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I. Ali et al. / Bioorg. Med. Chem. 21 (2013) 3808–3820
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of secondary structure of DNA. The occurrence of red shift indi-
cated the coordination of a compound with DNA through N7 posi-
tion of guanine.34 Overall, the outside groove binding is
characterized by no or minor change in UV–vis spectra; occasion-
ally with some hyperchromicity. Contrarily, outside binding with
self-stacking shows quite similar characteristics as the intercala-
tive binding mode but to a lesser extent.35–37 The absorption spec-
tra of compounds 4a–6d in the absence and presence of DNA are
shown in Figure FS1 (a–l) (Supplementary data). The absorption
spectra of compounds exhibited peaks in the range of 200–
500 nm. The compounds of series 4a–d had one absorption band
in the range of 251–266 nm, while compounds of series 5a–d
and 6a–d showed two bands (Supplementary data, Table TS1). In
series 5a–d, first and second bands ranged from 261 to 275 nm
and 355 to 380 nm. Similarly, in series 6a–d first and second bands
appeared at 265–370 nm and 350–450 nm, respectively (Supple-
mentary data, Table TS1). The band shifting was observed in the re-
gion of 200–450 nm by the addition of DNA. Small shifting of
second band of the compounds of series 6a–d was due to intra li-
15
10
5
0
4a
4b 4c
4d
5a 5b 5c
5d
6a
6b
6c
6d
Compound
Figure 3. Hemolysis assay of the synthesized compounds on rabbit RBC.
2.3. Pharamacological activities
gand p?
p⁄ transitions.38,39 The compounds with different substi-
2.3.1. Hemolytic assay
tuent’s showed different absorption bands, that is, ꢂ248–275 nm
for 4a–d (273 nm for 4a, 258 nm for 4b, 256 nm for 4c and
248 nm for 4d). For compounds 5a–6d, two absorptions peaks
were observed, one around 250–260 nm (for 5a–d) and another
in the region of 350–450 nm (for 6a–d). These data indicated bath-
ochromic shift of all the compounds due to the interactions with
DNA. It was also observed that with the addition of different con-
centrations of DNA [0.4–1.2 ꢀ 10ꢁ4 M], the absorption peaks
underwent hyper- and hypo-chromicities for compounds (4a–6d)
(Fig. FS1, Supplementary data), thus, indicating the formation of
DNA-compound adducts.35 Furthermore, it is interesting to note
that in all the cases, hyper and hypochromic effects were observed
with varying concentrations of DNA, which might be due to differ-
ent types of bonding (covalent and non-covalent).36 The hyper-
chromic shift at higher concentration of the bands might be due
to the uncoiling of DNA (more bases embedding in DNA ex-
posed).40 UV–vis data for compounds 4a–6d are given in Table 1
and Table TS1 (Supplementary data). More than one type of
DNA-compound interactions have been formed (partial intercala-
tion + electrostatic attraction) as indicated by the absence of any
fixed isobestic points in titration experiment.
In vitro hemolytic assay is the preliminary method to evaluate
the cytotoxicity of the new compounds.25 It is an acceptable
screening tool for gauging possible in vivo toxicity to the host
cells.26 Mammalian RBCs were used to determine the toxicity of
the synthesized compounds due to their freely availability and
easy detection of the lyses products.
As per the standard hemolytic index (ASTM), compounds with
0–2%, 2–10%, 10–20% and 20–40% are considered as non, slightly,
moderate and markedly hemolytic, respectively. On the other
hand, compounds with hemolytic index above 40% are supposed
as highly hemolytic in nature. The hemolytic activity of the synthe-
sized compounds, that is, pyrazolealdehydes (4a–d), Knoevenagel’s
condensates of curcumin (5a–d) and their Schiff’s bases (6a–d) are
shown in Figure 3. It is clear from this figure that 610%, 15%, 20%
and 25% toxicities were shown by 4a, 4c, 4d and 5a; 5c and 6a;
4b, 5b, 5d, 6c and 6d; 6b, respectively, at concentration100 lg/
mL. These results indicated the order of increasing toxicities as
6b > 5b > 4b > 6d > 6c = 5d > 5c > 6a = 5a > 4c > 4d > 4a. Standard
drug doxorubicin had 42% hemolysis activity at 100 lg/mL. There-
fore, it may be concluded that compounds 4a, 4c and 4d are
slightly hemolytic, 5a, 6a, 5c, 4b, 5d, 6c and 6d moderately hemo-
lytic and 5b and 6b markedly hemolytic in nature.
For a ready reference, the absorption spectra of first compound
(4a, 5a and 6a; 2.0 ꢀ 10ꢁ4 M) of all three series; in both absence
and presence (0.4–1.2 ꢀ 10ꢁ4 M) of calf-thymus DNA; are given
in Figure 4a–c. The values of DNA binding constants of these com-
pounds varied from 1.4 ꢀ 103 to 8.1 ꢀ 105 Mꢁ1, indicating good
interaction with DNA. The regression analysis was carried out
2.3.2. DNA binding
UV–vis spectroscopy is one of the most commonly used meth-
ods for the investigation of the interactions of a compound with
DNA.27 DNA is the primary pharmacological target for many anti-
tumor compounds. Therefore, the study of the interaction of the
new compounds with DNA is quite essential to assess their anti-
cancer activities and a possible mechanism of action. A compound
can bind to DNA either via covalent (in which a labile ligand is re-
placed with a nitrogen atom of DNA base, such as N7 of guanine) or
non-covalent (such as intercalative, electrostatic and groove bind-
ing) interaction. Normally, a compound bound to DNA through
intercalation results in hypochromism (decrease in absorbance)
and bathochromism (red shift). It is due to the fact that intercala-
tive mode involves a strong stacking interaction between aromatic
chromophore and the base pairs of DNA.28 It is believed that the
extent of hypochromism depends on the strength of intercala-
tion.29–32 Generally, electrostatic interaction of a compound with
DNA shows lower hypochromicity with no bathochromic shift33
Table 1
UV–vis spectral data of the compounds 4a–6d
a
Compounds
D
kmax (nm)
% Hypochromismb
Kb (Mꢁ1
)
4a
4b
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
8
2
2
2
4
1
1
3
1
3
4
—
7.7
6.2
5.2
5.6
12.3
13
29
8.1
8
1.9 ꢀ 103
1.4 ꢀ 103
2.5 ꢀ 103
7.6 ꢀ 104
1.4 ꢀ 104
2.1 ꢀ 104
9.4 ꢀ 104
7.8 ꢀ 105
2.6 ꢀ 104
3.0 ꢀ 104
9.1 ꢀ 104
8.1 ꢀ 105
10
9
11
(due to decrease of the
p?
p⁄ transition energy as p⁄ orbital of
a
the intercalated ligand couples with the orbital of the base pairs).
On the other hand, a compound bound to DNA through covalent
binding results in hyperchromism and red shift owing to breakage
For details of wavelength shifts, please see Supplementary data.
% Hypochromicity (H%) = [(Af ꢁ Ab)/Af] ꢀ 100, where Af and Ab represent the
b
absorbance of free and bound compounds.