New estrone oxime derivatives: Synthesis, cytotoxic evaluation and docking studies

Article abstract of DOI:10.3390/molecules26092687

The interest in the introduction of the oxime group in molecules aiming to improve their biological effects is increasing. This work aimed to develop new steroidal oximes of the estrane series with potential antitumor interest. For this, several oximes were synthesized by reaction of hydroxylamine with the 17-ketone of estrone derivatives. Then, their cytotoxicity was evaluated in six cell lines. An estrogenicity assay, a cell cycle distribution analysis and a fluorescence microscopy study with Hoechst 3358 staining were performed with the most promising compound. In addition, molecular docking studies against estrogen receptor α, steroid sulfatase, 17β-hydroxysteroid dehy-drogenase type 1 and β-tubulin were also accomplished. The 2-nitroestrone oxime showed higher cytotoxicity than the parent compound on MCF-7 cancer cells. Furthermore, the oximes bearing halogen groups in A-ring evidenced selectivity for HepaRG cells. Remarkably, the ?^9,11-estrone oxime was the most cytotoxic and arrested LNCaP cells in the G₂ /M phase. Fluorescence microscopy studies showed the presence of condensed DNA typical of prophase and condensed and fragmented nuclei characteristic of apoptosis. However, this oxime promoted the proliferation of T47-D cells. Interestingly, molecular docking studies estimated a strong interaction between ?^9,11-estrone oxime and estrogen receptor α and β-tubulin, which may account for the described effects.

Full text of DOI:10.3390/molecules26092687

molecules
Article
New Estrone Oxime Derivatives: Synthesis, Cytotoxic
Evaluation and Docking Studies
Catarina Canário 1 , Mariana Matias 1, Vanessa Brito 1, Adriana O. Santos 1, Amílcar Falcão 2,3
,
Samuel Silvestre 1,4,
*
and Gilberto Alves 1
1
CICS-UBI–Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilhã, Portugal;
catarina_canario@hotmail.com (C.C.); mariana.r.matias@gmail.com (M.M.);
vanessa_12_479@hotmail.com (V.B.); aos@ubi.pt (A.O.S.); gilberto@fcsaude.ubi.pt (G.A.)
Laboratory of Pharmacology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal;
acfalcao@ff.uc.pt
CIBIT-Coimbra Institute for Biomedical Imaging and Translational Research, University of Coimbra,
3000-548 Coimbra, Portugal
CNC–Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal
Correspondence: sms@ubi.pt
2
3
4
*
Abstract: The interest in the introduction of the oxime group in molecules aiming to improve their
biological effects is increasing. This work aimed to develop new steroidal oximes of the estrane
series with potential antitumor interest. For this, several oximes were synthesized by reaction of
hydroxylamine with the 17-ketone of estrone derivatives. Then, their cytotoxicity was evaluated in
six cell lines. An estrogenicity assay, a cell cycle distribution analysis and a fluorescence microscopy
study with Hoechst 3358 staining were performed with the most promising compound. In addition,
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
molecular docking studies against estrogen receptor
drogenase type 1 and -tubulin were also accomplished. The 2-nitroestrone oxime showed higher
cytotoxicity than the parent compound on MCF-7 cancer cells. Furthermore, the oximes bearing
halogen groups in A-ring evidenced selectivity for HepaRG cells. Remarkably, the
9,11-estrone
α, steroid sulfatase, 17β-hydroxysteroid dehy-
ꢀꢁꢂꢃꢄꢅꢆ
β
Citation: Canário, C.; Matias, M.;
Brito, V.; Santos, A.O.; Falcão, A.;
Silvestre, S.; Alves, G. New Estrone
Oxime Derivatives: Synthesis,
Cytotoxic Evaluation and Docking
Studies. Molecules 2021, 26, 2687.
https://doi.org/10.3390/
∆
oxime was the most cytotoxic and arrested LNCaP cells in the G2/M phase. Fluorescence microscopy
studies showed the presence of condensed DNA typical of prophase and condensed and fragmented
nuclei characteristic of apoptosis. However, this oxime promoted the proliferation of T47-D cells.
Interestingly, molecular docking studies estimated a strong interaction between ∆
9,11-estrone oxime
molecules26092687
and estrogen receptor α and β-tubulin, which may account for the described effects.
Academic Editor: Qiao-Hong Chen
Keywords: estrone; oximes; cytotoxicity; cancer; docking
Received: 25 March 2021
Accepted: 1 May 2021
Published: 4 May 2021
1. Introduction
Cancer is a major public health problem and is one of the leading causes of death world-
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published maps and institutional affil-
iations.
wide [
working in the development of new drugs with antitumor activity—namely, starting from
molecules that already exist in nature [ ]. In this context, steroids are natural compounds
that are usually involved in cell proliferation and consequently in cancer development [ ].
In the 90s, several steroids having very unusual and interesting structures were isolated
from marine sponges. Among these, steroidal oximes isolated by Rodriguez et al. [ ] from
Cinachyrella sponges showed relevant antiproliferative activity against several types of
cancer cells [ ]. The interesting results observed in these studies stimulated researchers to
1]. Therefore, over the years, medicinal chemists and other researchers have been
2
3
4
Copyright:
© 2021 by the authors.
5
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
prepare different series of steroidal oximes with potential anticancer interest. Classically,
the oxime functional group is usually introduced by condensation of an aldehyde or a ke-
tone, including of steroidal origin, with hydroxylamine affording, respectively, aldoximes
and ketoximes [6].
Among the different groups of steroidal oximes developed as anticancer agents, sev-
eral of them have endocrine activity, acting by inhibition of steroid sulfatase (ST), aromatase,
Molecules 2021, 26, 2687. https://doi.org/10.3390/molecules26092687
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2687
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17
hydrogenase type 1 (17
(E1) and therefore its inhibition can be of interest in breast cancer treatment. In this
context, Hejaz and co-workers [ ] synthesized the estrone oxime sulfamate (OMATE),
α
-hydroxylase-17,20-lyase (CYP45017α), 5
α
-reductase (5
α
-R) or 17β-hydroxysteroid de-
β-HSD1) enzymes [7–12]. ST converts estrone sulfate into estrone
8
which inhibited the ST enzyme and showed a potency similar to the observed with the
ketone analogue estrone sulfamate (EMATE). Later, modified 2-substituted estrogen-3-
O-sulfamate 17-oximes have also been prepared and showed significant antiproliferative
activity against breast MCF-7 cells [13]. 17β-HSD type 1 enzyme reduces the 17-ketone
of estrane steroids to the corresponding 17β-hydroxylated derivatives [14]. Relevant 17β-
HSD1 inhibition was also demonstrated by Allan et al. for 16-oxime and 6,17-oximes of E1
(% inhibition = 96% and 83%, respectively, at 10 µM) [9,12]. Interestingly, docking studies
performed by these authors showed that both the presence of 16 and 17-oximes were
associated to an improved 17β-HSD1 inhibition [9]. Also, estrone (E1)-16-oxime ethers
showed antiproliferative activities against cervix (HeLa), ovarian (A2780), MCF-7 and epi-
dermoid (A431) cancer cell lines, promoted an apoptotic cell death and modulated the cell
cycle progression (arrest at phase G1) [15]. Estrogen receptor (ER), which is a transcription
factor, was also involved in cell proliferation. Wendlandt et al. [16] showed that oxime
estrogen dimers are able to effectively enter cells and modulate ER
signaling. Otherwise, 2-methoxyestradiol (2ME2) is a naturally occurring E2 derivative
with antitumor and antiangiogenic properties, acting through the binding to -tubulin
α-mediated genomic
β
near the colchicine-binding site. In fact, 2ME2 inhibited microtubule polymerization and
induced mitotic arrest [17,18].
In another series of steroids, pregnenolone 20-oxime derivatives showed relevant activ-
ity as CYP45017α and 5α-R enzyme inhibitors [19–22]. The introduction of an oxime group
at C6 in androstane series was explored in the development of aromatase inhibitors [23].
Concerning antiproliferative assays, previous studies showed that the presence of
oximes in the steroid scaffold originated compounds with relevant potential anticancer
interest. For example, 6E-hydroxyimino cholest-4-ene derivatives isolated from ma-
rine sponges showed relevant antiproliferative activities against several types of cancer
cells [
growth and tubulin polymerization inhibitory effects. This series of compounds included
2-(2 ,2 ,2 -trifluoroethoxy)- and 2-ethoxy-6-oxoestradiol as well as their corresponding
4,5]. Later, Cushman et al. developed several 2ME2 analogues with cancer cell
´
´ ´
oximes, which demonstrated clearly higher antiproliferative effects than the ketone ana-
logues in several cancer cell lines, including breast, prostate and colon tumoral cells. In
addition, these compounds also inhibited tubulin polymerization and had low binding
affinities to ER
α
[
17
,24]. In estrane steroids, Rzheznikov and co-workers [25] synthe-
sized 9 -hydroxy,11
α
β-nitrooxyestrone-17-oxime and evidenced in vivo its antitumor effect
against breast cancers. However, when compared with the corresponding 17-ketone
analogue, the presence of the oxime group seemed to have low influence in this activ-
ity. In addition, these two compounds stimulated the tumor growth by the end of a
15-day treatment course, possibly due to their estrogenic effects [25]. Concerning sub-
stituted oximes, a large number of estrone-16-oxime ethers were synthesized and their
antiproliferative effects were in vitro evaluated against several cell lines. Of these, among
other compounds, interesting results were observed for 3-benzyloxy-16-propionyloximino-
13α-methylestrone and 3-(4-methoxybenzyloxy)-16-methoximinoestrone as well as for
the unsubstituted oximes 16-oximinoestrone and 3-sulfamoyloxy-16-oximinoestrone [15].
Sánchez-Sánchez et al. [26] also evidenced the antitumor effects of steroidal sapogenin
oximes on HeLa cell lines and showed that the antiproliferative activity was 2.3–2.8 times
higher than the observed with diosgenin. More recently, C20 oxime ester derivatives were
prepared from 16-dehydropregnenolone acetate and showed cytotoxicity against leukemia
(NB4), prostate (PC-3) and HeLa cancer cells [27].
In view of the therapeutic importance of steroidal oximes, and considering our interest
in developing modified estrane derivatives as anticancer agents [
7,28], the present study
focuses on the synthesis and antiproliferative evaluation of new E1 derivatives bearing

Molecules 2021, 26, 2687
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an oxime group at C17. Their cytotoxic activities were tested using breast (MCF-7, T47D),
prostate (LNCaP), liver (HepaRG), colon (Caco-2) and normal fibroblast (NHDF) cell lines.
For the most promising compounds the IC50 was determined and then an estrogenicity as-
say, cell cycle analysis by flow cytometry after propidium iodide staining and fluorescence
microscopy using Hoechst 3358 were performed. Molecular docking studies against the
ERα, ST, 17β-HSD1 and β-tubulin were also accomplished.
2. Results
2.1. Chemistry
Six steroidal oximes in estrane series were synthesized as shown in Scheme 1, five of
which for the first time (compounds 5, 6, 10–12), to the best of our knowledge. All com-
pounds were characterized by spectral analysis (IR, 1H- and 13C-NMR; in Supplementary
Materials) and HRMS was also obtained for the new prepared steroidal oximes. All spectral
data are in agreement with the presented structures. The NOH signal in 1H-NMR appeared
near 10 ppm. In 13C-NMR spectra, the signal of C17-ketone appeared near 220 ppm and
the C17-NOH near 168–172 ppm. The presence of
∆ 8 and 11)
9,11 double bond (compounds
was associated to the signal of C11-H that appeared at 6.06 in the 1H-NMR spectrum [29].
Scheme 1. Synthetic route to prepare estrone oxime derivatives. Reagents and conditions: (
NH2OH HCl, NaOH, EtOH, H2O, reflux; ( ) I2, morpholine, PhH, rt;
) HNO3, AcOH, T = 50 ◦C; (
(d) DDQ, MeOH, reflux; (e) N-bromosuccinimide, EtOH, rt.
a)
·
b
c

Molecules 2021, 26, 2687
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The nitration reaction was performed as described by Stubenrauch et al. [30], which
was applied by these authors to obtain 2-nitroestrone
nitrating agent was used, 2,4-dinitroestrone was also formed and the mixture of products
was separated by column chromatography. The yields of these nitro-steroids were similar
to the previously described ones [30]. The introduction of halogens (compounds and
and of ) were effected using methodologies already applied
9,11 double bond (compound
by us [28]. Finally, for the preparation of oximes we selected a method involving the use of
EtOH, NaOH and hydroxylamine hydrochloride [ ] as this is a more selective and greener
3. However, as an excess of the
4
7
9)
∆
8
8
strategy than other approaches that use, for example, pyridine [25]. In fact, these methods
involve more toxic reagents/solvents, are more time consuming, have complex workups
and can lead to lower reaction yields [31–33].
2.2. Biological Testing
2.2.1. Cell Proliferation Studies
All compounds were in vitro tested on MCF-7, T47D, LNCaP, HepaRG, Caco-2 and
NHDF cell lines by the MTT colorimetric assay. In this context, it is important to men-
tion that the results for non-oxime compounds 7–9 were described in our previous re-
search work [28].
Firstly, a screening study was performed at 30 µM for a first analysis of the cytotoxic
effect of these compounds (Figure 1). This screening showed that several oximes led to a
higher reduction in cell proliferation than the observed with parent compounds, which was
particularly evident for compounds
5 and 11 in most cell lines. In addition, these two com-
pounds and oxime were the most cytotoxic in these experimental conditions. Furthermore,
2
the cell lines mostly affected by all compounds were MCF-7 and HepaRG. On the other
hand, only compound 11 promoted a significant reduction of LNCaP cells proliferation.
MCF-7
T47-D
LNCaP
250
200
150
100
50
250
200
150
100
50
250
200
150
100
50
*
***
***
**
**
***
**
*
***
0
0
0
l
ro
l
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
ol
r
10
11
12
10
11
12
10
11
12
E1
E1
E1
ro
t
n
t
t
on
n
o
o
C
C
C
HepaRG
NHDF
Caco-2
250
200
150
100
50
250
200
150
100
50
250
200
150
100
50
***
***
*
***
*
***
***
***
***
0
0
0
l
ro
l
ro
2
3
4
5
6
2
3
4
5
6
2
3
4
5
6
ol
10
11
12
10
11
12
10
11
12
E1
E1
E1
r
t
t
on
t
n
n
o
o
C
C
C
Figure 1. Relative cell proliferation of hormone-dependent (MCF-7, T47-D, LNCaP) and hormone-
independent cancer cells (HepaRG, Caco-2 and NHDF) incubated for 72 h at 30 M with the synthe-
µ
sized compounds, determined by the MTT assay, spectrophotometrically quantifying formazan at
570 nm. Data are expressed as a percentage of cell proliferation relative to the negative control, are in-
dicated as means
** p < 0.01, * p < 0.05 vs. control (the Student t-test).
±
SD and are representative of at least two independent experiments. *** p < 0.001,
After the screening, for the cases where a reduction of cell proliferation was higher
than 50%, the IC50 was determined (Table 1). Generally, the estimated IC50 values were
in agreement with the results observed in the screening, confirming that the most potent

Molecules 2021, 26, 2687
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compounds were oximes
2
,
5
and 11. Of these, the most cytotoxic was
∆
9,11-estrone oxime
(compound 11) on LNCaP cells (IC50 = 3.59
µM). In addition, the highest selectivity index
was also observed for this derivative in these cells (Table 2). However, the variability
of MTT assays was higher in LNCaP cells, as we and others have experienced with this
poorly adherent cell line, and therefore the fit was less good and the uncertainty is higher
in this case.
Table 1. Estimated IC50 values (
µM) for various compounds in breast (MCF-7, T47-D), prostate
(LNcaP), liver (HepaRG), colon (Caco-2) and normal fibroblast cells (NHDF) a.
Compound
MCF-7
T47-D
LNCaP
HepaRG
Caco-2
NHDF
1
2
5
41.93
26.65
16.75
ND
25.63
ND
ND
29.26
ND
ND
43.45
ND
ND
ND
ND
ND
3.59
ND
7.79
29.53
16.94
12.32
28.00
18.35
21.94
1.78
42.69
ND
18.51
ND
24.33
ND
1.31
61.82
43.88
ND
ND
30.84
ND
10
11
12
5-FU
1.71
0.54
3.61
a
Cells were treated with different concentrations (0.1, 1, 10, 25, 50 and 100 µM) during 72 h. The cell proliferation
effects were determined by the MTT assay. The data shown are representative of at least two independent
experiments. 5-FU: 5-fluoruracil. ND: not determined.
Table 2. Selectivity index a of compounds 1, 2, 11 and 5-fluouracil (5-FU).
Compound
MCF-7
T47-D
LNCaP
HepaRG
Caco-2
1
2
11
1.47
1.65
1.20
2.11
ND
1.50
0.71
6.69
ND
ND
8.59
0.46
2.09
2.59
1.68
2.03
1.45
ND
1.27
2.76
5-FU
a
Selectivity index is the ratio of the IC50 values of the treatments of non-tumor cells (NHDF) and tumor cells
(MCF-7, T47-D, LNCaP, HepaRG and Caco); ND: not determined.
Although the structure of E1 oxime (compound 2) is widely known [8,9], few studies
concerning its biological activity have been published so far. Interestingly, our data showed
a good antiproliferative activity of this compound against HepaRG cells (IC50 = 16.94 µM).
9,11
When evaluating the effect of the presence of
pound
∆
double bond (compound 11 vs. com-
2
), it is interesting to note that the effect depends on the cell line, being observed
a higher cytotoxicity for compound 11 in MCF-7, LNCaP, Caco-2 and NHDF cells. Con-
cerning the effect of A-ring modifications in these oximes, the introduction of 2-nitro group
(compound
sence (steroid
bromination led to a lower cytotoxicity than the non-functionalized A-ring (compound
and 2-nitroestrone oxime . However, similarly to our previously published results with
5
) allowed an improvement of the cytotoxicity when comparing with its ab-
2) in MCF-7, HepaRG and Caco-2 cells. In the other hand, the iodination and
2)
5
2,4-diiodo- and 2,4-dibromoestrone [28], interesting IC50 values were observed for A-ring
halogenated E1 oximes 10 and 12 on HepaRG cancer cells. In this context, it is important
to mention that the nitro group(s) are susceptible to reduction by nitro reductases and
the
forces the importance of study its cytotoxic effect in hepatic cells [34]. On the other hand,
2,4-dinitroestrone oxime (compound ) displayed low antiproliferative activity. Finally,
∆
9,11 bond of compound 11 is prone to oxidation by the CYP P450 family, which rein-
6
considering the data on Figure 1 and Tables 1 and 2, it is clear that the majority of these
compounds had higher cytotoxicity against tumoral (MCF-7, T47D, LNCaP, HepaRG and
Caco-2) than non-tumoral (NHDF) cells.
To determine the potential estrogenic profile of the synthesized compound with the
most relevant anti-proliferative activity (steroid 11), its cell growing effect was measured
on the estrogen-sensitive breast cancer T47-D cells (ER+) in serum-free culture medium.
This proliferative/estrogenic activity was expressed as the difference between the cell
proliferation (in percentage) caused by a given compound and the basal cell proliferation

Molecules 2021, 26, 2687
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fixed at 100% (Figure 2) [35
,36]. E2 was also tested as reference compound. Unfortunately,
similarly to the observed with E2, compound 11 also stimulated the cell proliferation at
0.001 and 0.01 µM when compared with the negative control.
Figure 2. Proliferation of estrogen-sensitive T47-D cells incubated with 17
11 for 24 h. Each bar represents the mean SD (originated from two independent experiments).
*** p < 0.001, ** p < 0.01 vs. control (one way ANOVA post-hoc Bonferroni test).
β-estradiol and compound
±
In our previous study, it was also evidenced that ∆ 8) also stim-
9,11-E1 (compound
ulated the proliferation of T47-D cells [28] and therefore, its conversion into the oxime
analogue did not eliminate this effect. In this context, Palomino et al., [37] using X-ray
crystallography and molecular modeling studies, showed that the presence of ∆
9,11 dou-
ble bond caused a flattening of B, C and D rings and consequently reduced the binding
to ER in 1/5th in comparison with E2. Despite this, as evidenced by our results, the
presence of C9 = C11 double bond did not eliminate the estrogenic effect characteris-
tic of these compounds. In this context, other reports also showed that the presence of
an oxime group did not eliminate this effect. In fact, OMATE had a stimulatory effect
(0.15 g ± 0.01) on the uterine growth in ovariectomized rats, which was approximately
50% higher than that of EMATE (0.11 g
±
0.02) [
8]. In addition, and as previously referred,
despite that 9 -hydroxy,11
α
β-nitrooxyestrone-17-oxime had relevant anti-breast cancer ef-
fects, this compound also stimulated the tumor growth after a 15-day treatment period [25].
This preliminary study seemed to suggest the estrogenic activity of compound 11. Further
studies will be necessary to elucidate this activity (e.g., using a luciferase assay) [38].
2.2.2. Cell Survival, Cell Cycle Distribution Evaluation and Hoechst 33,258 Staining
The possible mechanism of action of compound 11 was studied by flow cytometry
after PI staining. This assay was performed in LNCaP cells at 24 h post treatment, and
5-FU was used as the positive control. In this cell line, it was observed that compound 11
led to 11% reduction in cell viability (Figure 3). This effect was similar to the one originated
by 5-FU (12%). In addition to this flow cytometry study, cells were also observed using an
optic microscope (Figure 4) and, after 24 h of treatment with compound 11, it was possible
to see small modifications in LNCaP cells, which lost their shape, becoming rounded, as it
is characteristic to happen during mitosis.
The arrest of cell cycle progression is one of the strategies used to stop cancer pro-
liferation [39]. In this context, some studies previously published showed the effect of
steroidal and non-steroidal oximes in cell cycle [26,40]. As example, it was evidenced that
E1-16-oxime ethers promoted the apoptotic HeLa cell death and modulated the cell cycle
progression (arrest at G1), leading to an increase in cellular shrinkage, nuclear condensation,
membrane permeability, sub-diploid population and caspase-3 activity [15]. In addition,
16
arrest and caspases-3 and 9 activation [41] and
HepaRG cell cycle [28]. Thus, the interference of compound 11 in cell cycle distribution
β
-triazolyl-17
α
-estradiol 3-benzyl ethers of the 13
α-E2 series led to a G2/M cell cycle
∆
9,11-E1 induced an arrest at G0/G1 in

Molecules 2021, 26, 2687
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was also evaluated by flow cytometry. Interestingly, it was found that the treatment with
this steroid oxime (50 µM, 24 h) induced a G2/M cell cycle arrest of LNCaP cells (Figure 5).
Also, LNCaP cells were not able to pass through to the S and G2/M phases treated by
compound 5-FU, which is in agreement with literature for these cells [42].
Figure 3. Percentage of LNCaP viable cells after 24 h treatment with 50
through propidium iodide flow cytometry assay. Control corresponds to untreated cells and 5-FU
(50 M) was used for comparison. The percentage of survival is the percentage of cells in R1 (live
cells) as compared to the total number of events in R2 (dead cells) and R3 (undetermined cells). Each
bar represents the mean SD (originated from two independent experiments). *** p < 0.001 vs.
control (one way ANOVA post-hoc Bonferroni test).
µM of compound 11 evaluated
µ
±
Figure 4. Photographs of the LNCaP cells (
5-FU (C) for 24 h. Amplification of 100x.
A, control) treated with 50 µM of compound 11 (B) and
Figure 5. Cell cycle distribution analysis of LNCaP cancer cells after treatment with compound
11 at 50 M for 24 h. Each bar represents the mean SD of four samples (originating from two
µ
±
independent experiments). ** p < 0.01 vs. control; *** p < 0.001 vs. control (one way ANOVA post-hoc
Bonferroni test).

Molecules 2021, 26, 2687
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Apoptosis is essential for maintaining the physiologic balance between cell death and
cell growth [43]. Therefore, studies for understanding the cancer cell cycle, particularly
the interplay with chromatin control, are providing opportunities for developing a new
range of anti-cancer drugs [44]. In this context, using a preliminary assay, the Hoechst
33,258 fluorescent dye was used by us to analyze nuclei morphology of LNCaP cells by
fluorescence microscopy after exposition to 50
Interestingly, it was observed the presence of condensed DNA, typical of prophase, and a
small proportion of condensed and fragmented nuclei, typical of apoptosis [45]. -Tubulin
µM of compound 11 during 24 h (Figure 6).
β
is a protein that polymerize into microtubules, which are involved in cell movement,
intracellular trafficking and mitosis [46]. Tubulin-binding drugs, such as paclitaxel and
docetaxel [47], kill cancerous cells by inhibiting microtubule dynamics leading to mitotic
arrest and cell death. In this context, as it was observed that compound 11 promoted a cell
cycle arrest at G2/M (Figure 5) and also led to the formation of condensed DNA typical of
prophase, plus condensed and fragmented nuclei typical of apoptosis (Figure 6), it can be
speculated that it can act by interference with
other steroids of the estrane series like as estramustine and 2ME2 [17
section, we studied the interaction of compound 11 and
β
-tubulin, similarly to which occurs with
24 48]. In the next
-tubulin by docking assay to try
,
,
β
to better understand the cell cycle arrest.
Figure 6. Fluorescence microscopy images of DNA staining (Hoechst 33258) in LNCaP cells treated
with vehicle (Control) or with compound 11 at 50 M for 24 h. Blue down-pointing arrows: condensed
µ
DNA onto visible chromosomes (prophase); Orange up-pointing arrow: condensed and fragmented
nuclei (apoptosis).
2.3. Molecular Docking Studies
Taking into account the enzymes involved in steroidogenesis, and given the structural
similarity of the compounds of our study and several of the above referred steroidal oximes
acting by interaction with the mentioned targets, we aimed to evaluate the affinities of the
steroids prepared by us and the proteins ERα, ST, 17β-HSD1 and β-tubulin. Molecular
docking is a standard computational tool that has been successfully employed in drug
design and discovery studies. Satisfactory docking results can be obtained when relatively
small ligands with few rotatable bonds are docked towards protein binding pockets in
which flexibility does not play an important role. However, for complex molecules (with
many rotatable bonds and flexibility), the use of methodology involving theoretical docking
and molecular dynamics techniques are important to overtake these limitations, because
they allow for evaluating and selecting the best molecule generated in the molecular
docking, which can affect the results [49–51]. The three-dimensional structural coordinates
of these three protein receptors were obtained from PDB, and molecular docking was
performed using the program AutoDock vina. To validate the standard docking method,
simulations were carried out between crystallized ligands/drugs with the respective
proteins and all control redocking simulations were able to reproduce the ligand-protein
interaction geometries presented in the respective crystal structures with a RMSD
Then, all compounds were docked for the referred targets, as observed in Table 3.
≤
2.0 Å.

Molecules 2021, 26, 2687
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Table 3. Predicted affinity energies of compounds 1–12 calculated against the estrogen receptor
(ST), 17 -hydroxysteroid dehydrogenase type 1 (17 -HSD1) and -tubulin by AutodockTools with vina executable. Binding
energies of co-crystalized ligand in the X-ray crystal structures were calculated by re-docking.
α (ERα), steroid sulfatase
β
β
β
Lowest Energy (kcal.mol−1
)
Compound
ERα
ST
17β−HSD1
β−Tubulin
−10.3 b
−9.7
−8.8
−6.6
−7.9
−5.3
−4.3 b
−10.9 b
−6.8 b
−5.8
−10.5
−5.0
−9.9 a
−
−6.2 b
−6.7
−7.0
−7.0
−6.9
−6.8
−6.0 b
−5.9 b
−6.3 b
−6.6
−6.5
−6.3
−
−8.1 b
−8.1
−8.2
−8.1
−8.3
−8.1
−7.7 b
−8.2 b
−8.1 b
−7.7
−8.3
−7.6
−
1
2
3
4
5
6
7
8
9
10
11
12
E2
−9.0
−8.9
−8.7
−8.5
−8.9
−8.4
−8.4
−9.0
−9.0
−8.5
−9.1
−8.5
−
N−acetyl−D−glucosamine
5α−Dihydrotestosterone
Colchicine
−7.2 a
−
−
−
−
−8.3 a
−
−
−
−
≤
−8.4 a
a
b
The RMSD between re-docked ligands and the corresponding X-ray crystal structure coordinates was
in a previous study [28].
2. These values were described
The results observed in the docking simulations with ER
compounds 7–9 are presented in our previous work [28]. According to the data presented
in Table 3, compound 11 can bind ER in a lower energy than the control (E2). In addition,
α, ST and 17β-HSD1 for
α
in Figure 7 can be observed that this compound can form two hydrogen bonds between
its oxime group at C17 and Hist 524 and between the hydroxyl group at C3 and Glu 353
of ERα target. These interactions are similar to the observed with E2. Therefore, globally,
these docking data seem to be in agreement with our experimental results (Figure 2).
The predicted affinity energies of the synthesized compounds for ST are all higher than
the energy obtained for co-crystalized ligand (Table 3). This suggested that these compounds
have a poor affinity to this macromolecule. Concerning the energy values obtained in the
docking studies with 17
the co-crystalized ligand (5
results, we also analyzed the interaction mode of the best ranked compounds,
with 17 -HSD1. It is already known from the literature that the main interactions between
β
-HSD1, generally they were very close to the determined affinity of
-dihydrotestosterone, DHT) (Table 3). Due to these interesting
and 11
α
5
,
β
DHT and this enzyme are a conventional hydrogen bond with Hist 221 residue and Van der
Waals interactions with Leu 149, Val 143 and Pro 187 residues [52]. However, the studied
compounds just exhibited the Van der Waals interactions, lacking the hydrogen bond with
Hist221, which can be determinant for their interaction with this target. Further in vitro
studies will be necessary to elucidate the significance of this interaction. The most interesting
binding was observed between compound 11 and
Besides the good affinity energy value, which was lower than the determined for colchicine,
compound 11 was also predicted to have the most important interactions with -tubulin,
β-tubulin, as shown in Figure 8 and Table 3.
β
such as the conventional hydrogen bond with Cys B 241, alkyl and Pi-alkyl interactions
with Leu B 248, and Van der Waals interaction with Val B 318 [53–55]. Interestingly, previous
studies suggested that the tubulin ligand interactions through amino acid residues Ala316
and Val318 are very crucial in inducing antitubulin effect [55]. The other studied compounds,
despite having good binding energies, did not establish the conventional bonds to tubulin.
Compounds 8 and 9 did not show a conventional hydrogen bond with Cys B 241, interacting
only through Van der Waals, alkyl and Pi-alkyl interactions, which explain that the affinity
energies may not be directly related to the established interactions required with the active site.
Therefore, the cell cycle arrest at G2/M originated by compound 11 (Figure 5), at prophase
(Figure 6), can perhaps occur due to
to prove this hypothesis.
β-tubulin inhibition. However, future studies are needed

Molecules 2021, 26, 2687
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Figure 7. Analysis of predicted ER
α binding orientations for the best raking compound, 11. (A) 3D
molecular and (B) 2D docking results showing the main interactions.
A
B
C
Figure 8. 2D diagram of the active interaction between colchicine and
diagram of predicted interactions between best ranked compound, 11, and
3D representation (panel C). ( ) Conventional hydrogen bonds are displayed in green, Van der
Waals interactions in light green, Pi-sigma interactions are displayed in purple and alkyl an Pi-alkyl
interactions in pink. ( ) All contact and hydrogen bonds are shown as dotted lines, and represented
with same colors above described.
β-tubulin (panel A). 2D
β-tubulin (panel B) and
A,B
C

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3. Materials and Methods
3.1. Chemistry
All chemicals received from suppliers were used without further purification. The
following reagents were purchased from: E1: Cayman Chemical (Ann Arbor, MI, USA);
hydroxylamine hydrochloride: Fluka (Buchs, Switzerland; methanol (MeOH): Fisher
Chemical (Hampton, MA, USA); N-bromosuccinimide (NBS): Alfa Aesar (Haverhill, MS,
USA); ethanol (EtOH) 99.9%: Manuel Vieira & C
ª
(Torres Novas, Portugal). In addi-
tion, 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), morpholine, 17β-estradiol (E2), and
dimethyl sulfoxide (DMSO) as well as the remaining chemical products referred in the text
were obtained from Sigma-Aldrich (St. Louis, MO, USA). Deuterated DMSO (DMSO-d6)
and deuterated chloroform (CDCl3) were purchased from Armar Chemicals (Leipzig, Ger-
many). All reactions were monitored by thin layer chromatography (TLC) using Al-backed
aluminum/silica gel plate 0.20 mm (Macherey-Nagel 60 F254, Duren, Germany). After
elution, plates were visualized in a CN-15.LC UV chamber under ultraviolet (UV) radiation
(254 nm). Then, the EtOH/concentrated sulfuric acid (95:5, v:v) solution was used, fol-
◦
lowed by heating at 120 C, to reveal the plates. The evaporation of solvents was achieved
by using a rotary vacuum drier from Büchi (R-215). Infrared (IR) spectra were collected
on a Thermoscientific Nicolet iS10 equipped with a diamond attenuated total reflectance
crystal at room temperature in the 4000–400 cm−1 range by averaging 16 scans at a spectral
resolution of 2 cm−1. Nuclear magnetic resonance (NMR) spectra (1H-NMR and 13C-NMR)
were acquired on a Bruker Avance 400 MHz spectrometer and were processed with the
software TOPSPIN 4.07 (Bruker, Fitchburg, WI, USA). Chemical shifts are reported in parts
per million (ppm) relative to tetramethylsilane (TMS) or solvent as an internal standard.
Coupling constants (J values) are reported in hertz (Hz) and splitting multiplicities are
described as s = singlet; brs = broad singlet; d = doublet and dd = double doublet. High
resolution mass spectrometry (ESI-HRMS) was performed by the microanalysis service on
a QSTAR XL instrument (Salamanca, Spain).
3.1.1. Procedures for the Synthesis of Intermediates—Compounds 3, 4, 7, 8 and 9
The intermediates
7, 8 and 9 were synthesized and structurally characterized as
previously described [28].
Synthesis of 3-hydroxy-2-nitroestra-1,3,5(10)-trien-17-one (3) and
3-hydroxy-2,4-dinitroestra-1,3,5(10)-trien-17-one (4)
E1 (541 mg, 2 mmol) was added to 16.3 mL of glacial acetic acid and the mixture
was vigorous stirred at 50 ◦C. Then, a solution of nitric acid 70% (178
µL) in glacial acetic
acid (540.6
µL) was added dropwise and the mixture was stirred at room temperature
for 48 h. After completion (TLC control), the reaction mixture was diluted in 150 mL of
ethyl acetate and the resulting solution was washed with 50 mL of saturated NaCl solution,
50 mL of saturated NaHCO3 solution and 50 mL of H2O. Next, the solution was dried over
anhydrous Na2SO4, filtered and concentrated under reduced pressure to yield the crude
product, which was purified by column chromatography (eluent: gradient of ethyl acetate
(EA)/petroleum ether (PE) 40–60 ◦C, 1:3; 1:1 and 3:1) to give compound 3 as yellow solid
(261 mg, 41%) and compound 4 as orange solid (341 mg, 47%) [30,56,57].
3-Hydroxy-2-nitroestra-1,3,5(10)-trien-17-one (3)
IR (υmax/cm−1): 896, 1258, 1306, 1372, 1429, 1478, 1519, 1563, 1628, 1733, 2858–2932,
3040, 3295; 1H-NMR (400 MHz, CDCl3)
δ
: 0.89 (s, 3H, C18-CH3), 6.84 (s, 1H, C4-H), 7.96 (d,
1H, J = 1.1 Hz, C1-H), 10.39 (s, 1H, OH); 13C-NMR (100 MHz, CDCl3)
δ: 13.97, 21.75, 25.90,
26.10, 29.83, 31.47, 35.98, 37.93, 43.66, 48.02, 50.56, 119.18, 121.77, 131.98, 133.31, 149.00,
153.12, 220.37.

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3-Hydroxy-2,4-dinitroestra-1,3,5(10)-trien-17-one (4)
IR (υmax/cm−1): 902, 1246, 1376, 1455, 1532, 1571, 1629, 1729, 2872–2939, 3185; H-
1
NMR (400 MHz, CDCl3)
δ
: 0.90 (s, 3H, C18-CH3), 8.14 (d, 1H, J = 1.0 Hz, C1-H), 10.60 (s,
1H, OH); 13C-NMR (100 MHz, CDCl3)
δ: 13.91, 21.64, 24.95, 25.06, 25.98, 31.33, 35.89, 37.21,
43.65, 47.85, 50.23, 122.87, 132.33, 133.72, 139.29, 141.87, 145.06, 219.76.
3.1.2. General Procedure for the Synthesis of Oximes
To a solution of parent compound in EtOH (8.1 mL/1 mmol of parent compound)
were added hydroxylamine hydrochloride (2.9 mmol), NaOH (2 mmol) and water (272 µL).
The mixture was heated under reflux for 3 h and upon cooling poured into an aqueous
solution of 1 N HCl (2.7 mL). The formed precipitate was filtered, washed with cold water,
and air-dried to give the corresponding product. After, the solid was recrystallized using
MeOH to afford the pure product [8]. As compounds 6 and 12 did not precipitate after
addition to HCl solution, the work up was performed as described below.
Synthesis of 17-hydroxyiminoestra-1,3,5(10)-trien-3-ol (2)
Compound 2 was prepared from compound 1 (270.37 mg, 1 mmol) to give white solid
(265 mg; 93%). After recrystallization compound 2 was obtained as colorless crystals [8]. IR
(
υmax/cm−1): 869, 918, 1151, 1237, 1283, 1348, 1373, 1460, 1497, 1583, 1618, 2868–2929, 3025,
3252, 3405; 1H-NMR (400 MHz, DMSO-d6)
δ: 0.85 (s, 3H, C18-CH3), 6.44 (d, 1H, J = 2.5 Hz,
C4-H), 6.51 (dd, 1H, J1 = 8.5 Hz, J2 = 2.5 Hz, C2-H), 7.04 (d, 1H, J = 8.5 Hz, C1-H), 8.99 (brs,
1H, C3-OH), 10.08 (brs, 1H, NOH); 13C-NMR (100 MHz, DMSO-d6)
δ: 17.32, 22.51, 24.89,
25.92, 26.84, 29.08, 34.27, 37.87, 43.36, 43.59, 52.49, 112.75, 114.94, 125.99, 130.15, 137.08,
154.96, 167.95.
Synthesis of 17-hydroxyimino-2-nitroestra-1,3,5(10)-trien-3-ol (5)
Compound 5 was prepared from compound 3 (157.68 mg, 0.5 mmol) to give a yellow
solid (120 mg, 73%). After recrystallization from MeOH, compound 5 was obtained as
yellow crystals. IR (υmax/cm−1): 927, 1017, 1095, 1259, 1298, 1373, 1433 1482, 1518, 1579,
1633, 2858–2961, 3283; 1H-NMR (400 MHz, DMSO-d6)
δ
: 0.85 (s, 3H, C18-CH3), 6.83 (s, 1H,
C4-H), 7.75 (s, 1H, C1-H), 10.11 (s, 1H, C3-OH), 10.54 (brs, 1H, NOH); 13C-NMR (100 MHz,
DMSO-d6) δ: 17.21, 22.46, 24.87, 25.52, 26.11, 28.83, 33.97, 37.04, 42.86, 43.23, 52.34, 118.49,
121.45, 131.91, 134.07, 145.96, 150.25, 167.74. HRMS (ESI-TOF): m/z [M+ + H] calcd for
C18H22N2O4: 330.1580; found 330.1573.
Synthesis of 17-hydroxyimino-2,4-dinitroestra-1,3,5(10)-trien-3-ol (6)
Compound 6 was prepared from compound 4 (180.04 mg, 0.5 mmol). As the crude did
not precipitate after the addition of HCl, the workup was performed in a different manner.
For this, the reactional mixture was diluted in 100 mL of dichloromethane and washed
with 50 mL of saturated NaCl solution, 50 mL of saturated NaHCO3 solution and 50 mL of
H2O, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure.
For analysis and use in cell studies, a sample was recrystallized from MeOH to afford
compound 6 as dark yellow crystals (130.7 mg, 73%). IR (υmax/cm-1): 927, 1025, 1184,
1259, 1305, 1356, 1377, 1436, 1468, 1537, 1577, 1630, 2931, 3240, 3612; 1H-NMR (400 MHz,
DMSO-d6) δ
: 0.86 (s, 3H, C18-CH3), 7.99 (s, 1H, C4-H), 10.12 (s, 1H, NOH); 13C-NMR
(100 MHz, DMSO-d6) δ: 17.17, 22.39, 23.94, 24.85, 24.96, 25.43, 33.87, 36.14, 42.76, 43.17,
52.01, 122.79, 133.01, 135.25, 135.99, 141.98, 142.92, 167.62. HRMS (ESI-TOF): m/z [M+ + H]
calcd for C18H21N3O6: 375.1430; found 375.1424.
Synthesis of 17-hydroxyimino-2,4-diiodooestra-1,3,5(10)-trien-3-ol (10)
Compound 10 was prepared from compound 7 (99.06 mg, 0.19 mmol) to give a beige
solid (69 mg, 34%). IR (υmax/cm−1): 800, 926, 1027, 1098, 1170, 1262, 1293, 1327, 1388, 1450,
2864–2927, 3270, 3451; 1H-NMR (400 MHz, CDCl3)
δ
: 0.94 (s, 3H, C18-CH3), 7.59 (s, 1H,
C1-H); 13C-NMR (100 MHz, CDCl3)
δ: 17.23, 23.02, 25.75, 26.63, 28.13, 33.95, 37.28, 37.29,

Molecules 2021, 26, 2687
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44.01, 44.54, 52.88, 78.57, 92.24, 136.01, 136.28, 140.82, 151.64, 172.12. HRMS (ESI-TOF): m/z
[M+ + H] calcd for C18H21I2NO2: 536.9662; found 536.9632.
Synthesis of 17-hydroxyimino-3-hydroxyestra-1,3,5(10),9(11)-tetraen-3-ol (11)
Compound 11 was obtained from compound 8 (67.1 mg, 0.25 mmol) to give a white
solid (55.2 mg, 78%). IR (υmax/cm−1): 926, 1155, 1246, 1279, 1354, 1426, 1465, 1494, 1575,
1613, 1627, 2831–2956, 3020, 3342; 1H-NMR (400 MHz, DMSO-d6)
δ: 0.86 (s, 3H, C18-CH3),
6.06 (m, 1H, C11-H), 6.45 (d, 1H, J = 2.5 Hz, C4-H), 6.54 (dd, 1H, J1 = 8.7 Hz, J2 = 2.5 Hz,
C2-H), 7.42 (d, 1H, J = 8.7, C1-H), 9.25 (br s, 1H, OH); 10.18 (br s, 1H, NOH). 13C-NMR
(100 MHz, DMSO-d6) δ: 18.03, 23.56, 25.32, 27.98, 29.32, 36.89, 37.47, 41.59, 49.88, 113.86,
114.87, 116.04, 125.11, 125.29, 135.18, 137.18, 156.18, 168.05. HRMS (ESI-TOF): m/z [M+ + H]
calcd for C18H21NO2: 283.1572; found 283.1564.
Synthesis of 17-hydroxyimino-2,4-dibromo-3-hydroxyestra-1,3,5(10)-trien-3-ol (12)
Compound 12 was obtained from compound 9 (107.4 mg, 0.25 mmol). As the crude
did not precipitate after the addition of HCl, the workup was performed in a different
manner. For this, the reactional mixture was diluted in 150 mL of dichloromethane and
washed with 50 mL of HCl 5%, 50 mL of saturated NaCl solution and 50 mL of H2O, dried
over anhydrous Na2SO4, filtered and concentrated under reduced pressure to produce a
white solid 12 (95 mg, 86%). IR (υmax/cm−1): 799, 927, 1019, 1097, 1173, 1260, 1398, 1463,
2869–2961, 3273, 3486; 1H-NMR (400 MHz, CDCl3)
C1-H); 13C-NMR (100 MHz, CDCl3)
: 17.28, 23.04, 25.55, 26.56, 27.36, 31.13, 33.98, 37.27,
δ: 0.93 (s, 3H, C18-CH3), 7.38 (s, 1H,
δ
44.10, 44.43, 52.92, 106.64, 113.43, 128.69, 135.41, 136.67, 147.43, 171.72. HRMS (ESI-TOF):
m/z [M+ + H] calcd for C18H21Br2NO2: 440.9939; found 440.9932.
3.2. Biology
3.2.1. Cell Culture
Breast (MCF-7, T47-D), prostatic (LNCaP) and colon (Caco-2) cancer cells, as well as
normal human dermal fibroblasts (NHDF), were obtained from American Type Culture
Collection (ATCC; Manassas, VA, USA). The hepatic (HepaRG) cell line was acquired
to Life Technologies-Invitrogen
™ (through Alfagene, Portugal). They were cultured in
◦
75 cm2 culture flasks at 37 C in a humidified air incubator with 5% CO2. MCF-7 cells
were maintained with high-glucose Dulbecco’s modified Eagle medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA) and 1%
antibiotic/antimycotic mixture (10,000 units/mL penicillin G, 100 mg/mL streptomycin
and 25 µg/mL amphotericin B) (Sigma-Aldrich, St Louis, MO, USA). High glucose DMEM
supplemented with 10% FBS and 1% of the antibiotic mixture of 10,000 units/mL penicillin
G and 100 mg/mL of streptomycin (Sigma-Aldrich, St Louis, MO, USA) was used for Caco-
2 cells. LNCaP and T47-D cells were cultured in RPMI 1640 medium with 10% FBS and 1%
antibiotic mixture. NHDF cells grew in RPMI 1640 medium supplemented with 10% FBS,
2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 1% antibiotic/antimycotic.
Lastly, hepatic cells were seeded in Williams’ E medium supplemented with 10% FBS,
1% antibiotic mixture, 5
(Sigma–Aldrich, St Louis, MO, USA).
µ
g/mL insulin, and 5
×
10−5 M hydrocortisone hemisuccinate
3.2.2. Preparation of Stock Solutions
The stock solutions of compounds were prepared in DMSO at 10 mM and stored at
4–8 ◦C. From these, the different diluted solutions of compounds were prepared in the
corresponding complete culture medium before each experiment. The maximum DMSO
concentration in cell studies was 1%.
3.2.3. Antiproliferative Activities against Six Cell Lines
The antiproliferative effect of compounds was evaluated by the 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St Louis, MO, USA) assay in

Molecules 2021, 26, 2687
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MCF-7, T47-D, LNCaP, HepaRG, Caco-2 and NHDF cells. After reaching near confluence,
cells were trypsinized and counted with a hemocytometer by means of the trypan-blue
exclusion of dead cells. Then, 100
96-well culture plates and left to adhere and growth during 48 h. After this period, the
medium was replaced by solutions of the compounds in study (30 M for screening assays
and 0.1, 1, 10, 25, 50 and 100 M for concentration-response studies) in the appropriate
cell culture medium for approximately 72 h. Then, cells were washed with 100 L of
phosphate buffer saline (PBS; NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM and KH2PO4
1.8 mM, pH 7.4), and 100 L of the MTT solution (5 mg/mL), prepared in the appropriate
µL of cell suspension (2
×
104 cells/mL) was seeded in
µ
µ
µ
µ
serum-free medium, was added to each well, followed by incubation for approximately
4 h at 37 ◦C. Afterward, MTT containing medium was removed and formazan crystals
were dissolved in DMSO. Absorbance was measured at 570 nm using a microplate reader
Bio-rad Xmark spectrophotometer. After background subtraction, cell proliferation values
were expressed as percentage relatively to the absorbance determined in negative control
cells. Untreated cells were used as the negative control and the clinical drug 5-fluorouracil
(5-FU) was used as positive control. Each experiment was performed in quadruplicate and
independently repeated.
3.2.4. E-Screening Assay in T47-D Cells
Breast T47-D cells (2
×
104 cells/mL; 100
µL) were seeded in 96-well culture plates in
RPMI 1640 medium supplemented with 10% FBS and allowed to attach. After overnight
incubation, the medium was replaced every 3 days with fresh phenol red free RPMI
1640 medium supplemented with 5% of dextran-coated charcoal-treated fetal calf serum
(DCC-FCS) and containing compound 11 (0.1, 0.01 and 0.001 µM). After 6 days of exposure,
the proliferation of T47-D cells was estimated by the MTT assay as described in the previous
section. Each experiment was performed in quadruplicate and independently repeated.
After background subtraction, cell proliferation values were expressed as percentage
relatively to the absorbance determined in negative control cells.
3.2.5. Analysis of LNCaP Cells Viability by Flow Cytometry
The analysis of LNCaP cells viability was performed by flow cytometry after staining
with propidium iodide (PI) (solution of PI 1 mg/mL in 0.1% of sodium azide and water;
Sigma Aldrich, St Louis, MO, USA). Briefly, 3 mL of cells suspension were seeded in 6-well
plates (5
×
104 cells/mL) in complete culture medium. After 48 h they were treated with
50 M of compound 11. Untreated cells were used as negative control and 5-FU (50 µM) was
µ
used as positive control. Each experiment was performed in duplicate and independently
repeated. At the end of 24 h of incubation, the supernatant of each well was collected,
cells were harvested by trypsinization and pooled with the supernatants. The resulting
cell suspension was kept on ice, pelleted by centrifugation and resuspended in 400
complete medium. Afterward, 395 L of the cell suspension was transferred to a FACS tube
and 5 L of PI and 0.5 L of ethylenediamine tetraacetic acid (EDTA, 0.123 M) were added.
µL of
µ
µ
µ
A minimum of 20,000 events was acquired using a BD Accuri C6 (San Jose, CA, USA)
flow cytometer in the channels forward scatter (FSC), side scatter (SSC) and fluorescence
channel-3 (FL3, for PI). Acquisition and analysis were performed with BD Accuri Software.
In the FSC/FL3 contour plot, three regions were created, one corresponding to viable cells
(R1), another to dead cells (R2) and a third to an indeterminate cell population between the
other two regions (R3) excluding debris that were not considered in the analysis (data not
shown). The percentage of viability is the percentage of cells in R1 as compared to the total
number of events in R1, R2 and R3.
3.2.6. Cell Cycle Distribution of LNCaP Cells
After 24 h of treatment with compound 11 at 50
LNCaP cells were collected and washed with PBS and resuspended in 450
solution of 0.5% bovine serum albumin (BSA; Amresco, Solon, OH, USA) and 1 mM EDTA
µ
M (6-well plates, 5
×
104 cells/mL),
L of a cold
µ

Molecules 2021, 26, 2687
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in PBS, followed by fixation with 70% EtOH and kept at
−
20 ◦C. Afterward, fixed cells
were washed twice with PBS and resuspended in a solution of PI (50
0.5% BSA and 1 mM EDTA in PBS and then incubated with Ribonuclease A from bovine
pancreas at a final concentration of 0.5 g/ L (solution in 50% glycerol, 10 mM Tris-HCl,
pH 8; Sigma Aldrich, St Louis, MO, USA) for 15 min in the dark. For comparison, untreated
cells were used as negative control and cells treated with 5-FU at 50 M were used as
µg/mL) prepared in
µ
µ
µ
positive control. Each experiment was performed in duplicate and independently repeated.
A minimum of 10,000 events was acquired using BD Accuri Software and analysis was
performed by Modfit software (Becton Dickinson, San Jose, CA, USA).
3.2.7. Fluorescence Microscopy in LNCaP Cells after DNA Staining
Near-confluent LNCaP cells were seeded in a 6-well plate (5
adherence and incubation for 24h with compound 11 (50 M), the dye Hoechst 33,258 was
added to the culture medium to achieve a final concentration of 1 g/mL. The cells were
×
104 cells/mL). After
µ
µ
incubated for 15 min at 37 ◦C and were then photographed by means of a Nikon Eclipse
microscope equipped with a fluorescence attachment containing the appropriate optical
blocks and a QCapture CCD camera. Apoptosis was revealed by nuclear changes such as
chromatin condensation and nuclear fragmentation.
3.2.8. Data Analysis
Data were expressed as mean
±
standard deviation (SD). t-Student test (two groups)
and one-way ANOVA (three groups) were used followed by Bonferroni post hoc tests
to determine statistically significant differences among the means. Difference between
groups was considered statistically significant for a p-value lower than 0.05 (p < 0.05).
The determination of IC50 was performed by sigmoidal fitting analysis [log(inhibitor) vs.
normalized response—Variable slope], considering a confidence level of 95%.
3.3. Molecular Docking Studies
3.3.1. Preparation of Proteins for Molecular Docking
The crystal structures of ER
α
(PDB ID: 1A52), ST (PDB ID: 1P49), 17β-HSD1 (PDB
ID: 3KLM) and -tubulin (PDB ID: 1SA0) were retrieved from Protein Data Bank [58
β
–
61].
The coordinates of all non-standard residues, including the co-crystalized ligand, were
deleted using the software Chimera (v. 1.10.1). Then, non-polar hydrogens were merged in
AutoDockTools (v. 1.5.6) and Kollman and Gasteiger partial charges were added. Lastly,
the prepared structure was converted from the PDB format to PDBQT for posterior use in
the docking simulations.
3.3.2. Preparation of Ligands
All ligands used in docking simulations were built using ChemDraw (v. 12.0) software.
Energy minimization and geometry optimization of these molecules were performed in
Chem3D (v. 12.0) and the obtained structures were saved as PDB file format. The process
of energy minimization was applied in a range from
ligands were completely prepared for docking choosing torsions and the structures were
converted into PDBQT format using the software AutoDockTools.
−
20 to
−
40 kcal.mol−1. Then, the
3.3.3. Grid Parameters
The grid parameters were selected using AutoDock vina and AutoDockTools based
on the coordinates of the co-crystalized ligand for each case: E2, N-acetyl-D-glucosamine,
5α
-dihydrotestosterone (DHT) and colchicine, with the respective macromolecule. The grid
box was centered on the ligand with the following coordinates: for ER , the coordinates
were x = 107.27, y = 13.94, z = 96.38; for ST were x = 62.033, y = 12.215, z = 52.512; for
17 -HSD1 were x = 11.643, y = 9.297, z = 11.887; and for -tubulin were x = 118.921,
y = 89.718, z = 5.932. The size of grid box was 20 × 20 × 20 with 1.0 Å of spacing.
α
−
β
−
β

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3.3.4. Docking Simulations
After the preparation of macromolecules and ligands, molecular docking simulations
were performed using AutoDock vina executable [62], which uses an iterated local search
global optimizer. The parameter exhaustiveness of performed experiments was defined as
15. The results of molecular docking were analyzed and visualized in Discovery Studio
Visualizer program from BIOVIA software.
3.3.5. Validation of the Molecular Docking Performance
Scoring functions are essential for molecular docking performance. In order to validate
the docking performance of AutoDock vina, the difference between the real and best-scored
conformations were analyzed by re-docking ERα with E2, ST with N-acetyl-D-glucosamine,
17 -HSD1 with DHT and -tubulin with colchicine. Low root-mean-square distance
β
β
(RMSD) values (<2.0 Å) were obtained for all the four cases, which means that the docking
process was reliable and validated [63].
4. Conclusions
Several E1 oxime derivatives were synthesized and revealed interesting effects against
the proliferation of several tumor cell lines when compared with parent ketone compounds.
Of these, oxime 11 showed the highest activity against LNCaP cancer cells, as well as a
very relevant selectivity index. In addition, it was also demonstrated that this compound
originated cell cycle arrest in G2/M on these cells in prophase and condensed and frag-
mented nuclei characteristic of apoptosis. However, in an E-screening assay this oxime
also promoted the proliferation of T47-D cells. Docking studies evidenced that compound
11 also showed relevant affinities for ERα and β-tubulin, which could explain its mecha-
nism of action and estrogenic effect. Interestingly, the oximes bearing halogens in A-ring
(2,4-diiodoestrone oxime 10 and 2,4-dibromoestrone oxime 12), evidenced a selectivity for
HepaRG cancer cells. Another A-ring functionalized derivative, 2-nitroestrone oxime, but
not its 2,4-dinitro analogue, showed higher cytotoxicity against HepaRG and MCF-7 cancer
cells. Thus, the presence of an oxime group at C17 in functionalized E1 scaffold was shown
to be a good strategy to obtain new molecules with relevant anticancer effects.
Supplementary Materials: The following are available online. Figure S1. 1H-NMR spectrum of
compound 5 5 in DMSO-d6. Figure S3.
in DMSO-d6. Figure S2. 13C-NMR spectrum of compound
1H-NMR spectrum of compound
6 6
in DMSO-d6. Figure S4. 13C-NMR spectrum of compound
in DMSO-d6. Figure S5. 1H-NMR spectrum of compound 10 in in CDCl3. Figure S6. 13C-NMR
1
spectrum of compound 10 in in CDCl3. Figure S7. H-NMR spectrum of compound 11 in DMSO-d6.
Figure S8. 13C-NMR spectrum of compound 11 in DMSO-d6. Figure S9. 1H-NMR spectrum of
compound 12 in in CDCl3. Figure S10. 13C-NMR spectrum of compound 12 in in CDCl3.
Author Contributions: Data curation, A.O.S., S.S. and G.A.; Formal analysis, C.C., M.M., V.B., A.O.S.
and S.S.; Funding acquisition, A.F., S.S. and G.A.; Investigation, C.C., M.M., V.B., A.O.S. and S.S.;
Methodology, C.C., M.M., A.O.S. and S.S.; Project administration, A.F. and S.S.; Resources, A.O.S.,
A.F. and G.A.; Software, V.B. and S.S.; Supervision, A.F., S.S. and G.A.; Validation, A.O.S., S.S. and
G.A.; Writing—original draft, C.C., V.B. and S.S.; Writing—review & editing, C.C., M.M., A.O.S., S.S.
and G.A. All authors have read and agreed to the published version of the manuscript.
Funding: The authors acknowledge the support provided by FEDER funds through the POCI-COMPETE
2020-Operational Programme Competitiveness and Internationalisation in Axis I-Strengthening research,
technological development and innovation (Project POCI-01-0145-FEDER-007491) and National Funds
by FCT-Foundation for Science and Technology (Project UID/Multi/00709/2013).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: C.C. would also like to thank Silvia Socorro’s group (CICS-UBI) for provid-
ing DCC-FCS.
Conflicts of Interest: The authors confirm that this article content has no conflict of interest.

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