Novel 1D chain Fe(III)-salen-like complexes involving anionic heterocyclic N-donor ligands. Synthesis, X-ray structure, magnetic, 57Fe Moessbauer, and biological activity studies

Article abstract of DOI:10.1039/b912676g

The iron(III) salen-type complexes [Fe(salen)(L)]_n (1-6) involving heterocyclic N-donor ligands HL {HL = 1H-imidazole (Himz), 1H-tetrazol-5-amine (Hatz), 5-methyl-1H-tetrazole (Hmtz), 1H-benzimidazole (Hbimz), 1H-1,2,4-triazole (Htriz) and 1H-b

Full text of DOI:10.1039/b912676g

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Novel 1D chain Fe(III)-salen-like complexes involving anionic heterocyclic
57
¨
N-donor ligands. Synthesis, X-ray structure, magnetic, Fe Mossbauer, and
biological activity studies†
a
a
a
b
a,c
ˇ
Radovan Herchel, Zdeneˇk Sindela´rˇ, Zdeneˇk Tra´vn´ıcˇek,* Radek Zborˇil and Ja´n Vancˇo
Received 26th June 2009, Accepted 10th September 2009
First published as an Advance Article on the web 2nd October 2009
DOI: 10.1039/b912676g
The iron(III) salen-type complexes [Fe(salen)(L)]n (1–6) involving heterocyclic N-donor ligands HL
{HL = 1H-imidazole (Himz), 1H-tetrazol-5-amine (Hatz), 5-methyl-1H-tetrazole (Hmtz),
1H-benzimidazole (Hbimz), 1H-1,2,4-triazole (Htriz) and 1H-benzotriazole (Hbtriz)} have been
prepared and characterised by elemental analysis, FT IR, CI mass and 57Fe Mo¨ssbauer spectroscopies,
and variable temperature magnetic measurements. Single crystal X-ray analysis of [Fe(salen)(btriz)]n (6)
revealed a 1D chain-polymeric structure of the complex with the btriz anion as a bridging ligand.
Magnetic data for all complexes were fitted using Fisher’s model (for S = 5/2) and also using a
heptanuclear closed ring model showing a weak antiferromagnetic interaction (J ª -1 to -2 cm-1), and
moreover, molecule-based magnet properties have been observed in the case of [Fe(salen)(atz)]n (2). The
exponential correlation between the magnetic properties (the isotropic exchange parameter J) and the
basicity of the free ligands (Kb) has been found. The antiferromagnetic ordering as well as a moderate
structural dissimilarity in the vicinity of iron atoms has been proved by the 57Fe Mo¨ssbauer
low-temperature (2 K) in-field (7 T) experiments in the case of (2), in which two sextets with the line
intensities (3/4/1/3/4/1) have been observed. The compounds have been tested for their SOD-like
activity, DNA cleavage activity, and in vitro cytotoxicity against two human cancer cell lines: chronic
myelogenous erythroleukemia (K562) and breast adenocarcinoma (MCF7). The best result regarding
the cytotoxicity has been achieved for the complex of [Fe(salen)(atz)]n (2), where IC50 = 6.4 mM against
K562.
[{Fe(salen)}2O],7 which are the subject of the above mentioned
studies, and moreover, they serve as the starting material for
advanced syntheses. To put our work into context, we will
summarize the chemistry of these two primary compounds with
selected N-donor based heterocyclic compounds.
Introduction
The coordination compounds of iron containing the salen ligand
{salen = N,N¢-ethylenbis(salicylaldimato) dianion} have been
known since 19331 and have been extensively studied due to their
specific physical2 and biological3 properties. The great potential
of the [Fe(salen)]+ cation lies in its ability to catalyze redox
reactions in organic syntheses4 e.g. oxidation, aldol condensation
and epoxidation. In addition to that, other practical applications
have been recently reported, such as its use in sensing electrodes
for glucose or uric acid5 or electrochemical sensors for the
determination of NO in solution.6
According to the Cambridge Structural Database (CSD),8
[{Fe(salen)}2O] forms adducts with pyridine (py)9 and
1-methylimidazole,10 in which the coordination number (c.n.)
of the central atom is equal to five. Moreover, octahe-
dral pyridine adducts of m-hydroxo-iron(III)-salen complexes,
e.g. [{Fe(salen)(OH)}2]·2py·2H2O, have also been structurally
characterized.9 Based on these facts, the formation of complexes
with N-donor basedheterocyclicligands coordinated to thecentral
atom of {Fe(salen)}2O subunit can be excluded.
In general, there are two iron(III)-salen starting compounds,
the mononuclear [Fe(salen)Cl] and dinuclear m-oxo-bridged
On the contrary, the reactions of [Fe(salen)Cl] with N-donor
based heterocyclic ligands (HL) led to formation of mononuclear
[Fe(salen)(H2O)(HL)]Y and [Fe(salen)(HL)2]Y complexes, where
HL is 1H-pyrazole, 1H-imidazole or 5-phenyl-1H-imidazole,
aDepartment of Inorganic Chemistry, Faculty of Science, Palacky´ Uni-
versity, Trˇ. 17. listopadu 12, CZ-77900, Olomouc, Czech Republic.
E-mail: zdenek.travnicek@upol.cz; Fax: 00420 585634954; Tel: 00420
585634944
bDepartment of Physical Chemistry, Faculty of Science, Palacky´ Univer-
sity, Trˇ. 17. listopadu 12, CZ-77900, Olomouc, Czech Republic. E-mail:
radek.zboril@upol.cz; Fax: 00420 58563 4954; Tel: 00420 58563 4947
and Y is Cl-, ClO4 , PF6 , NCS- or BPh4 ,11 however, the
heterocyclic N-donor molecule was coordinated as a neu-
tral ligand in all the latter-mentioned cases. In the case of
the mononuclear iron(III) complex containing 1H-imidazole
(Himz), [FeIII(salen)(Himz)2]ClO4, the spin crossover behaviour
was observed.12
-
-
-
cDepartment of Chemical Drugs, Faculty of Pharmacy, University of
Veterinary and Pharmaceutical Sciences Brno, Palacke´ho 1-3, CZ-612 42,
Brno, Czech Republic. E-mail: vancoj@vfu.cz; Fax: 00420 54121 9751;
Tel: 00420 54156 2929
† Electronic supplementary information (ESI) available: Experimental
details. CCDC reference number 737979. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/b912676g
Moreover, N-donor based heterocyclic compounds of the gen-
eral formula HL can be utilised as anionic bridging ligands to form
9870 | Dalton Trans., 2009, 9870–9880
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more complex iron species. Such compounds are very rare and only
a few are known, e.g. for imidazolato(1-),13 triazolato(1-)14 and
benzotriazolato(1-)15 anions. Introducing this kind of bridging
ligand into the iron-salen compounds would lead to the formation
of a novel group of polymeric iron-salen complex species with
most likely interesting chemical and physical properties. The
combination of the [FeIII(salen)]+ unit with anionic bridging
ligands may result in formation of one-dimensional chains, but
only two X-ray structures of such systems have been determined
Results and discussion
Synthesis
As a starting material for the synthesis of the iron complexes
(1–6), a dinuclear m-oxo-bridged complex [{Fe(salen)}2O] was
used, prepared according to the literature.7 This precursor was
dissolved in an organic solvent (n-butanol or nitrobenzene) and
the corresponding N-donor ligand HL was added in a large
excess. It has been found that HL behaves as a weak acid and a
general chemical equation leading to the formation of the resulting
complexes can be written as follows (Scheme 2):
up to now, i.e. [Fe(salen)(ac)]n and [Fe(salen)(dca)]n.17 In these
16
complexes, the iron atoms are linked via simple anions like acetato
(ac) and dicyanamide (dca) as bridging ligands. The magnetic
study of the latter complex showed a weak exchange interaction
to be present among iron(III) centres. Both the above mentioned
compounds were synthesized using [Fe(salen)Cl] as a starting
material.
[{Fe(salen)}2O] + 2 HL → 2 [Fe(salen)L] + H2O
Herein, we report a novel approach to the preparation of
polymeric iron(III)-salen-like compounds based on the fact that
[{Fe(salen)}2O] acts as a weak base and can readily react
with acids HL, such as 1H-imidazole (Himz), 1H-tetrazol-5-
amine (Hatz), 5-methyl-1H-tetrazole (Hmtz), 1H-benzimidazole
(Hbimz), 1H-1,2,4-triazole (Htriz), 1H-benzotriazole (Hbtriz)
(Scheme 1) to form one-dimensional chains [Fe(salen)(L)]n bridged
by heterocyclic aromatic N-donor anionic ligands. The prepared
compounds have been characterized by elemental analysis, mass,
infrared, electronic absorption and Mo¨ssbauer spectroscopies,
and single crystal X-ray analysis. Furthermore, the magnetic
and biological properties of the prepared compounds have been
studied in greater detail.
Scheme 2 Synthetic pathway for the preparation of complexes 1, 2 and
4–6.
The reaction mixture was heated under reflux and after cooling
down, the microcrystals of [Fe(salen)L]n, where HL = 1H-
imidazole (1), 1H-tetrazol-5-amine (2), 1H-benzimidazole (4),
1H-1,2,4-triazole (5), 1H-benzotriazole (6), formed and were
isolated.
The complex of [Fe(salen)(mtz)]n (3) was prepared by the
reaction of [Fe(salen)(N3)] with CH3CN under reflux. Under these
conditions, the complex involving the 5-methyltetrazolide anion
was formed.19
Structural analysis
Single crystals of [Fe(salen)(btriz)]n 6 suitable for X-ray diffraction
structural analysis were obtained by slow cooling of the reaction
mixture from 70 to 20 ◦C for seven days. The crystal data and
structure refinement for 6 are given in Table 1, while selected
bond distances and angles are presented in Table 2. Part of the
crystal structure of complex 6 showing the crystallographically
independent part of the unit cell as well as the 1D polymeric
nature of the complex is depicted in Fig. 1.
The structure of 6 consists of one-dimensional [Fe(salen)-
(btriz)]n chains, in which deformed square-planar [Fe(salen)]+ units
are bridged via the btriz anions—Fig. S1.† The geometry of donor
atoms in the vicinity of the central atom can be described as
a distorted-octahedron. There are three iron(III) centres in the
asymmetric unit, and surprisingly, each of the iron environments
show a differently deformed coordination octahedron. However,
the interatomic parameters in the vicinity of the middle iron
atom differ much more than those of both lateral ones, which
are more similar to each other, as can be supported e.g. by values
of N–Fe–O angles within the polymeric chain. These angles equal
Scheme 1 Schematic representations and abbreviations used for the
N-donor heterocyclic ligands HL. Numbers in parentheses correspond
to [Fe(salen)(L)]n compounds.
Transition metals, which contain unpaired electrons in their
valence shells, involved in polymeric chains often exhibit remark-
able magnetic properties and might be used as precursors for
the preparation of molecule-based magnets.18 Indeed, magnetic
ordering occurs for [Fe(salen)(atz)]n (2) below 3.5 K, probably
due to the presence of hydrogen bonding among one-dimensional
chains introduced through the amino group of the atz ligand.
Moreover, the combination of [FeIII(salen)]+ units with aromatic
heterocyclic ligands might result in compounds having notable
biological properties. Therefore, we tested the prepared complexes
for their SOD-like and DNA cleavage activities, and in vitro
cytotoxicity. In some cases, very promising and significant results
were achieved in connection with in vitro cytotoxicity and DNA
cleavage activity.
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Fig. 1 Crystallographically independent part of [Fe(salen)(btriz)]n (6). The shaded octahedrons represent the coordination chromophore [FeN4O2]. The
hydrogen atoms have been omitted for clarity.
◦
Table 1 Crystal data and structure refinement for [Fe(salen)(btriz)]n (6)
˚
Table 2 Selected bond lengths (A) and angles ( ) for [Fe(salen)(btriz)]n
(6)
Empirical formula
Formula weight
Temperature/K
C66 H54 Fe3 N15 O6
1320.79
110(2)
0.71073
Monoclinic
P21/a
17.0068(2)
19.5863(2)
17.7921(2)
90
93.7974(10)
90
5913.54(11)
4
1.484
Fe1–O2
Fe1–O1
Fe1–N1
Fe1–N2
Fe1–N3
Fe1–N5
Fe2–O3
Fe2–O4
Fe2–N8
Fe2–N9
Fe2–N7
Fe2–N10
Fe3–O5
Fe3–O6
Fe3–N14
Fe3–N13
Fe3–N12
Fe3–N15
1.9037(14)
1.9115(14)
2.1250(18)
2.1274(18)
2.1650(16)
2.1662(16)
1.8987(14)
1.9082(14)
2.0986(16)
2.1283(17)
2.1323(16)
2.1756(16)
1.8942(14)
1.9066(14)
2.1110(17)
2.1326(18)
2.1608(17)
2.2240(17)
˚
Wavelength/A
Crystal system
Space group
˚
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
3
˚
Volume/A
Z
Density (calculated)/Mg m-3
Absorption coefficient/mm-1
F(000)
0.795
2724
Crystal size/mm
0.35 ¥ 0.30 ¥ 0.25
2.59 to 25.00
-18 ≤ h ≤ 20, -23 ≤ k ≤ 22,
-19 ≤ l ≤ 21
49341
10461 (0.0199)
10461/0/813
1.159
R1 = 0.0283, wR2 = 0.0765
R1 = 0.0412, wR2 = 0.0890
0.504 and -0.371
q range for data collection/◦
Index ranges
O2–Fe1–O1
N1–Fe1–N2
N3–Fe1–N5
O3–Fe2–O4
N8–Fe2–N9
N7–Fe2–N10
O5–Fe3–O6
N14–Fe3–N13
N12–Fe3–N15
109.438(59)
76.487(68)
173.313(62)
106.413(60)
77.088(67)
176.021(62)
107.845(60)
77.045(66)
174.472(62)
Reflections collected
In dependent reflections (Rint)
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2s(I)]
R indices (all data)
-3
˚
Largest diff. peak and hole/e A
UV-VIS, FT IR and mass spectroscopy
173.31◦ (Fe1), 176.92◦ (Fe2) and 174.14◦ (Fe3). Non-uniformity
of each of the three iron atoms may also be evident from the
crystallographic point of view, from values of O–Fe–O angles,
which range from 106.4◦ (O3–Fe2–O4) to 109.4◦ (O2–Fe1–O1).
Next significant changes were also observed for bond lengths
UV-VIS spectra were measured in the solid state and only one d–d
transition at 540–580 nm was found and it may be assigned to
6
the 4A2← A1 transition presuming an elongated bipyramid of the
[FeN2O2N¢2] donor set. The other d–d bands were overlapped by
the charge-transfer transitions.
˚
between iron and axial nitrogen atoms, which range from 2.132 A
˚
(Fe2–N7) to 2.224 A (Fe3–N15). The one-dimensional chains are
Infrared spectroscopy confirmed the presence of the salen ligand
as well as the bridging HL ligands within the complexes. The
observed transitions in the range of 1626–1628 cm-1 may be
well separated and no hydrogen bonds or p–p stacking were found.
Only, the weak interactions of the C–H ◊ ◊ ◊ C and C–H ◊ ◊ ◊ O types
are present. (see Table 2).
=
assigned to the n(C N) vibration. The strong vibration of an
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Table 3 57Fe Mo¨ssbauer parameters obtained at room temperaturea
Compound
d (mm s-1)
EQ (mm s-1)
[Fe(salen)(imz)] (1)
[Fe(salen)(atz)] (2)
[Fe(salen)(mtz)] (3)
[Fe(salen)(bimz)] (4)
[Fe(salen)(triz)] (5)
[Fe(salen)(btriz)] (6)
[Fe(salen)(Himz)Cl]b
[Fe(salen)(Himz)2]ClO4
0.394(1)
0.407(2)
0.402(1)
0.417(5)
0.392(5)
0.397(3)
0.45(2)
0.36
1.109(3)
1.518(4)
1.725(3)
1.35(1)
1.243(8)
1.458(4)
1.60(2)
1.34
c
a All data are relative to natural iron foil. b Data taken from ref. 21. c Data
taken from ref. 11.
azide group found in the precursor, [Fe(salen)(N3)], disappeared
in the spectrum of the final product 3, confirming the formation of
the 5-methyltetrazolide. In the case of 2, the n(N–H) vibration of
the amine group in the 5-aminotetrazolide anion (atz) was found
at 3467 cm-1.
Chemical ionization mass spectroscopy (CI MS) identified the
[Fe(salen)]+ species (m/z = 322) and also the corresponding
[HL - H]+ cations of the bridging ligands, i.e. 69 m/z for Himz,
86 m/z for Hatz, 85 m/z for Hmtz, 119 m/z for Hbimz, 70 m/z
for Htriz and 120 m/z for Hbtriz.
Fig. 2 57Fe Mo¨ssbauer spectra of [Fe(salen)(atz)]n (2) at 300 K (top) and
25 K (bottom). Cross symbols correspond to experimental data and solid
lines to fitted data.
Mo¨ssbauer spectroscopy
The transmission 57Fe Mo¨ssbauer spectra were collected at room
temperature for all six compounds 1–6. The spectra consist of
the symmetric doublet with isomer shift d ranging from 0.39 to
0.42 mm s-1, which is consistent with high spin Fe(III) ions in a
N/O coordination environment.20
The quadrupole splitting EQ ranges from 1.11 to 1.73 mm s-1
(Table 3) and its relatively high value reflects the low-symmetry of
the coordination sphere [FeN4O2]. The isomer shift for 1 is very
close to values of similar compounds like [Fe(salen)(Himz)Cl]21
and [Fe(salen)(Himz)2]ClO4,11 thus confirming the oxidation state
as well as a type of polyhedron in the vicinity of the iron
centres. The difference in the quadrupole splitting between 1 and
[Fe(salen)(Himz)2]ClO4 can be explained by the replacement of
the Himz ligand by the imz anion—Table 3.
Fig. 3 57Fe Mo¨ssbauer spectra of [Fe(salen)(atz)]n (2) at 2 K and B = 7 T.
Cross symbols correspond to experimental data and the solid line to fitted
data. Dot and dashed lines correspond to fitted subspectra.
Further, low-temperature Mo¨ssbauer spectra at T = 25 K were
measured for 2 and resulted in d = 0.516(8) mm s-1 and EQ
=
1.491(3) mm s-1—Fig. 2. The change of the isomer shift upon
cooling Dd = 0.109 mm s-1 may be attributed to the second-order
Doppler effect.22
EQ2 = -0.651 mm s-1, Bhf 2 = 47.9 T for the second sextet.
The two structurally non-equivalent Fe(III) atoms are almost
indistinguishable in the spectrum and thus, they are fitted by
one sextet. This sextet possibly corresponds to lateral Fe(III)
atoms which are more similar to each other as proved by a single
crystal X-ray analysis. The spectrum area corresponding to these
“overlapped” Fe atoms is thus two-fold higher compared to the
other sextet with the lower hyperfine field and quadrupole shift
parameters. The second sextet should be clearly assigned to the
third structurally independent Fe(III) position, i.e. the middle iron
atom, exhibiting significantly different bond angles and generally
a higher symmetry of distribution of the electric charge in the
local environment of iron. The line intensities (3/4/1/3/4/1) of
both sextets reflect the perfect antiferromagnetic ordering in all Fe
positions, in accordance with the magnetic data.
In the quest to elucidate the degree of octahedron deformation
in the vicinity of each of the three Fe(III) ions, and moreover, to
explain a character of magnetic ordering within a 1D polymeric
chain, we performed Mo¨ssbauer spectra measurements of complex
2 at 2 K in an external magnetic field of 7 T. The in-field Mo¨ssbauer
spectrum of 2 including two fitted subspectra is depicted in Fig. 3.
Taking into account the above-mentioned results of single crystal
X-ray analysis, which clearly proved the fact that three iron atoms
are present within the crystallographically independent part of
the unit cell, we should have anticipated three subspectra (based on
an assumption that each iron possesses a different environment).
However, the best fit was achieved in the case of two sextets
with d1 = 0.540 mm s-1, EQ1 = -0.475 mm s-1, Bhf 1 = 42.9 T for
the first sextet with area fraction 34.5% and d2 = 0.550 mm s-1,
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Magnetic properties
derived under the assumption that the local spins are treated as
classical vectors,23
Magnetic properties of all complexes 1–6 were found to be very
similar. The magnetic susceptibility calculated as cmol = m0Mmol/B
(in SI units) increases upon cooling up to ca. 15 K. Below this
temperature a broad maximum is observed in the range of 9–14 K
and then the susceptibility is decreasing—Fig. 4.
⎛
⎜
⎜
⎞
⎛
⎜
⎜
⎞
⎟
⎟
JS(S +1)
kT
⎟
⎟
⎟
⎟
⎠
(3)
u = coth
−
⎟
⎟
⎠
⎜
⎜
⎝
⎜
⎝
kT
JS(S +1)
where u is the Langevin function (eq. 3).
The values of the isotropic exchange parameter in the 1D
chain J and g-factors are summarized in Table 4. The weakest
antiferromagnetic isotropic exchange was found for 5, J/hc =
-1.53 cm-1, and the strongest for 1, J/hc = -2.17 cm-1. This
difference reflects mostly the difference in electronic properties of
the bridging ligand. In order to identify some magneto-chemical
correlation, the constant of basicity Kb of the bridging ligands was
used (pKb = 7.01 (Himz), 8.07 (Hatz), 8.44 (Hmtz), 8.47 (Hbimz),
11.73 (Htriz) and 12.4 (Hbtriz)).24 The exponential correlation
(eq. 4) was found between J and pKb = -log Kb with the following
parameters,
(J/hc) = -1.53–42.5 ¥ exp(-pKb/1.67)
(4)
with a correlation coefficient R2 = 0.99 (Fig. 5). This simple
relationship results in the conclusion that the stronger the base
that is used as a bridging ligand, the stronger the antiferromagnetic
exchange that is observed.
Fig. 4 Magnetic properties of [Fe(salen)(imz)]n (1): (left) temperature
dependence of the effective magnetic moment, with the low-T region of
the molar susceptibility expanded in the inset; (right) field dependence of
magnetization at T = 2.0 K. Circles represent experimental points, solid
lines represent calculated values using the best-fit parameters for a 1D
chain (Table 4), the dashed line represents the Brillouin function for
S = 5/2.
The maximum at the susceptibility vs. T dependence is a
characteristic feature for a one-dimensional uniform chain with
antiferromagnetic isotropic exchange. The room temperature
values of the effective magnetic moment are in the range from 5.75
up to 5.87 meff/mB and are close to the theoretical value 5.92 mB for
an isolated iron(III) in a high-spin state with a g = 2.0 (Table 4).
First, the magnetic susceptibility was fitted to the one-
dimensional uniform Heisenberg chain model using the following
spin Hamiltonian (eq. 1)
ꢀ
ꢀ
ꢀ
ꢀ
chain
ˆ
(1)
H
= −J
Si ⋅Si+1 + mBg B
Si
∑
∑
i
i
Fig. 5 The exponential correlation of the isotropic exchange vs. basicity
of N-donor based bridging ligands. Empty circles correspond to the
experimental data and the solid line corresponds to eq. 4.
for which the well-known Fisher’s formula (eq. 2) for magnetic
susceptibility exists,
The field-dependences of the molar magnetization up to B =
7 T at T = 2 K were also measured for 1–6 and show significant
deviation from the Brillouin function for S = 5/2—Fig. 6. There
is no simple analytical formula for magnetization of a 1D uniform
chain as a function of the magnetic field. In order to interpret
these data, the spin Hamiltonian (eq. 5) for a heptanuclear ring
was postulated as
m0N AmB2 g2S(S +1) 1+u
(2)
cmol
=
3kT
1−u
Table 4 Spin Hamiltonian parameters resulting from fitting the magnetic
data either to Fisher’s model for a 1D uniform chain or to a heptanuclear
ringa
1D chain
Heptanuclear ring
meff/mB
meff/mB
6
7
⎡
⎢
⎢
⎤
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J/hc (cm-1)
g
J/hc (cm-1)
g
2 K
300 K
ring
⎥
ˆ
(5)
H
= −J
Si ⋅Si+1 + S7 ⋅S1 + mBg B
Si
⎥
∑
∑
⎢
⎣
⎥
⎦
i
i
1
2
3
4
5
6
-2.17
-1.89
-1.80
-1.79
-1.53
-1.59
2.01
2.01
2.02
2.01
2.00
2.01
-2.12
-1.78
-1.78
-1.73
-1.54
-1.48
2.00
2.00
2.02
2.01
2.01
2.00
1.12
1.25
1.23
1.29
1.32
1.37
5.75
5.81
5.82
5.81
5.78
5.87
The closed ring can mimic the magnetic properties of the 1D
chain, which was utilized several times to obtain approximated
analytical formulae for the magnetic susceptibility.25
The exchange coupling of seven Fe(III) centers, each with
local spin Si = 5/2, results in (2Si + 1)7 = 279 936 magnetic
states with total spins ranging from S = 1/2 to S = 35/2. For
a the effective magnetic moments are calculated per one iron(III).
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Fig. 6 Magnetic properties of [Fe(salen)(imz)]n (1): (left) temperature
dependence of the effective magnetic moment, with the low-T region of
the molar magnetization expanded in the inset; (right) field dependence
of the magnetization at T = 2.0 K. Circles represent experimental points,
solid lines represent calculated values using the best-fit parameters for a
heptanuclear ring (Table 4).
Fig. 7 The hysteresis loop of 2 measured at T = 2 K.
the postulated spin Hamiltonian (eq. 5) it is not possible to
obtain an analytical formula for the energy levels. Moreover, it
is not feasible to efficiently diagonalize such large interaction
matrices. To reduce the dimensions of the matrices, we have focused
on the total spin symmetry principle for which the conditions
(g-factors are equal for all magnetic centres and no non-isotropic
terms present) are fulfilled.26 In order to take advantage of this
method, it is necessary to calculate energy values in the coupled
basis set labelled as |aSM> using the irreducible tensor operators
technique,27 where a stands for the intermediate quantum numbers
denoting the coupling path. First, only the isotropic exchange
terms are involved, and the whole matrix is factorized into blocks
according to the final spin quantum number S. As a result, the
energies in zero magnetic field are obtained. The largest dimension
of the sub-matrix is 3150 for S = 9/2 (Table S1†). Consequently,
the energy levels in the non-zero magnetic field are calculated as
ei(aSM) = e0,j(aS) + mBgBM. From the energy levels, the molar
magnetization (eq. 6) can be easily calculated as
Fig. 8 Temperature variation of the field-cooled magnetization (FC), the
zero-field-cooled magnetization (ZFC) and the remanent magnetization
(RM) for 2 at B = 50 G.
is visible for the low frequencies only, which is not frequency
dependent, see Fig. S2.†
To summarize, the magnetic ordering was confirmed to take
place below 3.3 K for compound 2. This exceptional behaviour in
the presented series can be explained by the presence of the amine
group in the bridging ligand (5-aminotetrazolide anion), which
could be responsible for the hydrogen bonds among the chains in
the crystal structure.
⎡
⎤
M exp −e aSM /kT
⎥
⎦
(
)
⎢
⎣
i
∑
i
(6)
M mol = N AmBg
⎡
⎤
exp −e aSM /kT
⎥
⎦
(
)
⎢
⎣
i
∑
i
Biological activity studies
The experimental data sets (temperature dependence of the
magnetization at B = 1 T and field dependence of the magne-
tization at T = 2.0 K) were treated simultaneously during the
fitting procedure in order to unambiguously determine the fitted
parameters. The results are collected in Table 4 and Fig. 6. Values
of the isotropic exchange and g-factors are in good agreement with
the derived values from the Fisher’s formula for a 1D uniform
chain. The magnetic measurement taken at T = 2 K for 2 revealed
magnetic hysteresis with the coercive field Bc ª 50 G—Fig. 7. To
further magnetically characterize 2, the zero-field cooled and the
field-cooled magnetizations were measured—Fig. 8. The intercept
of these curves is an indicator of the ordering temperature Tc
ª 3.3 K. Also, the remanent magnetization drops to zero above
3 K. The ac susceptibility measurement taken at HDC = 10 Oe
and HAC = 1.5 Oe shows a maximum at 3.2 K in the in-phase
susceptibility. Similar maximum at out-of-phase susceptibility
SOD-mimic activity. The superoxide dismutase (SOD) mimic
activity of Fe(III)-salen complexes 1–6 was tested by a chemical
method based on the competitive reaction of the tetrazolium
salt XTT and the tested compound with superoxide anionic
radicals produced by a saturated DMSO solution of KO2. As a
parameter of antiradical activity, the IC50 values were determined
from the percentages of inhibition at four different concentration
levels. All tested Fe(III) complexes showed moderate SOD-mimic
activities in a relatively narrow range of values (IC50 ranges from
1.32 ¥ 10-5 to 5.40 ¥ 10-5 mol. dm-3), reaching up to 1% of
the antiradical activity of the native enzyme Cu,Zn-SOD (see
Table 5), and up to 10% of the known SOD-mimic compound
[Mn(salen)Cl]·H2O, also labelled as EUK-8, used as a standard.
These findings are in accordance with the published values of the
SOD-mimic activity of a Fe(III) complex with the tripodal ligand
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Table 5 A comparison of the SOD-mimic activity results of the com-
plexes (1–6) with [Fe(salen)Cl], [{Fe(salen)}2O], EUK-8 and Cu,Zn-SOD
expressed in the form of IC50 values with the corresponding standard
deviations
Table 6 DNA cleavage by the complexes (1–6), [Fe(salen)Cl],
[{Fe(salen)}2O] and EUK-8 in the presence of ascorbic acid at the 1:1
base pair (bp) molar ratioa
DI/percentage DII/percentage DNA cleavage
Complex
IC50 [mol. dm-3]
Compound
of CCC-form of OC-form
(%)
[Fe(salen)(imz)]n (1)
[Fe(salen)(atz)]n (2)
[Fe(salen)(mtz)]n (3)
[Fe(salen)(bimz)]n (4)
[Fe(salen)(triz)]n (5)
[Fe(salen)(btriz)]n (6)
[Fe(salen)Cl]
1.32 0.66 10-5
1.89 0.38 10-5
5.40 0.30 10-5
1.96 0.09 10-5
2.62 0.01 10-5
4.48 0.25 10-5
8.86 0.14 10-5
2.68 0.64 10-4
1.39 0.33 10-6
4.80 0.29 10-7
pRSET-B
3116/58.8
3297/59.3
1023/51.9
1293/55.4
380/7.2
10.9
18.2
42.4
44.6
21.8
35.2
30.6
62.9
65.6
43.7
[Fe(salen)(imz)]n (1)
[Fe(salen)(atz)]n (2)
[Fe(salen)(mtz)]n (3)
[Fe(salen)(bimz)]n (4) 4234/55.5
[Fe(salen)(triz)]n (5) 3836/49.1
[Fe(salen)(btriz)]n (6) 3772/50.0
[Fe(salen)Cl]
[{Fe(salen)}2O]
[Mn(salen)(Cl)]·H2O
(EUK-8)
734/13.2
753/38.2
1040/44.6
1180/15.5
2082/26.7
1663/22.0
1563/62.9
1158/65.6
1116/43.7
[{Fe(salen)}2O]
922/37.1
608/34.4
1440/56.3
[Mn(salen)(Cl)]·H2O (EUK-8)
Cu,Zn-SOD
Note: standard deviation calculated from three determinations.
control
3153/79.8
292/7.4
10.8
a DI, DII—integrated density of CCC-form and OC-form respectively; the
percentage of DNA cleavage was calculated by the following equation:
DNA cleavage = DII/(DI + DII).
tris(2-benzimidazolylmethyl)amine (ntb) [Fe(ntb)Cl2]ClO4.28 It is
also suggested in this source that the possible dismutation mecha-
nism could be similar to that of Fe-superoxide dismutase, where a
cyclic redox process occurs, in which the first stage is characterized
by the reduction of Fe(III) to Fe(II) and formation of oxygen
and consecutively, the second step generates a hydrogen peroxide
while the Fe(II) re-oxidizes.29 Results of mechanistic studies of
interactions of a superoxide radical with [Mn(salen)(Cl)]·H2O30
in the chemical system similar to ours identified that at higher
ratios of superoxide to complex, the oxo- and peroxo-complexes
are formed. The manganese(III) complex can also undergo
superoxide mediated reduction due to its specific borderline redox
properties,30 and thus, it can enter into the redox mechanism
of superoxide dismutation as described above. According to
the assumed facts, a similar concept is generally acceptable
for Fe(III)-salen complexes mimicking some monoogygenase
systems.31 The starting compound [{Fe(salen)}2O] could therefore
be considered as one of the stable intermediates of the interactions
between Fe(III)-salen and the superoxide radical, which may
be documented by a significantly lower SOD-mimic activity of
[{Fe(salen)}2O]. In the case of complexes 1–6, an interesting effect
of a bridging ligand on the potentiation of SOD-mimic activity was
found. It may be demonstrated by comparison of the SOD-mimic
activities of complexes 1–6 with a simple non-bridging chlorido-
complex [Fe(salen)Cl], in which SOD-mimic activity was found to
be up to 6-times lower than that of complexes 1–6. The effect of
the bridging ligand has already been documented in the case of
Fe(III)-porphyrin complexes with aromaticN-donor ligands and it
was attributed to a beneficial p-electron effect and the soft nature
of the N-donor ligands (i.e. they behave as soft-Lewis acids) in
these complexes which can facilitate the superoxide interaction.32
The redox based mechanism of superoxide dismutation could
be paradoxically one of the pathways responsible for the high
cytotoxicity of the tested complexes towards tumour cell lines.
There exists an accepted hypothesis that superoxide anion radicals
and hydrogen peroxide are overproduced in the stage of neoplasia
and at the same time play a vital role of apoptotic regulator.33 The
compounds possessing the SOD-mimic activity could therefore
be responsible for the local imbalance in superoxide/hydrogen
peroxide levels, leading to apoptosis of the target cells.34
a model molecule of dsDNA, represented by the circular plasmid
pRSET-B. The activity of the complexes to oxidatively cleave
the polynucleotide chains, in the presence of a high excess of
hydrogen peroxide, was monitored by gel electrophoresis, which
allowed qualitative and quantitative evaluation of different forms
of the modified DNA. The quantitative parameter of total DNA
cleavage was calculated as the percentage of the integrated density
of cleaved forms (OC-form + 2 ¥ L-form) from the sum of
integrated densities of all identified forms of DNA (CCC-form +
OC-form + 2 ¥ L-form). For a better understanding of the Fe(III)-
salen complex–DNA interaction, the actual final concentration in
the tested system was recalculated to express the molar ratio of
the tested compound to one base pair (bp) of plasmid DNA.
In the chemical system including the hydrogen peroxide, only
an insignificant increase of the DNA cleavage was identified,
thus excluding the “peroxidase-like” pathway connected with the
formation of the ferryl (Fe4+) species from the cleavage of DNA.
On the other hand, in the system with the hydrogen peroxide
supplemented with L-ascorbic acid as reducing agent, all the tested
complexes showed a significant increase in DNA cleavage on both
tested concentration levels up to 63% (See Table 6, Table 7, Fig. 9,
and ESI† for additional results).
Moreover, at relatively high concentration (120 mM, 5:1 bp
molar ratio), the formation of small fragments of DNA was ob-
served. At both concentration levels, the DNA cleavage caused by
the simple [Fe(salen)Cl] and the starting complex [{Fe(salen)}2O]
used for the synthesis of complexes 1–6 was significantly higher
than that of the complexes 1–6. On the other hand, the known
manganese based SOD-mimic compound EUK-8, cleaved the
DNA on a similar level as compared with the complexes 1–6.
The mechanism of DNA-cleavage is based on the well-known
hypothesis of sequence non-specific interaction of the soluble
cationic coordination unit [Fe(salen)]+ with the phosphate moieties
of double helical DNA,35 reduction of Fe(III) and Fenton reaction-
mediated production of reactive oxygen species that are able
to cleave the polynucleotide chains, probably at the C1¢ or C4¢
position of the adjacent deoxyribose moiety.36
DNA cleavage. All the Fe(III)-salen complexes 1–6 with
heterocyclic N-ligands were tested for their nuclease activity on
In vitro cytotoxicity. The in vitro cytotoxicity of the complexes
1–6 against K562 and MCF7 human cancer cell lines was
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Table 7 The DNA cleavage by the complexes (1–6), [Fe(salen)Cl],
[{Fe(salen)}2O] and EUK-8 in the presence of ascorbate at 5:1 bp molar
ratioa
Table 8 The cytotoxicity of the studied compounds, expressed as IC50
values (mM), assessed by a calcein AM assay for the two human cancer
cell linesa
DI/percentage DII/percentage DNA cleavage
Values of IC50 (mM) for the
complex/free ligand
Compound
of CCC-form
of OC-form
(%)
K562
MCF7
[Fe(salen)(imz)]n (1)
[Fe(salen)(atz)]n (2)
2963/45.6
266/22.6
1929/29.7
457/38.9
795/61.8
2467/29.4
3294/42.1
2905/36.8
1152/69.8
266/42.4
1330/45.1
39.4
63.3
62.1
41.3
54.9
47.9
69.8
68.6
45.1
[Fe(salen)(imz)]n (1)
[Fe(salen)(atz)]n (2)
[Fe(salen)(mtz)]n (3)
[Fe(salen)(bimz)]n (4)b
[Fe(salen)(triz)]n (5)
[Fe(salen)(btriz)]n (6)
[Fe(salen)Cl]
9.8 1.8/>100
6.4 1.2/>100
11.2 2.1/>100
>1/>50
10.1 1.7/>100
10.9 1.9/>100
—/—
23.7 3.6/>100
13.1 2.1/>100
20.6 3.1/>100
>1/>50
18.3 2.9/>100
16.9 2.8/>100
22.0 0.7c/—
18.0
[Fe(salen)(mtz)]n (3) 482/37.7
[Fe(salen)(bimz)]n (4) 3510/41.8
[Fe(salen)(triz)]n (5)
2706/34.6
[Fe(salen)(btriz)]n (6) 3162/40.0
[Fe(salen)Cl]
498/30.2
120/19.4
[{Fe(salen)}2O]
[Mn(salen)(Cl)]·H2O 1619/54.9
oxaliplatin
cisplatin
9.0
5.0
(EUK-8)
11.0
Control
2863/85
171/5.1
5.7
a K562–chronic myelogenous erythroleukemia, MCF7–breast adenocar-
cinoma. b The complex could not be tested in the desired concentration
range owing to the limited solubility in the testing medium. c The value
was obtained by an MTT test and taken from Ref. 40.
a DI, DII—integrated density of CCC-form and OC-form respectively; the
percentage of DNA cleavage was calculated by the following equation:
DNA cleavage = DII/(DI + DII).
that the efficient hydroxyl radical scavengers had no significant
effect on the iron influenced hydrogen peroxide toxicity in the
LLC-PK1 cells and deduced that other reactive iron-oxygen
species, such as ferryl (Fe4+), perferryl (Fe5+) and various other
forms are probably responsible for the cytotoxic effect. Although
the indirect dependence of the cytotoxicity of [Fe(salen)Cl],
[Fe(saloph)Cl]
and
[Fe(salnaphen)]
complexes,
where
H2salnaphen stands for Schiff base derived from salicylaldehyde
and naphthalene-2,3-diamine, against MFC7 cells on the in vitro
DNA-cleavage results has already been described,40 no such effect
could be found in the case of our experiments.
Fig. 9 An example of an electrophoreogram showing the effect of
concentration on the oxidative cleavage of plasmid DNA in the presence of
ascorbic acid. Lane (1) ladder 1kbp, (2) linearized plasmid (pure L-form,
cleaved by HInd III endonuclease), (3) supercoiled plasmid DNA (majority
of CCC-form), (4) complex 1 at the molar ratio 1:1 bp, (5) complex 1 at
the molar ratio 5:1 bp, (6) complex 4 at the molar ratio 1:1 bp, (7) complex
4 at the molar ratio 5:1 bp, (8) complex 6 at the molar ratio 1:1 bp, (9)
complex 6 at the molar ratio 5:1 bp.
Conclusions
In this work, six novel iron(III)-salen complexes with N-donor
aromatic heterocyclic bases (HL) were prepared and characterized
by a broad spectrum of physical techniques. X-ray analysis re-
vealed a one-dimensional molecular structure, in which the anionic
base acts as the bidentate bridging anionic ligand (L). Mo¨ssbauer
spectroscopy and magnetic measurements showed that iron(III)
centres are in the high-spin state over the whole temperature
interval and the isotropic exchange among them is in the range
from -1 to -2 cm-1. An exponential correlation was found between
the magnetic properties (the isotropic exchange parameter J) and
the electronic properties of the bridging ligand (basicity Kb).
Moreover, the [Fe(salen)(atz)]n 2 revealed magnetic properties
corresponding to a molecule-based magnet.
Further, biological activity studies revealed the great potential
of the prepared complexes to behave as promising anti-tumour
species, as proved by in vitro tests against K562 and MCF7 human
cancer cell lines. All tested compounds exhibited moderate SOD-
mimic activity, reaching up to 1% of Cu,Zn-SOD and up to
10% of a known SOD-mimic compound [Mn(III)(salen)Cl]·H2O
(EUK-8), used as a standard. Both in vitro methods of antioxidant
activity (i.e. SOD-mimic activity and DNA cleavage) suggested
the possible mechanism of reactive oxygen species related to
cytotoxicity, connected either with the Fe(III) complex mediated
imbalance in the superoxide/hydrogen peroxide ratio or Fenton-
like DNA-cleavage, or the formation of other reactive species
determined by a calcein acetoxymethyl (AM) assay. The activity
was expressed as the concentration (in mM) of the compounds
at which tumour cell growth showed 50% inhibition (IC50). The
results of the testing are summarized in Table 8.
The in vitro cytotoxic properties can be evaluated by comparison
of values found for the Fe(III) complexes against the starting
compounds, i.e. [{Fe(salen)}2O] and free HL ligands. It may
be noted that all the tested complexes show significant in
vitro cytotoxicity against both K562 and MCF7 cell lines.
The most promising cytotoxity has been found in the case
of complex 2 involving the 5-aminotetrazolide anion (atz).
In many cases, the effect of iron on the hydrogen peroxide
toxicity was explained by the initiation of the Fenton reaction.
The continuously broadened outlook for the mechanisms of
cytotoxicity mediated by Fe(III) complexes showed, however,
more sensitive and more specific pathways mediated by
the activation of specific genes.37 Indeed, the activation of
the mitochondrial pathway of apoptosis was discovered for
[Fe(salen)Cl] and [Fe(III)(saloph)(H2O)Cl] complexes.38 The
hypothesis of the multimodal effect of iron on the hydrogen
peroxide cytotoxicity was also supported by ref. 39 who found
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{e.g. ferryl (Fe4+) or perferryl (Fe5+) species} leading to apoptosis
[Fe(salen)(atz)]n (2). Yield: 0.09 g 74% (based on Fe). (Found:
C, 50.24; H, 4.04; N, 24.02. FeC17H16N7O2 requires C, 50.27;
H, 3.97; N, 24.17%); lmax(nujol)/nm: 576; nmax(KBr)/cm-1: 3467,
2918, 1630, 1547, 1468, 1444, 1392, 1335, 1291, 1150, 906, 797,
158, 620; CI-MS (m/z, amu): 322 (100%, [Fe(salen)]+), 86 (20%,
H2atz+).
of target cancer cells.
Experimental
All chemicals purchased were chemically pure and were of
analytical reagent grade and used without further purification.
Elemental analyses (C, H, N) were performed on a EA-1108
Analyzer Fisons Intrument. UV-VIS spectra were recorded on
a Perkin-Elmer Lambda 40 UV/VIS spectrometer in the solid
state using the nujol technique in the range of 9000–30 000 cm-1.
Infrared spectra of the complexes were recorded on a Perkin-
Elmer SPECTRUM One FT-IR spectrometer using KBr pellets in
the range 400–4000 cm-1, and on a ThermoNicolet NEXUS 670
FT-IR spectrometer using polyethylene discs in the range 200–
600 cm-1. The transmission 57Fe Mo¨ssbauer spectra were collected
using a Mo¨ssbauer spectrometer in a constant acceleration mode
with a 57Co(Rh) source. The measurements were carried out at
room temperature (for all complexes 1–6). Complex 2 was also
investigated at 2 K in an external magnetic field of 7 T applied
parallel to the g-ray propagation. CI mass spectra of the complexes
were obtained in the solid state using a direct probe on Thermo-
Electron Polaris Q mass spectrometer with an ion trap analyzer.
Isobutane was used as the ionization gas. The compounds were
heated in a temperature program from 40–450 ◦C. Electron energy
10 eV was applied. The mass monitoring interval was
50–1000 m/z. Variable temperature (T = 2–300 K, B = 1 T)
and variable magnetic field (B = 0–7 T, T = 2 K) magnetisation
measurements on polycrystalline samples were carried out with
a MPMS XL-7 Quantum Design SQUID magnetometer. Experi-
mental data were corrected for the diamagnetism of the constituent
atoms by using Pascal’s Tables constants and for the diamagnetism
of the sample holder.
[Fe(salen)(mtz)]n (3). Yield: 0.08 g 67% (based on Fe). (Found:
C, 53.08; H, 4.26; N, 21.17. FeC18H17N6O2 requires C, 53.35;
H, 4.29; N, 20.74%); lmax(nujol)/nm: 558; nmax(KBr)/cm-1: 1627,
1598, 1546, 1469, 1445, 1391, 1336, 1149, 907, 799, 758, 621; CI-
MS (m/z, amu): 322 (100%, [Fe(salen)]+), 85 (42, H2mtz+).
[Fe(salen)(bimz)]n (4). Yield: 0.09 g 70% (based on Fe). (Found:
C, 62.45; H, 4.76; N, 12.55. FeC23H19N4O2 requires C, 62.89;
H, 4.36; N, 12.75%); lmax(nujol)/nm: 540; nmax(KBr)/cm-1: 1627,
1599, 1543, 1467, 1446, 1336, 1298, 751, 618, 420; CI-MS (m/z,
amu): 322 (16%, [Fe(salen)]+), 119 (100, H2bimz+).
[Fe(salen)(triz)]n (5). Yield: 0.09 g 75% (based on Fe). (Found:
C, 55.41; H, 4.11; N, 17.62. FeC18H17N5O2 requires C, 55.26;
H, 4.38; N, 17.90%); lmax(nujol)/nm: 564; nmax(KBr)/cm-1: 1628,
1599, 1542, 1466, 1447, 1339, 1303, 757, 618, 421; CI-MS (m/z,
amu): 322 (100%, [Fe(salen)]+), 70 (62, H2triz+).
[Fe(salen)(btriz)]n (6). Yield: 0.08 g 61% (based on Fe). (Found:
C, 59.50; H, 4.05; N, 15.20. FeC22H18N5O2 requires C, 60.02;
H, 4.12; N, 15.91%); lmax(nujol)/nm: 540; nmax(KBr)/cm-1: 1627,
1599, 1543, 1468, 1446, 1336, 1298, 907, 751, 618, 423; CI-MS
(m/z, amu): 322 (100%, [Fe(salen)]+), 120 (15, H2btriz+).
Single crystal X-ray structure studies
X-ray measurements on the selected crystal of complex 6 was
performed on an Oxford Diffraction XcaliburTM2 equipped with
a Sapphire2 CCD detector using Mo-Ka radiation at 110 K. The
CrysAlis program package (version 1.171.32.11, Oxford Diffrac-
tion) was used for data collection and reduction. The molecular
structure of 6 was solved by direct methods SHELX-97 and all
non-hydrogen atoms were refined anisotropically on F2 using
the full-matrix least-squares procedure SHELXS-97 with weight:
Synthesis
The compounds of the general formula [Fe(salen)(L)]n (1, 2 and
4–6) were prepared according to the general procedure: an excess
of the heterocyclic N-donor base HL (4 mmol) was added to a
n-butanol (80 mL) solution of [(Fe(salen))2O] (0.10 g, 0.15 mmol)
prepared according to ref. 9. The mixture was heated to the
boiling temperature and 10–20 mL of solvent was evaporated.
After cooling of the resulting solution to room temperature,
microcrystals were precipitated, filtered off and washed with a
small amount of hot n-butanol and diethyl ether.
The compound [Fe(salen)(mtz)]n (3) was prepared by heating the
reaction mixture of 0.1 g [Fe(salen)(N3)]2 with acetonitrile (50 mL)
under reflux for 24 h. As a result, the 5-methyltetrazolide anion
was formed.19 After cooling down the resulting mixture to ambient
temperature, the microcrystals of product 3 formed, were isolated
by filtration and washed with small amounts of acetonitrile and
diethyl ether.
2
2
w = 1/[s (Fo)2 + (0.047P)2 + 3.122P], where P = (Fo2 + 2Fc )/3.41
All H atoms of 6 were found in differential maps of electron density
and their parameters were refined using the riding model with the
˚
C–H distance of 0.950 (CH), and 0.990 (CH2) A, respectively,
and with Uiso(H) = 1.2 Ueq(C). Additional calculations were
performed using the DIAMOND program.42 Crystal data and
structure refinement of 6 are summarized in Table 1 while selected
interatomic parameters are given in Table 2.
Cytotoxicity testing
The cytotoxicity of the free ligands and their iron(III) complexes
1–6 were tested in vitro by a calcein AM assay against the following
human cancer cell lines: chronic myologenous leukemia K562 and
breast adenocarcinoma cell line MCF7. The cells were treated
with a solution of the tested compound for 72 h at 37 ◦C, 5%
CO2; in the range of 0.41–100 mM for 1–3 and 5–6; in the range of
0.1–50 mM for 4. Further details regarding the biological activity
testing are detailed in ref. 43. The IC50 values are represented by
the arithmetic mean of three values.
[Fe(salen)(imz)]n (1). Yield: 0.08 g 70% (based on Fe). (Found:
C, 58.06; H, 4.01; N, 14.10. FeC19H17N4O2 requires C, 58.63;
H, 4.40; N, 14.40%); lmax(nujol)/nm: 572; nmax(KBr)/cm-1: 1627,
1599, 1542, 1467, 1446, 1341, 1304, 1085, 760, 616, 419; CI-MS
(m/z, amu): 322 (100%, [Fe(salen)]+), 69 (10, H2imz+).
9878 | Dalton Trans., 2009, 9870–9880
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SOD-mimic activity testing
1M6198959201) and the Academy of Sciences of the Czech
Republic (KAN115600801). Authors would like to thank Mrs.
Dita Parobkova´ for the cytotoxicity testing.
The SOD-mimic activities were measured by an indirect method
based on the competitive reaction of the tested compounds with
XTT dye [sodium 5,5¢-(5-(phenylcarbamoyl)-2H-tetrazole-3-ium-
2,3-diyl)bis(4-methoxy-2-nitrobenzenesulfonate)] with a saturated
DMSO solution of potassium superoxide (KO2). Generally, the
interactions of XTT dye with superoxide anion radical, led to
the formation of orange XTT-formazane. The scavengers of
superoxide radicals inhibit the formation of XTT-formazane and
the percentage of inhibition of this reaction was determined by
spectroscopic measurements at 480 nm. The IC50 values, calculated
from a linearized logarithmic curve of the concentration depen-
dence of the percentage of inhibition, represent the concentrations
of tested Fe(III) complexes which reduced formazane formation
by 50%.
Required amounts of DMSO solutions of the tested compounds
to provide 500, 100, 50, 25 and 10 mM solutions were added to
1.25 ml of 10 mM potassium phosphate buffer (pH 7.4) and 50 ml
of XTT solution in DMSO was added. The resulting solution
was mixed thoroughly. The reaction was started by addition of
100 ml of saturated KO2 solution in DMSO and the absorbance at
480 nm was measured against the blank prepared without addition
of XTT dye (value A). The same procedure was used to prepare
the control sample without the tested Fe(III) complexes and the
absorbance at 480 nm was measured against the solution of XTT
before the reaction with superoxide (value B). All samples were
incubated for 30 min at laboratory temperature. Three samples
of each concentration level of all Fe(III) complexes were tested
(n = 3). Percentages of inhibition (%INH) values were determined
according to the eq. 7. The IC50 values were calculated from a
linearized logarithmic curve of the concentration dependence of
the percentage of inhibition and represent the concentrations of
tested Fe(III) complexes which reduced the formazane formation
to 50%.
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⎛
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% INH = 100⋅ 1−
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Acknowledgements
We acknowledge the financial support from the Czech Min-
istry of Education, Youth and Sports (MSM6198959218,
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