Pregnenolone derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.steroids.2011.09.006

Pregnenolone (1) was used as a template to develop new anticancer compounds. Ring-D modification of 1 resulted in the synthesis of benzylidenes 2-17, pyrazolines 18-76, pyrazoles 85-91, hydrazones 77-84, and oximes 92-107 derivatives. The structure of com

Full text of DOI:10.1016/j.steroids.2011.09.006

Steroids 76 (2011) 1554–1559
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Pregnenolone derivatives as potential anticancer agents
M. Iqbal Choudhary a,b,c, M. Shahab Alam a, Atta-ur-Rahman a,b, Sammer Yousuf a, Yang-Chang Wu d,
An-Shen Lin d, F. Shaheen a,
⇑
a H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
c Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
d Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 March 2011
Received in revised form 28 August 2011
Accepted 8 September 2011
Available online 22 September 2011
Pregnenolone (1) was used as a template to develop new anticancer compounds. Ring-D modification of 1
resulted in the synthesis of benzylidenes 2–17, pyrazolines 18–76, pyrazoles 85–91, hydrazones 77–84,
and oximes 92–107 derivatives. The structure of compound 107 was also deduced through single crystal
X-ray diffraction studies. The inclusion of furanyl and pyridyl rings to pregnenolone skeleton increases
the cytotoxicity of all compounds significantly. Among benzylidene derivatives, only heterocyclic enone
8 (IC50 = 0.74 lM/mL against HepG2), and 17 (IC50 = 4.49 lM/mL against HepG2, IC50 = 5.01 lM/mL
Keywords:
Pyrazoline
Pregnenolone
Parallel synthesis
Benzylidene
against MDA-MB-230 cancer cell line) exhibited a significant activity. The cytotoxicity data of pyrazoline
derivatives 18–76 revealed that only furanyl bearing pyrazolines 40, 42–44, 48, and 49 exhibited signif-
icant activities. While all (O-carboxymethyl) oximes, hydazones, and pyrazoles derivatives of pregneno-
lone did not show any significant activity against both the cell lines. Thus the furanyl bearing enone 8
(IC50 = 0.74 lM/mL against HepG2), and its pyrazoline derivative 48 (IC50 = 0.91 lM/mL against MDA-
MB-230 cancer cell lines) were identified as the most active compounds in all derivatives of
MDA-MB-231
HepG2 cell lines
pregnenolone.
Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction
roids, bearing heterocyclic systems as a part of rings-A- and -D,
have exhibited diverse biological activities, such as anti-microbial,
Cancer has been a major threat to public health for many years.
Despite recent advances in early diagnosis, prevention, and ther-
apy, cancer still affects millions of people worldwide and is one
of the leading causes of death. Breast cancer is one of the most
common cancers among women in both developed and developing
countries. It is the malignancy with the highest incidence and
death rate in women [1,2]. On the other hand, hepatocellular car-
cinoma is the fifth most common form of cancer, and increasing
globally due to increased incidence of hepatitis [1]. However, the
efficacy of the existing drugs for the treatment of various cancers
is rather limited, and there is an urgent need to develop new ther-
apeutic agents to overcome the limitations with the current ther-
apy. In vitro studies, using a variety of human cell lines, have
been employed to evaluate the effectiveness of new medicinal
compounds against these cancers.
anti-inflammatory, hypotensive, hypocholesterolemic, and diuretic
activities. Among these derivatives, 16,17-condensed steroidal pyr-
azolines have immense pharmacological significance [6–9,16].
Pregnenolone (1), a naturally occurring steroid, is known as a
precursor to other hormones, including cortisone, estrogen, testos-
terone, and progesterone [10,11]. Previously different pregneno-
lone derivatives have been synthesized, and evaluated for various
biological activities. The pregnenolone analogs, hydroxylated at
C-20, are known to effect calcium-dependent processes, and also
affect the degree of depolarization of smooth muscles [12]. The
hemisuccinate of pregnenolone-derivative significantly increases
the perfusion pressure, and vascular resistance in isolated rat heart
[13]. The nitrochlorambucil ester of pregnenolone exhibited a sig-
nificant cytotoxic activity towards brain posterior fossa, medulla-
blastoma (Daoy), and lung large cell carcinoma (H460) cell lines
[14].
Steroids form an important class of biologically active com-
pounds which exhibit diverse biological activities. Upto now, sev-
eral steroidal derivatives have been investigated as new curative
agents for cancers, and other diseases [3–5]. Several modified ste-
In a recent report, benzylidene, and pyrazoline derivatives of
pregnenolone were synthesized, and activity of pyrazoline deriva-
tives against the HT-29, HCT-15, 502713 cancer cell lines, has been
evaluated [16]. This study, conducted by Banday et al. has shown
that pregnenolone pyrazoline derivatives (10 compounds) possess
significant cytotoxicity against HT-29, and HCT-15, 502713 cell
⇑
Corresponding author. Tel.: +92 21 34824924; fax: +92 21 4819018.
E-mail address: afnan.iccs@gmail.com (F. Shaheen).
0039-128X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2011.09.006

M. Iqbal Choudhary et al. / Steroids 76 (2011) 1554–1559
1555
lines. It was further demonstrated that the cytotoxicity of the com-
pounds against cancer cell lines increases significantly with the
changes in the substitutions on the aromatic ring [16]. To further
evaluate the potential of pyrazoline, and other heterocylic analogs
of pregnenolone, the present study was conducted in which pyraz-
olines 18–76, hydrazones 77–84, pyrazoles 85–91, and oximes
HO
O
21
OH
O
N
18
20
O
c
N
12
11
17
16
15
19
10
C 13
D
H
H
9
14
H
1
d
8
7
2
3
H
H
H
H
H
A
H
B
5
6
4
HO
HO
HO
Oxime 100
Pregnenolone (1)
(O-Carboxymethyl) oxime 107
5''
4''
R1
a
6''
3''
1''
2''
3'
2'
18
1'
O
12
H
17
16
15
13
14
19
10
11
9
1
4
8
7
2
3
H
H
5
6
Benzylidines 2-17
(R1 in Table-1)
HO
c
d
5''
R1
4''
5''
4''
2''
R1
HO
6''
1''
6''
b
3''
2''
1'''
2'''
3''
O
1''
3'
3'
OH
O
2'
2'
18
18
N
1'
N
1'
12
H
12
H
17
17
19
13
19
11
9
13
11
9
16
15
16
14
14
15
1
4
1
4
8
7
2
3
10
8
7
2
3
10
H
6
H
H
6
H
5
5
HO
HO
Oximes 92-99
(R1 in Table-5)
(O-Carboxymethyl) oximes 101-106
R1
(R1 in Table-6)
4''
4''
4''
5''
6''
5''
6''
5''
3''
3''
3''
3'''
3'''
6'''
R1
3'''
6'''
R1
6''
R1
2''
2''
2'''
1'''
2'''
1'''
4'''
R2
2''
2'''
1'''
4'''
4'''
1''
1''
1''
R2
R2
5'
5'''
5'
5'''
3'
5'''
N
N
HN
N
4'
4'
1'
1'
N 2'
6'''
2'
18
18
N 2'
18
13
3'
3'
1'
12
H
12
H
17
12
H
17
16
15
19
19
17
13
14
13
14
11
9
19
11
9
11
9
16
15
16
14
15
1
4
1
4
1
4
8
7
8
H
2
3
10
2
3
10
5
10
8
7
2
H
6
H
H
H
6
H
5
7
6
3
5
HO
HO
HO
Pyrazolines 18-76
(R1 and R2 in Table-2)
Hydrazones 77-84
(R1 and R2 in Table-3)
Pyrazoles 85-91
(R1 and R2 in Table-4)
Scheme 1. Synthesis of pregnenolone derivatives. Reagents and conditions: (a) EtOH (25 mL), NaOH (3 mL, 4 M, in H2O), R1CHO (0.6 mmol) r.t., 3 h; (b) EtOH (06 mL),
phenylhydrazine 1.5 eq. molar, HCl, 90 °C, 22 h; (c) NH2OH, pyridine, stirring for 22 h; (d) NH2OC.H2CO2H.HCl, pyridine, stirring for 24 h.

1556
M. Iqbal Choudhary et al. / Steroids 76 (2011) 1554–1559
4a0
92–107, and benzylidene 2–17 derivatives of pregnenolone were
synthesized, and evaluated against HepG2, and MDA-MB-230 cell
lines.
= 16.5–17.5 Hz), respectively. H-50 of the pyrazoline ring was
0
4a0
resonated as a double doublet between d 4.8–5.6 (J5 ,
= 8.2–
0
0
9.3 Hz, J5 , 4b = 10.5–11.2 Hz).
Reactions of benzylidene derivatives 2–17 with aromatic hydra-
zines, such as 2-nitro- and 2,4-dinitrophenyl hydrazines, produced
hydrazones 77–84 (Scheme 1; Table 3). The structures of all hydra-
zone derivatives were deduced from the IR and 1H NMR spectra. No
pyrazoline formation was detected in cases of dinitro derivatives
due to the strong electron withdrawing effect of NO2 groups in
phenylhydrazine, which decreases the electron density at NH,
and inhibits the formation of pyrazoline ring from hydrazone
derivatives.
The reactions of benzylidene derivatives 2–17 with phenylhydr-
azines bearing electron-donating groups such as 4-methoxy-, and
2-ethyl groups, and unsubstituted phenylhydrazine produced pyr-
azole derivatives 85–91 (Table 4). These products were character-
ized by IR and 1H NMR spectroscopy. In the 1H NMR spectra of the
pyrazoles, H-40 of the pyrazole ring was resonated as a singlet be-
tween d 6.2–6.4.
2. Results and discussion
By using pregnenolone as a template, libraries of pregnenolone
derivatives were created by parallel synthesis (Scheme 1). From
the screening of a total of 107 compounds, furanyl-bearing enone
8, and its oxime derivative 95 were found to be significantly active
against the Hep2 cell lines, while its pyrazoline derivative 48, was
strongly active against the MDA-MB-230 cell line.
3. Benzylidene derivatives 2–17
The synthesis of benzylidene derivatives of pregnenolone has
previously been reported [15,16]. In the present study, pregneno-
lone (1) was initially converted into a series of benzylidene deriv-
atives 2–17 by parallel synthesis. These derivatives were evaluated
against HepG2, and MDA-MB-231 cancer cell lines (Table 1). Deriv-
atives 2–17 were used in the synthesis of pregnenolone derivatives
18–106.
5. Pregnenolone oxime derivatives 92–100
Oxime derivatives 92–100 (Table 5) and the (O-carboxymethyl)
oxime derivatives 101–107 of pregnenolone (1) were synthesized
by adding hydroxylamine, and the (O-carboxymethyl) hydroxyl-
amine, respectively, in the presence of pyridine to the solution of
corresponding benzylidene derivatives 2–17, as shown in
Scheme 1. After the completion of the reaction, the mixtures were
poured in the acidic water to neutralize the pyridine. The products
were extracted with dichloromethane from acidic solution. The
analytical, and spectral data (1H NMR, and EI-MS) of all the prod-
ucts were in good agreement with the proposed structures.
4. Pregnenolone pyrazoline derivatives 18–76
The synthesis of 17-pyrazolinyl derivatives of pregnenolones
has previously been reported [15,16]. Banday et al. has also re-
ported the cytotoxicity of 17-pyrazolinyl derivatives of pregnenol-
ones against a panel of seven human cancer cell lines [16]. In the
reported study, a series of 10 N-acetyl pyrazolinyl derivatives of
pregnenolone were synthesized by reacting different benzylidene
derivatives of pregnenolone with hydrazine in the presence of ace-
tic acid. However in the present study, 59 pyrazoline derivatives of
pregnenolone 18–76 (Table 2) were synthesized by treating differ-
ently substituted phenylhydrazines with 16 different benzylidene
derivatives 2–17, in the presence of hydrochloric acid through par-
allel synthesis approach (Scheme 1). The pyrazoline derivatives
18–76 were obtained in moderate yields (40–60%). Analytical
and spectral data (IR, 1D, 2D-1H NMR and 13C NMR) of these deriv-
atives was in good agreement with the proposed structures. In the
1H NMR spectra of all pyrazolines, H-4a0 and H-4b0 resonated as a
6. X-ray structure of compound 107
Single-crystal X-ray diffraction analysis was carried out to de-
duce the structure of compound 107 (Scheme 1). The asymmetric
unit comprised on two independent molecules of 107. The ORTEP
diagrams of 107 (Fig. 1) showed four trans fused rings A, B, C,
and D with chair/half chair/chair, and envelop conformation, respec-
tively. The C-3 OH exists in equatorial orientation, while C-20/N-1
0
0
0
pair of a doublet of doublets between d 4.2–3.1 (J4a , 5 = 10.5 Hz, J4a ,
0
0
4b0
50
= 16.5–17.5 Hz), and 3.3–3.5 (J4b ,
= 10.5–11.5 Hz, J4b ,
Table 1
Benzylidene derivatives 2–17.
Product
R1
Yield %
IC50 (lM)
HepG2
MDA-MB-231
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Ph
94
72
93
85
47
42
89
43
56
78
73
72
81
89
87
88
16.25
>40.0
20.91
18.57
>40.0
>40.0
0.74
>40.0
>40.0
>40.0
>40.0
>40.0
20.02
12.49
>40.0
4.49
>40.0
>40.0
>40.0
30.83
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
15.80
>40.0
5.01
4-Phenoxyphenyl
4-Methylphenyl
3-Nitrophenyl
4-Methoxyphenyl
3-Chlorophenyl
2-Furyl
2-Naphthalenyl
2-Thienyl
3,4,5-Trimethoxyphenyl
4-Ethoxyphenyl
2-Chlorophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
2,4-Dichlorophenyl
2-Pyridinyl
Pregnenolone (1)
Doxorubicin
28.97
0.63
>40.0
1.23
Fig. 1. ORTEP diagram of final X-ray model of compound 107 (H-atoms are omitted
for clarity).

M. Iqbal Choudhary et al. / Steroids 76 (2011) 1554–1559
1557
oxime bond exists in Z configuration. All the bond angles and
lengths were within the normal range.
breast cancer cell line (MDA-MB-231). On the other hand, com-
pound 17 bearing a pyridinyl moiety, was highly cytotoxic against
both the cell lines.
The cytoxocity data of pyrazoline derivatives revealed that pyr-
azoline derivatives bearing furanyl ring (40, 42–44, and 49–50)
were active compounds. While the pyrazoline 48 bearing furanyl
7. SAR study
Pregnenolone (1) showed cytotoxicity against the HepG2
(IC50 = 28.97 lM/mL), while it was found to be inactive against
MDA-MB-230 cancer cell lines. Among the enone derivatives, com-
pound 8 with a furanyl group was most active against HepG2 cell
ring, was the most active compound (IC50 = 0.91 lM/mL) against
human breast cancer cell MDA-MB-231. All other pyrazolines, de-
rived from 4-methylpheny, 3-nitrophenyl, 3-chlorophenyl, 4-eth-
oxyphenyl, 2-chlorophenyl, and 3,4-dichlorophenyl-bearing
benzylidenes, were found to be inactive (Table 2). On the other
hand, all pyrazole and hydrazone derivatives did not show any sig-
nificant activity against both cancer cell lines (Tables 3 and 4).
Among the oxime derivatives (Table 5), compound 95 was
selectively active against the HepG2 cell line, while compound 98
showed activity against both the cancer cell lines. Interestingly all
(O-carboxymethyl) oxime derivatives were found to be inactive
against both the cancer cell lines (Table 6).
The purity level of cytotoxic compounds was determined by
Shimadzo analytical HPLC 10 A using UV Detector at the wave-
length of 256 nm (Table 7).
In conclusion, the synthesis and biological evaluation of a new
series of pregnenolone derivatives is reported. Among the enone,
pyrazoline, oxime, pyrazole, and hydrazone derivatives of pregnen-
line (IC50 = 0.74 lM/mL). It was found to be inactive against human
Table 2
Pyrazolines functionalized pregnenolones 18–76.
No.
R1
R2
IC50 (lM)
HepG2
MDA-MB-231
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3-Nitrophenyl
3-Nitrophenyl
3-Nitrophenyl
3-Nitrophenyl
3-Nitrophenyl
3-Nitrophenyl
3-Nitrophenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
4-Methylphenyl
2-Furyl
2-Furyl
2-Furyl
2-Furyl
2-Furyl
3-Chlorophenyl
3-Chlorophenyl
3-Chlorophenyl
2-Furyl
2-Furyl
2-Furyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
4-Ethoxyphenyl
2-Chlorophenyl
2-Chlorophenyl
2-Chlorophenyl
2-Chlorophenyl
2-Chlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
3,4-Dichlorophenyl
2-Pyridinyl
4-Bromophenyl
4-Chlorophenyl
2-Bromophenyl
4-Flourophenyl
2-Chlorophenyl
Phenyl
2-Nitrophenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methoxyphenyl
2-Chlorophenyl
2-Bromophenyl
Phenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methoxyphenyl
2-Chlorophenyl
2-Bromophenyl
Phenyl
2-NitroPhenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
2-Chlorophenyl
2-Bromophenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
Phenyl
2,4-Dichlorophenyl
4-Methylphenyl
4-Flourophenyl
2-Chlorophenyl
2-Bromophenyl
2-Nitrophenyl
4-Flourophenyl
4-Methoxyphenyl
2-Chlorophenyl
2-Bromophenyl
Phenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methoxyphenyl
2-Chlorophenyl
2-Bromophenyl
Phenyl
2-Ethylphenyl
2,4-Dichlorophenyl
4-Flourophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methoxyphenyl
2-Chlorophenyl
2-Bromophenyl
Phenyl
>40.0
33.77
>40.0
14.85
24.42
14.05
26.80
16.45
>40.0
>40.0
32.34
>40.0
26.73
30.34
>40.0
>40.0
>40.0
>40.0
27.69
>40.0
>40.0
22.81
14.25
>40.0
6.63
>40.0
20.83
>40.0
11.06
26.95
9.65
19.86
16.45
>40.0
>40.0
>40.0
>40.0
30.67
28.93
>40.0
>40.0
>40.0
>40.0
22.08
>40.0
>40.0
33.24
12.88
>40.0
11.12
18.87
6.65
Table 3
Hydrazone derivatives 77–84.
No.
R1
R2
HepG2
IC50 M)
MDA-MB-231
(
l
77
78
79
80
81
82
83
84
2-Furyl
2-Furyl
Phenyl
3-Nitrophenyl
3-Nitrophenyl
4-Methylphenyl
3,4-Dichlorophenyl
2-Pyridinyl
2-Nitrophenyl
29.68
>40.00
>40.00
>40.00
>40.00
>40.00
>40.00
16.28
19.66
>40.00
>40.00
>40.00
>40.00
>40.00
>40.00
16.28
2,4-Dichlorophenyl
2,4-Dinitrophenyl
2-Nitrophenyl
2,4-Dinitrophenyl
2,4-Dinitrophenyl
2-Nitrophenyl
2-Nitrophenyl
8.82
18.25
>40.0
>40.0
>40.0
>40.0
11.03
13.49
>40.0
29.10
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
16.12
32.47
23.56
>40.0
25.95
21.55
18.40
27.91
32.42
>40.0
>40.0
0.91
Table 4
Pyrazole derivatives 85–91.
No.
R1
R2
HepG2
MDA-MB-231
12.93
10.44
>40.0
29.10
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
>40.0
16.12
32.47
23.56
>40.0
25.95
23.21
18.38
18.49
IC50 (lM)
85
86
87
88
89
90
91
Phenyl
Phenyl
2-Furyl
2-Furyl
2-Furyl
3,4-Dichlorophenyl
2-Pyridinyl
4-Methoxyphenyl
Phenyl
4-Chlorophenyl
4-Methoxyphenyl
Phenyl
>40.00
25.57
24.43
>40.00
>40.00
>40.00
17.94
>40.00
18.69
18.64
>40.00
>40.00
>40.00
17.94
4-Methoxy
2-Ethylphenyl
Table 5
Cytotoxic activity of oximes 92–100.
S. no
R
IC50
(lM)
HepG2
MDA-MB-231
92
94
95
96
97
98
99
100
Phenyl
3-Nitrophenyl
2-Furyl
2-Chlorophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
2-Pyridinyl
–
13.30
25.32
5.09
26.69
>40.00
>40.00
a
a
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
2-Pyridinyl
–
–
18.05
4.50
>40.00
13.77
28.97
0.63
19.83
6.76
>40.00
29.30
>40.00
1.23
Pregnenolone (1)
Doxorubicin
2,4-Dichlorophenyl
a
Not tested.

1558
M. Iqbal Choudhary et al. / Steroids 76 (2011) 1554–1559
Table 6
ture was stirred for three hrs, and then neutralized by adding 2 M
HCl. The precipitates were filtered, washed with acidic water
(10 mL ꢀ 2), and neutral water (20 mL), and dried in vacuum.
Cytotoxicity of (O-carboxymethyl) oximes derivatives 101–106.
S. no.
R
IC50 (lM)
HepG2
MDA-MB-231
8.2. General procedure for pyrazoline derivatives 18–76
101
102
103
104
105
106
107
4-Methylphenyl
2-Chlorophenyl
3,4-Difluorophenyl
3,4-Dichlorophenyl
2-Pyridinyl
>40.00
>40.00
24.52
>40.00
>40.00
16.44
>40.00
28.97
0.63
>40.00
>40.00
34.52
>40.00
>40.00
26.92
>40.00
>40.00
1.23
One drop of hydrochloric acid (11.0 mol) was added to the solu-
tion of each benzylidene derivatives 2–17, each 100 mg) in ethanol
(6 mL). Phenyl hydrazine (1.5 eq molar) was added to the resulting
solution, and refluxed overnight at 90 °C. After completion of reac-
tion, the solvent was evaporated under vacuo, and purified by SiO2
gel column chromatography.
2,4-Dichlorophenyl
–
Pregnenolone (1)
Doxorubicin
8.3. General procedure for oxime derivatives 92–99
Each benzylidene derivative (100 mg) in pyridine (2.2 mL) was
combined with hydroxylamine hydrochloride (2.5 eq) and stirred
for 22 h. Reaction mixture was poured into a solution containing
pre-cooled 10% aq. HCl (10 mL). The product was extracted with
dichloromethane (3 ꢀ 15 mL). The organic extracts were com-
bined, and washed with saturated aqueous NaCl (3 ꢀ 10 mL), dried
over anhydrous magnesium sulfate, and the solvent was removed
in vacuo. Oximes 92–99 were purified by thin layer
chromatography.
Table 7
The purity level of the cytotoxic compounds.
Compound no.
% Purity
8
17
48
98
91.8
87.7
89.3
98.0
olone (1), the heterocyclic enone 8, and its pyrazoline derivative 48
were found to be the most active compounds. All (O-carboxy-
methyl) oxime, hydazones and pyrazole derivatives of pregneno-
lone did not show any significant activity against both cell lines.
8.4. (O-Carboxymethyl) oxime derivatives of enones 100–106
The benzylidene derivative (250 mg) in pyridine (100 mL) was
combined with amino oxy-acetic acid hemihydrochloride
(280 mg), and stirred at 23 °C. After 24 h, the reaction mixture was
poured into a solution containing 10% aq HCl (30 mL), and precoold
in ice. The product was extracted with ethyl acetate (3 ꢀ 50 mL). The
organic extract was washed with saturated solution of NaCl
(3 ꢀ 30 mL), dried over anhydrous MgSO4, and concentrated in
vacuo.
8. General experimental method
Parallel synthesizer (Smart Start Synthesizer with 16 reaction
vessels) was purchased from Chemspeed Ltd., Switzerland. Re-
agents were purchased from Wako Pure Chemical Industries Ltd.,
Japan. Pregnenolone (1) was supplied by Acros Organics, New
Jersey, USA, and CD3OD were purchased from the E. Merck
(Germany). The other organic solvents were obtained from Fisher
Scientific Limited, UK, and were used as received. Absolute ethanol
was obtained by distillation of ethanol over anhydrous calcium
oxide. Flash chromatography was performed on E. Merck (60
230–400 mesh silica gel). Thin layer chromatography was per-
formed on 0.25 mm silica gel plates (E. Merck, 60F254). Visualiza-
tion of the TLC plates was achieved under the UV light at 254
and 366 nm and by spraying with ceric sulfate reagent. The solvent
system n-hexane-ethyl acetate (3:2) was used as eluent. The IR
spectra were recorded on a Shimadzu FTIR-8900 spectrophotome-
Crystal data of 107: C23H36NO4, Mr = 390.53, monoclinic, space
group P21, a = 6.5950(11) Å, b = 11.3801(18) Å, c = 29.257(4) Å,
V = 2182.9(6) Å3, Z = 4,
q
calc = 1.185 mg/m3, F(000) = 848,
l
(Mo
Ka) = 0.71073 Å, max/min transmission 0.9960/0.9641 crystal
dimensions 0.46 ꢀ 0.23 ꢀ 0.05, 0.70 < h < 25.5, 12,988 reflections
were collected, of which 4240 reflections were judged
(Rint = 0.0477). The R values were: R1 = 0.0505, wR2 = 0.1165 for
I > 2r(I), and R1 = 0.0910, wR2 = 0.1476 for all data; max/min resid-
ual electron density: 0.223/ꢁ0.136 eÅꢁ3. Crystallographic data for
compound 107 has been deposited in the Cambridge Crystallo-
graphic Data Center. These information can directly be obtained free
of charge from CCDC data center (CCDC 836728 reference code).
ter. Melting point was taken on BUCHI 535. [a] was recorded on
D
Hitachi U-3200 spectrophotometer. EI- and HREI-MS were re-
corded on JMS HX 110 and on JMS-DA 500 mass spectrometers.
The 1H and 13C NMR spectra were recorded on Bruker NMR spec-
trometers, operating at 300, 400, and 500 (75, 100, and 125 for
13C). The chemical shifts values are reported in ppm (d) units,
and the coupling constants (J) are given in Hz. Single-crystal X-
ray diffraction data was collected on Bruker Smart APEX II, CCD
4-K area detector diffractometer [17]. Data reductions was per-
formed by using SAINT program. The structure was solved by di-
rect method [18], and refined by full-matrix least squares on F2
by using the SHELXTL-PC package [19]. The figures were plotted
with the aid of ORTEP program [20].
8.5. Cytotoxicity assay
Human breast (MDA-MB-231), and liver (HepG2) cancer cell
lines were obtained from the American Type Culture Collection
(ATCC), USA. Cell viability was measured by the MTT [3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide] colorimet-
ric method. After drug treatment, the attached cells were incubated
for 4 h with MTT (0.5 mg/mL, 1 h). The medium was removed, and
DMSO was added to dissolve the formazan crystals. The absor-
bance at 550 nm was then measured by using an ELISA microplate
reader.
Acknowledgment
8.1. General procedure for benzylidene derivatives 2–17
We are grateful to the Higher Education Commission, Pakistan,
for the research project entitled, ‘‘Development of New Antiviral
and Anticancer Steroids by Combinatorial Synthesis and High-
The ethanol (25 mL) and NaOH (4 M, 3 mL) were mixed, cooled in
an ice bath, and then compound 1 (0.3 mmol, 100 mg) was added.
The aromatic aldehyde (0.6 mmol) was added dropwise. The mix-
throughput Biological Screening-
a New Approach to Drug

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