Synthesis and evaluation of 4-triazolylflavans as new aromatase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.07.090

Aromatase is a target of pharmaceutical interest for the treatment of estrogen-dependent cancers. Azole derivatives such as letrozole or anastrozole have been developed for aromatase inhibition and are used for the treatment of breast tumors. In this paper, four 4-triazolylflavans were synthesized and were found to exhibit moderate to high inhibitory potency against aromatase.

Full text of DOI:10.1016/j.bmcl.2004.07.090

Bioorganic& Medicinal Chemistry Letters 14 (2004) 5215–5218
Synthesis and evaluation of 4-triazolylflavans as new
aromatase inhibitors
Samir Yahiaoui,a Christelle Pouget,a Catherine Fagnere,a Yves Champavier,b
a
Gerard Habrioux and Albert Jose Chulia
a,
*
´
´
a
´
´
UPRES EA 1085 ÔBiomolecules et Cibles Cellulaires Tumorales,Õ Faculte de Pharmacie, 2 rue du Docteur Marcland,
87025 Limoges cedex, France
b
´
´
Service Commun de RMN de l’Universite de Limoges, Faculte de Pharmacie, 2 rue du Docteur Marcland,
87025 Limoges cedex, France
Received 11 December 2003; revised 12 July 2004; accepted 15 July 2004
Abstract—Aromatase is a target of pharmacological interest for the treatment of estrogen-dependent cancers. Azole derivatives such
as letrozole or anastrozole have been developed for aromatase inhibition and are used for the treatment of breast tumors. In this
paper, four 4-triazolylflavans were synthesized and were found to exhibit moderate to high inhibitory activity against aromatase.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
oxy-4-imidazolylflavan B (Fig. 1), which were found to
present a significant anti-aromatase activity since they
were shown to be more potent than aminoglutethimide,
the first nonsteroidal inhibitor clinically used in breast
cancer therapy.7 Compounds A and B have to be consid-
ered as a result of the modulation of flavonoids, which
are natural compounds widely distributed in the plant
kingdom and with about the same inhibitory effect as
Breast cancer, being the first cause of death in women
between 40 and 50years old, is an important public
health problem. About 50% of breast cancers are con-
sidered to be estrogen dependent.1 The final and the rate
step in the production of estrogens is the conversion of
androgens, androstenedione, and testosterone to estrone
and estradiol, respectively, by aromatase, a cytochrome
P450 enzyme.2
aminoglutethimide
against
aromatase.8,9
Some
flavonoids are also known to be inhibitors of other
steroidogenicenzymes suhc as the 17 b-HSD type 1
(17b-hydroxysteroid dehydrogenase), which is involved
in the regulation of the reversible interconversion of
estrone to the potent estradiol.10,11
In postmenopausal women, aromatase inhibitors such
as letrozole and anastrozole, have been shown to be use-
ful in the second-line therapy of estrogen-dependent
breast cancer and have recently been approved as first-
line therapy in several countries.3 These compounds
are nonsteroidal inhibitors with an aza-hetero ring con-
taining a sp2 nitrogen that binds to the heme iron atom
of aromatase. Previous studies revealed that inhibitory
potency and selectivity for aromatase depend on the
number and position of sp2 nitrogen atoms in the hetero-
cycle.4–6
O
N
RO
In our search of new aromatase inhibitors, we synthe-
sized the 4-imidazolyl-7-methoxyflavan A and 7-hydr-
N
A : R = Me
B : R = H
Keywords: 4-Triazolylflavans; Aromatase inhibition; Breast cancer.
*
Corresponding author. Tel.: +33 (0) 555 435 834; fax: +33 (0) 555 435
910; e-mail: jose.chulia@unilim.fr
Figure 1. Structure of 4-imidazolylflavans A and B.
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.090

5216
S. Yahiaoui et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5215–5218
The structure of the two molecules A and B consists of
two parts, one being the imidazole part for interaction
with the heme of aromatase and the second, the flavan
skeleton, which mimics the steroid ring of the
substrate.
was obtained through reaction between thionyl chloride
and 1H-1,2,4-triazole.
For these triazole derivatives, two isomers could be iso-
lated: the 1H-triazole and the 4H-triazole isomers, both
with a 2,4-trans-configuration as previously established
for the 4-imidazolylflavans.7 For the 7-methoxy-substi-
tuted compounds, the 1H-triazole isomer 1a was
obtained in a 33% yield along with the 4H-triazole
isomer 1b in 34% yield, while for the 7-hydroxylated
counterparts, we obtained 24% yield for the compound
2a and 12% for the compound 2b. However, we noticed
that the 4H-triazole isomer 2b undergoes a rearrange-
ment to give the corresponding 1H-triazole derivative
2a, as described by Bentley et al. for 1,2,4-triazol-4-yl-
ethanols.13 The mechanism proposed for such a rear-
rangement involves the electron releasing effect of the
7-hydroxy group, which by mesomerism, leads to the
formation of the triazole anion (Fig. 3). Then, the nucleo-
philicattack of this anion, through the N-1 atom, pro-
vides the formation of the 1H-triazole derivative 2a.
With the aim to prepare new potent aromatase inhibi-
tors, we undertook the replacement of the imidazole
moiety by a triazole heterocycle, with the image of
letrozole and anastrozole containing such a group. In
this paper, we report the synthesis of the 4-triazolylfla-
vans 1a, 1b, 2a, and 2b and their inhibitory potency
against aromatase.
2. Chemistry
The syntheticpathway for compounds 1a, 1b, 2a, and 2b
is described in Figure 2. The reduction of the commer-
cially available 7-methoxyflavanone and 7-hydroxyflav-
anone was carried out with sodium borohydride in
ethanol as previously described.12 2,4-cis-7-Methoxyfla-
van-4-ol and 2,4-cis-7-hydroxyflavan-4-ol were respec-
tively, obtained and triazole moiety was introduced by
direct reaction of these two flavan-4-ols with 1,10-sul-
finylditriazole in dry acetonitrile. 1,10-Sulfinylditriazole
2.1. Experimental procedure
To an ice-cooled suspension of 1,2,4-triazole (4equiv) in
dry acetonitrile, was added a solution of thionyl chloride
3'
2'
1'
4'
5'
R
O
O
8
5
1
R
O
R
O
N
O
R
7
NaBH4
EtOH
6'
2
3
1,1'-sulphinylditriazole
6
CH3CN
4
OH
N
N
N
N
N
2,4-cis configuration
R = OMe
R = OH
1a
2a
1b
2b
2,4-trans configuration
Figure 2. Synthesis of 4-triazolylflavans.
O
N
H
O
O
H
O
4
1
N
3
5
N
5
2
N
2
N
1
N
3
4
2b
O
H
O
1
N
N
5
2
N
4
3
2a
Figure 3. Rearrangement of the 4H-triazole isomer 2b into the 1H-triazole derivative 2a.

S. Yahiaoui et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5215–5218
5217
(1equiv) in dry acetonitrile. The reaction was stirred
under nitrogen at room temperature for 1h. This mix-
ture was added dropwise to a solution of flavan-4-ol
(0.025equiv) in dry acetonitrile and the reaction mixture
was kept at room temperature under nitrogen atmos-
phere for 2h. Acetonitrile was evaporated under reduced
pressure and water was added to the residue. Extraction
was performed with chloroform and compounds were
purified via preparative TLC.
50), 130.8 (C-5), 140.1 (C-10), 143.5 (CH-triazole),
151.6 (CH-triazole), 155.7 (C-8a), 158.8 (C-7); ESP-
MS m/z, found [M+H]+: 294.1247. C17H16N3O2 requires
[M+H]+, 294.1243.
3.4. 2,4-trans-7-Hydroxy-4-4H-1,2,4-triazol-4-ylflavan
(2b)
1
Yield 12%; H NMR (400MHz; pyridine-d5): d 2.52–
2.56 (2H, m, H-3ax and H-3eq), 5.23 (1H, dd, J = 4.3
and 9.4Hz, H-2), 5.72 (1H, br t, J = 3.5Hz, H-4), 6.87
(1H, dd, J = 2.4 and 8.4Hz, H-6), 6.97 (1H, d,
J = 2.4Hz, H-8), 7.11 (1H, d, J = 8.4Hz, H-5), 7.32–
7.39 (5H, m, Ph), 8.89 (2H, s, H-triazole); 13C NMR
(100MHz; pyridine-d5): d 38.2 (C-3), 50.5 (C-4), 73.4
(C-2), 104.5 (C-8), 108.8 (C-4a), 111.2 (C-6), 126.9 (C-
20/60), 128.8 (C-40), 129.2 (C-30/50), 131.9 (C-5), 140.6
(C-10), 143.5 (2 ·CH-triazole), 157.2 (C-8a), 161.3 (C-7).
3. Structural analysis
3.1. 2,4-trans-7-Methoxy-4-1H-1,2,4-triazol-1-ylflavan
(1a)
1
Yield 33%; H NMR (400MHz; CDCl3): d 2.43 (1H,
ddd, J = 4.4, 12.0, and 14.5Hz, H-3ax), 2.73 (1H, br
dt, J = 2.2 and 14.6Hz, H-3eq), 3.82 (3H, s, OCH3),
4.88 (1H, dd, J = 1.9 and 12.0Hz, H-2), 5.53 (1H, dd,
J = 2.2 and 4.2Hz, H-4), 6.59 (1H, d, J = 2.4Hz, H-8),
6.61 (1H, dd, J = 2.5 and 8.4Hz, H-6), 7.11 (1H, d,
J = 8.4Hz, H-5), 7.32–7.41 (5H, m, Ph), 7.90 (1H, s,
H-triazole), 8.04 (1H, s, H-triazole); 13C NMR
(100MHz; CDCl3): d 36.0 (C-3), 54.3 (C-4), 55.4
(OCH3), 73.1 (C-2), 101.9 (C-8), 108.2 (C-4a), 109.4
(C-6), 126.1 (C-20/60), 128.4 (C-40), 128.7 (C-30/50),
131.3 (C-5), 139.5 (C-10), 143.2 (CH-triazole), 152.6
(CH-triazole), 156.8 (C-8a), 161.8 (C-7); ESP-MS m/z,
found: [M+H]+ 308.1400. C18H18N3O2 requires
[M+H]+, 308.1399.
4. Biological assay
Aromatase inhibitory activity of the compounds was
determined in vitro using human placental microsomes
and [1,2,6,7-3H] androstenedione as the substrate.8 The
IC50 values and the potencies (RP) relative to amino-
glutethimide of the triazolyl compounds and their corre-
sponding imidazolyl derivatives A and B are given in
Table 1. Compounds were tested in five appropriate
concentrations (5, 1, 0.5, 0.2, and 0.1lM for compounds
1a and 2a; 50, 25, 10, 5, and 1lM for compound 1b)
with each experiment performed in duplicate in order
to determine their IC50. The concentration of the sub-
strate [1,2,6,7-3H] androstenedione was 40nM.
3.2. 2,4-trans-7-Methoxy-4-4H-1,2,4-triazol-4-ylflavan
(1b)
1
Yield 34%; H NMR (400MHz; CDCl3): d 2.36 (1H, br
dt, J = 2.5 and 14.6Hz, H-3eq), 2.55 (1H, ddd, J = 4.4,
11.6, and 14.6Hz, H-3ax), 3.82 (3H, s, OCH3), 4.90
(1H, dd, J = 2.0 and 11.6Hz, H-2), 5.45 (1H, dd,
J = 3.0 and 3.9Hz, H-4), 6.59 (1H, d, J = 2.4Hz, H-8),
6.61 (1H, dd, J = 2.5 and 8.4Hz, H-6), 7.03 (1H, d,
J = 8.4Hz, H-5), 7.33–7.42 (5H, m, Ph), 8.16 (2H, s,
H-triazole); 13C NMR (100MHz; CDCl3): d 38.0 (C-
3), 50.2 (C-4), 55.5 (OCH3), 72.8 (C-2), 102.0 (C-8),
108.0 (C-4a), 109.7 (C-6), 126.1 (C-20/60), 128.7 (C-40),
128.8 (C30/50), 130.9 (C-5), 139.0 (C-10), 142.3 (2 · CH-
triazole), 156.7 (C-8a), 161.9 (C-7); ESP-MS m/z, found
[M+Na]+: 330.1220. C18H17N3O2Na requires [M+Na]+,
330.1218.
5. Results and discussion
The target compounds were tested for aromatase inhib-
itory activity except for compound 2b, which could not
be evaluated due to its rearrangement into the corre-
sponding 1H-triazole isomer. These derivatives demon-
strated moderate to high inhibitory activity against
aromatase since using our assay conditions, the IC50
ranged from 0.43 to 31.6lM while the IC50 of amino-
glutethimide was 5.2lM.
First, a marked difference of activity was observed be-
tween the two triazole isomers 1a and 1b; 4H-triazole
isomer was found 20 times less potent than the corre-
sponding 1H-triazole derivative. Similar results have
been already described by Marchand et al.14 for 3-(azol-
ylmethyl)-1H-indoles while Vinh et al.15 observed
that, in a benzofuran series, a 4H-triazolyl isomer
displayed greater activity than the 1H-isomer. Further
3.3. 2,4-trans-7-Hydroxy-4-1H-1,2,4-triazol-1-ylflavan
(2a)
1
Yield 24%; H NMR (400MHz; CD3 OD): d 2.45 (1H,
ddd, J = 4.8, 11.7, and 14.6Hz, H-3ax), 2.59 (1H, br dt,
J = 2.3 and 14.6Hz, H-3eq), 5.08 (1H, dd, J = 2.2 and
11.7Hz, H-2), 5.61 (1H, dd, J = 2.3 and 4.6Hz, H-4),
6.43 (1H, d, J = 2.4Hz, H-8), 6.47 (1H, dd, J = 2.4
and 8.4Hz, H-6), 7.03 (1H, d, J = 8.4Hz, H-5), 7.30–
7.38 (5H, m, Ph), 8.06 (1H, s, H-triazole), 8.28 (1H, s,
H-triazole); 13C NMR (100MHz; DMSO-d6): d 35.3
(C-3), 52.1 (C-4), 72.9 (C-2), 102.8 (C-8), 109.0 (C-4a),
109.2 (C-6), 121.1 (C-20/60), 128.0 (C-40), 128.4 (C-30/
Table 1. Aromatase inhibitory activity of 4-triazolylflavans
1a 1b
2a
0.43
A
B
IC50 (lM)
1.4 31.6
0.091
57.1
0.041
126.8
RP (/aminoglutethimide)a 3.7
0.16 12.1
a RP = relative potency calculated from the IC50 values.

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S. Yahiaoui et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5215–5218
investigations must be followed to determine the influ-
ence of the sp2 nitrogen position on the coordination
potential with the heme iron of aromatase. Then, it ap-
peared that the triazoles exhibited weaker inhibitory
activity than the corresponding imidazole analogues.
For example, compound 1a appeared to be 15 times less
potent than the corresponding 4-imidazolylflavan A
while inhibitory potency of compound 2a was 10 times
lower than that of derivative B. Such findings were pre-
viously demonstrated by Marchand et al.,14 Vinh et al.16
as well as by Lang et al.17 who compared, in a series of
cyanophenyl derivatives, the activity of letrozole with
that of the imidazolyl analogue.
Acknowledgements
The authors are grateful to the Region Limousin for the
grant awarded to S. Yahiaoui and to the CNRS central
service of analyses, Vernaison, France, for recording the
high resolution mass spectrometry.
References and notes
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a
N: ! Fe3+-heme coordinate link via the N-3 atom.
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However, it should be noted that compounds like letroz-
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extremely potent aromatase inhibitors, becoming essential
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replacement of the imidazole moiety by a triazole one
led on the one hand to lower in vitro inhibition and
on the other hand to higher in vivo activity.17 The
increased in vivo activity of triazoles compared with imi-
dazoles has been shown to result from increased meta-
18
bolicstability of the
triazole
derivatives.
Additionally, the triazole derived aromatase inhibitors
generally exhibit greater selectivity with respect to other
12. Pouget, C.; Fagnere, C.; Basly, J. P.; Leveque, H.; Chulia,
A. J. Tetrahedron 2000, 56, 6047.
17
steroidogenicand nonsteroidogenicP450 enzymes.
Therefore, further experiments on the in vivo inhibitory
potency and on the selectivity toward other steroido-
genicP450 enzymes will be performed regarding the po-
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Moreover, we demonstrated that the inhibitory effect de-
pends on the substitution pattern since exchanging the
7-methoxy group for a 7-hydroxy one increased signifi-
cantly the anti-aromatase activity. This result indicates
that it is not only the aza-heterocycle, which determines
activity but the overall ability of the entire structure to
fit in the P450 aromatase active site. Therefore, we are
currently investigating the influence of flavan skeleton
on anti-aromatase effect, particularly the substitution
of the phenyl group at C-2.