Twin applications of highly selective Cu2+ fluorescent chemosensor and cytotoxicity of 2-(2-phenylhydrazono)-1H-indene-1,3(2H)-dione and 2-(2-(4-methoxyphenyl)hydrazono)-1H-indene-1,3(2H)-dione: Molecular docking and DFT studies

Article abstract of DOI:10.1039/c4ra11006d

Fluorescent chemosensors based on 2-(2-phenylhydrazono)-1H-indene-1,3(2H)-dione (1) and 2-(2-(4-methoxyphenyl)hydrazono)-1H-indene-1,3(2H)-dione (2) with high sensitivity and selectivity toward paramagnetic Cu²⁺ were developed over other cation

Full text of DOI:10.1039/c4ra11006d

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Twin applications of highly selective Cu2+ fluorescent Chemosensor and Cytotoxicity of
2-(2-phenylhydrazono)-1H-indene-1,3(2H)-dione and 2-(2-(4-methoxyphenyl)hydrazono)-
1H-indene-1,3(2H)-dione: Molecular docking and DFT Studies
Annamalai Subhasria, Chinnadurai Anbuselvana* and Nagarajen Rajendraprasadb
aDepartment of Chemistry, Annamalai University, Annamalainagar − 608 002, India.
bDepartment of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar− 608
002, India.
*Corresponding author: Address
C. Anbuselvan
Assistant professor,
Department of Chemistry
Annamalai University
Annamalainagar 608 002
Eꢀmail: cas_amu@yahoo.co.in

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A fluorescent chemosensor based on 2ꢀ(2ꢀphenylhydrazono)ꢀ1Hꢀindeneꢀ1,3(2H)ꢀdione (1) and
2ꢀ(2ꢀ(4ꢀmethoxyphenyl)hydrazono)ꢀ1Hꢀindeneꢀ1,3(2H)ꢀdione (2) with high sensitivity and
selectivity toward paramagnetic Cu2+ was developed over other cations. The Cu2+ꢀinduced
fluorescence turn–on mechanism was revealed to be mediated by intramolecular charge transfer
1
13
from the ligand to the metal. The compounds were characterized by UVꢀVis, FTꢀIR, H and C
NMR techniques, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS),
elemental color mapping and fluorescence spectroscopy. Morphology changes of compound (1)
and (2) to complex with Cu2+ are also investigated by SEM. EDS analysis expose the occurrence
of copper in complexes. Elemental color mapping are also support the copper present in the
complexes of corresponding compound (1) and (2). The metal sensing (Chemosensing)
properties of compound (1) and (2) are also examined by fluorescence spectroscopy. The sensor
shows excellent selectivity with fluorescence enhancement to copper over other cations in
ethanolic solution. Chelating functionality of compound (1) and (2) were evaluated for their
inhibitory properties against various human cancer proteins like 4LRH, 4EKD, 4GIW and 4L9K
using online docking Server. From the cytotoxicity study, the ability of these compounds (1) and
(2) to inhibit the growth of KB cell lines. A combined experimental and theoretical studies were
carried out on the molecular structure using density functional methods (B3LYP) invoking
6ꢀ31G basis set. The density functional theory (DFT) calculation was carried out for both
compounds. Energy of the highest occupied molecular (HOMO) orbital and lowest unoccupied
(LUMO) molecular orbital have been predicted.

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Introduction
An improvement of expedient selective fluorescent chemosensors for the finding of
important species, such as certain transition, nonꢀtransition metal ions and anions is of current
interest because of a variety of biological and environmental evils which cause serious problems
for human health and ecology. Chemical sensing refers to the continuous monitoring of the
presence of chemical species.1ꢀ5 Many disciplines need sensing systems, including chemistry,
biology, clinical biology and environmental science.
Some money consuming and complicated methods, such as atomic absorption6 and
Inductively coupled plasma, atomic emission spectroscopy,7 for the detection of toxic metal ions
have been employed to detect. Compared with all of these sophisticated experimental methods,
fluorescent chemosensors are often used to detect many ions due to their simplicity and
sensitivity, low cost, high selectivity and instantaneous response.8, 9 The classical design of a
fluorescent indicator includes two moieties, a receptor responsible for the molecular recognition
of the analyte and a fluorophore responsible of signaling the recognition event. Copper is a one
of the vital element in the human body, a coꢀfactor in a variety of enzymes and copperꢀbased
pigments. Despite its significant roles in organisms, the accumulation of excess amounts of
copper ions or their misregulation can cause a series of cruel diseases. It is believed that the
disruption of copper homeostasis is implicated to some neurodegenerative disorders like
Alzheimer’s and Parkinson’s diseases.10, 11 The past years have seen increasing interest in design
and synthesis of fluorescent chemosensors with a Cu2+ induced “turnꢀon” fluorescence signal.
However, only few sensors are presently obtainable because the implementation of sensing
probes in functional devices without the loss of sensitivity is still very challenging.12 Lu et al.

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have reported a functional oligonucleotideꢀbased “turnꢀon” fluorescent probe for detection of
Cu2+ in aqueous solution with satisfying sensitivity and selectivity.13 Swamy et al, have
proposed a monoboronic acidꢀconjugated rhodamine based probe for Cu2+ with a reversible
fluorescence “turnꢀon” response and high selectivity, and they successfully applied it to image
Cu2+ in living cells and organisms.14
Aryl hydrazones are significant classes of compounds which have long fascinated
awareness, owing to their considerable biological and pharmacological properties, such as
antibacterial, antiviral, antineoplastic, and antimalarial behavior.15 The resonanceꢀassisted
hydrogen bond systems engage a synergistic reinforcement of hydrogen bonds by delocalization
of a pꢀconjugated chain connecting donor and acceptor atoms, they have been applied for the
activation of a carbon in αꢀposition to a carbonyl, induced enolization in keto–enol tautomerism,
controlled crystal packing, formation of Hꢀbonds in functional molecular materials, activation of
dinitriles towards formation of amidines, carboxamides and iminoesters, etc. while special
interest should be paid to the nature of the strong intramolecular O…H−N resonanceꢀassisted
hydrogen bond and its influence on the enolꢀazo
hydrazone transformation.16–19 The
rich tautomerism and isomerism of aryl hydrazones together with the intramolecular resonance
assisted hydrogen bond system can be applied for regulation of tautomerizationꢀisomerization,
activation of the carbon in αꢀposition to a carbonyl, antiferroelectric paraelectric transition,
regioselective activation of dinitriles, catalysis, ligand liberation, etc.20 In some cases hydrogen
bonding acts as an active site for initiation of chemical reaction. These results led to the
observation that the system exists in a conformation with a preferred orientation where the
stereoelectronic constraints and the stabilizing effect of hydrogen bonding are competing.

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In this article, we demonstrate the use of compound (1) and (2) as a highly efficient
fluorescent chemosensor for Cu2+. In terms of sensitivity concerns, chemosensors exhibiting
fluorescence enhancement (fluorescence “turn on”) upon Cu2+ ion complexation are favoured.
Among these detection approaches for the Cu2+ ion, fluorescence spectroscopy was used because
of its high sensitivity, high selectivity and low cost. Our sensor shows simple and good
21–28
selectivity compared to the recently developed Cu2+ sensors
association constants for the binding of Cu2+.
attributed to the very high
Experimental
All of the solvents were analytical reagent grade. The homogeneity of the compounds
was monitored by ascending thin layer chromatography (TLC) on silica gelꢀG (Merck) coated
aluminum plates, visualized by iodine vapor and UV light. IR spectra were recorded on an
Avatar Nicholet FTꢀIR spectrophotometer (range 4000–400 cmꢀ1) in KBr pellets (λmax in cmꢀ1).
13
1H and C NMR spectra for analytical purpose were recorded in CDCl3 on a Bruker instrument
at 400 MHz, chemical shifts are expressed in δꢀscale downfield from TMS as an internal
standard. SEM analysis was performed for surface morphology of compound (1), compound (2)
and corresponding complexes with Cu2+, on platinum coated samples using a JEOL JSMꢀ5610
SEM. EDS analysis was performed on using a JEOL JSMꢀ5610 SEM equipped with EDS. UV–
vis spectra were recorded on a SHIMADZU UVꢀ1650PC spectrophotometer by dissolving the
sample in spectral grade ethanol using a 1 cm path length quartz cell. A Perkin Elmer LS 55
fluorescence spectrometer was employed to record the fluorescence (FL) spectra at room
temperature. The choice of excitation wavelengths was based on the absorbance spectral
characteristics.

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Preparation of Compounds
Compounds were prepared by the general procedure (Scheme 1) which anilines (0.5
mmol) were dissolved separately in 1 N HCl (25 cm3) at 0ꢀ5 °C temperature and in each case
cooled aqueous solution (10 cm3) of NaNO2 (0.40 g) was added drop wise with stirring followed
by the addition of indeneꢀ1,3ꢀdione (0.73 g, 0.5 mmol) and sodium acetate (5.0 g) dissolved in
water (30 cm3). Corresponding mixtures were further stirred for 4 h at room temperature (25 °C).
Solids thus obtained were filtered and washed several times with water followed by ethanol and
then dried in vacuum. The crude products were recrystallized in ethanol. Compounds (1) and (2)
were purified by column chromatography by using benzene as eluent. Yield and melting points
of the derived compounds are mention below.
Computational details
Theoretical investigations on Compounds (1) and (2) , Compounds (1) and (2) ꢀCu2+ by
the GAUSSIAN 03W program package,29 invoking gradient geometry optimization at the
B3LYP level and the LANL2DZ effective core potential was used for all of the atoms.30 Initial
geometry generated from standard geometrical parameters was minimized without any constraint
in the potential energy surface at ab initio adopting the standard 6ꢀ31G basis set.
Results and discussion
2-(2-phenyl)hydrazono)-2H-indene-1,3-dione (1)
yellow solid, yield: 92 %, m. p. 197 °C; IR (KBr, cmꢀ1) 3421, 3083, 3043, 3018, 2922, 2850,
1
1718, 1664, 1539 (Fig. S1); H NMR (400 MHz, CDCl3) (δ): 7.22ꢀ7.96 (aromatic protons),

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13.48 (s, NH) (Fig. S3); 13C NMR (125 MHz, CDCl3) (δ) 116.3, 122.9, 123.2, 125.7, 126.3,
127.3, 128.3, 129.7, 135.0, 135.3, 140.6, 141.0, 144.1, 186.1, 188.0 (Fig. S4).
2-(2-(4-methoxyphenyl)hydrazono)-2H-indene-1,3-dione (2)
Orange solid, yield: 87 %, m. p. 190 °C; IR (KBr, cmꢀ1) 3431, 3154, 2962, 2923, 2851, 1711,
1
1671, 1592 (Fig. S2); H NMR (400 MHz, CDCl3) (δ): 3.85 (s, OCH3), 6.95ꢀ7.93 (aromatic
13
protons), 13.63 (s, NH) (Fig. S5); C NMR (125 MHz, CDCl3) (δ) 55.7, 115.0, 117.8, 122.6,
123.0, 129.9, 134.5, 134.7, 135.0, 138.7, 140.4, 158.5, 186.4, 188.9 (Fig. S6).
The arylhydrazones of 2ꢀ(2ꢀphenyl)hydrazono)ꢀ2Hꢀindeneꢀ1,3ꢀdione (1) and
2ꢀ(2ꢀ(4ꢀmethoxyphenyl)hydrazono)ꢀ2Hꢀindeneꢀ1,3ꢀdione (2) have been synthesized and their IR
spectra of 1 and 2 show ν(NH) vibration at 3421ꢀ3431 cm–1, while ν(C=O), ν(C=O...H) and ν(C=N) are
observed at 1718–1711, 1664–1671 and 1539–1592 cm–1 respectively (Fig. S1 and S2). These
bands can be related to the intramolecular hydrogen bonded hydrazone fragment. This
conclusion was further supported by the 1H and 13C NMR spectrum, the 1H NMR spectra of (1)
and (2) show signals at 13.48ꢀ13.63 ppm which can be assigned to the proton of the NH moiety
adjacent to aryl unit. In compound 2 the methoxy proton appeared at 3.85 ppm. In compound 1
and 2 the aromatic proton signals appear in the downfield region of 6.95–7.96 ppm with the
expected splitting patterns. Carbonyl sp2 carbon atoms appear as separate signals in the low field
region of about 186 and 188 ppm for Cꢀ(1) and Cꢀ(2), respectively. Strong intramolecular N–
H……O–C hydrogen bonding deshields Cꢀ(1) with respect to Cꢀ(2) to the extent of ca. 2 ppm.
Solvatochromism
The absorbtion spectra of compounds (1) and (2) were measured in various solvents of
different polarity and shown in figure S7A and S7B . The longest wavelength absorption band

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has been assigned to n – π* transition. This band is associated with a charge transfer from NꢀH
group to the carbonyl oxygen (C=O) due to intramolecular hydrogen bonding between them.
This intramolecular hydrogen bond is responsible for the stabilization of the charge transfer
band. A shoulder at shorter wavelength around 480 nm is due to π – π* transition. In particular
for hydazone derivatives charge transfer between a donor part, N–H (D) and an acceptor part,
indene1,3ꢀdione (C=O), has been considered. Both donor and acceptor appear to be localized in
the quasi aromatic rings (Fig. 9) containing the intramolecular hydrogen bonds. So, these
hydrogenꢀbonded quasiꢀaromatic rings contribute to the stabilization of the charge transfer band.
The emission spectra of compounds (1) and (2) were studied in various solvents. The
results are summarized in Table S1 and the spectrum of compound (1) and (2) in various solvents
are depicted in Fig. S8A and S8B. The compound (1) shows two emission bands: one band
occurs at around 460 nm and another band occurs at around 480 nm. It is assumed that the longer
one for Charge Transfer (CT) and the shorter one for Local Excitation (LE) 31,32,33. In both the
compounds the Photoinduced Electron Transfer(PET) from donor to the acceptor produces a low
lying anomalous CT state which leads to new longer wavelength fluorescence. Emission
maximum of the both the states found to be independent of the excitation Wavelength. In protic
solvents, protonation of the fluorophore increases oxidation state of donor group which results in
the LE of fluorescence only. The LE state is more enhanced in intensity when compared to CT
state due to the increased charge density on nitrogen.
Similarly compound (2) has LE at 485 nm and CT band at 515 nm. The variations in
solvatochromic properties of compounds (1) and (2) depend on the substitution pattern. The red
shift of the emission maximum increases in ꢀOCH3 substituted compound (2) when compared to
compound (1). This observation reveals the significant influence of Hꢀbonding on the

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solvatochromic properties, presumably as a result of the stabilization of the negative charge at
the carbonyl oxygen atoms during solvent relaxation.
To better understand the solvent polarity effect, the Lippert–Mataga relation was applied.
This relation has been widely used to correlate the energy difference between absorption (νa) and
emission (νf), also known as Stokes shift, with solvent polarity represented by ∆f. This relation
was given in equation. (1). It involves both the dielectric constant and the refractive index (n) of
the solvents.34, 35
(1)
In equation (1), νa and νf are the wavenumbers (cm−1) corresponding to the absorption and the
emission, respectively, h is Planck’s constant, c is the speed of light, and a is the radius of the
solvent cavity in which the fluorophore resides. For an elongated shape of the molecule, a is
usually estimated as 40% of its longest axis.36 The term (∆f) involving ε and n is called the
orientation polarizability, which only accounts for the spectral shifts due to the reorientation of
the solvent molecules. Therefore, the Lippert–Mataga relation is based on the assumption that
the energy difference is only proportional to the solvent orientation polarizability (known as the
general solvent effect). Inability of Stokes’ shift to increase linearly with ∆f usually implies that
specific solvent effects are involved.
(2)

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(Fig. S9). showed the Lippert–Mataga plots of compounds (1) and (2) in 13 organic solvents.
The estimation from the Lippert–Mataga equation is based on the assumption that the
photophysical properties of compounds (1) and (2) can be described by the theory of general
solvent effect; hence, it may not hold if specific solvent effects are involved. Stokes shift slightly
increases with increasing polarity of the solvent (as it is expected based on the CT character of
the excited state) and does not follow a linear dependence on f(ε, n) in all solvents used, ie.,
solvent specific interactions observed. The linear variation of the Stokes shift with ET (30) (with
solvent polarity parameter) has been shown in Fig. S10. The compounds (1) and (2) exhibit an
slightly increase or no increase of Stokes shift from nonꢀpolar to polar aprotic solvents mainly
due to the combined effect of increasing the polarity of the medium and intramolecular charge
transfer (CT) state and it confirmed that solvent polarity cannot affect the stoke shift of
compounds (1) and (2). The νA– νF value is unusually small in hexane and remarkably deviates
from the linear correlation. This deviation is particularly connected with decrease of λF in hexane
and indicates different charge redistribution of the excited state of 1 than in other solvents. This
suggested that the solvent polarity might not be the only factor affecting the spectral shifts.
Specific solvent effects including hydrogen bonding, acid–base chemistry, and charge transfer
interactions can also result in nonlinear Lippert–Mataga plots.
Chemosensor
The response of compounds (1) and (2) to various metal ions were investigated in
aqueous buffer. It displays a very distinctive selectivity for copper ions, as observed by the
fluorescence spectroscopy. When compounds (1) and (2) were treated with various metal ions,
such as Cd2+, Ni2+ , Pb2+, Zn2+, Ag+, Hg2+, Cu2+, Mn2+, Fe2+, Cr3+, K+, Fe2+, Fe3+, Al3+and Co2+ in
the buffer, complete fluorescence enhancement was observed only for Cu2+ ions not for other

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metal ions. The fluorescence responses of the sensors (1) and (2) to various cations and its
selectivity for Cu2+ were illustrated in Fig. 6A and 7A. The fluoroionophoric behavior of sensors
(1) and (2) were investigated in ethanol–water (1: 1, v/v) solution [sensor (1) and (2)] = 1 and 2
ꢁM, [Cu2+] = 1 ꢁM, as expected, sensors (1) and (2) showed a weak fluorescence in aqueous
solution. Upon addition of different metal ions, only with Cu2+, did sensors (1) and (2) show
remarkable fluorescence enhancement due to coordination to a paramagnetic Cu2+ centre (Fig.
1A and 2A).
Based on earlier studies37–39 and our own observation we propose the following
mechanism. Cu2+ is a well–known paramagnetic ion with an unfilled ‘d’ shell and could strongly
quench the fluorescence of the fluorophore near it via electron or energy transfer. Thus it is of
interest to us that the Cu2+ by compounds (1) and (2) does not quench the fluorescence. It is
known that the nitrogen lone pair electrons of compounds (1) and (2) can quench the
fluorescence of the indene 1,3ꢀdione moiety through photo induced electron transfer (PET) 40ꢀ44
.
Masking of the nitrogen lone pair electrons cause a suppression of their fluorescence quenching
and therefore, results increase in fluorescence intensity. It is evident from the emission spectra
that in the absence of Cu2+ the sensor exhibited weak fluorescence owing to the quenching of
compounds (1) and (2) fluorescence through PET. And also in the presence of the quenching
metal ions, the relative contribution of two opposing factors, the metalꢀion receptor binding that
leads to enhancement of the fluorescence signal and the fluorophoreꢀmetal ion interaction that
leads to fluorophore quenching determines the net effect. Therefore possible to observe
fluorescence enhancement in the presence of quenching ions when metal bindingꢀinduced
enhancement of fluorescence intensity is greater, than the quenchingꢀ induced reduction in the
fluorescence intensity. So, it is clear that for an already PET Quenched system, otherwise

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strongly quenching metal ion acts as a poor quencher. Obviously, in the absence of quenching
interaction, metal ion binding with receptor will only lead to fluorescence enhancement. Finally
we conclude that unavailability of the lone pair electrons of ‘N’ for PET caused fluorescence
enhancement.
The emission at around 500 nm in (1) and 510 nm in (2) did not change when the concentration
of Cu2+ increased from 1 ꢁmol/L compound (1) and (2) in 1:1(volume ratio) CH3CH2OH/H2O
(10 mmol/L 10 ꢁM HEPES buffer, pH=7.0) binding to Cu2+ with different concentrations ion.
It implying a 1:1 complexation of compounds (1) and (2) with Cu2+ ions (Fig. 1B and 2B). .The
maximum fluorescence enhancement was observed in the presence of 2 ꢁm (1) and 1.8 ꢁm in
(2). When the concentration of Cu2+ was less or equal to around 2 ꢁm, the binding of Cu2+ made
the polarization of the molecule larger, and it allowed only partial electron transfer, therefore the
fluorescence intensity was enhanced but the emission maximum did not change with the increase
of Cu2+ concentration; whilst the concentration of Cu2+ was more than 2 ꢁm, excess Cu2+
resulted in a sudden drop of relative intensity was observed, without question due to
concentration quenching45.
Furthermore, the relationship of fluorescence enhancement ratio(defined as I/I0, where I
and I0 are the fluorescence intensities of compound (1) and (2) in solution when binding with and
without Cu2+, respectively) with the binding concentration of Cu2+ was also investigated.
Apparently, the ratio was nearly proportional to the molar binding concentration of Cu2+ (R2=
0.9941 (1) and R2=0.9986 (2)) when the binding concentration of copper was in a range of
0―5.0 ꢁmol/L (Fig.1c and 2c).

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pH-Dependent Behavior
The influence of pH on chemosensor of compounds (1) and (2) were studied using UVꢀ
visꢀspectrocopy (Fig. S11). Over a pH range of 3ꢀ8, the visible absorption band centered at 480
nm in (1) and 450 nm in (2) were unchanged. But increase in pH from 9 to 11 engendered a shift
in the maximum absorption wavelength to 375 nm (1) and 380 nm (2) with color change of
yellow to blue. This difference were due to the dissociation of (1)ꢀCu2+ and (2)ꢀCu2+ complex,
which result in lower absorbance and color change from yellow to blue.
The pH emission spectra of sensors (1) and (2) and its ability to detect the Cu2+ cations
might be influenced by the pH of the solution because of the nitrogen atom of hydrazone moiety
with lone pair electrons in the sensors (1) and (2) .To verify this hypothesis, the pH effect on the
fluorescence intensity of sensors (1) and (2) in the unbound and bound of Cu2+ was studied. As
shown in Fig. S12, in the unbound of Cu2+ (curve b), sensors (1) and (2) presents a low
fluorescence intensity which remain stable and did not change with the pH. Upon addition of
Cu2+ (curve a), the emission intensity of compounds (1) and (2) with Cu2+ increased dramatically
from pH 3 to 5, resulting from the competition between the NꢀH proton and Cu2+ ion46, 47. In
particular, slight significant change in fluorescence spectra were observed in the range of pH 5ꢀ8
and decresed under alkaline condition with a color change of yellow to blue .The quenching at
higher pH could be well explained by the formation of Cu(OH)ꢀ or Cu(OH)2 and thus reducing
the concentration of Cu2+ꢀ (1) / (2)48 and therefore there are fewer ions as Cu2+ in solution that
are able to be complexed with compounds (1) and (2) which explains the decrease of the
fluorescence intensity. The results demonstrate that sensors (1) and (2) could be used in the
environment where the pH ranges from 5 to 8, which covers the physiological pH conditions.

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To prove the presence of C, O, N and Cu in the surface of the complexes (corresponds to
compound (1) and (2)) using Elemental mapping of FEꢀSEM was carried out. Fig. 3a and Fig. 4a
are exhibits FEꢀSEM color images of complexes (Compound (1)ꢀCu2+ and (2)ꢀCu2+), Fig. 3b–e
and 4bꢀe shows the elemental mapping for carbon, nitrogen, oxygen and copper respectively. It
is evident from that the Fig. 3b–d and 4b–d, carbon, nitrogen and oxygen are higher in density.
There is a homogenous distribution of carbon, nitrogen, oxygen and copper (Fig. 3a and 4a).
Thus elemental mapping shows that the complex of compound (1) and (2) contains Cu2+. This
also indicates the purity of the complexes (compound (1)ꢀCu2+ and (2)ꢀCu2+). EDS is generally
accurate up to trace amount of metal present in the surface of complexes. The EDS recorded
from the selected area is shown in Fig. 3f and 4f, which reveals the presence of C, O, N and Cu
in the complexes.
Docking Studies
Molecular docking technique is an eyeꢀcatching scaffold to understand between the
ligand and protein interactions. We have studied various cancer proteins like 4LRH, 4L9K,
4EKD and 4GIW from Protein data bank. Along with these proteins only 4LRH cancer protein
shows good results while compared to other binding energy values. Molecular docking
simulation was employed to predict interaction of compound (1) and (2) into 4LRH. We docked
compound (1) and (2) with 4LRH as shown in Fig. 5 and 6. The value of free energy binding
(∆E) and binding constant (ki) of compound (1) and (2) are given in table 1. The molecular
docking simulation shows that compound (1) has less activity compared with compound (2). The
above conclusion is proved from the hydrogen bond formations. The simulations for compound
(1) and (2) were carried out with other three cancer proteins (4L9K, 4EKD and 4GIW) as shown
in Figs. S17ꢀS22 respectively. From the figures it is evident that 4LRH docks strongly with

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compound (1) and (2). As shown in figures hydrogen bond distance between compound (2) and
important residue was smaller than hydrogen bond distance between compound (1) and residues.
In addition, hydrophobic interaction of compound (2)ꢀ 4LRH was more strongly than compound
(1)ꢀ 4LRH. The docked pose displayed in Fig. 5a, 5b, 6a and 6b reveals the subdomain IB of the
protein to be the favorable binding site for the drug. The principal hydrophobic binding sites in
4LRH are located in domains II and III, while domain I, characterized by a net negative charge,
normally serves as an suitable binding site for cationic probe molecules. The lowest binding
energy conformer was obtained from 4 different conformers for every docking simulation and
the consequential minimum energy conformation was used for further analysis. As is usual in a
blind docking simulation protocol, we obtained a number of binding sites and the corresponding
binding constants and free energies. Compelling evidence for the probableꢀbinding location of
the drug in subdomain IB of 4LRH was derived from the observation that the binding of
compounds (1) and (2) in the IB region was found to be characterized by a favorable binding
energy of – 8.25 kcal molꢀ1 and – 8.29 kcal molꢀ1 respectively (Table 1), a very low inhibition
constant (900.66 nM) for Compound (1) and (837.70 nM) for Compound (2). Fig. 5c marks the
protein residues in the near vicinity (within 4 Å) of the probe, which shows the presence of both
hydrophobic residues (TYRꢀ60, TRPꢀ171, and TYRꢀ85), as well as charged/polar residues (TRPꢀ
102 (cationꢀpi), TRPꢀ140 and HISꢀ135) for compound (1) and hydrophobic residues (TRPꢀ171,
TYRꢀ85, PHE‒62, TRPꢀ140 and TRPꢀ138) as well as charged/polar residues (TYRꢀ60 (cationꢀ
pi), HISꢀ135 (hydrogen bond), ARGꢀ103) for compound (2)(Fig. 6c) which evinces the binding
phenomenon to be mainly governed by hydrophobic forces with a significant contribution from
electrostatic interactions. Asymmetric charge distribution over the molecule because of the
presence of heteroatoms might have been responsible for such type of binding. Based on

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molecular docking results, we conclude that alkyl group has been more responsible into
biological activity than aromatic ring.
Cytotoxicity
The cytotoxicity responses of compound (1) and (2) with various concentrations added
are clearly evident from the cellular imaging. Hence, these results indicate that compound (1)
and (2) is an efficient candidate for monitoring changes in the intracellular concentration under
certain biological conditions; in order to test its cytotoxicity, we performed MTT assay in KB
cancer cells treated with various concentrations of compound (1) and (2) for up to 5 h. As shown
in Figs. 7 and 8, Compounds (1) and (2) did show significant cytotoxic effects on KB cancer
cells for at least up to 4 h. The synthesized compound (1) and (2) were examined for their
cytotoxic properties on KB cell line by means of MTT test that allows us to evaluate the toxic
effect of above compounds on cellular mitochondrial metabolism. Cells were tested for 48 h with
increasing concentrations of tested compounds. Microscopic images of control cancer cells and
apoptotic morphological changes in KB cell line treated with compound (1) and (2) are shown in
Fig. 7 and 8 respectively. The results indicate that the compound (1) showed minimum cell death
when compared to compound (2). The compounds (1) and (2) exhibit broad inhibition on the KB
cell lines with IC50 values of 32.78 and 40.08 respectively. The IC50 values of the compound
(Fig. S23) suggest that compound (2) possessed more potent inhibitory effect against the cancer
cells. Compound (2) carrying the –OCH3 group in para position and shows the highest IC50
value, convincing us to propose that the electronic effect may be one of the factors in
determining the anticancer activities of compound (2).49, 50

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Theoretical calculations
The geometries of the sensor compound (1) and compound (2) with Cu2+ ion were optimized
using DFTꢀB3LYP 6ꢀ31 G and LANL2DZ (d) levels respectively using Gaussian 03 package. In
Fig. S24 and S25 the optimized geometries of compound (1), compound (2) and its 1 : 1 complex
with Cu2+ ion are given, which displays the effective binding sites, namely –NH and C=O for
Cu2+ ion. To get an insight into the electronic behavior in the presence and absence of Cu2+ with
compound (1) and compound (2), TDꢀDFT calculations were carried out using the same level.
Plotting of HOMO and LUMO of compound (2) that the πꢀelectrons on the HOMO of ligand
focused on one of the ketone group of dione moity and lumo of the ligand focused on NꢀNH
group of hydrazone moiety. When Cu2+ is added, it shows a significant change in the distribution
of the πꢀelectrons on HOMO and LUMO. More significantly,the HOMO and LUMO densities in
compound (1) and (2) with Cu2+ are delocalized over the whole molecule except phenyl ring of
dione moiety in HOMO and methoxy phenyl ring in LUMO. The energies of HOMO and LUMO
of compound(1) and compound (2)ꢀCu2+ complex are slightly lower than that of compound(1)
and compound (2). More importantly, the decreasing energy in the LUMO is more obviously
than that of the HOMO. It suggests a smaller HOMOꢀLUMO gap and a bathochromic shift
compared with compounds in emission which may result from the increase of conjugation of the
complex.
Conclusion
In summary, the hydrazones of indeneꢀ1,3ꢀdione have been synthesized and characterized IR, 1H,
13C NMR spectra studies. The FTꢀIR, NMR, a promising analytical approach for detecting Cu2+
in ethanol : water solutions. When copper ion was added in (1) and (2) their morphology changes
were observed. We have prepared a simple type of fluorescent “turn–on” chemosensor based on

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hydrazones shows interesting properties such as high sensitivity for Cu2+. It possesses a high
affinity and selectivity for Copper ions relative to most other competitive metal ions by
enhancement of the monomer fluorescence emission of hydrazones in organic aqueous solution.
We expect that the present design strategy and the remarkable photophysical properties of this
sensor will help to extend applications of fluorescent sensors for metal ions. Compounds (1) and
(2) docking with 4LRH, 4L9K, 4EKD and 4GIW proteins only 4LRH cancer protein shows good
results while compared to other proteins binding energy values. Compound (1) and (2) has
potent invitro cytotoxic against KB cell line. In this present investigation molecular structure,
HOMO, LUMO, Mulliken charges and MEP analysis have been studied using ab initio DFT
B3LYP/6ꢀ31G calculation.
Acknowledgment
We are grateful to Dr. Nagarajen Rajendraprasad, Assistant Professor, Department of
Biochemistry and Biotechnology, Annamalai University, Annamalainagar, to study Cytotoxic
activity.

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Figure captions and figures
Fig. 1(A) Fluorescence emission spectra of chemosensors for compound (1) (1ꢀmol/L) with
different metal ions (perchlorate, chloride, or nitrate salts of (a) Cu2+, (b) Ni2+, (c) Co2+, (d)
Zn2+, (e) Pb2+, (f) Hg2+, (g) Mg2+, (h) Cr3+, (i) Na+, (j) K+, (k) Fe2+, (l) Fe3+, (m) Al3+, (n) Cd2+
and (o) Ag+) in aq CH3CH2OH (CH3CH2OH/H2O = 1:1, v/v, 10 ꢀM HEPES buffer, pH =
7.0), (B) Emission spectra of compound (1) in the presence of an increasing Cu2+
concentration Cu2+ /(ꢀmol Lꢁ1) from a to o 0, 0.2, 0.6, 0.8, 1.0, 1.2, 1.4, 1.2, 1.4, 1.6, 1.8, 2,
2.5, 3.5, 4.0, 4.5, 5.5 1 ꢀmol/L compound (1) in 1:1(volume ratio) CH3CH2OH/H2O (10
mmol/L 10 ꢀM HEPES buffer, pH=7.0) binding to Cu2+ with different concentrations.(c)
Fluorescence enhancement relationship of I/I0 with [Cu2+]/[compound(1)].
Fig. 2 (A) Fluorescence emission spectra of 10ꢀm solutions chemosensors for compound (2)
with different metal ions (perchlorate, chloride, or nitrate salts of (a) Cu2+, (b) Ni2+, (c) Co2+,
(d) Zn2+, (e) Pb2+, (f) Hg2+, (g) Mg2+, (h) Cr3+, (i) Na+, (j) K+, (k) Fe2+, (l) Fe3+, (m) Al3+, (n)
Cd2+ and (o) Ag+) in aq CH3CH2OH (CH3CH2OH/H2O = 4/1, v/v, 10 ꢀM HEPES buffer, pH
= 7.0), (B) Emission spectra of compound (2) in the presence of an increasing Cu2+
concentration Cu2+ /(ꢀmol Lꢁ1) from a to o 0, 0.2, 0.6, 0.8, 1.0, 1.2, 1.4, 1.2, 1.4, 1.6, 1.8, 2,
2.5, 3.5, 4.0, 4.5, 5.5 1 ꢀmol/L compound (2) in 1:1(volume ratio) CH3CH2OH/H2O (10
mmol/L 10 ꢀM HEPES buffer, pH=7.0) binding to Cu2+ with different concentrations ion.(c)
Fluorescence enhancement relationship of I/I0 with [Cu2+]/[compound(2)].
Fig. 3 FEꢁSEM elemental colour mapping image of compound (1)ꢁCu2+(a) survey spectrum,
(b) C, (c) N (d) O (e) Cu and (f) EDS spectra.
Fig. 4 FEꢁSEM elemental colour mapping image of compound (2)ꢁCu2+ (a) survey spectrum,
(b) C, (c) N, (d) O, (e) Cu and (f) EDS spectra.

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Fig. 5 Molecular docking studies of compound (1) with 4LRH.
Fig. 6 Molecular docking studies of compound (2) with 4LRH.
Fig. 7 Live Cell images of compound (1): (a) before and (b and c) after treatment with
Compound (1) examined by fluorescence microscopy.
Fig. 8 Live Cell images of compound (2): (a) before and (b and c) after treatment with
Compound (1) examined by fluorescence microscopy.
Fig. 9 Photoinduced Electron Transfer (PET) of compound (1) and compound (2) with Cu2+.
Scheme 1 Schematic representation of synthesis of Hydrozone.

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Fig. 1

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Fig. 2

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Fig. 3

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Fig. 4