← Back
New ruthenium compounds bearing semicarbazone 2-formylopyridine moiety: Playing with auxiliary ligands for tuning the mechanism of biological activity.
Accepted Manuscript
New
ruthenium
compounds
bearing
semicarbazone
2-formylopyridine moiety: Playing with auxiliary ligands for
tuning the mechanism of biological activity
Michał Łomzik, Olga Mazuryk, Dorota Rutkowska-Zbik,
Grażyna Stochel, Philippe C. Gros, Małgorzata Brindell
PII:
DOI:
Reference:
S0162-0134(17)30314-8
doi: 10.1016/j.jinorgbio.2017.07.006
JIB 10253
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
3 May 2017
4 July 2017
9 July 2017
Please cite this article as: Michał Łomzik, Olga Mazuryk, Dorota Rutkowska-Zbik,
Grażyna Stochel, Philippe C. Gros, Małgorzata Brindell , New ruthenium compounds
bearing semicarbazone 2-formylopyridine moiety: Playing with auxiliary ligands for
tuning the mechanism of biological activity, Journal of Inorganic Biochemistry (2017),
doi: 10.1016/j.jinorgbio.2017.07.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
ACCEPTED MANUSCRIPT
New Ruthenium Compounds bearing Semicarbazone 2Formylopyridine Moiety:
Playing with Auxiliary Ligands for Tuning the Mechanism of
CR
IP
T
Biological Activity
US
Michał Łomzik,#[a],[b] Olga Mazuryk,#[a] Dorota Rutkowska-Zbik,[c] Grażyna Stochel, [a] Philippe
AN
C. Gros,* [b] and Małgorzata Brindell*[a]
M
[a] Department of Inorganic Chemistry, Faculty of Chemistry, Jagiellonian University Ingardena
ED
3, 30-060 Krakow, Poland. brindell@chemia.uj.edu.pl
[b] Université de Lorraine, CNRS, UMR SRSMC, HecRIn, Boulevard des Aiguillettes
PT
Vandoeuvre-Lès-Nancy, France. philippe.gros@univ-lorraine.fr
CE
[c] Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences,
#
AC
Niezapominajek 8, 30-239 Krakow, Poland.
both authors contributed equally to this work
* corresponding authors: P.G. : email: philippe.gros@univ-lorraine.fr, phone: +33383684979 ;
M. B. : email: brindell@chemia.uj.edu.pl, phone: +48126632221.
Keywords ruthenium, polypyridine, semicarbazone, anticancer activity, apoptosis, auxiliary
ligands
1
ACCEPTED MANUSCRIPT
Abstract
Two ruthenium(II) complexes Ru1 and Ru2 bearing as a one ligand 2,2’-bipyridine substituted
IP
T
by a semicarbazone 2-formylopyridine moiety (bpySC: 5-(4-{4'-methyl-[2,2'-bipyridine]-4-
CR
yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone) and as the others 2,2’-bipyridine (bpy)
and 4,7-diphenyl-1,10-phenanthroline (dip), respectively, as auxiliary ligands have been
US
prepared. Their biological activity has been studied on murine colon carcinoma (CT26) and
human lung adenocarcinoma (A549) cell lines. The anti-proliferative activity was dependent on
AN
the presence of bpy or dip in the complex, with one order of magnitude higher cytotoxicity for
M
Ru2 (dip ligands). Ru1 (bpy ligands) exhibited a distinct increase in cytotoxicity going from 24
to 72 h of incubation with cells as was not observed for Ru2. Even though both studied
ED
compounds were powerful apoptosis inducing agents, the mechanism of their action was entirely
PT
different. Ru1-incubated A549 cells showed a notable increase in cells number in the S-phase of
the cell cycle, with concomitant decrease in the G2/M phase, while Ru2 promoted a cell
CE
accumulation in the G0/G1 phase. In contrast, Ru1 induced marginal oxidative stress in A549
AC
cell lines even upon increasing the incubation time. Even though Ru1 preferably accumulated in
lysosomes it triggered the apoptotic cellular death via an intrinsic mitochondrial pathway. Ru1incubated A549 cells showed swelling and enlarging of the mitochondria. It was not observed in
case of Ru2 for which mitochondria and endoplasmic reticulum were found as primarily
localization site. Despite this the apoptosis induced by Ru2 was caspase-independent. All these
findings point to a pronounced role of auxiliary ligands in tuning the mode of biological activity.
2
ACCEPTED MANUSCRIPT
1. Introduction
One of the most important issues in the field of medicinal inorganic chemistry concerns the
discovery of more effective and safer anticancer drugs. Many of the currently studied
metallodrugs are designed to be prodrugs and selectively activated in the cancer tissue. However,
T
a major advantage of this strategy can also be its main weakness, since several possible active
IP
molecules are produced and start to interact with biomolecules in the human body. Investigation
CR
of all these interactions is very challenging and is still the focus of many studies. Because of this,
US
the perspective about designing new anti-cancer drugs slowly shifted [1], and an increased
attention is paid to ruthenium compounds inert to substitution mainly those with polypyridyl ligands.
AN
Their electronic and structural properties can be tuned easily by the selection of appropriate ligands
giving rise to the formation of versatile compounds which might be designed either for therapeutic
ED
M
and/or optical imaging purpose.
The design of potential drugs based on organic compounds usually involves the modification of the
PT
lead structure in respect to the site of action, or otherwise new compounds are formed based on the
CE
known molecular targets (e.g. receptor proteins or enzymes) [2]. Even though many active rutheniumbased compounds have been discovered, their biological activity mechanism remains unclear and
AC
their targets still unidentified. The lack of this knowledge hampers rational design of new anticancer
agents based on these complexes. A new approach recently developed, is to design compounds
where one or more polypyridyl ligands around ruthenium center are modified by anchoring an
organic molecule with a known biological target or mechanism of action. The choice of the
organic targeting molecule is critical since it can have various functions such as i) tuning the
lipophilic character of the whole Ru complex for specific subcellular accumulation
(mitochondria, reticulum endoplasmic, nucleus, lysosome) [3, 4]; ii) targeting the specific
3
ACCEPTED MANUSCRIPT
receptors, enzymes or cellular processes [5, 6]; iii) oxygen-dependent accumulation in cells [7,
8]; iv) creating synergistic effects between the biologically active ligand systems and the metal
centre; v) tuning the photophysical/photochemical properties for an appropriate light activation
(photodynamic and photoactivated therapy) [9, 10]; vi) combining therapy and diagnosis
IP
T
(theranostics) [11] and others.
CR
In this context, we have designed two new ruthenium(II) polypyridyl complexes in which one
bipyridyl ligand is modified by a semicarbazone 2-formylopyridine (Scheme 1). α-N-heterocyclic
US
(thio)semicarbazones and their metal complexes are known as anticancer agents [12-14] therefore
AN
combining such moiety with Ru(II) polypyridyl complexes might bring additional benefits
towards their anticancer activity. The chosen semicarbazone is a structural analogue of 3-
M
aminopyridine-2-carboxyaldehyde thiosemicarbazone, which is currently clinically tested as an
ED
anticancer drug (Triapine®) [15, 16]. The anticancer activity of the Triapine® arises from its
ability to inhibit the action of ribonucleotide reductase (RR) [17]. The catalytically active centre
PT
of RR comprises a tyrosyl radical stabilized with two iron ions which might be a target for
CE
Triapine® having good metal chelating properties [18]. Ribonucleotide reductase is required for
DNA synthesis since it catalyses the reduction of ribonucleotides into deoxyribonucleotides.
AC
Consequently the inhibition of this enzyme interrupts cell proliferation. To take an advantage of
its chelating properties, semicarbazone 2-formylopyridine moiety was kept away from Ru centre
by a spacer based on an alkyne chain (Scheme 1). 2,2’-bipyridine (bpy) and 4,7-diphenyl-1,10phenanthroline (dip) were chosen as auxiliary ligands to evaluate the impact of their diverse
lipophilic character and size on the biological activity. The synthesis, photophysical properties
and biological evaluation on two cancer cell lines: murine colon carcinoma (CT26) and human
lung adenocarcinoma (A549) of the Ru1 and Ru2 complexes are reported. Their cytotoxicity,
4
ACCEPTED MANUSCRIPT
cellular distribution, induction of apoptosis, effect on cell-cycles, mitochondria potential,
intracellular calcium concentration as well as reactive oxygen production (ROS) are discussed in
relation to the parent ruthenium(II) complexes ([Ru(bpy)3]2+ and [Ru(dip)2(bpy)]2+) to understand
their mechanism of action. Our results prove that the choice of the auxiliary ligands might have a
T
predominate effect on the mode of action of ruthenium complexes having one ligand modified
Me
N
N
US
CR
IP
with biologically active organic substituent.
2+
AN
Ru
N
N
Me
N
N
N
N
N
N
N
N
O
N
NH2
Ru2
ED
Ru1
N
H
N
2+
Ru
M
O
N
N
N
H
NH2
CE
PT
Scheme 1. Ruthenium compounds studied in this work.
2. Materials and methods
AC
2.1. Synthetic procedures
All solvents were at analytical grade and were used without further purification. DMF was stirred
over calcium hydride for 12 h, filtrated and distilled from over potassium carbonate [19].
Diisopropylamine was distilled over NaH and stored under argon [19]. Reagents were purchased
from
Sigma-Aldrich
(ruthenium(III)
chloride,
n-butyllithium
solution
in
hexanes,
isopropylmagnesium solution in THF, 2,2’-bipyridyl, 4,7-diphenyl-1,10-phenantroline), Alfa
5
ACCEPTED MANUSCRIPT
aesar
(5-bromo-2-iodopyridine,
semicarbazide
hydrochloride,
3-(trimethylsilyl)propagyl
bromide) concentration of n-butyllithium solution was determined by titration of diphenylacetic
acid in dry THF [20].
T
2.1.1. Synthesis of 4-(3-(trimethylsilyl)prop-2-yn-1-yl)-4’-methyl-2,2’-bipyridyl (2). To a
IP
solution of freshly distilled diisopropylamine (0.19 mL, 1.3 mmol) in anhydrous THF (5 mL)
CR
under argon conditions at -78°C, a solution of n-butyllithium in hexane (0.75 mL, 1.2 mmol) was
added dropwise. The solution was stirred for 30 minutes at -78°C and then warmed up to 0°C. A
US
solution of 4,4’-dimethyl-2,2’-bipyridine (1) (0.184 g, 1 mmol) in dry THF (10 mL) was then
AN
added. After 2 h, 3-(trimethylsilyl)propagyl bromide (0.25 mL, 1.5 mmol) was added rapidly.
After another hour, the mixture was warmed to room temperature and quenched with water (30
M
mL). The organic phase was extracted with diethyl ether (3x20 mL) and the combined organic
ED
layers were washed twice with brine and dried over magnesium sulphate. The solvent was
removed under reduced pressure to give the pure product 2 (0.276 g, 92%). 2 was found too
PT
instable to be stored and was involved immediately in the next stage of the synthesis. 1H NMR
CE
(400 MHz, CDCl3): : 0.11 (s, 9H), 2.44 (s, 3H), 2.58 (t, J=7.2Hz, 2H), 2.91 (t, J=7.2 Hz, 2H),
7.12-7.14 (m, 1H), 7.18 (dd, J=4.8 and 1.6 Hz, 1H), 8.22 (s, 1H), 8.28 (s, 1H), 8.52 (d, J=5.2 Hz,
AC
1H), 8.58 (d, J=4.8 Hz, 1H) ppm (Fig. S4). 13C NMR (100 MHz, CDCl3): (ppm): -0.01, 21.05,
21.19, 34.40, 86.16, 105.52, 121.40, 121.98, 124.03, 124.68, 148.11, 148.95, 148.99, 150.34,
155.94, 156.27 ppm (Fig. S5).
2.1.2. Synthesis of 4-(prop-2-yn-1-yl)-4’-methyl-2,2’-bipyridyl (3). To a solution of 2 (0.276 g,
0.9 mmol) in methanol (5 mL) was added potassium carbonate (0.205 g, 1.5 mmol). The mixture
6
ACCEPTED MANUSCRIPT
was heated under argon at 50ºC for 2 h. After this time, the reaction was cooled down to 0°C,
neutralized with 2M HCl and extracted with diethyl ether (3x30 mL). The combined organic
layers were washed twice with brine and dried over magnesium sulphate. The solvent was
removed under reduced pressure to give the pure product as a brown solid (0.150 g, 72%). 1H
T
NMR (400 MHz, CDCl3): d 1.99 (t, J = 2.6 Hz, 1H), 2.44 (s, 3H), 2.54 (td, J =7.4 and 2.6 Hz,
IP
2H), 2.94 (t, J = 7.4 Hz, 2H), 7.13 (d, J=4.8 Hz, 1H), 7.20 (dd, J =5.0 and 1.8 Hz, 1H), 8.23 (d, ,
CR
J =0.8 Hz, 1H), 8.26 (d, J =0.8 Hz, 1H), 8.52 (d, J=5Hz, 1H), 8.58 (d, J=5Hz, 1H) ppm (Fig. S6).
13
C NMR (100 MHz, CDCl3): d 20.41, 21.12, 34.50, 68.87, 83.79, 120.71, 121.63, 124.04,
US
125.02, 147.99, 148.33, 148.90, 151.03, 155.89, 156.32 ppm. [HRMS – ESI] Calculated for
AN
C15H14N2 m/z : 223.1230 (M+H+), Found: m/z = 223.1239 (M+H+).
M
2.1.3. Synthesis of 5-bromo-2-formylpyridine (5) [21]. 5-bromo-2-formylpyridine was
ED
synthesized modifying the procedure described by Song et al. [21]. To a solution of 5-bromo-2iodopyridine (4) (0.586 g, 2 mmol) in anhydrous THF (10 mL) under argon conditions at -10°C,
PT
a solution of isopropylmagnesium chloride (1.5 mL, 2.17 mmol) was added dropwise. After 2 h,
CE
dry DMF (0.4 mL, 5.16 mmol) was added rapidly. The mixture was kept at -10°C for another
hour and then warmed up to room temperature over 2 h. The reaction was quenched with a
AC
saturated aqueous solution of NH4Cl (20 mL) and extracted with diethyl ether (3x25 mL). The
combined organic layers were washed twice with brine and dried over magnesium sulphate. The
solvent was removed under reduced pressure to give pure product as light yellow solid (0.306 g,
80%). 1H NMR (200 MHz, CDCl3): 7.84 (dd, J = 8.2 and 0.8 Hz, 1H), 8.02 (ddd, J = 8.2, 2.2
and 0.8 Hz, 1H), 8.85 (d, J=2 Hz, 1H), 10.04 (d, J=0.8 Hz, 1H) ppm.
7
ACCEPTED MANUSCRIPT
2.1.4. Synthesis of 2-[(5-bromopyridin-2-yl)methylidene]hydrazinecarboxamide (6). To a
suspension of semicarbazide hydrochloride (0.149 g, 1.3 mmol) in anhydrous ethanol (3 mL), a
solution of 5 (0.230 g, 1.2 mmol) in ethanol (9 mL) was added. The mixture was refluxed over 3
h and cooled down to 0°C. The solid was filtrated, washed twice with cold, anhydrous ethanol,
T
once with diethyl ether and dried in vacuum to give the pure product as light cream powder
IP
(0.300 g, 99%). 1H NMR (400MHz, DMSO-d6) 7.02 (brs, 2H), 7.86 (s, 1H), 8.10 (dd, J = 8.0
CR
and 1.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 1.6 Hz, 1H), 10.63 (s, 1H) ppm (Fig. S7).
13
CNMR (100MHz, DMSO-d6): 119.70, 121.57, 137.78, 139.73, 149.26, 152.25, 156.29 ppm
US
(Fig. S8). [HRMS-ESI]: Calculated for C7H7N4OBr: m/z 264.9701 (M+Na+) Found 264.9692
Synthesis
of
5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-
M
2.1.5.
AN
(M+Na+).
ED
carbaldehyde semicarbazone (bpySC). To a solution of 3 (0.170 g, 0.75 mmol) in acetonitrile
(6 mL), copper (I) iodide (0.05 g, 30 mol%) and Tetrakis(triphenylphosphine)palladium(0) (0.045
PT
g, 5 mol%) were added under argon conditions. Diisopropylamine (6 mL) was added and the
CE
mixture was stirred for 15 min. After that, a suspension of 6 (0.210 g, 0.9 mmol) in methanol (1
mL) was added. The mixture was refluxed for 18 hours. Concentrated ammonia (1 mL) was
AC
added to the brown suspension and the mixture was filtrated through Celite (followed by 70 mL
of acetonitrile). The solvent was removed under reduced pressure and the residue was dissolved
in dichloromethane/concentrated ammonia mixture. The organic phase was separated, washed
twice with concentrated ammonia (till all copper was removed), once with brine and dried over
magnesium sulfate. The solvent was removed under reduced pressure to give the crude product
which was dissolved in chloroform (15 mL) and treated with cyclohexane (60 mL). The mixture
8
ACCEPTED MANUSCRIPT
was kept at 4°C (fridge) and crystals were filtrated, washed with cyclohexane and dried under
vacuum to give the pure product as a white solid (0.132 g, 45%). 1H NMR (600 MHz, CDCl3): d
2.45 (s, 3H), 2.84 (t, J =7 Hz, 2H), 3.03 (t, J = 7 Hz, 2H), 7.14 (dd, J =16.2 and 4.8 Hz, 2H),
7.64-7.77 (m, 3H), 8.25 (s, 1H), 8.38 (s, 2H), 8.53-8.63 (m, 3H) ppm (Fig. S9). 13C NMR (150
T
MHz, CDCl3) d 20.67, 21.22, 34.17, 78.86, 93.77, 119.41, 120.83, 121.33, 122.04, 123.89,
IP
124.83, 138.90, 141.49, 148.23, 148.95, 149.18, 150.04, 151.14, 152.11, 155.79, 156.38, 156.79
CR
ppm (Fig. S10). [HRMS-ESI]: Calculated for C22H20N6O m/z 385.1771 (M+H+) Found 385.1771
US
m/z (M+H+).
AN
2.1.6. General procedure for synthesis of cis-Ru(NN)2Cl2. Ruthenium(III) chloride trihydrate
(0.262 g, 1 mmol), the ligand (2,2’-bipyridine or 4,7-diphenyl-1,10-phenantroline) (2 mmol) and
M
2 drops of N-ethylmorpholine were dissolved in degassed N,N-dimethylformamide (10 mL)
ED
under argon conditions. The mixture was put into a microwave reactor for 30 min (250W,
160ºC). The solvent was then removed under reduced pressure to c.a. 1 mL and acetone (20 mL)
PT
was added. After 24h at -20ºC, the dark solid obtained was filtrated and washed with cold
CE
acetone, twice with diethyl ether and dried under vacuum. The crude product was finally purified
product.
AC
by flash chromatography (neutral Al2O3, dichloromethane/methanol (5/95)) to give the pure
2.1.6. 1. cis-Ru(bpy)2Cl2 (8) [22]. 8 was obtained using 2,2'-bipyridine as ligand (0.181 g, 38%).
1
H NMR (200 MHz, DMSO-d6): 7.11 (t, J =8Hz, 2H), 7.51 (d, J=5.2Hz, 2H), 7.69 (t, 2H,
J=8Hz), 7.78 (t, J =7Hz, 2H), 8.08 (t, J =8Hz, 2H), 8.48 (d, J =8Hz, 2H), 8.64 (d, J =8Hz, 2H),
9.97 (d, J=5Hz, 2H) ppm. [HRMS-ESI]: Calculated for C20H16N4RuCl2: m/z 483.9784 m/z (M+)
Found 483.9791 (M+).
9
ACCEPTED MANUSCRIPT
2.1.6.2. cis-Ru(dip)2Cl2 (9) [23]. 9 was obtained using 4,7-diphenyl-1,10-phenantroline as ligand
(0.460 g, 55%). 1H NMR (600 MHz, DMSO-d6): 7.45-7.65 (m, 2H), 7.70 (t, J =7.8Hz, 4H),
7.81 (d, J =7.2Hz, 4H), 7.97 (d, J =9Hz, 2H), 8.25 (s, 1H), 8.27 (s, 1H) ppm. [HRMS-ESI]:
Calculated for C48H32N4RuCl2: m/z 859.0929 m/z (M+Na+) Found 859.0949 (M+Na+).
T
2.1.6.3. Synthesis of [Ru(bpy)2(bpySC)]Cl2 (Ru1). bpySC (0.023 g, 0.060 mmol) was
IP
dissolved in absolute ethanol (5 mL) under argon conditions. A solution of cis-Ru(bpy)2Cl2(8)
CR
(0.023 g, 0.048 mmol) in absolute ethanol (5 mL) was finally added. The dark violet mixture was
refluxed under argon for 16 h. After that, the solvent was removed under reduced pressure the
US
residue was dissolved in water (1 mL) and filtrated. The pure product was obtained after
AN
crystallization from water as dark red crystals (0.033 g, 80%). 1H NMR (600 MHz, CD3CN):
2.51 (s, 3H), 2.91 (t, J=6 Hz, 2H), 2.95 (t, J=6 Hz, 2H), 7.19 (s, 1H), 7.33-7.40 (m, 6H), 7.46 (d,
M
J=7.8 Hz, 1H), 7.51 (d, J=6 Hz, 1H), 7.61 (d, J=6 Hz, 1H), 7.71-7.74 (m, 6H), 7.80 (t, J=7.8 Hz,
ED
1H), 8.03-8.07 (m, 4H), 8.13 (s, 1H), 8.25 (d, J=6 Hz, 1H), 8.54-8.66 (m, 8H) ppm (Fig. S11).
[HRMS] Calculated for C42H36N10ORu: m/z 399.1052 (M+), Found 399.1058, (M+) Anal. Calc.
PT
for C42H36N10ORu: C (58.1%), H (4.2%), N (16.1%), Found: C (58.2%), H (4.4%), N (16.1%).
CE
2.1.6. 4. Synthesis of [Ru(dip)2(bpySC)]Cl2 (Ru2). To a solution of 9 (0.019 g, 0.023 mmol) in
absolute ethanol (10 mL) under argon conditions, was added a solution of bpySC (0.010 g, 0.026
AC
mmol) in absolute ethanol (5 mL). The mixture was refluxed for 22 h. After that time the solvent
was removed under reduced pressure. The crude product was dissolved in dichloromethane and
purified by flash chromatography (Al2O3 neutral, dichloromethane/Methanol(5/95 to 10/90
gradient) to give the pure product with yield (0.013 g, 47%). 1H NMR (600 MHz, CD3CN) 2.55
(s, 3H), 2.95 (t, J =6 Hz, 2H), 3.13 (t, J=6 Hz, 2H), 7.04 (s, 1H), 7.22 (d, J=6 Hz, 1H), 7.37 (dd,
J=15.6 Hz, 6 Hz, 2H), 7.57-7.63 (m, 20H), 7.67 (d, J=5.4 Hz, 2H), 7.73 (dd, J=5.4 and 2.4 Hz,
10
ACCEPTED MANUSCRIPT
2H), 7.79 (t, J=5.4 Hz, 1H), 8.13-8.20 (m, 6H), 8.23 (d, J=5.4 Hz, 1H), 8.26 (d, J=5.4 Hz, 1H),
8.33 (d, J=5.4 and 3.6 Hz, 1H), 8.80 (d, J=12.6 Hz, 1H), 8.90 (d, J=12.6 Hz, 1H) ppm (Fig. S12).
[HRMS]: m/z: Calculated for C70H52N10ORu: m/z 575.1679 (M+), Found 575.1658 (M+).
T
2.2. Spectroscopy measurements
IP
UV-Vis absorption spectra of ruthenium complexes were recorded at 25ºC in water using Perkin
CR
Elmer Lambda 35 spectrophotometer. Luminescence measurements were registered on Perkin
Elmer LS55 spectrofluorimeter in the range 480 - 900 nm upon excitation at the maximum of
US
charge transfer band for each ruthenium complex. The average of three scans was subjected to
AN
smoothing. For the determination of the quantum yield of luminescence (Φ), aqueous solutions of
[Ru(bpy)3]2+ with a small amount of DMSO (<0.008% v/v) were used as standards (Φ = 0.028
M
[24] and 0.042 [25] for air-equilibrated and deoxygenated conditions, respectively). The spectra
ED
for the ruthenium complexes were recorded at a concentration that had an absorbance less than
following equation:[26]
PT
0.05 units at the excitation wavelength. The quantum yield was calculated according to the
CE
Φ = Φref × [Aref/A] × [I/Iref] × [n2/nref2]
where I is the integrated luminescence intensity, A is the optical density, and n is the refractive
AC
index, ref refers to the values for the reference. The mean value was calculated from a minimum
of three independent experiments.
Luminescence lifetime experiments were performed at room temperature using Fluorolog-3
Horiba Jobin Yvon with single photon counting technique. The excitation wavelength was fixed
at 464 nm (NanoLED diode) and average luminescence lifetime was measured at emission
maximum. The instrument response functions were measured using a light scattering solution of
Ludox (colloidal silica, Sigma-Aldrich). The DAS6 software (HORIBA Scientific) was used for
11
ACCEPTED MANUSCRIPT
deconvolution of the obtained decays and for calculation of the lifetime values. Two or three
exponential fit were chosen based on χ2 parameter (the goodness of fit evaluation). A singleexponential fit was found to be an optimal description of the obtained results for the ruthenium
T
compounds.
IP
2.3. Computational characterization
CR
To obtain ground state geometries and electronic structures of Ru1 and Ru2 Density Functional
Theory (DFT) was applied using hybrid B3LYP functional [27-31] and 6-31G(d,p) basis set [32-
US
35] for all atoms except for ruthenium, for which LANL2DZ basis-set [36, 37] was applied. To
AN
compute electronic excitation spectra, Time Dependent – Density Functional Theory (TD-DFT)
was used with the same functional and basis sets for light atoms, with LANL2DZ pseudo-
M
potential for Ru. Eighty lowest laying electronic excitations were determined. All calculations
ED
were done with Gaussian 09 program [38], orbitals were visualized using GaussView [39]
PT
software.
CE
2.4. Cell Culture Conditions
Biological studies were performed using murine colon carcinoma CT26 and human lung
AC
adenocarcinoma A549 cell lines. Cells were maintained in DMEM medium supplemented with
10% fetal bovine serum (FBS) and 1% antibiotics – penicillin (100 units/mL) and streptomycin
(100 μg/mL). Cells were routinely cultured at 37 °C in a humidified incubator in a 5% CO2
atmosphere.
2.5. Cytotoxicity
12
ACCEPTED MANUSCRIPT
The evaluation of the cytotoxicity of the Ru complexes on A549 and CT26 cells, was conducted
using an Alamar Blue assay. The Alamar Blue test is based on the reduction of blue and nonfluorescent subtrate (resazurin) to a pink and highly fluorescent product (resorufin) by the alive
cells. Cells were seeded on a 96 wells plate with a density of 1.5 × 10 4 cells per cm2 one day
T
before the experiments. Then, cells were incubated with various concentrations of the Ru
IP
compounds and synthesized ligand L1 for 24/72 hours in the dark. All compounds were diluted in
CR
DMSO and then, added to the appropriate medium with or without 2% FBS to obtain the applied
concentrations. The final DMSO concentration was kept constant at 0.1% (v/v) in case of Ru2
US
and 0.5% (v/v) in case of Ru1 and bpySC. After the incubation, cells were washed with PBS and
AN
incubated in the resazurin sodium salt solution (25 µM) for 3 h. The cell viability was quantified
at 605 nm using 560 nm excitation light (Tecan Infinite 200 microplate reader). Experiments
M
were performed in triplicate and repeated at least three times to get the mean values ± standard
ED
deviation. The viability was calculated with respect to the untreated cells control. The IC50 values
PT
were determined using the Hill equation (Origin 9.0) [40]
(𝑦100 − 𝑦0 )[𝑐]𝐻
[𝐼𝐶50 ]𝐻 + [𝑐]𝐻
CE
y = y0 +
AC
2.6. Cellular imaging of accumulated Ru complexes
For co-localization experiments, A549 cells were seeded on a 6 well plate at a density of 3 × 104
cells per cm2 24 h prior to the staining. ER-Trackert Blue-White DPX, Mitotracker Green and
Lyso-Tracker Blue (Life Technologies) were used to image ER, mitochondria and lysosomes
according to the manufacturer’s protocols. The Ru complexes were incubated in basic medium
for 24 h ([Ru1] = 500 µM, [Ru2] = 2 µM), then rinsed twice with PBS. The chosen concentration
of the Ru complexes was not cytotoxic to the cells under these conditions. Images were acquired
13
ACCEPTED MANUSCRIPT
using a Olympus fluorescence microscope IX51 equipped with an XC10 camera with 470–495
and 530–550 nm emission filters.
2.7. Cell cycle
T
A549 cells were seeded into a 6 wells plate with a density of 3 × 104 cells per cm2. Cells were
IP
cultured in the full medium for 1 day. Then, the medium was removed and replaced with a basic
CR
medium containing various concentrations of the studied compounds, and incubated for 24 h.
Then, the cells were washed with PBS, detached by trypsin, fixed in cold methanol for 30 min,
US
stained with propidium iodide (PI) for 4 h in the dark, followed by analyzing using a BD
AN
LSRFortessa cytometer. The experiment was performed twice.
M
2.8. Apoptosis
ED
The early stage of apoptosis is manifested by the relocation of phosphatidylserine (PS) from the
inner side of the cellular membrane to the outer side. This process can be revealed by the staining
PT
cells with the labeled Annexin V. Annexin V is a cellular protein that in the presence of Ca2+
CE
ions, selectively binds to PS. DAPI was used instead of propidium iodide to assess the cell
necrosis, due to the overlapping excitation/emission parameter of the propidium iodide and the
AC
Ru complexes in live cells [41]. A549 cells were seeded into a 6 wells plate with a density of 3 ×
104 cells per cm2 and cultured in the full medium for 1 day. Afterwards, the medium was
removed and replaced by a medium containing different amounts of the studied compounds and
incubated for 24 h. Then, cells were washed with PBS and binding buffer. The cells were stained
with Annexin V-FITC for 10 min in the dark and then, with DAPI (0.5 µM) for 5 min. Cells were
analyzed by a BD LSRFortessa cytometer. As a positive control, H2O2 (300 µM) was used.
14
ACCEPTED MANUSCRIPT
2.9. Caspases activity
Activation of caspases 3/7, 8 and 9 was examined using luminescent Caspase Glo assays
(Promega) according to the manufacturer’s manual. A549 and CT26 cells were seeded on a white
96 wells plate with a density of 1.6 × 104 cells per cm2 24 h prior to the experiments. Next, the
T
compounds at different concentrations were added to the wells and incubated for 24 h ([Ru1] =
IP
200 µM, [Ru2] = 2 µM, [bpySC] = 100 µM). The luminescence intensity of the cells was
CR
measured using a Tecan Infinite 200 plate reader.
US
2.10. Mitochondrial morphology
AN
MitoTracker Green (ThermoFisher Scientific) was used to visualize mitochondria of A549 cells.
A549 cells were seeded on a 6 wells plate with a density of 3 × 104 cells per cm2 24 h prior to the
M
experiments. Next, the Ru complexes were added and incubated for 24 h. After the incubation,
ED
the Ru compounds were washed and cells were stained with MitoTracker Green (100 nM) for 30
min at 37 °C. After the staining, cells were visualized using an Olympus fluorescence microscope
CE
PT
IX51.
2.11. Cytosolic calcium homeostasis
AC
Cytosolic calcium concentration was measured using Fluo-8 AM probe (AAT Bioquest). A549
and CT26 cells were seeded on a 96 wells plate with a density of 1.5 × 104 cells per cm2 one day
before the experiments. Then, cells were incubated with various concentrations of the compounds
for 24 h in the dark. Next, cells were washed with PBS and stained with Fluo-8 AM (4 µM) for
30 min in the dark at 37 °C. After staining cells were washed twice with PBS and analyzed by a
Tecan Infinite 200 microplate reader measuring fluorescence of the probe at 525 nm using 490
15
ACCEPTED MANUSCRIPT
nm as an excitation wavelength. Experiment was performed three times in triplicate and the mean
values ± standard deviations were calculated.
2.12. Mitochondria membrane potential
T
Mitochondria membrane potential was evaluated using JC-1 probe (AAT Bioquest). A549 and
IP
CT26 cells were seeded on 96 wells plate with a density of 1.5 × 104 cells per cm2 one day before
CR
the experiments. Cells were incubated with various concentrations of the Ru compounds for 24 h
in the dark. Then, cells were washed with PBS and stained with JC-1 (10 µM) for 30 min in the
US
dark at 37 °C. Next, cells were washed with PBS and analyzed by a Tecan Infinite 200
AN
microplate reader measuring fluorescence of the probe at 525 nm and 590 nm using 490 nm as an
2.13. Total ROS production
ED
standard deviations were calculated.
M
excitation wavelength. Experiment was performed three times in triplicate and the mean values ±
PT
The level of the oxidative stress induced in cells after the incubation with the Ru complexes was
CE
conducted for A549 and CT26 cell lines using the cyto-ID Hypoxia/Oxidative stress detection kit.
A549 and CT26 cells were seeded with a density of 3 × 104 cells per cm2, respectively. A day
AC
later the compounds at different concentrations in medium without serum were added and
incubated in the dark for 24/72 h. Then, cells were washed with PBS, treated with trypsin and
analyzed by BD LSR cytometer. As a positive control, pyocyanin (300 µM) was used. The level
of the oxidative stress was determined as a percentage of the ROS positive cells of the whole cell
population.
16
ACCEPTED MANUSCRIPT
3. Results and discussion
3.1 Synthesis
T
The preparation of the target complexes Ru1 and Ru2 required the synthesis of ligand bpySC
IP
(Scheme 2) that was synthesised by a Sonogashira coupling between 4-(but-3-yn-1-yl)-4'-methyl-
CR
2,2'-bipyridine (3) and 5-bromopyridine-2-carbaldehyde semicarbazone (6). 6 was synthesised by
US
formylation of 5-bromo-2-iodopyridinefollowed by treatment with semicarbazide hydrochloride.
3 was synthesised by a selective monolithiation of 4,4’-dimethyl-2,2’-bipyridine (1) using LDA
AN
followed by reaction with 3-(trimethylsilyl)propagyl bromide and final deprotection using
M
potassium bicarbonate [21].
ED
Me
Me
TMS
Me
Me
N
N
Br
CE
1
PT
(a)
N
(c)
N
N
3
Br
(d)
Br
N
O
N
CHO
5
AC
4
N
2
N
I
(b)
N
N
H
NH2
6
Me
(e)
O
N
NH2
3+6
N
N
N NH
bpySC
17
ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of ligand bpySC Reagents and conditions: (a) (1) LDA, -78ºC, THF, 0.5h
then 0ºC, 2h; (3) 3-(trimethylsilyl)propagyl bromide, 1h; (b) K2CO3, MeOH, 50ºC, 2h; (c) (1)
iPrMgCl, -10ºC, THF, 2h; (2) DMF, -10ºC -> RT 2h; (d) NH2-NH-C(=O)-NH2·HCl, EtOH,
T
reflux, 3h; (e) CH3CN, CuI, Pd(PPh3)4, diisopropylamine, reflux, 18h.
IP
Complexes Ru1 and Ru2 were synthesised by refluxing cis-Ru(NN)2Cl2 complexes and bpySC
CR
in absolute ethanol. Dichlorobis(2,2'-bipyridine)ruthenium(II) (Ru(bpy)2Cl2) and dichlorobis(4,7diphenyl-1,10-phenantroline)ruthenium(II) (Ru(dip)2Cl2) were used for Ru1 and Ru2,
US
respectively. The cis-Ru(NN)2Cl2 complexes were prepared by microwave synthesis from
AN
ruthenium(III) chloride and the proper ligand in DMF according to previously described
procedures [42, 43]. Interestingly, the coordination of ruthenium occurred exclusively on the
M
bipyridine side of the bpySC and no trace of ruthenium coordination by the SC moiety was
8, L=bpy
9, L=dip
EtOH, Ar,
reflux, 22h
Ru1, L=bpy
Ru2, L=dip
PT
bpySC
L2RuCl2
ED
observed.
AC
CE
Scheme 3. Synthesis of Ru1 and Ru2.
3.2. Photophysical properties
The complexes have been characterized by UV-Vis spectroscopy using water as solvent. The
photophysical data are collected in Table 1. In order to measure the effect of bpySC on the
properties, complexes containing one bpy instead of bpySC were prepared and characterized. As
shown in Table 1 all complexes displayed intense 1MLCT (metal ligand charge transfer) bands in
the visible part of the spectrum, around 450 nm. Both Ru1 and Ru2 showed a slight red shift
18
ACCEPTED MANUSCRIPT
compared with their unsubstituted counterparts. In contrast, the emission band exhibited a notable
blue shift (11 nm for Ru1 and 10 nm for Ru2). The introduction of the bpySC ligand also came
with a decrease of the luminescence quantum yield (QY) that were 0.92% and 1.18% for Ru1
and Ru2 respectively under air-equilibrated conditions while argon-equilibrated conditions
T
allowed to increase these values to 1.63%. and 2.62%, respectively. A decay of the MLCT
IP
excited state lifetimes was also observed for both Ru1 and Ru2. Thus while globally affecting
CR
negatively the luminescence of the complexes, the bpySC ligand allowed to maintain QY and
AC
CE
PT
ED
M
AN
US
lifetimes exploitable for further use in imaging experiments.
19
ACCEPTED MANUSCRIPT
Table 1. Photophysical properties of the ruthenium(II) complexes in air-equilibrated and
deoxygenated aqueous solutions.
(air-equilibrated
(deoxygenated
conditions)
conditions)
λmax
ε
IP
λmax
Emission
T
Absorption
Emission
Φ
-1
[M cm ]
[nm]
τ [µs]
Φ
CR
[nm]
-1
438
19 000
461
18 500
286
108 600
15 400
455
18 100
279
CE
427
204 800
435
42 100
Ru2
457
43 000
0.57 ±
0.0420b
0.01
0.01
0.0367 ±
0.76 ±
0.1245 ±
2.51 ±
0.0004
0.01
0.0004
0.01
0.0092 ±
0.27 ±
0.0162 ±
0.36 ±
0.0009
0.03
0.0002
0.06
0.0118 ±
0.65 ±
0.0262 ±
1.72 ±
0.0003
0.03
0.0004
0.07
613
M
88 100
ED
279
AC
Ru1
14 000
PT
[Ru(dip)2(bpy)]2+ c
452
0.0280a
AN
628
US
0.37 ±
[Ru(bpy)3]2+
τ [µs]
617
623
data taken from a [24]; b [25];c [44]
3.3. Computational characterization
20
ACCEPTED MANUSCRIPT
Quantum chemical calculations within Density Functional Theory (DFT) approach allowed for
determination of the Ru1 and Ru2 geometry structures (compare Supplementary Information)
and characterization of their frontier orbitals (Fig. S1). In both complexes, HOMO spans over the
bpySC ligand, whereas LUMO is delocalised over pyridine atoms of the remaining ligands of
T
ruthenium: other bpys in Ru1 and bpy fragment of bpySC in Ru2. Further, the performed Time-
IP
Dependent DFT (TD-DFT) calculations allowed for the characterisation of the spectra of both
CR
complexes. The most intensive bands clearly show the MLCT character of their visible – see Fig.
1 for schematic representation of their main features. In case of Ru1, the most intensive bands are
US
centered around 421, 419 and 408 nm, and correspond to excitations from a mixture of Ru d and
AN
π bpy fragment of bpySC orbitals to π* orbitals of bpy ligands. In case of Ru2, they are present
at around 441, 438, 427, 405, and 393 nm, and they might be attributed to the excitations from a
AC
CE
PT
ED
M
mixture of Ru d and π orbitals of dip to π* orbitals of the bpy fragment of bpySC.
21
ACCEPTED MANUSCRIPT
AN
US
CR
IP
T
Ru1
AC
CE
PT
ED
M
Ru2
Fig. 1. Plots of orbitals responsible for the most intensive absorption bands in the visible range of
Ru1 and Ru2 spectra with schematic representation of the most intense absorption bands, as
computed with TD-DFT.
22
ACCEPTED MANUSCRIPT
3.4. Cytotoxicity of ruthenium complexes in vitro
The cytotoxic effect of the newly synthesised Ru complexes and their reference compounds was
determined on two cancer cell lines: murine colon carcinoma (CT26) and human lung
T
adenocarcinoma cell line (A549). Complexes were tested in serum free conditions (S-) and also
IP
in the presence of 2% serum (S+). The obtained results are presented in Table 2. Generally, the
CR
presence of serum reduced the cytotoxic effect of both Ru1 and Ru2. Such an effect has been
previously described [45] and might be related to a reduction in the Ru uptake due to binding to
US
the proteins present in the serum (see Table S1) impeding complexes accumulation. Ru
AN
complexes with dip ligands were much more cytotoxic in comparison with those having bpy
ones. Replacing one of the dip ligands with bpy or bpySC ligand decreases slightly the
M
cytotoxicity of the Ru compounds. The observed effect arises from the dependence of the Ru
ED
complexes accumulation and therefore their toxicity on their lipophilicity. In agreement with
some previous studies on Ru polypyridyl complexes [44], the poor cellular uptake of
PT
[Ru(bpy)3]2+ or Ru1 can be related to their weak lipophilic character.
CE
Increasing the incubation time from 24 to 72 h resulted in a distinct increase in an antiproliferative activity of Ru1, in particular against CT26 cells, while no significant change was
AC
observed in the cytotoxicity of Ru2. [Ru(bpy)3]2+ remains continuously non cytotoxic even after
72 h of incubation. At the same time a dramatic increase in the toxicity after 72 h was observed
for bpySC ligand alone (IC50 ca. 30 times higher). It suggests that this particular ligand might
play a crucial role in the anti-proliferative activity of Ru1. Furthermore, it can be assumed that an
appropriate selection of auxiliary ligands might lead to a change in action mechanism. For the
studied system, switching from two dip ligands to two bpy ones clearly highlighted the cytotoxic
23
ACCEPTED MANUSCRIPT
activity of bpySC ligand in Ru1, whereas in Ru2 compound these two auxiliary dip ligands
played a key role.
Table 2. IC50 of Ru complexes against two cancer cell lines A549 and CT26. Experiments were
T
performed in medium without (S-) or with (S+, 2%) serum.
-
3.3 ± 1.2
2.8 ± 0.8
Ru1
113 ± 154
202 ± 17
97 ± 14
138 ± 20
Ru2
6.8 ± 1.2
9.8 ± 2.3
9.0 ± 1.9
16 ± 2
[Ru(dip)2(bpy)]2+
4.0 ± 0.7
-
-
-
>320
>320
>320
>320
1.2 ± 0.1
-
-
-
96 ± 28
-
1.5 ± 0.3
2.0 ± 0.3
201 ± 25
233 ± 38
31 ± 11
50 ± 7.6
Ru2
4.6 ± 0.9
5.0 ± 0.1
5.7 ± 1.5
21 ± 5
[Ru(dip)2(bpy)]2+
3.4 ± 0.7
-
-
-
[Ru(bpy)3]2+
>320
>320
>320
>320
[Ru(dip)3]2+
1.2 ± 0.2
-
-
-
US
102 ± 59
CR
S+
AN
S-
bpySC
A549
M
[Ru(bpy)3]2+
bpySC
PT
Ru1
ED
[Ru(dip)3]2+
AC
CT26
S+
CE
S-
IC50 (µM) 72 h
IP
IC50 (µM) 24h
3.5. Cell accumulation and cellular targets
A549 cells were used to study the accumulation of the studied ruthenium complexes. A higher
cellular uptake was found for complex Ru2 in excellent agreement with the cytotoxicity data.
24
ACCEPTED MANUSCRIPT
Despite similar luminescence quantum yields for Ru2 and Ru1, much higher concentrations of
Ru1 were required to visualize its localization inside the cells. This further confirmed that the
accumulation of ruthenium polypyridyl complexes is strongly dependent on their lipophilicity
and that in the studied case it is controlled by bpy/dip ligands. Additionally, it points out the
T
superiority of the accumulation/lipophilicity parameters over luminescence parameters of Ru
IP
complexes in assessing the potential of the compounds as cellular dyes [44].
CR
To further investigate the accumulation of the Ru complexes, organelle-specific dyes were used.
Ru2 preferably accumulated into mitochondria and endoplasmic reticulum, as confirmed by
US
overlapping emission of organelle-specific dyes with the studied Ru complex (Fig. 2). Complex
AC
CE
PT
ED
M
localized within cellular lysosomes (Fig. 3).
AN
Ru1 exhibited rather small accumulation in those organelles (Fig. S2), while the majority of it is
25
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
PT
Fig. 2. Fluorescence images of A549 cells showing subcellular localization of Ru2 (2 µM,
incubation 24 h). (A,C) red colour comes from Ru2 complex emission; (B) ER-Tracker Blue was
CE
used to image endoplasmic reticulum, blue colour arises from organelle-specific dye while pink
AC
colour represents overlap of the red luminescence of Ru and blue emission from dye indicating
co-localization; (D) MitoTracker Green was used to image mitochondria and green colour arises
from organelle-specific dye, while yellow colour represents overlap of the red luminescence of
Ru and green emission from dye indicating co-localization. Scale bars 50 µm.
26
CR
IP
T
ACCEPTED MANUSCRIPT
US
Fig. 3. Fluorescence images of A549 cells showing subcellular localization of Ru1 (500 µM,
incubation 24 h). (A) Red colour comes from Ru1 complex emission; (B) LysoTracker Blue was
AN
used to image lysosomes, pink colour represents overlap of the red luminescence of Ru and blue
ED
3.6. Cell-cycle arresting properties
M
emission from dye indicating co-localization. Scale bars 50 µm.
PT
The influence of Ru1, Ru2 and bpySC on cell cycle was measured by flow-cytometry (Table 3).
The incubation of A549 cells with Ru1 or bpySC induced a significant increase in the number of
CE
cells in the S-phase of the cell cycle, with the corresponding decrease in the percentage of cells in
the G2/M phase. During the S-phase of the cell cycle deoxyribonucleotides, which are DNA
AC
precursors, are produced. In this phase, corresponding ribonucleotides need to be reduced by
ribonucleotide reductase. Inhibition of this enzyme should increase the number of cells arrested
in this phase [46, 47]. The S-phase arrest caused by Ru1 or bpySC suggests that their cellular
activities might be related to the inhibition of ribonucleotide reductase. Contrarily Ru2 caused
increased percentage of the cells in the G0/G1 phase, similarly to other Ru complexes containing
27
ACCEPTED MANUSCRIPT
dip ligands [45]. This further confirms the influence of the auxiliary ligands on the biological
AC
CE
PT
ED
M
AN
US
CR
IP
T
activity of Ru compounds.
28
ACCEPTED MANUSCRIPT
Table 3. Cell cycle distribution of A549 cells after 24 h treatment with the Ru polypyridyl
Control 57.5%
36.0%
6.5%
bpySC
22.2%
74.0%
3.8%
Ru1
47.5%
48.4%
4.1%
Ru2
76.9%
22.5%
0.6%
IP
G2/M
CR
S-phase
3.7. Reactive Oxygen Species (ROS) production
US
G0/G1
T
complexes and with ligand bpySC (representative analysis).
AN
The influence of Ru1 and Ru2 on reactive oxygen species (ROS) production in cancer cell lines
was evaluated. Ru2 as well as the attached ligand bpySC caused significant concentration
M
dependent increase in ROS production after 24 of h incubation in both A549 and CT26 cell lines
ED
(Fig. 4, Fig. S3). Increasing the incubation time 24 h to 72 h also increase the ROS level induced
by Ru2. However, Ru1 did not induce oxidative stress in the studied cell lines that could be
PT
partially explained by the lower accumulation of this complex. However increasing the
CE
incubation time with the compounds from 24 h to 72 h (that will also increase Ru complexes
accumulation level) did not induce any increase in ROS production in case of Ru1 as it did with
AC
Ru2 (Fig. 4). This strongly suggested that the Ru accumulation level is not linked to the lack of
ROS production in case of Ru1 and that completely different action mechanism of action
responsible for cytotoxic activity depending on whether bpy or dip ligand is present in the
ruthenium complex.
29
24 h
72 h
80
60
40
T
20
0
M
50 µ
100
µM
2 µM
Ru1
4 µM
M
50 µ
CR
nin
trol
cya
pyo
con
IP
ROS positive cells [%]
ACCEPTED MANUSCRIPT
µM
bpy-SC
US
Ru2
100
Fig. 4. The level of oxidative stress induced in A549 cells after 24 and 72 h treatment with the Ru
AN
polypyridyl complexes expressed as percentage of ROS positive cells in the whole cell
M
population (%). Pyocyanin was used as ROS positive control.
ED
3.8. Apoptosis inducing properties
PT
The determination of the cellular death mechanism was performed using flow cytometry. A549
cells were double stained with Annexin-V- FITC conjugate and DAPI after incubation with Ru1,
CE
Ru2 and bpySC for 24h. Incubation with all studied compounds significantly increased the
AC
population of Annexin V-positive cells, indicating cells undergoing early phase of apoptosis (Fig.
5), with no indication of the DAPI-positive cells that would suggest a necrotic cellular death.
Despite lesser accumulation of Ru1, that was previously confirmed by the cytotoxicity and
localization study, this compound was found to be powerful apoptosis inducing agent.
30
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Fig. 5. Apoptosis of A549 cells upon 24 h exposure to the Ru polypyridyl complexes. Cells were
labeled with Annexin V-FITC/DAPI.
3.9. Activation of the signalling pathways - caspases activity
31
ACCEPTED MANUSCRIPT
Early morphological signs of apoptosis such as translocation of phosphatidylserine are a result of
a cascade of biochemical changes occurring within the cell. To further explore the developed
changes and investigate the apoptotic mechanism of cellular death of cancer cell lines, caspases
activity was measured (Fig. 6). After incubation of A549 and CT26 cells with Ru2, no caspase
T
activation was observed, indicating that the observed apoptosis occurred via caspase-independent
IP
pathway. On the contrary, incubation of cells with Ru1 and bpySC induced significant increase
CR
in activity of initiator caspase 9 and effector caspases 3/7, while no activation of caspase 8 was
8
B
2.0
M
1.5
caspase activity [a.u]
AN
A
ED
1.0
0.5
0.0
control
Ru1
PT
caspase activity [a.u.]
mitochondrial pathway.
US
detected. This indicates that Ru1 and bpySC triggered apoptotic cellular death via intrinsic
Ru2
bpySC
7
6
5
4
3
2
1
0
control
Ru1
Ru2
bpySC
CE
Fig. 6. Caspases 3/7 (black), 8 (red) and 9 (blue) activities after 24 h exposure to the Ru
AC
polypyridyl complexes and ligand bpySC determined in A549 (A) and CT26 (B) cell lines.
This conclusion is further supported by the visualization of the mitochondria of the Ru treated
cells (Fig. 7). While no significant change is observed in mitochondria of Ru2 treated cell,
incubation of cells with Ru1 caused swelling and enlarging of mitochondria. Such process is
often observed in case of apoptotic cell death induced by mitochondria pathway.
32
CR
IP
T
ACCEPTED MANUSCRIPT
US
Fig. 7. Fluorescence images of A549 cells showing swelling and enlarging of mitochondria after
treatment with Ru1. MitoTracker Green was used to image mitochondria and green colour arises
AN
from a organelle-specific dye (A) untreated cells; (B) cells treated with Ru1 (500 µM) for 24 h.
M
Scale bars 50 µm.
ED
3.10. Mitochondrial membrane potential detection
PT
Since the studied Ru complexes either accumulated in mitochondria (Ru2) or changed the
mitochondria morphology (Ru1), their influence on alteration in mitochondria function was also
CE
studied by analyzing the mitochondria membrane potential. The loss of the mitochondria
membrane potential is often considered to be a prelude to apoptosis.
AC
The changes of mitochondrial membrane potential induced by the studied complexes were
determined using JC-1 mitochondrial probe. At low mitochondrial membrane potential, JC-1
exists in monomeric form and exhibits green fluorescence, but at high mitochondrial membrane
potential JC-1 forms aggregates, which exhibit red fluorescence. Two oligopeptides valinomycin
and gramicidin were used as positive controls. Valinomycin is K+ ionophore which allows the
potassium ions passing freely through the cell membrane and in this way causing the collapse of
33
ACCEPTED MANUSCRIPT
mitochondrial membrane potential. Gramicidin is another polypeptide which forms channels in
phospholipid membranes and allows ions to pass freely through the membrane leading to the
decrease in ΔΨm. The change of mitochondrial potential was represented as change of red/green
fluorescence ratio and the results are shown in Fig. 8. All tested compounds caused the decrease
T
in red/green fluorescence intensity ration, indicating depolarization of mitochondria membrane.
IP
The effect is stronger in CT26 cells. These results further confirm that Ru1 and bpySC induces
CR
apoptosis in cancer cells through the mitochondrial signal transduction pathway.
US
1.2
A549
CT26
AN
1.0
M
0.8
ED
0.6
PT
0.4
0.2
CE
0.0
control
AC
red/green fluorescence
intensity of JC-1 probe [a.u.]
1.4
50 µM
Ru1
100 µM
2 µM
4 µM
Ru2
50 µM
100 µM
Val
Gram
bpySC
Fig. 8. Effect of the Ru polypyridyl complexes on mitochondrial membrane potential (ΔΨm) in
A549 and CT26 cell lines after 24 h treatment measured using JC-1 probe. The results are
representative at least three independent experiments. Red fluorescence corresponds to the
fluorescence of J-aggregates, while green fluorescence is related to the fluorescence of Jmonomers. The decrease in a ratio of red to green fluorescence values indicates depolarization of
34
ACCEPTED MANUSCRIPT
mitochondria membrane potential. Valinomycin (Val) and gramicidin (Gram) were used as
positive controls.
3.11. Intracellular calcium concentration
T
Calcium ion is one of the most important signal transducers in cells, involved in numerous
IP
physiological and pathological processes. It is known that a variety of metals complexes are able
CR
to modify intracellular Ca2+ signalling and hence induce apoptosis or necrosis [48, 49]. Using
Fluo-8 AM probe the changes in intracellular calcium concentration ([Ca2+]C) in cancer cells after
US
24 h incubation with the studied compounds were monitored. The results are presented in Fig. 9.
AN
Alteration in calcium homeostasis upon treatment with the Ru complexes was cell line
dependent. In case of A549 cells, no significant changes in [Ca2+]C after treatment with Ru1 or
M
bpy-SC were observed. Only Ru2 accumulation caused a decrease in cytosolic calcium level,
containing dip ligands [50].
ED
and this effect had been already observed previously with other polypyridyl Ru complexes
PT
Accumulation of Ru1 in CT26 cell line resulted in concentration dependent decrease in
CE
intracellular calcium level, while Ru2 and bpySC caused the opposite effect - increasing the
calcium ions concentration. Such an increase in [Ca2+]C can cause a rise in mitochondrial calcium
AC
levels since those organelles serves as buffering units and might modulate Ca2+ feedbackinhibition or activation mechanisms. Increase in the mitochondrial calcium concentration can
cause the mitochondrial membrane depolarization and lead to higher amounts of reactive oxygen
species production. This can force the opening of the mitochondrial permeability pore causing
mitochondrial swelling and releasing cytochrome c and other pro-apoptotic factors, initiating
programmed cell death process.
35
ACCEPTED MANUSCRIPT
4
6
8
10
600
120
100
80
60
40
20
control 50 µM 100 µM 2 µM
Ru1
4 µM 50 µM 100 µM Val
500
400
300
200
100
0
control 50 µM 100 µM
2 µM
4 µM
bpy-SC
Ru1
Ru2
50 µM 100 µM
Val
Carb
bpy-SC
CR
Ru2
Carb
B
IP
0
Fluo-8AM
Fluorescence intensity [a.u.]
Fluo-8AM
Fluorescence intensity [a.u.]
2
A
T
0
140
Fig. 9. The effect of the Ru polypyridyl complexes on cytosolic calcium cations concentration
US
measured with Fluo-8AM in A549 (A) and CT26 (B) cell lines after 24 of incubation. The results
are representative for three independent experiments performed in triplicate. Valinomycin (Val)
M
AN
and carbachol (Carb) were used as positive controls.
4. Conclusions
ED
A new ligand based on semicarbazone 2-formylopyridine moiety conjugated with 2,2’-bipyridyl
PT
(bpySC) by an alkyne spacer has been developed. The two compounds produced as a result of
coordination by ruthenium(II) center by bpySC and either two bpy (Ru1) or two dip (Ru2)
CE
ligands have been obtained and characterized. They exhibited entirely different biological effects
AC
on cancer cell lines. The cytotoxic effect determined after 24 h of treatment with Ru2 was more
than an order of magnitude higher than that found for Ru1 as well as bpySC ligand alone against
both A549 and CT26 cancer cell lines. The extent of treatment up to 72 h resulted in the
increased anti-proliferative activity of both Ru1 and bpySC but not of Ru2. The Ru1 complex
accumulated preferentially in lysozyme, while Ru2 in mitochondria and endoplasmic reticulum.
Both Ru complexes and bpySC induced apoptosis. In the case of Ru2 it occurred via caspaseindependent pathway, while Ru1 and bpySC induced apoptosis in cancer cells through
36
ACCEPTED MANUSCRIPT
mitochondria signal transduction pathway, even though this organelle was not the primary
localization site. Both studied ruthenium complexes had an opposite effect on intracellular
calcium concentration. The increased in ROS production was evident upon treatment with Ru2
while negligible for Ru1 even after prolonged incubation time.
T
Taken together we have demonstrated that changing of bpy into dip ligands has a strong impact
IP
on the mechanism of biological activity of the ruthenium(II) complexes. Despite increasing the
CR
Ru complexes overall accumulation and cytotoxicity by choosing the more lipophilic dip ligands
the role of the semicarbazone 2-formylopyridine moiety in biological activity of the ruthenium
US
complex seems to be marginal. Therefore, a special attention must be paid in choosing
AN
appropriate auxiliary ligands in designing new ruthenium complexes to take advantage from
M
conjugation of biologically active molecules to polypyridyl ligands.
ED
Abbreviations
bpy – 2,2’-bipyridine;
–
semicarbazone;
5-(4-{4'-methyl-[2,2'-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde
CE
bpySC
PT
A549 – and human lung adenocarcinoma cell line;
AC
CT26 – murine colon carcinoma cell line;
DAPI – 4',6-diamidino-2-phenylindole;
DFT – Density Functional Theory;
dip – 4,7-diphenyl-1,10-phenanthroline;
DMEM – Dulbecco's Modified Eagle's medium;
DMF – dimethylformamide;
EtOH – ethanol;
37
ACCEPTED MANUSCRIPT
ER – endoplasmic reticulum;
ESI – electrospray ionization;
FBS – fetal bovine serum;
FITC – fluorescein isothiocyanate;
T
HOMO – highest occupied molecular orbital;
IP
HRMS – high resolution mass spectrometry;
CR
IC50 – concentration causing 50 % decrease in viability;
iPrMgCl – Isopropylmagnesium chloride;
US
JC-1 – 5,5,6,6-Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide;
AN
LDA – lithium diisopropylamide;
MeOH – methanol;
ED
MLCT – metal ligand charge transfer;
M
LUMO – lowest unoccupied molecular orbital;
QY – quantum yield;
PT
NMR – nuclear magnetic resonance;
CE
PBS – phosphate buffered saline;
PI – propidium iodide;
AC
ROS – reactive oxygen species;
RR – ribonucleotide reductase;
S- – in serum free conditions;
S+ – in the presence of 2%;
THF – tetrahydrofuran;
TD-DFT – Time-Dependent DFT.
38
ACCEPTED MANUSCRIPT
Acknowledgements
The CNRS and French Ministry of Research are thanked for their support. M. Ł. acknowledges
the French Embassy in Poland for providing a cotutelle Ph.D. grant. O. M. acknowledges the
financial support from the National Science Center (DEC-2013/11/N/ST5/01606). M. B.
the
financial
support
from
the
National
Science
Center
(DEC-
T
acknowledges
IP
2016/21/B/NZ7/01081). This work was carried out using the equipment purchased through
CR
financial support from the European Regional Development Fund in the framework of the Polish
US
Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08). This
AN
research was supported in part by PL-Grid Infrastructure.
M
References
[1] A. Bergamo, G. Sava, Dalton Trans. 40 (2011) 7817-7823.
ED
[2] C. Avendano, J.C. Menendez, Medicinal chemistry of anticancer drugs, Elsevier, 2008.
PT
[3] L.N. Lameijer, S.L. Hopkins, T.G. Breve, S.H.C. Askes, S. Bonnet, Chem. Eur. J 22 (2016)
18484-18491.
CE
[4] L. Blackmore, R. Moriarty, C. Dolan, K. Adamson, R.J. Forster, M. Devocelle, T.E. Keyes,
AC
Chem. Commun. 49 (2013) 2658-2660.
[5] K.K.W. Lo, T.K.M. Lee, J.S.Y. Lau, W.L. Poon, S.H. Cheng, Inorg. Chem. 47 (2008) 200208.
[6] R.R. Ye, Z.F. Ke, C.P. Tan, L. He, L.N. Ji, Z.W. Mao, Chem.-Eur. J. 19 (2013) 10160-10169.
[7] O. Mazuryk, M. Maciuszek, G. Stochel, F. Suzenet, M. Brindell, J. Inorg. Biochem. 134
(2014) 83-91.
[8] A. Son, A. Kawasaki, D. Hara, T. Ito, K. Tanabe, Chem. Eur. J 21 (2015) 2527-2525-2536.
39
ACCEPTED MANUSCRIPT
[9] G. Shi, S. Monro, R. Hennigar, J. Colpitts, J. Fong, K. Kasimova, H.M. Yin, R. DeCoste, C.
Spencer, L. Chamberlain, A. Mandel, L. Lilge, S.A. McFarland, Coord. Chem. Rev. 282 (2015)
127-138.
[10] J.X. Zhang, J.W. Zhou, C.F. Chan, D.W.J. Kwong, H.L. Tam, N.K. Mak, K.L. Wong, W.K.
T
Wong, BioConjugate Chem. 23 (2012) 1623-1638.
IP
[11] J. Yang, Q. Cao, W.-L. Hu, R.-R. Ye, L. He, L.-N. Ji, P.Z. Qinb, Z.-W. Mao, Dalton Trans.
CR
46 (2017) 445-454.
[12] S.M.H. Ali, Y.-K. Yan, P.P.F. Lee, K.Z.X. Khong, M.A. Sk, K.H. Lim, B. Klejevskajac, R.
US
Vilarc, Dalton Trans. 43 (2014) 1449-1459.
AN
[13] T.K. Venkatachalam, P.V. Bernhardt, C.J. Noble, N. Fletcher, G.K. Pierens, K.J. Thurecht,
D.C. Reutens, J. Inorg. Biochem. 162 (2016) 295-308.
M
[14] J.T. Wilson, X.H. Jiang, B.C. McGill, E.C. Lisic, J.E. Deweese, Chem. Res. Toxicol. 29
ED
(2016) 649-658.
[15] J. O’Neill, (2016).
PT
[16] C.A. Kunos, T. Radivoyevitch, S. Waggoner, R. Debernardo, K. Zanotti, K. Resnick, N.
CE
Fusco, R. Adams, R. Redline, P. Faulhaber, A. Dowlati, Gynecol. Oncol. 130 (2013) 75-80.
[17] N.S.H.N. Moorthy, N.M.F.S.A. Cerqueira, M.J. Ramos, P.A. Fernandes, Mini-Rev. Med.
AC
Chem. 13 (2013) 1862-1872.
[18] A.I. Matesanz, P. Souza, Mini-Rev. Med. Chem. 9 (2009) 1389-1396.
[19] W.L.E. Armarego, C.L.L. Chai, , , Purification of Laboratory Chemicals, 5th ed., Elsevier,
2003.
[20] M.C. Pirrung, The Synthetic Organic Chemist’s Companion, Wiley, New Jersey, 2007.
[21] J.J. Song, N.K. Yee, Z. Tan, J. Xu, S.R. Kapadia, C.H. Senanayake, Org. Lett. (2004) 49054907.
40
ACCEPTED MANUSCRIPT
[22] T. Togano, N. Nagao, M. Tsuchida, H. Kumakura, K. Hisamatsu, F. Howell, M. Mukaida,
Inorg. Chim. Acta 195 (1992) 221-225.
[23] R. Caspar, C. Cordier, J.B. Waern, C. Guyard-Duhayon, M. Gruselle, P. Le Floch, H.
Amouri, Inorg. Chem. 45 (2006) 4071-4078.
T
[24] J.B. Waern, C. Desmarets, L. Chamoreau, H. Amouri, A. Barbieri, C. Sabatini, B. Ventura,
IP
F. Barigelletti, Inorg. Chem. 47 (2008) 3340-3348.
CR
[25] R.B. Nair, B.M. Cullum, C.J. Murphy, Inorg. Chem. 36 (1997) 962-965.
[26] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Third Edition ed., Springer, 2006.
US
[27] P.A.M. Dirac, Proceedings of the Royal Society of London. Series A 123 (1929) 714-733.
AN
[28] J.C. Slater, Physical Review 81 (1951) 385-390.
[29] S.H. Vosko, L. Wilk, M. Nusair, Canadian Journal of Physics 58 (1980) 1200-1211.
M
[30] A.D. Becke, Physical Review A 38 (1988) 3098-3100.
ED
[31] C. Lee, W. Yang, R.G. Parr, Physical Review B 37 (1988) 785-789.
[32] R. Ditchfield, W.J. Hehre, J.A. Pople, J. Chem. Phys. 54 (1971) 724-728.
PT
[33] W.J. Hehre, R. Ditchfield, J.A. Pople, J. Chem. Phys. 56 (1972) 2257.
CE
[34] P.C. Hariharan, J.A. Pople, Theor. Chem. Acc. 28 (1973) 213-222.
[35] M.M. Francl, W.J. Pietro, W.J. Hehre, J.S. Binkley, D.J. DeFrees, J.A. Pople, M.S. Gordon,
AC
J. Chem. Phys. 77 (1982) 3654-3665.
[36] P.J. Hay, W.R. Wadt, J. Chem. Phys. 82 (1985) 270-283.
[37] W.R. Wadt, P.J. Hay, J. Chem. Phys. 82 (1985) 284.
[38] M. J. Frisch, G.W. Truck, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G.
Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, M.C. X. Li, A. Marenich, J. Bloino, B. G.
Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylow, J. L.
Sonnenberg, D. Williams-Young, F.L. F. Ding, F. Egidi, J. Goings, B. Peng, A. Petrone, T.
41
ACCEPTED MANUSCRIPT
Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H.
Nakai, T. Vreven, K. Throssell, J. A. Montgomery, J.E.P. Jr., F. Ogliaro, M. Bearpark, J. J. Heyd,
E. Brothers, K. N. Kudin, V. N. Staroverov, T. Keith, R. Kobayashi, J. Normand, K.
T
Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M.
IP
Klene, C. Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B.
CR
Foresman, D. J. Fox, , 2016., Gaussian, Inc., in, vol. Gaussian 09, Wallingford CT, 2016
[39] R. Dennington, T.A. Keith, J.M. Millam, GaussView, Version 6, , in, Semichem Inc.,
US
Shawnee Mission, KS, 2016.
AN
[40] J. Weyermann, D. Lochmann, Z. A., Int. J. Pharm. 288 (2005) 369-376.
[41] A. Lorents, P.K. Kodavali, N. Oskolkov, U. Langel, M. Hällbrink, M. Pooga, J. Biol. Chem.
M
287 (2012) 16880–16889.
ED
[42] D. Martineau, M. Beley, P.C. Gros, S. Cazzanti, S. Caramori, C.A. Bignozzi, Inorg. Chem.
46 ( 2007) 2272-2277.
PT
[43] O.V. Kharissova, B.I. Kharisov, U.O. Méndez, Microwave-assisted synthesis of
CE
coordination and organometallic compounds, in: G. Grundas (Ed.) Advances in induction and
microwave heating of mineral and organic materials, InTech, 2011.
AC
[44] O. Mazuryk, K. Magiera, B. Rys, F. Suzenet, C. Kieda, M. Brindell, J. Biol. Inorg. Chem. 19
(2014) 1305-1316.
[45] O. Mazuryk, F. Suzenet, C. Kieda, M. Brindell, Metallomics 7 (2015) 553-566.
[46] N. Sanvisens, R. de Llanos, S. Puig, Biomed. J. 36 (2013) 51-58.
[47] S. Erikssons, A. Graslund, S. Skog, L. Thelander, B. Tribukait, J. Biol. Chem. 259 (1984)
11695-11700.
[48] A.M. Florea, D. Büsselberg, Neurotoxicol. 30 ( 2009) 803-810.
42
ACCEPTED MANUSCRIPT
[49] G.R. Ash, F.L. Bygrave, Febs Lett. 78 (1977) 166-168.
[50] O. Mazuryk, O. Krysiak-Foria, A. Żak, F. Suzenet, A. Ptak-Belowska, T. Brzozowski, G.
AC
CE
PT
ED
M
AN
US
CR
IP
T
Stochel, M. Brindell, Eur. J. Pharm. Sci. 101 (2017) 43-55.
43
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
Graphical abstract
44
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
Appropriately chosen auxiliary ligands can regulate not only cytotoxicity and accumulation of
polypyridyl ruthenium complexes, but also determine the mechanism of cellular death and
highlight the effects of biologically active molecules.
45
ACCEPTED MANUSCRIPT
Highlights
T
IP
CR
US
AN
M
ED
PT
CE
New Ru complexes with semicarbazone 2-formylopyridine link to bipyridine were
synthesised.
As auxiliary ligands bipyridine and diphenylphenanthroline were chosen.
The studied compounds were powerful apoptosis inducing agents.
The anti-proliferative activity was strongly dependent on auxiliary ligands.
The auxiliary ligands determined effect of the complexes on cell cycle arrest and ROS
formation.
AC
46