Efficient construction of novel D-ring modified steroidal dienamides and their cytotoxic activities

Article abstract of DOI:10.1016/j.ejmech.2013.05.035

Two series of steroidal dienamides 4a-q and 5a-f were designed, synthesized and evaluated for cytotoxic activities against five human cancer cell lines (MGC-803, EC109, PC-3, SMMC-7721 and MCF-7). The protocol developed efficiently achieved the construction of carbon ecarbon double bond and selective conversion of nitrile group into carboxamide in one-pot procedure. Besides, compounds 4a-q and 5a-f showed moderate to excellent cytotoxic activities with the IC ₅₀ values ranging from 0.1 to 40 μM and most of them were more potent than 5-fluorouracil. Particularly, four compounds 4d, 4-, 4q and 5a showed excellent selectivity against MGC-803 with the IC₅₀ values less than 1 mM. Flow cytometry analysis demonstrated that compound 4c caused the cellular early apoptosis and cell cycle arrest in G2/M phase in a concentration-independent manner.

Full text of DOI:10.1016/j.ejmech.2013.05.035

European Journal of Medicinal Chemistry 66 (2013) 171e179
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Efficient construction of novel D-ring modified steroidal dienamides
and their cytotoxic activities
Bin Yu a, Xiao-Jing Shi a, Jing-li Ren a, Xiao-Nan Sun a, Ping-Ping Qi a, Yuan Fang a,
,
Xian-Wei Ye a, Meng-Meng Wang a, Jun-Wei Wang a, En Zhang a, De-Quan Yu b
,
*
,
Hong-Min Liu a
*
a School of Pharmaceutical Sciences and New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450001, PR China
b Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of steroidal dienamides 4aeq and 5aef were designed, synthesized and evaluated for cyto-
toxic activities against five human cancer cell lines (MGC-803, EC109, PC-3, SMMC-7721 and MCF-7). The
protocol developed efficiently achieved the construction of carbonecarbon double bond and selective
conversion of nitrile group into carboxamide in one-pot procedure. Besides, compounds 4aeq and 5aef
Received 18 April 2013
Received in revised form
14 May 2013
Accepted 25 May 2013
Available online 5 June 2013
showed moderate to excellent cytotoxic activities with the IC50 values ranging from 0.1 to 40
most of them were more potent than 5-fluorouracil. Particularly, four compounds 4d, 4e, 4q and 5a
showed excellent selectivity against MGC-803 with the IC50 values less than 1 M. Flow cytometry
mM and
m
Keywords:
Dehydroepiandrosterone
Dienamides
analysis demonstrated that compound 4c caused the cellular early apoptosis and cell cycle arrest in G2/M
phase in a concentration-independent manner.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Cytotoxicity
Apoptosis
Cell cycle arrest
1. Introduction
dienamides could be used as electron-rich or electron-deficient
dienes in DielseAlder reactions effectively [11], which have
Steroids are a class of important multi-cyclic compounds that
exhibit diverse biological activities in living organisms. It is proved
that a number of biologically important properties of modified
steroids are dependent upon structural features of the steroid ring
system [1] and side chain [2]. Chemical modification of the steroid
ring system and side chain provides a way to alter the functional
groups, and numerous structureeactivity relationships have been
established by such synthetic alterations [3]. These compounds
could be used for development of new potential therapeutics, as
well as tools for probing the spatial orientation of important
binding responsive features in target macromolecules.
already been applied to the synthesis of natural alkaloids [12] and
some interesting heterocycles [13e15]. Dienamides are also key
constituents in a number of pharmaceutically relevant units [16]
and have been reported to have antioxidant, cytotoxic [17] and
insecticidal activities [18]. For example, Saku et al. reported that
5,5-diarylpentadienamides (Fig. 1) as the transient receptor po-
tential vanilloid I (TRPVI) antagonists were under further evalua-
tion for clinical treatment of neuropathic pain [19]. Recently, Chen
and co-workers reported that (E, E)-2-(benzylaminocarbonyl)-3-
styrylacrylonitrile (Fig. 1) as the Mcl-1 protein inhibitor repre-
sented a 6-fold enhancement compared to its parent structure
Dienamides are also recognized as key reactive intermediates
due to their great diversities, potential synthetic values and
commonly existence in nature [4e6]. Examples of these include
Apicularen A [7,8], Salicylihalamide A [9] (Fig. 1). Among them,
Zampanolide represents potent cytotoxicity against P388, A549,
HT29 and MEL28 cell lines (IC50 ¼ 1e5 ng/mL) [10]. Besides,
(Kd ¼ 0.16
mM) [20].
Recently, we achieved the synthesis of steroidal dienamides
from dehydroepiandrosterone via rearrangement of 2H-pyrans
[21]. In view of the therapeutic importance of dienamides and
being involved in finding new biologically active modified steroids
[22e28], we are interested in the design, synthesis and biological
evaluation of novel steroidal dienamides. Here, we further deter-
mine the scope of the steroidal dienamides synthesis and evaluate
their cytotoxic activities against human cancer cell lines in vitro and
the effects toward the cell cycle and apoptosis.
* Corresponding authors. Tel./fax: þ86 371 67781739.
E-mail addresses: dqyu@imm.ac.cn (D.-Q. Yu), liuhm@zzu.edu.cn (H.-M. Liu).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.035

172
B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
Fig. 1. Some natural occurring and synthetic pharmaceutically active dienamides.
2. Results and discussion
condensation reaction of dehydroepiandrosterone 1 with malononi-
trile was also performed in ethanol catalyzed by ammonium acetate,
affording the corresponding product in low yield (about 50%) even
with prolonged reaction time (>12 h).
2.1. Chemistry
The protocol for the synthesis of D-ring modified steroidal dien-
amides (4aeq and 5aef) was very simple and straightforward
involving the vinylogous aldol reaction of steroidal a,a-dicyanoalkene
All the synthesized compounds were fully characterized by 1H,
13C NMR and high resolution mass spectra as described for 4a. In
the 1H NMR spectra, the olefinic proton of compound 4a appeared
3 and aldehydes, and then followed by rearrangement of the 2H-py-
ran intermediate (Scheme 1). The intermediate 3 was prepared in
at
at
d
¼ 7.25 ppm as a sharp singlet, where the NH2 protons resonated
d
¼ 6.02 and 5.77 ppm as two broad singlets. The amide carbon in
high yield via Aldol condensation of 3b-acetyl dehydroepiandroster-
13C NMR signaled at
d
¼ 164.92 ppm. In the HSQC spectra, the
one 2 with malononitrile in ethanol catalyzed by ammonium acetate.
According to our previously reported method [21], compounds 4aeq
were synthesized in moderate to high yields using NaOAc/EtOH sys-
tem. 3b-Hydroxyl steroids, as the most potent LXRs activators, would
function as a hydrogen bond acceptor and serve as anchor for binding
of either oxysterol or its analogs to receptor [29,30]. In light of these,
proton of the olefinic proton (
with the carbon (
d
¼ 7.25 ppm) had a direct correlation
d
¼ 98.51 ppm), revealing that the olefinic carbon
of 4a resonated at 98.51 ppm. The presence of a molecular ion peak
at m/z ¼ 485.2802 ([M þ H]þ) in the mass spectra (calcd. 485.2804)
further confirmed the structure of 4a.
A possible mechanism for the formation of compounds 4aeq
and 5aef was shown in Scheme 2. Under basic conditions, the
3b
-hydroxyl counterparts 5aef were also designed and synthesized
in high yields without further deprotection of 3 -acetyl group by
treatment compound 3 with Na2CO3 in boiling methanol. The direct
b
steroidal
a,a-dicyanoalkene 3 was first deprotonated to form a
nucleophile that then attacked the aldehyde via vinylogous aldol
Scheme 1. Synthesis of steroidal dienamides (4aeq and 5aef). Reagents and conditions: (a) (Ac)2O, DMAP, Et3N, DCM, r.t; (b) malononitrile, NH4OAc, EtOH, reflux; (c) aldehydes,
NaOAc, EtOH, reflux; (d) aldehydes, Na2CO3, MeOH, reflux.

B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
173
Scheme 2. Proposed mechanism for the formation of steroidal dienamides.
reaction. Subsequent intramolecular nucleophilic addition and
isomerization afforded the 2H-pyran intermediate I, which further
rearranged to the steroidal dienamides. Interestingly enough, this
protocol efficiently achieved the construction of carbonecarbon
double bond and selective conversion of nitrile group into car-
boxamide with another nitrile group intact in one-pot under mild
condition, which demonstrated that the formation of carboxamide
did not attribute to the conventional hydrolysis of nitrile group. It
should be noted that our final products with a conjugated dien-
amide group could be allowed to further modification into other
novel steroidal derivatives [13e15].
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), human prostate cancer cell line (PC-
3) and human esophageal cancer cell line (EC-109) were deter-
mined using the MTT assay. The results were listed in Table 1 and
the well-known anticancer drug 5-fluorouracil was used as positive
control.
As shown in Table 1, all the synthesized compounds showed
moderate to excellent cytotoxic activities against five human cancer
cell lines with the IC50 values ranging from 0.1 to 40
them were more potent than 5-fluorouracil. 3 -Hydroxyl steroidal
dienamides 5aef and their 3 -acetyl counterparts (compounds 4e,
mM and most of
b
However, different from our results, Elmegeed and coauthors
reported that they obtained A-ring fused steroidal 2H-pyrans, not
the steroidal dienamides via the intramolecular cyclization under
the same condition [31].
b
4m, 4f, 4o, 4p and 4q) had the similar cytotoxic activities against
EC109, MCF-7 and PC-3. However, compound 4e was about 9-fold
more potent than 5a against MGC-803 with the IC50 values of
0.58 and 5.13
less potent than 5f (6.34
showed excellent inhibitory effect against SMMC-7721
(IC50 ¼ 1.90 M), which was about 8- and 5-fold more potent
than 4m (IC50 ¼ 15.44 M) and 5-fluorouracil (IC50 ¼ 9.78 M),
respectively. Compounds 4j and 4k with heteroaryl groups (furyl
m
M, respectively. Compound 4q was about 3.5-fold
2.2. Biological evaluation
m
M versus 1.83 M). Compound 5b
m
2.2.1. Cytotoxic activity
The IC50 values (concentration required to inhibit tumor cell
proliferation by 50%) for the synthesized compounds against five
m
m
m
Table 1
Primary in vitro cytotoxic activities of the steroidal dienamides against five human cancer cell lines.
No
Ar
IC50
(
m
M)a
EC109
MGC-803
MCF-7
PC-3
SMMC-7721
4a
4b
4c
4d
4e
4f
4g
4h
4i
Phenyl
11.72 ꢀ 1.40
5.24 ꢀ 0.84
5.56 ꢀ 0.09
8.97 ꢀ 0.84
11.86 ꢀ 1.88
7.04 ꢀ 0.80
ndb
3.05 ꢀ 1.73
10.03 ꢀ 1.37
39.35 ꢀ 0.57
20.33 ꢀ 0.09
5.30 ꢀ 0.70
15.57 ꢀ 1.78
12.03 ꢀ 1.56
4.37 ꢀ 0.69
4.78 ꢀ 0.25
5.28 ꢀ 1.04
13.16 ꢀ 1.67
14.59 ꢀ 1.80
9.27 ꢀ 1.01
8.61 ꢀ 1.26
8.69 ꢀ 0.93
8.72 ꢀ 1.25
10.61 ꢀ 1.08
3.10 ꢀ 0.36
7.38 ꢀ 1.10
1.54
0.11
0.58
7.47 ꢀ 1.15
3.98 ꢀ 0.55
3.85 ꢀ 0.58
8.01 ꢀ 1.19
6.27 ꢀ 1.01
8.36 ꢀ 1.23
22.40 ꢀ 2.08
4.77 ꢀ 0.76
15.35 ꢀ 1.72
39.37 ꢀ 2.68
22.79 ꢀ 2.14
2.95 ꢀ 0.39
4.66 ꢀ 0.73
6.35 ꢀ 1.01
2.21 ꢀ 0.16
3.67 ꢀ 0.51
5.23 ꢀ 0.83
5.34 ꢀ 0.85
6.58 ꢀ 1.13
5.57 ꢀ 0.20
2.41 ꢀ 0.18
5.92 ꢀ 0.07
6.54 ꢀ 0.01
7.54 ꢀ 0.70
12.75 ꢀ 1.63
24.26 ꢀ 2.04
11.53 ꢀ 1.49
11.27 ꢀ 1.48
24.31 ꢀ 2.12
12.95 ꢀ 1.60
ndb
8.48 ꢀ 0.16
13.46 ꢀ 0.25
23.44 ꢀ 2.20
20.45 ꢀ 2.04
26.78 ꢀ 2.09
11.36 ꢀ 1.51
22.93 ꢀ 2.12
8.18 ꢀ 1.19
9.17 ꢀ 1.32
11.74 ꢀ 1.44
16.94 ꢀ 1.90
13.29 ꢀ 1.71
13.95 ꢀ 1.73
8.84 ꢀ 1.28
14.64 ꢀ 1.80
12.23 ꢀ 1.53
24.76 ꢀ 3.44
5.03 ꢀ 0.80
5.04 ꢀ 0.59
3.90 ꢀ 0.59
7.12 ꢀ 1.09
18.38 ꢀ 1.89
11.53 ꢀ 1.50
ndb
5.66 ꢀ 0.90
9.30 ꢀ 1.31
22.85 ꢀ 2.18
20.39 ꢀ 2.04
16.48 ꢀ 1.18
15.44 ꢀ 1.77
18.55 ꢀ 1.94
2.55 ꢀ 0.27
5.90 ꢀ 0.94
2.82 ꢀ 0.37
3.63 ꢀ 0.54
1.90
2,4-Dichlorophenyl
4-Chlorophenyl
3-Chlorophenyl
2-Chlorophenyl
3-Methoxylphenyl
2-Cyanophenyl
3,4-Difluorophenyl
4-Isoproplphenyl
Furyl
2.19 ꢀ 0.10
ndb
1.56
1.09
0.12
0.15
4j
4k
4l
Thienyl
4-Bromophenyl
2-Fluorophenyl
4-Methylphenyl
3,4-Dichlorophenyl
4-Fluorophenyl
3,4,5-Trimethoxylphenyl
2-Chlorophenyl
2-Fluorophenyl
3-Methoxylphenyl
3,4-Dichlorophenyl
4-Fluorophenyl
3,4,5-Trimethoxylphenyl
e
2.71 ꢀ 0.33
2.99 ꢀ 0.35
8.10 ꢀ 1.22
3.20 ꢀ 0.45
3.11 ꢀ 1.38
6.34 ꢀ 0.98
5.13 ꢀ 0.82
2.69 ꢀ 0.20
3.96 ꢀ 0.58
5.11 ꢀ 0.82
5.67 ꢀ 0.90
1.83
4m
4n
40
4p
4q
5a
5b
5c
5d
5e
5f
6.09 ꢀ 0.97
3.34 ꢀ 0.46
3.34 ꢀ 0.42
7.94 ꢀ 1.18
9.78 ꢀ 0.99
5-Fu
6.92 ꢀ 0.35
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 determined.

174
B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
and thienyl) at 16-position of steroidal skeleton showed weak
antiproliferative activity against EC109, MCF-7, PC-3 and SMMC-
propidium iodide (PI) and annexin-V-FITC in MGC-803 cells. After
treatment with compound 4c for 24 h at different concentrations
7721 (IC50 > 20
mM) but excellent inhibitory effect against MGC-
(0, 2.0, 4.0, 8.0 mM), MGC-803 cells were labeled with the two dyes,
803 with the IC50 values of 0.12 and 0.15
m
M, respectively (57-
and the resulting red (PI) and green (FITC) fluorescence was
monitored by flow cytometry. As shown in Fig. 2, after treatment
for 24 h, compound 4c caused the extent of early apoptosis to in-
crease significantly from 8.9% (DMSO control) to 80.9%. The results
showed that compound 4c markedly increased the cellular
apoptosis in a concentration-independent manner.
and 45-fold more potent than 5-Fu). This excellent and selective
inhibition against MGC-803 promoted us to perform further
investigation and the results will be reported in due course. By
contrast, when substituted phenyl groups were introduced into 16-
position of the steroidal skeleton, most of these compounds (4aei
and 5aef) represented moderate to excellent inhibitory activity, the
electronic effect and the position of substituent on the phenyl
group had a remarkable effect on their cytotoxic activity. To EC109,
Compounds 4i and 4n with electron-donating groups such as 4-
isopropyl and 4-methyl on the phenyl group had the moderate
2.2.3. Cell cycle analysis
Molecules that inhibit the growth of cancer cells invariably
cause alteration of cell cycle distribution, with preferential G2/M
blockade [32]. We therefore examined the effect of different con-
centrations of compound 4c on cell cycle progression with MGC-
803 cell line (Fig. 3). A cell-cycle cytotoxicity assay was per-
formed by treating MGC-803 cells at various concentrations of
inhibitory effect (10.03 and 12.03
pound 4p with the electron-withdrawing fluorine atom on the
phenyl group showed excellent inhibition (4.78 M). Compounds
mM, respectively), while com-
m
4c, 4d and 4e with a chlorine atom at the 4-, 3-and 2-position on
compound 4c (0, 2.0, 4.0, 8.0
mM) for 24 h. Compound 4c caused an
the phenyl group represented excellent inhibitory effect against
increase in the proportion of cells in G2/M phase in
a
MGC-803 with the IC50 values of 1.54, 0.11 and 0.58
tively. A similar trend was also observed to other cancer cell lines.
m
M, respec-
concentration-dependent manner with a concomitant decrease of
cells in other phases of the cell cycle. Specifically, the percentage of
cells in G2/M phase at different concentrations was 20.41%, 33.68%,
49.43% and 56.91%, respectively.
2.2.2. Apoptosis assay
Considering the excellent cytotoxic activities of these com-
pounds against all tested human cancer cell lines, compound 4c
was chosen to further explore its mechanism of action. In order to
better characterize the mode of cell death induced by compound
4c, we performed a biparametric cytofluorimetric analysis using
3. Conclusions
In summary, two series of steroidal dienamides 4aeq and 5aef
were designed and synthesized from dehydroepiandrosterone in
Fig. 2. Apoptosis effect on human MGC-803 cell line induced by compound 4c. Apoptotic cells were detected with Annexin V/PI double staining after incubation with compound 4c
(0, 2.0, 4.0, 8.0 M) 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
m
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.

B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
175
Fig. 3. Effect of compound 4c on the cell cycle distribution of MGC-803 cells. Cells were treated with different concentrations (0, 2.0, 4.0, 8.0
mM) for 24 h. Then the cells were fixed
and stained with PI to analyze DNA content by flow cytometry. The experiments were performed three times, and a representative experiment is shown.
high yields under mild conditions. This protocol efficiently ach-
ieved the construction of carbonecarbon double bond and selective
conversion of nitrile group into carboxamide in one-pot. Besides,
compounds 4aeq and 5aef were also screened for cytotoxic
activities against five human cancer cell lines, revealing that these
compounds showed moderate to excellent cytotoxic activities with
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 spec-
trometer by electrospray ionization (ESI).
the IC50 values ranging from 0.1 to 40 mM and most of them were
4.2. Synthesis of 3-acetyl dehydroepiandrosterone 2
more potent than 5-fluorouracil. It is worth mentioning that four
compounds 4d, 4e, 4q and 5a showed excellent inhibitory activity
A mixture of dehydroepiandrosterone (4.0 mmol), acetic anhy-
dride (4.4 mmol), 4-dimethylaminopyridine (DMAP, 0.02 mmol)
and Et3N (8.0 mmol) in dichloromethane (50 mL) was stirred for
about 5 h at room temperature. After completion of the reaction as
evident from TLC, the organic phase was washed with water and
brine, dried over Na2SO4. Removal of solvent afforded compound 2
quantitatively without further purification. White solid, mp 169.4e
against MGC-803 with the IC50 values less than 1
mM. Further
investigation showed that compound 4c caused the cellular early
apoptosis and an increase in the proportion of cells in G2/M phase
in a concentration-independent manner.
4. Experimental section
170.6 ꢁC. 1H NMR (400 MHz, CDCl3,
d
, ppm):
d
5.43 (d, J ¼ 5.1 Hz,
1H), 4.63 (m, 1H), 2.06 (s, 3H), 1.07 (s, 3H), 0.91 (s, 3H). HRMS (ESI):
4.1. General
m/z calcd for C21H30NaO3 (M þ Na)þ, 353.2093; found, 353.2094.
Reagents and solvents were purchased from commercial sour-
ces and were used without further purification. Thin-layer chro-
matography (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 chro-
matography over silica gel (Qingdao Haiyang Chemical Co., 200e
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
4.3. Synthesis of the D-ring modified steroidal dicyanoalkene 3
To a solution of compound 2 (3.0 mmol) in ethanol (10 mL)
containing ammonium acetate (0.5 g), malononitrile (4.5 mmol)
was added. The reaction mixture was heated under reflux for about
3 h until compound 2 had disappeared as indicated by TLC. The
solid product formed upon cooling at room temperature was
washed with ethanol, collected by filtration to yield white solid of
compound 3, yield: 95%, mp 187.9e188.9 ꢁC. 1H NMR (400 MHz,
standard in CDCl3 or DMSO-d6. Chemical shifts are given as
d ppm
values relative to TMS (Most of the peaks due to the steroidal
CDCl3,
d, ppm):
d
5.38 (d, J ¼ 5.1 Hz, 1H), 4.74e4.45 (m, 1H), 3.04e

176
B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
2.86 (m, 1H), 2.74 (m, 1H), 2.04 (s, 3H), 1.05 (s, 6H). 13C NMR
(100 MHz, CDCl3, , ppm): 196.08, 170.50, 139.93, 121.58, 112.26,
111.16, 79.73, 73.58, 55.22, 49.34, 48.91, 37.99, 36.83, 36.56, 34.73,
33.61, 31.39, 31.37, 27.63, 23.56, 21.40, 20.78, 19.27, 16.24. HRMS
(ESI): m/z calcd for C24H30N2NaO2 (M þ Na)þ, 401.2205; found,
401.2229.
4.4.5. 3b-Acetoxyl-5-en-16-(2-chlorobenzylidene)-androstano-17-
d
d
(2-amino-1-cyano-2-oxoethylidene) (4e)
Yellow solid, yield: 81%, mp 227.4e228.0 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm):
d
7.55 (s, 1H), 7.45 (d, J ¼ 7.2 Hz, 1H), 7.38 (t,
J ¼ 6.2 Hz, 1H), 7.33e7.17 (m, 2H), 6.14 (m, 2H), 5.35 (s, 1H), 4.70e
4.50 (m, 1H), 2.03 (s, 3H), 1.10 (s, 3H), 1.05 (s, 3H). 13C NMR
(100 MHz, CDCl3, d, ppm): d 173.27, 170.54, 164.76, 140.02, 139.50,
4.4. General procedure for the synthesis of the steroidal dienamides
4aeq
134.67, 131.61, 129.84, 129.70, 129.61, 126.60, 121.59, 116.17, 99.69,
73.69, 52.37, 49.44, 47.33, 38.02, 36.77, 36.59, 34.68, 31.37, 31.25,
30.96, 29.69, 27.64, 21.41, 21.06, 19.28, 16.60. HRMS (ESI): m/z calcd
for C31H35ClN2NaO3 (M þ Na)þ, 541.2234; found, 541.2232.
To a solution of compound 3 (1.0 mmol) in ethanol, aldehydes
(1.0 mmol) and sodium acetate (2.0 mmol) were added. The reac-
tion mixture was heated under reflux for about 3e7 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 with
ethyl acetate/petroleum (1/2) as the eluent to give the corre-
sponding steroidal dienamides.
4.4.6. 3b-Acetoxyl-5-en-16-(3-methoxylbenzylidene)-androstano-
17-(2-amino-1-cyano-2-oxoethylidene) (4f)
Yellow solid, yield: 90%, mp 146.1e147.8 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm)
d
7.28 (m, 1H), 7.20 (s, 1H), 7.01 (d, J ¼ 7.6 Hz, 1H),
6.93 (s, 1H), 6.86 (d, J ¼ 8.2 Hz, 1H), 6.37 (s, 2H), 5.39 (d, J ¼ 4.3 Hz,
1H), 4.69e4.51 (m, 1H), 3.81 (s, 3H), 2.03 (s, 3H), 1.04 (s, 6H). 13C
NMR (100 MHz, CDCl3, d, ppm): d 174.67, 170.63, 165.60, 159.59,
4.4.1. 3
b
-Acetoxyl-5-en-16-benzylidene-androstano-17-(2-amino-
140.00, 137.85, 137.55, 135.31, 129.60, 122.40, 121.66, 116.36, 115.29,
114.38, 98.80, 73.75, 55.32, 52.40, 49.44, 47.02, 38.02, 36.67, 34.69,
31.33, 29.68, 27.63, 21.41, 21.06, 19.28, 16.48. HRMS (ESI): m/z calcd
for C32H38N2NaO4 (M þ Na)þ, 537.2729; found, 537.2740.
1-cyano-2-oxoethylidene) (4a)
Yellow solid, yield: 76%, mp 134.1e135.8 ꢁC. 1H NMR (400 MHz,
CDCl3,
1H), 5.40 (d, J ¼ 4.7 Hz, 1H), 4.61e4.50 (m, 1H), 2.04 (s, 3H), 1.06 (s,
3H), 1.05 (s, 3H). 13C NMR (100 MHz, CDCl3,
, ppm): 175.44,
d, ppm): d 7.55e7.29 (m, 5H), 7.25 (s, 1H), 6.02 (s, 1H), 5.77 (s,
d
d
4.4.7. 3b-Acetoxyl-5-en-16-(2-cyanobenzylidene)-androstano-17-
170.54, 164.92, 140.09, 137.42, 136.19, 135.78, 129.90, 128.90, 128.67,
121.61, 116.36, 98.51, 73.70, 52.54, 49.51, 47.13, 38.05, 36.79, 36.61,
34.73, 31.48, 31.42, 31.29, 29.70, 27.66, 21.42, 21.10, 19.31, 16.51.
HRMS (ESI): m/z calcd for C31H37N2O3 (M þ H)þ, 485.2804; found,
485.2802.
(2-amino-1-cyano-2-oxoethylidene) (4g)
Brown solid, yield: 55%, mp 211.7e212.5 ꢁC. 1H NMR (400 MHz,
CDCl3,
6.06 (s, 1H), 5.38 (d, J ¼ 5.1 Hz, 1H), 4.73e4.50 (m, 1H), 2.05 (s, 3H),
1.12 (s, 3H),1.07 (s, 3H). 13C NMR (100 MHz, CDCl3,
, ppm): 172.42,
d, ppm): d 7.77e7.52 (m, 4H), 7.47e7.36 (m, 1H), 6.27 (s, 1H),
d
d
170.55, 164.21, 142.24, 140.12, 139.78, 133.21, 132.66, 129.67, 128.77,
128.49, 121.46, 117.68, 115.83, 112.96, 100.84, 73.67, 52.28, 49.42,
47.31, 38.03, 36.77, 36.60, 34.68, 31.41, 31.26, 29.70, 27.64, 21.41,
21.03, 19.30, 16.63. HRMS (ESI): m/z calcd for C32H35N3NaO3
(M þ Na)þ, 532.2576; found, 532.2578.
4.4.2. 3b-Acetoxyl-5-en-16-(2,4-dichlorobenzylidene)-androstano-
17-(2-amino-1-cyano-2-oxo-ethylidene) (4b)
Yellow solid, yield: 87%, mp 146.5e147.5 ꢁC. 1H NMR (400 MHz,
CDCl3,
1H), 6.38 (s, 1H), 5.35 (d, J ¼ 4.7 Hz, 1H), 4.68e4.52 (m, 1H), 2.03 (s,
3H), 1.08 (s, 3H), 1.04 (s, 3H). 13C NMR (100 MHz, CDCl3,
, ppm):
172.51, 170.60, 165.08, 140.04, 135.38, 134.70, 133.19, 130.36,
d, ppm): d 7.47 (s, 1H), 7.38 (dd, 2H), 7.25 (dd, 1H), 6.55 (s,
d
4.4.8. 3b-Acetoxyl-5-en-16-(3,4-difluorobenzylidene)-androstano-
d
17-(2-amino-1-cyano-2-oxo-ethylidene) (4h)
130.16, 129.64, 127.01, 121.55, 116.08, 100.11, 73.70, 52.33, 49.41,
47.31, 38.00, 36.75, 36.57, 34.67, 31.34, 31.24, 31.03, 27.63, 21.41,
21.22, 19.27, 16.59. HRMS (ESI): m/z calcd for C31H35Cl2N2O3
(M þ H)þ, 553.2025; found, 553.2007.
Yellow solid, yield: 85%, mp 143.2e145.5 ꢁC. 1H NMR (400 MHz,
CDCl3,
(s, 2H), 5.40 (d, J ¼ 4.6 Hz, 1H), 4.60 (m, 1H), 2.04 (s, 3H), 1.05 (s, 3H),
1.04 (s, 3H). 13C NMR (100 MHz, CDCl3,
, ppm): 174.28, 170.64,
d, ppm) d 7.20 (m, 4H, ArH and AreCH], overlapped), 6.38
d
d
165.23, 151.45, 148.96, 140.04, 138.41, 133.56, 132.96, 126.54, 121.55,
118.12, 117.94, 117.62, 117.45, 116.21, 99.23, 77.40, 77.08, 76.77, 52.36,
49.43, 47.10, 38.00, 36.76, 36.57, 34.69, 31.42, 31.24, 31.21, 27.62,
21.40, 21.05, 19.27, 16.49. HRMS (ESI): m/z calcd for C31H35F2N2O3
(M þ H)þ, 521.2616; found, 521.2614.
4.4.3. 3b-Acetoxyl-5-en-16-(4-chlorobenzylidene)-androstano-17-
(2-amino-1-cyano-2-oxoethylidene) (4c)
Yellow solid, yield: 86%, mp 161.0e162.1 ꢁC. 1H NMR (400 MHz,
CDCl3, d, ppm): d 7.34 (d, 4H), 7.19 (s, 1H), 6.32e5.96 (m, 2H), 5.39
(d, J ¼ 3.9 Hz, 1H), 4.63e4.58 (m, 1H), 2.04 (s, 3H), 1.05 (s, 6H). 13
C
NMR (100 MHz, CDCl3,
d
, ppm):
d
174.96, 170.58, 164.98, 140.07,
4.4.9. 3b-Acetoxyl-5-en-16-(4-isopropylbenzylidene)-androstano-
138.03, 134.69, 134.21, 130.99, 128.89, 121.56, 116.31, 98.83, 73.69,
52.44, 49.46, 47.14, 38.02, 36.68, 34.70, 31.58, 31.07, 27.64, 21.42,
21.07, 19.29, 16.51, 15.27. HRMS (ESI): m/z calcd for C31H36ClN2O3
(M þ H)þ, 519.2414; found, 519.2411.
17-(2-amino-1-cyano-2-oxoethylidene) (4i)
Yellow solid, yield: 70%, mp 142.2e143.6 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm):
d
7.38 (d, J ¼ 8.2 Hz, 2H), 7.29e7.23 (m, 3H, ArH and
AreCH], overlapped), 6.37 (m, 2H), 5.41 (d, J ¼ 4.4 Hz,1H), 4.60 (m,
1H), 2.93 (m 1H), 2.04 (s, 3H), 1.26 (s, 3H), 1.25 (s, 3H), 1.06 (s, 3H),
4.4.4. 3
b
-Acetoxyl-5-en-16-(3-chlorobenzylidene)-androstano-17-
1.04 (s, 3H). 13C NMR (100 MHz, CDCl3,
d, ppm): d 175.07, 170.63,
(2-amino-1-cyano-2-oxoethylidene) (4d)
165.84, 150.03, 140.01, 136.52, 135.59, 133.88, 130.08, 126.80, 121.70,
116.51, 98.26, 73.76, 52.50, 49.49, 47.00, 38.03, 36.68, 34.70, 33.98,
31.36, 29.69, 27.64, 23.79, 21.42, 21.09, 19.29,16.48. HRMS (ESI): m/z
calcd for C34H43N2O3 (M þ H)þ, 527.3274; found, 527.3271.
Yellow solid, yield: 84%, mp 147.0e148.2 ꢁC. 1H NMR (400 MHz,
CDCl3,
2H), 5.40 (d, J ¼ 4.7 Hz, 1H), 4.71e4.49 (m, 1H), 2.04 (s, 3H), 1.05 (s,
6H). 13C NMR (100 MHz, CDCl3,
, ppm): 174.60, 170.51, 164.71,
d, ppm): d 7.38 (s,1H), 7.36e7.24 (m, 3H), 7.18 (s,1H), 6.04 (m,
d
d
140.02, 138.89, 137.91, 134.55, 133.83, 129.80, 129.43, 128.65, 127.81,
121.52, 116.16, 99.18, 73.64, 54.81, 52.39, 49.42, 47.13, 37.99, 36.65,
34.67, 31.51, 31.05, 29.66, 27.60, 21.37, 21.03, 19.25, 16.48. HRMS
(ESI): m/z calcd for C31H35ClN2NaO3 (M þ Na)þ, 541.2234; found,
541.2230.
4.4.10. 3b-Acetoxyl-5-en-16-furylmethylene-androstano-17-(2-
amino-1-cyano-2-oxoethylidene) (4j)
Yellow solid, yield: 86%, mp 156.8e157.5 ꢁC. 1H NMR (400 MHz,
CDCl3,
d, ppm): d 7.51 (s, 1H), 7.11 (s, 1H), 6.50 (m, 4H), 5.40 (d,
J ¼ 3.9 Hz, 1H), 4.77e4.44 (m, 1H), 2.02 (s, 3H), 1.04 (s, 3H), 1.00 (s,

B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
177
3H). 13C NMR (100 MHz, CDCl3,
d, ppm):
d
174.01, 170.64, 165.58,
d 174.35, 170.60, 164.82, 140.06, 139.36, 136.17, 132.84, 132.70,
152.49, 144.44, 139.95, 134.60, 122.23, 121.77, 116.61, 114.60, 112.49,
97.92, 77.47, 77.24, 76.99, 73.78, 52.00, 49.45, 47.80, 38.01, 36.76,
36.56, 34.73, 31.64, 31.41, 31.26, 27.63, 21.40, 21.11, 19.28, 16.40,
14.18. HRMS (ESI): m/z calcd for C29H35N2O4 (M þ H)þ, 475.2597;
found, 475.2599.
131.22, 130.56, 128.74, 121.52, 116.16, 99.43, 73.68, 52.37, 49.42,
47.19, 38.01, 36.76, 36.58, 34.69, 31.42, 31.32, 31.27, 27.63, 21.43,
21.05, 19.30, 16.53. HRMS (ESI): m/z calcd for C31H35Cl2N2O3
(M þ H)þ, 553.2025; found, 553.2023.
4.4.16. 3b-Acetoxyl-5-en-16-(4-fluorobenzylidene)-androstano-17-
4.4.11. 3
b
-Acetoxyl-5-en-16-thienylmethylene-androstano-17-(2-
(2-amino-1-cyano-2-oxoethylidene) (4p)
amino-1-cyano-2-oxoethylidene) (4k)
Yellow solid, yield: 72%, mp 138.5e138.8 ꢁC. 1H NMR (400 MHz,
Yellow solid, yield: 84%, mp 153.3e154.3 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm)
d
7.41 (dd, J ¼ 8.7, 5.5 Hz, 2H), 7.22 (s, 1H), 7.08 (t,
CDCl3,
d
, ppm):
d
7.53 (s, 1H), 7.49 (d, J ¼ 4.8 Hz, 1H), 7.20 (d,
J ¼ 8.7 Hz, 2H), 6.02 (s, 1H), 5.72 (s, 1H), 5.40 (d, J ¼ 4.9 Hz, 1H), 4.60
(m, 1H), 2.79 (dd, J ¼ 15.9, 7.4 Hz, 1H), 2.75e2.64 (m, 1H), 2.04 (s,
J ¼ 3.2 Hz, 1H), 7.10 (dd, J ¼ 4.8, 3.2 Hz, 1H), 6.46 (s, 2H), 5.42 (d,
J ¼ 3.8 Hz,1H), 4.61 (m, 1H), 2.04 (s, 3H), 1.05 (s, 3H), 1.02 (s, 3H). 13
C
3H),1.06 (s, 6H). 13C NMR (100 MHz, CDCl3,
d, ppm) d 175.20, 170.59,
NMR (100 MHz, CDCl3,
d, ppm):
d
174.43, 170.65, 165.62, 140.50,
165.20, 163.96, 161.46, 140.07, 137.00, 134.39, 132.45, 131.72, 121.58,
116.39, 115.89, 115.68, 98.51, 73.69, 52.45, 49.46, 47.13, 38.02, 36.77,
36.59, 34.69, 31.43, 31.32, 31.26, 27.64, 21.43, 21.07, 19.30, 16.51.
HRMS (ESI): m/z calcd for C31H35FN2NaO3 (M þ Na)þ, 525.2529;
found, 525.2529.
140.01, 134.64, 131.50, 129.58, 128.67, 127.79, 121.70, 116.61, 97.87,
77.48, 77.17, 76.85, 73.77, 52.13, 49.44, 47.98, 38.02, 36.76, 36.58,
34.76, 31.65, 31.44, 31.27, 27.64, 21.42, 21.10, 19.30, 16.43. HRMS
(ESI): m/z calcd for C29H35N2O3S (M þ H)þ, 491.2368; found,
491.2365.
4.4.17. 3b-Acetoxyl-5-en-16-(3,4,5-trimethoxylbenzylidene)-andros
4.4.12. 3
b
-Acetoxyl-5-en-16-(4-bromobenzylidene)-androstano-
tano-17-(2-amino-1-cyano-2-oxoethylidene) (4q)
17-(2-amino-1-cyano-2-oxoethylidene) (4l)
Yellow solid, yield: 89%, mp 150.1e150.3 ꢁC. 1H NMR
Yellow solid, yield: 80%, mp 163.9e164.1 ꢁC. 1H NMR (400 MHz,
(400 MHz, CDCl3, d, ppm) d 7.17 (s, 1H), 6.65 (s, 2H), 6.34 (s, 1H),
CDCl3,
d
, ppm)
d
7.50 (d, J ¼ 8.5 Hz, 2H), 7.33e7.22 (d, J ¼ 8.5 Hz, 2H),
6.23 (s, 1H), 5.39 (d, J ¼ 4.5 Hz, 1H), 4.74e4.51 (m, 1H), 3.86 (s,
7.18 (s, 1H), 6.16 (s, 1H), 6.08 (s, 1H), 5.39 (d, J ¼ 4.5 Hz, 1H), 4.60 (m,
9H), 2.82 (dd, J ¼ 15.8, 6.2 Hz, 1H), 2.75e2.60 (m, 1H), 2.04 (s, 3H),
1H), 2.76 (dd, J ¼ 16.8, 6.5 Hz, 1H), 2.73e2.62 (m, 1H), 2.04 (s, 3H),
1.05 (s, 3H). 13C NMR (100 MHz, CDCl3,
d, ppm) d 174.88, 170.62,
1.06 (s, 3H), 1.05 (s, 3H). 13C NMR (100 MHz, CDCl3,
d
, ppm)
d
174.88,
165.52, 153.06, 140.15, 138.90, 136.68, 135.64, 131.82, 121.49,
116.43, 107.30, 98.41, 73.70, 60.95, 56.20, 52.39, 49.42, 46.99,
38.01, 36.75, 36.59, 34.66, 31.39, 31.22, 27.62, 21.42, 21.04, 19.28,
16.50. HRMS (ESI): m/z calcd for C34H42N2NaO6 (M þ Na)þ,
597.2941; found, 597.2939.
170.59, 165.04, 140.06, 138.19, 135.07, 134.24, 131.86, 131.22, 123.08,
121.57, 116.32, 98.88, 73.69, 52.42, 49.44, 47.14, 38.02, 36.77, 36.58,
34.69, 31.42, 31.26, 27.64, 21.44, 21.07, 19.30, 16.53. HRMS (ESI): m/z
calcd for C31H35BrN2NaO3 (M þ Na)þ, 585.1729; found, 585.1725.
4.4.13. 3
b
-Acetoxyl-5-en-16-(2-fluorobenzylidene)-androstano-17-
4.5. General procedure for the synthesis of the steroidal dienamides
(2-amino-1-cyano-2-oxoethylidene) (4m)
5aef
Yellow solid, yield: 65%, mp 140.1e140.3 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm)
d
7.53e7.39 (m, 2H), 7.29 (m, 1H), 7.15 (t, J ¼ 7.5 Hz,
To a solution of compound 3 (1.0 mmol) in methanol, aldehydes
(1.0 mmol) and sodium carbonate (2.0 mmol) were added. The
reaction mixture was heated under reflux for about 3e6 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
with ethyl acetate/petroleum (1/2) as the eluent to give the cor-
responding steroidal dienamides.
1H), 7.12e6.98 (m, 1H), 6.27 (s, 2H), 5.37 (d, J ¼ 4.7 Hz, 1H), 4.68e
4.50 (m, 1H), 2.80e2.61 (m, 2H), 2.04 (s, 3H), 1.07 (s, 3H), 1.05 (s,
3H). 13C NMR (100 MHz, CDCl3,
d, ppm) d 173.85, 170.60, 165.07,
161.93, 159.43, 140.00, 139.47, 130.38, 129.59, 126.92, 124.44, 124.31,
124.00, 121.63, 116.28, 115.86, 115.64, 99.47, 73.71, 52.33, 49.44,
47.21, 38.02, 36.75, 36.58, 34.65, 31.37, 31.25, 31.20, 27.64, 21.42,
21.05, 19.29, 16.58. HRMS (ESI): m/z calcd for C31H35FN2NaO3
(M þ Na)þ, 525.2529; found, 525.2531.
4.5.1. 3b-Hydroxyl-5-en-16-(2-chlorobenzylidene)-androstano-17-
4.4.14. 3
b
-Acetoxyl-5-en-16-(4-methylbenzylidene)-androstano-
(2-amino-1-cyano-2-oxo-ethylidene) (5a)
17-(2-amino-1-cyano-2-oxoethylidene) (4n)
White solid, yield: 75%, mp 228.1e228.3 ꢁC. 1H NMR (400 MHz,
Yellow solid, yield: 61%, mp 149.6e149.7 ꢁC. 1H NMR (400 MHz,
DMSO-d6, d, ppm) d 8.20 (s, 1H), 7.99 (s, 1H), 7.89e7.57 (m, 2H), 7.52
CDCl3,
d
, ppm)
d
7.32 (d, J ¼ 8.0 Hz, 1H), 7.22 (s, 1H), 7.19 (d,
(dd, J ¼ 13.9, 7.9 Hz, 1H), 7.49e7.31 (m, 2H), 5.26 (s, 1H), 4.63 (d,
J ¼ 8.0 Hz, 1H), 6.15 (s, 1H), 6.11 (s, 1H), 5.40 (d, J ¼ 4.5 Hz, 1H), 4.60
(m, 1H), 2.81 (dd, J ¼ 15.9, 6.1 Hz, 1H), 2.76e2.63 (m, 1H), 2.36 (s,
3H), 2.04 (s, 3H), 1.05 (s, 3H), 1.04 (s, 3H). 13C NMR (100 MHz, CDCl3,
J ¼ 4.2 Hz, 1H), 3.26 (d, J ¼ 4.7 Hz, 1H), 1.03 (s, 3H), 0.96 (s, 3H). 13
C
NMR (100 MHz, DMSO-d6, d, ppm) d 168.80, 164.11, 141.74, 141.52,
134.54, 133.82, 130.67, 130.38, 130.07, 127.77, 127.02, 120.44, 118.08,
100.57, 70.39, 52.29, 49.40, 47.77, 42.59, 36.48, 31.19, 19.57, 16.70.
HRMS (ESI): m/z calcd for C29H33ClN2NaO2 (M þ Na)þ, 499.2128;
found, 499.2125.
d, ppm) d 175.47, 170.58, 165.45, 140.04, 139.24, 136.40, 135.80,
133.46, 129.95, 129.43, 121.67, 116.51, 98.12, 73.73, 52.52, 49.49,
47.08, 38.04, 36.78, 36.59, 34.70, 31.44, 31.28, 29.70, 27.65, 21.43,
21.42, 21.09, 19.30, 16.48. HRMS (ESI): m/z calcd for C32H38N2NaO3
(M þ Na)þ, 521.2780; found, 521.2782.
4.5.2. 3b-Hydroxyl-5-en-16-(2-fluorobenzylidene)-androstano-17-
(2-amino-1-cyano-2-oxo-ethylidene) (5b)
4.4.15. 3
b-Acetoxyl-5-en-16-(3,4-dichlorobenzylidene)-androstano-
Yellow solid, yield: 82%, mp 173.0e173.2 ꢁC. 1H NMR (400 MHz,
17-(2-amino-1-cyano-2-oxoethylidene) (4o)
DMSO-d6,
d
, ppm)
d
8.19 (d, J ¼ 22.8 Hz, 1H), 7.93e7.57 (m, 3H),
Yellow solid, yield: 84%, mp 159.8e162.1 ꢁC. 1H NMR (400 MHz,
7.51e7.37 (m, 1H), 7.37e7.15 (m, 2H), 5.28 (s, 1H), 4.63 (dd, J ¼ 4.3,
CDCl3,
d
, ppm)
d
7.47 (d, J ¼ 1.7 Hz, 1H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.24
2.3 Hz, 1H), 3.33e3.16 (m, 1H), 0.99 (s, 3H), 0.96 (s, 3H). 13C NMR
(dd, J ¼ 8.4, 1.8 Hz, 1H), 7.14 (s, 1H), 6.20 (s, 1H), 6.10 (s, 1H), 5.39 (d,
J ¼ 4.6 Hz,1H), 4.60 (m,1H), 2.75 (dd, J ¼ 16.1, 5.8 Hz,1H), 2.72e2.63
(100 MHz, DMSO-d6, d, ppm) d 169.37, 168.32, 165.02, 164.06,
161.70, 159.23, 159.14, 141.86, 141.77, 141.43, 140.94, 130.94, 130.26,
130.15, 125.15, 125.08, 124.58, 124.46, 124.31, 124.20, 123.45, 122.27,
(m, 1H), 2.04 (s, 3H), 1.05 (s, 6H). 13C NMR (100 MHz, CDCl3,
d, ppm)

178
B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
120.43, 118.11, 116.95, 116.14, 116.10, 115.93, 115.89, 101.64, 100.31,
70.40, 52.27, 49.58, 49.46, 47.60, 46.59, 42.60, 37.16, 36.57, 36.50,
34.83, 33.59, 31.82, 31.49, 31.35, 31.23, 31.07, 19.57, 17.01, 16.68.
HRMS (ESI): m/z calcd for C29H33FN2NaO2 (M þ Na)þ, 483.2424;
found, 483.2426.
4.6. Effect of compounds on cell viability
Exponentially growing cells were seeded at 5 ꢂ 103 cells per well
into 96-well plates. After 24 h incubation at 37 ꢁC, the culture me-
dium was removed and replaced with fresh medium containing
the candidate compounds in different concentrations. The cells
4.5.3. 3
b
-Hydroxyl-5-en-16-(3-methoxylbenzylidene)-androstano-
were incubated for another 72 h. Then, 20 mL of MTT (3-(4,5-
17-(2-amino-1-cyano-2-oxo-ethylidene) (5c)
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution
Yellow solid, yield: 82%, mp 206.4e206.6 ꢁC. 1H NMR (400 MHz,
(5 mg/mL) was added to all wells and incubated for 4 h at 37 ꢁC. The
DMSO-d6,
d
, ppm)
d
8.13 (d, J ¼ 21.9 Hz, 1H), 7.73 (s, 1H), 7.62 (s, 1H),
medium containing MTT was discarded,150 mL of dimethyl sulfoxide
7.36 (dd, J ¼ 16.4, 8.3 Hz, 1H), 7.11 (d, J ¼ 8.0 Hz, 1H), 7.07e6.87 (m,
2H), 5.30 (d, J ¼ 2.8 Hz, 1H), 4.63 (dd, J ¼ 4.5, 2.3 Hz, 1H), 3.79 (s, 3H,
(DMSO) was added to each well and the plates agitated until the dark
blue crystals (formazan) had completely dissolved; the absorbance
was measured using a microplate reader at a wavelength of 570 nm.
Each concentration was analyzed in triplicate and the experiment
was repeated three times. The average 50% inhibitory concentration
(IC50) was determined from the concentrationeresponse curves ac-
cording to the inhibition ratio for each concentration.
d
, ppm), 3.27 (d, J ¼ 4.3 Hz,1H), 2.62e2.40 (m, 3H), 0.99 (s, 3H), 0.98
(s, 3H). 13C NMR (100 MHz, DMSO-d6,
d, ppm) d 170.78, 169.18,
165.26, 164.19, 159.79, 141.83, 139.69, 139.30, 138.09, 137.81, 132.25,
131.30, 130.27, 122.33, 120.49, 118.29, 117.03, 115.08, 114.92, 114.63,
100.84, 99.70, 70.41, 55.58, 52.36, 49.57, 47.38, 46.50, 42.62, 37.14,
36.54, 34.90, 33.55, 31.83, 31.32, 21.03, 19.57, 16.96, 16.61. HRMS
(ESI): m/z calcd for C30H36N2NaO3 (M þ Na)þ, 495.2624; found,
495.2627.
4.7. Analysis of cellular apoptosis
MGC-803 cells were plated in 6-well plates (1.0 ꢂ 106 cells/well)
and incubated at 37 ꢁC for 24 h. Exponentially growing cells were
then incubated for 24 h with complete medium (blank) or with the
compound 4c. Cells were then harvested and the Annexin-V-FITC/
PI apoptosis kit (Biovision) was used according to the manufac-
turer’s instructions to detect apoptotic cells. Ten thousand events
were collected for each sample and analyzed by Accuri C6 flow
cytometer.
4.5.4. 3b-Hydroxyl-5-en-16-(3,4-dichlorobenzylidene)-androstano-
17-(2-amino-1-cyano-2-oxo-ethylidene) (5d)
White solid, yield: 79%, mp 185.5e185.7 ꢁC. 1H NMR
(400 MHz, DMSO-d6,
d
, ppm)
d
8.14 (s, 1H), 7.76 (d, J ¼ 3.2 Hz,
2H), 7.69 (dd, J ¼ 8.3, 5.8 Hz, 1H), 7.56 (dd, J ¼ 15.9, 12.8 Hz, 2H),
5.30 (s, 1H), 4.65 (s, 1H), 3.27 (s, 1H), 2.77 (dd, J ¼ 16.3, 6.5 Hz,
1H), 2.73e2.57 (m, 1H), 0.97 (s, 3H), 0.96 (s, 3H). 13C NMR
(100 MHz, DMSO-d6, d, ppm) d 170.22, 168.67, 164.99, 163.95,
141.87, 141.81, 141.47, 137.37, 137.15, 131.96, 131.89, 131.37, 131.34,
131.23, 131.10, 129.77, 129.57, 128.71, 120.41, 118.05, 101.66,
100.54, 70.38, 52.29, 52.14, 49.51, 47.39, 46.55, 42.61, 37.12, 36.57,
36.51, 33.51, 31.81, 31.27, 31.09, 20.93, 19.59, 16.93, 16.56. HRMS
(ESI): m/z calcd for C29H32Cl2N2NaO2 (M þ Na)þ, 533.1739; found,
533.1735.
4.8. Flow cytometric analysis of cell cycle distribution
For flow cytometric analysis of DNA content, 5 ꢂ 105 MGC-803
cells in exponential growth were treated with different concen-
trations of the test compounds for 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).
4.5.5. 3b-Hydroxyl-5-en-16-(4-fluorobenzylidene)-androstano-17-
(2-amino-1-cyano-2-oxoethylidene) (5e)
Yellow solid, yield: 73%, mp 165.5e165.7 ꢁC. 1H NMR (400 MHz,
CDCl3,
d
, ppm)
d
7.50 (dd, J ¼ 8.5, 5.5 Hz,1H), 7.41 (dd, J ¼ 8.5, 5.5 Hz,
1H), 7.21 (s, 1H), 7.09 (q, J ¼ 9.1 Hz, 2H), 6.14 (s,1H), 6.03 (s, 1H), 5.37
(d, J ¼ 3.3 Hz, 1H), 3.71e3.40 (m, 1H), 2.78 (dd, J ¼ 15.7, 5.7 Hz, 1H),
2.74e2.57 (m, 1H), 1.06 (s, 3H), 1.05 (s, 3H). 13C NMR (100 MHz,
Acknowledgments
CDCl3, d, ppm) d 175.20, 165.41, 164.38, 163.94, 161.44, 141.18, 141.14,
This work was supported by the National Natural Sciences
Foundations of China (No. 8112937, U1204206), China Postdoctoral
Science Foundation (No. 20100480857, 201104402) and New
Teachers’ Fund for Doctor Stations, Ministry of Education (No.
20114101120013).
138.19, 137.03, 134.29, 132.79, 132.48, 132.45, 131.77, 131.73, 131.68,
120.66, 120.61, 118.29, 116.45, 115.89, 115.68, 98.49, 96.93, 71.51,
52.89, 52.49, 49.55, 48.24, 47.15, 42.09, 42.00, 37.03, 36.49, 34.73,
33.48, 31.46, 31.33, 31.27, 29.70, 27.02, 24.98, 21.12, 19.39, 16.53,
16.16. HRMS (ESI): m/z calcd for C29H34FN2O2 (M þ H)þ, 461.2604;
found, 461.2602.
Appendix A. Supplementary data
4.5.6. 3b-Hydroxyl-5-en-16-(3,4,5-trimethoxyl benzylidene)-andros
Supplementary data related to this article can be found at
http://dx.doi:10.1016/j.ejmech.2013.05.035.
tano-17-(2-amino-1-cyano-2-oxoethylidene) (5f)
Yellow solid, yield: 86%, mp 166.1e166.4 ꢁC. 1H NMR (400 MHz,
CDCl3, d, ppm) d 7.72 and 7.19 (1H), 6.76 (s, 1H), 6.67 (s, 1H), 6.17 (d,
References
J ¼ 12.1 Hz, 1H), 5.97 (d, J ¼ 25.6 Hz, 1H), 5.38 (s, 1H), 3.91 (s, 3H),
3.88 (s, 6H), 3.55 (s, 1H), 2.84 (dd, J ¼ 15.6, 6.9 Hz, 1H), 2.72 (d,
J ¼ 6.6 Hz, 1H), 1.12 (s, 3H), 1.07 (s, 3H). 13C NMR (100 MHz, CDCl3,
[1] R. Singh, G. Panda, An overview of synthetic approaches for heterocyclic
steroids, Tetrahedron 69 (2013) 2853e2884.
[2] D. Kakati, R.K. Sarma, R. Saikia, N.C. Barua, J.C. Sarma, Rapid microwave
assisted synthesis and antimicrobial bioevaluation of novel steroidal chal-
cones, Steroids 78 (2013) 321e326.
[3] N.T. Shahidi, A review of the chemistry, biological action, and clinical appli-
cations of anabolic-androgenic steroids, Clin. Ther. 23 (2001) 1355e1390.
[4] F. Babudri, V. Fiandanese, F. Naso, A. Punzi, A new straightforward and general
approach to dienamide natural products, Tetrahedron Lett. 35 (1994) 2067e2070.
[5] K. Fukuhara, H. Urabe, Iron-catalyzed 1, 6-addition of aryl Grignard reagents
to 2, 4-dienoates and -dienamides, Tetrahedron Lett. 46 (2005) 603e606.
d, ppm) d 177.84, 175.25, 165.40, 164.33, 153.14, 153.10, 141.31,
141.28, 138.99, 137.85, 136.67, 135.75, 134.11, 131.80, 120.55, 120.49,
118.35, 116.48, 107.38, 98.28, 96.62, 71.51, 60.98, 56.23, 52.85,
52.49, 49.54, 48.22, 47.07, 42.09, 41.98, 37.03, 36.51, 34.72, 33.49,
31.45, 31.31, 31.26, 29.69, 27.02, 24.98, 21.11, 19.37, 16.53, 16.17.
HRMS (ESI): m/z calcd for C32H40N2NaO5 (M þ Na)þ, 555.2835;
found, 555.2837.

B. Yu et al. / European Journal of Medicinal Chemistry 66 (2013) 171e179
179
[6] Y. Maekawa, T. Sakaguchi, H. Tsuchikawa, S. Katsumura, New type of aza-
cyclization: thermal preparation of 4, 6-disubstituted 2-piperidinone from N-
sulfonyldienamide and its substituent effect, Tetrahedron Lett. 53 (2012)
837e841.
[20] Z. Zhang, T. Song, X. Li, Z. Wu, Y. Feng, F. Xie, C. Liu, J. Qin, H. Chen, Novel
soluble myeloid cell leukemia sequence 1 (Mcl-1) inhibitor (E,E)-2-(benzyla-
minocarbonyl)-3-styrylacrylo-, nitrile (4g) developed using a fragment-based
approach, Eur. J. Med. Chem. 59 (2013) 141e149.
[7] A. Bhattacharjee, O.R. Seguil, J.K. De Brabander, Total synthesis and biological
evaluation of Apicularen A and synthetic analogs, Tetrahedron Lett. 42 (2001)
1217e1220.
[21] B. Yu, E. Zhang, X.N. Sun, J.L. Ren, Y. Fang, B.L. Zhang, D.Q. Yu, H.M. Liu, Facile
synthesis of novel D-ring modified steroidal dienamides via rearrangement of
2H-pyrans, Steroids 78 (2013) 494e499.
[8] K.C. Nicolaou, D.W. Kim, R. Baati, Stereocontrolled total synthesis of Apicu-
laren A and its D17,18 Z isomer, Angew. Chem. Int. Ed. 41 (2002) 3701e3704.
[9] K.L. Erickson, J.A. Beutler, J.H. Cardellina II, M.R. Boyd, Salicylihalamides A and
B, novel cytotoxic macrolides from the marine Sponge Haliclona sp, J. Org.
Chem. 62 (1997) 8188e8192.
[10] X.-W. Wang, P. Li, H. Xiao, S.-Z. Zhu, G. Zhao, A simple method for synthesis of
5-CF3 substituted dienamides via rearrangement of 2H-pyran derivatives,
Tetrahedron 67 (2011) 7618e7621.
[11] H. McAlonan, J.P. Murphy, M. Nieuwenhuyzen, K. Reynolds, P.K.S. Sarma,
P.J. Stevenson, N. Thompson, 4-Phenyloxazolidin-2-ones and isoindolin-1-
ones: chiral auxiliaries for DielseAlder reactions of N-substituted 1, 3-
dienes, J. Chem. Soc. Perkin Trans. 1 1 (2002) 69e79.
[22] L.H. Huang, Y.G. Wang, G. Xu, X.H. Zhang, Y.F. Zheng, H.L. He, W.Z. Fu, H.M. Liu,
Novel 4-azasteroidal N-glycoside analogues bearing sugar-like D ring: syn-
thesis and anticancer activities, Bioorg. Med. Chem. Lett. 21 (2011) 6203e
6205.
[23] L.H. Huang, Y.F. Zheng, Y.Z. Lu, C.J. Song, Y.G. Wang, B. Yu, H.M. Liu, Synthesis
and biological evaluation of novel steroidal[17,16-d][1,2,4]triazolo[1,5-a]py-
rimidines, Steroids 77 (2012) 710e715.
[24] L.H. Huang, Y.F. Zheng, C.J. Song, Y.G. Wang, Z.Y. Xie, Y.W. Lai, Y.Z. Lu, H.M. Liu,
Synthesis of novel D-ring fused 70-aryl-androstano[17,16-d][1,2,4] triazolo
[1,5-a]pyrimidines, Steroids 77 (2012) 367e374.
[25] H.P. Li, H.M. Liu, W.Z. Ge, L.H. Huang, L.H. Shan, Synthesis of 7
a
-hydroxy-
dehydro-, epiandrosterone and
7b-hydroxy-dehydroepiandrosterone, Ste-
[12] T. Yasukouchi, K. Kanematsu, An efficient approach to the basic skeleton of the
cis-trikentrins, J. Chem. Soc. Chem. Commun. 14 (1989) 953e954.
[13] X. Liu, X. Xin, D. Xiang, R. Zhang, S. Kumar, F. Zhou, D. Dong, Facile and effi-
cient synthesis of quinolin-2(1H)-ones via cyclization of penta-2, 4-
dienamides mediated by H2SO4, Org. Biomol. Chem. 10 (2012) 5643e5646.
[14] H. Tsuchikawa, Y. Maekawa, S. Katsumura, Palladium-catalyzed asymmetric
6-endo cyclization of dienamides with substituent-driven activation, Org. Lett.
12 (2012) 2326e2329.
roids 70 (2005) 970e973.
[26] L.H. Shan, H.M. Liu, K.X. Huang, G.F. Dai, C. Cao, R.J. Dong, Synthesis of 3
11 -trihydroxy-pregn-21-benzylidene-5-en-20-one derivatives and their
cytotoxic activities, Bioorg. Med. Chem. Lett. 19 (2009) 6637e6639.
b, 7a,
a
[27] S.M. Wang, Y.B. Zhang, H.M. Liu, G.B. Yu, K.R. Wang, Mild and selective
deprotection method of acetylated steroids and diterpenes by dibutyltin ox-
ide, Steroids 72 (2007) 26e30.
[28] H.W. Xu, G.Z. Liu, S.L. Zhu, G.F. Hong, H.M. Liu, Q. Wu, Digoxin derivatives
substituted by alkylidene at the butenolide part, Steroids 75 (2010) 419e423.
[29] T.A. Spencer, D. Li, J.S. Russel, J.L. Collins, R.K. Bledsoe, T.G. Consler, L.B. Moore,
C.M. Galardi, D.D. McKee, J.T. Moore, M.A. Watson, D.J. Parks, M.H. Lambert,
[15] X. Liu, N. Zhang, J. Yang, Y. Liang, R. Zhang, D. Dong, Hydrogen bond-assisted
6pi-azaelectrocyclization of penta-2,4-dienamides: synthesis of dihydropyr-
idin-2(3H)-ones, J. Org. Chem. 78 (2013) 3323e3328.
[16] T. Diyabalanage, C.D. Amsler, J.B. McClintock, B.J. Baker, A. Palmerolide,
A cytotoxic macrolide from the antarctic tunicate Synoicum adareanum, J. Am.
Chem. Soc. 128 (2006) 5630e5631.
T.M. Willson, Pharmacophore analysis of the nuclear oxysterol receptor LXRa,
J. Med. Chem. 44 (2001) 886e897.
[30] B.A. Janowski, M.J. Grogan, S.A. Jones, G.B. Wisely, S.A. Kliewer, E.J. Corey,
D.J. Mangelsdorf, Structural requirements of ligands for the oxysterol liver X
[17] A. Mollataghi, A.H.A. Hadi, S.-C. Cheah, (e)-Kunstleramide, a new antioxidant
and cytotoxic dienamide from the bark of beilschmiedia kunstleri gamble,
Molecules 17 (2012) 4197e4208.
receptors LXRa and LXRb, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 266e271.
[31] M. El-Far, G.A. Elmegeed, E.F. Eskander, H.M. Rady, M.A. Tantawy, Novel
modified steroid derivatives of androstanolone as chemotherapeutic anti-
cancer agents, Eur. J. Med. Chem. 44 (2009) 3936e3946.
[32] M.D. Vitorovic-Todorovic, A. Eric-Nikolic, B. Kolundzija, E. Hamel, S. Ristic,
I.O. Juranic, B.J. Drakulic, (E)-4-Aryl-4-oxo-2-butenoic acid amides, chalcone-
aroylacrylic acid chimeras: design, antiproliferative activity and inhibition of
tubulin polymerization, Eur. J. Med. Chem. 62 (2013) 40e50.
ꢀ
[18] M. Abarbri, J.-L. Parrain, A. Duchene, A synthetic approach to natural dien-
amides of insecticidal interest, Synth. Commun. 28 (1998) 239e249.
[19] O. Saku, H. Ishida, E. Atsumi, Y. Sugimoto, H. Kodaira, Y. Kato, S. Shirakura,
Y. Nakasato, Discovery of novel 5,5-diarylpentadienamides as orally available
transient receptor potential vanilloid 1 (TRPV1) antagonists, J. Med. Chem. 55
(2012) 3436e3451.