Redox-active metal(II) complexes of sterically hindered phenolic ligands: Antibacterial activity and reduction of cytochrome c. Part II. Metal(II) complexes of o-diphenol derivatives of thioglycolic acid

Article abstract of DOI:10.1016/j.poly.2011.07.008

The synthesis and physico-chemical characterization of Fe(II) and Mn(II) complexes of 2-[4,6-di(tert-butyl)-2,3-dihydroxyphenylsulfanyl]acetic acid (HL^I) and 2-[4,6-di(tert-butyl)-2,3-dihydroxyphenylsulfinyl]acetic acid (HL^II) were c

Full text of DOI:10.1016/j.poly.2011.07.008

Polyhedron 30 (2011) 2581–2591
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Redox-active metal(II) complexes of sterically hindered phenolic ligands:
Antibacterial activity and reduction of cytochrome c. Part II. Metal(II)
complexes of o-diphenol derivatives of thioglycolic acid
N.V. Loginova a, , T.V. Koval’chuk a,b, Y.V. Faletrov b, Y.S. Halauko a, N.P. Osipovich b, G.I. Polozov a,
⇑
R.A. Zheldakova c, A.T. Gres a, A.S. Halauko a, I.I. Azarko d, V.M. Shkumatov a, O.I. Shadyro a
a Faculty of Chemistry, Belarusian State University, Leningradskaya str. 14, 220030 Minsk, Belarus
b Research Institute for Physico-Chemical Problems of the Belarusian State University, Leningradskaya str. 14, 220030 Minsk, Belarus
c Faculty of Biology, Belarusian State University, Nezavisimosti Ave. 4, 220030 Minsk, Belarus
d Faculty of Physics, Belarusian State University, Bobruiskaya str. 5, 220030 Minsk, Belarus
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and physico-chemical characterization of Fe(II) and Mn(II) complexes of 2-[4,6-di(tert-
butyl)-2,3-dihydroxyphenylsulfanyl]acetic acid (HLI) and 2-[4,6-di(tert-butyl)-2,3-dihydroxyphenylsulfi-
nyl]acetic acid (HLII) were carried out. The investigation of the molecular and electronic structure of
Cu(II), Ni(II), Zn(II), Fe(II) and Mn(II) complexes has been performed within the density functional theory
(DFT) framework. The computed properties were compared to the experimental ones, and molecular
structures of the compounds were proposed based on the array of spectral data and quantum chemical
calculations. Antibacterial activity of the Fe(II) and Mn(II) complexes was evaluated in comparison with
Cu(II), Co(II), Ni(II) and Zn(II) complexes and three standard antibiotics; it was found to follow the
Received 24 March 2011
Accepted 6 July 2011
Available online 27 July 2011
Keywords:
Metal(II) complexes
Sterically hindered phenolic ligands
DFT calculations
Cytochrome c
Cytochrome P450-reductase
Antibacterial activity
order: (1) Cu(LI)2 > Mn(LI)2 > HLI > Ni(LI)2 > Zn(LI)2 > Fe(LI)2 > Co(H2O)2LI; (2) Cu(LII)2 > Co(LII)2 > Ni(LII)2
>
Mn(H2O)2(LII)2 > Fe(LII)2 > HLII > Zn(LII)2; their reducing ability (determined electrochemically) followed
the same order. Spectrophotometric investigation was carried out in order to estimate the rate of the
reduction of bovine heart cytochrome c with the ligands and their metal(II) complexes. The complexes
Cu(LI)2, Mn(LI)2 and Co(LII)2 with the high reducing ability were found to be characterized by the highest
rates of Cyt c reduction. NADPH:cytochrome P450-reductase had no substantial effect on the rate of cyto-
chrome c reduction with HLI and HLII ligands.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
of these compounds [5–7]. As the values of redox potentials gov-
ern the ability of compounds to participate in redox interactions,
Previously we have found that some mono- and di-substituted
derivatives of sterically hindered phenolic ligands as well as their
complexes with Cu(II), Co(II), Ni(II) and Zn(II) ions exhibit antimi-
crobial and antiviral activities, the complexes mostly demonstrat-
ing a higher level of activity than the parent ligands [1–6].
Furthermore, using the method of cyclic voltammetry, we have
shown these compounds to be also of a pronounced reducing
ability correlating with antimicrobial activity in a limited series
studying electrochemical properties of pharmacologically active
compounds may provide a useful approach to evaluation of their
ability to undergo redox transformations in biosystems and, par-
ticularly, mechanisms of antimicrobial activity. One of the possi-
ble types of biological macromolecular targets of redox-active
antimicrobial agents may be comprised by cytochromes c which
are components of mammalian and bacterial electron transport
chains [8–15].
All these considerations prompted us to extend our research to
other phenolic ligands and their metal(II) complexes. In the present
work the reduction of bovine heart cytochrome c (Cyt c), selected as
a plausible model of bacterial cytochrome c, with two derivatives of
sterically hindered sulfur-containing phenolic ligands, 2-[4,6-di
(tert-butyl)-2,3-dihydroxyphenylsulfanyl]acetic acid (HLI) and 2-
[4,6-di(tert-butyl)-2,3-dihydroxyphenylsulfinyl]acetic acid (HLII)
(see below),
⇑
Corresponding author. Tel.: +375 172 09 55 16; fax: +375 172 26 46 96/26 55
67.
E-mail addresses: loginonv@yahoo.com, loginonv@gmail.com (N.V. Loginova).
0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2011.07.008

2582
N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
ESR (Electron Spin Resonance) spectra of polycrystalline samples
were measured with ERS-220 X-band spectrometer (9.45 GHz) at
room temperature and at 77 K, using 100-kHz field modulation;
g factors were quoted relative to the standard marker 2,2-diphe-
nyl-1-picrylhydrazyl (DPPH). The magnetic moment measure-
ments of the investigated complexes were carried out using
SQUID magnetometer. Ultraviolet–Visible (UV–Vis) absorption
spectra were recorded with a SPECORD M500 spectrophotometer.
The molar conductance of 10ꢀ3 mol lꢀ1 solutions of the metal(II)
complexes in acetonitrile was measured at 20 °C using a TESLA
BMS91 conductometer (cell constant 1.0). The lipophilicity test
was made by determining the n-octanol/water partition coefficient
(Pow) [22]. Electrochemical measurements were performed under
dry nitrogen in a three-electrode two-compartment electrochemi-
cal cell using a glassy-carbon (GC) working electrode, Pt auxiliary
electrode and Ag|AgCl|0.1 mol lꢀ1 (C2H5)4NCl reference electrode.
The supporting electrolyte was 0.1 mol lꢀ1 (C2H5)4NClO4. The
Ag|AgCl|0.1 mol lꢀ1 (C2H5)4NCl reference electrode was calibrated
with the ferrocenium|ferrocene redox couple located at
E1/2 = +0.54 V. Acetonitrile was used as a solvent.
OH
OH
OH
OH
SCH2COOH
SCH2COOH
II
I
O
HL
HL
as well as with their redox-active transition metal (copper, cobalt,
nickel, zinc, manganese, and iron) complexes was investigated
spectrophotometrically. For the first time redox properties of
above-mentioned ligands and their metal complexes were deter-
mined electrochemically. We also report here the synthesis and
characterization of Fe(II) and Mn(II) complexes with aforesaid li-
gands in order to compare their coordinative behavior in relation
to Fe(II), Mn(II) ions with the results obtained earlier for their Cu(II),
Co(II), Ni(II) and Zn(II) complexes [16] and to assess the influence of
complexation on their biological activity and redox properties.
Accurate molecular geometries are prerequisite for a reliable
quantum chemical computation of properties. For molecules of
moderate size composed of light atoms geometrical parameters
can usually be calculated reliably at high ab initio levels. But for
complexes, especially sterically hindered ones, the molecular
geometries a priori can hardly be predicted for certain. The present
work was focused up on the elucidation of molecular structures of
mentioned complexes based on combination of spectral data and
quantum chemical calculations. To give the assignment of vibra-
tional spectra and to estimate molecular structures we applied
quantum chemical calculations at the DFT level of theory using
the B3PW91 hybrid functional with 6-31G(d) and LANL2DZ basis
sets (including similar effective core potential) respectively for
non-metals and metals. It has been proved earlier [17–21] that
such an approach is sufficiently effective.
2.2. Synthesis of the metal(II) complexes with 2-[4,6-di(tert-butyl)-
2,3-dihydroxyphenylsulfanyl]acetic acid (HLI) and 2-[4,6-di(tert-
butyl)-2,3-dihydroxyphenylsulfinyl]acetic acid (HLII)
Based on our previous findings [1,2,16], the preparation of me-
tal(II) complexes followed a common procedure. A solution of
0.05 mmol M(CH3COO)2 (M = Fe(II) and Mn(II)) in 10 ml of water
was added dropwise to a colorless solution of 0.100 mmol of a li-
gand dissolved in 10 ml of ethanol (molar ratio M(II):L = 1:2). As
these ligands can be oxidized by oxygen, argon was bubbled
through the solutions (pH ꢁ6) during the synthesis to ensure the
absence of oxygen. Precipitates of Fe(II) and Mn(II) complexes
formed instantaneously. After 1.5 h stirring, they were collected
on 0.2 lm membrane filters, washed with ethanol and water and
dried in vacuo (yield >70%).
The results obtained are discussed in the context of presumed
interconnection of the capacity of the compounds under study
for reducing Cyt c, their antibacterial activity, redox properties
determined electrochemically and other physico-chemical
characteristics.
2.2.1. Cu(LI)2, Co(H2O)2LI, Ni(LI)2 and Zn(LI)2 complexes
For elemental analyses data of these metal complexes see Ref.
[16].
2. Experimental
2.2.2. Fe(LI)2 complex
Blue. Yield: 73–75%. Anal. Calc. for C32H46S2O8Fe: C, 56.61; H,
6.84; S, 9.45; Fe, 8.23. Found: C, 56.54; H, 6.82; S, 9.38; Fe, 8.16%.
2.1. Materials and methods
Chemicals were purchased from commercial sources and were
used without further purification. 2-[4,6-Di(tert-butyl)-2,3-
dihydroxyphenylsulfanyl]acetic acid (HLI) and 2-[4,6-di(tert-bu-
tyl)-2,3-dihydroxyphenylsulfinyl]acetic acid (HLII) were prepared
according to the methods reported previously [16]. Synthesis of a
structural analog of o-diphenol derivatives of thioglycolic acid
(4,6-di(tert-butyl)-3-ethylsulfanyl-1,2-dihydroxybenzene) for the
biological assay was performed as described in Section 2.8. The
purity of compounds was checked by Thin Layer Chromatography
(TLC). Elemental analyses were carried out with an instrument
Vario EL (CHNS mode). Metals and sulfur were determined using
an atomic emission spectrometer with an inductively coupled plas-
ma excitation source (Spectroflame Modula). Infrared spectra of
solids were recorded with a Nicolet 380 spectrometer in the wave-
length range 4000–400 cmꢀ1 at room temperature, using ‘‘Smart
Performer’’; spectra in the range 400–50 cmꢀ1 were registered
using ‘‘Vertex 70’’ instrument (Bruker Optik GmbH). Thermal anal-
ysis was performed with a Simultaneous Thermal Analyzer STA
449 C. X-ray diffraction (XRD) analysis was carried out with an
2.2.3. Mn(LI)2 complex
White. Yield: 75–80%. Anal. Calc. for C32H46S2O8Mn: C, 56.69; H,
6.84; S, 9.47; Mn, 8.11. Found: C, 56.62; H, 6.78; S, 9.41; Mn, 8.05%.
2.2.4. Cu(LII)2, Co(LII)2, Ni(LII)2 and Zn(LII)2 complexes
For elemental analyses data of these metal complexes see Ref.
[16].
2.2.5. Fe(LII)2 complex
Blue. Yield: 70–72%. Anal. Calc. for C32H46S2O10Fe: C, 54.06; H,
6.53; S, 9.03; Fe, 7.86. Found: C, 55.98; H, 6.43; S, 8.92; Fe, 7.77%.
2.2.6. Mn(H2O)2(LII)2 complex
White. Yield: 75–80%. Anal. Calc. for C32H50S2O12Mn: C, 51.54;
H, 6.71; S, 8.59; Mn, 7.38. Found: C, 51.49; H, 6.77; S, 8.54; Mn,
7.32%.
HZG 4A diffractometer (Co K
a or Cu Ka radiation, MnO2-filter).

N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
2583
2.3. Physico-chemical characterization
645 (2.39), 590 (2.55), 343 (3.56), 315 (3.74), 297 (3.79), 258
(3.88), 228 (3.96). ESR data: giso = 2.02 (DH = 390 G). leff (RT,
BM) = 1.72.
2.3.1. Cu(LI)2, Co(H2O)2LI, Ni(LI)2 and Zn(LI)2 complexes
For physical and spectral characteristics of these metal com-
plexes see Refs. [1,16].
2.4. Computational details
2.3.2. Fe(LI)2 complex
All calculations were performed using the GAUSSIAN03 software
package [23]. MO calculations were carried out using the density
functional theory B3PW91 method. Optimized geometries and sin-
gle point energies of complexes were determined using the com-
bined 6-31G(d) + LANL2DZ [24] basis set (for non-metal and
metal atoms, respectively). In order to perform stationary points
characterization and to calculate zero-point vibrational energies
(ZPVE) and thermal corrections to Gibbs free energy, harmonic
vibrational frequencies were calculated for structures obtained at
the same level of theory. No special symmetry constraints were
implemented during the optimization process. Regarding the con-
formational flexibility of the complexes, their initial structures
were relaxed in TINKER program [25] prior to the DFT geometry opti-
mization. At the particular level of theory a scaling factor of 0.951
[26] was applied to the frequencies in order to obtain better agree-
ment with the experimental data.
Molar conductivity (in acetonitrile): Kmol=6.9
X .
ꢀ1 cm2 molꢀ1
Thermogravimetric/differential thermal analysis (TG/DTA) data:
no weight loss was observed until decomposition which began
about 170 °C, with an exothermic peak at 200 °C and an endother-
mic one at 335 °C, ultimately leaving FeO as the residue. The max-
imal weight loss of 88.6% corresponds to the loss of two ligand
molecules in the Fe(L)2 complex (Calc. 89.4%). Prominent IR
absorption bands
(m,
cmꢀ1): 3429m (O–H), 1383m (COOꢀ),
1473m (C@C arom), 1188m, 1113m and 1025m (C–O), 757w and
668m (C–S), 582m and 532m (Fe–O), 345w (Fe–S). UV–Vis data
(acetonitrile) (kmax, nm (log
(3.32), 310 (3.72), 300 (3.65), 240 (3.94), 225 (3.99). leff (RT,
e)): 665 (2.34), 600 (2.29), 420
BM) = 0.
2.3.3. Mn(LI)2 complex
Molar conductivity (in acetonitrile): Kmol = 10.3
X
ꢀ1 cm2 molꢀ1
.
To better understand the electronic structure of the complexes,
the Mulliken orbital population analysis was performed. The redox
properties were connected with the energies of the frontier orbitals
by using Koopmans theorem: ionization energy = ꢀE(HOMO) and
electron affinity = E(LUMO).
TG/DTA data: no weight loss was observed until decomposition
which began about 210 °C, with an endothermic peak at 285 °C,
ultimately leaving MnO as the residue. The maximal weight loss
of 88.3% corresponds to the loss of two ligand molecules in the
Mn(LI)2 complex (Calc. 89.5%). Prominent IR absorption bands (
m,
cmꢀ1): 3446w (O–H), 1395 m (COOꢀ), 1474w (C@C arom.),
1189m, 1175m, 1115m and 1026m (C–O), 668m (C–S), 582w,
531m and 509w (Mn–O), 335w (Mn–S). UV–Vis data (acetonitrile)
2.5. Antibacterial assays
The following test microorganisms (collection of Department of
Microbiology, Belarusian State University) were used: Escherichia
coli, Pseudomonasaeruginosa, Serratiamarcescens, Salmonella typhimu-
rium, Bacillus subtilis, Sarcina lutea, Staphylococcus saprophyticus,
Staphylococcus aureus, and Mycobacterium smegmatis.
(kmax, nm (log e)): 660 (2.39), 595 (2.55), 490 (3.56), 410 (3.65), 315
(3.74), 295 (3.79), 245(3.96). ESR data: giso = 2.015 (
leff (RT, BM) = 1.75.
DH = 270 G).
2.3.4. Cu(LII)2, Co(LII)2, Ni(LII)2 and Zn(LII)2 complexes
For physical and spectral characteristics of these metal com-
plexes see Ref. [16].
The antibacterial activity of the compounds was tested in vitro
using the twofold serial dilutions (from 200 to 3.1
quid broth method for determination of a minimum inhibitory
concentration (MIC,
g mlꢀ1) as described elsewhere [27]. The
l
g mlꢀ1) in li-
l
2.3.5. Fe(LII)2 complex
microorganisms stored in Muller–Hinton broth (Merck), were sub-
cultured for testing in the same medium and grown at 37 °C. Then
the cells were suspended, according to the McFarland protocol, in
saline solution, to produce a suspension of about 105 CFU/ml (col-
ony-forming units per ml). Serial dilutions of the test compounds,
previously dissolved in dimethyl sulfoxide (DMSO), were carried
out in test tubes to certain concentrations. Hundred milliliter of a
24 h old inoculum was added to each tube. The MIC, defined as
the lowest concentration of the test compound, which inhibits
the visible microbial growth, was determined after an incubation
period of 24 h at 37 °C for bacteria. Tests using DMSO as a negative
control were carried out in parallel. The amount of DMSO in the
medium was 1% and did not affect the growth of the tested micro-
organisms. Commonly used antibiotics (streptomycin, tetracycline
and chloramphenicol) were tested as positive controls. There were
three replicates for each dilution. Results were always verified in
three separate experiments.
Molar conductivity (in acetonitrile): Kmol = 4.9
X .
ꢀ1 cm2 molꢀ1
TG/DTA data: no weight loss was observed until decomposition
which began about 165 °C, with a broad exothermic peak at
230 °C, leaving FeO as the residue. The maximal weight loss of
88.9% corresponds to the loss of two ligand molecules in the
Fe(L)2 complex (Calc. 89.9%). Prominent IR absorption bands (m,
cmꢀ1): 3625s, 3391s and 3286m (O–H), 1577m and 1390m
(COOꢀ), 1485m (C@C arom.), 1168m and 1131m (C–O), 1023m
(S@O), 686w and 612m (C–S), 562m, 584m and 472m (Fe–O).
UV–Vis data (acetonitrile) (kmax, nm (log
(2.51), 338 (3.58), 305 (3.67), 255 (3.71), 225 (3.85). leff (RT,
BM) = 0.
e)): 650 (2.31), 560
2.3.6. Mn(H2O)2(LII)2 complex
Molar conductivity (in acetonitrile): Kmol = 11.0
X .
ꢀ1 cm2 molꢀ1
TG/DTA data: no weight loss was observed until decomposition
which began about 65 °C, with an endothermic peak at 107 °C
which corresponds to the loss of two water molecules (found:
4.27%, Calc. 4.83%) and with a broad exothermic peak in the tem-
perature range 180–300 °C, ultimately leaving MnO as the residue.
The maximal weight loss of 89.5% corresponds to the loss of two
ligand molecules in the Mn(H2O)2(LII)2 complex (Calc. 90.5%).
2.6. Reduction of cytochrome c
Bovine heart cytochrome c (Sigma) was used. Human P450R
was purified according to [28,29]. Spectrophotometric experiments
were performed with a Shimadzu UV-1202 spectrophotometer
using quartz cuvette with 1 cm optical path. Cyt c concentration
was determined on its interaction with excess sodium dithionite,
Prominent IR absorption bands (m
, cmꢀ1): 3625s, 3419w (O–H),
1575m (COOꢀ), 1494m (C@C arom.), 1171m, 1135m, 1085m and
1046m (C–O), 1027m (S@O), 696m and 617m (C–S), 582w, 531m
using the absorption coefficient
saturated acetonitrile solutions of the ligands and complexes under
e
550 = 21 mmolꢀ1 l cmꢀ1 [30]. Ar-
and 509w (Mn–O). UV–Vis data (acetonitrile) (kmax, nm (log e)):

2584
N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
study and Cyt c (7
l
mol lꢀ1) were used. Experiments were per-
(Section 2.3.6). The data of thermal analysis performed in nitrogen
atmosphere with identification of the final products by X-ray pow-
der diffraction have shown the agreement between the experimen-
tal and theoretical weight losses for the processes of thermal
decomposition of metal complexes (Sections 2.3.2, 2.3.3 and
2.3.5–2.3.6). These results confirm the above-mentioned two gen-
eral formulas of the metal complexes.
formed in 10 mmol lꢀ1 sodium phosphate buffer (pH 7.4) at
20 °C. Aliquots of the compounds under study were added to Cyt
c solution up to the final concentrations 35.0 or 17.4
necessary, P450R was first added to Cyt c. The initial rate of Cyt c
reduction ( ) was evaluated by the slope of the kinetic curve A550
l
mol lꢀ1; if
m
versus time according to [31,32]. The results were confirmed in
three independent experiments.
The complexes were insoluble in water, ethanol, methanol,
diethyl ether, but they were soluble in acetonitrile and dimethyl
sulfoxide. The conductivity data indicate their being essentially
non-electrolytes in acetonitrile [36] and suggest that the two
bidentate ligands may be coordinated to Fe(II) and Mn(II) ions as
monoanionic species.
No single crystals of Fe(II) and Mn(II) complexes suitable for X-
ray diffraction studies were obtained because of the well-known
difficulties of precipitating metal complexes with redox-active ste-
rically hindered ligands from solutions [37]. The geometrical
arrangement of the ligating atoms in the metal complexes was
investigated by several spectroscopic and magnetochemical meth-
ods. The experimental studies on the synthesized metal complexes
have been accompanied computationally by DFT which is com-
monly used to examine the electronic structure and geometrical
parameters of transition metal complexes [38,39].
2.7. Docking of the sterically hindered phenolic ligands with P450R
To evaluate a possible contribution of electrostatic repulsion
into interaction of P450R with sterically hindered phenolic ligands
under study, docking experiments of the above-mentioned ligand
structures in the active center of P450R were carried out. Auto-
mated docking simulations were conducted with Autodock 4.0
software; the Graphical User Interface program ‘‘AutoDockTools’’
(The Scripps Research Institute) was used to prepare, run and ana-
lyze the docking simulations [33,34]. P450R 3D-structure was from
RCSB protein database (PDB ID: 1b1c). Gasteiger partial charges
were calculated and assigned to the atoms of flavin mononucleo-
tide (FMN) and amino acid residues of the P450R [35]. The struc-
tures of the ligands were prepared using HyperChem
Most of the complexes under study can potentially exist as var-
ious geometrical isomers, where a metal ion is chelated by the li-
gand in different ways as shown in Fig. 1.
In order to identify the molecular structures and especially the
coordination polyhedron of the complexes discussed in this work,
we have analyzed their experimental vibrational spectra and com-
(Hypercube). The simulation space was defined as
a
60 ꢂ 60 ꢂ 60 Å3 box, which included FMN and closely disposed
amino acid residues. The ligands were docked by the Lamarckian
genetic algorithm default protocol. For each conformation interac-
tion constant (Kint) values were calculated from docking energy
(the sum of the intermolecular interaction energy and the ligand’s
internal energy) by AutoDock. Additionally, the least distances be-
tween phenolic oxygen atoms of the ligands and N5 atom of FMN
(Lmin(O–N))were calculated using AutoDockTools; both Lmin(O–N)
value for each ligand under study and the corresponding Kint value
were tabulated.
IOH
I
III
O
O
II
III
I
O
O
OH
IIIO
M
M
II
IV
OH
O
IVO
II
S
O
IVO
cis-/trans-
2.8. Synthesis of 4,6-di(tert-butyl)-3-ethylsulfanyl-1,2-dihydroxy-
benzene (HLV) – a structural analog of o-diphenol derivatives of
thioglycolic acid for the biological assay
IVO
IIO
IV
II
O
O
M
M
S
S
S
S
cis-/trans-
cis-/trans-
Ten millimolar of 3,5-di-tert-butyl-1,2-benzoquinone were
added to the solution of 13.5 mmol ethanethiol in 40 ml hexane
under stirring and cooling by ice water. An hour later the light-
green solution was boiled dry under vacuum, the residue was
dissolved in petroleum ether; the solution was filtered through a
silica gel layer. The filtrate was boiled dry under vacuum, and
the residue was recrystallized from hexane with the resulting
separation of white crystals (yield >55%). M.p.: 93 °C. Anal. Calc.
for C16H26O2S: C, 68.04; H, 9.28; S, 11.35. Found: C, 55.98; H,
6.43; S, 8.92%. Mass spectrum (m/z, I%): 281.05 (M+, 37), 267.05
(MꢀCH3, 100). 1H NMR dH (100 MHz, CDCl3): 6.93 [s, 1H, aromatic
H], 5.59 [s, 1H, OH], 2.66 [dd, 2H, CH2], 1.51 [s, 9H, Me3C], 1.42 [s,
9H, Me3C], 1.31 [t, 3H, Me].
Type A
IV
IV
III
O
O
O
IO
I
S
O
II
II
II
O
O
O
M
M
M
IIO
IIO
IIO
I
I
O
S
O
IVO
IIIO
IVO
Type B
IOH
I
III
O
O
II
III
I
O
O
OH
IIIO
M
M
IIO
IVO
IV
IVO
II
S
OH
O
VO
cis-/trans-
IVO
IIO
IV
II
O
O
3. Results and discussion
M
M
V
V
O
O
VO
VO
3.1. Physico-chemical characterization
cis-/trans-
Type C
cis-/trans-
IV
III
The solid products resulting from the interaction of Fe(II) and
Mn(II) ions with HLI and HLII ligands were well characterized by
means of elemental analysis, TG/DTA, FT-IR, UV–Vis, ESR, magnetic
susceptibility and conductance measurements. The elemental
analysis data for the Mn(II) and Fe(II) complexes with the ligand
HLI and the Fe(II) complex with the ligand HLII are in agreement
with the general formula ML2 (Sections 2.2.2, 2.2.3 and 2.2.5),
and for Mn(II) complex with the ligand HLII – Mn(H2O)2(LII)2
O
O
I
IO
IIO
O
II
II
O
O
M
M
IO
I
IIO
O
IVO
IIIO
Fig. 1. Plausible coordination cores for metal complexes of ML2 composition.

N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
2585
pared them with the calculated ones. It should be noted that this
approach allows one to predict the general coordination pattern
only, but it is very useful in our case for the lack of crystallographic
data. From the following discussion one can see that the coordina-
tion type can be confidently specified relying on IR spectra. As for
quantum chemical calculations, they allow one to distinguish be-
tween cis and trans configuration of the metal complex.
tively. According to [42,43], such parameters are characteristic of
Mn(II) complexes with a distorted octahedral coordination core
and with virtually no interaction between manganese atoms. No
signal of stabilized semiquinone radicals is present in ESR spectra
(g = 2.004–2.005) [44].
In the electronic absorption spectra of Fe(LI)2, Mn(LI)2, Fe(LII)2
and Mn(H2O)2(LII)2 complexes in acetonitrile solution the absorp-
tion maxima in the high-energy region 225–305 nm belong to
intraligand transitions (Sections 2.3.2, 2.3.3 and 2.3.5–2.3.6).
The absorption maxima appearing in the spectra of Fe(LI)2 and
Mn(LI)2 at 310–315 and 410–490 nm are indicative of the ligand-
to-metal(II) charge transfer transitions S ? MII and Ophenolate ? MII,
To specify the coordination cores in the Fe(II) and Mn(II) com-
plexes, we used IR spectroscopy (Sections 2.3.2, 2.3.3 and 2.3.5–
2.3.6). In the spectrum of the ligand HLI there are three broad
bands in the range from 3545 to 3349 cmꢀ1, which are evidence
of intermolecular hydrogen bonds involving phenolic hydroxyl
groups [40]. IR spectral assignments of the ligand HLI, Fe(LI)2 and
Mn(LI)2 complexes are indicative of metal(II) being coordinated
with hydroxyl of the ligand HLI: only one sharp band (3446 or
3429 cmꢀ1) is present in the spectra of metal complexes; the
absorption bands corresponding to the stretching C–O bond vibra-
tions are shifted to the lower frequency region, suggesting that the
ligand is coordinated in the form of monoanion [40]. The shift of
the bands at 775 and 677 cmꢀ1 (assigned to C–S bond vibrations)
in the spectra of Fe(LI)2, Mn(LI)2 complexes toward the low-fre-
quency region suggests that sulfur is involved in the complexation.
Instead of the characteristic bands of COOH group stretching vibra-
tions at 1760–1740 cmꢀ1 new bands appear in the spectra of these
metal complexes at 1600–1573 cmꢀ1, characteristic of COOꢀ-anion
coordinated to a metal ion. It should be noted that in the spectra of
Fe(LI)2 and Mn(LI)2 complexes there are new bands in the region
from 580 to 470 cmꢀ1, which may be assigned to the stretching
vibrations of M–O bonds [40]. M–S stretching vibration frequencies
O
carboxylate ? MII, respectively [45]. The spectra of the Fe(LII)2 and
Mn(H2O)2(LII)2 complexes exhibit absorption bands at 315–
345 nm which can be attributed to
carboxylate ? MII and
S@O ? MII charge transfer transitions [45,46].
According to the literature [45,47–49], the absorption maxima
O
O
observed in the spectra of the complexes Fe(LI)2 (600 nm) and Fe(-
LII)2 (560 nm) are due to d–d transitions characteristic of low-spin
Fe(II) complexes with the octahedral and square planar geometry
of FeS2O4 and FeO4 coordination cores, respectively [48,50]. This
conclusion is in agreement with ESR-spectroscopic data and values
of effective magnetic moments for Fe(LI)2 and Fe(LII)2 complexes,
indicative of their diamagnetism (Sections 2.3.2 and 2.3.5). Hence,
the field of the ligands HLI and HLII is strong enough for spin cou-
pling in Fe(II) ion, and they form low-spin complexes with this ion.
Spin coupling is also characteristic of Mn(II) ion in Mn(LI)2 and
Mn(H2O)2(LII)2 complexes, which is supported by the values of
effective magnetic moment leff of these complexes (Sections
2.3.3 and 2.3.6) [51]. Note that the absorption spectra of low-spin
Mn(II) complexes with redox-active ligands have d–d bands at
about 590–610 nm characteristic of the coordination octahedron
[43,45,51–53]. These bands were found in the spectra of Mn(LI)2
and Mn(H2O)2(LII)2 complexes, which may be indicative of the
octahedral geometry of their MnS2O4 and MnO6 chromophores
(Sections 2.3.3 and 2.3.6).
are registered in the region 350–330 cmꢀ1
.
In the IR spectrum of the ligand HLII there are three broad bands
at 3510, 3463, 3348 cmꢀ1, which undergo changes in the spectra of
its metal complexes (Sections 2.3.5 and 2.3.6), which may be due
to a free and a hydrogen-bond bound hydroxyl groups in metal
complex molecules [41]. A broad band in the range 3391–
3200 cmꢀ1 and a band at 899 cmꢀ1 assigned to M–OH2 vibrations
present in the spectrum of Mn(H2O)2(LII)2 complex substantiate
the participation of water molecules in forming the coordination
core of the complex and agree with the elemental analysis data
for this complex. In the spectra of metal complexes for the ligand
HLII neither the bands resulting from the stretching vibrations of
C–S bond change their position or intensity, nor the vibration
bands in the range 380–300 cmꢀ1 characteristic of M–S bond ap-
pear, thus suggesting that there is no coordination binding of sulfur
atom to metal ion. Besides, a band at 1580–1575 cmꢀ1 characteris-
tic of stretching vibrations of COOꢀ group appears in their spectra
instead of the band at 1715 cmꢀ1 (that of stretching vibrations of
COOH group) present in the spectrum of the parent HLII ligand.
In the spectra of Fe(LII)2 and Mn(H2O)2(LII)2 complexes a shift of
the characteristic band of S@O group is observed, which is due to
coordination binding of oxygen atom of this group. The strong
bands in the range 590–415 cmꢀ1 in the spectra of these com-
plexes belong to M–O bonds [40].
The absorption maxima appearing in the spectra of Fe(LI)2,
Mn(LI)2, Fe(LII)2 and Mn(H2O)2(LII)2 complexes at 640–665 nm are
indicative of the metal(II)-to-ligand charge transfer transitions
MII ? (
pO) characteristic of low-spin complexes of redox-active li-
gands with readily oxidized ions of transition metals, specifically,
Fe(II) and Mn(II) [45].
This view of coordination cores agrees with the data obtained
by physico-chemical methods for other metal(II) complexes of
the ligands HLI and HLII investigated previously [16]. The results
obtained are verified by quantum chemical calculation of elec-
tronic and molecular structure of Cu(II), Ni(II), Zn(II), Fe(II) and
Mn(II) complexes with these ligands. Co(II) complexes have not
been included in calculations because of Co(II) complex with HLI li-
gand being polymeric.
As quantum chemical calculations have auxiliary meaning with
the respect to the aims of investigation, their results are presented
in the discussion section in minimal necessary amount, but full re-
sults can be obtained from authors at request. As mentioned above,
the coordination type can be specified by IR spectra analysis, while
quantum chemical calculations provide a way of identifying cis or
trans configuration of the metal complex. The comparison of the
key low-frequency region in experimental and calculated for geo-
metric isomers IR spectra, comprising the most specific vibrations
of coordination core, was unambiguously indicative of trans config-
uration (Fig. 2).
The changes in the frequencies of (C@C) stretching vibrations of
aromatic ring in the spectra of all metal complexes compared to
those of the ligand HLI (1487 cmꢀ1
)
or the ligand HLII
(1602 cmꢀ1) also are evidence in favor of the coordination bond
formation.
The above-listed facts suggest that sulfanyl, sulfinyl and carbox-
ylate groups as well as deprotonated hydroxyls take part in form-
ing the MS2O4 or MO4 coordination cores in the metal(II)
complexes under study.
In the light of the spectral data, magnetic moment values, ana-
lytical results and quantum chemical calculations the general
mode of binding in the metal(II) complexes can be represented
as shown below:
The ESR spectra of Mn(LI)2 and Mn(H2O)2(LII)2 complexes show
a broad isotropic signal (270 and 390 G, respectively) at liquid
nitrogen temperature with giso values of 2.015 and 2.020, respec-

2586
N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
OH
OH
O
C
O
O
O
O
O
CH2
S
OH
OH
CH2
H
O
OH
C
S
C
S
CH2
OH2
O
O
M
O
M
M
O
O
O
O
S
O
O
O
OH2
OH
H
C
S
H2C
C
CH2
HO
S
CH2
C
O
HO
HO
HO
M=Cu(II), Ni(II), Fe(II), Mn(II)
M=Cu(II), Co(II), Ni(II), Fe(II)
M=Mn(II)
Cu(II), Ni(II) and Fe(II) complexes containing HLI have the struc-
ture of A type (Fig. 1) (extremely distorted octahedron), while
Mn(II) complex exists as B type structure, and the structure of
the Zn(II) complex undergoes tetrahedral distortion. The general
structure for complexes containing HLII is of C type. Cu(II) and
Fe(II) complexes are close to square planar ones, while those of
Ni(II) and Zn(II) are nearly tetrahedral, and the coordination core
of Mn(II) complex is octahedral (Table S1). When comparing
respective geometry parameters of a free and a bound ligand (for
example HLI and Ni(LI)2), one can see that noticeable changes occur
only for the atoms involved in coordination and their neighbors
(Table 1). Typical changes for the remote part of the ligand mole-
cule do not exceed 1 pm of bond length and 2° of bond angle. For
complexes of HLI it is the inequality of phenolic oxygen atoms that
I
attracts attention. The O moves off from benzene ring, while IIO
approaches it during complexation.
To analyze the peculiarities of electronic structure of phenolic
ligands and their complexes, the Mulliken population analysis
has been performed. The rearrangement of electron density during
complexation obviously must affect atomic charges; their evolu-
tion is given in Tables 2 and S2.
Table 1
Selected computed bond lengths (R, Å) and valence angles (
a
, °) for free ligands HLI,
HLII and their complexes with Ni(II) ions.a
Parameter
HLI
Ni(LI)2
Parameter
HLII
1.414
1.381
1.836
1.871
1.514
Ni(LII)2
R(C–IOH)
R(C–IIO)
R(Car–S)
R(Calk–S)
1.382
1.378
1.780
1.827
102.7
113.0
1.413
1.360
1.784
1.863
104.3
104.6
R(C–IOH)
R(C–IIOH)
R(Car–S)
R(Calk–S)
R(S–O)
1.401
1.357
1.828
1.822
1.561
99.8
a(C–S–C)
a(S–Calk–Ccarb
)
a
(C–S–C)
95.0
a
Selected computed bond lengths (R, Å) and valence angles (
a, °) for the com-
plexes with Cu(II), Zn(II), Fe(II) and Mn(II) ions are given in Table S1.
Table 2
Selected computed Mulliken atomic charges (in electron charge units) for free ligands
and their complexes with Ni(II) ions.a
Atom
HLI
Ni(LI)2
Atom
HLII
Ni(LII)2
Ni
0.271
ꢀ0.593
0.319
Ni
0.534
ꢀ0.659
0.949
IIO
ꢀ0.675
0.252
0.287
ꢀ0.289
ꢀ0.698
0.410
IIO
ꢀ0.620
0.974
0.327
ꢀ0.353
ꢀ0.746
0.396
S
S
C(–IIO)
0.357
C(–IIO)
Car(–S)
Calk(–S)
H(–IIO)
C(@O, –OH)
VO
0.337
Car(–S)
ꢀ0.293
ꢀ0.340
ꢀ0.670
0.461
Calk(–S)
ꢀ0.672
H(–IIO)
C(@O, –OH)
IIIO
0.681
0.649
ꢀ0.442
ꢀ0.575
0.640
0.642
ꢀ0.501
ꢀ0.514
ꢀ0.664
IVO
ꢀ0.549
IVO
ꢀ0.558
ꢀ0.530
a
Fig. 2. Comparison of theoretical and experimental IR spectra of NiLI2 complex: (a)
experimental, (b) trans, and (c) cis.
Selected computed atomic charges for the complexes with Cu(II), Zn(II), Fe(II)
and Mn(II) ions are given in Table S2.

N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
2587
A noticeable change of charge occurs only for the atoms involved
in coordination and adjacent atoms, which is rather logical. For phe-
nolic oxygen atoms of HLI it is only the deprotonated one that suffers
charge increase, while for those of HLII no major changes take place,
suggesting another binding mode. The main contribution in metal
electron density acquisitionis providedby sulfur, deprotonated oxy-
gen and adjacent carbon atoms for HLI and remaining phenolic
hydrogen atoms for both types of complexes.
expected. The frontier MO’s of ligands and complexes are given
in Fig. 3.
From the surfaces above one can see that for HLI the main con-
tribution into HOMO is made by oxygen and aromatic carbon
atoms basis functions. It means that these positions are preferable
for electrophilic attack, which is in agreement with HLI coordina-
tion mode. For HLII HOMO is localized mainly on sulfoxide group,
whereas in its complexes the main input into HOMO is from di-
rectly bound oxygen atoms as well as carbonyl. Sulfur atoms basis
functions make a negligible contribution into HOMO of both li-
gands and complexes, being presented in a deeper HOMOꢀ1. The
proximity of frontier orbitals of free ligands and metal complexes
is responsible for their rather close redox properties.
Regarding the mechanism of donor–acceptor bond formation,
the main input from ligand’s HOMO and metal’s LUMO can be
3.2. Electrochemical studies
The data on redox properties of the compounds HLI and HLII,
their metal complexes and 3,5-di(tert-butyl)pyrocatechin (HLIII)
are presented in Table 3 and Fig. 4. It was taken into consideration
that o-diphenol derivatives readily undergo electrochemical oxida-
tion to give respective o-semiquinones and o-benzoquinones
Table 3
Cyclic voltammetry data (anodic scan) for the ligands HLI, HLII and their metal(II)
complexes.
Compound
Epa
Epc
E1/2
Epa
Epc
E1/2
Epa
Epa
(V)
(V)
(V)
(V)
(V)
(V)
(V)
(V)
HLI
1.26
1.20
0.64
0.57
0.95
0.88
1.54a 1.88a
1.76a
Cu(LI)2
Co(H2O)2LI
Ni(LI)2
Zn(LI)2
Fe(LI)2
Mn(LI)2
HLII
0.41
0.14
0.28
1.77a
1.39
1.39
0.64
0.80
1.02
1.10
1.76a
1.76a
1.55a 1.81a
1.54a 1.83a
0.79
0.30
0.55
1.22
1.33
1.33
1.33
1.33
1.81
0.48
0.51
0.69
0.68
0.69
0.50
0.85
0.92
1.01
1.01
1.01
1.16
Cu(LII)2
Co(LII)2
Ni(LII)2
Zn(LII)2
Fe(LII)2
0.68
0.94
1.02
0.18
0.17
0.24
0.43
0.55
0.63
1.06
0.34
0.50
0.45
0.70
0.65
0.83
Mn(H2O)2(LII)2 0.80
1.30
0.65
0.90
HLIII
1.20
1.40a
1.77a
a
Irreversible process; it is just the anodic peak that is observed.
Fig. 4. Cyclic voltammograms (50 mV/s) of the ligands (1.36 mmol lꢀ1) HLI (dashed
line), HLII (dotted line), HLIII (dashed–dotted line) in 0.1 mol lꢀ1 (C2H5)4NClO4
acetonitrile solution on glassy-carbon electrode and background cyclic voltammo-
gram of glassy-carbon electrode (solid line).
Fig. 3. The representation of frontier orbitals0 surfaces for HLI (a and b), HLII (e and
f) and their Ni(II) complexes (c, d, j, and k).

2588
N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
[54,55]; besides, oxidation of sulfur atom in the side chain of the
ligands HLI and HLII is possible. The compound HLIII, lacking a sul-
fur-containing substituent, was used to carry out comparison stud-
ies of the redox conversions taking place.
arranged into a sequence according to their reducing ability, the
formal potential of the redox system E11=2 being used as its criterion
(according to [58]): Cu(LI)2 > Mn(LI)2 > HLI > Ni(LI)2 > Zn(LI)2 >
Fe(LI)2 > Co(H2O)2LI. (The formal potential is calculated as the
average potential of the peaks found by the cyclic voltammetry
method: E11=2 ¼ ðEp1a þ E1pcÞ=2Þ. On the basis of the electrochemical
findings the ligand HLII and its metal complexes can be graded in
their reducing ability as follows: Cu(LII)2 > Co(LII)2 > Ni(LII)2 >
Mn(H2O)2(LII)2 > Fe(LII)2 > HLII > Zn(LII)2. The data on redox proper-
ties of the ligands and their metal complexes were used in inter-
preting the results of the biological evaluation of the compounds
synthesized (Section 3.3).
On anodic potential scan o-diphenols HLI, HLII and HLIII undergo
electrochemical oxidation reactions (Fig. 4). Comparing electro-
chemical data for these o-diphenols, one can note an anodic peak
in the region 1.2–1.33 V, which is common for them, with a coun-
terpart on the reverse scan, that is, a cathodic peak corresponding
to reduction of oxidation products in the range of 0.45–0.64 V.
Controlled electrolysis of solutions of these o-diphenols demon-
strated that upon the anodic oxidation process associated with
the peak at 1.20–1.33 V, the quantity of electricity passed corre-
sponds to two electrons per molecule. In the absorption spectra
of solutions containing products of their electrochemical oxidation
there are bands at 400–470 nm which can be assigned to absorp-
tion of respective substituted o-benzoquinones [56]. Thus, it can
be concluded that at the potentials of the first peak the o-diphenol
derivatives HLI, HLII and HLIII undergo two successive one-electron
oxidation processes to give o-benzoquinones, the cathodic peak
corresponding to their reduction being observed in the range
0.45–0.64 V on the reverse scan. As this redox process for HLI, HLII
and HLIII involves phenolic hydroxyls, the potentials of respective
peaks are in a narrow range (Table 3), and it is only the compound
HLII that has a slightly different potential (1.33 V), which may be
due to the acceptor effect of the sulfoxide group [57].
3.3. Biological evaluation
3.3.1. Antibacterial activity
MIC values of the ligands HLI and HLII and their metal(II) com-
plexes are listed in Table 4. It was for the first time that the anti-
bacterial activity of Fe(II) and Mn(II) complexes of these ligands
was investigated. For the ligands and their Cu(II), Co(II), Ni(II)
and Zn(II) complexes, continuing our previous study [16], we have
expanded the spectrum of Gram-positive – (S. aureus, M. smegma-
tis), Gram-negative bacteria (S. typhimurium) and carried out their
microbiological investigation to compare the antibacterial activity
of all the metal complexes under unified conditions (see Section
2.5).
For the compounds HLII and HLIII the above-mentioned peaks at
the potentials E1pa and Ep1c are the only ones, while for the compound
HLI there are several anodic peaks more (Fig. 4 and Table 3). Taking
into account that the o-diphenol HLIII does not comprise sulfur-
containing substituent, it may be suggested that in the case of
o-diphenol derivative of thioglycolic acid HLI it is successive oxida-
tion processes involving sulfur atom and/or carboxylic group that
take place at 1.54 and 1.88 V. Unlike HLI, the compound HLII has a
sulfoxide lateral substitute, and a redox process involving sulfur
atom of this group does not occur any more. This results in only
one redox process involving hydroxyl groups of o-diphenol being
observed for HLII in the range under study (Fig. 4 and Table 3). No
peaks corresponding to reduction of the compounds HLI, HLII and
HLIII are observed upon cathodic polarization.
Evaluating the antibacterial activity of the ligands and their me-
tal(II) complexes, we can note that they demonstrated a low inhib-
iting ability toward Gram-negative bacteria: MIC >100 l
g mlꢀ1
(Table 4). Antibacterial activity of the ligands HLI and HLII and their
metal(II) complexes against Gram-positivebacteria (S. saprophiticus,
S. aureus, B. subtilis, S. lutea) is well higher than their activity against
Gram-negative ones, and in some tests it is comparable to that of
standard antibiotics (Table 4). The activity of the complexes Cu(LI)2,
Mn(LI)2, Cu(LII)2 and Co(LII)2 against most of Gram-positive bacteria
is higher than that of the ligands and other metal complexes under
study; in particular, the high activity of these metal(II) complexes
againstM. smegmatis is worthy of particular notice. The pronounced
change in activity resulting from complexation can be due to the
growth in their lipophilicity (Table 5).
Characteristics of the redox processes observed in voltammo-
grams of the metal complexes are presented in Table 3. The data
obtained allow one to make conclusions about the reducing ability
of the compounds under study. The investigation carried out
showed that the ligand HLI and its metal complexes can be
It has been found that the antibacterial activity of the metal(II)
complexes synthesized does not correlate with the toxicity of the
metal(II) ions to the bacteria tested, because none of the starting
metal salts acts against bacteria up to the dose of 200
The antibacterial activity of the compounds examined follows
l .
g mlꢀ1
Table 4
Antibacterial activity of the free ligands and their metal(II) complexes evaluated by minimum inhibitory concentration (MIC, g mlꢀ1).
Compound
Pseudomonas
aeruginosa
Serratia
marcescens
Salmonella
typhimurium
Escherichia Bacillus
Sarcina
lutea
Staphylococcus
saprophyticus
Staphylococcus Mycobacterium
coli
subtilis
aureus
smegmatis
HLI
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
6.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
12.5
6.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
3.1
50
25
25
25
6.2
50
25
25
50
25
100
12.5
25
50
50
50
50
6.2
6.2
6.2
25
6.2
50
25
25
100
12.5
50
6.2
12.5
25
50
50
50
6.2
3.1
6.2
12.5
6.2
100
25
100
50
6.2
50
6.2
6.2
25
Cu(LI)2
12.5
100
50
50
50
Co(H2O)2LI
Ni(LI)2
>100
>100
>100
100
50
>100
25
25
Zn(LI)2
Fe(LI)2
Mn(LI)2
HLII
12.5
50
Cu(LII)2
Co(LII)2
12.5
12.5
12.5
100
25
12.5
12.5
6.2
Ni(LII)2
25
Zn(LII)2
>100
>100
50
6.2
6.2
100
25
25
Fe(LII)2
Mn(H2O)2(LII)2
Streptomycin
Tetracycline
3.1
3.1
6.2
Chloramphenicol 12.5
6.2
3.1
12.5

N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
2589
Table 5
with the inhibiting effect of streptomycin, tetracycline and chlor-
amphenicol (Table 4).
Octanol/water partition coefficients (logPow) of the ligands
and metal complexes.a
Compound
logPow
3.3.2. Reduction of cytochrome c
Fe(LI)2
5.26
3.06
5.39
3.08
The results of spectrophotometric investigation of redox inter-
action of oxidized form of Cyt c with the HLI and HLII ligands and
their Cu(II), Co(II), Ni(II), Zn(II), Mn(II) and Fe(II) complexes are gi-
ven in Table 6. The characteristic absorption bands at 550 and
520 nm appearing when the ligands or their metal complexes are
added to the solution of the oxidized Cyt c bear witness to the abil-
ity of these compounds to reduce Cyt c in vitro [59,60].
Mn(LI)2
Fe(LII)2
Mn(H2O)2(LII)2
logPow(HLI) = 2.31; logPow(HLII) = ꢀ2.07; logPow of the
a
Cu(II), Co(II), Ni(II) and Zn(II) complexes with the ligands
HLI and HLII varies in the range 3–5 [16].
We have found that the rate of Cyt c reduction with HLII ligand
corresponds to that with HLI ligand (Table 3). Taking into account
the findings presented in [59–61], it can be suggested that the
most probable route of oxidation of the ligands HLI and HLII in
the system of interest in vitro under anaerobic conditions involves
single-electron stages of their anionic or molecular forms being
oxidized to o-benzoquinones upon interacting with Cyt c via inter-
mediate formation of respective o-benzosemiquinone (Fig. 5).
Almost equal rates of Cyt c reduction with HLI and HLII ligands can
be due first of all to similar reducing abilities of these compounds
(Table 3). Furthermore, their ionization constants, characterizing
their ability to form anions by hydroxyls being deprotonated, differ
but slightly (pK(HLI) = 8.3 and pK(HLII) = 7.9 [16]).
the order: (1) Cu(LI)2 > Mn(LI)2 > HLI > Ni(LI)2 > Zn(LI)2 > Fe(LI)2 >
Co(H2O)2LI;
(2)
Cu(LII)2 > Co(LII)2 > Ni(LII)2 > Mn(H2O)2(LII)2 >
Fe(LII)2 > HLII > Zn(LII)2; their reducing ability (determined electro-
chemically) follows the same order, as it was shown above
(Table 3).
Thus, Cu(LI)2, Mn(LI)2, Cu(LII)2 and Co(LII)2 complexes character-
ized by a strong antibacterial activity demonstrate a much higher
reducing ability than that of the ligands and the rest of the metal
complexes. These complexes may be considered as potential anti-
microbial agents, particularly when their activity is comparable
In the series of metal complexes with HLI ligand it is Cu(LI)2 and
Mn(LI)2 complexes that show the highest rate of Cyt c reduction,
higher than the rate of redox process involving the free ligand
(Table 6). It should be emphasized that, according to electrochem-
ical data, these complexes are the most active reducing agents in
the series of metal complexes with the ligand HLI (Table 3).
Co(H2O)2LI complex can reduce Cyt c in vitro in the system under
study faster than Ni(II), Zn(II), Fe(II) complexes and HLI ligand do
(Table 6), although it has the lowest reducing ability among the
complexes of this series (Table 3). It should be taken into account
that metal complexes can interact with Cyt c both in their molec-
ular form and via phenolate ligand and metal ions formed upon
their dissociation [60]. In this connection attention should be paid
to the values of stability constant of these metal complexes: Cu(LI)2
(logb = 7.1) and Cu(LII)2 (logb = 7.3) are the most stable ones among
the metal complexes, while Co(H2O)2LI is the most ready to
dissociate (logb = 3.5) [16].
Table 6
Rates of reduction of Cyt c (
m) with the ligands
HLI, HLII, and their metal(II) complexes.a
Compound
m )
(nmol minꢀ1
HLI
1.0
6.1
2.1
0.5
0.8
0.5
4.4
Cu(LI)2
Co(H2O)2LI
Ni(LI)2
Zn(LI)2
Fe(LI)2
Mn(LI)2
HLII
1.1
3.1
5.4
3.0
3.5
2.2
2.3
Cu(LII)2
Co(LII)2
Ni(LII)2
Zn(LII)2
Fe(LII)2
Mn(H2O)2(LII)2
a
All the metal complexes with the ligand HLII reduce Cyt c at a
rate several times higher than that of the redox process involving
the free ligand (Table 6). It is Co(LII)2 complex that is characterized
The final concentrations of Cyt c and the
complex (or the ligand) were respectively 7 and
35
mol lꢀ1
l
.
Fig. 5. Scheme of the reduction of Cyt c with the anion form of the ligand HLI; Fe(III)–Cyt c and Fe(II)–Cyt c – respectively the oxidized and reduced forms of Cyt c; the process
of disproportionation of the ligand HLI is depicted by a dashed line.

2590
N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
Table 7
4. Conclusions
Effect of electron-transfer proteins of P450-dependent monooxygenase systems on
the rate of Cyt c (7
l
mol lꢀ1) reduction (
m
) with HLI and HLII ligands (35
l
mol lꢀ1).
The complexes Cu(LI)2, Mn(LI)2 and Co(LII)2 with the high reduc-
ing ability (determined electrochemically) were found to be char-
acterized by the highest rates of Cyt c reduction. As a whole, the
reduction of Cyt c by the ligands and their metal complexes may
not be related solely to the facility of their oxidation, as it is possi-
ble that ionization of the compounds exerts a noticeable effect on
this process, too. The antibacterial activity of these compounds was
found to follow the order: (1) Cu(LI)2 > Mn(LI)2 > HLI > Ni(LI)2 >
Ligand
Enzyme/concentration (nmol lꢀ1
)
m
(nmol minꢀ1
)
HLI
HLI
HLI
HLII
HLII
HLII
0.9 0.1
0.8 0.1
0.9 0.1
P450R/170
P450R/270
1.0 0.1
0.9 0.1
0.9 0.1
P450R/170
P450R/270
Zn(LI)2 > Fe(LI)2 > Co(H2O)2LI;
(2)
Cu(LII)2 > Co(LII)2 > Ni(LII)2 >
Mn(H2O)2(LII)2 > Fe(LII)2 > HLII > Zn(LII)2; their reducing ability
(determined electrochemically) followed the same order. These se-
quences are not entirely the same as those characterizing the de-
crease of the rates of Cyt c reduction with the ligands and their
metal complexes. Despite the fact that in individual cases we have
found a correlation between the rates of Cyt c reduction and phys-
ico-chemical characteristics of the compounds under study, the
ability of bioactive compounds to interact with biomolecules has
a more intricate dependence on the structure and physico-chemi-
cal properties thereof.
Table 8
Calculated parameters for docked conformations of the ligands to P450R.
Ligand Lateral
groups
Lmin(O–N) (Å)/Kint
Kint range
(mmol lꢀ1
)
(mmol lꢀ1
)
HLI
–CO2H
–CO2H
–CH2OH
–CH3
3.1/86.9
6.4/63.3
3.5/1.4
2.9/5.2
17.7–147.6
14.1–152.3
1.4–14.2
HLII
HLIV
HLV
2.6–7.1
by the highest Cyt c reduction rate, thus being one of the strongest
reducing agents in this series of complexes. Cu(LII)2 complex, in
spite of the most cathodic ðEap1 þ Epc1Þ=2 value among these com-
pounds, ranks below Co(LII)2 complex in the rate of Cyt c reduction
(Table 6), which may be due to its being more stable to dissociation
as compared to the above-mentioned complexes. The rate of Cyt c
reduction with the rest of the metal complexes with the ligand HLII
varies in a very narrow range (2–3 nmol minꢀ1) and does not cor-
relate with their reducing ability determined electrochemically
(Tables 3 and 6).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.poly.2011.07.008.
References
[1] N.V. Loginova, T.V. Koval’chuk, R.A. Zheldakova, A.A. Chernyavskaya, N.P.
Osipovich, G.K. Glushonok, G.I. Polozov, V.L. Sorokin, O.I. Shadyro, Centr. Eur. J.
Chem. 4 (2006) 440.
[2] T.V. Koval’chuk, N.V. Loginova, G.I. Polozov, A.A. Chernyavskaya, D.A. Gursky,
O.I. Shadyro, Vestnik BGU. Ser. 2. Khim., Biol. Geogr. (2) (2008) 25 (in Russian).
[3] N.V. Loginova, T.V. Koval’chuk, R.A. Zheldakova, A.A. Chernyavskaya, N.P.
Osipovich, G.K. Glushonok, G.I. Polozov, V.N. Povalishev, V.L. Sorokin, O.I.
Shadyro, Polyhedron 25 (2006) 3603.
[4] N.V. Loginova, T.V. Koval’chuk, R.A. Zheldakova, N.P. Osipovich, V.L. Sorokin,
G.I. Polozov, G.A. Ksendzova, G.K. Glushonok, A.A. Chernyavskaya, O.I. Shadyro,
Bioorg. Med. Chem. Lett. 16 (2006) 5403.
[5] N.V. Loginova, T.V. Koval’chuk, N.P. Osipovich, G.I. Polozov, V.L. Sorokin, A.A.
Chernyavskaya, O.I. Shadyro, Polyhedron 27 (2008) 985.
[6] N.V. Loginova, T.V. Koval’chuk, G.I. Polozov, N.P. Osipovich, P.G. Rytik, I.I.
Kucherov, A.A. Chernyavskaya, V.L. Sorokin, O.I. Shadyro, Eur. J. Med. Chem. 43
(2008) 1536.
[7] N.V. Loginova, Y.V. Faletrov, T.V. Koval’chuk, N.P. Osipovich, G.I. Polozov, A.A.
Chernyavskaya, R.A. Zheldakova, I.I. Azarko, A.T. Gres’, O.I. Shadyro, V.M.
Shkumatov, Polyhedron 29 (2010) 1646.
[8] A. Albert, Selective Toxicity: Physico-Chemical Basis of Therapy, Chapman and
Hall, London, New York, 1985.
[9] S. Bollo, L.J. Núñez-Vergara, S. Kang, L. Zhang, H.I. Boshoff, C.E. Barry III, J.A.
Squella, C.S. Dowd, Bioorg. Med. Chem. Lett. 21 (2011) 812.
The influence of P450R on the rate of Cyt c reduction with HLI
and HLII ligands was studied. The addition of P450R had essentially
no effect on the initial rate of Cyt c reduction with the compounds
under study (Table 7), as opposed to the phenolic ligands 4,6-di(-
tert-butyl)-3[(2-ethyl)sulfanyl]-1,2-dihydroxybenzene (HLIV) and
2-amino-4,6-di(tert-butyl)phenol studied before [7]. This is indica-
tive of o-diphenol derivatives of thioglycolic acid being incapable
of redox interactions with P450R, responsible for an increase of
Cyt c reduction in vitro in the system described. In this connection
we have studied Cyt c reduction and P450R affecting this process
with the participation of a structural analog of o-diphenol deriva-
tives of thioglycolic acid, 4,6-di(tert-butyl)-3-ethylsulfanyl-1,2-
dihydroxybenzene (HLV), containing a lateral methyl group instead
of a carboxylic one. The rate of Cyt c reduction with this compound
was found to be 0.7 0.1 nmol Cyt c ꢂ minꢀ1, and it is about twice
as high as that with P450R added. Analyzing structure-functional
differences of these ligands, we can reveal probable reasons for this
effect: (i) a smaller reductive ability of the ligands HLI and HLII
compared to the o-diphenol and o-aminophenol derivatives stud-
ied before [7], (ii) lateral carboxylic groups of HLI and HLII ligands
being negatively charged at physiological pH 7.4.
It should be mentioned that redox-active FMN edge in P450R is
surrounded by several negatively charged residues of Glu and Asp
amino acids located on the protein surface. To evaluate the sup-
posed contribution of electrostatic repulsion into P450R interac-
tion with the ligands under study, we carried out docking
experiments of the above-mentioned ligand structures in the ac-
tive center of P450R according to the procedure described in
[33–35]. Millimolar order of Kint values calculated from the docking
results suggests the non-specific character of P450R interaction
with all the compounds described above, but the compounds HLIV
and HLV were more effectively bound in the vicinity of FMN than o-
diphenol derivatives of thioglycolic acid HLI and HLII under in silico
conditions (Table 8).
[10] B. Wen, K.J. Coe, P. Rademacher, W.L. Fitch, M. Monshouwer, S.D. Nelson,
Chem. Res. Toxicol. 21 (2008) 2393.
[11] J. Simon, FEMS Microbiol. Rev. 26 (2002) 285.
[12] J. Hendriks, A. Oubrie, J. Castresana, A. Urbani, S. Gemeinhardt, M. Saraste,
Biochim. Biophys. Acta 1459 (2000) 266.
[13] L. De Smet, S.N. Savvides, E. Van Horen, G. Pettigrew, J.J. Van Beeumen, J. Biol.
Chem. 281 (2006) 4371.
[14] I. Bertini, G. Cavallaro, A. Rosato, Chem. Rev. 106 (2006) 90.
[15] L. Thony-Meyer, Microbiol. Mol. Biol. Rev. 61 (1997) 337.
[16] N.V. Loginova, T.V. Koval’chuk, G.I. Polozov, N.P. Osipovich, A.A.
Chernyavskaya, V.L. Sorokin, O.I. Shadyro, Redox-active antimicrobial metal
complexes with sterically hindered o-diphenol and o-aminophenol
derivatives, in: G. Blanc, D. Moreau (Eds.), Biometals: Molecular Structures,
Binding Properties, Nova Science Publisher’s, Hauppauge, New York, 2010, pp.
59–90.
[17] M. Buhl, H. Kabrede, J. Chem. Theory Comput. 2 (2006) 1282.
[18] T. Wang, G. Brudvig, V.S. Batista, J. Chem. Theory Comput. 6 (2010) 755.
[19] Y. Yang, M.N. Weaver, K.M. Merz, J. Phys. Chem. A 113 (2009) 9843.
[20] Z. An, J. Zhang, R. Wang, N. Koga, Polyhedron 26 (2007) 2126.
[21] K. Malek, H. Kozlowski, L.M. Proniewicz, Polyhedron 24 (2005) 1175.
[22] A. Leo, C. Hansch, D. Elkins, Chem. Rev. 71 (1971) 525.
[23] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman,
J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar,
J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A.
Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa,

N.V. Loginova et al. / Polyhedron 30 (2011) 2581–2591
2591
M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox,
H.P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann,
O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K.
Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S.
Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K.
Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Clifford, J.
Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L.
Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M.
Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, C. Gonzalez, J.A.
Pople, GAUSSIAN 03, Revision C, Gaussian Inc., Pittsburgh, PA, 2003.
[41] L.J. Bellamy, The Infrared Spectra of Complex Molecules, Methuen, London,
1958.
[42] S. Belaid, S. Djebbar, O. Benali-Baitich, M. Khan, G. Bouet, J. Inorg. Biochem. 102
(2008) 63.
[43] A. Carrington, E. MacLachlan, Introduction to Magnetic Resonance with
Applications to Chemistry and Chemical Physics, Harper and Row, New York,
1967.
[44] A.D. Shultz, A.K. Boal, D.J. Driscoll, J.R. Kitchin, G.N. Tew, J. Org. Chem. 60
(1995) 3578.
[45] A.B.P. Lever, E.I. Solomon (Eds.), Inorganic Electronic Structure and
Spectroscopy, John Wiley and Sons, New York, 2006.
[24] P.J. Hay, W.R. Wadt, J. Chem. Phys. 82 (1985) 299.
[25] J.W. Ponder, Tinker Software, Version 5.0, 2010, http://dasher.wustl.edu/tinker.
[46] A. Pui, J.-P. Mahy, Polyhedron 26 (2007) 3143.
[26] D.C. Young, Computational Chemistry:
A
Practical Guide for Applying
[47] A.M. Shaker, M.Sh. Adam, Synth. React. Inorg. Met.-Org. Chem. 33 (2003) 1081.
[48] M.J. Sever, J.J. Wilker, J. Chem. Soc., Dalton Trans. (2004) 1061.
[49] M.L. Childers, J. Cho, C.A.S. Regino, M.W. Brechbiel, A.G. DiPasquale, A.L.
Rheingold, S.V. Torti, F.M. Torti, R.P. Planalp, J. Inorg. Biochem. 102 (2008) 150.
[50] N.R. Perron, J.N. Hodges, M. Jenkins, J.L. Brumaghim, Inorg. Chem. 47 (2008)
6153.
Techniques to Real-World Problems, Wiley Interscience, New York, 2001.
[27] A.V. Aleshukina, Medicinal Microbiology, Phoenix, Rostov-on-Don, 2003 (in
Russian).
[28] A.A. Akhrem, N.V. Lapko, A.G. Lapko, V.M. Shkumatov, V.L. Chashchin, Acta
Biol. Med. Ger. 38 (1979) 257.
[29] S. Tamura, K.R. Korzekwa, S. Kimura, H.V. Gelboin, F.J. Gonzalez, Arch.
Biochem. Biophys. 293 (1992) 219.
[30] C.C. Winterbourn, Biochem. J. 207 (1982) 609.
[31] G.F.Z. da Silva, W.M. Tay, Li-J. Ming, J. Biol. Chem. 280 (2005) 16601.
[32] N. Rahman, Y. Ahmad, S.N.H. Azmi, AAPS PharmSciTech. 6 (2005) 543.
[33] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J.
Olson, J. Comput. Chem. 19 (1998) 1639.
[51] A. Saha, P. Majumdar, S. Goswami, J. Chem. Soc., Dalton Trans. (2000) 1703.
[52] M.J. Sabater, A. Corma, J.V. Folgado, H. García, J. Phys. Org. Chem. 13 (2000) 57.
[53] J.J. Alexander, H.B. Gray, J. Am. Chem. Soc. 90 (1968) 4260.
[54] G. Speier, Z. Tyeklar, P. Toth, E. Speier, S. Tisza, A. Rochen-bauer, A.M. Whalen,
N. Alkire, C.G. Pierpont, Inorg. Chem. 40 (2001) 5653.
[55] C.G. Pierpont, Coord. Chem. Rev. 216–217 (2001) 99.
[56] F. Zippel, F. Ahlers, R. Werner, W. Haase, H.F. Nolting, B. Krebs, Inorg. Chem. 35
(1996) 3409.
[34] M.F. Sanner, J. Mol. Graphics Mod. 17 (1999) 57.
[35] J. Gasteiger, M. Marsili, Tetrahedron 36 (1980) 3219.
[36] W.J. Geary, Coord. Chem. Rev. 7 (1971) 81.
[57] N.T. Berberova, I.V. Smolyaninov, A.O. Okhlobystin, N.N. Letichevskaya, E.V.
Shinkar, Mend. Chem. J. 59 (5) (2005) 67 (in Russian).
[37] C.N. Verani, Rational Synthesis and Characterization of Paramagnetic
Heteropolynuclear Systems, Ph.D. Thesis, Ruhr-Universität Bochum,
Mülheim an der Ruhr, 2000.
[38] C.J. Cramer, D.G. Truhlar, Phys. Chem. Chem. Phys. 11 (2009) 10757.
[39] W. Koch, M.C. Holthausen, A Chemist’s Guide to Density Functional Theory,
Wiley-VCH, 2000.
[58] F. Scholz, Electroanalytical Methods: Guide to Experiments and Applications,
Springer-Verlag, Berlin, 2002.
[59] H.T. Nagasawa, H.R. Gutmann, M.A. Morgan, J. Biol. Chem. 234 (1958) 1600.
[60] M.M.M. Saleem, M.T. Wilson, Biochem. J. 201 (1982) 433.
[61] E.L. Cavalieri, K.M. Li, N. Balu, M. Saeed, P. Devanesan, S. Higginbotham, J. Zhao,
M.L. Gross, E.G. Rogan, Carcinogenesis 6 (2002) 1071.
[40] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds: Theory and Applications in Inorganic Chemistry, John Wiley and
Sons Inc., New York, 1997.