Synthesis and biological in vitro evaluation of the effect of hydroxyimino steroidal derivatives on breast cancer cells

Article abstract of DOI:10.1016/j.steroids.2020.108787

Breast cancer is the most common cause of cancer death in women, according to Global Cancer Observatory. This fact forces scientists to continue in the search for effective treatments against this aggressive type of cancer. Breast cancer frequently metast

Full text of DOI:10.1016/j.steroids.2020.108787

Steroids 166 (2021) 108787
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological in vitro evaluation of the effect of hydroxyimino
steroidal derivatives on breast cancer cells
Alan Carrasco-Carballo , María Guadalupe Hern ´a ndez-Linares c, Maura C a´ rdenas-Garcíad,
a
b,
a,*
Jesús Sandoval-Ramírez
a
Facultad de Ciencias Químicas, Laboratorio de Elucidaci ´o n y Síntesis en Química Org a´ nica, Benem ´e rita Universidad Aut o´ noma de Puebla, 72570 Puebla, Mexico
Herbario y Jardín Bot a´ nico Universitario, Benem ´e rita Universidad Aut ´o noma de Puebla, Ciudad Universitaria, 72570 Puebla, Mexico
Centro de Química, Instituto de Ciencias, Benem ´e rita Universidad Aut ´o noma de Puebla, 72570 Puebla, Mexico
b
c
d
Facultad de Medicina, Benem ´e rita Universidad Aut ´o noma de Puebla, 72570 Puebla, Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Breast cancer is the most common cause of cancer death in women, according to Global Cancer Observatory. This
fact forces scientists to continue in the search for effective treatments against this aggressive type of cancer.
Breast cancer frequently metastasizes to other organs, most often the bones, lungs, and liver. Breast cancer is
normally associated with estrogen and progestogen levels and can be hormone or non-hormone dependent. In
current experiments herein reported, some hydroxyimino spirostan derivatives showed great potential against
MCF-7 breast cancer, a Luminal-A cancer. On the other hand, a set of synthesized 6-hydroxyimino-22-oxocholes-
tane compounds had excellent activity against the MDA-MB-231 breast cancer cell line. The synthesis of
hydroxyamino derivatives from spirostan and 22-oxocholestane compounds was improved. The hydroxyimino
compounds enhanced the bioactivity when compared with their parent carbonyl skeletons.
Hydroxyimino steroids
In vitro antiproliferative assays
Spirostan
2
2-oxocholestane
1
. Introduction
Breast cancer is a pathology with high incidence and mortality in the
cancer, as it is able to decrease the progression of cancer [14,15]. For-
mestane (3) and EMATE (4) are drugs used to avoid the production of
estrogen- and androgen-dependent cancer [16–18].
female population worldwide [1]. Current treatments are successful if
tumors are detected during the early stages. Chemotherapy shows low
selectivity and severe damage to the immune system. Current anti-
cancer drugs are highly cytotoxic and also cause the death of healthy
cells [2–5].
The non-hormone dependent breast cancer (triple-negative) presents
a lower incidence rate but the worst prognosis. Surgery and chemo-
therapy have been considered the only viable option. Several studies
have been focused on the analysis of this type of cancer. ROS can induce
a pro-tumorigenic effect by damaging nucleic acids and promoting ge-
netic instability. Antioxidants can act as a double-edged sword in the
treatment of cancer [19,20]. On the other hand, reported in vitro assays
with MDA-MB-231 cell line allowed the establishment of a connection
with steroids, highlighting that diosgenin (5) is not only a single in-
hibitor but also inhibits actin polymerization, VaV2 phosphorylation,
and Cdc42 activation [16,19,21–26].
Breast cancer can be clinically classified based on the presence or
absence of estrogen and progesterone receptors (HR+/HR-). The in-
crease or decrease of human epidermal growth factor receptor 2
(
HER2+/HER2-) allows the disease to be classified as hormone or non-
hormone dependent [6,7]. The most diagnosed type of breast cancer is
estrogen receptor-positive (ER+) [7]. The anti-estrogen drugs 1–4
(
Fig. 1), are receptor blockers and have high affinity to ER [8–10].
Natural 6-hydroxyimino-4-en-3-one steroids extracted from the
marine sponges Cinachyrella alloclada and C. apion [27] showed potent
cytotoxic activity against P-388, A-549, HT-29, and MEL-28 tumor cells
Trilostane (1) is an approved drug for postmenopausal breast cancer,
that acts as an allosteric estrogen receptor modulator and blocks estra-
diol at the ERβ in MCF-7 breast cancer cells [9,11–13]. Compound ful-
vestrant (2), a steroidal selective estrogen receptor degrader (SERD), is
one of the most commonly used drugs to treat metastatic ER + breast
(IC50 between 1.25 and 5.5
act such as steroid sulfatase, aromatase, 17
reductase, and 17β-hydroxy dehydrogenase type 1 inhibitory activities
μ
g/mL) [28]. Other hydroxyamino steroids
α
-hydroxy-17,20-lyase, 5 -
α
*
Corresponding author.
E-mail address: jesus.sandoval@correo.buap.mx (J. Sandoval-Ramírez).
https://doi.org/10.1016/j.steroids.2020.108787
Received 11 September 2020; Received in revised form 15 December 2020; Accepted 21 December 2020
Available online 28 December 2020
0
039-128X/© 2020 Elsevier Inc. All rights reserved.

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
[
29–33].
The research on new drugs against breast cancer is being focused on
oxime 10. It is known that when regenerating the hydroxyl group at C-3,
a stereospecific reaction occurs [39] giving rise to the 3β-OH and 5
α
highly selective compounds against non-hormone-dependent cancers. It
is worth mentioning that in both hormonal- and non-hormonal-
dependent cancers, steroids have shown potential applications as
effective drugs. In this work, we describe the activity of hydroxyimino
laxogenin [34] and 22-oxocholestane derivatives, tested using in vitro
antiproliferative assays on MCF-7 and MDA-MB-231 breast cancer lines.
configurations. This was confirmed by the wide signal (W1/2 = 30 Hz) at
3.6 ppm in the 1H NMR spectra (due to large axial coupling 11.5 Hz
constants of H-3
α and H-2β and H4β), corresponding to the proton H-3α
(see Fig. S14 at Supplementary Material).
The hydroxyamino compound 10 could also be produced from the
ketone 8, in 93% yield (Scheme 3) by addition of hydroxylamine.
The 22-oxocholestane derivatives 11, 12, and 13 were obtained via
the fission of the tetrahydrofuran and the tetrahydropyran spirostan
2
. Results and discussion
rings. The spiroketal fission of 8 or 10, using Ac
2
O/Et
2 3
O⋅BF , produced
2
.1. Synthesis and characterization
11 and 12 in 42 and 50%, respectively [41]. To our knowledge, com-
pounds 9, 10, and 12 are synthesized for the first time. Starting from 8,
besides the target 22-oxocholestane compound 11, compound 13 was
obtained; in this step, the ketone at C-6 was rapidly tautomerized to the
enol form and this was immediately acetylated, to produce 11 and 13 in
42 and 45%, respectively. In general, when a spirostan compound is
treated under the above conditions, acetylation reaction occurs first at
C-16, then at C-3, and if the reaction takes a long time, it is acetylated at
C-26. If the reaction is stopped at no long time, water from work-up
attacks at C-26 (Reference 41), and an alcohol group is obtained at C-
26. The Attempts to hydrolyze selectively the enol ester failed; the basic
medium produced in fact the ketone group at C-6, but at the same time
the spiroketal was re-formed, recovering 8. Differently, when the spi-
roketal moiety of 10 was treated by the same Lewis acid, in the same
conditions as above, 12 was obtained in 50% yield and an analog
holding an acetate at C-26 was produced in 45% yield. In this case, the
time reaction was longer. This latter compound could be chemo-
selectively hydrolyzed at C-26, by a dilute solution of KOH/MeOH,
rising the production of 12 to 95% yield. No acetylation of the hydroxyl
group of the oxime was detected.
Hydroxyimino derivatives derived from spirostan and 22-oxocholes-
tane compounds were synthesized from diosgenin (5). For the synthesis
of laxogenin (8) from 5 (Fig. 2), 3 routes were explored: a) oxidative
hydroboration of 5; b) oxidation of the double bond between C-5 and C-
6
of 5 to produce a β-oxirane as a key intermediate, and c) through the
formation of the i-diosgenin derivative (6) For route a, the yield was
improved [35,36] obtaining an 82% overall yield of 8 (Scheme 1). Route
b shows the formation, as a first step, of the β-oxirane; the best overall
yield for 8 was 57% [37].
Route c (Scheme 1) was meticulously analyzed to improve the
overall yield. In the first step, after tosylation of 5 [38,39], and hydro-
lysis of the tosylate, 6 was obtained in 87% yield. The axial alcohol at C-
6
5
of 6 was oxidized with N-bromosuccinimide to produce the 3,5-cyclo-
-spirostan-6-one (7) in 96% yield. The acidic hydrolysis of 7, pro-
α
duced 8, in 93% yield; under this sequence of reactions, 8 was produced
with an overall yield of 77%. The cyclopropane ring of 6 was protonated
at C-5 at the
sition C-3 by the β-face [40]. In the latter reaction, the intermediate
carbocation at C-3 also led to a minute mixture (7%) of (25R)-5 -spirost-
-en-6-one and (25R)-5 -spirost-4-en-6-one, through elimination of
protons at C-2 and C-4, respectively.
α
-face, and water attacked the resulted carbocation at po-
α
The characterization data for derivatives 5–8 and 11, fit with those
2
α
reported in the literature [37,39]. In the case of the 3 ,5-cyclosteroid
α
derivatives, tridimensional figures are presented in the Supplementary
Information. From these figures, different magnetic influences from
hydroxyl, carbonyl, and hydroxyamino groups at C-6 over C-4 protons
are appreciated. For compound 6, the cyclopropane ring produces a
magnetic effect that exerts high influence over protons at C-4. In this
On the other hand, 7 reacted with an excess of hydroxylamine hy-
drochloride, in the presence of KOAc, directing to the oxime 9 in 92%
yield. When the latter compound was treated with sulfuric acid, the
cyclopropane ring in ring A was opened, but the hydroxylamino group at
ring B was not hydrolyzed. This fact is explained by the fast protonation
of the nitrogen atom producing a quaternary nitrogen atom that pro-
motes the cyclopropane fission between C-3 and C-5, recovering the
nitrogen atom an electron pair. Consequently, a stabilized homoallylic
carbonium ion among C-3, C-5, and C-6 is produced (Scheme 2). A
molecule of water attacked in a concerted manner the carbonium ion at
C-3, by the steroidal β-face, as already established [40], generating the
sense, at its 1H NMR spectra, a shielding effect is observed: H-4
is
α
highly shielded, appearing at 0.23 ppm (dd, J = 8.1, 4.8 Hz), and H-4β at
0.43 ppm (dd, J = 4.8, 4.3 Hz). The carbonyl and hydroxyamino groups
1
at C-6 distort the influence of the cyclopropane ring; at the H NMR
spectra of 7 and 9, protons H-4
and protons H-4β are slightly displaced, to 0.73 and 0.53, respectively.
For compounds 7 and 9, protons H-4 lie close to the lone pair of the
α
, appear at 1.19 and 1.09 respectively,
α
Fig. 1. Anti-estrogen drugs: trilostane (1), fulvestrant (2), formestane (3), and EMATE (4).
2

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
Fig. 2. Dose-response curves of the antiproliferative effect of compounds 7 and 12, with ANOVA test p < 0.05, minitab 18.
Scheme 1. Routes for to the synthesis of laxogenin (8) from diosgenin (5).
Scheme 2. Reaction mechanism for the opening of the cyclopropane ring in 9.
carbonyl or the hydroxyamino group provoking a down-field shift for
(See S9 spectra). For compound 11, the 1H NMR spectra (S-8 spectra),
shows a doublet signal towards 3.35 ppm for the diastereotopic protons
H-26. The acidic treatment of the spiroketal of 11 directs, as described
previously [41], to the fission of the E ring, generating an oxonium ion at
the dihydropyranic moiety, in a retro-biosynthetic manner. Derived from
those protons.
When the spiroketal moiety of 8 or 10 was opened, a carbonyl group
1
3
was produced at C-22. This could be corroborated in the C NMR
spectra of 11 where a new carbonyl signal, towards 210 ppm is present
3

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
Scheme 3. General pathways for the synthesis of hydroxyamino targets.
the latter, a nucleophile attack at C-26 is possible: by an acetate group
present in the reaction medium or by water during the work-up and
forming the carbonyl group at C-22. The fission of the spiroketal group
occurs rapidly, and it is not possible to avoid the formation of the acetate
at C-26. Protons H-26 are diastereotopic; firstly, because they are placed
next to the chiral stereocenter C-25. Secondly, the 22-oxocholestane
side-chain is able to be folded to establish a proton bridge between
the carbonyl group at C-22 and the hydroxyl group at C-26; in this way,
protons at C-26 are placed near the C-20 stereocenter; therefore, the
magnetic environment for both protons is different. This folding is
clearly observed when hecogenin was treated in the same acidic con-
ditions [42]. For oxime compounds 9, 10, and 12, their structures were
confirmed when analyzing their 13C spectra (S-11, S-13, and S-15
spectra). The corresponding carbonyl group signal of the hydroxyamino
group at C-6 appears towards 160 ppm while carbonyl of acetates at C-3
and C-16 appear towards 170 ppm (S-15 spectra), and the carbonyl at C-
hydroxyimino group at C-6. A complementary task was the evaluation of
their activity on the breast cancer cell lines MCF-7 Luminal-A (that ex-
presses progesterone, estrogen
α and β receptors) and MDA-MB-231,
classified as Triple Negative (a kind of breast cancer that does not
have any expression on estrogen, progesterone or HER2 receptors).
The antiproliferative activity of derivatives 7–12 using cell line MCF-
7 Luminal-A and MDA-MB-231 was determined by the cell feasibility kit
XTT obtaining their mean inhibitor concentration IC50. A blank using
cultured cells without compounds was also carried out. Table 1 shows
the observed IC50 for compounds 7, 8, 9, 10 versus MCF-7 and MDA-MB-
231 cancer cells. Spirostan compounds 7–10 had a remarkable positive
activity at concentrations below 10 µM, over the MDA-MB-231 cancer
line. From these tests, the natural compound laxogenin (8), was the most
effective. On the other hand, a satisfactory result against MCF-7 was
provided by the 6-hydroxyimino-22-oxocholestane derivative 12 at a
dose of 9.3 µM. Fig. 2 shows the dose–response curves of the new
compounds 7 and 12, evaluated for the first time.
2
2 appears towards 214 ppm (S-15 spectra). The formation of the oxime
compounds was highly chemoselective at C-6; no attack at the C-22
carbonyl group was observed; this fact is explained by the high steric
hindrance at C-22.
When comparing the effects on the two studied lines, MCF-7 (having
hormone receptors) and MDA-MB-231 (without hormone receptors), an
exciting relationship was found: the 22-oxocholestanic derivatives were
active towards Luminal-A and the spirostan compounds towards the
TNBC, indicating promising selective treatments. An important effect
was shown after the introduction of the hydroxyimino functional group,
which increased the affinity for hormonal receptors, reaffirming facts
stated in a previous report [50].
2
.2. Biological activity
It has been reported that steroidal derivatives have great potential as
inhibitors of cell proliferation, in different cell lines [43–48] and as anti-
inflammatory agents, repressing the expression of proinflammatory
genes TNF-a, COX-2, and IL-6, that in most cases are closely related to
tumor development. For the latter, it has been reported that the presence
of a hydroxyimino group at C-26 of a 22-oxocholestane skeleton, or at C-
Table 1
2
5 position in 27-nor-22-oxocholestane framework has a great activity
IC50 of spirostan, and 22-oxocholestane derivatives over cancer cells.
[
49]. The hydroxyimino group confers a certain advantage in the
treatment of cancer; recently, it was reported that the hydroxyimino
derivative of the 23-acetyldiosgenin acetate exhibited an IC50 of 5.6 µg/
mL on the cervical cancer HeLa line, and of 5.4 µg/mL when CaSki
cervical cancer cells were treated [50]. Laxogenin derivatives, as its
glucoside, showed an apoptotic effect in cervical cancer cells Hela,
CaSki, and ViBo, but non on T lymphocyte cells [34].
Steroid
IC50
(
μ
M)
MCF-7
MDA-MB-231
7
Not reached after 100
2.3
8
Not reached after 100
1.9
9
78.5
78.0
11.8
9.3
9.3
10
9.3
1
1
1
2
Not reached after 100
Not reached after 100
Due to the importance of hydroxyimino steroids, some derivatives
were synthesized from spirostans and 22-oxocholestanes, bearing the
4

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
2
.3. In silico studies
for the following reaction. Data fit with that reported in the literature
◦
◦
[
39]: mp 174–175 C. [
α
]
D
= ꢀ 93.5 (CHCl
3
–
, c = 1.0). IR: 2947 (CH),
ꢀ
1
A first approach to explain the selectivity shown by the spirostan
1558 (Ar), 1243 (C
–
O), 1039, 987 (O
C
–
O) cm . Values for
+
+
1
derivatives towards the MDA-MB-231 cell line (classified within the MSL
subtype) is associated with various proteins, highlighting EGFR, mTOR,
TGFB1, and PDGF [51–54]. These proteins are targets for the modula-
tion of this type of cancer, related to the molecular mechanism. The
inhibition of these proteins, in particular EGFR, can be analyzed by in
silico experiments. Table 2 shows that the spirostan derivatives 7–10
have better coupling energies on the receptor domain, compared to
Erlotinib, the reference inhibitor.
C
34
H
48
O
5
S + H = [M + H] found: 569.3285, Calc.: 569.3295. H NMR
δ (500 MHz): 7.81 (2H, d, Jo-m = 8.32, Hortho), 7.35 (2H, d, Jm-o = 8.32,
meta), 5.31 (1H, m, H-6), 4.41 (1H, m, H-16), 4.32 (1H, tt, J2e-3a = J3a-4e
= 4.74, J2a-3a = J3a-4a = 11.5, H-3), 3.49 (1H, dd, Jgem = 10.9, J25a-26e
5.6, H-26e), 3.38 (1H, dd, Jgem = 10.9, J25a-26a = 6.5, H-26a), 2.49 (3H, s,
pCH
Ar), 2.43(1H, m, H-4a), 2.29 (1H, ddd, Jgem = 13.76, J3a-4e = 4.74,
2e-4e = 2.1, H-4e), 1.08 (3H, d, J20-21 = 7.1, CH -21), 0.97 (3H, d, J25-27
= 6.8, CH -27), 0.88 (3H, s, CH -18), 0.82 (3H, s, CH
H
=
3
J
3
1
3
3
3
3
-19). C NMR δ
On the other hand, in the case of studies carried out with nuclear
receptors, it can be concluded that the compounds do not have the
ability to inhibit the receptors at the energy level, but it is possible that
these can be modulated in different ways, given the case of modulation
at the enzymatic level affecting the proteins that are in charge of the
production of estrogens, such as CYP19-A1 among others. Further
studies will be launched before proposing a mechanism of action.
(125 MHz): 144.1 (Cpara), 134.6 (Cipso), 129.8 (Cortho),127.6 (Cmeta),
37.2 (C-1), 31.4 (C-2), 80.7 (C-3), 42.2 (C-4), 138.8 (C-5), 123.3 (C-6),
32.0 (C-7), 31.6 (C-8), 50.0 (C-9), 36.6 (C-10), 20.8 (C-11), 39.7 (C-12),
40.2 (C-13), 56.5 (C-14), 31.8 (C-15), 82.0 (C-16), 62.0 (C-17), 16.3 (C-
18), 19.4 (C-19), 41.6 (C-20), 14.5 (C-21), 109.14 (C-22), 31.3 (C-23),
28.7 (C-24), 30.3 (C-25), 66.8 (C-26), 17.1 (C-27), 21.4 (CH
2nd step. Potassium acetate (1.0 g, 12.5 mmol) was added to a so-
lution of diosgenin tosylate (2.2 g, 3.87 mmol) in 30 mL acetone/H
3
Ar).
2
O
3
. Material and methods
(4:1) and the reaction was stirred to reflux for 5 h. The acetone was
eliminated by means of a rotary evaporator and the solid was dissolved
in EtOAc (2x15 mL), washed with water (2x25mL), and dried under
3
.1. General procedures and materials
2 4
anhydrous Na SO . The solvent was evaporated, obtaining a crude that
Melting points were determined using a Celsius scale in a Melt Temp
was purified using a chromatography column containing silica gel and a
mobile phase of a 9:1 hexane:EtOAc solution, under moderate air
apparatus, using open capillary tubes. The optical rotations were
measured on a Perkin Elmer 241 polarimeter, using a microcell 10 cm
long, chloroform solutions, and the sodium line D (λ 589 nm). Con-
centrations are expressed in g/100 mL, at room temperature. IR spectra
were recorded on an Agilent Cary 630 FTIR spectrophotometer, using a
diamond ATR sensor interface. High Resolution Mass spectra data were
obtained from an Agilent coupled HPLC-TOF-MS (compounds 6–11),
◦
pressure. A white powder (1.44 g, 89%) was obtained: mp 166–167 C.
◦
[
α
]
D
= ꢀ 85 (CHCl
3
, c = 1.0). IR: 3473 (O
–
H), 2947 (C H), 1444
–
ꢀ 1
+
(C
–
O), 1011, 897 (O C O) cm . Values for C27H O + H = [M +
– –
42 3
+
1
H] found: 415.3128, Calc.; 415.3112. H- NMR δ (500 MHz): 4.33 (1H,
m, H-16), 3.41 (1H, dd, Jgem = 10.9, J25-26e = 5.6, H-26e), 3.32 (1H, dd,
J
gem = 10.9, J25-26a = 6.5, H-26a), 3.20 (1H, t, J6-7 = 2.91, H-6), 1.08
1
and from a JEOL MStation spectrometer (compounds 12–13). H and
(3H, d, J21-20 = 7.1, CH
(3H, s, CH -18), 0.82 (3H, s, CH
4.3, H-4β), 0.23 (1H, dd, J ,3 = 8.1, Jgem = 4.8, H-4α
3
-21), 0.97 (3H, d, J27-25 = 6.8, CH
-19), 0.43 (1H, dd, Jgem = 4.8, J4β,3
). 13C NMR δ (125
3
-27), 0.88
1
3
C NMR spectra were recorded of 500 MHz on a Bruker FT-NMR
3
3
=
spectrometer. Chemical shifts (δ) are reported in parts per million
4α
1
(
ppm), referenced in H spectra by the signal of TMS proton resonance,
MHz): 37.2 (C-1), 31.4 (C-2), 45.8 (C-3), 42.2 (C-4), 42.5 (C-5), 74.0 (C-
6), 32.0 (C-7), 31.6 (C-8), 50.0 (C-9), 36.6 (C-10), 20.8 (C-11), 39.7 (C-
12), 40.2 (C-13), 56.5 (C-14), 31.8 (C-15), 81.5 (C-16), 62.0 (C-17), 16.3
(C-18), 19.4 (C-19), 41.6 (C-20), 14.5 (C-21), 109.5 (C-22), 31.3 (C-23),
1
3
3
and in C spectra by the central signal of CDCl . Coupling constants (J)
are reported in Hertz. Spectrophotometric readings were performed in a
Thermo scientific multiskan FC microplate reader, Thermo Fisher.
2
8.7 (C-24), 30.3 (C-25), 66.8 (C-26), 17.1 (C-27).
25R)-3 ,5-Cyclo-5 -spirostan-6-one (7)
(
α
α
3
.2. Chemical synthesis and Characterization.
25R)-3 ,5-Cyclo-5
-spirostan-6β-ol (6)
A solution of i-diosgenin (6, 1.8 g, 4.34 mmol) in 50 mL of dioxane
◦
was cooled to 0 C and treated with 1.07 g of NBS (6 mmol) and 2 mL of
AcOH, at room temperature; the reaction was followed by TLC. At the
(
α
α
1
st step. Partridge’s procedure was followed [38]. A solution of
2 2 4
end of the reaction, 10 mL of a 10% aq solution of Na S O was added,
diosgenin (5, 2.0 g, 4.82 mmol) in anhydrous pyridine (20 mL) was
and the dioxane phase separated. Dioxane was removed by evaporation,
and the residue was re-dissolved in dichloromethane, washed with 10
prepared to which was added 1.0 g of TsCl (5.24 mmol). After stirring for
6
h at room temperature, the solution was slowly poured into 10% po-
tassium hydrogen carbonate solution. The precipitate was collected by
filtration, redissolved in CH Cl , washed with water 5%, HCl aqueous
solution (2x20 mL), brine (2x25 mL), distilled water (2x25 mL), dried
with anhydrous Na SO and evaporated to dryness. A white solid of
mL of concentrated solution of NaHCO
3
, distilled water (2x25 mL), dried
over anhydrous Na SO and concentrated under vacuum to obtain, after
2
4
2
2
chromatographic purification (9:1 hexane:EtOAc) a white powder (1.71
◦
◦
g, 96%): mp 183–184 C. [
α
]
D
= ꢀ 49.5 (CHCl
3
, c = 1.0). IR: 2947 (CH),
2
4
ꢀ
1
1
674 (CO), 1064 (C
–
O), 987, 901 (O
–
C
–
O) cm . Values for
diosgenin tosylate (2.68 g, 97.7%) was obtained, and used immediately
+
+
1
C
27
H
40
O
3
+ H = [M + H] found: 413.2968, Calc.; 413.2950. H NMR
δ (500 MHz): 4.41 (1H, m, H-16), 3.47 (1H, ddd, J26e-24 = 2.0, J26e-25a
=
Table 2
4
.4, Jgem = 10.8, H-26e), 3.36 (1H, dd, J26a,25a = Jgem = 10.8, H-26a),
.46 (1H, dd, J7e,7a = 16.4, J7e,8 = 4.4, H-7e), 2.11 (1H, m, H-8), 1.01
-19), 0.98 (3H, d, J21,20 = 6.8, CH -21),
-18), 0.79 (3H, d, J27,25 = 6.4, CH -27), 0.73 (1H, dd,
Coupling energies (kcal/mol) obtained by molecular docking of couples of
compound-protein (associated with the selected cell lines).
2
(
1H, m, H-4pe), 1.02 (3H, s, CH
3
3
Compound
Protein
EGFR
0
.83 (3H, s, CH
3
3
1
3
Er
_
α
PR
J
gem = 4.8, J4β,3 = 4.3, H-4β). C NMR δ (125 MHz) 33.5 (C-1), 25.9 (C-
Erlotinib
ꢀ 7.3
_
_
2), 35.5 (C-3), 11.8 (C-4), 46.8 (C-5), 208.9 (C-6), 44.9 (C-7), 34.3 (C-8),
46.1 (C-9), 40.7 (C-10), 22.7 (C-11), 39.8 (C-12), 46.3 (C-13), 56.7 (C-
Estradiol
ꢀ 8.7
_
_
Progesterone
_
ꢀ 9.3
ꢀ 8.4
ꢀ 9.1
ꢀ 8.7
ꢀ 9.1
ꢀ 7.1
ꢀ 7.4
1
4), 31.6 (C-15), 80.5 (C-16), 62.0 (C-17), 16.6 (C-18), 19.9 (C-19), 41.7
7
8
9
ꢀ 9.1
ꢀ 8.3
ꢀ 8.9
ꢀ 8.9
ꢀ 7.4
ꢀ 7.9
ꢀ 8.0
ꢀ 8.4
ꢀ 8.1
ꢀ 8.3
ꢀ 8.2
ꢀ 8.0
(
C-20), 14.6 (C-21), 109.2 (C-22), 31.4 (C-23), 28.8 (C-24), 30.3 (C-25),
6
6.8 (C-26), 17.2 (C-27)
1
1
1
0
1
2
(25R)- 3β-Hydroxy-5
α
-Spirostan-6-one (Laxogenin, 8)
A solution of compound 7 (1.5 g, 3.64 mmol) in dioxane (30 mL) was
2 4
prepared to which was added a solution of 0.2 mL of concentrated H SO
5

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
and 1.0 mL of water. The reaction was stirred at reflux temperature for
CH
3
-27), 0.88 (3H, s, CH
3
-18), 0.82 (3H, s, CH
3
-19). 13C NMR δ (125
3
0 min, then the acidic solution was neutralized with a saturated solu-
MHz): 37.2 (C-1), 31.4 (C-2), 45.8 (C-3), 42.2 (C-4), 42.5 (C-5), 156.0
(C-6), 32.0 (C-7), 31.6 (C-8), 50.0 (C-9), 36.6 (C-10), 20.8 (C-11), 39.7
(C-12), 40.2 (C-13), 56.5 (C-14), 31.8 (C- 15), 80.8 (C-16), 62.0 (C-17),
16.3 (C-18), 19.4 (C-19), 41.6 (C-20), 14.5 (C-21), 109.3 (C-22), 31.3 (C-
23), 28.7 (C-24), 30.3 (C-25), 66.8 (C-26), 17.1 (C-27).
tion of NaHCO solution (2x5 mL). The remaining dioxane was elimi-
3
nated under vacuum and the aqueous medium was extracted with
EtOAc. The organic phase was washed with water (2x50 mL) to obtain,
after chromatographic purification (1:1 hexane:EtOAc eluating solu-
◦
◦
tion) a white powder (1.46, 93%): mp 210–211 C. [
α]
D
= ꢀ 86 (CHCl
3
,
(25R)-26-Hydroxy-6,22-dioxo-5
etate (11).
A solution of 2.5 mL of Ac
α-cholestane-3β,16β-diyl diac-
ꢀ 1
c = 1.0). IR: 3468 (OH), 2947 (CH), 1690 (CO) cm . Values for
+
+
1
C
27
H
42
O
4
+ H = [M + H] found: 431.3087, Calc.: 431.3056. H NMR
2
O and 3 mL of Et
2
O⋅BF
3
was prepared at
◦
δ (500 MHz): 4.41 (1H, m, H-16), 3.57 (1H, m, H-3), 3.47 (1H, dd, Jgem
=
0 C, and added to a solution of 1.0 g (2.32 mmol) of laxogenin (8) in 10
◦
1
2
1
0.9, J26e-25 = 5.6, H-26e), 3.36 (1H, dd, Jgem = 10.9, J26a-25 = 6.5, H-
6a), 2.35 (1H, dd, Jgem = 8.54, J7a-8 = 2.27, H-7e), 2.2 (1H, m, H-5),
.98 (1H, d, Jgem = 8.54, H-7a), 1.08 (3H, d, J21-20 = 7.1, CH -21), 0.97
-27), 0.88 (3H, s, CH -18), 0.82 (3H, s, CH -19).
mL of CH
2
Cl
2
, cooled to 0 C. The reaction was kept under strong stirring
for 15 min, then poured into iced water and extracted with CH
mL). The crude was washed with a saturated solution of NaHCO
mL), distilled water (2x25 mL), dried with anhydrous Na
2
Cl
2
(3x25
(2x20
3
3
(
1
3H, d, J27-25 = 6.8, CH
3
3
3
2
SO
4
and
3
C NMR δ (125 MHz): 37.2 (C-1), 31.4 (C-2), 45.8 (C-3), 42.2 (C-4),
2.5 (C-5), 210.5 (C-6), 32.0 (C 7), 31.6 (C-8), 50.0 (C-9), 36.6 (C-10),
0.8 (C-11), 39.7 (C-12), 40.2 (C-13), 56.5 (C-14), 31.8 (C- 15), 80.8 (C-
6), 62.0 (C-17), 16.3 (C-18), 19.4 (C-19), 41.6 (C-20), 14.5 (C-21),
09.3 (C-22), 31.3 (C-23), 28.7 (C-24), 30.3 (C-25), 66.8 (C-26), 17.1 (C-
7).
filtered. The filtrate was concentrated under reduced pressure, and the
crude was purified using a silica gel packed chromatographic column
and a 2:3 (hexane:EtOAc) mobile phase system obtaining 519 mg (42%)
of a white powder that corresponds to derivative 11, and 600 mg (45%)
4
2
1
1
2
◦
◦
to derivative 13. Data for 11: mp 160–161 C. [
α
]
D
= ꢀ 119 (CHCl
3
–
, c =
–
1.0). IR: 3468 (OH), 2947 (CH), 1715, 1709, 1681 (C O), 1253 (C O)
–
ꢀ
1
+
+
(
25R)-3
The steroidal ketone 7 (500 mg, 1.2 mmol) was dissolved in EtOH
20 mL), to which was added NH
OH⋅HCl (200 mg, 2.4 mmol) and KOAc
180 mg, 1.83 mmol). The reaction was stirred under reflux for 2 h, then
α
,5-Cyclo-6-hydroxyimino-5
α
-spirostan (9).
cm . Values for C31
H
48
O
7
+ H = [M + H] Found: 533.3398 Calc.:
1
533.3378. H NMR δ (500 MHz): 4.94 (1H, m, H-3), 4.59 (1H, m, H-16),
3.35 (2H, d, J26-25 = 6.0, H-26), 2.98 (1H, m, H-20), 2.62 (1H, m, H-23a),
(
(
2
2.21 (2H, m, H-5, H-7a), 1.96 (3H, s, CH
3
CO
-21), 1.01 (3H, s, CH
27), 0.85 (3H, s, CH
-19). 13C NMR δ (125 MHz): 36.8
2
-16), 1.9 (3H, s,CH
3
CO
2
-3),
the solvent was evaporated under vacuum. The crude was dissolved in
EtOAc and washed with 5% aq HCl (20 mL), water (2x30 mL), dried with
1.13 (3H, d, J21,20 = 7.1, CH
27,25 = 6.7, CH
3
3
-18), 0.90 (3H, d,
J
3
3
anhydrous Na
2
SO
4
. After elimination of the solvent, 10 was obtained as
(C-1), 27.6 (C-2), 75.7 (C-3), 38.0 (C-4), 59.8 (C-5), 214.1 (C-6), 31.5 (C-
7), 31.2 (C-8), 49.7 (C-9), 36.5 (C-10), 20.7 (C-11), 39.5 (C-12), 41.8 (C-
13), 53.8 (C-14), 34.8 (C-15), 73.8 (C-16), 55.0 (C-17), 13.2 (C-18), 19.3
(C-19), 43.5 (C-20), 16.8 (C-21), 210.7 (C-22), 38.5 (C-23), 26.1 (C-24),
◦
◦
a white powder (477 mg, 92%), mp 179–180 C. [
α]
D
= ꢀ 58 (CHCl
3
, c
=
1.0). IR: 3468 (OH), 2947 (CH), 1690 (CO), 1243 (C O), 987, 901
–
ꢀ 1
+
+
(
O
–
C
–
O) cm . Values for C27
H41NO
3
+ H = [M + H] found:
1
4
28.3050, Calc.: 428.3056. H NMR δ (500 MHz): 7.4(1H, m, OH), 4.41
35.4 (C-25), 67.4 (C-26), 16.6 (C-27), 169.9 (CH
(CH COO-16), 21.4 (CH COO-3), 21.1 (CH COO-16).
-cholestane-
3
COO-3), 170.5
(
1H, m, H-16), 3.47 (1H, ddd, J26e,24 = 2.0, J26e,25 = 4.4, Jgem = 10.8, H-
3
3
3
2
6e), 3.36 (1H, dd, J26a,25 = Jgem = 10.8, H-26a), 2.46 (1H, dd, J7e,7a
6.4, J7e,8 = 4.4, H-7e), 2.11 (1H, m, H-8), 1.19 (1H, m, H-4pe), 1.02
-19), 0.98 (3H, d, J21,20 = 6.8, CH -21), 0.83 (3H, s, CH -18),
.79 (3H, d, J27,25 = 6.4 Hz, CH -27), 0.53 (1H, dd, Jgem = 4.8, J4β,3
.3, H-4β). C NMR δ (125 MHz):33.5 (C-1), 25.9 (C-2), 35.5 (C-3), 11.8
=
(25R)-26-Hydroxy-6-hydroxyimino-22-oxo-5
3β,16β-diyl diacetate (12)
Method A.
α
1
(
3H, s, CH
3
3
3
0
4
3
=
1.0 g (2.32 mmol) of laxogenin oxime (10) was dissolved in 10 mL of
1
3
◦
CH
of Et
stirring, the reaction was poured into iced water and extracted with
CH Cl (3x25 mL) and the crude was washed with a saturated solution of
NaHCO (3x20 mL) and distilled water (2x25 mL). The organic phase
was dried with Na SO and concentrated under reduced pressure. The
2
Cl
2
and cooled to 0 C. Then, a solution of 2.5 mL of Ac
2
O and 3 mL
(
C-4), 46.8 (C-5), 160.1 (C-6), 44.9 (C-7), 34.3 (C-8), 46.1 (C-9), 40.7 (C-
2
3
O⋅BF was prepared and added to the steroid solution. After 15 min
1
8
2
1
0), 22.7 (C-11), 39.8 (C-12), 46.3 (C-13), 56.7 (C-14), 31.6 (C- 15),
0.5 (C-16), 62.0 (C-17), 16.6 (C-18), 19.9 (C-19), 41.7 (C-20), 14.6 (C-
1), 109.2 (C-22), 31.4 (C-23), 28.8 (C-24), 30.3 (C-25), 66.8 (C-26),
7.2 (C-27).
2
2
3
2
4
(
25R)-6-Hydroxyimino-5
Method A.
A solution of 9 (500 mg, 1.12 mmol) in dioxane (30 mL) was pre-
pared to which was added 1.0 mL of water and 0.2 mL of concentrated
SO . The reaction was stirred at reflux temperature for 30 min, then,
the acidic solution was neutralized with a saturated Na CO solution
3x10 mL) and with brine (3x10 mL). The dioxane was eliminated under
vacuum and the crude was extracted with EtOAc, washed with water
2x50 mL), and concentrated to obtain 10, after chromatographic pu-
α
-spirostan-3β-ol (10).
crude product was purified by chromatography using a silica gel column
and a 2:3 hexane:EtOAc mobile phase system, obtaining 629 mg (50%
yield) of a white powder that corresponds to derivative 12. In this re-
action, a second compound, holding an acetate group at C-26 isolated in
45% yield, was chemoselectively hydrolyzed using a 5% solution of KOH
in MeOH. The reaction was followed by TLC to stop the reaction when a
more polar spot appeared (hydrolysis of acetate at C-3). In this way the
yield of 12 was improved to 95%.
H
2
4
2
3
(
(
Method B.
rification (5:5 (hexane:EtOAc), as a white powder (495 mg, 95%).
Method B.
The ketone 11 (500 mg, 1.1 mmol) was dissolved in EtOH (20 mL),
and then NH
2
OH⋅HCl (200 mg, 2.4 mmol) and KOAc (180 mg, 1.83
The steroidal ketone 8 (500 mg, 1.2 mmol) was dissolved in EtOH
mmol) were added. The reaction was stirred under reflux for 2 h, then
the solvent was evaporated under vacuum. The crude was dissolved in
EtOAc, washed with water (2x30 mL), and dried with anhydrous
(
20 mL), to which was added NH
2
OH⋅HCl (200 mg, 2.4 mmol) and KOAc
(
180 mg, 1.83 mmol). The reaction was stirred under reflux for 2 h, then
the solvent was evaporated under vacuum. The crude was dissolved in
EtOAc and washed with water (2x30 mL), dried with anhydrous Na SO
Na
2
SO
4
. After elimination of the solvent, 12 was obtained as a white
◦
◦
2
4
.
powder in 95% yield. mp 159–160 C. [
IR: 3567 (OH), 2938 (CH), 1728, 1735, 1638 (CO), 1253 (C O) cm .
α
]
D
= ꢀ 122 (CHCl
3
–
, c = 1.0).
ꢀ
1
After elimination of the solvent, 10 was obtained as a white powder
◦
◦
+
+
(
495 mg, 93%). mp 198–199 C, [
α]
D
= ꢀ 86 (CHCl
3
, c = 1.0) IR: 3468
Values for C31
H49NO
7
+ H = [M + H] found: 548.3515, Calc.:
ꢀ 1
1
(
OH), 2977 (CH), 1590 (CO), 1243 (N
–
O) cm . Values for C27
H
43NO
4
548.3512.. H NMR δ (500 MHz): 4.92 (1H, m, H-3), 4.58 (1H, m, H-16),
3.18 (2H, d, J25-26 = 6.0, H-26), 2.94 (1H, m, H-20), 2.62 (1H, m, H-
+
= [M + H] found: Calc.: 446.3165. 1H NMR δ (500 MHz):4.41
+
+
H
(
1H, m, H-16), 3.57 (1H, m, H-3), 3.47 (1H, dd, Jgem = 10.9, J26e-25 = 5.6
23a), 2.21 (1H, m, H-5, H-7a), 1.96 (3H, s, CH
3
CO
-21), 1.01 (3H, s, CH
-27), 0.85 (3H, s, CH
-19). 13C NMR δ (125
MHz): 36.8 (C-1), 27.6 (C-2), 75.7 (C-3), 38.0 (C-4), 59.8 (C-5), 159.5
2
-16), 1.90 (3H, s,
Hz, H-26e), 3.36 (1H, dd, Jgem = 10.9, J26a-25 = 6.5, H-26a), 2.35 (1H, dd,
gem = 8.54, J7a-8 = 2.27, H-7e), 2.2 (1H, m, H-5), 1.98 (1H, d, Jgem
.54, H-7a), 1.08 (3H, d, J21-20 = 7.1, CH -21), 0.97 (3H, d, J27-25 = 6.8,
CH
3
CO
2
-3), 1.13 (3H, d, J20,21 = 7.1, CH
3
3
-18), 0.90
J
=
(3H, d, J25,27 = 6.7, CH
3
3
8
3
6

A. Carrasco-Carballo et al.
Steroids 166 (2021) 108787
(
(
C-6), 31.5 (C-7), 31.2 (C-8), 49.7 (C-9), 36.5 (C-10), 20.7 (C-11), 39.5
C-12), 41.8 (C-13), 53.8 (C-14), 34.8 (C-15), 73.8 (C-16), 55.0 (C-17),
independent runs to produce 20 final docked poses. These poses were
clustered according to the RMSD with a cut-off of 2.0 Å.
1
2
3
3.2 (C-18), 19.3 (C-19), 43.5 (C-20), 16.8 (C-21), 213.7 (C-22), 38.5 (C-
3), 26.1 (C-24), 35.4 (C-25), 67.4 (C-26), 16.6 (C-27), 169.9 (CH COO-
), 170.5 (CH COO-16), 21.4 (CH COO-3), 21.1 (CH COO-16).
25R)-26-Hydroxy-22-oxocholest-5-ene-3β,6,16β-triyl triacetate
13).
Compound 13 was identified by: mp 159–160 ◦C. [
CHCl
CO), 1343 (C
3
4
. Conclusions
3
3
3
(
The antiproliferative activity of spirostan and 22-oxocholestane
(
compounds on breast cancer cells was evaluated. The spirostan com-
pounds 7, 8, 9, and 10 showed selectivity towards the Triple-Negative
MDA-MB-231 breast cancer cell line, while the 22-oxocolestanic de-
rivatives 11 and 12 demonstrated an enhanced activity on the MCF-7
cell line breast cancer, Luminal-A. The experimental and in silico re-
sults of the new steroidal 6-hydroxyamino derivatives 9, 10, and 12 are
a good motivation to test the best resulting compounds in an in vivo
model. The cyclosteroidal compounds 7 and 9 showed excellent results
on the two cancer cell lines, described above.
α]
D
= ꢀ 114◦
(
(
3
, c = 1.0). IR: 3468 (OH), 2947 (CH), 1795, 1752, 1735, 1682
ꢀ
1
–
O) cm . Values for C33H50O8 = [M] + . found:
1
574.3509, Calc.: 574.3507. H NMR δ (500 MHz): 5.01 (1H, m, H-3),
4.68 (1H, m, H-16), 3.9 (2H, d, J26-25 = 6.0, H-26), 2.98 (1H, m, H-20),
2.62 (1H, m, H-23a), 2.31 (2H, m, H-4, H-7a), 2.09 (3H, s, CH CO -16),
2.05 (3H, s, CH CO -6), 1.93 (3H, s, CH CO -3), 1.13 (3H, d, J21,20
7.1, CH -21), 1.01 (3H, s, CH
-18), 0.90 (3H, d, J27,25 = 6.7, CH
0.85 (3H, s, CH
3
2
3
2
3
2
=
3
3
3
-27),
-19). 13C NMR δ (125 MHz): 36.8 (C-1), 27.6 (C-2), 75.7
3
(
(
C-3), 38.0 (C-4), 124.8 (C-5), 154.5 (C-6), 31.5 (C-7), 31.2 (C-8), 49.7
C-9), 36.5 (C-10), 20.7 (C-11), 39.5 (C-12), 41.8 (C-13), 53.8 (C-14),
Declaration of Competing Interest
3
2
6
4.8 (C15), 73.8 (C-16), 55.0 (C-17), 13.2 (C-18), 19.3 (C-19), 43.5 (C-
0), 16.8 (C-21), 213.7 (C-22), 38.5 (C-23), 26.1 (C-24), 35.4 (C-25),
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
7.4 (C-26), 16.6 (C-27), 172.5 (CH
3
COO-6),169.9 (CH
3
COO-3), 170.5
COO-6).
(
CH
3
COO-16), 21.4 (CH COO-3), 21.1 (CH
3
3
COO-16), 20.2 (CH
3
3
3
.3. Biological evaluation
.3.1. Cell cultures
Acknowledgments
We acknowledge CONACYT for financial support (Grant PN-2015-
Human breast cancer TN cell line MDA-MB-231 was obtained from
0
1-331) and Scholarship for ACC (Grant CVU 698207). We greatly
ATCCR HTB-26 and routinely cultured in DMEM in phenol red (51435,
MERCK) supplemented with 10% fetal bovine serum (FBS) (Biowest,
SOUTH AMERICA, S15836) in the presence of 100U/ml penicillin and
appreciate Allen J. Coombes (Curator of BUAP-Botanic Garden) for
correcting the English.
1
3
00 µg/mL streptomycin (P4333, MERCK). Cells were incubated at
◦
Appendix A. Supplementary data
7
2
C with 5% CO , Human Luminal breast cancer MCF-7 cell line,
R
obtained from ATCC HTB-22, an incubate in some conditions but in
media culture MEM in phenol red (M2279, MERCK) with 1% Insulin
transferrin-selenium (ITS) (I3146, MERCK). All cells used for the treat-
ment experiments were found during their linear phase of growth.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.steroids.2020.108787.
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.3.3. Statistical analysis
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[
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The preparation of three-dimensional structure coordinates was
generated and optimized using MM2 force-field-steepest Decent algo-
rithm [55] implemented in Chem3D 15.0 (Perkin Elmer). All structures
were further optimized through the PM7 semi-empirical method
implemented in MOCAP 201 code (http://OpenMOPAC.net); All pro-
teins were searched in the Protein Data Bank (PDB), prepared in
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