Novel C,N-Cyclometalated Benzimidazole Ruthenium(II) and Iridium(III) Complexes as Antitumor and Antiangiogenic Agents: A Structure-Activity Relationship Study

Article abstract of DOI:10.1021/acs.jmedchem.5b01194

A series of novel C,N-cyclometalated benzimidazole ruthenium(II) and iridium(III) complexes of the types [(η⁶-p-cymene)RuCl(κ²-N,C-L)] and [(η⁵-C₅Me₅)IrCl(κ²-N,C-L)] (HL = methyl 1-butyl-2-

Full text of DOI:10.1021/acs.jmedchem.5b01194

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Article
Novel C,N-cyclometalated Benzimidazole Ruthenium(II) and
Iridium(III) Complexes as Antitumor and Antiangiogenic
Agents: a Structure-Activity Relationship Study
Jyoti G Yellol, Sergio Alejo Pérez, Alicia Buceta, Gorakh S. Yellol, Antonio Donaire, Piotr Szumlas,
Patrick J Bednarski, Gamall Makhloufi, Christoph Janiak, Arturo Espinosa Ferao, and José Ruiz
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01194 • Publication Date (Web): 27 Aug 2015
Downloaded from http://pubs.acs.org on September 3, 2015
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Novel C,N-cyclometalated Benzimidazole Ruthenium(II) and
Iridium(III) Complexes as Antitumor and Antiangiogenic Agents:
a Structure-Activity Relationship Study
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†
†
†
†
Jyoti Yellol, Sergio A. Pérez, Alicia Buceta, Gorakh Yellol, Antonio Donaire, Piotr
†
#
Szumlas, Patrick J. Bednarski, Gamall Makhloufi, Christoph Janiak, Arturo
#
‡
‡
§
,†
Espinosa, and José Ruiz*
†
Departamento de Química Inorgánica and Regional Campus of International Excellence
“
Campus Mare Nostrum”, Universidad de Murcia, and Institute for Bio-Health Research of
Murcia IMIB-Arrixaca, E-30071 Murcia, Spain
#
Pharmaceutical and Medicinal Chemistry, Institut für Pharmazie, EMA-University of
Greifswald, D-17487 Greifswald, Germany
‡
Institut für Anorganische Chemie und Strukturchemie, Heinrich-Heine-Universität
Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
§
Departamento de Química Orgánica, Universidad de Murcia, E-30071 Murcia, Spain
ABSTRACT
A series of novel C,N-cyclometalated benzimidazole ruthenium(II) and iridium(III)
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2
5
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complexes of the types [(
(HL = methyl 1-butyl-2-arylbenzimidazole carboxylate) with varying substituents (H, Me, F,
CF , MeO, NO , and Ph) in the R position of the phenyl ring of 2-phenyl-benzimidazole
5 5
η -p-cymene)RuCl(κ -N,C-L)] and [(η -C Me )IrCl(κ -N,C-L)]
3
2
4
chelating ligand of the ruthenium (3a−3g) and iridium complexes (4a−4g) have been
prepared. The cytotoxic activity of the new ruthenium(II) and iridium(III) compounds have
been evaluated in a panel of cell lines (A2780, A2780cisR, A427, 5637, LCLC, SISO and
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HT29) in order to investigate structure−activity relationships. Phenyl substitution at the R
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position shows increased potency in both Ru and Ir complexes (3g and 4g, respectively) as
compared to their parent compounds (3a and 4a) in all cell lines. In general, ruthenium
complexes are more active than the corresponding iridium complexes. The new ruthenium
and iridium compounds increased caspase-3 activity in A2780 cells, as shown for 3a,d and
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4a,d. Compound 4g is able to increase the production of ROS in A2780 cells. Furthermore,
all the new compounds are able to overcome the cisplatin resistance in A2780cisR cells. In
addition, some of the metal complexes effectively inhibit angiogenesis in the human
umbilical vein endothelial cell line EA.hy926 at 0.5
ꢀM, being the ruthenium derivatives 3g
(
Ph) and 3d (CF ) the best-performers. QC calculations performed on some ruthenium model
3
complexes showed only moderate or slight electron depletion at the phenyl ring of the C,N-
cyclometalated ligand and the chlorine atom on increasing the electron withdrawing effect of
the R substituent.
INTRODUCTION
1
The discovery of the anticancer activity of cisplatin by Rosenberg et al. not only
transformed cancer chemotherapy and broadened the range of routinely applied
2
chemotherapeutics from organic drugs to metal-based compounds but also stimulated the use
3
of other metals in chemotherapeutics. Since then the potential of different metal-based
anticancer agents has been widely explored and especially Ru, Ir and Os complexes have
been widely studied in drug discovery.3−13 In the course of metal based anticancer drug
development programmes, organometallic, and especially half-sandwich metal complexes,
13−35
have increasing demonstrated their potential.
Their hydrophobic arene ligand is thought
to facilitate the diffusion through the cell membrane while the remaining coordinating sites
can be varied with different ligands to modulate biological and pharmacological
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properties.
In addition, angiogenesis is a hallmark of tumor development and metastasis,
36,37
and thus a possible target.
Furthermore, the limited survival benefits of antiangiogenic
drugs have stimulated the interest in the combination of antiangiogenic drugs with established
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chemotherapies.
Since the ruthenium compound NAMI-A demonstrated antimetastatic
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activity in vivo, more non-toxic ruthenium(II),
and iridium(III) complexes have also
been probed as angiogenesis inhibitors.
On the other hand, while recent development of Pt and Ru anticancer compounds were
carried out, it was envisaged that structural modifications in ligands around the metal center
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5,46
can modulate the anticancer activity of the complex.
Concomitantly, Sadler et al.
established a relationship between the hydrolysis rate for Ru(II) arene compounds and
47
anticancer activity.
Ru(II) complexes containing benzimidazole (a widely used
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52
pharmacophore
) exhibited outstanding potency in anticancer,
antiangiogenic, anti-
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3
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Alzheimer, anion-sensing and photophysical
studies. In addition, an ester functionality
was installed as a handle for intended functionalization of metallodrugs. Likewise, our
rationally designed neutral benzimidazole C,N-cyclometalated ruthenium(II) and iridium(III)
complexes exhibited good anticancer activity against HT29, T47D, A2780, and A2780cisR
4
6
cancer cell lines, compared to that of other relevant “piano‐stool” ruthenium(II) analogues
2
9,56
recently published.
An increasing interest for new half sandwich iridium C,N-
3
0
cyclometalated anticancer agents has been recently shown.
Thus, it was supposed that the electronic effect on the metal coordinated ligand might
influence the strength of metal−carbon or metal−nitrogen bond as well as the rate of metal
hydrolysis which, in the end, affects the anticancer activity of the complex. With this
hypothesis, we have designed the 2-phenyl-benzimidazole complexes where the phenyl ring
is substituted in the para position to study structure-activity relationship (SAR) of
benzimidazole analogs of ruthenium and iridium complexes. The influence of electron-
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donating or electron-withdrawing groups, aromatic and neutral substituents on 4-position of
phenyl ring of 2-arylbenzimidazole ruthenium/iridium complexes has been studied (Figure
1). SAR with respect to cytotoxicity, apoptosis and cell cycle arrest in a panel of human
cancer cells, and antiangiogenesis studies in the endothelial cell line EA.hy926 have been
investigated.
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Figure 1. Design of the 2-phenyl-benzimidazole ligand for preparation of metallodrugs.
RESULTS AND DISCUSSION
1. Synthesis and Characterization of the Cyclometallated Compounds
1.1. Molecular Design and Synthesis of the Modified 2-Arylbenzimidazole Ligands.
The key intermediate 1 was efficiently obtained from 4-chloro-3-nitro-benzoic acid by using
46,53
reported procedures
with a few modifications (Scheme 1). Acid catalyzed methyl
esterification, nucleophilic aromatic substitution of the chloro group by butyl amine followed
by reduction of the nitro group with zinc and ammonium formate affords 1 in an overall 60%
yield. The final ligands were synthesized by condensation of intermediate 1 with
appropriately substituted benzaldehyde in ethanol with catalytic amount of trifluoroacetic
acid at room temperature for 16−24 h (Scheme 1). Methyl substitution was preferred initially
to understand the electronically neutral but space filling substituent effect. Comparatively, a
methoxy group as a strong electron donating agent and a nitro group as a strong electron
withdrawing group were considered to determine related effect on activity through
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physiological effect. On the other hand, fluoro and trifluoromethyl groups were also chosen
for SAR studies as fluorine can considered "the medicinal chemist's magic element”. In fact,
fluorine is the most electronegative of the elements, but is also relatively small in size, which
means that replacing for example a hydrogen atom with a fluorine atom can have in general
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profound electronic effects on a molecule without greatly changing its overall size.
In
addition, fluorine increases lipid solubility (bioavailability) and strength C−F bonds are
resistant to metabolic processes.
All the ligands with different substitutions were synthesized with same condensation
method with a 50−76% yield.
O
O
H N
2
NO2
O
NEt , CH Cl , RT, 12h
3 2 2
NH2
O
Zn, HCOONH4,
Cl
N
H
MeOH:EtOAc,
RT, 10 h
1
O
O
O
H
N
N
R
R
TFA, MgSO ,EtOH
4
RT, 24 h
R = 2a, H;
2b, CH3;
2
2
2
c, F;
2d, CF3;
e, OCH ; 2f, NO ;
3
2
g, Ph
Scheme 1. Synthesis of main ligands for SAR.
1
.2. Synthesis of the New Cyclometalated Complexes. Stimulated by the initial results
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derived from the introduction of negatively-charged C,N-chelated benzimidazole ligands,
the synthesis of new cyclometalated benzimidazole ruthenium(II) and iridium(III) complexes
was considered for SAR studies. All the ruthenium complexes were synthesized using the
generalized procedure shown in Scheme 2. The benzimidazole ligand 2a−2g was treated with
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2 2
para-cymene ruthenium(II) [(p-cymene)RuCl ] and sodium acetate in dichloromethane at
room temperature for 24 h to obtain the corresponding ruthenium metal complex (3a−3g) in
moderate to good yield. The structures of the new ruthenium complexes were confirmed by
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spectroscopic methods. In the H NMR spectra of 3a−3g the disappearance of one aromatic
proton of the 2-arylbenzimidazole ligand is observed (Supporting Information, Figure S1 for
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3d). In addition, resonances of the
η
-p-cymene protons were resolved to distinct peaks due
to desymmetrization of the arene ligand upon complexation of the anionic chelating
benzimidazole ligand at the Ru center. The positive-ion ESI-MS spectra displayed the [M +
+
1
] peaks with the expected isotopic distribution pattern (Supporting Information, Figure S2
for 3d). Similarly, the half-sandwich iridium(III) complexes (4a−4g) were prepared using a
similar method starting from the corresponding pentamethylcyclopentadienyl chlorido
iridium(III) in good yields. The structures were also established by spectroscopic and
analytical methods, including COSY, HSQC NMR, and ESI-MS techniques (Supporting
Information, Figures S3−S7 for 4d). The new compounds are soluble in solvents such as
3
CDCl , DMSO or DMF.
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O
N
N
O
R
2
a-g
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O
Ru Cl
O
Ir Cl
N
N
O
N
N
R
O
R
3
3
3
a. H
4a. H
b. CH3
c. F
4b. CH3
4c. F
3d. CF3
4d. CF3
4e. OMe
3e. OMe
3
f. NO2
4f. NO2
3
g. Ph
4g. Ph
Scheme 2. Synthesis of metal complexes.
1.3. Structures Determination. Single crystals suitable for X-ray diffraction analysis were
2
obtained from the slow diffusion of hexane into a saturated solution of 3d and 4d in CH Cl /
2
toluene. Their structures and atom numbering schemes are shown in Figure 2.
Crystallographic data are shown in Tables S1 and S4 in the Supporting Information, and
selected bond lengths and angles are listed in Tables S2 and S5 in the Supporting
Information. The unit cell of 3d shows the two possible enantiomers (R and S ) resulting
Ru
Ru
from the stereogenic nature of the metal center. As shown in Figure 2a, complex 3d adopts
the classical pseudooctahedral three-legged piano-stool arrangement and, hence, the arene
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ring displays the common π-bonded
η
-coordination mode, whereas the arylbenzimidazole-
2
type ligand assumes a bidentate chelate coordination mode (
κ
-C,N), occupying two
coordination positions. Both Ru−cymcentroid distance for 3d [1.712(1) Å] and Ru−Cl bond
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distance [2.4191(4) Å] are standard compared to alike complexes. The orthometalated
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Ru−C distance [Ru−C = 2.0508(18) Å] is shorter than Ru−N [Ru−N = 2.0666(14) Å] but
similar to the Ru−C distances of related complexes. These features result from the strong σ-
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donor nature of the negatively charged C atom. The packing in the structure of 3d is
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organized by intermolecular C−H···F, C−H···Cl, C−H···O, and C-H···
π interactions,
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(
Supporting Information, Table S3 and Figure S8). There are no interactions.
π-π
Figure 2. Molecular structures with atom numbering schemes for (a) 3d (50% thermal
ellipsoids) and (b) 4d (50% thermal ellipsoids). Selected bond lengths (Å) and angles (deg)
for 3d: Ru−C29 = 2.0508(18), Ru−N1 = 2.0666(14), Ru−Cl = 2.4191(4), Ru−cym(centroid)
= 1.712(1), C29−Ru−N1 = 76.64(6), N1−Ru−Cl = 86.77(4), C29−Ru−Cl = 87.68(5),
cym(centroid)−Ru−Cl
=
128.26(6),
cym(centroid)−Ru−N1
=
131.30(7),
cym(centroid)−Ru−C29 = 129.02(7). Selected bond lengths (Å) and angles (deg) for 4d:
Ir−C29 = 2.059(14), Ir−N1 = 2.060(11), Ir−Cl = 2.406(3), Ir−Cp*(centroid) = 1.81(2),
C29−Ir−N1 = 76.8(5), N1−Ir−Cl = 85.6(3), C29−Ir−Cl = 88.7(4), Cp*(centroid)−Ir−Cl =
125.6(7), Cp*(centroid)−Ir−N1 = 133.1(7), Cp*(centroid)−Ir−C29 = 130.4(7).
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Figure 2b depicts the ORTEP diagram of complex 4d, confirming its “piano-stool”
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structure with the pentamethylcyclopentadienyl group displaying the common
π-bonded η
coordination mode, whereas the 1-butyl-2-phenyl-benzimidazole carboxylate assumes a
bidentate-chelate coordination mode (κ2
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-C,N), the two rings of the benzimidazole and
phenyl moieties being not strictly coplanar. The orthometalated Ir−C distance [2.059(14)] is
similar to that of Ir−N 2.060(11) Å. The Ir−centroid distance for 4d is 1.81(2) Å. The Ir Cl
−
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bond distance [2.406(3) Å] is similar to that previously observed [2.4050(11) Å] for 4a.
1.4. Electrostatic Potential Surface. With the aim of exploring the little influence
observed in activities upon peripheral substitution at the C,N-cyclometalated ligand, QC
calculations were performed on ruthenium model complexes 3'a-b,d featuring a methyl
(instead of butyl) group at N, as well as an electron donating (3'b, R = CH ) or withdrawing
3
group (3'd, R = CF
3
), or none (3'a, R = H) at the derivatization site. Electrostatic potential
(ESP) isocontour plots at the plane orthogonal to the Ru-Cl bond and containing the
ruthenium atom (Figure 3a-c) show little differences in the proximity of the metal atom
among the three substitution patterns, in agreement with the roughly same natural charge at
N
Ru (q
= 0.275, 0.275 and 0.273
e
, for 3'b, 3'a and 3'd, respectively). Also, mapping of ESP
at a electron density isosurface (0.04 au) for the same three model complexes (Figure 3d-f)
evidence systematic higher electron density at the formally C-anionic phenyl ligand in
comparison to the N-donor benzoimidazole ring system. Main observable differences along
the series 3'b/3'a/3'd consist of moderate or slight electron depletion at the phenyl ring and
the chlorine atom on increasing the electron withdrawing effect of the R substituent, which
N
parallels the decrease of negative charge at Cl (
q
= -0.495, -0.494 and -0.489
e
, for 3'b, 3'a
and 3'd, respectively) therefore resulting in a slightly less-polarized stronger Ru−Cl bond
(WBI= 0.521, 0.521 and 0.525, ρ(r) = 0.0740, 0.0742 and 0.0746 au, for 3'b, 3'a and 3'd,
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respectively). The p-cymene ligand buffers most of the electron effect of the substituent, as
N
observed by the larger charge differences along the (p-cymene)RuCl moiety: q = 0.015,
0.017 and 0.041 e, for 3'b, 3'a and 3'd, respectively.
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9
0
Figure 3. Computed (B3LYP-D3/def2TZVPPecp) ESP contour plots (0.005, 0.01, 0.05,
0.1, 0.5 and 1 au isovalues) at the plane containing the metal and orthogonal to the Ru-Cl
bond, for a) 3'b, b) 3'a and c) 3'd. Second row (d-f): ESP maps (from -0.02 au in red, to
+0.25 au in blue) drawn onto an electron density isosurface (0.04 au) for the same
compounds.
1.5. Hydrolysis Studies. The easy hydrolysis of some metal half-sandwich complexes has
35,65
been previously reported.
Methanol was required to ensure the solubility of the
1
complexes in aqueous solution. The H NMR spectra of complex 3b (ca. 0.5 mM) in MeOD-
d and in 25% MeOD-d /75% D O (v/v) at 37 °C are shown in Figures 4A and 4B,
4
4
2
respectively, indicating that hydrolysis took place even in the presence of adventitious water.
On the other hand, the ESI-MS of 3b in 25% v/v methanol/water (in the presence of
ammonium formiate) show a set of peaks centered at m/z 557.17 showing the Ru isotope
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pattern, which is compatible with the formation of the aqua ruthenium(II) complex ([M –
+
H
2
O] ). In order to check the reversibility of the hydrolysis, NaCl (0.5 mM and 1 mM) was
4 2
added to two different samples of solution of 3b in MeOD-d /D O, however the same pattern
of signals than in Figure 4B was again observed (Figures 4C and 4D, respectively), and
abundant precipitation of the complexes was observed. Similar behavior was found when 3b
and 3g were dissolved in 25% MeOD-d /75% D O (v/v). No iridium complexes were studied
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4
2
by NMR due to their low solubility.
1
Figure 4. H NMR (600 MHz) spectra of complex 3b (ca. 0.5 mM) in (A) MeOD-d
4
(
containing adventitious water) and (B) 25% MeOD-d /75% D O (v/v) after 10 min at 37 °C.
4
2
Addition of NaCl 0.5 mM (C) and NaCl 1 mM (D), respectively, to two different samples of (B).
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1.6. Relative Hydrophobicity. The RP-UPLC of complexes 3b,c,g (ca. 10 µM) dissolved
in 10% v/v methanol in water (to ensure solubility of the complexes) have been undertaken in
order to determine the relative hydrophobicities (the mobile phases used were A = H
2
O with
0
.2% HCOOH and B = acetonitrile). The more hydrophobic complexes should have longer
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R
retention times (t ). As expected the order of retention time follows (Figure 5) the
sequence: 3c < 3b < 3g. The QTOF-MS of the ∼5.9 min of 3g shows the ruthenium isotopic
+
pattern at m/z 561.15, which could assigned to [3g − Cl] or to the loss of H
2
O from the
corresponding aqua-ruthenium complex (see Section 1.5). A similar behaviour was observed
for 3b and 3c.
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Figure 5. RP-UPLC chromatograms with UV detection at 320 nm of 10 µM 3b,c,g
solutions in mobile phase. Mass spectra for UPLC fractions also shown for 3c and 3g. An
Acquity UPLC BEH C18 1.7 µm particle size analytical column 50 mm × 2.1 mm (Waters)
was used. The mobile phases used were A = H O with 0.2% HCOOH and B = acetonitrile.
2
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The percentage of organic modifier (B) was changed linearly as follows: 0 min, 20%; 1 min,
30%; 8 min, 99%; 8.50 min, 99%; 9.50 min, 20%. Curves were obtained within 4 h after
preparation of the 3b,c,g solutions at room temperature.
2. Biological Activity
2.1. Cytotoxicity Studies. The cytotoxicity of the new complexes 3a−3g and 4a−4g was
evaluated towards a panel of human cancer cell lines, including A2780 (human ovarian
cancer cells), A2780cisR (acquired resistance to cisplatin, CDDP), 5637 (human bladder
cancer cells), A427, LCLC-103H (both human lung carcinoma cells), SISO (human uterine
cervical adenocarcinoma cells), and HT29 (human colorectal adenocarcinoma cells). Due to
the low aqueous solubility of the complexes, the tested compounds were dissolved in DMF
first and then serially diluted in complete culture medium such that the effective DMF
66
content did not exceed 0.4%, as previously described. CDDP, diluted in water, was used as
positive control. Table 1 reports the IC50 values of the compounds. On the other hand, IC50
values for 3a−3g and 4a−4g in the human umbilical vein endothelial EA.hy926 cell line were
also calculated (Table 1) with the aim to verify that the anti-angiogenic effect (vide infra) was
not due to a cytotoxic effect but rather to their anti-angiogenic potential.
Table 1. IC50 (µM) for 2a, 3a−3g and 4a−4g and Cisplatin at 48 h. Results are
averages ± standard errors deviations of three independent experiments.
Compound
A2780
A2780cisR
2.37 ± 0.10
1.46 ± 0.14
3.26 ± 0.32
5637
A427
LCLC
SISO
HT29
EA.hy926
7.05 ± 1.07
5.49 ± 1.57
> 8
3a
1.82 ± 0.35
1.48 ± 0.35
1.36 ± 0.07
2.90 ± 0.11
2.53 ± 0.57
1.87 ± 0.32
2.58 ± 0.64
2.99 ± 0.24
1.85 ± 0.29
3.52 ± 0.58
3.50 ± 0.40
2.16 ± 0.20
3.92 ± 0.29
3.73 ± 0.34
2.05 ± 0.09
2.88 ± 0.55
3.31 ± 0.04
2.74 ± 0.11
3
b
3
c
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3
d
1.24 ± 0.45
1.56 ± 0.48
1.30 ± 0.45
1.07 ± 0.34
1.78 ± 0.85
3.60 ± 0.28
1.98 ± 0.23
2.18 ± 0.22
2.52 ± 0.16
0.96 ± 0.23
2.59 ± 0.42
5.65 ± 0.78
1.25 ± 0.17
2.27 ± 0.23
2.66 ± 0.19
1.56 ± 0.50
5.56 ± 0.25
1.28 ± 0.07
1.84 ± 0.60
1.47 ± 0.18
1.24 ± 0.07
3.78 ± 0.41
1.41 ± 0.40
2.45 ± 0.46
2.32 ± 0.03
1.31 ± 0.08
9.86 ± 1.04
1.39 ± 0.21
2.34 ± 0.46
2.28 ± 2.26
1.82 ± 0.17
1.93 ± 0.07
2.76 ± 0.31
2.35 ± 0.27
1.99 ± 0.08
1.37 ± 0.38
3.35 ± 0.04
5.10 ± 0.25
7.21 ± 0.04
1.67 ± 0.03
2.85 ± 0.10
> 8
3
e
3
f
3
g
4a
5.49 ± 0.93
13.87 ±
0.17
4
b
0
1
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0
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2
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9
0
9
.37 ± 0.53
7.25 ± 0.28
4.28 ± 0.55
2.62 ± 0.56
2.35 ± 0.62
2.34 ± 0.06
1.59 ± 0.34
> 50
15.14 ± 0.49
5.58 ± 0.33
6.63 ± 0.88
5.10 ± 0.33
3.47 ± 0.13
3.61 ± 0.21
> 20
8.68 ± 0.92
9.50 ± 0.75
3.77 ± 0.31
6.27 ± 0.69
5.73 ± 0.43
4.01 ± 0.56
> 25
4
c
2.31 ± 0.39
2.70 ± 0.13
2.78 ± 0.12
2.29 ± 0.46
1.22 ± 0.41
> 15
3.37 ± 0.41
1.93 ± 0.15
1.70 ± 0.38
3.76 ± 0.64
1.21 ± 0.48
> 15
2.95 ± 0.71
2.45 ± 0.10
3.70 ± 0.09
5.15 ± 0.12
1.48 ± 0.88
> 20
4.76 ± 0.87
6.65 ± 0.04
3.42 ± 0.26
3.61 ± 0.93
3.79 ± 0.31
> 20
2.99 ± 0.09
5.22 ± 0.88
1.19 ± 0.05
1.98 ± 0.40
5.95 ± 0.92
> 15
4d
4
e
4
f
4
g
2a
CDDP
1.90 ± 0.20
19.57 ± 1.82
3.39 ± 0.05
6.09 ± 1.49
1.92 ± 0.54
0.98 ± 0.07
7 ± 0.07
9.86 ± 0.64
The cell growth inhibitory potency of all the metal complexes was noticeably much higher
than their parent ligands (e.g., compared with 2a). Most of the complexes were even more
active than CDDP in A427 and HT29 cell lines. As depicted in Table 1 we investigated the
3 2 4
effect of H, Me, F, CF , MeO, NO , and Ph substituents in the R position of the phenyl ring
of the 2-phenyl-benzimidazole chelating ligand of the ruthenium (3a−3g) and iridium
complexes (4a−4g) on the cytotoxicity. Methyl substitution produces no effect on potency
compared to the unsubstituted compound (3a) in ruthenium series but it loses some potency
in iridium complexes for most of cell lines. The “magic” fluoro influence was observed with
both Ru and Ir complexes, where activity of fluoro-substituted complex was enhance
somewhat in all studied cell lines except the HT29 cell line for iridium 4c. Trifluoromethyl
substitution enhances the activity of Ru complex 3d in almost all the cell lines while for the
iridium complex 4d there was no effect on potency in LCLC, SISO and HT29 cell lines.
Activity enhances by electron donating methoxy substitution in A427, LCLC and SISO cell
lines for the ruthenium complex 3e. Similarly, electron donating nitro substitution also
increases potency of 3f in A427, LCLC and SISO cell lines as compared to the parent
ruthenium complex 3a, while it shows mixed behaviour in the iridium complexes with
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respect to all cell lines. Certainly no ample discrepancy was detected with donating electronic
effect as compared with withdrawing electronic effect in ruthenium complexes taking into
account all studied cancer cell lines. Interestingly, phenyl substitution shows increased
potency in both Ru and Ir complexes (3g and 4g, respectively) as compared to their parent
compounds in all cell lines. In general, ruthenium complexes are more active than the
corresponding iridium complexes.
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2
.2. Complexes 3a−3g and 4a−4g Overcome the Acquired Resistance to CDDP. As it is
well known A2780cisR encompasses all of the known major mechanisms of resistance to
67
cisplatin: reductions in intracellular of drug levels, enhanced DNA repair and/or increased
damage tolerance, elevated cellular thiol (GSH) level, and failure of cell-death pathways. The
ability of the compound to overcome the acquired resistance to cisplatin was determined from
the resistance factor, RF, defined as the ratio between the IC50 values of the resistant and the
sensitive lines. An RF of <2 is considered as the limit beyond which the compound denotes
6
8
non-crossresistance. In general, all the new compounds studied here are able to overcome
cisplatin resistance; their RF are much lower than the RF of cisplatin (Figure 6). As it has
been recently shown by using a tracer technique, the ruthenium complex 3a is able to reduce
the uptake and increase the release of the organic osmolyte taurine in Ehrlich Lettre´ Ascites
5
0
(ELA) cells, with innate resistance to CDDP, and in A2780 human ovarian cancer cells.
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Figure 6. Resistance factors, RF, for CDDP and all the complexes studied toward CDDP-
resistant A2780 human ovarian cancer cells, A2780cisR.
2.3. Accumulation Studies. The cellular accumulation of the Ru(II) complexes 3b,c,g in
the A2780 ovarian cancer cell lines was studied to investigate the possible relationship
between cellular uptake and cytotoxicity. Cellular concentrations were determined by AAS
after 24 h of exposure to 3b, 3c and 3g at 2 ꢀM. The results are summarized in Figure 7. As
shown, for these compounds a direct relationship between cellular-ruthenium accumulation in
A2780 ovarian cells and cytotoxicity is not observed.
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3
b
3g
3c
Figure 7. Intracellular ruthenium accumulation in A2780 cells at 24 h incubation with
complexes 3b, 3c and 3g. Experiments were made in triplicate.
2.4. DNA-bound metal in cells. Iridium and ruthenium levels on nuclear DNA were
determined after the exposure of A2780 cells to 2 µM complexes 3g and 4g for 24 h. The
iridium content of nuclear DNA extracted from the A2780 cells treated with 4g was found to
be 0.74 ± 0.05 pg Ir per ng DNA, which is a very small value compared to that of the iridium
5
+
complex [(η -C
5
Me
4
C
6
H
4
C
6
H
5
)Ir(phen)Cl] (phen = phenanthroline), which has been shown
69
to cause both nuclear DNA damage and mitochondrial dysfunction in ovarian cancer cells.
The ruthenium content of nuclear DNA extracted from the A2780 cells treated with 3g was
found to be also low (1.1 ± 0.09 pg Ru per ng DNA), a result that agrees with the intracellular
distribution observed for the ruthenium parent compound 3a in Ehrlich Lettre´ Ascites (ELA)
cells, with an elevated content of 3a in the cytosol (78 %) compared to nuclei and
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2.5. Apoptosis Studies. Apoptotic studies of ruthenium complexes 3d and 3f, and iridium
complexes 4d and 4f were carried out with 5637 and SISO cell lines. Exposure of
phosphatidylserine (PS) to the outer side of the cell membrane, an early sign of apoptosis,
was followed by a flow cytometric assay based on the Annexin V-FLUOS, propidium iodide
(PI) method, which also can identify necrotic or late apoptotic cells. The results are shown in
Figure 8. The four typical quadrants identifying the living (lower left quadrant D3, not
stained cells), the early apoptotic (lower right quadrant D4, only the annexin-V stained cells),
the necrotic (upper left quadrant D1, only PI stained cells), and the late apoptotic (upper right
quadrant D2, cells stained with both fluorescent dyes) cells appear in these diagrams. As
depicted in Figure 8, it is clear that complexes 3d, 3f, 4d and 4f all induce a high incidence of
early to late stage apoptosis in both 5637 and SISO cells at 24 h without increasing necrotic
population.
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Figure 8. Flow cytometry analysis of 5637 and SISO cancer cells after treatment with 3d, 3f,
4d and 4f as detected by annexin V/PI. Density plots for untreated cells (control) and for cells
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treated with 5 µM (24 h) of 3d, 3f, 4d and 4f. The experiments were performed in triplicate.
2.6. Enhanced Activation of Caspase 3 by Complexes 3a,d and 4a,d. Caspases, in
general, are a family of cysteine proteases that play essential roles in apoptosis, necrosis, and
70
inflammation. Colorimetric methods can be used to measure its activation when the DEVD
71
sequence is labeled with p-NA. As shown in Figure 9, complexes 3a,d and 4a,d increased
caspase-3 activity in A2780 cells (monitored spectrophotometrically using the substrate
DEVD-pNA).
Figure 9. Caspase 3 activation in A2780 ovarian cancer cells caused by 24 h exposure to
complexes 3a,d and 4a,d at 37 °C. Results are expressed as absorbance at 410 nm (detection
of p-nitroaniline after cleavage from the labelled substrate DEVD-pNA). Concentrations of
the complexes used were 2
be statistically significant.
ꢀM. Cont: control cells not treated. *p < 0.05 was considered to
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2.7. Cell Cycle Arrest. To understand the effect of complexes 3d and 3f on cell growth
their effect on the cell cycle by FACS (Fluorescence Activated Cell Sorter) analysis in 5637
and SISO cancer cells was examined. After treatment of 5637 cell lines with 5
ꢀM of 3d and
3
f for 24 h (Figure 10 and Supporting Information, Figure S10) we found different
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behaviours of compounds within the same as well as different cell lines. Compound 3d
arrests the cycle in the G2/M phase in both cell lines. However, complex 3f arrests the cell
cycle in the G0/G1 phase of 5637 cells but arrests the cell cycle in G2/M phase in the SISO
cell line.
Figure 10. Cell cycle analysis of 5637 and SISO cancer cells after 24 h treatment with 1
of compounds 3d and 3f at 37 °C.
ꢀM
2
.8. Intracellular Reactive Oxygen Species (ROS) determination. 2’,7’-
dichlorodihydrofluorescein diacetate (DCFH-DA) was used to measure the effect of 3g and
g and, for comparative purposes, also of cisplatin on the production of ROS by A2780 cells.
The DCFH-DA method is designed to provide a highly sensitive, quantifiable, real-time
4
6
9,72,73
assessment of ROS production.
To evaluate ROS formation, A2780 cells untreated or
treated with 3g, 4g or cisplatin were incubated with 25 µM DCFH-DA for 30 min in the dark.
DCFH-DA is cleaved by cellular esterases, oxidized by ROS and yields a fluorescent
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product. After incubation, the resulting fluorescence was measured and the data are plotted in
Figure 12.
Treatment of A2780 cells with 4g at 2 ꢀM resulted in an increase of
fluorescence, indicating the production of ROS.
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Figure 11. Generation of ROS in A2780 cells induced by various concentrations of 3g, 4g
or cisplatin at concentrations of one ROS 3g, 4g and cisplatin. *p < 0.05 was considered to
be statistically significant. Experiments were made in triplicate.
3. Interaction with biomacromolecules
3.1. Interaction of Ruthenium Complex 3d with HSA. Reactions of anticancer
metallodrugs with proteins are of considerable interest as these interactions might feature
processes that are crucial for the biodistribution, the toxicity, and even the mechanism of
action. HSA is the most abundant of the blood serum proteins and serves as a transport
vehicle for a wide variety of ligands, including metal ions and various pharmaceuticals.32,74−77
It comprises a single chain with 585 amino acids, including a single tryptophan residue (Trp
214) responsible for most of the intrinsic fluorescence of the protein. Upon excitation at 295
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nm, HSA strongly fluoresces at λ = 350 nm, which can be attenuated by binding of a small
molecule at or near local tryptophan environment. The HSA emission spectra in the presence
of different concentrations of the benzimidazole complexes were recorded in the wavelength
range 300−580 nm by exciting at 295 nm. As seen in Figure 6, the fluorescence intensities of
the protein are increasingly decreased with the increasing concentration of the probed
compound, indicating the binding of the complex to the protein. Furthermore, the
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fluorescence quenching mechanism can be described by the Stern−Volmer equation (Eq. 1),
where F and F represent the fluorescence intensities in the absence and the presence of a
0
quencher at a given quencher concentration, respectively. KSV and [Q] are the Stern–Volmer
quenching constant, and the quencher concentration, respectively.
F
0
/F = 1 + KSV[Q]
(1)
0
For a homogeneously emitting solution eq 1 predicts a linear plot of F /F vs [Q] (Table 2
and Supporting Information, Figure S11).
For a static quenching process, the association constant (K
sites (n) can be calculated using eq 2.
A
) and the number of binding
Here F and F are the fluorescence intensities in the absence and presence of complexes,
0
respectively; [Q
Supporting Information, Figure S12 shows the plots of log (F
F)[P ]/F )} for the interaction between HSA and complex 3d. K
A
t
] and [P
t
] are the total concentrations of complexes and HSA, respectively.
F)/F versus log {1/([Q
and n obtained from
0
−
t
] −
(F
0
−
t
0
the plots and KSV are listed in Table 2.
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3.2. Site-Selective Binding of Complex 3d on HSA. There is growing interest in the
nature of binding of metallodrugs to biomolecules in order to improved the design of next-
generation drug candidates. To identify the binding site location of benzimidazole metal
complexes on the region of HSA, competitive binding experiments were carried out by using
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warfarin (WF), a characteristic marker for site I and dansyl glycine (DG) as one for site II.
The weak warfarin fluorescence is enhanced upon binding with HSA because of its
interaction with Trp214 in HSA when excited at λ = 320 nm. Likewise, the fluorescence of
warfarin in its bound state to HSA decreases if a second ligand competes for the same site.
Complex 3d binding site has been evaluated by monitoring the changes in the fluorescence
spectra as a function of the gradual increase in complex 3d concentration in an HSA−WF-
complex solution. Figure 13B depicts a decrease in the emission intensity of warfarin when
complex 3d is added. The quenching of warfarin reflects a release of warfarin from HSA; i.e,
both warfarin and complex 3d compete for the same binding site. Therefore, we conclude that
3
d binding occurs at least in part at the warfarin site I. Similarly, a regular decrease in the
HAS−DG fluorescence when complex 3d concentrations are increased also indicates the
binding of complex to the protein at the dansyl glycine site II.
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Figure 13. Emission spectra of HSA, HSA−WF and HSA−DG in presence of increasing
amounts of complex 3d. λex = 295 nm, [HSA] = 5.0 µM; λex = 320 nm, [HSA−WF] = 5.0: 5.0
µM; λex = 320 nm, [HSA−DG] = 5.0: 5.0 µM; [Complex 3d] : 0−50 µM (top to bottom
gradual increments). Temperature = 296 K. pH 7.4.
Table 2. Quenching and Binding Parameters for the interaction of
complex 3d with HSA, HSA−WF, and HSA−DG at 298 K.
-4
−1
-4
−1
Study
10 K (M )
R
10 K (M )
n
SV
A
HSA
3.33
3.85
2.82
0.9979
0.9989
0.9808
6.46
5.69
4.14
1.13
1.54
1.38
HSA−WF
HSA−DG
3.3. Hoechst 33258 Displacement from DNA by Ruthenium Complex 3d. In order to
clarify the binding mode between the new metal complexes and DNA, a fluorescence
competition experiment with Hoechst 33258 was employed. This ligand is able to bind via
electrostatic interactions to the minor groove of duplex DNA (with marked preference for
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AT-rich regions) at high DNA/Hoechst ratio. Due to the fluorescence yield of Hoechst
3
3258 increasing significantly in the presence of DNA, an intensity reduction with complexes
addition is interpreted as the displacement of Hoechst bound to ct-DNA. When complex 3d is
added to a Hoechst-DNA solution a decrease (∼81%) is observed in the fluorescence as
shown in Figure 14 and Supporting Information (Figure S13). This suggests that complex 3d
is capable of weakly binding to the minor groove of DNA.
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Figure 14. A) Changes in fluorescence spectra of Hoechst−ctDNA in presence of increasing
amounts of complex 3d.
λex = 450 nm, [DNA] = 10 µM; [Complex 3d]: 0−150 µM (top to
bottom gradual increments). 296 K.
4
. Inhibition of Tube Formation by Benzimidazole Metal Complexes in EA.hy926
Endothelial Cells
Angiogenesis is often assessed by the ability of endothelial cells to sprout, migrate and
form vascular tubes in vitro on Matrigel matrix, a gelatinous protein mixture secreted by
3
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Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.
In vitro angiogenesis was measured 16 h after incubating EA.hy926 cells with compounds
a−3g and 4a−4g at concentrations 0.5 µM (Figure 15). The low concentration was used in
3
order to work at its sub-cytotoxic concentration in EA.hy926 endothelial cells (Table 1). The
resulting formation or inhibition of capillary-like structures was quantified (Figure 15a) by
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using a combination of the three most representative parameters: total length (µm), number of
meshes (network or polygonal structures) and total meshes area (area occupied by the
2
meshes, µm ). Only those compounds that gave significant differences in all the studied
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5
parameters were considered antiangiogenic. At 0.5 µM all complexes inhibit the formation
of vascular tubes. Compounds 3d and 3g are the most active angiogenic inhibitors in
EA.hy926 cells at sub-cytotoxic concentrations, and at the same time they are also strongly
cytotoxic in the assayed cancer cell lines (Table 1). Thus, we conclude that 3d and 3g are
dual cytotoxic and antiangiogenic compounds.
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Figure 15. Effect of the benzimidazole metal complexes in the endothelial cell tube
formation assay. (a) Typical images after an ImageJ process of EA.hy926 cells added to 24-
well plates precoated with Matrigel for 16 h: for culture medium as a control and with 3d, 0.5
µM. Tube formation of EA.hy926 cells were photographed under an inverted phase-contrast
microscope. (b) Quantification after the ImageJ process of number of meshes, total meshes
area, and total length of the tubular structure of EA.hy926 cells under 0.5 µM of compounds
3a−3g and 4a−4g and CDDP for 16 h. Error bars represent the standard deviation of three
independent experiments. *p < 0.05 was considered to be statistically significant.
CONCLUSIONS
We achieved a successful divergent synthesis of novel C,N-cyclometalated benzimidazole
6
2
ruthenium(II) and iridium(III) complexes of the types [(
η
-p-cymene)RuCl(
-N,C-L)] (HL = modified 2-arylbenzimidazole) with various substituents
, MeO, NO , and Ph) in the R position of the phenyl ring of the 2-phenyl-
κ -N,C-L)] and
5
2
5 5
[(η -C Me )IrCl(κ
(
H, Me, F, CF
3
2
4
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benzimidazole chelating ligand of the ruthenium (3a−3g) and iridium complexes (4a−4g).
1
Hydrolysis of the ruthenium-chlorido bond is relatively rapid as observed in 3b,c,g by H
NMR in MeOD-d
4 4 2
(containing adventitious water) and 25% MeOD-d /75% D O (v/v) at 37
°C. The cytotoxic activity of the new ruthenium(II) and iridium(III) compounds has been
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evaluated in a panel of human cancer cell lines (A2780, A427, 5637, LCLC, SISO and HT29)
in order to establish a structure−activity relationship in these families of compounds. Most of
the new compounds are more active than CDDP in A427 and HT29 cell lines. Phenyl
substitution shows increased potency in both Ru and Ir complexes (3g and 4g, respectively)
as compared to their parent compounds in all cell lines. The relative hydrophobicities,
according to RP-UPLC-QTOF-MS studies, follow the sequence: 3c < 3b < 3g. In general,
ruthenium complexes are more active than the corresponding iridium complexes. The new
ruthenium and iridium compounds increased caspase-3 activity in A2780 cells, as shown for
3a,d and 4a,d. Furthermore, all the new compounds are able to overcome the cisplatin
resistance in A2780cisR cells. Compound 4g is able to increase the production of ROS in
A2780 cells at 2 ꢀM. The metal content of nuclear DNA extracted from the A2780 cells
treated with 3g and 4g was found to be low. In addition, some of the metal complexes
effectively inhibit angiogenesis in the human umbilical vein endothelial cell line EA.hy926 at
3
0.5 ꢀM, with the ruthenium derivatives 3g (Ph) and 3d (CF ) being the best inhibitors. Tube
formation in EA.hy926, as quantified with the ImageJ software, has also been observed. QC
calculations performed on ruthenium model complexes 3'a,b,d showed only moderate or
slight electron depletion at the phenyl ring of the C,N-cyclometalated ligand and the chlorine
atom on increasing the electron withdrawing effect of the R substituent, which explains the
little influence observed in activities upon peripheral substitution at the C,N-cyclometalated
ligand. Complex 3d is also capable of weakly binding to the minor groove of DNA and
interacts with HSA both at the warfarin site I and dansyl glycine site II. Thus, these novel
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Ru(II) and Ir(III) organometallic compounds possess a variety of interesting biological effects
that make them attractive as potential anticancer drugs.
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EXPERIMENTAL SECTION
General Methods and Starting Materials. The C, H and N analyses were performed with
1
13
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a Carlo Erba model EA 1108 microanalyzer. The H, C and F spectra were recorded on a
Bruker AC 300E, Bruker AV 400 or Bruker AV 600 NMR spectrometer. Chemical shifts are
1
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4 3
cited relative to SiMe ( H and C, external) and CFCl ( F, external). ESI mass (positive
mode) analyses were performed on a HPLC/MS TOF 6220. The isotopic distribution of the
heaviest set of peaks matched very closely that calculated for the formulation of the complex
cation in every case. UV/vis spectroscopy was carried out on a Perkin Elmer Lambda 750 S
spectrometer with operating software. Fluorescence measurements were carried out with a
Perkin Elmer LS 55 50 Hz Fluorescence Spectrometer.
All synthetic manipulations were carried out under an atmosphere of dry, oxygen-free
6
nitrogen using standard Schlenk techniques. Solvents were dried by the usual methods. [(η -
5
p-cymene)RuCl ] , [(η -C Me )IrCl ] , substituted benzaldehydes, trifluoroacetic acid,
2 2
5
5
2 2
magnesium sulfate, sodium sulfate, sodium salt of calf thymus DNA, ethidium bromide (EB),
Hoechst 33258 and human serum albumin (HSA) were obtained from Sigma-Aldrich
(
Madrid, Spain). Deuterated solvents were obtained from Euriso-top. The purity of all
biologically evaluated molecules, based on elemental analysis, is >95%.
Preparation of Compound 1. 4-Chloro-3-nitrobenzoic acid was dissolved in methanol.
2 4
Concentrated H SO (1 mL) was added dropwise in reaction mixture. The reaction mixture
was refluxed overnight (18 h), allowed to cool and then concentrated in vacuo. The resulting
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