Biological evaluation of dinuclear copper complex/dichloroacetic acid cocrystal against human breast cancer: Design, synthesis, characterization, DFT studies and cytotoxicity assays

Article abstract of DOI:10.1039/c7ra08262b

Binuclear copper(ii) cocrystal "[Cu₂(valdien)₂?2Cl₂CHCOOH]," 1 was synthesized from H₂valdien scaffold and anticancer drug pharmacophore "dichloroacetic acid" embedded with two Cu(ii) connected via a hydrogen bo

Full text of DOI:10.1039/c7ra08262b

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Biological evaluation of dinuclear copper complex/
dichloroacetic acid cocrystal against human breast
cancer: design, synthesis, characterization, DFT
studies and cytotoxicity assays†
Cite this: RSC Adv., 2017, 7, 47920
a
Mohammad Usman,a Farukh Arjmand,
Rais Ahmad Khan,b Ali Alsalme,b
a
Musheer Ahmadc and Sartaj Tabassum
*
Binuclear copper(II) cocrystal “[Cu2(valdien)2/2Cl2CHCOOH],” 1 was synthesized from H2valdien scaffold
and anticancer drug pharmacophore “dichloroacetic acid” embedded with two Cu(II) connected via
a hydrogen bonded network. [Cu2(valdien)2/2Cl2CHCOOH] 1 was thoroughly characterized by single-
crystal XRD and by other spectroscopic techniques. The non-covalent interaction (NCI) index and
Hirshfeld surface analysis were used to study the various kinds of interactions (O/H, N/H, H/Cl, Cu/
H, C/O, N/O, C/Cl, and O/Cl, etc.) responsible for the stabilization of crystal lattice. [Cu2(valdien)2/
2Cl2CHCOOH] 1 was validated as potential antitumor drug entity by studying its DNA binding profile,
cleavage mechanism with pBR322 by gel electrophoretic assay and in vitro cytotoxicity on MCF-7 cancer
cell lines. The mechanistic pathways were also deduced via dual staining AO/EB of cancer cells which
confirmed the potential of the cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 to act as effective anticancer
agent towards breast cancers.
Received 26th July 2017
Accepted 28th September 2017
DOI: 10.1039/c7ra08262b
rsc.li/rsc-advances
strategy, because monotherapy (i.e., targeting
a specic
1. Introduction
receptor) is not much efficacious in the treatment of chronic
diseases, such as HIV/AIDS, cancer, diabetes, and cardiovas-
cular disorders etc.17–24 In the present contribution, an example
of unique cocrystallized bis(o-valdien copper) complex with
dichloroacetic acid (DCA) is described. To the best of our
knowledge, there is no cocrystal of a metal complex having drug
moiety ‘dichloroacetic acid’ as one of the components.
Bio-inorganic and Pharmaceutical chemists are striving
constantly to improve the physical and pharmacokinetic
features of active pharmaceutical ingredients (APIs) viz., crys-
tallinity, solubility, hygroscopicity, stability, particle size, ow,
lterability, density etc.1–6 Current approaches involved in
changing properties of APIs include the exploitation of
hydrates, solvates, salts, and more recently, cocrystals. Cocrys-
tals are single phase crystalline materials comprising of two or
more different molecular and/or ionic compounds in a stoi-
chiometric ratio, which are generally neither solvates nor
simple salts.7 They are distinct from two conformers due to non-
covalent interactions in the new material.
Although the number of possible organic cocrystals is
virtually innite, the cocrystals of only metal complexes and
containing metal complex-drug are rare.8–16 The combinatorial
synthetic approach to obtain a single unit drug motif from
conformers has invoked much interest in drug development
Dichloroacetic acid (DCA) is directly involved in cell
apoptosis and works synergistically with additional cancer
therapies, viz., radio, gene, and viral therapy.25–28 Since it gets
easily absorbed by the body and can permeate through blood–
brain barrier; it poses risks of unexpected and severe neuro-
logical effects.29 Therefore, a cocrystals based prodrugs are
designed for sustained release of free haloacetates in the
circulatory system, which can be cleaved to release the hal-
oacetates in a selective manner inside the cancer cell. The pro-
drug molecule self-assembled in the presence of a dinuclear
copper complex to generate stabilized cocrystals. It is worth
mentioning here that many metalloenzymes and proteins
contain in their active sites two copper ions that operate coop-
eratively.30 Thus, a great deal of consideration has been given to
bimetallic copper compounds with two metal ions in close
proximity because they provide an opportunity to study intra-
molecular binding, bimetallic catalysts, magnetic exchange
interactions, multi-electron redox reactions and activity
mimicking the possible activation of small substrate molecules
aDepartment of Chemistry, Aligarh Muslim University, Aligarh-202002, India. E-mail:
tsartaj62@yahoo.com; Tel: +91 9358255791
bDepartment of Chemistry, College of Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Saudi Arabia
cDepartment of Applied Chemistry, Aligarh Muslim University, Aligarh-202002, India
† Electronic supplementary information (ESI) available. CCDC 1402001. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c7ra08262b
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by enzymes.31 In addition, the current studies showed that the consistent with the distorted square planar environment
binuclear Cu(II) compounds possess high anticancer activities.32 around Cu(II).39
Therefore, we attempt to synthesized binuclear copper(II)
complex as an anticancer chemotherapeutic.
Emission spectra of cocrystal [Cu2(valdien)2/2Cl2CHCOOH]
1 (Fig. S2†) recorded at room temperature in MeOH, exhibited
The designing of the ligand motifs has an important role in uorescence at 458 nm when excited at 371 nm, while a weak
tuning and regulating the drug candidate specicity towards band appears at 332 nm along with 458 nm upon excitation at
the biological target molecules. Among the several organic 271 nm. The emission corresponds to intra-ligand charge
ligands, our interest stems in Schiff bases derived from o- transfer (ILCT), a ligand to ligand charge transfer (LLCT) or
vanillin and their metal complexes are known to exhibit diverse a combination of both.
biological applications viz., antitumor, anti-inammatory,
To obtain the evidence for the stability of the dinuclear
antiviral, antibacterial and cell imaging agents.33 The cocrystal [Cu2(valdien)2/2Cl2CHCOOH], 1 in solution, ESI-MS,
synthetic strategy to obtain bioactive cocrystal fundamentally and UV-vis spectra were measured. The ESI-MS shows an m/z of
depends on the use of polydentate bridging ligand as in the case 1122.93 [M + H+] (Fig. S3†), corresponding to the stability of
of transition-metal therapeutics34,35 which can mediate syner- cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 in solution phase.40
gistic interactions between the drug conformer dichloroacetic Also, the UV-vis spectra (Fig. S4†) of 1 in Tris–HCl buffer under
acid (DCA) via hydrogen bonding and its dinuclear copper physiological conditions (pH 7.3 and T 296 K) was measured
complex. Copper demonstrates high affinity for nucleobases over different time intervals (0, 1, 3, 6, 12, 24, 48 and 72 h) using
and has exhibited broad anticancer activity due to the selective UV-vis spectrophotometer. The small variation in measured
permeability of cancer cell membranes to copper complexes.36,37 spectra (ca. 10 nm blue shi with 0.2 hyperchromic shi), was
Besides this, copper is a physiologically important endogenous observed which may be attributed to the solvation effect of
metal ion that plays a signicant role in redox reactions and [Cu2(valdien)2/2Cl2CHCOOH] under physiological conditions
thus triggers the production of reactive oxygen species (ROS) (Tris–HCl buffer, pH 7.3 & T 310 K). This spectral variation
which induce apoptosis in cancer cells.38
without appearing any new absorption band indicated the
A novel dinuclear Cu(II) cocrystal “[Cu2(valdien)2/2Cl2- stability of the cocrystal in Tris–HCl buffer medium.
CHCOOH]” as a potent antitumor drug motif has been achieved
and thoroughly characterized via single crystal XRD and other
spectroscopic methods. The chemotherapeutic potential was
ascertained by binding with CT-DNA employing UV-visible,
2.2. Crystal structure description of [Cu2(valdien)2/
2Cl2CHCOOH] cocrystal (1)
uorescence, and circular dichroism techniques. DNA The dinuclear copper(II) cocrystal “[Cu2(valdien)2/2Cl2-
cleavage experiments were carried out to study the mechanistic CHCOOH] 1 was structurally characterized by single-crystal
insight into the binding phenomena at the target site. 1 was X-ray crystallography (Fig. 1). Details of the crystallography
tested against MCF-7 breast cancer cells line to validate its data and renement parameters are summarized in Table 1.
cytotoxicity.
Selected bond angles and distances are listed in Table S1 and
S2.† Particulars of the hydrogen bonding parameters are shown
in Table S3.† Single-crystal X-ray structural study revealed that 1
2. Results and discussion
2.1. Synthesis and characterization
ꢀ
crystallized in the triclinic P1 space group possessing the lattice
ꢁ
ꢁ
ꢁ
parameters, a ¼ 12.790(5) A, b ¼ 14.351(5) A, c ¼ 15.034(5) A,
The compartmental acyclic Schiff base H2valdien ligand was ob- a ¼ 109.079(5), b ¼ 91.480(5), g ¼ 114.551(5), per unit cell. The
tained from the condensation reaction of o-vanillin and dieth- asymmetric unit of 1 contains one [Cu2(valdien)2] and two DCA
ylenetriamine. To obtain [Cu2(valdien)2], Cu(CH3COO)2$H2O was molecules, in which [Cu2(valdien)2] moiety link each other
reacted with H2valdien in 1 : 1 ration in the presence of triethyl- through the C33–H33/O12, C11–H11B/O9, C12–H12A/C13/
amine in MeOH. The cocrystallization process involved Cl31 hydrogen bonds to form
a one-dimensional chain
combining [Cu2(valdien)2] with dichloroacetic acid (DCA) (1 : 2) (Fig. S5†), two adjacent chains are connected by dichloroacetic
in methanol/acetonitrile (4 : 1) solution. The resulting solution acid (DCA) molecules through C7–H7B/O10 hydrogen bonds
was subjected to controlled evaporation to help crystallization. to generate a 2D sheet (Fig. S5†). The 2D sheets are further held
Aer 2–3 months, dark brown colored rectangular-shaped crys- together by a network of C36–H36/Cl3/Cl31 hydrogen bonds to
tals were obtained (Scheme 1).
form the 3D framework of 1 (Fig. S5†).
The IR spectrum of cocrystal [Cu2(valdien)2/2Cl2CHCOOH]
Furthermore, [Cu2(valdien)2] unit consists of two indepen-
1 exhibited strong absorption bands between 1655 cmꢀ1 and dent valdien2ꢀ ligands, adopting h1:h1:h1:h1:m2 coordination
1414 cmꢀ1 attributed to carboxylic acid groups. Broad bands in mode (Fig. S6†) in bridging fashion to synchronize both the
the region 3510–3100 cmꢀ1 indicate the presence of N–H group. metal centers Cu1 and Cu2 through their phenoxide atoms O1,
The sharp peak at 1617 cmꢀ1 was attributed to the imine bond. O3, O5, O6 and their iminic nitrogen atoms N1, N2, N5, N6.
The electronic spectrum (Fig. S1†) of cocrystal Each copper ion is, therefore, tetra-coordinated in a distorted
[Cu2(valdien)2/2Cl2CHCOOH] 1 exhibited an intense transi- square planar N2O2 environment which deviated signicantly
tion at 271 nm attributed to the intraligand p–p* transition and from planarity. The phenyl rings of the ligands are parallel two
the absorption band at 404 nm characteristic of the LMCT was by two, giving rise to p/p stacking interactions with centroid/
ꢁ
observed. The low energy d–d band appeared at 559 nm centroid distances of 3.515 and 3.884 A (Fig. 2).
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Scheme 1 Schematic representation of the ligand and dinuclear copper(II) cocrystal “[Cu2(valdien)2/2Cl2CHCOOH]”.
2.3. Hirshfeld surface analyses
The Hirshfeld surfaces of the title cocrystal are illustrated in
Fig. 3, showing that have been mapped over dnorm (ꢀ0.27 to 1.54
ꢁ
ꢁ
ꢁ
A), shape index (ꢀ1.0 to 1.0 A) and curvedness (ꢀ4.0 to 0.40 A).
The surfaces are shown in a similar orientation for 1, around
which they are calculated. It is clear that the information
present in the histogram (Fig. S7†) is summarized effectively in
these spots, with large circular depressions (deep red) visible on
the front and back views of the surfaces indicative of hydrogen
bonding contacts. Other visible spots in the surfaces are
because of H/H contacts.
The 2D ngerprint plots and the relative contribution of the
important intermolecular contacts of dichloroacetic acid (DCA)
in the cocrystal are shown in Fig. 4. The most signicant
Fig. 1 X-ray crystal structure of dinuclear cocrystal [Cu2(valdien)2/ contributions come from H/H, Cl/H, C/H and H/O
2Cl2CHCOOH] 1. Hydrogen atoms are omitted for clarity.
contacts, which correspond to the van der Waals interactions
and hydrogen bonds, respectively. Since the dnorm values for
these contacts are close (48.6%, 16.2%, 16.2% and 9.40% for
The hydrogen atoms on N3 & N4 are involved in intra-molecular
hydrogen bonds with O1, O2 & O8, O7, respectively [N3–H3A/O1:
H/H, H/Cl, C/H and H/O, respectively), the lattice is
stabilized equally by both H-bonds and dispersion forces.
The synthon interactions, which have the closest interatomic
distances of all non-covalent interactions, are seen on the
ngerprint plot as two distinct spikes as in the case of C/H and
N/H contacts. The H/H contacts, being less directed are
presented on 2D ngerprint plots as bulk central areas. In
contrast to H/O, C/H and H/Cl contacts, are attractive while
ꢁ
ꢁ
ꢁ
2.84 (11) A, N3–H3A/O2: 2.97 (12) A, N4–H4A/O7: 2.90 (11) A,
ꢁ
ꢁ
N4–H4A/O8: 3.01 (12) A, C10–H10B/O3: 2.92 (11) A, C10–
ꢁ
ꢁ
H10B/N6: 3.23 (13) A, C12–H12A/O7: 2.87 (11) A, C30–H30B/
ꢁ
ꢁ
O5: 2.94 (12) A, C30–H30B/N1: 3.22 (13) A and C32–H32/O1:
ꢁ
2.88 (11) A] (see Fig. 2), which along with the stacking interactions,
help stabilizing the conformation of the [Cu2(valdien)2] moiety.
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Table 1 Crystal data with refinement parameters for [Cu2(valdien)2/
2Cl2CHCOOH] cocrystal 1
between the parallel aromatic ring of the copper-coordinated
validienꢀ2 ligand moiety which comprises 1.2% to the total
Hirshfeld surfaces. Blue triangles represent convex regions
because of the ring carbon atoms of the molecule inside the
surface, while red triangles represent concave regions resulting
from the carbon atoms of the p-stacked moiety above it.
The directional C/H/H/C close contacts that are associ-
ated with p/H interaction between metal complex and
dichloroacetic acid (DCA) and metal complexes of adjacent
layers are observed as broad spikes on each side in the nger-
print plot (Fig. 4). Interestingly, in the C–H/p interaction alkyl,
C–H and O–H (DCA) are involved along with the C–H of
aromatic rings, this could be due to the delocalization of elec-
tron density of nitrogen atoms to interacting alkyl carbon atoms
of the valdienꢀ2 ligand moiety. Additionally, some other non-
traditional interactions (Cu/H, C/O, N/O, N/H, C/Cl,
and O/Cl) to are also observed which are lesser contributing to
Hirshfeld surface. Finally, this example underlines the utility of
Hirshfeld surfaces, and in particular, ngerprint-plot analysis
for “visual screening” and rapid detection of unusual crystal
structures features through a whole structure view of intermo-
lecular interactions.
Parameters
1
Formula
C88H100C19Cu4N12O24
Fw (g molꢀ1
)
2283.00
Triclinic
Crystal system
Space group
ꢀ
P1
ꢁ
a (A)
12.790(5)
ꢁ
b (A)
14.351(5)
ꢁ
c (A)
15.034(5)
a (deg)
b (deg)
g (deg)
109.079(5)
91.480(5)
114.551(5)
ꢁ3
U (A )
2329.8(14)
2.4. Theoretical insight into intermolecular interactions via
NCI approach
Z
1
rcalc (g cmꢀ3
)
1.627
The non-covalent interaction (NCI) index based on the rela-
tionship between the electron density and reduced density
gradient (RDG) has been introduced by Yang and co-
workers.57,58 This technique is based on the analysis and the
graphical interpretation of the electronic density (r) and its
derivatives, namely the l2 eigenvalues of its Hessian and its
reduced gradient s(r):
m (mmꢀ1
)
1.241
F(000)
1173
Crystal size (mm)
Temp (K)
0.28 ꢁ 0.21 ꢁ 0.15
296(2)
1
jVrj
s ¼
Measured rens
Unique rens
GOFa
27 813
1=3
r4=3
2ð3p2Þ
7323
The non-covalent interaction (NCI) index was carried out
based on the properties of the electronic density of the mole-
cule, to visualize both attractive (hydrogen bonding, van der
Waals) and repulsive (steric) interactions.41 An inter- or intra-
molecular interaction causes a signicant alteration in the
reduced gradient of density (S) in between the interacting atoms
resulting in density critical points and interacting fragments. In
the 2D plots of S vs. r, the troughs represent critical points.42 To
analyze the non-covalent interaction is attractive or repulsive,
the sign of an eigenvalue l2 of the electronic density Hessian
matrix, V2r ¼ l1+ l2 + l3 (l1 < l2 < l3) can be utilized.43 The
reduced gradient of density S troughs are used to recognize non-
1.079
Final Rb indices [I > 2s(I)]
Rb indices (all data)
CCDC
R1 ¼ 0.0829, wR2 ¼ 0.2178
R1 ¼ 0.0898, wR2 ¼ 0.2229
1402001
P
a
2
GOF is dened as { [w(F0 ꢀ Fc2)]/(n ꢀ p)}1/2 where n is the number of
P
P
data and p is the number of parameters. b R ¼ { rrF0r ꢀ rFcrr/ rF0r},
P
P
wR2 ¼ { w(F0 ꢀ Fc2)2/ w(F02)2}1/2
.
2
the H/H contacts reside in the part of the Hirshfeld surface covalent interactions, point at which the value of S is
with positive dnorm, and thus should be considered repulsive. approaching zero, the strength of interaction is dened by
The bright colored area in the upper right part of the quantity sign (l2)r, i.e., higher the value, stronger will be
ngerprint plot and large at region across the molecule in interaction and ꢀve sign (attractive interaction) vs. +ve sign
Hirshfeld surface, which is most clearly visible on the curved- (repulsive interaction).
ness surface corresponds to p–p stacking contacts. The red and
The NCI index calculation of 1 revealed various kinds of
blue triangles on the same region of the shape index surface is intra- and inter-molecular non-covalent interactions within the
another characteristic of p–p stacking (C/C) interaction cocrystal which are responsible for the stabilization of crystal
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Fig. 2 Non-covalent interaction of cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1. (a) Inter-molecular C–H/O and C–H/Cl hydrogen bonding
interaction. (b) C–H/N, C–H/O, and N–H/O, intra-molecular hydrogen bonding interaction. (c) p/p stacking interactions.
lattice. These interactions identied in the 2D plots as spikes CH/Cl hydrogen-bond and p/p stacking interactions in the
with cutoff value (RDG ¼ 2 and r ¼ ꢀ0.25 to 0.25) and their cocrystal, respectively.
corresponding electronic density map with color coding. The
Interestingly, we observed various sites where electronic
electronic density map of the title compound not only revealed density was distributed between the various fragments of the
the attractive and previously identied non-covalent contacts premise of the co-crystal revealing not only hydrogen bonding
but also display valuable information about the destabilization (H/O, H/N, H/Cl), CH/p and p/p stacking interaction
factor as steric clashes which are repulsive in nature and responsible for the stabilization of geometrical conformation of
responsible for the geometrical distortion.
the co-crystal, in fact various other non-traditional dispersion
These stabilization and destabilization contacts differ in the forces between the atom pairs (Cu/H, C/O, N/O, C/Cl, and
shape of isosurfaces whose color code reveals the nature of the O/Cl) were also responsible. These non-traditional dispersion
interaction, where red is used for destabilizing, blue for stabi- forces are derived due the extensive delocalization of the elec-
lizing and green for delocalized weak interactions.
tronic charge density within the cavity of the [Cu2(valdien)2]
Fig. 5 displays the S vs. sign (l2)r in the [Cu2(valdien)2/ complex as green colored NCI surface is a signature for the
2Cl2CHCOOH] cocrystal exhibited four characteristic spikes delocalization of electronic density (Fig. 6). It is found that the
with density ꢀ0.05, ꢀ0.03, ꢀ0.01, 0.006 which are correspond- result of NCI index calculation is in good agreement with the
ing to the strong intra-molecular, inter-molecular, H/O, H/N, Hirshfeld surface analysis results.
Fig. 3 Hirshfeld surface mapped with dnorm (left), shape index (middle), and curvedness (right) for the title cocrystal [Cu2(valdien)2/2Cl2-
CHCOOH] 1.
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Fig. 4 2D fingerprinting plots the cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1.
interaction of the cationic core to the polyanionic phosphate
backbone of the DNA double helix, in addition to the active
participation of ligand chromophores via partial intercalation.44,45
1 was quantitatively ascertained by intrinsic binding constant, Kb,
value which was found to be 2.60 ꢁ 104 Mꢀ1 (Fig. S9†).
2.5. DNA binding prole and pBR322 nuclease activity
The binding study of 1 with CT-DNA was analyzed by spectro-
scopic optical methods via UV-vis, uorescence, and circular
dichroism. The band centered at 266 nm exhibited ‘hyper-
chromism’ with strong blue shi of 5–7 nm and the weak band at
399 nm exhibited ‘hypochromism’ of about 33% and 56%,
respectively (Fig. S8a†) revealing the favorable electrostatic
From the emissive titration spectra, it is apparent that on the
addition of increasing concentration of CT DNA (0.00–1.8 ꢁ
Fig. 5 Plot of the reduced density gradient versus the electronic density multiplied by the sign of the second Hessian eigenvalue of cocrystal
[Cu2(valdien)2/2Cl2CHCOOH] 1.
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Fig. 6 NCI electronic density distribution (isovalue: 0.8) in the [Cu2(valdien)2/2Cl2CHCOOH] 1 cocrystal.
10ꢀ4 M) to xed amount of 1 (Fig. S8c and S10†), displayed 276 nm (UV: lmax, 260 nm, CD [mdeg] 0.9602) because of base
hyperchromism of about 73%, respectively at 458 nm with no stacking and a negative band at 245 nm (CD [mdeg] ꢀ0.8893) due
shi in the wavelength was observed. The intrinsic binding to the right-handed helicity of the B-DNA form (with a zero-
constant K for 1 determined from Scatchard equation from the crossover around 254 nm) which are quite sensitive to the
plot of r/CF versus r (¼ CB/[DNA]) were found to be 5.8 ꢁ mode of DNA interactions with small molecules.47 Upon succes-
104 Mꢀ1. Furthermore, upon addition of increasing concentra- sive addition of 1, the positive band decreased in intensity with
tion of 1 to pretreated CT DNA with EB ([DNA]/[EB] ¼ 1) solu- a concomitant red shi to 280 nm while the negative band
tion, the emission band at 596 nm exhibited quenching of the (245 nm) increased in intensity, (Fig. S8b†) which ascertains the
emission intensity more than 70% of the initial uorescence potential of 1 to unwind the DNA by loosing its helicity.
intensity when the molar ratio of the complex to DNA (r ¼
The ability of 1 to cleave DNA was examined by incubating
[Complex]/[DNA]) range from 1.6 to 15.0 (Fig. S8d and S11†). different concentrations of 1 with supercoiled pBR322 DNA in
The observed quenching of DNA-EB uorescence indicated that the absence of reducing agents using standard physiological
the EB molecule was displaced by 1 from their DNA binding conditions (5 mM Tris–HCl/50 mM NaCl buffer, pH 7.2, incu-
sites.46 The quenching efficiency (KSV) was evaluated, and bation time 1 h). On increasing concentration of 1, Form I of
quenching data is in agreement with the classical linearꢀS1tern– pBR322 DNA gets converted into Form II (Lane 2–4), gradually,
Volmer equation,46 which was found to be 5.30 ꢁ 104 M
.
starting from 5 mM to 20 mM. But, when the concentration of 1
The CD spectrum of DNA exhibits characteristic B-type reached to 25 mM (Lane 6), Form II appeared with the formation
signature in the UV part of the spectra with a positive band at
Fig. 7 (a) The cleavage patterns of the agarose gel electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by 1 at 310 K
after 40 min of incubation; Lane 1, DNA control; Lane 2, 5 mM of 1 + DNA; Lane 3: 10 mM of 1 + DNA; Lane 4: 15 mM of 1 + DNA; Lane 5: 20 mM of 1
+ DNA; Lane 6: 25 mM of 1 + DNA. (b) Gel electrophoresis assay of 1 (15 mM) with pBR322 in presence of different activating (0.4 mM) and
scavenging agents (0.4 mM); Lane 1, DNA control; Lane 2, DNA + 1 + Asc; Lane 3, DNA + 1 + H2O2; Lane 4, DNA + 1 + GSH; Lane 5, DNA + 1 +
MPA; Lane 6, DNA + 1 + MG; Lane 7, DNA + 1 + DAPI; Lane 8, DNA + 1 + DMSO; Lane 9, DNA + 1 + tBuOH; Lane 10, DNA + 1 + NaN3; Lane 11,
DNA + 1 + SOD.
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Fig. 8 Molecular docked model of cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 with DNA [dodecamer duplex of sequence d(CGCGAATTCGCG)2
(PDB ID: 1BNA)].
of Form III, which migrates in between SC and NC form (Fig. 7a)
revealing double strand DNA cleavage.48
Cu(II)-complex + DNA / Cu(II)-complex DNA
Cu(II)-complex + eꢀ / Cu(I)-complex
To ascertain whether any adventitious agents present in the
reaction mixture could account for the increased DNA degra-
dation by “[Cu2(valdien)2/2Cl2CHCOOH],” 1, the DNA cleavage
activity was studied in the presence of activators. The nuclease
efficiency of the copper(II) complexes are known to depend on
the activators used for initiating the DNA cleavage. Thus, the
further activity of “[Cu2(valdien)2/2Cl2CHCOOH],” 1 has been
studied with different activators viz. H2O2 as an oxidizing agent,
ascorbate (Asc), 3-mercaptopropionic acid (MPA) and gluta-
thione (GSH) as reducing agents. The signicant enhancement
in the cleavage activity was noticed and followed the order H2O2
> GSH > MPA > ASC (Fig. 7b).
ꢀ
Cu(I)-complex + O2 / Cu(II)-complex + O2
O2 + 2H+ / O2 + H2O2
ꢀ
In Fig. 7b, the DNA cleavage by 1 was also carried out in the
presence of DAPI (minor groove binder) and methyl green, MG
(major groove binder). The cleavage pattern obtained showed
no inhibition in case of MG whereas DAPI resulted in apparent
inhibition, implicates the minor groove binding affinity of the
complex 1.
To get insight into the pathway of nuclease activity, (Fig. 7b)
1 was treated with DMSO and EtOH (hydroxyl radical scaven-
gers), SOD (superoxide scavenger) and NaN3 (singlet oxygen
quencher). No apparent inhibition was noticed upon addition
of DMSO and EtOH. However, in the presence of NaN3, signif-
icant inhibition was observed which revealed that ROS species
2.6. Molecular docking studies
To account for the topology of target-specic binding between
cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 with DNA, blind
molecular docking was carried out to mimic the interaction
modes. From the resultant docked structures (Fig. 8), it is clear
that 1 has slightly twisted the hydrophobic surface (interior) of
1
viz., singlet oxygen; O2 was responsible for the DNA cleavage
process via oxidative pathway.49
DNA in such
a
way that planar part of appended
Table 2 Non-covalent interactions of 1 with the DNA
Binding affinity (kcal molꢀ1
)
ꢁ
Name
Distance (A)
Category
Type
1: H11-B: DC21: O2
A: DA5: H3-1: O12
1: H26-B: DC23: O30
1: H32-A: DC3: O2
1: H32-A: DG4: O40
1: H1-B: DC23: O2
1.92
2.19
2.36
3.06
2.65
2.52
Conventional
Conventional
C–hydrogen bond
C–hydrogen bond
C–hydrogen bond
C–hydrogen bond
Hydrogen bond
ꢀ9.20
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Table
3 In vitro cytotoxicity of cocrystal [Cu2(valdien)2/2Cl2-
the MCF-7 cell, control as well as upon treatment are shown in
Fig. 9. The control cell exhibited uniform green uorescence
with the standard feature. However, the treatment with
cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1, showed red uo-
rescence with apoptotic features viz., cell shrinkage, chromatin
condensation, nuclear fragmentation and apoptotic body
formation. These typical features are ascribed to the efficient
DNA binding and nucleolytic properties of cocrystal
[Cu2(valdien)2/2Cl2CHCOOH] 1 as discussed and studied
above. In some of the cells, swelling and lysis were also wit-
nessed, may be attributed to ROS generation induced by coc-
rystal [Cu2(valdien)2/2Cl2CHCOOH] 1. To ascertain the ROS
role in the cytotoxicity, we further studied the [Cu2(valdien)2/
CHCOOH] 1 on MCF 7 breast cancer cell lines
IC50 values (mM)
(upon treatment of 24 h)
Compounds/synthetic drug
[Cu2(valdien)2/2Cl2CHCOOH] 1
Dichloroacetic acid (DCA)
Cisplatin
15.0 ꢂ 0.15
>100
20.2 ꢂ 0.05
pharmacophore ligand makes favorable stacking interactions 2Cl2CHCOOH] 1 in MCF-7 cell lines.
between DNA base pairs and 1 lead to attractive charge elec-
2.7.3. ROS generation. The cytotoxicity is also linked with
trostatic interaction with the sugar-phosphate backbone which the potential of the drug candidate to generate reactive oxygen
denes the stability of groove, detail description given in Table species (ROS) like O2cꢀ, OHc, and H2O2. Cu+ is known to convert
2. The binding energy of the docked structures cocrystal H2O2 to OHc, while O2cꢀ or glutathione reduces Cu2+ to Cu+. It is
[Cu2(valdien)2/2Cl2CHCOOH] 1 was found to ꢀ9.2 kcal molꢀ1
.
well known that copper facilitates the generation of ROS like
Therefore, it can be concluded that it is in good agreement with OHꢄ regardless of the oxidation state Cu2+ or Cu+, in which it is
the experimental studies.
entered into the body. Intracellular ROS generation surplus
leads to DNA damage and triggers various signals that lead to
the apoptosis. Our results of AO/EB staining had shown
swelling and lysis of the cells, necrosis that can be associated
with the ROS species generation inside the cell, so we studied it
using DCF-uorescence. The results exhibited by the MCF-7
breast cancer cells on treating with increasing concentration
2.7. Cytotoxicity studies
2.7.1. MTT assay. The cytotoxic effect of cocrystal
[Cu2(valdien)2/2Cl2CHCOOH] 1 was investigated on cultured
MCF 7 cell line (breast cancer) as well as non-cancerous human
embryonic kidney (HEK293) cells, by treating the cells by the
complex (1–20 mM) for 24 h. The dinuclear cocrystal 1 showed
a dose- and a cell-dependent decrease in cell viability of the
cancer cells. The IC50 value was found to 15.2 ꢂ 0.15 mM which
is signicantly lower than the value of standard cisplatin
(ꢃ20 mM). The IC50 values are tabulated in Table 3. Morpho-
logical changes are quite evident in the Fig S12,† on treatment
with cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 over MCF-7
breast cancer cell lines.
of
1 showed signicant ROS generation and exhibited
concentration-dependent pattern (Fig. 10). These results
conrm the above results of the role of ROS in cytotoxicity.
2.7.2. Extent of apoptosis in MCF-7 cells induced by
[Cu2(valdien)2/2Cl2CHCOOH]. The results obtained by the
dual staining of acridine orange (AO)/ethidium bromide (EB) of
Fig. 9 The AO/EB dual staining of the cocrystal [Cu2(valdien)2/ Fig. 10 (A) Images are showing the generation of ROS upon treatment
2Cl2CHCOOH] 1, induced apoptosis in MCF7 cells. The graph shows with complex 1 in MCF7 cancer cell lines (B) Histogram representing
the apoptotic cell count in percentage (mean % ꢀ SD% of triplicate concentration dependent ROS generation in MCF-7 cells following the
experiments).
exposure of 1–20 mM for 24 h.
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4.2. Synthesis of ligand (H2valdien)
3. Conclusions
The pro-ligand H2valdien was synthesized from the reaction of
o-vanillin (2 mmol, 0.304 g) and diethylenetriamine (1 mmol,
0.108 ml) according to the procedure reported earlier.50
A novel dinuclear copper(II) cocrystal “[Cu2(valdien)2/2Cl2-
CHCOOH],” 1 was synthesized from H2valdien scaffold and
dichloroacetic acid embedded with two Cu(II) connected via
hydrogen bonded network. [Cu2(valdien)2/2Cl2CHCOOH] 1
was thoroughly characterized by single-crystal X-ray crystallog-
raphy and by other spectroscopic techniques viz. FT-IR, UV-vis,
ESI-mass and uorescence spectroscopy. The non-covalent
interaction (NCI) index and Hirshfeld surface analysis
revealed the stabilization of cocrystal [Cu2(valdien)2/2Cl2-
CHCOOH] lattice from various kinds of non-covalent interac-
tions (O/H, N/H, H/Cl, Cu/H, C/O, N/O, C/Cl, and O/
Cl, etc.). In vitro interaction studies of [Cu2(valdien)2/2Cl2-
CHCOOH] with ct-DNA revealed good binding propensity which
was reected by its higher Kb, K and KSV values. The nuclease
activity of cocrystal [Cu2(valdien)2/2Cl2CHCOOH] with pBR322
plasmid DNA was ascertained by the gel electrophoretic
mobility assay which revealed an efficient cleaving ability via the
oxidative pathway involving singlet oxygen and the superoxide
anion as the ROS responsible for mediating the double
stranded DNA break reactions. The in vitro antiproliferative
activity was ascertained via the MTT assay, and the IC50 values
were found to be 15.0 ꢂ 0.15 (treatment upon 24 h) for MCF-7
cancer cell lines. Furthermore, on exposure of cocrystal
[Cu2(valdien)2/2Cl2CHCOOH] 1 to MCF-7 cancer cell lines,
ROS level was signicantly increased. Thus, the results of
in vitro antiproliferative activity validated that cocrystal
[Cu2(valdien)2/2Cl2CHCOOH] 1 is a potent chemotherapeutic
molecular drug entity for MCF-7 carcinomas, and it warrants
further in vivo investigations.
4.3. Synthesis of the cocrystal [Cu2(valdien)2/
2Cl2CHCOOH] (1)
To a stirred solution of H2valdien (1 mmol, 0.371 g) and
Cu(CH3COO)2$H2O (1 mmol, 0.199 g) in 20 ml of MeOH,
triethylamine (2 mmol, 0.278 ml) was added. The resulting clear
dark green solution was stirred for 30 min then ltered. To the
ltered solution dichloroacetic acid (2 mmol, 0.164 ml) in 5 ml
MeCN was added. Then, the solution was placed in a refriger-
ator for slow evaporation at 4 ꢅC. Aer 2–3 month, rectangular-
shaped brown crystals were collected. Yields ¼ 65–70%. EA, IR,
UV-vis, Fluorescence and ESI-mass data are given in the ESI.†
4.4. Computational details
The computational programme “ORCA” package was used to
carry out density functional theory (DFT) calculations51,52 using
the B3LYP functionals53 with Aldrich's def2-TZVP basis set for
copper atoms and def2-SVP basis set for C, H, O, N atoms for the
geometry optimization and single-point calculations.54–56 Start-
ing geometry was obtained from single crystal X-ray data.
Atomic coordinates of optimized structure (Fig. S13†) of
cocrystal [Cu2(valdien)2/2Cl2CHCOOH] 1 is given in Table S4,†
in good agreement with single crystal X-ray structure. To
calculate the density of the electron for all atoms to get NCI
index measurements, def2-TZVP basis set were used for the
structure of 1.57–59 To speed up the calculations we employed the
resolution of identity (RI) approximation with the decontracted
auxiliary def2-SVP/J and def2-TZV/J Coulomb tting basis sets
and the chain-of-spheres approximation to exact exchange with
empirical van der Waals correction as executed in ORCA.60,61
The molecular docking studies were carried out by using
Autodock Vina version 1.1.2.62,63 All rotatable bonds inside the
ligand were allowed to rotate freely, and receptor was consid-
ered rigid. From the http://www.rcsb.org./pdb (protein data
bank), the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID:
1BNA) crystal structure was retrieved The Discovery Studio 4.1,
and PyMol64,65 were used to visualize minimum energy favorable
docked poses. Hirshfeld surface was mapped using Crystal
Explorer66 soware using crystal structure coordinates of CIF
les.
4. Experimental section
4.1. Materials and measurements
Cu(CH3COO)2$H2O (Merck), o-vanillin (Sigma-Aldrich),
dichloroacetic acid (uka), 6X loading dye (Fermentas Life
Science), and supercoiled pBR322 plasmid DNA (Sigma-Aldrich)
were utilized as received. The disodium salt of CT-DNA was
ꢅ
purchased from Sigma Chem. Co. and was stored at 4 C.
The diagnostic kits, reagents, and other specied chemicals,
for cytotoxic studies, were procured from Sigma Chemical
Company Pvt. Ltd, St Louis, MO, USA. DMEM, antibiotics/
antimycotics solution and FBS were purchased from Invi-
trogen, Life Technologies (USA). Plastic and Culture wares
consumables used in this study were procured from Nunc,
Denmark.
Carbon, hydrogen, and nitrogen contents were carried out
on CHN Elemental Analyzer (model: Elementar Vario EL III).
4.5. Crystal structure determination
The Fourier-transform infrared (FT-IR) spectra were done on Bruker SMART APEX CCD diffractometer was used to collect
Spectrum Two Perkin-Elmer FT-IR spectrometers. The ESI-MS single crystal X-ray data of cocrystal [Cu2(valdien)2$$$$2Cl2-
spectrum was recorded on Micromass Quattro II triple quad- CHCOOH] 1 at 100 K on a using graphite monochromatic MoKa
ꢁ
rupole mass spectrometer. Electronic spectra were recorded on radiation (l¼ 0.71073 A). The linear absorption coefficients,
PerkinElmer Lambda 35 UV-vis spectrometer in MeOH using scattering factors for the atoms and the anomalous dispersion
1 cm path length cuvette, and data were reported in lmax/nm. corrections were referred for X-ray crystallography from the
Fluorescence measurements were determined on an RF-5301 International Tables.67 The data integration and reduction were
PC spectrouorophotometer (Shimadzu).
analyzed by using SAINT soware.68 Empirical absorption
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correction was applied to the collected reections with
SADABS,69 and the space group was determined using XPREP.70
By using the direct methods SHELXTL-2016, the structure
was solved and rened on F2 by full-matrix least-squares using
the SIR-97 program package.71 Only a few H atoms could be
located in the difference Fourier maps in the structure. The
remaining were positioned in calculated positions using ideal-
ized geometries (riding model) and assigned xed isotropic
displacement parameters. All non-H atoms were rened aniso-
tropically. The renement and crystal data are presented in
Table 1.
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4.6. Methodology for biological studies
Nucleic acid binding experiments were performed in Tris–HCl/
NaCl (5 : 50) buffer at pH 7.2 which included absorption spec-
tral traces, emission spectroscopy and circular dichroism
measurements confronted to the standard methods and prac-
tices previously adopted by our laboratory.72–76 While measuring
the absorption spectra an equal amount of DNA was added to
the reference solution to eliminate the absorption of the CT-DNA
itself, and the absorbance of the Tris buffer was subtracted
through baseline correction. The cleavage experiments of
supercoiled pBR322 DNA (300 ng) in Tris–HCl/NaCl (5 : 50 mM)
buffer at pH 7.2 was studied by agarose gel electrophoresis. The
ꢅ
samples were incubated for 45 min at 37 C, and the cleavage
process with and without ROS was monitored using agarose gel
electrophoresis. A loading buffer comprising of 25% bromo-
phenol blue, 0.25% xylene cyanol, and 30% glycerol was added,
and electrophoresis was done at 30 V for 1 h in Tris–HCl buffer
1% agarose gel containing 1.0 mg mlꢀ1 ethidium bromide.
In vitro experiment on MCF7 (human, mammary gland, meta-
static site, epithelial cell, breast cancer) cell line viz., cell prolif-
eration, cell culture, MTT assay, AO/EB staining and ROS
generation assays were carried by adopting standard
methods77–79 with slight modication as reported earlier by us
and references therein.80–85
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The cell lines was purchased from ATCC.
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Conflicts of interest
There are no conicts to declare.
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CrystEngComm, 2011, 13, 4358–4364.
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Acknowledgements
We gratefully acknowledge the DST–PURSE program, DST–FIST
and DRS–1 (SAP) from UGC, New Delhi, India. The authors
extend their appreciation to the Deanship of Scientic Research
at King Saud University for funding this work through research
group No. RG-1438-006. Authors are thankful to the staff of IIT
KANPUR for the assistance in Single Crystal X-ray studies.
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