Design, synthesis, and biological evaluation of imidazolyl derivatives of 4,7-disubstituted coumarins as aromatase inhibitors selective over 17-α-hydroxylase/C17-20 lyase

Article abstract of DOI:10.1021/jm101120u

The design, synthesis, and biological evaluation of a series of new aromatase (AR, CYP19) inhibitors bearing an imidazole ring linked to a 7-substituted coumarin scaffold at position 4 (or 3) are reported. Many compounds exhibited an aromatase inhibitory potency in the nanomolar range along with a high selectivity over 17-α-hydroxylase/C17-20 lyase (CYP17). The most potent AR inhibitor was the 7-(3,4-difluorophenoxy)-4-imidazolylmethyl coumarin 24 endowed with an IC₅₀ = 47 nM. Docking simulations on a selected number of coumarin derivatives allowed the identification of the most important interactions driving the binding and clearly indicated the allowed and disallowed regions for appropriate structural modifications of coumarins and closely related heterocyclic molecular scaffolds.

Full text of DOI:10.1021/jm101120u

ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Imidazolyl Derivatives
of 4,7-Disubstituted Coumarins as Aromatase Inhibitors Selective over
17-r-Hydroxylase/C17-20 Lyase
Angela Stefanachi,† Angelo D. Favia,‡ Orazio Nicolotti,† Francesco Leonetti,† Leonardo Pisani,†
Marco Catto,† Christina Zimmer,§ Rolf W. Hartmann,§ and Angelo Carotti*,†
†Dipartimento Farmaco-Chimico, Universitꢀa degli Studi di Bari “Aldo Moro”, via Orabona 4, I-70125 Bari, Italy
‡D3 Department, Istituto Italiano di Tecnologia (IIT), via Morego 30, I-16163 Genoa, Italy
§Pharmaceutical and Medicinal Chemistry, Saarland University & Helmholtz Institute for Pharmaceutical Research Saarland
(HIPS), P.O. Box 15 11 50, D-66041 Saarbr€ucken, Germany
ABSTRACT: The design, synthesis, and biological evaluation of
a series of new aromatase (AR, CYP19) inhibitors bearing an
imidazole ring linked to a 7-substituted coumarin scaffold at position
4 (or 3) are reported. Many compounds exhibited an aromatase
inhibitory potency in the nanomolar range along with a high
selectivity over 17-R-hydroxylase/C17-20 lyase (CYP17). The
most potent AR inhibitor was the 7-(3,4-difluorophenoxy)-4-imi-
dazolylmethyl coumarin 24 endowed with an IC50 = 47 nM.
Docking simulations on a selected number of coumarin derivatives
allowed the identification of the most important interactions driving
the binding and clearly indicated the allowed and disallowed regions
for appropriate structural modifications of coumarins and closely related heterocyclic molecular scaffolds.
’ INTRODUCTION
fewer side effects than ER antagonists because of the lack of
estrogenic activity on uterus and vasculature.13
Seventy-five percent of breast cancers in postmenopausal
women are estrogen-dependent (ERþ).1 While in premenopau-
sal women estrogens are principally produced by ovaries, during
postmenopause, they continue to be synthesized by nonovarian
tissues,2 such as breast tissue.3 This peripheral synthesis allows
estrogens to reach locally a concentration 4-6 times higher than
in the serum and equivalent to breast tissue levels recorded in
premenopausal women.4,5
Estrogens are biosynthesized from circulating androgens through
an aromatization reaction of the steroidal A ring, catalyzed by the
enzyme aromatase (AR, CYP19). Because in malignant tissues AR
activity and estrogen levels are higher than in healthy tissues, two
main strategies have been devised by medicinal chemists to control
or block the pathological activity in ERþ cancer.6
The first one implies the block of the binding of estrogens to
their biological receptor, the estrogen receptor (ER), with
antagonists7 among which tamoxifen and raloxifen have proven
to be very effective drugs in the treatment of breast cancer.8
Unfortunately, in some tissues, such as the uterus and vascula-
ture, such compounds also present an estrogenic action, less
pronounced with raloxifen,9 and this results in an increased risk
of endometrial cancer10 and stroke.11 Hence, more selective
estrogen receptor modulators (SERMs) are eagerly pursued.12
Furthermore, a second strategy was devised by directly targeting
AR, a key enzyme in the biosynthesis of estrogens. Aromatase
inhibitors (ARIs), acting on the estrogen biosynthesis, present
Localized in the endoplasmic reticulum of cells, AR is a
multienzymatic complex consisting of two main components,
a form of cytochrome P450 (CYP19) and a NADPH-cyto-
chrome P450 flavoprotein reductase. AR catalyzes the con-
version of androgens to estrogens through the transformation
of the steroidal enone A ring into the aromatic phenolic ring
with the concomitant loss of the C19 methyl group.14-17 The
earliest ARIs were discovered in the early 1970s, and formes-
tane (Chart 1) was the first selective ARI approved for the
treatment of breast cancer. Nowadays, exemestane, whose
structure closely resembles that of formestane (Chart 1),
anastrozole and letrozole, both sharing a nonsteroidal 1,2,4-
triazolyl structure, have been approved by FDA.18-20 The
effectiveness of the triazolyl inhibitors in blocking the enzy-
matic activity resides in their ability to coordinate the iron
atom of the AR heme group by means of the lone pair carried
on the triazole nitrogen at position 4. Anastrozole and
letrozole have been recommended by the FDA as first-line
drugs in the therapy of breast carcinoma.21
Despite their selectivity, the prolonged clinical use of ARIs
eventually leads to severe side effects, arising mainly from the
concomitant, unwanted inhibition of other CYP enzymes, and
Received: August 30, 2010
Published: February 22, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
ARTICLE
Chart 1. Chemical Structures of Some ARIs
coumarin 34 was designed to verify the importance of a regioselec-
tive interaction within the AR binding site. In this regard, all of the
designed inhibitors might also serve as valuable molecular probes to
gain insights into main structural features of the binding site regions
of AR.
In addition, the recently solved crystal structure of human
AR38,39 was exploited to better rationalize the outcomes of the
biological assays. To this aim, docking simulations of selected
ligands were performed on a suitable refined model of the
enzyme to reproduce likely induced fit phenomena occurring
upon binding. The analysis of their putative binding conforma-
tions helped to shed light on the key structural features respon-
sible for high inhibitory potencies.
’ CHEMISTRY
Compounds 1-13 (Scheme 1) were synthesized from
4-chloromethyl-7-hydroxycoumarin,30,40 through the benzyla-
tion of its phenolic group followed by the nucleophilic substitu-
tion of the chlorides with imidazole. Compound 26 was obtained
directly from 4-chloromethyl-7-hydroxycoumarin by a nucleo-
philic substitution with imidazole. As described in Scheme 1, for
the synthesis of compounds 14-24, the phenolic group was first
arylated by a copper acetate-mediated coupling,41 and then, the
imidazole ring was introduced, as above.
The synthesis of compound 25 (Scheme 2) started with the von
Pechmann reaction between (3-hydroxyphenyl)carbamic acid eth-
yl ester intermediate 25a42 and ethyl 4-chloroacetoacetate. After
deprotection, the amino group was arylated by a copper acetate-
mediated coupling,43 and then, final compound 25 was obtained by
a nucleophilic substitution reaction with imidazole.
To obtain compound 28 (Scheme 3), 4-benzyl-7-hydroxy-
coumarin 28a44 was methylated by CH3I and NaH in DMF,
brominated by N-bromosuccinimide (NBS) in CCl4, and reacted
with imidazole.
Intermediates 29b, 31b, and 32b (Scheme 4) were obtained
by von Pechmann cyclocondensation of 3-phenoxyphenol with
the corresponding ethyl phenylacetoacetates 29a, 31a, and 32a,
respectively.45 As before, methylene bromination with NBS
followed by reaction with imidazole yielded the desired final
compounds 29, 31, and 32.
The synthesis of compound 30 (Scheme 5) started with the
arylation of the phenolic intermediate 28a followed by methy-
lene bromination and reaction with imidazole.
this calls for new, potent, more selective, and less toxic CYP19
inhibitors.22-29 Along this research line, we have begun a
systematic, long-term study, aiming at the design and synthesis
of new ARIs endowed with improved biological, pharmacologi-
cal, and toxicological profiles. Our first study dealing with the
design, synthesis, and biological evaluation as CYP19 inhibitors
of a series of 4-imidazolylmethyl-7-substituted coumarins was
reported in 200430 followed by the publication at the wwPDB31
of a three-dimensional homology model of the human AR
enzyme32 later used as a valuable tool for structure-based ligand
design.27,33,34
Biological and modeling results from these two papers30,32
suggested 4-imidazolylmethyl-7-substituted coumarin derivatives 1
and 14 (Table 1) as promising leads for further structural modifica-
tions aimed to deepen our understanding of structure-affinity
relationships (SAFIRs) and to discover new molecules with potent
AR inhibition along with good selectivity over 17-R-hydroxylase/
C17-20 lyase (CYP17) and therapeutic potential in breast cancer.
CYP17 is a cytochrome P450-dependent enzyme, involved in the
development of prostatic cancer, and huge efforts are currently
devoted to the synthesis of selective CYP17 enzyme inhibitors, which
may halt the progression of this tumor toward pharmacologically
untreatable advanced forms.35-37
Developing molecules that act selectively toward one of the two
strictly related CYP17 and CYP19 enzymes represents nowadays a
very important task for medicinal chemists and may constitute a
logical follow up of the present work.
Starting from the remarkable AR inhibitory activity and CYP19
over CYP17 selectivity exhibited by compounds 1 and 14, two new
series of coumarin derivatives, 2-13 and 15-26 (Table 1), were
designed, prepared, and tested to explore the substituent effects on
the 7-benzyloxy and 7-phenoxy aromatic rings, respectively. More-
over, the synthesis of derivatives 28-32 (Table 1) was also under-
taken to analyze the effect on affinity and selectivity resulting from
the introduction of a phenyl and substituted phenyl rings on the
4-methylene bridge of lead compound 14. Eventually, 3-substituted
The synthesis of compound 34 (Scheme 6), a 3,7-disubstitut-
ed coumarin derivative, was accomplished starting from inter-
mediate 34a,46 which was demethylated by BBr3, arylated by a
copper acetate-mediated coupling, and brominated by NBS in
CCl4. The subsequent nucleophilic substitution with imidazole
gave compound 34.
’ BIOLOGICAL ASSAYS
All of the molecules in Table 1 were tested for their inhibitory
activities against AR (CYP19) and 17R-hydroxylase/C17,20-
lyase (CYP17), two related P450 enzymes, which are responsible
for catalyzing the final step in estrogen and androgen biosynth-
esis, respectively. In the case of CYP19, human placental micro-
somes were used as a source of the enzyme and [1β-3H]
androstenedione as a substrate as described by Thompson and
Siiteri,14 with slight modifications.47,48 For the determination of
CYP17 inhibition, microsomes from Escherichia coli-expressing
human CYP17 and progesterone as substrate were applied;48,49
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Table 1. Chemical Structures and Inhibition Data of Coumarin Derivatives 1-34
entry
R
R1a
CYP19 inhibitionb
CYP17 inhibitionc (%)
130
2
C6H5CH2O
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.150
0.114
0.113
0.130
0.235
0.207
0.141
0.267
0.178
0.127
0.481
0.169
0.165
0.051
0.072
0.072
0.292
0.690
0.112
0.164
0.296
0.081
0.070
0.047
0.105
3.75
3
0
30-CH3C6H4CH2O
30-FC6H4CH2O
30-ClC6H4CH2O
30-CF3C6H4CH2O
30-OCF3C6H4CH2O
30-NO2C6H4CH2O
40-FC6H4CH2O
40-ClC6H4CH2O
40-OCH3C6H4CH2O
40-OCF3C6H4CH2O
30,50-F2C6H3CH2O
30,40-F2C6H3CH2O
C6H5O
3
0
4
3
5
0
6
3
7
3
8
0
9
1
10
11
12
13
1430
15
16
17
18
19
20
21
22
23
24
25
26
2730
28
29
30
31
32
33d30
34d
0.5
0.5
2
1
26
1
30-FC6H4O
30-ClC6H4O
2
30-OCH3C6H4O
40-CH3C6H4O
40-ClC6H4O
40-CNC6H4O
40-COCH3C6H4O
40-N(CH3)2C6H4O
30,50-F2C6H3O
30,40-F2C6H3O
C6H5NH
2
1
5
16
4
3
1
14
3
HO
7
CH3O
0.280
0.455
0.067
0.317
0.532
4.01
14
3
CH3O
C6H5
C6H5O
30,40-F2C6H3O
C6H5
4
C6H5
1
C6H5O
4-ClC6H4
4-CNC6H4
H
5
C6H5O
3
CH3O
2.82
43
C6H5O
C6H5
0.313
13
a Chiral compounds carrying a phenyl substituent on the methylene bridge at position 4 or 3 were tested as racemic mixtures. b IC50 values (μM) coming
from three independent experiments; the SEMs were always <10%; substrate, 500 nM 1β 3H androstenedione/androstenedione. c Percent at 2.5 μM;
substrate, 25 μM progesterone; enzyme, bacterial membranes containing recombinantly expressed human CYP17; reference compounds, 1. IC50, 0.15
μM for CYP19; ketoconazole IC50, 4.5 μM for CYP17. d Compounds 33 and 34 are 3-substituted coumarin derivatives.
the inhibition data are shown in Table 1 as a percentage of
inhibition at the indicated concentration. Compounds 1 and 24
also were tested as inhibitors of the two enzymes catalyzing the
final step of corticosteroids biosynthesis, namely, CYP11B1,50
for cortisol and CYP11B251-54 for aldosterone. The assay was
carried out on V79MZ cells expressing human CYP11B1 and
CYP11B2 genes by using [1,2-3H]11-deoxycorticosterone/11-
deoxycorticosterone as the radioactive substrate; the inhibition
data are reported in Table 2.
’ RESULTS AND DISCUSSION
Chemical structures and inhibition data of the newly synthe-
sized and others already described molecules, used herein as
reference (i.e., 1, 14, 27, and 33), are reported in Table 1.
Inhibition data were expressed as IC50 (μM) for CYP19 and as
percentage of inhibition at a 2.5 μM concentration for CYP17.
At a glance, biological data reported in Table 1 indicated that
all of the designed compounds showed a very high CYP19/
CYP17 selectivity. In fact, the percentage of CYP17 inhibition at
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ARTICLE
Scheme 1a
a Reagents and conditions: (i) K2CO3, EtOH, reflux, 3 h. (ii) Cu(OAc)2, Et3N, molecular sieves 4 Å, CH2Cl2, room temperature, 18 h. (iii) K2CO3,
THF, reflux, 5-7 h.
Scheme 2a
a Reagents and conditions: (i) Ethyl 4-chloroacetoacetate, H2SO4 70%, room temperature, 4 h. (ii) H2SO4 96%, glacial acetic acid, reflux, 4 h. (iii)
Cu(OAc)2, Et3N, CH2Cl2, room temperature, 72 h. (iv) K2CO3, THF, reflux, 5-7 h.
2.5 μM concentration was generally lower than 10%, with very
few exceptions. Conversely, IC50 values lower than 0.30 μM were
exhibited at CYP19 by most of the new compounds.
position. Indeed, compound 11 bearing the larger substituent at the
para position was the least potent compound of the 7-benzyloxy
series. The 3,5- and 3,4-difluorobenzyloxy derivatives 12 and 13
displayed a slightly weaker activity than reference compound 1.
Unfortunately, no clear SAFIR emerged from the analysis of the
electronic, hydrophobic, and steric substituent effects of 7-benzyloxy
derivatives 1-13.
Inhibition data of the 7-phenoxy derivatives 15-24 fell in the
0.047-0.690 μM range, and only one compound, that is, the
3,4-difluorophenoxy derivative 24, exhibited an inhibitory po-
tency equal or slightly better than the reference compound 14
(0.047 vs 0.051 μM). Similarly to what was observed in the
7-benzyloxy series, the meta-chloro congener 16 exhibited an
inhibitory potency higher than the corresponding para derivative
19. Also, for the series of 7-phenoxy coumarin derivatives, no
clear electronic, hydrophobic, and steric effects on the affinity
could be detected. In fact, the almost isolipophilic compounds 18
To gain clear insights into the SAFIRs, data in Table 1 were
examined taking into account separately the two homologous
series of 7-benzyloxy-4-imidazolylmethylcoumarin derivatives
2-13 and 7-phenoxy-4-imidazolylmethylcoumarin derivatives
15-24. The already reported highly potent inhibitors 1 and
14 were taken as reference compounds for the evaluation of the
SAFIRs of the 7-benzyloxy and 7-phenoxy series, respectively.
Inhibition data of the 7-benzyloxy derivatives 2-13 fell in the
0.113-0.481 μM range. In comparison with reference compound 1,
meta-substituted derivatives 2, 3, and 4 and the para-substituted
derivative 10 showed a slightly increased affinity. Para-substituted
derivatives are less active than the corresponding meta-substituted
analogues (compare 8 vs 3, 9 vs 4, and 11 vs 6), and this may be
indicative of a reduced enzymatic accessibility for substituents in that
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Scheme 3a
Scheme 5a
a Reagents and conditions: (i) NaH, CH3I, DMF, 0 °C, 1 h. (ii) NBS,
DBP, CCl4, reflux, 1 h. (iii) K2CO3, THF, reflux, 7 h.
a Reagents and conditions: (i) Cu(OAc)2, Et3N, molecular sieves 4 Å,
CH2Cl2, room temperature, 4 h. (ii) NBS, DBP, CCl4, reflux, 4 h. (iii)
CH3CN, reflux, 6 h.
Scheme 4a
also observed for compound 25 that carries a 7-anilino substituent
much more hydrophilic than the 7-phenoxy isosteric substituent of 14
(hydrophobic substituent constant π = 2.08 and 1.37, respectively)55
suggested that beside the substituent lipophilicity, a conserved
binding topology, likely placing the two phenyl rings in similar
enzymatic pockets, also plays a key role in the ligand binding process.
To extend the SAFIRs to other regions of the enzyme, a phenyl
group was introduced on the methylene bridge at position 4 of lead
compound 14. This structural modification left the affinity nearly
unchanged (29, IC50 = 0.067 μM vs IC50 = 0.051 μM of 14). The
good affinity of compound 29 and the presence of a chiral center on
its 4-substituent prompted us to explore possible enantioselective
interactions at the AR active site by measuring the individual activity
of the two separated enantiomers. Initially, the separation of the
enantiomers of 29 was investigated by using amylose and cellulose
packed columns, as well as glycopeptide antibiotics-based stationary
phases.56 The best enantiomeric separation was obtained on a
Chiralpak IA column by using MeOH as the mobile phase
(R = 1.6, Rs = 2.0). Regrettably, the strong acidity of the 4-methylene
proton caused a rapid racemization of both of the separated pure
enantiomers in solution, and this precluded the execution of any
enzymatic inhibition assay. Docking simulations following these
discouraging results suggested similar binding energies for the top-
scoring poses of the two enantiomers of inhibitor 29.
The introduction of two fluorine atoms at 3- and 4-positions
on the 7-phenoxy ring of 29 led to a 5-fold decrease of affinity
(30, IC50 = 0.317 μM), and this might indicate either some steric
clash or the overcome of some molecular lipophilicity limit. The
same explanation might apply to interpret the higher decrease
of affinity (IC50 from 0.067 to 0.532 μM) resulting from the
introduction of a para-chloro substituent on the 4-benzylic
moiety of 29, resulting in inhibitor 31. The even more dramatic
drop of affinity observed in the para-cyanophenyl derivative 32
seemed to indicate a more likely steric effect at the para position,
with the cyano substituent much less lipophilic than chlorine
(π = -0.57 vs 0.71).55 The affinity decrease, more evident for the
para-cyano congener 32 than for the para-chloro congener 31,
might be ascribed to a different steric effect arising from the two
differently shaped substituents.
a Reagents and conditions: (i) H2SO4 96%, 120 °C, 30 min. (ii) NBS,
DBP, CCl4, reflux, 1 h. (iii) K2CO3, THF, reflux, 5-7 h.
and 19 showed a striking different affinity, and the same consi-
deration holds when comparing the affinity of inhibitors 18 and
22, bearing Me and N(Me)2 substituents of comparable electron
donor ability (σp- = -0.15 and -0.12, respectively).55 However,
it is worth noting that inhibitors carrying halogen substituents in
the para and/or meta position (i.e., 15, 16, 19, 23, and 24)
exhibited the highest affinity within the whole 7-phenoxy series.
Despite its hydrophobic character, the introduction of a para-
methyl substituent on lead compound 14 determined a dramatic
drop of affinity, with compound 18 being the weakest ligand of
the 7-phenoxy coumarin series. No sound explanation could be
provided to interpret this unexpected result.
The favorable effect of lipophilic substituents at position 7 was
quite evident when comparing the affinity of the 7-OH (26, IC50 =
3.75 μM), 7-OMe (27, IC50 = 0.280 μM), and 7-OPh (14, IC50 =
0.051 μM) derivatives. However, the good affinity(IC50 =0.105μM)
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Scheme 6a
a Reagents and conditions: (i) BBr3, CH2Cl2, 0 °C f room temperature, 18 h. (ii) Cu(OAc)2, Et3N, molecular sieves 4 Å, CH2Cl2, room temperature, 1
h. (iii) NBS, DBP, CCl4, reflux, 4 h. (iv) CH3CN, reflux, 6 h.
Table 2. Inhibition Data of Coumarin Derivatives 1 and 24 on the Indicated CYPs
entry
CYP19 inhibitiona
CYP17 inhibitionb (%)
CYP11B1 inhibitionc
CYP11B2 inhibitionc
1
0.150
0.047
0.052
3
14
2
0.072
0.933
0.010
0.289
2.19
24
(()-fadrozole
0.001
a IC50 values (μM) coming from 3 independent experiments; the SEM were always <10%; substrate: 1β 3H androstenedione/androstenedione 500 nM.
b % at 2.5 μM; substrate: progesterone 25 μM; enzyme: bacterial membranes containing recombinantly expressed human CYP17; c IC50 values (μM)
coming from 3 independent experiments; the SEM were always <15%; substrate: [1,2-3H]11-deoxycorticosterone/11-deoxycorticosterone, 100 nM.
To shed light on the structural determinants responsible for
some of the recorded activity variation, compounds 1, 14, and 29
were docked at the binding site of the recently solved model of
human AR.38,39 To overcome the well-known flexibility-related
issues given by the use of a single protein structure,57 the AR
model was subjected to a focused minimization protocol similar
in spirit to the ones recently reported by others.58,59 As observed
for many CYP inhibitors, the binding driving interaction was the
heme iron coordination that, for the studied compounds, was
mediated by the electron-rich nitrogen of the imidazole ring.
As it can be inferred from Figure 1A, where the putative
binding modes of lead compounds 1 and 14 are shown, the
coumarin scaffolds were almost perpendicular to the plane of the
imidazole ring and stabilized by one hydrogen bond (HB)
between the lactone carbonyl and the hydroxyl of S478. In
addition, a convenient allocation of the phenoxy and benzyloxy
groups in a hydrophobic accessory site constituted of I70, L228,
I229, L372, M374, I395, and L477 may be observed. This three-
site interaction seemed to be better fulfilled when the coumarin
ring carried at position 4 an imidazolylmethyl group and a
phenoxy substituent at position 7, and this may justify the better
affinity of compound 14 as compared to 1. Indeed, the longer
benzyloxy group at position 7 seemed to induce some modifica-
tion of the heme coordination and of the HBs pattern. In
particular, the distance between the imidazole nitrogen and the
lactone carbonyl in both series of compounds is likely to play a
major role in the affinity regulation. This hypothesis was fully
corroborated by the fact that when the phenyl-imidazolylmethyl
substituent was moved to position 3 of the coumarin ring, a
substantial affinity drop was observed (compare compound 29
with 34). The effects of a substitution on the methylene bridge at
position 4 could be less easily rationalized due to the lack of
inhibition data on separated enantiomers.
However, the investigated binding poses of this class of mole-
cules, illustrated in Figure 1B, where compound 29 is shown as a
prototypical case, indicated that both enantiomers bind in a similar
fashion, consistent with the above illustrated three-site binding
hypothesis. In addition, a local displacement of some amino acids at
the bottom of the binding area was needed to allocate correctly the
ligand. The nonspecific interactions occurring between the phenyl
group of the ligand and the hydrophobic side chains of the protein
seem to compensate for the imperfect heme coordination (more
pronounced for the S-enantiomer), resulting in an IC50 value
comparable with that of its achiral precursor 14. Furthermore,
the drop of affinity observed upon substitution on the phenyl ring
(i.e., 31) can be ascribed to a restricted accessibility of the active site
to that position as well as to the induced changes in the electronic
features of the aromatic ring, thus limiting an effective binding. This
effect was even more pronounced when a longer and more
hydrophilic group such as the cyano substituent was present
(i.e., 32), although in such a case also the suboptimal allocation
of the group in a region defined mostly by hydrophobic or
negatively charged amino acids (i.e., F134, F221, W224, I305,
and D309) was likely to play a significant role.
To evaluate the selectivity of our ARIs over other CYPs involved
in the biosynthesis of steroids, one representative compound from
both series (i.e., compounds 1 and 24 from the 7-benzyloxy and
7-aryloxy series) was selected and tested toward CYP11B1 (steroid
11β-hydroxylase) and CYP11B2 (aldosterone synthase; Table 2).
Interestingly, these enzymes could also be targets for the treatment
of Cushing's syndrome or metabolic disease (CYP11B1)50 as well
as for hyperaldosteronism, congestive heart failure, and myocardial
fibrosis (CYP11B2).51-54 On the other hand, it is worth noting
that an unselective inhibition of these two CYPs by ARIs also could
lead to unwanted side effects such as hyponatremia, hyperkalemia,
adrenal hyperplasia, and hypovolemic shock.
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ARTICLE
Figure 1. (A) Putative binding mode of compounds 1 and 14 at the enzyme binding site, shown as stick models C-colored gray and cyan, respectively.
Helix I of the enzyme is highlighted to help interpretation. (B) Binding models of the two enantiomers of compound 35 at AR active site. The R- and S-
enantiomers are colored cyan and orange, respectively. The yellow blob represents the vdW surface of the aminoacids highlighted in A, not including
C437. In both figures, the protein backbone is depicted as ribbons, the green dashed lines are drawn between the atoms likely involved in hydrogen bond
or heme coordination, and selected relevant residues are C-colored white. In both cases, the protein backbone is depicted as ribbons, the dashed lines are
drawn between the atoms likely involved in HB or heme coordinations, and selected relevant residues are C-colored white.
Inhibition data in Table 2 indicated that lead compound 1, with
an IC50 on CYP19 of 0.150 μM, is a strong inhibitor of both
CYP11B1 and CYP11B2 enzymes with IC50 values of 0.072 and
0.289 μM, respectively. However, as compared to the well-known
ARI fadrozole (IC50 = 0.052 μM on CYP19), which exhibited IC50
values of 0.01 μM for CYP11B1 and 0.001 μM for CYP11B2, the
CYP19 over CYP11B2 selectivity of 1 was improved nearly 100-
fold. Compound 24, the most potent CYP19 inhibitor of the whole
series, showed IC50 values of 0.933 and 2.19 μM for CYP11B1 and
CYP11B2, respectively, with an ameliorated selectivity profile as
compared to fadrozole. These data might be indicative of less
adverse effects potentially arising from the inhibition of the aldos-
terone biosynthesis.60 As a consequence, inhibitors of the 7-aryloxy
series appeared safer and more suitable for further preclinical tests as
compared to the 7-benzyloxy congeners.
of properly chosen substituents all around the coumarin ring. Thus,
an easy variation of the local and global physicochemical properties of
the selected molecules may be possible.
Docking simulations of a selected number of coumarin
derivatives allowed the identification of the most important
interactions underlying the inhibitor binding and suggested the
positions of the coumarin ring suitable for further structural
modifications. This molecular mapping might be used to guide
the design of new, original inhibitors even based on molecular
scaffolds different from coumarin.
’ EXPERIMENTAL SECTION
Chemistry. High analytical grade chemicals and solvents were from
commercial suppliers. When necessary, solvents were dried by standard
techniques and distilled. After extraction from aqueous layers, the
organic solvents were dried over anhydrous magnesium or sodium
sulfate. Thin-layer chromatography (TLC) was performed on aluminum
sheets precoated with silica gel 60 F254 (0.2 mm) type (E. Merck).
Chromatographic spots were visualized by UV light or Hanessian
reagent.66 Purification of crude compounds was carried out by flash
column chromatography on silica gel 60 (Kieselgel 0.040-0.063 mm, E.
Merck) or by crystallization. Melting points (uncorrected) for fully
purified products (see below) were determined in a glass capillary tube
on a Stuart Scientific electrothermal apparatus SMP3. 1H NMR spectra
were recorded in CDCl3 (unless otherwise indicated) at 300 MHz on a
Varian Mercury 300 instrument. All of the detected signals were in
accordance with the proposed structures. Chemical shifts (δ scale) are
reported in parts per million (ppm) relative to the central peak of the
solvent. Coupling constant (J values) are given in Hertz (Hz). Spin
multiplicities are given as: s (singlet), s br (broad singlet), d (doublet),
dd (double doublet), t (triplet), q (quadruplet), or m (multiplet). ESI-
MS was performed with an electrospray interface ion trap mass spectro-
meter (1100 series LC/MSD Trap System Agilent, Palo Alto, Ca). The
purity of all of the intermediates, checked by 1H NMR and HPLC, was
always better than 90%. The purity of all tested final products,
’ CONCLUSION
In summary, a number of new coumarin imidazolyl derivatives
described in this paper proved to be remarkably potent ARIs with
a high selectivity over CYP17. CYP11B1 and CYP11B2 inhibi-
tion data of lead compounds 1 and 24 suggested that 7-aryloxy
derivatives may be safer and more suitable for further develop-
ment than the 7-benzyloxy congeners.
In comparison with already reported ARIs of similar potency,
coumarin derivatives present several advantages, such as a facile and
straightforward synthesis and a likely favorable ADMET and toxico-
logical profile as the coumarin scaffold is present in many natural and
dietary products61 and in some drugs as well.62 However, some
suitably functionalized coumarins may constitute good substrates/
inhibitors for some P450 metabolic enzymes.63,64 The SAFIR that
recently emerged also at this level65 may help the design of new
compounds with the desired P450 potency, selectivity, and ADMET
properties. Simple coumarin chemistry may facilitate the attainment
of such important goals since it may allow the introduction of a variety
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Journal of Medicinal Chemistry
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determined by analytical HPLC, was always greater than 95%. Reverse
phase HPLC analyses were performed on a system equipped with
automatic injector and a Waters Breeze 1525 high performance liquid
chromatography (HPLC) pump coupled with a Waters 2489 UV
Detector (Waters Corp., Milford, MA) using a Waters XTerra RP 5
μm C8 column (150 mm ꢀ 3.0 mm i. d.). The UV detection was
measured at 254 and 280 nm. Each tested compound was analyzed by
elution with two different mobile phase systems: In system 1, com-
pounds were eluted using a 80/20 methanol/water mixture at a flow rate
of 0.5 mL/min; in system 2, compounds were eluted using a 65/35
acetonitrile/water mixture at a flow rate of 0.5 mL/min. The synthesis of
compounds 1, 14, 27, and 33 have been described in a previous paper.30
General Procedure for the Preparation of 7-Benzyloxy-
4-(chloromethyl)-2H-chromen-2-one Derivatives 2a-13a. To
a solution of 4-(chloromethyl)-7-hydroxycoumarin (0.32 g, 1.5 mmol)
in EtOH (15 mL), K2CO3 (0.62 g, 4.5 mmol) and the appropriate benzyl
bromide (4.5 mmol) were added. The reaction was stirred at reflux for 3 h.
After the solution was cooled, K2CO3 was filtered off, and the solution was
evaporated to dryness. The oily residue was treated with ether obtaining a
precipitate that was filtered and used without further purification.
4-(Chloromethyl)-7-[(3-methylbenzyl)oxy]-2H-chromen-2-one
(2a). Yield, 57%. 1H NMR δ: 7.56 (d, J = 8.8 Hz, 1H), 7.29-7.15 (m,
4H), 6.98-6.91 (m, 2H), 6.40 (s, 1H), 5.10 (s, 2H), 4.62 (s, 2H), 2.38
(s, 3H).
(m, 3H), 6.97-6.93 (m, 1H), 6.89-6.88 (m, 1H), 6.42 (s, 1H), 5.08 (s,
2H), 4.62 (s, 2H).
General Procedure for the Preparation of 7-Aryloxy-
4-(chloromethyl)-2H-chromen-2-one Derivatives 15a-
24a41. 4-(Chloromethyl)-7-hydroxycoumarin (0.21 g, 1.0 mmol),
Cu(OAc)2 (0.18 g, 1.0 mmol), arylboronic acid (3.0 mmol), and
powdered 4 Å molecular sieves were suspended in 10 mL of CH2Cl2,
and triethylamine (0.70 mL, 5.0 mmol) was added. The reaction mixture
was stirred under ambient atmosphere for 18 h at room temperature.
The mixture was filtered over Celite, and the organic filtrate was
evaporated to dryness and purified by flash chromatography using a
mixture of CH2Cl2/petroleum ether, 8/2 (v/v), as the eluent.
4-(Chloromethyl)-7-(3-fluorophenoxy)-2H-chromen-2-one (15a). The
product was used for the following reaction without purification.
The presence of the desired compound was certified by ESI-MS m/z
305 [M þ H]þ.
4-(Chloromethyl)-7-(3-chlorophenoxy)-2H-chromen-2-one (16a). Yield,
30%. 1H NMR δ: 7.64 (d, J = 8.8 Hz, 1H), 7.35-7.20 (m, 3H), 7.09 (s,
1H), 7.01-6.92 (m, 2H), 6.47 (s, 1H), 4.63 (s, 2H).
4-(Chloromethyl)-7-(3-methoxyphenoxy)-2H-chromen-2-one
(17a). Yield, 41%. 1H NMR δ: 7.61 (d, J = 8.8 Hz, 1H), 7.64-7.42 (m,
3H), 7.00-6.80 (m, 3H), 6.44 (s, 1H), 4.65 (s, 2H), 3.81 (s, 3H).
4-(Chloromethyl)-7-(4-methylphenoxy)-2H-chromen-2-one (18a).
Yield, 44%. 1H NMR δ: 7.58 (d, J = 8.8 Hz, 1H), 7.22 (d, J = 8.8 Hz,
2H), 6.99-6.93 (m, 3H), 6.84 (d, J = 2.5 Hz, 1H), 6.42 (s, 1H), 4.62
(s, 2H), 2,38 (s, 3H).
4-(Chloromethyl)-7-(4-chlorophenoxy)-2H-chromen-2-one (19a). Yield,
42%. 1H NMR δ: 7.62 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 9.0 Hz, 2H), 7.03 (d,
J = 9.0 Hz, 2H), 6.91-6.89 (m, 2H), 6.46 (s, 1H), 5.28 (s, 2H).
4-{[4-(Chloromethyl)-2-oxo-2H-chromen-7-yl]oxy}benzonitrile
(20a). Yield, 49%. 1H NMR δ: 7.68-7.72 (m, 3H), 7.13 (d, J = 9.0 Hz,
2H), 7.04-7.01 (m, 2H), 6.51 (s, 1H), 4.64 (s, 2H).
7-(4-Acetylphenoxy)-4-(chloromethyl)-2H-chromen-2-one (21a). Yield,
44%. 1H NMR δ: 8.01 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.8 Hz, 1H), 7.12
(d, J = 8.5 Hz, 2H), 7.02-7.03 (m, 2H), 6.49 (s, 1H), 4.64 (s, 2H), 2.61
(s, 3H).
4-(Chloromethyl)-7-[(3-fluorobenzyl)oxy]-2H-chromen-2-one
(3a). Yield, 55%. 1H NMR δ: 7.58 (d, J = 8.8 Hz, 1H), 7.41-7.34 (m,
1H), 7.25-7.13 (m, 2H), 7.07-7.01(m, 1H), 6.97(dd, J1 =2.5Hz, J2 =8.8
Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.41 (s, 1H), 5.13 (s, 2H), 4.62 (s, 2H).
7-[(3-Chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one (4a).
Yield, 52%. 1H NMR δ: 7.64 (d, J = 8.8 Hz, 1H), 7.58-7.55 (m, 1H),
7.43-7.26 (m, 3H), 7.00-6.89 (m, 2H), 6.37 (s, 1H), 5.12 (s, 2H), 4.62
(s, 2H).
4-(Chloromethyl)-7-{[3-(trifluoromethyl)benzyl]oxy}-2H-chromen-
1
2-one (5a). Yield, 54%. H NMR δ: 7.43-7.26 (m, 5H), 7.04-6.90
(m, 2H), 6.42 (s, 1H), 5.18 (s, 2H), 4.63 (s, 2H).
4-(Chloromethyl)-7-{[3-(trifluoromethoxy)benzyl]oxy}-2H-chromen-
2-one (6a). Yield, 51%. 1H NMR δ: 7.59 (d, J = 8.9 Hz, 1H), 7.49-7.18
(m, 4H), 6.97 (dd, J1 = 2.6 Hz, J2 = 8.9 Hz, 1H), 6.91 (d, J = 2.6 Hz, 1H),
6.41 (s, 1H), 5.14 (s, 2H), 4.63 (s, 2H).
4-(Chloromethyl)-7-[(3-nitrobenzyl)oxy]-2H-chromen-2-one (7a). Yield,
59%. 1H NMR δ: 8.33 (s, 1H), 8.21 (d, J = 8.8 Hz, 1H), 7.78 (d, J = 7.7Hz,
1H), 7.63-7.58 (m, 2H), 7.02-6.91 (m, 2H), 6.43 (s, 1H), 5.23 (s, 2H),
4.63 (s, 2H).
4-(Chloromethyl)-7-[(4-fluorobenzyl)oxy]-2H-chromen-2-one
(8a). Yield, 58%. 1H NMR δ: 7.63 (d, J = 8.8 Hz, 1H), 7.42-7.39 (m,
2H), 7.13-7.07 (m, 2H), 6.97- 6.90 (m, 2H), 6.41 (s, 1H), 5.10 (s, 2H),
4.62 (s, 2H).
[(4-Chlorobenzyl)oxy]-4-(chloromethyl)-2H-chromen-2-one (9a). Yield,
52%. 1H NMR δ: 7.57 (d, J = 9.0 Hz, 1H), 7.33-7.29 (m, 4H), 6.91-6.87
(m, 2H), 6.41 (s, 1H), 5.10 (s, 2H), 4.55 (s, 2H).
4-(Chloromethyl)-7-[4-(dimethylamino)phenoxy]-2H-chromen-2-
one (22a). Yield, 48%. 1H NMR δ: 7.54 (d, J = 9.0 Hz, 1H), 6.99-6.90
(m, 3H), 6.79-6.68 (m, 3H), 6.39 (s, 1H), 4.60 (s, 2H), 2.96 (s, 6H).
4-(Chloromethyl)-7-(3,5-difluorophenoxy)-2H-chromen-2-one (23a).
The residue was used for the following reaction without purification.
The presence of the desired compound was certified by ESI-MS m/z 345
[M þ Na]þ.
4-(Chloromethyl)-7-(3,4-difluorophenoxy)-2H-chromen-2-one
(24a). Yield, 43%. 1H NMR δ: 7.64 (d, J = 8.8 Hz, 1H), 7.21-7.23 (m,
1H), 6.93-6.97 (m, 4H), 6.47 (s, 1H), 4.63 (s, 2H).
General Procedure for the Preparation of (1H-Imidazol-1-
ylmethyl)coumarin Derivatives 2-13, 15-24, and 26. To a
solution of imidazole (0.081 g, 1.2 mmol) in THF (2 mL), K2CO3 (0.17
g, 1.2 mmol) and the suitable (chloromethyl)coumarin derivative (0.41
mmol) were added. The reaction mixture was refluxed for 5-7 h. After it
was cooled, the inorganic residue was filtered off, and the solution was
evaporated to dryness. The crude residue was recrystallized from
ethanol.
4-(Chloromethyl)-7-[(4-methoxybenzyl)oxy]-2H-chromen-2-one
(10a). Yield, 35%. 1H NMR δ: 7.59 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.5
Hz, 2H), 7.26-7.24 (m, 4H), 6.40 (s, 1H), 5.06 (s, 2H), 4.55(s, 2H), 3.82
(s, 3H).
4-(1H-Imidazol-1-ylmethyl)-7-[(3-methylbenzyl)oxy]-2H-chromen-2-
one (2). Yield, 69%; mp 118-120 °C. 1H NMR δ: 7.58 (d, J = 8.8 Hz,
1H), 7.31-7.15 (m, 7H), 6.98-6.91 (m, 2H), 5.86 (s, 1H), 5.72 (s, 2H),
5.09 (s, 2H), 2.37 (s, 3H). ESI-MS: m/z 347 [M þ H]þ.
4-(Chloromethyl)-7-[(4-trifluoromethoxybenzyl)oxy]-2H-chromen-
2-one (11a). Yield, 54%. 1H NMR δ: 7.56 (d, J = 8.8 Hz, 1H), 7.36 (d,
J = 8.5 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H), 6.96 (dd, J1 = 2.4 Hz, J2 = 8.8
Hz, 1H), 6.91 (d, J = 2.4 Hz, 1H), 6.41 (s, 1H), 5.13 (s, 2H), 4.63
(s, 2H).
7-[(3-Fluorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (3). Yield, 65%; mp 175-178 °C. 1H NMR δ: 8.50 (s, 1H), 7.50
(d, J = 8.8 Hz, 1H), 7.38-7.27 (m, 3H), 7.18-6.90 (m, 5H), 5.76
(s, 1H), 5.49 (s, 2H), 5.12 (s, 2H). ESI-MS: m/z 351 [M þ H]þ.
7-[(3-Chlorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (4). Yield, 62%; mp 127-129 °C. 1H NMR δ: 8.83 (s, 1H), 7.54
4-(Chloromethyl)-7-[(3,5-difluorobenzyl)oxy]-2H-chromen-2-one
(12a). Yield, 56%. 1H NMR δ: 7.59 (d, J = 8.9 Hz, 1H), 6.99-6.95 (m,
3H), 6.87-6.68 (m, 2H), 6.37 (s, 1H), 5.27 (s, 2H), 5.10 (s, 2H).
4-(Chloromethyl)-7-[(3,4-difluorobenzyl)oxy]-2H-chromen-2-one
1
(13a). Yield, 55%. H NMR δ: 7.50 (d, J = 8.8 Hz, 1H), 7.19-7.15
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ARTICLE
(d, J = 8.8 Hz, 1H), 7.42 (s, 1H), 7.35-7.29 (m, 4H), 7.06 (s, 1H), 6.95
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 6.91 (d, J = 2.5 Hz, 1H), 5.81 (s, 1H),
5.57 (s, 2H), 5.11 (s, 2H). ESI-MS: m/z 367 [M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-{[3-(trifluoromethyl)benzyl]oxy}-2H-
chromen-2-one (5). Yield, 52%; mp 136-138 °C. 1H NMR δ: 8.80 (s
br, 1H), 7.70 (s, 1H), 7.63-7.61 (m, 2H), 7.56-7.51(m, 3H), 6.99-6.92
(m, 3H), 5.79 (s, 1H), 5.51 (s, 2H), 5.18 (s, 2H). ESI-MS: m/z 401
[M þ H]þ.
(d, J= 8.8 Hz, 1H), 7.38 (d, J =8.8Hz, 2H), 7.03 (d, J= 8.8 Hz, 2H), 6.95-
6.85 (m, 4H), 5.80 (s, 1H), 5.42 (s, 2H). ESI-MS: m/z 353 [M þ H]þ.
4-{[4-(1H-Imidazol-1-ylmethyl)-2-oxo-2H-chromen-7-yl]oxy}ben-
zonitrile (20). Yield, 61%; mp 145-147 °C. 1H NMR δ: 8.71 (s, 1H),
7.69 -7.66 (m, 3H), 7.15-7.13 (m, 3H), 6.99-6.98 (m, 3H), 5.85
(s, 1H), 5.58 (s, 2H). ESI-MS: m/z 344 [M þ H]þ.
7-(4-Acetylphenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-one
(21). Yield, 62%; mp 157-160 °C. 1H NMR δ: 8.04 -8.00 (m, 4H),
7.87 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 9.0 Hz, 2H), 7.12 (dd, J1 = 2.6 Hz, J2 =
8.8 Hz, 1H), 7.02-6.99 (m, 1H), 6.91 (d, J = 2.6 Hz, 1H), 5.84 (s, 1H),
5.41 (s, 2H), 2.61 (s, 3H). ESI-MS: m/z 361 [M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-{[3-(trifluoromethoxy)benzyl]oxy}-
2H-chromen-2-one (6). Yield, 57%; mp 102-104 °C. 1H NMR δ: 8.01
(s, 1H), 7.60 (s, 1H), 7.47-7.44 (m, 2H), 7.42-7.37 (m, 1H), 7.29 (s,
1H), 7.22-7.17 (m, 2H), 6.98-6.92 (m, 2H), 5.72 (s, 1H), 5.29 (s, 2H),
5.15 (s, 2H). ESI-MS: m/z 417 [M þ H]þ.
7-[4-(Dimethylamino)phenoxy]-4-(1H-imidazol-1-ylmethyl)-2H-
chromen-2-one (22). Yield, 67%; oily product. 1H NMR δ: 7.60
(s, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.17 (s, 1H), 6.98-6.95 (m, 3H),
6.76-6.73 (m, 4H), 5.70 (s, 1H), 5.27 (s, 2H), 2.96 (s, 6H). ESI-MS: m/z
362 [M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-[(3-nitrobenzyl)oxy]-2H-chromen-2-
one (7). Yield, 62%; mp 218-221 °C. 1H NMR δ: 8.29 (s, 1H), 8.14-
8.20 (m, 2H), 7.75 (d, J = 8.8 Hz, 1H), 7.49-7.61 (m, 2H), 7.20 (s, 1H),
6.91-7.04 (m, 3H), 5.73 (s, 1H), 5.42 (s, 2H), 5.21 (s, 2H). ESI-MS: m/
z 378 [M þ H]þ.
7-(3,5-Difluorophenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-
one (23). Yield, 29%; mp 119-120 °C.1H NMR δ: 7.68 (s, 1H), 7.51
(d, J = 9.6Hz, 1H), 7.21 (s, 1H), 7.10-6.90 (m, 3H), 6.80-6.50 (m, 3H),
5.80 (s, 1H), 5.32 (s, 2H). ESI-MS: m/z 353 [M - H]-.
7-[(4-Fluorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (8). Yield, 66%; mp 138-140 °C. 1H NMR δ: 9.66 (s, 1H), 7.64
(d, J = 8.8 Hz, 1H), 7.38-7.41 (m, 3H), 7.17-7.25 (m, 3H), 7.09
(m, 2H), 5.85 (s, 1H), 5.81 (s, 2H), 5.09 (s, 2H). ESI-MS: m/z 351
[M þ H]þ.
7-(3,4-Difluorophenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-
1
one (24). Yield, 66%; mp 80-83 °C. H NMR δ: 7.70 (s, 1H), 7.59
(s, 1H), 7.48 (d, J = 8.8 Hz, 1H), 7.26-7.17 (m, 3H), 6.88-6.97 (m, 3H),
7-[(4-Chlorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (9). Yield, 20%; mp >220 °C. 1H NMR δ: 7.58 (s, 1H), 7.42-
7.36 (m, 5H), 7.18 (s, 1H), 6.96-6.92 (m, 3H), 5.73 (s, 1H), 5.27 (s,
2H), 5.10 (s, 2H). ESI-MS: m/z 365 [M - H]-.
5.76 (s, 1H), 5.30 (s, 2H). ESI-MS: m/z 355 [M þ H]þ.
7-Hydroxy-4-(1H-Imidazol-1-ylmethyl)-2H-chromen-2-one (26). Yield,
74%; mp > 250 °C. 1H NMR (DMSO-d6) δ: 10.55 (s, 1H), 7.83 (s, 1H)
7.87-7.76 (m, 1H), 7.26 (s, 1H), 7.01 (s, 1H), 6.83-6.73 (m, 2H), 5.51
(s, 2H), 5.38 (s, 1H). ESI-MS: m/z 241 [M - H]-.
4-(1H-Imidazol-1-ylmethyl)-7-[(4-methoxybenzyl)oxy]-2H-chromen-2-
one (10). Yield, 20%; mp 209-211 °C. 1H NMR δ: 7.58 (s, 1H), 7.28-
7.34(m, 4H), 7.18(s, 1H), 6.96-6.92(m, 4H), 5.72(s, 1H), 5.26(s, 2H),
5.06 (s, 2H), 3.82 (s, 3H). ESI-MS: m/z 361 [M - H]-.
4-Chloromethyl-2-oxo-2H-chromen-7-yl)-carbamic Acid Ethyl Ester
(25b). Ethyl chloroacetoacetate (1.3 g, 7.8 mmol) and 25a42 (1.0 g, 5.5
mmol) were suspended in 13 mL of 70% H2SO4 and were stirred at room
temperature for 4 h. The mixture was poured in 50 mL of ice water, giving
a white precipitate that was crystallized from absolute EtOH. Yield, 83%.
1H NMR δ: 8.62 (s, 1H), 7.54-7.40 (m, 3H), 6.37 (s, 1H), 4.59 (s, 2H),
4.17 (q, J = 7.4 Hz, 2H), 1.25 (t, J = 7.4 Hz, 3H).
7-Amino-4-chloromethyl-2H-chromen-2-one (25c). Derivative 25b
(0.43 g, 1.5 mmol) was dissolved in a solution of 20 mL of concentrated
H2SO4 and 20 mL of glacial acetic acid, and the mixture was heated at
reflux for 4 h. After it was cooled, the mixture was poured in 400 mL of ice
water. At 0 °C, 50% NaOH was added until the solution resulted slightly
alkaline. The obtained precipitate was filtered and washed with ice water
(3 ꢀ 50 mL). Yield, 62%. 1H NMR δ: 7.45 (d, J = 8.8 Hz, 1H), 6.58 (dd, J1
= 2.2 Hz, J2 = 8.8 Hz, 1H), 6.41 (d, J = 2.2 Hz, 1H), 6.22 (s, 2H), 6.16 (s,
1H), 4.85 (s, 2H).
4-Chloromethyl-7-phenylamino-2H-chromen-2-one (25d). Com-
pound 25c (0.11 g, 0.50 mmol), benzeneboronic acid (0.12 g, 1.0
mmol), Cu(OAc)2 (0.090 g, 0.50 mmol), and triethylamine (0.14 mL,
1.0 mmol) were dissolved in 10 mL of CH2Cl2, and the mixture was
stirred at room temperature for 72 h. The solvent was evaporated to
dryness, and the product was purified by flash chromatography by using
CH2Cl2 as the eluent. Yield, 35%. 1H NMR δ: 8.98 (s, 1H), 7.64 (d, J =
8.8 Hz, 1H), 7.36-7.31 (m, 2H), 7.22-7.19 (m, 2H), 7.02-6.99 (m,
2H), 6.88 (s, 1H), 6.32 (s, 1H), 4.92 (s, 2H).
7-Anilino-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-one (25). To
a solution of imidazole (0.082 g, 1.2 mmol), in THF (2.0 mL), K2CO3
(0.33 g, 2.4 mmol), and 25d (0.12 g, 0.41 mmol) were added. The mixture
was heated at reflux for 7 h. After the mixture was cooled, the inorganic
residue was filtered off, and the organic solution was dried under vacuum.
The crude product was purified by flash chromatography using a mixture
of CHCl3/MeOH, 9:1 (v/v) as the eluent. Yield, 60%; oily residue.
1H NMR δ: 8.99 (s, 1H), 7.84 (s, 1H), 7.66 (d, J = 8.8 Hz, 1H), 7.37-
7.32 (m, 3H), 7.23-7.10 (m, 2H), 7.02-6.96 (m, 3H), 6.88 (d,
J = 2.2 Hz, 1H), 5.48 (s, 1H), 5.31 (s, 2H). ESI-MS: m/z 318
[M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-[(4-trifluoromethoxybenzyl)oxy]-2H-chr-
1
omen-2-one (11). Yield, 51%; mp 102-104 °C. H NMR δ: 7.67
(s, 1H), 7.48-7.41 (m, 2H), 7.26-7.20 (m, 4H), 7.17 (s, 1H), 6.96-6.92 (m, 2H),
5.75 (s, 1H), 5.29 (s, 2H), 5.13 (s, 2H). ESI-MS: m/z 415 [M - H]-.
7-[(3,5-Difluorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (12). Yield, 22%; mp 170-172 °C. 1H NMR δ: 7.57
(s, 1H), 7.43 (d, J = 9.0 Hz, 1H), 7.20 (s, 1H), 6.95-6.80 (m, 5H),
6.81-6.79 (m, 1H), 5.78 (s, 1H), 5.20 (s, 2H), 5.08 (s, 2H). ESI-MS:
m/z 367 [M - H]-.
7-[(3,4-Difluorobenzyl)oxy]-4-(1H-imidazol-1-ylmethyl)-2H-chromen-
2-one (13). Yield, 63%; mp 91-93 °C. 1H NMR δ: 9.61 (s, 1H), 7.60 (d,
J = 8.8 Hz, 1H), 7.25-7.15 (m, 4H), 7.17-6.89 (m, 3H), 5.74 (s, 1H),
5.18 (s, 2H), 5.08 (s, 2H). ESI-MS: m/z 369 [M þ H]þ.
7-(3-Fluorophenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-
one (15). Yield, 25%; mp 108-110 °C. 1H NMR δ: 8.08 (s, 1H), 7.45
(d, J = 9.0 Hz, 1H), 7.40-7.30 (m, 2H), 7.00-6.80 (m, 4H), 5.80
(s, 1H), 5.40 (s, 2H), two imidazole protons were not detected. ESI-MS:
m/z 335 [M - H]-.
7-(3-Chlorophenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-
one (16). Yield, 62%; mp 137 °C (dec). 1H NMR δ: 7.99 (s, 1H), 7.51
(d, J = 8.8 Hz, 1H), 7.32-7.35 (m, 4H), 7.09-6.92 (m, 4H), 5.81 (s, 1H),
5.39 (s, 2H). ESI-MS: m/z 353 [M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-(3-methoxyphenoxy)-2H-chromen-
2-one (17). Yield, 67%; amorphous solid. 1H NMR δ: 8.85 (s, 1H), 7.58
(d, J = 8.8 Hz, 1H), 7.35-6.65 (m, 8H), 5.84 (s, 1H), 5.70 (s, 2H), 3.81 (s,
3H). ESI-MS: m/z 349 [M þ H]þ.
4-(1H-Imidazol-1-ylmethyl)-7-(4-methylphenoxy)-2H-chromen-2-
one (18). Yield, 32%; mp 106-108 °C. 1H NMR δ: 9.96 (s, 1H), 7.65
(d, J = 8.5 Hz, 1H), 7.41 (s, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.15 (s, 1H),
6.98-6.94 (m, 3H), 6.84 (d, J = 2.5 Hz, 1H), 5.94 (s, 1H), 5.83 (s, 2H),
2.38 (s, 3H). ESI-MS: m/z 333 [M þ H]þ.
7-(4-Chlorophenoxy)-4-(1H-imidazol-1-ylmethyl)-2H-chromen-2-
1
one (19). Yield, 65%; mp >250 °C. H NMR δ: 8.03 (s, 1H), 7.51
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4-Benzyl-7-methoxy-2H-chromen-2-one (28b). Derivative 28a46
(11 mmol, 2.8 g) was dissolved in DMF (45 mL), and the solution
was cooled to 0 °C. NaH (0.60 g, 25 mmol) was slowly added to the
solution followed by 20 min of stirring. CH3I (1.6 mL, 25 mmol) was
then added to the reaction mixture that slowly reached room tempera-
ture. After 1 h, the mixture was poured onto ice and extracted with
EtOAc (3 ꢀ 10 mL). The organic layer was extracted with a 2 N solution
of NaOH (3 ꢀ 5 mL) and water (3 ꢀ 5 mL), dried over Na2SO4, and
then concentrated under vacuum to give the desired derivative. Yield,
43%. 1H NMR δ: 7.47 (d, J = 8.8 Hz, 1H), 7.35-7.16 (m, 5H), 6.84-
6.75 (m, 2H), 6.00 (s, 1H), 4.05 (s, 2H), 3.85 (s, 3H). ESI-MS: m/z 251
[M - H]-.
4-[Bromo(phenyl)methyl]-7-methoxy-2H-chromen-2-one (28c). To
the solution of 28b (0.18 g, 0.69 mmol) in CCl4 (2.5 mL) were added N-
bromosuccinimide (0.15 g, 0.83 mmol) and a catalytic amount of benzoyl
peroxide. The reaction mixture was refluxed until the disappearance of the
starting material (about 2 h). The succinimide was rapidly filtered off, and
the desired solid product was recovered after cooling and used in the next
step without further purification. Yield, 38%. 1H NMR δ: 7.53 (d, J = 8.8
Hz, 1H), 7.46-7.34 (m, 5H), 6.84-6.80 (m, 2H), 6.48 (s, 1H), 6.33
(s, 1H), 3.85 (s, 3H).
4-[1H-Imidazol-1-yl(phenyl)methyl]-7-methoxy-2H-chromen-2-one
(28). To a solution of imidazole (0.082 g, 1.2 mmol) in THF (2 mL)
were added K2CO3 (0.17 g, 1.2 mmol) and 28c (0.15 g, 0.41 mmol). The
reaction mixture was refluxed for 5-7 h. After the mixture was cooled, the
inorganic residue was filtered off, and the solution was evaporated to
dryness. Yield, 45%; amorphous solid. 1H NMR δ: 8.23 (s br, 1H), 7.58
(d, J = 8.8 Hz, 1H), 7.46-7.18 (m, 7H), 6.89-6.85 (m, 2H), 5.64 (s, 1H),
3.85 (s, 3H), one imidazole proton was not detected. ESI-MS: m/z 333
[M þ H]þ. The synthesis of derivatives 31a and 32a was done as
described for compound 29a.45
filtrate was evaporated to dryness. The residue was purified by flash
chromatography by using CHCl3 as the eluent.
4-[Bromo(phenyl)methyl]-7-phenoxy-2H-chromen-2-one (29c). Yield,
33%. 1H NMR δ: 8.08 (d, J = 7.2Hz, 1H), 7.83-7.25 (m, 8H), 7.09-7.05
(m, 2H), 6.86-6.83 (m, 2H), 6.43 (s, 1H), 6.33 (s, 1H).
4-[Bromo(4-chlorophenyl)methyl]-7-phenoxy-2H-chromen-2-one
(31c). Yield, 47%. 1H NMR δ: 7.50 (d, J = 8.7 Hz, 1H), 7.46-7.34 (m,
7H), 7.07 (d, J = 7.5 Hz, 2H), 6.87 (m, 2H), 6.50 (s, 1H), 6.29 (s, 1H).
4-[Bromo(2-oxo-7-phenoxy-2H-chromen-4-yl)methyl]benzonitrile
(32c). Yield, 81%. 1H NMR δ: 8.04 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 8.7
Hz, 2H), 7.80-7.59 (m, 2H), 7.42-7.35 (m, 2H), 7.15-7.01 (m, 2H),
6.90-6.75 (m, 3H), 6.26 (s, 1H).
General Procedure for the Preparation 4-[1H-Imidazol-1-
yl(aryl)methyl]-7-phenoxy-2H-chromen-2-ones 29, 31, and
32. The appropriate bromide (0.085 mmol) was dissolved in 5 mL of
dry acetonitrile. Imidazole (0.058 g, 0.85 mmol) was added, and the
mixture was refluxed for 5 h. The solvent was evaporated to dryness, and
the residue was purified by chromatography using as the eluent a mixture
of CHCl3/MeOH, 9/1 (v/v).
4-[1H-Imidazol-1-yl(phenyl)methyl]-7-phenoxy-2H-chromen-2-one
(29). Yield, 95%; mp 92-94 °C. 1H NMR δ: 9.62 (s, 1H), 7.98 (s, 1H),
7.58-7.30 (m, 10H), 7.05-7.01 (m, 3H), 6.90-6.79 (m, 2H), 5.69
(s, 1H). ESI-MS: m/z 395 [M þ H]þ.
4-[(4-Chlorophenyl)(1H-imidazol-1-yl)methyl]-7-phenoxy-2H-chromen-
2-one (31). Yield, 41%; mp 86-88 °C. 1H NMR δ: 9.88 (s, 1H), 8.24 (s,
1H), 7.44-7.38 (m, 10H), 7.05 (d, J = 9.0 Hz, 1H), 6.84 (m, 2H), 5.68 (s,
1H), one imidazole proton was not detected. ESI-MS: m/z 451 [M þ
Na]þ.
4-[1H-Imidazol-1-yl(2-oxo-7-phenoxy-2H-chromen-4-yl)methyl-
1
]benzonitrile (32). Yield, 69%; amorphous solid. H NMR δ: 9.82 (s,
1H), 8.34 (s, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2H), 7.39
(m, 4H), 7.25 (s, 1H), 7.04 (d, J = 7.2Hz, 2H), 6.96 (s, 1H), 6.85 (m, 2H),
5.72 (s, 1H). ESI-MS: m/z 420 [M þ H]þ.
4-(4-Chlorophenyl)-3-oxobutanoic Acid Ethyl Ester (31a). Yield,
50%. 1H NMR δ: 7.29 (d, J = 9.0 Hz, 2H), 7.24 (d, J = 9.0 Hz, 2H), 4.16
(q, J = 7.2 Hz, 2H), 3.80 (s, 2H), 3.44 (s, 2H), 1.25 (t, J = 7.2 Hz, 3H).
4-(4-Cyanophenyl)-3-oxobutanoic Acid Ethyl Ester (32a). Yield,
Synthesis of 4-Benzyl-7-(3,4-difluorophenoxy)-2H-chromen-2-one
(30a). Intermediate 28a (0.050 g, 0.20 mmol) was suspended in 2 mL
of dry CH2Cl2. 3,4-F2-benzeneboronic acid (0.11 g, 0.70 mmol),
Cu(OAc)2 (0.036 g, 0.20 mmol), and triethylamine (0.14 mL, 1.0
mmol) were added. The reaction was stirred at room temperature for 4
h. The mixture was filtered over Celite. The filtrate was evaporated to
dryness giving a residue that was purified by flash chromatography by using
CHCl3 as the eluent. Yield, 20%. 1H NMR δ: 7.59 (d, J = 9.1 Hz, 1H),
7.37-7.17 (m, 6H), 6.98-6.78 (m, 4H), 6.07 (s, 1H), 4.08 (s, 2H).
4-[Bromo(phenyl)methyl]-7-(3,4-difluorophenoxy)-2H-chromen-
2-one (30b). Coumarin 30a (0.021 g, 0.051 mmol) and NBS (0.011 g,
0.060 mmol) were dissolved in 2 mL of CCl4. The mixture was heated at
reflux, and a catalytic amount of DBP was added. After 4 h, the hot mixture
was filtered, and the filtrate was evaporated to dryness. The residue
obtained was used without purification for the next reaction.
1
52%. H NMR δ: 7.61 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H),
4.17 (q, J = 7.2 Hz, 2H), 3.92 (s, 2H), 3.48 (s, 2H), 1.25 (t, J = 7.2 Hz,
3H).
General Procedure for the Synthesis of 7-Phenoxy-4-
arylmethyl-2H-cromen-2-ones 29b, 31b, and 32b. The ap-
propriate ethyl phenylacetoacetate (6.4 mmol) was charged in a flask,
and 3-phenoxyphenol (1.0 g, 5.4 mmol) was added. The mixture was
stirred at 120 °C, and 2 drops of concentrated H2SO4 were added. After
30 min, the mixture was allowed to reach room temperature, and later, it
was poured in ice water (100 mL). The product was extracted with
CHCl3, and the organic layer was dried over Na2SO4 and evaporated to
dryness to give an oil that was purified by flash chromatography using a
mixture of CHCl3/n-hexanes, 7:3 (v/v), as the eluent.
4-Benzyl-7-phenoxy-2H-chromen-2-one (29b). Yield, 54%. 1H
NMR δ: 7.98 (d, J = 8.5 Hz, 1H), 7.71-7.21 (m, 8H), 7.11-7.06
(m, 2H), 6.95-6.78 (m, 2H), 6.03 (s, 1H), 4.07 (s, 2H).
7-(3,4-Difluorophenoxy)-4-[1H-imidazol-1-yl(phenyl)methyl]-2H-
chromen-2-one (30). Intermediate 30b (0.022 g, 0.050 mmol) was
dissolved in 2 mL of dry acetonitrile, and imidazole (0.034 g, 0.50 mmol)
was added. The mixture was heated at reflux for 6 h. The solvent was
evaporated to dryness, and the residue was purified by flash chromatog-
raphy using as the eluent a mixture of CHCl3/MeOH, 9:1 (v/v). Yield,
21%; mp86-88°C. 1H NMR δ: 7.96 (s, 1H), 7.72-7.69(m, 1H), 7.54-
7.51 (m, 1H), 7.47-7.44 (m, 2H), 7.31-7.28 (m, 2H), 7.24-7.15 (m,
3H), 7.00 (s, 1H), 6.94-6.87 (m, 2H), 6.84-6.75 (m, 2H), 5.78 (s, 1H).
ESI-MS: m/z 429 [M - H]-.
3-Benzyl-7-hydroxy-2H-chromen-2-one (34b). 3-Benzyl-7-methoxy-
2H-chromen-2-one 34a46 (0.27 g, 1.0 mmol) was dissolved in 2 mL of dry
CH2Cl2, and 1 M BBr3 in dry CH2Cl2 (2.5 mL) was added dropwise at 0 °C.
The mixture was allowed to reach room temperature, and after it was stirred
for 18 h, it was poured in ice water, stirring vigorously. The obtained
precipitate was filtered and used without further purification. Yield, 99%.
4-(4-Chlorobenzyl)-7-phenoxy-2H-chromen-2-one (31b). Yield,
1
20%. H NMR δ: 7.49 (d, J = 9.0 Hz, 1H), 7.42-7.38 (m, 3H), 7.31
(d, J = 8.9 Hz, 2H), 7.33-7.30 (m, 2H), 7.15 (d, J = 8.9 Hz, 2H),
7.06-7.04 (m, 1H), 6.85-6.82 (m, 1H), 6.01 (s, 1H), 4.04 (s, 2H).
4-(2-Oxo-7-phenoxy-2H-chromen-4-ylmethyl)-benzonitrile (32b). Yield,
35%. 1H NMR δ: 7.64 (d, J = 8.1 Hz, 2H), 7.50-7.30 (m, 5H), 7.24 (s,
1H), 7.06 (d, 2H, J = 8.1 Hz), 6.82 (m, 2H), 6.01 (s, 1H), 4.13 (s, 2H).
General Procedure for the Preparation of 4-[Bromo-
(aryl)methyl]-7-phenoxy-2H-chromen-2-ones 29c, 31c, and
32c. The chosen coumarin (0.61 mmol) and N-bromosuccinimide
(0.13 g, 0.73 mmol) were dissolved in 2 mL of CCl4. The mixture was
heated at reflux, and a catalytic amount of dibenzoyl peroxide was added.
The mixture was refluxed for 1 h. Succinimide was filtered off, and the
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Journal of Medicinal Chemistry
ARTICLE
1H NMR δ: 10.42 (s, 1H), 7.68 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.35-7.15
(m, 5H), 6.80-6.65 (m, 2H), 3.72 (s, 2H).
structure was mildly minimized (rmsd convergence criterion set to 0.30 Å) to
eliminate, where present, steric clashes. At first, lead compound 14 was
allowed to look for favorable poses within a box centered on the androste-
nedione (the cognate ligand in the X-ray structure) and spanning 20 Å in each
of the three directions so to contain entirely the protein active site. During the
run, the van der Walls radii of nonpolar atoms were scaled by a factor of 0.3
both for the protein and for the ligand to induce a permissive allocation of the
ligand. Moreover, a metal bond constraint was used to funnel the search
protocol toward iron-coordinating poses and hydroxyl groups of amino acid
side chains enclosed in the box were allowed to rotate. The main purpose of
this step was to provide a reasonable starting complex with which to work.
The final model, comprising compound 14 top-ranked pose and the enzyme,
was subsequently energy minimized, using MacroModel,69 upon one distance
(Fe-coordinating N equilibrium distance set to 2.4 Å) and one angle (Fe-
coordinating N and noncoordinating N of the imidazole ring equilibrium
angle set to 180°) constraints to explicitly account for the iron coordination.
The so obtained relaxed complex was then used to dock selected compounds
at the active site. The rmsd, calculated over all of the heavy atoms within 5 Å of
lead compound 14, between the so obtained model and the starting structure
was as small as 1.2 Å. The docking simulations were performed through the
Induced Fit Docking70 (IFD) protocol as implemented in the Schr€odinger
suite of programs to account for likely adapted fit effects. During the initial
Glide docking of the IFD run, the van der Walls radii of nonpolar atoms were
scaled by a factor of 0.8, while I133, W224, and D309 were mutated into
alanine. At the end of this step, 20 poses were retained, and residues 133, 224,
and 309 were mutated back into the original amino acids and optimized along
with the residues within 5 Å of the docked pose, to reach the closest energy
minimum. This is the central engine of IFD workflows that allows first a
comfortable allocation of the ligand within a wider active site and then let the
original side chains adapt to the generated pose. At this stage, the ligands were
docked back into the rigid induced protein structure and ranked accordingly
to the IFDScore, which is a sum of the SP score from the redocking and 5% of
the Prime energy from the refinement calculation.
3-Benzyl-7-phenoxy-2H-chromen-2-one (34c). Intermediate 34b
(0.20 g, 0.80 mmol) was suspended in 2 mL of dry CH2Cl2. Benzene-
boronic acid (0.98 g, 0.80 mmol), Cu(OAc)2 (0.15 g, 0.80 mmol), and
triethylamine (0.56 mL, 4.0 mmol) were added. The reaction was stirred
at room temperature for 1 h. The mixture was filtered over Celite. The
filtrate was evaporated to dryness giving a residue that was purified by
flash chromatography using a mixture of petroleum ether/CHCl3, 3/7
1
(v/v), as the eluent. Yield, 29%. H NMR δ: 7.42-7.18 (m, 10H),
7.12-7.03 (m, 2H), 6.90-6.81 (m, 2H), 3.85 (s, 2H).
3-[Bromo(phenyl)methyl]-7-phenoxy-2H-chromen-2-one (34d).
Coumarin 34c (0.072 g, 0.22 mmol) and N-bromosuccinimide (0.047 g,
0.26 mmol) were dissolved in 4 mL of CCl4. The mixture was heated at
reflux, and a catalytic amount of DBP was added. After 4 h, the hot mixture
was filtered, and the filtrate was evaporated to dryness. The residue was
purified by flash chromatography by using CHCl3 as the eluent. Yield, 30%.
1H NMR δ: 7.82 (s, 1H), 7.45-7.18 (m, 9H), 7.10-7.01 (m, 2H), 6.92
(dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 6.86 (d, J = 2.5 Hz, 1H), 5.50 (s, 1H).
3-[1H-Imidazol-1-yl(phenyl)methyl]-7-phenoxy-2H-chromen-2-
one (34). Intermediate 34d (0.074 g, 0.184 mmol) was dissolved in 4 mL
of dry acetonitrile, imidazole (0.037 g, 0.55 mmol) was added, and the
mixture was refluxed for 6 h. The solvent was evaporated to dryness, and
the residue purified by flash chromatography using a mixture of CHCl3/
MeOH, 9:1 (v/v), as the eluent. Yield, 41%; mp 88-90 °C. 1H NMR δ:
8.07 (s, 1H), 7.48-7.38 (m, 7H), 7.34 (s, 1H), 7.28 (s, 1H), 7.26-7.24
(m, 2H), 6.91 (dd, J1 = 2.5 Hz, J2 = 8.8 Hz, 1H), 6.85 (d, J = 2.5 Hz, 1H),
6.77 (s, 1H). ESI-MS: m/z 417 [M þ Na]þ.
Biological Assays. Cellular Assays for Testing CYP19 and CYP17
Inhibition. As a source of the enzymes, used were the following microsomal
preparations: for CYP19, human placenta,14,47 for CYP17, E. coli-expressing
human CYP17.48,49 The CYP19 assay was performed as described using the
3H2O method: [1β-3H]androstenedione/androstenedione (0.5 μM)47 was
used as substrate. The CYP17 assay was performed with nonlabeled
progesterone, and an HPLC procedure was employed for the separation of
the substrate and androstenedione using UV detection.48,67
Cellular Assays for Testing CYP11B1 and CYP11B2 Inhibition.
V79MZh11B1 and V79MZh11B2 cell lines were cultivated in Dulbec-
co's modified Eagle's medium supplemented with 5% fetal calf serum,
penicillin (100 U/mL), streptomycin (100 μg/mL), glutamine (2 mM),
and sodium pyruvate (1 mM) at 37 °C in 5% CO2 in air.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þþ39-0805442782. Fax: þþ39-0805442230. E-mail:
carotti@farmchim.uniba.it.
’ ACKNOWLEDGMENT
V79MZ cells expressing human CYP11B1 and human CYP11B2 genes,
respectively, were grown on 24-well cell culture plates (8 ꢀ 105 cells per well)
with 1.9 cm2 culture area per well in 1 mL of DMEM culture medium until
confluence. Before testing, the DMEM culture medium was removed, and
450 μL of fresh DMEM, containing the inhibitor, was added to each well.
Every value was determined at least three times. After a preincubation step of
60 min at 37 °C, the reaction was started by the addition of 50 μL of DMEM
containing the substrate 11-deoxycorticosterone (containing 0.15 μCi of [1,2-
3H]-11-deoxycorticosterone, dissolved in ethanol; final concentration, 100
nM). The V79MZh11B1 cells were incubated for 25 min, the V79MZh11B2
cells were incubated for 50 min. Controls were treated in the same way
without inhibitors. The maximum DMSO concentration in each well was 1%.
Enzyme reactions were stopped by extracting the supernatant with 500 μL of
ethyl acetate. Samples were centrifuged (10000g, 2 min), and the solvent was
pipetted into fresh cups. The solvent was evaporated, and the steroids were
redissolved in 40 μL of methanol and analyzed by HPLC using radioflow
detection.
We thank the Universitꢀa degli Studi di Bari “Aldo Moro” and
the Fonds der Chemischen Industrie, respectively, for financial
support. We also thank Prof. Saverio Cellamare (Dipartimento
Farmacochimico, Universitꢀa degli Studi di Bari “Aldo Moro”,
Italy) for the development of chiral HPLC analyses.
’ ABBREVIATIONS USED
ER, estrogen receptor; CYP, cytochrome P450; AR, aromatase; ARI,
aromatase inhibitor; SAFIR, structure-affinity relationship; SERM,
selective estrogen receptor modulators; HER2, human epidermal
growth factor receptor 2; hmAb, human monoclonal antibody
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