Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

Article abstract of DOI:10.1016/j.bioorg.2019.01.066

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are curr

Full text of DOI:10.1016/j.bioorg.2019.01.066

BioorganicChemistry86(2019)401–409
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Benzophenones as xanthone-open model CYP11B1 inhibitors potentially
useful for promoting wound healing
⁎
Silvia Gobbia, ,1, Qingzhong Hub, Giacomo Foschia, Elena Catanzaroc, Federica Bellutia,
⁎
Angela Rampaa, Carmela Fimognaric, Rolf W. Hartmannd, Alessandra Bisia, ,1
a Department of Pharmacy and Biotechnology, Alma Mater Studiorum-University of Bologna, Via Belmeloro, 6, I-40126 Bologna, Italy
b Guangzhou University of Chinese Medicine, Guangzhou, China
c Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna, corso d'Augusto 237, 47921 Rimini, Italy
d Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Universitätscampus E8 1, 66123 Saarbrücken, Germany
A R T I C L E I N F O
A B S T R A C T
InChIKey:
The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the man-
agement of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and
CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate
cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes
in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the
identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound
healing and cardiovascular diseases.
YZPWQVXUAGIWFK-UHFFFAOYSA-
NKeywords:
Benzophenone
CYP11B1 (11-β-hydroxylase)
CYP11B2 (aldosterone synthase)
Wound healing
Cortisol
In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1
and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously re-
ported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible
scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective
towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold,
leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a
phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in se-
lectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives
also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.
Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential
of this compound as a wound-healing promoter.
Steroidogenesis
1. Introduction
and to the onset of pathologies such as Cushing’s Syndrome (CS) and
impaired wound healing [1]. CS is a life-threatening disease char-
Cortisol, a steroidal glucocorticoid hormone biosynthesized in the
adrenal gland under the control of the hypothalamus-pituitaryadrenal
axis, plays a crucial role in regulating many essential physiological
functions, including stress response, suppression of immune system,
various metabolic processes and neurogenesis. Its production and re-
lease are regulated by two main signals, corticotropin-releasing hor-
mone (CRH)a and adrenocorticotropic hormone (ACTH), and a dysre-
gulation of this refined control mechanism leads to hypercortisolism
acterized by a vast array of symptoms, including hypertension, obesity
and diabetes, which could ultimately lead to fatal complications. The
currently applied therapeutic strategy includes the use of glucocorticoid
receptor antagonists, nonselective inhibitors of adrenal steroidogenesis
or drugs able to reduce ACTH secretion, often showing limited efficacy
and unacceptable incidence of side effects [2]. On the other hand, the
occurrence of chronic wounds, affecting around 1–2% of the popula-
tion, is generally a comorbid condition, often arising in combination
⁎ Corresponding authors.
E-mail addresses: silvia.gobbi@unibo.it (S. Gobbi), alessandra.bisi@unibo.it (A. Bisi).
1 These authors contributed equally to this work.
a CRH (corticotropin-releasing hormone); ACTH (adrenocorticotropic hormone); CS (Cushing’s Syndrome); CYP11B1 (11-beta hydroxylase); CYP11B2 (aldosterone
synthase); CYP (cytochrome P450); CYP19 (aromatase); CYP17 (17α hydroxylase/17,20 lyase); 1-ImiXs (1-imidazolylmethylxanthones); 3-ImiXs (3-imidazo-
lylmethylxanthones); NBS (N-bromosuccinimide); BPO (Benzoylperoxyde).
https://doi.org/10.1016/j.bioorg.2019.01.066
Received 5 September 2018; Received in revised form 21 January 2019; Accepted 28 January 2019
Availableonline30January2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

S. Gobbi et al.
BioorganicChemistry86(2019)401–409
with diabetes and obesity. Due to the increased incidence of these
diseases and to the aging of population, the number of patients is ex-
pected to progressively increase. The onset of chronic wounds is
probably linked to a cutaneous overexpression of cortisol, leading to an
impaired inflammatory response, blocking fibroblast proliferation, and
collagen synthesis, and finally to an increased risk of infection [3].
Innovative treatments for this unpleasant disease involve stem cell
applications and skin transplantation, techniques that proved to be
expensive and often unsatisfactory. In this framework, drugs able to
reduce cortisol concentration at skin level by reducing its biosynthesis
could represent a valuable therapeutic option.
of the substitution pattern was proved to change the orientation of the
molecule, leading to different inhibitory activities towards the two
highly homologue enzymes. Some derivatives endowed with low-na-
nomolar potency for the inhibition of CYP11B1 were obtained and
noteworthy, in the 6-substituted 3-ImiXs series, more than in pre-
viously reported ones, the nature of substituents seemed negligible for
activity on this isoform.
In this study, with the aim to evaluate whether the planar and
conformationally constrained xanthone moiety is a critical feature for
optimal interaction with these enzymes, its ether oxygen was removed
and a small series of flexible imidazolylmethylbenzophenones (Fig. 1)
was designed and synthesized. A similar strategy had been successfully
applied in the search for aromatase inhibitors, enabling us to obtain
potent compounds [18], and offered the advantage of a large synthetic
accessibility, which also allowed for an easy identification of the best
positioning of the imidazole nitrogen on the benzophenone scaffold.
Indeed, in that series the position of the imidazole ring on the benzo-
phenone was varied, and substituents were also introduced in different
positions.
CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase)
are cytochrome P450 (CYP) isoforms mainly expressed in the adrenal
cortex, CYP11B1 in the zona fasciculata and CYP11B2 in the zona
glomerulosa, where they catalyse key steps in the biosynthesis of cor-
tisol and aldosterone, respectively. Like CYP19 (aromatase), re-
sponsible for the production of estrogens and CYP17 (17α hydroxylase/
17,20 lyase), involved in the biosynthesis of androgens, CYP11B1 and
B2 represent validated drug targets for treating disorders related to
abnormally increased levels of adrenal steroidal hormones. These iso-
forms share a high degree of sequence homology (up to 94%), differing
by only 30 residues, all located away from the substrate recognition
site. Therefore, the identification of specific features allowing the de-
sign of selective inhibitors becomes particularly challenging. Despite
some exciting improvements in the search for selective compounds able
to inhibit CYP11B1 [4–6], no specific inhibitor has been approved to
treat hypercortisolism. Yet, some compounds have been reported [4],
for which the ability to inhibit CYP11B1 with high potency could be
exploited to promote chronic wound healing, overcoming the drawback
of low selectivity vs CYP11B2. Indeed, the biosynthesis of CYP11B1 was
found to be up-regulated in injured tissues [7], so that an excess of
cortisol impairing wound healing is produced, while CYP11B2 isoform
has not been observed in skin cells [8]. On the other hand, in the last
years a number of potent and selective inhibitors of CYP11B2 have been
reported [9–12], highlighting the crucial role of a nitrogen-containing
heterocyclic function (pyridine or imidazole) for a proper inhibition. It
is indeed well known that CYPs inhibition takes advantage of the pre-
sence of a nitrogenated heterocycle, able to coordinate the heme iron
establishing the primary interaction with the enzyme, while a properly
decorated suitable scaffold could discriminate between different related
cytochromes.
For the new series reported here, the unsubstituted 2-, 3- and 4-
imidazolylmethylbenzophenone already tested for aromatase inhibition
[18] (1–3, Fig. 2) were screened for activity towards CYP11B enzymes
and were found to be quite potent inhibitors of CYP11B1, while
showing lower efficacy towards CYP11B2 (Table 1, see also Biological
evaluation section). Among them 3, carrying the imidazolylmethyl
chain in position 4, proved to be the most potent and selective for
CYP11B1 over CYP11B2, while appreciable but not extremely high
potency on aromatase was previously reported. Taking into account
that the corresponding xanthone derivative 3-ImiX (Fig. 2) had been
the most promising of its series as regards selectivity for CYP11B1
(Table 1) [17], 3 was selected for further modifications. Therefore, this
compound was decorated with different substituents in position 4′,
mimicking the 6-substituted parent 3-ImiXs (Fig. 1). In particular,
methoxy and phenyl moieties were selected to evaluate the roles of
different size and lipophilicity on activity; hydroxyl groups were also
introduced to study the effect of H-bond donors. Finally, a fluorine
atom was appended, due to its known ability to influence pharmaco-
kinetic and physicochemical properties such as metabolic stability,
membrane permeation and intrinsic potency, when properly introduced
into potential drugs, since it could affect the metabolism of the adjacent
hydroxyl group. The new compounds, whose structures are reported in
Fig. 3, were tested for inhibition of CYP11B1/2 and for inhibition of the
related steroidogenic enzymes CYP19 and 17, to establish their se-
lectivity.
In our long lasting experience in the field of steroidogenic cyto-
chromes inhibitors, we investigated the ability of appropriately sub-
stituted oxaheterocyclic cores, structurally related to natural com-
pounds (xanthones, chromones and flavones), to inhibit different CYPs
[13–15]. Key feature for inhibition appeared to be an imidazolylmethyl
moiety, able to interact with the heme-iron in the active site of these
enzymes, while appropriate and correctly positioned substituents could
account for fine-tuning of activity. In particular, selective modulation of
the potency towards different cytochromes was obtained with a suitable
pattern of substitution on the xanthone scaffold, whose large con-
jugated π system would conveniently form π-π interactions with several
aromatic aminoacid residues in the active sites and contribute, together
with the selected substituent, to the inhibitory activity [15]. This het-
erocycle again confirmed to be a highly versatile “privileged structure”,
whose activity can be directed to different targets when appropriately
functionalized.
2. Results and discussion
2.1. Chemistry
The compounds were prepared according to Scheme 1. The synth-
esis of the benzophenone nucleus was performed via Friedel-Craft
acylation of biphenyl or the selected anisole with p-toluylchloride in
dry methylene chloride, using AlCl3 as Lewis acid. The 4-methylben-
zophenone intermediates 9–12 were then brominated with NBS in the
presence of benzoyl peroxide (BPO) and the bromomethyl derivatives,
without purification, were then reacted with imidazole in refluxing
acetonitrile under nitrogen to provide the intermediates 13 and 14 and
the final compounds 4 and 8. The hydroxy derivatives 5–7 were ob-
tained by demethylation of 4, 13 and 14 by refluxing in HBr 48% so-
lution.
In recent papers, the design and synthesis of series of 1- and 3-
imidazolylmethylxanthones (1-ImiXs and 3-ImiXs, Fig. 1) carrying
different substituents in positions 4 or 6 of the central core, as potent
inhibitors of CYP11B1-2 enzymes, was reported [15–17]. Notably,
moving the imidazolylmethyl moiety from position 1 to position 3 led
to a remarkable shift in activity towards CYP11B1. Considering the
primary interaction of the sp2 hybridized imidazole nitrogen of the li-
gands via coordination of the heme iron, which normally shows a
perpendicular setting and serves as an anchor to binding, modification
2.2. Biological evaluation
The testing results for CYP11B1-2 inhibition of the previously re-
ported unsubstituted imidazolylmethylbenzophenones 1–3 are col-
lected in Table 1, together with earlier data obtained for CYP19 and
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BioorganicChemistry86(2019)401–409
Fig. 1. Design strategy.
results obtained when the position of the chain was varied on the
xanthone nucleus [17], where 3-ImiX proved to be one of the most
active compounds and the most selective for CYP11B1. In fact, the re-
moval of the ether oxygen and the consequent increase in flexibility of
the molecule proved to enhance activity on both CYP11B enzymes.
Compound 3 also showed appreciable selectivity towards CYP19 and
CYP17, making it a suitable compound for further modifications.
The results obtained with the introduction of substituents on the 4′-
position of the scaffold of 3, collected in Table 2, show that hydrophilic
groups (as in compounds 5, 6 and 7) led to a general decrease in in-
hibitory activity and selectivity with respect to the unsubstituted 3. In
contrast, compounds 4 and 8, bearing more lipophilic substituents
(namely methoxy and phenyl, respectively), were endowed with low-
nanomolar potency towards both CYP11B1 (IC50 values of 16.7 nM and
8.6 nM, respectively) and CYP11B2 (29.9 nM and 17.2 nM, respec-
tively). In particular, these substituents proved to positively influence
the inhibition of CYP11B2 to a higher extent with respect to CYP11B1,
for which an appreciable increase was observed only with the in-
troduction of a phenyl group (compound 8). As a consequence, se-
lectivity between the CYP11B isoforms was somewhat lost compared to
the parent compound 3. Remarkably, while comparing the un-
substituted 3 to the 4′-methoxy derivative 4 only an increase in activity
towards CYP11B2 could be seen (IC50 values of 103.5 nM vs 29.9 nM,
respectively), the presence of a phenyl group in compound 8 was able
to affect the activities on different CYPs, leading to an increase in po-
tency for inhibition of CYP11B1 and –B2 and a 10-fold decrease for
CYP19. This behavior could represent a favorable feature for the de-
velopment of agents to be used for the promotion of wound healing, for
which the selectivity between the two CYP11B isoforms does not seem
to be a crucial issue, while the inhibition of CYP19 could be detrimental
for activity. Indeed, the expression of CYP19 and CYP17 in epidermis
tissue has been reported, and estrogens in the skin could have a
moisturizing effect, thus preventing aging and favorably influencing
wound healing [19].
Fig. 2. Benzophenones 1–3 previously tested on aromatase and 3-ImiX as re-
ference compound.
Table 1
Inhibition of CYP11B, CYP19 and CYP17 for previously reported unsubstituted
benzophenones and 3-ImiX.
Compound
CYP11B1a
CYP11B2a
CYP19c
CYP17d
SFe
IC50 (nM)b
IC50 (nM)b
IC50 (nM)b
% inhib. 2.5 µM
B2/B1
1
66.2
69.5
17.3
70.8
105.9
178.7
103.5
344.1
560f
7.3f
252f
390
NAf
NAf
NAf
33%
1.6
2.6
6.0
4.9
2
3
3-ImiXg
a
The deviations were within 25%. Hamster fibroblasts expressing human
CYP11B1/B2; substrate: deoxycorticosterone 100 nM.
b
c
The given values are mean values of at least three experiments.
The deviations were within 5%. Human placental CYP19; substrate an-
drostenedione, 500 nM.
d
e
f
g
Escherichia coli expressing human CYP17; substrate progesterone, 25 μM.
From a structural point of view, these data seem to indicate that an
increase in flexibility allowed the compounds to properly fit in the
enzyme active site and to maintain a relevant activity, compared to the
more rigid xanthone-based compounds. However, the potency seemed
to be related to the lipophilicity and/or size of the substituent in po-
sition 4′, as demonstrated by the progressive reduction in activity no-
ticed by shifting from the 4′-phenyl (8) to the 4′-methoxy (4), the un-
substituted (3) and the 4′-hydroxy (5) derivative. Moreover, the
simultaneous introduction of two substituents (3′ and 4′-positions) led
to a marked decrease in activity on both enzymes (6 and 7 with respect
to 5), which was more significant with the introduction of an additional
Selectivity factor: IC50CYP11B2/ IC50CYP11B1.
See Ref. [18].
See Ref. [17]. NA = no activity detected.
CYP17 inhibition. In this small series, while the compounds showed the
same range of activity on CYP11B2, a small difference could be seen for
inhibition of CYP11B1. Compound 3, in which the nitrogen-containing
chain is located in position 4, proved to be the most potent and selective
inhibitor of this isoform (IC50 = 17.3 nM and SFB2/B1 = 6.0), showing
that this site is the most appropriate for the positioning of the imida-
zolylmethyl group on this scaffold. This finding is in line with the
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BioorganicChemistry86(2019)401–409
Fig. 3. Newly synthetized benzophenones 4–8.
hydroxyl group than with a fluorine atom, very similar in size to the
hydrogen. Finally, while selectivity between CYP11B1 and –B2 was
somewhat decreased by the introduction of substituents with respect to
3, some of the new compounds proved to be considerably selective
towards related steroidogenic enzymes, showing a micromolar potency
on CYP19 and being devoid of CYP17 inhibiting activity.
energy values of −9.13 and −9.39 Kcal/mol were observed for com-
pounds 3 and 8, respectively, indicating high binding affinities. Not
surprisingly, the compounds coordinate to the heme iron in perpendi-
cular manners (Fig. 4). With such an anchor, both molecules lean
against the β2-sheet, however, in different modes: the benzophenone
core of compound 3 stretches along the β2-sheet, in contrast to the cross
position that compound 8 forms with its additional phenyl moiety. The
binding of both compounds is riveted by the π-π interactions between
their conjugating body and Phe130, Phe381 and Phe487, while an
To further investigate the binding of the synthesized inhibitors in
CYP11B1, compounds 3 and 8 were docked into a human CYP11B1
homology model [20]. In the most favorable binding poses, free binding
Reagents and conditions: i) AlCl3, dry CH2Cl2, rt; ii) NBS, CCl4, hν, BPO; iii) imidazole, CH3CN,
N2; iv) HBr 48%.
Scheme 1. Synthesis of the studied compounds.
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BioorganicChemistry86(2019)401–409
Table 2
Inhibition of CYP11B, CYP19 and CYP17 for the studied benzophenones.
Compound
R
R′
CYP11B1a
IC50 (nM)b
CYP11B2a
IC50 (nM)b
CYP19c
CYP17d
SFe
IC50 (nM)b
% inhib. 10 µM
B2/B1
3
4
5
6
7
8
H
H
17.3
16.7
44.7
232.4
808.1
8.6
103.5
29.9
252f
NAf
6.0
1.8
2.5
1.1
1.3
2
H
OCH3
OH
OH
OH
Ph
237.8
1100
1237.5
5275
2531
17.5%
34.4%
31%
H
113.5
256.9
1028.1
17.2
F
OH
H
3.4%
16.6%
a
The deviations were within 25%. Hamster fibroblasts expressing human CYP11B1/B2; substrate: deoxycorticosterone 100 nM.
The given values are mean values of at least three experiments.
b
c
d
e
f
The deviations were within 15%. Human placental CYP19; substrate androstenedione, 500 nM.
Escherichia coli expressing human CYP17; substrate progesterone, 25 μM.
Selectivity factor (SF): IC50CYP11B2/ IC50CYP11B1.
See Ref. [18].
Fig. 4. Docking of compounds 3 (A, magenta) and 8 (B, cyan) in a CYP11B1 homology model.
additional π-π interaction between the far-end phenyl group and
63.62
31.52
1.99%, respectively, of wound healing compared to
0.95% of untreated cells. These preliminary data seem to
Phe239 is observed for compound 8. Furthermore, the ketones point
into distinct directions: in compound 3, it forms a net of hydrogen-
bonds with the phenol and amide H of Tyr485 as well as Phe381 and
Leu380, respectively, whereas only the interaction with Arg110 is
present for compound 8. These hydrogen bonds contribute significantly
to the stability of this binding mode and thus to the affinity for the
target enzyme.
validate the ability of this compound to act as a wound-healing pro-
moter, making this class of derivatives worth of further in-depth stu-
dies.
3. Conclusions
Finally, in the light of the obtained results and aiming at evaluating
the wound healing potential of the newly synthetized molecules, an in
vitro scratch test was performed on the most active compound 8 em-
ploying the HT-29 cell line, a human colon cancer cell line expressing
relatively high levels of CYP11B1 [21]. This inexpensive and straight-
forward assay allows obtaining a first insight on the ability of com-
pounds to positively influence new tissue formation, promoting cell
migration following a purposely induced injury in a cell monolayer,
mimicking what happens during wound healing in vivo [22]. First, the
cytotoxicity of the compound was measured with the 4-methy-
lumbelliferyl heptanoate (MUH) assay to select the concentrations to be
used in the scratch test. The compound exhibited a cytotoxic effect only
at the highest tested concentration (100 µM) (data not shown). Thus, 4
and 40 nM treatments were chosen to perform the scratch test.
As reported in Fig. 5, the compound stimulated the filling of the
scratch with healthy cells starting after 4 days of treatment. A more
pronounced effect was recorded after 8 days. By then, at 4 and 40 nM
In this study, taking advantage of the positive results obtained in the
field of CYP19 inhibitors, the role of the increase in flexibility obtained
by moving from the xanthone to the benzophenone scaffold was also
evaluated for the homologues enzymes CYP11B1 and –B2. The best
positioning of the key imidazolylmethyl chain for interaction with
CYP11B1 was identified and an optimization process was attempted
with the synthesis of a purposely designed small series of 4′-substituted
derivatives. An evident preference for lipophilic groups was noticed, as
activity progressively increased shifting from hydroxyl to hydrogen,
methoxy and phenyl substituents on the benzophenone core.
Compound 8, bearing an additional phenyl ring and thus em-
bodying a biphenyl moiety, emerged as the most promising derivative
of the series, being endowed with low nanomolar potency on CYP11B1
and a remarkable selectivity towards CYP19 and CYP17. The potential
use of this compound for topical application, supported by the results of
a preliminary in vitro assay, would circumvent the low selectivity for
the highly omologue isoform CYP11B2, making it a suitable drug can-
didate for the acceleration of chronic wound healing.
treatments, the compound provoked 47.71
1.20% and
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BioorganicChemistry86(2019)401–409
Fig. 5. Percentage of wound healing and representative images of the progression of wound closure after 4 and 8 days of treatment with compound 8. Original
magnification ×10. Data shown are means
SEM, **p < 0.01; ***p < 0.001.
4. Experimental
4.1. Chemistry
naming algorithm developed by CambridgeSoft Corporation and used
in ChemDraw Professional 15.0.
4.1.2. General method for the preparation of benzophenones 9–12
To a solution of the selected anisole or biphenyl (13.0 mmol) in
30 mL of dry CH2Cl2 at 0° C, p-toluoylchloride (2 g, 13.0 mmol), and
AlCl3 (3 g, 22.05 mmol) were added and the mixture was stirred at rt
overnight. After addition of ice, the mixture was diluted with CH2Cl2,
washed with NaHCO3 saturated solution, dried and evaporated to
dryness.
4.1.1. General methods
All melting points were determined in open glass capillaries using a
Büchi apparatus and are uncorrected. 1H NMR and 13C NMR spectra
were recorded in CDCl3, unless otherwise indicated, on a Varian VXR
Gemini spectrometer 400 MHz and 101 MHz, respectively. Chemical
shifts are reported in parts per million (ppm) relative to tetra-
methylsilane (TMS) as internal standard and spin multiplicities are
given as s (singlet), d (doublet), t (triplet), m (multiplet) or br (broad).
Direct infusion ES-MS spectra were recorded on a Waters Micromass ZQ
4000 apparatus. Chromatographic separations were performed by flash
chromatography on silica gel columns (Kieselgel 40, 0.040–0.063 mm;
Merck). Organic solutions were dried over anhydrous sodium sulfate.
Elemental analysis was used to determine purity of the described
compounds. Analyses indicated by the symbols of the elements were
within 0.4% of the theoretical values. All chemicals were purchased
from Aldrich Chemistry, Milan (Italy), or from Alfa Aesar, Milan (Italy),
and were of the highest purity. Compounds were named relying on the
4.1.2.1. (4-methoxyphenyl)(p-tolyl)methanone 9. Using the previously
reported procedure and starting from 1.4 g of anisole, 2.41 g of 9 were
obtained (82%), mp 91–92 °C (lit. [23] mp 94–95 °C). 1H NMR: δ 2.46
(s, 3H, CH3), 3.85 (s, 3H, OCH3), 7.09 (d, J = 8.8 Hz, 2H arom.), 7.37
(d, J = 8.4 Hz, 2H arom.), 7.66 (d, J = 8.0 Hz, 2H arom.), 7.75 (d,
J = 8.4 Hz, 2H arom.).
4.1.2.2. (3-fluoro-4-methoxyphenyl)(p-tolyl)methanone 10. Using the
previously reported procedure and starting from 1.64 g (1.46 mL) of
2-fluoroanisole, 2.47 g of 10 were obtained (78%), mp 78–80 °C (lit.
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BioorganicChemistry86(2019)401–409
[23] mp 79–81 °C). 1H NMR: δ 2.45 (s, 3H, CH3), 3.98 (s, 3H, OCH3),
7.11 (s, 1H arom.), 7.24 (d, J = 8.4 Hz, 2H arom) 7.46 (d, J = 7.2 Hz,
2H arom.), 7.75 (d, J = 8.0 Hz, 2H arom.).
4.1.4. General method for demethylation
To the selected methoxy derivative (2.0 mmol) an excess of HBr
48% (20 mL) was added, and the mixture was heated under reflux for
8 h. After dilution with H2O, the solution was basified with NaOH
pellets, washed with CH2Cl2, acidified to pH 6 with HCl 37%. The solid
was collected by filtration.
4.1.2.3. (3,4-dimethoxyphenyl)(p-tolyl)methanone
11. Using
the
previously reported procedure and starting from 1.8 g of 1,2-
dimethoxybenzene, 2.65 g of 11 were obtained (80%), mp 105–106 °C
(lit. [23] mp 105–107 °C). 1H NMR: δ 2.42 (s, 3H, CH3), 3.95 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 7.18 (s, 1H arom.), 7.28 (d, J = 8.4 Hz, 2H
arom) 7.53 (d, J = 7.2 Hz, 2H arom.), 7.81 (d, J = 8.0 Hz, 2H arom.).
4.1.4.1. (4-((1H-imidazol-1-yl)methyl)phenyl)(4-hydroxyphenyl)
methanone 5. Using the previously reported procedure and starting
from 0.58 g of 4, 5 was obtained, yield 58%, mp 120 °C (d). 1H NMR
(DMSO): δ 5.32 (s, 2H, CH2-imi), 6.88 (d, J = 8.8 Hz, 2H arom.), 6.95
(s, 1H, imi), 7.25 (s, 1H, imi), 7.36 (d, J = 8.0 Hz, 2H arom.), 7.62–7.76
(m, 4H arom), 7.81 (s, 1H, imi), 10.43 (s, 1H, OH). 13C NMR δ: 49.99,
115.35, 127.65, 127.79, 129.66, 132.54, 137.81, 162.14, 193.89. MS
(ES) m/z: 279 (M+H), 301 (M + Na). Anal. C17H14N2O2 (C, H, N).
4.1.2.4. [1,1′-biphenyl]-4-yl(p-tolyl)methanone 12. Using the previously
reported procedure and starting from 2.0 g of biphenyl, 2.54 g of 12
were obtained (72%), mp 127–129 °C (lit. [24] mp 121–122 °C). 1H
NMR: δ 2.47 (s, 3H, CH3), 7.31 (d, J = 8.0 Hz, 2H arom.), 7.42–7.51
(m, 3H arom), 7.66 (d, J = 7.2 Hz, 2H arom.), 7.71 (d, J = 8.0 Hz, 2H
arom.), 7.77 (d, J = 8.0 Hz, 2H arom.), 7.88 (d, J = 8.4 Hz, 2H arom).
4.1.4.2. (4-((1H-imidazol-1-yl)methyl)phenyl)(3-fluoro-4-hydroxyphenyl)
methanone 6. Using the previously reported procedure and starting
from 0.62 g of 13, 6 was obtained, yield 52%, mp 232–235 °C. 1H NMR
(DMSO): δ 5.56 (s, 2H, CH2-imi), 7.09 (t, J = 8.4 Hz, 1H arom.),
7.43–7.45 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H arom.), 7.51–7.56 (m, 3H
arom + imi), 7.72–7.74 (m, 3H arom + imi), 7.84 (s, 1H, imi), 9.28 (s,
1H, arom), 10.99 (s, 1H, OH). 13C NMR δ: 51.22, 117.44, 117.67,
120.69, 122.23, 128.04, 128.21 129.87, 135.82, 137.67, 139.00,
149.37, 150.04, 150.16, 151.79, 193.06. MS (ES) m/z: 297 (M+H).
Anal. C17H13FN2O2 (C, H, N).
4.1.3. General method for the preparation of imidazole derivatives 4, 8, 13,
14
A mixture of the selected benzophenone (5.0 mmol) and N-bromo-
succinimide (0.85 g, 5.0 mmol) in CCl4 (50 mL), in the presence of a
catalytic amount of benzoyl peroxide, was refluxed for 5 h and then hot
filtered. The solvent was evaporated to dryness to give the corre-
sponding bromomethyl derivative, which was used without further
purification.
A mixture of this derivative and imidazole (0.9 g,
14 mmol) in CH3CN (40 mL) was refluxed under N2 for 6 h. The solvent
was evaporated to dryness and the residue was purified by flash chro-
matography (toluene/acetone 4:1) to give the desired compound (yield
15–21%).
4.1.4.3. (4-((1H-imidazol-1-yl)methyl)phenyl)(3,4-dihydroxyphenyl)
methanone 7. Using the previously reported procedure and starting
from 0.64 g of 14, 7 was obtained, yield 48%, mp 250 °C (d). 1H NMR
(DMSO): δ 5.38 (s, 2H, CH2-imi), 6.84 (d, J = 7.6 Hz, 1H arom.),
7.03–7.08 (m, 2H arom), 7.22 (s, 1H, imi), 7.31 (s, 1H, imi), 7.37 (d,
J = 7.6 Hz, 2H, arom), 7.63 (d, J = 7.2 Hz, 2H, arom), 7.96 (s, 1H, imi),
9.42 (s, 1H, OH), 9.92 (s, 1H, OH). 13C NMR δ: 50.04, 116.04, 116.68,
125.65, 127.82, 129.66, 132.54, 133.42, 137.81, 145.70, 152.14,
193.98. MS (ES) m/z: 295 (M+H). Anal. C17H14N2O3 (C, H, N).
4.1.3.1. (4-((1H-imidazol-1-yl)methyl)phenyl)(4-methoxyphenyl)
methanone 4. Using the previously reported procedure and starting
from 1.13 g of 9, 4 was obtained, mp 103–106 °C (ligroin). 1H NMR
(DMSO): δ 3.85 (s, 3H, OCH3), 5.32 (s, 2H, CH2-imi), 6.94 (s, 1H, imi),
7.08 (d, J = 8.8 Hz, 2H arom.), 7.24 (s, 1H, imi), 7.37 (d, J = 8.4 Hz,
2H arom.), 7.67 (d, J = 8.0 Hz, 2H arom.), 7.73 (d, J = 8.4 Hz, 2H
arom.), 7.80 (s, 1H, imi). 13C NMR δ: 49.14, 55.63, 113.98, 119.78,
127.34, 128.90, 129.37, 129.77, 132.23, 137.16, 137.65, 141.94,
163.06, 194.05. MS (ES) m/z: 293 (M+H), 315 (M + Na). Anal.
4.2. Biological methods
4.2.1. Inhibition of CYP11B1 and CYP11B2
C
18H16N2O2 (C, H, N).
V79 MZh cells expressing human CYP11B1 or CYP11B2 were grown
on six-well cell culture plates with 9.6 cm2 culture area per well until
confluence. The reaction was subsequently started by the addition of
[1,2-3H]-11-deoxycorticosterone as substrate and corresponding in-
hibitor at different concentrations. After incubations of 6 h for
V79MZh11B1 and 30 min for V79MZh11B2 cells, respectively, enzyme
reactions were stopped by extracting the supernatant with chloroform.
Samples were centrifuged (10,000g, 10 min) and the solvent was pi-
petted into fresh cups. The solvent was evaporated, the steroids were re-
dissolved in chloroform and analysed by HPTLC [25,26].
4.1.3.2. (4-((1H-imidazol-1-yl)methyl)phenyl)(3-fluoro-4-
methoxyphenyl)methanone 13. Using the previously reported procedure
and starting from 1.22 g of 10, 13 was obtained as oily compound. 1H
NMR: δ 4.01 (s, 3H, OCH3), 5.25 (s, 2H, CH2-imi), 6.95–7.18 (m, 2H
arom + imi), 7.28 (d, J = 8.4 Hz, 2H arom) 7.53–7.55 (m, 3H,
arom + imi), 7.62 (s, 1H, imi), 7.81 (d, J = 8.0 Hz, 2H arom.).
4.1.3.3. (4-((1H-imidazol-1-yl)methyl)phenyl)(3,4-dimethoxyphenyl)
methanone 14. Using the previously reported procedure and starting
from 1.28 g of 11, 14 was obtained as oily compound. 1H NMR: δ 3.95
(s, 3H, OCH3), 3.98 (s, 3H, OCH3), 5.31 (s, 2H, CH2-imi), 6.96–7.18 (m,
2H, arom + imi), 7.28–7.31 (m, 3H, arom + imi), 7.53 (d, J = 7.2 Hz,
2H arom.), 7.62 (s, 1H, imi), 7.81 (d, J = 8.0 Hz, 2H arom.).
4.2.2. CYP17 preparation and assay
Human CYP17 was co-expressed with rat NADPH-P450 reductase in
E. coli, which was subsequently treated with lysozyme, incubated on ice
with continuous shaking for 30 min and sonicated (50,000g, 20 min) at
4 °C to break cell wall and obtain membrane pellet preparations. A
solution of progesterone as substrate, NADPH generating system
(10 mM NADP+, 100 mM glucose-6-phosphate, and 2.5 units of glu-
cose-6-phosphate dehydrogenase) and inhibitors of various concentra-
tions were pre-incubated at 37 °C for 5 min before a diluted membrane
suspension was added to start the reaction. After an incubation of
30 min at 37 °C, it was quenched with 50 mL 1 N HCl and steroids were
extracted with ethyl acetate. The 17α-hydroxylase activity of CYP 17
was determined by measuring the conversion of progesterone into 17α-
hydroxyprogesterone and by-product 16α-hydroxyprogesterone
[27,28].
4.1.3.4. (4-((1H-imidazol-1-yl)methyl)phenyl)([1,1′-biphenyl]-4-yl)
methanone 8. Using the previously reported procedure and starting
from 1.36 g of 12, 8 was obtained, mp 123–125 °C. 1H NMR: δ 5.24 (s,
2H, CH2-imi), 6.96 (s, 1H, imi), 7.16 (s, 1H, imi),7.26 (d, J = 8.0 Hz, 2H
arom.), 7.42–7.51 (m, 3H arom), 7.62 (s, 1H, imi), 7.65 (d, J = 7.2 Hz,
2H arom.), 7.71 (d, J = 8.0 Hz, 2H arom.), 7.83 (d, J = 8.0 Hz, 2H
arom.), 7.88 (d, J = 8.4 Hz, 2H arom). 13C NMR δ: 50.61, 119.50,
127.15, 127.20, 127.45, 128.42, 129.14, 130.19, 130.82, 136.03,
137.67, 137.90, 140.01, 140.67, 145.66, 195.68. MS (ES) m/z: 339
(M+H), 361 (M + Na). Anal. C23H18N2O (C, H, N).
407

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BioorganicChemistry86(2019)401–409
4.2.3. CYP19 preparation and assay
Appendix A. Supplementary material
Human CYP19 was obtained from the microsomal fraction of freshly
delivered human term placental tissue. The incubation tubes containing
[7] androstenedione as the substrate, NADPH, glucose-6-phosphate,
glucose-6-phosphate dehydrogenase and inhibitors of various con-
centrations in phosphate buffer (0.05 M, pH 7.4) were pre-incubated at
30 °C in a shaking water bath for 5 min before microsomal protein was
added to start the reaction. After further incubation for 3 h, the reaction
was terminated with ethyl acetate and aliquots were pipetted into cold
HgC1 solution (1 mM). After addition of aqueous dextran-coated char-
coal (DCC) suspension (2%), the vials were shaken for 20 min and
centrifuged at 1500g for 5 min to separate the charcoal-adsorbed ster-
oids. Inhibition of enzyme activity was determined by measuring the
formation of 3H2O [29].
Representative NMR spectra (compounds 4 and 8). Supplementary
data to this article can be found online at https://doi.org/10.1016/j.
bioorg.2019.01.066. These data include MOL files and InChiKeys of the
most important compounds described in this article.
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