Synthesis and cytotoxicity of 17E-(2-aryl-2-oxo-1-ethylidene)-5α- androstane derivatives

Article abstract of DOI:10.1016/j.steroids.2011.10.003

The efficient synthesis of some 17E-(2-aryl-2-oxo-1-ethylidene)-5α- androstan-3β-ols was investigated. 17-Alkynyl-3,17-androstanediols were prepared through the nucleophilic addition of epiandrosterone using the corresponding 1-alkynes in the presence of

Full text of DOI:10.1016/j.steroids.2011.10.003

Steroids 76 (2011) 1615–1620
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxicity of 17E-(2-aryl-2-oxo-1-ethylidene)-5
derivatives
a-androstane
⇑
Yan Li, Jie Huang, Jinliang Liu, Peiyun Yan, Hui Liu, Qian Sun, Xingbin Wang, Cunde Wang
School of Chemistry and Chemical Engineering, Yangzhou University, 180 Siwangting Street, Yangzhou 225002, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 July 2011
Received in revised form 6 October 2011
Accepted 10 October 2011
Available online 17 October 2011
The efficient synthesis of some 17E-(2-aryl-2-oxo-1-ethylidene)-5a-androstan-3b-ols was investigated.
17-Alkynyl-3,17-androstanediols were prepared through the nucleophilic addition of epiandrosterone
using the corresponding 1-alkynes in the presence of a strong base n-BuLi firstly. The Meyer–Schuster
rearrangement of 17-alkynyl-3,17-androstanediols was carried out efficiently catalyzed by 10% H2SO4
and HgSO4 in THF. This strategy offered a very straightforward and efficient method for access to conju-
gated
a,b-unsaturated ketone 17E,5a-androstan-3b-ols from the 17-alkynyl-3,17-androstanediols in
Keywords:
Epiandrosterone
17E-(2-aryl-2-oxo-1-ethylidene)-5
androstane
Meyer–Schuster rearrangement
Mercury sulfate
good overall yields, which are key intermediates for the preparation of some biologically important mod-
ified 17-side chain steroids. Evaluation of the synthesized compounds for cytotoxicity against A549,
SKOV3, MKN-45 and MDA-MB-435 cell lines showed that 17E-(2-aryl-2-oxo-1-ethylidene)-5a-andros-
tanes possessing a hydroxyl groups at C-3 and fluoro-substituted group of aromatic ring in the side chain
have significant inhibition activity.
a-
Anticancer activity
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
activities is not fully understood, a great number of the modified
steroids containing a
a,b-unsaturated carbonyl function as antican-
The modifications of the natural steroids and the studies on the
biological activity of these steroidal derivatives have attracted con-
siderable attentions from synthetic organic chemists and pharma-
ceuticals researchers. The modified steroidal derivatives have been
a rich source of candidates with potential pharmaceutical applica-
tions that have encouraged the design and synthesis of new ana-
logs with increased pharmacological activity. Recently, in the
field of chemical modifications about the natural steroids, studies
have revealed that a number of biologically important properties
of modified steroids are dependent upon structural features of
the steroid D-ring [1–10]. Chemical modification of the steroid
D-ring provides a way to alter the functional groups, sizes, and ste-
reochemistry of the D-ring, and numerous structure–activity rela-
tionships have been established by such synthetic alterations. For
example, some 16E-arylidene androstene derivatives modified in
D-ring have exhibited a broad range of biological activities as
potent antimicrobial agents and anticancer agents [5–10].
cer agents were described [12–14]. Moreover,
a
,b-unsaturated car-
bonyl steroids fused a heterocyclic ring such as a pyrazole and an
isoxazole ring were indicated as an aromatase inhibitors to resist
breast cancers [15].
The modified D-ring steroid containing a
bonyl function are differ from the modified A, B or C-ring steroids.
a,b-unsaturated car-
Having such varied pharmacological activities, on the synthesis of
D ring substituted steroidal analogs containing a
a,b-unsaturated
carbonyl function, these compounds mainly possess an exocyclic
double bond. Some researches proved 16-exocyclic double bond
of the steroid ring was achieved easily [5–10]. On the contrary,
the researches about 17E alkylidene androstane have rarely been re-
ported [16–18]. It is known that the Meyer–Schuster rearrangement
is a means of preparing
a,b-unsaturated carbonyl compounds as
part of a two-stage olefination strategy [19,20]. Due to the impor-
tant application in the synthesis of the complex compounds, the
research and development of the Meyer–Schuster rearrangement
were further promoted [21–23]. Based on this above information,
The preliminary studies on the mechanism of antitumor activi-
ties of the modified steroid derivatives revealed that compounds
the C-17 alkylidene group and the
function of ring D were designed and five 17E-(2-aryl-2-oxo-1-eth-
ylidene)-5 -androstan-3b-ols and two 3b-acetoxy-17E-(2-aryl-2-
oxo-1-ethylidene)-5 -androstanes were synthesized via the
a,b-unsaturated carbonyl
containing a
a,b-unsaturated carbonyl function can form adducts
with reactive thiol groups of proteins to induce protein modification
and mis-folding, which might be responsible for the observed anti-
tumor activities [11]. Although the mechanism of their antitumor
a
a
Meyer–Schuster rearrangement of 17-substituted ethynyl steroidal
17-ols as a key reaction to investigate their cytotoxic effects on
tumor cell lines. In the present paper, we describe a good yielding,
convergent strategy for the synthesis of the17E alkylidene
⇑
Corresponding author. Tel.: +86 514 8797 5568; fax: +86 514 8797 5244.
E-mail address: wangcd@yzu.edu.cn (C. Wang).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.10.003

1616
Y. Li et al. / Steroids 76 (2011) 1615–1620
androstanes with a
a,b-unsaturated carbonyl function and evaluate
2.2.1.3. 17a-(2-(4-Methylphenyl)-1-ethynyl)-5a-androstane-3b,17b-
the anticancer activity of these compounds. Our results may provide
useful information for the design of chemotherapeutic drugs.
diol (2c). Following the above procedure using 1-ethynyl-4-meth-
ylbenzene as a starting material, the title compound 2c (81% in
yield) was obtained as a white solid, mp 93–95 °C (EtOAc–hex-
anes); IR (KBr, cmÀ1
) m 3400, 2928, 2857, 2220, 1578, 1510,
1451, 1379, 1283, 1136, 1043, 817; 1H NMR (CDCl3, 600 MHz) d
(ppm): 7.33 (d, J = 6.6 Hz, 2H), 7.12 (d, J = 6.6 Hz, 2H), 3.61–3.56
2. Experimental
(m, 1H, 3 -H), 2.35 (s, 3H, Ar–CH3), 0.88 (s, 3H), 0.83 (s, 3H); 13C
a
2.1. General
NMR (CDCl3, 150 MHz) d (ppm): 137.27, 130.54 (2C), 128.00 (2C),
119.00, 91.21, 84.87, 79.37, 70.32, 53.09, 49.72, 46.41, 43.89,
38.10, 37.23, 36.04, 35.25, 34.58, 32.05, 30.61, 30.55, 27.61,
22.25, 20.43, 20.00, 12.00, 11.35; MS (EI): 407 (M+1, 28%); Anal.
Calcd. for C28H38O2: C, 82.71; H, 9.42; found C, 82.63; H, 9.55.
All melting points were determined in a Yanaco melting point
apparatus and are uncorrected. IR spectra were recorded in a Nico-
let FT-IR 5DX spectrometer. The 1H NMR (600 MHz) and 13C NMR
(150 MHz) spectra were recorded in a Bruker AV-600 spectrometer
with TMS as internal reference in CDCl3 solutions. The J values are
given in hertz. Only discrete or characteristic signals for the 1H
NMR are reported. The MS spectra were obtained on a ZAB-HS
mass spectrometer with 70 eV. The elemental analyses were per-
formed in a Perkin–Elmer 240C instrument. Flash chromatography
was performed on silica gel (230–400 mesh) eluting with ethyl
acetate–hexanes mixture.
2.2.1.4. 17a-(2-(4-Fluorophenyl)-1-ethynyl)-5a-androstane-3b,17b-
diol (2d). Following the above procedure using 1-ethynyl-4-fluoro-
benzene as a starting material, the title compound 2d (72% in
yield) was obtained as a white solid, mp 87–89 °C (EtOAc–hex-
anes); IR (KBr, cmÀ1
) m 3400, 2930, 2857, 2218, 1591, 1507,
1452, 1380, 1227, 1144, 1043, 837; 1H NMR (CDCl3, 600 MHz) d
(ppm): 7.36–7.34 (m, 2H), 6.94 (t, J = 7.8 Hz, 2H), 3.54–3.49 (m,
2.2. Organic synthesis
1H, 3 -H), 0.81 (s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 150 MHz) d
a
(ppm): 161.42 (d, J = 247.5 Hz, 1C), 132.52 (d, J = 7.5 Hz, 2C),
118.07, 114.50 (d, J = 22.5 Hz, 2C), 91.55, 83.73, 79.33, 70.28,
53.04, 49.76, 46.38, 43.84, 38.05, 37.16, 35.99, 35.19, 34.56,
32.05, 30.58, 30.49, 27.55, 22.24, 19.95, 11.98, 11.34; MS (EI):
411 (M+1, 23%); Anal. Calcd. for C27H35FO2: C, 78.99; H, 8.59; found
C, 79.20; H, 8.50.
2.2.1. Preparation of 17
diols (2a–e) (Scheme 1, Table 1)
2.2.1.1. 17 -(2-Phenyl-1-ethynyl)-5
a
-(2-aryl-1-ethynyl)-5
a
-androstane-3b,17b-
a
a-androstane-3b,17b-diol (2a). To
a solution of 1-ethynylbenzene (0.66 mL, 6 mmol) in dried THF
(10 mL) was added n-BuLi (2.7 M THF solution, 2.22 mL, 6 mmol) at
À20 °C under nitrogen. After the resultant mixture was cooled to
À78 °C, the 3b-hydroxy-5a-androstan-17-one (0.58 g, 2 mmol) in
2.2.1.5. 17a-(2-(3-Fluorophenyl)-1-ethynyl)-5a-androstane-3b,17b-
dried THF (20 mL) was added drop wise. Then the reaction mixture
was warmed to room temperature, the mixture was stirred at this
temperature for 3 h and was monitored on TLC. After completion of
the reaction, the cold water (20 mL) was poured into the reaction
mixture and the majority of solvent was evaporated under reduced
pressure. The product was extracted with methylene chloride
(2 Â 15 mL). The combined extractswerewashedwith water andsat-
urated brine, dried over Na2SO4, and evaporated under reduced pres-
sure to give a residue. The residue was purified by preparative TLC
(EtOAc:hexanes = 1:10) to furnish compound 2a as a white solid
diol (2e). Following the above procedure using 1-ethynyl-4-fluoro-
benzene as a starting material, the title compound 2e (70% in yield)
was obtained as a white solid, mp 139–141 °C (EtOAc–hexanes); IR
(KBr, cmÀ1
) m 3373, 2934, 2858, 2221, 1578, 1475, 1440, 1377,
1290, 1140, 1045, 871, 782, 681; 1H NMR (CDCl3, 600 MHz) d
(ppm): 7.22–7.19 (m, 1H), 7.15 (d, J = 7.2 Hz, 1H), 7.06 (d,
J = 9.0 Hz, 1H), 6.96–6.93 (m, 1H), 3.54–3.49 (m, 1H, 3
a-H), 0.81
(s, 3H), 0.76 (s, 3H); 13C NMR (CDCl3, 150 MHz) d (ppm): 162.34
(J = 244.5 Hz, 1C), 129.83 (J = 9.0 Hz, 1C), 127.55, 124.89
(J = 10.5 Hz, 1C), 118.47 (J = 22.5 Hz, 1C), 115.52 (J = 21.0 Hz, 1C),
93.95, 84.61, 80.32, 71.30, 54.04, 50.83, 47.46, 44.84, 39.04,
38.16, 36.99, 36.20, 35.57, 33.08, 31.61, 31.50, 28.57, 23.27,
20.96, 13.00, 12.36; MS (EI): 411 (M+1, 36%); Anal. Calcd. for
(0.61 g, 78%); mp 115–118 °C (EtOAc–hexanes); IR (KBr, cmÀ1
) m
3430, 2956, 2926, 2856, 2202, 1602, 1460, 1379, 1263, 1039, 741,
698; 1H NMR (CDCl3, 600 MHz) d (ppm): 7.37 (d, J = 6.6 Hz, 2H),
7.26–7.24 (m, 3H), 3.54–3.49 (m, 1H, 3
a-H), 0.81 (s, 3H), 0.76 (s,
C27H35FO2: C, 78.99; H, 8.59; found C, 79.13; H, 8.42.
3H); 13C NMR (CDCl3, 150 MHz) d (ppm): 130.63 (2C), 127.24 (2C),
127.17, 122.01, 91.89, 84.74, 79.32, 70.29, 53.02, 49.71, 46.39,
43.82, 38.03, 37.15, 35.98, 35.19, 34.54, 32.02, 30.58, 30.48, 27.56,
22.24, 19.96, 12.00, 11.34; MS (EI): 393 (M+1, 38%); Anal. Calcd. for
2.2.2. Preparation of 17E-(2-aryl-2-oxo-1-ethylidene)-5a-androstan-
3b-ols (3a–e)
2.2.2.1. 17E-(2-phenyl-2-oxo-1-ethylidene)-5a-androstan-3b-ol (3a). To
C27H36O2: C, 82.61; H, 9.24; found C, 82.83; H, 9.10.
the mixture of 17 -(2-phenyl-1-ethynyl)-5
a
a-androstane-3b,17b-diol
(2a) (0.392 g, 1 mmol) in THF (10 mL) was added 10% H2SO4 (0.6 mL,
0.6 mmol) and HgSO4 (30 mg, 0.1 mmol). The resulting mixture was
stirred at 30 °C for 48 h and was monitored on TLC. Then the reaction
was terminated and the majority of solvent was evaporated under re-
duced pressure. Distilled water (15 mL) was added into the reaction
mixture, and the product was extracted with methylene chloride
(2 Â 15 mL). The combined extracts were washed with water, satu-
rated NaHCO3 and saturated brine, dried over Na2SO4, and evaporated
under reduced pressure to give a residue. The residue was subjected to
chromatography using ethyl acetate/dichloromethane (1:30) as the
eluent to give 0.225 g of product 3a (65%) as a white solid, mp 206–
2.2.1.2. 17a-(2-(4-Methoxyphenyl)-1-ethynyl)-5a-androstane-3b,17b-
diol (2b). Following the above procedure using 1-ethynyl-4-
methoxybenzene as a starting material, the title compound 2b (85%
in yield) was obtained as a white solid, mp 81–83 °C (EtOAc–hex-
anes); IR (KBr, cmÀ1
) m 3333, 2931, 2855, 2219, 1571, 1510, 1445,
1383, 1247, 1132, 1035, 831; 1H NMR (CDCl3, 600 MHz) d (ppm):
7.30 (d, J = 6.6 Hz, 2H), 6.76 (d, J = 6.6 Hz, 2H), 3.73 (s, 3H, Ar–OMe),
3.52–3.47 (m, 1H, 3 -H), 0.80 (s, 3H), 0.74 (s, 3H); 13C NMR (CDCl3,
a
150 MHz) d (ppm): 158.51, 132.07 (2C), 114.28, 112.90 (2C), 90.66,
84.52, 79.27, 70.24, 54.29, 53.08, 49.70, 46.37, 43.87, 38.12, 37.16,
36.03, 35.24, 34.56, 32.07, 30.61, 30.48, 27.61, 22.26, 20.00, 12.04,
11.34; MS (EI): 423 (M+1, 52%); Anal. Calcd. for C28H38O3: C, 79.58;
H, 9.06; found C, 79.80; H, 9.17.
209 °C (EtOAc); IR (KBr, cmÀ1
) m 3475, 3069, 2924, 2860, 1660, 1610,
1447, 1372, 1259, 1220, 1040, 706, 676; 1H NMR (CDCl3, 600 MHz) d
(ppm): 7.86 (d, J = 7.8 Hz, 2H), 7.45–7.43 (t, J = 7.8 Hz, 1H), 7.38–7.36

Y. Li et al. / Steroids 76 (2011) 1615–1620
1617
(t, J = 7.8 Hz, 2H), 6.63 (s, 1H, 20-H), 3.56–3.51 (m, 1H, 3
a-H), 2.98–2.81
J = 22.5 Hz, 1C), 110.75, 70.21, 53.50, 52.50, 46.00, 43.86, 37.16,
36.02, 34.64, 34.57, 34.28, 30.92, 30.81, 30.48, 27.56, 23.50,
20.19, 17.63, 11.35; EI-MS: 411 (M+1, 16%); Anal. Calcd. for
(m, 2H, 16-CH2), 0.81 (s, 3H), 0.78 (s, 3H); 13C NMR (CDCl3, 150 MHz) d
(ppm): 190.17, 177.32, 138.69, 131.01, 127.41 (2C), 126.99 (2C),
111.19, 70.24, 53.60, 52.60, 45.87, 43.94, 37.23, 36.08, 34.68 (2C),
34.36, 30.97, 30.61, 30.54, 27.62, 23.55, 20.25, 17.67, 11.36; EI-MS:
393 (M+1, 35%); Anal. Calcd. for C27H36O2: C, 82.61; H, 9.24; found C,
82.59; H, 9.08.
C27H35FO2: C, 78.99; H, 8.59; found C, 78.89; H, 8.66.
2.2.3. Esterification of 17E-(2-aryl-2-oxo-1-ethylidene)-5a-
androstan-3b-ols (3b–c)
2.2.3.1. 17E-(2-(4-methoxyphenyl)-2-oxo-1-ethylidene)-3b-acetyloxy-
-androstane (4b). The mixture of 17E-(2-(4-methoxyphenyl)-2-
oxo-1-ethylidene)-5 -androstan-3b-ol (3b) (0.422 g, 1 mmol) and
2.2.2.2. 17E-(2-(4-methoxyphenyl)-2-oxo-1-ethylidene)-5
3b-ol(3b). Followingtheaboveprocedureusing17 -(2-(4-methoxy-
phenyl)-1-ethynyl)-5 -androstane-3b,17b-diol (2b) as a starting
material, the title compound 3b(63%inyield)was obtainedasa white
solid, mp 198–200 °C (EtOAc); IR (KBr, cmÀ1
3476, 3080, 2925,
2858, 1659, 1608, 1510, 1440, 1350, 1265, 898, 706, 675; 1H NMR
(CDCl3, 600 MHz) (ppm): 7.93 (d, J = 8.4 Hz, 2H), 6.93 (d,
a
-androstan-
5a
a
a
a
acetic anhydride (0.204 g, 2 mmol) in dried pyridine (3.5 mL) was
stirred for12 h atroomtemperature. After completion ofthe reaction,
the cold water (20 mL) was poured into the reaction mixture. The
resulting mixture was extracted with methylene chloride (2 Â
15 mL). The combined extracts were washed with water and satu-
rated brine, dried over Na2SO4, and evaporated under reduced pres-
sure to give a residue. The residue was purified by preparative TLC
(EtOAc:hexanes = 1:3) to furnish compound 4b as a white solid
)
m
d
J = 8.4 Hz, 2H), 6.67 (s, 1H, 20-H), 3.87 (s, 3H, Ar–OCH3), 3.64–3.58
(m, 1H), 3.04–2.86 (m, 2H, 16-CH2), 0.87 (s, 3H), 0.85(s, 3H); 13C
NMR (CDCl3, 150 MHz) d (ppm): 188.80, 176.22, 169.58, 161.62,
131.80, 129.02 (2C), 112.80 (2C), 112.00, 70.14, 54.52, 53.50, 46.88,
44.80, 38.17, 37.12, 35.64, 35.10, 31.92, 31.68, 31.33, 30.91, 28.60,
24.48, 21.26, 18.60, 12.32; EI-MS: 423 (M+1, 29%); Anal. Calcd. for
(0.441 g, 95%); mp 105–109 °C (EtOAc/hexanes); IR (KBr, cmÀ1
) m
3684, 2943, 2878, 1731, 1655, 1602, 1369, 1242, 1169, 1027, 838;
1H NMR (CDCl3, 600 MHz) d (ppm): 7.87 (d, J = 7.2 Hz, 2H), 6.86 (d,
C28H38O3: C, 79.58; H, 9.06; found C, 79.59; H, 9.14.
J = 7.2 Hz, 2H), 6.60 (s, 1H, 20-H), 4.65–4.60 (m, 1H, 3
a-H), 3.80 (s,
3H, Ar–OMe), 2.96–2.80 (m, 2H, 16-CH2), 1.96 (s, 3H, 3b-OAc), 0.80
(s, 6H, 2XCH3); 13C NMR (CDCl3, 150 MHz) d (ppm): 188.84, 176.07,
169.63, 161.79, 131.56, 129.19 (2C), 112.59 (2C), 111.00, 72.60,
54.40, 53.42, 52.46, 45.71, 43.70, 35.79, 34.64 (2C), 34.29, 33.00,
30.84, 30.40, 27.47, 26.47, 23.53, 20.42, 20.17, 17.65, 11.25; EI-MS:
465 (M+1, 35%); Anal. Calcd. for C30H40O4: C, 77.55; H, 8.68; found
C, 77.43; H, 8.60.
2.2.2.3. 17E-(2-(4-methylphenyl)-2-oxo-1-ethylidene)-5
3b-ol (3c). Following the above procedure using 17 -(2-(4-meth-
ylphenyl)-1-ethynyl)-5 -androstane-3b,17b-diol (2c) as a starting
material, the title compound 3c (65% in yield) was obtained as a
white solid, mp 167–170 °C (EtOAc); IR (KBr, cmÀ1
3472, 3080,
2926, 2850, 1660, 1606, 1510, 1445, 1352, 1270, 898, 710, 678;
1H NMR (CDCl3, 600 MHz) d (ppm): 7.83 (d, J = 7.8 Hz, 2H), 7.24
(d, J = 7.8 Hz, 2H), 6.68 (s, 1H, 20-H), 3.65–3.58 (m, 1H), 3.04–2.86
(m, 2H, 16-CH2), 0.88 (s, 3H), 0.85(s, 3H); 13C NMR (CDCl3,
a-androstan-
a
a
)
m
2.2.3.2. 17E-(2-(4-methylphenyl)-2-oxo-1-ethylidene)-3b-acetyloxy-
5a
-androstane (4c). Following the above procedure using 17E-(2-
150 MHz)
d
(ppm): 189.56, 176.60, 169.60, 141.52, 136.10,
(4-methylphenyl)-2-oxo-1-ethylidene)-5 -androstan-3b-ol (3c)
a
128.18 (2C), 127.10 (2C), 111.16, 70.59, 53.48, 52.02, 45.88,
43.72, 35.68, 34.70, 34.20, 33.00, 30.78, 30.36, 27.46, 26.48,
23.50, 20.44, 20.12, 17.60, 11.26; EI-MS: 407 (M+1, 47%); Anal.
Calcd. for C28H38O2: C, 82.71; H, 9.42; found C, 82.60; H, 9.38.
as a starting material, the title compound 4c (93% in yield) was
obtained as a white solid; mp 103–107 °C (EtOAc/hexanes); IR
(KBr, cmÀ1
) m 3687, 2944, 2878, 1736, 1661, 1611, 1511, 1451,
1370, 1243, 1180, 1034, 838; 1H NMR (CDCl3, 600 MHz) d (ppm):
7.76 (d, J = 7.8 Hz, 2H), 7.17 (d, J = 7.8 Hz, 2H), 6.61 (s, 1H, 20-H),
2.2.2.4. 17E-(2-(4-fluorophenyl)-2-oxo-1-ethylidene)-5
3b-ol (3d). Following the above procedure using 17 -(2-(4-fluoro-
phenyl)-1-ethynyl)-5 -androstane-3b,17b-diol (2d) as a starting
material, the title compound 3d (50% in yield) was obtained as a
a
-androstan-
4.65–4.60 (m, 1H, 3
a
-H), 2.97–2.80 (m, 2H, 16-CH2), 2.33 (s, 3H,
Ph–Me), 1.95 (s, 3H, 3b-OAc), 0.81 (s, 3H), 0.80 (s, 3H); 13C NMR
(CDCl3, 150 MHz) (ppm): 189.84, 176.62, 169.63, 141.63,
a
a
d
136.07, 128.09 (2C), 127.11 (2C), 111.18, 72.59, 53.41, 52.45,
45.76, 43.69, 35.79, 34.64, 34.60, 34.29, 33.00, 30.84, 30.50,
27.46, 26.47, 23.53, 20.55, 20.42, 20.16, 17.64, 11.25; EI-MS: 449
(M+1, 18%); Anal. Calcd. for C30H40O3: C, 80.31; H, 8.99; found C,
80.49; H, 9.20.
white solid, mp 211–213 °C (EtOAc); IR (KBr, cmÀ1
) m 3685, 2929,
2853, 1664, 1603, 1509, 1453, 1372, 1224, 1157, 1041, 840; 1H
NMR (CDCl3, 600 MHz) d (ppm): 7.90–7.87 (m, 2H), 7.06–7.03
(m, 2H), 6.59 (s, 1H, 20-H), 3.58–3.53 (m, 1H, 3
a-H), 2.97–2.80
(m, 2H, 16-CH2), 0.81 (s, 3H), 0.79 (s, 3H); 13C NMR (CDCl3,
150 MHz) d (ppm): 189.55, 178.96, 165.16 (d, J = 252 Hz, 1C),
135.88, 130.51 (d, J = 9 Hz, 2C), 115.44 (d, J = 22.5 Hz, 2C), 117.73,
71.24, 54.49, 53.50, 46.92, 44.86, 38.16, 37.01, 35.64, 35.28,
31.93, 31.68, 31.48, 30.94, 28.56, 24.52, 21.20, 18.65, 12.36; EI-
MS: 411 (M+1, 15%); Anal. Calcd. for C27H35FO2: C, 78.99; H,
8.59; found C, 78.82; H, 8.58.
2.2.4. Cytotoxic activity against human lung carcinoma cell line A549,
human ovarian carcinoma cell line SKOV3, human gastric adenocar
cinoma cell line MKN-45 and human breast carcinoma cell line MDA-
MB-435
All tumor cell lines tested were purchased from Shanghai Insti-
tute of Cell Biology, Chinese Academy of Science. The cell lines
were cultured in RPMI 1640 medium with 10% newborn calf serum
serum. It was maintained in a humidified incubator with an atmo-
sphere of 95% air and 5% CO2 at 37 °C. The cells were continuously
passaged once every 3–4 days. Growing cells were collected on
experiments. DMSO was used as latent solvent with the highest
concentration less than 0.1% in solution of the drug. The control
groups of blank (1640) and DMSO solvent were set up at the same
time. Proliferative activity was evaluated by colorimetric sulforho-
damine B (SRB) assay. Briefly, cells were plated in 96-well plates.
After cell adhering, they were treated with different compounds
in a dose-dependent way for 44 h. Then the cells were fixed by
10% TDA for 1 h and stained by SRB for 10 min. After washed with
acetic acid to remove the excess dye, protein bounding dye were
2.2.2.5. 17E-(2-(3-fluorophenyl)-2-oxo-1-ethylidene)-5
3b-ol (3e). Following the above procedure using 17 -(2-(3-fluoro-
phenyl)-1-ethynyl)-5 -androstane-3b,17b-diol (2e) as a starting
material, the title compound 3e (49% in yield) was obtained as a
white solid, mp 205–208 °C (EtOAc); IR (KBr, cmÀ1
3477, 2930,
a-androstan-
a
a
)
m
2853, 1664, 1611, 1585, 1445, 1372, 1262, 1161, 1043, 882, 811,
725; 1H NMR (CDCl3, 600 MHz) d (ppm): 7.64 (d, J = 7.8 Hz, 1H),
7.53 (d, J = 7.8 Hz, 1H), 7.35 (s, 1H), 7.14 (t, J = 7.8 Hz, 1H), 6.57 (s,
1H, 20-H), 3.56–3.51 (m, 1H, 3
a-H), 2.97–2.80 (m, 2H, 16-CH2),
0.81 (s, 3H), 0.78 (s, 3H); 13C NMR (CDCl3, 150 MHz) d (ppm):
188.59, 178.74, 165.10 (d, J = 252 Hz, 1C), 140.75, 129.02 (d,
J = 7.5 Hz, 1C), 122.63, 117.97 (d, J = 21.0 Hz, 1C), 113.81 (d,

1618
Y. Li et al. / Steroids 76 (2011) 1615–1620
Table 1
dissolved in 10 mM Tris and detected by a Model Elx 800 Autoplate
reader (Bio-Tek Instruments, USA). All the data of the experiment
were compiled and analyzed according to SPSS 15.0 software. Mea-
surement data were expressed as the mean S. D.
Preparation of target compounds (3a–e, 4b–c).
Compd.
Ar
Yield%a
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4b
4c
C6H5
78
85
81
72
70
65
63
65
50
49
95
93
p-MeOC6H4
p-MeC6H4
p-FC6H4
m-FC6H4
C6H5
p-MeOC6H4
p-MeC6H4
p-FC6H4
m-FC6H4
p-MeOC6H4
p-MeC6H4
3. Results and discussion
3.1. Chemistry
In general,
condensation from ketones or aldehydes with an active
lene group. Thus, firstly, the aldol reaction of (3b,5 )-3-hydroxyan-
a,b-unsaturated ketones could be prepared via aldol
a-methy-
a
drostan-17-one and acetophenone was carried out to synthesize the
desired product in the presence of sodium hydroxide or sodium hy-
dride at room temperature or under refluxing, but the desired 17E-
(2-phenyl-2-oxo-1-ethylidene)-5a-androstan-3b-ol (3a) was not
Table 2
Various catalysts effect on the reaction.a
afforded. Considering the synthesis of these complex steroids, we
needed an active intermediate for the formation of steroidal
17E(20)-alkenes. Fortunately, the Meyer–Schuster rearrangement
Entry
Catalyst
Yield%b
1
2
3
4
5
6
7
Co(OAc)2
Zn(OAc)2
Hg(OAc)2
Cu(OAc)2
Ni(OAc)2
Fe2(SO4)3
HgSO4
10
16
46
10
Trace
Trace
45
of an alkyne with
a-hydroxy group as a starting material in the pres-
ence of Lewis acid could give the desired
a,b-unsaturated ketone.
To initiate our studies, we first prepared five 17-alkynyl-3,
17-androstanediols (2a–e) in 70–85% yields through the nucleo-
philic addition of epiandrosterone using the corresponding 1-al-
kynes in the presence of a strong base n-BuLi (Schemes 1 and 2,
Table 1).
a
Reaction conditions: 10% mol catalyst, 10% H2SO4, acetone,
60 °C, 48 h.
To achieve this goal for the formation of the designed a,b-unsat-
b
Isolated yields.
urated ketone, at first, the Lewis acids were used for the prepara-
tion of compound 3a from the model compound 2a. We observed
the formation of the desired product 3a (Table 2) when the reac-
tion was carried out using compound 2a, 10% H2SO4 (0.6 mL,
0.6 mmol) and acetone (5 mL) in the presence of various catalysts
at 60 °C for 48 h. A comparison of the method using Hg(OAc)2 or
HgSO4 as a catalyst (Table 2, entry 3, 46%; and entry 7, 45% in
yield), with selected other Lewis acid catalyst such as Co(OAc)2,
Zn(OAc)2, Cu(OAc)2, Ni(OAc)2 and Fe2(SO4)3 (Table 2, entry 1, 10%
in yield; entry 2, 16% in yield; entry 4, 10% in yield; entry 5, trace;
entry 6, trace, respectively) that were examined is collected in Ta-
ble 2 demonstrated that the method using Hg(OAc)2 or HgSO4 as a
catalyst is indeed superior to several of the other protocols. Thus,
Hg(OAc)2 and HgSO4 were found to be the better choice for this
reaction.
To optimize the reaction conditions, reaction time, reaction tem-
perature and solvent were varied. Firstly the reaction time in the
preparation of compound 3a from the model compound 2a, and
10% H2SO4 in the presence of Hg(OAc)2 (0.10 mmol) was varied.
Among the reaction time tested (Table 3, entries 1–5), the reaction
time from 48 to 60 h gave the best result. The result showed that
the reaction time at 48 and 60 h gave the product 3a in 45% yield.
In the second set of experimental, the model reaction with com-
pound 2a, and 10% H2SO4 in the presence of Hg(OAc)2 (0.10 mmol)
in acetone was carried out by varied reaction temperature. After
some experimentation (Table 4, entries 1–4), it was found that
the model reaction using reaction temperature 30 °C produced
the corresponding compound 3a in 56% yield.
Ar
O
OH
Ar
H
H
n-BuLi, THF, -78oC to r.t. 3 h
70-85%
HO
H
H
H
H
HO
H
H
2a-e
1
O
O
Ar
Ar
Ac2O,Py,
HgSO4, H2SO4,
THF, 30 oC,48 h
H
H
r.t. 12 h
H
H
93-95%
AcO
H
H
49-65%
HO
H
H
4b-c
3a-e
Furthermore, among the solvents tested (Table 5, entries 1–5),
THF and acetone gave the best result. The result showed that
THF, acetone gave the product 3a in 65%, 56% yield, respectively.
Scheme 1. Preparation of target compounds (3a–e, 4b–c).
O
Table 3
Various reaction time effect on the reaction.a
OH
Yield%b
H
Entry
Time h
H
reaction conditions
1
2
3
4
5
12
24
36
48
60
<5
18
33
45
45
H
H
H
H
HO
HO
H
H
2a
3a
a
Reaction conditions: 10% mol HgSO4, 10% H2SO4, acetone,
60 °C.
Scheme 2. Various reaction conditions for the Meyer–Schuster rearrangement of
compound 2a.
b
Isolated yields.

Y. Li et al. / Steroids 76 (2011) 1615–1620
1619
Table 4
Table 6
Various reaction temperature effect on the reaction.a
The in vitro cytotoxic activity (IC50, in l
mol/L) of compounds 3a–e, 4b–c.a
Entry
T °C
Yield%b
Compound
Cancer cell linesb
1
2
3
4
10
30
40
60
40
56
48
45
A549
SKOV3
MKN-45
MDA-MB-435
3a
3b
3c
3d
3e
4b
4c
Cisplatin
58.1 3.4
38.5 1.6
42.3 4.6
22.1 1.8
41.5 2.3
45.0 0.8
205.9 8.8
9.14
16.5 1.6
12.3 0.8
15.6 2.2
3.4 0.2
4.7 0.4
20.1 1.4
74.5 4.5
10.22
74.5 4.2
32.2 0.6
42.2 1.8
34.3 2.0
13.1 1.2
101.6 5.6
343.6 8.2
18.25
27.7 1.3
28.2 2.6
36.6 3.2
16.7 1.6
28.6 0.8
44.6 3.8
104.3 6.8
33.20
a
Reaction conditions: 10% mol HgSO4, 10% H2SO4, ace-
tone, 48 h.
b
Isolated yields.
a
Table 5
The results are the average mean of eight replicate determinations SD.
Used as reference. A549: human lung carcinoma, SKOV3: human ovarian car-
Various solvents effect on the reaction.a
b
cinoma, MKN-45: human gastric adenocarcinoma, MDA-MB-435: human breast
carcinoma.
Entry
Solvent
Yield%b
1
2
3
4
5
Dioxane
THF
Acetone
Methanol
Ethanol
40
65
56
Trace
Trace
and 4b–c synthesized as described above were subjected to
in vitro cytotoxic evaluation against A549 (human lung carci-
noma), SKOV3 (human ovarian carcinoma), MKN-45 (human gas-
tric adenocarcinoma), MDA-MB-435 (human breast carcinoma)
a
Reaction conditions: 10% mol HgSO4, 10% H2SO4, 30 °C,
48 h.
cell lines. The results are summarized as IC50 values in
Table 6.
lmol/L in
From the data shown in Table 6, most of the new compounds
showed a measurable anti-cancer activity against A549, SKOV3,
MKN-45, and MDA-MB-435 cell lines tested. Although this is a
preliminary screening, the results showed all steroids tested
showed a higher cytotoxicity against SKOV3 cells than against
A549, MKN-45, and MDA-MB-435 cell lines. Compounds 3a–e,
with same 3-hydroxyl structure and different types of substituted
group of aromatic ring in the side chain, showed a distinct differ-
ence in their cytotoxicity against these cancer cells. Compound
3d and e with a fluoro-substituted group of aromatic ring in the
side chain had a better cytotoxicity than compounds 3a–3c against
SKOV3 cells, and also had a better cytotoxicity than cisplatin which
has been introduced into clinical use for couples of years. The ana-
logs 4b and c, with an acetoxy at C-3, remarkably decreased their
cytotoxic activity against A549, SKOV3, MKN-45, and MDA-MB-
435 cells in comparison with the analogs 3b and c, which have a
hydroxyl groups at C-3. Compound 4c containing an acetoxy at
C-3 and 4-methylphenyl substituted group in the side chain was
found inactive to A549, MKN-45, and MDA-MB-435 cells tested
Thus, with these results in hand, we synthesized five 17E-(2-
aryl-2-oxo-1-ethylidene)-5a-androstan-3b-ols (3a–e) in yield
varying from 49% to 65% by the Meyer–Schuster rearrangement
of 17-alkynyl-3,17-androstanediols (2a–e) (1.0 mmol), H2SO4
(0.6 mL, 0.6 mmol) and HgSO4 (0.10 mmol) in THF (10 ml) at
30 °C for 48 h (Scheme 1, Table 1).
We obtained an X-ray crystal structure of 4b that is presented in
Fig. 1. The configuration at C-17 has been assigned E. Crystallo-
graphic data for 4b have been deposited with the Cambridge Crys-
tallographic Data Centre with the deposition number CCDC
808601. These data can be obtained free of charge on application
to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax (+44) 1223
336033, e-mail: deposit@ccdc.cam.ac.uk].
In order to study the effect on the cytotoxicity of a hydroxyl or
acetoxy at C-3 of steroidal 17(20)E-alkenes, esterification of com-
pound 3b–c was carried out by treatment with acetic anhydride
and pyridine to furnish the corresponding esters 4b–c in 93–95%
yields (Scheme 1, Table 1).
(IC50 value >80 lmol/L). The above results indicated that a hydro-
xyl groups at C-3 must be present to retain cytotoxic activity. This
may indicate that the anticancer properties depend not only on a
hydroxyl groups at C-3 but also on the moieties attached to the
side chain.
3.2. Biological activity
Recently some studies showed those natural and synthetic ste-
roids with
a,b-unsaturated ketone core gave the potency against
human cancer cell lines [12,24–26]. Thus, all compounds 3a–e
4. Conclusion
In summary, we have successfully developed a novel and
operationally simple reaction for highly efficient synthesis of 17E-
(2-aryl-2-oxo-1-ethylidene)-5a-androstan-3b-ols. Firstly, 17-alky-
nyl-3,17-androstanediols were prepared through the nucleophilic
addition of epiandrosterone using the corresponding 1-alkynes in
the presence of a strong base n-BuLi. The Meyer–Schuster rear-
rangement reaction of 17-alkynyl-3,17-androstanediols was carried
out efficiently catalyzed by H2SO4 and HgSO4 in THF. This strategy
offered a very straightforward and efficient method for access to
conjugated a,b-unsaturated ketone 17E,5a-androstan-3b-ols from
the 17-alkynyl-3,17-androstanediols in good overall yields, which
can be key intermediates for the preparation of some biologically
important modified 17-side chain steroids. The preliminary results
showed that those 17E-(2-aryl-2-oxo-1-ethylidene)-5
a-andros-
Fig. 1. X-ray crystal structure of 4b.
tanes possessing a hydroxyl groups at C-3 and different types of

1620
Y. Li et al. / Steroids 76 (2011) 1615–1620
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impact on inhibiting human lung carcinoma cell line A549, human
ovarian carcinoma cell line SKOV3, human gastric adenocarcinoma
cell line MKN-45 and human breast carcinoma cell line MDA-MB-
435. Compounds 3d, e were found to be more potent compounds,
especially the compound 3d. Due to the structural features of our
novel compounds, the mechanism of action cannot be discerned.
Further research on the structure–activity relationship, their possi-
ble mechanism of inhibiting proliferation of cancer cell lines and the
development of 17E-(2-aryl-2-oxo-1-ethylidene)-5a-androstanes
as promising anticancer agents are ongoing.
Acknowledgments
We are grateful to theNSF of Jiangsu Province (GrantBK2008216)
and the National Natural Science Foundation of China (Project No.
21173181) for financial support. We thank Mr. Chenggang Jiang
for helpful work.
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