Structure, DNA- and albumin-binding of the manganese(II) complex with the non-steroidal antiinflammatory drug niflumic acid

Article abstract of DOI:10.1016/j.poly.2013.01.049

The manganese(II) complex with the non-steroidal antiinflammatory drug niflumic acid has been synthesized and characterized. The crystal structure of the complex [Mn(O-niflumato)₂(methanol)₄] has been determined by X-ray crystallography, where a monodentate coordination of niflumato ligand was revealed. Niflumic acid and its Mn(II) complex exhibit good binding affinity to human or bovine serum albumin proteins with high binding constant values. UV study of the interaction of the compounds with calf-thymus DNA (CT DNA) has shown that the compounds can bind to CT DNA and [Mn(O-niflumato)₂(methanol)₄] exhibits higher binding constant to CT DNA than free niflumic acid. The compounds can bind to CT DNA via intercalation as concluded by DNA solution viscosity measurements. Competitive studies with ethidium bromide (EB) have shown that the compounds can displace the DNA-bound EB suggesting strong competition with EB.

Full text of DOI:10.1016/j.poly.2013.01.049

Polyhedron 53 (2013) 215–222
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Polyhedron
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Structure, DNA- and albumin-binding of the manganese(II) complex with the
non-steroidal antiinflammatory drug niflumic acid
Paulina Tsiliki a, Franc Perdih b, Iztok Turel b, George Psomas a,
⇑
a Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece
b Faculty of Chemistry and Chemical Technology, University of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
a r t i c l e i n f o
a b s t r a c t
Article history:
The manganese(II) complex with the non-steroidal antiinflammatory drug niflumic acid has been synthe-
sized and characterized. The crystal structure of the complex [Mn(O-niflumato)2(methanol)4] has been
determined by X-ray crystallography, where a monodentate coordination of niflumato ligand was
revealed. Niflumic acid and its Mn(II) complex exhibit good binding affinity to human or bovine serum
albumin proteins with high binding constant values. UV study of the interaction of the compounds with
calf-thymus DNA (CT DNA) has shown that the compounds can bind to CT DNA and [Mn(O-niflumato)2(-
methanol)4] exhibits higher binding constant to CT DNA than free niflumic acid. The compounds can bind
to CT DNA via intercalation as concluded by DNA solution viscosity measurements. Competitive studies
with ethidium bromide (EB) have shown that the compounds can displace the DNA-bound EB suggesting
strong competition with EB.
Received 18 December 2012
Accepted 29 January 2013
Available online 8 February 2013
Keywords:
Manganese(II) complexes
Non-steroidal antiinflammatory drugs
Niflumic acid
Interaction with DNA
Interaction with albumins
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
diseases and relieve acute pain and it is effective against period
pains, pain after surgery, and fever [7,8]. The crystal structures of
Non-steroidal antiinflammatory drugs (NSAIDs), among the
most used analgesic, antiinflammatory and antipyretic agents [1],
act through inhibition of the cyclo-oxygenase (COX)-mediated pro-
duction of prostaglandins [2], while they have also presented a
synergistic role on the activity of certain antitumor drugs leading
a series of cancer cell lines to cell death via apoptosis [3]. As a
means to explain the tentative anticancer and antiinflammatory
activity of the NSAIDs and their complexes, the interaction with
DNA is considered of great importance and should be further eval-
uated, although few relevant reports on the interaction of NSAIDs
and their complexes with DNA have been published so far [4,5].
The chemical classes of NSAIDs comprise phenylalkanoic acids,
salicylate derivatives, anthranilic acids, oxicams, sulfonamides and
furanones [6]. Niflumic acid (=Hnif, Scheme 1) is a NSAID of N-
phenylanthranilic acid derivatives and resembles chemically to
mefenamic, tolfenamic and flufenamic acid and other fenamates
in clinical use [6]. Hnif is used to treat inflammatory rheumatoid
two dinuclear [8,9] and two mononuclear copper(II) complexes
[10,11] and a silver(I) [12] complex of niflumic acid have been re-
ported in the literature.
Manganese, one of the most significant biometals, is found in the
active center of many enzymes of diverse functionality [13,14]. It is
known that, like many other metal ions, also hydrated manga-
nese(II) ions interact with DNA [15]. The crystal structure of oligonu-
cleotide in the presence of manganese(II) revealed among other the
involvement of the metal in the formation of cross-links between
neighbor duplexes [16]. The crystal structure of nucleosome core
particle has shown that manganese(II) interacts with N7 atom of
guanines and adenines [17]. Manganese-containing compounds
SC-52608 and Teslascan are used in medicine as anticancer and
MRI contrast agents, respectively [18], and an increasing number
of manganese complexes exhibit biological interest showing anti-
bacterial [19,20], anticancer [21–23] and antifungal [24] activity.
Furthermore, a thorough search of the literature has not revealed
any structurally characterized manganese complexes with NSAIDs.
The interaction of carboxylate-containing non-steroidal antiin-
flammatory drugs with Co2+, Cu2+ and Zn2+ [25–32] has been the
subject of our recent studies covering the characterization of the
resultant complexes and the interaction of these metal complexes
with biomolecules such as DNA and serum albumin proteins, in an
attempt to examine their mode of binding and possible biological
relevance. Having in mind the significance of NSAIDs in medicine,
the activity of manganese complexes and potential synergetic
Abbreviations: bipy, 2,20-bipyridine; CT, calf-thymus; bipyam, 2,20-bipyridyl-
amine; BSA, bovine serum albumin; COX, cyclo-oxygenase; DMF, N,N-dimethyl-
formamide; DMSO, dimethylsulfoxide; EB, ethidium bromide, 3,8-diamino-5-ethyl-
6-phenyl-phenanthridinium bromide; Hnif, niflumic acid, 2-[3-(trifluoro-
methyl)anilino]nicotinic acid; HSA, human serum albumin; NSAID, non-steroidal
antiinflammatory drug; phen, 1,10-phenanthroline; py, pyridine; SA, serum albu-
min; sh, shoulder; vs, very strong;
D, masym(CO2)–msym(CO2).
⇑
Corresponding author. Tel.: +30 2310997790; fax: +30 2310997738.
E-mail address: gepsomas@chem.auth.gr (G. Psomas).
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.01.049

216
P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
1 h. The solution was added dropwise to a methanolic solution
(10 mL) of MnCl2ꢀ4H2O (0.2 mmol, 40 mg). Colorless crystals of
[Mn(nif)2(MeOH)4] 1 suitable for X-ray structure determination
were collected after a few days. Yield: 105 mg, 70%. Anal. Calc.
for [Mn(nif)2(MeOH)4] (C30H32F6MnN4O8) (MW = 745.54): C,
48.33; H, 4.33; N, 7.52. Found: C, 47.69; H, 4.12; N, 7.25%. IR
(KBr pellet):
CO2): 1389 (vs);
nm (
/Mꢁ1 cmꢁ1) as nujol mull: 327, 296; in DMSO: 331 (sh)
(4500), 295 (22500).
m
max/cmꢁ1
asym(CO2)–
m
asym(CO2): 1606 (very strong (vs));
msym(-
D
=
m
m
sym(CO2): 217 cmꢁ1; UV–Vis: k/
e
leff = 5.95 BM at room temperature. The com-
Scheme 1. Niflumic acid (=Hnif).
plex is soluble in DMF and DMSO (KM = 7 mho cm2 molꢁ1, in 1 mM
DMSO solution).
effects, we present the synthesis and the structural characteriza-
tion of the mononuclear Mn(II) complex with the NSAID niflumic
acid [Mn(nif)2(MeOH)4], 1. The crystal structure of complex 1 has
been determined by X-ray crystallography. Additionally, the bio-
logical properties of complex 1 including its binding to CT DNA
investigated by UV spectroscopy and viscosity measurements, its
ability to displace ethidium bromide (EB) as a means to investigate
the existence of a potential intercalation to CT DNA in competition
to the classical DNA-intercalator EB studied by fluorescence spec-
troscopy, and its affinity to bovine (BSA) and human serum albu-
min (HSA) – binding to these proteins involved in the transport
of metal ions and metal–drug complexes through the blood stream
may result in lower or enhanced biological properties of the origi-
nal drug, or new paths for drug transportation – investigated by
fluorescence spectroscopy, have been evaluated and compared to
those of free niflumic acid.
The addition of an N,N0-donor ligand such as bipy (0.2 mmol,
31 mg), phen (0.2 mmol, 36 mg) or bipyam (0.2 mmol, 34 mg) to
the reaction solution has resulted in the isolation of crystalline
product of complex 1, and no coordination of the N,N0-donor was
observed.
2.3. X-ray structure determination
Single-crystal X-ray diffraction data were collected at room
temperature on an Agilent Technologies SuperNova Dual with an
Atlas detector using mirror-monochromatized Mo K
a radiation
(k = 0.71073 Å). The data were processed using CRYSALIS PRO [33].
Structure was solved by direct methods implemented in SIR97
[34] and refined by a full-matrix least-squares procedure based
on F2 with SHELXL-97 [35]. All non-hydrogen atoms were refined
anisotropically. All hydrogen atoms were readily located in differ-
ence Fourier maps and were subsequently treated as riding atoms
in geometrically idealized positions with Uiso(H) = kUeq(C, N),
where k = 1.5 for NH and methyl groups, which were permitted
to rotate but not to tilt, and 1.2 for all other H atoms. Hydrogen
atoms bonded to methanol O3 and O4 atoms were refined using
DFIX instruction. The –CF3 group is disordered over two positions
in ratio 0.59:0.41. Crystallographic data are listed in Table 1.
2. Experimental
2.1. Materials – instrumentation – physical measurements
Niflumic acid, MnCl2ꢀ4H2O, 2,20-bipyridylamine (=bipyam), 1,10-
phenanthroline (=phen), 2,20-bipyridine (=bipy), KOH, trisodium cit-
rate, NaCl, CT DNA, BSA, HSA and EB were purchased from Sigma–Al-
drich Co and all solvents were purchased from Merck. All chemicals
and solvents were reagent grade and were used as purchased.
DNA stock solution was prepared by dilution of CT DNA to buf-
fer (containing 150 mM NaCl and 15 mM trisodium citrate at pH
7.0) followed by exhaustive stirring at 4 °C for three days, and kept
at 4 °C for no longer than a week. The stock solution of CT DNA
gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of
1.88, indicating that the DNA was sufficiently free of protein con-
tamination. The DNA concentration was determined by the UV
2.4. Albumin binding studies
Protein binding studies have been performed by tryptophan
fluorescence quenching experiments using bovine (BSA, 3
lM) or
human serum albumin (HSA, 3 M) in buffer (containing 15 mM
l
trisodium citrate and 150 mM NaCl at pH 7.0). The quenching of
the emission intensity of BSA or HSA tryptophan residues at
343 nm or 351 nm, respectively, was monitored using Hnif and
complex 1 as quenchers with increasing concentration (up to
absorbance at 260 nm after 1:20 dilution using
[26–28].
e
= 6600 Mꢁ1 cmꢁ1
Table 1
Infrared (IR) spectra (400–4000 cmꢁ1) were recorded on a Nico-
let FT-IR 6700 spectrometer with samples prepared as KBr pellets.
UV–Visible (UV–Vis) spectra were recorded as nujol mulls and in
solution at concentrations in the range 10ꢁ5–10ꢁ3 M on a Hitachi
U-2001 dual beam spectrophotometer. Room temperature mag-
netic measurements were carried out on a magnetic susceptibility
balance of Sherwood Scientific (Cambridge, UK). C, H and N ele-
mental analysis were performed on a Perkin–Elmer 240B elemen-
tal analyzer. Molar conductivity measurements were carried out in
1 mM DMSO solution of the complexes with a Crison Basic 30 con-
ductometer. Fluorescence spectra were recorded in solution on a
Hitachi F-7000 fluorescence spectrophotometer. Viscosity experi-
ments were carried out using an ALPHA L Fungilab rotational vis-
cometer equipped with an 18 mL LCP spindle.
Crystallographic data for complex 1.
Complex 1
Formula
Fw
T (K)
Crystal system
Space group
a (Å)
C30H32F6MnN4O8
745.54
293(2)
orthorhombic
Pbca
16.1718(3)
9.8618(2)
21.0311(4)
90.00
90.00
90.00
3354.10(11)
4
1.476
b (Å)
c (Å)
a
(°)
b (°)
(°)
c
Volume (Å3)
Z
D(calc) (Mg mꢁ3
)
Abs. coef.,
GOF on F2
R1=
l
(mmꢁ1
)
0.481
1.046
2.2. Synthesis of [Mn(nif)2(MeOH)4], 1
0.0364a
0.0907
wR2=
A
methanolic solution (10 mL) containing niflumic acid
a
3023 reflections with I > 2r(I).
(0.4 mmol, 112 mg) and KOH (0.4 mmol, 22 mg) was stirred for

P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
217
2.2 ꢂ 10ꢁ5 M) [36]. Fluorescence spectra were recorded from 300
of the carboxylato group, respectively. The difference
C@O)–msym(C@O)] is a useful characteristic tool for determining
the coordination mode of the carboxylato ligands and has a value
of 217 cmꢁ1 which is indicative of monodentate binding mode
for the niflumato ligands [37].
The UV–Vis spectra of complex 1 have been recorded as nujol
mull and in DMSO solution (Fig. S1) and are similar suggesting that
it retains its structure in solution. In these spectra, two bands
attributed to intraligand transitions appeared. In addition, the fact
that the complex has the same UV–Vis spectral pattern in nujol and
in DMSO solution as well as in the presence of the buffer solution
used in the biological experiments in combination to the molar
conductivity measurements suggests that it keeps its integrity in
solution [28].
D
[=
m
asym(-
to 500 nm at an excitation wavelength of 296 nm. The compounds
do not emit any significant fluorescence under the same experi-
mental conditions. The Stern–Volmer and Scatchard equations
and graphs have been used in order to study the interaction of each
quencher with serum albumins.
2.5. DNA-binding studies
The interaction of Hnif and complex 1 with CT DNA has been
studied with UV spectroscopy in order to investigate the possible
binding modes to CT DNA and to calculate the binding constants
to CT DNA (Kb). In UV titration experiments, the spectra of CT
DNA in the presence of each compound have been recorded for a
constant CT DNA concentration in diverse [compound]/[CT DNA]
mixing ratios (r). The binding constants, Kb, of the compounds with
CT DNA have been determined using the UV spectra of the com-
pound recorded for a constant concentration in the absence or
presence of CT DNA for diverse r values. Control experiments with
DMSO were performed and no changes in the spectra of CT DNA
were observed.
The observed value of leff (=5.95 BM) for the complex is close to
the spin-only value (=5.92 MB) at room temperature and typical
for mononuclear high-spin Mn(II) complexes with d5 configuration
(S = 5/2) [38,39].
3.2. Description of the crystal structure of [Mn(niflumato)2(MeOH)4]
A diagram of 1 is shown in Fig. 1, and selected bond distances
and angles are listed in Table 2. The complex is mononuclear with
the niflumato ligand behaving as a monodentate deprotonated li-
gand coordinated to manganese atom via a carboxylate oxygen.
The structure of the complex is centrosymmetric, the manga-
nese(II) ion is sitting on a center of symmetry and is coordinated
to two niflumato ligands and four methanol molecules related by
the inversion center. Thus, the manganese atom is six-coordinate
and displays an octahedral geometry. All the Mn–O distances are
typical of Mn(II)–O bond distances with the Mn–Ocarboxylate
(Mn(1)–O(1) = 2.1287(11) Å) being shorter than the Mn–Omethanol
(Mn(1)–O(3) = 2.1923(12), Mn(1)–O(4) = 2.2148(13) Å). Taking
into account the differences found in the Mn–O distances in
combination with the angles around manganese (O(1)–Mn(1)–
O(3) = 92.20(5)°, O(1)–Mn(1)–O(4) = 89.38(5)° and O(3)–Mn(1)–
Viscosity experiments were carried out using an ALPHA L Fun-
gilab rotational viscometer equipped with an 18 mL LCP spindle
and the measurements were performed at 100 rpm. The viscosity
of a DNA solution has been measured in the presence of increasing
amounts of the compounds. The relation between the relative solu-
tion viscosity (
L/L0 = (
1/3, where L0 denotes the apparent molecular length in
the absence of the compound [27–29]. The obtained data are pre-
g/g0) and DNA length (L/L0) is given by the equation
g/g0)
1/3
sented as (
g/g0)
versus r, where g is the viscosity of DNA in the
presence of the compound, and g0 is the viscosity of DNA alone in
buffer solution.
The competitive studies of each compound with EB have been
investigated with fluorescence spectroscopy in order to examine
whether the compound can displace EB from its CT DNA–EB com-
plex. The CT DNA–EB complex was prepared by adding 20
lM EB
O(4) = 90.56(5)°), the octahedron displays
a slight distortion.
and 26 M CT DNA in buffer (150 mM NaCl and 15 mM trisodium
l
citrate at pH 7.0). The intercalating effect of the compounds with
the DNA–EB complex was studied by adding a certain amount of
a solution of the compound step by step into the solution of the
DNA–EB complex. The influence of the addition of each compound
to the DNA–EB complex solution has been obtained by recording
the variation of fluorescence emission spectra.
3. Results and discussion
3.1. Synthesis and spectroscopy
Complex 1 has been synthesized under aerobic conditions with
the addition of MnCl2 to deprotonated niflumic acid. The complex
has been also isolated in the presence of an N,N0-donor ligand such
as bipy, phen or bipyam; the N,N0-donor ligand was not coordi-
nated to manganese as concluded by IR spectroscopy and X-ray
crystallography. Complex 1 is soluble in DMSO and DMF and is
not electrolyte (KM = 7 mho cm2 molꢁ1, in 1 mM DMSO solution;
a value indicative of non-dissociation in DMSO solution).
Fig. 1. The molecular structure of 1 with only the heteroatoms labelled. Hydrogens
and atoms in disorder are omitted for clarity.
Table 2
Selected bond distances and angles for complex 1.
Distance
(Å)
Distance
(Å)
Mn(1)–O(1)
Mn(1)–O(4)
O(2)–C(1)
2.1287(11)
2.2148(13)
1.253(2)
Mn(1)–O(3)
2.1923(12)
The deprotonation and binding mode of niflumic acid has been
confirmed by IR spectroscopy. In the IR spectrum of Hnif, the
O(1)–C(1)
O(3)–C(14)
1.2615(19)
1.406(2)
absorption band at 3379(br, m) cmꢁ1, attributed to the
m
(H–O)
stretching vibration disappears upon binding to manganese. The
bands at 1663(s) cmꢁ1 and 1284(s) cmꢁ1 attributed to
(C@O)car-
and (C–O)carboxylic stretching vibrations of the carboxylic
O(4)–C(15)
1.420(3)
Angle
(°)
Angle
(°)
O(1)–Mn(1)–O(1)0
O(1)–Mn(1)–O(3)
O(1)–Mn(1)–O(3)0
O(1)–Mn(1)–O(4)
O(1)–Mn(1)–O(4)0
180.00(7)
92.20(5)
87.80(5)
89.38(5)
90.62(5)
O(3)–Mn(1)–O(3)0
O(3)–Mn(1)–O(4)0
O(3)–Mn(1)–O(4)
O(4)–Mn(1)–O(4)0
180.00(8)
89.44(5)
90.56(5)
180.00(9)
m
m
boxylic
moiety (–COOH), respectively, shift in the IR spectra of the complex
at 1606 cmꢁ1 and 1389 cmꢁ1 assigned to the antisymmetric,
m
asym(C@O), and the symmetric, msym(C@O), stretching vibrations

218
P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
Table 3
Hydrogen bonding interactions in 1.
D–Hꢀ ꢀ ꢀA
D–H (Å)
Hꢀ ꢀ ꢀA (Å)
1.96
1.972(11)
0.832(10)
Dꢀ ꢀ ꢀA (Å)
2.6264(18)
2.7916(18)
2.6254(16)
D–Hꢀ ꢀ ꢀA (°)
133.6
171(3)
161(2)
Symmetry transformation for acceptors
N(2)–H(2A)ꢀ ꢀ ꢀO(2)
O(3)–H(1)ꢀ ꢀ ꢀN(1)
O(4)–H(2)ꢀ ꢀ ꢀO(2)
0.86
0.826(10)
0.832(10)
x, y, z
x, y + 1, z
x, y, z
Fig. 2. Hydrogen bonding in 1. Disorder on –CF3 group and hydrogen atoms not involved in the motif shown have been omitted for clarity. Symmetry codes: (i) ꢁx, ꢁy, ꢁz; (ii)
x, y + 1, z.
The carboxylate group is asymmetrically bound to manganese
(C(1)–O(1) = 1.2615(9) Å and C(1)–O(2) = 1.253(2) Å). Similar
arrangement of a monodentate carboxylate NSAID around the
500
400
central metal ion has been also observed in the crystal structure
of [Co(O-mefenamato)2(MeOH)4] [26].
The structure of 1 is stabilized by intramolecular O–Hꢀ ꢀ ꢀO and
N–Hꢀ ꢀ ꢀO hydrogen bonding between methanolic or NH group as
hydrogen-bond donors and the carbonyl moiety of the niflumato
ligand as hydrogen bonding acceptor (Table 3). An infinite chain
is formed due to O–Hꢀ ꢀ ꢀN intermolecular hydrogen bonding be-
tween methanolic group and the aromatic nitrogen atom of the
niflumato ligand (Fig. 2).
300
200
100
0
300
350
400
450
500
λ
(nm)
3.3. Interaction with serum albumins
Fig. 3. Emission spectra (kexit = 295 nm) of HSA ([HSA] = 3 lM) in buffer solution in
the absence and presence of increasing amounts of complex 1 (r = [1]/[HSA] = 0–6).
The arrow shows the changes of intensity upon increasing amounts of 1.
Serum albumin (SA) is the most abundant protein in plasma; its
main role is the transport of metal ions, drugs and their metal com-
plexes through the blood stream [40]. Human serum albumin
(HSA) has a tryptophan at position 214 and its most extensively
studied structural homolog bovine serum albumin (BSA) has two
tryptophans, Trp-134 and Trp-212 [41]. HSA and BSA solutions ex-
hibit, when excited at 295 nm, an intense fluorescence emission
with kem,max = 351 nm and 343 nm, respectively, which is attrib-
uted to the tryptophans [36]. Hnif and complex 1 do not emit
any significant fluorescence under the same experimental condi-
tions and the quenching occurring in the HSA or BSA fluorescence
emission spectra upon addition of Hnif or 1 (Fig. 3) are primarily
due to change in protein conformation, subunit association, sub-
strate binding or denaturation [41].
Addition of Hnif and complex 1 to SA solution results in moder-
ate to significant quenching of HSA fluorescence at k = 351 nm
(Fig. 4(A)) (quenching of 62% of the initial fluorescence intensity
for Hnif and 79% for 1) and to a much more enhanced quenching
of the BSA fluorescence at k = 343 nm (Fig. 4(B)) (quenching of
86% of the initial fluorescence intensity for Hnif and 98% for 1).
The observed quenching may be due to possible changes in protein
secondary structure leading to changes in tryptophan environment
of HSA, and thus indicating the binding of each complex to the
albumins [42].
The Stern–Volmer and Scatchard equations and graphs are used
in order to evaluate the interaction of a quencher with serum albu-
mins. From Stern–Volmer quenching equation [36]:
Io
I
¼ 1 þ kqt0½Qꢃ ¼ 1 þ KSV½Qꢃ
ð1Þ
where Io = the initial tryptophan fluorescence intensity of SA, I = the
tryptophan fluorescence intensity of SA after the addition of the
quencher (i.e. Hnif and complex 1), kq = the quenching rate con-
stants of SA, KSV = the dynamic quenching constant, so = the average
lifetime of SA without the quencher and [Q] = the concentration of
the quencher, the dynamic quenching constant (KSV, in Mꢁ1) can
Io
I
be obtained by the slope of the diagram versus [Q] (Figs. S2 and
S3). From the equation KSV = kqso and taking as fluorescence lifetime
(
s
o) of tryptophan in SA at ꢄ10ꢁ8 s [43], the approximate quenching
constant (kq, in Mꢁ1 sꢁ1) may also be calculated. The calculated val-
ues of Ksv and kq for the interaction of the compounds with HSA and
BSA are given in Table 4 suggesting good binding propensity of the
compounds with complex 1 exhibiting higher SA quenching ability
than free Hnif. The kq values (>1012 Mꢁ1 sꢁ1) of the compounds
are higher than diverse kinds of quenchers for biopolymers

P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
219
(A)
(B)
100
100
80
60
40
20
0
80
Hnif
Hnif
60
1
1
40
20
0
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
r = [Compound]/[HSA]
r = [Compound]/[BSA]
Fig. 4. Plot of % relative fluorescence intensity at (A) kem = 351 nm (%) versus r (r = [complex]/[HSA]) and (B) kem = 343 nm (%) versus r (r = [complex]/[BSA]), for Hnif and
complex 1 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0).
Table 4
The SA constants and parameters derived for Hnif and complex 1.
Compound
Ksv (Mꢁ1
)
kq (Mꢁ1 sꢁ1
)
K (Mꢁ1
)
n
HSA
BSA
Hnif
7.48( 0.35) ꢂ 104
7.48( 0.35) ꢂ 1012
4.14( 0.36) ꢂ 105
0.66
0.89
[Mn(nif)2(H2O)4], 1
1.96( 0.08) ꢂ 105
1.96( 0.08) ꢂ 1013
3.80( 0.16) ꢂ 105
Hnif
3.09( 0.21) ꢂ 105
3.11( 0.07) ꢂ 106
3.09( 0.21) ꢂ 1013
3.11( 0.07) ꢂ 1014
1.18( 0.07) ꢂ 106
2.89( 0.09) ꢂ 106
0.88
1.00
[Mn(nif)2(H2O)4], 1
fluorescence (ꢄ2.0 ꢂ 1010 Mꢁ1 sꢁ1) indicating the existence of a sta-
tic quenching mechanism [41].
and complexes can bind to DNA via a covalent (a labile ligand of
the complex is replaced by a nitrogen base of DNA, e.g. guanine
N7) and/or a noncovalent (intercalation, electrostatic or groove
binding) interaction [25–30].
From the Scatchard equation [36]:
D
½Qꢃ
I=Io
DI
Io
¼ nK ꢁ
K
ð2Þ
The UV spectra of a CT DNA solution with standard concentra-
tion (2 ꢂ 10ꢁ4 M) have been recorded upon addition Hnif or com-
plex 1 at different [compound]/[DNA] mixing ratios (r). The
changes observed are similar and UV spectra of a CT DNA solution
upon addition of Hnif are shown representatively in Fig. 5(A). The
decrease of the intensity at kmax = 257 nm is accompanied with a
red-shift of the kmax up to 263 nm for both compounds, indicating
that the interaction with CT DNA results in the direct formation of
a new complex with double-helical CT DNA [44]. The observed
where n is the number of binding sites per albumin and K is the
association binding constant, K (in Mꢁ1) may be calculated from
D
I=Io
D
I
the slope in plots
versus (Figs. S4 and S5) and n is given by
½Qꢃ
the ratio of y intercept to thIeo slope [36]. The results concerning
the K and n values are given in Table 4, showing that for HSA Hnif
exhibits higher K value and for BSA 1 does. Additionally, the n value
of Hnif increases for the SA when coordinated to Mn(II).
hypochromism may be attributed to
p ?
p⁄ stacking interaction
In conclusion, the compounds exhibit higher K values for BSA
than for HSA. In general, the binding constant of a compound to
a protein such as an albumin should be at an optimum range; it
should be (a) high enough to allow binding and possible transfer
by the protein and (b) not too high so that it can be released upon
arrival at its target(s). Bearing that in mind, the K values of the
compounds may be considered to be within such a range; high en-
ough (3.80 ꢂ 105–2.89 ꢂ 106 Mꢁ1) to allow the binding of the com-
plexes to SAs and also significantly below the association constant
of one of strongest known non-covalent bonds for the interaction
between avidin and ligands (K ꢅ 1015 Mꢁ1), suggesting a possible
release from the serum albumin to the target cells [42].
between the aromatic chromophore from niflumato ligands and
DNA base pairs consistent with the intercalative binding mode
[45] and the accompanying bathochromism may be considered
an evidence of stabilization of CT DNA duplex [46].
In the UV region of the spectra (10ꢁ5 M) of the compounds, the
observed absorption bands are mainly attributed to intraligand
transitions of the NSAID ligands [25–30]. The existence and the
possible mode of interaction between each compound and CT
DNA may be revealed by the changes in the intraligand centered
spectral transitions upon addition of CT DNA solution in diverse r
values. More specifically, in the UV spectrum of Hnif (Fig. 5(B)),
the band centered at 340 nm exhibits in the presence of increasing
amounts of CT DNA a significant hypochromism up 30% suggesting
tight binding to CT DNA probably by intercalation. Further addition
of DNA results in a gradual elimination of this band. A distinct isos-
bestic point at 335 nm appears upon addition of CT DNA. In the UV
spectrum of 1 (Fig. 5(C)), the bands centered at 331 nm (band I)
and 295 nm (band II) exhibit in the presence of increasing amounts
of CT DNA a slight hypochromism as an evidence of tight binding.
Although the exact mode of binding cannot be merely proposed
by UV spectroscopic titration studies, the results collected from the
UV titration experiments suggest that both compounds can bind to
3.4. Interaction with calf-thymus DNA
The potential anticancer and the antiinflammatory activity of
the NSAIDs and their complexes may be often related to their abil-
ity to interact with DNA [4,5]. Nevertheless, the number of such
studies so far is limited; the interaction of DNA with a series of
copper(II) and cobalt(II) complexes with the NSAIDs naproxen, dic-
lofenac, mefenamic acid and diflunisal has been recently reported
by our lab [25–30], while complexes of oxicams are reported to
bind to DNA via intercalation [5]. As known, transition metal ions

220
P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
Table 5
2.0
1.5
1.0
0.5
0.0
(A)
The DNA binding constants (Kb) and Stern–Volmer constants (KSV
fluorescence for Hnif and complex 1.
)
of EB–DNA
)
Compound
Kb (Mꢁ1
)
KSV (Mꢁ1
Hnif
7.60( 0.13) ꢂ 105
1.54( 0.03) ꢂ 106
[Mn(nif)2(H2O)4], 1
8.67( 0.22) ꢂ 105
1.82( 0.03) ꢂ 106
and eb = the extinction coefficient for the compound in the fully
bound form. The values of Kb for the compound, as calculated by
Eq. (3) and the plots in Fig. S6, are given in Table 5. The Kb values
are considered high suggesting a strong binding of the compounds
to CT DNA [26–29]. Upon coordination of niflumic acid to Mn(II), an
increase of the Kb value may be observed. The Kb values of both
compounds are higher than that of the classical intercalator EB
(Kb = 1.23( 0.07) ꢂ 105 Mꢁ1) [26–29].
240
260
280
300
320
λ (nm)
2.5
2.0
1.5
1.0
0.5
0.0
(B)
The measurement of the viscosity of DNA solution upon addi-
tion of a compound may provide significant aid to clarify the inter-
action mode of a compound with DNA, since DNA viscosity is
sensitive to DNA length changes [26–29]. In the case of classic
intercalative binding mode, the insertion of the compound in be-
tween the DNA base pairs results in an increase in the separation
of base pairs at intercalation sites in order to host the bound com-
pound; therefore, the increase of the length of the DNA helix will
be obvious through an increase of DNA viscosity, the magnitude
of which is usually in accordance to the strength of the interaction.
Furthermore, the binding of a compound to DNA grooves via a par-
tial or non-classic intercalation (i.e. electrostatic interaction or
external groove-binding) may provoke a bend or kink in the DNA
helix and subsequently a shortening of its effective length; as a re-
sult, the viscosity of the DNA solution may show a slight decrease
or may remain unchanged [49,50].
Viscosity measurements were carried out on CT DNA solutions
(0.1 mM) upon addition of increasing amounts of the compounds.
The addition of the compounds results in an increase of the relative
viscosity of DNA (Fig. 6) which may be an evidence of the existence
of an intercalative binding mode between DNA and each com-
pound [25–29]; a conclusion in accordance to that derived from
UV spectroscopic studies.
300
320
340
360
380
400
λ (nm)
1.5
1.0
0.5
0.0
(C)
Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phe-
nanthridinium bromide) is a typical indicator of intercalation
[51]; it can intercalate to CT DNA via its planar EB phenanthridine
ring between adjacent base pairs on the double helix, and, as a re-
sult of such an intercalation, intense fluorescence is emitted in the
presence of CT DNA. Therefore, the changes observed in the fluo-
rescence emission spectra of a solution containing EB bound to
CT DNA may be used to study the interaction between DNA and
280
300
320
340
360
λ (nm)
Fig. 5. (A) UV spectra of CT DNA (2 ꢂ 10ꢁ4 M) in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0) in the absence or presence of Hnif. The arrow
shows the changes upon increasing amounts of Hnif. (B) and (C) UV spectra of a
DMSO solution (10ꢁ5 M) of (B) Hnif and (C) [Mn(nifl)2(H2O)4], 1 in the presence of
CT DNA at increasing amounts. The arrows show the changes upon increasing
amounts of CT DNA.
1.5
1.4
1.3
1.2
CT DNA [47]. The observed hypochromic effect may be considered
as first evidence of tight binding to CT DNA probably by intercala-
tion and a stabilization of the DNA double helix [48].
The magnitude of the binding strength of compounds to CT DNA
can be evaluated through the calculation of the binding constant
Kb, which is obtained by monitoring the changes in the absorbance
at the corresponding kmax with increasing concentrations of CT
1.1
Hnif
DNA. Kb (in Mꢁ1) is given by the ratio of slope to the y intercept
1
1.0
½DNAꢃ
in plots ð
Þ versus [DNA] (Fig. S6), according to the equation [45]:
eA
ꢁ
ef
0.00 0.05 0.10 0.15 0.20 0.25 0.30
r = [Compound] / [DNA]
½DNAꢃ
½DNAꢃ
1
¼
þ
ð3Þ
ð
eA
ꢁ
ef
Þ
ð
eb
ꢁ
ef
Þ
Kbðeb
ꢁ
ef
Þ
Fig. 6. Relative viscosity of CT DNA (g/go) in buffer solution (150 mM NaCl and
15 mM trisodium citrate at pH 7.0) in the presence of Hnif and complex 1 at
where [DNA] is the concentration of DNA in base pairs,
eA = Aobsd/
[compound], f = the extinction coefficient for the free compound
e
increasing amounts (r).

P. Tsiliki et al. / Polyhedron 53 (2013) 215–222
221
(A)
(B)
100
5000
4000
3000
2000
1000
0
Hnif
1
80
60
40
20
0
550
600
650
λ (nm)
700
750
0.00
0.01
0.02
0.03
0.04
r = [Compound]/[DNA]
Fig. 7. (A) Emission spectra (kexit = 540 nm) for EB–DNA ([EB] = 20 lM, [DNA] = 26 lM) in buffer solution in the absence and presence of increasing amounts of complex 1.
The arrow shows the changes of intensity upon increasing amounts of 1. (B) Plot of EB–DNA relative fluorescence intensity (%I/Io) at kem = 592 nm versus r (r = [compound]/
[DNA]) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence of Hnif or complex 1.
other compounds, since the addition of a compound which could
intercalate to DNA equally or more strongly than EB, should result
in a quenching of the DNA-induced EB fluorescence emission
(Fig. 7(A)) [52].
1 has higher quenching ability for the albumins’ fluorescence than
free Hnif.
UV spectroscopy studies and viscosity measurements have re-
vealed the ability of the compounds to bind to CT DNA. The binding
strength of the complexes with CT DNA calculated with UV spec-
troscopic titrations have shown that the complex [Mn(nif)2(-
MeOH)4] exhibits higher Kb value than that of free niflumic acid;
both compounds have higher Kb values than that of EB. Competi-
tive binding studies with EB have revealed the ability of the com-
pounds to displace the typical intercalator EB from the EB–CT DNA
complex indicating intercalation as a possible mode of their inter-
action with CT DNA. The intercalative binding mode has been also
confirmed by viscosity measurements of CT DNA solutions in the
presence of the compounds.
The emission spectra of EB bound to CT DNA in the absence and
presence of each compound have been recorded for [EB] = 20
lM,
[DNA] = 26 M for increasing amounts of the compound. The addi-
l
tion of Hnif or complex 1 at diverse r values results in a significant
decrease of the intensity of the emission band of the DNA–EB sys-
tem at 592 nm (the final fluorescence is up to 24% of the initial EB–
DNA fluorescence intensity for Hnif and 20% for complex 1,
(Fig. 7(B))) indicating the competition of the compounds with EB
in binding to DNA. The observed significant quenching of DNA–
EB fluorescence by the compounds suggests that they can displace
EB from the DNA–EB complex, thus probably interacting with CT
DNA by the intercalative mode [26–30].
Acknowledgements
The Stern–Volmer constant, KSV (in Mꢁ1), is used to evaluate the
quenching ability of each compound according to the equation (Eq.
(4)):
Financial support from the Slovenian Research Agency (ARRS)
through project P1-0175 is gratefully acknowledged. The project
was also supported by COST Action CM1105 and partially by the
infrastructure of the EN-FIST, Center of Excellence, Ljubljana,
Slovenia.
Io
I
¼ 1 þ KSV½Qꢃ
ð4Þ
where Io and I are the emission intensities in the absence and the
presence of the quencher, respectively, [Q] is the concentration of
the quencher (Hnif or complex 1). KSV (in Mꢁ1) is obtained by the
Appendix A. Supplementary data
Io
slope of the diagram versus [Q] in Stern–Volmer plots of DNA–
I
CCDC 916035 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: +44 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.poly.2013.01.049.
EB. The experimental data (Fig. S7) indicate that the quenching of
EB bound to DNA provoked by the compound is in good agreement
(R = 0.99) with the linear Stern–Volmer equation (Eq. (4)). The rela-
tively high KSV (Table 5) values of the compounds show that they
can bind tightly to the DNA [25–30] with complex 1 exhibiting
higher KSV value than free niflumic acid.
4. Conclusions
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