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New uses for old drugs: attempts to convert quinolone antibacterials into potential anticancer agents containing ruthenium.
Article
pubs.acs.org/IC
New Uses for Old Drugs: Attempts to Convert Quinolone
Antibacterials into Potential Anticancer Agents Containing
Ruthenium
Jakob Kljun,†,‡ Ioannis Bratsos,§,∥ Enzo Alessio,§ George Psomas,⊥ Urška Repnik,⊗ Miha Butinar,⊗
Boris Turk,†,⊗,# and Iztok Turel*,†,‡
†
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškerčeva 5, SI-1000 Ljubljana, Slovenia
EN→FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
§
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Trieste, Via L. Giorgieri 1, I-34127 Trieste, Italy
∥
Department of Physical Chemistry, NCSR “Demokritos”, GR-15310 Ag. Paraskevi, Athens, Greece
⊥
Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, P.O. Box 123,
GR-54124 Thessaloniki, Greece
⊗
Jozef Stefan Institute, Jamova c. 39, SI-1000 Ljubljana, Slovenia
#
CIPKEBIP Centre of Excellence, Jamova c. 39, SI-1000, Ljubljana
‡
S Supporting Information
*
ABSTRACT: Continuing the study of the physicochemical and biological
properties of ruthenium-quinolone adducts, four novel complexes with the
general formula [Ru([9]aneS3)(dmso-κS)(quinolonato-κ2O,O)](PF6), containing the quinolones levofloxacin (1), nalidixic acid (2), oxolinic acid (3),
and cinoxacin (4), were prepared and characterized in solid state as well as in
solution. Contrary to their organoruthenium analogues, these complexes are
generally relatively stable in aqueous solution as substitution of the
dimethylsulfoxide (dmso) ligand is slow and not quantitative, and a minor
release of the quinolonato ligand is observed only in the case of 4. The
complexes bind to serum proteins displaying relatively high binding
constants. DNA binding was studied using UV−vis spectroscopy, cyclic
voltammetry, and performing viscosity measurements of CT DNA solutions
in the presence of complexes 1−4. These experiments show that the
ruthenium complexes interact with DNA via intercalation. Possible
electrostatic interactions occur in the case of compound 4, which also
shows the most pronounced rate of hydrolysis. Compounds 2 and 4 also
exhibit a weak inhibition of cathepsins B and S, which are involved in the
progression of a number of diseases, including cancer. Furthermore, complex 2 displayed moderate cytotoxicity when tested on
the HeLa cell line.
■
INTRODUCTION
Cisplatin is the first and by far the most widely used metalbased anticancer drug.1 It is a pro-drug which is activated by
hydrolysis of the two cis-bonded chlorido ligands that allows
the binding of the cis-{Pt(NH3)2}2+ fragment to nuclear DNA.
This creates a kink in the DNA tertiary structure which
prevents the replication of cancer cells. In the development of
metal-based anticancer drugs platinum compounds still occupy
the most important position. However, a number of
coordination compounds of various other metals have also
been evaluated.2 Among them, the ruthenium complexes
occupy the most prominent position as two compounds,
namely, NAMI-A ([imH]trans-[RuCl4(dmso-S)(im)], im =
imidazole) and KP1019 ([indH]trans-[RuCl4(ind)2], ind =
indazole), have been tested in clinical trials.3,4 In the past
© XXXX American Chemical Society
decade the research has shifted mainly toward half-sandwich
organoruthenium(II) complexes, and compounds bearing a
wide variety of ligands were studied and evaluated for possible
biological or medical applications.5
It has also been shown that the π-bonded aromatic ligands
are not essential for the anticancer activity of ruthenium(II)
complexes. The aromatic core can be substituted by another 6electron face-capping ligand like the crown thioether 1,4,7trithiacyclononane ([9]aneS3), yet the complex still retains the
properties that are needed to be suitable as a candidate for
cancer treatment.6,7
Received: May 16, 2013
A
dx.doi.org/10.1021/ic401220x | Inorg. Chem. XXXX, XXX, XXX−XXX
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The exact mechanism of action of ruthenium compounds is
not fully understood; however, recent research seems to favor
the hypothesis that their interactions with proteins and
enzymes specific for processes of cancer development and/or
progress are crucial.2 Most importantly, unlike the platinum(II)
compounds, the different types of ruthenium compounds
exhibit very different affinity and reactivity toward biomacromolecules and lower selectivity, since they readily interact and
form adducts with various targets, including DNA (to a minor
degree).8−10 It is thus quite possible that ruthenium complexes
hit multiple targets, and a combination of their action
contributes to the observed beneficial properties. It is therefore
important to investigate interactions with various potential
targets to gain insight into the possible modes of action of these
compounds and thus to be able to design novel and more
effective drug candidates.
In this paper we present the continuation of our research on
the physicochemical and biological properties of rutheniumquinolone complexes. Quinolones are a family of synthetic
antibacterial agents widely used in clinical practice. Nalidixic
acid was the first quinolone introduced to clinical use in 1962
and since then more than 10,000 related compounds have been
synthesized and tested as potential antibacterial agents, and
more than thirty were, or still are, in clinical or veterinary use.11
Besides their antibacterial activity, some quinolone derivatives
with the extended aromatic ring system, namely, the
quinobenzoxazines, were also shown to exhibit antitumor
properties.12
Despite their widespread application, the exact mechanism of
action of the quinolones is not fully understood. It is known
that the quinolones bind to DNA, thus inhibiting the bacterial
topoisomerase and preventing the replication of bacteria.11
However, the binding mode of the quinolone to DNA is still
uncertain. Various binding modes have been proposed13 and
the recent discovery of a topoisomerase-DNA-quinolone
complex suggests that the role of metal ions is very important.
In the aforementioned complex a magnesium ion binds the
quinolone β-ketocarboxylate group and four water molecules,
which in turn form hydrogen bonds with DNA nucleobases.14
Several metal complexes of compounds belonging to the
quinolone family were reported in the past decades,15 and their
interactions with important biological macromolecules and
their biological activity were investigated.16,17 Our group
recently reported the structures, hydrolytic and biological
properties of organoruthenium complexes of quinolones that
could potentially be used as anticancer agents.9,18,19 The
complex with the quinolone ofloxacin has shown binding to
DNA which provoked a considerable shrinkage of the DNA
tertiary structure and has proven to be moderately effective
against the CH1 ovarian cancer cells. Moreover, the organoruthenium complex of the thionated derivative of the quinolone
nalidixic acid showed considerable stability in aqueous solution
and increased cytotoxicity toward various cancer cell lines and
inhibition of cathepsins, enzymes involved in the development
and progression of cancer and other diseases.9,18,19
Four novel ruthenium(II) complexes bearing the facecapping sulfur macrocycle [9]aneS3, S-bonded dimethylsulfoxide (dmso-κS), and a quinolonato ligand (quinolonatoκ2O,O) with the general formula [Ru([9]aneS3)(dmso-κS)(quinolonato-κ2O,O)](PF6), where the quinolone ligand is
levofloxacin (levo, 1), nalidixic acid (nal, 2), oxolinic acid (oxol,
3), and cinoxacin (cin, 4), respectively (Figure 1), were
synthesized. The crystal structures of compounds 2 and 4 were
Figure 1. Schematic representation of the general formula of the new
half sandwich [Ru([9]aneS3)(dmso-κS)(quinolonato-κ2O,O)](PF6)
compounds 1−4 and of the quinolonato ligands in anionic form
used in the present work, with the numbering scheme used for the
NMR characterization.
determined by X-ray diffraction analysis, and the stability of 1−
4 in water solution was investigated by means of 1H NMR
spectroscopy. The interaction of complexes 1−4 with calfthymus DNA (CT DNA) was studied, and a competitive study
of the intercalative agent ethidium bromide (EB) was
performed. The affinity of 1−4 toward bovine and human
serum albumin (BSA and HSA) was investigated, and their
binding constants were determined. The inhibitory potency of
the compounds against cathepsins B and S, two enzymes of the
cathepsin family, was also evaluated, as well as their cytotoxicity
on two cancer cell lines (HeLa and A549).
1. EXPERIMENTAL PART
1.1. Materials and Instrumentation. All solvents, the quinolone
ligands, CT DNA, BSA, HSA, EB, NaCl, tetraethylammonium
perchlorate (TEAP), and trisodium citrate were purchased from
Sigma-Aldrich. The ruthenium precursor [Ru([9]aneS3)(dmso)3](PF6)2 (P1) was prepared according to the reported procedure.20 All
the chemicals and solvents were reagent grade and were used as
purchased.
Infrared spectra (ATR; Attenuated total reflectance) were recorded
on a Perkin-Elmer Spectrum 100 spectrometer. The measurements
were made in the range from 4000 to 600 cm−1. Elemental analyses
were performed on a Perkin-Elmer Elemental analyzer 2400 CHN. Xray diffraction data were collected on a Nonius Kappa CCD
diffractometer at 150 K for compound 2·3H2O and at room
temperature for compound 4·EtOH using graphite monochromated
Mo-Kα radiation and processed using DENZO.21 The structures were
solved using SIR92.22 A full-matrix least-squares refinement on F
magnitudes with anisotropic displacement factors for all non-hydrogen
atoms using SHELXL was employed.23 The drawings, the analysis of
bond lengths, angles, and intermolecular interactions was done using
Mercury and Platon.24 Hydrogen atoms were placed in geometrically
calculated positions and were refined using a riding model.
Mono- (1H (400 or 500 MHz), 13C (101 or 126 MHz)) and
bidimensional (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) NMR
spectra were recorded on a JEOL Eclipse 400FT or on a Varian 500
spectrometer (Trieste) or on a Bruker Avance III 500 spectrometer
(Ljubljana). 1H chemical shifts in D2O were referenced to the internal
standard 2,2-dimethyl-2,2-silapentane-5-sulfonate (DSS) at δ = 0.00,
while in CD 3NO2 were referenced to the peak of residual
nondeuterated solvent (δ = 4.33); 13C chemical shifts were referenced
to the peak of residual nondeuterated nitromethane (δ = 62.8).
UV−visible (UV−vis) spectra were recorded in solution at
concentrations in the range 10−5−10−3 M on a Hitachi U-2001 dual
beam spectrophotometer. Fluorescence spectra were recorded in
solution on a Hitachi F-7000 fluorescence spectrophotometer.
Viscosity experiments were carried out using an ALPHA L Fungilab
B
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Selected IR resonances (cm−1, ATR): 2989, 1622, 1604, 1573, 1492,
1447, 1258, 1080, 835, 804. CHN: Calc. for
C20H29F6N2O4PRuS4·3H2O C, 30.42; H, 4.47; N, 3.55. Found C,
30.27; H, 4.71; N, 3.69.
NMR: 1H NMR (400 MHz, D2O) δ 9.22 (s, 1H, C2H), 8.71 (d, J =
8.3 Hz, 1H, C5H), 7.55 (d, J = 8.3 Hz, 1H, C6H), 4.77−4.60 (m, 2H,
C12H2), 3.19 (s, 3H, CH3 dmso-S), 3.16 (s, 3H, CH3 dmso-S), 3.14−
2.54 (m, 12H, CH2 [9]aneS3), 2.74 (s, 3H, C14H3), 1.51 (t, J = 7.1 Hz,
3H, C13H3).
1
H NMR (500 MHz, CD3NO2) δ 9.24 (s, 1H, C2H), 8.72 (d, J =
8.3 Hz, 1H, C5H), 7.52 (d, J = 8.3 Hz, 1H, C6H), 4.81−4.68 (m, 2H,
C12H2), 3.10 (s, 3H, CH3 dmso-S), 3.02 (s, 3H, CH3 dmso-S), 3.09−
2.53 (m, 12H, CH2 [9]aneS3), 2.75 (s, 3H, C14H3), 1.56 (t, J = 7.2 Hz,
3H, C13H3).
13
C NMR (101 MHz, CD3NO2) δ 180.2 (C4O), 167.0 (C11O),
166.5 (C7), 153.9 (C2H), 148.3 (C9), 137.5 (C5H), 124.0 (C6H), 121.6
(C10), 115.4 (C3), 49.1 (C12H2), 43.8 (CH3 dmso-S), 43.6 (CH3 dmsoS), 36.3 (CH2 [9]aneS3), 35.4 (CH2 [9]aneS3), 34.3 (CH2 [9]aneS3),
33.4 (CH2 [9]aneS3), 31.4 (CH2 [9]aneS3), 31.0 (CH2 [9]aneS3), 25.5
(C14H3), 15.6 (C13H3).
1.2.3. [Ru([9]aneS3)(dmso-κS)(oxol-κ2O,O)](PF6) (3). This complex
was prepared according to the general procedure, slightly modified,
using 25.9 mg of oxolinic acid (0.099 mmol). After refluxing in
MeOH, the product started to precipitate as pale yellow solid upon
cooling. The precipitation was completed after the concentration of
the solution to about 4 mL affording 62.3 mg of 3 (Yield 82.8%).
Selected IR resonances (cm−1, ATR): 3000, 1636, 1583, 1563, 1466,
1264, 1084, 1033, 1022, 831. CHN: Calc. for C21H28F6NO6PRuS4 C,
32.98; H, 3.61; N, 1.83. Found C, 32.32; H, 3.98; N, 1.71.
NMR: 1H NMR (400 MHz, D2O) δ 8.95 (s, 1H, C2H), 7.81 (s, 1H,
5
C H), 7.39 (s, 1H, C8H), 6.21 (s, 2H, C14H2), 4.49 (dd, J = 14.7, 7.7
Hz, 2H, C12H2), 3.18 (s, 3H, CH3 dmso), 3.15 (s, 3H, CH3 dmso),
3.13−2.54 (m, 12H, CH2 [9]aneS3), 1.51 (t, J = 7.1 Hz, 3H, C13H3).
1
H NMR (500 MHz, CD3NO2) δ 8.98 (s, 1H, C2H), 7.81 (s, 1H,
5
C H), 7.31 (s, 1H, C8H), 6.22 (s, 2H, C14H2), 4.52 (q, J = 7.2 Hz, 2H,
C12H2), 3.09 (s, 3H, CH3 dmso), 3.02 (s, 3H, CH3 dmso), 3.08−2.52
(m, 12H, CH2 [9]aneS3), 1.58 (t, J = 7.2 Hz, 3H, C13H3).
13
C NMR (126 MHz, CD3NO2) δ 177.8 (C4O), 167.4 (C11O),
155.1 (C7), 151.5 (C2H), 149.3 (C6), 136.9 (C9), 125.3 (C10), 105.0
(C14H2), 104.2 (C5H), 97.0 (C8H), 52.0 (C12H2), 43.8 (CH3 dmso-S),
43.5 (CH3 dmso-S), 36.2 (CH2 [9]aneS3), 35.4 (CH2 [9]aneS3), 34.3
(CH2 [9]aneS3), 33.4 (CH2 [9]aneS3), 31.4 (CH2 [9]aneS3), 31.1
(CH2 [9]aneS3), 14.9 (C13H3).
1.2.4. [Ru([9]aneS3)(dmso-κS)(cin-κ2O,O)](PF6)·EtOH (4·EtOH).
This complex was prepared according to the general procedure
using 26.0 mg of cinoxacin (0.099 mmol) affording 74.4 mg of 4 as a
yellow-orange solid (Yield 92.9%). Crystals suitable for X-ray analysis
were obtained from a MeOH/EtOH 1:1 solution upon slow
evaporation at room temperature.
Selected IR resonances (cm−1, ATR): 2981, 1618, 1601, 1519, 1468,
1272, 1089, 1034, 829, 784. CHN: Calc. for
C20H27F6N2O6PRuS4·C2H6O C, 29.96; H, 3.90; N, 3.50. Found C,
30.33; H, 3.87; N, 3.34.
NMR: 1H NMR (500 MHz, D2O) δ 7.74 (s, 1H, C5H), 7.49 (s, 1H,
C8H), 6.29 (s, 2H, C14H2), 4.76 (overlapped with HOD, C12H2), 3.22
(s, 3H, CH3 dmso-S), 3.19 (s, 3H, CH3 dmso-S), 3.15−2.55 (m, 12H,
CH2 [9]aneS3), 1.56 (t, J = 7.3 Hz, 3H, C13H3).
1
H NMR (500 MHz, CD3NO2) δ 7.74 (s, 1H, C5H), 7.37 (s, 1H,
C8H), 6.29 (d, J = 2.1 Hz, 2H, C14H2), 4.75 (q, J = 7.3 Hz, 2H, C12H2),
3.14 (s, 3H, CH3 dmso-S), 3.07 (s, 3H, CH3 dmso-S), 3.11−3.04 (m,
1H, CH2 [9]aneS3), 3.02−2.69 (m, 9H, CH2 [9]aneS3), 2.67−2.60 (m,
1H, CH2 [9]aneS3), 2.60−2.53 (m, 1H, CH2 [9]aneS3), 1.59 (t, J = 7.3
Hz, 3H, C13H3).
13
C NMR (126 MHz, CD3NO2) δ 172.0 (C4O), 165.3 (C11O),
156.6 (C7), 151.4 (C6), 139.8 (C9), 138.5 (C3H), 127.0 (C10), 105.6
(C14H2), 102.2 (C5H), 95.9 (C8H), 55.7 (C12H2), 43.7 (CH3 dmso-S),
43.6 (CH3 dmso-S), 36.2 (CH2 [9]aneS3), 35.6 (CH2 [9]aneS3), 34.2
(CH2 [9]aneS3), 33.5 (CH2 [9]aneS3), 31.3 (CH2 [9]aneS3), 31.2
(CH2 [9]aneS3), 14.2 (C13H3).
rotational viscometer equipped with an 18 mL LCP spindle, and the
measurements were performed at 100 rpm.
Cyclic voltammetry studies were performed on an Eco chemie
Autolab Electrochemical analyzer. Cyclic voltammetry experiments
were carried out in a 30 mL three-electrode electrolytic cell. The
working electrode was platinum disk, a separate Pt single-sheet
electrode was used as the counter electrode, and a Ag/AgCl electrode
saturated with KCl was used as the reference electrode. The cyclic
voltammograms of the complexes were recorded in 0.4 mM
dimethylsulfoxide (DMSO) solutions and in 0.4 mM (1:2)
DMSO:buffer solutions at v = 100 mV s−1 with TEAP and the buffer
solution being the supporting electrolytes, respectively. Oxygen was
removed by purging the solutions with pure nitrogen which had been
previously saturated with solvent vapors. All electrochemical measurements were performed at 25.0 ± 0.2 °C.
1.2. Syntheses and Characterization. General Synthetic
Procedure for 1−4. An 80.0 mg portion of the ruthenium precursor
[Ru([9]aneS3)(dmso)3](PF6)2 (P1, 0.099 mmol) and 1 equiv of the
appropriate quinolone (0.099 mmol) in acidic form with 5.0 mg of
NaOMe (0.093 mmol), or just 1 equiv of nalidixic acid sodium salt
hydrate in case of 2, were suspended in 15 mL of MeOH. The
suspension gradually dissolved as the reaction mixture was heated
yielding a clear yellow solution, which was then refluxed for 2 h. The
solvent was then rotary-evaporated, replaced by acetone, and NaPF6
was removed by filtration. The solvent was again replaced by EtOH,
and the formed product was collected by filtration, washed with EtOH
and diethyl ether, and vacuum-dried.
1.2.1. [Ru([9]aneS3)(dmso-κS)(levo-κ2O,O)](PF6) (1). This complex
was prepared according to the general procedure using 35.9 mg of
levofloxacin (0.099 mmol) affording 42.5 mg of 1 as yellow solid
(Yield 49.5%). Crystals of complex 1 were obtained by slow
evaporation of a methanol/ethanol solution.
Selected IR resonances (cm−1, ATR): 3651, 2971, 1624, 1578, 1515,
1467, 1272, 1087, 830, 804. CHN: Calc. for
C26H37F7N3O5PRuS4·1,5H2O C, 35.01; H, 4.52; N, 4.71. Found C,
35.18; H, 4.37; N, 4.79.
NMR: 1H NMR (400 MHz, D2O) δ 8.98 (s, 1H, C2H), 7.79 (d, J =
12.7 Hz, 1H, C5H), 4.81 (overlapped with the peak of HOD, C1″H),
4.60 (d, J = 11.8 Hz, 1H, C2″Ha), 4.40 (m br, 1H, C2″Hb), 3.51 (s br,
4H, C2′H2/C6′H2), 3.19 (s, 3H, CH3 dmso), 3.15 (s, 3H, CH3 dmso),
3.11−2.57 (m, 19H, C3′H2/C5′H2, CH2 [9]aneS3, C7′H3), 1.56 (t, J =
5.9 Hz, 3H, C3″H3).
The presence of two diasteromers is evident in the NMR spectra of
1 in CD3NO2, where almost all the resonances consist of two closely
spaced peaks. The isolation and the full assignment of each isomer was
not attempted.
1
H NMR (500 MHz, CD3NO2) δ 8.98 and 8.97 (2s, 2 × 1H, C2H),
7.74 (d br, J = 12.9 Hz, 2 × 1H, C5H), 4.77 (m br, 2 × 1H, C1″H),
4.62 (d, J = 12.0 Hz, 2 × 1H, C2″Ha), 4.45 and 4.43 (2d, J = 10.0 Hz, 2
× 1H, C2″Hb), 3.57 (m br, 2 × 4H, C2′H2/C6′H2), 3.10 and 3.09 (2s,
2 × 3H, CH3 dmso), 3.08−3.03 (m, 2 × 4H, C3′H2/C5′H2), 3.02 and
3.01 (2s, 2 × 3H, CH3 dmso), 2.99−2.54 (m, 2 × 12H, CH2
[9]aneS3), 2.70 (s br, 2 × 3H, C7′H3), 1.64 and 1.62 (2d, J = 6.8
Hz, 2 × 3H, C3″H3).
13
C NMR (126 MHz, CD3NO2) δ 178.4 (C4O), 167.3 (C11O),
158.8 and 156.9 (C6H), 150.3 (C2H), 141.7 (C8), 132.4 and 132.4
(C7), 125.0 (C9), 124.0 and 124.0 (C10), 113.9 and 113.8 (C3), 105.5
and 105.3 (C5H), 69.8 and 69.8 (C2″H2), 57.6 and 57.6 (C1″H), 56.7
(C3′H2/C5′H2), 50.4 (C2′H2/C6′H2), 46.0 (C7′H3), 43.8 (CH3 dmsoS), 43.6 (CH3 dmso-S), 36.2 and 36.2 (CH2 [9]aneS3), 35.6 and 35.5
(CH2 [9]aneS3), 34.3 and 34.2 (CH2 [9]aneS3), 33.5 and 33.4 (CH2
[9]aneS3), 31.4 and 31.3 (CH2 [9]aneS3), 31.2 and 31.1 (CH2
[9]aneS3), 18.5 and 18.4 (C3″H3).
1.2.2. [Ru([9]aneS3)(dmso-κS)(nal-κ2O,O)](PF6)·3H2O (2·3H2O).
This complex was prepared according to the general procedure
using 27.0 mg of nalidixic acid sodium salt hydrate (0.099 mmol)
affording 62.4 mg of 2 as yellow solid (Yield 79.8%). Crystals of 2
suitable for X-ray analysis (yellow needles) were obtained by
dissolving the complex in a EtOH/MeOH mixture (ca. 5 mL; 4:1)
followed by slow evaporation.
C
dx.doi.org/10.1021/ic401220x | Inorg. Chem. XXXX, XXX, XXX−XXX
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1.3. Bioassays. To study the biological behavior (interactions with
DNA, serum albumins, cathepsin inhibition, and cytotoxicity) of
complexes 1−4, they were initially dissolved in DMSO (1 mM), and
the resultant solutions were used for no longer than 2 h. Mixing of
such solutions with the aqueous solutions used in biological studies
never exceeded 5% DMSO (v/v) in the final solution, which was
needed because of low aqueous solubility of some of the complexes
and ligands. Control experiments performed with 5% DMSO showed
no influence on the final results.
1.3.1. DNA Binding Studies. DNA stock solution was prepared by
dilution of CT DNA in the buffer (containing 15 mM trisodium citrate
and 150 mM NaCl at pH 7.0) followed by exhaustive stirring for 3
days, and kept at 4.0 °C for no longer than a week. The stock solution
of CT DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/
A280) of 1.89, indicating that the DNA was sufficiently free of protein
contamination. The DNA concentration per nucleotide was
determined by UV absorbance measurements at 260 nm after 1:20
dilution using ε = 6600 M−1 cm−1.17
The interaction of the quinolones and complexes 1−4 with CT
DNA has been studied with UV spectroscopy to investigate the
possible binding modes and to calculate the binding constants (Kb). In
UV titration experiments, the spectra of CT DNA in the presence of
each compound have been recorded for a constant CT DNA
concentration at diverse [compound]:[DNA] ratios (r). The DNAbinding constants of the compounds, Kb, have been determined
according to Supporting Information, eq S1 using the UV spectra
recorded for a constant concentration of the compounds in the
absence or presence of CT DNA for diverse r values.17 Control
experiments with DMSO were performed, and no changes in the
spectra of CT DNA were observed.
Interaction of complexes 1−4 with CT DNA has been also
investigated by monitoring the changes observed in the cyclic
voltammogram of a 0.40 mM 1:2 DMSO:buffer solution of complex
upon addition of CT DNA at diverse r values. The buffer was also used
as the supporting electrolyte, and the cyclic voltammograms were
recorded at ν = 100 mV s−1.
The viscosity of a DNA solution has been measured in the presence
of increasing amounts of complexes 1−4. The relation between the
relative solution viscosity (η/η0) and DNA length (L/L0) is given by
the equation L/L0 = (η/η0)1/3, where L0 denotes the apparent
molecular length in the absence of the compound.25 The obtained data
are presented as (η/η0)1/3 versus r, where η is the viscosity of DNA in
the presence of the compound, and η0 is the viscosity of DNA alone in
buffer solution.
The competitive studies of each compound with EB have been
investigated with fluorescence spectroscopy to examine whether the
compound can displace EB from its EB-DNA complex. The EB-DNA
complex was prepared by adding 20 μM EB and 26 μM CT DNA in
buffer (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). The
possible intercalating effect of the quinolones and complexes 1−4 with
the DNA was studied by adding a certain amount of a solution of the
compound step by step into the solution of the EB-DNA complex.
The influence of the addition of each compound to the EB-DNA
complex solution has been obtained by recording the variation of
fluorescence emission spectra. The Stern−Volmer equation (Supporting Information, eq S2) is used to evaluate the quenching efficiency for
each compound.17
1.3.2. Albumin Binding Studies. The protein binding study was
performed by tryptophan fluorescence quenching experiments using
bovine (BSA, 3 μM) or human serum albumin (HSA, 3 μM) in buffer
(containing 15 mM trisodium citrate and 150 mM NaCl at pH 7.0).
Quenching of the emission intensity of tryptophan residues of BSA at
342 nm or HSA at 351 nm was monitored using the quinolones and
complexes 1−4 as quenchers with increasing concentration (up to 2.2
× 10−5 M).17,26 Fluorescence spectra were recorded in the range 300−
500 nm at an excitation wavelength of 296 nm. The fluorescence
spectra of the compounds in buffer solutions were recorded under the
same experimental conditions, and no fluorescence emission was
recorded. The Stern−Volmer and Scatchard equations (Supporting
Information, eqs S3−S5) and graphs have been used to study the
interaction of the compounds with serum albumins and calculate the
corresponding constants.17
1.3.3. Cytotoxicity Studies. Human cervix carcinoma (HeLa) and
non small cell lung carcinoma (A549) cells used in the experiment
were obtained from the ECACC (European Collection of Cell
Cultures, Public Health England; HeLa) or DSMZ (Leibniz-Institute
DSMZ-Deutche Sammlung von Mikroorganismen and Zellkulturen
GmbH; A549) and maintained in MEM (Minum Essential Medium,
PAA) supplemented with 10% fetal bovine serum (Sigma), 2 mM Lglutamine (Invitrogen), 100 units of penicillin, 100 μg/mL
streptomycin (Invitrogen), and 1% nonessential amino acids
(Invitrogen) at 37.0 °C in a humidified 5% CO2 atmosphere. Cells
were harvested by trypsinization and seeded in 96-well transparent
plates (PTT) at a final volume of 100 μL and densities of 4 × 103 cells
per well. Cytotoxicity was then determined by the colorimetric MTT
assay (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;
Thiazolyl blue, Sigma Aldrich) in triplicate essentially as described
previously.9
1.3.4. Cathepsin Inhibition Studies. Recombinant human cathepsins B and S were prepared as described earlier.27 Initial solutions of
the compounds were prepared in DMSO. Subsequent dilutions were
made in 100 mM phosphate buffer, pH 6.0. Inhibition of cathepsins
was then determined essentially as described previously.9
2. RESULTS AND DISCUSSION
2.1. Synthesis and Characterization of the Complexes.
Reaction of [Ru([9]aneS3)(dmso)3](PF6)2 (P1) with one
molar equivalent of the appropriate quinolone in refluxing
methanol and in the presence of sodium methoxide led to the
isolation of cationic compounds of the general formula
[Ru([9]aneS3)(dmso-κS)(O-O′)](PF6), where O-O′ = levo
(1), oxol (3), and cin (4). Compound 2 was prepared in a
similar manner, but using the sodium salt of nalidixic acid (nalNa) because of its commercial availability. Attempts to isolate
the neutral coordination compounds with general formula
[Ru([9]aneS3)Cl(O-O′)], analogues of the previously described organometallic complexes [Ru(η6-p-cym)Cl(O-O′)]
(O-O′ = oflo, nal, cin),18,19 by reacting the neutral precursor
[Ru([9]aneS3)Cl2(dmso-κS)] with the appropriate quinolone
ligand in the presence of a base were unsuccessful and often led
to the isolation of mixtures of the dmso derivative [Ru([9]aneS3)(dmso-κS)(O-O′)](Cl) and of the aqua derivative
[Ru([9]aneS3)(OH2)(O-O′)](Cl), as proven by NMR experiments. All new complexes were characterized by elemental
analysis and IR spectroscopy in solid state and by NMR
spectroscopy in solution state. The solid state structures of 2
and 4 were also determined by X-ray crystallography
(Supporting Information, Table S1).
Crystals of compound 1 were obtained, and the structure was
partially solved which confirmed the proposed structure.
However, they could not be adequately refined because of
the low crystal quality, the presence of various disordered
solvate molecules, and the presence of rotational disorder on
the anionic species. On the other hand, the structures of
complexes 2 and 4 were unambiguously refined. In all analyzed
complex cations, the Ru center displays the expected slightly
distorted octahedral geometry, and the coordination sphere is
composed of the tridentate [9]aneS3 ligand in facial geometry,
of one quinolonato ligand acting as chelating ligand through the
pyridone, and the carboxylato oxygen atoms and of one dmso
molecule bonded through sulfur (Figure 2 and 3).
The Ru−S bond length values in complexes 2 and 4 are
comparable to those in the previously published crystal
structures of [Ru([9]aneS3)(dmso-κS)(O,O′-ligand)] complexes bearing dianionic dicarboxylate ligands (oxalate,
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while in the case of the carboxylate oxygen a slight elongation is
observed (Table 1)
As mentioned before, the quality of the crystals of compound
1 did not permit adequate refinement, yet it was evident from
the presence of a different number of counterions, that upon
standing in a saturated solution for few weeks compound 1
crystallized as a dicationic complex with the ligand levo in its
zwitterionic form, with the protonated N4′ piperazine nitrogen
atom.
Complex 4·EtOH presents a fairly simple structure (Figure
3) devoid of supramolecular motifs, except for a hydrogen bond
between the solvate ethanol molecule and the unbound
carboxylate oxygen atom of the cinoxacinato ligand. On the
other hand, in complex 2·3H2O, two of the cocrystallized water
molecules (O51 and O52) are bound by hydrogen bonds which
results in the formation of a helix about the screw axis. The
cationic complex species of 2 is directly linked to the helix
through a hydrogen bond to the ruthenium-bound carboxylate
oxygen. The free carboxylic oxygen forms an additional
hydrogen bond to the third water molecule (O50) which is
then bound to the second helix-forming water molecule (Figure
4). The hydrogen bond lengths and angles are given in
Supporting Information, Table S1b. The structure is further
stabilized by weak hydrogen interactions between the O50
water molecule and the hexafluorophosphate anion.
The new complexes are soluble in H2O, although they show
a remarkable difference in their solubility: 10 mM aqueous
solutions of complexes 1 and 4 can easily be prepared, while the
solubility of complexes 2 and 3 is one- and two orders of
magnitude lower, respectively. 1H NMR studies were thus
performed mainly in the biologically relevant D2O solvent at
about 1 mM except for 3, for which a saturated solution was
prepared by brief gentle heating and sonication followed by the
filtration of the undissolved product. However, since these
compounds hydrolyze in water (see below) it was not possible
to record the 13C NMR spectra of the intact species in D2O.
Therefore, proton and, in particular, carbon NMR spectra were
recorded also in CD3NO2, where complexes are stable and well
soluble. The full assignment of the proton and carbon NMR
spectra of all complexes was performed by combination of 1D
(1H and 13C) and 2D (1H-1H COSY, phase sensitive 1H-13C
HSQC and 1H-13C HMBC) NMR experiments in either D2O
or CD3NO2 (see Supporting Information, Figures S1−S13).
All products share a common structural feature: the
tridentate thioether macrocycle 1,4,7-trithiacyclononane ([9]aneS3) coordinated to the metal center in a facial manner. The
methylene protons of this face-capping ligand provide a
characteristic pattern of resonances in the 1H NMR spectrum,
a manifold of partially overlapping multiplets in the region
3.20−2.50 ppm. This pattern, with minor differences, is
observed in the NMR spectra of all the studied complexes
and will not be discussed further. In addition, as the O-O′
ligands are not symmetrical, in all complexes the two methyl
groups of the dmso are diastereotopic and resonate as two
singlets in the region typical for S-bound dmso.
The 1H NMR spectrum of 1 in D2O displays two aromatic
resonances: one singlet (δ = 8.98) attributed to the C2H (see
Figure 1 in the Introduction section for atom numbering) and
one doublet (δ = 7.79), because of the coupling with the
adjacent F, assigned to the C5H. The diastereotopic protons of
C2″H2 resonate as one doublet and one broad multiplet (δ =
4.60 and 4.40, respectively) that are coupled to each other in
the 1H-1H COSY spectrum (Supporting Information, Figure
Figure 2. Crystal structure of compound [Ru([9]aneS3)(dmsoκS)(nal)](PF6)·3H2O (2·3H2O) with heteroatom labeling. The
hydrogen atoms and water molecules are omitted for clarity. The
thermal ellipsoids are shown at 20% probability level. Selected bond
lengths: Ru−S1′ = 2.256(2) Å, Ru−S1 = 2.351(2) Å, Ru−S2 =
2.294(2) Å, Ru−S3 = 2.289(2) Å, Ru−O4 = 2.088(2) Å, Ru−Oh =
2.104(2) Å.
Figure 3. Crystal structure of compound [Ru([9]aneS3)(dmsoκS)(cin)](PF6)·EtOH (4·EtOH) with heteroatom labeling. The
hydrogen atoms, the EtOH, and the hexafluorophosphate anion are
omitted for clarity. The thermal ellipsoids are shown at 20%
probability level. Selected bond lengths: Ru−S1′ = 2.269(3) Å, Ru−
S1 = 2.345(3) Å, Ru−S2 = 2.284(3) Å, Ru−S3 = 2.281(3) Å, Ru−O4
= 2.097(2) Å, Ru−Oh = 2.085(3) Å.
malonate) which are structurally the most similar complexes
with known crystal structure so far, even though they have a
different charge.7
In comparison to the organoruthenium complexes with
quinolones previously described by our group, the Ru−O bond
lengths in the case of the pyridone oxygen remain the same
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Table 1. Selected Bond Distances (Å) and Angles (deg) in Ruthenium-Quinolone Complexes
[(η6-cymene)Ru(nal)Cl]19
[(η6-cymene)Ru(cin)Cl]19
[(η6-cymene)Ru(oflo)Cl]18
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
Ru−O4
Ru−Oh
O4−Ru−Oh
2.087(2)
2.099(2)
2.071(2)
2.088(2)
2.097(2)
2.070(2)
2.071(2)
2.069(2)
2.104(2)
2.085(3)
87.30(8)
84.94(8)
85.30(7)
88.92(7)
87.02(11)
Figure 5. Perspective view of the two diastereomers formed by the
chiral chelating ligand levo on the Ru center. The chiral center on the
levo ligand is indicated with (*).
almost all the resonances are split into two closely spaced
singlets (Supporting Information, Figure S2). For example, the
methylene carbons of the [9]aneS3 ligand resonate as 12 well
resolved peaks.
The 1H NMR spectrum of 2 in D2O or in CD3NO2
(Supporting Information, Figure S3) is consistent with the
molecular structure found in solid state. The nal ligand displays
the expected pattern of resonances: one singlet and two
doublets in the aromatic region attributed respectively to the
C2H, C5H, and C6H, one multiplet (δ = 4.70 in D2O and 4.74
in CD3NO2) assigned to the C12H2, and two intense
resonances, one singlet and one triplet, in the upfield region
for the methyl groups C14H3 and C13H3, respectively. In D2O,
all resonances of the nal are shifted downfield compared to the
free ligand because of its binding on the Ru center. For easier
analysis and comparison of the 1H and 13C NMR spectra see
Supporting Information, Tables S2 and S3.
The 13C NMR spectrum of 2 in CD3NO2 (Supporting
Information, Figure S4) is consistent also with the geometry
and the low symmetry of the complex: six resonances for the
unequivalent methylene carbons of the [9]aneS3 ligand and two
signals (very closely spaced) for the carbons of the
diastereotopic methyl groups of the dmso ligand together
with the expected peaks of the bound nal.
The oxol ligand is similar to nal with the differences that it
bears a dioxolyl moiety at C6 and C7 and that the N8 in nal is
replaced by C8H in oxol (see Figure 1). Accordingly, the 1H
spectrum of 3, either in D2O or in CD3NO2 (Supporting
Information, Figure S5), is similar to that of 2 with few
differences: the resonance of C5H is now a singlet, while the
doublet for C6H and the singlet for the C14H3 in the spectrum
of 2 have been replaced by two singlets, for C8H and for C14H3
respectively, in the spectrum of 3. The 13C NMR spectrum of 3
in CD3NO2 (Supporting Information, Figure S6) is consistent
also with the proposed geometry, and the low symmetry, of the
complex.
Complex 4 bears the cin ligand, which is a quinolone very
similar to oxol: only the C2H of oxol is replaced by a nitrogen
atom in cin. Owing to this similarity, the 1H and 13C NMR
spectra of complex 4 in D2O and in CD3NO2 are very similar to
Figure 4. Hydrogen bonds in the crystal structure of compound
2·3H2O with heteroatom. Non relevant hydrogen atoms and the
carbon atoms of the ligand [9]aneS3 are omitted for clarity. The
thermal ellipsoids are shown at 20% probability level.
S9) and to the same carbon resonance in the 1H-13C HSQC
spectrum (Supporting Information, Figure S10), while the
C1″H is overlapped with the intense resonance of the HOD. In
the upfield region, besides the dmso (singlets at δ = 3.19 and
3.15 ppm) and the [9]aneS3 peaks the spectrum displays also a
broad singlet (δ = 3.51, 4H) attributed to the C2′H2 and C6′H2
of the piperazine ring and a broad triplet assigned to the methyl
C3″H3; the peaks of C3′H2 and C5′H2 of the piperazine and the
methyl C7′H3 are partially overlapped with the multiplets of
[9]aneS3 and were identified by the phase sensitive 1H-13C
HSQC spectrum (Supporting Information, Figure S10). Worth
noting, all resonances of levo are downfield shifted compared to
the free ligand and in particular those of C2H and C5H (δ =
8.35 and 7.54, respectively, in free levo), which is indicative for
the binding of levo on the Ru center.
Binding of the chiral ligand levo (i.e., (S)-enantiomer) on the
Ru center is expected to produce two equally abundant
diastereomers (Figure 5), since Ru also becomes chiral. The
formation of both isomers was not obvious from the NMR
experiments in D2O, where only one set of resonances was
observed. Conversely, the presence of both isomers is evident
when NMR spectroscopy was carried out in CD3NO2. In the
1
H NMR spectrum of 1 in CD3NO2, although the pattern is
similar to that found in D2O, almost all peaks are split in two
equally intense sets, each attributed to one of the two
diastereomers; this splitting is predominantly evident for the
singlets of the methyl groups of the dmso ligand (Supporting
Information, Figure S1). The presence of both diasteromers is
more obvious in the 13C NMR spectrum of 1, where again
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those of 3, with the obvious absence of the C2H resonance
(Supporting Information, Figures S7 and S8).
Complexes 1−4 display similar IR spectra, with bands
attributed to the asymmetric (1636−1618 cm −1 ) and
symmetric (1468−1447 cm−1) stretching modes for the
carboxyl groups and to the CO (1578−1519 cm−1) of the
quinolone ligands, which are in agreement with the binding
mode of the quinolones, that is, chelating through the oxygens
of the carboxylate group and of the pyridone. In addition, the
bands in the region 1089−1080 cm−1 and 835−829 cm−1
attributed to the SO stretching of the S-bonded dmso and to
the PF6¯ counterion, respectively, are clearly distinguishable.
The fluorescence emission spectra of the complexes have
been recorded in DMSO solution. As an example, the
fluorescence excitation and emission spectra for complexes 3
and 4 are shown in Supporting Information, Figure S14. The
complexes exhibit an intense emission band with λem,max lying in
the region 444−495 nm when excited at λexc,max 343−430 nm
(Supporting Information, Table S4).
2.2. Chemical Behavior in Aqueous Solution. The
chemical behavior of compounds 1−4 in aqueous solution was
investigated by 1H NMR spectroscopy at room temperature in
view of their potential biological activity and interactions with
biological macromolecules. The chemical changes in the spectra
were monitored mainly through the aromatic resonances of the
quinolone ligands and the dmso resonances, since the
multiplets of [9]aneS3 are unsuitable for monitoring purposes.
All new complexes exhibit very similar behavior in aqueous
solution. They are relatively stable: no rapid changes were
observed in their 1H NMR spectra after dissolution in D2O.
Slow formation of the aqua species [Ru([9]aneS3)(OH2)(OO′)]+ (O-O′ = levo (1a), nal (2a), oxol (3a), and cin (4a);
Figure 6) was indicated by the gradual decrease of the two
behavior in aqueous solution: dmso release was observed within
about the first 2 h, while after 2 weeks the release of the cin
ligand was detected by the growth of a new set of resonances
attributed to the free ligand. At equilibrium the ratio between 4,
4a, and free cinH was 13.8: 5.2: 1 (Supporting Information,
Figure S13).
In general, compared to the neutral organometallic [Ru(η6-pcym)Cl(quinolonato)] complexes previously studied by
us,9,18,19 compounds 1−4 open up a coordination position
more slowly and, above all, not quantitatively. In addition,
contrary to what was observed for the organometallic
quinolonato complexes, in the new coordination compounds,
with exception of 4, quinolones remained tightly bound to the
metal center in aqueous solution even after a prolonged time,
only in the case of 4 do we observe a partial release of the
quinolone.
2.3. Bioassays. 2.3.1. DNA-Binding Studies. Studies of the
interaction of quinolones and their complexes with DNA are of
great importance since their activity as antibacterial drugs
presumably depends on the inhibition of DNA replication by
targeting essential type II bacterial topoisomerases such as
DNA gyrase and topoisomerase IV.15 DNA can provide three
distinct binding modes for quinolone metal complexes: binding
to the groove, electrostatic binding to phosphate groups, and
intercalation.
The changes observed in the UV spectra upon titration may
provide evidence of the existing interaction mode, since a
hypochromism due to π → π* stacking interactions may appear
in the case of the intercalative binding mode, while red-shift
(bathochromism) may be observed when the DNA duplex is
stabilized.28
The UV spectra have been recorded for a constant CT DNA
concentration at different [complex]:[DNA] mixing ratios (r).
The UV spectra of CT DNA in the presence of 1 at diverse r
values (up to 0.3) are shown as examples in Figure 8A. Similar
spectral changes were obtained when CT DNA was treated
with complexes 2−4, cinH and nalNa. The decrease of the
intensity at λmax = 258 nm is accompanied by a red-shift of the
λmax up to 265 nm for all compounds, suggesting that the
interaction with CT DNA results in the direct formation of a
new adduct with double-helical CT DNA.29 The observed
hypochromism may be attributed to stacking interaction
between the aromatic chromophore of the compound and
DNA base pairs consistent with the intercalative binding mode,
while the red-shift is an evidence of the stabilization of the CT
DNA duplex.17,30
The changes in the UV spectrum of a 2 × 10−5 M solution of
the quinolones and complexes 1−4 during the titration upon
addition of CT DNA in diverse r values may be observed in
Figure 8 and Supporting Information, Figure S14. In the UV
region, the intense absorption bands observed in the spectra of
the complexes are attributed to the intraligand transition of the
coordinated groups of quinolone ligands and could be
perturbed in the case of interaction with CT DNA.17,30
In the UV spectrum of 2 × 10−5 M DMSO solution of cinH,
the initial band at 357 nm displays a slight hypochromism upon
addition of increasing amounts of CT DNA (up to 1/r = 2). An
additional band of increasing intensity appears at 375 nm,
suggesting formation of a new adduct, which is also supported
by the existence of a distinct isosbestic point at 367 nm (Figure
8B). In the UV spectrum of nalNa, the initial two bands at 333
and 345 nm (as a shoulder), respectively, present a hyperchromism upon addition of increasing amounts of CT DNA
Figure 6. Chemical behavior of compounds 1−4 in aqueous solution.
dmso-S singlets and the corresponding growth of the free dmso
resonance at δ = 2.71 ppm. After several hours the systems
reached equilibria between the intact and aquated species. The
time to reach equilibrium and the ratio between the intact and
aqua species varies, depending mainly on the quinolone ligand.
Complex 1 reaches equilibrium after about 24 h with a 7:3 ratio
between 1 and 1a, according to the integration of the aromatic
resonances (Supporting Information, Figure S11). The same
ratio was detected between 2 and 2a, but equilibrium was
reached after about 2 days (Figure 7). The aquation of 3 is
more prolonged compared to that of 1 and 2, and equilibrium
is reached after several days with 45% of 3 to be hydrolyzed
(Supporting Information, Figure S12). During the observation
times, no release of the quinolone ligand from complexes 1−3
was detected. On the other hand, compound 4 has different
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Figure 7. Time evolution 1H NMR spectra of complex 2 (ca. 2.0 mM) in D2O at room temperature. Selected resonances of the aqua species 2a are
indicated with an asterisk (*).
Figure 8. (A) UV spectra of CT DNA (1.44 × 10−4 M) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the absence or
presence of 1. The arrow shows the changes upon increasing amounts of complex (up to r = 0.3). (B) UV spectra of cinH (2 × 10−5 M) in DMSO
solution in the presence of CT DNA at increasing amounts (up to 1/r = 2). The arrows show the changes upon increasing amounts of CT DNA.
(up to 1/r = 2) accompanied by a red-shift of 3 and 2 nm (to
336 and 347 nm), respectively (Supporting Information, Figure
S15(A)). The observed spectral phenomena may suggest
binding to CT DNA and stabilization.
In the UV spectrum of 1, the band at 317 nm exhibits a slight
hyperchromism while the absorbance of the band at 355 nm
decreases slightly upon addition of CT DNA (Supporting
Information, Figure S15(B)). In the UV spectrum of 2, the
band at 360 nm exhibits a slight hypochromism upon addition
of CT DNA (Supporting Information, Figure S15(C)). In the
UV spectrum of 3, the band at 344 nm exhibits a slight
hyperchromism upon addition of CT DNA followed by a blueshift of 2 nm (to 342 nm) while the absorbance of a second
band at 352 nm initially decreases and is followed by a slight
increase of the absorbance accompanied by a 2 nm red-shift (to
354 nm) (Supporting Information, Figure S15(D)). In the UV
spectrum of 4, the band at 325 nm attributed to the intraligand
transition in the quinolone ligand presents slight hyperchromism which is also observed for the band at 378 nm
(Supporting Information, Figure S15(E)).
The results derived from the UV titration experiments
suggest that all complexes can bind to CT DNA, although the
exact mode of binding cannot be reliably proposed on the basis
of UV spectroscopic titration studies.17,30 The spectral behavior
of the complexes upon addition of CT DNA suggests binding
to CT DNA and stabilization while the existing differences may
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Table 2. DNA-Binding Constants (Kb) and Stern−Volmer Constants (KSV) of the EB-DNA Quenching for the Quinolones and
Complexes 1−4
compound
Kb (M−1)
Ksv
[Ru([9]aneS3)(dmso-κS)(levo)](PF6), 1
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(oxol)](PF6), 3
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
levofloxacin. levoH17
sodium nalidixicate, nalNa
oxolinic acid, oxolH32
cinoxacin, cinH
2.02(±0.20) × 104
1.23(±0.30) × 104
0.78(±0.03) × 104
1.55(±0.12) × 104
4.36(±0.11) × 104
1.33(±0.12) × 104
0.30(±0.01) × 104
1.26(±0.30) × 104
3.58(±0.22) × 105
7.57(±0.39) × 105
1.83(±0.07) × 105
4.47(±0.25) × 105
11.10(±0.30) × 105
3.53(±0.08) × 105
4.99(±0.21) × 105
Table 3. Cathodic and Anodic Potentials (in mV) for the Redox Couples (1) Ru(II)/Ru(I) and (2) Ru(II)/Ru(III) in DMSO
Solution of the Complexes
a
complex
Epca
Epaa
Epab
Epcb
Epc1′c
Epa1′c
Epa2′c
[Ru([9]aneS3)(dmso-κS)(levo)](PF6), 1
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(oxol)](PF6), 3
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
−905
−885(lc)d
−835
−1360
−745
−705
−710
−1275
−490
−540(lc)d
−460
−615
−492
−512
−505
−505
−1050
−1095
−1155
−1335
−535
−920
−590
−850
−360
−374
−375
−375
Epc/a of the redox couple Ru(II)/Ru(I). bEpc/a of the redox couple Ru(II)/Ru(III). cEpc/a 1/2′ potentials that appear in second scan. dlc = low current.
Table 4. Cathodic and Anodic Potentials (in mV) for the Redox Couple Ru(II)/Ru(I) in 1:2 DMSO:Buffer Solution of the
Complexes in the Absence and Presence of CT DNA
a
complex
Epc(f)a
Epc(b)b
ΔEpcc
Epa(f)a
Epa(b)b
ΔEpac
[Ru([9]aneS3)(dmso-κS)(levo)](PF6), 1
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(oxol)](PF6), 3
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
−705
−703
−692
−697
−695
−688
−685
−680
+10
+15
+7
+17
−550
−554
−565
−550
−550
−554
−555
−557
0
0
+10
−7
Epc/a(f) in the absence of CT DNA. bEpc/a(b) in the presence of CT DNA. cΔEpc/a = Epc/a(b) − Epc/a(f).
electrochemical behavior in DMSO and the corresponding
potentials are given in Table 3. In few cases, the current of the
oxidation Ru(I) → Ru(II) is low and cannot be clearly
detected.
In the cyclic voltammograms of the complexes in 0.4 mM 1:2
DMSO:buffer solution, only the potentials (Epc(f) and Epa(f), for
the free complex) for the quasi-reversible redox couple Ru(II)/
Ru(I) have been determined, and their values are given in
Table 4. The changes of this redox couple Ru(II)/Ru(I) (up to
r = 0.15) (Supporting Information, Figure S18) have been
studied upon the addition of CT DNA (Supporting
Information, Figure S17) and the potentials (Epc(b) and Epa(b),
for the complex bound to CT DNA) as well as the
corresponding shifts (ΔEpc and ΔEpa) are given in Table 4.
In general, the electrochemical potential of a small molecule
shifts positively when it intercalates into the DNA double helix,
whereas it will shift to a negative direction in the case of
electrostatic interaction with DNA. Furthermore, when two or
more potentials exist, a positive shift of Ep1 and a negative shift
of Ep2 may imply that the molecule can bind to DNA by both
intercalation and electrostatic interaction.34 No new redox
peaks appeared after the addition of CT DNA to each complex,
while the current of all peaks decreased significantly suggesting
the existence of an interaction between each complex and CT
DNA. The decrease in current can be explained in terms of an
equilibrium mixture of free and DNA-bound complex on the
electrode surface. For increasing amounts of CT DNA (up to r
= 0.15), at least one of the cathodic (Epc) or the anodic (Epa)
potentials of complexes showed a positive shift (ΔEpc = (+7)−
(+17) mV) (Table 4) suggesting the existence of an
be attributed to the quinolones. The existence of hypochromism and bathochromism in the spectra of CT DNA upon
addition of 1−4 may be considered as first evidence that their
binding via intercalation of the aromatic ligand accompanied by
stabilization of the CT DNA duplex cannot be ruled out.28
The DNA-binding constant values, Kb, (Table 2) as
calculated by Supporting Information, eq S1 and plots in
Supporting Information, Figure S16 suggest a moderate binding
of complexes 1−4 to CT DNA.17,30 Comparison of the Kb
values of complexes 1−4 with the corresponding quinolones
(Table 2) shows that the values are of the same magnitude,
with complex 1 exhibiting the highest Kb value among the
complexes (2.02(±0.20)·× 104 M−1). The Kb values of the
complexes are of the same order of magnitude as the previously
reported Ni(II) and Zn(II) complexes with oxolinato and
levofloxacinato ligands.17,30 However, they are lower than that
of the classical intercalator EB, whose binding affinity for CT
DNA is given by Kb = 1.23(±0.07)·105 M−1.17,30,31
The cyclic voltammograms of 1 in DMSO solution
(Supporting Information, Figure S17) exhibit a cathodic wave
at −905 mV (Epc1) followed by two anodic waves at −500 mV
(Epa1) and −320 mV (Epa2), while in the second scan a new
second cathodic wave appears at −490 mV (Epc2) and the initial
potentials Epc1′, Epa1′ and Epa2′ shift to −1050, −535, and −360
mV, respectively. The wave with potentials Epc1 and Epa1
constitutes an irreversible redox process attributed to the
Ru(II)/Ru(I) redox couple, while Epa2 and Epc2 exhibit much
higher current intensity and can be attributed to a quasireversible Ru(II)/Ru(III) redox couple,33 since the quinolones
are electrochemically inactive. Complexes 2−4 exhibit similar
I
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intercalative binding mode, while for 4 the second potential
shifted slightly to more negative values and the coexistence of
electrostatic interaction cannot be ruled out.17,30,32,35,36
DNA viscosity is sensitive to DNA length change; therefore,
its measurement upon addition of a compound provides a
reliable evidence for the binding mode to DNA.25,37 The
addition of increasing amounts (up to r = 0.25) of complexes
1−4 to a DNA solution (0.1 mM) results in an increase in the
relative viscosity of DNA (Figure 9), which is much more
or electron transfer are the two mechanisms proposed to
account for the decrease of the emission intensity.38
The emission spectra of EB bound to CT DNA in the
absence and presence of each compound have been recorded
for [EB] = 20 μM, [DNA] = 26 μM for increasing amounts of
each compound up to the value of r = 0.08. The addition of
each the quinolones or complexes 1−4 at diverse r values
results in significant decrease of the intensity of the emission
band (Figure 10) of the EB-DNA system at 592 nm indicating
Figure 9. Relative viscosity (η/ηo)1/3 of CT DNA (0.1 mM) in buffer
solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in
the presence of the quinolones or complexes 1−4 at increasing
amounts (r).
Figure 10. Plot of EB relative fluorescence intensity at λem = 592 nm
(%) vs r (r = [Compound]/[DNA]) for complexes 1−4 and the
quinolones (27% of the initial fluorescence intensity for 1, 16% for 2,
33% for 3, 25% for 4, 22% for cinH, 26% for nalNa, 100% for oxolH,
and 23% for levoH) in buffer solution (150 mM NaCl and 15 mM
trisodium citrate at pH 7.0).
pronounced upon addition of complexes 1, 2, and 4. In the case
of classic intercalation, DNA base pairs are separated to host
the bound compound resulting in increased DNA viscosity, the
magnitude of which is usually in accordance to the strength of
the interaction, because of the lengthening of the DNA helix.
Upon binding by means of partial and/or nonclassic
intercalation in DNA grooves, the DNA solution viscosity is
decreased or remains unchanged, since the DNA helix bend or
kink may reduce slightly its effective length.37 Therefore, the
observed viscosity increase may be explained by an increase in
overall DNA length provoked by the insertion of the
compounds in between the DNA base pairs due to interaction
via intercalation through the aromatic chromophore of
quinolone ligands in the complexes.30,32,35
2.3.1.1. Competitive Studies with Ethidium Bromide.
Ethidium bromide (EB = 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide) is a typical indicator of intercalation
since it forms soluble complexes with nucleic acids and emits
intense fluorescence in the presence of DNA because of the
intercalation of the planar phenanthridine ring between the
adjacent base pairs on the DNA double helix. Compounds 1−4
do not show any significant fluorescence at room temperature
in solution or in the presence of CT DNA, when excited at 540
nm. Furthermore, the addition of complexes 1−4 to a solution
containing EB does not provoke quenching of free EB
fluorescence, and no new peaks appear in the spectra. The
changes observed in the spectra of EB on its binding to CT
DNA are often used for studying the interaction between DNA
and other compounds, such as metal complexes, since the
addition of a compound, capable of intercalating DNA equally
or more strongly than EB, could result in a quenching of the
EB-DNA fluorescence emission. The displacement of EB and/
the competition of the compounds with EB in binding to DNA.
The observed quenching of EB-DNA fluorescence for the
compounds suggests that they can displace EB from the EBDNA complex and that they can probably interact with CT
DNA by the intercalative mode.30,32,35
The Stern−Volmer plots of the EB-DNA quenching
(Supporting Information, Figure S19) illustrate that the
quenching provoked by the compounds is in good agreement
(R = 0.99) with the linear Stern−Volmer equation (Supporting
Information, eq S2) The Stern−Volmer constants values, KSV,
of the compounds are moderate to high (Table 2) showing that
they can displace EB and bind relatively tightly to
DNA.30,32,35,39
In general, the quenching of the EB-DNA fluorescence
provoked by the complexes is similar to the queching provoked
by the Zn(II)-quinolone complexes and more pronounced than
by the Ni(II)-quinolone complexes reported.17,30,32,39 The KSV
values of the Ru(II)-quinolone complexes are of the same order
as the Zn(II)- and Ni(II)-quinolone complexes reported,
although a direct correlation cannot be performed because of
different coligands and metal environment.39
2.3.2. Albumin Binding Studies. The serum albumins (SAs)
are the most abundant proteins in the circulation system and
play important roles in the delivery of many pharmaceuticals to
the sites of disease.40 It is therefore important to study the
interactions of biologically active compounds with these
transport proteins since binding to these proteins may lead to
loss or enhancement of the biological properties of the original
drug, or provide paths for drug transportation. Interactions of
metallodrugs with proteins are crucial for their biodistribution,
toxicity, and even for their mechanism of action. For one of the
J
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Figure 11. Plot of % relative fluorescence intensity at (A) λem = 351 nm (%) vs r (r = [complex]/[HSA]) for the quinolones and complexes 1−4
(48% of the initial fluorescence intensity for 1, 88% for 2, 92% for 3, 87% for 4, 87% for cinH, 91% for nalNa, 45% for oxolH, and 38% for levoH)
and (B) λem = 342 nm (%) vs r (r = [complex]/[BSA]) for the quinolones and complexes 1−4 (37% of the initial fluorescence intensity for 1, 83%
for 2, 63% for 3, 62% for 4, 75% for cinH, 38% for nalNa, 50% for oxolH, and 30% for levoH) in buffer solution (150 mM NaCl and 15 mM
trisodium citrate at pH 7.0).
Table 5. SA Constants and Parameters (Ksv, kq, K, n) Derived for Complexes 1−4 and the Free Quinolones
compound
[Ru([9]aneS3)(dmso-κS)(levo)](PF6), 1
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(oxol)](PF6), 3
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
levofloxacin. levoH17
sodium nalidixicate, nalNa
oxolinic acid, oxolH32
cinoxacin, cinH
[Ru([9]aneS3)(dmso-κS)(levo)](PF6), 1
[Ru([9]aneS3)(dmso-κS)(nal)](PF6), 2
[Ru([9]aneS3)(dmso-κS)(oxol)](PF6), 3
[Ru([9]aneS3)(dmso-κS)(cin)](PF6), 4
levofloxacin. levoH17
sodium nalidixicate, nalNa
oxolinic acid, oxolH32
cinoxacin, cinH
KSV (M−1)
BSA
8.89(±0.39) × 104
0.82(±0.01) × 104
3.09(±0.10) × 104
3.07(±0.28) × 104
9.47(±0.59) × 104
7.31(±0.09) × 104
5.01(±0.22) × 104
1.70(±0.09) × 104
HSA
5.98(±0.25) × 104
0.15(±0.02) × 104
0.31(±0.03) × 104
0.80(±0.03) × 104
0.84(±0.02) × 104
0.22(±0.03) × 104
6.39(±0.26) × 104
0.84(±0.02) × 104
most representative ruthenium anticancer drug candidates,
KP1019, it was found that rapid binding occurs in a
noncovalent manner at the hydrophobic binding sites of
HSA, but longer exposure results in a protein-coordinated
form.41 We have previously studied the interactions between
HSA and the organoruthenium-quinolone complexes by means
of ICP-MS coupled with capillary zone electrophoresis and
have found that protein binding occurs rapidly with more than
90% of the ruthenium being bound within 20 min.19 Since
complexes 1−4 have shown notable differences in their
physicochemical properties compared to their organometallic
counterparts, the main difference being the kinetic inertness to
ligand substitution in aqueous solution, we were interested to
see if the complexes still show the affinity toward proteins. In
this study, the interaction between BSA/HSA (bovine/human
serum albumin) and the ruthenium-quinolone complexes was
investigated by fluorescence spectroscopy as this method allows
a quantitative assessment of the binding strength.
BSA is the most extensively studied serum albumin because
of its high structural homology with HSA. BSA has two
tryptophan residues, Trp-134 and Trp-212, whereas HSA has a
kq (M−1s−1)
K (M−1)
n
8.89(±0.39) × 1012
0.82(±0.01) × 1012
3.09(±0.10) × 1012
3.07(±0.28) × 1012
9.47(±0.59) × 1012
7.31(±0.09) × 1012
5.01(±0.22) × 1012
1.70(±0.09) × 1012
8.59 × 104
1.50 × 104
2.04 × 104
3.77 × 104
3.59 × 104
31.0 × 104
10.9 × 104
1.57 × 104
0.99
0.51
1.34
0.74
1.71
0.71
0.75
1.07
5.98(±0.25) × 1012
0.15(±0.02) × 1012
0.31(±0.03) × 1012
0.80(±0.03) × 1012
0.84(±0.02) × 1012
0.22(±0.03) × 1012
6.39(±0.26) × 1012
0.84(±0.02) × 1012
2.01 × 104
43.6 × 104
12.4 × 104
7.59 × 104
11.5 × 104
58.1 × 104
11.3 × 104
0.47 × 104
0.92
0.10
0.07
0.21
0.89
0.09
0.60
0.85
single tryptophan located at position 214. Both BSA and HSA
can bind reversibly to a large number of compounds.42 BSA and
HSA solutions exhibit an intense fluorescence emission with a
peak at 342 and 351 nm, respectively, due to the tryptophan
residues, when excited at 295 nm.26 The changes and the
quenching observed in the fluorescence emission spectra of
tryptophan in BSA or HSA upon addition of complexes 1−4
are primarily due to changes in protein conformation, subunit
association, substrate binding, or denaturation.
Addition of the quinolones or complexes 1−4 to a SA
solution (up to r values of 7) results in a moderate quenching
of the HSA fluorescence at λ = 351 nm for 1, oxolH and levoH
and lower quenching for complexes 2−4, cinH and nalNa
(Figure 11A) while in the case of BSA the quenching of the
fluorescence at λ = 342 nm is more significant (Figure 11B).
The observed quenching may be attributed to changes in
protein tertiary structure leading to changes in tryptophan
environment of SA, and thus indicating the binding of each
compound to the albumins.42
The calculated values of Stern−Volmer quenching constant
(Ksv) and quenching rate constant (kq) for the interaction of
K
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the compounds with the SAs (from Supporting Information,
eqs S3 and S4 and Figures S20 and S21) are given in Table 5
and indicate relatively good quenching ability of the SA
fluorescence, with 1 exhibiting the highest kq value for both
albumins. Furthermore, the kq values (>1011 M−1 s−1) are
higher than diverse kinds of quenchers for biopolymer
fluorescence (2.0 × 1010 M−1 s−1) indicating the existence of
a static quenching mechanism.42
The values of the SA-binding constant (K) and the number
of binding sites per albumin (n), as calculated from the
Scatchard equation (Supporting Information, eq S5)43 and
Scatchard plots (Supporting Information, Figures S22 and S23)
for the compounds are given in Table 5. The highest binding
constant to BSA (K) is found for complex 1 and to HSA for
complex 2. Additionally, complexes 3 and 1 present the highest
number of binding sites per albumin (n) among the complexes
for BSA and HSA, respectively. Comparing the affinity of
complexes 1−4 for BSA and HSA (K values), it is obvious
(Table 5) that complexes 2−4 show higher affinity for HSA
than BSA, while 1 exhibits higher binding constant for BSA
than for HSA.
The values of the quenching rate constant (kq) and the SA
binding constant (K) found for complexes 1−4 are similar to
the values reported for a series of zinc and nickel complexes
bearing oxolinato and levofloxacinato ligand, although a more
detailed comparison cannot be performed since the coligands
are different (N-donor ligands).17,30,32,39 The SA-binding
constants (K) of all compounds studied are within the range
which could be considered optimal; they are high enough so
that the compounds bind to SA to get transport, but
nevertheless they are sufficiently low (i.e., below the value of
1015 M−1, which is the association constant of avidin with
diverse ligands; this interaction is considered the strongest
among known noncovalent interactions) so that the compounds can be released from the albumin upon arrival at the
target cells.39,43
2.3.3. Cathepsin Inhibition and Cytotoxicity of the
Compounds. Having found that the complexes 1−4 can bind
to serum proteins and DNA, we were interested to evaluate
whether they can bind to enzymes involved in cancer
progression. Among these are also proteases from the cysteine
cathepsin family, which are known to have a major role in
various stages of cancer progression, as demonstrated by
inhibition studies and gene ablation studies in animal cancer
models.44 Moreover, they are known to bind to various metal
complexes because of their free cysteine in the active site.10,45
The studies were focused on cathepsins B and S, which are
among the cathepsins most often linked with cancer
progression. Compounds 2 and 4 exhibited weak inhibition
of the two cathepsins, whereas compounds 1 and 3 were
inactive (Table 6). These interactions are substantially weaker
than that observed earlier for the organoruthenium complex
with 4-thionalidixic acid but may not be relevant for the in vivo
studies, suggesting that organoruthenium complexes may have
other/additional, perhaps more important targets in vivo.9
In vitro cytotoxicity assays were performed on human cervix
carcinoma (HeLa) and non small cell lung carcinoma (A549)
cells. All four tested ruthenium compounds exhibited modest
activity against HeLa cell line. However, only the precursor P1
showed moderate activity against the A549 cell line, whereas
compounds 1, 2, and 4 exhibited only a very low cytotoxic
activity over the concentration range used and only an estimate
(IC50 > 600 μM) could be given (Table 7). These results are
Table 6. IC50 (μM) Values for Inhibition of Human
Cathepsins B and S by Compounds 1−4 at pH 6.0a
1
2
3
4
cathepsin B
cathepsin S
540 ± 57
377 ± 22
>500
236 ± 23
>500
200 ± 22
>500
318 ± 10
a
All experimental conditions were as described in the Materials and
Methods section. IC50 values are given together with their standard
errors.
Table 7. IC50 (μM) Values for the Cytotoxicity of the
Compounds 1, 2, 4, and P1 on HeLa and A549 Human
Tumor Cell Linesa
1
2
4
P1
HeLa
A549
227 ± 40
475 ± 115
394 ± 98
440 ± 94
>600
>600
>600
454 ± 44
a
All experimental conditions were as described in the Materials and
Methods section. IC50 values are given together with their standard
errors.
similar to those of the organoruthenium complexes tested
before.9 However, all these cell lines are derived from primary
tumors and may not be the best models for metastasis spread,
where ruthenium complexes were found to be the most
efficient in vivo. This has been clearly shown for the compound
NAMI-A, which is not cytotoxic in vitro but highly active
against metastases in vivo. Therefore, the high IC50 values alone
are not a sufficient reason to discard a compound as a potential
drug candidate.3,46 It can be suggested that further in vivo
studies would be needed to evaluate whether these compounds
exhibit antitumor/antimetastatic effect.
3. CONCLUSION
A study of the stability and behavior in aqueous solution of
novel metal compounds with potential anticancer activity is
essential for further studies of interactions with potential targets
including DNA, serum proteins, and enzymes. In our previous
work we have reported the synthesis and physicochemical
characterization of four neutral organoruthenium compounds
with quinolones of the general formula [Ru(η6-cymene)(quinolonato-κ2O,O)Cl], and we have thoroughly investigated
their stability and behavior in aqueous solution.9,18,19 The
organometallic compounds undergo a fast release of the
chlorido ligand forming the corresponding [Ru(η6-cymene)(O,O′-quinolonato)(OH2)]+ species which, in turn, slowly
release the quinolone ligand, reaching an equilibrium after
about 24 h in which 10−50% of the quinolone is free. The
trithiacyclononane complexes of the general formula [Ru([9]aneS3)(dmso-κS)(quinolonato-κ2O,O)](PF6) reported herein
have much higher stability since the release of the auxiliary
ligand dmso is very slow compared to the chlorido ligand in the
organometallic counterparts, and the quinolone is not released
either. UV spectroscopy, viscosity measurements, and cyclic
voltammetry studies have revealed the ability of the complexes
to bind to CT DNA. The UV spectroscopic titrations have
shown that complexes 1−4 exhibit similar Kb values with CT
DNA. Competitive binding studies with EB have revealed the
ability of the complexes to displace EB from the EB-DNA
L
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complex, and cyclic voltammetry titration studies as well as
viscosity measurements have confirmed intercalation as the
most probable binding mode to DNA. All complexes show
good binding affinity to BSA and HSA, with relatively high
binding constants. Compounds 2 and 4 also exhibit moderate
inhibitory properties toward cathepsins B and S, two members
of the cathepsin family involved in cancer development and
progression. All four tested compounds exhibited modest
cytotoxicity against one of the tested cancer cell lines (HeLa).
This study provides additional confirmation that ruthenium
complexes could indeed have multiple targets and mechanisms
of action. Finding new uses for old drugs47 and incorporation of
a bioactive functionality into a structure that can operate in a
synergistic way with the metal moiety48 are two well established
concepts in modern drug design. Our studies have clearly
confirmed that quinolone antibacterial agents may be converted
to ruthenium complexes that exert interesting physicochemical
and biological properties. Moreover, our results also show that
even minor changes in the structure of the quinolone ligands
may substantially change the physicochemical and biological
properties of their Ru complexes. This is the case of 3 and 4,
whose quinolone ligands differ only in one nitrogen atom, yet
display significantly different aqueous solubility and hydrolytic
behavior. Interestingly, these differences do not influence much
the type and strength of the interactions with the biomolecules
used in our study. At this stage it is still difficult to find a clear
correlation between the structure and function data, and more
work is needed for better understanding.
■
ASSOCIATED CONTENT
■
AUTHOR INFORMATION
S Supporting Information
*
Tables S1a and S1b as well as cif files giving crystallographic
data for compounds 2 and 4. Figures (S1−S23) and Tables
(S2−S4) containing NMR, UV, and CV data for the
compounds as well as the plots and equations (S1−S5) used
for the quantitative assessment of the interactions with DNA,
HSA, and BSA and fluorescence experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
The cif files were submitted to CCDC and have been allocated
the deposition numbers CCDC 947546 and 947547 for 2 and
4, respectively.
Corresponding Author
*E-mail: iztok.turel@fkkt.uni-lj.si.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We are grateful to the Slovenian Research Agency (ARRS) for
the junior research grants for J.K. and M.B., the project grant
J1-4131 (IT), and the program grant P1-0140 (BT). The
project was supported by COST actions CM1105 and D39, in
particular for a short term scientific mission of I.B. to Ljubljana.
■
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