Novel highly potent and selective nonsteroidal aromatase inhibitors: Synthesis, biological evaluation and structure-activity relationships investigation

Article abstract of DOI:10.1021/jm100319h

In further pursuing our search for potent and selective aromatase inhibitors, a new series of molecules was designed and synthesized, exploring possible structural modifications of a previously identified xanthone scaffold. Among them, highly potent compounds, with inhibitory activity in the low nanomolar range, were found. In particular, substitution of the heterocyclic oxygen atom in the xanthone core by a sulfur atom and/or increase in structure flexibility seemed to be favorable for the interaction with the enzyme.

Full text of DOI:10.1021/jm100319h

J. Med. Chem. 2010, 53, 5347–5351 5347
DOI: 10.1021/jm100319h
Novel Highly Potent and Selective Nonsteroidal Aromatase Inhibitors: Synthesis, Biological Evaluation
and Structure-Activity Relationships Investigation
Silvia Gobbi,*,† Christina Zimmer,‡ Federica Belluti,† Angela Rampa,† Rolf W. Hartmann,‡ Maurizio Recanatini,† and
Alessandra Bisi†
†Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, I-40126 Bologna, Italy, and ‡Pharmaceutical and Medicinal
Chemistry, Saarland University, P.O. Box 151150, & Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), D-66041 Saarbru€cken,
Germany
Received February 24, 2010
In further pursuing our search for potent and selective aromatase inhibitors, a new series of molecules
was designed and synthesized, exploring possible structural modifications of a previously identified
xanthone scaffold. Among them, highly potent compounds, with inhibitory activity in the low
nanomolar range, were found. In particular, substitution of the heterocyclic oxygen atom in the
xanthone core by a sulfur atom and/or increase in structure flexibility seemed to be favorable for the
interaction with the enzyme.
Introduction
Chart 1. Most Representative Aromatase Inhibitors
Breast cancer is one of the leading causes of cancer-related
mortality among women worldwide.1 In a high percentage of
cases, it proves to be hormone-dependent because tumor
progression is dependent on high levels of circulating estro-
gens, which play a critical role in cancer cell proliferation.
Moreover, in postmenopausal women, biologically active
estrogens are locally produced from circulating inactive ster-
oids in an intracrine mechanism in breast cancer tissues and
confer estrogenic activities to carcinoma cells.2 A series of
enzymes are involved in this intratumoral or in situ produc-
tion of estrogens in breast carcinoma tissues, but aromatase
(CYP19a), a member of the cytochromeP450family, is the key
enzyme involved in their synthesis, promoting the aromatiza-
tion of the A ring of androgen precursors.3 Different strategies
have been devised to control or block the progression of
hormone-dependent breast cancer, and one of the main app-
roaches involves reduction of estrogen levels by inhibition of
CYP19. Although third generation aromatase inhibitors (AIs),
such as letrozole, anastrozole, and exemestane (Chart 1), are
now considered a valid alternative to tamoxifen as first line
treatment of advanced breast cancer,4,5 the search for potent
and selective AIs still remains an attractive subject.6-9 Moreover,
alternative strategies such as development of multipotent
compounds10,11 are being evaluated from different research
groups to deal with the complexity and multiplicity of factors
involved in the development of hormone-dependent breast
cancer.12
Chart 2. Design Strategy for the New Compounds
In the first paper of this series, we reported on some mole-
cules featuring different oxygenated heterocycles such as flavone,
chromone, and xanthone13 as aromatase inhibitors. Then,
with the aim to increase potency and selectivity versus other
CYP enzymes, mainly 17R-hydroxylase/17,20-lyase (CYP17),
a cytochrome P450 involved in the synthesis of androgens,
several concerted structural modifications on these scaffolds
were introduced and very potent and selective AIs were
synthesized.14-16 Among early synthesized molecules,13 high
inhibitory activity was shown by appropriately substituted
xanthones such as 1 (Chart 2), confirming the importance of
*To whom correspondence should be addressed. Phone/Fax:þ39 051
2099732/34. E-mail: silvia.gobbi@unibo.it.
a Abbreviations: CYP19, aromatase; AIs, aromatase inhibitors; CYP17,
17R-hydroxylase/17,20-lyase; PPA, polyphosphoric acid; SAR, structure-
activity relationships.
r
2010 American Chemical Society
Published on Web 06/22/2010
pubs.acs.org/jmc

5348 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 1. Structures and Biological Profile of the New Compounds
Gobbi et al.
CH2-imid
position
CYP19a IC50 nMc or %
CYP17b IC50 nMc or %
compd
R
X
Y
inhib (10 μM)
inhib (2.5 μM)
1
4
4
4
4
4
4
4
3
2
1
1-NO2
2-NO2
3-NO2
1-NO2
1-NO2
H
O
O
O
S
CH
CH
CH
CH
N
40
53
4%
2
NA
9%
3
1900
16.5
101
4
5%
5
O
O
S
NA
17%
28%
33%
11%
880
6
CH
CH
CH
CH
CH
17
7
H
3.98
390
8
H
O
O
O
O
S
9
H
7900
150
10
11
12
13
H
NO2
NO2
H
11.45
5.59
389.2
52
NA
17%
8%
O
fadrozole
ketoconazole
NA
2780
17.7%
a Human aromatase, placental microsomes, substrate 1β[3H]androstenedione 500 nM. b Human CYP17 expressed in E. coli, substrate progesterone
25 μM. c The given values are mean values of at least three experiments. The deviations were within (5%. “NA” = no activity detected.
Scheme 1a
this class of heterocycles in several medicinal chemistry re-
search fields.17,18
Thus, from further exploring possible structural modifica-
tions on the scaffold of 1 in order to investigate the effect on
aromatase inhibition and to confirm the hypothesis of an
H-bond network of these ligands withthe enzyme,13,16 wehere
report on xanthones, aza- and thioxanthones, and phenoxy-
and phenylsulfanylbenzylimidazoles, lacking the ketone moi-
ety, which can be regarded as bioisosteres and “open” analo-
gues of the parent structure 1, respectively, with or without
an H-bond accepting substituent (Chart 2). In particular,
the appropriate distances between the imidazole nitrogen, the
nitro and the carbonyl moieties were explored shifting the
position of the nitro group on the xanthone nucleus (com-
pounds 2 and 3). Thioxanthone and azaxanthone bioisosteres
(4 and 5, respectively) were also synthesized, and finally, the
roles of the putative H-bond accepting groups on the different
scaffolds were evaluated by removing the nitro group (6 and
7), varying the position of the imidazole with respect to the
carbonyl (8-10) or removing the carbonyl itself (11-13). The
structures of the new compounds are shown in Table 1.
Chemistry
The xanthen- and thioxanthen-9-one derivatives 2-3 and
6-10 (Scheme 1) were obtained by reaction of bromo deri-
vatives 22-28 with imidazole. While compounds 23-28 were
previously described19-21 and synthesized according to litera-
a Reagents and conditions: (a) PPA, 120 °C; (b) NBS, CCl4, benzoyl
peroxide, reflux; (c) imidazole, N2, CH3CN, reflux.
ture procedures, compound 22 was obtained by bromination
of the corresponding methyl derivative 15. This intermediate
was synthesized by cyclization in PPA of 14, obtained via the
Ullmann reaction, heating o-chlorobenzoic acid and 2-methyl-
4-nitrophenol with potassium carbonate, Cu, and CuI. 3-
Imidazol-1-ylmethylxanthen-9-one 8, described in a previous
paper,13 was also included in this series.
methyl-5-nitrophenylsulfanyl)benzoic acid 29, prepared via
the Ullmann reaction from 2-mercaptobenzoic acid and 2-
bromo-1-methyl-4-nitrobenzene, followed by bromination
and reaction with imidazole. The same intermediate was
decarboxylated by heating at 220 °C with a catalytic amount
of Cu and then treated as above to obtain compound 12.
In Scheme 2, the synthesis of compounds 4, 11, and 12 is
depicted: compound 4 was obtained by cyclization of the 2-(2-

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5349
Compound 11 was prepared as described for 12, starting
from 2-(2-methyl-5-nitrophenoxy)benzoic acid 30, prepared
via the Ullmann reaction from salicylic acid. The same proce-
durewasapplied to preparecompound 13 (Scheme3), starting
from 2-o-tolyloxybenzoic acid.22
biological activities. Their interaction with different biological
targets appears to be dependent on the substitution pattern on
the heterocyclic scaffold because the nature and position of
different substituents could direct the biological effect of the
molecule.17,18 Recently, some natural occurring prenylated
xanthones, derived from the pericarp of Garcinia mangostana,
have shown aromatase inhibition properties.28 As regards
synthetic derivatives, a xanthone scaffold bearing an imida-
zole ring able to contact the heme iron of the enzyme and a
group capable of establishing H-bond interactions with the
enzyme, in particular the nitro group, have proved to be
critical features for antiaromatase activity.13 In this study,
the role of each element in the assessment of the inhibitory
potency of the compounds was evaluated. Table 1 shows that
some of the new compounds were highly potent AIs, with an
inhibitory activity in the low nanomolar range. Moreover, all
the compounds showed high selectivity for aromatase with
respect to lyase. In particular, only very low percentages of
inhibition of CYP17 were seen at the highest dose tested
(2.5 μM) for most of the studied compounds, while for some
of them no activity could be detected. Interestingly, the only
compound for which an IC50 for inhibition of lyase could be
determined (10) is the only one that carries the imidazole
substituent in position 1, confirming that this pattern of
substitution is related to a higher affinity for this enzyme, as
seen with a previous series of molecules.13
Finally, compound 5 (Scheme 4) was synthesized starting
from 8-methyl-9-oxa-1-aza-anthracen-10-one39,23 which was
nitrated, brominated, and then reacted with imidazole. All the
1
final compounds were characterized by H and 13C NMR,
mass spectra, and HPLC.
Biological Evaluation
The new compounds were tested for inhibition of aromatase
using human placental microsomes24 incubated with 1β[3H]-
androstenedione and measuring the tritiated water formed
during the aromatization of the substrate, as previously des-
cribed.25 The inhibitory activity of the compounds toward
17R-hydroxylase/17,20-lyase (CYP17) was also assessed in
order to evaluate their selectivity toward a related enzyme.
For the CYP17 inhibition tests human CYP17 expressed in
Escherichia coli and P450 reductase were used.25 In these
experiments, fadrozole26 (Chart 1) was used as a positive control
for aromatase and ketoconazole27 as a positive control for
17R-hydroxylase/17,20-lyase.
Results and Discussion
Hereafter, we report on the results of the structure-activity
relationships (SAR) analysis.
Xanthones are well-known natural and synthetic chemical
entities that were found to be endowed with several important
Role of the Heteroatom on the Potency of the Compounds.
The insertion of different heteroatoms (X, Y in Table 1) on
the central core of the molecule, maintaining the nitro group
in the same position as for the parent compound 1, strongly
influenced the inhibitory potency. While the introduction of
a nitrogen, to give the azaxanthone 5, caused a significant
drop in the activity, the thioxanthone 4, in which the oxygen
was substituted by a sulfur leading to a more lipophilic
derivative, showed an increase in potency with respect to 1.
Role of the Nitro Group on the A-Ring. Taking into account
the relative distances between the imidazole nitrogen and the
nitro group, it can be noticed that moving the latter from
position 1 to position 2 on the xanthone nucleus (compound
2) did not seem to modify the potency of the compounds,
while a further repositioning closer to the imidazole (3) led to
a significant decrease in activity. Here, the occurrence of
steric hindrance between these key features of the molecules
could be responsible for this drop in activity, leading to a less
favorable interaction with the heme iron of aromatase.
Successively, the nitro group was removed from both the
xanthone and the thioxanthone scaffolds (compounds 6 and
7, respectively) in order to explore the possibility for the
ketone to act as H-bond acceptor, mimicking the NO2 of 1
and 4, as seen for previous series of AIs.16 Both compounds
showed an increase in activity, confirming the role of the
ketone that proved to be in the proper position to interact
with the enzyme as postulatedabove. Again, the thioxanthone
Scheme 2a
a Reagents and conditions: (a) Cu, CuI, K2CO3, nitrobenzene, 180 °C;
(b) PPA, 120 °C; (c) Cu, 220 °C; (d) NBS, CCl4, benzoyl peroxide, reflux;
(e) imidazole, N2, CH3CN, reflux.
Scheme 3a
a Reagents and conditions: (a) Cu, 220 °C; (b) NBS, CCl4, benzoyl peroxide, reflux; (c) imidazole, N2, CH3CN, reflux.

5350 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Gobbi et al.
Scheme 4a
significant increase in activity. In particular, substitution of
the oxygen by a sulfur atom always enhanced the potency,
likely due to an increase in lipophilicity. The nitro group
proved not to be essential in xanthone derivatives, as the
ketone itself was in the proper position to form H-bonds with
the enzyme when no nitro group was present. On the other
hand, when the ketone was removed, the NO2 became essen-
tial for H-bond interaction, stabilizing the more flexible
diphenyl-ether derivatives and leading to a slight increase in
potency. Among the new compounds, 7 and 12 (IC50 3.98 and
5.59 nM, respectively) resulted the most potent AIs synthe-
sized in this project. Considering that the high activity of these
compounds was achieved in in vitro testing, further develop-
ment strategies for the present molecules, together with pre-
vious obtained potent AIs, could involve in depth investi-
gations, further biological evaluation, and assessment of their
selectivity toward other significant P450 enzymes.
a Reagents and conditions: (a) HNO3, H2SO4, 0-5 °C; (b) NBS, CCl4,
benzoyl peroxide, reflux; (c) imidazole, N2, CH3CN, reflux.
showed higher activity with respect to the oxygen analogue.
Considering compounds 1, 4, 5, 6, and 7, a close relationship
between decrease in lipophilicity and loss of potency could be
suggested, pointing out the important role of this physico-
chemical property for high aromatase inhibition. Moreover,
the increase in activity for 6 and 7 could also be a conse-
quence of reduced overall steric hindrance due to the absence
of the nitro group competing with the ketone for the H-bond
with aromatase.
Experimental Section
Spatial Relationship between Ketone and Imidazole. To
further evaluate the spatial relationship between the ketone
and the imidazole ring, the position of the imidazolylmethy-
lene on the xanthone scaffold was varied. While a decrease of
a log unit in activity was noticed moving the chain from
position 4 to position 3 and again moving it to position 2
(compounds 8 and 9, respectively), some potency was regai-
ned when the chain was placed in position 1 on the xanthone
(compound 10). Still, none of these modifications could
increase the activity, leading to the identification of position
4 as the most favorable for an optimal positioning of the side
chain with respect to the ketone.
Influence of Conformational Flexibility and the Role of the
Nitro Group on the Diphenyl-Ether and -Thioether. Finally,
the ketone itself was removed, keeping the nitro group from
both the xanthone and the thioxanthone leading to the corres-
ponding more flexible diphenyl-ether 11 and -thioether 12,
respectively. This modification allowed us to obtain deriva-
tives for which a closer similarity to clinically available
compounds, such as letrozole and anastrozole, could be
observed. Both compounds proved to be very potent aro-
matase inhibitors, showing activity in the low nanomolar
range. When compared to the corresponding cyclic deriva-
tives 1 and 4, it clearly appeared that the increase in flexibility
led to an increase in activity for both molecules. On the other
hand, these flexible derivatives did not show any significant
improvement with respect to the most potent compound of
the series (7). Again, the substitution of the oxygen by a
sulfur atom proved to be beneficial for the inhibition and
compound 12 resulted one of the most potent AIs synthe-
sized so far. The importance of the nitro group as H-bond
acceptor in this series of molecules, due to the absence of the
ketone moiety, was confirmed by the synthesis of compound
13, in which the NO2 was removed, which showed very low
activity.
Chemistry. General Methods. See Supporting Information.
General Method for Preparation of Imidazol-1-yl Derivatives
2-13. A mixture of the selected bromomethyl derivative (0.001
mol) and imidazole (0.003 mol) in 50 mL of acetonitrile was
refluxed for 7 h under nitrogen. The solvent was removed under
reduced pressure, and the residue was purified by flash chroma-
tography.
4-Imidazol-1-ylmethyl-2-nitroxanthen-9-one (2). Starting from
22, 0.22 g of 2 (71%) were obtained (toluene/acetone 3:2), mp
179 °C (dec). 1H NMR: δ 5.70 (s, 2H, CH2-imi), 7.00-8.40 (m,
9H, Arþimi). 13C NMR: δ 44.85, 117.12, 121.32, 121.86, 122.48,
125.78, 126.54, 127.03, 127.43, 127.69, 129.76, 132.01, 140.03,
141.27, 157.89, 164.45, 186.98. ES-MS m/s: 322 (MHþ).
4-Imidazol-1-ylmethyl-3-nitroxanthen-9-one (3). Starting from
23,19 0.20 g of 3 (63%) were obtained (toluene/acetone 3:2), mp
153-155 °C. 1H NMR: δ 5.80 (s, 2H, CH2-imi), 7.20-8.40 (m,
9H, Arþimi). 13C NMR: δ 37.03, 116.11, 116.98, 121.36, 122.58,
125.88, 126.50, 127.68, 129.84, 131.87, 132.67, 139.78, 152.43,
157.93, 159.35, 187.01. ES-MS m/s: 322 (MHþ).
4-Imidazol-1-ylmethyl-1-nitrothioxanthen-9-one (4). Starting
from 32, 0.22 g of 4 (66%) were obtained (toluene/acetone 3:2),
mp 189-191 °C. 1H NMR: δ 5.45 (s, 2H, CH2-imi), 6.90-8.45
(m, 9H, Arþimi). 13C NMR: δ 48.35, 121.55, 122.54, 126.03,
126.67, 130.64, 133.08, 133.35, 134.33, 134.97, 135.29, 139.66,
140.54, 146.11, 147.80, 186.95. ES-MS m/s: 338 (MHþ).
8-Imidazol-1-ylmethyl-5-nitro-9-oxa-1-azaanthracen-10-one (5).
Starting from 41, 0.12 g (38%) of 5 were obtained (toluene/
acetone 9.5:0.5), mp 202-207 °C. 1H NMR: δ 5.40 (s, 2H, CH2-
imi), 7.00-8.55 (m, 8H, Arþimi). 13C NMR: δ 45.72, 117.98,
119.46, 119.97, 120.76, 122.48, 126.03, 135.21 136.55, 139.76, 140.39,
147.44, 151.66, 157.00, 164.76, 187.21. ES-MS m/s: 323 (MHþ).
4-Imidazol-1-ylmethylxanthen-9-one (6). Starting from 25,20
0.14 g (50%) of 6 were obtained (toluene/acetone 3:2), mp
195-196 °C. 1H NMR: δ 5.55 (s, 2H, CH2-imi), 6.60-8.30 (m,
10H, Arþimi). 13C NMR: δ 45.53, 116.89, 121.11, 121.43,
122.51, 125.80, 126.21, 126.44, 126.66, 126.90, 129.87, 132.09,
132.58, 139.79, 157.59, 158.33, 187.00. ES-MS m/s: 277 (MHþ).
4-Imidazol-1-ylmethylthioxanthen-9-one (7). Starting from
24,21 0.17 g (58%) of 7 were obtained (toluene/acetone 3:2),
mp 112-115 °C. 1H NMR: δ 5.38 (s, 2H, CH2-imi), 6.98-8.30
(m, 10H, Arþimi). 13C NMR: δ 48.17, 121.98, 126.05, 126.45,
126.87, 128.04, 129.89, 132.23, 132.67, 133.04, 134.12, 139.78,
139.89, 140.23, 140.43, 186.96. ES-MS m/s: 293 (MHþ).
2-Imidazol-1-ylmethylxanthen-9-one (9). Starting from 27,20
0.16 g (62%) of 9 were obtained (toluene/acetone 3:2), mp
175-176 °C. 1H NMR: δ 5.60 (s, 2H, CH2-imi), 6.80-8.10 (m,
10H, Arþimi). 13C NMR: δ 55.23, 116.85, 117.03, 121.23,
122.56, 126.01, 126.55, 126.78, 129.80, 130.67, 131.98, 132.54,
139.78, 154.54, 157.56, 187.11. ES-MS m/s: 277 (MHþ).
Conclusions
Xanthones have long been recognized as an important class
of natural and synthetic compounds, endowed with a wide
range of interesting biological activities. In particular, they
proved to be useful molecular scaffolds that could be functio-
nalized in order to meet the structural requirements of differ-
ent biological systems. In pursuing our search for novel
improved aromatase inhibitors, we introduced some modifi-
cations on a previously reported xanthone (1) that led to a

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14 5351
1-Imidazol-1-ylmethylxanthen-9-one (10). Starting from 28,20
0.11 g (42%) of 10 were obtained (toluene/acetone 3:2), mp
169-170 °C. 1H NMR: δ 5.45 (s, 2H, CH2-imi), 6.85-8.15 (m,
10H, Arþimi). 13C NMR: δ 49.13, 114.13, 117.36, 121.21,
121.97, 122.56, 125.87, 126.60, 127.45, 129.89, 131.76, 131.98,
138.89, 139.76, 157.34, 157.65, 186.69. ES-MS m/s: 277 (MHþ).
1-(4-nitro-2-phenoxybenzyl)-1H-imidazole (11). Starting from
35, 0.18 g (62%) of 11 were obtained (toluene/acetone 4:1), mp
107-110 °C. 1H NMR: δ 5.35 (s, 2H, CH2-imi), 7.00-7.90 (m,
11H, Arþimi). 13C NMR: δ 45.85, 112.45, 116.53, 117.11,
121.34, 122.65, 126.02, 128.11, 129.78, 132.56, 139.91, 144.89,
155.98, 157.87. ES-MS m/s: 296 (MHþ).
1-(4-Nitro-2-phenylsulfanylbenzyl)-1H-imidazole (12). Start-
ing from 36, 0.21 g (71%) of 12 were obtained (toluene/acetone
3:2), mp 115-116 °C. 1H NMR: δ 5.30 (s, 2H, CH2-imi), 6.90-
8.00 (m, 11H, Arþimi). 13C NMR: δ 48.21, 120.98, 122.65,
126.02, 126.12, 127.04, 129.21, 130.70, 130.98, 131.76, 133.54,
139.76, 145.89, 146.43. ES-MS m/s: 312 (MHþ).
B. V. Highly Potent First Examples of Dual Aromatase-Steroid
Sulfatase Inhibitors based on a Biphenyl Template (dagger).
J. Med. Chem. 2010, 53, 2155–2170.
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oil (lit.29 mp hydrochloric salt 144-145 °C). 1H NMR: δ 5.15 (s,
2H, CH2-imi), 6.98-7.60 (m, 12H, Arþimi). 13C NMR: δ 45.67,
117.21, 117.39, 121.76, 121.90, 122.87, 125.76, 126.13, 126.54,
128.43, 128.98, 139.53, 156.23, 156.78. ES-MS m/s: 251 (MHþ).
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Acknowledgment. This work was supported by grants from
the University of Bologna.
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Supporting Information Available: Experimental and spectro-
scopic details of intermediate compounds 14, 15, 22, 29-38, 40,
41. This material is available free of charge via the Internet at
http://pubs.acs.org.
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