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Comparative solution equilibrium and structural studies of half-sandwich ruthenium(II)(η6-toluene) complexes of picolinate derivatives.
Comparative solution equilibrium and structural studies of half-sandwich
ruthenium(II)(6-toluene) complexes of picolinate derivatives
Jelena M. Poljarević,a,b Tamás G. Gál,c Nóra V. May,c Gabriella Spengler,d Orsolya
Dömötör,a Aleksandar R. Savić,b Sanja Grgurić-Šipka,b Éva A. Enyedya*
a
Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7. H-6720 Szeged, Hungary
b
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
c
Research Centre for Natural Sciences Hungarian Academy of Sciences, Magyar tudósok körútja 2, H-1117
Budapest, Hungary
d
Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10, H-6720 Szeged,
Hungary
Keywords: Stability constants; X-ray crystal structures; Half-sandwich complexes; Speciation;
Antiproliferative activity
* Corresponding author.
E-mail address: enyedy@chem.u-szeged.hu (É.A. Enyedy).
ABSTRACT
Five Ru(II)(6-toluene) complexes formed with 2-picolinic acid and its various derivatives have
been synthesized and characterized. X-ray structures of four complexes are also reported.
Complex formation processes of [Ru(II)(6-toluene)(H2O)3]2+ organometallic cation with the
metal-free ligands were studied in aqueous solution in the presence of chloride ions by the
combined use of 1H NMR spectroscopy, UV-visible spectrophotometry and pH-potentiometry.
Solution stability, chloride ion affinity and lipophilicity of the complexes were characterized
together with the in vitro cytotoxic and antiproliferative activity in cancer cell lines being
sensitive and resistant to classic chemotherapy and in normal cells as well. Formation of mono
complexes such as [Ru(6-toluene)(L)(Z)] (L: completely deprotonated ligand; Z = H2O/Cl‒)
with high stability and [Ru(6-toluene)(L)(OH)] was found in solution. The pKa values (8.38.7) reflect the formation of low amount of mixed hydroxido species at pH 7.4 at 0.2 M KCl
ionic strength. The complexes are fairly hydrophilic and show moderate chloride ion affinity
and fast chloride-water exchange processes. The studied complexes exhibit no cytotoxic
activity in human cancer cells (IC50 > 100 M), only complexes formed with 2-picolinic acid
1
(1) and its 3-methyl derivative (2) represented a moderate antiproliferative effect (IC50 = 84.8
(1), 79.2 μM (2)) on a multidrug resistant (MDR) colon adenocarcinoma cell line revealing
considerable MDR selectivity. Complexes 1 and 2 are bound to human serum albumin
covalently and relatively slowly with moderate strength at multiple binding sites without ligand
cleavage.
2
1. Introduction
Ruthenium complexes have emerged as attractive alternatives to platinum based
compounds such as cisplatin, carboplatin and oxaliplatin which are undoubtedly successful
anticancer drugs but have several drawbacks such as serious side-effects and lack of activity
(drug resistance) against certain types of cancer. Ruthenium compounds have different physicochemical and pharmacokinetic properties compared to the platinum drugs, and they have
different mechanism of action as well, this is the reason why they are the subject of extensive
drug
discovery
efforts
[1-3].
Imidazolium
trans-
[tetrachlorido(DMSO)(imidazole)ruthenate(III)] (NAMI-A) was the first Ru(III) complex
reached clinical trials [4], while sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)]
(NKP-1339, IT-139) is one of the most promising investigational non-Pt drugs in current
clinical development. NKP-1339 is active against solid malignancies such as non-small cell
lung cancer, colorectal carcinoma and the treatment is accompanied by minor side effects [5,6].
While cisplatin induces DNA damage via adduct formation [7], endoplasmic reticulum stress
and reactive oxygen species-related effects were found to be involved in the mechanism of
action of NKP-1339 [5,8]. Ru(III) complexes are considered as prodrugs that are activated by
reduction and it provides the impetus for the development of various Ru(II) anticancer
compounds [5]. It is noteworthy that a novel Ru(II) compound [Ru(4,4’-dimethyl-2,2’bipyridine)2-(2-(2’,2’’:5’’,2’’’-terthiophene)-imidazo[4,5-f][1,10]phenanthroline)]Cl2 (TLD1433) has entered a human clinical trial recently as nontoxic photosensitizing agent [9]. Ru(II)
is often stabilized in the +2 oxidation state by the coordination of η6-arene type ligands and
there are two main prototypes of Ru(II)-arene complexes [3]: i) RAPTA compounds contain
1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane (PTA) such as [Ru(6-p-cymene)(PTA)Cl2]
(RAPTA-C) possessing significant antimetastatic property and is ready for translation into
clinical evaluation [10,11]; ii) RAED complexes bear the bidentate 1,2-ethylenediamine (en)
ligand such as [Ru(6-biphenyl)(en)Cl]PF6 (RM175) that has a similar cytotoxic activity to
cisplatin [12,13]. In most of the half-sandwich organoruthenium(II) compounds a bidentate
ligand with an (O,O), (O,S), (O,N), (N,N) or (N,S) binding mode is coordinated and a chloride
ion acts as the leaving group [3,14-16]. Aquation (replacement of the chlorido ligand by a water
molecule) facilitates the reaction with biological macromolecules such as proteins or DNA,
therefore the strength of the Ru-Cl bond and the rate of its cleavage have a strong impact on the
bioactivity of the Ru(II)-arene complexes [17]. Notably, the chemical and pharmacological
3
properties of the Ru(II)-arene half-sandwich compounds can be fine-tuned by variation of the
coordinating ligand, the arene ring and the leaving group [1,3,10]. Although a large number of
Ru(II)-arene compounds has been developed and extensively investigated, information about
their solution speciation and stability constants is still limited in the literature. Most of the
solution equilibrium studies are focused on [Ru(6-p-cymene)(X,Y)Cl] type complexes [1824]. For the better understanding of the pharmacokinetic properties and mechanisms of action
of these metal complexes, the knowledge of the aqueous chemistry and the most plausible
chemical forms in water, especially at physiological pH, is a mandatory prerequisite.
In our previous works we have studied the biological activity of Ru(II)(6-p-cymene)
complexes of various pyridine derivatives [25-28] and moderate-to-low cytotoxicity was found
in six tumor cell lines; although the complex of 2-picolinic acid (picH) represents an enhanced
antiproliferative activity (e.g. IC50 = 82 M in HeLa cells, 36 M in FemX cells [27]) and
antimetastatic effect based on wound migration assay [25]. The solution speciation of
Ru(II)(6-p-cymene) picolinate complexes was also studied by some of us revealing the
formation of mono-ligand complexes with high stabilities [23]. Notably, the Os(II) congener of
the picolinate complex showed very high in vitro cytotoxic activity [29].
As the physico-chemical and biological properties can be modified by the exchange of
the arene ring, in this work we have prepared and structurally characterized Ru(II)(6-toluene)
complexes formed with picH and its 3-methyl (3-Me-picH), 5-bromo (5-Br-picH), 2,4dicarboxylic (2,4-dipicH2) and 2,5-dicarboxylic (2,5-dipicH2) derivatives (Chart 1). In addition
to the determination of the solid phase structures of the four complexes by X-ray
crystallography, solution speciation of these Ru(II)(6-toluene) complexes in water was
revealed by pH-potentiometry,
1
H NMR
spectroscopy and UV-visible (UV-vis)
spectrophotometry involving studies on their stability and chloride ion affinity. The
antiproliferative and cytotoxic effectiveness of these complexes in multidrug resistant/nonresistant human cancer lines wasere also tested. Interactions between human serum albumin
and the complexes showing antiproliferative effect were monitored using fluorometry and
ultrafiltration.
4
O
(a)
-O
Br
O-
(b)
O
N
N
N
N
N
N
Ru
Cl
O
O
O-
pic
O-
O
3-Me-pic
O
O-
5-Br-pic
O
O-
2,4-dipic
O
O-
2,5-dipic
[Ru( 6-toluene)(L)(Cl)]
Chart 1. Chemical structures of the ligands in their completely deprotonated forms (a) and the general
formula of the prepared [Ru(6-toluene)(L)(Cl)] complexes.
2. Experimental
2.1. Chemicals
All solvents were of analytical grade and used without further purification. Pyridine-2carboxylic acid (2-picolinic acid, picH), 3-methylpyridine-2-carboxylic acid (3-Me-picH), 5bromo-2-pyridinecarboxylic acid (5-Br-picH), 2,4-pyridinedicarboxylic acid monohydrate
(2,4-dipicH2·H2O), 2,5-pyridinedicarboxylic acid (2,5-dipicH2), RuCl3·3H2O, KCl, HCl, KOH,
4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), 1-methylimidazole (N-MeIm), human
serum albumin (HSA, as lyophilized powder with fatty acids, A1653), KH2PO4,
NaH2PO4·2H2O, Na2HPO4·2H2O were purchased from Sigma-Aldrich in puriss quality. Doubly
distilled Milli-Q water was used for preparation of samples. The purity of the ligands and the
exact concentration of their stock solutions were determined by pH-potentiometric titrations
and by the computer program HYPERQUAD [30]. [Ru(η6-toluene)Cl2]2 was prepared
according to a well known procedure [31]. A stock solution of [Ru(η6-toluene)(Z)3], where Z
is H2O or Cl‒, was obtained by dissolving [Ru(η6-toluene)Cl2]2 in water and the exact
concentration of this stock was determined with pH-potentiometric titrations. The modified
phosphate-buffered saline (PBS’) contains 12 mM Na2HPO4, 3 mM KH2PO4, 1.5 mM KCl and
100.5 mM NaCl; and the concentration of the K+, Na+ and Cl‒ ions corresponds to that of the
human blood serum. HSA solution was freshly prepared before the experiments and its
concentration was estimated from its UV absorption: 280 nm(HSA) = 36850 M−1cm−1 [32]. Stock
solution of N-MeIm was prepared on a weight-in-volume basis in PBS’ solution.
2.2. Synthesis of the complex [(η6-toluene)RuCl(μ-Cl)] 2 with different picolinic acids
2.2.1. Synthesis of the precursor [Ru(η6-toluene)Cl(μ-Cl)] 2
5
[Ru(η6-toluene)Cl(μ-Cl)]2 was prepared according the literature procedure used for the
analogous [Ru(η6-benzene)Cl(μ-Cl)]2 [31] by adding 5 mL of 1-methyl-1,4-cyclohexadiene to
a solution of 0.5 g RuCl3·3H2O (1.9 mmol) in 40 mL of absolute ethanol. This mixture was
refluxed for 8 h. The reddish brown precipitate formed during the synthesis was filtered off,
washed with diethyl ether and left to dry in exsiccator. Yield: 85%, 0.450 g; 1H NMR
(500.26 MHz, DMSO-d6, d, ppm): 2.12 (3H, s, CH3), 5.68 (3H, m, C2, C4, C6 arene), 5.97 (2H,
m, C3, C5 arene); 13C NMR (125.79 MHz MHz, DMSO-d6) 18.73 (CH3), 82.22 (C4 arene),
84.83 (C5, C3 arene), 89.28 (C6, C2 arene), 105.82 (C1 arene).
2.2.2. Synthesis of chlorido[(pyridine-κN-2-carboxylato-κO)(η6-toluene)ruthenium(II)] (1):
To a warm solution of [Ru(η6-toluene)Cl2]2 (0.030 g, 0.057 mmol) in 25 mL of 2-propanol, was
added a solution of picH (0.015 g, 0.13 mmol) in 2 mL of 2-propanol. The reaction mixture was
stirred at room temperature for 7 days and the yellow-range precipitate was formed. Solution
was filtered off and product was dried in exsiccator. Yield: 58%, 0.023 g; 1H NMR (500.26
MHz, DMSO-d6, , ppm): 2.15 (3H, s, CH3), 5.60 (2H, m, C2, C6 arene), 5.70 (1H, m, C4
arene), 5.99 (2H, m, C3, C5 arene), 7.72 (2H, m, C3, C4 ligand), 8.06 (1H, t, C5 ligand), 9.29
(1H, d, C6 ligand); 13C NMR (125.79 MHz, DMSO-d6) 18.57 (CH3), 77.07 (C4 arene), 78.44
(C5 arene), 79.71 (C3 arene), 86.15 (C6 arene), 88.06 (C2 arene), 101.01 (C1 arene), 125.31
(C3 ligand), 128.09 (C5 ligand), 139.64 (C4 ligand), 150.89 (C2 ligand), 153.88 (C6 ligand).
ESI/MS (m/z): [M‒Cl]+ = 315 and [M‒Cl‒COO+H+]+ = 272.
2.2.3. Synthesis of complexes of chlorido[(3-methylpyridine-κN-2-carboxylato-κO)(η6toluene)ruthenium(II)]
(2),
chlorido[(5-bromopyridine-κN-2-carboxylato-κO)(η6-
toluene)ruthenium(II)]
(3),
chlorido[(4-carboxylate-pyridine-κN-2-carboxylato-κO)(η6-
toluene)ruthenium(II)]
(4),
chlorido[(5-carboxylate-pyridine-κN-2-carboxylato-κO)(η6-
toluene)ruthenium(II)] (5):
Methanolic solution of the ligand (3-Me-picH (10.4 mg, 0.076 mmol) or 5-Br-picH (15.4 mg,
0.076 mmol) or 2,4-dipicH2·H2O (14.1 mg, 0.076 mmol) or 2,5-dipicH2 (12.7 mg, 0.076 mmol))
was slowly added in the methanolic (5 mL) solution of [Ru(η6-p-toluene)Cl2]2 (20.0 mg, 0.038
mmol) and reaction mixture was stirred for 3 h, at 40°C. Then, reaction volume was reduced
to half and desired orange complex was precipitated. Solution was filtered off and product was
dried in exsiccator.
6
2: Yield: 57%, 0.016 g; 1H NMR (500.26 MHz, DMSO-d6, , ppm): 2.16 (3H, s, arene CH3),
2.54 (3H, s, ligand CH3), 5.57 (1H, d,C2, arene), 5.60 (1H, d, C6, arene), 5.68 (1H, t, C4 arene),
5.97 (2H, dd, C3, C5 arene), 7.59 (1H, dd, C5 ligand), 7.89 (1H, d, C4 ligand), 9.22 (1H, d, C6
ligand); 13C NMR (125.79 MHz, DMSO-d6) 18.37 (CH3, ligand), 18.65 (CH3, arene), 77.11
(C4 arene), 78.88 (C5 arene), 79.21 (C3 arene), 86.62 (C6 arene), 88.43 (C2 arene), 101.18 (C1
arene), 126.88 (C5 ligand), 137.92 (C4 ligand), 142.70 (C6 ligand), 147.29 (C3 ligand), 152.48
(C2 ligand), 170.89 (COO-Ru). ESI/MS (m/z): [M‒Cl]+ = 330 and [M‒Cl‒COO+H+]+ = 287.
3: Yield: 52%, 0.017 g; 1H NMR (500.26 MHz, DMSO-d6, , ppm): 2.17 (3H, s, CH3), 5.63
and 5.67 (2H,dd ,C2, C6 arene), 5.77 (1H, t,C4 arene), 6.06 (2H, m, C3, C5 arene), 7.68 (1H,
d, C3 ligand), 8.34 (1H, d, C4 ligand), 9.52 (1H, s, C6 ligand); 13C NMR (125.79 MHz, DMSOd6) 18.41 (CH3), 76.98 (C4 arene), 78.54 (C5 arene), 79.21 (C3 arene), 86.58 (C6 arene), 88.16
(C2 arene), 101.60 (C1 arene), 122.95 (C5 ligand), 126.30 (C3 ligand), 142.28 (C4 ligand),
149.76 (C2 ligand), 154.04 (C6 ligand), 169.69 (COO-Ru). ESI/MS (m/z): [M‒Cl]+ = 395 and
[M‒Cl‒COO+H+]+ = 352.
4: Yield: 56%, 0.017 g; 1H NMR (500.26 MHz, DMSO-d6, , ppm): 2.18 (3H, s, CH3), 5.66
(2H,dd ,C2, C6 arene),5.75 (1H, t,C4 arene), 6.06 (2H, m, C3, C5 arene), 8.06 (2H, m, C3, C5
ligand), 9.51 (1H, d, C6 ligand), 14.22 (1H, s, free COOH ligand); 13C NMR (125.79 MHz,
DMSO-d6) 18.41 (CH3), 77.27 (C4 arene), 78.94 (C5 arene), 79.68 (C3 arene), 86.61 (C6
arene), 88.17 (C2 arene), 101.54 (C1 arene), 123.82 (C3 ligand), 126.63 (C5 ligand), 140.93
(C4 ligand), 151.88 (C6 ligand), 155.17 (C2 ligand), 164.66 (COO-Ru), 169.73 (COOH).
ESI/MS (m/z): [M‒Cl]+ = 360 and [M‒Cl‒COO+H+]+ = 317.
5: Yield: 50%, 0.015 g; 1H NMR (500.26 MHz, DMSO-d6, , ppm): 2.18 (3H, s, CH3), 5.66(1H,
d ,C2 arene), 5.70(1H, d, C6 arene),5.80(1H, t, C4 arene), 6.08 (2H, m, C3, C5 arene), 7.89
(1H, d, C4 ligand), 8.51 (1H, d, C3 ligand), 9.56 (1H, s, C6 ligand), 14.20 (1H, s, free COOH
ligand); 13C NMR (125.79 MHz, DMSO-d6) 18.43 (CH3), 77.11 (C4 arene), 78.72 (C5 arene),
79.32 (C3 arene), 86.53 (C6 arene), 87.99 (C2 arene), 101.46 (C1 arene), 125.29 (C3 ligand),
130.63 (C4 ligand), 140.24 (C5 ligand), 153.28 (C6 ligand), 154.32 (C2 ligand), 164.42 (COORu), 169.56 (COOH). ESI/MS (m/z): [M‒Cl]+ = 360 and [M‒Cl‒COO+H+]+ = 317.
For the characterization of the prepared complexes 1H and 13C NMR spectroscopy and
electrospray ionization mass spectrometry (ESI-MS) were used. NMR spectra were recorded
on a Bruker Avance III 500 spectrometer or a Bruker Ultrashield 500 Plus instrument, and
DMSO-d6 was used as solvent. ESI-MS measurements were performed using a Micromass Q7
TOF Premier (Waters MS Technologies) mass spectrometer equipped with electrospray ion
source.
2.3. Crystallographic structure determination
Single crystals suitable for X-ray diffraction experiment of compounds [Ru(6-toluene)(pic)Cl]
(1), [Ru(6-toluene)(3-Me-pic)Cl]∙H2O (2∙H2O), [Ru(6-toluene)(5-Br-pic)Cl] (3) and [Ru(6toluene)(2,5-dipic)Cl] (5) were grown from methanol solution of the solid complexes.
Orange (1) and yellow (2∙H2O, 3, 5) single crystals were mounted on loops and transferred
to the goniometer. X-ray diffraction data were collected at ‒170 °C (for 1, 2∙H2O) or 20 °C (for
3, 5) on a Rigaku RAXIS-RAPID II diffractometer using Mo-K radiation. A numerical
absorption correction [33] was carried out using the program CrystalClear [34]. Sir2014 [35]
and SHELXL [36] under WinGX [37] software were used for structure solution and refinement,
respectively. The structures were solved by direct methods. The models were refined by fullmatrix least squares on F2. Refinement of non-hydrogen atoms was carried out with anisotropic
temperature factors. Hydrogen atoms were placed into geometric positions (except for water
hydrogens which were constrained). They were included in structure factor calculations but
they were not refined. The isotropic displacement parameters of the hydrogen atoms were
approximated from the U(eq) value of the atom they were bonded to. The summary of data
collection and refinement parameters are collected in Table S1. Selected bond lengths and
angles of compounds were calculated by PLATON software [38]. The graphical representation
and the edition of CIF files were done by Mercury [39] and PublCif [40] softwares, respectively.
The crystallographic data files for the complexes have been deposited with the Cambridge
Crystallographic Database as CCDC x, CCDC x, CCDC x and CCDC x.
2.4. pH-potentiometric measurements and data evaluation
The pH-potentiometric measurements determining the proton dissociation and formation
constants were carried out at 25 ± 0.1°C and an ionic strength I = 0.20 M (KCl) in order to keep
the activity coefficient constant. The titrations were performed in a carbonate-free KOH
solution (0.20 M). The exact concentrations of HCl and KOH were determined by pHpotentiometric titrations. An Orion 710A pH-meter equipped with a Metrohm combined
electrode (type 6.0234.100) and Methrom 665 Dosimat burette were used for the pHpotentiometric measurements. The electrode system was calibrated to the pH= ‒ log[H+] scale
by means of black titrations (strong acid HCl vs. strong base KOH), as suggested by Irving et
8
al. [41]. The average water ionization constant, pKw, was determined as 13.76 ± 0.01, which
corresponds well to the literature data [42]. The reproducibility of the titration points included
in the calculations was within 0.005 pH. The pH-potentiometric titrations were performed in
the pH range 2.0 to 11.5. The initial volume of the samples was 5 mL. The ligand concentration
was 2 mM and metal to ligand ratios of 1:1 and 1:2 were used. The accepted fitting between the
measured and calculated titration data points regarding the volume of the titrant was < 10 µL.
The samples were degassed by bubbling purified argon through them for 10 min prior the
measurements and the argon was also passed over the solutions during the titrations.
The computer program HYPERQUAD [30] was utilized to establish the stoichiometry of
the complexes and to calculate the overall stability constants. β(MpLqHr) is defined for the
general equilibrium:
pM +qL+rH ⇌ MpLqHr as β(MpLqHr) ⇌ [MpLqHr]/[M]p[L]q[H]r (1)
where M denotes the metal moiety [Ru(η6-toluene)(Z)3] (Z = H2O/Cl‒) and L the completely
deprotonated ligand. In all calculations exclusively titration data were used from experiments
in which no precipitate was visible in the reaction mixture. As equilibrium constants were
determined in the presence of 0.2 M chloride ion, they are considered as conditional constants.
log values for the various hydroxido complexes[(Ru(6-toluene))2(2-OH)i](4-i)+ (i=2,3) were
calculated based on the pH-potentiometric titration data in the presence of chloride ions and
were found to be in fairly good agreement with previously published data [43].
2.5. UV-vis spectrophotometric and 1H NMR spectroscopic titrations, and determination of the
distribution coefficients
A Hewlett Packard 8452A diode array spectrophotometer was used to record the UV-vis spectra
in the interval 200 – 800 nm. The path length was 1 cm. Equilibrium constants (proton
dissociation, stability constants and H2O/Cl− exchange constants) and the individual spectra of
the species were calculated with the computer program PSEQUAD [44]. The
spectrophotometric titrations were performed in pure water on samples containing the ligands
with or without the organometallic cation and the concentration of the ligands was 120 μM. The
organometallic cation was also titrated (120 M) separately. The metal-to-ligand ratios were
1:1 in the pH range from 2 to 11.5 at 25.0±0.1 °C at an ionic strength of 0.20 M (KCl).
Measurements for 1:1 metal-to-ligand systems were also carried out by preparing individual
samples in which KCl was partially or completely replaced by HCl; pH values, varying in the
range ca.0.7–2.5, were calculated from the strong acid content. The absorbance data were
9
always recorded after 4 h of incubation. UV-vis spectra recorded as a function of chloride
concentrations (0–252 mM) were used to investigate the H2O/Cl− exchange processes of
complexes [Ru(6-toluene)(L)(H2O)] at pH 7.40 (using 20 mM phosphate buffer).
1
H NMR titrations were carried out on a Bruker Ultrashield 500 Plus instrument using
WATERGATE water suppression pulse scheme. DSS was used as an internal NMR standard.
1
H NMR spectra of samples containing [Ru(II)(η6-toluene)(H2O)3]2+ (1 mM) and ligand picH
(1 mM) in D2O at various pH values were recorded after 4 h of incubation (25 °C, I = 0.20 M
(KCl)). Titration of 2 mM solution of [Ru(η6-toluene)(Z)3] was also performed separately. To
study the interaction with HSA and N-MeIm 1H NMR spectra were recorded for samples
containing precursor [Ru(η6-toluene)Cl(μ-Cl)]2 or complex 1 (1 mM), with or without half
equivalent of HSA or N-MeIm. Samples were prepared in PBSʹ buffer and incubated for 24 h
at 25 °C.
Distribution coefficients at physiological pH (D7.4) of the complexes 1–5 and the ligands
as well as the Ru precursor were determined by the traditional shake-flask method in noctanol/buffered aqueous solution at pH 7.40 at various chloride concentrations using UV-vis
detection as described in our former work [24].
2.6. Fluorescence and membrane ultrafiltration/UV-vis studies with HSA
Fluorescence spectra were recorded on a Hitachi-F4500 fluorometer in 1 cm quartz cell at 25.0
± 0.1 °C. All solutions were prepared in PBS’ (pH 7.4) and were incubated for 24 h following
a time-dependence experiment. Samples contained 1 M HSA, and various HSA-to- Ru(6toluene) or 1 or 2 ratios (from 1:0 to 1:10) were used. The excitation wavelength was 295 nm
and the emission was read in the range of 310-500 nm. The quenching (KQ’) constants were
calculated with the computer program PSEQUAD [44] using the same approach applied in our
previous works [45,46].
Samples (0.50 mL) used for the ultrafiltration studies contained 40 M HSA and Ru(6toluene) or 1 or 2 (up to 1:10 protein-to-complex ratio) in PBS’ buffer (pH 7.4) at 25.0 ± 0.1 °C
and were incubated for 24 h. Samples were separated by ultrafiltration through 10 kDa
membrane filters (Millipore Amicon Ultra-0.5 centrifugal filter unit) in low (LMM) and high
molecular mass (HMM) fractions with the help of a temperature controlled centrifuge (Sanyo,
10000 rpm, 10 min). The LMM fraction containing the non-bound metal complex was separated
from the protein and its adducts in the HMM fraction. The concentration of the non-bound
10
compounds in the LMM fractions was determined by UV-vis spectrophotometry by comparing
the recorded spectra to those of reference samples without the protein.
2.7. Cell lines
Human colonic adenocarcinoma cell lines Colo 205 doxorubicin-sensitive (ATCC-CCL-222)
and Colo 320/MDR-LRP multidrug resistant overexpressing ABCB1 (MDR1)-LRP (ATCCCCL-220.1) were purchased from LGC Promochem, Teddington, UK. The cells were cultured
in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM Lglutamine, 1 mM sodium pyruvate and 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES). The cell lines were incubated at 37 °C, in a 5% CO2, 95% air atmosphere. The
semi-adherent human colon cancer cells were detached with Trypsin-Versene (EDTA) solution
for 5 min at 37 C.
MRC-5 human embryonal lung fibroblast cell lines (ATCC CCL-171) wasere purchased
from LGC Promochem, Teddington, UK. The cell line was cultured in Eagle’s Minimal
Essential Medium (EMEM, containing 4.5 g/L glucose) supplemented with a non-essential
amino acid mixture, a selection of vitamins and 10% heat-inactivated fetal bovine serum. The
cell lines were incubated at 37 °C, in a 5% CO2, 95% air atmosphere.
2.8. Assay for cytotoxic effect
In the study MRC-5 non-cancerous human embryonic lung fibroblast and human colonic
adenocarcinoma cell lines (doxorubicin-sensitive Colo 205 and multidrug resistant Colo 320
colonic adenocarcinoma cells) were used to determine the effect of compounds on cell growth.
The effects of increasing concentrations of compounds (complexes 1-5, the metal-free ligands,
the precursor [Ru(η6-toluene)Cl(μ-Cl)]2 and cis-[Pt(NH3)2(Cl)2], and the positive control
(cisplatin (, Teva)) on cell growth were tested in 96-well flat-bottomed microtiter plates. The
compounds were diluted in a volume of 100 μL of medium.
The adherent human embryonal lung fibroblast cells were cultured in 96-well flatbottomed microtiter plates, using EMEM supplemented with 10% heat-inactivated fetal bovine
serum. The density of the cells was adjusted to 2×104 cells in 100 μL per well, the cells were
seeded for 24 h at 37 C, 5% CO2, then the medium was removed from the plates containing
the cells, and the dilutions of compounds previously made in a separate plate were added to the
cells in 200 μL.
In case of the colonic adenocarcinoma cells, the two-fold serial dilutions of compounds
were prepared in 100 μL of RPMI 1640, horizontally. The semi-adherent colonic
11
adenocarcinoma cells were treated with Trypsin-Versene (EDTA) solution. They were adjusted
to a density of 2×104 cells in 100 μL of RPMI 1640 medium, and were added to each well, with
the exception of the medium control wells. The final volume of the wells containing compounds
and cells was 200 μL.
The culture plates were incubated at 37 °C for 24 h; at the end of the incubation period,
20 μL of MTT (thiazolyl blue tetrazolium bromide, Sigma-Aldrich) solution (from a stock
solution of 5 mg/mL) were added to each well. After incubation at 37 ˚C for 4 h, 100 μL of
sodium dodecyl sulphate (SDS) (Sigma-Aldrich) solution (10% in 0.01 M HCI) were added to
each well and the plates were further incubated at 37 °C overnight. Cell growth was determined
by measuring the optical density (OD) at 540/630 nm with Multiscan EX ELISA reader
(Thermo Labsystems, Cheshire, WA, USA). Inhibition of the cell growth was determined
according to the formula below:
OD sample OD medium control
IC50 = 100
100
OD cell control OD medium control
Results are expressed in terms of IC50, defined as the inhibitory dose that reduces the growth of
the cells exposed to the tested compounds by 50%.
2.9. Assay for antiproliferative effect
The method is similar to the one described in the assay described in Section 2.8 and
antiproliferative effect of complexes 1-5, the metal-free ligands, the precursor [Ru(η6toluene)Cl(μ-Cl)]2 and cisplatin was determined. In the assay testing the inhibition of cell
proliferation, 6×103 colon adenocarcinoma cells were distributed in 100 μL of medium with the
exception of the medium control wells. The culture plates were incubated at 37 °C for 72 h and
after the incubation time the plates were stained with MTT according to the experimental
protocol applied for the cytotoxicity assay vide supra.
3. Results and discussion
3.1. Synthesis, characterization and X-ray diffraction analysis of organometallic Ru(II)
complexes
The Ru(II) precursor [Ru(η6-toluene)Cl(μ-Cl)]2 and the complexes of picH, 3-Me-picH, 5-BrpicH, 2,4-dipicH2 and 2,5-dipicH2 (Chart 1) were obtained according to the literature procedure
used for the analogous [Ru(η6-p-cymene) complexes [25-28]. Pure compounds (1-5) were
12
isolated from methanol or 2-propanol with moderate yields 50-58%. The organometallic Ru(II)
complexes were characterized by means of standard analytical methods (1H , 13C NMR and
ESI-MS). The 1H NMR spectra of complexes confirm the coordination of the ligands
manifesting itself in downfield or upfield shifts of the pyridine protons (e.g. in the case of 1 the
C3, C4 protons of the ligand are upfield while C5, C6 are downfield shifted upon coordination
as shown in Fig. S1). Similar observations were made for the analogous Ru(II)(6-p-cymene)
complex of picH [27]. In general, signals representing protons next to the pyridine nitrogen
were shifted distinctly upon coordination.
Single crystals of complexes 1, 2∙H2O, 3 and 5 were obtained by the slow diffusion
method from methanol and their structures were determined by single crystal X-ray diffraction.
The ORTEP representations of these complexes are depicted in Fig. 1. The complexes 1 and
2∙H2O crystallized in monoclinic crystal systems in space group P21/n and P21, respectively.
Figure 1. Molecular structures of ruthenium complexes in crystal 1 (a) in crystal 2 (b) in crystal 3 and (c)
in crystal 5 (d). Displacement parameters are drawn at 50% probability level; hydrogen atoms and water
molecule for 2 are omitted for clarity.
The crystals 3 and 5 crystallized in triclinic crystal systems in space group P-1. All of the
complexes adopt the so-called “piano stool” configuration, whereby toluene forms the seat and
the chelating picolinate ligand as well as the chlorido leaving group constitute the chair legs. In
these half-sandwich complexes the ligand is coordinated through the pyridine nitrogen and the
carboxylate oxygen. In these structures Ru(II) is a chiral centre. In crystals 1, 3 and 5 both
13
enantiomers were crystallized in non-chiral space groups. On the other hand complex 2
crystallized together with a solvate water molecule and only one enantiomer could be found in
the chiral space group P21. The absolute configuration RRu could be determined according to
CIP convention [47], the Flack parameter is 0.01(5). The molecular structures of the studied
complexes were directly compared to that of the benzene derivative [Ru(6-C6H6)(pic)(Cl)]
defined previously (Ref code OHUFUT [48]) which crystallized without solvate inclusion in
triclinic P-1 space group (Fig. 2.) Selected bond distances and angles are collected in Table 1 for
comparison. Distances between the toluene ring and the Ru ion are within the range of observed
other ruthenium arene half-sandwich complexes (2.079(11)-2.392(7) Å) [49]. Bond lengths and
angles do not show significant differences compared to each other (Table 1).
Figure 2. Comparison of molecular structures of Ru(II)(η6-toluene) picolinate complexes 1 (colored by
element), 2 (orange), 3 (yellow), 5 (violet) together with [Ru(6-C6H6)(pic)(Cl)] (CSD Ref. code
OHUFUT) (cyan) [48]. Atoms Ru1, Cl1, N1 and O1 are superimposed.
However, the angles between planes of CgA and CgB (where Cg is the centre of gravity
calculated for rings A and B, respectively) show slight differences (Table 1 and Fig. 2). The
methyl groups of the toluene molecule are almost in the same position for crystals 1, 2∙H2O and
3 (the torsion angle O1-Ru1-Cg(A)-C7 is 5.5 o, 13.3o and -7.7o degree for 1, 2∙H2O and 3,
respectively). However, there is a significant difference in crystal 5 where the methyl group turns
to the side of the chloride ion and this torsion angle is 116.2o).
14
Table 1. Selected bond distances (Å) and angles (o) of the studied Ru(II)(η6-toluene) picolinate
complexes in crystals 1-3, 5 and [Ru(6-C6H6)(pic)(Cl)] (OHUFUT [48])
1
2∙H2O
3
5
OHUFUT
Ru1-Cl1
2.4133(5)
2.415(2)
2.405(4)
2.396(3)
2.4133(6)
Ru1-O1
2.074(1)
2.063(6)
2.085(9)
2.093(6)
2.075(2)
Ru1-N1
2.089(2)
2.092(8)
2.11(1)
2.095(7)
2.087(2)
Ru1-C1
2.183(2)
2.179(9)
2.17(1)
2.21(1)
2.178(3)
Ru1-C2
2.194(2)
2.18(1)
2.23(1)
2.17(1)
2.179(3)
Ru1-C3
2.185(2)
2.18(1)
2.16(1)
2.14(1)
2.191(3)
Ru1-C4
2.187(2)
2.17(1)
2.16(1)
2.16(1)
2.190(3)
Ru1-C5
2.148(2)
2.159(8)
2.21(1)
2.14(1)
2.168(3)
Ru1-C6
2.174(2)
2.172(9)
2.16(1)
2.16(1)
2.160(3)
Ru1-Cg(A)a
1.6564(9)
1.656(4)
1.662(6)
1.659(5)
1.662
O1-Ru1-N1
77.04(6)
76.8(3)
77.0(4)
77.4(3)
77.54(9)
O1-Ru1-Cl1
87.44(4)
84.9(2)
85.9(3)
87.0(2)
87.04(6)
N1-Ru1-Cl1
85.73(4)
83.7(2)
83.6(3)
84.2(2)
84.20(7)
Cg(A)-Ru1-O1 a
127.69(5)
129.3(2)
129.6(4)
128.4(3)
128.77
Cg(A)-Ru1-N1 a
132.73(5)
133.6(2)
134.2(4)
132.8(3)
132.55
Cg(A)-Ru1-Cl1 a
128.74(4)
129.66(17)
128.2(2)
129.1(2)
128.97
Cg(A)-Cg(B)b
52.94(9)
64.1(5)
61.9(7)
58.9(6)
55.30
5.5
13.3
-7.8
116.2
-
Bond length (Å)
Bond angles (o)
O1-Ru1-Cg(A)-Cl1
a
Cg is the centre of gravity calculated for ring A. b Angles between planes calculated for
rings A and B.
The positions of the picolinate ligands are slightly different in the studied complexes due
to secondary interactions with adjacent molecules as different molecular arrangements and
solvate inclusion (for crystal 2∙H2O) realized in these crystal structures. The packing
arrangements are shown in Figs. S2-S4 viewing along selected crystallographic axes. The main
secondary interactions between molecules are C-H…O hydrogen bonds between the toluene
hydrogens and the carboxylate oxygen (O1) of the picolinate ligand. Beside the hydrogen bonds
considerable secondary interactions are formed between neighboring complexes by C-H…Cl
interactions (e.g. C12-H12…Cl1 in 2∙H2O and C5-H5…Cl1 in 4, Table S2 and Figs. S3 and S5).
15
3.2. Proton dissociation processes of the studied ligands and hydrolysis of [Ru(6toluene)(H2O)3] 2+ organometallic cation
Proton dissociation constants of the ligands picH, 3-Me-picH, 5-Br-picH, 2,4-dipicH2 and
2,5-dipicH2 (Chart 1) were determined by pH-potentiometric and UV-vis spectrophotometric
titrations performed in the pH range from 2 up to 11.5 (Table 2). Molar absorbance spectra of
the ligand species in the different protonation states were calculated via the deconvolution of
the spectra recorded at various pH values as it is shown in Fig. S6 for 5-Br-picH. The pKa value
picH and the calculated molar absorbance spectra of the HL and L‒ forms are in reasonably
good agreement with data reported previously [23,50]. The protonated compounds picH, 3-MepicH, 5-Br-picH possess two, while 2,4-dipicH2 and 2,5-dipicH2 have three dissociable protons.
It was found in all cases that the first deprotonation step assigned to the carboxylic group at
position 2 takes place in a fairly acidic range and no pKa values could be determined for this
process. Therefore this carboxylate remains deprotonated in the whole studied pH range. pKa
determined for picH, 3-Me-picH, 5-Br-picH can be attributed to the deprotonation of the
pyridinium (NH+) group as well as the higher pKa of 2,4-dipicH2 and 2,5-dipicH2. The lower
pKa of the latter two ligands belongs to the carboxylic group at position 4 and 5, respectively.
Comparing the pKa values to that of Hpic, it is worth mentioning that the methyl substituent has
no measurable effect at position 3, while the bromo and the carboxylic groups decrease the
pKa (NH+) significantly due to the electron withdrawing power of the halogen substituent and
the mesomeric effect of the COO‒ moiety.
Based on the determined pKa values it can be declared that all the studied ligands are
present in their completely deprotonated forms (L‒: pic, 3-Me-pic, 5-Br-pic; L2‒: 2,4-dipic, 2,5dipic) at pH 7.4 resulting in their strongly hydrophilic character (logD7.4 < ‒2).
Table 2. Proton dissociation constants (pKa) of the studied ligands determined by pH-potentiometric
and UV-vis spectrophotometric titrations; max and molar absorptivity () values for the ligand species
in the different protonation states. {T = 25.0˚C, I = 0.20 M (KCl)}
Method
pKa
pKa (NH+)
max (nm) / (M-1cm-1)
(COOH)
pic
3-Me-pic
pH-metry
<1
5.13 ±0.03
HL: 263 / 7100
UV-vis
<1
5.07 ±0.01
L‒: 263 / 3900
pH-metry
<1
5.16 ±0.03
HL: 274 / 6820
UV-vis
<1
5.16 ±0.03
L‒: 268 / 4400
16
5-Br-pic
2,4-dipic
pH-metry
<1
3.44 ±0.02
HL: 278 / 6570; 240 / 9770
UV-vis
<1
3.34 ±0.04
L‒: 268 / 4400; 232 / 10650
pH-metry
1.84 ±0.05
4.70 ±0.02
H2L: 278 / 5100
UV-vis
1.9 ±0.1
4.56 ±0.08
HL‒: 274 / 5980
L2‒: 276 / 3700
2,5-dipic
pH-metry
2.19 ±0.05
4.63 ±0.04
H2L: 272 / 6900
UV-vis
2.16 ±0.02
4.57 ±0.01
HL‒: 272 / 7100
L2‒: 272 / 5500
Hydrolytic behavior of the organometallic cation [Ru(6-toluene)(H2O)3]2+ has been
already studied by Buglyó et al. in the presence and in the absence of chloride ions [43]. In the
latter case the fast hydrolysis of the aquated organoruthenium cation yields the species [(Ru(6toluene))2(μ2-OH)3]+ that becomes predominant at pH > 5. When 0.2 M KCl was used as the
background electrolyte, as in our studies, formation of various chlorido and mixed
chlorido/hydroxido species as intermediates was found in addition to the major hydrolysis
product [(Ru(6-toluene))2(μ2-OH)3]+. In a good accordance with their findings based on the
combined use of 1H NMR spectroscopy and ESI-MS, we have also detected three different
species based on the 1H NMR spectra recorded at various pH values (Fig. S7). Namely, the
identified species are [Ru(6-toluene)(H2O)2Cl]+ (= M), [(Ru(6-toluene))2(-OH)2Cl]+ (=
[M2(OH)2]) and [(Ru(6-toluene))2(-OH)3]+ (= [M2(OH)3]+). Overall stability constants for
the dinuclear hydrolysis products [(Ru(6-toluene))2(2-OH)i](4-i)+ (i=2,3) were determined by
pH-potentiometric and UV-vis spectrophotometric titrations at 0.2 M chloride ion concentration
(Table 3) and are in good agreement with data obtained by Buglyó et al. using pH-potentiometry
[43]. Notably these are conditional stability constants being valid only at 0.2 M KCl ionic
strength. Concentration distribution curves were computed on the basis of the stability constants
determined by pH-potentiometry showing that the hydrolysis is suppressed somewhat due to
the presence of chloride ions, since [M2(OH)3] dominates only at pH > 6 (Fig. S8). The 1H
NMR signals of the three kinds of species (M, [M2(OH)2], [M2(OH)3]) could be integrated and
distribution of the organometallic fragment was calculated showing an acceptable match
between the two kinds of methods.
17
3.3. Complex formation equilibria of [Ru(6-toluene)(H2O)3] 2+ with the picolinate ligands:
stability, deprotonation, chloride ion affinity and lipophilicity
Complexation processes were studied by the combined use of pH-potentiometric, UV-vis
spectrophotometric titrations and 1H NMR spectroscopy in a 0.2 M chloride-containing
medium. Therefore the formation (logK [ML]) and deprotonation (pKa [ML]) constants
determined herein are considered as conditional stability constants. The complex formation
between [Ru(6-toluene)(H2O)3]2+ and the studied bidentate picolinate ligands follows a fairly
simple scheme (Chart S1). Namely a mono complex [Ru(6-toluene)(L)(Z)] (=[ML]) is formed,
and a mixed hydroxido species [ML(OH)] appears by the deprotonation of the coordinated H2O
molecule and/or by the displacement of the chlorido co-ligand by OH‒ in the basic pH range,
similarly to the behavior of analogous half-sandwich Ru(6-p-cymene) complexes [22,23]. The
complex formation of the organometallic cation with the picolinate ligands was found to be a
rather slow process. E.g. the steady state could be reached after more than 35 min in the [Ru(6toluene)(H2O)3]2+ ‒ picH system at pH 2.8 as the time-dependence of the UV-vis spectra
indicates (Fig. 3). This slow reaction hindered the application of conventional pHpotentiometric titrations to determine the logK [ML] values. In order to solve this problem,
individual samples were prepared by the addition of different amount of strong base under
argon, and the UV-vis spectra and the actual pH values were measured after 4 h. Based on the
recorded spectra it could be concluded that the complex formation proceeds in a great extent
already at pH 2 in all cases. As a consequence logK [ML] constants were determined from the
UV-vis spectral changes of the metal-to-ligand charge-transfer (Ru 4d6→π*) and ligand (π
→π*) transition bands in the pH range from 0.7 to 3.0 in the case of 3-Me-pic and 2,4-dipic
(Table 2). On the other hand, the spectra were unchanged from pH 3 down to pH 0.7 in the
[Ru(6-toluene)(H2O)3]2+ – pic/5-Br-pic/2,5-dipic systems showing negligible decomposition
of the complexes under such strongly acidic conditions. Thus for the logK [ML] constants only
a lower limit could be estimated (Table 3). Based on these findings the complexation of pic
with [Ru(6-p-cymene)(H2O)3]2+ was reinvestigated using longer incubation times (4 h) needed
to reach steady state in the presence of chloride ions (0.2 M KCl) and a higher logK [ML] value
(>11.5) was obtained than previously published [23].
18
Abs. at 310 nm
Absorbance
0.8
0.6
0.4
34 min
0.3
0.2
0.1
0
10
20
30
40
t / min
0.2
5 min
0.0
220
280
340
400
/ nm
460
520
Figure 3. Time-dependence of UV-vis absorption spectra recorded for the [Ru(6-toluene)(H2O)3]2+ ‒
picH (1:1) system in the presence of chloride ions. The inset shows the absorbance changes at 310 nm.
{cRu = 102 M; T = 25 ˚C; I = 0.20 M (KCl); ℓ = 1.0 cm}.
Table 3. Stability constants logK [ML], pKa [ML] values of the [Ru(6-toluene)(H2O)3]2+ complexes
formed with picolinate ligands in 0.2 M chloride-containing aqueous solutions determined by various
methods; H2O/Cl− exchange constants (logK’) for the [Ru(6-toluene)(L)(H2O)]+ complexes and pM*
values at pH = 7.4 (pM* = −log([M] + [M2(OH)3] + [M2(OH)2]) at cM = 100 μM). {T = 25.0 ˚C, I =
0.20 M (KCl)}
logK [ML]
pKa [ML]
pKa [ML]
pM* logK’
(H2O/Cl−)
ligand
complex UV-vis
UV-vis
pH-metry
pic
1
3-Me-pic
>10.6 a
8.53 ±0.01 b
8.47 ±0.01 b
>5.8
1.33 ±0.01
2
9.87 ±0.01
8.71 ±0.01
8.68 ±0.05
5.3
1.32 ±0.01
5-Br-pic
3
> 8.9 a
8.47 ±0.01
8.41 ±0.03
>4.7
1.50 ±0.01
2,4-dipic
4
11.22 ±0.07
8.44 ±0.01
8.37 ±0.06
6.2
1.23 ±0.01
2,5-dipic
5
> 11.9 a
8.58 ±0.01
8.38 ±0.07
>6.7
1.09 ±0.01
a
UV-vis
Estimated values based on UV-vis spectrum recorded at pH 0.7; b pKa [ML] values based on 1H NMR
titrations: 8.52 ±0.09 (0.2 M KCl) and 7.87 ±0.09 (0 M KCl)
Increasing the pH values the studied [ML] complexes may undergo a combination of
deprotonation and decomposition. Deprotonation of the coordinated water molecule (and/or Cl‒
→OH‒ exchange) results in the formation of mixed hydroxido [ML(OH)] complexes, while
decomposition can yield unbound ligand and metal ion in hydrolyzed forms depending on the
actual pH. The recorded UV-vis spectra were the same in a wide pH range (e.g. in the [Ru(619
toluene)(H2O)3]2+ ‒ 3-Me-picH system at pH between 3.1 and 7.6 shown in Fig. 4) , while
significant spectral changes are observed at pH > 8 due to the formation of [ML(OH)]. The
appearance of isosbestic points suggests that the metal complexes do not decompose under
these conditions; merely they are deprotonated almost in all cases. It should be noted that the
complex of 5-Br-pic showed a low extent of decomposition in the basic pH-range. Based on
these spectral changes pKa [ML] constants were determined for the complexes (Table 3).
Notably, the spectra of the complexes did not change over a 24 h period at both pH 7.4 and 11
values, and the deprotonation process was found to be rather fast. Therefore pH-potentiometric
titrations were also performed to determine pKa [ML] constants (Table 3) started from pH ~4
but only after a 4 h waiting period whilst the formation of [ML] becomes complete. pKa [ML]
Absorbance
0.8
Abs. 306 nm
constants obtained by the two kinds of methods are in a good agreement.
ligand
pH = 2.0
0.6
0.45
0.40
0.35
0.30
0.5
pH = 3.0
3.5
6.5
9.5
pH
0.4
11.0
Ru(6-toluene)(H2O)3]2+
pH = 2.0
0.2
0.0
230
290
350
/ nm
410
470
Figure 4. UV-vis absorption spectra recorded for the [Ru(6-toluene)(H2O)3]2+ ‒ 3-Me-picH (1:1)
system in the presence of chloride ions in the pH range from 3 up to 11. The inset shows the absorbance
changes at 306 nm at pH between 0.7 and 11. {cRu = 102 M; T = 25 ˚C; I = 0.20 M (KCl); ℓ = 1.0 cm}.
In addition 1H NMR spectra were also recorded for the [Ru(6-toluene)(H2O)3]2+ – pic
system in the presence of 0.2 M chloride ions at a 1:1 metal-to-ligand ratio at various pH values
using 4 h incubation time (Fig. 5). The spectra undoubtedly reveal that neither a free metal ion
nor a ligand is present in the whole pH range studied (pH = 2 – 11.5), which means that the
complex does not suffer from decomposition at 1 mM concentration due to its high stability.
The aqua [ML(H2O)] and the chlorinated [ML(Cl)] complexes were identified in the acidic pH
range. An upfield shift of all peaks belonging to the [ML(H2O)] complex is observed in the
basic pH range due to the fast exchange process on the NMR time scale between the aquated
and the mixed hydroxido [ML(OH)] species. In the meanwhile the intensity of the peaks
20
belonging to the [ML(Cl)] complex is decreased. Based on the integrals of the CH(6) toluene
proton in the acidic pH range the [ML] complex is mainly chlorinated (~83% [ML(Cl)]). As
the [ML(OH)] starts to be formed the three species are present together in the solution, and their
equilibrium concentrations cannot be simply calculated due to the fast exchange process.
[ML(OH)]
[ML(H2O)]+
(a)
9.5
(c)
(b)
pH =
11.40
10.43
9.56
8.96
8.60
8.02
7.56
6.96
6.14
3.59
2.54
[MLCl]
9.0 8.5
8.0 7.5
6.0
/ ppm
5.5
2.38
2.15
Figure 5. 1H NMR spectra of [Ru(6-toluene)(H2O)3]2+ ‒ picH (1:1) system in aqueous solution in the
presence of 0.2 M chloride ions at the indicated pH values in the regions of the ligand protons (a), the
toluene CH protons (b) and the toluene CH3 protons (c). {cRu = 1 mM; T = 25 ˚C; I = 0.20 M (KCl);
D2O; pH = pD×0.93+0.40 [51]}.
Therefore, the pKa of the aqua [ML(H2O)] was determined (pKa = 7.87 ±0.09) based on the pHdependent chemical shift (δ) values of [ML(H2O)] and [ML(OH)] species. (Notably this value
equals to the pKa [ML] in the chloride-free medium.) Using this constant the ratio of the latter
two species can be calculated at any chosen pH and then the actual concentrations of all the
three complexes could be computed (Fig. 6). From the ratio of the summed concentration of
[ML(Cl)] and [ML(H2O)] (as [ML] species) and that of [ML(OH)] pKa [ML] in the 0.2 M
chloride-containing medium was calculated (Table 3) representing a good match to the data
obtained by the other two methods.
21
Ru(II)(6-toluene) %
100
[ML(Cl)]
80
[ML(OH)]
60
40
[ML(H2O)]+
20
0
2
4
6
pH
8
10
Figure 6. Distribution of Ru(6-toluene) in the [Ru(6-toluene)(H2O)3]2+ ‒ picH (1:1) system in the
presence of 0.2 M chloride ions in the pH range from 2 up to 10 based on the 1H NMR peak integrals
for the CH(6) toluene proton of species identified based on Fig. 5. The ratio of the [ML(H2O]+ and
[ML(OH)] at a given pH is calculated using the pKa [ML] of the aqua complex. {cRu = 1 mM; T = 25 ˚C;
I = 0.20 M (KCl)}.
In order to compare the stability of the studied Ru(6-toluene) complexes of the different
picolinates to each other pM* values were computed using the experimentally determined
equilibrium constants (Table 3). pM is the negative logarithm of the equilibrium concentration
of the unbound metal ion, and a higher pM value indicates a stronger metal ion binding ability
of the ligand under given circumstances. Due to the hydrolysis of the Ru(6-toluene) fragment
pM* was computed reflecting the unbound fraction of the metal ion where pM* = −log([M] +
[M2(OH)2] + [M2(OH)3]). These pM* values indicate the formation of relatively high stability
complexes suggesting the following stability order at pH 7.4: 5 > 4 > 1 > 2 > 3. E.g.
decomposition of 1% and 20% are estimated for complexes 1 and 3 at 100 μM concentration,
respectively. Based on the speciation data it can be concluded that the complexes are present
mainly in their [ML] forms at pH 7.4, and they are only partly deprotonated ([ML(OH)] ~ 10%)
in the 0.2 M chloride-containing medium.
The ratio of the chlorinated and aqua complexes ([ML(Cl)] and [ML(H2O)]) can be
characterized by the H2O/Cl‒ exchange constant, which was determined by UV-vis
spectrophotometry using the same approach that we used in our previous works for analogous
Rh(5-C5Me5) complexes [52,53]. Representative UV-vis spectra recorded at various chloride
ion concentrations for the complex 1 and the measured and fitted absorbance values are shown
in Fig. S9. Notably a lower H2O/Cl‒ exchange constant allows an easier replacement of Cl‒ by
water or by donor atoms of biomolecules. The logK’ (H2O/Cl−) values (Table 3) obtained for
22
1-5 reflect a moderate affinity towards chloride ions which is much lower compared to e.g. the
analogous Rh(5-C5Me5) picolinate complexes [52,53]. The dependence of cytotoxicity on
chloride ion affinity has been reported for several Ru(η6-arene) complexes [54], however many
other factors such as lipophilicity have a strong influence on the pharmacological activity.
Therefore, distribution coefficients at pH 7.4 (logD7.4) were determined for the complexes 1-5,
for the metal-free ligands and for the precursor [Ru(η6-toluene)Cl(μ-Cl)]2 at various chloride
ion concentrations according to the chloride content of blood serum: ~100 mM, cell plasma:
~24 mM and cell nucleus: ~4 mM. The precursor, the ligands, the complexes 2, 4 and 5 were
found to be very hydrophilic at each studied chloride ion concentration (logD7.4 < ‒2.5). logD7.4
values only for complexes 2 and 3 could be determined accurately by the applied n-octanolwater partitioning (Fig. 7), and they exhibit increasing lipophilicity with increasing chloride ion
concentration, although even at 100 mM they are considered as fairly hydrophilic compounds.
They have stronger hydrophilic character in the presence of less chloride ions since they are
more aquated and the complex turns to be charged ([ML(Cl)] → [ML(H2O]+).
c (Cl-):
-1.4 0.2
4 mM
-2.4 0.2
-1.16 0.06
-1.60 0.07
24 mM
-0.97 0.04
100 mM
-1.16 0.01
-2.5
-2
-1.5
-1
-0.5
0
logD7.4 of complexes
Figure 7. n-Octanol/water distribution coefficients at pH 7.4 (logD7.4) for complexes 2 (white bars) and
3 (grey bars) at various chloride ion concentrations {T = 25 °C, pH = 7.4 (20 mM phosphate buffer)}
3.4. Cytotoxic and antiproliferative activity in human cancer cell lines
In order to evaluate the biological effects of complexes 1-5, antiproliferative and cytotoxicity
assays were applied in doxorubicin-sensitive (Colo 205) and multidrug resistant (Colo 320)
human colonic adenocarcinoma cell lines. The resistance of Colo 320 cells is primarily
mediated by the overexpression of ABCB1 (P-glycoprotein), a member of the ATP-binding
cassette (ABC) transporter family, which pumps out xenobiotics from the cells. Cytotoxicity
was measured in normal human embryonal lung fibroblast cells (MRC-5) as well. In addition
the corresponding free ligands and the precursor [Ru(η6-toluene)Cl(μ-Cl)]2 were tested for
23
comparison. In case of the antiproliferative assay, a low cell number (6×103 cells/well) was
chosen and the incubation period of the MTT assay was longer (72 h). Using these conditions
information can be provided about the activity of the complexes to inhibit cell proliferation. In
case of the cytotoxicity assay, a high cell number (2×104 cells/well) was used and the inhibition
of cell growth was determined after 24 h by MTT assay. The latter assay is an important tool to
investigate the toxicity of the complexes. In both assays cisplatin was used as a positive control.
IC50 values are collected in Table S3. The ligands and the precursor did not show either
cytotoxic or antiproliferative activities (IC50 >100 μM).
The complexes 1-5 did not possess any cytotoxic activity on the colon adenocarcinoma
cell lines and on the normal MRC-5 human embryonic fibroblast cells. On the other hand the
complexes 1 and 2 showed a moderate antiproliferative effect on the MDR Colo 320 colon
adenocarcinoma cell line with IC50 values of 84.84 ± 4.79 and 79.19 ± 6.71 μM, respectively.
Interestingly, these complexes had greater activity on the MDR cell line than on the sensitive
Colo 205 cell line implying the selectivity of these complexes towards the MDR colon
adenocarcinoma cell line.
Table 4. pKa of the complexes [ML(H2O)]+ in the absence and in the presence of chloride ions at 0.2 M
ionic strength, the Cl‒/H2O exchange constants (logK’ (H2O/Cl‒) for the [ML(H2O)]+ + Cl‒ ⇌ [ML(Cl)]
+ H2O equilibrium, estimated ratio of the chlorinated complex [ML(Cl)] at 4 and 100 mM chloride ion
concentrations, and representative IC50 values measured in human cancer cells for the complexes of
[Ru(6-toluene)(pic)Cl],
[Ru(6-p-cymene)(pic)Cl],
[Os(6-p-cymene)(pic)Cl]
and
[Rh(5-
C5Me5)(pic)Cl].
1
[Ru(6-p-
[Os(6-p-
[Rh(5-
cymene)(pic)Cl]
cymene)(pic)Cl]
C5Me5)(pic)Cl]
pKa (0 M Cl‒)
7.87
8.00 b
6.67 d
9.32 e
pKa (0.2 M Cl‒)
8.53
8.90 b
n.d.
10.44 e
logK’ (H2O/Cl‒)
1.33
1.83 b
n.d.
2.20 e
rate of Cl‒/H2O
fast
fast b
slower d
fast e
t1/2 ~ 12 min
[ML(Cl)] fraction
c(Cl‒) = 4 mM 68%
87% b
100% d
94% e
c(Cl‒) = 100 mM 8%
22% b
28% d
36% e
24
IC50 (M)
84.84±4.79 82 (HeLa) c
(Colo320)
36 (FemX) c
17 (A549) d
343 (A549) e
4.5 (A2780) d
258 (CH1) e
a
a
Antiproliferative activity; b Data taken from Ref. 23.; c Data taken from Ref. 27.; d Data taken from Ref. 29.; e
Data taken from Ref. 52.
Among the half-sandwich organometallic complexes of picolinic acid reported in the
literature [Os(6-p-cymene)(pic)Cl] has the highest cytotoxic effect [29], [Ru(6-pcymene)(pic)Cl] is moderately cytotoxic [27], while compounds [Ru(6-toluene)(pic)Cl] (1)
and [Rh(5-C5Me5)(pic)Cl] [52] possess much lower activity. In order to compare these
complexes for getting insight their different biological activity some physico-chemical
properties such as pKa [ML], logK’ (H2O/Cl‒) are collected in Table 4. A low pKa [ML] is
generally considered to be unfavorable as the chance for the formation of the ternary mixed
hydroxido [ML(OH)], that is believed to be less prone to interact with biomolecules [55],
becomes higher at pH 7.4. In this context the Os(II) complex would be expected to be the least
active. The effect of a strong chloride ion affinity (higher logK’ (H2O/Cl‒), thus higher fraction
of the chlorinated complex) can be dual. If the affinity is high the complex can retain the original
chlorido ligand coordinated more efficiently in the serum and the neutral [ML(Cl)] complex
can go across the cell membrane easier via passive transport. Additionally the lipophilicity of
the complex should be also optimal; however no logD7.4 values are available for most of these
complexes. On the other hand, after entering the cell, it is assumed that the lower intracellular
chloride content can induce partial aquation of the complexes leading to the formation of the
active aqua complex. When the chloride affinity is high, the replacement of Cl‒ by water or
donor atoms of proteins is aggravated. Besides these properties the reaction rate of the
displacement reaction is also an important factor. Based on these parameters it seems that
relatively slow kinetics of the Os(II) complex is advantageous. Whilst the strong hydrophilic
character, fast Cl‒/water exchange process of the [Ru(6-toluene)(picolinate)Cl] studied in this
work can be at least partly responsible due to the lack of their cytotoxicity.
3.5. Interaction of complexes 1 and 2 with human serum albumin
HSA is the most abundant plasma protein and serves as a transport vehicle for a wide variety
of endogenous compounds and pharmaceuticals. Binding to HSA has a strong impact on the
pharmacokinetic properties of drugs. In addition HSA-bound drugs are known to accumulate
in solid tumors as a consequence of the enhanced permeability and retention effect, which can
25
be an operative way of selective tumor targeting [56]. This protein has various metal binding
sites such as the N-terminal site, the reduced Cys34 residue, the multi-metal binding site and
certain side chain donor atoms such as imidazole nitrogens of His are also able to coordinate to
the metal ions [57,58]. On the other hand nonspecific binding pockets located in subdomains
IIA and IIIA are willing to accommodate compounds of a wide variety [58]. In all diversified
binding modes are possible for potential metallodrugs.
Interaction of complexes 1 and 2 representing moderate antiproliferative activity (see
Section 3.4) towards HSA was studied by mainly ultrafiltration/UV-vis and spectrofluorometric
methods. All measurements were performed at pH 7.4 at 25 ºC using a modified phosphate
buffered saline (PBS’) in which the concentration of the chloride ions corresponds to that of the
human blood serum. First of all binding of 1 to HSA was monitored by 1H NMR spectroscopy.
Spectra were recorded for 1 in the absence or in the presence of the protein after a 24 h
incubation period (Fig. S10). (This incubation time was chosen as the preliminary timedependence studies showed that the reaction is relatively slow, depending on the conditions
several hours are needed to reach the equilibrium state.) It was found that the signal of the
toluene methyl group is shifted in the presence of HSA and no free ligand was detected. These
observations strongly suggest the formation of ternary adducts with the protein without ligand
cleavage. Then the direct interaction of complexes 1, 2 and the [Ru(η6-toluene)Cl(μ-Cl)]2
precursor was followed by ultrafiltration. The unbound, low molecular mass (LMM) fractions
after separation were analyzed by UV-vis quantification. Analysis of the recorded spectra
confirmed that the complexes 1, 2 are intact upon binding as we could not detect free ligand in
the LMM fraction (Fig. S11). Comparing the spectra recorded after the separation to reference
spectra the ratio of the bound compounds per HSA was calculated and plotted against the ratio
[bound complex] / c (HSA)
of the total concentrations of the complexes and the protein (Fig. 8).
7
Ru precursor
6
5
2
4
3
1
2
1
0
0
2
4
6
8
c (complex) / c (HSA)
26
10
Figure 8. Ratio of the bound complexes (Ru precursor, 1 and 2) and HSA plotted against the ratio of
the total concentrations of the complexes and HSA calculated from the UV-vis spectra recorded for the
LMM fractions of the ultrafiltered samples. {Original sample composition: HSA: 40 µM; complexes: 0400 µM; T = 25 ˚C; pH = 7.4 in PBS’; incubation time: 24 h}.
These formation curves show the binding at multiple sites for the Ru precursor and for the
complexes, although no saturation could be achieved up to the applied 10-fold complex excess.
The binding of the precursor is almost quantitative, but realized at a lower level compared to
the Rh(5-C5Me5) precursor [45]. The binding of 1 is somewhat weaker compared to 2;
however at least 3 or 5 binding sites are feasible for them, respectively.
100
I/I0 %
80
60
40
20
0
0
2
4
6
8
10
c (complex) / c (HSA)
Figure 9. Changes of fluorescence emission intensities at 338 nm plotted against the complex-to-HSA
ratios for 1 (●), 2 (×) and the Ru precursor (▲) using 295 nm excitation and 340 nm emission
wavelengths. {cHSA = 1 µM; complexes: 0-10 µM; T = 25 ˚C; pH = 7.4 in PBS’; incubation time: 24 h}.
In order to obtain preliminary information about the binding sites the interaction of 1, 2
and the Ru(II) precursor were monitored by fluorometry. HSA contains a single Trp (214)
residue near site I (at subdomains IIA) that is responsible for the majority of the intrinsic
fluorescence of the protein. Upon excitation at 295 nm its emission can be attenuated by a
binding event close to Trp214 [58,59]. It is worth mentioning that coordination of protein side
chains such as histidine nitrogens (e.g. His242) [59] located nearby this site to the ruthenium
complexes by the substitution of the chlorido/aqua ligand at the third coordination site is very
feasible. Addition of the Ru(II) compounds to HSA quenches the Trp214 fluorescence emission
(Fig. 9) indicating that the conformation of the hydrophobic binding pocket is significantly
affected upon their binding. Based on the emission intensity changes quenching constants were
computed. LogKQʹ values of 5.25 ±0.01, 4.16 ±0.01 and 4.18 ±0.01 were obtained for the Ru
precursor, 1 and 2, respectively. These values reflect fairly strong binding of the precursor, and
27
a moderate and similar binding of 1 and 2 at this particular site of HSA. As more than one
binding sites are suggested on the basis of the ultrafiltration measurements, the complexes 1
and 2 (as well as the precursor) should be bound on other sites beside site I as well, such as the
more accessible surface donors. Among the side chain donors His, Met and Cys residues are
suggested to be responsible to coordinate to Ru complexes [60,61]. The prominent role of His
was pointed out in the case of Rh(5-C5Me5) complexes in our former work [45]. Therefore
interaction of 1 and the precursor with 1-methylimidazole (N-MeIm), a monodentate model
compound of His, was screened by 1H NMR spectroscopy. It was found that 95% the Ru(II)
precursor is bound to N-MeIm at 1:1 ratio (Fig. S12), while 100% of the analogous [Rh(5C5Me5)Cl(μ-Cl)]2 precursor is bound under the same condition [45]. In the case of complex 1
the original picolinate ligand was not replaced by the model compound but formation of ternary
[Ru(η6-toluene)(pic)(N-MeIm)] complex of significant fraction (1: 85%) was observed (Fig.
S13). This observation confirms the feasible coordination of the imidazole nitrogen of His at
the third coordination site of the studied picolinate complexes.
4. Conclusions
Metal complexes of 2-picolinic acid and its 3-methyl, 5-bromo, 4-carboxylic, 5-carboxylic
derivatives formed with Ru(6-toluene) organometallic fragment were synthesized and
characterized in solid phase and in solution. The structures of four complexes were also
determined by single-crystal X-ray diffraction showing a pseudo-octahedral “pianostool”
geometry, and the deprotonated picolinates bind in a bidentate mode via (N,O) donor atoms and
the coordination sphere is completed by a chlorido ligand. Complex formation equilibrium
processes were studied in aqueous solution by the combined use of UV-visible
spectrophotometry, pH-potentiometry and 1H NMR spectroscopy in the presence of chloride
ions in addition to the characterization of the proton dissociation equilibria of the ligands. The
complex formation reached a significant extent already at pH 0.7 representing prominently high
stability and was found to be relatively slow (ca. 35 min); while deprotonation of the complex
and water/chloride exchange processes took place fast. By means of these methods we could
demonstrate exclusive formation of mono complexes such as [Ru(6-toluene)(L)(Z)] (L:
completely deprotonated ligand; Z = H2O/Cl‒) and [Ru(6-toluene)(L)(OH)] in solution.
Moderate pKa values (8.3-8.7) were obtained reflecting the formation of ca. 10% mixed
hydroxido species at pH 7.4 in the presence of 0.2 M KCl. The chloride ion affinity of the
complexes was characterized by moderate H2O/Cl− co-ligand exchange equilibrium constants
28
(logK’ H2O/Cl− = 1.1-1.5) which are lower than those of the analogous Ru(6-p-cymene) and
Rh(5-C5Me5) compounds.
All the studied metal complexes exhibit a rather hydrophilic character at 100 mM
chloride concentration and become even more hydrophilic at lower chloride content. The
studied complexes were not cytotoxic against colon adenocarcinoma cell lines and normal
MRC-5 human embryonic fibroblast cells. However, the complexes formed with 2-picolinic
acid (1) and its 3-methyl derivative (2) represented a moderate antiproliferative effect (IC50 =
84.84, 79.19 μM) on the multidrug resistant Colo 320 colon adenocarcinoma cell line revealing
considerable MDR selectivity. Interaction of complexes 1 and 2 with the blood transport protein
HSA was investigated by ultrafiltration and fluorometry. The binding is relatively slow and no
ligand cleavage was observed, thus formation of ternary adducts with the protein via
coordination bonds at several binding sites (at least 3-5) is suggested. Complex 1 represents a
somewhat weaker overall binding compared to 2, while their binding at site I is fairly similar
based on the Trp(214) quenching studies. 1-methylimidazole binds efficiently to these
complexes at the third coordination site suggesting the probable binding of imidazole nitrogens
of the protein with non-dissociative characteristics.
Abbreviations:
5-Br-picH
5-bromo-2-pyridinecarboxylic acid
cisplatin
cis-[Pt(NH3)2(Cl)2]
D7.4
distribution coefficients at physiological pH
2,4-dipicH2
2,4-pyridinedicarboxylic acid
2,5-dipicH2
2,5-pyridinedicarboxylic acid
DSS
4,4-dimethyl-4-silapentane-1-sulfonic acid
EMEM
Eagle’s Minimal Essential Medium
HSA
human serum albumin
MDR
multidrug resistance
3-Me-picH
3-methylpyridine-2-carboxylic acid
MTT
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
NKP-1339
sodium trans-[Ru(III)Cl4(Ind)2], Ind = indazole; IT-139
N-MeIm
1-methylimidazole
PBS’
modified phosphate-buffered saline
picH
pyridine-2-carboxylic acid, 2-picolinic acid
UV-vis
UV-visible
29
Acknowledgements
This work was supported by National Research, Development and Innovation Office-NKFIH through
projects GINOP-2.3.2-15-2016-00038, OTKA FK 124240, OTKA K 115762, the New National
Excellence Program UNKP-17-4-III-SZTE-13 (E.A.E.), the J. Bolyai Research Scholarship of the
Hungarian Academy of Sciences (N.V.M.) and Ministry of Education, Science and Technological
development – Republic of Serbia (MPNTR 172035 and MPNTR postdoctoral grant (J. M. P.)).
Appendix A. Supplementary data
Supplementary data associated with this article can be found online at…
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