Studies on novel D-ring substituted steroidal pyrazolines as potential anticancer agents

Article abstract of DOI:10.1016/j.steroids.2010.02.014

An efficient and facile synthesis of 17-pyrazolinyl derivatives of pregnenolone and their evaluation as potential anticancer agents against various human cancer cell lines are reported. The scheme involves the transformation of the starting pregnenolone acetate into pregnenolone, conversion of pregnenolone to the corresponding benzylidine derivatives and finally the conversion of this derivative to the stable steroidal 17-pyrazoline. Various compounds 4b, 4c, 4e, 4f, 4h and 4j showed significant cytotoxic activity especially against HT-29, HCT-15, 502713 cell lines.

Full text of DOI:10.1016/j.steroids.2010.02.014

Steroids 75 (2010) 805–809
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Studies on novel D-ring substituted steroidal pyrazolines as
potential anticancer agents
Abid H. Bandaya,b,∗, Bilal P. Mira, Imtiyaz H. Lonea, K.A. Surib, H.M. Sampath Kumarb
a Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India
b Synthetic Chemistry Division, Indian Institute of Integrative Medicine, Canal Road, Jammu-Tawi 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient and facile synthesis of 17-pyrazolinyl derivatives of pregnenolone and their evaluation as
potential anticancer agents against various human cancer cell lines are reported. The scheme involves
the transformation of the starting pregnenolone acetate into pregnenolone, conversion of pregnenolone
to the corresponding benzylidine derivatives and finally the conversion of this derivative to the stable
steroidal 17-pyrazoline. Various compounds 4b, 4c, 4e, 4f, 4h and 4j showed significant cytotoxic activity
especially against HT-29, HCT-15, 502713 cell lines.
Received 14 December 2009
Received in revised form 23 February 2010
Accepted 24 February 2010
Available online 4 March 2010
Keywords:
Pyrazoline
© 2010 Elsevier Inc. All rights reserved.
Pregnenolone
Benzylidines
Dipolar cycloaddition
1. Introduction
also possess antidepressant, anti-inflammatory and antirheumatic
activities [6,8,9]. Besides Pyrazolines are also used as potent antidi-
Recent years have seen an extensive focus of research directed
towards the rational modification of steroid molecules. This is prob-
ably because of the various advantages associated with steroid
based chemotherapeutics. These compounds turn out to be non-
toxic, less vulnerable to multi-drug resistance (MDR) and highly
bioavailable because of being capable of penetrating the cell wall.
This is pertinently true of modified steroids bearing heterocyclic
systems as a part of their skeleton. This has been proved by dif-
ferent ring modification studies of steroidal molecules involving
the A- and D-ring whereby incorporation of heteroatom (N or O)
has been reported to enhance the biological activities of these
molecules. Such systems have shown a lot of different biological
activities such as anti-microbial, anti-inflammatory, hypotensive,
hypocholesterolemic and diuretic activities [1–5]. As a result, a
number of different heterocyclic systems have been introduced into
the core structure of steroids with pyrazoles, pyrazolines, isoxa-
zoles, isoxazolines, thiazoles, thiadiazoles, pyridines, pyrimidines,
imidazoles, etc. as the notable ones. Amongst these heterocycles,
pyrazolines are an interesting group of compounds, many of which
possess wide spread pharmacological properties such as anal-
gesic, antipyretic and antiandrogenic activities [6,7]. Pyrazolines
abetic agents [10,11]. Recently, pyrazolines were reported as a
DP-IV inhibitors and antitumor agents [12–14]. Nitrogen heterocy-
cles have also recently been reported to exhibit antiparkinsonian
[15] anticancer [16–18], antimicrobial [19–21] activities.
In view of the therapeutic importance of these steroidal hete-
rocycles, we, in continuation of our programme on the synthesis
of D-ring substituted steroidal heterocyles, aimed to investi-
gate the synthesis of steroidal pyrazolines starting from readily
available 20-keto pregnenanes [22]. In spite of few preliminary
reports about the synthesis of steroidal diphenyl pyrazolines,
no literature precedants were found describing the synthesis of
steroidal pyrazolines through the strategy we have employed.
Keeping all these facts into consideration, we herein report
a very facile and high yielding approach for the synthesis of
steroidal D-ring substituted pyrazolines starting from 20-keto
pregnenanes involving the intermediacy of the corresponding
benzylidines prepared by condensation with various aromatic
aldehydes.
2. Experimental
2.1. General methods
Melting points were recorded on Buchi Melting point appa-
ratus D-545; IR spectra (KBr discs) were recorded on Bruker
Vector 22 instrument. NMR spectra were recorded on Bruker
DPX200 instrument in CDCl3 with TMS as internal standard for
∗
Corresponding author at: Department of Chemistry, Islamia College of Science
and Commerce, Hawal, Srinagar 190009, Jammu and Kashmir, India.
Tel.: +91 969 7301630; fax: +91 194 2429014.
E-mail address: abidrrl@gmail.com (A.H. Banday).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.02.014

806
A.H. Banday et al. / Steroids 75 (2010) 805–809
(m, 2H). 13C NMR (CDCl3, 125 MHz): ı 12.24, 18.40, 19.95,
20.82, 23.58, 28.60, 30.59, 31.03, 35.54, 36.27, 37.38, 41.23,
45.07, 50.69, 55.38, 57.66, 70.64, 111.11, 113.16, 120.30,
129.37, 139.86, 143.79, 159.69, 164.58, 167.71. ESI-MS: 479
(M++H). Anal. Calcd. for C30H39FN2O2: C, 75.28; H, 8.21; N,
5.85; found C, 75.43; H, 8.03; N, 6.04.
protons and solvent signals as internal standard for carbon spec-
tra. Chemical shift values are mentioned in ı (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 cm × 5 cm pre-
coated silica gel 60 F254 plates of thickness of 0.25 mm (Merck).
The chromatograms were visualized under UV 254–366 nm and
iodine.
(3) 1-(5-(4-Fluorophenyl)-4,5-dihydro-3-
((10R,13S)2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-
yl) pyrazol-1-yl)ethanone (4c): Colourless solid powder. Yield
82%. M.p: 156–158 ◦C. [˛]D25—25.0 (ca. 0.20, CHCl3). IR (KBr,
cm−1): 3375, 3166, 2928, 1721, 1404, 1042, 756. 1H NMR
(CDCl3, 200 MHz): ı 0.65 (s, 3H), 1.05 (s, 3H), 1.82–1.90 (m,
6H), 2.17 (s, 3H), 2.76 (m, 2H), 3.26 (m, 1H), 3.35 (m, 1H), 5.35
(s, 1H), 5.37 (m, 1H), 7.04 (d, 2H, J = 8.6), 7.14 (d, 2H, J = 8.4).
13C NMR (CDCl3, 125 MHz): ı 12.34, 19.40, 19.65, 20.82, 23.58,
28.60, 30.59, 31.03, 35.54, 36.27, 38.38, 41.23, 42.91, 45.07,
51.69, 56.38, 57.66, 70.64, 111.11, 113.16, 120.30, 129.37,
139.86, 143.79, 159.15, 162.71, 164.56, 167.71. ESI-MS: 479
(M++H). Anal. Calcd. For C30H39FN2O2: C, 75.28; H, 8.21; N,
5.85; found C, 75.51; H, 8.05; N, 6.09.
2.2. Chemical synthesis
2.2.1. General procedure for the synthesis of pyrazoline
derivatives
To a solution of pregnenolone 1 (0.316 g, 1 mmol, 1 equiv.) in
ethanol (10 ml) was added a conc. aq. solution of KOH (2 equiv.).
Then aldehyde 2 (1.2 equiv.) was charged into the reaction mixture
to get the corresponding benzylidine derivative 3. After completion,
the reaction mixture was precipitated with water. The precipitate
was filtered, dried and 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 condi-
tions. Thus the study was restricted to the use of aromatic aldehydes
only. The condensation product 3 (1.0 g, 2.4 mmol) was refluxed
in ethanol in the presence of hydrazine hydrate (0.24 g, 4.8 mmol)
so as to yield the desired pyrazolines. However the products thus
obtained were very unstable and they decomposed even at ambient
temperature conditions probably because of the inherent instabil-
ity associated with pyrazolines. The solvent thus used was replaced
by acetic acid so as to ensure the formation of N-acetyl pyrazoline
4 (0.99 g, 2.2 mmol, 90%) which was highly stable. The product was
precipitated by charging the reaction mass into excessive amounts
of ice-cold water. After filtration under suction, the product was
obtained in high yields as colourless powder which was later dried
in vacuo. The same procedure was followed for the synthesis of all
other analogs. The spectral data of various compounds is given as
under (most of the peaks due to steroidal skeleton were merged
and could not be differentiated. Thus ı values of only those peaks
that distinguish the product and could easily be differentiated are
reported):
(4) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)-5-p-tolylpyrazol-1-
yl)ethanone (4d): Greyish powder. Yield 79%. M.p: 130–133 ◦C.
[˛]D25—33.3 (ca. 0.20, CHCl3). IR (KBr, cm−1): 3416, 2936,
1719, 1642, 1455, 1087, 756. 1H NMR (CDCl3, 200 MHz): ı
0.61 (s, 3H), 1.08 (s, 3H), 1.82–1.92 (m, 6H), 2.05 (s, 3H), 2.22
(s, 3H), 2.67 (t, 1H, J = 8.8), 2.77 (m, 2H), 3.40 (m, 1H), 3.48
(m, 1H), 5.36 (m, 2H), 7.80 (dd, 4H, J = 6.3). 13C NMR (CDCl3,
125 MHz): ı 12.87, 20.54, 22.46, 23.38, 25.83, 31.13, 33.04,
33.19, 33.48, 37.99, 38.72, 39.94, 43.68, 45.30, 47.38, 47.69,
51.53, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78, 128.84,
130.28, 142.29, 161.65, 162.53. ESI-MS: 497 (M++Na). Anal.
Calcd. For C31H42N2O2: C, 78.44; H, 8.92; N, 5.90; found C,
78.67; H, 8.73; N, 6.13.
(5) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)-5-o-tolylpyrazol-1-yl)
ethanone (4e): Colourless solid. Yield 83%. M.p: 124–126 ◦C.
[˛]D25—23.4 (ca. 0.20, CHCl3). IR (KBr, cm−1): 3406, 2941,
1729, 1642, 1455, 1077, 753. 1H NMR (CDCl3, 200 MHz): ı
0.61(s, 3H), 1.09 (s, 3H), 1.84–1.93 (m, 6H), 2.10 (s, 3H), 2.66
(t, 1H, J = 8.8), 2.74 (m, 2H), 3.40 (m, 1H), 3.44 (m, 1H), 5.22
(s, 1H), 5.29 (m, 1H), 6.84 (m, 1H), 7.06 (m, 3H). 13C NMR
(CDCl3, 125 MHz): ı 11.76, 14.85, 20.85, 22.40, 23.32, 25.83,
31.13, 33.04, 33.19, 33.48, 37.99, 36.72, 39.84, 43.68, 45.80,
47.35, 47.69, 51.53, 53.18, 57.94, 60.74, 73.11, 123.80, 126.78,
127.84, 130.28, 145.29, 161.63, 166.62, ESI-MS: 497 (M++Na).
Anal. Calcd. For C31H42N2O2: C, 78.44; H, 8.92; N, 5.90; found
C, 78.27; H, 8.85; N, 6.09.
(1) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)-5-phenylpyrazol-1-yl)
ethanone (4a): Colourless solid powder. Yield 76%. M.p:
123–125 ◦C. [˛]D25—16.9 (ca. 0.20, CHCl3). IR (KBr, cm−1):
3384, 2926, 1717, 1646, 1404, 1042, 699. 1H NMR (CDCl3,
200 MHz): ı 0.63 (s, 3H), 1.06 (s, 3H), 1.82–1.90 (m, 6H), 2.17
(s, 3H), 2.65 (t, 1H, J = 8.8), 2.79 (m, 2H), 3.26 (m, 1H), 3.49 (m,
1H), 5.33 (s, 1H), 5.44 (m, 1H), 7.15 (d, 2H, J = 6.5), 7.22–7.32
(m, 3H). 13C NMR (CDCl3, 125 MHz): ı 14.85, 20.85, 22.40,
23.32, 25.83, 31.13, 33.04, 33.48, 37.99, 38.72, 39.94, 43.68,
45.30, 47.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78,
128.84, 130.28, 142.29, 160.63, 164.62. ESI-MS: 483 (M++Na).
Anal. Calcd. For C30H40N2O2: C, 78.22; H, 8.75; N, 6.08; found
C, 78.47; H, 8.83; N, 6.21.
(6) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)-5-m-tolylpyrazol-1-yl)
ethanone (4f): Colourless solid powder. Yield 74%. M.p:
120–122 ◦C. [˛]D25—25.5 (ca. 0.20, CHCl3). IR (KBr, cm−1):
3374, 2939, 1719, 1638, 1404, 1041, 756, 702. 1H NMR (CDCl3,
200 MHz): ı 0.67 (s, 3H), 1.03 (s, 3H), 1.81–1.92 (m, 6H),
2.19 (s, 3H), 2.20–2.23 (m, 3H), 3.40 (m, 1H), 3.47 (m, 1H),
5.35 (m, 2H), 7.03 (d, 1H, J = 7.19), 7.17 (d, 1H, J = 7.19). 13C
NMR (CDCl3, 125 MHz): ı 12.35, 20.85, 22.40, 23.32, 25.83,
31.13, 33.04, 33.19, 33.48, 37.99, 39.72, 39.94, 43.68, 45.30,
46.38, 47.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78,
128.84, 131.35, 143.23, 161.63, 167.62. ESI-MS: 475 (M++H).
Anal. Calcd. For C31H42N2O2: C, 78.44; H, 8.92; N, 5.90; found
C, 78.24; H, 8.84; N, 6.11.
(2) 1-(5-(3-Fluorophenyl)-4,5-dihydro-3-
((10R,13S)2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-
3-hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-
yl) pyrazol-1-yl)ethanone (4b): Colourless solid. Yield 79%.
M.p: 118–121 ◦C. [˛]D25—19.2 (ca. 0.20, CHCl3). IR (KBr,
cm−1): 3408, 2936, 1718, 1448, 1021, 756. 1H NMR (CDCl3,
200 MHz): ı 0.63 (s, 3H), 1.06 (s, 3H), 1.82–1.90 (m, 6H), 2.15
(s, 3H), 2.67 (t, 1H, J = 8.8), 2.77 (m, 2H), 3.26 (m, 1H), 3.35 (m,
1H), 5.30 (s, 1H), 5.39 (m, 1H), 6.80–6.91 (m, 2H), 7.30–7.34

A.H. Banday et al. / Steroids 75 (2010) 805–809
807
(7) 1-(5-(Furan-2-yl)-4,5-dihydro-3-((10R,13S)-
(Breast) and SF-295 (CNS) and were obtained from National Can-
cer 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 stan-
dard Sulforhodamine B assay. Briefly, cells in their log phase of
growth were harvested, counted and seeded (104 cells/well in
100 ␮L medium) in 96-well microtitre plates. After 24 h of incu-
bation at 37 ◦C and 5% CO2 to allow cell attachment, cultures were
treated with varying concentrations (0.1–100 ␮M) of test samples
made with 1:10 serial dilutions. Four replicate wells were set up
for each experimental condition. Test samples were left in con-
tact 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
Sulforhodamine 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 experimen-
tal set. Percent growth in presence of test material was calculated
considering the growth in absence of any test material as 100%
and in turn percent growth inhibition in presence of test material
was calculated. Finally the IC50 values (Table 1) 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.
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-hydroxy-
10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)pyrazol-
1-yl)ethanone (4g): Colourless powder. Yield 82%. M.p:
127–129 ◦C. [˛]D25—25.8 (ca. 0.20, CHCl3). IR (KBr, cm−1):
3386, 2936, 1727, 1647, 1451, 1043, 754. 1H NMR (CDCl3,
200 MHz): ı 0.67 (s, 3H), 1.02 (s, 3H), 1.81–1.90 (m, 6H), 2.17
(s, 3H), 3.10–3.15 (m, 3H), 3.52 (m, 1H), 5.30 (s, 1H), 5.37 (s,
1H), 5.51(m, 1H), 6.28 (m, 2H), 7.30 (s, 1H). 13C NMR (CDCl3,
125 MHz): ı 13.85, 20.85, 22.90, 22.32, 25.84, 31.13, 33.04,
33.19, 33.48, 37.99, 38.82, 39.94, 44.68, 45.30, 47.38, 48.69,
51.78, 53.18, 57.94, 60.56, 73.11, 122.80, 126.78, 128.84,
130.28, 142.29, 160.61, 164.62. ESI-MS: 473 (M++Na). Anal.
Calcd. For C28H38N2O3: C, 74.63; H, 8.50; N, 6.22; found C,
74.47; H, 8.71; N, 6.47.
(8) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
cyclopenta[a]phenanthren-17-yl)-5-(4-methoxyphenyl)
pyrazol-1-yl)ethanone (4h): Colourless solid. Yield 84%.
M.p: 103–105 ◦C. [˛]D25—28.4 (ca. 0.20, CHCl3). IR (KBr,
cm−1): 3406, 2930, 2871, 1719, 1642, 1419, 1041, 757. 1H
NMR (CDCl3, 200 MHz): ı 0.67 (s, 3H), 1.01 (s, 3H), 1.82–1.88
(m, 6H), 2.03 (s, 3H), 2.53–2.73 (m, 2H), 3.20 (m, 1H), 3.29
(m, 1H), 3.76 (s, 3H), 5.34 (m, 2H), 6.82 (d, 2H, J = 8.1), 7.05
(d, 2H, J = 8.1). 13C NMR (CDCl3, 125 MHz): ı 13.38, 20.85,
22.44, 23.32, 25.83, 31.13, 33.04, 33.19, 34.48, 37.99, 38.72,
39.94, 43.68, 45.30, 47.38, 47.69, 51.55, 53.18, 57.94, 60.56,
73.11, 122.80, 126.78, 127.84, 130.28, 142.29, 160.63, 165.62.
ESI-MS: 491 (M++H). Anal. Calcd. For C31H42N2O3: C, 75.88;
H, 8.63; N, 5.71; found C, 75.63; H, 8.81; N, 5.87.
3. Results and discussion
(9) 1-(4,5-Dihydro-3-((10R,13S)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-3-hydroxy-10,13-dimethyl-1H-
Though the importance of different steroidal D-ring heterocy-
cles is now well validated, only few efforts have been reported
for their efficient synthesis, to the best our knowledge. This spe-
cially refers to the pyrazoline based heterocycles at the D-ring of
steroids including the very important class of pregnanes. Though
there are reports for the synthesis of other such heterocycles, the
same is not true for the pyrazoline derivatives at the D-ring. Taking
inspiration from the number of reported biological activities asso-
ciated with structurally related analogs, we, in continuation of our
efforts towards the synthesis of novel D-ring heterocycles, herein
report a novel efficient and simple synthesis of D-ring pyrazoline
derivatives of 20-keto pregnenanes and their evaluation as poten-
tial anticancer agents. The preparation of the latter involves the
following synthetic approach (Scheme 1).
cyclopenta[a]phenanthren-17-yl)-5-(2-methoxyphenyl)
pyrazol-1-yl)ethanone (4i): Colourless solid. Yield 80%. M.p:
130–132 ◦C. [˛]D25—33.1 (ca. 0.20, CHCl3). IR (KBr, cm−1):
3399, 2936, 2871, 1719, 1642, 1419, 1039, 757. 1H NMR
(CDCl3, 200 MHz): ı 0.66 (s, 3H), 1.01 (s, 3H), 1.81–1.88 (m,
6H), 2.04 (s, 3H), 2.54–2.73 (m, 2H), 3.20 (m, 1H), 3.29 (m, 1H),
3.83 (s, 3H), 5.35 (m, 2H), 6.85–6.93 (m, 2H), 7.24–7.33 (m,
2H). 13C NMR (CDCl3, 125 MHz): ı 13.67, 20.85, 22.40, 23.32,
25.83, 31.13, 31.04, 33.19, 33.48, 37.99, 38.72, 39.94, 43.68,
45.30, 47.38, 43.69, 51.53, 53.18, 57.94, 60.56, 73.11, 122.80,
126.78, 128.84, 130.28, 142.29, 160.73, 164.62. ESI-MS: 491
(M++H). Anal. Calcd. For C31H42N2O3: C, 75.88; H, 8.63; N,
5.71; found C, 75.99; H, 8.41; N, 5.65.
(10) 1-(5-(2-Chlorophenyl)-4,5-dihydro-3-((10R,13S)-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-3-
3.1. Biology
hydroxy-10,13-dimethyl-1H-cyclopenta[a]phenanthren-17-yl)
pyrazol-1-yl)ethanone (4j): Colourless white powder. Yield
79%. M.p: 128–130 ◦C. [˛]D25—25.6 (ca. 0.20, CHCl3). IR
(CHCl3, cm−1): 3386, 2944, 1720, 1640, 1492, 1407, 1090,
962, 756. 1H NMR (CDCl3, 200 MHz): ı 0.67 (s, 3H), 1.08
(s, 3H), 1.81–1.88 (m, 6H), 2.04 (s, 3H), 2.57–2.65 (m, 2H),
3.20 (m, 1H), 3.50 (m, 1H), 5.34–5.42 (m, 2H), 7.09 (d, 2H,
J = 8.4), 7.37 (d, 2H, J = 8.4). 13C NMR (CDCl3, 125 MHz): ı
12.27, 12.46, 18.40, 19.61, 20.80, 23.36, 23.55, 30.53, 30.72,
31.01, 35.53, 36.25, 37.51, 41.17, 45.11, 49.23, 50.68, 55.46,
57.52, 70.66, 120.33, 125.82, 127.98, 132.14, 139.80, 158.60,
166.63, 167.71, 173.88. ESI-MS: 517.4 (M++Na). Anal. Calcd.
For C30H39ClN2O2: C, 72.78; H, 7.94, Cl, 7.16,; N, 5.66; found
C, 72.96; H, 8.21; Cl, 7.01; N, 5.79.
The following table gives the cancer cell inhibitory data obtained
after treating different cancer cell lines with test doses of the dif-
ferent steroidal pyrazoline derivatives and the values are reported
in terms of IC50
.
A number of correlations can be made from the data given in the
table above. It is clear from the IC50 values, that the compounds 4b,
4c, 4e, 4f and 4h showed significant cytotoxic activity especially
against HT-29, HCT-15, 502713 cell lines. Moreover the cytotoxi-
city of the compounds for certain cancer cell lines increases more
than hundred times when just the substitutions are changed over
the aromatic ring (IC50 values of compounds 4i and 4j may be
compared for HT-29 and HCT-15 cancer cell lines). It is also evi-
dent from the data that even the change in the position of same
substituent on the aromatic ring influences the relative toxicity.
This can be attributed to their differences in either polarity which
changes their lipophilicity or the conformation which alters the tar-
get protein binding properties present within the cell or on the cell
membrane (for comparison see the IC50 values of 4b and 4c). Com-
2.3. Biology
The human cancer cell lines used for the test were HT-29,
HCT-15 (Colon), 502713 (Colon), HOP-62, A-549 (Lung), MCF-7

808
A.H. Banday et al. / Steroids 75 (2010) 805–809
Table 1
IC50 values (␮M) of 17-pyrazolinyl derivatives of pregnenolone against a panel of human cancer cell lines.
Compound
HT-29
HCT-15
502713
HOP-62
A-545
MCF-7
0.43
SF-295
0.63
R
4a
6.35
2.31
0.56
1.46
11.0
4b
0.44
0.53
0.32
0.50
1.42
1.60
3.79
4c
0.53
6.25
1.46
5.11
0.72
20.8
4.18
ND
1.67
8.11
1.84
4.84
8.86
3.65
4d
4e
4f
0.34
0.35
0.62
0.31
0.41
0.43
0.69
0.57
0.64
0.56
0.79
1.60
1.0
1.67
4g
4h
11.8
2.37
23.4
0.65
44.2
0.46
50.0
0.81
48.1
0.7
ND
50.6
5.44
1.91
4i
4j
25.7
0.24
21.8
0.25
28.5
37.0
32.9
50.0
29.2
32.2
23.1
17.5
49.5
30.2
ND, not determined. Cell lines: HT-29, HCT-15 (Colon), 502713 (Colon), HOP-62 (Lung), A549 (Lung), MCF-7 (Breast) and SF-295 (CNS).
Scheme 1. Synthesis of D-ring substituted steroidal pyrazolines.
pound 4j, which contained an o-chloro substitution on the aromatic
ring, showed significant cytotoxicity, selective against HT-29 and
HCT-15 cell lines.
Acknowledgements
The authors thank Principal ICSC and Director IIIM, for their inter-
est and encouragement.
4. Conclusion
References
A series of novel 17-pyrazolinyl derivatives of pregnenolones
were synthesized and screened for anticancer activity against a
panel of seven human cancer cell lines. From the data it was found
that all the compounds are having promising anticancer activity
and the compound 4j was found to be the most active especially
against HT-29 and HCT-15 cancer cell lines indicating the high
degree of selectivity of this o-chloro phenyl compound against the
said cell lines.
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