Design, synthesis and evaluation of 4-imidazolylflavans as new leads for aromatase inhibition

Article abstract of DOI:10.1016/S0960-894X(02)00565-6

Two 4-imidazolylflavans were synthesized and their relative stereochemistry was established by ¹H and ¹³C NMR data. These compounds were tested for their activity to inhibit aromatase. It was observed that the introduction of an imidazolyl group at carbon 4 on flavan nucleus led to potent molecules.

Full text of DOI:10.1016/S0960-894X(02)00565-6

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2859–2861
Design, Synthesis and Evaluation of 4-Imidazolylflavans as
New Leads for Aromatase Inhibition
Christelle Pouget, Catherine Fagnere, Jean-Philippe Basly, Gerard Habrioux
and Albert Jose Chulia*
´ ´
UPRES EA 1085, ‘Biomolecules et cibles cellulaires tumorales’, Faculte de Pharmacie, 2 rue du Docteur Marcland,
87025 Limoges cedex, France
Received 29 April 2002; revised 8 July 2002; accepted 17 July 2002
1
Abstract—Two 4-imidazolylflavans were synthesized and their relative stereochemistry was established by H and 13C NMR data.
These compounds were tested for their activity to inhibit aromatase. It was observed that the introduction of an imidazolyl group at
carbon 4 on flavan nucleus led to potent molecules.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
about the same activity as aminoglutethimide.3ꢀ6 Thus,
we demonstrated that 7-methoxyflavanone and
1
Non steroidal aromatase inhibitors are known to pre-
vent the conversion of androgens to estrogens and thus
play a significant role in the treatment of advanced
breast cancer in postmenopausal women.1 Today, a
broad range of inhibitors has already been studied.
Aminoglutethimide was the first non steroidal inhibitor
clinically available. Then, the second generation of aro-
matase inhibitors, like the non steroidal fadrozole and
7-hydroxyflavanone 2 were able to block aromatase
activity (IC50=8.0 and 3.8 mM, respectively). Our own
interest in both flavonoids and anti-breast cancer agents
led us to design new aromatase inhibitors by taking the
flavan nucleus as molecular skeleton on which to insert
an imidazole ring supposed to be critical for the binding
to the heme iron atom of cytochrome P450 of aromatase.
the
steroidal
substrate
analogue
4-hydroxy-
In the present paper, we report the synthesis and struc-
tural analysis of the 4-imidazolylflavans 5 and 6 and
their biological evaluation against aromatase.
androstenedione, was launched. Recently, a third gen-
eration of azole compounds with greater selectivity and
potency has been developed including letrozole and
anastrozole.1 These two compounds have become the
established second-line treatment for metastatic breast
cancer after tamoxifen and have recently been approved
as first-line therapy in several countries.2 The structure
of these non steroidal aromatase inhibitors can be
regarded as consisting of two parts, one being the azole
part with a nitrogen atom which binds to the heme iron
atom of cytochrome P450 of aromatase and the other,
the bulky aryl part which mimics the steroid ring of the
substrate.
Chemistry
The synthesis of the target compounds 5 and 6 is
described in Scheme 1 and consisted in introduction of
an imidazolyl group at carbon 4 on flavan nucleus. Ste-
reoselective reduction by NaBH4 of 7-methoxy-
flavanone 1 and 7-hydroxyflavanone 2 gave respectively
2,4-cis-7-methoxyflavan-4-ol 3 and 2,4-cis-7-hydroxy-
flavan-4-ol 4 as previously described.7 Treatment of
these two compounds with 1,10-carbonyldiimidazole in
THF led to the azole derivatives 5 (38%) and 6 (37%).
Besides synthetic aromatase inhibitors, some flavonoids
which are natural compounds widely distributed in the
plant kingdom, were found to inhibit aromatase with
Structural Analysis
The key signals and the associated coupling constants in
1
*Corresponding author. Tel.: +33-555-435-834; fax: +33-555-435-
910; e-mail: jose.chulia@unilim.fr
the H NMR spectra of the compounds 5 and 6 are
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00565-6

2860
C. Pouget et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2859–2861
Scheme 1.
given in Table 1 except for the coupling constant
between H-3eq and H-4 which could not be measured
due to the broadening of the signals for these two pro-
tons. The assignment of stereochemistry to these 4-imi-
a quasi-equatorial position for the proton H-4 and
allowed to determine a 2,4-trans configuration for these
two compounds. This configuration was confirmed by
the occurrence of the H-4 signal as a triplet; indeed, in
the 2,4-trans-4-substituted flavans, J3ax,4 and J3eq,4 are
sufficiently close for the H-4 signal to appear as a triplet.8
1
dazolylflavans was readily made on the basis of the H
NMR vicinal coupling constants. As previously descri-
bed for 4-substituted flavans, the coupling constants for
these two compounds were consistent with either the
half-chair (a) or sofa (b) conformation of the hetero-
cyclic ring in which the 2-aryl group was equatorial and
H-2 was axial (Scheme 2).7,8
All 13C NMR signals could be assigned by the C–H
correlation and long-range C–H COSY techniques. The
C-4 resonance for compounds 5 and 6 at about 50 ppm
was characteristic for a C–N type bond.
For the compound 5, the occurrence of four protons at
d 2.38 (br dt, J=2.6 and 14.5 Hz), 2.48 (ddd, J=4.4,
11.2 and 14.5 Hz), 4.97 (dd, J=2.2 and 11.2 Hz) and
5.34 (br t, J=3.7 Hz) was characteristic for the hetero-
cyclic ring of a 4-substituted flavan. The two highest
field signals were assigned to H-3eq and H-3ax for
which the geminal coupling constant was 14.5 Hz.
Among the other constants for these two protons, the
value 11.2 Hz was the only one which could arise from a
trans-diaxial coupling and therefore corresponded to the
constant between H-2 and H-3ax. This allowed H-2 to
be assigned as the signal at 4.97 ppm and H-3ax as the
signal at 2.48 ppm. Consequently, H-4 resonated at d
5.34 ppm. In the same way, for the compound 6, the
signal at 5.02 ppm was assigned to H-2 and the signal at
2,4-trans-4-Imidazolyl-7-methoxyflavan 5. Yield 38%;
1H NMR (400 MHz; CDCl3): d 2.38 (1H, br dt, J=2.6
and 14.5 Hz, H-3eq), 2.48 (1H, ddd, J=4.4, 11.2 and
14.5 Hz, H-3ax), 3.81 (3H, s, OCH3), 4.97 (1H, dd,
J=2.2 and 11.2 Hz, H-2), 5.34 (1H, br t, J=3.7 Hz, H-
4), 6.57 (1H, d, J=1.9 Hz, H-8), 6.59 (1H, dd, J=2.6
and 9.0 Hz, H-6), 6.94 (1H, br s, H-400), 7.04 (1H, d,
J=9.2 Hz, H-5), 7.12 (1H, br s, H-500), 7.34–7.40 (5H,
m, Ph), 7.48 (1H, br s, H-200); 13C NMR (100 MHz;
CDCl3): d 38.3 (C-3), 51.0 (C-4), 55.4 (OCH3), 73.1 (C-
2), 101.6 (C-8), 109.2 (C-6), 109.6 (C-4a), 118.3 (C-400),
126.1 (C-20/60), 128.4 (C-40), 128.7 (C-30/50), 129.7 (C-
500), 131.2 (C-5), 136.8 (C-200), 139.6 (C-10), 156.4 (C-8a),
161.4 (C-7); m/z (EI), M+ 306, (Found: M+, 306.1354.
C19H18N2O2 requires M, 306.1368).
1
5.42 ppm to H-4. Therefore, in the H NMR spectra of
both compounds 5 and 6, the lowest field signal was
assigned to H-4 which was in accordance with previous
results about 4-substituted flavans.9 Then, the coupling
constant between H-4 and H-3ax, which was equal to
4.4 Hz for both compounds 5 and 6, was consistent with
2,4-trans-7-Hydroxy-4-imidazolylflavan 6. Yield 37%;
1H NMR (400 MHz; DMSO-d6): d 2.39 (1H, br dt,
J=3.2 and 14.4 Hz, H-3eq), 2.48 (1H, ddd, J=4.4, 10.4
and 14.4 Hz, H-3ax), 5.02 (1H, dd, J=2.8 and 10.8 Hz,
H-2), 5.42 (1H, t, J=4.0 Hz, H-4), 6.38 (1H, d, J=2.0
Hz, H-8), 6.42 (1H, dd, J=2.0 and 8.4 Hz, H-6), 6.91
(1H, d, J=8.4 Hz, H-5), 6.93 (1H, br s, H-400), 7.13 (1H,
br s, H-500), 7.34–7.40 (5H, m, Ph), 7.57 (1H, br s, H-200),
9.43 (1H, br s, OH); 13C NMR (100 MHz; DMSO-d6): d
37.6 (C-3), 50.2 (C-4), 73.3 (C-2), 103.5 (C-8), 109.9 (C-
6), 110.3 (C-4a), 119.0 (C-400), 126.6 (C-20/60), 128.4 (C-
40), 128.9 (C-30/50), 129.1 (C-500), 131.3 (C-5), 137.1 (C-
200), 140.6 (C-10), 156.1 (C-8a), 159.3 (C-7); m/z (EI),
M+ 292 (found: M+, 292.1200. C18H16N2O2 requires
M, 292.1211).
Table 1. Selected 1H NMR data for compounds 5 and 6
d H-3eq d H-3ax d H-2 d H-4 J3ax-3eq J2ꢀ3ax J2ꢀ3eq J4ꢀ3ax
5
6
2.38
2.39
2.48
2.48
4.97
5.02
5.34
5.42
14.5
14.4
11.2
10.8
2.2
2.8
4.4
4.4
Biological Assay
The inhibitory activities of the compounds 5 and 6
towards aromatase were determined in vitro using human
placental microsomes and [1,2,6,7-3H]-androstenedione
Scheme 2.

C. Pouget et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2859–2861
Table 2. Aromatase inhibitory activity of compounds 5 and 6
2861
In conclusion, we have described the synthesis of new
4-imidazolylflavans and biochemical studies have shown
that compounds 5 and 6 were highly potent as aroma-
tase inhibitors. Therefore, they can be considered as
new leads and further investigation is currently under-
going to explore the flavanone scaffold as skeleton of
our molecules for inhibiting aromatase. In particular,
we will carry out the substitution of the phenyl group at
C-2 to reinforce the interaction between the nitrogen
atom of the inhibitors and the heme iron atom of cyto-
chrome P450 of aromatase. The full details of the
structure–activity relationships, chemical synthesis,
molecular modeling and biochemical studies of this
novel series of compounds will be the subject of future
publications from these laboratories.
Compd
5
6
IC50 (mM)
0.091
57
88
0.041
127
93
RPa/aminoglutethimide
RPa/flavanone
aRP, relative potency calculated from the IC50 values.
as previously described.5 The IC50 values and the
potencies of the compounds 5 and 6, relative to amino-
glutethimide (IC50=5.2 mM) and to the corresponding
flavanones, are given in Table 2. To determine IC50
values, compounds 5 and 6 were tested in five appro-
priate concentrations with each experiment performed
in duplicate.
Acknowledgements
The authors are grateful to the Region Limousin for its
financial support and to Y. Champavier for running the
NMR spectra.
Results and Discussion
The target compounds 5 and 6 were tested for in vitro
inhibitory activity against aromatase; these two deriva-
tives demonstrated high potential against aromatase
since under our assay conditions, the IC50 were 0.091
and 0.041 mM, respectively. They proved to be more
potent than aminoglutethimide, exhibiting relative
potencies RP=57 and 127, respectively. The IC50 value
for compound 6 was not significantly different from that
obtained for the well-known non steroidal inhibitor
fadrozole (IC50=0.019 mM).
References and Notes
1. Brodie, A. M. H.; Njar, V. C. O. Steroids 2000, 65, 171.
2. Goss, P. E.; Strasser, K. J. Clin. Oncol. 2001, 19, 881.
3. Kellis, J. T.; Vickery, L. E. Science 1984, 225, 1032.
4. Ibrahim, A. R.; Abul-Hajj, Y. J. J. Steroid Biochem. Mol.
Biol. 1990, 37, 257.
5. Le Bail, J. C.; Laroche, T.; Marre-Fournier, F.; Habrioux,
G. Cancer Lett. 1998, 133, 101.
6. Jeong, H. J.; Shin, Y. G.; Kim, I. H.; Pezzuto, J. M. Arch.
Pharm. Res. 1999, 22, 309.
7. Pouget, C.; Fagnere, C.; Basly, J. P.; Leveque, H.; Chulia,
A. J. Tetrahedron 2000, 56, 6047.
8. Clark-Lewis, J. W. Aust. J. Chem. 1968, 21, 2059.
9. Bolger, B. J.; Hirwe, A.; Marathe, K. G.; Philbin, E. M.;
Vickars, M. A. Tetrahedron 1966, 22, 621.
Comparing the azole derivatives with their correspond-
ing flavanones, it became apparent that replacement of
the carbonyl function by an imidazolyl group led to a
strong enhancement in enzyme inhibition. Further, the
inhibitory potency depended on the substitution pat-
tern; thus, exchanging the 7-methoxy group for a
hydroxy substituent resulted in a marked increase of
inhibitory potency. This result is in agreement with that
described for flavanones.5