# Cu(II) Complexes of Isoniazid Schiff Bases: DNA/BSA Binding and Cytotoxicity Studies on A549 Cell Line

Authors: Pulipaka Ramadevi; Rinky Singh; Akhilesh Prajapati; Sarita Gupta; Debjani Chakraborty
Journal: Advances in Chemistry
 (2014)

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## Abstract

A series of isonicotinoyl hydrazones have been synthesized via template method and were complexed to Cu(II). The ligands are coordinated to Cu(II) ion through the enolic oxygen and azomethine nitrogen resulting in a square planar geometry. The CT-DNA and bovine serum albumin binding propensities of the compounds were determined spectrophotometrically, the results of which indicate good binding propensity of complexes to DNA and BSA with high binding constant values. Furthermore, the compounds have been investigated for their cytotoxicities on A549 human lung cancer cell. Also the mode of cell death was examined employing various staining techniques and was found to be apoptotic.


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## Body

## 1. Introduction

A wide range of Schiff’s bases with their reactive azomethine linkage show interesting inhibitory activity against tumor cells. Schiff’s bases could be hydrolyzed selectively by the tumor cells to act as alkylating agents; at the same time the active amine becomes free to act as antimetabolite [1]. Studies show that the metal complexes of Schiff’s base ligands have better antimicrobial and anticancer activities as compared to Schiff’s bases [2].Copper is well known as a bioessential element and its complexes have proven to be excellent candidates for biological applications due to their binding ability and positive redox potential [3–6]. Cu(II) complexes containing heterocyclic bases have been extensively explored in virtue of their strong interactions with DNA and cytotoxic activity [7–9]. They can act as chemical nucleases [10, 11] and their cytotoxicity has been proposed to be caused by their ability to bind and cleave DNA that leads to cell cycle arrest and apoptosis or generation of reactive oxygen species (ROS) that in turn leads to cell death [12]. Literature has shown that Cu(II)-based complexes exhibit antineoplastic potency towards human ovarian carcinoma (CH1), murine leukemia (L1210), and various cervicouterine carcinomas which is comparable to or even higher than that of cisplatin [13, 14]. Fei et al. in their article [15] have reported the in vitro cytotoxicity of two copper-2,2′-bipyridine complexes towards five selected tumor cell lines HepG-2, HeLa, NCI-H460, MCF-7, and HL-6 with IC50 value at a concentration of 50 μM. Cu(II) complexes of Schiff bases can show their antibacterial and antiproliferative activities because of the properties of the metal center or the coordinated ligands alone, as well as the structural and electronic properties which is ascribed to the coordination [16–21]. Several Schiff base ligands and their Cu(II) complexes have been investigated to show apoptotic activity against various cancer cell lines [22]. Oxoquinoline based Schiff base copper complexes have been synthesized and investigated upon and their IC50 values were found to be in the range of 8–19 μM against HeLa and HL-60 cells [23].INH is a first generation antitubercular drug whose derivatives and their metal complexes have been subjected to a lot of research for their antitubercular activities but their potentials as anticancer agents have remained largely unexplored. Their potency as anticancer agent is reflected from the little work that has been published so far wherein a few researchers have synthesized hydrazones of INH and their iron complexes and evaluated them for their antitumoral activity suggesting a mechanism of inhibition of ribonucleotide reductase [24].The research interest in the present work stems from the aforementioned points. With this view point we herein report Ni(II) mediated synthesis and characterization of four Schiff’s bases of isonicotinoyl hydrazine (INH) by its condensation with four different aromatic aldehydes and their Cu(II) complexes. The synthesized isonicotinoyl hydrazones and their Cu(II) complexes were studied for their interactions with DNA and BSA. The results were indicative of better binding efficiencies of the copper bound ligands compared to the ligands themselves. Based on the results of their binding studies the ligands and their copper complexes were evaluated for their anticancer activity against human lung carcinoma A549 cell lines. Furthermore the mode of interaction of these complexes with A549 cells has been evaluated employing various staining techniques to assess the mechanism of cell death, via excessive ROS generation and/or nuclear condensation.


## 2. Results and Discussion

### 2.1. Synthesis and Characterization

In a general sense, transition metal-based catalysis can be viewed as template reactions where reactants coordinate to adjacent sites on the metal ion and, owing to their adjacency, the two reactants interconnect (insert or couple) either directly or via the action of another reagent. Template effects may arise from stereochemistry imposed by metal ion coordination of some of the reactants, promoting a series of controlled steps and characteristically providing routes to products not formed in the absence of the metal ion. Appreciable amount of work has been done on one pot multicomponent synthesis of Schiff bases using transition metal ions as templates [25–28]. Following this insight we carried out the Ni(II) mediated Schiff base synthesis (Figure 1) and found that the reaction time had reduced and product yields had increased markedly.Figure 1
Schematic representation of synthetic route for the ligandsL1–L4 and their Cu(II) complexesC5–C8.The IR spectra of complexesC5–C8 lack the strong secondary amide carbonyl absorption at 1650–1700 cm−1 that is typically seen in the spectra of the free ligandsL1–L4. In all the complexes, the νC=N band is shifted to lower frequency between 1570 and 1610 cm−1 indicating coordination of the Schiff bases through the azomethine nitrogen [29]. Moreover, the enolate structure of the coordinated ligand is supported by a band at 1059-1060 cm−1 due to the enolic C-O stretching [30]. The N-H stretching bands in the range 3150–3250 cm−1 found in the IR spectra ofL1–L4 are completely lost in the spectra of theirC5–C8 which is also indicative of the enolization and deprotonation on coordination.The UV spectra of the ligands and their complexes show bands in the wavelength range 200–350 nm. In the UV spectra of the ligands (Figure2) the first band appearing within 200–270 nm region can be assigned to the medium energy π-π* transition of the aromatic rings. The second band observed within 280–340 nm region is due to the excitation of the electrons of the azomethine group which corresponds to an intraligand n→π* transition [31]. In case ofL2 (Figure S1 in the Supplementary Material) there is a third band located at 326 nm that can be ascribed to charge transfer within the entire Schiff base molecule. This band is commonly observed in o-hydroxyl Schiff bases [32] and is based on strong intramolecular hydrogen bonding between the hydroxyl group of the salicylidene and the azomethine nitrogen [33]. The UV spectra of complexes (Figure 2) show the first band at nearly the same wave length as in the spectra of their corresponding ligands which reveal that these (π-π*) transitions are not significantly affected by chelation. On the other hand the band corresponding to azomethine showed broadening and a shift to longer wavelength on going from ligand to complex, indicating coordination of ligand to metal through the azomethine moiety [34].Figure 2
UV spectra of ligandL1and corresponding Cu(II) complexC5 recorded at a concentration of 2.5 mM in DMSO with path length 1 cm.The mass spectra of ligandsL1–L4 show peaks corresponding to the molecular ions M+ atm/z = 225.09, 241.07, 271.09, and 255.09, respectively (Figure S2 in the Supplementary Material, available online at http://dx.doi.org/10.1155/2014/630575). In addition the mass spectra of all the ligands show prominent peaks atm/z = 106 and 78 corresponding to (pyridin-4-ylmethylidyne)oxonium and pyridin-4-ylium fragments, respectively. The ESI mass spectra of copper complexesC5–C8 show peaks atm/z = 511.7(M+), 546.1(M+ + 2), 603.7(M+), and 571.7(M+), respectively (Figure S3 in the Supplementary Material). Them/z values of the molecular ion peaks forC5–C8 indicate that two ligands are coordinated to the metal centre. Detailed interpretation of the mass fragments has been summarized in Table S1 in the Supplementary Material.The ESR spectra ofC6 andC7were recorded in solid state at 77 K (Figure S4 in the Supplementary Material) and that ofC8 (Figure 3) was recorded in DMSO at 120 K using 100 KHz field modulation and the g factors were quoted relative to the standard marker TCNE (g=2.00277). The g∥, g⊥, gav, and G values have been calculated and summarized in Table 1. The spectrum of the four coordinatedC8 in frozen DMSO resolved four copper hyperfine lines, characteristic of monomeric copper(II) complexes [35]. The observed g∥>g⊥>ge (2.0023) values indicate that the unpaired electron is predominantly in the dx2-y2 orbital giving a 2B1g ground state [36, 37] and the complexes have distorted square planar geometry. ForC6 andC7, the G-values are <4, suggesting the monomeric nature of copper(II) centres and consistent with a dx2-y2 ground state having a small exchange coupling in the solid state while forC8, G=4.7 indicates that there are no spin exchange interactions between the copper centres in solution [38]. The gav values for the complexes were found to be in the range 2.10–2.17. The deviation of the gav from that of the free electron (2.0023) is due to the covalent nature of the complexes [39].Table 1
ESR spectral parameters of complexesC6, C7, and C8.
Complex
g
∥
g
⊥
g
av
G
C6 (solid state, 77 K)
2.185
2.066
2.106
2.828
C7 (solid state, 77 K)
2.185
2.068
2.107
2.752
C8 (in DMSO, 120 K)
2.368
2.079
2.175
4.747Figure 3
ESR spectrum ofC8 in DMSO at 120 K using 100 KHz field modulation.Thermogravimetric analyses ofL1–C8 were carried out at 10°C per min in a temperature range 50–850°C in N2 atmosphere. The thermogram ofL1given in Figure 4 showed 99.6% weight loss in the range 200°C–320°C attributed to the total decomposition of the molecule. A continuous weight loss in the temperature range 200°C–750°C is observed in case ofC5 (Figure 4) attributed to the removal of the ligands leaving behind CuO2N2 as residue.Figure 4
Thermal degradation curves of ligandL1and complexC5 at heating rate of 10°C per minute under N2 atmosphere.The spectral (IR, EPR, UV, Mass) and the thermogravimetric analyses data of the ligands and the complexes are in full agreement with the proposed structures (Figure1).


### 2.2. DNA Binding Studies

#### 2.2.1. Absorption Studies

Transition metal complexes can bind to DNA through covalent bonding via replacement of a labile ligand of the complex by a nitrogen base of DNA such as guanine N7 [40] or noncovalent interactions such as intercalation, electrostatic, or groove binding [41]. Electronic absorption spectroscopy is one of the most useful techniques in DNA binding studies [42] since the observed changes in the spectra may give indication of the mode of interaction.Theπ-π* intraligand absorption bands with λmax⁡ in the region 200–270 nm were used to monitor the interaction of compoundsL1–C8 with double helical CT-DNA. The UV spectra have been recorded for a constant compound concentration in diverse [DNA]/[compound] ratios(r). The changes observed in the intraligand transition of the compoundsL1–C8 upon addition of CT-DNA solution in diverse r values may reveal the existence of interaction between each compound and DNA and may indicate the possible mode of binding.Upon increasing the CT-DNA concentration, the UV spectra of compound-DNA solutions show a gradual decrease in the intensities of the absorption bands (hypochromism) (Figure5) for all compounds without any apparent red shift. The observed hypochromism may be attributed to stacking interaction between the aromatic chromophores of the free and bound ligands and DNA base pairs probably due to intercalation.Figure 5
UV absorption spectra ofC5(12.5 μM) in tris buffer solution at increasing concentrations of CT-DNA. The arrow shows the change in the intensity of absorption upon addition of increasing amounts of CT-DNA at a [complex]/[DNA] ratio in the range 0–4.2.Inset. Plot of [DNA]/(εA-εf) versus [DNA] forC5. The slope to intercept ratio gave Kb values.Among the free ligands,L4 exhibits the maximum hypochromism of 15% followed byL1 andL2, each showing a hypochromism of 10%. The absorption bands of complexesC5,C6,C7, andC8 exhibit a maximum of 48, 24, 28, and 27% hypochromicity, respectively (Table 2). The hypochromism is more pronounced forC5. The complexes exhibit distinctively greater hypochromism compared to the free ligands which indicates that presence of a metal ion facilitates binding.Table 2
DNA binding constants (Kb), Stern-Volmer quenching constants, λmax⁡, and % hypochromism for the interaction of ligands L1–L4 and complexes C5–C8 with DNA.
Compound
K
b  (M−1)
λ
max
⁡ (nm)
% hypochromism
L1
5.7
×
10
4
299
11
L2
4.3
×
10
4
288
10
L3
1.7
×
10
4
326
9
L4
2.0
×
10
4
316
15
C5
1.8
×
10
4
325
48
C6
3.4
×
10
4
293
24
C7
1.4
×
10
4
332
28
C8
4.2
×
10
4
274
27The magnitude of binding strength to CT-DNA may be determined through the calculation of binding constantKb which is obtained by monitoring the changes in the absorbance at the corresponding λmax⁡ with increasing concentrations of CT-DNA. Kb is given by the ratio of slope to the y intercept in plots [DNA]/(εA-εf) versus [DNA] according to [43]
(1)[DNA](εA-εf)=[DNA](εb-εf)+1Kb(εb-εf),
where [DNA] is the concentration of DNA in base pairs, εA=Aobsd/[compound], εf is the extinction coefficient for the unbound compound (L1–C8), and εb is the extinction coefficient for the compound in the fully bound form. The values for Kb as calculated by (1) are given in Table 2 and the representative [DNA]/(εA-εf) versus [DNA] plot forC5 in the inset of Figure 5. Plots of remaining compounds are given in Figure S5 (Supplementary Material).The binding constants of the compounds range from 5.7 × 104 to 1.4 × 104 M−1 suggesting that the Schiff bases and their complexes strongly bind to CT-DNA. The highest Kb value (5.7 × 104 M−1) among the ligands was observed forL1 whereasC8 exhibited the highest Kb value (4.2 × 104 M−1) among the complexes. However the overall Kb values of the compounds are lower than those observed for classical intercalator EB (1.23 × 105 M−1) with a proven DNA binding mode involving intercalation of the planar phenanthridine ring between the adjacent base pairs on the double helix. This is indicative of the binding ofL1–C8 with CT-DNA with an affinity less than EB.The results derived from the UV-titration experiments suggest that all the compounds can bind to CT-DNA although the exact mode of binding cannot be merely proposed by UV-spectroscopic titration studies. In most cases, the existence of hypochromism could be considered as first evidence that the binding of the compounds involving intercalation between the base pairs of CT-DNA cannot be ruled out.


#### 2.2.2. DNA Ladder Assay

DNA ladder assay was carried out on A549 cells with two complexesC5 andC8 at three different concentrations (50 μM, 75 μM, and 100 μM) and a marker for reference (Figure 6), to further support the binding of compounds to DNA and to study its effect on the cancer cells. The results presented in Figure 6 show that both complexesC5 andC8 provoke DNA fragmentation in A549 cells. The first band seen in all the six lanes at different concentrations of the two complexes is of the intact DNA, while a smear can be seen in the lower half of the gel which is of fragmented DNA. Though the fragmentation process is weak, it is conclusive of an apoptotic effect of the compounds on the cancer cells.Figure 6
Photogenic view of cleavage of A549 cells with complexesC5andC8using 1.5% agarose gel containing ethidium bromide. Complexes were incubated for 24 h at 37°C.


### 2.3. BSA Binding Studies

Serum albumins are proteins involved in binding and transport of drugs through the blood stream [44]. Their binding with a drug may actually result in increase or decrease in the drug’s efficacy making it important to investigate the interactions of prospective drugs with serum albumins. BSA solutions exhibit a strong fluorescence emission with a peak at 343 nm, due to the tryptophan residues, when excited at 296 nm. The changes and quenching occurring in the fluorescence emission spectra of BSA upon addition of compoundsL1–C8 are primarily due to change in protein conformation, subunit association, substrate binding, or denaturation because of their binding [45].Addition ofL1–C8 to a solution of BSA results in a decrease of the fluorescence intensity (Figure 7). It is observed that the complexes show more % fluorescence quenching compared to the ligands (Figure 7, inset) indicating stronger protein binding of the ligands on complexation.Figure 7
Plot of fluorescence emission intensityI versus wavelength λ for BSA at different concentrations of compoundL1. The arrow shows the change in intensity of BSA emission on increasing amount of the compound at a [BSA]/[complex] ratio in the range 0–5.Inset. Plot of BSA % relative fluorescence intensity at λmax⁡ = 343 nm versus r (r=[Q]/[BSA]) for ligandsL1–L4 and complexesC5–C8 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) showing that the Cu complexes have better binding propensity than the ligands.The values of the Stern-Volmer quenching constants and the quenching rate constants for the compoundsL1–C8 interacting with BSA are calculated by Stern-Volmer quenching equation (2) [46] and the corresponding Stern-Volmer plots Io/I versus [Q] (Figure 8):
(2)IoI=1+KSV[Q]=1+kqτ0[Q],
where Io is the initial tryptophan fluorescence intensity of BSA, I the tryptophan fluorescence intensity of BSA after the addition of the quencher, KSV (M−1) the quenching constant, kq (M−1 s−1) the quenching rate constants, τ0 the average fluorescence lifetime (10−8 s) [47] of BSA without the quencher, and [Q] the concentration of the quencher. The quenching constant (KSV M−1) can be obtained from the slope of the plot Io/I versus [Q] and has been tabulated in Table 3 forL1–L4 and in Table 4 forC5–C8.Table 3
Stern-Volmer quenching constant (KSV), quenching rate constant kq, association binding constant (Ka), and number of binding sites n calculated from double logarithmic plots for the interaction of ligands L1–L4 with BSA.
Compound
K
SV (M−1)
k
q (M−1s−1)
K
a (M−1)
n
L1
3.7
×
10
4
3.7
×
10
12
0.98
×
10
4
0.88
L2
2.6
×
10
4
2.6
×
10
12
0.31
×
10
4
0.814
L3
3.2
×
10
4
3.2
×
10
12
0.72
×
10
4
0.88
L4
1.7
×
10
4
1.7
×
10
12
0.11
×
10
4
0.75Table 4
Stern-Volmer quenching constant (KSV) and effective quenching constant/binding constant (Ka) calculated from modified Stern-Volmer plots for the interaction of complexes C5–C8 with BSA.
Compound
K
SV (M−1)
k
q (M−1s−1)
K
a (M−1)
C5
2.2
×
10
5
2.2
×
10
13
8.53
×
10
4
C6
1.1
×
10
5
1.1
×
10
13
7.49
×
10
4
C7
2.8
×
10
5
2.8
×
10
13
7.25
×
10
4
C8
4.3
×
10
5
4.3
×
10
13
3.48
×
10
4Stern-Volmer quenching plot of BSA for (a) ligandsL1–L4 and (b) their Cu(II) complexesC5–C8.
(a)
(b)The double-logarithm equation (3) [48] has been employed to determine the binding constant (Ka) and the number of binding sites n for ligand-BSA interaction:
(3)log⁡(Io-I)I=log⁡Ka+nlog⁡[Q].
The plot of log [(Io-I)/I] versus log [Q] for each of the ligand is linear (Figure 9) and the values of Ka and n have been obtained from the intercept and slope, respectively (Table 3).Figure 9
Double logarithmic plot for the quenching of BSA fluorescence by ligandsL1–L4to determine the binding constant Ka and number of binding sitesn.Due to the nonlinearity in the Stern-Volmer plots ofC5–C8 (Figure 8(b)), the binding constants (Ka) have been obtained from modified Stern-Volmer (MSV) equation [49]:
(4)Io(Io-I)=1(faKa[Q])+1fa,
where fa is the fraction of the fluorophore that is initially accessible to the complex. The linearity in the modified Stern-Volmer plot (Io/Io-I) versus 1/[Q] (Figure 10) indicates a probable ground state complex formation betweenC5–C8 and BSA, which is static quenching mechanism [50]. The ratio of intercept to slope of MSV plots gave Ka values (Table 4) for complex-BSA binding.Figure 10
Modified Stern-Volmer plot for the quenching of BSA fluorescence byC5–C8.Considering the Stern-Volmer constant of about 105 M−1 and the lifetime of albumins, 10−8 s [51], we obtain a value of 1013 M−1 s−1 for the rate constant (kq) of the bimolecular quenching process, which largely overrides the accepted limit of the rate constant (2 × 1010 M−1 s−1) of the diffusional quenching implying biopolymers. This observation supports the fact that the experimental quenching of albumin fluorescence is due to a predominantly static process [52].


### 2.4. Cytotoxicity

In vitro cytotoxicity tests were performed on the lung cancer (A549) and normal mouse adiposities (3T3L1). The IC50 values of compoundsL1–C8 are listed in Table 5. The cell viabilities (%), obtained for A549 cells with continuous exposure to the compounds for 24 h, are depicted in Figure 11 whereas the IC50 values of the compounds on A549 and 3T3L1 cells have been compared in Figure 12. All the compounds were found to be very less toxic to normal cell line. Copper(II) complexation enhanced the cytotoxicity of the ligands asC5,C7, andC8 were more toxic to A549 cells than their ligand counter parts thoughL2 was more toxic than its Cu(II) complexC6. Nevertheless the compounds were found to be less toxic than cis-platin (IC50 = 26 μM).Table 5
IC
50 values of ligands L1–L4 and their corresponding Cu(II) complexes C5–C8 obtained from MTT assay on A549 cells.
Compounds
L1
L2
L3
L4
C5
C6
C7
C8
cis-Platin
IC
50  (µM)
500
340
503
325
60
475
92
<50
26Figure 11
% cell viability in presence of ligandsL1–L4 and complexesC5–C8 for A549 human lung cancer cell lines. Each point is the mean ± standard error obtained from three independent experiments.Figure 12
Graph showing comparison between the IC50 (concentration at 50% cell death) values ofL1–C8 on A549 and 3T3L1 cell lines treated for 24 h at 37°C which clearly depicts the low cytotoxicity of the compounds on normal mouse adipocytes than on lung cancer cells at same doses of treatment.


### 2.5. Evaluation of Mode of Cell Death via Staining Techniques

Cell staining is a valuable and accurate tool for the assessment of apoptosis permitting identification of the classical hallmarks of apoptosis such as cell shrinkage, membrane blebbing, and formation of apoptotic bodies.


#### 2.5.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells. Acridine orange (AO) permeates all cells and makes the nuclei appear green. Ethidium bromide (EB) is only taken up by cells when cytoplasmic membrane integrity is lost and stains the nucleus red. EB also dominates over AO. Thus live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis have a structurally normal orange nucleus [53]. The cells were treated with complexesC5–C8 at 100 μM concentration for 16 h. The confocal images of control cells show that the live cells are stained with AO and hence emit green fluorescence whereas the treated cells bearing bright green nucleus indicate early apoptosis and cells bearing orange nucleus indicate late apoptosis (Figure 13). Moreover the treated cells as seen in the phase contrast images (Figure 14(f)) undergo a distinct alteration in cell shape and morphology. The majority of the cells appear to be rounded and shrunken due to apoptosis.Figure 13
Confocal images of A549 cells treated with (100μM)C5–C8 for 16 h followed by dual staining with AO/EB. The arrows indicate early apoptotic cells bearing bright green nucleus with late apoptotic cells bearing orange nucleus.Figure 14
Images of A549 cells observed under fluorescence microscope: (a, b) control cells and treated cells stained with DCFDA; (c, d) control cells and treated cells stained with DAPI; (e, f) control and treated A549 cells observed under phase contrast microscope, where in treated cells they show distinct rounding indicative of apoptosis.


#### 2.5.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However when ROS production increases (cellular or external) and overwhelms the cellular antioxidant capacity, it can induce macromolecular damage (by reacting with DNA, proteins, and lipids) and thus contributes to a broad spectrum of diseases and pathological conditions. In the first instance, damage can lead to apoptosis or necrosis. DCFDA (2′,7′-dichlorofluorescein diacetate) is a cell permeable fluorescent dye that measures hydroxyl, peroxyl, and other ROS activities within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 2′,7′-dichlorofluorescein (DCF), a highly fluorescent dye. In the present study, the images taken using fluorescence microscopy of A549 cells treated with complexesC5–C8 at a concentration of 100 μM after 24 h incubation showed that the cells are distinctly stained with the DCF dye indicating excessive ROS generation (Figure 14(b)) whereas the control cells were faintly stained by the dye or not stained at all (Figure 14(a)). The ROS generation seen here can be attributed to the fact that the schiff base derivative of INH produces carbon, nitrogen, or oxygen centered free radicals on interaction with catalase-peroxidases in the cell. This observation confirms an oxidative cleavage of DNA as is evident from the DNA ladder assay.


#### 2.5.3. Staining Nucleic Acid Using DAPI Stain

The morphological assessment of nuclei using a nucleophilic dye DAPI was performed in A549 cells untreated and treated withC5–C8 (100 μM for 24 h). Observations revealed that a large population of the treated cells showed nuclear condensation and associated nuclear anomalies (Figure 14(d)). These observations provide further evidence on nuclear dysfunction induced byC5–C8 in A549 cells.


## 2.1. Synthesis and Characterization

In a general sense, transition metal-based catalysis can be viewed as template reactions where reactants coordinate to adjacent sites on the metal ion and, owing to their adjacency, the two reactants interconnect (insert or couple) either directly or via the action of another reagent. Template effects may arise from stereochemistry imposed by metal ion coordination of some of the reactants, promoting a series of controlled steps and characteristically providing routes to products not formed in the absence of the metal ion. Appreciable amount of work has been done on one pot multicomponent synthesis of Schiff bases using transition metal ions as templates [25–28]. Following this insight we carried out the Ni(II) mediated Schiff base synthesis (Figure 1) and found that the reaction time had reduced and product yields had increased markedly.Figure 1
Schematic representation of synthetic route for the ligandsL1–L4 and their Cu(II) complexesC5–C8.The IR spectra of complexesC5–C8 lack the strong secondary amide carbonyl absorption at 1650–1700 cm−1 that is typically seen in the spectra of the free ligandsL1–L4. In all the complexes, the νC=N band is shifted to lower frequency between 1570 and 1610 cm−1 indicating coordination of the Schiff bases through the azomethine nitrogen [29]. Moreover, the enolate structure of the coordinated ligand is supported by a band at 1059-1060 cm−1 due to the enolic C-O stretching [30]. The N-H stretching bands in the range 3150–3250 cm−1 found in the IR spectra ofL1–L4 are completely lost in the spectra of theirC5–C8 which is also indicative of the enolization and deprotonation on coordination.The UV spectra of the ligands and their complexes show bands in the wavelength range 200–350 nm. In the UV spectra of the ligands (Figure2) the first band appearing within 200–270 nm region can be assigned to the medium energy π-π* transition of the aromatic rings. The second band observed within 280–340 nm region is due to the excitation of the electrons of the azomethine group which corresponds to an intraligand n→π* transition [31]. In case ofL2 (Figure S1 in the Supplementary Material) there is a third band located at 326 nm that can be ascribed to charge transfer within the entire Schiff base molecule. This band is commonly observed in o-hydroxyl Schiff bases [32] and is based on strong intramolecular hydrogen bonding between the hydroxyl group of the salicylidene and the azomethine nitrogen [33]. The UV spectra of complexes (Figure 2) show the first band at nearly the same wave length as in the spectra of their corresponding ligands which reveal that these (π-π*) transitions are not significantly affected by chelation. On the other hand the band corresponding to azomethine showed broadening and a shift to longer wavelength on going from ligand to complex, indicating coordination of ligand to metal through the azomethine moiety [34].Figure 2
UV spectra of ligandL1and corresponding Cu(II) complexC5 recorded at a concentration of 2.5 mM in DMSO with path length 1 cm.The mass spectra of ligandsL1–L4 show peaks corresponding to the molecular ions M+ atm/z = 225.09, 241.07, 271.09, and 255.09, respectively (Figure S2 in the Supplementary Material, available online at http://dx.doi.org/10.1155/2014/630575). In addition the mass spectra of all the ligands show prominent peaks atm/z = 106 and 78 corresponding to (pyridin-4-ylmethylidyne)oxonium and pyridin-4-ylium fragments, respectively. The ESI mass spectra of copper complexesC5–C8 show peaks atm/z = 511.7(M+), 546.1(M+ + 2), 603.7(M+), and 571.7(M+), respectively (Figure S3 in the Supplementary Material). Them/z values of the molecular ion peaks forC5–C8 indicate that two ligands are coordinated to the metal centre. Detailed interpretation of the mass fragments has been summarized in Table S1 in the Supplementary Material.The ESR spectra ofC6 andC7were recorded in solid state at 77 K (Figure S4 in the Supplementary Material) and that ofC8 (Figure 3) was recorded in DMSO at 120 K using 100 KHz field modulation and the g factors were quoted relative to the standard marker TCNE (g=2.00277). The g∥, g⊥, gav, and G values have been calculated and summarized in Table 1. The spectrum of the four coordinatedC8 in frozen DMSO resolved four copper hyperfine lines, characteristic of monomeric copper(II) complexes [35]. The observed g∥>g⊥>ge (2.0023) values indicate that the unpaired electron is predominantly in the dx2-y2 orbital giving a 2B1g ground state [36, 37] and the complexes have distorted square planar geometry. ForC6 andC7, the G-values are <4, suggesting the monomeric nature of copper(II) centres and consistent with a dx2-y2 ground state having a small exchange coupling in the solid state while forC8, G=4.7 indicates that there are no spin exchange interactions between the copper centres in solution [38]. The gav values for the complexes were found to be in the range 2.10–2.17. The deviation of the gav from that of the free electron (2.0023) is due to the covalent nature of the complexes [39].Table 1
ESR spectral parameters of complexesC6, C7, and C8.
Complex
g
∥
g
⊥
g
av
G
C6 (solid state, 77 K)
2.185
2.066
2.106
2.828
C7 (solid state, 77 K)
2.185
2.068
2.107
2.752
C8 (in DMSO, 120 K)
2.368
2.079
2.175
4.747Figure 3
ESR spectrum ofC8 in DMSO at 120 K using 100 KHz field modulation.Thermogravimetric analyses ofL1–C8 were carried out at 10°C per min in a temperature range 50–850°C in N2 atmosphere. The thermogram ofL1given in Figure 4 showed 99.6% weight loss in the range 200°C–320°C attributed to the total decomposition of the molecule. A continuous weight loss in the temperature range 200°C–750°C is observed in case ofC5 (Figure 4) attributed to the removal of the ligands leaving behind CuO2N2 as residue.Figure 4
Thermal degradation curves of ligandL1and complexC5 at heating rate of 10°C per minute under N2 atmosphere.The spectral (IR, EPR, UV, Mass) and the thermogravimetric analyses data of the ligands and the complexes are in full agreement with the proposed structures (Figure1).


## 2.2. DNA Binding Studies

### 2.2.1. Absorption Studies

Transition metal complexes can bind to DNA through covalent bonding via replacement of a labile ligand of the complex by a nitrogen base of DNA such as guanine N7 [40] or noncovalent interactions such as intercalation, electrostatic, or groove binding [41]. Electronic absorption spectroscopy is one of the most useful techniques in DNA binding studies [42] since the observed changes in the spectra may give indication of the mode of interaction.Theπ-π* intraligand absorption bands with λmax⁡ in the region 200–270 nm were used to monitor the interaction of compoundsL1–C8 with double helical CT-DNA. The UV spectra have been recorded for a constant compound concentration in diverse [DNA]/[compound] ratios(r). The changes observed in the intraligand transition of the compoundsL1–C8 upon addition of CT-DNA solution in diverse r values may reveal the existence of interaction between each compound and DNA and may indicate the possible mode of binding.Upon increasing the CT-DNA concentration, the UV spectra of compound-DNA solutions show a gradual decrease in the intensities of the absorption bands (hypochromism) (Figure5) for all compounds without any apparent red shift. The observed hypochromism may be attributed to stacking interaction between the aromatic chromophores of the free and bound ligands and DNA base pairs probably due to intercalation.Figure 5
UV absorption spectra ofC5(12.5 μM) in tris buffer solution at increasing concentrations of CT-DNA. The arrow shows the change in the intensity of absorption upon addition of increasing amounts of CT-DNA at a [complex]/[DNA] ratio in the range 0–4.2.Inset. Plot of [DNA]/(εA-εf) versus [DNA] forC5. The slope to intercept ratio gave Kb values.Among the free ligands,L4 exhibits the maximum hypochromism of 15% followed byL1 andL2, each showing a hypochromism of 10%. The absorption bands of complexesC5,C6,C7, andC8 exhibit a maximum of 48, 24, 28, and 27% hypochromicity, respectively (Table 2). The hypochromism is more pronounced forC5. The complexes exhibit distinctively greater hypochromism compared to the free ligands which indicates that presence of a metal ion facilitates binding.Table 2
DNA binding constants (Kb), Stern-Volmer quenching constants, λmax⁡, and % hypochromism for the interaction of ligands L1–L4 and complexes C5–C8 with DNA.
Compound
K
b  (M−1)
λ
max
⁡ (nm)
% hypochromism
L1
5.7
×
10
4
299
11
L2
4.3
×
10
4
288
10
L3
1.7
×
10
4
326
9
L4
2.0
×
10
4
316
15
C5
1.8
×
10
4
325
48
C6
3.4
×
10
4
293
24
C7
1.4
×
10
4
332
28
C8
4.2
×
10
4
274
27The magnitude of binding strength to CT-DNA may be determined through the calculation of binding constantKb which is obtained by monitoring the changes in the absorbance at the corresponding λmax⁡ with increasing concentrations of CT-DNA. Kb is given by the ratio of slope to the y intercept in plots [DNA]/(εA-εf) versus [DNA] according to [43]
(1)[DNA](εA-εf)=[DNA](εb-εf)+1Kb(εb-εf),
where [DNA] is the concentration of DNA in base pairs, εA=Aobsd/[compound], εf is the extinction coefficient for the unbound compound (L1–C8), and εb is the extinction coefficient for the compound in the fully bound form. The values for Kb as calculated by (1) are given in Table 2 and the representative [DNA]/(εA-εf) versus [DNA] plot forC5 in the inset of Figure 5. Plots of remaining compounds are given in Figure S5 (Supplementary Material).The binding constants of the compounds range from 5.7 × 104 to 1.4 × 104 M−1 suggesting that the Schiff bases and their complexes strongly bind to CT-DNA. The highest Kb value (5.7 × 104 M−1) among the ligands was observed forL1 whereasC8 exhibited the highest Kb value (4.2 × 104 M−1) among the complexes. However the overall Kb values of the compounds are lower than those observed for classical intercalator EB (1.23 × 105 M−1) with a proven DNA binding mode involving intercalation of the planar phenanthridine ring between the adjacent base pairs on the double helix. This is indicative of the binding ofL1–C8 with CT-DNA with an affinity less than EB.The results derived from the UV-titration experiments suggest that all the compounds can bind to CT-DNA although the exact mode of binding cannot be merely proposed by UV-spectroscopic titration studies. In most cases, the existence of hypochromism could be considered as first evidence that the binding of the compounds involving intercalation between the base pairs of CT-DNA cannot be ruled out.


### 2.2.2. DNA Ladder Assay

DNA ladder assay was carried out on A549 cells with two complexesC5 andC8 at three different concentrations (50 μM, 75 μM, and 100 μM) and a marker for reference (Figure 6), to further support the binding of compounds to DNA and to study its effect on the cancer cells. The results presented in Figure 6 show that both complexesC5 andC8 provoke DNA fragmentation in A549 cells. The first band seen in all the six lanes at different concentrations of the two complexes is of the intact DNA, while a smear can be seen in the lower half of the gel which is of fragmented DNA. Though the fragmentation process is weak, it is conclusive of an apoptotic effect of the compounds on the cancer cells.Figure 6
Photogenic view of cleavage of A549 cells with complexesC5andC8using 1.5% agarose gel containing ethidium bromide. Complexes were incubated for 24 h at 37°C.


## 2.2.1. Absorption Studies

Transition metal complexes can bind to DNA through covalent bonding via replacement of a labile ligand of the complex by a nitrogen base of DNA such as guanine N7 [40] or noncovalent interactions such as intercalation, electrostatic, or groove binding [41]. Electronic absorption spectroscopy is one of the most useful techniques in DNA binding studies [42] since the observed changes in the spectra may give indication of the mode of interaction.Theπ-π* intraligand absorption bands with λmax⁡ in the region 200–270 nm were used to monitor the interaction of compoundsL1–C8 with double helical CT-DNA. The UV spectra have been recorded for a constant compound concentration in diverse [DNA]/[compound] ratios(r). The changes observed in the intraligand transition of the compoundsL1–C8 upon addition of CT-DNA solution in diverse r values may reveal the existence of interaction between each compound and DNA and may indicate the possible mode of binding.Upon increasing the CT-DNA concentration, the UV spectra of compound-DNA solutions show a gradual decrease in the intensities of the absorption bands (hypochromism) (Figure5) for all compounds without any apparent red shift. The observed hypochromism may be attributed to stacking interaction between the aromatic chromophores of the free and bound ligands and DNA base pairs probably due to intercalation.Figure 5
UV absorption spectra ofC5(12.5 μM) in tris buffer solution at increasing concentrations of CT-DNA. The arrow shows the change in the intensity of absorption upon addition of increasing amounts of CT-DNA at a [complex]/[DNA] ratio in the range 0–4.2.Inset. Plot of [DNA]/(εA-εf) versus [DNA] forC5. The slope to intercept ratio gave Kb values.Among the free ligands,L4 exhibits the maximum hypochromism of 15% followed byL1 andL2, each showing a hypochromism of 10%. The absorption bands of complexesC5,C6,C7, andC8 exhibit a maximum of 48, 24, 28, and 27% hypochromicity, respectively (Table 2). The hypochromism is more pronounced forC5. The complexes exhibit distinctively greater hypochromism compared to the free ligands which indicates that presence of a metal ion facilitates binding.Table 2
DNA binding constants (Kb), Stern-Volmer quenching constants, λmax⁡, and % hypochromism for the interaction of ligands L1–L4 and complexes C5–C8 with DNA.
Compound
K
b  (M−1)
λ
max
⁡ (nm)
% hypochromism
L1
5.7
×
10
4
299
11
L2
4.3
×
10
4
288
10
L3
1.7
×
10
4
326
9
L4
2.0
×
10
4
316
15
C5
1.8
×
10
4
325
48
C6
3.4
×
10
4
293
24
C7
1.4
×
10
4
332
28
C8
4.2
×
10
4
274
27The magnitude of binding strength to CT-DNA may be determined through the calculation of binding constantKb which is obtained by monitoring the changes in the absorbance at the corresponding λmax⁡ with increasing concentrations of CT-DNA. Kb is given by the ratio of slope to the y intercept in plots [DNA]/(εA-εf) versus [DNA] according to [43]
(1)[DNA](εA-εf)=[DNA](εb-εf)+1Kb(εb-εf),
where [DNA] is the concentration of DNA in base pairs, εA=Aobsd/[compound], εf is the extinction coefficient for the unbound compound (L1–C8), and εb is the extinction coefficient for the compound in the fully bound form. The values for Kb as calculated by (1) are given in Table 2 and the representative [DNA]/(εA-εf) versus [DNA] plot forC5 in the inset of Figure 5. Plots of remaining compounds are given in Figure S5 (Supplementary Material).The binding constants of the compounds range from 5.7 × 104 to 1.4 × 104 M−1 suggesting that the Schiff bases and their complexes strongly bind to CT-DNA. The highest Kb value (5.7 × 104 M−1) among the ligands was observed forL1 whereasC8 exhibited the highest Kb value (4.2 × 104 M−1) among the complexes. However the overall Kb values of the compounds are lower than those observed for classical intercalator EB (1.23 × 105 M−1) with a proven DNA binding mode involving intercalation of the planar phenanthridine ring between the adjacent base pairs on the double helix. This is indicative of the binding ofL1–C8 with CT-DNA with an affinity less than EB.The results derived from the UV-titration experiments suggest that all the compounds can bind to CT-DNA although the exact mode of binding cannot be merely proposed by UV-spectroscopic titration studies. In most cases, the existence of hypochromism could be considered as first evidence that the binding of the compounds involving intercalation between the base pairs of CT-DNA cannot be ruled out.


## 2.2.2. DNA Ladder Assay

DNA ladder assay was carried out on A549 cells with two complexesC5 andC8 at three different concentrations (50 μM, 75 μM, and 100 μM) and a marker for reference (Figure 6), to further support the binding of compounds to DNA and to study its effect on the cancer cells. The results presented in Figure 6 show that both complexesC5 andC8 provoke DNA fragmentation in A549 cells. The first band seen in all the six lanes at different concentrations of the two complexes is of the intact DNA, while a smear can be seen in the lower half of the gel which is of fragmented DNA. Though the fragmentation process is weak, it is conclusive of an apoptotic effect of the compounds on the cancer cells.Figure 6
Photogenic view of cleavage of A549 cells with complexesC5andC8using 1.5% agarose gel containing ethidium bromide. Complexes were incubated for 24 h at 37°C.


## 2.3. BSA Binding Studies

Serum albumins are proteins involved in binding and transport of drugs through the blood stream [44]. Their binding with a drug may actually result in increase or decrease in the drug’s efficacy making it important to investigate the interactions of prospective drugs with serum albumins. BSA solutions exhibit a strong fluorescence emission with a peak at 343 nm, due to the tryptophan residues, when excited at 296 nm. The changes and quenching occurring in the fluorescence emission spectra of BSA upon addition of compoundsL1–C8 are primarily due to change in protein conformation, subunit association, substrate binding, or denaturation because of their binding [45].Addition ofL1–C8 to a solution of BSA results in a decrease of the fluorescence intensity (Figure 7). It is observed that the complexes show more % fluorescence quenching compared to the ligands (Figure 7, inset) indicating stronger protein binding of the ligands on complexation.Figure 7
Plot of fluorescence emission intensityI versus wavelength λ for BSA at different concentrations of compoundL1. The arrow shows the change in intensity of BSA emission on increasing amount of the compound at a [BSA]/[complex] ratio in the range 0–5.Inset. Plot of BSA % relative fluorescence intensity at λmax⁡ = 343 nm versus r (r=[Q]/[BSA]) for ligandsL1–L4 and complexesC5–C8 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) showing that the Cu complexes have better binding propensity than the ligands.The values of the Stern-Volmer quenching constants and the quenching rate constants for the compoundsL1–C8 interacting with BSA are calculated by Stern-Volmer quenching equation (2) [46] and the corresponding Stern-Volmer plots Io/I versus [Q] (Figure 8):
(2)IoI=1+KSV[Q]=1+kqτ0[Q],
where Io is the initial tryptophan fluorescence intensity of BSA, I the tryptophan fluorescence intensity of BSA after the addition of the quencher, KSV (M−1) the quenching constant, kq (M−1 s−1) the quenching rate constants, τ0 the average fluorescence lifetime (10−8 s) [47] of BSA without the quencher, and [Q] the concentration of the quencher. The quenching constant (KSV M−1) can be obtained from the slope of the plot Io/I versus [Q] and has been tabulated in Table 3 forL1–L4 and in Table 4 forC5–C8.Table 3
Stern-Volmer quenching constant (KSV), quenching rate constant kq, association binding constant (Ka), and number of binding sites n calculated from double logarithmic plots for the interaction of ligands L1–L4 with BSA.
Compound
K
SV (M−1)
k
q (M−1s−1)
K
a (M−1)
n
L1
3.7
×
10
4
3.7
×
10
12
0.98
×
10
4
0.88
L2
2.6
×
10
4
2.6
×
10
12
0.31
×
10
4
0.814
L3
3.2
×
10
4
3.2
×
10
12
0.72
×
10
4
0.88
L4
1.7
×
10
4
1.7
×
10
12
0.11
×
10
4
0.75Table 4
Stern-Volmer quenching constant (KSV) and effective quenching constant/binding constant (Ka) calculated from modified Stern-Volmer plots for the interaction of complexes C5–C8 with BSA.
Compound
K
SV (M−1)
k
q (M−1s−1)
K
a (M−1)
C5
2.2
×
10
5
2.2
×
10
13
8.53
×
10
4
C6
1.1
×
10
5
1.1
×
10
13
7.49
×
10
4
C7
2.8
×
10
5
2.8
×
10
13
7.25
×
10
4
C8
4.3
×
10
5
4.3
×
10
13
3.48
×
10
4Stern-Volmer quenching plot of BSA for (a) ligandsL1–L4 and (b) their Cu(II) complexesC5–C8.
(a)
(b)The double-logarithm equation (3) [48] has been employed to determine the binding constant (Ka) and the number of binding sites n for ligand-BSA interaction:
(3)log⁡(Io-I)I=log⁡Ka+nlog⁡[Q].
The plot of log [(Io-I)/I] versus log [Q] for each of the ligand is linear (Figure 9) and the values of Ka and n have been obtained from the intercept and slope, respectively (Table 3).Figure 9
Double logarithmic plot for the quenching of BSA fluorescence by ligandsL1–L4to determine the binding constant Ka and number of binding sitesn.Due to the nonlinearity in the Stern-Volmer plots ofC5–C8 (Figure 8(b)), the binding constants (Ka) have been obtained from modified Stern-Volmer (MSV) equation [49]:
(4)Io(Io-I)=1(faKa[Q])+1fa,
where fa is the fraction of the fluorophore that is initially accessible to the complex. The linearity in the modified Stern-Volmer plot (Io/Io-I) versus 1/[Q] (Figure 10) indicates a probable ground state complex formation betweenC5–C8 and BSA, which is static quenching mechanism [50]. The ratio of intercept to slope of MSV plots gave Ka values (Table 4) for complex-BSA binding.Figure 10
Modified Stern-Volmer plot for the quenching of BSA fluorescence byC5–C8.Considering the Stern-Volmer constant of about 105 M−1 and the lifetime of albumins, 10−8 s [51], we obtain a value of 1013 M−1 s−1 for the rate constant (kq) of the bimolecular quenching process, which largely overrides the accepted limit of the rate constant (2 × 1010 M−1 s−1) of the diffusional quenching implying biopolymers. This observation supports the fact that the experimental quenching of albumin fluorescence is due to a predominantly static process [52].


## 2.4. Cytotoxicity

In vitro cytotoxicity tests were performed on the lung cancer (A549) and normal mouse adiposities (3T3L1). The IC50 values of compoundsL1–C8 are listed in Table 5. The cell viabilities (%), obtained for A549 cells with continuous exposure to the compounds for 24 h, are depicted in Figure 11 whereas the IC50 values of the compounds on A549 and 3T3L1 cells have been compared in Figure 12. All the compounds were found to be very less toxic to normal cell line. Copper(II) complexation enhanced the cytotoxicity of the ligands asC5,C7, andC8 were more toxic to A549 cells than their ligand counter parts thoughL2 was more toxic than its Cu(II) complexC6. Nevertheless the compounds were found to be less toxic than cis-platin (IC50 = 26 μM).Table 5
IC
50 values of ligands L1–L4 and their corresponding Cu(II) complexes C5–C8 obtained from MTT assay on A549 cells.
Compounds
L1
L2
L3
L4
C5
C6
C7
C8
cis-Platin
IC
50  (µM)
500
340
503
325
60
475
92
<50
26Figure 11
% cell viability in presence of ligandsL1–L4 and complexesC5–C8 for A549 human lung cancer cell lines. Each point is the mean ± standard error obtained from three independent experiments.Figure 12
Graph showing comparison between the IC50 (concentration at 50% cell death) values ofL1–C8 on A549 and 3T3L1 cell lines treated for 24 h at 37°C which clearly depicts the low cytotoxicity of the compounds on normal mouse adipocytes than on lung cancer cells at same doses of treatment.


## 2.5. Evaluation of Mode of Cell Death via Staining Techniques

Cell staining is a valuable and accurate tool for the assessment of apoptosis permitting identification of the classical hallmarks of apoptosis such as cell shrinkage, membrane blebbing, and formation of apoptotic bodies.


### 2.5.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells. Acridine orange (AO) permeates all cells and makes the nuclei appear green. Ethidium bromide (EB) is only taken up by cells when cytoplasmic membrane integrity is lost and stains the nucleus red. EB also dominates over AO. Thus live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis have a structurally normal orange nucleus [53]. The cells were treated with complexesC5–C8 at 100 μM concentration for 16 h. The confocal images of control cells show that the live cells are stained with AO and hence emit green fluorescence whereas the treated cells bearing bright green nucleus indicate early apoptosis and cells bearing orange nucleus indicate late apoptosis (Figure 13). Moreover the treated cells as seen in the phase contrast images (Figure 14(f)) undergo a distinct alteration in cell shape and morphology. The majority of the cells appear to be rounded and shrunken due to apoptosis.Figure 13
Confocal images of A549 cells treated with (100μM)C5–C8 for 16 h followed by dual staining with AO/EB. The arrows indicate early apoptotic cells bearing bright green nucleus with late apoptotic cells bearing orange nucleus.Figure 14
Images of A549 cells observed under fluorescence microscope: (a, b) control cells and treated cells stained with DCFDA; (c, d) control cells and treated cells stained with DAPI; (e, f) control and treated A549 cells observed under phase contrast microscope, where in treated cells they show distinct rounding indicative of apoptosis.


### 2.5.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However when ROS production increases (cellular or external) and overwhelms the cellular antioxidant capacity, it can induce macromolecular damage (by reacting with DNA, proteins, and lipids) and thus contributes to a broad spectrum of diseases and pathological conditions. In the first instance, damage can lead to apoptosis or necrosis. DCFDA (2′,7′-dichlorofluorescein diacetate) is a cell permeable fluorescent dye that measures hydroxyl, peroxyl, and other ROS activities within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 2′,7′-dichlorofluorescein (DCF), a highly fluorescent dye. In the present study, the images taken using fluorescence microscopy of A549 cells treated with complexesC5–C8 at a concentration of 100 μM after 24 h incubation showed that the cells are distinctly stained with the DCF dye indicating excessive ROS generation (Figure 14(b)) whereas the control cells were faintly stained by the dye or not stained at all (Figure 14(a)). The ROS generation seen here can be attributed to the fact that the schiff base derivative of INH produces carbon, nitrogen, or oxygen centered free radicals on interaction with catalase-peroxidases in the cell. This observation confirms an oxidative cleavage of DNA as is evident from the DNA ladder assay.


### 2.5.3. Staining Nucleic Acid Using DAPI Stain

The morphological assessment of nuclei using a nucleophilic dye DAPI was performed in A549 cells untreated and treated withC5–C8 (100 μM for 24 h). Observations revealed that a large population of the treated cells showed nuclear condensation and associated nuclear anomalies (Figure 14(d)). These observations provide further evidence on nuclear dysfunction induced byC5–C8 in A549 cells.


## 2.5.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells. Acridine orange (AO) permeates all cells and makes the nuclei appear green. Ethidium bromide (EB) is only taken up by cells when cytoplasmic membrane integrity is lost and stains the nucleus red. EB also dominates over AO. Thus live cells have a normal green nucleus; early apoptotic cells have bright green nucleus with condensed or fragmented chromatin; late apoptotic cells display condensed and fragmented orange chromatin; cells that have died from direct necrosis have a structurally normal orange nucleus [53]. The cells were treated with complexesC5–C8 at 100 μM concentration for 16 h. The confocal images of control cells show that the live cells are stained with AO and hence emit green fluorescence whereas the treated cells bearing bright green nucleus indicate early apoptosis and cells bearing orange nucleus indicate late apoptosis (Figure 13). Moreover the treated cells as seen in the phase contrast images (Figure 14(f)) undergo a distinct alteration in cell shape and morphology. The majority of the cells appear to be rounded and shrunken due to apoptosis.Figure 13
Confocal images of A549 cells treated with (100μM)C5–C8 for 16 h followed by dual staining with AO/EB. The arrows indicate early apoptotic cells bearing bright green nucleus with late apoptotic cells bearing orange nucleus.Figure 14
Images of A549 cells observed under fluorescence microscope: (a, b) control cells and treated cells stained with DCFDA; (c, d) control cells and treated cells stained with DAPI; (e, f) control and treated A549 cells observed under phase contrast microscope, where in treated cells they show distinct rounding indicative of apoptosis.


## 2.5.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

Reactive oxygen species (ROS) are involved in the regulation of many physiological processes. However when ROS production increases (cellular or external) and overwhelms the cellular antioxidant capacity, it can induce macromolecular damage (by reacting with DNA, proteins, and lipids) and thus contributes to a broad spectrum of diseases and pathological conditions. In the first instance, damage can lead to apoptosis or necrosis. DCFDA (2′,7′-dichlorofluorescein diacetate) is a cell permeable fluorescent dye that measures hydroxyl, peroxyl, and other ROS activities within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a nonfluorescent compound, which is later oxidized by ROS into 2′,7′-dichlorofluorescein (DCF), a highly fluorescent dye. In the present study, the images taken using fluorescence microscopy of A549 cells treated with complexesC5–C8 at a concentration of 100 μM after 24 h incubation showed that the cells are distinctly stained with the DCF dye indicating excessive ROS generation (Figure 14(b)) whereas the control cells were faintly stained by the dye or not stained at all (Figure 14(a)). The ROS generation seen here can be attributed to the fact that the schiff base derivative of INH produces carbon, nitrogen, or oxygen centered free radicals on interaction with catalase-peroxidases in the cell. This observation confirms an oxidative cleavage of DNA as is evident from the DNA ladder assay.


## 2.5.3. Staining Nucleic Acid Using DAPI Stain

The morphological assessment of nuclei using a nucleophilic dye DAPI was performed in A549 cells untreated and treated withC5–C8 (100 μM for 24 h). Observations revealed that a large population of the treated cells showed nuclear condensation and associated nuclear anomalies (Figure 14(d)). These observations provide further evidence on nuclear dysfunction induced byC5–C8 in A549 cells.


## 3. Conclusions

The synthesis and characterization of four isonicotinoyl Schiff bases and their Cu(II) complexes (L1–C8) have been realized with physicochemical and spectroscopic methods. In all the complexes, the Schiff bases are bound to the metal via the enolic oxygen and the azomethine nitrogen adapting a square planar geometry around the metal centre. The interaction of the complexes with CT DNA and BSA is supported by the following observations: (i) high values of Kb in the range of 5.7(±0.17)×104–1.4(±0.4)×104 and hypochromism of the intraligand absorption bands of the compoundsL1–C8, (ii) high values of Ksv in the range of 1.79(±0.006)×104–4.3(±0.013)×105, and (iii) efficient cleavage of CT-DNA. Cytotoxicity studies show that the compounds are less toxic than the clinically used drug cis-platin. The metal complexes of ligandsL1,L3 andL4 show greater cytotoxicity as compared to the ligands. Moreover the results obtained from staining techniques indicate that the mode of cell death is essentially apoptosis, probably via ROS generation.


## 4. Experimental

### 4.1. Materials and Methods

All the chemicals and solvents used for synthesis and characterization of ligands and complexes are of analytical grade and were used as purchased. INH (isonicotinoyl hydrazine) was purchased from Acros organics. Aldehydes, CT-DNA, Tri-sodium citrate, and EB (ethidium bromide) were purchased from SRL (Sisco research laboratory, Mumbai, India.). The metal salts and solvents were purchased from Merck.1H NMR spectra were recorded on Bruker 400 MHz NMR Spectrophotometer. ESI Mass spectra of the ligands were recorded on Thermoscientific DSQ-II Mass spectrometer and those of the complexes were recorded on Applied Biosystem API 2000 Mass spectrometer. C, H, and N elemental analyses were performed on a Perkin-Elmer 240B elemental analyzer. Infrared spectra (400–4000 cm−1) were recorded on Perkin Elmer RX-1 FTIR with samples prepared as KBr pellets. UV spectra were recorded in DMSO solution at concentrations in the range 10−6–10−3M on Perkin Elmer Lambda-35 dual beam UV-Vis spectrophotometer. Thermogravimetric analyses were carried out with EXSTAR model SII TG/DTA 6300. Solid state as well as solution state EPR spectra at liquid nitrogen temperature were recorded on Bruker Biospin Gn. Fluorescence spectra were recorded in solution on JASCO FP-6300 fluorescence spectrophotometer. The % copper content in each of the complexes was determined by complexometric titration method after decomposition of the complexes.


### 4.2. Synthesis of LigandsL1–L4

A mixture of INH, aldehyde (benzaldehyde/salicylaldehyde/vanillin/p-anisaldehyde) and NiCl2·6H2O were taken in the mole ratio of 1 : 1 : 0.5 in 20 mL dry methanol. A few drops of acetic acid were added to adjust the pH to 5 and the reaction mixture was refluxed for 1-2 h. The product obtained was filtered and recrystallized from absolute ethanol.


#### 4.2.1.  (N′-Benzylideneisonicotinohydrazide) (L1)

L1 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and benzaldehyde (3.6 mmol, 386 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 84.14%, mp 197°C; M Wt 225.25 g/mol; Anal. Calc. for C13H11N3O: C 69.32, H 4.92, N 18.66. Found: C 69.24, H 4.89, N 18.61%; MSm/z: 225.09(M+); δH (400 MHz, DMSO-d6) 12.10 (s, 1H, NH), 8.79 (d,J5.2, 2H, pyridine α-H), 8.47 (s, 1H, N=CH), 7.83 (d,J 4.4, 2H, pyridine β-H), 7.77–7.75 (dd,J1 7.6,J2 2, 2H, Ar-H), 7.51–7.47 (m, 3H, Ar-H) (Figure S6,(a) in the Supplementary Material); δC (400 MHz, DMSO-d6) 162.1, 150.8, 149.4, 140.9, 134.4, 130.8, 129.3, 127.7, 122.0 (Figure S7,(a) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νN-H 3197(b), νC=O 1694(s).


#### 4.2.2. (N′-(2-Hydroxybenzylidene)isonicotinohydrazide) (L2)

L2was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and salicylaldehyde (3.6 mmol, 445 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg). Yellow crystals were obtained. Solubility: DMSO, DMF; Yield 87.2%; mp 248°C; M Wt 241.25 g/mol; Anal. Calc. for C13H11N3O2: C 64.72, H 4.60, N 17.42. Found: C 64.69, H 4.58, N 17.55%; MSm/z: 241.07 (M+); δH (400 MHz, DMSO-d6) 12.31 (s, 1H, NH), 11.08 (s, 1H, OH), 8.80 (d,J4.4, 2H, pyridine α-H), 8.68 (s, 1H, N=CH), 7.85 (d,J4.4, 2H, pyridine β-H), 7.62–7.60 (dd,J1 7.6,J2 1.6, 1H, Ar-H), 7.34–7.29 (t,J8.4, 1H, Ar-H), 6.95–6.91 (m, 2H, Ar-H) (Figure S6,(b) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.7, 157.9, 150.8, 149.3, 140.4, 132.2, 129.6, 121.9, 119.8, 119.1, 116.9 (Figure S7,(b) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1613(s), νN-H 3179(b), νC=O 1679(s).


#### 4.2.3. (N′-(4-Hydroxy-3-methoxybenzylidene)-N-methylisonicotinohydrazide) (L3)

L3was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and vanillin (3.6 mmol, 554 mg) in presence of NiCl2.6H2O (1.8 mmol, 433 mg). Yellowish brown crystals were obtained. Solubility: DMSO, DMF; Yield 79.06%; mp 226°C; M Wt 271.27 g/mol; Anal. Calc. for C14H13N3O3: C 61.99, H 4.83, N 15.49. Found: C 61.94, H 4.80, N 15.42%; MSm/z: 271.09(M+); δH (400 MHz, DMSO-d6) 11.91 (s, 1H, OH), 9.65 (s, 1H, NH), 8.78 (d,J5.2, 2H, pyridine α-H), 8.35 (s, 1H, N=CH), 7.81 (d,J6.0, 2H, pyridine β-H), 7.33 (d,J1.2, 1H, Ar-H), 7.13–7.10 (dd,J1 8.0,J2 1.6, 1H, Ar-H), 6.85 (d,J8, 1H, Ar-H), 3.83 (s, 3H, OCH3) (Figure S6,(c) in the Supplementary Material);δC (400 MHz, DMSO-d6) 161.8, 150.7, 150.0, 149.7, 148.5, 141.1, 125.8, 122.9, 121.9, 115.9, 109.4, 55.9 (Figure S7,(c) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νN-H 3231(b), νC=O 1649(s), νO-H 3416(b).


#### 4.2.4. (N′-(4-Methoxybenzylidene)-N-methylisonicotinohydrazide) (L4)

L4 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) andp-anisaldehyde (3.6 mmol, 495 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 89.37%; mp 138°C; M Wt 255.27 g/mol; Anal. Calc. for C14H13N3O2: C 65.87, H 5.13, N 16.46. Found: C 65.81, H 5.09, N 16.43%; MSm/z: 255.09(M+). δH (400 MHz, DMSO-d6) 11.96 (s, 1H, NH), 8.78 (d,J4.4, 2H, pyridine α-H), 8.40 (s, 1H, N=CH), 7.82 (d,J4.4, 2H, pyridine β-H), 7.70 (d,J8.8, 2H, Ar-H), 7.03 (d,J6.8, 2H, Ar-H), 3.81 (s, 3H, OCH3) (Figure S6,(d) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.8, 161.5, 150.7, 149.3, 141.0, 129.3, 126.9, 121.9, 114.8, 55.7 (Figure S7,(d) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1611(s), νN-H 3242(b), νC=O 1662(s).


### 4.3. Synthesis of Cu(II) ComplexesC5–C8

The synthesized ligands and CuCl2·2H2O were taken in 2 : 1 ratio in 15 mL methanol and refluxed for 6 h. The precipitated Cu(II) complexes were filtered, washed with water followed by methanol, and dried in air.


#### 4.3.1.  [Cu (C13H11N3O)2] (C5)

C5 was prepared by adding drop-wise a methanolic solution (10 mL) ofL1 (1 mmol, 225 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.45 g (88.06%); M Wt 511.09 g/mol; Anal. Calc. for C26H20CuN6O2: C 60.99, H 3.94, N 16.41, Cu 12.41. Found: C 60.95, H 3.90, N 16.35, Cu 12.39%; MS (ESI)m/z: 511.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1573(s), νC-O(enolic) 1059(m).


#### 4.3.2.  [Cu (C13H11N3O2)2] (C6)

C6 was prepared by adding drop-wise a methanolic solution (10 mL) ofL2 (1 mmol, 241 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Yellow brown precipitates were obtained. Solubility: DMSO, DMF; Yield 0.51 g (93.75%); M Wt 544.02 g/mol; Anal. Calc. for C26H20CuN6O4: C 57.40, H 3.71, N 15.45, Cu 11.68. Found: C 57.37, H 3.69, N 15.43, Cu 11.65%; MS (ESI)m/z: 546.1(M+ + 2); νmax⁡   (KBr)/cm−1 νC=N 1607(s), νC-O(enolic) 1060(m).


#### 4.3.3.  [Cu (C14H13N3O3)2] (C7)

C7 was prepared by adding drop-wise a methanolic solution (10 mL) ofL3 (1 mmol, 271 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Brown coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.47 g (77.8%); M Wt 604.07 g/mol; Anal. Calc. for C28H24CuN6O6: C 55.67, H 4.00, N 13.91, Cu 10.52. Found: C 55.61, H 4.02, N 13.88, Cu 10.47%; MS (ESI)m/z: 603.7(M+); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νC-O(enolic) 1059(m), νO-H 3382(b).


#### 4.3.4. [Cu (C14H13N3O2)2] (C8)

C8 was prepared by adding drop-wise a methanolic solution (10 mL) ofL4 (1 mmol, 255 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Dark green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.43 g (75.1%); M Wt 572.07 g/mol; Anal. Calc. for C28H24CuN6O4: C 58.79, H 4.23, N 14.69, Cu 11.11. Found: C 58.73, H 4.21, N 14.67, Cu 11.05%; MS (ESI)m/z: 571.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νC-O(enolic) 1059(m).The entire synthetic route has been schematically represented in Figure1. The 1H NMR spectra of ligandsL1–L4 have been given in Figure S1 (Supplementary Material).


### 4.4. DNA Binding Studies

#### 4.4.1. Absorption Studies

The interaction of the compounds (L1–C8) with CT DNA has been studied with UV spectroscopy in order to investigate the possible binding modes to CT-DNA and to calculate the binding constant (Kb). Absorption studies were performed with fixed compound concentrations while varying the CT-DNA concentration within. For recording the absorption spectra of compounds under the effect of DNA, a calculated amount of the stock solution (2.5 mM) was diluted with tris buffer to get the desired concentration and equal increments of CT-DNA were added to the compound solution. The ratio of UV absorbance at 260 and 280 nm of DNA solution was 1.8, indicating that the DNA was sufficiently free of protein.


#### 4.4.2. DNA Ladder Assay

The DNA ladder assay was carried out on A549 cell lines where the cells were incubated with the compounds at three different concentrations (50μM, 75 μM, and 100 μM) for 24 hr at 37°C in a 5% CO2 incubator. Following this treatment the A-549 cells were collected, washed with PBS, and lysed (lysis solution: 10 mMTris-HCl pH 7.4, 10 mM EDTA, and 0.5% Triton X-100). Cell lysates were then incubated with RNase A (200 mg mL−1; Bioline, USA) for 1 h, followed by proteinase K (200 mg mL−1; Bioline, USA) for 1 h at 37°C. After treating the samples with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) followed by chloroform, DNA was precipitated in two volumes of ethanol in the presence of 0.3 M sodium acetate at −20°C. Finally the DNA samples were run on 1.5% agarose gel at 60 V and visualized by ethidium bromide staining under UV light.


### 4.5. BSA Binding Studies

The protein-binding study was performed by tryptophan fluorescence quenching experiments using bovine serum albumin. The quenching of emission intensity of the tryptophan residues of BSA at 343 nm was monitored in the presence of increasing concentrations ofL1–C8 as quenchers [54]. The quencher was added in equal increments with concentration increasing from 1.5 μM to 15 μM to a fixed concentration of BSA. Fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 296 nm.


### 4.6. Cytotoxicity

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] cell viability assay was performed to assess the cytotoxicity of the compounds [55] on the human lung cancer cell line (A549) and normal mouse adipose cell line (3T3L1). Cells (5.0×103 cells well−1) were placed in 96-well culture plates (Tarson India Pvt. Ltd.) and grown overnight at 37°C in a 5% CO2 incubator. Compounds to be tested were then added to the wells to achieve final concentrations ranging from 50 to 750 μM. Control wells were prepared by addition of culture medium without the compounds. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Upon completion of the incubation, MTT dye solution was added to each well to a final concentration of 500 μg/mL. After 4 h incubation, the culture media were discarded and the wells were washed with phosphate buffer saline (Hi-Media, India Pvt. Ltd.), followed by addition of DMSO and subsequent incubation for 30 min. The optical density of each well was then measured on ELX800 Universal Microplate Reader at a wavelength of 540 nm. The IC50 values were determined by plotting the percentage viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remained viable relative to the control. Each experiment was repeated at least three times to obtain mean values.


### 4.7. Staining Techniques

#### 4.7.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 16 h treatment with 100 μM metal complexesC5–C8, followed by PBS wash, the cells were stained with 1 μL of acridine orange (AO) and ethidium bromide (EB) dye mixture (1 mg/mL of each in PBS) in 9 μL of media. The cover slip was then mounted on a clean glass slide and immediately photographed confocal microscope [56].


#### 4.7.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

A549 cells (1.0 × 105 cells well−1) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration, the cells were incubated with 7.5 × 10−6M DCFDA stain at 37°C for 30 min, observed, and photographed [57].


#### 4.7.3. Staining Nucleic Acid Using DAPI Stain

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration the cells were washed with PBS and stained with 100 μL DAPI (300 μM) followed by incubation at 37°C for 30 min. The cover slip was mounted on a clean glass slide and immediately photographed [58].


## 4.1. Materials and Methods

All the chemicals and solvents used for synthesis and characterization of ligands and complexes are of analytical grade and were used as purchased. INH (isonicotinoyl hydrazine) was purchased from Acros organics. Aldehydes, CT-DNA, Tri-sodium citrate, and EB (ethidium bromide) were purchased from SRL (Sisco research laboratory, Mumbai, India.). The metal salts and solvents were purchased from Merck.1H NMR spectra were recorded on Bruker 400 MHz NMR Spectrophotometer. ESI Mass spectra of the ligands were recorded on Thermoscientific DSQ-II Mass spectrometer and those of the complexes were recorded on Applied Biosystem API 2000 Mass spectrometer. C, H, and N elemental analyses were performed on a Perkin-Elmer 240B elemental analyzer. Infrared spectra (400–4000 cm−1) were recorded on Perkin Elmer RX-1 FTIR with samples prepared as KBr pellets. UV spectra were recorded in DMSO solution at concentrations in the range 10−6–10−3M on Perkin Elmer Lambda-35 dual beam UV-Vis spectrophotometer. Thermogravimetric analyses were carried out with EXSTAR model SII TG/DTA 6300. Solid state as well as solution state EPR spectra at liquid nitrogen temperature were recorded on Bruker Biospin Gn. Fluorescence spectra were recorded in solution on JASCO FP-6300 fluorescence spectrophotometer. The % copper content in each of the complexes was determined by complexometric titration method after decomposition of the complexes.


## 4.2. Synthesis of LigandsL1–L4

A mixture of INH, aldehyde (benzaldehyde/salicylaldehyde/vanillin/p-anisaldehyde) and NiCl2·6H2O were taken in the mole ratio of 1 : 1 : 0.5 in 20 mL dry methanol. A few drops of acetic acid were added to adjust the pH to 5 and the reaction mixture was refluxed for 1-2 h. The product obtained was filtered and recrystallized from absolute ethanol.


### 4.2.1.  (N′-Benzylideneisonicotinohydrazide) (L1)

L1 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and benzaldehyde (3.6 mmol, 386 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 84.14%, mp 197°C; M Wt 225.25 g/mol; Anal. Calc. for C13H11N3O: C 69.32, H 4.92, N 18.66. Found: C 69.24, H 4.89, N 18.61%; MSm/z: 225.09(M+); δH (400 MHz, DMSO-d6) 12.10 (s, 1H, NH), 8.79 (d,J5.2, 2H, pyridine α-H), 8.47 (s, 1H, N=CH), 7.83 (d,J 4.4, 2H, pyridine β-H), 7.77–7.75 (dd,J1 7.6,J2 2, 2H, Ar-H), 7.51–7.47 (m, 3H, Ar-H) (Figure S6,(a) in the Supplementary Material); δC (400 MHz, DMSO-d6) 162.1, 150.8, 149.4, 140.9, 134.4, 130.8, 129.3, 127.7, 122.0 (Figure S7,(a) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νN-H 3197(b), νC=O 1694(s).


### 4.2.2. (N′-(2-Hydroxybenzylidene)isonicotinohydrazide) (L2)

L2was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and salicylaldehyde (3.6 mmol, 445 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg). Yellow crystals were obtained. Solubility: DMSO, DMF; Yield 87.2%; mp 248°C; M Wt 241.25 g/mol; Anal. Calc. for C13H11N3O2: C 64.72, H 4.60, N 17.42. Found: C 64.69, H 4.58, N 17.55%; MSm/z: 241.07 (M+); δH (400 MHz, DMSO-d6) 12.31 (s, 1H, NH), 11.08 (s, 1H, OH), 8.80 (d,J4.4, 2H, pyridine α-H), 8.68 (s, 1H, N=CH), 7.85 (d,J4.4, 2H, pyridine β-H), 7.62–7.60 (dd,J1 7.6,J2 1.6, 1H, Ar-H), 7.34–7.29 (t,J8.4, 1H, Ar-H), 6.95–6.91 (m, 2H, Ar-H) (Figure S6,(b) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.7, 157.9, 150.8, 149.3, 140.4, 132.2, 129.6, 121.9, 119.8, 119.1, 116.9 (Figure S7,(b) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1613(s), νN-H 3179(b), νC=O 1679(s).


### 4.2.3. (N′-(4-Hydroxy-3-methoxybenzylidene)-N-methylisonicotinohydrazide) (L3)

L3was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and vanillin (3.6 mmol, 554 mg) in presence of NiCl2.6H2O (1.8 mmol, 433 mg). Yellowish brown crystals were obtained. Solubility: DMSO, DMF; Yield 79.06%; mp 226°C; M Wt 271.27 g/mol; Anal. Calc. for C14H13N3O3: C 61.99, H 4.83, N 15.49. Found: C 61.94, H 4.80, N 15.42%; MSm/z: 271.09(M+); δH (400 MHz, DMSO-d6) 11.91 (s, 1H, OH), 9.65 (s, 1H, NH), 8.78 (d,J5.2, 2H, pyridine α-H), 8.35 (s, 1H, N=CH), 7.81 (d,J6.0, 2H, pyridine β-H), 7.33 (d,J1.2, 1H, Ar-H), 7.13–7.10 (dd,J1 8.0,J2 1.6, 1H, Ar-H), 6.85 (d,J8, 1H, Ar-H), 3.83 (s, 3H, OCH3) (Figure S6,(c) in the Supplementary Material);δC (400 MHz, DMSO-d6) 161.8, 150.7, 150.0, 149.7, 148.5, 141.1, 125.8, 122.9, 121.9, 115.9, 109.4, 55.9 (Figure S7,(c) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νN-H 3231(b), νC=O 1649(s), νO-H 3416(b).


### 4.2.4. (N′-(4-Methoxybenzylidene)-N-methylisonicotinohydrazide) (L4)

L4 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) andp-anisaldehyde (3.6 mmol, 495 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 89.37%; mp 138°C; M Wt 255.27 g/mol; Anal. Calc. for C14H13N3O2: C 65.87, H 5.13, N 16.46. Found: C 65.81, H 5.09, N 16.43%; MSm/z: 255.09(M+). δH (400 MHz, DMSO-d6) 11.96 (s, 1H, NH), 8.78 (d,J4.4, 2H, pyridine α-H), 8.40 (s, 1H, N=CH), 7.82 (d,J4.4, 2H, pyridine β-H), 7.70 (d,J8.8, 2H, Ar-H), 7.03 (d,J6.8, 2H, Ar-H), 3.81 (s, 3H, OCH3) (Figure S6,(d) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.8, 161.5, 150.7, 149.3, 141.0, 129.3, 126.9, 121.9, 114.8, 55.7 (Figure S7,(d) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1611(s), νN-H 3242(b), νC=O 1662(s).


## 4.2.1.  (N′-Benzylideneisonicotinohydrazide) (L1)

L1 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and benzaldehyde (3.6 mmol, 386 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 84.14%, mp 197°C; M Wt 225.25 g/mol; Anal. Calc. for C13H11N3O: C 69.32, H 4.92, N 18.66. Found: C 69.24, H 4.89, N 18.61%; MSm/z: 225.09(M+); δH (400 MHz, DMSO-d6) 12.10 (s, 1H, NH), 8.79 (d,J5.2, 2H, pyridine α-H), 8.47 (s, 1H, N=CH), 7.83 (d,J 4.4, 2H, pyridine β-H), 7.77–7.75 (dd,J1 7.6,J2 2, 2H, Ar-H), 7.51–7.47 (m, 3H, Ar-H) (Figure S6,(a) in the Supplementary Material); δC (400 MHz, DMSO-d6) 162.1, 150.8, 149.4, 140.9, 134.4, 130.8, 129.3, 127.7, 122.0 (Figure S7,(a) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νN-H 3197(b), νC=O 1694(s).


## 4.2.2. (N′-(2-Hydroxybenzylidene)isonicotinohydrazide) (L2)

L2was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and salicylaldehyde (3.6 mmol, 445 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg). Yellow crystals were obtained. Solubility: DMSO, DMF; Yield 87.2%; mp 248°C; M Wt 241.25 g/mol; Anal. Calc. for C13H11N3O2: C 64.72, H 4.60, N 17.42. Found: C 64.69, H 4.58, N 17.55%; MSm/z: 241.07 (M+); δH (400 MHz, DMSO-d6) 12.31 (s, 1H, NH), 11.08 (s, 1H, OH), 8.80 (d,J4.4, 2H, pyridine α-H), 8.68 (s, 1H, N=CH), 7.85 (d,J4.4, 2H, pyridine β-H), 7.62–7.60 (dd,J1 7.6,J2 1.6, 1H, Ar-H), 7.34–7.29 (t,J8.4, 1H, Ar-H), 6.95–6.91 (m, 2H, Ar-H) (Figure S6,(b) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.7, 157.9, 150.8, 149.3, 140.4, 132.2, 129.6, 121.9, 119.8, 119.1, 116.9 (Figure S7,(b) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1613(s), νN-H 3179(b), νC=O 1679(s).


## 4.2.3. (N′-(4-Hydroxy-3-methoxybenzylidene)-N-methylisonicotinohydrazide) (L3)

L3was prepared by condensation of isoniazide (3.6 mmol, 500 mg) and vanillin (3.6 mmol, 554 mg) in presence of NiCl2.6H2O (1.8 mmol, 433 mg). Yellowish brown crystals were obtained. Solubility: DMSO, DMF; Yield 79.06%; mp 226°C; M Wt 271.27 g/mol; Anal. Calc. for C14H13N3O3: C 61.99, H 4.83, N 15.49. Found: C 61.94, H 4.80, N 15.42%; MSm/z: 271.09(M+); δH (400 MHz, DMSO-d6) 11.91 (s, 1H, OH), 9.65 (s, 1H, NH), 8.78 (d,J5.2, 2H, pyridine α-H), 8.35 (s, 1H, N=CH), 7.81 (d,J6.0, 2H, pyridine β-H), 7.33 (d,J1.2, 1H, Ar-H), 7.13–7.10 (dd,J1 8.0,J2 1.6, 1H, Ar-H), 6.85 (d,J8, 1H, Ar-H), 3.83 (s, 3H, OCH3) (Figure S6,(c) in the Supplementary Material);δC (400 MHz, DMSO-d6) 161.8, 150.7, 150.0, 149.7, 148.5, 141.1, 125.8, 122.9, 121.9, 115.9, 109.4, 55.9 (Figure S7,(c) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νN-H 3231(b), νC=O 1649(s), νO-H 3416(b).


## 4.2.4. (N′-(4-Methoxybenzylidene)-N-methylisonicotinohydrazide) (L4)

L4 was prepared by condensation of isoniazide (3.6 mmol, 500 mg) andp-anisaldehyde (3.6 mmol, 495 mg) in presence of NiCl2·6H2O (1.8 mmol, 433 mg) as mentioned above. Pale yellow crystals were obtained. Solubility: DMSO, DMF; Yield 89.37%; mp 138°C; M Wt 255.27 g/mol; Anal. Calc. for C14H13N3O2: C 65.87, H 5.13, N 16.46. Found: C 65.81, H 5.09, N 16.43%; MSm/z: 255.09(M+). δH (400 MHz, DMSO-d6) 11.96 (s, 1H, NH), 8.78 (d,J4.4, 2H, pyridine α-H), 8.40 (s, 1H, N=CH), 7.82 (d,J4.4, 2H, pyridine β-H), 7.70 (d,J8.8, 2H, Ar-H), 7.03 (d,J6.8, 2H, Ar-H), 3.81 (s, 3H, OCH3) (Figure S6,(d) in the Supplementary Material); δC (400 MHz, DMSO-d6) 161.8, 161.5, 150.7, 149.3, 141.0, 129.3, 126.9, 121.9, 114.8, 55.7 (Figure S7,(d) in the Supplementary Material); νmax⁡ (KBr)/cm−1 νC=N 1611(s), νN-H 3242(b), νC=O 1662(s).


## 4.3. Synthesis of Cu(II) ComplexesC5–C8

The synthesized ligands and CuCl2·2H2O were taken in 2 : 1 ratio in 15 mL methanol and refluxed for 6 h. The precipitated Cu(II) complexes were filtered, washed with water followed by methanol, and dried in air.


### 4.3.1.  [Cu (C13H11N3O)2] (C5)

C5 was prepared by adding drop-wise a methanolic solution (10 mL) ofL1 (1 mmol, 225 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.45 g (88.06%); M Wt 511.09 g/mol; Anal. Calc. for C26H20CuN6O2: C 60.99, H 3.94, N 16.41, Cu 12.41. Found: C 60.95, H 3.90, N 16.35, Cu 12.39%; MS (ESI)m/z: 511.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1573(s), νC-O(enolic) 1059(m).


### 4.3.2.  [Cu (C13H11N3O2)2] (C6)

C6 was prepared by adding drop-wise a methanolic solution (10 mL) ofL2 (1 mmol, 241 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Yellow brown precipitates were obtained. Solubility: DMSO, DMF; Yield 0.51 g (93.75%); M Wt 544.02 g/mol; Anal. Calc. for C26H20CuN6O4: C 57.40, H 3.71, N 15.45, Cu 11.68. Found: C 57.37, H 3.69, N 15.43, Cu 11.65%; MS (ESI)m/z: 546.1(M+ + 2); νmax⁡   (KBr)/cm−1 νC=N 1607(s), νC-O(enolic) 1060(m).


### 4.3.3.  [Cu (C14H13N3O3)2] (C7)

C7 was prepared by adding drop-wise a methanolic solution (10 mL) ofL3 (1 mmol, 271 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Brown coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.47 g (77.8%); M Wt 604.07 g/mol; Anal. Calc. for C28H24CuN6O6: C 55.67, H 4.00, N 13.91, Cu 10.52. Found: C 55.61, H 4.02, N 13.88, Cu 10.47%; MS (ESI)m/z: 603.7(M+); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νC-O(enolic) 1059(m), νO-H 3382(b).


### 4.3.4. [Cu (C14H13N3O2)2] (C8)

C8 was prepared by adding drop-wise a methanolic solution (10 mL) ofL4 (1 mmol, 255 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Dark green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.43 g (75.1%); M Wt 572.07 g/mol; Anal. Calc. for C28H24CuN6O4: C 58.79, H 4.23, N 14.69, Cu 11.11. Found: C 58.73, H 4.21, N 14.67, Cu 11.05%; MS (ESI)m/z: 571.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νC-O(enolic) 1059(m).The entire synthetic route has been schematically represented in Figure1. The 1H NMR spectra of ligandsL1–L4 have been given in Figure S1 (Supplementary Material).


## 4.3.1.  [Cu (C13H11N3O)2] (C5)

C5 was prepared by adding drop-wise a methanolic solution (10 mL) ofL1 (1 mmol, 225 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.45 g (88.06%); M Wt 511.09 g/mol; Anal. Calc. for C26H20CuN6O2: C 60.99, H 3.94, N 16.41, Cu 12.41. Found: C 60.95, H 3.90, N 16.35, Cu 12.39%; MS (ESI)m/z: 511.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1573(s), νC-O(enolic) 1059(m).


## 4.3.2.  [Cu (C13H11N3O2)2] (C6)

C6 was prepared by adding drop-wise a methanolic solution (10 mL) ofL2 (1 mmol, 241 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Yellow brown precipitates were obtained. Solubility: DMSO, DMF; Yield 0.51 g (93.75%); M Wt 544.02 g/mol; Anal. Calc. for C26H20CuN6O4: C 57.40, H 3.71, N 15.45, Cu 11.68. Found: C 57.37, H 3.69, N 15.43, Cu 11.65%; MS (ESI)m/z: 546.1(M+ + 2); νmax⁡   (KBr)/cm−1 νC=N 1607(s), νC-O(enolic) 1060(m).


## 4.3.3.  [Cu (C14H13N3O3)2] (C7)

C7 was prepared by adding drop-wise a methanolic solution (10 mL) ofL3 (1 mmol, 271 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Brown coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.47 g (77.8%); M Wt 604.07 g/mol; Anal. Calc. for C28H24CuN6O6: C 55.67, H 4.00, N 13.91, Cu 10.52. Found: C 55.61, H 4.02, N 13.88, Cu 10.47%; MS (ESI)m/z: 603.7(M+); νmax⁡ (KBr)/cm−1 νC=N 1595(s), νC-O(enolic) 1059(m), νO-H 3382(b).


## 4.3.4. [Cu (C14H13N3O2)2] (C8)

C8 was prepared by adding drop-wise a methanolic solution (10 mL) ofL4 (1 mmol, 255 mg) to a methanolic solution (5 mL) of CuCl2·2H2O (0.5 mmol, 85 mg) and refluxed for 6 h. Dark green coloured precipitates were obtained. Solubility: DMSO, DMF; Yield 0.43 g (75.1%); M Wt 572.07 g/mol; Anal. Calc. for C28H24CuN6O4: C 58.79, H 4.23, N 14.69, Cu 11.11. Found: C 58.73, H 4.21, N 14.67, Cu 11.05%; MS (ESI)m/z: 571.7 (M+); νmax⁡ (KBr)/cm−1 νC=N 1598(s), νC-O(enolic) 1059(m).The entire synthetic route has been schematically represented in Figure1. The 1H NMR spectra of ligandsL1–L4 have been given in Figure S1 (Supplementary Material).


## 4.4. DNA Binding Studies

### 4.4.1. Absorption Studies

The interaction of the compounds (L1–C8) with CT DNA has been studied with UV spectroscopy in order to investigate the possible binding modes to CT-DNA and to calculate the binding constant (Kb). Absorption studies were performed with fixed compound concentrations while varying the CT-DNA concentration within. For recording the absorption spectra of compounds under the effect of DNA, a calculated amount of the stock solution (2.5 mM) was diluted with tris buffer to get the desired concentration and equal increments of CT-DNA were added to the compound solution. The ratio of UV absorbance at 260 and 280 nm of DNA solution was 1.8, indicating that the DNA was sufficiently free of protein.


### 4.4.2. DNA Ladder Assay

The DNA ladder assay was carried out on A549 cell lines where the cells were incubated with the compounds at three different concentrations (50μM, 75 μM, and 100 μM) for 24 hr at 37°C in a 5% CO2 incubator. Following this treatment the A-549 cells were collected, washed with PBS, and lysed (lysis solution: 10 mMTris-HCl pH 7.4, 10 mM EDTA, and 0.5% Triton X-100). Cell lysates were then incubated with RNase A (200 mg mL−1; Bioline, USA) for 1 h, followed by proteinase K (200 mg mL−1; Bioline, USA) for 1 h at 37°C. After treating the samples with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) followed by chloroform, DNA was precipitated in two volumes of ethanol in the presence of 0.3 M sodium acetate at −20°C. Finally the DNA samples were run on 1.5% agarose gel at 60 V and visualized by ethidium bromide staining under UV light.


## 4.4.1. Absorption Studies

The interaction of the compounds (L1–C8) with CT DNA has been studied with UV spectroscopy in order to investigate the possible binding modes to CT-DNA and to calculate the binding constant (Kb). Absorption studies were performed with fixed compound concentrations while varying the CT-DNA concentration within. For recording the absorption spectra of compounds under the effect of DNA, a calculated amount of the stock solution (2.5 mM) was diluted with tris buffer to get the desired concentration and equal increments of CT-DNA were added to the compound solution. The ratio of UV absorbance at 260 and 280 nm of DNA solution was 1.8, indicating that the DNA was sufficiently free of protein.


## 4.4.2. DNA Ladder Assay

The DNA ladder assay was carried out on A549 cell lines where the cells were incubated with the compounds at three different concentrations (50μM, 75 μM, and 100 μM) for 24 hr at 37°C in a 5% CO2 incubator. Following this treatment the A-549 cells were collected, washed with PBS, and lysed (lysis solution: 10 mMTris-HCl pH 7.4, 10 mM EDTA, and 0.5% Triton X-100). Cell lysates were then incubated with RNase A (200 mg mL−1; Bioline, USA) for 1 h, followed by proteinase K (200 mg mL−1; Bioline, USA) for 1 h at 37°C. After treating the samples with phenol/chloroform/isoamyl alcohol (25 : 24 : 1, v/v/v) followed by chloroform, DNA was precipitated in two volumes of ethanol in the presence of 0.3 M sodium acetate at −20°C. Finally the DNA samples were run on 1.5% agarose gel at 60 V and visualized by ethidium bromide staining under UV light.


## 4.5. BSA Binding Studies

The protein-binding study was performed by tryptophan fluorescence quenching experiments using bovine serum albumin. The quenching of emission intensity of the tryptophan residues of BSA at 343 nm was monitored in the presence of increasing concentrations ofL1–C8 as quenchers [54]. The quencher was added in equal increments with concentration increasing from 1.5 μM to 15 μM to a fixed concentration of BSA. Fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 296 nm.


## 4.6. Cytotoxicity

The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] cell viability assay was performed to assess the cytotoxicity of the compounds [55] on the human lung cancer cell line (A549) and normal mouse adipose cell line (3T3L1). Cells (5.0×103 cells well−1) were placed in 96-well culture plates (Tarson India Pvt. Ltd.) and grown overnight at 37°C in a 5% CO2 incubator. Compounds to be tested were then added to the wells to achieve final concentrations ranging from 50 to 750 μM. Control wells were prepared by addition of culture medium without the compounds. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h. Upon completion of the incubation, MTT dye solution was added to each well to a final concentration of 500 μg/mL. After 4 h incubation, the culture media were discarded and the wells were washed with phosphate buffer saline (Hi-Media, India Pvt. Ltd.), followed by addition of DMSO and subsequent incubation for 30 min. The optical density of each well was then measured on ELX800 Universal Microplate Reader at a wavelength of 540 nm. The IC50 values were determined by plotting the percentage viability versus concentration on a logarithmic graph and reading off the concentration at which 50% of cells remained viable relative to the control. Each experiment was repeated at least three times to obtain mean values.


## 4.7. Staining Techniques

### 4.7.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 16 h treatment with 100 μM metal complexesC5–C8, followed by PBS wash, the cells were stained with 1 μL of acridine orange (AO) and ethidium bromide (EB) dye mixture (1 mg/mL of each in PBS) in 9 μL of media. The cover slip was then mounted on a clean glass slide and immediately photographed confocal microscope [56].


### 4.7.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

A549 cells (1.0 × 105 cells well−1) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration, the cells were incubated with 7.5 × 10−6M DCFDA stain at 37°C for 30 min, observed, and photographed [57].


### 4.7.3. Staining Nucleic Acid Using DAPI Stain

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration the cells were washed with PBS and stained with 100 μL DAPI (300 μM) followed by incubation at 37°C for 30 min. The cover slip was mounted on a clean glass slide and immediately photographed [58].


## 4.7.1. Apoptosis and Necrosis Evaluation by Fluorescent Double Staining with AO/EB

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 16 h treatment with 100 μM metal complexesC5–C8, followed by PBS wash, the cells were stained with 1 μL of acridine orange (AO) and ethidium bromide (EB) dye mixture (1 mg/mL of each in PBS) in 9 μL of media. The cover slip was then mounted on a clean glass slide and immediately photographed confocal microscope [56].


## 4.7.2. Measurement of Intracellular ROS Generation Using DCFDA Stain

A549 cells (1.0 × 105 cells well−1) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration, the cells were incubated with 7.5 × 10−6M DCFDA stain at 37°C for 30 min, observed, and photographed [57].


## 4.7.3. Staining Nucleic Acid Using DAPI Stain

A549 cells (1.0 × 105 cells/well) were grown on cover slips using 6-well cell culture plates for 12 h. After a 24 h treatment withC5–C8 at 100 μM concentration the cells were washed with PBS and stained with 100 μL DAPI (300 μM) followed by incubation at 37°C for 30 min. The cover slip was mounted on a clean glass slide and immediately photographed [58].



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*Source: 630575-2014-07-14.xml*