Design, synthesis and biological evaluation of imidazole and triazole-based carbamates as novel aromatase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.113115

In the search for novel aromatase inhibitors, a series of triazole and imidazole-based carbamate derivatives were designed and synthesized. Final compounds were thus evaluated against human aromatase by in vitro kinetic experiments in a fluorimetric assay in comparison with letrozole. The effect of most active derivatives 13a and 15c was then evaluated in vitro on the human breast cancer cell line MCF7 by MTT assay, cytotoxicity assay (LDH release) and cell cycle analysis, revealing a dose-dependent inhibition profile of cell viability and low micromolar IC₅₀ values. In addition, docking simulations were also carried out to elucidate at a molecular level of detail the binding modes adopted to target human aromatase.

Full text of DOI:10.1016/j.ejmech.2020.113115

European Journal of Medicinal Chemistry 211 (2021) 113115
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of imidazole and triazole-
based carbamates as novel aromatase inhibitors
,
Alessandra Ammazzalorso a *, Marialucia Gallorini b, Marialuigia Fantacuzzi a,
Nicola Gambacorta c, Barbara De Filippis a, Letizia Giampietro a, Cristina Maccallini a,
Orazio Nicolotti c, Amelia Cataldi b, Rosa Amoroso a
a Unit of Medicinal Chemistry, Department of Pharmacy, “G. D’Annunzio” University, Chieti, Italy
b Unit of Anatomy, Department of Pharmacy, “G. D’Annunzio” University, Chieti, Italy
c Unit of Medicinal Chemistry, Department of Farmacia-Scienze Del Farmaco, “A. Moro” University, Bari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
In the search for novel aromatase inhibitors, a series of triazole and imidazole-based carbamate de-
rivatives were designed and synthesized. Final compounds were thus evaluated against human aro-
matase by in vitro kinetic experiments in a fluorimetric assay in comparison with letrozole. The effect of
most active derivatives 13a and 15c was then evaluated in vitro on the human breast cancer cell line
MCF7 by MTT assay, cytotoxicity assay (LDH release) and cell cycle analysis, revealing a dose-dependent
inhibition profile of cell viability and low micromolar IC50 values. In addition, docking simulations were
also carried out to elucidate at a molecular level of detail the binding modes adopted to target human
aromatase.
Received 4 November 2020
Received in revised form
7 December 2020
Accepted 15 December 2020
Available online 25 December 2020
Keywords:
Aromatase inhibitors
Breast cancer
Cytochrome P450
Carbamates
© 2020 Elsevier Masson SAS. All rights reserved.
Imidazole
Triazole
1. Introduction
High levels of aromatase have been found in ovary of premen-
opausal women, in placenta of pregnant women, but also in pe-
Tamoxifen has been the standard endocrine therapy for the
treatment of estrogen-dependent breast cancer since the early ‘70s
[1]. Acting as a selective estrogen receptor modulator, this drug was
very effective for the treatment of early and advanced breast cancer
despite the occurrence of resistance and several side effects
affected the efficacy of the therapy [2,3].
In more recent years, the endocrine therapy for breast cancer
benefited of the development of a different class of drugs, able to
inhibit the estrogen formation. Aromatase inhibitors, in fact, repre-
sent a novel strategy in endocrine therapy, based on the suppression
of estrogen biosynthesis that is relevant for the aromatase activity
[4e6]. Member of cytochrome P450 enzymes, aromatase (CYP19) is a
monooxygenase responsible for the conversion of androgens (an-
drostenedione and testosterone) to estrogens (estradiol and estrone).
ripheral adipose tissue of postmenopausal women and men. An
increased expression of aromatase in breast cancer has been re-
ported by several studies, and it is recognized its pivotal role in
stimulating the growth of tumors in both autocrine and paracrine
manner [7e9].
Extensive research over the last decades led to the identification
of several aromatase inhibitors, classified as steroidal and non-
steroidal (Fig. 1), acting by a competitive or not competitive
mechanism of action [10e13].
Steroidal aromatase inhibitors decrease the estrogen biosyn-
thesis by competing with the substrate androstenedione for the
binding to the active site of the enzyme. From a structural point of
view, they show the androstenedione nucleus, with substitutions in
different position of the steroid skeleton. Exemestane is the most
representative member of this class, endowed with an irreversible
mechanism of inhibition.
Non-steroidal inhibitors act by a reversible mechanism of inhi-
bition, by coordinating the heme iron in the catalytic site of aro-
matase. Starting from the structure of aminoglutetimide, more
* Corresponding author. Department of Pharmacy, “G. d’Annunzio” University,
Via dei Vestini 31, 66100, Chieti, Italy.
E-mail address: alessandra.ammazzalorso@unich.it (A. Ammazzalorso).
https://doi.org/10.1016/j.ejmech.2020.113115
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
Fig. 1. Chemical structures of third generation aromatase inhibitors exemestane, letrozole and anastrozole.
selective compounds were generated, referred as “second and third
generation” aromatase inhibitors. They include the imidazole de-
rivative fadrazole and triazole derivatives anastrozole and letrozole
[14]. Anastrozole and letrozole showed improved efficacy and a
superior toxicity profile compared to tamoxifen [15,16], and they
currently are first-line drugs in postmenopausal women with
advanced breast cancer.
All final compounds, synthesized as racemates, were evaluated
against aromatase, in a fluorimetric in vitro assay, and compared to
letrozole; the most active derivatives were then submitted to cell
viability and cytotoxicity evaluation on MCF7 breast cancer cells by
MTTand LDH assays, followed by cell cycle analysis. Docking studies
were also performed on most promising compounds to shed light on
their binding mode in the human aromatase active site.
In these compounds, a key role in the mechanism of inhibition is
played by the heterocyclic nitrogen, involved in the interaction
with heme iron; indeed, aromatase inhibitors devoid of the nitro-
gen were discovered, as the family of flavonoids possessing the
benzopyranone ring [17,18]. In addition to heterocyclic nitrogen,
the presence of hydrogen bond acceptors in these structures is
believed an important feature providing auxiliary interactions in-
side the binding pocket.
In the search for novel aromatase inhibitors, we focused our
attention on an extensive virtual screening study by Caporuscio
et al., in which several structures containing azoles were analyzed
for their aromatase inhibition [19]. Starting from this virtual study,
we performed chemical modifications on the sulfonamide com-
pound SYN20028567, producing imidazole and indole derivatives
endowed with good inhibition properties [20,21].
2. Results and discussion
2.1. Chemistry
The first series of carbamates was synthesized as depicted in
Scheme 1. Triazole was reacted with 1,2-epoxy-3-phenoxypropane
to obtain racemic alcohol 1 [23]; the reaction of 1 with isocyanates
afforded carbamates 2a-e.
We decided to explore the possibility of substitution of both
phenyl rings, introducing in para a chlorine or a methoxy group. To
obtain final carbamates 6 and 9 (Scheme 2), epibromohydrin 3 was
reacted with p-cloro or p-methoxyphenol to obtain intermediates
4,7. The treatment of 4,7 with triazole afforded racemic alcohols 5,8,
that were transformed to carbamates 6 and 9 by reaction with
isocyanates.
Carbamates of third series, with a two-carbon linker, were ob-
tained following the same synthetic scheme, by reacting triazole
with styrene oxide (Scheme 3).
Imidazole-based derivatives 13a-c and 15a-c were obtained as
depicted in Scheme 4.
In the current study, a novel group of compounds were designed
by modification of BAS02077837, reported as nanomolar aromatase
inhibitor [19,22]. Overall, we decided to modify different portions
of the molecule, as briefly summarized in Fig. 2: the azole ring, the
length of the linker connecting the azole and the aromatic, the ester
and the p-substitution of both aromatic rings.
In a first series of compounds (Fig. 3), the ester of BAS02077837
was replaced by a carbamate keeping unaltered the propanoxy
linker. The p-substitution on aromatic rings was then explored in
the series II, where a chlorine or a methoxy group were introduced
in para to both phenyl rings. The last series (III) was then synthe-
sized by modifying the linker length from three to two carbon units
and by removing ether oxygen atom.
2.2. In vitro aromatase inhibition assay
The human aromatase inhibitory activity of the synthesized
compounds was determined by using an in vitro fluorescence-
based assay [Aromatase (CYP19A) Inhibitor Screening Kit (Fluori-
metric) BioVision]. This assay employs a fluorogenic substrate that
is converted into a highly fluorescent metabolite detected in the
visible range (Ex/Em ¼ 488/527 nm). Target compounds were
Finally, starting from general structures of Series I and III, some
carbamates containing imidazole were also prepared.
dissolved in acetonitrile (1
mM), and the results were compared to
letrozole (1
mM), used as reference compound. The activity was
measured each minute up to 60 min; the value obtained for each
compound at 10 min was registered. Only compounds displaying
inhibition percentages higher than 20 were reported (Fig. 4).
Triazole-based carbamates induced a limited ability to inhibit
aromatase when compared to letrozole, inducing percentages of
inhibition lesser than 20%; only compounds 6a and 9b showed a
moderate inhibition, with 46% and 49%, respectively. Imidazole-
based carbamates 13a-c and 15a-e showed a greater inhibition
ability, with six derivatives inducing a valuable inhibition profile:
13a and 15c induced, respectively, inhibition of 81 and 72%
compared to letrozole.
Fig. 2. Chemical modifications performed on lead compound BAS02077837.
2

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
Fig. 3. Triazole-based carbamates designed in this study.
Scheme 1. Synthesis of triazole-based carbamates 2a-e.
Reagents and conditions: a) 1,2-epoxy-3-phenoxypropane, THF, 60 ꢀC, 24 h; b) sodium hydride, ReNCO, dry ACN, N2, r.t., 24 h.
Scheme 2. Synthesis of triazole carbamates 6a-c and 9a-c.
Reagents and conditions: a) p-chlorophenol (for 6) or p-methoxyphenol (for 9), potassium carbonate, refluxing ACN, 7 h; b) triazole, THF, 60 ꢀC, 24 h; c) sodium hydride, ReNCO, dry
ACN, N2, r.t., 24 h.
We decided to calculate the IC50 for the aromatase inhibition of
compounds displaying a percentage inhibition higher than 70%
compared to letrozole: 13a and 15c were selected for this evalua-
tion. Concentration-response curves for both compounds were
derived in the in vitro fluorimetric assay, testing each compound at
activity relationships could be drawn. As far as triazole derivatives
are concerned, we observed a moderate aromatase inhibition only
for compounds bearing substituents on the aromatic rings, as in the
case of the chloro and methoxy derivatives 6a and 9b. Other tested
derivatives showed inhibition percentages lesser than 20%.
concentration from 0.01 to 100
potency was obtained for both compounds, with IC50 values of 0.82
and 0.86 M, respectively (Table 1).
Based on enzymatic assay results, some preliminary structure-
m
M. A submicromolar inhibition
Imidazole derivatives gave satisfactory results in the enzymatic
assay, displaying better inhibition percentages, despite lower
compared to letrozole. In the case of short chain derivatives 13,
benzylic derivative 13a showed better activity (81%) than phenyl
m
3

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
Scheme 3. Synthesis of triazole-based carbamates 11a-c.
Reagents and conditions: a) styrene oxide, THF, 60 ꢀC, 24 h; b) sodium hydride, ReNCO, dry ACN, N2, r.t., 24 h.
Scheme 4. Synthesis of imidazole-based carbamates 13a-c and 15a-e.
Reagents and conditions: a) styrene oxide (for 12) or 1,2-epoxy-3-phenoxypropane (for 14), THF, 60 ꢀC, 24 h; b) sodium hydride, ReNCO, dry ACN, N2, r.t., 24 h.
Fig. 4. Aromatase inhibition results for synthesized carbamates. Letrozole and synthesized compounds were tested at 1 mM. Experiments were repeated in triplicate.
derivatives 13b (61%) and 13c (62%). Among long chain derivatives 15,
best activity occurred for p-chloro (15c, 72%), with a slight decrease of
activity for p-methoxy (15d, 61%) and a drastic drop for phenyl (15b,
27%) and the o,p-dimethoxy phenyl derivative (15e, 25%).
evaluation on the breast cancer cell line MCF7 by MTT assay,
cytotoxicity assay (LDH release) and cell cycle analysis. DMSO-
dissolved compounds were assayed within a broad range of con-
centrations (10e250 mM) and the metabolic activity of cells as a
measurement of cell viability was quantified after 24, 48, and 72 h
of treatment (Fig. 5A). As for compound 13a, the percentage of
metabolic activity is set at 43.67% after the administration of 10 mM
2.3. In vitro evaluation of the biological effects on human MCF7
breast cancer cells
after 24 h, without any dose-dependent significant fluctuations at
the highest doses. This trend is maintained also for the 48 h
Most active compounds 13a and 15c underwent an in vitro
4

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
Table 1
evolves later than 13a (Fig. 5A). More in details, cell metabolic ac-
tivity is assessed around 50% after 24 h, independently from the
In vitro human aromatase inhibition profile for 13a and 15c.
Cpd
Aromatase inhibition %a
81
IC50
0.82 0.02
(m
M)b,c
concentration tested. On the other hand,
decrease of the amount of metabolically active cells and a dra-
matic drop beyond 100 M (10.39%) is observed after 48 h of
treatment. Likewise, the downgrade of cell metabolic activity at
72 h is registered starting from compound 15c 50 M (25.18%).
a dose-dependent
m
m
After having investigated cell viability, the cytotoxic effect of
both compounds was measured by the lactate dehydrogenase ac-
tivity of MCF7 cells in response to loading concentrations of 13a and
15c (Fig. 6). The lactate released is around 4 folds with respect to
the one secreted from cultures exposed only to the DMSO (vehicle),
independently from the compound concentration. There is only a
slight increase (4.8 fold) in the LDH activity in the presence of both
72
0.86 0.05
compounds at 250 mM. These results let us to hypothesize that both
a
Inhibition percentage was calculated with respect to letrozole (1 mM), taken as
13a and 15c are cytotoxic for MCF7 cells, but they exert a different
outcome on cell metabolic activity being 15c apparently more
effective in decreasing the percentage of metabolically active cells.
The relationship between cellular metabolism and the cell cycle
machinery is well known and the ability of a cell to enter the cell
cycle critically depends on the availability of metabolites [25]. In
this respect, the cell cycle profile of MCF7 cells in the presence of
increasing concentrations of compounds 13a and 15c was after-
wards analyzed (Fig. 7).
Percentages of cells in the various phases of their cycle in a
routine culture (T0) are assessed at 71.88% for the G1 phase, at
16.99% for the S phase and at 11.12% for the G2/M phase, behaving
congruently as highly proliferating cells (Fig. 7B). After having
100%.
b
c
Compounds were tested at concentration from 0.01 to 100
The values represent mean of triplicate determinations standard deviation.
mM.
administration, except for 250
slight but significant increase of cell metabolic activity is registered
in the concentration range 10e50 M after 72 h of treatment
(50.16%, 55.06% and 46.70%, respectively), which could be ascribed
to MCF7 cell chemoresistance mechanisms [24]. By contrast, cell
response towards compound 15c is time and dose-dependent,
starting from 48 h of treatment. Indeed, this compound seems to
be more effective in reducing cell metabolic activity, but its action
mM 13a (30.75%). On the opposite, a
m
Fig. 5. A) Cell metabolic activity of MCF7 human breast cancer cells in the presence of increasing concentrations of compounds 13a and 15c. Data shown are the means standard
deviation of three replicates and are expressed as percentages of cultures in the presence of vehicle (DMSO) set as 100%. (***p < 0.0001 between compounds and DMSO; xp < 0.05
between the same doses of each compound; xxxp < 0.0001 between the same doses of each compound). B) Light microscopy images of MCF7 cells cultivated in 6 well plates
(2.5 ꢁ 105/well) in the presence of DMSO (control) and loading concentrations of compounds 13a and 15c for 72 h.
5

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
an increase of G1 phase and a decrease of S phase observed after 6
days of treatment [27]. More deepen molecular studies on 13a and
15c might eventually clarify the biological actions driving the
observed and promising antiproliferative effects.
2.4. Computational studies
To complement experimental studies, a number of computa-
tional investigations were carried out in order to predict the entire
pharmacological profile of 13a and 15c and to gain insights into the
causative molecular interactions behind the effective binding to the
active site of aromatase.
2.4.1. Target prediction analysis
By employing our recently developed MuSSel web server avail-
able upon a free license [28e30], 13a and 15c were screened in the
attempt to profile their spectrum of putative protein drug targets.
Although retrospective, this analysis has been carried out to better
support the quality of our experimental results and to enlarge the
potential of these candidate compounds for future drug reposition-
ing studies. Satisfactorily, MuSSel returned the aromatase (e.g. Cy-
tochrome P450 19A1, Homo Sapiens) for 13a and 15c as the top-three
with a score equal to 14.88% and, more interestingly, as the top-one
with a score equal to 32.42%, respectively. Provided with a wealth of
experimental information, nine and four high similar compounds
were found for 13a and 15c, this amount of additional data is now
being exploited for more focused structure-activity relationship an-
alyses for future lead optimization studies. The interested reader can
find a comprehensive view of these analyses enclosed as Supple-
mentary Information. In addition, the bioactivity pIC50 values of 13a
and 15c against aromatase were predicted equal to 6.23 ( 2.39) and
6.25 ( 2.51), respectively, by using our eMuSSel approach, the latest
update of our multi-searching similarity algorithm. Further details
are reported as Supplementary Information.
Fig. 6. Lactate dehydrogenase released (LDH) by MCF7 human breast cancer cells in
the presence of increasing concentrations of compounds 13a and 15c after 24 h. Data
shown are the means S.D. of three replicates and are expressed as the fold increase
on the LDH released by cultures in the presence of vehicle (DMSO) set as 1.
(**p < 0.002 between compounds and DMSO).
administered 13a for 48 h, a significant dose-dependent increase of
the S phase at the expense of the G2/M phase is registered in the
concentration range 10e50 mM. This event let us to hypothesize a
block in between the G1 and the S phase. Conversely, when cells are
exposed to loading concentrations of 15c, the S phase increases in
parallel to a G1 phase decrease and an enlargement of the G2/M
proportion, mainly at 100 and 250
blocked in the G2 phase.
mM, suggesting that cells are
These results are in agreement with MTT data. However, the
different behaviour displayed by 13a and 15c on cell cycle is not
surprising, as described in literature for other aromatase inhibitors
[26]. In addition, it is widely reported that the effects on cell cycle
exerted by classic aromatase inhibitors are time-dependent, with
Fig. 7. Cell cycle analysis of MCF7 human breast cancer cells in the presence of increasing concentrations of compounds 13a and 15c after 48 h. A) Histograms (number of cells vs
propidium iodide fluorescence/DNA content) represent the cell cycle profiles of MCF7 cells at T0 (routine culture), in the presence of vehicle (DMSO) and 10 M of compounds 13a
m
and 15c. B) Data shown are the means standard deviation. of three replicates and are expressed as cell population percentages in the various phases of the cell cycle (G1, S and G2/
M phases). (***p < 0.0001 between compounds and DMSO).
6

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
2.4.2. Molecular docking studies
induced a good enzymatic inhibition, with percentages of 81 and
72%, respectively, compared to reference drug letrozole, and IC50 of
0.82 and 0.86 mM.
Molecular docking studies were carried out on the X-ray com-
plex, including human placental aromatase cytochrome and an-
drostenedione, available from PDB with the code 3EQM. These
studies were conducted to predict the putative binding mode of the
most active compounds 13a and 15c and to shed light on their
They were then submitted to an in vitro evaluation on MCF7
breast cancer cells by MTT assay, cytotoxicity assay and cell cycle
analysis. A sensible reduction of cell viability and cytotoxicity was
assessed for our compounds, together with the arrest of cell cycle.
Molecular docking experiments returned for 13a and 15c novel
compounds a binding mode comparable to letrozole. On the other
hand, drug target prediction studies confirmed the role of 13a and
15c for aromatase activity inhibition and open new perspectives for
further studies.
experimentally measured IC50 inhibition values, equal to 0.82
and 0.86 M, respectively. As shown in Fig. 8A, the top scored
(ꢂ6.738 kcal/mol) docking pose of 13a was able to engage
mM
m
p-p
contacts with residues Phe148, Phe134 and the large tetrapyrrole
ring of the heme group. Interestingly, a distance of 2.33 Å occurred
between the nitrogen atom of the imidazole ring of 13a and the iron
ion of heme. Moreover, hydrophobic contacts with the backbone of
the following residues Arg115, Ile132, Ile133, Phe134, Leu152,
Phe221, Trp224, Glu302, Met303, Ala306, Thr310, Val370, Val373,
Met374, Leu477 were also detected.
4. Experimental section
4.1. Chemistry
As far as 15c is concerned, the top scored (ꢂ6.715 kcal/mol)
docking poses was able to engage
p-p contact mostly with the
Melting points were determined with a Buchi Melting Point B-
450 and were uncorrected. NMR spectra were recorded on a Varian
Mercury 300 spectrometer with 1H at 300.060 MHz and 13C at
75.475 MHz. Proton chemical shifts were referenced to the TMS
internal standard. Chemical shifts are reported in parts per million
residue Phe148. Likewise for 13a, an equal distance of 2.33 Å
occurred between the nitrogen atom of the imidazole ring of 15a
and the iron ion of heme. Hydrophobic contacts with the backbone
of Arg115, Ile132, Ile133, Phe134, Leu152, Phe221, Trp224, Phe278,
Glu302, Met303, Ile305, Ala306, Thr310, Val370, Leu372, Val373,
Met374, Leu477 and Ser478 were also detected. Interestingly, the
chlorine atom was able to engage a halogen bond with the back-
bone of Met374 (Fig. 8B).
Docking studies demonstrated that the binding modes and
docking score values of the 13a and 15c are very much comparable
thus reflecting their IC50 experimental values. Interestingly, the
imidazole ring of both 13a and 15c was preferably oriented to make
a coordination bond with the iron ion of the heme group as
observed in the case of azole-based aromatase inhibitors [19].
Moreover, the two phenyl rings are located in similar areas to
(ppm,
d units). Coupling constants are reported in units of Hertz
(Hz). Splitting patterns are designed as s, singlet; d, doublet; t,
triplet; q, quartet; dd, double doublet; m, multiplet; b, broad.
Elemental analyses were recorded on a PerkinElmer 240 B micro-
analyzer, obtaining results within 0.4% of the theoretical values.
The purity of all compounds was over 98%. All commercial chem-
icals and solvents were reagent grade and were purchased from
Sigma Aldrich; they are used without further purification, unless
otherwise specified. Reactions were monitored by thin layer chro-
matography on silica gel plates (60F-254, Sigma Aldrich) and the
analysis of the plates was carried out using a UV lamp 254/365 nm.
Flash chromatography was performed on silica gel 60 (Merk). The
following solvents and reagents have been abbreviated: chloroform
(CHCl3), dichloromethane (DCM), ethanol (EtOH), ethyl acetate
(EtOAc), methanol (MeOH), tetrahydrofuran (THF). All reactions
were carried out with the use of the standard techniques.
interface the side chains of residues Phe134 and Phe148 through
p-
p
contacts. In addition, 13a and 15c share several hydrophobic
contacts with residues Arg115, Ile132, Ile133, Phe134, Leu152,
Phe221, Trp224, Glu302, Ile305, Ala306, Thr310, Val370, Val373,
Met374 and Leu477. Noteworthy, these hydrophobic contacts along
with the coordination bond with the iron ion of heme group were
experienced also by letrozole [31], a well-known inhibitor of
aromatase.
4.1.1. General procedure for the synthesis of alcohols 1,10
Mixtures of triazole (1 mmol) and 1,2-epoxy-3-phenoxypropane
(for 1) or styrene oxide (for 10) (1 mmol) in THF (4 ml) were heated
at 60 ꢀC and stirred for 24 h. After this time, the solvent was
evaporated at reduced pressure, and obtained crude mixtures were
purified by column on silica gel (chloroform/methanol) or crystal-
lization to obtain desired products.
3. Conclusion
In conclusion, novel imidazole and triazole-based compounds
were synthesized and tested against human aromatase in an in vitro
fluorescence enzymatic assay. Imidazole derivatives 13a and 15c
The same reaction scheme was applied to obtain triazole
Fig. 8. Zoomed in view of the binding site of aromatase. 13a (Panel A) and 15c (Panel B) are rendered in green stick representation. The most relevant residues are rendered as white
sticks. The blue arrows indicate contacts. The orange arrow indicates the coordination bond to the iron ion. The green arrow indicates the halogen bonds. The iron ion is
p-p
represented as an orange sphere and the tetrapyrrole ring of heme as a dark grey stick model. (For interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
7

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
alcohols 5,8 and imidazole alcohols 12,14.
CHAr); 13C NMR (CDCl3)
d 44.7, 50.2, 55.6, 69.4, 114.6, 115.6, 152.6,
154.1.
4.1.1.1. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
1.
Colorless needles, 59% yield; m.p. 90e91 ꢀC; 1H NMR (CDCl3)
4.1.3. General procedure for the synthesis of carbamates 2, 6, 9, 11,
13, 15
d
3.92e4.03 (m, 2H, NCH2), 4.12 (s, 1H, OH), 4.36e4.53 (m, 3H,
CHOH and CH2O), 6.88 (d, 2H, J ¼ 8.7 Hz, CHAr), 6.98 (t, 1H,
Sodium hydride (1 mmol) was added to a solution of alcohols
(1 mmol) in dry acetonitrile (8 ml) at room temperature, under
nitrogen atmosphere, and the reaction was stirred for 2 h. Then the
proper isocyanate (1.5 mmol) was added to the mixture, and the
reaction was allowed to stir for 24 h. After this time, the solvent was
removed under reduced pressure, and crude materials were puri-
fied by column chromatography (eluent cyclohexane/ethyl acetate
or chloroform/methanol) or crystallization.
J ¼ 7.8 Hz, CHAr), 7.29 (t, 2H, J ¼ 8.7 Hz, CHAr), 7.94 (s, 1H, CHAr), 8.28
(s, 1H, CHAr); 13C NMR (CDCl3)
d 52.5, 68.4, 68.5, 114.4, 121.5, 129.6,
144.0, 151.2, 158.0.
4.1.1.2. 1-Phenyl-2-(1H-1,2,4-triazol-1-yl)ethanol 10. White solid,
54% yield; m.p. 103e105 ꢀC; 1H NMR (CDCl3)
4.22e4.38 (m, 3H,
d
NCH2 and OH), 5.08 (m, 1H, CHOH), 7.31e7.37 (m, 5H, CHAr), 7.84 (s,
1H, CHAr), 7.99 (s, 1H, CHAr); 13C NMR (CDCl3)
d 56.8, 72.2, 125.8,
127.2, 128.4, 128.7, 143.9, 151.7.
4.1.3.1. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl)propan-2-yl benzylcar-
bamate 2a. White needles, 47% yield; m.p. 83e84 ꢀC; 1H NMR
4.1.1.3. 1-(4-Chlorophenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-ole
5. White solid, 52% yield; m.p. 94e96 ꢀC; 1H NMR (CDCl3)
3.19 (bs,
(CDCl3)
d
4.09 (dq, 2H, J ¼ 10.5 Hz, J ¼ 5.1 Hz, NCH2), 4.33 (d, 2H,
d
J ¼ 6.0 Hz, CH2NH), 4.59 (d, 2H, J ¼ 5.7 Hz, CH2O), 5.21 (bs, 1H, NH),
5.41e5.49 (m, 1H, CH), 6.90 (d, 2H, J ¼ 7.5 Hz, CHAr), 6.98 (t, 1H,
J ¼ 7.2 Hz, CHAr), 7.22e7.35 (m, 7H, CHAr), 7.94 (s, 1H, CHAr), 8.10 (s,
1H, OH), 3.88e4.00 (m, 2H, CH2N), 4.30e4.49 (m, 3H, CHO and
CH2O), 6.81 (d, 2H, J ¼ 9.3 Hz, CHAr), 7.23 (d, 2H, J ¼ 9.3 Hz, CHAr),
7.90 (s, 1H, CHAr), 8.12 (s, 1H, CHAr); 13C NMR (CDCl3)
d
52.2, 68.4,
1H, CHAr); 13C NMR (CDCl3)
d 45.0, 49.6, 66.1, 70.3, 114.5, 121.5,
69.0, 115.7, 126.4, 129.5, 144.1, 151.9, 156.6.
127.5, 127.6, 128.7, 129.6, 137.9, 144.2, 151.8, 155.1, 157.9. Calcd for
19H20N4O3: C, 64.76; H, 5.72; N, 15.90. Found: C, 64.61; H, 5.71; N,
C
4.1.1.4. 1-(4-Methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
15.87.
ole 8. White solid, 55% yield; m.p. 106e108 ꢀC; 1H NMR (CDCl3)
d
3.75 (s, 3H, OCH3), 3.88e3.99 (m, 2H, CH2N), 4.33e4.49 (m, 3H,
4.1.3.2. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl)propan-2-yl
phenyl-
CHO and CH2O), 6.82 (s, 4H, CHAr), 7.93 (s, 1H, CHAr), 8.13 (s, 1H,
carbamate 2b. White solid, 51% yield; m.p. 154e156 ꢀC; 1H NMR
CHAr); 13C NMR (CDCl3)
d
52.1, 55.7, 68.6, 69.3, 114.7, 115.4, 144.1,
(acetone-d6)
d
4.23 (dq, 2H, J ¼ 10.5 Hz, J ¼ 4.5 Hz, CH2N), 4.68e4.80
151.9, 152.1, 154.3.
(m, 2H, CH2O), 5.51e5.55 (m, 1H, CH), 6.93e7.05 (m, 3H, CHAr),
7.24e7.33 (m, 5H, CHAr), 7.52 (d, 2H, J ¼ 7.5 Hz, CHAr), 7.91 (s, 1H,
CHAr), 8.42 (s, 1H, CHAr), 8.92 (bs, 1H, NH); 13C NMR (acetone-d6)
4.1.1.5. 2-(1H-imidazole-1-yl)-1-phenylethanol 12. White needles,
58% yield; m.p. 145e147 ꢀC; 1H NMR (CD3OD)
4.11e4.25 (dq, 2H,
d
d 49.2, 66.6, 70.5, 114.5, 118.4, 121.1, 121.9, 122.8, 128.6, 128.7, 129.5,
J ¼ 14.1 Hz, J ¼ 7.2 Hz, NCH2), 4.88e4.93 (m, 2H, CHOH and OH), 6.88
144.7, 151.6, 158.5. Calcd for C18H18N4O3: C, 63.89; H, 5.36; N, 16.56.
Found: C, 63.80; H, 5.37; N, 16.59.
(s, 1H, CHAr), 7.05 (s, 1H, CHAr), 7.29e7.32 (m, 5H, CHAr), 7.48 (s, 1H,
CHAr); 13C NMR (CD3OD)
d 53.8, 72.8, 120.0, 125.6, 126.9, 127.4,
128.0, 137.6, 141.5.
4.1.3.3. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl)propan-2-yl(4-
chlorophenyl)carbamate 2c. White needles, 61% yield; m.p.
4.1.1.6. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-ol
solid, 75% yield; m.p. 109e110 ꢀC; 1H NMR (CDCl3)
14. White
3.85e4.03 (dq,
128e130 ꢀC; 1H NMR (CDCl3)
CH2N), 4.63 (d, 2H, J ¼ 5.4 Hz, CH2O), 5.45e5.51 (m, 1H, CH), 6.91 (d,
2H, J ¼ 7.8 Hz, CHAr), 6.99 (t, 1H, J ¼ 7.5 Hz, CHAr), 7.23e7.32 (m, 6H,
CHAr), 7.41 (bs,1H, NH), 7.97 (s,1H, CHAr), 8.12 (s,1H, CHAr); 13C NMR
d
4.14 (dq, 2H, J ¼ 10.5 Hz, J ¼ 4.5 Hz,
d
2H, J ¼ 9.3 Hz, J ¼ 4.8 Hz, NCH2), 4.10e4.14 (m, 2H, CHOH and OH),
4.23e4.29 (m, 2H, CH2O), 6.88e7.01 (m, 5H, CHAr), 7.25e7.32 (m,
2H, CHAr), 7.55 (s, 1H, CHAr); 13C NMR (CDCl3)
d
50.5, 68.5, 68.8,
(CDCl3) d 49.7, 66.0, 70.4, 114.5, 119.9, 121.5, 121.7, 129.0, 129.7, 136.1,
114.4, 120.0, 121.4, 127.9, 129.6, 137.5, 158.1.
144.2, 151.4, 157.8, 158.0. Calcd for C18H17ClN4O3: C, 57.99; H, 4.60;
N, 15.03. Found: C, 57.78; H, 4.60; N, 15.02.
4.1.2. General procedure for the synthesis of oxiranes 4,7
Epibromohydrin (2 mmol), p-methoxyphenol or p-chlorophenol
(2 mmol) and potassium carbonate (3 mmol) were dissolved in
acetonitrile (10 ml) and stirred at reflux for 7 h. The mixtures were
cooled and diluted with ethyl acetate and washed with water.
Crude products were then purified by chromatography on silica gel,
eluent chloroform.
4.1.3.4. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl)propan-2-yl (4-
methoxyphenyl)carbamate 2d. White solid, 55% yield; m.p.
103e105 ꢀC; 1H NMR (CDCl3)
d 3.77 (s, 3H, OCH3), 4.14 (dq, 2H,
J ¼ 10.5 Hz, J ¼ 4.5 Hz, NCH2), 4.63 (d, 2H, J ¼ 5.4 Hz, CH2O), 5.46 (q,
1H, J ¼ 5.4 Hz, CH), 6.82e7.01 (m, 6H, CHAr), 7.22e7.32 (m, 4H,
CHAr), 7.96 (s, 1H, CHAr), 8.12 (bs, 1H, NH); 13C NMR (CDCl3)
d 47.4,
55.4, 66.1, 70.6, 110.0, 114.3, 114.5, 120.8, 121.6, 129.6, 144.1, 152.2.
Calcd for C19H20N4O4: C, 61.95; H, 5.47; N, 15.21. Found: C, 61.89; H,
5.46; N, 15.23.
4.1.2.1. 2-[(4-Chlorophenoxy)methyl]oxirane 4. Colorless oil, 59%
yield; 1H NMR (CDCl3)
d
2.75 (dd, 1H, J 4.8 Hz, J ¼ 2.4 Hz, OCHH),
2.90 (dd, 1H, J ¼ 5.4 Hz, J ¼ 4.2 Hz, OCHH), 3.31e3.36 (m, 1H, CHO),
3.89 (dd, 1H, J ¼ 11.1 Hz, J ¼ 6.0 Hz, CHHO), 4.21 (dd, 1H, J ¼ 11.1 Hz,
J ¼ 2.7 Hz, CHHO), 6.84 (d, 2H, J ¼ 8.7 Hz, CHAr), 7.22 (d, 2H,
4.1.3.5. 1-Phenoxy-3-(1H-1,2,4-triazol-1-yl) propan-2-yl (2,4-
dimethoxyphenyl) carbamate 2e. Pale pink oil, 80% yield; 1H NMR
J ¼ 8.7 Hz, CHAr); 13C NMR (CDCl3)
d
44.6, 50.0, 69.0, 115.9, 126.1,
(CDCl3)
d
3.77 and 3.81 (both s, 3H, OCH3), 4.13 (dq, 2H, J ¼ 10.5 Hz,
129.3, 157.0.
J ¼ 4.8 Hz, NCH2), 4.64 (d, 2H, J ¼ 5.1 Hz, CH2O), 5.48 (q, 1H, J 5.1 Hz,
CH), 6.43e6.47 (m, 2H, CHAr), 6.90e7.00 (m, 3H, CHAr), 7.08 (s, 1H,
CHAr), 7.27e7.31 (m, 2H, CHAr), 7.85 (bd, 1H, NH), 7.96 (s, 1H, CHAr),
4.1.2.2. 2-[(4-Methoxyphenyl)methyl]oxirane 7. Colorless solid, 64%
yield; m.p. 49e51 ꢀC; 1H NMR (CDCl3)
d
2.73 (dd, 1H, J ¼ 5.4 Hz,
8.14 (s, 1H, CHAr); 13C NMR (CDCl3)
d
49.5, 55.5, 55.6, 66.2, 70.5, 98.7,
103.8, 114.5, 119.2, 121.6, 129.6, 144.2, 152.2, 157.9. Calcd for
20H22N4O5: C, 60.29; H, 5.57; N, 14.06. Found: C, 60.18; H, 5.55; N,
14.02.
J ¼ 3.0 Hz, OCHH), 2.88 (t, 1H, J ¼ 3.9 Hz, OCHH), 3.31e3.35 (m, 1H,
CHO), 3.76 (s, 3H, OCH3), 3.90 (dd, 1H, J ¼ 11.1 Hz, J ¼ 6.0 Hz, CHHO),
4.16 (dd, 1H, J ¼ 11.4 Hz, J ¼ 3.0 Hz, CHHO), 6.84 (d, 4H, J ¼ 4.8 Hz,
C
8

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
4.1.3.6. 1-(4-Chlorophenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
74.0, 114.7, 121.0, 126.5, 126.9, 127.7, 127.9, 128.5, 128.7, 128.9, 144.1,
152.1. Calcd for C18H18N4O2: C, 67.07; H, 5.63; N, 17.38. Found: C,
66.89; H, 5.62; N, 17.33.
ylphenylcarbamate 6a. White solid, 44% yield; m.p. 143e145 ꢀC; 1H
NMR (CDCl3)
d
4.11 (dq, 2H, J ¼ 10.8 Hz, J ¼ 4.5 Hz, CH2N), 4.62 (d,
2H, J ¼ 5.4 Hz,CH2O), 5.47 (q, 1H, J ¼ 5.4 Hz, CHO), 6.84 (d, 2H,
J ¼ 9.3 Hz, CHAr), 7.08 (t, 1H, J ¼ 6.9 Hz, CHAr), 7.19 (bs, 1H, NH),
7.23e7.36 (m, 6H, CHAr), 7.97 (s, 1H, CHAr), 8.12 (s, 1H, CHAr); 13C
4.1.3.13. 1-Phenyl-2-(1H-1,2,4-triazol-1-yl)ethyl
11b. Pale yellow oil, 68% yield; 1H NMR (CDCl3)
J ¼ 6.0 Hz, NCH2), 6.13 (t,1H, J ¼ 6.0 Hz, CH), 7.04e7.37 (m, 11H, CHAr
and NH), 7.94 (s, 2H, CHAr); 13C NMR (CDCl3)
54.1, 74.3,118.6,123.7,
126.1, 127.1, 129.0, 129.1, 129.2, 136.4, 137.3, 143.8, 151.9. Calcd for
17H16N4O2: C, 66.22; H, 5.23; N, 18.17. Found: C, 66.02; H, 5.22; N,
phenylcarbamate
d
4.56 (d, 2H,
NMR (CDCl3) d 49.4, 66.5, 70.4, 115.8, 118.7, 124.0,126.6, 129.1, 129.5,
144.2, 152.2, 156.5. Calcd for C18H17ClN4O3: C, 57.99; H, 4.60; N,
15.03. Found: C, 57.90; H, 4.59; N, 14.98.
d
C
4.1.3.7. 1-(4-Chlorophenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
18.15.
yl(4-chlorophenyl)carbamate 6b. Pale yellow solid, 39% yield; m.p.
128e129 ꢀC; 1H NMR (CDCl3)
d
4.12 (dq, 2H, J ¼ 10.8 Hz, J ¼ 4.5 Hz,
4 .1. 3 .14. 1- P h e nyl -2-( 1H- 1, 2 , 4-t r i az ol - 1-yl )e t hyl 4-
CH2N), 4.62 (d, 2H, J ¼ 5.4 Hz,CH2O), 5.47 (q, 1H, J ¼ 5.4 Hz, CHO),
chlorophenylcarbamate 11c. Colorless oil, 47% yield 1H NMR
6.83 (d, 2H, J ¼ 8.7 Hz, CHAr), 7.23e7.29 (m, 6H, NH and CHAr), 7.98
(CDCl3)
7.11e7.38 (m, 9H, CHAr and NH), 8.04 (s, 1H, CHAr), 8.23 (s, 1H, CHAr);
13C NMR (CDCl3)
56.0, 70.3, 114.5, 119.9, 121.7, 125.1, 125.6, 125.8,
d
4.83 (dq, 2H, J ¼ 6.0 Hz, NCH2), 5.74 (t, 1H, J ¼ 6.0 Hz, CH),
(s, 1H, CHAr), 8.11 (s, 1H, CHAr); 13C NMR (CDCl3)
d 49.3, 66.4, 70.6,
115.7, 119.9, 126.7, 129.1, 129.6, 135.7, 144.1, 151.9, 152.3, 156.4. Calcd
for C18H16Cl2N4O3: C, 53.09; H, 3.96; N, 13.76. Found: C, 52.98; H,
3.95; N, 13.72.
d
129.1, 129.7, 137.5, 152.1, 157.8. Calcd for C17H15ClN4O2: C, 59.57; H,
4.41; N, 16.34. Found: C, 59.48; H, 4.41; N, 16.31.
4.1.3.8. 1-(4-Chlorophenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
4.1.3.15. 2-(1H-imidazole-1-yl)-1-phenylethyl benzylcarbamate 13a.
Pale orange solid, 93% yield; m.p. 124e127 ꢀC; 1H NMR (CDCl3)
yl(4-methoxyphenyl)carbamate 6c. White solid, 50% yield; m.p.
148e149 ꢀC; 1H NMR (CDCl3)
d
3.77 (s, 3H, OCH3), 4.11 (dq, 2H,
d
4.32 (dq, 4H, J ¼ 10.5 Hz, J ¼ 4.5 Hz, NCH2), 5.44 (bs, 1H, NH), 5.92
(t, 1H, J ¼ 5.4 Hz, CH), 6.74 (s, 1H, CHAr), 6.96 (s, 1H, CHAr), 7.18e7.34
(m, 11H, CHAr); 13C NMR (CDCl3)
45.1, 51.8, 74.9, 119.6, 126.1, 126.9,
127.1, 127.5, 127.6, 128.5, 128.7, 128.8, 129.1, 137.8, 152.2. Calcd for
19H19N3O2: C, 71.01; H, 5.96; N, 13.08. Found: C, 70.91; H, 5.95; N,
J ¼ 10.8 Hz, J ¼ 4.5 Hz, CH2N), 4.62 (d, 2H, J ¼ 5.4 Hz,CH2O), 5.44 (q,
1H, J ¼ 5.4 Hz, CHO), 6.83 (d, 4H, J ¼ 8.7 Hz, CHAr), 6.95 (bs, 1H, NH),
d
7.24 (d, 4H, J ¼ 8.7 Hz, CHAr), 7.97 (s, 1H, CHAr), 8.13 (s, 1H, CHAr); 13
C
NMR (CDCl3)
d
49.4, 55.4, 66.5, 70.4, 114.3, 115.8, 120.8, 126.5, 129.5,
C
144.2, 152.2, 156.5. Calcd for C19H19ClN4O4: C, 56.65; H, 4.75; N,
13.91. Found: C, 56.58; H, 4.73; N, 13.87.
13.04.
4.1.3.16. 2-(1H-imidazole-1-yl)-1-phenylethyl phenylcarbamate 13b.
4.1.3.9. 1-(4-Methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
White solid, 48% yield; m.p.194e196 ꢀC; 1H NMR (CDCl3)
d 4.36 (dq,
ylphenylcarbamate 9a. White solid, 53% yield; m.p. 118e119 ꢀC; 1H
2H, J ¼ 10.5 Hz, J ¼ 4.5 Hz, NCH2), 5.98 (m,1H, CH), 6.76 (s,1H, CHAr),
7.00 (s, 2H, CHAr), 7.19e7.40 (m, 7H, CHAr and NH), 7.51 (d, 2H,
J ¼ 8.1 Hz, CHAr), 8.42 (s, 1H, CHAr), 8.87 (s, 1H, CHAr); 13C NMR
NMR (CDCl3)
d
3.76 (s, 3H, OCH3), 4.09 (dq, 2H, J ¼ 10.5 Hz,
J ¼ 4.5 Hz, CH2N), 4.64 (d, 2H, J ¼ 5.1 Hz,CH2O), 5.46 (q, 1H,
J ¼ 4.8 Hz, CHO), 6.85 (s, 4H, CHAr), 6.95 (bs, 1H, NH), 7.10 (t, 1H,
J ¼ 6.9 Hz, CHAr), 7.27e7.36 (m, 4H, CHAr), 7.97 (s, 1H, CHAr), 8.15 (s,
(CDCl3)
d 52.6, 74.2, 118.8, 120.2, 123.3, 123.4, 125.9, 128.7, 128.9,
136.5, 138.0, 138.5, 152.2. Calcd for C18H17N3O2: C, 70.34; H, 5.58; N,
13.67. Found: C, 70.26; H, 5.57; N, 13.60.
1H, CHAr); 13C NMR (CDCl3)
d 49.4, 55.7, 66.9, 70.7,114.7,115.5,129.1,
137.1, 144.2, 151.7, 152.2, 154.4. Calcd for C19H20N4O4: C, 61.95; H,
5.47; N, 15.21. Found: C, 61.90; H, 5.46; N, 15.10.
4 .1. 3 .17 . 2 - ( 1 H - i m i d a z o l e - 1 - y l ) - 1 - p h e n y l e t h y l 4 -
chlorophenylcarbamate 13c. Pale yellow solid, 52% yield; m.p.
4.1.3.10. 1-(4-Methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
192e194 ꢀC; 1H NMR (CD3OD)
d
4.44 (d, 2H, J ¼ 5.7 Hz, NCH2), 5.98
(t, 1H, J ¼ 6.0 Hz, CH), 6.91 (s, 1H, CH Ar), 7.11 (bs, 1H, NH), 7.21e7.40
(m, 10H, CHAr), 7.54 (s, 1H, CHAr); 13C NMR (CD3OD)
51.2, 75.0,
yl(4-chlorophenyl) carbamate 9b. Pale yellow solid, 47% yield; m.p.
117e119 ꢀC; 1H NMR (CDCl3)
d
3.78 (s, 3H, OCH3), 4.08 (dq, 2H,
d
J ¼ 10.5 Hz, J ¼ 4.5 Hz, CH2N), 4.63 (d, 2H, J ¼ 5.1 Hz,CH2O), 5.46 (q,
119.6,120.1,125.6,125.8,127.3,128.2,128.3,128.4,137.3,137.7,152.4.
Calcd for C18H16ClN3O2: C, 63.25; H, 4.72; N, 12.29. Found: C, 63.19;
H, 4.71; N, 12.27.
1H, J ¼ 4.8 Hz, CHO), 6.85 (s, 5H, CHAr), 7.23e7.32 (m, 5H, NH and
CHAr), 7.99 (s, 1H, CHAr), 8.14 (s, 1H, CHAr); 13C NMR (CDCl3)
d 49.4,
55.7, 66.8, 70.8, 104.2, 114.7, 115.5, 119.8, 129.1, 134.7, 135.9, 152.0,
154.4. Calcd for C19H19ClN4O4: C, 56.65; H, 4.75; N, 13.91. Found: C,
56.53; H, 4.74; N, 13.87.
4.1.3.18. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-yl benzylcarba-
mate 15a. Yellow oil, 51% yield; 1H NMR (CDCl3)
d 3.79 and 4.08
(dd, 2H, J ¼ 7.5 Hz, NCH2), 4.33 (d, 2H, J ¼ 6.0 Hz, CH2NH), 4.39 (d,
2H, J ¼ 4.5 Hz, CH2O), 5.25e5.29 (m, 1H, CH), 6.22 (bt, 1H, J ¼ 5.7 Hz,
NH), 6.87e7.06 (m, 5H, CHAr), 7.24e7.32 (m, 7H, CHAr), 8.17 (s, 1H,
4.1.3.11. 1-(4-Methoxyphenoxy)-3-(1H-1,2,4-triazol-1-yl)propan-2-
yl(4-methoxyphenyl)carbamate 9c. White solid, 53% yield; m.p.
99e100 ꢀC; 1H NMR (CDCl3)
d
3.76 and 3.77 (both s, 3H, OCH3), 4.08
CHAr); 13C NMR (CDCl3)
d
45.0, 46.9, 65.0, 70.4, 114.4, 120.1, 121.6,
127.5, 127.6, 128.5, 128.7, 129.6, 138.1, 138.2, 155.2, 157.7. Calcd for
20H21N3O3: C, 68.36; H, 6.02; N, 11.96. Found: C, 68.20; H, 6.00; N,
(dq, 2H, J ¼ 10.5 Hz, J ¼ 4.5 Hz, CH2N), 4.62 (d, 2H, J ¼ 5.1 Hz,CH2O),
5.44 (q, 1H, J ¼ 4.8 Hz, CHO), 6.81e6.94 (m, 7H, CHAr), 7.23e7.28 (m,
2H, NH and CHAr), 7.97 (s, 1H, CHAr), 8.13 (s, 1H, CHAr); 13C NMR
C
11.92.
(CDCl3)
d 49.4, 55.4, 55.7, 66.9, 70.6, 114.2, 114.7, 115.5, 120.7, 130.2,
144.2, 152.0, 152.2, 154.4. Calcd for C20H22N4O5: C, 60.29; H, 5.57; N,
14.06. Found: C, 60.09; H, 5.56; N, 14.00.
4.1.3.19. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-yl
phenyl-
carbamate 15b. White needles, 66% yield; m.p. 125e127 ꢀC; 1H
NMR (CDCl3)
d
3.79 and 4.16 (dd, 2H, J ¼ 7.5 Hz, NCH2), 4.45 (d, 2H,
4.1.3.12. 1-Phenyl -2-(1H-1,2,4-triazol-1-yl)ethyl benzylcarbamate
J ¼ 4.8 Hz, CH2O), 5.33e5.38 (m, 1H, CH), 6.90e7.07 (m, 6H, CHAr),
7.22e7.33 (m, 4H, CHAr), 7.52 (d, 2H, J ¼ 7.5 Hz, CHAr), 8.31 (s, 1H,
11a. Colourless oil, 44% yield; 1H NMR (CDCl3)
d 4.27 (d, 2H,
J ¼ 5.1 Hz, NCH2), 4.48 (d, 2H, J ¼ 5.7 Hz, CH2NH), 5.41 (bt, 1H,
CHAr), 8.81 (bs, 1H, NH); 13C NMR (CDCl3)
d 47.4, 64.6, 69.9, 114.4,
J ¼ 5.7 Hz, NH), 6.06 (t, 1H, J ¼ 6.0 Hz, CH), 7.17e7.35 (m, 10H, CHAr),
118.9,120.5,121.7, 123.5, 126.9, 128.9,129.7, 138.7, 152.4, 157.6. Calcd
for C19H19N3O3: C, 67.64; H, 5.68; N, 12.46. Found: C, 67.51; H, 5.67;
7.87 (s, 1H, CHAr), 8.02 (s, 1H, CHAr); 13C NMR (CDCl3)
d 45.0, 54.3,
9

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
N, 12.43.
were incubated for additional 20 min at 37 ꢀC and 5% CO2 and af-
terwards gently swirled for 10 min at room temperature. The op-
4.1.3.20. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-yl(4-
tical density was measured at 540 nm by means of a
chlorophenyl)carbamate 15c. White solid, 75% yield; m.p.
spectrophotometer (Multiskan GO, Thermo Scientific, Monza,
Italy). Results were expressed as the percentage of cells in the
presence of vehicle (DMSO) set as 100% and each experiment was
performed in triplicate (n ¼ 3).
120e122 ꢀC; 1H NMR (CDCl3)
d
3.80 and 4.16 (dd, 2H, J ¼ 7.5 Hz,
NCH2), 4.43 (d, 2H, J ¼ 4.8 Hz, CH2O), 5.31e5.37 (m, 1H, CH),
6.90e7.06 (m, 5H, CHAr), 7.19e7.33 (m, 4H, CHAr), 7.45 (d, 2H,
J ¼ 9.0 Hz, CHAr), 8.00 (s, 1H, CHAr), 9.50 (bs, 1H, NH); 13C NMR
(CDCl3)
d
47.3, 64.6, 70.0, 114.4, 120.1, 120.5,121.7, 127.4, 128.9, 129.7,
4.2.2.3. Cytotoxicity assay. MCF7 cells were seeded and stimulated
as previously described for the MTT test. After the exposure time,
cell supernatants were collected, centrifugated at 450ꢁg for 4 min
and stored on ice. In order to quantify the cytotoxicity of loading
136.8, 138.4, 152.5, 157.6. Calcd for C19H18ClN3O3: C, 61.38; H, 4.88;
N, 11.30. Found: C, 61.27; H, 4.87; N, 11.26.
4.1.3.21. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-yl (4-
concentrations of compounds (0e250 mM), the CytoTox 96® Non-
methoxyphenyl)carbamate 15d. White solid, 75% yield; m.p.
Radioactive Cytotoxicity Assay (Promega Corporation, WI, USA)
was performed as previously described [21]. Optical densities ob-
tained were normalized on the ones obtained from the MTT assay
and results were expressed as the fold increase on the LDH released
from cultures in the presence of DMSO only (vehicle). Each exper-
iment was performed in triplicate (n ¼ 3).
118e120 ꢀC; 1H NMR (CDCl3)
d 3.77 (s, 3H, OCH3), 3.90 and 4.08
(both dd, 2H, J ¼ 10.2 Hz, J ¼ 4.2 Hz, NCH2), 4.39 (d, 2H, J ¼ 5.1 Hz,
CH2O), 5.32 (m, 1H, CH), 6.83e7.04 (m, 7H, CHAr), 7.27e7.33 (m, 4H,
CHAr), 7.51 (s, 1H, CHAr), 7.80 (bs, 1H, NH); 13C NMR (CDCl3)
d 46.6,
55.4, 65.1, 70.5, 114.2, 114.5, 119.9, 120.9, 121.7, 129.3, 129.7, 138.1,
157.8. Calcd for C20H21N3O4: C, 65.38; H, 5.76; N, 11.44. Found: C,
65.22; H, 5.74; N, 11.40.
4.2.2.4. Cell cycle analysis. In order to measure the proliferation
rate of MCF7 cells exposed to loading concentrations of compounds
13a and 15c, cell cycle progression was assessed by flow cytometry.
Cells were seeded (2.5 ꢁ 105/well) in a 6-well tissue culture-treated
plate (Falcon®, Corning Incorporated, NY, USA) and let them adhere
for 24 h. Then, growth medium was removed and replaced with
4.1.3.22. 1-(1H-imidazole-1-yl)-3-phenoxypropan-2-yl (2,4-
dimethoxyphenyl)carbamate 15e. Pale pink oil, 86% yield; 1H NMR
(CDCl3)
d 3.78 and 3.84 (both s, 3H, OCH3), 3.92 and 4.04 (both dd,
2H, J ¼ 9.9 Hz, J ¼ 3.9 Hz, NCH2), 4.41 (d, 2H, J ¼ 5.4 Hz, CH2O), 5.33
(q, 1H, J ¼ 4.2 Hz, CH), 6.45e6.49 (m, 2H, CHAr), 6.90e7.08 (m, 6H,
CHAr), 7.27e7.33 (m, 2H, CHAr), 7.54 (s, 1H, CHAr), 7.88 (bs, 1H, NH);
fresh medium mixed with compounds (0e250 mM) for 48 h. After
the exposure time, cells were washed twice with PBS without
calcium and magnesium, harvested with Trypsin EDTA 1X (all
purchased by EuroClone S.p.a., Milan, Italy) and counted by means
of Trypan blue exclusion test (Sigma Aldrich, Milan, Italy).
Approximately 5 ꢁ 105 cells/experimental condition were fixed
with cold ethanol 70% v/v and kept overnight at 4 ꢀC. Cells were
then resuspended in the staining solution made by PBS without
calcium and magnesium, 1 mg/ml Propidium Iodide (final con-
13C NMR (CDCl3)
d 46.5, 55.5, 55.6, 65.1, 70.8, 98.7, 103.8, 114.4,
119.9, 121.7, 129.4, 129.7, 138.0, 157.9. Calcd for C21H23N3O5: C,
63.46; H, 5.83; N, 10.57. Found: C, 63.29; H, 5.82; N, 10.55.
4.2. Biological assays
4.2.1. Aromatase activity inhibition assay and IC50 calculation
The aromatase inhibitory activity of the novel compounds was
determined using a commercial fluorimetric assay (Aromatase
CYP19A Inhibitor Screening kit, BioVision, Milpitas, CA, USA). The
assay utilizes a fluorogenic aromatase substrate, that is converted
into a highly fluorescent metabolite detected in the visible range
(Ex/Em ¼ 488/527 nm), ensuring a high signal-to-background ratio
with little interference by autofluorescence.
centration 10
mg/ml) and 10 mg/ml RNAse (final concentration
4
100
m
g/ml) and kept overnight at 4 ꢀC. Cell cycle profiles (1 ꢁ 10
events/sample) were finally analyzed with a CytoFLEX Flow Cy-
tometer (Beckman Coulter, FL, USA) and data were quantified using
the ModFit LT™ software (Verity Software House, Topsham, ME,
USA).
The inhibition assay and the IC50 calculation were performed as
reported in a previous work [21]. Concentration-response curves
were fitted with GraphPad Prism 5.0 [32].
4.2.2.5. Statistics
Data are presented as mean values standard deviations (S.D.)
of three independent experiments. Statistical differences were
determined by one-way ANOVA and post hoc Tukey multiple
comparison tests using the Prism software (version 5.0, GraphPad
Software, San Diego, CA, USA). P values < 0.05 were considered
statistically significant.
4.2.2. Cell viability evaluation
4.2.2.1. Cell culture. Human breast cancer MCF7 (HTB-22 ™) cells
were purchased from ATCC and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) high glucose (EuroClone, Milan, Italy)
supplemented with 10% of fetal bovine serum (FBS) and 1% of
penicillin/streptomycin (Gibco - Thermofisher Scientific, MD, USA).
4.3. Molecular docking
The crystal structure was fetched from the Protein Data Bank
[33] and the protein preparation wizard available from the
4.2.2.2. MTT assay. Cell metabolic activity of MCF7 human breast
cancer cells was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-Diphenyltetrazolium Bromide) test (Sigma Aldrich, Milan,
Italy). Cells were seeded (0.1 ꢁ 105/well) in a 96-well tissue culture-
treated plate (Falcon®, Corning Incorporated, NY, USA) and let them
adhere for 24 h. Next, medium was removed and the cell monolayer
was incubated in the presence of loading concentrations of com-
€
Schrodinger suite [34] was used for the optimization and the
elimination of the X-ray crystal ambiguities. Next, the ligand
structures to be docked were prepared and optimize using the
LigPrep tool [35] allowing the generation of the putative ionization
states at physiological pH as well as the enumeration of the possible
tautomers.
pounds 13a and 15c (0e250
established exposure times, cells were incubated with 100
m
M) for 24, 48 and 72 h. After the
l/well
As a first step, a standard energetic grid was centered on the
heme group of 3EQM X-ray solved structure by using default set-
tings. A metal constraint was employed to better sample those
ligand poses in which the nitrogen atom of the imidazole ring was
able to coordinate the iron ion of the heme group as indicated in a
m
of MTT (5 mg/ml) 1:10 with fresh growth medium (final concen-
tration 0.5 mg/ml) for 4 h at 37 ꢀC and 5% CO2. Finally, the MTT
solution was removed and replaced with 100 ml/well of DMSO. Cells
10

A. Ammazzalorso, M. Gallorini, M. Fantacuzzi et al.
European Journal of Medicinal Chemistry 211 (2021) 113115
inhibitors of aromatase from the structure-function perspective, J. Med. Chem.
59 (2016) 5131e5148.
recent work [19]. Glide Standard Precision (SP) was used for
docking studies by setting 50000 poses per ligand for the initial
phase and 4000 poses per ligand for energy minimization.
[15] B. Thurlimann, A. Keshaviah, A.S. Coates, H. Mouridsen, L. Mauriac, J.F. Forbes,
M. Paridaens, M. Castiglione-Gertsch, R.D. Gelber, M. Rabaglio, I. Smith,
A. Wardley, K.N. Price, A. Goldhirsch, A comparison of letrozole and tamoxifen
in postmenopausal women with early breast cancer, N. Engl. J. Med. 353
(2005) 2747e2757.
Funding
[16] R. Jakesz, W. Jonet, M. Gnant, M. Mittleboeck, R. Greil, C. Tausch, J. Hilfrich,
W. Kwasny, C. Menzel, H. Samonigg, Switching of postmenopausal women
with endocrine-responsive early breast cancer to anastrozole after 2 years’
adjuvant tamoxifen: combined results of ABCSG trial 8 and ARNO 95 trial,
Lancet 366 (2005) 455e462.
[17] C. Pelissero, M.J. Lenczowski, D. Chinzi, B. Davail-Cuisset, J.P. Sumpter,
A. Fostier, Effects of flavonoids on aromatase activity, an in vitro study,
J. Steroid Biochem. Mol. Biol. 57 (1996) 215e223.
This work was supported by FAR funds (Italian Ministry for In-
struction, University and Research) assigned to Alessandra
Ammazzalorso and Amelia Cataldi, and by “4FRAILTY e Sensoristica
intelligente, infrastrutture e modelli gestionali per la sicurezza di
soggetti fragili”, codex ARS01_00345, assigned to Orazio Nicolotti.
[18] L. Banjare, S.K. Verma, A.K. Jain, S. Thareja, Design and pharmacophoric
identification of flavonoid scaffold-based aromatase inhibitors, J. Heterocycl.
Chem. 57 (2020) 3483e3492.
Declaration of competing interest
[19] F. Caporuscio, G. Rastelli, C. Imbriano, A. Del Rio, Structure-based design of
potent aromatase inhibitors by high-throughput docking, J. Med. Chem. 54
(2011) 4006e4017.
[20] M. Di Matteo, A. Ammazzalorso, F. Andreoli, I. Caffa, B. De Filippis,
M. Fantacuzzi, L. Giampietro, C. Maccallini, A. Nencioni, M.D. Parenti,
D. Soncini, A. Del Rio, R. Amoroso, Synthesis and biological characterization of
3-(imidazole-1-ylmethyl) piperidine sulfonamides as aromatase inhibitors,
Bioorg. Med. Chem. Lett 26 (2016) 3192e3194.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Appendix A. Supplementary data
[21] M. Fantacuzzi, B. De Filippis, M. Gallorini, A. Ammazzalorso, L. Giampietro,
C. Maccallini, Z. Aturki, E. Donati, R.S. Ibrahim, E. Shawky, A. Cataldi,
R. Amoroso, Synthesis, biological evaluation, and docking study of indole aryl
sulfonamides as aromatase inhibitors, Eur. J. Med. Chem. 185 (2020) 111815.
[22] S.J. Arkinstall, A. Arulandanam, X. Jiang, S. Magar, R. Nabioullin, J.Y. Zhang,
P. Blume-Jensen, Preparation of N-(imidazolylmethyl)benzamides and
Imidazolylalkyl-Benzoates as MEK-1 and ERK-2 Kinase Inhibitors, 2002.
WO2003035626, October 23.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.113115.
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