Modulation of cytochromes P450 with xanthone-based molecules: From aromatase to aldosterone synthase and steroid 11β-hydroxylase inhibition

Article abstract of DOI:10.1021/jm301844q

Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized.

Full text of DOI:10.1021/jm301844q

Article
pubs.acs.org/jmc
Modulation of Cytochromes P450 with Xanthone-Based Molecules:
From Aromatase to Aldosterone Synthase and Steroid 11β-
Hydroxylase Inhibition
Silvia Gobbi,*,† Qingzhong Hu,‡ Matthias Negri,‡ Christina Zimmer,‡ Federica Belluti,† Angela Rampa,†
Rolf W. Hartmann,‡ and Alessandra Bisi*,†
†Department of Pharmacy and Biotechnologies, University of Bologna, Via Belmeloro, 6, I-40126 Bologna, Italy
‡Pharmaceutical and Medicinal Chemistry, Saarland University, P.O. Box 151150, and Helmholtz Institute for Pharmaceutical
Research Saarland (HIPS), D-66041 Saarbrucken, Germany
̈
S
* Supporting Information
ABSTRACT: Imidazolylmethylflavones previously reported by us as aromatase
inhibitors proved to be able to interact with aldosterone synthase (CYP11B2), a
cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone
aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in
the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual
screening of a small compounds library of our earlier synthesized aromatase inhibitors
was performed and, according to the results and the corresponding biological data, led to
the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl
substituent in position 1 and different substituents in position 4. Some very potent
inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on
CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be
directed to different targets when appropriately functionalized.
researchers’ attention as potential therapeutic targets,2 since
INTRODUCTION
■
overproduction of cortisol can cause diseases such as
The search for inhibitors of cytochrome P450 (CYP) enzymes
catalyzing the biosynthesis of steroid hormones has been
hypercortisolism and Cushing’s syndrome,3 while hyper-
aldosteronism could be responsible for hypertension, and
intensely pursued during the past several decades, since
aldosterone has been more recently found to play a role in
abnormally increased levels of these hormones, sharing closely
other cardiac diseases such as congestive heart failure or
related synthetic pathways, have proved to be involved in the
development of several hormone-related diseases. Inhibition of
the aromatase enzymatic complex (CYP19), which promotes
the aromatization of the A ring of androgen substrates leading
myocardial fibrosis.4
Recently, some imidazolylmethylflavones previously de-
scribed by our group as aromatase inhibitors (Chart 1)5,6
have been shown to be able to interact with aldosterone
synthase when tested in a compound library screening.7 They
were then used as part of a training set in order to generate an
extended pharmacophore model for this enzyme, in which the
earlier described pharmacophoric points8 were confirmed and a
novel large hydrophobic area was identified.7 After absorbing
to estrogen hormones, is one of the approaches currently
exploited for the treatment of estrogen-dependent breast
cancer, aiming to reduce the estrogen concentration in
blood.1 Closely related 17α-hydroxylase/C17,20-lyase
(CYP17), a key enzyme of androgen biosynthesis, is considered
to be an interesting target for the treatment of benign prostatic
hyperplasia and prostate cancer.2
Chart 1. Reference Structures
Two other steroidogenic CYP enzymes, namely, 11β-
hydroxylase (CYP11B1) and aldosterone synthase
(CYP11B2), isoforms sharing a high degree of sequence
homology (more than 90%), are involved in the biosyntheses of
cortisol and aldosterone, respectively. In particular, the final
steps of aldosterone biosynthesis are performed by these two
enzymes that catalyze the 11-hydroxylation of 11-deoxycorti-
costerone to corticosterone, which is then further hydroxylated
to 18-hydroxycorticosterone (18OH-B). In man, only the
CYP11B2 isoform can perform the final oxidation of C18 to
produce aldosterone, while CYP11B1 is mainly responsible for
glucocorticoid formation.3 These enzymes have raised
Received: December 14, 2012
Published: January 31, 2013
© 2013 American Chemical Society
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more CYP11B2 inhibitors that have been reported in the latest
patents into the training set, the pharmacophore model was
further improved to be a more powerful tool aiding the
identification of new CYP11B2 inhibitors.9 Inspired by the
interesting finding that imidazolylmethylflavones inhibit
CYP11B2, a small compound library of earlier synthezised
aromatase inhibitors was virtually screened with this novel
pharmacophore model as query using MOE. The pharmaco-
phore search returned four compounds, characterized by the
structurally related xanthone scaffold, that could be inhibitors of
CYP11B2. In particular, these molecules (Chart 1 and Table 1,
enzymes, with the unsubstituted molecule 1a showing lower
activity with respect to the substituted ones (Table 1). In
contrast, compound 1c, substituted by a 4-Br, showed the
highest potency, suggesting that apolar substituents in position
4 might lead to potent inhibition. To explore this option, a
series of related xanthones derivatives have now been designed
and synthesized, carrying groups with different polarity and size
in position 4, in order to explore the chemical space, consider
their role in the interaction with the enzyme, and investigate
the requirements of this hydrophobic area. The new
compounds (1e−p), collected in Table 1, were tested for
their inhibition of CYP11B2 and CYP11B1. Due to the close
structural resemblance to compounds originally designed for
aromatase inhibition and in order to examine their potential
selectivity, the new compounds were then further evaluated for
inhibition of the other steroidogenic enzymes CYP19 and
CYP17.
Table 1. Structures and Biological Profiles of the New
Compounds
CHEMISTRY
■
The key intermediates for the synthesis of the studied
compounds, 4-substituted-1-methylxanthones 4e−o, were
prepared starting from 4-amino-1-methylxanthone10 2 by
reacting it with NaNO2 and coupling the obtained diazonium
salt with CuCl or NaI or H2SO4 to give 4e, 4f, and 3,
respectively, or via formation of arenediazonium tetrafluor-
oborate and coupling with potassium trifluoro(phenyl)borate
to give 4g.12 Intermediate 3 was then alkylated with dimethyl
sulfate or selected alkyl halides to obtain 4h−o (Scheme 1). 1-
Methylxanthones (4e−o) were then brominated with NBS, and
by reaction with imidazole, compounds 1e−n were obtained, as
depicted in Scheme 2 (6 was deprotected by refluxing it in
acetic acid to give 1o). Finally, 1p was obtained by
hydrogenation of the previously described 1-imidazolylmeth-
yl-4-nitroxanthen-9-one10 1b (Scheme 3).
RESULTS AND DISCUSSION
■
Following the results obtained with imidazolylmethylflavones
that were selected among earlier synthesized aromatase
inhibitors, a pharmacophore search was performed on an in-
house compound library of previously synthesized CYP19
inhibitors with the recently described 3D-pharmacophore
model of CYP11B2.9 This pharmacophore model consisted
of six hydrophobic/aromatic features (HY0−HY5) and two
acceptor atom features (AA1 and AA2) (Figure 1). Among
them, HY0 and AA1 together were attributed to the heterocycle
that coordinated the heme iron with its sp2 hybrid N, while
HY1 represented the aromatic core. These pharmacophores
were deemed essential for the CYP11B2 inhibition. Since the
pharmacophore model was developed on the basis of various
structurally diversified CYP11B2 inhibitors, which made the
model more powerful in identifying potential CYP11B2
inhibitors, the possession of the rest of the pharmacophores
may increase the affinity or improve the selectivity. However, it
is not necessary to occupy as many pharmacophores as possible
for the sake of ligand efficiency. This model was employed as
the pharmacophore query to filter the 3D conformation
databases generated from the small CYP19 inhibitor library
by comparing the pharmacophore features of the query with
the ligand annotation points of each conformation. If all the
attributions, distances and constrains satisfied and the rigid-
body superposition passed, a hit was identified. Only one hit for
each compound was conserved according to the ranking of
a
Hamster fibroblasts expressing human CYP11B1; substrate deoxy-
b
corticosterone, 100 nM. Hamster fibroblasts expressing human
CYP11B2; substrate deoxycorticosterone, 100 nM. SF = selectivity
factor. Mean value of at least three experiments; relative standard
deviation less than 25%. See ref 11. See ref 10.
c
d
e
f
1a−d)10,11 have a common feature, the imidazolylmethyl
substituent in position 1, for coordinating the heme iron of
these CYP enzymes, and different substituents (or no
substituent) in position 4. The compounds were then
experimentally tested for inhibition of CYP11B2 and
CYP11B1 and were shown to be potent inhibitors of these
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a
Scheme 1
a
Reagents and conditions: (i) H2SO4, NaNO2, −5 °C to rt, then H2SO4, 80 °C; (ii) dimethyl sulfate, K2CO3, acetone or NaH, DMSO, alkyl halide;
(iii) HCl, NaNO2, −5 °C to rt, then CuCl, 80 °C; (iv) H2SO4, NaNO2, −5 °C to rt, then NaI, 80 °C; (v) NaBF4, HCl, H2O, NaNO2, −5 °C, then
Pd(CH3COO)2, potassium trifluoro(phenyl)borate, dry dioxane, rt.
a
rmsd. Since the prediction ability of the model has been fully
validated before,9 no further refining of the query was
performed. The virtual screening revealed that some molecules
(1a−d),10,11 characterized by a xanthone scaffold, could fit into
the model, indicating potential inhibition of CYP11B2.
Although only differing for the groups in position 4 of the
xanthone core, these four compounds exhibited two different
poses in the pharmacophore model. Both poses occupied HY0
and AA1 with the imidazolyl moiety, whereas the positions of
the xanthone cores were different (Figure 1). Compounds 1a,
1c and 1d showed a similar occupational pattern, with ring A of
the xanthone nucleus covering the essential feature HY1 and
ring C fitting the recently identified feature HY3 (pose 1,
Figure 1a; compound 1a as representative, violet). However,
compound 1b employed ring B and C to match features HY1
and HY2b, respectively (pose 2, Figure 1b, green). Strikingly,
this second pose was only observed for compound 1b, with the
polar NO2 group in position 4 filling HY3. The compounds
were then tested and found to be potent inhibitors of both
CYP11B1 and CYP11B2 enzymes (Table 1). The presence of a
substituent in position 4 in the xanthone scaffold proved to be
favorable for interaction, the unsubstituted 1a having lower
activity with respect to the substituted 1b−d. In particular, a
slight improvement in potency could be seen going from 1b,
substituted with a nitro group in position 4, to 1d, featuring a
cyano moiety, to 1c, carrying a bromine, which showed activity
in the low nanomolar range. However, no significant selectivity
was observed between the two enzymes, and the compounds
were slightly better inhibitors of CYP11B1 with respect to
CYP11B2. Some selectivity could indeed be noted when these
results were compared with those obtained for inhibition of
CYP19 and CYP1710,11 (Table 2), mainly for substituted
compounds. In particular, 1b showed a 30−40-fold selectivity
for CYP11B1 and CYP11B2 with respect to CYP19, and for 1c
and 1d, the selectivity ratios against CYP19 were 120−140 and
120−180, respectively, while selectivity toward CYP17 was
generally lower.
Scheme 2
a
Reagents and conditions: (i) NBS, CCl4, benzoyl peroxide, hν, reflux;
(ii) N2, imidazole, reflux; (iii) CH3COOH, H2SO4, reflux.
a
Scheme 3
a
Reagents and conditions: (i) H2, Pd/C, THF, rt.
Since xanthones with common pose 1 occupied HY3 with
their C-ring, similar to the pattern of previously reported
CYP11B2 inhibitors,7 we were encouraged by this similarity to
further modify this structure for more potent CYP11B2
inhibition. When tested, compound 1c (pose 1) showed the
highest potency, suggesting that apolar substituents in position
4 that could fit the hydrophobic regions HY2a or HY2b of the
Figure 1. The fitting pattern of xanthone compounds 1a (violet, A)
and 1b (green, B) in the pharmacophore model.
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fold selectivity for CYP11B1 over CYP11B2, 1b and 1f were
not able to discriminate between the two enzymes.
Table 2. Inhibition of Human CYP19 and CYP17 for the
New Compounds 1e−p
Notably, an increase in steric hindrance in position 4 on the
xanthone nucleus seemed not to be easily accepted by these
enzymes, especially CYP11B1, where the insertion of a phenyl
group (compound 1g) led to a decrease in activity. On the
other hand, the introduction of alkoxy substituents, both linear
or branched, seemed to enhance activity, particularly toward
CYP11B2, with the only exception of 1n carrying the large
pentyloxy group, which again showed similar or lower potency
with respect to the unsubstituted 1a for CYP11B2 and
CYP11B1, respectively. In particular, activity toward aldoster-
one synthase increased with respect to 1a with the introduction
of a methoxy group (compound 1h, IC50 = 41.1 nM) and again
with an ethoxy one (1i, IC50 = 14.8 nM), but further
lengthening and branching of the chain did not lead to any
significant improvement. Somewhat different behavior was
noted for inhibition of CYP11B1; the introduction of the small
methoxy substituent (1h) was not significant for activity, which
was increased with an ethoxy substituent (1i, IC50 = 25.4 nM)
but decreased by branching this short chain, as in 1j (IC50
=
92.5 nM), and the introduction of a longer branched alkoxy
group did not significantly improve potency, with the only
exception of 1m, carrying a cyclopropylmethoxy group (IC50
=
5.5 nM). Regarding selectivity of these alkoxy derivatives, it
could be noted that shorter chains seemed to better
differentiate between CYP11B2 and CYP11B1; for example,
1j with an isopropoxy substituent was almost 5-fold more
potent on aldosterone synthase. In contrast, compound 1m
showed a 4-fold selectivity for 11β-hydroxylase.
Finally, the introduction in position 4 of the xanthone
nucleus of hydrophilic moieties such as OH and NH2
(compounds 1o and 1p, respectively) led to a significant
decrease in activity for both CYP11B2 and CYP11B1.
Taking into account the close similarity with our previously
reported aromatase inhibitors, to further investigate the
selectivity of the new synthesized compounds, the potential
of compounds 1e−p to inhibit CYP19 and CYP17 was also
evaluated, and results are collected in Table 2. The compounds
generally proved to be poor inhibitors of CYP17. In contrast to
the lower selectivity of the synthesized compounds toward
CYP11B2 when compared to fadrozole (SF = 7.9, Table 1),
they exhibited higher selectivity than the reference compound
for CYP11B2 and CYP11B1 with respect to CYP19, although
their inhibition of CYP19 are within micro- or submicromolar
range. Above all, compounds 1e and 1i showed 2−3 orders of
magnitude higher activity on the target enzymes with respect to
CYP19 and CYP17. For a quantitative outline of the selectivity
of the new compounds for the different CYPs evaluated, a table
reporting selectivity factors (SF) can be found in the
Supporting Information (Table 1 SI).
a
Human placental CYP19; substrate androstenedione, 500 nM.
b
Escherichia coli expressing human CYP17; substrate progesterone,
c
25 μM. Mean value of at least three experiments; relative standard
deviation less than 25%. See ref 11. See ref 10.
d
e
pharmacophore model might lead to potent inhibition. New
compounds (1e−p) were then designed and synthesized in
order to investigate the effect of different substitutions on this
part of the molecule and to try to address the selectivity issue.
The results of the testing on CYP11B2 and CYP11B1 are
presented in Table 1 and show remarkable activity for most of
the new compounds. Considering the substitution with halogen
atoms (compounds 1c, 1e, and 1f), it should be noted that it
was proved to be highly favorable, leading to very potent
compounds with activity in the low nanomolar range. In
particular, inhibition seemed to slightly increase going from the
bulky iodine (compound 1f) to the chlorine containing 1e,
which showed an IC50 of 3.0 nM on CYP11B2. Moreover,
activity on CYP11B1 showed the same tendency for these
derivatives, with 1e (IC50 = 0.5 nM) as the best inhibitor of this
enzyme in the whole series. While this compound showed a 6-
CONCLUSIONS
■
In the search for potential ligands for CYP11B2 and CYP11B1,
a series of molecules characterized by a xanthone scaffold,
structurally related to previously reported aromatase inhibitors,
has been designed and synthezised, on the basis of a recently
described pharmacophore model of CYP11B2.9 These
compounds carry an imidazolylmethyl substituent in position
1 and different substituents in position 4. Among them, some
very potent inhibitors were obtained, in particular, the 4-
chlorine derivative 1e proved to be active in the low nanomolar
or subnanomolar range on CYP11B2 and CYP11B1,
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(0.6 g, 5.7 mmol), HCl (4 mL), and H2O (8 mL), and then NaNO2
(0.31 g, 4.44 mmol) in water was added dropwise keeping the
temperature below −5 °C. The mixture was stirred for 1 h, and the
precipitate formed was filtered and dried to obtain a crude solid (1.25
g, 88%) (mp: 180−181 °C), which was added, under nitrogen, to a
mixture of Pd(CH3COO)2 (44 mg, 0.2 mmol) and potassium
trifluoro(phenyl)borate (0.86 g, 4.69 mmol) (see below for
preparation). Dry dioxane (15 mL) was added and the mixture was
stirred at rt for 5 h, diluted with diethyl ether, washed with brine, and
evaporated to dryness. The crude was purified by flash chromatog-
raphy (petroleum ether/ethyl acetate 9:1) to obtain 4g (0.3 g, 26%).
respectively. Generally speaking, the introduction of hydro-
phobic substituents of appropriate size seemed to enhance
affinity for both enzymes, as expected from the docking
simulations studies. It should be noted that the selectivity issue
regarding these highly homologous enzymes still remains to be
solved, although some of the new compounds could
discriminate between the two: in particular, compound 1j,
carrying an isopropoxy group, showed a 5-fold selectivity for
CYP11B2, and 1e and 1m, bearing chlorine and cyclo-
propylmethyl groups, respectively, were found to be the most
selective for CYP11B1 (6- and 4-fold, respectively). On the
other hand, a remarkable degree of selectivity was observed
toward CYP19 and CYP17, indicating that a modulation of
different steroidogenic cytochromes P450 can be obtained with
an appropriate pattern of substitution on the xanthone nucleus.
These results confirm that the xanthone can be considered as
an excellent scaffold, whose activity can be directed to different
targets when appropriately functionalized.
1
Mp: 95−97 °C. H NMR: δ 2.85 (s, 3H, CH3), 7.35−7.55 (m, 8H,
arom), 7.59−7.65 (m, 1H, arom), 7.70−7.80 (m, 1H, arom), 8.34−
8.40 (m, 1H, arom).
Potassium trifluoro(phenyl)borate was obtained from phenyl-
boronic acid (0.9 g, 7.2 mmol) in CH3OH by adding dropwise an
aqueous solution of KHF2 (1.86 g, 23.7 mmol) and stirring for 10 min;
the solvent was evaporated and the residue was extracted with acetone
and evaporated to obtain a crude solid (0.95 g, 72%). Mp: 295−298
°C.
4-Methoxy-1-methylxanthen-9-one (4h). To a solution of 3 (1 g,
4.42 mmol) in acetone (100 mL) were added K2CO3 (1.2 g, 8.69
mmol) and dimethyl sulfate (0.88 g, 7.93 mmol). The mixture was
refluxed for 7 h, hot-filtered, and evaporated to dryness to obtain 4h
(1.07 g, 90%). Mp: 169−170 °C (lit.13 mp: 168−169 °C). 1H NMR: δ
2.85 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 7.32−7.45 (m, 4H, arom),
7.63−7.75 (m, 1H, arom), 8.25−8.36 (m, 1H, arom).
General Procedure for the Alkylation of 3. NaH (60%, 7.5
mmol) was suspended in DMSO (30−40 mL) and heated to 70 °C for
1 h under nitrogen. 3 (5 mmol) was added and the mixture was stirred
for another 3 h, and then the selected alkyl halide (5 mmol) was added
and the reaction was kept at the same temperature for 8 h. The
mixture was then poured into ice, extracted with DCM, washed with
water, dried over Na2SO4 and evaporated to dryness to obtain
compounds 4i−n (see the Supporting Information).
4-Methoxymethoxy-1-methylxanthen-9-one (4o). 3 (1.4 g, 6.19
mmol) in dry THF was added to a suspension of 60% NaH (0.18 g,
7.40 mmol) in dry THF at 0 °C under nitrogen. MOM-Cl (0.54 mL,
7.40 mmol) was added dropwise and the resulting solution was stirred
at 0 °C for 1 h and then at rt overnight. The mixture was then poured
into ice and filtered and the solid washed with water. The crude was
purified by flash chromatography (toluene/acetone 9.75:0.25), to
obtain 4o (55%). Mp: 96−98 °C. 1H NMR: δ 2.93 (s, 3H, CH3), 3.62
(s, 3H, OCH3), 5.26 (s, 2H, OCH2), 7.30−7.51 (m, 4H, arom), 7.63−
7.76 (m, 1H, arom), 8.24−8.36 (m, 1H, arom).
General Procedure for the Bromination of 4e−o. To a
solution of the selected methyl derivative (2 mmol) in CCl4 (10−20
mL) were added N-bromosuccinimide (2 mmol) and a catalytic
amount of benzoyl peroxide, and the mixture was refluxed for 5−7 h.
The mixture was hot-filtered and evaporated to dryness to give a crude
product (compounds 5e−o; see the Supporting Information) that was
used without further purification.
General Procedure for the Synthesis of Imidazolylmethyl
Compounds 1e−n, 6o. A mixture of the selected bromomethyl
derivative (1 mmol) and imidazole (3 mmol) in acetonitrile (40−50
mL) was refluxed for 7 h under nitrogen. The solvent was removed
under reduced pressure and the residue was purified by flash
chromatography.
1-Imidazolylmethyl-4-chloroxanthen-9-one (1e). Eluent: petro-
leum ether/ethyl acetate 1:4. Yield: 30%. Mp: 223−224 °C. 1H NMR:
δ 6.19 (s, 2H, CH2), 6.96 (s, 1H, imi), 7.15 (s, 1H, imi), 7.38−7.55
(m, 3H, arom), 7.71−7.82 (m, 3H, arom + imi), 8.32−8.38 (m, 1H,
arom). 13C NMR: δ 43.99, 117.49, 119.52, 120.60, 120.98, 122.19,
124.58, 127.07, 128.51, 131.81, 134.59, 135.33, 135.73, 138.14, 154.94,
156.34, 177.58. ES-MS: 311 (M + 1). Anal. (C17H11ClN2O2): C, H, N
(see the Supporting Information).
EXPERIMENTAL SECTION
■
Chemistry. General Methods. All melting points were
determined in open glass capillaries using a Buchi apparatus and are
̈
1
uncorrected. H and 13C NMR spectra were recorded on a Varian
Gemini 300 spectrometer in CDCl3 solutions unless otherwise
indicated, with Me4Si as the internal standard. Mass spectra were
recorded on a V.G. 7070 E spectrometer or on a Waters ZQ 4000
apparatus operating in electrospray (ES) mode. Silica gel (Merck,
230−400 mesh) was used for purification with flash chromatography.
Chemical purities of the tested compounds were determined by
elemental analysis (C, H, N) and confirmed ≥95% purity (see the
Supporting Information). Compounds were named following IUPAC
rules as applied by AUTONOM, a PC software for systematic names
in organic chemistry (Beilstein-Institut and Springer).
4-Hydroxy-1-methylxanthen-9-one (3). 4-Amino-1-methyl-9-xan-
thenone10 (2) (3 g, 13.5 mmol) was dissolved in H2SO4 (40 mL), and
NaNO2 (0.93 g, 13.5 mmol) in water was added dropwise keeping the
temperature below −5 °C. The mixture was stirred for 30 min, allowed
to go to room temperature (rt) for another 30 min, and poured into
warm H2SO4 1:2 (300 mL) with stirring. The mixture was heated to
80 °C until the evolution of N2 ceased and poured into ice and the
precipitate was filtered to obtain 3 (0.9 g, 31%). Mp: 243−245 °C. 1H
NMR (acetone-d6): δ 2.81 (s, 3H, CH3), 7.31−7.32 (m, 1H, arom),
7.38−7.42 (m, 2H, arom), 7.49−7.52 (m, 1H, arom), 7.76−7.79 (m,
1H, arom), 8.21−8.23 (m, 1H, arom), 9.72 (s, 1H, OH).
4-Chloro-1-methylxanthen-9-one (4e). 210 (1 g, 4.44 mmol) was
dissolved in HCl 1:2 (12 mL), and NaNO2 (0.31 g, 4.44 mmol) in
water was added dropwise keeping the temperature below −5 °C. The
mixture was stirred for 30 min, allowed to go to rt for further 30 min,
and added to a cold solution of CuCl (0.53 g, 5.33 mmol) in HCl (3
mL). The mixture was heated to 80 °C until the evolution of N2
ceased and poured into ice, and the precipitate was filtered to obtain
1
4e (0.45 g, 42%, ligroin). Mp: 151−153 °C. H NMR: δ 3.05 (s, 3H,
CH3), 7.30−7.48 (m, 3H, arom), 7.65−7.76 (m, 2H, arom), 8.25−
8.32 (m, 1H, arom).
4-Iodo-1-methylxanthen-9-one (4f). 210 (1 g, 4.44 mmol) was
dissolved in H2SO4 (13 mL), and NaNO2 (0.31 g, 4.44 mmol) in
water was added dropwise keeping the temperature below −5 °C. The
mixture was stirred for 30 min and allowed to go to rt for 30 min. A
solution of NaI (0.66 g, 4.44 mmol) in water was added, and the
mixture was warmed to 80 °C for 1 h and then poured into ice. The
precipitate was filtered, dissolved in DCM, washed with Na2S2O3
solution, and evaporated to dryness to obtain 4f (0.4 g, 27%, ligroin).
Mp: 188−190 °C. 1H NMR: δ 3.11 (s, 3H, CH3), 7.08−7.15 (m, 1H,
arom), 7.31−7.45 (m, 2H, arom), 7.63−7.75 (m, 1H, arom), 8.06−
8.12 (m, 1H, arom), 8.24−8.31 (m, 1H, arom).
1-Imidazolylmethyl-4-iodoxanthen-9-one (1f). Eluent: toluene/
acetone 1:1. Yield: 26%. Mp: 105−107 °C (ethyl acetate/petroleum
1
1-Methyl-4-phenylxanthen-9-one (4g). 4-Amino-1-methyl-9-xan-
ether). H NMR: δ 6.18 (s, 2H, CH2), 6.99 (s, 1H, imi), 7.10 (s, 1H,
thenone10 (2) (1 g, 4.44 mmol) was dissolved in a mixture of NaBF4
imi), 7.38−7.51 (m, 3H, arom), 7.70−7.80 (m, 3H, arom + imi),
1727
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8.25−8.32 (m, 1H, arom). 13C NMR: δ 43.12, 82.14, 119.16, 121.12,
122.11, 122.43, 124.08, 126.92, 128.82, 131.13, 134.01, 135.87, 137.99,
142.48, 156.65, 168.81, 176.11. ES-MS: 403 (M + 1). Anal.
(C17H11IN2O2): C, H, N (see the Supporting Information).
1.80 (m, 4H, 2 × CH2), 1.86−2.03 (m, 4H, 2 tme CH2), 4.85−4.97
(m, 1H, OCH), 6.03 (s, 2H, CH2), 6.96 (s, 1H, imi), 7.21 (s, 1H, imi),
7.32−7.57 (m, 4H, arom), 7.65−7.78 (m, 1H, arom), 7.81 (s, 1H,
imi), 8.25−8.35 (m, 1H, arom). 13C NMR: δ 24.03 (2 C), 32.99 (2 C),
40.54, 80.76, 117.42, 119.80, 119.97, 120.51, 121.98, 123.80, 124.29,
124.40, 126.87, 127.65, 134.79, 138.06, 151.35, 152.60, 155.25, 178.76.
ES-MS: 361 (M + 1). Anal. (C22H20N2O3): C, H, N (see the
Supporting Information).
1-Imidazolylmethyl-4-phenylxanthen-9-one (1g). Eluent: petro-
leum ether/ethyl acetate 1:4. Yield: 21%. Mp: 178−180 °C (toluene).
1H NMR: δ 5.95 (s, 2H, CH2), 6.75 (s, 1H, imi), 6.90 (s, 1H, imi),
7.15−7.80 (m, 11H, arom + imi), 8.22−8.33 (m, 1H, arom). 13C
NMR: δ 44.70, 117.42, 119.09, 119.20, 119.56, 122.54, 124.19, 126.97,
128.06, 128.17, 128.32, 128.71 (2 C), 129.43 (2 C), 134.11, 134.93,
136.68, 139.22, 140.72, 155.04, 157.12, 178.25. ES-MS: 353 (M + 1).
Anal. (C23H16N2O2): C, H, N (see the Supporting Information).
1-Imidazolylmethyl-4-methoxyxanthen-9-one (1h). Eluent: tol-
uene/acetone 4:1. Yield: 40%. Mp: 198−201 °C. 1H NMR: δ 3.98 (s,
3H, OCH3), 6.01 (s, 2H, CH2), 6.92 (s, 1H, imi), 7.20 (s, 1H, imi)
7.33−7.78 (m, 5H, arom), 7.81 (s, 1H, imi), 8.32−8.35 (m, 1H,
arom). 13C NMR: δ 40.12, 52.14, 116.98, 119.35, 119.99, 120.37,
120.97, 122.11, 123.72, 124.92, 126.58, 128.12, 134.28, 138.01, 151.29,
152.10, 155.34, 178.24. ES-MS: 307 (M + 1). Anal. (C18H14N2O3) C,
H, N (see the Supporting Information).
1-Imidazolylmethyl-4-methoxymethoxyxanthen-9-one (6o). Elu-
1
ent: toluene/acetone 4:1. Yield: 75%. Mp: 148−150 °C. H NMR: δ
3.62 (s, 3H, OCH3), 5.46 (s, 2H, OCH2), 6.19 (s, 2H, CH2), 6.94 (s,
1H, imi), 7.23 (s, 1H, imi), 7.31−7.80 (m, 5H, arom), 7.85 (s, 1H,
imi), 8.31−8.43 (m, 1H, arom).
1-Imidazolylmethyl-4-hydroxyxanthen-9-one (1o). A solution of
6o (0.3 g, 0.9 mmol) in CH3COOH 1:1 (10 mL) with a few drops of
H2SO4 was refluxed for 15 min and poured into ice. The mixture was
neutralized with K2CO3 and the precipitate formed was filtered and
purified by flash chromatography (toluene/acetone 3:2) to obtain 1o
1
(0.21 g, 80%). Mp: 203−206 °C. H NMR (DMSO): 5.81 (s, 2H,
CH2), 6.78 (s, 1H, imi), 7.18 (s, 1H, imi), 7.23−7.55 (m, 5H, arom +
imi), 7.63−7.89 (m, 1H, arom), 8.18−8.26 (m, 1H, arom). 13C NMR
(DMSO): δ 40.61, 118.71, 120.32, 120.84, 121.11, 121.30, 122.35,
125.13, 125.22, 127.21, 128.12, 136.36, 138.25, 151.50, 153.85, 155.80,
179.10. ES-MS: 293 (M + 1). Anal. (C17H12N2O3): C, H, N (see the
Supporting Information).
1-Imidazolylmethyl-4-ethoxyxanthen-9-one (1i). Eluent: toluene/
1
acetone 3:2. Yield: 12%. Mp: 170−173 °C . H NMR: δ 1.47 (t, J =
4.10 Hz, 3H, CH3), 4.12−4.24 (q, J = 4.10 Hz, 2H, OCH2), 6.03 (s,
2H, CH2), 6.96 (s, 1H, imi), 7.15−7.72 (m, 6H, arom + imi), 7.86 (s,
1H, imi), 8.21−8.38 (m, 1H, arom). 13C NMR: δ 16.91, 40.77, 68.34,
117.24, 119.01, 119.87, 120.52, 120.88, 122.56, 123.28, 124.73, 126.15,
128.79, 134.41, 137.69, 151.38, 152.35, 155.03, 178.44. ES-MS: 321
(M + 1). Anal. (C19H16N2O3): C, H, N (see the Supporting
Information).
1-Imidazolylmethyl-4-aminoxanthen-9-one (1p). 1-Imidazolyl-
methyl-4-nitroxanthen-9-one10 (1b) (0.45 g, 1.4 mmol) was dissolved
in THF and hydrogenated at room temperature using a catalytic
amount of Pd/C. After filtration, the solvent was removed under
reduced pressure and the residue was crystallized from toluene to
obtain 1p (0.27 g, 70%). Mp: 218−220 °C (dec). 1H NMR: δ 5.90 (s,
2H, CH2), 7.01−7.15 (m, 2H, arom), 7.20 (s, 1H, imi), 7.35−7.65 (m,
3H, arom + imi), 7.68 (s, 1H, imi), −7.73−7.85 (m, 1H, arom), 8.25−
8.34 (m, 1H arom). 13C NMR: δ 40.27, 118.34, 120.15, 120.58,
121.52, 121.71, 122.62, 125.28, 125.36, 127.38, 128.42, 136.03, 138.47,
149.58, 151.89, 155.56, 178.76. ES-MS: 292 (M + 1). Anal.
(C17H13N3O2): C, H, N (see the Supporting Information).
Biological Methods. Inhibition of CYP11B1 and CYP11B2.
V79MZh cells expressing human or rat CYP11B1 or CYP11B2 were
incubated with [1,2-3H]-11-deoxycorticosterone (100 nM) as the
substrate and the inhibitor at different concentrations.14 The assay was
performed as previously described.15
CYP17 Preparation and Assay. Human CYP17 was expressed in E.
coli16 (coexpressing human CYP17 and NADPH-P450 reductase), and
the assay was performed using the method previously described with
progesterone (25 μM) as the substrate and NADPH as the
cofactor.17,18
CYP19 Preparation and Assay. Human CYP19 was obtained from
microsomal preparations of human placenta19 and the assay was
performed using the 3H2O method as previously described with
[1β-3H]androstenedione (500 nM) as the substrate.20
1-Imidazolylmethyl-4-isopropoxyxanthen-9-one (1j). Eluent: tol-
uene/acetone 4:1. Yield: 50%. Mp: 147−149 °C. 1H NMR: δ 1.41 (s,
3H, CH3), 1.46 (s, 3H, CH3), 4.65−4.75 (m, 1H, OCH), 6.08 (s, 2H,
CH2), 6.97 (s, 1H, imi), 7.22 (s, 1H, imi), 7.34−7.71 (m, 5H, arom.),
7.82 (s, 1H, imi), 8.33−8.39 (m, 1H, arom). 13C NMR: δ 22.10 (2 C),
40.38, 71.50, 117.40, 119.80, 119.91, 120.01, 120.90, 122.01, 123.80,
125.05, 126.86, 128.06, 134.75, 138.32, 151.48, 152.39, 155.21, 178.72.
ES-MS: 335 (M + 1). Anal. (C20H18N2O3): C, H, N (see the
Supporting Information).
1-Imidazolylmethyl-4-(3-methylbutoxy)xanthen-9-one (1k). Elu-
1
ent: toluene/acetone 3:2. Yield: 12%. Mp: 146−148 °C. H NMR: δ
0.98 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.75−1.82 (m, 2H, CH2), 2.58−
2.70 (m, 1H, CH), 4.15 (t, J = 4.20 Hz, 2H, OCH2), 6.01 (s, 2H,
CH2), 6.95 (s, 1H, imi), 7.20 (s, 1H, imi), 7.31−7.70 (m, 5H, arom),
7.87 (s, 1H, imi), 8.28−8.36 (m, 1H, arom). 13C NMR: δ 22.98 (2 C),
24.01, 38.20, 40.82, 63.12, 116.91, 118.87, 119.73, 120.32, 120.85,
122.37, 123.51, 125.38, 126.78, 128.61, 134.28, 138.17, 151.93, 152.88,
155.03, 178.54. ES-MS: 335 (M + 1). Anal. (C22H22N2O3): C, H, N
(see the Supporting Information).
1-Imidazolylmethyl-4-s-butoxyxanthen-9-one (1l). Eluent: tol-
1
uene/acetone 3:2. Yield: 32%. Mp: 150−153 °C. H NMR: δ 1.03
(t, J = 4.40 Hz, 3H, CH3), 1.35 (s, 3H, CH3), 1.72−1.85 (m, 2H,
CH2), 4.31−4.45 (m, 1H, OCH), 6.01 (s, 2H, CH2), 6.92 (s, 1H, imi),
7.20 (s, 1H, imi), 7.35−7.65 (m, 5H, arom), 7.81 (s, 2H, imi), 8.34−
8.41 (m, 1H, arom). 13C NMR: δ 10.01, 20.25, 31.11, 40.51, 73.16,
117.17, 119.15, 119.74, 120.27, 120.98, 122.28, 123.52, 125.38, 126.73,
128.29, 134.57, 138.46, 151.67, 152.29, 155.17, 178.21. ES-MS: 349
(M + 1). Anal. (C21H20N2O3): C, H, N (see the Supporting
Information).
Pharmacophore Search. The previously reported pharmaco-
phore model9 was employed as the pharmacophore query.
Compounds were built and energy minimized in the MMFF94s
force field with MOE 2008. 3D-conformational database was
subsequently constructed with a strain limit of 4 kcal/mol using the
Conformation Import module in MOE. Only one matched conformer
was conserved for each compound according to the ranking of rmsd.
No further refining of the query was performed.
1-Imidazolylmethyl-4-cyclopropylmethoxyxanthen-9-one (1m).
Eluent: toluene/acetone 3:2. Yield: 14%. Mp: 276−179 °C. 1H
NMR: δ 0.39−0.45 (m, 2H, CH2), 0.68−0.80 (m, 2H, CH2), 1.37−
1.44 (m, 1H, CH), 3.91 (d, J = 4.30 Hz, 2H, OCH2), 6.12 (s, 2H,
CH2), 6.95 (s, 1H, imi), 7.25−7.51 (m, 4H, arom), 7.62−7.78 (m, 1H,
arom), 7.94 (s, 1H, imi), 8.21−8.32 (m, 1H, arom). 13C NMR: δ 3.01
(2 C), 10.92, 40.13, 78.38, 117.22, 119.38, 119.56, 120.38, 121.02,
122.37, 123.11, 125.27, 126.36, 128.15, 134.48, 138.26, 151.74, 152.32,
155.28, 178.53. ES-MS: 347 (M + 1). Anal. (C21H18N2O3): C, H, N
(see the Supporting Information).
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental and spectroscopic details for intermediate
compounds 4i−n and 5e−o, elemental analyses of target
compounds 1e−p, and Table 1 SI. This material is available free
of charge via the Internet at http://pubs.acs.org.
1-Imidazolylmethyl-4-cyclopentyloxyxanthen-9-one (1n). Eluent:
toluene/acetone 4:1. Yield: 45%. Mp: 200−201 °C. 1H NMR: δ 1.61−
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(13) Valenti, P.; Ceccarelli, G.; Da Re, P. NMR structure
determination of some new 4-hydroxyxanthone derivatives. J. Chem.
Eng. Data 1970, 15 (4), 4.
(14) Ehmer, P. B.; Bureik, M.; Bernhardt, R.; Muller, U.; Hartmann,
R. W. Development of a test system for inhibitors of human
aldosterone synthase (CYP11B2): Screening in fission yeast and
evaluation of selectivity in V79 cells. J. Steroid. Biochem. Mol. Biol.
2002, 81 (2), 173−179.
AUTHOR INFORMATION
Corresponding Author
*E-mail: silvia.gobbi@unibo.it; alessandra.bisi@unibo.it.
Phone/Fax: +39 051 2099732/34.
■
Notes
The authors declare no competing financial interest.
(15) Yin, L.; Lucas, S.; Maurer, F.; Kazmaier, U.; Hu, Q.; Hartmann,
R. W. Novel imidazol-1-ylmethyl substituted 1,2,5,6-
tetrahydropyrrolo[3,2,1-ij]quinolin-4-ones as potent and selective
CYP11B1 inhibitors for the treatment of Cushing’s syndrome. J.
Med. Chem. 2012, 55 (14), 6629−6633.
(16) Ehmer, P. B.; Jose, J.; Hartmann, R. W. Development of a
simple and rapid assay for the evaluation of inhibitors of human
17alpha-hydroxylase-C(17,20)-lyase (P450cl7) by coexpression of
P450cl7 with NADPH-cytochrome-P450-reductase in Escherichia coli.
J. Steroid. Biochem. Mol. Biol. 2000, 75 (1), 57−63.
(17) Hutschenreuter, T. U.; Ehmer, P. B.; Hartmann, R. W. Synthesis
of hydroxy derivatives of highly potent non-steroidal CYP 17
inhibitors as potential metabolites and evaluation of their activity by
a non cellular assay using recombinant human enzyme. J. Enzyme
Inhib. Med. Chem. 2004, 19 (1), 17−32.
(18) Hu, Q.; Negri, M.; Olgen, S.; Hartmann, R. W. The role of
fluorine substitution in biphenyl methylene imidazole-type CYP17
inhibitors for the treatment of prostate carcinoma. ChemMedChem
2010, 5 (6), 899−910.
ABBREVIATIONS USED
■
CYP, cytochrome P450; CYP19, aromatase; CYP17, 17α-
hydroxylase/17,20-lyase; CYP11B1, 11β-hydroxylase;
CYP11B2, aldosterone synthase; 18OH-B, 18-hydroxycorticos-
terone.
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