DNA cleavage and antimicrobial studies of 17-membered schiff base macrocyclic triazoles: Synthesis and spectroscopic approach

Article abstract of DOI:10.1007/s10847-010-9794-4

A novel series of 17-membered complexes [MLCl₂] (M = Co²⁺, Ni²⁺ and Cu²⁺) have been synthesized with newly derived biologically active ligands (L^I-L^IV). These ligands were synthesized by the condensation of 3-subtituted-4-amino-5-hydrazino-1,2,4-triazole with bis(phthalaldehyde)ethylenediamine precursor. The structure of the complexes has been proposed by elemental analyses, IR, EPR, electronic spectral studies, conductivity, magnetic, thermal and electrochemical studies. All the complexes are soluble in DMF and DMSO and are non-electrolytes. All these Schiff bases and their complexes have been screened for their antibacterial (Escherichia coli, Staphylococus aureus, Salmonella typhi, Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus and Cladosporium) by the Agar and Potato dextrose agar diffusion method. The DNA cleavage study was done by Agarose gel electrophoresis technique.

Full text of DOI:10.1007/s10847-010-9794-4

J Incl Phenom Macrocycl Chem (2010) 68:347–358
DOI 10.1007/s10847-010-9794-4
ORIGINAL ARTICLE
DNA cleavage and antimicrobial studies of 17-membered schiff
base macrocyclic triazoles: synthesis and spectroscopic approach
Udaykumar V. Kamble
Prema S. Badami
•
Sangamesh A. Patil
•
Received: 28 November 2009 / Accepted: 27 April 2010 / Published online: 11 May 2010
Ó Springer Science+Business Media B.V. 2010
Abstract A novel series of 17-membered complexes
[
FAB
Fast atom bombardment
2
MLCl ] (M = Co , Ni and Cu ) have been synthesized
?
2?
2?
TG/DTG Thermogravimetric/differential
thermogravimetric analysis
2
I
IV
with newly derived biologically active ligands (L –L ). These
ligands were synthesized by the condensation of 3-subtituted-4-
amino-5-hydrazino-1,2,4-triazole with bis(phthalaldehyde)-
ethylenediamine precursor. The structure of the complexes has
been proposed by elemental analyses, IR, EPR, electronic
spectral studies, conductivity, magnetic, thermal and electro-
chemical studies. All the complexes are soluble in DMF and
DMSO and are non-electrolytes. All these Schiff bases andtheir
complexes have been screened for their antibacterial (Esche-
richia coli, Staphylococus aureus, Salmonella typhi, Pseudo-
monas aeruginosa) and antifungal activities (Aspergillus niger,
Aspergillus flavus and Cladosporium) by the Agar and Potato
dextrose agar diffusion method. The DNA cleavage study was
done by Agarose gel electrophoresis technique.
DMF
DMSO
IR
N,N dimethyl formamide
Dimethylsulphoxide
Infra red
MIC
NMR
MRI
TMS
Minimum inhibitory concentration
Nuclear magnetic resonance
Magnetic resonance imaging
Tetramethylsilane
Introduction
The field of macrocyclic chemistry of metals is developing
very fast because of its variety of applications and impor-
tance in the area of coordination chemistry [1, 2]. The
rational design and construction of inorganic and organo-
metallic metallomacrocycles by transition metal directed
multicomponent self assembly has major impact on
supramolecular chemistry [3, 4]. The incorporation of
metal centres into supramolecular system gives rise to
novel electronic and/or magnetic properties as well as
fascinating structural features. The importance of macro-
cyclic ligands and their complexes is obvious when seen in
relationship to natural products such as metalloprotein,
vitamin B12 and chlorophyll [5]. A number of nitrogen
donor macrocyclic derivatives have long been used in
analytical, industrial and medical applications [6]. Macro-
cyclic metal complexes are of great importance due to their
resemblances to many natural systems such as porphyrins
and cobalamines. Macrocyclic nickel complexes find use in
DNA recognition and oxidation while the macrocyclic
Keywords Phthalaldehyde ꢀ Triazole ꢀ Ethylenediamine ꢀ
Macrocycle ꢀ Biologically active
Abbreviations used
BM
Bohr Magneton
EPR
Electron paramagnetic resonance
Electronic supplementary material The online version of this
article (doi:10.1007/s10847-010-9794-4) contains supplementary
material, which is available to authorized users.
U. V. Kamble ꢀ S. A. Patil (&)
P.G. Department of Chemistry, Karnatak University, Dharwad
580003, Karnataka, India
e-mail: patil1956@rediffmail.com
P. S. Badami
Department of Chemistry, Shri Sharanabasaveshwar College
of Science, Gulbarga 585 102, Karnataka, India
123

3
48
J Incl Phenom Macrocycl Chem (2010) 68:347–358
copper complexes find use in DNA binding and cleavage
7, 8]. Macrocyclic metal complexes of the lanthanides e.g.
were recorded on a JEOL SX 102/DA-600 mass spec-
trometer/data system using argon/xenon (6 kV, 10 Am) as
the FAB gas. The accelerating voltage was 10 kV and the
spectra were recorded at room temperature by using m-
nitro benzyl alcohol as matrix. The mass spectrometer was
operated in the positive ion mode. The EPR spectrum of
the copper(II) complex in polycrystalline state was recor-
ded using VARIAN E-4 X-band EPR spectrometer with
cylindrical quartz sample tube operating at microwave
frequency *9.1 GHz. Field calibration was checked using
tetracynoethylene(TCNE) free radical for which g =
2.00277 at room temperature. Thermogravimetric analyses
were measured from room temperature to 1000 °C at
heating rate of 10 °C/min. The data were obtained by using
a PERKIN-ELMER DIAMOND TG/DTG instrument.
Molar conductivity measurements were recorded on a
ELICO-CM-82T conductivity bridge with cell having cell
constant 0.51 and magnetic moment was carried out on
Faraday balance.
[
?
3
Gd etc. are used as MRI contrast agents [9–11]. Several
macrocyclic complexes with tetraaza macrocyclic ligand,
such as cyclen, cyclam or bicyclam were reported to
exhibit antitumour activity [12]. The chemistry of macro-
cyclic complexes is also important due to their use as dyes
and pigments as well as NMR shift reagents [13]. Macro-
cyclic complexes are also well known for their antibacterial
and antifungal activities [14, 15]. 1,2,4-Triazole display
biological activity such as inhibition of cholinesterase,
interference with mitosis and reversible denaturation of
serum proteins [16] and its derivatives have become very
useful compounds in medicine, agriculture and in many
fields of technology [17]. Chemistry of 1,2,4-triazole
derivatives have attracted widespread attention due to their
diverse biological activities and they are a new class of
antimicrobial agents viz. Fluconazole and Itraconazole are
used as antimicrobial drugs. The mercapto derivatives of
triazoles have been proved to be effective pesticides and
insecticides. 3-Amino-1,2,4-triazole under the trade name
Amizol as a commercial herbicide and 4n-butyl-4H-1,2,4-
triazole as a systemic protective fungicide against leaf rust
for both spring and winter wheat. Recently, several com-
plexes of various transition and inner transition metals with
substituted 1, 2, 4-triazole ligands have been reported from
our laboratory [18, 19]. There is growing interest in the
studies on the metal complexes of macrocyclic Schiff
bases derived from substituted triazoles which are well
known bactericides, pesticides, insecticides and potential
fungicides [20]. However, paucity of literature on the
metal complexes of macrocyclic ligands derived from
bis(phthalaldehyde)ethylenediamine and 3-subtituted-4-
amino-5-hydrazino-1, 2, 4-triazole encourage us to syn-
thesize the divalent cobalt, nickel and copper macrocyclic
complexes.
Synthesis
All the chemicals used for preparing triazoles and their
Schiff bases were of reagent grade. The 3-substituted-4-
amino-5-mercapto-1, 2, 4-triazoles were prepared as
reported [21, 22].
Synthesis of Bis(phthalaldehyde)ethylenediamine
A mixture of phthalaldehyde and ethylenediamine in 2:1 M
proportions in an ethanolicmedium (40 mL each) containing
few drops of concentrated HCl was refluxed for 3–4 h. The
product formed was filtered, washed with ethanol and
recrystalized form EtOH.
Brown; Yield, 71%, M.P.192 °C, Selected IR Data (KBr
-
1
m cm ): 3206 m (NH), 1712 m (C = O), 1634 m (C = N),
1
1
443, 1091, 748 m(Ring vibrations). H-NMR-(300 MHz
DMSO-d ): d (ppm) = 3.26 (s, 4H, CH –CH ), 7.24 (m,
6
2
2
Experimental
9H, Ar–H), 8.20 (s, 2H, –CH = N), 13.1 (s, 1H, –NH),
?
.98 (s, 2H –CHO). MS: m/z (M ) = 292. Elemental
9
Physical measurements
analyses: Found. C, 73.97%; H, 5.47%; N, 9.58%; Calcd.
C, 74.91%; H, 5.41%; N, 9.51%.
Carbon, hydrogen and nitrogen were estimated by using
Carlo Erba EA1108 Elemental analyzer. Chloride was
determined by Volhard’s method. The IR spectra of the
Schiff bases and their complexes were recorded on HIT-
Synthesis of 3-substituted-4-amino-5-hydrazino-1, 2,
4-triazole
-
1
ACHI-270 IR spectrometer in the 4000–250 cm region
in KBr discs. The electronic spectra of the complexes were
recorded in DMF on VARIAN CARY 50-BIO UV-spec-
trophotometer in the region of 200–1100 nm. The proton
A mixture of 3-subtituted-4-amino-5-mercapto-1, 2, 4 tri-
azole and N H .H O in 1:1 M proportion in an ethanolic
2
4
2
medium (40 mL each) was boiled under reflux for 4–5 h.
on water bath. The reaction mixture was cooled at room
temperature; within 1 h the compound separated from the
clear solution. It was filtered, washed and recrystalized
from EtOH.
NMR spectra of the ligands were recorded in DMSO-d on
6
a BRUKER 300 MHz spectrometer at room temperature
using TMS as an internal reference. FAB mass spectra
1
23

J Incl Phenom Macrocycl Chem (2010) 68:347–358
349
For Compound C H N Colorless Solid; Yield, 78%,
2
6 6,
-
1
M.P. 98 °C, Selected IR Data (KBr m cm ): 3209 m (NH),
288 m asym. (NH ), 3107 m sym. (NH ), 890 m (N–N) of
3
2
2
N
N
N
1
the hydrazone residue. H-NMR-(300 MHz DMSO-d ): d
6
(
ppm) = 6.40 (s, 1H,), 4.95 (br, 2H, NH of NHNH ), 5.55
2 2
?
s, 2H, NH ), 8.81 (br, 1H, –NH). MS: m/z (M ) = 114.
Elemental analyses: Found. C, 21.05%; H, 5.26%; N,
(
2
N
7
3.68%. Calcd. C, 20.96%; H, 5.19%; N, 73.59%.
HN
N
R
For Compound C H N Colorless Solid; Yield, 77%,
3
8 6,
-
1
M.P.132 °C, Selected IR Data (KBr m cm ): 3207 m (NH),
291 m asym. (NH ), 3108 m sym. (NH ), 892 m (N–N) of
N
N
3
2
2
1
the hydrazone residue. H-NMR-(300 MHz DMSO-d ): d
R = H, CH C H and C H
3, 2 5 3 7
6
(
ppm) = 2.51 (s, 3H, CH ), 4.96 (br, 2H, NH of NHNH ),
3 2 2
Fig. 1 Synthesized macrocyclic Schiff bases
5
.57 (s, 2H, NH ), 8.83 (br, 1H, –NH). MS: m/z
2
?
M ) = 128. Elemental analyses: Found. C, 28.12%; H,
(
6
6
.25%; N, 65.62%. Calcd. C, 28.06%; H, 6.19%; N,
5.61%.
64.78%; H, 4.78%; N, 30.22%. Calcd. C, 64.86%; H,
4.86%; N, 30.27%;
II
For Compound L , (C H N ), Cream; Yield, 69%,
For Compound C H N Colorless Solid; Yield, 80%,
4
10 6,
21 20 8
-
1
-1
M.P. 201 °C, Selected IR Data (KBr m cm ): 3212 m (NH),
M.P.162 °C, Selected IR Data (KBr m cm ): 3209 m (NH),
368 m asym. (NH ), 3155 m sym. (NH ), 891 m (N–N) of
3
2924 m (C–H), 1630 m (C = N), 1440, 1095, 745 m(Ring
2
2
1
1
the hydrazone residue. H-NMR-(300 MHz DMSO-d ): d
vibrations). H-NMR-(300 MHz DMSO-d ): d (ppm) =
6
6
(
ppm) = 1.42 (t, 3H, CH ), 2.96 (q, 2H, CH ), 4.98 (br,
3
3.38 (s, 4H, CH –CH ), 7.58 (m, 8H, Ar–H), 8.30 (s, 1H,
2 3
2
2
H, NH of NHNH ), 5.81 (s, 2H, NH ), 8.92 (br, 1H,
2
–CH = N), 13.4 (s, 1H, –NH), D O exchangeable. MS: m/z
2
2
2
?
NH). MS: m/z (M ) = 142. Elemental analyses: Found.
C, 33.80%; H, 7.04%; N, 59.15%. Calcd. C, 33.62%; H,
?
(M ) = 384. Elemental analyses: Found. C, 65.48%; H,
–
5.18%; N, 29.11%. Calcd. C, 65.62%; H, 5.20%; N,
7
.01%; N, 59.11%.
29.16%.
III
For Compound C H N Colorless Solid; Yield, 79%,
12 6,
For Compound L , (C22H N ), Cream; Yield, 73%,
22 8
-1
5
-
1
M.P.172 °C, Selected IR Data (KBr m cm ): 3210 m (NH),
376 m assym. (NH ), 3168 m sym. (NH ), 894 m (N–N) of
the hydrazone residue. MS: m/z (M ) = 156. H-NMR-
M.P. 206 °C, Selected IR Data (KBr m cm ): 3211 m (NH),
3
2926 m (C–H), 1635 m (C = N), 1400, 1130, 755 m(Ring
2
2
?
1
1
vibrations). H-NMR-(300 MHz DMSO-d
): d (ppm) =
–CH ), 3.34 (s,
4H, –CH –CH ), 7.61 (m, 8H, Ar–H), 8.31 (s, 4H,
6
(
300 MHz DMSO-d ): d (ppm) = 1.01(t, 3H, CH ), 1.65–
1.31 (t, 3H, CH
2
–CH
3
), 2.42 (q, 2H, CH
2
3
6
3
1
.90 (m, 2H, ß-CH ), 2.97 (t, 2H, a-CH ), 4.95 (br, 2H,
2 2
2
2
NH of NHNH ), 5.62 (s, 2H, NH ), 8.89 (br, 1H, –NH).
2
–CH = N), 13.60 (s, 1H, –NH, D O exchangeable). MS:
2
2
2
?
m/z (M ) = 398. Elemental analyses: Found. C, 66.36%;
H, 5.48%; N, 28.01%. Calcd. C, 66.33%; H, 5.52%; N,
Elemental analyses: Found. C, 38.46%; H, 7.69%; N,
3.84%. Calcd. C, 38.41%; H, 7.61%; N, 53.81%.
5
2
8.14%;
For Compound L , (C23
12 °C, Selected IR Data (KBr m cm ): 3210 m (NH),
I IV
Synthesis of Schiff bases (L –L )
IV
H N ), Yellow, 75%, M.P.
24 8
-
1
2
A mixture of bis(phthalaldehyde)ethylenediamine and
3
2928 m (C–H), 1633 m (C = N), 1460, 1100, 770 m (Ring
vibrations). H-NMR-(300 MHz DMSO-d ): d (ppm) =
6
1
-subtituted-4-amino-5-hydrazino-1, 2, 4-triazole in 1:1 M
proportion in an alcoholic medium containing few drops of
concentrated HCl was refluxed for 3–4 h. The mixture was
cooled to room temperature and the solvent removed under
reduced pressure until solid formed that was washed with
cold ethanol and dried under vacuum. The synthesized
Schiff bases have shown in Fig. 1.
1.01 (t, 3H, –CH –CH –CH ), 1.72 (m, 2H, –CH –CH –
2
2
2
3
2
CH
CH
), 2.62 (t, 2H, –CH
–CH
–CH
), 3.37 (s, 4H, –CH
–
3
2
2
2
3
2
), 7.80 (m, 8H, Ar–H), 8.40 (s, 1H, –CH = N), 13.5(s,
?
1H, –NH, D O exchangeable). MS: m/z (M ) = 412.
2
Elemental analyses: Found. C, 66.48%; H, 5.96%; N,
27.02%. Calcd. C, 66.99%; H, 5.82%; N, 27.02%.
I
For Compound L , (C H N ), Pale yellow; Yield,
2
0 18 8
-
1
7
1%, M.P.198 °C, Selected IR Data (KBr m cm ): 3210 m
Synthesis of the complexes (1–16)
(
NH), 2940 m (C–H), 1630 m (C = N), 1420, 1080, 760
m(Ring vibrations). H-NMR-(300 MHz DMSO-d ): d
1
I
IV
Co(II), Ni(II) and Cu(II) complexes of the ligands (L –L )
were prepared by adapting template method owing to
the insolubility of the ligands in common organic solvents.
To a stirred solution of bis(phthalaldehyde)ethylenediamine
6
(
ppm) = 3.29 (s, 4H, CH –CH ), 7.26 (m, 9H, Ar–H), 8.10
2 2
(
s, 2H, –CH = N), 13.4 (s, 1H, –NH), D O exchangeable.
2
?
MS: m/z (M ) = 370. Elemental analyses: Found. C,
123

3
50
J Incl Phenom Macrocycl Chem (2010) 68:347–358
(
0.01 mol), and respective metal chloride (0.01 mol) in meth-
which may reasonably be assigned to m(M–Cl) [26]. The
two moderately strong bonds appearing at 890 and
anol(40 mL each) was added drop wise methanolic solution of
-
1
3
-subtituted-4-amino-5-hydrazino-1, 2, 4-triazole (0.01 mol
0 mL). After the addition was completed the mixture was
720 cm can be attributed respectively to m(N–N) of the
hydrazone residue and inplane deformation of the triazole
ring [27, 28].
4
refluxed for 4–5 h. The separated complexes were collected by
filtration, washed with hot methanol and dried under vacuum
over CaCl2.
1
13
H and C NMR spectra
1
Analyses
The H NMR spectral data of the macrocyclic Schiff bases
give some important information to conclude to the for-
mation of macrocyclic ligands. The disappearance of the
primary amino proton signal and the appearance of a sin-
glet observed in the region 3.29–3.37 ppm in the ligands
may be assigned to methylene protons adjacent to nitrogen
indicated that the proposed macrocyclic skeleton has been
formed [29]. This fact was also supported by the disap-
pearance of peaks around 9.98 ppm corresponding to
The metal contents were estimated gravimetrically by the
standard method [23]. Carbon, hydrogen, nitrogen were
estimated using a C, H, N analyzer. The results of ele-
mental analyses and molar conductance values are listed in
Table S1 (Supplementary information).
1
Result and discussion
aldehydic protons [30]. The H NMR spectra of all Schiff
bases exhibited signals in the range 13.6 and 13.4 ppm due
All the Co(II), Ni(II) and Cu(II) complexes are colored,
stable in air and non-hygroscopic solids. They are soluble
in DMF and DMSO. The elemental analyses show that, the
Co(II), Ni(II) and Cu(II) complexes have 1:1 stoichiometry
to NH protons these protons are D O exchangeable and
2
confirming the assignment [31]. Multiplets observed in the
range of 7.2–7.8 ppm have been assigned to the aromatic
protons (Table 1).
2
?
2?
2?
13
of the type [MLCl ] (M = Co , Ni and Cu ). The
2
The C NMR spectra of the ligands exhibited signals in
-
3
molar conductance values at the 10 M concentrations are
too low to account for any dissociation of the complexes in
DMF. Hence, the Co(II), Ni(II) and Cu(II) complexes may
be regarded as non-electrolytes.
the range 152.22–154.65 ppm indicating the presence of
carbon which is doubly bonded to nitrogen [32]. The ligand
which is having methylenic carbon adjacent to nitrogen
atom contains signal in the range of 45.3–59.4 ppm indi-
cate the presence of N-CH linkage [33]. The aryl carbons
2
Infrared spectra
are resonated in the range of 131.8–140.1 ppm.
Infrared spectra frequencies of Schiff bases and their
Co(II), Ni(II) and Cu(II) complexes are presented in Table
S2 and S3 respectively (Supplementary information). The
free ligands showed a medium intensity band in the 3212–
Electronic absorption spectra and magnetic studies
The electronic spectra of the macrocyclic Co(II) complexes
showed two bands in the region 14260–14400 and 21150–
-
1
-1
4
3
210 cm region assigned to m NH vibrations [24], which
21500 cm , which may be reasonably assigned to the T
1g
-
1
4
4
4
has been observed in the 3210–3207 cm region in the
case of complexes. It can be observed that, there is no
considerable shift in the m NH vibrations in case of com-
plexes compared to the ligands, indicates non-involvement
of NH group in the coordination. The ligands coordinate to
the metal ions through the nitrogen of C = N group is
supported by the presence of the mC = N band in the 1605–
(F) ? A (F) and T1g (F) ? T (P) transitions,
2
g
1g
1 I_ IV
Table 1 H NMR spectroscopic data of the of Schiff bases (L L )
Schiff
base
NMR data
C H N 3.29(s, 4H, –CH –CH ), 7.26(m, 9H, Ar–H), 8.10(s, 4H, –
18
2
0
8
2
2
-
1
1
595 cm range in the case of macrocyclic Co(II), Ni(II)
CH = N), 13.4(s, 1H, –NH, D
2
O exchangeable).
and Cu(II) complexes. However, these bands appeared in
C
21
C
22
C
23
H
H
H
20
N
N
N
8
2.12 (s, 3H, CH ), 3.33(s, 4H, –CH
3
2
–CH ), 7.58(m, 8H,
2
-
the 1635–1630 cm range in the IR spectra of the mac-
1
Ar–H), 8.30(s, 1H, –CH = N) 13.4(s, 1H, –NH, D O
2
exchangeable).
rocyclic ligands [25]. The presence of a medium-intensity
-
1
22
24
8
8
1.31(t, 3H, CH
CH –CH ), 7.61(m, 8H, Ar–H), 8.31(s, 4H, –
CH = N), 13.60(s, 1H, –NH, D O exchangeable).
1.01(t, 3H, –CH –CH –CH ), 1.72(m, 2H, –CH –CH
), 2.62(t, 2H, –CH –CH –CH ), 3.37(s, 4H, –CH
), 7.80(m, 8H, Ar–H), 8.40(s, 1H, –CH = N),
O exchangeable).
2 3 2 3
–CH ), 2.42(q, 2H, CH –CH ), 3.34(s, 4H,
band at 440–410 cm region corresponding to the m (M–
N) vibration further confirms the formation of macrocyclic
complex. All the complexes show bands in the 1460–1400,
–
2
2
2
2
2
3
2
2
–
-
1
1
130–1080 and 760–740 cm regions assigned to phenyl
CH
CH
3
2
2
2
3
2
–
ring vibrations. The coordination of the chloro group has
-
been ascertained by band in the 310–280 cm region,
1
13.5(s, 1H, –NH, D
2
1
23

J Incl Phenom Macrocycl Chem (2010) 68:347–358
351
respectively, suggesting an octahedral geometry around the
cobalt (II) ion [34, 35]. However, the third band expected
exchange interaction is negligible, but if G \ 4 consider-
able interaction occurs in the complexes [36]. The calcu-
lated G value is larger than four suggesting that there is no
interaction between the copper centers.
-
1
around 8000 cm could not be properly resolved. The
magnetic moment values of 4.57 and 4.56 BM further
support the electronic spectral observations. The ligand
0
field parameters Dq, B , m /m b, b% have been calculated
Thermogravimetric analyses
2
1,
and presented in Table 2. The electronic spectra of the
macrocyclic nickel(II) complexes exhibited two bands in
their electronic spectra in the regions 11,400–11,600 and
TG and DTG studies were carried out for some of the
complexes. These complexes decompose gradually with
the formation of respective metal oxide above 500 °C. All
the metal complexes showed only a single decomposition
curve between 309 and 345 °C corresponding to the loss of
organic moiety. Above 500 °C, organic moieties in these
macrocyclic compounds were decomposed leading to the
formation of metal oxide.
-
1
3
1
8,100–18,300 cm , which may be ascribed to A2g
3
3
3
(
F) ? T (F) and A2g (F) ? T (P) transitions,
1
g
1g
respectively, are consistent with an octahedral geometry
around nickel(II)ion [35]. Further confirmation regarding
the octahedral environment around the nickel(II) ion has
been obtained from magnetic moment values of 3.15 and
3
.18 BM.
II;
FAB mass spectra of Schiff base L and its Ni(II)
The electronic spectra of the macrocyclic Copper (II)
complexes showed band in the 16,250–16,400 and 20,300–
complex (2)
-
0,500 cm , regions which may be ascribed to B
1
2
2
?
1
g
2
2
2
II
The FAB mass spectrum of Schiff base (L ) showed a
E and B ? B transitions, respectively, correspond-
g
1g
2g
ing to distorted octahedral geometry around Cu(II)ions.
The magnetic moment values in the range 1.78–1.82 fur-
ther support the electronic spectral data.
molecular ion peak at m/z 384 which is equivalent to its
molecular weight. The fragments in the spectrum leading
?
to the formation of the species [C H N ] . The FAB
2
1 20 8
mass spectrum of Ni(II) complex (2) exhibited molecular
?
ion peaks [M] at m/z 513 [M] , 515[M ? 2] and
?
?
EPR spectrum of copper (II) complex (12)
?
17[M ? 4] which are equivalent to its molecular weight
5
The EPR spectrum of polycrystalline macrocyclic Cu(II)
of the Ni(II) complex (2). Some other peaks appeared at m/
?
z 238, 340 and 436 corresponds to the [Ni(C H N )Cl] ,
(12) complex studied here recorded at 25 °C did not show
any hyperfine splitting, it exhibited only single signal. The
9
10 2
?
?
[Ni(C H N )Cl] and [Ni(C H N )Cl] species which
16 14 3 19 16 7
analyses of the spectrum gives g = 2.178, g\ = 2.040,
are resulted from the parent compound (Fig. 2). The two
peaks appeared at m/z 443 and 478 due to loss of two
chlorine atoms and one chlorine atom respectively. All
these fragmentation patterns are well observed in the FAB
mass spectra.
||
2
2
which support that d - may be the ground state. The
x
y
observed g value for the complex is less than 2.3 which is
|
|
in agreement with the covalent character of the metal–
ligand bond [36]. The g (2.178) [ g\ (2.040) observed
||
was found to be in accordance with the criterion of Ki-
Fab mass spectral results of complexes: Co(C H
0 18
2
?
N )Cl : m/z (M ) = 498, m/z [M ? 2] = 500, m/z
?
velson and Neiman, implying the presence of unpaired
8
2
2
2
?
?
electron is localized in dx - y orbital for the Cu(II) ion
characteristic of the axial symmetry. Tetragonally elon-
gated structure is thus confirmed for Cu(II) complex. The
parameter G was calculated by using the expression i.e.
G = (g - 2)/(g\ - 2). According to Hathaway, the G
[M ? 4] = 502. Co(C H N )Cl : m/z (M ) = 513, m/z
21 20 8 2
?
[M ? 2] = 515, m/z [M ? 4] = 517. Co(C H N )
?
2
2 22 8
?
?
Cl : m/z (M ) = 527, m/z [M ? 2] = 529, m/z [M ?
2
?
4] = 531. Co(C H N )Cl : m/z (M ) = 541, m/z
?
2
3
24
8
2
?
?
[M ? 2] = 543, m/z [M ? 4] = 545. Ni(C H N )Cl
2
|
|
20 18
8
?
: m/z (M ) = 498, m/z [M ? 2] = 500, m/z [M ? 4] =
?
?
value measures the exchange interaction between metal
?
502. Ni(C H N )Cl : m/z (M ) = 513, m/z [M ?
centers in polycrystalline solids. In case G [ 4, the
2
1
20
8
2
I
Table 2 Ligand field parameters of Co(II) (1–4) complexes of the Schiff bases(L –L
IV
)
-
1
)
0
-1
)
Complex no.
Transitions
Dq (cm
B (cm
2
m /m
1
LFSE
b
b%
m
1
m
2
(Cacd.)
m
3
1
2
3
4
14260
14310
14352
14400
29623
29728
29817
29929
21150
21230
21350
21500
1536.37
1541.82
1546.77
1552.94
532.91
535.21
541.18
548.63
2.077
2.077
2.077
2.078
35.11
35.24
35.35
35.49
0.5511
0.5534
0.5596
0.5673
44.889
44.651
44.035
44.264
123

3
52
J Incl Phenom Macrocycl Chem (2010) 68:347–358
Fig. 2 FAB-mass spectrum of Ni (II) (6) complex
?
?
2
] = 515, m/z [M ? 4] = 517. Ni(C H N )Cl : m/z
22 22 8 2
?
?
?
M ) = 527, m/z [M ? 2] = 529, m/z [M ? 4] = 531.
(
?
Ni(C H N )Cl : m/z (M ) = 541, m/z [M ? 2] = 543,
?
2
3
24
8
2
?
m/z [M ? 4] = 545. Cu(C H N )Cl : m/z (M ) =
?
2
0
18
8
2
?
?
5
03, m/z [M ? 2] = 505, m/z [M ? 4] = 507. Cu(C
21
?
?
H N )Cl : m/z (M ) = 531, m/z [M ? 2] = 533, m/z
2
0
8
2
?
M ? 4] = 535. Cu(C H N )Cl :: m/z (M ) = 545, m/z
?
[
22 22 8 2
?
M ? 2] = 547, m/z [M ? 4] = 549.
?
[
Electrochemical studies
Electrochemical properties of the complexes were studied on
a CHI1110A-Electrochemical analyzer in N,N-dimethyl-
formamide(DMF) containing 0.05 M n-Bu NClO as the
4
4
supporting electrolyte. A cyclic voltammogram of Co(II) (3)
displays a reduction peak at Epc = -1.0400 V with corre-
sponding oxidation peak at Epa = -0.5922 V (Fig. 3). The
Fig. 3 Cyclic voltamogram of Co (II) (3) complex
peak separation (DE ) of this couple is 0.44 V at scan rate
p
0
.1 V and increases with scan rate. The most significant
feature of the Co(II) complex is the Co(II)/Co(I) couple
which is a quasi-reversible one electron oxidation. The ratio
of cathodic to anodic peak height was less than one; however,
the peak current increases with increase of the square root of
the scan rate, establishing a diffusion controlled electrode
process. The separation in peak potentials increases at higher
scan rates consistent with quasi-reversibility of the Co(II)/
Co(I) couple. The Cu(II) (9) complex exhibits a reduction
peak at Epc = 0.5824 with direct re-oxidation peak at
Epa = 0.3120 V corresponding to the Cu(II)/Cu(I) couple
(
Fig. 4). The peak separation (DE ) is 0.270 V. This Cu(II)
p
complex is also quasi-reversible as the separation in peak
potential is higher than 59 mV and the peak current rise with
Fig. 4 Cyclic voltamogram of Cu (II) (9) complex
1
23

J Incl Phenom Macrocycl Chem (2010) 68:347–358
353
increasing square root of the scan rate [37]. The difference
between forward and backward peak potentials can provide a
rough evaluation of the degree of the reversibility of one
electron transfer reaction. The analyses of cyclic voltam-
metric responses with the scan rate varying 50–250 mV/s
gives the evidence for quasi-reversible one electron
oxidation.
order to clarify any effect of solvent DMF on the biological
screening, separate studies were carried out with solvent
DMF only and it showed no activity against any microbial
strains.
Minimum inhibitory concentration (MIC)
Some compounds showing promising antibacterial/anti-
fungal activities were selected for minimum inhibitory
concentration studies [40, 41].
Biological activities
In vitro antibacterial and antifungal assay
Antimicrobial results
The synthesized Schiff bases and their corresponding
Co(II), Ni(II) and Cu(II) complexes were screened for their
biological activities by using four bacteria, namely E. coli,
S. aureus, S. typhi and P. aeruginosa and three fungi
namely A. niger, A. flavus and Cladosporium by the
reported method [38, 39]. The bacteria were subcultured in
agar medium. The Petri dishes were incubated for 24 h at
The microbial results were systematized in Tables 3 and 4.
The antibacterial and antifungal studies suggested that, all
the Schiff bases were found to be biologically active and
their metal(II) complexes showed significantly enhanced
antibacterial and antifungal activities. It is, however,
known [42, 43] that, chelation tends to make the Schiff
bases act as more powerful and potent bactereostatic
agents, thus inhibiting the growth of bacteria and fungi
more than the parent Schiff bases. It is suspected that,
factors such as solubility, conductivity, dipole moment and
cell permeability mechanism (influenced by the presence of
metal ions) may be the possible reasons for the increase in
activity. In case of bacteriological studies, the results were
compared with the standard drug (Gentamycine). It was
observed that, some of the Schiff bases were found
3
7 °C. The standard antibacterial drug (Gentamycine) was
also screened under similar conditions for comparison. The
fungi were subcultured in potato dextrose agar medium.
Standard antifungal drug (Fluconazole) was used for
comparison. The Petri dishes were incubated for 48 h at
3
7 °C. The wells were dug in the agar media using sterile
metallic borer. Activity was determined by measuring the
diameter of the zone showing complete inhibition (mm).
Growth inhibition was compared with standard drugs. In
I
Table 3 Antibacterial and antifungal results of Schiff bases (L –L
IV
)
Schiff
bases
Conc.
(lg mL
Anti bacterial activity (zone of inhibition in %)
Antifungal activity (zone of inhibition in %)
-1
)
E. coli
S. aureus
S.typhi
P.aeruginosa
A. flavus
Cladosporium
A. niger
I
L
100
50
49
50
58
58
57
61
58
59
81
80
81
6
45
44
44
59
59
58
57
57
56
60
59
60
6
48
49
50
43
42
43
57
56
57
46
46
45
6
67
66
66
60
59
59
69
70
71
63
62
61
6
85
84
83
88
87
86
87
86
88
74
75
74
6
74
73
72
71
69
70
51
50
51
72
72
71
6
75
74
73
78
76
77
84
71
74
86
88
88
6
5
3
0
0
II
L
100
5
3
0
0
III
L
100
5
3
0
0
IV
L
100
5
3
0
0
DMF
100
5
3
0
0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Standard
100
99
100
98
99
99
99
99
100
100
99
100
99
98
100
99
98
98
99
100
99
100
5
3
0
0
123

3
54
J Incl Phenom Macrocycl Chem (2010) 68:347–358
Table 4 Anti bacterial and anti fungal results of Co(II), Ni(II) and Cu(II) complexes (1–12) and standard
Complex
no.
Conc.
(lg mL
Anti bacterial activity (zone of inhibition in %)
Antifungal activity (zone of inhibition in %)
-
1
)
E. coli
S. Aureus
S.typhi
P.aeruginosa
A. flavus
Cladosporium
A. niger
1
2
3
4
5
6
7
8
9
100
51
54
54
68
67
66
70
69
69
84
85
85
81
80
80
69
69
68
71
71
70
81
81
81
54
54
54
69
69
68
68
68
68
80
80
80
6
54
55
54
60
61
61
62
60
61
68
68
69
56
60
61
62
64
59
64
64
64
73
71
72
60
59
62
64
64
64
65
65
65
71
72
70
6
62
63
62
50
51
50
68
69
70
50
50
53
69
67
68
58
58
58
68
69
70
61
64
61
65
65
65
54
54
56
70
70
71
64
63
65
6
70
71
70
64
63
62
73
74
74
69
67
68
80
80
81
65
65
66
74
74
73
82
84
82
72
69
70
66
68
68
74
75
75
81
83
81
6
86
84
85
86
88
87
89
88
88
74
75
78
86
87
85
89
88
88
89
90
91
89
89
89
88
88
88
90
90
91
89
90
90
90
89
88
6
79
80
80
71
73
74
59
61
61
85
84
85
78
79
80
80
81
81
58
57
57
82
80
81
81
82
83
83
82
81
58
62
59
83
81
82
6
79
78
78
79
80
81
79
78
80
98
99
97
80
80
81
84
82
81
84
81
82
89
88
88
82
82
79
80
81
80
81
83
84
91
89
90
6
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
1
1
1
0
1
2
100
5
3
0
0
100
5
3
0
0
100
5
3
0
0
DMF
100
5
3
0
0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Standard
100
99
100
98
99
99
99
99
100
100
99
100
99
98
100
99
98
98
99
100
99
100
5
3
0
0
potentially active against all bacterial strains. Compound
III
also screened against the same bacterial strains. It was
evident that, overall potency of the uncoordinated com-
pounds was enhanced on coordination with metal ions. In
case of antifungal activity, the results were compared with
(
L ) shows high activity against all bacterial strains
especially with P.aeruginosa and E. coli, where as meta-
I IV
l(II) complexes (1–12) of these Schiff bases (L –L ) were
1
23

J Incl Phenom Macrocycl Chem (2010) 68:347–358
355
the standard drug (Fluconazole). All Schiff bases were
I
show high activity against fungal species. Compound (L )
Potato dextrose broth (potato, 250; dextrose, 20, (g/L)) was
used for the culture of A. niger. The 50 mL media was
prepared and autoclaved for 15 min at 121 °C under 15 lb
pressure. The autoclaved media was inoculated with the
seed culture E. coli was incubated for 24 h and A. niger for
48 h at 37 °C.
IV
and (L ) show very high activity, an interesting feature is
IV
that, the compound (L ) shows high activity against
A. niger, however, the Co(II), Ni(II) and Cu(II) complexes
(
1–12) of these Schiff bases showed much enhanced
activity as compared to the uncoordinated compounds. It
was evident from the data that, this activity significantly
increased on coordination. This enhancement in the activ-
ity may be rationalized on the basis of the presence of
C = N bond. It has been suggested that, chelation/coordi-
nation reduces the polarity of the metal ion mainly because
of partial sharing of its positive charge with a donor group
within the whole chelate ring system [44, 45]. This process
of chelation thus increases the lipophilic nature of the
central metal atom, which in turn, favors its permeation
through the lipoid layer of the membrane thus causing the
metal complex to cross the bacterial membrane more
effectively so increasing the activity of the complexes [46].
It has also been observed that some moieties such as the
azomethine linkage or heteroaromatic nucleus introduced
into such compounds exhibited extensive biological activ-
ities that may be a result of the increase in hydrophobic
character and liposolubility of the molecules in crossing the
cell membrane of the microorganism and enhance the
biological utilization ratio and activity of complexes [47].
The minimum inhibitory concentration 10 lg/mL was
Isolation of DNA
The fresh bacterial culture (1.5 mL) is centrifuged to
obtain the pellet, which was then dissolved in 0.5 mL of
lysis buffer (100 mM tris pH 8.0, 50 mM EDTA, 10%
SDS). To this 0.5 mL of saturated phenol was added and
incubated at 55 °C for 10 min. Then, it was centrifuged at
10,000 rpm for 10 min and equal volume of chloroform :
isoamyl alcohol (24:1) and 1/20th volume of 3 M sodium
acetate (pH 4.8) was added to this supernatant and centri-
fuged at 10,000 rpm for 10 min. To this supernatant three
volumes of chilled absolute alcohol was added. The pre-
cipitated DNA was separated by centrifugation. Dried the
pellet and dissolved in TE buffer (10 mM tris pH 8.0,
1
mM EDTA) and stored in cold condition.
Agarose gel electrophoresis
Cleavage products were analyzed by agarose gel electro-
phoresis method. Test samples (1 mg/mL) were prepared in
DMF. The samples (25 lg) were added to the isolated DNA
of E. coli and A. niger. The samples were incubated for 2 h at
37 °C and then 20 lL of DNA sample (mixed with bromo-
phenol blue dye at 1:1 ratio) was loaded carefully into the
electrophoresis chamber wells along with standard DNA
marker containing TAE buffer (4.84 g Tris base, pH 8.0,
0.5 M EDTA/1 L) and finally loaded on agarose gel and
passed the constant 50 V of electricity for around 30 min.
Removed the gel and stained with 10.0 lg/mL ethidium
bromide for 10–15 min and the bands observed under UV
transilluminator and photographed to determine the extent of
DNA cleavage and the results were compared with standard
DNA marker.
IV
shown by compound L against E. coli and A. niger and
compound 8 against A. flavus and P.aeruginosa compound
5
shown MIC 10 lg/mL against S. typhi and A. flavus. In
all other cases, the compounds exhibited MICs ranging
from 10–100 lg/mL against all the microbial strains and
some of are given in Table 5.
DNA cleavage experiment
Preparation of Culture media
DNA cleavage experiments were done according to the
literature [48]. Nutrient broth (peptone, 10; Yeast extract,
5
; NaCl, 10 in (g/L)) was used for culturing of E. coli and
-
1
Table 5 Minimum inhibitory concentration (lg mL ) results for some compounds
Compds.
Anti bacterial activity (zone of inhibition in %)
Antifungal activity (zone of inhibition in %)
E. coli
S. Aureus
S.typhi
P.aeruginosa
A. flavus
Cladosporium
A. niger
IV
5
10
15
15
10
20
25
20
25
25
25
25
20
15
10
15
25
20
20
10
15
15
15
25
20
20
10
20
15
20
15
15
15
15
20
10
15
10
25
25
10
20
15
6
8
1
1
0
2
123

3
56
J Incl Phenom Macrocycl Chem (2010) 68:347–358
Cl
N
N
N
M
N
Cl
HN
N
R
Fig. 5 M: Standard Molecular weight Marker; C-E. coli- Control
IV
DNA of E. coli; Lane 1–4: E. coli DNA treated with Schiff base (L
and it’s Cu(II) Co(II) and Ni(II) complexes respectively. Lane 5–8: A.
)
N
N
IV
niger DNA treated with Schiff base (L ) and it’s Cu(II) Co(II) and
Ni(II) complexes respectively. C-niger is Control DNA of A. niger
R = H, CH C H and C H
7
3
,
2
5
3
M = Co(II), Ni(II) and Cu(II)
Electrophoretic analysis result
Fig. 6 Proposed structure for metal (II) complexes
II
The Schiff base (L ) and its Co(II), Ni(II) and Cu(II)
was confirmed by the analytical, IR, electronic, magnetic,
EPR, FAB mass, thermal and electrochemical studies. In
biological results it confirms that, all the Schiff bases are
biologically active and their metal(II) complexes have
shown more promising activities than the Schiff bases. The
interaction of these complexes with DNA was investigated
by gel electrophoresis technique. From the observation, it
complexes were studied for their DNA cleavage activity by
agarose gel electrophoresis method and presented in the
Fig. 5. DNA cleavage reactions generally proceed via two
major pathways (I) Oxidative cleavage of the sugar and/or
nucleobase moiety and (II) hydrolytic pathway involving
the phosphate group. Iron and copper complexes are known
to be useful for oxidative cleavage of DNA involving
nucleobase oxidation and/or degradation of sugar by
abstraction of deoxyribose hydrogen atoms while com-
plexes containing strong Lewis acids like copper (II) and
Zinc (II) are suitable for hydrolytic cleavage of DNA.
Sigman and co-worker have reported bis(phen)copper (I)
complex as first ‘‘copper based chemical nuclease’’ that
cleaves the DNA in presence of H O and thiol [49].
IV
was found that Schiff base (L ) and its metal (II) com-
plexes cleave DNA more efficiently. All these observations
put together lead us to propose the following structure
shown in Fig. 6 in which, the complex having the stoi-
chiometry of the type [MLCl ] (M = Co(II), Ni(II) and
2
Cu(II)).
2
2
Acknowledgements The authors are grateful to the Chairman,
Department of Chemistry, Karnatak University, Dharwad for the
facilities. One of the authors (UVK) grateful to the University Grant
Commission, New Delhi for grant of Research Fellowship in Science
for Meritorious Students.
Similarly, the anticancer antibiotic bleomycins containing
iron cleave DNA in an oxidative manner [50, 51].
The gel after electrophoresis clearly revealed that, both
IV
the Schiff base (L ) and it’s complexes have acted on
DNA as there was molecular weight difference between the
control and the treated DNA samples. The difference was
observed in the bands (Lane 1–8) compared to the control
DNA of E. coli and A. niger. This shows that, the control
DNA alone does not show any apparent cleavage where as
complexes shown. However, the nature of reactive inter-
mediates involved in the DNA cleavage by the complexes
has not been clear. The results indicated the important role
of metal in these isolated DNA cleavage reactions. As the
compound was observed to cleave the DNA, it can be
concluded that the compound inhibits the growth of the
pathogenic organism by cleaving the genome.
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