS (ESI) m=e 324
(Mþ).
1H NMR (200 MHz, DMSO-d6) 3.73 (s, 3H, Ar–O–
CH3), 4.07–4.20 (dd, J ¼ 5:02 Hz, J ¼ 9:29Hz, 1H, Ar–
CH–CO), 4.60–4.82 (m, 2H, Ar–O–CH2–CH–CO),
6.80–6.96 (d+m, J ¼ 2:01 Hz, 4H, Ar-H), 7.15–7.25 (d,
J ¼ 8:79Hz, 2H, Ar- H), 7.83–7.89(dd, J ¼ 8:29Hz,
J ¼ 0:50 Hz, 1H, Ar-H); MS (ESI) m=e 354 (Mþ).
6.17. 2,2-Dimethyl-propanoic acid 3,4-dihydro-3-(3-
methoxyphenyl)-4-oxo-2H-1-benzopyran-7-yl ester (3h)
The title compound was prepared from 2h by a method
similar to that described for 3a (yield ¼ 55%).
6.13. 2,2-Dimethyl-propanoic acid 3,4-dihydro-3-(4-
methylphenyl)-4-oxo-2H-1-benzopyran-7-yl ester (3d)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
3.73 (s, 3H, Ar–O–CH3), 4.12–4.25 (m, Ar–CH–CO),
4.60–4.88 (m, 2H, Ar–O–CH2–CH), 6.76–6.94 (m, 5H,
Ar-H), 7.18–7.34 (m, 1H, Ar-H), 7.80–7.90 (dd,
J ¼ 0:56 Hz, J ¼ 8:40 Hz, 1H, Ar-H); MS (ESI) m=e 354
(Mþ).
The title compound was prepared from 2d by a method
similar to that described for 3a (yield ¼ 75%).
1H NMR (200 MHz, DMSO-d6) 1.03 (s, 9H, 3CH3),
2.28 (s, 3H, Ar–CH3), 4.10–4.20 (dd, J ¼ 5:11 Hz,
J ¼ 9:02 Hz, Ar–CH–CO), 4.56–4.88 (m, 2H, Ar–CH2–
CH–CO), 6.81–6.90 (d+m, J ¼ 2:10 Hz, 2H, Ar-H), 7.15
(s, 4H, Ar-H), 7.81–7.88 (dd, J ¼ 0:60 Hz, J ¼ 8:11 Hz,
1H, Ar-H); MS (ESI) m=e 338 (Mþ).
6.18. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(2-methoxyphenyl)-4-[[4-(phenylmethoxy)phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (5a)
6.14. 2,2-Dimethyl-propanoic acid 3-(4-butoxyphenyl)-
3,4-dihydro-4-oxo-2H-1-benzopyran-7-yl ester (3e)
A solution of 4 (6 g; 25.8 mmol) in dry THF (37 mL) was
added dropwise to Mg turnings (1.8 g; 75 mmol) heating
to reflux. The reaction was carried out under nitrogen
flux. On completion of the addition, the mixture was left
to reach 30 ꢁC, and then further cooled to )20 ꢁC. A
solution of compound 3a (3.5 g; 10 mmol) in dry THF
(20 mL) was added dropwise in 20 min keeping
t ¼ À20 ꢁC. On completion of the addition, the mixture
was kept stirred until rt was reached, and it was then
quenched with water (5 mL) and filtered.
The title compound was prepared from 2e by a method
similar to that described for 3a (yield ¼ 60%).
1H NMR (200 MHz, DMSO-d6) 0.88–0.99 (t,
J ¼ 7:23 Hz, 3H, CH3), 1.30 (s, 9H, C–[CH3]), 1.33–1.53
(m, 2H, CH3–CH2–CH2), 1.58–1.78 (m, 2H, CH3–
CH2CH2), 3.88–3.99 (t, J ¼ 6:65 Hz, 2H, Ar–O–CH2),
4.08–4.18 (dd, J ¼ 5:21 Hz, J ¼ 9:26 Hz, 1H, Ar–CH–
CO), 4.59–4.82 (m, 2H, Ar–O–CH2–CH), 6.80–6.94 (m,
4H, Ar-H), 7.15–7.22 (d, J ¼ 8:67 Hz, 2H, Ar-H), 7.81–
The solution was evaporated to dryness under vacuum,
and ethyl acetate (20 mL) was added to the residue; the

3772
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
Wurtz coupling by-product of the Grignard reaction ([[4-
[2-[4-(phenylmethoxy)phenyl]ethyl]phenoxy]methyl]ben-
zene) crystallized and was filtered off. The solution
was evaporated to dryness and purified by flash chro-
matography (petroleum ether/ethyl acetate ¼ from 90/10
to 60/40). Compound 5a (1.85 g, 3.35 mmol) were
obtained as a viscous oil (yield ¼ 34%).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.85–3.00 (d, J ¼ 14:03 Hz, 1H, Ar–CH–C–OH), 3.25–
3.40 (d, J ¼ 14:03 Hz, 1H, Ar–CH–C–OH), 3.58–3.70
(s+m, 4H, Ar–CH–CH2, Ar–O–CH3), 3.98–4.34 (m, 2H,
Ar–O–CH2–CH), 5.02 (s, 2H, Ar–O–CH2–Ar), 5.40 (s,
1H, C–OH), 6.47–6.51 (d, J ¼ 2:47 Hz, 1H, Ar-H),
6.62–6.71 (dd, J ¼ 2:47 Hz, J ¼ 8:52 Hz, 1H, Ar-H),
6.73–6.77 (s, 4H, Ar-H), 6.80–7.00 (m, 2H, Ar-H), 7.15–
7.47 (m, 6H, Ar-H), 7.53–7.62 (d, J ¼ 8:52 Hz, 1H, Ar-
H), 7.66–7.74 (dd, J ¼ 1:65 Hz, J ¼ 7:70 Hz, 1H, Ar-H);
MS (ESI) m=e 552 (Mþ).
6.21. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydro-
xy-3-(4-methylphenyl)-4-[[4-(phenylmethoxy)phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (5d)
The title compound was prepared in 30% yield from 3d
by a method similar to that described for 5a.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH2),
2.27 (s, 3H, Ar–CH3), 2.66–2.82 (d, J ¼ 14:46 Hz, 1H,
Ar–CH–C–OH), 3.00–3.14 (dd, J ¼ 3:43 Hz, J ¼
9:56 Hz, 1H, Ar–CH–C–OH), 3.16–3.30 (d, J ¼
14:46 Hz, 1H, Ar–CH–C–OH), 3.99–4.12 (dd,
J ¼ 3:43 Hz, J ¼ 9:56 Hz, 1H, Ar–O–CH–CH–C–OH),
4.25–4.42 (t, J ¼ 10:05 Hz, 1H, Ar–O–CH–CH–C–OH),
5.02 (s, 2H, Ar–O–CH2–Ar), 5.29(s, 1H, C–O H), 6.50–
6.73 (2m, 6H, Ar-H), 7.03–7.40 (m, 9H, Ar-H), 7.51–
7.60 (d, J ¼ 8:58 Hz, 1H, Ar-H), 9.12 (s, 1H, Ar–OH);
MS (ESI) m=e 536 (Mþ).
6.22. 2,2-Dimethyl-propanoic acid 3-(4-butoxyphenyl)-
3,4-dihydro-4-hydroxy-4-[[4-(phenylmethoxy)phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (5e)
6.19. 2,2-Dimethyl-propanoic acid 4-[7-(2,2-dimethyl-1-
oxopropoxy)-3,4-dihydro-4-hydroxy-4-[[4-(phenylmeth-
oxy)phenyl]methyl]-2H-1-benzopyran-3-yl]phenyl ester
(5b)
The title compound was prepared in 35% yield from 3e
by a method similar to that described for 5a.
The title compound was prepared in 30% yield from 3b
by a method similar to that described for 5a.
1H NMR (200 MHz, DMSO-d6) 0.85–1.02 (t,
J ¼ 7:25 Hz, 3H, CH3), 1.30 (s, 9H, 3CH3), 1.34–1.54
(m, 2H, CH3–CH2–CH2), 1.59–1.78 (m, 2H, CH3–CH2–
CH2), 2.72–2.90 (d, J ¼ 14:50 Hz, 1H, Ar–CH–C–OH),
2.99–3.12 (dd, J ¼ 3:34 Hz, J ¼ 9:47 Hz, 1H, Ar–CH–
CH2–O), 3.22–3.39(d, J ¼ 14:50 Hz, 1H, Ar–CH–C–
OH), 3.86–4.15 (t+dd, J ¼ 6:41 Hz, J ¼ 3:34 Hz,
J ¼ 10:59Hz, 3H, Ar–O–C H2–CH2, Ar–O–CH–CH–
Ar), 4.24–4.43 (t, J ¼ 10:03 Hz, 1H, Ar–O–CH–CH–
Ar), 4.97–5.05 (s, 2H, Ar–O–CH2–Ar), 5.34 (s, 1H, C–
OH), 6.50–6.57 (d, J ¼ 2:23 Hz, 1H, Ar-H), 6.65–6.74
(dd, J ¼ 2:23 Hz, J ¼ 8:36 Hz, 1H, Ar-H), 6.77–6.89(br,
6H, Ar-H), 7.14–7.24 (d, J ¼ 8:92 Hz, 2H, Ar-H), 7.30–
7.47 (m, 5H, Ar-H), 7.52–7.63 (d, J ¼ 8:36 Hz, 1H, Ar-
H); MS (ESI) m=e 594 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.30–1.31 (2s, 18H,
6CH3), 2.75–2.88 (d, J ¼ 14:52 Hz, 1H, Ar–CH–C–OH),
3.08–3.20 (dd, J ¼ 3:41 Hz, J ¼ 11:12 Hz, 1H, OH–C–
CHAr), 3.26–3.39(d, J ¼ 14:52 Hz, 1H, Ar–CH–C–
OH), 4.08–4.44 (dd+t, J ¼ 3:41 Hz, J ¼ 11:12 Hz, 1H,
Ar–O–CH2–CH), 5.02 (s, 2H, Ar–O–CH2–Ar), 5.48 (s,
1H, Ar–CH2–COH), 6.53–6.57 (d, J ¼ 2:49Hz, 1H, Ar-
H), 6.65–6.74 (dd, J ¼ 2:49Hz, J ¼ 8:40 Hz, 1H, Ar-H),
6.78–6.85 (br, 3H, Ar-H), 6.96–7.05 (d, J ¼ 8:62 Hz, 2H,
Ar-H), 7.24–7.47 (m, 8H, Ar-H), 7.51–7.61 (d,
J ¼ 8:62 Hz, 1H, Ar-H); MS (ESI) m=e 622 (Mþ).
6.20. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(4-methoxyphenyl)-4-[[4-(phenylmethoxy)phenyl]-meth-
yl]-2H-1-benzopyran-7-yl ester (5c)
6.23. 2,2-Dimethyl-propanoic acid 3-(3,4-dimethoxyphen-
yl)-3,4-dihydro-4-hydroxy-4-[[4-(phenylmethoxy)phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (5f)
The title compound was prepared in 50% yield from 3c
by a method similar to that described for 5a.
The title compound was prepared in 35% yield from 3f
by a method similar to that described for 5a.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.74–2.88 (d, J ¼ 14:19Hz, 1H, Ar–C H–C–OH), 2.98–
3.12 (dd, J ¼ 3:60 Hz, J ¼ 9:91 Hz, 1H, O–CH2–CH–
Ar), 3.22–3.37 (d, J ¼ 14:19Hz, 1H, Ar–C H–C–OH),
3.73 (s, 3H, Ar–O–CH3), 4.00–4.13 (dd, J ¼ 3:60 Hz,
J ¼ 9:91 Hz, 1H, O–CH–CH–Ar), 4.25–4.43 (t,
J ¼ 10:14 Hz, 1H, O–CH–CH–Ar), 5.02 (s, 2H, Ar–O–
CH2–Ar), 5.35 (s, 1H, Ar–CH2–C–OH), 6.51–6.55 (d,
J ¼ 2:26 Hz, 1H, Ar-H), 6.65–6.73 (dd, J ¼ 2:26 Hz,
J ¼ 8:56 Hz, 1H, Ar-H), 6.78–6.90 (s+d, J ¼ 8:78 Hz,
6H, Ar-H), 7.16–7.25 (d, J ¼ 8:78 Hz, 2H, Ar-H), 7.29–
7.44 (m, 5H, Ar-H), 7.53–7.61 (d, J ¼ 8:56 Hz, Ar-H);
MS (ESI) m=e 552 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.77–2.90 (d, J ¼ 14:10 Hz, 1H, Ar–CH–C–OH), 2.98–
3.10 (dd, J ¼ 3:52 Hz, J ¼ 10:14 Hz, 1H, Ar–O–CH2–
CH–C–OH), 3.27–3.40 (d, J ¼ 14:10 Hz, 1H, Ar–CH–
C–OH), 3.66 (s, 3H, Ar–O–CH3), 3.73 (s, 3H, Ar–O–
CH3), 4.02–4.15 (m, 1H, Ar–OCH–CH–C–OH), 4.29–
4.42 (t, J ¼ 10:36 Hz, 1H, Ar–O–CH–CH–C–OH), 5.02
(s, 2H, Ar–O–CH2–Ar), 5.34 (s, 1H, C–OH), 6.52–6.57
(d, J ¼ 2:21 Hz, 1H, Ar-H), 6.65–6.73 (dd, J ¼ 2:21 Hz,
J ¼ 8:37 Hz, 1H, Ar-H), 6.77–6.91 (m, 8H, Ar-H), 7.31–
7.43 (m, 4H, Ar-H), 7.53–7.61 (d, J ¼ 8:37 Hz, 1H, Ar-
H); MS (ESI) m=e 582 (Mþ).

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3773
6.24. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-phenyl-4-[[4-(phenylmethoxy)phenyl]methyl]-2H-1-
benzopyran-7-yl ester (5g)
7.63 (d, J ¼ 8:57 Hz, 1H, Ar-H), 7.66–7.76 (d,
J ¼ 6:49Hz, 1H, Ar- H), 9.10 (s, 1H, Ar–OH); MS (ESI)
m=e 462 (Mþ).
The title compound was prepared in 30% yield from 3g
by a method similar to that described for 5a.
6.27. 2,2-Dimethyl-propanoic acid 4-[7-(2,2-dimethyl-1-
oxopropoxy)-3,4-dihydro-4-hydroxy-4-[(4-hydroxyphenyl)-
methyl]-2H-1-benzopyran-3-yl]phenyl ester (6b)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.75–2.90 (d, J ¼ 14:38 Hz, 1H, Ar–CH–C–OH), 3.06–
3.20 (dd, J ¼ 3:53 Hz, J ¼ 9:22 Hz, 1H, Ar–CH–CH2–
O), 3.24–3.40 (d, J ¼ 14:38 Hz, 1H, Ar–CH–C–OH),
4.06–4.21 (dd, J ¼ 3:52 Hz, J ¼ 10:85 Hz, 1H, Ar–
OCH–CH–Ar), 4.30–4.47 (t, J ¼ 10:04 Hz, 1H, Ar–O–
CH–CH–Ar), 5.02 (s, 2H, Ar–O–CH2–Ar), 5.41 (s, 1H,
C–OH), 6.51–6.59(d, J ¼ 2:17 Hz, 1H, Ar-H), 6.65–6.75
(dd, J ¼ 2:17 Hz, J ¼ 8:68 Hz, 1H, Ar-H), 6.82 (s, 4H,
Ar-H), 7.20–7.48 (m, 10H, Ar-H), 7.52–7.63 (d,
J ¼ 8:68 Hz, 1H, Ar-H); MS (ESI) m=e 522 (Mþ).
The title compound was prepared in quantitative yield
from 5b by a method similar to that described for 6a.
1H NMR (200 MHz, DMSO-d6) 1.30–1.31 (2s, 18H,
6CH3), 2.70–2.82 (d, J ¼ 13:20 Hz, 1H, Ar–CH–C–OH),
3.11–3.21 (dd, J ¼ 3:21 Hz, J ¼ 10:35 Hz, 1H, OH–C–
CH–Ar), 3.23–3.35 (d, J ¼ 13:20 Hz, 1H, Ar–CH–C–
OH), 4.07–4.44 (dd+t, J ¼ 3:21 Hz, J ¼ 10:35 Hz, 2H,
Ar–O–CH2–CH), 5.43 (s, 1H, Ar–CH2–C–OH), 6.50–
6.75 (m, 6H, Ar-H), 6.97–7.06 (d, J ¼ 8:57 Hz, 2H, Ar-
H), 7.27–7.38 (d, J ¼ 8:57 Hz, 2H, Ar-H), 7.52–7.62 (d,
J ¼ 8:56 Hz, 1H, Ar-H), 9.14 (s, 1H, Ar–OH); MS (ESI)
m=e 532 (Mþ).
6.25. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydr-
oxy-3-(3-methoxyphenyl)-4-[[4-(phenylmethoxy)phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (5h)
The title compound was prepared in 32% yield from 3h
by a method similar to that described for 5a.
6.28. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
4-[(4-hydroxyphenyl)methyl]-3-(4-methoxyphenyl)-2H-1-
benzopyran-7-yl ester (6c)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.78–2.93 (d, J ¼ 14:36 Hz, 1H, Ar–CH–C–OH), 3.00–
3.15 (dd, J ¼ 3:33 Hz, J ¼ 8:97 Hz, 1H, Ar–CH–CH2–
O), 3.26–3.40 (d, J ¼ 14:36 Hz, 1H, Ar–CH–C–OH),
3.70 (s, 3H, Ar–O–CH3), 4.07–4.20 (dd, J ¼ 3:33 Hz,
J ¼ 10:00 Hz, 1H, Ar–O–CH–CH–Ar), 4.28–4.45 (t,
J ¼ 9:74 Hz, 1H, Ar–O–CH–CH–Ar), 4.98–5.05 (s, 2H,
Ar–O–CH2–Ar), 5.40 (s, 1H, C–OH), 6.53–6.58 (d,
J ¼ 2:31 Hz, 1H, Ar-H), 6.65–6.75 (dd, J ¼ 2:31 Hz,
J ¼ 8:47 Hz, 1H, Ar-H), 6.80–6.90 (m, 4H, Ar-H), 7.15–
7.25 (t, J ¼ 8:21 Hz, 1H, Ar-H), 7.30–7.45 (m, 3H, Ar-
H), 7.52–7.60 (d, J ¼ 8:47 Hz, 1H, Ar-H); MS (ESI) m=e
552 (Mþ).
The title compound was prepared in quantitative yield
from 5c by a method similar to that described for 6a.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.67–2.81 (d, J ¼ 14:39Hz, 1H, Ar–C H–C–OH), 2.99–
3.12 (dd, J ¼ 3:47 Hz, J ¼ 9:68 Hz, 1H, O–CH2–CH–C–
OH), 3.24–3.34 (d, J ¼ 14:39Hz, 1H, Ar–C H–C–OH),
3.73 (s, 3H, Ar–O–CH3), 3.99–4.10 (dd, J ¼ 3:47 Hz,
J ¼ 9:68 Hz, 1H, O–CH–CH–Ar), 4.24–4.39(t,
J ¼ 10:42 Hz, 1H, O–CH–CH–Ar), 5.28 (s, 1H, Ar–
CH2–C–OH), 6.49–6.58 (m, 3H, Ar-H), 6.63–6.73 (m,
3H, Ar-H), 6.81–6.89(d, J ¼ 8:68 Hz, 2H, Ar-H), 7.17–
7.25 (d, J ¼ 8:68 Hz, 2H, Ar-H), 7.51–7.60 (d,
J ¼ 8:70 Hz, 1H, Ar-H); MS (ESI) m=e 462 (Mþ).
6.26. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydr-
oxy-4-[(4-hydroxyphenyl)methyl]-3-(2-methoxyphenyl)-
2H-1-benzopyran-7-yl ester (6a)
Compound 5a (3.5 g; 6.3 mmol) was dissolved in ethyl
acetate (45 mL), Pd 5%/C (50% water) (1.8 g) was added
and the mixture was hydrogenated (40 psi, rt) for 3 h.
After filtering, the solution was evaporated under vac-
uum and diethyl ether was added and then completely
evaporated. A white foam of the title compound (2.9g;
6.3 mmol) was obtained (quantitative yield). The prod-
uct was used for the subsequent steps without any fur-
ther purification.
6.29. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
4-[(4-hydroxyphenyl)methyl]-3-(4-methylphenyl)-2H-1-
benzopyran-7-yl ester (6d)
The title compound was prepared in quantitative yield
from 5d by a method similar to that described for 6a.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH2),
2.27 (s, 3H, Ar–CH3), 2.66–2.82 (d, J ¼ 14:46 Hz, 1H,
Ar–CH–C–OH), 3.00–3.14 (dd, J ¼ 3:43 Hz, J ¼
9:56 Hz, 1H, Ar–CH–C–OH), 3.16–3.30 (d, J ¼
14:46 Hz, 1H, Ar–CH–C–OH), 3.99–4.12 (dd,
J ¼ 3:43 Hz, J ¼ 9:56 Hz, 1H, Ar–O–CH–CH–C–OH),
4.25–4.42 (t, J ¼ 10:05 Hz, 1H, Ar–O–CH–CH–C–OH),
5.29(s, 1H, C–O H), 6.50–6.73 (2m, 6H, Ar-H), 7.03–
7.23 (m, 4H, Ar-H), 7.51–7.60 (d, J ¼ 8:58 Hz, 1H, Ar-
H), 9.12 (s, 1H, Ar–OH); MS (ESI) m=e 446 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.82–2.97 (d, J ¼ 14:29Hz, 1H, Ar–C H–C–OH), 3.20–
3.37 (d, J ¼ 14:29Hz, 1H, Ar–C H–C–OH), 3.61–3.73
(m+s, 4H, Ar–CH–CH2, Ar–O–CH3), 3.98–4.38 (m, 2H,
Ar–O–CH2–CH), 5.33 (s, 1H, C–OH), 6.42–6.74 (m, 6H,
Ar-H), 6.80–6.90 (m, 1H, Ar-H), 6.93–7.04 (d,
J ¼ 7:53 Hz, 1H, Ar-H), 7.16–7.30 (m, 1H, Ar-H), 7.52–

3774
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
6.30. 2,2-Dimethyl-propanoic acid 3-(4-butoxyphenyl)-
3,4-dihydro-4-hydroxy-4-[(4-hydroxyphenyl)methyl]-2H-
1-benzopyran-7-yl ester (6e)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.72–2.86 (d, J ¼ 14:38 Hz, 1H, Ar–CH–C–OH), 3.00–
3.15 (dd, J ¼ 3:23 Hz, J ¼ 9:10 Hz, 1H, Ar–CH–CH2–
O), 3.19–3.35 (d, J ¼ 14:38 Hz, 1H, Ar–CH–C–OH),
3.70 (s, 3H, Ar–O–CH3), 4.05–4.18 (dd, J ¼ 3:23 Hz,
J ¼ 10:80 Hz, 1H, Ar–O–CH–CH–Ar), 4.28–4.44 (t,
J ¼ 9:70 Hz, 1H, Ar–O–CH–CH–Ar), 5.32 (s, 1H, C–
OH), 6.50–6.60 (m, 3H, Ar-H), 6.65–6.75 (m, 3H, Ar-
H), 6.80–6.90 (m, 3H, Ar-H), 7.13–7.26 (t, J ¼ 7:93 Hz,
1H, Ar-H), 7.51–7.60 (d, J ¼ 8:80 Hz, 1H, Ar-H), 9.13
(s, 1H, Ar–OH); MS (ESI) m=e 462 (Mþ).
The title compound was prepared in quantitative yield
from 5e by a method similar to that described for 6a.
1H NMR (200 MHz, DMSO-d6) 0.87–0.99 (t,
J ¼ 7:22 Hz, 3H, CH3), 1.30 (s, 9H, C–[CH3]3), 1.35–
1.53 (m, 2H, CH3–CH2), 1.58–1.77 (m, 2H, CH3–CH2–
CH2), 2.70–2.80 (d, J ¼ 14:13 Hz, 1H, Ar–CH–C–OH),
2.98–3.10 (dd, J ¼ 3:31 Hz, J ¼ 9:62 Hz, 1H, Ar–O–
CH2–CH–C–OH), 3.18–3.30 (d, J ¼ 14:13 Hz, Ar–CH–
C–OH), 3.85–4.13 (t+m, J ¼ 6:62 Hz, 3H, Ar–O–CH2,
Ar–O–CH–C–OH), 4.25–4.40 (t, J ¼ 10:22 Hz, 1H, Ar–
O–CH–CH–C–OH), 5.28 (s, 1H, C–OH), 6.48–6.60 (m,
3H, Ar-H), 6.65–6.73 (m, 3H, Ar-H), 6.80–6.88 (d,
J ¼ 8:72 Hz, 2H, Ar-H), 7.15–7.24 (d, J ¼ 8:72 Hz, 2H,
Ar-H), 7.53–7.60 (d, J ¼ 8:70 Hz, 1H, Ar-H), 9.12 (s,
1H, Ar–OH); MS (ESI) m=e 504 (Mþ).
6.34. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydr-
oxy-3-(2-methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-yl ester (7a)
To a solution of 6a (2.9g; 6.3 mmol) in acetone (30 mL),
K2CO3 (1.3 g; 9.4 mmol) was added. The solution was
taken to reflux and N-chloroethyl-piperidine (1.12 g of
free base, previously extracted from the corresponding
hydrochloride salt; 7.6 mmol) was added. The reaction
was completed in 4 h; the salt was filtered off and the
solution was evaporated to dryness. The residue was
dissolved in ethyl acetate and extracted with a 5%
CH3COOH aqueous solution. The organic layer was
dried (Na2SO4) and evaporated to dryness. The residue
was purified by flash chromatography (methylene chlo-
ride/methanol ¼ from 99/1 to 90/10) obtaining the title
6.31. 2,2-Dimethyl-propanoic acid 3-(3,4-dimethoxyphen-
yl)-3,4-dihydro-4-hydroxy-4-[(4-hydroxyphenyl)methyl]-
2H-1-benzopyran-7-yl ester (6f)
The title compound was prepared in quantitative yield
from 5f by a method similar to that described for 6a.
1H NMR (200MHz, DMSO-d6) 1.30 (s, 9H, 3CH3), 2.70–
2.82 (d, J ¼ 14:24 Hz, 1H, Ar–CH–CH–C–OH), 2.96–
3.10 (dd, J ¼ 3:49Hz, J ¼ 9:58 Hz, 1H, Ar–O–CH2–CH–
C–OH), 3.21–3.32 (d, J ¼ 14:24 Hz, 1H, Ar–CH–CH–C–
OH), 3.67 (s, 3H, Ar–O–CH3), 3.73 (s, 3H, Ar–O–CH3),
4.00–4.12 (m, 1H, Ar–O–CH–CH–C–OH), 4.25–4.42 (t,
J ¼ 10:25 Hz, 1H, Ar–O–CH–CH–C–OH), 5.27 (s, 1H,
C–OH), 6.49–6.58 (m, 3H, Ar-H), 6.61–6.73 (m, 3H, Ar-
H), 6.80–6.91 (3H, Ar-H), 7.51–7.60 (d, J ¼ 8:43 Hz, 1H,
Ar-H), 9.12 (s, 1H, Ar–OH); MS (ESI) m=e 492 (Mþ).
compound (2.17 g; 3.78 mmol) as
a
yellow oil
(yield ¼ 60%).
1H NMR (200 MHz, DMSO-d6) 1.28 (s, 9H, 3CH3),
1.35–1.73 (m, 6H, 3CH2), 2.76–2.90 (m, 4H, N–(CH2)2),
2.92–3.10 (m, 2H, N–CH2), 3.28–3.42 (d, J ¼ 13:84 Hz,
1H, Ar–CH–CH2), 3.57–3.76 (m+s, 5H, Ar–CH2–C–
OH, Ar–O–CH3), 3.97–4.40 (m, 4H, Ar–O–CH2–CH,
Ar–O–CH2–CH2), 6.47–6.53 (d, J ¼ 2:30 Hz, 1H, Ar-
H), 6.60–7.04 (m, 7H, Ar-H), 7.16–7.30 (m, 1H, Ar-H),
7.54–7.63 (d, J ¼ 8:60 Hz, 1H, Ar-H), 7.68–7.80 (dd,
J ¼ 1:47 Hz, J ¼ 7:55 Hz, 1H, Ar-H); MS (ESI) m=e 573
(Mþ).
6.32. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydr-
oxy-4-[(4-hydroxyphenyl)methyl]-3-phenyl-2H-1-benzo-
pyran-7-yl ester (6g)
The title compound was prepared in quantitative yield
from 5g by a method similar to that described for 6a.
6.35. 2,2-Dimethyl-propanoic acid 4-[7-(2,2-dimethyl-1-
oxopropoxy)-3,4-dihydro-4-hydroxy-4-[[4-[2-(1-piperid-
inyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-3-yl]phenyl
ester (7b)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
2.69–2.84 (d, J ¼ 14:27 Hz, 1H, Ar–CH–C–OH), 3.05–
3.17 (dd, J ¼ 3:44 Hz, J ¼ 9:25 Hz, 1H, Ar–O–CH2–
CH), 3.19–3.36 (d, J ¼ 14:27 Hz, 1H, Ar–CH–C–OH),
4.05–4.16 (dd, J ¼ 3:44 Hz, J ¼ 10:83 Hz, 1H, Ar–O–
CH–CH–Ar), 4.28–4.43 (t, J ¼ 10:83 Hz, Ar–O–CH–
CH–Ar), 5.35 (s, 1H, C–OH), 6.48–6.76 (m, 6H, Ar-H),
7.18–7.36 (br, 5H, Ar-H), 7.50–7.60 (d, J ¼ 8:72 Hz, 1H,
Ar-H), 9.15 (s, 1H, Ar–OH); MS (ESI) m=e 432 (Mþ).
The title compound was prepared from 6b by a method
similar to that described for 7a (yield ¼ 50%).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 18H, 6CH3),
1.38–1.80 (m, 6H, 3CH2), 2.77–2.89(d, J ¼ 14:58 Hz,
1H, Ar–CH–C–OH), 2.90–3.03 (br, 4H, N–[CH2]2),
3.07–3.23 (m, 3H, N–CH2, OH–C–CH–Ar), 3.26–3.41
(d, J ¼ 14:58 Hz, 1H, Ar–CH), 4.08–4.45 (m+t,
J ¼ 5:13 Hz, 4H, Ar–O–CH2–CH2, Ar–OCH2), 5.44–
5.61 (br, 1H, Ar–CH2–C–OH), 6.52–6.58 (d,
J ¼ 2:44 Hz, 1H, Ar-H), 6.65–6.89(m+d, J ¼ 5:94 Hz,
5H, Ar-H), 6.95–7.05 (d, J ¼ 8:64 Hz, 2H, Ar-H), 7.26–
7.37 (d, J ¼ 8:64 Hz, 2H, Ar-H), 7.53–7.62 (d,
J ¼ 8:38 Hz, 1H, Ar-H); MS (ESI) m=e 643 (Mþ).
6.33. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydr-
oxy-4-[(4-hydroxyphenyl)methyl]-3-(3-methoxyphenyl)-
2H-1-benzopyran-7-yl ester (6h)
The title compound was prepared in quantitative yield
from 5h by a method similar to that described for 6a.

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3775
6.36. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(4-methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (7c)
6.39. 2,2-Dimethyl-propanoic acid 3-(3,4-dimethoxyphen-
yl)-3,4-dihydro-4-hydroxy-4-[[4-[2-(1-piperidinyl)etho-
xy]-phenyl]methyl]-2H-1-benzopyran-7-yl ester
(compound 7f)
The title compound was prepared from 6c by a method
similar to that described for 7a (yield ¼ 54%).
The title compound was prepared from 6f by a method
similar to that described for 7a (yield ¼ 62%).
1H NMR (200 MHz, DMSO-d6+CF3COOH-d1) 1.30 (s,
9H, 3CH3), 1.32–1.57 (m, 6H, 3CH2), 2.32–2.45 (t,
J ¼ 4:56 Hz, 4H, N–[CH2]2), 2.54–2.64 (t, J ¼ 6:08 Hz,
2H, N–CH2), 2.72–2.87 (d, J ¼ 13:93 Hz, Ar–CH–C–
OH), 2.99–3.10 (dd, J ¼ 3:30 Hz, J ¼ 9:62 Hz, 1H, Ar–
CH–CH2–O), 3.17–3.31 (d, J ¼ 13:93 Hz, 1H, Ar–CH–
C–OH), 3.70 (s, 3H, Ar–O–CH3), 3.88–4.10 (t,
J ¼ 5:82 Hz, 3H, Ar–O–CH2–CH2–N, O–CH–CH–C–
OH), 4.19–4.37 (t, J ¼ 10:64 Hz, 1H, O–CH–CH–C–
OH), 6.45–6.53 (d, J ¼ 2:54 Hz, 1H, Ar-H), 6.60–6.90
(m, 7H, Ar-H), 7.08–7.22 (d, J ¼ 8:87 Hz, 2H, Ar-H),
7.45–7.57 (d, J ¼ 8:61 Hz, 1H, Ar-H); MS (ESI) m=e 573
(Mþ).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
1.38–1.71 (m, 6H, 3CH2), 2.71–2.83 (m, 4H, N–[CH2]2),
2.85–3.10 (m, 4H, N–CH2, Ar–CH–CH–C–OH, Ar–O–
CH2–CH–C–OH), 3.27–3.40 (d, J ¼ 13:89Hz, 1H, Ar–
CH–C–OH), 3.68 (s, 3H, Ar–O–CH3), 3.73 (s, 3H, Ar–
O–CH3), 4.03–4.21 (m, 3H, Ar–O–CH–CH–C–OH, Ar–
O–CH2–CH2–N), 4.28–4.45 (t, J ¼ 10:14 Hz, 1H, Ar–
O–CH–CH–C–OH), 6.52–6.55 (d, J ¼ 2:21 Hz, 1H, Ar-
H), 6.65–6.73 (dd, J ¼ 2:21 Hz, J ¼ 8:37 Hz, 1H, Ar-H),
6.75–6.89(m, 7H, Ar- H), 7.53–7.61 (d, J ¼ 8:37 Hz, 1H,
Ar-H); MS (ESI) m=e 603 (Mþ).
6.40. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]-methyl]-
2H-1-benzopyran-7-yl ester (7g)
6.37. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(4-methylphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (7d)
The title compound was prepared from 6g by a
method similar to that described for 7a (yield ¼
50%).
The title compound was prepared from 6d by a method
similar to that described for 7a (yield ¼ 50%).
1H NMR (200 MHz, DMSO-d6) 1.21–1.57 (s+m, 15H,
3CH3, 3CH2), 2.33–2.45 (t, J ¼ 5:90 Hz, 4H, N–[CH2]2),
2.54–2.65 (t, J ¼ 5:90 Hz, 2H, N–CH2), 2.75–2.88 (d,
J ¼ 14:34 Hz, 1H, Ar–CH–C–OH), 3.05–3.17 (dd,
J ¼ 3:38 Hz, J ¼ 9:28 Hz, 1H, Ar–O–CH2–CH), 3.23–
3.36 (d, J ¼ 14:34 Hz, 1H, Ar–CH–C–OH), 3.91–4.03 (t,
J ¼ 5:90 Hz, 2H, Ar–O–CH2–CH2), 4.06–4.18 (dd,
J ¼ 3:37 Hz, J ¼ 10:69Hz, 1H, Ar–O–C H–CH–Ar),
4.28–4.46 (t, J ¼ 10:69Hz, 1H, Ar–O–C H–CH–Ar),
5.39(s, 1H, C–O H), 6.50–6.57 (d, J ¼ 2:25 Hz, 1H,
Ar-H), 6.10–6.87 (m, 5H, Ar-H), 7.18–7.33 (br, 5H,
Ar-H), 7.52–7.60 (d, J ¼ 8:44 Hz, 1H, Ar-H); MS (ESI)
m=e 543 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.29(9H, 3C H3), 1.33–
1.65 (m, 6H, 3CH2), 2.27 (s, 3H, Ar–CH3), 2.54–2.71 (m,
4H, N–[CH2]2), 2.73–2.91 (m, 3H, Ar–CH–C–OH, N–
CH2), 3.00–3.13 (dd, J ¼ 3:54 Hz, J ¼ 9:72 Hz, 1H, Ar–
O–CH2–CH–C–OH), 3.23–3.37 (d, J ¼ 14:15 Hz, Ar–
CH–C–OH), 4.00–4.16 (m, 3H, Ar–O–CH–CH–C–OH,
Ar–O–CH2–CH2–N), 4.27–4.43 (t, J ¼ 10:32 Hz, Ar–O–
CH–CH–C–OH), 5.25–5.48 (br, 1H, C–OH), 6.51–6.54
(d, J ¼ 2:36 Hz, 1H, Ar-H), 6.65–6.72 (dd, J ¼ 2:36 Hz,
J ¼ 8:84 Hz, 1H, Ar-H), 6.73–6.84 (m, 4H, Ar-H), 7.00–
7.20 (m, 4H, Ar-H), 7.52–7.61 (d, J ¼ 8:84 Hz, 1H, Ar-
H); MS (ESI) m=e 557 (Mþ).
6.38. 2,2-Dimethyl-propanoic acid 3-(4-butoxyphenyl)-
3,4-dihydro-4-hydroxy-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-yl ester (7e)
6.41. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydro-
xy-3-(3-methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-yl ester (7h)
The title compound was prepared from 6e by a method
similar to that described for 7a (yield ¼ 58%).
The title compound was prepared from 6h by a method
similar to that described for 7a (yield ¼ 65%).
1H NMR (200 MHz, DMSO-d6) 0.87–0.99 (t,
J ¼ 7:39Hz, 3H, C H3), 1.30 (s, 9H, C–[CH3]3), 1.33–
1.76 (m, 10H, CH3–[CH2]2, 3CH2), 2.57–2.72 (m, 4H,
2CH2–N), 2.76–2.90 (m, 3H, CH2–N, Ar–CH–C–OH),
2.98–3.11 (m, 1H, Ar–CH–C–OH), 3.22–3.36 (m, 1H,
Ar–CH–C–OH), 3.87–3.99 (t, J ¼ 6:39Hz, 2H, Ar–O–
CH2–CH2–N), 4.02–4.15 (m, 3H, Ar–O–CH2, Ar–O–
CH–CH–C–OH), 4.25–4.41 (t, J ¼ 10:09Hz, 1H, Ar–
O–CH–CH–C–OH), 5.26–5.42 (br, 1H, C–OH), 6.51–
6.54 (d, J ¼ 2:21 Hz, 1H, Ar-H), 6.65–6.95 (m, 7H, Ar-
H), 7.15–7.25 (d, J ¼ 8:62 Hz, 2H, Ar-H), 7.52–7.60 (d,
J ¼ 8:61 Hz, 1H, Ar-H); MS (ESI) m=e 615 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
1.34–1.70 (m, 6H, 3CH2), 2.62–2.77 (m, 4H, N–(CH2)2),
2.80–2.95 (m, 3H, Ar–CH–C–OH, N–CH2), 3.05–3.16
(dd, J ¼ 3:34 Hz, J ¼ 9:24 Hz, 1H, Ar–CH–CH2–O),
3.27–3.40 (d, J ¼ 14:10 Hz, 1H, Ar–CH–C–OH), 3.70 (s,
3H, Ar–OCH3), 4.05–4.20 (m, 3H, Ar–O–CH–CH–Ar,
Ar–O–CH2–CH2–N), 4.30–4.46 (t, J ¼ 9:24 Hz, 1H, Ar–
O–CH–CH–Ar), 6.53–6.57 (d, J ¼ 2:31 Hz, 1H, Ar-H),
6.65–6.95 (m, 8H, Ar-H), 7.11–7.26 (t, J ¼ 8:21 Hz, 1H,
Ar-H), 7.50–7.62 (d, J ¼ 8:72 Hz, 1H, Ar-H); MS (ESI)
m=e 573 (Mþ).

3776
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
6.42. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(4-methoxyphenyl)-4-[[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (7i)
7.27 (d, J ¼ 8:81 Hz, 2H, Ar-H), 7.50–7.62 (d,
J ¼ 8:80 Hz, 1H, Ar-H); MS (ESI) m=e 587 (Mþ).
The title compound was prepared from 6c and N-chlo-
roethyl-pyrrolidine by a method similar to that de-
scribed for 7a (yield ¼ 36%).
6.45. 2,2-Dimethyl-propanoic acid 4-[[4-[2-(hexahydro-1H-
azepin-1-yl)ethoxy]phenyl]methyl]-3,4-dihydro-4-hydroxy-
3-(4-methoxyphenyl)-2H-1-benzopyran-7-yl ester (7l)
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
1.80–1.93 (m, 4H, 2CH2), 1.73–1.90 (t, J ¼ 14:29Hz,
1H, Ar–CH–C–OH), 2.97–3.42 (2m, 8H, Ar–CH–C–
OH, Ar–CH–CH2–O, N(CH2)3), 3.73 (s, 3H, Ar–O–
CH3), 4.00–4.11 (dd, J ¼ 3:46 Hz, J ¼ 10:61 Hz, 1H,
Ar–O–CH–CH–Ar), 4.13–4.41 (m, 3H, Ar–O–CH–CH–
Ar, Ar–O–CH–CH2), 5.37 (s, 1H, C–OH), 6.48–6.56 (d,
J ¼ 2:17 Hz, 1H, Ar-H), 6.64–6.73 (dd, J ¼ 2:17 Hz,
J ¼ 8:44 Hz, 1H, Ar-H), 6.75–6.95 (m, 6H, Ar-H), 7.16–
7.25 (d, J ¼ 8:87 Hz, 2H, Ar-H), 7.53–7.61 (d,
J ¼ 8:44 Hz, 1H, Ar-H); MS (ESI) m=e 559(M þ).
The title compound was prepared from 6c and
2-(hexamethyleneimino)ethyl chloride by a method
similar to that described for 7a (yield ¼ 38%).
1H NMR (200MHz, DMSO-d6) 1.30 (s, 9H, 3CH3), 1.48–
1.82 (br, 6H, 3CH2), 2.75–2.88 (d, J ¼ 14:40 Hz, 1H, Ar–
CH–C–OH), 2.98–3.10 (dd, J ¼ 3:27 Hz, J ¼ 9:60 Hz,
1H, Ar–CH–CH2–O), 3.22–3.46 (br d, J ¼ 14:40 Hz, 7H,
Ar–CH–C–OH, N–[CH2]3), 3.73 (s, 3H, Ar–O–CH3),
4.00–4.12 (dd, br, J ¼ 3:27 Hz, J ¼ 10:25 Hz, 3H, Ar–O–
CH–CH, Ar–O–CH2–CH2), 4.25–4.40 (d, J ¼ 10:25 Hz,
1H, Ar–O–CH–CH), 5.36 (s, 1H, C–OH), 6.50–6.55 (d,
J ¼ 2:18 Hz, Ar-H), 6.65–6.90 (m, 7H, Ar-H), 7.16–7.26
(d, J ¼ 8:72 Hz, 2H, Ar-H), 7.52–7.62 (d, J ¼ 8:50 Hz,
1H, Ar-H); MS (ESI) m=e 587 (Mþ).
6.43. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-phenyl-4-[[4-[3-(1-piperidinyl)propoxy]phenyl]-methyl]-
2H-1-benzopyran-7-yl ester (7j)
6.46. 3,4-Dihydro-3-(2-methoxyphenyl)-4-[[4-[2-(1-piper-
idinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol
(8a)
The title compound was prepared from 6g and N-chlo-
ropropyl-piperidine by a method similar to that de-
scribed for 7a (yield ¼ 40%).
To a solution of 7a (3.6 g; 6.3 mmol) in methanol
(100 mL), K2CO3 (1.4 g; 10.1 mmol) was added and the
mixture was stirred at rt for 3 h. The volume of the
solution was then reduced under vacuum to 10 mL and
the solution was added dropwise to water (150 mL)
under stirring.
1H NMR (200 MHz, DMSO-d6) 1.30 (s, 9H, 3CH3),
1.31–1.58 (m, 6H, 3CH2), 1.69–1.89 (m, 2H, O–CH2–
CH2–CH2–Nþ), 2.22–2.39(m, 6H, N þ–[CH2]3), 2.74–
2.89(d, J ¼ 14:07 Hz, 1H, Ar–CH–C–OH), 3.06–3.17
(dd, J ¼ 3:37 Hz, J ¼ 9:51 Hz, 1H, OH–C–CH–Ar),
3.22–3.38 (d, J ¼ 14:07 Hz1H, Ar–CH–C–OH), 3.84–
3.97 (t, J ¼ 6:34 Hz, 2H, Ar–O–CH2–CH2), 4.05–4.19
(dd, J ¼ 3:37 Hz, J ¼ 9:51 Hz, 1H, Ar–O–CH–CH–Ar),
4.29–4.46 (t, J ¼ 10:50 Hz, 1H, Ar–O–CH–CH–Ar),
6.51–6.56 (d, J ¼ 2:38 Hz, 1H, Ar-H), 6.65–6.85 (m, 5H,
Ar-H), 7.17–7.34 (br, 5H, Ar-H), 7.51–7.60 (d,
J ¼ 8:52 Hz, 1H, Ar-H); MS (ESI) m=e 557 (Mþ).
The mixture was left at +4 ꢁC overnight and a white
amorphous solid precipitated. The solid was filtered,
washed with water (10 mL), and purified by flash chro-
matography (methylene chloride/methanol ¼ from 99/1
to 90/10) obtaining 2.8 g (5.7 mmol) of the title com-
pound (yield ¼ 90%) as a yellow oil.
1H NMR (200 MHz, DMSO-d6) 1.27–1.56 (m, 6H, 3CH2),
2.31–2.45 (t, J ¼ 5:38 Hz, 4H, N–(CH2)2), 2.53–2.63 (t,
J ¼ 6:01 Hz, 2H, N–CH2), 2.76–2.90 (d, J ¼ 13:9 2 Hz,
1H, Ar–CH–C–OH), 3.20–3.35 (d, J ¼ 13:9 2 Hz, 1H, Ar–
CH–COH), 3.50–3.70 (m+s, 4H, Ar–CH–CH2–O, Ar–O–
CH3), 3.82–4.03 (m, 3H, Ar–O–CH2–CH2, Ar–O–CH–
CH), 4.10–4.27 (t, J ¼ 10:12 Hz, 1H, Ar–O–CH–CH),
4.90–5.25 (br, 1H, C–OH), 6.00–6.15 (d, J ¼ 2:22 Hz, 1H,
Ar-H), 6.32–6.42 (dd, J ¼ 2:22 Hz, J ¼ 8:54 Hz, 1H, Ar-
H), 6.60–6.76 (m, 4H, Ar-H), 6.78–7.00 (m, 2H, Ar-H),
7.15–7.25 (m, 1H, Ar-H), 7.30–7.40 (d, J ¼ 8:85 Hz, 1H,
Ar-H), 7.72–7.82 (dd, J ¼ 1:58 Hz, J ¼ 7:59Hz, 1H, Ar-
H); MS (ESI) m=e 489(M þ).
6.44. 2,2-Dimethyl-propanoic acid 3,4-dihydro-4-hydroxy-
3-(4-methoxyphenyl)-4-[[4-[3-(1-piperidinyl)propoxy]phenyl]-
methyl]-2H-1-benzopyran-7-yl ester (7k)
The title compound was prepared from 6c and N-chlo-
ropropyl-piperidine by a method similar to that de-
scribed for 7a (yield ¼ 30%).
1H NMR (200 MHz, DMSO-d6) 1.22–1.56 (s+m, 15H,
3CH3, 3CH2), 1.70–1.90 (m, 2H, Ar–O–CH2–CH2),
2.22–2.40 (m, 6H, N–(CH2)3), 2.72–2.87 (d, J ¼
14:16 Hz, 1H, Ar–CH–C–OH), 2.98–3.12 (dd, J ¼
3:46 Hz, J ¼ 9:75 Hz, 1H, Ar–CH–CH2–O), 3.20–3.39
(d, J ¼ 14:16 Hz, 1H, Ar–CH–C–OH), 3.73 (s, 3H, Ar–
O–CH3), 3.83–3.97 (t, J ¼ 6:29Hz, 2H, Ar–O–C H2–
CH2), 4.00–4.13 (dd, J ¼ 2:83 Hz, J ¼ 10:07 Hz, 1H,
Ar–O–CH–CH–Ar), 4.24–4.43 (t, J ¼ 10:07 Hz, 1H,
Ar–O–CH–CH–Ar), 5.34 (s, 1H, C–OH), 6.48–6.50 (d,
J ¼ 2:20 Hz, 1H, Ar-H), 6.33–6.93 (m, 7H, Ar-H), 7.15–
6.47. 3,4-Dihydro-3-(4-hydroxyphenyl)-4-[[4-[2-(1-piper-
idinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol
(8b)
The title compound was prepared in 60% yield from 7b
by a method similar to that described for 8a.

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3777
1H NMR (200 MHz, DMSO-d6) 1.25–1.62 (m, 6H,
3CH2), 2.29–2.45 (m, 4H, 2CH2), 2.53–2.63 (t,
J ¼ 6:90 Hz, 2H, CH2–N), 2.64–2.78 (d, J ¼ 14:19Hz,
1H, Ar–CH–C–OH), 2.83–2.97 (dd, J ¼ 2:97 Hz,
J ¼ 10:89Hz, 1H, OH–C–C H–Ar), 3.13–3.34 (d,
1H NMR (200 MHz, DMSO-d6) 0.87–0.98 (t,
J ¼ 7:55 Hz, 3H, CH3), 1.27–1.55 (m, 8H, 4CH2), 1.60–
1.78 (m, 2H, CH2), 2.34–2.44 (t, J ¼ 4:84 Hz, 4H,
2CH2–N), 2.54–2.64 (t, J ¼ 6:04 Hz, 2H, CH2–N), 2.65–
2.75 (d, J ¼ 14:20 Hz, 1H, Ar–CH–C–OH), 2.88–3.00
(dd, J ¼ 3:32 Hz, J ¼ 10:27 Hz, 1H, Ar–O–CH2–CH),
3.18–3.31 (d, J ¼ 14:20 Hz, Ar–CH–C–OH), 3.82–4.03
(m, 5H, Ar–O–CH2–CH2–N, Ar–O–CH2–[CH2]2, Ar–
O–CH–CH–C–OH), 4.18–4.33 (t, J ¼ 10:58 Hz, 1H,
Ar–CH–CH–C–OH), 5.04 (s, 1H, C–OH), 6.11–6.16 (d,
J ¼ 2:42 Hz, 1H, Ar-H), 6.36–6.45 (dd, J ¼ 2:42 Hz,
J ¼ 8:46 Hz, 1H, Ar-H), 6.65–6.88 (m+d, J ¼ 8:77 Hz,
6H, Ar-H), 7.13–7.23 (d, J ¼ 8:77 Hz, 2H, Ar-H), 7.31–
7.40 (d, J ¼ 8:46 Hz, 1H, Ar-H), 9.34 (s, 1H, Ar-H); MS
(ESI) m=e 531 (Mþ).
J ¼ 14:19Hz,
1H, Ar–C H), 3.80–4.06
(dd+t,
J ¼ 2:97 Hz, J ¼ 10:89Hz, J ¼ 11:88 Hz, 3H, Ar–O–
CH2, Ar–O–CH–CH–Ar), 4.16–4.34 (t, J ¼ 11:88 Hz,
1H, Ar–O–CH–CH–Ar), 4.82–5.12 (br, 1H, Ar–CH2–
C–OH), 6.09–6.18 (d, J ¼ 1:98 Hz, 1H, Ar-H), 6.35–6.47
(dd, J ¼ 1:98 Hz, J ¼ 8:25 Hz, 1H, Ar-H), 6.59–6.81 (m,
6H, Ar-H), 7.00–7.13 (d, J ¼ 8:57 Hz, 2H, Ar-H), 7.30–
7.42 (d, J ¼ 8:25 Hz, 1H, Ar-H), 8.73–9.77 (br, 2H, Ar–
OH); MS (ESI) m=e 475 (Mþ).
6.48. 3,4-Dihydro-3-(4-methoxyphenyl)-4-[[4-[2-(1-piperid-
inyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol (8c)
6.51. 3-(3,4-Dimethoxyphenyl)-3,4-dihydro-4-[[4-[2-(1-
piperidinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-
diol (8f)
The title compound was prepared from 7c by a method
similar to that described for 8a (yield ¼ 70%).
The title compound was prepared from 7f by a method
similar to that described for 8a (yield ¼ 90%).
1H NMR (200 MHz, DMSO-d6) 1.25–1.56 (m, 6H,
3CH2), 2.31–2.44 (t, J ¼ 4:54 Hz, 4H, N–[CH2]2), 2.53–
2.63 (t, J ¼ 5:67 Hz, 2H, N–CH2), 2.64–2.76 (d,
J ¼ 14:19Hz, 1H, Ar–C H–C–OH), 2.89–3.01 (dd,
J ¼ 3:41 Hz, J ¼ 10:79Hz, 1H, OH–C–C H–Ar), 3.17–
3.31 (d, J ¼ 14:19Hz, 1H, Ar–C H–C–OH), 3.73 (s, 3H,
Ar–O–CH3), 3.83–4.02 (d+m, J ¼ 3:41 Hz, 3H, Ar–O–
CH–C–OH, Ar–O–CH2–CH2–N), 4.18–4.34 (t, J ¼
10:79Hz, 1H, Ar–O–C H–CH–C–OH), 4.78–5.30 (br,
1H, Ar–CH2–C–OH), 6.10–6.20 (d, J ¼ 2:55 Hz, 1H, Ar-
H), 6.35–6.45 (dd, J ¼ 2:55 Hz, J ¼ 8:52 Hz, 1H, Ar-H),
6.65–6.79(m, 4H, Ar- H), 6.80–6.89(d, J ¼ 8:80 Hz, 2H,
Ar-H), 7.14–7.25 (d, J ¼ 8:80 Hz, 2H, Ar-H), 7.30–7.40
(d, J ¼ 8:52 Hz, 1H, Ar-H); MS (ESI) m=e 489(M þ).
1H NMR (200 MHz, DMSO-d6) 1.25–1.57 (m, 6H,
3CH2), 2.33–2.44 (t, J ¼ 5:50 Hz, 4H, N–[CH2]), 2.54–
2.64 (t, J ¼ 5:80 Hz, 2H, N–CH2O), 2.66–2.78 (d,
J ¼ 14:04 Hz, 1H, Ar–CH–C–OH), 2.87–2.98 (dd,
J ¼ 306 Hz, J ¼ 10:68 Hz, Ar–O–CH2–CH–C–OH),
2.20–2.35 (d, J ¼ 14:04 Hz, 1H, Ar–CH–C–OH), 3.68 (s,
3H, Ar–O–CH3), 3.73 (s, 3H, Ar–CH3), 3.84–4.02 (m+t,
J ¼ 5:80 Hz, 3H, Ar–O–CH–CH–C–OH, Ar–O–CH2–
CH2–N), 4.20–4.36 (t, J ¼ 10:68 Hz, 1H, Ar–O–CH–
CH–C–OH), 4.89–5.17 (br, 1H, C–OH), 6.11–6.17 (d,
J ¼ 2:44 Hz, 1H, Ar-H), 6.36–6.46 (dd, J ¼ 2:44 Hz,
J ¼ 8:54 Hz, 1H, Ar-H), 6.67–6.90 (2m, 7H, Ar-H), 7.31–
7.41 (d, J ¼ 8:54 Hz, 1H, Ar-H); MS (ESI) m=e 519(M þ).
6.49. 3,4-Dihydro-3-(4-methylphenyl)-4-[[4-[2-(1-piperid-
inyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol (8d)
6.52. 3,4-Dihydro-3-phenyl-4-[[4-[2-(1-piperidinyl)eth-
oxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol (8g)
The title compound was prepared in 80% yield from 7d
by a method similar to that described for 8a.
The title compound was prepared in 90% yield from 7g
by a method similar to that described for 8a.
1H NMR (200 MHz, DMSO-d6) 1.25–1.56 (m, 6H,
3CH2), 2.27 (s, 3H, Ar–CH3), 2.32–2.44 (t, J ¼ 5:34 Hz,
4H, N–[CH2]2), 2.54–2.63 (t, J ¼ 5:91 Hz, 2H, N–CH2),
2.64–2.76 (d, J ¼ 14:35 Hz, 1H, Ar–CH–C–OH), 2.90–
3.02 (dd, J ¼ 3:09Hz, J ¼ 10:13 Hz, 1H, Ar–O–CH2–
CH–C–OH), 3.17–3.30 (d, J ¼ 10:13 Hz, Ar–CH–C–
OH), 3.82–4.06 (m+t, J ¼ 5:91 Hz, 3H, ArO–CH2–
CH2–N, Ar–O–CH–CH–C–OH), 4.18–4.37 (t, J ¼
10:40 Hz, 1H, Ar–O–CH–CH–C–OH), 6.09–6.18 (d,
J ¼ 2:25 Hz, 1H, Ar-H), 6.34–6.46 (dd, J ¼ 2:25 Hz,
J ¼ 8:44 Hz, 1H, Ar-H), 6.64–6.80 (m, 4H, Ar-H), 6.99–
7.23 (m, 4H, Ar-H), 7.29–7.41 (d, J ¼ 8:44 Hz, 1H, Ar-
H); MS (ESI) m=e 473 (Mþ).
1H NMR (200 MHz, DMSO-d6) 1.28–1.58 (m, 6H,
3CH2), 2.31–2.43 (t, J ¼ 5:60 Hz, 4H, N–[CH2]2), 2.54–
2.68 (t, J ¼ 5:82 Hz, 2H, N–CH2), 2.70–2.82 (d,
J ¼ 9:72 Hz, 1H, Ar–CH–C–OH), 2.95–3.15 (dd,
J ¼ 2:71 Hz, J ¼ 4:34 Hz, 1H, Ar–CH–CH2–O), 3.18–
3.34 (d, J ¼ 9:72 Hz, 1H, Ar–CH–C–OH), 3.45–4.10 (m,
3H, Ar–O–CH–CH, Ar–O–CH2–CH2), 4.18–4.42 (t,
J ¼ 8:34 Hz, 1H, Ar–OCH–CH), 3.60–4.80 (very br,
OH), 6.05–6.15 (d, J ¼ 2:22 Hz, 1H, Ar-H), 6.35–6.50
(dd, J ¼ 2:22 Hz, J ¼ 8:45 Hz, 1H, Ar-H), 6.55–6.80 (q,
AA0BB0 system, 4H, Ar-H), 7.15–7.35 (br, 6H, Ar-H);
MS (ESI) m=e 459(M þ).
6.53. 3,4-Dihydro-3-(3-methoxyphenyl)-4-[[4-[2-(1-piper-
idinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol
(8h)
6.50. 3-(4-Butoxyphenyl)-3,4-dihydro-4-[[4-[2-(1-piperid-
inyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol (8e)
The title compound was prepared from 7e by a method
similar to that described for 8a (yield ¼ 30%).
The title compound was prepared from 7h by a method
similar to that described for 8a (yield ¼ 90%).

3778
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
1H NMR (200 MHz, DMSO-d6) 1.25–1.60 (m, 6H,
3CH2), 2.32–2.45 (t, J ¼ 5:45 Hz, 4H, N–9(CH2)2),
2.54–2.64 (t, J ¼ 5:73 Hz, 2H, N–CH2), 2.67–2.80 (d,
J ¼ 14:33 Hz, 1H, Ar–CH–C–OH), 2.92–3.05 (dd,
J ¼ 2:87 Hz, J ¼ 10:31 Hz, 1H, Ar–CH–CH2–O), 3.20–
3.33 (d, J ¼ 14:33 Hz, 1H, Ar–CH–C–OH), 3.70 (s, 3H,
Ar–O–CH3), 3.88–4.04 (m, 3H, Ar–O–CH–CH–Ar, Ar–
O–CH2–CH2–N), 4.20–4.36 (t, J ¼ 10:31 Hz, 1H, Ar–
O–CH–CH–Ar), 4.95–5.20 (br, 1H, C–OH), 6.10–6.20
(d, J ¼ 2:30 Hz, 1H, Ar-H), 6.35–6.45 (dd, J ¼ 2:30 Hz,
J ¼ 8:60 Hz, 1H, Ar-H), 6.63–6.91 (m, 7H, Ar-H), 7.12–
7.24 (t, J ¼ 8:02 Hz, 1H, Ar-H), 7.30–7.40 (d,
J ¼ 8:60 Hz, 1H, Ar-H); MS (ESI) m=e 489(M þ).
2.58 (m, 6H, N–(CH2)3), 2.77–2.90 (d, J ¼ 14:18 Hz, 1H,
Ar–CH–C–OH), 3.00–3.12 (dd,
J ¼ 10:11 Hz,
J ¼ 3:49Hz, 1H, Ar–C H–CH2–O), 3.40 (s, 1H, Ar–CH–
C–OH), 3.77 (s, 3H, Ar–O–CH3), 3.90–4.02 (m, 3H, Ar–
O–CH2–CH2, Ar–CH–CH–O), 4.20–4.35 (t, J ¼
10:48 Hz, 1H, Ar–CH–CH–O), 6.21–6.25 (d, J ¼
2:58 Hz, 1H, Ar-H), 6.42–6.50 (dd, J ¼ 2:58 Hz,
J ¼ 8:64 Hz, 1H, Ar-H), 6.65–6.90 (m, 6H, Ar-H), 7.15–
7.23 (d, J ¼ 8:83 Hz, 2H, Ar-H), 7.33–7.40 (d,
J ¼ 8:64 Hz, 1H, Ar-H); MS (ESI) m=e 503 (Mþ).
6.57. 4-[[4-[2-(Hexahydro-1H-azepin-1-yl)ethoxy]phenyl]-
methyl]-3,4-dihydro-3-(4-methoxyphenyl)-2H-1-benzopy-
ran-4,7-diol (8l)
6.54. 3,4-Dihydro-3-(4-methoxyphenyl)-4-[[4-[2-(1-pyrrol-
idinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol
(8i)
The title compound was prepared in 90% yield from 7l
by a method similar to that described for 8a.
1H NMR (200 MHz, CH3OH-d4) 1.46–1.64 (br, 8H,
4CH2), 2.60–2.70 (m, 4H, N–(CH2)2), 2.72–2.85 (m, 3H,
N–CH2, Ar–CH–C–OH), 2.90–3.01 (dd, J ¼ 3:41 Hz,
J ¼ 10:23 Hz, 1H, Ar–O–CH2–CH–C–OH), 3.16–3.33
(d, J ¼ 13:98 Hz, 1H, Ar–CH–C–OH), 3.73 (s, 3H, Ar–
O–CH3), 3.83–4.00 (m, 3H, Ar–O–CH–CH, Ar–O–
CH2–CH2), 4.18–4.35 (t, J ¼ 10:23 Hz, 1H, Ar–O–CH–
CH), 4.90–5.18 (br, 1H, C–OH), 6.11–6.18 (d,
J ¼ 2:38 Hz, 1H, Ar-H), 6.36–6.46 (dd, J ¼ 2:38 Hz,
J ¼ 8:52 Hz, 1H, Ar-H), 6.65–6.90 (m+d, J ¼ 8:53 Hz,
6H, Ar-H), 7.15–7.25 (d, J ¼ 8:86 Hz, 2H, Ar-H), 7.30–
7.40 (d, J ¼ 8:52 Hz, 1H, Ar-H), 9.00–10.00 (br, 1H,
Ar–OH); MS (ESI) m=e 503 (Mþ).
The title compound was prepared in 85% yield from 7i
by a method similar to that described for 8a.
1H NMR (200 MHz, DMSO-d6) 1.55–1.80 (m, 4H,
2CH2), 2.60–2.80 (m, 1H, Ar–CH–C–OH), 2.88–3.01
(m, 1H, Ar–O–CH2–CH–Ar), 3.14–3.35 (m, 1H, Ar–
CH–C–OH), 3.73 (s, 3H, Ar–OCH3), 3.87–4.05 (m, 3H,
Ar–O–CH2–CH2, Ar–O–CH–CH–Ar), 4.17–4.36 (t,
J ¼ 10:22 Hz, 1H, Ar–O–CH–CH–Ar), 5.05 (s, 1H, C–
OH), 6.10–6.18 (d, J ¼ 2:34 Hz, 1H, Ar-H), 6.35–6.46
(dd, J ¼ 2:34 Hz, J ¼ 8:46 Hz, 1H, Ar-H), 6.63–6.90 (m,
6H, Ar-H), 7.13–7.25 (d, J ¼ 8:76 Hz, 2H, Ar-H), 7.30–
7.40 (d, J ¼ 8:46 Hz, 1H, Ar-H), 9.34 (s, 1H, Ar–OH);
MS (ESI) m=e 475 (Mþ).
6.55. 3,4-Dihydro-3-phenyl-4-[[4-[3-(1-piperidinyl)prop-
oxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol (8j)
6.58. 3-(2-Methoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (9a)
The title compound was prepared from 7j by a method
similar to that described for 8a (yield ¼ 40%).
Compound 8a (2.8 g; 5.7 mmol) was dissolved in boiling
acetonitrile (20 mL), and HCl 37% (0.5 mL) was added.
The solution was stirred at reflux for 1 h, after which the
volume was reduced under a nitrogen flux; the solution
was kept at +4 ꢁC for several hours and the title com-
pound crystallized. The white solid was filtered and
washed with acetonitrile and diethyl ether, obtaining
300 mg of the title compound (mp 134–136 ꢁC;
yield ¼ 10%).
1H NMR (200 MHz, DMSO-d6) 1.20–1.90 (m, 6H,
3CH2), 2.80–3.09(m, 2H, N þ–CH2), 3.34–3.68 (m, 6H,
Nþ–(CH2)2, Ar–CH2–C@C), 3.80 (s, 3H, Ar–O–CH3),
4.25–4.40 (t, J ¼ 4:92 Hz, 2H, Ar–O–CH2–CH2), 4.66–
4.82 (br, 2H, Ar–O–CH2–C@C), 6.18–6.27 (m, 2H, Ar-
H), 6.77–6.98 (m, 4H, Ar-H), 7.01–7.20 (m, 4H, Ar-H),
7.23–7.38 (m, 1H, Ar-H); MS (ESI) m=e 471 (Mþ free
base); HRMS (ESþ) m=e calcd for C30H34NO4 (MHþ)
472.2488, found 472.2482.
1H NMR (200 MHz, DMSO-d6) 1.26–1.57 (m, 6H,
3CH2), 1.70–1.90 (m, 2H, O–CH2–CH2–CH2–N), 2.22–
2.40 (m, 6H, N–[CH2]3), 2.63–2.95 (d, J ¼ 14:14 Hz, 1H,
Ar–CH–C–OH),
2.95–3.08
(dd,
J ¼ 3:41 Hz,
J ¼ 9:69Hz, 1H, Ar–O–CH 2–CH–Ar), 3.17–3.34 (d,
J ¼ 14:14 Hz, 1H, Ar–CH–C–OH), 3.83–4.03 (m, 3H,
Ar–O–CH–CH–Ar, Ar–O–CH2–CH2), 4.21–4.39(t,
J ¼ 10:22 Hz, Ar–O–CH–CH–Ar), 5.11 (s, 1H, Ar–
CH2–COH), 6.13–6.18 (d, J ¼ 2:62 Hz, 1H, Ar-H),
6.37–6.46 (dd, J ¼ 2:62 Hz, J ¼ 8:39Hz, 1H, Ar- H),
6.65–6.80 (m, 4H, Ar-H), 7.20–7.30 (s, 5H, Ar-H), 7.32–
7.39(d, J ¼ 8:39Hz, 1H, Ar- H), 9.35 (s, 1H, Ar–OH);
MS (ESI) m=e 473 (Mþ).
6.56. 3,4-Dihydro-3-(4-methoxyphenyl)-4-[[4-[3-(1-piper-
idinyl)propoxy]phenyl]methyl]-2H-1-benzopyran-4,7-diol
(8k)
The title compound was prepared in 85% yield from 7k
by a method similar to that described for 8a.
6.59. 3-(4-Hydroxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (9b)
1H NMR (200 MHz, CH3OH-d4) 1.38–1.60 (m, 6H,
3CH2), 1.85–2.04 (m, 2H, Ar–O–CH2–CH2–CH2), 2.37–
The title compound was prepared from 8b by a method
similar to that described for 9a. The reaction was con-

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3779
ducted in ethanol and the product was precipitated by
diethyl ether (mp 153–164 ꢁC dec; yield ¼ 70%).
1H NMR (200 MHz, DMSO-d6) 0.83–0.98 (t,
J ¼ 7:31 Hz, 3H, CH3–[CH2]3–O), 1.27–1.88 (2m, 10H,
0
þ
–
3CH2 CH3–[CH2]2–CH2–O), 2.82–3.09(m, 2H, N
1H NMR (200 MHz, DMSO-d6) 1.20–1.90 (2m, 6H,
3CH2), 2.77–3.12 (m, 2H, N–CH2), 3.33–3.54 (m, 4H, N–
[CH2]2), 3.72 (s, 2H, Ar–CH2), 4.24–4.41 (t, J ¼ 5:92 Hz,
2H, Ar–O–CH2), 4.85 (s, 2H, Ar–O–CH2–C@C), 6.16–
6.26 (d+br, J ¼ 2:37 Hz, 2H, Ar-H), 6.69–6.80 (d,
J ¼ 8:59Hz, 2H, Ar- H), 6.81–6.92 (m, 3H, Ar-H), 7.03–
7.17 (dd, J ¼ 2:66 Hz, J ¼ 8:59Hz, 4H, Ar- H), 9.51 and
9.54 (2s, 2H, Ar–OH), 10.12–10.50 (br, 1H, Nþ–H); MS
(ESI) m=e 457 (Mþ free base); HRMS (ESþ) m=e calcd
for C29H32NO4 (MHþ) 458.2331, found 458.2330.
CH2), 3.35–3.54 (m, 4H, Nþ–[CH2]2), 3.65–3.79(br, 2H,
Ar–CH2–C@C), 3.87–4.02 (t, J ¼ 6:35 Hz, 2H, Ar–
OCH2–[CH2]2–CH3), 4.24–4.40 (t, J ¼ 4:77 Hz, 2H, Ar–
O–CH2–CH2–Nþ), 4.88 (s, 2H, Ar–O–CH2–C@C),
6.17–6.27 (d+m, J ¼ 2:22 Hz, 2H, Ar-H), 6.81–6.97 (m,
5H, Ar-H), 7.04–7.28 (2d, J ¼ 8:90 Hz, J ¼ 8:58 Hz, 4H,
Ar-H), 9.53 (s, 1H, Ar–OH), 10.06 (br, 1H, Nþ–H); MS
(ESI) m=e 513 (Mþ free base); HRMS (ESþ) m=e calcd
for C33H40NO4 (MHþ) 514.2957, found 514.2949.
6.63. 3-(3,4-Dimethoxyphenyl)-4-[[4-[2-(1-piperidinyl)eth-
oxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride
(9f)
6.60. 3-(4-Methoxy)phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (9c)
The title compound was prepared from 8c by a method
similar to that described for 9a (mp 199–205 ꢁC dec;
yield ¼ 85%).
The title compound was prepared from 8f by a method
similar to that described for 9a (mp 209–214 ꢁC dec;
yield ¼ 50%).
1H NMR (200 MHz, DMSO-d6) 1.20–1.96 (2m, 6H,
3CH2), 2.80–3.11 (m, 2H, Nþ–CH2), 3.34–3.55 (m, 4H,
Nþ–[CH2]2), 3.69–3.75 (br+s, 5H, Ar–O–CH3, Ar–CH2–
C@C), 4.29–4.40 (t, J ¼ 4:95 Hz, 2H, Ar–O–CH2–CH2–
Nþ), 4.88 (s, 2H, Ar–O–CH2–C@C), 6.18–6.28 (d+m,
J ¼ 2:32 Hz, 2H, Ar-H), 6.80–6.98 (2d, J ¼ 8:72 Hz, 5H,
Ar-H), 7.04–7.28 (2d, J ¼ 8:72 Hz, 4H, Ar-H), 9.58 (s,
1H, Ar–OH), 10.52–10.78 (br, 1H, Nþ–H); MS (ESI)
m=e 471 (Mþ free base); HRMS (ESþ) m=e calcd for
C30H34NO4 (MHþ) 472.2488, found 472.2487.
1H NMR (200 MHz, DMSO-d6) 1.19–191 (2m, 6H,
3CH2), 2.80–3.09(m, 2H, N þ–CH2), 3.34–3.53 (m, 4H,
Nþ–[CH2]2), 3.60 (s, 3H, Ar–O–CH3), 3.69–3.78 (br, 5H,
Ar–O–CH3, Ar–CH2–C@C), 4.27–4.38 (t, J ¼ 5:29Hz,
2H, Ar–O–CH2–CH2–Nþ), 4.91 (s, 2H, Ar–O–CH2–
C@C), 6.18–6.29(d+br, J ¼ 2:49Hz, 2H, Ar- H), 6.78–
7.00 (m, 6H, Ar-H), 7.05–7.18 (d, J ¼ 8:41 Hz, 2H, Ar-
H), 9.55 (s, 1H, Ar–OH), 10.22–10.48 (br, 1H, Nþ–H);
MS (ESI) m=e 501 (Mþ free base); HRMS (ESþ) m=e
calcd for C31H36NO5 (MHþ) 502.2593, found 502.2595.
6.61. 3-(4-Methylphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (9d)
6.64. 3-Phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]-
methyl]-2H-1-benzopyran-7-ol hydrochloride (9g)
The title compound was prepared in 10% yield from 8d
by a method similar to that described for 9a. The solu-
tion was then evaporated and the product crystallized
from a mixture of ethanol and diethyl ether (mp 175–
182 ꢁC dec; yield ¼ 10%).
1H NMR (200 MHz, DMSO-d6) 1.21–1.87 (2m, 6H,
3CH2), 2.29(s, 3H, Ar–C H3), 2.83–3.11 (m, 2H, Nþ–
CH2), 3.34–3.55 (m, 4H, Nþ–[CH2]2), 3.72 (s, 2H, Ar–
CH2–C@C), 4.28–4.38 (t, J ¼ 5:24 Hz, 2H, Ar–CH2–
CH2–Nþ), 4.89(s, 2H, Ar–O–C H2–C@C), 6.16–6.27
(d+m, J ¼ 2:46 Hz, 2H, Ar-H), 6.80–6.93 (2d,
J ¼ 2:46 Hz, J ¼ 8:63 Hz, 3H, Ar-H), 7.05–7.13 (d,
J ¼ 8:63 Hz, 2H, Ar-H), 7.18 (s, 4H, Ar-H), 9.54 (s, 1H,
Ar-H), 10.05–10.30 (br, 1H, Nþ–H); MS (ESI) m=e 455
(Mþ free base); HRMS (ESþ) m=e calcd for C30H34NO3
(MHþ) 456.2539, found 456.2526.
The title compound was first prepared as the free base in
a 70% yield from 8g by a method similar to that de-
scribed for 9a, although the reaction was conducted in
more diluted conditions. The free base of 9g was isolated
by adding a 7-fold excess of K2CO3 when the reaction
was finished (by HPLC analysis). After 10 min stirring,
the salt was filtered off and the solution was evaporated
to dryness. The residue was treated with acetone,
obtaining a light yellow solid which was filtered. The
free base of 9g was then salified with HCl (1:1 ratio) in
ethyl acetate (500 vol), obtaining the title compound
crystallizing as a white solid (mp 175–180 ꢁC dec;
yield ¼ 90%).
1H NMR (200 MHz, DMSO-d6) 1.25–1.90 (2m, 6H,
3CH2), 2.80–3.10 (m, 2H, Nþ–CH2), 3.32–3.55 (m, 4H,
Nþ–[CH2]2), 3.72 (s, 2H, Ar–CH2–C@C), 4.24–4.42 (t,
J ¼ 5:18 Hz, 2H, Ar–O–CH2–CH2–Nþ), 4.91 (s, 2H,
Ar–O–CH2–C@C), 6.17–6.31 (m, 2H, Ar-H), 6.81–6.96
(m, 3H, Ar-H), 7.04–7.17 (d, J ¼ 8:63 Hz, 2H, Ar-H),
7.23–7.47 (m, 5H, Ar-H), 9.58 (s, 1H, Ar–OH), 10.18–
10.48 (br, 1H, Nþ–H); MS (ESI) m=e 441 (Mþ free
base); HRMS (ESþ) m=e calcd for C29H32NO3 (MHþ)
442.2382, found 442.2364.
6.62. 3-(4-n-Butoxyphenyl)-4-[[4-[2-(1-piperidinyl)ethoxy]-
phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (9e)
The title compound was prepared in ethanol from 8e by
a method similar to that described for 9a (mp 129–
134 ꢁC dec; yield ¼ 50%).

3780
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
6.65. 3-(3-Methoxyphenyl)-4-[[4-[2-(1-piperidinyl)eth-
oxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride
(9h)
(Mþ); HRMS (ESþ) m=e calcd for C30H34NO3 (MHþ)
456.2539, found 456.2527.
The title compound was prepared from 8h by a method
similar to that described for 9a. The product was pre-
cipitated by diethyl ether (mp 104–112 ꢁC; yield ¼ 30%).
6.68. 3-(4-Methoxyphenyl)-4-[[4-[3-(1-piperidinyl)prop-
oxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride
(9k)
1H NMR (200 MHz, DMSO-d6) 1.20–1.90 (m, 6H,
3CH2), 2.77–3.14 (m, 2H, Nþ–CH2), 3.34–3.55 (m, 4H,
(CH2)2), 3.61–3.81 (s+br, 5H, Ar–CH2–C@C, Ar–O–
CH3), 4.24–4.41 (t, J ¼ 4:59Hz, 2H, Ar–O–C H2–CH2),
4.90 (s, 2H, Ar–O–CH2–C@C), 6.18–6.28 (m, 2H, Ar-
H), 6.76–6.96 (m, 5H, Ar-H), 7.04–7.37 (m, 4H, Ar-H),
9.57 (s, 1H, Ar–OH), 9.98–10.41 (br, 1H, Nþ–H); MS
(ESI) m=e 471 (Mþ free base); HRMS (ESþ) m=e calcd
for C30H34NO4 (MHþ) 472.2488, found 472.2490.
The title compound was prepared in ethanol from 8k by
a method similar to that described for 9a (mp 164–
169 ꢁC dec; yield ¼ 40%).
1H NMR (200 MHz, DMSO-d6) 1.23–1.87 (2m, 6H,
3CH2), 2.03–2.25 (m, 2H, Ar–O–CH2–CH2), 2.72–2.96
(m, 2H, Nþ–CH2–CH2), 3.02–3.20 (m, 2H, Nþ–CH2),
3.34–3.50 (m, 2H, Nþ–CH2), 3.70–3.78 (br+s, 5H, Ar–
OCH3, Ar–CH2–C@C), 3.90–4.04 (t, J ¼ 5:95 Hz, 2H,
Ar–O–CH2–CH2), 4.88 (s, 2H, Ar–O–CH2–C@C), 6.17–
6.28 (m, 2H, Ar-H), 6.75–6.98 (m, 5H, Ar-H), 7.02–7.12
(d, J ¼ 8:64 Hz, 2H, Ar-H), 7.18–7.28 (d, J ¼ 8:63 Hz,
2H, Ar-H), 9.55 (s, 1H, Ar–OH), 10.18–10.48 (br, 1H,
Nþ–H); MS (ESI) m=e 485 (Mþ free base); HRMS (ESþ)
m=e calcd for C31H36NO4 (MHþ) 486.2644, found
486.2632.
6.66. 3-(4-Methoxyphenyl)-4-[[4-[2-(1-pyrrolidinyl)eth-
oxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride
(9i)
The title compound was prepared in ethanol from 8i by
a method similar to that described for 9a (mp 231–
232 ꢁC dec; yield ¼ 55%).
6.69. 4-[[4-[2-(Hexahydro-1H-azepin-1-yl)ethoxy]phenyl]-
methyl]-3-(4-methoxyphenyl)-2H-1-benzopyran-7-ol
hydrochloride (9l)
1H NMR (200 MHz, DMSO-d6) 1.75–2.09(br, 4H,
2CH2), 2.95–3.20 (br, 2H, Nþ–CH2), 3.41–3.63 (t,
J ¼ 5:09Hz, 4H, N þ–(CH2)2), 3.67–3.80 (br+s, 5H, Ar–
CH2–C@C, Ar–O–CH3), 4.20–4.30 (t, J ¼ 4:79Hz, 2H,
Ar–O–CH2–CH2), 4.88 (s, 2H, Ar–O–CH2–C@C), 6.18–
6.28 (m, 2H, Ar-H), 6.80–7.00 (m, 5H, Ar-H), 7.05–7.15
(d, J ¼ 8:68 Hz, 2H, Ar-H), 7.18–7.30 (d, J ¼ 8:68 Hz,
2H, Ar-H), 9.52 (s, 1H, Ar–OH), 10.35–10.56 (br, 1H,
Nþ–H); MS (ESI) m=e 457 (Mþ free base); HRMS (ESþ)
m=e calcd for C29H32NO4 (MHþ) 458.2331, found
458.2334.
The title compound was prepared from 8l by a method
similar to that described for 9a. The product was iso-
lated by crystallization from ethanol (mp 129–137 ꢁC;
yield ¼ 30%).
1H NMR (200 MHz, DMSO-d6) 1.50–1.95 (2m, 8H,
4CH2), 3.02–3.58 (m, 6H, Nþ–(CH2)3), 3.65–3.82 (br+s,
5H, Ar–CH–C@C, Ar–O–CH3), 4.25–4.42 (t, J ¼
4:87 Hz, 2H, Ar–O–CH2–CH2), 4.88 (s, 2H, Ar–O–
CH2–C@C), 6.18–6.28 (d+m, J ¼ 2:24 Hz, 2H, Ar-H),
6.80–6.98 (m, 5H, Ar-H), 7.04–7.15 (d, J ¼ 8:61 Hz, 2H,
Ar-H), 7.19–7.29 (d, J ¼ 8:61 Hz, 2H, Ar-H), 9.55 (s,
1H, Ar–OH), 10.40–10.65 (br, 1H, Nþ–H); MS (ESI)
m=e 485 (Mþ free base); HRMS (ESþ) m=e calcd for
C31H36NO4 (MHþ) 486.2644, found 486.2651.
6.67. 3-Phenyl-4-[[4-[3-(1-piperidinyl)propoxy]phenyl]-
methyl]-2H-1-benzopyran-7-ol (9j)
The title compound was first prepared as the free base in
a 45% yield from 8j by a method similar to that de-
scribed for 9a, although the reaction was conducted in
more diluted conditions than those described for 9a. The
free base of the product was isolated by adding a 7-fold
excess of K2CO3 when the reaction was finished (by
HPLC analysis). After 10 min stirring, the salt was fil-
tered off and the solid was washed with water to com-
pletely eliminate the inorganic salts. The white solid was
dried under vacuum (mp 107–109 ꢁC dec; yield ¼ 45%).
1H NMR (200 MHz, DMSO-d6) 1.34–1.79(m, 6H,
3CH2), 1.89–2.14 (br, 2H, O–CH2–CH2–CH2–Nþ),
2.57–3.12 (br, 6H, Nþ–[CH2]3), 3.70 (s, 2H, Ar–CH2–
C@C), 3.88–4.03 (t, J ¼ 6:04 Hz, 2H, Ar–O@CH2–
CH2), 4.91 (s, 2H, Ar–O–CH2–C@C), 6.18–6.28 (m, 2H,
Ar-H), 6.76–6.85 (d, J ¼ 8:72 Hz, 2H, Ar-H), 6.86–6.94
(d, J ¼ 8:38 Hz, 1H, Ar-H), 7.01–7.12 (d, J ¼ 8:72 Hz,
2H, Ar-H), 7.24–7.44 (m, 5H, Ar-H); MS (ESI) m=e 455
6.70. 3-Phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]-
methyl]-7-methoxy-2H-1-benzopyran methansulfonate
(10a)
The title compound was prepared from 9g as free base.
The free base of compound 9g (1.2 g; 2.7 mmol) was
dissolved in DMF (5 mL). NaH 80% (154 mg; 5.4 mmol)
and methyl iodide (3.6 mmol; 22.4 mL of a 0.16 M
solution in DMF) were added and the mixture was
stirred at rt for 2 h. After quenching with water (20 mL)
the product was purified by flash chromatography
(CH2Cl2/CH3OH from 80/20 to 90/10). The residue was
dissolved in acetone and an equimolar amount of
methansulfonic acid was added. The title compound was
precipitated as the methansulfonate salt by adding di-
ethyl ether (mp 122–124 ꢁC dec; yield ¼ 35%).

G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
3781
1H NMR (200 MHz, CH3OH-d4) 1.23–1.91 (2m, 6H,
3CH2), 2.33 (s, 3H, CH3–SO3), 2.85–3.12 (m, 2H, Nþ–
CH2), 3.38–3.58 (m, 4H, Nþ–[CH2]2), 3.69–3.75 (2s, 5H,
Ar–O–CH3, Ar–CH2–C@C), 4.21–4.34 (t, J ¼ 4:62 Hz,
2H, Ar–O–CH2–CH2–Nþ), 4.96 (s, 2H, Ar–O–CH2–
C@C), 6.33–6.48 (d+m, J ¼ 244 Hz, 2H, Ar-H), 6.82–
6.94 (d, J ¼ 8:72 Hz, 2H, Ar-H), 6.96–7.05 (d,
J ¼ 8:47 Hz, 1H, Ar-H), 7.06–7.17 (d, J ¼ 8:72 Hz, 2H,
Ar-H), 7.24–7.48 (m, 5H, Ar-H), 9.10–10.48 (br, 1H,
Nþ–H); MS (ESI) m=e 455 (Mþ free base); HRMS (ESþ)
m=e calcd for C30H34NO3 (MHþ) 456.2539, found
456.2537.
Nonestrogenic controls were given vehicle alone. After
the final dose, the animals fasted overnight. The rats were
autopsied 24 h after the final dose. At autopsy, the uterine
wet weight was determined, and uterine weight/body
weight ratios (UWR) were calculated for each animal.
The inhibition percentage of the estrogen-induced re-
sponse was then calculated by the following formula: %
inhibition ¼ 100 · [(UWREE2 ) UWRtest agent)/ (UWREE2
·
UWRcontrol)].
6.72. Molecular modeling
Molecular modeling studies were performed with Sybyl
6.8,32 running on a Silicon Graphics O2 workstation.
The geometry of the ligands was optimized in vacuo
using the MMFF94s force field,33 with the Powell
6.71. Pharmacology
6.71.1. Human ERa and ERb binding. ERa and b bind-
ing analysis was performed as previously described.23
Briefly, the standard assay was performed in a volume of
100 lL, containing a final concentration of 0.5 nM [3H]-
17b-estradiol (PerkinElmer Life Science, Boston, MA),
increasing concentration of unlabeled test compounds
(0.01–100 nM), 5 lL of diluted (1:100 in binding buffer)
human recombinant ERa or b (insect Sf9cells) and
95 lL of binding buffer (10 mM Tris pH 7.5, 10% gly-
cerol, 1 mM dithiothreithol, 1 mg/mL bovine serum
albumin). The Ki of [3H]-17b-estradiol at ERa and ERb
were 0.018 and 0.015 nM, respectively. Incubation was
carried out at room temperature for 3 h. After incuba-
tion, 100 lL of 50% hydroxyapatite slurry (equilibrated
in 50 mM Tris pH 7.4, 1 mM EDTA) was added to each
tube and vortexed three times over 15 min. One milliliter
of wash buffer (40 mM Tris pH 7.4, 1 mM EGTA, 1 mM
EDTA, and 100 mM KCl) was added to each reaction
and was centrifuged at 10,000g for 5 min, and the
supernatant was aspirated. The wash step was repeated
two more times and then the hydroxyapatite pellet was
resuspended in 400 lL of ethanol, transferred to a
scintillation vial, and counted. Nonspecific binding was
defined as that which occurred in the presence of 1 lM
diethylstilbestrol and represented 10–15% of the total
binding. Ki values were calculated from the equation of
Cheng and Prusoff30 using the observed half maximal
inhibition concentration (IC50) of the tested compound,
the concentration of radioligand used in the assay, and
the dissociation constant value of the ligand. The data
were also fitted by an iterative program (RECEPT) for
nonlinear regression analysis31 both to a one-site and to
a two-site model. The one-site model was then chosen
when it yielded the best correlation coefficient and when
the improvement of goodness-of-fit for the two-site
model was not statistically significant ðP < 0:05Þ
according to the F -test on the sums of squared errors.
method34 to an energy gradient of 0.05 kcal/mol A using
ꢁ
MMFF94 charges, with the dielectric constant set at 1.
The same force field was applied to proteins and com-
plexes, with energy minimization performed to a gradi-
ꢁ
ent of 0.10 kcal/mol A. ERa and ERb molecular models
were built starting from the crystal coordinates of
receptor-ligand complexes found in the PDB.21 The
BIOPOLYMER module of Sybyl was employed to add
the missing side chains and hydrogen atoms, and for
other protein-building processes. Ligand structures
within the complexes were refined by the standard
commands of Sybyl.
The model of human ERa was built starting from the
coordinates of the complex ERa/OHT (PDB code:
3ERT35). After the addition of missing side chains and
hydrogen atoms, the model was submitted to three cy-
cles of energy minimization: at first, only the position of
the hydrogen atoms was optimized, and then the opti-
mization process was extended to the protein side chains
and to the ligand, and, finally, to the whole complex.
The model of human ERb was built starting from the
coordinates of the complex between rat ERb and
raloxifene (PDB code: 1QKN15). Hydrogen atoms and
missing side chains were added and then the last 25
amino acids of the ligand-binding domain (473–497,
partially unresolved in 1QKN) were replaced by the
corresponding amino acids taken from our ERa refined
structure. The replaced sequence included His475 (cor-
responding to His524 in ERa), the loop connecting
His475 to helix 12 and helix 12. On the basis of a
homology modeling approach, the alignment performed
by ClustalX36 between rat and human ERb amino acid
sequences was employed to build the human model of
ERb, by replacing the appropriate amino acids. The
ligand raloxifene was replaced by the 4-hydroxytamox-
ifen (OHT) structure, taken from the refined model of
ERa; OHT was fitted in the ligand-binding domain
of ERb by superposition of its stiryl portion to that of
raloxifene. The resulting complex, ERb-OHT, was
submitted to a cycle of energy minimizations as de-
scribed above for ERa. The amino acid numeration of
ERb, which we referred to in the text, is that of the
1QKN crystal structure, as reported by Pike et al.15 Both
the refined models of ERa and ERb were analyzed with
6.71.2. Immature female rat study. Female Sprague-
Dawley rats (21 days old), weighing approximately 40–
50 g (Charles River Italica, Calco, Italy) were treated by
oral gavage with vehicle (0.5% methylcellulose, 3 mL/kg),
test compounds (0.001–10 mg/kg day), or EE2 at 0.05 mg/
kg day for 3 days. The compounds under investigation
were also administered 15 min before the EE2 gavage,
used as estrogenic stimulus to increase uterine weight.

3782
G. Amari et al. / Bioorg. Med. Chem. 12 (2004) 3763–3782
the program Procheck v. 3.5.437 and resulted acceptable
from a biophysical point of view, with no bad contacts
and proper values of phi, psi, and chi angles.
14. Brzozowski, A. M.; Pike, A. C. W.; Dauter, Z.; Hubbard,
€
R. E.; Bonn, T.; Engstrom, O.; Ohman, L.; Greene, G. L.;
€
Gustafsson, J.; Carlquist, M. Nature 1997, 389, 753.
15. Pike, A. C. W.; Brzozowski, A. M.; Hubbard, R. E.;
€
Bonn, T.; Thorsell, A.-G.; Engstrom, O.; Gustaffson,
J.-A.; Carlquist, M. EMBO J. 1999, 18, 4608.
The minimum-energy conformations of the ERa and
ERb receptors were employed for docking studies, after
deletion of the ligand OHT atoms. Docking studies were
performed using the Dock command of Sybyl 6.8, on
benzopyran derivatives whose basic side chain was
considered in its protonated state. The minimization of
the interaction energy was performed by keeping the
protein structure fixed and that of the ligand flexible,
applying the MMFF94s force field, to a gradient of
ꢁ
16. Grese, T. A.; Sluka, J. P.; Bryant, H. U.; Cullinan, G. J.;
Glasebrook, A. L.; Jones, C. D.; Matsumoto, K.; Palko-
witz, A. D.; Sato, M.; Termine, J. D.; Winter, M. A.;
Yang, N. N.; Dodge, J. A. Proc. Natl. Acad. Sci. 1997, 94,
14105.
17. Giossi, M.; Caruso, P. L.; Civelli, M.; Bongrani, S. Calcif.
Tissue Int. 1996, 58, 419.
18. Galbiati, E.; Armani, E.; Caruso, P.; Delcanale, M.;
Giossi, M.; Civelli, M. Pharmacol. Res. 1999, 39, 9 3.
19. Delcanale, M.; Amari, G.; Armani, E.; Lipreri, M.; Civelli,
M.; Galbiati, E.; Giossi, M.; Caruso, P.; Crivori, P.;
Carrupt, P.-A.; Testa, B. Helv. Chim. Acta 2001, 84, 2417.
20. Galbiati, E.; Caruso, P. L.; Amari, G.; Armani, E.;
Ghirardi, S.; Delcanale, M.; Civelli, M. J. Pharmacol. Exp.
Ther. 2002, 300, 802.
ꢁ
0.10 kcal/mol A.
Acknowledgements
21. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.;
Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E.
Nucleic Acids Res. 2000, 28, 235.
22. Greene, T. W.; Wuts, P. G. M. In Protective Groups in
Organic Synthesis, 3rd ed.; John Wiley: New York, 1999;
pp 246–292.
ꢀ
The C.I.M. (Centro Interfacolta Misure) and C.C.E.
(Centro di Calcolo Elettronico) of the University of
Parma are gratefully acknowledged for providing the
Sybyl software license.
23. Obourn, J. D.; Koszewski, N. J.; Notides, A. C. Biochem-
istry 1993, 32, 6229.
References and notes
24. Eppenberger, U.; Wosikowski, K.; Kung, W. Am. J. Clin.
Oncol. 1991, 14(Suppl. 2), S5.
1. Jordan, V. C. J. Med. Chem. 2003, 46, 883.
2. Ciocca, D. R.; Vargas Roig, L. M. Endocr. Rev. 1995, 16,
35.
3. Lyndsay, R. In Osteoporosis Etiology Diagnosis and
Management; Riggs, B. L., Melton, L. G., Eds.; Raven:
New York, 1988; pp 333–358.
4. Ross, R. K.; Pike, M. C.; Mack, T. M.; Henderson, B. E.
In HRT and Osteoporosis; Drife, G. O., Studd, J. W. W.,
Eds.; Springer: London, 1990; pp 209–222.
5. Vesey, M. P. J. R. Soc. Med. 1984, 77, 542.
6. Jacobs, H. S. Endocr. Relat. Cancer 2000, 7, 53.
7. Session, D. R.; Kelly, A. C.; Jewelewicz, R. Fertil. Steril.
1993, 2, 277.
25. Egner, U.; Heinrich, N.; Ruff, M.; Gangloff, M.; Mueller-
Fahrnow, A.; Wurts, J.-M. Med. Res. Rev. 2001, 21, 523.
26. Pike, A. C. W.; Brzozowski, A. M.; Walton, J.; Hubbard,
R. E.; Thorsell, A.; Yi-Lin, L.; Gustafsson, J.; Carlquist,
M. Structure 2001, 9, 145.
27. Renaud, J.; Bischoff, S. F.; Buhl, T.; Floersheim, P.;
Fournier, B.; Halleux, C.; Kallen, J.; Keller, H.; Schlaeppi,
J. M.; Stark, W. J. Med. Chem. 2003, 46, 2945.
28. Galbiati, E.; Caruso, P. L.; Amari, G.; Armani, E.;
Ghirardi, S.; Delcanale, M.; Civelli, M. J. Pharmacol. Exp.
Ther. 2002, 303, 196.
29. Ghirardi, S.; Armani, E.; Amari, G.; Delcanale, M.;
Civelli, M.; Caruso, P.; Galbiati, E. Drug Future 2002,
27(Suppl. A), 454.
30. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
8. Wakeling, A. E.; Valcaccia, B. J. Endocr. 1983, 99, 445.
9. Miller, C. P.; Komm, B. S. Ann. Rep. Med. Chem. 2001,
36, 149.
10. Black, L. J.; Sato, M.; Rowley, E. R.; Magee, D. E.;
Bekele, A.; Williams, D. C.; Cullinan, G. J.; Bendele, R.;
Kauffman, R. F.; Bensch, W. R.; Frolik, C. A.; Termine,
J. D.; Bryant, H. U. J. Clin. Invest. 1994, 93, 63.
11. Miller, C. P. Curr. Pharm. Des. 2002, 8, 2089.
12. Grese, T. A.; Cho, S.; Finley, D. R.; Godfrey, A. G.;
Jones, C. D.; Lugar, C. W., III; Martin, M. J.; Matsu-
moto, K.; Pennington, L. D.; Winter, M. A.; Adrian, M.;
Cole, H. W.; Magee, D. E.; Phillips, D. L.; Rowley, E. R.;
Short, L. L.; Glasebrook, A. L.; Bryant, H. U. J. Med.
Chem. 1997, 40, 146.
31. Benfenati, F.; Guardabasso, V. In Principles and Methods
in Receptor Binding; Cattabeni, F., Nicosia, S., Eds.;
Plenum: New York, 1985; pp 41–63.
32. Sybyl 6.8, Tripos Inc., 1699 South Hanley Rd., St. Louis,
Missouri 63144, USA.
33. Halgren, T. A. J. Comput. Chem. 1999, 20, 720.
34. Powell, M. J. D. Math. Program. 1977, 12, 241.
35. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.;
Kushner, P. G.; Agard, D. A.; Green, G. L. Cell 1998, 95,
927.
36. Thompson, J. D.; Gibson, T. J.; Plewniak, F.; Jeanmou-
gin, F.; Higgins, D. G. Nucleic Acids Res. 1997, 24, 4876.
37. Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.;
Thornton, J. M. J. Appl. Cryst. 1993, 26, 283.
13. Minutolo, F.; Bertini, S.; Papi, C.; Carlson, K. E.;
Katzenellenbogen, J. A.; Macchia, M. J. Med. Chem.
2001, 44, 4288.