Synthesis, characterization, antimicrobial, DNA binding, and oxidative cleavage activities of Cu(II) and Co(II) complexes with 2-(2- hydroxybenzylideneamino)isoindoline-1,3-dione

Article abstract of DOI:10.1080/00958972.2011.554544

(E)-2-(2-hydroxybenzylideneamino)isoindoline-1,3-dione (Hbid) was prepared by condensation of N-aminophthalimide and salicylaldehyde and characterized by elemental analysis, IR, ¹H-NMR, and mass spectral studies. Mononuclear complexes [(phen)Cu

Full text of DOI:10.1080/00958972.2011.554544

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Synthesis, characterization,
antimicrobial, DNA binding, and
oxidative cleavage activities of Cu(II)
and Co(II) complexes with 2-(2-
hydroxybenzylideneamino)isoindoline-1,3-
dione
a
a
L. Shiva Kumar & H.D. Revanasiddappa
a
Department of Chemistry , University of Mysore ,
Manasagangotri, Mysore – 570 006, Karnataka, India
Published online: 06 Feb 2011.
To cite this article: L. Shiva Kumar & H.D. Revanasiddappa (2011) Synthesis, characterization,
antimicrobial, DNA binding, and oxidative cleavage activities of Cu(II) and Co(II) complexes with
2
6
-(2-hydroxybenzylideneamino)isoindoline-1,3-dione, Journal of Coordination Chemistry, 64:4,
99-714, DOI: 10.1080/00958972.2011.554544
To link to this article: http://dx.doi.org/10.1080/00958972.2011.554544
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Journal of Coordination Chemistry
Vol. 64, No. 4, 20 February 2011, 699–714
Synthesis, characterization, antimicrobial, DNA binding, and
oxidative cleavage activities of Cu(II) and Co(II) complexes
with 2-(2-hydroxybenzylideneamino)isoindoline-1,3-dione
L. SHIVA KUMAR and H.D. REVANASIDDAPPA*
Department of Chemistry, University of Mysore, Manasagangotri,
Mysore – 570 006, Karnataka, India
(
Received 26 July 2010; in final form 22 November 2010)
(E)-2-(2-hydroxybenzylideneamino)isoindoline-1,3-dione (Hbid) was prepared by condensation
of N-aminophthalimide and salicylaldehyde and characterized by elemental analysis, IR,
1
II
H-NMR, and mass spectral studies. Mononuclear complexes [(phen)Cu (ꢀ-Hbid)
2
H
2
O] (1),
II
(phen)Co (Cl)
[
2
(ꢀ-Hbid)]6H
2
O (2) (phen ¼ 1,10-phenanthroline) and binuclear complexes
II
2
II
[
2
Cu (ꢀ-Hbid)] (3), and [Co (ꢀ-Hbid)] (4) with Hbid were prepared and characterized by
elemental analysis, IR, UV-Vis, molar conductance, and thermogravimetric (TG) techniques.
DNA-binding properties of 1–4 were investigated by UV spectroscopy, fluorescence spectros-
copy, and viscosity measurements. The results suggest that 1 and 2 bind to DNA by partial
intercalation, whereas 3 and 4 find different groove-binding sites. The cleavage of these
complexes with super coiled pUC19 has been studied using gel electrophoresis; all the
complexes displayed chemical nuclease activity in the absence and presence of H O via an
2 2
oxidative mechanism. Complexes 1–4 inhibit the growth of both Gram-positive and Gram-
negative bacteria.
Keywords: 2-(2-Hydroxybenzylideneamino)isoindoline-1,3-dione; Cu(II)/Co(II) complexes;
DNA binding; Gel electrophoresis; Antimicrobial activity
1. Introduction
The toxicity of cisplatin by forming covalent bond with adjacent purine base of DNA
led to a number of multinuclear complexes bridged by polydentate ligands, which can
interact with DNA in different modes, being prepared as metal-based chemotherapeutic
agents [1–3]. Interaction of transition metal complexes with DNA has long been
intensively investigated for applications in molecular biology, biotechnology, and
medicine [4, 5]. Since DNA is the intracellular target in treating a wide range of
anticancer cells, binding of small molecules with DNA is extremely useful in
understanding drug–DNA interactions. Transition metal complexes capable of cleaving
DNA are important for their use as new structural probes in nucleic acid chemistry
and as therapeutic agents [6, 7]. A large number of metal complexes mediate
*
Corresponding author. Email: hdrevanasiddappa@yahoo.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
DOI: 10.1080/00958972.2011.554544

7
00
L. Shiva Kumar and H.D. Revanasiddappa
DNA oxidation. Bis(1,10-phenanthroline)copper(I) showed efficient DNA cleavage
activity, useful in foot-printing applications and for sequence specific binding to DNA
[
8]. Ternary Cu(II)/Co(II) complexes of 1,10-phenanthroline and Schiff base hydrolyt-
ically cleave DNA and exhibit cytotoxicity [9]. Applications of such complexes focus on
interaction with the fully planar ligand which helps to bind DNA through an
intercalative mode [10]. A Schiff base containing copper(II)/cobalt(II) complexes shows
efficient DNA cleavage by oxidative and hydrolytic pathways [11].
Copper and cobalt are bio-essential elements in all living systems. Different groups
12, 13] have reported Cu(II)/Co(II) complexes showing antifungal and antibacterial
[
properties against several pathogenic fungi and bacteria depending on reaction with the
central DNA system. Compounds containing an amide have ability to form metal
complexes and exhibit a wide range of biological activities [14]. The distinctive DNA-
binding ability [15] of phen has been the basis for selection as another bioactive ligand
for the preparation of ternary complexes. This study reports the synthesis, structure,
DNA binding and cleavage studies, and antimicrobial activities of mono- and binuclear
copper(II) and cobalt(II) complexes with Hbid.
2
2
. Experimental
.1. Chemicals
All reagents were procured from Merck and Sigma Aldrich. The solvents used for
electrochemical and spectroscopic studies were purified by standard procedures [16].
Supercoiled (SC) pUC19 (cesium chloride purified) DNA was purchased from
Bangalore Genei (India). Tris-HCl buffer solution was prepared using deionized,
sonicated triply distilled water.
2.2. Physical measurement
Elemental analyses were carried out on an Elemental Vario EL elemental analyzer.
ꢀ1
DMF solution at room
ꢀ3
Molar conductance data were recorded in 10 mol L
temperature using an Elico CM-180 conductometer. The cell constant of the
ꢀ
1
conductivity cell was 0.5 cm . Electrospray ion mass spectra (ESIMS) were recorded
on an API 2000 Applied Bio-system triple quadruple mass spectrometer. The solution
ꢀ
1
was introduced into the ESI source through a syringe pump at the rate 5 mL min
H-NMR spectra were recorded on a Bruker AV-400 MHz FT NMR spectrometer in
.
1
ꢀ1
DMSO-d . Infrared spectra were recorded from 4000–400 cm on a Jasco FT/IR-4100
6
FT-IR spectrometer as Nujol mulls. Electronic spectra were obtained with a Hitachi
U-2000, Japan, recording spectrophotometer in DMF at a concentration of
ꢀ
3
ꢀ1
1
0
mol L . Magnetic measurements were carried out by the Gouy method at room
ꢂ
temperature (28 ꢁ 2 C) using Hg[Co(SCN) ] as the calibrant. TG analyses and DTA
4
were performed by using Universal V4.3A TA Instruments. Fluorescence spectra were
recorded on a Shimadzu RF-5301pc spectrofluorimeter.

Isoindoline-1,3-dione
701
2
.3. Synthesis of Hbid
Salicylaldehyde (1.221 g, 10 mmol) was dissolved in 20 mL ethanol and added to a
0 mL hot ethanolic solution containing N-aminophthalimide (1.621 g, 10 mmol) with
3
continuous stirring. The mixture was further stirred and refluxed for 5 h and allowed to
stand overnight. The formed yellow crystalline Hbid was collected after evaporation of
solvent at room temperature. The reaction pathway of the preparation of Hbid is given
ꢂ
ꢀ1
in scheme 1. [Hbid]: Yield: (89%); m.p. 192 C; IR ꢁ(Nujol mull, cm ): 1768 and 1725
1
(
C¼O), 1617 (HC¼N), 3386 (Ph–OH). H-NMR (DMSO-d , ppm): 10.67 (s, 1H, Ph–
6
þ
OH), 9.45 (s, 1H, HC¼N), 6.92–7.91 (m, 4H, Ar–H). FAB-MS: m/z ¼ 267 [Mþ1] .
Anal. Calcd for C H N O : C, 67.67; H, 3.79; N, 10.52; O, 18.03. Found: C, 67.05;
1
5
10
2
3
H, 3.81; N, 10.68; O, 17.98.
II
II
.4. Synthesis of [(phen)Cu (l-Hbid)2H O] (1) and [(phen)Co (Cl)
2
2
2
(
l-Hbid)]6H O (2)
2
Copper(II)/cobalt(II) complexes were prepared by following a general procedure. An
ethanolic solution (10 mL) of 1 mmol (0.171 g) quantity of CuCl ꢃ 2H O was reacted
2
2
with 1 mmol (0.1980 g) of the heterocyclic base (phen) in 10 mL MeOH under stirring at
ꢂ
3
(
0 C for 0.5 h. The resulting solution was then reacted with 1.0 mmol (0.266 g) of ligand
Hbid) in 10 mL hot MeOH. The mixture was refluxed with stirring for 6.0 h and
cooled. After slow evaporation at room temperature, a light green solid (1) was
crystallized in ꢄ70% yield. Complex 2 was synthesized by the same method using
(
1 mmol, 0.236 g) CoCl ꢃ 6H O instead of CuCl ꢃ 2H O.
2
2
2
2
ꢂ
ꢀ1
Complex 1: Yield: (67%); m.p. 4 300 C. IR ꢁ(Nujol mull, cm ): 1716 (C¼O); 1650
þ
(
HC¼N); 3460 (H O); 506, 518 (M–N); and 402 (M–O). FAB-MS m/z: 547 [M þ 1] .
2
ꢀ
1
2
cm mol ), 8.4.
ꢀ1
Conductance (Ã,
ꢀ
ꢀ
(BM), 1.86. Anal. Calcd for
eff
[
C, 59.45; H, 4.09; Cu, 11.76; N, 10.32; O, 14.23.
C H CuN O ] (%): C, 59.39; H, 4.06; Cu, 11.64; N, 10.26; O, 14.65. Found:
27 22 4 5
ꢂ
ꢀ1
Complex 2: Yield: (63%); m.p. 4 300 C. IR ꢁ(Nujol mull, cm ): 1715 (C¼O); 1618
(
HC¼N); 3423 (H O); 509, 523 (M–N); 400 (M–O); and 353 (2Cl). FAB-MS m/z: 684
þ ꢀ1 ꢀ1
eff
2
2
[
M þ 1] . Conductance (Ã, ꢀ cm mol , 10.2. ꢀ (BM), 4.84. Anal. Calcd for
[
C, 47.34; H, 4.37; Co, 8.96; N, 8.22; O, 21.45.
CoC H Cl N O ] (%): C, 47.38; H, 4.42; Co, 8.61; N, 8.19; O, 21.04. Found:
2
7
30
2
4
9
II
.5. Synthesis of [Cu (l-Hbid)] (3) and [Cu (l-Hbid)] (4)
II
2
2
2
Methanolic solution (15 mL) of Hbid (0.266 g, 1 mmol) was added to CuCl ꢃ 2H O
2
2
(1 mmol, 0.171 g) dissolved in 10 mL MeOH. This solution was refluxed for 5 h and let
Scheme 1. Proposed reaction scheme for the synthesis of ligand (Hbid).

7
02
L. Shiva Kumar and H.D. Revanasiddappa
stand for a day at room temperature. A light green crystal product was formed for 3.
The product was separated by filtration, purified by washing with cold methanol and
then with ether. Complex 4 was synthesized by the same method using (1 mmol, 0.236 g)
CoCl ꢃ 6H O replacing CuCl ꢃ 2H O.
2
2
2
2
ꢂ
ꢀ1
Complex 3: Yield: (66%); m.p. 4 300 C. IR ꢁ(Nujol mull, cm ): 1720 (C¼O); 1603
þ
(
HC¼N); 510 (M–N); 427 (M–O). FAB-MS m/z: 658 [M þ 1] . Conductance (Ã,
–
1
2
ꢀ1
cm mol ) 6.7; ꢀ (BM) 0.59. Anal. Calcd for [C H Cu N O ] (%): C, 54.80; H,
eff 30 18 2 4 6
ꢀ
2.76; Cu, 19.33; N, 8.52; O, 14.60. Found: C, 54.34; H, 2.71; Co, 19.66; N, 8.12;
O, 14.54.
ꢂ
ꢀ1
Complex 4: Yield (75%); m.p. 4 300 C. IR ꢁ(Nujol mull, cm ): 1715 (C¼O); 1620
þ
–1
2
ꢀ1
)
(
HC¼N); 506 (M–N). FAB-MS m/z: 650 [M þ 2] . Conductance (Ã, ꢀ cm mol
7
N, 8.64; O, 14.81. Found: C, 55.22; H, 2.81; Co, 18.36; N, 8.32; O, 14.45.
.4; ꢀ (BM) 4.12. Anal. Calcd for [C H Co N O ] (%): C, 55.57; H, 2.80; Co, 18.18;
eff 30 18 2 4 6
2
.6. DNA binding and cleavage activity
2
5
.6.1. Electronic absorption titration. All spectroscopic titrations were carried out in
mmol L Tris-HCl buffer (pH 7.1) containing 50 mmol L NaCl at room temper-
ꢀ ꢀ1
1
ature. Before adding CT-DNA to 1–4, their behavior in the buffer solution at room
temperature was monitored by a UV-Vis spectrometer for 24 h. Liberation of the ligand
was not observed under these conditions, suggesting that the complexes are stable under
the studied conditions. A solution of CT-DNA in the buffer gave a ratio of UV
absorbance at 260 and 280 nm of 1.84 : 1, indicating that the DNA was sufficiently free
of protein [17]. The DNA concentration per nucleotide was determined by absorption
ꢀ
1 ꢀ1 ꢀ1
spectroscopy using the molar absorption coefficient (6600 (mol L
ꢂ
)
18]. Stock solutions were stored at 4 C and used within 4 days. Titration experiments
cm ) at 260 nm
[
ꢀ
1
were performed by using 50 mmol L complex while varying the concentration of
ꢀ
1
CT-DNA from 0 to 400 mmol L . While measuring the absorption spectra, equal
quantity of CT-DNA was added to both the complex solution and the reference
solution to eliminate absorbance of CT-DNA itself. The complex–DNA solutions were
allowed to equilibrate for 5 min before spectra were recorded. The intrinsic binding
constant (K ) for interaction of the complexes with CT-DNA was determined from a
b
plot of [DNA]/(" ꢀ " ) versus [DNA] using absorption spectral titration data [19] and
a
f
the following equation:
½
where [DNA] is the concentration of DNA, the apparent absorption coefficients " , " ,
DNAꢅ=ð"a ꢀ " Þ ¼ ½DNAꢅ=ð" ꢀ " Þ þ 1=K ð" ꢀ " Þ,
f
b
f
b
b
f
a
f
and " correspond to Aobsd/[complex], the extinction coefficient for the free metal
b
complex and the extinction coefficient for the metal complex in the fully bound form,
respectively. The K value is given by the ratio of the slope to the intercept.
b
2
.6.2. Fluorescence spectral study. The relative binding of the ternary complexes to
CT-DNA was studied by the fluorescence spectral method using EB bound CT-DNA
solution in Tris-HCl/NaCl buffer (pH 7.2). Emission intensity measurements were
carried out on a Shimadzu RF-5301pc spectrofluorimeter. Tris buffer was used as the

Isoindoline-1,3-dione
703
blank to make preliminary adjustments. For all fluorescence measurements, the
entrance and exit slits were maintained at 10 nm each. Fluorescence intensities at
6
02 nm (478 nm excitation) were measured at different complex concentrations. DNA
ꢂ
was pretreated with EB in the ratio [DNA/EB] ¼ 1 for 30 min at 27 C. The metal
ꢀ1
complexes with increasing concentration (0–50 mmol L ) were then added to the DNA-
EB mixture and their effect on emission intensity measured.
2
.6.3. Viscosity measurements. Viscosity measurements were carried out with an
ꢂ
Ubbelodhe viscometer maintained at 28.0 ꢁ 0.1 C in a thermostated bath. DNA
samples of ca 200-bp average length were prepared by sonication in order to minimize
complexities arising from DNA flexibility [20]. The flow time was measured with a
digital stopwatch, and each sample was tested three times to get an average calculated
1
/3
flow time. Data were presented as (ꢂ/ꢂ0) versus binding ratio [21], where ꢂ is the
viscosity of DNA in the presence of complex, ꢂ0 being the viscosity of free
DNA solution alone. The viscosity values were calculated from the observed flow
time of CT-DNA containing solutions (t) duly corrected for that of the buffer alone
(
t ), ꢂ ¼ (t ꢀ t )/t .
0
0
0
2
.6.4. Cleavage efficiency. DNA cleavage activity of the metal complexes was studied
ꢀ
1
by using agarose gel electrophoresis. SC plasmid pBR19 DNA (50 mmol L ) was
dissolved in a 50 mmol L (Tris-HCl) buffer (pH 7.2) containing 50 mmol L NaCl
and different concentrations of complexes (100, 200, 300, and 400 mmol L ). The
ꢀ1
ꢀ1
ꢀ1
ꢂ
mixtures were incubated at 37 C for 24 h and then mixed with the loading buffer (2 mL)
containing 25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol. Each
sample (5 mL) was loaded into 0.8% w/v agarose gel. Electrophoresis was undertaken
for 1 h at 50 V in Tris-acetate-EDTA (TAE) buffer. The gel was stained with EB for
5
min after electrophoresis and then photographed under UV light. The proportion of
DNA in each fraction was quantitatively estimated from the intensity of each band with
the Alpha Innotech Gel documentation system (AlphaImager 2200). To enhance DNA
ꢀ
1
cleaving ability by the complexes, hydrogen peroxide (100 mmol L ) was added into
ꢀ
1
each complex (400 mmol L ). The cleavage mechanism was further investigated by
using scavengers for the hydroxyl radical (4 mL, DMSO) and the singlet oxygen species
ꢀ1
(
100 mmol L , NaN ). All experiments were carried out in triplicate under the same
3
conditions.
2
.7. Antimicrobial activities
The complexes and ligand were tested for in vitro antibacterial activity against
Escherichia coli, Staphylococcus aureus, Xanthomonas vesicatoria, and Ralstonia
solanacearum strains. Initial screening was performed by using the disc diffusion
method. Suspensions in sterile distilled water from 24 h cultures of microorganisms were
adjusted to 0.5 McFarland. Muller–Hinton Petri dishes of 90 mm were inoculated using
these suspensions. Paper discs (5 mm in diameter) containing 100 mL of the substance to
ꢀ1
be tested (at a concentration of 2 mg mL in DMF) were placed at the edge of the Petri
plates containing Nutrient agar (NA). One 5 mm diameter agar disc of test organism
from a 1-week old NA culture was placed in the center of the plate. Incubation of the

7
04
L. Shiva Kumar and H.D. Revanasiddappa
ꢂ
plates was done at 37 C for 24 h. Reading of the results was done by measuring the
diameters of the inhibition zones generated by the tested substances, using a ruler.
Chloramphenicol was used as a reference substance.
The in vitro antifungal assay was performed by the disc diffusion method. The
complexes and ligand were tested against Aspergillus niger and Aspergillus flavus,
cultured on potato dextrose agar as medium. In a typical procedure, a well was made at
the center on the agar medium inoculated with the fungi. The well was filled with the
ꢂ
test solutions (100 mL) using a micropipette and the plate was incubated at 37 C for
7
2 h. During this period, the test solution diffused and growth of the inoculated fungi
was affected. The inhibition zone developed on the plate was measured. Fluconazole
was used as a reference compound.
The prepared compounds were further used to determine minimum inhibitory
concentration (MIC) in 96 well, sterile, flat bottom microtiter plates, based on broth
micro dilution assay, which is an automated colorimetric method, using the absorbance
(
optical density) of cultures in microtiter plates [22]. Each well of microtiter plates
was filled with 200 mL of nutrient broth/potato dextrose broth, 1 mL of test organism
and 15 mL of different concentration of selected compounds. For bacteria and fungi,
ꢂ
the microtiter plates were incubated at 35 ꢁ 2 C for 24 h. After the incubation period,
the plates were read at 610 nm using an ELISA reader (ELX 800 MS, Biotek
Instruments, Inc., USA). MIC, which was determined as the lowest concentration of
compound inhibiting the growth of the organism, was determined based on the
optical density.
3
3
. Results and discussion
.1. Chemistry
The synthesis, spectroscopic, and analytical data of Hbid and its complexes are
presented in section 2. The elemental analyses are consistent with the composition
suggested for the mixed (1 and 2) and binary (3 and 4) copper(II)/cobalt(II) complexes.
All complexes are soluble in DMSO and DMF. Conductivity was measured in DMF
ꢀ
3
ꢀ1
(
1 ꢆ 10 mol L ). The molar conductivity (Ã) values of the complexes fall in the range
ꢀ ꢀ1
1
2
6.7–10.2 ꢀ cm mol , in agreement with non-electrolytes [23]. The ligand and its
complexes are very stable at room temperature in the solid state.
1
1
3
.1.1. H-NMR and IR spectra. The H-NMR spectra of Hbid show a singlet at
0.67 ppm for OH of salicylidene. A peak at 9.46 ppm was assigned to imine (HC¼N),
1
which is a very sensitive site for coordination to metal ions. A multiplet assigned to
1
aromatic ring is 6.92–7.91 ppm. The H-NMR spectra of the complexes were not
obtained due to paramagnetism.
IR spectrum of the free ligand was compared with spectra of metal complexes. Hbid
ꢀ1
shows prominent absorption peaks at 1768 and 1725 cm corresponding to two (C¼O)
ꢀ
1
groups of N-aminophthalimide. The carbonyl peak at 1768 cm is diminished in all
ꢀ
1
spectra of complexes, while the peak at 1725 cm shifts to lower frequency 1716–
ꢀ1
1
715 cm , indicating coordination of the latter. Hbid has a characteristic (C¼N) band

Isoindoline-1,3-dione
705
ꢀ
1
ꢀ1
at 1617 cm , which shifts in all the complexes (1650–1603 cm ), indicating that the
azomethine nitrogen coordinates. IR spectra of the complexes show new bands at
ꢀ ꢀ1
1
5
The weak band at 352 cm for 2 is probably due to M–Cl bonds [25]. The ligand
06–510 cm and 400–427 cm , due to M–N and M–O bonds, respectively [24].
ꢀ1
ꢀ
1
shows a broad OH band at 3386 cm
due to phenolic O–H. The position and
broadness of the band are indicative of intramolecular hydrogen bonding between the
phenolic proton and the imine nitrogen. This OH band disappeared upon complexation
of the ligand with metal due to loss of the phenolic proton in 3 and 4. Complexes 1 and
ꢀ
1
2
show a broad band at 3460–3423 cm indicating the coordination of water to the
metal ion. Thus, Hbid is tridentate, coordinating through the azomethine nitrogen and
phenolic and carbonyl oxygens to one Cu(II)/Co(II) ion to give ONO coordination
sphere for 3 and 4. In 1 and 2, Hbid is bidentate, coordinating through azomethine N
and carbonyl O.
3
.1.2. Electronic spectra and magnetic moments. The electronic spectra of Hbid and
ꢀ3
ꢀ1
complexes were recorded in DMF (10 mol L ). In spectra of Hbid, two intense bands
at 289 and 334 nm were observed. The first is assigned to a ꢃ ! ꢃ* transition. The
second is assigned to the ꢃ ! ꢃ* transition associated with C¼N and C¼O of Hbid
[
1
4
26, 27]. Intense UV bands assigned to ligand-centered ꢃ ! ꢃ* transitions appeared for
and 3 at 336–305 nm, and for 2 and 4 at 332–300 nm [28]. Another intense band at
28–368 nm for 1 and 3, and 417–352 nm for 2 and 4 is from coincidence of charge
transfer d ! ꢃ* and L ! M transition [29].
2
2
The electronic spectra of 1 show a d–d band at 688 nm assigned to a B ! A
1
g
2g
transition, suggesting a distorted octahedral geometry. The reflectance spectrum of 2
showed a broad band at 656 nm and a shoulder at 485 nm, besides the ligand
4
4
absorptions. The former band would be due to a T ! A electronic transition,
1
g
2g
indicating an octahedral configuration around Co(II). Electronic spectra of 3 contain a
low intensity d–d band at 643 nm, indicating four-coordinated copper. In the visible
region, 4 displayed a broad band at 620 nm corresponding to the d–d transition of
tetrahedral cobalt complex [30].
The effective magnetic moment per metal atom was calculated from the expression,
1
=2
ꢀ
eff ¼ 2:84½ꢄmTꢅ BM,
where ꢄm is the molar susceptibility of the complex after applying the diamagnetic
corrections by use of Pascal’s constants. Hg[Co(SCN) ] was used as a calibrant.
4
Copper(II) complexes exhibit a wide range of geometries, often with low symmetry and
in most geometries electronic spectra exhibit a very broad band, which contains all the
expected transitions. Copper(II) complex 1 shows an effective magnetic moment
ꢀeff 1.86 BM, which is higher than that of the spin only value (1.73 BM) and indicates
the presence of an unpaired electron for mononuclear copper ion [31]. Thus, the
magnetic moment value and spectral data support distorted octahedral geometry for 1.
Copper complex 3 exhibits a very low magnetic moment, 0.59 BM (nearly diamagnetic),
per copper center. Clearly, the single unpaired electron on the coppers couple
antiferromagnetically to produce a low-lying singlet (diamagnetic) [32]. Spectral and
magnetic studies indicate that 3 has a tetragonal distortion (distorted to a square-planar
geometry) [33]. The cobalt complex 2 with magnetic moment value 4.84 BM is
consistent with the presence of a high-spin octahedral Co(II), while cobalt complex 4 at

7
06
L. Shiva Kumar and H.D. Revanasiddappa
4
.12 BM suggests a spin quartet state S ¼ 3/2 in a tetrahedral geometry [30]; this may be
attributed to dimerization of the cobalt complex [34]. The expected structures for the
complexes are presented in figure 1.
3.1.3. FAB mass and thermal studies. The analyses of FAB mass spectra, without any
ambiguity, conclude the mononuclear nature for 1 and 2, binuclear for 3 and 4,
paralleling the results of elemental analyses. The FAB mass spectra of Hbid showed
þ
molecular ion peak at m/z 267[M þ 1] (43%). Complex 1 shows two molecular ion
þ
þ
peaks at m/z 545[M] (11%) and 547[M þ 2] (5%) corresponding to copper isotopes,
6
3
65
þ
þ
þ
i.e., Cu and Cu, respectively. Fragment of 2 shows 684[M] (6%), 686[M þ 2]
þ
þ
(
(
2%), 688[M þ 4] (1%) indicating the presence of two Cl’s, whereas 3 showed 658[M]
þ
5%), 660[M þ 2] (3%), 662[M þ 4] (1%), due to the presence of copper in its two
þ
isotopic forms. For 4, the molecular ion peak appeared at 650 [M þ 1 þ H] (12%).
O
OH
O
OH
N
N
Cu
N
N
N
H O
2
Cl
O
O
6H O
2
Co
N
N
N
OH2
Cl
1
2
O
O
N
N
N
N
O
O
Cu
Co
O
O
Co
C u
O
O
O
O
N
N
N
N
O
O
3
4
II
Figure 1. Structures of [(phen)Cu (ꢀ-Hbid)2H
Hbid)} ] (3), and [{Co (ꢀ-Hbid)} ] (4) from spectral and analytical techniques.
2 2
II
O] (1), [(phen)Co (Cl)
II
O (2), [{Cu (ꢀ-
2
2
(ꢀ-Hbid)]6H
2
II

Isoindoline-1,3-dione
707
The results reveal different interactions between copper(II)/cobalt(II) ion, (Hbid) and
1,10-phenanthroline.
Room temperature stabilities for 1 and 4 were revealed by their TG analysis, carried
ꢂ
out from ambient temperature to 700 C in nitrogen. Cu(II) complex 1 is thermally
ꢂ
stable to 132 C, undergoing decomposition beyond this temperature, as indicated by
ꢂ
the first mass loss in the TG curve (Supplementary material). The mass loss from 134 C
ꢂ
to 196 C corresponds to elimination of two coordinated H O (Calcd/found: 6.59%/
2
ꢂ
6
.95%). Further decomposition starts at 200 C corresponding to Hbid and phen
ꢂ
(Calcd/found: 80.68%/78.97%), which ends at 316 C. After decomposition, the mass
loss at 507–610 C corresponds to the formation of CuO (Calcd/found: 14.5%/15.06%).
ꢂ
The cobalt(II) complex 4 shows an initial weight loss of 78.2% (Calcd 81.45%) at
ꢂ
203–328 C, attributed to loss of organic moiety. The decomposition pattern rules out
the possibility of lattice held water molecules (Supplementary material). The next
ꢂ
ꢂ
decomposition step occurs from 420 C to 510 C corresponding to the formation of
CoO (Calcd/found: 11.52%/10.2%). These observations further support the composi-
tion of Cu(II) and Co(II) complexes.
3
3
.2. Biological activities
.2.1. Electronic absorption titration. Electronic absorption spectroscopy is the most
useful technique for DNA-binding studies of metal complexes [35]. A complex binding
to DNA through intercalation usually results in hypochromism and bathochromism,
due to intercalation involving a strong ꢃ–ꢃ* stacking interaction between an aromatic
chromophore and the base pairs of DNA. The extent of hypochromism in the UV band
is consistent with the strength of intercalative interaction [36, 37].
UV-Vis titrations of 1–4 with CT-DNA have been studied in Tris buffer from 800 to
00 nm. With increasing concentration of DNA, there is no appreciable change in the
2
position of the charge transfer band of 1 (ꢄ5 nm) and 2 (ꢄ3 nm) at 263 and 267 nm,
respectively, but the intensity of the band decreases for 1 (17.6%) and 2 (12.4%).
Absorption spectra of the copper complex in the absence and presence of CT-DNA are
shown in figure 2. This suggests that the ꢃ* orbital of the intercalated phen can couple
with the ꢃ orbital of DNA base pairs and that the coordinated phen partially inserts
between the base pairs of DNA. The results suggest an association of the compound
with DNA, and it is likely that 1 and 2 significantly bind to CT-DNA helix by
intercalation [38]. The K values obtained from the absorption spectral technique are
b
5
4
ꢀ1 ꢀ1
1
.4 ꢆ 10 and 4.02 ꢆ 10 (mol L
) for 1 and 2, respectively.
The binary complexes 3 and 4, which lack any DNA binding phen, show only minor
hypochromism (3 (2.2%); 4 (1.4%)) upon increasing DNA concentration and a minor
shift of the bands (ꢄ2 nm) at 304 and 324 nm occurs, indicating non-intercalation of the
complexes, with primarily groove-binding [39, 40]. The K values obtained from the
b
2
2
ꢀ1 ꢀ1
) for 3 and 4,
respectively. However, the observed K values are much lower than those observed for
absorption spectral technique are 2.12 ꢆ 10 and 1.52 ꢆ 10 (mol L
b
6
ꢀ1 ꢀ1
typical classical intercalators (EB, K , 1.4 ꢆ 10 (mol L
) [41]). Complexes 3 and 4
b
show moderate binding to CT-DNA, while 1 and 2 are better DNA binders than
some well-established intercalation agents [42, 43], due to the presence of additional
planar phen.

7
08
L. Shiva Kumar and H.D. Revanasiddappa
Figure 2. Absorption spectra of complex 1 in Tris-HCl buffer upon addition of increasing concentration of
ꢀ "
ꢀ
1
ꢀ1
CT-DNA [0–400 mmol L ]. [Complex] ¼ 50 mmol L . The inner plot is of ("
titration of DNA with complex 1.
a
f
)/("
b
ꢀ "
f
) vs. [DNA] for the
3.2.2. Viscosity measurements. Interactions between the complexes and DNA were
also investigated by viscosity measurements. Optical photophysical probes provided
necessary, but not sufficient clues to support a binding model. Hydrodynamic
measurements that are sensitive to length change (i.e., viscosity and sedimentation)
are the least ambiguous and the most critical tests of binding mode in solution in the
absence of crystallographic structural data [44]. A classical intercalation model
lengthens the DNA helix, as base pairs are separated to accommodate the binding
ligand leading to increasing DNA viscosity. In contrast, a partial, non-classical
intercalation of ligand could bend (or kink) the DNA helix, reduce its effective length
and, concomitantly, its viscosity [45]. As seen in figure 3, the viscosity of CT-DNA
increases with increase in the ratio of 1 and 2 to CT-DNA, resembling the binding mode
of EB to CT-DNA. For 3 and 4, the relative viscosity of the DNA solution was
unchanged due to electrostatic forces or groove-binding [46]. The results parallel the
spectroscopic results.
3
.2.3. Fluorescence spectral studies. EB was non-emissive in Tris-buffer medium due
to fluorescence quenching by solvent [47]. In the presence of CT-DNA, it shows
enhanced emission intensity due to intercalative binding to DNA. A competitive
binding of the copper(II)/cobalt(II) complexes to CT-DNA results in the reduction of
the emission intensity due to the displacement of bound EB and/or quenching of the
fluorescence of EB by metal complexes. The quenching of the emission spectra of EB
bound to DNA by 1 is shown in figure 4.
According to the classical Stern–Volmer equation [48], I /I ¼ 1 þ K [Q], I and I
0
SV
0
are the fluorescence intensities in the absence and presence of the quencher,
respectively. KSV is a linear Stern–Volmer quenching constant, [Q] is the concentration
of the quencher. The quenching plot illustrates that the quenching of EB bound to

Isoindoline-1,3-dione
709
Figure 3. Effect of increasing the concentration of the complexes 1 (g), 2 (
ꢂ
ꢇ
), 3 (*), 4 (m), and EB (^) on
the relative viscosities of CT-DNA at 28.0 ꢁ 0.1 C in 5 mmol L Tris-HCl buffer (pH 7.2).
ꢀ1
Figure 4. Emission spectrum of EB bound to DNA in the presence of increasing complex 1 concentration
(
[EB] ¼ 10 mmol Lꢀ , [DNA] ¼ 10 mmol L , [complex] ¼ 0–50 mmol L , ꢅex ¼ 478 nm).
1
ꢀ1
ꢀ1
DNA by complex is in agreement with the linear Stern–Volmer equation, indicating
that the complex binds to CT-DNA. The Stern–Volmer constant KSV used to evaluate
the quenching efficiency is obtained as the slope of the linear I /I versus complex
0
4
ꢀ1 ꢀ1
concentration plot. From figure 5, the K value for 1 and 2 is 6.5 ꢆ 10 (mol L
SV
) and
) , respectively, due to the complex interaction with DNA through
4
ꢀ1 ꢀ1
3
.85 ꢆ 10 (mol L
intercalation, thus releasing some free EB from the EB-DNA complex, which is
consistent with the above absorption spectral result.

7
10
L. Shiva Kumar and H.D. Revanasiddappa
Figure 5. Plot of I
0
/I vs. [complex], drawn by using the classical Stern–Volmer equation I
0
/I ¼ 1 þ KSV [Q],
for the titration of the complexes (^) 1, (g) 2, (m) 3, and ( ) 4 with the CT-DNA-EB system.
ꢇ
Complexes 3 and 4, which are shown by viscosity measurements not to be involved in
intercalative binding, also stops the enhancement in emission of DNA-bound EB very
3
ꢀ1 ꢀ1
3
ꢀ1 ꢀ1
faintly. The KSV values for 3 and 4 are 2.8 ꢆ 10 (mol L
)
and 2.35 ꢆ 10 (mol L
) ,
respectively. This shows that 3 and 4 are unable to accommodate an intercalating
molecule like EB but are suitable for groove-binding of the complex facilitated by the
hydrophobic interaction of the aromatic carbonyl groups with DNA [49].
3
.2.4. DNA cleavage activity. DNA cleavage activity is controlled by relaxation of SC
circular conformation of pUC19 DNA to nicked circular and/or linear conformation.
When electrophoresis is applied to circular plasmid DNA, fastest migration will be
observed for DNA of closed circular conformations (Form I). If one strand is cleaved,
the SC will relax to produce a slower moving nicked conformation (Form II). If both
strands are cleaved, a linear conformation (Form III) will be generated that migrates in
between [50, 51].
Incubating plasmid pUC19 DNA with complex concentration of 100, 200, 300, and
ꢀ
1
4
00 mmol L under physiological conditions produced the outcome shown in figure 6.
Both copper(II) and cobalt(II) complexes behave as chemical nucleases by nicking the
DNA Form I into Form III. Increasing concentrations of complexes led to a gradual
diminish in band intensity of Form I, while that of Form III progressively increased.
Many copper(II)/cobalt(II) complexes cleave DNA more efficiently in the presence of
exogenous agents, such as hydrogen peroxide, which act as a reducing agent [52]. On
ꢀ1
incubation of 1–4 (400 mmol L ) with pUC19 DNA in the presence of hydrogen
peroxide (figure 7a and b, lanes 4 and 8), the amount of Form III obtained was
considerably greater than in the presence of the complexes alone. This indicates that
hydrogen peroxide aids the complexes in DNA degradation by oxidative cleavage.
To gain further insights of the DNA cleavage mechanism, hydroxyl radical scavengers
(DMSO) and a singlet oxygen scavenger (NaN ) were added into the samples under the
same conditions (figure 7a and b). The experiments reveal that hydroxyl radical
3
inhibitor prevents DNA degradation by 1 and 2, but not completely. The presence of
NaN efficiently inhibits DNA scission. Complexes 1 and 2 may cut the DNA strand at
3
two positions. Complexes 3 and 4 affect the cleavage more by the dominant singlet
oxygen species, and almost negligible activity by hydroxyl radical scavenger, consistent
with groove-binding. Thus, in this study, both hydroxyl radical and the dominant

Isoindoline-1,3-dione
711
ꢀ
1
ꢀ1
Figure 6. Cleavage of SC pUC19 DNA (30 mmol L ) by different concentrations of 1–4 (100–400 mmol L
in 10 mmol L Tris-HCl/1 mmol L EDTA buffer (pH 8.0): (a) lane 1, DNA Marker; lane 2, DNA control;
)
ꢀ1
ꢀ1
ꢀ
1
ꢀ1
lanes 3–6, DNA þ 1 (100–400 mmol L ); and lanes 7–10, DNA þ 2 (100–400 mmol L ) and (b) lane 1, DNA
ꢀ1
Marker; lane 2, DNA control; lanes 3–6, DNA þ 3 (100–400 mmol L ); and lanes 7–10, DNA þ 4 (100–
ꢀ
1
4
00 mmol L ).
ꢀ
1
ꢀ1
2 2
Figure 7. Cleavage of SC pUC19 DNA (30 mmol L ) by 1–4 (400 mmol L ) in the presence of H O
(
ꢀ1
ꢀ1
ꢀ1
100 mmol L ) in 10 mmol L Tris-HCl/1 mmol L EDTA buffer (pH 8.0). Strong inhibitions of DNA
cleavage were observed in the presence of the hydroxyl radical scavenger DMSO and singlet oxygen scavenger
NaN
: (a) lane 1, DNA Marker; lane 2, DNA control; lane 3 and 7, DNA þ complex 1 and DNA þ 2,
respectively; lane respectively; lanes and 9,
3
4
and 8, DNA þ 1 þ H
2
O
2
and DNA þ 2 þ H
2
O
2
,
5
DNA þ 1 þ H
2
O
O
2
2
þ DMSO (4 mL) and DNA þ 2 þ H
2
O
2
þ DMSO (4 mL), respectively; and lanes 6 and 10,
ꢀ1
ꢀ1
DNA þ 1 þ H
2
þ NaN
3
(100 mmol L ) and DNA þ 2 þ H
2
O
2
þ NaN
3
(100 mmol L ), respectively and (b)
lane 1, DNA Marker; lane 2, DNA control; lanes 3 and 7, DNA þ 3 and DNA þ 4, respectively; lanes 4 and 8,
DNA þ 3 þ H
DNA þ 4 þ H
2
O
O
2
2
and DNA þ 4 þ H
2
O
2
, respectively; lanes 5 and 9, DNA þ 3 þ H
2
O
2
þ DMSO (4 mL) and
ꢀ
3
1
2
þ DMSO (4 mL), respectively; and lanes 6 and 10, DNA þ 3 þ H
2
O
2
þ NaN
(100 mmol L )
ꢀ1
and DNA þ 4 þ H
2
O
2
þ NaN
3
(100 mmol L ), respectively.
singlet oxygen species are involved in oxidative cleavage. Possible reaction mechanisms
of DNA damage by copper(II)/cobalt(II) complexes in the presence of hydrogen
peroxide have been proposed [53].
The cleavage efficiency of 1 and 2 is slightly greater than that of 3 and 4, from the
presence of aromatic diimine ligand providing intercalation with CT-DNA. An
additional binding site like groove-binding is provided by carbonyl containing Hbid,
which is supported by viscosity experiment, thus resulting in a stronger interaction [54].
These results correspond to their DNA binding capabilities from K values.
b
3
.2.5. Antimicrobial studies. The antibacterial studies inferred that Hbid has moderate
activity against X. vesicatoria and R. solanacearum, but low activity against S. aureus
and E. coli. All the Cu(II) and Co(II) complexes show more activity against all the
bacterial strains (table 1) than the free ligand. For antifungal activity, Hbid and its
Cu(II) and Co(II) complexes were active with metal complexes more active than Hbid.
This higher antimicrobial activity of the metal complexes compared to Hbid may be due

7
12
L. Shiva Kumar and H.D. Revanasiddappa
Table 1. Antimicrobial results of Hbid and its metal complexes.
Inhibition against bacteria (%)
Inhibition against fungi (%)
Compounds
S. aureus X. vesicatoria R. solanacearum E. coli
A. niger
A. flavus
Hbid
1
2
3
4
13
34
30
28
25
100
–
22
39
33
30
30
100
–
26
48
46
33
32
100
–
15
31
28
26
22
100
–
24
53
50
43
38
–
28
58
53
45
36
–
Chloramphenicol
Fluconazole
100
100
Results obtained are the average of three replicates.
ꢀ1
Table 2. MICs of the synthesized compounds (in mg mL ).
Compound
S. aureus
X. vesicatoria
R. solanacearum
E. coli
A. niger
A. flavus
Hbid
1
2
3
4
4100
45
50
100
4100
20
4100
38
47
4100
35
42
4100
64
67
4100
4100
20
100
37
42
52
70
–
100
33
40
49
65
–
77
85
20
60
66
20
Chloramphenicol
Fluconazole
–
–
–
–
20
20
Results obtained are the average of three replicates.
to chelating making metal complexes more powerful and potent bacteriostatic agents,
inhibiting growth of the microorganisms [55].
The MICs of the synthesized compounds against bacteria and fungi were determined.
These compounds were active in inhibiting growth of the tested organisms starting from
ꢀ
1
3
5 mg mL (table 2). Complexes 1 and 2 have broad spectrum with mild to moderate
activity toward most strains. These complexes can further be explored as specific
antimicrobial drugs due to their activities against all experimental strains.
4
. Conclusion
This study demonstrates new complexes of Hbid with Cu(II) and Co(II) chlorides.
Elemental analyses, spectral studies and molar conductance studies reveal that the
complexes are both mono and binuclear and are non-electrolytes. Hbid is monobasic,
tridentate ‘‘ONO’’ donor in 3 and 4, whereas in 1 and 2 it is bidentate ‘‘ON’’ donor.
Intercalation of phen between diagonal nucleobases on opposite DNA strands or
adjacent bases on the same DNA strand fix the orientation of the chelated Hbid
for interaction with adjacent nucleobases. Besides this DNA-binding selectivity, the
mixed ligand complexes cleave DNA efficiently. The complexes exhibit biological
activities.

Isoindoline-1,3-dione
713
Acknowledgments
One of the authors, HDR, is grateful to the University of Mysore for providing minor
research project. The other author, LSK, is grateful to the UGC, New Delhi, for the
award of Rajiv Gandhi National Fellowship. They thank the Head of the Department
of Biotechnology for providing help in carrying out antimicrobial and nuclease
activities.
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