Synthesis, crystal structure, DNA binding and cleavage studies of copper(II) complexes with isoxazole Schiff bases

Accepted Manuscript

Synthesis, Crystal structure, DNA binding and cleavage studies of Copper(II)

complexes with Isoxazole Schiff bases

Marri Pradeep Kumar, Somapangu Tejaswi, Aveli Rambabu, Veerendra Kumar

A. Kalalbandi, Shivaraj

PII:

S0277-5387(15)00407-6

DOI:

http://dx.doi.org/10.1016/j.poly.2015.07.052

POLY 11438

Reference:

Polyhedron

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Received Date:

Accepted Date:

13 April 2015

20 July 2015

Please cite this article as: M.P. Kumar, S. Tejaswi, A. Rambabu, V.K.A. Kalalbandi, Shivaraj, Synthesis, Crystal

structure, DNA binding and cleavage studies of Copper(II) complexes with Isoxazole Schiff bases, Polyhedron

(2015), doi: http://dx.doi.org/10.1016/j.poly.2015.07.052

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Synthesis, Crystal structure, DNA binding and cleavage studies of Copper(II)

complexes with Isoxazole Schiff bases

Marri Pradeep Kumar^a, Somapangu Tejaswi^a, Aveli Rambabu^a, Veerendra Kumar A.

Kalalbandi^band Shivaraj^a*

a) Department of Chemistry, Osmania University, Hyderabad, Telangana-500007, India

b) Department of Chemistry, Karnatak University, Dharwad-580003, India

Email: shivaraj_sunny@yahoo.co.in

Abstract:

Mobile No: +91 7842572128

Three novel binary copper(II) complexes 1 [Cu(L¹)₂], 2 [Cu(L²)₂] and 3 [Cu(L³)₂]

where, L¹(1-((E)-(3,5-dimethylisoxazol-4-ylimino)methyl)naphthalen-2-ol, C₁₆H₁₄N₂O₂), L²

(2-((E)-(3,5-dimethylisoxazol-4-ylimino)methyl)-4-methoxyphenol, C₁₃H₁₄N₂O₃) and L³(2-

((E)-(3,5-dimethylisoxazol-4-ylimino)methyl)-4-bromophenol, C₁₂H₁₁BrN₂O₂) have been

synthesized. All Cu(II) complexes have been characterized by elemental analysis, FT-IR, ESI

mass, UV-Visible, ESR, TG-DTA, magnetic moments and single crystal X-ray diffraction

studies. Based on spectral studies square planar geometry is assigned for all Cu(II)

complexes. Ligand L¹, [Cu(L¹)₂] and [Cu(L²)₂] are crystallized and found to be triclinic,

orthorhombic and monoclinic crystal systems respectively. The Schiff bases and their Cu(II)

complexes have been screened for antibacterial activity and antifungal activity by paper disc

method. It is observed that all Cu(II) complexes showed more activity than corresponding

Schiff bases. DNA binding and cleavage studies of Cu(II) complexes have also been

investigated. It is observed an intercalation mode of binding with CT-DNA and cleavage of

supercoiled pBR322 DNA in the presence of H₂O₂and UV light.

Key words: Schiff base, Binary Cu(II) complex, Crystal structure, Antimicrobial activity,

DNA interaction.

1. Introduction

The transition metal complexes of Schiff bases have tendency to interact with DNA

because of their cationic character and three dimensional structural profiles. It has been

proved that azomethine linkage (C=N) of Schiff base provides an opportunity for the

stupendous biological activities such as antitumor, antibacterial, antifungal and herbicidal

1

activities [1]. Metal complexes can bind to DNA through non-covalent interactions, such as

electrostatic binding, groove binding, intercalative binding and partial intercalative binding

[2]. Copper as an essential element for human beings with its bio essential activity and

oxidative nature has attracted numerous inorganic chemists to address medicinal applications

of Cu(II) complexes [3-8]. Copper(II) complexes containing heterocyclic bases have been

extensively explored in virtue of their strong interactions with DNA via surface associations

or intercalation [9-12] and potential DNA cleavage activities via hydrolytic, photolytic and

oxidative mechanisms [13-18]. Square planar complexes containing an aromatic moiety can

bind to DNA by intercalation and stabilize the DNA double helix [19, 20]. Synthesis,

characterization, biological activity and DNA interaction of Cu(II) complexes of isoxazole

Schiff bases were reported earlier from our laboratory [21].

In continuation of our earlier work, herein we report synthesis, characterisation, DNA

binding and cleavage studies of copper(II) complexes 1, 2 and 3. The antimicrobial property

of ligands and their complexes have also been assessed against selected bacteria and fungi.

2. Experimental

2.1. Materials and instrumentation

All reagents used in this study were analytical reagent grade obtained from Merck, Hi

Media Ltd., and Sigma-Aldrich Chemicals. Solvents such as water, methanol, acetone,

petroleum ether and chloroform were purified by standard procedures. The CT-DNA and

supercoiled pBR322 DNA were purchased from Genie, Bangalore and stored at 4^oC. Double

distilled water was used for preparing all the solutions for the DNA binding and cleavage

studies. A Tris-buffer solution of CT-DNA gave a ratio of 1.8–1.9 of UV absorbance at 260

and 280 nm, indicating that the DNA was sufficiently free of protein [22]. The DNA

concentration per nucleotide in base pairs was determined spectrophotometrically by

employing a molar absorptivity (6600 M^-1cm^-1) at 260 nm [23]. CT-DNA stock solution was

prepared by diluting DNA to Tris–HCl/NaCl buffer (pH = 7.2, 5 mM Tris–HCl, 50 mM

NaCl).

¹H-NMR and ¹³C-NMR spectra of the Schiff bases were recorded on Bruker 400 MHz

NMR instrument using TMS as internal standard. ESI mass spectra were recorded on VG

AUTOSPEC mass spectrometer. Electronic spectra of all compounds were recorded on

Schimadzu UV-VIS 1601 spectrophotometer. Magnetic susceptibilities of Cu(II) complexes

were determined on Gouy balance model 7550 using Hg[Co(NCS)₄] as standard. The

diamagnetic corrections of the complexes were computed using Pascal’s constants. Melting

2

points of the ligands and decomposition temperature of complexes were determined on

Polmon instrument (Model No. MP-96). IR spectra of the compounds were recorded using

KBr pellets in the range 4000-400 cm^-1on Perkin-Elmer Infrared model 337. The percentage

composition of C, H, N of the compounds were determined by using micro analytical

techniques on Perkin Elmer 240C (USA) elemental analyzer. Copper content of the

complexes was estimated by atomic absorption spectroscopy after decomposing the

complexes with concentrated HNO₃using GBC Avanta 1.0 AAS. The ESR spectra were

recorded by using JES-FA200 ESR Spectrometer (JEOL-Japan) at liquid nitrogen

temperature. The thermal studies were carried out in a dynamic nitrogen atmosphere (20ml

min^-1) with a heating rate of 10^oC min^-1using a Shimadzu TGA-50H in the temperature range

of 27-1000^oC. The high resolution X-ray diffraction data for compounds were collected on

Bruker SMART APEX2 CCD diffractometer using Mo Kα radiation (λ=0.71073Å) at 296K.

Viscosity measurements were performed using an Ostwald viscometer (Vensil).

2.2. Conventional and Microwave synthesis of Schiff bases

The synthetic route of Schiff bases L¹, L²and L³were described in Scheme 1. 3, 5-

dimethyl-4-amino- isoxazole (1.0 mmol) was dissolved in hot methanol to which methanolic

solution of 2-hydroxy naphthalene-1-carbaldehyde / 2-hydroxy-5-methoxy benzaldehyde / 2-

hydroxy-5-bromo benzaldehyde (1.0 mmol) was added and the resulting mixture was

refluxed with stirring at 60-80^oC for 3 hours. Microwave assisted synthesis was also carried

out using few drops of methanol in a domestic oven (LG, 1300W, 28L capacity). The

reaction mixture was subjected to microwave irradiation at 320W for the period of 60 to 90

sec. The dark yellow product formed was washed with petroleum ether and recrystallized

from methanol. Purity of the compounds was checked by TLC. Microwave assisted synthesis

brought down the reaction time from 3 hours to 90 sec and improved the yield from 70–75 to

90–95% in comparison with the conventional method. The suitable crystals for single crystal

X-ray study were grown in (1:1) methanol and dichloromethane mixture.

2.2.1. Ligand L¹: Yield 75% and 95%. M.P: 170^oC. Mol Wt: 266. Anal. Calc.

(C₁₆H₁₄N₂O₂)(%): C, 72.16; H, 5.30; N, 10.52. Found: C, 72.32; H, 5.39; N, 10.45. IR

(KBr)(cm^-1): υ_(OH)3454; υ_(CH=N)1603; υ_(C-O)1184. UV (CHCl₃) λ_max/nm(cm^-1): 374 (26738);

1

330 (30303); 246 (40650). H NMR (400 MHz, CDCl₃): δ = 14.65 (s, 1 H), 9.46 (s, 1 H),

8.04 (d, J = 8.4 Hz, 1 H), 7.87 (d, J = 9.0 Hz, 1 H), 7.80 (d, J = 8.1 Hz, 1 H), 7.58-7.55 (m, 1

H), 7.41-7.38 (m, 1 H), 7.20 (d, J = 9.0 Hz, 1 H), 2.56 (s, 3 H), 2.47 (s, 3 H), Fig. S1 (see

3

Supplementary). ¹³C NMR (400 MHz, CDCl₃): δ = 163.3, 159.8, 159.4, 154.8, 135.3, 132.5,

129.4, 128.1, 127.9, 125.6, 123.7, 119.8, 119.0, 109.4, 11.4, 10.8. MS (ESI): m/z = 266.9

[M+H]⁺.

2.2.2. Ligand L²: Yield 73% and 91%. M.P: 153^oC. Mol Wt: 246. Anal. Calc.

(C₁₃H₁₄N₂O₃)(%): C, 63.40; H, 5.73; N, 11.38. Found: C, 63.54; H, 5.69; N, 11.45. IR

(KBr)(cm^-1): υ_(OH)3443; υ_(CH=N)1583; υ_(C-O)1043. UV (CHCl₃) λ_max/nm(cm^-1): 368 (27174);

1

302 (33113); 252 (39683). H NMR (400 MHz, CDCl₃): δ = 12.27 (s, 1 H), 8.55 (s, 1 H),

7.02-6.94 (m, 2 H), 6.83 (d, J = 3.0 Hz, 1 H), 3.81 (s, 3 H), 2.50 (s, 3 H), 2.40 (s, 3 H), Fig.

S2 (see Supplementary). ¹³C NMR (400 MHz, CDCl₃): δ = 162.7, 160.3, 154.9, 154.7,

152.4, 125.3, 120.5, 118.7, 118.1, 115.0, 55.9, 11.3, 10.8. MS (ESI): m/z = 247.0 [M+H]⁺.

2.2.3. Ligand L³: Yield 70% and 90%. M.P: 161^oC. Mol Wt: 295. Anal. Calc.

(C₁₆H₁₄N₂O₂)(%): C, 48.84; H, 3.76; N, 9.49. Found: C, 48.96; H, 3.65; N, 9.58. IR

(KBr)(cm^-1): υ_(OH)3412; υ_(CH=N)1605; υ_(C-O)1230. UV (CHCl₃) λ_max/nm(cm^-1): 352 (28409);

1

301 (33223); 248 (40323). H NMR (400 MHz, CDCl₃): δ = 12.71 (s, 1 H), 8.52 (s, 1 H),

7.46-7.43 (m, 2 H), 6.91 (d, J = 8.28 Hz, 1 H), 2.51 (s, 3 H), 2.41 (s, 3 H), Fig. S3 (see

Supplementary). ¹³C NMR (400 MHz, CDCl₃): δ = 161.3, 160.9, 159.6, 154.6, 135.8, 134.0,

125.0, 120.5, 119.3, 110.8, 11.4, 11.0. MS (ESI): m/z = 297.0 [M+2]⁺.

2.3. Synthesis of binary Cu(II) complexes

The preparation of all Cu(II) complexes 1-3 were described in scheme. 1.

2.3.1. [Cu(L¹)₂](1): Hot methanolic solution of Cu(CH₃COO)₂.H₂O (0.5 mmol) (10 mL) was

added drop wise to a solution of L¹(1 mmol) in hot methanol (20 mL) and the reaction

mixture was refluxed with stirring at 60-80^oC for 5 hours. A dark colored solid product

obtained was filtered and washed with cold methanol and petroleum ether, dried in vacuum

desiccator over P₄O₁₀. Yield 80%. M.P: 208^oC. Mol Wt: 594. Anal. Calc.

(C₃₂H₂₆CuN₄O₄)(%): C, 64.69; H, 4.41; N, 9.43; Cu, 10.70. Found: C, 64.65; H, 4.44; N,

9.39; Cu, 10.67. IR (KBr)(cm^-1): υ_(C=N)1577, υ_(C-O)1168, υ_(M-O)541, υ_(M-N)464. ESR:

g_║=2.2935, g_⊥=2.0619, G=4.885. UV-Vis (DMSO) λ_max/nm(cm^-1): 223 (44843), 308

(32468), 416 (24038), ε (M^-1cm^-1) (0.013×10⁶). µ_eff(BM):1.82. Mass(m/z): 612.0 [M+NH₄]⁺.

2.3.2. [Cu(L²)₂](2): Following the same procedure as described in synthesis of 1 received

complex 2 with L². Yield 78%. M.P: 205^oC. Mol Wt: 554. Anal. Calc. (C₂₆H₂₆CuN₄O₆)(%):

4

C, 56.36; H, 4.73; N, 10.11; Cu, 11.47. Found: C, 56.32; H, 4.75; N, 10.08; Cu, 11.49. IR

(KBr)(cm^-1): υ_(C=N)1533, υ_(C-O)1033, υ_(M-O)559, υ_(M-N)459. ESR: g_║=2.2412, g_⊥=2.0532,

G=4.6935. UV-Vis (DMSO) λ_max/nm(cm^-1): 222 (45045), 296 (33784), 356 (28090), ε (M^-

¹cm^-1) (0.015×10⁶). µ_eff(BM):1.74. Mass(m/z): 554.1 [M] ⁺.

2.3.3. [Cu(L³)₂](3): Following the same procedure as described in synthesis of 1 received

complex

3

with L³. Yield 75%. M.P: 197^oC. Mol Wt: 652. Anal. Calc.

(C₂₄H₂₀Br₂CuN₄O₄)(%): C, 44.23; H, 3.09; N, 8.60; Cu, 9.75. Found: C, 44.25; H, 3.06; N,

8.66; Cu, 9.71. IR (KBr)(cm^-1): υ_(C=N)1600, υ_(C-O)1222, υ_(M-O)559, υ_(M-N)462. ESR:

g_║=2.2613, g_⊥=2.0466, G=5.8465. UV-Vis (DMSO) λ_max/nm(cm^-1): 224 (44643), 343

(29155), 399 (25063), ε (M^-1cm^-1) (0.004×10⁶); µ_eff(BM):1.77. Mass(m/z): 652.0 [M] ⁺.

5

Scheme. 1. Synthesis of Schiff bases and their copper(II) complexes

2.4. X-ray crystallographic procedures

The single crystal of the size of 0.30x0.20x0.20 mm was selected under a polarizing

microscope and it was mounted on a glass fiber for x-ray diffraction data collection. The high

resolution X-ray diffraction data sets for both Schiff bases and complexes were collected on

Bruker SMART APEX2 CCD diffractometer using Mo Kα radiation (λ = 0.71073 Å) at 296

K. The crystal-to-detector distance was fixed at 40 mm. The diffraction data have been scaled

for absorption effect by the multi-scanning method. Data collection has been performed by

6

applying the apex-II Software system. Structures were solved by direct methods and refined

on F²by weighted full-matrix least-squares. Programs SHELXS97 and SHELXL97

integrated in the WinGX v. 1.70.01 [24] software system were used to solve and refine the

structure. All non-hydrogen atoms were refined anisotropically. The ORTEP, planes and

packing diagrams were generated using the Mercury 3.3 software.

2.5. Antimicrobial activity

The in vitro antibacterial activity of the ligands L¹, L²and L³and their copper(II)

complexes 1, 2 and 3 were tested against the gram negative bacteria Escherichia coli (E.coli),

Pseudomonas putida (P.putida) and Klebsiella pneumoniae (K.pneumoniae) and gram

positive bacteria Bacillus subtilis (B.subtilis) and Staphylococcus aureus (S.aureus). The

antifungal activity was tested against Aspergillus niger (A.niger) and Candida albicans

(C.albicans). The paper disc technique was employed [25] for antimicrobial activity using

nutrient agar as the medium. The stock solution was prepared by dissolving the 1 mg of

sample in 10 mL of DMSO to give the concentration of 100µg/mL. The nutrient agar

medium was inoculated with the test organisms. The sterilized blank paper discs of 6 mm

diameter were impregnated with tested compounds and placed on the surface of the agar

plates previously spread with 0.1 mL of overnight culture of microorganisms. The incubation

was carried out for 24 hours at 30^oC. During this period, the test solution diffused and the

growth of the inoculated microorganisms were affected.

2.6. DNA-binding experiments

2.6.1. Absorption study

Absorption titration experiments were performed with fixed complex concentrations

(10 µM) while varying the CT-DNA concentration within 0-10 µM. Because of their low

solubility in the buffer solution the copper(II) complexes were dissolved in DMSO first to get

a stock solution. While measuring the absorption, equal increments of CT-DNA were added

to both the complex solution and the reference solution to eliminate the absorbance of CT-

DNA itself.

2.6.2. Viscosity measurements

Viscosity experiments were carried out by using an Ostwald capillary viscometer

immersed in a thermostatic water bath with the temperature setting at 30 0.1^oC. In this

7

experiment DNA concentration was kept constant and concentration of complexes solution

increased gradually. The flow time of the solution through the capillary was determined by a

digital stop-watch. This flow time measurement was repeated three times for the same

complexes solution to get the average flow time. To study the effect of DNA binding on the

viscosity of the complex, a plot was made between (η/η_o)^1/3and the ratio of the concentration

of the complexes to CT-DNA, where η is the viscosity of CT-DNA in the presence of the

complex and η_ois the viscosity of CT-DNA alone. Viscosity values were calculated from the

experimental flow time of CT-DNA containing solutions corrected from the flow time of

buffer alone.

2.7. DNA cleavage experiments

Interactions between all Cu(II) complexes and supercoiled pBR322 DNA were

studied using agarose-gel electrophoresis at pH 7.5 in Tris–HCl buffer solution. Oxidative

DNA cleavage in the presence of H₂O₂and photolytic cleavage in the presence of UV light

were monitored by treating with supercoiled pBR322 DNA by varying concentration of

Cu(II) complexes and followed by dilution with 5mM Tris-HCl / 5mM NaCl buffer to a total

volume of 16µL. The samples were incubated at 37°C for 2 hours. DNA cleavage activity

was evaluated by monitoring the conversion of supercoiled pBR322 DNA (Form I) to nicked

circular DNA (Form II) and linear DNA (Form III).

3. Results and discussion

All the Cu(II) complexes are coloured, stable at room temperature and non-

hygroscopic. On heating they decompose at high temperatures. These complexes are

insoluble in water, alcohol and chloroform but soluble in acetonitrile, DMF and DMSO. The

analytical data obtained for all complexes are consistent with the formation of mononuclear

copper complexes and the metal to ligand ratio is 1:2.

3.1. X-ray diffraction analysis

Single crystals of ligand L¹and complexes 1 and 2 were grown by slow evaporation

of methanol and dichloromethane mixture of solvents and their crystal structures were solved

and refined by SHELXS-97 [24]. The ORTEP diagrams of the L¹, the mononuclear unit of

complexes 1 and 2 are presented in Fig. 1. The crystal refinement data is given in Table. 1.

The L¹is almost planar as the dimethyl isoxazole ring is not making any significant dihedral

8

angle with naphthalenol moiety. All the bond lengths and bond angles are observed to be in

normal ranges. There is hydrogen bonding between hydrogen atom attached to O1 and N1 of

Schiff base. In crystal packing of L¹it is observed that two molecules were packed in the unit

cell.

Fig. 1. Thermal ellipsoidal plot of L¹, complexes 1 and 2 with atom labeling scheme.

Displacement ellipsoids are drawn at 50% probability level except for the H atoms, which are

shown as circles of arbitrary radius.

Table. 1. Crystal structure refinement data of ligand L¹, complexes 1 and 2.

Parameter

L¹

1

2

Empirical formula

Formula weight

Temperature

C₁₆H₁₄N₂O₂

266.29

C₃₂H₂₆CuN₄O₄

594.12

C₂₆H₂₆CuN₄O₆

554.05

296K

Crystal size

0.42x0.22x0.18mm

Triclinic

0.30x0.20x0.20 mm

Orthorhombic

0.30x0.20x0.20 mm

Monoclinic

Crystal system

9

Space group

P1

̅

Pbca

P2₁/n

A

B

C

α

7.5360 (8)(Å)

9.0395 (11) (Å)

10.8864 (11) (Å)

74.071 (10)˚

10.5512 (3)(Å)

15.2811 (5) (Å)

16.8042 (6) (Å)

90˚

10.6396 (7) (Å)

10.6082 (7) (Å)

11.9303 (8) (Å)

90˚

β

86.689 (9)˚

90˚

104.258 (4)˚

90˚

γ

69.227 (11)˚

90˚

-8<=h<=8,

-9<=k<=10,

-12<=l<=11

-13<=h<=13,

-16<=k<=18,

-8<=l<=21

-13<=h<=13,

-13<=k<=13,

-14<=l<=14

Limiting indices

Volume

666.12 (13) (Å³)

2, 1.328 mg/m³

4114

2709.41 (15) Å³

4, 1.466 mg/m³

12575

1305.06 (15) Å³

2, 1.425 mg/m³

9270

Z, Calculated density

Measured reflections

No. of independent

reflections

2335

3006

2588

θ

range for data θ_max= 24.99°,

θ_max= 27.31°,

θ_min= 2.42°

θ_max= 27.31°,

θ_min= 2.42°

collection

θ_min= 2.72°

Final R indices

[I> 2σ(I)]

R1 = 0.0525,

wR2 = 0.1246

R1 = 0.0360,

wR2 = 0.1052

R1 = 0.0389,

wR2 = 0.1036

R1 = 0.0964,

wR2 = 0.1619

R1 = 0.0530,

wR2 = 0.1163

R1 = 0.0542,

wR2 = 0.1117

R indices (all data)

Data

parameters

/

restraints

/

2335 / 0 / 184

SHELXS-97

3006 / 0 / 189

SHELXS-97

2588 / 0 / 172

SHELXS-97

Structure

Determination

Refinement

Full-matrix

least-squares on F²

Difference

density Min/max/rms

electron

-0.189/0.138/0.038

CCDC-936590

-0.504/0.238/0.050

CCDC 1005830

-0.471/0.232/0.052

CCDC 1005831

CCDC number

The crystal units of complexes 1 and 2 have crystallographic inversion centre located

through the Cu atom. In both complexes, Cu(II) ion is surrounded by two molecules of

respective ligands and each ligand acts as a mono negative bidentate chelating agent. The

structures of complexes 1 and 2 reveal that Cu(II) is in a square planar environment having

trans-N₂O₂donor set. However, the complex 1 is observed to be slightly distorted from

10

square planar when compared to that of complex 2 as evidenced by bond angles [N1-Cu1-O2

89.95(7)º (θ₁), O2-Cu1-N1 90.05(7)º (θ₂) for complex 1 and N1-Cu1-O1 88.11(8)º (θ₁),

O1-Cu1-N1a 91.89(8)º (θ₂) for complex 2]. Further, trans L-M-L angles θ₁and θ₂are equal

to 180º in both complexes [26]. The Cu···N distances in complexes 1 and 2 are 1.969(17) Ǻ

and 1.988(19) Ǻ respectively while the Cu···O distances in complexes 1 and 2 are 1.895(15)

Ǻ and 1.870(17) Ǻ respectively. Upon examination of metal-ligand distances showed that the

Cu···N distances in complex 1 are shorter than that in complex 2, whereas the Cu···O

distances in complex 1 are longer than that observed in complex 2. Further, the Cu···N

distances are longer than Cu···O distances in both complexes. Typical Cu···N distances are

ranging from 1.939 to 2.019 Ǻ while the Cu···O distances ranging from 1.879 to 1.893 Ǻ

[27-30].

From the crystal structure of the ligand L¹it is evident that the ligand molecule is

almost planar, but during complexation the five membered dimethyl isoxazole ring [O1 N2

C2 C3 C1] forms a dihedral angle of 41.64º with the mean plane of naphthalenol moiety [C11

C12 C13 C14 C15 C16] to stabilize the complex structure Fig. 2., while in complex 2 both

the central metal ion and phenol rings are in the same plane whereas the five membered

dimethyl isoxazole ring forms a dihedral angle of 79.20º with the mean plane of the phenol

ring to stabilize the complex structure Fig. 2. Further, no classical hydrogen bonding is

observed in both complexes. However, the molecules of both complexes are stabilized by

several weak intermolecular interactions in the solid state, Table. S1 (see Supplementary).

The crystal packing structures of complexes 1 and 2 are shown in Fig. S4 (see

Supplementary).

Fig. 2. Dihedral angles formed in complexes 1 and 2.

11

3.2. Spectral characterization

3.2.1. FT-IR spectra

The coordination mode and sites of the ligands to the metal ions have been

investigated by comparing the infrared spectra of the free ligands with their corresponding

Cu(II) complexes. There are some significant shifts in stretching frequencies of Cu(II)

complexes when compared to free ligand upon chelation as expected. The IR data is

presented in Table. 2. The Schiff bases exhibit a characteristic strong band around 1605–

-1

1583 cm^-1due to the υ(C=N). This band is shifted by 5–50 cm to a lower wave number in

complexes indicating coordination of azomethine nitrogen atom of Schiff bases [31-34]. A

broad band in the range of 3412-3454 cm^-1is due to phenolic -OH group of Schiff base

disappeared in their copper(II) complexes confirming the participation of oxygen of phenolic

group in coordination to the metal ion. This is also confirmed by the shift in the υ(C–O) band

at 1043-1230 cm⁻¹to lower frequency to the extent of 8–16 cm⁻¹in respective Cu(II)

complexes [35]. The coordination of the azomethine nitrogen and phenolic oxygen are further

supported by the appearance of two non ligand bands at 541–559 cm^-1and 459–464 cm^-1due

to υ(Cu-O) and υ(Cu-N) respectively [36, 37], Figs. S5 and S6 (see Supplementary).

Table. 2. Some important IR Absorption frequencies (cm^-1) of Schiff bases and their Cu(II)

complexes.

COMPOUND

L¹

v(OH) v(HC=N) v (C-O)

v (M-O) v (M-N)

3454

1603

1577

1583

1533

1605

1600

1184

1168

1043

1033

1230

1222

-

464

-

[Cu(L¹)₂]

L²

-

3443

-

541

-

[Cu(L²)₂]

559

-

459

-

L³

3412

-

[Cu(L³)₂]

559

462

12

3.2.2. Electronic spectra and magnetic susceptibility

The magnetic susceptibility values (µ_eff) of all the Cu(II) complexes are in the range

of 1.74–1.82 BM which is consistent with the presence of a single unpaired electron [38, 39].

Three absorption bands for L¹, L²and L³Schiff base ligands are observed at 252–246 nm,

330–301 nm and 374–352 nm respectively, Figs. S7 and S8 (see Supplementary). The bands

at higher energies (252–246 nm) and (330–301 nm) are attributed to the π-π* transitions of

the benzene ring while the other low energy bands (374–352) are assigned to the π-π*

transitions of the azomethine chromophore respectively [40, 41]. These absorptions are also

present in the spectra of copper(II) complexes but they undergone bathochromic shift. This

shift in the spectra of the complexes supported the coordination of the ligands to Cu(II) ion.

In addition to these bands all the Cu(II) complexes exhibit an additional and much important

2

characteristic broad d-d transition band at 416-356 nm due to B_1g→²E_gtransition. From the

electronic spectral data and magnetic susceptibility values a square planar geometry is

assigned to all Cu(II) complexes [42].

3.2.3. ESR spectra

The ESR spectra of Cu(II) complexes provide information about the extent of the

delocalization of unpaired electron. The X-band ESR spectra of Cu(II) complexes were

recorded in liquid state at liquid nitrogen temperature (77K), their g_║, g_⊥, ∆g, g_avand G have

been calculated. ESR spectra of complexes 1, 2 and 3 are given in Fig. 3. The values of ESR

parameters g_║, g_⊥, g_av, ∆g and G for complex 1 are found to be 2.2935, 2.0619, 2.1391,

0.2316 and 4.885 respectively, for complex 2 are 2.2412, 2.0532, 2.116, 0.188, and 4.6935

respectively and for complex 3 are 2.2613, 2.0466, 2.118, 0.2147 and 5.8465 respectively.

2

The values g_║>g_⊥>g_e(2.0023) revealed that the unpaired electron is localized in d_x-_yorbital

2

with B_1gground state of Cu(II) square planar complex [43]. The g_║values for these

complexes are found to be less than 2.3 suggesting covalent environment [44]. In all Cu(II)

complexes the G values are found to be within the range of 4.6935-5.8465 indicating

negligible exchange interaction of Cu-Cu in the complex according to Hathaway [45].

13

Fig. 3. ESR spectra of complexes 1, 2 and 3.

3.2.4. ESI mass spectral studies

The mass spectra of ligands L¹, L^2,L³and complexes 1, 2 and 3 are recorded in

positive mode exhibited peaks at ESI mass: 266.9, 247.0, 297.0, 612.0, 554.1 and 652.0

respectively, Figs. S9-S14 (see Supplementary) indicating the presence of [M+H]⁺, [M+H]⁺,

[M+2]⁺, [M+NH₄]⁺, [M]⁺and [M]⁺ions respectively. These molecular ions of complexes

confirm the stoichiometry of copper complex as [Cu(L)₂] type.

3.2.5. Thermal analysis

Thermo gravimetric (TG) and differential thermal analysis (DTA) are useful

techniques to determine the thermal stability of the metal complexes. In the present study

heating rate is suitably controlled at 10^oC/min under nitrogen atmosphere and the loss in

weight is measured up to 1000^oC. Thermo grams of complexes 1, 2 and 3 are shown in Fig.

4. The results showed that all the Cu(II) complexes have a similar decomposition process

mainly in two steps. These complexes exhibit thermal stability up to 200^oC which confirms

14

that these complexes are free from coordinated water molecules. In the first step, these

complexes showed weight loss around 215–350^oC associated with the partial loss of ligand.

In the second decomposition step mass loss is appeared at the temperature range 409–790^oC

corresponding to the complete thermal decomposition of the complexes and the loss of their

organic portion resulting CuO as final products. This process is accompanied by an

exothermic process in the range of 311°C to 349°C in DTA curves of the complexes [46],

Fig. S15 (see Supplementary).

Fig. 4. Thermal analysis curves of complexes 1, 2 and 3.

3.3. Antimicrobial activity

In vitro biological screening effects of the ligands L¹, L², L³and their Cu(II)

complexes 1, 2 and 3 are tested against the bacterial species such as E. coli, P. putida, K.

pneumoniae, B. subtilis and S. aureus as well as fungal species such as A. niger and C.

albicans by the paper disc method. The inhibition zone values (mm) of the compounds are

summarized in Table. 3. A comparative study of the ligands and their complexes indicates

that complexes exhibited higher antimicrobial activity than the free ligands. The increase in

15

antimicrobial activity of the complexes can be explained on the basis of Overtone’s concept

[47] and Tweedy’s Chelation theory [48, 49]. According to Overtone’s concept of cell

permeability, the lipid membrane that surrounds the cell favors the passage of only the lipid

soluble materials makes which liposolubility as an important factor which controls the

antimicrobial activity. Moreover, coordination reduces the polarity of the metal ion

essentially because of the partial sharing of its positive charge with the donor groups [50]

within the chelate ring system formed during coordination. This process, in turn, increases the

lipophilic nature of the central metal atom which favors its permeation more effectively

through the lipid layer of microorganism [51] thus destroying them more aggressively.

Table. 3. Antimicrobial activity results of Schiff bases and their Cu(II) complexes at

100µg/mL concentration.

Compound

Bacterium(mm)

Fungi(mm)

Gram-negative bacteria

Gram-positive bacteria

A.niger C.albicans

E.coli P.putida K.pneumoniae

B.subtilis

S.aureus

L¹

10

12

11

22

23

24

2

9

11

9

8

10

7

11

10

25

23

24

2

9

L²

11

8

10

11

21

22

20

-

7

L³

9

8

24

25

-

1

20

24

21

-

20

21

19

3

23

20

-

2

3

Cu(NO₃)₂.3H₂O

Ampicillin

Ketoconazole

30

-

31

-

30

-

33

-

31

-

32

31

16

3.4. DNA binding studies

3.4.1. UV–Vis spectroscopic studies

The binding activities of DNA–metal complexes have been a clue of paramount

importance for understanding the mechanism of effective metal based chemotherapeutic

drugs [52]. Electronic absorption spectroscopy is an effective method to examine the binding

mode of DNA with metal complexes [53, 54]. The binding behavior of the metal complexes

to DNA helix is often investigated by the changes in the absorbance and shift in the

wavelength. A complex bound to DNA through intercalation is characterized by the change

in absorbance (hypochromism) and red shift in wavelength due to the intercalative mode

involving a stacking interaction between the aromatic chromophore and the DNA base pairs.

Metallo-intercalators are metal complexes bearing planar aromatic side groups of the

coordinating ligands. As the name suggests, these ligands, oriented parallel to the base pairs

and protruding away from the metal center, can readily π-stack in the DNA duplex. Often, in

such complexes the metal ion is part of the planar portion [55]. After the complexes

intercalate to the base pair of DNA, the π * orbital of the intercalated ligand on the complexes

can couple with π orbital of the base pairs, thus decreasing the π-π * transition energies. On

the other hand, the coupling π * orbital are partially filled by electrons, thus decreasing the

transition probabilities [56, 57]. The extent of hypochromism is commonly consistent with

the strength of the intercalative interaction [58]. In the present investigation, the Cu(II)

complexes show a band in the range of 416–356 nm attributed to a d-d transition band. On

addition of DNA, the intensity of the d-d transition band decreases by 31–73%

(hypochromism) with a red shift of 2–16 nm in the absorption spectra. The absorption spectra

of complexes 1, 2 and 3 in absence and presence of CT-DNA are shown in Figs. 5, 6 and 7.

The intrinsic binding constant K_bis determined from a plot of [DNA]/(ε_a-ε_f) vs [DNA]

using the equation: [DNA]/(ε_a-ε_f) = [DNA]/(ε_b-ε_f) +1/K_b(ε_b-ε_f), where [DNA] is the

concentration of DNA in base pairs, and the apparent absorption coefficients ε_a, ε_fand ε_b

correspond to A_obs/[complex], the extinction coefficient for the free compound and for the

compound in the fully bound form respectively [59]. K_bis given by the ratio of slope to the

intercept. From the absorption data, the binding constant K_bfor complex 1 is 7.43 0.02×10⁶

M^-1, complex 2 is 3.38 0.03×10⁵M^-1and complex 3 is 2.92 0.02×10⁵M^-1. The K_bvalues are

comparable with the potential intercalators like ethidium bromide (EB) (K_b=7×10⁷M^-1) [60].

These results show that the strength of interaction of the complexes with CT-DNA.

17

Fig. 5. Absorption spectra of 1 in the absence (dashed line) and presence (solid line) of

increasing amounts of CT-DNA in Tris HCl buffer (pH 7.2). Conditions: [1] =10 µM, [DNA]

↓) shows the absorbance changes upon increasing DNA concentration.

= 0–10 µM. Arrow (

Inset: linear plot for the calculation of the intrinsic DNA binding constant, K_b.

18

Fig. 6. Absorption spectra of 2 in the absence (dashed line) and presence (solid line) of

increasing amounts of CT-DNA in Tris HCl buffer (pH 7.2). Conditions: [2] =10 µM, [DNA]

↓) s

= 0–10 µM. Arrow (

hows the absorbance changes upon increasing DNA concentration.

Inset: linear plot for the calculation of the intrinsic DNA binding constant, K_b.

19

Fig. 7. Absorption spectra of 3 in the absence (dashed line) and presence (solid line) of

increasing amounts of CT-DNA in Tris HCl buffer (pH 7.2). Conditions: [3] =10 µM, [DNA]

= 0–10 µM. Arrow (↓) shows the absorbance changes upon increasing DNA concentration.

Inset: linear plot for the calculation of the intrinsic DNA binding constant, K_b.

3.4.2. Viscosity measurements

The viscometric measurement is also an important tool to find the nature of binding of

metal complexes to the DNA. Hydrodynamic measurements (i.e., viscosity and

sedimentation) sensitive to length changes are the most critical and least ambiguous tests of

binding in solution without crystallographic data [61, 62]. A classical intercalation molecule,

such as EB, results in the lengthening of the DNA helix. This leads to an increase in DNA

viscosity, which is caused by an increase in the separation of base pairs at the interaction site

and an increase in overall double helix length. By contrast, a partial or non classical

intercalation of the complex results in bending of the DNA helix, which reduces the effective

length of DNA, as well as the DNA viscosities. The relative specific viscosity of DNA is

determined by varying the concentration of the added metal complexes. The effects of all the

20

synthesized Cu(II) complexes on the viscosity of DNA at 30 0.1^oC are shown in Fig. 8. The

experimental results clearly showed that all the complexes can intercalate between adjacent

DNA base pairs as also evidenced by UV-Vis spectroscopic results. By increasing the

complex concentration to DNA causing an extension in the helix and thus increase the

viscosity of DNA [63]. The complexes can intercalate strongly leading to a greater increase

in viscosity of the DNA with an increasing concentration of complexes. The increase in the

degree of viscosity follows the order of complex 1> 2>3. This may be due to affinity of the

complex towards DNA.

Fig. 8. Effect of increasing amounts of EB, complexes 1, 2 and 3 on the relative viscosity of

CT-DNA at 30 0.1^oC.

3.5. DNA cleavage

The DNA cleavage of super coiled pBR322 DNA is investigated by agarose gel

electrophoresis method with synthesized Cu(II) complexes at different concentrations in

presence of H₂O₂and UV light. The results are shown in Figs. 9, 10 and Figs. S16, 17 (see

21

Supplementary). The ability of the complexes in DNA cleavage is estimated by the

conversion of DNA from Form I to Form II and Form III. The fastest migration is detected in

the supercoiled form (Form I). If only one strand is cleaved, the super coils relax to convert

into a slower-moving nicked form (Form II). If both strands are cleaved, a linear form (Form

III) is produced which migrates in between Form I and Form II [64].

After incubation of supercoiled pBR322 DNA with the Cu(II) complexes in presence

of H₂O₂(oxidative cleavage) and UV irradiation at 345 nm (photo cleavage) [13], for control

experiments no DNA cleavage is observed as there is no complex(lane 1). With increasing

concentrations of the Cu(II) complexes, the percentage of super coiled DNA (Form I)

diminished gradually where as nicked circular DNA (Form II) and linear DNA (Form III)

increased. These results indicate that the Cu(II) complexes can degrade supercoiled pBR322

DNA. Further it is observed that complex 1 promote the cleavage of supercoiled pBR322

DNA more efficiently than complexes 2 and 3.

3.5.1. Mechanism for Oxidative Cleavage:

On the basis of above observation, the mechanism of DNA cleavage mediated by 1-3

may be proposed as follows: DNA cleavage is redox-mediated. The complexes would first

interact with DNA by intercalation to form a Cu^II· · · DNA adduct species, followed by its

reduction by the external agent (H₂O₂) to a Cu^I· · · DNA adduct, which then generates HO^•

radicals on reaction with O₂. These HO^•radicals would then attack DNA, causing strand

scission (Scheme 2). A similar pathway was proposed by Sigman for the oxidative cleavage

reaction of the bis(phen)copper complex [64].

Scheme 2. A Possible Mechanism for the Oxidative Cleavage of DNA by complexes 1–3 in

the Presence of H₂O₂.

22

3.5.2. Mechanism for Photolytic Cleavage:

Observation of DNA photolytic cleavage mediated by 1-3 indicates the involvement of

singlet oxygen (¹O₂) as the reactive species. As suggested by Toshima et al., the triplet states

resulting from the n-p* and π-π* photo-excitation in the UV radiation could activate oxygen

to form ¹O₂that cleaves DNA [65].

Fig. 9. Oxidative cleavage of supercoiled pBR322 DNA(0.2 µg, 33.3 µM) at 37 °C in 5mM

Tris. HCl/5 mM NaCl buffer by the Cu(II) complexes 1 and 3. (a) Lane 1, DNA control;

Lane 2, DNA + H₂O₂(1mM); Lane 3–8, DNA + H₂O₂(1mM) + complex 1, [complex] = 20,

30, 40, 50, 60 and 70 µM, respectively. (b) Lane 1, DNA control; Lane 2, DNA + H₂O₂

(1mM); Lane 3–8, DNA + H₂O₂(1mM) + complex 3, [complex] = 20, 30, 40, 50, 60 and 70

µM, respectively.

Fig. 10. Photoactivated cleavage of supercoiled pBR322 DNA (0.2 µg, 33.3 µM) at 37 °C in

5mM Tris. HCl/5 mM NaCl buffer by the Cu(II) complexes 1 and 2 using UV irradiation of

wavelength 345 nm. (a) Lane 1, DNA control; Lane 2–7, DNA + complex 1, [complex] = 20,

23

30, 40, 50, 60 and 70 µM, respectively. (b) Lane 1, DNA control; Lane 2–7, DNA + complex

2, [complex] = 20, 30, 40, 50, 60 and 70 µM, respectively.

Conclusion

The synthesized Schiff bases and their binary Cu(II) complexes have been

characterized by analytical and spectral analysis. The crystal structures of ligand L¹,

complexes 1 and 2 have also been determined by X-ray diffraction studies. From the data it is

observed that in solid state all Cu(II) complexes adopt a square planar geometry. In vitro

antibacterial and antifungal activities of Schiff base ligands and their Cu(II) complexes have

been screened. It is observed that all the Cu(II) complexes exhibit better activity than the

corresponding Schiff base ligands and also some complexes showed comparable activity with

the standard drug. The interaction between the Cu(II) complexes and CT-DNA have been

investigated by UV-Vis spectra and viscosity measurements. The respective results of the

binding studies revealed that an intercalative mode of binding. The DNA cleavage studies of

Cu(II) complexes revealed that these complexes can effectively cleave supercoiled pBR322

DNA in the presence of H₂O₂and also in the presence of UV light. Further it is observed that

among the complexes, complex 1 exhibits good antimicrobial, DNA binding and cleaving

properties.

Acknowledgements

We express our sincere thanks to the Head, Department of the Chemistry for

providing the necessary facilities. We are thankful to the Director, CFRD, Osmania

University, Hyderabad, and the Director, IICT, Hyderabad, and the SAIF, IIT Bombay for

providing spectral and analytical data. We are thankful to the Department of Chemistry,

Karnatak University, Dharwad for providing single crystal X-ray diffraction analysis. We are

also thankful to CSIR, New Delhi, DST-SERB and UGC-UPE (FAR) for providing financial

assistance.

Appendix A. Supplementary material

CCDC 936590, 1005830 and 1005831 contain the supplementary crystallographic

data for L¹, complex 1 and 2, respectively. These data can be obtained free of charge via

http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic

24

Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e-mail:

deposit@ccdc.cam.ac.uk.

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