Synthesis and characterization of steroidal heterocyclic compounds, DNA condensation and molecular docking studies and their in vitro anticancer and acetylcholinesterase inhibition activities

Article abstract of DOI:10.1039/c5ra11049a

A facile, convenient and efficient approach for the synthesis of a new series of steroidal heterocyclic compounds (4-12) by reacting a mixture of compounds (1e-3e) with o-aminothiophenol/o-aminophenol/o-phenylenediamine is reported. The structural assignment of products is confirmed on the basis of IR, ¹H NMR, ¹³C NMR, MS and analytical data. The compounds obey the Lipinski's 'Rule of Five' analysis based on computational prediction and pharmacokinetic properties. The anticancer activity has been tested in vitro against three cancer cell lines Hep3B (human hepatocellular carcinoma), MCF7 (human breast adenocarcinoma), HeLa (human cervical carcinoma) and one non-cancer normal cell i.e. PBMCs (peripheral blood mononuclear cell) by MTT assay. In addition, the synthesized compounds are also tested for their in vitro antioxidant activity by various reported methods in which compounds 10-12 exhibited good antioxidant activity. Nonenzymatic degradation of DNA has been investigated. The acetylcholinesterase (AChE) inhibitor activities of the steroidal derivatives are also evaluated using Ellman's method. Moreover, the application of compound 6 as a DNA gene transporter is evaluated by DNA condensation and ascertained by employing TEM and AFM, which illustrate that the compound 6 induces the condensation of CT-DNA. Molecular docking studies further characterize the interaction of the synthesized compounds with DNA. 2015

Full text of DOI:10.1039/c5ra11049a

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PAPER
Synthesis and characterization of steroidal
heterocyclic compounds, DNA condensation and
molecular docking studies and their in vitro
anticancer and acetylcholinesterase inhibition
activities†
Cite this: RSC Adv., 2015, 5, 75964
Abad Ali,a Mohd Asif,a Hena Khanam,a Ashraf Mashrai,a Mohd Asif Sherwani,b
b
a
*
Mohammad Owais and Shamsuzzaman
A facile, convenient and efficient approach for the synthesis of a new series of steroidal heterocyclic
compounds (4–12) by reacting mixture of compounds (1e–3e) with o-aminothiophenol/o-
a
aminophenol/o-phenylenediamine is reported. The structural assignment of products is confirmed on
the basis of IR, 1H NMR, 13C NMR, MS and analytical data. The compounds obey the Lipinski's ‘Rule of
Five’ analysis based on computational prediction and pharmacokinetic properties. The anticancer activity
has been tested in vitro against three cancer cell lines Hep3B (human hepatocellular carcinoma), MCF7
(human breast adenocarcinoma), HeLa (human cervical carcinoma) and one non-cancer normal cell i.e.
PBMCs (peripheral blood mononuclear cell) by MTT assay. In addition, the synthesized compounds are
also tested for their in vitro antioxidant activity by various reported methods in which compounds 10–12
exhibited good antioxidant activity. Nonenzymatic degradation of DNA has been investigated. The
acetylcholinesterase (AChE) inhibitor activities of the steroidal derivatives are also evaluated using
Ellman's method. Moreover, the application of compound 6 as a DNA gene transporter is evaluated by
DNA condensation and ascertained by employing TEM and AFM, which illustrate that the compound 6
induces the condensation of CT-DNA. Molecular docking studies further characterize the interaction of
the synthesized compounds with DNA.
Received 10th June 2015
Accepted 2nd September 2015
DOI: 10.1039/c5ra11049a
www.rsc.org/advances
modifying of their molecular properties. The major advantage of
steroids is their rigid skeleton, variability of substitution and
their similarity to naturally occurring structures.7 The synthesis
Introduction
Steroidal compounds have drawn attention not only due to
unusual and interesting chemical structures, but also due to
their widespread application as anti-inammatory, diuretic,
anabolic, contraceptive, antiandrogenic, progestational and
anticancer agents.1 In the global drug market, steroid drugs rank
second only aer antibiotics.2 Most steroidal drugs in use today
are semi-synthetic compounds and widely used in traditional
medicines by the modication of the steroid ring system and
side chains.3–6 Steroids regulate a wide variety of physiological
and developmental processes in vertebrates and are used as the
building block in the construction of larger molecules with
sufficient rigidity and with suitable substitution for the further
and physiological activity of hetero-steroids have received a lot of
attention over the decades by medicinal chemists.8,9 The inter-
esting structural and stereochemical features of the steroid
nucleus provide additional fascination to the researchers, and
thereby the introduction of heteroatom, heterocycle or replace-
ment of one or more carbon atoms in the steroidal skeleton has
been envisaged to discover new chemical entities with a poten-
tial to afford some promising drugs of the future and brings
notable modications of its biological activity.10–12 The incorpo-
ration of a heterocyclic ring and/or a heteroatom in the steroid
backbone affects the chemical properties of a steroid, the
development of new types of activity mainly depending on the
position of heterocyclic ring on the steroid nucleus and oen
results in useful alterations in its biological activities. As a result,
researchers are on a continuous search to design and produce
better hetero-steroids.13,14 Based on all these facts and in
continuation of our earlier studies of hetero-steroidal
compounds,15 herein we report the synthesis of steroidal
heterocyclic compounds (4–12) which are also investigated for
aSteroid Research Laboratory, Department of Chemistry, Aligarh Muslim University,
Aligarh 202 002, India. E-mail: shamsuzzaman9@gmail.com; Tel: +91 9411003465
bInterdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002,
India
† Electronic supplementary information (ESI) available: Copy of 1H NMR, 13C
NMR and HRMS spectra of all synthesized compounds. See DOI:
10.1039/c5ra11049a
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their antioxidant, anticancer, AChE inhibition, DNA gene 1e–3e with o-aminothiophenol/o-aminophenol/o-phenylenedi-
transporter and molecular docking studies.
amine in DMSO.16
The structures of the products were established by means of
their IR, 1H NMR, 13C NMR, MS and analytical data. The
selected diagnostic bands in the IR spectra of synthesized
compounds provide useful information for determining their
structures. The absorption bands at 1627–1637 cmꢀ1 (C]N),
682–687 cmꢀ1 (C–S–C), 1050–1061 cmꢀ1 (C–O) and 1310–1329
cmꢀ1 (C–N) conrmed the formation of thiazole, oxazole and
imidazole ring systems in products (4–12). The absorption
bands at 1560–1569 cmꢀ1, 1387–1397 cmꢀ1, 3081–3089 cmꢀ1
Results and discussion
Chemistry
Development of highly functional molecules from simple
building blocks has always attracted the curiosity of synthetic
chemists. So we herein report a convenient route for the
synthesis of steroidal heterocyclic compounds 4–12. All the
compounds (Scheme 1) were prepared by reuxing compounds
Scheme 1 Schematic pathway for the formation of steroidal compounds (4–12).
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are attributed to the aromatic ring in the products 4–12 and the Lipinski rules due to a calculated C log P value above the limit of
bands in the range 3315–3342 cmꢀ1 conrm the presence of NH 5 and the molecular weight above 500 (Table 1).
in compounds 10–12. In their 1H NMR study the downeld singlet
On the basis of the above results, we can say the compounds
at d 5.3–4.4 was ascribed to NH of imidazole ring. In 13C NMR obeyed the Lipinski's ‘Rule of Five’ analysis based on compu-
spectra, the signals at d 159.2–156.4, 153.5–150.2, 149.2–145.6, tational prediction of molecular and pharmacokinetic proper-
160.5–137.6 conrm the presence of N]C–S, N]C–O, N]C–NH ties,18 it was found that the synthesized compounds have good
and C]N groups in the products. A conceivable reaction mech- oral absorption. The exceptions to the Lipinski's rule are
anism for the synthesis of compounds 4–12 is represented in recognized and involve anticancer drugs such as doxorubicin.19
Scheme 2. Finally the presence of distinct molecular ion peak The physico-chemical properties of the synthesized compounds
[M+c] at m/z: 679, 655/657, 621, 663, 639/641, 605, 662, 638/640, are reasonable starting points for a drug discovery effort. The
604 and 574, 550/552, 516 in the MS spectra also proved the
formation of the compounds (4–12 and 1e–3e, respectively). This
strategy can also be applied to further modications on the
steroidal substituted heterocyclic systems.
Table 1 Calculated physicochemical properties of steroidal hetero-
cyclic derivatives (4–12)
Comp.
Mw
C log P
HBD
HBA
TPSA
No violations
Pharmacology
4
5
6
7
8
9
10
11
12
680.01
656.42
621.97
663.94
640.35
605.91
662.96
639.37
604.92
9.59
9.69
9.72
9.45
9.57
9.60
9.42
9.54
9.57
0
0
0
0
0
0
1
1
1
5
3
3
6
4
4
6
4
4
63.92
37.62
37.62
77.06
50.76
50.76
79.71
53.41
53.41
2
2
2
2
2
2
2
2
2
Rule of Five and bioactivity score. The use of Lipinski's rule
as a lter to choose the reasonable scaffolds for biological
activity is well known.17 The rule states that most molecules with
good membrane permeability have log P # 5, molecular weight
# 500, number of hydrogen bond acceptors # 10, number of
hydrogen bond donors # 5 and polar surface area less than
2
˚
140 A . The synthesized compounds showed two violations of
Scheme 2 A provisional mechanism for the synthesis of steroidal compounds (4–12).
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Table 2 Bioactivity score of steroidal heterocyclic derivatives (4–12)
Comp.
GPCR ligand
Ion channel
Kinase inhibitor
Protease inhibitor
Nuclear receptor ligand
Enzyme inhibitor
4
5
6
7
8
9
10
11
12
ꢀ0.65
ꢀ0.37
ꢀ0.33
ꢀ0.66
ꢀ0.39
ꢀ0.35
ꢀ0.48
ꢀ0.20
ꢀ0.16
ꢀ1.48
ꢀ1.14
ꢀ0.97
ꢀ1.48
ꢀ1.14
ꢀ0.97
ꢀ1.29
ꢀ0.93
ꢀ0.76
ꢀ1.34
ꢀ0.99
ꢀ0.84
ꢀ1.41
ꢀ1.06
ꢀ0.91
ꢀ1.20
ꢀ0.84
ꢀ0.68
ꢀ0.46
ꢀ0.38
ꢀ0.32
ꢀ0.55
ꢀ0.47
ꢀ0.42
ꢀ0.35
ꢀ0.27
ꢀ0.21
ꢀ0.90
ꢀ0.58
ꢀ0.43
ꢀ0.79
ꢀ0.46
ꢀ0.31
ꢀ0.81
ꢀ0.49
ꢀ0.34
ꢀ0.67
ꢀ0.40
ꢀ0.30
ꢀ0.69
ꢀ0.42
ꢀ0.32
ꢀ0.57
ꢀ0.29
ꢀ0.18
bioactivity scores of the synthesized compounds were also found to be effective against Hep3B cells. It might be concluded
calculated for six criteria, GPCR ligand activity, ion channel that substituents at 3b-position as well as heterocyclic moiety (S,
modulation, kinase inhibition activity, protease inhibitor, O, NH) played a key role in determining activity. It is manifested
enzyme inhibitor and nuclear receptor ligand activity (Table 2). from the data that chloro derivative (compound 5) with S in the
As a general rule, larger is the bioactivity score, higher is the heterocyclic ring showed marked inhibitory activity against
probability that investigated compound will be active. For Hep3B and MCF7 cell lines (IC50: 09.03 mM; Hep3B, 09.83 mM;
organic molecules if the bioactivity score is (>0.00), then the MCF7) while delta-5 derivative containing oxygen was most
compound is active, but if it is between ꢀ0.50 to 0.00 then the active against MCF7 (compound 9, IC50: 08.26 mM). This might
compound is moderately active20 and if the compound (<ꢀ0.50), be attributed to their differences in either polarity which
then it is inactive compound.21 The results of the present study in changes their lipophilicity or the conformation which alters the
Table 2, demonstrated that the some investigated compounds target protein binding properties present within the cell or on
are biologically moderately active molecules and some are inac- the cell membrane. Although the exact reason of such kind of
tive molecules.
behaviour of compounds is unknown and there is no denite
trend in structure activity relationship. Yet it gives useful
information regarding SARS.
Anticancer activity
The in vitro anticancer screening of steroidal heterocyclic
compounds (4–12) was done using human cancer cell lines
Hep3B, MCF7, HeLa and non-cancer PBMCs cells. Doxorubicin
(Dox) and 5-uorouracil (5-Fu) were used as cytotoxic drugs of
reference. A period of 48 h of drug exposure was chosen to test
cytotoxicity. The cytotoxicity (IC50) is the concentration in ‘mM’
required for 50% inhibition of cell growth as compared to that
of untreated control. The growth inhibitory effect of compounds
(4–12) towards the cancer cells was measured by means of MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay in which the cell viability was measured with the purple
formazan that was metabolized from MTT by mitochondrial
dehydrogenase, which is active only in live cells. IC50 of the
synthesized steroidal heterocyclic compounds 4–12 against
cancer cell lines as well as normal cells are detailed in Table 3,
whereas the bar graph of dose-dependent effects of the
compounds (4–12) as displayed in Fig. 1.
A number of correlations can be made from the data given in
the Table 3. It is evident from the IC50 values, that all the
compounds showed moderate to good activity while
compounds 5, 6, 8, 9 and 12 elicited a marked inhibitory activity
(IC50 < 19 mM) against all three cell lines.
All the compounds were found to be nontoxic to normal cells
(IC50 > 50 mM). It is noteworthy point that compound 6 was
found to be active against all the three cancer cell lines.
Compound 8 was specic to HeLa cells and compound 9
showed selectivity towards MCF7 cells while compound 12 was
Antioxidant activity
By DPPH assay. The in vitro antioxidant activity and scav-
enging effects of steroidal compounds 4–12 were evaluated by
using different reactive species assay containing DPPH radical
scavenging activity. The effect of antioxidants on DPPH radical
scavenging was thought to be due to their hydrogen donating
ability.22 DPPH is a stable free radical and accepts an electron or
hydrogen radical to become a stable diamagnetic molecule.23
Table 3 The cytotoxicity data of steroidal heterocyclic derivatives
(4–12)a
IC50 (mmol Lꢀ1
)
Comp.
Hep3B
MCF7
HeLa
PBMC
4
5
6
7
8
9
16.94 ꢁ 1.6
09.03 ꢁ 1.3
12.70 ꢁ 1.6
21.20 ꢁ 1.2
21.18 ꢁ 1.4
19.12 ꢁ 1.7
19.05 ꢁ 1.6
29.19 ꢁ 2.5
11.80 ꢁ 2.3
04.16 ꢁ 1.6
01.95 ꢁ 2.5
21.70 ꢁ 1.5
09.83 ꢁ 2.1
15.06 ꢁ 2.2
23.60 ꢁ 2.4
18.21 ꢁ 1.7
08.26 ꢁ 1.6
21.20 ꢁ 2.5
24.17 ꢁ 2.4
17.80 ꢁ 1.6
06.18 ꢁ 2.1
04.52 ꢁ 1.3
12.20 ꢁ 1.3
19.40 ꢁ 1.5
12.30 ꢁ 1.4
24.40 ꢁ 2.1
11.11 ꢁ 1.5
18.80 ꢁ 1.3
22.30 ꢁ 3.1
22.13 ꢁ 1.7
20.05 ꢁ 2.3
06.80 ꢁ 2.5
02.78 ꢁ 1.1
51.31 ꢁ 2.1
53.32 ꢁ 1.3
52.22 ꢁ 1.5
54.12 ꢁ 2.4
50.17 ꢁ 1.8
51.11 ꢁ 1.7
56.38 ꢁ 1.5
52.24 ꢁ 1.1
54.21 ꢁ 1.9
—
10
11
12
Doxa
5-Fub
—
a
Standard drugs used for reference: adoxorubicin, b5-uorouracil.
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Fig. 1 Dose-dependent effects of steroidal heterocyclic compounds (4–12) on cell viability of HeLa, Hep3B, MCF7 cancer cell lines and PBMCs
cell. Data shown are mean ꢁ standard error of at least three independent experiments.
The reduction capability of DPPH radicals was determined by a that cysteine, glutathione, ascorbic acid, tocopherol, poly-
decrease in their absorbance at 517 nm induced by antioxi- hydroxy aromatic compounds (e.g., hydroquinone, pyrogallol,
dants. The decrease in absorbance of DPPH radical caused by gallic acid), reduce and decolorize 1,1-diphenylpicrylhydrazyl
antioxidants, because of reaction between antioxidant mole- (DPPH) by their hydrogen donating capabilities. The free radical
cules and radical, progresses, which result in the scavenging of scavenging activity of all the synthesized compounds 4–12 were
the radical by hydrogen donation. It is visually noticeable as a evaluated through their ability to quench the DPPH. Using
discoloration from purple to yellow. It has been documented ascorbic acid as a reference. The potencies for the antioxidant
activity of compounds 4–12 to the reference compound are
shown in Table 4 and Fig. 2.
Table 4 The DPPH antioxidant activity of compoundsa (4–12)
% inhibition
Comp.
25 mg mLꢀ1
50 mg mLꢀ1
75 mg mLꢀ1
100 mg mLꢀ1
4
5
6
7
8
9
10
11
12
18.12 ꢁ 1.20 23.41 ꢁ 1.60 25.32 ꢁ 1.10 29.14 ꢁ 0.91
16.23 ꢁ 1.20 21.14 ꢁ 1.40 26.31 ꢁ 1.50 29.34 ꢁ 1.30
16.87 ꢁ 0.81 22.34 ꢁ 1.20 26.13 ꢁ 1.70 31.42 ꢁ 1.10
17.23 ꢁ 0.90 20.33 ꢁ 1.20 25.31 ꢁ 1.50 32.56 ꢁ 1.10
18.13 ꢁ 1.40 23.52 ꢁ 1.30 27.56 ꢁ 1.70 32.54 ꢁ 1.20
14.74 ꢁ 1.40 19.45 ꢁ 1.10 25.12 ꢁ 1.50 29.13 ꢁ 0.91
18.84 ꢁ 1.30 26.34 ꢁ 0.79 32.76 ꢁ 1.10 36.13 ꢁ 1.30
18.92 ꢁ 1.20 22.45 ꢁ 1.50 27.24 ꢁ 1.30 32.34 ꢁ 1.20
19.89 ꢁ 1.30 27.67 ꢁ 1.40 31.94 ꢁ 1.10 39.34 ꢁ 1.50
Standard 38.23 ꢁ 1.20 39.00 ꢁ 1.10 47.23 ꢁ 1.30 55.23 ꢁ 1.50
Controlb
—
—
—
—
Fig. 2 Concentration dependent antioxidant activities of steroidal
heterocyclic compounds (4–12). Data shown are mean ꢁ standard
error of at least three independent experiments.
a
Values represent the mean ꢁ standard error mean (SEM) of three
experiments. b No inhibition, standard: ascorbic acid.
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Table
5 The reducing power scavenging antioxidant activity of
scavenging activity of all the synthesized compounds 4–12 were
evaluated through their ability by radical quenching (H transfer)
using trolox as a reference and the free radical scavenging power
of compounds increased with an increasing concentration. The
reducing power scavenging activity of the compounds 4–12 to the
reference compound are shown in Table 5 and Fig. 3.
Among the tested compounds, many compounds showed
signicant reducing power capacities. But compounds 10–12
showed good reducing power capacities while all other
compounds exhibited moderate activity in comparison with the
standard trolox. The good reducing power capacity of these
compounds might be due to the presence of imidazole
functionalities.
compoundsa (4–12)
% inhibition
25 mg mLꢀ1
Comp.
50 mg mLꢀ1
75 mg mLꢀ1
100 mg mLꢀ1
4
5
6
7
8
9
10
11
12
23.12 ꢁ 3.20 35.41 ꢁ 2.60 52.32 ꢁ 3.10 62.14 ꢁ 3.91
24.23 ꢁ 2.20 44.14 ꢁ 3.40 58.91 ꢁ 2.50 63.34 ꢁ 3.30
20.87 ꢁ 2.81 33.54 ꢁ 2.20 57.53 ꢁ 3.70 65.56 ꢁ 4.10
21.23 ꢁ 1.90 35.33 ꢁ 2.20 52.31 ꢁ 3.50 65.16 ꢁ 4.10
22.13 ꢁ 2.40 43.32 ꢁ 2.30 56.56 ꢁ 2.70 62.24 ꢁ 3.90
18.74 ꢁ 2.40 39.45 ꢁ 2.10 52.12 ꢁ 3.50 72.13 ꢁ 3.91
26.84 ꢁ 1.30 51.34 ꢁ 2.79 58.76 ꢁ 3.10 74.13 ꢁ 3.30
22.92 ꢁ 2.20 44.45 ꢁ 3.50 55.24 ꢁ 2.30 67.34 ꢁ 3.20
29.89 ꢁ 2.30 49.45 ꢁ 2.40 69.34 ꢁ 3.10 79.34 ꢁ 3.50
Standard 39.23 ꢁ 1.20 51.01 ꢁ 1.10 77.23 ꢁ 1.30 85.23 ꢁ 1.50
Controlb
—
—
—
—
By ABTS assay
a
Values represent the mean ꢁ standard error mean (SEM) of three
experiments. b No inhibition, standard: trolox.
The ABTS radical cation decolorization assay is an excellent tool
for determining the antioxidant activity of hydrogen-donating
antioxidants and chain-breaking antioxidants.27,28 ABTS is
In general, all the synthesized compounds were less potent
than ascorbic acid as the reference. Among these compounds,
10, 11 and 12 exhibited a slightly better antioxidant activity,
with the strongest being observed in compound 10 and 12.
Table 6 The radical scavenging antioxidant activity of compoundsa
(4–12)
% inhibition
By ferric reducing antioxidant power (FRAP) assay
Comp.
25 mg mLꢀ1
50 mg mLꢀ1
75 mg mLꢀ1
100 mg mLꢀ1
The FRAP assay mainly depends on the reducing capacity of Fe3+
to Fe2+ conversion and serves as a signicant indicator of its
potential antioxidant activity. The antioxidant activities have been
attributed to various reactions, binding of transition metal ion
catalysts, decomposition of peroxides, prevention of continuous
proton abstraction and radical scavenging activity.24 It has been
also proved that the potential antioxidants through in vitro ferric
reducing antioxidant power assay increased the total antioxidant
capacity of blood plasma.25 In addition, the reducing capacity
measures the ease of the compounds in donating electrons that
terminates the oxidation chain reaction by reducing the oxidized
intermediates into the stable form.26 The reducing power
4
5
6
7
8
9
10
11
18.32 ꢁ 1.2
19.23 ꢁ 2.4
20.87 ꢁ 2.7
20.23 ꢁ 1.9
22.13 ꢁ 2.4
19.84 ꢁ 2.7
23.14 ꢁ 3.3
21.94 ꢁ 2.3
23.29 ꢁ 2.5
37.25 ꢁ 1.2
—
29.51 ꢁ 1.9
34.14 ꢁ 2.7
31.54 ꢁ 2.4
27.33 ꢁ 2.6
29.32 ꢁ 2.5
29.47 ꢁ 2.5
41.24 ꢁ 2.7
34.45 ꢁ 3.1
49.67 ꢁ 3.3
52.01 ꢁ 1.1
—
39.32 ꢁ 2.8
48.91 ꢁ 2.9
41.52 ꢁ 3.3
42.33 ꢁ 3.4
36.56 ꢁ 2.4
39.42 ꢁ 3.5
51.16 ꢁ 3.3
45.54 ꢁ 3.3
57.14 ꢁ 2.9
61.24 ꢁ 1.3
—
54.14 ꢁ 2.81
58.34 ꢁ 3.80
55.56 ꢁ 3.20
55.17 ꢁ 3.90
53.24 ꢁ 3.60
56.13 ꢁ 3.30
64.43 ꢁ 3.50
61.53 ꢁ 3.40
68.24 ꢁ 3.10
75.23 ꢁ 1.50
—
12
Standard
Controlb
a
Values represent the mean ꢁ standard error mean (SEM) of three
experiments. b No inhibition, standard: ascorbic acid.
Fig. 3 Concentration dependant FRAP assay of steroidal heterocyclic Fig. 4 Concentration dependant ABTS assay of steroidal heterocyclic
compounds (4–12). Data shown are mean ꢁ standard error of at least compounds (4–12). Data shown are mean ꢁ standard error of at least
three independent experiments.
three independent experiments.
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another widely used synthetic radical for both the polar and DNA condensation to compound
6
was determined by
non-polar samples. The ABTSc+ scavenging abilities of the employing various visualization techniques viz. transmission
compounds 4–12 were also evaluated and reported in Table 6.
electron microscopy (TEM) and atomic force microscopy
The radical has a relatively stable blue-green color, which (AFM).31 These techniques have been used here to analyze the
equates to absorbance at 734 nm. Antioxidant compounds reduce morphology of the steroidal-DNA complex condensates. The
the intensity of this color to a degree that is proportionate to their TEM images of the compound are given in Fig. 6.
antioxidant concentration or activity. Fig. 4, depicts the scav-
enging activities of the compounds 4–12 against ABTS radicals.
The shape of the particles is irregular and the particles are
of variable diameter ranging from 15.2 to 697 nm. The particles
These data demonstrate that scavenging activity increased as size increases when CT-DNA condensed to it indicating that
the concentration increased. The compounds 10–12 had a the compound 6 can condense fragments of CT-DNA to
maximum scavenging activity among all in the order of 12 > 10 > compact solid DNA drug condensate as shown in Fig. 6(a–d).
11. It was interesting to note that this order is in concordance Several numbers of drugs are unable to be effective due to the
with the DPPH and FRAP assay.
lack of information of their structural and morphological
details.32 To obtain the morphological and structural infor-
mation of the compound and the condensates, tapping mode
AFM experiments were performed using commercially etched
silicon tips as AFM probes. Two and three dimensional AFM
images of the compound and the condensate materials are
shown in Fig. 7.
The change in the morphological structure of compound 6
and its condensates with CT-DNA clearly validate that the
complex is facilitating the DNA condensation.
Fluorescence microscopy
HeLa cells treated with compounds 4–12 were observed under
Fluorescence Microscope and microscopic examination of gross
morphology of the treated and untreated cancer cells are shown
in Fig. 5. These images clearly designated reduction in cancer
cell count as well as cytotoxicity potential of above mentioned
steroids, in the order of 8 > 4 > 6 > 9 > 5 > 12 > 11 > 10 > 7 and
arrow indicates apoptosis of cells.
DNA condensation
Nonenzymatic DNA damage
DNA condensation is an essential process to transport a thera- The mechanisms governing the anticancer actions of steroid
peutic gene to its target. It includes the collapse of extended derivatives are not fully known. A number of pathways have been
DNA chain into compact, orderly particles containing one or studied regarding the cytotoxic activity of steroids.33 Our exper-
more molecule.29 Steroidal compounds are able to recognize iments suggested that the cell death may be due to cleavage or
and bind to single or double-stranded DNA with high affinity fragmentation of DNA of cancer cells and the active species
and selectivity due to structural hydrophobicity.30 In this study, responsible for this are ROS (cOH) which is conrmed from the
Fig. 5 Micrographs showing effect of steroidal compounds 4–12 on HeLa cells. Micrographs captured after 48 h of incubation in bright field
under Fluorescence Microscope. A ¼ untreated control, B ¼ treated with compound 7, C ¼ treated with compound 10, D ¼ treated with
compound 11, E ¼ treated with compound 12, F ¼ treated with compound 5, G ¼ treated with compound 9, H ¼ treated with compound 6,
I ¼ treated with compound 4 and J ¼ treated with compound 8.
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Fig. 6 The TEM images of the compound 6 before CT-DNA condensates (a) and after CT-DNA condensates (b–d).
in vitro reaction of different concentrations of compound 6 with promote DNA oxidation and the resulting damage enhanced by
copper in presence of thiobarbituric acid (Fig. 8).
packaging of DNA as a nucleosome.39
The results conrmed the relatively higher rate of formation
of hydroxyl radicals and correlated with the rate of DNA degra-
dation by the compound 6. The proposed mechanism of DNA
In vitro acetylcholinesterase inhibition activity
damage is believed to occur via redox cycling (Scheme 3). In the The anti-cholinesterase effects of the compounds (4–12) were
DNA cleavage reactions mediated by various antioxidants in the determined by modied Ellman's spectrophotometric method
presence of Cu(II), it has been well known that Cu(II) is reduced using AChE from Electrophorus electricus with tacrine as
to Cu(I) by the antioxidant which is an essential intermediate in reference compound. All the compounds were carefully
the DNA cleavage reactions.34–36 It is also generally understood measured and the results are shown in Table 7. The results were
that DNA cleavage by various antioxidants and Cu(II) is the result collected from at least three independent measurements. From
of the generation of hydroxyl radicals. Cu(II) is reduced to Cu(I) the results obtained, compound 4, 7 and 10 (IC50 ¼ 0.31 ꢁ 0.10,
and the re-oxidation of Cu(I) to Cu(II) by molecular oxygen gives 0.37 ꢁ 0.02 and 0.39 ꢁ 0.03, respectively) exhibited signicant
rise to superoxide anion which in turn leads to the formation of inhibition on AChE among all the compounds. The improved
H2O2.37 Studies have also shown that Cu(II) has a high affinity to activity of the acetoxy derivatives in comparison to all synthe-
DNA, preferentially binding to guanine residues at the N7 sized compounds can be explained on the basis of its skeleton
position.38 This Cu(II)–DNA interaction has been shown to and electronic properties at 3b-position of the cholestane ring.
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crystallography or NMR spectroscopic data. The conformational
diversity of DNA allows for large deformations upon binding of
ligands to it. Drug–DNA interactions are of high pharmaceutical
interest since the mode of action of various drugs is directly
associated with their binding to DNA. A reliable prediction of
drug–DNA binding at the atomic level by molecular docking
methods provides the basis for the design of new drug
compounds.40,41 Targeting the minor groove of DNA by small
molecules has long been considered as an important tool in
molecular recognition of specic DNA sequence.42,43 Molecular
modeling allows the exibility within the ligand to be modeled
that can utilize more detailed molecular mechanics to calculate
the relative binding energy of the ligand–receptor complex in the
context of the putative active site. In our experiment, structure of
compounds (4–12) was made exible to attain different confor-
mations in order to predict the best t orientation of compounds
with DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer
(PDB ID: 1BNA) which provides an energetically favorable docked
pose. Table 8 shows the minimized orientation of minor groove
interaction of compounds 4–12 with DNA and the resulting
relative binding energy of the docked pose. The minimized
conformation of only compound 10 sitting in the groove of the
sequence d(CGCGAATTCGCG)2 show hydrogen bonding with the
NH of the imidazole ring in the docked pose of the dodecamer.
While all other compounds do not show any possible hydrogen
bonding with the base pairs of the DNA dodecamer. Generally,
the more negative the relative binding energy, the stronger is the
interaction between molecule and DNA and more stable is the
complex formed. Thus the complex formed between compound
7 and DNA with relative binding energy of ꢀ8.338 kcal molꢀ1
might be having highest stability.44–47 The relative binding
energies of compound 10 and 4 with DNA are almost equal
suggesting that both are forming equally stable complex with
DNA and the presence of hydrogen bonds between compound 10
and DNA might not be affecting the stability of the complex.
Irrespective of the absence of any net positive charge on
compounds 4–12, negative values of the relative binding energies
indicated a higher binding potential of these compounds with
DNA. Hence, these docking results provide valuable information
about the mode of interaction of our compounds with DNA and
the conformation constraints for adduct formation.
Fig.
7 The AFM images of the compound 6 before CT-DNA
condensates (a) 2D (two dimensions) and (b) 3D (three dimensions)
and after CT-DNA condensates (c) 2D (two dimensions) and (d) 3D
(three dimensions).
Fig. 8 Determination of hydroxyl radical production of compound 6
by assay thiobarbituric acid.
Experimental
Materials and methods
The presence of acetoxy group at C-3 of cholestane skeleton
increases activity due to the formation of additional non-
classical bonds with amino acid residues of the protein and
easily performs as guest relation with receptor protein (host).
The synthesized compounds were found to be fairly active with
respect to the reference drug, tacrine.
All glass apparatus were oven-dried prior to use. Chemicals and
solvents used in this study were of ACS grade and used directly
without additional steps of purication. Melting points were
determined on a Biogen digital auto melting point apparatus.
The IR spectra were recorded on KBr pellets with Perkin Elmer
FT-IR Spectrometer spectrum Two and values are given in cmꢀ1
.
Molecular docking
1H and 13C NMR spectra were run in CDCl3 on a Bruker Avance
1
Molecular docking is one of the important computational II 400 NMR Spectrometer (operating at 400 MHz for H and at
chemistry techniques that are routinely used for drug discovery 100 MHz for 13C NMR) with TMS as internal standard and
processes. Molecular modelling studies of nucleic acids and values are given in parts per million (ppm) (d). Mass spectra
their complexes can provide valuable information that is not were recorded on a JEOL D-300 Mass spectrometer. Elemental
available by other experimental techniques such as X-ray analyses were recorded on Perkin Elmer 2400 CHN Elemental
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Scheme 3 Proposed mechanism of oxidative DNA damage of steroidal compounds.
Analyzer. Thin layer chromatography (TLC) plates were coated cold conditions. Aer completion of the reaction (monitored by
with silica gel G and exposed to iodine vapours to check the TLC), the precipitate thus obtained was ltered, washed with
homogeneity as well as the progress of reaction. Sodium sulfate water, air dried and recrystallized from methanol to afford
(anhydrous) was used as a drying agent.
respective products (1d–3d).51
General method for the synthesis of steroidal ketones (1c–3c)
General method for the synthesis of steroidal hydrazone
derivatives (1e–3e)
3b-Acetoxy-5a-cholestan-6-one,48 3b-chloro-5a-cholestan-6-one,49
5a-cholestan-6-one50 and compounds 1(a, b), 2(a, b) and 3(a, b)
were prepared according to the literature procedure.15f
To a solution of steroidal hydrazone (1d–3d) (1.5 mmol) in
absolute ethanol (20 mL), phthalaldehyde (1.5 mmol) was added.
Then the reaction mixture was stirred at 70 ꢂC. Aer completion
of reaction (monitored by TLC), solvent was evaporated under
General method for the synthesis of steroidal hydrazones (1d–3d)
The steroidal ketones (1c–3c) reacted with hydrazine hydrate in reduced pressure. The residue was puried by recrystallization to
ethanol solution in the presence of few drops of acetic acid in afford the corresponding target products (1e–3e).
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Table
7 In vitro anti-AChE activities data of the synthesized
anal. calcd for C35H52N2O; C, 81.34; H, 10.14; N, 5.42 found; C,
81.37; H, 10.12; N, 5.41; MS (EI): m/z 516 [M+c].
compounds (4–12) and reference drug (tacrine)
Comp.
AChE inhibitor IC50a (mM)
General method for the synthesis of steroidal heterocyclic
4
5
6
7
8
9
10
11
0.31 ꢁ 0.10
0.52 ꢁ 0.30
0.53 ꢁ 0.01
0.37 ꢁ 0.02
0.49 ꢁ 0.02
0.59 ꢁ 0.10
0.39 ꢁ 0.03
0.48 ꢁ 0.02
0.56 ꢁ 0.20
0.29 ꢁ 0.06
compounds (4–12)
A mixture of steroidal hydrazone derivatives (1e–3e) (1 mmol)
and o-aminothiophenol/o-aminophenol/o-phenylenediamine
(1.25 mmol, 1.25 equiv.) in DMSO (30 mL) was reuxed for 2–6
h.16 The progress as well as completion of reaction was moni-
tored by TLC. Aer completion of the reaction, the reaction
mixture was poured into water. The precipitate thus obtained
was ltered, washed with water and air dried. The crude product
was puried by column chromatography on silica gel column
using light petroleum ether/ether (10 : 1). For further purica-
tion, the compounds were recrystallized from methanol to give
desirable product (4–12).
12
Tacrine
a
The values are mean of three independent experiments ꢁ SD.
3b-Acetoxy-5a-cholestan-6-(iminohydrazono)-2-
methylbenzaldehyde (1e)
3b-Acetoxy-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-thiazole (4)
Yield (82%), mp: 166–168 ꢂC; IR (KBr, n cmꢀ1): 3080, 1567, 1394
(C–H aromatic), 1735 (OCOCH3), 1630 (C]N), 1057, 1059 (C–O),
1696 (–CHO); 1H NMR (400 MHz, CDCl3, ppm): d 7.9–8.3 (m, 5H,
aromatic), 10.4 (s, 1H, aldehyde), 4.77 (m, 1H, C3-aH, W1/2 ¼ 17
Hz, axial), 2.01 (s, 3H, OCOCH3), 1.15 (C10–CH3), 0.73 (C13–CH3),
0.91 & 0.84 (other methyl protons); 13C NMR (100 MHz, CDCl3,
ppm): d 194.6, 172.3, 162.7, 144.3, 137.6, 133.3, 131.9, 129.2,
128.7, 127.4, 71.1, 60.6, 58.3, 53.8, 43.6, 40.5, 39.2, 38.8, 36.3,
35.7, 34.6, 32.3, 31.9, 30.6, 29.2, 26.7, 25.4, 24.8, 23.5, 22.6, 21.4,
20.1, 19.5, 17.4, 16.3, 15.4, 13.7; anal. calcd for C37H54N2O3; C,
77.31; H, 9.47; N, 4.87 found; C, 77.33; H, 9.49; N, 4.85; MS (EI):
m/z 574 [M+c].
Yield (75%), mp: 156–158 ꢂC; IR (KBr, n cmꢀ1): 3085, 1569, 1396
(C–H aromatic), 1739 (OCOCH3), 1635 (C]N), 1050, 1054 (C–O),
687 (C–S–C); 1H NMR (400 MHz, CDCl3, ppm): d 7.7–8.0 (m, 5H,
aromatic), 4.75 (m, 1H, C3-aH, W1/2 ¼ 16 Hz, axial), 2.03 (s, 3H,
OCOCH3), 1.15 (C10–CH3), 0.73 (C13–CH3), 0.91 & 0.84 (other
methyl protons); 13C NMR (100 MHz, CDCl3, ppm): d 171.4,
161.2, 159.2, 151.1, 142.6, 135.7, 132.1, 130.2, 127.4, 126.8,
126.1, 125.9, 123.7, 122.9, 120.3, 119.9, 70.2, 58.3, 57.1, 53.8,
44.1, 42.5, 40.1, 38.1, 37.5, 35.9, 35.6, 33.2, 32.9, 28.8, 27.9, 26.1,
25.2, 24.9, 24.0, 22.9, 21.3, 21.9, 20.3, 19.7.0, 18.9, 15.1, 11.9;
anal. calcd for C43H57N3O2S; C, 75.95; H, 8.45; N, 6.18 found; C,
75.91; H, 8.49; N, 6.15; MS (EI): m/z 679 [M+c].
3b-Chloro-5a-cholestan-6-(iminohydrazono)-2-
methylbenzaldehyde (2e)
Yield (79%), mp: 160–162 ꢂC; IR (KBr, n cmꢀ1): 3081, 1560, 1388
(C–H, aromatic), 1627 (C]N), 740 (C–Cl), 1700 (–CHO); 1H NMR
(400 MHz, CDCl3, ppm): d 7.9–8.3 (m, 5H, aromatic), 10.5 (s, 1H,
3b-Chloro-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-thiazole (5)
Yield (72%), mp: 162–164 ꢂC; IR (KBr, n cmꢀ1): 3086, 1562, 1393
(C–H, aromatic), 1632 (C]N), 743 (C–Cl), 685 (C–S–C); 1H NMR
(400 MHz, CDCl3, ppm): d 7.90–8.2 (m, 5H, aromatic), 3.91 (m,
1H, C3-aH, W1/2 ¼ 14 Hz, axial), 1.15 (C10–CH3), 0.73 (C13–CH3),
0.91 & 0.84 (other methyl protons); 13C NMR (100 MHz, CDCl3,
ppm): d 160.3, 157.7, 152.6, 140.8, 135.7, 134.0, 130.3, 129.1,
127.0, 127.9, 126.7, 124.2, 122.9, 120.0, 118.2, 59.6, 57.6, 55.0,
52.4, 41.4, 40.7, 39.2, 38.5, 37.9, 36.5, 35.9, 34.6, 34.0, 33.9, 31.2,
29.5, 27.2, 26.8, 23.0, 22.9, 21.6, 20.1, 20.9, 17.3, 15.1, 13.7; anal.
calcd for C41H54ClN3S; C, 75.02; H, 8.29; N, 6.40 found; C, 75.05;
H, 8.25; N, 6.44; MS (EI): m/z 655/657 [M+c].
aldehyde), 3.71 (m, 1H, C3-aH, W1/2 ¼ 15 Hz, axial), 1.15 (C10
–
CH3), 0.73 (C13–CH3), 0.91 & 0.84 (other methyl protons); 13C
NMR (100 MHz, CDCl3, ppm): d 196.7, 159.8, 142.3, 139.4, 137.3,
135.8, 132.2, 127.6, 125.3, 58.7, 57.4, 55.8, 52.2, 47.4, 45.2, 41.5,
40.2, 37.1, 36.3, 35.7, 34.6, 32.4, 32.9, 31.1, 29.9, 26.7, 25.1, 23.3,
21.7, 20.6, 19.2, 17.8, 16.5, 14.9, 11.5; anal. calcd for
C
35H51ClN2O; C, 76.26; H, 9.33; N, 5.08 found; C, 76.24; H, 9.35;
N, 5.09; MS (EI): m/z 550/552 [M+c].
5a-Cholestan-6-(iminohydrazono)-2-methylbenzaldehyde (3e)
Yield (77%), mp: 155–157 ꢂC; IR (KBr, n cmꢀ1): 3085, 1564, 1391
5a-Cholestan-6-(iminohydrazonomethylphenyl-2-yl)-benzo-
[d]-thiazole (6)
(C–H, aromatic), 1631 (C]N), 1702 (–CHO); 1H NMR (400 MHz, Yield (71%), mp: 154–156 ꢂC; IR (KBr, n cmꢀ1): 3082, 1565, 1389
CDCl3, ppm): d 7.9–8.3 (m, 5H, aromatic), 10.4 (s, 1H, aldehyde), (C–H, aromatic), 1629 (C]N), 682 (C–S–C); 1H NMR (400 MHz,
1.15 (s, 3H, C10–CH3), 0.73 (s, 3H, C13–CH3), 0.91 & 0.84 (other CDCl3, ppm): d 7.90–8.2 (m, 5H, aromatic), 1.15 (s, 3H, C10–
methyl protons); 13C NMR (100 MHz, CDCl3, ppm): d 195.2, CH3), 0.73 (s, 3H, C13–CH3), 0.91 & 0.84 (other methyl protons);
160.5, 141.7, 135.2, 134.7, 132.2, 129.8, 127.4, 126.1, 57.9, 53.3, 13C NMR (100 MHz, CDCl3, ppm): d 158.7, 156.4, 151.7, 139.7,
43.0, 42.9, 39.2, 38.7, 37.9, 36.4, 34.8, 33.2, 32.4, 32.0, 30.7, 29.8, 135.9, 132.4, 130.9, 129.1, 127.4, 126.5, 125.9, 123.9, 122.6,
27.6, 26.1, 25.8, 22.9, 21.5, 20.4, 19.6, 18.5, 17.7, 15.3, 12.1, 11.2; 120.7, 118.9, 54.2, 52.9, 50.1, 45.3, 41.6, 40.2, 39.0, 37.7, 36.4,
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35.3, 34.8, 33.9, 30.3, 28.1, 27.3, 26.7, 24.1, 22.9, 21.6, 21.1, 20.6, protons); 13C NMR (100 MHz, CDCl3, ppm): d 169.9, 159.9,
19.5, 17.2, 16.9, 15.9, 11.5; anal. calcd for C41H55N3S; C, 79.18; 147.3, 144.6, 143.8, 139.2, 138.4, 132.7, 129.3, 128.1, 126.5,
H, 8.91; N, 6.76 found; C, 79.21; H, 8.87; N, 6.77; MS (EI): m/z 621 124.6, 123.1, 122.8, 113.7, 111.5, 71.5, 54.2, 52.6, 50.3, 42.8, 40.3,
[M+c].
39.2, 38.5, 37.5, 36.2, 35.9, 34.2, 33.7, 30.5, 29.7, 28.2, 26.4, 25.2,
24.9, 23.6, 22.5, 21.8, 20.4, 19.7, 18.2, 16.8, 14.2; anal. calcd for
C
43H58N4O2; C, 77.90; H, 8.82; N, 8.45 found; C, 77.86; H, 8.85;
3b-Acetoxy-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-oxazole (7)
N, 8.48; MS (EI): m/z 662 [M+c].
Yield (78%), mp: 175–177 ꢂC; IR (KBr, n cmꢀ1): 3083, 1564, 1390
(C–H, aromatic), 1736 (OCOCH3), 1631 (C]N), 1053, 1059 (C–
O); 1H NMR (400 MHz, CDCl3, ppm): d 8.0–8.3 (m, 5H,
3b-Chloro-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-imidazole (11)
aromatic), 4.79 (m, 1H, C3-aH, W1/2 ¼ 17 Hz, axial), 2.02 (s, 3H, Yield (77%), mp: 152–154 ꢂC; IR (KBr, n cmꢀ1): 3335 (NH), 3084,
OCOCH3), 1.15 (C10–CH3), 0.73 (C13–CH3), 0.91 & 0.84 (other 1566, 1387 (C–H, aromatic), 1633 (C]N), 1317 (C–N), 744 (C–
methyl protons); 13C NMR (100 MHz, CDCl3, ppm): d 173.4, Cl); 1H NMR (400 MHz, CDCl3, ppm): d 4.7 (s, 1H, NH,
163.4, 153.5, 145.2, 141.2, 139.5, 135.6, 130.1, 129.3, 127.9, exchangeable with D2O), 7.9–8.2 (m, 5H, aromatic), 3.72 (m, 1H,
126.4, 123.4, 121.5, 119.4, 115.5, 108.4, 69.6, 53.2, 52.7, 50.1, C3-aH, W1/2 ¼ 14 Hz, axial), 1.15 (C10–CH3), 0.73 (C13–CH3), 0.91
42.8, 40.2, 38.4, 37.2, 37.9, 36.1, 35.7, 34.2, 32.9, 30.9, 29.0, 28.6, & 0.84 (other methyl protons); 13C NMR (100 MHz, CDCl3, ppm):
26.3, 25.1, 23.9, 22.1, 21.7, 20.9, 19.7, 17.9, 16.6, 15.4, 10.9; anal. d 160.2, 149.2, 141.5, 140.8, 139.4, 138.0, 133.7, 130.9, 129.7,
calcd for C43H57N3O3; C, 77.79; H, 8.65; N, 6.33; found: C, 77.81; 128.5, 125.4, 124.8, 120.3, 112.9, 110.5, 59.5, 57.1, 55.9, 52.7,
H, 8.62; N, 6.35; MS (EI): m/z 663 [M+c].
45.2, 43.7, 40.2, 39.5, 38.8, 37.4, 36.1, 35.4, 34.7, 33.8, 32.5, 29.3,
27.5, 26.7, 25.9, 24.1, 23.6, 22.1, 20.6, 17.3, 16.7, 14.2; anal. calcd
for C41H55ClN4; C, 77.02; H, 8.67; N, 8.76 found; C, 77.05; H,
8.70; N, 8.75; MS (EI): m/z 638/640 [M+c].
3b-Chloro-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-oxazole (8)
Yield (76%), mp: 181–183 ꢂC; IR (KBr, n cmꢀ1): 3081, 1561, 1395
(C–H, aromatic), 1637 (C]N), 1056 (C–O), 741 (C–Cl); 1H NMR
(400 MHz, CDCl3, ppm): d 7.9–8.3 (m, 5H, aromatic), 3.81 (m,
5a-Cholestan-6-(iminohydrazonomethylphenyl-2-yl)-benzo-
[d]-imidazole (12)
1H, C3-aH, W1/2 ¼ 16 Hz, axial), 1.15 (C10–CH3), 0.73 (C13–CH3), Yield (81%), mp: 164–166 ꢂC; IR (KBr, n cmꢀ1): 3342 (NH), 3089,
0.91 & 0.84 (other methyl protons); 13C NMR (100 MHz, CDCl3, 1568, 1388 (C–H, aromatic), 1628 (C]N), 1329 (C–N); H NMR
1
ppm): d 156.1, 151.3, 148.6, 143.5, 140.2, 135.9, 130.3, 129.4, (400 MHz, CDCl3, ppm): d 4.4 (s, 1H, NH, exchangeable with
128.1, 126.9, 124.3, 122.5, 121.2, 117.9, 109.9, 59.6, 55.3, 54.6, D2O), 7.9–8.3 (m, 5H, aromatic), 1.15 (s, 3H, C10–CH3), 0.73 (s,
53.9, 41.5, 40.9, 38.7, 37.5, 36.9, 36.7, 35.4, 34.9, 33.8, 32.7, 31.4, 3H, C13– CH3), 0.91 & 0.84 (other methyl protons); 13C NMR (100
29.9, 27.1, 26.3, 24.9, 22.9, 21.7, 20.2, 19.1, 18.8, 15.7, 13.1; anal. MHz, CDCl3, ppm): d 162.5, 147.6, 145.6, 143.2, 140.3, 137.6,
calcd for C41H54ClN3O; C, 76.90; H, 8.50; N, 6.56; found: C, 134.2, 129.6, 128.1, 125.9, 124.7, 123.2, 122.8, 117.3, 112.6, 59.2,
76.87; H, 8.52; N, 6.59; MS (EI): m/z 639/641 [M+c].
57.5, 55.2, 49.5, 44.7, 39.1, 37.9, 36.9, 35.4, 34.7, 32.5, 30.8, 29.7,
27.9, 26.6, 25.3, 24.7, 23.4, 22.8, 21.2, 20.5, 19.8, 18.2, 17.6, 15.9,
13.6; anal. calcd for C41H56N4; C, 81.41; H, 9.33; N, 9.26 found;
C, 81.44; H, 9.29; N, 9.21; MS (EI): m/z 604 [M+c].
5a-Cholestan-6-(iminohydrazonomethylphenyl-2-yl)-benzo-
[d]-oxazole (9)
Yield (80%), mp: 176–178 ꢂC; IR (KBr, n cmꢀ1): 3088, 1560, 1397
(C–H, aromatic), 1634 (C]N), 1058 (C–O); H NMR (400 MHz,
1
Pharmacology
CDCl3, ppm): d 7.9–8.3 (m, 5H, aromatic), 1.15 (C10–CH3), 0.73
Rule of Five and bioactivity score. The physicochemical
(C13–CH3), 0.91 & 0.84 (other methyl protons); 13C NMR (100 parameters including octanol partition coefficients (C log P),
MHz, CDCl3, ppm): d 157.3, 150.2, 149.4, 140.7, 137.6, 136.7, Mw, HBD, HBA and TPSA were calculated using ChemBioOffice
132.2, 128.5, 127.2, 126.5, 124.6, 122.1, 121.5, 118.7, 111.5, 58.2, 2008. The Bioactivity score calculated using molinspiration
56.7, 55.4, 48.7, 41.5, 40.1, 39.2, 38.5, 37.9, 36.5, 34.3, 33.2, 29.0, server (http://www.molinspiration.com/cgi-bin/properties).
27.9, 26.4, 25.2, 24.9, 23.6, 22.7, 21.4, 20.1, 19.8, 18.3, 17.7, 15.9,
13.2; anal. calcd for C41H55N3O; C, 81.27; H, 9.15; N, 6.94;
Anticancer activity
found: C, 81.31; H, 9.20; N, 6.95; MS (EI): m/z 605 [M+c].
MTT assay. The cancerous cell lines (HeLa/Hep3B/MCF7)
and non-cancerous cell (PBMCs) were maintained in RPMI-
1640 culture medium supplemented with 10% heat-
inactivated fetal calf serum (FCS) and antibiotic antimycotic
3b-Acetoxy-5a-cholestan-6-(iminohydrazonomethylphenyl-2-
yl)-benzo-[d]-imidazole (10)
Yield (79%), mp: 167–169 ꢂC; IR (KBr, n cmꢀ1): 3331 (NH), 3087, solution. The cells were plated at a density of 5 ꢃ 103 cells per
1563, 1391 (C–H aromatic), 1738 (OCOCH3), 1636 (C]N), 1061, well in a 96-well plate, and cultured for 24 h at 37 ꢂC. The cells
1065 (C–O), 1310 (C–N); 1H NMR (400 MHz, CDCl3, ppm): d 5.3 were subsequently exposed to drugs. The plates were incubated
(s, 1H, NH, exchangeable with D2O), 7.9–8.2 (m, 5H, aromatic), for 48 h, and cell proliferation was measured by adding 20 mL of
4.71 (m, 1H, C3-aH, W1/2 ¼ 18 Hz, axial), 2.01 (s, 3H, OCOCH3), MTT dye (5 mg mLꢀ1 in phosphate buffered saline) per well. The
1.15 (C10–CH3), 0.73 (C13–CH3), 0.91 & 0.84 (other methyl plates were incubated for a further 4 h at 37 ꢂC in a humidied
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chamber containing 5% CO2. Formazan crystals formed due to reagent composed of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine)
the reduction of dye by viable cells in each well were dissolved in (10 mL), 20 mM FeCl3$6H2O (10 mL), and 300 mM acetate buffer
150 mL dimethyl sulfoxide (DMSO), and absorbance was read at (100 mL) in ratio of 1 : 1 : 10 were freshly prepared. 300 mM
570 nm. The absorption values were expressed as the cell acetate buffer (pH 3.6) was prepared by dissolving sodium acetate
viability (%), according to the control group as 100%. The trihydrate (3.1 g) in distilled water (500 mL) then glacial acetic
concentration required for 50% inhibition of cell viability (IC50
)
acid (16 mL) was added and made up to the mark of 1 L with
distilled water and checked for its pH. 10 mM TPTZ solution was
prepared in 40 mM HCl and 20 mM FeCl3$6H2O was prepared in
distilled water. Different concentrations of compounds (25, 50,
75 and 100 mg mLꢀ1) were taken and an increase in the absor-
bance by [FeIII(TPTZ)2]3+ complex was measured at 593 nm aer
5 min of incubation at 37 ꢂC. Trolox (6-hydroxy-2,5,7,8-tetra-
methylchroman-2-carboxylic acid) was used as the standard
solution and the concentration of FRAP content in the
compounds were expressed in terms of mg trolox-equivalent (TE)
per g deduced from the standard curve of trolox.57,58
was calculated using the soware “Prism 3.0”.52
Blood peripheral mononuclear cell isolation
The blood sample (20–15 mL) was diluted with the same volume
of phosphate buffer saline (PBS). Aer that, the diluted blood
sample was layered on Ficoll-Histopaque. The mixture was
ꢂ
centrifuged at 400 g for 30 min at 20–22 C. The undisturbed
lymphocyte layer was transferred out. The lymphocyte was
washed and pelleted down with three volumes of PBS twice and
resuspended RPMI-1640 media with antibiotic and antimycotic
solution 10%, v/v FCS. Cell counting was performed to determine
the PBMCs cell number with equal volume of trypan blue.53,54
By ABTS assay
The total antioxidant activity of compounds 4–12 were
measured by ABTS [20,20-azinobis(3-ethylbenzothiazoline-6-
sulfonic acid)] radical cation decolorization assay with some
modication.59 A stock solution of ABTS (2 mM) was prepared
by dissolving the ABTS in phosphate-buffered saline (PBS,
50 mL) to produce a solution with a pH of 7.4. The ABTSc+ was
generated by reacting 50 mL of the stock solution with 200 mL
of a 70 mM aqueous K2S2O8 solution. The resulting ABTSc+
solution was diluted with PBS to obtain a reasonable absor-
bance at 734 nm. A 100 mL of drug solution at different
concentration (25, 50, 75 and 100 mg mLꢀ1) was mixed with
ABTSc+ solution (1.9 mL), and the absorbance of the mixed
solution was read at ambient temperature aer 3 min. The PBS
solution was used as a blank sample.60 The experiment was
done in triplicates and the radical scavenging activity of the
sample was calculated with the following formula:
Antioxidant activity
By DPPH assay. Steroidal heterocyclic compounds (4–12)
were tested for their antioxidant property by 1,1-diphenylꢀp1i-
crylhydrazyl (DPPH) method.55 Drug stock solution (1 mg mL
)
was diluted to nal concentration of 2, 4, 6, 8, 10 and 12 mg
mLꢀ1 in methanol. Methanolic DPPH solution (1 mL, 0.3 mmol)
was added to 3.0 mL of drug solution of different concentra-
tions. The mixture was shaken vigorously and incubated in dark
at an ambient temperature for 30 min and the absorbance was
measured as the decrease in the absorbance of DPPH at 517 nm
resulting from the color change from purple to yellow. The
decrease in absorbance is because of formation of stable
molecule of DPPH on reaction with an antioxidant through
donation of hydrogen or electron by an antioxidant. The free
electron on DPPH radical is responsible for giving absorbance
peak at 517 nm and appears purple in color. The antioxidant
agent pair up through donation of electron or release of
hydrogen with the free electron on DPPH radical and form
stable molecule of DPPH-H. The change of color from purple to
yellow is attributed to decrease of molar absorptivity of DPPH
radical when odd electron of DPPH pair up with the antioxidant
agent. The resulting decrease in color is also stoichiometric
with number of electrons captured. The DPPH radical-
scavenging activity (%) was calculated by the following formula:
BO ꢀ BS
SC% ¼
ꢃ 100
BO
where, BO and BS are the absorbance values of the blank and of
the tested samples, respectively. The assay was carried out on
ascorbic acid, which served as a standard.
Fluorescence microscopy
The HeLa cell line was maintained in RPMI-1640 culture
medium supplemented with 10% heat-inactivated FCS. The
cells were plated at a density of 5 ꢃ 103 cells on glass cover slips,
and cultured for 24 h at 37 ꢂC. These were subsequently exposed
with compounds for 48 h. Cells were xed by 2% para-
formaldehyde for 2 h followed by washing with Hank's Balanced
Salt Solution (HBSS). The HeLa cells treated with compounds
4–12 were observed under Fluorescence Microscope.
[% inhibition ¼ [(Acontrol ꢀ Asample/Acontrol) ꢃ 100]
where Acontrol is the absorbance of the L-ascorbic acid (standard)
and Asample is the absorbance of different compounds. The
methanolic DPPH solution (1 mL, 0.3 mM) was used as a
negative control whereas L-ascorbic acid was used as a positive
control.
Preparation for TEM and AFM
By FRAP assay
The sample for TEM analysis was prepared by placing a drop of
FRAP assay measures the antioxidant capacity by the reduction of colloidal solution on a copper grid coated with a carbon lm
ferric 2,4,6-tripyridyl-s-triazine complex [FeIII(TPTZ)2]3+ to the and allowed to equilibrate for 3–5 min. The solution was wiped
intensely blue colored ferrous complex [FeII(TPTZ)2]2+.56 The off with lter paper and allowed to air dry. Images were taken
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using TEM, JEOL JEM-2100 Electron Microscope set at an of DNA.64 The initial structures of the steroidal compounds 4–12
accelerating voltage of 200 kV to determine the size and were generated by ChemBioDraw Ultra 12.0 soware. The
morphology of the condensate material. The samples for AFM structures of compounds were optimized for use in the
analysis of the compound 6 and its condensate with CT-DNA following docking study. The parameters that were used for
were prepared from the solution of the compound 6 (prepared docking include: correlation type-shape only, FFT mode-3D,
in DMSO) and equimolar mixture of compound 6 and CT-DNA grid dimension-0.6, receptor range-180, ligand range-180,
(dissolved in 0.01 M Tris–HCl buffer pH 7.2). The samples were twist range-360, distance range-40. The crystal structure of the
placed drop wise on a mica wafer, then air dried at room B-DNA dodecamer d(CGCAAATTTCGC)2 (PDB ID: 1BNA) were
temperature for 12 h and the images were recorded with Agilent downloaded from the protein data bank (http://www.rcsb.org./
Model 5500 using tapping mode AFM, where cantilever and pdb). All calculations were carried out on an Intel CORE i5,
AFM tip oscillate and are scanned over the surface of the dry 2.6 GHz based machine running MS Windows 8 as the oper-
sample.
ating system. Visualization of the docked pose have been done
using PyMol molecular graphics program.65
Nonenzymatic DNA damage
In order to identify and determine the possible role of ROS, the
reaction mixture (0.5 mL) containing Tris–HCl (10 mM, pH 7.5),
CT-DNA (200 mg), Cu(II) (100 mM) and increasing concentrations
of compound 6 (12.5 mM, 25 mM, 50 mM, 75 mM, 100 mM, 200 mM,
400 mM, 600 mM). The volume was made up to 1 mL by buffer
solutions and incubated at 37 ꢂC for 60 min. Reaction was
stopped using 0.5 mL of trichloroacetic acid solution (TCA)
(28%) and 0.5 mL of 1% thiobarbituric acid (TBA) was added,
boiled for 15 min and cooled to room temperature. The inten-
sity was read at 532 nm.61
Conclusion
In conclusion, a series of new steroidal heterocyclic compounds
with signicant anti-tumor and antioxidant activities were
successfully designed and synthesized. Their anti-tumor activity
in vitro was evaluated against Hep3B (human hepatocellular
carcinoma), MCF7 (human breast adeno carcinoma), HeLa
(human cervical carcinoma) cancer cell lines and on normal
cells PBMCs (peripheral blood mononuclear cell). The results
demonstrated that most of the synthesized derivatives exhibited
signicant anti-tumor activity; however compounds 5, 6, 8, 9
and 12 exhibited excellent activity with IC50 < 19 mM against all
the cancer cell lines. In addition, compounds 10–12 were found
to be good antioxidant. Nonenzymatic degradation of DNA has
also been investigated. More importantly, the application of
compounds 6 as DNA gene transporter was evaluated by DNA
condensation and ascertained by employing TEM and AFM,
which illustrated that the compound 6 induces the condensa-
tion of CT-DNA. Lipinski's ‘Rule of Five’ analysis predicted good
oral absorption of the synthesized compounds. Moreover, the
acetylcholinesterase (AChE) inhibitor activities of the steroidal
derivatives were also evaluated using Ellman's method. From
the results obtained we deduced that compound 4, 7 and 10
(IC50 ¼ 0.31 ꢁ 0.10, 0.37 ꢁ 0.02 and 0.39 ꢁ 0.03, respectively)
exhibited signicant inhibition on AChE among all the
synthesized compounds. The molecular docking studies
undertaken in the present work are in total agreement, with the
primary mode of binding of the synthesized compounds with
DNA, although the H-bonding and other types of interactions
can also be argued. These ndings demonstrated that among
steroidal heterocyclic derivatives one can nd compounds with
interesting therapeutic properties that could become a new
group of potential anti-tumor and antioxidant agents.
In vitro acetylcholinesterase inhibition activity
The ability of synthesized steroidal compounds (4–12) to inhibit
AChE activity was assessed by Ellman's method.62 AChE stock
solution was prepared by dissolving human recombinant AChE
(EC:3.1.1.7) lyophilized powder in 0.1 M phosphate buffer (pH
8.0) containing Triton X-100 (0.1%). Five increasing concen-
trations of test compounds were assayed to obtain percent
inhibition of the enzymatic activity in the range of 20–80. The
assay solution consisted of a 0.1 M phosphate buffer pH 8.0,
with the addition of 340 mM 5,50-dithio-bis(2-nitrobenzoic acid),
0.02 unit per mL of human recombinant AChE from human
serum and 550 mM of substrate (acetylthiocholine iodide,
ATCh). Increasing concentration of tested inhibitor were added
to the assay solution and pre-incubated for 5 min at 37 ꢂC with
the enzyme followed by the addition of substrate. Initial rate
assays were performed at 37 ꢂC with a Jasco V-530 double beam
spectrophotometer. Absorbance value at 412 nm was recorded
for 5 min and enzyme activity was calculated from the slope of
the obtained linear trend. Assays were carried out with a blank
containing all components except AChE to account for the non-
enzymatic reaction. The reaction rates were compared and the
percent inhibition due to the presence of tested inhibitors was
calculated. Each concentration was analyzed in triplicate and
IC50 values were determined graphically from log concentra-
tion–inhibition curves (GraphPad Prism 4.03 soware, Graph-
Pad Soware Inc.). Tacrine was used as a standard inhibitor.
Acknowledgements
Authors thank the Chairman, Department of Chemistry,
A.M.U., Aligarh, for providing necessary research facilities.
Facilities provided by SAP (DRS-II), FIST & PURSE for their
Molecular docking
The rigid molecular docking studies were performed using HEX generous research support are gratefully acknowledged.
8.0.0 soware.63 HEX is an interactive molecular graphics University Sophisticated Instrumentation Facility (USIF),
program for calculating and displaying feasible docking modes A.M.U., Aligarh is acknowledged for TEM analysis and SAIF
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