Tetranuclear cubane Cu4O4 complexes as prospective anticancer agents: Design, synthesis, structural elucidation, magnetism, computational and cytotoxicity studies

Article abstract of DOI:10.1016/j.ica.2017.12.039

Two new homometallic Cu₄O₄ cubane clusters 1 and 2 have been synthesized by self-assembly of copper(II) acetate and ligand, 2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-2-hydroxymethyl-propane-1,3-diol (H₄L) and characteriz

Full text of DOI:10.1016/j.ica.2017.12.039

Accepted Manuscript
Research paper
Tetranuclear Cubane Cu O complexes as prospective Anticancer Agents: De-
4
4
sign, Synthesis, Structural Elucidation, Magnetism, Computational and Cyto-
toxicity Studies
Mohammad Usman, Farukh Arjmand, Rais Ahmad Khan, Ali Alsalme, Musheer
Ahmad, Mousumi Sen Bishwas, Sartaj Tabassum
PII:
S0020-1693(17)31439-1
https://doi.org/10.1016/j.ica.2017.12.039
ICA 18073
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
14 September 2017
21 November 2017
29 December 2017
Please cite this article as: M. Usman, F. Arjmand, R.A. Khan, A. Alsalme, M. Ahmad, M.S. Bishwas, S. Tabassum,
Tetranuclear Cubane Cu O complexes as prospective Anticancer Agents: Design, Synthesis, Structural Elucidation,
4
4
Magnetism, Computational and Cytotoxicity Studies, Inorganica Chimica Acta (2017), doi: https://doi.org/10.1016/
j.ica.2017.12.039
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Tetranuclear Cubane Cu O complexes as prospective Anticancer Agents:
4
4
Design, Synthesis, Structural Elucidation, Magnetism, Computational and
Cytotoxicity Studies
1
1
2
2
3
Mohammad Usman, Farukh Arjmand, Rais Ahmad Khan, Ali Alsalme, Musheer Ahmad,
4
1
Mousumi Sen Bishwas, and Sartaj Tabassum. *
1
Department of Chemistry, Aligarh Muslim University, Aligarh-202002, India
2
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh
1451, KSA.
1
3
Department of Applied Chemistry, ZHCET, Aligarh Muslim University, Aligarh, 202002, India.
Physical and Material Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi
Bhabha Road, Pune 411008, India.
4
*
Corresponding Author:
Prof. Sartaj Tabssum, Tel: +91 9358255791, Email: tsartaj@yahoo.com
Abstract
Two new homometallic Cu
of copper(II) acetate and ligand, 2-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-2-
hydroxymethyl-propane-1,3-diol (H L) and characterized thoroughly by various spectroscopic
4
4
O cubane clusters 1 and 2 have been synthesized by self-assembly
4
techniques and single crystal X-ray diffraction analysis. Temperature-dependent magnetic
susceptibility measurements have been performed to elucidate the antiferromagnetic and
ferromagnetic nature in Cu
4
4
O clusters 1 and 2, respectively. In vitro DNA binding studies of
cubane clusters were carried out by employing optical spectroscopic techniques. Gel
electrophoretic mobility assay performed to examine the nuclease activity of the complexes 1
and 2 with pBR322 DNA, and results revealed oxidative DNA cleavage via reactive oxygen
•
1
species (ROS) species viz., O
calculate the cytotoxicity of complexes 1 and 2. The IC50 evaluated were ~ 20 µM in MCF-7
Breast) and ~ 30-35 µM in HepG2 (Liver) cancer cell lines. Additionally, in the presence of 1
2
‾, O
2
, etc. In vitro cell proliferation via MTT assay was studied to
(

and 2, ROS and TBARS (Thiobarbituric acid reactive substance) levels amplified significantly,
coupled with GSH (glutathione) levels in cancer lines. Hence, the results exhibited the major role
of ROS in apoptosis induced by 1 and 2 clusters and validate their prospective to be efficient
anticancer drug entities.
Keywords: Cu
4
4
O cubane complexes, Magnetism, DFT, DNA binding, Nuclease activity,
Cytotoxicity.
1. Introduction
The synthetic design of multinuclear transition metal complexes is a thriving thrust area of
research from the point of view of i) bioinorganic and ii) supramolecular chemistry [1].
Polynuclear complexes emerging from the aggregation of homo- or hetero-metal centers are the
result of spontaneous self-assembly processes, capable of distinct recognition phenomenon
towards the substrate. Interestingly, these complexes have shown remarkable biological, catalytic
and magnetic properties [2].
Among the high nuclearity transition metal complexes, tetranuclear copper clusters having
cubane-like Cu
4
O
4
core are well studied and have received considerable attention [3, 4].
cubane assembly.
Tridentate Schiff base ligands are commonly used for the building of Cu
4
O
4
Three coordination sites of Cu(II) ion are filled by tridentate ligand, and a bridging ligand (azide,
halides, hydroxide, pseudo-halides, oxalate, etc.) or µ-bridging alkoxo/phenoxo oxygen (self-
assembly process) is used to obtain cubane structural frameworks [5].
Cu
4
4
O cubane clusters have been classified into structural types as a function of their topology
and degree of distortion [6]. More recently, Alvarez et al. proposed a classification based on the
dispersal of the six Cu···Cu distances with (2+4), (4+2) and (6+0) classes [3b]. The (2+4) and
(
4+2) cubane usually resembles systems in which Cu(II) ion has elongated octahedral or a square

pyramidal geometry, whereas (6+0) cubanes are assigned to the six equivalent faces unusual
cores having geometry around Cu(II) ion trigonal bipyramidal in general. To date, only a limited
number of alkoxo/phenoxo-bridge Cu(II) cubane structures have been reported [3c]. Apart from
the structural novelty of cubane cores, the multinuclearity, and Cu
4
4
O cubane core are of great
importance to the biologists in the area of drug design [7]. High metal nuclearity enhances the
positive charge which in turn shows a greater propensity for negatively charged phosphate-sugar
back bone of DNA and synchronized tuning between two or more metal centers enabling these
complexes to bind nucleic acids multifold more selectively via non-covalent mode; further, it
promotes DNA cleavage efficiency to achieve target specific chemotherapeutic agents [8]. In
drug design strategy, high nuclearity component modulates pharmacological parameters and
bears a disposition to alter the efficacy of the drug [7c].
Herein, we have designed and prepared Cu
area of drug design, particularly as an antitumor chemotherapeutic candidate. A very few
literature reports have demonstrated the antitumor activity of Cu cubane tetranuclear clusters
9]. However, DNA binding and cleavage studies of some cubane complexes have been reported,
yet there is much scope for more consolidated studies on design and synthetic strategy of novel
cubane antitumor compounds [7a,9,10]. The presently synthesized Cu cubane clusters
4
O
4
open cubane 1 cluster for the application in the
4
O
4
[
4
O
4
satisfactorily meet pharmacological drug discovery criteria i) high water solubility, ii) exhibit
high DNA-binding propensity, snugly fit in the major groove of DNA helix as validated by
docking iii) efficient DNA cleaving activity via ROS generation. The cytotoxic activity of
cubane clusters 1 and 2 against HepG2 and MCF7 cancer cell lines were carried out by MTT
assay, revealed good selectivity towards MCF7 breast cancer cell. As 1 showed selectivity

towards MCF7 breast cancer cell line, so we have explored the mechanistic pathways by
carrying out assays to evaluate ROS generation, GSH depletion, and lipid peroxidation.
2. Experimental section
2
.1. Materials and methods
2
-hydroxy-3-methoxybenzaldehyde, 2-Amino-2-(hydroxymethyl)-1,3-propanediol, ethidium
bromide (EB) and trimethylamine (Sigma-Aldrich), copper acetate monohydrate (Merck),
supercoiled pBR322 DNA and 6X loading dye (Genie), were utilized as received and solvent
were used as obtained. The disodium salt of calf thymus DNA was obtained from Sigma-
Aldrich.
The chemicals, reagents, and diagnostics used for cytotoxic assays were purchased from Sigma-
Aldrich. From Invitrogen, Life Technologies (USA), DMEM, solution of
antibiotics/antimycotics and FBS were obtained.
On ThermoFinnigan CHN analysis was done. Eutech con 510 electronic (conductivity bridge)
utilized to get the molar conductance at room temperature. PerkinElmer FT-IR spectrometers,
was used to record FT-IR spectra. The EPR spectra were obtained by JEOL FA-200 continuous-
wave spectrometer using Manganese (Mn) as standard (g = 2.0036). The absorption spectra were
noted on PerkinElmer Lambda 35 UV/VIS spectrometer. Emission spectra were recorded on a
Shimadzu RF5301PC spectrofluorophotometer using slit widths of 10/10 nm.
Magnetic readings were taken of polycrystalline powder sample by the help of a SQUID VSA dc
magnetometer of Magnetic Property Measurement System (MPMS) from Quantum Design Inc.,
San Diego, CA, USA, armed with a superconducting magnet (7T). To affect our data accuracy,
the magnetic value from the holder was insignificant. The dc magnetization M vs. T spectra were
taken at 100 Oe with heating/cooling @ 2K/min. Magnetization vs. field loops was taken in a

field sweep from -50 to + 50 k Oe, @ 50 Oe/s. Axygen electrophoresis held with Genei power
supply with a potential range of 50-100 V used for electrophoretic experiments, visualized and
photographed by a Vilber-INFINITY gel documentation system.
2
4
.2. Synthesis of ligand (H L)
A methanolic solution of 2-Amino-2-(hydroxymethyl)-1,3-propanediol (0.121g, 1 mmol) was
added dropwise to the methanolic solution of 2−hydroxy−3−methoxybenzaldehyde (0.152 g, 1
mmol). The resulting mixture was refluxed at 80˚C for about 2 h until a pale yellow solid
compound precipitated, filtered, washed with hexane and dried in vacuo.
Yield: 78%, M.P. 105 °C. Analysis Calculated for [C12
H
17NO
5
] (%): C, 56.46; H, 6.71; N, 5.49;
); 2920 υ(C-
Found: C, 56.42; H, 6.78; N, 5.51. FT-IR (KBr pellet): 1643 υ(C=N); 1023 υ(O-CH
3
–
4
H), 1023 ν(-OCH ). UV–vis (1 x 10 M, MeOH, λmax nm): 240, 292 and 416.
3
2
.3. Synthesis of [Cu
4
(H
2
L)
4
·2H
2
2
O]·5H O (1)
A methanolic solution of 2-Amino-2-(hydroxymethyl)-1,3-propanediol(0.121g, 1 mmol) was
added dropwise to the methanolic solution of 2−hydroxy−3−methoxybenzaldehyde (0.152 g, 1
mmol). The mixture solution was stirred for another 2 h at 80 °C and gives a clear dark yellow
solution. Cu(CH
3
COO)
2
·H
2
O (0.199 g, 5 mmol) was added to the above reaction mixture in the
O (90:10), which was further refluxed
presence of triethylamine (TEA) at pH 8-9 in MeOH/H
2
for 6h until a deep green solution resulted. After the reaction completion, the volume was
reduced by heating on a hot plate and filtered hot. Then, the filtrate was left for slow evaporation,
resulted in deep green colored crystals over a period of 6-7 days. These crystals were filtered,
washed and air dried (Scheme 1).

Yield: 73%, M.P. 130 °C. Analysis calculated for [Cu
4
(H
2
L)
4
·2H
2
O]·5H O (%): C, 40.21; H,
2
-1
8
.02; N, 3.91; Found: C, 40.37; H, 8.11; N, 4.02. FT-IR (KBr pellet): 1624 cm υ(C=N); 3373
-1
-1
-4
cm υ(O-H); 2936 cm υ(Ar-H). UV-vis (1×10 M, MeOH, λmax nm): 234, 280, 375, 645 nm.
-3
-1
2
-1
Molar conductance:
ʌ
M
(1 × 10 M, DMSO): 6.03 Ω cm mol (non-electrolyte).
2
.4. Synthesis of [Cu
4
(H
2
L)
4
2
·4H O] (2)
A methanolic solution of 2-Amino-2-(hydroxymethyl)-1,3-propanediol(0.121g, 1 mmol) was added
dropwise to the methanolic solution of 2−hydroxy−3−methoxybenzaldehyde (0.152 g, 1 mmol).
The solution mixture was allowed to stirr for 2 h at 80 °C to obtain a deep yellow clear solution.
To this yellow solution, Cu(CH
3
COO)
2
·H
2
O (0.199 g, 1 mmol) solution in DMF and piperazine
(
0.086 g, 1 mmol) was added and refluxed ~4 h, until resulted into a dark green solution. When
reaction got completed, the solution reduced in volume on a hot plate and filtered hot. Then, the
filtrate is left for slow evaporation and after a period of 2-3 weeks, the deep green colored crystal
was obtained. The crystals obtained were washed and air dried (Scheme 1).
Yield: 58%, M.P. 137 °C. Analysis calculated for [Cu
4
(H
2
L)
4
·4H
2
O] (%): C, 42.16; H, 7.08; N,
-
1
-1
4
2
.10; Found: C, 42.07; H, 7.12; N, 4.17. FT-IR (KBr): 1627 cm υ(C=N); 3454 cm υ(O-H);
-
1
-4
907 cm υ(Ar-H). UV-vis (1 × 10 , MeOH, λmax nm): 234, 280, 375, 625 nm. Molar
-3
-1
2
-1
conductance:
ʌ
M
(1 × 10 M, DMSO): 6.16 Ω cm mol (non-electrolyte).
2
.5. Computational methodology
ORCA computational package was used for all the studies performed.[11] The single X-ray
data was utilized to get geometry. By hybrid B3LYP functional by means of basis sets viz,
’
Aldrich s def2-TZVP (for copper atom) and def2-SVP (for C, H, O, N atoms), single point
energy calculations were done [12]. The computation was accelerated, by using RI (the

resolution of identity approximation) with the decontracted auxiliary coulomb fitting basis sets
def2-TZV/J or def2-SVP/J) and RIJCOSX (the chain-of-spheres approximation) to exact
(
exchange as employed in ORCA [13].
The Autodock Vina (version 1.1.2.) [14] was used to perform molecular docking studies with B–
DNA dodecamer (PDB ID: 1BNA) d(CGCGAATTCGCG) was saved from the protein data
2
bank (http://www.rcsb.org./pdb). For the minimum energy docked pose visualization, the
Discovery Studio 4.1 and PyMol were used [15].
2
.6. X-ray data collection and structure refinement
Single crystal X-ray data of synthesized Cu cubane clusters 1 and 2 were collected using
MoK radiation (λ = 0.71073 Å) in diffractometer (Bruker SMART APEX CCD). The
International Tables for Crystallography was inferred for the corrections in scattering factors
atoms), linear absorption coefficients, and anomalous dispersion [16]. The SAINT software was
4
O
4
α
(
used to study the data integration and reduction [17]. The collected reflections were applied to
empirical absorption correction with SADAB [18], and space group was determined using
XPREP [19]. The direct methods using SHELXL-97 and SHELXL-2016/6 were used to solve
2
the Cu
4
O
4
cubane clusters 1 and 2 respectively, which were refined of F by full-matrix least-
squares using the SIR-97 programme package [20]. Only a few H atoms could be located in the
difference Fourier maps in the structure. The rest were placed in calculated positions using
idealized geometries (riding model) and assigned fixed isotropic displacement parameters. All
non-H atoms were refined anisotropically. Several DFIX commands were used for fixing some
bond distances in both complexes. The crystal and refined data are collected in Table 1 while
selective bond distances and angles are given in Supporting Information, Table S1-S4.

Table 1. Crystal and structure refinement data for Cu
4
O
4
cubane clusters 1 and 2.
Parameters
1
2
Formula
C
48
H
60Cu
4
N
4
O
27
C
48
H
1335.2
64Cu
4
N
4
O
24
–
1
Fw (g mol )
crystal system
space group
a (Å)
b (Å)
c (Å)
1379.16
Monoclinic
P21/n
20.828(10)
13.202(6)
21.331(10)
90
Tetragonal
I41/a
18.609(2)
18.609(2)
15.425(2)
90
α (deg)
β(deg)
97.643(8)
90
5813(5)
4
90
90
γ (deg)
3
U (Å )
5341.6(13)
16
Z
3
ρ
calc (g/cm )
1.576
1.532
1.660
1.660
–
1
µ (mm )
F(000)
2832
2752
Temp (K)
296(2)
27375
4181
296(2)
25013
2016
measured reflns
unique reflns
a
GOF
1.000
R1 = 0.0718
1.204
R1 = 0.1088
wR2 = 0.2245
R1 = 0.1240
b
Final R indices
[
I>2σ(I)]
wR2 = 0.2090
R1 = 0.1740
wR2 = 0.2435
b
R indices
(
all data)
wR2 = 0.2325
CCDC
1003913
1556830
a
2
2
1/2
b
GOF is defined as {Σ[w(F
0
–F
c
2 2
)]/(n–P)} ; n no. of data & “p” no. of parameters. R= {Σ||F |–
0
2
2
2 2 1/2
|
F
c
||/Σ|F
0
|, wR = {Σ w(F
0
–F
c
) / Σ w(F ) } .
0
2
.7. DNA interaction and cleavage experiments
The DNA binding studies were performed in Tris-HCl/NaCl (5:50) buffer at pH 7.4, using the
standard protocols and with slight modification adopted by our laboratory in the past [21-24].
2
.8. Biological experiments
All the biological experiments were carried out by our group with slight modifications in the
standard protocols [22,25,26]
2
.8.1. Cell lines and culture

In Brief, Both the cells HepG2 and MCF-7 cancer cells were cultured in DMEM in addition with
FBS (10%), 0.2% NaHCO , and 1 mL/100 mL of 100X medium (antibiotic/antimycotic
solution). At 37 °C with high humidity under 5% CO -95% air, the cells were maintained.
.8.2 Cytotoxicity by the MTT assay
3
2
2
The protocol followed for MTT assay was previously reported by us [22]. Briefly, In 96 well
4
culture plate, 1x 10 cells adhered to 24h in CO
2
incubator. Then, the from the stock of 5mg/mL
in PBS of MTT added 10 µL to each cell suspension and incubated for 4h. After this supernatant
discarded and DMSO (200 µL) to each well and gently mixed and read at 550 nm. Identical
conditions were used for control, and IC50 values were calculated.
2
.8.3. Cell Morphology
Inverted phase contrast microscope was used to take images of morphological changes observed
by the compounds on MCF7 and HepG2 cells after treatment for 24 h.
2
.8.4. Reactive oxygen species (ROS) generation
The protocol used to examine ROS generation is described earlier [22], using DCFH-DA dye as
the fluorescent probe. After treatment cells for 24h washed with PBS and then were incubated
with DCFH-DA (20 µM) for 1h in the dark. Lastly, by the help of fluorescence microscope, the
cells were analyzed.
2
.8.5. Intracellular Glutathione Depletion
The protocol used to evaluate Intracellular glutathione depletion was described earlier [22,25],
Concisely, after cellular treatment protein is extracted from sonication with 1 mL TCA (10%)
and cooled on ice for 1h and then centrifuged at 3000 rpm for 10 min. In 2 mL solution of Tris
buffer (0.4 M) with 00.2 M EDTA supernatant is added and then 0.01M DTNB used to make up

to 3 mL. Later, at 37 °C for 10 min kept on shaking water bath, and the absorbance was read at
4
12 nm.
2
.8.6. Lipid peroxidation assay
For this study, TBARS protocol was adopted [26]. Precisely, after treatment cells were
centrifuged and then sonicated in 1.15% potassium chloride (ice cold) solution and centrifuged at
3
00 rpm for 10 min. Then 1 ml of supernatant is added to a mixture of TCA (15%), TBA (0.7%)
and HCl (0.25N) called as TBA reagent (2 mL) and at 100 °C for 15 min was heated, in boiling
bath. Further cooled and again centrifuges for 10 min at 1000rpm and the absorbance was
measured at 550 nm.
3. Result and discussion
3
.1. Synthesis and characterization
The multi-dentate Schiff base ligand H
hydroxy-3-methoxybenzaldehyde with 2-Amino-2-(hydroxymethyl)-1,3-propanediol in MeOH.
At room temperature in 1:1 molar ratio, H L with Cu(CH COO) ·H O were mixed and added
triethylamine in methanol-water (90:10) at pH 8-9 yielded dark green colored cubane clusters 1
Scheme 1). The reaction of H L with Cu(CH COO) ·H O along with piperazine in a 1:1:1
4
L was synthesiszed by simple condensation of 2-
4
3
2
2
(
4
3
2
2
molar ratio and triethylamine at pH 8-9 in MeOH-DMF (80:20) at ambient temperature afforded
a light green color compound that was crystallized into a new cubane cluster 2 (Scheme 1). Our
efforts to control self-assembly process of POM-based clusters, using organo-cation is vital and
perhaps could provide the basis to design, employing a crystal engineering approach. To achieve
this target, we used organic amine cation viz. piperazine in the synthesis of complex 2. The use
of piperazine helps to isolate self-assembled POM species in one-pot reaction systems thus
preventing their rapid aggregation into clusters having more stable uniform symmetrical

topology. The molecular structures of 1 and 2 were elucidated by using a single crystal X-ray
diffraction study. The analytical techniques which were employed in the characterization of
complexes include elemental analysis, solution electrical conductivity, FT-IR, UV-vis, X-band
EPR and ESI-MS techniques.
CHO
O
OH
OH
HO
OH
OH
OH
O
MeOH
Reflux
OH N
HO
H N
2
H4L
H4L
MeOH+H2O, Reflux
OH
MeOH+DMF, RT
OH
HN
NH
OH
OH
N
N
O
O
Cu
O
O
O
Cu
O
H O
2
O
Cu
Cu
O
O
Cu
O
O
O
N
Cu
O
OH
OH
O
Cu
O
Cu
N
OH2
H O
2
O
HO
HO
1
2
Scheme 1: Synthetic route of Cu
4
O
4
open cubane 1 and Cu
4
O closed cubane 2 clusters.
4
2
-
Coordinating ligand (H
2
L ) of two copper center is omitted for clarity.
3
.2. Crystal structure of [Cu
4
(H
2
L)
4
·2H
2
2
O]·5H O (1)
The Single X-ray crystal structural technique revealed that complex 1 crystallized in the P21/n
monoclinic space group possessing lattice parameters, a = 20.828 (10) Å, b = 13.202 (6) Å, c =
ο
2
1.331 (10) Å, α = γ = 90 , β = 97.643 (8) Å. The asymmetric unit consists of four Cu(II) ion,

2
-
four H
2
L ligand, two coordinated and five lattice water molecules (Fig. 1). The complex 1
possesses open cubane-like core in which the four copper atoms are bridged by four oxygen
2
-
atoms, two phenolate oxygen (O7 and O12) and two alkoxo oxygen atoms (O5, O20) from H
ligands. Selected bond angles and lengths are given in Supporting Information, Table. S1 and S2.
The Cu cluster comprises of four symmetry non-equivalent Cu(II) species, Cu1 and Cu4
cores reveal CuN coordination arrangement distorted octahedral shape, as evident by the
2
L
4
O
4
1
O
5
ο
ο
angles around them, which deviate from 180 and 90 . The Cu1 atom is basically has alike to
Cu4, and bonded with equatorial O2, N1, O5, and O7 atoms [Cu1-O6 2.373 Å; Cu1-O20 2.576
2
-
Å] of the H L ligand moieties. The Cu4 atom has same coordination arrangement as ascribed
2
for Cu1 atom and equitorial sites are satisfied by O20, N4, O17, O12 atoms [Cu4-O20 1.930 Å;
Cu4-N4 1.976 Å; Cu4-O17 1.887 Å; Cu4-O12 2.001 Å], axial sites completed with O5 and O11
[
Cu4-O5 2.588 Å; Cu4-O11 2.354 Å].
The penta-coordinated Cu2 signify CuN
coordinated from O10, N2, O7, and O20 atoms [Cu2-O10 2.008 Å; Cu2-N2 1.958 Å; Cu2-O7
1
O distorted square pyramidal geometry with τ = 0.02,
4
2
-
1
.958; Cu2-O20 1.930Å] in equatorial positions of ligand H
engaged by O2W [Cu2-O2W 2.334 Å] of water molecule. Also, Cu3 metal core displayed
CuN distorted square pyramidal coordination with τ = 0.12, the equitorial position bonded to
2
L ligand moieties, while axial site
1
O
4
O15, N3, O12, and O5 [Cu3-O15 2.030 Å; Cu3-N3 1.984 Å; Cu3-O12 1.951 Å; Cu3-O5 1.939
Å] and O1W [ Cu3-O1W 2.233 Å] of water molecule in an axial position.
In the Cu
two oxygen alkoxo (µ
cubane core [Cu (µ -O)
4
O
4
core, metal cores are interconnected through two oxygen phenoxo (µ
1
-O7/O12) and
L ligand to produce a distorted single-open
] with idealized S4-symmetry with Cu···Cu separation in the
2
-
2
-O5/O20) atoms of four H
(µ -O)
2
4
1
2
2
2

3
.218 – 3.301 Å range (avg. 3.247), which are quite comparable to the values of similar Cu
cubane clusters reported in the literature [18-20].
.3. Crystal structure of [Cu (H L) ·4H O] (2)
4
O
4
3
4
2
4
2
Ssingle X-ray crystal exhibited that the complex 2 crystallized in the tetragonal I 41/a space
ο
group possessing lattice parameters, a = b = 18.609 (2) Å, c = 15.425 (2) Å, α = β = γ = 90 per
2
-
unit cell. The asymmetric unit consists of four Cu(II) ion, four ligand H
2
L moieties and four
coordinated water molecules (Fig. 1b). The complex 2 portrayed as a closed cubane-like core in
2
-
which four Copper atoms are linked by four alkoxo bridges oxygen atoms of ligands H L .
2
Selected bond angles and lengths are given in Table. S3 and S4.
The Cu
4
O
4
closed cubane clusters consist of four symmetry-equivalent Cu atoms, in a CuN
1
O
5
coordination environment arranged in octahedral geometries. The coordination environment
around Cu is filled by three alkoxo bridge oxygen atoms [Cu1-O3 1.933(7) Å], one phenolate O2
2
-
and an imine N1 atoms [Cu1-N1 1.946(10) Å; Cu1-O2 1.933(7) Å] of the H L ligand and sixth
2
coordination position was completed by a water molecule [Cu1-O1W 2.719 Å].
The four copper centers in the cluster are mutually interconnected via four alkoxo oxygen (µ1-
2
-
O3) atoms of four ligand H
2
L moieties to generate a single-closed cubane core [Cu
4
(µ
1
-O) ]
4
with Cu···Cu separations in the range of 3.171- 3,473 Å, well corroborated with earlier reported

results
[3].
Fig. 1 X-ray molecular structures (a) Cu
4
O
4
open cubane cluster, 1 (b) Cu
4
O
4
closed cubane
cluster, 2. For clarity hydrogen atoms and lattice molecules have been omitted.
3
.4. Spectroscopy
The IR spectrum of the free H
4
L ligand exhibited IR absorptions at 1644, 1023, and 3342-
(-OCH ) and (O-H) vibrational modes, respectively, Supporting
Information, Fig. S1 and S2 [20]. The
-
1
3
205cm due to
ν(C=N),
ν
3
ν
ν(C=N) vibrational mode of complexes 1 and 2 w
-1
-1
observed at 1624 and 1627cm respectively, which are slightly lowered (15-20 cm ) relative to
2
-
the free H
2
L ligand, revealing the coordination of imine nitrogen, Supporting Information, Fig.
S3 and S4 [21]. Another substantial downward shift was observed in the stretching vibration of
−
1
the C−OCH
atom. While an upward shift detected from 959 to 983 cm in 2, revealing the cubane core free
from −OCH coordination. Additionally, in the far-IR spectra of 1, Supporting Information (Fig.
S5), several absorption bands attributed to
originating from vibrations in the [Cu ] core were observed [21a].
3
group, from 959 to 923 cm in 1 thereby indicating its coordination with the Cu
−
1
3
-1
ν(Cu-O) frequencies viz.685, 399, 282 and 207 cm
4
O
4

The electronic spectra of 1 and 2 recorded in a MeOH solution, Supporting Information (Fig.
S6), show very close resemblance and revealed three well-resolved absorption maxima at 234,
*
2
80 and 375 nm. The two sharp absorption bands centered at 234 and 280 nm correspond to π-π
intraligand transitions while a broadband at 375 nm was assigned to the charge-transfer (LMCT)
transitions of the phenolate or alkoxo oxygens to the copper ions. The bands centered at 645 nm
and 625 in 1 and 2, respectively, were assigned to the d-d transitions, Fig. S7 [21c].
The X-band EPR spectra for the polycrystalline powder of the Cu
4
4
O open cubane and closed
cubane clusters 1 and 2 at room temperature (r.t) and liquid nitrogen (LN) temperature was
recorded, Supporting Information, Fig. S8. No significant shift was observed in the g-values. In
spectra of 1 an axial symmetrical signal with g|| at 2.33, g
⊥
at 2.03 and gav at 2.13 {computed
)} was observed. The calculated values g|| and g for 1 show
> 2.0023 which is indicative of the presence of an unpaired electron in the
dx -y orbital. The closed cubane cluster 2 exhibited a broad isotropic signal with g at 2.50 at
7K however, no signal was observed at room temperature. All these observations are in good
agreement with the solid-state structure as determined by X-ray crystallography.
2
2
2
from the formula gav = 1/3(g|| +2g
⊥
⊥
the order as g|| > g
⊥
2
2
7
To obtain evidence for the stability of the Cu
4
O
4
cluster 1 and 2 in solution, ESI-MS and UV-vis
+
spectra were analysed. For 1, ESI-MS exhibited m/z values of 1308.27 [M-4H
2
O] and 1336.16
+
[
M+H ] for 2 (Fig. S9 and S10), corresponding to the stability of both the complexes in solution.
The Fig. S11 of UV-vis spectra of 1 and 2 in Tris-HCl buffer at pH 7.4 and T 296 K, was
recorded at various time intervals. The results displayed no significant changes nor in the
intensity or position of absorption bands, and also demonstrating the stability of the Cu
4
O
4
clusters 1 and 2 in solution phase.
3
.5. Magnetic Properties

In 1, χ
m
increases monotonically from 300 K to 100 K and then a broad hump around 50 K was
observed. Below this temperature, a very weak bifurcation in the ZFC-FC curve was observed,
which is suggestive of antiferromagnetic interaction in the system. The χ T value at room
m
3
temperature (300 K) was found to be 2.8 cm /mol.K which decreased monotonically as the
3
temperature decreases and reached a value of 0.1 cm /mol.K at 3 K. The 1/χ
m
versus T plot
indicates a very strong antiferromagnetic coupling with Weiss constant of -240 K as depicted in
Fig. 2 and 3.
Similarly, in 2, χ increased rapidly below 50 K till it reaches 3K. A very feeble bifurcation
m
around 60 K was observed in the ZFC-FC magnetization data (Fig. 2, inset) which implicated the
presence of extremely weak ferromagnetic interaction between the Cu(II) ions in the cubane
3
complex. The observed χ
m
T value at room temperature (300 K) was found to be 3.2 cm /mol.K
which almost remained constant till the temperature decreased to 100 K and then after a short
3
increase the χ
m
T value decreased sharply to reach the value of 2.6 cm /mol.K at 3 K. The 1/χ
m
versus T plot in the 2 indicated that this complex obeyed the Curie-Weiss law in the whole
measured temperature range and the Weiss constant was found to be ∼-1 K showing an
extremely weak interaction.

Fig. 2 The plot of molar susceptibility χ
applied external magnetic field for Cu
lower temperature showing very feeble bifurcation between ZFC-FC magnetization curves.
m
versus temperature from 3 to 300 K with 100 Oe
4
O
4
clusters 1 and 2. The inset shows the zoomed view at
Fig. 3 M-H data at temperature 100 K for Cu
4
4
O clusters 1 and 2. No coercivity was observed at
1
00 K and magnetization follows a linear relationship with the externally applied field.

3
.6. Preliminary DNA binding studies
DNA is often a pharmacological target of most of the known anticancer drugs used for the
treatment of solid cancers [27]. DNA-metal complex interactions play a significant role to dictate
the possible mechanism of cell inhibition of drug entities and to evaluate the potential of
chemical entities to act as drug candidates [28]. The binding of the complexes to CT-DNA/RNA
and proteins is a pre-criteria for drug design and more insight into the cell death mechanism can
be obtained by other enzymatic and cytotoxicity assays. We have studied copper cubane cluster 1
and 2 by many biophysical techniques viz. absorption, fluorescence, circular dichroism and
thermal denaturation to validate their potential as drug entities. Briefly, we have summarized
these preliminary studies in this section.
The binding propensity of 1 and 2 with CT-DNA was examined by employing absorption
-4
spectroscopy. After the successive addition of CT-DNA concentraions (0.0 – 2.0 × 10 M) to a
-5
*
Cu
4
O cubane clusters fixed concentrations (6.67 × 10 M), the π-π intraligand absorption band
4
at 280 nm exhibited ‘hyperchromism’ along with a pertinent blue shift of 8-12 nm whereas
LMCT band at 375 nm exhibited ‘hypochromism’ Fig. S12 and S13.
The strong hyperchromic effect (58 % and 62 % in 1 and 2, respectively) whereas feeble %
hypochromic effect of 24 % (1) and 30 % (2), revealed favorable electrostatic interaction of
cationic polymetallic core to oxygen of sugar-phosphate (negatively charged) backbone of the
DNA helix, as well as with the lively contribution of aromatic chromophores thorugh partial
intercalation [29]. The cubane cluster topology gave a distinct isosbestic point at 299 nm in the
presence of 1 and 2 indicative of equilibrium between the interacted Cu
double helix. Further, qualitative estimation of binding strengths of 1 and 2 with CT-DNA was
established by the K values (intrinsic binding constant), obtained through Wolfe-Shimmer
4
4
O clusters and DNA
b

equation [30]. The intrinsic binding constant K values for complex 1 and 2 were found to be
b
4
−1
4
−1
1
.48 ± 0.14 ×10 M and 2.54 ± 0.11 × 10 M , respectively, implicating strong binding affinity
of Cu clusters with CT−DNA.
The fluorescence spectra of 1 and 2 exhibited a detectable fluorescence at ca. 350 nm when λex
80 nm. Further enhancement of the fluorescence was observed on substantial addition of CT-
4
O
4
=
2
-4
DNA (0.0 – 1.8 × 10 M) without any significant emission maxima shift (Fig. S14).
The binding strength of 1 and 2 with CT-DNA was determined by binding constant (K) values
4
-1
4
-1
via Scatchard’s equation [31] and were 4.41 ± 0.17 × 10 M and 8.78 ± 0.12 × 10 M ,
respectively. The two-fold larger extent of binding propensity of 2 in comparison to 1 has been
warranted by studying the frontier molecular orbital (HOMO and LUMO) analysis acquired
through B3LYP/DFT calculations. Earlier literature accounts have established the evidence that
the large HOMO energy of DNA and small LUMO energy of the molecules in contact directs
quite higher binding affinity, since “electronic charge density” relocate with ease from DNA
molecules (HOMO) to LUMO of molecule interacts and thus, a stronger interaction between
DNA and the molecule happens [32]. Therefore, it was established that lower LUMO energy of
the complex greater would be the binding affinity and here, complex 2 has lower LUMO energy
(
-1.60 eV) in comparison to complex 1 (-1.39 eV) as depicted in Fig. 4.

Fig. 4 Isodensity surfaces (isovalue 0.03) for the MOs of Cu
4
4
O clusters 1 and 2 were produced
from the Kohn-Sham orbitals.
EB alone emits weak luminescence but in the presence of DNA, the EB-DNA complex emits
strong luminescence that is because of intercalation of EB molecule planar phenenthridinium
ring in between the adjacent base pairs of DNA. When 1 and 2 were added to DNA pretreated
with EB, the induced emission intensity at 596 nm was quenched, Supporting Information, Fig.
S15. The quenching of the fluorescence intensity upon the addition of the second molecule
(
complex 1 or 2) was either due to the replacement of the bound EB or damage of the secondary
structure of DNA. Since EB was not completely displaced, electrostatic interaction mode in

addition to the partial intercalation cannot be ruled out. Moreover, fluorescence quenching extent
quantification was revealed by the Stern-Volmer equation [33]. The Ksv value for Cu clusters
and 2 was 2.42 ± 0.09 × 10 M and 4.13 ± 0.06 × 10 M , respectively following a similar
trend as observed in K values.
The circular dichroic studies were employed to study the conformational changes observed due
to Cu cubane clusters –DNA exchanges. The CD spectrum of CT-DNA exhibits
characteristic B-type signature due to base stacking in the UV spectra at 276 nm (+ve band, UV:
max, 260 nm, CD [medg] 0.9602)) and 245 nm (-ve band, CD [medg] -0.8893) due to the B-
4
O
4
4
-1
4
-1
1
b
4
O
4
λ
DNA right-handed helicity (with a zero-crossover around 254 nm) which are quite sensitive to
describe the mode of binding [34]. Upon successive addition of 1 and 2, the +ve band intensity
get reduced, (Fig. S16) which ascertain that 1 and 2 unwinds the DNA double helix and losses
the helicity. The closed cubane cluster 2 showed more reduction in intensity of the CD band than
1
at the same concentration thus confirmed that topology of 2 (closed cubane clusters) is more
efficient in disturbing the structure of DNA.
The amount of double-helical DNA strands transformed to single strands reduces to half at a
particular temperature is known as the melting temperature “T ”. The unfolding of ds-DNA into
m
ss-DNA determined by observed “hyperchromism” in the electronic absorption. At 260 nm, the
molar extinction coefficient of ss-DNA is greater in comparison to ds-DNA [35]. The range of
∆
T
m
lies in between the value tempted for intercalative and electrostatic mode of binding. The
intercalation stabilizes the DNA through π-π stacking that leads to substantial rise in the T of
DNA, whereas the groove binding/electrostatic mode brings an slight deviation i.e. of 1-3 ˚C.
36].
m
[

For free CT-DNA, the T
presence of 1 and 2 were 84 ± 0.5 and 88 ± 0.5 ˚C. The observed ∆T
similar to another groove binder along with the partial intercalators [37]. The melting profile of
DNA in absence and presence of Cu cubane clusters 1 and 2 is depicted in Fig. S17.
.7. DNA cleavage studies
m
value was 80 ± 0.5 ˚C. The melting temperature (T
m
) values in the
m
value of 4-8 ˚C was
4
O
4
3
To evaluate the DNA cleavage efficiency of Cu
was treated with different concentrations of 1 and 2 in 5 mM Tris-HCl/NaCl buffer at pH 7.4 for
h without adding any reducing agents. With concomitant increase of 1 and 2, Form I i.e.,
supercoiled form (SC) gets converted gradually into Form II i.e., nicked circular form (NC)
Lane 2-8 for 1 and 9-15 for 2). At 35 µM concentration of 1 and 2 (Lane 6 for 1 and 11 for 2),
Form I was tranformed to Form II deprived of the formation of Form III i.e., linear circular form
LC) (Fig. 5). Thus, confirms the single-strand DNA cleavage by the complexes1 and 2 [38].
The mechanistic pathway of cleavage of DNA by complexes 1 and 2 was studied being with the
activators viz. hydrogen peroxide (H ), 3-mercaptopropionic acid (MPA), ascorbic acid
ASC), and glutathione (GSH). The DNA cleavage in presence of activators was significantly
enhanced and follows order H > GSH > MPA > ASC (Fig. S18). Moreover, to enlighten the
4
O
4
cubane clusters 1 and 2, the plasmid DNA
1
(
(
2
O
2
(
2
O
2
DNA cleavage mechanism, the interaction of 1 and 2 were studied in the company of ROS
scavenger’s viz. DMSO (dimethyl sulfoxide) and EtOH (ethanol) as hydroxyl radical scavengers,
NaN (sodium azide) as singlet oxygen quencher and SOD (superoxide dismutase) as superoxide
3
scavengers (Fig. S19). From the electrophoretic pattern, it was perceived that DMSO and EtOH
do not exhibits any deceptive inhibition of the DNA, thus rules out the prospect of diffusible
hydroxyl radical’s involvement in cleavage process. However, in addition of NaN noteworthy
3

1
inhibition was noticed which confirms the presence of singlet oxygen, O
2
. Thus, it confirms that
DNA cleavages via oxidative path [39].
To further endorse binding of 1 and 2 with DNA, the cleavage reactions with, 4',6-diamidino-2-
phenylindole (DAPI), a minor groove binder and methyl green (MG), a major groove binder,
were studied (Fig. S19). No inhibition with DAPI was noticed while substantial inhibition of
DNA cleavage with methyl green was observed point to the binding of Cu
4
4
O cubane clusters to
the major groove of DNA. These results were further corroborated via molecular modelling
studies.
Fig. 5 Gel electrophoresis assay of Cu
4
4
O clusters 1 and 2 with pBR322 DNA in 5 mM Tris-
HCl/NaCl buffer (pH, 7.4) after 1h incubation time. (a) Lane 1: DNA control; Lane 2: 5 µM 1 +
DNA; Lane 3: 10 µM 1 + DNA; Lane 4: 15 µΜ 1 + DNA; Lane 5: 20 µM 1 + DNA; Lane 6: 25
µM 1 + DNA, Lane 7: 30 µM 1 + DNA, Lane 8: 35 µM 1 + DNA. (b) Lane 9: 5 µM 2 + DNA;
Lane 10: 10 µM 2 + DNA; Lane 11: 15 µΜ 2 + DNA; Lane 12: 20 µM 2 + DNA; Lane 13: 25
µM 2+ DNA, Lane 14: 30 µM 2+ DNA, Lane 15: 35 µM 2+ DNA.
3
.8. Molecular docking
To rationalize the topology of the specific binding interaction of Cu
4
4
O cubane clusters with
DNA, the blind molecular docking was done, to simulate the interaction mode. In the present
study, the structures of 1 and 2 were retained flexible to attain various conformations to predict
best feasible minimum energy docked complex. The study revealed that major groove binding
makes intimate contacts with the groove surface and as an outcome of this interaction numerous

electrostatic and van der Waals interactions occur between Cu
4
4
O cubane clusters and DNA
bases and its sugar-phosphate backbone. By ensuing docked structures (Fig. 6-8), it is evident
that both the cubane clusters 1 and 2, slightly twists the interior hydrophobic surface of DNA in a
way to make favorable contacts, the planar part of the aromatic rings stack between DNA base
pairs and flexible alkyl alcohol arms lead to van der Waals and hydrophobic interactions with the
outer surface of DNA, which are responsible for the stability of groove, detail are tabulated in
Table S6. The relative binding propensity of docked structures was 1 (-4.8 kcal/mol) and 2 (-5.2
kcal/mol). Higher negative binding energy indicates a more stable association with DNA. Thus,
it is concluded that the binding ability of 2 with the major groove of DNA is stronger than that of
1
.
Fig. 6 Molecular docked structures of (a) Cu
4
O
4
cluster 1, and (b) Cu
4
4
O cluster 2 in the cavity
of major groove of DNA.

Fig. 7 Binding site non-covalent interactions minimum energy conformation of (a) Cu
and (b) Cu cluster 2 with major groove residues.
4
4
O cluster
1
4
O
4

Fig. 8 Minimum energy docked pose of conformational binding site interaction differences
between Cu clusters 1 and 2 in the major groove of the DNA.
4
O
4
3
.9. Cytotoxicity activity
The cytotoxicity of copper cubane clusters 1 and 2 were tested on two human cancer cell lines,
HepG2 and MCF-7 in concentration dependent fashion of cubane complexes (5–50 µM) by
exposing cell for 24 h by MTT assay. Interestingly, our results demonstrate that 1 and 2
deliberated significant cytotoxic activity (IC50 value 20 and 17 µM, respectively) selectively
towards MCF-7 breast cancer cell line. On the other hand, cytotoxicity activity of 1 and 2 on
HepG2 cell line was moderate (IC50 value 35 and 30 µM, respectively). The changes observed in
the morphology of the cell lines in MCF-7 and HepG2 cells on treatment with 1 and 2 after
incubation 24 h are shown in Fig. 9.

(a)
Control MCF7 cell
Complex 1
(b)
Control HepG2 cell
Complex 2
Fig. 9 (a) Morphological changes in MCF7 cells and (b) HepG2 cells, following the exposure of
Cu clusters 1 and 2 for 24 h. Images were taken under the phase contrast inverted microscope
4
O
4
at 20x magnification.
The morphology of MCF-7 and HepG2 cells on treatment with cubane clusters was found to be
altered when observed in phase contrast inverted microscope. Upon exposure of compound to
MCF-7 and HepG2 cells, there is a severe drop in the standard morphology and the cell adhesion
capacity was noticed as compared with the control in concentration dependent pattern. However,
the cytotoxicity of the compounds towards MCF-7 breast cancer cells is quite significant
attributable to the particular target-oriented selectivity that can be ascribed to the ligand and the

Cu
facilitated [40].
.10. ROS generation in cell apoptosis
4
4
O metallic core high electron density, by which the DNA-binding propensity also gets
3
It is well known that metallo-anticancer drugs, mainly copper complexes, exhibit their anticancer
prpoperty because of reactive oxygen species (ROS) generation, viz. superoxide anion radical
•−
•
(
O ), and hydroxyl radical (OH ) [41]. Generally, Cu(I) ions reduces hydrogen peroxide to form
2
•
•−
hydroxyl radical (OH ) whereas Cu(II) ions get reduced to Cu(I) by the superoxide anion (O )
2
•
or glutathione (GSH). The formation of ROS viz. OH is copper compelled, irrespective of the
oxidation state of copper hosted inside the body. The existence of additional intracellular ROS
could lead to severe DNA damage in cells. The intracellular ROS generation extent in MCF-7
cells was observed upon treatment of 1 and 2 and by monitoring fluorescence intensity of
DCFH-DA. The results showed triggered ROS generation and the pattern exhibited was also
dose-dependent (Fig. 10), which confirmed that ROS generation and their role in the cell
apoptosis.
Complex 1
Complex 2
Control
Fig. 10 ROS generation in MCF-7 (breast cancer cell lines) after the treatment of 20 µM of the
complexes 1 and 2 for 24 h.
3
.11. Intracellular glutathione depletion

For the oxidative stress inside the cells, an intracellular antioxidant glutathione (GSH) is present
for protection. In vivo, Cu(II) ions get reduced to Cu(I) by GSH / superoxide anion (O2•−). A
specific copper transporter (hCtr) is known to take Cu(I) inside the cell. This enables the
independent penetration of copper complexes, without the need of any binding to any other
agent. To find out the role of oxidative stress in the cytotoxicity, the impact on intracellular GSH
level is an important parameter. It is well known that the effect of Cu(II) complex produces a
noteworthy drop in GSH level. The GSH/GSSG ratio affects the regulation of cell cycle, DNA
synthesis, and mutagenic processes in cancer cells. The GSH level is found to be in the higher
range in cancer cells in comparison to normal cells. The GSH level of 1 and 2 at concentrations
of 15 and 20 µM was quantified in MCF-7 cell lines after exposure for 24 h. The results are
depicted in the histogram (Fig. 11). The depletion in the level of GSH was found upon treatment
of 1 and 2 which was attributed to the presence of four Cu(II) ions in Cu
4
4
O cubane core
clusters. Interestingly, 2 which is a closed cubane cluster and possess relatively less energy
separation in HOMO and LUMO ( as evidenced by DFT) exhibited more significant results and
showed the reduced intracellular GSH level to 45% at a concentration of 20 µM, thus endorsing
the important role of oxidative stress by cubane clusters.