Synthesis and conversion of primary and secondary 2-aminoestradiols into A-ring-integrated benzoxazolone hybrids and theirin vitroanticancer activity

Article abstract of DOI:10.1039/d1ra01889b

Hybrid systems are often endowed with completely different and improved properties compared to their parent compounds. In order to extend the chemical space toward sterane-based molecular hybrids, a number of estradiol-derived benzoxazol-2-ones with combined aromatic rings were synthesizedviathe corresponding 2-aminophenol intermediates. 2-Aminoestradiol was first prepared from estrone by a two-step nitration/reduction sequence under mild reaction conditions. Subsequent reductive aminations with different arylaldehydes furnished secondary 2-aminoestradiol derivatives in good yields. The proton dissociation processes of the aminoestradiols were investigated in aqueous solution by UV-visible spectrophotometric titrations to reveal their actual chemical forms at physiological pH. The determined pK₁and pK₂values are attributed to the⁺NH₃or⁺NH₂R and OH moieties, and both varied by the different R substituents of the amino group. Primary and secondary 2-aminoestradiols were next reacted with carbonyldiimidazole as a phosgene equivalent to introduce a carbonyl group with simultaneous ring-closure to give A-ring-fused oxazolone derivatives in high yields. The novel aminoestradiols and benzoxazolones were subjected toin vitrocytotoxicity analysis and were found to exert cancer cell specific activity.

Full text of DOI:10.1039/d1ra01889b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Synthesis and conversion of primary and secondary
2
-aminoestradiols into A-ring-integrated
benzoxazolone hybrids and their in vitro anticancer
activity†‡
Cite this: RSC Adv., 2021, 11, 13885
a
b
b
b
Ferenc Kovacs,
´
E´ va A. Enyedy
Mohana K. Gopisetty, Dora I. Adamecz, Monika Kiricsi,
´ ´
cd
a
and E´ va Frank
*
Hybrid systems are often endowed with completely different and improved properties compared to their
parent compounds. In order to extend the chemical space toward sterane-based molecular hybrids,
a number of estradiol-derived benzoxazol-2-ones with combined aromatic rings were synthesized via
the corresponding 2-aminophenol intermediates. 2-Aminoestradiol was first prepared from estrone by
a
two-step nitration/reduction sequence under mild reaction conditions. Subsequent reductive
aminations with different arylaldehydes furnished secondary 2-aminoestradiol derivatives in good yields.
The proton dissociation processes of the aminoestradiols were investigated in aqueous solution by UV-
visible spectrophotometric titrations to reveal their actual chemical forms at physiological pH. The
+
+
1 2 3 2
determined pK and pK values are attributed to the NH or NH R and OH moieties, and both varied
by the different R substituents of the amino group. Primary and secondary 2-aminoestradiols were next
reacted with carbonyldiimidazole as a phosgene equivalent to introduce a carbonyl group with
simultaneous ring-closure to give A-ring-fused oxazolone derivatives in high yields. The novel
aminoestradiols and benzoxazolones were subjected to in vitro cytotoxicity analysis and were found to
exert cancer cell specific activity.
Received 10th March 2021
Accepted 5th April 2021
DOI: 10.1039/d1ra01889b
rsc.li/rsc-advances
Thanks to their attractive structural, physicochemical and
Introduction
biological features, natural sex steroids are oen used to
5
–7
Molecular hybridization is a very promising approach in drug construct molecular hybrids for anticancer indication, either
8
design and can offer therapeutic options for a wide variety of by covalent attachment to another pharmacophore directly or
1,2
9
diseases. The main goal of hybrid development is to enhance via a spacer, or by integrating key functional elements to form
10
or favourably modulate the pharmacokinetic and/or pharma- a new chemical entity. However, any residual hormonal effect
codynamic properties of a molecule through the synergistic of the steroid moiety is undesirable in these cases, and should
effects of combined structural elements; although combating be signicantly reduced or, preferably, eliminated during
multidrug resistance and minimizing unwanted side effects are hybrid formation.
also important considerations. Occasionally, new, unpredict-
For estrane-based hybrids, the best way to avoid hormonal
able bioactivities may arise which are different from those of the side effects is to modify the OH groups at positions C-3 and C-
3
,4
individual molecules that constitute the hybrid compounds.
17, which are primarily responsible for the hormone receptor
11
binding via H-bonding interactions. Decreased affinity to the
target protein, due to electronic and steric reasons, has also
been demonstrated for a number of 2-substituted estradiol
a
Department of Organic Chemistry, University of Szeged, D ´o m t ´e r 8, H-6720 Szeged, derivatives (e.g., 2-methoxyestradiol), which, however, are
Hungary. E-mail: frank@chem.u-szeged.hu; Tel: +36-62-544-275
b
effective in inhibiting the proliferation of various human cancer
Department of Biochemistry and Molecular Biology, Doctoral School of Biology,
University of Szeged, K ¨o z ´e p fasor 52, H-6726 Szeged, Hungary
12–16
cells.
Although the cytotoxic effect of 2-nitroestradiol ob-
tained by direct nitration of estradiol was weaker than that of 2-
methoxyestradiol, the attachment of a piperidine or morpho-
line ring to the phenolic-O through a spacer led to particularly
c
Department of Inorganic and Analytical Chemistry, Interdisciplinary Excellence
Centre, University of Szeged, D ´o m t ´e r 7, H-6720 Szeged, Hungary
d
MTA-SZTE Lend u¨ let Functional Metal Complexes Research Group, University of
17
Szeged, D ´o m t ´e r 7, H-6720 Szeged, Hungary
potent cytotoxic compounds. Interestingly, there are very few
examples in the literature for the preparation of A-ring-modied
estrane-based derivatives containing a [2,3]-fused heterocyclic
†
‡
1
Dedicated to Prof. Gyula Schneider on his upcoming 90th birthday.
Electronic supplementary information (ESI) available. See DOI:
0.1039/d1ra01889b
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13885

View Article Online
RSC Advances
Paper
1
8,19
26–30
moiety.
Elimination of the phenolic OH group in parallel derivatives
(Fig. 1B). All other synthetic methods, e.g. those
31–36
37,38
with the substitution at C-2 makes it even more likely that the depicted in Fig. 1A
and Fig. 1C,
involve several steps and
compound will not be able to bind to hormone receptors, and require protection/deprotection of the phenolic OH group,
possibly other major bioactivities can predominate without expensive reagents or harsh conditions, and suffer from selec-
undesired side effects.
Since 2-aminophenols are valuable building blocks for many yields than route B.
tivity problems that ultimately lead to the nal product in lower
20
biologically active compounds and can be used as starting
In addition to studying a number of methods that may be
materials for the synthesis of pharmacologically important N,O- suitable for the most efficient and scalable access to 2-amino-
21–23
heterocycles,
our primary aim was to introduce an amino estradiol, our next goal was to further convert the compound to
substituent at the C-2 position of estradiol. In view of the high secondary aminophenols by reductive amination. In order to
reactivity of the phenolic A-ring of estrogens toward electro- characterize the actual chemical forms (and charges) of the
philes and the two available ortho positions (C-2 and C-4), the novel compounds in solution focusing on physiological pH, and
regioselective introduction of a substituent into C-2 that can be to explore the effect of the various substituents on the aromatic
easily converted to an amino group is a challenging task. ring of the benzylamino moiety in the case of secondary amines,
a
Although C-2 attack is slightly favoured, in most of the cases, the proton dissociation constants (K ) of the amphoteric ami-
e.g. during halogenation, nitration or formylation, mixtures of nophenols were determined by UV-visible spectrophotometric
mono- and disubstituted products – which are oen difficult to titrations. Finally, bifunctional derivatives were subjected to
separate – were usually obtained, and the yield of the 2-func- CDI-induced ring-closure reactions with the simultaneous
24
tionalized derivative was only moderate. Accordingly, the rst incorporation of a carbonyl group from the reagent to furnish
step of the seemingly simplest process involving nitration and various novel estradiol–benzoxazolone molecular hybrids. The
subsequent reduction for the synthesis of primary 2-amino cytotoxicity of all synthesized derivatives was tested in vitro by
estradiol oen suffered from the above-mentioned the
colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-
25,26
problems.
diphenyltetrazolium bromide (MTT) assay using human MCF-
When forming a 2-aminophenol moiety on estrogens, given 7 breast, HeLa cervical as well as DU145 and PC-3 prostate
the high cost of sex hormones, it is desirable to develop cancer cell lines, and simultaneously non-cancerous MRC-5
a process that consists of only few reaction steps and provides broblast cells. Based on the primary toxicity screen, ve
the product in acceptable yield. Moreover, if the amino deriva- potent compounds were selected and subjected to further
tive is to be used as a starting material for the preparation experiments to obtain their IC50 values on different cell lines.
of N,O-heterocycles, it must be synthesized in a reproducible
manner in larger quantities. Although the task does not seem
difficult from an organic chemical point of view, it is no coin-
Result and discussion
cidence that there are only few literature examples for the
Synthetic studies
preparation of 2-aminoestrone or 2-aminoestradiol, most of
Based on the literature background, we selected the two-step
method for the preparation of 2-aminoestradiol; however,
both the nitration of estrone and the following reduction were
which are based on a two-step procedure, i.e. nitration and
subsequent reduction aer separation of the mono- and dinitro
Fig. 1 Possible routes for the synthesis of 2-amino estrogens.
13886 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
investigated separately. Since mononitration seemed unavoid- proved to be suitable, aer reduction, for the synthesis of A-ring
able at both the C-2 and C-4 positions of the sterane core, we [2,3]-fused and [3,4]-fused 2-oxazolones.
tried to nd a proper experimental condition where at least
As a continuation, conversion of 2 and 3 to aminoestradiols
over-nitration and thus the formation of the dinitro product was investigated. An insoluble red precipitate was formed as
could be eliminated. A number of literature procedures a by-product when 2-nitrophenols (2 or 3) were reduced with
describing the selective mononitration of phenol or estrone iron lings under classical B ´e champ conditions, probably due
29
28
either by using 65% (m/m) HNO
to be reproduced in different solvents (such as AcOH, dioxane, adding NaOH was also unsuccessful thanks to the high
EtOH, CH Cl or CHCl ), however, signicant amount of 2,4- susceptibility of sodium phenolates to oxidation. Therefore,
3
or metal nitrates were tried to metal complex formation, and then complex dissociation by
2
2
3
dinitro derivative reducing the yield of the desired product, was another hydrogenation method had to be found. In order to
obtained in all cases. Furthermore, unreacted estrone remained avoid introducing ammable hydrogen into the reaction
in the reaction mixtures when the nitrating agent was not used mixture from a gas cylinder, hydrogen generated in situ from
41,42
in excess, which further complicated the separation and puri- NaBH
cation process. was used for catalytic reduction of 2 and 3 (Scheme 1). This
Since the selective nitration of phenol and substituted method was also suitable to reduce not only the NO but also
4
in a protic solvent (MeOH) in the presence of Pd–C

2
phenols to the corresponding mononitro compounds was re- the 17-(C]O) group stereoselectively, thus resulting in 2- (4) or
ported earlier under mild conditions in a liquid–liquid two- 4-aminoestradiol (6) in a single step with excellent isolated
phase system with diluted nitric acid (6% m/m) and in the yields. In view of the amphoteric character of the products,
presence of tetrabutylammonium bromide as phase-transfer adjustment of neutral pH during work-up was of crucial
39
catalyst, we tried to adapt this method on the substitution importance.
reaction of estrone (1). However, given the poor solubility of
In the following, unsubstituted (8a) and para-substituted
estrone relative to phenol in water-immiscible organic solvents, benzylamino estradiols (8b–h) were prepared from 4 with
a relatively large amount had to be used to prepare a homoge- benzaldehyde and its derivatives by reductive amination
neous solution, so that the volume of water (added with (Table 1). Imine formation was performed in the presence of
a diluted HNO solution) was signicantly smaller than that of a catalytic amount of AcOH and molecular sieves by boiling the
3
the organic phase. Therefore, a phase transfer catalyst was not reactants in MeOH for 1 h. Secondary aminophenols (8a–h)
required even during the scale-up synthesis; the reaction pro- were obtained in good to excellent yields by reducing the
ceeded with 1 equiv. of nitrating reagent in boiling CH Cl
unstable imines in situ with NaBH . With the only exception of
4
2
2
under vigorous stirring for 1 h to give exclusively a ca. 1 : 1 the p-nitro-substituted compound (8f), all derivatives were
mixture of 2- and 4-nitroestrone in 92% overall yield (Scheme 1). found to be stable both in the solid state and in organic solvents
The mononitro derivatives were easily separated by column (Table 1).
chromatography owing to their large polarity difference. First, 2-
Both the prepared primary (4 and 6) and secondary amines
nitroestrone (2) eluted from the column; its more apolar nature (8a–h) were further converted to estradiol–benzoxazolone
is due to the intramolecular hydrogen bonding interaction hybrids by cyclization and incorporation of a carbonyl group
between the phenolic OH and the NO group. In contrast, the from the applied 1,1-carbonyldiimidazole (CDI) reagent in the
2
more polar character of 4-nitroestrone (3) can be attributed to presence of triethylamine (TEA) as base by boiling in THF
the fact that due to its sterically hindered environment, the (Scheme 1 and Table 1). The reactions provided the products (5,
40
nitro group twists out of the plane of the sterane skeleton,
7 and 9a–h) in good yield (67–94%) within 2 h.
therefore, intramolecular interaction masking the polarity of
the functional groups cannot develop.
The structures of all compounds prepared in our experi-
mental work were conrmed by H and C NMR spectroscopic
1
13
Although mononitration was not regioselective, both and high-resolution electrospray ionization mass spectrometry
successfully separated nitro compounds (2 and 3) subsequently (ESI-HRMS) methods. Due to the nature of the transformations
Scheme 1 Two-step synthesis of monoamino-estradiol regioisomers and their conversion to A-ring-fused 2-oxazolones.
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13887

View Article Online
RSC Advances
Paper
Table 1 Synthesis of secondary aminoestradiols by reductive amination and their conversion to A-ring-fused N-substituted 2-oxazolones
a
a
Entry
R
Aminoestradiol
Yield (%)
Benzoxazolone
Yield (%)
1
2
3
4
5
6
7
8
H
F
Cl
Br
CN
NO
CH
8a
8b
8c
8d
8e
8f
76
76
91
75
96
82
72
94
9a
9b
9c
9d
9e
9f
69
76
94
77
92
92
78
90
b
2
3
8g
8h
9g
9h
OMe
a
b
Aer purication by column chromatography. Proved to be sensitive to oxidation even in the solid state.
1
performed, the range of H NMR spectra above chemical shi compound in a 30% (v/v) DMSO/H O solvent mixture. However,
2
values of 3 ppm was informative; below 3 ppm, the signals of the the halogen and CN substituents in 8b–e resulted in worse
skeletal protons and the C-13 angular methyl group could be solubility, and lower compound concentration (10 mM) had to
observed. The most important difference between 2- and 4- be applied to avoid the appearance of precipitate. Since 2-ami-
nitroestrone (2, 3) and 2- and 4-aminoestradiol (4, 6) derivatives nophenol (2AP) and its derivatives can easily undergo oxidation,
1
43,44
can be noticed in the aromatic range of the H NMR spectra. For especially in their completely deprotonated forms,
a strong
the 2,3-disubstituted compounds (2, 4), the C-1 and C-4 protons argon ow was applied during the titrations to exclude the
give singlet signals, while the adjacent C-1 and C-2 protons of oxygen. Deprotonation processes of 2AP as a simpler model
the 3,4-disubstituted analogs (3, 6) appear as doublets with the
same coupling constant. In the spectra of the secondary ami-
nophenols (8), the appearance of the extra signals between 7
2
and 8 ppm as well as the presence of the benzyl-CH at 4.3 ppm
are indicative for the incorporation of the aromatic ring from
the arylaldehyde reagents and the success of the reductive
amination. As for the oxazolone (5, 7, 9) derivatives, the negative
carbon signal of the introduced C]O group can be seen at
1
3
around 154 ppm in the C NMR spectra (J-MOD), which indi-
cated the heterocyclization of the aminophenols upon treat-
ment with CDI.
Proton dissociation processes of primary and secondary
aminoestradiols
The methods (NMR, ESI-HRMS) used for the characterization of
2-aminoestradiols (4 and 8a–h) provide information about their
purity and chemical structure in the solid phase and in solution
of organic solvents. However, knowledge on their behaviour in
aqueous solution and proton dissociation is essential for
a deeper understanding of their pharmacological properties
and structure–activity relationships. Therefore, we aimed to
a
determine the proton dissociation constants (K ) of the
compounds to predict their actual chemical forms at pH 7.4 in
solution, and to reveal the inuence of the various substituents.
The studied sterane-based derivatives (4 and 8a–h) have limited
solubility in pure water, thus UV-visible spectrophotometric
Fig. 2 (a) UV-visible spectra recorded for 4 at various pH values. (b)
Concentration distribution curves calculated for 4 and the measured
absorbance values at 310 nm (C) with the fitted values (blue solid line).
ꢀ
titrations were performed on samples containing 50 mM {c ¼ 50 mM; l ¼ 2 cm; 30% (v/v) DMSO/H
2
O; T ¼ 25 C; I ¼ 0.1 M (KCl)}.
13888 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
compound were also investigated for comparison. The studied
compounds displayed strong absorption bands in the UV region
mostly due to p–p* transitions of the benzene rings. Repre-
sentative UV-visible spectra recorded for 4 at various pH values
are shown in Fig. 2a. Two well-separated processes can be
observed upon increasing the pH; namely, the rst step is
accompanied by an increase of the absorbance and the lmax
from 282 nm to 296 nm in the pH range from 2 to 7, while the
second deprotonation step, taking place at pH > 9.5 results in
a further increase in the lmax (310 nm) with the appearance of
a novel band in the wavelength range 350–470 nm. As the
deprotonation steps are not overlapping, clear-cut isosbestic
+
2
points could be found at 284 nm and 302 nm in case of the H L
+
ꢁ
+
#
HL + H and HL # L + H equilibrium processes, respec-
ꢁ
tively, where L is the completely deprotonated form of the
compound. This nding also indicates that no disturbing
processes such as oxidation took place during the titration of 4
under the ow of the inert gas. Proton dissociation constants
(
Ka) were determined by the deconvolution of the measured
spectra and are collected in Table 2.
Based on the determined pK values, concentration distri-
bution curves were computed (Fig. 2b) showing the predomi-
nant formation of the HL species in the pH range ca. 6–9.5. The
a
Fig. 3 (a) UV-visible spectra recorded for 8a at various pH values.
Inserted figure shows the measured absorbance values at 312 nm (C)
with the fitted values (solid line) (b) concentration distribution curves
pK
a
values were also determined for 2AP and sterane-based calculated for 8a. {c ¼ 50 mM; l ¼ 2 cm; 30% (v/v) DMSO/H O; T ¼
2
ꢀ
2
5 C; I ¼ 0.1 M (KCl)}.
compounds based on the recorded spectra (Table 2, see repre-
sentative spectra and concentration distribution curves for 8a in
Fig. 3). Notably, the spectra recorded for the compounds with
the methoxy (8h), nitrile (8e), chlorine (8c) and bromine
substituents (8d) showed a certain extent of oxidation in the water (pK
basic pH range (as no clear isosbestic point was found at pH > obtained in this work using 30% (v/v) DMSO/H
As it is expected, the pK
values determined for 2AP in pure
a
45
1
¼ 4.91 and pK
2
¼ 9.87 ) differ somewhat from those
O solvent
2
a 3
values could not be determined mixture. Namely, the pK of the NH group is lower, while that
2
+
1
0); therefore, the pK
accurately.
Similarly to the case of 2AP, the pK
of the phenolic-OH is higher in the presence of the DMSO than
values are attributed to in pure water. DMSO has a lower charge neutralization
45
1
+
+
the amino moiety (NH3 or NH ) in all cases, while the pK
deprotonation/protonation processes are more favourable in
2
2
values belong to the phenolic OH group. Since the pK and pK
the more apolar medium. Comparing the pK values to each
a
1
2
values are in the range 3.85–4.82 and 10.34–11.1, respectively, it other (Table 2), it can be concluded that the incorporation of
can be concluded that all the studied compounds are in their 2AP into the sterane core resulted in an increase in both of the
neutral HL form at the physiological pH, and become air pK
sensitive only in the basic pH range (pH > 9) when the depro- bouring B-ring of the sterane core (c.f. 2AP and 4). Derivatization
tonation of the phenolic OH group starts.
on the amino group resulted in secondary amines, and the
introduction of the benzyl functional group led to lower pK
a
values due to the electron-donating effect of the neigh-
a
Table 2 Proton dissociation constants (pK
a
) of the compounds (c
O}
L
¼ 10 or 50 mM) determined by UV-visible spectrophotometric titrations. {T ¼
ꢀ
2
5 C; I ¼ 0.1 M (KCl); 30% (v/v) DMSO/H
2
+
Compound
R
pK
a
(NHamino
)
pK
a
(OHphenolic
)
c
L
2
AP
—a
4.41 ꢂ 0.02
10.36 ꢂ 0.02
50 mM
4
8
8
8
8
8
8
8
H
4.82 ꢂ 0.01
4.58 ꢂ 0.03
4.23 ꢂ 0.04
4.34 ꢂ 0.05
3.94 ꢂ 0.02
3.85 ꢂ 0.01
4.75 ꢂ 0.03
4.76 ꢂ 0.03
10.63 ꢂ 0.02
10.44 ꢂ 0.04
10.36 ꢂ 0.02
ꢃ10.8
50 mM
50 mM
10 mM
10 mM
10 mM
10 mM
50 mM
50 mM
a
b
c
d
e
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
-Ph
-(p-F)C H
6 4
-(p-Cl)C
-(p-Br)C
-(p-CN)C
-(p-CH )C
-(p-MeO)C
6 4
H
6
H
4
ꢃ11.1
6
H
4
ꢃ11.0
g
h
3
6
H
4
10.34 ꢂ 0.05
ꢃ10.6
6
H
4
a
Reference compound.
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13889

View Article Online
RSC Advances
Paper
values (c.f. 4 and 8a). The pK
a
values of all the other derivatives Table 3 IC50 (ꢂSD) values of some selected estrane-based derivatives
and of cisplatin assessed on non-cancerous MRC-5 cells as well as on
(8b–e, 8g and 8h) are compared to those of 8a as a reference
HeLa and DU145 cancer cell lines
compound. The electron-donating methyl (8g) and methoxy
substituents (8h) slightly increased the pK of the amino group,
while the electron-withdrawing CN (8e) and halo substituents
a
IC50 (mM)ꢂSD
Compound
MRC-5
HeLa
DU145
(8b–d) decreased it according to the expectations. Notably, the
largest effect was observed in the case of the nitrile and bromo
substituents. Differences were also observed in the case of the
pK values of the phenolic OH group. This group is located
a
2
5
7
>2.5 ꢂ 1.35
>2.5 ꢂ 1.41
>2.5 ꢂ 1.02
>2.5 ꢂ 1.02
>2.5 ꢂ 1.09
31.8 ꢂ 1.09
1.1 ꢂ 1.01
0.09 ꢂ 1.39
1.2 ꢂ 1.01
—
—
0.7 ꢂ 1.01
—
further away from the benzyl ring, and the inuence of 8c
1.1 ꢂ 1.01
1.2 ꢂ 1.01
117.8 ꢂ 1.02
9
b
—
substituents on pK needs some explanations. Effect of the CH ,
2
3
Cisplatin
347.3 ꢂ 1.08
MeO and F groups was negligible, while signicantly higher pK2
values were obtained in the case of the other substituents,
47
although these values are more questionable due to the recog- enzyme required to orchestrate programmed cell death. The
nized oxidation process. Most probably, the substituent effect observed ndings imply that the toxicity induced by these
can be realized via the possible N/OH hydrogen bond between compounds might depend on functional caspase-3, however,
the deprotonated amino group and the phenolic OH, and the further studies are required to prove this notion.
electron-withdrawing groups might result in
a
stronger
Based on the data of the overall toxicity screen, we selected
some derivatives that induced the strongest toxic effects in one
or more cancer cell lines. Therefore, we chose compounds 5, 8c
and 9b to be tested further on DU145, compounds 2, 5, and 7 for
a more profound examination on HeLa, and nally, all the
hydrogen bond leading to the increase of the pK values.
2
Pharmacological studies of the synthesized compounds
Cytotoxicity of all the synthesized derivatives was tested in vitro aforementioned molecules (namely 2, 5, 7, 8c and 9b) were
on cancerous cell lines MCF-7, PC-3, DU145, and HeLa and also examined on MRC-5 cells to assess their IC50 values. Since none
on non-cancerous MRC-5 broblasts. For the preliminary of these molecules showed cytotoxicity in MCF-7 breast cancer
screen, compounds were applied in 1.5 mM concentration for cells, and only two of them were slightly effective on PC-3
7
2 h, and a heat map (Fig. 4, ESI, Table S1‡) was constructed prostate cancer cells, these cell lines were omitted from
with the mean cytotoxicity values obtained from three inde- further analyses.
pendent experiments. The results of the screen indicated that at
For IC50 determination, the selected steroids or cisplatin
.5 mM concentration, all the compounds, except for 8a, 8e, 9a (positive control) were applied at different concentrations on
1
and 9h induced cytotoxicity in at least one or in more cancerous the cell lines, and at the end of the treatments cell viability was
cells (PC-3, DU145, and HeLa), but not in non-cancerous MRC-5 measured via MTT assay. Dose-response curves were tted on

broblasts, suggesting that most of the synthesized compounds the obtained viability data (ESI, Fig. S1‡), and subsequently the
exhibit a cancer cell-selective toxic feature. Interestingly, none corresponding IC50 values were calculated (Table 3).
of the compounds showed cytotoxicity on MCF-7 breast cancer
In agreement with the preliminary screen, all the tested
46
cells. These cancer cells lack caspase-3, a crucial cellular compounds, notably, 2-nitroestrone (2), the A-ring-fused 2-
oxazolones (5, 7), the secondary aminoestradiol 8c and also the
A-ring-fused-N-substituted 2-oxazolone 9b were effective on
cancerous cells at several fold lower concentrations (Table 3,
Fig. S1 and S2‡) compared to positive control cisplatin.
Unlike the tested steroid derivatives, cisplatin did not show
any cancer cell specic cytotoxicity, which implies a potential
advantage of these compounds as candidates for future thera-
peutic developments. The most potent molecule on HeLa cells
proved to be the A-ring-fused 2-oxazolone (5), whereas the
secondary aminoestradiol 8c and the benzoxazolone 9b were
the most effective agents on DU145 prostate cancer cells despite
being ineffective on HeLa cells. It is noteworthy that compound
8c exerted the strongest effect also on the other prostate cancer
cell line PC-3.
Conclusions
Fig. 4 The primary cytotoxic effect of the synthesized aminoestradiols
and benzoxazolones on various human cancerous cell lines and on
non-cancerous MRC-5 fibroblasts shown on a heat map (c ¼ 1.5 mM;
In summary, we developed an efficient methodology for the
mononitration of estrone at C-2 and C-4 positions. Reduction of
nitroestrones by in situ generated hydrogen under catalytic
conditions resulted in 2- and 4-aminoestradiol derivatives in
7
2 h incubation time). Control represents the viability of untreated
cells.
13890 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
a single step. Primary and secondary aminoestradiols, the latter that was used in positive and negative ion mode. Samples
obtained by reductive amination of benzaldehyde and p- introduced with FIA (ow injection analysis) method, eluent
substituted arylaldehydes with 2-aminoestradiol, were sub- stream (water, acetonitrile in 1 : 1 volume ratio with 0.1% for-
jected to cyclization with CDI to afford A-ring-integrated estra- mic acid) was provided by a Waters Acquity I-class liquid
diol–benzoxazolone hybrids in good to excellent yields.
Compounds 4, 8a–h were characterized by two pK
that belong to the amino (pK ) and the phenolic-OH (pK
moieties, respectively. Based on the determined values (pK
.9–4.8; pK
¼ 10.3–11.1), all the studied compounds are
chromatograph (Waters).
a
values
1
2
)
Synthetic procedures
1
¼
3
2
Nitration of estrone (1). To a solution of estrone (1, 5.41 g, 20
present in their neutral form in a wide pH range, including the mmol) in CH Cl (540 mL), nitric acid (6% (m/m), 15.4 mL, 1
2
2
physiological pH. These aminophenol derivatives become air- equiv.) was added dropwise and the reaction mixture was
sensitive as the deprotonation of the OH functional group heated at reux temperature for 1 h, aer which TLC moni-
starts, thus in the basic pH range (pH > 9). The substituents on toring indicated the completion of the reaction and the
the benzyl function of the secondary amines (8) have an impact formation of two new products (2 and 3). The solvent was
on the proton dissociation processes mainly of the amino removed in vacuo, and the remaining crude product was puri-
group, namely, the electron-donating CH
increase pK , while CN and the halogens decrease it.
Based on a preliminary cytotoxicity screen indicating
a possible cancer cell-selective pharmacological effect of the bright yellow crystals; Mp 258–260 C; ESI-HRMS: m/z 314.1394
synthesized compounds, the 5 most potent agents – A-ring- [M ], 314.1398 calcd for C H NO ; H NMR (500 MHz, CDCl ):
3
and MeO groups ed by column chromatography on silica gel (eluent: EtOAc/
1
CH Cl
2
2
¼ 2 : 98).
2-Nitroestra-1,3,5(10)-triene-17-one (2). Yield: 3.03 mg (48%);
ꢀ
ꢁ
1
1
8
21
4
3
fused 2-oxazolones (5, 7), the 2-nitroestrone (2), the secondary d 0.91 (3H, s, 18-CH ), 1.39–1.69 (6H, m), 1.96–2.10 (3H, m), 2.15
3
aminoestradiol 8c and also the A-ring-fused-N-substituted 2- (1H, dt, J 18.9, 8.9), 2.18–2.27 (1H, m), 2.38–2.46 (1H, m), 2.51
oxazolone 9b – were selected for more thorough testing. The (1H, m), 2.85–3.02 (2H, m, 6-H ), 6.86 (1H, d, J 1.1, 4-H), 7.97
2
1
3
chosen derivatives were effective at lower concentrations on (1H, d, J 1.5, 1-H), 10.40 (1H, s, 3-OH); C NMR (125 MHz,
HeLa and DU145 cancerous cells than cisplatin, highlighting CDCl ): d 13.9 (18-CH ), 21.7 (CH ), 25.8 (CH ), 26.0 (CH ), 29.7
their potential as lead compounds for anticancer drug (CH ), 31.4 (CH ), 35.9 (CH ), 37.8 (8-CH), 43.6 (9-CH), 47.9 (13-
3
3
2
2
2
2
2
2
development.
C), 50.5 (14-CH), 119.1 (4-CH), 121.7 (1-CH), 131.9 (2-C), 133.2
10-C), 148.9 (5-C), 153.0 (3-C), 220.3 (17-C).
(
4
-Nitroestra-1,3,5(10)-triene-17-one (3). Yield: 2.86 g (45%);
Experimental
Materials and methods
ꢀ
yellowish white powder; Mp 165–166 C; ESI-HRMS: m/z
14.1394 [M ], 314.1398 calcd for C H NO ; H NMR (500
ꢁ
1
3
18 21 4
6 3
Chemicals, reagents, and solvents were purchased from MHz, DMSO-d ): d 0.83 (3H, s, 18-CH ), 1.28–1.45 (3H, m), 1.42–
commercial suppliers (Sigma-Aldrich and Alfa Aesar) and used 1.62 (3H, m), 1.71–1.82 (1H, m), 1.95 (2H, m), 2.06 (1H, dt, J
without further purication. Melting points (Mp) were deter- 18.9, 8.8), 2.19 (1H, td, J 10.4, 4.7), 2.28–2.39 (1H, m), 2.39–2.48
mined on an SRS Optimelt digital apparatus and are uncor- (1H, m), 2.61 (1H, m), 2.70 (1H, m), 6.86 (1H, d, J 8.6, 2-H), 7.30
1
3
rected. The transformations were monitored by TLC using (1H, d, J 8.7, 1-H), 10.45 (1H, s, 3-OH); C NMR (125 MHz,
.25 mm thick Kieselgel-G plates (Si 254 F, Merck). The DMSO-d ): d 13.4 (18-CH ), 20.9 (CH ), 23.4 (CH ), 24.8 (CH ),
compound spots were detected by spraying with 5% phospho- 25.4 (CH ), 31.1 (CH ), 35.2 (CH ), 36.9 (8-CH), 43.1 (9-CH), 47.1
0
6
3
2
2
2
2
2
2
molybdic acid in 50% aqueous phosphoric acid. Flash chro- (13-C), 49.2 (14-CH), 114.4 (2-CH), 127.8 (5-C), 127.8 (1-CH),
matographic purications were carried out on silica gel 60, 40– 131.1 (10-C), 140.3 (4-C), 146.3 (3-C), 219.2 (17-C).
6
5
3 mm (Merck). NMR spectra were recorded with a Bruker DRX
00 instrument at room temperature in CDCl or DMSO-d
Reduction of nitroestrone derivatives. 2- (2) or 4-nitroestrone
3
6
(3) (1.89 g, 6 mmol) was dissolved in MeOH/THF ¼ 1 : 1 (100
ꢀ
using residual solvent signals as an internal reference. Chem- mL) and cooled to 0 C, followed by the addition of 10% Pd–C
ical shis are reported in ppm (d scale), and coupling constants (200 mg). To this suspension, NaBH
4
(0.91 g, 24 mmol, 4 equiv.)
1
(J) are given in Hz. Multiplicities of the H signals are indicated was added in small portions. Aer 30 min, TLC indicated
as a singlet (s), a doublet (d), doublet of doublets (dd), doublet complete conversion, so the solution was neutralized with
of triplets (dt) a triplet (t), triplet of doublets (td), or a multiplet diluted HCl, ltered through a Cellite® pad for the removal of
1
3
1
(
m). C NMR spectra are H-decoupled and the J-MOD pulse the Pd–C catalyst, and the solvent was evaporated under
sequence was used for multiplicity editing. In this spin-echo reduced pressure. To the crude product MeOH (20 mL) was
type experiment, the signal intensity is modulated by the added, and the solution was poured onto ice-cold water. The
different coupling constants J of carbons depending on the forming white precipitate was ltered off, washed with water,
number of attached protons. Both protonated and unproto- and dried.
nated carbons can be detected (CH
3
and CH carbons appear as
2-Aminoestra-1,3,5(10)-triene-3,17b-diol (4). Substrate: 2,
ꢀ
positive signals, while CH and C carbons as negative signals). yield: 1.78 g, (97%, white solid); Mp 188–190 C, ESI-HRMS: m/z
2
+
1
ESI-HRMS spectra were recorded on a Q Exactive Plus hybrid 288.1969 [M + H] , 288.1958 calcd for C H NO ; H NMR (500
1
8
25
2
quadrupole-orbitrap mass spectrometer (Thermo Scientic) MHz, DMSO-d
6 3
): d 0.67 (3H, s, 18-CH ), 1.03–1.36 (6H, m), 1.38
equipped with a heated electrospray ionization (HESI-II) probe (1H, m), 1.52–1.62 (1H, m), 1.74 (1H, m), 1.79–1.86 (1H, m),
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13891

View Article Online
RSC Advances
Paper
1.83–1.93 (1H, m), 2.01 (1H, s), 2.13 (1H, dt, J 12.1, 3.3), 2.55 (1H, 36.6 (CH
2 2
), 38.8 (8-CH), 42.7 (13-C), 43.8 (9-CH), 46.2 (N–CH ),
dt, J 10.3, 5.1), 2.61 (1H, m), 3.52 (1H, td, J 8.5, 4.6, 17a-H), 4.15 49.5 (14-CH), 80.0 (17-CH), 107.6 (1-CH), 113.7 (4-CH), 114.7,
(
(
2H, s, 2-NH ), 4.39 (1H, d, J 4.8, 17-OH), 6.32 (1H, s, 1 H), 6.52 114.8, 123.2 (5-C), 128.9 (CH), 128.9 (CH), 130.3 (10-C), 134.6 (2-
2
1
3
1H, s, 4 H), 8.52 (1H, s, 3-OH); C NMR (125 MHz, DMSO-d6): C), 136.8, 136.9, 142.1 (3-C), 160.0, 161.9.
0
d 11.1 (18-CH
3
), 22.7 (CH
2
), 26.2 (CH
2
), 27.3 (CH
2
), 28.3 (CH
2
),
2-(4 -Chlorobenzylamino)estra-1,3,5(10)-triene-3,17b-diol
2
9.9 (CH ), 36.6 (CH
2
2
), 38.8 (8-CH), 42.7 (C-13), 43.7 (9-CH), 49.6 (8c). The synthesis was carried out according to the general
(
14-CH), 80.0 (17-CH), 111.6 (4-CH), 114.4 (1-CH), 123.8 (5-C), procedure, using p-chlorobenzaldehyde (154 mg). Yield: 376 mg
ꢀ
1
30.5 (2-C), 133.8 (10-C), 142.0 (3-C).
-Aminoestra-1,3,5(10)-triene-3,17b-diol (6). Substrate: 3, 412.2049 [M + H] , 412.2038 calcd for C25
yield: 1.68 g, (92%, brownish solid); the product was subjected to (500 MHz, DMSO-d ): d 0.62 (3H, s, 18-CH ), 1.00–1.28 (6H, m),
(91%); yellowish white solid; Mp 180–183 C; ESI-HRMS: m/z
+
+ 1
4
2
H31ClNO ; H NMR
6
3
ring-closure directly aer formation without characterization.
1.30–1.41 (1H, m), 1.54 (1H, m), 1.67–1.74 (1H, m), 1.74–1.80
(
(
1H, m), 1.86 (1H, m), 1.91–2.06 (2H, m), 2.51–2.65 (2H, m), 3.48
1H, td, J 8.5, 4.7, 17a-H), 4.25 (2H, d, J 5.7, N–CH ), 4.43 (1H, d, J
.8, 17-OH), 5.02 (1H, t, J 6.3, –NH), 6.28 (1H, s, 1 H), 6.33 (1H, s,
2
General procedure for the synthesis of secondary
aminoestradiols (8a–h)
4
4
0
0
0
0
H), 7.35 (4H, s, 2 -H, 3 -H, 5 -H and 6 -H), 8.92 (1H, s, 3-OH);
1
3
To a solution of 2-aminoestradiol (4, 287 mg, 1 mmol) in MeOH
(
C NMR (125 MHz, DMSO-d
), 27.2 (CH ), 28.4 (CH
added, followed by the addition of (p-substituted) benzaldehyde CH), 42.8 (13-C), 43.9 (9-CH), 46.1 (N–CH
6
): d 11.2 (18-CH
), 29.9 (CH ), 36.6 (CH
), 49.5 (14-CH), 80.0
3
), 22.7 (CH
2
), 26.1
5 mL), catalytic amount of AcOH and molecular sieves were (CH
2
2
2
2
2
), 38.8 (8-
2
0
(
1.1 equiv.). The mixture was kept at reux temperature for 1 h, (17-CH), 107.5 (1-CH), 113.7 (4-CH), 123.3 (5-C), 128.1 (3 -CH
(76 mg, 2 and 5 -CH), 128.9 (2 -CH and 6 -CH), 130.3 (4 -C), 130.9 (10-C),
equiv.). Aer complete conversion of the Schiff-base (30 min, 134.5 (2-C), 140.0 (1 -C), 142.2 (3-C).
0
0
0
0
cooled to room temperature and reduced with NaBH
4
0
0
TLC), the reaction was quenched with water, MeOH was
2-(4 -Bromobenzylamino)estra-1,3,5(10)-triene-3,17b-diol
removed, and the remaining residue was extracted with EtOAc (8d). The synthesis was carried out according to the general
(
3 ꢄ 5 mL). The combined organic phase was washed with water procedure, using p-bromobenzaldehyde (204 mg). Yield: 342 mg
ꢀ
and brine, dried over Na
2
SO
4
, and concentrated in vacuo. The (75%); white solid; Mp 172–175 C; ESI-MS: m/z 456.1542 [M +
+
+
1
crude product was puried by column chromatography using H] , 456.1533 calcd for C25
EtOAc/CH Cl (5 : 95) as eluent.
DMSO-d ): d 0.63 (3H, s, 18-CH
-(Benzylamino)estra-1,3,5(10)-triene-3,17b-diol (8a). The (5H, m), 1.36 (1H, m), 1.55 (1H, m), 1.68–1.78 (1H, m), 1.75–1.81
synthesis was carried out according to the general procedure, (1H, m), 1.86 (1H, m), 1.92–2.06 (2H, m), 2.52–2.64 (2H, m), 3.49
using benzaldehyde (110 mL, 117 mg). Yield: 286 mg (76%); (1H, td, J 8.5, 4.8, 17a-H), 4.24 (2H, d, J 5.5, N–CH ), 4.38 (1H, d, J
white solid; Mp 171–173 C; ESI-HRMS: m/z 378.2440 [M + H] , 4.8, 17-OH), 4.98 (1H, t, J 6.4, –NH), 6.29 (1H, s, 1-H), 6.34 (1H, s,
H
31BrNO
; H NMR (500 MHz,
2
), 1.00–1.09 (1H, m), 1.11–1.27
2
2
6
3
2
2
ꢀ
+
+
1
0
0
0
3
78.2428 calcd for C25
H
32NO
2
; H NMR (500 MHz, DMSO-d
), 0.99–1.29 (6H, m), 1.36 (1H, m), 1.55 (1H, 5 -H), 8.86 (1H, s, 3-OH); C NMR (125 MHz, DMSO-d
), 22.7 (CH ), 26.0 (CH ), 27.1 (CH ), 28.3 (CH
), 36.6 (CH ), 38.8 (8-CH), 42.7 (13-C), 43.8 (9-CH), 46.2 (N–
), 49.5 (14-CH), 80.0 (17-CH), 107.5 (1-CH), 113.7 (4-CH),
6
): 4-H), 7.30 (2H, d, J 8.3, 2 -H and 6 -H), 7.48 (2H, d, J 8.3, 3 -H and
0
13
d 0.63 (3H, s, 18-CH
3
6
): d 11.1
), 29.9
m), 1.75 (2H, m), 1.86 (1H, m), 1.97 (1H, td, J 10.7, 4.0), 2.04 (1H, (18-CH
dt, J 10.7, 3.4), 2.55 (1H, dd, J 6.1, 2.1), 2.60 (1H, m), 3.49 (1H, td, (CH
J 8.5, 4.5, 17a-H), 4.26 (2H, d, J 5.3, N–CH
), 4.38 (1H, d, J 4.8, 17- CH
OH), 4.85 (1H, t, J 6.2, –NH), 6.35 (2H, d, J 5.0, 1 H, 4 H), 7.18– 119.2 (4 -C), 123.3 (5 C), 129.3 (2 -CH and 6 -CH), 130.3 (10 C),
7
(
3
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
.25 (1H, m, 4 -H), 7.27–7.37 (4H, m, 2 -H, 3 -H, 5 -H, 6 -H), 8.86 130.9 (3 -CH and 5 -CH), 134.5 (2-C), 140.4 (3-C), 142.2 (1 -C).
1
3
0
2-(4 -Cyanobenzylamino)estra-1,3,5(10)-triene-3,17b-diol
1H, s, 3-OH); C NMR (125 MHz, DMSO-d ): d 11.1 (18-CH ),
6
3
2
2.7 (CH
2
), 26.0 (CH
2
), 27.2 (CH
2
), 28.3 (CH
2
), 29.9 (CH
2
), 36.6 (8e). The synthesis was carried out according to the general
(
CH
2
), 38.8 (8-CH), 42.7 (13-C), 43.8 (9-CH), 47.0 (N–CH
2
), 49.5 procedure, using p-cyanobenzaldehyde (144 mg). Yield: 386 mg
ꢀ
(
14-CH), 80.0 (17-CH), 107.5 (4-CH), 113.7 (1-CH), 123.1 (5-C), (96%); white solid; Mp 184–187 C; ESI-HRMS: m/z 403.2391 [M
0
0
0
0
0
+
+
1
1
1
26.4 (4 -C), 127.1 (2 -CH and 6 -CH), 128.1 (3 -CH and 5 -CH), + H] , 403.2380 calcd for C26
30.3 (10-C), 134.8 (2-C), 140.7 (1 -C), 142.1 (3-C).
2
H
31
N
2
O
2
;
H NMR (500 MHz,
), 0.99–1.35 (7H, m), 1.32–1.40
(1H, m), 1.54 (1H, m), 1.67–1.78 (1H, m), 1.85 (1H, m), 1.89–2.04
0
DMSO-d ): d 0.61 (3H, s, 18-CH
6 3
0
-(4 -Fluorobenzylamino)estra-1,3,5(10)-triene-3,17b-diol
(
8b). The synthesis was carried out according to the general (2H, m), 2.51–2.64 (2H, m), 3.48 (1H, td, J 8.5, 4.7, 17a-H), 4.37
procedure, using p-urobenzaldehyde (118 mL, 137 mg). Yield: (2H, d, J 6.0, N–CH
2
), 4.42 (1H, d, J 4.8, 17-OH), 5.21 (1H, t, J 6.4,
ꢀ
0
3
3
01 mg (76%); orange solid; Mp 163–165 C; ESI-MS: m/z –NH), 6.21 (1H, s, 1-H), 6.34 (1H, s, 4-H), 7.52 (2H, d, J 8.0, 2 -H
+
+
1
0
0
0
96.2341 [M + H] , 396.2333 calcd for C25
): d 0.63 (3H, s, 18-CH ), 1.01–1.27 (6H, m),
.31–1.42 (1H, m), 1.55 (1H, m), 1.69–1.92 (3H, m), 1.97 (1H, td, (CH
H31FNO ; H NMR and 6 -H), 7.76 (2H, d, J 8.3, 3 -H and 5 -H), 8.96 (1H, s, 3-OH);
2
1
3
(500 MHz, DMSO-d
6
3
C NMR (125 MHz, DMSO-d
), 27.2 (CH ), 28.4 (CH
6
): d 11.2 (18-CH
), 29.9 (CH ), 36.6 (CH
2
3
), 22.7 (CH
), 38.8 (8-
), 49.5 (14-CH), 80.0
2
), 26.0
1
2
2
2
2
J 10.3, 3.6), 2.04 (1H, dd, J 12.8, 4.1), 2.52–2.64 (2H, m), 3.49 (1H, CH), 42.7 (13-C), 43.8 (9-CH), 46.4 (N–CH
2
0
0
td, J 8.5, 4.7, 17a-H), 4.22–4.27 (2H, m, N–CH
2
), 4.38 (1H, d, J 4.8, (17-CH), 107.5 (1-CH), 109.1 (4 -C), 113.8 (4-CH), 119.0 (4 -CN),
0
0
0
1
(
7-OH), 4.90 (1H, s, –NH), 6.33 (2H, d, J 7.5, 1-H and 4-H), 7.11 123.4 (5-C), 127.9 (2 -CH and 6 -CH), 130.3 (10-C), 132.1 (3 -CH
0 0 0 0
0 0
2H, t, J 8.9, 7.6, 3 -H and 5 -H), 7.37 (2H, dd, J 8.5, 2 -H and 6 - and 5 -CH), 134.3 (2-C), 142.3 (3-C), 147.4 (1 -C).
1
3
0
H), 8.85 (1H, s, 3-OH); C NMR (125 MHz, DMSO-d
6
): d 11.1 (18-
2-(4 -Nitrobenzylamino)estra-1,3,5(10)-triene-3,17b-diol (8f).
CH ), 22.7 (CH ), 26.0 (CH ), 27.2 (CH ), 28.3 (CH ), 29.9 (CH
3
2
2
2
2
2
), The synthesis was carried out according to the general
13892 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
6
procedure, using p-nitrobenzaldehyde (166 mg). Yield: 346 mg DMSO-d ): d 0.67 (3H, s), 1.06–1.45 (7H, m), 1.58 (1H, m), 1.79 (1H,
(82%); yellow solid. Due to stability issues it was immediately m), 1.88 (2H, m), 2.16 (1H, td, J 10.9, 4.2), 2.23 (1H, dt, J 12.9, 3.6),
converted into 9f without purication.
2.80 (2H, dt, J 8.1, 4.1, 6-H ), 3.52 (1H, t, J 8.5, 17a-H), 4.45 (1H, s,
17-OH), 6.92 (2H, d, J 7.4, 1-H and 4 H), 11.32 (1H, s, NH). C NMR
2
0
13
2-(4 -Methylbenzylamino)estra-1,3,5(10)-triene-3,17b-diol
(
8g). The synthesis was carried out according to the general (125 MHz, DMSO-d
procedure, using p-tolualdehyde (132 mg). Yield: 346 mg (72%); (CH ), 29.0 (CH ), 29.8 (CH
white solid; Mp 151–153 C; ESI-HRMS: m/z 392.2596 [M + H] , 43.8 (9-CH), 49.6 (14-CH), 79.9 (17-CH), 106.2 (CH), 109.1 (CH),
6
): d 11.1 (18-CH
3
), 22.7 (CH
2 2
), 26.1 (CH ), 26.6
2
2
2
), 36.5 (CH
2
), 38.2 (8-CH), 42.7 (13-C),
ꢀ
+
+
1
0
3
92.2584 calcd for C26
H
34NO
2
; H NMR (500 MHz, DMSO-d
6
): 128.2, 130.0, 135.6, 141.5, 154.6 (2 -C).
0
0
0
d 0.64 (3H, s, 18-CH
3
), 1.06–1.24 (6H, m), 1.31–1.42 (1H, m), 1.55
Oxazolo[4 ,5 :4,3]estra-1,3,5(10)-triene-17b-ol-2 -one
(7).
(
1H, m), 1.69–1.76 (1H, m), 1.79 (1H, dt, J 12.3, 3.1), 1.87 (1H, Substrate: 6 (144 mg); eluent: EtOAc/CH Cl ¼ 50 : 50; yield:
2
2
ꢀ
m), 1.93–2.02 (1H, m), 2.04–2.11 (1H, m), 2.27 (3H, s), 2.47–2.66 105 mg (67%); white powder; Mp 258–260 C; ESI-HRMS: m/z
+
+ 1
(2H, m), 3.50 (1H, td, J 8.5, 4.3, 17a-H), 4.20 (2H, s, N–CH ), 4.38 314.1757 [M + H] , 314.1751 calcd for C H NO ; H NMR (500
2
19 24
3
(
(
6 3
1H, d, J 4.8, –NH), 4.77 (1H, s, 17-OH), 6.33 (1H, s, 1-H), 6.37 MHz, DMSO-d ): d 0.67 (3H, s, 18-CH ), 1.07–1.44 (7H, m), 1.60
0 0
1H, s, 4-H), 7.11 (2H, d, J 7.8, 3 -H and 5 -H), 7.23 (2H, d, J 8.0, (1H, m), 1.88 (3H, m), 2.17 (1H, td, J 11.1, 4.3), 2.29 (1H, m),
0
0
13
2
-H and 6 -H), 8.83 (1H, s, 3-OH); C NMR (125 MHz, DMSO- 2.59–2.70 (1H, m), 2.73–2.81 (1H, m), 3.53 (1H, td, J 8.5, 4.3, 17a-
): d 11.1 (18-CH ), 20.5 (CH ), 22.7 (CH ), 26.1 (CH ), 27.2 H), 4.43 (1H, d, J 4.8, 17-OH), 7.00 (2H, s, 1-H and 4-H), 11.44
CH ), 28.3 (CH ), 29.9 (CH ), 36.6 (CH ), 38.8 (8-CH), 42.7 (13- (1H, s, NH); C NMR (125 MHz, DMSO-d
C), 43.8 (9-CH), 46.8 (N–CH ), 49.5 (14-CH), 80.0 (17-CH), 107.5 (CH ), 24.1 (CH ), 25.8 (CH ), 26.2 (CH ), 29.8 (CH ), 36.5 (CH ),
d
(
6
3
2
2
2
1
3
2
2
2
2
6 3
): d 11.1 (18-CH ), 22.7
2
2
2
2
2
2
2
0 0 0
(
(
(
1-CH), 113.6 (4-CH), 123.1 (4 -C), 127.1 (3 -CH and 5 -CH), 128.6 38.0 (8-CH), 42.6 (13-C), 43.6 (9-CH), 49.4 (14-CH), 79.9 (17-CH),
0
0
0
2 -CH and 6 -CH), 130.3 (5-C), 134.80 (1 -C), 135.4 (10-C), 137.5 106.3 (2-CH), 118.3 (1-CH), 119.1 (5-C), 128.4 (4-C), 135.7 (3-C),
0
2-C), 142.1 (3-C).
140.8 (10-C), 154.9 (2 -C).
0
0
0
0
0
2
-(4 -Methoxybenzylamino)estra-1,3,5(10)-triene-3,17b-diol
3 -Benzyloxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-17b-ol-2 -one
Cl
¼ 5 : 95;
(
8h). The synthesis was carried out according to the general (9a). Substrate: 8a (189 mg); eluent: EtOAc/CH
2
ꢀ
2
procedure, using p-anisaldehyde (134 mL, 150 mg). Yield: yield: 139 mg (69%); white powder; Mp 148–150 C; ESI-HRMS:
3
4
ꢀ
+
+ 1
84 mg (94%); white solid; Mp 135ꢁ137 C; ESI-HRMS: m/z m/z 404.2228 [M + H] , 404.2220 calcd for C26
H
30NO
3
; H NMR
+
+ 1
08.2551 [M + H] , 408.2533 calcd for C H NO ; H NMR (500 (500 MHz, CDCl ): d 0.77 (3H, s), 1.17 (1H, m), 1.23–1.54 (7H,
2
6
34
3
3
MHz, DMSO-d ): d 0.64 (3H, s, 18-CH ), 0.99–1.28 (6H, m), 1.36 m), 1.70 (1H, m), 1.88 (1H, m), 1.94 (1H, m), 2.06–2.23 (3H, m),
6
3
(
1
1
1H, m), 1.50–1.61 (1H, m), 1.68–1.92 (3H, m), 1.98 (1H, td, J 2.79–2.94 (2H, m), 3.72 (1H, t, J 8.5, 17a-H), 4.90–5.03 (2H, m, N–
0
0
0.8, 4.0), 2.05–2.12 (1H, m), 2.57 (2H, m), 3.49 (1H, td, J 8.5, 4.7, CH
2
), 6.75 (1H, s, 1-H), 6.91 (1H, s, 4-H), 7.31 (1H, dt, 4 -CH),
0
00
00
00
00
13
7a-H), 3.72 (3H, s, 4 -OMe), 4.17 (2H, d, J 6.0, N–CH
2
), 4.43 (1H, 7,31–7.39 (4H, m, 2 -H, 3 -H, 5 -H and 6 -H); C NMR (125
): d 11.2 (18-CH ), 23.3 (CH ), 26.7 (CH ), 27.2 (CH ),
), 30.8 (CH ), 36.8 (CH ), 38.7 (8-CH), 43.3 (13-C), 44.4
-H and 6 -H), 8.88 (1H, s, 3-OH); C NMR (125 MHz, DMSO- (9-CH), 46.2 (N–CH ), 50.3 (14-CH), 81.9 (17-CH), 105.9 (4-CH),
d, J 5.1, –NH), 4.75 (1H, t, J 6.2, 17-OH), 6.32 (1H, s, 1-H), 6.36 MHz, CDCl
3
3
2
2
2
0
0
(1H, s, 4-H), 6.83–6.90 (2H, m, 3 -H and 5 -H), 7.23–7.28 (2H, m, 29.85 (CH
2
2
2
0
0
13
2
2
0
0
00
00
00
d ): d 11.2 (18-CH ), 22.8 (CH ), 26.1 (CH ), 27.3 (CH ), 28.4 110.1 (1-CH), 127.8 (3 -CH and 5 -CH), 128.3 (4 -CH), 129.1 (1 -
(
CH), 46.5 (N–CH ), 49.5 (14-CH), 55.0 (4 -OMe), 80.0 (17-CH), C), 141.2 (3-C), 155.3 (2 -C).
1
and 6 -CH), 130.3 (1 -C), 132.5 (10-C), 134.8 (2-C), 142.2 (3-C), 17b-ol-2 -one (9b). Substrate: 8b (198 mg); eluent: EtOAc/CH
1
6
3
2
2
2
00 00
CH ), 29.9 (CH ), 36.7 (CH ), 38.8 (8-CH), 42.8 (13-C), 43.9 (9- C), 129.1 (2 -CH and 6 -CH), 131.6 (2-C), 135.2 (5-C), 136.4 (10-
2 2 2
0
0
2
0
0
0
0
0
00
0
0
0
07.6 (CH), 113.6 (3 -CH and 5 -CH), 123.1 (5-C), 128.4 (2 -CH
3 -(4 -Fluorobenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
0
2
Cl
ꢀ
2
0
58.0 (4 -C).
¼ 5 : 95; yield: 160 mg (76%); white powder; Mp 146–148 C;
+
ESI-HRMS: m/z 422.2136 [M + H] , 422.2126 calcd for
+
1
26 3 3 3
C H29FNO ; H NMR (500 MHz, CDCl ): d 0.77 (3H, s, 18-CH ),
General procedure for the synthesis of oxazolone hybrids (5, 7
and 9a–h)
1
2
.13–1.75 (9H, m), 1.88 (1H, m), 1.96 (1H, dt, J 12.2, 2.9), 2.07–
.24 (3H, m), 2.80–2.94 (2H, m), 3.73 (1H, t, J 8.5, 17a-H), 4.88–
The primary (4 or 6) or secondary amine (8a–h) (0.5 mmol) was 4.99 (2H, m, N–CH
dissolved in THF/CH Cl
¼ 1 : 4 (5 mL), CDI (89 mg, 1.1 equiv.) (2H, td, J 8.5, 1.4), 7.29–7.38 (2H, m); C NMR (125 MHz,
and TEA (140 mL, 1 mmol, 2 eq.) were added and the mixture CDCl ): d 11.2 (18-CH ), 23.3 (CH ), 26.7 (CH ), 27.2 (CH ), 29.8
was kept at reux temperature for 2 h. The solvent was removed (CH ), 30.8 (CH ), 36.8 (CH ), 38.7 (8-CH), 43.3 (13-C), 44.4 (9-
under reduced pressure, the crude product was suspended in CH), 45.4 (N–CH ), 50.3 (14-CH), 81.9 (17-CH), 105.7 (1-CH),
), 6.74 (1H, s, 1-H), 6.91 (1H, s, 4-H), 7.04
2
1
3
2
2
3
3
2
2
2
2
2
2
2
water (5 mL), acidied with diluted HCl and extracted with 110.3 (4-CH), 116.0, 116.2, 128.8 (2-C), 129.5, 129.6, 131.0,
0
EtOAc (3 ꢄ 5 mL). The combined organic phase was washed 131.00, 131.8 (5-C), 136.4, 141.1, 155.2 (2 -C), 161.7, 163.7.
0
00
0
0
0
with water and brine, dried over anhydrous Na
2
SO
4
and
3 -(4 -Chlorobenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
concentrated in vacuo. The product was puried by column 17b-ol-2 -one (9c). Substrate: 8c (206 mg); eluent: EtOAc/CH
Cl
ꢀ
2
2
chromatography.
¼ 5 : 95; yield: 205 mg (94%); white powder; Mp 128–130 C;
0
0
0
+
Oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-17b-ol-2 -one (5). Sub- ESI-HRMS: m/z 438.1840 [M + H] , 438.1830 calcd for
+
1
strate: 4 (144 mg); eluent: EtOAc/CH Cl ¼ 50 : 50; yield: 120 mg
C
26
H
29ClNO
), 1.05–1.43 (7H, m), 1.57 (1H, m), 1.78 (1H, m), 1.87 (2H,
3
M + H] , 314.1751 calcd for C19H24NO ; H NMR (500 MHz, m), 2.14 (1H, td, J 11.0, 4.2), 2.28 (1H, m), 2.77–2.83 (2H, m),
3 6
; H NMR (500 MHz, DMSO-d ): d 0.65 (3H, s, 18-
2
2
ꢀ
(
[
76%); white powder; Mp 280–282 C; ESI-HRMS: m/z 314.1761 CH
3
+
+ 1
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13893

View Article Online
RSC Advances
.52 (1H, td, J 8.5, 4.9, 17a-H), 4.47 (1H, d, J 4.8, 17-OH), 5.02 CH
Paper
3
2
Cl
2
¼ 5 : 95; yield: 163 mg (78%); white powder; Mp 129–
ꢀ
+
(
2H, s, N–CH ), 7.01 (1H, s, 4-H), 7.13 (1H, s, 1-H), 7.36–7.45 (4H, 131 C; ESI-HRMS: m/z 418.2385 [M + H] , 418.2377 calcd for
2
00
00
00
00
13
+ 1
3
m, 2 -H, 3 -H, 5 -H and 6 -H); C NMR (125 MHz, DMSO-d6): C H NO ; H NMR (500 MHz, DMSO-d ): d 0.66 (3H, s, 18-CH ),
27
32
6
3
d 11.2 (18-CH ), 22.7 (CH ), 26.1 (CH ), 26.6 (CH ), 29.1 (CH ), 1.04–1.47 (7H, m), 1.58 (1H, m), 1.69–1.82 (1H, m), 1.87 (2H, m),
3
2
2
2
2
2
9.9 (CH
2
), 36.5 (CH
2
), 38.2 (8-CH), 42.7 (13-C), 44.0 (over- 2.15 (1H, td, J 11.2, 10.7, 4.0), 2.26 (4H, m), 2.80 (2H, dt, J 8.4, 3.6, 6-
), 49.5 (14-CH), 79.9 (17-CH), 106.2 (1- ), 3.52 (1H, td, J 8.5, 4.9, 17a-H), 4.42 (1H, d, J 4.8, 17-OH), 4.96
CH), 109.5 (4-CH), 128.6 (2 C), 128.7 (3 -CH and 5 -CH), 129.4 (2H, s, N–CH ), 7.00 (1H, s, 4-H), 7.09 (1H, s, 1-H), 7.16 (2H, d, J 7.8,
lapping, 9-CH and N–CH
2
H
2
0
0
00
2
00
00
00
00
00
00
00
00
13
(2 -CH and 6 -CH), 130.9 (4 -C), 132.4 (5-C), 134.9 (1 -C), 136.0 3 -H and 5 -H), 7.25 (2H, d, J 8.0, 2 -H and 6 -H); C NMR (125
0
(10-C), 140.2 (3-C), 154.1 (2 -C).
MHz, DMSO-d
6 3 2 2
): d 11.1 (18-CH ), 20.5 (CH ), 22.6 (CH ), 26.1
0
00
0
0
0
3
-(4 -Bromobenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
(CH ), 26.5 (CH ), 29.0 (CH ), 29.8 (CH ), 36.4 (CH ), 38.1 (8-CH),
2
2
2
2
2
1
¼
7b-ol-2 -one (9d). Substrate: 8d (228 mg); eluent: EtOAc/CH Cl
42.6 (13 C), 43.9 (9-CH), 44.5 (N–CH ), 49.5 (14-CH), 79.9 (17-CH),
2
2
2
ꢀ
00
00
5 : 95; yield: 186 mg (77%); white powder; Mp 159–161 C; 106.1 (1-CH), 109.3 (4-CH), 127.4 (2 -CH and 6 -CH), 128.6 (2 C),
+
00
00
00
00
ESI-HRMS: m/z 482.1335 [M + H] , 482.1325 calcd for 129.1 (3 -CH and 5 -CH), 130.7 (1 -C), 132.7 (5 C), 135.9 (4 -C),
+
1
0
C
CH
26
H29BrNO
3
; H NMR (500 MHz, CDCl ): d 0.77 (3H, s, 18- 136.9 (10-C), 140.1 (3-C), 154.1 (2 -C).
3
0
00
0
0
0
3
), 1.13–1.54 (8H, m), 1.70 (1H, m), 1.88 (1H, m), 1.96 (1H,
3 -(4 -Methoxybenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
m), 2.07–2.24 (3H, m), 2.80–2.94 (2H, m), 3.73 (1H, t, J 8.5, 17a- 17b-ol-2 -one (9h). Substrate: 8h (204 mg); eluent: EtOAc/CH
2
Cl
2
ꢀ
H), 4.85–4.98 (2H, m), 6.72 (1H, s, 4 H), 6.92 (1H, s, 1-H), 7.22 ¼ 2 : 98; yield: 195 mg (90%); white powder; Mp 136–138 C;
00 00 00 00
2H, s, J 8.4, 2 -H and 6 -H), 7.48 (2H, d, J 8.4, 3 -H and 5 -H); ESI-MS: m/z 434.2335 [M + H] , 434.2326 calcd for C H NO ;
27 32 4
+
+
(
1
3
1
C NMR (125 MHz, CDCl ): d 11.2 (18-CH ), 23.3 (CH ), 26.7
H NMR (500 MHz, DMSO-d ): d 0.66 (3H, s, 18-CH ), 1.10–1.43
6 3
3
3
2
(
CH ), 27.2 (CH ), 29.9 (CH ), 30.8 (CH ), 36.8 (CH ), 38.6 (8- (7H, m), 1.57 (1H, m), 1.78 (1H, m), 1.87 (2H, m), 2.15 (1H, td, J
2
2
2
2
2
CH), 43.3 (13-C), 44.4 (9-CH), 45.5 (N–CH
2
), 50.3 (14-CH), 81.9 10.9, 4.0), 2.25–2.33 (1H, m), 2.79 (2H, dt, J 8.4, 3.4), 3.52 (1H, td,
0
0
0
0
(
1
1
17-CH), 105.6 (1-CH), 110.3 (4-CH), 122.4 (4 -C), 128.8 (2-C), J 8.5, 4.7, 17a-H), 3.72 (3H, s, 4 -OMe), 4.48 (1H, d, J 4.8, 17-OH),
00 00 00 00 00 00
2
29.4 (2 -CH and 6 -CH), 131.9 (5-C), 132.3 (3 -CH and 5 -CH), 4.94 (2H, s, N–CH ), 6.91 (2H, d, J 8.6, 3 -H and 5 -H), 7.00
00
0
00 00
(1H, s, 4-H), 7.12 (1H, s, 1-H), 7.32 (2H, d, J 8.6, 2 -H and 6 -H);
34.2 (1 -C), 136.5 (10-C), 141.1 (3-C), 155.2 (2 -C).
0
00
0
0
0
13
3
-(4 -Cyanobenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
C NMR (125 MHz, DMSO-d
6 3 2
): d 11.2 (18-CH ), 22.7 (CH ), 26.2
1
7b-ol-2 -one (9e). Substrate: 8e (201 mg); eluent: EtOAc/CH Cl
(CH ), 26.6 (CH ), 29.1 (CH ), 29.9 (CH ), 36.5 (CH ), 38.2 (8-
2
2
2
2
2
2
2
ꢀ
¼
10 : 90; yield: 197 mg (92%); white powder; Mp 134–137 C; CH), 42.7 (13-C), 44.0 (9-CH), 44.2 (N–CH ), 49.5 (14-CH), 55.1
2
+
00
00
ESI-HRMS: m/z 429.2181 [M + H] , 429.2173 calcd for (4 -OMe), 79.9 (17-CH), 106.3 (1-CH), 109.4 (4-CH), 114.1 (3 -CH
+
1
00 00 00 00
C
27
H
29
N
2
O
3
; H NMR (500 MHz, DMSO-d
), 1.04–1.43 (7H, m), 1.57 (1H, m), 1.74–1.93 (3H, m), 2.15 130.7 (5-C), 135.9 (10-C), 140.1 (3-C), 154.1 (2 -C), 158.9 (4 -C).
1H, td, J 10.9, 4.0), 2.23–2.31 (1H, m), 2.81 (2H, dd, J 8.0, 3.9),
6
): d 0.65 (3H, s, 18- and 5 -CH), 127.8 (1 -C), 128.6 (2 C), 129.0 (2 -CH and 6 -CH),
0
00
CH
(
3
3.52 (1H, td, J 8.5, 4.8 17a-H), 4.47 (1H, d, J 4.8, 17-OH), 5.14
UV-visible spectrophotometric titrations
(
8
(
2
(
1
2H, s, N–CH
2
), 7.04 (1H, s, 4-H), 7.15 (1H, s, 1-H), 7.53 (2H, d, J
Stock solutions of tested compounds were prepared in DMSO at
00 00 00 00
13
.3, 2 -H and 6 -H), 7.83 (2H, d, J 8.3, 3 -H and 5 -H); C NMR
125 MHz, DMSO-d ): d 11.2 (18-CH ), 22.7 (CH ), 26.2 (CH ),
5
mM concentration. UV-visible spectra were recorded for
compounds in order to determine proton dissociation constants
pK ) in the pH range 2–12 in a 30% (v/v) DMSO/H O mixture at
6 3 2 2
6.6 (CH ), 29.1 (CH ), 29.9 (CH ), 36.4 (CH ), 38.2 (8-CH), 42.7
2
2
2
2
(
a
2
13-C), 44.0 (9-CH), 44.3 (N–CH
2
), 49.5 (14-CH), 79.9 (17-CH),
00 00 00
ꢀ
2
5.0 ꢂ 0.1 C. Ionic strength of 0.10 M (KCl) was used in order to
06.1 (1-CH), 109.5 (4-CH), 110.5 (4 -CN), 118.5 (4 -C), 128.2 (2 -
keep the activity coefficients constant. The titrations were per-
00
00
00
CH and 6 -CH), 128.6 (2-C), 131.0 (5-C), 132.6 (3 -CH and 5 -
CH), 136.1 (10-C), 140.2 (3-C), 141.5 (1 -C), 154.1 (2 -C).
formed with carbonate-free KOH solution of known concentration
0
0
0
(
0.10 M) with 30% (v/v) DMSO content. The electrode system was
0
00
0
0
0
3
-(4 -Nitrobenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene-
+
calibrated to the pH ¼ ꢁlog[H ] scale by means of blank titrations
1
5
1
C
7b-ol-2 -one (9f). Substrate: 8f (211 mg); eluent: EtOAc/CH Cl ¼
2
2
48
(HCl vs. KOH) according to the method suggested by Irving et al.
: 95; yield: 206 mg (92%); yellowish white powder; Mp 168–
The average water ionization constant (pK
w
) is 14.52 ꢂ 0.05 in the
ꢀ
+
70 C; ESI-HRMS: m/z 449.2082 [M + H] , 449.2071 calcd for
3
0% (v/v) DMSO/H O mixture. Argon was always passed over the
2
+
1
26 29 2 5 6 3
H N O ; H NMR (500 MHz, DMSO-d ): d 0.65 (3H, s, 18-CH ),
solutions during the titrations. An Agilent Cary 8454 diode array
spectrophotometer was used to record the UV-visible spectra at an
interval of 200–800 nm. The path length was 2 or 5 cm. Spectro-
photometric titrations were performed on samples containing the
compounds at 10 or 50 mM concentration. Proton dissociation
1
1
8
.06–1.43 (7H, m), 1.58 (1H, m), 1.74–1.93 (3H, m), 2.15 (1H, td, J
0.9, 3.9), 2.24–2.32 (1H, m), 2.81 (2H, dd, J 8.1, 3.9), 3.51 (1H, td, J
.5, 4.9, 17a-H), 4.47 (1H, d, J 4.8, 17-OH), 5.20 (2H, s, N–CH ), 7.05
2
00 00
1H, s, 4-H), 7.17 (1H, s, 1-H), 7.60 (2H, d, J 8.8, 2 -H and 6 -H), 8.22
(
00 00
2H, d, J 8.8, 3 -H and 5 -H); C NMR (125 MHz, DMSO-d ): d 11.2
6
13
(
a
constants (K ) of the compounds and the UV-visible spectra of the
(18-CH ), 22.7 (CH ), 26.2 (CH ), 26.6 (CH ), 29.1 (CH ), 29.9 (CH ),
3 2 2 2 2 2
individual species in the differently protonated forms were
3
6.4 (CH ), 38.2 (8-CH), 42.7 (13-C), 44.0 (9-CH), 44.1 (N–CH ), 49.5
2
2
49
calculated by the computer program PSEQUAD.
00
(14-CH), 79.9 (17-CH), 106.2 (1-CH), 109.6 (4-CH), 123.8 (3 -CH and
00
00 00
-CH), 128.5 (2 -CH and 6 -CH), 128.6 (2-C), 131.1 (5-C), 136.2 (10-
5
0
0
00
0
Cell culture
C), 140.2 (3-C), 143.6 (1 -C), 147.0 (4 -C), 154.1 (2 -C).
0
00
0
0
0
3
-(4 -Methylbenzyl)oxazolo[4 ,5 :2,3]estra-1,3,5(10)-triene- All the cell lines were purchased from ATCC. MCF-7, DU145 and
1
7b-ol-2 -one (9g). Substrate: 8g (196 mg); eluent: EtOAc/ PC-3 cells were maintained in RPMI-1640 (LONZA) complete
13894 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry

View Article Online
Paper
RSC Advances
medium (10% FBS, 2 mM glutamine and penicillin–strepto-
2 G. B ´e rub ´e , Expert Opin. Drug Discovery, 2016, 11, 281.
3 K. Nepali, S. Sharma, M. Sharma, P. M. S. Bedi and
K. L. Dhar, Eur. J. Med. Chem., 2014, 77, 422.
ꢀ
mycin) at 37 C and 5% CO . HeLa and MRC-5 cells were
2
ꢀ
maintained in EMEM (LONZA) complete medium at 37 C and
5
% CO . Treatments were applied in cell specic complete
4 G. Mehta and V. Singh, Chem. Soc. Rev., 2002, 31, 324.
5 P. Borah and P. Chowdhury, Curr. Drug Ther., 2017, 12, 3.
2
medium described above.
6
7
8
9
P. Saha, C. Debnath and G. B ´e rub ´e , J. Steroid Biochem. Mol.
Biol., 2013, 137, 271.
A. Gupta, B. Sathish Kumar and A. S. Negi, J. Steroid Biochem.
Mol. Biol., 2013, 137, 242.
I. Scherlitz-Hofmann, M. Dubs, R. Krieg, B. Sch ¨o necker,
M. Kluge and D. Sicker, Helv. Chim. Acta, 1997, 80, 2345.
S. D. Kuduk, F. F. Zheng, L. Sepp-Lorenzino, N. Rosen and
S. J. Danishefsky, Bioorg. Med. Chem. Lett., 1999, 9, 1233.
MTT cytotoxicity assay
Each synthesized compound was dissolved in cell culture grade
DMSO (Sigma) to a nal concentration of 10 mM. Cytotoxicity of
these compounds on cancerous (MCF-7, HeLa, DU145, PC-3)
and non-cancerous (MRC-5) cell lines was tested using MTT
cell viability assay. Briey, in a 96-well plate cells were seeded at
5000 cells per well density, and were le to grow overnight in 5%
ꢀ
10 L. F. Tietze, G. Schneider, J. W ¨o ling, T. N ¨o bel, C. Wulff,
I. Schubert and A. R u¨ beling, Angew. Chem., Int. Ed., 1998,
CO atmosphere at 37 C. Cells were incubated for 72 hours at
2
ꢀ
3
7 C under 5% CO with each synthesized compound sepa-
2
37, 2469.
rately in 1.5 mM concentration upon the primary screen and
only with the selected compounds in concentrations 0.5, 1, 1.5,
1
1
1
1
1 G. M. Anstead, K. E. Carlson and J. A. Katzenellenbogen,
Steroids, 1997, 62, 268.
2
and 2.5 mM for IC50 determination. At the end of treatment,
´
2 E. Frank and G. Schneider, J. Steroid Biochem. Mol. Biol.,
cell viability was measured by MTT assay as described previ-
19
2013, 137, 301.
ously. The positive control cisplatin was applied for 24 h in 20,
0, 80, 160, and 330 mM concentrations. Final results were ob-
3 R. Dutour, J. Roy, F. Cort ´e s-Ben ´ı tez, R. Maltais and
D. Poirier, J. Med. Chem., 2018, 61, 9229.
4 G. Panchapakesan, V. Dhayalan, N. Dhatchana Moorthy,
N. Saranya and A. K. Mohanakrishnan, Steroids, 2011, 76,
4
tained by three independent replicates. GraphPad Prism 7
soware was used to perform statistics and to construct the heat
map.
1491.
1
1
5 T. V. Petrasheuskaya, M. A. Kiss, O. D ¨o m ¨o t ¨o r, T. Holczbauer,
Author contributions
ˇ
ˇ
´ ´
N. V. May, G. Spengler, A. Kincses, A. C. Gasparovic, E. Frank
´
´
´
and E. A. Enyedy, New J. Chem., 2020, 44, 12154.
Conceptualization, E. F.; E. A. E. and M. K.; chemical synthesis
and optimization experiments, F. K.; pharmacological
studies, M. K. G. and D. I. A.; UV-visible spectrophotometric
6 M. Numazawa, M. Ando, Y. Watari, T. Tominaga, Y. Hayata
and A. Yoshimura, J. Steroid Biochem. Mol. Biol., 2005, 96, 51.
´
17 D. Bandyopadhyay, G. Rivera, J. L. Sanchez, J. Rivera,
J. C. Granados, A. M. Guerrero, F.-M. Chang, R. K. Dearth,
J. D. Short and B. K. Banik, Eur. J. Med. Chem., 2014, 82, 574.
titrations, E. A. E.; structural analysis, F. K.; formal analysis and
interpretation of data, M. K. G. and D. I. A.; methodology,
resources, supervision, E. F.; M. K. and E. A. E.; writing—orig-
inal dra preparation, F. K.; M. K. G. and D. I. A.; writing—
review and editing, E. F.; M. K. and E. A. E. All authors have read
and agreed to the published version of the manuscript.
´
´
18 B. Xiao, T.-J. Gong, Z.-J. Liu, J.-H. Liu, D.-F. Luo, J. Xu and
L. Liu, J. Am. Chem. Soc., 2011, 133, 9250.
´
´
1
9 B. Moln ´a r, M. K. Gopisetty, D. I. Adamecz, M. Kiricsi and
´
E. Frank, Molecules, 2020, 25, 4039.
2
2
0 X. Yang, G. Shan and Y. Rao, Org. Lett., 2013, 15, 2334.
1 J. Poupaert, P. Carato and E. Colacino, Curr. Med. Chem.,
Conflicts of interest
2005, 12, 877.
There are no conicts to declare.
2
2
2 M. Smist and H. Kwiecien, Curr. Org. Synth., 2014, 11, 676.
3 S. Bilginer, H. I. Gul, F. S. Erdal, H. Sakagami, S. Levent,
I. Gulcin and C. T. Supuran, J. Enzyme Inhib. Med. Chem.,
2019, 34, 1722.
Acknowledgements
This work was supported by National Research, Development
and Innovation Office-NKFIA (Hungary) through projects FK 24 G. Neef, Estrogens and Antiestrogens I, Handbook of
124240, GINOP-2.3.2-15-2016-00038, GINOP-2.3.2-15-2016-
Experimental
Pharmacology,
Springer-Verlag
Berlin
00035, and Ministry of Human Capacities (Hungary) grant
Heidelberg, 1999, Vol 135/I, ch. 2, p. 21.
TKP-2020. The New National Excellence Program of the Ministry 25 A. Cornelis, P. Laszlo and P. Pennetreau, J. Org. Chem., 1983,
48, 4771.
and the J ´a nos Bolyai Research Scholarship of the Hungarian 26 A. Bose, W. P. Sanjoto, S. Villarreal, H. Aguilar and
´
for Innovation and Technology (UNKP-20-5-SZTE-655 for M. K.)
Academy of Sciences (BO/00878/19/8 for M. K.) are gratefully
acknowledged.
B. K. Banik, Tetrahedron Lett., 2007, 48, 3945.
27 Y. Wang, Z. Zhan, Y. Zhou, M. Lei and L. Hu, Monatsh.
Chem., 2018, 149, 527.
2
2
8 J.-M. Poirier and C. Vottero, Tetrahedron, 1989, 45, 1415.
9 L. W. Lawrence Woo, B. Leblond, A. Purohit and
B. V. L. Potter, Bioorg. Med. Chem., 2012, 20, 2506.
References
1
L. F. Tietze, H. P. Bell and S. Chandrasekhar, Angew. Chem.,
Int. Ed., 2003, 42, 3996.
©
2021 The Author(s). Published by the Royal Society of Chemistry
RSC Adv., 2021, 11, 13885–13896 | 13895

View Article Online
RSC Advances
Paper
30 T. M. Lavallee, V. S. Pribluda, J. Simons, N. Mabjeesh, 40 Y. A. Mostafa, B. Kralt, P. P. N. Rao and S. D. Taylor, Bioorg.
P. Giannakakou, US Pat., WO 2003073985 A2, 2003.
1 P. N. Rao and J. W. Cessac, Steroids, 2002, 67, 1065.
2 P. Crabb ´e , L. A. Maldonado and I. S ´a nchez, Tetrahedron, 42 S. I. El-Hout, S. M. El-Sheikh, H. M. A. Hassan, F. A. Harraz,
Med. Chem., 2015, 23, 5681–5692.
41 N. Al Soom and T. Thieman, Int. J. Org Chem., 2016, 6, 1–11.
3
3
1
971, 27, 711.
I. A. Ibrahim and E. A. El-Sharkawy, Appl. Catal., A, 2015, 503,
176–185.
43 S. C. Mitchell, P. Carmichael and R. Waring, Kirk-Othmer
Encyclopedia of Chemical Technology, ed. 2003.
3
3
3
3 A. Loupy and S. R ´e gnier, Tetrahedron Lett., 1999, 40, 6221.
4 N. Ganguly and P. Mondal, Synthesis, 2010, 3705.
5 G. Wu, P. N. Ibrahim, Y. Zhou, S. Mamo, S. J. Gillette,
Y.-L. Zhu, J. Liu, C. Zhang, K. Zhang, D. R. Artis, US Pat., 44 J. Kaizer, R. Csonka and G. Speier, J. Mol. Catal., 2002, 180,
WO 2010129467, A1, 2010. 91–96.
6 M. Armitage, G. Bret, B. M. Choudary, M. Kingswood, 45 A. Vaidyan and P. Bhattacharya, Indian J. Chem., 1994, 33,
3
M. Lo, S. Moore, S. Smith and M. W. J. Urquhart, Org.
Process Res. Dev., 2012, 16, 1626.
103–1007.
46 R. U. Janicke, Breast Cancer Res. Treat., 2009, 117, 219–221.
3
7 I. Bacsa, D. Szemer ´e di, J. W ¨o ling, G. Schneider, L. Fekete 47 A. G. Porter and R. U. J ¨a nicke, Cell Death Differ., 1999, 6, 99–
and E. Merny ´a k, Beilstein J. Org. Chem., 2018, 14, 998.
104.
8 I. Bacsa, R. J ´o j ´a rt, G. Schneider, J. W ¨o ling, P. Mar ´o ti, 48 H. M. Irving, M. G. Miles and L. D. Petit, Anal. Chim. Acta,
3
B. E. Herman, M. Sz ´e csi and E. Merny ´a k, Steroids, 2015,
04, 230–236.
9 A. V. Joshi, M. Baidoosi, S. Mukhopadhyay and Y. Sasson,
1967, 38, 475–488.
1
49 L. Z ´e k ´a ny and I. Nagyp ´a l, Computational Methods for the
Determination of Stability Constants, Plenum Press, New
York, 1985.
3
Org. Process Res. Dev., 2003, 7, 95–97.
13896 | RSC Adv., 2021, 11, 13885–13896
© 2021 The Author(s). Published by the Royal Society of Chemistry