JIB-09029; No of Pages 7

Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

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Journal of Inorganic Biochemistry

j o u r n a l h o m e p a ge : w w w . e l s e v i e r . c o m / l o c a t e / j i n o r gb i o

Synthesis, characterization, and anticancer activity of ruthenium-pyrazole

complexes
Solene David a, Richard S. Perkins a, Frank R. Fronczek b, Sahba Kasiri c,
Subhrangsu S. Mandal c, Radhey S. Srivastava a,
a
b
c

Department of Chemistry, University of Louisiana at Lafayette, LA 70504

Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803
Department of Chemistry and Biochemistry, University of Texas at Arlington, TX 76019

article

info

Article history:
Received 26 November 2011
Received in revised form 23 February
2012 Accepted 24 February 2012
Available online 4 March 2012
Keywords:
Ruthenium(III)pyrazole complexes
Crystal structure cyclic voltammetry
in vitro cytotoxicity

abstract
A series of new water soluble Ru(III) pyrazole complexes mer-[RuCl3(DMSO-S)(pyz)2] 1, mer-[RuCl3(DMSOS) (DMSO-O)(pyz)] 2, mer-[RuCl3(bpy)(dmpyz)] 3, and mer-[RuCl3(DMSO-S)(dmpyz)2] 4 (pyz
=pyrazole; dmpyz = 3,5-dimethylpyrazole, bpy = 2,2-bipyridine) have been synthesized and
characterized by use of a combination of spectroscopy (IR and UV-visible), X-ray diffraction, and
cyclic voltammetry. The molecular X-ray structure of all reported compounds ( 1-4) revealed
distorted octahedral coordination around
ruthenium. The cytotoxicity assay on human breast cancer cells (MCF7) demonstrated that
compounds 1 and 4 affect cell viability, whereas compounds 2 and 3 do not show appreciable activity.
The IC50 values for 1 and 4 lie within the range of 71-32 M in MCF7 cells.
2012 Elsevier Inc. All rights reserved.

1. Introduction

The success of platinum-derived anticancer drugs has

stimulated a
renaissance of medicinal inorganic chemistry. Cisplatin is one
of the most widely used drugs in clinical use. However, its
effectiveness is limited due to its severe toxicity, resistance to
the drug, and unwanted side effects [1-5]. This has led to the
search for complexes of other transition metals with
interesting biological properties [6-17], wider ranges of
activity, and lower systematic toxicities. Ruthenium complexes are increasingly gaining interest as potential
alternatives to platinum-based chemotherapeutic agents.
Some
ruthenium
complexes have been shown to be effective against cancers not
readily treated by cisplatin [18]. Among all rutheniumbased anticancer
agents, ruthenium-DMSO complexes are believed to have great
potential because of their selectivity for solid tumor metastases
and
low
host toxicity
[19]. Furthermore, several ruthenium
complexes
have
been found to display a significantly higher degree of
selectivity
towards cancerous cells than the leading commercially
available
platinum derived drugs, which results in reduced damage to
healthy
tissues [19]. The mechanism of selective delivery of Ru(III)
complexes
to specific cancer cells is widely argued. One widely accepted
possibility
is the ability of ruthenium(III) complexes to mimic iron in

effective means for ruthenium complexes to accumulate in cancer

cells [22]. Two ruthenium complexes, KP1019 {[IndH][trans-RuCl4(reversible
binding to plasma proteins such as transferrin, which is
correlated
with the overexpressed concentration of receptors for
this
protein
on
the surface of cancer cells [20-22]. This mechanism
provides an

Corresponding author. Tel.: + 1 337 482 5677; fax: + 1

337
482
5676.
E-mail address: rss1805@louisiana.edu (R.S. Srivastava).
0162-0134/$ - see front matter 2012 Elsevier Inc. All
rights reserved. doi:10.1016/j.jinorgbio.2012.02.022

Ind)2] (Ind = indazole)} [23] and NAMI-A {[ImH][trans-RuCl4(DMSO)(Im)] (Im = imidazole)} [24], are known to bind to
iron(III) binding sites of transferrin [25].
NAMI-A and KP1019 are under clinical trial against metastatic and
colon cancers, respectively [26,27]. They are scheduled for phase 2
trials [28-30]. The low general toxicity of these complexes is
ascribed to the ability of ruthenium to collect specifically in
cancer tissues, possibly via the transferrin mechanism [2022,31,32].
Several platinum and ruthenium complexes with Nheterocyclic
ligands have
been
reported
of
which
some
showed
significant
cytotoxicity. For example one of the ligands in NAMI-A is imidazole,
a heterocyclic amine. Pyrazole, a five membered N-heterocycle, also
belongs to the same class of compound as imidazole. Pyrazole is a
poor -electron acceptor and a better -donor and hence acts as a
hard donor site [33]. Pyrazoles have long been applied in
agrochemical
and pharmaceutical industries as herbicides and active
pharmaceuticals. Pyrazole moieties are known for their antimicrobial,

anticancer,
ACE (angiotensin-converting-enzyme) inhibitory, antiviral and
antiinflammatory activities. However, pyrazole complexes are
less
explored. Pyrazole type heterocycles also represent the important
class
of non-leaving ligands because of their rich electronic property
which
can be altered by appropriate choice of substituents on the
pyrazole
ring. This in turn enables optimization of the electronic
properties
on
the metal. Also, pyrazole ligands are less basic than imidazole.
This
reduces loss of DMSO from the complex and increases the
antitumor
activity [34]. Additionally, all the complexes reported
herein
are
water soluble, which makes them attractive for being
developed
as
drugs. It is therefore pertinent to explore the chemistry of
ruthenium

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes,
J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022

S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

I > 2(I)
Refinement method
full-matrix least squares on F2
to anti-cancer properties.
R [F > 2(F)]
0.031
0.022
Rint
0.011
0.019
and cytotoxic effects of
wR(F)
0.058
0.052

(III) pyrazole complexes with regard

Herein, we report synthesis, structure,
these
plexes (1-4) on human breast cancer cells MCF7.

com-

2. Experimental section
2.1. Materials
All reagents were of analytical grade. Hydrated
ruthenium(III) chloride and silver triflate were purchased
from
Strem
chemicals. DMSO,
pyrazole, 3,5dimethylpyrazole, and solvents, including
anhydrous solvents, were purchased from Sigma-Aldrich.
Reagent grade solvents were dried and stored over 4
molecular sieves.

2.2. Cyclic voltammetry

Cyclic voltammetry was carried out with a three electrode
system described by us previously [35]. Redox potentials
were calculated using Gaussian 03. DFT (Density functional
theory) calculations
were done using the BP3LP method and a LANL2DZ basis
set. Optimized gas phase structures were used to calculate
energies in solution using the IEFCPM solvation model.

2.3. Physical measurements

The IR spectra were measured on a JASCO 480 Plus
spectrophotometer with samples in compressed KBr discs. UV-visible
(UV-vis) spectra were obtained on a JASCO V-550
spectrophotometer.
Elemental analysis was performed by Atlantic Microlab,
Norcross, Georgia. The diffraction data were collected at low
temperature on a Nonius KappaCCD equipped with Mo K
( = 0.71073 ) and a Bruker Kappa Apex II equipped with
Cu ( = 1.54178 ) radiation source diffractometer, a
graphite monochromator, and an Oxford Cryostream lowtemperature device. Absorption collections were made by
the multi-scan method. Crystal data and details of data
collection and refinement are given in Tables 1-3.

Table 1
Crystal data and structure refinement for compounds 1 and 2.
1

Formula
C8H14Cl3N4ORuS
C7H16Cl3N2O2RuS2.CH2Cl2
Formula weight
421.71
516.68
Crystal system
Monoclinic
Monoclinic
Space group
Cc
P21/n
T (K)
90
90
a ()
8.921 (2)
13.2179 (10)
b ()
13.054 (2)
8.3150 (5)
c ()
12.856 (3)
16.5031 (14)
()
98.090
90.084 (3)
3
V (A )
1482.2 (5)
1813.8 (2)
Z
4
4
Dx (Mg m- 3)
1.890
1.892
(mm- 1)
1.73
1.83
F(000)
836
1028
Crystal size (mm)
0.15 x 0.10 x 0.03
0.35 x 0.22 x 0.12
range ()
4.8-57.1
2.5- 33.1
h, k, l limits
-12 12, -18 17, -18 18 -2020, -1212,
-2525
Reflections measured
10419 67756
Unique data with
3765
6176

Table 2
Crystal data and structure refinement for compounds 3 and 4.
3

Formula
C15H16Cl3N4Ru
C12H22Cl3N4ORuS.H2O
Formula weight
459.74
495.83
Crystal system
Monoclinic
Monoclinic
Space group
P21/n
P21/n
T (K)
90
90
a ()
7.8024 (16)
8.9841 (10)
b ()
9.965 (2)
13.7983 (15)
c ()
21.753 (5)
30.823 (3)
()
92.801 (19)
95.453 (5)
V (A3)
1689.3 (6)
3803.7 (7)
Z
4
8
-3
Dx (Mg m )
1.808
1.732
-1
(mm )
11.90
11.69
F(000)
916
2008
Crystal size (mm)
0.03 x 0.02 x 0.02
0.28 x 0.10 x 0.04
range ()
2.5-30.5
4.1-68.2
h, k, l limits
-88, -1110, -2313 -109, -1616, -3636
Reflections measured
12076
32623
Unique data with I > 2(I)
13496195
Refinement method
full-matrix least squares on F2
R [F > 2(F)]
0.058
0.035
Rint
0.172
0.044
wR(F)
0.163
0.073

2.4. Cell viability assay and IC 50 measurements

Human breast cancer cells (MCF7 purchased from ATCC)
were grown in Dulbeco's modified Eagles media (DMEM)
that was
supplemented with 10% heat inactivated fetal bovine serum
(FBS,
Sigma-Aldrich), 2 mM L-glutamine and penicillin/streptomycin
(100
units and 0.1 mg/mL) in humidified CO 2 incubators with 5% CO2
as
described by us previously [36,37]. The cytotoxicity of
ruthenium
metal complexes was determined by MTT
(3-(4,5dimethylthiazol2-yl)-2,5- diphenyltetrazolium bromide) assay described
previously
[38,39]. In brief, approximately 10,000 cells were seeded into
each
well of a 96-well micro titer plate and incubated for 24 h in
150
L
DMEM. An additional 50 L DMEM containing the required
amount

of each metal complex to obtain 0.05 to 60 M final

concentration
(in 200 L final volume) were added in each well. Each
experiment
was carried out in 5 replicate wells. Control wells were treated
with
equivalent amounts of
DMSO
(the solvent used for
dissolving
the
metal-complex stock solution). After 96 h of incubation, 20
L
of
MTT (5 mg/mL) was added into each well and cell viability
was
assayed by measuring the formazan absorption at 560 nm by
using
a 96 well plate reader, Flowstar-Omega (BMG labtech). The
absorbance
(at
560 nm)
values
were
plotted
against
concentration
of
metal-complexes to determine the IC 50. The concentration
of
the
metal-complexes at which the conversion of
MTT to
formazan
by
viable cells is reduced by 50% compared to control cells is
defined
as
the IC50. The experiments were repeated at least twice
with 5
replicates each time.
2.5. Synthesis of starting complexes
Complexes [H(DMSO)2]trans-[RuCl4(DMSO)2] and mer-[RuCl3
(DMSO-S)2(DMSO-O)] were prepared by the literature method [40].
2.6. Synthesis of ruthenium-pyrazole complexes
2.6.1. Synthesis of mer-[RuCl3(DMSO-S)2(DMSO-O)]
(1): A solution of mer-[RuCl3(DMSOS)2(DMSO-O)] (0.1
g,
0.23 mmol) and pyrazole
(0.118 g,
1.74 mmol) in
dichloromethane
(20 mL) was heated to reflux for 24 h. The volume of solution
was
reduced to 10 mL and then a small amount of diethyl
ether
was
added. The mixture was kept refrigerated to yield a dark red
crystalline solid (yield 0.172 mmol, 76%). Anal. Calcd. for
C8H14Cl3N4ORuS

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes,
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S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

Table 3
bond length and bond angle of compounds 1, 2, 3, and 4.
Compound

Bond length ()

Ru1-N3
Ru1-N1
Ru1-S1
Ru1-Cl1
Ru1-Cl2
Ru1-Cl3

2.064 (3)
2.114 (4)
2.2783 (12)
2.3378 (9)
2.3439 (10)
2.3508 (9)

Ru1-N1
Ru1-S1
Ru1-O2
Ru1-Cl1
Ru1-Cl2
Ru1-Cl3
S2-O2

2.0958
2.2777
2.0656
2.3477
2.3400
2.3456
1.5592

Ru1-N1
Ru1-N2
Ru1-N3
Ru1-Cl1
Ru1-Cl2
Ru1-Cl3

2.024
2.063
2.095
2.352
2.366
2.327

Ru1-N1
Ru1-N3
Ru1-S1
Ru1-Cl1
Ru1-Cl2
Ru1-Cl3

2.095 (3)
2.106 (3)
2.3050 (9)
2.3356 (9)
2.3466 (9)
2.3411 (9)

(11)
(3)
(9)
(3)
(3)
(3)
(10)

(11)
(9)
(9)
(4)
(3)
(3)

Bond angle ()
N3-Ru1-N1
N3-Ru1-S1
N1-Ru1-S1
N3-Ru1-Cl1
N1-Ru1-Cl1
S1-Ru1-Cl1
N3-Ru1-Cl2
N1-Ru1-Cl2
S1-Ru1-Cl2
Cl1-Ru1-Cl2
N3-Ru1-Cl3
N1-Ru1-Cl3
S1-Ru1-Cl3
Cl1-Ru1-Cl3
Cl2-Ru1-Cl3
O2-Ru1-S1
N1-Ru1-Cl2
N1-Ru1-S1
O2-Ru1-N1
N1-Ru1-Cl3
S1-Ru1-Cl1
O2-Ru1-Cl2
O2-Ru1-Cl1
S1-Ru1-Cl2
N1-Ru1-N2
N1-Ru1-N3
N2-Ru1-N3
N1-Ru1-Cl3
N2-Ru1-Cl3
N3-Ru1-Cl3
N1-Ru1-Cl1
N2-Ru1-Cl1
N3-Ru1-Cl1
Cl3-Ru1-Cl1
N1-Ru1-Cl2
N2-Ru1-Cl2
N3-Ru1-Cl2
Cl3-Ru1-Cl2
Cl1-Ru1-Cl2
N1-Ru1-N3
N1-Ru1-S1
N3-Ru1-S1
N1-Ru1-Cl1
N3-Ru1-Cl1
N3-Ru1-Cl3
Cl3-Ru1-Cl1
N1-Ru1-Cl2
Cl3-Ru1-Cl2
Cl1-Ru1-Cl2

86.54 (13)
94.69 (9)
178.57 (11)
87.35 (9)
90.15 (8)
89.19 (3)
176.78 (9)
90.27 (11)
88.49 (4)
92.20 (3)
87.97 (9)
88.71 (8)
92.05 (3)
175.25 (4)
92.42 (3)
94.59 (3)
88.95 (3)
179.75 (3)
85.59 (4)
89.48 (3)
90.083 (13)
174.28 (3)
86.42 (3)
90.856 (12)
79.4 (4)
98.2 (4)
177.5 (4)
84.6 (3)
91.7 (3)
88.6 (3)
92.4 (3)
88.1 (3)
91.5 (3)
177.03 (12)
171.5 (3)
94.1 (3)
88.4 (3)
90.24 (12)
92.73 (12)
88.81 (12)
92.28 (9)
176.62 (9)
91.14 (9)
91.36 (9)
90.11 (9)
178.38 (3)
176.83 (9)
89.24 (3)
90.13 (3)

(424.71): C, 22.78; H, 3.35; N, 13.29. Found: C, 22.93; H,

3.24; N,
13.25. Selected IR absorption bands in KBr (cm- 1):
SO1054
(S-DMSO), C=C (pyz) 1629, C=N (pyz) 1400. UV-vis (H2O):
364 nm ( = 32.60 M- 1 cm- 1); 426 nm ( = 10.60 M- 1 cm- 1).
2.6.2. Synthesis of mer-[RuCl3(DMSO-S)(DMSO-O)(pyz)]
(2): mer-[RuCl3(DMSO-S)2(DMSO-O)] (0.1 g, 0.23 mmol)
and
pyrazole
(0.017 g,
0.25 mmol)
were
dissolved
in
dichloromethane
(10 mL) and stirred at room temperature for 20 h. The
solution
was
evaporated to 3 mL and 10 drops of hexane were added.
The
flask
was kept in a refrigerator. The resulting solution yields red
crystals
(yield
0.184
mmol,
80%).
Anal.
Calcd.
for
C7H16Cl3N2O2RuS2CH2Cl2
(516.68) C,18.6; H, 3.5; N, 5.4. Found: C, 18.87; H, 3.82;
N,
5.11.
Selected IR absorption bands in KBr (cm - 1): SO1103, (SDMSO),
909 (O-DMSO), C=C (pyz) 1627, C=N (pyz) 1409. UV-vis

dimethylpyrazole. However, the X-ray structure revealed that the

(H2O):
386 nm ( = 27.11 M- 1 cm- 1); 525 nm ( = 8.34 M- 1 cm- 1).
2.6.3. Synthesis of mer-[RuCl3(bpy)(dmpyz]
(3). Our aim was to synthesize mer-[RuCl2(bpy)
(DMSO-S)
(dmpyz)]CF3SO3 by refluxing mer-[RuCl3(DMSO-S)(bpy)]
with
equimolar amounts of silver triflate and an excess
amount of 3,5-

isolated product is mer-[RuCl3(bpy)(dmpyz)] 3, not what we

expected. Compound 3 was also synthesized by the direct reaction
of
mer-[RuCl3(DMSO-S)(bpy)] with an excess amount of 3,5-dimethylpyrazole. mer-[RuCl3(DMSO-S)(bpy)] (0.1 g, 0.23 mmol), 3,5-dimethylpyrazole (0.167 g, 1.74 mmol) and acetone (35 mL) were placed in a
flask,
which was heated to reflux for 24 h. The volume of solution
was
reduced to 3 mL and 10 drops of hexane were added. On cooling at
4C the resulting solution yields orange crystalline solid (yield
0.156 mmol, 68%). Calcd. for C15H16Cl3N4Ru (459.74) C, 39.2; H, 3.5;
N, 12.2. Found: C, 38.8; H, 3.41; N, 11.81. Selected IR absorption bands
in KBr (cm - 1): C=C, 1573 (m), C=N, 1448 (s) (pyz), C-N (bpy) 1607
(m), UV-vis (H2O): 435 nm ( = 47.89 M- 1 cm- 1); 375 nm
( = 8.29 M- 1 cm- 1). Unfortunately, our attempt to synthesize the
similar complex with pyrazole ligand was unsuccessful.

2.6.4. Synthesis of mer-[RuCl3(DMSO-S)(dmpyz)2]

(4): A solution of mer-[RuCl3(DMSO-S)2(DMSO-O)] (0.2 g,
0.45 mmol) and 3,5-dimethylpyrazole (0.334 g, 3.48 mmol) in
dichloromethane (15 mL) was heated to reflux for 9 h. The
brown
solution was evaporated to 3 mL and then 1 mL of hexane was
added. On cooling at 4C a reddish-orange solid was separated
(yield 0.262 mmol, 58%). Product was purified by column
chromatography with ethyl acetate and hexane (40/60) as eluent. Anal.
Calcd.
C12H20Cl3N4ORuSH2O (495.83): C, 29.06; H, 4.95, N, 11.35. Found:
C, 29.18.; H, 5.24; N, 10.95. Selected IR absorption bands in KBr
(cm- 1): SO1086 (S-DMSO), C=C (pyz) 1632, C=N (pyz) 1398.
UV-vis (H2O): 362 nm ( = 12.20 M- 1 cm- 1).

3. Results and discussion

3.1. Syntheses and general properties

All the complexes were formed by the displacement of

either one or two DMSO ligands from the precursor, mer[RuCl3(DMSO-S)2 (DMSO-O)]
with
pyrazole
or 3,5dimethylpyrazole ligands. The
spectroscopic
features
of
the
sulfoxides
in
these
complexes
are
comparable to those found for the corresponding complexes
with
N-monodentate ligands [41]. The pyrazole (Fig. 1) as a ligand,
coordinates to metals and metalloids through the N2 [42]. The
presence
of
a
strong IR band in the range assignable to N-H vibrations in the
spectra
of all the complexes (1-4) confirms the coordination of
pyrazole
via
N2
(Fig.
1) in the neutral monodentate form. The
characteristic
IR
bands of C =C and C =N stretching modes are observed at ca.
1640,
and 1558 cm- 1 in uncomplexed pyrazole and are shifted to
considerably lower frequencies. This is fairly consistent with longer C =C
and
C =N bonds in 1-4 compared with the same distances in the
uncomplexed ligand [43]. The composition of all the complexes
reported
herein has been determined by various physical methods,
especially
single crystal analysis.

N2
N1
H
Fig. 1. Structure of pyrazole.

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes,
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S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

Fig. 2. Ortep diagram of mer-[RuCl3(DMSO)(pyz)2] 1.

3.2. Crystallographic analysis

The single crystals suitable for X-ray diffraction of the
complexes
1,2,3, and 4 were obtained by slow diffusion of hexane into
CH2Cl2
and diethyl ether into acetone solutions. Figs. 2-5 depict
the
molecular structures of these complexes. Data collection
parameters and selected bond angles and lengths are listed
in Tables 1-3 for all the complexes.
Compound 1 crystallizes in the monoclinic space group
Cc that adopts a distorted octahedral geometry with
meridional arrangement (Fig. 2). Both pyrazole ligands are
coordinated to ruthenium through the tertiary ring nitrogen
atom N2 (Fig. 1), The average Ru-Cl bond length, 2.344(9)
is comparable to other related Ru(III) complexes [41,44,45].
The Ru-S bond distance, 2.2783(12) is appreciably shorter than
the average value of 2.34 (1) found for the Ru(III)-S
compounds trans to DMSO-S in the precursor [46,47].
Likewise
the
average bond length of Ru-N, 2.089 is comparable to
Ru(III)-N
(imidazole), 2.079 [41]. The bond angles of N1Ru1S1,
178.57(11);

Fig. 3. Ortep diagram of mer-[RuCl3(DMSO)2(pyz)] 2.

Fig. 4. Ortep diagram of mer-[RuCl3(bpy)(dmpyz)2] 3.

and Cl2Ru1N3, 176.78(9) are slightly smaller than the known

values of octahedral complexes of ruthenium (III).
Compound 2 was crystallized in the monoclinic space group
P21/n
with distorted octahedral geometry having meridional
arrangement
(Fig. 3). The pyrazole ligands coordinated to ruthenium
through
N2
(Fig. 1). The average Ru-Cl bond length, 2.344(9) is
similar to
compound 1. The Ru-S bond distance, 2.2777(3) is very
close
to
compound 1. The bond length of Ru-N, 2.0958 is slightly
smaller
than Ru(III)-N (imidazole), 2.079 [41] but significantly
shorter
than 1 (2.114 ). The Ru-O bond length 2.065 is slightly
shorter
than the precursor, mer-[RuCl3(DMSO-S)2(DMSO-O)] (2.077 ).
The
S-O bond distance (1.559 ) of the O-bonded DMSO is
noticeably
longer than those of the S- bonded DMSO ligands (1.479 ),
which

shows a considerable decrease of the double-bond character

of
the
sulfur-oxygen bond upon coordination to the metal atom via
oxygen.

Fig. 5. Ortep diagram of mer-[RuCl3(DMSO)(dmpyz)2] 4.

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S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

Compound 3 was crystallized in the monoclinic space group

P21/c.
The bite angle in compound 3
(Fig. 4) involving
coordinated
bipyridine is N1RuN2, 79.4(4), typically found in 2, 2bipyridine
coordinated to second and third row transition metal ions
[48].
The
bond angle of the nonchelating ligand, N3Ru1Cl2 is 88.4
(3)
and is much larger than the chelating ligand bond angle,
N1RuN2, 79.4 (4). The average Ru-N (bipyridine) bond
length, 2.0435 , is
shorter than the Ru-N(pyrazole) bond length, 2.095 , and
distinctive
bond length of 2.099 reported for Ru(II, III) complexes [4749].
This
is probably
due to chelation
through bipyridine
nitrogens.
The
average Ru-Cl bond length of 2.348 is comparable to the
related
Ru(III) complexes. The bond angles of N2RuN3, 177.5(4);
N1RuCl2,
171.5 (3) are smaller than the known values of
octahedral
complexes. Likewise, the bond angles N2RuCl3, 91.7(3);
N1RuCl1, 92.4 (3); Cl1RuN3, 91.5(3); N2RuCl2, 94.1(3)
vary from 91 to
94. These distortions from ideal octahedral geometry are
probably
caused by the acute (~ 79.4) bite angle of the chelating
bipyridine
ligand.
Compound 4 was crystallized in the monoclinic space group
P21/n.
The X-ray structure of 4 shows a distorted octahedral
geometry
(Fig. 5) with three chlorine atoms in a mer configuration. The
average
Ru-Cl bond length, 2.3411(9) is slightly shorter than
that
of
compound 1. The Ru-S bond distance, 2.3050(9) is shorter
than
the average value of 2.34(1) found for the Ru(III)-S
compounds
trans to DMSO-S in the precursor [40,46]. The average bond
length
of Ru-N, 2.1005(3) , is longer than Ru(III)-N (imidazole),
2.079

[44,45], and the average bond length (Ru-N) of 1 (2.089

).
The
bond angles of N1Ru1S1,
176.62(9) and N1RuCl2,
176.83(3)
are
close to the known values of octahedral complexes of
ruthenium
(III).
The average values of the Ru-Cl bond lengths are
comparable in all four complexes (2.344(9) for 1; 2.344(9)
for 2; 2.348 for 3, and 2.3411 (9) for 4) and are close to the
average value of 2.357 found
in related ruthenium (III) complexes [44]. These distances are
shorter than those found in the trans-Cl-Ru(II)-Cl (av. 2.41 )
[40,44,50].

3.3. Electrochemical study

Figures of cyclic voltammetry (Figures S1-S7) are given
in
supporting information. Cyclic voltammetry results were

obtained
for 1 in both aqueous and DMSO solutions at sweep
rates
ranging
from 100 mV/s to 3011 mV/s. Sweeps were similar at all
rates.
A
dominant reduction and corresponding oxidation are evident.
In
addition
there is a weak and broad oxidation peak. The values of the
separation
of the dominant oxidation and reduction peaks (E p)
ranged
from
79 mV at 100 mV/s to 137 mV at 3011 mV/s and are
assumed
to
be
due to a quasireversible oxidation-reduction couple.
The
formal
potential (E) calculated as the average of the
oxidation and
reduction peak values ranged from 0.059 to 0.061 V with
an average value of 0.060 V vs. SCE (saturated calomel
electrode). Semiderivative analysis [51-53] was also
done with these sweeps. The average formal potential
obtained this way was 0.061 V.
Using an extended range in the anodic direction
gives an additional anodic/cathodic pair of peaks. The
characteristics of this voltammagram are consistent
with an electron-chemical-electron (ECE) mechanism as
shown generally though not exactly by simulation [54] as given in Fig. S3. The chemical reaction
involved in the ECE mechanism may be due to ligand
replacement by DMSO similar to that discussed for
compound 2 below.
Compound 1 gives similar sweeps in aqueous solution.
Voltammagrams at rates from 100 to 3011 mV/s were similar. For
the largest pair of peaks, an average value of E p =
83 mV and an average E = 0.333 V were found from
the voltammagrams. Semiderivative analysis gives E
= 0.337 mV. The pair of peaks is assumed to be due to
the quasireversible Ru(III)/Ru(II) redox couple.

The cyclic voltammagram for 2 shows a large reduction peak at

around 0.20 V but only a small corresponding oxidation peak
that
is barely discernible at low sweep rates. Further oxidation to
about
0.6 V on the anodic sweep produces a well defined oxidation
peak
and corresponding reduction on the following anodic sweep. When
cyclic voltammetry is initiated in the anodic direction the peak
at
0.6 V does not appear on the first sweep. Only after reduction at

0.2 V does the more positive redox couple appear (Fig. S5).
This
behavior is interpreted as being due to reduction of 2 as Ru(III) goes
to Ru(II). Oxidation of 2 on the following anodic sweep competes
with chemical reactions between 2 and DMSO. The predominant
compound that forms is then the basis for the redox couple seen at
more positive potentials. There may be other compounds formed
as
well. These would account for redox couples that give small
(but
quite distinct when viewed using semiderivative analysis)
oxidation
and reduction peaks.
Lacking a large anodic signal, it is possible to give only an
estimate of the characteristics of the redox couple of 2. The
intermediate between Eox and Ered was calculated and extrapolated
to zero sweep rate for both the CVs and the semiderivative graphs.
These
extrapolations give an average value of 0.15 V. For the other large
redox peaks, extrapolation of CV data gives a formal potential of
0.545 V and extrapolation of semiderivative data gives 0.546 V.
The identity of the main compound formed from 2 is assumed to
be the product of ligand displacement by DMSO. In 2, one to four
ligands could be displaced. In addition, DMSO can bond through S
or O atoms. This gives rise to many possibilities. One possibility can
be checked with available CV data. Alessio et al. [40] obtained
the
CV of mer-[RuCl3(DMSO-S)2(DMSO-O)] and observed a redox couple
at 0.070 V This then would not be responsible for our large
peaks
around 0.55 V but it may be responsible for one of the small
peaks
around zero volts in our data. The redox potentials of this compound
as well as 2 were calculated using Gaussian 03. The value obtained for
mer-[RuCl3(DMSO-S)2(DMSO-O)] was found to be only 0.3 V more
positive than that for 2. Calculations were not done for every
possible
compound but calculation for mer-[RuCl3(DMSO-S)2(DMSO-O)] gives

a redox potential about 1.1 V more positive than calculated for

2.
Cyclic voltammetry was done for 3 in DMSO (Fig. S6) (No
peaks
were found in aqueous solution). Five subsequent segments
were
recorded and are nearly the same as the second and third
segments
in the figure. Voltammagrams in DMSO were done at rates of
100 to
3011 mV/s and were all similar in appearance. Two redox
couples
are seen. The formal potential averaged over all sweep rates for
one
couple was found to be 0.175 V from the cyclic
voltammagrams
and 0.180 V as determined from semiderivative analysis.
For the
other complex the formal potential was found to be 0.129 V
from
cyclic voltammetry and 0.125 V from semiderivative
analysis. The
couples are assumed to be due to sequential one-electron
processes
involving the + 1, + 2, and + 3 oxidation states of Ru.
Cyclic voltammetry was done for 4 in aqueous and DMSO
solutions. In aqueous solution a single redox couple is observed.
Anodic
and cathodic peaks of the couple are broad and shallow. At
some
sweep rates the signal is only a shoulder to the water
reduction
area of the graph. Though only approximate, values obtained
indicate
a separation between anodic and cathodic peaks to be about
0.15 V
and an average of the anodic and cathodic potentials is
around
0.1 V. Cyclic voltammetry in DMSO shows four cathodic peaks
and
five anodic peaks. Near-coincidence of potential values for
cathodic
and anodic peaks when using semiderivative analysis
indicates
redox couples at these potentials: -0.41 V; -0.15 V; + 0.44 V
(Fig.
S7). Many in the array of peaks for 4 are likely due to other
compounds formed through exchange of dmpyz with dmso and
through
bonding rearrangement through the O and S atoms of DMSO
ligands.
The electrochemical study of metal complexes explains the
change
of structural arrangement of the ligand around metal ions. It is
worth
mentioning that the cyclic voltammetry of compounds 1-4
displayed

Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes,
J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022

S. David et al. / Journal of Inorganic Biochemistry 111 (2012) xxx-xxx

120

increasing ease of reduction [63]. We observed that the reduction potential of compound 2 is 2.0 V, which probably suggests that reduction of Ru(III) to Ru(II) is slow compared to compounds 1 and 3.

100

4. Conclusions

80
60

1
2

40

20

0
0

20

40

60

80

100

We have reported, herein, the syntheses and characterization of 4

new neutral water soluble ruthenium-DMSO-pyrazole complexes. In
all cases, the synthesis of these complexes involves substitution of
one or more DMSO molecules with pyrazoles. All members are fully
characterized by various physical techniques as mononuclear distorted octahedral with meridonial arrangement. The result of in
vitro cytotoxicity testing showed that compounds 1 and 4 show significant cytotoxicity to the MCF7 breast cancer cells, whereas compounds 2 and 3 do not show appreciable anticancer activity to
tested cancer cell lines.

Concentration (M)
Fig. 6. Effect of ruthenium-pyrazole complexes on cell viability- IC50
Values of
compounds: 1. 71.0 M (+ 2.1); 2. > 100 M; 3. > 100 M; 4. 32.2 M (+ 1.3).

reversible Ru(III)/Ru(II) reduction waves. The observed redox

potentials fall in the biologically relevant and accessible range. The
known
value of reduction potential is 0.48 vs. SCE [55,56] in the
proliferating
cells, while inside the tumor cells it is up to 100 mV lower [57],
suggesting that biological reducing agents (e.g., glutathione or
ascorbic
acid)
are capable of reducing the compounds to the corresponding
Ru(II)
species [58]. Moreover, redox potentials, which are obtained
by
cyclic
voltammetry were interconnected with protein binding and
cytotoxic
activity.
3.4. Cell viability assay and IC 50 measurement
The metal ion coordination environment in biomolecules
consists of nitrogen and oxygen-donor atoms that are
provided by histidine, aspartates, or glutamates. Since
pyrazole resembles imidazole, it has been incorporated in
stable chelates to obtain ligand systems applicable in the
design of new drugs.
In order to evaluate the antitumor potential of the
metal complexes, we measured their cytotoxicity toward human breast
cancer
cells, MCF7. In brief, cells were incubated with varying
concentrations
of the metal complexes for 96 h at 37 C and 5% CO2. Viable
cells were
then quantified using MTT assay as described previously
[59,60]. The
cell viability is plotted vs. concentration of ruthenium
complexes
(Fig. 6). These studies demonstrated that compounds 1 and 4
showed
significant cytotoxicity toward MCF7 cells with IC 50 values 71
(+ 2.1)
M and 32.2 (+ 1.3) M respectively (Fig. 6). In contrast 2 and
3 were
not effective (IC50 > 100 M) in inducing cell death in MCF7
cells. We
also determined the IC50 (~ 18 M for MCF7) for cisplatin as a
positive
control and it is in agreement with previously reported

Acknowledgements
values.
It is interesting to note that compounds 1 and 4 showed
significant
cytotoxicity in MCF7 cells with IC50 values 71 ( 2) M
and 32.2
( 1.3) M whereas, the Ru-bipyridine complex 3 is not
effective
(IC50 > 100 M) in inducing cell death in MCF7 cells. Some Rupolypyridine complexes like mer-[Ru(terpy)Cl3] are known to
form
DNA-interstrand crosslinks, whereas the inactive cis[Ru(bpy)2Cl2]
appears to exhibit no such interactions [61,62]. We believe
that compound 3, which contains polypyridine (bipyridine) ligand
follows a
similar trend. A possible explanation of discrimination
between
mer-[Ru-(terpy)Cl3] and cis-[Ru(bpy)2Cl2] and [Ru(terpy)
(bpy)CI]CI
or compound 3 is intracellular ruthenium uptake [61]. The
biological
diversity between compounds 1, 4 and 2, 3 may be due to their
different redox properties [62]. There are multiple factors such
as pKa
values of the ligands, protein binding, and redox potential
of compounds, etc. that are responsible for the cytotoxic effect of
the compounds. However, cytotoxic ability is expected to
increase with

We are grateful for financial support provided by the Louisiana

Board of Regents [LEQSF(2009-12)-RD-B-08]. Research in Dr.
Mandal's lab is supported by the American Heart Association and
the
Texas
Advanced Research Program.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10. 1016/j.jinorgbio.2012.02.022.
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Please cite this article as: S. David, et al., Synthesis, characterization, and anticancer activity of ruthenium-pyrazole complexes,
J. Inorg. Biochem. (2012), doi:10.1016/j.jinorgbio.2012.02.022