On the antitumor properties of novel cyclometalated benzimidazole Ru(ii), Ir(iii) and Rh(iii) complexes

Article abstract of DOI:10.1039/c3cc46239k

Smart design and efficient synthesis of benzimidazole Ru, Ir and Rh cyclometalated complexes are reported with promising cytotoxic activity against HT29, T47D, A2780 and A2780cisR cancer cell lines. Their apoptosis, accumulation, cell cycle arrest, protei

Full text of DOI:10.1039/c3cc46239k

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On the antitumor properties of novel cyclometalated
benzimidazole Ru(II), Ir(III) and Rh(III) complexes†
Cite this: Chem. Commun., 2013,
49, 11533
Gorakh S. Yellol,a Antonio Donaire,a Jyoti G. Yellol,a Vera Vasylyeva,b
Received 15th August 2013,
Accepted 17th October 2013
b
a
´
Christoph Janiak and Jose Ruiz*
DOI: 10.1039/c3cc46239k
www.rsc.org/chemcomm
Smart design and efficient synthesis of benzimidazole Ru, Ir and Rh
cyclometalated complexes are reported with promising cytotoxic
activity against HT29, T47D, A2780 and A2780cisR cancer cell lines.
Their apoptosis, accumulation, cell cycle arrest, protein binding and
DNA binding effects are also discussed.
Since the end of the 1970s, platinum based metallodrugs such as
cisplatin, carboplatin and oxaliplatin have become established chemo-
therapeutics for applying to various types of cancers.1 Despite their
remarkable versatility, platinum drug resistance to tumor represents a
Fig. 1 Design of a novel ligand for metallodrugs.
significant limiting factor and a continuing challenge.2 Consequently, a fused heterocyclic skeleton that has a substantial intellectual appeal.6
the discovery of novel metallodrugs with distinct structural and Hence the benzimidazole moiety linked with a phenyl ring is selected
mechanistic profiles for drug development plays an important role in as the main core of the design. As shown in Fig. 1, the C–N site is
cancer drug research. To enhance the traditional paradigm of metallo- readily available for cyclometalation to construct organometallic com-
drug discovery, organometallic compounds with properties somewhat plexes. Gratifyingly, the presence of an NH-functionality on benz-
intermediate between classical inorganic and organic drugs have imidazole can allow a simple installation of different moieties to
recently been considered as promising alternatives. Moreover, organo- modulate hydrophilicity. Easy derivatization of the phenyl ring can be
metallic compounds are suitable for rational drug design and thus they achieved for probable SAR studies. The ester functionality was
could solve many of the challenges in turning a structural lead into a installed as a handle for intended functionalization of metallodrugs.
drug candidate with improved efficacy and tolerability.3 Concomitantly Accordingly, in continued efforts to develop novel better metallo-
with platinum based metallodrugs, substantial efforts have been drugs,7 here we disclose a novel series of C,N-cyclometalated ruthe-
dedicated to develop Ru, Ir, Os and Rh organometallodrugs.4
nium, iridium and rhodium antitumor complexes containing
Synthesis of small drug-like heterocyclic compounds and the use benzimidazole ligands. To the knowledge of the authors, the present
of these molecules as chelating ligands for synthesis of organometallic strategy with these polyvalent ligands is completely innovative in the
complexes have been very well realized for generating promising bibliography, and these studies open a new panoramic view for
anticancer metallodrugs. The design concept of the presently synthe- modeling metal drugs with diverse and simultaneous functions.
sized target has originated from the recognition of the biological role
The key intermediate 1 was efficiently synthesized from 4-fluoro-3-
of the benzimidazoles which exhibits a wide range of pharmacological nitro-benzoic acid using reported procedures with few modifications
properties including anticancer and HIV-1 integrase inhibition.5 More- (Scheme 1).8 Acid catalyzed methyl esterification and nucleophilic
over, the benzimidazole core can be easily tuned for the generation of aromatic substitution of the fluoro group by butyl amine affords
methyl 4-(butylamino)-3-nitrobenzoate. The butyl group was chosen
initially aiming to determine lipophilic properties of the final complex.
a
´
´
Departamento de Quımica Inorganica and Regional Campus of International
Subsequently, reduction of the nitro group using zinc and ammonium
formate in methanol affords 1 in 60% overall yield. The final ligand 2
was synthesized with construction of a benzimidazole ring by con-
densation of 1 and benzaldehyde. Reaction was carried out in acidic
ethanol at room temperature for 24 h to obtain benzimidazole ligand
2 in good (67%) yield. The formation of a benzimidazole moiety was
confirmed by spectroscopic methods. The downfield shifting of three
Excellence (Campus Mare Nostrum), Universidad de Murcia and Instituto
´
Murciano de Investigacion Biosanitaria, Murcia, Spain. E-mail: jruiz@um.es;
Fax: +34 868884148; Tel: +34 868887477
b
¨
¨
¨
Institut fur Anorganische Chemie und Strukturchemie Universitat Dusseldorf,
¨
D-40204 Dusseldorf, Germany
† Electronic supplementary information (ESI) available: Synthesis, characteriza-
tion data and biological study details. CCDC 950001. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c3cc46239k
c
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Scheme 1 Synthesis of a phenyl benzimidazole ligand.
Fig. 2 X-ray crystallographic ORTEP diagram of complex 4.
Scheme 2 Synthesis of cyclometalated Ru–Ir–Rh complexes.
phenyl protons and the appearance of new phenyl ring peaks indicate
formation of a benzimidazole ring.
Scheme 3 Synthesis of analogs of metal complexes.
With the key benzimidazole ligand in hand, we next focused on than those corresponding to ruthenium complexes (65–75%), while
synthesis of organometallic complexes with ruthenium, iridium and rhodium complexes are obtained in medium yields (50–60%). The
rhodium metals. Cyclometalation was achieved by slight modification of structures of all complexes were confirmed by 1H NMR (1D and 2D)
methods used for other ligands.9 Ligand 2 was treated with para-cymene and ESI-MS while their high degree of purity was determined by
ruthenium(II) [RuCl2( p-cymene)]2 and sodium acetate in dichloro- elemental analysis (see the ESI†). Moreover stability of metal com-
methane at room temperature for 24 h to obtain ruthenium complex 3 plexes was determined in DMSO, DMSO–water and 100 mM chloride
in 72% yield (Scheme 2). Formation of a ruthenium complex was ion concentration in DMSO–water for 24 h at 37 1C.
1
confirmed by spectroscopic methods. In the H NMR spectrum of 3,
The cytotoxicity of all the compounds was evaluated toward a panel of
disappearance of one aromatic proton and introduction of four doublets human cancer cell lines representative of epithelial ovarian carcinoma
at 6.5–6.7 ppm, a singlet at 2.2 ppm, and two doublets at 0.9 ppm for six A2780 and A2780cisR (acquired resistance to cisplatin), breast cancers
protons corresponding to p-cymene depicts the formation of ruthenium (T47D) and colon cancers (HT29). For comparison purposes the cytotoxi-
complex 3. Similarly, half-sandwich iridium(III) complex 4 and rhodium- city of cisplatin and the free ligand 2 was also evaluated. As depicted in
(III) complex 5 were prepared using a similar method starting from the Table 1, the majority of complexes are more active than cisplatin towards
corresponding pentamethylcyclopentadienyl chlorido iridium(III) and HT29 and T47D. Noteworthy, complexes 4 and 9 are about as active as
rhodium(III) dimers, respectively, in good yield. The structures of 4 and cisplatin towards A2780. On the other hand, A2780cisR encompasses all
5 were also established by spectroscopic and analytical methods.
of the known major mechanisms of resistance to cisplatin: reduced drug
In addition, the structure of representative iridium complex 4 was transport, enhanced DNA repair/tolerance, and elevated GSH levels.10
unambiguously confirmed by the X-ray crystallographic study. Fig. 2 The ability of most of the new complexes to circumvent cisplatin acquired
depicts the ORTEP diagram of complex 4 (X-ray data are supplied in
ESI†). The single crystal X-ray analysis of compound 4 confirmed its
‘‘piano-stool’’ structure, showing that the two rings of the benz-
imidazole and phenyl moieties are not coplanar.
Table 1 Cytotoxic activity of complexes expressed as IC50 values [mM]
Comp
HT29
T47D
A2780
A2780cisRa
With the successful convergent synthesis of three organometallic
complexes, our next intention was to synthesize analogs of these
complexes to generalize the method. The first emphasis was placed
on effects of different substitution on the benzimidazole nitrogen
which probably modulates the lipophilicity and hydrophilicity of the
metal complex and ultimately the cytotoxicity. As a model study we
first chose the methyl and benzyl groups for N-substitution. Accord-
ingly, a small series of complexes 6 to 11 was synthesized as depicted
in Scheme 3 following a similar protocol. It is noteworthy to mention
that yields for synthesis of iridium complexes are higher (85–90%)
2
3
4
5
6
7
8
9
>50
>100
>100
>100
2.18 Æ 0.39
0.98 Æ 0.02
7.76 Æ 0.04
>50
2.40 Æ 0.13
9.16 Æ 0.98
2.40 Æ 0.07
>50
5.48 Æ 0.17
2.27 Æ 0.04
6.41 Æ 0.23
15 Æ 1
6.61 Æ 0.12
1.87 Æ 0.04
7.12 Æ 0.14
69 Æ 7
6.42 Æ 0.13 (1.0)
1.77 Æ 0.04 (0.9)
4.67 Æ 0.07 (0.7)
36 Æ 2 (0.5)
4.37 Æ 0.11
6.67 Æ 0.29
2.34 Æ 0.22
8.97 Æ 0.24
22 Æ 1
7.40 Æ 0.04
6.09 Æ 0.18
1.82 Æ 0.02
8.05 Æ 0.09
6.64 Æ 0.08
1.54 Æ 0.07
7.46 Æ 0.12 (1.0)
4.43 Æ 0.12 (0.7)
2.07 Æ 0.06 (1.1)
5.27 Æ 0.16 (0.6)
4.36 Æ 0.08 (0.6)
15 Æ 1 (9.7)
10
11
CisPt
5.37 Æ 0.02
9.5 Æ 0.2
38 Æ 2
a
The numbers in parentheses are the resistance factors.
c
11534 Chem. Commun., 2013, 49, 11533--11535
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(5–10 mM) for 48 h, accumulation of Ru, Ir and Rh inside the cell
was determined by atomic absorption spectroscopy. Compounds 3 and
4 display higher levels of metal accumulation (ca. 2–3 times) than that
we have recently found for cisplatin in this cell line.7a It is remarkable
that Rh accumulation is lower than either Ru or Ir compounds. Thus a
relationship between cytotoxicity and the mode of action of the drug is
not evident; very similar for the three compounds, and the accumula-
tion in the cell.
Reactions of anticancer metallodrugs with proteins and DNA are of
considerable interest as they play a crucial role in the biodistribution,
toxicity, and their mechanism of action.12 We studied the interaction of
these complexes with HSA and the ct-DNA by means of competition
experiments using fluorescence spectroscopy. Complexes 3 and 4
interact with HSA at site I (warfarin binding) as well as at site II (dansyl
glycine site). They are also able to bind DNA at the minor groove, since
both displace Hoechst 33258 (ESI,† Fig. S8–S13 and Table S3).
In conclusion, we have successfully synthesized a series of benz-
imidazole cyclometalated complexes exhibiting good anticancer activity
against HT29, T47D, A2780 and A2780cisR cancer cell lines. Represen-
tative complexes show high apoptosis, good accumulation and S-phase
cell arrest and strongly bind to HSA at sites I and II and also weakly
bind to DNA at the minor groove.
This work was financially supported by the European Union Seventh
Framework Programme–Marie Curie COFUND (FP7/2007–2013) under
U-IMPACT Grant Agreement 267143, the Spanish Ministerio de Econ-
Fig. 3 Results corresponding to the arrest cell cycle (A), apoptosis (B) with HT29
cells and metal accumulation (C) assays in T47D cell line (C). Right panels in A and
B correspond to the experimental data for compound 4. In the three panels
brown, blue, orange and green colors correspond to control and complexes 3, 4,
and 5, respectively. For cell cycle experiments (A) the results are given as the
percentage of DNA found in each of the phases. Metal accumulation (C) shows
the mg of metal inside the cell per one million cells when either 10 or 5 mM of the
compounds 3, 4, and 5 were added to the RPMI 1640 medium.
´
´
omıa y Competitividad and FEDER (Project SAF2011-26611), Fundacion
Seneca (Projects 08666/PI/08 and 15354/PI/10) and COST ACTION
´
CM1105. HT29 cells were provided by Dr Martınez-Lacaci (Hospital
Virgen de la Arrixaca).
Notes and references
´
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resistance was determined from the resistance factor (RF) defined as the
ratio of the IC50 resistant line to the IC50 parent line, a very low RF value
being observed at 48 h (RF = 0.5, Table 1). An RF value of o2isconsidered
to denote noncross-resistance.11 Notably, butyl substituted complexes are
more active than their benzyl and methyl derivatives in all studied cell
lines. The IC50 value of free ligand 2was higher than 50 mMinallstudied
cancer cell lines.
To understand the impact of the new complexes on cell growth we
examined the effect of the most active compounds (i.e. N-butyl
substituted complexes) on the cell cycle. Treatment of HT29 cell lines
with compounds 3, 4 and 5 at their IC50 concentrations led to a 11%,
10% and 20% decrease, respectively, in the number of cells in the
G0/G1 phase (Fig. 3A). The number of cells accumulated in the
S-phase improved from 27% (control cells) to 36% (cell treatment
with 3), 34% (4) and 39% (5) at 48 h. These results show that these
compounds are able to arrest the S cell cycle.
´
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Apoptotic studies were also carried out with HT29 cells by flow
cytometric assay following exposure of phosphatidylserine with the
propidium iodide/Annexin V-FLUOS staining kit (Roche). The results
are shown in Fig. 3B. Compounds 3, 4, and 5 show nearly one third of
the total population of cells (33.20, 31.51, and 37.44%, respectively) in
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(Fig. 3C). After exposing a T47D cell with complexes 3, 4, and 5
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c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 11533--11535 11535