Design, synthesis and biological evaluation of novel steroidal spiro-oxindoles as potent antiproliferative agents

Article abstract of DOI:10.1016/j.jsbmb.2014.01.015

Two series of novel steroidal spiro-pyrrolidinyl oxindoles 3a-t and 6a-c were designed and synthesized from dehydroepiandrosterone using the 1,3-dipolar cycloaddition as the key step and further evaluated for their antiproliferative activities for four human cancer cell lines (MGC-803, EC109, SMMC-7721 and MCF-7). This protocol achieved the formation of two CC bonds, one CN bond and the creation of one new five-membered pyrrolidine ring and three contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized steroidal spiro-pyrrolidinyl oxindoles possessed moderate to good antiproliferative activities against the tested cell lines and some of them were more potent than 5-Fu. Particularly, compound 3g showed good antiproliferative activity against SMMC-7721 (IC₅₀ = 0.71 μM). Steroid dimer 6b showed improved antiproliferative activities against SMMC-7721 and MCF-7 with the IC₅₀ values of 4.30 and 2.06 μM, respectively. Flow cytometry analysis demonstrated that compound 3n caused the cellular early apoptosis and cell cycle arrest at G2/M phase in a concentration- and time-independent manner.

Full text of DOI:10.1016/j.jsbmb.2014.01.015

Accepted Manuscript
Title: Design, synthesis and biological evaluation of novel
steroidal spiro-oxindoles as potent antiproliferative agents
Author: Bin Yu Xiao-Jing Shi Ping-Ping Qi De-Quan Yu
Hong-Min Liu
PII:
S0960-0760(14)00026-0
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jsbmb.2014.01.015
SBMB 4143
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date:
Revised date:
Accepted date:
6-11-2013
26-1-2014
28-1-2014
Please cite this article as: B. Yu, X.-J. Shi, P.-P. Qi, D.-Q. Yu, H.-M. Liu, Design,
synthesis and biological evaluation of novel steroidal spiro-oxindoles as potent
antiproliferative agents, Journal of Steroid Biochemistry and Molecular Biology (2014),
http://dx.doi.org/10.1016/j.jsbmb.2014.01.015
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Design, synthesis and biological evaluation of novel steroidal spiro-oxindoles as
potent antiproliferative agents
Bin Yu, Xiao-Jing Shi, Ping-Ping Qi, De-Quan Yu *, Hong-Min Liu∗
School of Pharmaceutical Sciences and New Drug Research & Development Center, Zhengzhou
University, Zhengzhou 450001, P. R. China.
Abstract: Two series of novel steroidal spiro-pyrrolidinyl oxindoles 3a-t and 6a-c were designed
and synthesized from dehydroepiandrosterone using the 1, 3-dipolar cycloaddition as the key step
and further evaluated for their antiproliferative activities for four human cancer cell lines
(MGC-803, EC109, SMMC-7721 and MCF-7). This protocol achieved the formation of two C-C
bonds, one C-N bond and the creation of one new five-membered pyrrolidine ring and three
contiguous stereocenters in a single operation. Biological evaluation showed that these synthesized
steroidal spiro-pyrrolidinyl oxindoles possessed moderate to good antiproliferative activities against
the tested cell lines and some of them were more potent than 5-Fu. Particularly, compound 3g
showed good antiproliferative activity against SMMC-7721 (IC50 = 0.71 µM). Steroid dimer 6b
showed improved antiproliferative activities against SMMC-7721 and MCF-7 with the IC50 values
of 4.30 and 2.06 µM, respectively. Flow cytometry analysis demonstrated that compound 3n caused
the cellular early apoptosis and cell cycle arrest at G2/M phase in a concentration- and
time-independent manner.
Keywords: Dehydroepiandrosterone; Spiro-oxindoles; 1, 3-Dipolar cycloaddition; Antiproliferative
activity; Apoptosis; Cell cycle arrest
1. Introduction
Steroids are a family of molecules that play a crucial role in a wide range of biological processes
and in human physiology. Their hormonal action via binding to a specific receptor is well known
and has led to the development of antagonists to treat certain hormone-dependent diseases [1]. It is
proved that a number of biologically important properties of modified steroids depend upon
∗ First author: Bin Yu, Tel: +8613937109715, e-mail: zzuyubin@hotmail.com; Corresponding authors: (1) Tel.
/fax: +86 371 67781739, e-mail: liuhm@zzu.edu.cn (H.-M. Liu); (2) E-mail: dqyu@imm.ac.cn (D.-Q. Yu).
1
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structural features of the steroid ring system [2] and side chain [3]. Chemical modifications of the
steroid ring system and side chain provide a way to alter the functional groups, and numerous
structure activity relationships (SARs) have been established by such synthetic alterations [4].
Among them, dimeric steroids are a special group of compounds, which have recently received
significant attention [5, 6]. There is evidence that dimerization of steroid skeleton offers some
unique characteristics that are applicable in different areas, especially in pharmacology [7-9].
The specificity of biological activities found in natural compounds is generally in connection with
the characteristic structural complexity and well-defined stereo-architecture [10, 11]. The biological
activity and structural complexity found in nature has stimulated generations of synthetic chemists
to design strategies for assembling challenging structures found in natural products [12, 13].
Particularly intriguing are the spirocyclic oxindole scaffolds, which feature in a large number of
natural and unnatural compounds with important biological activities [14-18] and can also serve as
key intermediates for the synthesis of alkaloids and many kinds of pharmaceuticals or drug
precursors [19]. Among them, the spiro-pyrrolidinyl oxindoles possess a myriad of biological
activities. For example, coerulescine [20], the simplest spirooxindole-pyrrolidine hybrid found in
nature, displays local anesthetic effect. Pteropodine modulates the function of muscarinic serotonin
receptors [21]. The spirotryprostatins have antibiotic properties and are of interest as anticancer lead
compounds [22], and the recently discovered small-molecule MDM2 inhibitor MI-219 and its
analogues are in advanced preclinical development for cancer therapeutics [14, 16] (Fig. 1).
Fig. 1
Recently, we have achieved the synthesis and antiproliferative evaluation of steroidal dienamides
from dehydroepiandrosterone [23, 24]. Inspired by the varied and significant biological activities of
spiro-oxindole skeletons [25-30] and being involved in finding new biologically active modified
steroids [31, 32], we are interested in the design, synthesis and biological evaluation of novel
steroidal spiro-pyrrolidinyl oxindoles. In this paper, we report the efficient and catalyst-free
construction of steroidal spiro-oxindoles with spirotricyclic skeleton via a one-pot three-component
protocol and evaluate their antiproliferative activities against human cancer cell lines in vitro and
the effects toward the cell cycle and apoptosis.
2. Experimental
2
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2.1. General
Reagents and solvents were purchased from commercial sources and were used without further
purification. 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 spectrometer with TMS as internal
standard in CDCl3 or DMSO-d6. Chemical shifts are given as δ ppm values relative to TMS.
High-resolution mass spectra (HRMS) were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionization (ESI).
2.2. General procedure for the synthesis of 16-arylidene-17-ketosteroids (2)
A mixture of dehydroepiandrosterone (DHEA, 1) (2.0 mmol), aromatic aldehydes (2.1 mmol) and
KF/Al2O3 (2.0 mmol) in EtOH (20 mL) was heated under reflux for about 1 h. After completion of
the reaction as evident from TLC, the slurry was filtered and the residue was washed thoroughly
with CH2Cl2. The filtrate was condensed under reduced pressure, and the solid obtained was
crystallized from EtOH or MeOH to yield the corresponding 16-arylidene-17-ketosteroids 2. All the
intermediates 2 were synthesized following the procedure previously reported by our group [31].
All the intermediates were reported before by our group [31] and Kumar [43], so spectral data for a
representative compound 2a was given below. White solid, yield 93%. 1H NMR (400 MHz,
CDCl3): δ 7.99 (d, J = 8.3 Hz, 2H, ArH), 7.71 (d, J = 8.4 Hz, 2H, ArH), 7.46 (s, 1H, Ar-CH=),
5.45–5.36 (m, 1H, 6-H), 3.60–3.50 (m, 1H, 3α-H), 3.09 (s, 3H, Ar-SO2CH3), 2.94 – 2.82 (m, 1H),
2.50 (ddd, J = 16.0, 12.8, 3.0 Hz, 1H), 2.40 – 2.32 (m, 1H), 2.32 – 2.26 (m, 1H), 2.26 – 2.15 (m,
1H), 2.01 (dd, J = 9.6, 3.1 Hz, 1H), 1.86 (ddd, J = 15.8, 12.0, 4.3 Hz, 2H), 1.80 – 1.63 (m, 4H), 1.57
(ddd, J = 14.5, 11.9, 3.3 Hz, 2H), 1.47 – 1.33 (m, 2H), 1.19 – 1.11 (m, 1H) , 1.09 (s, 3H, 19-CH3),
1.01 (s, 3H, 18-CH3). HRMS (ESI): m/z calcd for C27H34O4SNa (M+Na)+, 477.2075; found,
477.2075.
2.3. General procedure for the synthesis of steroidal spiro-pyrrolidinyl oxindoles (3a-t)
A mixture of 16-arylidene-17-ketosteroids (2a-t, 1mmol), (substituted) isatin (1.5 mmol) and
sarcosine (2.0 mmol) in methanol/1, 4-dioxane mixture (v/v = 1/1, 20 mL) was refluxed for about
3
Page 3 of 40

5h. After completion of the reaction as evident from TLC (petroleum ether/ethyl acetate = 2/1), the
mixture was evaporated and purified by column chromatography on silica gel using petroleum
ether/ethyl acetate = 2/1(v/v) as the eluent to obtain the pure products 3a-t.
2.3.1. Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-methylsulfonyl phenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3a)
o
White solid, yield: 89%, m. p. 262.0-264.2 C; 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H, NH),
7.92 (d, J = 8.2 Hz, 2H, ArH), 7.70 (d, J = 6.7 Hz, 2H, ArH), 7.28 – 7.15 (m, 1H, ArH), 7.23 (t, J =
5.9 Hz, 1H, ArH), 7.07 – 6.96 (m, 1H, ArH), 6.85 (d, J = 7.7 Hz, 1H, ArH), 5.10 (d, J = 4.0 Hz, 1H,
6-H), 4.11 (t, J = 8.7 Hz, 1H，12’-H), 3.92 (t, J = 9.4 Hz, 1H, 11’-H), 3.54 (t, J = 8.1 Hz, 1H, 11’-H),
3.51 – 3.39 (m, 1H, 3α-H), 3.09 (s, 3H, CH3SO2-), 2.17 (s, 3H, N-CH3), 2.28 – 2.14 (m, 1H), 2.14 –
1.89 (m, 3H), 1.79 (dd, J = 10.7, 6.3 Hz, 2H), 1.63 (dd, J = 29.1, 12.9 Hz, 2H), 1.44 (dd, J = 18.6,
8.1 Hz, 1H), 1.38 – 1.11 (m, 3H), 0.98 – 0.86 (m, 1H), 0.82 (s, 3H, 19-CH3),0.81 (dd, J = 18.4, 6.6
Hz, 1H), 0.75 – 0.64 (m, 1H), 0.52 (s, 3H, 18-CH3), 0.41 (td, J = 11.6, 4.9 Hz, 1H), 0.30 (dt, J =
12.7, 5.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 178.64, 146.62, 142.11, 141.16, 139.00, 131.34,
129.66, 128.67, 127.53, 126.95, 122.80, 120.44, 109.58, 78.08, 71.47, 67.44, 60.56, 51.25, 50.20,
47.79, 47.02, 44.55, 42.16, 36.94, 36.51, 34.81, 33.36, 31.32, 31.04, 30.76, 30.20, 19.79, 19.26,
14.82. HRMS (ESI): m/z calcd for C35H45N2O5S (M+H)+, 629.3049; found, 629.3043.
2.3.2 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-nitrophenyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3b)
Yellow solid, yield: 91%, m. p. 202.3-204.3 oC; 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 7.8 Hz,
2H, ArH), 7.68 (d, J = 5.9 Hz, 2H, ArH), 7.25 (d, J = 7.6 Hz, 1H, ArH), 7.12 (d, J = 7.6 Hz, 1H,
ArH), 7.05 (t, J = 7.5 Hz, 1H, ArH), 6.85 (d, J = 7.7 Hz, 1H, ArH), 5.22 (s, 1H, 6-H), 3.93 (t, J = 9.4
Hz, 1H, 12’-H), 3.59 (t, J = 8.4 Hz, 1H, 11’-H), 3.55 – 3.37 (m, 2H, 11’-H and 3α-H), 2.19 (s, 3H,
N-CH3), 2.30 – 2.07 (m, 2H), 1.88 (d, J = 16.2 Hz, 2H), 1.79 (d, J = 12.1 Hz, 2H), 1.66 (dd, J =
23.1, 13.0 Hz, 2H), 1.46 – 1.31 (m, 3H), 1.31 – 1.25 (m, 1H), 1.01 – 0.88 (m, 1H), 0.85 (s, 3H,
19-CH3), 0.73 (m, 2H), 0.57 (s, 3H, 18-CH3), 0.43 (td, J = 11.6, 4.9 Hz, 1H), 0.39 – 0.28 (m, 1H);
13C NMR (100 MHz, CDCl3) δ 178.51, 147.71, 147.11, 141.82, 141.16, 131.16, 129.72, 128.75,
126.92, 123.70, 122.98, 120.47, 109.48, 78.02, 71.52, 67.45, 65.88, 60.44, 51.19, 50.20, 47.88,
47.07, 42.14, 36.92, 36.50, 34.79, 33.36, 31.42, 31.09, 30.74, 30.23, 19.21, 14.82. HRMS (ESI):
4
Page 4 of 40

m/z calcd for C36H42N3O5 (M+H)+, 596.3124; found, 596.3126.
2.3.3 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(3’’-nitrophenyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3c)
o
1
Yellow solid, yield: 93%, m. p. 189.5-193.8 C; H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H, NH),
8.59 (s, 1H, ArH), 8.11 (dd, J = 8.2, 1.3 Hz, 1H, ArH), 7.75 (s, 1H, ArH), 7.50 (t, J = 7.9 Hz, 1H,
ArH), 7.34 – 7.27 (m, 1H, ArH), 7.11 (d, J = 7.3 Hz, 1H, ArH), 7.04 (t, J = 7.5 Hz, 1H, ArH), 6.96
(d, J = 7.7 Hz, 1H, ArH), 4.93 (s, 1H, 6-H), 4.12 (m, 1H, 12’-H), 3.95 (t, J = 9.3 Hz, 1H, 11’-H),
3.64 (t, J = 8.5 Hz, 1H, 11’-H), 3.60 – 3.45 (m, 1H, 3α-H), 2.22 (s, 3H, N-CH3), 2.20 – 1.99 (m, 2H),
1.76 (m, 3H), 1.65 (t, J = 13.5 Hz, 2H), 1.48 – 1.31 (m, 2H), 1.31 – 1.21 (m, 2H), 1.16 (ddd, J =
15.5, 9.3, 4.4 Hz, 1H), 0.96 (dd, J = 19.6, 7.6 Hz, 1H), 0.82 (s, 3H, 19-CH3), 0.89 – 0.67 (m, 2H),
0.58 (s, 3H, 18-CH3), 0.50 – 0.30 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 179.33, 148.66, 142.30,
141.99, 141.06, 136.50, 129.84, 129.34, 128.43, 126.75, 125.11, 122.91, 122.15, 120.27, 109.99,
78.22, 71.31, 67.30, 60.77, 51.05, 49.94, 47.67, 47.06, 42.01, 36.75, 36.50, 34.85, 33.31, 31.53,
31.01, 30.68, 30.28, 19.21, 14.87. HRMS (ESI): m/z calcd for C36H42N3O5 (M+H)+, 596.3124;
found, 596.3126.
2.3.4 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(3’’-methoxylphenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3d)
o
1
Yellow solid, yield: 86%, m. p. 174.0-175.8 C; H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H, NH),
7.23 (t, J = 7.8 Hz, 2H, ArH), 7.13 (d, J = 7.5 Hz, 2H, ArH), 7.02 (t, J = 7.6 Hz, 2H, ArH), 6.81 (dd,
J = 11.0, 5.1 Hz, 2H, ArH), 5.19 (d, J = 4.9 Hz, 1H, 6-H), 4.09 – 3.89 (m, 2H, 12’-H and 11’-H),
3.82 (s, 3H, CH3O-), 3.55 (t, J = 7.4 Hz, 1H, 11’-H), 3.48 (m, 1H, 3α-H), 2.19 (s, 3H, N-CH3), 2.28
– 2.10 (m, 2H), 2.03 (m, 1H), 1.95 – 1.74 (m, 3H), 1.74 – 1.57 (m, 2H), 1.46 (dd, J = 18.7, 8.0 Hz,
1H), 1.40 – 1.31 (m, 2H), 1.24 – 1.16 (m, 1H), 0.92 (dt, J = 12.4, 9.0 Hz, 2H), 0.86 (s, 3H, 19-CH3),
0.75 – 0.63 (m, 1H), 0.6 (s, 3H, 18-CH3), 0.43 (td, J = 11.6, 5.0 Hz, 1H), 0.34 (td, J = 12.7, 4.2 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 178.69, 159.70, 141.92, 141.22, 141.02, 129.41, 129.31,
128.78, 127.40, 122.74, 122.62, 120.69, 115.38, 112.84, 109.26, 78.17, 71.54, 67.77, 60.43, 60.35,
55.20, 51.59, 50.27, 47.96, 47.09, 42.13, 36.96, 36.51, 34.90, 33.19, 31.52, 31.14, 30.83, 30.27,
19.23, 14.61. HRMS (ESI): m/z calcd for C37H45N2O4 (M+H)+, 581.3379; found, 581.3376.
5
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2.3.5 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-fluorophenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3e)
o
1
Yellow solid, yield: 90%, m. p. 178.4-180.6 C; H NMR (400 MHz, CDCl3) δ 8.91 (s, 1H, NH),
7.43 (s, 2H, ArH), 7.22 (t, J = 7.6 Hz, 1H, ArH), 7.11 (d, J = 7.5 Hz, 1H, ArH), 7.00 (dd, J = 15.7,
7.9 Hz, 3H, ArH), 6.85 (d, J = 7.7 Hz, 1H, ArH), 5.17 (s, 1H, 6-H), 4.06 – 3.93 (m, 1H, 12’-H),
3.87 (t, J = 9.4 Hz, 1H, 11’-H), 3.62 – 3.39 (m, 2H, 11’-H and 3α-H), 2.15 (s, 3H, N-CH3), 2.59 –
2.17 (m, 3H), 2.01 (d, J = 4.1 Hz, 1H), 1.83 (dd, J = 20.4, 16.3 Hz, 2H), 1.65 (dd, J = 28.5, 12.8 Hz,
2H), 1.52 – 1.39 (m, 1H), 1.36 (d, J = 20.7 Hz, 2H), 1.23 – 1.14 (m, 1H), 0.92 (dd, J = 16.7, 12.1
Hz, 2H), 0.85 (s, 3H, 19-CH3), 0.74 – 0.62 (m, 1H), 0.55 (s, 3H, 18-CH3), 0.43 (td, J = 11.6, 5.0 Hz,
13
1H), 0.38 – 0.26 (m, 1H); C NMR (100 MHz, CDCl3) δ 179.10, 163.11, 160.67, 142.15, 141.07,
135.34, 135.31, 131.77, 131.69, 129.52, 128.65, 127.23, 122.76, 120.65, 115.39, 115.18, 109.46,
78.21, 71.50, 67.57, 60.68, 50.86, 50.21, 47.89, 47.06, 42.14, 36.93, 36.51, 34.84, 33.21, 31.46,
31.08, 30.76, 30.27, 19.24, 14.56. HRMS (ESI): m/z calcd for C36H42FN2O3 (M+H)+, 569.3179;
found, 569.3176.
2.3.6 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(2’’-fluorophenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3f)
o
1
White solid, yield: 79%, m. p. 189.4-192.5 C; H NMR (400 MHz, CDCl3) δ 7.97 – 7.82 (m, 2H,
ArH and NH), 7.26 – 7.09 (m, 4H, ArH), 7.07 – 6.93 (m, 2H, ArH), 6.80 (d, J = 7.7 Hz, 1H, ArH),
5.21 (d, J = 5.0 Hz, 1H, 6-H), 4.52 (dd, J = 9.7, 8.3 Hz, 1H, 12’-H), 3.91 (t, J = 9.4 Hz, 1H, 11’-H),
3.50 (t, J = 8.5 Hz, 1H, 11’-H), 3.47 – 3.39 (m, 1H, 3α-H), 2.18 (s, 3H, N-CH3), 2.28 – 2.08 (m, 2H),
2.07 – 1.96 (m, 1H), 1.93 – 1.82 (m, 1H), 1.78 (d, J = 11.2 Hz, 2H), 1.73 – 1.56 (m, 2H), 1.48 –
1.38 (m, 1H), 1.38 – 1.17 (m, 3H), 0.98 – 0.87 (m, 2H), 0.85 (s, 3H, 19-CH3), 0.74 – 0.63 (m, 1H),
0.55 (s, 3H, 18-CH3), 0.42 (td, J = 11.2, 5.4 Hz, 1H), 0.31 (td, J = 12.6, 4.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 207.10, 178.49, 162.47, 160.03, 141.85, 141.11, 132.03, 129.46, 128.96, 128.40,
128.32, 127.30, 126.50, 126.37, 124.40, 124.36, 122.81, 120.66, 115.15, 114.92, 109.21, 78.05,
71.56, 67.05, 59.56, 50.27, 47.95, 47.20, 42.14, 36.94, 36.52, 34.88, 33.20, 31.52, 31.15, 30.95,
30.90, 30.29, 19.81, 19.23, 14.78. HRMS (ESI): m/z calcd for C36H42FN2O3 (M+H)+, 569.3179;
found, 569.3178.
2.3.7 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-chlorophenyl)-tetrahydro-1H-
6
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pyrrolo-dehydroandrosterone (3g)
o
1
Yellow solid, yield: 86%, m. p. 197.3-199.7 C; H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H, NH),
7.39 (s, 2H, ArH), 7.27 (d, J = 8.4 Hz, 2H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.09 (d, J = 7.5 Hz,
1H, ArH), 7.00 (t, J = 7.6 Hz, 1H, ArH), 6.83 (d, J = 7.7 Hz, 1H, ArH), 5.19 (d, J = 3.7 Hz, 1H,
6-H), 3.97 (t, J = 8.8 Hz, 1H, 12’-H), 3.86 (t, J = 9.4 Hz, 1H, 11’-H), 3.49 (m, 2H, 11’-H and 3α-H),
2.13 (s, 3H, N-CH3), 2.52 (s, 1H), 2.31 – 2.16 (m, 2H), 2.02 (d, J = 4.1 Hz, 1H), 1.84 (dd, J = 29.2,
14.0 Hz, 2H), 1.65 (dd, J = 29.8, 12.8 Hz, 2H), 1.45 (dd, J = 23.0, 10.2 Hz, 1H), 1.33 (t, J = 13.3 Hz,
2H), 1.21 (d, J = 4.8 Hz, 1H), 1.00 – 0.87 (m, 2H), 0.85 (s, 3H, 19-CH3), 0.76 – 0.61 (m, 1H), 0.55
(s, 3H, 18-CH3), 0.43 (td, J = 11.5, 4.9 Hz, 1H), 0.34 (td, J = 12.5, 3.9 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 178.97, 142.16, 141.11, 138.18, 132.80, 131.61, 129.51, 128.67, 128.61, 127.20,
122.74, 120.61, 109.45, 78.16, 71.50, 67.50, 50.99, 50.21, 47.89, 47.07, 42.17, 36.94, 36.53, 34.80,
33.22, 31.49, 31.09, 30.77, 30.28, 21.07, 19.82, 19.23, 14.63. HRMS (ESI): m/z calcd for
C36H42ClN2O3 (M+H)+, 585.2884; found, 585.2882.
2.3.8 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(3’’-chlorophenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3h)
o
1
White solid, yield: 83%, m. p. 227.4-229.5 C; H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H, NH),
7.56 (s, 1H, ArH), 7.31 (s, 1H, ArH), 7.28 – 7.17 (m, 3H, ArH), 7.10 (d, J = 7.6 Hz, 1H, ArH), 7.01
(t, J = 7.5 Hz, 1H, ArH), 6.87 (d, J = 7.6 Hz, 1H, ArH), 5.11 (s, 1H, 6-H), 4.03 – 3.94 (m, 1H,
12’-H), 3.94 – 3.82 (m, 1H, 11’-H), 3.54 (t, J = 8.1 Hz, 1H, 11’-H), 3.48 (m, 1H, 3α-H), 2.17 (s, 3H,
N-CH3), 2.28 – 2.09 (m, 2H), 2.09 – 2.01 (m, 1H), 1.99 (s, 1H), 1.83 (dd, J = 29.1, 14.6 Hz, 2H),
1.65 (dd, J = 27.4, 13.0 Hz, 2H), 1.50 – 1.39 (m, 1H), 1.35 (d, J = 10.8 Hz, 2H), 1.20 (dd, J = 12.8,
8.1 Hz, 1H), 0.94 (dd, J = 18.4, 8.5 Hz, 2H), 0.85 (s, 3H, 19-CH3), 0.75 – 0.63 (m, 1H), 0.55 (s, 3H,
18-CH3), 0.43 (td, J = 11.6, 5.0 Hz, 1H), 0.35 (td, J = 13.0, 4.2 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 178.96, 142.07, 141.91, 140.90, 134.36, 130.18, 129.66, 129.53, 128.63, 128.51, 127.26,
127.16, 122.77, 120.66, 109.50, 78.14, 77.37, 77.06, 76.74, 71.52, 67.52, 51.23, 50.20, 47.86, 47.04,
42.13, 36.94, 36.51, 34.84, 33.17, 31.54, 31.13, 30.77, 30.29, 19.82, 19.24, 14.20. HRMS (ESI):
m/z calcd for C36H42ClN2O3 (M+H)+, 585.2884; found, 585.2886.
2.3.9 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(2’’-chlorophenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3i)
7
Page 7 of 40

White solid, yield: 81%, m. p. 208.7-210.7 oC; 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H, NH),
7.94 (d, J = 7.7 Hz, 1H, ArH), 7.42 (dd, J = 7.3, 3.5 Hz, 2H, ArH), 7.35 – 7.17 (m, 2H, ArH), 6.95
(t, J = 7.5 Hz, 1H, ArH), 6.84 (dd, J = 16.0, 7.6 Hz, 2H, ArH), 5.16 (s, 1H, 6-H), 4.57 (dd, J = 12.8,
6.8 Hz, 2H, 12’-H and 11’-H), 3.70 (t, J = 9.2 Hz, 1H, 11’-H), 3.17 (dd, J = 10.0, 5.3 Hz, 1H, 3α-H),
2.09 (s, 3H, N-CH3), 2.08-2.04 (m, 1H), 1.98-1.92 (m, 1H), 1.73 (d, J = 16.4 Hz, 1H), 1.57 (d, J =
10.0 Hz, 2H), 1.41 (d, J = 12.2 Hz, 1H), 1.31 – 1.07 (m, 5H), 0.77 (s, 3H, 19-CH3), 0.73-0.59 (m,
3H), 0.55 (s, 3H, 18-CH3), 0.24 (td, J = 11.2, 5.1 Hz, 1H), 0.18 – 0.07 (m, 1H); 13C NMR (100
MHz, DMSO-d6) δ 178.40, 143.94, 141.90, 137.64, 134.32, 132.61, 130.02, 129.61, 129.05, 128.30,
127.87, 127.35, 121.94, 119.99, 109.85, 77.71, 70.22, 66.79, 60.58, 50.69, 47.61, 46.99, 45.29,
42.47, 37.14, 36.49, 34.63, 33.63, 31.71, 31.17, 31.07, 30.96, 30.08, 19.37, 15.51. HRMS (ESI):
m/z calcd for C36H42ClN2O3 (M+H)+, 585.2884; found, 585.2881.
2.3.10 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(3’’, 4’’-dichlorophenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3j)
o
1
Yellow solid, yield: 88%, solid, m. p. 201.4-203.8 C; H NMR (400 MHz, CDCl3) δ 8.90 (s, 1H,
NH), 7.64 (s, 1H, ArH), 7.37 (d, J = 8.0 Hz, 1H, ArH), 7.32 (d, J = 6.2 Hz, 1H, ArH), 7.23 (t, J =
7.5 Hz, 1H, ArH), 7.09 (d, J = 7.4 Hz, 1H, ArH), 7.01 (t, J = 7.5 Hz, 1H, ArH), 6.86 (d, J = 7.7 Hz,
1H, ArH), 5.15 (s, 1H, 6-H), 3.94 (t, J = 8.7 Hz, 1H, 12’-H), 3.84 (t, J = 9.3 Hz, 1H, 11’-H), 3.63 –
3.43 (m, 2H, 11’-H and 3α-H), 2.14 (s, 3H, N-CH3), 2.53 – 2.18 (m, 3H), 1.84 (dd, J = 33.8, 13.9
Hz, 2H), 1.65 (dd, J = 25.2, 12.9 Hz, 2H), 1.55 – 1.39 (m, 1H), 1.35 (d, J = 10.6 Hz, 2H), 1.31 –
1.16 (m, 1H), 1.00 – 0.87 (m, 2H), 0.85 (s, 3H, 19-CH3), 0.75 – 0.62 (m, 1H), 0.58 (s, 3H, 18-CH3),
0.49 – 0.30 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 179.01, 142.14, 141.05, 140.24, 132.46,
132.03, 131.03, 130.38, 129.71, 129.64, 128.56, 126.98, 122.82, 120.55, 109.61, 78.10, 71.48,
67.28, 50.73, 50.18, 47.85, 47.09, 42.16, 36.93, 36.52, 34.77, 33.21, 31.49, 31.09, 30.74, 30.29,
26.90, 21.08, 19.23, 14.78. HRMS (ESI): m/z calcd for C36H41Cl2N2O3 (M+H)+, 619.2494; found,
619.2493.
2.3.11 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-phenyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3k)
White solid, yield: 85%, m. p. 246.9-248.0 oC; 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H, NH),
7.34 (dd, J = 12.8, 5.5 Hz, 4H, ArH), 7.29 – 7.18 (m, 2H, ArH), 6.93 (t, J = 7.5 Hz, 1H, ArH), 6.87
8
Page 8 of 40

(d, J = 7.4 Hz, 1H, ArH), 6.80 (d, J = 7.7 Hz, 1H, ArH), 5.16 (d, J = 4.2 Hz, 1H, 6-H), 4.57 (d, J =
4.6 Hz, 1H, 12’-H), 3.80 (m, 2H, 12’-H and 11’-H), 3.18 (m, 1H, 3α-H), 2.09 (s, 3H, N-CH3),
2.09-2.06 (m, 2H), 2.01-1.93 (m, 1H), 1.77 (d, J = 16.3 Hz, 1H), 1.58 (d, J = 10.2 Hz, 2H), 1.42 (d,
J = 12.4 Hz, 1H), 1.33 – 1.19 (m, 4H), 1.14-1.08 (m, 2H), .0.77 (s, 3H, 19-CH3), 0.71-0.63 (m, 2H),
0.44 (s, 3H, 18-CH3), 0.26 (td, J = 11.5, 4.8 Hz, 1H), 0.17 (td, J = 12.5, 4.0 Hz, 1H); 13C NMR (100
MHz, DMSO-d6) δ 178.38, 143.92, 141.91, 140.04, 130.26, 129.88, 128.85, 128.20, 127.55, 127.42,
121.82, 119.99, 109.73, 77.74, 70.24, 67.66, 60.37, 51.17, 50.70, 47.67, 46.74, 42.48, 37.15, 36.49,
34.72, 33.24, 31.73, 31.16, 31.04, 30.96, 30.12, 19.36, 14.77. HRMS (ESI): m/z calcd for
C36H43N2O3 (M+H)+, 551.3274; found, 551.3268.
2.3.12 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-methylphenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3l)
o
1
Yellow solid, yield: 82%, m. p. 184.8-186.6 C; H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H, NH),
7.33 (d, J = 5.5 Hz, 2H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.12 (d, J = 7.6 Hz, 3H, ArH), 7.00 (t,
J = 7.5 Hz, 1H, ArH), 6.83 (d, J = 7.5 Hz, 1H, ArH), 5.20 (s, 1H, 6-H), 4.06 – 3.95 (m, 1H, 12’-H),
3.96 – 3.80 (m, 1H, 11’-H), 3.60 – 3.36 (m, 2H, 11’-H and 3α-H), 2.33 (s, 3H, CH3Ph-), 2.15 (s, 3H,
N-CH3), 2.28 – 2.17 (m, 2H), 2.10 – 2.01 (m, 1H), 1.84 (dd, J = 33.5, 14.3 Hz, 2H), 1.64 (dd, J =
30.2, 12.9 Hz, 2H), 1.51 – 1.29 (m, 4H), 1.24 – 1.12 (m, 1H), 0.96-0.87 (m, 2H), .0.86 (s, 3H,
19-CH3), 0.74 – 0.62 (m, 1H), 0.44 (s, 3H, 18-CH3), 0.43 (td, J = 11.6, 5.0 Hz, 1H), 0.33 (td, J =
12.6, 4.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 179.00, 178.91, 142.17, 142.09, 141.04, 136.55,
136.40, 130.13, 129.37, 129.16, 128.75, 127.47, 122.65, 120.75, 109.38, 78.23, 71.53, 67.77, 51.30,
50.23, 47.96, 47.06, 42.17, 36.95, 36.53, 34.87, 33.15, 31.53, 31.11, 30.83, 30.29, 21.14, 19.25,
14.53. HRMS (ESI): m/z calcd for C37H45N2O3 (M+H)+, 565.3430; found, 565.3426.
2.3.13 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’isopropylphenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3m)
o
1
Yellow solid, yield: 73%, m. p. 186.4-188.7 C; H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H, NH),
7.36 (d, J = 6.4 Hz, 2H, ArH), 7.27 – 7.06 (m, 4H, ArH), 7.01 (t, J = 7.6 Hz, 1H, ArH), 6.83 (d, J =
7.6 Hz, 1H, ArH), 5.23 (d, J = 3.5 Hz, 1H, 6-H), 4.04 – 3.96 (m, 1H, 12’-H), 3.96 – 3.87 (m, 1H,
11’-H), 3.49 (m, 2H, 11’-H and 3α-H), 2.87 (heptet, J = 6.9 Hz, 1H, -CH(CH3)2), 2.46-2.18 (m, 4H),
2.15 (s, 3H, N-CH3), 2.10 – 2.00 (m, 1H), 1.91 (d, J = 16.8 Hz, 1H), 1.81 (d, J = 11.4 Hz, 1H), 1.69
9
Page 9 of 40

(d, J = 13.3 Hz, 1H), 1.62 (d, J = 12.3 Hz, 1H), 1.53 – 1.29 (m, 3H), 1.25 (d, J = 6.9 Hz, 6H,
-CH(CH3)2), 1.00 – 0.88 (m, 2H), 0.86 (s, 3H, 19-CH3), 0.75 – 0.63 (m, 1H), 0.57 (s, 3H, 18-CH3),
0.44 (td, J = 11.3, 4.9 Hz, 1H), 0.34 (td, J = 12.6, 4.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
178.89, 147.44, 142.12, 141.10, 136.68, 130.11, 129.36, 128.75, 127.49, 126.47, 122.66, 120.77,
109.34, 78.22, 71.54, 67.82, 51.28, 50.24, 47.98, 47.07, 42.15, 36.97, 36.56, 34.86, 33.66, 33.11,
31.52, 31.13, 30.82, 30.32, 23.98, 19.86, 19.28, 14.57. HRMS (ESI): m/z calcd for C39H49N2O3
(M+H)+, 593.3743; found, 593.3742.
2.3.14 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-tert-buthylphenyl)-tetrahydro-1H-
pyrrolo-dehydroandrosterone (3n)
o
1
Yellow solid, yield: 83%, m. p. 205.4-207.3 C; H NMR (400 MHz, CDCl3) δ 8.35 (s, 1H, NH),
7.43 – 7.28 (m, 4H, ArH), 7.21 (m, 1H, ArH), 7.12 (d, J = 7.5 Hz, 1H, ArH), 7.00 (t, J = 7.7 Hz, 1H,
ArH), 6.81 (d, J = 7.6 Hz, 1H, ArH), 5.23 (d, J = 4.7 Hz, 1H, 6-H), 3.99 (dd, J = 9.8, 7.6 Hz, 1H,
12’-H), 3.96 – 3.86 (m, 1H, 11’-H), 3.48 (m, 2H, 11’-H and 3α-H), 2.14 (s, 3H, N-CH3), 2.30 – 2.07
(m, 4H), 2.08 – 1.99 (m, 1H), 1.96 – 1.85 (m, 1H), 1.79 (d, J = 11.6 Hz, 1H), 1.69 (d, J = 13.4 Hz,
1H), 1.61 (d, J = 12.6 Hz, 1H), 1.47 (dd, J = 19.1, 8.6 Hz, 1H), 1.31 (s, 9H, -C(CH3)3), 1.25-1.21
(m, 1H), 0.99 – 0.87 (m, 2H), 0.86 (s, 3H, 19-CH3), 0.74 – 0.63 (m, 1H), 0.57 (s, 3H, 18-CH3), 0.43
13
(td, J = 11.5, 5.0 Hz, 1H), 0.33 (td, J = 12.9, 4.7 Hz, 1H); C NMR (100 MHz, CDCl3) δ 178.76,
149.73, 142.03, 141.12, 136.30, 129.82, 129.35, 128.78, 127.50, 125.31, 122.67, 120.77, 109.28,
78.19, 71.55, 67.82, 51.14, 50.25, 47.99, 47.07, 42.14, 36.97, 36.56, 34.88, 34.45, 33.10, 31.51,
31.39, 31.15, 30.82, 30.31, 19.28, 14.59. HRMS (ESI): m/z calcd for C40H51N2O3 (M+H)+,
607.3900; found, 607.3884.
2.3.15 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-naphthyl)-tetrahydro-1H-pyrrolo
-dehydroandrosterone (3o)
White solid, yield: 79%, m. p. 262.0-264.2 oC; 1H NMR (400 MHz, DMSO-d6) δ 10.78 (s, 1H, NH),
7.94 (t, J = 6.9 Hz, 2H, ArH), 7.83 (dd, J = 16.4, 8.0 Hz, 2H, ArH), 7.61 (t, J = 7.6 Hz, 1H, ArH),
7.49 (d, J = 3.7 Hz, 2H, ArH), 7.25 (t, J = 7.4 Hz, 1H, ArH), 7.11 – 6.88 (m, 2H, ArH), 6.82 (d, J =
7.6 Hz, 1H, ArH), 5.11 (s, 1H, 6-H), 4.88 (t, J = 8.6 Hz, 1H, 12’-H), 4.55 (d, J = 4.3 Hz, 1H, -OH),
4.13 – 3.90 (m, 1H, 11’-H), 3.42 (t, J = 7.7 Hz, 1H, 11’-H), 3.16 (d, J = 3.7 Hz, 1H, 3α-H), 2.05 (s,
3H, N-CH3), 1.99-1.91 (m, 2H), 1.68 (d, J = 16.5 Hz, 1H), 1.53 (d, J = 13.3 Hz, 2H), 1.37 (d, J =
10
Page 10 of 40

11.8 Hz, 1H), 1.31 – 1.11 (m, 4H), 1.11 – 0.90 (m, 2H), 0.78 – 0.58 (m, 2H), 0.67 (s, 3H, 19-CH3),
0.51 (t, J = 13.9 Hz, 1H), 0.18 (t, J = 11.1 Hz, 1H), 0.17 (s, 3H, 18-CH3), 0.09-0.04 (m, 1H); 13C
NMR (100 MHz, DMSO-d6) δ 178.19, 143.98, 141.82, 136.00, 133.91, 132.62, 129.99, 129.34,
128.32, 128.10, 127.64, 127.55, 126.67, 126.17, 126.06, 123.45, 121.93, 120.02, 109.82, 99.99,
77.81, 70.21, 67.98, 60.71, 50.69, 47.50, 47.19, 42.91, 42.45, 37.13, 36.44, 34.76, 33.48, 31.70,
31.03, 29.95, 19.83, 19.23, 15.81. HRMS (ESI): m/z calcd for C40H45N2O3 (M+H)+, 601.3430;
found, 601.3428.
2.3.16 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’ -pyridyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3p)
White solid, yield: 87%, m. p. 189.9-191.4 oC; 1H NMR (400 MHz, CDCl3) δ 8.51 (dd, J = 4.8, 0.9
Hz, 1H, ArH), 7.98 – 7.82 (m, 2H, ArH and NH), 7.69 (m, 1H, ArH), 7.22 (m, 1H, ArH), 7.19 –
7.11 (m, 2H, ArH), 7.08 – 6.92 (m, 1H, ArH), 6.80 (d, J = 7.6 Hz, 1H, ArH), 5.20 (d, J = 5.1 Hz,
1H, 6-H), 4.29 (dd, J = 9.8, 8.3 Hz, 1H, 12’-H), 4.03 (t, J = 9.5 Hz, 1H, 11’-H), 3.64 (t, J = 8.6 Hz,
1H, 11’-H), 3.52 – 3.38 (m, 1H, 3α-H), 2.18 (s, 3H, N-CH3), 1.97 (dd, J = 13.5, 4.3 Hz, 1H), 1.89 (d,
J = 21.5 Hz, 1H), 1.84 – 1.72 (m, 2H), 1.72 – 1.56 (m, 2H), 1.49 – 1.38 (m, 1H), 1.38 – 1.14 (m,
5H), 0.96 – 0.81 (m, 1H), 0.85 (s, 3H, 19-CH3),0.78 (d, J = 13.7 Hz, 1H), 0.70 (s, 3H, 18-CH3),
0.67-0.63 (m, 1H), 0.47 – 0.27 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 178.55, 160.28, 148.87,
141.85, 141.15, 136.56, 129.46, 128.93, 127.22, 124.22, 122.78, 121.93, 120.63, 109.23, 78.10,
71.54, 67.21, 58.90, 53.39, 50.24, 47.99, 47.24, 42.13, 36.94, 36.52, 34.97, 32.83, 31.53, 31.13,
30.95, 30.92, 30.33, 19.24, 14.90. HRMS (ESI): m/z calcd for C35H42N3O3 (M+H)+, 552.3226;
found, 552.3223.
2.3.17 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-furyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3q)
o
1
Yellow solid, yield: 88%, m. p. 180.0-181.6 C; H NMR (400 MHz, CDCl3) δ 7.94 (s, 1H, NH),
7.34 (d, J = 1.0 Hz, 1H, ArH), 7.21 (dd, J = 11.0, 4.2 Hz, 1H, ArH), 7.08 (d, J = 7.4 Hz, 1H, ArH),
6.99 (t, J = 7.5 Hz, 1H, ArH), 6.79 (d, J = 7.7 Hz, 1H, ArH), 6.41 (d, J = 3.1 Hz, 1H, ArH), 6.38 –
6.31 (m, 1H, ArH), 5.23 (d, J = 4.7 Hz, 1H, 6-H), 4.12 (m, 1H, 12’-H), 3.96 (t, J = 9.3 Hz, 1H,
11’-H), 3.51 (t, J = 8.5 Hz, 1H, 11’-H), 3.48 – 3.41 (m, 1H, 3α-H), 2.16 (s, 3H, N-CH3), 2.28 – 2.09
(m, 2H), 2.01 (d, J = 4.6 Hz, 1H), 1.97 – 1.74 (m, 3H), 1.74 – 1.58 (m, 1H), 1.50 – 1.30 (m, 2H),
11
Page 11 of 40

1.04 (t, J = 14.1 Hz, 1H), 0.98 – 0.84 (m, 1H), 0.89 (s, 3H, 19-CH3), 0.79 (s, 3H, 18-CH3),
0.66-0.61 (m, 1H), 0.49 – 0.32 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 178.19, 154.35, 141.96,
141.52, 141.05, 129.49, 128.50, 126.97, 122.70, 120.77, 110.59, 109.27, 108.07, 77.83, 71.56,
67.02, 57.20, 50.25, 47.93, 47.62, 44.10, 42.15, 36.97, 36.54, 34.95, 32.09, 31.53, 31.12, 30.91,
30.40, 21.07, 19.28, 15.10. HRMS (ESI): m/z calcd for C34H41N2O4 (M+H)+, 541.3066; found,
541.3063.
2.3.18 Spiro [2'.16]-1'-methyl-2'-(indolin-2-one)-4'-(4’’-thienyl)-tetrahydro-1H-pyrrolo-
dehydroandrosterone (3r)
o
1
Yellow solid, yield: 89% m. p. 192.6-193.3 C; H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H, NH),
7.27 – 7.18 (m, 2H, ArH), 7.09 (dd, J = 13.9, 5.2 Hz, 2H, ArH), 7.05 – 6.94 (m, 2H, ArH), 6.84 (d,
J = 7.7 Hz, 1H, ArH), 5.22 (t, J = 12.9 Hz, 1H, 6-H), 4.31 (t, J = 8.8 Hz, 1H, 12’-H), 3.97 (t, J = 9.4
Hz, 1H, 11’-H), 3.64 (t, J = 8.6 Hz, 1H, 11’-H), 3.55 – 3.41 (m, 1H, 3α-H), 2.18 (s, 3H, N-CH3),
2.30 – 2.10 (m, 2H), 2.05 – 1.98 (m, 1H), 1.95 – 1.74 (m, 4H), 1.67 (dd, J = 25.4, 13.0 Hz, 2H),
1.49-1.35 (m, 3H), 1.10 (t, J = 14.1 Hz, 1H), 1.00 – 0.81 (m, 1H), 0.88 (s, 3H, 19-CH3), 0.76 – 0.61
13
(m, 1H), 0.70 (s, 3H, 18-CH3), 0.47-0.36 (m, 2H); C NMR (100 MHz, CDCl3) δ 178.52, 142.96,
142.08, 140.98, 129.53, 128.48, 127.17, 127.04, 126.92, 124.78, 122.75, 120.76, 109.40, 77.98,
71.55, 67.51, 61.12, 50.24, 47.83, 47.27, 45.75, 42.15, 36.96, 36.52, 34.87, 32.60, 31.52, 31.12,
30.81, 30.32, 21.07, 19.26, 14.21. HRMS (ESI): m/z calcd for C34H41N2O3S (M+H)+, 557.2838;
found, 557.2836.
2.3.19 Spiro [2'.16]-1'-methyl-2'-(5’’’-fluoro-indolin-2-one)-4'-(4’’-chlorophenyl)-tetrahydro-1H
-pyrrolo-dehydroandrosterone (3s)
Yellow solid, yield: 79%, m. p. 187.5-189.2 oC; 1H NMR (400 MHz, CDCl3) δ 8.11 (Brs, 1H, NH),
7.39 (d, J = 6.8 Hz, 2H, ArH), 7.30 (s, 1H, ArH), 7.27 (d, J = 2.6 Hz, 1H, ArH), 7.03 – 6.82 (m, 2H,
ArH), 6.76 (dd, J = 8.4, 4.2 Hz, 1H, ArH), 5.21 (d, J = 4.0 Hz, 1H, 6-H), 3.96 (dd, J = 9.7, 8.0 Hz,
1H, 12’-H), 3.84 (t, J = 9.4 Hz, 1H, 11’-H), 3.51 (t, J = 8.4 Hz, 1H, 11’-H), 3.48 – 3.39 (m, 1H,
3α-H), 2.16 (s, 3H, N-CH3), 2.33 – 2.09 (m, 2H), 2.02 (dd, J = 14.5, 5.1 Hz, 1H), 1.89 (d, J = 15.2
Hz, 1H), 1.79 (d, J = 11.9 Hz, 2H), 1.70 (dd, J = 13.2, 3.2 Hz, 3H), 1.45-1.38 (m, 2H), 1.36 – 1.19
(m, 1H), 1.00 – 0.90 (m, 1H), 0.86 (s, 3H, 19-CH3), 0.81 – 0.74 (m, 1H), 0.72 – 0.59 (m, 1H), 0.57
(s, 3H, 18-CH3), 0.51 – 0.31 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 178.58, 160.14, 157.74,
12
Page 12 of 40

141.16, 137.92, 137.72, 132.95, 131.58, 129.34, 129.26, 128.67, 120.52, 116.97, 116.71, 116.07,
115.83, 109.86, 109.78, 78.26, 71.57, 67.78, 60.53, 51.06, 50.32, 48.29, 47.16, 42.14, 36.95, 36.53,
34.83, 33.14, 31.50, 31.11, 30.95, 30.28, 22.66, 19.84, 19.22, 14.57. HRMS (ESI): m/z calcd for
C36H41ClFN2O3 (M+H)+, 603.2790; found, 603.2783.
2.3.20 Spiro [2'.16]-1'-methyl-2'-(5’’’-bromo-indolin-2-one)-4'-(4’’-chlorophenyl)-tetrahydro-
1H-pyrrolo-dehydroandrosterone (3t)
o
1
White solid, yield: 76%, m. p. 203.4-207.9 C; H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H, NH),
7.46 – 7.32 (m, 3H, ArH), 7.28 (d, J = 8.1 Hz, 2H, ArH), 7.24 (d, J = 1.9 Hz, 1H, ArH), 6.75 (d, J =
8.2 Hz, 1H, ArH), 5.19 (d, J = 4.6 Hz, 1H, 6-H), 4.04 – 3.89 (m, 1H, 12’-H), 3.83 (t, J = 9.4 Hz, 1H,
11’-H), 3.62 – 3.41 (m, 2H, 11’-H and 3α-H), 2.15 (s, 3H, N-CH3), 2.42 (brs, 1H), 2.30 – 2.09 (m,
2H), 2.01 (dd, J = 13.9, 4.3 Hz, 1H), 1.94 – 1.78 (m, 2H), 1.78 – 1.67 (m, 2H), 1.47 (dd, J = 26.0,
12.5 Hz, 2H), 1.34 (d, J = 9.9 Hz, 2H), 1.02 – 0.89 (m, 2H), 0.87 (s, 3H, 19-CH3), 0.71 – 0.60 (m,
1H), 0.58 (s, 3H, 18-CH3), 0.51-0.38 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 178.42, 141.17,
141.12, 137.89, 132.94, 132.37, 131.78, 131.57, 129.49, 128.68, 120.54, 115.58, 110.90, 78.05,
71.57, 67.74, 51.01, 50.26, 48.27, 47.17, 42.17, 36.95, 36.56, 34.89, 33.12, 31.51, 31.11, 30.65,
30.29, 19.26, 14.64. HRMS (ESI): m/z calcd for C36H41BrClN2O3 (M+H)+, 663.1989; found,
663.1990.
2.4. General procedure for the synthesis of phenyl-linked 16-methylene-17-ketosteroid dimer (5)
A mixture of dehydroepiandrosterone (DHEA, 1) (2.0 mmol), isophthalaldehyde (1.1 mmol) and
KF/Al2O3 (1.0 mmol) in EtOH (20 mL) was heated under reflux for about 1 h. After completion of
the reaction as evident from TLC (petroleum ether/ethyl acetate = 3/1), the slurry was filtered and
the residue was washed thoroughly with CH2Cl2. The filtrate was condensed under reduced pressure,
and the solid obtained was crystallized from EtOH to yield the phenyl linked
o
1
16-methylene-17-ketosteroid dimer 5. White solid, yield 90%, mp 222.9-224.0 C. H NMR (400
MHz, CDCl3): δ 7.72 (s, 1H, Ar-H), 7.55 (d, J = 7.8 Hz, 2H, Ar-H), 7.52–7.48 (m, 1H, Ar-H), 7.46
(s, 2H, Ar-CH=), 5.41 (d, J = 4.8 Hz, 2H, 6-H), 3.61–3.50 (m, 2H, 3α-H), 2.92 (dd, J = 16.0, 5.6 Hz,
2H), 2.55 – 2.42 (m, 2H), 2.40 – 2.26 (m, 4H), 2.27 – 2.16 (m, 2H), 2.01 (d, J = 12.5 Hz, 2H), 1.95
– 1.79 (m, 6H), 1.79 – 1.64 (m, 4H), 1.64 – 1.48 (m, 4H), 1.49 – 1.34 (m, 4H), 1.18 – 1.04 (m, 4H),
1.10 (s, 6H, 19-CH3), 1.01 (s, 6H, 18-CH3); 13C NMR (100 MHz, CDCl3): δ 209.53, 141.19, 136.72,
13
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136.08, 132.43, 131.90, 130.84, 129.08, 120.75, 71.59, 50.32, 49.78, 47.39, 42.22, 37.14, 36.76,
31.59, 31.56, 31.27, 31.04, 29.51, 20.40, 19.46, 14.24; HRMS (ESI): m/z cacld for C46H58O4Na
(M+Na) +, 697.4233; found, 697.4235.
2.5. General procedure for the synthesis of dimeric steroidal spiro-oxindoles (6a-c)
A mixture of 16-methylene-17-ketosteroid dimer (5, 0.5 mmol), (substituted) isatin (2.5 mmol)
and sarcosine (2.5 mmol) in methanol/1, 4-dioxane mixture (v/v = 1/1, 20 mL) was refluxed for
about 5h. After completion of the reaction as evident from TLC (petroleum ether/ethyl acetate=2/1),
the mixture was evaporated and purified by column chromatography on silica gel using petroleum
ether/ethyl acetate = 2/1(v/v) as the eluent to obtain the pure products 6a-c.
2.5.1. Steroid dimer 6a
o
1
White solid, yield: 73%, m. p. 233.2-235.9 C; H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H, NH),
7.88 – 7.59 (m, 1H, ArH), 7.46 (s, 1H, ArCH=), 7.36 (m, 3H, ArH), 7.23 (t, J = 7.6 Hz, 1H, ArH),
7.12 (d, J = 7.4 Hz, 1H, ArH), 7.02 (t, J = 7.5 Hz, 1H, ArH), 6.83 (d, J = 7.7 Hz, 1H, ArH), 5.40 (s,
1H, 6-H), 5.20 (d, J = 4.1 Hz, 1H, 6-H), 4.11 – 4.00 (m, 1H, 12’-H), 3.93 (t, J = 9.5 Hz, 1H, 11’-H),
3.51 (m, 3H, 11’-H and 3α-H), 2.17 (s, 3H, N-CH3), 2.86 (dd, J = 16.1, 6.0 Hz, 1H), 2.63 (d, J = 6.9
Hz, 1H), 2.37 – 2.19 (m, 4H), 2.06 – 1.94 (m, 3H), 1.87-1.83 (m, 5H), 1.73 – 1.47 (m, 7H), 1.47 –
1.30 (m, 7H), 1.08 (s, 3H, 19-CH3), 1.00 (s, 3H, 19-CH3), 0.99 – 0.91 (m, 2H), 0.83 (s, 3H, 18-CH3),
0.81 – 0.75 (m, 1H), 0.76 – 0.64 (m, 1H), 0.54 (s, 3H, 18-CH3), 0.48 – 0.37 (m, 1H), 0.32 (td, J =
12.7, 4.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 209.88, 178.46, 141.98, 141.38, 141.25, 140.11,
136.32, 135.88, 133.13, 131.42, 129.48, 128.84, 128.75, 127.33, 122.75, 120.83, 120.52, 109.34,
78.04, 71.57, 71.48, 67.74, 60.51, 51.61, 50.36, 50.21, 50.06, 47.98, 47.45, 47.04, 42.25, 42.13,
38.97, 37.13, 36.93, 36.77, 36.53, 34.84, 33.40, 31.59, 31.44, 31.22, 30.81, 30.26, 29.69, 29.66,
29.52, 22.66, 20.44, 19.84, 19.47, 19.22, 19.18, 14.64, 14.34. HRMS (ESI): m/z calcd for
C56H69N2O5 (M+H)+, 849.5206; found, 849.5208.
2.5.2. Steroid dimer 6b
o
1
White solid, yield: 71%, m. p. 240.3-242.3 C; H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H, NH),
7.73 (s, 1H, ArCH=), 7.52 – 7.29 (m, 4H, ArH), 7.04 – 6.86 (m, 2H, ArH), 6.78 (dd, J = 8.4, 4.2 Hz,
1H, ArH), 5.39 (s, 1H, 6-H), 5.20 (d, J = 3.9 Hz, 1H, 6-H), 4.08 – 3.98 (m, 1H, 12’-H), 3.90 (t, J =
9.4 Hz, 1H, 11’-H), 3.65 – 3.37 (m, 3H, 11’-H and 3α-H), 2.18 (s, 3H, N-CH3), 2.86 (dd, J = 16.0,
14
Page 14 of 40

5.8 Hz, 1H), 2.68 – 2.48 (m, 1H), 2.40 – 2.20 (m, 5H), 2.15 – 2.07 (m, 2H), 2.00 – 1.83 (m, 8H),
1.70 (d, J = 13.0 Hz, 4H), 1.64 – 1.48 (m, 3H), 1.47 – 1.31 (m, 6H), 1.08 (s, 3H, 19-CH3), 1.00 (s,
3H, 19-CH3), 0.84 (s, 3H, 18-CH3), 0.81 – 0.75 (m, 1H), 0.75 – 0.62 (m, 1H), 0.56 (s, 3H, 18-CH3),
0.44 (ddd, J = 24.8, 12.1, 6.6 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ 209.90, 178.37, 160.10,
157.70, 141.39, 141.32, 139.89, 137.91, 136.33, 135.92, 133.11, 131.41, 129.51, 129.38, 129.30,
128.79, 120.82, 120.43, 116.98, 116.72, 116.05, 115.82, 109.88, 109.80, 78.18, 71.58, 71.50, 67.90,
51.69, 50.34, 50.28, 50.03, 48.30, 47.45, 47.14, 42.25, 42.11, 38.97, 38.74, 37.12, 36.93, 36.76,
36.54, 34.85, 34.12, 33.31, 31.59, 31.46, 31.22, 30.96, 30.27, 29.66, 29.49, 27.95, 22.96, 22.66,
20.44, 20.15, 19.85, 19.48, 19.23, 19.17, 14.58, 14.41, 14.33, 14.16, 14.11, 11.40. HRMS (ESI): m/z
calcd for C56H68FN2O5 (M+H)+, 867.5112; found, 867.5114.
2.5.3. Steroid dimer 6c
White solid, yield: 68%, m. p. 233.5-235.9 oC; 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 48.3 Hz,
1H, NH), 7.69 (s, 1H, ArCH=), 7.55 – 7.32 (m, 4H, ArH), 7.32 – 7.20 (m, 1H, ArH), 7.02 (dt, J =
15.6, 7.6 Hz, 2H, ArH), 5.42 (s, 1H, 6-H), 5.22 (s, 1H, 6-H), 4.11 – 3.98 (m, 1H, 12’-H), 3.92 (t, J =
9.5 Hz, 1H, 11’-H), 3.66 – 3.42 (m, 3H, 11’-H and 3α-H), 2.19 (s, 3H, N-CH3), 2.87 (dd, J = 16.1,
6.0 Hz, 1H), 2.60 (dd, J = 27.3, 13.8 Hz, 1H), 2.42 – 2.21 (m, 5H), 2.12 (dd, J = 14.2, 4.3 Hz, 2H),
1.95 (dd, J = 41.1, 11.4 Hz, 9H), 1.75 – 1.52 (m, 6H), 1.40 – 1.30 (m, 4H), 1.10 (s, 3H, 19-CH3),
1.02 (s, 3H, 19-CH3), 1.00 – 0.91 (m, 3H), 0.85 (s, 3H, 18-CH3), 0.71 – 0.57 (m, 1H), 0.55 (s, 3H,
18-CH3), 0.48 (td, J = 11.7, 5.0 Hz, 1H), 0.33 (td, J = 12.6, 4.2 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 209.82, 177.63, 141.41, 141.06, 139.78, 139.54, 136.36, 135.94, 133.02, 131.35, 129.51,
129.33, 128.93, 128.84, 127.07, 123.56, 120.75, 120.47, 114.43, 79.07, 71.58, 71.38, 67.95, 60.48,
51.78, 50.34, 50.17, 49.99, 48.29, 47.43, 47.09, 42.25, 42.08, 37.14, 36.89, 36.79, 36.59, 34.91,
33.36, 31.61, 31.56, 31.39, 31.29, 31.23, 31.17, 30.89, 30.34, 29.50, 26.91, 22.66, 20.44, 19.79,
19.50, 19.22, 14.57, 14.31, 14.12. HRMS (ESI): m/z calcd for C56H68ClN2O5 (M+H)+, 883.4817;
found, 883.4816.
2.6. 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
o
and 95% air at 37 C. Cancer cells were maintained in RPMI1640 medium. All cell lines were
15
Page 15 of 40

purchased from the China Center for Type Culture Collection (CCTCC, Shanghai, China). For
pharmacological investigations, 10 mM stock solutions of the tested compounds 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.
2.7. Antiproliferative 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 concentrations. The cells were incubated
for another 72 h. Then, 20 µL of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) solution (5 mg/mL) was added to all wells and incubated for 4 h at 37 °C. Discarded the
suspension and added 150 µL 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 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.
2.8. Analysis of cellular apoptosis
MGC-803 cells were plated in 6-well plates (5.0 × 106 cells/mL) and incubated at 37 °C for 12 or
24 h. Exponentially growing cells were then incubated for 12 or 24 h with complete medium (blank)
or with the compound 3n. 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.
2.9. 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 compounds for 12 or 24 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, Dickinson). Data obtained from the
flow cytometer was analyzed using the FlowJo software (Tree Star, Inc., Ashland, OR, USA).
3. Results and discussion
16
Page 16 of 40

3.1. Chemistry
As shown in Fig. 2, Our approach involved the 1, 3-dipolar cycloaddition between
(E)-3β-hydroxy-5-en-16-arylidene-17-ketosteroids (2) and azomethine ylides generated in situ from
substituted isatins and sarcosine. Initially, (E)-3β-hydroxy-5-en-16-arylidene-17-ketosteroids were
prepared by the KF/Al2O3-catalyzed Claisen-Schmidt condensation of dehydroepiandrosterone
(DHEA, 1) with aromatic aldehydes in excellent yields following the procedure previously reported
by our group [31]. Subsequent 1, 3-dipolar cycloaddition of azomethine ylides, which were
generated from the decarboxlative condensation of isatins and sarcosine, with 2 afforded novel
steroidal spiro-pyrrolidinyl oxindoles 3 (Scheme 1). Apart from the expected products, some
uncharacterisable impurities were also generated in this reaction.
Fig. 2 and Scheme 1
Solvent-optimization for this cycloaddition was investigated by the choosing the model
three-component reaction between 16-(4-methylsulfonylbenzylidene)-17-ketosteroid (2a, 1 mmol),
isatin (1 mmol) and sarcosine (1 mmol) in toluene, methanol, methanol/H2O mixture (v/v = 1/1),
acetonitrile, 1, 4-dioxane, methanol/1, 4-dioxane mixture (v/v = 1/1) under reflux. As can be seen
from Table 1, the best result was obtained by refluxing the reaction mixture in methanol/1,
4-dioxane for 5h to furnish steroidal spiro-pyrrolidinyl oxindole 3a in 75% yield (Table 1, entry 6),
12%, 31% and 15% higher than those in 1, 4-dioxane, toluene and acetonitrile, respectively (Table
1, entries 3-5). This could be ascribed to the diminished stabilization of the polar transition states
involved in this reaction in 1, 4-dioxane, toluene and acetonitrile. The yields were very low when
this reaction was performed in methanol or methanol/H2O mixture due to the poor solubility of
17-ketosteroid 2a (Table 1, entries 1-2). The reaction when performed with 2 moles of sarcosine
and 1.5 moles isatin per 1 mole of 17-ketosteroid 2a afforded a remarkably enhanced 89% yield
(Table 1, entry 7). The optimum yield was obtained in methanol/1, 4-dioxane when the ratio of
17-ketosteroid, isatin and sarcosine was 1: 1.5: 2.0. Having established the optimal condition, we
further investigated the scope and reproducibility of this methodology by applying the same
reaction condition to other substrates.
Table 1
All the synthesized compounds were fully characterized by 1H, 13C NMR and high-resolution mass
1
spectra as described for 3a. The H NMR spectrum of 3a has four singlets at 3.10, 2.18, 0.83 and
0.53 ppm for 17’-CH3, N-CH3 of the pyrrolidine ring, 19-CH3 and 18-CH3, respectively. The
17
Page 17 of 40

multiplet at 3.43-3.51 ppm is assigned to the 3α-H of steroid A-ring. The hydrogens of the N-CH3
of the pyrrolidine ring have the HMBC correlations with a 11’-CH2 methylenic carbon and the
quaternary carbon at 3’-position (78.08 ppm), which also shows the HMBC correlation with the NH
proton of the oxindole ring appears at 8.59 ppm (Fig. 3). From the HMBC and HSQC correlations
of 11’-CH2, the triplets at 3.90-3.95 ppm (J = 9.4 Hz) and 3.53-3.57 ppm (J = 8.4 Hz) are assigned
to 11’-CH2 with its carbon signal at 60.56 ppm. The multiplicity of H-12’ and its H-H COSY
correlation with the 11’-CH2 hydrogens and its HMBCs with C-16, C-13’ and C-12’ enables the
assignment of the former to the multiplet at 4.09-4.15 ppm. The hydrogen attached to C-6 appears
as a singlet at 5.11 ppm and the aromatic hydrogens appears as doublets and multiplets at 6.85-6.94
ppm. Associated with DEPT135 spectrum, the quaternary carbon signals of C-16 and C-3’ appears
at 67.45 and 78.08 ppm. These signal assignments are consistent with the HMBCs and H-H COSY
correlations depicted in Fig. 3. No trace of the other possible regioisomer 4 was observed (Scheme
2). The benzylic proton (H-12’) appears as a multiplet at 4.09-4.15 ppm, which clearly confirms the
regiochemistry of the cycloaddition reaction. If other regioisomer 4 was formed, the benzylic proton
1
would appear as a singlet in the H NMR spectrum (Scheme 2). The presence of a molecular ion
peak at m/z = 629.3049 ([M+H]+) in the mass spectrum (calcd. 629.3043) further confirms the
structure of 3a.
Fig. 3
The mechanism proposed to rationalize the formation of the novel steroidal spiro-pyrrolidinyl
oxindoles is summarized in Scheme 2. Decarboxylative condensation of the isatin with sarcosine
gives the azomethine ylide, which then undergoes 1, 3-dipolar cycloaddition reaction with the
dipolarophile 2 in a regioselective manner. The formation of other possible regioisomer was not
observed. 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 2.
The steroidal spiro-pyrrolidinyl oxindoles were also formed with complete stereoselectivity,
affording one diastereomer exclusively, despite the presence of three newly formed stereocenters in
the product. The carbonyl group linked to the steroid ring system and the amide carbonyl are in
trans-relationship with respect to C2-C3 bond of the pyrrolidine ring system, this is presumably
ascribable to 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 pyrrolidine ring adjacent to the spiro carbon of 3 is also the trans-relationship to the
18
Page 18 of 40

carbonyl of the steroid skelton with respect to C3-C4 bond of the pyrrolidine ring system due to the
E-configuration of exocyclic double bond at the 16-position of 17-ketosteroids 2. It should be noted
that three stereocenters (two quaternary carbon centers), two C-C bonds and one C-N bond were
formed in one-pot procedure under catalyst-free condition. This complete regioselectivity and
stereoselectivity was also observed by other groups [33-37].
Scheme 2
Natural products such as cephalostatins, ritterazines and Crellastatins are classical dimeric steroids
with potent cytotoxicity [38, 39]. However, the natural supply of dimeric steroids with any direct
pharmacological action is extremely limited, the synthesis of natural steroid dimers and their
analogs has drawn wide attention [40, 41]. Considering this, we also synthesized three steroid
dimers with dispiropyrrolidine skeleton (Scheme 3). The protocol involved the condensation
reaction of dehydroepiandrosterone (DHEA, 1) with isophthalaldehyde via Claisen-Schmidt
condensation catalyzed by KF/Al2O3 in ethanol, affording a novel phenyl-linked ketosteroid dimer
5 in 90% yield, followed by the [3+2] cycloaddition with substituted isatin and sarcosine and finally
gave compounds 6a-c. Unexpectedly, compounds 7 were not obtained even 5 equivalents of isatin
and sarcosine were used probably due to the steric hindrance.
Scheme 3
3.2. Biological evaluation
3.2.1. Antiproliferative activity
The IC50 values (concentration required to inhibit tumor cell proliferation by 50%) for the
synthesized compounds against four human cancer cell lines including human gastric cancer cell
line (MGC-803), human breast cancer cell line (MCF-7), human liver cancer cell line (SMMC-7721)
and human esophageal cancer cell line (EC-109) were determined using the MTT assay. The
well-known anticancer drug 5-fluorouracil was evaluated in parallel as a positive control and the
results were listed in Table 2.
Table 2
As shown in Table 2, most of the synthesized compounds showed moderate to good
antiproliferative activities against four human cancer cell lines with the IC50 values ranging from
0.7 to 43 µM and some of them were more potent than 5-Fu. The structure-activity relationships
were probed through altering substituents in the isatin nucleus and those attached to the 4-position
of pyrrolidine. Compounds 3a-d with electron-withdrawn groups had moderate inhibitory effect
19
Page 19 of 40

towards most of the tested cancer cell lines with the IC50 values less than 28 µM. However,
compound 3a showed weak inhibition against EC109 and SMMC-7721 (IC50 > 42 µM). It should
be noted that compound 3b having a nitro substituent at the 4-position of phenyl group was more
potent than 5-Fu against EC109 and MGC803 with the IC50 values of 5.06 and 5.09 µM,
respectively. Compared to compounds 3a-d, halogenated compounds 3e-j represented similar
antiproliferative activities against EC109, SMMC-7721 and MCF-7. To MGC-803, compounds 3e-j
demonstrated improved antiproliferative activities with the IC50 values ranging from 5.21 to 10.02
µM. Among them, compounds 3e and 3h-j were more potent than 5-Fu. Particularly, compound 3g
showed the most potent inhibitory effect against SMMC-7721 (IC50 = 0.71 µM). Compared to 3k,
compounds 3l-n with electron-donating groups displayed enhanced antiproliferative activities
against EC109, MGC-803 and MCF-7, but to SMMC-7721, compound 3k was about 2.3-fold more
potent than 5-Fu (IC50 = 4.33 µM). Compounds 3q-r with heteroaryl groups (furyl and thienyl)
exhibited similar antiproliferative activities against the tested cancer cell lines with those having
substituted phenyl group. Substituents on the isatin nucleus had remarkable effect on their
antiproliferative activities toward SMMC-7721 cells. Incorporation of a fluoro or bromo substituent
at the 5-position of isatin nucleus was found to be negative for activity. Compounds 3s and 3t were
about 19- and 15-fold less potent than 3g. Steroid dimers 6a-c did not represent significant
improvement on their antiproliferative activities toward the cancer cell lines. Among them,
compound 6b had the moderate inhibition against EC109, SMMC-7721 and MCF-7 with the IC50
values of 7.01, 4.30 and 2.06 µM, respectively.
3.2.2. Apoptosis assay
Considering the moderate to good antiproliferative activities of these compounds against all tested
human cancer cell lines, compound 3n was chosen to further explore its mechanism of action. In
order to better characterize the mode of cell death induced by compound 3n, we performed a
biparametric cytofluorimetric analysis using propidium iodide (PI) and annexin-V-FITC in
MGC-803 cells. After treatment MGC-803 cells with compound 3n for 12 or 24h at different
concentrations (0, 2.5, 5.0, 10.0 µM), MGC-803 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 3n caused the remarkable early apoptosis. Specifically, after treatment for 12 h,
the early apoptosis rate was 8.7% for the control group, the early apoptosis rate increased gradually
20
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with the increase of the concentration of Compound 3n and finally amounted to 32.1% for the high
concentration group (10.0 µM) (Fig. 4A). A similar trend was also observed after treatment for 24h
and the early apoptosis rate increased significantly and stood at 65.7% for the high concentration
group (10.0 µM) (Fig. 4B). Besides, the late apoptosis rate also witnessed a slight increase. The
results showed that compound 3n markedly increased the cellular apoptosis in a concentration-and
time-dependent manner.
Figure 4A and Figure 4B
3.2.3. 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 3n,
a cell cycle progression was performed by treating MGC-803 cells at different concentrations of
compound 3n (0, 2.5, 5.0, 10 µM). Treatment MGC-803 cells for 12 h, the percentage of cells at
G2/M phase increased to 35.87% for the high concentration group (23.18% for the control group)
(Fig. 5A), whereas when treatment for 24 h, the percentage of cells at G2/M phase increased
remarkably from 22.96% to 50.9% (Fig. 5B). The results revealed that 3n caused an obvious G2/M
arrest pattern in a concentration- and time-dependent manner with a concomitant decrease of cells
in other phases of the cell cycle.
Figure 5A and Figure 5B
4. Conclusions
In summary, we have developed an efficient and catalyst-free protocol for the synthesis of novel
steroidal spiro-pyrrolidinyl oxindoles using the (E)-3β-hydroxy-5-en-16-arylidene-17-ketosteroids
as the dipolarophiles. The method involved the one-pot, three-component 1, 3-dipolar cycloaddition
of azomethine ylides generated in situ from decarboxylative condensation of isatin with 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, three of which are quaternary. Biological evaluation showed that
these synthesized steroidal spiro-pyrrolidinyl oxindoles possessed moderate to good
antiproliferative activities against a panel of cell lines and some of them were more potent than 5-Fu.
Particularly, compound 3g showed good antiproliferative activity against SMMC-7721 (IC50 = 0.71
µM). Steroid dimer 6b showed significantly improved antiproliferative activities against
21
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SMMC-7721 and MCF-7 with the IC50 values of 4.30 and 2.06 µM, respectively. Further
investigation showed that compound 3n caused the cellular early apoptosis and an increase in the
proportion of cells at G2/M phase in a concentration-and time-independent manner.
Acknowledgments
This work was supported by the Natural Science Foundation of China (No. 21372206).
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Legends
Fig. 1. Some naturally occurring and synthetic spiro-pyrrolidinyl oxindoles
Fig. 2. The strategy for the synthesis of steroidal spiro-pyrrolidinyl oxindoles
Scheme 1. Synthesis of steroidal spiro-pyrrolidinyl oxindoles (3). Reagents and conditions: (a)
Aldehydes, KF/Al2O3, EtOH, reflux; (b) 1, 4-Dioxane/MeOH (v/v=1/1), 100 oC.
Table 1. Optimization for the synthesis of 3a
Fig. 3. Selected HMBC (A and B) and H-H COSY (C) correlations of 3a (arbitrary numbering)
Scheme 2. Plausible mechanism for the formation of steroidal spiro-pyrrolidinyl oxindole 3a
Scheme 3. Protocol for the Synthesis of steroid dimers (6a-c). Reagents and conditions: (a)
Isophthalaldehyde, KF/Al2O3, EtOH, reflux; (b) Substituted isatin, sarcosine, MeOH/dioxane (v/v =
1/1), 100 oC.
Table 2. Preliminary in vitro antiproliferative activities of steroidal spiro-oxindoles against four
human cancer cell lines.
Figure 4. Apoptosis effect on human MGC-803 cell line induced by compound 3n. Apoptotic cells
were detected with Annexin V-FITC/PI double staining after incubation with compound 3n (0, 2.5,
5.0, 10.0 µM) for 12 h or 24h. (A) Incubated for 12 h; (B) incubated 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.
Figure 5. Effect of compound 3n on the cell cycle distribution of MGC-803 cells. Cells were
treated with different concentrations (0, 2.5, 5.0, 10.0 µM) for 12 h or 24 h. Then the cells were
fixed and stained with PI to analyze DNA content by flow cytometry. (A) Incubated for 12 h; (B)
incubated for 24 h. The experiments were performed three times, and a representative experiment is
shown.
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Table 1 Optimization for the synthesis of 3a
Entry
Solvent
Yield a (%)
1
2
3
4
5
6
7b
Methanol
Methanol/H2O (1:1)
Toluene
Acetonitrile
1, 4-Dioxane
13
0
44
60
63
75
89
Methanol/1, 4-dioxane (1:1)
Methanol/1, 4-dioxane (1:1)
a Isolated yield after purification by column chromatography. b The ratio of 2a, isatin and sarcosine
was 1: 1.5: 2.0.
27
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Table 1
Preliminary in vitro antiproliferative activities of steroidal spiro-oxindoles against four human cancer
cell lines.
IC50 (µM)a
EC109
Compounds Ar
R
MGC-803 SMMC-7721 MCF-7
3a
3b
3c
3d
3e
3f
4-Methylsulfonylphenyl H
42.02±1.62 17.82±1.13 101.51±2.00 18.15±1.26
4-Nitrophenyl
3-Nitrophenyl
3-Methoxylphenyl
4-Fluorophenyl
2-Fluorophenyl
4-Chlorophenyl
3-Chlorophenyl
2-Chlorophenyl
3, 4-Dichlorophenyl
Phenyl
H
5.06±0.40 5.09±0.71 14.14±0.85
12.91±0.81 9.64±0.98 13.08±0.82
24.21±1.08 12.99±1.00 16.66±0.92
14.68±0.87 6.62±0.82 18.77±0.97
22.16±1.04 10.02±1.06 6.94±0.54
10.45±0.72 7.26±0.86 0.71±0.11
11.77±0.77 5.21±0.72 14.13±0.85
9.16±0.66 6.12±0.79 12.36±0.79
9.11±0.66 5.93±0.77 9.79±0.69
23.44±1.07 14.46±1.06 4.33±0.34
12.79±0.81 11.87±1.07 10.12±0.70
3.51±0.24 4.22±0.63 14.13±0.85
13.57±0.83 5.79±0.76 6.05±0.48
13.72±1.14 8.40±0.92 19.86±1.30
20.49±1.31
25.26±1.40
27.71±1.43
18.31±1.27
11.41±1.06
14.27±1.15
14.37±1.16
17.87±1.25
18.00±1.26
23.35±1.37
14.65±1.17
37.97±1.58
10.25±1.01
11.82±1.07
ns
H
H
H
H
H
3g
3h
3i
H
H
H
3j
H
3k
3l
4-Methylphenyl
4-Isopropylphenyl
4-t-butylphenyl
1-Naphthyl
2-Pyridyl
H
H
3m
3n
3o
3p
3q
3r
3s
H
H
nsb
ns
ns
H
Furyl
H
8.95±0.65 11.10±1.05 14.30±0.85
16.49±0.92 8.00±0.90 12.46±0.79
6.78±0.83 9.79±0.99 13.24±1.12
10.32±1.01 7.58±0.88 10.62±1.03
29.76±1.47 17.03±1.23 27.73±1.44
7.01±0.85 15.38±1.19 4.30±0.63
13.74±1.14 16.81±1.23 26.56±1.42
10.61±1.08 6.92±0.35 9.78±0.99
34.11±1.53
28.02±1.45
8.09±0.91
5.54±0.74
15.28±1.18
2.06±0.31
6.86±0.84
7.54±0.70
Thienyl
H
4-Chlorophenyl
4-Chlorophenyl
-
5-F
5-Br
H
3t
6a
6b
6c
5-Fu
-
5-F
7-Cl
-
-
-
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 Not soluble in DMSO.
28
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Highlights
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Two series of novel steroidal spiro-oxindoles were efficiently synthesized
Most of these compounds exhibited broad-spectrum antiproliferative activities
Some of them were more potent than 5- Fu against four human cancer cell lines
compound 3g showed good antiproliferative activity against SMMC-7721 (IC50 = 0.71 μM)
Compound 3n showed cell cycle arrest at G2/M phase and induced early apoptosis
29
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