Synthesis and antileukemic activity of 16E-[4-(2-carboxy)ethoxybenzylidene] -androstene amides

Article abstract of DOI:10.1016/j.steroids.2012.01.020

In order to determine the structural requirements for cytotoxicity against various tumor cell lines, a new series of 16E-arylidene androstene amides with varying degrees of unsaturation in ring A has been synthesized. Characterization and in vitro cytotox

Full text of DOI:10.1016/j.steroids.2012.01.020

Steroids 77 (2012) 552–557
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antileukemic activity of
16E-[4-(2-carboxy)ethoxybenzylidene]-androstene amides
⇑
Ranju Bansal , Pratap Chandra Acharya
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to determine the structural requirements for cytotoxicity against various tumor cell lines, a new
series of 16E-arylidene androstene amides with varying degrees of unsaturation in ring A has been syn-
thesized. Characterization and in vitro cytotoxic studies of the newly synthesized compounds are dis-
cussed. The compounds on evaluation against various tumor cell lines exhibited significant growth
inhibition on leukemia cell lines. 3-Chloro-16E-{[4-(4-methylpiperazin-1-yl)-2-oxoethoxy]benzyli-
dene}androst-5-en-17-one (10) emerged as the most potent compound of the series with GI50 values
Received 20 November 2011
Received in revised form 20 January 2012
Accepted 30 January 2012
Available online 10 February 2012
Keywords:
Cytotoxic agents
of 3.94, 2.61, 6.90 and 1.79
respectively.
l
M against CCRF-CEM, K-562, RPMI-8226 and SR leukemia cell lines,
1
6-Arylidenosteroids
Ó 2012 Elsevier Inc. All rights reserved.
Steroidal amides
Antileukemic activity
1
. Introduction
Cancer is the leading cause of death in economically developed
Developmental Therapeutic Program of NCI, Bethesda, USA after
successfully passing the first two stages i.e. 60 cell line assay and
in vivo hollow fiber assay [14]. This encouraged us to further
explore the structural motif responsible for the anticancer proper-
ties of 16-arylidene androstenes. As reported earlier [14], presence
of tertiary amino groups enhances the cytotoxic potential of the
16-arylidene compounds; we decided to synthesize amides of 16-
arylidene steroidal acid with the anticipation that it may result
countries and the second leading cause of death in developing
countries [1]. In spite of the large number of available chemother-
apeutic antineoplastic agents, the medical need is still largely un-
met. The main reasons are lack of selectivity of conventional
drugs leading to toxicity, the metastatic spreading implying early
tumor implantation in organs other than primary site, the hetero-
geneity of the disease comprising about 100 types of cancer and
the intrinsic or acquired resistance to chemotherapy developed
after few therapeutic cycles i.e. multi-drug resistance [2]. Use of
steroids is a common practice for the treatment of cancer. Many
steroid hormones as well as their agonists and antagonists, such
as glucocorticoids (e.g., prednisone), androgens (e.g., fluoxymester-
one), antiandrogen (e.g., cyproterone acetate), estrogens (e.g., stil-
3
into enhanced activity. Replacement or substitution of C –OH func-
tion has a prominent impact on cytotoxic effects of 16-(para-
substituted)benzylidene series of anticancer compounds [13,14].
Hence it was also envisaged to study the effect of such changes
on cytotoxicity profile.
2
. Experimental
bestrol), aromatase inhibitors (e.g., formestane) and 5a-reductase
2.1. Chemistry
inhibitors (e.g., finasteride) are already in clinical use and are
known to produce beneficial results in the prevention and treat-
ment of cancer [3–9].
Melting points were determined on a Veego melting point appa-
À1
ratus and are uncorrected. Infrared (wavenumbers in cm ) spectra
were recorded on Perkin–Elmer RX1 FTIR spectrophotometer mod-
Several androstene derivatives with substitution at position C-
À1
1
1
6 have been described in the literature as potent antineoplastic
el using potassium bromide pellets (
were obtained on a Bruker Avance II 400 MHz spectrometer using
deuterated-chloroform (CDCl ) or deuterated dimethylsulfoxide
DMSO-d ) as solvents containing tetramethylsilane as internal
mmax in cm ). H NMR spectra
agents [10,11]. Recently some interesting 16E-arylidene andro-
stene derivatives (1) have been reported from our laboratory as
strong in vitro inhibitors of the growth of many types of human tu-
mor cells [12–14]. One of the compounds possessing diethylamino
group (2, Fig. 1) reached up to in vivo xenograft testing stage of
3
(
6
standard (chemical shifts in d, ppm). Mass spectra were obtained
on an Applied Biosystems API 2000™ Mass spectrophotometer.
Elemental analyses were carried out on a Perkin Elmer-2400 model
CHN analyzer. Plates for thin layer chromatography (TLC) were
prepared with silica gel G according to method of Stahl (E. Merck)
⇑
Corresponding author. Tel.: +91 172 2541142; fax: +91 172 2543101.
E-mail address: ranju29in@yahoo.co.in (R. Bansal).
0
039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2012.01.020

R. Bansal, P.C. Acharya / Steroids 77 (2012) 552–557
553
Fig. 1. Strucutres of 16-benzylidene steroids.
using ethyl acetate as solvent and activated at 110 °C for 30 min.
Iodine was used to develop the TLC plates. Anhydrous sodium sul-
fate was utilized as drying agent. All solvents were distilled prior to
use according to standard procedures.
2.1.2.2.
3-Chloro-16E-[4-(2-morpholin-4-yl-2-oxoethoxy)benzyli-
dene]androst-5-en-17-one (8) (RB-431). White solid. Yield 44%.
m.p. 224–226 °C. FT-IR: 2935, 2851, 1710, 1667, 1510, 1442,
1
1236, 1110, 1026. H NMR (CDCl
3
): d 0.96 (s, 3H, 18-CH
– and –O–(CH –, morpho-
-H), 4.74 (s, 2H, –OCH –), 5.42 (d, 1H, 6-CH, J
5.04 Hz), 6.99 (d, 2H, –CH, J = 8.68 Hz, aromatic), 7.39 (s, 1H,
vinylic-H, 16-arylidene), 7.50 (d, 2H, –CH, J = 8.72 Hz, aromatic).
ESI-MS m/z: 538.2 [M ], 540.1 [M+2] . Anal. calcd for C32 Cl:
C, 71.42; H, 7.49; N, 2.60; found: C, 71.67; H, 7.32; N, 2.52.
3
), 1.09
(
s, 3H, 19-CH
3
), 3.66 (m, 8H, –N–(CH
)
2 2
2 2
)
line), 3.77 (m, 1H, 3
a
2
2.1.1. Synthesis of 16E-[4-(carboxymethoxy)benzylidene]-3b-hydroxy-
=
o
androst-5-en-17-one (5) (RB-490)
Methyl chloroacetate (1 ml, in excess) was added to a stirred
and refluxing suspension of 4-hydroxybenzaldehyde (0.5 g,
o
+
+
H40NO
3
4
.09 mmol) and anhydrous potassium carbonate (2 g) in ethyl
methyl ketone (100 ml). The reaction mixture was further refluxed
for 6 h with continuous stirring. The completion of the reaction
was monitored by TLC. The reaction mixture was cooled, filtered
and the excess of solvent was removed under reduced pressure
to obtain 4-formylphenoxyacetic acid methyl ester (3) as an oily
residue, which was used as such for further reaction.
2
.1.2.3.
3-Chloro-16E-[4-(2-oxo-2-piperidin-1-yl-ethoxy)benzyli-
dene]androst-5-en-17-one (9) (RB-432). White solid. Yield 43.5%.
m.p. 230–233 °C. FT-IR: 2934, 2857, 1707, 1647, 1600, 1512,
1
1
3
3
443, 1250, 748. H NMR (CDCl
H, 19-CH
3
): d 0.96 (s, 3H, 18-CH
–, piperidine), 3.73 (m, 1H,
–), 5.42 (d, 1H, 6-CH, J = 5.44 Hz), 6.99
= 8.32 Hz, aromatic), 7.39 (s,1H, vinylic-H, 16-arylid-
ene), 7.50 (d, 2H, –CH, J = 8.16 Hz, aromatic). ESI-MS m/z: 536.2
M ], 538 [M+2] . Anal. calcd for C33 Cl: C, 73.93; H, 7.90;
N, 2.61; found: C, 74.05; H, 7.71; N, 2.59.
3
), 1.09 (s,
3 2 2
), 3.53 (m, 4H, –N–(CH )
a-H), 4.72 (s, 2H, –OCH
2
A
solution of dehydroepiandrosterone (4; DHA) (0.5 g,
.73 mmol), above obtained oily residue and sodium hydroxide
1 g) in methanol (20 ml) was stirred at room temperature for
2 h and the completion of reaction was monitored by TLC. Cold
(
d, 2H, –CH, J
o
1
(
1
o
+
+
[
H42NO
3
water was added to the reaction mixture and the precipitate
obtained was filtered, washed with water, dried and recrystallized
from methanol to yield compound 5 (64.9%), m.p. 308–310 °C. FT-
2.1.2.4. 3-Chloro-16E-{[4-(4-methylpiperazin-1-yl)-2-oxoethoxy]ben-
1
zylidene}androst-5-en-17-one (10) (RB-433). White solid. Yield 52%.
m.p. 186–188 °C. FT-IR: 2942, 2857, 1711, 1666, 1510, 1440, 1251,
IR: 3401, 2937, 1712, 1601, 1510, 1422, 1232, 1055, 832. H NMR
(
3
DMSO-d
-H), 4.13 (s, 2H, –OCH
brs, 2H, –CH, aromatic), 7.22 (s, 1H, vinylic-H, 16-arylidene), 7.5
6
): d 0.87 (s, 3H, 18-CH
3 3
), 1.00 (s, 3H, 19-CH ), 3.33 (m, 1H,
1
1
CH
175, 828. H NMR (CDCl
3
): d 0.89 (s, 3H, 18-CH
piperazine), 2.46 (m, 4H, CH
piperazine), 3.59 (m, 4H, –N–(CH ) –, piperazine), 3.71 (m, 1H, 3
3
), 1.02 (s, 3H, 19-
a
2
–), 5.32 (d, 1H, 6-CH, J = 4.80 Hz), 6.87
3
), 2.10 (s, 3H, –N–CH
3
3
–N–(CH –,
2 2
)
(
(
+
a
-
brs, 2H, –CH, aromatic). ESI-MS m/z: 451.1 [M ]. Anal. calcd for
: C, 74.64; H, 7.61; found: C, 74.80; H, 7.72.
2 2
H), 4.66 (s, 2H, –OCH
2
–), 5.35 (d, 1H, 6-CH, J = 5.16 Hz), 6.92 (d, 2H,
28 34 5
C H O
–
7
5
5
CH, J
o
= 8.80 Hz, aromatic), 7.32 (s, 1H, vinylic-H, 16-arylidene),
+
.42 (d, 2H, –CH, J
o
= 8.80 Hz, aromatic). ESI-MS m/z: 551.2 [M ],
33 [M+2] . Anal. calcd for C33 Cl: C, 71.91; H, 7.86; N,
.08; found: C, 71.75; H, 7.91; N, 5.12.
2.1.2. General method for the preparation of compounds 7–11
+
43 2 3
H N O
A mixture of aldol product 5 (0.2 g, 0.44 mmol) and thionyl
chloride (3 ml) was stirred at room temperature for 3 h. The excess
of thionyl chloride was vacuum evaporated. To the chlorinated res-
idue, 3-chloro-16E-[4-(chlorocarbonyl)methoxybenzylidene]and-
rost-5-en-17-one (6), dichloromethane (20 ml) was added
followed by requisite secondary amine (0.5 ml) and the mixture
was stirred overnight at room temperature. Solvent was evapo-
rated and water was added to the obtained residue. The solid so
obtained was filtered, washed with water, dried and recrystallized
from a mixture of chloroform/methanol (30:70) to yield the corre-
sponding steroidal amide 7–11.
2
.1.2.5. 3-Chloro-16E-{4-[2-(N,N-diethylamino)-2-oxoethoxy]benzyli-
dene}androst-5-en-17-one (11) (RB-434). White solid. Yield 65%.
m.p. 195–197 °C. FT-IR: 2942, 2852, 1711, 1662, 1509, 1445,
1
1
1
251, 1075. H NMR (CDCl
3
): d 0.90 (s, 3H, 18-CH
CH ) 1.14 (t, 3H, –N–CH
), 3.70 (m, 1H, 3 -H), 4.65 (s, 2H, –OCH
.36 (d, 1H, 6-CH, J = 5.44 Hz), 6.92 (d, 2H, –CH, J = 8.40 Hz, aro-
matic), 7.32 (s, 1H, vinylic-H, 16-arylidene), 7.43 (d, 2H, –CH, J
3
), 1.03 (s, 3H,
CH ), 3.33
–),
9-CH
3
), 1.07 (t, 3H, –N–CH
CH
2
3
2
3
(
m, 4H, –N(CH
2
3
)
2
a
2
5
o
o
+
+
=
8.28 Hz, aromatic). ESI-MS m/z: 524 [M ], 526.1 [M+2] . Anal.
calcd for C32 Cl: C, 73.33; H, 8.27; N, 2.67; found: C, 73.41;
H, 8.40; N, 2.72.
H42NO
3
2.1.2.1.
3-Chloro-16E-[4-(2-oxo-2-pyrrolidin-1-yl-ethoxy)benzyli-
dene]androst-5-en-17-one (7) (RB-430). White solid. Yield 65.2%.
m.p. 190–192 °C. FT-IR: 2941, 1712, 1661, 1510, 1447, 1256,
2.1.3. Synthesis of 3b-hydroxy-16E-[4-(2-methoxy-2-
oxoethoxy)benzylidene]androst-5-en-17-one (12)
A mixture of compound 5 (0.4 g, 0.88 mmol), methanol (50 ml)
2 4
and concentrated H SO (5 ml) was heated under reflux for 4 h on a
water bath. Methanol was vacuum evaporated, cold water
added and resultant solution was neutralized with solid sodium
bicarbonate. The solid product so obtained was filtered off, washed
with water, dried and recrystallized from methanol to yield the
1
1
1
4
086, 829. H NMR (CDCl
3
): d 0.89 (s, 3H, 18-CH
, pyrrolidine), 3.70 (m, 1H, 3
–), 5.35 (d, 1H, 6-CH, J = 4.8 Hz), 6.92 (d, 2H, –
= 8.64 Hz, aromatic), 7.32 (s, 1H, vinylic-H, 16-arylidene),
3
), 1.02 (s, 3H,
9-CH
3
), 3.46 (m, 4H, –N(CH
2
)
2
a
-H),
.59 (s, 2H, –OCH
2
CH, J
o
+
7
5
2
.43 (d, 2H, –CH, J
o
= 8.64 Hz, aromatic). ESI-MS m/z: 522.2 [M ],
24 [M+2] . Anal. calcd for C32 Cl: C, 73.61; H, 7.72; N,
.68; found: C, 73.50; H, 7.71; N, 2.63.
+
H40NO
3

5
54
R. Bansal, P.C. Acharya / Steroids 77 (2012) 552–557
compound 12 (85%), m.p. 195–197 °C. FT-IR: 3452, 2933, 1769,
142 °C. FT-IR: 2932, 1712, 1648, 1598, 1507, 1428, 1239, 1176,
1
1
1
710, 1604, 1511, 1437, 1226, 1178, 831. H NMR (CDCl
s, 3H, 18-CH ), 1.07 (s, 3H, 19-CH ), 3.54 (m, 1H, 3 -H), 3.82 (s,
), 4.68 (s, 2H, –OCH –), 5.40 (d, 1H, 6-CH, J = 4 Hz),
.94 (d, 2H, –CH, J = 8.80 Hz, aromatic), 7.39 (s, 1H, vinylic-H,
6-arylidene), 7.50 (d, 2H, –CH, J = 8.80 Hz aromatic). Anal. calcd
Cl: C, 74.97; H, 7.81; N, 17.22; found: C, 74.71; H,
3
): d 0.95
1090. H NMR (CDCl
CH ), 3.53 (m, 4H, –N(CH
5.76 (s, 1H, 4-CH), 6.99 (d, 2H, –CH, J
(s, 1H, vinylic-H, 16-arylidene), 7.49 (d, 2H, –CH, J
3
): d 1.00 (s, 3H, 18-CH
, pyrrolidine), 4.67 (s, 2H, –OCH
= 8.80 Hz, aromatic), 7.40
= 8.80 Hz aro-
matic). ESI-MS m/z: 502.3 [M ]. Anal. calcd for C32 : C,
76.61; H, 7.84; N, 2.79; found: C, 76.42; H, 7.87; N, 2.82.
3
), 1.25 (s, 3H, 19-
(
3
3
a
3
2
)
2
2
–),
3
6
1
H, –COOCH
3
2
o
o
o
+
o
H39NO
4
for C32
H42NO
3
7
.91; N, 2.79.
2
4
.1.5.2. 16E-[4-(2-morpholin-4-yl-2-oxoethoxy)benzylidene]androst-
-ene-3,17-dione (17) (RB-438). White solid. Yield: 54%, m.p. 196–
2
.1.4. General method for the preparation of compounds 13–15
A mixture of compound 12 (0.2 g, 0.43 mmol) and appropriate
1
1
1
4
=
2
98 °C. FT-IR: 2932, 2852, 1713, 1661, 1596, 1507, 1435, 1228,
secondary amine (2 ml) was thermally fused at 100 °C for 5 h.
The reaction was monitored by TLC for completion. Cold water
was added and solid product so obtained was filtered, washed,
dried and recrystallized from a mixture of chloroform/methanol
1
097, 1026. H NMR (CDCl
9-CH ), 3.65 (m, 8H, –N–(CH
.74 (s, 2H, –OCH –), 5.76 (s, 1H, 4-CH), 6.99 (d, 2H, –CH, J
8.80 Hz, aromatic), 7.40 (s, 1H, vinylic-H, 16-arylidene), 7.50 (d,
3
): d 1.00 (s, 3H, 18-CH
3
), 1.25 (s, 3H,
3
2
)
2
– and –O–(CH –, morpholine),
2 2
)
2
o
(
30:70) to yield the corresponding amide derivative 13–15.
+
H, –CH, J
o
= 8.80 Hz, aromatic). ESI-MS m/z: 518.3 [M ]. Anal. calcd
for C32
N, 2.75.
H39NO : C, 74.25; H, 7.59; N, 2.71; found: C, 74.43; H, 7.61;
5
2
.1.4.1. 3b-Hydroxy-16E-[4-(2-oxo-2-pyrrolidin-1-yl-ethoxy)benzyli-
dene]androst-5-en-17-one (13) (RB-435). White solid. Yield 69%.
m.p. 270–273 °C. FT-IR: 2929, 2862, 1708, 1653, 1511, 1445,
1
1
3
255, 1090, 831. H NMR (CDCl
3
): d 0.97 (s, 3H, 18-CH
, pyrrolidine and 3
–), 5.40 (d, 1H, 6-CH, J = 5.24 Hz), 6.70 (d, 2H, –CH,
= 8.76 Hz, aromatic), 7.39 (s, 1H, vinylic-H, 16-arylidene), 7.50
3
), 1.07 (s,
a-H), 4.67
2
4
1
.1.5.3. 16E-[4-(2-oxo-2-piperidin-1-yl-ethoxy)benzylidene]androst-
-ene-3,17-dione (18) (RB-440). White solid. Yield: 31%, m.p. 188–
90 °C. FT-IR: 2933, 2854, 1714, 1661, 1598, 1508, 1441, 1246, 826.
H, 19-CH
3
), 3.53 (m, 5H, –N(CH
2
)
2
(
s, 2H, –OCH
2
J
o
1
H NMR (CDCl
H, –N–CH –, piperidine), 3.56 (t, 2H, –N–CH
s, 2H, –OCH –), 5.76 (s, 1H, 4-CH), 6.99 (d, 2H, –CH, J
aromatic), 7.40 (s, 1H, vinylic-H, 16-arylidene), 7.49 (d, 2H, –CH,
3
): d 1.00 (s, 3H, 18-CH
3
), 1.25 (s, 3H, 19-CH
–, piperidine), 4.73
= 8.80 Hz,
3
), 3.49 (t,
+
(
d, 2H, –CH, J
o
= 8.00 Hz aromatic). ESI-MS m/z: 504.3 [M ]. Anal.
H41NO : C, 76.31; H, 8.20; N, 2.78; found: C, 76.45;
2
(
2
2
calcd for C32
H, 8.26; N, 2.71.
4
2
o
+
J
o
= 8.80 Hz, aromatic). ESI-MS m/z: 516.2 [M ]. Anal. calcd for
33 4
H41NO : C, 76.86; H, 8.01; N, 2.72; found: C, 76.52; H, 8.06; N,
2
.1.4.2. 3b-Hydroxy-16E-[4-(2-morpholin-4-yl-2-oxoethoxy)benzyli-
dene]androst-5-en-17-one (14) (RB-437). White solid. Yield 62.5%.
m.p. 220–222 °C. FT-IR: 3466, 2928, 1711, 1654, 1600, 1512,
C
2
.75.
1
1
1
442, 1244, 1180, 1033. H NMR (CDCl
3
): d 0.95 (s, 3H, 18-CH
-H), 3.68 (m, 8H, –N–(CH
–, morpholine), 4.74 (s, 2H, –OCH
-CH, J = 5.16 Hz), 6.99 (d, 2H, –CH, J = 8.80 Hz, aromatic), 7.39
s, 1H, vinylic-H, 16-arylidene), 7.51 (d, 2H, –CH, J = 8.84 Hz, aro-
: C, 73.96;
3
),
.07 (s, 3H, 19-CH
3
), 3.59 (m, 1H, 3
a
2
)
2
–
3. Results and discussion
and –O–(CH
6
(
2
)
2
2
–), 5.40 (d, 1H,
o
3.1. Chemistry
o
+
matic). ESI-MS m/z: 520.3 [M ]. Anal. calcd for C32
H41NO
5
The synthesis of various 16E-arylidenoandrostene derivatives
has been carried out as depicted in Schemes 1 and 2. Aldol conden-
sation (Claisen–Schmidt reaction) [15] of DHA (4) with 4-formyl-
phenoxyacetic acid methyl ester (3) at room temperature in
alkaline medium afforded 16E-benzylidene steroidal acid 5. It
was observed that during the course of reaction, alkaline hydroly-
sis of methyl ester took place due to the presence of sodium
hydroxide and resulted in the formation of 5 with free carboxylic
acid group. The methine-bridged proton at C16 appeared at d
H, 7.95; N, 2.70; found: C, 74.01; H, 7.90; N, 2.74.
2
.1.4.3. 3b-Hydroxy-16E-[4-(2-oxo-2-piperidin-1-yl-ethoxy)benzyli-
dene]androst-5-en-17-one (15) (RB-439). White solid. Yield 75%.
m.p. 242–244 °C. FT-IR: 3414, 2935, 2856, 1707, 1644, 1511,
1
1
1
444, 1249, 1079, 746. H NMR (CDCl
3
): d 0.97 (s, 3H, 18-CH
–, piperidine and 3
–), 5.40 (d, 1H, 6-CH, J = 5.2 Hz), 6.99 (d,
= 8.80 Hz, aromatic), 7.39 (s, 1H, vinylic-H, 16-arylid-
ene), 7.49 (d, 2H, –CH, J = 8.80 Hz, aromatic). ESI-MS m/z: 518.3
: C, 76.56; H, 8.37; N, 2.71; found:
3
),
.07 (s, 3H, 19-CH
3
), 3.50 (m, 5H, –N–(CH
2
)
2
a-
H), 4.72 (s, 2H, –OCH
H, –CH, J
2
1
2
o
7.22 ppm in the H NMR spectrum of 5. The configuration at C16
with respect to the carbonyl at C17 has been assigned E on the basis
of earlier reports from our laboratory [14,16,17].
o
+
[
M ]. Anal. calcd for C33
H43NO
4
C, 76.67; H, 8.40; N, 2.69.
The carboxylic acid 5 was chlorinated using thionyl chloride,
which subsequently yielded 3-chloro-16E-[4-(chlorocarbonyl)
methoxybenzylidene]androst-5-en-17-one (6). Further treatment
with requisite secondary amines such as pyrolidine, morpholine,
piperidine, N-methyl piperazine and diethylamine yielded corre-
sponding amides 7–11. IR absorption band of carbonyl function
2.1.5. General method for the preparation of compounds 16–18
The compounds 13–15 (1.92 mmol) were dissolved in a mixture
of cyclohexanone (5 ml) and dry toluene (150 ml). Azeotropic dis-
tillation was continued at a slow rate while adding a solution of
aluminium isopropoxide (1 g) in dry toluene (15 ml) drop wise.
The reaction mixture was then refluxed for 5 h and allowed to
stand at room temperature overnight. The slurry was filtered and
the residue was washed thoroughly with dry toluene. The com-
bined filtrate and the washings were subjected to steam distilla-
tion until the complete removal of toluene and cyclohexanone.
The solid product obtained was filtered, washed with water, dried
and recrystallised from chloroform/methanol (3: 7) to yield com-
pounds 16–18, respectively.
À1
of parent carboxylic acid shifted from 1721 cm to lower wave-
À1
number ꢀ1660 cm in compounds 7–11 suggesting formation of
amide. The methine-bridged proton of amides appeared at a down-
1
field value at ꢀd 7.35 in the H NMR spectra in comparison to par-
ent acid, where it resonated at d 7.22 ppm. Similarly, the –OCH
2
proton appeared downfield (d 4.59–4.74 ppm) in compounds 7–
11 as compared to the parent aldol compound (d 4.10 ppm) indi-
cating amide formation. The 3a-H was also observed at an upfield
1
d 3.61–3.77 ppm position in the H NMR spectra, which confirmed
the conversion of 3-hydroxy substitution to chloro moiety. A
+
2
4
.1.5.1. 16E-[4-(2-oxo-2-pyrrolidin-1-yl-ethoxy)benzylidene]androst-
-ene-3,17-dione (16) (RB-436). White solid. Yield 35%. m.p. 140–
[M+2] peak due to presence of chlorine was also observed in the
mass spectra of compounds 7–11.

R. Bansal, P.C. Acharya / Steroids 77 (2012) 552–557
555
3 2
Scheme 1. Reagents and reaction conditions: (a) CH OH, NaOH, (b) SOCl , (c) requisite amine, dichloromethane, RT.
Scheme 2. Reagents and reaction conditions: (a) H
2
SO
4
, CH
3
OH, reflux 4 h, (b) requisite amine, fusion, 100 °C, 6 h, (c) Al(t-BuO)
3
, cyclohexane, toluene, reflux, 4 h.
À1
An alternative synthetic pathway was followed to prepare com-
pounds with enhanced oxidation in ring A as shown in Scheme 2.
The 16E-benzylidene steroid 5 was esterified to its methyl ester
carbonyl function to lower wavenumber ꢀ1660 cm
was ob-
served for these amide derivatives.
Fusion of methyl ester 12 with N-methyl piperazine gave a
sticky mass, which could not be crystallized. Also attempts to fuse
diethyl amine with the methyl ester 12 remained unsuccessful.
In view of the literature reports [14,18,19] that 3,17-diketo ste-
roids with higher degree of unsaturation in A-ring exhibit potent
cytotoxicity, Oppenauer oxidation of 13–15 was carried out to af-
1
2 4
2 in methanol using H SO as dehydrating agent. IR absorption
bands of carbonyl of ester function appeared at a higher wavenum-
À1
À1
ber (1769 cm ) than parent carboxylic acid (1721 cm ) and a
1
3
singlet of –OCH appeared at d 3.82 ppm in the H NMR spectrum
of compound 12. Steroidal ester 12 was further thermally fused
with various cyclic secondary amines like pyrolidine, morpholine
and piperidine at 100 °C for about 5 h to yield 3-hydroxy substi-
tuted target amides 13–15. Shifting of IR vibrational frequency of
ford
a
,b-unsaturated-3-keto steroids 16–18, respectively. A singlet
-H
was observed in the H NMR spectra of all the Oppenauer products.
for the 4-CH proton at about d 5.76 ppm and disappearance of 3
a
1

5
56
R. Bansal, P.C. Acharya / Steroids 77 (2012) 552–557
3.2. Biology
Table 1
Percentage growth of cancer cells at 10 lM concentration of compounds.
The compounds 8, 10, 11 and 16 were selected by National Can-
Cell lines
Percentage growth
10
cer Institute, Bethesda, Maryland, USA for evaluation of in vitro
antineoplastic activity. The screening is a two-stage process, begin-
ning with the evaluation of selected compounds against the 60 cell
lines, known as NCI-60 cell lines and include cells from eight mel-
anomas, six leukemia, eight breast cancers, two prostate, nine lung,
seven colon, six ovary, eight kidney, and six central nervous system
8
13
16
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
72.57
66.59
69.33
71.39
64.55
73.74
49.29
93.60
49.01
38.25
18.33
59.29
53.53
0.22
104.50
95.30
98.38
99.37
97.94
14.42
43.99
34.52
41.14
50.97
cancers, at a single dose of 10 lM. A 48 h continuous drug
exposure protocol was used, and a sulforhodamine B (SRB) protein
assay was used to estimate the cell viability or growth. Com-
pounds, which exhibit significant growth inhibition in the single
dose were evaluated against the 60 cell panel at five concentration
levels in the next phase [20].
The output from the single dose screen is reported as a mean
graph and is summarized in table 1. The compounds 8, 10, 11
and 16 exhibited significant growth reduction against five leuke-
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
78.99
77.12
64.97
68.22
96.75
77.19
71.22
85.71
17.19
61.51
87.66
95.92
98.83
99.22
92.69
93.48
103.82
91.19
59.85
69.38
99.16
65.76
76.42
75.25
75.95
66.88
109.05
84.82
84.72
84.47
91.89
76.09
NCI-H522
mia cell lines at a dose of 10 lM. The compounds displayed a mean
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
growth percentage of 58.14, 36.49, 98.18 and 39.00 against these
cell lines, respectively. The mean growth percentages against all
the 60 cell lines were found to be 90.78%, 66.33%, 101.70% and
100.80
102.03
81.69
93.58
76.41
89.79
96.08
60.50
90.60
34.58
54.37
À9.75
72.87
40.38
100.39
101.67
104.36
104.08
114.56
106.90
108.04
63.00
89.75
54.04
61.41
38.24
68.08
87.09
6
8.63%, respectively, for steroidal derivatives 8, 10, 11 and 16
suggesting that compound 10 and 16 have considerable inhibitory
effect on all the cell lines at 10 M.
l
Of all, compound 10 was selected by NCI for further evaluation
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
against the 60 cell panel at five concentration levels at ten fold
88.51
92.47
113.31
117.28
101.44
95.51
73.17
82.78
93.61
98.04
86.78
34.13
93.21
101.91
108.59
113.18
93.08
75.13
82.18
101.68
85.04
82.14
71.47
À4
dilutions, the highest being 10 M. Mean dose response parame-
ters against all 60 cell lines such as GI50 (drug concentration result-
ing in a 50% reduction in the net protein increase), TGI (drug
concentration of total growth inhibition) and LC50 (concentration
of drug resulting in a 50% reduction in the measured protein at
the end of the drug treatment as compared to that at the begin-
ning) are summarized in Table 2. It was observed that the selected
compound inhibited growth of leukemia cancer cell lines at very
low concentration. The GI50 value for compound 10 against four
leukemia cell lines CCRF-CEM, K-562, RPMI-8226 and SR were
104.82
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
84.59
96.32
103.97
104.32
76.46
109.12
76.48
81.25
78.91
23.46
73.83
À13.00
56.59
89.82
103.13
80.37
38.82
89.99
101.05
114.20
107.55
96.70
112.25
103.66
99.50
57.41
84.45
92.20
87.36
72.27
98.32
32.40
50.55
63.97
found to be 3.94, 2.61, 6.90 and 1.79
lM, respectively.
As proposed earlier, replacement or substitution of C
3
–OH func-
81.83
110.15
tion has a prominent impact on cytotoxic effect, it was found that
retaining the hydroxyl group in 3b position of androstene skeleton
leads to reduction in growth inhibition as observed for compound
3 which is also in accordance with our previous reports [14].
Whereas, 3,17-diketo compound 16 with higher degree of
oxidation in ring A displayed enhanced antiproliferative activity.
3
Incorporation of a chloro group at C as in compound 10 also re-
sulted in increased activity. Significant growth inhibition was ob-
served for compounds 10 and 16 containing N-methyl piperazine
and pyrolidine as tertiary amino function with mean %growth of
Ovarian cancer
IGROV1
112.86
97.19
117.69
106.98
93.90
86.64
76.09
70.73
99.69
78.91
69.21
85.83
97.51
110.60
108.19
109.11
105.10
93.41
80.04
70.04
51.30
96.78
72.61
71.34
93.56
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
1
82.28
107.99
105.24
Renal cancer
786-0
91.60
87.19
92.34
78.40
104.99
90.52
87.09
107.09
70.25
75.25
80.08
70.14
78.98
79.75
88.21
99.92
106.69
88.24
113.23
95.46
117.42
–
83.47
–
A498
6
6.33% and 68.63%, respectively. Compound 10 with N-methyl
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
87.14
74.72
77.46
66.52
76.24
87.03
piperazine group was found to be the most potent compound in
comparison to compounds having other tertiary amino function.
In conclusion, it is evident from the results depicted in Tables 1
and 2 that the synthesized amide derivatives of 16E-[4-(2-
carboxy)ethoxybenzylidene]androstene exhibit potent antileuke-
mic activity, rather than having a generalized cytotoxic effect on
the full panel of 60 cell lines as is observed for compound 2 [14].
This may be attributed to the presence of amino function as a labile
amide linkage in these compounds. Among the synthesized steroi-
100.39
101.52
Prostate cancer
PC-3
DU-145
72.00
94.62
71.35
74.94
90.35
97.52
45.87
78.60
Breast cancer
MCF7
85.02
122.00
87.92
87.25
91.75
90.78
83.87
75.25
87.33
71.23
66.55
66.33
99.65
119.62
99.05
102.18
102.65
101.70
76.08
77.09
36.31
40.23
49.49
68.63
dal amides, compound 10 with C
3
–Cl group was found to have sig-
MDA-MB-31/ATCC
BT-549
T-47D
MDA-MB-468
Mean % growth
nificant antileukemic activity. This study provides valuable
information regarding structural requirements to design and de-
velop more potent steroidal anticancer agents in the androstene
series.

R. Bansal, P.C. Acharya / Steroids 77 (2012) 552–557
557
Table 2
Acknowledgements
Dose response parameters such as GI50, TGI and LC50 of the 60-cell line assay for
compound 10.
The financial support provided by the UGC, New Delhi, India for
this project is gratefully acknowledged. The authors express appre-
ciation to National Cancer Institute, Bethesda, Maryland, USA for
evaluation of compounds for antineoplastic activity and Cipla
Ltd., Bombay, India for the generous supply of steroids.
Cell lines
GI50
(lM)
TGI (
l
M)
LC50 (lM)
Leukemia
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
3.94
2.61
–
6.90
1.79
>100
>100
>100
>100
–
>100
>100
>100
>100
>100
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