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Naphthoquinones of natural origin: Aqueous chemistry and coordination to half-sandwich organometallic cations
Journal of Organometallic Chemistry xxx (xxxx) 121070
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Journal of Organometallic Chemistry
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journal homepage: http://ees.elsevier.com
Naphthoquinones of natural origin: Aqueous chemistry and coordination to
half-sandwich organometallic cations
János P. Mészáros a ,b ,1 , Heiko Geisler c ,1 , Jelena M. Poljarević a ,d , Alexander Roller c , Maria S. Legina c ,
Michaela Hejl c , Michael A. Jakupec c ,e , Bernhard K. Keppler c ,e , Wolfgang Kandioller c ,e , Éva A. Enyedy a ,b ,∗
a
Department of Inorganic and Analytical Chemistry, Interdisciplinary Excellence Centre, University of Szeged, Dóm tér 7, H-6720, Szeged, Hungary
MTA-SZTE Momentum Functional Metal Complexes Research Group, University of Szeged, Dóm tér 7, H-6720, Szeged, Hungary
c
Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 42, A-1090, Vienna, Austria
d Faculty of Chemistry, University of Belgrade, Studentski trg. 12-16, 11000, Belgrade, Serbia
e Research Cluster “Translational Cancer Therapy Research”, University of Vienna, Währinger Str. 42, A-1090, Vienna, Austria
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b
ARTICLE INFO
ABSTRACT
Article history:
Received 28 October 2019
Received in revised form 5 December 2019
Accepted 6 December 2019
Available online xxx
Half-sandwich organometallic complexes featuring Ru(II), Os(II) and Rh(III) metal centers and naturally occurring bidentate 2-hydroxy-[1,4]-naphthoquinone ligands (lawsone and phthiocol) have been synthesized and characterized in both solid state and solution phase by analytical, spectroscopic, electrochemical and single crystal
X-ray diffraction techniques. Comparative studies revealed the influence of the respective metal center (Ru, Os,
Rh), leaving group (Cl, Br, I) and arene (p-cymene, toluene, pentamethylcyclopentadienyl), as well as the naphthoquinone ligand on the structural properties and solution speciation. Additionally, cytotoxicity was tested in
SW480, CH1/PA-1 and A549 human cancer cell lines showing a broad range of IC50 values.
Keywords
Solution stability
X-ray crystal structures
Cytotoxicity
Natural ligands
Half-sandwich complexes
Abbreviations
BOLD-100 sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)], KP-1339
CV
cyclic voltammetry
deferiprone 1,2-dimethyl-3-hydroxy-pyridin-4(1H)-one
DMF
dimethylformamide
Hlaw
lawsone, 2-hydroxy-[1,4]-naphthoquinone
Hphth
phthiocol, 3-methyl-2-hydroxy-3-methyl-[1,4]-naphthoquinone
maltol 3-hydroxy-2-methyl-pyran-4(1H)-one
menadione 3-methyl-[1,4]-naphthoquinone
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
NAMI-A trans-[tetrachlorido(DMSO)(imidazole)ruthenate(III)
ROS
reactive oxygen species
∗ Corresponding
author. Department of Inorganic and Analytical Chemistry,
Interdisciplinary Excellence Centre, University of Szeged, Dóm tér 7, H-6720, Szeged,
Hungary.
E-mail address: enyedy@chem.u-szeged.hu (É.A. Enyedy)
1
Both authors contributed equally to this work.
https://doi.org/10.1016/j.jorganchem.2019.121070
0022-328/© 2019.
TGA
© 2019
thermogravimetric analysis
1. Introduction
Due to the reoccurring limitations with well-established Pt(II) drugs
(e.g. cisplatin) in cancer treatment, much research effort has been focused on the search for other metallodrugs with different activity profiles and lower toxicity [1,2]. In this context, metal center variation
brought to light the possible suitability of Ru based drugs. At this time
Ru(III) compounds, as well as Ru(II) “piano-stool” complexes are extensively investigated. Two well-known examples of Ru(III) drug candidates are BOLD-100 (sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)], formerly KP-1339 or IT-139), which is about to
enter clinical phase II studies, and NAMI-A (trans-[tetrachlorido(DMSO)(imidazole)ruthenate(III)]). Both of these complexes highlight the importance of the metal's coordination sphere (Fig. 1). While
BOLD-100 features two indazole ligands and is active against primary
tumors as well as metastases, its structurally related congener NAMI-A
only shows activity against metastases [3,4].
On the other hand, organometallic Ru(II) complexes often feature a
stabilizing arene moiety, and mono-, or bidentate ligands, as well as a
leaving group. The name of these so-called “piano-stool” complexes is
2
J.P. Mészáros et al. / Journal of Organometallic Chemistry xxx (xxxx) 121070
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Herein we report the synthesis, characterization, detailed solution
stability and complex formation properties of six new “piano-stool”
complexes. These organometallics feature either 3-methyl-2-hydroxy-3-methyl- [1,4]-naphthoquinone (phthiocol, Hphth) or its
non-methylated analogue 2-hydroxy- [1,4]-naphthoquinone (lawsone,
Hlaw) as O,O-chelates for Ru(II)-, Os(II)- and Rh(III)-arene organometallic ions. Additionally, cytotoxicity tests in human cancer cell lines were
conducted in order to assess the anticancer potency of these compounds.
2. Results and discussion
2.1. Synthesis
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Fig. 1. Structural formulae of Ru(III) and Ru(II) drug candidates, which are currently undergoing or have undergone (pre)-clinical trials.
Lawsone is commercially available, phthiocol was synthesized in
a two-step synthesis, starting from commercially available 3-methyl[1,4]-naphthoquinone (menadione), according to literature with minor
modifications [21,22]. Firstly, an epoxide intermediate was formed, using aqueous hydrogen peroxide solution (30%). Ring opening reaction
was performed with concentrated sulfuric acid and silica gel in THF and
provided good yields (72%) over two steps (Scheme S1).
The phthiocol-based Ru(II)-p-cymene complex (KP2048) showed
promising anticancer activity in vitro and in vivo experiments [14].
Within this work, we wanted to investigate the effect of different metal
centers (Ru(II), Os(II), Rh(III)), halide leaving groups (Cl−, Br−, I−),
arenes (p-cymene, toluene, C5Me5-) and ligands on the solution behavior
and cytotoxic potency.
The desired complexes (1–7) were obtained by the deprotonation
of the 2-hydroxy-naphthoquinone (phthiocol or lawsone) with sodium
methoxide in dry methanol, followed by the addition of the respective dimeric metal precursor ([MX2(arene)]2) (see Scheme 1). Following the standard purification reported in literature [16], the complexes were isolated in moderate-to-good yields. Two different methods were applied to obtain the desired complexes. In method A: the
starting materials were reacted under standard conditions (20–40 °C) for
2–24 h. The other method (method B) included microwave irradiation
for 6–14 min at 40–50 °C. Both methods were used for all complexes.
However, the microwave assisted method reduced the reaction time of
complexes 2, 4 and 5 considerably. After purification, yields between 15
and 90% were obtained.
derived from the resulting geometrical form, which resembles a three
legged stool, where the arene is the seat and the other ligands are
the legs. The most advanced and extensively studied drug candidates,
RM175 and RAPTA-C (Fig. 1) are believed to have completely different modes of action. While Sadler et al. postulated that RM175 interacts
with DNA via intercalation of the biphenyl moiety and covalent binding
to nucleobases; activity of RAPTA-C is ascribed to protein interaction
[5]. However, no “piano-stool” complex has been evaluated in clinical
studies so far.
Another approach is the synthesis of Ru(II) “piano-stool” complexes
featuring bioactive natural products with anticancer properties. Many
natural compounds such as quinones and naphthoquinones are found in
plants, algae, and bacteria and used as versatile building blocks for organic synthesis of medicinal drugs [6,7]. Other members of this family
are phthiocol and lawsone, which are considered as derivatives of vitamin K (Fig. 2) [6,8,9].
Naphthoquinones readily act as reducing agents leading to the formation of radicals and reactive oxygen species (ROS) [10]. In the past,
several quinones and naphthoquinones were biologically tested and revealed cytostatic properties [6,7,11–13]. Half-sandwich organometallic
complexes containing 2-hydroxy-[1,4]-naphthoquinones as ligand scaffold have already been reported in literature with promising results
[14–16]. Furthermore, heterobimetallic compounds with a pendant ferrocene moiety have been investigated for their anticancer properties
[17,18]. Physicochemical properties (e.g. solubility, lipophilicity), biologic parameters (e.g. IC50 values, mode of action) as well as solution
equilibrium constants are crucial factors in drug development. One of
the main advantages of using “piano-stool” complexes is the possibility
to fine-tune these physicochemical properties, namely stability, redox
properties, solubility via ligand modification and variation of the metal
center or the leaving group [19,20].
Fig. 2. Structural formulae of vitamin K3 (menadione) and its naphthoquinone derivatives: phthiocol, lawsone, and lapachol (from left to right).
2.2. Crystal structures of phthiocol complexes
Single crystals of five complexes (1–3, 5–6) were obtained by liquid-liquid diffusion from dichloromethane/n-hexane (see Figs. 3 and
4). These complexes exhibit a pseudo-octahedral geometry with a ligand-to-metal ratio of 1:1. The arene, the bidentate naphthoquinone ligand (Hphth) and the monodentate halide ion are coordinated to the
metal center and adopt the characteristic “piano-stool” configuration.
Scheme 1. Synthetic pathway for complex synthesis.
3
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Fig. 3. Molecular structures of 1, 2 and 3 drawn at 50% probability level. Solvent molecules were omitted for clarity. Where both enantiomers crystallized one of them was omitted as
well.
Fig. 4. Molecular structures of 5 and 6 drawn at 50% probability level. Solvent molecules were omitted for clarity. Where both enantiomers crystallized one of them was omitted as well.
Complexes 2, 3, and 6 crystallized in the triclinic space group P1̅. Compounds 1 and 5 crystallized in the monoclinic space group P21/c.
The bond lengths between the donor atoms of phthiocol and the
metal center and bond angles are shown in Table 1. The variation of
the metal center Ru(II) (1) to Os(II) (5) has no relevant impact on the
bond lengths and angles. However, the Rh(III) complex 6 exhibits a
slight elongation of the coordination bonds, compared to the Ru(II) analog 1.
Based on these data the variation of halide ions has no relevant effect
on geometric parameters in the coordination sphere. These organometallic complexes possess a chiral metal center and both enantiomers were
found in the unit cell of complexes 2, 3 and 6.
J.P. Mészáros et al. / Journal of Organometallic Chemistry xxx (xxxx) 121070
Table 1
Selected bond lengths, bond angles and torsion angles for the phthiocol complexes: (1–3,
5–6).
1
2
Bond distances [Å]
M-Cg
1.651
M-O1
2.119(2)
M-O2
2.092(2)
M-X
2.403(1)
Bond angles [°]
O1-M-O2
76.31(8)
b
O-M-X
84.89(7)
a
b
a
3
a
5
6
a
1.646
2.123(2)
2.082(2)
2.532(4)
1.651
2.122(3)
2.085(3)
2.711(4)
1.651
2.119(3)
2.092(4)
2.394(1)
1.743
2.160(4)
2.095(5)
2.390(2)
76.39(8)
85.61(5)
76.18(9)
86.17(8)
75.60(1)
83.66(8)
76.25(2)
87.63(4)
Calculated average of the two isomers in one unit cell.
Calculated average value of O1-M-X and O2-M-X.
2.3. In vitro anticancer activity of compounds
Table 2
Cytotoxicity of ligands, their corresponding half-sandwich organometallic complexes (1,
4–7) and cisplatin and RAPTA-C for comparison in three different human cancer cell lines
using 96 h incubation time.
IC50 [μM]
a
1
4
5
6
Hphth
7
Hlaw
b
cisplatin
c
RAPTA-C
a
Investigation of solution behavior and determination of stability constants of bioactive metal complexes are key steps in drug development
processes since from speciation data the biologically active chemical
forms can be predicted. Thus this kind of information can help in deepening the understanding of pharmacokinetic properties and the mechanism of action. Currently no speciation data have been reported for
half-sandwich organometallic complexes of 2-hydroxy-[1,4]-naphthoquinones so far. Only a few studies on the time-dependent stability in
buffered solutions can be found in literature [15].
In this work we investigated systematically the aqueous solution
chemistry of metal-ligand systems containing phthiocol and
organometallic triaqua complex cation ([Ru(η6-p-cymene)(H2O)3]2+,
[Ru(η6-toluene)(H2O)3]2+ and [Rh(η5-C5Me5)(H2O)3]2+) in the presence of 0.2 M chloride ions. pH-potentiometry is not suitable for determining stability constants for these naphthoquinone complexes due to
their low water solubility and high concentration demand of the technique. Therefore UV–visible (UV–Vis) titrations were performed where
lower concentrations can be employed. Though the osmium complex 5
was synthesized and its in vitro cytotoxicity was determined, the solution chemical properties were not characterized here since the more pronounced and slower hydrolytic behavior of [Os(η6-p-cymene)(H2O)3]2+
compared
with
[Ru(η6-p-cymene)(H2O)3]2+
or
5
2+
[Rh(η -C5Me5)(H2O)3]
would make the studies fairly difficult
[27,28]. Lawsone was also involved the studies to reveal the impact of
the methyl group on the ligand scaffold on the solution chemical properties.
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The cytotoxicity of 2-hydroxy- [1,4]-naphthoquinones depends on
the nature of alkyl substituents at position 3 [13]. Cytotoxic activities of
Hphth, Hlaw and the respective complexes 1, 4–7 were determined in
human colon carcinoma (SW480), ovarian teratocarcinoma (CH1/PA-1)
and non-small cell lung carcinoma (A549) cells by means of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (Table 2). No IC50 values could be determined for complexes 2 and 3, due to the poor solubility in aqueous systems.
All tested complexes 1, 4–7 showed at least a tendency for increased cytotoxicity in the intrinsically chemo-resistant (P-glycoprotein-expressing) human cancer cell line SW480, compared to the free ligands (Hphth, Hlaw). The combination of Ru(II) and p-cymene yields a
clearer increase of cytotoxicity in all cancer cell lines employed, whereas
the other variants with a different metal center (6) or arene ligand (4)
showed only marginally increased cytotoxicity in the cell line SW480
and no enhanced activity in A549 and CH1/PA-1 cells.
Based on these data we could conclude that the tested compounds
exhibited a broad range of anticancer potency with IC50 in the range
of 15 μM to >200 μM. Generally the dimeric organometallic precursors
do not exhibit strong cytotoxicity (>100 μM [RuCl2(η6-p-cymene)]2 reported for several cancer cell lines [25,26], and both naphthoquinone
ligands showed IC50 values > 100 μM. The arene change resulted in a
loss of activity compared to the p-cymene derivative 1, as did the metal
center variation from Ru(II) to Os(II) or Rh(III). Introduction of the lawsone ligand was not beneficial with regard to cytotoxicity either, but
complex 7 was still more active than the free ligand Hlaw.
2.4. Solution chemistry
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4
A549
SW480
CH1/PA-1
47 ± 4
IC50 > 100
111 ± 23
IC50 > 200
210 ± 32
98 ± 24
157 ± 13
6.2 ± 1.2
>500
15 ± 3
87 ± 6
81 ± 18
96 ± 17
116 ± 37
86 ± 20
247 ± 17
3.3 ± 0.2
170 ± 60
31 ± 10
IC50 > 100
59 ± 12
IC50 ~ 200
129 ± 29
84 ± 15
246 ± 24
0.077 ± 0.006
65 ± 15
2.4.1. Proton dissociation processes of naphthoquinones and hydrolysis of
the organometallic cations
The pKa value is a key parameter of bioactive compounds, as it has
a large effect on the pharmacokinetic properties considering the actual
protonation state (and charge), and it also affects the pH-dependence
of the lipophilicity. with the help of these constants it is possible to
calculate the distribution of different protonated species and the average charge at a given pH. phthiocol and lawsone have one dissociable
proton, as only the hydroxyl group can lose a proton with increasing
pH. although the structures are similar, the methyl group in the third
position has a great impact on the neighboring hydroxyl group. proton dissociation constants of lawsone and phthiocol were determined
by UV–Vis titrations (I = 0.2 M KCl, Fig. s1) and deprotonation occurs
at much lower pH values in the case of lawsone (pKa (Hl) = 3.90 and
5.08 for lawsone and phthiocol, respectively, see Table 3). These data
Table 3
Proton dissociation constants (pKa (HL)) of Hlaw and Hphth and stability constants (logΚ
[M(arene)L]) of their complexes. {T = 25.0 °C, I = 0.2 M (KCl)}.
Hlaw
Hphth
Data are taken from Ref. [14].
Data are taken from Ref. [23].
c
Data are taken from Ref. [24].
b
a
b
Ru(η 6-pcymene)
Ru(η 6toluene)
Rh(η 5C5Me5)
3.45 ± 0.01
3.90 ± 0.01
3.31 ± 0.02
<3.1
0.01%
<0.01%
6.6%
4.04 ± 0.03
5.08 ± 0.01
3.78 ± 0.04
3.52 ± 0.03
0.04%
<0.01%
14.5%
a
pΚa (HL)
log Κ
a
[M(arene)L]
% of
b
[M(arene)L]
a
pΚa (HL)
log Κ
a
[M(arene)L]
% of
b
[M(arene)L]
From UV–Vis spectrophotometric titrations.
Calculated at c(M(arene)) = c(L) = 100 μM, pH = 7.40.
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2.4.2. Complex formation equilibria of naphthoquinones with
organometallic ions
Based on the crystallographic structures and literature data of other
half-sandwich Ru(II) and Rh(III) complexes with (O,O) donors, mono
complex formation is the most probable process under the applied conditions. The complexes of half-sandwich Rh and Ru complexes with
(O,O) donors usually reaches the equilibrium state rather fast
[28,32–34]. Because of the extended delocalized electron system, the
complexes of phthiocol and lawsone have absorbance in the UV–Vis region, so spectrophotometric titration is a suitable method for following the complex formation and spectra were recorded at 1:1 and 1:2
metal-to-ligand ratios.
In Fig.
5a the pH-dependent UV–Vis spectra of the
[Ru(η6-toluene)(H2O)3]2+ – lawsone (1:1) system are shown. The spectra above 590 nm (see Fig. 5a inserted diagram) give the most valuable
informa
tion: neither the metal ion nor the ligand has absorption in that region. Spectral changes here directly show the formation of the formed
metal complex. In the system with 1:2 metal-to-ligand ratio the absorbance change is relatively high making the determination of the
stability constants more precise (Table 3). In Fig. 5b the concentration distribution curves calculated with the stability constants and the
absorbance at 590 nm are shown. The maximum of the formed complex is 11.4% at pH 4.72, which demonstrates low solution stability.
With [Ru(η6-p-cymene)(H2O)3]2+ the spectral changes are similar (Fig.
S2) and the formation of the [M(arene)L] complex is detectable also
around 590–600 nm. However, with Rh(η5-C5Me5) no complex formation occurred under the used conditions. Titration of lawsone with the
organometallic ion at pH 7.4 shows no interaction between them, and
the linearity of the curve in Fig. S3 proves the lack of complex formation in the system. Only an upper limit could be provided for the stability constant of this complex in Table 3.
The
pH-dependence
of
UV–Vis
spectra
of
[Ru(η6-p-cymene)(H2O)3]2+, phthiocol and their 1:1 metal-to-ligand ratio system is shown in Fig. 6a. The detection of complex formation
in the case of phthiocol is more difficult: there is only a minor spectral change at ~600 nm with a maximum of 0.02 absorbance (see Fig.
6a inserted figure); in this region only the metal complex absorbs photons. Based on this absorbance change, the complex formation shows
a maximum at pH 5.1. Calculations approved the low stability of the
formed complex as the concentration distribution curves represent in
Fig. 6b; [M(arene)L] has a maximum with 26.7% at 100 μM concentration and at pH 5.06. With [Ru(η6-toluene)(H2O)3]2+ the spectral
changes and the extent of complex formation are even smaller than with
[Ru(η6-p-cymene)(H2O)3]2+ (shown in Fig. S4). Additionally, the absorbance at 600 nm reaches only 0.015, which makes the detection of
the complex formation even more problematic. The formation of the
half-sandwich Rh(η5-C5Me5) complex of phthiocol is also observable at
600 nm and the absorbance changes are negligible for this system (ΔA
~0.015, see Fig. 7) (see Fig. 8).
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are in good agreement with literature data, although different conditions
were used (I = 0.5 M KCl) [29,30].
Hydrolysis
of
the
[Rh(η5-C5Me5)(H2O)3]2+,
6
2+
[Ru(η -p-cymene)(H2O)3]
and
[Ru(η6-toluene)(H2O)3]2+
organometallic complex cations has been characterized in detail previously [27,28]. In these organometallic triaqua cations and (their studied complexes) the bond between the metal ion and the arene is stable under the used conditions (pH = 2.0–11.5). Increasing the pH μ-hydroxido bridged dinuclear species are formed with the formula
[(M(arene))2(OH)2]2+ and [(M(arene))2(OH)3]+ [27,28]. Although
Bíró et al. examined extensively the species formed from
[Ru(η6-p-cymene)(H2O)3]2+ in chloride ion containing medium and determined the overall stability constants for five different species, all the
titrations can be described sufficiently by only the overall stability constants determined for [(M(arene))2(OH)2]2+ and [(M(arene))2(OH)3]+
(I = 0.2 M
KCl)
[31].
These
constants
are
log
β
[(M(arene))2(OH)2] = −6.50,
−7.12,
−11.12
and
log
β
[(M(arene))2(OH)3] = −10.56,
−11.88,
−19.01
for
M(arene) = Ru(η6-toluene), Ru(η6-p-cymene) and Rh(η5-C5Me5), respectively, and these data were used in this work as well [27,28].
5
Fig. 5. a) UV–Vis absorption spectra of the [Ru(η6-tol)(H2O)3]2+ – lawsone 1:1 system at pH = 2.0–11.5. b) Concentration distribution curves calculated with the determined constants
from Table 3. Absorbance at 590 nm (○) shows the complex formation {c(lawsone) = c([Ru(η6-tol)(H2O)3]2+) = 200 μM; I = 0.2 M (KCl); T = 25.0 °C; l = 2 cm}.
Fig. 6. a) UV–Vis absorption spectra of phthiocol, [Ru(η6-p-cymene)(H2O)3]2+ and the [Ru(η6-p-cymene)(H2O)3]2+ – phthiocol 1:1 system at pH = 2.0–11.5. b) Concentration distribution curves calculated with the determined constants from Table 3. Absorbance at 600 nm (○) shows the complex formation {c(phthiocol) = c([Ru(η6-p-cymene)(H2O)3]2+) = 100 μM;
I = 0.2 M (KCl); T = 25.0 °C; l = 2 cm}.
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Fig. 7. a) Molar absorbance spectra calculated from the spectrophotometric titration of the [Rh(η5-C5Me5)(H2O)3]2+ – phthiocol system at pH = 2.0–11.5. b) Concentration distribution curves calculated with the determined constants from Table 3. Absorbance at 600 nm (□) shows the formation of [M(arene)L] complex {c(phthiocol) = c([Rh(η5-C5Me5)(H2O)3]2+) = 100 μM; I = 0.2 M (KCl); T = 25.0 °C; l = 1 cm}.
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of the method introduced by Raymond et al. is used, namely the direct comparison of pM* values [36]. The original pM is the negative
logarithm of the free metal ion concentration, while pM* is calculated
for the nonbound metal ion concentration: not only the free metal ion
but the hydroxido species are taken into account as well. The higher
pM* value shows higher stability. The calculated pM* curves are shown
in Fig. 8 and Fig. S6, illustrating the stability difference between
the complexes of 2-hydroxy-[1,4]-naphthoquinones (Table 3), 2,4-diketonates (acetylacetone), 3-hydroxy-2-methyl-pyran-4(1H)-one (maltol)
and
1,2-dimethyl-3-hydroxy-pyridin-4(1H)-one
(deferiprone)
[28,32–34]. The stability constants for the Ru(η6-toluene) – deferiprone
system were missing for this comparison, thus pH-potentiometric titrations were performed in this work to determine these data, which
are:
log
K
[M(arene)L] = 11.74 ± 0.08
and
pKa
[M(arene)L] = 9.34 ± 0.09. The trend of stability is the same independently from the type of the organometallic cations: deferiprone > maltol > acetylacetone > 2-hydroxy- [1,4]-naphthoquinones is the order
in a wide pH range.
Fig. 8. Calculated pM*-curves obtained for the [Rh(η5-C5Me5)(H2O)3]2+ ‒ (O,O) bidentate ligand systems plotted against the pH. L = lawsone (- - -); phthiocol (‒‒); acetylacetone
(-∙-∙-);
maltol
(-∙∙-∙∙-);
deferiprone
(∙∙∙∙∙∙∙)
[28,32–34]
{c(L) = c([Rh(η5-C5Me5)(H2O)3]2+) = 100 μM; T = 25.0 °C; I = 0.2 M (KCl)}.
Comparison of the stability constants for both ligands shows a clear
trend. The stability constant is the highest for Ru(η6-p-cymene) complexes, which is followed by Ru(η6-toluene) and the Rh(η5-C5Me5) complexes have the lowest stability constants (Table 3). The same tendency
has been found previously in the case of other (O,O) donor bidentate
ligands [33]. Based on the determined stability constants (Table 3) the
calculated concentration distribution curves show different characteristics for the [Rh(η5-C5Me5)(H2O)3]2+ – phthiocol (1:1) system (Fig. 7b);
namely the complex is present in a wider range (>5% at pH 4.4–8.0)
than the ruthenium analogues (>5% at pH 3.8–6.0). For the interpretation of the biological activity data, Table 3 shows the amount of the
[M(arene)L] complex formed at pH = 7.4. It can be concluded that only
the [Rh(η5-C5Me5)(phth)Cl] complex is present in the solution in more
than 10% and it is the least cytotoxic of them.
Quinones and naphthoquinones are redox active molecules and their
redox chemistry is well characterized [35]. As complex formation often
has a great impact on the ligand's redox chemistry cyclic voltammetry
(CV) was used to follow the changes of the voltammograms upon complexation in a 9:1 solvent mixture of dimethylformamide (DMF) and water due to the low solubility of the compounds in water (Fig. S5). However, variation of the metal center had only a small effect on the shape
of the voltammograms, most probably as a result of the low fraction of
complex formation under the applied conditions.
2.4.3. Comparison the stability of naphthoquinone complexes with other
(O,O) donors
To compare the stability of complexes of the same organometallic
ion formed with ligands of different pKa values, herein a modification
3. Experimental
3.1. Chemicals
All solvents were of analytical grade and used without further purification. Dry solvents were used for synthesis and stored under argon.
Lawsone, menadione (Acros Organics), RuCl3 × xH2O, RhCl3 × xH2O,
IrCl3 × xH2O, OsO4 (Johnson Matthey), α-terpinene (Alfa Aesar),
1,2,3,4,5-pentamethylcyclopentadiene (TCI Europe), deferiprone, H2O2
(30%), KCl, HCl, KOH, KH-phthalate, NaOMe, Na2CO3, KH2PO4,
NaH2PO4, Na2HPO4, MeOH, CHCl3, CH2Cl2, DMF and DMSO (Sigma
Aldrich) were purchased from commercially available suppliers. For
aqueous solutions Milli-Q water was used. The exact concentrations of
the metal precursor stock solutions were determined by pH-potentiometric titrations with the aim of Hyperquad 2013 program [37], employing the stability constants for hydroxido complexes from (I = 0.2 M
KCl) [27,28]. Stock solutions of the ligands were prepared on a
weight-in-volume basis. In the case of buffered samples, 20 mM phosphate buffer was used instead of water with pH = 7.40.
For characterization of the compounds NMR spectra (Figs. S7–13)
were recorded with a Bruker FT-NMR spectrometer Avance III™
500 MHz with an UltraShieldPlus magnet at 25 °C. The measurement
frequency for proton NMR (1H) was 500.10 MHz and for carbon NMR
(13C{1H}) 125.75 MHz CDCl3 was used as solvent. Elemental analysis
for carbon, hydrogen, nitrogen, sulfur and oxygen was performed at the
Microanalytical Laboratory of the University of Vienna with Eurovector EA 3000 CHNS–O elemental analyzer (2009) equipped with a high
temperature pyrolysis furnace (HT, Hekatech, Germany, 2009). Thermogravimetric analysis (TGA) was carried out on a TGA/SDTA851e in
J.P. Mészáros et al. / Journal of Organometallic Chemistry xxx (xxxx) 121070
7
electrodes: one was the working and another was the counter electrode,
the reference electrode was an Ag/AgCl/(1 M) KCl electrode. The system was calibrated for 0.01 M ferrocene before every experiment and
gave a potential at +0.688 V vs. NHE.
3.2. pH-potentiometric measurements
3.5. Synthesis phthiocol (Hphth)
pH-potentiometric measurements determining concentrations of
[Rh(η5-C5Me5)(H2O)3]2+,
[Ru(η6-p-cymene)(H2O)3]2+
and
6
2+
[Ru(η -toluene)(H2O)3] were carried out at 25.0 °C ± 0.1 °C in water
and at a constant ionic strength of 0.2 M KCl. The titrations were performed with a carbonate-free KOH solution (0.20 M). The exact concentrations of HCl and KOH solutions were determined by pH-potentiometric titrations. An Orion710A pH-meter equipped with a Metrohm electrode (type 6.0234.100) filled with 3 M KCl and a Metrohm 665 Dosimat burette was used. The volume resolution of the burette is 0.001 mL
and its precision is 0.002 mL. The electrode system was calibrated to
the pH = -log [H+] scale by means of blank titrations (strong acid vs.
strong base: HCl vs. KOH), as suggested by the Irving method [38].
The water ionization constant (pKw) was determined as 13.76 ± 0.01 at
25.0 °C ± 0.1 °C, I = 0.2 M (KCl) [39]. The reproducibility of the titration points included in the calculations was within 0.005 pH units. The
pH-potentiometric titrations were performed in the pH range between
2.0 and 11.5. The initial volume of the samples was 5.0 mL. The metal
ion concentration was 2.0 mM. The goodness-of-fit measured in Hyperquad2013 [37] by sigma (σ) represents the overall goodness-of-fit derived from the sum of squared residuals (calculated-experimental titration data). The model was accepted when σ was close to one (<1.5).
The standard deviation of the log β values of species included into
the model was always lower than 0.1. Samples were degassed by bubbling purified argon through them for about 10 min prior to the measurements and the inert gas was also passed over the solutions during the titrations. Log β values for the various hydroxido complexes
[(Rh(η5-C5Me5))2(μ-OH)i](4−i)+, [(Ru(η6-p-cymene))2(μ-OH)i](4−i)+ and
[(Ru(η6-toluene))2(μ-OH)i](4−i)+ (i = 2 or i = 3) were calculated based
on the pH-potentiometric titration data in the presence of chloride ions
and were found to be in good agreement with the previously published
data [27,28]. Stability constants for (M(arene))pLqHr complexes cannot
be determined by pH-potentiometry because of solubility problems of
the ligands except the case of the deferiprone complexes.
Menadione (1.00 g, 5.81 mmol, 1 eq.) was dissolved in methanol
(10 mL) and stirred under ice cooling. Na2CO3 (0.207 g, 1.95 mmol,
0.3 eq.) and H2O2 solution (36%, 1 mL, 32.6 mmol, 1.7 eq.) were dissolved in H2O (10 mL). The aqueous solution was added dropwise to
the yellow suspension. After complete addition, the mixture was stirred
at room temperature for 1 h. H2O (100 mL) was added to the mixture and the formed precipitate was separated by filtration and dried in
vacuo. The colorless solid was suspended in water (20 mL) and concentrated sulfuric acid was added until complete dissolution. The dark red
solution was diluted with water and extracted with dichloromethane.
The organic layer was separated and extracted with saturated NaHCO3
solution. The aqueous layer was acidified with concentrated HCl and
extracted with dichloromethane. Afterwards, the yellow solution was
dried over anhydrous Na2SO4, evaporated and dried in vacuo. Yield:
0.788 g (4.19 mmol, 72%), yellow powder. Characterization: 1H NMR
(500.10 MHz, CDCl3) δ = 2.11 (s, 3H, H1’), 7.29 (s, 1H, HOH), 7.68
(ddd, J = 8 Hz, 1 Hz, 1H, H6/7.), 7.75 (ddd, J = 8 Hz, 1 Hz, 1H, H6/7.),
8.08 (dd, J = 8 Hz, 1H, H5/8), 8.13 (dd, J = 8 Hz, 1H, H5/8).
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strument with a Sample Robot TS0801R0 from Mettler Toledo (Figs.
S14–16). The recorded TGA curves revealed thermal decomposition at
T < 100 °C, which did not allow the determination of the water content
of the samples by this method.
3.3. UV–Vis spectrophotometric measurements
An Agilent Cary 8454 diode array spectrophotometer was used to
record the UV–Vis spectra in the interval 200–800 nm. The path length
was 1 or 2 cm. Equilibrium constants (proton dissociation and stability
constants) and the individual spectra of the species were calculated with
the computer program PSEQUAD [40]. The spectrophotometric titrations were performed in aqueous solution on samples containing the ligands with or without the organometallic cations and the concentration
of the ligands was 50–200 μM. The metal-to-ligand ratio was 1:1 in the
pH range from 2 to 11.5 at 25.0 °C ± 0.1 °C, I = 0.2 M (KCl).
3.4. Cyclic voltammetric measurements
Electrochemical experiments of phthiocol containing samples were
performed on an Autolab-PGSTAT 204 potentiostat/galvanostat and
monitored with Metrohm's Nova software. Measurements of the ligand (1 mM) in the presence and the absence of the metal ions (1 mM)
were obtained in a 9:1 DMF/water mixture because of low solubility of phthiocol. The aqueous part contained 20 mM phosphate buffer
(pH = 7.40). The supporting electrolyte was 0.2 M [n-Bu4N][BF4]. A
three-electrode configuration cell was used with two glassy carbon
3.6. Synthesis of dimeric metal precursors
The dimeric metal precursors [RuCl2(η6-p-cymene)]2 [41],
[RuCl2(η6-tolouene)]2
[42],
[RuBr2(η6-p-cymene)]2
[43],
[RuI2(η6-p-cymene)]2
[43],
[RhCl2(η5-C5Me5)]2
[44]
and
[OsCl2(η6-p-cymene)]2 [45] were synthesized according to literature.
3.7. General procedure
Phthiocol (3 eq.) and sodium methoxide (3.2 eq.) were dissolved
in dry methanol (12 mL) and stirred for 10 min. Afterwards, the respective metal precursor dimer (1 eq.) was added to the dark red mixture. Depending on the desired complex, two different methods were
applied. Method A: The mixture was stirred at room temperature or
at 40 °C for 2–24 h. Method B: The mixture was stirred at 40–50 °C
under microwave irradiation for 6–14 min. In both methods, the purification procedure was conducted in the same way. The precipitate
was separated from the supernatant, which was further evaporated. The
solid were dissolved in dichloromethane, filtrated, and the solvent was
evaporated. The remaining black residues were combined, dissolved in
dichloromethane and n-hexane was added until precipitation started.
The mixture was cooled to ensure complete crystallization of the desired
complexes. The black crystals were separated and dried in vacuo. Minor
changes to the general procedure are described in the respective synthesis. 1H NMR spectra recorded for complexes are shown in Figs. S7–13
with the general numbering scheme for clarity (Chart S1).
3.7.1. Synthesis of Ru(η6-p-cymene)(phth)Cl](1)
The synthesis of complex 1 was introduced earlier [14] and the
reaction was performed according to this procedure (method A), using [Ru(η6-p-cym)Cl2]2 (150 mg, 0.24 mmol, 1 eq.), Hphth (135 mg,
0.72 mmol, 3 eq.) and sodium methoxide (42 mg, 0.77 mmol, 3.2 eq.).
The mixture was stirred at room temperature for 2 h. Yield: 122 mg
(0.27 mmol, 56%), black crystals. 1H NMR: (500.10 MHz, CDCl3)
δ = 1.41 (d, J = 7 Hz, 3H, Hg), 1.43 (d, J = 7 Hz, 3H, Hg), 2.10 (s, 3H,
H1’), 2.41 (s, 3H, He), 2.96–3.03 (m, 1H, Hf), 5.48 (d, J = 6 Hz, 2H, Hb),
5.75 (d, J = 5 Hz, 2H, Hc), 7.52 (dd, J = 8 Hz, J = 8 Hz, 1H, H8), 7.68
(dd, J = 8 Hz, J = 8 Hz, 1H, H5), 7.94–7.99 (m, 2H, H6/7).
J.P. Mészáros et al. / Journal of Organometallic Chemistry xxx (xxxx) 121070
13C NMR (125.76 MHz, CDCl ) δ = 196.1 (C ), 183.4 (C ), 169.3 (C ),
3
1
4
3
136.1 (C8), 133.0 (C4a), 131.4 (C5), 128.1 (C8a), 126.6 (C7), 126.5 (C6),
123.5 (C2), 100.7 (Cd), 96.8 (Ca), 82.1 (Cc), 81.1 (Cc), 79.5 (Cb), 78.5
(Cb), 31.6 (Cf), 22.6 (Cg), 22.6 (Cg), 18.8 (Ce), 9.0 (C1‘). Anal. Calc.
for C21H21ClO3Ru·0.2H2O: C 54.65%, H 4.67%, O 11.09%. Found: C
54.52%, H 4.56%, N < 0.05% S < 0.02%, O 11.07%.
3.7.6. Synthesis of [Rh(η5-C5Me5)(phth)Cl](6)
The reaction was performed according to method B, using compound [RhCl2(η5-C5Me5)]2 (100 mg, 0.16 mmol, 1 eq.), Hphth (90 mg,
0.48 mmol, 3 eq.) and sodium methoxide (28 mg, 0.51 mmol, 3.2 eq.).
The mixture was stirred under microwave irradiation at 50 °C for 6 min
(10–20 W). Yield: 111 mg (0.24 mmol, 75%), black crystals. 1H NMR
(500.10 MHz, CDCl3) δ = 1.79 (s, 15H, Hb), 2.10 (s, 3H, H1’), 7.49 (ddd,
J = 8 Hz, J = 7 Hz, J = 1 Hz, 1H, H7), 7.67 (ddd, J = 8 Hz, J = 8 Hz,
J = 1 Hz, 1H, H6), 7.96–8.02 (m, 2H, H5/8). 13C NMR (125.76 MHz,
CDCl3) δ = 194.2 (C1), 183.7 (C4), 169.2 (C3), 135.7 (C6), 133.6 (C4a),
131.2 (C7), 129.2 (C8a), 126.4 (C8), 126.4 (C5), 122.6 (C2), 93.1(Ca),
9.2 (Cb), 9.2 (C1’). Anal. Calc. for C21H22ClO3Rh·0.1H2O: C 54.53%, H
4.84%, N < 0.05% S < 0.02%, O 10.72%. Found: C 54.15%, H 4.75%,
N < 0.05% S < 0.02%, O 10.34%.
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3.7.2. Synthesis of [Ru(η6-p-cymene)(phth)Br](2)
The reaction was performed according to method B, using compound
[RuBr2(η6-p-cymene)]2 (150 mg, 0.19 mmol, 1 eq.), Hphth (107 mg,
0.57 mmol, 3 eq.) and sodium methoxide (33 mg, 0.61 mmol, 3.2 eq.).
The mixture was stirred under microwave irradiation at 50 °C for 15 min
(10–20 W). Yield: 126 mg (0.25 mmol, 66%), black crystals. 1H NMR
(500.10 MHz, CDCl3) δ = 1.41 (d, J = 7 Hz, 3H, Hf), 1.44 (d, J = 7 Hz,
3H, Hf), 2.08 (s, 3H, H1’), 2.41 (s, 3H, He), 3.01 (hept, J = 7 Hz, 1H,
Hg), 5.48 (d, J = 6 Hz, 1H, Hb), 5.50 (d, J = 6 Hz, 1H, Hb), 5.74 (d,
J = 6 Hz, 1H, Hc), 5.77 (d, J = 6 Hz, 1H, Hc), 7.52 (dd, J = 8 Hz,
J = 8 Hz, 1H, H8), 7.68 (dd, J = 8 Hz, J = 8 Hz, 1H, H5), 7.94–8.00
(m, 2H, H6/7). 13C NMR (125.76 MHz, CDCl3) δ = 196.2 (C1), 183.3
(C4), 169.4 (C3), 136.1 (C8), 132.9 (C4a), 131.4 (C5), 128.0 (C8a), 126.5
(C7), 126.5 (C6) 123.5 (C2), 100.9 (Cd), 96.5 (Ca), 82.3 (Cc), 80.8 (Cc),
80.3 (Cb), 78.6 (Cb), 31.7 (Cf), 22.7 (Cg), 22.6 (Cg), 19.0 (Ce), 8.9 (C1’).
Anal. Calc. for C21H21BrO3Ru: C 50.21%, H 4.21%, O 9.55%. Found: C
49.90%, H 4.07%, N < 0.05%, S < 0.02%, O 9.80%.
3.7.5. Synthesis of [Os(η6-p-cymene)(phth)Cl](5)
The reaction was performed according to method A, using
[Os(η6-p-cymene)Cl2]2 (126 mg, 0.159 mmol, 1 eq.), Hphth (63 mg,
0.335 mmol, 2.2 eq.) and sodium methoxide (21 mg, 0.382 mmol, 2.4
eq.). The mixture was stirred at 40 °C for 1.5 h. Yield: 26 mg
(0.048 mmol, 15%), black crystals. 1H NMR (500.10 MHz, CDCl3)
δ = 1.37 (d, J = 7 Hz, 3H, Hg), 1.39 (d, J = 7 Hz, 3H, Hg), 2.15 (s,
3H, H1’), 2.44 (s, 3H, He), 2.79–2.89 (m, 1H, Hf), 5.93 (d, J = 5 Hz,
1H, Hb), 5.96 (d, J = 5 Hz, 1H, Hb), 6.22 (d, J = 5 Hz, 1H, Hc), 6.25
(d, J = 5 Hz, 1H, Hc), 7.55 (dd, J = 8 Hz, J = 8 Hz, 1H, H8), 7.73
(dd, J = 8 Hz, J = 8 Hz, 1H, H5), 8.00–8.04 (m, 2H, H6/7). 13C NMR
(125.76 MHz, CDCl3) δ = 198.0 (C1), 183.5 (C4), 170.2 (C3), 136.4
(C5), 133.0 (C2), 131.6 (C8), 128.0 (C8a), 126.8 (C6/7), 126.7 (C6/7),
124.2 (C4a), 91.3 (Cd), 88.2 (Ca), 73.7 (Cc), 72.7 (Cc), 70.3 (Cb), 69.3
(Cb), 32.3 (Cf), 23.0 (Cg), 22.9 (Cg), 19.2 (Ce), 9.0 (C1’). Anal. Calc.
for C21H21ClO3Os·0.5H2O: C 45.36%, H 3.99%, O 10.07%. Found: C
45.07%, H 3.98%, N 0.07%, O 9.67%.
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3.7.3. Synthesis of [Ru(η6-p-cymene)(phth)I](3)
The reaction was performed according to method A, using
[RuI2(η6-p-cymene)]2 (120 mg, 0.12 mmol, 1 eq.), Hphth (68 mg,
0.36 mmol, 3 eq.) and sodium methoxide (21 mg, 0.61 mmol, 3.2 eq.).
The mixture was stirred at 40 °C for 23 h. Yield: 96 mg (0.17 mmol,
71%), black crystals. 1H NMR (500.10 MHz, CDCl3) δ = 1.42 (d,
J = 7 Hz, 3H, Hg), 1.45 (d, J = 7 Hz, 3H, Hg), 2.05 (s, 3H, H1’), 2.41
(s, 3H, He), 2.97–3.07 (m, 1H, Hf), 5.50 (d, J = 6 Hz, 1H, Hb), 5.58
(d, J = 6 Hz, 1H, Hb), 5.76 (d, J = 6 Hz, 1H, Hc), 5.84 (d, J = 5.8,
1H, Hc), 7.52 (dd, J = 8 Hz, J = 8 Hz, 1H, H8), 7.69 (dd, J = 8 Hz,
J = 8 Hz, 1H, H5), 7.92–8.01 (m, 2H, H6/7). 13C NMR (125.76 MHz,
CDCl3) δ = 196.2 (C1), 183.3 (C4), 169.6 (C3), 136.0 (C5), 132.8 (C2),
131.4 (C8), 127.9 (C8a), 126.5 (C6/7), 126.4 (C6/7), 123.4 (C4a), 101.0
(Cd), 96.0 (Ca), 82.7 (Cc), 81.6 (Cc), 80.7 (Cb), 79.1 (Cb), 31.8 (Cf), 22.8
(Cg), 22.7 (Cg), 19.3 (Ce), 8.9 (C1’). Anal. Calc. for C21H21IO3Ru·0.1H2O:
C 45.76%, H 3.88%, O 9.00%. Found: C 45.43%, H 3.86%, N < 0.05%
S < 0.02%, O 8.66%.
3.7.4. Synthesis of [Ru(η6-toluene)(phth)Cl](4)
The reaction was performed according to method B, using Hphth
(85 mg, 0.454 mmol, 2.4 eq.) and sodium methoxide (27 mg,
0.491 mmol, 2.6 eq.). They were dissolved in 6 mL dry methanol and
stirred for 10 min at room temperature. [Ru(η6-toluene)Cl2]2 (100 mg,
0.189 mmol, 1 eq.) was added and the mixture was stirred under microwave irradiation at 40 °C for 7 min. Afterwards, the dark mixture was
stored in the fridge overnight for complete precipitation. The black solid
was separated, washed twice with 2 mL ice cold methanol, twice with
4 mL n-hexane and dried in vacuo. 141 mg (0.339 mmol, 90%). 1H NMR
(500.10 MHz, CDCl3) δ = 7.99 (ddd, J = 7.6, 4.0, 1.2 Hz, 2H), 7.69
(ddd, J = 7.6, 1.3 Hz, 1H), 7.53 (ddd, J = 7.6, 1.3 Hz, 1H), 6.01–5.96
(m, 2H), 5.71 (t, J = 5.6 Hz, 1H), 5.50–5.47 (m, 2H), 2.41 (s, 3H), 2.11
(s, 3H). 13C NMR (151 MHz, CDCl3) δ 196.5 (C1), 183.5 (C4), 169.2 (C2),
136.3(C6/7), 132.9 (C8a/4a), 131.5 (C6/7), 128.1(C8a/4a), 126.6 (2xC5/
8), 123.9 (C3), 99.9 (Ca), 86.4 (Cc), 85.7 (Cc), 78.3 (Cb), 75.9 (Cb),
19.17 (Cg), 9.0 (C1’). Anal. Calc. for C16H15ClO3Ru·0.1H2O: C 51.77%, H
3.67%, N < 0.05% S < 0.02%, O 11.88%. Found: C 51.47%, H 3.54%,
N < 0.05% S < 0.02%, O 12.22%.
3.7.7. Synthesis of [Ru(η6-p-cymene)(law)Cl](7)
The complex of lawsone was synthesized according to method A.
A solution of [Ru(η6-p-cymene)Cl2]2 (200 mg, 0.33 mmol, 1 eq.) in
CH2Cl2 (10 mL) was added to a solution of Hlaw (124 mg, 0.73 mmol,
2 eq.) and sodium methoxide (43 mg, 0.80 mmol, 2.2 eq.) in methanol
(15 mL). The reaction mixture was stirred at room temperature and under argon atmosphere for 3 h. The reaction mixture was filtered and solvent was evaporated under reduced pressure. The residue (deep violet
solid) was extracted with dichloromethane and concentrated. The solid
was dissolved in 2 mL MeOH and precipitated by addition of ether and
n-hexane. Yield: 202 mg (0.455 mmol, 69%). 1H NMR (500.10 MHz,
CDCl3): δ 1.41 (dd, J = 6.9, 2.4 Hz, 6H, Hg), 2.40 (s, 3H, He), 3.00
(hept, J = 6.7 Hz, 1H, Hf), 5.53–5.49 (m, 2H), 5.80–5.74 (m, 2H), 6.13
(s, 1H, H1’), 7.58 (ddd, J = 7.6, 1.2 Hz, 1H, H6/7), 7.75 (ddd, J = 7.6,
1.3 Hz, 1H, H6/7), 8.04–7.99 (m, 2H, H5/8). 13C NMR (126 MHz, CDCl3)
δ 197.8 (C1), 183.7 (C4), 172.0 (C3), 136.8 (Carom.), 133.1 (Carom.), 131.7
(Carom.), 128.3 (Carom.), 127.0 (Carom.), 126.7 (Carom.), 113.0 (C2), 101.3
(Cd), 96.8 (Ca), 81.4 (2xCc), 79.2 (2xCb), 31.6 (Cf), 22.6 (2xCg), 19.0
(Ce). Anal. Calc. for C20H19ClO3Ru·0.25H2O: C 53.57%, H 4.38%. Found:
C 53.71%, H 4.04%, N < 0.05% S < 0.02%.
3.8. Single-crystal X-ray structure analysis
The X-ray intensity data were measured on a Bruker D8 Venture
diffractometer equipped with multilayer monochromators, Mo K/α INCOATEC micro focus sealed tubes and Oxford system. The structures
were solved by direct methods and refined by full-matrix least-squares
techniques. Non-hydrogen atoms were refined with anisotropic displacement parameters. Hydrogen atoms were inserted at calculated positions
J.P. Mészáros et al. / Journal of Organometallic Chemistry xxx (xxxx) 121070
3.9. Cell lines, culture conditions and cytotoxicity tests in cancer cell lines
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3.9.1. Cell lines and culture conditions
CH1/PA-1 cells (identified via STR profiling as PA-1 ovarian teratocarcinoma cells by Multiplexion, Heidelberg, Germany) were a gift
from Lloyd R. Kelland, CRC Center for Cancer Therapeutics, Institute
of Cancer Research, Sutton, UK. SW480 (human adenocarcinoma of
the colon), and A549 (human non-small cell lung cancer) cells were
provided by Brigitte Marian (Institute of Cancer Research, Department
of Medicine I, Medical University of Vienna, Austria). All cell culture
reagents were obtained from Sigma-Aldrich and plasticware from Starlab (Germany). Cells were grown in 75 cm2 culture flasks as adherent monolayer cultures in minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (Gibco™, Thermo
Fisher),1 mM sodium pyruvate, 4 mM l-glutamine, and 1% non-essential amino acids (from 100 × ready-to-use stock). Cultures were maintained at 37 °C in humidified atmosphere composed of 95% air and 5%
CO2.
tion with metal ions, such as Ru(II)-, Os(II)- and Rh(III)-arene is a
promising approach for the development of novel organometallic compounds with anticancer properties. Within this work, six naphthoquinone-based complexes with different metal centers, arenes, chelating ligands and leaving groups were synthesized and characterized.
The analysis of the single-crystal X-ray data of the phthiocol-based
organometallic complexes showed that the exchange of the metal center
with a higher charged metal ion affects the bond lengths of the coordination bonds considerably. The cytotoxic properties of the compounds
in three human cancer cell lines and their solution chemistry were investigated. The performed structural variations did not lead to an increase
in cytotoxicity in the used cancer cells compared to the most cytotoxic
compound 1.
Solution equilibrium chemistry was investigated systematically for
Ru(η6-p-cymene), Ru(η6-toluene) and Rh(η5-C5Me5) complexes of both
ligands with natural origin, and stability constants were determined.
The observed trend of the stability constants is the following:
Ru(η6-p-cymene) > Ru(η6-toluene) > Rh(η5-C5Me5), which is the same
as it was found earlier for other (O,O) bidentate ligand complexes. Despite this trend the [Rh(η5-C5Me5)(phth)Cl] complex has the highest solution stability at physiological pH among the studied complexes. Notably, the tested complexes have generally lower stability compared
with those of other ligands bearing (O,O) donor set.
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and refined with riding model. The following software was used: Bruker
SAINT software package [46] using a narrow-frame algorithm for frame
integration, SADABS [47] for absorption correction, OLEX2 [48] for
structure solution, refinement, molecular diagrams and graphical
user-interface, SHELXLE [49] for refinement and graphical user-interface SHELXS-2015 [50] for structure solution, SHELXL-2015 [51] for
refinement, Platon [52] for symmetry check and π-π interactions. Experimental
data
for
complexes
[Os(η6-p-cymene)(phth)Cl],
[Ru(η6-p-cymene)(phth)Cl],
[Ru(η6-p-cymene)(phth)Br],
[Ru(η6-p-cymene)(phth)I] and [Rh(η5-C5Me5)(phth)Cl] (available online:
http://www.ccdc.cam.ac.uk/conts/retrieving.html,
codes:
1961117–1961121) can be found in Table S1. Crystal data and structure refinement details for complexes are given in Tables S1–S6.
3.9.2. MTT assay
Cytotoxic effects were determined by means of a colorimetric microculture assay (MTT assay). For this purpose, cells were harvested from
culture flasks by trypsinization and seeded in 100 μL/well aliquots into
96-well microculture plates. Cell densities of 1.0 × 103 cells/well (CH1/
PA-1), 2.0 × 103 cells/well (SW480), and 3.0 × 103 cells/well (A549)
were chosen in order to ensure exponential growth of untreated controls
throughout the experiment. Cells were allowed to settle and resume exponential growth in drug-free complete culture medium for 24 h. The
compounds were then dissolved in DMF (compounds 4, 6) or DMSO (all
other compounds) first, diluted in complete culture medium and added
to the plates where the final DMF/DMSO content did not exceed 0.5%.
After 96 h of exposure, all media were replaced with 100 μL/well of a
1:7 MTT/RPMI 1640 solution (six parts of RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 4 mM l-glutamine; one part of 5 mg/mL MTT reagent in phosphate-buffered saline
(PBS)). After incubation for 4 h, the supernatants were removed and the
formazan crystals formed by viable cells were dissolved in 150 μL DMSO
per well. Optical densities at 550 nm were measured with a microplate
reader (BioTek ELx808) using a reference wavelength of 690 nm to correct for unspecific absorption. The quantity of viable cells was expressed
as percentage of untreated controls, and 50% inhibitory concentrations
(IC50) were calculated from concentration-effect curves by interpolation.
Evaluation is based on means from at least three independent experiments, each comprising three replicates per concentration level.
4. Conclusions
Phthiocol and lawsone are 2-hydroxy-[1,4]-naphthoquinones with
pronounced bioactivity and redox chemistry; therefore the complexa
9
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This work was supported by National Research, Development and
Innovation Office-NKFIA through projects GINOP-2.3.2-15-2016-00038,
FK 124240 and FIKP program TUDFO/47138–1/2019-ITM.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jorganchem.2019.121070.
References
[1] I. Ott, R. Gust, Arch. Pharm. 340 (2007) 117–126, doi:10.1002/
ardp.200600151.
[2] N. Muhammad, Z. Guo, Curr. Opin. Chem. Biol. 19 (2014) 144–153,
doi:10.1016/j.cbpa.2014.02.003.
[3] R. Trondl, P. Heffeter, C.R. Kowol, M.A. Jakupec, W. Berger, B.K. Keppler,
Chem. Sci. 5 (2014) 2925–2932, doi:10.1039/C3SC53243G.
[4] E. Alessio, Eur. J. Inorg. Chem. 2017 (2017) 1549–1560, doi:10.1002/
ejic.201600986.
[5] J. Coverdale, T. Laroiya-McCarron, I. Romero-Canelón, Inorganics 7 (2019)
31–45, doi:10.3390/inorganics7030031.
[6] Y. Kumagai, Y. Shinkai, T. Miura, A.K. Cho, Annu. Rev. Pharmacol. Toxicol. 52
(2012) 221–247, doi:10.1146/annurev-pharmtox-010611-134517.
[7] C.O. Salas, M. Faúndez, A. Morello, J.D. Maya, R.A. Tapia, Curr. Med. Chem. 18
(2011) 144–161, doi:10.2174/092986711793979779.
[8] H. Babich, A. Stern, J. Appl. Toxicol. 13 (1993) 353–358, doi:10.1002/
jat.2550130510.
[9] Q. Zhang, J. Dong, J. Cui, G. Huang, Q. Meng, S. Li, Chem. Pharm. Bull. 66
(2018) 612–619, doi:10.1248/cpb.c18-00013.
[10] K.W. Wellington, RSC Adv. 5 (2015) 20309–20338, doi:10.1039/C4RA13547D.
[11] I.T. Crosby, D.G. Bourke, E.D. Jones, P.J. de Bruyn, D. Rhodes, N. Vandegraaff,
S. Cox, J.A.V. Coates, A.D. Robertson, Bioorg. Med. Chem. 18 (2010)
6442–6450, doi:10.1016/j.bmc.2010.06.105.
[12] O. Pawar, A. Patekar, A. Khan, L. Kathawate, S. Haram, G. Markad, V. Puranik,
S. Salunke‒Gawali, J. Mol. Struct. 1059 (2014) 68–74, doi:10.1016/
j.molstruc.2013.11.029.
[13] S.H. Wang, C.Y. Lo, Z.H. Gwo, H.J. Lin, L.G. Chen, C.D. Kuo, J.Y. Wu, Molecules 20 (2015) 11994–12015, doi:10.3390/molecules200711994.
[14] C.M. Hackl, B. Schoenhacker‒Alte, M.H.M. Klose, H. Henke, M.S. Legina, M.A.
Jakupec, W. Berger, B.K. Keppler, O. Brüggermann, I. Teasdale, P. Heffeter, W.
Kandioller, Dalton Trans. 46 (2017) 12114–12124, doi:10.1039/C7DT01767G.
[15] M. Kubanik, W. Kandioller, K. Kim, R.F. Anderson, E. Klapproth, M.A. Jakupec,
A. Roller, T. Söhnel, B.K. Keppler, C.G. Hartinger, Dalton Trans. 45 (2016)
13091–13103, doi:10.1039/c6dt01110a.
[16] W. Kandioller, E. Balsano, S.M. Meier, U. Jungwirth, S. Göschl, A. Roller, M.A.
Jakupec, W. Berger, B.K. Keppler, C.G. Hartinger, Chem. Commun. 49 (2013)
3348‒3350, doi:10.1039/c3cc40432c.
[17] L. Tabrizi, H. Chiniforoshan, J. Organomet. Chem. 822 (2016) 211–220,
doi:10.1016/j.jorganchem.2016.09.003.
[18] L. Tabrizi, H. Chiniforoshan, Dalton Trans. 46 (2017) 2339–2349, doi:10.1039/
C6DT04339A.
[19] S.J. Dougan, A. Habtemariam, S.E. McHale, S. Parsons, P.J. Sadler, Proc. Natl.
Acad. Sci. U.S.A. 105 (2008) 11628–11633, doi:10.1073/pnas.0800076105.
[20] M.A. Jakupec, M. Galanski, V.B. Arion, C.G. Hartinger, B.K. Keppler, Dalton
Trans. (2008) 183–194, doi:10.1039/b712656p.
[21] L. Kathawate, S.P. Gejji, S.D. Yeole, P.L. Verma, V.G. Puranik, S.
Salunke‒Gawali, J. Mol. Struct. 1088 (2015) 56–63, doi:10.1016/
j.molstruc.2015.01.053.
[22] R. Zhu, L. Xing, X. Wang, C. Cheng, B. Liu, Y. Hu, Synlett 2007 (2007)
2267–2271, doi:10.1055/s-2007-985584.
[23] H.P. Varbanov, S. Göschl, P. Heffeter, S. Theiner, A. Roller, F. Jensen, M.A.
Jakupec, W. Berger, M. Galanski, B.K. Keppler, J. Med. Chem. 57 (2014)
6751–6764, doi:10.1021/jm500791c.
[24] M.V. Babak, S.M. Meier, K.V.M. Huber, J. Reynisson, A.A. Legin, M.A. Jakupec,
A. Roller, A. Stukalov, M. Gridling, K.L. Bennett, J. Colinge, W. Berger, P.J.
Dyson, G. Superti-Furga, B.K. Keppler, C.G. Hartinger, Chem. Sci. 6 (2015)
2449–2456, doi:10.1039/C4SC03905J.
[25] S. Grgurić-Šipka, I. Ivanović, G. Rakić, N. Todorović, N. Gligorijević, S.
Radulović, V.B. Arion, B.K. Keppler, Eur. J. Med. Chem. 45 (2010) 1051–1058,
doi:10.1016/j.ejmech.2009.11.055.
[26] I. Bratsos, D. Urankar, E. Zangrando, P. Genova Kalou, J. Košmrlj, E. Alessio, I.
Turel, Dalton Trans. 40 (2011) 5188–5199, doi:10.1039/C0DT01807D.
[27] L. Bíró, A.J. Godó, Z. Bihari, E. Garribba, P. Buglyó, Eur. J. Inorg. Chem. (2013)
3090–3100, doi:10.1002/ejic.201201527.
[28] O. Dömötör, S. Aicher, M. Schmidlehner, M.S. Novak, A. Roller, M.A. Jakupec,
W. Kandioller, C.G. Hartinger, B.K. Keppler, É. Enyedy, J. Inorg. Biochem. 34
(2014) 57–65, doi:10.1016/j.jinorgbio.2014.01.020.
[29] E.B. Ball, J. Biol. Chem. 106 (1934) 515–524.
[30] A. Beauchamp, R.L. Benoit, Can. J. Chem. 44 (1966) 1607–1613, doi:10.1139/
v66-244.
[31] L. Bíró, E. Farkas, P. Buglyó, Dalton Trans. 41 (2012) 285–291, doi:10.1039/
c1dt11405k.
[32] É. Enyedy, O. Dömötör, C.M. Hackl, A. Roller, M.S. Novak, M.A. Jakupec, B.K.
Keppler, W. Kandioller, J. Coord. Chem. 68 (2015) 1583–1601, doi:10.1080/
00958972.2015.1023195.
[33] J.P. Mészáros, J.M. Poljarevic, G.T. Gál, N.V. May, G. Spengler, É. Enyedy, J.
Inorg. Biochem. 195 (2019) 91–100, doi:10.1016/j.jinorgbio.2019.02.015.
[34] L. Bíró, E. Farkas, P. Buglyó, Dalton Trans. 39 (2010) 10272–10278,
doi:10.1039/c0dt00469c.
[35] M. Aguilar‒Martínez, N.A. Marcías‒Ruvalcaba, J.A. Bautista‒Martínez, M.
Gómez, F.J. González, I. González, Curr. Org. Chem. 8 (2004) 1721–1738,
doi:10.2174/1385272043369548.
[36] K.N. Raymond, C.J. Carrano, Acc. Chem. Res. 12 (1979) 183–190, doi:10.1021/
ar50137a004.
[37] P. Gans, A. Sabatini, A. Vacca, Talanta 43 (1996) 1739–1753, doi:10.1016/
0039-9140(96)01958-3.
[38] H.M. Irving, M.G. Miles, L.D. Petit, Anal. Chim. Acta 38 (1967) 475–488,
doi:10.1016/S0003-2670(01)80616-4.
[39] SCQuery, The IUPAC stability constants database, academic software (version
5.5), R. Soc. Chem (1993–2005).
[40] L. Zékány, I. Nagypál, in: D.L. Leggett (Ed.), Computational Methods for the Determination of Stability Constants, Plenum Press, New York, 1985, pp.
291–353.
[41] L. Ma, R. Ma, Z. Wang, S.M. Yiu, G. Zhu, Chem. Commun. 52 (2016)
10735–10738, doi:10.1039/C6CC04354B.
[42] L.E. Heim, S. Vallazza, D. van der Waals, M.H.G. Prechtl, Green Chem. 18
(2016) 1469–1474, doi:10.1039/C5GC01798J.
[43] M.G. Mendoza‒Ferri, C.G. Hartinger, A.A. Nazarov, R.E. Eichinger, M.A. Jakupec, K. Severin, B.K. Keppler, Organometallics 28 (2009) 6260–6265,
doi:10.1021/om900715j.
[44] J.W. Kang, K. Moseley, P.M. Maitlis, J. Am. Chem. Soc. 91 (1969) 5970–5977,
doi:10.1021/ja01050a008.
[45] W.A. Kiel, R.G. Ball, W.A.G. Graham, J. Organomet. Chem. 383 (1990)
481–496, doi:10.1016/0022-328X(90)85149-S.
[46] B. AXS, Bruker SAINT v8.38A/B & v7.56/7.68A, 2005-2019.
[47] G.M. Sheldrick, SADABS, University of Göttingen, Germany, 1996.
[48] O.V. Dolomanov, L.J. Bourhis, R.J. Gildea, J.A.K. Howard, H. Puschmann, J.
Appl. Crystallogr. 42 (2009) 339–341, doi:10.1107/S0021889808042726.
[49] C.B. Hubschle, G.M. Sheldrick, B. Dittrich, J. Appl. Crystallogr. 44 (2011)
1281–1284, doi:10.1107/S0021889811043202.
[50] G.M. Sheldrick, SHELXS v 2016/4, University of Göttingen, Germany, 2015.
[51] G.M. Sheldrick, SHELXL v 2016/4, University of Göttingen, Germany, 2015.
[52] A.L. Spek, Acta Crystallogr. D65 (2009) 148–155, doi:10.1107/
S0021889802022112.
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