Dicopper(II) and dizinc(II) complexes with nonsymmetric dinucleating ligands based on indolo[3,2-c]quinolines: Synthesis, structure, cytotoxicity, and intracellular distribution

Article abstract of DOI:10.1021/ic401573d

Dicopper(II) and dizinc(II) complexes [Cu₂( ^MeOOCL^COO)(CH₃COO)₂] (1) and [Zn₂(^MeOOCL^COO)(CH₃COO) ₂] (2) were synthesized by reaction of Cu(CH

Full text of DOI:10.1021/ic401573d

Article
pubs.acs.org/IC
Dicopper(II) and Dizinc(II) Complexes with Nonsymmetric
Dinucleating Ligands Based on Indolo[3,2‑c]quinolines: Synthesis,
Structure, Cytotoxicity, and Intracellular Distribution
Michael F. Primik,† Simone Goschl,† Samuel M. Meier,† Nadine Eberherr,† Michael A. Jakupec,†
̈
‡,§
Eva A. Enyedy, Ghenadie Novitchi,∥ and Vladimir B. Arion*,†
́
†Institute of Inorganic Chemistry, University of Vienna, Wahringer Strasse 42, A-1090 Vienna, Austria
̈
‡Department of Inorganic and Analytical Chemistry, University of Szeged, Dom
́
ter
7, H-6720 Szeged, Hungary
iques Intenses-CNRS, 25 Avenue des Martyrs, F-38042 Grenoble Cedex 9, France
́
7, H-6720 Szeged, Hungary
§HAS-USZ Bioinorganic Chemistry Research Group, Dom
∥Laboratoire National des Champs Magnet
́
ter
́
́
S
* Supporting Information
ABSTRACT: Dicopper(II) and dizinc(II) complexes [Cu2(MeOOCLCOO)-
(CH3COO)2] (1) and [Zn2(MeOOCLCOO)(CH3COO)2] (2) were
synthesized by reaction of Cu(CH3COO)2·H2O and Zn(CH3COO)2·
2H2O with a new nonsymmetric dinucleating ligand EtOOCHLCOOEt
prepared by condensation of 6-hydrazinyl-11H-indolo[3,2-c]quinoline
with diethyl-2,2′-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)-
diacetate. The design and synthesis of this elaborate ligand was
performed with the aim of increasing the aqueous solubility of
indolo[3,2-c]quinolines, known as biologically active compounds, and
investigating the antiproliferative activity in human cancer cell lines and
the cellular distribution by exploring the intrinsic fluorescence of the
indoloquinoline scaffold. The compounds have been comprehensively
characterized by elemental analysis, spectroscopic methods (IR, UV−vis,
1H and 13C NMR spectroscopy), ESI mass spectrometry, magnetic
susceptibility measurements, and UV−vis complex formation studies (for 1) as well as by X-ray crystallography (1 and 2). The
antiproliferative activity of EtOOCHLCOOEt, 1, and 2 was determined by the MTT assay in three human cancer cell lines, namely,
A549 (nonsmall cell lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon adenocarcinoma), yielding IC50 values in
the micromolar concentration range and showing dependence on the cell line. The effect of metal coordination on cytotoxicity of
EtOOCHLCOOEt is also discussed. The subcellular distribution of EtOOCHLCOOEt and 2 was investigated by fluorescence microscopy,
revealing similar localization for both compounds in cytoplasmic structures.
drugs, paullones remain at an early preclinical stage mainly
because of their low aqueous solubility and bioavailability.
Metal coordination was suggested as a means to overcome
these problems. However, the original paullones did not
contain suitable binding sites for metal ions, and these had to
be introduced by chemical modification. A library of paullone-
based ligands with a broad structural diversity and the
respective complexes with copper(II), gallium(III), ruthenium-
(II), and osmium(II) have been reported.12−16
In an effort to elucidate novel structure−activity relationships
(SARs), the folded seven-membered azepine ring of paullones
has been replaced by a pyridine ring, leading to another class of
biologically active compounds, namely, indolo[3,2-c]quinolines,
with an essentially planar structure. Indolo[3,2-c]quinolines and
the structurally related indolo[3,2-b]quinolines are known
INTRODUCTION
■
Cancer is a disease that is difficult to treat, and novel drugs are
still highly demanded.1−3 Synthesis of metal complexes with
biologically active ligands is a promising approach in developing
anticancer drugs, as metal ions can significantly alter the
physical and biological properties of these ligands.4−7 Indolo-
[3,2-d]benzazepines, also referred to as paullones, are one class
of potential cyclin-dependent kinase (Cdk) inhibitors, identi-
fied in a comparative database search at the National Cancer
Institute (NCI; NCI60 screen). Thereby, the lead compound
kenpaullone exhibited an activity profile similar to that of
flavopiridol,8,9 the first clinically studied Cdk inhibitor. Within a
series of paullones, however, the antiproliferative activity did
not parallel the Cdk inhibitory potencies.10 As a result, other
intracellular targets for this class of compounds, e.g., glycogen
synthase kinase 3β (Gsk3β) or mitochondrial malate
dehydrogenase (mMDH), have been suggested.11 Despite
marked efforts to develop these compounds as anticancer
Received: June 20, 2013
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of EtOOCHLCOOEt, 1, and 2, and Atom-Numbering Schemes for Modified Indoloquinolines
a
Reagents and conditions: (i) 35% formaldehyde solution, conc. HCl;23,24 (ii) diethyl-2,2′-iminodiacetate, triethylamine, dry THF, room
temperature, 3 h (95%); (iii) methanol, room temperature, 2 h (95%); (iv) copper(II) acetate monohydrate or zinc(II) acetate dihydrate, methanol,
room temperature, 30 min [1 (37%), 2 (33%)].
guanosine 5′-triphosphate were received from Sigma-Alrdich. L-
Histidine, formaldehyde solution (35%), copper(II) acetate mono-
hydrate, and zinc(II) acetate dihydrate were received from Merck,
while tetrahydrofuran (THF) and methanol (both analytical reagent
grade) were received from Fisher Scientific. THF was dried prior to
use by a standard protocol. Dimethyl sulfoxide (DMSO) was received
from Acros, ammonium bicarbonate, formic acid, and L-glutamic acid
were received from Fluka, and L-aspartic acid was received from Serva.
Milli-Q water (18.2 MΩ, Millipore Advantage A10, 185 UV Ultrapure
Water System, Molsheim, France) and methanol (Fisher, HPLC
grade) were used for ESI-MS experiments. 6-Hydrazinyl-11H-
indolo[3,2-c]quinolone (D, Scheme 1) was synthesized according to
the published protocol.19 Details of the synthesis and 1H NMR
characterization of D are given in the Supporting Information. 3-
(Chloromethyl)-2-hydroxy-5-methylbenzaldehyde (B) was obtained
from 5-methyl-2-hydroxybenzaldehyde (A) via a previously described
chloromethylation reaction.23,24
phytochemicals found in the roots of the West African climbing
shrub Cryptolepis sanguinolenta that is used in traditional African
medicine. Both exhibit a broad spectrum of biological
properties, including antibacterial, antitumor, as well as anti-
inflammatory activity.17 In contrast to indolo[3,2-b]quinolines,
few studies have addressed indolo[3,2-c]quinolines. Like
paullones, indoloquinolines do not contain binding sites for
metal ions. They can, however, be introduced with synthetic
tools essentially different from those applied for paullones. The
first ruthenium(II), osmium(II), and copper(II) complexes
with modified indolo[3,2-c]quinoline ligands were derived from
structurally related paullone complexes using distinct chemical
transformations.18−21 In particular, it has been found that
complexes of indolo[3,2-c]quinolines exhibit higher cytotoxicity
than their paullone counterparts, thus clearly establishing the
effect of replacing the azepine ring in paullones by a pyridine
ring in indoloquinolines. In addition, it was shown that SARs of
complexes with modified paullones do not necessarily apply to
indolo[3,2-c]quinoline-based compounds.18,20 Current efforts
by us are focused on investigation of the underlying
mechanisms of their antiproliferative activity by exploiting the
intrinsic fluorescence of indolo[3,2-c]quinolines.22
Recently, we reported on the syntheses of highly
antiproliferative copper(II) complexes with modified indolo-
[3,2-c]quinolines.20 Herein, we report on the synthesis of a
more elaborate bioconjugate EtOOCHLCOOEt with two distinct
binding sites and the dinuclear copper(II) and zinc(II)
complexes 1 and 2, respectively. The new ligand is sufficiently
soluble in biological media and intrinsically fluorescent when
light irradiated at λex = 395 nm. These properties permitted us
to track the intracellular distribution of EtOOCHLCOOEt and 2.
Moreover, the ligand design led to assembly of homometallic
dinuclear complexes with distinct compartments (Scheme 1), a
feature not explored by us so far in the development of
anticancer metal complexes.
Diethyl-2,2′-((3-formyl-2-hydroxy-5-methylbenzyl)-
azanediyl)diacetate (C). To a stirred solution of 3-(chloromethyl)-
2-hydroxy-5-methylbenzaldehyde (0.10 g, 0.54 mmol) in dry THF (30
mL) was added diethyl-2,2′-iminodiacetate (85 μL, 0.48 mmol) under
argon atmosphere. Upon addition of triethylamine (281 μL, 2.0
mmol) the colorless solution turned bright yellow and a white
precipitate formed. After stirring for 3 h at room temperature, the
precipitate was filtered off. The yellow filtrate was concentrated under
reduced pressure to give an orange oil, which was dried in vacuo
1
overnight. Yield: 0.17 g, 95%. H NMR 500.13 MHz (DMSO-d6, δH,
4
ppm): 10.60 (s, 1H, OH), 10.23 (s, 1H, C1), 7.43 (d, 1H, J(HC5) =
2.0 Hz, C7), 7.32 (d, 1H, 4J(HC7) = 2.1 Hz, C5), 4.12 (q, 4H,
3J(HC16, C18) = 7.1 Hz, C15, C17), 3.90 (s, 2H, C9), 3.54 (s, 4H, C11,
C13), 2.26 (s, 3H, C8), 1.20 (t, 6H, 3J(HC15, C17) = 7.1 Hz, C16, C18).
13C{1H} NMR 125.76 MHz (DMSO-d6, δC, ppm): 192.9 (C1), 171.2
(C12, C14), 158.3 (C3), 137.9 (C5), 129.1 (C7), 128.6 (C6), 125.2
(C4), 122.3 (C2), 60.8 (C15, C17), 54.4 (C11, C13), 53.6 (C9), 20.3
(C8), 14.5 (C16, C18).
Diethyl-2,2′-((3-(((5H-indolo[3,2-c]quinolin-6(11H)-ylidene)-
hydrazono)methyl)-2-hydroxy-5-methylbenzyl)azanediyl)-
diacetate (EtOOCHLCOOEt). To a solution of C (0.78 g, 2.3 mmol) in
methanol (40 mL) was added D (0.54 g, 2.2 mmol). The reaction
mixture turned into a bright yellow suspension, which was stirred for 2
h under argon atmosphere and allowed to stand at 4 °C overnight.
The yellow product was filtered off, washed with cold methanol (2 × 4
mL), and dried in vacuo overnight. Yield: 0.77 g, 95%. Anal. Calcd for
C32H33N5O5·0.75H2O (Mr = 581.15): C, 66.14; H, 5.98; N, 12.05.
EXPERIMENTAL SECTION
■
Materials. All chemicals were purchased from commercial
suppliers and used without further purification. Hydrochloric acid, 2-
hydroxy-5-methylbenzaldehyde (A), diethyl-2,2′-iminodiacetate, 4-
((2-hydroxyethyl)-piperazin-1-yl)ethanesulfonic acid (HEPES), and
1
Found: C, 66.25; H, 6.08; N, 12.03. H NMR 500.13 MHz (DMSO-
d6, δH, ppm): 12.46 (s, 1H, N11), 10.72 (s, 1H, N5), 10.63 (s, 1H,
B
dx.doi.org/10.1021/ic401573d | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
OH), 8.77 (s, 1H, C14), 8.43 (d, 1H, 3J(HC8) = 7.8 Hz, C7), 8.11 (d,
Agilent 8453 spectrophotometer in the 190−1000 nm window using
samples dissolved in methanol at 10 μM concentrations. IR spectra
were measured with a Bruker Vertex 70 Fourier transform IR
spectrometer by means of the attenuated total reflection (ATR)
technique. Fluorescence excitation and emission spectra were recorded
with a Horiba FloroMax-4 spectrofluorimeter and processed using the
FluorEssence v3.5 software package. Samples of EtOOCHLCOOEt and 2
were prepared from a 1 mM solution of each in DMSO and dilution
with HEPES buffer (20 mM, pH = 7.4) to give samples at 10 μM
concentrations with a maximum content of 1% DMSO (v/v).
Crystallographic Structure Determination. X-ray diffraction
measurements were performed on a Bruker X8 APEXII CCD
diffractometer. Single crystals were positioned at 40 mm from the
detector, and 1312 and 722 frames were measured, each for 60 and 90
s over 1° scan width for 1·3CH3OH and 2·2CH3OH, correspondingly.
Data were processed using SAINT software.25 Crystal data, data
collection parameters, and structure refinement details are given in
Table 1. Structures were solved by direct methods and refined by full-
3
3
1H, J(HC2) = 7.8 Hz, C1), 7.80 (d, 1H, J(HC3) = 8.3 Hz, C4), 7.61
(d, 1H, 3J(HC9) = 8.3 Hz, C10), 7.59 (s, 1H, C20), 7.50−7.44 (m, 1H,
C3), 7.40−7.35 (m, 1H, C9), 7.30−7.22 (m, 2H, C8, C2), 7.10 (s, 1H,
3
C18), 4.13 (q, 4H, J(HC29, C31) = 7.1 Hz, C28, C30), 3.94 (s, 2H,
C22), 3.57 (s, 4H, C24, C26), 2.30 (s, 3H, C21), 1.21 (t, 6H,
3J(HC28, C30) = 7.1 Hz, C29, C31). 13C{1H} NMR 125.76 MHz
(DMSO-d6, δC, ppm): 171.2 (C25, C27), 154.0 (C16), 152.1 (C14),
150.1 (C6), 138.6 (C10a), 138.5 (C11a), 138.3 (C4a), 132.1 (C18),
129.5 (C3), 129.2 (C20), 127.8 (C19), 124.3 (C9), 124.2 (C17),
124.1 (C6b), 122.9 (C7), 122.2 (C1), 121.9 (C2), 121.2 (C8), 120.8
(C15), 117.1 (C4), 113.6 (C11b), 112.0 (C10), 105.2 (C6a), 60.6
(C28, C30), 54.5 (C24, C26), 53.1 (C22), 20.7 (C21), 14.6 (C29,
C31). ESI-MS (methanol), positive m/z 379 [EtOOCHLCOOEt −N-
(CH2COOEt)2]+, 568 [EtOOCHLCOOEt + H+]+, 590 [EtOOCHLCOOEt
+
Na+]+; negative m/z 566 [EtOOCHLCOOEt − H+]−, 603 [EtOOCHLCOOEt
+ Cl−]−. UV−vis (methanol), λmax (ε, M−1 cm−1): 226 (43 300), 260
(32 350), 273 sh (25 300), 306 (22 400), 348 (15 250), 365 sh (15
900), 382 (17 300). ATR-IR, selected bands, cm−1: 3640, 3375, 2976,
1730, 1608, 1461, 1192, 1002.
Table 1. Crystal Data and Details of Data Collection for 1·
3CH3OH and 2·2CH3OH
Di(μ-acetato-κ2O,O′)-(2-((3-((2-(11H-indolo[3,2-c]quinolin-6-
yl-κN5)hydrazono-κN13)methyl)-5-methyl-2-oxidobenzyl-κO1)-
(2-methoxy-2-oxoethyl-κO5)amino κN23)acetato-κO2)-
dicopper(II) (1). To a suspension of EtOOCHLCOOEt (0.20 g, 0.35
mmol) in methanol (15 mL) was added copper(II) acetate
monohydrate (0.16 g, 0.78 mmol). After stirring for 30 min the
dark-green solution was allowed to stand at 25 °C to evaporate slowly.
After 3 days, green crystals formed were filtered off, dried in vacuo
overnight, and stored under argon atmosphere. Yield: 0.11 g, 37%.
Anal. Calcd for C33H31Cu2N5O9·1.5H2O (Mr = 795.72): C, 49.81; H,
4.31; N, 8.80. Found: C, 49.57; H, 4.30; N, 8.64. ESI-MS (methanol):
positive m/z 604 unidentified, 648 [1 − (HOAc) − (OAc)]+, 680 [1
− (OAc)2 + (CH3O)]+. UV−vis (methanol), λmax (ε, M−1 cm−1): 235
(60 800), 272 (41 000), 296 (24 540), 354 (18 900), 420 sh (20 800),
441 (22 700). ATR-IR, selected bands, cm−1: 1737, 1583, 1540, 1385,
1217, 1028. X-ray diffraction-quality single crystals were picked from
the reaction vessel prior to filtration.
1·3CH3OH
2·2CH3OH
empirical formula
fw
C36H43Cu2N5O12
864.83
C35H39N5O11OZn2
836.45
space group
a [Å]
P−1
P−1
11.1929(5)
11.3582(5)
15.4454(7)
71.745(2)
76.682(3)
81.086(2)
1807.32(14)
2
10.7024(5)
11.6277(5)
15.4646(8)
99.404(3)
105.532(3)
94.840(3)
1812.59(15)
2
b [Å]
c [Å]
α [deg]
β [deg]
γ [deg]
V [Å3]
Z
λ [Å]
0.71073
0.71073
ρcalcd [g cm−3
]
1.589
1.533
Di(μ-acetato-κ2O,O′)-(2-((3-((2-(11H-indolo[3,2-c]quinolin-6-
yl-κN5)hydrazono-κN13)methyl)-5-methyl-2-oxidobenzyl-κO1)-
(2-methoxy-2-oxoethyl-κO5)amino κN23)acetato-κO2)dizinc(II)
(2). To a suspension of EtOOCHLCOOEt (0.14 g, 0.25 mmol) in
methanol (15 mL) was added zinc(II) acetate dihydrate (0.12 g, 0.57
mmol). After stirring for 30 min the yellow solution was allowed to
stand at 25 °C to evaporate slowly. After 4 days cold pentane was
added and the mixture allowed to stand at 4 °C for 3 h. The yellow
precipitate formed was filtered off, dried in vacuo overnight, and stored
under argon atmosphere. Yield: 0.07 g, 33%. Anal. Calcd for
C33H31N5O9Zn2·CH3OH·H2O (Mr = 822.46): C, 49.65; H, 4.53; N,
8.52. Found: C, 49.96; H, 4.35; N, 8.25. 1H NMR 500.13 MHz
(DMSO-d6, δH, ppm): 12.33−11.60 (bs, 2H, N11, N12), 8.62−6.71
(bm, 11H, C1−4, 7−10, 14, 18, 20), 4.00−3.53 (bm, 9H, C22, 24, 26,
28), 2.22 (s, 3H, C21), 1.88 (bs, 6H, CH3COO). ESI-MS (methanol),
positive: m/z 668 [2 − (OAc)2 − (CH3) + (CH3OH)]+, 682 [2 −
(OAc)2 + (CH3O)]+, 710 [2 − (OAc)]+, 724 unidentified, 784
unidentified. UV−vis (methanol), λmax (ε, M−1 cm−1): 230 (44 400),
258 (45 700), 290 (27 900), 309 (31 000), 330 (17 900), 346 (18
200), 394 (18 900). ATR-IR, selected bands, cm−1: 1744, 1706, 1583,
1407, 1216, 1012. X-ray diffraction-quality single crystals were picked
from the reaction vessel prior to addition of pentane.
cryst size [mm3]
0.20 × 0.10 × 0.02
120(2)
0.15 × 0.15 × 0.10
120(2)
T [K]
μ [mm−1
]
1.249
1.533
a
R1
0.0418
0.0485
b
wR2
0.1242
0.1420
c
GOF
1.071
1.084
a
b
2
2
R1 = Σ∥Fo| − |Fc∥/Σ|Fo|. wR2 = {Σ[w(Fo − Fc2)2]/Σ[w(Fo )2]}1/2
.
c
GOF = {Σ[w(Fo − Fc2)2]/(n − p)}1/2, where n is the number of
2
reflections and p is the total number of parameters refined.
matrix least-squares techniques. Non-hydrogen atoms were refined
with anisotropic displacement parameters, while H atoms were
inserted in calculated positions and refined with a riding model. The
following software programs were used: structure solution, SHELXS-
97; refinement, SHELXL-97;26 molecular diagrams, ORTEP;27
computer, Intel CoreDuo.
Magnetic Studies. Magnetic measurements were carried out on a
microcrystalline sample of 1 with a Quantum Design SQUID
magnetometer (MPMS-XL). Variable-temperature (2−300 K) direct
current (dc) magnetic susceptibility was measured under an applied
magnetic field of 0.1 T. All data were corrected for the contribution of
the sample holder and diamagnetism of the samples estimated from
Pascal’s constants.28,29 Analysis of the magnetic data was carried out by
fitting the χMT(T) and χM(T) thermal variations including temper-
ature-independent paramagnetism (TIP), impurity contribution (ρ),
and intermolecular interaction (zJ′)29−31 according to the expression
(eq 1)
Physical Measurements and Instrumentation. 1H, 13C, and
two-dimensional 1H−1H COSY, 1H−1H TOCSY, 1H−13C HSQC,
and 1H−13C HMBC NMR spectra were recorded on a Bruker Avance
III spectrometer (Ultrashield Magnet) in DMSO-d6 at 25 °C using
1
standard pulse programs at 500.13 (1H) and 125.76 (13C) MHz. H
and 13C NMR chemical shifts are quoted relative to the residual
solvent signals. Elemental analyses were carried out at the Micro-
analytical Service of the Faculty of Chemistry, University of Vienna.
Electrospray ionization mass spectrometry was performed on a Bruker
Esquire 3000 instrument (Bruker Daltonic, Bremen, Germany) on
samples dissolved in methanol. UV−vis spectra were recorded with an
C
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Ng2β2
3kT
supra) into 96-well plates in volumes of 100 μL/well. Depending on
the cell line, different cell densities were used to ensure exponential
growth of the untreated controls during the experiment: 1.0 × 103
(CH1), 2.0 × 103 (SW480), and 3.0 × 103 (A549) cells per well. In
the first 24 h, the cells were allowed to settle and resume exponential
growth. Then the test compounds were dissolved in DMSO, serially
diluted in medium, and added to the plates in volumes of 100 μL/well
so that the DMSO content did not exceed 1%. Due to limited
solubility of EtOOCHLCOOEt and 1, the highest concentration was
applied in volumes of 200 μL/well after replacing the original medium.
After continuous exposure for 96 h (in the incubator at 37 °C and
under 5% CO2), the medium was replaced with 100 μL/well RPMI
1640 medium (supplemented with 10% heat-inactivated fetal bovine
serum and 4 mM L-glutamine) and MTT solution (MTT reagent in
phosphate-buffered saline, 5 mg/mL) in a ratio of 6:1 and plates were
incubated for further 4 h. Then the medium/MTT mixture was
removed and the formed formazan was dissolved in DMSO (150 μL/
well). Optical densities at 550 nm were measured (reference
wavelength 690 nm) with a microplate reader (ELX880, BioTek).
The quantity of viable cells was expressed as a percentage of untreated
controls, and 50% inhibitory concentrations (IC50) were calculated
from the concentration−effect curves by interpolation. Every test was
repeated in at least three independent experiments, each consisting of
three replicates per concentration level.
χ (T)
d
d
χM(T) =
(1 − ρ) + ρ
S(S + 1)
[1−2zJ′χ (T)/Ng2β2]
+ TIP
(1)
UV−Vis Titration Studies. Complex formation was studied by
UV−vis titration of 10 and 250 μM solutions of EtOOCHLCOOEt in
methanol with 10 μL aliquots of 0.5 and 6.25 mM stock solutions of
copper(II) acetate monohydrate, respectively. One aliquot was added
at 2 min intervals followed by homogenization of the solutions as
within this period the equilibrium could be reached. An Agilent 8453
spectrophotometer was used to record UV−vis spectra in the 190−
1000 nm window. The path length was 1 cm. Stability constants and
molar absorbance spectra of the individual copper(II) complexes were
calculated by the computer program PSEQUAD.32
ESI−MS Studies. Electrospray ionization mass spectra were
recorded on an AmaZon SL ion trap mass spectrometer (Bruker
Daltonics GmbH, Bremen, Germany). Experimental data and provided
simulations were acquired using Compass 1.3 software and processed
using Data Analysis 4.0 (Bruker Daltonics GmbH, Bremen, Germany).
The experimentally obtained mass signals include a maximum standard
deviation of m/z
0.06 for each species. General instrument
parameters were set as follows: Positive-ion mode (HV −4.5 kV, RF
level 89%, trap drive 74.4, dry temperature 250 °C, nebulizer 8 psi, dry
gas 6 L/min and average accumulation time 144 μs), negative-ion
mode (HV 4.5 kV, RF level 89%, trap drive 63.8, dry temperature 250
°C, nebulizer 8 psi, dry gas 6 L/min and average accumulation time 2
ms). Samples were diluted with water:methanol (50:50) or water:-
methanol:formic acid (50:50:0.2) to a final metal concentration of 5−
10 μM and measured by direct infusion into the mass spectrometer at
a flow rate of 4 μL/min. Stock solutions of 1 and 2 in DMSO (10
mM) were prepared and stored at −20 °C in the dark. Each
compound was diluted in ammonium carbonate buffer (20 mM, pH =
7.95) to give a solution of 100 μM of each compound (with 1%
DMSO content). Furthermore, a solution containing L-histidine (His),
L-aspartic acid (Asp), L-glutamic acid (Glu), and guanosine 5′-
triphosphate (GTP) in equimolar amounts (100 μM each) and a
solution containing His, Asp, Glu, and GTP (each 100 μM) and
ascorbic acid (Asc, 400 μM) were prepared in the same buffer. Metal-
containing solutions were diluted with buffer or mixed with the
solutions containing the amino acids and Asc at equimolar ratios to
give a final metal concentration in each incubation mixture of 50 μM.
Reaction mixtures were incubated at 37 °C, and aliquots were
measured directly after mixing and after 1, 3, 5, and 24 h after 10-fold
dilution of each with water:methanol (1:1). The slightly acidic
tetramethylammonium acetate buffer (20 mM, pH = 6) was avoided
because partial release of the metal was observed. Finally, dilution with
water only resulted in a low ionization in the positive- and negative-ion
modes.
Fluorescence Microscopy. SW480 cells were seeded in medium
on coverslips in 6-well plates and allowed to settle and resume
exponential growth for 24 h. Then cells were incubated for 1−2 h with
5 μM of 2 or 10 μM of EtOOCHLCOOEt in medium. Co-staining with
ER-Tracker Red and Lyso-Tracker Red (Invitrogen) was performed
according to the manufacturer’s instructions. After staining, each slide
was washed three times in PBS. A fluorescence microscope BX40
̂
(Olympus) with F-View CCD Camera (Olympus), CellF fluorescence
imaging software (Olympus), and 60× magnification oil immersions
objective lens were used.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. Syntheses of the ligand
EtOOCHLCOOEt and the copper(II) and zinc(II) complexes 1 and
2, respectively, were carried out as shown in Scheme 1. We
prepared a potentially hexadentate nonsymmetric ligand
consisting of two chelating arms, one flexible able to provide
facial coordination to an octahedral metal ion, while the second
is rigid and provides a meridional binding. Ester functionalities
are frequently introduced into the structure of organic
molecules to improve their aqueous solubility and bioavail-
ability.33
Recently, our group reported on the conjugation of L- and D-
proline to 3-(chloromethyl)-2-hydroxy-5-methylbenzaldehyde
(B) after chloromethylation of 2-hydroxy-5-methylbenzalde-
hyde (A) (Scheme 1).34 Similarly, we reacted 3-(chlorometh-
yl)-2-hydroxy-5-methylbenzaldehyde (B) with diethyl-2,2′-
iminodiacetate and triethylamine in dry THF at room
temperature, obtaining diethyl-2,2′-((3-formyl-2-hydroxy-5-
methylbenzyl)azanediyl)-diacetate (C) as an orange oil in
excellent yield (95%). The ligand EtOOCHLCOOEt was obtained
by reacting C with 6-hydrazinyl-11H-indolo[3,2-c]quinoline
(D)19 in methanol at room temperature, again in excellent yield
(95%). Complexes 1 and 2 were synthesized in 37% and 33%
yields starting from the ligand EtOOCHLCOOEt and copper(II)
acetate monohydrate and zinc(II) acetate dihydrate, respec-
tively, in methanol at room temperature. The complexation
reaction is in both cases accompanied by hydrolysis of one ethyl
ester group and transesterification of another ethyl ester
function with formation of a new ligand MeOOCHLCOOH. Both
generated donor arms are involved in coordination to
copper(II) and zinc(II) in 1 and 2, respectively, via the
Cell Lines and Cell Culture Conditions. For cytotoxicity
determination, three different human cancer cell lines were used:
A549 (nonsmall cell lung cancer) and SW480 (colon carcinoma) from
the American Type Culture Collection (ATCC, Manassas, VA), both
kindly provided by Brigitte Marian, Institute of Cancer Research,
Department of Medicine I, Medical University Vienna, Austria, as well
as CH1 (ovarian carcinoma), established and kindly provided by the
laboratory of Lloyd R. Kelland, CRC Centre for Cancer Therapeutics,
Institute of Cancer Research, Sutton, U.K. Cells were grown as
adherent monolayer cultures in 75 cm2 culture flasks (StarLab,
CytoOne) in Minimal Essential Medium supplemented with 10%
heat-inactivated fetal bovine serum (Invitrogen), 1 mM sodium
pyruvate, 1% (v/v) nonessential amino acids (from 100× ready-to-use
stock), and 4 mM L-glutamine but without antibiotics at 37 °C under a
moist atmosphere containing 5% CO2 and 95% air. All cell culture
media and reagents were purchased from Sigma-Aldrich Austria unless
indicated otherwise.
Cytotoxicity Assay. Cytotoxicity was determined by the
colorimetric MTT assay (MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide) as described previously.20 Briefly,
cells were harvested by trypsinization and seeded in medium (vide
D
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deprotonated carboxylate group and the carbonyl oxygen of the
methyl ester group (see Scheme 1, Figures 1 and 2).
indicate that the ligand adopts a configuration with an exocyclic
C6N12 double bond. Moreover, ESI mass spectra of 1 and 2
in methanol showed peaks that confirmed formation of dimetal
complexes. The most abundant peaks at m/z 680 and 682 for 1
and 2, correspondingly, were assigned to [1/2 − (OAc)2 +
(CH3O)]+.
UV−vis spectra of EtOOCHLCOOEt, 1, and 2 in methanol are
depicted in Figure S1 (Supporting Information). Metal
coordination led to pronounced changes in the visible range
of the ligand spectrum, namely, to evolution of an absorption
band at ca. 400 nm for 2 and formation of a broad charge-
transfer band at 440 nm for 1.
X-ray Crystallography. Results of X-ray diffraction studies
of 1·3CH3OH and 2·2CH3OH shown in Figures 1 and 2,
respectively, confirm formation of dinuclear complexes with the
two copper(II) ions and zinc(II) ions bridged by the phenolate
oxygen and two exogenous μ2-η1:η1 acetato ligands.35 Both
copper(II) ions in 1 are distorted square pyramidal with τ =
0.2736 for Cu1 with the bridging phenolate oxygen O1 in an
apical position and tertiary amine N23, atoms O6 and O8 of
the two bridging acetates, and one aminoacetate O2 in the basal
plane. We do not describe the coordination environment
around Cu1 as octahedral, since the interaction between Cu1
and O5 of the dangling methyl ester group is extremely weak
(Cu1···O5 2.946(2) Å). For Cu2 a distorted square-pyramidal
coordination geometry (τ = 0.22) was realized with a bridging
phenolate oxygen O1, quinoline nitrogen N5, hydrazinic
nitrogen N13, one oxygen atom O9 of bridging acetate in a
basal plane, and another bridging acetate oxygen atom O7 in
apical position.
Figure 1. ORTEP view of [Cu2(MeOOCLCOO)(CH3COO)2] with
thermal ellipsoids drawn at the 50% probability level. Selected bond
distances (Angstroms) and bond angles (degrees): Cu1−O1 2.323(2),
Cu1−O2 1.937(2), Cu1−O6 1.946(2), Cu1−O8 1.936(2), Cu1−N23
2.093(3), Cu1···O5 2.946(2), Cu2−O1 1.913(2), Cu2−O7 2.194(2),
Cu2−O9 2.002(2), Cu2−N5 2.038(3), Cu2−N13 1.956(3), Cu1−
O1−Cu2 101.47(10).
Unlike 1, the coordination environments of zinc(II) ions in 2
differ from each other. Zn1 has an octahedral environment
comprised of the bridging phenolate oxygen O1, tertiary amine
donor N23, methyl ester oxygen O5, and atom O6 of the
bridging acetate in equatorial positions and two oxygen atoms,
one aminoacetate O2, and a second O8 of a bridging acetate in
apical positions. Zn2 in contrast to Cu2 shows a more
pronounced tendency toward a trigonal-bipyramidal coordina-
tion geometry (τ = 0.47) of the same donor atoms. Cu2 lies in
the mean plane through Cu2N5C6N12N13 in 1, while Cu1
comes out from this plane by 1.307 Å. In 2 the deviation of Zn1
from the mean plane through Zn2N5C6N12N13 is markedly
smaller (0.820 Å), while distortion from planarity of the
indoloquinoline moiety is more evident than in 1. The bridging
of copper(II) ions via phenolate oxygen results in distinct
Cu1−O1 and Cu2−O1 bond distances. The difference between
them (0.39 Å) is larger than in other nonsymmetrically μ-
phenoxido bridged dicopper(II) complexes, in which the two
copper(II) ions in addition are bridged by at least one
exogenous μ2-η1:η1 acetato group.37,38 The Cu1−O6 bond
distance is markedly shorter than Cu2−O7, and Cu1−O8 is
also shorter than Cu2−O9 (see caption to Figure 1). The
Cu1···Cu2 distance in the complex is 3.2897(6) Å, which is
comparable with Cu···Cu distances of 3.297(3)39 and 3.263(2)
Å40 in dicopper(II) complexes with symmetric dinucleating
ligands, containing a di-μ-acetato-μ-phenolatodicopper(II)
core.
Figure 2. ORTEP view of [Zn2(MeOOCLCOO)(CH3COO)2] with
thermal ellipsoids drawn at the 50% probability level. Selected bond
distances (Angstroms) and bond angles (degrees): Zn1−O1 2.057(3),
Zn1−O2 2.105(3), Zn1−O6 2.004(3), Zn1−O8 2.079(3), Zn1−N23
2.149(4), Zn1−O5 2.322(3), Zn2−O1 2.027(3), Zn2−O7 1.991(3),
Zn2−O9 1.993(3), Zn2−N5 2.087(4), Zn2−N13 2.097(4), Zn1−
O1−Zn2 103.86(14).
The ligand EtOOCHLCOOEt and its zinc(II) complex 2 have
been characterized by one- and two-dimensional NMR
spectroscopy, ESI mass spectrometry, elemental analysis,
UV−vis, and ATR-IR spectroscopy, while copper(II) complex
1 was studied by magnetic susceptibility measurements, ESI
mass spectrometry, and optical spectroscopy. Additionally, both
complexes have been characterized by X-ray crystallography.
1H and 13C NMR spectral data of intermediate C, ligand
EtOOCHLCOOEt, and zinc(II) complex 2 along with their
assignments are given in the Experimental Section. The
The Zn1−O1 bond distance is only slightly longer than
Zn2−O1, as also observed in other complexes with nonsym-
metrical dinucleating ligands with a di-μ-acetato-μ-
phenolatodizinc(II) core.41 The Zn1−O6 bond distance is
only slightly longer than Zn2−O7, while the difference between
Zn1−O8 and the shorter bond Zn2−O9 is more pronounced
1
presence of a proton at N5 in the H NMR spectra and the
chemical shift of neighboring C6 in the 13C NMR spectra
E
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2NgC2uβ2
TkB
(see caption to Figure 2). The interaction between Zn1 and O5
of the dangling methyl ester group is markedly stronger than
comparable interaction in 1. The Zn1···Zn2 distance in the
complex is at 3.2154(7) Å, which is similar to the Zn···Zn
distance of 3.29(1) Å41a in a dizinc(II) complex with a
nonsymmetrical hybrid ligand.
Magnetic Properties. The magnetic behavior of a
polycrystalline sample of 1·3CH3OH in the temperature
range 2−300 K in a field of 0.1 T is shown in Figure 3. The
1
χd =
×
3 + e−2J/k T
(3)
B
The fitting procedure results in an excellent agreement between
the experimental data and the calculated curve (R = 1.4 × 10−6;
Figure 3). The parameters extracted from the fit are J = 3.49(3)
cm−1, g = 2.24(1), and zJ′ = −0.08(1) cm−1 and correspond to
ferromagnetic interaction between copper(II) ions. The
temperature-independent paramagnetism (TIP) and impurity
contribution (ρ) have values close to zero, and both were fixed
at zero in the final fit. The presence of ferromagnetic
interaction was confirmed by magnetization measurements at
low temperature (see inset picture in Figure 3). The fitting of
magnetization vs field using the Brillouin function indicates the
presence of spin ground state S = 1 (g = 2.203(2)) in 1·
3CH3OH, which is consistent with the results obtained from
analysis of the temperature dependence of magnetic suscept-
ibility.
The nature of magnetic interaction in dinuclear copper(II)
complexes has been extensively studied from both theoretical
and experimental points of view.43−51 The magnetic interaction
in 1·3CH3OH occurs via three bridges: two μ2-η1:η1 acetato
ligands and one bridging phenolate.
According to the literature,47 the acetate bridges mediate the
antiferromagnetic interactions, while the phenolate bridge in
dinuclear copper(II) complexes can promote both antiferro-
magnetic as well as ferromagnetic interactions. The character of
magnetic interaction depends on geometrical features,
especially on the Cu−O−Cu angle α, out-of-plane deviation
angle φ (see Figure 4), and torsion angle Cu−O−Cu−O. For α
angles < 99° and φ angles > 30°, a strong ferromagnetic
interaction can be expected.47 In the case of 1·3CH3OH with α
= 101.47° and φ = 30.04°, the presence of a weak
ferromagnetic interaction (J = 3.49 cm−1) is justified. We can
conclude that due to the out-of-plane deviation of the phenol
group relative to Cu−O−Cu plane the resulting magnetic
interaction between the triple-bridged Cu(II) ions is weakly
ferromagnetic. Comparable weakly ferromagnetic interactions
were reported for other dinuclear copper(II) complexes with
two50,52 or three different bridges.51,53−57
Complex Formation Studies. To elucidate whether the
two binding sites in EtOOCHLCOOEt show different affinities to
copper(II), complex formation was studied for 1 via UV−vis
titrations of the ligand EtOOCHLCOOEt at two different
concentrations with copper(II) acetate monohydrate in
methanol at room temperature (Figures 5 and S2, Supporting
Information).
Figure 3. Plots of χMT vs T and magnetization vs H (inset) at 2 and 3
K for 1·3CH3OH. Solid lines correspond to the best fit with
parameters quoted in the text.
value of χMT is 0.952 cm3 K mol−1 at 300 K. This value is
slightly higher than the expected χMT value (0.750 cm3 K
mol−1) for two noninteracting copper(II) ions (d9, g = 2.0, S =
1/2). The value of χMT continuously increases with decreasing
temperature and reaches a value of 1.174 cm3 K mol−1 at 3 K.
This behavior suggests the presence of ferromagnetic
interactions in 1·3CH3OH. According to X-ray diffraction
data, complex 1·3CH3OH has a dinuclear structure, in which
the two copper(II) ions are connected by a phenolate oxygen
atom and two bidentate bridging acetato ligands (Figure 1).
Therefore, the magnetic behavior can be analyzed by using the
classical spin Hamiltonian (eq 2):29,30,42
H = −2JS1S2
(2)
where J is the exchange coupling constant and S1 = S2 = 1/2.
In this case, the Van Vleck equation leads to the following
analytical expression (eq 3)
Development of a broad charge-transfer band at ca. 440 nm
was observed upon addition of up to ∼1.5 mol equiv of
Figure 4. Coordination core in 1·3CH3OH showing the angles α and φ.
F
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release over time. Products of ester hydrolysis are detected
directly after dissolving the compounds in buffer, and the ester
is quantitatively hydrolyzed within 24 h. The major
thermodynamic products after this period correspond to ions
[M2(L−Me)(OH) − H+]− and [M(L−Me) − H+]−, where M
= Cu or Zn and L = MeOOCLCOO, detected in the negative-ion
mode (Figure 7). The latter mass signal suggests that release of
Figure 5. UV−vis absorbance spectrum of the ligand EtOOCHLCOOEt
(dashed trace) and its changes by addition of copper(II) acetate
monohydrate (solid traces) in methanol (cL = 10 μM; cCu = 0−22.5
μM; T = 298 K; l = 1 cm). (Inset) Calculated molar absorption spectra
of the copper(II) complexes.
copper(II). Then a small shift of λmax occurred (Figure 5).
Characteristic spectral changes have also been registered in the
range of the d−d transitions (Figure S2, Supporting
Information). A wide band with λmax at 664 nm overlapped
partly with the charge-transfer band was seen upon addition of
copper(II). This absorption band is slightly red shifted upon
addition of more than 1 equiv of copper(II). On the basis of the
spectral changes in the wavelength range 230−520 nm (Figure
5), overall stability constants have been calculated for the
mono- [CuL] (log β = 7.17
species (log β = 13.13
0.08) and dinuclear [Cu2L]
0.24; log K = 5.96). The molar
absorbance spectra of the ligand, [CuL], and [Cu2L] complexes
were also calculated (Figure 5). The goodness-of-fit between
measured and calculated absorbance values is shown in Figure
6. Stability constants obtained by using the changes of the d−d
Figure 7. ESI mass spectra in negative-ion mode are shown for 1 (A)
and 2 (B) in methanol over a period of 24 h. (C) Glu- and Asc-
adducts of 1, which were only detected directly after mixing. Samples
were diluted with water:methanol (50:50).
specifically one metal can occur from both 1 and 2.
Interestingly, these signals are detected at 95% and 38%
intensities relative to [M2(L−Me)(OH) − H+]− for 1 and 2,
respectively, i.e., the Cu complex 1 releases the metal to a
greater extent. Therefore, complex 2 appears to be slightly
more stable in aqueous solution, which also seems to be of
relevance for the cytotoxicity. Additionally, 2 does not ionize in
the positive-ion mode, suggesting stable bonds between Zn ions
and the acetato ligands (Figure S3, Supporting Information).
Note that acetato complexes were not detected in the mass
spectra of 1 or 2. Furthermore, the isotopic distributions of the
major mass signals of 1 and 2 are in good agreement with
simulated patterns (Figure S4, Supporting Information). Both
complexes were exposed to mixtures containing equimolar
amounts of L-histidine (His), L-aspartic acid (Asp), L-glutamic
acid (Glu), and guanosine 5′-triphosphate (GTP). The
complexes did not react with any of the biological nucleophiles,
and similar mass spectra were observed compared to solutions
containing only the respective metal. Addition of 4 equiv of
ascorbic acid (Asc) to the amino acids resulted in transient
formation of Glu and Asc adducts with 1 in a small amount;
however, they were only detected immediately after mixing
(Figure 7C) and absent for 2. Free ascorbate was consumed
within 1 h but had no impact on the overall reactivity of the
complexes.
Figure 6. Measured and calculated (dashed lines) absorbance values at
382 ( ) and 440 nm (×) at various EtOOCHLCOOEt -to-copper(II)
⧫
ratios (cL = 10 μM; cCu = 0−22.5 μM; T = 298 K; l = 1 cm, methanol).
transition bands were in good agreement with those obtained
by monitoring the charge-transfer band within 0.2 log unit.
Stepwise formation constant of the [Cu2L] species is merely ∼1
log unit lower than that of the [CuL] showing the overlapping
binding of the metal ions. Therefore, it can be concluded that
both binding sites in ligand EtOOCHLCOOEt coordinate with a
similar affinity and no preference for either of them can be
perceived.
ESI-MS Studies. The stability of complexes 1 and 2 in
aqueous solution and their reactivity toward small biomolecules
was studied by ESI mass spectrometry (ESI-MS) since it
proved to be effective for characterizing also complex
metallodrug interactions with biomolecules.58−60 Both com-
plexes display a very similar aqueous solution behavior, which is
characterized by ester hydrolysis of the ligand and partial metal
G
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An interesting feature of both compounds is their ability to
release a metal ion also in a pH-dependent manner (Figure S5,
Supporting Information). The samples incubated at pH = 7.95
for 24 h displayed only partial metal release. Lowering the pH
of this incubation solution by dilution with 0.1% formic acid
resulted in immediate and quantitative release of one metal
from both dimetallic complexes. It is suggested that the
carboxylates are prone to protonation under these conditions,
leading to release of the coordinated metal.
Fluorescence Properties. Fluorescence spectra of
EtOOCHLCOOEt and 2 were recorded in HEPES-buffered
solutions (20 mM; pH = 7.4) with a 1% (v/v) content of
DMSO (Figure S6, Supporting Information). Fluorescence
excitation spectra (λem = 470 nm) were measured in the range
between 260 and 460 nm and emission spectra (λex = 395 nm)
in the range from 410 to 710 nm. The emission maximum of
the ligand was observed at 532 nm. Coordination to zinc(II)
led to a blue shift of the emission band by 54 nm, with the
maximum at 466 nm in the spectra of 2. EtOOCHLCOOEt was
found fluorogenic, as excitation and emission spectra strongly
increased in intensity upon binding to zinc(II).
Fluorescence Microscopy. On the basis of the fluo-
rescence properties of EtOOCHLCOOEt and dizinc(II) complex 2,
their subcellular localization was studied by fluorescence
microscopy in human cancer cells including their colocalization
with organelle-specific dyes. For visualization of the compounds
in live SW480 cells, the U-MWU2 filter (Olympus Japan,
excitation filter BP330-385, emission filter BA420) was used,
while the costaining dyes were recorded using the U-MWG2
filter (Olympus Japan, excitation filter BP510-550, emission
filter BA590). The compounds do not show interference in the
U-MWG2 channel, and autofluorescence of the cells was not
observed with the used filters. Microscopic images of cells
treated with EtOOCHLCOOEt and 2, as shown in Figure 8,
Cytotoxicity in Cancer Cells. The cytotoxicity of
EtOOCHLCOOEt, 1, and 2 was determined by the MTT assay
in three human cancer cell lines, namely, A549 (nonsmall cell
lung carcinoma), CH1 (ovarian carcinoma), and SW480 (colon
adenocarcinoma), all yielding IC50 values in the micromolar
concentration range (Table 2). Values for a simple copper(II)
salt, CuCl2, are given for comparison.
Table 2. Cytotoxicity of Ligand EtOOCHLCOOEt, Complexes 1
and 2 in Three Human Cancer Cell Lines
a
IC50 (μM), 96 h
b
EtOOCHLCOOEt
28
2.2 0.3
16
1
2
CuCl2·2H2O
A549
1
29
6.8 1.4
22
4
12
1
153
43
8
CH1
1.6 0.4
7.8 0.3
3
SW480
2
2
>160
a
Fifty percent inhibitory concentrations (means standard deviations
from at least three independent experiments), as obtained by the MTT
b
assay using exposure times of 96 h. Taken from ref 12.
Figure 8. Fluorescence microscopy images of live SW480 cells. Cells
were costained with 10 μM of EtOOCHLCOOEt (A) or 5 μM of 2 (B)
and ER-Tracker Red (500 nM) and Lyso-Tracker Red (1 μM),
respectively. Magnification of areas marked by squares are shown as
insets. Scale bars are 20 μm.
CH1 is the most sensitive cell line to all tested compounds,
whereas A549, a more chemoresistant cell line equipped with
multidrug-resistance-mediating proteins,61 is the least sensitive
one, with IC50 values up to 13 times higher than in CH1 cells.
Whereas complexation with copper(II) has either little effect on
cytotoxicity (A549, SW480 cells) or yields 3-fold decreased
potency (CH1 cells), complexation with zinc(II) results in
about 2-fold enhancement of cytotoxicity, compared to the
metal-free ligand EtOOCHLCOOEt in all three cell lines. In
comparison to the dicopper(II) complex 1, the dizinc(II)
complex 2 is up to three times more active in SW480 and four
times more active in CH1 cells (see also Figure S7, Supporting
Information). This might be directly related to the lower
tendency of 2 to release a metal in aqueous media compared to
1 as observed in the ESI-MS experiments. On the basis of these
observations, it can be concluded that complexation to zinc(II)
results in higher cytotoxicity but also better solubility in
biocompatible media compared to the metal-free ligand as well
as copper(II) complex 1. Cytotoxic potency of a simple
copper(II) salt, CuCl2, is lower, with IC50 values being at least
five times higher than those of complex 1.
revealed localization of fluorescence in diffuse voluminous as
well as distinct small cytoplasmic structures but no discernible
uptake into the nucleus. The highest accumulation matches
with both the ER-Tracker Red and the Lyso-Tracker Red
staining, suggesting that the endoplasmic reticulum as well as
lysosomes are potential target compartments of EtOOCHLCOOEt
or that lysosomes are involved in sequestration and/or
detoxification of the compound. The same may apply to 2,
provided that the complex is sufficiently stable throughout its
passage through the cell, as it cannot be ruled out that the
fluorescence distribution originates from dissociated ligand
molecules.
CONCLUSION
■
Condensation of 6-hydrazinyl-11H-indolo[3,2-c]quinoline with
diethyl-2,2′-((3-formyl-2-hydroxy-5-methylbenzyl)azanediyl)-
diacetate afforded a new nonsymmetric dinucleating ligand
H
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EtOOCHLCOOEt with increased aqueous solubility and fluores-
cence properties. Complexes 1 and 2 were obtained upon
treatment of the ligand with 2 equiv of Cu(CH3COO)2·H2O
and Zn(CH3COO)2·2H2O in methanol, respectively. Complex-
ation reaction in both cases is accompanied by hydrolysis of
one ethyl ester group and transesterification of another ethyl
ester function with formation of MeOOCHLCOOH. Dinuclear
structure in 1·3CH3OH and 2·3CH3OH is supported by three
bridges: two acetato ligands and one phenolato bridge from
nonsymmetric MeOOCHLCOOH ligand. The temperature depend-
ence and field dependence magnetic measurements for 1·
3CH3OH indicate a weak ferromagnetic interaction (J = 3.49
cm−1) between copper(II) ions. All three compounds show
respectable antiproliferative activity in human cancer cell lines
(A549, CH1, SW480) with IC50 values in the low micromolar
concentration range. It seems that the increased resistance of 2
toward metal release in aqueous solution compared to 1 may be
responsible for the higher cytotoxicity. Localization of
EtOOCHLCOOEt and 2 in cytoplasmic structures has been
found by fluorescence microscopy, suggesting that the
endoplasmic reticulum as well as the lysosomes can be
potential target compartments of these compounds.
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ASSOCIATED CONTENT
311.
■
(13) Dobrov, A.; Arion, V. B.; Kandler, N.; Ginzinger, W.; Jakupec,
S
* Supporting Information
́
M. A.; Rufinska, A.; Graf von Keyserlingk, N.; Galanski, M.; Kowol, C.;
Synthesis and NMR characterization of intermediates, UV−vis
spectra of EtOOCHLCOOEt, 1, and 2 in methanol, UV−vis
absorbance spectrum of the ligand EtOOCHLCOOEt and its
changes by addition of copper(II) in methanol, ESI mass
spectra of 1 and 2, details on isotopic distributions of the major
detected mass signals of 1 and 2 with simulations, mass spectra
measured at lower pH, fluorescence excitation and emission
spectra of EtOOCHLCOOEt and 2 at physiological pH,
concentration-effect curves of EtOOCHLCOOEt and complexes 1
and 2 in human cancer cell lines, experimental masses and their
assignment, crystallographic data in CIF format. This material is
available free of charge via the Internet at http://pubs.acs.org.
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̈
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(19) Filak, L. K.; Muhlgassner, G.; Bacher, F.; Roller, A.; Galanski,
̈
M.; Jakupec, M. A.; Keppler, B. K.; Arion, V. B. Organometallics 2011,
30, 273−283.
AUTHOR INFORMATION
■
(20) Primik, M. F.; Goschl, S.; Jakupec, M. A; Roller, A.; Keppler, B.
̈
Corresponding Author
*E-mail: vladimir.arion@univie.ac.at.
K.; Arion, V. B. Inorg. Chem. 2010, 49, 11084−11095.
(21) Filak, L. K.; Goschl, S.; Hackl, S.; Jakupec, M. A.; Arion, V. B.
̈
Notes
Inorg. Chim. Acta 2012, 393, 252−260.
The authors declare no competing financial interest.
(22) Filak, L. K.; Goschl, S.; Heffeter, P.; Ghannadzadeh Samper, K.;
̈
Egger, A. E.; Jakupec, M. A; Keppler, B. K.; Berger, W.; Arion, V. B.
Organometallics 2013, 32, 903−914.
ACKNOWLEDGMENTS
■
(23) Huisman, M.; Koval, I. a.; Gamez, P.; Reedijk, J. Inorg. Chim.
Acta 2006, 359, 1786−1794.
We thank Alexander Roller for collection of X-ray data for 1
and 2. This work was financially supported by the University of
Vienna within the doctoral program “Initiativkolleg Functional
Molecules” (IK I041-N) and the Austrian Science Fund
(Project no. P22339-N19).
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DEDICATION
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Dedicated to Prof. Dr. Manfred T. Reetz on the occasion of his
70th birthday.
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