Design, synthesis and crystal structure determination of dinuclear copper-based potential chemotherapeutic drug entities; In vitro DNA binding, cleavage studies and an evaluation of genotoxicity by micronucleus test and comet assay

Article abstract of DOI:10.1039/c2md20141k

Copper-based potential chemotherapeutic complexes 1 and 2 were designed, synthesized and evaluated for in vitro DNA binding, cleaving capability and in vivo genotoxicity. The structural elucidation of complexes was done using elemental and spectroscopic data while the (R)-enantiomer of Cu(ii) complex 1 was studied by single crystal diffraction. In vitro DNA binding profiling of both (R)- and (S)-enantiomers of complexes 1 and 2 was carried out to evaluate their enantioselectivity, exhibiting a remarkable degree of enantioselectivity in their interaction with DNA, with the (R)-enantiomer exhibiting greater DNA binding propensity. Interaction between complexes and pBR322 DNA was evaluated by agarose gel electrophoresis assay; both the (R)-enantiomeric complexes exhibit effective DNA cleavage and proceed via an oxidative pathway. Furthermore, the in vivo genotoxicity of the (R)-enantiomer of complex 1 was evaluated by micronucleus testing on bone marrow cells and comet assay in peripheral blood lymphocytes. These results support our contention that the (R)-enantiomer of complex 1 is a suitable chemotherapeutic drug candidate showing reduced toxic effects on normal cells as compared to cisplatin and an antioxidant (EVOO). The Royal Society of Chemistry.

Full text of DOI:10.1039/c2md20141k

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Design, synthesis and crystal structure determination of
dinuclear copper-based potential chemotherapeutic
drug entities; in vitro DNA binding, cleavage studies and
an evaluation of genotoxicity by micronucleus test and
comet assay†
Cite this: DOI: 10.1039/c2md20141k
a
Farukh Arjmand, Mohd Muddassir,a Yusra Zaidib and Debashis Rayc
*
Copper-based potential chemotherapeutic complexes 1 and 2 were designed, synthesized and evaluated
for in vitro DNA binding, cleaving capability and in vivo genotoxicity. The structural elucidation of
complexes was done using elemental and spectroscopic data while the (R)-enantiomer of Cu(II) complex
1 was studied by single crystal diffraction. In vitro DNA binding profiling of both (R)- and (S)-
enantiomers of complexes 1 and 2 was carried out to evaluate their enantioselectivity, exhibiting a
remarkable degree of enantioselectivity in their interaction with DNA, with the (R)-enantiomer
exhibiting greater DNA binding propensity. Interaction between complexes and pBR322 DNA was
evaluated by agarose gel electrophoresis assay; both the (R)-enantiomeric complexes exhibit effective
DNA cleavage and proceed via an oxidative pathway. Furthermore, the in vivo genotoxicity of the (R)-
enantiomer of complex 1 was evaluated by micronucleus testing on bone marrow cells and comet assay
in peripheral blood lymphocytes. These results support our contention that the (R)-enantiomer of
complex 1 is a suitable chemotherapeutic drug candidate showing reduced toxic effects on normal cells
as compared to cisplatin and an antioxidant (EVOO).
Received 30th May 2012
Accepted 27th November 2012
DOI: 10.1039/c2md20141k
www.rsc.org/medchemcomm
Genotoxicity of CDDP and other anticancer drugs which exhibit a
mechanism of action similar to cisplatin is a serious problem
Introduction
Cisplatin, cis-diamminedichloroplatinum(II) or CDDP, is the
most striking example of an anticancer drug widely used clini-
cally in modern medicinal chemistry.1 CDDP is highly effective
in treating a variety of solid tumors, including testicular (cancer
which was incurable prior to cisplatin therapy) for which the
overall cure rate exceeds 90%.2
Cisplatin exerts its mode of action as a consequence of cova-
lent bonding to the natural structure of double helix DNA. More
than 90% of CDDP adducts are DNA cross-links. If enough of
these adducts are produced without repair, the cell undergoes
apoptotic death. This process is not selective, cisplatin interacts
with non-cancerous (healthy or normal) cells and biomolecules
producing secondary effects that limit the dose that can be
administered.3 In addition, cisplatin is also associated
with systemic toxicity and inherent or intrinsic resistance.
due to the possibility of inducing secondary tumors or ‘metas-
tasis’.4 To address this issue, optimization of the chemical enti-
ties to exert specic chemotherapeutic action in vitro and in vivo
is being carried out by many researchers worldwide.5–7
Chiral drugs are at the forefront of pharmaceutical drug
research as introduction of chirality not only enforces stereo-
selective specic drug interaction but also promotes the
formation of active compounds with therapeutic benets as
most of the biotargets viz., DNA (the primary intracellular
target) are chiral in nature. Enantioselective interaction plays a
pivotal role in drug DNA adducts formation and provides clues
on metal complex coordination to DNA helix (binding mode).
Furthermore, the use of stereochemistry can give clear insight
into the mechanism of action allowing the discrimination
between unspecic interaction which is common to both
enantiomers and specic contacts that give rise to enantiose-
lectivity. In view of this, we have designed copper-based
complexes 1 and 2 which are chiral in nature derived from
reduced Schiff base ligands of salicylaldehyde/benzaldehyde
and (R)- and (S)-2-amino-1-propanol. These complexes bear
novelty because of (i) reduced Schiff base skeletons which are
more exible and stable, (ii) chiral stereogenic carbon centres,
aDepartment of Chemistry, Aligarh Muslim University, Aligarh-202002, India. E-mail:
farukh_arjmand@yahoo.co.in; Tel: +91 5712703893
bDepartment of Zoology, Aligarh Muslim University, Aligarh-202002, India
cDepartment of Chemistry, Indian Institute of Technology, Kharagpur, 721302, India
† Electronic supplementary information (ESI) available. CCDC 765605. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c2md20141k
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(iii) bio-essential copper centres which show high nucleobase both enantiomeric complexes was carried out to evaluate their
affinity capable of efficient DNA cleavage, (iv) dinuclear redox enantioselective preference.
active copper centres (redox activity of metal complexes is
correlated with their anti-proliferative properties) and (v)
exceptional DNA cleaving capability. We have validated DNA
binding potential and cleavage ability of (R)- and (S)-enantio-
mers of 1 and 2 to evaluate their enantiopreferential selectivity.
Apart from the modulation of the drug design, the possible role
of dietary antioxidants (EVOO) in combination with anticancer
agents can be promising to minimize or avoid primary genetic
damage and therefore the effects of olive oil in the presence of
the complex 1b as a potential chemotherapeutic drug candidate
and also with CDDP have been evaluated. Considering the role
of DNA damage in some diseases, the evaluation of the geno-
toxicity and protective effects of small molecules or therapeutic
compounds has become very important. The micronucleus
(MN) assay on peripheral blood or bone marrow cells is
considered the primary assay for assessing in vivo genotoxic
potential.8 The alkaline comet assay complements the MN test.9
We have analyzed the in vivo genotoxicity of these complexes by
micronucleus testing on bone marrow cells and comet assay in
peripheral blood lymphocytes. This is the rst report to the best
of our knowledge on the protective effect of EVOO against
damage induced by CDDP and complex 1b. Overall, these chiral
complexes exhibit potential to act as promising specic anti-
tumor drugs with reduced toxicity.
Synthesis and characterization
IR spectroscopy is one of the useful techniques used to assign
the carboxylate coordination modes, and the following trend is
generally accepted as a guideline to differentiate various
carboxylate coordination modes, Dn chelating < Dn bridging <
Dn ionic < Dn monodentate.10 Complex 1 revealed intense bands
at 1632 cmꢀ1 for nasym (OCO) and at 1391 cmꢀ1 for nsym (OCO),
respectively. The Dn ¼ 241 cmꢀ1 value of complex 1 was higher
than that reported for sodium acetate (Dn ¼ 164 cmꢀ1), and thus
indicates a monodentate acetate coordination mode.11 Complex
2 revealed one band at 1636 cmꢀ1 assignable to nasym (OCO) and
one band at 1479 cmꢀ1 assignable to nsym (OCO) and the
calculated Dn ¼ 157 cmꢀ1 corresponded to syn–syn bidentate
bridging.12 The characteristic HC]N stretching vibration,
which appears at the 1575–1630 cmꢀ1 range, was absent in the
ligands, clearly supporting the reduction of the imine bond by
NaBH4. A broad band around 3450 cmꢀ1 suggested the presence
of lattice water in 1.13 Furthermore, diagnostic IR bands at 3120,
3262 cmꢀ1 and 1546, 1543 cmꢀ1 due to n(–NH) and d(–NH),
respectively, for complex 1 and 2 were observed.14 The presence
of bound aqua ligand in the case of 2 has been ascertained by a
medium intensity band at 3753 cmꢀ1
.
Both complexes 1 and 2 exhibited a broad d–d transition
envelope at 661 and 676 nm, respectively consistent with ve-
coordinate Cu(II) centers having square-pyramidal geometry.15
The stronger absorption bands at 393 nm of complex 1 were
ascribed to a charge transfer transition from the phenolate
moieties to the terminal copper ions, however, these bands were
absent in complex 2, because of the presence of the benzalde-
hyde moiety. The bands around 247 nm and 251 nm were
ascribed to p–p* transition for 1 and 2, respectively.
EPR spectroscopy has proven to be a valuable tool for the
elucidation of the molecular structure of the binuclear copper
complexes.16 The X-band EPR spectra of 1 and 2 were recorded
at room and different temperatures in the solid as well as liquid
state (ESI, Fig. S1†). The spectra (solid) at room temperature
show no hyperne splitting of 1 and 2 irrespective of tempera-
ture variation. The g values are given in Table 4.
Results and discussion
The synthesis involves in situ reduction (by NaBH4) of a Schiff
base of salicylaldehyde/benzaldehyde and (R)- and (S)-2-amino-
1-propanol to yield the ligand L1 and L2 (a and b), respectively.
Subsequently, complexes 1 and 2 were synthesized and thor-
oughly characterized (Scheme 1). Complexes 1 and 2 are stable
toward air and moisture and soluble in H2O and other common
solvents. Molar conductance values of complexes reveal their
non-electrolytic nature. In vitro DNA binding and cleavage of
In solution state (methanol) four hyperne lines in the
spectra of 1 and 2 were observed reecting the coupling with the
Cu(II) nucleus (I ¼ 3/2), indicative of the existence of Cu(II)–Cu(II)
interactions.17 No effect of temperature was observed in the
liquid state as it remains the same with the same hyperne
splitting. The trend g > gt > 2.02 reveals that the unpaired
k
electron is present in the dx2ꢀy2 orbital.18 For a Cu(II) complex, g
k
is a parameter sensitive enough to indicate covalence. For a
covalent complex, g < 2.3 and for an ionic environment, g ¼ 2.3
k
k
or more. In the present complexes g < 2.3 which indicates an
k
appreciable metal–ligand covalent character.
Description of the crystal structure of 1b
The single crystal X-ray structure determination of complex 1b
Scheme 1 Synthetic scheme of ligands L1, L2 and complexes 1 and 2.
was undertaken. All calculations were performed using
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SHELXL-97 implemented in the WINGX system of programs.
ORTEP views of complex 1b with atom numbering scheme and
thermal ellipsoids are drawn at 30% probability level and are
given in Fig. 1 and 2. The crystallographic data are given in
Table 1, and the selected bond lengths and angles for the
asymmetric unit are given in Tables 2 and 3. The asymmetric
part of the unit cell consists of two crystallographically inde-
pendent and chemically identical units of complex 1b, units 1(a)
and 1(b), respectively and four symmetrically H-bonded water
molecules. The asymmetric part represents a [Cu2] complex
bridged by phenolate oxygen (Oph) atoms. Two tridentate
deprotonated [OphNamO1aH]ꢀ ligands bind the CuII ion in a
facial mode. In the square-pyramidal coordination environ-
ment, depending on the axial or equatorial positions, the Cu–O
bond distances in the Cu–m–Oph unit vary within a wide range of
˚
˚
1.936–1.900 A for 1a and 1.927–1.981 A for 1b, respectively. The
combination of two short and two long bonds clearly indicates
the presence of an asymmetric Cu2O2 diamond motif.
Further coordination from amine nitrogen (Nam), terminal
alkoxido oxygen (Oal) and acetato oxygen (Oac) atoms completes
the distorted N2O3 square pyramidal environment around each
CuII. Within the diamond core the Cu1–Oph–Cu2 angles are
102.51ꢁ, 102.16ꢁ for 1(a) and 101.08ꢁ, 103.48ꢁ for 1(b). The solid
angle around the bridging Oph atoms ranges from 356.2ꢁ to
359.84ꢁ for a trigonal planar arrangement compared to a
Fig. 2 One unit of labeled ORTEP view of 1b with atom numbering scheme.
H atoms and water molecules are omitted for clarity.
˚
distance of 1.936 A for greater bonding interaction with the
trigonal pyramidal structure. The bridging phenolate (Oph
)
bridging phenolate oxygen atom. On the basis of the coordi-
nation sphere symmetry, it was proposed that at the square
pyramid coordination limit (near s ¼ 0) the magnetic orbitals
are mainly dx2ꢀy2 and weak ferromagnetic interactions should
groups provide an intramolecular CuII/CuII separation of 3.063
˚
˚
A and 3.049 A for 1(a) and 1(b), respectively. The calculation of
the least-squares planes dened by the atoms of the square-
planar coordination plane including the central metal atom
shows the copper ions to be displaced from the basal planes
result.20 The Cu–Nam distances (av. 2.007 A for 1(a) and av. 2.005
˚
˚
A for 1(b)) are in the normal range. The av. apical Cu–OalH
˚
toward the alkoxido oxygen atom by 0.764 (Cu1) and 0.253 A
˚
˚
distances (2.236 A in 1(a) and 2.298 A in 1(b)) are longer than the
˚
(Cu2) of 1(a) and 0.208 (Cu3) and 0.317 A (Cu4) of 1(b),
˚
˚
av. Cu–Oac distances (2.006 A in 1(a) and 1.964 A in 1(b)). The
terminal monodentate acetate groups having pendant carboxyl
oxygen atoms show an extensive H-bonding network with four
respectively. The symmetry of the coordination spheres around
the two CuII ions measured by the Addison parameter (s)19 is
best described by distorted square pyramids with s values for
Cu1, Cu2, Cu3 and Cu4 as 0.22, 0.33, 0.17 and 0.10,respectively.
Thus, the Cu2 center is more distorted with a shorter Cu–Oph
Table 1 Crystallographic data for complex 1ba
CCDC no.
Color
CCDC 765605
Blue
Molecular formula
Molecular weight
Crystal system
Space group
C24H34N2O10Cu2
637.63
ꢀ
P1
Orthorhombic
12.104(3)
16.437(4)
27.634(7)
5498(2)
˚
a (A)
˚
b (A)
˚
c (A)
3
˚
U (A )
Dc (g cmꢀ3
Z
)
1.531
8
F(000)
2608
Crystal size (mm)
0.06 ꢂ 0.09 ꢂ 0.19
m (mmꢀ1
)
1.603
q range (ꢁ)
2.09–28.43
0.0650, 0.0995
1.064
R1, wR2 [I > 2s(I)]
Goodness-of-t on F2
ꢀ3
˚
Final difference map max., min. (e A
)
1.210, ꢀ0.838
P
P
P
P
a
(||Fo| ꢀ |Fc||)/ |Fo|; wR2 ¼ [ w(|Fo| ꢀ |Fc|)2/ w(Fo)2]1/2
;
Fig. 1 ORTEP view of 1b with atom numbering scheme. H atoms are omitted
R1
¼
w ¼ 0.75/(s2(Fo) + 0.0010Fo2).
for clarity.
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˚
˚
˚
˚
Table 2 Selected bond lengths [A] in 1b
O14 ¼ 2.778 A, O14/O4W ¼ 2.828 A; O16/O4 ¼ 2.577 A]. All
these distances are comparable to the O/O distances in ice
Cu(4)–O(9)
Cu(4)–O(11)
Cu(4)–N(3)
Cu(4)–O(10)
Cu(4)–Cu(3)
Cu(3)–O(9)
Cu(3)–O(11)
Cu(3)–N(4)
1.956(5)
1.967(5)
2.018(5)
2.282(5)
3.0487(13)
1.927(4)
1.981(4)
1.993(5)
Cu(1)–O(2)
Cu(1)–O(1)
Cu(1)–O(3)
Cu(2)–O(4)
Cu(2)–N(2)
Cu(2)–O(3)
Cu(2)–O(1)
Cu(3)–O(12)
2.271(5)
1.990(4)
1.958(4)
2.201(5)
1.999(5)
1.979(4)
1.936(4)
2.315(5)
21
˚
(about 2.74 A). The (H2O)3 trapped supramolecular 1b/
(H2O)3/1b arrangement is shown in Fig. S4.†
DNA binding prole
UV-vis absorption titrations. The absorption spectra of the
complexes in the absence and presence of CT DNA at constant
concentration of complexes “66.6 ꢂ 10ꢀ6 M” are depicted in
Fig. 3 (1a and 1b). With increasing CT DNA (0–33.3 ꢂ 10ꢀ6 M),
the absorption bands are affected exhibiting “hyperchromism”
26–39% which reects higher binding propensity of the
complexes for DNA. The labile acetate ion (or water molecule for
2a and 2b) of the complexes could be replaced by a nucleophile
in DNA, usually a nitrogeneous base such as guanine N7,
leading to a strong DNA binding of the complexes.22 In some
cases, binding to the adenine N7 nitrogen has also been
Table 3 Selected bond angles [ꢁ] in 1b
O(13)–Cu(4)–O(9)
O(9)–Cu(4)–O(11)
O(9)–Cu(4)–N(3)
O(13)–Cu(4)–O(10)
O(11)–Cu(4)–O(10)
O(13)–Cu(4)–Cu(3)
O(11)–Cu(4)–Cu(3)
O(10)–Cu(4)–Cu(3)
O(9)–Cu(3)–N(4)
O(9)–Cu(3)–O(15)
N(4)–Cu(3)–O(15)
O(11)–Cu(3)–O(12)
O(15)–Cu(3)–O(12)
O(11)–Cu(3)–Cu(4)
O(15)–Cu(3)–Cu(4)
O(1)–Cu(2)–O(3)
O(3)–Cu(2)–N(2)
O(3)–Cu(2)–O(8)
O(1)–Cu(2)–O(4)
N(2)–Cu(2)–O(4)
O(3)–Cu(1)–O(1)
O(1)–Cu(1)–O(5)
O(1)–Cu(1)–N(1)
O(3)–Cu(1)–O(2)
O(5)–Cu(1)–O(2)
C(11)–O(3)–Cu(1)
Cu(1)–O(3)–Cu(2)
Cu(2)–O(1)–Cu(1)
C(25)–O(9)–Cu(4)
C(35)–O(11)–Cu(4)
Cu(4)–O(11)–Cu(3)
162.7(2)
76.87(18)
93.1(2)
O(13)–Cu(4)–O(11)
O(13)–Cu(4)–N(3)
O(11)–Cu(4)–N(3)
O(9)–Cu(4)–O(10)
N(3)–Cu(4)–O(10)
O(9)–Cu(4)–Cu(3)
N(3)–Cu(4)–Cu(3)
O(9)–Cu(3)–O(11)
O(11)–Cu(3)–N(4)
O(11)–Cu(3)–O(15)
O(9)–Cu(3)–O(12)
N(4)–Cu(3)–O(12)
O(9)–Cu(3)–Cu(4)
N(4)–Cu(3)–Cu(4)
O(12)–Cu(3)–Cu(4)
O(1)–Cu(2)–N(2)
O(1)–Cu(2)–O(8)
N(2)–Cu(2)–O(8)
O(3)–Cu(2)–O(4)
O(8)–Cu(2)–O(4)
O(3)–Cu(1)–O(5)
O(3)–Cu(1)–N(1)
O(5)–Cu(1)–N(1)
O(1)–Cu(1)–O(2)
N(1)–Cu(1)–O(2)
C(11)–O(3)–Cu(2)
C(1)–O(1)–Cu(1)
C(25)–O(9)–Cu(3)
Cu(3)–O(9)–Cu(4)
C(35)–O(11)–Cu(3)
98.0(2)
90.3(2)
169.0(2)
108.36(19)
80.3(2)
88.9(2)
106.84(17)
132.59(17)
39.62(13)
118.29(12)
169.5(2)
37.92(13)
129.74(16)
77.21(19) observed but to a lesser extent. Complexes 1a and 1b undergo
92.4(2)
159.64(19)
103.18(18)
81.2(2)
blue shi of 5 and 3 nm in p–p*, with a concomitant increase of
molar absorptivity, suggesting strong structural and confor-
mational changes involving coordination sphere binding with
DNA, while complexes 2a and 2b (ESI, Fig. S5†) display a slight
96.91(19)
92.7(2)
111.36(17)
88.91(18)
39.29(13)
129.55(13)
77.62(17)
93.66(19)
151.59(19)
101.79(18)
81.43(19)
76.85(16)
154.49(18)
91.4(2)
105.60(17)
93.17(18)
131.4(4)
102.16(19)
102.51(18)
126.9(4)
38.60(13)
130.98(17) increase in molar absorptivity with no appreciable shi.
117.79(12)
171.24(19)
97.24(18)
90.81(19)
117.4(2)
91.0(2)
95.43(18)
168.2(2)
95.0(2)
Nevertheless, in both complexes an aromatic moiety is present
so the binding of these complexes could also involve partial
intercalation of an aromatic ring between the base pairs of DNA.
To further illustrate the enantioselective approach of the
complexes, the quantitative comparison of the DNA binding
affinities of 1–2 (a and b) with CT DNA, the intrinsic binding
constant Kb values of the complexes were determined by
monitoring the change in the absorbance of the p–p* bands
with increasing concentration of CT DNA. Furthermore, the Kb
values (Table 5) clearly indicate the enantioselective approach
of the complexes emphasizing the stronger binding affinity of
the (R)-enantiomer for DNA as compared to the (S)-enantiomer.
Interaction with nucleotides. To obtain concrete informa-
tion and to determine the coordination of the metal ion to the
specic site at the molecular target, interactions with low
molecular building blocks of large DNA molecules viz., guano-
sine-50-monophosphate, adenosine-50-monophosphate, cyti-
dine-50-monophosphate and thymine-50-monophosphate were
carried out with the (R)-enantiomer of complexes 1 and 2. On
interaction of 1b with 50-GMP, there was a remarkable large red
shi (13 nm) in the ligand eld band accompanied by a change
in the intensity of the bands clearly indicating coordinate
covalent binding mode while with other nucleotides viz.,
50-AMP, 50-CMP and 50-TMP relatively less blue shi of 6, 6
and 8 nm, respectively, was observed. Interestingly, with CT
112.30(18)
79.2(2)
122.7(4)
126.9(4)
129.0(4)
103.5(2)
127.7(4)
131.1(4)
101.08(19)
Table 4 EPR parameters of complexes 1b and 2b
Complex
g
gt
giso
G
k
1b (solid)
1b (liquid)
2b (solid)
2b (liquid)
2.0950
2.0700
2.0780
2.0700
2.0379
2.0579
2.0470
2.0480
2.0569
2.0610
2.0570
2.0550
2.5066
1.2089
1.6595
1.4583
lattice water molecules. A hexacopper net was generated around DNA a red shi of 5 nm was observed (Fig. 4). This reveals that
three (O1w, O2w and O3w) H-bonded guest water molecules complex 1b shows preferential interaction toward the guano-
(ESI, Fig. S3†).
sine residue by the coordination to the N7 atom of the guanine
Two [Cu2] complexes trap four water molecules within, base of DNA double helix. Our results are in agreement with
which show an intricate H-bonding network with CuII bound other interaction studies of Cu(II) complexes with DNA nucleo-
acetate oxygen atoms [O1W/O5 ¼ 2.828 A; O1W/O7 ¼ tides using absorption and EPR measurements.23
˚
˚
˚
˚
2.778 A, O1W/O3W ¼ 2.828 A; O2W/O3W ¼ 2.778 A, O3W/
Nonetheless, in the case of complex 2b, there was relatively
˚
˚
˚
O8 ¼ 2.828 A; O12/O7 ¼ 2.778 A, O2W/O2 ¼ 2.828 A; O2W/ small shi on interaction with the nucleotides as well as CT
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Fig. 3 Absorption spectral traces of 1a and 1b (a and b) in Tris–HCl buffer upon addition of CT DNA at 25 ꢁC. Inset: plots of [DNA]/3b vs. [DNA] for the titration of CT
DNA with complexes -, experimental data points; full lines, linear fitting of the data. [Complex] 6.67 ꢂ 10ꢀ6 M, [DNA] 0–33.3 ꢂ 10ꢀ6 M.
accordance with observations of UV-vis absorption titrations of
the complex with CT DNA.
Table 5 The binding constant (Kb) values of complexes with the DNA (mean
standard deviation)
Emission spectral studies. Enantiomericcomplexes1aand1b,
2a and 2b emit luminescence in Tris–HCl buffer at room temper-
ature, when excited with light of 246 nm. On addition of increasing
concentration of CT DNA to the xed amount of complexes, there
was enhancement of the emission intensity indicative of strong
interaction of the complexes with CT DNA as depicted in Fig. 5 (1a
and 1b) and ESI, Fig. S6† (2a and 2b). The enhancement of the
emission intensity is largely due to the change in the environment
of the metal complex and is related to the extent to which the
complex gets into a hydrophobic environment inside the DNA.24
Complex
Kb (Mꢀ1
)
% Hyperchromism
Blue Shi
1a
1b
2a
2b
1.11 ꢂ 104(ꢃ0.03)
5.56 ꢂ 104(ꢃ0.04)
0.32 ꢂ 104(ꢃ0.04)
1.30 ꢂ 104(ꢃ0.03)
28
39
26
38
3
5
0
1
DNA (50-GMP, 50-CMP, 50-TMP display a red shi of 1–2 nm,
while AMP and CT DNA display a blue shi of 2–3 nm) (Fig. 4).
All these parameters are consistent with groove binding in
Fig. 4 Absorption spectra of 1b and 2b (1 ꢂ 10ꢀ3 M) (a) in Tris–HCl buffer, on interacting with guanosine-50-monophosphate (b), adenosine-50-monophosphate (c),
cytidine-50-monophosphate (d), thymine-50-monophosphate (e) and CT DNA (f) at 10ꢀ4 M at 25 ꢁC.
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Fig. 5 Emission spectra of 1a and 1b (a and b) in Tris–HCl buffer DNA in the presence of DNA at 25 ꢁC. [DNA] 0–33.3 ꢂ 10ꢀ6 M. Arrows show the intensity changes
upon increasing concentration of the complexes.
This effect arises because, in the presence of DNA, the metal
complex is bound in a relatively non-polar environment
compared to water. The binding constant K as quantied by the
Scatchard equation25 for complexes 1a, 1b, 2a and 2b is found
Circular dichroism. Circular dichroism (CD) is a powerful
and sophisticated tool for identifying conformational changes
of DNA. The secondary structure of DNA is greatly inuenced by
interaction of (R)- and (S)-enantiomers and changes in confor-
mation of DNA from B / Z or B / A. CT DNA in the B
conformation shows two conservative CD bands, a positive
band at 275 nm due to base stacking and a negative band at
245 nm due to right handed helicity. These bands are sensitive
towards binding of any small molecule or drug and hence can
be exploited to investigate the binding of small molecules to
DNA.26 Simple groove binding and electrostatic interaction of
the complexes with DNA show less or no perturbations on the
base stacking and helicity bands while the intercalator
enhances the intensities of both the bands. On incubation of
present complexes 1a and 1b, 2a and 2b with CT DNA, the CD
spectrum of DNA undergoes changes in both positive and
negative bands. Upon the addition of complex 1a, the CD
spectrum of CT DNA displayed an increase in positive and
negative ellipticity bands with a small red shi (ꢄ2 nm), while
to be 1.14 ꢂ 104, 5.50 ꢂ 104, 0.31 ꢂ 104 and 1.34 ꢂ 104 Mꢀ1
,
respectively.
EB competition assay. EB emits intense uorescence in the
presence of DNA due to its strong intercalation between the
adjacent DNA base pairs. However, the enhanced uorescence
can be quenched evidently when there is a second molecule that
can replace the bound EB or break the secondary structure of
DNA. The addition of the complexes 1a and 1b to DNA pre-
treated with EB causes an appreciable reduction in the uo-
rescence intensity (Fig. 6).
This suggests that complexes 1a and 1b compete with EB in
binding to DNA.23 In contrast to complex 1a and 1b, emission
intensity of complexes 2a and 2b (ESI, Fig. S7†) decreases to a
smaller extent. But in both complexes (R)-enantiomers 1b and
2b exhibited a greater decrease as compared to (S)-enantiomers.
Fig. 6 Fluorescence emission spectra of the EB–CT DNA system in the absence and presence of complexes 1a and 1b (a and b) at 25 ꢁC. [Complex], [EB], [DNA] ¼ 10ꢀ5 M.
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in the case of 1b both positive base stacking and negative hel- complex is capable of performing direct double-strand scission,
icity bands showed a sharp decrease in comparison to CT DNA while many other copper complexes are only able to cleave
alone clearly underlining the enantioselective approach (Fig. 7). single strands successively.28 The results demonstrate that both
Similarly, upon the addition of complex 2a, the CD spectrum of complexes 1b and 2b are good cleaving agents, however,
CT DNA revealed an increase in positive and negative bands complex 1b exhibited higher activity than complex 2b.
without shi in the band position, while in the case of 2b a Enhancement in hydrophobicity conferred by salicylaldehyde
reverse effect was observed (as in the case of 1b) (ESI, Fig. S8†). would increase the mobility of the reactive Cu(II) species on
Therefore, striking differences were observed in CD spectra of DNA, which would also contribute to the higher cleavage
complexes on incubating with CT DNA attenuating the effect of activity.29 The behavior observed for the electrophoretic
enantiomers on biological activity.
mobility of these complexes indicates that some conforma-
tional changes have occurred. This means that the degree of
superhelicity of the DNA molecules has been altered.
DNA cleavage properties
In order to identify recognition elements (groove binding)
and ROS that are responsible for the DNA cleavage reaction,
experiments in the presence of methyl green, DAPI and different
common scavengers such as tert-butyl alcohol and DMSO
(hydroxyl radical scavengers), NaN3 (singlet oxygen scavenger),
D2O (which is known to increase the lifetime of the O2) and SOD
(superoxide anion radical, O2_ꢀ) were carried out, respectively
with the R-enantiomer. As shown in Fig. 8(a) and (b), complex
1b inhibits the methyl green (lane 2) as well as DAPI (lane 3) of
the DNA digestion, whereas 2b shows inhibition on methyl
green and did not show any effect on DAPI (Fig. 8b). Under these
conditions, complex 1b may bind to both the major as well as
the minor groove, whereas complex 2b binds only to the major
groove.
The cleavage activity of 1b is reduced dramatically by the
presence of hydroxyl radical scavengers tert-butyl alcohol and
DMSO (lanes a4 and a5), indicating that the freely diffusible
hydroxyl radical is one of the intermediates involved in the DNA
scission process, while complex 2b has a very slight effect on
hydroxyl radical scavengers (lanes b4 and b5).
The interactions of pBR322 DNA with (R)-enantiomers of
complexes 1b and 2b were studied in order to determine the
DNA cleavage efficiency. When a circular plasmid DNA is sub-
jected to agarose gel electrophoresis, the fastest migration will
be observed for the supercoiled form (form I). If one strand is
cleaved, the supercoils will relax to produce a slower moving
open circular form (form II). If both strands are cleaved, a linear
form (form III) will be generated that migrates in between form I
and form II .The complexes 1b and 2b convert form I into form
II, while form III appeared at 0.10 mM for 1b (lanes 3); however,
at the same concentration, other complexes cleave form I DNA
into form II but linearized DNA was not observed suggesting
that the cleavage may occur randomly over DNA since a
signicant portion of the plasmid already appears to be
transformed into form II without concurrent formation of
form III (Fig. S9†).27
The most impressive cleavage feature observed for 1b is that
form III DNA appears before the disappearance of form I DNA
(lanes 3 and 6) (Fig. S9a†). This phenomenon indicates that this
Singlet oxygen scavenger NaN3 signicantly diminishes the
cleavage activity of both the complexes (lanes a6 and b6), sug-
gesting that 1O2 also takes part in the cleavage mechanism. The
involvement of 1O2 is also demonstrated by the remarkable
enhancement of the cleavage activity in D2O (lanes a7 and b7),
where the lifetime of 1O2 is signicantly longer than that in
water.28 Addition of SOD to the reaction mixture shows signi-
cant quenching of the cleavage revealing that the superoxide
Fig. 8 Agarose gel electrophoresis pattern for the cleavage of pBR322 plasmid
DNA (300 ng) by complex 1b (a) and 2b (b) (0.20 mmol) in the presence of DNA
minor groove binding agent DAPI, major groove binding agent methyl green and
different ROS at 25 ꢁC after incubation for 30 minutes. Lane 1, DNA control; lane
2, DNA + metal complex + methyl green (2.5 mL of a 0.01 mg mLꢀ1 solution); lane
3, DNA + metal complex + DAPI (8 mM); lane 4, DNA + metal complex + tert-butyl
alcohol (0.4 mM); lane 5, DNA + metal complex + DMSO (0.4 mM); lane 6, DNA +
metal complex + NaN3 (0.4 mM); lane 7, DNA + metal complex + D2O (70%); lane
8, DNA + metal complex + superoxide dismutase (15 units), respectively.
Fig. 7 CD spectra of CT DNA alone and in the presence of 1a and 1b in Tris–HCl
buffer at 25 ꢁC. [Complex] ¼ 10ꢀ7 M, [DNA] ¼ 10ꢀ7 M. The sky blue line represents
DNA alone, the dark blue line represents (S)-enantiomer interacting with DNA
and the red line represents (R)-enantiomer interacting with DNA.
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anion is also the active species (lanes a8 and b8). In summary,
these complexes seem to follow some similar pathways in the
cleavage process, in which hydroxyl radicals, singlet oxygen and
the superoxide anion are crucial ROS responsible for the
cleavage reactions.
Biological studies
In vivo genotoxic studies. We have analyzed the genotoxicity
of the complex 1b in vivo rat system by micronucleus (MN)
testing on bone marrow cells and comet assay in peripheral
blood lymphocytes and compared the results with CDDP. The
use of these two tests in conjugation provides additional data
indicating whether the DNA damage detected in comet assay
resulted in changes that can be detected in the MN test. The
data presented here are the rst documentation of genotoxic
comparison of a new chemotherapeutic metal-based drug
candidate with CDDP, furthermore the protective effect of extra
virgin olive oil (EVOO) was also studied. CDDP is widely used as
a positive control in in vivo experiments due to its genotoxic and
mutagenic properties.30–32
Micronucleus test (MNT). In bone marrow cells, the
frequencies of micronucleated polychromatic erythrocytes
(MNPCEs) (Fig. 9) signicantly increased in CDDP and complex
1b groups as compared to the control group (p < 0.05) but the
MNPCEs frequency increased by 1.67 fold in the CDDP group as
compared to the 1b group demonstrating signicantly reduced
toxicity of complex 1b (Fig. 10).
Evangelista et al. observed that a single dose of EVOO in vivo
has an anticlastogenic effect against CDDP-induced chromo-
somal damage in bone marrow cells.33 The EVOO is remarkably
rich in effective phenolic antioxidants that could provide
protection by inhibiting oxidative damage.34,35 Therefore, the
effect of EVOO in the presence of CDDP and complex 1b was
also evaluated. EVOO oral post-treatment for 7 consecutive days
induced signicant decrease of micronucleus frequencies in
CDDP as well as 1b groups as compared to CDDP and 1b group
alone, respectively (p < 0.05). This is the rst report to the best of
our knowledge on the protective effect of EVOO against damage
induced by CDDP and complex 1b. We have also seen the
protective effects of EVOO on the formation of MN induced by
CDDP and complex 1b by observing percentage reductions in
DNA damage obtained in the micronucleus assay. The results
were calculated and are presented in Fig. 11. The reduction in
Fig. 10 Frequency of PCEs with micronuclei (fMNPCEs) in a total of 2000
analysed cells per rat in each treatment group to evaluate the mutagenicity of
CDDP and 1b complex and the anti-mutagenicity of the extra virgin olive oil
(EVOO). Values are expressed as mean ꢃ SD (n ¼ 5). Values that do not share a
common superscript letter differ significantly at p < 0.05 (one-way ANOVA fol-
lowed by Tukey's test).
micronucleated cells was more expressive (83.79%) in the group
treated with the complex 1b along with the EVOO and less
expressive (33.30%) for the CDDP group orally post treated with
EVOO, which validates the effectiveness of antioxidants in
reducing the MNPCEs frequency in complex 1b as compared to
CDDP.
Comet assay. The comet assay is a highly sensitive technique
for analysis of DNA damage induced by a drug. CDDP reacts
with the nuclear DNA to yield a variety of adducts that include
intra- and interstrand DNA–DNA cross-links, as well as DNA–
protein crosslinks.36–39 Under these experimental conditions,
analyses of the mean comet tail length (TL) and olive tail
moment (OTM) of lymphocytes of CDDP and complex 1b
treated groups were studied as depicted in Fig. S10.† When
isolated rat lymphocytes were treated with CDDP and complex
1b, a greater extent of cellular DNA damage with clear tail
lengths was observed in the lymphocytes of CDDP and 1b
treated rats as compared to the control group but the DNA
breakage with clear tail length and OTM was signicantly
higher in lymphocytes of CDDP treated rats as compared to that
of the complex 1b treated group (p < 0.05).
Fig. 11 Percentage of DNA damage reduction promoted by the extra virgin
olive oil (EVOO) in the presence of CDDP and 1b complex, calculated according to
the formula of Manoharan and Banerjee and Waters et al. The parameters shown
are the frequency of micronucleated polychromatic erythrocytes (MNPCEs) and
olive tail moment (OTM).
Fig. 9 The micronucleus in polychromatic erythrocytes of bone marrow.
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Fig. 12 Representative photographs of comets of peripheral blood lymphocytes treated with CDDP, complex 1b, and their combination with EVOO.
We also evaluated the antioxidant potential of EVOO in acid (MPA) (Sigma-Aldrich) and supercoiled plasmid DNA
providing protection to lymphocytes against CDDP and complex pBR322 (Genei) were utilized as received.
1b induced oxidative injury. The increase in DNA damage was
Carbon, hydrogen and nitrogen contents were determined
recovered when the rats were orally post treated with EVOO using Carlo Erba Analyzer Model 1108. Molar conductance was
(5 mL per kg body weight) for 7 consecutive days aer the CDDP measured at room temperature on a Digisun Electronic
and complex 1b treatment. Combined treatment of EVOO with Conductivity Bridge. Fourier-transform infrared (FTIR) spectra
CDDP caused signicant inhibition of DNA breakage as evi- were recorded on an Interspec 2020 FTIR spectrometer in Nujol
denced by decreased tail length and OTM by 1.69 and 2.03 fold, mull. Electronic spectra were recorded on a UV-1700 Phar-
respectively as compared to the CDDP group alone while the maSpec UV-vis spectrophotometer (Shimadzu). Data were
oral administration of EVOO in complex 1b treated group, the reported in lmax (nm). The EPR spectra of the Cu(II) complexes
decrease in DNA breakage in terms of tail length and OTM were were acquired on a Varian E 112 spectrometer using X-band
1.08 and 1.94 fold, respectively as compared to the 1b group frequency (9.1 GHz) at liquid nitrogen temperature in the solid
1
alone (Fig. 12). The observations of genotoxicity clearly support state. The H and 13C NMR spectra were obtained on a Bruker
the contention that copper-based drug candidate complex 1b DRX-400 spectrometer. Optical rotations of chiral complexes
ꢁ
exhibits less cellular DNA damage as compared to the cisplatin were determined on a Polarimeter Rudolf Autopol III at 25 C
drug and these results are well corroborated with the observa- using the sodium D line in DMSO. ESI-MS spectra were recor-
tions of MNT.
ded on a Micromass Quattro II triple quadrupole mass
spectrometer.
Experimental section
Synthesis
Materials and measurements
Synthesis of (S)- and (R)-2-((1-hydoxypropan)-2-ylamino)
All reagents were of the best commercial grade and were used methyl) phenol (L1). 2-Amino-1-propanol (0.75 g, 10 mmol) [(S)-
without further purication. (S)-/(R)-2-Phenylglycinol, (S)-/(R)-2- 2-amino-1-propanol a; (R)-2-amino-1-propanol b] and salicy-
amino-1-propanol (Aldrich), salicylaldehyde (Alfa Aesar), NaBH4, laldehyde (1.22 g, 10 mmol) were stirred together in methanol
Cu(CH3COO)2$H2O, Tris-(hydroxymethyl)aminomethane–HCl (15 mL) for 1 h at room temperature. The resulting yellow
(Tris–HCl) (E. Merck) were used as received. Disodium salt of calf colored solution was monitored by TLC for the completion of
thymus DNA (CT DNA) was purchased from Sigma Chemical Co the reaction and formation of Schiff base. To this solution,
and was stored at 4 ꢁC. Adenosine-50-monophosphate disodium NaBH4 (0.46 g, 12 mmol) was added in portions and was
salt (50-AMP), cytidine-50-monophosphate disodium salt hydrate reuxed for 2 h, the solution changed color slowly from yellow
(50-CMP), guanosine-50-monophosphate disodium salt (50-GMP) to colorless. The solvent was removed under vacuum and the
and thymine-50-monophosphate (50-TMP) were purchased from residue was dissolved in water (30 mL) and was extracted with
Fluka and were stored at ꢀ20 ꢁC. 6X loading dye (Fermental Life CHCl3 (3 ꢂ 30 mL). The CHCl3 layers were combined and dried
Science), agarose, ascorbic acid, sodium azide (NaN3), super- under vacuum. Colorless liquid was formed upon introduction
oxide dismutase (SOD), methyl green, DAPI, mercaptopropionic of dry HCl.
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(S-enantiomer) a: [a]2D5 ¼ +72 (c 0.05 in MeOH).
(R-enantiomer) b: [a]2D5 ¼ ꢀ71 (c 0.05 in MeOH).
Both enantiomers exhibited identical IR, Mass, UV-vis and
NMR spectra.
n(Cu–O), 426 n(Cu–N); UV-vis [CH3OH; lmax nmꢀ1] 246 (26140),
389 (23890), 661 (90).
Synthesis of (R)- and (S)-[Cu2II(m–OAc)2 (L2$H2O)2] (2). To a
methanolic solution (10 mL) of L2 (a and b) (0.330 g, 2 mmol)
Selected IR data on KBr pellet (n/cmꢀ1): 3217 n(N–H), 1565 was added a solution of Cu(CH3COO)2$H2O (0.398 g, 2 mmol) in
d(N–H), 2842 n(CH2), 1475 n(C–N), 1390 n(C–C), 1067 n(C–Oalcohol), methanol (10 mL). The solution was stirred for 4 hours with
ꢁ
1250 n(C–Ophenole), 751 n(Ar); ESI-MS (m/z) 181 (51%) heating at 80 C. On completion of the reaction, the reaction
[C10H15NO2]+; UV-vis [CH3OH; lmax nmꢀ1] 308.
mixture was kept at room temperature which yielded a sky blue
dH (400 MHz; DMSO-d6): 1.08 (d, 3H, –CH3), 2.29 (s, –OH, colored product, washed with hexane and driedꢁin vacuo.
1H), 2.85 (m, chiral –CH, 1H), 3.29 (d, –CH2, 2H), 3.59 (d, –CH2,
(S-enantiomer) 2a: yield 45%; m.p. 224–226 C; Anal. Calc.
2H), 4.64 (s, –NH), 7.22–7.33 (multiplet, 5H, Ar–H), 7.57 (s, (%) for C24H40N2O8Cu2: C, 47.28; H, 6.28; N 4.59. Found: C,
phenol –OH); dC (100 MHz; DMSO-d6): 157.26 (Phenol–OH), 47.19; H, 6.25; N, 4.61; [a]2D5 ¼ +68 (c 0.05 in DMSO); ESI-MS
128.53, 128.17, 127.13, 126.99, 126.17 (Ar C), 65.52 (–CH2OH), (m/z): 609 [C24H38N2O8Cu2]+.
ꢁ
53.78 (chiral C), 51.12 (–CH2NH).
(R-enantiomer) 2b: yield 44%; m.p. 222–224 C; Anal. Calc.
Synthesis of (S)- and (R)-2-(benzylamino)propan-1-ol (L2). (%) for C24H40N2O8Cu2: C, 47.28; H, 6.28; N 4.59. Found: C,
This was prepared according to a procedure similar to that of L1 47.27; H, 6.31; N, 4.62; [a]2D5 (DMSO) ꢀ67 (c 0.05 in DMSO); ESI-
except that 2-amino-1-propanol (0.75 g, 10 mmol) [(S)-2-amino- MS (m/z): 610 [C24H38N2O8Cu2 +H]+.
1-propanol a; (R)-2-amino-1-propanol b] was mixed with benz-
Both enantiomeric metal complexes exhibited identical IR,
aldehyde (1.09 g, 10 mmol) in methanol (15 mL), followed by UV-vis and EPR spectra.
in situ reduction by NaBH4 and extraction with CHCl3. A color-
less liquid was formed upon introduction of dry HCl.
(S-enantiomer) a: [a]2D5 ¼ +119 (c 0.05 in MeOH).
(R-enantiomer) b: [a]2D5 ¼ ꢀ120 (c 0.05 in MeOH).
Both enantiomers exhibited identical IR, Mass, UV-vis and
NMR spectra.
Selected IR data on KBr pellet (n/cmꢀ1): 3753 n(coordinated
water), 3262 n(–NH), 2864 n(–CH2), 1636 cmꢀ1 for nasym(OCO),
1479 cmꢀ1 for nsym(OCO), 1543 d(N–H), 1440 n(C–N), 1397
n(C–C), 1040 n(C–Oalkoxide), 762 n(Ar), 545 n(Cu–O), 432 n(Cu–N).
UV-vis [CH3OH; lmax nmꢀ1] 251 (25240), 676 (80).
Selected IR data on KBr pellet (n/cmꢀ1): 3219 n(N–H), 1557
d(N–H), 2853 n(CH2), 1478 n(C–N), 1401 n(C–C), 1043 n(C–Oalcohol),
755 n(Ar); ESI-MS(m/z) 165 [C10H14NO+2H]+; UV-vis [CH3OH;
lmax nmꢀ1] 313.
DNA binding experiments
DNA binding experiments which include absorption spectral
titrations, luminescence, circular dichoric experiments and
DNA cleavage conformed to the standard methods40–42 and
practices previously adopted by our laboratory.43 While
measuring the absorption spectra an equal amount of DNA was
added to both the compound solution and the reference solu-
tion to eliminate the absorbance of the DNA itself.
dH (400 MHz; DMSO-d6): 1.10 (d, 3H, –CH3), 2.26 (s, –OH,
1H), 2.69 (m, chiral –CH, 1H), 3.27 (d, –CH2, 2H), 3.59 (d, –CH2,
2H), 4.81 (s, –NH), 7.23–7.34 (multiplet, 5H, Ar H); dC (100 MHz;
DMSO-d6): 126.95, 126.77, 126.09, 125.83, 125.44 (Ar C), 67.92
(–CH2OH), 54.98 (chiral C), 54.87 (–CH2NH).
Synthesis of (R)- and (S)-[Cu2II(m–L1)2(OAc)2]$2H2O (1). To a
methanolic solution (10 mL) of L1 (a and b) (0.362 g, 2 mmol)
Animals and treatment
was added a solution of Cu(CH3COO)2$H2O (0.398 g, 2 mmol) in Adult male Wistar rats, obtained from the animal house of
methanol (10 mL) under reuxing condition for 3 hours. On JNMC, Aligarh Muslim University, Aligarh, were used for this
completion of the reaction, the reaction mixture was kept at study. Rats (150 ꢃ 50 g) were housed in polycarbonate cages
room temperature which yielded a blue colored product, with steel wire tops (ve animals per group) at standard room
ꢁ
washed with hexane and dried in vacuo. Aer ltration, crystals temperature (22 ꢃ 2 C) and humidity (55 ꢃ 10%) with a 12 h
suitable for X-ray diffraction were obtained by slow evaporation light–dark cycle, and received standard food and water ad libi-
of the ltrate aer several days.
tum. The day of administration is designated as day 0. The
ꢁ
(S-enantiomer) 1a: yield 49%; m.p. 218–221 C; Anal. Calc. animals were randomly separated and grouped according to age
(%) for C24H38N2O10Cu2: C, 44.92; H, 5.97; N 4.37. Found: C, and weight into the following four groups of ve rats each.
45.03; H, 5.95; N, 4.41; [a]2D5 ¼ +53 (c 0.05 in DMSO); ESI-MS Group I: which received a single dose of DMSO i.p. (intra peri-
(m/z): 641 [C24H38N2O10Cu2]+.
toneal) served as control. Group II: received a single dose of
ꢁ
(R-enantiomer) 1b: yield 46%; m.p. 217–219 C; Anal. Calc. CDDP (5 mg per kg body weight) i.p. Group III: received a single
(%) for C24H38N2O10Cu2: C, 44.92; H, 5.97; N 4.37. Found: C, dose of complex 1b (5 mg per kg body weight) i.p. Group IV:
44.86; H, 5.94; N, 4.40; [a]2D5 ¼ ꢀ53 (c 0.05 in DMSO); ESI-MS EVOO was administrated orally for 7 consecutive days aer a
(m/z): 641 [C24H38N2O10Cu2]+.
single dose of cisplatin. Group V: administration of EVOO orally
Both enantiomeric metal complexes exhibited identical IR, for 7 consecutive days aer a single dose of complex 1b. Group
UV-vis and EPR spectra.
VI: administration of EVOO orally for 7 consecutive days alone.
Selected IR data on KBr pellet (n/cmꢀ1): 3450 n(lattice water), At the end of experiments, the rats of each group were sacri-
3120 n(–NH), 2865 n(–CH2), 1632 cmꢀ1 for nasym(OCO), ced. By 7 days aer drugs and antioxidant administration,
1391 cmꢀ1 for nsym(OCO), 1546 d(N–H), 1438 n(C–N), 1354 blood samples from the rats were collected in tubes containing
n(C–C), 1038 n(C–Oalkoxide), 1252 n(C–Ophenolate), 752 n(Ar), 521 heparin for comet assay and bone marrow from femur bones
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was sampled for micronucleus assay. The experimental proto-
cols for this study were approved by the Local Ethics Committee
for Animal of JNMC, AMU, Aligarh.
Conclusion
Enantiomeric dinuclear copper-based potential chemotherapeutic
drug candidates 1 and 2 were designed and synthesized to
ascertain the structure–activity relationship of these complexes
towards DNA. Both enantiomers of 1 and 2 were thoroughly
characterized by microanalysis and spectroscopically while the
structure of the (R)-enantiomer of 1 was elucidated by single
crystal X-ray crystallography, which revealed dinuclear Cu(II)
complexes exhibiting an unusual acetate bridging structure and
diamond core topology. The complexes exhibited square pyra-
midal geometry around both the Cu(II) centers as ascertained by
UV-vis, EPR spectroscopic studies, and by X-ray data in the case
of (R)-enantiomer 1b.
Metal complex–DNA interactions were studied by UV-vis,
uorescence, and circular dichroism in both enantiomers of 1
and 2 to validate their potential to act as cancer chemothera-
peutic agents as DNA is the primary cellular target of most of the
anticancer drugs. Both the complexes bind to DNA by coordi-
nate covalent and groove binding, respectively. Nevertheless,
(R)-enantiomers bind more strongly in comparison to (S)-
enantiomers. Therefore, DNA cleavage activity of (R)-enantio-
mers of 1 and 2 was ascertained with pBR322 DNA by agarose
gel electrophoretic assay and they followed an oxidative mech-
anistic pathway in the cleavage process, involving hydroxyl
radicals, singlet oxygen and superoxide radicals. The genotox-
icity of (R)-enantiomer complex 1b was evaluated in vivo by
micronucleus (MN) testing on bone marrow cells and comet
assay in peripheral blood lymphocytes and the results were
compared with CDDP. The protective effect of extra virgin olive
oil (EVOO) was also studied. The observations of genotoxicity
clearly support the contention that copper-based drug candi-
date complex 1b exhibits less cellular DNA damage as compared
to the cisplatin drug. The present study demonstrate that
studying the enantiomeric forms of biologically active copper
complexes can provide a rationale for efficacious, less toxic and
target-specic drug design therapy.
Micronucleus test
Bone marrow smears and staining were done following the
method of Schmid.44 Briey, both femurs were removed, and
the bone marrow was ushed out into a centrifuge tube with
fetal calf serum (FCS). The bone marrow cells were collected by
centrifugation at 1000 rpm for 10 min, and the pellet was
resuspended in 1 mL FCS for preparation of the slides. A drop
of this suspension was smeared on a clean slide, air-dried,
xed in absolute methanol for 5 min and stained the following
day with May-Gruenwald and Geimsa stains. Two thousand
polychromatic erythrocytes (PCEs) were analyzed for each
animal and the number of micronucleated PCEs (MNPCEs)
was recorded.
Comet assay
Blood was obtained from different treatment groups via cardiac
puncture immediately before sacrice. Samples were heparin-
ized and lymphocytes were isolated from blood by density
gradient centrifugation using Histopaque 1077, and the cells
were nally suspended in RPMI 1640. The lymphocytes were
checked for their viability before the start and aer the end of
the reaction using the trypan blue exclusion test.45 The viability
of the cells was found to be greater than 93%.
The alkaline version of the comet assay was performed
according to the guidelines proposed by Singh, McCoy, Tice,
and Scheinder (1998),46 with a slight modication. Microscope
slides pre-coated with 1% normal-melting-temperature agarose
were used. Around 1 ꢂ 105 cells mixed with 75 mL of 1% LMPA
was then layered over the rst layer, and le to harden for 5
minutes at 4 ꢁC. Finally a third layer of 0.5% LMPA (75 mL) was
layered and le to solidify and covered immediately by a
coverslip. This was followed by immersion in ice-cold alkaline
lysing solution [2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1%
ꢁ
Triton X-100, nal pH 10.0] for at least 1 h at 4 C. The slides
Acknowledgements
were then incubated for 30 min in ice-cold electrophoresis
solution (0.3 M NaOH, 1 mM EDTA, pH > 13), followed by
electrophoresis at 0.7 V cmꢀ1: 300 mA current. Aer electro-
phoresis, the slides were then neutralized (Tris 0.4 M, pH 7.5)
and stained with ethidium bromide (75 microlitre). Fiy cells
per mouse (25 cells analysed in each slide) were scored at 100ꢂ
magnication using an image analysis system (Komet 5.5,
Kinetic Imaging, Liverpool, UK) attached to an Olympus
(CX 41) uorescent microscope and a COHU 4910 integrated
CC camera.
We express our gratitude to Department of Biotechnology, New
Delhi, for generous nancial support (scheme no. BT/PR9205/
Med/30/13/2007). Our thanks are also due to (RSIC, Panjab
University, Chandigarh) for NMR and ESIMS facility, RSIC,
CDRI, Lucknow for providing the elemental analysis and IIT
Mumbai for EPR measurements.
Notes and references
1 G. G. Hovhannisyan, T. S. Haroutunyan and
R. M. Arutyunyan, Exp. Oncol., 2004, 26, 240–242.
2 C. X. Zhang and S. J. Lippard, Curr. Opin. Chem. Biol., 2003, 7,
481–489.
3 E. A. P. Gonzalez and A. G. Fuchs, Dose-Response, 2009, 7,
234–246.
Statistical analysis
Results are presented as mean values with their standard
deviation for experiments. Comparison of mean values between
groups was assessed by one-way ANOVA followed by a post-hoc
Tukey's test. P values of less than 0.05 were considered signi-
cant. Data were analyzed using an SPSS statistical soware
package (SPSS for Windows, version 16).
4 V. Srb, E. Kubzova and K. Kubikova, Neoplasma, 1986, 33,
615–620.
This journal is ª The Royal Society of Chemistry 2012
Med. Chem. Commun.

View Article Online
MedChemComm
Concise Article
5 C. Sanchez-Cano and M. J. Hannon, Dalton Trans., 2009, 24 L.-F. Tan, H. Chao, K.-C. Zhen, J.-J. Fei, F. Wang, Y.-F. Zhou
10702–10711. and L.-N. Ji, Polyhedron, 2007, 26, 5458–5468.
6 C. G. Hartinger, S. Zorbas-Seifried, M. A. Jakupec, B. Kynast, 25 G.-D. Liu, J.-P. Liao, Y.-Z. Fang, S.-S. Huang, G.-L. Sheng and
H. Zorbas and B. K. Keppler, J. Inorg. Biochem., 2006, 100,
891–904.
7 E. Tomlinson, Adv. Drug Delivery Rev., 1987, 1, 87–198.
8 S. Thyagarajan, N. N. Murthy, A. A. N. Sarjeant, K. D. Karlin
and S. E. Rokita, J. Am. Chem. Soc., 2006, 128, 7003–7008.
9 P. U. Maheswari, K. Lappalainen, M. Sfregola, S. Barends,
R.-Q. Yu, Anal. Sci., 2002, 18, 391–395.
26 B. Norden and F. Tjerneld, Biopolymers, 1982, 21, 1713–1734.
27 K. J. Humphreys, A. E. Johnson, K. D. Karlin and S. E. Rokita,
JBIC, J. Biol. Inorg. Chem., 2002, 7, 835–842.
28 X. Dong, X. Wang, M. Lin, H. Sun, X. Yang and Z. Guo, Inorg.
Chem., 2010, 49, 2541–2549.
P. Gamez, U. Turpeinen, I. Mutikainen, G. P. van Wezel 29 T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii,
and J. Reedijk, Dalton Trans., 2007, 3676–3683; S. Dhar,
P. A. N. Reddy, M. Nethaji, S. Mahadevan, M. K. Saha and
A. R. Chakravarty, Inorg. Chem., 2002, 41, 3469–3476.
M. Chikira, P. T. Selvi and M. Palaniandavar, J. Inorg.
Biochem., 2005, 99, 1205–1219.
30 T. L. Overbeck, J. M. Knight and D. J. Beck, Mutat. Res., 1996,
362, 249–259.
10 K. Nakamoto, Infrared and Raman Spectra of Inorganic and
´
Coordination Compounds, Wiley-Interscience, New York, 31 L. M. Antunes, M. C. Araujo, J. D. Darin and M. L. Bianchi,
1986. Mutat. Res., 2000, 465, 131–137.
11 G. Du, A. Ellern and L. K. Woo, Inorg. Chem., 2003, 42, 873– 32 L. M. Antunes, L. M. Pascoal, M. De. L. Bianchi and
877. F. L. Dias, Mutat. Res., 2005, 585, 113–119.
12 U. Kumar, J. Thomas and N. Thirupathi, Inorg. Chem., 2010, 33 C. M. Evangelista, L. M. Antunes, H. D. Francescato and
49, 62–72. M. L. Bianchi, Food Chem. Toxicol., 2004, 42, 1291–1297.
13 U. Mukhopadhyay and D. Ray, Proc. – Indian Acad. Sci., Chem. 34 R. W. Owen, A. Giacosa, W. E. Hull, R. Haubner,
Sci., 1998, 110, 517–526.
B. Spiegelhalder and H. Bartsch, Eur. J. Cancer, 2000, 1235–
14 R. Singh, A. Banerjee, E. Colacio and K. K. Rajak, Inorg.
Chem., 2009, 48, 4753–4762.
1247.
35 F. Visioli and C. Galli, Crit. Rev. Food Sci. Nutr., 2002, 42, 209–
15 S. Banerjee, S. Mondal, S. Sen, S. Das, D. L. Hughes,
221.
C. Rizzoli, C. Desplanches, C. Mandal and S. Mitra, Dalton 36 K. Wozniak, A. Czechowska and J. Blasiak, Chem.-Biol.
Trans., 2009, 6849–6860. Interact., 2004, 147, 309–318.
16 A. Ozarowski, I. B. Szymanska, T. Muzio and J. Jezierska, 37 A. M. J. Fichtinger-Schepman, A. T. van Oosterom,
´
J. Am. Chem. Soc., 2009, 131, 10279–10292.
17 M. Trivedi, D. S. Pandey and N. P. Rath, Inorg. Chim. Acta,
2009, 362, 284–290.
P. H. M. Lohman and F. Berends, Cancer Res., 1987, 47,
3000–3004.
38 M. Yoshida, A. Khokhar, Y. Kido, F. Ali-Osman and Z. Siddik,
Biochem. Pharmacol., 1994, 17, 793–799.
18 E. V. R. Akimova, A. Y. Nazarenko, L. Chen, P. W. Krieger,
A. M. Herrera, V. V. Tarasov and P. D. Robinson, Inorg. 39 Y. Sedletska, M.-J. Giraud-Panis and J.-M. Malinge, Curr.
Chim. Acta, 2001, 324, 1–15. Med. Chem.: Anti-Cancer Agents, 2005, 5, 251–265.
19 A. W. Addison, T. N. Rao, J. Reedijk, J. van Rijn and 40 J. Marmur, J. Mol. Biol., 1961, 3, 208–214.
G. C. Verschoor, J. Chem. Soc., Dalton Trans., 1984, 1349– 41 A. Wolfe, G. H. Shimer and T. Meehan, Biochemistry, 1987,
1356.
20 R. D. Willett, G. Pon and C. Nagy, Inorg. Chem., 2001, 40, 42 J. R. Lakowiez and G. Webber, Biochemistry, 1973, 12, 4161–
4342–4352. 4170.
21 Y. Zubavichus, M. Zharnikov, Y.-J. Yang, O. Fuchs, 43 F. Arjmand and M. Aziz, Eur. J. Med. Chem., 2009, 44, 834–
26, 6392–6396.
E. Umbach, C. Heske and M. Grunze, Langmuir, 2006, 22,
7241–7247.
844.
44 W. Schmid, Mutat. Res., 1975, 31, 9–15.
22 N. Grover, T. W. Welch, T. A. Fairley, M. Cory and 45 B. L. Pool-Zobel, C. Guigas, R. Klein, C. H. Neudecker,
H. H. Thorp, Inorg.Chem., 1994, 33, 3544–3548.
23 A. Raja, V. Rajendiran, P. U. Maheswari, R. Balamurugan,
H. W. Renner and P. Schmezer, Food Chem.Toxicol., 1993,
31, 184–191.
C. A. Kilner, M. A. Halcrow and M. Palaniandavar, J. Inorg. 46 N. P. Singh, M. T. McCoy, R. R. Tice and E. L. Schnieder, Exp.
Biochem., 2005, 99, 1717–1732.
Cell Res., 1998, 175, 184–191.
Med. Chem. Commun.
This journal is ª The Royal Society of Chemistry 2012