Morphological and pharmacological investigation on some biopotent materials derived from substituted pyrimidine and imidazole enzyme constituents

Article abstract of DOI:10.1016/j.saa.2014.01.089

Coordinating behavior of novel N₂O type mixed ligand complexes (1-6) have been synthesized from substituted fluoropyrimidine [5-Fluorouracil (5-FU; A)] with biopotent imidazole enzyme constituents (B) viz., imidazole(him) and benzimidazole(bim) in the presence of Ni(II), Cu(II) and Zn(II) ions. Synthesized complexes were characterized by chemical analysis, spectral studies, magnetic moment and conductivity measurements. The results of chemical analysis and the observed low molar conductance values propose their stoichiometry to be 1:1:1 (M:A:B) with non-electrolytic nature. From the spectral data, it is inferred that the ligands A & B coordinate with M(II) ions in bi and monodentate approach through C₍₄₎O, N₍₃₎ and imidazole ring N₍₃₎ atoms respectively. The thermogravimetric analysis shows the dehydration, decomposition and thermal stability of mixed ligand complexes. XRD and SEM patterns show sharp crystalline peaks with homogeneous morphology. In vitro antimicrobial activities of free ligands (A & B) and their metal complexes were screened against some pathogenic strains by well diffusion technique. Absorption and gel electrophoresis experiments on the interaction of mixed ligand complexes with DNA suggest that all the complexes can bind as well as cleave the DNA by intercalation between chromophores and DNA base pairs. In addition, in vitro antioxidant activities were tested by DPPH free radical scavenging model.

Full text of DOI:10.1016/j.saa.2014.01.089

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Morphological and pharmacological investigation on some biopotent
materials derived from substituted pyrimidine and imidazole enzyme
constituents
c
d
Sutha Shobana a, Perumal Subramaniam b, , Jeyapraksh Dharmaraja , Arvind Narayan
⇑
a Department of Chemistry, Division of Science and Humanities, Rajas International Institute of Technology for Women, Ozhuginaserri, Nagercoil 629 001, Tamil Nadu, India
b Department of Chemistry and Research Centre, Aditanar College of Arts and Science, Virapandianpatnam, Thiruchendur 628 216, Thoothukudi District, Tamil Nadu, India
c Department of Chemistry, Division of Science and Humanities, Sree Sowdambika College of Engineering, Chettikurichi, Aruppukottai 626 134, Tamil Nadu, India
d Division of Science and Humanities, K.N.S.K. College of Engineering, Therekalputhoor, Nagercoil 629 001, Kanyakumari District, 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
Novel N2O type mixed ligand complexes (1–6) have been synthesized from substituted fluoropyrimidine
[5-Fluorouracil (5-FU: A)] with imidazole(him) and benzimidazole(bim) enzyme constituents(B) in the
presence of M(II) ions [where M(II) = Ni(II), Cu(II) and Zn(II)]. Synthesized complexes (1–6) were charac-
terized by chemical analysis and various spectral studies. In vitro antimicrobial activities of 5-Fluoroura-
cil(A) and their mixed ligand complexes were screened against some bacterial and fungal strains by well
diffusion technique. Electronic absorption and oxidative cleavage studies of the chelates with DNA under
aerobic conditions show remarkable activities. Also, the absorption binding studies of CT DNA with the
M(II)-5-FU(A)-him(B) complexes show decrease of 5–15% intensity with minor red shift along with sig-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Substituted halopyrimidine
derivatives are used in colorectal
cancer chemotherapy.
5-Fluorouracil plays a crucial role in
Food and Drug Administration
systems.
The metal complexes show
homogeneous morphology with
microcrystalline environment.
Gel electrophoresis shows CT DNA
undergoes oxidative cleavage with
the complexes.
In vitro antimicrobial and antioxidant
assessments show high inhibiting
potential.
nificant hypochromicity and the free energy change values (
act with DNA in a spontaneous manner.
D
àG) indicate the M(II) complexes can inter-
a r t i c l e i n f o
a b s t r a c t
Article history:
Coordinating behavior of novel N2O type mixed ligand complexes (1–6) have been synthesized from
substituted fluoropyrimidine [5-Fluorouracil (5-FU; A)] with biopotent imidazole enzyme constituents
(B) viz., imidazole(him) and benzimidazole(bim) in the presence of Ni(II), Cu(II) and Zn(II) ions. Synthe-
sized complexes were characterized by chemical analysis, spectral studies, magnetic moment and con-
ductivity measurements. The results of chemical analysis and the observed low molar conductance
values propose their stoichiometry to be 1:1:1 (M:A:B) with non-electrolytic nature. From the spectral
data, it is inferred that the ligands A & B coordinate with M(II) ions in bi and monodentate approach
Received 29 December 2013
Accepted 22 January 2014
Available online 18 February 2014
Keywords:
Fluoropyrimidine
⇑
Corresponding author. Tel.: +91 94435 00381; fax: +91 4639 245247.
E-mail addresses: Subramaniam.perumal@gmail.com, psubramaniam.ac@
gmail.com (P. Subramaniam).
http://dx.doi.org/10.1016/j.saa.2014.01.089
1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
243
Imidazole enzyme constituents
Mixed ligand complexes
Spectral studies
Pharmacological evaluation
DNA studies
through C(4)@O, N(3) and imidazole ring N(3) atoms respectively. The thermogravimetric analysis shows
the dehydration, decomposition and thermal stability of mixed ligand complexes. XRD and SEM patterns
show sharp crystalline peaks with homogeneous morphology. In vitro antimicrobial activities of free
ligands (A & B) and their metal complexes were screened against some pathogenic strains by well diffu-
sion technique. Absorption and gel electrophoresis experiments on the interaction of mixed ligand com-
plexes with DNA suggest that all the complexes can bind as well as cleave the DNA by intercalation
between chromophores and DNA base pairs. In addition, in vitro antioxidant activities were tested by
DPPH free radical scavenging model.
Ó 2014 Elsevier B.V. All rights reserved.
Introduction
The interactions of transition metal ions with DNA are signifi-
cant, often changing the structure and function of genetic materi-
The heterocyclic ring systems containing bioactive donor of N/O
atoms present in pyrimidine moieties are the basic building blocks
in modern drug designing architecture. These derivatives have
occupied a unique and significant position in the field of medicinal,
biological and therapeutical applications since they possess anti-
bacterial, fungicidal, antitumor, antimitotic, antithyroid and sur-
face anaesthesia activities [1,2]. Further, these derivatives are
essential antimicrobial, antineoplastic, antiviral, antitumor, anti-
HIV, antiinflammatory, antimalarial and cardiovascular agents in
chemotherapeutical and agricultural areas [3–6]. They also bring
into play an imperative role in hypnotic drugs for the nervous sys-
tem, antagonists of the human A2A adenosine and calcium sensing
receptors [7]. It is well known that pyrimidine derivatives of uracil,
thymine and cytosine are the fundamental components in nucleic
acids of DNA and RNA. In adjuvant chemotherapy, substituted
pyrimidine derivatives alone or with leucovorin are functional to
standard healing for high-risk Duke’s C colon cancer (Stage II or
Stage III). At very low concentration (<20 ppm), 5-Fluorouracil
exhibits a cheerful inspiring effect on plant tumor growth which
is produced by F1 hybrid of Nicotiana glauca Grah and Nicotiana
langsdorffii Weinm [8]. Further, 5-Fluorouracil is a novel oral tu-
mor-activated and tumor-elective fluoropyrimidine carbamate
and an oral chemotherapeutic agent which is also used in the treat-
ment of breast, esophageal and larynx, gastrointestinal and genito-
urinary tract cancers. The diazole ring moieties of imidazole
derivatives are suggested as effective antifungal drugs (bifonazole,
butoconazole, clotrimazole, miconazole, etc.,) and they possess
some medicinal applications which include anticancer, b-lacta-
mase inhibitors, antiaging agents, anticoagulants, antimalarial,
antiinflammatory, antidiabetic and antitubercular activities
[9,10]. Benzimidazole moiety acts as a fraction of the nucleotide
fragment in vitamin B12 and in a range of biological and pharmaco-
logical actions [11].
Copper is an elementary micronutrient, necessary for all the liv-
ing organisms and it is ubiquitous in catalytic co-factor for a range
of metalloenzymes, electron transfer and oxygen transport pro-
teins [12]. The copper containing proteins play a dynamic role in
three major cellular processes namely amino acid metabolism,
coagulation cascade and tyrosine metabolism with melanin bio-
synthesis. In addition to this, it concerned with many biological
and physiological processes such as hemoglobin regulation, iron
metabolism, mitochondrial respiration, biosynthesis of neuro-
transmitter, free radical detoxification, development of embryo
and connective tissue, nerve coverings and bone [12]. Nickel is nec-
essary to human and higher animal species like chickens, rats, pigs,
cows, sheep and goats [13]. It also stabilizes DNA and RNA against
thermal denaturation [13] and activates many enzymes. Zinc is the
second richest trace element in human body which is intracellular
in bone, muscle, skin, hair and liver [14]. It plays a chief role in all
the biochemical pathways, perpetuation of genetic materials and
in the function of retina, retinal pigment epithelium (RPE) and
choroid.
als. Mixed ligand complexes have been introduced into DNA in
order to gain the knowledge about the binding and/or cleaving
mechanism, in particular to understand pre-requisites of specific-
ity [15]. In addition, they have been used in the development of
new target molecules for bio-reactivity of these metal complexes
with DNA [16]. In absorption studies, the change in the intensities
can be used to explain the nature and strength of the stacking
interaction between chromospheres and DNA base pairs. In a se-
quel to our attempt [17–23], the novel N2O type mixed ligand com-
plex systems of Ni(II)/Cu(II)/Zn(II)-5-FU(A)-him/bim(B) were
synthesised and characterized by means of analytical and spectral
techniques. The in vitro antimicrobial and antioxidant activities of
5-Fluorouracil and their complexes have been studied. Electronic
absorption and oxidative cleavage interaction of 5-Fluorouracil(A)
and their metal complexes with DNA was also studied.
Experimental
Materials and reagents
All the ligands are extra pure Sigma Aldrich, Fluka (Puriss) prod-
ucts and they are used without further purification. Solvents for
the physical measurements were of analytical grade and purified
according to literature methods [24]. DNA was purchased from
Genie (Bangalore, India), agarose (molecular biology grade) and
ethidium bromides (EB) were obtained from Sigma Aldrich (USA).
2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid (AA) were
purchased from Sigma Aldrich.
Instruments
Melting points (m.p.) of all the mixed ligand complexes were
determined on Gallenkamp apparatus in open glass capillaries
and are uncorrected. C, H and N analytical data were performed
on Elementar Vario EL III CHNS analyzer. Metal content of the
mixed ligand complexes were estimated gravimetrically by the
standard procedure. Molar conductances of the mixed ligand com-
plexes (1 ꢁ 10ꢂ3 mol) were measured using an Elico CM 180
conductivity bridge by using 0.01 M KCl solution as calibrant. Fast
atomic bombardment mass spectra (FAB-MS) were recorded using
a
VGZAB-HS spectrometer in a 3-nitrobenzylalcohol matrix.
Magnetic susceptibility measurements were carried out on a Gouy
balance at room temperature using mercuric tetra(thiocya-
nato)cobaltate(II) as the calibrant. Diamagnetic corrections were
applied in compliance with Pascal’s constant [25]. Electronic
absorption spectra were recorded with a Hitachi U-2000 double
beam spectrophotometer in the 200–1100 nm range. Vibrational
spectra were recorded using KBr pellets on a JASCO FT/IR-410
spectrometer, in the 4000–400 cmꢂ1 range. 1H NMR and 13C NMR
spectra of the diamagnetic Zn(II) complexes were carried out in
DMSO-d6 at room temperature using TMS as internal standard on

244
S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
a Perkin Elmer R-32 spectrometer. X-band EPR spectra of Cu(II)
complexes, at room temperature and liquid nitrogen conditions
in DMSO medium, were recorded on a Varian ESR spectrometer
using DPPH as internal standard. Thermal stabilities of the com-
plexes were recorded in dynamic nitrogen atmosphere (flow rate
20 cm3/min) with a heating rate of 10 K/min using a Perkin Elmer
(TGS-2 model) thermal analyzer. Powder X-ray diffraction (XRD)
patterns were recorded with a Bruker AXS D8 advance powder
X-ray diffractometer (X-ray source: Cu, Wavelength 1.5406 Å)
using Si(Li)PSD as the detector. Scanning Electron Micrography
with Energy Dispersive Spectrometry associated (SEM/EDS) using
JSM-5610 scanning electron microscope was used for morphologi-
cal investigation.
Interaction between mixed ligand complexes with DNA
DNA experiments, with mixed ligand complexes in DMSO med-
ium, were done in buffer (50 mmol Tris–HCl/NaCl, pH = 7.2)
[28,29]. DNA concentration per nucleotide was determined by
absorption spectroscopy using the molar absorption coefficient
(e = 6600 molꢂ1 cmꢂ1) at 260 nm. The purity of DNA was checked
from its absorbance values at 260 and 280 nm and the ratio,
A260/A280, was found to be indicating that CT DNA was sufficiently
free from protein contamination [30]. Stock solution of CT DNA
concentration per nucleotide [C(p)] in the buffer medium was
measured from its absorption intensity at 260 nm after 1:100 dilu-
tion with known e = 6600 molꢂ1 cmꢂ1 value. Stock solutions were
kept at 4 °C and used after not more than four days. Doubly dis-
tilled carbonate free water was used to prepare the buffer and
other required solutions.
In vitro antimicrobial activities
Absorption spectral titration experiments were done by keeping
the concentration of mixed ligand complexes (1–3) as constant
(30 mmol) while varying the CT DNA concentration ranged from
0 to 50 or more equivalents by maintaining the total volume of
the solution as kept constant. After the addition of CT DNA, the
resulting mixture was shaken up and allowed to equilibrate for
5–10 min at room temperature. The slow and successive additions
of CT DNA to the complexes showed both hypochromicity and red-
shifted charge transfer peak with maxima in the spectra. The titra-
tion processes were repeated until there was no change in the
spectra indicating that binding saturation had been achieved.
Absorption readings corresponding to the changes at maximum
absorption with increasing concentration of CT DNA were recorded
and the binding strength of mixed ligand complexes were evalu-
ated quantitatively [31] as:
In vitro antimicrobial activities of 5-Fluorouracil(A) and their
M(II)-5-FU(A)-him/bim(B) complexes in DMSO medium were
tested against three Gram-positive pathogenic bacterial strains:
Bacillus subtilis, Staphylococcus saphyphiticus and Staphylococcus
aureus, two Gram-negative bacterial strains: Escherichia coli and
Pseudomonas aeruginosa using Muller Hinton agar nutrient, and
three fungal strains namely Aspergillus niger, Enterobacter species
and Candida albicans using potato dextrose agar as the medium
by well diffusion technique [26]. About 2–8 h, older microorganism
species inoculums containing approximately 104–106 colony
forming units (CFU) per cm3 were used in these analyses. All the
analyses were made in three replicate for each and the detailed
procedure for measuring the zone of inhibition was followed as de-
scribed earlier [18,19]. The results were recorded as zone of inhibi-
tion in mm and compared with the commercially available
tetracycline and amphotricine, used as antibacterial and antifungal
control respectively. Zone of inhibition is given as the average of
three independent determinations.
½DNAꢃ
½DNAꢃ
1
¼
þ
ð
ea
ꢂ
ef
Þ
ð
eb
ꢂ
ef
Þ
Kbðeb
ꢂ
ef
Þ
where ef, eb and ea are the molar extinction coefficients of the free
complex, completely bound form with CT DNA and with DNA
respectively at a definite concentration. The observed absorption
data was then fit into the above equation to obtain the intrinsic
binding constant (Kb) by the plot of [DNA]/(ea ꢂ ef) versus [DNA],
where the slope and y-intercept of the above equation are 1/(eb ꢂ ef)
and 1/Kb(eb ꢂ ef)respectively. The intrinsic binding constant (Kb) was
determined from the ratio of the slope to y-intercept.
In vitro antioxidant activity
In vitro antioxidant activities of 5-Fluorouracil(A) and their
mixed ligand complexes have been tested under physiological con-
ditions by DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scav-
enging assay method at 37 °C. DPPH free radical scavenging effect
was carried out according to the Blois method [27]. The different
concentrations of 5-Fluorouracil(A) (10, 20, 30, 40 and 50 lmol)
Oxidative DNA cleavage studies
and their metal complexes were prepared in DMSO medium.
1 cm3 of each compound solution having different concentrations
were taken in different test tubes and 4 cm3 of a 0.1 mmol DPPH
solution in DMSO was added to each test tube and the reaction
mixture was shook vigorously for about 2–3 min. Each tube was
then incubated in the dark room for 20–30 min at room tempera-
ture. A blank DPPH solution prepared without compound was used
for the baseline correction. After incubation, the absorbance value
of all compounds were measured at a ðkmaxÞ of 517 nm, using an
UV–vis. spectrophotometer. There was a decrease in the absor-
bance values ðkmaxÞ which indicated that the mixed ligand com-
plexes show moderate free radical scavenging activities. Ascorbic
acid (AA) was used as the reference or positive control. Free radical
scavenging effects in percentage was calculated using the formula,
DNA cleavage activities of 5-Fluorouracil(A) and their mixed li-
gand complexes were monitored by agarose gel electrophoresis on
CT DNA. Gel electrophoresis experiments were performed under
aerobic condition with an oxidant (H2O2) by incubation at 35 °C
for 2 h as follows: CT DNA 30
50 mol of oxidant in 50 mmol Tris–HCl/NaCl buffer (pH = 7.2)
containing 50 mmol NaCl solutions. After incubation, 1
cm3 of
lmol, 50 lmol of each complex,
l
l
loading buffer (bromophenol blue in water) was added to each
tube and the samples were loaded on 1% agarose gel. Samples were
electrophoresed at a constant voltage (50 V) for 2 h in Tris–acetic
acid–EDTA buffer (pH 8.3). After electrophoresis, the gel was
stained for 30 min by immersing it in 1 l
g/cm3 ethidium bromide
(EB) solution. DNA cleavage were visualized by viewing the gel
under Ultraviolet (UV) light and photographed.
ꢀ
ꢁ
ðAcontrol ꢂ Asample
Þ
Scavenging effects ð%Þ ¼
ꢁ 100
Acontrol
General procedure for synthesis of mixed ligand complexes (1–6)
where Acontrol is the absorbance of the control without the ligand or
the complex and Asample is the absorbance of the ligand or the com-
plex. All the analyses were made in three replicate for each and the
results were compared with control.
All the six mixed ligand complexes were synthesised by the
following general procedure: 5-Fluorouracil (0.013 g, 10 mmol)
was dissolved in aqueous (10 cm3) solution containing a few drops

S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
245
of concentrated ammonia and get a clear solution on stirring. An
appropriate metal salt solution (10 cm3) of 10 mmol, 0.025 g
Ni(CH3COO)2ꢄ4H2O or 0.020 g Cu(CH3COO)2ꢄH2O or 0.022 g Zn
(CH3COO)2ꢄ2H2O), was added dropwise to the above solution and
stirred at room temperature (Scheme 1). To this, aqueous solution
(10 cm3) of imidazole (0.007 g, 10 mmol) or benzimidazole
(0.012 g, 10 mmol) was added and the reaction mixture was
refluxed for 6–8 h on a water bath. The pH (6.8) of the reaction
mixture was adjusted by adding few drops of aqueous Na2CO3
solution. The resulting solutions were reduced to 1/3 of its original
volume and kept aside. On standing, the mixed ligand complexes
were obtained and collected by vacuum filtration, washed several
times with cold water, ethanol and anhydrous ether. The mixed
ligand complexes were obtained as powder and dried in air and
stored in vacuo over anhydrous CaCl2 at room temperature. The
yield of the isolated complexes were found to be 60–75%.
kmax = 26,738 cmꢂ1
;
l
eff = Diamagnetic; KM (10ꢂ3 mol in
DMSO) = 20.15 S cm2 molꢂ1
.
Complex (4). [5-Fluoro-8,8-dihydroxy-8-(3H-1k4,3-benzodiazol-3-yl)-
2-oxo-7k3-oxa-1,3-diaza-8-nickel(II)abicyclo[4.2.0]octa-4,6-dien-8-yl
acetate]
Yield: 68%; m.p.: 243 °C; Color: Lustrous dark green; Mol. for.:
[C13H15FN4O6Ni]; Mol. wt.: 400.97; Selected FT-IR data in cmꢂ1
ꢀ
(KBr disk):
m
¼ 3600—3500; 1512; 1415 (br,
m(NH) vib. of N1H &
N3H group in 5-FU), 3450–3300, 835, 729 (br, OH2 molecule),
3353, 1456 (br, NH, imidazole ring), 1701 and 1659 (d, C@O str.,
pyrimidine ring), 1537 (s, C@N, imidazole ring), 1570 and 1347
(m, ACOO, acetate group), 1468 (m, C5AF vib. of 5-FU, 518 (w,
NiAN), 457 (w, NiAO) cmꢂ1; FAB-MS: m/z = 402.03 [M + 1]; UV–
vis., (10ꢂ3 mol in DMSO): kmax = 33,898, 26,455, 16,920 and
10,225 cmꢂ1
;
l
eff = 3.15 BM; KM (10ꢂ3 mol in DMSO) =
.
19.53 S cm2 molꢂ1
Complex (1). [5-Fluoro-8,8-dihydroxy-8-(3H-1k4,3-imidazol-3-yl)-2-
oxo-7k3-oxa-1,3-diaza-8-nickel(II)abicyclo[4.2.0]octa-4,6-dien-8-yl
acetate]
Complex (5). [5-Fluoro-8-(3H-1k4,3-benzodiazol-3-yl)-2-oxo-7k3-
oxa-1,3-diaza-8-cupra(II)abicyclo[4.2.0]octa-4,6-dien-8-yl acetate]
Yield: 74%; m.p.: 250 °C; Color: Pale brown; Mol. for.: [C13H11
FN4O4Cu]; Mol. wt.: 369.80; Selected FT-IR data in cm–1 (KBr disk):
ꢀ
m(NH) vib. of N1H & N3H group in
Yield: 68%; m.p.: 247 °C; Color: Lustrous dark green; Mol. for.:
[C9H13FN4O6Ni]; Mol. wt.: 350.92; Selected FT-IR data in cmꢂ1
ꢀ
m(NH) vib. of N1H &
(KBr disk):
m
¼ 3600—3500; 1515; 1409 (br,
m
¼ 3600—3500; 1517; 1411 (br,
N3H group in 5-FU), 3450–3300, 846, 716 (br, OH2 molecule),
3375, 1472 (br, NH, imidazole ring), 1698 and 1666 (d, C@O str.,
pyrimidine ring), 1568 (s, C@N, imidazole ring), 1575 and 1358
(m, ACOO, acetate group), 1472 (m, C5AF vib. of 5-FU), 522 (w,
NiAN), 461 (w, NiAO); FAB-MS: m/z = 352.01 [M + 1]; UV–vis.,
(10ꢂ3 mol in DMSO): kmax = 32,258, 25,840, 17,153 and
5-FU), 3351, 1454 (br, NH, imidazole ring), 1705 and 1662 (d, C@O
str., pyrimidine ring), 1541 (s, C@N, imidazole ring), 1566 and 1348
(m, ACOO, acetate group), 1471 (m, C5AF vib. of 5-FU), 539
(w, CuAN), 466 (w, CuAO) cmꢂ1; FAB-MS: m/z = 371.01 [M + 1];
UV–vis., (10ꢂ3 mol in DMSO): kmax = 25,707 and 13,680 cmꢂ1
;
l
eff = 1.94 BM; KM (10ꢂ3 mol in DMSO) = 20.39 S cm2 molꢂ1
.
10,362 cmꢂ1
DMSO) = 18.19 S cm2 molꢂ1
;
l
eff = 3.09
BM;
KM
(10ꢂ3 mol
in
Complex (6). [5-Fluoro-8-(3H-1k4,3-benzodiazol-3-yl)-2-oxo-7k3-
.
oxa-1,3-diaza-8-zinc(II)abicyclo[4.2.0]octa-4,6-dien-8-yl acetate]
Complex (2). [5-Fluoro-8-(3H-1k4,3-imidazol-3-yl)-2-oxo-7k3-oxa-
1,3-diaza-8-cupra(II)abicyclo[4.2.0]octa-4,6-dien-8-yl acetate]
Yield: 72%; m.p.: 263 °C; Color: White; Mol. for.: [C13H11FN4O4
Zn]; Mol. wt.: 371.66; Selected FT-IR data in cmꢂ1 (KBr disk):
ꢀ
m
¼ 3600—3500; 1514; 1408 (br,
m(NH) vib. of N1H & N3H group
Yield: 70%; m.p.: 266 °C; Color: Pale brown; Mol. for.: [C9H9FN4
O4Cu]; Mol. wt.: 319.74; Selected FT-IR data in cmꢂ1 (KBr
in 5-FU), 3354, 1457 (br, NH, imidazole ring), 1703 and 1676 (d,
C@O str., pyrimidine ring), 1535 (s, C@N, imidazole ring), 1545
and 1321 (m, ACOO, acetate group), 1469 (m, C5AF vib. of 5-FU),
512 (w, ZnAN), 451 (w, ZnAO) cmꢂ1; FAB-MS: m/z = 372.04
disk): = 3600–3500, 1514, 1406 (br, m(NH) vib. of N1H & N3H group
in 5-FU), 3372, 1473 (br, NH, imidazole ring), 1695 and 1653 (d,
C@O str., pyrimidine ring), 1572 (s, C@N, imidazole ring), 1585
and 1362 (m, ACOO, acetate group), 1473 (m, C5AF vib. of 5-FU),
538 (w, CuAN), 470 (w, CuAO); FAB-MS: m/z = 320.98 [M + 1];
[M + 1]; UV–vis., (10ꢂ3 mol in DMSO): kmax = 27,624 cmꢂ1
; leff =
Diamagnetic; KM (10ꢂ3 mol in DMSO) = 21.85 S cm2 molꢂ1
.
UV–vis., (10ꢂ3 mol in DMSO): kmax = 26,455 and 13,441 cmꢂ1
;
l
eff = 1.96 BM; KM (10ꢂ3 mol in DMSO) = 19.62 S cm2 molꢂ1
.
Results and discussion
Complex (3). [5-Fluoro-8-(3H-1k4,3-imidazol-3-yl)-2-oxo-7k3-oxa-
Physico-chemical properties of the metal complexes (1–6)
1,3-diaza-8-zinc(II)abicyclo[4.2.0]octa-4,6-dien-8-yl acetate]
Yield: 68%; m.p.: 278 °C; Color: Lustrous colorless; Mol. for.:
The synthesised mixed ligand complexes are highly air-stable
and non-hygroscopic in nature. They are insoluble in water and
in common organic solvents like benzene, acetone, petroleum
ether etc., but soluble in DMSO, DMF and Dioxan. The obtained
chemical analysis (C, H, N) result with its molecular formula and
other spectral data values are summarized in the experimental
part. From the analytical data, the stoichiometry of M(II): 5-Fluoro-
uracil: imidazole/benzimidazole is to be 1:1:1 in MAB type
[C9H9FN4O4Zn]; Mol. wt.: 321.60; Selected FT-IR data in cmꢂ1
ꢀ
(KBr disk):
m
¼ 3600—3500; 1516; 1411 (br,
m(NH) vib. of N1H &
N3H group in 5-FU), 3374, 1470 (br, NH, imidazole ring), 1699
and 1671 (d, C@O str., pyrimidine ring), 1566 (s, C@N, imidazole
ring), 1560 and 1341 (m, ACOO, acetate group), 1470
(m, C5AF vib. of 5-FU), 521 (w, ZnAN), 455 (w, ZnAO); FAB-MS:
m/z = 322.91
[M + 1];
UV–vis.,
(10ꢂ3 mol
in
DMSO):
Scheme 1. Formation of mixed ligand complexes (MAB).

246
S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
complexes and this analytical values are in good concord with the
calculated values (Table 1). The observed low molar conductance
values of the metal complexes (1 ꢁ 10ꢂ3 mol, DMSO solution) at
room temperature are reliable with the non-electrolytic nature
due to the absence of counter ions in the proposed structures
[32]. Fast atomic bombardment mass spectrum (FAB-MS) of the
synthesized mixed ligand complexes show the molecular ion
(m/z) peaks which confirms the stoichiometry [M(II)-5-FU(A)-
him/bim(B)(OAc)(OH2)x = 0/2] of the metal chelates. This type of
stoichiometry is further confirmed by the observed analytical
and their spectral data of the metal chelates.
Electronic absorption spectra coupled with magnetic moment values
The electronic absorption spectra of complexes were recorded
in (1 ꢁ 10ꢂ3 mol) DMSO medium at room temperature (Fig. 1)
and the electronic absorption spectral data of the mixed ligand
complexes are presented in Table 3. Also, various spectral param-
eters (B, B0, b, b (%) and LFSE) for the Ni(II) complexes are calculated
by applying band energies on Tanabe–Sugano diagrams.
Ni(II)-5-FU(A)-him/bim(B) complexes display four spin allowed
bands at 33,898–32,258, 26,455–25,840, 17,153–16,920 and
3
10,362–10,225 cmꢂ1, which may be due to LMCT (n ? p⁄), A2g
3
3
(F) ? 3T1g(P), A2g(F) ? 3T1g(F) and A2g(F) ? 3T2g(F) transitions
3
respectively, with an distorted octahedral geometry with A2g as
Infrared spectra with mode of binding
ground state [36,37]. The ratio of m2/m1 values lie in between 1.66
The characteristic IR spectral data (KBr pellet, cmꢂ1) of Ni(II)/
Cu(II)/Zn(II)-5-FU(A)-him/bim(B) complexes were represented in
the experimental part. In order to study the mode of coordination
between free ligands (A & B) with the M(II) ions in mixed ligand
complexes, the IR spectrum of 5-FU(A) and him/bim(B) ligands
were compared to the spectra of their mixed ligand complexes
and are listed in Table 2. From the data, it is found that 5-FU(A) acts
as bidentate which form metal chelates through the deprotonated
N3 and C4@O of carbonyl oxygen atoms. Similarly, him/bim(B) li-
gands bind the M(II) ions in unidentate manner via imidazole ring
AN3 atom. In addition, two new peaks were appeared in the far
and 1.65, as expected for octahedral geometry, and the magnetic
moment values, 3.09 and 3.15 BM for the complexes indicating
two unpaired electrons with the high spin six-coordinated octahe-
dral environment around the Ni(II) ion [36].
The copper complexes (2 & 5) exhibit one LMCT band, at
26,455 cmꢂ1 for Cu(II)-5-FU(A)-him(B) and at 25,707 cmꢂ1 for
Cu(II)-5-FU(A)-bim(B) complex and an unsymmetrical broad
absorption band which is centered at 13,441 and 13,680 cmꢂ1
respectively that can be assigned to d–d electronic transition
[36]. Such type of low energy absorption band with less inten-
sity is commonly found in distorted tetrahedral environment
which is caused by Jahn–Teller distortion. The observed mag-
netic moment values, 1.96 and 1.94 BM are also well agreed
within the predictable region of 1.92–2.00 BM, found for dis-
torted tetrahedral environment [36,37]. However, the complexes
of Zn(II)-5-FU(A)-him/bim(B) show one broad band middled at
26,738 and 27,624 cmꢂ1 respectively in the UV region due to
L ? M charge transfer transition in tetrahedral environment.
Further, due to diamagnetic nature of Zn(II) ion, Zn(II) com-
plexes do not show any d–d transitions in the visible region
[36,37].
infrared region at 470–451 cmꢂ1 and 539–512 cmꢂ1 for
and (MAN) modes respectively [33,34]. This (MAO) and
(MAN) peak values follow Irving-William order of stability as:
m(MAO)
m
m
m
Cu > Ni > Zn and also in comparable with the crystal field stabiliza-
tion energies [35]. The Ni(II)-5-FU(A)-him/bim(B) complexes show
a broad peak in the region 3450–3300 cmꢂ1 and new peaks ap-
peared in 846–835 cmꢂ1 and 739–716 cmꢂ1 are attributed to the
stretching, rocking and wagging mode of
m(OH) vibration of the
coordinated water (OH2) molecule in the complexes [33,34]. The
absence of coordinated water molecules in the heated synthesised
complexes (1 & 4) at 85–140 °C is further confirmed from the ab-
sence of any broad band in the IR spectra around at ca. 3450–
From Tanabe–Sugano diagrams, the stabilization parameters
like ligand field splitting energy (Dq), Racah interelectronic repul-
sion parameter (B), the nephelauxetic ratio (b), percentage of cov-
alency (% b) and LFSE values for Ni(II)-5-FU(A)-him/bim(B)
complexes (1 & 4) were calculated and the values are 1036 and
1022 cm–1, 793.62 and 846.71, 0.77 and 0.82, 22.95 and 17.79,
and 145.83 and 146.88 kJ molꢂ1 respectively for complexes
Ni(II)-5-FU(A)-him/bim(B) complexes. The observed ligand field
parameters and magnetic moment values also support the covalent
character of distorted octahedral geometry around the Ni(II) ion
with D4h symmetry [38,39]. Also, due to the mixing of ground state
(3A2g) with the excited state (3T2g) in octahedral Ni(II) complexes
3300 cmꢂ1
.
Also, all these complexes show an additional characteristic
bands in the region 1585–1545 cmꢂ1 and 1362–1321 cmꢂ1 are as-
cribed to
m(CAO) of asymmetric and symmetric vibration of the
acetate (COOꢂ) group respectively [33], indicating the participation
of the carboxylate oxygen in the mixed ligand complex formation.
The magnitude of
Dm
values fall in the range 224–217 cmꢂ1 sug-
gesting the unidentate coordination of carboxylate acetate (OAc)
group in the complexation. Based on the conductivity measure-
ments, magnetic moment, spectral studies (IR spectra, 1H & 13C
NMR spectra) and thermal analyses of mixed ligand complexes,
we confirm that Ni(II) complexes (1 & 6) are of distorted octahe-
dral environment with a stoichiometry of [Ni(II)-5-FU(A)-him/
bim(B)(OH2)2(OAc)] and Cu(II)/Zn(II) complexes are distorted tet-
8k0
give the Lunde’s factor, i.e., g ¼ 2 ꢂ 10Dq, (where k0 = spin-orbit cou-
pling constant, ꢂ368 cmꢂ1). In general, the hexaaquo Ni(II) com-
plexes show the g value of 2.25 [38], while Ni(II)-5-FU(A)-him/
bim(B) complexes (1 & 4) show the g values of 2.246 and 2.252
respectively which supports the octahedral environment around
the Ni(II) ions.
rahedral
geometry
with
[M(II)-5-FU(A)-him/bim(B)(OAc)]
stoichiometry.
Table 1
Elemental analysis, molar conductivity and melting point of mixed ligand complexes (1–6).
Compound
Color
Empirical Formula
Yield (%)
Elemental analysis, found (calculated) %
Km (S cm2 molꢂ1
)
m.p. (°C)
M
C
H
N
(1)
(2)
(3)
(4)
(5)
(6)
Lustrous dark green
Pale brown
Lustrous colorless
Lustrous dark green
Pale brown
NiC9H13FN4O6
CuC9H9FN4O4
ZnC9H9FN4O4
NiC13H15FN4O6
CuC13H11FN4O4
ZnC13H11FN4O4
68
70
68
68
74
72
16.59 (16.73)
19.52 (19.87)
20.10 (20.34)
14.28 (14.64)
16.95 (17.18)
17.41 (17.60)
30.68 (30.80)
33.45 (33.81)
33.38 (33.61)
38.56 (38.94)
41.96 (42.22)
41.89 (42.01)
3.54 (3.73)
2.63 (2.84)
2.66 (2.82)
3.58 (3.77)
2.86 (3.00)
2.73 (2.98)
16.14(15.97)
17.28 (17.52)
17.15 (17.42)
13.76 (13.97)
14.89 (15.15)
14.87 (15.07)
18.19
19.62
20.15
19.53
20.39
21.85
247
266
278
243
250
263
White

S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
247
Table 2
IR spectral data (in cmꢂ1) of 5-Fluorouracil(A), imidazole(B), benzimidazole(B) and their mixed ligand complexes (1–6).
Complex
m
(MAO)
m
(MAN)
m
(OH2)
m
(C@O) of C2 and
d(NAH) of N1 and
N3 pyrimidine
ring
m
(C5AF)
m
(N1AH)
m
(C@N)
m
(COOꢂ)as
m
(COOꢂ)s
C4 pyrimidine
ring
pyrimidine
ring
imidazole
ring
imidazole
ring
5-Fluorouracil(5-FU:
A)
–
–
–
1704, 1685
1513, 1430
1470
–
–
–
–
Imidazole(him: B)
Benzimidazole(bim:
B)
–
–
–
–
–
–
–
–
–
–
–
–
3380, 1475 1585
3355, 1460 1552
–
–
–
–
Ni(II)-5-FU(A)-
him(B)
461
522
3450–
3300, 846,
716
1698, 1666
1515,1409
1472
3375, 1472 1568
1575
1358
Cu(II)-5-FU(A)-
him(B)
Zn(II)-5-FU(A)-
him(B)
Ni(II)-5-FU(A)-
bim(B)
470
455
457
538
521
518
–
1695, 1653
1699, 1671
1701, 1659
1514, 1406
1516,1411
1512,1415
1473
1470
1468
3372, 1473 1572
3374, 1470 1566
3353. 1456 1537
1585
1560
1570
1362
1341
1347
–
3450–
3300, 835,
729
Cu(II)-5-FU(A)-
bim(B)
Zn(II)-5-FU(A)-
bim(B)
466
451
539
512
–
1705, 1662
1703, 1676
1517,1411
1514,1408
1471
1469
3351,1454
3354,1457
1541
1535
1566
1545
1348
1321
–
monodentate acetate ion can act as one of the ligating agent and
binds in the inner coordination sphere of the mixed ligand com-
plexes. This is further confirmed from the observed molar conduc-
tivity values i.e., non-electrolytic nature of the complexes. Thus,
the NMR study of Zn(II) complexes confirm the conclusions drawn
from the IR spectra on the mode of binding of the ligands.
EPR spectra of Cu(II) complexes (2 & 5)
EPR spectra of polycrystalline Cu(II)-5-FU(A)-him/bim(B) com-
plexes (2 & 5) were recorded on X-band at ambient and low
(300–77 K) temperatures in DMSO medium and the EPR spectrum
of Cu(II)-5-FU(A)-him(B) complex is shown in Fig. S1 (Supplemen-
tary data file). At 300 K, the EPR spectra of the complexes exhibit
well-resolved hyperfine structures due to magnetic coupling be-
tween the unpaired electron with effective 63,65Cu nuclei (I = 3/2).
In the case of frozen solution (77 K), the spectra show anisotropic
pattern for the powder samples in the parallel and perpendicular
regions, i.e., four well-resolved peaks and this may be assignable
to monomeric nature of the complexes [40]. This is further sup-
ported from the observed magnetic moment values of the Cu(II)
complexes in solid state. Axial symmetric with g-tensor values of
Hamiltonian parameters follow the order as:
g
(2.15) > g\
k
Fig. 1. Electronic absorption spectra of M(II)-5-FU(A)-him/bim(B) complexes (1–6)
(2.04) > ge (2.0023) for complex (2) and g (2.11) > g\ (2.03) > ge
k
in DMSO medium at room temperature (10ꢂ3 mol).
(2.0023) for complex (5) respectively. It clearly indicates that the
unpaired electron is localized in dx2
orbital [40] with 3d9 config-
ꢂy2
uration. The observed g-tensor values are less than 2.3, indicating
considerable covalent environment in the CuAL bond [40] which
is in conformity with the presence of mixed CuAN and CuAO
bonds in chelates. Further, the deviation of calculated g-tensor val-
ues 2.08 and 2.05 from the free electron value (ge = 2.0023) sup-
ports the covalent property. The absence of any half field signal
1H and 13C NMR spectra of Zn(II) complexes (3 & 6)
Both 1H and 13C NMR spectra of diamagnetic Zn(II)-5-FU(A)-
him/bim(B) complexes (3 & 6) were recorded in DMSO-d6 using
tetramethylsilane (TMS) as internal standard at room temperature.
The representative structure of the Zn(II)-5-FU(A)-him/bim(B)
complexes (3 & 6) are compared with the free ligands, 5-FU(A)
and him/bim(B) which are shown in Fig. 2. All the protons are
established to be in their predictable regions [39]. From the NMR
spectra it is proved that the 5-FU(A) and him/bim(B) ligands act
as bi and monodentate coordination around the Zn(II) ion via,
deprotonated AN3, carbonyl oxygen (AC4@O) and imidazole ring
AN3, atoms respectively. In addition, there is a peak found at
d = 1.81 and 1.85 ppm in the metal chelates indicate that the ace-
tate (OAc) molecule is coordinated with the Zn(II) ion [39]. Thus
at 1600 G corresponding to
magnetic exchange i.e., CuACu interactions in the complexes.
Bonding parameter values (
2 = 0.59 & 0.61 and b2 = 0.89 &
0.87) clearly indicate that the – bonding is more covalent than
in-plane
DMs = 2 transitions, ruling out any
a
r
p
– bonding [41,42]. Also the observed A|| values (138
and 132 ꢁ 10ꢂ4 cm) are comparable to the reported distorted tet-
rahedral Cu(II) complexes [42]. The geometry of the complexes
(2 & 5) are further inveterate by the empirical factor (f) i.e.,
g
f ¼ k , an index of tetragonal distortion. The reported (f) value for
N2OAkdonor groups around the copper ion for square planar com-

248
S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
Table 3
Absorption spectral data (in DMSO) and the magnetic moment values of mixed ligand complexes (1–6).
Complex
kmax
(cmꢂ1
Band
assignments
Geometry
leff
(BM)
Ligand field parameter
)
Dq
B (cmꢂ1
)
b
b (%)
LFSE
m2
m1
/
(cmꢂ1
)
(kJ molꢂ1
)
Ni(II)-5-FU(A)-
him(B)
10,362
17,153
25,840
32,258
3A2g(F) ? 3T2g(F) Distorted
3A2g(F) ? 3T1g(F) octahedral
3A2g(F) ? 3T1g(P)
3.09
1036
793.62 (B0 for free Ni(II)
ion = 1030)
0.77 22.95 145.83
1.66
LMCT (n ? pꢅ
)
Cu(II)-5-FU(A)-
him(B)
13,441
26,455
d–d Envelope
Distorted
tetrahedral
1.96
Dia.
3.15
–
–
–
–
–
–
–
–
–
LMCT (n ? pꢅ
)
Zn(II)-5-FU(A)-
him(B)
26,738
(b)
LMCT (M N)
Distorted
tetrahedral
–
–
–
Ni(II)-5-FU(A)-
bim(B)
10,225
16,920
26,455
33,898
3A2g(F) ? 3T2g(F) Distorted
3A2g(F) ? 3T1g(F) octahedral
3A2g(F) ? 3T1g(P)
1022
846.71
0.82 17.79 146.88
1.65
LMCT (n ? pꢅ
)
Cu(II)-5-FU(A)-
bim(B)
13,680
25,707
d–d Envelope
Distorted
tetrahedral
1.94
Dia.
–
–
–
–
–
–
–
–
–
–
–
–
LMCT (n ? pꢅ
)
Zn(II)-5-FU(A)-
bim(B)
27,624
(b)
LMCT (M N)
Distorted
tetrahedral
Fig. 2. 1H NMR and 13C NMR of the free ligands (A & B) and their Zn(II)-5-FU(A)-him/bim(B) complexes (3 & 6) in DMSO-d6 medium at room temperature.
plexes are in the order of 100–125 ꢁ 10ꢂ4 cm where as in Cu(II)
complexes (2 & 5) the observed (f) value is in the range, 130–
150 ꢁ 10ꢂ4 cm and the calculated empirical factor value (f = 156
and 159 ꢁ 10ꢂ4 cm) confirm distorted tetrahedral arrangement.
This is in agreement with the other reported values for distorted
tetrahedral environment around the copper(II) complexes
[41,42]. This is further supported from the calculated G-tensor va-
lue [43], which is a measure of the exchange interaction between
metal centers in polycrystalline states, is calculated by using the
perature range of ambient to 1100 °C under stable air condition.
Thermal decomposition of the metal complexes (1 & 6) occur in
four and three steps respectively. In Ni(II)-5-FU(A)-him(B) complex
(1), the first loss in weight occurs in the range of 80–150 °C, indi-
cating the presence of coordinated water molecules (H2O). These
results explain accordingly the analytical suggestions [44,45], i.e.,
(i) Lattice water, free ions and organic fragments that are not di-
rectly coordinated to the metal ions are found to leave the complex
at earlier stages compared with coordinated fragments, (ii) The
heating range (ambient to 1100 °C temperature) produces incom-
plete decomposition of M(II) complexes and (iii) the final products
are dependent on the type of M(II) ion and on (M ? L) affinity
which reflects the stability of complexes [44].
h
i
ðgkꢂge
Þ
Þ
expression G ¼
. Since the observed G values (3.92 and
ðg?ꢂge
3.89) are <4, the exchange interaction is considerable in the pres-
ent case and it may be due to the aligned parallel or slightly misa-
ligned environment of local tetragonal axes [43].
In the decomposition of Ni(II) complex (1), the first step corre-
sponds to the removal of two coordinated water molecules fol-
lowed by the removal of one acetate moiety in the second step.
In the third and fourth steps, the subsequent elimination of imida-
zoles (B) and 5-Fluorouracil(A) ligand moieties take place and
Thermogravimetric analyses
Thermogravimetric analyses were performed for Ni(II)-5-FU(A)-
him(B) and Zn(II)-5-FU(A)-bim(B) complexes (1 & 6) in the tem-

S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
249
finally it leaves air stable metal oxide (NiAO) as residue [44] at
very high temperatures, above 720 °C. The results are well agreed
with the composition of metal chelate and the final product is con-
firmed by IR studies. The thermogravimetric degradation of Ni(II)
complex (1) is represented in Scheme 2.
The Zn(II)-5-FU(A)-bim(B) complex (6) does not show any loss
in weight up to 180 °C, reveals that crystal water molecules or
coordinated water molecules are absent in the complex [45]. Also,
at 180 °C, the complex is further analysed by IR spectral studies
and their IR spectra exhibits absence of any broad band around
at ca. 3450–3300 cmꢂ1 which confirms the absence of water mol-
ecule in this complex. The TGA/DTA diagram of Zn(II)-5-FU(A)-
bim(B) complex (6) is shown in Fig. S2 (Supplementary data file).
The decomposition of Zn(II) complex (6) occurs in three steps.
The first step corresponds to the removal of coordinated acetate
moiety followed by the subsequent elimination of ligands, benz-
imidazole (B) and 5-Fluorouracil(A) at 294–343.71 and 382–
417.53 °C respectively. Finally, it leaves the air stable metal oxide
(ZnAO) as residue at very high temperatures from 700 °C onwards.
The results are in accord with the composition of metal chelates.
Further, the kinetic order of decomposition was calculated by the
Fig. 3. Proposed structure of M(II)-5-FU(A)-him/bim(B) complexes (1–6).
presence of 5-FU(A), imidazoles(B) moiety and acetate/water mol-
ecules in the mixed ligand complexes [47].
In the diffractogram, the strong peaks corresponding to 2(h) ap-
peared at 18.6, 25.6 and 34.8, for Ni(II) complex (1), at13.5, 22.5 &
29.8 for Cu(II) complex (2) and at 11.8, 19.6 & 28.7 for Zn(II) com-
plex (3), confirm the mixed ligand complex formation. Also there
are a large number of feeble peaks observed in the mixed ligand
complexes indicate uniform phase with no impurity. The XRD pat-
tern shows microcrystalline nature of the complexes and this
behavior is due to the incorporation of water and acetate mole-
cules into the coordination sphere. The average crystalline size of
the synthesised mixed ligand complexes are found to be 57.9 nm,
66.6 nm and 61.8 nm for Ni(II), Cu(II) and Zn(II) respectively as cal-
culated from the main X-ray diffraction peaks using the Scherre’s
equation [47],
h
i
ðWsꢂWf Þ
modified Horowitz and Metzger equation [46], Cs ¼
and
ðWoꢂWf Þ
the observed Cs values (0.64–0.60) of the mixed ligand complexes
(1 & 6) indicate that the decomposition follows first order kinetics.
Based on the conductivity measurements, magnetic moment,
spectral studies (IR spectra, 1H and 13C NMR spectra) and thermal
analyses of mixed ligand complexes (1–6), it is confirmed that
Ni(II) complexes (1 & 4) have distorted octahedral environment
with stoichiometry [Ni(II)-5-FU(A)-him/bim(B)(OH2)2 (OAc)] while
Cu(II)/Zn(II) complexes have distorted tetrahedral geometry with
[M(II)-5-FU(A)-him/bim(B)(OAc)] stoichiometry (Fig. 3).
D ¼ 0:9k=b cos h
where k is the wavelength of X-ray radiation (k = 0.154 nm), b is the
full width half maximum of the characteristic peak (in radians) cor-
rected for instrumental broadening, h is Bragg diffraction angle for
hkl plane and D is the crystalline size (nm). This crystalline value
suggests that the synthesized mixed ligand complexes are in micro-
crystalline state which confirms the proposed composition.
Morphology and particle size of the MAB type mixed ligand
complexes of Ni(II) and Cu(II)-5-FU(A)-bim(B) complexes (4 & 5)
were illustrated from the scanning electron microscopic picto-
graph (Fig. 5). From the SEM pictographs, it is inferred that the
mixed ligand complexes have uniform matrix with smooth inter-
face having perfect regular shape of homogeneous phase material.
A mixture of wrecked brick and crumb like shapes is observed in
Powder X-ray diffraction and SEM analysis
Powder X-ray diffraction studies and SEM analysis are useful to
determine the structure, particle size and morphology of the syn-
thesized mixed ligand complexes. The X-ray diffractogram of the
synthesised mixed ligand complexes of Ni(II)/Cu(II)/Zn(II)-5-
FU(A)-him(B) complexes (1–6) are shown in Fig. 4.
The observed d-space values for the mixed ligand complexes
are compared with the standard d-space values of the free metal(II)
ions, free ligands (A & B), M(II)-5-substituted uracil derivatives,
Ni(II)-(him)2 (NO3)2 and Cu(II)-(him)2 Cl2 complexes from JCPDS
data files. The experimental d-space values of the mixed ligand
complexes match with the JCPDS data d-space values which are
shown in Table S1 (Supplementary data file) which confirm the
the Ni(II) complex (4) with the particle size 35
of Cu(II) complex (5) is obtained as 20 m with huge structure of
regular micro-crystalline shape of germ like phase. This leads to
lm. Particle size
l
Scheme 2. Proposed thermogravimetric depiction of Ni(II)-5-FU(A)-him(B) complex (3).

250
S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
In vitro antimicrobial activity
In vitro antimicrobial activities of the free ligands viz., of
5-Fluorouracil(A), imidazole(B), benzimidazole(B) and their
M(II)-5-FU(A)-him/bim(B) complexes (1–6) were tested against
five pathogenic bacteria and three fungal strains by well diffusion
method using agar as nutrient (concentration of each com-
plex = 30 lg in DMSO solution). Measured zone of inhibition (in
mm) against the growth of bacterial and fungal for the above sys-
tems are given in Table S2 (Supplementary data file) and are shown
in Fig. 6. The antimicrobial activity of the free ligands and their
complexes are compared with the commercially available standard
drugs, tetracycline (antibacterial control) and amphotricine (anti-
fungal control). The antimicrobial activities of the mixed ligand
complexes depend upon the size, charge distribution, shape and
redox potential of the M(II) ions [48].
All the mixed ligand complexes show moderate activity than
the corresponding free ligands against different types of microor-
ganism. Variations in the effectiveness of different biocidal species
Fig. 4. X-ray diffraction patterns of M(II)-5-FU(A)-him(B) complexes (1–3).
believe that the synthesized mixed ligand complexes have homo-
geneous phase material.
Fig. 6. Biological activities of (a) free ligands (A & B) & M(II)-5-FU(A)-him(B)
complexes (1–3) and (b) free ligands (A & B) & M(II)-5-FU(A)-bim(B) complexes
(4–6) with different microorganisms.
Fig. 5. SEM photographs of (a) Ni(II)-5-FU(A)-bim(B) complex (4) and (b) Cu(II)-5-
FU(A)-bim(B) complex (5).

S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
251
against microorganisms depend on the impermeability of the cell
Interaction of DNA with ligands and their mixed ligand complexes
DNA binding studies by electronic absorption method
of the microbes or the changes in the nature of ribosomes of micro-
bial cells [49]. Higher inhibition zones of mixed ligand complexes
can be explained on the basis of Overtone’s concept and Tweedy’s
chelation theory [50,51]. The significance of this work lies that,
these mixed ligand complexes could be applied reasonably in heal-
ing of some common diseases like septicaemia, gastroenteritis, uri-
nary tract illness and hospital acquired infections caused by
Bacillus subtilis and Escherichia coli [52]. In addition, the distinc-
tive possessions of the investigated mixed ligand complexes could
be applied safe and sound in the treatment of infections caused by
any of these particular strains. All the mixed ligand complexes
show more significant antibacterial and less pronounced anti-
fungal activities. This enhanced activities may be due to the pres-
ence of additional electron withdrawing [19,20] group present in
5-Fluorouracil (C5AF) and also the imidazole moieties in them.
The results clearly show that the order of antimicrobial activity,
among the mixed ligand complexes, is Control > (5) > (2) >
(1) > (4) > (3) > (6).
Electronic absorption spectral technique is used to investigate
the interaction of the complexes (1–3) with CT DNA. Electronic
spectra of the Ni(II), Cu(II) and Zn(II)-5-FU(A)-him(B) complexes
(1–3) in the absence and presence of CT DNA are shown in
Fig. S3 (Supplementary data file). When the magnitude of CT
DNA is increased, the absorption bands of the complexes are also
affected (Table 5). The binding of the metal complexes to DNA
through intercalation can be observed usually from the reduction
in the intensity of the charge transfer bands to the extent of 5–
15% along with the red shift (bathochromic shift). In general, a
red shift is associated with the strong intercalative binding be-
tween the metal complexes and the base pairs of CT DNA helix
[54].
The intrinsic binding constant (Kb) is determined from the plot
of [DNA]/(ea ꢂ ef) versus [DNA] from the absorption maxima in the
range of 240–320 nm. The intrinsic binding constant for Ni(II)/
Cu(II)/Zn(II)-5-FU(A)-him(B) complexes (1–3) are 3.215, 5.922
and 2.469 ꢁ 104 molꢂ1 respectively. From the observed values, it
is clear that Cu(II)-5-FU(A)-him(B) complex (2) has significantly
enhanced binding affinity when compared to Ni(II) and Zn(II) com-
plexes. In addition, these binding constant values follow the order
of stability. The higher binding constant value observed in the
Cu(II) complex (2) may be due to its ability to counterbalance
the dinegative charge of the phosphate sugar backbone of the
DNA helix to some extent [55]. From the intrinsic binding constant
In vitro antioxidant activity
Antioxidant activities of 5-Fluorouracil(A) and its mixed ligand
complexes Ni(II)/Cu(II)/Zn(II)-5-FU(A)-him/bim(B), (1–6) at 37 °C
were studied in vitro by DPPH free radical scavenging methods.
DPPH is a stable free radical that is often used for finding the rad-
ical scavenging activity in chemical analysis [53]. Ascorbic acid
(AA) is used as the reference or positive control. All the analyses
were done in three replicates and the results are averaged
(Table 4).
(Kb), the free energy change (D
àG) values are also calculated
(Table 5) and these results indicate that the mixed ligand com-
plexes can interact with the DNA in a spontaneous manner.
Reduction capability of DPPH radical is determined by the de-
crease in its absorbance at 517 nm (blank) which can be induced
by the antioxidant. From the results, it is seen that the mixed li-
gand complexes have higher activities than free ligands which
may be due to the presence of the metal moiety and 5-Fluoroura-
cil(A) which itself shows some activity [53].
Oxidative DNA cleavage studies by gel electrophoresis method
Cleavage efficiency of the mixed ligand complexes with CT DNA
is studied by gel electrophoresis method in the presence of an oxi-
dant (H2O2). A representative cleavage pictograph of 5-Fluoroura-
cil(A) and their mixed ligand complexes (1–6) are shown in
Fig. S4 (Supplementary data file). The DNA cleavage efficiency of
Table 4
In vitro antioxidant activities of 5-Fluorouracil(A) and M(II)-5-FU(A)-him/bim(B) complexes (1–6) by DPPH free radical scavenging assay method at different concentrations (in
mol).
l
Complex
Scavenging activity (%)a
10 mol
l
20
lmol
30
l
mol
40
lmol
50 lmol
Ascorbic acidb
5-Fluorouracil(A)
85 0.17
22 0.25
36 0.20
–
31 0.19
34 0.24
–
88 0.18
26 0.28
39 0.26
46 0.30
35 0.25
37 0.29
41 0.41
28 0.20
90 0.20
32 0.27
41 0.31
50 0.32
38 0.36
42 0.32
49 0.44
33 0.34
92 0.20
34 0.29
46 0.37
53 0.34
42 0.43
44 0.35
54 0.46
37 0.42
95 0.21
36 0.33
51 0.41
58 0.33
48 0.42
48 0.39
57 0.48
44 0.49
Ni(II)-5-FU(A)-him(B)
Cu(II)-5-FU(A)-him(B)
Zn(II)-5-FU(A)-him(B)
Ni(II)-5-FU(A)-bim(B)
Cu(II)-5-FU(A)-bim(B)
Zn(II)-5-FU(A)-bim(B)
–
a
Standard deviation (average of three replicates) and ‘‘–’’ denotes less activity.
Ascorbic acid is used as the standard control.
b
Table 5
Intrinsic binding constant (Kb) and free energy change (D
àG) of mixed ligand M(II)-5-FU(A)-him(B) complexes with variation of CT DNA at 37 °C.
a
b
Complex
kmax (nm)
D
k (nm)
H (%)
Kb /mol–1
(D )
àG) (kJ molꢂ1
Free
Bound
Ni(II)-5-FU(A)-him(B)
Cu(II)-5-FU(A)-him(B)
Zn(II)-5-FU(A)-him(B)
263
260
261
268
267
265
5
7
4
12.09
18.41
11.82
3.215 ꢁ 104
5.922 ꢁ 104
2.469 ꢁ 104
ꢂ86.29
ꢂ91.32
ꢂ84.10
h
i
ðAfree ꢂAfound
Þ
a
Hð%Þ ¼
ꢁ 100%.
Afree
b
Kb = Intrinsic DNA binding constant.

252
S. Shobana et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 126 (2014) 242–253
the M(II)-5-FU(A)-him/bim(B) complexes are due to the different
extent of affinity of the complexes towards DNA. From the picto-
graph, it is shown that the nature of M(II) ions play an important
role in the cleavage of isolated DNA. Control experiment using CT
DNA alone (Lane C) does not show any significant cleavage even
on longer exposure time. In general, the oxidative cleavage is pro-
posed to account the cleavage of CT DNA caused by M(II) ions
reacting with the co-reactant H2O2 to produce the molecular oxy-
gen or peroxy derivatives or diffusible hydroxyl radicals via the
abstraction of a hydrogen atom from sugar units (usually at C04 po-
sition). This also predicts the release of specific residues arising
from transformed sugars, depending on the position from which
the hydrogen atom is removed and finally cleaves CT DNA [56].
Further, the reaction is modulated by a metallo complex bound
to a peroxo species generated from the co-reactant H2O2, which
may damage DNA through Fenton type chemistry [57]. The results
from the pictograph shows that three out of seven systems includ-
ing the free 5-FU(A) ligand have the ability to cleave the CT DNA in
the presence of oxidant. In the present experiment, the mixed li-
gand complexes show a considerable extent of cleavage than the
free ligand(A).
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Acknowledgements
All of us dedicate this paper to our beloved late Professors Dr.
T.C. Manohar and K. Natesan, Department of Chemistry and Re-
search Centre, South Travancore Hindu College, Nagercoil, India.
PSM and SS thank the Management of Aditanar College of Arts
and Science, Tiruchendur for providing Research facilities. We are
grateful to STIC, CUSAT, Cochin for providing the analytical
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