Discovery of novel steroidal pyran-oxindole hybrids as cytotoxic agents

Article abstract of DOI:10.1016/j.steroids.2014.05.022

A series of novel steroidal pyran-oxindole hybrids were efficiently synthesized in a single operation through the vinylogous aldol reaction of vinyl malononitrile 3 with substituted isatins involving the construction of C-C and C-O bonds. Some compounds displayed moderate to good cytotoxicity against T24, SMMC-7721, MCF-7 and MGC-803 cells. Compounds 4f and 4i were more potent than 5-Fu against T24 and MGC-803 cells with the IC₅₀ values of 4.43 and 8.45 μM, respectively. Further mechanism studies indicated that compound 4i induced G2/M arrest and early apoptosis in a concentration- and time-dependent manner.

Full text of DOI:10.1016/j.steroids.2014.05.022

Steroids 88 (2014) 44–52
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Discovery of novel steroidal pyran–oxindole hybrids as cytotoxic agents
1
1
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Bin Yu , Ping-Ping Qi , Xiao-Jing Shi, Li-Hong Shan , De-Quan Yu , Hong-Min Liu
School of Pharmaceutical Sciences and New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel steroidal pyran–oxindole hybrids were efficiently synthesized in a single operation
through the vinylogous aldol reaction of vinyl malononitrile 3 with substituted isatins involving the con-
struction of C–C and C–O bonds. Some compounds displayed moderate to good cytotoxicity against T24,
SMMC-7721, MCF-7 and MGC-803 cells. Compounds 4f and 4i were more potent than 5-Fu against T24
Received 18 March 2014
Received in revised form 14 May 2014
Accepted 25 May 2014
Available online 11 June 2014
and MGC-803 cells with the IC50 values of 4.43 and 8.45 lM, respectively. Further mechanism studies
indicated that compound 4i induced G2/M arrest and early apoptosis in a concentration- and time-
Keywords:
dependent manner.
Pyran–oxindole hybrids
Cytotoxicity
Ó 2014 Elsevier Inc. All rights reserved.
Apoptosis
Cell cycle arrest
1. Introduction
activations of caspase-9/-3, cleavage of MDM2 as well as upregula-
tion of the expressions of p53 and Bax [9].
Steroids are extensively available in nature and well known for
their profound biological activities due to their high ability to pen-
etrate cells and bind to nuclear and membrane receptors. Chemical
modifications of the steroid ring system and side chain provide a
way to alter the functional groups and numerous structure–activ-
ity relationships have been established by such synthetic altera-
tions [1,2]. It is proved that a number of biologically important
properties of modified steroids are dependent upon structural fea-
tures of the steroid ring system and side chain [3–6]. In recent
years, a number of steroidal heterocycles with interesting activities
have been isolated or synthesized. For example, spironolactone, as
the mineralocorticoid antagonist, is a clinically used drug for
congestive heart failure (Fig. 1) [7]. A recent report from Puranik
Purushottamachar et al. described that galeterone analogue had
potential for development as new drugs for the treatment of all
The oxindole nucleus is a privileged scaffold that is highly pre-
valent in natural and synthetic compounds of medicinal interest
[10]. Molecules containing an oxindole nucleus display a diverse
range of biological activities [11], such as inhibitors of the
MDM2-p53 interaction [12], antimicrobial activities [13], cholines-
terase inhibitors [14,15], anticancer properties [16], protein kinase
activators [17] and so on. Particularly intriguing is the NITD609,
which has been studied as an antimalarial drug candidate that acts
through the distinct mechanism of action as existing antimalarial
drugs have [18]. Besides, oxindoles can also serve as synthetic
intermediates for alkaloids and many kinds of pharmaceuticals or
drug precursors [19]. Fox example, coerulescine, the simplest spi-
rooxindole–pyrrolidine hybrid found in nature, displays local anes-
thetic effect [10]. The spirotryprostatins have antimitotic
properties and are of interest as anticancer lead compounds [20],
and the recently discovered small-molecule MDM2 inhibitor MI-
219 and its analogues are in preclinical development for cancer
therapeutics (Fig. 2) [12,21]. Additionally, the six-membered pyran
ring system, as an important structural motif, is also found in a
wide range of biologically active compounds and is known to have
wide applications in medicinal chemistry [22]. For example, pyr-
rolo [2, 3-h] chromenes (Fig. 2) showed excellent anticancer activ-
ity with an EC50 value of 13 nM against T47D cells [23,24].
forms of prostate cancer with an IC50 value of 0.87 lM through
degrading both full-length and truncated ARs in CWR22rv1 human
prostate cancer cells [8]. Our group recently reported that [1,2,4]
triazolo [1,5-a] pyrimidine-based phenyl-linked steroid dimer
induced apoptosis through the mitochondrial pathway accompa-
nied with the decrease of mitochondrial membrane potential,
On the basis of the above-mentioned observations regarding the
potential of oxindoles and pyrans and our own previous observa-
tions regarding [25] the importance of the steroidal oxindoles in
conferring cytotoxic activities, a series of steroidal pyran–oxindole
hybrids were designed, incorporating both oxindole and
⇑
Corresponding authors. Tel./fax: +86 371 67781739.
E-mail addresses: zzuyubin@hotmail.com (B. Yu), shlh@zzu.edu.cn (L.-H. Shan),
dqyu@imm.ac.cn (D.-Q. Yu), liuhm@zzu.edu.cn (H.-M. Liu).
1
These authors made equal contributions.
http://dx.doi.org/10.1016/j.steroids.2014.05.022
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

B. Yu et al. / Steroids 88 (2014) 44–52
45
Fig. 1. Selective biologically active steroidal compounds.
2-imino-3-cyano pyran key scaffolds, with an aim to obtain potent
and selective cytotoxic agents. Besides, we also explored the effects
toward the cell cycle and possible mechanism of inducing apoptosis.
with malononitrile in ethanol catalyzed by ammonium acetate
according to our previously reported method [4,6].
2.3. General procedure for the synthesis of steroidal pyran–oxindole
hybrids 4a–j
2. Experimental section
To a solution of compound 3 (1.0 mmol) and substituted isatin
(1.0 mmol) in ethanol, 1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU)
(2.0 mmol) was added. The reaction mixture was stirred at room
temperature for about 1 h. The solvent was removed and CH2Cl2
was added, the organic phase was washed with water and brine,
dried over Na2SO4. After removal of the solvent, the residue was
purified by silica gel chromatography using acetone/petroleum
ether (1/2) as the eluent to give the corresponding steroidal
pyran–oxindole hybrids.
2.1. General
Reagents and solvents were purchased from commercial
sources and were used without further purification. Isatins used
were purchased from Aladdin Company (www.aladdin-e.com).
Thin-layer chromatography (TLC) was carried out on glass plates
coated with silica gel (Qingdao Haiyang Chemical Co., G60F-254)
and visualized by UV light (254 nm). The products were purified
by column chromatography over silica gel (Qingdao Haiyang
Chemical Co., 200-300 mesh). Melting points were determined
on a X-5 micromelting apparatus and are uncorrected. All the
NMR spectra were recorded with a Bruker DPX 400 MHz spectrom-
eter with TMS as internal standard in CDCl3. Chemical shifts are
given as d ppm values relative to TMS (Most of the peaks due to
the steroidal skeleton are merged and could not be differentiated.
Thus d values of only those peaks that distinguish the product and
could easily be differentiated are reported). High-resolution mass
spectra (HRMS) were recorded on a Waters Micromass Q-T of
Micromass spectrometer by electrospray ionization (ESI).
2.3.1. Compound 4a
White solid, yield: 75%, m. p. 230.7–232.0 °C. 1H NMR
(400 MHz, CDCl3) d 9.41 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.10 (d,
J = 7.9 Hz, 1H), 7.06 (d, J = 5.5 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H),
5.29 (s, 1H), 4.69–4.51 (m, 1H), 3.96 (s, 1H), 2.04 (s, 3H), 1.56 (s,
3H), 1.06 (s, 3H). 13C NMR (100 MHz, CDCl3) d 191.66, 175.50,
170.57, 169.41, 143.96, 139.93, 131.45, 127.39, 124.40, 121.25,
119.99, 112.20, 110.23, 104.38, 73.49, 72.66, 57.16, 56.05, 49.28,
47.70, 37.97, 36.74, 36.64, 34.60, 32.27, 30.45, 29.28, 27.60,
21.40, 19.98, 19.29, 15.57. HRMS (ESI): m/z calcd for C32H34BrN3-
NaO4 (M + Na)+, 626.1630; found, 626.1654.
2.2. Synthesis of steroidal a, a-dicyanoalkene (3)
2.3.2. Compound 4b
The steroidal
a
,
a
-dicyanoalkene (3) was efficiently prepared
Yellow solid, yield: 78%, m. p. 232.1–233.7 °C. 1H NMR
(400 MHz, CDCl3) d 9.51 (s, 1H), 7.19 (s, 1H), 7.18–7.10 (m, 1H),
via Aldol condensation of 3b-acetyl dehydroepiandrosterone 2
Fig. 2. Designed strategy of steroidal pyran–oxindole hybrids. The dashed boxes indicate the key core structure present in previously reported molecules.

46
B. Yu et al. / Steroids 88 (2014) 44–52
7.01 (d, J = 7.8 Hz, 1H), 6.47 (d, J = 7.2 Hz, 1H), 5.30 (s, 1H), 4.61
(s, 1H), 4.03 (d, J = 6.3 Hz, 1H), 2.04 (s, 3H), 1.53 (s, 3H), 1.06
(s, 3H). 13C NMR (100 MHz, CDCl3) d 191.50, 175.58, 170.54,
169.29, 143.79, 139.94, 131.27, 131.23, 124.34, 121.97, 121.27,
112.05, 109.57, 103.66, 73.50, 72.63, 57.24, 55.12, 49.35, 47.43,
37.98, 36.75, 36.64, 34.52, 32.45, 32.26, 30.44, 27.61, 21.40,
20.01, 19.31, 15.14. HRMS (ESI): m/z calcd for C32H35ClN3O4
(M + H)+, 560.2316; found, 560.2316.
2.3.7. Compound 4g
White solid, yield: 84%, m. p. 242.5–247.6 °C. 1H NMR
(400 MHz, CDCl3)
d 7.48–7.34 (m, 1H), 7.26–7.15 (m, 3H),
7.12–7.00 (m, 2H), 6.96 (d, J = 7.5 Hz, 1H), 6.67 (d, J = 7.9 Hz, 1H),
6.35 (s, 1H), 5.39 (s, 1H), 5.07 (t, J = 16.8 Hz, 1H), 4.86 (dd,
J = 16.5, 8.6 Hz, 1H), 4.70–4.52 (m, 1H), 4.10 (s, 1H), 2.04 (s, 3H),
1.47 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz, CDCl3) d 189.05,
174.38, 170.51, 167.98, 143.52, 139.85, 133.03, 132.56, 130.03,
129.97, 129.80, 129.13, 128.10, 127.35, 125.92, 122.72, 122.04,
121.43, 111.81, 109.91, 104.49, 73.54, 71.06, 57.56, 55.48, 49.63,
45.89, 41.77, 37.98, 36.78, 36.72, 36.66, 34.36, 33.68, 32.45,
30.28, 27.62, 21.41, 20.07, 19.37, 14.70. HRMS (ESI): m/z calcd for
2.3.3. Compound 4c
White solid, yield: 83%, m. p. 254.3–255.6 °C. 1H NMR
(400 MHz, CDCl3) d 7.27 (m, 6H), 7.01 (t, J = 7.6 Hz, 1H), 6.92
(d, J = 7.4 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.21 (s, 1H), 5.39
(s, 1H), 5.14 (d, J = 15.7 Hz, 1H), 4.72–4.52 (m, 2H), 4.05 (s, 1H),
2.04 (s, 3H), 1.46 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 189.05, 174.28, 170.48, 167.96, 143.73, 139.85, 135.44, 129.69,
129.00, 127.92, 127.29, 125.86, 122.51, 122.13, 121.43, 111.87,
109.81, 104.42, 73.53, 71.10, 57.54, 55.37, 49.62, 45.86, 44.08,
37.99, 36.79, 36.66, 34.36, 33.68, 32.45, 30.27, 27.63, 21.40,
20.08, 19.36, 14.68. HRMS (ESI): m/z calcd for C39H41N3NaO4
(M + Na)+, 638.2995; found, 638.2994.
C
39H40ClN3NaO4 (M + Na)+, 672.2605; found, 672.2600.
2.3.8. Compound 4h
White solid, yield: 69%, m. p. 227.1–228.9 °C. 1H NMR
(400 MHz, CDCl3) d 7.33–7.20 (m, 3H), 7.19–6.97 (m, 3H), 6.92
(d, J = 7.4 Hz, 1H), 6.82 (dd, J = 25.3, 7.9 Hz, 1H), 6.40 (s, 1H), 5.38
(d, J = 4.2 Hz, 1H), 4.96 (dd, J = 27.2, 15.7 Hz, 1H), 4.80
(dd, J = 23.7, 15.7 Hz, 1H), 4.60 (dt, J = 10.7, 5.6 Hz, 1H), 4.04
(s, 1H), 2.04 (s, 3H), 1.45 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 190.32, 189.07, 174.22, 171.56, 170.52, 168.42, 168.07,
144.00, 143.45, 140.25, 139.84, 129.98, 129.85, 129.74, 125.82,
124.96, 124.70, 124.66, 124.19, 123.64, 123.18, 122.55, 122.47,
122.32, 122.15, 122.07, 121.44, 121.05, 115.94, 115.73, 115.56,
115.34, 112.07, 111.86, 109.95, 109.64, 104.42, 102.78, 99.99,
73.54, 71.11, 57.55, 56.95, 55.26, 49.62, 45.84, 45.56, 37.98,
36.78, 36.71, 36.66, 34.35, 34.06, 33.59, 32.49, 32.43, 30.33,
30.27, 27.62, 21.40, 20.07, 19.39, 19.36, 14.84, 14.66. HRMS (ESI):
2.3.4. Compound 4d
White solid, yield: 80%, m. p. 245.4–247.6 °C. 1H NMR
(400 MHz, CDCl3) d 7.18 (t, J = 7.6 Hz, 3H), 7.08 (d, J = 4.6 Hz, 1H),
7.00 (td, J = 7.3, 2.6 Hz, 1H), 6.94 (d, J = 7.3 Hz, 1H), 6.92–6.85 (m,
1H), 6.58–6.42 (m, 1H), 5.37 (d, J = 3.6 Hz, 1H), 5.14 (dd, J = 16.6,
8.1 Hz, 1H), 4.68–4.54 (m, 1H), 4.55–4.33 (m, 1H), 4.08 (s, 1H),
2.40 (s, 3H), 2.04 (s, 3H), 1.47 (s, 3H), 1.09 (s, 3H). 13C NMR
m/z calcd for
C
39H40FN3NaO4 (M + Na)+, 656.2901; found,
(100 MHz, CDCl3)
d
189.27, 174.18, 170.49, 144.10, 139.82,
656.2902.
135.68, 132.98, 130.67, 129.63, 127.51, 126.31, 125.75, 122.19,
121.47, 111.93, 110.04, 104.38, 100.00, 73.54, 57.66, 55.31, 49.63,
45.85, 42.13, 37.99, 36.78, 36.66, 34.39, 33.59, 32.44, 30.26,
27.63, 21.41, 20.08, 19.37, 14.67. HRMS (ESI): m/z calcd for
2.3.9. Compound 4i
White solid, yield: 65%, m. p. 263.9–265.3 °C. 1H NMR
(400 MHz, CDCl3) 7.29 (dd, J = 13.8, 5.8 Hz, 1H), 7.23
d
C
40H44N3O4 (M + H)+, 630.3332; found, 630.3329.
(t, J = 7.6 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.18 (m, 1H), 5.39
(d, J = 4.4 Hz, 1H), 5.06–4.89 (m, 2H), 4.70–4.54 (m, 1H), 4.01
(s, 1H), 2.04 (s, 3H), 1.45 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 189.00, 173.52, 170.50, 167.93, 167.87, 162.72, 162.65,
160.25, 160.17, 143.06, 139.81, 130.37, 130.27, 129.80, 125.82,
122.50, 122.02, 121.47, 112.03, 111.87, 111.79, 110.82, 110.66,
109.12, 104.42, 73.55, 71.06, 71.02, 57.53, 55.09, 49.62, 45.84,
37.98, 36.78, 36.65, 34.35, 33.61, 32.43, 32.25, 30.27, 27.63,
21.40, 20.07, 19.35, 14.67. HRMS (ESI): m/z calcd for C39H39F2N3
NaO4 (M + Na)+, 674.2806; found, 674.2804.
2.3.5. Compound 4e
White solid, yield: 79%, m. p. 236.7–239.0 °C. 1H NMR
(400 MHz, CDCl3) d 7.23–7.17 (m, 2H), 7.07 (t, J = 6.4 Hz, 3H),
7.00 (d, J = 7.5 Hz, 1H), 6.92 (d, J = 7.4 Hz, 1H), 6.75 (d, J = 8.1 Hz,
1H), 6.18 (s, 1H), 5.38 (d, J = 3.4 Hz, 1H), 5.10 (d, J = 15.7 Hz, 1H),
4.63 (m, 1H), 4.57 (d, J = 15.7 Hz, 1H), 4.05 (s, 1H), 2.31 (s, 3H),
2.04 (s, 3H), 1.46 (s, 3H), 1.09 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 189.06, 174.29, 170.49, 167.97, 143.83, 139.85, 138.81, 135.39,
129.69, 128.85, 128.69, 127.89, 124.34, 122.45, 122.14, 121.44,
111.90, 109.84, 104.41, 73.53, 71.13, 57.54, 55.36, 49.63, 45.84,
44.05, 37.99, 36.78, 36.72, 36.66, 34.36, 33.67, 32.44, 30.27,
27.63, 21.41, 20.07, 19.36, 14.69. HRMS (ESI): m/z calcd for
2.3.10. Compound 4j
White solid, yield: 64%, m. p. 216.6–218.5 °C. 1H NMR
(400 MHz, CDCl3) d 7.72 (d, J = 7.4 Hz, 1H), 7.40 (dt, J = 14.9,
7.3 Hz, 2H), 7.20 (t, J = 7.7 Hz, 1H), 7.06 (t, J = 7.7 Hz, 2H), 6.99
(d, J = 7.4 Hz, 1H), 6.57 (s, 1H), 6.52 (d, J = 7.8 Hz, 1H), 5.38
(d, J = 4.8 Hz, 1H), 5.26 (d, J = 17.3 Hz, 1H), 4.89 (d, J = 17.2 Hz,
1H), 4.70–4.54 (m, 1H), 4.15 (s, 1H), 2.04 (s, 3H), 1.49 (s, 3H),
1.10 (s, 3H). 13C NMR (100 MHz, CDCl3) d 189.03, 174.59, 170.51,
168.12, 143.34, 139.86, 133.57, 132.49, 129.82, 128.02, 127.67,
126.61, 126.50, 126.45, 126.03, 125.67, 122.89, 122.04, 121.41,
111.70, 109.72, 104.57, 73.54, 71.12, 57.58, 55.57, 49.63, 45.91,
40.60, 37.98, 36.78, 36.66, 34.35, 33.68, 32.46, 30.27, 27.62,
26.92, 21.40, 20.08, 19.37, 14.71. HRMS (ESI): m/z calcd for C40H41
F3N3O4 (M + H)+, 684.3049; found, 684.3044.
C
40H44N3O4 (M + H)+, 630.3332; found, 630.3331.
2.3.6. Compound 4f
White solid, yield: 80%, m. p. 218.1–220.5 °C. 1H NMR
(400 MHz, CDCl3) d 7.22 (d, J = 7.4 Hz, 1H), 7.20–7.14 (m, 2H),
7.11 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 7.3 Hz, 1H), 6.90 (d, J = 7.4 Hz,
1H), 6.76 (d, J = 7.9 Hz, 1H), 6.60 (s, 1H), 5.37 (d, J = 4.9 Hz, 1H),
5.14 (d, J = 16.0 Hz, 1H), 4.61 (dd, J = 16.0, 9.8 Hz, 2H), 4.04 (s,
1H), 2.30 (s, 3H), 2.04 (s, 3H), 1.45 (s, 3H), 1.09 (s, 3H). 13C NMR
(100 MHz, CDCl3)
d 189.21, 174.27, 170.50, 168.28, 143.84,
139.81, 137.57, 132.53, 129.60, 129.42, 127.45, 127.32, 125.74,
122.38, 122.15, 121.47, 111.92, 109.86, 104.38, 93.86, 73.54,
71.30, 57.57, 55.30, 49.62, 45.82, 43.93, 37.98, 36.79, 36.65,
36.50, 34.35, 33.59, 32.44, 30.23, 27.63, 21.41, 21.10, 19.36,
14.66. HRMS (ESI): m/z calcd for C40H44N3O4 (M + H)+, 630.3332;
found, 630.3329.
2.4. 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 atmosphere of 5% CO2

B. Yu et al. / Steroids 88 (2014) 44–52
47
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, Shanghai, China). For pharmaco-
logical investigations, 10 mM stock solutions of the tested com-
pounds were prepared with dimethyl sulfoxide (DMSO). The
highest DMSO concentration of the medium (0.1%) did not have
any substantial effect on the determined cellular functions.
Dickinson). Data obtained from the flow cytometer was analyzed
using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).
3. Results and discussion
3.1. Chemistry
The protocol for the synthesis of steroidal pyran–oxindole
hybrids 4a–j was straightforward using DBU-catalyzed cascade
Aldol/cyclization process as the key step (Scheme 1). The interme-
diate 3 was prepared in high yield via Aldol condensation of
3b-acetyl dehydroepiandrosterone 2 with malononitrile in ethanol
catalyzed by ammonium acetate following our previously reported
methods [4,6]. In the presence of base, compounds 4a–j were effi-
ciently synthesized via the cascade Aldol/cyclization process of
compound 3 and substituted isatins. Besides, we also investigated
the effects of bases (Et3N, DMAP, imidazole, pyridine, piperidine,
NaHCO3 and Na2CO3), solvents (toluene, DCM, EtOAc and MeOH)
and the molar ratio toward the reaction between compound 3
and substituted isatins at room temperature, revealing that the
optimized reaction condition was 3 (1.0 mmol), substituted isatin
(1.0 mmol) and DBU (2.0 mmol) in EtOH (10 mL).
2.5. Cytotoxic activity assays
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 the candidate compounds in different concen-
trations. The cells were incubated for another 72 h. Then, 20 lL of
MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-
mide) solution (5 mg/mL) was added to all wells and incubated
for 4 h at 37 °C. Discarded the suspension and added 150 lL of
dimethyl sulfoxide (DMSO) to each well and shook the plates to
dissolve the dark blue crystals (formazan); the absorbance was
measured using a microplate reader at a wavelength of 490 nm.
Each concentration was analyzed in triplicate and the experiment
was repeated three times. The average 50% inhibitory concentra-
tion (IC50) was determined from the dose-response curves accord-
ing to the inhibition ratio for each concentration.
All the synthesized compounds were characterized by 1H, 13C
NMR and high resolution mass spectra as described for compound
4i (Fig. 3). In the 1H NMR spectrum of compound 4i, the signals of
protons attached to CH3CO-, C-19 and C-18 appeared at 2.04, 1.45
and 1.09 ppm respectively as sharp singlets. The signal of H-6
appeared at 5.38 ppm as a doublet (J = 4.4 Hz). The multiplet at
2.6. Analysis of cellular apoptosis
4.58–4.62 ppm was assigned to the 3a-H of steroid A-ring. The
MGC-803 cells were plated in 6-well plates (5.0 ꢀ 106 cells/mL)
and incubated at 37 °C for 24 or 48 h. Exponentially growing cells
were then incubated for 24 or 48 h with complete medium (blank)
or with the compound 4i. 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 by
Accuri C6 flowcytometer.
benzyl protons showed their peaks at 4.89–5.06 ppm as a multiplet
(two protons). A broad peak that appeared at 6.14–6.21 ppm was
assigned to the N–H proton due to the fact that there was no any
correlation in the HSQC spectrum. The aromatic hydrogens
appeared as doublets and multiplets at 6.75–7.32 ppm. Interest-
ingly enough, the signal of H-16 appeared at 4.01 ppm as a sharp
singlet and no coupling with protons attached to C-15 was
observed although it was adjacent to a secondary carbon (C-15)
as indicated in DEPT135, H–H COSY and HMBC spectra. This was
probably attributed to the special space orientation between the
H-16 and H-15 and there was no interaction between the H-16
and other protons as indicated in NOE spectrum. From the HSQC
spectrum together with 1H and 13C NMR spectra, some direct
C–H correlations were observed, confirming that the signals of
the benzyl carbon, C-3, C-16, CH3CO-, C-18 and C-19 appeared at
32.43, 73.55, 55.09, 21.40, 14.67 and 19.35 ppm, respectively. From
the HMBC spectrum, we clearly found that both the N–H and H-16
had HMBC correlation with a quaternary carbon atom (indicated in
2.7. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 5.0 ꢀ 106
MGC-803 cells in exponential growth were treated with different
concentrations of the test compound 4i for 24 or 48 h. After an
incubation period, the cells were collected, centrifuged and fixed
with ice-cold ethanol (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,
Scheme 1. Synthesis of steroidal pyran–oxindole hybrids 4a–j. Reagents and conditions: (a) (Ac) 2O, DMAP, Et3N, DCM, r.t; (b) malononitrile, NH4OAc, EtOH, reflux; (c) DBU,
EtOH, r.t.

48
B. Yu et al. / Steroids 88 (2014) 44–52
Fig. 3. Selected 1H NMR chemical shifts of compound 4i.
Scheme 2. Proposed reaction mechanism for the synthesis of steroidal pyran–oxindole hybrids 4a–j.
DEPT135 spectrum) with its signal appearing at 71.06 ppm,
proving that this carbon atom was the spiro carbon that linked
the pyran and oxindole motif. A carbon atom whose signal
appeared at 189.00 ppm had HMBC correlations with the N–H,
H-15 and H-18, a phenomenon that in turn confirmed the chemical
shift of C-17. Besides, the N–H proton had HMBC correlations with
C-10 (167.93 ppm) and C-20 (104.42 ppm). The downfield chemical
shift of C-17 and relatively upfield chemical shift of C-20 were
probably due to the inductive effects of the electron-withdrawing
cyano group and 2-imino ester group attached to C-20. The pres-
ence of a molecular ion peak at m/z = 674.2804 ([M + Na]+) in the
mass spectrum (calcd. 674.2806) further confirmed the structure
of compound 4i. For all the spectra of compound 4i, please refer
to the supporting information.
A possible mechanism for the formation of compounds 4a–j
was proposed in Scheme 2. In the presence of base, the first step
Table 1
Preliminary in vitro cytotoxic activities of steroidal pyran–oxindole hybrids against four human cancer cell lines.
Compounds
R1
R2
IC50
T24
(l
M)a
SMMC-7721
MCF-7
MGC-803
4a
4b
4c
4d
4e
4f
4 g
4 h
4i
4-Br
4-Cl
H
H
H
H
H
H
H
H
H
49.00 1.69
52.89 1.72
>128
>128
>128
4.43 0.86
>128
10.84 1.04
32.52 1.51
49.46 1.69
7.14 0.85
>128
80.53 1.91
>128
>128
>128
117.87 2.07
64.35 1.81
30.84 1.49
>128
>128
4.29 0.63
24.12 1.38
14.78 1.20
57.52 1.76
113.46 2.01
69.14 1.84
66.37 1.07
46.31 1.67
17.42 1.24
12.31 0.98
56.21 0.81
10.53 1.56
108.51 2.04
14.45 1.16
30.95 1.49
40.17 1.60
82.30 1.92
89.88 1.95
57.74 1.76
10.05 0.56
8.45 0.93
>128
Benzyl
20-Methylbenzyl
30-Methylbenzyl
40-Methylbenzyl
20-Chlorobenzyl
20-Fluorobenzyl
20, 60-Difluorobenzyl
30-Trifluoromethylbenzyl
–
4j
5-Fu
H
–
9.13 0.96
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
presented as the means SDs of three independent experiments.

B. Yu et al. / Steroids 88 (2014) 44–52
49
Fig. 4. Apoptosis effect on human MGC-803 cell line induced by compound 4i. Apoptotic cells were detected with Annexin V-FITC/PI double staining after incubation with
compound 4i (0, 4.0, 8.0, 16.0 M) for 24 h or 48 h. (A) Incubated for 24 h; (B) Incubated for 48 h. The lower left quadrants represent live cells, the lower right quadrants are
l
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.

50
B. Yu et al. / Steroids 88 (2014) 44–52
Fig. 5. Effect of compound 4i on the cell cycle distribution of MGC-803 cells. Cells were treated with different concentrations (0, 4.0, 8.0, 16.0 lM) for 24 h or 48 h. Then the
cells were fixed and stained with PI to analyze DNA content by flow cytometry. (A) Incubated for 24 h; (B) Incubated for 48 h. The experiments were performed three times,
and a representative experiment is shown.

B. Yu et al. / Steroids 88 (2014) 44–52
51
involved facile deprotonation of vinyl malononitrile (3) to furnish a
8.45 to 17.42
lM. Particularly, compound 4i was the most cytotoxic
nucleophile that attacked the isatin to form an aldol adduct (A) fol-
lowed by the intramolecular cyclization in a tandem procedure,
yielding the title compounds 4a–j. Different from the findings
reported by Thelagathoti Hari Babu and co-authors [26], further
isomerization of compounds 4a–j was not observed probably due
to the relatively mild acidity of H-16. Functionalized steroidal
pyran–oxindole hybrids were synthesized through the vinylogous
aldol reaction of vinyl malononitrile with various isatin derivatives,
involving tandem construction of C–C and C–O bonds and two
continuous chiral centers (one of them was the quaternary carbon).
The spiro-oxindole framework occupies a special place in heterocy-
clic chemistry because of the presence of this framework in a
number of natural products, such as surugatoxin, horsfiline, etc.
Aza-spiro derivatives are well known [27], but the preparation of
the corresponding oxa-analogues has evolved at a relatively slow
pace [28]. To the best of our knowledge, it is the first time to report
the synthesis of functionalized steroidal oxa spiro-oxindoles.
one with the IC50 value of 8.45 0.93 lM and potent than 5-Fu. On
the basis of those discussed above, we can conclude that the elec-
tronic effect and positions of substituents on the oxindole and N-
benzyl group has a remarkable but different effect on the cytotox-
icity against all the cancel cells tested.
3.2.2. Apoptosis assay
Due to the good cytotoxic activity against some of tested human
cancer cell lines, compound 4i was chosen to be further
investigated regarding its mechanism of action. In order to better
characterize the mode of cell death induced by compound 4i, we
performed a biparametric cytofluorimetric analysis using propidi-
um iodide (PI) and Annexin V-FITC in MGC-803 cells. After treat-
ment with compound 4i for 24 or 48 h at different concentrations
(0, 4.0, 8.0, 16.0 lM), MGC-803 cells were labeled with two dyes,
and the resulting red (PI) and green (FITC) fluorescence was
monitored by flow cytometry. It can be observed from Fig. 4 that
compound 4i caused the early apoptosis. Specifically, after treat-
3.2. Biological evaluation
ment for 24 h, when the concentration was 4.0
tosis rate was 12.6%, and for the high concentration group
(16.0 M), the early and late apoptosis rates were 26.1% and 5.2%,
respectively (6.5% and 0.5% for the control group) (Fig. 4A). Simi-
larly, the early rate for the high concentration group (16.0 M)
lM, the early apop-
3.2.1. Cytotoxic activities
l
The IC50 values (concentrations that are required to inhibit
tumor cell proliferation by 50%) for the synthesized compounds
against four human cancer cell lines including human bladder can-
cer cell line (T24), human liver cancer cell line (SMMC-7721),
human breast cancer cell line (MCF-7) and human gastric cancer
cell line (MGC-803), were determined using the MTT assay. The
well-known anticancer drug 5-fluorouracil was also evaluated in
parallel as a positive control and the results were listed in Table 1.
The structure–activity relationships (SARs) were probed
through altering substituents in the oxindole and N-benzyl moie-
ties. As shown in Table 1, most of the compounds showed moderate
cytotoxic activities against four human cancer cell lines, and a rel-
atively good selectivity was observed, compounds 4a–j were gener-
ally found to be more active against MCF-7 and MGC-803 cells. It is
also clear that substituents on the oxindole and N-benzyl moieties
had remarkable effect on their cytotoxicity. Specifically, compound
4a showed moderate inhibitory effect against MCF-7 cells
l
was 34.5% after treatment for 48 h. The results showed that
compound 4i markedly increased the cellular apoptosis in a
concentration- and time-dependent manner.
3.2.3. Cell cycle analysis
To better understand the cytotoxic activity of compound 4i, a
cell-cycle assay was performed by treating MGC-803 cells at differ-
ent concentrations of compound 4i (0, 4.0, 8.0, 16.0 lM). After
treatment MGC-803 cells with compound 4i for 24 h, the percent-
age of cells in G2/M phase at different concentrations were 10.08%,
14.45%, 19.81% and 25.52%, respectively (Fig. 5A), whereas when
treatment for 48 h, the percentage of cells in G2/M phase were
15.41%, 25.00%, 30.89% and 48.23%, respectively (Fig. 5B). The
results suggested that 4i caused an obvious G2/M arrest in a con-
(IC50 = 24.12 1.38
MGC-803 cells. Similarly, compound 4b displayed weak inhibition
against T24 and SMMC-7721 cells (IC50 > 52 M), but showed mod-
erate inhibition against MCF-7 and MGC-803 cells with the IC50 val-
ues ranging from 14.15 to 24.12 M. For MGC-803 cells, compound
4b was about 7–8 times more potent than compound 4a with the
IC50 values of 14.45 1.16 and 108.51 2.04 M, respectively.
lM), but was inactive to T24, SMMC-7721 and
centration- and time-dependent manner with
a concomitant
decrease in terms of the number of cells in other phases of the cell
cycle.
l
l
4. Conclusion
l
For T24 cells, compounds 4a–j showed distinguishing inhibitory
effect, among them, compound 4f with 40-methyl substituent on
the phenyl group exhibited excellent cytotoxicity (IC50 = 4.43
In summary, we first reported the efficient one-pot synthesis of
novel steroidal pyran–oxindole hybrids under mild conditions
through the vinylogous Aldol reaction of vinyl malononitrile with
various isatin derivatives. This procedure involved cascade con-
struction of C–C and C–O bonds. Some of these compounds dis-
played weak to moderate cytotoxicity against T24, SMMC-7721,
MCF-7 and MGC-803 cells and were sensitive to MCF-7 and
MGC-803 cells. Compounds 4f and 4i were more potent than 5-
Fu against T24 and MGC-803 cells with the IC50 values of 4.43
0.86
By contrast, compounds 4c–e were found to be inactive to T24 cells
(IC50 > 128 M), showing that the position of substituent had a
lM) and was more potent than 5-Fu (IC50 = 7.14 0.85 lM).
l
remarkable effect on the cytotoxicity. In addition, compound 4g
with 20-chloro substituent on the phenyl group also showed no
activity as compound 4d did. While compound 4h with 20-fluoro
substituent showed significantly enhanced cytotoxic activity
and 8.45 lM respectively. Further mechanism studies indicated
(IC50 = 10.84 1.04 lM) compared with compound 4g. Further-
that compound 4i induced G2/M arrest and early apoptosis in a
concentration- and time-dependent manner. Synthesis of more
analogues, SAR studies and further mechanism investigations are
under way and will be reported in due course.
more, when an additional fluoro atom (4i) or a trifluoromethyl
group (4j) was introduced, the activity was decreased to varying
degrees. For SMMC-7721 cells, all the compounds showed weak
to moderate inhibition and were less potent than 5-Fu. Compound
4f with the most excellent activity against T24 cells had weak
inhibitory effect against MCF-7 and MGC-803 cells with the IC50
Acknowledgment
values of 66.37 1.07 and 89.88 1.95 lM, respectively. Fluori-
nated compounds 4h–i displayed excellent cytotoxicity activities
against MCF-7 and MGC-803 cells with the IC50 values ranging from
This work was supported by the National Natural Science
Foundation of China (No. 21372206).

52
B. Yu et al. / Steroids 88 (2014) 44–52
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