Ruthenium(II)-arene complexes with dibenzoylmethane induce apoptotic cell death in multiple myeloma cell lines

Article abstract of DOI:10.1016/j.ica.2016.04.031

A series of ruthenium(II) arene complexes (arene = p-cymene, hexamethylbenzene and benzene) containing dibenzoylmethane ligand (DBMH) of formula [(arene)Ru(DBM)Cl] and their ionic derivatives bearing PTA (1,3,5-triaza-7-phosphaadamantane), of formula [(arene)Ru(DBM)(PTA)]X (X = PF₆- or SO₃CF₃-) have been synthesized and fully characterized. The solid-state structures of five complexes have been determined by single-crystal X-ray diffraction. These ruthenium materials exhibit intense photoluminescence emission at room temperature in the solid state. All complexes effectively bind to calf thymus DNA through intercalative/electrostatic interactions with more affinity for DNA minor groove. Finally, the antitumor activity of both ligand and complexes was evaluated against the U266 and RPMI human multiple myeloma cell lines, and some of them showed a cytotoxic and pro-apoptotic effect toward both cell lines.

Full text of DOI:10.1016/j.ica.2016.04.031

Inorganica Chimica Acta xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Ruthenium(II)-arene complexes with dibenzoylmethane induce
apoptotic cell death in multiple myeloma cell lines
b
a
a
a
Riccardo Pettinari a, , Fabio Marchetti , Agnese Petrini , Claudio Pettinari , Giulio Lupidi ,
⇑
Belén Fernández c, Antonio Rodríguez Diéguez c, Giorgio Santoni a, Massimo Nabissi a
a School of Pharmacy, University of Camerino, via S. Agostino 1, 62032 Camerino, MC, Italy
b School of Science and Technology, University of Camerino, via S. Agostino 1, 62032 Camerino, MC, Italy
c Departamento de Química Inorgánica, Universidad de Granada, Avda Fuentenueva s/n, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ruthenium(II) arene complexes (arene = p-cymene, hexamethylbenzene and benzene) contain-
ing dibenzoylmethane ligand (DBMH) of formula [(arene)Ru(DBM)Cl] and their ionic derivatives bearing
PTA (1,3,5-triaza-7-phosphaadamantane), of formula [(arene)Ru(DBM)(PTA)]X (X = PF6- or SO3CF3-) have
been synthesized and fully characterized. The solid-state structures of five complexes have been deter-
mined by single-crystal X-ray diffraction. These ruthenium materials exhibit intense photoluminescence
emission at room temperature in the solid state. All complexes effectively bind to calf thymus DNA
through intercalative/electrostatic interactions with more affinity for DNA minor groove. Finally, the anti-
tumor activity of both ligand and complexes was evaluated against the U266 and RPMI human multiple
myeloma cell lines, and some of them showed a cytotoxic and pro-apoptotic effect toward both cell lines.
Ó 2016 Elsevier B.V. All rights reserved.
Received 26 January 2016
Received in revised form 11 April 2016
Accepted 18 April 2016
Available online xxxx
Keywords:
Ru(II)-arene
Dibenzoylmethane
RAPTA
DNA interaction studies
Protein binding studies
Multiple myeloma
1. Introduction
b-diketonates, containing also the complex [(p-cym)Ru(DBM)Cl]
(1) reported in this work, possess good activity toward A2780
Half-sandwich
g
6-arene ruthenium complexes are currently
human ovarian cells [23]. The combination of dibenzoylmethane
and PTA ligands was also investigated by Dyson, who reported a
series of cationic arene-Ru(II) complexes, quite soluble in water
with a high cytotoxicity towards A2780 human ovarian cancer cells
and also against A549 lung carcinoma [24].
attracting increasing attention for their potential applications as
metallopharmaceuticals in cancer therapy [1–4]. The hydrophobic
arene ligand can facilitate the diffusion of the complexes through
the lipophilic cell membrane and the three remaining Ru coordina-
tion sites can be filled with several kinds of ligands [5,6]. Recent
developments on this field by several authors deal mainly on the
use of chelating N,N0-donor ligands, such as complexes from
Sadler’s group [7–10], or complexes with the P-donor ligand PTA
investigated by Dyson’s group [11–14]. However, also reports on
arene Ru(II) complexes with O,O0-donor ligands are attracting
increasing attention, as many different possibilities are available
to tune both the chelating ring from four to six-membered and
the steric crowing of such ligands [15–20]. Some papers have
focused on b-diketonate complexes, firstly investigated by Sadler
who reported that replacement of neutral ethylenediamine (en)
by anionic acetylacetonate (acac) as the chelating ligand not only
increases the rate and extent of hydrolysis but also changes
the nucleobase specificity [21,22]. Some of these arene-Ru(II)
Multiple myeloma (MM) remains almost an incurable disease,
and several new drugs are currently undergoing evaluation for dis-
ease control [25]. As a part of our continuing research in ruthenium
chemistry, here we report the synthesis of a series of arene-Ru(II)
complexes (arene = p-cymene, hexamethylbenzene, and benzene)
with dibenzoylmethane (DMBH) and their ionic derivatives with
the PTA ligand and two external counterions, namely PF6 and
SO3CF3 anions. The choice to use different counterions with the
same cationic complex was planned with the goal to investigate
their potential influence on cytotoxicity. Their radical scavenging
ability (DPPH), DNA and BSA-protein interactions and also the
cytotoxicity toward U266 and RPMI multiple myeloma (MM) cell
lines have been evaluated for the first time, differently from previ-
ous studies [23,24].
Our interest on these tumor lines deals on the fact that they
have been previously characterized for their cytogenetic abnormal-
ities (loss of 17p), which is associated with a poor outcome. In fact,
⇑
Corresponding author. Tel.: +39 0737402338.
E-mail address: riccardo.pettinari@unicam.it (R. Pettinari).
http://dx.doi.org/10.1016/j.ica.2016.04.031
0020-1693/Ó 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: R. Pettinari et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.04.031

2
R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
del(17p) is the most important molecular finding for prognostica-
tion as it linked to an aggressive disease phenotype [26].
2.2.2. [(hmb)Ru(DBM)Cl] (2)
The synthesis was performed as for 1 using a modified literature
method [27] (yield 86%). Compound 2 is soluble in alcohols, ace-
tone, acetonitrile, chlorinated solvents, DMF and DMSO and
slightly soluble in diethyl ether. Mp: 279–281 °C. Anal. Calc. for
2. Experimental
C
27H29ClO2Ru: C, 62.12; H, 5.60. Found: C, 62.15; H, 5.66. KM
(CD3CN, 298 K, 10ꢀ4 mol/L): 47 S cm2 molꢀ1. IR (cmꢀ1): 3013w
(CAH), 1592 m, 1543s, 1532vs (C@O; C@C), 1485m, 1454m,
1397s, 1373m, 1305m, 757s, 286m
(RuACl). 1H NMR (CDCl3,
293 K): d 2.15 (s, 18H, CH3(hmb)), 6.45 (s, 1H, CH of DBM), 7.40
(m, 6H, Ph of DBM), 7.96 (d, 4H, Ph of DBM, 3J = 7.2 Hz). 13C NMR
(CDCl3, 293 K): d 15.5 (s, CH3(hmb)), 90.7 (s, C6(hmb)), 93.0
(s, CH of DBM), 127.3, 128.3, 131.0, 139.5 (s, Ph of DBM), 180.9
(s, C@O, DBM). ESI-MS (+) CH3OH (m/z (relative intensity, %)):
487 (100) [(hmb)Ru(DBM)]+.
2.1. Materials and methods
m
m
The dimers [(arene)RuCl2]2 (arene = p-cym, hmb, benzene) and
dibenzoylmethane were purchased from Aldrich and were used
as received. All other materials were obtained from commercial
sources and were used as received. IR spectra were recorded from
4000 to 30 cmꢀ1 on a PerkinElmer Frontier FT-IR instrument. 1H,
13C and 31P NMR spectra were recorded on a 400 Mercury Plus Var-
ian instrument operating at room temperature (400 MHz for 1H
and 100 MHz for 13C relative to TMS, and 162 MHz relative to
85% H3PO4). Positive and negative ion electrospray ionization mass
spectra (ESI-MS) were obtained on a Series 1100 MSI detector HP
spectrometer using methanol as the mobile phase. Solutions
(3 mg/mL) analysis were prepared using reagent-grade methanol.
Masses and intensities were compared to those calculated using
IsoPro Isotopic Abundance Simulator, version 2.1.28. Melting
points are uncorrected and were recorded on a STMP3 Stuart
scientific instrument and on a capillary apparatus. Samples for
microanalysis were dried in vacuo to constant weight (20 °C, ca.
0.1 Torr) and analyzed on a Fisons Instruments 1108 CHNS-O
elemental analyzer. Electrical conductivity measurements
m
2.2.3. [(benz)Ru(DBM)Cl] (3)
The synthesis was performed as for 1 using [(benz)RuCl2]2 and
keeping the reaction stirring at reflux for 24 h (yield 85%). Com-
pound 3 is soluble in alcohols, acetone, acetonitrile, chlorinated sol-
vents, DMF and DMSO and slightly soluble in diethyl ether. Mp:
223–225 °C. Anal. Calc. for C21H17ClO2Ru: C, 57.60; H, 3.91. Found:
C, 57.49; H, 3.88. KM (CD3CN, 298 K, 10ꢀ4 mol/L): 19 S cm2 molꢀ1
.
IR (cmꢀ1): 3062w
1485m, 1451m, 1380s, 1373m, 1306w, 722s, 270s
m(CAH), 1591m, 1545s, 1522vs
m
(C@O; C@C),
m
(RuACl). 1H
NMR (CDCl3, 293 K): d 5.74 (s, 6H, C6H6(benz)), 6.44 (s, 1H, CH of
DBM), 7.41 (m, 6H, Ph of DBM), 7.90 (d, 4H, Ph of DBM,
3J = 7.2 Hz). 13C NMR (CDCl3, 293 K): 82.8 (s, C6H6(benz)), 94.0
(s, CH of DBM), 127.6, 128.3, 131.2, 139.1 (s, Ph of DBM), 182.1
(s, C@O, DBM). ESI-MS (+) CH3OH (m/z (relative intensity, %)):
403 (100) [(benz)Ru(DBM)]+.
(
KM, reported as S cm2 molꢀ1) of acetonitrile solutions of the
complexes were recorded using a Eutech Instruments CON2700
at room temperature. UV–Vis spectra of the proligands and com-
plexes were performed with a Varian Cary1 spectrometer at
20 °C. The fluorescence of the proligands and complexes was ana-
lyzed using a Hitachi F-4500 spectrofluorimeter at 20 °C.
2.2.4. [(p-cym)Ru(DBM)(PTA)][SO3CF3] (4)
2.2. Synthesis of ruthenium complexes
Compound 1 (271.7 mg, 0.55 mmol) was dissolved in methanol
(20 mL) and AgSO3CF3 (141.3 mg, 0.55 mmol) was added. The mix-
ture was stirred for 1 h at room temperature and filtered to remove
AgCl. PTA (PTA = 1,3,5-triaza-7-phosphaadamantane; 86.4 mg,
0.55 mmol) was then added to the filtrate which was stirred for
24 h at room temperature. Then the solution was dried by rotary
evaporation and the crude product was obtained by precipitation
using a mixture of dichloromethane and n-hexane. The yellow-
brown powder obtained (319.6 mg, 0.42 mmol, yield 76%) was
identified as 4. It is soluble in alcohols, acetone, acetonitrile, chlo-
rinated solvents, DMF and DMSO and rather soluble in water. Mp:
107–109 °C. Anal. Calc. for C32H37F3N3O5PRuS: C, 50.26; H, 4.88; N,
5.49. Found: C, 50.29; H, 4.92; N, 5.53. KM (CD3CN, 298 K,
2.2.1. [(p-cym)Ru(DBM)Cl] (1)
The compound was synthetized using a modified literature
method [23]. Dibenzoylmethane (DBMH, 224.0 mg, 1.00 mmol)
was dissolved in methanol (20 mL) and KOH (56.1 mg, 1.00 mmol)
was added. The mixture was stirred for 1 h at room temperature
and then [(p-cym)RuCl2]2 (244.9 mg, 0.40 mmol) was added. The
resulting mixture was stirred for 24 h at room temperature. The
solvent was removed under reduced pressure, dichloromethane
(10 mL) was added and the mixture was filtered to remove potas-
sium chloride. The solution was concentrated to 2 mL and then an
excess of n-hexane resulted in precipitation of a red powder
(332.0 mg, 0.67 mmol, yield 84%) which was identified as 1. It is
soluble in alcohols, acetone, acetonitrile, chlorinated solvents,
DMF and DMSO and slightly soluble in diethyl ether. Mp: 197–
198 °C. Anal. Calc. for C25H25ClO2Ru: C, 60.79; H, 5.10. Found: C,
10ꢀ4 mol/L): 126 S cm2 molꢀ1. IR (cmꢀ1): 3064-2938w
1590m, 1519vs (C@O; C@C), 1477m, 1374s, 1258vs, 1152s,
1030vs
(SO3CF3), 971s, 946vs, 723s, 636s. 1H NMR (CD3CN,
m(CAH),
m
m
293 K): d 1.31 (d, 6H, CH3C6H4CH(CH3)2, 3J = 6.8 Hz), 2.08 (s, 3H,
CH3C6H4CH-(CH3)2), 2.71 (sept, 1H, CH3C6H4CH(CH3)2, 3J = 6.8 Hz),
4.16 (s, 6H, NCH2P, PTA), 4.46 (s, 6H, NCH2N, PTA), 5.89 (d, 2H, CH3-
C6H4CH(CH3)2, 3J = 6.4 Hz), 5.94 (d, 2H, CH3C6H4CH(CH3)2,
3J = 6.4 Hz), 6.88 (s, 1H, CH of DBM), 7.53 (m, 4H, Ph of DBM),
7.63 (m, 2H, Ph of DBM), 8.00 (d, 4H, Ph of DBM, 3J = 8.0 Hz). 13C
NMR (CD3CN, 293 K): d 17.4 (s, CH3C6H4CH(CH3)2), 22.4 (s, CH3C6-
H4CH(CH3)2), 31.3 (s, CH3C6H4CH(CH3)2), 52.0 (d, PCH2N, PTA,
JCP = 13.0 Hz), 73.1 (d, NCH2N, PTA, JCP = 7.7 Hz), 89.3, 90.7 (s, CH3-
C6H4CH(CH3)2), 96.5 (s, CH of DBM), 98.5, 105.5 (s, CH3C6H4CH
(CH3)2), 128.4, 129.7, 133.1, 138.4 (s, Ph of DBM), 184.4 (s, C@O,
DBM). 31P NMR (CD3CN, 293 K): d ꢀ27.9 (s, PTA). ESI-MS (+) CH3OH
(m/z (relative intensity, %)): 616 (100) [(p-cym)Ru(DBM)(PTA)]+,
459 (20) [(p-cym)Ru(DBM)]+.
60.74; H, 5.08. KM (CD3CN, 298 K, 10ꢀ4 mol/L): 36 S cm2 molꢀ1
IR (cmꢀ1): 3063-2871w
(CAH), 1591m, 1520vs (C@O; C@C),
1371s, 1306s, 724s, 280m
(RuACl). 1H NMR (CDCl3, 293 K): d
.
m
m
m
1.41 (d, 6H, CH3C6H4CH(CH3)2, 3J = 7.2 Hz), 2.34 (s, 3H, CH3C6H4
CH-(CH3)2), 3.02 (sept, 1H, CH3C6H4CH(CH3)2, 3J = 7.2 Hz), 5.31 (d,
2H, CH3C6H4CH(CH3)2, 3J = 6.0 Hz), 5.59 (d, 2H, CH3C6H4CH(CH3)2,
3J = 6.0 Hz), 6.44 (s, 1H, CH of DBM), 7.39 (m, 6H, Ph of DBM),
7.91 (d, 4H, Ph of DBM, 3J = 7.2 Hz). 13C NMR (CDCl3, 293 K):
d 18.3 (s, CH3C6H4CH(CH3)2), 22.7 (s, CH3C6H4CH(CH3)2), 31.1
(s, CH3C6H4CH(CH3)2), 79.6, 83.4 (s, CH3C6H4CH(CH3)2), 93.6
(s, CH of DBM), 97.7, 100.0 (s, CH3C6H4CH(CH3)2), 127.5, 128.4,
131.2, 139.2 (s, Ph of DBM), 181.7 (s, C@O, DBM). ESI-MS (+)
CH3OH (m/z (relative intensity, %)): 459 (100) [(p-cym)Ru(DBM)]+.
Please cite this article in press as: R. Pettinari et al., Inorg. Chim. Acta (2016), http://dx.doi.org/10.1016/j.ica.2016.04.031

R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
3
2.2.5. [(p-cym)(RuDBM)(PTA)][PF6] (5)
equipped with graphite monochromated Mo
K
a
radiation
The synthesis was performed as for 4 using AgPF6 (yield 73%). 5
is soluble in alcohols, acetone, acetonitrile, chlorinated solvents,
DMF and DMSO and slightly soluble in water. Mp: 138–140 °C.
Anal. Calc. for C31H37F6N3O2P2Ru: C, 48.95; H, 4.90; N, 5.52. Found:
C, 48.91; H, 4.88; N, 5.49. KM (CD3CN, 298 K, 10ꢀ4 mol/L): 129 S
(k = 0.71073 Å). The data reduction was performed with the APEX2
[28] software and corrected for absorption using SADABS [29]. Crystal
structures were solved by direct methods using the SIR97 program
[30] and refined by full-matrix least-squares on F2 including all
reflections using anisotropic displacement parameters by means
of the WINGX crystallographic package [31,32]. Generally, anisotro-
pic temperature factors were assigned to all atoms except for
hydrogen atoms, which are riding their parent atoms with an iso-
tropic temperature factor arbitrarily chosen as 1.2 times that of the
respective parent. It should be noted that all crystals undergo
degradation when they are removed from the mother liquor which
has a high impact on the quality of the data. Several crystals of
compounds 3–7 were measured and the structures were solved
from the best data we were able to collect. For this reason isotropic
temperature factors were assigned to some non H atoms n the
structures. Attempts to solve disorder problems with one (SO3CF3)-
cm2 molꢀ1. IR (cmꢀ1): 3059-2925w
(C@O; C@C), 1476m, 1373s, 1260m, 972s, 947s, 833vs
m
(CAH), 1590m, 1519vs
m
m
(PF6),
722s, 556s. 1H NMR (CD3CN, 293 K): d 1.31 (d, 6H, CH3C6H4CH
(CH3)2, 3J = 6.8 Hz), 2.07 (s, 3H, CH3C6H4CH-(CH3)2), 2.71 (sept,
1H, CH3C6H4CH(CH3)2, 3J = 6.8 Hz), 4.16 (s, 6H, NCH2P, PTA), 4.45
(s, 6H, NCH2N, PTA), 5.88 (d, 2H, CH3C6H4CH(CH3)2, 3J = 6.4 Hz),
5.94 (d, 2H, CH3C6H4CH(CH3)2, 3J = 6.4 Hz), 6.88 (s, 1H, CH of
DBM), 7.54 (m, 4H, Ph of DBM), 7.63 (m, 2H, Ph of DBM), 8.00
(d, 4H, Ph of DBM, 3J = 8.0 Hz). 13C NMR (CD3CN, 293 K): d 17.5
(s, CH3C6H4CH(CH3)2), 22.5 (s, CH3C6H4CH(CH3)2), 31.5 (s, CH3C6-
H4CH(CH3)2), 52.4 (d, PCH2N, PTA, JCP = 13.0 Hz), 73.4 (d, NCH2N,
PTA, JCP = 7.7 Hz), 89.4, 90.7 (s, CH3C6H4CH(CH3)2), 96.7 (s, CH of
DBM), 98.9, 105.8 (s, CH3C6H4CH(CH3)2), 128.5, 129.7, 133.2,
138.6 (s, Ph of DBM), 184.7 (s, C@O, DBM). 31P NMR (CD3CN,
293 K): d ꢀ28.2 (s, PTA), ꢀ143.5 (sept, PF6, JPF = 706.4 Hz). ESI-MS
(+) CH3OH (m/z (relative intensity, %)): 616 (100) [(p-cym)Ru
(DBM)(PTA)]+, 459 (5) [(p-cym)(RuDBM)]+.
ꢀ
molecule and one chloroform solvent molecule failed in com-
pounds 4 and 6, respectively. Instead, a new set of F2 (hkl) values
with the contribution from solvent molecules withdrawn was
obtained by the SQUEEZE procedure implemented in PLATON-94 [33].
Final R(F), wR(F2) and goodness of fit agreement factors, details
on the data collection and analysis can be found in Table 1.
2.2.6. [(hmb)Ru(DBM)(PTA)][SO3CF3][CHCl3]3. (6)
2.4. Luminescence measurement
The synthesis was performed as for 4 using precursor 2 (yield
82%). 6 is soluble in alcohols, acetone, acetonitrile, chlorinated sol-
vents, DMF and DMSO and slightly soluble in water. Mp: 248–
250 °C. Anal. Calc. for C37H44Cl9F3N3O5PRuS: C, 38.61; H, 3.85; N,
3.65. Found: C, 38.57; H, 3.45; N, 3.36. KM (CD3CN, 298 K,
A Varian Cary-Eclipse fluorescence spectrofluorometer was
used to obtain the fluorescence spectra. The spectrofluorometer
was equipped with a xenon discharge lamp (peak power equiva-
lent to 75 kW), Czerny–Turner monochromators, and a R-928 pho-
tomultiplier tube which is red sensitive (even 900 nm) with
manual or automatic voltage controlled using Cary Eclipse soft-
ware for Windows 95/98/NT system. The photomultiplier detector
voltage was 700 V and the instrument excitation and emission slits
were set at 5 and 5 nm, respectively.
10ꢀ4 mol/L): 125 S cm2 molꢀ1. IR (cmꢀ1): 2926w
1519vs (C@O; C@C), 1475m, 1374s, 1260vs, 1148s, 1030vs
m
(CAH), 1589m,
(SO3-
m
m
CF3), 971s, 947vs, 722m, 636m. 1H NMR (CD3CN, 293 K): d 2.12 (s,
18H, CH3(hmb)), 4.07 (s, 6H, NCH2P, PTA), 4.39 (m, 6H, NCH2N,
PTA), 6.89 (s, 1H, CH of DBM), 7.55 (m, 4H, Ph of DBM), 7.64 (m,
2H, Ph of DBM), 8.08 (d, 4H, Ph of DBM, 3J = 8.0 Hz). 13C NMR (CD3-
CN, 293 K): d 16.4 (s, CH3(hmb)), 50.5 (d, PCH2N, PTA, JCP = 12.2 Hz),
73.5 (d, NCH2N, PTA, JCP = 7.2 Hz), 96.2 (s, C6(hmb)), 99.6 (s, CH
DBM), 128.3, 130.0, 133.2, 139.3 (s, Ph of DBM), 184.1 (s, C@O of
DBM). 31P NMR (CD3CN, 293 K): d ꢀ35.2 (s, PTA). ESI-MS (+) CH3OH
(m/z (relative intensity, %)): 644 (100) [(hmb)Ru (DBM)(PTA)]+, 487
(15) [(hmb)Ru(DBM)]+.
2.5. DNA interaction studies
Stock solutions of calf thymus DNA (ct-DNA) (Sigma–Aldrich)
were prepared by dissolving the solid material overnight in
10 mM phosphate buffer pH 7.4 containing 5 mM NaCl at 4 °C.
The solution of ct-DNA in phosphate buffer gave a ratio of UV
absorbance at 260 and 280 nm, A260/A280, of ca. 1.9, indicating that
the DNA was sufficiently free from protein. The concentration of ct-
DNA solution was determined by UV absorbance at 260 nm. The
2.2.7. [(hmb)Ru(DBM)(PTA)][PF6][CHCl3]][H2O] (7)
The synthesis was performed as for 6 using AgPF6 (yield 79%). 7
is soluble in alcohols, acetone, acetonitrile, chlorinated solvents,
DMF and DMSO and slightly soluble in water. Mp: 184–185 °C.
Anal. Calc. for C33H44Cl3F6N3O3P2Ru: C, 44.10; H, 4.79; N, 4.54.
Found: C, 43.93; H, 4.69; N, 4.42. KM (CD3CN, 298 K, 10ꢀ4 mol/L):
molar absorption coefficient,
e
260, was taken as 6600 Mꢀ1 cmꢀ1
[34]. Complexes and ligands were dissolved in DMSO.
2.6. Electronic absorption titration
138 S cm2 molꢀ1. IR (cmꢀ1): 2924w
m
(CAH), 1589m, 1519vs
(C@O; C@C), 1475m, 1374s, 1279m, 1242m, 1014m, 972s, 947s,
834vs
(PF6), 722s, 556s. 1H NMR (CD3CN, 293 K): d 2.12 (s, 18H,
m
Electronic absorption spectra were carried out at 25 °C by fixing
the concentration of the complex or ligands, with DNA concentra-
tion ranging from 0 to 35 10ꢀ6 M. Absorption spectra were mea-
sured at 200–600 nm in 50 mM phosphate buffer pH 7.4. The
complex solution was titrated with the DNA, and changes on the
intensity of the bands of the complex were monitored. The intrin-
sic binding constant (Kb) for the interaction of the complexes with
m
CH3(hmb)), 4.07 (s, 6H, NCH2P, PTA), 4.39 (s, 6H, NCH2N, PTA),
6.89 (s, 1H, CH of DBM), 7.56 (m, 4H, Ph of DBM), 7.63 (m, 2H,
Ph of DBM), 8.07 (d, 4H, Ph of DBM, 3J = 8.0 Hz). 13C NMR (CD3CN,
293 K): d 16.9 (s, CH3(hmb)), 51.1 (d, PCH2N, PTA, JCP = 12.2 Hz),
74.0 (d, NCH2N, PTA, JCP = 7.1 Hz), 96.7 (s, C6(hmb)), 100.1 (s, CH
of DBM), 128.8, 130.5, 133.7, 139.9 (s, Ph of DBM), 184.6 (s, C@O
of DBM). 31P NMR (CD3CN, 293 K): d ꢀ35.3 (s, PTA), ꢀ143.5 (sept,
PF6, JPF = 706.4 Hz). ESI-MS (+) CH3OH (m/z (relative intensity,
%)): 644 (100) [(hmb)Ru (DBM)(PTA)]+, 487 (15) [(hmb)Ru(DBM)]+.
ct-DNA was determined from a plot of [DNA]/(ea
using absorption spectral titration data and the following equation
ꢀ
ef) versus [DNA]
[35]:
½DNAꢁ
½DNAꢁ
1
eb
¼
þ
ð1Þ
2.3. X-ray crystallography
j
ea
ꢀ
ef
j
j
eb
ꢀ
ef
j
Kb
j
ꢀ
ef
j
Red crystals for 3–7 were mounted on a glass fibre and used for
where [DNA] is the concentration of DNA, the apparent absorption
data collection on a Bruker D8 Venture with Photon detector
coefficients ea ef and eb correspond to Aobsd/[complex], the
,
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4
R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
Table 1
Crystallographic data and structural refinement details for 3–7.
Compound
3
4
5
6
7
Chemical formula
CCDC
C
21H17ClO2Ru
C32H37F3N3O5RuS
1449968
764.74
100
C31H37F6N3O2P2Ru
1449969
760.64
100
C37H44Cl9F3N3O5RuS
1449970
1150.90
100
C34H44Cl3F6N3O3P2Ru
1449966
926.08
1449967
437.86
100
M/gmolꢀ1
T (K)
100
k (Å)
0.71073
monoclinic
P21/c
11.1175(9)
11.6046(9)
13.8839(10)
104.575(3)
1733.6(2)
4
0.71073
orthorhombic
Pbca
10.169(3)
21.587(6)
30.539(9)
90
6704(3)
8
1.515
0.71073
orthorhombic
Pbca
10.0960(19)
20.469(4)
30.285(5)
90
6259(2)
8
1.615
0.71073
monoclinic
P21/c
9.1857(4)
24.3202(11)
19.8960(8)
100.877(2)
4364.9(3)
4
0.71073
monoclinic
P21/c
9.5940(9)
36.361(5)
11.4295(11)
97.265(4)
3955.2(7)
4
Crystal system
Space group
a (Å)
b (Å)
c (Å)
b (°)
V (Å3)
Z
q
l
(g cmꢀ3
(mmꢀ1
)
)
1.678
1.069
1.751
1.055
1.555
0.746
0.638
0.674
Unique reflections
6024
0.055
1.011
0.035
28956
0.096
0.962
0.051
0.123
38237
0.149
1.262
0.133
21909
0.116
1.055
0.092
12710
0.088
1.098
0.089
Rint
Goodness-of-fit (GOF) on F2
R1 [I > 2
wR2 [I > 2
r
(I)]a
(I)]a
r
0.059
0.306
0.193
0.171
a
R(F) = å||Fo| ꢀ |Fc||/å|Fo|; wR(F2) = [åw(Fo2 ꢀ Fc2)2/åwF4]1/2
.
extinction coefficient for the free metal complex and the extinction
coefficient for the metal complex in the fully bound form, respec-
tively. The Kb value is given by the ratio of the slope to the intercept.
2.7.2. Minor groove displacement assay
A minor groove binders, 40,6-diamidino-2-phenilindole (DAPI),
was used to perform the minor groove displacement assays. In a
typical experiment, the changes in the emission spectra of DAPI
complex with the DNA in 50 mM phosphate buffer pH 7.4, were
monitored upon addition of increasing concentrations of com-
pounds at room temperature. Fluorescence quenching data were
evaluated after excitation of DAPI-ct-DNA complex at 338 nm
and recording the spectra from 400 to 600 nm. Values of Ksv
constants were evaluated by using Eq. (3), after that all quenching
fluorescence values obtained were corrected according to the Eq.
(2). In the presence of enhancement of fluorescent, the values of
Ksv were evaluated as previously described [38,39].
2.7. Competitive binding studies
2.7.1. Major groove displacement assay
A well-known major groove binder, ethidium bromide (EB), was
used to perform the major groove displacement assays. The com-
petitive binding experiment was carried out by maintaining the
ethidium bromide (EB) and ct-DNA concentration at 5
55.7 M, respectively, while increasing the concentration of differ-
ent complexes. Fluorescence quenching spectra were recorded
using Hitachi 4500 spectrofluorimeter with an excitation
lM and
l
a
2.8. BSA binding studies
wavelength of 490 nm and emission spectrum in the range
500–700 nm. The fluorescence decrease in the emission spectra
was corrected according to Eq. (2):
The protein-binding study was performed by tryptophan fluo-
rescence quenching experiments using bovine serum albumin
(BSA). Bovine serum albumin (Sigma–Aldrich) stock solutions were
prepared in 50 mM potassium phosphate buffer at pH 7.4. Fluores-
cence measurements were carried out with a Hitachi 4500 spec-
trofluorometer by keeping the concentration of BSA constant
(15 ꢂ 10ꢀ6 M) while varying the complex concentration from were
added to protein solution from 0 to 40 ꢂ 10ꢀ6 M, at room temper-
atures. Fluorescence intensities were recorded after each succes-
sive addition of complex solution and equilibration (ca. 5 min).
Fluorescence spectra were recorded from 300 to 450 nm at an exci-
tation wavelength of 285 nm. The values of Stern Volmer constant
Ksv and the bimolecular quenching rate constant Kq of the metal
complexes to BSA were evaluated following the Eq. (3) considering
þA
Þ
2
Fc ¼ Fm ꢂ eðA
ð2Þ
1
2
where Fc and Fm are the corrected and measured fluorescence,
respectively [36] and A1 and A2 are the absorbances of tested com-
pounds at the exciting and emission wavelengths. For fluorescence
quenching experiments, Stern–Volmer’s Eq. (3) was used:
F0
Fc
¼ 1 þ kqs0½Cꢁ ¼ 1 þ Ksv½Cꢁ
ð3Þ
where F0 and Fc represent the fluorescence intensity in the absence
and presence of the metal complexes, respectively, [C] is the con-
centration of the complex and Ksv is the Stern–Volmer constant that
can be obtained from fluorescence values plotted as F0/Fc versus the
complex concentration [36]. All experiments involving ct-DNA were
performed at room temperature in phosphate buffer solution
(50 mM, pH 7.4). The apparent binding constant (Kapp) of the metal
complexes has been calculated by using the Eq. (4):
that s0 was the average fluorescence lifetime of the fluorophore in
the absence of drug with a value of about 10ꢀ8 s [37]. All fluores-
cent values were corrected by Eq. (2).
Considering the existence of similar and independent binding
sites in the BSA for the static quenching interaction, the binding
constant (Kb) and the number of the binding sites (n) for the differ-
ent complexes on BSA can be estimated using the Scatchard Eq. (5):
[40,41]
KEB½EBꢁ ¼ Kapp½Cꢁ
ð4Þ
ꢀ
ꢁ
F0ꢀFC
ꢂ
ꢃ
in which the complex concentration [C] is the value at a 50% reduc-
tion of the fluorescence intensity of EB and KEB = 1.0 ꢂ 107 Mꢀ1
F0
F0 ꢀ FC
¼ nKb ꢀ Kb
ð5Þ
½Cꢁ
F0
([EB] = 5.0 lM) [37].
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R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
5
where n is the number of binding sites for protein, Kb (Mꢀ1) is the
association binding constant that can be calculated from the slope
of plot of ((F0 ꢀ FC)/F0)/[C] versus ((F0 ꢀ FC)/F0), n is given by the
ratio of the y intercept to the slope.
arene
Cl
O
O
MeOH
KOH
[Ru(arene) Cl2]2
Ru
+
O
O
2.9. Cell culture and inhibition of cell growth
2.9.1. Cells
RPMI8226 (RPMI) and U266 MM cell lines (purchased from
ATCC, LGC Standards, Milan, IT). Cell authentication was performed
by IST (Genova, Italy). Cell lines were cultured in RPMI medium
(Lonza, Milan, IT) supplemented with 10% fetal bovine serum
1
, arene = p-cym
2, arene = hmb
3, arene = benz
Scheme 1. Synthesis of complexes 1–3.
(FBS), 2 mM L-glutamine, 100 IU/ml penicillin, 100 lg strepto-
mycin and 1 mM sodium pyruvate. Cell lines were maintained at
37 °C with 5% CO2 and 95% humidity.
By substitution of chloride ligand with PTA in the ruthenium
coordination environment, achieved by adding PTA and silver salts,
i.e. AgX where X = SO3CF3 or PF6, cationic complexes 4–7 were iso-
lated (Scheme 2). Complexes 1–7 are air stable and soluble in alco-
hols, acetone, acetonitrile and DMSO and chlorinated solvents but
cationic complexes 4–7 are also partially soluble in water. The IR
2.9.2. MTT assay
3 ꢂ 103 cells/well were seeded in 96-well plates, in a final vol-
ume of 100 lL. After 1 incubation day, DBM complexes or vehicle
were added and cell viability was evaluated up to 72 h. Four repli-
cate wells were used for each treatment. At the indicated time
point, cell viability was assessed by adding 0.8 mg/ml of MTT
(Sigma–Aldrich) to the media. After 3 h, the plates were cen-
trifuged, the supernatant was discharged, and the pellet was solu-
spectra of 1–7 display the typical shift of
after metal coordination [42,43]. For complexes 1–3 also the
(RuACl) has been detected in the far-IR region. By contrast, the
m(C@O) of b-diketones
m
IR spectra of 5 and 7 display a strong, sharp absorption at 825
and 826 cmꢀ1 due to the PF6 counteranion [44,45] whereas the
spectra of 4 and 6 contain a characteristic absorption pattern in
the region 1000–1200 cmꢀ1, typical of a not-coordinated O3SCF3
anion [46]. The 1H NMR spectra of 1–7 display all the expected sig-
nals due to the coordinated arene rings, DBM ligand and for 4–7
also of PTA. The resonances due to the PTA ligand are shifted to
lower field with respect to those of uncoordinated PTA confirming
its coordination to the ruthenium center [47]. The 31P NMR spectra
of 4 and 5, containing the p-cymene moiety, display a singlet cen-
tered at ca. ꢀ28 ppm due to PTA, whereas 6 and 7 with the hexam-
ethyl moiety show the PTA resonance at ca. ꢀ35 ppm, in a range
typical of related compounds and in accordance with the existence
of only one species in solution [48].
bilized with 100 ll/well DMSO. The absorbance of the samples
against a background control (medium alone) was measured at
570 nm using an ELISA reader microliter plate (BioTek Instruments,
Winooski, VT).
2.9.3. Cell cycle analysis
3 ꢂ 104 cells/ml were incubated with 25
lM of the DBM com-
plexes, for 48 h. Then, cells were fixed for 1 h by adding ice-cold
70% ethanol and washed with staining buffer (PBS, 2% FBS and
0.01% NaN3). Next, the cells were treated with 10
ase A solution (Sigma– Aldrich), incubated for 30 min at 37 °C,
stained for 30 min at room temperature with 20 g/ml of propid-
lg/ml ribonucle-
l
ium iodide (Sigma–Aldrich) and finally analyzed by flow cytometry
using linear amplification.
The ESI mass spectra of 1–3 show a main peak corresponding to
[Ru(arene)(DBM)]+ fragment. Whereas the ESI mass spectra of 4–7
show two main peak envelopes, that of highest relative intensity
corresponding to the intact [Ru(arene)(DBM)(PTA)]+ species, the
other being due to the [Ru(arene)(DBM)]+ fragment formed upon
PTA dissociation. For complexes 1–3, conductivity measurements
in acetonitrile indicate a slight dissociation of the chloride at room
temperature. Whereas, in acetonitrile, ionic derivatives 4–7 display
conductivity values within the range typical of 1:1 electrolytes
[49].
2.9.4. Apoptosis assays
The exposure of phosphatidylserine on MM cells was detected
by Annexin V staining and cytofluorimetric analysis. Briefly,
3 ꢂ 104/ml cells were treated with 25
lM of the appropriate drugs
for 72 h. Four replicates were used for each treatment. After treat-
ment, the cells were stained with 5 ll of Annexin V FITC (Vinci
Biochem, Vinci, Italy) for 10 min at room temperature, washed
once with binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2) and analyzed on a FACScan flow cytometer
using CellQuest software.
The stability of 6 (the most cytotoxic compound, see Section 3.4)
was also determined under pseudo-pharmacological conditions in
5 mM NaCl solution (being a model for the low intracellular
2.9.5. Statistical analysis
The data presented represent the mean and standard deviation
arene
-
+
(SD) of at least
3 independent experiments. The statistical
X
arene
⁄
#
significance was determined by Student’s t-test;
,
,
§p < 0.01.
The statistical analysis of IC50 levels was performed using Prism
5.0a (Graph Pad).
Ru
Ru
MeOH
O
Cl
O
P
N
N
O
1. AgX
2. PTA
O
N
3. Results and discussion
3.1. Synthesis and characterization
4 arene = p-cym, X = SO3CF3
5, arene = p-cym, X = PF6
The arene-Ru(II) complexes 1–3 were prepared in high yield in a
single step (Scheme 1), by interaction of [(arene)RuCl2]2 (arene =
p-cymene (p-cym), hexamethylbenzene (hmb) and benzene(benz))
with dibenzoylmethane DBMH in basic solution.
6
, arene = hmb, X = SO3CF3
7, arene = hmb, X = PF6
Scheme 2. Synthesis of complexes 4–7.
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6
R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
chloride concentration in cells) and in 100 mM NaCl solution
(approximating to the higher chloride levels in blood). Solutions
of the complexes (c = 2.0 mM) in aqueous NaCl (c = 5 mM or
100 mM in D2O containing 10% of [D6]DMSO) were prepared and
maintained at 37 °C for 7 days. The decomposition of the
complexes was monitored by 1H and 31P NMR spectroscopy. The
complex [(hmb)Ru(DBM)(PTA)][SO3CF3] did not undergo hydroly-
sis in either 5 and 100 mM NaCl solutions, i.e. the 1H and 31P
NMR spectra of 6 remained unchanged after 7 days (see Fig. 1).
3.2. Crystal structures
Fig. 2. Perspective view of
omitted for clarity.
3 [(benz)RuCl(DBM)]. Hydrogen atoms have been
The solid state structures of 3–7 were established by X-ray crys-
tallography (see Section 2). Their structures are shown in Figs. 2–6
and principal bond parameters are given in the Table S1. In general,
the structures consist in ‘‘piano stool” distribution formed by the
ruthenium-arene unit bound to the chloride, PTA and DBM ligand.
The distances of ruthenium atom and arene rings are 1.650, 1.700,
1.705, 1.710 and 1.695 Å for compounds 3, 4, 5, 6 and 7, respec-
tively, in good agreement with others published distances
[50,51]. Compound 3 crystallizes in P21/c monoclinic group. In
mononuclear complex, the metal is coordinated with a pseudo-
octahedral geometry to one benzene, formally occupying three
contiguous coordination positions, one chloride atom and two
positions corresponding to one DBM ligand (Fig. 2). The RuACl
distance has a value of 2.425(1) Å while DBM coordinates in a che-
late mode with RuAO bond distances of 2.055(3) and 2.068(3) Å.
The chloride atom and DBM molecule occupy a fac disposition with
angles in the range 84.03(8)–88.34(11). These mononuclear units
present staking interactions (3.597 Å) between the arene and one
of the benzene pertaining to DBM of a neighboring molecule.
Compound 4 crystallizes in Pbca orthorhombic group. In this
case, coordination geometry about the metal center ruthenium like
those in 3, is typical ‘‘piano stool” with one position occupied by
phosphorous atom from PTA, two oxygen atoms from DBM ligand
and arene ring in ɳ6-manner. The RuAP distance has a value of
2.3140(17) Å while DBM coordinates in a quelate mode with RuAO
bond distances of 2.071(4) and 2.078(4) Å. The phosphorous atom
and DBM molecule occupy a fac disposition with angles in the
range 86.11(11)–88.26(15). In this molecule, planes containing
benzene rings pertaining to DBM ligand present a dihedral angle
of 37.63°. These mononuclear compounds present partial staking
interactions (3.440 Å) between the arene rings of neighboring
molecules due to the steric impediments of methyl groups. Com-
pound 5 crystallizes in Pbca orthorhombic group. In this case, coor-
dination geometry about the metal center ruthenium is similar to
4. One position is occupied by phosphorous atom from PTA and the
others two positions have oxygen atoms from DBM ligand. The
principal different of this compound with 4 is the presence of
(PF6)ꢀ anion instead (SO3CF3)ꢀ.
The RuAP distance has a value of 2.302(7) Å while DBM coordi-
nates in a chelate mode with RuAO bond distances of 2.061(15)
and 2.067(15) Å. The phosphorous atom and DBM molecule occu-
pies a fac disposition with angles in the range 86.0(5)–89.0(5). In
this molecule, planes containing benzene rings pertaining to
DBM ligand present a dihedral angle of 34.53°. These mononuclear
compounds present partial staking interactions (3.351 Å) between
the arene rings due to the steric impediments of methyl groups.
Compound 6 crystallizes in P21/c monoclinic group. In this case,
coordination geometry about the metal center ruthenium like
those in the above compounds, with one position occupied by
phosphorous atom from PTA, two oxygen atoms from DBM ligand
Fig. 1. 31P NMR spectra of 6 in 5 mM (a) and in 100 mM (b) aqueous NaCl solution.
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R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
7
Fig. 5. View of 6 [(hmb)Ru(DBM)(PTA)](SO3CF3)(CHCl3)3. One chloroform solvent
molecule and hydrogen atoms have been omitted for clarity.
Fig. 3. View of 4 [(p-cym)Ru(DBM)(PTA)](SO3CF3). Anion and hydrogen atoms have
been omitted for clarity.
Fig. 6. View of 7 [(hmb)Ru(DBM)(PTA)][PF6][CHCl3]][H2O]. Hydrogen atoms have
been omitted for clarity.
occupies a fac disposition with angles in the range 81.40(19)–
88.12(19). In this molecule, planes containing benzene rings per-
taining to DBM ligand present a dihedral angle of 35.67°. These
mononuclear compounds presents staking interactions (3.314 Å)
between the arene and one of the benzene pertaining to DBM of
a neighbouring molecule.
Compound 7 crystallizes in P21/c monoclinic group. In this case,
coordination geometry about the metal center ruthenium like
those in 6. One position is occupied by phosphorous atom from
PTA and the others two positions have oxygen atoms from DBM
ligand. The principal different of this compound with 6 is the pres-
ence of (PF6)ꢀ anions and crystallization water molecules. The
RuAP distance has a value of 2.336(3) Å and DBM coordinates in
a chelate mode with RuAO bond distances of 2.055(8) and 2.096
(7) Å. The phosphorous atom and DBM molecule occupies a fac dis-
position with angles in the range 82.8(2)–87.1(2). In this molecule,
planes containing benzene rings pertaining to DBM ligand present
a dihedral angle of 35.67°. These mononuclear compounds present
staking interactions (3.379 Å) between the arene and one of the
benzene pertaining to DBM of a neighboring molecule. There is a
strong hydrogen bond (2.911 Å) between the water molecule and
the nitrogen atom N3B pertaining to the PTA ligand.
Fig. 4. View of complex 5 [(cym)Ru(DBM)(PTA)](PF6). Hydrogen atoms have been
omitted for clarity.
and hexamethylbenzene in ɳ6-manner. This mononuclear com-
pound contains one (SO3CF3)ꢀ anion and three chloroform solvent
molecules. The RuAP distance has a value of 2.297(3) Å while DBM
coordinates in a chelate mode with RuAO bond distances of 2.073
(6) and 2.079(6) Å. The phosphorous atom and DBM molecule
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8
R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
Table 2
3.3. Luminescence studies
DBM complexes reduce cell viability in U266 and RPMI human multiple myeloma
(MM) cell lines. U266 and RPMI cell lines, were cultured up to 72 h with different
doses (0-to 500 M) of DBM complexes. Cell viability was determined by MTT assay.
Data shown are expressed as mean SE of three separate experiments. IC50 values
were calculated using Prism 5.0, at 72 h post-treatments.
Due to its extended aromaticity, the DBMH ligand is a good can-
didate for enhanced emissive properties. For this reason, room
temperature solid state emission spectra of the synthesized com-
pounds 1–7 have been recorded. As previously reported [52], coor-
dination compounds based on dibenzoylmethane ligand exhibit
broad emission bands with the maxima peaking around 350 nm,
respectively. When excited at 310 nm, the main emissions of com-
pounds 1–7 (Fig. 7) are slightly shifted to 360 and 375 nm com-
pared to the DBMH ligand.
l
Compound
RPMI (IC50
lM) S.E.
U266 (IC50 lM) S.E.
1
2
3
4
5
6
7
16.9 0.2
19.8 0.6
37.8 1.8
20.2 1.8
21.3 1.7
16.7 1.7
40.2 2.5
463.2 12
16.6 0.1
20.1 1.6
39.2 2.7
19.4 0.6
23.1 1.2
15.5 1.1
38.4 3.1
448.2 18.2
The similarity of the emission spectra of 1–7 is in agreement
with the explanation, which indicates that ligand centred
p–
p⁄
DBMH
excitation is responsible for the emission. On the other hand, it
can be presumed that all these emissions originate from p⁄ ? or
and/or on cell death induction, in MM cell lines. First, the effects of
DBM complexes (at 25 M dose), on cell cycle phases, was ana-
even p⁄
? p transitions in which the energy gap slightly decreases
l
(red shift or bathochromic emission) according to the coordination
of the ligands to metal centres due to the increased rigidity pro-
vided to the system [53].
lyzed at 24 and 48 h post-treatments: while at 24 h no effects were
observed (results not shown), at 48 h post-treatments an effect on
cell cycle phases was demonstrated. In fact, we found that all the
DBM complexes induce an accumulation in G1 and in Sub-G1
(indicating DNA fragmentation) phases of cell cycle compared with
vehicle, with 5 showing the higher percentage of Sub-G1 and G1
cell accumulation (Fig. S3, Supporting information). Similar results
were obtained in both MM cell lines. These results indicate that
complexes 1–7 are able to induce cell cycle arrest and to increase
the percentage of cells in sub-G1 phase, suggesting that DBM-trea-
ted cells activated a mechanism of cell death.
3.4. Biological studies
3.4.1. Cytotoxicity
U266 and RPMI human multiple myeloma cell lines were resis-
tant to a 48 h exposure to cisplatin at 1
by Gougelet [54]. The ability of 1–7, used at different doses
(1–500 M) and incubation times (from 24 up to 72 h), to affect
lg/mL, as recently described
l
the viability of U266 and RPMI human multiple myeloma cells,
has been evaluated by MTT assay. As shown in Table 2, all ruthe-
nium derivatives were effective in reducing the viability of both
MM cells, in a time- and dose-dependent manner starting from
24 h post-treatments (Figs. S1 and S2, Supporting information).
IC50 values, calculated at 72 h post-treatments, indicate differ-
ent efficacy of DBM complexes in reducing cell viability. All com-
plexes are comparatively more effective than the free neutral
DBMH proligand. Among the similar chloride species 1–3, com-
pound 3, containing benzene on Ru, is the less effective toward
both tumour cell lines while 1 with p-cymene on Ru displays the
highest efficacy. Previous studies from Sadler and Dyson on p-cym-
ene-Ru b-diketonates against the A2780 cell line, reported that the
introduction of PTA on the complex increased their activity [23,24],
here we observe a reduction of the efficacy for complex 4 and 5
with respect to neutral complex 1. The hmb-Ru(II) complex 2 is
slightly less active than p-cym-Ru(II) 1. In the case of ionic hmb
complexes 6 and 7, compared to 2, we observe an increase and a
reduction of activity, respectively. The SO3CFꢀ3 counterion seems
to improve the activity in 4 and 6, with respect to PFꢀ6 in 5 and 7.
The reduced cell viability effect, observed with DBM complexes,
was evaluated by investigating their effect on cell cycle regulation
To further investigate the Sub-G1 cells status, after DBM com-
plexes treatments we stained DBM-treated MM cell lines with
Annexin-V and apoptotic cells death was determined. As shown
(Fig. S4, Supporting information) DBM complexes are able to
induce, with different percentage, apoptotic cell death with similar
effects in both MM cell lines.
3.4.2. Antioxidant activity
The in vitro antioxidant activities for the proligand DBMH and
complexes 1–7 were determined by the DPPH assay [55,56] and
results are given in Table 3.
Complexes 1–5 present higher scavenging activity against DPPH,
compared to the DBMH proligand and 1, 3 and 5 are also more effi-
cient than Trolox used as control, while 6 and 7 show a much lower
efficacy. Among the neutral complexes 1–3, that containing the ben-
zene moiety displays the highest antioxidant activity. The introduc-
tion of PTA decreases the antioxidant activity in all type of
complexes, with a strong effect for hmb-Ru complexes 6 and 7.
3.5. DNA-binding properties
3.5.1. Electronic absorption titration
The interaction of proligand DBMH and complexes 1–7 with ct-
DNA was followed by spectrophotometric titrations. In absence of
ct-DNA, UV–Vis spectra of all compounds display intense bands
around 240–280 and 300–350 nm (see Supplementary material,
Table 3
Antioxidant activity of DBMH and complexes 1–7.
Compound
DPPH IC50 10ꢀ6
M
1
2
3
15.85 ( 0.20)
40.12 ( 0.80)
6.48 ( 0.10)
4
5
6
7
50.64 ( 0.29)
19.14 ( 0.10)
439.39 ( 7.25)
293.09 ( 5.18)
155.52 ( 3.10)
21.80 ( 0.80)
DBMH
Trolox
Fig. 7. The experimental emission spectra of compounds 1–7 after excitation at
310 nm in solid state at room temperature.
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R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
9
Table 5
Fig. S5) due to the phenyl groups of DBM scaffold, and a medium
intensity band close to 400 nm arising from a metal to ligand charge
transfer transition, typical of these d6 arene-Ru(II) complexes [57].
From the absorption titration spectrum (Fig. S5, Supporting infor-
mation) it is apparent that upon addition of ct-DNA to fixed concen-
trations of complexes 1–7 and DBMH, the intra ligand band
(251 nm) displayed hyperchromism along with a negligible red
shift. On the other hand bands in the range 300–350 nm exhibit
hypochromism without any significant shift. The presence of isos-
bestic points in the titration curve indicate the presence of more
than two species in the medium. To compare the binding affinity
of the complexes with ct-DNA, the equilibrium binding constants
(Kb) have been evaluated and data reported in Table 4: a rather
strong binding of DBMH, 4 and 6 to ct-DNA has been found. Among
the neutral complexes 1–3 the one containing the hmb moiety (2)
displays the greater affinity for ct-DNA. The introduction of PTA
in ionic compounds with SO3CFꢀ3 counterion (complexes 4 and 6)
strongly increases the affinity for ct-DNA with respect to the other
ionic complexes 5 and 7 with the PF-6 counterion.
Protein quenching constant (Ksv), binding constant (Kb), and number of binding sites
(n) for the interaction of DBMH and complexes 1–7 with BSA.
Complex
BSA binding
Ksv 105 Mꢀ1
kq 1012 Mꢀ1 sꢀ1
Kb 105 Mꢀ1 sꢀ1
n
1
2
3
4
5
6
7
1.22 ( 0.07)
0.53 ( 0.03)
0.62 ( 0.04)
0.70 ( 0.01)
0.55 ( 0.02)
0.49 ( 0.01)
0.21 ( 0.02)
0.07 ( 0.01)
12.2 ( 0.3)
5.3 ( 0.3)
6.2 ( 0.4)
7.0 ( 0.1)
5.5 ( 0.2)
4.9 ( 0.1)
2.1 ( 0.2)
0.7 ( 0.1)
0.61 (0.04)
0.52 (0.07)
0.38 (0.05)
0.19 (0.03)
0.25 (0.02)
0.24 (0.03)
0.17 (0.02)
0.02 (0.00)
1.23
1.01
1.21
1.65
1.40
1.51
1.14
1.25
DBMH
on BSA fluorescence intensity is shown in Fig. S8 (Supporting infor-
mation). The strong fluorescence emission peak at 346 nm of BSA
decreased when it was titrated with different amounts of 1–7. From
the value of Ksv it appears that all complexes show more affinity for
the protein with respect to DBMH proligand. Neutral complexes 1–3
are more active than their ionic derivatives and all the compounds
containing the p-cymene moiety show a higher binding affinity to
BSA than the others. The value of bimolecular quenching constant
(kq), reported in Table 5 for all tested compounds, falls in the range
0.7–12.2 1012 L molꢀ1 sꢀ1, which is higher than the maximum possi-
ble value for dynamic quenching (2.0 1010 L molꢀ1 sꢀ1) [50]. These
results might indicate that the quenching could be initiated not by
dynamic collision but through the formation of the complex
between quencher and fluorophore suggesting the involvement of
a static quenching mechanism. The data of the binding constant
(Kb) together with the number of the binding sites (n) that were
found close to 1, suggest that there is only one binding site for these
compounds on the BSA molecule.
3.5.2. Competitive binding studies
In order to provide further evidence for the interaction mode of
1–7 with ct-DNA, EtBr displacement experiments have been per-
formed by competitive binding fluorescence assay. The displace-
ment of EtBr from the major grove of ct-DNA by a metal complex
leads to a decrease in the fluorescence intensity. Quenching
parameters for all compounds, Ks and Kapp are reported in Table 4.
By addition of increasing amounts of complexes 1–7 and DBMH,
the fluorescence spectra display a decrease of intensity of the emis-
sion band at 590 nm due to the EB-ct-DNA system (Fig. S6, Support-
ing information), thus indicating the displacement of EB from the
major grove of the ct-DNA. The compound 2 with hmb moiety
shows higher activity with respect to p-cymene and benzene
derivatives 1 and 3 respectively. Among the ionic derivatives, 4
and 6 containing the SO3CFꢀ3 counterion display lower binding
activity with respect to PF6 derivatives 5 and 7. Further evidence
for the interaction mode of DBMH and complexes 1–7 to ct-DNA
was evaluated by using a displacement assay with DAPI [58]. All
complexes show good binding affinity for the minor grove of DNA
(Fig. S7, Supporting information). In more detail, all complexes
are less effective than the free DBMH proligand and among the neu-
tral complexes 1–3, the one containing the p-cymene moiety (1) is
slightly more effective. Lower differences have been observed for
ionic complexes 4–7. From the observed quenching binding param-
eters we can hypothesize that all complexes bind to DNA via inter-
calation mode with more affinity for the minor grove.
4. Conclusions
A series of neutral ruthenium(II) arene complexes with diben-
zoylmethane and their ionic derivatives embedding PTA and differ-
ent counterions have been synthesized. The complexes were fully
characterized by analytical and spectroscopic methods and the
solid-state structures of neutral and ionic complexes have been
determined by single-crystal X-ray diffraction. The stability under
pseudo-physiological conditions was also investigate for the most
cytotoxic complex (6) showing that it is stable in these environ-
ments up to seven days. All physicochemical techniques tested
demonstrated that all the compounds effectively bind with DNA
through intercalative/electrostatic interactions with higher bind-
ing affinity through minor DNA groove.
The protein binding properties of the compounds suggest that
they interact with protein (BSA) and the binding affinity increases
in all complexes with an affinity from 3 to 15-fold higher to that
reported for the free DBMH ligand. The higher antioxidant activity
of complexes 1, 3 and 5 with respect to Trolox suggests a possible
application of these compounds in the elimination of the hydroxyl
radical. All compounds reported here possess a good cytotoxicity
toward both U266 and RPMI human multiple myeloma cell lines.
In the case of ionic complexes, the SO3CFꢀ3 counterion increases
the in vitro anticancer activity with respect to PFꢀ6 . In conclusion
the coordination of dibenzoylmethane to the arene-ruthenium(II)
moiety is able to preserve the interaction with DNA, but increases
the binding to BSA, the free radical scavenger properties and the
cytotoxicity towards tumor cell lines.
3.6. Protein binding studies
To understand the mechanism of interaction of 1–7 and DBMH
with Bovine Serum Albumin (BSA), fluorescence quenching experi-
ments have been carried out. The effect of complexes 1–7 and DBMH
Table 4
Binding constant (Kb), Stern–Volmer constant (Ksv) and the apparent binding constant
(Kapp) for DBMH complexes 1–7 for ct-DNA and EB-ct-DNA, DAPI-ct-DNA complexes.
Complex
ct-DNA
EB-ct-DNA
DAPI-ct-DNA
Kb 104 Mꢀ1
Ksv 104 Mꢀ1
Kapp 105 Mꢀ1
Ksv 104 Mꢀ1
1
2
3
4
5
6
7
3.59 ( 0.09)
4.99 ( 0.10)
2.86 ( 0.10)
11.48 ( 0.15)
3.82 ( 0.10)
17.91 ( 0.23)
2.55 ( 0.39)
9.37 ( 0.19)
0.56 ( 0.02)
0.66 ( 0.05)
0.47 ( 0.01)
0.48 ( 0.03)
0.63 ( 0.04)
0.17 ( 0.01)
0.60 ( 0.05)
0.20 ( 0.06)
2.80 ( 0.10)
3.30 ( 0.25)
0.85 ( 0.05)
2.40 ( 0.15)
3.15 ( 0.20)
0.85 ( 0.05)
3.00 ( 0.25)
1.00 ( 0.30)
1.86 ( 0.09)
0.56 ( 0.01)
0.91 ( 0.02)
1.26 ( 0.02)
1.08 ( 0.03)
1.48 ( 0.02)
1.47 ( 0.05)
2.84 ( 0.13)
Author contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
DBMH
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10
R. Pettinari et al. / Inorganica Chimica Acta xxx (2016) xxx–xxx
[22] M. Melchart, A. Habtemariam, S. Parsons, P.J. Sadler, J. Inorg. Biochem. 101
Notes
(2007) 1903.
[23] A. Habtemariam, M. Melchart, R. Fernández, S. Parsons, I.D.H. Oswald, A.
Parkin, F.P.A. Fabbiani, J.E. Davidson, A. Dawson, R.E. Aird, D.I. Jodrell, P.J.
Sadler, J. Med. Chem. 49 (2006) 6858.
The authors declare no competing financial interest.
[24] C.A. Vock, A.K. Renfrew, R. Scopelliti, L. Juillerat-Jeanneret, P.J. Dyson, Eur. J.
Inorg. Chem. 2008 (2008) 1661.
Acknowledgments
[25] A. Romano, C. Conticello, M. Cavalli, C. Vetro, C. Di Raimondo, V. Di Martina, E.
Schinocca, A. La Fauci, N.L. Parrinello, A. Chiarenza, F. Di Raimondo, BioMed
Res. Int. 2014 (2014) 14.
We thank the University of Camerino for financial support.
[26] M.B. Morelli, M. Offidani, F. Alesiani, G. Discepoli, S. Liberati, A. Olivieri, M.
Santoni, G. Santoni, P. Leoni, M. Nabissi, Int. J. Cancer 134 (2014) 2534.
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Absorption Correction of Area Detector Data 1996.
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Appendix A. Supplementary material
CCDC 1449966–1449970 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2016.04.031.
[31] G.M. Sheldrick, Program for Crystal Structure Refinement, University of
Göttingen, Göttingen, Germany, 2014.
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