Benzylidine pregnenolones and their oximes as potential anticancer agents: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.steroids.2014.03.010

The present study reveals the anticancer activity of benzylidine pregnenolones and their oxime derivatives. The synthesis of the analogs of both series is very simple and involves aldol condensation in the first step followed by nucleophillic addition of hydroxylamine across carbonyl in the second step. Quantitative yields of more than 80% are obtained in both the steps. All the compounds were tested for their cytotoxic activities against a panel of six human cancer cell lines. Amongst all the compounds of both the series screened for their cytotoxic activity, compound 3e, 3f and 4e are very potent especially against HCT-15 and MCF-7 cancer cell lines.

Full text of DOI:10.1016/j.steroids.2014.03.010

Steroids 84 (2014) 64–69
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Benzylidine pregnenolones and their oximes as potential
anticancer agents: Synthesis and biological evaluation
Abid H. Banday a,b, , S.M.M. Akram a,c, Shameem A. Shameem a
⇑
a Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India
b Department of Chemistry and Biochemistry, University of Arizona, Tucson 85721, USA
c Department of Chemistry, University of Kashmir, Srinagar 190002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study reveals the anticancer activity of benzylidine pregnenolones and their oxime deriva-
tives. The synthesis of the analogs of both series is very simple and involves aldol condensation in the
first step followed by nucleophillic addition of hydroxylamine across carbonyl in the second step. Quan-
titative yields of more than 80% are obtained in both the steps. All the compounds were tested for their
cytotoxic activities against a panel of six human cancer cell lines. Amongst all the compounds of both the
series screened for their cytotoxic activity, compound 3e, 3f and 4e are very potent especially against
HCT-15 and MCF-7 cancer cell lines.
Received 8 November 2013
Received in revised form 5 March 2014
Accepted 15 March 2014
Available online 1 April 2014
Keywords:
Benzylidine pregnenolone
Aldol condensation
Nucleophillic addition
Cytoxicity
Ó 2014 Elsevier Inc. All rights reserved.
Dehydration
1. Introduction
based anticancer compounds such as vinblastine (Velban), vincris-
tine (Oncovin), vinorelbine (Navelbine), etoposide (VP-16), tenipo-
Over the past twenty years, medicinal chemistry has gained
enormous popularity for its role in drug discovery. A number of
natural products, their semi synthetic analogs and small molecules
have been discovered and evaluated for their role in the treatment
and cure of various fatal diseases such as cancer, diabetes, micro-
bial infections and cardiovascular diseases etc. [1,2]. However be-
cause of the drug resistance and drug tolerance problems, there
is always scope for the design and development of new and mod-
ified analogs as more efficient drug candidates. This has been done
through the rational design and synthesis of receptor based lead
molecules which still remains an open area. Natural products have
extensively been used as starting tools for the design and synthesis
of lead therapeutic scaffolds. The use of natural products for curing
human ailments dates back to 1550 BC, since natural products
have been used in medicine especially for the treatment of cancer
and related diseases. A number of plants have been used as folklore
medicinal agents. The modern drug discovery program has made a
great success as a number of lead compounds have been developed
from the traditionally used medicinal plants. A number of plant
side (VM-26), Taxol (paclitaxel), and most recently Taxotere
(docetaxel), topotecan (Hycamtin), and irinotecan (Camptosar)
have been approved for use as anticancer drugs by the US FDA
[3]. Steroids as natural products have been extensively studied as
their biological and clinical importance is now well validated. Ste-
roids as well as their derivatives have been found to have the po-
tential to be developed as drugs for the treatment of a large
number of diseases including cardiovascular [4], autoimmune dis-
eases [5], brain tumours, breast cancer, prostate cancer, osteoar-
thritis, etc. [6]. The promise of using steroids for development of
lead molecules lies in their regulation of a variety of biological pro-
cesses and being a fundamental class of signaling molecules [7].
We have been interested in the area of steroid based medicinal
chemistry for the past few years and we have successfully evalu-
ated various steroid based congeners as potent pharmacological
agents showing activities such as cytotoxic, antimicrobial, antioxi-
dant etc. [8]. This is particularly true of pregnenolone which hap-
pens to be a precursor of most other steroids and has been
extensively studied for various biological activities. It has been
found that the molecule holds a great promise against brain disor-
ders, memory loss, schizophrenia, insulin related disorders and
cancer [9]. Thus in continuation of our program, we herein report
the anticancer activities of two series of our compounds I,e benzyl-
idine pregnenolones and their oxime derivatives. We are reporting
⇑
Corresponding author at: Department of Chemistry, Islamia College of Science
and Commerce, Srinagar 190009, India. Fax: +91 194 2429014.
E-mail address: abidrrl@gmail.com (A.H. Banday).
http://dx.doi.org/10.1016/j.steroids.2014.03.010
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.

A.H. Banday et al. / Steroids 84 (2014) 64–69
65
Table 1
the primary in vitro results as the mechanistic studies are in pro-
gress and will be communicated in due course of time.
Nature of group ‘‘R’’ in the compounds 3a–3h.
Entry
Nature of R
Entry
Nature of R
2. Experimental
3a
3e
F
2.1. General methods
3b
3c
3d
3f
F
Melting points were recorded on Buchi Melting point apparatus
D-545; IR spectra (KBr discs) were recorded on Bruker Vector 22
instrument. NMR spectra were recorded on Bruker DPX200 instru-
ment in CDCl3 with TMS as internal standard for protons and sol-
vent signals as internal standard for carbon spectra. Chemical
shift values are mentioned in d (ppm) and coupling constants are
given in Hz. Mass spectra were recorded on EIMS (Shimadzu)
and ESI-esquire 3000 Bruker Daltonics instrument. The progress
of all reactions was monitored by TLC on 2 Â 5 cm pre-coated silica
gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromato-
grams were visualized under UV 254–366 nm and iodine.
3g
3h
OMe
MeO
Table 2
2.2. Chemical synthesis
Nature of group ‘‘R’’ in the compounds 4a–4g.
Entry
Nature of R
Entry
Nature of R
2.2.1. General procedure for the synthesis of Benzylidine
pregnenolones (3)
4a
4e
NO2
To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 eq.) in eth-
anol (10 ml) was added a conc. aq. solution of KOH (2 eq.). Then
aldehyde 2 (1.2 eq.) was charged into the reaction mixture to get
the corresponding benzylidine derivative 3 (Scheme 1). After com-
pletion as revealed by thin layer chromatography (TLC) in an aver-
age span of around 1 h, the reaction mixture was precipitated using
water because of the limited solubility. The precipitate was fil-
tered, dried and monitored through TLC for the purity. Thin layer
chromatography revealed just a single spot which proved the pres-
ence of a single product. For further purification, the product was
recrystallized from EtOAc:Hexane to give product as solid white
powder. It is to be mentioned that when non-aromatic aldehydes
were used, the product was formed in a very minor quantity and
that too not stable enough at ambient conditions. Thus the study
was restricted to the use of aromatic aldehydes only (Tables 1
and 2).
4b
4c
4f
F
F
4g
OMe
MeO
4d
Cl
(86%). M.p.: 128–131 °C; IR (KBr) cmÀ1
: 3425, 2938, 1804,
1637,1403, 1041, 689; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s,
3H),1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H), 2.82 (t, J = 8.80, 1H);
3.51 (m, 1H); 6.78 (s, J = 16.00, 1H), 7.39 (m, 3H), 7.55 (m, 3H);
13C NMR (500 MHz, CDCl3): d 13.33, 19.26, 21.07, 22.67, 24.62,
31.13, 31.78, 31.99, 37.22, 41.81, 45.11, 48.61, 48.78, 48.95,
49.12, 49.29, 50.04, 57.14, 61.97, 71.22, 121.24, 126.69, 128.29,
128.90, 130.41, 134.62, 140.85, 141.96, 201.32; ESI-MS: 405
(M+H); Anal. Calcd. for C28H36O2: C, 83.12; H, 8.97; Found C,
83.37; H, 8.83.
2.2.2. General procedure for the synthesis of Benzylidine pregnenolone
oximes (4)
To an ethanolic solution of compound 3d (0.418 g, 1 mmol) was
added hydroxylamine hydrochloride (0.139 g, 2 mmol) and the
reaction was stirred for 2 h at room temperature. A precipitate of
the oxime was obtained as a single spot as revealed by the TLC.
The reaction mixture was worked up by first evaporating the eth-
anol and then extracting the solid with EtOAc: H2O (3 Â 25 ml).
Quantitative yields of 90–93% were obtained for all the com-
pounds.The spectral data of various compounds from both the ser-
ies is given as under.
2.2.3.2. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[
thren-17(b)-yl)-3-o-tolylprop-2-en-1-one (3b). Yellow
a
]phenan-
powder
(89%). M.p.: 208–210 °C; IR (KBr) cmÀ1: 3405, 2941, 1774, 1674,
1403, 1041, 757; 1H NMR (CDCl3): d 0.62 (s, 3H), 1.00 (s, 3H),
1.63–1.90 (m, 6H), 2.22–2.35 (m, 3H), 2.38 (s, 3H), 2.82 (t,
J = 8.43, 1H); 3.52 (m, 1H), 5.32 (s, 1H), 6.69 (d, J = 15.78, 1H),
7.32 (m, 3H), 7.57 (d, J = 6.83, 1H), 7.82 (d, J = 15.78,1H); 13C
NMR (500 MHz, CDCl3): d 13.54, 19.46, 19.95, 21.21, 2.84, 24.77,
2.2.3.1. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[
thren-17(b)-yl)-3-phenylprop-2-en-1-one (3a). White
a
]phenan-
powder
R
R
O
HON
O
OHC
R
NH2OH.HCl
EtOH
2
EtOH/ KOH
HO
HO
HO
4
3
1
Scheme 1. Synthesis of D-ring substituted benzylidine pregnenolones and their oximes.

66
A.H. Banday et al. / Steroids 84 (2014) 64–69
31.67, 31.92, 32.09, 36.61, 37.27, 37.42, 39.32,42.32, 45.00, 50.13,
57.25, 62.17, 71.70, 71.76, 121.48, 121.72, 126.29, 126.37, 128.01,
129.10, 130.08, 130.93, 133.81, 138.27, 139.06, 140.53, 140.86,
200.51; ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21;
H, 9.15; Found C, 83.32; H, 8.83.
2.2.3.7. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[ ]phenan-
a
thren-17(b)-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3g). Solid-
white powder (87%). M.p.: 200–203 °C; IR (KBr) cmÀ1: 3415,
2926, 2853, 1803, 1708, 1637, 1508, 1255, 1036, 762; 1H NMR
(CDCl3): d 0.60 (s, 3H), 1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.36
(m, 3H), 2.83 (t, J = 8.89, 1H), 3.53 (m, 1H), 3.87 (s, 3H), 5.36 (s,
1H), 6.65 (d, J = 15.92, 1H), 6.91 (d, J = 8.72, 2H), 7.51 (d, J = 8.72,
2H), 7.53 (d, J = 15.84, 1H); 13C NMR (500 MHz, CDCl3): d 12.13,
18.31, 21.11, 20.48, 21.78, 24.10, 30.62, 30.81, 31.23, 34.52,
36.27, 38.14, 43.26, 43.96, 49.22, 56.21, 60.99, 71.73, 120.44,
124.94, 127.29, 128.65, 133.16, 139.63, 140.54, 196.54; ESI-MS:
457 (M+Na); Anal. Calcd. for C29H38 O3: C, 80.14; H, 8.81; Found
C, 80.32; H, 8.67.
2.2.3.3. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenan-
thren-17(b)-yl)-3-m-tolylprop-2-en-1-one (3c). Solid white powder
(85%). M.p.: 211–213 °C; IR (KBr) cmÀ1: 3415, 2934, 1804, 1773,
1638, 1403, 1042, 779; 1H NMR (CDCl3): d 0.63 (s, 3H), 1.00 (s,
3H), 1.61–1.90 (m, 6H), 2.20–2.34 (m, 3H), 2.44 (s, 3H), 2.86 (t,
J = 8.73,1 H); 3.54 (m, 1H), 5.36 (s, 1H), 6.75 (d, J = 15.93, 1H),
7.27 (m, 4H), 7.51 (d, J = 15.93, 1H); 13C NMR (500 MHz, CDCl3):
d 12.36, 19.38, 20.13, 20.48, 21.78, 24.10, 30.62, 30.86, 31.03,
35.54, 36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73,
120.44, 124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 198.42;
ESI-MS: 441 (M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15;
Found C, 83.03; H, 9.33.
2.2.3.8. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[
a
]phenan-
thren-17(b)-yl)-3-(2-methoxyphenyl)prop-2-en-1-one
(3h). Grey
powder (76%). M.p.: 225–227 °C; IR (KBr) cmÀ1: 3425, 2940,1728,
1636, 1598, 1296, 1024, 753; 1H NMR (CDCl3): d 0.63 (s,3H),
1.00 (s, 3H), 1.65–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.89 (t,
J = 8.85, 1H), 3.52 (m, 1H), 3.91 (s, 3H), 5.37 (s, 1H), 6.94 (d,
J = 16.16, 1H), 6.98 (m, 2H), 7.36 (m, 1H), 7.55 (d, J = 6.37, 1H),
7.88 (d, J = 16.16, 1H); 13C NMR (500 MHz, CDCl3): d 12.34,
18.34, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54,
36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 118.30,
124.94, 127.29, 128.65, 131.18, 139.63, 140.54, 195.23; ESI-MS:
457 (M+Na); Anal. Calcd. for C29H38O3: C, 80.14; H, 8.81; Found
C, 80.02; H, 8.97.
2.2.3.4. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenan-
thren-17(b)-yl)-3-p-tolylprop-2-en-1-one (3d). Solid yellowish
powder (78%). M.p.: 243–245 °C; IR (KBr) cmÀ1: 3397, 2941,
1804, 1773, 1637, 1403, 1216, 1039, 758; 1H NMR (CDCl3): d
0.63 (s, 3 H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.38 (m, 3H),
2.45 (s, 3H), 2.85 (t, J = 8.43, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.74
(d, J = 15.98, 1H), 7.22 (d, J = 7.82, 2H), 7.48 (d, J = 7.82, 2H), 7.54
(d, J = 15.98, 1H); 13C NMR (500 MHz, CDCl3): d 12.42, 18.38,
20.13, 20.48, 21.78, 23.70, 30.62, 30.86, 31.03, 35.54, 36.27,
38.14, 41.27, 43.96, 49.11, 56.21, 60.99, 70.73, 120.44, 124.94,
127.29, 128.65, 131.08, 139.73, 140.55, 199.42; ESI-MS: 441
(M+Na); Anal. Calcd. for C29H38O2: C, 83.21; H, 9.15; Found C,
83.31; H, 8.95.
2.2.3.9. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
thren-17-yl)-3-p-methylphenylprop-2-en-1-imine
a
]phenan-
(4a). Grey
powder (90%). M.p.: 235–240 °C; IR (KBr) cmÀ1: 3525, 3385,
2948, 1814, 1617, 1512, 1423, 1051, 609; 1H NMR (CDCl3): d
0.78 (s, 3H), 1.200 (s, 3H), 1.54–2.0 (m, 6H), 2.41–2.48 (m, 3H),
2.42 (t, J = 8.80, 1H); 3.21 (m, 1H); 5.98 (s, J = 16.00, 1H), 6.89 (m,
3H), 7.05 (m, 3H), 8.68 (s, 1H); 13C NMR (500 MHz, CDCl3): d
14.23, 21.06, 22.07, 24.02, 31.03, 31.48, 32.09, 36.52, 42.11,
45.21, 48.01, 48.75, 49.32, 49.79, 50.84, 57.44, 61.07, 73.12,
123.26, 127.69, 128.89, 131.41, 135.72, 142.15, 158.6, 201.12;
ESI-MS: 420 (M+H); Anal. Calcd. for C28H37NO2: C, 80.15; H, 8.89;
N, 3.34; O, 7.63; Found C, 80.10; H, 8.71; N, 3.04; O, 7.52.
2.2.3.5. (2E)-3-(3-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclo-
penta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3e). Solid yellow-
ish powder (84%). M.p.: 228–231 °C; IR (KBr) cmÀ1: 3407, 2940,
1773, 1678, 1508, 1232, 1040, 756; 1H NMR (CDCl3): d 0.62 (s,
3H), 1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.32 (m, 3H), 2.84 (t,
J = 8.92, 1H); 3.52 (m, 1H), 5.36 (s, 1H), 6.70 (d, J = 15.93, 1H),
7.08 (m, 2H), 7.53 (m, 3H); 13C NMR (500 MHz, CDCl3): d 13.06,
19.28, 20.13, 20.48, 21.78, 25.10, 30.62, 30.86, 31.03, 35.54,
36.27, 38.14, 43.26, 43.96, 49.11, 56.21, 60.99, 71.73, 122.44,
124.94, 126.49, 129.02, 132.18, 139.63, 140.53, 196.92; ESI-MS:
445 (M+Na); Anal. Calcd. for C28H35F O2: C, 79.58; H, 8.35, F,
4.50; Found C, 79.62; H, 8.52, F, 4.32.
2.2.3.10. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
a
]phenan-
thren-17-yl)-3-(3-fluorophenyl)-prop-2-en-1-imine
(4b). White
powder (91%). M.p.: 240–245 °C; IR (KBr) cmÀ1: 3407, 3312,
2940, 1773, 1618, 1518, 1232, 1040, 781; 1H NMR (CDCl3): d
0.66 (s, 3H), 1.00 (s, 3H), 1.81–2.00 (m, 6H), 2.40–2.62 (m, 3H),
2.94 (t, J = 8.92, 1H); 4.12 (m, 1H), 5.36 (s, 1H), 6.90 (d, J = 15.93,
1H), 7.18 (m, 2H), 7.73 (m, 3H) 9.36 (s, 1H); 13C NMR (500 MHz,
CDCl3): d 12.16, 19.18, 20.43, 22.78, 25.90, 30.82, 30.96, 31.13,
35.74, 36.87, 38.24, 43.56, 44.96, 49.71, 56.81, 60.99, 72.73,
122.14, 124.14, 126.79, 129.12, 132.98, 159.73, 196.92; ESI-MS:
438 (M+H); Anal. Calcd. for C28H36FNO2: C, 76.85; H, 8.29; N,
3.02; O, 7.31 F, 4.34; Found: C, 76.76; H, 8.21; N, 2.92; O, 7.11 F,
4.14.
2.2.3.6. (2E)-3-(4-fluorophenyl)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,
14,15,16,17-tetradecahydro-3(b)-hydroxy-10,13-dimethyl-1H-cyclo-
penta[a]phenanthren-17(b)-yl) prop-2-en-1-one (3f). Solid White
powder (80%). M.p.:196–198 °C; IR (KBr) cmÀ1: 3417, 2946, 1772,
1678, 1508, 1232, 1045, 755; 1H NMR (CDCl3): d 0.62 (s, 3H),
1.00 (s, 3H), 1.61–1.90 (m, 6H), 2.20–2.33 (m, 3H), 2.83 (t,
J = 8.61,1 H), 3.63 (m, 1H), 5.37 (s, 1H), 6.75 (d, J = 15.84, 1H),
7.08 (m, 2H), 7.24–7.32 (m, 2H), 7.50 (d, J = 15.84, 1H); 13C NMR
(500 MHz, CDCl3): d 12.22, 19.26, 20.43, 20.48, 21.78, 23.15,
30.62, 30.86, 31.03, 35.54, 36.27, 38.14, 43.26, 43.96, 49.11,
56.21, 60.99, 71.73, 120.52, 124.94, 127.29, 128.65, 131.18,
139.63, 140.54, 196.12; ESI-MS: 445 (M+Na); Anal. Calcd. for
2.2.3.11. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
thren-17-yl)-3-(4-methoxyphenyl)prop-2-en-1-imine
a
]phenan-
(4c). Solid
powder (90%). M.p.: 250–255 °C; IR (KBr) cmÀ1: 3405, 3322,
2916, 2853, 1803, 1637, 1521, 1275, 1036, 769; 1H NMR (CDCl3):
d 0.68 (s, 3H), 1.2(s, 3H), 1.7–1.90 (m, 6H), 2.3–2.56 (m, 3H), 2.93 (t,
J = 8.89, 1H), 3.13 (m, 1H), 4.17 (s, 3H), 5.46 (s, 1H), 6.55 (d,
C28H35F O2: C, 79.58; H, 8.35, F, 4.50; Found C, 79.42; H, 8.52, F,
4.67.

A.H. Banday et al. / Steroids 84 (2014) 64–69
67
J = 15.92, 1H), 6.81 (d, J = 8.72, 2H), 7.61 (d, J = 8.72, 2H), 7.83 (d,
J = 15.84, 1H), 9.36 (s, 1H); 13C NMR (500 MHz, CDCl3): d 12.23,
19.31, 21.21, 21.78, 24.23, 31.51, 32.13, 34.42, 36.17, 38.24,
43.56, 44.96, 49.12, 56.31, 61.89, 75.83, 121.54, 124.74, 127.19,
128.45, 132.46, 139.63, 160.14, 199.94; ESI-MS: 450 (M+H); Anal.
Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12; O, 10.68 Found:
C, 77.38; H, 8.64; N, 3.02; O, 10.56.
450 (M+H); Anal. Calcd. for C29H39NO3: C, 77.47; H, 8.74; N, 3.12;
O, 10.68; Found: C, 77.38; H, 8.64; N, 3.02; O, 10.56.
2.3. Cell culture and bio-assays
The human cancer cell lines used for the test were HT-29, HCT-
15 (Colon), SF-295 (CNS), HOP-62, A-549 (Lung) and MCF-7
(Breast). All these cancer cell lines were obtained from National
cancer institute (NCI), biological testing branch, Federick Research
and Development Centre, USA. Cellular viability in the presence
and absence of experimental agents was determined using the
standard Sulforhodamine B assay. Briefly, cells in their log phase
of growth were harvested, counted and seeded (104 cells/well in
2.2.3.12. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
a]phenan-
thren-17-yl)-3-(4-chlorophenyl)-prop-2-en-1-imine
(4d). Grey
powder (91%). M.p.: 240–250 °C; IR (KBr) cmÀ1: 3425, 3300,
2917, 1718, 1611, 1513, 1296, 1024, 763; 1H NMR (CDCl3): d
0.61 (s, 3H), 1.03 (s, 3H), 1.65–1.92 (m, 6H), 2.22–2.54 (m, 3H),
2.76 (t, J = 9.12, 1H), 3.62 (m, 1H), 5.45 (s, 1H), 6.74 (d, J = 15.31,
1H), 6.96–7.21 (m, 4H), 7.54 (d, J = 15.31, 1H), 9.96 (s, 1H), 13C
NMR (500 MHz, CDCl3): d 13.13, 19.58, 20.23, 24.04, 25.21,
30.92, 31.66, 35.04, 36.17, 38.44, 43.16, 45.76, 49.21, 57.28,
61.29, 75.03, 121.64, 124.04, 127.19, 129.45, 130.98, 142.13,
160.04, 199.83; ESI-MS: 455 (M+H); Anal. Calcd. for C28H36ClNO2:
C, 74.07; H, 7.99; N, 3.08; O, 7.05; Cl, 7.81; Found C, 74.01; H, 7.89;
N, 3.01; O, 6.99; Cl, 7.78.
100
tion at 37° and 5% CO2 to allow cell attachment, cultures were trea-
ted with varying concentrations (0.1–100 M) of test samples
lL medium) in 96-well microtitre plates. After 24 h of incuba-
l
made with 1:10 serial dilutions. Four replicate wells were set up
for each experimental condition. Test samples were left in contact
with the cells for 48 h under same conditions. Thereafter, cells
were fixed with 50% chilled trichloroacetic acid (TCA) and kept at
4 °C for 1 h, washed and air dried. Cells were stained with Sulforho-
damine B dye. The adsorbed dye was dissolved in Tris-Buffer and
plates were gently shaken for 10 min on a mechanical shaker.
The optical density (OD) was recorded on ELISA reader at
540 nm. The cell growth was calculated by subtracting mean OD
value of respective blank from the mean OD value of experimental
set. Percent growth in presence of test material was calculated con-
sidering the growth in absence of any test material as 100% and in
turn percent growth inhibition in presence of test material was cal-
culated. Finally the IC50 values (Table 3) were calculated using
Microsoft Office Excel. The different steroidal derivatives (test
material) were dissolved in a mixture of DMSO:Water (1:1) and
then introduced into the medium containing the cancer cell lines.
2.2.3.13. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenan-
thren-17-yl)-3-p-nitrophenylprop-2-en-1-imine (4e). White powder
(91%). M.p.: 255–260 °C; IR (KBr) cmÀ1: 3410, 3315, 2941, 1764,
1651, 1323, 1061, 797; 1H NMR (CDCl3): d 0.71 (s, 3H), 1.09 (s,
3H), 1.93–2.0 (m, 6H), 2.26–2.39 (m, 3H), 2.31 (s, 3H), 3.12 (t,
J = 8.43, 1H); 3.82 (m, 1H), 6.12 (s, 1H), 7.01 (d, J = 15.78, 1H),
7.72 (m, 3H), 7.89 (d, J = 6.83, 1H), 8.12 (d, J = 15.78, 1H), 9.31 (s,
1H), 13C NMR (500 MHz, CDCl3): d 13.94, 19.96, 20.15, 21.11,
22.14, 24.87, 31.07, 32.19, 36.71, 37.37, 37.82, 39.82,42.12,
45.18,51.23, 57.75, 63.27, 71.30, 71.86, 121.82, 126.57, 128.11,
129.18, 130.58, 131.93, 134.81, 138.87, 139.76, 140.53, 160.3,
200.51; ESI-MS: 465 (M+H); Anal. Calcd. for C28H36N2O4: C,
72.39; H,7.81; N, 6.03; O, 13.77; Found: C, 72.09; H, 7.68; N,
5.83; O, 13.57.
2.4. Results and discussion
2.4.1. Benzylidines Steroidal benzylidines and their oximes
Benzylidines of natural products are considered as resourceful
molecules as for as their pharmacological activities are concerned.
In reference to steroidal benzylidines, we in our earlier communi-
cations [8] have shown their therapeutic potential. As we are inter-
ested in thorough pharmacological studies of various steroidal
analogs, we examined the anticancer potential of such analogs
and the results are presented in this manuscript. Though the
importance of different chalcone based natural product analogs is
now well validated, only few efforts have been reported for their
efficient synthesis and biological evaluation as far as steroids are
concerned [10]. Modifications at the D-ring of steroids are essen-
tially important and as per the accepted trend as such alterations
result in effective receptor binding or the increased bioavailability.
Though there are reports for the synthesis of other such analogs
[11], the same is not true for the benzylidine derivatives at the
D-ring. Taking inspiration from the number of reported biological
activities associated with structurally related analogs, we, in con-
tinuation of our efforts towards the synthesis of novel D-ring semi-
synthetic analogs [8], herein report an efficient and simple
synthesis of D-ring benzylidine derivatives of 20-keto pregnenanes
and their oximes as effective cytotoxic agents. The preparation the
benzylidine analogs (3a–3h) and the oxime derivatives (4a–4g) in-
volves the following synthetic approach.
2.2.3.14. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
a]phenan-
thren-17-yl)-3-(4-fluorophenyl)-prop-2-en-1-imine
(4f). White
powder (92%). M.p.:243–250 °C; IR (KBr) cmÀ1: 3417, 3315, 2946,
1774,1641, 1518, 1232, 1045, 759; 1H NMR (CDCl3): d 0.67 (s,
3H), 1.09 (s, 3H), 1.71–1.99 (m, 6H), 2.23–2.53 (m, 3H), 2.83 (t,
J = 8.61, 1H), 3.73 (m, 1H), 5.57 (s, 1H), 6.65 (d, J = 15.84, 1H),
7.18 (m, 2H), 7.14–7.22 (m, 2H), 7.52 (d, J = 15.84, 1H) 8.86 (s,
1H); 13C NMR (500 MHz, CDCl3): d 12.13, 19.36, 20.23, 20.58,
21.68, 23.45, 30.42, 31.86, 32.03, 35.34, 36.17, 38.24, 43.16,
44.96, 49.21, 56.01, 61.99, 70.73, 121.52, 125.04, 127.19, 128.35,
132.08, 139.53, 158.14, 196.82; ESI-MS: 438 (M+H); Anal. Calcd.
for C28H36FNO2: C, 76.85; H, 8.29; N, 3.02; O, 7.31 F, 4.34; Found:
C, 76.74; H, 8.22; N, 2.91; O, 7.12 F, 4.14.
2.2.3.15. (2E)-1-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetra-
decahydro-3-hydroxy-10,13-dimethyl-1H-cyclopenta[
a]phenan-
thren-17-yl)-3-(2-methoxyphenyl)prop-2-en-1-imine
(4g). Grey
powder (93%). M.p.: 255–260 °C; IR (KBr) cmÀ1: 3485, 3330,
2950, 1718, 1623, 1528, 1246, 1014, 763; 1H NMR (CDCl3): d
0.70 (s, 3H), 1.70 (s, 3H), 1.9–2.10 (m, 6H), 2.60–2.72 (m, 3H),
2.99 (t, J = 8.85,1H), 3.52 (m, 1H), 3.98 (s, 3H), 5.57 (s, 1H), 6.84
(d, J = 16.16, 1H), 7.01 (m, 2H), 7.46 (m, 1H), 7.59 (d, J = 6.37, 1H),
7.98 (d, J = 16.16, 1H), 10.06 (s, 1H),; 13C NMR (500 MHz, CDCl3):
d 13.34, 19.14, 20.38, 21.78, 25.11, 30.67, 30.88, 31.03, 35.44,
36.27, 38.44, 43.56, 44.76, 49.01, 55.29, 61.89, 71.13, 118.75,
124.44, 127.09, 128.15, 130.38, 140.93, 160.34, 199.93; ESI-MS:
The in vitro cytotoxicity studies of various bezylidine pregne-
nolones and their oxime derivatives revealed that these derivatives
are cell specific as these were found to be active mostly against the
HCT-15 and MCF-7 cell lines. Further the oxime derivatives of ste-
roids were more potent than the benzylidines. This may be attrib-
uted to the more hydrophilicity and thus more bioavalibilty of the

68
A.H. Banday et al. / Steroids 84 (2014) 64–69
Table 3
IC50 values (
l
M) of benzylidine pregnenolones and their oxime derivatives against a panel of human cancer cell lines.
Entry
HT-29
5.25
HCT-15
2.12
SF-295
1.43
HOP-62
2.46
A-545
11.0
MCF-7
2.46
R
3a
3b
3c
2.44
1.53
2.43
1.46
3.79
8.86
1.51
4.48
3.42
1.67
1.63
1.84
3d
3e
6.25
3.34
5.11
1.02
3.65
11.0
ND
8.11
1.74
4.84
0.79
10.69
F
3f
4.55
11.8
2.37
0.81
23.4
2.64
1.67
5.44
5.44
3.57
6.81
6.81
1.56
2.17
2.17
1.00
1.91
1.91
F
3g
3h
OMe
MeO
4a
4b
2.44
2.43
3.53
1.46
3.79
8.86
1.5.
1.42
1.97
1.90
1.34
4.18
F
4c
4d
4e
4f
6.25
3.44
2.35
11.8
2.37
5.11
1.62
0.31
1.4
3.65
2.00
1.67
50.6
5.44
ND
8.11
10.64
3.56
4.84
10.79
0.60
OMe
Cl
3.69
3.57
50.0
0.81
NO2
F
48.1
0.93
4g
0.65
7.17
1.91
MeO
ND = not determined.
Cell lines: HT-29, HCT-15 (Colon), SF-295 (CNS), HOP-62 (Lung), A-549 (Lung) and MCF-7 (Breast).
oxime derivatives. As compounds 3e, 3f and 4e were found to be
more active than other analogs, it can be assumed that electron
withdrawing groups have an effect over the activity. It is worth
to mention that although the oximes formed may be a mixture
of syn and anti isomers, however only one of the two was obtained
keeping the steric constraints into consideration.
1.2
1
0.8
0.6
0.4
0.2
0
HCT
MCF
2.4.2. Cell culture and bio-assays
The following table gives the cancer cell inhibitory data ob-
tained after treating different cancer cell lines with test doses of
the different steroidal benzylidine and oxime derivatives and the
values are reported in terms of IC50.
3e
3f
4e
Acꢀve Compounds
It is clear from the IC50 values, that the compounds 3e, 3f and 4e
showed significant cytotoxic activity especially against HCT-15 and
MCF-7 cancer cell lines. Fig. 1 represents the standard error in the
IC50 values of the most potent compounds against HCT and MCF
cell lines. It is evident from the IC50 data that even the position
of substituent on the aromatic ring influences the relative cytotox-
icity which can be attributed to their differences in either the bio-
availability or the protein binding properties. As already discussed
Fig. 1. Standard error values (% error) of the most active compounds against HCT-
15 and MCF-7.
it can be assumed that the strong electron withdrawing groups
such as flouro- and nitro- increase the in vitro cytotoxicity. Overall
the cytotoxicity of these compounds is cancer cell specific as the
IC50 values make it evident.

A.H. Banday et al. / Steroids 84 (2014) 64–69
69
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3. Conclusion
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data it was found that all the compounds are having promising
anticancer activity and the compound 4e was found to be the most
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