Synthesis and biological evaluation of imidazolylmethylacridones as cytochrome P-450 enzymes inhibitors

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Abstract
Two positional isomers with the general formula 1H-imidazol-1-ylmethylacridin-9(10H)-one were synthesized (5, 6) and evaluated for their inhibitory activity versus CYP11B1, CYP11B2, CYP17 and CYP19. Compound 5 was more effective than letrozole in inhibiting CYP19 (aromatase). Interestingly, compound 5 also inhibited CYP11B1 with an IC 50 of 21.2 nM. On the other hand compound 6 was almost completely inactive against all CYPs; this indicates that the positioning and spatial orientation of the imidazolylmethyl moiety is of paramount importance to activity. Sequence alignment of the four steroidogenic CYP enzymes and docking studies with 5, 6 and letrozole supported this finding and suggested Ser478 to be an essential residue for both inhibition and selectivity. These novel compounds may have benefits for the treatment of Cushing's syndrome, hypertension, congestive heart failure and myocardial fibrosis and breast cancer.
Synthesis and biological evaluation of imidazolylmethylacridones as
cytochrome P-450 enzymes inhibitors
Ashraf H. Abadi,
*
ab
Sahar M. Abou-Seri,
b
Qingzhong Hu,
c
Matthias Negri
d
and Rolf W. Hartmann
cd
Received 16th March 2012, Accepted 10th April 2012
DOI: 10.1039/c2md20072d
Two positional isomers with the general formula 1H-imidazol-1-ylmethylacridin-9(10H)-one were
synthesized (5, 6) and evaluated for their inhibitory activity versus CYP11B1, CYP11B2, CYP17 and
CYP19. Compound 5 was more effective than letrozole in inhibiting CYP19 (aromatase). Interestingly,
compound 5 also inhibited CYP11B1 with an IC
50
of 21.2 nM. On the other hand compound 6 was
almost completely inactive against all CYPs; this indicates that the positioning and spatial orientation
of the imidazolylmethyl moiety is of paramount importance to activity. Sequence alignment of the four
steroidogenic CYP enzymes and docking studies with 5, 6 and letrozole supported this finding and
suggested Ser478 to be an essential residue for both inhibition and selectivity. These novel compounds
may have benefits for the treatment of Cushing’s syndrome, hypertension, congestive heart failure and
myocardial fibrosis and breast cancer.
Introduction
Cytochromes (CYPs) are metalloenzymes involved in the
metabolism of xenobiotics and biosynthesis of hormones.
Among the prominent CYPs are CYP11B1, which is involved in
the biosynthesis of cortisol; CYP11B2, involved in the biosyn-
thesis of aldosterone; CYP17, which catalyzes 2 steps in the
biosynthesis of testosterone and CYP19 (aromatase), responsible
for ring A aromatization and formation of estradiol from the
androgen testosterone. Potential applications of these CYP
inhibitors are for the treatment of Cushing’s syndrome, hyper-
tension, congestive heart failure and myocardial fibrosis, pros-
tate cancer and breast cancer.
1–4
Among the CYP11B1 and CYP19 inhibitors available on the
market are metyrapone (1) and letrozole (2), respectively. It is
believed that the lone pair of electrons of the pyridine and tri-
azole moieties of the above two drugs are responsible for
chelation of the iron atom of the respective CYPs, while the
remaining part of the molecule is responsible for modulating the
selectivity through appropriate interaction with the residues of
the apo-protein.
5,6
In two previous reports from our group we described the
CYP inhibitory profile of imidazolylmethylxanthone and imi-
dazolylmethylthiaxanthone derivat ives, and the best derivatives
(3, 4) were found to be potent aromatase inhibitors with an IC
50
of 40 nM and 16.5 nM, respectively. To explore the scope and
limitations of this class of co mpounds as CYP inhibitors and to
minimize their potential toxicity, herein we are reporting the
activity of the imidazolylm ethylacridone derivatives as tricyclic
planar systems with an NH group, the latter moiety can behave
as a hydrogen bond donor as com pared to the acceptor func-
tionali ty of the oxygen and sulfur atoms in 3 and 4, respectively.
The nitro group present in 3 (Fig. 1) was removed to avoid its
potential toxicity. The positioning of the imidazolylmethyl
moiety relative to the carbonyl and NH groups was explored by
designing two isomers in which the imidazolylmethyl is at
position 4 (5) or position 2 (6) of the acridone ring. The s elec-
tivity profile to inhibit different CYPs was determined for both
compounds 5 and 6.
7,8
For the semi-empirical (AM1) geometry-
optimized three-dimensional structures of 2, 5 and 6, the
distances between the sp
2
hybridized N of the imidazole/triazole
and the carbonyl O (for 2 the nitrile N was considered) (d
O]C
),
and between the sp
2
N and the NH (d
NH
), as well as the angle
between the sp
2
N, methylene carbon betw een the two rings, and
carbonyl O/nitrile N (a) were measured. Finally, 2, 5 and 6 were
docked into the CYP19 cry stal structure, and their activity and
selectivity profiles discus sed based on binding mode, sequence
comparison (different CYPs), and geometric differences
(Fig. 2). The role of Ser478, an essential residue important to be
targeted for dual inhibition (in this case CYP19–CYP11B1), is
explore d.
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and
Biotechnology, German University in Cairo, Cairo 11835, Egypt.
E-mail: ashraf.abadi@guc.edu.eg; Fax: +20 2-27581041 ; Tel: +20 2-
27590716
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo
University, Cairo 11562, Egypt
c
Pharmaceutical and Medicinal Chemistry, Saarland University, Campus
C2.3, 66123 Saarbr
ucken, Germany
d
Department of Drug Design and Optimization, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Campus C2.3, 66123
Saarbr
ucken, Germany
Electronic supplementary information (ESI) available: See DOI:
10.1039/c2md20072d
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Results and discussion
Chemistry
The synthesis of 5 and 6 is depicted in Scheme 1. In brief, Ullman
copper-catalyzed condensation of 2-chlorobenzoic acid and the
appropriate toluidine yielded the diphenylamine derivative.
9
This
was followed by cyclization to the methylacridone derivative
with H
2
SO
4
. Our attempts to synthesize the bromomethyl
derivative from the methylacridone using N-bromosuccinimide
were unsuccessful. We modified our scheme by reaction of the
methylacridone with POCl
3
to give the chloroacridine derivative,
which is then reacted with NBS/CCl
4
to yield the bromomethyl
derivative, followed by heating with 10% HCl to yield the bro-
momethylacridone. The last step involved condensation of the
latter with imidazole to obtain the final product.
Biology
Compounds 5, 6, letrozole 2 and metyrapone 1 were evaluated
for their inhibitory effect on CYP11B1, CYP11B2, CYP17 and
CYP19. Screening was carried out in two stages, single dose
screening at a relatively high concentration, followed by exact
IC
50
determination for the active compounds. The results are
shown in Table 1.
Compound 5 showed dual activity by inhibiting both CYP19
and CYP11B1, but with no significant inhibition of CYP11B2
and CYP17. Compound 5 inhibited CYP19 more than the
reference letrozole, while it was less active than metyrapone
versus CYP11B1. As seen from the docking pose (Fig. 3) 5 was
stabilized by p-stacking with Phe134, Phe221 and Trp224 and by
a hydrogen bond between its carbonyl moiety and Ser478.
Notably, the potent antiestrogenic letrozole 2 was also found to
interact with these very same residues,
15
it formed a hydrogen
bond to the backbone of Met374 (Fig. 1, ESI†). Compound 6
had no significant inhibitory effect on the four CYPs, high-
lighting the importance of a precise spatial orientation between
e.g. the imidazolylmethyl function and the carbonyl (Fig. 2 and
Table 2). The inactivity of 6 can be explained by the dissimilarity
in angle and distances compared to the active compounds 2 and
5, as reported in Table 2 (compare d
O]C
, d
NH
and angle
a between compounds 2, 5 and 6).
Previous studies proposed the circadian cortisol rhythm as
a biomarker for the prognosis of breast cancer. Moreover,
women with advanced breast cancer have significant elevations
in levels of basal cortisol, which is released in response to stress.
Such individuals are significantly more likely to die than patients
with normal levels of the hormone.
10–12
In addition, elevated
cortisol concentration down-regulates the tumor suppressor gene
BRCA1A7 and therefore accelerates the progress of breast
cancer.
13
Recent studies also revealed that excessive cortisol
mediates the resistance to paclitaxel by a CDK-1-dependent
pathway.
13,14
Thus, in this case, the dual inhibition reported for
Fig. 1 Chemical structures of metyrapone (1) and letrozole (2), xanthone derivative (3) and thiaxanthone derivative (4), and the newly reported
acridones 5 and 6.
Fig. 2 Energy minimized forms of compounds 2, 5 and 6, shown left to right, showing the orientation and distance of the imidazole function relative to
the aryl part.
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Scheme 1
Table 1 Biological results for CYP inhibition by compounds 5, 6 and the reference compounds letrozole 2 and metyrapone 1
a
Cpd
CYP11B1 CYP11B2 CYP17 CYP19
%inhibition
at 500 nM IC
50
(nM)
%inhibition
at 500 nM IC
50
(nM)
%inhibition
at 2 mMIC
50
(nM)
%inhibition
at 2 mMIC
50
(nM)
5 97.5 1.2 21.2 1.1 40.8 1.4 ND 1.4 0.3 ND 95.5 1.7 14.4 1.1
6 40.7 4.7 ND 3.4 1.3 ND 0.0 0.0 ND 0.0 0.0 ND
2 16.4 2.6 ND 25.6 1.6 ND 6.8 1.0 ND 99.0 0.5 36.2 2.0
1 98.0 0.8 14.6 1.6 95.0 1.9 71.8 2.1 0.0 0.0 ND 0.0 0.0 ND
a
Results are the average of 3 experiments, each done in duplicate, the results are expressed as the mean SD. ND ¼ not determined.
Fig. 3 Binding mode of compound 5 in the crystal structure of human aromatase (PDB 3EQM). Compound 5 is stabilized by a hydrogen bond to
Ser478 (with the carbonyl), by heme-complexation (the imidazolyl function faces the porphyrin iron with a distance of 1.8
A), and by pp stacking with
F134, F221 and W224. Additional hydrophobic interactions occur with Val370, Leu477, Met374 and Ile133.
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the first time has to be seen as a positive addition and not as an
unwanted side-effect. Since there are no pure CYP11B1 inhibi-
tors in clinical use, it might be beneficial to develop selective dual
inhibitors of CYP19 and CYP11B1. According to the docking
results as well as the sequence alignment we might come to the
identification of Ser478 as an essential residue both in terms of
inhibition of CYP19, and selectivity versus other CYP enzymes,
which have to be targeted whenever a dual-inhibition is wanted.
Molecular modeling
Docking. Compounds 2, 5 and 6 were docked into the CYP19
crystal structure PDB-ID 3EQM by means of the docking soft-
ware GOLD v5.01 and the CYP dedicated GOLDSCORE
function goldscore.p450_pdb.params.
In its main pose compound 5 was found to dock with the
imidazolyl plane perpendicular to the heme with the sp
2
N at 1.8–
2.1
A from the iron. The carbonyl oxygen pointed toward the C-
terminal loop of CYP19 forming a hydrogen bond with the –OH
of Ser478. The tricyclic core (7.75
A length) was oriented
almost perpendicular to the longitudinal axis of the I-helix
occupying the whole width of the active site (distance Ca
Met374
to
Ca
Thr310
z 16
A), with the imidazolyl moiety-bearing ring facing
the I-helix residues Ile305–Thr310. Thereby, 5 was stabilized by
p-stacking with Phe134, Phe221 and Trp224 and by hydrophobic
interactions with Ile133, Val370, Met374 and Leu477.
Also, letrozole (2) was found to have the triazole sp
2
N
pointing perpendicular to the heme iron and p-stacked with
Phe134, Phe221 and Trp224. One of the p-CN-phenyl rings
pointed towards the hydrophobic loop opposite to the I-helix,
while the other p-CN-phenyl ring was placed on the left of the C-
terminal loop. Contrary to 5, a hydrogen bond was formed
between the nitrile N and the backbone NH of Met374 and not
with the –OH of Ser478. However, changing the crystallographic
rotamer of Ser478 resulted in the formation of a second hydrogen
bond, this time between the nitrile of the second p-CN-phenyl
and Ser478.
Compound 6 was also docked into the CYP19 active site.
However, no pose was found with the imidazolyl nitrogen
pointing to the heme, which would be expected for potent
inhibitors.
Sequence alignment. In order to understand the selectivity seen
for compound 5 between the four CYP enzymes we aligned their
sequences (for CYP17 and CYP19 also a superimposition was
done) using T-coffee and manual refinement. According to the
crucial polar interaction with Ser478 (missing for 6 and partially
also for 2) we focused on the C-terminal turn regions (Fig. 4).
Notably, while for CYP11B1 the counterpart to Ser478 (of
CYP19) was also a Ser (Ser492), for CYP17 and CYP11B2
a hydrophobic (Val483) and an apolar (Gly492) residue was
found, both incapable of hydrogen bond formation with their
side-chains.
Acknowledgements
The authors are grateful for the STDF-Egypt and DAAD for
a mobility grant to Ashraf Abadi in Rolf Hartmann’s lab.
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Table 2 Spatial orientation of the imidazolylmethyl group of compound
5 and 6 relative to the carbonyl and NH groups. d
O]C
is the distance
between the sp
2
hybridized nitrogen of the imidazole/triazole and the
carbonyl oxygen or the nitrile nitrogen, d
NH
the distance between the sp
2
N and the NH of the acridone scaffold, and a is the angle between the sp
2
N, the bridge-methylene group and the carbonyl oxygen or the nitrile
nitrogen
d
O]C
d
NH
a
2 9.18
A 116
5 8.02
A 4.41
A 107.3
6 6.75
A 7.66
A 98.8
Fig. 4 Sequence alignment of CYP19, CYP17, CYP11B1 and
CYP11B2. Zoomed view of the C-terminal turn region with Ser478, an
important interaction partner for CYP19-inhibitors, marked with the red
sign.
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