Lasing the DNA fragments through β-diketimine framed Knoevenagel condensed Cu(II) and Zn(II) complexes - An in vitro and in vivo approach

Article abstract of DOI:10.1016/j.saa.2013.09.109

The syntheses, structures and spectroscopic properties of Cu(II) and Zn(II) complexes having Knoevenagel condensate β-diketimine Schiff base ligands have been investigated in this paper. Characterization of these complexes was carried out using FTIR, NMR, UV-Vis, elemental analysis, mass and EPR techniques. Absorption titration, electrochemical analyses and viscosity measurements have also been carried out to determine the mode of binding. The shift in ΔEp, E_1/2 and Ip_c values explores the interaction of CT DNA with the above metal complexes. Interaction of ligands and their complexes with DNA revealed an intercalative mode of binding between them. Antimicrobial studies showed an effective antimicrobial activity of the metal ions after coordination with the ligands. The antioxidant properties of the Schiff base ligands and their complexes were evaluated in a series of in vitro tests by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and H ₂O₂ free radical scavengers. In vivo and in vitro antitumor functions of the complexes against Ehrlich ascites carcinoma tumor model have also been investigated. All the results support that β-diketone derived Knoevenagel condensate Schiff base complexes may act as novel antitumor drugs and suggest that their potent cell life inhibition may contribute to their anti-cancer efficacy.

Full text of DOI:10.1016/j.saa.2013.09.109

Accepted Manuscript
Lasing the DNA fragments through β-diketimine framed Knoevenagel con-
densed Cu(II) and Zn(II) complexes – An in vitro and in vivo approach
Natarajan Raman, Narayanaperumal Pravin
PII:
S1386-1425(13)01116-5
http://dx.doi.org/10.1016/j.saa.2013.09.109
SAA 11088
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Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
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10 September 2013
26 September 2013
Please cite this article as: N. Raman, N. Pravin, Lasing the DNA fragments through β-diketimine framed
Knoevenagel condensed Cu(II) and Zn(II) complexes – An in vitro and in vivo approach, Spectrochimica Acta Part
A: Molecular and Biomolecular Spectroscopy (2013), doi: http://dx.doi.org/10.1016/j.saa.2013.09.109
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Lasing the DNA fragments through β-diketimine framed Knoevenagel condensed Cu(II)
and Zn(II) complexes – An in vitro and in vivo approach
Natarajan Raman,* Narayanaperumal Pravin
Research Department of Chemistry, VHNSN College, Virudhunagar-626 001, Tamil Nadu, India,
E-mail: ramchem1964@gmail.com Tel: +091-92451-65958
E-mail: pravinknp2012@gmail.com Tel: +091-9677846333
___________________________________________________________________________________________
ABSTRACT
_____________________________________________________________________________
The syntheses, structures and spectroscopic properties of Cu(II) and Zn(II) complexes
having Knoevenagel condensate β-diketimine Schiff base ligands have been investigated in this
paper. Characterization of these complexes was carried out using FTIR, NMR, UV–Vis.,
elemental analysis, mass and EPR techniques. Absorption titration, electrochemical analyses and
viscosity measurements have also been carried out to determine the mode of binding. The shift in
∆Ep, E1/2 and Ipc values explores the interaction of CT DNA with the above metal complexes.
Interaction of ligands and their complexes with DNA revealed an intercalative mode of binding
between them. Antimicrobial studies showed an effective antimicrobial activity of the metal ions
after coordination with the ligands. The antioxidant properties of the Schiff base ligands and
their complexes were evaluated in a series of in vitro tests by using 1,1-diphenyl-2-
picrylhydrazyl (DPPH•) and H2O2 free radical scavengers. In vivo and in vitro antitumor
functions of the complexes against Ehrlich ascites carcinoma tumor model have also been
investigated. All the results support that β–diketone derived Knoevenagel condensate Schiff base
complexes may act as novel antitumor drugs and suggest that their potent cell life inhibition may
contribute to their anti-cancer efficacy.
______________________________________________________________________________
Keywords:
β-diketimine; intercalative mode; antimicrobial studies; antioxidant properties; antitumor activity
______________________________________________________________________________
*Corresponding author: E-mail: ramchem1964@gmail.com (N.Raman)
Tel.: +091-092451-65958; Fax: +091-4562-281338
1

1. Introduction
Medicinal researchers are continuing all over world in order to explore a safe and
effective metal based biologically active compound as potential antitumor drug which is of great
urgency to overcome the drug-induced cellular resistance and the efficacy of each drug against
certain cancers. The treatment involves the administration of multiple drugs as it is clear that
chemotherapy leads to the development of resistance. As the first generation of the platinum-
based antitumor agent, cisplatin is widely used in the treatment of head and neck, testicular,
small lung cell and ovarian cancers [1–4]. In spite of its success, the clinical application of
cisplatin is greatly restricted by its toxicity, low water-solubility, instinct and acquired drug
resistance [5,6]. Thousands of platinum compounds have been synthesized and screened for their
antitumor activity, but little success has been achieved so far in finding novel platinum-based
drugs active towards cisplatin resistant/refractory tumors [7,8]. Non-platinum active compounds
are likely to have mechanism of action, bio distribution and toxicity which are different from
those of platinum drugs and might be effective against human cancers that are poor
chemosensitive or have become resistant to conventional platinum drugs. Among the different
therapeutic strategies to eradicate cancer cells through DNA damage, the view of using transition
metal complexes, capable of oxidative or hydrolytic DNA cleavage as anti-cancer drugs, is a
challenging topic in bioinorganic chemistry [9, 10]. Recently, there has been tremendous interest
in studies related to the interaction of transition metal ions with nucleic acid because of their
relevance in the development of new reagents for biotechnology and medicine [11]. These
studies are also important to understand the toxicity of drugs containing metal ions [12].
β-Diketimines are versatile ligand systems which have been known to form complexes with
almost every metal ion and metalloid [13]. Earlier reports indicate that the attempts to prepare
highly symmetrical and conjugated macrocyclic systems derived from β-diketones condensed
with organic amines have not led to the desired results. Condensation of the active methylene
group of the β-diketone with an aldehydic group gives a non-enolisable Knoevenagel
condensate, which can effectively react with amines to form Schiff bases [14]. Metal complexes
of β-diketone derivatives have played an important role in coordination chemistry [15–17] and
have been widely used in various aspects of industries, such as organic electroluminescent
technology, luminescent materials and sensors for bioinorganic applications [18–20]. With an
2

aim to develop cis-platin type anti-tumor response of β-diketone, different aromatic aldehydes
are introduced in active methylene group by Knoevenagel condensation. β-Diketone derived
complexes show antimicrobial, anti-malarial and antitumorous activities, antioxidant, insecticidal
activity. Recently transition metal complexes have received considerable interest in nucleic acid
chemistry because of their chelating nature. The coordination behavior of β-diketimine also has
significant influences in the relative stabilities of the Schiff base complexes as well as their use
in biomedicine [21–24]. Thus, scientists are now engaged to explore the transition metal based
complexes. Hence, the higher degree of conjugated versatile ligand systems of the Knoevenagel
condensate β-diketimine as Schiff bases containing electron releasing/electron withdrawing
groups and their low molecular weight copper(II) and zinc(II) complexes have been synthesized.
It is therefore of interest to carryout investigations on model compounds to understand how a
ligand environment could affect the redox properties of the central metal and thereby, the
spectral properties and also interested to explore the DNA binding and DNA cleavage activity of
synthesized complexes. This result would be helpful for understanding the binding mode of the
complexes to DNA and it also lays a foundation for developing new useful DNA probes and
effective inorganic complex nucleases.
2. Experimental protocols
2.1. Reagents and instruments
All reagents, benzaldehyde, acetylacetone, para substituted anilines and metal(II)
chlorides were of Merck products and they were used as supplied. Commercial solvents were
distilled and then used for the preparation of ligands and their complexes. DNA was purchased
from Bangalore Genei (India). Microanalyses (C, H and N) were performed in Carlo Erba 1108
analyzer at Sophisticated Analytical Instrument Facility (SAIF), Central Drug Research Institute
(CDRI), Lucknow, India. Molar conductivities in DMF (10−3 M) at room temperature were
measured by using Systronic model-304 digital conductivity meter. Magnetic susceptibility
measurement of the complexes was carried out by Gouy balance using copper sulphate
pentahydrate as the calibrant. Infrared spectra (4000–350 cm−1 KBr disc) of the samples were
recorded on an IR Affinity-1 FT-IR Shimadzu spectrophotometer. NMR spectra were recorded
on a Bruker Avance Dry 300 FT-NMR spectrometer in DMSO-d6, using TMS as the internal
reference. Mass spectrometry experiments were performed on a JEOL-AccuTOF JMS-T100LC
3

mass spectrometer equipped with a custom-made electrospray interface (ESI). EPR spectra were
recorded on a Varian E 112 EPR spectrometer in DMSO solution both at room temperature (300
K) and liquid nitrogen temperature (77 K) using TCNE (tetracyanoethylene) as the g-marker.
The absorption spectra were recorded by using Shimadzu model UV-1601 spectrophotometer at
room temperature.
2.2. Synthesis of 3-benzylidenepentane-2,4-dione
The non-enolisable β-diketone was prepared by employing the modified procedure
reported earlier [25]. Pentane-2,4-dione (10 mmol) was mixed with benzaldehyde (10 mmol) and
piperidine (0.2 mL), and the reaction mixture was stirred thoroughly for ca. 5 h with occasional
cooling. Yellow colored crystalline solid was obtained after two weeks on keeping in the
refrigerator which was filtered, washed with ethanol followed by an excess of petroleum-ether to
remove any unreacted reagent. Washing was repeated for two or three times and then the
compound was recrystallized from ethylacetate–petroleum ether mixture to get a pure yellow
solid, Knoevenagel condensate [3-benzylidenepentane-2,4-dione]. It was used as the starting
material for the preparation of Schiff base.
Yield: 73%. Anal. Calc. for C12H12O2 : C, 76.6; H, 6.5; Found: C, 75.8; H, 6.1 (%). IR
1
(KBr pellet, cm-1): 1718 ν(–C=O), 1494 ν(–HC=C). H–NMR (): (aromatic, 6H) 7.0–7.4 (m);
(–C=CH–, 1H), 8.3 (s); (–CH3), 2.3 (s). 13C–NMR (): 126.0–128.0 (C1 to C3), 134.7 (C4), 129.0
(C5), 140.6 (C6), 143.7 (C7), 198.6 (C8), 28.3. MS m/z (%): 189 [M+1]+.
2.3. Synthesis of Schiff bases
The Schiff base was prepared by dissolving 3-benzylidenepentane-2,4-dione (10 mmol)
in ethanol and refluxed with p-substituted (X) aniline where X = –NO2 (L1), –H (L2), –OH (L3)
and –OCH3 (L4) (20 mmol) in ethanol after the addition 0.5 g of anhydrous K2CO3 for ca. 6 h.
The K2CO3 was filtered off from the reaction mixture. The dark brown solution was set aside to
evaporate and the dark brown solid that separated was filtered off and recrystallized from ethanol
and dried in vacuo.
L1. Yield: 68%. Anal. Calc. for C24H20N4O4: C, 67.3; H, 4.7; N, 13.1 (%); Found: C, 66.5; H,
4.5; N, 12.4 (%). IR (KBr pellet, cm-1): 1631 ν(–C=N); 1502 ν(–HC=C);1473, 1307,840 ν(–C–N
ring str; –NO2).1H–NMR (): (aromatic) 6.9–7.3 (m); (–CH3, 6H), 2.1 (s). 13C–NMR (): 125.0–
4

127.0 (C1 to C3), 134.1 (C4), 136.6 (C5), 114.3 (C6), 175.4 (C7), 20.4 (C8), 153.4 (C9), 123.2
(C10), 125.1 (C11) 146.4 (C12). MS m/z (%): 429 [M+1]+. max (cm–1) in EtOH, 45857, 27132.
L2. Yield: 66%. Anal. Calc. for C24H22N2: C, 85.2; H, 6.6; N, 8.3; Found: C, 85.1; H, 6.5; N, 8.1
1
IR (KBr pellet, cm-1): 1509 ν(–HC=C–); 1632 ν(–C=N). H–NMR (, ppm): (aromatic) 6.9–7.2
13
(m); (–CH3, 6H), 2.3 (s). C–NMR (, ppm):125.0–129.0 (C1 to C3), 133.4 (C4), 136.2 (C5),
112.4 (C6), 173.9 (C7), 20.3 (C8), 137.1 (C9), 119.6 (C10), 131.4 (C11), 128.7 (C12). MS m/z (%):
339 [M+1]+. max (cm–1) in EtOH, 42783, 28324.
L3. Yield: 63%. Anal. Calc. for C24H22N2O2: C, 77.8; H, 5.9; N, 7.6; Found: C, 77.3; H, 5.8; N,
1
7.4 (%). IR (KBr pellet, cm-1): 3430 ν(–OH); 1638 ν(–C=N); 1525 ν(–HC=C). H–NMR (,
13
ppm): (aromatic) 6.9-7.4 (m); (–OH, 1H) 10.3 (s); (–CH3, 6H), 2.2 (s). C–NMR (, ppm):
125.2–128.0 (C1 to C3), 134.3 (C4), 136.2 (C5), 114.1 (C6), 175.2 (C7), 20.2 (C8), 147.6 (C9),
122.9 (C10), 118.4 (C11), 154.8 (C12). MS m/z (%): 371 [M+1]+. max (cm–1) in EtOH, 44768,
28237.
L4. Yield: 64%. Anal. Calc. for C26H26N2O2: C, 78.4; H, 6.6; N, 7.0; Found: C, 78.2; H, 6.4; N,
1
6.7 (%). IR (KBr pellet, cm-1): 1635 ν(–C=N); 1541 ν(–HC=C); 1273, 1083 ν(–C–O–C–). H–
13
NMR (): (aromatic) 6.9–7.3 (m); (–CH3, 6H), 2.4 (s); 3.8 (–OCH3, 6H). C–NMR ():125.0–
129.0 (C1 to C3), 132.9 (C4), 137.2 (C5), 112.7 (C6), 172.8 (C7), 20.6 (C8), 142.4 (C9), 122.1
(C10), 116.5 (C11), 158.6 (C12), 58.3 (C13). MS m/z (%): 399 [M+1]+. max (cm–1) in EtOH,
41656, 28014.
2.4. Synthesis of metal complexes
To a stirred ethanolic solution of the above Schiff base(s) (5 mmol), a solution of
copper(II)/zinc(II) chloride (5 mmol) in ethanol was added dropwise. The reaction solution was
refluxed for 2 h. After cooling the reaction mixture to an ambient temperature, the formed solid
was filtered, washed with diethyl ether and finally dried in vacuum.
[CuL1Cl2]. Yield: 65%. Anal. Calc. for C24H20N4O4CuCl2: Cu, 11.3; C, 51.2; H, 3.6; N, 9.9;
Found: Cu, 11.2; C, 51.1; H, 3.6; N, 9.8 (%). IR (KBr pellet, cm-1): 1603 ν(–C=N); 1497
ν(–HC=C); 1472, 1306, 843 ν(–C–N str; –NO2); 423 (M–N). MS m/z (%): 562 [M+1]+.
ΛM 10-3(ohm-1 cm2 mol-1) = 5.5. max in DMF, 43928, 35116, 17457. μeff (BM): 1.84.
[CuL2Cl2]. Yield: 62%.). Anal. Calc. for C24H22N2CuCl2: Cu, 13.4; C, 61.0; H, 4.7; N, 5.9;
Found: Cu, 13.4; C, 60.9; H, 4.6; N, 5.8 (%). IR (KBr pellet, cm-1): 1608 ν(C=N); 1507
5

ν(–HC=C); 437 (M–N). MS m/z (%): 472 [M+1]+. ΛM 10-3 (ohm-1 cm2 mol-1) = 9.2 max (cm–1)
in DMF, 40263, 29359, 18264. μeff (BM): 1.87.
[CuL3Cl2]. Yield: 59%.). Anal. Calc. for C24H22N2O2CuCl2: Cu, 12.6; C, 57.1; H, 4.4; N, 5.6;
Found: Cu, 12.5; C, 56.9; H, 4.4; N, 5.5 (%). IR (KBr pellet, cm-1): 1612 ν(–C=N); 1522
ν(–HC=C); 3430 ν(–OH); 441 (M–N). MS m/z (%): 504 [M+1]+. ΛM 10-3 (ohm-1 cm2 mol-1) =
4.3. max (cm–1) in DMF, 41543, 33245, 17176. μeff (BM): 1.83.
[CuL4Cl2]. Yield: 51%. Anal. Calc. for C26H26N2O2CuCl2: Cu, 11.9; C, 59.0; H, 4.9; N, 5.3;
Found: Cu, 11.8; C, 58.5; H, 4.9; N, 5.2 (%). IR (KBr pellet, cm-1): 1609 ν(–C=N); 1538
ν(–HC=C); 1269, 1081,ν(–C–O–C–); 428 (M–N). MS m/z (%): 532 [M+1]+. ΛM 10-3 (ohm-1 cm2
mol-1) = 3.9. max (cm–1) in DMF, 41727, 31429, 18426. μeff (BM): 1.81
[ZnL1Cl2]. Yield: 63%. Anal. Calc. for C24H20N4O4ZnCl2: Zn, 11.6; C, 51.0; H, 3.6; N, 10.0;
Found: Zn, 11.5; C, 50.9; H, 3.5; N, 9.9 (%). IR (KBr pellet, cm-1): 1610 ν(–C=N); 1501
1
ν(–HC=C); 1469, 1309, 838 ν(–C–N str; –NO2); 427 (M–N). H–NMR (): (aromatic) 6.9–7.2
13
(m); (–CH3, 6H), 2.0 (s). C–NMR (): 124.9–126.2 (C1 to C3), 134.1 (C4), 136.4 (C5), 114.1
(C6), 169.2 (C7), 20.1 (C8), 153.2 (C9), 123.1 (C10), 124.9 (C11) 146.3 (C12). MS m/z (%): 563
[M+1]+. ΛM 10-3 (ohm-1 cm2 mol-1) = 3.7. max (cm–1) in DMF, 42254, 32397. μeff (BM):
diamagnetic.
[ZnL2Cl2]. Yield: 58%. Anal. Calc. for C24H22N2ZnCl2: Zn, 13.8; C, 60.7; H, 4.7; N, 5.9; Found:
Zn, 13.7; C, 60.6; H, 4.6; N, 5.9 (%). IR (KBr pellet, cm-1): 1608 ν(–C=N); 1524 ν(–HC=C);
1
442 (M–N). H–NMR (, ppm): (aromatic) 6.8–7.2 (m); (–CH3, 6H), 2.2 (s). 13C–NMR (,
ppm):125.6–128.7 (C1 to C3), 133.3 (C4), 136.1 (C5), 112.2 (C6), 168.4 (C7), 20.2 (C8), 137.0
(C9), 119.5 (C10), 131.4 (C11), 128.7 (C12). MS m/z (%): 473 [M+1]+.ΛM 10-3 (ohm-1 cm2 mol-1)
= 5.9. max (cm–1) in DMF, 40453, 29467. μeff (BM): diamagnetic.
[ZnL3Cl2]. Yield: 54%. Anal. Calc. for C24H22N2O2ZnCl2: Zn, 13.0; C, 56.9; H, 4.4; N, 5.5;
Found: Zn, 12.9; C, 56.8; H, 4.3; N, 5.5 (%). IR (KBr pellet, cm-1): 1618 ν(–C=N); 1526
1
ν(–HC=C); 3430 ν(–OH); 445 (M–N). H–NMR (, ppm): (aromatic) 6.9-7.2 (m); (–OH, 1H)
10.1 (s); (–CH3, 6H), 2.1 (s). 13C–NMR (, ppm): 125.1–127.8 (C1 to C3), 134.2 (C4), 136.1 (C5),
114.1 (C6), 169.8 (C7), 20.1 (C8), 147.4 (C9), 122.8 (C10), 118.3 (C11), 154.8 (C12). MS m/z (%):
505 [M+1]+. ΛM 10-3 (ohm-1 cm2 mol-1) = 4.5. max (cm–1) in DMF, 41567, 33946. μeff (BM):
diamagnetic.
6

[ZnL4Cl2]. Yield: 53%. Anal. Calc. for C26H26N2O2ZnCl2: Zn, 12.2; C, 58.4; H, 5.0; N, 5.2;
Found: Zn, 12.2; C, 58.3; H, 5.0; N, 5.1 (%). IR (KBr pellet, cm-1): 1613 ν(–C=N); 1540
ν(–HC=C); 1267, 1083, ν(–C–O–C–); 433 (M–N). 1H–NMR (): (aromatic) 6.8–7.2 (m); (–CH3,
13
6H), 2.2 (s); 3.6 (–OCH3, 6H). C–NMR ():124.7–128.8 (C1 to C3), 132.8 (C4), 137.2 (C5),
112.6 (C6), 167.6 (C7), 20.5 (C8), 142.3 (C9), 122.0 (C10), 116.5 (C11), 158.5 (C12), 58.1 (C13).
MS m/z (%): 533 [M+1]+. ΛM 10-3 (ohm-1 cm2 mol-1) = 8.4. max (cm–1) in DMF, 42285, 31518.
μeff (BM): diamagnetic.
2.5. DNA binding experiments
The interaction between metal complexes and DNA was studied using electronic
absorption, viscosity and electrochemical methods. Disodium salt of calf thymus DNA was
stored at 4°C. All the experiments involving the interaction of the complexes with calf thymus
(CT) DNA were carried out in Tris–HCl buffer (50 mM Tris–HCl, pH 7.2) containing 5 %
DMSO at room temperature. A solution of CT DNA in the buffer gave a ratio of UV absorbance
at 260 and 280 nm of about 1.89:1, indicating that the CT DNA was sufficiently free from
protein [26]. The concentration of DNA was measured by using its extinction coefficient at 260
nm (6600 M-1cm-1) after 1: 100 dilution. Stock solutions were stored at 4°C and used not more
than 4 days. Doubly distilled water was used to prepare solutions. Concentrated stock solutions
of the complexes were prepared by dissolving the complexes in DMSO and diluting properly
with the corresponding buffer to the required concentration for all the experiments.
Absorption titration experiment was performed by keeping the concentration of the metal
complex as constant at 50 µM while varying the concentration of the CT DNA within
40–400 µM. While measuring the absorption spectrum, equal quantity of CT DNA was added to
both the complex solution and the reference solution to eliminate the absorbance of CT DNA
itself. From the absorption data, the intrinsic binding constant (Kb) was determined from the plot
of [DNA]/(εa-εf) vs. [DNA] using the equation (1):
[DNA]/(εa–εf) = [DNA]/(εb–εf) + [Kb(εb–εf)]–1 ------------------ (1)
where [DNA] is the concentration of CT DNA in base pairs. The apparent absorption
coefficients εa, εf and εb correspond to Aobs./[M], the extinction coefficient for the free metal(II)
complex and extinction coefficient for the metal(II) complex in the fully bound form,
respectively [27]. Kb is given by the ratio of slope to the intercept.
7

Cyclic voltammetry studies were performed on a CHI 620C electrochemical analyzer
with three electrode system of glassy carbon as the working electrode, a platinum wire as
auxiliary electrode and Ag/AgCl as the reference electrode. Solutions were deoxygenated by
purging with N2 prior to measurements. Viscosity experiments were carried on an Ostwald
viscometer, immersed in a thermostated water-bath maintained at a constant temperature at
30.0 ± 0.1°C. CT DNA samples of approximately 0.5 mM were prepared by sonicating in order
to minimize complexities arising from CT DNA flexibility [28]. Flow time was measured with a
digital stopwatch three times for each sample and an average flow time was calculated. Data
were presented as (η/η0)1/3 versus the concentration of the metal(II) complexes, where η is the
viscosity of CT DNA solution in the presence of complex, and η0 is the viscosity of CT DNA
solution in the absence of complex. Viscosity values were calculated after correcting the flow
time of buffer alone (t0), η = (t – t0)/ t0 [29].
2.6. Interaction with pBR322 plasmid DNA
The extent of pBR322 DNA cleavage in the absence and presence of an activating agents
H2O2, groove binding agents, distamycin, methyl green (MG) and SOD (superoxide) radical
scavenger was monitored using agarose gel electrophoresis. In reactions using super coiled
pBR322 plasmid DNA Form I (2 μL,10 μM) in Tris–HCl buffer (50 mM) with 50 mM NaCl (pH
7.2) which was treated with the metal complex (30 μM) and activating agents (10 μM) followed
by dilution with the Tris–HCl buffer to a total volume of 20 μL. The samples were incubated for
1 h at 37 ºC. A loading buffer containing 25% bromophenol blue, 0.35% xylene cyanol, 30%
glycerol (3μl) was added and electrophoresis was performed at 40 V for each hour in Tris-
Acetate-EDTA (TAE) buffer using 1% agarose gel containing 1.0 μg/mL ethidium bromide. The
gel was visualized by photographing the fluorescence of intercalated ethidium bromide under a
UV illuminator. The cleavage efficiency was measured by determining the ability of the complex
to convert the super coiled (SC) DNA to nicked circular form (NC) and linear form (LC).
Inhibition reactions were carried out by prior incubation of the SC pBR322 DNA (40 mM) with
DMSO (2 mL).
2.7. Evaluation of antimicrobial activity
Qualitative determination of antimicrobial activity was done using the disc diffusion
method. The biological activities of synthesized Schiff base and its metal complexes were
8

studied for their antibacterial and antifungal activities in DMF solvent against bacterial and fungi
species. Suspensions in sterile peptone water from 24 h cultures of microorganisms were
adjusted to 0.5 McFarland. Muller–Hinton petri discs of 90 mm were inoculated using these
suspensions. Paper discs (6 mm in diameter) containing 10 μL of the substance to be tested were
placed in a circular pattern in each inoculated plate. DMF impregnated discs were used as
negative controls. Toxicity tests of the solvent, DMF, showed that the concentration used in
antibacterial activity assays did not interfere with the growth of the microorganisms.
2.8. Determination of MIC
The in vitro antimicrobial activity was performed against Gram-positive bacteria:
Staphylococcous aureus, Bacillus subtilis and Salmonella typhi Gram-negative bacteria:
Escherichia coli, Pseudomonas aeruginosa and fungal strains: Rhizoctonia bataticola, Fusarium
Solani, Candida albicans, Culvularia lunata and Aspergillus niger. The standard and test
samples were dissolved in DMF to give a concentration of 100 μg/mL. The minimum inhibitory
concentration (MIC) was determined by broth microdilution method [30]. Dilutions of test and
standard compounds were prepared in nutrient broth (bacteria) or Sabouraud dextrose broth
(fungi) [31]. The samples were incubated at 37◦C for 24 h (bacteria) and at 25◦C for 48 h (fungi),
respectively, and the results were recorded in terms of MIC (the lowest concentration of test
substance which inhibited the growth of microorganisms).
2.9. Antioxidant assays
The ability of all the synthesized complexes to act as free radical scavengers was tested
by conducting a series of in vitro antioxidant assays involving DPPH radical, hydrogen peroxide,
metal chelating assay and the results were compared with that of standard antioxidants including
natural antioxidant Vitamin C and synthetic antioxidant BHT (Butylated Hydroxytoluene)..
2.10. DPPH˙ scavenging assay
The DPPH radical scavenging activity of the compounds was measured according to the
method of Blios [32]. The DPPH radical is a stable free radical and due to the presence of an odd
electron, it shows a strong absorption band at 517 nm in visible spectrum. If this electron
becomes paired off in the presence of a free radical scavenger, this absorption vanishes resulting
in decolorization stoichiometrically with respect to the number of electrons taken up. Various
concentrations of the experimental complexes were taken and the volumes were adjusted to 100
9

mL with DMSO. About 5 mL of 0.1 mM methanolic solution of DPPH was added to the aliquots
of samples and standards (BHT and Vitamin C) and shaken vigorously. Negative control was
prepared by adding 100 mL of methanol in 5 mL of 0.1 mM methanolic solution DPPH. The
tubes were allowed to stand for 20 min at 27ºC. The absorbance of the sample was measured at
517 nm against the blank (DMSO).
2.11. H2O2 scavenging assay
The ability of the complexes to scavenge hydrogen peroxide was determined using the
method of Ruch et al. [33]. A solution of hydrogen peroxide (2.0 mM) was prepared in
phosphate buffer (0.2 M, pH-7.4) and its concentration was determined spectrophotometrically
from absorption at 230 nm with molar absorptivity 81 M-1 cm-1. The complexes (100 mg/mL),
BHT and Vitamin C (100 mg/mL) were added to 3.4 mL of phosphate buffer together with
hydrogen peroxide solution (0.6 mL). An identical reaction mixture without the sample was
taken as negative control. Absorbance of hydrogen peroxide at 230 nm was determined after 10
min against the blank (phosphate buffer).
2.12. Measurement of SOD like activity
Superoxide dismutase (SOD)-like activity was investigated using Beauchamp and
Fridovich’s method – as improved by Iwamoto et al [34]. This method is based on the inhibitory
effect of SOD on the reduction of nitrobluetetrazolium (NBT) by the O2˙ˉ generated by the
xanthine/xanthine oxidase system. The assay was carried out in the assay buffer containing
50mM Tris HCl, pH 8.0, 0.1 mM DTPA and 0.1 mM hypoxanthine. Radical detector consisted
of a tetrazolium salt and was diluted by assay buffer. Similarly, the solutions of SOD standards
and xanthine oxidase were prepared in sample buffer consisting of 50mM Tris-HCl, pH 8.0. All
the studied complexes were dissolved in DMSO and absorbance was reported for each set of
concentrations after 15 min interval. Results were graphed as % inhibition of NBT reduction
taken at various concentrations (0 μM-20 μM) for each of the studied complexes. IC50 values are
reported equivalent concentrations (μM) to 1U bovine erythrocyte superoxide dismutase (native
SOD).
2.13. Antitumor studies
Adult Swiss female albino mice (20-25 g) were procured from Animal house,
Vivekananda College of Pharmacy, Trichengodu, Tamil Nadu, India and used throughout the
10

study. They were housed in microlon boxes in controlled environment (temperature 25±2ºC and
12 h dark/light cycle) with standard laboratory diet and water ad libitum. The experiments were
performed in accordance with guidelines established by the European community for the care
and use of laboratory animals, and were approved by the Institutional Animal Ethics Committee
(IAEC) of Vivekananda College of Pharmacy, Tamil Nadu, India.
2.14. Cells
Ehrlich Ascites Carcinoma (EAC) cells were obtained through the courtesy of Amala
Cancer Research Centre, Trissur, India. They were maintained by weekly intraperitoneal
inoculation of 106 cells/mouse [35].
2.15. Mean survival time [36]
Animals were inoculated with 1×106 cells/mouse on day ‘0’ and treatment with
synthesized complexes started 24 h after inoculation, at a dose of 100 mg/kg/day, p.o. The
control group was treated with the same volume of 0.9% sodium chloride solution. All the
treatments were given for nine days. The median survival time (MST) of each group, consisting
of 10 mice was noted. The antitumor efficacy of complexes was compared with that of
5-fluorouracil (Dabur Pharmaceutical Ltd, India; 5-FU, 20 mg/kg/day, i.p. for 9 days).
2.16. Hematological parameters [36]
The hematological parameters like total red blood cell (RBC), white blood cells (WBC),
lymphocytes (LYM), hematocrit (HCT), hemoglobin (HGB) and MID cells (less frequently
occurring and rate cells correlating to monocytes, eosinophils, basophils etc.) were determined
using a blood automatic analyzer (Celldyn, Abbot Inc. USA). In order to detect the influence of
complexes on the hematological status of EAC-bearing mice, a comparison was made among
three groups (n=5) of mice on the 14th day after inoculation. The groups comprised of (1) tumor-
bearing mice (2) tumor-bearing mice treated with complexes (100 mg/kg/day, p.o. for the first 9
days) and (3) control mice (normal). Blood was drawn from each mouse by the retro orbital
plexus method and the white blood cell count (WBC), red blood cell count (RBC), hemoglobin
and protein were determined [37-40].
2.17. Cytotoxicity
Human cervical cancer cell lines (HeLa), Human laryngeal epithelial cancer (Hep2),
Human liver cancer (HepG2) and Human breast cancer (MCF-7) cells were obtained from
11

National Centre for Cell Science (Pune, India). Stock cells of HeLa, Hep2, HepG2 and MCF-7
cell lines were cultured in RPMI-1640 or DMEM supplemented with 10% in activated new born
calf serum, penicillin (100 IU/mL), streptomycin (100 mg/mL), and amphotericin-B (5 mg/mL)
under a humidified atmosphere of 5% CO2 at 37oC until confluent. The cells were dissociated in
0.2% trypsin, 0.02% EDTA in phosphate buffer saline solution. The stock culture was grown in
25 cm2 tissue-culture flasks, and cytotoxicity experiments were carried out in 96-well microtiter
plates (Tarsons India, Kolkata, India).
Cell lines in the exponential growth phase were washed, trypsinized and resuspended in
complete culture media. Cells were plated at 10,000 cells / well in 96-well microtiter plates and
incubated for 24 h, during which a partial monolayer formed. They were then exposed to various
concentrations of the complexes (0.1−100 mg/mL) and cisplatin. Control wells received only
maintenance medium. The plates were incubated at 37oC in a humidified incubator with 5% CO2
for a period of 72 h at the end of 72 h, viability was determined by MTT assay.
3. Results and discussion
Synthetic approaches for the preparation of different biosensitive compounds are depicted
in Scheme 1. The ligands and their complexes are found to be stable in air. The ligands are
soluble in common organic solvents but their complexes are soluble only in DMF and DMSO.
3.1. Infrared spectra
The coordination mode and sites of the ligand to the metal ions were investigated by
comparing the infrared spectra of the free ligand with its metal complexes. The spectra of free
ligands show a band in the region 1638-1631 cm–1 characteristics of the (C=N) stretching mode
[41-43] indicating the formation of the Schiff base products. This band is shifted towards lower
frequency by ca.30 cm−1 on complexation compared to free ligands indicating the involvement
of the azomethine nitrogen in chelation with the metal ion, the coordination of the nitrogen to the
metal ion would be expected to reduce the electron density of the azomethine link and thus
causes a shift in the (C=N) group [44]. Coupled with this, the absence of a band around
1655–1710 cm–1 [45], characteristic of ν(–C=O) in 3-benzylidenepentane-2,4-dione, suggests
that the condensation of the keto groups is complete. The free –OH group of the ligand L3
vibrated at ca. 3430 cm–1 [45] does not show any significant shift on complex formation.
Conclusive evidence of the bonding is also shown by the observation that new bands appear in
12

the spectra of all metal complexes in the low frequency regions at 420–447 and 360–389 cm-1
characteristic for M–N and M–Cl vibrations, respectively, which are not observed in the spectra
of free ligands.
3.2. Molar conductivity
The complexes are found to be non-electrolytic nature in 10–3 M DMF solution, implying
the coordination of chloride anion to the central metal ion. The lower conductance values (3.7–
9.2 Ω−1 cm−2 mol−1) of the complexes support their non-electrolytic nature. The elemental
analysis results of the metal complexes also agree with the calculated values, showing that the
complexes have 1:1 metal/ligand ratio.
3.3. Magnetic moments and electronic spectra
The geometry of the metal complexes has been deduced from electronic spectra and
magnetic data of the complexes. The electronic spectra of the complexes were recorded in
DMSO solution. The free ligands exhibit two intense bands in 45857–41656 and 28361-27138
cm–1 region due to π → π* and n → π* transitions [46], respectively. In all the metal complexes,
the absorption bands at 43928-40263 and 29359-35116 cm−1 are due to π → π* and n → π*
transitions that are observed in the spectra of the free ligands L1, L2, L3 and L4. These transitions
are shifted to blue or red frequencies due to the coordination of the ligand with metal ions. The
electronic spectra of Cu(II) complexes show two broad, low intensity shoulder bands in the
visible region, around 17176– 18426 cm-1, which are assigned to the combination of 2B1g  2A1g
2
2
and B1g  Eg transitions respectively. The electronic spectral data suggest a square-planar
geometry around the Cu(II) ion. The observed magnetic moment of the Cu(II) complexes (1.81-
1.87 B.M) at room temperature indicates the non-coupled mononuclear complexes of
magnetically diluted d9 system with S=1/2 spin-state. The monomeric nature of the complexes is
further supported by the microanalytical and ESI mass spectral data. The electronic absorption
spectra of the diamagnetic Zn(II) complexes show the bands at 40453–42285 and 29467–32946
cm–1 which are assigned to intra-ligand charge transfer transitions [47].
3.4. NMR spectra of zinc complexes
1
In H NMR, the aromatic region is a set of multiplets in the range of 6.8-7.4 ppm for all
the ligands and their Zn(II) complexes. The phenolic –OH proton for L3 ligand and its zinc
complex was observed as a singlet at ca. 9.3 ppm. It is suggesting that phenolic –OH group is not
13

1
taking part in the complexation. H NMR spectra of aliphatic methyl protons exhibit at 2.0-2.4
ppm for all the Schiff base ligands and their Zn(II) complexes. There is no appreciable change in
all other signals of the complex.
13
The C NMR spectrum of the ligand showed aromatic carbons at 119–129 ppm. The
ligand also showed the C=N carbons at 172.8–175.4 ppm, which were shifted to downfield at
167.6–169.2 ppm, upon coordination indicating that the C=N groups participate in complex
formation. Comparison of all carbon peaks of the ligands with those of zinc complexes shows
some upfield and downfield shifts, but these shifts are not large. This indicates the coordination
of the ligand to the metal ion.
3.5. Mass spectra
The ESI-mass spectra of synthesized ligands and their complexes were recorded and the
obtained molecular ion peaks confirm the proposed formulae. The mass spectrum of L1 ligand
shows M+1 peak at m/z 428 (86.4 %) corresponding to [C24H20N4O4]+ ion. Also, the spectrum
exhibits the fragments at m/z 102, 77 and 66 corresponding to [C6H5NO]+, [C6H5]+ and [C5H6]+
respectively. The mass spectrum of [CuL1Cl2] shows peaks at m/z 561 with 92.2% abundances,
respectively. The strongest peaks (base beak) at m/z 428 represent the stable species
C24H20N4O4. Moreover, the spectrum exhibits the fragments at m/z 102, 77 and 66 corresponding
to [C6H5NO]+, [C6H5]+ and [C5H6]+ respectively. The m/z of all the fragments of ligands and
their complexes confirm the stoichiometry of the complexes as [MLCl2]. The observed peaks are
in good agreement with their formulae as expressed from microanalytical data. Thus, the mass
spectral data reinforce the conclusion drawn from the analytical and conductance values.
3.6. EPR spectra
The EPR spectrum of copper complex provides information which is important in
studying the metal ion environment. The X-band EPR spectra of all copper(II) complexes have
been recorded in DMSO at liquid nitrogen temperature and at room temperature. The spectrum
of the copper complexes at RT shows one intense absorption band in the high field and is
isotropic due to the tumbling motion of the molecules. However, this complex at LNT shows
well resolved peaks with low field region. The spin Hamiltonian parameters of the complexes
were calculated and are summarized in Table 1. From this spectral data, it is found that A
2
(131-138) > A (39-66); g (2.13-2.26) > g(2.01-2.05) > ge 2.0027, which support the dx -y2 as
14

the ground state, characteristic of square-planar geometry and axially symmetric. Further, in an
axial symmetry, the g-values are related by the expression,
G = (g - 2.0027)/( g-2.0027)
which measures the exchange interaction between the copper centers in polycrystalline solid.
The G values lie within the range 4.6–5.2 for all the copper complexes indicating negligible
exchange interaction of Cu–Cu in the complexes according to Hathaway [48, 49].
For the present copper complexes, the g/A values found in the range of 154–174 cm–1
are in agreement with significant deviation from planarity which is further confirmed by the
bonding parameter 2 whose value is less than unity.
The covalency parameters α2 (covalent in-plane σ-bonding) and β2 (covalent in-plane π-
bonding) have been calculated using the following equations [48]. If α2 = 1.0, it indicates
complete ionic character whereas α2 = 0.5 denotes 100% covalent bonding, with assumption of
negligible small values of the overlap integral.
2 = (A /0.036) + (g  2.0027) + 3/7 (g 2.0027) + 0.04
2 = (g  2.0027) (E / -82 )
From Table 1, the α2 and β2 values indicate that there is a substantial interaction in the in-plane
σ-bonding whereas the in-plane π-bonding is almost ionic. The lower value of α2 compared to β2
indicates that the in-plane σ-bonding is more covalent than in-plane π-bonding. These data are
well in accordance with other reported values [48]. Based on the above observations, a square-
planar geometry is proposed for the complexes. The EPR study of the copper(II) complexes has
provided supportive evidence to the conclusion obtained on the basis of electronic and magnetic
moment values.
3.7. Electronic absorption titration
The absorption spectra of the ligand L1and its complexes of Cu(II) and Zn(II) in the
presence or without DNA were mutually compared, which is shown in Fig. 1a-1c. In the UV
region of the spectra, all the ligands exhibited an absorption band around 312.2-323.8 nm (due
to π–π* transition) and their copper complexes exhibited an intense absorption around 340–364.5
nm and zinc complexes showed a band at 341.5–369.5 nm (due to n–π* transition). With
15

increasing concentration of DNA, both the ligands and their complexes showed hypochromicity
and a red-shifted charge transfer peak maxima in the absorption spectra. In the case of ligands,
Fig. 1a showed hypochromicity in the range 6-8% and slight red-shift in the range 0.3-0.6 nm
observed in the absorption spectra. The hypochromicity values of all the complexes observed in
the presence of DNA were in the range 6–10%, and their red shifts in the region 0.8–2.0 nm. The
change in the absorbance values with increasing amount of DNA was used to evaluate the
intrinsic binding constant Kb for the present complexes, the values of which are given in Table 2.
The change in hypochromicity may be attributed to the nature of the binding of the complexes
with DNA, which is significant due to π-stacking or hydrophobic interactions of the aromatic
phenyl rings [50]. However, the metal ions play crucial role in DNA binding by these complexes.
The binding strength of the synthesized complexes with DNA is shown as in the following order:
–NO2 > –H > –OH > –OCH3. The strong binding affinity of the metal complexes is due to
additional π-π interaction through the aromatic phenyl rings and central metal ions as compared
with Schiff base ligands. From the results ligands itself act as a pathetic intercalators as
compared to complexes which act as strong intercalators. These results suggest that intercalative
ligands with extended aromatic plane, good conjugation effect and electron-withdrawing
substituted groups can greatly promote the DNA-binding ability of their Knoevenagel
condensate complexes.
3.8. Viscosity measurements
The relative specific viscosity of DNA is determined by varying the concentration of the
added metal complexes. Measuring the viscosity of DNA is a classical technique used to analyze
the DNA binding mode in solution. Under appropriate conditions, intercalation of drugs, such as
ethidium bromide [EB], causes a significant increase in the viscosity of a DNA solution due to
the increase in the separation of the base pairs of the intercalation sites and hence, results in an
increase in the overall DNA contour length, as shown in Fig. 2. The viscosity of the DNA
solution increased with increasing ratio of both the copper and zinc complexes to DNA. As
expected, the known DNA-intercalator EB increased the relative viscosity of DNA due to its
strong intercalation. Compared with EB, complexes exhibit minor increase in the relative
viscosity of CT-DNA, suggesting an intercalation mode between the complex and DNA. In case
of ligand L1 exhibits pathetic intercalation, as compared with EB. This result further suggests an
16

intercalating binding mode of the complexes with DNA and also parallels the above
spectroscopic results, such as hypochromism and red shift of the complexes in the presence of
DNA. The viscosity studies provide a strong evidence for intercalation. The increase in viscosity
of DNA is ascribed to the intercalative binding mode of the drug because this could cause the
effective length of the DNA to increase [51].
3.9. Electrochemical studies
Cyclic voltammetric technique provides a useful complement to the previously used
methods of investigation like absorption spectral titration and viscosity studies. The cyclic
voltammograms of the glassy carbon electrode in solutions containing [CuL1Cl2] in the absence
and in the presence of varying amounts of DNA are shown in Fig. 3a and 3b. Presence of DNA
causes a considerable decrease in the voltammetric current of the redox wave with a slight shift
in E1/2 to positive potential. The drop of the voltammetric currents in the presence of DNA may
be attributed to slow diffusion of the metal complex bound to CT DNA. This in turn indicates the
extent of binding affinity of the complex to DNA. The net shift in E1/2 can be used to estimate the
ratio of equilibrium constants for the binding of 2+ and 1+ ions to DNA. The quasi-reversible
redox couples for Cu(II) and Zn(II) complexes in DMF: buffer solution that have been studied
upon addition of CT-DNA and the shifts of the cathodic (Epc) and anodic (Epa) potentials are
given in Table 3. No new redox peaks appeared after the addition of CT-DNA to each complex,
but the current intensity of all the peaks decreased significantly, suggesting the existence of an
interaction between each complex and CT-DNA. The decrease in current intensity can be
explained in terms of an equilibrium mixture of free and DNA-bound complexes to the electrode
surface [52]. Finally, the conclusion derived from the CV study is that copper and zinc
complexes can bind to DNA by intercalative binding mode.
3.10. Antimicrobial activity
The minimal inhibitory concentrations of tested compounds against certain bacteria and
fungi are shown in Tables 4 and 5. The ligands (L1–L4) and their metal complexes were prepared
and tested for their in vitro antimicrobial activity against the five strains of bacteria (gram
negative and gram positive), and five strains of fungi. Few metal complexes of the functionalized
β-diketimines showed high in vitro antimicrobial activity. All the Cu(II) and Zn(II) metal
complexes showed significant antibacterial and antifungal activities compared to free ligands,
17

But the activity was lesser than the standard drugs. It is clearly depicted in Fig. 4a and 4b. Such
increased activity of the complexes can be explained on the basis of Overtone’s concept [53] and
the Tweedy’s Chelation theory [54]. The presence of electron-withdrawing nitro group on the
aromatic ring in general increases the antimicrobial activities of the tested metal complexes
compared to complexes having no substituent. The nature of metal ion also plays a decisive role
in determining antimicrobial properties. In the present study, the order of the antimicrobial
activity of the synthesized compounds (based on the substituent present in the phenyl ring) is as
follows: –NO2 > –H > –OCH3 > –OH. It is inferred from the results that electron-withdrawing
nitro group has effective and direct impact on selective antimicrobial activities against both
bacteria and fungi. In the complexes, the ligands have few uncoordinated hetero atoms which
enhance the activity of the complexes by bonding with trace elements present in microorganisms
may combine with the uncoordinated site and may inhibit the growth of fungi. The mode of
action of the compounds may involve the formation of a hydrogen bond through the azomethine
group with the active centers of cell constituents, resulting in interferences with the normal cell
process [55, 56].
3.11. Cleavage activity of pBR322 plasmid DNA
Gel electrophoresis experiments using pBR322 circular plasmid DNA were performed
with the ligands and complexes in the presence and absence of H2O2 as an oxidant. At
micromolar concentrations for a 2 h incubation period, the ligands exhibited moderate cleavage
activity in presence of the oxidant (H2O2). The nuclease activity was greatly enhanced by the
incorporation of the metal ion into the respective ligands. The cleavage activities of Cu(II) and
Zn(II) complexes of β-diketimines on pBR322 DNA are shown in Fig. 5. In Fig. 5, lane 1 is
control while the other lanes contain complexes in presence of oxidant (H2O2). It is clear that the
parent ligands show an apparent cleavage activity in presence of the oxidant. The other lanes
contain metal complexes in form I (super coiled) converted into form II (nicked) and form III
(linear). The cleavage activity of the complexes is significantly increased in the presence of the
oxidant (H2O2). This may be attributed to the formation of hydroxyl free radicals, which oxidize
+2 to +3, presumably through Fenton-type reactions, resulting in the formation of reactive
oxygen species, which could then cause oxidative damage to DNA [57]. In order to clarify the
intercalative mechanism of pBR322DNA introduced by metal(II)complexes, the investigation
18

has been carried out further on adding distamycin and methyl green (groove binders). It is found
that no inhibition of DNA cleavage is observed indicating that hydroxyl radical is not involved in
the cleavage process in the presence of groove binders (Fig. 6 and Fig. 7). These prompted us to
investigate the SOD activity of the metal complexes with pBR322 DNA. While in presence of
SOD, a facile superoxide anion radical O2•− quencher, the cleavage is improved which indicates
•−
•−
that O2 might be an inhibitor in the cleavage of the plasmid and reducing the amount of O2
could improve the cleavage effect (Fig. 8a and 8b). Thus, freely diffusible OH• is considered as
the active species responsible for pBR322 DNA cleavage. From these results, we infer that the
metal complexes act as potent nuclease agents. As the compounds are observed to cleave the
DNA, it can be concluded that, the compound inhibits the growth of the pathogenic organism by
cleaving the genome. Therefore, the compounds can be used as promising antitumor agents in
vivo to inhibit the DNA replication in the cancer cells and not allow the tumor for further growth.
3.12. Anti-oxidative activity
The IC50 values of all the complexes (Table 6) obtained from different types of assay
experiments strongly support that they possess excellent antioxidant activities, which are better
than those of standard antioxidants including the natural antioxidant vitamin C and the synthetic
antioxidant BHT (butylated hydroxytoluene). From the results, it is seen that the metal
complexes have higher activities than the free ligands due to the presence of the central metal ion
moiety in the complexes. At 10 μM concentration, Cu(II) complex of L1 showed a stronger
H2O2 scavenging activity (65.5 μM) than other ligands and their complexes. The positive
controls, BHT and vitamin C showed 215.3 and 143.6 μM H2O2 radical scavenging, respectively.
The metal complexes with similar structures showed almost comparable antioxidant activities.
Cu(II) complexes, which increase the capacity to stabilize the unpaired electrons and thereby
scavenge the free radicals. The hydroxyl radical scavenging power of the tested complexes is the
greatest. Being a potent scavenger of hydroxyl radicals, the complexes could capture hydrogen
peroxide, as evident from the IC50 values [58]. Although the radical scavenging mechanism of
the complexes under study remains unclear, the experimental results are helpful in designing
more effective antioxidant agents against free radicals.
The DPPH method is described as a simple, rapid and convenient method independent of
sample polarity for screening many samples for radical scavenging activity [59]. The DPPH
19

radical has been widely used to test the ability of compounds as free radical scavengers or
hydrogen donors to evaluate the antioxidant activity. The antioxidant activity of complexes is
presented in Table 6. At 10 μM concentration, Cu(II) complex of L1 showed a stronger DPPH
scavenging activity (69.6 μM) than other ligands and their complexes. The positive controls,
BHT and vitamin C showed 87.4 and 146.8 μM DPPH scavenging, respectively. The marked
antioxidant activity of copper complex of L1, in comparison to free ligands and other complexes,
could be due to the coordination of copper in the condensed ring system, increasing its capacity
to stabilize unpaired electrons and, thereby, to scavenge free radicals. In addition, incorporation
of aromatic moiety in the β-diketone derivatives further enhanced the antioxidant activity. Cu(II)
complex of L1 showed higher antioxidant activity than vitamin C which is due to the
introduction of p-substituted (–NO2) phenyl moiety in the β-diketimine. It implied that copper
complex of L1 might be considered as new promising lead candidate for further design and
synthesis of antioxidants.
3.13. SOD mimetic assay
The O2˙¯ scavenging activity (IC50) of the complexes was determined by NBT assay
using UV-Vis spectroscopy. All Cu(II) complexes demonstrated SOD activity in the micromolar
range (IC50) varying from 2.96-7.83 μM as shown in Table 7. Cu(II) Complex (L1) possesses
superior SOD mimetic activity among all the studied complexes. Although, the IC50 value of the
Cu(II) complex is higher than those reported for native bovine erythrocyte SOD (0.04 μM), it is
in good agreement with the IC50 values of previously reported synthetic SOD mimics [60,61].
These findings fulfill the prerequisite for complex [CuL1Cl2] to act as a potent SOD mimic.
3.14. Antitumor activity against Ehrlich ascite carcinoma cells in mice [62,63]
3.14.1 Effect on biochemical and haematological parameters [62]
On the day 14, the biochemical and haematological parameters, as regard to haemoglobin
level, erythrocytes and leucocytes counts, were compared in the group treated with the standard
drug 5-FU and the group treated with the newly synthesized Cu(II) compound of L1, which has
shown the highest % increase in lifespan over control, with values obtained from normal and
control groups. The EAC tumor cells from the ascitic fluid of different treatment groups were
stained with Lieshman stain. They showed marked cytological changes and cytolytic activity
when compared to the tumor control cells as shown in Fig. 9a-9c. Similar results were observed
20

in the present study in animals of the EAC tumor control group. It is found that the cell cycle of
normal cells was not affected during the treatment with our complexes whereas the tumor cells
were affected in which degenerative changes have been observed in the form of membrane
blebbing, vacuolated cytoplasm and a reductive in the staining intensity. As shown in Table 8,
the biochemical and haematological parameters in the group treated with the Cu(II) compound of
L1 have been nearly recovered completely to be within the normal values. The values are
comparable to those obtained from the group treated with 5-FU.
3.14.2. Effect on survival time
The first measure that can be used to compare the anti-neoplastic activities for the tested
compounds is the increase in survival time for each treated group over the control group. The
mean survival time (MST) for each group was calculated by dividing the total survival times for
all the mice in that group by the number of mice in the same group, then the percent increase in
lifespan for each group over the control group was calculated as follows [62]:
MST of treated group
%Increase in lifespan over control = ————————— × 100 – 100
MST of control group
Comparing the % increase in lifespan of the control group in each treated group (Table 9)
revealed that the [CuL1Cl2] has shown the same increase in lifespan produced by the standard
drug 5-FU. The maximum increase in lifespan was obtained by [CuL1Cl2], which nearly cured
the animals, expanding their lifespan to be approached to the normal non-diseased group.
3.14.3. Effect of complexes on EAC cell lines
The IC50 values of all the synthesized complexes are given in Table 10. This table shows
that the complexes demonstrate different anti-tumor activities. The results show excellent
potential of [CuL1Cl2] complex towards EAC cell lines with IC50 value (113.87 μg/mL), very
sensitive with the value obtained for the standard drug 5-Flurouracil (5-FU), which is taken as
positive control. It suggests that the β-diketimine moiety and its metal ions have important effect
on cytotoxicity. Amongst all the complexes tested, [CuL1Cl2] has lowest IC50 value of 113.87
μg/mL when compared to other complexes. These results indicate that the complexes exert
cytotoxic effects against tested carcinoma cell lines.
21

3.15. Cytotoxicity
A biological assay (MTT assay), which measures the mitochondrial dehydrogenase
activity as an indication of cell viability, is carried out. The experiment is performed on four
cancer cell lines, HeLa, Hep-2, HepG-2 and MCF-7cells. Cis-Pt(NH3)2Cl2 is used as the control,
and it shows high cytotoxicity, which is in accordance with the literature reports [64-66]. The
Cytotoxicity of complexes is presented in Fig. 10. The IC50 values have been calculated after
48 h of incubation with complexes and are listed in Table 11. As shown in Table 11, HeLa and
MCF-7cells are more sensitive to Cu(II) complexes of L1 and L2 than Hep-G2 and Hep-2 cells,
and the cytotoxicities of Cu(II) complex of L1 against HeLa and MCF-7 cells are higher
compared with other complexes and cis-Pt(NH3)2Cl2. It is well known that cis-Pt(NH3)2Cl2
always displays IC50 value in the micromolar range. Although higher complex concentration
reduces the percentages of cell survival, there is a significant difference in susceptibility between
the complexes and cis-Pt(NH3)2Cl2. Due to exist leaving group (chloride ion) in cis-Pt(NH3)2Cl2,
its cytotoxic effects through covalent binding to DNA form cis-DNA adducts, which interferes
with DNA replication and transcription and ultimately induces cell death. According to the
results, we speculate that the antitumor activity of all the complexes not only may be related to
inserting DNA, but also related to the specific molecular shape of the complex and the chemical
structure and nature of the inserted ligand. Synthetically considering these results, the antitumor
activity of both complexes are dependent on changing from the parent ligands of electron-
withdrawing and electron-releasing substituent at p-position, consistent with the affinity of their
complexes binding with DNA, and the ability of both complexes effecting on the cells apoptosis
are dependent on the values of IC50 of complexes against different tumor cell.
4. Conclusion
In this paper, few novel β–diketone derived Schiff bases and their Cu(II) and Zn(II)
complexes have been synthesized and characterized by spectral and analytical data. The IR,
electronic transition and g tensor data lead to the conclusion that the central metal ion assumes a
square planar geometry. The absorption spectroscopy, viscosity measurement and cyclic
voltammetry studies have been carried out on the interaction of the complexes with DNA.
Comparative DNA binding studies of ligands with their Cu(II) and Zn(II) complexes reveal that
the complexes exhibit higher binding affinity towards DNA as compared to ligands. The Cu(II)
22

complex of L1 shows the highest binding affinity. The plasmid pBR322 DNA cleavage activity
of the complexes is significantly increased in the presence of the oxidant (H2O2). No inhibition
of DNA cleavage is observed indicating that hydroxyl radical is not involved in the cleavage
process in the presence of groove binders which confirms the intercalative mode of binding. We
have evaluated in vitro the antibacterial, antifungal and antioxidant activities of newly
synthesized Schiff base ligands and their metal complexes. The copper(II) complex of L1 has
higher potency against Gram-positive bacteria than Gram-negative bacteria. Copper may easily
interact with radicals, especially with molecular oxygen. Enzymatic behavior fulfills the
prerequisite for complex [CuL1Cl2] to act as a potent SOD mimic. Mononuclear Cu(II) and
Zn(II) complexes together with β–diketimine ligands are tested against several human tumor
cells which reveal significant antitumor activity and lower resistance of tumor cells in vitro than
cisplatin. The results suggest that all the complexes produce a potent antitumor and cytotoxic
effect against EAC.
Acknowledgements
The authors express their heartfelt thanks to the Science & Engineering Research Board
(SERB), DST, New Delhi for financial Support. They also express their gratitude to the College
Managing Board, Principal and Head of the Department of Chemistry, VHNSN College,
Virudhunagar for providing research facilities. They thank Prof. R. Senthil Kumar, Department
of Pharmaceutical Chemistry, Swami Vivekanandha College of Pharmacy, Elayampalayam,
Tiruchengodu, for in vitro and in vivo anti-cancer studies.
.
23

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27

Figure Captions
Scheme 1. Synthesis of Schiff base ligands and their metal complexes.
0
Fig. 1a. Absorption spectrum of ligand L1 in buffer pH = 7.2 at 25 C in presence of
increasing amount of DNA.
0
Fig. 1b. Absorption spectrum of [CuL1Cl2] in buffer pH = 7.2 at 25 C in presence of
increasing amount of DNA.
0
Fig. 1c. Absorption spectrum of [ZnL1Cl2] in buffer pH = 7.2 at 25 C in presence of
increasing amount of DNA.
Fig. 2. Effect of increasing amounts of [EB] (■), [CuL1Cl2] (♦), [CuL2Cl2] (●), [CuL3Cl2]
(▲), [CuL4Cl2] (), [ZnL1Cl2] (●), [ZnL2Cl2] (ж), [ZnL3Cl2] (▲), [ZnL4Cl2] () and [L1]
(♦) on the relative viscosity of DNA.1/R = [Complex]/[DNA] or [EB]/[DNA].
0
Fig. 3a. Cyclic voltammogram of [ZnL1Cl2] in buffer pH = 7.2 at 25 C in presence of
increasing amount of DNA.
0
Fig. 3b. Cyclic voltammogram of [CuL1Cl2] in buffer pH = 7.2 at 25 C in presence of
increasing amount of DNA.
Fig. 4a. Minimum inhibitory concentration of the synthesized compounds against the
growth of bacteria (μg/mL).
Fig. 4b. Minimum inhibitory concentration of the synthesized compounds against growth
of fungi (μg/mL).
Fig. 5. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA
(10 μM) by complexes Cu(II) and Zn(II). Lane 1: DNA alone; lane 2: 60 μM [L1] + DNA
+ H2O2; lane 3: 60 μM [CuL1Cl2] + DNA + H2O2; lane 4: 60 μM [CuL2Cl2] + DNA
+H2O2; lane 5: 60 μM [CuL3Cl2] + DNA + H2O2; lane 6: 60 μM [CuL4Cl2] + DNA +
H2O2; lane 7: 60 μM [ZnL1Cl2] + DNA + H2O2; lane 8: 60 μM [ZnL2Cl2] + DNA + H2O2;
lane 9: 60 μM [ZnL3Cl2] + DNA + H2O2; lane 10: 60 μM [ZnL4Cl2] + DNA + H2O2.
Fig. 6. Agarose gel electrophoresis pattern showing the cleavage of pBR322 (10μM)
plasmid DNA (10 μM) by complexes 60 μM in presence of DNA minor groove binding
agent distamycin (100 μM) at 310 K after incubation for 30 min. Lane 1: DNA alone;
lane 2: 60 μM [CuL1Cl2] + DNA + distamycin; lane 3: 60 μM [CuL2Cl2] + DNA
+distamycin; lane 4: 60 μM [CuL3Cl2] + DNA + distamycin; lane 5: 60 μM [CuL4Cl2] +
DNA + distamycin; lane 6: 60 μM [ZnL1Cl2] + DNA + distamycin; lane 7: 60 μM

[ZnL2Cl2] + DNA + distamycin; lane 8: 60 μM [ZnL3Cl2] + DNA + distamycin; lane 9:
60 μM [ZnL4Cl2] + DNA + distamycin.
Fig. 7. Agarose gel electrophoresis pattern showing the cleavage of pBR322 (10μM)
plasmid DNA (10 μM) by complexes 60 μM in presence of DNA major groove binding
agent methyl green (MG) (2.5 mL of a 0.01 μg/mL solution) at 310 K after incubation for
30 min. Lane 1: DNA alone; lane 2: 60 μM [CuL1Cl2] + DNA + MG; lane 3: 60 μM
[CuL2Cl2] + DNA + MG; lane 4: 60 μM [CuL3Cl2] + DNA + MG; lane 5: 60 μM
[CuL4Cl2] + DNA + MG; lane 6: 60 μM [ZnL1Cl2] + DNA + MG; lane 7: 60 μM
[ZnL2Cl2] + DNA + MG; lane 8: 60 μM [ZnL3Cl2] + DNA + MG; lane 9: 60 μM
[ZnL4Cl2] + DNA + MG.
Fig. 8a. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA (10
μM) by Cu(II) complexes of L1-L4 in presence of SOD (1U). Lane 1: DNA alone; lane 2:
60 μM [CuL1Cl2] + DNA + SOD (1U); lane 3: 60 μM [CuL2Cl2] + DNA + SOD (1U);
lane 4: 60 μM [CuL3Cl2] + DNA + SOD (1U); lane 5: 60 μM [CuL4Cl2] + DNA + SOD
(1U);
Fig. 8b. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA (10
μM) by Zn(II) complexes of L1-L4 in presence of SOD (1U). Lane 1: DNA alone; lane 2:
60 μM [ZnL1Cl2] + DNA + SOD (1U); lane 3: 60 μM [ZnL2Cl2] + DNA + SOD (1U);
lane 4: 60 μM [ZnL3Cl2] + DNA + SOD (1U); lane 5: 60 μM [ZnL4Cl2] + DNA + SOD
(1U).
Fig. 9a. Smear showing matured EAC control cells with definite cell wall and structure
without degeneration.
Fig. 9b. EAC tumor cells showing degenerative changes in the form of membrane
blebbing, cell wall fragmentation and low staining intensity.
Fig. 9c. Cu(II) complex of L1 treated EAC Cells showing degenerative changes like
membrane blebbing, vacuolated cytoplasm and low staining intensity with complete
destruction of cells.
Fig. 10. IC50 (mg/mL) values of complexes and cisplatin against various cancer cell
lines.