Novel mixed ligand complexes of bioactive Schiff base (E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol and 2-aminophenol/2-aminobenzoic acid: Synthesis, spectral characterization, antimicrobial and nuclease studies

Article abstract of DOI:10.1016/j.saa.2013.07.096

A novel bidentate Schiff base ligand has been synthesized using 2,4-dihydroxybenzophenone and aniline. Its mixed ligand complexes of MAB type [M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA = Schiff base and B = 2-aminophenol/2-aminobenzoic acid] have

Full text of DOI:10.1016/j.saa.2013.07.096

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel mixed ligand complexes of bioactive Schiff base
(E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol
and 2-aminophenol/2-aminobenzoic acid: Synthesis,
spectral characterization, antimicrobial and nuclease studies
d
P. Subbaraj a, A. Ramu b, N. Raman c, , J. Dharmaraja
⇑
a Department of Chemistry, Devanga Arts College, Aruppukottai 626 101, Tamil Nadu, India
b Department of Inorganic Chemistry, School of Chemistry, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India
c Research Department of Chemistry, VHNSN College, Virudhunagar 626 001, Tamil Nadu, India
d Department of Chemistry, Sree Sowdambika College of Engineering, Aruppukottai 626 134, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The (E)-4-(phenyl (phenylimino) methyl) benzene-1,3-diol derived Schiff base mixed ligand complexes
act as good DNA binding and DNA cleaving agents. They are good antimicrobial activators.
ꢀ
ꢀ
ꢀ
The synthesized metal complexes are
good intercalators.
The complexes are microcrystalline
nature.
Better antimicrobial active agents
than the free ligand.
ꢀ
ꢀ
Effective DNA cleavage activators.
The Cu(II) complex is an excellent
DNA binder.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 March 2013
Received in revised form 22 July 2013
Accepted 31 July 2013
Available online 8 August 2013
A novel bidentate Schiff base ligand has been synthesized using 2,4-dihydroxybenzophenone and aniline.
Its mixed ligand complexes of MAB type [M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA = Schiff base and
B = 2-aminophenol/2-aminobenzoic acid] have been synthesized and characterized on the basis of spectral
data UV–Vis, IR, 1H NMR, FAB-Mass, EPR, SEM and magnetic studies. All the complexes were soluble in DMF
and DMSO. Elemental analysis and molar conductance values indicate that the complexes are non-electro-
lytes. HA binds with M(II) ions through azomethine and deprotonated phenolic group and B binds through
the primary amine group and deprotonated phenolic/carboxylic groups. Using FAB-Mass the cleavage pat-
tern of the ligand (HA) has been established. All the complexes adopt octahedral geometry around the
metal ions. It has been confirmed with the help of UV–Vis, IR, 1H NMR and FAB-Mass spectral data. DNA
binding activities of the complexes 1d and 2d are studied by UV–Vis spectroscopy and cleavage studies
of Schiff base ligand and its complexes 1d and 2d have been by agarose gel electrophoresis method. In vitro
biological activities of the free ligand (HA) and their metal complexes (1a–1e and 2a–2e) were screened
against few bacteria, Escherichia coli, Staphylococcus saphyphiticus, Staphylococcus aureus, Pseudomonas
aeruginosa and fungi Aspergillus niger, Enterobacter species, Candida albicans by well diffusion technique.
Ó 2013 Elsevier B.V. All rights reserved.
Keywords:
Benzophenone
2-Aminophenol
2-Aminobenzoic acid
Metal complexes
Antimicrobial activity
DNA studies
⇑
Corresponding author. Tel.: +91 92451 65958; fax: +91 4562 281338.
E-mail address: drn_raman@yahoo.co.in (N. Raman).
1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2013.07.096

66
P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
was carried out by the Gouy balance. Electronic spectra of free Schiff
Introduction
base ligand and the metal complexes were recorded in DMSO
medium (1 ꢁ 10ꢂ4 M) using Shimadzu UV-1601 spectrophotometer
at ambident temperature. 1H NMR spectra of the ligand (HA) and its
diamagnetic zinc complexes (1e and 2e) in DMSO-d6 medium were
recorded on a Bruker Advance DRX 300 FT-NMRspectrometer using
TMS as internal standard. Fast atomic bombardment mass spectra
(FAB-MS) of the ligand (HA) and their metal complexes were
recorded in VGZAB HS spectrometer using 3-nitrobenzoylalcohol
matrix. X-band EPR spectra of Cu(II) complexes were recorded in
DMSO at room temperature (300 K) and liquid nitrogen conditions
(77 K) on VARIAN E-112 EPR spectrometer using TCNE as internal
standard. Thermogravimetric (TGA) analysis of the mixed ligand
complexes was recorded on Perkin Elmer (TGS-2 model) thermal
analyzer with heating rate of 10 K/min at N2 atmosphere (flow rate
20 mL/min). Scanning Electron Micrography (SEM) was used for
morphological evaluation.
Design and synthesis of new drugs of fewer side effects with un-
ique properties is vital in medicinal inorganic chemistry [1]. At
present, the bioactive novel Schiff base ligands containing N,O/S
donor sites with transition metal complexes have considerable
attention in the treatment of cancer cells in chemotherapeutic field
[2–5]. Interaction of nucleic acid with metal complexes has been
investigated to design the new types of pharmaceutical architec-
ture, the mechanism involved in the site specific recognition of
DNA and to determine the principles governing the recognition
[6–9]. Mixed ligand complexes containing transition metals have
considerable attention due to their effective DNA binding and
cleavage activities under certain physiological conditions [10–
14]. Hydroxybenzophenone and its derivatives exhibit good
binding and cleavage behavior against DNA [15]. Their metal com-
plexes show broad spectrum of pharmacological and clinical activ-
ities such as antibacterial, antifungal, antitumor and herbicidal
[16–20]. Now-a-days greater attention has been given to the
interaction of nucleic acids with Cu(II) and Ru(II) metal complexes
[21] than the other metal complexes. These view encouraged us to
design and synthesize novel bioactive mixed ligand complexes
using NO type Schiff base ligand [(E)-4-(phenyl(phenylimi-
no)methyl)benzene-1,3-diol; HA] with secondary ligand of 2-ami-
nophenol/2-aminobenzoic acid (B).
In the sequel of our work [22], we have synthesized a novel
bioactive bidendate NO type Schiff base ligand (HA) [(E)-4-(phe-
nyl-phenylimino-methyl)-benzene-1,3-diol; obtained by the
condensation of 2,4-dihydroxybenzophenone with aniline]. More-
over, we synthesized MAB type of mixed ligand complexes using
this Schiff base ligand (HA) as primary ligand and 2-aminophe-
nol/2-aminobenzoic acid(B) as co-ligand (s) with few transition
bivalent M(II) ions [M = Mn, Co, Ni, Cu and Zn]. They are character-
ized by both physico-chemical and various spectral methods. DNA
binding behavior of the metal complexes has been explored by
electronic absorption spectral technique and cleavage are done
by gel electrophoresis method under oxidative condition. Further,
we have accounted for the biocidal activities against few patho-
genic bacterial and fungal strains.
Synthesis of Schiff base ligand (HA) and its mixed ligand complexes
Synthesis of Schiff base ligand [4-(phenyl-phenylimino-methyl)-
benzene-1,3-diol; HA]
Hot ethanolic solution (20 mL) of 2,4-dihydroxybenzophenone
(2.14 g, 10 mmol) was added drop wise into an ethanolic solution
(20 mL) of aniline (0.93 g, 10 mmol). The resultant mixture was re-
fluxed for 6 h under stirring (Scheme 1) (Supplementary data file).
Then it was reduced to 1/3 of its original volume and kept aside at
room temperature for evaporation. The yellow crystalline solid
product separated was recrystallized from the same solvent. Yield:
85%.
Synthesis of complexes (1a–1e)
To 10 mL of ethanolic solution of Schiff base ligand (0.287 g,
1 mmol), 10 mL of ethanolic solution of metal(II) chloride (1 mmol)
was added drop wise. The mixture was stirred continuously for 2 h.
To this solution, 10 mL of hot ethanolic solution of 2-aminophenol
(0.109 g, 1 mmol) was added and refluxed for 4–6 h. The resultant
solution was reduced to one-third of its volume, filtered and evap-
orated to dryness. The obtained solid precipitate was washed with
water and cold ethanol. It was dried in vacuo.
Experimental
Synthesis of complexes (2a–2e)
To 10 mL of ethanolic solution of Schiff base ligand (0.287 g,
1 mmol) was added drop wise to the ethanolic solution (10 mL)
of metal(II) chlorides (1 mmol) and stirred for 2 h. To this solution,
10 mL of hot ethanolic solution of 2-aminobenzoic acid (0.137 g,
1 mmol) was added and refluxed for 4–6 h. The resultant solution
was reduced to one-third of its volume, filtered and evaporated to
dryness. The solid product thus obtained was washed with water
followed by cold ethanol and dried in vacuo.
Materials and methods
2,4-Dihydroxybenzophenone (extra pure) and aniline were pur-
chased from Sigma Aldrich and S.D. Fine, respectively. 2-aminophe-
nol, 2-aminobenzoic acid and metal chlorides were obtained from
Merck products. In this study, all the chemicals have been used as
such without any further purification. All the solvents were purified
according to the literature. Calf Thymus (CT) DNA and pUC-19 DNA
were obtained from Genei, Bangalore. Agrose (Molecular Biology
grade), ethidium bromide (EB) were from Sigma (USA). Tris-
(hydroxymethyl)aminomethane-HCl (Tris–HCl) buffer solution
was prepared using deionized and sonicated triple distilled water
using a quartz water distillation setup. Melting points of all the
mixed ligand complexes were determined in open glass capillaries
and taken as such. C, H and N analyses of the free Schiff base ligand
and its mixed ligand complexes were performed in CHN analyzer
Calrlo Erba 1108, Heraeus. Metal contents were analyzed by the
standardprocedure. Elico conductivity bridge (Model No. CM-180)
was used to measure the molar conductance of the free Schiff base
ligand (HA) and the metal complexes in DMSO (1 ꢁ 10ꢂ3 M).
Vibration spectra were recorded in KBr disc medium in the range
400–4000 cmꢂ1 in Perkin–Elmer 783 series spectrophotometer.
Magnetic susceptibility measurement of the powdered samples
Antimicrobial studies
In vitro biological screening effects of the synthesized free Schiff
base ligand and the mixed ligand complexes were done against the
bacterial strains, Escherichiacoli, Staphylococcussaphyphiticus,
Staphylococcusaureus, Pseudomonas aeruginosa and fungal strains,
Aspergillusniger, Enterobacterspecies, Candida albicans by well diffu-
sion method [23]. The same procedure [24] was followed for the
determination of zone inhibition of the synthesized free ligand
(HA) and the mixed ligand complexes against standard controls.
Nuclease studies
The concentration of CT DNA was determined by UV absorbance
at 260 nm
(e
= 6600 Mꢂ1cmꢂ1). CT DNA free from protein

P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
67
contamination was confirmed from its absorbance values at
Results and discussion
260 nm, 280 nm and the ratio A260/A280 was found to be 1.87
[25]. Stock solutions were kept at 4 °C and used after not more than
four days. To prepare buffer and other solutions redistilled water
free from CO2 have been used.
The bidentate NO type of Schiff base ligand (HA) and its mixed
ligand complexes with 2-aminophenol/2-aminobenzoic acid (B)
were synthesized and characterized by various spectral tech-
niques. The synthesized mixed ligand complexes were found to
be air stable, amorphous nature, moisture free and soluble only
in DMF and DMSO.
Absorption studies
The electronic absorption spectra of mixed ligand complexes
were recorded in the absence and presence of CT-DNA in DMSO
medium. Absorption titrations were done by keeping the complex
concentration (in 10ꢂ5 M) as constant and varying the DNA con-
Elemental analysis and conductivity measurements
The synthesized Schiff base ligand (HA) and the mixed ligand
complexes of MAB type were investigated using various physio-
chemical properties like melting point (m.p.), color, yield, elemen-
tal analysis and conductance values which are given in Table 1. The
elemental analytical data of ligand (HA) and its MAB complexes
agreed well with the calculated values, showing the 1:1:1
(M:A:B) stoichiometry ratio of the complexes (Fig. 1). The low mo-
lar conductance of the complexes indicates their non-electrolytic
nature [29].
centration (0–50 lM). Binding experiments were done in DMSO
solution [26–28] using Tris–HCl/NaCl buffer (5 mM Tris–HCl,
5 mM NaCl, pH = 7.2). Absorption spectra were recorded after suc-
cessive addition of CT-DNA to the complex solution. The intrinsic
binding constant (Kb) was calculated according to the following
equation:
½DNAꢃ
½DNAꢃ
1
¼
þ
ð
ea
ꢂ
ef
Þ
ð
eb
ꢂ
ef
Þ
Kbðeb
ꢂ
ef
Þ
Vibrational spectral studies
where ef
, eb and ea are the molar extinction coefficients of the free
Vibrational spectra of free Schiff base (HA) and 2-aminophenol
(2-AP)/2-aminobenzoic acid (2-ABA) ligands (B) were compared to
investigate the mode of binding present in the synthesized mixed
ligand complexes. The ligand showed strong bands at 1245 and
1598 cmꢂ1 which are assigned to deprotonated phenolic-O and
azomethine-N atoms and these bands were shifted to lower re-
gions in the complexes which indicate that the ligand acts as
bidentate manner in the metal chelates [30]. Free ligand (B) of 2-
complex in solution, complex in the completely bound form with
CT-DNA and complex bound to DNA at a definite concentration
respectively. In the plot of [DNA]/(ea
calculated.
ꢂ
ef) versus [DNA], Kb was
Cleavage studies
pUC-19 DNA at pH 7.5 in Tris–HCl buffered solution was used to
perform the gel electrophoresis technique. Oxidative cleavage of
DNA was examined keeping the concentration of the complexes
aminophenol showed
1265 cmꢂ1 for
(OH) vibrations. Similarly, 2-aminobenzoic acid
(B) showed a band at 3360 cmꢂ1 for (NH2) and 1498 cmꢂ1 for
(COOH) vibrations. On complexation, the phenolic-O band was
a m(NH2) and
band at 3380 cmꢂ1 for
m
30
ume to 16
The resulting solution was incubated at 37 °C for 2 h after the addi-
tion of DNA. 2 L of 25% bromophenol blue was added to quench
lM and 2
l
L of pUC-19 DNA was added and made up the vol-
m
lL using 5 mM Tris–HCl/5 mM NaCl buffer solution.
m
shifted to higher region (1275–1262 cmꢂ1) in all the complexes
and the azomethine-N band was shifted to the lower frequency re-
gion (1582–1560 cmꢂ1). The ligands (B) bind with the M(II) ions in
a bidentate manner through amine-N and deprotonated phenolic-
O or caroxylate-O atoms, respectively. In 1a–1e and 2a–2e com-
plexes, HA and B form stable 6, 5 and 6, 6 membered chelation
l
the reaction. The samples were electrophoresed for 2 h at 50 V in
Tris–acetate–EDTA (TAE) buffer using 1% agarose gel containing
1.0
lg/mL ethidium bromide (EB) and photographed under UV
light.
Table 1
Elemental and physical data of ligand and its complexes.
Complex Empirical
formula
Formula
weight
Color
m.p.
(°C)
Yield
(%)
Elemental analysis Found (Calc.)%
KM
(X
ꢂ1 molꢂ1 cm2)
M
–
C
H
N
HA
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
C
19H17NO2
289
487
491
491
496
497
514
519
519
524
525
Yellow
85
65
58
60
55
62
54
61
69
70
63
78.79
4.64
11.34
–
(79.10)
61.32
(61.51)
60.81
(61.10)
60.54
(60.85)
60.18
(60.45)
60.04
(60.31)
60.36
(60.57)
59.86
(60.12)
59.93
(4.79)
4.73
(4.80)
4.79
(4.92)
4.76
(4.85)
4.65
(4.78)
4.69
(4.76)
4.64
(4.75)
4.53
(4.65)
4.52
(11.25)
5.74 (5.62)
C25H24MnN2O5
C25H24CoN2O5
C25H24NiN2O5
C25H24CuN2O5
C25H24ZnN2O5
C26H24MnN2O6
C26H24CoN2O6
C26H24NiN2O6
C26H24 CuN2O6
C26H24 ZnN2O6
Reddish
brown
Pink
280
10.88
(11.10)
11.12
(11.25)
11.55
(11.72)
12.52
(12.75)
12.86
(13.12)
10.42
(10.66)
11.09
(11.35)
11.16
12.5
14.1
19.3
16.8
20.1
16.4
13.6
18.2
15.9
21.8
286
5.49 (5.58)
5.78 (5.65)
5.51 (5.60)
5.64 (5.53)
5.58 (5.44)
5.29 (5.40)
5.52 (5.35)
5.18 (5.31)
5.05 (5.25)
Pale green
Brown
>280
>280
>280
272
Pale yellow
Brown
Violet
279
Green
284
(11.31)
11.80
(12.12)
12.21
(60.15)
59.34
(59.58)
59.07
(4.66)
4.49
(4.61)
4.44
Dark brown
Yellow
>285
>285
(12.44)
(59.38)
(4.60)

68
P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
Fig. 1. Proposed structure of mixed ligand complexes.
Table 2
Vibration spectral data for free ligand (HA) and mixed ligand complexes in KBr disk (cmꢂ1).
Complex
m
(H2O)
m
(OH)phen
m
(C@N)
m
(NH2)
m
(OH)
m
(COOH)
m
(M-O)
m
(M-N)
2-APa
2-ABAb
HAc
1a
1b
1c
1d
1e
2a
2b
–
–
–
–
–
–
–
3380,946
3360,924
–
1265
–
–
3188,1281
3192,1284
3196,1277
3185,1286
–
1498
–
–
–
–
–
–
1466
1472
1469
1460
1468
–
–
–
–
–
–
1245
1266
1270
1262
1269
1274
1269
1275
1266
1271
1268
1598
1576
1582
1578
1580
1579
1570
1574
1580
1576
1579
3335,810,708
3320,822,714
3340,830,710
3320,840,716
3345,825,715
3330,838,720
3355,825,709
3340,844,712
3335,838,714
3320,850,710
3365,925
3350,918
3361,922
3355,920
3359,916
3344,912
3338,906
3332,904
3342,914
3349,909
421
445
465
449
440
432
451
460
449
464
551
569
573
585
571
559
564
570
563
575
3189,1295
–
–
–
–
–
2c
2d
2e
a
b
c
2-AP = 2-aminophenol.
2-ABA = 2-aminobenzoic acid.
HA = Schiff base ligand.
respectively. Further, the two new bands appeared in the far infra-
red region at 421–465 and 551–585 cm–1 were assigned to
medium using TMS (tetramethylsilane) as internal standard. The
free ligand (HA) showed a group of multiplet signals at 6.4–7.6
due to phenyl moiety. The signals at 11.94 (s, 1H, OH) and
10.75 ppm (s, 1H, OH) are due to phenolic-OH group present in
Schiff base ligand. Disappearance of peak at 10.75 ppm in the com-
plexes (1e and 2e) is due to the deprotonated phenolic-O group in-
volved in the chelation. Downfield shifting of azomethine
(AHC@NA) group in free ligand from 8.45 ppm to 8.27 and
8.22 ppm in the complexes of 1e and 2e respectively indicatesthat
the azomethine group is involved in the complexation. New peaks
appeared between 4.72 and 4.85 ppm in the complexes indicate
the presence of coordinated water molecules.
m
(MAO) and m(MAN) respectively [31]. The broad diffuse band at
3320–3355 cmꢂ1 and few bands appeared at 810–850 cmꢂ1 and
708–720 cmꢂ1 are attributed to the stretching, rocking and wag-
ging mode of m(OH) vibration of the coordinated water (OH2) mol-
ecules present in the complexes [31]. These are presented in
Table 2.
FAB mass spectra
FAB mass spectrum (Fig. S1) (Supplementary data file) of the
isolated Schiff base ligand (HA) showed the fragmentation pattern
along with m/z value observed at 287 [M+1] and the most intense
peaks (m/z) at 215, 185, 138 and 104, corresponding to the frag-
ments of [C13H10NO2], [C13H11N], [C7H5NO2] and [C6H5O2]+ respec-
tively (Fig. S2) (Supplementary data file). This fragmentation
pattern confirmed the proposed structure of the ligand (HA). The
mass spectra of the complexes (1c–1d and 2c–2d) exhibited m/z
peaks at 493, 498, 521 and 524 with [M+2] pattern respectively
and which evidently support the composition of the mixed ligand
complexes. Thus, the FAB spectral results confirm the stoichiome-
try composition presenting in free ligand (HA) and MAB complexes
which reinforce the conclusion drawn from the elemental analysis
data.
Electronic spectra and magnetic moment values
Electronic spectra of HA and the mixed ligand complexes were
recorded (10ꢂ3 M, in DMSO solution) at room temperature.
Absorption regions, band assignments, proposed geometry and
the ligand field parameters are given in Table 3. HA showed three
bands at 40,816, 34,364 and 29,411 cmꢂ1, the first two bands are
attributed to
p ?
p⁄ transition of benzene moieties and the other
band is due to intra molecular charge transfer transition of n ? p⁄
respectively [29,30]. Mn(II) complexes (1a and 2a) exhibited
three weak absorption bands at 24,155–24,245, 20,284–20,452
and 13,805–13,650 cmꢂ1 regions which are assigned to A1g
?
6
4-
T1g(4G) ( 3), 6A1g ? 4T2g(4G)( 2) and 6A1g ? 4A1g 4Eg (4G) (m1
m m , )
1H NMR spectra
transitions respectively [30]. The observed magnetic moment val-
ues of the above complexes (5.44–5.63 BM) are slightly lower
than high spin complexes (6.0 BM) expected for five unpaired
electron with an octahedral environment around Mn(II) ion
Proton NMR spectrum of HA was compared with the diamaga-
netic mixed ligand Zn(II) complexes of 1e and 2e in DMSO-d6

P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
69
Table 3
Electronic absorption spectral data (in DMSO) and magnetic susceptibility of the mixed ligand complexes (1a–1e and 2a–2e) at 310 K.
Complex
kmax (cmꢂ1
)
Band assignments
Geometry
leff (BM)
Ligand field parameter
Dq (cmꢂ1
)
B (cmꢂ1
)
b
b (%)
LFSE (kJ molꢂ1
)
m2/m1
40,816
34,364
29,411
24,155
20,284
13,805
19,685
14,599
9900
34,722
25,794
16,891
10,162
14,528 (b)
26,254
24,245
20,452
13,650
19,157
14,771
9766
p ?
pꢅ
HA
1a
2b
1c
n ? pꢅ
–
–
–
–
–
–
–
–
INCT
6A1g T1g (4G)
4
6A1g
6A1g
?
?
4T2g (4G)
Octahedral
Octahedral
Octahedral
5.44
5.63
3.18
–
–
–
–
–
–
4A1g 4Eg (4G)
4T1g (F) ? 4T1g (P)
4T1g (F) ? 4A2g (F)
4T1g (F) ? 4T2g (F)
1520.3
1016
783 (971 for free ion)
825 (1030 for free ion)
081
0.80
19.34
19.88
145.59
145.97
1.47
1.66
LMCT (n ? pꢅ
)
3A2g (F) ? 3T1g (P)
3A2g (F) ? 3T1g (F)
3A2g (F) ? 3T2g (F)
1d
1e
2Eg
?
2T2g
Octahedral
Octahedral
1.87
Dia.
–
–
–
–
–
–
–
–
–
–
–
Dia.
LMCT (n ? pꢅ
)
6A1g
6A1g
6A1g
?
?
?
4T1g (4G)
2a
2b
4T2g (4G)
Octahedral
Octahedral
5.63
5.75
–
–
–
–
–
–
4A1g 4Eg (4G)
4T1g (F) ? 4T1g (P)
4T1g (F) ? 4A2g (F)
4T1g (F) ? 4T2g (F)
1089.6
701 (971 for free ion)
0.72
27.76
104.34
1.51
2c
34,843
25,840
16,639
10,020
14,311 (b)
26,458
LMCT (n ? pꢅ
)
Octahedral
3.14
1002
828 (1030 for free ion)
0.80
19.62
143.93
1.66
3A2g (F) ? 3T1g (P)
3A2g (F) ? 3T1g (F)
3A2g (F) ? 3T2g (F)
2d
2e
2Eg
?
2T2g
Octahedral
Octahedral
1.84
Dia.
–
–
–
–
–
–
–
–
–
–
–
Dia.
LMCT (n ? pꢅ
)
[32–33]. Complexes 1b and 2b showed three bands in the regions
EPR spectra
9766–9900, 14,599–14,771 and 19,157–19,685 cmꢂ1, which are
4
4
4
assigned to T1g (F) ? 4T2g (F), T1g (F) ? 4A2g (F) and T1g
(F) ? 4T1g (P) respectively. The leff values of Co(II) complexes
1b and 2b (5.63 and 5.75 BM) indicate the presence of four un-
paired electrons in the high spin six-coordinated octahedral Co(II)
complexes [33].
The EPR spectra of the Cu(II) complexes (1d and 2d) were re-
corded at room temperature and liquid nitrogen temperature
(77 K). The g-tensor values were computed from the spectrum
using tetracyanoethylene (TCNE) free radical as ‘g’ marker. From
the observed spectrum, Cu(II) complexes (1d and 2d) follow the
g-tensor values as g|| (2.27 and 2.29) > g\ (2.01 and 2.03) > ge
(2.0027) which indicate that the unpaired electron is localized
The complexes (1c and 2c) showed four absorption bands at
10,020–10,162, 16,639–16,891, 25,840–25,974 and 34,843–
3
3
34,722 cmꢂ1 which may be assigned to A2g (F) ? 3T2g (F), A2g
in dx2
orbital of the Cu(II) ion with 3d9 configuration [36] with
ꢂy2
(F) ? 3T1g (F), A2g (F) ? 3T1g (P) and LMCT transitions respec-
considerable covalent nature [37] present in CuAL. The observed
gavg value of these complexes is equal to 2.06–2.10 and deviated
from ge (2.0027) value due to the covalence property. The g|| value
was well agreed with the other reported N2O2 type complexes
[37]. These values also give evidence for the elemental and
vibrational studies of metal chelating environment. Absence of
3
3
tively with octahedral geometry with A2g as ground state in
D4h symmetry [32,33]. The water molecules occupied on z-axis
of Cartesian coordinate made them hexa coordination around
Ni(II) ion. Absence of any band below 10,000 cmꢂ1 ruled out
the possibility of a tetrahedral. The observed magnetic moment
of Ni(II) complexes were 3.14–3.18 BM which correspond to
two unpaired electrons per Ni(II) ion for six-coordinated octahe-
dral geometry [33]. From the absorption bands, the ligand field
parameters such as B, B0, Dq/B0, 10 Dq and b for Co(II) and Ni(II)
complexes were calculated from Tanaba Saugano diagrams. The
observed Racah parameter (B) value is less than the free ion
(971 cmꢂ1 for Co(II) ion and 1030 cmꢂ1 for Ni(II) ion; b (0.72–
0.81) and b (%) (19.34–27.76) values also support the covalent
character of Co(II) and Ni(II) complexes with distorted octahedral
geometry [34,35]. Complexes of 1d and 2d exhibited only one
broad band centered at 14,528 and 14,311 cmꢂ1 which may be
assigned to the 2Eg ? 2T2g transition in an octahedral geometry
respectively [33]. The observed leff values (1.87 and 1.84 BM)
for complexes 1d and 2d indicate that the unpaired electron lies
any half field signal at 1600 G corresponding to
DMs = 2 transi-
tions, ruling out any magnetic exchange i.e., CuACu interactions
in the complexes. The bonding parameters of a2 and b2 values
were calculated using Kivelson and Neimann formula and the
observed values reflect higher covalence of the LACu bonding
present in the mixed ligand complexes. Hyperfine structure ob-
served corresponds to N2O2 coordination mode in distorted octa-
hedral environment present in Cu(II) complexes. Hathaway [38]
pointed out that for the pure
p
-bonding, K|| > K\ ꢄ 0.77 and for
in-plane -bonding K|| < K\, while for out-of-plane
p
p
-bonding
K|| > K\ the following simplified expressions were used to
calculate K|| and K\:
ꢀ
ꢁ
gjj ꢂ 2:0023
8k0
g? ꢂ 2:0023
K
K
¼
¼
ꢁ d—d transition
jj
predominantly in dx2
ground state with octahedral structure
ꢂy2
ꢀ
ꢁ
[31]. Diamagnetic nature of Zn(II) ion did not show any d–d tran-
sition in the visible region. However, the complexes (1e and 2e)
showed only one broad band at 26,254 and 26,458 cmꢂ1 due to
L ? M charge transfer in a distorted octahedral environment with
sp3d2 hybridization [33].
ꢁ d—d transition
?
2k0
The observed K|| > K\ relation indicates the absence of significant
in-plane -bonding.
p

70
P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
Thermogravimetric analysis
TGA of mixed ligand complexes are used to (i) get information
about the thermal stability, (ii) to decide whether the water mole-
cules (if present) are inside or outside the coordination sphere and
(iii) thermal decomposition of chelates [39]. In this present study,
heating rates were 10 °C minꢂ1 under nitrogen atmosphere. The
Cu(II) complexes (1d and 2d) were fairly stable at room tempera-
ture and by increasing the temperature up to 200 °C, there was a
gradual weight loss up to 5–6% indicating the coordinated water
molecules are present in the complexes. Above 200 °C, there was
a sudden weight loss up to 400 °C which indicates the evaporation
of organic ligand moiety. The decomposition of ligand molecules
continued and leading to formation of air stable metal oxide as
the end product at about 780 °C (Fig. S3) (Supplementary data file).
SEM morphology analysis
The particle size and the surface structure of the mixed ligand
complexes have been shown from the scanning electron micro-
graph (SEM) pictograph. Fig. S4 (Supplementary data file) illus-
trates the SEM photographs of the mixed ligand Ni(II) complexes
(1c and 2c). From the pictographs, the complexes have homoge-
neous matrix with even interface having ideal shape of uniform
phase material. A bundle of irregularly broken straw like shape
was observed for the complex (1c) with the particle size of
Fig. 2. Electronic absorption spectrum of complex (1d) in the absence (-----) and
presence (—) of increasing amount of CT DNA.
and this higher zone of inhibition can be explained on the basis
of Overtone’s concept and Tweedy’s chelation theory [41–42]. All
the mixed ligand complexes showed more considerable antibacte-
rial than antifungal activities. A representative graph for the bio-
logical activity of Schiff base ligand (HA) and the MAB complexes
is shown Fig. S5 (Supplementary data file). Biological activities of
the metal complexes were found to be increased as the stability
of the mixed ligand complexes increased and the activity of free
Schiff base ligand (HA) and the MAB complexes followed the order
which is given below:
ꢆ9
lm. Particle size of the complex (2c) was obtained as 10 lm
with broken stemless mushroom pieces like shape. This leads to
believe that we are dealing with homogeneous phase material.
DNA binding studies
DNA binding interaction of mixed ligand complexes (1d and 2d)
were carried out by using electronic absorption techniques at
37 °C. Absorption spectra of synthesized Cu(II) complex (1d) in
the absence and presence of DNA are shown in Fig. 2. With
[DNA], the absorption intensity of the complexes decreased (hypo-
chromic effect) [40] and the kmax values were shifted to red region
(bathochromic shift). The interaction of metal complexes with DNA
leads to both hypochromic and bathochromic effects which indi-
cate the intercalative binding mode, due to strong intercalation be-
tween the metal complexes and the base pairs of DNA. In this
binding, there was a strong stacking interaction between the plan-
ner aromatic chromophores of the ligand with DNA base pairs In
the UV region, Cu(II) complexes (1d and 2d) exhibited three bands
at ca. 265–268, 310–315 and 360–368 nm under the same experi-
mental conditions. The intrinsic binding constant values for Cu(II)
complexes (1d and 2d) were 4.96 ꢁ 105 Mꢂ1 and 5.24 ꢁ 105 Mꢂ1
respectively. From the intrinsic binding constant (Kb), the free en-
ergy change values for the Cu(II) complexes (1d and 2d) were cal-
culated (ꢂ29.44 and ꢂ31.10 kJ molꢂ1 respectively) and these
results indicate that the mixed ligand complexes can interact with
DNA in a spontaneous manner.
Control > ð2dÞ > ð2cÞ > ð2bÞ > ð2eÞ > ð2aÞ > HA and
Control > ð1dÞ > ð1cÞ > ð1bÞ ꢄ ð1eÞ > ð1aÞ > HA complexes
Oxidative cleavage
The oxidative cleavage activities of free Schiff base ligand (HA)
and its Cu(II) complexes have been studied by gel electrophoresis
and a representative pictograph is shown in Fig. S6 (Supplemen-
tary data file). The results showed that the supercoiled pUC19
DNA in buffer medium (pH = 7.2; Tris–HCl/NaCl) was converted
into open circular form due to the formation of metal chelation.
During the cleavage process, the smallest fragments moved quickly
towards anode than the larger fragments. Bromophenol blue was
used as a photosensitizer that can be activated on irradiation by
UV. The completion of gel electrophoresis experiment clearly indi-
cated that the intensity of the treated DNA samples has diminished
due to the cleavage of DNA. The complex 1d cleaved the DNA mod-
erately than the complex 2d and the Schiff base ligand. These re-
sults indicated that the metal ions played an important role in
the cleavage of DNA.
Biological activity
In vitro microbial activities of free Schiff base ligand (HA) and
MAB mixed ligand complexes (1a–1e and 2a–2e) were tested
against few pathogenic bacterial and fungal strains by well diffu-
sion method using agar as nutrient at different concentrations
Conclusion
In this paper, a novel bidentate Schiff base ligand (HA) was syn-
thesized using 2,4-dihydroxybenzophenone and aniline. Complexes
of MAB type (M = Mn(II), Co(II), Ni(II), Cu(II) and Zn(II); HA = Schiff
base; B = 2-aminophenol/2-aminobenzoic acid) were also synthe-
sized and characterized by the spectral and analytical techniques.
The UV–Vis, IR, 1H NMR, EPR and FAB-Mass data showed that all
the complexes adopt octahedral geometry. XRD and SEM analyses
indicated that the complexes are microcrystalline nature. From
(25, 50 and 75 lg). Commercially available standard drugs Ampi-
cillin and Nystain were used as antibacterial and antifungal
controls respectively and the zone inhibition measurements
against the controls are listed in Table 3. From Table 3, it is ob-
served that all the complexes showed a remarkable biological
activity at higher concentration (75 lg) against microorganisms

P. Subbaraj et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 117 (2014) 65–71
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