Antiproliferative, cytotoxic, and apoptotic activity of steroidal oximes in cervicouterine cell lines

Article abstract of DOI:10.3390/molecules21111533

Steroidal sapogenins have shown antiproliferative effects against several tumor cell lines; and their effects on human cancer cells are currently under study. Changes in the functionality on the steroidal structure make it possible to modify the biological activity of compounds. Herein, we report the synthesis and in vitro antitumor activity of two steroidal oxime compounds on cervical cancer cells. These derivatives were synthesized from the steroidal sapogenin diosgenin in good yields. The in vitro assays show that the steroidal oximes show significant antiproliferative activity compared to the one observed for diosgenin. Cell proliferation, cell death, and the cytotoxic effects were determined in both cervical cancer cells and human lymphocytes. The cancer cells showed apoptotic morphology and an increased presence of active caspase-3, providing the notion of a death pathway in the cell. Significantly, the steroidal oximes did not exert a cytotoxic effect on lymphocytes.

Full text of DOI:10.3390/molecules21111533

molecules
Article
Antiproliferative, Cytotoxic, and Apoptotic Activity
of Steroidal Oximes in Cervicouterine Cell Lines
1
2,
3
Luis Sánchez-Sánchez , María Guadalupe Hernández-Linares *, María L. Escobar ,
1
4,5
6
Hugo López-Muñoz , Edgar Zenteno , María A. Fernández-Herrera ,
Gabriel Guerrero-Luna 2 , Alan Carrasco-Carballo
,7
2,7
and Jesús Sandoval-Ramírez 7
1
Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México,
9230 Ciudad de México, Mexico; luisss@unam.mx (L.S.-S.); hugogris@hotmail.com (H.L.-M.)
Laboratorio de Investigación, Jardín Botánico Universitario, Benemérita Universidad Autónoma de Puebla,
2570 Puebla, Pue., Mexico; gabriel.guerrero@alumno.buap.mx (G.G.-L.);
0
2
7
alan.carballo@alumno.buap.mx (A.C.-C.)
Departamento de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México,
3
4
0
4510 Ciudad de México, Mexico; escobarluisa@ciencias.unam.mx
Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México,
4510 Ciudad de México, Mexico; ezenteno@unam.mx
0
5
6
Centro de Investigación UNAM-UABJO, 68120 Oaxaca, Oax., Mexico
Departamento de Física Aplicada, Centro de Investigación y de Estudios Avanzados—Unidad Mérida,
km 6 Antigua Carretera a Progreso, Cordemex, 97310 Mérida, Yuc., Mexico; mfernandez@cinvestav.mx
Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, 72570 Puebla, Pue., Mexico;
jesus.sandoval@correo.buap.mx
7
*
Correspondence: guadalupe.mghl@correo.buap.mx; Tel./Fax: +52-222-295-500 (ext. 7039)
Academic Editor: Margaret Brimble
Received: 17 September 2016; Accepted: 7 November 2016; Published: 14 November 2016
Abstract: Steroidal sapogenins have shown antiproliferative effects against several tumor cell lines;
and their effects on human cancer cells are currently under study. Changes in the functionality on
the steroidal structure make it possible to modify the biological activity of compounds. Herein, we
report the synthesis and in vitro antitumor activity of two steroidal oxime compounds on cervical
cancer cells. These derivatives were synthesized from the steroidal sapogenin diosgenin in good
yields. The in vitro assays show that the steroidal oximes show significant antiproliferative activity
compared to the one observed for diosgenin. Cell proliferation, cell death, and the cytotoxic effects
were determined in both cervical cancer cells and human lymphocytes. The cancer cells showed
apoptotic morphology and an increased presence of active caspase-3, providing the notion of a death
pathway in the cell. Significantly, the steroidal oximes did not exert a cytotoxic effect on lymphocytes.
Keywords: steroidal oximes; 23-acetyldiosgenin; (25R)-spirost-4-en-3,6-dione; apoptosis;
antiproliferative activity
1
. Introduction
Cancers figure among the leading causes of morbidity and mortality worldwide, with therapies
based primarily on surgery, radiation therapy, and chemotherapy which, to date, are not successful
interventions, and the complete removal of the cancer without damage to other tissues is the ideal
goal of treatment. Sometimes this can be accomplished by surgery, but the propensity of cancers
to invade adjacent tissues or to spread to distant sites by microscopic metastasis often limits its
effectiveness. On the other hand, chemotherapy and radiotherapy may have a negative effect on
normal cells [1]. Antitumor research is a very active field, and a large amount of information dealing
with clinical aspects of cancer chemotherapy is generated; there is, however, a continuing need for
Molecules 2016, 21, 1533; doi:10.3390/molecules21111533
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1533
2 of 17
new treatments inspired by medicinal chemistry and drug design. In this field, synthetic chemistry
has been broadly employed to modify drug targets, especially those of natural origin, in order to
find a solution to the problem of the limited supply of natural products by developing synthetic
strategies. Sapogenins constitute a class of natural products that occur widely in their glycoside form
and promote diverse health properties; among them the steroidal sapogenins (spirostans) are the
most important bioactive compounds from several plants. Many biological functions of steroidal
sapogenins have been reported, e.g., anticarcinogenic, antithrombotic, antiviral, hypocholesterolemic,
and hypoglycemic [
2–
21]. Diosgenin (
1, Figure 1) is one of the most available steroidal sapogenins;
it was first isolated by Takeo Tsukamoto in 1936 from Dioscorea tokoro [22]. Spirostan
1
is the major
constituent in fenugreek seeds (Trigonella foenum-graecum L.) and in wild yams (Dioscorea villosa L.)
which are consumed as food ingredients or condiments by some populations in Latin America, Eastern
Europe, and Asia [23,24].
Figure 1. Diosgenin (1); radicicol (2); and radicicol oxime derivative (3).
Diosgenin (
of steroids [25]. It has the ability to penetrate cell membranes and bind to specific receptors [26].
However, mammals are unable to convert into important steroidal metabolites, such as cholesterol,
1) is used in the pharmaceutical industry as the main precursor in the synthesis
1
pregnanes, androstanes, etc., due to the lack of the appropriate enzymes involved in steroid hormone
biosynthesis. In that sense, there is no evidence to validate the claim of an estrogenic potency
of diosgenin in humans [24
,
25
,27]. The trustworthiness of its consumption by humans has been
established [28]. The beneficial effect of diosgenin (1) on human health has so far been limited to its
efficacy in preventing metabolic diseases like hypercholesterolemia [29]. However, it was recently
found that 1 exerts antiproliferative activity against cell lines; as HeLa (cervical cancer) [30], HEL
and K562 (erythroleukemia) [31], osteosarcoma 1547 [32], HepG2, C3A, and HUH-7 (hepatocellular
carcinoma) [33], and MCF-7 (breast cancer) [34] among others. Some studies have determined that
diosgenin is also an apoptosis inducer and may act as a chemopreventive agent [11]. In addition,
many naturally occurring and synthetic derivatives with an oxime moiety became important because
of their broad biological activity profile: anti-inflammatory, antifungal, antibacterial, anticancer, or
antiviral [35
oxime derivatives (
Figure 1).
This fact has stimulated the search for new routes to synthesize steroidal derivatives with optimal
,
36]. For example, radicicol (
2) shows antitumor activity in in vivo assays, but radicicol
3
) exhibit a higher antitumor activity in both in vivo and in vitro assays [37
,38]
(
biological activity from this pharmacophore on a large scale. In 2009, Cui et al. reported the synthesis
and cytotoxic activity of a series of oximes derived from a cholestane, campestane, and stigmastane
skeletons [39,40]. They suggested that the presence of the oxime group on ring B, a hydroxyl group on
ring A or B, and a cholesterol-type side chain, resulted in high cytotoxicity when evaluated against a
diversity of cancer cells such as Sk-Hep-1, H-292, PC-3, and Hey-1B [41]. In this work, we modified
the spirostane side chain inserting the hydroxyimino group in the A/B rings (C-3 and C-6) and in the
side chain (at C-23). The ketones, as precursors of
showed important cytotoxic activity in various cancer cell lines [42
of the spirostan oximes and and the evaluation of their antiproliferative, apoptotic, and necrotic
activity in cervical cancer cells and human peripheral blood lymphocytes.
α
,
β-unsaturated oximes, as well as the oxime esters,
,
43]. Herein, we report the synthesis
4
5

Molecules 2016, 21, 1533
3 of 17
2
. Results and Discussion
2
.1. Synthesis
The strategy to synthesize the 23-hydroxyimino derivative
4
was based on the introduction of an
acetyl group at C-23 of sapogenin
1 and a further oximation of the (23R)-23-acetyldiosgenin acetate by
treatment with NH OH
·
HCl. The preparation of oximes at A/B rings was accomplished in two steps;
the strategy was to obtain the diketone by oxidation of 1 with Jones Reagent, and to generate the
dioxime 5 derivative by treatment with hydroxylamine hydrochloride, as described in Scheme 1.
2
9
Scheme 1. Synthesis of oxime of (23R)-acetyldiosgenin acetate (
,6-dione (5).
4) and dioxime of (25R)-spirost-4-en-
3
2
.2. NMR Analysis
The structure of products was confirmed by 2D NMR and HRMS analyses (see Supplementary
1
Data). Analysis of the H-NMR spectrum of
4 showed the axial proton H-23 at 2.62 ppm as a doublet
of doublets presenting coupling constants with protons H-24 (J23a,24a = 12.4 Hz, J23a,24e = 5.8 Hz). The
axial proton H-23 of
The 13C spectrum of
8 is located at 2.51 ppm, a similar position for the same proton in compound 7.
1
1
4
showed the C-23 at 159.6 ppm is different from the C-23 in 8, which is located
at 209.1 ppm, and that these two effects result from the formation of the hydroxylamine moiety; the
data were confirmed with the HMBC experiment (Figure 2).
Figure 2. HMBC correlations for the oxime (4) and dioxime (5).

Molecules 2016, 21, 1533
4 of 17
The NMR spectra show a single set of signals for oxime
4. The stereochemistry of the oxime
4
was corroborated by a NOESY experiment, which shows that
position with regard to the alkyl residue. Furthermore, the C-23 is in the same plane as the H in the
4
has the hydroxyl group in an anti
2
C-23 position.
1
Analysis of the H-NMR spectrum of
5
9
showed a single signal at 6.58 ppm for H-4, which was
, observed at 6.18 ppm. In the 13C-NMR of
two signals
around 156 ppm corresponded to C-3 and C-6, the carbon atoms of the oximes. These signals are
up-field compared with those of carbonyls at the derivative (201.8 ppm and 199.4 ppm). The HMBC
shifted downfield compared to the H-4 of
5
9
experiment showed that H-4 correlated with the carbons 3 and 6. Other correlations in the HMBC
experiment between C-3 with and H-2, and C-6 with H-7 are found. Furthermore, correlations among
H-17, H-20, H-21, and H-23 with C-22 are clearly exhibited (Figure 2).
For the synthesis of
3-acetylsapogenins, i.e., in a previous work, we reported the preparation of
of the 22,26-epoxycholestane 6 (Scheme 1), which is obtained from 1 [44] by the treatment with Ac O
4, several synthetic routes are available for the large-scale preparation of
2
7
through the hydrolysis
2
and Et O BF , at r.t., followed by quenching with water. Subsequently, the (25R,23R)-acetyldiosgenin
·
2
3
acetate (
8
) was obtained in excellent yield through a standard acetylation. Next, the oximation of
8 using
NH OH
·
HCl afforded the expected 23-hydroxyimino derivative . This structural addition of nitrogen
4
2
at the spirostane side chain, promoted important changes for the higher bioactivity in comparison to
other structural motifs as observed in the literature. The oxime function in the steroidal framework
has been studied by several authors and first isolated from marine sponges [41
reports, the (6E)-hydroxyimino derivatives exhibited a selective cytotoxic activity against several
types of cancer cells [46 48]. Furthermore, this group of steroids was also reported to show a high
,45]. In different
–
affinity for human placental aromatase and to function as a competitive inhibitor of this enzyme [48].
The structural features, which appear to be important for the higher bioactivity in comparison to other
structural motifs, is the existence of a ketone functionality at C3, and an elevated degree of oxidation in
ring A [49]. The configuration of the oxime does not represent an influence on cytotoxic activity since
it has shown similar activity patterns [50]. Thus, we produced the dioxime derivative
5 through the
oximation of the 3,6-diketone derivative . The synthesis of oximes and was conceived to evaluate
9
4
5
them in cervical cancer cell lines and analyze their antiproliferative activity and cytotoxicity in both
cellular types: cancer cells and normal cells (lymphocytes).
2
.3. Biological Activity
2
.3.1. Antiproliferative Activity on HeLa and CaSki Cervical Cancer Cell Lines
In order to determine the antiproliferative activity of steroidal compounds, diosgenin (1) was
dissolved in EtOH:DMSO 3:1 and compounds
4 and 5 were dissolved in DMSO, and screened at a
range of concentrations against cervicouterine cancer cells HeLa and CaSki. Their antiproliferative
activity as measured by a decrease in cell population (IC50) was determined after 24 h by crystal violet
staining and the dose-response curves are shown in Figure 3 (Table 1).
The inhibitory effect of steroidal oximes
occurred in a dose-dependent manner. Diosgenin (
while those for the oximes were between 5.3 and 5.6
g/mL for compound (Table 1). These results suggest that the antiproliferative activity of
increased 2.3–2.8 times due to the oxime group present in the spiroketalic side chain in ; while the
4
and
) presented IC50 values between 15 and 13
g/mL for compound ; and between 19 and
5
on the proliferation of HeLa and CaSki cells
1
µg/mL,
µ
4
22
µ
5
1
4
oxime groups present on the steroidal A/B rings decreased antiproliferative activity 1.2–1.7 times.
Interestingly, in the literature we notice that the double bond between positions C4 and C5 on the
steroidal ring A, confers a negative cytotoxic effect on the cancer cells for 3-hydroxyimino-substituted
compounds, in contrast with double bond in C5–C6 [35]. These data suggest that the results in
antiproliferative activity may be mediated at least to some extent, by a different behavior between
steroidal oximes 4 and 5, related to the conformational difference on the rings A/B (a double bond shift).

Molecules 2016, 21, 1533
5 of 17
Figure 3. Dose-response curves of the antiproliferative effect of compounds
cancer cell lines HeLa and CaSki. Cisplatin was used as positive control.
1, 4, and 5 on cervicouterine
Table 1. IC50 values of compounds 1, 4, and 5 on cervicouterine cancer cell lines HeLa and CaSki.
IC50 µg/ML (µM)
Cell line
Diosgenin (1)
Oxime (4)
Dioxime (5)
HeLa
CaSki
15 (36.17)
13 (31.35)
5.6 (10.9)
5.4 (10.51)
19 (41.61)
22 (48.18)
2
.3.2. Determination of Cytotoxic Activity on Cervicouterine Cancer Cells
In order to determine whether compounds , and induce necrosis, their cytotoxic activity
1
,
4
5
was evaluated (Figure 4). Considering that the loss of the integrity of the cytoplasmic membrane is
a hallmark of necrotic cell death [51], both HeLa and CaSki cell cultures were stimulated with the
previously determined IC50 values. The amount of lactate dehydrogenase (LDH) released into the
culture supernatant was used as a measure of the loss of plasma-membrane integrity. The positive
control consisted of treating the cells with Triton X-100 to induce cell membrane disruption.
The two tumor cell lines HeLa and CaSki were induced with Triton X-100 in independent
experiments, and the LDH released was adjusted to 100% as a control. The cytotoxicity values
for compounds
at 20%–30%. Taken together, these findings indicate that the cytotoxic activity of compound
increased with the incorporation of the oxime group present in compound . In addition, these
results suggest that the decrease observed in the cell populations of the cultures treated with the
different compounds—diosgenin , monooxime , or dioxime —is affected through a cell death
1 and 4 were less than, or equal to, 15%, while for compound 5 they were higher
1
5
1
4
5
pathway distinct from necrosis, and another event different from necrosis decreases the cell number
observed in cultures.

Molecules 2016, 21, 1533
6 of 17
Figure 4. Evaluation of cytotoxicity for compounds
were seeded in 96-well tissue culture plates at 7500 cells/well. After 24 h the medium was removed and
cells were exposed to compounds , and at the respective IC50 concentrations or with EtOH/DMSO
:1 or DMSO (10 L/mL) and evaluated after 24 h by the amount of LDH released in the culture
supernatant. Experimental data are described as mean S.D. of three independent experiments with
1, 4, and 5 on cervicouterine cancer cell lines. Cells
1,
4
5
3
µ
±
three repetitions * p < 0.05 versus ethanol or DMSO (Student’s t-test).
2
.3.3. Apoptotic Effect on Tumor Cells
Under normal conditions, cells are controlled by a balanced cell cycle such that they proliferate,
lapse after a certain time, and then die, in order to maintain histological homeostasis. Apoptosis is
an important, well-controlled form of cell death that is observed under a variety of physiological
and pathological conditions. Unlike necrotic cell death, apoptosis does not induce an inflammatory
response, thus allowing cells to be removed without producing side effects. As mentioned above,
inducing apoptosis in tumor cells has become an important therapeutic target [52
for example, that inducing cells to cell-cycle arrest constitutes one of the most prevalent strategies
used to stop or limit cancer spreading [54 62]. The decrease in the evaluated cultures suggests that
compounds presumably affect the cell-cycle in one of the phases (checkpoints), causing cell death.
This is a matter worthy of in depth study in future work.
,53]. It is well known,
–
4–5
2
.3.4. Apoptotic Bodies-DAPI Staining
Apoptosis is characterized by morphological changes that include cytoplasmic and chromatin
condensation that produces smaller, more compact nuclei than those present in non-apoptotic
cells. Highly-compacted chromatin is a key feature of apoptotic bodies and a hallmark of the
apoptotic process. To evaluate the capacity of compounds 1, 4, and 5 to induce apoptotic cell
death, HeLa and CaSki cell cultures were stimulated with them. Morphological changes and
chromatin condensation, including the formation of apoptotic bodies, were identified by staining
with fluorochrome 4,6-diamidino-2-phenylindole (DAPI) [63], and recognized under epifluorescence

Molecules 2016, 21, 1533
7 of 17
microscopy (Figure 5). Results showed that the HeLa and CaSki cells treated with compound
1
changed their morphology, showing decreased size and a spherical shape. Additionally, the nuclei
had condensed chromatin, but the presence of clearly-identifiable apoptotic bodies was not confirmed.
Similarly, compounds
4 and 5 caused cell contraction, but also induced DNA fragmentation and
generated clear apoptotic bodies.
Figure 5. Apoptotic morphology induced by 1, 4, and 5 compounds on cervical cancer HeLa and
CaSki cell lines. Phase contrast allows identifying the normal polyhedral morphology in control and
vehicle treated cells; the normal chromatin is evident with the DAPI staining. Colchicine treated cells
(positive control to apoptosis) have a spherical shape, with cytoplasmic and nuclear compaction.
Compounds , and cause cell morphological changes corresponding to the apoptosis morphology.
1,
4
5
Apoptotic bodies are more evident in 4 and 5 than in 1 (arrows). Scale bar 50 microns.
2
.3.5. Detection of Active Caspase-3
Apoptotic cell death is a programmed process that is dependent on proteases called caspases.
These are a family of cysteine proteases that are crucial mediators in the process of apoptosis. Caspase-3
is perhaps the best understood of the mammalian caspases in terms of its specificity and role in
apoptosis. It is also required for some typical hallmarks of apoptosis; for example, it is indispensable
for apoptotic chromatin condensation and DNA fragmentation, and plays an essential role in certain
processes associated with the formation of apoptotic bodies [64]. While this morphological evidence
of the apoptotic process is well-documented, it is still necessary to detect the activation of effector
caspases, such as active caspase-3, to establish the apoptotic process. In this study, we detected
active caspase-3 by flow cytometry in order to obtain quantitative results. Our findings revealed the
presence of active caspase-3 in the HeLa and CaSki cell cultures treated with all compounds (Figure 6),
indicating that they induce the activation of this caspase and, therefore, that apoptosis could be
triggered through a caspase-dependent process. In addition, these results, together with the effect on

Molecules 2016, 21, 1533
8 of 17
cell morphology, where only compound
1
induced apoptotic morphology without fragmented nuclei
(
i.e., fragmented chromatin), suggest that compounds
4
and
5 may have the capacity to increase the
apoptotic effect of
1. In the present work, we evaluated the antiproliferative activity of steroidal oxime
derivatives; however, it is not the aim of this study to describe aspects of the molecular mechanisms
that regulate the observed antitumor effects. We only addressed the potential molecular basis of
these interactions, recent literature provides plausible pathways for the antitumor action of various
steroidal oxime derivatives [65
mechanisms of diosgenin ( ) to induce the cell death that include caspase-3 activation, PARP cleavage,
DNA fragmentation, and cell shrinking [70]. Our results show that compounds and induce
morphological changes such as cell shrinking and DNA compaction. Considering that active caspase-3
is present after the treatment with compounds and , we can hypothesize that these compounds
–69]. Likewise, previous reports have proposed diverse molecular
1
4
5
4
5
trigger a molecular mechanism to induce apoptosis that is mediated by the activation of caspase-3 and
this, in turn, activates cytoplasmic DNAase, which is responsible for DNA fragmentation.
Figure 6. Induction of active caspase-3 by compounds 1, 4, and 5 on HeLa and CaSki cell cultures.
2
.3.6. Evaluation of Antiproliferative Activity on Non-Tumoral Cells
Several compounds used currently in chemotherapy present problems for selective activity
towards malignant cells and produce undesirable secondary effects. It is crucial to determine the
selectivity of tested compounds using the antiproliferative and cytotoxic assays in order to derive
any conclusions on the potential for anti-cancer treatment. For this reason, the effect of
on the proliferation of peripheral blood lymphocytes was assessed. Lymphocytes were exposed to
compounds (15 g/mL), (5.6 g/mL), and (22 g/mL). It is well known that during chemotherapy
the immune system is usually affected; thus, the proliferation of an enriched lymphocyte population
ELP) was evaluated with compounds , and . ELPs from a normal blood donors were labeled with
(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE), stimulated with phytohaemagglutinin
PHA), or treated with compounds , or and cultured for 72 h. After treatment, the cells were
1, 4, and 5
1
4
5
(
1,
4
5
5
(
1,
4
5
harvested, analyzed by flow cytometry (FACS ARIA-II, BD, San Jose, CA, USA), and the data were
processed by FACSDiva 4.0 software (BD, Piscataway, NJ, USA). Considering that lymphocytes were
treated longer with compounds for longer (72 h), the effect of the compounds on the proliferative
potential of ELPs (Figure 7) indicates that they all slightly affected the proliferative potential of
lymphocytes. Compound 1 decreased the proliferation of lymphocytic cells by 23%, while the decreases

Molecules 2016, 21, 1533
9 of 17
for compounds
4
and
5
were 35% and 27%, respectively. These results suggest a greater degree of
antiproliferative selectivity towards malignant cell lines than lymphocytes.
Figure 7. Antiproliferative activity of 1, 4, and 5 compounds on human lymphocyte cell cultures at the
respective concentrations (1 (15 g/mL), 4 (5.6 g/mL), and 5 (22 g/mL)).
2
.3.7. Determination of Cytotoxic Activity on Lymphocyte Cells
In order to determine whether necrosis is induced by these sapogenins in non-tumor cells, their
cytotoxicity on human lymphocytes was evaluated (Figure 8). The application of this model is very
important since the results obtained allowed us to determine lower toxicity and, hence, properly
demonstrate the antiproliferative activity with minor secondary effects. The lymphocytes were
activated with phytohaemagglutinin (PHA) and stimulated at the level of the IC50 values determined
for compounds 1, 4, and 5. Results indicated that these concentrations were not cytotoxic for human
lymphocytes, which suggests that these steroidal oximes were not cytotoxic in non-tumor cells, so the
decrease in cell populations was caused by a mechanism distinct from necrosis.
Figure 8. Evaluation of cytotoxicity for compounds 1, 4, and 5 on human lymphocyte cell cultures.
Surprisingly, lower cytotoxicity values were found in all cases, even with regard to the cytotoxicity
on non-tumoral cells (human lymphocytes). These results suggest that the observed cell decrease in
treated cultures is not a necrotic process.

Molecules 2016, 21, 1533
10 of 17
3
. Materials and Methods
3
.1. General Procedures and Materials
◦
Melting points were determined on a melt-temp apparatus at 24 C by the open capillary method
◦
and are uncorrected. Optical rotations were measured at 24 C in a Perkin-Elmer 241 polarimeter
Perkin Elmer Instruments LLC., Beaconsfield, UK) in chloroform solutions. H- and C-NMR spectra
1
13
(
were recorded at 400 and 100 MHz in CDCl , respectively, on a Varian MERCURY NMR instrument
3
1
(
Palo Alto, CA, USA). The H-NMR spectra were referenced to residual protonated solvent and for
C-NMR the central signal of the triplet of CDCl . Chemical shifts are given in ppm (δ-scale) and
1
3
3
coupling constants are expressed in Hertz (Hz). All assignments were confirmed with the aid of DEPT,
COSY, HSQC, HMBC and NOESY experiments. Spectra processing was performed using MestReNova
software (10.0, Santiago de Compostela, Spain). High resolution mass spectra were obtained by the
ESI technique using a JEOL JMS AX-505 HA mass spectrometer. IR spectra were recorded on an
−
1
Agilent Cary 630 FT-IR spectrophotometer (range: 4000–600 cm ). Column chromatography was
performed using Merck silica gel 60 m (230–400 mesh) by flash chromatography and analytical TLC
was performed on aluminum plates pre-coated with silica gel 60F-254. The preparation of compounds
µ
6
3
3
and 7 (Scheme 1) was performed according to the literature [44].
.2. Chemical Synthesis and Characterization
.2.1. 23-Acetyldiosgenin Acetate 8
A solution of 23-acetyldiosgenin
7 (1.0 g, 2.19 mmol) in acetic anhydride (10 mL) was stirred at
reflux for a period of 3 h. After that time, the crude product was poured into cold water and the
precipitate filtered. The crude product was purified by column chromatography using a mixture of
◦
hexane/EtOAc (8:2) as eluent, to afford compound
8
as a white solid (1.04 g, 95%). m.p. 171–172 C.
◦
1
[
α
] =
D
−
135 (c = 0.2). υmax 2920, 1732, 1678, 1636, 1033. H-NMR (CDCl )
δ
: 5.23 (1H, d, J = 5 Hz,
3
H-6), 4.46 (1H, m, H-3), 4.28 (1H, m, H-16), 3.36 (1H, m, H-26a); 3.28 (1H, dd, J26e,26a, J26e,25a = 11 Hz,
H-26e), 2.51 (1H, dd, J = 12.00 Hz, H-23a), 2.28 (1H, ddd, J2
= 13 Hz, J24a,23a = 13 Hz, J24a,25e = 5 Hz,
4a,24e
2
H-24a), 2.06 (3H, s, 23 ), 1.89 (3H, s,
C
H COO-3), 0.89 (3H, s, CH -19), 0.85 (3H, d, J
3
= 6.0 Hz,
21,20
δ
3
1
3
CH -21), 0.73 (3H, d, J
= 6.0 Hz, CH -27), 0.54 (3H, s, CH -18). C-NMR (CDCl )
: 14.1 (C-21),
6.0 (C-18), 16.9 (C-27), 19.2 (C-19), 20.6 (C-11), 21.3 (CH COO-3), 27.6 (C-23 ), 28.4 (C-24), 29.6 (C-6),
3
3
27,25
3
3
3
2
1
3
1.0 (C-7), 31.4 (C-2 and C-15), 31.9 (C-1 and C-12), 36.5 (C-8), 36.8 (C-5), 37.9 (C-9), 38.2 (C-13), 39.5
(
1
C-20), 40.3 (C-24), 49.7 (C-25), 54.7 (C-23), 56.1 (C-14), 61.0 (C-17), 65.8 (C-26), 73.5 (C-3), 80.8 (C-16),
1
07.6 (C-22), 121.8 (C-6), 139.3 (C-5), 169.9 (CH COO-3), 209.4 (C-23 ).
3
3
.2.2. Oxime of 23-Acetyldiosgenin Acetate 4
Hydroxylamine chlorhydrate (0.108 g, 1.53 mmol) was added to a solution of
8 (1.0 g, 1.92 mmol)
in pyridine (30 mL) and allowed to stir at reflux. After a period of 1 h organic extraction was performed
CH Cl , 2 10 mL), the organic phase was washed with distilled water (2 15 mL), dried with
(
×
×
2
2
Na SO and concentrated under reduced pressure. Compound
solid, recrystallized from CH Cl . m.p. 180–181 C (CH Cl ). [
1
4
α
was obtained (1.01 g, 93%) as a white
2
4
◦
◦
] =
−
24 (c = 0.3). υmax 3309, 1732,
: 5.34 (1H, d, J = 5.6 Hz, H-6), 4.58 (1H, m, H-3) 4.38 (1H, m, H-16),
2
2
2
2
D
1
666, 1636, 949. H-NMR (CDCl )
δ
3
3.48 (1H, dd, J26a,26e = 14 Hz, J26a,25 = 4 Hz, H-26a); 3.38 (1H, dd, J26e,26a = 14 Hz, H-26e), 2.62 (1H, dd,
J23a,24a = 12.4 Hz, J
= 5.8 Hz, H-23a), 2.31(2H, m, H-4), 2.17 (1H, dd, J = 12.4 Hz, H-24a), 2.02
23a,24e
2
(
3H, s, CH -23 ), 1.90 (3H, s,
C
H COO-3), 1.60 (1H, dd, J = 5.8 Hz H-24e), 1.00 (3H, s, CH -19), 0.94
3
3
3
13
(3H, d, J = 6.0 Hz, CH -21), 0.82 (3H, d, J = 6.0 Hz, CH -27), 0.67 (3H, s, CH -18). C-NMR (CDCl )
3 3 3 3
2
−
δ: 11.9 (CH -COO-3), 14.1 (C-21), 16.1 (C-18), 17.0 (C-27), 19.3 (C-19), 20.8 (C-11), 21. 5 (C-23 ), 27.7
3
C-24), 30.4 (C-8), 31.2 (C-9), 31.7 (C-1), 32.1 (C-2), 32.4 (C-10), 36.7 (C-7), 36.9 (C-15), 37.7 (C-4), 38.1
C-13), 39.7 (C-20), 40.4 (C-24), 45.6 (C-23), 49.8 (C-25), 56.3 (C-14), 61.1 (C-17), 66.1 (C-26), 73.8 (C-3),
(
(

Molecules 2016, 21, 1533
11 of 17
1
C
OO-3). HRMS-EI: [M + H]+
8
1.0 (C-16), 109.7 (C-22), 122.1 (C-6), 139.4 (C-5), 159.3 (C-23 ), 170.3 (CH3
calcd. for C H O N, 513.3454; found: 513.3445.
31
47
5
3
.2.3. (25R)-Spirost-4-en-3,6-dione 9
◦
10 C to a solution of 1 (1.0 g, 2.41 mmol) in
CH Cl (40 mL) and acetone (132 mL). The temperature of the reaction mixture should not exceed
Jones Reagent (10 mL) was added dropwise at
−
2
◦
2
1
0 C. At the end of the reaction some drops of isopropanol were added and the stirring continued,
at room temperature, for 3 h, then filtered through florisil. The crude product was neutralized with
saturated aqueous solution of NaHCO , and the organic phase was washed with brine (2 50 mL)
50 mL). The organic phase was dried with Na SO , concentrated under
×
3
and distilled water (2
×
2
4
reduced pressure, and finally purified on a silica gel column, with hexane/EtOAc (8:2) as eluent,
◦
◦
106.2 (c = 0.0014). υmax 2949,
to afford
685. H-NMR (CDCl )
9
(0.87 g, 85%) as a white solid. m.p. 193–194 C. [
α
] =
D
−
1
1
δ
: 6.18 (1H, s, H-4), 4.43 (1H, m, H-16), 3.48 (1H, m, H-26e), 3.36 (1H, dd,
3
J26a,26e = J26a,25a = 10.4 Hz, H-26a), 2.71 (2H, d, J7,8 = 12.0 Hz, H-7), 1.19 (3H, s, CH -19), 0.99 (3H, d,
3
13
J21,20 = 6.8 Hz, CH -21), 0.84 (3H, s, CH -18), 0.80 (3H, d, J
= 6.0 Hz, CH -27). C-NMR (CDCl3) δ:
3
3
3
27,25
3
3
6.6 (C-1), 33.9 (C-2), 199.4 (C-3), 125.5 (C-4), 160.6 (C-5), 201.8 (C-6), 32.1 (C-7), 35.1 (C-8), 53.7 (C-9),
8.6 (C-10), 20.8 (C-11), 39.6 (C-12), 40.3 (C-13), 55.6 (C-14), 31.6 (C-15), 80.6 (C-16), 61.9 (C-17), 17.3
(
1
C-18), 16.3 (C-19), 41.6 (C-20), 14.5 (C-21), 109.2 (C-22), 31.3 (C-23), 28.7 (C-24), 30.2 (C-25), 66.8 (C-26),
7.1 (C-27).
3
.2.4. Dioxime of (25R)-Spirost-4-en-3,6-dione 5
To a solution of (1.70 g, 4.00 mmol) in ethanol (30 mL) and pyridine (1 mL), NH OH
0.560 g, 8.00 mmol) was added. The reaction mixture was stirred at reflux for 1 h. The organic phase
was separated, washed with distilled water (2 15 mL), dried with Na SO and concentrated under
reduced pressure. The reaction crude was purified on silica gel column, with hexane/EtOAc (6:4)
9
·
HCl
2
(
×
2
4
◦
◦
α] = +37.8 (c = 0.0067), υmax
D
as eluent, to afford
5
(1.41 g, 77%) as white crystals, Mp 150–152 C, [
: 6.58 (1H, s, H-4), 4.43 (1H, m, H-16), 3.48 (1H, dd, J = 9.2, J = 2.8 Hz,
1
3
313, 1681. H-NMR (CDCl )
δ
3
H-26e), 3.37 (1H, dd, J = 11.2, J = 11.2 Hz, H-26a), 3.07 (2H, dd, J2,2 = 18, J2,1 = 3.6 Hz H-2), 3.44 (2H,
m, H-7), 1.06 (3H, s, CH -19), 0.98 (3H, d, J = 6.8 Hz, CH -21), 0.82 (3H, s, CH -18), 0.79 (3H, d,
3
21,20
3
3
13
J27,25 = 6 Hz, CH -27). C-NMR (CDCl3) δ: 39.3 (C-1), 18.7 (C-2), 156.4 (C-3), 119.3 (C-4), 147.3 (C-5),
3
156.6 (C-6), 46.0 (C-7), 32.7 (C-8), 49.8 (C-9), 38.8 (C-10), 21.1 (C-11), 33.4 (C-12), 40.3 (C-13), 61.8 (C-14),
3
1.4 (C-15), 80.6 (C-16), 56.3 (C-17), 16.2 (C-18), 19.0 (C-19), 41.6 (C-20), 14.4 (C-21),109.3 (C-22), 31.3
+
(C-23), 28.7 (C-24), 30.2 (C-25), 66.9 (C-26), 17.1 (C-27). HRMS-EI: [M + H] calcd. for C H O N ,
27
40
4
2
4
56.2988; found: 456.2982.
.3. Biological Evaluation
.3.1. Cell Cultures
3
3
HeLa and CaSki cell lines were purchased from the American Type Culture Collection
ATCC, Rockville, MD, USA) and cultured in RPMI-1640 medium (GIBCO BRL, Grand Island, NY,
(
USA) containing 5% newborn calf serum (NCS, GIBCO BRL) with red phenol supplemented by
◦
benzylpenicillin. Cultures were maintained in a humidified atmosphere with 5% CO at 37 C.
2
All cell-based assays were performed using cells in the exponential growth phase.
3
.3.2. Cell Proliferation Assays
Assays were performed by seeding 7500 cells/well in 96-well tissue culture plates at a volume of
100
µL of RPMI-1640 medium supplemented with 5% NCS per well. Cells were allowed to grow for
2
4 h in the culture medium prior to exposure to the compounds. Cisplatin was used as positive control.
Additionally, 1% of vehicle (DMSO/EtOH, 1:1) was added to the control cells. Antiproliferative

Molecules 2016, 21, 1533
12 of 17
activity (IC ) was determined after 24 h by crystal violet staining [71]. Cell counts were performed by
50
measuring absorbance at 590 nm in an ELISA plate reader (ChroMate, Palm, FL, USA).
3
.3.3. Determination of Cytotoxicity
Cytotoxic activity was determined by means of LDH Cytotoxicity Assay Kit (Cyto Tox 96®
Promega, Madison, WI, USA) following the manufacturer’s instructions. LDH oxidizes lactate to
pyruvate, which then reacts with the INT to produce formazan. The increased amount of formazan
generated in the culture supernatant directly correlates to the increase in the number of lysed cells.
Formazan dye is water-soluble and can be detected with a spectrophotometer at 500 nm [51].
3
.3.4. CFSE-Labeling Assay
Heparinized blood samples were obtained from healthy human volunteers. PBMCs were isolated
using standard Hypaque (Sigma-Aldrich, St. Louis, MO, USA) density gradient centrifugation.
The PBMCs were then washed twice with RPMI 1640 (GIBCO BRL) medium containing 10% NCS,
penicillin (100 U/mL), and streptomycin (100
enriched (ELP) by eliminating adherent cells (cells were incubated at 37 C, 5% CO for 1 h, and
µ
g/mL). The lymphocyte population was further
◦
2
non-adherent cells were harvested). ELPs were re-suspended in RPMI-1640 medium at a concentration
6
of 1
2
×
10 cells/mL. CFSE (Sigma-Aldrich) was added to the cell suspension at a final concentration of
mM and then incubated for 15 min at room temperature in the dark. Labeling was completed by
adding the same volume of NCS during 5 min at room temperature to quench the free CFSE. Labeled
cells were washed five times with sterile PBS containing 10% NCS, counted, and then re-suspended in
6
RPMI-1640 medium at 1
×
10 cells/mL [72]. Unstimulated, PHA-stimulated, or treated, cells were
5
plated at 2
×
10 cells/well in 96-well, flat-bottomed cell culture plates, and five replicate samples for
each treated amount were prepared. Cells were incubated in a 5% CO incubator at 37 C for 72 h.
◦
2
Cultured cells were harvested, washed twice with PBS, fixed with 1% formaldehyde, then analyzed by
flow cytometry, acquiring a minimum of 20,000 events from each sample. Data analysis was performed
using FACSDiva software (4.0, Piscataway, NJ, USA).
3
.3.5. Detection of Active Caspase-3
HeLa and CaSki cells were seeded at 10 cells/mL in 60-mm tissue culture plates and allowed
to grow for 24 h in culture medium before treatment with their respective IC . Cells were harvested
5
50
with versene solution. For active caspase-3 immunodetection, they were fixed and permeabilized
in 50% methanol in PBS, washed in PBS, incubated with primary anti-body anti-active-caspase-3
(
ABCAM, Cambridge, MA, USA), and diluted in PBS (1:500) for 18 h. Next, the cells were washed
and incubated with the secondary goat anti-rabbit antibody with FITC diluted to 1:200 in PBS for
h. The samples were analyzed by flow cytometry, acquiring a minimum of 10,000 events from each
sample. Data analysis was performed using FACSDiva 4.0 software.
2
3
.3.6. Statistical Analysis
Means and standard deviations (SD) were calculated using Excel (Microsoft Office, Version 2010,
Redmond, WA, USA). Statistical analysis of differences was carried out by ANOVA using SPSS 10.0 for
Windows. A p-value < 0.05 (Student’s t-test) was considered significant.
4
. Conclusions
Two new steroidal sapogenins were synthesized, with the incorporation of the functional group
oxime to measure their antiproliferative activity, in the search for compounds with an increased broad
biological activity profile. The assays showed that the steroidal oximes and generate interesting
results in the antiproliferative activity in vitro compared to the activity observed with diosgenin ( ).
The antiproliferative activity of was 2.3–2.8 times that of due to the oxime group present in the
4
5
1
4
1

Molecules 2016, 21, 1533
13 of 17
spiroketalic side chain of
4; the oxime groups present on the steroidal A/B rings of
5
decreased
the antiproliferative activity 1.2–1.7 times, suggesting that the oxime group improves the activity
of spirostans if it is present in the side chain. A relevant aspect of compounds and is that they
4
5
produce low-cytotoxicity effects in two cellular types: cancer cells and normal cells (lymphocytes),
since apoptosis, more than necrosis, was the pathway for cancer cell elimination with evidenced
apoptotic morphology and an increased presence of active caspase-3. The fact that compounds
4 and 5
did not affect proliferation of normal lymphocytes confers a certain advantage to these compounds.
It has been shown that the double bond between positions C4 and C5 on the steroidal ring A, confers a
negative cytotoxic effect on cancer cells for 3-hydroxyimino-substituted compounds [35]. This suggests
that antiproliferative activity may also be mediated, at least to some extent, by a different behavior
between steroidal oximes
4
and
5, related to the conformational difference on the rings A/B (a double
bond shift) and it is possible that compound
4
may be recognized more easily by the active site in
the cell by binding to protein- associated signaling molecules [73]. From this information our future
work will focus on the search for new routes to synthesize steroidal derivatives with oxime moieties
on a larger scale and on the search for molecular mechanisms involved in antiproliferative activity.
The information obtained may be useful for the design of novel chemotherapeutic drugs.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
1
1/1533/s1, Figure S1–Figure S22.
Acknowledgments: This work was supported by CONACyT grants RET-250025, 255881, and 253979. We thank
VIEP and CUVyTT-BUAP for financial support. M.G.H.-L. thanks BUAP-PRODEP-NPTC-DSA/103.5/16/10420
support. We thank PAPIIT IN222114 and IN220916 for academic and financial support. We are thankful to
CONACyT for the scholarships to GGL-573785 and ACC-598465. This paper constitutes a partial fulfilment of the
Ph. D. degree in Biological and Medical Sciences from Universidad Autónoma Benito Juárez de Oaxaca, México.
M.A.F.-H. thanks the “For Women in Science Program” for the L’Oréal
−
UNESCO
−
Conacyt AMC fellowship.
−
The authors would like to thank Allen J. Coombes and Paul C. Kersey Johnson for proofreading the manuscript.
Author Contributions: The list authors contributed to this work as follows: L.S.-S., M.L.E., H.L.-M., and
E.Z.: developed the biological analysis; L.S.-S. and M.L.E.: draft of biological area. A.C.-C.: Synthesis and
purification of oxime of (23R)-acetyldiosgenin acetate (
4). G.L.-G.: Synthesis and purification of dioxime of
(
25R)-spirost-4-en-3,6-dione (5). M.A.F.-H.: NMR analysis of oximes and precursors and drafting of chemical
area. M.G.H.-L.: elucidation, analyzed the data, wrote the paper and acquired funding of chemical area. J.S.-R.:
polished the paper and acquired funding.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
References
1.
2.
3.
4.
5.
6.
7.
8.
Enger, E.D.; Ross, F.C.; Bailey, D.B. Concepts in Biology, 12th ed.; McGraw-Hill: New York, NY, USA, 2007;
pp. 171–173.
Kato, A.; Miura, T.; Fukunaga, T. Effects of steroidal glycosides on blood glucose in normal and diabetic
mice. Biol. Pharm. Bull. 1995, 18, 167–168. [CrossRef] [PubMed]
Gurib-Fakim, A. Medicinal plants: Traditions of yesterday and drugs of tomorrow. Mol. Asp. Med. 2006, 27,
1
–93. [CrossRef] [PubMed]
Butler, M.S. Natural products to drugs: Natural product derived compounds in clinical trials. Nat. Prod. Rep.
005, 22, 162–195. [CrossRef] [PubMed]
2
Man, S.; Gao, W.; Zhang, Y.; Huang, L.; Liu, C. Chemical study and medical application of saponins as
anti-cancer agents. Fitoterapia 2010, 81, 703–714. [CrossRef] [PubMed]
Shi, J.; Arunasalam, K.; Yeung, D.; Kakuda, Y.; Mittal, G.; Jiang, Y. Saponins from edible legumes: Chemistry,
processing, and health benefits. J. Med. Food 2004, 7, 67–78. [CrossRef] [PubMed]
Djerassi, C.; Rosenkranz, G.; Pataki, J.; Kaufmann, S. Steroids, XXVII. Synthesis of allopregnane-3
7α-, 20β, 21-pentol from cortisone and diosgenin. J. Biol. Chem. 1952, 194, 115–118. [PubMed]
Raju, J.; Mehta, R. Cancer chemopreventive and therapeutic effects of diosgenin, a food saponin. Nutr. Cancer
008, 61, 27–35. [CrossRef] [PubMed]
β, 11β,
1
2

Molecules 2016, 21, 1533
14 of 17
9
.
Yue, L.; Chen, L.; Kou, J.P.; Yu, B.Y. Recent advances of diosgenin in its pharmacological activities and
mechanism. Chin. J. Clin. Pharmacol. Ther. 2010, 2, 233–237.
1
0. Corbière, C.; Liagre, B.; Bianchi, A.; Bordji, K.; Dauça, M.; Netter, P.; Beneytout, J.L. Different contribution of
apoptosis to the antiproliferative effects of diosgenin and other plant steroids, hecogenin and tigogenin, on
human 1547 osteosarcoma cells. Int. J. Oncol. 2003, 22, 899–906. [CrossRef] [PubMed]
1
1
1
1
1
1. Corbière, C.; Liagre, B.; Terro, F.; Beneytout, J.L. Induction of antiproliferative effect by diosgenin through
activation of p53, release of apoptosis-inducing factor (AIF) and modulation of caspase-3 activity in different
human cancer cells. Cell Res. 2004, 14, 188–196. [CrossRef] [PubMed]
2. Wang, W.C.; Liu, S.F.; Chang, W.T.; Shiue, Y.L.; Hsieh, P.F.; Hung, T.J.; Hung, C.Y.; Hung, Y.J.; Chen, M.F.;
Yang, Y.L. The effects of diosgenin in the regulation of renal proximal tubular fibrosis. Exp. Cell Res. 2014
23, 255–262. [CrossRef] [PubMed]
,
3
3. Chen, P.S.; Shih, Y.W.; Huang, H.C.; Cheng, H.W. Diosgenin, a steroidal saponin, inhibits migration and
invasion of human prostate cancer PC-3 cells by reducing matrix metalloproteinases expression. PLoS ONE
2
011, 6, e20164. [CrossRef] [PubMed]
4. Mao, Z.J.; Tang, Q.J.; Zhang, C.A.; Qin, Z.F.; Pang, B.; Wei, P.W.; Liu, B.; Chou, Y.N. Anti-proliferation and
anti-invasion effects of diosgenin on gastric cancer BGC-823 cells with HIF-1 shRNAs. Int. J. Mol. Sci. 2012
3, 6521–6533. [CrossRef] [PubMed]
α
,
1
5. Kang, T.H.; Moon, E.; Hong, B.N.; Choi, S.Z.; Son, M.; Park, J.H.; Kim, S.Y. Diosgenin from Dioscorea nipponica
ameliorates diabetic neuropathy by inducing nerve growth factor. Biol. Pharm. Bull. 2011, 34, 1493–1498.
[CrossRef] [PubMed]
1
1
6. Huang, C.H.; Ku, C.Y.; Jan, T.R. Diosgenin attenuates allergen-induced intestinal inflammation and IgE
production in a murine model of food allergy. Planta Med. 2009, 75, 1300–1305. [CrossRef] [PubMed]
7. Jan, T.R.; Wey, S.P.; Kuan, C.C.; Liao, M.H.; Wu, H.Y. Diosgenin, a steroidal sapogenin, enhances
antigen-specific IgG2a and interferon-
γ expression in ovalbumin-sensitized BALB/c mice. Planta Med.
2
007, 73, 421–426. [CrossRef] [PubMed]
1
1
8. Huang, C.H.; Liu, D.Z.; Jan, T.R. Diosgenin, a plant-derived sapogenin, enhances regulatory T-cell immunity
in the intestine of mice with food allergy. J. Nat. Prod. 2010, 73, 1033–1037. [CrossRef] [PubMed]
9. Gao, M.; Chen, L.; Yu, H.; Sun, Q.; Kou, J.; Yu, B. Diosgenin down-regulates NF-
pathways and attenuates acute lung injury induced by lipopolysaccharide in mice. Int. Immunopharmacol.
013, 15, 240–245. [CrossRef] [PubMed]
κB p65/p50 and p38MAPK
2
2
2
0. Zhang, R.; Huang, B.; Du, D.; Guo, X.; Xin, G.; Xing, Z.; Liang, L.; Chen, Y.; Chen, Q.; He, Y.; et al.
Anti-thrombosis effect of diosgenyl saponins in vitro and in vivo. Steroids 2013, 78, 1064–1070. [CrossRef]
[PubMed]
1. He, Z.; Tian, Y.; Zhang, X.; Bing, B.; Zhang, L.; Wang, H.; Zhao, W. Anti-tumour and immunomodulating
activities of diosgenin, a naturally occurring steroidal saponin. Nat. Prod. Res. 2012, 26, 2243–2246. [CrossRef]
[PubMed]
2
2
2
2
2
2
2
2
2. Tsukamoto, T.; Ueno, Y.; Ohta, Z. On the constitution of diosgenin. I. Glucoside of Dioscorea tokoro makino.
J. Pharm. Soc. Jpn. 1936, 56, 931–940.
3. Taylor, W.G.; Elder, J.L.; Chang, P.R.; Richards, K.W. Microdetermination of diosgenin from fenugreek
(Trigonella foenum-graecum) seeds. J. Agric. Food Chem. 2000, 48, 5206–5210. [CrossRef] [PubMed]
4. Hooker, E. Final report of the amended safety assessment of Dioscorea villosa (Wild Yam) root extract.
Int. J. Toxicol. 2004, 23, 49–54.
5. Marker, R.E. Sterols, C.V. The preparation of testosterone and related compounds from sarsasapogenin and
diosgenin. J. Am. Chem. Soc. 1940, 62, 2543–2547. [CrossRef]
6. Tietze, L.F.; Bell, H.P.; Chandrasekhar, S. Natural product hybrids as new leads for drug discovery.
Angew. Chem. Int. Ed. 2003, 42, 3996–4028. [CrossRef] [PubMed]
7. Komesaroff, P.A.; Black, C.V.; Cable, V.; Sudhir, K. Effects of wild yam extract on menopausal symptoms,
lipids and sex hormones in healthy menopausal women. Climacteric 2001, 4, 144–150. [CrossRef] [PubMed]
8. Yan, C.; You-Mei, T.; Su-Lan, Y.; Yu-Wei, H.; Jun-Ping, K.; Bao-Lin, L.; Bo-Yang, Y. Advances in the
pharmacological activities and mechanisms of diosgenin. Chin. J. Nat. Med. 2015, 13, 578–587.
9. Juarez-Oropeza, M.A.; Diaz-Zagoya, J.C.; Rabinowitz, J.L. In vivo and in vitro studies of hypocholesterolemic
effects of diosgenin in rats. Int. J. Biochem. 1987, 19, 679–683. [CrossRef]

Molecules 2016, 21, 1533
15 of 17
3
0. Huo, R.; Zhou, Q.; Wang, B.; Tashiro, S.; Onodera, S.; Ikejima, T. Diosgenin induces apoptosis in HeLa cells
via activation of caspase pathway. Acta Pharmacol. Sin. 2004, 25, 1077–1082.
1. Raju, J.; Bird, R.P. Diosgenin, naturally occurring furostanol saponin suppresses
-hydroxy-3-methylglutaryl CoA reductase expression and induces apoptosis in HCT-116 human
3
a
3
colon carcinoma cells. Cancer Lett. 2007, 255, 194–204. [CrossRef] [PubMed]
3
2. Moalic, S.; Liagre, B.; Corbière, C.; Bianchi, A.; Dauça, M.; Bordji, K.; Beneytout, J.L. A plant steroid,
diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells. FEBS Lett. 2001, 506,
2
25–230. [CrossRef]
3
3. Li, F.; Fernandez, P.P.; Rajendran, P.; Hui, K.M.; Sethi, G. Diosgenin, a steroidal saponin, inhibits STAT3
signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular
carcinoma cells. Cancer Lett. 2010, 292, 197–207. [CrossRef] [PubMed]
3
3
3
3
4. Li, J.; Liu, X.; Guo, M.; Liu, Y.; Liu, S.; Yao, S. Electrochemical study of breast cancer cells MCF-7 and its
application in evaluating the effect of diosgenin. Anal. Sci. 2005, 21, 561–564. [CrossRef] [PubMed]
5. Bansal, R.; Acharya, P.C. Man-made cytotoxic steroids: Exemplary agents for cancer therapy. Chem. Rev.
2
014, 114, 6986–7005. [CrossRef] [PubMed]
6. Díaz, J.; Durán, N.; Robles, J.; Loaiza, A. Síntesis y evaluación “in vitro” de la actividad antifúngica de
oximas, éteres de oxima e isoxazoles. Univ. Sci. 2011, 16, 294–302. [CrossRef]
7. Soga, S.; Neckers, I.M.; Schulte, T.W.; Shiotsu, Y.; Akasaka, K.; Narumi, H.; Agatsuma, T.; Ikuina, Y.;
Murakata, C.; Tamaoki, T.; et al. KF25706, a novel oxime derivative of Radicicol, exhibits in vivo antitumor
activity via selective depletion of Hsp90 binding signaling molecules. Cancer Res. 1999, 59, 2931–2938.
[PubMed]
3
8. Sato, H.; Yamada, R.; Yanagihara, M.; Okuzawa, H.; Iwata, H.; Kurosawa, A.; Ichinomiya, S.; Suzuki, R.;
Okabe, H.; Yano, T.; et al. New 2-aryl-1,4-naphthoquinone-1-oxime methyl ether compound induces
microtubule depolymerization and subsequent apoptosis. J. Pharmacol. Sci. 2012, 118, 467–478. [CrossRef]
[PubMed]
3
4
4
4
4
4
9. Cui, J.; Fan, L.; Huang, Y.; Xin, Y.; Zhou, A. Synthesis and evaluation of some steroidal oximes as cytotoxic
agents: Structure/activity studies (I). Steroids 2009, 74, 62–72. [CrossRef] [PubMed]
0. Cui, J.; Fan, L.; Huang, Y.; Xin, Y.; Zhou, A. Synthesis and evaluation of some steroidal oximes as cytotoxic
agents: Structure/activity studies (II). Steroids 2009, 74, 989–995. [CrossRef] [PubMed]
1. Rodríguez, J.; Nuñez, L.; Peixinho, S.; Jiménez, C. Isolation and synthesis of the first natural 6-hydroximino
4
-en-3-one-steroids from the sponges Cinachyrella spp. Tetrahedron Lett. 1997, 38, 1833–1836. [CrossRef]
2. Ning-Juan, F.; Jiang-Jiang, T.; He, L.; Xiao-Jun, L.; Bo, L.; Jin-Ming, G. Synthesis and cytotoxicity of some
novel steroidal C-17 pyrazolinyl derivatives. Eur. J. Med. Chem. 2013, 69, 182–190.
3. Ning-Juan, F.; Shao-Peng, W.; Jin-Ming, G.; Jiang-Jiang, T. Potential insecticidal activity of steroidal C-17
pyrazolinyl derivatives. J. Braz. Chem. Soc. 2015, 26, 389–392.
4. Meza-Reyes, S.; Sandoval-Ramírez, J.; Montiel-Smith, S.; Hernández-Linares, G.; Viñas-Bravo, O.;
Martínez-Pascual, R.; Fernández-Herrera, M.A.; Vega-Báez, J.L.; Merino-Montiel, P.; Santillán, R.L.;
et al.
β-Alkoxy-α,β-unsaturated ketone systems in steroidal frameworks, and their conversion to
2
3,24-bisnorcholane lactones. Arkivoc 2005, vi, 307–320.
4
4
4
4
4
5
5. Kovganko, N.V.; Chernov, Y.G. 13C-NMR Spectra of 6-hydroximinosteroids of the stigmastane series.
Chem. Nat. Compd. 2001, 37, 351–355. [CrossRef]
6. Cui, J.; Huang, L.; Fan, L.; Zhou, A. A facile and efficient synthesis of some (6E)-hydroximino-4-en-3-one
steroids, steroidal oximes from Cinachyrella spp. sponges. Steroids 2008, 73, 252–256. [CrossRef] [PubMed]
7. Kovganko, N.B.; Chernov, Y.G. Novel synthesis of (24R,6E)-24-ethylcholest-6-hydroxyimino-4-en-3-one, a
steroidal oxime from Cinachyrella spp. sponges. Chem. Nat. Compd. 2000, 36, 189–191. [CrossRef]
8. Holland, H.L.; Kumaresan, S.; Tan, L.; Njar, V.C.O. Synthesis of 6-hydroximino-3-oxo steroids, a new class of
aromatase inhibitor. J. Chem. Soc. Perkin Trans. 1 1992, 5, 585–587. [CrossRef]
9. Deive, N.; Rodríguez, J.; Jiménez, C. Synthesis of cytotoxic 6E-hydroximino-4-ene steroids: Structure/activity
studies. J. Med. Chem. 2001, 44, 2612–2618. [CrossRef] [PubMed]
0. Poza, J.; Rega, M.; Paz, V.; Alonso, B.; Rodríguez, J.; Salvador, N.; Fernández, A.; Jiménez, C. Synthesis and
evaluation of new 6-hydroximinosteroid analogs as cytotoxic agents. Bioorg. Med. Chem. 2007, 15, 4722–4740.
[CrossRef] [PubMed]

Molecules 2016, 21, 1533
16 of 17
5
1. Legrand, C.; Bour, J.M.; Jacob, C.; Capiaumont, J.; Martial, A.; Marc, A.; Wudtke, M.; Kretzmer, G.;
Demangel, C.; Duval, D.; et al. Lactate dehydrogenase (LDH) activity of the number of dead cells in
the medium of cultured eukaryotic cells as marker. J. Biotechnol. 1992, 25, 231–243. [CrossRef]
2. Koff, J.L.; Ramachandiran, S.; Bernal-Mizrachi, L. A Time to Kill: Targeting Apoptosis in Cancer. Int. J.
Mol. Sci. 2015, 16, 2942–2955. [CrossRef] [PubMed]
5
5
3. Fulda, S. Targeting extrinsic apoptosis in cancer: Challenges and opportunities. Semin. Cell Dev. Biol. 2015
9, 20–25. [CrossRef] [PubMed]
,
3
5
5
4. Shapiro, H.M. Practical Flow Cytometry, 2nd ed.; Alan R. Liss, Inc.: New York, NY, USA, 1988.
5. Hartwell, L.H.; Weinert, T.A. Checkpoints: Controls that ensure the order of cell cycle events. Science 1989
,
2
46, 629–634. [CrossRef] [PubMed]
5
6. Darzynkiewicz, Z. Nucleic Acid Analysis; Robinson, Ed.; Current Protocols in Cytometry, John Wiley & Sons,
Inc.: New York, NY, USA, 1997; Chapter 7. (In Japanese)
5
7. Carlson, B.; Lahusen, T.; Singh, S.; Loaiza-Perez, A.; Worland, P.J.; Pestell, R.; Albanese, C.; Sausville, E.A.;
Senderowicz, A.M. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast
carcinoma cells induced by flavopiridol. Cancer Res. 1999, 59, 4634–4641. [PubMed]
5
5
8. Sherr, C.J. The Pezcoller lecture: Cancer cell cycles revisited. Cancer Res. 2000, 60, 3689–3695. [PubMed]
9. Senderowicz, A.M.; Sausville, E.A. Preclinical and clinical development of cyclin-dependent kinase
modulators. J. Natl. Cancer Inst. 2000, 92, 376–387. [CrossRef] [PubMed]
6
0. Jackson, J.R.; Gilmartin, A.; Imburgia, C.; Winkler, J.D.; Marshall, L.A.; Roshak, A. An indolocarbazole
inhibitor of human checkpoint kinase (Chk1) abrogates cell cycle arrest caused by DNA damage. Cancer Res.
2
000, 60, 566–572. [PubMed]
1. Hirose, Y.; Berger, M.S.; Pieper, R.O. Abrogation of the Chk1-mediated G2 checkpoint pathway potentiates
temozolomide-induced toxicity in a p53- independent manner in human glioblastoma cells. Cancer Res. 2001
1, 5843–5849. [PubMed]
6
,
6
6
2. McLaughlin, F.; Finn, P.; La Thangue, N.B. The cell cycle, chromatin and cancer: Mechanism-based
therapeutics come of age. Drug Discov. Today 2003, 8, 793–802. [CrossRef]
6
3. Krajewska, M.; Wang, H.G.; Krajewski, S.; Zapata, J.M.; Shabaik, A.; Gascoyne, R.; Reed, J.C.
Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease.
Cancer Res. 1997, 57, 1605–1613. [PubMed]
6
6
4. Li, Y.; Yao, J.; Chang, M.; Cuendet, M.; Bolton, J.L. Altered apoptotic response in MCF 10A cells treated with
the equine estrogen metabolite, 4-hydroxyequilenin. Toxicol. Lett. 2004, 154, 225–233. [CrossRef] [PubMed]
5. Zhag, H.; Qian, Z.D.; Sun-Tan, Y.; Lee, K.; Gao, P.; Ren, R.Y.; Rey, S.; Hammers, H.; Chang, D.; Pili, R.; et al.
Digoxin and other cardiac glycosides inhibit HIL-1 synthesis and block tumor growth. Med. Sci. 2008, 105,
1
9579–19586.
6
6. Dimas, K.; Papadopoulou, N.; Baskakis, C.; Prousis, C.K.; Tsakos, M.; Alkahtani, S.; Honisch, S.; Lang, F.;
+
+
Calogeropoulou, T.; Alevizopoulos, K.; et al. Steroidal cardiac Na /K ATPase inhibitors exhibit stong
anti-cancer potential in vitro and in prostate and lung cancer xenegrafts in vivo. Anti-Cancer Agents
Med. Chem. 2014, 14, 762–770. [CrossRef]
+
+
6
6
7. Alevizopoulos, K.; Calogeropoulou, T.; Lang, F.; Stournaras, C. Na /K ATPase Inhibitors in Cancer.
Curr. Drug Targets 2014, 15, 988–1000. [PubMed]
8. Gobbini, M.; Armanoli, S.; Banfi, L.; Benicchio, A.; Carzana, G.; Fedrizzzi, G.; Ferrari, P.; Giacalone, G.;
+
+
Giubileo, M.; Narazzi, G.; et al. Novel analogues of Istaroxime a potent inhibitor of Na /K -ATPase:
Synthesis and structure-activity relationship. J. Med. Chem. 2008, 51, 4601–4608. [CrossRef] [PubMed]
9. Alevizopoulos, K.; Dimas, K.; Papadopoulou, N.; Schmidt, E.M.; Tsapara, A.; Alkahtani, S.; Honisch, S.;
Prousis, C.K.; Alarifi, S.; Calogeropoulou, T.; et al. Funtional characterization and anti-cancer action of the
6
+
+
clinical phase II cardiac Na /K ATPase inhibitor istaroxime: In vitro and in vivo properties and cross talk
with the membrane androgen receptor. Oncotarget 2016, 7, 24415–24428. [PubMed]
7
7
0. Raju, J.; Rao, C.V. Diosgenin, a Steroid Saponin Constituent of Yams and Fenugreek: Emerging Evidence for
Applications in Medicine. Bioactive Compounds in Phytomedicine, Prof; Rasooli, I., Ed.; InTech.: Rijeka, Croatia,
2
012; pp. 125–142.
1. Kueng, W.; Silber, E.; Eppenberger, U. Quantification of cells cultured on 96-well plates. Anal. Biochem. 1989
82, 16–19. [CrossRef]
,
1

Molecules 2016, 21, 1533
17 of 17
7
2. Lyons, A.B.; Hasbold, J.; Hodgkin, P.D. Chapter 17 Flow cytometric analysis of cell division history
using dilution of carboxyfluorescein diacetate succinimidyl ester, a stably integrated fluorescent probe.
Methods Cell Biol. 2001, 63, 375–398. [PubMed]
7
3. Thareja, S. Steroidal 5
α-Reductase inhibitors: A comparative 3D-QSAR study review. Chem. Rev. 2015, 115,
2
883–2894. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 1, 4–9 are available from the authors.
©
2016 by the authors; 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 (http://creativecommons.org/licenses/by/4.0/).