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Synthesis, chemical characterization and preliminary in vitro antitumor activity evaluation of new ruthenium(II) complexes with sugar derivatives
Polyhedron 30 (2011) 1671–1679
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, chemical characterization and preliminary in vitro antitumor activity
evaluation of new ruthenium(II) complexes with sugar derivatives
Gianfranco Fontana a, Michele Abbate b,⇑, Girolamo Casella b, Claudia Pellerito b, Alessandro Longo c,
Francesco Ferrante b
a
b
c
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Università degli Studi di Palermo, Viale delle Scienze, Parco d’Orleans II, Ed. 17, 90128 Palermo, Italy
Dipartimento di Chimica ‘‘Stanislao Cannizzaro’’, Università degli Studi di Palermo, Viale delle Scienze, Parco d’Orleans II, Ed. 17, 90128 Palermo, Italy
C.N.R. – I.S.M.N. via Ugo La Malfa 153, 90146 Palermo, Italy
a r t i c l e
i n f o
Article history:
Received 5 May 2010
Accepted 23 March 2011
Available online 14 April 2011
Keywords:
Ruthenium(II)
Carbohydrates
Anti-cancer
Melanoma A375
a b s t r a c t
Three new complexes of Ru(II), namely [RuCl2(Glun-N,O)2]Na2 (I; Glun = glucosaminate), [RuCl2(1Tglu)(EtOH)2]Na (II; 1-Tglu = 1-thio-b-D-glucose) and [Ru2(EtOH)6(AL)Cl4] (III; AL = 60 -aminolactose)
were prepared from the same Ru(II) precursor, [RuCl2(DMSO)4] (DMSO = dimethyl sulfoxide). The characterization of the complexes was carried out by elemental analysis, FT-IR, ES-MS, NMR, EXAFS and
DFT calculations. The effectiveness of the complexes on metastatic melanoma A 375 was investigated.
The results show that complex II is the most active species.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Ruthenium complexes are increasingly attracting the interest of
researchers due to their promising pharmacological properties
[1a,b]. Particularly with ligands of biological relevance, the preferred
octahedral coordination for the common (II) and (III) oxidation
states in aqueous solution, together with adequate substitution
rates and redox potentials for biological interactions and a demonstrated low toxicity make ruthenium a particularly attractive choice
for the development of new metallopharmaceuticals [2–11].
The first representative of bioactive ruthenium complexes is the
Ru(III) complex trans-[RuCl4(Im)(DMSO)][ImH] (NAMI-A; Im =
imidazole), synthesized and characterized by Sava et al. in Trieste,
which possesses outstanding antimetastatic activity [12] and it has
completed phase I clinical trials [13]. Further, trans-[RuCl4(Ind)2][IndH] (KP1019; Ind = indazole) [14–16] possesses a high activity against colon cancer [17]. The effectiveness of NAMI-A on the
metastatic melanoma B16 has recently been demonstrated and it
was suggested that the metastasis inhibition is due to the negative
modulation of tumor cell invasion processes, a mechanism in
which the reduction of the gelatinolytic activity of tumor cells
plays a crucial role [18].
Some interesting examples of bioactive Ru(II) complexes are
Ru(II)–arene/ethylenediamine and related derivatives as well as
Ru(II)–arene–PTA complexes (PTA = 1,3,5-triaza-7-phosphaadamantane) [19]. Further, cis- and trans-[RuCl2(DMSO)4], two neutral
octahedral Ru(II) compounds, showed significant antitumor activity [20–22]. The trans isomer is markedly more toxic and reactive
than the cis one [21,23].
The aim of this work was to prepare three new Ru(II) complexes
with carbohydrate-based ligands and to investigate their cytotoxicity in vitro on melanoma A375 cell lines. This biological target was
chosen considering that A375 cells are relatively resistant to many
standard cancer therapies [24–26] and the Ru(II)-derived complex
[Ru(bbdo)(dppz)](ClO4)2 (bbdo = 1,8-bis(benzimidazol-2-yl)-3,6dithiaoctane,
dppz = dipyrido[3,2a:20 ,30 -c]phenazine)
showed
selective inhibition activity against this cell line [27]. The structures
of the ligands employed are reported in Fig. 1. To the best of our
knowledge, little of work has been done to investigate carbohydrate
based ruthenium complexes [28], though the use of these ligands
may imply potentially interesting consequences for the metabolism, bioavailability and administration procedure of the drugs [1b].
2. Experimental
⇑ Corresponding author. Tel.: +39 91 590367; fax: +39 91 427584.
E-mail addresses: fontgian@unipa.it (G. Fontana), micheleabbate@unipa.it (M.
Abbate), gcasella@unipa.it (G. Casella), bioinorg@unipa.it (C. Pellerito), alex@pa.ismn.cnr.it (A. Longo), f.ferrante@unipa.it (F. Ferrante).
0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2011.03.046
2.1. Materials
All the reagents used were chemically pure or analytical reagent
grade. Ethanol was from J.T. Beaker (Italy). D-Glucosaminic acid
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G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
NH2
O
O
OH
HO
NH2
ONa
HO
OH OH O
a
O
HO
HO
O
O
O
HO
O
SNa
O
O
OH
O
O
c
b
Fig. 1. Chemical structures of D-glucosaminic acid sodium salt (a), 1-thio-b-D-glucose sodium salt (b) and 60 -aminolactose precursor (c).
was from Fluka (Italy) and the 1-thio-b-D-glucose sodium salt was
from Aldrich (Italy).
The starting complex, [RuCl2(DMSO)4], was prepared by reported literature methods [20–22]. Glucosaminic acid was deprotonated according to the following procedure: 390 mg (2 mmol)
of the acid were dissolved in 5 mL of ethanol, then NaOH
(120 mg, 3 mmol) was added in one portion. The solution was stirred for 30 min, then the solvent removed by rotary evaporation.
The residual water was removed by several co-evaporation steps
with toluene.
2.2. Physical measurements
2.2.1. Elemental analysis
The recrystallized solids were analyzed for C, H, N and S, using a
Vario EL III CHNS elemental analyzer (Elementar Analysensysteme
GmbH, Hanau, Germany) in our laboratory. Chlorine was determined by the Schöniger W. method [29].
2.2.2. FT-IR spectroscopy
FT-IR spectra were registered, as KBr pellets or in Nujol mulls in
CsI windows, on a Mod. Spectrum One Perkin–Elmer FT-IR spectrophotometer, in the 4000–300 cm1 region.
2.2.3. Mass spectroscopy
Electrospray ionization (ESI) MS spectra were recorded on a
Finnigan LCQ-Duo ion trap electrospray mass spectrometer (Bremen, Germany). Sample solutions were introduced into the ESI
source via 100 lm i.d. fused silica, from a 250 lL syringe. The
experimental conditions for the spectra, acquired in positive ion
mode, were as follows: needle voltage 5 kV; flow rate 5 lL/min;
source temperature 150 °C; m/z range 200–2000; cone potential
46 V; tube lens offset 15 V.
High-resolution (HR) MS were recorded using an Autospec Ultima o-TOF (Micromass, Manchester, UK) mass spectrometer connected with a gas chromatography (GC) system HP 6890 series
(Hewlett Packard, Palo Alto, CA, USA).
2.2.4. NMR spectroscopy
One-dimensional 1H, 13C{1H} and two-dimensional 1H-COSY,
1
( H–13C)-gHSQC, (1H–13C)-gHMBC spectra were acquired at 298 K
on a Bruker ARX 300 (7.04 T) spectrometer at 300.13 and
76.45 MHz with spectral widths corresponding to windows of 10
and 200 ppm, respectively. The 1H and 13C{1H} spectra of the free
ligands were acquired in CDCl3 and calibrated by the solvent’s proton and carbon resonances (1H, d = 7.26 ppm; 13C, d = 77.16 ppm).
For compounds I and II, the 1H NMR spectra were acquired in
D2O and D2O/EtOH-d6 (1:1 V/V) solutions, respectively, and the
external reference for 1H and 13C were the resonances of 4,4-dimethyl 4-silapentane sodium sulfonate (DSS) (1H, d = 0.00 ppm;
13
C, d = 0.00 ppm). For compound III, the 1H NMR spectrum was acquired in DMSO-d6 solution and calibrated upon the solvent’s proton and carbon resonances (1H, d = 2.51 ppm; 13C, d = 39.52 ppm).
13
C{1H} spectra were acquired with broadband proton powergated decoupling. The concentrations for all the samples were in
the 0.05–0.10 M range.
2.2.5. Optical rotations
Optical rotations were recorded on a Jasco P-1010 polarimeter.
The specific rotation was obtained in CHCl3 at 29 °C.
2.2.6. Flash chromatography (FC): Si gel Merck LiChroprepÒ 15–25/
25–40 lm 1/1 was used for flash chromatography (elution under
0.7 psi of Ar)
2.2.6.1. EXAFS spectroscopy. EXAFS spectra were recorded in the
transmission mode at beam line XAFS at the ELETTRA synchrotron
(Trieste, Italy). Spectra at the Ru K-edge at 22.117 eV were acquired
at low temperature (80 K) using a Si(1 1 1) monochromator. For Ru
K-edge measurements a ruthenium metal foil was used as a reference. Detection was performed by using three ionization chambers, reading the intensity before and after the sample and after
the reference sample, respectively. The energy was calibrated to
the first inflection point of the Ru-metal foil. A total of 2–4 scans
were collected for each sample for an analysis time of about 1 h
in order to get an acceptable signal-to-noise ratio. Extraction and
fitting of the EXAFS signal were performed by standard procedures
using the HORAE software suite [30]. Fourier transforms (FT) of the
w(k)k2v (k) EXAFS data were carried out in the range 2–12 Å1.
The phase shift and backscattering amplitude factor were corrected by using the calculated values of FEFF6 [31], for Ru as an absorber and Cl, S, O and N as scatterers.
2.2.7. Computational details
Density functional calculations have been performed with
GAUSSIAN 03 [32] on a number of isomers of complexes I–III using
the B3LYP functional [33] (in restricted and unrestricted forms)
joined with the correlation consistent polarized valence double
zeta, cc-pVDZ, basis set for light atoms and the Relativistic Small
Core Stuttgart ‘97 Effective Core Potential for ruthenium, with
the corresponding valence basis set. Various spin multiplicities
have been investigated for the three ruthenium complexes, and
the singlet state was always the most stable. The character of the
minimum in the potential energy surface of all the optimized
geometries has been checked by inspection of the harmonic vibrational frequencies; accordingly all energy values here reported
have been corrected for the zero point vibrational contribution.
2.3. Synthesis
2.3.1. Synthesis of the ligand c
2.3.1.1. 60 -Phtalimido-2,3:5,6:30 ,40 -tri-O-isopropylidenelactose dimethyl acetal (c00 , Scheme 1). About 280 lL (288 mg, 1.35 mmol) of
diisopropyl azodicarboxylate were added dropwise to a stirred
solution of 200 mg (0.393 mmol) of compound c0 , 360 mg
(1.37 mmol) of triphenylphosphine and 200 mg (1.36 mmol) of
phtalimide at 0 °C under Ar. Then the solution was warmed to
G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
room temperature (RT) and stirred overnight. The solvent was removed under reduced pressure and the residue was partitioned between brine and ethyl acetate. The organic phase was washed with
water and dried over Na2SO4. The solvent was removed by a rotary
evaporator and the residue was purified by flash chromatography
(FC) (elution with EtOAc–petroleum ether in gradient from 3:7 to
7:3) to give pure c00 .
Compound c00 : amorphous solid; yield: 55.0%; ½a29
D + 8.9 (c
0.336, CHCl3); IR (KBr) mmax cm1: 3428, 3029, 2990, 1718, 1216,
1071, 1103. 1H NMR (300 MHz, CDCl3) d galacto moiety: 3.80
(dd, J6A0 –6B0 = 14.0, J6A0 –50 = 10.3, 1H, H-60 A), 4.06 (m, 1H, H-40 ),
4.08 (dd, J20 –10 = J20 –30 = 7.8, 1H, H-20 ), 4.11 (m, 1H, H-30 ), 4.12 (m,
1H, H-50 ), 4.18 (dd, J60 B–60 A = 10.3, J60 B–50 = 5.3, 1H, H-60 B), 4.21 (d,
J10 –20 = 8.0, 1H, H-10 ); gluco moiety: 3.53 (m, 1H, H-5), 3.73 (m,
1H, H-2), 3.90 (m, 1H, H-6A), 3.92 (d, J1–2 = 4.3, 1H, H-1), 3.97 (m,
2H, H-6B + H-4), 4.00 (m, 1H, H-3); other signals: 1.27 (s, 6H,
2 CH3 isopropylidene), 1.31 (s, 3H, CH3 isopropylidene), 1.34 (s,
3H, CH3 isopropylidene), 1.47 (s, 3H, CH3 isopropylidene), 1.57 (s,
3H, CH3 isopropylidene), 3.22 (s, 3H, OCH3), 3.31 (s, 3H, OCH3),
7.47–8 (m, 4H, CH aryl). 13C NMR (75.00 MHz, CDCl3) d galacto
moiety: 39.2 (t, C-60 ), 71.00 (d, C-50 ), 73.5 (d, C-30 ), 75.5 (d, C-20 ),
77.9 (d, C-30 ), 104.4 (d, C-10 ); gluco moiety: 65.0 (t, C-6), 74.4 (d,
C-5), 77.1 (d, C-2), 77.5 (d, C-4), 79.1 (d, C-3), 104.8 (d, C-1); other
signals: 25.2–28.5 (6 peaks, q, C(CH3)2), 54.4 (q, OMe), 57.5 (q,
OMe), 108.5 (s, C(CH3)2), 110.0 (s, C(CH3)2), 110.7 (s, C(CH3)2),
123.8 (d, aromatic C), 131.5 (s, aromatic C), 134.3 (d, aromatic C),
168.5 (s, imidic carbonyl). HRMS: m/z 637.6717, calculated for
C31H43NO13: 637.2734.
2.3.1.2. 60 -Amino-2,3:5,6:30 ,40 -tri-O-isopropylidenelactose dimethyl
acetal (c, Scheme 1). To a solution of 640 mg (1 mmol) of compound c0 0 in 2 mL of absolute EtOH, 11 lL of monohydrate hydrazine were added in one portion. The resulting solution was
refluxed for 3.5 h and then slowly cooled to 0 °C and kept at this
temperature for 12 h. A precipitate appeared that was filtered off.
The solution was reduced to a small volume by rotary distillation
and treated with 1:1 HCl until a pH value of 4 was reached. Then
10 mL of brine were added to the solution, which was then extracted with 2:1 CHCl3/EtOH. The solvent was removed by rotary
distillation and the residue, still containing large amount of
‘‘phthalate derivate’’ impurities was suspended in 2:1 EtOH/H2O
and treated with 1 N NaOH until a pH value of 12 was obtained,
and then it was stirred for 1 h. Afterward the solvent was removed
by rotary distillation and the residual slurry was crushed and stirred into 10 mL of ethyl acetate. Finally the organic phase was
passed through a funnel containing a short pad of Celite under a
pad of dry Na2SO4 between two layers of sea sand. The subsequent
purification by FC (light petroleum/EtOAc 1:1, then pure EtOAc,
then EtOAc/MeOH 8:2) afforded compound c, see Fig. 1 (67% yield).
Compound c: caramel colored amorphous solid; ½a29
D + 29.8 (c
0.420, CHCl3); IR (KBr) mmax cm1: 3365, 3304, 3184, 1253, 1216,
1075; 1H NMR (300 MHz, CDCl3) d galacto moiety: 2.86 (dd, J60 A–
0
60 B = 13.3, J60 A–50 = 2.0, 1H, H-6 A), 3.06 (dd, J60 B–60 A = 13.3, J60 B–
0
0
0
0 = 9.6, 1H, H-6 B), 3.45 (m, 1H, H-2 ), 3.66 (m, 1H, H-5 ), 3.99
5
(m, 1H, H-30 ), 4.25 (m, 1H, H-40 ), 4.37 (d, J10 –20 = 8.2, 1H, H-10 ); gluco
moiety 3.84 (m, 1H, H-3), 3.95 (m, 1H, H-6A), 3.98 (m, 1H, H-5),
4.00 (m, 1H, H-4), 4.13 (dd, J6B–6A = 8.0, J6B–5 = 6.0, 1H, H-6B),
4.28 (d, J1–2 = 6.0, 1H, H-1), 4.45 (dd, J2–1 = J2–3 = 6.9, 1H, H-2);
other signals: 1.25 (s, 6H, 2 CH3 isopropylidene), 1.32 (s, 6H,
2 CH3 isopropylidene), 1.43 (s, 6H, 2 CH3 isopropylidene),
3.92 (s, 3H, OCH3), 3.41 (s, 3H, OCH3).
13
C NMR (75.00 MHz, CDCl3) d galacto moiety: 43.0 (t, C-60 ),
74.5 (d, C-20 ), 74.9 (d, C-50 ), 78.2 (d, C-40 ), 79.6 (d, C-30 ), 103.8 (d,
C-10 ); gluco moiety 64.8 (t, C-6), 75.2 (d, C-4), 76.1 (d, C-5), 76.2
(d, C-2), 78.5 (d, C-3), 107.1 (d, C-1); other signals 24.9–29.1 (q, 6
peaks, C(CH3)2), 54.1 (q, OCH3), 56.5 (q, OCH3), 107.7 (s, C(CH3)2),
1673
109.3 (s, C(CH3)2), 109.6 (s, C(CH3)2). HRMS: m/z 507.2665, calculated for C23H41NO11: 507.2680.
2.3.2. Synthesis of the Ru(II) complexes
2.3.2.1. [RuCl2(Glun-N,O)2] Na2 (I). In a Schlenk flask, [RuCl2(DMSO)4] (484.5 mg, 1 mmol) was dissolved in 25 mL of dried ethanol
and gently refluxed for 2 h. Then a solution of glucosaminic acid
sodium salt (434 mg, 2 mmol) dissolved in 10 mL of ethanol was
added and the mixture was heated under reflux for four days.
The color of the solution changed from brown-green to brown.
The product precipitated from the solution after five days of isothermal evaporation of the solvent in air. The product was filtered
out and washed with diethyl ether. The complex was recrystallized
from ethanol and dried in a vacuum over P4O10. Yield: 63%. (Anal.
Calc. for C12H24Cl2N2Na2O12Ru (Mr = 606.3) C, 23.77; H, 3.99; N,
4.62; Cl, 11.70. Found: C, 23.91; H, 4.15; N, 4.69; Cl, 11.83%). Selected IR bands (KBr, cm1) (s = strong, m = medium, and
w = weak): 3372 (m, mOH), 3239 (w, mNH), 1656 (s, mC@O), 404 (m,
mRuO), 314 (s, mRuCl), 520 (mRuN) [34]. 1H NMR (d ppm, 300 MHz,
D2O): 3.95 (m, 1H, H-2), 4.32 (m, 1H, H-3), 3.75 (m, 1H, H-4),
3.70 (m, 1H, H-5), 3.63 (m, 2H, H-6).
2.3.2.2. [RuCl2(1-Tglu)(EtOH)2]Na (II). In a Schlenk flask, [RuCl2(DMSO)4] (484.5 mg, 1 mmol) was dissolved in 25 mL of dried ethanol
and gently refluxed for 30 min. Afterwards, 1-thio-b-D-glucose sodium salt (654.6 mg, 3 mmol) dissolved in 20 mL of ethanol was
added in one portion. The solution was refluxed for two days and
its color changed from orange to black. A brown-black precipitate
appeared in the solution after a few days of isothermal evaporation
of the solvent in air. The product was filtered out and washed with
diethyl ether. The complex was recrystallized from ethanol and
dried in a vacuum over P4O10. Yield: 63%. (Anal. Calc. for
C10H23Cl2NaO7RuS (Mr = 482.3) C, 24.90; H, 4.81; S, 6.65; Cl,
14.70. Found: C, 24.56; H, 4.87; S, 6.78; Cl, 15.35%). Selected IR
bands (KBr, cm1): 3400 (m, mOH), 422 (w, mRuS), 325 (s, mRuCl). 1H
NMR (d ppm, 300 MHz, D2O/EtOH-d6): 3.27 (m overlapped, 3H,
H-3 + 2H-6), 3.32 (m, 1H, H-4), 3.45 (m, 2H, H-2 + H-5), 4.70 (d,
J = 8.5 Hz, 1H, H-1).
2.3.2.3. [Ru2(EtOH)6(AL)Cl4] (III). A 100 mL Schlenk flask equipped
with a reflux condenser was charged with 40 mL of dried ethanol
and [RuCl2(DMSO)4] (484.5 mg, 1 mmol). A freshly prepared solution of the 60 -aminolactose precursor c (1.523 g, 3.0 mmol) was
added and the resulting mixture was refluxed for five days. The
hot solution produced a brown solid which was separated by filtration, washed several times with cold diethyl ether and dried in a
vacuum. Yield: 55%. (Anal. Calc. for C24H59Cl4NO16Ru2
(Mr = 961.7) C, 29.97; H, 6.18; N, 1.46; Cl, 14.75. Found: C, 29.52;
H, 6.01; N, 1.51; Cl, 15.07%). Selected IR bands (KBr, cm1): 3383
(s, mOH); 2894 (w, mNH); 1151 (m, mCN); 1055 (m, mC–O); 540 (w,
mRuN); 458 (m, mRuO); 3134 (s, mRuCl). 1H NMR (d ppm, 300 MHz,
DMSO-d6) (see Section 3).
2.4. Cytotoxicity tests
2.4.1. Cell cultures
The A375 melanoma cell line is a highly invasive cell line from a
solid metastatic tumor (ATCC-CRL-1619) [35]. Cells were cultured
in RPMI supplemented with 10% FCS and 1% penicillin–streptomycin (10 000 U/ml and 10 000 lg/ml, respectively) in 5% CO2 at
37 °C. The cell viability was determined by the trypan-blue exclusion dye test, the most common stain used to distinguish viable
cells from non-viable cells. In brief, cells were seeded into 12-well
plates at 1.5 105 cells/well in 1.5 mL of growth medium. Cultures
were incubated for 24 h, after which the appropriate concentrations of the test substances were added. [RuCl2(DMSO)4] and the
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G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
ligands were used at 104, 105 and 106 M. Adherent and nonadherent cells were pooled after 24, 48 and 72 h treatment and a
small sample of each cell suspension was diluted 1:1 in trypan blue
and counted under normal light microscopy. The effects of the
treatment were quantified as the percentage of cell growth using
untreated cells as a control.
2.4.2. Invasivity cells test
About 3 106 cells were inoculated into a p/100 plate and left
to grow for 24 h. Afterwards, the cell layer was scraped and a
104 M solution of compound II was added to the plate. The number of cells that reached the scraped area were counted after 24, 48
and 72 h in the treated and untreated plates.
3. Results and discussion
Glucosaminic acid was deprotonated by treatment with 1 eq. of
NaOH in ethanolic solution to give its sodium salt a (Fig. 1), then
[RuCl2(DMSO)4] was added and the resulting solution was refluxed
for 4 days, after which time the color changed from dark-green to
brown. Complex I (Fig. 2) precipitated as a brown solid upon cooling and concentrating the solution.
Complex II (Fig. 2) was prepared by mixing 3 eq. of commercial 1thio-b-D-glucose sodium salt b with 1 eq. of [RuCl2(DMSO)4] and
refluxing the solution for 2 days. The color changed from orange to
brown and a solid precipitated from the concentrated cooled solution.
The ligand c was synthesized by the following procedure: b-lactose was transformed to the known O-protected derivative c0 by
the two steps-one pot procedure developed by Barili et al. [36].
Then this compound was converted under Mitsunobu conditions,
by phthalimide, diisopropyl azodicarboxylate (DIAD) and triphenylphosphine (TPP), to give the 60 -phtalimidoderivative, c00 , in a
55% yield (Scheme 1). It is worth noting that this transformation
is quite regioselective as the C-2 hydroxy function remains unaffected, likely due to the high steric hindrance at this position.
The compound c00 was in turn converted to the 60 -aminogalacto
derivative c by treatment with hydrazine in a reasonable yield of
67%. It must be emphasized that the isolation and work-up procedures for compound c (see Section 2) are crucial in order to obtain
a satisfactory reaction yield. Double sequential treatment with
aqueous acid and base to remove acid- and alkali-soluble impurities is necessary. The application of the standard protocol consisting of organic/aqueous phases partitioning could involve
significant loses of product and the lack of reproducibility of the final yield. This is probably caused by the amphipolar nature of the
aminoalcohol c.
The compound c, the precursor of the 60 -aminolactose, was reacted with [RuCl2(DMSO)4], in boiling ethanol. The complex III
(Fig. 2) was obtained as a brown precipitate.
Elemental analysis and ESI-MS confirmed the formation of complexes I–III with a definite stoichiometry. The analysis of the higher mass region of the mass spectra did not show any signal
attributable to the presence of oligomers in the sample. The metal/organic ligand ratios observed are: 1:2 for the complex I, 1:1
for the complex II and 2:1 for the complex III (i.e. a bimetallic species). A significant reduction in the carbon content of complex III
with respect to the free ligand c led us to suspect a deep structural
modification of the organic ligand before coordination (see below).
3.1. IR spectra
The infrared spectra of complexes I–III show the following
bands in the region attributable to ruthenium-ligand bonds (only
unambiguous assignments are reported): the spectra of complexes
I and III show the m(Ru–N) stretching at 520 and 540 cm1, respectively, while the m(Ru–O) stretching for compounds I and II appears
at 404 and 458 cm1, respectively [37–39]. The expected absorption for the Ru–S bond in the spectrum of complex II is found at
422 cm1. Finally, the Ru–Cl stretching vibrations of the three complexes give signals at around 300 cm1 [40,41].
3.2. 1H NMR
In the 1H NMR spectra of the complexes I and II, the signals of
the ligands do not show major chemical shift variations with respect to their free form, indicating the lack of any Ru(II) to Ru(III)
oxidation process. With regards to complex III, a chemical transformation of the free ligand c before coordination occurs, as will be
discussed in detail later.
Fig. 2. Proposed structures for the complexes I–III.
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G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
HO
OH
OH
(a)
HO
O
O
HO
HO
HO
O
O
O
OH
O
O
HO
O
O
HO
O
O
O
O
c'
(b)
NH2
O
O
O
O
O
(c)
O
HO
O
O
O
O
O
O
N
O
O
O
O
O
HO
O
O
O
O
O
c
c''
Scheme 1. Synthesis of the ligand c. Reagents and conditions: (a) DMP/PTSA (cat.), MeOH, 80 °C; (b) Phtalimide, TPP, DIAD, THF, 0 °C; (c) NH2NH2, EtOH, 25 °C.
For complex I, a sensible chemical shift variation in the signals
of the sugar moiety, concerning in particular protons H-2 and H-3,
seems to confirm a coordination mode (Fig. 2) in which the organic
ligand is bound via its N atom and the negatively charged carboxylate. The proton 1H NMR spectrum of complex II also shows signals corresponding to the sugar moiety with chemical shift
variations due to coordination. However, a fast exchange due to
the solvent employed (D2O/EtOH-d6) prevent the observation of
proton signals of the coordinated ethanol. On the other hand, the
presence of two molecules of ethanol is supported by the ESI-MS
and elemental analysis data.
The 1H NMR spectrum of complex III is worthy of further comment. It shows a group of overlapping signals in the oxygenated
proton region (3.30–3.90 ppm) and distinct signals assignable to
the CH2NH2 moiety (3.10 ppm) and the two peaks corresponding
to the acetylic protons of the sugar (4.32 and 4.72 ppm). It is worth
noting the disappearance of the isopropilidenic signals; both the
NMR data and the Mr obtained from the MS and the elemental
analysis show that the complete deprotection of the sugar occurs.
This fact can be explained on the ground of the well known Lewis
acid character of ruthenium and the nucleophilicity of the solvent
that synergistically promote the acetal opening process displayed
in Fig. 3. Hence the actual ligand in complex III is a molecule of
60 -aminolactose, formed in situ from compound c.
A group of four methyl signals (Fig. 4) from the coordinated ethanol molecules appears in the aliphatic region of the 1H NMR spectrum; six signals are not distinguishable because they are
overlapping, while six peaks in the 3.2–3.8 ppm interval of the
Fig. 4. Details of the 1H NMR and 1H–1H COSY spectra of complex III.
COSY spectrum, two of which are overlapped but still distinguishable, are very indicative of the presence of the six diastereotopic
coordinated alcohols in the structure proposed for complex III.
3.3. EXAFS investigation
Fig. 3. Acetal opening process.
The lack of X-ray crystallographic data due to the non-crystalline nature of the obtained solids does not allow the assignment
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G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
Fig. 5. Experimental (circle) and calculated (solid line) EXAFS functions and their corresponding Fourier transform plots for the different ruthenium(II) complexes measured
at the Ru K-edge.
Table 1
EXAFS determined structural parameters at the Ru K-edge.
Complex
A-Bsa
Nb
rc (Å)
rd (Å)
EFe
I
Ru–O/N
Ru–Cl
Ru–O–Oshort
Ru–O–Olong
4
2
4
21
2.16(2)
2.46(2)
3.26(3)
4.56(5)
0.003(1)
0.003(2)
0.003(1)
0.008(3)
4.68
III
Ru–O/Cl/N
Ru–Cl–Cl
6
12
2.39(3)
4.60(2)
0.014(3)
0.007(1)
4.01
a
Absorber (A) – backscatters (Bs).
Coordination number.
Interatomic distance.
d
Debye–Waller factor r with its calculated deviation.
e
Fermi energy.
b
c
of the exact tridimensional structure of the complexes, such as the
exact octahedral environment (i.e. mer or fac, cis or trans), and the
conformation of the sugar ligand. Attempts of chemical derivatization of the sugar ligands in form of acetates and benzyl ethers with
the purpose of obtaining crystalline compounds failed. This could
be the consequence of the Lewis acid character of the ruthenium
chloride that implies breakage of both the ether and the ester linkages, as one could have been anticipated considering the fate of the
precursor c. However, the local structure and the coordination
geometry of complexes I and III were determined by extended Xray absorption fine structure (EXAFS) spectroscopy.
EXAFS spectroscopy provides information on the coordination
number, the nature of the scattering atoms surrounding the
Fig. 6. Treated/control A375 cells growth with 104 M solutions of complexes I–III.
absorbing atom, the interatomic distances between the absorbing
atom and the backscattering atoms and the Debye–Waller factor,
which accounts for disorders due to static displacements and thermal vibrations [42,43]. In the fitting procedure, the coordination
numbers are held fixed for different backscatterers surrounding
the excited atom, and the other parameters, i.e. the interatomic
distances, the Debye–Waller factor and the Fermi energy value,
G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
1677
Fig. 7. A375 cells vitality test with a 104 M solution of complex II.
are varied by iteration. The experimental and calculated EXAFS
functions in k-space and their Fourier transforms for the ruthenium complexes measured at the Ru–K edge are shown in Fig. 5.
The corresponding structural parameters derived from the fit are
collected in Table 1. In the analysis of the complex [RuCl2(GlunN,O)2]Na2, I, the first shell consisting of nitrogen and oxygen backscatterers from the coordinating ligands are fitted with a coordination number of four at about 2.16 Å. Owing to the similar
backscattering behavior of the neighbors occurring at nearly the
same distance, a separate fit of N and O shells cannot be performed,
so a single shell model is used with nitrogen amplitude and phase
functions. The observed ruthenium–nitrogen and ruthenium–oxy-
gen distances are in agreement with those of analogue ruthenium
complexes reported in the literature [44,45]. A second shell is
determined at about 2.45 Å and consists of two chlorine atoms
[46]; additionally, two shells attributable to oxygen backscatterers
are found at 3.26 and 4.56 Å. As regards to the neutral complex
[Ru2(EtOH)6(AL)Cl4], III, a fit of the first shell at 2.39 Å is possible
only if a coordination number of six (N/O/Cl backscatterers) is considered. This problem could originate from the sterically congested
environment of the metal centers in complex III, which could involve variations of the interatomic distances such that distinct
coordination shells for N/O and Cl backscatterers cannot be determined. This would be mirrored in the high value of the Debye–
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G. Fontana et al. / Polyhedron 30 (2011) 1671–1679
Waller factor (0.014), witnessing a certain degree of disorder
around the ruthenium atoms. A second shell attributed to chlorine
backscatterers is determined at 4.60 Å. The EXAFS results are in
agreement with 6-fold coordination geometries around the ruthenium atoms in the compounds I and III.
3.4. Computational results
According to the results of the DFT calculations in the isolated
state, the geometries of the most stable isomers of compounds I–
III are reported in Fig. 2. These calculations focused only on the
coordination around the ruthenium atom, neglecting the occurrence of different conformations of the ligand molecules. The compound [RuCl2(Glun-N,O)2]2 (Ia) has an isomer which is clearly
preferred to others: in its coordination geometry the chlorine
atoms are trans with respect to the aminic nitrogen atoms and
cis to each other. Within 20 kJ/mol there are two other isomers:
the first, where two ligands coordinate symmetrically on the equatorial plane leaving two axial chlorine atoms, is 7 kJ/mol higher in
energy, while the second, which shows the chlorine atoms trans to
the oxygen atoms and cis to each other, has an energy value 17 kJ/
mol higher. The other two isomers have much higher energy. Calculated vibrational frequencies for the isomer Ia are: mRuCl at 212
and 245 cm1, mRuO at 364 cm1 and mRuN at 509 cm1. In the case
of the species [RuCl2(1-Tglu)(EtOH)2], the characterization of
structural isomers is not so clearly defined: three isomers of the
six structures investigated lie within 1.5 kJ/mol. The most stable
isomer (IIa in Fig. 2) has two EtOH molecules trans with respect
to the 1-Tglu ligand; in the second isomer (IIb, 1.1 kJ/mol higher
in energy) the above trans positions are occupied by chlorine,
and in the third isomer (IIc, 1.4 kJ/mol higher) Cl and EtOH are
trans to the S and O atoms of 1-Tglu, respectively. According to
the Boltzmann distribution, in gas phase the population of the
three isomers should be 45%, 30% and 25%, respectively. Globally,
the representative normal modes of these structures have the following frequency values: mRuCl in the range 240–280 cm1, and
mRuO in the range 453–511 cm1. Seven isomers of compound III
have been investigated with regards to the coordination of the ligands around the two ruthenium centers. Due to the conformational flexibility of the 60 -aminolactose ligand, small energy
differences between structural isomers could be not indicative of
a preferred coordination. On the other hand, the only isomers
which should have a significant population in the gas phase share
the same coordination sphere around the ruthenium atom bound
to the aminic moiety of 60 -aminolactose (RuI) and slightly differ
for the ligands arrangement around the second ruthenium atom,
the one coordinated to the –OH group of the disaccharide. In this
kind of isomers, where the EtOH ligands around the RuI atom are
in a mer arrangement and the chlorine atoms are trans to each
other, the mRuCl stretching frequencies are in the range 243–
315 cm1, mRuO in the range 341–374 cm1 and mRuN is at 530 cm1.
3.5. Biological activity
The cytotoxicity of complexes I–III was investigated on a A375
cell line target. The cell cultures were incubated with 104, 105
and 106 M solutions of complexes I–III and the cell growth was
determined with respect to the untreated cells (control). The results obtained with 104 M are reported in Fig. 6. The other solutions were inactive.
Of the three compounds tested, only complex II shows a 50%
growth inhibition activity after 72 h, with an extrapolated IC50 value of (5.08 ± 1.21) 104 M. The in vitro cytotoxicity of this compound against the A375 cell line is comparable to that reported by
Zorzet et al. [47] for NAMI-A against MCF-7, LoVo, KB and TS/A tumor cells in the same range of concentration. These authors also
showed a significant inhibition of the invasiveness of the TS/A adenocarcinoma cells in vitro, associated with an inhibitory effect –
both in the number and in the weight of the outbreaks – toward
the metastasis of the Lewis lung carcinoma in mice. The combination of these results was proposed as a pre-requisite for a ruthenium compound to possess a selective anti-metastasis effect
in vivo [47]. In light of this, we were encouraged to investigate
the residual mobility and proliferative capacity of A375 cells after
treatment with the active complex II. Hence an invasiveness test
was performed at 24, 48 and 72 h (Fig. 7). As reported in Fig. 7, cells
treated with complex II demonstrate a significant reduction in
their invasion capacity with respect to the untreated cells. Considering the mechanism of action of the metastasis-inhibiting ruthenium compounds [18,47], the partial inhibition of the cell
mobility can be considered as a valuable starting point for further
specific biological studies.
4. Conclusions
Two new mononuclear and one binuclear ruthenium(II) complexes were synthesized and structurally characterized by elemental analysis, IR, NMR, ESI-MS, EXAFS and DFT calculations. For
complex I, the data show the coordination of two molecules of glucosaminic acid sodium salt acting as a bidentate ligand, probably
through the aminic and the carboxylate group with an ester type
coordination. For complex II, a five-membered metallocycle has
been postulated in which a strong Ru–S bonds should stabilize
the structure. Complex III has a more complicated bond pattern
that should include two metallic centers with similar environments. Compound II exhibits promising biological properties
in vitro: despite a weak direct cytotoxicity, this complex shows
anti-invasive activity against A375 melanoma cell lines. The combination of these observed biological properties should encourage
further investigations into the in vivo antitumor activity of this
compound.
Acknowledgements
Financial supports of Università degli Studi di Palermo are
gratefully acknowledged (OIPA078W7F, OIPA0737W2). We thank
XAFS beamline scientist, Dr. Luca Olivi, for assistance during data
collection. Sincrotrone Elettra, Trieste is gratefully acknowledged
for financial support during the campaign of EXAFS measurements.
We wish to thank Dr. ssa M. A. Costa and Dr. ssa G. Barbieri of the
Istituto di Biomedicina e Immunologia Molecolare ‘‘Alberto Monroy’’, Consiglio Nazionale delle Ricerche (CNR), Palermo, Italy, for
their helpful suggestions and support.
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