Design and synthesis of heterobimetallic topoisomerase I and II inhibitor complexes: In vitro DNA binding, interaction with 5′-GMP and 5′-TMP and cleavage studies

Article abstract of DOI:10.1016/j.jphotobiol.2010.06.009

New potential cancer chemotherapeutic complexes Cu-Sn₂/Zn-Sn₂ 3 and 4 were designed and prepared as topoisomerases inhibitors; their in vitro DNA binding studies were carried out which reveal strong electrostatic binding via phosphat

Full text of DOI:10.1016/j.jphotobiol.2010.06.009

Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
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Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
Design and synthesis of heterobimetallic topoisomerase I and II inhibitor
complexes: In vitro DNA binding, interaction with 50-GMP and 50-TMP and
cleavage studies
*
Farukh Arjmand , Mohd. Muddassir
Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New potential cancer chemotherapeutic complexes Cu–Sn2/Zn–Sn2 3 and 4 were designed and prepared
as topoisomerases inhibitors; their in vitro DNA binding studies were carried out which reveal strong
electrostatic binding via phosphate backbone of DNA helix, in addition to other binding modes viz. coor-
dinate covalent and partial intercalation. To throw insight to molecular binding event at the target site,
UV–vis titrations of 3 and 4 with mononucleotides of interest, viz, 50-GMP and 50-TMP were carried out,
(in case of 4) by 1H and 31P NMR. Cleavage studies employing gel electrophoresis demonstrate both the
complexes 3 and 4 are efficient cleavage agents and are specific groove binders (complex 3 binds to both
major and minor groove while complex 4 is specifically minor groove binder only). In addition, the com-
plexes show high inhibition activity against topoisomerase I and II. However, complex 4 exhibits signif-
Received 22 March 2010
Received in revised form 11 June 2010
Accepted 21 June 2010
Available online 30 June 2010
Keywords:
Heterobimetallic complexes
In vitro binding studies
50-GMP
50-TMP
Cleavage studies
Topoisomerases inhibition
icant inhibitory effects on the Topo I activity at a very low concentration ꢀ2.5
lM.
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
Topo II is closely regulated in normal cells, but tumours main-
tain high level of topo II, a target for several antitumour agents
[18]. There is a clear relationship between qualitative alterations
of topo II and during sensitivity in a series of in vitro biological sys-
tems. In the recent past, an antitumour copper salicylaldoxime
(CuSAL) complex has been reported which induces topo II to form
single strand nicks in DNA and poisons its activity [19]. Routier and
coworkers synthesized a functionalized Cu-salen complex and
investigated the DNA-binding and DNA-cleaving properties of
Cu-salen complex. It has been shown that the complex induces sin-
gle-strand breaks via an interaction within one of the grooves of
the double helix [20]. Tin(IV) and organotin(IV) complexes of Schiff
bases have also attracted considerable interest [21,22]. Tin(IV)
complexes show high in vitro activity against P388 leukemia in
mice as well as some human tumour cell lines [23,24]. In our pre-
vious work, we have demonstrated that the heterobimetallic com-
plexes of Cu(II)–Sn(IV) induce apoptosis via p53 mitochondrial
pathway [25]. Insertion of second metal, Sn(IV) (a hard Lewis acid)
neutralizes the dinegative charge of phosphate sugar, further
tunes/modulates the monometallic complexes to behave as site-
specific oriented cancer chemotherapeutic agents. The unique sig-
nature of transition metal ions, Cu(II)/Zn(II) together with Sn(IV) –
a non transition metal ion exhibits dual mode of action in vivo due
to its preferential selectivity inside the cells [26]. Cu(II) have dis-
tinct preference for covalent mode of binding via N7 of guanine
while Zn(II) effectively function to recognize N3 of thymine
Salicylaldimine Schiff bases have emerged as an important class
of ligands not only due to their novel structural features but also in
view of their immense biological properties [1,2]. Metal complexes
of Schiff bases have been designed and synthesized to explore their
pharmacological activity and more recently antitumour activity
[3–5]. 5-Aminosalicylic acid (5-ASA) has been utilized to combat
many inflammatory diseases such as Crohns disease and ulcerative
colities. [6]. Anti-inflammatory drugs have been shown to be active
for the treatment or prevention of cancer in particular; colorectal
cancer [7]. It has been shown that copper accumulates in tumours
due to the selective permeability of cancer cell membranes to cop-
per compounds [8]. Because of this, a number of copper complexes
have been screened for anti-cancer activity and some of them were
found active both in vivo and in vitro [9].
DNA topoisomerase I and II are established molecular targets of
anti-cancer drugs [10–13]. Topoisomerases are crucial for cellular
genetic process such as DNA replication, transcription, recombina-
tion and chromosome segregation at mitosis [14,15] and several
class of topoisomerase inhibitors have been introduced into cancer
clinics as potent anti-cancer drugs [16,17].
* Corresponding author. Tel.: +91 5712703893.
E-mail address: farukh_arjmand@yahoo.co.in (F. Arjmand).
1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotobiol.2010.06.009

38
F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
nucleobase whereas Sn(IV) prefers to bind to oxygen atom of the
phosphate sugar backbone of DNA helix [27–29]. Besides this,
copper and zinc complexes have established biological properties;
they are the most abundant trace elements present in biological
systems and several metalloprotiens contain one or both elements
[30,31]. We report herein, new heterobimetallic topoisomerase I
and II inhibitors derived from salicylaldimine pharmacophore
containing Cu(II)–Sn2(IV)/Zn(II)–Sn2(IV) metallic cores which are
major and minor groove binders, respectively. Since DNA topoiso-
merase I and II are the ultimate molecular targets of anti-cancer
drugs, the interaction studies of the complexes 3 and 4 were
carried out with CT DNA, nucleotides viz, 50-GMP and 50-TMP (in
comparison to their monometallic complexes and the Schiff base
ligand) and inhibitory activity with topoisomerases was examined
.
dine, 0.1 mg/mL BSA, 0.25
plexes (3 and 4). These reaction mixtures were incubated at 37 °C
for 30 min, and the reaction was terminated by addition of 4 L of
5ꢁ stop solution consisting of 0.25% bromophenol blue, 4.5% SDS
and 45% glycerol. The samples were electrophoresed through 1%
agarose in TBE at 30 V for 8 h. Then the gel was photographed un-
der UV light. The concentration of the inhibitor that prevented 50%
of the supercoiled DNA from being converted into relaxed DNA
(IC50 values) were calculated by the midpoint concentration for
drug-induced DNA unwinding.
lg pBR322 DNA, 2 Unit Topo I and com-
l
DNA topoisomerase II
no further purification was performed. One unit of the enzyme was
defined as completely relax 0.3 g of negatively supercoiled
pBR322 DNA in 15 min at 37 °C under the standard assay condi-
tions. The reaction mixture (30 L) contained 10 mM Tris–HCl
(pH 7.9), 50 mM NaCl, 50 mM KCl, 5.0 mM MgCl2, 0.1 mM
Na2H2edta, 15 g/mL BSA, 1.0 mM ATP, 0.25 g pBR322 DNA, 5
a (Topo II) was purchased from Sigma and
l
l
l
l
2. Material and methods
Unit Topo II, and complexes (3 and 4). The reaction mixtures were
incubated at 30 °C for 15 min. Reactions were stopped, processed,
and subjected to gel electrophoresis as above.
2.1. Materials and physical measurements
Zinc chloride anhydrous (Rankem), Copper chloride dihydrate,
Zinc acetate dihydrate, Copper acetate monohydrate (E. Merck),
Tin tetrachloride pentahydrate (Lancaster), 5-aminosalicylic acid
(Fluka) and Salicylaldehyde (Alfa Aesar) were used as received. 6ꢁ
loading dye (Fermental Life Science) and Super coiled plasmid DNA
pBR322 (Genei) were utilized as received. Calf thymus DNA (CT
DNA)guanosine50-monophosphatedisodiumsalt(50-GMP)andthy-
mine 50-monophosphate disodium salt (50-TMP) were purchased
from Sigma chemical Co. and Fluka, respectively. All reagents were
of the best commercial grade and were used without further purifi-
cation. Carbon, hydrogen and nitrogen contents were determined
using Carlo Erba Analyzer Model 1106. Molar conductances were
measured at room temperature on a Digsun Electronic conductivity
Bridge. Fourier-transform IR (FTIR) spectra were recorded on an
Interspec 2020 FTIR spectrometer. Electronic spectra were recorded
on UV-1700 PharmaSpec Uv–vis spectrophotometer (Shimadzu).
Datawerereportedinkmax/nm. TheEPRspectrumofthecoppercom-
plex was acquired on a Varian E 112 spectrometer using X-band fre-
quency (9.1 GHz) at liquid nitrogen temperature in solid state. The
1H, 13C NMR and 119Sn spectra were obtained on a Bruker DRX-400
spectrometer operating at room temperature. TGA/DSC was per-
formed with a Universal V3.8 B TA SDT Q600 Build 51 Thermal Ana-
lyzer undernitrogen atmosphereusingaluminapowderas reference
material. FAB mass spectra were recorded on Micromass Quattro II.
The visualization of spots on TLC plates was effected by exposure to
iodine or spraying with 10% H2SO4 and charring.
2.4. Synthesis
2.4.1. Synthesis of ligand
5-{[(1E)-(2-hydroxyphenyl)methylene]amino}-2-hydroxyben-
zoic acid (L).
The ligand L was synthesized with 5-aminosalicylic acid (0.5 g,
3.26 mmol) and salicylaldehyde (0.44 g, 3.60 mmol) in ethanol on a
reflux for 2 h according to the procedure reported earlier [1].
2.4.2. Monometallic complexes 1 and 2
Monometallic complexes 1 and 2 was synthesized by mixing
Cu(CH3COO)2 (0.20 g, 1.10 mmol)/Zn(CH3COO)2 (0.20 g, 1.09 mmol)
and ligand L (0.59 g, 2.29 mmol) in ethanol on a reflux for 3 h
according to the procedure reported earlier [1].
2.4.3. Heterobimetallic complex 3 (Cu–Sn2)
The complexes were prepared by a general synthetic method in
which a solution of monometallic complexes 1 (0.576 g, 1 mmol)
was added to NaOH (0.08 g, 2 mmol) in ethanol (15 mL) with stir-
ring for 10 min until the solution became clear. This was followed
by addition of a MeOH solution (20 mL) of SnCl4ꢂ5H2O (0.356 g,
2 mmol) and the reaction mixture was put to reflux for a period
of 2 h. The resulting yellow solution which precipated immediately
was isolated by filtration washed with ether and EtOH in portion
and dried in vacuo. Yield, 60%, m.p. 300 °C, Anal. Calc. for
C
28H24N2O12CuSn2Cl4 (%) C, 32.87; H, 2.36; N, 2.74, Found: C,
32.91; H,2.37; N, 2.76 IR (KBr, cmꢃ1) 3300–3400
(H2O), 1608
(C@N), 1690 (symCOO); 1335 (antisym.COO); 787 (Ar), 433
m
2.2. DNA binding and cleavage experiments
m
m
(CuAO), 538
m(CuAN), 650
m
(SnAO), 270
m
(SnACl). Molar Conduc-
tance, KM (1 ꢁ 10ꢃ3 M, DMSO): 12.0
X
ꢃ1 cm2 molꢃ1 (non-electro-
DNA binding experiments which include absorption spectral
traces, luminescence and viscosity conformed to the standard
methods and practices previously adopted by our laboratory [32–
35]. The number of metal cluster bound per DNA in fluorescence
were calculated [36,37] whereas DNA cleavage experiment has
been performed by the standard protocol as described in [38].
lyte). UV–vis (DMSO, nm): 262 nm; 592 nm.
2.4.4. Complex 4 (Zn–Sn2)
This complex was synthesized with monometallic complex 2
(0.578 g, 1 mmol) according to the method outlined above.
Yield, 62%, m.p. 300 °C (decompose) Anal. Calc. for
2.3. Topoisomerase inhibition assay
C
28H24N2O12ZnSn2Cl4ꢂC2H5OH (%) C, 33.65; H 2.83 N 2.62, Found:
C, 33.69; H, 2.86; N, 2.64, IR (KBr, cmꢃ1) 3300–3400
(H2O), 1608
m(C@N), 1592 (sym.COO); 1336 (antisym.COO); 787 (Ar), 462
m
DNA topoisomerase I, Human (Topo I) was purchased from
CALBIOCHEM.
(ZnAN); 522 (ZnAO); 650 m(SnAO), 270 m(SnACl).
And no further purification was performed. One unit of the en-
Molar Conductance, KM (1 ꢁ 10ꢃ3 M, DMSO): 15.0
X
ꢃ1 cm2
zyme was defined as completely relax 1
coiled pBR322 DNA in 30 min at 37 °C under the standard assay
conditions. The reaction mixture (30 L) contained 35 mM Tris–
HCl (pH 8.0), 72 mM KCl, 5 mM MgCl2, 5 mM DTT, 2 mM spermi-
l
g of negatively super-
molꢃ1 (non-electrolyte) UV–vis (1 ꢁ 10ꢃ4 M, DMSO) kmax/nm
318 nm; 390 nm.
l
1H NMR (DMSO-d6, ppm): 8.75 (s, 2H, C(H) = N), 8.03 (S, 2H,
Ar), 7.83 (d, 2H, Ar), 7.77 (d of d, 4H, Ar), 6.99–6.84 (ov, m, 6H, Ar),

F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
39
3.7–4.0 (br, s, 8H, H2O), 2.15 (s, 1H, –OH), 1.15 (s, 5H, C2H5), 13C
NMR (DMSO-d6 ppm): 171.62 (C@O), 160.8 (C@N), 160.4 (CO),
160.2 (CO), 139 (ArC), 132.4 (ArC), 131.9 (ArC), 128.59 (ArC),
124.1 (ArC), 122.4 (ArC), 122.1 (ArC), 118 (ArC), 116 (ArC), 112.9
(ArC) (119Sn NMR, DMSO-d6,ppm): ꢃ622.33.
revealed by the presence of medium intensity (Cu/Zn–N), (Cu/
Zn–O) and (Sn–Cl) bands around ꢀ460, ꢀ520 and ꢀ330 cmꢃ1
,
respectively in the far IR region [42,43].
3.2. NMR spectral studies
3. Results and discussion
The 1H NMR spectrum of complex 4 displays several interesting
features in comparison to the free ligand L and complex 2 depicted
in Fig. S1. In 1H NMR spectrum of complex 4, signals due to free
carboxylic (COOH) and phenolic (OH) groups at 12.0–10.0 and
13.36 ppm, respectively were absent indicating the coordination
of monometallic complex to Sn(IV) atom via deprotonation [40].
Coupled with these observations, the characteristic signals at
8.75, 8.03–6.94, 4.0–3.7 corresponding to CH@N, aromatic proton
[44], coordinated H2O groups, respectively (Supplementary infor-
mation, Fig. S1).
All the complexes are stable towards air and moisture and sol-
uble in DMSO and DMF. In complexes 1–4, the coordination geom-
etry of central metal ion copper(II) and zinc(II)) were square planar
and tetrahedral, respectively while the Sn(IV) atom was present in
hexacoordinated environment (Scheme 1), which was proposed on
the basis of 119Sn NMR and other spectroscopic studies ‘‘see
below”, following IR spectroscopic studies. DNA binding profile of
the heterobimetallic complexes 3 and 4 were studied in compari-
son to the monometallic complexes 1 and 2 and the ligand L
employing UV–vis and fluorescence spectroscopy. DNA binding
modes were evaluated by the interaction of both 3 and 4 to 50-
GMP and 50-TMP by UV–vis spectroscopy and in case of 4 by 1H
and 31P NMR techniques. In addition, the complexes 3 and 4 show
high inhibition activity against topoisomerase I and II.
The 13C NMR spectrum of the ligand L exhibits 172.2 (C@O),
162.2 (C@N), 160.8 (CO), 160.7 (CO) in addition to aromatic carbon
signals which appeared at 114.1–139.7 ppm.
The 13C NMR spectrum of complex 4 exhibits various reso-
nances due to OAC@O, CH@N, C–O–Sn and C–O–Zn carbons at
171.62, 160.8, 160.4 and 160.2 ppm, respectively. In addition, aro-
matic carbon signals were observed at 112.9–139 ppm.
119Sn NMR spectroscopy has been found to be a useful tech-
nique for structure elucidation and the nature of coordination of
tin atom in complexes. The 119Sn chemical shift, d (119Sn) is sensi-
tive to the chemical environments of the tin atom. 119Sn NMR spec-
trum of the complex 4 displayed one sharp signal at ꢃ622.33, due
to the presence of two tin metal centers, but in the same environ-
ment [45]. These chemical shift values are consistent with those
reported for complexes with hexacoordinated geometry of tin me-
tal atom [46].
3.1. Infrared spectra
The IR spectrum of free ligand L displayed a strong characteris-
tic band at 1610 cmꢃ1 due to
m(C@N) which shifted slightly to
1608 cmꢃ1 in the monometallic complexes 1 and 2 implying the
coordination of N atom of the ligand to the metal center.
In heterobimetallic complexes 3 and 4, coordination of the sec-
ond metal ion Sn(IV) through the O atom of both the phenolic
groups by the elimination of NaCl/HCl [39]. The bands around
1590 cmꢃ1 and 1335 cmꢃ1 observed in the free monometallic com-
plexes 1 and 2 attributed to antisymmetric and symmetric –C@O
stretching vibrations were shifted to lower frequencies, suggesting
the terminal coordination mode of carboxylate group of monome-
3.3. Mass spectroscopy
tallic complexes to the Sn(IV) through deprotonation. The
as(CO2)–
ing of carboxylate to tin(IV) atom. In general, the difference in
D
m
The FAB mass spectrum of complex 3 showed a prominent peak
at m/z 954 which corresponds to the fragment [3-Cl2+2H+]. The
appearance of other peaks at m/z 885, 631 and 579 (50%) were
ascribed to the species [3-Cl4+4H+], [3-Sn2Cl4ꢂ2H2O+7H+] and [3-
Sn2Cl4ꢂ4H2O+7H+], respectively. The spectrum of 4 revealed molec-
ular ion peak at m/z 1073 (20%) due to [4-C2H5OH+2H]+. The
appearance of peaks at m/z 989, 938, 919 and 613 (50%), respec-
tively were ascribed to the species [4-C2H5OH–Cl]+, [4-C2H5
OH–Cl2–H2O+3H]+, [4-C2H5OH–Cl2–2H2O+4H]+ and [4-C2H5OH–
Cl4–2H2O–Sn2+H]+. These spectral features confirm the formation
of heterobimetallic complexes. The isotopic distribution patterns
of the above peaks match well with the theoretical results. These
observations indicate that the metal-ligated skeletons of these
complexes are stable in solution.
(m
m
s(CO2)) value was used to determine the nature of bind-
D
m
between asymmetric (mas(CO2)) and symmetric (ms(CO2)) absorp-
tion frequency below 200 cmꢃ1 suggests the bidentate carboxylate
moiety while greater than 200 cmꢃ1 implies the unidentate car-
boxylate moiety [40]. In heterobimetallic complexes 3 and 4, the
Dm
values were 255 and 258 cmꢃ1 indicative of unidentate carbox-
ylate ligation. The coordination of the water molecules to Sn(IV)
metal ions was further supported by the appearance of non-ligand
band in the region 840–851 cmꢃ1 attributed to rocking mode of
water [41]. Other medium intensity bands at 749–755 cmꢃ1 were
attributed to the characteristic signatures of aromatic ring vibra-
tion. The heterobimetallic complex formation of 3 and 4 was also
Cl
Cl
COOH
OH2
Sn
HO
O
H
H2O
C O
C
N
O
H
C
O
M
N
M
N
NaOH, SnCl4.5H2O
CH3OH
O
N
O
COOH
OH
O
C
H
O
O
C
OH2
C
H
Sn Cl
Cl
O
H2O
M = Cu (II) & Zn (II)
Scheme 1. Synthesis of heterobimetallic complexes 3 and 4.

40
F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
3.4. EPR studies
The X-band EPR spectrum of complex 3 at LNT consists of a very
broad axial symmetrical signal with g|| at 2.18 and g\ at 2.073 and
giso at 2.10 computed from the formula giso = gk+2g?=3. These
parameters are consistent with the square planar geometry of
the central metal ion viz, copper(II) and are quite similar to the val-
ues reported for other related square planar copper(II) systems.
The trend g|| > g\ > ge (2.0023) further confirm that the ground
state of the copper(II) is predominantly dx2 ꢃ y2 orbital [47].
3.5. Thermal studies
Thermogravimetric analysis of heterobimetallic complex 4 was
carried out to study the pyrolysis pattern in temperature range 25–
800 °C. The thermogram of 4 is shown in Supplementary informa-
tion, Fig. S2, exhibited weight loss in three steps over the temper-
ature range 75–90, 210–400 and 415–650 °C. These were in close
agreement with the proposed structure. The weight loss in the
temperature range 75–90 °C confirmed the removal of C2H5OH
(4.2%) while at 210–400 °C (12.48%) the noticeable weight loss
indicated the removal of both the coordinated water molecule
and the chloride ions [48]. At 510 °C, the compound exhibited
approximately 41% of the weight loss corresponding to the decom-
position of aryl group and its substituents [49]. Finally, the TGA
curve showed a plateau above 710 °C corresponding to the forma-
tion of Sn-oxide and Zn-oxide as the final products.
3.6. Electronic spectra
The absorption spectra of the complexes were recorded in
DMSO, except ligand L which was soluble in EtOH. The electronic
spectrum of L exhibits two bands at 210 nm and 335 nm, which
Fig. 1. Absorption spectral traces of: (a) heterobimetallic complex 3, (b) heterobi-
metallic complex 4 in 5%DMSO/5 mM Tris HCl/50 mM NaCl buffer at pH 7.2 buffer
upon addition of CT DNA. Inset: plots of [DNA]/ea–
ef (m2 cm) vs. [DNA] for the
has been assigned to (
three broad bands at 255 nm, 300 nm and 550 nm, which were as-
signed to (
*), ( *) and 2B1 g ? 2A1 g, transitions, respectively
p–p p–p
*) and ( *). The complex 1 reveals
titration of CT DNA with complexes j, experimental data points; full lines, linear
fitting of the data. [complex] 0.16 ꢁ 10ꢃ4 M, [DNA] 0–1 ꢁ 10ꢃ4 M.
p–p
p–p
while complex 2 revealed two bands at 240 and 334 due to intral-
igand and LMCT transitions, respectively. The complex 3 revealed
two bands at 262 nm and 592 nm which have been assigned to
covalent binding to N7/N3 of the nucleobases. These complexes
show dual mode of action at the molecular target site and exhibit
novelty due to preferential selectivity and recognition towards
nucleobases inside the cells [53]. To quantify the extent of DNA
binding, the intrinsic binding constant Kb of ligand and the com-
plexes were determined [25] (Table 1), follows the order
4 > 3 > 2 > 1 > L which is in conformity with our hypothesis that
heterobimetallic complexes are more prominent DNA binders than
the monometallic in comparison to the free ligand. It is well estab-
lished in literature that Zn(II) complexes effectively function to
recognize thymine base and Zn(II) complexes in contrast to Cu(II)
break the hydrogen bonds of A–T base pairs of DNA helix to selec-
tively bind thymine; altering the local structure of the AT-region of
DNA [53]. These observations are also validated by interaction with
50-GMP and 50-TMP by UV–vis and in case of 4 by 1H and 31P NMR
spectroscopy.
(p–p
*) and 2B1 g ? 2A1 g. These results are consistent with the
square planar environment around Cu(II) metal ion [50]. Similarly,
the absorption spectrum of complex 4 revealed bands at 318 nm
and 390 nm due to intraligand and LMCT transitions, respectively.
4. DNA binding studies
4.1. Absorption titrations
The Uv–vis spectra of L, monometallic complexes 1 and 2 (Sup-
plementary information, Fig. S3a–c) and their heterobimetallic
complexes 3 and 4 depicted in Fig. 1a and b at room temperature
in DMSO exhibited strong bands in the UV region at 210–340 nm
attributed to
p– p–
p* or intraligand transitions. Changes in the p*
transition of L and 1–4 in 5% DMSO/5 mM Tris HCl/50 mM NaCl
buffer at pH 7.2 were determined as a function of added CT DNA
concentration (0–1 ꢁ 10ꢃ4). The ‘‘hyperchromic effect” is typical
of cationic complexes involving ligand with extended hydrophobic
regions or surfaces usually bind to DNA noncovalently [51]. Elec-
trostatic interaction, intercalation and groove binding are three
major noncovalent binding modes for the small molecules inter-
acting with DNA [52]. The cationic core of heterobimetallic 3 and
4 could exert a stronger electrostatic attraction to the anionic
phosphate backbone of DNA due to the presence of Sn(IV) which
has preferential selectivity for phosphate [25], in addition to the
presence of second metal ion Cu(II)/Zn(II) which prefers coordinate
Table 1
The binding constant (Kb) values of complexes including ligand with the DNA (mean
standard deviation of 0.12).
Complex
Kb (Mꢃ1
)
Monitored at
(nm)
%
Red shift
(nm)
Hyperchromism
L
1
2
3
4
0.76 ꢁ 103
2.20 ꢁ 103
5.50 ꢁ 103
1.67 ꢁ 104
4.00 ꢁ 104
210
334
334
257
265
23
51
57
20
55
00
02
02
03
02

F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
41
4.2. Interaction with 50-GMP and 50-TMP
Usually, Cu complexes specifically binds to GMP, (which is more
basic than AMP) through its N7 nitrogen [54] and Zn specifically
binds to TMP, through its N3 nitrogen [53] therefore, further inter-
action has been done with 50-GMP and 50-TMP. In the complexes 3
and 4, a sharp increase in absorbance intensity ‘‘hyperchromic
effect” was observed in the UV region which was followed by con-
comitant decrease in intensity ‘‘hypochromic effect” (Fig. 2 and 3a
and b). Hyperchromism is indicative of electrostatic mode of bind-
ing while the hypochromism could be attributed to the partial
intercalation which is also evidenced by a sharp decrease in the
fluorescence intensity of complexes 3 and 4 in EB replacement as-
say. The Kb values further revealed that the magnitude of binding
of 3 was higher than 4 in case of 50-GMP, while it was reverse in
case of 50-TMP (Table 2). When thymine binds to 4, hydrogen
bonds could be favored between nitrogen atoms of ligand with car-
bonyl oxygen of thymine. Secondly, the presence of electron with-
drawing two oxo groups at the C2 and C3 position of the thymine
lowers the energy of the lone pair orbital at N3 of thymine base
which results in stronger molecular orbital interaction in compar-
ison to guanine [55]. These results can be corroborated with the
observation of UV–visible titration with CT DNA and demonstrate
Fig. 3. Absorption spectral traces of: (a) complex 3 and (b) complex 4 in 5%DMSO/
5 mM Tris HCl/50 mM NaCl buffer upon addition of 50-TMP. [Complex] 0.16 ꢁ 10ꢃ4
M, [50-GMP] 0–1 ꢁ 10ꢃ4 M.
the specific recognition of heterobimetallic complex 4 for thymine.
Our results are consistent with the earlier reports on preferential
binding to N3 position of thymine in the zinc(II) complexes [56].
To validate our hypothesis the binding of 4 to 50-GMP and 50-
TMP was examined by 1H and 31P NMR techniques at physiological
pH 7.3. The 50-GMP exhibited H8 signal at 8.07 ppm and H10-H50
ribose proton signals of 3.85–5.80 ppm, respectively. In the presence
Table 2
The binding constant (Kb) values of complexes 3 and 4 with the nucleotides.a.
Complex 50-GMP
(105 Mꢃ1
50-TMP
Monitored Hyperchromism Red
)
(105 Mꢃ1
)
at (nm)
(%)
shift
(nm)
3
3
3
4
4
4
4
0.69
0.33
–
0.13
0.12
–
–
–
0.77
–
–
245
708
247
328
390
255
300
40
04
26
05
37
22
07
2
1
3
2
3
4
2
1.32
1.30
Fig. 2. Absorption spectral traces of: (a) complex 3 and (b) complex 4 in 5% DMSO/
5 mM Tris HCl/50 mM NaCl buffer upon addition of 50-GMP. [Complex] 0.16 ꢁ 10ꢃ4
M, [50-GMP] 0–1 ꢁ 10ꢃ4 M.
–
a
50-GMP and 50-TMP (mean standard deviation of 0.08).

42
F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
of complex 4, there was no significant shift in H8 signal of guanine.
At different time intervals, the spectra remains unaltered after
24 h, which was attributed to non-participation of guanine on
binding with 4 through coordination mode. However, the ribose
proton H10–H50 signals have shifted downfield ꢀ0.06 ppm, which
indicate the preference for ribose sugar due to the presence of
Sn(IV), hard Lewis acid center. The 31P NMR signal appeared in
free 50-GMP at 3.67 ppm which was shifted upfield to 1.67 ppm
indicating clearly that Sn(IV) was bound to phosphate group.
(Supplementary information, Fig. S4a and b). On interaction of 4
with 50-TMP, the signal of N3–H atom of 50-TMP observed at
7.64 ppm disappeared due to deprotonation of the N3 proton.
The signal due to C6–H atom of 50-TMP at 6.2 ppm shifted to
6.9 ppm with increased intensity. The T–CH3 peaks at 1.95 shifted
to slightly higher frequency relative to that of free TMP. The rest of
the signals associated with free 50-TMP merged with the signals of
complex 4. This has resulted in the enhancement of the intensity
of signals of the metal bound 50-TMP. At different time intervals,
the spectra remain unaltered and even after 24 h no changes were
observed in the spectral pattern ((Supplementary information,
Fig. S5a). The 31P NMR signal appeared in free 50-TMP at
3.67 ppm which was shifted upfield to 1.70 ppm. The 119Sn NMR
spectrum of the complex 4 displayed one sharp signal at –622.33
attributed to hexacoordinate environment around the tin atoms
[45]. On interaction with 50-TMP, the signal at ꢃ622.33 shifts to
ꢃ868.61 ppm (Supplementary information, Fig. S5b). The large
chemical shift was attributed to the obvious change in coordina-
tion geometry from hexa to heptacoordinate due to the binding
of Sn(IV) to the oxygen atom of the thymine nucleobases.
4.3. Fluorescence spectroscopic studies
The ligand L and complexes 1–4 exhibited luminescence either
in DMSO or in presence of CT DNA. Hence, binding studies of L and
the complexes 1–4 to CT DNA were carried out by fluorescence
spectral titration in absence of any fluorophore [57]. Upon addition
of CT DNA to the L and complexes 1–4 there is enhancement in the
fluorescence–emission intensity (Supplementary information,
Fig. S6a–e). Since it is found that complexes with increased ligand
hydrophobicities show greater increase in emission intensities
upon binding to polyelectrolytes. Thus, we conclude that there is
some interaction between the CT DNA and complexes that occurs
due to the hydrophobicity of both molecules. The binding constant
estimated for L and the complexes 1–4 by Scatchard equation were
8.3 ꢁ 103 Mꢃ1, 1.3 ꢁ 104 Mꢃ1, 1.5 ꢁ 104 Mꢃ1, 3.5 ꢁ 104 Mꢃ1 and
4.8 ꢁ 104 Mꢃ1, respectively. The binding constant K values were
of the order 4 > 3 > 2 ꢄ 1 > L, and the number of metal complex
including ligand L bound per DNA (n) calculated [36,37] was found
to be 0.61, 1.04, 1.15, 1..57 and 1.81, respectively. These results are
consistent with the findings obtained from UV-vis spectral studies.
Fig. 4. Emission quenching spectra of: (a) complex 3 and (b) complex 4, with
increasing concentration of quencher ethidium bromide, in the absence and
presence of CT DNA in buffer 5 mM Tris–HCl/50 mM NaCl, pH = 7.2 at room
temperature.
The Ksr values for 3 and 4 were found to be 0.98 ꢁ 105 Mꢃ1and
1.56 ꢁ 105 Mꢃ1, respectively. Since EB was not completely dis-
placed, partial intercalation in addition to the electrostatic mode
of binding cannot be ruled out.
4.5. Viscosity measurements
Measurement of DNA viscosity is regarded as the least ambigu-
ous and the most essential analysis of the binding mode of DNA in
solution. In the absence of crystallographic structural data, viscos-
ity measurements are regarded as most critical test of a DNA bind-
ing model in solution [60]. To clarify further the mode of
interaction between the complexes 3, 4 and DNA, viscosity mea-
4.4. Ethidium bromide displacement assay
To further investigate the mode of binding of the complexes 3
and 4, the ethidium bromide displacement assay was carried out
[58]. The extent of quenching of the fluorescence of EthBr bound
to DNA would reflect the extent of DNA binding of 3 and 4. EB is
an intercalator that gives a significant increase in fluorescence
emission when bound to DNA and its displacement from DNA re-
sults in a decrease in fluorescence intensity [59]. On addition of
complexes 3 and 4 to CT DNA pretreated with EthBr ([DNA]/
[EB] = 1) there was a sharp decrease in emission intensity (Fig. 4a
and b). Addition to the EB-DNA system induces notable fluores-
cence changes, which indicate that EB molecule, have been re-
placed by the complexes 3 and 4. Further, the quenching extents
were evaluated qualitatively by employing Stern–Volmer equation.
surements were performed and the results were presented as (g/
g
0) vs. binding ratio, where
g
is the viscosity of DNA in the pres-
0 is the viscosity of DNA alone. Since the rel-
0) of DNA reflects the increase in
ence of complex and
ative specific viscosity (g/g
g
contour length associated with separation of DNA base pairs
caused by intercalation, a classical intercalator such as EB could
cause a significant increase in viscosity of DNA solutions. In con-
trast, a partial and/or non-classical intercalation of the ligand could
bend or kink DNA resulting in a decrease in its effective length with
a concomitant increase in its viscosity [61]. The plots of relative

F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
43
strate. The activity of 3 and 4 was assessed by the conversion of
DNA from Form I to Form II or Form III. A concentration-dependent
DNA cleavage by 3 and 4 was first performed. Briefly, pBR322 DNA
was mixed with different concentration of 3 and 4 and the mixture
was incubated at 310 K for 45 min Fig. 6a. With the increase of con-
centration of 3 and 4, DNA was converted from Form I (Supercoiled
Form) to Form II (Nicked Circular Form) and then to Form III (Lin-
ear Form).
Since the nuclease efficiency of complexes is usually dependent
on activators [64]. Therefore, DNA cleavage activity of 3 and 4 in
presence of H2O2, ascorbate, 3-mercaptopropionic acid and gluta-
thione was evaluated (Fig. 6b). The cleavage activity of 3 and 4
were significantly enhanced by these activators and follows the or-
der MPA > H2O2 > Asc > GSH for 3, while 4 follows the order
MPA > Asc > GSH > H2O2.
4.7.2. DNA cleavage in presence of minor and major groove binding
agent
Fig. 5. Effect of increasing amount of 3 and 4 on the relative viscosities (g/go) of CT
DNA in Tris–HCl buffer (pH 7.2).
Minor groove binding agent DAPI [65] and major groove bind-
ing agent methyl green [66] were used to probe the potential inter-
acting site of complex 3 and 4 with plasmid pBR322 DNA. The DNA
was treated with DAPI or methyl green prior to the addition of 3
and 4. The patterns presented in the Supplementary information
Fig. S8, demonstrated that 3 prefers both major and minor groove,
whereas 4 prefers minor.
viscosities with R = [M]/[DNA] are shown in Fig. 5. The relative vis-
cosity of DNA increases with increase in the concentration of the
complexes 3 and 4 but less compared to that of potential classical
intercalators, e.g. ethidium bromide. This is consistent with the ob-
served trend by other optical methods and suggesting primarily
electrostatic and/or groove binding nature of the complex. Since
the increase is not very high, therefore a combination of both elec-
trostatic as well as groove binding may not be ignored.
4.7.3. Reactive oxygen species responsible for DNA cleavage
The interaction between metal complexes and dioxygen or re-
dox reagents are believed to be a major cause of DNA damage
[67]. To probe the potential mechanism of DNA cleavage mediated
by complex 3 and 4, some standard radical scavengers were used
prior to the addition of 3 and 4 to DNA solution. The involvement
of ROS was investigated using tert-butyl alcohol and DMSO [68] as
a hydroxyl radical scavenger, sodium azide [69] as singlet oxygen
scavenger and SOD as superoxide oxygen scavenger under identi-
cal conditions. On adding DMSO and tert-butyl alcohol (Lanes 2,
3 for 3 and Lanes 6, 7 for 4) inhibit the DNA cleavage suggestive
of involvement of diffusible (ÅOH) hydroxyl radicals as one of the
ROS responsible for DNA breakage. (Fig. 6c). On the other hand,
addition of sodium azide (singlet oxygen scavenger) decreases
the cleavage efficiencies, this reveals that 1O2 is also the activated
oxygen intermediate responsible for the cleavage (Lane 4 for 3 and
Lane 8 for 4). Addition of superoxide dismutase (superoxide scav-
enger) to the reaction mixture show no significant quenching of
the cleavage reaction revealing that superoxide anion is not the ac-
tive species responsible for the cleavage (Lane 5 for 3 and Lane 9
4.6. Interaction of metal complexes with DNA by IR spectroscopy
Shift ofthe guanineband at1710 cmꢃ1 (guanine–C = O stretch)to
a lower frequencyat 1695 cmꢃ1 for 3-DNA and 1685 cmꢃ1 for 4-DNA
complexes indicate the metal–DNA interaction [62], (Supplemen-
tary information, Fig. S7). An increase in the intensity of the band
at 1685 cmꢃ1 for 4-DNA complex was also observed while 3-DNA
complex has no change in intensity. The spectral changes observed
for the band at 1710 cmꢃ1 are due to complexes–DNA interaction
via guanine bases. Thymine band which is observed at 1661 cmꢃ1
(thymine–O2) showed a small shift for 4-DNA complexes from
1661 to 1658 cmꢃ1 whereas no major change observed for 3-DNA
interaction. The negative and positive features centered at 1627–
1644 cmꢃ1 in the spectra, complex–DNA interaction is may be be-
cause of thymine interaction or from H2O bending mode, as it is very
difficult to differentiate between them [63]. Similarly, the adenine
band at 1610 cmꢃ1 (adenine–N7) exhibits no shifting and intensity
changes due to a lack of metal complex–adenine interaction. No ma-
jor alterations of cytosine band at 1491 cmꢃ1 can also be attributed
to no direct metal complex–cytosine binding.
DNA is characterized by the spectral alterations of the PO2
asymmetric and symmetric bands at 1225 and 1088 cmꢃ1, respec-
tively [63]. The PO2 asymmetric band at 1225 cmꢃ1 and symmetric
band at 1088 cmꢃ1 shifted towards lower frequencies at 1224 and
1082 (3-DNA), 1228 and 1081 (4-DNA), respectively. The observed
spectral shifting was accompanied by intensity variations of the
phosphate bands. The spectral changes observed are due to a major
analogue-phosphate interaction, because of Sn(IV) which specifi-
cally binds to phosphate backbone of DNA and thus contributes
to the stabilization of metal complex–DNA interaction.
Fig. 6a. The cleavage patterns of the agrose gel electrophoresis diagram showing
cleavage of pBR322 supercoiled DNA (300 ng) by 3 and 4 at 310 K after 45 min of
4.7. DNA cleavage activity
incubation; Lane 1, DNA control; Lane 2, DMSO control; Lane 3, 20
complex + DNA; Lane 4: 40 of complex + DNA; Lane 5: 60 of com-
plex + DNA; Lane 6: 80 M of complex + DNA; Lane 7: 100 M of complex + DNA;
Lane 8: 120 M of complex + DNA.
lM of
4.7.1. DNA cleavage without added reductant
The DNA cleavage ability of 3 and 4 was studied by agarose gel
electrophoresis using supercoiled pBR322 plasmid DNA as a sub-
lM
lM
l
l
l

44
F. Arjmand, Mohd. Muddassir / Journal of Photochemistry and Photobiology B: Biology 101 (2010) 37–46
Fig. 6b. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 3 and 4 in presence of different activating agents at 310 K after
incubation for 45 min. Lane 1: DNA control; Lane 2: 120
(0.4 M) + DNA; Lane 3: 120 M of complex 3 + Ascorbic acid (0.4 M) + DNA; Lane 4:
120 M of complex 3 + MPA (0.4 M) + DNA; Lane 5: 120 M of complex 3 + GSH
(0.4 M) + DNA; Lane 6: 120 M of complex 4 + H2O2 (0.4 M) + DNA; Lane 7: 120
of complex 4 + Ascorbic acid (0.4 M) + DNA; Lane 8: 120 M of complex 4 + MPA
(0.4 M) + DNA; Lane 9: 120 M of complex 4 + GSH (0.4 M) + DNA.
lM of complex 3 + H2O2
Fig. 7. The cleavage patterns of the agrose gel electrophoresis diagram showing
effect of different concentration of 3 and 4 on the activity of DNA topoisomerase II
(Topo II); Lane 1, Topo II control; Lane 2, DNA control; Lane 3, 2.5 M of complex
3 + DNA + Topo II; Lane 4: 5.0 M of complex 3 + DNA + Topo II; Lane 5: 7.5 M of
complex 3 + DNA + Topo II; Lane 6: 10 M of complex 3 + DNA + Topo II; Lane 7:
12.5 M of complex 3 + DNA + Topo II; Lane 8, 2.5 M of complex 4 + DNA + Topo II;
Lane 9: 5.0 of complex 4 + DNA + Topo II; Lane 10: 7.5 of complex
4 + DNA + Topo II; Lane 11: 10 of complex 4 + DNA + Topo II; Lane 12:
12.5 M of complex 4 + DNA + Topo II.
l
a
l
l
l
l
lM
l
l
l
l
l
l
l
lM
lM
lM
l
33258 [75], Topostatin [76],
D
-[Ru(bpy)2(uip)]2,+
[Ru(bpy)2(appo)]2+ [13].
K-[Ru(b-
py)2(uip)]2+ [77], [Ru(bpy)2(dppz)]2+
,
These findings implicate that complexes 3 and 4 are catalytic
Fig. 6c. Agarose gel electrophoresis pattern for the cleavage of pBR322 supercoiled
DNA (300 ng) by 4 in presence of Reactive oxygen species at 310 K after incubation
inhibitors of human topoisomerses II.
for 45 min. Lane 1: DNA control; Lane 2: 120
(0.4 M) + DNA; Lane 3: 120 of complex 3 + DMSO (0.4 M) + DNA; Lane 4:
120 M of complex 3 + sodium azide (0.4 M) + DNA; Lane 5: 120 M of complex
3 + SOD (15 units) + DNA; Lane 6: 120 of complex 4 + tert-butyl alcohol
(0.4 M) + DNA; Lane 7: 120 of complex 4 + DMSO (0.4 M) + DNA; Lane 8:
120 M of complex 4 + sodium azide (0.4 M) + DNA; Lane 9: 120 M of complex
4 + SOD (15 units) + DNA.
lM of complex 3 + tert-butyl alcohol
l
M
5. Conclusion
l
l
l
M
The in vitro DNA binding studies of novel heterobimetallic com-
plexes 3 and 4 reveal an electrostatic mode of binding as well as
major/minor groove binding, respectively. The complex 3 exhib-
ited an outstanding ability to affect DNA double strand scission
in oxidative as well as free hydroxyl radical manner. Our spectro-
scopic results demonstrate that complex 4 is an avid and more
strong DNA binder which is well corroborated with the literature
reports In addition, both the complexes exhibit high inhibitory ef-
fect on topoisomereses, in particular complex 4 shows high inhibi-
tion activity against Topo I at a very low concentration. This work
features design of novel metal-based topoisomerase inhibitors,
also specific DNA groove binders, and since such inhibitions are re-
lated to DNA binding therefore, they can act as potent antitumour
drugs.
lM
l
l
for 4) [70]. Thus, freely diffusible oxygen intermediate or hydroxyl
radical is involved in the strand scission and hence a simple diffus-
ible radical mechanism is applicable.
4.8. Topoisomerase inhibition
Metal complexes have been reported to inhibit topoisomerase I
and II despite their moderate DNA binding [71]. The mechanism of
human Topo I (topoisomerase I) has been well-established [72,73].
The results of Topo I inhibition assay by different concentration of
3 and 4 are shown in Supplementary information Fig. S9. Both
complexes inhibited the ability of Topo I to relax negatively super-
Acknowledgement
coiled plasmid DNA (IC50 is >12
l
M for 3 and ꢀ2.5
lM for 4). These
We express our gratitude to Department of Biotechnology, New
Delhi, for generous financial support (Scheme No. BT/PR9208/
Med/30/13/2007). Thanks to RSIC, Panjab University, Chandigarh
for providing CHN analysis data and NMR, and IIT Bombay for
EPR measurements.
findings imply that both complexes may block the DNA strand pas-
sage event of the enzyme, and serve as catalytic inhibitor of Topo I.
Since Topo I removes only the unconstrained positive supercoils,
the negatively supercoiled DNA product would be identical to the
topological state of the original plasmid substrate. In this case,
the complexes will also appear to inhibit enzyme catalysis. The
DNA topoisomerase inhibitory activity of the complexes under
study was found to be concentration-dependent. Surprisingly,
complex 4 displays a significant inhibitory action on Topo I rather
than complex 3 (which display a significant concentration-depen-
dent DNA cleavage and further exhibited strong cleavage in pres-
ence of different activators).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jphotobiol.2010.06.009.
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creases complexes 3 and 4 successively inhibited the activity of
Topo II, and finally inhibited the activity of Topo II at a concentra-
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