From hydrolytically labile to hydrolytically stable Ru(II)-arene anticancer complexes with carbohydrate-derived co-ligands.

Abstract

The synthesis, characterization, reactivity and in vitro anticancer activity of a series of Ru(II)-arene complexes with carbohydrate-derived phosphite and biscarboxylato co-ligands are reported. The compounds were characterized by NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, and the molecular structures of oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside)ruthenium(II) and oxalato(η(6)-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofuranoside)ruthenium(II) were determined by X-ray diffraction analysis. In contrast to their dichlorido counterparts, the biscarboxylato complexes did not exhibit significant reactivity towards biomolecules, such as cysteine, methionine, ubiquitin or the DNA model 5'-GMP, and resist hydrolysis; no hydrolytic species were detected by (1)H and (31)P{(1)H} NMR spectroscopy over several days. These structural alterations led to a decrease in the tumor-inhibiting potency of the compounds in human cancer cell lines.

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From hydrolytically labile to hydrolytically stable RuII–arene anticancer complexes
with carbohydrate-derived co-ligands
Muhammad Hanifa, Samuel M. Meiera,c, Wolfgang Kandiollera, Anna Bytzeka, Michaela Hejla,
Christian G. Hartingera,c,⁎, Alexey A. Nazarova,b,⁎, Vladimir B. Ariona,c, Michael A. Jakupeca,c,
Paul J. Dysonb, Bernhard K. Kepplera,c
aUniversity of Vienna, Institute of Inorganic Chemistry, Waehringer Str. 42, A-1090 Vienna, Austria
bInstitut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
cUniversity of Vienna, Research Platform “Translational Cancer Therapy Research”, Waehringer Str. 42, A-1090 Vienna, Austria
a b s t r a c t a r t i c l ei n f o
Article history:
Received 13 August 2010
Received in revised form 30 September 2010
Accepted 6 October 2010
Available online 14 October 2010
Keywords:
Anticancer activity
Aquation
Bioorganometallic chemistry
Carbohydrate ligands
Ruthenium
The synthesis, characterization, reactivity and in vitro anticancer activity of a series of RuII–arene complexes
with carbohydrate-derived phosphite and biscarboxylato co-ligands are reported. The compounds were
characterized by NMR spectroscopy and electrospray ionization (ESI) mass spectrometry, and the molecular
structures of oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranoside)ru-
thenium(II) and oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofurano-
side)ruthenium(II) were determined by X-ray diffraction analysis. In contrast to their dichlorido
counterparts, the biscarboxylato complexes did not exhibit significant reactivity towards biomolecules,
such as cysteine, methionine, ubiquitin or the DNA model 5′-GMP, and resist hydrolysis; no hydrolytic species
were detected by1H and31P{1H} NMR spectroscopy over several days. These structural alterations led to a
decrease in the tumor-inhibiting potency of the compounds in human cancer cell lines.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
Organometallic medicinal chemistry is an emerging research area
with considerable potential [1]. In recent years many examples of
organometallic compounds appeared that exhibit potent anticancer
activity [1–4], including compounds that selectively inhibit enzymes
[5–9], block receptors [2], or have been used to label peptides or other
biomolecules [10,11]. Indeed, titanocene dichloride, Cp2TiCl2, entered
clinical trials as an anticancer agent [12,13], and other compounds are
expected to follow.
One of the most investigated class of organometallic compounds
are based on the RuII–arene unit linked to ethylenediamine (en)
[14,15], 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane (pta) [16], pyr-
(id)ones [17–19] or bioactive groups [5,6,8,9,20]. For example, the so-
called RAPTA compounds are a large family of organometallic
compounds with general formula [RuII(η6-arene)(pta)Cl2] (for
RAPTA-C, see Fig. 1), which have been extensively investigated for
their tumor-inhibiting properties. In vivo studies on some RAPTA
compounds have shown excellent inhibition of metastases growth in
CBA mice bearing the MCa mammary carcinoma while exhibiting
selective, but mild cytotoxicity towards the TS/A tumorigenic cell line
as opposed to the HBL-100 non-tumorigenic cell line in in vitro
anticancer assays [16]. RAPTA complexes undergo aquation of the
chloride ligand(s) in aqueous solution [21]. The extent of the aquation
depends on the concentration of the complex in solution, the amount
of chloridepresent andthe pH. Hydrolysiswas foundto be suppressed
at the high chloride concentration as in blood plasma (ca. 100 mM),
but occurs at chloride concentrations typical of cytoplasm (ca. 4 mM),
representing a possible activation pathway.
In order to improve efficacy, a number of structural modifications
havebeen madetothebasicRAPTAframeworksuchas introduction of
H-bonding functionalities [22], change of the arene ligand [16] or the
leaving group (Fig. 1) [23] and tethering to bioactive segments such as
human serum albumin which resulted in a twentyfold increase in
cytotoxicity with respect to prototype RAPTA-C [24].
By replacing the pta moiety with sugar-phosphite ligands with
similar hydrophilicity [25,26], RAPTA-like analogues were obtained,
potentially capable of targeting cancer cells by exploiting the in-
creased glucose uptake resulting from overexpression of glucose
transporters and glycolytic enzymes [25,27]. This concept led to the
development of compounds with the general formula [RuII(η6-p-
cymene)(3,5,6-bicyclophosphite-α-D-glucofuranoside)Cl2]. The
complexes showed moderate cytotoxicity, with certain selectivity
for tumor cells over non-tumorigenic cells, and importantly activity in
Journal of Inorganic Biochemistry 105 (2011) 224–231
⁎ Corresponding authors. University of Vienna, Institute of Inorganic Chemistry,
Waehringer Str. 42, A-1090 Vienna, Austria. Tel.: +43 1 4277 52609; fax: +43 1 4277
52680.
E-mail addresses: christian.hartinger@univie.ac.at (C.G. Hartinger),
alex.nazarov@univie.ac.at (A.A. Nazarov).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.10.004
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio

Page 2

cisplatin-resistant cells [26]. The cytotoxicity appears to be deter-
mined, to some extent, by lipophilicity, i.e., the most lipophilic com-
poundwasthemostcytotoxic.Thesecompoundsarepronetoaquation/
hydrolysis similar to RAPTA compounds in that they undergo aquation
of the first halido ligand in aqueous solution, but additionally the
hydrolysis of a P–O bond of the phosphite ligand, and ultimately the
formation of inert dinuclear species takes place [26,28].
Use of anionic chelating ligands such as oxalate instead of halido
ligands has already been employed to overcome the problems of
solubility and stability associated with platinum compounds and
titanocenes [1,3,29]. The replacement of the two chlorido ligands in
RAPTA-C by oxalate resulted in a complex which resists aquation [23].
In this paper we describe the influence of the leaving group on
hydrolysis, binding to biomolecules, and cytotoxicity for a series of
carbohydrate–ruthenium(II)-arene complexes, with oxalato and
malonato leaving groups, and compare it to data obtained for
structurally similar Os compounds [30].
2. Experimental
All reactions were carried out in dry solvents under argon
atmosphere. Chemicals were obtained from commercial suppliers
and used as received. Ubiquitin from bovine red blood cells was
purchased from Sigma, L-cysteine from Fluka and L-methionine from
Sigma-Aldrich and used as obtained. MeOH (VWR Int., 20864.320,
HiPerSolv CHROMANORM), formic acid (Fluka) and milliQ H2O were
used as solvents for MS studies. For the microemulsion electrokinetic
chromatography studies sodium dodecyl sulfate (SDS), dodecano-
phenone, 1-butanol and sodium monohydrogenphosphate were
purchased from Sigma-Aldrich. Sodium hydroxide solution (0.1 M),
hydrochloric acid, heptane and sodium dihydrogenphosphate were
obtained from Fluka, and DMSO from Fisher Scientific. Methanol and
CH2Cl2 used in synthesis were dried using standard procedures.
Dichlorido(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropyli-
dene-α-D-glucofuranoside)ruthenium(II) 1 [26], the dimer bis-
[dichlorido(η6-p-cymene)ruthenium(II)] {[RuII(cym)Cl(μ-Cl)]2 a
[31]}, 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofura-
noside I [32], 3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-
glucofuranoside II [32], 1,2-O-(2,2,2-trichloroethylidene)-α-D-
glucofuranose 3,5,6-bicyclophosphite III [33], disilver oxalate and
malonate [23] and oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-
O-isopropylidene-α-D-glucofuranoside)osmium(II) 2Osand oxalato
(η6-p-cymene)-(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-
D-glucofuranoside)osmium(II) 3Os[23] were synthesized using
literature procedures.1H,13C{1H} and31P{1H} NMR spectra were
recorded at 25 °C on a Bruker FT NMR spectrometer Avance III
500 MHz at 500.10 (1H), 125.75 (13C{1H}) and 202.44 MHz (31P
{1H}). The31P NMR spectra were referenced to 85% phosphoric acid.
2D NMR spectra were collected in a gradient-enhanced mode.
Melting points were measured on a Büchi B-540 apparatus and are
uncorrected. Elemental analysis was determined by the Laboratory
for Elemental Analysis, Faculty of Chemistry, University of Vienna,
on a Perkin-Elmer 2400 CHN Elemental Analyzer. Electrospray
ionization mass spectra were recorded on a Bruker esquire3000.
2.1. General procedure for the synthesis of 2–6
The dimer [(η6-cymene)RuCl(μ-Cl)]2 (122 mg, 0.20 mmol) and
silver dicarboxylate (0.44 mmol) were stirred in water (25 mL) for
12 h. The mixture was filtered through a bed of Celite to remove the
AgCl precipitate. The solvent was removed under vacuum, and the
residue was redissolved in methanol (25 mL) and the carbohydrate
ligand (0.40 mmol) was added. The reaction mixture was stirred for
2 h, then the volume of the solution was reduced to 5 mL and diethyl
ether (25 mL) was added. The slurry was cooled to 4 °C for 12 h to
afford a yellow crystalline product that was isolated by filtration,
washed with diethyl ether (2×5 mL) and dried under vacuum.
Oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopropyli-
dene-α-D-glucofuranoside)ruthenium(II) 2 was prepared following
the general procedure, using silver oxalate (133 mg, 0.44 mmol) and
3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-glucofuranose
(100 mg,0.40 mmol).CrystalssuitableforX-raydiffractionstudieswere
grownbyslowdiffusionfromdiethyletherandmethanol.Yield:190 mg
(83%), m.p.175–177 °C (decomp.), Elementalanalysis,found % C, 43.37;
H, 4.91; calcd. for C21H27O10PRu·0.5H2O, C, 43.45; H, 4.86. MS (ESI+):
m/z [Found (Calcd)]: 572.7 (573.0) [M+H]+, 595.1 (595.0) [M+Na]+;
1H NMR (500.10 MHz, CDCl3, 25 °C, d = doublet, m = multiplet, s =
singlet, brs = broad singlet): δ=6.08 (d, J=4 Hz, 1H; H-1), 5.88 (d,
J=6 Hz, 1H; H–Ar), 5.86 (d, J=6 Hz, 1H; H–Ar), 5.74 (d, J=5 Hz, 1H;
H–Ar), 5.71 (d, J=5 Hz, 1H; H–Ar), 5.15–5.18 (m, 1H; H-5), 4.71 (brs,
1H; H-3), 4.60 (d, J=4 Hz, 1H; H-2), 4.56 (brs, 1H; H-6), 4.34 (brs, 2H;
H-4, H-6′), 2.77–2.82 (m, 1H; CH(CH3)2), 2.22 (s, 3H; CH3), 1.46 (s, 3H;
C(CH3)2), 1.30 (s, 3H; C(CH3)2), 1.29 (d, J=7 Hz, 3H; CH(CH3)2), 1.28
(d, J=7 Hz, 3H; CH(CH3)2)ppm;
25 °C): δ=165.2 (J=4 Hz; C=O), 112.6 (C(CH3)2), 109.3 (C–Ar), 105.7
(C-1), 105.0 (C–Ar), 89.8 (CH–Ar), 89.6 (CH–Ar), 89.0 (CH–Ar), 88.2
(CH–Ar), 83.6 (J=5 Hz; C-2), 78.6 (J=8 Hz; C-3), 76.9 (J=6 Hz; C-4),
74.7 (J=5 Hz; C-5), 69.3 (J=8 Hz; C-6), 31.2 (CH(CH3)2), 26.9 (C
(CH3)2), 26.2 (C(CH3)2), 22.5 (CH(CH3)2), 22.4 (CH(CH3)2), 18.3 (CH3)
ppm;31P{1H} NMR (202.44 MHz, CDCl3, 25 °C): δ=134.1 ppm.
Oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclohex-
ylidene-α-D-glucofuranoside)ruthenium(II) 3 waspreparedfollow-
ingthegeneralprocedure,usingsilveroxalate(133 mg,0.44 mmol)and
3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-glucofuranose
(115 mg,0.40 mmol).CrystalssuitableforX-raydiffractionstudieswere
grownbyslowdiffusionfromdiethyletherandmethanol.Yield:191 mg,
(78%), m.p.181–183 °C (decomp.), Elementalanalysis,found % C, 46.24;
H, 4.87; calcd. for C24H31O10PRu·0.5H2O, C, 46.45; H, 5.19. MS (ESI+):
m/z [Found (Calcd)]: 612.8 (613.1) [M+H]+, 635.3 (635.1) [M+Na]+;
1H NMR (500.10 MHz, CDCl3, 25 °C): δ=6.08 (d, J=3 Hz, 1H, H-1),
5.87 (d, J=6 Hz, 1H; H–Ar), 5.85 (d, J=6 Hz, 1H; H–Ar), 5.72 (d,
J=5 Hz, 1H; H–Ar), 5.70 (d, J=5 Hz, 1H; H–Ar), 5.14–5.17 (m, 1H; H–
5), 4.70 (brs 1H, H-3), 4.61 (d, J=3 Hz, 1H; H-2), 4.51–4.53 (m, 1H; H-
6), 4.32 (brs, 2H; H-4, H-6′), 2.78–2.83 (m, 1H; CH(CH3)2), 2.22 (s, 3H;
CH3), 1.59–1.65 (m; 4H, C6H10), 1.51–1.55 (m, 6H; C6H10), 1.29 (d,
J=7 Hz, 3H; CH(CH3)2), 1.28 (d, J=7 Hz, 3H; CH(CH3)2)ppm;13C{1H}
NMR (125.75 MHz, CDCl3, 25 °C): δ=165.1 (J=4 Hz; C=O), 113.3 (C
(CH3)2),109.3(C–Ar),105.3(C-1),105.0(C–Ar),89.7(C–Ar),89.5 (C–Ar),
88.9 (C–Ar), 88.2 (C–Ar), 83.1 (J=6 Hz; C-2), 78.7 (J=8 Hz; C-3), 76.8
(J=8 Hz;C-4),74.7(J=6 Hz;C-5),69.2(J=6 Hz;C-6),36.5(C6H10),35.6
13C{1H} NMR (125.75 MHz, CDCl3,
Fig. 1. Structures of anticancer RAPTA complexes and an analogous carbohydrate-derived compound.
225
M. Hanif et al. / Journal of Inorganic Biochemistry 105 (2011) 224–231

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(C6H10), 31.1
22.5 (CH(CH3)2–Ar), 22.4 (CH(CH3)2–Ar), 18.3 (CH3–Ar)ppm;31P{1H}
NMR (202.44 MHz, CDCl3, 25 °C): δ=134.1 ppm.
Oxalato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-(2,2,2-
trichloroethylidene-α-D-glucofuranoside)ruthenium(II) 4 was
prepared following the general procedure, using silver oxalate
(133 mg, 0.44 mmol) and 3,5,6-bicyclophosphite-1,2-O-(2,2,2-
trichloroethylidene)-α-D-glucofuranose (135 mg, 0.40 mmol).
Yield: 177 mg (67%), m.p. 251–252 °C (decomp.), Elemental analysis,
found% C, 35.34; H, 3.26; calcd. for C20H22O10Cl3PRu·0.75H2O, C, 35.62;
H, 3.51. MS (ESI+): m/z [Found (Calcd)]: 662.6 (662.9) [M+H]+;1H
NMR (500.10 MHz, DMSO-d6, 25 °C): δ=6.29 (d, J=4 Hz, 1H; H-1),
6.14 (d, J=5 Hz, 1H; H–Ar), 6.12 (d, J=5 Hz, 1H; H–Ar), 5.92 (d,
J=6 Hz, 1H; H–Ar), 5.90 (d, J=6 Hz, 1H; H–Ar), 5.57 (s, 1H; CHCCl3),
5.20–5.24(m,1H;H-5),5.04(d,J=3 Hz,1H;H-3),4.84(d,J=3 Hz,1H;
H-2), 4.77–4.81 (m, 1H; H-6), 4.71 (brs, 1H; H-4), 4.06–4.10 (m, 1H; H-
6′),2.63–2.68(m,1H;CH(CH3)2), 2.07 (s, 3H; Ar–CH3), 1.20 (d, J=7 Hz,
6H; CH(CH3)2)ppm;
δ=164.6 (J=6 Hz; C=O), 108.6 (C–Ar), 106.9 (C-1), 106.5 (CH–Ar),
104.8 (C–Ar), 97.4 (CCl3), 90.8 (CH–Ar), 89.0 (CH–Ar), 88.6 (CH–Ar),
86.0 (J=6 Hz; C-2), 78.6 (J=5 Hz; C-3), 77.9 (J=8 Hz; C-4), 74.4
(J=5 Hz; C-5), 69.1 (J=9 Hz; C-6), 31.1 (CH(CH3)2), 24.5 (CHCCl3),
22.5 (CH(CH3)2), 22.4 (CH(CH3)2), 18.1 (CH3) ppm;
(202.44 MHz, CDCl3, 25 °C): δ=135.0 ppm.
Malonato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-isopro-
pylidene-α-D-glucofuranoside)ruthenium(II) 5 was prepared fol-
lowing the general procedure, using silver malonate (140 mg,
0.44 mmol) and 3,5,6-bicyclophosphite-1,2-O-isopropylidene-α-D-
glucofuranose (100 mg, 0.40 mmol). Yield: 168 mg (72%), m.p.N200 °C
(decomp.), Elemental analysis, found% C, 43.62; H, 5.05; calcd. for C22
H29O10PRu·H2O, C, 43.78; H, 5.18. MS (ESI+): m/z [Found (Calcd)]:
608.9 (609.0) [M+Na]+;
δ=6.09 (d, J=4 Hz, 1H; H-1), 5.81 (d, J=7 Hz, 1H; H–Ar), 5.80 (d,
J=7 Hz, 1H; H–Ar), 5.68 (d, J=6 Hz, 2H; H–Ar), 5.64 (d, J=6 Hz,
2H; H–Ar), 5.17–5.20 (m, 1H; H-5), 4.73 (brs, 1H; H-3), 4.66 (d,
J=3.5 Hz, 1H; H-2), 4.52–4.56 (m, 1H; H-6), 4.33–4.34 (m, 2H; H-4
and H-6′), 3.41 (d, J=17 Hz, 1H; CH2COO), 3.20 (d, J=17 Hz, 1H,
CH2COO), 2.80–2.86 (m, 1H; CH(CH3)2), 2.19 (s, 3H; CH3), 1.49 (s,
3H; C(CH3)2), 1.32 (s, 3H; C(CH3)2), 1.26 (d, J=7 Hz, 3H; CH
(CH3)2)ppm;13C{1H} NMR (125.75 MHz, CDCl3, 25 °C): δ=175.4
(J=10 Hz; C=O), 112.7 (C(CH3)2), 109.6 (C–Ar), 106.7 (C–Ar),
105.7 (C-1), 90.0 (C–Ar), 89.7 (C–Ar), 89.4 (C–Ar), 88.9 (C–Ar), 83.4
(J=5 Hz; C-2), 78.7 (J=8 Hz; C-3), 76.7 (J=10 Hz; C-4), 74.7
(J=5 Hz; C-5), 69.0 (J=9 Hz; C-6), 46.8 (CH2COO), 30.7 (CH
(CH3)2), 26.8 (C(CH3)2), 26.1 (C(CH3)2), 22.2 (CH(CH3)2), 18.1
(CH3) ppm;
δ=137.4 ppm.
Malonato(η6-p-cymene)(3,5,6-bicyclophosphite-1,2-O-cyclo-
hexylidene-α-D-glucofuranoside)ruthenium(II) 6 was prepared
following the general procedure, using silver malonate (140 mg,
0.44 mmol) and 3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-D-
glucofuranose (115 mg, 0.40 mmol). Yield: 185 mg (74%), m.p.N
200 °C (decomp.), Elemental analysis, found% C, 46.39; H, 5.75;
calcd. for C25H33O10PRu·H2O, C, 46.65; H, 5.48. MS (ESI+): m/z [Found
(Calcd)]: 648.8 (649.1) [M+Na]+;
25 °C): δ=6.09 (d, J=4 Hz, 1H; H-1), 5.80 (d, J=7 Hz, 1H; H–Ar),
5.79 (d, J=7 Hz, 1H; H–Ar), 5.67 (d, J=6 Hz, 2H; H–Ar), 5.62 (d,
J=6 Hz, 2H; H–Ar), 5.16–5.19 (m, 1H; H-5), 4.74 (brs, 1H; H-3), 4.65
(d, J=4 Hz, 1H; H-2), 4.50–4.54 (m, 1H; H-6), 4.33 (brs, 2H; H-4 and
H-6′), 3.40 (d, J=17 Hz, 1H; CH2COO), 3.20 (d, J=17 Hz, 1H;
CH2COO), 2.81–2.86 (m, 1H; CH(CH3)2), 2.20 (s, 3H; CH3), 1.64–1.67
(m, 4H; C6H10), 1.53–1.55 (m, 6H, C6H10), 1.26 (d, J=7 Hz, 3H; CH
(CH3)2), 1.25 (d, J=7 Hz, 3H; CH(CH3)2)ppm;
(125.75 MHz, CDCl3, 25 °C): δ=175.4 (J=9 Hz; C=O), 113.4
(C(CH3)2), 109.6 (C–Ar), 106.7 (C–Ar), 105.3 (C-1), 90.1 (C–Ar),
89.7 (C–Ar), 89.4 (C–Ar), 88.8 (C–Ar), 83.0 (J=5 Hz; C-2), 78.8
(CH(CH3)2), 24.7 (C6H10), 23.8 (C6H10), 23.5 (C6H10),
13C{1H} NMR (125.75 MHz, CDCl3, 25 °C):
31P{1H} NMR
1H NMR (500.10 MHz, CDCl3, 25 °C):
31P{1H} NMR (202.44 MHz, CDCl3, 25 °C):
1H NMR (500.10 MHz, CDCl3,
13C{1H} NMR
(J=8 Hz;
(J=9 Hz; C-6), 46.8 (CH2COO), 36.4 (C6H10), 35.6 (C6H10), 30.7 (CH
(CH3)2), 24.7 (C6H10), 23.8 (C6H10), 23.4 (C6H10), 22.2 (CH(CH3)2–Ar),
22.4 (CH(CH3)2–Ar), 18.1 (CH3–Ar) ppm;31P{1H} NMR (202.44 MHz,
CDCl3, 25 °C): δ=137.5 ppm.
C-3), 76.7 (J=5 Hz; C-4), 74.7 (J=5 Hz; C-5), 68.9
2.2. X-ray diffraction analysis
X-ray diffraction measurements of 2 and 3 were performed on a
Bruker X8 APEXII CCD diffractometer at 100 K. The crystals were
positioned at 40 and 35 mm from the detector and 1386 and 1345
frames, each for 10 and 80 s over 1°, were measured. The data were
processed using the SAINT software package [34]. Crystal data, data
collection parameters, and structure refinement details are given in
Table 1. The structures were solved by direct methods and refined by
full-matrix least-squares techniques. Non-hydrogen atoms were
refined with anisotropic displacement parameters. H atoms were
inserted at calculated positions and refined with a riding model. One
of the four co-crystallized methanol molecules was refined with 75%
occupancy. The following computer programs were used: structure
solution SHELXS-97, refinement SHELXL-97 [35], molecular diagrams
ORTEP-3[36]. Crystallographic data for the structural analysis of 2 and
3 has been deposited with the Cambridge Crystallographic Data
Centre, CCDC 785120 and 785121, respectively. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
2.3. Reactions with biomolecules
2.3.1. NMR spectroscopy
Solutions of 1 or 2 containing either Cys, Met (both 1:1) or 5'-GMP
(1:2) were prepared in D2O (pH 4–5) and kept at room temperature
for 5 days. The reaction progress was monitored by1H and31P{1H}
NMR spectroscopy at 0.5, 48 and 120 h. ESI-MS.
Table 1
Crystal data and details of data collection for 2 and 3.
Compound
23
Chemical formulaC22H31O11PRuC25.88H38.50O11.88PRu
M (g mol−1)
Temperature (K)
Crystal size (mm)
Crystal color, habit
Crystal system
Space group
a (Å)
b (Å)
c (Å)
V (Å3)
Z
Dc(g cm−3)
μ (cm−1)
F(000)
Θ range for data collection (°)
h range
k range
l range
No. refls. used in refinement
No. parameters
Rint
R1(obs.)a
wR2(all data)b
Flack parameter
Sc
603.51
100(2)
0.20×0.10×0.03
yellow, plate
orthorhombic
P212121
8.2719(7)
12.9548(14)
22.785(2)
2441.7(4)
4
1.642
7.67
1240
2.92 to 26.00
−10/10
−15/15
−28/28
4781
319
0.1242
0.0503
0.1266
0.01(5)
1.053
671.61
100(2)
0.25×0.08×0.04
orange, stick
monoclinic
P21
15.1978(9)
13.0153(7)
15.7673(9)
2964.0(3)
4
1.505
6.42
1391
2.72 to 26.00
−18/18
−16/16
−19/19
11628
731
0.0775
0.0374
0.0837
−0.05(2)
1.006
aRefinement was by full-matrix least-squares (Fo2) for all reflections, R1=Σ||Fo|–
|Fc||/Σ|Fo|.
bwR2={Σ[w(Fo2–Fc2)2]/Σ[w(Fo2)2]}1/2.
cGoodness of fit, S={Σ[w(Fo2–Fc2)2]/(n–p)}1/2.
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M. Hanif et al. / Journal of Inorganic Biochemistry 105 (2011) 224–231

Page 4

2.3.2. ESI-MS
The complexes were incubated at 37 °C with Cys and Met at a
molar ratio of 1:1 and with ubiquitin at 2:1 molar ratio in milliQ
water (18.2 MΩ; Millipore Synergy 185 UV Ultrapure Water system;
Molsheim, France). The samples containing amino acids were diluted
with MeOH, and the protein incubation mixtures were diluted with
H2O/MeOH/formic acid (50:50:0.1). Mass spectra were recorded after
3, 6, 24, 72 h and 7 days at concentrations of 1–10 μM, using a Bruker
esquire3000 ion trap mass spectrometer with direct infusion at a flow
rate of 5 μl/min. The experimental conditions were as follows:
capillary 4.5 kV, end plate offset 0.5 kV, skim1 45.5 V, skim2 6 V,
cap exit offset 78.4 V and dry temperature 200 °C. The spectra were
recorded using ESI Compass 1.3 (Bruker) and DataAnalysis 4.0
software. Deconvolution was obtained by the maximum entropy
algorithm with a 0.1 m/z-mass step and an instrument peak width
of 1.
2.4. Aquation studies
For hydrolysis studies, the compounds were dissolved in D2O and
the samples were analyzed by31P{1H} NMR spectroscopy once a day
over 15 days. The31P{1H} NMR spectra were recorded on a Bruker FT
NMR spectrometer Avance III 500 MHz at 202.44 MHz.
2.5. Cytotoxicity studies
2.5.1. Cell lines and culture conditions
CH1 (ovarian carcinoma, human) cells were a gift from Lloyd R.
Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer
Research, Sutton, UK. SW480 (adenocarcinoma of the colon, human)
and A549 (non-small cell lung cancer, human) cells were provided by
Brigitte Marian (Institute of Cancer Research, Department of Medicine
I, Medical University of Vienna, Austria). All cell culture reagents were
purchased from Sigma-Aldrich. Cells were grown in 75 cm² culture
flasks (Iwaki) as adherent monolayer cultures in Eagle's Minimal
Essential Medium (MEM) supplemented with 10% heat-inactivated
fetal calf serum, 1 mM sodium pyruvate and 2 mM L-glutamine at
37 °C under a humidified atmosphere containing 95% air and 5% CO2.
2.5.2. MTT assay conditions
Cytotoxicity was determined by the colorimetric MTT (3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Fluka)
microculture assay. For this purpose, cells were harvested from
culture flasks by trypsinization and seeded in 100 μL aliquots of MEM
supplemented with 10% heat-inactivated fetal calf serum, 1 mM
sodium pyruvate, 4 mM L-glutamine and 1% non-essential amino
acids (100×) into 96-well microculture plates (Iwaki). The following
cell densities were chosen to ensure exponential growth of untreated
controls throughout the experiment: 1.5×103cells/well (CH1),
2.5×103cells/well (SW480) and 4×103cells/well (A549). Cells
were allowed to settle and resume exponential growth for 24 h. The
test compounds were dissolved and serially diluted in the same
medium and added in aliquots of 100 μL/well. After exposure for 96 h,
all media were replaced by 100 μL/well RPMI1640 medium (supple-
mented with 10% heat-inactivated fetal calf serum) plus 20 μL/well
MTT in phosphate-buffered saline (5 mg/mL). After incubation for 4 h,
the supernatants were removed, and the formazan crystals formed by
viable cells were dissolved in 150 μL DMSO per well. Optical densities
at 550 nm were measured with a microplate reader (Tecan Spectra
Classic), using a reference wavelength of 690 nm to correct for
unspecific absorption. The quantity of viable cells was expressed as
percentages of untreated controls, and 50% inhibitory concentrations
(IC50) were calculated from concentration–effect curves by interpo-
lation. The results given are mean values from at least three or two
independent experiments in case of activity or inactivity, respectively,
each comprising three replicates per concentration level.
2.6. Microemulsion electrokinetic chromatography (MEEKC) studies
CE separations were carried out on an HP3DCE system (Agilent,
Waldbronn, Germany) equipped with an on-column diode-array
detector and the analytes were detected by UV absorption at 200 nm.
For all measurements, capillaries of 48.5 cm total length (40 cm
effective length; 50 μm ID) were used (Polymicro Technologies,
Phoenix, AZ, USA). Capillary and sample tray were thermostatted at
25 °C. The sample injection was carried out by hydrodynamic
injection at 10 mbar for 15 s with a separation voltage of 25 kV.
New capillaries were conditioned with 0.1 M HCl, water, 0.1 M NaOH,
and again with water (10 min each). The capillary was flushed with
0.1 M NaOH, water, and BGE for 5 min each before the first run each
run. Before each injection the capillary was purged with 0.1 M NaOH,
water and the BGE for 2 min each.
The following component ratios (wt.%) were used to establish a
stable microemulsion: 91.22% sodium phosphate buffer (20 mM, pH
7.4), 0.84% heptane, 1.47% SDS, and 6.48% 1-butanol. For the
preparation of the MEEKC solutions, surfactant, alcohol, oil, and a
small portion of buffer were mixed by sonication for 5 min. The
residual buffer was then slowly added to the mixture. Afterwards, the
solution was kept for 30 min at room temperature before use. The
analytes were dissolved in the microemulsion to give concentrations
between0.45 and 0.6 mg/ml.DMSO anddodecanophenonewereused
as markers for the electroosmotic flow (EOF) and the microemulsion
droplets, respectively: to 1 ml of the sample solution 1 μl DMSO and
20 μl dodecanophenone (18 mg/ml in methanol) were added.
The capacity factor, k, is defined as the ratio nme/naq(nme=total
number of moles of solute in the microemulsion phase, naq=total
number of moles of solute in the aqueous phase) and this mass
distribution coefficient can be calculated according to Eq. (1)
[to=migration time of an unretained substance (EOF marker),
tme=migration time of the microemulsion, tR=solute migration
time] [37,38].
k =
tR−t0
t01−tR?
tme
??
ð1Þ
3. Results and discussion
In previous work, we prepared RAPTA-C analogues by linking
various carbohydrate-derived ligands to a RuII–arene scaffold, and
some examples such as dichlorido(η6-p-cymene)(3,5,6-bicyclopho-
sphite-1,2-O-isopropylidene-α-D-glucofuranoside)ruthenium(II) 1
were more active than RAPTA-C in in vitro assays. The complexes
were found to exhibit aquation of the first halido ligand in aqueous
solution,followed by hydrolysis of a P–O bond of thephosphiteligand,
and finally formation of dinuclear species [26]. In order to study the
influence of chelating ligands on the hydrolysis, reactivity towards
biomolecules and cytotoxicity of the complexes, we synthesized
carbohydrate containing RuII–arene dicarboxylato compounds. This
concept proved successful in case of RAPTA complexes [23], which
undergo rapid hydrolysis in aqueous media, although they can be
stabilized by high concentrations of NaCl [21]. These dicarboxylate
species were found to be highly soluble and kinetically more stable
than their RAPTA precursors with the two chlorido ligands and
notably,theanticanceractivitywasmaintained, aswastheirreactivity
with biomolecules [23].
3.1. Synthesis and characterization
The preparation of the RuII–arene biscarboxylato complexes, 2–6,
is summarized in Scheme 1, and follows a literature procedure [23]. In
the first step, [biscarboxylato(η6-p-cymene)(H2O)ruthenium(II)] was
formed by reacting bis[dichlorido(η6-p-cymene)ruthenium(II)] with
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Page 5

disilver oxalate or malonate (obtained by reacting 2.2 eq. of AgNO3
with disodium oxalate or malonate) in water. In the second step the
aqua species was stirred for 2 h in CH3OH with the respective
carbohydrate-basedphosphorus ligand.This reactionsequenceavoids
side reactions involving the silver ions and the phosphorus ligand.
The complexes were characterized by 1D and 2D NMR spectro-
scopy, ESI-MS, and elemental analysis (see Experimental for full
details).31P NMR spectroscopy is very informative, as coordination of
the P-containing sugar ligands to the metal center results in a
significant change of the chemical shift from 117 ppm to 134–
137 ppm. These values are in the same range as those observed for the
analogous Ru compounds with chlorido ligands [26]. In the1H NMR
spectra of malonato Ru–arene complexes, the methylene protons of
the malonic acid become diastereotopic upon complexation and give
two doublets centered at about δ=3.4 and 3.2 ppm with a geminal
coupling constant2J(H,H)=17 Hz, as expected for an AB system. The
13C{1H} NMR spectra of 2–6 contain prominent signals at ca. 165 and
175 ppm that correspond to coordinated carboxylates of oxalato and
malonato ligands, respectively.
Single crystals of 2 and 3 suitable for X-ray diffraction studies were
grownby slow diffusion of diethyl ether into a solution of the complex
in methanol. Their molecular structures are shown in Fig. 2, and as
expected, confirm the piano-stool geometry typical of such systems
[5,18,23,39]. The ruthenium–centroidarenedistances are 1.703(3) and
1.704(2) Å in 2 and 3, which are similar to that observed in the
dichlorido analogue (1.711 Å) [26]. Both 2 and 3 contain methanol
which forms H-bonds with a carbonyl oxygen of the oxalato ligand.
The key bond lengths, such as Ru–P and Ru–O, are similar to those of
oxaloRAPTA-C (see Table 2) [23].
3.2. Aquatic stability
In order to study the influence of chelating biscarboxylate ligands
on the behavior of carbohydrate-based RuII–arene complexes in
aqueous solution, 2 and 5 were dissolved in D2O and the reaction was
monitored by
expected, the exchange of the chlorido ligands by biscarboxylato
resulted in enhanced stability of the compounds. Both the oxalato and
malonato derivatives are stable towards aquation over more than two
weeks and no aquation products were identified. In the case of the
dichloridoRuII–areneanaloguesmono aqua speciesare formed within
a few hours followed by cleavage of the P–O(C5) bond of the sugar-
derived phosphorus ligand, terminating in the formation of dinuclear
species [26]. The same trends were basically confirmed by ESI-MS, but
in addition, a peak at m/z 564.88±0.02 that may be assigned to
[(cym)2Ru2(μ-OMe)3]+(19%, exact mass 565.08) was observed. This
species presumably results from the exchange of the hydroxido
ligands in [(cym)2Ru2(μ-OH)3]+for the methanol added to assist the
spraying/ionization process.
1H and
31P{1H} NMR spectroscopy over 15 days. As
3.3. Reactivity with biomolecules
As shown above, the complexes are inert in water, but previous
studies provided evidence that in the presence of other ligands (such
as in biological media) reaction may occur more rapidly [23,26].
Consequently, the reaction of 1 and 2 with the sulfur-containing
amino acids Cys and Met, with the small protein ubiquitin as well as
5′-GMP as a DNA model were carried out in aqueous solution. In
particular, the binding to proteinaceous targets appears to be of
relevance to the mode of action of organometallic RuII–arene
complexes [9,40–44], although they are also known to have high
affinity to DNA [44,45].
Analysis of samples containing 1 and Met by ESI-MS revealed
adduct formation during the first hours of incubation, confirmed by
the presence of a signal at m/z 649.84±0.02 (relative intensity 76%,
Fig. 2. Molecular structures of 2 and one of the two crystallographically independent molecules of 3 (3A); thermal ellipsoids are drawn at 50% probability level and solvent molecules
were omitted for clarity.
Scheme 1. Synthetic route to biscarboxylato RuII–arene compounds with sugar-derived
co-ligands.
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M. Hanif et al. / Journal of Inorganic Biochemistry 105 (2011) 224–231

Page 6

exact mass 650.11) assignable to the ion [1–2Cl+Met+OH]+
(Table 3). With time the main peak observed in the spectrum is
found at m/z 383.83±0.02 (100% after 72 h, exact mass 384.06),
which corresponds to [(cym)Ru(Met)–H]+.
Incubation of 1 with Cys leads to defined products only within the
first 24 h where the most abundant signal at m/z 500.83±0.02
corresponds to [1−2Cl+OH]+, followed by m/z 355.90 (14%, exact
mass 356.04; [(cym)Ru(Cys)−H]+) and m/z 855.95 (37%, exact mass
856.06; [(cym)2Ru2(Cys)3−2 H+Na]+and the hydrolysis products
discussed before. Over time the intensities of those peaks decrease
dramatically and after 72 h they are not assignable to defined reaction
products anymore, probably due to Cys decomposing the RuII–arene
complexes present in solution.
In the spectrum of a solution containing 2 and Met recorded after
24 h of reaction, there were only traces of Ru–Met adducts visible in
themass spectrum(Table 3), indicatinga muchlowerreaction ratefor
the dicarboxylato species. After 72 h, a signal attributable to [(cym)
Ru-(Met)−H]+(24%) was observed, which increases in relative
intensity over 7 days of incubation to become the most abundant
species, similar to the reaction of 1 with Met. In contrast to the
reaction with Met, there is no significant interaction of 2 with Cys
indicated by mass spectrometric methods over 7 days of incubation.
These observations were also confirmed by NMR spectroscopy.
The reaction of 1 and 2 with the model protein Ub (8564.2±
0.2 Da) was monitored over 7 days, and in the case of the dichlorido
complex 1, a steady increase in relative intensity of the mono-adduct
[(cym)Ru+Ub] at m/z 8797.4±0.6 Da was observed in the deconvo-
luted mass spectrum (Fig. 3). The phospite ligand is lost during the
binding process, as also observed for other RuII–arene complexes [28].
Although 1 was present in a 2-fold excess no peaks corresponding to
bis-adducts were observed. Furthermore, the protein appears to
undergodegradation over time, as indicated by the appearance of, e.g.,
the [y58+H]+fragment (51% after 72 h). When incubating 2 with Ub,
no evidence for adduct formation was found during 7 days of
incubation.
Table 2
Selected bond lengths (Å) and bond angles (°) of 1–3 and the structurally related compound oxaloRAPTA-C for comparison purposes.
1a
23c
OxaloRAPTA-Cb
AB
Ru–O1
Ru–O2
Ru–P
Ru-centroid
O–Ru–O
O1–Ru–P
O2–Ru–P
–
–
2.2406 (11)
1.711
–
–
–
2.073 (4)
2.109 (4)
2.2491 (16)
1.703 (3)
79.11 (17)
85.95 (12)
89.37 (14)
2.071 (3)
2.087 (3)
2.2500 (11)
1.701 (2)
78.89 (11)
86.25 (8)
89.05 (9)
2.071 (3)
2.088 (3)
2.2492 (12)
1.704 (2)
78.62 (12)
85.40 (8)
89.21 (9)
2.093 (2)
2.095−(1)
2.310 (1)
1.690
78.43 (7)
82.83 (5)
88.79 (5)
a,b)Taken from Refs. [26] and [23], respectively.
c)There are two crystallographically independent molecules of 3 in the asymmetric unit.
Table 3
Relative abundance of selected adducts between 1 and 2 and Met, Cys and Ub
referenced to the most abundant species in the ESI mass spectra recorded after 24, 72
and 168 h of incubation.
Bioligand CompoundAdduct Relative abundance / %
24 h72 h 168 h
Met
1
[(cym)Ru(Met)−H]+
[1−2Cl+OH]+
[(cym)Ru(Met)−H]+
[2+H]+
[(cym)Ru(Cys)−H]+
[1−2Cl+OH]+
[(cym)Ru(Cys)−H]+
[2+H]+
[Ub+{Ru(cym)}]+
[Ub]+
[y58]+
[Ub+2]+
[Ub]+
[y58]+
37
100
100
26
24
100
100
0
2
3 100
100
10
100
9
Cys
1
0
–a
–a
100
2
040
100
66
100
38
100
100
51
17
100
100
31
59
Ub
1
2
000
100
13
100
16
100
34
aCysteine appears to decompose the complexes completely within 168 h.
Fig. 3. The reaction of 1 with Ub studied by ESI-MS over a period of 3 days. Upon adduct formation, the carbohydrate ligand of 1 is cleaved.
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The reactivity of 2 with 5′-GMP was evaluated by means of1H and
31P{1H} NMR spectroscopy and compared to 1. Samples containing
molar ratios of 1:2 (metal complexes : 5′-GMP) were prepared in D2O
and analyzed after 0.5, 48 and 120 h. These studies reveal low
reactivity for 2 with 5′-GMP, whereas 1 and other dichlorido Ru
compounds are relatively reactive and form adducts via the N7 atoms
of guanine [26,46].
3.4. Evaluation of the in vitro anticancer activity
The antiproliferative potential of 2, 3, 5 and 6 (4 is not sufficiently
soluble in water to be included in the biological assay) was
determined in human SW480 colon adenocarcinoma, CH1 ovarian
cancer and A549 non-small cell lung cancer cells using the MTT assay,
and the obtained results are compared to those of the analogous
dichlorido compound 1 and the Os analogues of 2 and 3, i.e., 2Osand
3Os(Table 4) [30]. In general, only modest antiproliferative activity
was observed for this series of sugar compounds, which is not
unexpected considering recent literature reports on the tumor-
inhibiting properties of different carbohydrate–metal conjugates
[25]. Low in vitro cytotoxicity is common for many ruthenium drug
candidates, including NAMI-A and some RuII–arene compounds,
which nevertheless are often potent in vivo, e.g., certain RAPTA
complexes against tumor metastases [16,47,48].
In the series of compounds reported here, the replacement of the
labile chlorido ligands by chelating biscarboxylates significantly alters
the biological activity of the compounds. The IC50values in the CH1 and
SW480 cells are approximately 5–7 times lower for the biscarboxylato
complexes compared to their dichlorido counterparts [26], with the
malonato complexes 5 and 6 being somewhat more active than their
oxalato analogues 2 and 3, suggesting an influence of ligand stability on
cytotoxic potency (Fig. 4). In contrast, similar modifications in the
RAPTA-type complexes had only a minor effect on their tumor-
inhibiting potencies [23]. The variation of the phosphite ligand has a
minorinfluenceonanticanceractivity,althoughpotentialbindingtothe
glucose receptor might by influenced by the substituents at the
carbohydrate-derived moiety.
In order to estimate the relative lipophilicity of the compounds,
being of relevance for the cellular uptake and therefore for the
intracellular concentration, microemulsion electrokinetic chromatog-
raphy (MEEKC) experiments were conducted and the capacity factors
logk weredeterminedfor 2,3,4, 6andtheOsanalogues of2 and3,i.e.,
2Osand 3Os(Table 4), using a literature method [25,49]. The
separation in MEEKC is not only based on the analytes electrophoretic
mobilitybutontheirhydrophobic interactionwiththemicroemulsion
droplets. The most active compound 6 was found to have the highest
lipophilicity whereas the log k values of the other compounds are in a
rather similar range, as are their IC50values.
4. Conclusions
RAPTA-type complexes with their P-based pta ligand are among
the best studied and most promising RuII–arene anticancer agents
that appear to interfere with enzymatic targets and in vivo they show
selective antitumor activity. By maintaining the structural features
around the Ru center, but modifying the carrier ligand, we have
preparedclosely related sugar-derived RuII–arene complexes. In order
to study the role of the leaving group, the dichlorido ligands were
replaced by chelating biscarboxylates, i.e., oxalate and malonate.
These structural modifications have a dramatic influence on the
chemical and biological properties of the compounds. As expected,
and in analogy to the RAPTA counterparts, hydrolysis is markedly
inhibited. The reactivity of the organometallic species to the biological
nucleophiles Met, Cys, Ub and 5′-GMP is significantly reduced, and
compared to the respective dichlorido complexes, essentially the
same adducts are formed, but at a significantly lowered rate. Since the
introduction of the biscarboxylate ligands resulted in significantly
reduced in vitro anticancer activity in all tested cell lines, it appears
that covalent binding to biological targets is a prerequisite for this
type of compounds to exert their desired biological properties.
Acknowledgements
We thank the Higher Education Commission of Pakistan, the
Austrian Exchange Service (ÖAD), the Hochschuljubiläumsstiftung
Vienna, the Theodor-Körner-Fonds, the FFG—Austrian Research
Promotion Agency (811591), the Austrian Council for Research and
Technology Development (IS526001), COST D39 and CM0902 and the
Austrian Science Fund for the financial support. This research was
supported by a Marie Curie Intra European Fellowship within the
7th European Community Framework Programme project 220890-
SuRuCo (A.A.N.). We gratefully acknowledge Alexander Roller for
collecting the X-ray diffraction data and Prof. Markus Galanski for
recording the 2D NMR spectra.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.jinorgbio.2010.10.004.
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