Stereoselective synthesis of novel antiproliferative steroidal (E, E) dienamides through a cascade aldol/cyclization process

Article abstract of DOI:10.1016/j.steroids.2013.08.001

The stereoselective and metal-free protocol involving a cascade aldol/cyclization process for the synthesis of steroidal (E, E) dienamides from steroidal α, α-dicyanoalkene was reported. This protocol efficiently achieved the construction of CC bond and selective conversion of cyano group into carboxamide in one-pot procedure under mild condition. Further biological evaluation showed that some of these compounds had moderate to excellent cytotoxic activities against all the tested cancer cell lines and were more potent than well-known drug 5-fluorouracil. Particularly, compound 3c represented excellent inhibitory effect against MCF-7 (IC₅₀ = 0.76 μM), which was about 10-fold more potent than 5-fluorouracil.

Full text of DOI:10.1016/j.steroids.2013.08.001

Steroids 78 (2013) 1134–1140
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Stereoselective synthesis of novel antiproliferative steroidal (E, E)
dienamides through a cascade aldol/cyclization process
Bin Yu a,1, Xiao-Nan Sun a,1, Xiao-Jing Shi a, Ping-Ping Qi a, Yuan Fang a, En Zhang a, De-Quan Yu a,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:
Received 17 June 2013
Received in revised form 27 July 2013
Accepted 1 August 2013
Available online 14 August 2013
The stereoselective and metal-free protocol involving a cascade aldol/cyclization process for the synthesis
of steroidal (E, E) dienamides from steroidal -dicyanoalkene was reported. This protocol efficiently
achieved the construction of C@C bond and selective conversion of cyano group into carboxamide in
one-pot procedure under mild condition. Further biological evaluation showed that some of these com-
pounds had moderate to excellent cytotoxic activities against all the tested cancer cell lines and were
more potent than well-known drug 5-fluorouracil. Particularly, compound 3c represented excellent
a, a
Keywords:
Pregnenolone
Dienamides
Stereoselective synthesis
Cytotoxic activities
inhibitory effect against MCF-7 (IC50 = 0.76
fluorouracil.
lM), which was about 10-fold more potent than 5-
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Despite their synthetic utility and biological potential, the syn-
thetic routes available for the synthesis of dienamides are rela-
tively limited [16,17]. A number of recent approaches for the
synthesis of dienamides are mainly transition metal-based proto-
cols, which require rather severe catalytic reaction conditions
[18]. This can make these preparative methods environmentally
unfriendly and less cost effective. Moreover, heavy metals may
contaminate the final products, a problem which is frequently
encountered in the pharmaceutical industry [19]. The development
of efficient, mild and metal-free methods for the synthesis of more
versatile and new dienamides is also desirable. Recently, Zhao [20]
and Perumal [21] reported a cascade one-pot approach to a variety
Dienamides have long been recognized as key reactive interme-
diates due to their great diversities, potential synthetic values and
commonly existence in nature [1–3] and have been reported to
have antioxidant, cytotoxic [4] and insecticidal activities [5]. Acy-
clic dienamides are also key constituents in a number of biologi-
cally active natural products and pharmaceutically relevant units.
Examples of these include Apicularen A [6,7], Salicylihalamide A
[8], 5,5-diarylpentadienamides [9] and (E, E)-2-(benzylaminocar-
bonyl)-3-styrylacrylonitrile [10] (Fig. 1). For example, Saku et al.
reported that 5,5-diarylpentadiena-mides as the transient receptor
potential vanilloid I (TRPVI) antagonists are under further evalua-
tion for clinical treatment of neuropathic pain [9]. Recently, Chen
and co-workers reported that (E, E)-2-(benzylaminocarbonyl)-3-
styrylacrylonitrile as the Mcl-1 protein inhibitor represented a 6-
of dienamides from a,a-dicyanoalkenes and aldehydes via vinylo-
gous aldol reaction by the electrocyclic ring opening of the initially
formed pyran derivatives under mild basic condition with good
diastereoselectivity, respectively. In view of the therapeutic impor-
tance of dienamides and being involved in finding new biologically
active modified steroids [22–25], we are interested in the design,
synthesis and biological evaluation of novel steroidal dienamides.
Our group recently reported that dehydroepiandrosterone-based
steroidal dienamides showed excellent cytotoxic activities with
fold enhancement compared to its parent structure (Kd = 0.16 lM)
[10]. Besides, dienamides could be used as electron-rich or elec-
tron-deficient dienes in Diels–Alder reactions effectively [11],
which have already been applied to the total synthesis of natural
alkaloids [12] and some interesting heterocycles [13–15].
the IC50 values ranging from 0.1 to 40 lM and caused the cellular
early apoptosis and cell cycle arrest in G2/M phase in a concentra-
tion-independent manner (Scheme 1) [26]. To better understand
the structure-activity relationship of steroidal dienamides, herein
we report the stereoselective and metal-free synthesis of steroidal
⇑
Corresponding authors at: School of Pharmaceutical Sciences and New Drug
Research & Development Center, Zhengzhou University, Zhengzhou 450001, PR
China. Tel./fax: +86 371 67781739.
(E, E) dienamides from steroidal
a,a-dicyanoalkene through a
E-mail addresses: dqyu@imm.ac.cn (D.-Q. Yu), liuhm@zzu.edu.cn (H.-M. Liu).
These authors made equal contributions to this work.
1
cascade aldol/cyclization process, affording a series of conjugated
0039-128X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.08.001

B. Yu et al. / Steroids 78 (2013) 1134–1140
1135
heated under reflux for about 3 h until compound 1 disappeared
as indicated by TLC (petroleum ether/ethyl acetate = 9/1). The solid
product formed upon cooling at room temperature was washed
with ethanol, collected by filtration to yield compound 2. White so-
lid; mp 205.2–205.3 °C; Yield: 95%; 1H NMR (400 MHz, CDCl3) d
5.38 (d, J = 5.0 Hz, 1H), 4.70-4.53 (m, 1H), 3.03 (t, J = 8.9 Hz, 1H),
2.28 (s, 3H), 2.04 (s, 3H), 1.02 (s, 3H), 0.73 (s, 3H). 13C NMR
(100 MHz, CDCl3)
d 183.47, 170.53, 139.81, 122.05, 112.74,
112.39, 86.79, 73.74, 57.38, 56.20, 50.22, 49.74, 38.04, 37.65,
36.92, 36.63, 31.88, 31.75, 27.68, 25.45, 25.09, 21.86, 21.45,
20.72, 19.33, 14.25. HRMS (ESI): m/z calcd for C26H34N2NaO2
(M + Na)+, 429.2518; found, 429.2516.
2.3. General procedure for the synthesis of compounds 3a–k
Fig. 1. Some natural occurring and synthetic pharmaceutically active dienamides.
To a solution of compound 2 (407 mg, 1.0 mmol) in ethanol,
aldehyde (1.2 mmol) and sodium acetate (405 mg, 2.0 mmol) were
added. The reaction mixture was heated under reflux for about 3–
7 h. The solvent was removed and CH2Cl2 was added, the organic
phase was washed with water and brine, dried over Na2SO4. After
removal of the solvent, the residue was purified by silica gel
chromatography using ethyl acetate/petroleum ether (1/2) as the
eluent to give the corresponding steroidal dienamides.
steroidal dienamides in moderate yields. Besides, we also evalu-
ated their cytotoxic activities against MGC-803, MCF-7, EC109
and SMMC-7721 cancer cell lines.
2. Experimental
2.1. General remarks
2.3.1. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
phenyl-(2E, 4E)-penta-2,4-dienoic acid amide (3a)
Reagents and solvents were purchased from commercial
sources and were used without further purification. Melting points
were determined on a X-5 micromelting apparatus and are uncor-
rected. 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 d ppm values relative to
TMS (Most of the peaks due to the steroidal skeleton are merged
and could not be differentiated. Thus, d values of only those peaks
that distinguish the product and could easily be differentiated are
reported). Signal patterns are indicated as s, singlet; d, doublet; dd,
double doublet; t, triplet; m, multiplet; bs, broad singlet. Coupling
constant (J) is given in Hertz (Hz). High-resolution mass spectra
(HRMS) were recorded on a Waters Micromass Q–T of Micromass
spectrometer by electrospray ionization (ESI).
White solid; mp 109.9–110.0 °C; Yield: 75%; 1H NMR (400 MHz,
CDCl3) d 7.51-7.40 (m, 2H), 7.41-7.29 (m, 3H), 7.20 (d, J = 16.3 Hz,
1H), 6.86 (d, J = 16.3 Hz, 1H), 6.03 (s, 1H), 5.67 (s, 1H), 5.39 (d,
J = 5.0 Hz, 1H), 4.94-4.27 (m, 1H), 3.13 (t, J = 9.2 Hz, 1H), 2.03 (s,
3H), 1.02 (s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDCl3) d
170.55, 166.53, 164.01, 139.80, 137.48, 135.78, 129.38, 128.91,
127.44, 125.42, 122.24, 117.99, 106.98, 73.83, 56.56, 56.38, 49.94,
48.65, 38.14, 38.07, 36.96, 36.68, 31.99, 31.85, 27.71, 25.58,
25.12, 21.43, 20.84, 19.32, 14.44. HRMS (ESI): m/z calcd for
C
33H41N2O3 (M + H)+, 513.3117; found, 513.3118.
2.3.2. 2-Ccyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-chlorophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3b)
White solid; mp 199.0–199.1 °C; Yield: 82%; 1H NMR (400 MHz,
CDCl3) d 7.38 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H), 7.18 (d,
J = 16.3 Hz, 1H), 6.79 (d, J = 16.3 Hz, 1H), 6.13 (s, 1H), 5.99 (s, 1H),
5.39 (d, J = 4.6 Hz, 1H), 4.70-4.53 (m, 1H), 3.12 (t, J = 9.1 Hz, 1H),
2.04 (s, 3H), 1.02 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 170.52, 166.31, 164.00, 139.71, 135.74, 135.06, 134.24, 129.06,
2.2. 20-Dicyanometylidene-5-pregnen-3b-ol acetate (2)
To a solution of compound 1 (1.076 g, 3.0 mmol) in ethanol
(10 mL) containing ammonium acetate (0.5 g, 6.49 mmol), malon-
onitrile (297 mg, 4.5 mmol) was added. The reaction mixture was
Scheme 1. Design of pregnenolone-based dienamides.

1136
B. Yu et al. / Steroids 78 (2013) 1134–1140
128.50, 126.05, 122.17, 117.89, 107.09, 73.75, 56.50, 56.29, 49.84,
48.64, 38.06, 37.99, 36.89, 36.60, 31.91, 31.77, 27.64, 25.48,
25.05, 21.40, 20.77, 19.26, 14.37. HRMS (ESI): m/z calcd for C33H40-
ClN2O3 (M + H)+, 547.2727; found, 547.2729.
2.3.7. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-fluorophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3g)
White solid; mp 226.1–226.3 °C; Yield: 83%; 1H NMR (400 MHz,
CDCl3) d 7.51-7.37 (m, 2H), 7.14 (d, J = 16.3 Hz, 1H), 7.05 (t,
J = 8.6 Hz, 2H), 6.82 (d, J = 16.3 Hz, 1H), 6.08 (s, 1H), 5.78 (s, 1H),
5.39 (d, J = 4.9 Hz, 1H), 4.69-4.54 (m, 1H), 3.12 (t, J = 9.2 Hz, 1H),
2.04 (s, 3H), 1.02 (s, 3H), 0.80 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 170.80, 166.80, 164.80, 164.27, 162.31, 140.03, 136.32, 132.31,
132.28, 129.45, 129.36, 125.55, 122.47, 118.28, 116.33, 116.11,
107.08, 74.05, 56.76, 56.62, 50.16, 48.91, 38.39, 38.29, 37.19,
36.90, 32.21, 32.08, 29.94, 27.94, 25.81, 25.36, 21.68, 21.08,
19.56, 14.67. HRMS (ESI): m/z calcd for C33H40FN2O3 (M + H)+,
531.3023; found, 531.3023.
2.3.3. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(200-chlorophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3c)
Yellow solid; mp 121.2–121.5 °C; Yield: 90%; 1H NMR
(400 MHz, CDCl3) d 7.69-7.54 (m, 1H), 7.38 (dd, J = 7.2, 1.9 Hz,
1H), 7.32-7.23 (m, 2H), 7.20 (d, J = 2.7 Hz, 2H), 6.16 (s, 1H), 5.88
(s, 1H), 5.39 (d, J = 4.3 Hz, 1H), 4.60 (m, 1H), 3.15 (t, J = 9.2 Hz,
1H), 2.04 (s, 3H), 1.02 (s, 3H), 0.80 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 170.56, 167.20, 163.73, 139.78, 134.32, 134.15, 132.72,
130.01, 129.87, 128.31, 127.39, 127.21, 122.25, 117.93, 107.58,
73.83, 56.84, 56.34, 49.89, 48.79, 38.23, 38.06, 36.95, 36.68,
31.99, 31.85, 27.71, 25.54, 25.17, 21.45, 20.87, 19.33, 14.59. HRMS
(ESI): m/z calcd for C33H40ClN2O3 (M + H)+, 547.2727; found,
547.2726.
2.3.8. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(300,400-difluorophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3h)
Yellow solid; mp 122.7–122.8 °C; Yield: 85%; 1H NMR
(400 MHz, CDCl3) d 7.32-7.23 (m, 1H), 7.15 (d, J = 7.3 Hz, 2H),
7.12 (d, J = 10.3 Hz, 1H), 6.71 (d, J = 16.3 Hz, 1H), 6.09 (s, 1H),
5.72 (s, 1H), 5.39 (d, J = 4.9 Hz, 1H), 4.61 (m, 1H), 3.11 (t,
J = 9.2 Hz, 1H), 2.04 (s, 3H), 1.02 (s, 3H), 0.79 (s, 3H). 13C NMR
2.3.4. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(300-chlorophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3d)
(100 MHz, CDCl3)
d 170.57, 166.70, 163.60, 139.79, 134.49,
126.70, 123.88, 122.21, 117.88, 117.83, 117.65, 115.69, 115.51,
107.26, 73.80, 56.76, 56.35, 49.91, 48.79, 38.12, 38.06, 36.96,
36.67, 31.98, 31.83, 27.70, 25.47, 25.10, 21.45, 20.83, 19.33,
Yellow solid; mp 197.7–197.8 °C; Yield: 84%; 1H NMR
(400 MHz, CDCl3) d 7.43 (s, 1H), 7.39-7.22 (m, 3H), 7.18 (d,
J = 16.3 Hz, 1H), 6.73 (d, J = 16.3 Hz, 1H), 6.06 (s, 1H), 5.69 (s, 1H),
5.39 (d, J = 4.5 Hz, 1H), 4.73-4.48 (m, 1H), 3.11 (t, J = 9.1 Hz, 1H),
2.04 (s, 3H), 1.02 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 170.57, 166.68, 163.63, 139.79, 137.65, 135.29, 134.89, 130.12,
129.12, 127.10, 126.95, 125.57, 122.23, 117.82, 107.45, 73.81,
56.78, 56.34, 49.91, 48.78, 38.11, 38.06, 36.95, 36.67, 31.98,
31.83, 27.71, 25.48, 25.10, 21.45, 20.83, 19.33, 14.47. HRMS (ESI):
m/z calcd for C33H40ClN2O3 (M + H)+, 547.2727; found, 547.2725.
14.45. HRMS (ESI): m/z calcd for
C
33H39F2N2O3 (M + H)+,
549.2929; found, 549.2926.
2.3.9. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
300, 400,500-trimethoxylphenyl)-(2E, 4E)-penta-2,4-dienoic acid amide
(3i)
White solid; mp 201.2–201.4 °C; Yield: 80%; 1H NMR (400 MHz,
CDCl3) d 7.17 (d, J = 16.2 Hz, 1H), 6.80 (d, J = 16.2 Hz, 1H), 6.67 (s,
2H), 6.09 (s, 1H), 5.74 (s, 1H), 5.39 (d, J = 4.6 Hz, 1H), 4.70-4.52
(m, 1H), 3.89 (s, 6H), 3.86 (s, 3H), 3.14 (t, J = 9.1 Hz, 1H), 2.04 (s,
3H), 1.02 (s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, CDCl3) d
170.50, 166.43, 164.09, 153.45, 139.73, 139.43, 137.53, 131.37,
124.76, 122.18, 118.12, 106.51, 104.60, 73.75, 56.18, 49.87, 48.58,
38.11, 38.00, 36.90, 36.61, 31.92, 31.80, 29.65, 27.65, 25.56,
25.22, 25.09, 24.70, 21.39, 20.78, 19.26, 14.45. HRMS (ESI): m/z
calcd for C36H47N2O6 (M + H)+, 603.3434; found, 603.3433.
2.3.5. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-bromophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3e)
White solid; mp 176.8–177.0 °C; Yield: 79%; 1H NMR (400 MHz,
CDCl3) d 7.48 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 7.22 (d,
J = 16.3 Hz, 1H), 6.77 (d, J = 16.3 Hz, 1H), 6.06 (s, 1H), 5.71 (s, 1H),
5.39 (d, J = 4.7 Hz, 1H), 4.75-4.50 (m, 1H), 3.12 (t, J = 9.2 Hz, 1H),
2.04 (s, 3H), 1.02 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, CDCl3)
d 170.79, 166.88, 164.03, 140.03, 136.05, 135.00, 132.31, 129.06,
126.50, 123.64, 122.46, 118.21, 107.29, 74.04, 56.85, 56.61, 50.16,
48.97, 38.39, 38.30, 37.19, 36.91, 32.22, 32.08, 29.95, 28.35,
27.94, 26.64, 25.76, 25.35, 21.69, 21.08, 14.68. HRMS (ESI): m/z
calcd for C33H39BrN2NaO3 (M + Na)+, 613.2042; found, 613.2044.
2.3.10. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-furyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3j)
Yellow solid; mp 145.3–145.6 °C; Yield: 86%; 1H NMR
(400 MHz, CDCl3) d 7.45 (d, J = 1.2 Hz, 1H), 7.12 (d, J = 16.1 Hz,
1H), 6.75 (d, J = 16.1 Hz, 1H), 6.48 (d, J = 3.3 Hz, 1H), 6.46-6.35
(m, 1H), 6.03 (s, 1H), 5.82 (s, 1H), 5.39 (d, J = 4.7 Hz, 1H), 4.61 m,
1H), 3.08 (t, J = 9.2 Hz, 1H), 2.04 (s, 3H), 1.02 (s,3H), 0.81 (s, 3H).
13C NMR (100 MHz, CDCl3) d 170.80, 165.33, 164.64, 152.01,
144.45, 140.02, 125.50, 123.36, 122.49, 118.42, 113.34, 112.50,
106.55, 74.07, 56.64, 56.30, 50.17, 48.77, 38.39, 38.30, 37.19,
36.91, 32.19, 32.10, 29.94, 27.95, 25.96, 25.38, 21.68, 21.07,
19.56, 14.60. HRMS (ESI): m/z calcd for C31H39N2O4 (M + H)+,
503.2910; found, 503.2912.
2.3.6. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-nitrophenyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3f)
Brown solid; mp 138.9–139.0 °C; Yield: 86%; 1H NMR
(400 MHz, CDCl3) d 8.24 (d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.8 Hz, 2H),
7.62 (d, J = 8.8 Hz, 2H), 7.59 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 16.4 Hz,
1H), 7.15 (d, J = 16.2 Hz, 1H), 6.97 (d, J = 16.2 Hz, 1H), 6.76 (d,
J = 16.4 Hz, 1H), 6.19 (d, J = 25.9 Hz, 2H), 5.73 (d, J = 9.0 Hz, 2H),
5.38 (s, 2H), 4.61 (d, J = 5.9 Hz, 2H), 4.06 (t, J = 9.4 Hz, 1H), 3.13
(t, J = 9.1 Hz, 1H), 2.04 (s, 6H), 1.02 (s, 6H), 0.79 (s,6H). 13C NMR
2.3.11. 2-Cyano-3-[(30b,170b)-30-(acetyloxy) androst-50-en-170-yl]-5-
(400-thienyl)-(2E, 4E)-penta-2,4-dienoic acid amide (3k)
(100 MHz, CDCl3)
d 170.57, 170.37, 168.55, 167.15, 165.65,
163.28, 163.21, 163.12, 150.95, 147.97, 147.69, 144.94, 142.16,
141.56, 139.90, 139.80, 139.74, 136.60, 135.70, 133.25, 130.72,
130.27, 127.86, 127.79, 124.24, 124.20, 122.26, 122.17, 120.01,
118.18, 117.61, 117.52, 115.30, 108.02, 106.04, 73.79, 57.20,
56.31, 56.24, 50.97, 49.90, 49.85, 49.01, 48.74, 38.05, 36.97,
36.89, 36.66, 32.02, 31.94, 31.81, 29.71, 27.70, 26.18, 25.33,
25.14, 25.08, 21.44, 20.84, 20.71, 19.32, 14.56, 14.51. HRMS (ESI):
m/z calcd for C33H39N3NaO5 (M + Na)+, 580.2787; found, 580.2787.
Yellow solid; mp 124.0–124.3 °C; Yield: 85%; 1H NMR
(400 MHz, CDCl3) d 7.32 (d, J = 4.9 Hz, 1H), 7.18-7.11 (m, 1H),
7.09 (s, 2H), 7.02 (dd, J = 5.0, 3.7 Hz, 1H), 6.05 (s, 1H), 5.72 (s,
1H), 5.39 (d, J = 4.9 Hz, 1H), 4.71-4.50 (m, 1H), 3.10 (t, J = 9.2 Hz,
1H), 2.03 (s, 3H), 1.02 (s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz,
CDCl3) d 170.56, 165.29, 164.19, 141.36, 139.80, 131.06, 129.32,
128.09, 127.53, 124.52, 122.25, 118.26, 106.20, 73.83, 56.42,
56.18, 49.94, 48.55, 38.19, 38.07, 36.96, 36.68, 31.97, 31.87,

B. Yu et al. / Steroids 78 (2013) 1134–1140
1137
Scheme 2. Synthesis of steroidal dienamides (3a–k). Reagents and conditions: (a) malononitrile, NH4OAc, EtOH, reflux; (b) aldehydes, NaOAc, EtOH, reflux.
Table 1
Optimization for the synthesis of 3a.a
Entry
Solvent
Base
Yieldb (%)
1
2
3
4
5
6
7
8
EtOH
EtOH
EtOH
HOCH2CH2OH
DCM
EtOH
EtOH
EtOH
Et3N (1.0 eq)
Trace
24
67
No reaction
No reaction
30
56
82
Piperidine (1.0 eq)
Na2CO3 (1.0 eq)
Et3N (1.0 eq)
Et3N (1.0 eq)
NaOAc (1.0 eq)
NaOAc (2.0 eq)
NaOAc (5.0 eq)
a
Unless otherwise noted, the reaction was carried out with 2 (0.5 mmol) and 4-chlorobenzaldehyde (0.6 mmol) under reflux in solvent (5 mL) for 12 h.
Isolated yields.
b
27.71, 25.71, 25.16, 21.44, 20.85, 19.32, 14.40. HRMS (ESI): m/z
2 and 4-chlorobenzaldehyde was selected as a model reaction for
catalyst and solvent evaluation (Table 1). While using Et3N as the
organic base, the yield was very low or no reaction was observed
regardless of the solvent used (Table 1, entries 1, 4 and 5). When
piperidine was used, the yield increased to 24% (Table 1, entry
2). As shown in Table 1, the inorganic bases were beneficial to this
reaction. Specifically, one equivalent of NaOAc was used, the yield
was about 30% (Table 1, entry 6), when the amount of NaOAc in-
creased, the yield increased correspondingly (Table 1, entries 7
and 8). However, when Na2CO3 was used, the yield decreased to
67% probably due to the formation of the competitive dehydration
product 4a (Table 1, entry 3). The present reaction was best per-
formed with 5.0 equivalents of NaOAc in EtOH under reflux for
about 12 h.
calcd for C31H39N2O3S (M + H)+, 519.2681; found, 519.2679.
2.4. Cytotoxic activity assays
Exponentially growing cells were seeded into 96-well plates at
a concentration of 5 ꢀ 103 cells per well. After 24 h incubation at
37 °C, the culture medium was removed and replaced with fresh
medium containing the candidate compounds in different concen-
trations. The cells were incubated for another 72 h. Then, 20 lL of
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) solution (5 mg/mL) was added to all wells and incubated
for 4 h at 37 °C. Discarded the suspension and added 150 lL of di-
methyl sulfoxide (DMSO) to each well and shook the plates to dis-
solve the dark blue crystals (formazan); the absorbance was
measured using a microplate reader at a wavelength of 490 nm.
Each concentration was analyzed in triplicate and the experiment
was repeated three times. The average 50% inhibitory concentra-
tion (IC50) was determined from the dose-response curves accord-
ing to the inhibition ratio for each concentration.
Having established the optimal condition, the scope and repro-
ducibility of this methodology was explored by applying the same
reaction condition to other aldehydes and the representative re-
sults were listed in Table 2. Excellent yields were obtained irre-
spective of the position of the substituents on the phenyl ring
(3a–k). Besides, when heteroaromatic aldehydes such as furfural
and 2-thiophenecarboxaldehyde were used, the corresponding
products (3j and 3k) were also obtained in high yields. It should
be noted that the electronic nature of the substituents on the phe-
nyl ring had a remarkable effect on the yield. When 4-morpholinyl
benzaldehyde, 4-dimethylamino benzaldehyde, pyrrole-2-carbox-
aldehyde, indole-3-formaldehyde and aliphatic aldehydes were
used, the reaction gave a complex reaction mixture. Besides, no
reaction was observed with compound 2 recovered quantitatively
when ketones such as acetone and acetophenone were used due
to their low reactivity.
3. Results and discussion
3.1. Chemistry
The protocol for the synthesis of steroidal (E, E) dienamides 3a–
k was very simple and straightforward involving the tandem aldol/
cyclization process of steroidal a,a-dicyanoalkene 2 and aldehydes
(Scheme 2). The intermediate 2 was prepared in high yield via aldol
condensation of 3b-acetyl pregnenolone 1 with malononitrile in
ethanol catalyzed by ammonium acetate according to our previ-
ously reported method [25].
In order to improve the yields of final products, we evaluated
the key step of this protocol. Initially, the reaction of compound
All the synthesized compounds were fully characterized by 1H,
13C NMR and high-resolution mass spectra as described for 3a. In
the 1H NMR spectra of 3a (Fig. 2), the NH2 protons resonated at d
6.03 and 5.67 ppm as two singlets. The two olefinic protons of acy-
clic dienamide gave two doublets at d 7.20 (J = 16.3 Hz, 1H) and d

1138
B. Yu et al. / Steroids 78 (2013) 1134–1140
Table 3
Table 2
Preliminary in vitro cytotoxic activities of compounds 1, 2 and 3a–k against four
human cancer cell lines.
Synthesis of steroidal (E, E) dienamides 3a–ka.
Compounds
IC50 (l
M)a
SMMC-7721
MCF-7
EC109
MGC-803
1
2
124.05 1.62
>128
36.90 1.57
>128
11.08 1.05
>128
>128
72.80 1.86
14.40 1.16
10.15 1.01
>128
55.80 1.67
21.30 1.33
9.78 0.99
73.48 1.87
15.30 1.19
91.05 1.96
9.11 0.96
0.76 0.12
9.58 0.98
13.21 1.12
5.23 0.72
2.82 0.45
2.59 0.41
6.05 0.78
4.59 0.66
2.18 0.34
7.54 0.70
32.03 1.51
>128
24.80 1.40
61.67 1.79
80.04 1.90
34.76 1.54
20.71 1.32
25.69 1.41
68.75 1.84
31.01 1.49
26.19 1.42
19.33 1.29
32.54 1.51
27.64 1.44
24.17 1.38
6.92 0.35
3a
3b
3c
3d
3e
3f
3 g
3 h
3i
50.66 1.71
38.19 1.58
5.57 0.75
38.54 1.59
54.94 1.74
49.14 1.69
9.66 0.99
9.78 0.99
63.13 1.8
26.16 1.42
4.78 0.68
10.61 1.08
Compounds
Ar
Yieldb (%)
75
3a
3b
3c
82
90
3j
3k
5-Fu
a
Inhibitory activity was assayed by exposure for 72 h to substances and
3d
84
expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50).
Data are presented as the means SDs of three independent experiments.
mass spectrum (calcd. 513.3117) further confirmed the structure
of 3a.
3e
3f
79
86
A possible mechanism for the formation of compounds 3a-k
was proposed in Scheme 3. In the presence of base, the first step
involved facile deprotonation of vinyl malononitrile 2 to furnish
a nucleophile that attacked the aldehyde via vinylogous aldol reac-
tion and subsequent intramolecular nucleophilic addition, afford-
ing the intermediate A. Then, there were two possible pathways
to form the final products. (A) Isomerization of intermediate A
afforded intermediate B, which underwent electrolytic ring open-
ing to give the final products (path A); (B) A second deprotonation
of active hydrogen of the intermediate A catalyzed by NaOAc oc-
curred via a stable chair conformation and generated the products
(path B). The process forming the intermediate A involved the tan-
dem construction of the C–C and C–O bonds (highlighted in bold in
scheme 3). Interestingly enough, this protocol efficiently achieved
the construction of carbon–carbon double bond and selective con-
version of cyano group into carboxamide with another cyano group
intact in one-pot procedure under mild condition, which demon-
strated that the formation of carboxamide did not attribute to
the conventional hydrolysis of cyano group. The final products
were isolated as single diastereomers with (E, E) configuration. It
should be noted that our final products with a conjugated diena-
mide group could be allowed to convert into other novel steroidal
derivatives [13–15].
3g
3h
83
85
3i
80
3j
86
85
3k
a
Unless otherwise noted, the reaction was carried out with 2 (1.0 mmol), alde-
hyde (1.2 mmol) and NaOAc (5.0 mmol) under reflux in EtOH for 12 h.
b
Isolated yields.
3.2. Bioactivity
With these compounds in hand, we next performed tests of
their cytotoxic activities against human cancer cell lines and preli-
minary structure-activity relationship (SAR) study. 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 results were listed in
Table 3 and the well-known cytotoxic drug 5-fluorouracil was used
as positive control.
Fig. 2. Selected 1H NMR chemical shifts of compound 3a.
6.86 (J = 16.3 Hz, 1H), showing that the newly formed double bond
had the E-configuration. The aromatic protons appeared at d 7.52-
7.42 (m, 2H) and d 7.42-7.29 (m, 3H), respectively. The proton of
As represented in Table 3, compounds 1 and 2 showed weak
inhibitory effect against all the tested cancer cell lines with the
3a-H appeared as a multiplet in the region of d 4.70-4.52 ppm.
IC50 values ranging from 15 to 128 lM. Besides, all the compounds
The protons attached to C-18, C-19 and acetyl group occurred at
d 0.81 (s, 3H), 1.02 (s, 3H) and 2.03 (s, 3H), respectively. The pres-
ence of a molecular ion peak at m/z = 513.3118 ([M + H]+) in the
were less potent than 5-Fu against MGC-803 and SMMC-7721.
Among them, compound 3h was the most potent one with the
IC50 values of 10.05 and 19.33 lM, respectively. Compared with

B. Yu et al. / Steroids 78 (2013) 1134–1140
1139
Scheme 3. Possible mechanism for the synthesis of steroidal (E, E)-dienamides.
compounds 3b–k, compound 3a had the relatively weak inhibitory
effect against all the tested cancer cell lines with the IC50 values
more than 36 lM, indicating that aromatic ring and the substitu-
toral Science Foundation (No. 20100480857, 201104402) and
New Teachers’ Fund for Doctor Stations, Ministry of Education
(No. 20114101120013).
ents on the phenyl ring had the remarkable effect on their cyto-
toxic activities. Specifically, compounds 3b–d with chlorine atom
on the 4-, 2- and 3-position on the phenyl ring had different inhi-
bition against the tested cell lines, compound 3c was relatively
more potent than compounds 3b and 3d and showed excellent
inhibitory effect against MCF-7 and EC109 cells with the IC50 val-
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2013.
08.001.
ues of 0.76 and 5.57
than 5-Fu). Compound 3e had the moderate inhibition against
MCF-7 (IC50 = 13.21 M) and showed weak inhibitory effect
lM, respectively (about 10- and 2-fold potent
References
l
against the remaining three cancer cell lines. Compounds 3f–h
with electron withdrawing groups such as nitro and fluorine atom
on the phenyl ring had the moderate inhibitory effect against
SMMC-7721 and MGC-803. However, to MCF-7 and EC109 cell
lines, these compounds represented excellent inhibition with the
IC50 values ranging from 2.5 to 10 lM, which were also more
potent than 5-Fu. Compound 3i with electron donating group
demonstrated excellent cytotoxic activities against MCF-7
(IC50 = 6.05 lM) but weak inhibition against MGC-803, EC109
[1] Babudri F, Fiandanese V, Naso F, Punzi A. A new straightforward and general
approach to dienamide natural products. Tetrahedron Lett 1994;35:2067–70.
[2] Fukuhara K, Urabe H. Iron-catalyzed 1,6-addition of aryl Grignard reagents to
2,4-dienoates and -dienamides. Tetrahedron Lett 2005;46:603–6.
[3] Maekawa Y, Sakaguchi T, Tsuchikawa H, Katsumura S. New type of
azacyclization: thermal preparation of 4,6-disubstituted 2-piperidinone from
N-sulfonyldienamide and its substituent effect. Tetrahedron Lett
2012;53:837–41.
[4] Mollataghi A, Hadi AHA, Cheah S-C. (ꢁ)-Kunstleramide, a new antioxidant and
cytotoxic dienamide from the bark of beilschmiedia kunstleri gamble. Molecules
2012;17:4197–8.
ˇ
[5] Abarbri M, Parrain J-L, Duchene A. A synthetic approach to natural dienamides
and SMMC-7721. Compounds 3j and 3k with heteroaryl groups
(furyl and thienyl) had the similar inhibitory effect with com-
pounds 3f–h against the tested cell lines and were more potent
than 5-Fu. This excellent inhibition of these compounds, especially
compound 3c, against MCF-7 promoted us to perform further
investigation and the results will be reported in due course.
of insecticidal interest. Synth Commun 1998;28:239–49.
[6] Bhattacharjee A, Seguil OR, De Brabander JK. Total synthesis and biological
evaluation of apicularen
2001;42:1217–20.
A and synthetic analogs. Tetrahedron Lett
[7] Nicolaou KC, Kim DW, Baati R. Stereocontrolled total synthesis of Apicularen A
and its d17,18 Z isomer. Angew Chem Int Ed 2002;41:3701–4.
[8] Erickson KL, Beutler JA, Cardellina IIJH, Boyd MR. Salicylihalamides A and B,
novel cytotoxic macrolides from the marine sponge haliclona sp. J Org Chem
1997;62:8188–92.
[9] Saku O, Ishida H, Atsumi E, Sugimoto Y, Kodaira H, Kato Y, Shirakura S,
Nakasato Y. Discovery of novel 5,5-diarylpentadienamides as orally available
transient receptor potential vanilloid 1 (TRPV1) antagonists. J Med Chem
2012;55:3436–51.
4. Conclusion
In conclusion, we have reported the stereoselective and metal-
[10] Zhang Z, Song T, Li X, Wu Z, Feng Y, Xie F, Liu C, Qin J, Chen H. Novel soluble
free synthesis of steroidal (E, E) dienamides from steroidal a, a-dic-
myeloid cell leukemia sequence
1
(Mcl-1) inhibitor (E, E)-2-
yanoalkene through a cascade aldol/cyclization process. This pro-
tocol efficiently achieved the construction of C@C bond and
selective conversion of cyano group into carboxamide with another
cyano group intact in one-pot procedure under mild condition.
Further biological evaluation showed that some of these com-
pounds had moderate to excellent cytotoxic activities against all
the tested cancer cell lines and were more potent than well-known
drug 5-Fu. Particularly, compound 3c represented excellent inhib-
(benzylaminocarbonyl)-3-styryl-acrylonitrile (4g) developed using
a
fragment-based approach. Eur J Med Chem 2013;59:141–9.
[11] McAlonan H, Murphy JP, Nieuwenhuyzen M, Reynolds K, Sarma PKS,
Stevenson PJ, Thompson N. 4-Phenyloxazolidin-2-ones and isoindolin-1-
ones: chiral auxiliaries for Diels–Alder reactions of N-substituted 1,3-dienes.
J Chem Soc Perk Trans 2002;1:69–79.
[12] Yasukouchi T, Kanematsu K. An efficient approach to the basic skeleton of the
cis-trikentrins. J Chem Soc Chem Commun 1989;14:953–4.
[13] Liu X, Xin X, Xiang D, Zhang R, Kumar S, Zhou F, Dong D. Facile and efficient
synthesis of quinolin-2(1H)-ones via cyclization of penta-2,4-dienamides
mediated by H2SO4. Org Biomol Chem 2012;10:5643–56.
[14] Tsuchikawa H, Maekawa Y, Katsumura S. Palladium-catalyzed asymmetric 6-
endo cyclization of dienamides with substituent-driven activation. Org Lett
2012;12:2326–9.
[15] Liu X, Zhang N, Yang J, Liang Y, Zhang R, Dong D. Hydrogen bond-assisted 6pi-
azaelectrocyclization of penta-2,4-dienamides: synthesis of dihydropyridin-
2(3H)-ones. J Org Chem 2013;78:3323–8.
[16] Mathieson JE, Crawford JJ, Schmidtmann M, Marquez R. Fast and efficient one
step synthesis of dienamides. Org Biomol Chem 2009;7:2170–5.
[17] Shakil Hussain SM, Suleiman R, Ali BE. New conjugated dienamides via
palladium-catalyzed selective aminocarbonylation of enynes. Tetrahedron Lett
2012;53:6535–9.
itory effect against MCF-7 (IC50 = 0.76 lM), which was about 10-
fold more potent than 5-Fu. Preliminary SAR analysis showed that
the cytotoxic activities varied greatly depended on the position and
electronic nature of substituents on the phenyl ring. Further inves-
tigation of mechanism of action is under way and will be reported
in due course.
Acknowledgments
This work was supported by the National Natural Sciences
Foundations of China (No. 8112937, U1204206), China Postdoc-
[18] Tsujita H, Ura Y, Matsuki S, Wada K, Mitsudo TA, Kondo T. Regio- and
stereoselective synthesis of enamides and dienamides by ruthenium-catalyzed

1140
B. Yu et al. / Steroids 78 (2013) 1134–1140
co-oligomerization of N-vinylamides with alkenes or alkynes. Angew Chem Int
Ed 2007;46:5160–3.
[23] Huang LH, Zheng YF, Lu YZ, Song CJ, Wang YG, Yu B, Liu HM. Synthesis and
biological evaluation of novel steroidal[17,16-d][1,2,4]triazolo[1,5-
a]pyrimidines. Steroids 2012;77:710–5.
[24] Huang LH, Zheng YF, Song CJ, Wang YG, Xie ZY, Lai YW, Lu YZ, Liu HM.
Synthesis of novel D-ring fused 70-aryl-androstano[17,16-d][1,2,4]
triazolo[1,5-a]pyrimidines. Steroids 2012;77:367–74.
[25] Yu B, Zhang E, Sun XN, Ren JL, Fang Y, Zhang BL, Yu DQ, Liu HM. Facile
synthesis of novel D-ring modified steroidal dienamides via rearrangement of
2H-pyrans. Steroids 2013;78:494–9.
[26] Yu B, Shi XJ, Ren JL, Sun XN, Qi PP, Fang Y, Ye XW, Wang MM, Wang JW, Zhang
E, Yu DQ, Liu HM. Efficient construction of novel D-ring modified steroidal
dienamides and their cytotoxic activities. Eur J Med Chem 2013;66:171–9.
[19] Li H, Wang L, Zhang Y, Wang J. Transition-metal-free synthesis of pinacol
alkylboronates from tosylhydrazones. Angew Chem Int Ed 2012;51:2943–6.
[20] Wang XW, Li P, Xiao H, Zhu SH, Zhao G. A simple method for synthesis of 5-CF3
substituted dienamides via rearrangement of 2H-pyran derivatives.
Tetrahedron 2011;67:7618–21.
[21] Babu TH, Pawar S, Muralidharan D, Perumal PT. Rearrangement of pyran
derivatives obtained from vinyl malononitriles and aldehydes via vinylogous
aldol reaction: A novel facile method for the synthesis of dienamides. Synlett
2010;2010:2125–9.
[22] Huang LH, Wang YG, Xu G, Zhang XH, Zheng YF, He HL, Fu WZ, Liu HM. Novel
4-azasteroidal N-glycoside analogues bearing sugar-like D ring: synthesis and
anticancer activities. Bioorg Med Chem Lett 2011;21:6203–5.