Synthesis and biological evaluation of the thionated antibacterial agent nalidixic acid and its organoruthenium(II) complex

Article abstract of DOI:10.1021/om300424w

The thionated derivative of the antibacterial agent nalidixic acid and its organoruthenium complex were prepared, and their crystal structures were determined. The aqueous stability of the complex was studied and, unlike the case for the nalidixicato complex, increased stability of the ruthenium complex in aqueous solution was observed with only a minor degree of thionalidixicato ligand dissociated within 1 week. While the derivatization caused the antibacterial activity of the ligand against E. coli to decrease, the cytotoxicity of the complex against three cancer cell lines was significantly increased and the inhibitory potency against two enzymes of the cathepsin family was increased by 10-fold.

Full text of DOI:10.1021/om300424w

Article
pubs.acs.org/Organometallics
Synthesis and Biological Evaluation of the Thionated Antibacterial
Agent Nalidixic Acid and Its Organoruthenium(II) Complex
†
,‡
†
§
∥
†,∥,⊥
Rosana Hudej, Jakob Kljun, Wolfgang Kandioller, Ursk
̌
a Repnik, Boris Turk,
Christian G. Hartinger,§,# Bernhard K. Keppler, Damijan Miklavci
§
c,
‡
and Iztok Turel*
,†,$
̌
̌
†
‡
§
∥
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ask
Faculty of Electrical Engineering, University of Ljubljana, Trzaska c. 25, SI-1000 Ljubljana, Slovenia
Institute of Inorganic Chemistry, University of Vienna, Wahringer Straße 42, A-1090 Vienna, Austria
Jozef Stefan Institute, Jamova c. 39, SI-1000 Ljubljana, Slovenia
̌ ̌
erceva c. 5, SI-1000 Ljubljana, Slovenia
̌
̌
̈
⊥
CIPKEBIP Centre of Excellence, Jamova c. 39, SI-1000 Ljubljana, Slovenia
#
School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
$
EN→Fist Centre of Excellence, Dunajska c. 156, SI-1000 Ljubljana, Slovenia
*
S Supporting Information
ABSTRACT: The thionated derivative of the antibacterial agent nalidixic acid and its
organoruthenium complex were prepared, and their crystal structures were determined.
The aqueous stability of the complex was studied and, unlike the case for the
nalidixicato complex, increased stability of the ruthenium complex in aqueous solution
was observed with only a minor degree of thionalidixicato ligand dissociated within 1
week. While the derivatization caused the antibacterial activity of the ligand against E.
coli to decrease, the cytotoxicity of the complex against three cancer cell lines was
significantly increased and the inhibitory potency against two enzymes of the cathepsin
family was increased by 10-fold.
1
. INTRODUCTION
S,O-chelates resulted in metal complexes with increased
stability and antiproliferative properties.
7
Transition-metal complexes have played an important role in
the treatment of cancer since the discovery of cisplatin in the
Quinolones are a family of synthetic antibacterial agents that
possess a β-ketocarboxylate moiety and can thus coordinate to
metal cations. Many coordination compounds of metal cations
with quinolone ligands display interesting biological proper-
1
1
960s. Among the non-platinum compounds, ruthenium
complexes have been studied most extensively and two of
them, namely NAMI-A and KP1019/KP1339, have successfully
entered clinical trials. Recent developments in the field of
ruthenium medicinal chemistry show that organometallic “half-
sandwich” ruthenium complexes have promising anticancer
activity in vitro and in vivo (Figure 1). Furthermore, the
combination of ruthenium chemotherapeutics and electro-
poration was shown to be a promising concept, as the
antitumor activity of KP1339 (an analogue of KP1019
containing sodium instead of indazolium as the counterion)
was enhanced in vitro and in vivo. On the other hand, NAMI-
A, which is not cytotoxic but is active against metastases in in
vivo setups, exhibits moderate cytotoxic properties in
combination with electroporation.
Many metal complexes have been evaluated as anticancer
agents, and also Ru compounds bearing O,O-chelating ligands,
which range from simple diketonates or carboxylates to
bioactive small molecules, were tested as potential anticancer
drugs. It was found that derivatization of the ligand to form
8
9
2
ties, and they are a hot topic in current research. Notably, the
anticancer activity of quinolone derivatives has also been
10
reported.
In recent years, it has become evident that interactions with
various proteins are of greater importance in the mechanism of
action of non-platinum anticancer drugs than binding to
3
4
12
DNA. A series of pta-based organoruthenium complexes was
therefore evaluated as inhibitors of thioredoxin reductase and
cathepsin B, two enzymes that are believed to be important
targets in cancer chemotherapy and considered to be highly
sensitive to metallodrugs. Cysteine cathepsins are a group of
primarily lysosomal proteases, which in humans comprises 11
5
6
13
different enzymes. The cathepsins were found to have a major
Special Issue: Organometallics in Biology and Medicine
Received: May 17, 2012
Published: July 13, 2012
©
2012 American Chemical Society
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role in a number of diseases, including, cancer, rheumatoid
14
arthritis, atherosclerosis, and osteoporosis. On the basis of
animal model studies, genetic ablation of several cathepsins, in
particular cathepsins B, S, and L, significantly reduced cancer
progression in, for example, the pancreatic islet model and the
15
mammary gland model. Moreover, pharmacological inhib-
ition of cathepsins by broad-spectrum cathepsin inhibitors (i.e.,
JPM-OEt) also resulted in delayed tumor growth in the same
1
5a,16
mouse models
or in significant sensitization to other
17
therapies, suggesting that cathepsins are valid targets for
anticancer therapy. In addition, intracellular cathepsins are also
involved in apoptosis triggering and in autophagy that is
promoting tumorigenesis, thereby exhibiting a dual mode of
action, which may be of further importance for cancer
treatment. Therefore, an active search for molecules that
would inhibit cathepsins in vivo may be one of the future
priorities in anticancer therapy.
18
13b,14b
Organoruthenium−pta
compounds were found to inhibit cathepsin B with IC values
5
0
in the low-μM range, suggesting that such organometallics may
represent a novel class of cathepsin inhibitors that could be
19
used in cancer therapy. In addition to the organoruthenium
20
compounds, organotellurium(IV) compounds and cyclo-
21
metalated Pd(II), oxorhenium(V), and gold complexes were
found to be potent inhibitors of various cysteine cathepsins,
including cathepsins B, S, V, and K.
Herein, we report the preparation of the thionated derivative
of the synthetic antibacterial agent nalidixic acid and its
organoruthenium complex. The compounds were characterized
using different physicochemical methods, and their crystal
structures were determined by single-crystal X-ray diffraction
analysis. The antimicrobial activity of the novel nalidixic acid
derivative and its organoruthenium complex was evaluated in
comparison to the parent compound nalidixic acid and the
Figure 1. Biologically active ruthenium complexes: (top left) KP1019;
(
(
top right) NAMI-A; (bottom left) organoruthenium complex with
7
thio)maltol; (bottom right) organoruthenium complex with
11
quinolone nalidixic acid (4-O,O).
Scheme 1. Synthetic Route to Compounds 3-S,O and 4-S,O
5
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related organoruthenium complex (4-O,O; see Figure 1). Their
cytotoxicity against human cancer cell lines was compared, and
the effect of electroporation on cytotoxicity in a murine
melanoma cell line was studied.
2. RESULTS AND DISCUSSION
2.1. Syntheses and Characterization. Thionalidixic acid
was prepared using sodium nalidixicate hydrate as the starting
material, which was first converted into the colorless nalidixic
acid ethyl ester (1) under water-free conditions. Use of a
Dean−Stark trap improved the conversion rate from 20−30%
to 60−65%. Compound 1 was reacted with Lawesson’s reagent
1
in dry THF to give the red thionalidixic acid ethyl ester (2). H
NMR spectra of crude reaction mixtures showed a conversion
rate of 75−80%, which could not be improved either by
increasing the reaction time up to 24 h or by using an excess of
the thionating reagent. The ester was hydrolyzed under alkaline
conditions, and the free thionalidixic acid (3-S,O) was
precipitated as a yellow powder by dropwise addition of
concentrated HCl (Scheme 1). The organoruthenium complex
Figure 2. Crystal structures of 3-S,O (left) and 4-S,O (right). The
ellipsoids are drawn at the 50% probability level, and the hydrogen
atoms of 4-S,O are omitted for clarity. Selected bond lengths (Å):
4
-S,O, Ru−Cl = 2.421(1), Ru−O = 2.069(4), Ru−S = 2.326(2), S−C4
= 1.680(5); 3-S,O, S−C = 1.699(2).
4
4
-S,O was obtained by deprotonation of 3-S,O with sodium
methoxide followed by reaction with the ruthenium precursor
bis[dichlorido(η -p-cymene)ruthenium(II)] (P1). After filtra-
tion of the byproduct NaCl and other insoluble impurities over
Celite, the complex was precipitated from a concentrated
dichloromethane solution upon addition of n-hexane (Scheme
explained by the bulkier frame of the ligand. The structure is
further stabilized by π−π interactions between partially stacked
quinolonato ligands. Additional crystallographic data are
provided in the Supporting Information.
6
The behavior of 4-S,O in aqueous solution was investigated
1
1) which yielded a red-violet powder.
by means of H NMR spectroscopy. Similar to the case for the
Crystals suitable for X-ray diffraction analysis were obtained
by slow evaporation of ethyl acetate (1 and 2), chloroform
3-S,O), and dichloromethane/n-hexane solutions (4-S,O).
Compound 1 exhibits π−π interactions with a distance
between planes defined by the atoms of the planar
naphthyridine core of 3.426(3) Å and considerable overlap of
the stacked molecules. The close packing and the absence of
bulkier substituents results in a very narrow unit cell with a =
previously investigated ruthenium complex bearing nalidixic
6
2
acid as a ligand, i.e., chlorido(η -p-cymene)(nalidixicato-κ -
11b
(
O,O)ruthenium(II) (4-O,O), the chlorido ligand is quickly
released and the aquated species is formed. The aqua species is,
in contrast to 4-O,O, stable in aqueous solution and only a
minor release (<3%) of the thioquinolone ligand was observed
after 1 week (∼50% in the case of 4-O,O; cf. ref 11b).
2.2. Antimicrobial Activity. The antimicrobial activity of
both quinolones (3-O,O and 3-S,O) and their ruthenium
complexes (4-O,O and 4-S,O) against the bacterial strain E. coli
K12 was evaluated using the agar diffusion test. While the
nalidixic acid and its ruthenium complex showed moderate
activity inhibiting bacterial growth at 40 μM, the thionated
derivative and its complex were inactive in the tested
concentration range (up to 1 mM). The derivatization of
nalidixic acid to thionalidixic acid lowers the antimicrobial
activity, which is in agreement with one of the two proposed
mechanisms of action for quinolones; this suggests the
formation of a magnesium(II) tetraaquaquinolonato complex
which binds to the phosphate backbone of bacterial DNA
through hydrogen bonds and prevents the replication of
4
.6074(3) Å and a relatively small tilting of the carboxylic group
with an angle of 14.24(2)° between planes defined by the
aromatic core and the carboxylic carbon and two oxygen atoms.
On the other hand, compound 2 presents only weak π−π
stacking with the aromatic backbones of the thionalidixic acid
ethyl ester molecules, forming a staircase motif, which in turn
allows the tilting of the carboxyl group at an angle of 54.90(2)°.
The structure of compound 3-S,O (see Figure 2) features an
intramolecular hydrogen bond between the carboxylic acid
hydrogen atom and the sulfur atom, which in turn causes the
near-coplanarity of the carboxylic group with the aromatic
quinolone backbone (φ = 1.18(1)°). Compound 4-S,O
presents a “piano stool” geometry with a π-bonded cymene, a
chlorido ligand, and the thionalidixicato ligand coordinated to
the Ru center via the thiocarbonyl sulfur and one carboxylic
oxygen (Figure 2).
22
bacteria by inhibiting DNA gyrase. The lower affinity of the
sulfur atom toward magnesium(II) cations in comparison to
that toward the oxygen atom may explain the ineffectiveness of
the novel derivative. Interestingly, complexation of the
quinolone to a ruthenium center does not affect the
antimicrobial activity as was the case for other multivalent
metal cations, which are commonly found in over-the-counter
drugs and/or food supplements such as magnesium, aluminum,
In comparison with the previously published structures of
organometallic ruthenium quinolone complexes, the Ru−
cymene centroid distance is longer (1.668(2) Å vs 1.633(3)−
1
1
1
1
.64(2) Å). However, H NMR spectra indicate that in
chloroform solution the Ru−cymene bonding is stronger. In
comparison to the organoruthenium complex of the non-
thionated ligand, a lower chemical shift of one of the doublets
corresponding to aromatic cymene protons (Δδ = −0.16 ppm)
was observed, whereas the shifts assigned to the other group of
aromatic cymene hydrogens remain unchanged. The bite angle
of the quinolonato ligand is increased as compared to those in
structurally related complexes (90.2° vs 85.3−87.3°), which is
22b
calcium, iron, and zinc.
2.3. Cytotoxicity. In vitro cytotoxicity assays were
performed on human non small cell lung carcinoma (A549),
colon adenocarcinoma (SW480), and human ovarian carcino-
ma (CH1) cells. From the five tested compounds only 4-S,O
showed moderate activity against these human cell lines, while
both ligands (3-O,O and 4-S,O), the ruthenium precursor P1,
5
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a
and 4-O,O were inactive over the concentration range used
Table 2. IC50 Values (μM) for Inhibition of Recombinant
Human CatB and CatS by 4-O,O and 4-S,O
(
IC50 > 640 μM) (Table 1).
IC50 (μM)
Table 1. IC50 Values (μM) of P1, 3-S,O and 4-S,O for
Different Human Tumor Cell Lines
compd
-O,O
4-S,O
CatB
≥800
137 ± 10
CatS
4
≥800
66 ± 15
IC50 (μM)
a
compd
A549
>640
CH1
>640
SW480
IC50 values are given together with the standard errors.
P1
>640
3
4
-S,O
-S,O
>640
>640
>640
540 ± 112
364 ± 17
302 ± 24
In order to improve the antiproliferative activity of the
complexes, electroporation was applied. Electroporation is a
process in which exposing cells to specific electrical pulses
results in temporary formation of hydrophilic pores in the cell
membrane. Temporally increased cell permeability thus enables
extracellular molecules with otherwise hampered transmem-
brane transport to enter cells. It has been demonstrated that
combining electroporation with chemotherapy potentiates the
cytotoxicity of drugs when the drug’s efficacy is limited by its
uptake into the cell. This procedure is clinically approved and is
4
highly effective in the treatment of solid tumors. Furthermore,
promising results were also found when applied in combination
5
,6
with the treatment of cells with ruthenium complexes.
However, electroporation did not influence the cytotoxicity of
test compounds used in this work. All five compounds were
inactive on murine melanoma B16F1 cells with and without
electroporation, whereas electroporation improved the cytotox-
5
,6
icity of the ruthenium compounds NAMI-A and KP1339. It
needs to be stressed, however, that in vivo antitumor activity of
ruthenium compounds cannot always be predicted from in vitro
experiments. For example, NAMI-A is not cytotoxic but
possesses antimetastatic activity in vivo. Subsequent experi-
ments in vivo are thus required to fully evaluate the antitumor
activity of the compound class alone and in combination with
electroporation.
2e
Figure 3. Time dependence of CatB and CatS inhibition by 4-S,O.
CatB and CatS were incubated with increasing concentrations of 4-S,O
(10 nM to 400 μM) for 3, 6, 14, and 24 h before measuring the initial
velocities of the substrate hydrolysis. For more experimental details see
the Experimental Section. The solid lines crossing the experimental
data points were calculated with the standard three-parameter
equation for IC50 calculations using GraFit software (Erithacus
Software, U.K.).
2
.4. Cathepsin Inhibition. The mode of action of
ruthenium-based metallodrugs is widely unknown. Due to the
presence of a free cysteine in the active site and the ability of
various metals to inhibit their activity, cysteine cathepsins have
1
9,23
been proposed as potential targets of Ru compounds.
Table 3. Time Dependence of Recombinant Human CatB
a
Therefore, the inhibitory activity of 4-O,O and 4-S,O against
cathepsin B (CatB) and cathepsin S (CatS), two proteases
involved in cancer progression, was evaluated. Recombinant
cathepsins B and S were incubated with 4-O,O and 4-S,O for 14
and CatS Inhibition by 4-S,O at pH 6.0
IC50 (μM)
Ki (μM)
1
9
incubation time (h)
CatB
CatS
CatS
h, similar to the procedure in previous studies. The
ruthenium thionalidixicato complex 4-S,O inhibited cathepsins
B and S with IC50 values of 137 and 66 μM, whereas its
nonthionated counterpart 4-O,O exhibited essentially no
inhibition of the two enzymes. Obviously the replacement of
the pyridone O4 by sulfur improved the inhibition of both
cathepsins by at least 1 order of magnitude (Table 2).
3
6
204 ± 16
167 ± 14
137 ± 10
110 ± 11
98 ± 21
107 ± 23
66 ± 15
66 ± 12
1
4
4
19.5 ± 4.4
19.5 ± 3.6
2
a
The experimental conditions are described in the Experimental
Section. The best estimates for the IC50 values are given together with
19
the standard errors and were calculated as described. K values for the
Due to the very slow inhibition of cathepsin B, the time
dependence of cathepsin B and S inhibition was monitored for
i
inhibition of CatS by 4-S,O were also calculated following the
24
correction for substrate competition using a K value of 21 μM.
m
24 h (Figure 3). The maximum inhibition of cathepsin B was
seen after 24 h, whereas inhibition of cathepsin S was already
completed after 14 h, yielding a K value of 19.5 μM (Table 3)
i
24
after a correction for substrate competition (21 μM). The
inhibition of cathepsin S was thus significantly stronger than
that of cathepsin B and was almost comparable to the inhibition
as only crude cathepsin B extract of bovine origin that may
contain other components which could have influenced the
1
9
inhibition was used in that study. This is, however,
substantially lower in comparison to the case for other
organometallic compounds, such as gold, tellurium, and
19
of cathepsin B by other organoruthenium compounds.
However, a direct comparison of the results is not possible,
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2
palladium compounds, which exhibit IC values for cathepsin
refined by a full-matrix least-squares procedure based on F using
5
0
2
8
2
0,21
SHELXL-97. All non-hydrogen atoms were refined anisotropically.
The hydrogen atoms were placed at calculated positions and treated
using appropriate riding models. The programs Mercury, ORTEP, and
Platon were used for data analysis and figure preparation. The crystal
structures of compounds 1, 2, 3-S,O, and 4-S,O have been submitted
to the CCDC and have been allocated the deposition numbers CCDC
79554−879557 for compounds 3-S,O, 2, 4-S,O, and 1, respectively.
.2. Bioassays. 4.2.1. Antimicrobial Activities. The antimicrobial
activities of the test compounds were evaluated on the bacterial strain
Escherichia coli K12 (ER1821, New England Biolabs) using the agar
diffusion test. Bacteria were allowed to grow overnight, and their
concentration was then determined. The bacterial culture was
incorporated into Luria Broth nutrient agar with the final
B in the low μM to nM range.
In addition to the IC /K values, it is important to know
5
0
i
how rapidly the inhibitors block the protease activity. A k /K
2
1
29
−1
−1
value of ∼1.00 ± 0.26 M
s
was determined for cathepsin S
inhibition by 4-S,O, which is almost 10-fold higher than that for
−1
−1
cathepsin B (0.130 ± 0.068 M s ). Although this is
substantially slower than inhibition by other well-known
cathepsin inhibitors, such as epoxides or some other organo-
8
4
2
0,21
metallic compounds,
it may still contribute to the
antiproliferative effect, especially at relatively high inhibitor
concentration. Cysteine cathepsins have a half-life between 14
and 21 h in lysosomes, whereas they are destabilized within
7
2
5
concentration of bacteria being approximately 10 CFU/mL (colony
1
0−90 min at neutral pH, as found in the extracellular space.
forming unit per mL). Inoculated medium (20 mL) was poured into
Petri dishes and kept at 4 °C until use. Circles of agar (0.8 cm) were
cut out from the cooled medium. The MIC (minimal inhibitory
concentration) values of thionalidixic acid, sodium nalidixicate, and 4-
S,O were determined, using nalidixic acid as a reference substance.
MIC represents the lowest concentration of an antibiotic that inhibits
the growth of a tested organism. For estimating the MIC, the
antibacterial substances were diluted gradually in distilled water. A 100
μL portion of each dilution was poured into the holes cut into the
inoculated medium, and the system was kept at 37 °C for 24 h. Finally,
the plates were examined for the occurrence of inhibition zones.
The only exception is CatS, which is quite stable under these
2
6
conditions. It is therefore important to develop inhibitors that
act rapidly. Note that inhibition of cysteine cathepsins alone
does not trigger cancer cell death. However, blocking the
cathepsins in vivo by, for example, epoxides significantly slows
down tumor growth and invasiveness by blocking the signaling
pathways triggered by the cathepsins. Therefore, these results
suggest that the potentially beneficial anticancer effect might be
a combination of cytotoxicity of the compounds and inhibition
of cysteine cathepsins.
16
4
.2.2. Cytotoxicity. The human non small cell lung carcinoma
(
A549) and colon adenocarcinoma (SW480) cell lines were provided
3
. CONCLUSION
by Brigitte Marian (Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Austria). The human
ovarian carcinoma cell line CH1 was a gift from Lloyd R. Kelland
As a part of an ongoing study on ruthenium quinolone
complexes and their physicochemical and biological properties,
we have successfully devised a synthetic pathway for the
preparation of thionated quinolones, where the pyridone
oxygen atom is substituted by a sulfur atom. The thionated
derivative of nalidixic acid (3-S,O) and its organoruthenium
complex chlorido(η -p-cymene)(thionalidixicato-κ S,O)-
ruthenium(II) (4-S,O) were thus prepared. As was previously
found in the case of complexes bearing hydroxypyrone
(
CRC Centre for Cancer Therapeutics, Institute of Cancer Research,
2
Sutton, U.K.). Cells were grown in 75 cm culture flasks (Iwaki/Asahi
Technoglass, Gyouda, Japan) in complete medium (Minimum
Essential Medium supplemented with 10% heat-inactivated fetal
bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine, and 1%
nonessential amino acids (100×)) as adherent monolayer cultures. All
media and supplements were purchased from Sigma-Aldrich (Vienna,
Austria). Cultures were maintained at 37 °C under a humidified
atmosphere containing 5% CO2.
6
2
3
,7
ligands, the change of the ruthenium coordination sphere
from Ru(η -C )ClO to Ru(η -C )ClOS led to a significantly
Cytotoxicity was determined by a colorimetric assay (MTT assay,
MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide, Fluka). For this purpose, cells were harvested from culture flasks
by trypsinization and seeded in 100 μL per well into 96-well plates
6
6
6
2
6
more stable complex in aqueous solution with improved
cytotoxicity. The novel compound also exhibited a substantial
increase by at least 1 order of magnitude in the potency of
inhibition of two enzymes, cathepsins B and S, which are
involved in various pathological processes, including cancer
progression.
3
(Iwaki/Asahi Technoglass, Gyouda, Japan) in cell densities of 4 × 10
3
3
(A549), 1.5 × 10 (CH1), and 2.5 × 10 cells per well (SW480),
respectively. These cell numbers ensure exponential growth of
untreated controls throughout drug exposure of treated microcultures.
Cells were allowed to adhere and resume proliferation in drug-free
complete culture medium for 24 h. Drugs were dissolved in complete
medium and appropriately diluted, and instantly 100 μL of the drug
dilutions was added per well. After exposure for 96 h at 37 °C and 5%
4. EXPERIMENTAL SECTION
4.1. Materials and Methods. The starting materials and solvents
that were used in the syntheses were purchased from Sigma-Aldrich,
Acros Organics, or TCI and were used as received. NMR spectra were
recorded at 25 °C using a Bruker Avance III 500 MHz FT-NMR
CO , drug solutions were replaced by 100 μL/well RPMI 1640 culture
2
medium (supplemented with 10% heat-inactivated fetal bovine serum
and 4 mM L-glutamine) plus 20 μL/well MTT solution in phosphate-
buffered saline (5 mg/mL) and incubated for 4 h. Subsequently, the
medium/MTT mixture was removed and the formazan crystals that
were formed in vital cells were dissolved in 150 μL of DMSO
(dimethyl sulfoxide) per well. Optical densities were measured with a
microplate reader (Tecan Spectra Classic) at 550 nm (and a reference
wavelength of 690 nm) to yield relative quantities of viable cells as
percentages of untreated controls, and 50% inhibitory concentrations
(IC ) were calculated from concentration-effect curves by inter-
polation. Calculations are based on at least three independent
experiments, each consisting of three replicates per concentration
level.
4.2.3. Electroporation Experiments. The cytotoxicity of the test
compounds was determined on the murine melanoma cell line B16F1
(European Collection of Cell Cultures, U.K.) in combination with or
without electroporation. Cell suspensions were prepared from
1
spectrometer. H NMR spectra were measured in DMSO-d or CDCl
6
3
at 500.10 MHz and 13C{ H} NMR spectra at 125.75 MHz. Infrared
spectra were recorded with a Perkin-Elmer Spectrum 100 FTIR
spectrometer, equipped with a Specac Golden Gate Diamond ATR as
a solid sample support. Elemental analyses (C, H, N) were performed
with a Perkin-Elmer 2400 Series II CHNS/O analyzer. HRMS were
measured on an Agilent 6224 Accurate Mass TOF LC mass
spectrometer. X-ray diffraction data for 1 was collected on an Oxford
Diffraction SuperNova diffractometer with Mo microfocus X-ray
source (Kα radiation, λ = 0.710 73 Å) with mirror optics and an Atlas
detector. X-ray diffraction data for compounds 2, 3-S,O, and 4-S,O
were collected on a Nonius Kappa CCD diffractometer equipped with
a Mo anode (Kα radiation, λ = 0.710 73 Å) and a graphite
monochromator at 150(2) K (2, 4-S,O) and 293(2) K (3-S,O). The
1
50
2
7
structures were solved by direct methods implemented in SIR92 and
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3
confluent cell cultures by trypsinization. The final concentration of
cells in 0.9% NaCl was 2.2 × 10 cells/mL. The test compounds were
Hz, 2H, OCH CH ), 2.63 (s, 3H, ArCH ), 1.37 (t, J(H,H) = 8 Hz,
2 3 3
7
3
13
3H, NCH CH ), 1.29 (t, J(H,H) = 8 Hz, 3H, OCH CH ). C NMR
2
3
2
3
dissolved in 0.9% NaCl and prepared in concentrations from 0 to 1
mM. The compounds were added to the cell suspensions to reach final
concentrations of 0, 0.01, and 0.1 mM. Immediately after incubation
(DMSO-d , 126 MHz): δ 173.1, 164.3, 162.4, 149.1, 148.2, 135.9,
6
121.0, 120.5, 111.2, 59.8, 45.6, 24.6, 14.8, 14.3. IR (cm , ATR): 2991,
−1
1682, 1625, 1604, 1544, 1439, 1255, 1209, 1092, 799. HRMS-ESI (m/
+
(
<0.5 min), a drop of cell suspension was placed between two flat
z): [M + H] calcd for C H N O 261.1239, found 261.1235. Anal.
14
17
2
3
parallel stainless-steel electrodes 2 mm apart. A train of electric
rectangular unipolar pulses (8 pulses, 800 V/cm, 100 μs, 1 Hz) was
applied with an Cliniporator electroporator (Igea, Carpi, Italy). The
same procedure without electric pulses was used for cells exposed to
the tested compounds only. In addition, we tested the cytotoxicity of
compounds after prolonged incubation time (60 min, without
electroporation). Nonelectroporated cells incubated in 0.9% NaCl
served as controls (c). Cell viability was measured 72 h after treatment
using the MTS-based Cell Titer 96 AQueous One Solution Cell
Proliferation Assay (Promega, USA). Absorption at 490 nm
wavelength (A490) was measured with a Tecan Infinite M200
spectrophotometer (Tecan, Switzerland). Statistical analysis was
performed using One-Way ANOVA test and SigmaPlot v. 11.0
statistical software (SPSS, Chicago, IL).
Calcd for C H N O : C, 64.60; H, 6.20; N, 10.77. Found: C, 64.33;
14
16
2
3
H, 6.57; N, 11.02.
.3.2. Thionalidixic Acid Ethyl Ester (2; C H N O S). Compound
4
14 16 2 2
1
(250 mg, 0.91 mmol, 1 equiv) and Lawesson’s reagent (213 mg, 0.50
mmol, 1.1 equiv) were suspended in 10 mL of dry THF. The
suspension was refluxed under argon for 5 h. After a few minutes the
suspension turned into a deep red solution. Compound 2 was isolated
from the reaction mixture on a silica column using a n-hexane/ethyl
acetate (2/1) mixture as eluent (R = 0.65, 164 mg, 62%). Single
f
crystals suitable for X-ray diffraction analysis were obtained by slow
1
evaporation of an ethyl acetate solution. H NMR (DMSO-d , 500.10
6
3
MHz, 25 °C): δ 8.94 (d, J(H,H) = 8 Hz, 1H, H5), 8.55 (s, 1H, H2),
.50 (d, J(H,H) = 8 Hz, 1H, H6), 4.52 (q, J(H,H) = 8 Hz, 2H,
NCH CH ), 4.27 (q, J(H,H) = 8 Hz, 2H, OCH CH ), 2.65 (s, 3H,
ArCH ), 1.41 (t, J(H,H) = 8 Hz, 3H, NCH CH ), 1.30 (t, J(H,H) =
3
3
7
3
2
3
2
3
4
.2.4. Cathepsin Inhibition Assays. Initial solutions of 50 mM 4-
3
3
3
2
3
O,O and 10 mM 4-S,O were prepared in DMSO. Final DMSO
concentrations never exceeded 4% (v/v), which does normally not
affect cathepsin activity. Subsequent dilutions were made in 100 mM
phosphate buffer, pH 6.0. Recombinant human cathepsins were
13
8
1
1
Hz, 3H, OCH CH ). C NMR (DMSO-d , 126 MHz): δ 190.4,
2
3
6
65.9, 163.6, 144.1, 139.4, 138.7, 128.5, 127.2, 122.8, 60.8, 46.4, 24.8,
4.9, 14.0. IR (cm , ATR): 2986, 1723, 1600, 1439, 1368, 1267, 1176,
1123, 1046, 785. HRMS-ESI (m/z): [M + H] calcd for C H N O S
−
1
+
3
0
31
14 17 2 2
prepared as described earlier in E. coli (CatB) or P. pastoris
(
32
277.1011, found 277.1009. Anal. Calcd for C H N O S; C, 60.85; H,
14 16 2 2
CatS) expression systems. The assay buffer for enzyme kinetics
5
.84; N, 10.14. Found: C, 61.11; H, 5.99; N, 10.02.
.3.3. Thionalidixic Acid (3-S,O; C H N O S). Compound 2 (200
measurements was 100 mM sodium phosphate, pH 6.0, supplemented
with 1 mM EDTA, 0.1% (w/v) polyethylene glycol (PEG), and 1 mM
dithiothreitol (DTT). The experimental conditions, i.e. pH 6.0 and the
use of PEG, were selected in order to improve the stability of the
enzymes and to minimize protein adsorption to the surface.
Cathepsins were activated in the assay buffer supplemented with 5
mM DTT for 5 min at room temperature. The cleavage of the
fluorogenic substrate Z-Phe-Arg-AMC (Bachem) was used to assess
the enzyme activity. Kinetic measurements were performed with a
fixed cathepsin concentration of 25 nM and a fixed substrate
concentration of 50 μM. Inhibitor concentrations ranged from 10
nM to 400 μM: 10 nM/100 nM/500 nM/1 μM/5 μM/10 μM/25
μM/50 μM/100 μM/200 μM/400 μM. Prior to the addition of
substrate, compounds were incubated with CatB or CatS for the
designated time (3−24 h) at room temperature. Cathepsin activity was
then measured at room temperature over 3 min at excitation and
emission wavelengths of 370 and 460 nm, respectively. All measure-
ments were performed in duplicate, using a Tecan Saphire plate reader
4
12 12 2 2
mg, 0.72 mmol, 1 equiv) and NaOH (115 mg, 2.88 mmol, 4 equiv)
were dissolved in 30 mL of a THF/water (1/1) mixture and stirred
vigorously for 2 h at room temperature. Concentrated HCl was added
dropwise until a yellow precipitate formed (167 mg, 93%). Single
crystals of 3-S,O suitable for X-ray diffraction analysis were obtained by
slow evaporation of a chloroform solution. 1H NMR (DMSO-d6,
5
00.10 MHz, 25 °C): δ 15.07 (s, 1H, OH), 9.35 (s, 1H, H2), 9.08 (d,
3
3
J(H,H) = 8 Hz, 1H, H5), 7.71 (d, J(H,H) = 8 Hz, 1H, H6), 4.74 (q,
3
3
J(H,H) = 8 Hz, 2H, NCH CH ), 2.73 (s, 3H, ArCH ), 1.46 (t,
2
3
13
3
J(H,H) = 8 Hz, 3H, NCH CH ). C NMR (DMSO-d , 126 MHz): δ
2
3
6
1
1
1
2
87.6, 166.0, 165.2, 146.8, 143.8, 138.7, 126.4, 124.2, 121.7, 48.0, 24.9,
−1
5.0. IR (cm , ATR): 3061, 2976, 2391, 1701, 1597, 1422, 1364,
+
343, 1126, 786. HRMS-ESI (m/z): [M + H] calcd. for C H N O S
1
2
13
2
2
49.0698, found: 249.0695. Anal. Calcd for C H N O S: C, 58.05;
12
12
2
2
H, 4.87; N, 11.27. Found: C, 57.87; H, 5.01; N, 11.39.
6
2
4
.3.4. Chlorido(η -p-cymene)(thionalidixicato-κ -S,O)ruthenium-
6
(
Tecan Group, Switzerland). Initial velocities of the enzyme activity
(
II) (4-S,O; C H ClN O RuS). [Ru(η -p-cymene)Cl(μ-Cl)] (40.0 mg,
22 25 2 2 2
reactions were calculated and plotted against the inhibitor concen-
trations. IC50 values were calculated using the standard three-
parameter equation for determination of IC50 values by the GraFit
0
.065 mmol, 0.5 equiv), 3-S,O (32.4 mg, 0.130 mmol), and NaOMe
(6.9 mg, 0.128 mmol, 0.98 equiv) were dissolved in 25 mL of a
MeOH/chloroform (1/1) mixture and refluxed overnight under an
argon atmosphere. The solvent was evaporated, and the remaining
solid was dissolved in dichloromethane and filtered over Celite to
remove NaCl and other insoluble impurities. The solution was
concentrated to 3 mL, and the product (4-S,O; 55.4 mg, 82%) was
precipitated by addition of hexane (20 mL). Single crystals of 4-S,O
program (Erithacus Software, U.K.). The k /K1 (association rate
2
constant) values for the interaction between both cathepsins and 4-S,O
were then calculated from the IC50 curves using the assumption that
this is a pseudo-first-order reaction with a large excess of the inhibitor.
Due to a significant loss of activity observed for both enzymes,
especially at the 24 h time point (30−40%; Figure 3), a correction for
spontaneous inactivation of the enzymes was necessary.
suitable for X-ray diffraction analysis were obtained by slow
1
evaporation of a dichloromethane/n-hexane (1/1) solution.
H
4
.3. Syntheses and Characterization. 4.3.1. Nalidixic Acid Ethyl
3
NMR (CDCl , 500.10 MHz, 25 °C): δ 9.18 (d, 1H, J(H,H) = 8
3
Ester (1; C H N O ). Sodium nalidixicate hydrate (500 mg, 1.84
1
4
16
2
3
3
Hz, H5), 9,14 (s, 1H, H2), 7.45 (d, J(H,H) = 8 Hz, 1H, H6), 5.48−
mmol, 1 equiv) was suspended in 40 mL of chloroform, and
p-toluenesulfonic acid hydrate (1067 mg, 5.61 mmol, 3.05 equiv) and
5
2
2
.33 (m, 4H, ArH cymene), 4.70−4.65 (m, 2H, NCH CH ), 2.97−
2
3
.88 (m, 1H, ArCH(CH ) , cymene), 2.74 (s, 3H, ArCH nalidixic),
1
mL of ethanol were added. The reaction mixture was refluxed
3
2
3
3
.16 (s, 3H, ArCH3 cymene), 1.58 (t, 2H, J(H,H) = 8 Hz,
NCH CH ), 1.20 (d, 6H, J(H,H) = 7 Hz, ArCH(CH ) , cymene).
C NMR (DMSO-d , 126 MHz): δ 179.8, 165.8, 163.6, 146.0, 141.7,
overnight. The crude product was treated with approximately 20 mL
of saturated sodium carbonate solution in a separation funnel. The
organic layer was collected, washed with distilled water (2 × 10 mL),
and dried with sodium sulfate, and the solvent was removed under
vacuum (291 mg; 55%). Single crystals suitable for X-ray diffraction
3
2
3
3 2
13
6
137.4, 124.1, 122.3, 101.7, 99.4, 83.4, 83.2, 82.8, 80.6, 79.5, 47.6, 29.6,
−1
24.2, 21.7, 21.2, 17.3, 14.3. IR (cm , ATR): 2963, 1618, 1599, 1447,
+
analysis were obtained by slow evaporation of an ethyl acetate
1352, 1337, 1249, 1117, 1027, 793. HRMS-ESI (m/z): [M − Cl]
1
solution. H NMR (DMSO-d , 500.10 MHz, 25 °C): δ 8.81 (s, 1H,
calcd for C22H N O RuS 483.0680, found 483.0679. Anal. Calcd for
25 2 2
6
3
3
H2), 8.44 (d, J(H,H) = 8 Hz, 1H, H5), 7.41 (d, J(H,H) = 8 Hz, 1H,
H6), 4.48 (q, J(H,H) = 8 Hz, 2H, NCH CH ), 4.23 (q, J(H,H) = 8
C H ClN O RuS: C, 51.01; H, 4.87; N, 5.41. Found: C, 51.31; H,
22 25 2 2
4.42; N, 5.72.
3
3
2
3
5
872
dx.doi.org/10.1021/om300424w | Organometallics 2012, 31, 5867−5874

Organometallics
Article
4
.4. Aquation Experiments. Complex 4 (1−2 mg/mL) was
(6) Bice
istry 2007, 71, 113−117.
̌
k, A.; Turel, I.; Kanduse
̌
r, M.; Miklavci
̌
c,
̌
D. Bioelectrochem-
1
dissolved in 10% DMSO-d /D O, and H NMR spectra were recorded
immediately after dissolution, after 24 h, and after 1 week.
6
2
(7) (a) Hanif, M.; Henke, H.; Meier, S. M.; Martic, S.; Labib, M.;
Kandioller, W.; Jakupec, M. A.; Arion, V. B.; Kraatz, H. B.; Keppler, B.
K.; Hartinger, C. G. Inorg. Chem. 2010, 49, 7953−7963. (b) Mendoza-
Ferri, M. G.; Hartinger, C. G.; Mendoza, M. A.; Groessl, M.; Egger, A.
E.; Eichinger, R. E.; Mangrum, J. B; Farrell, N. P.; Maruszak, M.;
Bednarski, P. J.; Klein, F.; Jakupec, M. A.; Nazarov, A. A.; Severin, K.;
Keppler, B. K. J. Med. Chem. 2009, 52, 916−925. (c) Peacock, A. F. A.;
Melchart, M.; Deeth, R. J.; Habtemariam, A.; Parsons, S.; Sadler, P. J.
Chem. Eur. J. 2007, 13, 2601−2613. (d) Melchart, M.; Habtemariam,
A.; Parsons, S.; Sadler, P. J. J. Inorg. Biochem. 2007, 101, 1903−1912.
ASSOCIATED CONTENT
Supporting Information
■
*
S
A table and CIF files giving crystallographic data for 1, 2, 3-S,O,
and 4-S,O and figures giving crystal structures of compounds 1
and 2 and a numbering scheme for NMR assignments. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(
e) Kandioller, W.; Hartinger, C. G.; Nazarov, A. A.; Kuznetsov, M. L.;
AUTHOR INFORMATION
Corresponding Author
Fax: (+) 386 1 24 19 220. E-mail: iztok.turel@fkkt.uni-lj.si.
John, R. O.; Bartel, C.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K.
Organometallics 2009, 28, 4249−4251. (f) Kandioller, W.; Hartinger,
C. G.; Nazarov, A. A.; Bartel, C.; Skocic, M.; Jakupec, M. A.; Arion, V.
B.; Keppler, B. K. Chem. Eur. J. 2009, 15, 12283−12291.
■
*
Notes
(8) Turel, I. Coord. Chem. Rev. 2002, 232, 27−47.
The authors declare no competing financial interest.
(9) (a) Skyrianou, K. C.; Perdih, F.; Papadopoulos, A. N.; Turel, I.;
Kessissoglou, D. P.; Psomas, G. J. Inorg. Biochem. 2011, 105, 1273−
1285. (b) Tarushi, A.; Polatoglou, E.; Kljun, J.; Turel, I.; Psomas, G.;
Kessissoglou, D. P. Dalton Trans. 2011, 40, 9461−9473. (c) Saraiva,
R.; Lopes, S.; Ferreira, M.; Novais, F.; Pereira, E.; Feio, M. J.; Gameiro,
P. J. Inorg. Biochem. 2010, 104, 843−850.
ACKNOWLEDGMENTS
■
We are grateful for financial support from a bilateral
Slovenian−Austrian project, project J1-4131 (I.T.), junior
researcher grants for R.H. and J.K., and the program grant
P1-0140 (B.T.) of the Slovenian Research Agency (ARRS). We
acknowledge the support of the Mahlke-Obermann Founda-
(10) (a) Fan, J. Y.; Sun, D.; Yu, H. T.; Kerwin, S. M.; Hurley, L. H. J.
Med. Chem. 1995, 38, 408−424. (b) Zeng, Q. P.; Kwok, Y.; Kerwin, S.
M.; Mangold, G.; Hurley, L. H. J. Med. Chem. 1998, 41, 4273−4278.
(11) (a) Turel, I.; Kljun, J.; Perdih, F.; Morozova, E.; Bakulev, V.;
Kasyanenko, N.; Byl, J. A. W.; Osheroff, N. Inorg. Chem. 2010, 49,
̈
tion, the Hochschuljubilaumsstiftung Vienna and the Austrian
̈
Exchange Service (OAD). The project was also supported by
COST D39 and COST CM1105, in particular by a short-term
scientific mission (STSM D39-6067) for J.K. We thank Amalija
1
0750−10752. (b) Kljun, J.; Bytzek, A. K.; Kandioller, W.; Bartel, C.;
Jakupec, M. A.; Hartinger, C. G.; Keppler, B. K.; Turel, I.
Organometallics 2011, 30, 2506−2512.
Golobic
̌
and Marta Kasunic
̌
for help and advice in crystal
Hodzi for
(12) (a) Casini, A.; Hartinger, C. G.; Gabbiani, C.; Mini, E.; Dyson,
structure solution and refinement. We thank Dusa
̌
̌
c
́
P. J.; Keppler, B. K.; Messori, L. J. Inorg. Biochem. 2008, 102, 564−575.
b) Ang, W. H.; De Luca, A.; Chapuis-Bernasconi, C.; Juillerat-
Jeanneret, L.; Lo Bello, M.; Dyson, P. J. ChemMedChem 2007, 2,
799−1806. (c) Casini, A.; Mastrobuoni, G.; Ang, W. H.; Gabbiani,
C.; Pieraccini, G.; Moneti, G.; Dyson, P. J.; Messori, L. ChemMedChem
help and insights on the laboratory work with bacterial strains
and Dr. Michael Jakupec and the cell biology team of the
Institute of Inorganic Chemistry at the University of Vienna for
conducting the MTT assays.
(
1
2
007, 2, 631−635.
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