Efficient synthesis of novel antiproliferative steroidal spirooxindoles via the [3+2] cycloaddition reactions of azomethine ylides

Article abstract of DOI:10.1016/j.steroids.2015.08.003

Abstract A series of novel steroidal spirooxindoles 3a-h were synthesized from pregnenolone in a high regioselective manner using the 1,3-dipolar cycloaddition as the key step. This protocol resulted in the formation of two C-C bonds, one C-N bond and the creation of one pyrrolidine ring and three contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized steroidal spirooxindoles exhibited moderate to good antiproliferative activity against the tested cell lines and some of them were more potent than 5-FU. Among them, compounds 3e and 3f displayed the best antiproliferative activity against MCF-7 cells with the IC₅₀ values of 4.0 and 3.9 μM, respectively. Flow cytometry analysis demonstrated that compound 3d caused the cellular apoptosis and cell cycle arrest at G2/M phase in a concentration-dependent manner. Docking results indicated that compound 3d fitted well into the MDM2 active site 1RV1 by interacting with Lys94 and Thr101 residues.

Full text of DOI:10.1016/j.steroids.2015.08.003

Accepted Manuscript
Efficient synthesis of novel antiproliferative steroidal spirooxindoles via the
[3+2] cycloaddition reactions of azomethine ylides
Bin Yu, Xiao-Nan Sun, Xiao-Jing Shi, Ping-Ping Qi, Yi-Chao Zheng, De-Quan
Yu, Hong-Min Liu
PII:
S0039-128X(15)00216-0
http://dx.doi.org/10.1016/j.steroids.2015.08.003
STE 7820
DOI:
Reference:
Steroids
To appear in:
Received Date:
Accepted Date:
2 August 2015
4 August 2015
Please cite this article as: Yu, B., Sun, X-N., Shi, X-J., Qi, P-P., Zheng, Y-C., Yu, D-Q., Liu, H-M., Efficient synthesis
of novel antiproliferative steroidal spirooxindoles via the [3+2] cycloaddition reactions of azomethine ylides,
Steroids (2015), doi: http://dx.doi.org/10.1016/j.steroids.2015.08.003
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Efficient synthesis of novel antiproliferative steroidal spirooxindoles via the [3+2]
cycloaddition reactions of azomethine ylides
Bin Yu a, Xiao-Nan Sun b, Xiao-Jing Shi a, Ping-Ping Qi a, Yi-Chao Zheng a, De-Quan
Yu a*, Hong-Min Liu a∗
a School of Pharmaceutical Sciences and New Drug Research & Development Center,
Zhengzhou University, Zhengzhou 450001, P. R. China.
b
The Affiliated Hospital of Huanghe Sanmenxia of Henan University of Science and
Technology, Sanmenxia 472000, P. R. China.
Abstract: A series of novel steroidal spirooxindoles 3a-h were synthesized from
pregnenolone in a high regioselective manner using the 1,3-dipolar cycloaddition as
the key step. This protocol resulted in the formation of two C-C bonds, one C-N bond
and the creation of one pyrrolidine ring and three contiguous stereocenters in a single
operation. Biological evaluation showed that these synthesized steroidal
spirooxindoles exhibited moderate to good antiproliferative activities against the
tested cell lines and some of them were more potent than 5-FU. Among them,
compounds 3e and 3f displayed the best antiproliferative activity against MCF-7 cells
with the IC50 values of 4.0 and 3.9 µM, respectively. Flow cytometry analysis
demonstrated that compound 3d caused the cellular apoptosis and cell cycle arrest at
G2/M phase in a concentration-dependent manner. Docking results indicated that
compound 3d fitted well into the MDM2 active site 1RV1 by interacting with Lys94
and Thr101 residues.
Keywords: Pregnenolone; Spirooxindoles; Antiproliferative activity; Apoptosis; Cell
cycle arrest; Docking studies
1. Introduction
Steroids, an important class of polycyclic compounds, have been proved to possess
1
diverse biological activities. Chemical modifications on the steroid nucleus and side
chains have been recognized as efficient strategies to access biologically important
molecules. Particularly, the incorporation of heterocycles into the steroid scaffolds has
2
always been an intriguing topic in the field of steroids, because the introduction of
biologically relevant heterocycles always endows these new hybrids with novel
3
properties or improved potency.
For example, N-heterocycle-containing
4
abiraterone has been used for the treatment of advanced prostate cancer in clinic
while galeterone has successfully finished its phase clinical development. Among
5
these heterocycles, spirooxindoles (Fig. 1) have drawn wide attention in consequence
of their unique structural scaffolds that are prevalent in numerous naturally occurring
7
oxindole alkaloids 6 and in synthetic molecules with promising biological activities.
Three representative drug candidates CFI-400945, SAR405838 and NITD609,
8
9
10
∗
Corresponding authors: (1) Tel. /fax: +86 371 67781739, E-mail: zzuyubin@hotmail.com (B.
Yu); E-mail: liuhm@zzu.edu.cn (H.-M. Liu); (2) E-mail: dqyu@imm.ac.cn (D.-Q. Yu)
1

have entered clinical trials for the treatment of human cancers and malaria,
respectively (Fig. 1). Therefore, the introduction of spirooxindole skeleton into the
steroid core would be an interesting strategy to construct compound library with
structural novelty and biological potential.
O
N
OMe
HN
O
CFI-400945
Anticancer agent targeting PLK4
N
Cl
N
H
H
N
F
Cl
NH
F
O
O
NH
H
N
N
Cl
OH
H
N
H
Cl
NITD609
Antimalarial agent targeting PfATP4
O
SAR405838
Anticancer agent targeting MDM2
O
N
H
Spirooxindole scaffold
Present work
HN
Previous work
O
Ar
O
O
N
H
N
H
Ar
H
O
NH
HO
H
H
R
Steroidal spirooxindoles
IC50 = 0.71 M against SMMC-7721
induce apoptosis and cell cycle arrest
HO
Fig. 1 Spirooxindole-containing biologically active molecules
The broad and promising biological activities of spirooxindole-containing molecules
together with intriguing structural architectures have been a major driving force for
11
the development of new synthetic approaches, especially the asymmetric synthesis,
as some diastereoisomer always represents markedly enhanced activity compared to
its racemates or other diastereoisomers as shown by CFI-400945, SAR405838 and
NITD609. The 1,3-dipolar cycloaddition reactions between azomethine ylides and
dipolarophiles have been recognized as efficient strategies for the synthesis of
12
pyrrolidines with high yields and stereoselectivity. Very recently, we reported that
steroidal spirooxindoles (Fig. 1) can potently inhibit the proliferation of several
human cancer cells (IC50 = 0.71 µM against SMMC-7721 cells) and induce apoptosis
13
and cell cycle arrest at G2/M phase in a time-/concentration-dependent manner. As
part of our efforts toward the identification of novel heterocycles with anticancer
14
potential, we herein report the synthesis of a new series of steroidal spirooxindoles
from pregnenolone using the 1,3-cycloaddition reaction as the key step and their in
vitro antiproliferative activities against several human cancer cell lines. Besides, we
also investigated the effects toward the cell cycle and apoptosis. Docking studies were
also performed to illustrate the possible binding model of MDM2.
2. Results and discussion
2

2.1. Chemistry
As shown in Fig. 2, our strategy involved the 1,3-dipolar cycloaddition between
21-arylidenepregnenolone derivatives (2a-h) and azomethine ylide generated in situ
from isatin and sarcosine.
Fig. 2 Strategy employed for the synthesis of steroidal spirooxindoles
21-arylidenepregnenolone derivatives 2a-h were initially synthesized from
pregnenolone and the corresponding aromatic aldehydes in excellent yields following
13
our previously reported method,
which were then subjected to the 1,3-dipolar
cycloaddition reactions with azomethine ylide generated from the decarboxylative
condensation of isatin and sarcosine, affording steroidal spirooxindoles 3a-h in
moderate to good yields (Scheme 1). During optimization of the reaction condition,
the mixture of 1,4-dioxane and MeOH (1/1) was found to be the best solvent system
for this key transformation. Excess of isatin and sarcosine were necessary for
complete conversion of 21-arylidenepregnenolone derivatives 2a-h. With this
optimized condition in hand, we further investigated the scope and reproducibility of
this methodology.
Scheme 1 Synthesis of steroidal spirooxindoles. Reagents and conditions: (a)
Aromatic aldehyde, KF/Al2O3, EtOH, reflux; (b) Isatin, sarcosine, 1,4-dioxane/MeOH
(1/1), reflux.
All final compounds 3a-h were fully characterized by NMR and high-resolution mass
spectra (HRMS) as described for compound 3g. Three singlets at 2.21, 0.89 and 0.12
ppm correspond the peaks of N-CH3 of the pyrrolidine ring, 19-CH3 and 18-CH3,
respectively. The multiplet at 3.46-3.54 ppm is assigned to the 3α-H of steroid A-ring;
The proton attached to C-6 appears as a doublet at 5.23 ppm with a coupling constant
1
of 4 Hz. The broad peak at 2.72 ppm is assigned to H-17 when compared to the H
3

NMR spectrum of pregnenolone. The peak of the N-H proton at the oxindole ring
appears as a singlet at 8.80 ppm (no direct correlation with any carbon is observed in
the HSQC spectrum). The aromatic protons appear as doublets and multiplets at
6.93-8.39 ppm. It is evident from the 13C NMR spectrum of compound 3g that the
peaks of C-20 and carbonyl carbon of oxindole appear at 207.09 and 180.29 ppm,
respectively. From the HSQC spectrum, a secondary carbon at 59.95 ppm has direct
correlations with two protons at around 3.61 and 3.41 ppm, respectively. Besides,
correlations between the methylenic protons (H-3') and the carbon of N-CH3 are also
observed from the HMBC spectrum (correlations between methylenic protons and
protons of N-CH3 are also observed in the H-H COSY spectrum). All these indicate
that the peak at 59.95 ppm can be assigned to the methylenic carbon (C-3') of the
pyrrolidine ring. From the HMBC spectrum, a doublet at 3.76 ppm has correlations
with C-20, amide carbon and a quaternary carbon at 72.56 ppm, indicating that this
doublet is the peak of H-1' and the quaternary carbon is the spiro carbon (C-4'). The
proton with the peak at 4.42-4.49 ppm as a multiplet has correlations with H-1' and
H-3' from the H-H COSY spectrum, this means the multiplet is the peak of benzylic
proton (H-2'). These signal assignments are consistent with the H-H COSY
correlations depicted in Fig. 3 (please see the supporting information for 2D spectra of
compound 3g). The presence of a molecular ion peak at m/z = 624.3441 ([M+H]+) in
the mass spectrum (calcd. 624.3437) further confirms the structure of 3g. The crystal
structures of steroidal spirooxindoles synthesized using a similar method have been
recently reported. 15
Fig. 3 Selected H–H COSY correlations of compound 3g (arbitrary numbering)
The possible mechanism for the formation of steroidal spirooxindoles is outlined in
Scheme 2. The key azomethine ylide intermediate was formed through the direct
decarboxylation of iminium salt formed from isatin and sarcosine (Path A) or the
formation of lactone intermediate (Path B). The azomethine ylide then underwent
1,3-dipolar cycloaddition reactions with dipolarophiles 2a-h in a regioselective
manner. The regioselectivity of the reaction is explicable by the preference of the
electron-rich carbon of the dipole adding to the electron-deficient β-carbon of the
α,β-unsaturated moiety of 2a-h. The steroidal spirooxindoles were also formed with
high stereoselectivity although three stereocenters were formed. The carbonyl group
linked to the steroid ring system and the amide carbonyl are in trans-relationship with
respect to C1-C2 bond of the pyrrolidine ring system, this is presumably ascribable to
4

the preferred spatial arrangement of the dipolarophile and the azomethine ylide in the
cycloaddition step, which would minimize the repulsion between the carbonyl groups.
The aryl ring attached to the C-3 position is also the trans-relationship to the carbonyl
of the steroid skeleton with respect to C2-C3 bond of the pyrrolidine ring system due
to the E-configuration of the double bond of 21-arylidenepregnenolone derivatives
2a-h. Besides, it should be noted that three continuous stereocenters (one quaternary
carbon centers), two C-C bonds and one C-N bond were formed in one-pot procedure
under catalyst-free condition. The high regioselectivity and stereoselectivity were also
observed in our previous work 13 and by other groups. 16
Scheme 2 The possible mechanism for the formation of steroidal spirooxindoles.
2.2. Biological evaluation
2.2.1. Antiproliferative activity
13
In our previous work, we found that steroidal spirooxindoles showed moderate to
good inhibitory activity against EC109, MGC-803, SMMC-7721 and MCF-7 cell
lines. Structure-activity relationships (SARs) studies revealed that substituents
attached to aryl groups (Fig. 1) played an important role in the activity. To explore the
SARs further, we designed and synthesized another new series of steroidal
spirooxindoles (3a-h) from pregnenolone by varying the steroid nucleus and the aryl
groups compared to our previous work. Steroidal spirooxindoles 3a-h were then
subjected to antiproliferative evaluation against four human cancer cell lines (T24,
SMMC-7721, MCF-7 and MGC-803) using the MTT assay. The well-known
anticancer drug 5-fluorouracil (5-FU) works principally through irreversible inhibition
of thymidylate synthase, while some steroidal derivatives such as estradiol,
progesterone, DHEA, testosterone could also markedly inhibit thymidylate synthase.
17
Due to the similar mode of action, 5-FU was used as the positive control in the
antiproliferative assay.
5

Table 1 In vitro antiproliferative activity against four human cancer cell lines
IC50 (µM) a
No
Ar
T24
SMMC-7721 MCF-7
MGC-803
8.3 0.9
Phenyl
23.3 1.4
80.9 1.9
4.1 0.6
6.1 0.8
15.4 1.2
14.4 1.2
8.8 1.0
44.1 1.6
7.1 0.9
18.8 1.3
35.2 1.6
43.1 1.6
6.7 0.8
5.1 0.7
21.8 1.3
17.7 1.3
4.6 0.7
4.0 0.6
3.9 0.6
10.2 1.1
9.9 1.0
10.5 1.6
3a
3b
3c
3d
3e
3f
15.6 1.2
16.1 1.2
10.2 1.0
6.2 0.8
4-i-Propylphenyl
2-Fluorophenyl
3-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
3-Nitrophenyl
1-Naphthyl
18.5 1.3
9.3 1.0
11.0 1.0
13.5 1.1
14.0 1.2
9.1 1.0
3g
3h
44.0 1.6
12.2 1.1
4.3 0.6
---
5-FU
a
Inhibitory activity was assayed by exposure for 72 h to substances and expressed as
concentration required to inhibit tumor cell proliferation by 50% (IC50). Data are shown as the
means SDs of three independent experiments.
As shown in Table 1, all the synthesized compounds showed moderate to good
antiproliferative activities against four human cancer cell lines with IC50 values
ranging from 3.9 to 80.9 µM and some of them were more potent than 5-FU. The
SARs were probed by altering the substituents on the phenyl ring. It is evident that the
electronic nature of substituents on the phenyl ring had remarkable effect on the
activity. For T24 cells, compounds 3a, 3b and 3h with electron-rich aromatic ring
represented markedly decreased inhibitory effect than those with electron-deficient
substituents (3c-g). Compounds 3c and 3d with a fluoro atom showed the best
inhibition against T24 cells with the IC50 values of 4.1 and 6.1 µM, respectively and
were slightly more potent than 5-FU. By contrast, all the compounds showed
moderate antiproliferative activity against SMMC-7721 cells. However, none of them
had better inhibition than 5-FU. Intriguingly, most of these compounds were more
active than 5-FU against MCF-7 cells. Among them, compounds 3e and 3f with a
chloro and bromo atom had the best but comparable activity with the IC50 values of
4.0 and 3.9 µM, respectively. Compound 3d with a fluoro atom at the 3-position (IC50
= 4.6 µM) was more than 3-fold potent than compound 3c with a fluoro atom at the 2
position. It is evident that compounds with an elecron-withdrawing group were
generally more potent than those having an elecron-donating group toward MCF-7
cells. Besides, the moderate inhibitory effect was observed for the tested compounds
against MGC-803 cells. However, it should be noted that all the compounds had
comparable antiproliferative activity with 5-FU regardless of the electronic nature of
their substituents. Inspired by this finding, the synthesis of more analogs and further
investigation of modes of action are in progress in our group.
2.2.2. Apoptosis assay
Considering the moderate to good antiproliferative activities of these compounds
against the tested human cancer cell lines, compound 3d was chosen to further
explore its mechanism of action. In order to characterize the mode of cell death
6

induced by compound 3d, we performed a biparametric cytofluorimetric analysis
using propidium iodide (PI) and annexin-V-FITC in MCF-7 cells. After treatment
MCF-7 cells with compound 3d for 24h at different concentrations (0, 2.0, 4.0, 8.0
µM), MCF-7 cells were labeled with the two dyes, and the resulting red (PI) and
green fluorescence (FITC) was monitored by flow cytometry. As shown in Fig. 4,
compound 3d caused the remarkable early apoptosis in a concentration-dependent
manner. The apoptosis rate was 4.8% for the control group, while the rate was
increased to 21.4% at the concentration of 8.0 µM.
Fig. 4 Apoptotic effect of compound 3d toward MCF-7 cells. Apoptotic cells were
detected with Annexin V-FITC/PI double staining after incubation with compound 3d
at different concentrations (0, 2.0, 4.0, 8.0 µM) for 24 h. The lower left quadrants
represent live cells, the lower right quadrants are for early/primary apoptotic cells,
upper right quadrants are for late/secondary apoptotic cells, while the upper left
quadrants represent cells damaged during the procedure.The experiments were
performed three times, and a representative experiment is shown.
2.2.3. Cell cycle analysis
Molecules that inhibit the growth of cancer cells invariably cause alteration of cell
cycle distribution, with preferential G2/M blockade. To better understand the
antiproliferative activity of compound 3d, a cell cycle progression was performed by
treating MCF-7 cells at different concentrations of compound 3d (0, 2.0, 4.0, 8.0 µM).
7

As shown in Fig. 5, when treating MCF-7 cells for 12 h, the percentage of cells at
G2/M phase was increased from 18.9% of the control sample to 45.4% at the highest
concentration of 3d. The result revealed that 3d caused an obvious G2/M arrest in a
concentration-dependent manner with a concomitant decrease of cells in other phases
of the cell cycle.
Fig. 5 Effect of compound 3d on the cell cycle distribution of MCF-7 cells. Cells
were treated at different concentrations (0, 2.0, 4.0, 8.0 µM) for 12 h. Then the cells
were fixed and stained with PI to analyze DNA content by flow cytometry. The
experiments were performed three times, and a representative experiment is shown.
2.3.3 Docking studies
Spirooxindoles have been reported to be able to potently inhibit MDM2-p53
interaction, extensive modifications conducted by Wang’s group yielded anticancer
9
drug candidate SAR405838 (also known as MI-77301, Ki = 0.88 nmol/L) and its
18
second-generation MI-1061 (Ki = 0.16 nmol/L). Inspired by this interesting finding
and considering that our structures share the same spiro-pyrrolidinyl oxindole scaffold
with SAR405838 and MI-1061, the docking simulations in the active sites of MDM2
19
were performed by the AutoDock tools and Autodock Vina program. The docking
results would help us visualize the potential receptor-ligand interactions of steroidal
spirooxindoles with MDM2 and guide further SAR studies. The crystal structure of
MDM2 (PDB: 1RV1) was extracted from RCSB Protein Data Bank; the target protein
structure of 1RV1 was docked with the most active compound 3d. The lowest energy
pose of compound 3d at MDM2’s active site is shown in Fig. 6. Compound 3d fitted
well into this pocket showing two hydrogen binding interactions with Lys94 and
Thr101 residues. The F-H and N-O distance are 2.81 and 2.90 Å, respectively.
8

Interestingly, compound 3d docked into the same active pocket with the well-known
MDM2 inhibitor Nutlin-2. 20
Fig. 6 Binding models of compound 3d with MDM2 active site 1RV1.
3. Conclusions
In summary, a series of novel steroidal spirooxindoles were efficiently synthesized
from commercially available pregnenolone using the 21-arylidenepregnenolone
derivatives as the dipolarophiles following our previously reported method. The
method involved the one-pot, three-component 1,3-dipolar cycloaddition of
azomethine ylide generated in situ from the decarboxylative condensation of isatin
and sarcosine and afforded the final products with high regio- and stereoselectivity.
This protocol achieved the formation of two C-C bonds, one C-N bond and the
creation of one new five-membered ring and three contiguous stereocenters, one of
which is quaternary. Biological evaluation showed that these synthesized steroidal
spirooxindoles possessed moderate to good antiproliferative activities against four
human cancer cell lines and some of them were more potent than 5-FU. Among them,
compounds 3e and 3f showed the best antiproliferative activity against MCF-7 cells
(IC50 = 4.0 and 3.9 µM, respectively). Further investigation showed that compound 3d
caused the cellular early apoptosis and cell cycle arrest at G2/M phase in a
concentration-dependent manner. Docking analysis showed that compound 3d may
interrupt the p53-MDM2 interaction by forming two hydrogen bonds in the active
pocket that the previously reported MDM2 inhibitor Nutlin-2 targets.
4. Experimental section
4.1. General
Reagents were purchased and used without further purification. Thin layer
chromatography (TLC) was carried out on glass plates coated with silica gel and
visualized by UV light (254 nm). The final products were purified by column
chromatography. Melting points were determined on an X-5 micromelting apparatus.
All the NMR spectra were recorded with a Bruker DPX 400 MHz spectrometer with
9

TMS as the internal standard in CDCl3. Chemical shifts are given as δ ppm values
relative to TMS (Most of the peaks due to the steroidal skeleton are merged and could
not be differentiated. Thus, the δ values of only those peaks that distinguish the
product and could easily be differentiated are reported). High-resolution mass spectra
were recorded on a Waters Micromass Q-T of Micromass spectrometer by
electrospray ionization (ESI).
4.2. General procedure for the synthesis of 21-arylidenepregnenolone derivatives
(2a-h)
A mixture of pregnenolone (2.0 mmol), aromatic aldehyde (2.1 mmol) and
KF/Al2O3 (2.0 mmol) in EtOH (20 mL) was heated under reflux for about 2 h. Upon
completion of the reaction (monitored by TLC, petroleum ether/EtOAc = 2/1), the
slurry was filtered and the residue was washed thoroughly with CH2Cl2. The filtrate
was concentrated under reduced pressure, and the solid obtained was crystallized from
EtOH or MeOH to give the 21-arylidenepregnenolone derivatives 2a-h. All the
intermediates are known compounds and only characterized by 1H and 13C NMR.
4.2.1. (21E)-3β-Hydroxy-21-benzylidenepregn-5-en-20-one (2a)
1
White solid, yield: 90 %, mp 107.1-109.2 °C. H NMR (400 MHz, CDCl3, δ, ppm):
7.55 (dd, J = 9.7, 5.9 Hz, 2H), 6.49 (d, J = 16.0 Hz, 1H), 7.43-7.32 (m, 3H), 6.78 (d, J
= 16.0 Hz, 1H), 5.36 (s, 1H), 3.61-3.44 (m, 1H), 2.86 (t, J = 8.7 Hz, 1H), 1.01 (s, 3H),
0.63 (s, 3H). 13C NMR (100 MHz, CDCl3, δ, ppm): 200.4, 141.5, 140.8, 134.8, 130.3,
128.9, 128.3, 126.8, 121.4, 71.7, 63.7, 62.1, 57.2, 50.0, 45.0, 44.0, 42.2, 38.8, 37.3,
36.5, 32.0, 31.9, 31.6, 24.5, 22.8, 21.1, 19.4, 13.2.
4.2.2. (21E)-3β-Hydroxy-21-(4’-isopropylbenzylidene) pregn-5-en-20-one (2b)
1
White solid, yield: 90 %, mp 89.9-90.8 °C. H NMR (400 MHz, CDCl3, δ, ppm):
7.59-7.43 (m, 3H), 7.25 (d, 2H), 6.73 (dd, J = 15.8, 12.1 Hz, 1H), 5.35 (s, 1H),
3.62-3.40 (m, 1H), 2.95-2.90 (m, 1H), 1.00 (s, 3H), 0.63 (s, 3H). 13C NMR (100 MHz,
CDCl3, δ, ppm): 199.7, 150.9, 140.8, 140.1, 131.7, 127.7, 126.3, 125.3, 120.7, 71.0,
61.2, 58.3, 56.5, 50.2, 49.4, 48.8, 45.4, 44.2, 41.5, 38.4, 36.5, 35.8, 34.8, 33.4, 31.3,
31.1, 30.9, 25.4, 24.0, 23.5, 22.0, 20.4, 20.3, 18.7, 12.7.
4.2.3. (21E)-3β-Hydroxy-21-(2’-fluorobenzylidene) pregn-5-en-20-one (2c)
1
White solid, yield: 92 %, mp 130.8-131.7°C. H NMR (400 MHz, CDCl3, δ, ppm):
7.66 (d, J = 16.1 Hz, 1H), 7.61-7.52 (m, 1H), 7.36 (dd, J = 7.3, 1.5 Hz, 1H), 7.16 (t, J
= 7.5 Hz, 1H), 7.13-7.05 (m, 1H), 6.87 (d, J = 16.1 Hz, 1H), 5.35 (t, J = 6.1 Hz, 1H),
3.59-3.44 (m, 1H), 2.88 (t, J = 8.8 Hz, 1H), 1.00 (s, 3H), 0.64 (s, 3H). 13C NMR (100
MHz, CDCl3) δ 200.6, 161.7 (JC-F = 253 Hz), 140.9, 134.2, 131.7 (JC-F = 9 Hz), 129.4
(JC-F = 7 Hz), 124.5, 123.0 (JC-F = 12 Hz), 121.7, 121.5, 116.3 (JC-F = 22 Hz), 116.2,
71.7, 62.0, 57.3, 50.1, 45.1, 42.3, 39.1, 37.3, 36.6, 32.1, 31.9, 31.7, 24.8, 22.8, 21.2,
19.5, 13.5.
4.2.4. (21E)-3β-Hydroxy-21-(3’-fluorobenzylidene) pregn-5-en-20-one (2d)
10

White solid, yield: 92 %，mp 154.9-155.8°C. 1H NMR (400 MHz, CDCl3) δ 7.50 (d, J
= 15.9 Hz, 1H), 7.35 (ddd, J = 18.6, 9.3, 6.7 Hz, 2H), 7.25 (dd, J = 9.7, 2.0 Hz, 1H),
7.13 – 7.04 (m, 1H), 6.76 (d, J = 15.9 Hz, 1H), 5.36 (d, J = 5.2 Hz, 1H), 3.61 – 3.47
(m, 1H), 2.84 (t, J = 8.8 Hz, 1H), 1.00 (s, 3H), 0.64 (s, 3H). 13C NMR (100 MHz,
CDCl3, δ, ppm): 200.1, 163.0(JC-F = 245 Hz), 140.7, 140.0, 137.1(JC-F = 7 Hz),
130.4(JC-F = 8 Hz), 127.8, 124.3(JC-F = 3 Hz), 121.3, 117.1(JC-F = 22 Hz), 114.3(JC-F
=
21 Hz),71.6, 62.2, 57.1, 50.0, 45.0, 42.2, 39.1, 37.2, 36.5, 32.0, 31.8, 31.6, 24.6, 22.7,
21.1, 19.3, 13.4.
4.2.5. (21E)-3β-Hydroxy-21-(4’-chlorobenzylidene) pregn-5-en-20-one (2e)
1
White solid, yield: 94%, mp 130.6-131.7°C. H NMR (400 MHz, CDCl3, δ, ppm):
7.51-7.47 (m, 3H), 7.36 (d, J = 8 Hz, 1H), 6.73 (d, J = 16.0 Hz, 1H), 5.35 (t, J = 6.4
Hz, 1H), 3.51 (m, J = 1H), 2.84 (t, J = 8.8 Hz, 1H), 1.00 (s, 3H), 0.64 (s, 3H). 13C
NMR (100 MHz, CDCl3, δ, ppm): 199.4, 140.1, 139.3, 135.4, 132.6, 128.7, 128.4,
126.4, 120.7, 71.0, 61.5, 56.5, 49.3, 44.3, 41.5, 38.4, 36.5, 35.8, 31.3, 31.1, 30.9, 23.9,
22.0, 20.4, 18.7, 12.7.
4.2.6. (21E)-3β-Hydroxy-21-(4’-bromobenzylidene) pregn-5-en-20-one (2f)
1
White solid, yield: 95%, mp 156.2-157.8°C. H NMR (400 MHz, CDCl3, δ, ppm):
7.52 (d, J = 8.4 Hz, 2H), 7.47 (d, J = 15.9 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 6.76 (d, J
= 15.9 Hz, 1H), 5.36 (d, J = 4.9 Hz, 1H), 3.53 (s, 1H), 2.84 (t, J = 8.7 Hz, 1H), 1.00 (s,
3H), 0.64 (s, 3H). 13C NMR (100 MHz, CDCl3, δ, ppm): 200.2, 140.8, 140.1, 133.7,
132.1, 129.6, 127.2, 124.5, 121.4, 71.6, 62.2, 57.1, 50.0, 45.0, 42.2, 39.1, 37.2, 36.5,
32.0, 31.8, 31.6, 24.6, 22.7, 21.1, 19.4, 13.4.
4.2.7. (21E)-3β-Hydroxy-21-(3’-nitrobenzylidene) pregn-5-en-20-one (2g)
1
White solid, yield: 95%, mp 168.0-169.1°C. H NMR (400 MHz, CDCl3, δ, ppm):
8.40 (s, 1H ), 8.23 (dd, J = 8.2, 1.3 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.58 (dd, J =
16.0, 8.2 Hz, 2H), 6.88 (d, J = 16.0 Hz, 1H), 5.36 (d, J = 5.1 Hz, 1H), 3.62-3.42 (m,
1H), 2.87 (t, J = 8.7 Hz, 1H), 1.00 (s, 3H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3,
δ, ppm): 200.1, 149.0, 141.0, 138.7, 136.9, 134.3, 130.2, 129.4, 124.7, 122.6, 121.6,
71.9, 62.7, 57.4, 50.3, 45.4, 42.5, 39.4, 37.5, 36.8, 32.3, 32.1, 31.8, 24.9, 23.0, 21.3,
19.6, 13.8.
4.2.8. (21E)-3β-Hydroxy-21-(1’-naphthylidene) pregn-5-en-20-one (2h)
White solid, yield: 95%，mp 92.5-93.2°C. 1H NMR (400 MHz, CDCl3, δ, ppm): 8.40
(d, J = 15.7 Hz, 1H), 8.21 (d, J = 8.2 Hz, 1H,), 7.88 (t, J = 7.7 Hz, 2H), 7.78 (d, J =
7.2 Hz, 1H), 7.62-7.44 (m, 3H), 6.86 (d, J = 15.7 Hz, 1H), 5.36 (d, J = 5.1 Hz, 1H),
3.58-3.43 (m, 1H), 2.90 (t, J = 8.7 Hz, 1H), 0.99 (s, 3H), 0.69 (s, 3H). 13C NMR (100
MHz, CDCl3, δ, ppm): 200.3, 140.8, 138.4, 133.7, 132.3, 131.8, 130.6, 129.4, 128.8,
126.9, 126.3, 125.5, 124.9, 123.5, 121.4, 71.7, 62.3, 57.2, 50.1, 45.1, 42.3, 39.3, 37.3,
36.6, 32.1, 31.9, 31.6, 24.7, 22.8, 21.2, 19.4, 13.6.
4.3. General procedure for the synthesis of steroidal spirooxindoles (3a-h)
11

To
a
mixture of 1,4-dioxane/MeOH (10 mL/10 mL) were added the
21-arylidenepregnenolone (1 mmol), isatin (1.5 mmol) and sarcosine (2.0 mmol), and
then the solution was kept under reflux for about 5 h. Upon completion of the reaction
(monitored by TLC, petroleum ether/EtOAc = 2/1), the solvent was removed under
vacuum, the resulting residue was subjected to column chromatography to give the
corresponding product.
4.3.1. Compound 3a
White solid, yield: 65 %, mp 162.1-162.7 oC; 1H NMR (400 MHz, CDCl3) δ 8.82 (brs,
1H), 7.48 (d, J = 8.0 Hz, 2H), 7.34-7.26 (m, 2H), 7.22 (dd, J = 7.4, 6.1 Hz, 2H),
7.09-6.98 (m, 2H), 6.89 (dd, J = 7.5, 2.7 Hz, 1H), 5.21 (s, 1H), 4.29 (dt, J = 8.5, 6.3
Hz, 1H), 3.77 (d, J = 9.5 Hz, 1H), 3.65-3.57 (m, 1H), 3.54-3.42 (m, 1H), 3.35 (t, J =
8.0 Hz, 1H), 2.19 (s, 3H), 0.87 (s, 4H), 0.07 (s, 5H). 13C NMR (100 MHz, CDCl3) δ
207.6, 180.7, 141.4, 140.9, 140.7, 129.3, 128.5, 128.4, 127.6, 127.3, 126.8, 123.1,
121.2, 109.7, 72.8, 71.7, 69.6, 63.8, 57.0, 49.8, 45.0, 44.2, 42.1, 38.8, 37.3, 36.4, 34.7,
31.6, 30.9, 24.2, 22.4, 19.3, 13.3. HRMS (ESI): m/z calcd for C38H47N2O3 (M+H)+,
579.3587; found, 579.3581.
4.3.2. Compound 3b
o
White solid, yield: 80 %, mp 158.9-159.4 C; 1H NMR (400 MHz, CDCl3) δ 8.99 (s,
1H), 7.40 (d, J = 8.1 Hz, 2H), 7.20 (m, 1H), 7.15 (d, J = 8.1 Hz, 2H), 7.07 (d, J = 7.2
Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 6.88 (d, J = 7.5 Hz, 1H), 5.22 (s, 1H), 4.27 (m, 1H),
3.72 (d, J = 9.5 Hz, 1H), 3.63 (t, J = 9.5 Hz, 1H), 3.49 (dd, J = 9.5, 6.1 Hz, 1H), 3.34
(dd, J = 8.4, 7.6 Hz, 1H), 2.87 (dt, J = 13.7, 6.8 Hz, 1H), 2.19 (s, 3H), 1.21 (d, J = 6.9
Hz, 6H), 0.87 (s, 3H), 0.08 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 208.2, 181.1,
147.7, 141.2, 140.9, 139.0, 129.5, 128.6, 127.8, 127.7, 126.7, 123.3, 121.5, 109.9,
73.1, 71.9, 70.2, 64.2, 60.2, 57.2, 50.0, 45.3, 44.2, 42.4, 38.8, 37.6, 36.6, 35.0, 34.0,
31.9, 31.8, 27.2, 24.4, 24.3, 22.6, 21.1, 19.5, 13.4. HRMS (ESI): m/z calcd for
C41H53N2O3 (M+H)+, 621.4056; found, 621.4049.
4.3.3. Compound 3c
o
White solid, yield: 70 %, mp 163.3-163.8 C; 1H NMR (400 MHz, CDCl3) δ 9.45 (s,
1H), 7.64 (t, J = 6.9 Hz, 1H), 7.21 (dd, J = 15.5, 7.8 Hz, 2H), 7.16-7.11 (m, 1H),
7.11-7.05 (m, 1H), 7.02 (m, 2H), 6.92 (d, J = 7.5 Hz, 2H), 5.21 (s, 1H), 4.67 (dd, J =
16.9, 9.5 Hz, 1H), 3.89 (d, J = 9.4 Hz, 1H), 3.64-3.44 (m, 2H), 3.33 (t, J = 7.9 Hz,
1H),2.17 (s, 3H), 0.90 (s, 3H), 0.11 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.0,
180.8, 162.2, 159.7, 141.1, 140.7, 123.0, 129.9, 129.4, 128.3, 128.1, 127.9, 127.5,
127.3, 124.3, 123.1, 121.22, 115.6, 115.4, 109.9, 72.7, 71.6, 67.8, 63.6, 57.1, 49.9,
44.9, 42.1, 39.0, 37.4, 36.7, 36.4, 34.7, 31.6, 31.5, 24.2, 22.5, 19.3, 13.4. HRMS (ESI):
m/z calcd for C38H46FN2O3 (M+H)+, 597.3492; found, 597.3488.
4.3.4. Compound 3d
White solid, yield: 74 %, mp 171.9-172.2 oC; 1H NMR (400 MHz, CDCl3) δ 9.27 (brs,
1H), 7.35-7.16 (m, 4H), 7.05-6.99 (m, 2H), 6.98-6.83 (m, 2H), 5.20 (s, 1H), 4.32 (dd,
12

J = 16.9, 9.5 Hz, 1H), 3.73 (d, J = 9.3 Hz, 1H), 3.57 (t, J = 9.5 Hz, 1H), 3.50 (td, J =
10.4, 4.9 Hz, 1H), 3.35 (t, J = 8.0 Hz, 1H), 2.18 (s, 3H), 0.91 (s, 3H), 0.12 (s, 3H). 13C
NMR (100 MHz, CDCl3) δ 206.6, 180.1, 163.5, 161.0, 143.6 140.4, 140.0, 129.2,
128.7, 126.7, 126.4, 123.5, 122.4, 120.4, 114.5, 114.3, 113.1, 112.9, 109.2, 72.0, 71.0,
68.6, 63.1, 59.4, 56.4, 49.1, 44.3, 43.0, 41.4, 38.3, 36.7, 35.7, 33.9, 30.8, 23.4, 21.7,
18.5, 12.7. HRMS (ESI): m/z calcd for C38H46FN2O3 (M+H)+, 597.3492; found,
597.3490.
4.3.5. Compound 3e
o
White solid, yield: 80 %, mp 165.4-165.9 C; 1H NMR (400 MHz, CDCl3) δ 8.71 (s,
1H), 7.43 (d, J = 8.4 Hz, 2H), 7.30-7.25 (d, , J = 8.4 Hz, 2H), 7.25-7.18 (m, 1H), 7.02
(m, 2H), 6.88 (d, J = 7.7 Hz, 1H), 5.23 (d, J = 4.7 Hz, 1H), 4.34-4.20 (m, 1H), 3.70 (d,
J = 9.4 Hz, 1H), 3.50 (m, 2H), 3.32 (m, 1H), 2.17 (s, 3H), 0.90 (s, 3H), 0.11 (s, 3H).
13C NMR (100 MHz, CDCl3) δ 207.4, 180.5, 140.9, 140.7, 140.0, 132.5, 129.8, 129.4,
128.6, 127.4, 127.3, 123.2, 121.2, 109.7, 72.7, 71.7, 69.3, 63.7, 60.2, 57.0, 49.8, 45.1,
45.0, 43.41, 42.10, 38.9, 37.3, 36.4, 34.7, 31.6, 31.5, 24.2, 22.5, 21.0, 19.3, 13.5.
HRMS (ESI): m/z calcd for C38H46ClN2O3 (M+H)+, 613.3197; found, 613.3193.
4.3.6. Compound 3f
o
White solid, yield: 80 %, mp 170.4-171.1 C; 1H NMR (400 MHz, CDCl3) δ 8.63 (s,
1H), 7.43 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.26-7.12 (m, 1H), 7.02 (q, J =
7.3 Hz, 2H), 6.88 (d, J = 7.7 Hz, 1H), 5.23 (d, J = 4.6 Hz, 1H), 4.27 (dd, J = 16.8, 9.6
Hz, 1H), 3.70 (d, J = 9.2 Hz, 1H), 3.50 (m, 2H), 3.32 (t, J = 8.0 Hz, 1H), 2.17 (s, 3H),
0.90 (s, 3H), 0.12 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.9, 180.9, 141.4, 141.2,
141.1, 132.1, 130.7, 129.9, 127.9, 127.8, 123.7, 121.7, 121.1, 110.2, 73.2, 72.2, 69.8,
64.3, 61.0, 57.6, 50.3, 45.6, 44.0, 42.6, 39.4, 37.8, 36.9, 35.2, 32.1, 32.0, 24.7, 23.0,
21.6, 21.5, 19.8, 14.0. HRMS (ESI): m/z calcd for C38H46BrN2O3 (M+H)+, 657.2692;
found, 657.2686.
4.3.7. Compound 3g
o
White solid, yield: 82 %, mp 170.8-171.0 C; 1H NMR (400 MHz, CDCl3) δ 8.80 (s,
1H), 8.39 (t, J = 1.8 Hz, 1H), 8.15-8.08 (m, 1H), 7.89 (d, J = 7.8 Hz, 1H), 7.52 (t, J =
7.9 Hz, 1H), 7.29-7.22 (m, 1H), 7.10-7.00 (m, 2H), 6.94 (d, J = 7.7 Hz, 1H), 5.23 (d, J
= 3.9 Hz, 1H), 4.46 (td, J = 9.6, 7.3 Hz, 1H), 3.76 (d, J = 9.3 Hz, 1H), 3.61 (t, J = 9.4
Hz, 1H), 3.56-3.44 (m, 1H), 3.41 (dd, J = 8.7, 7.3 Hz, 1H), 2.72 (s, 1H), 2.21 (s, 3H),
0.89 (s, 3H), 0.12 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.1, 180.3, 147.7, 143.1,
140.2, 140.0, 134.1, 128.9, 128.7, 126.4, 122.5, 122.4, 121.3, 120.4, 109.2, 72.6, 71.8,
71.0, 68.3, 63.0, 59.2, 56.3, 49.1, 44.4, 42.8, 41.4, 38.4, 35.6, 33.9, 30.8, 30.7, 23.4,
21.8, 20.3, 18.5, 12.9. HRMS (ESI): m/z calcd for C38H46N3O5 (M+H)+, 624.3437;
found, 624.3441.
4.3.8. Compound 3h
o
White solid, yield: 81 %, mp 182.3-183.4 C; 1H NMR (400 MHz, CDCl3) δ 9.05 (s,
1H), 8.77 (d, J = 8.6 Hz, 1H), 8.06 (d, J = 7.1 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.90
13

(d, J = 8.1 Hz, 1H), 7.77-7.70 (m, 1H), 7.64 (t, J = 7.7 Hz, 2H), 7.45-7.36 (m, 1H),
7.33 (d, J = 7.3 Hz, 1H), 7.20 (dd, J = 16.0, 8.9 Hz, 1H), 7.08 (d, J = 7.7 Hz, 1H),
5.42 (dd, J = 16.7, 9.1 Hz, 1H), 5.34 (d, J = 3.6 Hz, 1H), 4.24 (d, J = 9.1 Hz, 1H),
3.86 (t, J = 9.2 Hz, 1H), 3.64 (dd, J = 17.2, 8.6 Hz, 2H), 2.39 (s, 3H), 0.98 (s, 3H),
0.24 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 207.1, 179.7, 140.0, 139.7, 137.2, 132.9,
131.2, 128.4, 127.6, 126.6, 126.4, 126.2, 125.1, 124.6, 124.5, 124.4, 123.1, 122.2,
120.2, 108.8, 72.1, 70.8, 68.5, 62.9, 60.5, 56.2, 48.9, 44.0, 41.2, 37.9, 37.6, 36.4, 35.4,
35.3, 33.9, 31.2, 30.6, 23.2, 21.6, 20.0, 18.3, 12.1. HRMS (ESI): m/z calcd for
C42H49N2O3 (M+H)+, 629.3743; found, 629.3742.
4.4. Cell culturing
Human cancer cell lines were maintained in minimal essential medium supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified
o
atmosphere of 5% CO2 and 95% air at 37 C. Cancer cells were maintained in
RPMI1640 medium. All cell lines were purchased from the China Center for Type
Culture Collection (CCTCC, China). For pharmacological investigations, 10 mM
stock solutions of the tested compounds were prepared with DMSO. The highest
DMSO concentration of the medium (0.1%) does not have any substantial effect on
the determined cellular functions.
4.5. Antiproliferative assay
Exponentially growing cells were seeded into 96-well plates at a concentration of 5 ×
103 cells per well. After 24 h incubation at 37 °C, the culture medium was removed
and replaced with fresh medium containing tested compounds at different
concentrations. The cells were incubated for another 72 h. Then, 20 µL of MTT
solution (5 mg/mL) was added to all wells and incubated for 4 h at 37 °C. The
suspension was discarded and 150 µL of DMSO was added to each well. The plate
was shaken to dissolve the dark blue crystal (formazan); the absorbance was measured
using a microplate reader at the wavelength of 490 nm. Each concentration was
analyzed in triplicate and the experiment was repeated three times. The average 50%
inhibitory concentration (IC50) was determined from the dose-response curves
according to the inhibition ratio for each concentration 21.
4.6. Analysis of cellular apoptosis
MCF-7 cells were plated in 6-well plate (5.0 × 106 cells/mL) and incubated at 37 °C
for 24 h. Exponentially growing cells were then incubated for 24 h with complete
medium (blank) or with compound 3d. Cells were then harvested and the
Annexin-V-FITC/PI apoptosis kit (Biovision) was used according to the
manufacturer’s instructions to detect apoptotic cells. Ten thousand events were
collected for each sample and analyzed byAccuri C6 flow cytometer.
4.7. Flow cytometric analysis of cell cycle distribution
14

For flow cytometric analysis of DNA content, 5.0 × 106 MCF-7 cells in exponential
growth were treated with different concentrations of compound 3d for 12 h. After an
incubation period, the cells were collected, centrifuged and fixed with ice-cold EtOH
(70%). The cells were then treated with buffer containing RNAse A and 0.1% Triton
X-100 and then stained with PI. Samples were analyzed on Accuri C6 flow cytometer
(Becton, Dickinson). Data obtained from the flow cytometer was analyzed using the
FlowJo software (Tree Star, Inc., Ashland, OR, USA).
4.7. Molecular modeling
The structure of the MDM2 binding site was obtained from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb). Docking simulations were carried out in two stages
using AutoDock Vina designed and implemented by Dr. Oleg Trott in the Molecular
Graphics Lab at The Scripps Research Institute. To run autodock, we used a searching
grid extended over the selected target proteins; polar hydrogens were added to the
ligand moieties. Kollman charges were assigned and atomic solvation parameters
were added. Polar hydrogen charges of the Gasteiger-type were assigned and the
nonpolar hydrogens were merged with the carbons and the internal degrees of
freedom and torsions were set.
Acknowledgements
We are grateful for the financial support from the Natural Science Foundation of
China (No. 21372206, 81430085, 81172937) ; the Development Foundation of
Priority, Ministry of Education (No. 20134101130001); 2013 Yong Teacher
Foundation from Zhengzhou University (No. 000001307615); Key Scientific
Research Project for Higher Education by He'nan Educational Committee (No.
15A350018).
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18

Highlights
ꢀ Spirooxindoles are privileged scaffolds in drug discovery
ꢀ A series of novel steroidal spirooxindoles were synthesized
ꢀ Most of these compounds exhibited moderate to good antiproliferative activity
ꢀ Some of them were more potent than 5-FU
ꢀ Compound 3d caused cell cycle arrest at G2/M phase and early apoptosis
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