Synthesis and aromatase inhibitory activity of flavanones

Article abstract of DOI:10.1023/A:1014490817731

Purpose. Aromatase inhibitors are known to prevent the conversion of androgens to estrogens and play a significant role in the treatment of estrogen dependent diseases such as breast cancer. Some flavonoids have been reported as potent aromatase inhibitors; therefore, in an effort to develop novel anti breast cancer agents, B ring substituted flavanones with a 7-methoxy group on A ring were synthesized and tested to assess their ability to inhibit aromatase activity and to determine the optimal B ring substitution pattern. Methods. A series of flavanones was prepared by cyclisation of 2′hydroxychalcones previously obtained by Claisen-Schmidt condensation and the aromatase inhibitory activity of these compounds was investigated using human placental microsomes and radiolabeled [1,2,6,7-³H]-androstenedione as substrate. Results. Almost all flavanones exhibited inhibitory effect on the aromatase activity but their potency was dependent on their B ring substitution pattern. Hydroxylation at position 3′ and/or 4′ enhanced the anti-aromatase activity; thus, 3′,4′-dihydroxy-7-methoxyflavanone was found to be twice more potent than aminoglutethimide, the first aromatase inhibitor clinically used. Conclusions. These results indicated that these flavanones could be considered as potential anti breast cancer agents through the inhibition of aromatase activity and allowed us to select some of these compounds as skeleton for the development of flavonoid structurally-related aromatase inhibitors.

Full text of DOI:10.1023/A:1014490817731

Pharmaceutical Research, Vol. 19, No. 3, March 2002 (© 2002)
Research Paper
ring and synthetic flavonoids, which are ubiquitous natural
phenolic compounds and mediate a host of biologic activities
(2), were found to demonstrate inhibitory effects on aro-
matase (3–6). The aim of the present study was to investigate
the structural requirements on B-ring of flavanones for inhi-
bition of aromatase activity, both to identify an optimal can-
didate among synthesized compounds, as well as to ascertain
potential directions for synthetic lead-optimization studies. It
has been previously demonstrated that 7-methoxyflavanone
was a potent aromatase inhibitor (7) and also an effective
antiproliferative agent against MCF-7 breast cancer cells (8).
Moreover, this flavanone was found to be non-estrogenic un-
like other flavonoids (9,10). Therefore, we undertook the
pharmacomodulation of this lead. In particular, the substitu-
tion of its B-ring by hydroxy and methoxy groups was carried
out and the influence of halogen atoms and dimethylamino or
cyano groups on the 4’-position was also investigated. Finally,
the structure-activity relationships were discussed.
Synthesis and Aromatase Inhibitory
Activity of Flavanones
Christelle Pouget,1 Catherine Fagnere,1
Jean-Philippe Basly,1 Anne-Elise Besson,1
Yves Champavier,2 Gerard Habrioux,1 and
Albert-Jose Chulia1,3
Received October 24, 2001; accepted November 30, 2001
Purpose. Aromatase inhibitors are known to prevent the conversion
of androgens to estrogens and play a significant role in the treatment
of estrogen dependent diseases such as breast cancer. Some flavo-
noids have been reported as potent aromatase inhibitors; therefore,
in an effort to develop novel anti breast cancer agents, B ring sub-
stituted flavanones with a 7-methoxy group on A ring were synthe-
sized and tested to assess their ability to inhibit aromatase activity
and to determine the optimal B ring substitution pattern.
Chemistry
Methods. A series of flavanones was prepared by cyclisation of 2’-
hydroxychalcones previously obtained by Claisen-Schmidt condensa-
tion and the aromatase inhibitory activity of these compounds was
investigated using human placental microsomes and radiolabeled
[1,2,6,7-3H]-androstenedione as substrate.
Results. Almost all flavanones exhibited inhibitory effect on the aro-
matase activity but their potency was dependent on their B ring sub-
stitution pattern. Hydroxylation at position 3Ј and/or 4’ enhanced the
anti-aromatase activity; thus, 3Ј,4’-dihydroxy-7-methoxyflavanone
was found to be twice more potent than aminoglutethimide, the first
aromatase inhibitor clinically used.
Conclusions. These results indicated that these flavanones could be
considered as potential anti breast cancer agents through the inhibi-
tion of aromatase activity and allowed us to select some of these
compounds as skeleton for the development of flavonoid structurally-
related aromatase inhibitors.
The synthesis of flavanones 1b-23b called on the follow-
ing sequence (Scheme 1). The Claisen-Schmidt condensation
of 2-hydroxy-4-methoxyacetophenone with appropriate benz-
aldehydes afforded various 2’-hydroxychalcones 1a-23a
(Table I). Benzaldehydes with a 4-hydroxy group and the
3,5-dihydroxybenzaldehyde were protected as tetrahydropy-
ranyl ethers before condensation. 2’-hydroxychalcones re-
acted with H2SO4 / MeOH to give corresponding flavanones
1b-23b (Table I) in a good yield. Structural analysis for com-
pounds 1b-23b are based on mass spectrometry (Table I),
1H-NMR (Table II) and 13C-NMR (Table III) data.
MATERIALS AND METHODS
General Experimental Procedures
KEY WORDS: flavanones; aromatase; breast cancer; structure-
activity relationships.
NMR spectra were recorded on a Bruker 400 MHz spec-
trometer with Me4Si as internal standard. ESI-MS spectra
were performed on a Waters Alliance system equipped with
an API-MS interface. Compounds were purified by prepara-
tive thin layer chromatography on Macherey-Nagel silica gel.
INTRODUCTION
Aromatase, which catalyses the final step in the steroido-
genesis pathway of estrogens, has been the target for the
design of inhibitors as agents in the treatment of breast cancer
for postmenopausal women. Several classes of aromatase in-
hibitors such as substrate androstenedione analogs, nonste-
roidal aminoglutethimide and imidazole or triazole deriva-
tives have been designed over the past twenty years (1).
Besides the development of synthetic compounds, the
potential of various classes of natural products to inhibit aro-
matase was evaluated in order to discover novel breast cancer
chemopreventive agents. As a result, several naturally occur-
General Procedure for Obtaining Chalcones
and Flavanones
2’- hydroxy-2,4’-dimethoxychalcone (1a)
2-hydroxy-4-methoxyacetophenone (0.136 g, 0.82 mmol)
and 2-methoxybenzaldehyde (1.2 eq) were dissolved in EtOH
and partially dehydrated barium hydroxide octahydrate (0.2
g) was slowly added. The reaction mixture was stirred at re-
flux for 2 h and then concentrated in vacuo. Water was added
to the mixture that was acidified with 2M HCl (→ pH 5) and
extracted with diethyl ether. The organic layer was separated,
washed with water, dried over Na2SO4 and evaporated
in vacuo. Purification of the residue via preparative TLC
(Hexane 19 / EtOAc 1) yielded 2’-hydroxy-2,4’-dimethoxy-
chalcone (0.155 g, 0.55 mmol).The same procedure was ap-
plied to obtain all the chalcone derivatives except for com-
pounds 4a, 6a, 8a, and 9a.
1 UPRES EA 1085, “Biomolécules et Cibles Cellulaires Tumorales -
Prolifération cellulaire et inhibition enzymatique” Faculté de Phar-
macie, 2 rue du Dr. Marcland, 87025 Limoges Cedex, France.
2
Service Commun de Résonance Magnétique Nucléaire de
l’Université de Limoges, 2 rue du Dr. Marcland, 87025 Limoges
Cedex, France.
To whom correspondence should be addressed. (e-mail:
3
jose.chulia@unilim.fr)
0724-8741/02/0300-0286/0 © 2002 Plenum Publishing Corporation
286

Synthesis and Aromatase Inhibitory Activity of Flavanones
287
Scheme 1.
2’,4 –dihydroxy-4’-methoxychalcone (4a)
droxy-4-methoxyacetophenone (0.05 g, 0.3 mmol) and 4-(tet-
rahydropyran-2-yloxy)benzaldehyde (1.2 eq) were treated as
in 1a to give the 2’-hydroxy-4’-methoxy-4-(tetrahydropyran-
2-yloxy)chalcone. This compound (0.05 g) and p-
toluenesulfonic acid (3 mg, 0.1 eq) were dissolved in MeOH
(20 ml). The reaction mixture was stirred for 4 h at room
temperature and then evaporated in vacuo. After water had
been added, the mixture was neutralized with 5% NaHCO3
and extracted with EtOAc. The organic layer was separated,
washed with water, dried over Na2SO4 and evaporated in
vacuo. Purification of the residue via preparative TLC (Tolu-
4-hydroxybenzaldehyde (0.1 g, 0.82 mmol) and pyri-
dinium p-toluenesulfonate (0.02 mmol) were dissolved in
methylene chloride (10 ml) and 3,4-dihydro-␣-pyran (0.206 g,
2.4 mmol) in methylene chloride (5 ml) was added dropwise.
The reaction mixture was stirred at room temperature for 24
h, then washed with water, dried on Na2SO4 and evaporated
in vacuo. The residue was purified by preparative TLC (Hex-
ane 8 / EtOAc 2) to obtain the corresponding 4-(tetrahy-
dropyran-2-yloxy)benzaldehyde (0.075 g, 0.36 mmol). 2-hy-
Table I. Chalcones 1a-23a and Flavanones 1b-23b
Flavanones (b)
Yield (%)
Chalcones (a)
Yield (%)
Compound
R1
R2
R3
R4
R5
m/z
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
OMe
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
H
H
OH
OMe
H
H
H
H
OH
OMe
OH
OMe
OH
H
H
OMe
H
H
OMe
H
H
H
H
H
H
H
H
H
OH
H
H
OMe
H
H
OMe
H
OMe
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
67
50
54
37
58
35
50
24
28
59
48
36
20
50
30
33
50
70
84
72
85
86
66
60
62
50
67
62
30
52
30
62
50
20
50
30
43
70
52
40
82
65
63
69
42
68
284
270
284
270
284
286
300
300
286
314
314
314
314
314
314
344
344
344
333
288
272
279
297
H
OH
OH
OMe
OH
OMe
H
H
H
OMe
OMe
OMe
H
OMe
H
H
H
H
H
H
OMe
OMe
H
H
H
H
H
H
OMe
OMe
OMe
Br
Cl
F
H
H
H
H
CN
N(CH3)2
H
H
H

288
Pouget et al.

Synthesis and Aromatase Inhibitory Activity of Flavanones
289

290
Pouget et al.
Table IV. Inhibition of Human Placental Aromatase by Flavanones
ene 19 / Et2O 1) afforded 2’,4-dihydroxy-4’-methoxychalcone
4a (0.03 g, 0.11 mmol).
The same procedure was applied to obtain chalcones 6a,
8a, and 9a starting from respectively 3,4-dihydroxybenzalde-
hyde, 4-hydroxy-3-methoxybenzaldehyde and 3,5-dihydroxy-
benzaldehyde.
Compound
IC50 (␮M)a
AG
7-OMe F
1b
5.2
8.0
6.6
2b
3.5
3b
4b
(43.6)
3.7
4’-hydroxy-7-methoxyflavanone (4b)
4b
6b
7b
8b
(43.8)
2.5
6.2
5.4
3.5
A mixture of 2’,4-dihydroxy-4’-methoxychalcone (0.03g,
0.11 mmol) and 50 ml of 10% H2SO4 / MeOH was refluxed
for 7 h and then evaporated in vacuo. Water was added to the
mixture which was neutralized with 5% NaHCO3 and ex-
tracted with EtOAc. The organic layer was separated, washed
with water, dried over Na2SO4 and evaporated in vacuo. Pu-
rification of the residue via preparative TLC (Toluene 8 /
Et2O 2) afforded 4’-hydroxy-7-methoxyflavanone (0.02 g,
0.074 mmol).
9b
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
6.6
8.0
(26.3)
(25.7)
(42.7)
(31.9)
4.8
(29.3)
(33.9)
(41.2)
(47.3)
(42.3)
(38.3)
(16.6)
The same procedure was applied to obtain all the flava-
none derivatives starting from appropriate 2’-hydroxychal-
cones.
Biological Methods
Preparation of Human Placental Microsomes
Microsomes were obtained from human placenta after
normal full-term delivery, prepared as previously described
(11) and stored at −80°C.
a When 50% inhibition could not be reached at 10 ␮M, the % of
inhibition is given in parentheses.
Aromatase Assay
All enzymatic studies were performed in 0.1 M phos-
phate buffer (0.1 M KH2PO4, 1 mM dithiothreitol DTT, pH
7.4). The final incubation volume was 1 ml. Aromatase incu-
bation mixture contained 3 mM glucose-6-phosphate, 0.5 mM
NADP, 0.1 unit glucose-6-phosphate dehydrogenase, 40 nM
[1,2,6,7-3H] androstenedione and 0.05 mg microsomal pro-
tein. Stock solutions of each inhibitor were freshly prepared
in ethanol and then diluted into the culture medium. The
steroids were incubated at 37°C, extracted, identified and
quantified as previously described (7).
␮M) and 7-methoxyflavanone (7-OMe F, IC50 ס 8.0 ␮M)
were used as reference compounds.
All the flavanones tested caused dose-dependent inhibi-
tion of aromatase activity but their potency was dependent on
the B ring substitution pattern. Compounds 2b, 4b, 6b, and
9b, the most active flavanones, were found to be more potent
than aminoglutethimide and 7-methoxyflavanone. Therefore,
the B ring substitution by hydroxy groups enhanced the in-
hibitory effect on aromatase activity. The 3Ј and 4’-hydroxy
groups seemed to act synergistically since flavanone 6b is
more potent than flavanones 2b and 4b. On the contrary,
methylation of these hydroxy groups decreased the inhibitory
effect since flavanones 3b, 5b, 14b, and 15b appeared to be
weak aromatase inhibitors relative to their hydroxylated
counterparts. Surprisingly, the presence of a 2’-methoxy
group seemed to be responsible for an increase in anti-
aromatase activity; the importance of such a substituent was
suggested by comparison of activities of flavanones 1b, 10b,
11b, and 16b respectively with those of flavanones 7OMe F,
3b, 5b, and 14b, where the only difference between these two
Percentage of Inhibition Measurement
Aromatase inhibition percentage corresponded to the
percentage of unchanged radiolabeled androstenedione. Val-
ues reported represented the average of two separate deter-
minations. IC50 values were obtained by graphical determi-
nation.
RESULTS
In a previous work (12), we demonstrated that some series of compounds was the presence of an additional 2’-
chalcones were potent inhibitors of aromatase activity and methoxy group in the first subset. However, the weak anti-
that hydroxylations at position 2’, 4’ and 6’ on A ring may be aromatase activity of flavanones 12b and 13b relative to the
required to obtain high aromatase inhibitory effect. In con- 2’,7-dimethoxyflavanone 1b clearly indicated that the pres-
trast, we showed that the presence of a 4’-methoxy group ence of an additional methoxy group at position 5Ј or 6’ dras-
decreased the anti-aromatase activity. Therefore, the chal- tically reduced the inhibitory effect.
cones here synthesized (1a-23a), which had this type of sub-
stitution, were not further evaluated.
Finally, contrary to a 4’-hydroxy group, the presence of
halogen atoms as well as a cyano group at position 4’ did not
The anti-aromatase activities of flavanones 1b-23b are influence the inhibitory effect while the substitution by a di-
reported in Table IV. Aminoglutethimide (AG, IC50 ס 5.2 methylamino group led to a less more potent flavanone.

Synthesis and Aromatase Inhibitory Activity of Flavanones
DISCUSSION
291
catechols abolished estrogenic effect (13). Therefore, besides
the potent aromatase inhibitory effect exhibited by 3Ј,4’-
dihydroxy-7-methoxyflavanone, this compound might be
without estrogenic activity and could actually be considered
as a potential anti-breast cancer agent.
Finally, on the basis of the studies reported herein, 3Ј-
hydroxy-7-methoxyflavanone 2b and 3Ј,4’-dihydroxy-7-
methoxyflavanone 6b have been selected for further modu-
lation on C ring in order to develop flavonoid structurally-
related aromatase inhibitors.
Different studies have demonstrated that several natu-
rally occurring flavonoids such as 7-hydroxyflavanone, 7-
hydroxyflavone, naringenin (5,7,4’-trihydroxyflavanone),
apigenin (5,7,4’-trihydroxyflavone), luteolin (5,7,3Ј,4’-tetra-
hydroxyflavone) or eriodictyol (5,7,3Ј,4’-tetrahydroxyflava-
none) were potent aromatase inhibitors (3-7,12). However,
most of these compounds were also found to display estro-
genic activity (9,13). Structure-activity relationships clearly
indicated that a 7-hydroxy group was the substituent essential
for enhanced aromatase inhibitory activity but flavonoids
with such a substituent were found to be invariably estro-
genic.
In contrast, 7-methoxyflavanone did not display estro-
genic activity (9,10) and exhibited an aromatase inhibitory
effect (7) as well as an antiproliferative activity on MCF-7
breast cancer cells (8). These data may suggest that pharma-
comodulation of the B ring of this flavanone could lead to
compounds having a balance between different anti breast
cancer activities.
The present results demonstrated that some of the
synthesized flavanones such as compounds 2b, 4b, 6b, or 9b
were potent aromatase inhibitors which indicated that hy-
droxylations on B ring may be required to obtain high aro-
matase inhibitory effect. Thus, the activity exhibited by 3Ј-
hydroxy-7-methoxyflavanone 2b and 3Ј,4’-dihydroxy-7-
methoxyflavanone 6b relative to 7-methoxyflavanone and 4’-
hydroxy-7-methoxyflavanone 4b respectively, clearly showed
that the presence of an additional 3Ј-hydroxy group enhanced
the anti-aromatase activity. These findings are consistent with
the increased potency we observed with eriodictyol (5,7,3Ј,4’-
tetrahydroxyflavanone) relative to naringenin (5,7,4’-trihy-
droxyflavanone) (12). Among all the flavanones tested, 3Ј,4’-
dihydroxy-7-methoxyflavanone 6b was found to be the most
active and twice more potent than aminoglutethimide, the
first aromatase inhibitor clinically used. Therefore, structure-
activity relationships with flavanones seem to indicate that
two hydroxy groups at position 3Ј and 4’ are the optimal
pattern of B ring substitution that gives rise to anti-aromatase
activity.
ACKNOWLEDGMENTS
The authors are grateful to the Region Limousin for its
financial support and for the grant awarded to C. Pouget.
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