DNA binding and cytotoxicity of some Cu(II)/Zn(II) complexes containing a carbohydrazone Schiff base ligand along with 1,10-phenanthroline as a coligand

Article abstract of DOI:10.1016/j.ica.2017.06.068

We report the synthesis, characterization and biological activities of the complexes [Cu(o-phen)HL] (1), [Cu(o-phen)LCu(OAc)] (2), and [Zn(o-phen)LCu(OAc)] (3), where, H₃L?=?o-HOC₆H₄C(H)[dbnd]N–NH–C(OH)[dbnd]N–N[dbnd]C(H)–C₆H₄OH-o, o-phen?=?1,10-phenanthroline, and OAc?=?CH₃COO^?. The free ligand and compound 3 have been characterized by X-ray crystallography. A four-line EPR pattern originating from the interaction of the unpaired electron with the central ^63/65Cu nucleus (I?=?3/2) with the isotropic coupling constant (A_iso) value of 80?±?1.5?G at RT for compound 1 in DMF suggests its monomeric nature in solution. These compounds undergo irreversible oxidation-reduction. Biological studies show intercalative DNA binding and remarkable cell cytotoxicity as well as anticancer activity. The IC₅₀ values of 1, 2 and 3 for the human lung cancer A549 cell line (0.44, 0.22, and 4.80?μM, respectively) and for the breast cancer MCF7 cell line (2.7, 4.1, and 3.6?μM, respectively) are found to be very promising and appear to be more potent than some anticancer drugs tested for these cell lines. Most importantly, 3 is found to be remarkably less toxic for HaCaT and L132 normal cell lines as evident from the cell viabilities of these two cell lines in presence of this compound.

Full text of DOI:10.1016/j.ica.2017.06.068

Accepted Manuscript
Research paper
DNA binding and cytotoxicity of some Cu(II)/Zn(II) complexes containing a
carbohydrazone Schiff base ligand along with 1,10-phenanthroline as a coligand
Manjuri K. Koley, Natarajan Duraipandy, Manikantan Syamala Kiran, Babu
Varghese, Periakaruppan T. Manoharan, Aditya P. Koley
PII:
S0020-1693(17)30679-5
http://dx.doi.org/10.1016/j.ica.2017.06.068
ICA 17717
DOI:
Reference:
Inorganica Chimica Acta
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Received Date:
Revised Date:
Accepted Date:
30 April 2017
25 May 2017
28 June 2017
Please cite this article as: M.K. Koley, N. Duraipandy, M.S. Kiran, B. Varghese, P.T. Manoharan, A.P. Koley, DNA
binding and cytotoxicity of some Cu(II)/Zn(II) complexes containing a carbohydrazone Schiff base ligand along
with 1,10-phenanthroline as a coligand, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.
2017.06.068
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DNA binding and cytotoxicity of some Cu(II)/Zn(II) complexes containing a
carbohydrazone Schiff base ligand along with 1,10-phenanthroline as a coligand
Manjuri K. Koleya,*, Natarajan Duraipandyb, Manikantan Syamala Kiranb, Babu Varghesec,
Periakaruppan T. Manoharand,* and Aditya P. Koleye,*
a Department of Chemical Engineering, Birla Institute of Technology and Science-Pilani, K.K. Birla
Goa Campus, Goa 403726, India
b Biological Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai 600
020, India
c Sophisticated Analytical Instrument Facility, Indian Institute of Technology-Madras, Chennai 600
036, India
d Department of Chemistry, Indian Institute of Technology-Madras, Chennai 600 036, India
e Department of Chemistry, Birla Institute of Technology and Science-Pilani, K.K. Birla Goa
Campus, Goa 403 726, India
________________________________________________________________________________
Abstract
We report the synthesis, characterization and biological activities of the complexes [Cu(o-phen)HL]
(1), [Cu(o-phen)LCu(OAc)] (2), and [Zn(o-phen)LCu(OAc)] (3), where, H3L = o-
HOC6H4C(H)=N−NH−C(OH)=N−N=C(H)−C6H4OH-o, o-phen = 1,10-phenanthroline, and OAc =
CH3COO−. The free ligand and compound 3 have been characterized by X-ray crystallography. A
four-line EPR pattern originating from the interaction of the unpaired electron with the central 63/65Cu
nucleus (I = 3/2) with the isotropic coupling constant (Aiso) value of 80 1.5 G at RT for compound 1
in DMF suggests its monomeric nature in solution. These compounds undergo irreversible oxidation-
reduction. Biological studies show intercalative DNA binding and remarkable cell cytotoxicity as
well as anticancer activity. The IC50 values of 1, 2 and 3 for the human lung cancer A549 cell line
(0.440, 0.220, and 4.80 µM, respectively) and for the breast cancer MCF7 cell line (2.7, 4.1, and 3.6
µM, respectively) are found to be very promising and appear to be more potent than some anticancer
drugs tested for these cell lines. Most importantly, 3 is found to be remarkably less toxic for HaCaT

2
and L132 normal cell lines as evident from the cell viabilities of these two cell lines in presence of
this compound.
_______________________________
Keywords: Cu(II)/Zn(II) complex; EPR and electronic spectra; DNA binding; MTT assay;
Cytotoxicity and anticancer activity
_______________________________
*Corresponding authors. Email: manjuri@goa.bits-pilani.ac.in (M. K. Koley); ptm@iitm.ac.in (P.T.
Manoharan); koleyap@yahoo.co.in (A.P. Koley)
1. Introduction
In recent years there have been increasing efforts to design and develop metal complexes that show
DNA binding property, nucleic acid cleavage activity as well as cytotoxicity in order to explore the
possibility of finding new metallodrugs that may serve as anticancer agents [1−13]. Many of these
works [14−22] are based on bio-essential metal ions, Zn(II) and Cu(II) in particular since these are
the second and third most abundant transition metal ions that are present in the cellular body after
iron. DNA is an important target for anticancer drugs and the nature of the ligand present in the
metallodrugs plays an important role in the binding of the metal complex to DNA (or protein) [23,
24]. Metal complexes with extended planar aromatic ligands bind to DNA in a non-covalent [25−27]
manner, either through intercalative or partial intercalative binding, but metal complexes with non-
planar ligands are normally found to bind to DNA but not by intercalation. The metal ion also may
play an important role in the efficacy of the metal complex as a drug. Cu(II) being a redox active
metal ion may promote transformation of DNA or protein through an oxidative pathway whereas
Zn(II), being redox inactive is expected to operate in a different pathway [15b]. In our continuing
interest and effort in designing and developing metal complexes with suitable donor ligands with
additional heterocyclic N-donors [28], that are capable of effectively binding and cleaving DNA, we
have synthesized a few Cu(II)/Zn(II) complexes with a carbohydrazone ligand because
carbohydrazones (and also thiocarbohydrazones) are known to be a class of fascinating ligand

3
systems in the field of coordination chemistry with their versatile structural conformations and
possess several donor sites [29]. Here, we present the synthesis, spectral (ESI-MS, UV-visible, IR,
and EPR), electrochemical and the biological activities of three compounds of Cu(II)/Zn(II)
containing the enol form of the Schiff base ligand 1,5-bis(salicylidene)carbohydrazide and 1,10-
phenanthroline as a coligand.
2. Results and discussion
We have studied the reactions of Cu(OAc)2⋅H2O and Zn(OAc)2⋅2H2O with 1,5-Bis(salicylidene)-
carbohydrazide (A) in methanol to prepare the complexes of the corresponding Schiff base B or B′ as
shown in Fig.1 in its enol form in 1:1 or 2:1 (M:L) ratio to prepare the corresponding mononuclear or
dinuclear complexes, respectively. The ligand in its enol form can exist either in syn or anti with two
distinctly different coordination sites [29]. The site 1 is characterized by one phenolate O−, one
enolate O− and one imine N, while the site 2 is characterized by one phenolate O−, one imine N, and
another secondary NH donor atom. The different forms of the ligand are shown in Fig. 1.
Fig. 1. Structure of the ligand in its keto and enol forms.

4
The complexes are poorly soluble in common organic solvents like methanol, ethanol, acetonitrile or
dichloromethane but readily soluble in DMSO and DMF. Conductivity measurements with fresh
solutions of the compounds 1 - 3 in DMF show these are nonelectrolyte in nature. The ESI-MS
spectra (in the positive-ion mode) for complexes 1–3 have been recorded in DMSO and the results
are presented in Fig. S1 (Supplementary material). Complex 1 showed peaks at m/z 540.33 [M]+,
m/z 579.19 [M + K]+, compound 2 showed peak at m/z 701.77 [M + K + H]+ while complex 3
showed peak at m/z 701.70 [M + K]+, respectively. Based on their physico-chemical properties,
elemental analyses, mass spectra and X-band RT solution EPR results (vide infra) the proposed
structures of 1-3 are shown in Fig. 2.
Fig. 2. Proposed structures of the compounds 1-3.
The IR spectrum of the free ligand (figure not shown) clearly displays the ν(C=O) at 1690 cm−1 and
ν(C=N) at 1625 cm−1, respectively, clearly suggesting it exists in its keto form [29] and is confirmed
from its crystal structure (vide infra). The ν(C=O) band at 1690 cm−1 is found to be absent in all three

5
metal complexes and the ν(C=N) of the free ligand at 1625 cm−1 is shifted to 1605 cm−1 in the IR
spectra of all these complexes indicating the coordination of the imine N to the metal ion. This is
confirmed from the crystal structure of 3. The IR spectra of compounds 2 and 3 in the region 4000-
400 cm−1 are found to be very similar (Fig. S2, Supplementary material), indicating their structural
similarities and similar ligand environments but the IR spectra of 1 and 2 (or 3), as expected, are
different (figure not shown). This difference is originating from the absence of metal ion along with
the acetate group in the second site in compound 1. For example, a sharp peak observed at 3266 cm−1
for 1 which is most likely originating from ν(N−H) is found to be absent in the IR spectrum of 2.
Similarly, the peak observed for 2 at 1545 cm−1, possibly associated with the coordinated acetate
group is absent in the spectrum of 1. It is to be mentioned here that there is another possibility for
compound 1 having an alternate structure [29] with slight difference in coordination or ligand
environment as shown in 1B of Fig. 2. However, it is difficult to differentiate among these two
structures (1A or 1B) at this stage in the absence of any crystal structure, though we suggest the
possibility of having structure 1A based on the analogy with the crystal structure of 3. Moreover, the
differences in the electronic structures of compounds 1-3 are clearly reflected from their electronic
spectra shown in Fig. S3 (Supplementary material) as well as from their EPR spectra (vide infra).
These compounds exhibit strong LMCT in the visible region, however, the weak ligand field
transitions are observed in the 750-550 nm region as evident from Fig. S3 (Supplementary material).
Interestingly, these complexes exhibit significant catalytic activity toward the oxidation of catechol
in DMSO medium in presence of air. The catecholase activity of the complexes 1 and 2 were studied
through the oxidation of model substrate 3,5-di-tert-butyl-catechol (3,5-DTBC) under excess
substrate conditions. The oxidation product 3,5 -di-tert-butylquinone (3,5-DTBQ) has a
characteristic absorption band maxima at ~ 401 nm . The electronic spectral change due to the
formation of 3,5-DTBQ from the catalytic oxidation of 3,5-DTBC in presence of 1 and 2 were
monitored with time and observed spectral changes are shown in Fig. S4 (Supplementary material).

6
Fig. 3 shows the plot of absorbance change at ~401 nm for the formation of 3,5-DTBQ with time
from the catalytic oxidation of 3,5-DTBC in presence of air with 1 in DMSO [0.90 × 10−4 M] (blue
circles), 2 in DMSO [0.72 × 10−4 M] (black squares), and also 2 in DMSO:H3OH (1:9) [0.75 × 10−4
M] (red triangles) and keeping the 3,5-DTBC concentration same [1.10 × 10−2 M] for all three cases.
It is evident from Fig. 3 that the catalytic activity of 1 in DMSO toward 3,5-DTBC oxidation in
presence of air is slower than that of 2 in DMSO (keeping all other parameters same) based on the
rate of absorbance change near 401 nm (Fig. S4, Supplementary material) for the formation of 3,5-
DTBQ as reflected in Fig. 3. However, this catalytic oxidation process is found to be much faster and
reached the steady state within 10 h when the same reaction was carried out in 9:1 methanol:DMSO
medium keeping other parameters same, clearly showing the role of the solvent on their reactivity.
Fig. 3. Plot of absorbance change at ~401 nm for the formation of 3,5-DTBQ from the catalytic
oxidation of 3,5-DTBC in presence of air with (1) 0.90 × 10−4 M 1 and 1.10 × 10−2 M 3,5-DTBC in
DMSO (blue circles), (2) 0.72 × 10−4 M 2 and 1.10 × 10−2 M 3,5-DTBC in DMSO (black squares),
(3) 0.75 × 10−4 M 2 and 1.10 × 10−2 M 3,5-DTBC in CH3OH : DMSO (9:1) (red triangles).

7
2.1. Crystal structures of 1,5-Bis(salicylidene)carbohydrazide, (o-HOC6H4CH=NNH)2C=O and
[Zn(o-phen)LCu(OAc)]0.5H2O⋅0.5CH3OH
The structure of 1,5-bis(salicylidene)carbohydrazide and compound 3 have been determined by X-
ray crystallography. Fig. 4 shows the ORTEP representation of the molecule 1,5-bis(salicylidene)-
carbohydrazide with 40% probability ellipsoids [30]. The crystal is indexed in orthorhombic system
with space group 'Aba2' and lattice parameters a = 14.5381(18) Å, b = 9.3483(11) Å, c =
10.2927(11) Å, α = 90ο, β = 90ο, γ = 90ο. The bond length and bond angles are given in Table S1 of
the Supplementary material. The very symmetrical structure (Fig. 4) of this compound with both
same C(8)-N(2) bond distances of 1.365(4) Å clearly suggest its keto form when not coordinated to
metal ion. The packing of the molecules in the unit cell is shown in Fig. S5 (Supplementary
material).
Fig. 4. ORTEP diagram of the compound 1,5-bis(salicylidene)carbohydrazide.
On the other hand, compound 3 crystallizes in triclinic system with the space group Pī. Fig. 5 shows
the ORTEP representation of the molecule with 40% probability ellipsoids [30] and packing of the
molecules is shown in Fig. S6 (Supplementary material). There are two molecules in the asymmetric
unit along with one molecule of methanol and one molecule of water (Fig. 5B). Methanol and water
molecules are two-fold disordered (i.e., 50% occupancy each). Cu atoms of both molecules (Cu1 and
Cu2) have near square-planar coordination with two nitrogen atoms, one phenolate O− and one
acetate O− donors (N4N6O3O4 for Cu1 and N10N12O8O9 for Cu2), there is a fifth weak
coordination for Cu from the second acetate oxygen (Cu1−O4 = 2.646 (4) Å, Cu2−O10 = 2.668(6)

8
Å). The mean plane calculation of the basal square plane coordination shows the coordinating N and
O atoms to have small tetrahedral distortion (from the ideal square plane). However, the Cu atoms
stay in the mean plane (no deviation from the mean plane). Both Zn atoms are five-coordinated. The
geometry of coordination is approximately square pyramidal with N1O1N3O2 atoms forming the
basal plane for Zn1 and N7O7N9O6 forming the basal plane for Zn2. Both the Zn atoms are slightly
shifted in the direction of fifth coordination (from the basal mean plane, domed toward the fifth
ligand). All the hydrogens could be located in the difference Fourier map. However, it was decided
to fix the hydrogens at geometrically meaningful positions. These atoms were refined by riding
model. The packing of molecules in the unit cell shows solvent mediated H-bonding interactions
stabilizing the lattice. It is evident from the bond distances of C(20)-N(5) = 1.27(3) Å and C(20)-
N(4) = 1.38(3) Å, respectively that the Schiff base ligand is no longer symmetrical after its
enolization and coordination to the metal ion(s). Also there are significant differences in bond
lengths of C(21)-N(6) = 1.27(3) Å and C(19)-N(3) = 1.36(3) Å, suggesting the differences in the two
metal binding sites.
Fig. 5. ORTEP diagram of the compound Zn(o-phen)LCu(OAc)⋅0.5H2O⋅0.5CH3OH . Selected bond
lengths (Å) and angles (ο): N(1)-Zn(1), 1.97(3); N(2)-Zn(1), 2.21(3); N(3)-Zn(1), 1.88(3); O(1)-
Zn(1), 1.91(2); O(2)-Zn(1), 2.009(18); N(4)-Cu(1), 1.98(2); N(6)-Cu(1), 1.96(2); O(3)-Cu(1),

9
1.83(2); O(4)-Cu(1), 1.98(2); N(7)-Zn(2), 1.99(2); N(8)-Zn(2), 2.24(2); N(9)-Zn(2), 1.86(2); O(6)-
Zn(2), 1.92(2); O(7)-Zn(2), 2.003(19); N(10)-Cu(2), 1.93(2); N(12)-Cu(2), 1.99(2); O(8)-Cu(2),
1.84(2); O(9)-Cu(2), 1.95(2); N(3)-Zn(1)-O(1), 95.6(11); N(3)-Zn(1)-N(1), 166.7(11); O(1)-Zn(1)-
N(1), 97.5(10); N(3)-Zn(1)-O(2), 79.9(10); O(1)-Zn(1)-O(2), 161.8(8); N(1)-Zn(1)-O(2), 87.0(9);
N(3)-Zn(1)-N(2), 97.5(10); O(1)-Zn(1)-N(2), 112.4(9); N(1)-Zn(1)-N(2), 79.4(10); O(2)-Zn(1)-N(2),
85.7(8); N(9)-Zn(2)-O(6), 96.3(11); N(9)-Zn(2)-N(7), 166.4(11); O(6)-Zn(2)-N(7), 96.5(9); N(9)-
Zn(2)-O(7), 78.0(10); O(6)-Zn(2)-O(7), 162.5(9); N(7)-Zn(2)-O(7), 88.4(9); N(9)-Zn(2)-N(8),
99.6(10); O(6)-Zn(2)-N(8), 110.6(9); N(7)-Zn(2)-N(8), 80.0(10); O(7)-Zn(2)-N(8), 86.8(8); O(3)-
Cu(1)-N(6), 90.0(10); O(3)-Cu(1)-N(4), 167.6(9); N(6)-Cu(1)-N(4), 81.4(10); O(3)-Cu(1)-O(4),
94.2(9); N(6)-Cu(1)-O(4), 169.9(9); N(4)-Cu(1)-O(4), 95.7(9); O(8)-Cu(2)-N(10), 166.8(9); O(8)-
Cu(2)-O(9), 93.3(9); N(10)-Cu(2)-O(9), 95.8(9); O(8)-Cu(2)-N(12), 90.4(10); N(10)-Cu(2)-N(12),
82.1(10); O(9)-Cu(2)-N(12), 170.8(9).
2.2. EPR results
The powder EPR spectra for the compounds 1-3 were recorded at room temperature (RT) as well as
at liquid nitrogen temperature (LNT) at X-band frequency and are shown in Fig. 6. 1 at RT has a
broad line at a g-value of 2.08 (an averaged out g–value) and also a weak forbidden line around 155
mT indicating that it is possibly a dimer. But its EPR at LNT, i.e. 1 at LNT not only shows the
forbidden ∆Ms = 2 line at around the same field but also its main g-region is resolved though not
clearly. One can possibly account for a seven line not-so-well resolved EPR spectrum in the parallel
component, but g┴ does not show any hyperfine line because of line broadening and low value
expected for the hyperfine coupling constants in this direction. The EPR spectra of 2 at RT and at
LNT almost appear to be the same as the corresponding 1 spectrum. The presence of forbidden ∆Ms
= 2 line around g~4 and a reduced hyperfine coupling constant value for Cu63,65 make both of them
resulting from a dimer of a Cu-Cu interaction. While this is true for the molecule of 2, it is not

10
possible for 1 to have such an interaction since there is no such intra-molecular dimer moiety as part
of its structural characterization which has only one copper. However, if the molecular packing
involves a dimer from two different molecules, then it can also give such an exchange interaction.
Fig. 6. X-band EPR spectra of the compounds. (a)-(c) are RT spectra for 1-3 powder samples, (d)-(f)
are LNT spectra for 1-3 powder samples, (g)-(i) are RT solution spectra for 1-3 in DMF, and (j)-(l)
are LNT frozen glass spectra for 1-3 in DMF, respectively. * indicates the existence of the forbidden
∆Ms = 2 line around g~4.
On the other hand, 3 at both the RT and LNT do not show either any forbidden line or hyperfine
lines. It is understandable from the structure of 3 that the interacting centres involve Cu-Zn instead
of Cu-Cu in 2. Possibly due to line broadening effects due to dipolar interactions in solid state 3 has

11
just given a broad line both in parallel and perpendicular directions. RT solution EPR shows
compound 1 is a monomer of Cu2+ and the not so well resolved isotropic hyperfine lines from copper
nuclei with Aiso being approximately 8 mT with g value around 2.06, typical of a monomeric Cu2+
squareplanar or at the most C2V symmetry. The same compound in DMF at LNT gives A║ value of
~20 mT with g║ = 2.17 and g⊥ = 2.04, respectively. But the same at RT and LNT give similar line
but broadened to a whopping width of ~36mT due to dipolar interaction. Compound 2 in DMF,
likely to be a dimer, exhibited spectra both at RT and LNT which are clearly one line spectrum with
a width of about 35 mT at RT and about 10 mT at LNT. Higher intensity at LNT coupled with
sharpening of the line clearly indicates the possibility of its being a weak ferromagnet due to
intramolecular exchange coupling. But in solid state, as already mentioned, at RT it gives a strong
line with a width of ~35 mT and the existence of the forbidden ∆Ms = 2 line around g~4 resulting
from the same intramolecular dimer with exchange coupling due to Cu-Cu interaction. Compound 3
in DMF gives a weak EPR line with unresolved hyperfine at RT but at LNT it becomes a strong line
with resolved hyperfine values specially in the parallel section. Possibly it indicates that most of the
spin is located in Cu2+ probably not leaking to Zn2+. But the powder at RT gives a strong one
anisotropic line with no hyperfine lines. The EPR parameters are presented in the following Table 1.
Table 1. EPR parameters derived from the spectra at RT and LNT.
_________________________________________________________________________________
Compound
g║
g┴
g / gav
A║
∆Ms = 2
_________________________________________________________________________________
1-RT
-
-
2.082
-
1-LNT
2.17
2.040
2.083(gav )
11.5 mT
Yes
Yes
2-RT
-
-
2.092
2-LNT
3-RT
2.19
2.19
2.17
2.10
2.12 (gav )
2.11 (gav)
2.097 (gav)
13.5 mT
2.07
2.06
3-LNT
_________________________________________________________________________________

12
2.3. DNA binding studies
DNA binding studies were performed using UV-Vis spectroscopy. In order to get the binding
constants for the compounds 1-3, electronic absorption titration experiments were performed with
incremental amounts of calf thymus DNA (ct-DNA) in 10 mM Tris–HCl buffer (pH 7.45) and by
keeping the individual complex concentration constant. The binding of complexes to the ct-DNA
exhibits hypochromism (Fig. 7) with a minor red shift of the band at around 394 nm for 1, 395 nm
for 2, and 380 nm for 3, respectively, indicating intercalative binding, resulting in strong stacking
between the DNA base and aromatic chromophore [31]. The binding constant Kb has been calculated
from the kinetic data obtained using the following equation (1).
(1)
where, [DNA] is the concentration of ct-DNA used, and εa, εf and εb correspond to apparent
extinction coefficients for the complex i.e., Abs/[complex] in the presence of DNA, in the
absence of DNA and to fully bound DNA, respectively. A plot of [DNA]/(εa − εf) versus
[DNA] yields a slope = 1/(εb − εf), and the intercept = 1/Kb(εb − εf), respectively. The binding
constant Kb is calculated from the ratio of the slope to the intercept. The binding constant Kb is found
to be 1.18 × 105 M−1 for 1, 2.63 × 105 M−1 for 2, and 2.53 × 105 M−1 for 3, respectively. The Kb
values are comparable to that reported for a classical intercalator such as ethidium bromide (Kb =
1.40 × 105 M−1) in 25 mM Tris-HCl buffer (pH 7.33) [32]. The higher Kb values for 2 and 3 as
compared to 1 suggest that 2 and 3 possessing two metal centres interact with DNA more strongly
than complex 1. Also the Kb values for 2 and 3 are found to be higher than those we reported [28]
recently for the mononuclear compounds [Cu(pabt)(Imz)](ClO4) and [Cu(pabt)(N-MeImz)](ClO4),
(Hpabt = N-(2-mercaptophenyl)-2'-pyridylmethylenimine, Imz = imidazole, N-MeImz = N-

13
methylimidazole) both of which were distorted square planar containing a tridentate NNS− Schiff
base ligand and imidazole or N-methylimidazole as the coligand in the fourth position.
Fig. 7. Change in electronic absorption spectra of 1-3 [5 × 10−5 M] upon titration with ct-DNA (0–40
µM) dissolved in 10 mM Tris–HCl buffer (pH 7.45). The arrow shows the decrease in absorbance
with respect to an increase in the concentration of ct-DNA. The figure in inset shows the linear fit of
[DNA]/(εa − εf ) versus [DNA]. The binding constant (Kb) was calculated using the eqn. 1.

14
2.4. Results on biological activity
2.4.1. Cytotoxicity
The cytotoxicity profiling of the three compounds were evaluated by MTT assay using A549 lung
cancer cell line, MCF7 breast cancer cell line, L132 human lung embryonic normal cell line and
human keratinocyte HaCaT normal cell line. The cell viability was determined by the reduction of
yellow MTT into purple formazan product by the enzymatic activity of mitochondrial dehydrogenase
in the live cells. Dose dependent cell death was induced by all three compounds as evidenced from
Fig. 8. It was observed that for L132 cell line, compound 2 showed high toxicity at concentration as
low as 2.5 µM having cell viability of ∼24% while compound 1 showed moderate effect at 2.5 µM
when compared to compound 2. Compound 3 showed much less toxicity even at concentration as
high as 10 µM concentration with cell viability of ∼55% for the same cell line. Similar result is also
obtained with HaCaT cell line in which ∼65% cell viability is observed at 10 µM concentration of 3,
whereas 1 and 2 were found to be very toxic even at 2.5 µM concentration. In MCF7 cell line, all
three compounds were found to be less toxic at 2.5 µM concentration with cell viability when
compared to L132 cell line indicating that these compounds did not have any specificity against
breast cancer cell. A549 cell line, compounds 1 and 2 are found to be toxic even at 0.5 µM
concentration with cell viabilities of 46% and 39.5%, respectively, while at 2.5 µM concentration the
cell viabilities are 30.9% for 1 and 34.25% for 2, and 62.3% for 3, respectively. At 5 µM
concentration the cell viabilities are 26.6% for 1 and 25.6% for 2, and 49.1% for 3, respectively. At
10 µM concentration the cell viabilities are 22.6% for 1 and 24.5% for 2, and 36.7% for 3,
respectively. The cell viability for 3 at 10 µM concentration was found to be as much as 65% (Fig.
8) in HaCaT cell line. Thus, though compounds 1 and 2 are found to be quite toxic for all these cell
lines, 3 is found to be less toxic for L132 and HaCaT but possesses good potency for MCF7 and
A549 cell lines.

15
Fig. 8. MTT assay for the compounds 1-3. Panel A. HaCaT cell line, panel B. L132 cell line, panel
C. A549 cell line, and panel D. MCF7 cell line.
Fig. 9 shows the plot of cell viability versus concentrations of the compounds 1, 2, and 3 for all these
cell lines. The IC50 values for 1-3 calculated from this plot for both the cancer cell lines A549 and
MCF7 are presented in Table 2. It is to be noted here that the IC50 values for A549 cells are
comparable to those reported by Lu et al. [2c] and better than the IC50 value reported for cisplatin
(4.13 µM for A549). On the other hand, the IC50 values for 1-3 for MCF7 cell line are found to be
higher than that (0.46 µM) reported by Lu et al. [2c] but comparable with that reported for cisplatin
(3.92 µM for MCF7) and much lower than that reported for carboplatin (36.65 µM for MCF7) [2c].

16
Fig. 9. Plot of cell viability versus concentrations of the compounds 1, 2, and 3: panel A. HaCaT cell
line, panel B. L132 cell line, panel C. A549 cell line, and panel D. MCF7 cell line.
Table 2. IC50 values for the compounds 1-3 calculated from the plot of cell viability versus
concentration in the MTT assay.
Compound
IC50 in A549 cell line
IC50 in MCF7 cell
line
1
2
3
0.44 µM
0.22 µM
4.80 µM
2.7 µM
4.1 µM
3.6 µM
2.4.2. Dual-Fluorescence Viability: AO/PI Dual Staining assay for A549 and HaCaT cells
This dual staining assay using the fluorescence microscopy is used to analyze the induction of cell
apoptosis in presence of the compounds 1-3. Acridine orange (AO) and propidium iodide (PI) are
nucleic acid binding dyes, AO is permeable to both live and dead cells, staining all nucleated cells to

17
generate green fluorescence but PI enters only in dead, dying, and necrotic nucleated cells with
compromised membranes and stains them all generating red fluorescence. But cells stained with both
AO and PI together, all dead nucleated cells fluoresce red and all live nucleated cells fluoresce green
due to Förster resonance energy transfer (FRET). In this staining method, the following
morphological changes were observed (Figs. 10 – 12) in the treated cells: (a) the viable cells with
highly organized nuclei fluoresce green as seen from all the control in Figs. 10 – 12; (b) early
apoptotic cells with nuclear condensation, that emit orange-green fluorescence; (c) late apoptotic
cells with highly condensed or fragmented chromatin and fluoresce orange to red, observed in the
treatment with 1 µM concentration of the respective compounds; and (d) necrotic cells fluoresce red
with no indication of chromatin fragmentation as clearly evident (Figs. 10 – 11) in the cases of A549
cells treated with 2 µM of each of 1 and 2, while such red fluorescence is not observed (Fig. 12) with
2 µM concentration for 3 suggesting no or very little DNA damage at this concentration. This is
consistent with the cell viability observed from its MTT assay (Fig. 8 panels A, C). All these
morphological changes indicate that these complexes might have induced cell death via both
apoptosis and necrosis [10b] though the majority of the cells appear to be rounded and shrunken due
to apoptosis [1].

18
Fig. 10. AO/PI staining results for compound 1 with A549 and HaCaT cell lines. Scale bar: 20 µm.
Fig. 11. AO/PI staining results for compound 2 with A549 and HaCaT cell lines. Scale bar: 20 µm.
Fig. 12. AO/PI staining results for compound 3 with A549 and HaCaT cell lines. Scale bar: 20 µm.

19
It is to be noted here that recently Nagababu et al. [1c] have reported the anticancer activity of four
mononuclear Cu(II) complexes with some polypyridyl ligands, two of which were found to be highly
potent for the anticancer activity. However, these two compounds showed cytotoxicity toward
cancerous cell as well as noncancerous cell; and these were comparable to the results obtained with
doxorubicin, a FDA approved anticancer drug, which also showed cytotoxicity toward both
cancerous and noncancerous cells equally strongly. Based on this, these authors have argued that the
possibility of using the compounds synthesized by them as a therapeutic agent cannot be completely
ruled out. We also have a very similar situation in our present study, though compound 3, being
much less toxic toward normal cells might be more suitable, but all three compounds can be potential
candidates to be tested as therapeutic agents.
2.5. Electrochemical results
The electrochemical behaviour of compounds 1, 2 and 3 have been studied in DMF containing 0.1 M
[N(n-Bu)4]ClO4 at a platinum working electrode using cyclic voltammetry (CV) in dinitrogen
atmosphere. For an initial negative scan compounds 1 and 3 do not show (Fig. 13) any specific
reduction wave within 0 to −1.5 V, while compound 2 shows only a broad reduction wave near −1.21
V (versus Ag/AgCl), but no corresponding oxidation peak is observed up to 0.0 V on scan reversal.
Compound 1 undergoes two successive oxidations at +0.86 and +1.2 V, while 2 undergoes
oxidations at +0.16, +0.89 and +1.30 v, respectively. Compound 3, on the other hand shows the
oxidation waves at +0.55 and +1.28 V, respectively. Reversal of these scans between +1.5 and 0.0 V
yielded reduction waves at +0.159 V for 1, +0.16 V for 2, and +0.35 V for 3, respectively (Fig. 13).
Scanning between −0.5 and +0.5 V did not show any redox couple in any of these cases (figure not
shown). Hence, all these three compounds undergo only irreversible oxidations and reductions.

20
Fig. 13. Cyclic voltammogram of the compounds (1.5 × 10−3 mol L−1) in DMF containing 0.1 M
[N(n-Bu)4]ClO4 at a scan rate of 100 mV s−1. Panel A for 1, panel B for 2, and panel C for 3.
3. Conclusions
All the three compounds found to have efficient DNA binding capabilities with binding constants
ranging from 1.18 × 105 M−1 to 2.63 × 105 M−1 and they bind to DNA by intercalation mode as
revealed from their DNA binding study with calf thymus DNA. The compounds 1, 2 and 3 exhibited
in vitro cytotoxicity against human breast cancer MCF7 cell line and human lung cancer A549 cell

21
line; as evident from significant decrease in cell viability for doses 0.5-10 µM for both the cell lines;
and they are found to be highly potent for both these cell lines as revealed from their IC50 values.
AO/PI dual staining results strongly suggest the induction of apoptotic pathway for the anticancer
activity of these complexes.
4. Experimental
4.1. Chemicals
Carbohydrazide and salicylaldehyde were obtained from Aldrich. Calf thymus DNA was obtained
from Sigma, N,N-dimethylformamide (DMF, GR), absolute ethanol, methanol (GR), DMSO, 1,10-
phenanthroline monohydrate, Zn(OAc)2⋅2H2O and Cu(OAc)2⋅H2O (GR) were obtained from Merck.
4.2. Preparation of 1,5-Bis(salicylidene)carbohydrazide, (o-HOC6H4CH=NNH)2C=O (H3L)
This was prepared by modifying the reported method of Bustos et al. [29]. Salicylaldehyde (20
mmol) was added to a solution of carbohydrazide (10 mmol) dissolved in 50 mL of methanol, and
the resulting reaction mixture was stirred at RT for 2 h, filtered through a G-3 crucible and the pale
white solid was washed well with methanol and air dried. Yield 80%; mp 226 oC. Anal. Calc. for
C15H14N4O3: C, 60.40; H, 4.73; N, 18.78. Found: C, 61.01; H, 4.68; N, 18.51%. Recrystallization of
this compound from methanol-ethanol (1:1) mixture led to formation of colorless single crystals
suitable for X-ray diffraction. We have characterized it by X-ray crystal structure (vide infra).
4.2. Preparation of the complexes
4.2.1. [Cu(o-phen)(HL)] (1)
1,10-phenanthroline monohydrate (0.2 g, 0.001 mol) in methanol (10 mL) was very slowly added to
a solution of Cu(OAc)2⋅H2O (0.2 g, 0.001 mol) in methanol (15 mL) during 20 min with stirring
below room temperature (∼15 °C) and the dark blue solution was stirred for another 40 min at RT.
This blue solution was then added very slowly to a solution of 1,5-bis(salicylidene)carbohydrazide
(0.358 g, 0.0012 mol) in 75 mL of methanol during 30 min while the reaction mixture turned bright
green. This was stirred for 24 h at RT and the green compound separated from the solution was

22
filtered through a G-3 sintered glass crucible, washed very well with methanol, air dried, then
washed thoroughly with water, and finally washed again with methanol and dried in vacuo. Yield
~65%. Anal. Calc. for C27H20N6O3Cu: C, 60.05; H, 3.73; N, 15.56. Found: C, 59.98; H, 3.75; N,
15.61%. ESI−MS in DMSO: m/z 540.33[M]+, m/z 579.19 [M + K]+.
4.2.2. [Cu(o-phen)LCu(OAc)] (2)
1,10-phenanthroline monohydrate (0.2 g, 0.001 mol) in methanol (10 mL) was very slowly added to
a solution of Cu(OAc)2⋅H2O (0.2 g, 0.001 mol) in methanol (15 mL) during 20 min with stirring
below room temperature (∼15 °C) and the dark blue solution was stirred for another 40 min at RT.
This blue solution was then added very slowly to a solution of 1,5-bis(salicylidene)-carbohydrazide
(0.298 g, 0.001 mol) in 50 mL of methanol during 30 min while the reaction mixture turned bright
green. This was stirred for 3 h, then Cu(OAc)2⋅H2O (0.2 g, 0.001 mol) in methanol (15 mL) was
slowly added to this reaction mixture when the solution turned from bright green to dark green.
Stirring was continued at RT for 24 h and the dark green compound was filtered, washed well with
methanol followed by water and dried in vacuo. Yield: 85%. Anal. Calc. for C29H22N6O5Cu2: C,
52.65; H, 3.35; N, 12.70. Found: C, 52.51; H, 3.37; N, 13.01%. ESI-MS in DMSO: m/z 701.77 [M +
K + H]+ .
4.2.3. [Zn(o-phen)LCu(OAc)] (3)
1,10-phenanthroline monohydrate (0.2 g, 0.001 mol) in methanol (10 mL) was very slowly added to
a solution of Zn(OAc)2⋅2H2O (0.219 g, 0.001 mol) in methanol (15 mL) during 30 min with stirring
below room temperature (∼15 °C) and the solution was stirred for another 40 min at RT. This
colorless solution was then added very slowly to a solution of 1,5-bis(salicylidene)-carbohydrazide
(0.298 g, 0.001 mol) in 50 mL of methanol during 30 min while the reaction mixture turned yellow.
This was stirred for 6 h, then Cu(OAc)2⋅H2O (0.2 g, 0.001 mol) in methanol (15 mL) was slowly
added to this reaction mixture when the solution turned dark green. Stirring was continued at RT for
24 h and the dark green compound was filtered through a G-3 sintered glass crucible, and the green

23
mother filtrate was collected in a 50 mL beaker. The green solid in the crucible was washed very
well with methanol, air dried. Then this was washed thoroughly with water, and finally washed again
with methanol and dried in vacuo. Anal. Calc. for C29H22N6O5ZnCu: C, 52.50; H, 3.34; N, 12.67.
Found: C, 52.41; H, 3.34; N, 12.70%. ESI−MS in DMSO: m/z 701.70 [M + K]+.
The green mother filtrate collected in a 50 mL beaker was covered with a watch glass and left for
slow evaporation at RT when very thin dark blue crystals were obtained within 3-4 days and were
used for X-ray crystallography. This is to be noted here that on long standing (or in vacuum) these
crystals lose their solvent of crystallization (both methanol and water) and become amorphous.
4.3. Physical measurements
Microanalyses (C, H, N) were performed in a Perkin-Elmer 240C elemental analyzer. Mass spectra
were recorded on a Perkin Elmer (USA) Flexer SQ 300 MS Mass Spectrometer operating in ESI
mode. Infrared and far-infrared spectra were measured with a Jasco IR report-100 and a Shimadzu IR
Affinity - 1 FT-IR spectrometer using KBr pellet. Electronic absorption spectra were recorded on a
Jasco V-570 UV/VIS/NIR spectrophotometer using a pair of matched quartz cell of path length of 1
cm. Electron paramagnetic resonance spectra were recorded on a JEOL, Japan Model: JES - FA200
ESR spectrometer with X and Q band. Room temperature solution EPR spectra were recorded using
an aqueous cell and frozen glass spectra were recorded in liquid nitrogen using a quartz dewer.
Conductivity measurement was done using a Mettler Toledo dual conductivity/pH meter model
SevenMulti equipped with Inlab 730 and Inlab 413 electrodes. Electrochemical measurements were
done with the help of a Bioanalytical system CV-27 electrochemical analyzer and a BAS model X-Y
recorder at 298K under dinitrogen. A standard three electrode cell consisting of a platinum working
electrode, a platinum auxiliary electrode and a Ag/AgCl reference electrode was used.
Tetrabutylammonium perchlorate ([N(n-Bu)4]ClO4) was used as supporting electrolyte.
4.4. X-ray crystallography

24
4.4.1. 1,5-Bis(salicylidene)carbohydrazide, (o-HOC6H4CH=NNH)2C=O
Colourless crystals were obtained from the slow evaporation of a methanolic-ethanol (1:1) solution
of 1,5-Bis(salicylidene)carbohydrazide (LIGAND) at room temperature. A crystal with approximate
size of 0.3 × 0.2 × 0.2 mm was mounted on a Bruker Axs Kappa Apex2 diffractometer equipped with
graphite-monochromated [Mo Kα, λ = 0.71073 Å] radiation. The unit cell parameters (Table 3) were
determined by the method of difference vectors using reflections scanned from three different zones
of the reciprocal lattice. The intensity data were measured using ω and φ scan with frame width of
0.5°. The frames integration and data reduction were performed using Bruker SAINT-Plus (Version
7.06a) software [33]. The multi-scan absorption corrections were applied to the data using SADABS
software. The crystal is indexed in Orthorhombic system with space group Aba2 and lattice
parameters a = 14.5381(18) Å, b = 9.3483(11) Å, c = 10.2927(11) Å, α = 90ο, β = 90ο , γ = 90ο.
SIR-92 program [34] was used for solving the structure. Structure was refined using SHELXL-2014
(Sheldrick, 2014) program [35]. Successive difference fourier map showed the positions of all
hydrogen atoms. However, the hydrogen positions were geometrically fixed and refined through
riding model. The full matrix structure refinement was carried out through minimization of the
function∑(w(F2 − F2 ))2, where w = [σ2 (F2 ) + (0. 0.0389P)2 + 0.6887P]−1 and P = (F2 + 2F2 )/3,
o
c
o
o
c
F2 is the measured intensity (i.e., intensity observed) and F2 is the intensity calculated. The final
o
c
residual factors were R = 0.03 and wR = 0.0893. The largest difference map peak was 0.139 e/Å3.
4.4.2. [Zn(o-phen)LCu(OAc)]0.5H2O⋅0.5CH3OH (COMPLEX 3)
Blue crystals were obtained from the slow evaporation of a methanolic solution of compound 3 at
room temperature. A crystal with approximate size of 0.3 × 0.2 × 0.2 mm was mounted on a Bruker
Axs Kappa Apex2 diffractometer equipped with graphite-monochromated [Mo Kα, λ = 0.71073 Å]
radiation. The unit cell parameters (Table 3) were determined by the method of difference vectors
using reflections scanned from three different zones of the reciprocal lattice. The intensity data were
measured using ω and φ scan with frame width of 0.5°. The frames integration and data reduction

25
were performed using Bruker SAINT-Plus (Version 7.06a) software [33]. The multi-scan absorption
corrections were applied to the data using SADABS software. The crystal is indexed in triclinic
system with space group Pī and lattice parameters a = 12.473(2) Å, b = 15.955(3) Å, c = 16.000(3)
Å, α = 77.052(11)ο, β = 67.897(9)ο , γ = 89.979(10)ο. SIR-92 program [34] was used for solving the
structure. Structure was refined using SHELXL-2014 (Sheldrick, 2014) program [35]. Successive
difference fourier map showed the positions of all hydrogen atoms. However, the hydrogen positions
were geometrically fixed and refined through riding model. The full matrix structure refinement was
carried out through minimization of the function∑(w(F2 − F2 ))2, where w = [σ2 (F2 ) + (0.1626P)2 +
o
c
o
116.3643P]−1 and P = (F2 + 2F2 )/3, F2 is the measured intensity (i.e., intensity observed) and F2 is
o
c
o
c
the intensity calculated. The final residual factors were R = 0.11 and wR = 0.3351. The largest
difference map peak was 0.853 e/Å3.
Table 3. Crystal data and structure refinement for LIGAND and COMPLEX 3.
________________________________________________________________________________________
Identification code
Empirical formula
Formula weight
Temperature
LIGAND
C15H14N4O3
298.30
COMPLEX 3
C29.50H24CuN6O6Zn
687.46
293(2) K
293(2) K
Wavelength
0.71073 Å
Orthorhombic
A b a 2
0.71073 A
Crystal system
Space group
Triclinic
Pī
Unit cell dimensions
a = 14.5381(18) Å
b = 9.3483(11) Å
c = 10.2927(11) Å
α = 90°
a = 12.473(2) Å
b = 15.955(3) Å
c = 16.000(3) Å
α = 77.052(11)°
β = 67.897(9)°
γ = 89.979(10)°
β = 90°
γ = 90°
3
3
Volume
1398.8(3) Å
2862.8(9) Å
Z
4
4
1.595 Mg/m3
1.636 mm-1
1400
3
Density (calculated)
Absorption coefficient
F(000)
1.416 Mg/m
-1
0.102 mm
624

26
3
3
Crystal size
0.300 x 0.200 x 0.200 mm
2.802 to 24.996°.
0.300 x 0.200 x 0.200 mm
1.315 to 17.653°
Theta range for data collection
Index ranges
-17<=h<=17, -11<=k<=10, -12<=l<=12 -10<=h<=10, -13<=k<=13,
-13<=l<=13
Reflections collected
8223
17755
Independent reflections
Completeness to theta
1228 [R(int) = 0.0308]
= 25.000° 99.7 %
3296 [R(int) = 0.1213]
= 25.242° 31.8 %
Absorption correction
Semi-empirical from equivalents
Semi-empirical from
equivalents
Max. and min. transmission
Refinement method
0.971 and 0.942
0.762 and 0.601
Full-matrix least-squares on F2
2
Full-matrix least-squares on F
Data / restraints / parameters
1228 / 1 / 109
3296 / 573 / 821
2
Goodness-of-fit on F
1.115
1.094
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0339, wR2 = 0.0795
R1 = 0.0448, wR2 = 0.0893
n/a
R1 = 0.1062, wR2 = 0.2724
R1 = 0.1755, wR2 = 0.3351
0.0059(12)
Extinction coefficient
0.853 and -0.797 e.Å-3
-3
Largest diff. peak and hole
0.139 and -0.141 e.Å
________________________________________________________________________________________
4.5. Methods for biological activity
4.5.1. DNA binding studies
Electronic spectra of the compounds 1-3 with incremental amounts of calf thymus DNA (ct-DNA)
have been recorded using a Jasco V-570 UV/VIS/NIR spectrophotometer in order to get the binding
constant for the compounds. The absorption titration experiments were performed by keeping the
complex concentration constant and by varying the concentration of ct-DNA from 0 to 40 µM in 10
mM Tris–HCl buffer (pH 7.43).
4.5.2. Cytotoxicity studies
4.5.2.1. Cell culture and treatment
The human cancer cell lines A549, MCF7 and normal human keratinocyte HaCaT cells and human
lung embryonic L132 cells were purchased from NCCS Pune. All the cell lines were cultured in T-
25 mm NUNC Cell culture flasks with DMEM High glucose medium with 10% Fetal Bovine Serum

27
(GIBCO performance plus grade, US origin) supplemented with penicillin (100 units/ml),
streptomycin (100 µg/ml) Gentamycin 50µg/mL and amphotericin B (2.5 µg/ml) at 37oC (5% CO2
and 95 % O2) in a CO2 incubator. The cells were sub cultured after they had reached 70-80 %
confluence by trypsinization and used for further experimental purpose.
4.5.2.2. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
MTT assay was performed to evaluate the cytotoxicity of the compounds on cancer and normal cell
lines. In this assay, cell viability was determined by the reduction of yellow MTT into purple
formazan product by the enzymatic activity of mitochondrial dehydrogenase in the live cells based
on the method of Mabley et al. [36]. The cells were plated on 96 well plates at a density of 4 × 104
per well and treated with complexes 1, 2, and 3 as described at varying concentration starting from
0.25µM, 0.5µM, 0.75µM, 1 µM, 2.5µM, 5µM and 10µM was added with DMEM culture medium
containing 10%FCS. The MTT assay was carried out in triplicates. After 24 hour incubation, the
cells were examined under phase contrast microscope and observed the cells morphology and
photographed using Leica systems. Subsequently, the medium was removed and the cells were
treated with 0.5mg/ml of MTT (Thiazolyl Blue Tetrazolium Bromide salt) in 1X PBS (150µL/well)
and incubated for 4 hours in a CO2 incubator. Following 4 incubation with MTT solution, the
solution was removed and blue colored formazan crystal was solubilized with 200 µL DMSO. The
absorbance was measured at 570 nm using BioRad ELISA plate reader.
4.5.2.3. Cellular morphology assessment. Acridine orange (AO)/ Propidium iodide (PI) dual staining
A549 cells and HaCaT cells were plated in 6-well plates at a density of 5×104, and were allowed to
grow at 37°C in a humidified CO2 incubator until they were 70–80% confluent. The cells were then
treated with different concentrations of complexes 1-3 (0.5, 1, and 2 µM) for 24 h. The culture
medium was aspirated from each well and cells were gently rinsed twice with PBS at room
temperature. Equal volumes of cells from control and metal complex treated were stained with 1

28
µg/mL of acridine orange and propidium iodide and incubated in the dark for 30 min, then viewed
with Leica fluorescence microscope.
Acknowledgements
We thank the SAIF, IIT-Madras for XRD facilities; and SAIF, IIT-Bombay for recording EPR of the
compounds 1-3 using JEOL, Japan Model: JES - FA200 ESR spectrometer. A.P.K. thanks Mr. Atanu
Banerjee and Dr. Rupam Dinda, Department of Chemistry, National Institute of Technology,
Rourkela, India for help with the ESI-MS results. A.P.K. thanks Mr. Srimanta Haldar and Dr. P.
Bhavana, Department of Chemistry, BITS-Pilani K. K. Birla Goa Campus for help with the
electrochemical studies. P.T.M. thanks the SERB/DST, Govt. of India for a research Scheme
EMR/2016/000745 and the INSA for the scheme SP/IHS/2016.
Appendix A. Supplementary material
Fig. S1. ESI-MS of the compounds 1-3. Fig. S2. IR spectra of compounds 2 and 3 in KBr. Fig. S3.
Electronic spectra of the compounds. Fig. S4. Electronic spectral change for the reaction of the
compounds 1-2 with 3,5-DTBC. Fig. S5. Packing of the 1,5-Bis(salicylidene)carbohydrazide
molecules in the unit cell. Fig. S6. Packing of Zn(o-phen)LCu(OAc)⋅0.5H2O⋅0.5CH3OH in the unit
cell. Tables S1-S6. Crystal data and structure refinement for LIGAND, complex 3 and their bond
lengths and bond angles. Crystallographic data for the structural analysis have been deposited with
the Cambridge Crystallographic Data Center, numbers are CCDC 1546215 for the ligand and CCDC
1546227 for complex 3, respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: (+044) 1223-336-033; or E-mail:
deposit@ccdc.cam.ac.uk.
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