Synthesis of novel metal (II) complexes tailored from 9-oxo-9H-fluorene-1-carboxylic acid via green protocol: DNA cleavage and anticancer studies

Article abstract of DOI:10.1016/j.ica.2019.119210

Despite being a polycyclic aromatic compound, fluorenone is not classified as carcinogenic. Some of the derivatives of fluorenone and metal complexes embedded with fluorenone based ligands exhibited challenging biological activities, including anticancer activity. The current investigation hence focuses on synthesis, characterization and anticancer activities of fluorenone based ligands and their coordinate compounds of some first row transition metals. The stoichiometry of all the synthesized metal complexes is found to be 1: 2 (metal: ligand) with the general formula [M(L)₂], L = singly deprotonated ligand, whereas the geometry of all the metal complexes is found to be octahedral. The synthesized ligands and their respective metal (II) complexes were found to cleave the pBR322 DNA, during gel electrophoresis studies. The inhibition of cancer cell growth has been confirmed by fluorescence imaging techniques using DAPI as staining dye. The IC50 values of ligands and their metal (II) complexes suggest that synthesized compounds are more active towards A549 (human lung adenocarcinoma) cell line when compared with the standard drug Paclitaxel. In case of MCF-7 (human breast carcinoma) cell line, compound Cu(L²)₂ is found to be more active than the standard drug Paclitaxel.

Full text of DOI:10.1016/j.ica.2019.119210

Journal Pre-proofs
Synthesis of Novel Metal (II) Complexes Tailored from 9-Oxo-9H-fluorene-1-
carboxylic Acid via Green Protocol: DNA Cleavage and Anticancer Studies
Mahantesh Kumbar, Sangamesh A. Patil, Shivakumar S. Toragalmath, Shailaja
S. Jawoor, Arun Shettar
PII:
S0020-1693(19)31024-2
https://doi.org/10.1016/j.ica.2019.119210
ICA 119210
DOI:
Reference:
Inorganica Chimica Acta
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Revised Date:
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22 July 2019
29 September 2019
14 October 2019
Please cite this article as: M. Kumbar, S.A. Patil, S.S. Toragalmath, S.S. Jawoor, A. Shettar, Synthesis of Novel
Metal (II) Complexes Tailored from 9-Oxo-9H-fluorene-1-carboxylic Acid via Green Protocol: DNA Cleavage and
Anticancer Studies, Inorganica Chimica Acta (2019), doi: https://doi.org/10.1016/j.ica.2019.119210
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Synthesis of Novel Metal (II) Complexes Tailored from 9-Oxo-9H-fluorene-1-carboxylic
Acid via Green Protocol: DNA Cleavage and Anticancer Studies
Mahantesh Kumbara, Sangamesh A. Patila*, Shivakumar S. Toragalmathb, Shailaja S. Jawoora,
Arun Shettarc
aP. G. Department of Chemistry, Karnatak University, Dharwad 580 003, Karnataka, India.
bK L E’s, P. C. Jabin Science College, Autonomous, Vidyanagar, Hubballi 580 031, Karnataka,
India.
cP. G. Department of Biotechnology and Microbiology, Karnatak University, Dharwad 580 003,
Karnataka, India.
*Corresponding author e-mail: patil1956@rediffmail.com Contact no: +91 9986265906
Abstract
Despite being a polycyclic aromatic compound, fluorenone is not classified as
carcinogenic. Some of the derivatives of fluorenone and metal complexes embedded with
fluorenone based ligands exhibited challenging biological activities, including anticancer
activity. The current investigation hence focuses on synthesis, characterization and anticancer
activities of fluorenone based ligands and their coordinate compounds of some first row
transition metals. The stoichiometry of all the synthesized metal complexes is found to be 1: 2
(metal: ligand) with the general formula [M(L)2], L = singly deprotonated ligand, whereas the
geometry of all the metal complexes is found to be octahedral. The synthesized ligands and their
respective metal (II) complexes were found to cleave the pBR322 DNA, during gel
electrophoresis studies. The inhibition of cancer cell growth has been confirmed by fluorescence
imaging techniques using DAPI as staining dye. The IC50 values of ligands and their metal (II)
complexes suggest that synthesized compounds are more active towards A549 (human lung
adenocarcinoma) cell line when compared with the standard drug Paclitaxel. In case of MCF-7
(human breast carcinoma) cell line, compound Cu(L2)2 is found to be more active than the
standard drug Paclitaxel.
Keywords: 9-oxo-9H-fluorene-1-carboxylic acid, green synthesis, DNA cleavage, anticancer,
cytotoxicity.

Highlights
 The manuscript describes the synthesis of two new hydrazone Schiff base ligands.
 Synthesis of metal complexes from greener protocol.
 Structure of synthesized compounds was confirmed by analytical and spectroscopic methods.
 DNA cleavage, DAPI staining and anticancer activity studies were carried out.
 Study confirms that, synthesized compounds are more active towards A549 cell line when
compared to that of standard drug Paclitaxel.
2

1. Introduction
Cardiovascular disease and cancer are the top two medical challenges for the 21st-century
scientific community, which are the serious cause of mortality worldwide [1]. Despite advances
in biomedical research and technology, cancer is a growing public health issue, particularly in
developed countries [2, 3]. By 2030, the global burden due to cancer incidence is expected to
grow to 21.7 million [4]. The increasing incidence of cancer worldwide could be largely due to
environmental factors, such as tobacco smoking, urbanization and its associated pollution,
changing diet patterns and extended post-reproductive life span [5]. The cancer spreads when
genetic changes interfere with the orderly process of mitosis and apoptosis. Cells start to grow
uncontrollably and they may form a mass called ‘tumor’, which could be cancerous or benign. A
cancerous tumor is malignant; it can grow and spread to other parts of the body whereas benign
tumor can grow but will not spread [6, 7].
Current treatment includes gold standard strategies like surgical excision, chemotherapy,
and radiotherapy which are adopted individually or in combination [8]. Many therapeutic agents
have been isolated from animals, plants and minerals, as well as major of them, are synthesized
in the laboratory. Chemotherapeutic agents like doxorubicin, paclitaxel, cabazitaxel, granisetron,
docetaxel, letrozole and platinum based drugs like carboplatin, nedaplatin, oxaliplatin, lobaplatin
remain as a principal mode of treating various cancers. However, most of them suffer from
various side effects including fatigue, appetite loss, sore mouth, changes in taste, fever, infection,
anxiety, depression, nausea and vomiting which are the prime setbacks in their clinical utility [9,
10]. In order to get rid of these disadvantages of side effects, current effort in the development of
novel metallodrugs [11, 12] focused more and more on the use of transition metal complexes
with unconventional and unprecedented organic ligands. Hence, the current expedition focuses
on synthesizing new organic moieties with suitable donor atoms and their metal complexes,
which may help better in treating vulnerable cancer. The literature survey reveals that,
fluorenone being a polycyclic aromatic compound, has exhibited reasonable to challenging
biological activities including anticancer properties [13, 14]. Hence, the current study focuses on
synthesizing new fluorenone based hydrazone Schiff bases and their metal (II) complexes owing
to their excellent biological activity and as anticancer agents. The fluorenone derivatives were
identified as a histamine H3 inhibitor [15]. 1-methoxydiazofluorene can inhibit the proliferation
of HeLa cells [16]. Fluorenone-carboxamide scaffolds are proved to be potent and have selective
3

binding properties for tetraplex DNA [17]. Structure-activity relationship for the carboxamide
group present in various N-aryl-9-oxo-9H-fluorene-1-carboxamides have been studied and N-(2-
(1H-pyrazol-1-yl)phenyl)-9-oxo-9H-fluorene carboxamide is identified as a lead compound with
increased aqueous solubility, was found to retain the broad activity in the caspase activation
assay and in the cell growth inhibition assay [18, 19]. The antiproliferative activity of tri- and
tetraazabenzo[3,2-a]fluorene-5,6-dione derivatives is comparable with that of standard drugs
Mitoxantrone and Doxorubicin [20]. In the present study, we have successfully incorporated
above discussed functional groups viz., carboxamide, carboxylic acid and hydrazone Schiff base
into our newly designed fluorenone based ligands and synthesized transition metal (II)
complexes from these ligands and characterized thoroughly using analytical and spectroscopic
techniques. The metal (II) complexes and their respective ligands were screened for their potency
towards anticancer activities for A549 and MCF-7 cell lines and mode of cell death through
fluorescence imaging using DAPI [4ʹ,6-diamidino-2-phenylindole] staining method. The DNA
cleavage studies were carried out with gel electrophoresis technique.
2. Experimental
2.1 Materials and Instrumentation
All the raw materials including reagents, catalysts, solvents and drying agents were of
fine chemical grade and utilized as received by the vendor.
Infrared (IR) spectra of the synthesized materials were recorded in the range of 4000-550
cm-1 with PerkinElmer Spectrum Two FT-IR spectrometer. By using TMS as internal reference
compound, 1H and 13C NMR spectra were recorded in DMSO-d6 solvent on JEOL 400 MHz and
BRUKER 400 MHz spectrometer at room temperature. Using Thermo Quest elemental analyzer,
elemental analysis of the compounds is carried out. Electronic spectra of ligands and their
metal(II) complexes were recorded on a PerkinElmer LAMBDA 365 UV-Vis spectrophotometer
in the range of 1100-200 nm. Thermal analysis of the metal complexes was carried out with an
SDT Q600 V20.9 analyzer, by increasing the temperature from RT to 1000 °C at the rate of 10
°C min-1. The molar conductivity measurements were made on ELICO CM 180 conductivity
meter with a cell constant of 1.0 after calibration with standard KCl solution at 25 °C. LC-ESI-
MS spectra were recorded on LCMS 2010A, SHIMADZU instrument.
4

2.2 Synthetic Procedure
2.2.1 Procedure for the synthesis of 9-oxo-9H-fluorene-1-carboxylic acid
9-Oxo-9H-fluorene-1-carboxylic acid is prepared from fluoranthene using potassium
dichromate as an oxidizing agent as per the protocol reported in our previous publication [21].
Yield: 99%. Color: Orange. M. P: 194-196 °C (Lit.: 196-198 °C). Scheme 1 depicts the synthetic
route for 9-oxo-9H-fluorene-1-carboxylic acid.
O
O
K2Cr2O7/H2SO4
OH
Reflux
AcOH/H2O
Scheme 1: Schematic for the synthesis of 9-oxo-9H-fluorene-1-carboxylic acid.
2.2.2 General procedure for the synthesis of hydrazone Schiff base
An equimolar mixture (2.5 g, 11.15 mmol) of 9-oxo-9H-fluorene-1-carboxylic acid and
benzohydrazide (1.52 g, 11.15 mmol) is refluxed in methanol at 70 °C for 8 h [22]. The contents
are partially soluble at RT. About 30 min of heating at 70 °C, the reaction mixture turns orange
to yellow and yellow solid gets separated. The reaction is monitored by TLC in 9: 1 chloroform:
methanol. The solids are filtered and washed with cold methanol and dried under vacuo at 40 °C.
Scheme 2 represents the synthesis of hydrazone Schiff bases. Yield: LH1: Yield: 3.55 g, 93%.
Color: Yellow. LH2: Yield: 3.60 g, 90%. Color: Yellow.
R
O
O
OH
O
O
O
OH
NH
N
MeOH
NH2
N
H
70 °C, 8 h
R
R = H (LH1)
OH (LH2)
Scheme 2: Schematic for the synthesis of hydrazone Schiff base.
5

2.2.3 Green procedure for the synthesis of transition metal complexes
Copper complex: The copper(II) complexes are synthesized in water at room temperature.
Equimolar mixtures of ligand (500 mg, 1.46 mmol), copper (II) chloride (250 mg, 1.46 mmol)
and sodium acetate (120 mg, 1.46 mmol) are stirred in DI (deionized) water for 8 h at RT. The
solid separated is filtered, washed with water and dried under vacuo at 50 °C. Cu(L1)2: Yield:
283 mg, 52%. Color: Green. Cu(L2)2: Yield: 315 mg, 58%. Color: Brown.
Nickel/Cobalt complexes: The Co(II) and Ni(II) complexes are synthesized in water under
reflux conditions. Equimolar mixtures of ligand (500 mg, 1.46 mmol), metal (II) chloride
(CoCl2.6H2O/NiCl2.6H2O) and sodium acetate (120 mg, 1.46 mmol) are refluxed in DI
(deionized) water for 8 h. The solid separated is filtered, washed with water and dried under
vacuo at 50 °C. Scheme 3 represents the synthesis of transition metal (II) complexes. Co(L1)2:
Yield: 260 mg, 48%. Color: Yellow. Co(L2)2: Yield: 270 mg, 50%. Color: Brownish yellow.
Ni(L1)2: Yield: 243 mg, 45%. Color: Yellow. Ni(L2)2: Yield: 226 mg, 42%. Color: Yellow.
R
R
O
O
O
MCl2 . xH2O
O
NH
N
O
OH
NH
N
M
N
HN
O
O
CH3COONa/H2O
O
R
M = Cu(II), Ni(II), Co(II)
R = H, OH
Scheme 3: Schematic for the synthesis of transition metal (II) complexes.
6

2.3. Bioassay Procedure
2.3.1 Cytotoxicity: MTT Cell Proliferation Assay
The cells were seeded at a density of approximately 5×103 cellswell-1 in a 96-well flat-
bottom microtitre plate and maintained at 37 °C overnight in 95% humidity and 5% CO2.
Different concentrations (50, 40, 30, 20, 10, 5 µM) of samples were treated and the cells were
incubated for another 48 h. The cells in well were washed twice with phosphate buffer saline
(PBS) and 20 µL of the MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide]
staining solution (5 mgmL-1 in phosphate buffer saline) was added to each well and plate was
incubated at 37 °C. After 4 h, 100 µL of DMSO was added to each well to dissolve the formazan
crystals and absorbance was recorded at 570 nm using micro plate reader [23].
2.3.2 Photocleavage Study of pBR322 DNA
Supercoiled pBR322 DNA (200 ng) in TAE buffer (40 mM Tris-Acetate, 1 mM
EDTA) was treated with synthesized ligands and their respective metal (II) complexes to yield a
total volume of 10 μL and then incubated in dark for 30 min at 37 °C. Different concentrations of
compounds were tested; 30, 40 and 50 µM. The reaction was quenched by the addition of 3 μL
loading buffer and then the resulting solutions were loaded on a 2% Agarose gel. Electrophoresis
was carried out at 80 V for 1 hr in TAE buffer (pH 8.0). DNA bands were visualized under UV
light and photographed [24].
2.3.3 Cell Apoptosis and Fluorescence Imaging
The cells were seeded in a 24-well flat bottom micro plate containing cover slips and
maintained at 37 °C in CO2 incubator overnight and treated with 200 µgmL-1 of compound for
48 h. After the incubation, cells were washed with PBS (phosphate buffer saline) and fixed with
4% paraformaldehyde for 30 min. Then, 20 µL of DAPI [4ʹ,6-diamidino-2-phenylindole] was
added to the cells, which were incubated for 5 min at room temperature in the dark and images
were taken under fluorescent microscope (Olympus BX41 microscope). Randomly selecting the
fields in the microscope and counted the number of cells undergone apoptosis and the percentage
of apoptotic cells was calculated [25].
7

3. Results and Discussion
3.1 Spectral Characterization
3.1.1 9-Oxo-9H-fluorene-1-carboxylic acid (1): The IR spectrum of 9-oxo-9H-fluorene-1-
carboxylic acid (spectrum 1) betray two characteristic bands, one due to ν(C=O) group of
carboxylic acid at 1668 cm-1, which has shifted to lower frequency due to conjugation with
ν(C=C) of fluorenone ring, another sharp absorption band at 1746 cm-1 is due to ketone ν(C=O)
group vibrations. The sharp medium absorption band is observed at 1279 cm-1 infers the
presence of C–O linkage in carboxylic acid functional group. The weak absorption bands at 1604
cm-1 and 1574 cm-1 are attributed to aromatic ν(C=C) vibrations.
1
The H NMR spectrum of 9-oxo-9H-fluorene-1-carboxylic acid (spectrum 2) exhibit a
broad peak centered at 3.6 ppm due to the proton attached to carboxylic acid group. Remaining
protons are aromatic in nature and are resonated between 7.0-9.0 ppm. 1H NMR (400MHz,
DMSO-d6) /ppm: 3.6(br, 1H, –OH), 7.33(t, 1H, J = 7.8 Hz), 7.46(d, 1H, J = 7.6 Hz), 7.56(m,
3H), 7.67(d, 1H, J = 7.6 Hz), 7.76(d, 1H, J = 7.6 Hz).
3.1.2 IR Spectral Studies of Ligands and Their Respective Metal (II) Complexes
To identify the bonding mode as well as the complexation behavior, the IR spectra were
recorded for the synthesized hydrazone Schiff base ligands and their respective metal complexes.
The IR spectrum of ligand 1 (LH1) is reproduced in spectrum 3. The sharp intense band at 1698
cm-1 is assigned to ν(C=O) of carboxylic acid group. Another sharp band at 1659 cm-1 is due to
ν(C=O) vibrations of amide group. One more band at 1631 cm-1 is due to hydrazone ν(C=N)
group. The weak broad bands at 3189 cm-1 and 3032 cm-1 are due to ν(N–H) and ν(O–H)
respectively.
Similar observations are made for ligand 2 (LH2, spectrum 4) as well, the spectrum
exhibit two weak broad bands at 3265 and 3209 cm-1 due to phenolic ν(O–H) and carboxylic
ν(O–H) vibrations. The sharp intense band at 1675 cm-1 is assigned to ν(C=O) of carboxylic acid.
Sharp band appearing at 1652 cm-1 is due to amide ν(C=O) group vibrations. Hydrazone ν(C=N)
group vibrations are seen at 1637 cm-1. The band due to ν (N–H) is masked by the two broad
ν(O–H) vibrations in the region 3100-3500 cm-1. The diagnostic IR frequencies of ligands and
their copper (II) complexes are compiled in Table 1.
8

In all the metal (II) complexes the hydrazone ν(C=N) band is shifted to lower wave
number, which infers the involvement of nitrogen atom of hydrazone group in coordination to
the central metal ion [26]. The absence of broad band in the region 3200-3650 cm-1 infers the
coordination of carboxylic acid oxygen via deprotonation. Further, the strong band due to
ν(C=O) of carboxylic acid group has disappeared upon complex formation, since in carboxylate
ion the negative charge is equally distributed on two oxygen atoms due to resonance, thereby
decreasing the bond order. This implies that, the carboxylate ion is involved in bond formation
[27]. Simultaneously, two new bands have appeared in the range 1580-1610 cm-1 and 1360-1380
cm-1 which are assigned to the asymmetric and symmetric stretching frequencies of coordinated
carboxylate ion respectively. The amide carbonyl stretching frequency ν(C=O) observed at 1659
and 1652 cm-1 in LH1 (spectrum 3) and LH2 (spectrum 4) respectively have shifted to the lower
wave number value of 1628 and 1632 cm-1 in Cu(L1)2 (spectrum 5) and Cu(L2)2 (spectrum 6)
respectively indicating the coordination of amide carbonyl oxygen to the central metal ion. The
medium vibrations due to ν (M–O) are observed between 550-600 cm-1 [28, 29]. IR data suggests
the ONO donor tridentate behavior of the ligands.
Table 1. Prominent infrared frequencies (in cm-1) of ligands (LH1 and LH2) and copper (II)
complexes.
m = medium, s = strong, br = broad
ν(C=O)s
Carboxylic
acid
ν(COO)
νasym νsym
ν(C=O)s
Amide
Phenolic
ν(O–H)br
Acidic
Compound
ν(N–H)br ν(C=N)s
ν(M–O)m
ν(O–H)br
3189 &
1631
LH1
1698
disappeared
1675
1659
1628
1652
1602 1306
1590 1373
1606 1323
---
---
3032
disappeared
3209
---
559
---
3163
3185 &
3159
Cu(L1)2
LH2
1600
Masked
1637
1613
3265
3538 &
3429
Cu(L2)2
disappeared
1632
1586 1375
3245
disappeared
577
9

3.1.3 1H and 13C NMR Spectral Studies
The 1H NMR spectrum of ligand 1 (LH1, spectrum 7) shows a broad singlet at 15.20 ppm
due to the proton attached to carboxylic acid oxygen [30]. Another broad singlet at 12.25 ppm is
due to the amido proton (–NH–). Remaining protons are aromatic in nature and appeared as
1
multiplets between 7.0-9.0 ppm. H NMR (400MHz, DMSO-d6) /ppm: 7.69(m, 6H), 8.05(m,
13
2H), 8.17(m, 4H), 12.25(s, 1H, -NH), 15.20(s, 1H, –OH). C NMR spectrum of ligand 1 (LH1,
spectrum 9) shows a characteristic peak due to carboxylic acid carbon at 167 ppm. The peak at
163 ppm is due to the presence of amide carbon. The bridging carbon of fluorene ring is
resonating at 155 ppm. Remaining carbon nuclei are part of the aromatic ring and are resonating
in the range 120-150 ppm [31].
1
The H NMR spectrum of ligand 2 (LH2, spectrum 8) shows broad singlets at 15.48,
12.05 and 10.32 ppm due to the proton attached to carboxylic acid oxygen, the amido proton (–
NH–) and the phenolic –OH respectively. Aromatic protons appeared as multiplets in the range
6.0-9.0 ppm. 1H NMR (400MHz, DMSO-d6) /ppm: 6.96(d, 2H, J = 8.36 Hz), 7.57(t, 1H, J = 7.6
Hz), 7.66(m, 2H), 8.02(m, 4H), 8.17(m, 2H), 10.32(br, 1H, Phe-H), 12.02(s, 1H, –NH), 15.48(br,
13
1H, acidic-H). C NMR spectrum of ligand 2 (LH2, spectrum 10) displays a characteristic peak
due to carbon attached to carboxylic acid group at 170 ppm. The peak at 165 ppm is due to the
presence of amide carbon. The carbon attached to phenolic –OH is deshielded and is resonating
downfield at 161 ppm. The bridging carbon of fluorene ring is resonating at 155 ppm. Remaining
carbon nuclei are part of the aromatic ring and are resonating between 110-150 ppm [31].
3.1.4 Mass Spectral Studies
The mass spectral data of ligands 1 (LH1) and 2 (LH2) and their copper (II) complexes
[Cu(L1)2 and Cu(L2)2] are depicted in spectrum 11, 12, 13 and 14 respectively. The ligands LH1
and LH2 exhibit [M]+ and [M+1]+ peaks at m/z 342, 359 respectively. The fragmentation peaks at
m/z 325 and 341 in LH1 and LH2 respectively, are due to the loss of OH radical. Electrospray
ionization mass spectral study (ESI-MS) of metal (II) complexes supports 1 : 2 [(ML)2]
stoichiometry. Copper (II) complexes, Cu(L1)2 and Cu(L2)2 show [M+1]+ and [M-1]+ peaks at
m/z 746 and 776 respectively; which are matching very well with the predicted molecular
10

weights of the compounds and this confirms the formation of metal (II) complexes in each case
[32, 33].
3.1.5 Electronic Spectral Studies
Electronic spectra of ligands 1 (LH1, spectrum 15) and 2 (LH2, spectrum 16) were
recorded in methanol and their respective metal (II) complexes were recorded in DMF. The free
ligands absorb strongly at 348 and 355 nm due to intra ligand π
π* transitions. Absorptions
π* transitions
at 412, 432 nm (in LH1) and 415, 440 nm (in LH2) are attributed to n
associated with carbonyl groups and hydrazone (C=N) group. The bathochromic (red) shift is
observed in all the cases upon complex formation due to the donation of charge density to the
central metal ion for the coordination. This confirms that, the nitrogen atom of hydrazone group,
the oxygen of amide and carboxylic acid group are involved in the bond formation with the
central metal ion. IR spectra and mass spectral studies of the metal (II) complexes support the
observations made from electronic spectral studies [34, 35].
The absorption spectrum of Cu(L1)2 and Cu(L2)2 show one broad peak due to d-d
2
transition centered at 726 and 724 nm respectively, assignable to T2g
2Eg as is the
consequence of distorted octahedral environment around copper (II) ion [36]. Electronic spectra
of Cu(L1)2 and Cu(L2)2 complexes are reproduced in spectrum 17 and 18 respectively.
Table 2 shows the solution electronic absorption spectral data of ligands and their
respective copper (II) complexes. In case of nickel and cobalt complexes d-d transitions are
rarely recorded or too small to be observed, since these transitions are Laporte forbidden.
11

Table 2. Solution electronic absorption spectral data of ligands (LH1 and LH2) and their copper
(II) complexes.
Compound
LH1
λmax in cm-1 (nm)
Band assignments
Geometry
---
28,735 (348),
24,272 (412),
23,148 (432)
π
n
n
π*,
π*,
π*
Cu(L1)2
LH2
13,774 (726)
2T2g
2Eg
Octahedral
---
28,169 (355),
24,096 (415),
22,727 (440)
π
n
n
π*,
π*,
π*
Cu(L2)2
13,812 (724)
2T2g
2Eg
Octahedral
3.1.6 Thermal Analysis Studies
Thermal behavior of all the metal complexes was studied over a temperature range of 25-
1000 °C under nitrogen atmosphere. Thermograms of Cu(L1)2, Ni(L1)2 and Co(L1)2 complexes
are reproduced in entry 19 of supporting information. The copper (II) complex is found to be
highly stable up to 225 °C. Later on from 255 – 350 °C, the metal complex decomposes in only
one significant step; which infers that, only ligand moiety is present in the complex has lost, after
which the formation of metal oxide is observed [37]. This result is in line with the IR, electronic
spectral and ESI-MS data. Similar plot is observed in case of Ni(L1)2, Co(L1)2 and second set of
complexes as well. Thus, the thermal data of the metal (II) complexes give no evidence for the
presence of any other coordinated moieties like water, chloride, methanol, DMF etc.,
3.1.7 Molar Conductivity Studies
Conductivity measurement of metal complexes in solution state is a powerful tool to
ascertain their electrolytic nature. Molar conductivity (Λm) of all the metal (II) complexes was
measured at 25 °C by preparing mmol solutions in DMF using the conductivity bridge (G* = 1).
The values obtained for the metal (II) complexes did not exceed 20 Ω-1 cm2 mol-1. These results
confirm that, the metal (II) complexes are non-electrolytic in nature, which is due to the absence
of counter ions in their structures [38]. The molar conductivity values have been reproduced in
12

Table 3 along with the elemental analysis data. The detailed experimental results have been
tabulated in Table 4. Molar conductivity studies support the observations made in ESI-MS and
thermal
studies.
13

Table 3. Elemental analysis of ligands (LH1 and LH2) and their respective metal (II) complexes along with molar conductivity data.
Molar
M%
C%
H%
N%
Empirical
formula
Color/
% yield
conductivity
value, Λm
Compound
Obsd. Calcd. Obsd. Calcd. Obsd. Calcd. Obsd. Calcd.
(Ω-1cm2mol-1)
LH1
C21H14N2O3
C42H26CuN4O6
C42H26NiN4O6
C42H26CoN4O6
C21H14N2O4
--
--
73.72
67.71
68.11
68.14
70.44
64.93
73.68
67.60
68.04
68.02
70.39
64.82
4.18
3.60
3.62
3.66
3.99
3.49
4.12
3.51
3.53
3.53
3.94
3.37
8.25
7.62
7.67
7.59
7.88
7.31
8.18
7.51
7.56
7.55
7.82
7.20
Yellow/93
Green/52
--
Cu(L1)2
Ni(L1)2
Co(L1)2
LH2
8.63
8.01
8.04
--
8.52
7.92
7.95
--
16.3
12.9
15.1
--
Yellow/45
Yellow/48
Yellow/90
Brown/58
Cu(L2)2
C42H26CuN4O8
8.29
8.17
19.5
Ni(L2)2
Co(L2)2
C42H26NiN4O8
C42H26CoN4O8
7.67
7.77
7.59
7.62
64.31
65.34
65.23
65.21
3.50
3.48
3.39
3.39
7.33
7.35
7.24
7.24
Yellow/42
13.6
16.0
Brownish
yellow/50

Table 4. Molar conductivity (Λm) data of synthesized metal (II) complexes.
Molar conductivity
(Λm)
Conductance (G)
at 25 °C
Conductivity (κ)
in μΩ-1
Complexes
in Ω-1cm2mol-1
κ = G (∵ G*=1.0)
16.3 x 10-6
12.9 x 10-6
15.1 x 10-6
19.5 x 10-6
13.6 x 10-6
16.0 x 10-6
Λm = (1000 κ)/c
Cu(L1)2
Ni(L1)2
Co(L1)2
Cu(L2)2
Ni(L2)2
Co(L2)2
16.3 x 10-6
12.9 x 10-6
15.1 x 10-6
19.5 x 10-6
13.6 x 10-6
16.0 x 10-6
16.3
12.9
15.1
19.5
13.6
16.0
Cell constant (G*) of ELICO CM 180 conductivity meter = 1.0.
Conductance (G) values are obtained from the experiment in μΩ-1 or μS.
1mM solutions of metal (II) complexes are prepared in DMF and their conductance (G) is recorded.
3.2 Bioassay Results
3.2.1 Cytotoxicity- in vitro Studies
In order to find out the potency and effectiveness of the synthesized compounds as
chemotherapeutic drugs, it is important to evaluate the anticancer activity using MTT assay. The
in vitro anticancer activity of ligands and their metal (II) complexes against A549 and MCF-7
cell lines have been tested by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
Bromide] assay, using the untreated cells as control (NC). Ligands and all the metal (II)
complexes exhibit concentration-dependent anticancer activity which is evident from the bar plot
1 and 2 (Tables 5 and 6). Each entry in the plot is the mean of triplicate results obtained by
carrying out separate experiments. Results are expressed as mean± standard deviation (n = 3). At
5 μM concentration all the compounds show highest cell survival when compared to that at
higher concentrations [39-41].

Table 5. Cell viability data of ligands LH1, LH2 and six metal (II) complexes for A549 cell line
at six different concentrations.
A549
Concentration
in µgmL-1
LH1
Co(L1)2
Cu(L1)2
Ni(L1)2
LH2
Co(L2)2
Cu(L2)2
Ni(L2)2
5
98.36±0.1643 94.85±0.1715 96.67±0.2694 95.49±0.2876 97.60±0.3226 91.41±0.3171 88.84±0.4009 92.48±0.3151
92.43±0.2017 87.34±0.5530 90.48±0.2041 89.42±0.2465 91.52±0.2641 84.37±0.2947 77.24±0.1685 88.39±0.2369
80.48±0.2379 78.47±0.3054 81.60±0.2166 73.52±0.2524 81.59±0.1902 77.60±0.2046 67.43±0.3844 78.50±0.2046
66.75±0.1515 73.48±0.2449 53.93±0.2120 67.26±0.2597 53.39±0.2120 70.36±0.2531 52.44±0.2416 67.63±0.3260
61.24±0.1574 54.96±0.2089 43.36±0.2206 59.48±9.2169 43.36±0.2206 47.49±0.3542 32.58±0.3193 56.45±0.3127
54.37±0.1968 44.71±0.5213 36.45±0.2293 48.30±0.1470 36.45±0.2293 44.53±0.2562 22.43±0.3785 50.39±0.3111
10
20
30
40
50
A549
120
100
80
60
40
20
0
LH1
Co(L1)2
Cu(L1)2
Ni(L1)2
LH2
Co(L2)2
Cu(L2)2
Ni(L2)2
5
10
20
30
40
50
Concentration μgmL-1
Plot 1. Bar graph showing the % cell viability of ligands and their metal (II) complexes for A549
cell line v/s concentration in μgmL-1.
16

Table 6. Cell viability data of ligands LH1, LH2 and six metal (II) complexes for MCF-7 cell line
at six different concentrations.
MCF-7
Concentration
in µgmL-1
LH1
Co(L1)2
Cu(L1)2
Ni(L1)2
LH2
Co(L2)2
Cu(L2)2
Ni(L2)2
5
97.40±0.3559 98.78±0.2904 88.49±0.2248 86.43±0.3007 64.51±0.3353 72.52±0.0623 66.59±0.3950 89.65±0.2658
88.46±0.3374 96.28±0.1557 66.35±0.2458 77.34±0.2458 59.53±0.2455 61.29±0.1498 56.36±0.2795 84.44±0.1948
81.56±0.3026 91.62±0.2135 60.40±0.2763 74.45±0.2782 56.35±0.1498 59.54±0.1883 34.57±0.2169 80.52±0.3408
77.39±0.2223 66.47±0.2409 57.44±0.1899 70.32±0.2416 50.50±0.2458 51.82±0.0980 32.36±0.2740 77.30±0.1569
65.26±0.1146 56.58±0.2164 56.39±0.2583 65.45±0.1730 45.41±0.0492 46.43±0.3182 30.84±0.1271 71.34±0.1586
57.42±0.3627 48.61±0.3704 51.34±0.1407 56.39±0.2583 41.68±0.1818 35.48±0.1552 29.34±0.1186 56.31±0.1236
10
20
30
40
50
MCF-7
100
90
80
70
60
50
40
30
20
10
0
LH1
Co(L1)2
Cu(L1)2
Ni(L1)2
LH2
Co(L2)2
Cu(L2)2
Ni(L2)2
5
10
20
30
40
50
Concentration μgmL-1
Plot 2. Bar graph showing % cell viability of ligands and their metal (II) complexes for MCF-7
cell line v/s concentration in μgmL-1.
The % cell viability (% cell survival) of the cells is calculated using the relation,
% Viability = (Mean OD of test compound/Mean OD of negative control) x 100
Inhibiting cells (%) =100 - Surviving cells.
17

The IC50 was extrapolated from the dose-response curve. The drug concentration that
reduced the viability of cells by 50% (IC50) was determined by plotting triplicate data points
over a concentration range and calculating the values using GraphPad Prism Ver. 5.1 program.
The IC50 (Half maximal inhibitory concentration) values of ligands and their metal (II)
complexes suggest that synthesized compounds are more active towards A549 cell line when
compared with the standard drug Paclitaxel. In case of MCF-7 cell line, compound Cu(L2)2 is
found to be more active than the standard drug Paclitaxel. Further, activity of the metal (II)
complexes is more compared to that of free ligands which is evident from their lower IC50
values. IC50 values of ligands and their respective metal (II) complexes in µgmL-1 for both the
cell lines tested are compiled in Table 7. The cell images of anticancer activity are reproduced in
Fig. 1 (LH1 and its metal (II) complexes) and Fig. 2 (LH2 and its metal (II) complexes). The
results obtained from these images confirm apoptosis induced in cancer cells (characterized by
distorted cytoplasm, compactness of cancer cells, ruptured cell walls) when treated with ligands
and their metal (II) complexes; it is found to be more effective when compared with that of
standard drug Paclitaxel [42-44].
Table 7. IC50 values of ligands and their respective metal (II) complexes in µgmL-1 for A549
and MCF-7 cell lines tested.
Cell line
Compound
A549
MCF-7
(μgmL-1)
(μgmL-1)
LH1
Co(L1)2
Cu(L1)2
Ni(L1)2
LH2
54.740
47.390
35.560
50.330
56.610
44.140
27.220
51.040
73.25
66.950
46.380
52.010
99.510
27.580
27.870
10.940
92.610
23.476
Co(L2)2
Cu(L2)2
Ni(L2)2
Paclitaxel
18

Fig. 1. In vitro anticancer activity of ligands LH1, LH2 and six metal (II) complexes for A549
cell line.
19

Fig. 2. In vitro anticancer activity of ligands LH1, LH2 and six metal (II) complexes for MCF-7
cell line.
20

3.2.2 DNA Cleavage Studies
Designing small molecules help in targeting specific sites on a DNA strand and can lead
to novel therapeutic agents. Photo cleavage study of DNA also helps in various applications like
photodynamic therapy of cancers, DNA foot printing agents and in genomic research [45-47].
The ligand and their metal (II) complexes were tested at different concentrations (5, 10, 20, 40,
50 μM) to evaluate their cleaving ability. They displayed DNA degradation effect which verifies
their binding affinity to the supercoiled pBR322 DNA. The synthesized metal (II) complexes
exhibited the enhanced cleavage compared to free ligands (LH1 and LH2) [48, 49]. The DNA
cleavage images are reproduced in Fig. 3.
Fig. 3. Gel electrophoresis images of ligands 1 and 2 (LH1 and LH2) and their respective metal
(II) complexes showing the effect on pBR322 DNA compared with negative control.
21

3.2.3 Cell Morphology Studies by Fluorescence Microscopy
DAPI [4ʹ,6-diamidino-2-phenylindole] is a fluorescent dye; a DNA specific probe which
enhances the fluorescence and forms a complex with DNA by preferentially binding to the
Adenine-Thymine (A-T) regions of double stranded DNA. The fluorescence enhancement is
particularly good for mitochondrial DNA which is rich in A-T sequences. DAPI is a DNA
specific fluorescent stain and it is not sensitive to RNA. The fluorescence enhancement of DAPI
in the presence of DNA is the basis for a simple and rapid method for DNA microassay in the
presence of RNA, proteins and cellular homogenates [50]. This technique is employed in
studying the cell apoptosis induced by the synthesized ligands and their respective metal (II)
complexes, by staining the ‘cell treated’ and ‘untreated (NC)’ with DAPI. The cell death was
characterized by shrinkage of cell walls, nuclear fragmentation and DNA condensation [51-54].
These results confirm the inference made in case of MTT assay. The fluorescence images are
reproduced in Figs. 4 and 5.
Cells treated: Cancer cells treated with synthesized ligands and their respective metal (II)
complexes.
Untreated cells: Cancer cells untreated with synthesized compounds are used as negative
control (NC).
22

Fig. 4. Fluorescence microscopy images of cells stained with DAPI, showing untreated control
cells (NC) and cells treated with ligand 1 (LH1) and its metal (II) complexes.
23

Fig. 5. Fluorescence microscopy images of cells stained with DAPI, showing untreated control
cells (NC) and cells treated with ligand 2 (LH2) and its metal (II) complexes.
24

4. Conclusions
In the current study two new ligands were synthesized; the key precursors, ligands and
13
their transition metal (II) complexes were characterized by spectral (1H, C NMR, IR, GC-MS,
electronic), elemental analysis, thermal and molar conductivity measurements. The ligands and
their transition metal (II) complexes were screened for DNA cleavage, fluorescence imaging
using DAPI staining, anticancer activity with apoptotic effect. The hydrazone Schiff bases and
their metal (II) complexes successfully cleaved the pBR322 DNA. Cell apoptosis is confirmed
by the fluorescence images obtained upon treatment of cancer cells (A549 and MCF-7) with the
synthesized compounds. Concentration-dependent cell survival rate was observed during in vitro
anticancer activity, which decreases with increase in concentration. The IC50 values of ligands
and their metal (II) complexes suggest that synthesized compounds are more active towards
A549 cell line when compared with the standard drug Paclitaxel. In case of MCF-7 cell line,
compound Cu(L2)2 is found to be more active than the standard drug Paclitaxel.
Acknowledgements
The author acknowledges University Grants Commission, New Delhi, India, for
providing the financial support through BSR-RFSMS fellowship [F. NO: 25-1/2014-15(BSR)/7-
100/2007/(BSR) Dated: 05/11/2015]. Authors are grateful to the Chairman, Department of
Chemistry, Karnatak University Dharwad, for providing the laboratory facilities. Authors are
thankful to University Scientific Instrumentation Centre (USIC) and SAIF K. U. D, SAIF IIT
Madras and IISC Bangaluru, for providing instrumental facilities. The authors also express their
gratitude to Maratha Mandal’s Central Research Laboratory Belagavi, Karnataka, India, for their
valuable support for bioassay related study.
Disclosure Statement
Authors do not have any conflicts of interest to declare.
25

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