Biological evolution of titanium(IV) complex [{(NNO)2Ti}3O3] bearing bidentate heteroditopic Schiff base ligand: Synthesis, structure and biological studies

Article abstract of DOI:10.14233/ajchem.2020.22464

Titanium(IV)-complex of chemical composition [{(NNO)2Ti}3O3] (2) bearing bidentate heteroditopic Schiff base [(C5H4OH)-N=CHC4H3-NH] (L1) ligand in titanium coordination sphere was reported and its biological significance was evaluated. The in vitro cytoto

Full text of DOI:10.14233/ajchem.2020.22464

Asian Journal of Chemistry; Vol. 32, No. 2 (2020), 441-450
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22464
Biological Evolution of Titanium(IV) Complex [{(NNO)
2
Ti}
3
O ] Bearing Bidentate
3
Heteroditopic Schiff Base Ligand: Synthesis, Structure and Biological Studies
1
,*,
1,
1,
2,
RAVI K. KOTTALANKA
, ESWAR PAGADALA , SHIVA K. LOKE , S. REX JEYA RAJKUMAR
,
1,
2
VENKATESAN SRINIVASADESIKAN
and BALAJEE RAMCHANDRAN
1
Division of Chemistry, Department of Sciences and Humanities, Vignan′s Foundation for Science, Technology and Research, Vadlamudi-
22213, India
Department of Biotechnology, Vignan's Foundation for Science, Technology and Research, Vadlamudi-522213, India
5
2
*
Corresponding author: E-mail: ravikottalanka@gmail.com
Received: 24 September 2019;
Accepted: 16 November 2019;
Published online: 30 December 2019;
AJC-19740
Titanium(IV)-complex of chemical composition [{(NNO)
2
Ti}
3
O
3
] (2) bearing bidentate heteroditopic Schiff base [(C
5
H
4
OH)-N=CH-
C
4
3
H -NH] (L1) ligand in titanium coordination sphere was reported and its biological significance was evaluated. The in vitro cytotoxicity
of L1 and 2 were evaluated by using MTT assay on cancer cell lines (MCF-7 & A549) and observed significant cytotoxicity. Further, the
LDH and NO assay studies on both L1 and 2 on cancer cell lines revealed that the enhanced cytotoxicity compared to standard anticancer
drug i.e. cisplatin. The DNA binding studies of tested compounds with Ct-DNA molecule by using UV-visible and fluorescence spectra
and molecular docking studies revealed that moderate to good binding interactions with test molecules. Thus, the present work contributes
and implies the biological significance of Ti-complex (2) and the correlation between the structure and the biological activities of such
Ti-complexes supported by Schiff base systems opens up opportunities for further exploitation of similar biologically active titanium systems.
Keywords: Titanium(IV) complex, Bidentate heteroditopic Schiff base, Biological activity, DNA Binding, Docking studies.
were developed three decades later and found that high efficacy
both in vivo and in vitro and markedly improved stability in
biological solution [24-35]. Further, structure-activity relation-
ship (SAR) studies on various Salan type substituted diamino-
bis(phenolato)titanium(IV) complexes revealed that a negative
effect of steric bulk and a positive effect of ortho-halogenation
on hydrolytic stability and cytotoxicity. Further, mechanistic
insight of various bis(phenolato)titanium(IV) complexes of
INTRODUCTION
Tetravalent titanium coordination complexes were eval-
uated as first alternative non-platinum anticancer-drugs and
had reached the clinical trial stages [1-7]. The enormous benefit
of titanium is that, it′s low toxicity, high catalytic efficacy [8],
therapeutic activity and the hydrolysis of titanium(IV) coordi-
nation complexes in biological environment yield inert TiO .
2
After the discovery of platinum based anticancer metallodrugs,
two classes of titanium(IV) compounds supported by cyclo-
pentadienide and diketonato ancillary ligands were evaluated
as efficient anticancer drugs and they have been showed subs-
tantial activity and moderate to good toxicity in vivo [9-22].
However, the rapid hydrolysis of these Ti(IV) compounds to
form multiple aggregates and low solubility of these complexes
under biological environment disadvantaged further advance-
ment in that area [14,23]. However, Salan type diaminobis-
the type [LTiX ] based on DNA binding, protein binding and
2
enzyme binding studies suggested that labile ligands are not
essential for reactivity and being the first to hydrolyze, they
reduce the overall hydrolytic stability of the complexes. Thus,
design and synthesis of new water stable therapeutic Ti(IV)
metal complexes, with no labile ligands, are highly desirable
for anticancer applications and clinical trails.
In this work, we report a new water stable titanium(IV)
complex of composition [{(NNO)
tate heteroditopic Schiff base [(C
(L1) ligand in titanium coordination sphere. The obtained
2
Ti}
3 3
O ] (2) bearing biden-
(
phenolato)titanium(IV) coordination complexes [LTiX
2
] type;
H
5 4
OH)-N=CH-C
4
3
H -NH]
where L = diaminobis(phenolato) ligand and X = labile group]
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442 Kottalanka et al.
Asian J. Chem.
Ti-complex (2) and the ligand (L1) were characterized using
spectroscopic and analytical techniques to conform the struc-
tures and composition of the compounds. The in vitro cyto-
toxicity of ligand (L1) and Ti(IV) complex (2) were further
evaluated by using MTT assay dilution method on cancer cell
lines (MCF-7 & A549) and observed significant cytotoxicity.
Furthermore, lactose dehydrogenase assay (LDH) and nitric
oxide assay (NO) studies on both ligand (L1) and titanium(IV)
complex (2) on cancer cell lines (MCF-7 and A549) revealed
that the enhanced cytotoxicity compared to the standard anti-
cancer therapeutic drug i.e. cisplatin by releasing significant
amount of LDH and NO from the cancer cells. The ligand and
its Ti(IV) complex were further examined on various Gram-
positive and Gram-negative bacterial and fungal strains under
in vitro condition, exhibited significant antibacterial and anti-
fungal activity. The DNA binding studies were carried out to
evaluate the binding interactions of these compounds with
Ct-DNA helix by using UV-visible and fluorescence spectral
measurements and molecular docking studies. DNA binding
and molecular docking studies revealed that Ti-complex (2) is
more active towards protein molecules having multiple binding
interactions with more than one type bindings such as inter-
chelation and electrostatic binding interactions.
Scheme-I: Synthesis of heteroditopic Schiff base ligand (L1)
Synthesis of [{(NNO)
solution (10 mL) of bidentate heteroditopic Schiff base ligand
L1) (1.0 g, 5.30 mmol), toluene solution (10 mL) of titanium
2
Ti}
3
O
3
] (2): To a dried toluene
(
isopropoxide (0.7g, 2.65 mmol) was added under stirring
condition at room temperature. The reaction mixture was then
allowed to proceed for another 12 h under inert atmospheric
conditions and then all volatile materials and solvent were
removed under vacuum to afford titanium(IV) complex of
composition [(C
yellow solid. The obtained titanium(IV) compound (1) was
further treated with H O in 1:10 molar ration for 12 h under
5
H
4
O)N=CHC
4
H
3
2 2
NH) Ti(OiPr) ] (1) as reddish-
2
constant stirring at room temperature. Volatile materials and
solvents were removed under vacuum to give deep red colour
solid and then it was washed with n-hexane (Scheme-II). The
recrystallization of compound 2 from hot toluene gave the
required compound as crystalline product in 89 % yield (3.57 g).
1
H NMR (400 MHz, CDCl ): δ 12.45 (br, 1H, N-H), 7.36 (s,
3
1
1
1
(
1
H, N=C-H), 6.81 (m, 1H, ArC), 6.69 (m, 1H, ArC), 6.58 (m,
EXPERIMENTAL
H, ArC), 6.34 (m, 1H, ArC), 6.27-6.29 (d, 2H pyr), 5.88 (d,
All manipulations of air-sensitive materials were per-
formed with the rigorous exclusion of oxygen and moisture in
flame dried Schlenk-type glassware on a dual manifold
13
H, 5-pyr). C NMR (100 MHz, CDCl
3
): δ159.2 (N=CH), 149.6
ArC), 140.9 (ArC), 127.2 (ArC), 124.6 (p-ArC), 119.2 (PyC),
15.4 (PyC), 114.3 (PyC), 109.6 (PyC). FT-IR (selected freque-
-
4
Schlenk line, interfaced to a high vacuum (10 torr) line.
-1
ncies, KBr, νmax, cm ): 3145 (br, N-H), 2860 (w,ArC-H), 1654
Hydrocarbon solvents (toluene and n-hexane) were distilled
(
(
s, C=N), 1245(s, C-O). Elemental analysis of C66
H
54
N
12
O
9
Ti
3
under nitrogen from LiAlH
4
and stored under N atmosphere.
2
m.w. 1302.83), calcd. (found): C 60.85 (60.82), H 4.18 (4.21),
N 12.90 (12.86).
Detection method: in vitro Cytotoxicity activity studies
were performed by using MTT assay dilution method and
further confirmed by using LDH and NO assay methods [37,38].
The antibacterial and antifungal studies were evaluated by
determining the MIC studies on various Gram-positive and
Gram-negative bacterial and fungi strains.
HPLC grade methanol was purchased from Sigma-Aldrich and
it was further dried by using 3 Å molecular sieves and followed
1
13
by distillation. H NMR (400 MHz) and C NMR (100 MHz)
spectra were recorded on aVARIAN INOVA 400 spectrometer
atAccuAnalytical, Hyderabad, India. Perkin-Elmer spectrum
IR version 10.6.0 and Agilent Technologies FT-IR was used
-1
for FT-IR measurement fitted in the 4000-450 cm range.
Elemental analyses were performed on a Truespec Micro CHNS
Biological studies
2
09-190. Electronic absorption spectra were obtained on a
Cell cultures: The cancer cell lines used in present study
include human lung carcinoma (A549) and human breast
carcinoma (MCF-7), whereas the normal cell lines used were
human Keratinocytes (HaCaT).All cells were purchased from
National Centre for Cell Science (NCCS), Pune, India. A549
were grown in RPMI-1640 (Roswell Park Memorial Institute),
while MCF-7 and HaCaT were grown in DMEM (Dulbecco
modified eagles medium), respectively. To ensure growth and
viability of the cells, the mediums were supplemented with
Shimadzu Lambda-1800 UV-Vis spectrophotometer. The
starting materials like pyrrole-2-carbaldehyde, 2-aminophenol
(
99 % pure), Titanium(IV) isopropoxide (97 % pure) were
purchased from Sigma-Aldrich and used without further purifi-
cation.
Synthesis of Schiff based ligand (L1):A bidentate hetero-
5 4
ditopic Schiff base ligand [(C H OH)-N=CH-C
4
H -NH] (L1)
3
was synthesized as per the literature procedure [36]. In brief,
a solution of pyrrole-2-aldehyde (2.0 g, 21.0 mmol) was added
drop by drop to a boiling solution of 2-aminophenol (2.29 g,
1
0 % FBS (HiMedia, India) and incubated in a humidified
atmosphere with 5 % CO at 37 ºC.
2
21 mmol) in 50 mL MeOH. The reaction mixture was refluxed
3
MTT assay:All the three cells (2.5 × 10 ) were seeded in
for 1 h and then concentrated to 30 mL.After cooling, the solu-
tion was mixed with 30 mL of water (Scheme-I). The preci-
96-well plates and allowed to adhere overnight at 37 ºC. Briefly,
following treatment of cells with cisplatin and all the other
compounds for 24 h, MTT reagent [3-(4,5-dimethylthiazol-
pitate was recrystallized from MeOH/H
5 %. FT-IR (selected frequencies, KBr, νmax, cm ): 3410br
N-H), 3061w (ArC-H), 1618s (C=N). Elemental analysis of
(m.w. 610.40), calcd. (found) %: C 82.58 (82.45), H
.25 (8.22), N 9.17 (9.13).
2
O (1:2): (2.06 g) yield:
-1
8
(
2-yl)-2,5-diphenyltetrazolium bromide] was added to each well
and incubated for 4 h at 37 ºC. Dimethyl sulphoxide was used
as a control.Absorbance was recorded at 595 nm and sensitivity
C
42
H
50
N
4
8

Vol. 32, No. 2 (2020) Biological Evolution of Titanium(IV) Complex [{(NNO)
2
Ti}
3
O ] Bearing Bidentate Heteroditopic Schiff Base Ligand 443
3
Scheme-II: Synthesis of tri-nuclear Ti-Schiff base complex [{NNO)
2
Ti}
3
3
O ] (2)
to cisplatin and the compounds were calculated based on cell
proliferation measurements at 24 h.
Lactate dehydrogenase release: LDH assay was perfor-
med according to the literature method as described byWacker
et al. [37].
Nitric oxide assay: Nitric oxide assay was performed
according to the literature method [38].
in vitroAntibacterial and antifungal activities: The test
microorganisms used for screening four bacterial pathogens
Each experiment was performed in triplicate and the results
are represented as an average zone of inhibition and minimum
inhibitory concentration of all the test pathogens.
Minimal inhibitory concentration (MIC) determination:
The antibacterial and antifungal activity of bidentate hetero-
ditopic Schiff base (L1) and its titanium(IV) complex (2) were
determined using sterile 2 mL 96-well plates [39]. The 12
wells of each row were filled with 0.5 mL sterilized Mueller
Hinton agar. Sequentially, wells 2-11 received an additional
0.5 mL of a mixture of culture medium and plant extract serially
diluted to create a concentration sequence from 0.512 mL to
0.008 mL. Well 1 served as growth control, well 12 as antibiotic
control. Gentamycin (10 µg/mL) and ketoconazole (10 µg/mL)
were used as controls for the bacteria and fungi, respectively.
The respective antibiotics were chosen because they are often
employed as first line antibiotics in the respective bacterial
and fungal infections. The deep wells were incubated for 24 h
at 37 ºC. The resulting turbidity was observed and after 24 h
MIC was determined to be where growth was no longer visible
by assessment of turbidity by optical density readings at 600
nm with a UV-Vis spectrophotometer.
(
Gram-positive and Gram-negative) and 5 fungal pathogens
which were procured from Institute of Microbial Technology
IMTECH-CSIR), Chandigarh, India. The pure bacterial stock
(
cultures were maintained on nutrient agar medium, whereas
the fungal stock cultures were maintained on potato dextrose
agar (PDA) medium. Each bacterial and fungal culture was
further maintained by sub-culturing regularly on the same
medium and stored at 4 ºC. The antibacterial and antifungal
activities of both bidentate heteroditopic Schiff base (L1) and
its Ti-complex (2) were evaluated by using standard agar
diffusion method as reported, by taking Gram-positive and
Gram-negative pathogens viz. S. aureus, S. pneumoniae, P.
aeruginosa, A. baumannii and fungal pathogens viz. C. albicans,
T. rubrum, A. fumigatus, A. niger and C. tropicalis. Gentamycin
and ketoconazole were used as positive controls to study the
antibacterial and antifungal activities, respectively. The anti-
microbial activity of the test compounds was checked with
various concentrations (25, 50 and 100 µg/mL) against all the
test pathogens.
Molecular docking: The Schrodinger molecular model-
ling suite was employed to carry out the molecular docking
protocols. The binding affinity plays a crucial role to determine
the biological activity between the receptor and inhibitor. It
was influenced by the geometrical positions, steric and the
physical properties. The small molecule was synthesized in
our laboratory. It was re-drawn using ACD/Chemsketch ver

444 Kottalanka et al.
Asian J. Chem.
1
2.0, it was geometrically optimized and converted into 3D
-C-O bond stretching.All other pyrrole ring and aromatic ring
C-H vibrations were best fitted with the literature reports [40-
44]. The H NMR spectrum of Ti-complex (2), clearly showed
format. the PDBID:3G7B was extracted from Protein Data
Bank (www.rcsb.org). Later, the protein molecule was prepared
using Protein preparation wizard, Schrodinger. The molecule
was optimized, and it was minimized with the OPLS-2005
force field.
Docking methodologies: The molecular docking was
performed using GLIDE, Schrodinger with the vdW scaling
of 0.8 and partial cut-off of 0.15 to soften the potential for
non-polar sites and implemented without the constraints. The
docking was performed using GLIDE-SP. An attention paid
to obtain the better GLIDE energy and interactions to favour
the outcome.
1
the broad resonance signal at δ 12.45 ppm, could be best assi-
gned to the N-H proton and a singlet signal observed at δ 7.36
ppm could be assigned to imine -N=C-H proton, respectively.
The resonance signals for pyrrole ring protons were obtained
in the region of δ 5.88-6.29 ppm are in good agreement with
literature reports [44,45]. The aromatic ring protons as well as
13
C NMR spectrum of Ti-complex (2) were showed resonance
signals at expected regions and fully supported the formation
of Ti-complex (2) of composition [{(NNO)
2
Ti}
3
O ] in good
3
yield.
X-ray crystallographic studies:The single crystals suitable
for X-ray diffraction analysis were obtained in hot toluene
after two days. The X-ray crystallographic studies showed that
RESULTS AND DISCUSSION
The bidentate heteroditopic Schiff base ligand of che-
mical composition [(C
5
H
4
OH)-N=CH-C
4
H
3
-NH] (L1) was
Ti-complex (2) crystallizes in monoclinic I /a space group
2
successfully reproduced accordingly the literature method [36].
having four molecules in the unit cell. However, we couldn't
obtained suitable crystallographic data miserably, due to the
decomposition of crystal when it is exposed to X-rays for long
period of time. The obtained crystallographic data was used
for structure refinement by using (SIR92) [46] and refined on
i
The Schiff base ligand (L1) was then treated with Ti(O Pr)
4
in 2:1 molar ratio in dry toluene. Under inert atmospheric
conditions, titanium(IV) complex of chemical composition
i
[(C
H
5 4
O)N=CHC
4
3
H NH)
2
Ti(O Pr)
2
] (1) was first isolated as
2
an intermediate compound (reddish-yellow solid). Without any
F by full-matrix least-squares methods using SHELXL-97
further purification the obtained titanium(IV) complex (1) was
[47]. The Diamond 3 and Mercury 4.1 programs were used to
draw the solid-state structures of Ti-complex (2) and those are
shown in Fig. 1.
immediately treated with H O in 1:10 molar ratio at atmos-
2
pheric conditions. The resultant deep red colour compound
so obtained was recrystallized in hot toluene to afford the
The crystallographic data reveals that each titanium(IV)
required Ti-complex of composition [{(NNO)
good yield (89 %).
2
Ti}
3
O
3
] (2) in
cationic center present in the Ti-complex [{(NNO)
2
Ti}
3
O ] (2)
3
is six-coordinated due to the mono- and bidentate chelation
from four oxygen atoms (i.e. two are from phenolic oxides and
two are bridging oxides derived from replacement of isoprop-
oxide groups) and two imine nitrogen atoms of the two ligand
moieties. Furthermore, no coordination from N-atoms of the
pyrrole rings were also observed. Therefore, at each Ti(IV)
cationic center there exists distorted octahedral geometry and
a six-membered ring is constructed in a trinuclear form having
The FT-IR spectrum of Ti-complex (2) showed a broad
-1
absorption band at 3145 cm , which indicate the presence of
N-H bond stretching. The enhanced broadness is due to the
inter-molecular hydrogen bonding exists between the pyrrole
N-H and adjacent phenolic oxygen atom (N-H---O) of the ligand
moiety. Further, in FT-IR spectra, the absorption band at 1654
-1
cm could be ascribed to the -C=N- (imine) bond stretching
-1
and absorption band at 1245 cm could be best ascribed to the
3
D symmetry with three Ti-O units. To support this result similar
Fig. 1. Solid-state structure of [{(NNO)
2
Ti}
3
3
O ] (2) obtained by diamond (left) and mercury (right)

Vol. 32, No. 2 (2020) Biological Evolution of Titanium(IV) Complex [{(NNO)
2
Ti}
3
O ] Bearing Bidentate Heteroditopic Schiff Base Ligand 445
3
kind of trinuclear six-membered ring was also observed in Ti-
over ligand. Ligand (L1) and Ti-complex (2) were screened
Cl-H
Cl-H
complex of chemical composition [(L
)
3 3 3
Ti O ] (where L
for their cytotoxic activity on the normal cell lines viz. HaCaT
and it was found to be non-toxic in nature as shown in Fig. 2.
The enrichment of cytotoxic activity of Ti-complex (2) can be
corroborate with structure-activity relation (SAR) where electron
donating N-terminal substitution on the coordinated ligand
play major role. Ti-complex (2) owning un-coordinated nitrogen
as a terminal N-substitution in the coordinated ligand and prob-
ably, which is responsible for the higher cytotoxic ability than
the titanium complexes bearing only coordinated nitrogen
ligands. In addition, anticancer effectiveness of new Ti-complex
=
p-Cl-C -CH-N=N-CH-C -(o-O)) in the literature [48].
6
H
4
6
H
4
In addition, the crystallographic studies also revealed that each
titanium octahedral core is vertex shared with other two titanium
octahedral cores through bridging oxide ligands. The obtained
structure was best fitted with the NMR data further indicate
that trinuclear ring structure was still remains in the solution
state as well.
Biological activity studies
in vitro Cytotoxicity activity: Bidentate heteroditopic
Schiff base ligand (L1) and Ti-complex (2) were subjected to
MTT assay towards, the normal human Keratinocytes (HaCaT),
human lung carcinoma (A549) and human breast carcinoma
(
2) was found to be better than Ti-complexes already reported
in the literature [11-14].
LDH assay: The cancer cell lines MCF-7 andA549 were
treated with the different amounts of the Ti-complex (2), ligand
(
MCF-7) cell lines. After 24 h of incubation, ligand and metal
(
2
L1) and standard anticancer drug (cisplatin) for a period of
4 h. A significant amount of LDH release was observed in the
complex showed good cytotoxic reduction in the cell viability
on comparing with cisplatin. The cytotoxic ability of synthe-
sized ligand and Ti-complex occurred in a dose-dependent
manner as shown in Fig. 2 and furthermore, the IC50 values
were also calculated. The metal complex was found to have
potential cytotoxic effect than the ligand against both cancer
cell lines tested.
culture medium (Fig. 3) by Ti-complex (2) compared to cisplatin
indicates the efficiency of Ti-complex (2) by inducing the cell
death of membrane integrity is collapsed. Compared to cisplatin,
Ti-complex (2) showed a good level of leakage of LDH in
MCF-7 and A549 cells. From the present findings, it can be
determine that Ti-complex (2) was found to be more effective
than the corresponding Schiff base ligand (L1).
The bidentate chelation of the ligand with Ti(IV) might
be responsible for the cytotoxic nature of the metal complex
HaCaT
Ligand
Complex
Cisplatin
Ligand
Complex
100
100
A549
50
50
0
0
Cisplatin
Ligand
Complex
5
4
3
2
1
0
Cisplatin
Ligand
Ti(IV) complex
100
MCF7
IC50
50
0
Fig. 2. in vitro Cytotoxicity activity of ligand; Ti-complex with standard drug (i.e., cisplatin)

446 Kottalanka et al.
Asian J. Chem.
2
2
1
1
5
0
5
0
5
0
Cisplatin
Ligand
Complex
80
60
40
20
0
S, 10 µg
25 µg
50 µg
100 µg
S. aureus S. pneumoniae P. aeruginosa
S. typhi
Fig. 5. Antibacterial activity of Schiff base ligand and Ti-complex (2)
MCF7
A549
considerably reduce the polarity of the central atom by partial
sharing of its positive charge with donor groups and possible
π-electron delocalization within the whole chelating rings. This
eventually increases the lipophilic nature of metal complexes
[50].
Therefore, an increase in the lipophilic character of Ti(IV)-
complex (2) seems to be responsible for their enhanced anti-
bacterial activity. The antibacterial activity results also displayed
all the compounds possess higher inhibitory activity against
the bacterial species at 100 µg/mL concentration than at 50
µg/mL concentration.
in vitro Antifungal activity: in vitro Antifungal activity
of newly synthesized heteroditopic Schiff base ligand (L1)
and its Ti-complex (2) were carried out against five different
fungi cells such as Candida albicans, Trichophyton rubrum,
Aspergillus niger, Aspergillus fumigatus and Candida tropicalis
and compared with standard antifungal drug ketoconazole at
minimum (10 µg/mL) concentration. Antifungal activity data
is shown in Fig. 6, which indicates that Ti-complex (2) was
found to be more toxic than heteroditopic Schiff base ligand,
whereas at higher (100 µg/mL) concentrations both Schiff base
ligands and Ti-complex (2) were found to be more toxic towards
fungi cells. The activity is greatly enhanced at the higher concen-
tration.
Fig. 3. Amount of LDH released by the MCF-7 and A549 cells after an
incubation period of 24 h with the Ti-complex (2)
NO assay: In this study, amount of nitric oxide released
by Ti-complex (2), when it is treated with MCF-7 and A549
cancer cells was evaluated. The determination of nitrite released
in the cell media by Griess assay [38] is one of the cost effective
measurements. The Griess assay showed that Ti-complex (2)
exceptionally effective to release high amount of NO in culture
medium than standard anticancer drug i.e. cisplatin and corres-
ponding ligand (L1) (Fig. 4), hence NO assay study useful to
conform the cytotoxicity activity of the studied Ti-complex
(
2).
6
0
0
0
0
Cisplatin
Ligand
Complex
4
2
25
20
15
10
5
0
MCF7
A549
Fig. 4. Nitrite released (n moles) by the MCF7 andA549 after an incubation
period of 24 h with the test compounds
S, 10 µg
2
5 µg
0 µg
in vitroAntibacterial activity: The antibacterial activities
of heteroditopic Schiff base ligand (L1) and its Ti-complex
2) were tested with reference to gentamycin (10 µg) on Gram-
5
(
100 µg
positive bacteria: S. aureus & S. pneumonie and Gram-negative
bacteria: P. aeruginosa & S. typhi. The antibacterial activities
of the ligand (L1) and its Ti-complex (2) were evaluated by
measuring the inhibition zone observed around the tested
materials (Fig. 5). The maximum zone of inhibition was obser-
ved for Ti-complex (2) when compared to corresponding hetero-
ditopic Schiff base ligand and it can effectively decrease the
population of bacterial species. This is mainly because of chel-
ation effect [49], where the donor groups of chelating ligands
S. albicans T. rubrum A. niger A. fumigatus C. tropicalis
Fig. 6. Antifungal activity of Schiff base ligand and Ti-complex (2)
DNA binding studies: The DNA binding studies were
carried out by using UV-absorption and fluorescence emission
spectral investigations. The amount of Schiff base ligand (L1)

Vol. 32, No. 2 (2020) Biological Evolution of Titanium(IV) Complex [{(NNO)
2
Ti}
3
O ] Bearing Bidentate Heteroditopic Schiff Base Ligand 447
3
and Ti-complex (2) were kept constant at [Test sample] = 2.26
nm in 300-400 nm region and less intense fluorescence band
-4
3
×
10 mol/dm and the aliquots of Ct-DNA were added up to
at 410 to 436 nm was also observed. On addition of Ct-DNA,
there is no change in 310 fluorescence band and little down
field (towards higher energy) shift was observed in 355 nm
fluorescence band with increased intensity. A considerable
quenching of fluorescence band at 410 to 436 nm was observed
at higher concentration of added Ct-DNA. On the other hand,
fluorescence spectra of Ti-complex (2) displayed three intense
fluorescence bands viz. 309, 355 and 415 to 440 nm. On addition
of Ct-DNA, there is no change in 309 fluorescence band. The
intensity of 355 nm band was slightly decreased and little down
filed shift towards higher energy or shorter wavelength side
was observed. However, a very significant quenching of fluore-
scence band of 415 to 440 nm was observed at higher concen-
tration of added Ct-DNA. These results clearly indicate that
Ti-complex (2) was more active towards DNA helix compared
to the corresponding Schiff base ligand (L1) with more than
one mode of binding.
-4
3
a final concentration of [Ct-DNA] = 0.121 × 10 mol/dm . As
the addition of Ct-DNA increases, the absorption band at 261
nm enhances (Fig. 7). The band at 261 nm is mainly because
of the absorption of light by Ct-DNA helix and hence, this blue
shift and the absorbance values were not applied for the calcu-
lation of the binding constant in case of ligand and Ti-complex
(
2) with Ct-DNA. However, in addition to absorption band at
2
3
61 nm, we observed another less intense absorption band at
14 nm in case of Ti-complex (2). The addition of portion of
Ct-DNA does not alter this band due to the enrichment of the
61 nm band which defeats the intense changes in the 314 nm
2
band. However, no such kind of less intense absorption band
was observed in case of Schiff base ligand (L1) when it treated
with different aliquots of Ct-DNA.
The binding titrations were performed using fluorescence
emission spectroscopy. The fluorescence emission spectra of
both Schiff base ligand (L1) and Ti-complex (2) with different
amounts of Ct-DNA are shown in Fig. 8. The ligand fluore-
scence spectra displayed two emission bands viz. 310 and 355
It is known that the fluorescence phenomenon is being
sensitive to the medium, nature of fluorophore and polarity
change results in shift of the spectrum [51]. The mechanism
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0
0.50
(
a) Ligand (L1
)
(b) Ti-complex (2)
0
.45
.40
0
0
–
6
0
1
2×10
1×10
–
6
6
6
6
6
–6
×10
0.35
.30
0.25
.20
0.15
2×10–
6
–
3×10–
6
0
–
4×10–6
5×10–6
3
4
5
6
7
8
9
×10
×10
×10
×10
×10
×10
×10
–
6
7
×10–6
×10–6
–
0
–6
8×10–6
9×10
–
6
6
6
–
0
.10
.05
0
–
0
-
0.05
-0.05
200
2
00
300
400
500
600
700
800
900
300
400
500
600
700
800
Wavelength (nm)
Wavelength (nm)
Fig. 7. (a) & (b). Absorption spectra of Schiff ligand (L1) and Ti-complex (2) with different concentrations of added Ct-DNA
1
200000
0
1
250000
(
a) Ligand (L1
)
0
1
2
(b) Ti-complex (
2)
–6
–
–
–
–
–
–
–
–
–
6
6
6
6
6
6
6
6
6
1×10
2×10
3×10
4×10
×10
×10
–6
–6
–6
–6
–6
–6
–6
–6
1000000
3×10
1000000
750000
4×10
5×10
6×10
5×10
6×10
7×10
8×10
9×10
8
6
4
2
00000
00000
00000
00000
7×10
8
9
×10
×10
500000
250000
0
300
3
00
325
350
375
400
425
450
475
500
325
350
375
400
425
450
475
500
Wavelength (nm)
Wavelength (nm)
Fig. 8. (a) & (b). Fluorescence spectra of Schiff ligand (L1) and Ti-complex (2) with different concentrations of added Ct-DNA

448 Kottalanka et al.
Asian J. Chem.
1
1
1
.20
.15
.10
(
a) Ligand (L1
)
(b) Ti-complex (2)
1.0
0.9
0.8
0.7
0.6
1.05
1.00
0.95
0.90
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
–
6
–3
–6
–3
[
Q] × 10 (mol dm )
[Q] × 10 (mol dm )
Fig. 9. Stern-Volmer plots of Schiff base (L1) and Ti-complex (2) with concentration of added Ct-DNA
of interaction of Ct-DNA with the binder involves the insertion
of the binder into the active base pairs of DNA helix. The freely
rotating small (binder) molecule probably deactivates the excited
states and hence there will be fluorescence enhancement [52].
Further, narrow-cut binding agents which experience electro-
static interactions with Ct-DNA get close to the backbone of
Ct-DNA viz. sugar phosphates groups and this leads to the
significant quenching of fluorescence activity. In the current
fluorescence spectral studies, it is noticed that quenching and
the enhancement of fluorescence bands of Ti-complex (2),
which indicates that when Ti-complex (2) treated with Ct-DNA
helix probably two type of interactions namely electrostatic
and intercalative binding were involved. The Stern-Volmer
equation was utilized to find the quenching constant for the
interaction of binders such as Schiff base ligand (L1) and Ti-
complex (2) with Ct-DNA.
the conformation. The complex results the docking score of
-3.224 kcal/mol with the hydrogen bonding between theAsp81
with the distance of 1.85 Å as shown in Fig. 10.
0
F
=
1+ k [Q]
sv
F
0
where, F = Initial intensity of fluoresence of binder without
Ct-DNA; F = intensity of fluoresence of binder with Ct-DNA.
In the presence of quenching agent (ligand/Ti-complex),
Fig. 10. Binding interactions of heteroditopic Schiff base ligand (L1) with
protein molecule having non-covalent interactions with Asp81
k
sv is the quenching constant and [Q] is the concentration of
Therefore, residues are considered as an active site to
proceed further. The docking was performed with the ciprofl-
oxacin which shows as an interaction with Asp81 and Ser129
respectively, with the distance ~2.0 Å. The test molecule was
docked with the reference of the existing molecules. The test
molecule revealed that it has an interaction with Asp81 with
1.85 Å, by showing -3.224 kcal/mol. The test molecule clearly
shows that it contains strong hydrogen bonding network with
N-H...O type interaction. However, corresponding trinuclear
quenching agent (ligand/Ti-complex). The Stern-Volmer quen-
ching studies were illustrated in Fig. 9. The Stern-Volmer plot
of ligand (L1) with varying concentration of Ct-DNA repre-
sents the non-linear downward curve due to the lesser binding
capability of ligand (L1), whereas the corresponding Ti-complex
(
2) plot represents the upward straight line which is indicative
of static fluorescence quenching process due to the formation
of stable complex with binder (Ti-complex) molecules. The
quenching constant ksv value obtained as a slope from the plot
Ti-complex [{(NNO)
2
Ti}
3
O ] (2) is having three Ti(IV) cationic
3
0
4
–1
of [Q] vs. F /F was found to be 2.9 × 10 M . These results
further support the strong binding ability of Ti-complex (2)
over the Schiff base ligand (L1) towards the Ct-DNA helix.
Molecular docking analysis: The study aimed to identify
the significance of the synthesized small molecule against anti-
bacterial target [53,54]. The molecular docking has initiated
with reproducing the crystal structure of 3G7B. The hydrogen
bonding interactions have been observed from the in-sights of
centers and each cation is surrounded by two such type of small
molecule having uncoordinated N-H groups. Therefore, Ti-
complex (2) perhaps having multiple interactions with protein
used for docking studies and may have approximately -10-18
kcal/mol energy as docking score. It clearly shows that Ti-
complex (2) is more active towards protein molecules and
therefore, it can be considered as active antibacterial/fugal and
anticancer agents.

Vol. 32, No. 2 (2020) Biological Evolution of Titanium(IV) Complex [{(NNO)
2
Ti}
3
O ] Bearing Bidentate Heteroditopic Schiff Base Ligand 449
3
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4
Conclusion
In this study, synthesis of tri-nuclear Ti(IV)-complex of
type [{(NNO) Ti} ] (2) was well demonstrated. To evaluate
biological significance of synthesized Ti-complex (2) along
with corresponding bidentate heteroditopic Schiff base ligand
2
O
3 3
8
.
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5 4
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4
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3
in vitro cytotoxicity activity by using MTT assay on cancer
cell lines viz. human breast carcinoma (MCF-7) and human
lung carcinoma (A549) and normal cell lines such as human
keratinocytes (HaCaT). The results demonstrated that Ti(IV)-
complex (2) displayed significant cytotoxicity compared to
standard anticancer drug (i.e. cisplatin). The enhanced cyto-
toxicity of Ti-complex (2) was further supported by the LDH
and nitric oxide assay by releasing significant amount LDH
and NO. The in vitro antibacterial and antifungal activity
studies revealed that Ti-complex (2) having enhanced anti-
bacterial activity due to its increased lipophilic character and
it was found to be more toxic than Schiff base ligand (L1). At
higher concentration (100 µg/mL) both Schiff base ligand (L1)
and Ti-complex (2) were found to be more toxic towards fungi
cells. Furthermore, DNA binding studies were performed by
using UV-visible and fluorescence investigations to evaluate
the binding capacity of the tested compounds with Ct-DNA
helix revealed that Ti-complex (2) is having significant inter-
actions than the heteroditopic Schiff base ligand (L1). The
molecular docking studies also conformed that significant
hydrogen bonding interactions (N-H---O type) with Asp81
were observed when heteroditopic Schiff base ligand (LI) was
subjected to docking studies with protein molecule. From all
these studies, it is concluded that the synthesized tri-nuclear
1
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ACKNOWLEDGEMENTS
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(
This work is supported by the VFSTR (Deemed to be
University), Vadlamudi, India under the scheme of seed grant
for research faculty. Two of the authors, Eswar and Shiva thanks
to VFSTR for providing facilities and fellowship.
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CONFLICT OF INTEREST
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The authors declare that there is no conflict of interests
regarding the publication of this article.
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