Journal of Saudi Chemical Society (2016) 20, 69–80

King Saud University

Journal of Saudi Chemical Society

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis, characterization, biological activity

and DNA cleavage studies of tridentate Schiff bases
and their Co(II) complexes
P. Kavitha a, M. Rama Chary b, B.V.V.A. Singavarapu c, K. Laxma Reddy a,*

a
Department of Chemistry, National Institute of Technology, Warangal 506004, Andhra Pradesh, India
b
L.B. College, Warangal 506 007, Andhra Pradesh, India
c
Vimta Labs Ltd., Hyderabad 500 078, Andhra Pradesh, India

Received 11 January 2013; accepted 23 March 2013

Available online 4 April 2013

KEYWORDS Abstract In the present study a series of Co(II) complexes of formyl chromone Schiff bases have
Anticancer; been synthesized characterized by analytical, molar conductance, IR, electronic, magnetic suscepti-
Antioxidant; bility, thermal, fluorescence and powder XRD measurements and screened for various biological
DNA cleavage; activities (antimicrobial, antioxidant, nematicidal, DNA cleavage and cytotoxicity). In all the Co(II)
Fluorescence; complexes 1:2 metal to ligand molar ratio was obtained from analytical data. The molar conduc-
Formyl chromone; tance data confirm that all complexes are non-electrolytic in nature. Based on the electronic and
Nematicidal activity magnetic data, an octahedral geometry is ascribed for all the Co(II) complexes. Thermal behaviour
of the synthesized complexes illustrates the general decomposition patterns of the complexes. The
X-ray analysis data show that all the Co(II) complexes have triclinic crystal system with different
unit cell parameters. Metal complexes have greater antimicrobial activity than ligands. Antioxidant
and nematicidal activities indicate that the ligands exhibit greater activity when compared to their
respective Co(II) complexes. All ligands and Co(II) complexes of HL1 and HL2 showed consider-
able anticancer activity against Raw, MCF-7 and COLO 205 cell lines. All ligands and their Co(II)
complexes showed more pronounced DNA cleavage activity in the presence of H2O2.
ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.

1. Introduction

* Corresponding author. Tel.: +91 870 2462663; fax: +91 The Co(II) complexes have gained more significant applica-
8702459547. tions in the field of biology (Shahabadi et al., 2010; Hettich
E-mail address: [email protected] (K. Laxma Reddy). and Schneider, 1997; Dixon et al., 1996; Peng et al., 2007).
Peer review under responsibility of King Saud University. These complexes have also been used as catalysts in the oxygen-
ation reactions of organic molecules (Pui et al., 2007). In recent
years, great efforts have been made on the design, synthesis and
development of new luminescent materials for a great array of
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applications in sensor technology, electroluminescent devices,
1319-6103 ª 2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.jscs.2013.03.005
70 P. Kavitha et al.

photo molecular switches and dyes. Some of the transition 2. Experimental

metal complexes are used as luminescent markers. For the
coordination of metal ions, ligand systems have been developed 2.1. Materials
which combine good donor abilities with fluorescent properties
(Funston et al., 2003).
O-hydroxy acetophenone, phosphorous oxychloride, 2-amino
Chromone contains c-pyrone nucleus fused to benzene ring
phenol, 2-amino 3-hydroxy pyridine, 2-amino benzoic acid,
at the 5th and 6th positions. The benzopyron ring system is
2-amino thiophenol, cobalt(II) acetate, DPPH (1,1-Diphenyl
central in important classes of bioactive natural products as
2-picryl hydrazyl), BHT (Butylated Hydroxy Toluene) and sol-
coumarines, chromones and flavonoids. Chromones are the
vents used were of AR grade.
most widespread class of naturally occurring compounds.
They are minor constituents of the human diet and have been
2.2. Physical measurements
reported to exhibit a wide range of biological properties
(Kabalka and Mereddy, 2005; Martens and Methofer, 2005).
These biological properties are antimicrobial, antitumor, anti- Elemental analysis (C, H, N, and S) was performed using Perkin
oxidant, antiHIV, antiviral, antiallergenic and nematicidal Elmer CHNS analyzer. IR spectra of the ligands and their
(Grassmann et al., 2002; Wu et al., 2003; Seijas et al., 2005; Co(II) complexes were recorded on Tensor 2 FTIR spectropho-
Shakil et al., 2008; azarenkow et al., 2012). They are also used tometer within the range of 4000–400 cm1 using KBr disc. Mo-
in the synthesis of many hetero cyclic compounds, xanthones lar conductance of the complexes was measured using a Digisun
and transition metal chelates (Arjunan et al., 2004). Moreover, conductivity meter in DMF. The electronic absorption spectra
p-electron conjugation system containing organic molecules of the ligands and their Co(II) complexes were recorded using
like chromone can produce non-linear optical (NLO) materials JASCO V-670 Spectrophotometer from 250–1400 nm. Mag-
(Kalanithi et al., 2011). netic susceptibilities of the complexes were measured on a Guoy
DNA is very stable and the half life of dimethyl phosphate balance at room temperature, by making diamagnetic correc-
of DNA backbone is reported to be approximately tions using Pascal’s constant. Thermal studies of the complexes
**130,000 years via spontaneous hydrolysis in a neutral solu- were carried out on a Perkin Elmer diamond TGA instrument at
tion at room temperature (Radzicka and Wolfenden, 1995; Li a heating rate of 10 C and a nitrogen flow rate of 20 ml/min.
et al., 2008). Therefore, from the last decade onwards, DNA The fluorescence spectra of ligands and their complexes were re-
degradation studies have been of great interest for biologists corded on FLUOROLOG – FL3–11 spectroflourimeter. The X-
and chemists. Many transition metal complexes that cleave ray patterns of the complexes were recorded on Xpert-Pro X-ray
DNA under physiological conditions have been developed diffractometer with Cu Ka radiation (k = 1.5406 Å). The dif-
as artificial nucleases (Raman and Sobha, 2012; Patil et al., fraction data are integrated on using the Nakamuta programme.
2011). The nature of the ligand and metals is of main impor-
tance in the interaction of the complexes with the DNA mol- 2.3. Synthesis of ligands
ecule, which would help in the design of newer drugs and
develop new selective, efficient DNA recognition and cleaving All the Schiff base ligands (HL1, HL2, HL3, and HL4) were
agents. A large number of transition metal complexes have prepared as per the literature procedures and structures of
been found to promote DNA cleavage because of their redox the ligands are given in Fig. 1 (Khan et al., 2009; Sigg et al.,
properties. Some of the Co(II) complexes are known to func- 1982; Kavitha et al., 2013).
tion as specific probe for DNA bulges due to its ability to
cleave DNA specifically (Cheng et al., 1999). Earlier studies 2.4. Synthesis of Co(II) complexes
of formyl chromone Schiff bases and their metal complexes
have been focused on DNA binding studies (Li et al., 2010; To a hot methanolic solution of ligands (20 ml, 2 mM), a hot
Wang and Yang, 2008). Information on DNA binding methanolic solution of Co(CH3COO)2Æ4H2O (10 ml, 1 mM)
parameters of these molecules explores their potential appli- was added drop wise with constant stirring. The change of
cations, since their binding mode could be associated with color was observed immediately upon the addition of
their ability to cause DNA damage. This DNA damage is Co(CH3COO)2Æ4H2O. The mixture was stirred continuously
leading to the inhibition of uncontrolled growth of cancerous for 1–2 h at room temperature. The precipitate thus formed
cells. was filtered off, washed with methanol and dried in vacuum.
In continuation of our ongoing interest is the synthesis,
characterization and biological activity studies (antimicrobial,
antioxidant, nematicidal, anticancer and DNA cleavage) of 2.5. Biological studies
formyl chromone Schiff bases and their metal complexes.
(Kavitha et al., 2013; Kavitha and Laxma Reddy, 2012). Herein 2.5.1. Antimicrobial screening
we reported the synthesis of a series of Co(II) complexes, In vitro antimicrobial activity of the synthesized ligands and
structural characterization and biological activity studies i.e. their Co(II) complexes were screened against Proteus vulgaris,
antimicrobial, nematicidal, antioxidant, DNA cleavage and Klebsiella pnuemoniae, Staphylococcus aureus, Bacillus subtili
anticancer activities of formyl chromone Schiff bases such as and Candida albicans (fungi). The activity was carried out
3-((2-hydroxy phenyl imino)methyl)-4H-chromen-4-one (HL1), using disc diffusion method. The antibiotics kanamycin and
2-((4-oxo-4H-chromen-3-yl)methylneamino) benzoicacid (HL2), clotrimazole are the standards for antibacterial and antifungal
3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one activity studies. A standard inoculum, 1–2 · 107 cfu/ml 0.5 Mc
(HL3) and 3-((2-mercaptophenylimino)methyl)-4H-chromen- Farland standards (Stemper and Matsen, 1970) was intro-
4-one (HL4). duced onto the surface of sterile nutrient agar plate and evenly
Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases 71

O OH cotton wool filter paper and incubated at 28 ± 2 C to obtain

H
C N freshly hatched juveniles (J2). Juveniles collected within 48 h
were used for screening nematicidal activity of the compounds.
O
2.5.2.1. Mortality test. The ligands and their Co(II) complexes
3-(-(2-hydroxyphenylimino)methyl)-4 H-chromen-4-one
(HL1) were initially dissolved in dimethyl sulfoxide (DMSO) and
then in distilled water to make dilutions of 250, 150, and
HO 50 lg/ml. Experiments were performed under laboratory con-
O C O ditions at 28 ± 2 C. About 100 freshly hatched second stage
H
C N juveniles were suspended in 5 ml of each diluted compound
and incubated. Distilled water with nematode larvae was taken
O as control. The dead nematodes were observed under an in-
2-((4-oxo-4H-chromen-3-yl)methyleneamino)benzoic acid verted binocular microscope. After an incubation of 24 and
(HL2) 48 h, percentage of mortality was calculated. Nematodes were
considered dead, if they did not move when probed with a fine
O OH needle (Cayrol et al., 1989).
H
C N
2.5.3. DPPH radical scavenging activity
N
O The free radical scavenging activity of the ligands and their
3-((3-hydroxypyridin-2-ylimino)methyl)-4H-chromen-4-one Co(II) complexes were determined by using DPPH free radical
(HL3) scavenging method according to the literature (Braca et al.,
2001; Saha et al., 2008). Compounds were dissolved in methanol
O SH (10 mg/10 ml) and used as stock solutions. From the stock solu-
H
C N tions, 1 ml of each compound solution with different concentra-
tions (0.25–1.00 lg) was added to 3 ml of methanolic DPPH
O (0.004%) solution. After 30 min, the absorbance of the test com-
3-(-(2-mercaptophenylimino)methyl)-4H-chromen-4-one pounds was taken at 517 nm using UV–VIS spectrophotometer.
(HL4) BHT used as standard, DPPH solution was used as control with-
out the test compounds and methanol was used as blank. The
Figure 1 Structures of ligands. percentage of scavenging activity of DPPH free radical was mea-
sured by using the following formula:
 
ðA0  Ai Þ
spread by using a sterile glass spreader. Sterile antibiotic discs Scavenging activityð%Þ ¼
A0
(6 mm in diameter, prepared using Whatmann No. 1 paper)
were placed over the nutrient agar medium. Each disc was where, A0 is the absorbance of the control and Ai is the absor-
spread with 100 lg of the compounds (initially dissolved in bance of the sample.
DMSO). The plates were incubated with bacterial cultures
for 24 h at 37 C and fungal cultures at 25 C for 48 h. The 2.5.4. Anticancer activity
activity of the compounds was determined by measuring the Raw 264.7 cell line (murine macrophage cell line), MCF-7 (hu-
diameter of inhibition zone in ‘millimetres’ and compared with man breast carcinoma cell line) and COLO 205 (human colon
standard antibiotics. DMSO (which has no activity) and stan- carcinoma cell line) were obtained from National Center for
dard antibiotics were used as negative and positive controls for Cell Science (NCCS), Pune, India. The cell lines were cultured
antimicrobial activity studies. All experiments were carried out in Dulbecco’s Modified Eagle Medium (DMEM) supple-
in triplicates and the average zone of inhibition was calculated. mented with 10% fetal bovine serum (FBS), amphotericin
Minimum inhibitory concentrations (MIC) of the ligands (3 lg/ml), gentamycin (400 lg/ml), streptomycin (250 lg/ml)
and Co(II) complexes which exhibited antimicrobial activity and penicillin (250 units/ml) in a carbon dioxide incubator at
were determined using literature method (Shankar et al., 5% CO2. About 700 cells/wells were seeded in 96 well plate
2009). All compounds diluted within the range of 100–10 lg/ using culture medium. The viability of the cells was tested
ml were mixed in the nutrient broth and 0.1 ml of active inocu- using trypan blue dye with the help of haemocytometer and
lums was added to each tube. The tubes were incubated aero- 95% of viability was confirmed. After 24 h, the new medium
bically at 37 C for bacteria and 25 C for fungi for 24 h. The with compounds in the concentration of 100, 10, and 1 lg/ml
lowest concentration of the compound that completely inhib- was added at respective wells and kept in incubation for
ited bacterial growth (no turbidity) in comparison to control 48 h. After incubation the following assay was performed.
was regarded as MIC.
2.5.4.1. MTT assay. After 48 h of the compounds treatment,
2.5.2. Nematicidal activity the medium was changed again for all groups and 10 ll of
Nematicidal activity of the ligands and their Co(II) complexes MTT (5 mg/ml stock solution) was added and the plates were
was carried out on Meloidogyne incognita. Fresh egg masses of incubated for an additional 4 h. The medium was discarded
M. incognita collected from stock culture were maintained on and the formazan blue, which was formed in the cells, was dis-
tomato (Lycopersicon esculentum) root tissues and kept in solved with 50 ll of DMSO. The optical density was measured
water for egg hatching. The egg suspension was poured on a at 570 nm. Cisplatin was used as a standard. The percentage of
72 P. Kavitha et al.

toxicity was calculated by using the following formula (Sargent

Molar conductivity
(mho1 mol1 cm2)
and Taylor, 1989).
 
O:D of treatedcells
% Cytotoxicity ¼ 1   100
O:D of untreatedcells
The IC50 (concentration of drug required to inhibit growth

20
21
14
15
of 50% of the cancer cells) values of all the compounds were
calculated using GraphPad Prism software tool.

(10.19)
(9.74)
(8.54)
(9.43)
2.5.5. DNA cleavage studies
The DNA cleavage activity of the Co(II) complexes was stud-

10.11

10.82
10.24
9.30
ied by agarose gel electrophoresis method. pUC19 plasmid was

Co
cultured, isolated and used as DNA for the experiment. Test
samples (1 mg/ml) were prepared in DMF. 25 lg of the test

10.25 (10.26)
samples was added to the isolated plasmid and incubated for
2 h at 37 oC. After incubation, 30 ll of plasmid DNA sample
mixed with bromophenol blue dye (1:1) was loaded into the
electrophoresis chamber wells along with the control DNA,

S
5 M FeSO4 (treated with DNA) and standard DNA marker
containing TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M

(4.62)
(3.73)
(8.96)
(4.49)
EDTA/1 L). Finally, it was loaded onto an agarose gel and
electrophoresed at 50 V constant voltage up to 30 min. After

4.39
4.15
9.51
4.38
the run, gel was removed and stained with 10.01 lg/ml ethi-

Elemental analysis %found (%calcd.)

N
dium bromide and the image was taken in Versadoc (Biorad)
imaging system. The results were compared with standard

(3.64)
(3.30)
(3.52)
(3.20)
DNA marker. The same procedure was followed in the pres-
ence of H2O2 also.

3.75
3.49
3.04
3.19
H
3. Results and discussion

(63.48)
(58.55)
(57.61)
(61.58)
The physical and analytical data of Co(II) complexes are de-
picted in Table 1. All Co(II) complexes were colored and very
stable at room temperature, soluble in DMF and DMSO. Ana- 63.19
61.59
60.55
61.54
lytical data confirm the metal to ligand molar ratio is 1:2 in all
C

the Co(II) complexes. The molar conductance measurements

Physical, analytical and molar conductivity data of Co() complexes.

of the complexes were recorded in DMF (1 · 103 M). The re-

Color (% Yield)

sults indicate their non-electrolytic nature (Geary, 1971).

Brick Red (75)

Dark Red (80)

Orange (65)

Black (76)

3.1. FTIR spectral studies

The FTIR spectral data containing relevant vibrational bands

of the ligands and their Co(II) complexes are listed in Table 2.
The ligands showed a band in the range of 1620–1650 cm1
Mol. Wt.

which is due to t(C‚O) group of the chromone moiety. This

band was shifted to lower wavenumber region 5–48 cm1 in
604
696
624
654

their corresponding Co(II) complexes, indicating the coordina-

tion of oxygen atom of carbonyl group of the chromone moi-
ety (Dziewulska-Kuaczkowska, 2010). The stretching
[Co(L4)2]Æ2H2O [Co(C16O2NSH10)2]Æ2H2O
[Co(L2)2]Æ3H2O [Co(C17O4NH10)2]Æ3H2O
[Co(L3)2]Æ2H2O [Co(C15O3N2H9)2]Æ2H2O

vibration of the azomethine group (C‚N) was observed in

[Co(L1)2]ÆH2O [Co(C16O3NH10)2]ÆH2O

the range of 1605–1555 cm1 in all the ligands. This band

was shifted to 30–40 cm1 lower wavenumber region in their
cobalt(II) complexes, indicating the participation of nitrogen
atom of azomethine group in coordination to the metal ion
(Rosu et al., 2010). A broad band which appeared in HL1
and HL3 at 3241 and 3246 cm1, respectively, is attributed to
the t(O–H) group. The absence of this band in their Co(II)
complexes is due to the involvement of oxygen atom of (OH)
Compound

group in coordination with the metal ion (Singh et al., 2010).

Table 1

In HL2 ligand, a strong band appeared at 1365 cm1 due to

the t(C–O) of carboxylic group. In its Co(II) complex, it was
shifted by 14 cm1, indicating the coordination of the oxygen
Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases 73

atom of carboxylic group with the metal ion (Padmaja et al., as well as the observed and calculated mass loss percentages
2011). The stretching vibrations of S–H have no significant of all Co(II) complexes are illustrated in Table 4. The TG
role, since its band is very weak in HL4 ligand. However, par- graphs of [Co(L1)2]ÆH2O and [Co(L4)2]Æ2H2O complexes are
ticipation of the SH group in chelation is ascertained from the given in Fig. 3. The results show a good agreement with the
shift of t(C–S) at 790 cm1 in the ligand to lower frequencies theoretical formula as suggested from the analytical data
by 40 cm1 in the complex (Soliman and Mohamed, 2004). (Table 1). The data reveal the following findings; [Co(L1)2]ÆH2O
Further, the coordination of nitrogen and oxygen atoms was was thermally decomposed in three steps. The first decomposi-
supported by the appearance of a non-ligand bands at 600– tion step with estimated mass loss 3.17% (cald. mass loss
400 cm1 region due to the t(Co–N) and t(Co–O), respec- 2.98%) within the temperature range of 30–89 C, may be
tively. From the above spectral data, it was concluded that attributed to the liberation of hydrated molecule. The second
Schiff base ligands acts as monobasic tridentate ligands with and third steps were found within the temperature range of
ONO/ONS donor sites. 90–456 C with an estimated mass loss of 82.73% (cald. mass
loss 84.64%), which corresponds to the loss of total organic
3.2. Electronic and Magnetic studies part present in the complex. The final step horizontal plateau
was observed greater than 457 C with estimated mass loss
The electronic spectra and magnetic measurements were con- of 14.10% (cald. mass loss 12.38%) regarded as CoO as resi-
ducted in order to obtain the geometry of the complexes. due. In the case of [Co(L2)2]Æ3H2O complex, the first step at
The electronic absorption spectra of the ligands and their 30–76 C range by estimated mass loss of 8.00% (cald. mass
Co(II) complexes were recorded in solid state at room temper- loss 7.75%), may be attributed to the loss of three water
ature. The electronic spectral data and magnetic moments of molecules. The second and third decomposition steps are with-
the complexes are summarized in Table 3. The electronic spec- in temperature range 77–571 C, with an estimated mass loss
tra of HL4 and its Co(II) complex are shown in Fig. 2. The of 81.32% (cald. mass loss 81.5%), accounting for the expul-
electronic spectra of all ligands showed two bands. One band sion of the total organic part. The remaining mass loss is
within the range of 25,000–27,000 cm1 is attributed to the 10.68% (cald. mass loss 10.75%), regarded as CoO residue.
n fi p* transitions. Another band within the range of 31,000– [Co(L3)2]Æ2H2O complex decomposed in three successive steps.
37,000 cm1 is due to the p fi p* transitions. The electronic The first step corresponds to the mass loss of two water mole-
spectra of all the Co(II) complexes showed three bands cules with an estimated mass loss of 5.98% (cald. mass loss
around. 8000, 16,000, and 24,000 cm1 assigned to the 4T1g 5.76%) at the temperature range of 30–80 C. The second
(F) fi 4T2g (F) (m1), 4T1g (F) fi 4A2g (F) (m2) and 4T1g and third steps occur within the temperature range of 81–
(F) fi 4T1g (P) (m3) transitions, respectively which are charac- 457 C with an estimated mass loss of 81.74% (cald. mass loss
teristic of octahedral geometry (AbouEl-Enein et al., 2007). 82.25%), which may be attributed to the loss of organic part.
The octahedral geometry of Co(II) complexes is further sup- The remaining mass loss is 12.28% (cald. mass loss 11.99%),
ported by the ratio of m2/m1, which lies in the range of 1.8– regarded CoO as residue. The TG curve of [Co(L4)2]Æ2H2O
2.0. Various ligand field parameters like 10Dq, B and b were complex also decomposed in three stages. The first estimated
calculated for all the Co(II) complexes. The nephelauxetic mass loss of 5.59% (cald. mass loss 5.49%) observed in the
parameter b is calculated from the B(complex)/B(free ion) where temperature range of 30–99 C is due to the loss of hydrated
B(free ion) is 1124 cm1. The values of b are in the range of molecules. The second and third steps are found in the temper-
0.81–0.88, indicating that the covalent character of metal li- ature range of 100–483 C with an estimated mass loss of
gand r-bond is low. At room temperature, the magnetic mo- 82.72% (cald. mass loss 83.07%), corresponding to the loss
ment values of the Co(II) complexes lie in the range of 4.0– of total organic molecules. The final product obtained in this
5.0 BM. These values are in good agreement with those re- complex also may be due to the formation of CoO.
ported for the octahedral structure (Ajibade et al., 2006). Based on all the spectral data, an octahedral geometry has
been proposed for all Co(II) complexes (Fig. 4.).
3.3. Thermal studies
3.4. Fluorescence spectra
Thermogravimetric studies (TG) for the Co(II) complexes were
carried out from room temperature to 800 C. The stages of The emission spectra of all ligands and their Co(II) complexes
decomposition, temperature range, decomposition products have been measured in the solid state at room temperature.

Table 2 IR spectral data of ligands and their Co(II) complexes.

Compound t(O–H) t(C‚O)(c pyrone) t(C‚N) t(CO) (carboxylate) t(C–S) t(M–N) t(M–O)
HL1 3241 1643 1605
[Co(L1)2]ÆH2O 1638 1569 532 439
HL2 1651 1605 1365
[Co(L2)2]Æ3H2O 1641 1570 1351 528 449
HL3 3246 1647 1591
[Co(L3)2]Æ2H2O 1599 1555 530 446
HL4 1622 1597 790
[Co(L4)2]Æ2H2O 1599 1564 754 536 469
74 P. Kavitha et al.

Table 3 Magnetic, electronic spectral data and ligand field parameters of Co(II) complexes.
Compound Absorption maxima Tentative assignments Magnetic moment m2/m1 10Dq (cm1) B (cm1) b
(cm1) (B.M)
4
[Co(L1)2]ÆH2O 8853 T1g(F) fi 4T2g(F) (m1) 4.05 1.82 7328 918 0.94
4
16181 T1g(F) fi 4A2g(F) (m2)
4
24154 T1g(F) fi 4T1g(P) (m3)
4
[Co(L2)2]Æ3H2O 8679 T1g(F) fi 4T2g(F) (m1) 4.99 1.86 7524 915 0.81
4
16205 T1g(F) fi 4A2g(F) (m2)
4
23562 T1g(F) fi 4T1g(P) (m3)
4
[Co(L3)2]Æ2H2O 8980 T1g(F) fi 4T2g(F) (m1) 5.01 1.95 8533 955 0.98
4
17513 T1g(F) fi 4A2g(F) (m2)
4
23752 T1g(F) fi 4T1g(P) (m3)
4
[Co(L4)2]Æ2H2O 8912 T1g(F) fi 4T2g(F) (m1) 4.98 1.86 7671 949 0.97
4
16583 T1g(F) fi 4A2g(F) (m2)
4
24390 T1g(F) fi 4T1g(P) (m3)

Figure 2 Electronic spectra of (a) HL4 (b) Co(II) complex of HL4.

Table 4 TG data of Co(II) complexes.

Compound Temperature range (C) Mass loss found (%cacld.) Assignment
[Co(L1)2]ÆH2O 30–89 3.17(2.98) H2O
[Co(C16O3NH10)2]ÆH2O 90–284 17.31(17.52) C6H4ON
284–456 65.42(67.12) C16O2NH10+C10O2H6
>457 14.10(12.38) CoO (Residue)
[Co(L2)2]Æ3H2O 30–76 8.00(7.75) 3H2O
[Co(C17O4NH10)2]Æ3H2O 76–334 18.91(19.22) C7H4O2N
335–571 62.41(62.28) C17O3NH10 + C10H6O2
>571 10.68(10.75) CoO (Residue)
[Co(L3)2]Æ2H2O 30–80 5.98(5.76) 2H2O
[Co(C15O3N2H9)2]Æ2H2O 81–323 25.77(25.58) C10H6O2
324–457 55.97(56.67) C15O3N2H9 + C5ON2H3
>457 12.28(11.99) CoO (Residue)
[Co(L4)2]Æ2H2O 30–99 5.59(5.49) 2H2O
[Co(C16O2NSH10)2]Æ2H2O 100–390 40.22(40.31) C16ONSH10
391–483 42.50(42.76) C16O2NSH10
>483 11.69(11.44) CoO (Residue)
Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases 75

Figure 3 TG graph of (a) [Co(L1)2]ÆH2O (b) [Co(L4)2]Æ2H2O.

O
A
NC
X H O
Co
.nH2O
O H X
CN
A
O
A=C and X=O/S/COO
A=N and X=O

Figure 4 Proposed structure of all Co(II) complexes.

The fluorescence spectra of HL4 and its Co(II) complex are

shown in Fig. 5. The HL1 ligand showed an intense emission
band at 598 nm upon photo excitation at 398 nm. However,
its Co(II) complex exhibits a strong emission band at 642 nm
and a weak band at 538 nm (kex = 438 nm). HL2 and its
Figure 5 Fluorescence spectra of HL4 and its Co(II) complex.
Co(II) complex show emission bands at 492 nm
(kex = 391 nm) and (553, 514 and 475 nm) (kex = 438 nm),
respectively. The HL3 and its Co(II) complex exhibit emission Co(II) complexes show triclinic crystal system. The unit cell
bands at 524 nm (kex = 395 nm) and (746, 621, and 551 nm) parameters of all complexes are as follows; [Co(L1)2]ÆH2O :
(kex = 453 nm), respectively. HL4 ligand shows strong emis- a = 7.2909 Å, b = 7.6443 Å, c = 5.09 Å, a = 105.1,
sion band at 492 nm (kex = 308 nm). Two emission bands were b = 102.79, c = 90.375, V = 283.68 Å3; [Co(L2)2]Æ3H2O:
observed in its Co(II) complex at 461 and 413 nm a = 5.6634 Å, b = 6.2966 Å, c = 5.8533 Å, a = 99.303,
(kex = 438 nm). The fluorescence intensity of the Schiff base li- b = 92.249, c = 96.429, V = 204.3 Å3; [Co(L3)2]Æ2H2O:
gands decreased dramatically when coordinated with the metal a = 5.4042 Å, b = 6.9164 Å, c = 5.3034 Å, a = 90.174,
ions. Decrease in the emission maxima was also observed in b = 90.136, c = 109.312 , V = 198.22 Å3; [Co(L4)2]Æ2H2O:
the metal complexes when compared to their corresponding a = 6.176 Å, b = 8.1453 Å, c = 6.5487 Å, a = 93.929,
ligands. b = 103.401, c = 94.586, V = 318.1 Å3. The crystallite size
of the Co(II) complexes is calculated from Scherer’s formula
3.5. Powder XRD studies (Warren, 1990).
0:94k
The X-ray diffraction patterns of [Co(L2)2]Æ3H2O and [Co D¼
b cos h
(L4)2]Æ2H2O complexes are given in Fig. 6. Single crystals of
the complexes could not be isolated from any solvents. The where, k is the wavelength of X-ray radiation, b is the full
powder XRD patterns of Co(II) complexes show sharp crystal- width at half maximum of diffraction line and h is the diffrac-
line peaks indicating its crystalline nature. Phase and unit cell tion angle. Using the full width at half maximum intensity of
parameters were found using trial and error methods. All the the patterns, the average sizes of the crystals are around 36,
76 P. Kavitha et al.

Figure 6 Powder XRD pattern of (a) [Co(L2)2]Æ3H2O (b) [Co(L4)2]Æ2H2O.

Table 5 MIC values (lg/ml) for antimicrobial activity of ligands and their Co(II) complexes.
Compound Bacillus subtilis Staphylococcus aureus Proteus vulgaris Klebsiella pneumoniae Candida albicans
HL1 – – – – –
HL2 80 80 80 80 80
HL3 – – – – –
HL4 – – – – –
[Co(L1)2]ÆH2O 80 80 80 80 80
[Co(L2)2]Æ3H2O 30 20 30 20 30
[Co(L3)2]Æ2H2O 50 50 50 50 55
[Co(L4)2]Æ2H2O 80 80 – 80 –
Kanamysin 04 10 08 11 –
Clotrimazole – – – – 10

Table 6 Nematicidal activity (% mortality) values of ligands and their Co(II) complexes.
Compound After 24 h After 48 h
250 (lg/ml) 150 (lg/ml) 50 (lg/ml) 250 (lg/ml) 150 (lg/ml) 50 (lg/ml)
HL2 51 35 18 70 51 28
HL3 55 38 20 77 57 33
[Co(L1)2]ÆH2O 7 – – 10 – –
[Co(L2)2]Æ3H2O – – – – – –
[Co(L3)2]Æ2H2O 5 – – 7 – –
[Co(L4)2]Æ2H2O – – – – – –

35, 40, and 30 nm for Co(II) complexes of HL1, HL2, HL3 and bacteria and fungi strains. However [Co(L4)2]Æ2H2O complex
HL4, respectively. does not show activity against P. vulgaris and C. albicans. In
comparison, the activity of all the strains was increased by li-
3.6. Biological studies gands when coordinated with Co(II) ion. Many literature re-
ports reveal that, inactive or less active compounds become
3.6.1. Antimicrobial activity more active upon coordination/complexation (Kulkarni
The MIC values of the antimicrobial activity of the ligands and et al., 2011; Nejo et al., 2010). This can be explained based
their Co(II) complexes are presented in Table 5. From the re- on the Overtones concept and Chelation theory. According
sults, the ligands either exhibited no activity or having low to to the Overtones concept, chelation/coordination reduces the
moderate activity against bacteria and fungi strains. But their polarity of the metal ion mainly because of partial sharing of
Co(II) complexes showed moderate to good activity compared its positive charge with donor groups within the whole chelate
to the standard antibiotics. Among all the Co(II) complexes, ring system. This process of chelation thus increases the lipo-
[Co(L2)2]Æ3H2O complex showed effective activity against all philic nature of the central metal atom, which in turn, favors
Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases 77

Table 7 IC50 values of DPPH radical scavenging activity of Table 8 IC50 values (lg/ml) of anticancer activity of ligands
ligands and their Co(II) complexes. and their Co(II) complexes.
Compound IC50(lg/ml) Compound Raw MCF-7 COLO 205
HL3 1.27 HL1 46.8 24.5 20.1
HL4 0.40 HL2 56.1 34.2 39.1
[Cu(L2)2]Æ3H2O 2.32 HL3 52.8 29.2 15.2
BHT 0.67 HL4 46.9 30.4 68.1
[Co(L1)2]ÆH2O 42.2 41.9 51.6
[Co(L2)2]Æ3H2O 52.1 15.4 27.1
[Co(L3)2]Æ2H2O 199.8 231.7 63.9
its permeation through the lipoid layer of the membrane thus [Co(L4)2]Æ2H2O 246.5 405.5 56.7
causing the metal complex to cross the bacterial membrane Cisplatin 1.5 1.7 5.6
more effectively, which in turn increases the activity of the
complexes. These complexes also disturb the respiration pro-
cess of the cell and thus block the synthesis of proteins, which complex. The free radical scavenging activity of the compounds
restricts further growth of organisms. Besides this, many other depends on the structural factors such as the phenolic hydroxyl,
factors such as solubility, dipole moment and conductivity of carboxylic groups and other structural features (Bhuiyan et al.,
complexes may be the reason for remarkable antimicrobial 2009). The order of antioxidant activity of ligands and their
activities of the complexes (Coombs et al., 2005; Chohan Co(II) complexes according to their IC50 values is as follows
et al., 2001, 2010). HL4 > HL3 > ½CoðL2 Þ2   3H2 O
From the results, none of the compounds are effective
against the tested microorganisms when compared with the
standard antibiotics like kanamycin and clotrimazole. 3.6.4. Anticancer activity studies
The anticancer activity of the ligands and their Co(II) com-
3.6.2. Nematicidal activity. Plant parasitic nematodes are the plexes was determined by MTT assay. The IC50 values of li-
main pathogens on most fibre crops, horticultural, food and gands and their Co(II) complexes are presented in Table 8.
vegetable crops and without adequate control, they cause loss The pharmacological testing has proved that the cytotoxic ef-
of yield and quality. Nematode Meloidogyne species is known fect of the ligands and their Co(II) complexes was considerably
to attack almost all types of plants and cause considerable moderate to less pronounced compared to the standard drug
damage (Adekunle and Akinlua, 2007). M. incognita produces cisplatin, since calculated IC50 values were in the range of
galls on the roots of many host plants and is also responsible 15–400 lg/ml. Among all ligands and their Co(II) complexes
for 44.87% of yield loss in brinjal (Kapoor et al., 2012). The evaluated, the Co(II) complex of HL2 showed the highest anti-
past literature works concerning nematode problems have cancer activity against MCF-7 (IC50 = 15.4 lg/ml;
indicated that there is a need to check this pest by control prac- IC50 = 1.7 lg/ml for Cisplatin). Co(II) complex of HL1
tices, using various chemicals. showed the lowest IC50 value among all the ligands and their
The nematicidal activity of all the ligands and their Co(II) Co(II) complexes against Raw cell lines. Furthermore HL3 li-
complexes were evaluated against M. incognita with different con- gand exhibited cytotoxicity with low concentration
centrations after 24 and 48 h and the details are given in Table 6. (IC50 = 15.2 lg/ml; IC50: 5.6 lg/ml for Cisplatin) compared
The results revealed that, all the ligands except HL2 and HL3, and to all ligands and complexes against COLO 205 cell lines.
their Co(II) complexes showed very less activity. The ligands HL2 However the ligands have a higher inhibitory effect than their
and HL3 showed more than 70% mortality in 250 lg/ml concen- corresponding Co(II) complexes. Several compounds in partic-
tration after 48 h. The highest activity was observed at higher con- ular HL3 and Co(II) complex of HL2 were endowed with sig-
centrations and activity also increased with time. nificant cytotoxic potency and can be viewed as new lead
compounds for further modifications.
3.6.3. DPPH radical scavenging activity
In DPPH free radical scavenging activity, antioxidants are 3.6.5. DNA cleavage studies
reacting with the stable free radical 1,1-diphenyl-2-picryl- The interaction of plasmid pUC19 DNA with Co(II) com-
hydrazyl (DPPH) producing a colorless 1,1-diphenyl-2-picryl- plexes was studied using gel electrophoresis in the presence
hydrazine. When DPPH receives an electron or hydrogen and absence of oxidizing agent H2O2. DNA cleavage was
radical to become more stable, its absorption decreases achieved by monitoring the gel electrophoresis for naturally
(Konzen et al., 2006). The DPPH scavenging activity was occurring, covalently closed circular form (Form I) transition
expressed as IC50, whose concentration is sufficient to obtain to the nicked circular (Form II) and linear forms (Form III).
50% of maximum scavenging activity. The IC50 values of When circular plasmid DNA is subjected to electrophoresis,
ligands and their Co(II) complexes are depicted in Table 7. relatively fast migration will be observed for the super coil
BHT was used as standard. From the results, ligands HL3 form (Form I), slower migration will be observed for nicked
(IC50 = 1.27 lg) and HL4 (IC50 = 0.4 lg) showed good activ- circular form (Form II) and linear form occurred between
ity, where as remaining ligands HL1 and HL2 did not show any the super coiled and nicked circular forms (Li et al., 2011;
antioxidant activity. The HL4 (IC50 = 0.40 lg) ligand showed Kashanian et al., 2012). The gel electrophoresis pictures are
effective activity when compared to standard drug BHT shown in Fig. 7. In the absence of H2O2, control DNA (In
(IC50 = 0.67 lg). All the Co(II) complexes did not show Fig. 7a. Lane control) does not show any activity. FeSO4
prominent activity except [Co(L2)2]Æ3H2O (IC50 = 2.32 lg) was used as standard, disappearance of bands was observed
78 P. Kavitha et al.

Figure 7 (a) DNA cleavage in the absence of H2O2 Lane 1: DNA+HL1; Lane 2: DNA+HL2; Lane 3: DNA+HL3; Lane 4:
DNA+HL4; Lane 5: DNA+[Co(L1)2].H2O; Lane 6: DNA+[Co(L2)2].3H2O; Lane 7: DNA+[Co(L3)2].2H2O; Lane 8: DNA+
[Co(L4)2].2H2O; Lane 9: DNA+FeSO4; Lane C: DNA alone (b) DNA cleavage in the presence of H2O2 Lane 1: DNA+ HL1+H2O2;
Lane 2: DNA+HL2+H2O2; Lane 3: DNA+HL3+H2O2; Lane 4: DNA+HL4+H2O2; Lane 5: DNA+[Co(L1)2].H2O+H2O2; Lane 6:
DNA+[Co(L2)2].3H2O+H2O2; Lane 7: DNA+[Co(L3)2].2H2O+H2O2; Lane 8: DNA+[Co(L4)2].2H2O+H2O2; Lane 9: DNA+Fe-
SO4+H2O2; Lane C1: DNA+H2O2; Lane C2: DNA alone. *Lane 1-4 in figure (a) and (b) from Kavitha et al., 2013.

in its lane, indicating the DNA cleavage. All the ligands exhib- physical and various spectroscopic techniques. The results re-
ited significant activity in the presence of H2O2 (Fig. 7b, Lanes vealed that all ligands coordinated in a tri dentate manner to
1–4), in the absence of H2O2, ligands do not show cleavage the metal ions and the geometry of the complexes is found to
activity (Fig. 7a, Lanes 1–4) (Kavitha et al., 2013). All the be octahedral. From the thermo grams the number of lattice
Co(II) complexes showed a decrease in the concentration of water molecules present in the complexes is calculated. Triclinic
the super coiled form and increase in the concentration of system has been proposed for all the complexes. Complexes
the nicked circular form. It indicates the DNA cleavage activ- exhibited good fluorescence. The antimicrobial activity results
ity of the complexes without any external reagents i.e. H2O2. revealed that all the Co(II) complexes have higher antimicro-
Interestingly, the complexes cleaved DNA hydrolytically in bial activity than the ligands. HL3 ligand showed effective anti-
the absence of any reducing agents and light. In the presence oxidant and nematicidal activities when compared to the
of an oxidizing agent H2O2, the DNA cleavage activity oc- remaining ligands and complexes. All ligands and their Co(II)
curred as evidenced by the total disappearance of DNA complexes showed certain cytotoxic activity against tested
[Fig. 7b, Lanes 5–8]. On the basis of these results, it is con- cancer cell lines except Co(II) complexes of HL3 and HL4.
cluded that prominent DNA cleavage activity was observed The ligands and their Co(II) complexes can effectively cleave
in the presence of an oxidizing agent H2O2. plasmid DNA in the presence of an oxidizing agent H2O2.

4. Conclusions Acknowledgments

In summary, a series of Co(II) complexes of 3-formyl chromone The authors are thankful to Dr. Anil Gopala, Application
Schiff bases have been prepared and characterized using Leader, India, Perkin Elmer Pvt. Ltd. for providing the
Synthesis, characterization, biological activity and DNA cleavage studies of tridentate Schiff bases 79

TG-DTA facility and the Director, CIF, Pondicherry Central measurable 10 million-fold rate increase. J. Am. Chem. Soc. 119,
University, Pondicherry, India for recording fluorescence 5638–5647.
spectra. The authors also acknowledge the Department of Kabalka, G.W., Mereddy, A.R., 2005. Microwave assisted synthesis of
Biochemistry, Sri Venkateswara University, Tirupati, functionalized flavones and chromones. Tetrahydron Lett. 46,
6315–6317.
India, for providing the antioxidant activity data and
Kalanithi, M., Kodimunthiri, D., Rajarajan, M., Tharmaraj, P., 2011.
Dr. M. Ramanathan, PSG College of Pharmacy, Coimbatore, Synthesis, characterization and biological activity of some new
Tamilnadu for anticancer activity studies. VO(IV), Co(II), Ni(II), Cu(II) and Zn(II) complexes of chromone
based NNO Schiff base derived from 2-aminothiazole. Spectro-
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