Synthesis, cytotoxicity, and DNA interactions of new cisplatin analogues containing substituted benzimidazole ligands

Article abstract of DOI:10.1021/jm8000983

Six new platinum(II) complexes with 1-H or methyl-2-chloromethyl or acetoxymethyl or 2′-hydroxyethyl- benzimidazole carrier ligands were synthesized and evaluated for their reactivity against model nucleophile I ^-, cellular uptake, and in vitro antiproliferative activities against the human MCF-7 breast and HeLa cervix cancer cell lines. The effect of the compounds on pBR322 plasmid DNA was studied by gel electrophoretic mobility measurements. Flow cytometric analysis was also carried out to study the effect of representative compounds 1 and 2, bearing 2-chloromethyl or -acetoxymethylbenzimidazole carrier ligands, on the cell cycle distribution of MCF-7 and HeLa cells, respectively. In general, it was found that Pt(II) complexes were less cytotoxic than cisplatin and were comparable to carboplatin. The results of the plasmid DNA interaction and the restriction studies suggest that changing the chemical structure of the benzimidazole ligands may modulate DNA binding mode and the sequence selectivity. Compounds 1 and 2 had no significant effect on the cell cycle profile of the cells used. However, compound 2 induced a significant increase in the SubG1 cell population at a concentration of 20 μM.

Full text of DOI:10.1021/jm8000983

J. Med. Chem. 2009, 52, 1345–1357
1345
Synthesis, Cytotoxicity, and DNA Interactions of New Cisplatin Analogues Containing
Substituted Benzimidazole Ligands
†
‡
§
‡
∞
Fatma Gu¨mu¨s¸,*,† Go¨kc¸en Eren, Leyla Ac¸ık, Ayten C¸ elebi, Fatma Oztu¨rk, S¸u¨kran Yılmaz, Rahs¸an Ilıkc¸ı Sagˇkan,#
Sibel Gu¨r,
¨
,×
|
|
¨
Aykut Ozkul, Ayhan Elmalı, and Yalc¸ın Elerman
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi UniVersity, 06330 Ankara, Turkey, Department of Biology,
Faculty of Art and Science, Gazi UniVersity, 06500 Ankara, Turkey, Department of Biology, Faculty of Art and Science, Kirikkale UniVersity,
71450 Kirikkale, Turkey, Cell and Virus Bank Department, Foot and Mouth Disease Institute, Ankara, Turkey, Department of Immunology,
Gulhane Military Medicinal Academia, Ankara, Turkey, Department of Virology, Faculty of Veterinary Medicine, Ankara UniVersity,
Ankara, Turkey, and Department of Engineering Physics, Faculty of Engineering, Ankara UniVersity, 06100 Ankara, Turkey
ReceiVed January 31, 2008
Six new platinum(II) complexes with 1-H or methyl-2-chloromethyl or acetoxymethyl or 2′-hydroxyethyl-
benzimidazole carrier ligands were synthesized and evaluated for their reactivity against model nucleophile
I-, cellular uptake, and in vitro antiproliferative activities against the human MCF-7 breast and HeLa cervix
cancer cell lines. The effect of the compounds on pBR322 plasmid DNA was studied by gel electrophoretic
mobility measurements. Flow cytometric analysis was also carried out to study the effect of representative
compounds 1 and 2, bearing 2-chloromethyl or -acetoxymethylbenzimidazole carrier ligands, on the cell
cycle distribution of MCF-7 and HeLa cells, respectively. In general, it was found that Pt(II) complexes
were less cytotoxic than cisplatin and were comparable to carboplatin. The results of the plasmid DNA
interaction and the restriction studies suggest that changing the chemical structure of the benzimidazole
ligands may modulate DNA binding mode and the sequence selectivity. Compounds 1 and 2 had no significant
effect on the cell cycle profile of the cells used. However, compound 2 induced a significant increase in the
SubG1 cell population at a concentration of 20 µM.
Introduction
site of damaged DNA recognition proteins, such as HMG
domain proteins.6 Binding of the HMG domain proteins to
cisplatin-DNA lesions has been suggested to mediate the
antitumor activity of the drug.7,8 The specific binding of HMG
domain proteins to the widened, hydrophobic minor groove can
shield the major 1,2-intrastrand adducts from access of the
nucleotide excision repair (NER) complex, thus preventing
resistance.9 An interesting concept for the design of new
platinum antitumor drugs has been introduced on the basis of
preventing resistance by enhancing this mechanism using more
hydrophobic platinum compounds.10
Cisplatin (cis-diamminedichloroplatinum(II)) is a widely used
chemotherapeutic agent for the treatment of testicular cancer,
and it is used in combination regimens for a variety of other
tumors, including ovarian, cervical, bladder, lung, and those of
the head and neck.1 The clinical success of cisplatin is limited
by significant side effects and acquired or intrinsic resistance.
Therefore, much attention has focused on designing new pla-
tinum compounds with improved pharmacological properties
and a broader range of antitumor activity.2
Although there is some evidence to suggest that other
biological targets may be important in the mechanism, it is
generally accepted that DNA is the primary biological target
of the drug.3 The platinum atom of cisplatin forms covalent
bonds to the N7 positions of purine bases to afford primarily
1,2- or 1,3-intrastrand adducts and a lower number of interstrand
cross-links.4 It is generally believed that biological activity of
cisplatin is associated with the recognition of its DNA adducts
by cellular proteins such as repair enzymes, transcription factors,
histones, and high mobility group (HMG) domain proteins.5 It
has been shown that major 1,2-intrastrand d(GpG) cross-link
can create a hydrophobic notch at the site of the cross-link
generated by the destacking of platinated guanines. This
hydrophobic notch serves as a high affinity and specific binding
There is evidence that other cellular repair mechanisms, such
as mismatch repair (MMR), can affect antitumor efficacy of
cisplatin and that dysfunction of this type of DNA repair may
result in cisplatin resistance or tolerance.5 Two models have
been proposed to explain how MMR induces cell death. In the
first model, MMR of mismatches that contain cisplatin-DNA
guanine adducts leads to the removal and resynthesis of the
paired cytidine base in the newly synthesized strand instead of
removing the cisplatin-DNA lesions, which ultimately induces
cell death. In the second model, recognition and binding of the
MMR proteins to cisplatin adducts alone could be a signal that
triggers cell cycle arrest or programmed cell death.4,5
In metal-DNA binding the kinetics of the metal-ligand
binding are more important than the thermodynamic binding.11
In the amine part of cisplatin derivatives, substitutions allow
the introduction of DNA binding or repelling side arms so that
the kinetics of the DNA binding can be influenced by charge,
hydrogen-bonding, or steric effects. The stabilization after
platinum binding at guanines can be influenced in this way.11
* To whom correspondence should be addressed. Phone and Fax: +90-
312-2120236. E-mail: fgumus@gazi.edu.tr or gumus53@gmail.com.
†
Department of Pharmaceutical Chemistry, Gazi University.
Department of Biology, Gazi University.
Kirikkale University.
Foot and Mouth Disease Institute.
‡
§
∞
#
Gulhane Military Medicinal Academia.
The carrier ligand of the platinum DNA-damaging agents may
affect the biodistribution, rates and type of DNA adduct
formation, and recognition of damaged DNA by repair enzymes
or regulatory/binding proteins.12 The use of sterically demanding
Department of Virology, Ankara University.
Current Address: Department of Virology, Faculty of Veterinary
×
Medicine, Kocatepe University, Afyon, Turkey.
|
Department of Engineering Physics, Ankara University.
10.1021/jm8000983 CCC: $40.75
2009 American Chemical Society
Published on Web 02/16/2009

1346 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Gu¨mu¨s¸ et al.
Scheme 1
diamines as carrier ligands as an alternative compound to
cisplatin can slow or block repair enzymes.13 In addition,
platinum complexes with DNA binding modes distinctively
different from that of cisplatin may provide higher antitumor
activity against cisplatin-resistant cancer cells.14
In previous studies, taking into consideration the fact that
variations in the chemical structure of the ammine groups of
the cisplatin can have significant effects on the cytotoxic activity
and toxicity of platinum complexes, we synthesized some
platinum(II) complexes with the 2-substituted benzimidazole
ligands.15–20 It was determined that some of these platinum
complexes have in vitro cytotoxic activities on RD,16 HeLa,19
and MCF-717,19 cell lines. It was also determined that the DNA
platinated with cis-[Pt(L2)Cl2]·H2O, where L is 5(6)-nonsub-
stituted or 5(6)-chloro-substituted 2-hydroxymethylbenzimida-
zole, was specifically recognized by the HMG domain protein
HMG1.21
In the present study, six platinum(II) complexes having 1-H
or methyl-2-chloromethyl or acetoxymethyl or 2′-hydroxyeth-
ylbenzimidazole carrier ligands were designed and synthesized
as potential antitumor compounds on the basis of previous fin-
dings on some Pt(II) complexes of benzimidazole deriva-
tives.19–21 One of the major goals in the design of the new
platinum(II) complexes was to produce possibly different types
of adducts than those of cisplatin on the DNA, which may
recognized and processed differently by cellular proteins. It was
also aimed to investigate the role of the substituents on position
2 of the benzimidazole carrier ligands and the necessity of the
free N1-H moiety of these ligands on the cytotoxic activity of
platinum(II) complexes.
derivatives, and it is structurally similar to purin bases. (ii) The
substituents on position 1 and/or position 2 of the benzimidazole
ligands can induce notable changes in the electronic, steric, and
hydrophobic properties of the compounds. (iii) The nonplaner
benzimidazole ligands are flexible and bulky enough to affect
the kinetics and cytotoxicities of the corresponding platinum(II)
complexes. (iv) The benzimidazole ligands having acetoxy,
hydroxyl, and/or free N1-H moiety which would have hydrogen-
bond donor and/or acceptor properties, could facilitate novel
types of lesions with cellular DNA, and might exhibit sequence
selectivity.
In this paper, we report the synthesis and the initial results
on the reactivity against model nucleophile I-, the cellular
uptake, in vitro testing of the preliminary antiproliferative
activities on human MCF-7 breast and HeLa cervix cancer cell
lines, and the plasmid DNA interactions of the new dichloro
monosubstituted or disubstituted benzimidazole Pt(II) com-
plexes. We also report the effect of the representative com-
pounds on the cell cycle modification.
Results and Discussion
Chemistry. The ligands L1-L6, used as carrier ligands in
the structure of the new Pt(II) complexes 1-6 were prepared
according to the published procedures as shown in Scheme 1
and their melting points were in accordance with the literature.22–27
The Pt(II) complexes 1-6 were synthesized (Scheme 2) and
characterized by elemental analysis, fast atom bombardment
1
mass spectrometry (FAB-MS), infrared (IR) spectroscopy, H
nuclear magnetic resonance (1H NMR) spectrometry, and
electrospray ionization liquid chromatography/mass spectrom-
etry (ESI-LC/MS). For the characterization of the complexes
Benzimidazole ligands, chosen as the carrier ligands of the
platinum(II) complexes, have four main features that could be
important in the interaction of their platinum(II) complexes with
DNA. (i) The benzimidazole nucleus is found in a variety of
naturally occurring compounds such as vitamin B12 and its
1
by comparison with the data of the ligands, IR and H NMR
data of the ligands were also obtained. The structure of
compound 2, which was the only compound that could be

Cytotoxicity of Substituted Benzimidazole-Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1347
Scheme 2
crystallized among the compounds synthesized, was also
determined by X-ray crystallography. Melting points and H
N1-H acid character after platinum binding.31 The presence
of the bulky ligands in the structures of compounds 1-6 has a
large influence on the chemical shifts as evidenced by the
multiplicity of some of the signals in the 1H NMR spectra, since
the rotation around the Pt-N bound is limited. The existence
of only one set of signals in the 1H NMR spectrum of compound
2, which was recrystallized from DMF, suggests that the
recrystallization procedure did not cause conversion to the trans
isomer. For complexes having the N1-methyl group, two
conformations, syn and anti, are believed to exist. For com-
pounds 5 and 6 the integrated areas under these signals
correspond to the ratios ∼1:2 and 2:1, respectively.
1
NMR data of ligands L1-L6, elemental analysis, IR, 1H NMR,
FAB-MS, and ESI-LC/MS data of compounds 1-6 and crystal-
lographic data of compound 2 are presented in Supporting
Information. For compounds 1, 3, 5, 6, and 2, molecules of
water and DMF were included as justified by the analytical
results. All major species were identified by ESI-LC/MS. Both
the retention times and the MS spectra of the peaks in samples
are evidence of the purity and the expected structures of the
compounds. From the IR data, the presence of hydration lattice
water in the structure of these compounds except for compound
3 can be inferred.
The synthesized compounds except 4 (compound 4 has
limited solubility in the conditions of tests applied) were
investigated for their biological activities.
The IR spectra of the compounds were measured in the region
4000-200 cm-1 and showed characteristic changes when
compared to those of the free ligands. The Pt-N and Pt-Cl
vibrations are considered characteristic for diamine-dichloro
platinum complexes. However, the metal-nitrogen stretching
bands could not be distinguished from other ring skeleton
vibrations present in the spectra. According to the kinetic trans
effect,28 the synthesis method used is expected to yield
complexes with cis geometry. In the far-IR region of the
compounds’ spectra a new broadband with a half-width of about
30 cm-1 appeared assigned to ν (Pt-Cl) centered at ∼325 cm-1.
It is well-known that cis-dichloro complexes should show two
bands of medium intensity because the vibrations are additive,
but in a lot of cases the second band is only a shoulder.29 In
some cases, cis-dichloro complexes show only one band due
to low resolutions independent from cis or trans-configurations.30
Although no shoulder was apparently observable at the Pt-Cl
stretching band of the compounds 1-6, the broad nature of the
band suggested the presence of the bands overlapped in this
domain. In addition, the position of the ν (Pt-Cl) bands of the
complexes was at ∼325 cm-1 and the geometry can be assigned
as cis.
Characterization of Compound 2 in the Solid State. The
structure of compound 2 consists of discrete cis-[Pt(L2)2Cl2]
molecules and two crystallization DMF molecules. An ORTEP
view of the molecule with the DMF molecules is shown in
Figure 1, and a selection of bond distances and angles are given
in Table 1. The coordination geometry of the Pt center is square-
planar, and Pt is surrounded by two N and two Cl atoms. The
PtN2Cl2 square has a cis configuration, which is consistent with
the preparation method. The most interesting feature of this
structure is its head to head orientation exhibited by the two
AcOMBim moieties. The dihedral angle formed by the two
coordination planes of AcOMBim moieties is 73.55(9)°, and
the whole molecule is not planar. The Pt-N and Pt-Cl bond
lengths are comparable with the bond lengths reported in other
analogous platinum complexes.32,33
The insolubility of the compounds in the other organic
solvents made it necessary to record 1H NMR spectra in
dimethyl sulfoxide-d6 (DMSO-d6). All 1H NMR measurements
were recorded immediately in order to avoid ligand exchange
reactions between the Pt(II) complexes and DMSO-d6. The 1H
NMR spectrum of the Pt(II) complexes in DMSO-d6 was
indicative of complex formation and exhibited only one set of
signals for the carrier ligands protons. The large downfield shifts
in the imidazole N1-H signal in the spectra of the complexes
with respect to their ligands are a result of an increase in the
Figure 1. ORTEP view of compound 2, cis-[Pt(AcOMBim)2Cl2] ·
2DMF.

1348 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Gu¨mu¨s¸ et al.
Table 1. Selected Bond Distances and Angles for Compound 2,
cis-[Pt(AcOMBim)2Cl2]·2DMF
distance, Å
Pt-Cl1
Pt-Cl2
Pt-N1
Pt-N1
2.2975(8)
2.3150(10)
2.077(3)
2.099(3)
angle, deg
N1-Pt1-N2
N2-Pt1-Cl1
N2-Pt1-Cl2
N1-Pt1-Cl1
N1-Pt1-Cl2
Cl1-Pt1-Cl2
90.32(11)
179.26(8)
88.44(8)
89.90(8)
178.57(8)
91.35(4)
Determination of the Reactivity of the Compounds against
Model Nucleophile I-. The antitumor platinum drugs are readily
degraded into nonactive complexes by both glutathione and
methionine.34 This degradation occurs through the ability of the
sulfur atoms to displace both the chloride and the monodentate
amine ligands of some cis-Pt(II) complexes.34,35 These interactions
are generally believed to play a role in mechanisms underlying
tumor resistance to platinum compounds, their inactivation, and
side effects.
Figure 2. Time dependence of the formation of PtI2Cl2 after incubation
of the compounds cis-[PtL2Cl2] (1-3, 5, and 6) (L ) L1-L3, L5, and
L6) with excess aqueous KI solution at 37 °C. For compounds: 1 (2);
2 (grey box); 3 (grey star); 5 (black cross); 6 (b).
When the compounds were reacted with excess KI for 30,
60, 90, 120, and 1440 min, for all compounds tested two major
peaks were observed that were due to the [PtI2Cl2] species ([M
- 2L + 2I + Na]+ and unchanged [PtL2Cl2]. For compounds
1, 2, and 3, which are the N1-H free ligands, [M - 2Cl +
2I]+, [M - 2Cl + 2I - 2OAc]+, and [M - 2Cl + 2I]+ species,
respectively, could also be identified. Under the employed
reaction conditions aquated intermediates are not detectable.
Mass spectrometry also revealed the formation of other minor
degradation species. The relative amounts of [PtI2Cl2] species,
as a percentage of total Pt, are shown in Figure 2.
Although it was not in a significant amount, the initial reaction
of the compounds tested with KI leads to loss of the carrier
ligands and rapid formation of [PtI2Cl2] species. It is noteworthy
that for all the compounds tested except for compound 1 the
amount of the [PtI2Cl2] species did not change significantly after
the incubation time of 30 min.
On the basis of the % amount of [PtI2Cl2], the relative order
of the reactivity of the compounds tested assessed from the 24 h
values is 1 . 3 > 6 > 2 > 5.
Compounds 5 and 6 having N1-methyl moiety in their
structures were slightly more stable compounds against model
nucleophile I- than their N1-H free analogues. This finding
must be taken into consideration for the design of new
benzimidazole-platinum(II) complexes in future studies. The
less reactivity of compounds 5 and 6 may be partly related to
electron releasing effect of their methyl groups.
The specific interactions between the two carrier ligands may
also play a role in the stability of the complexes. Compound 1
lost its carrier ligands in a higher amount than the other
compounds tested. The driving force for the dissociation of
ClMBim carrier ligands from compound 1 in the test conditions
used might be the steric and electronic repulsion between the
two chloromethyl substituents.
Cytotoxicity. The preliminary in vitro antiproliferative activi-
ties of the new sterically hindered benzimidazole-platinum(II)
complexes 1-3, 5, and 6 and cisplatin and carboplatin used as
reference compounds were determined on the human MCF-7
breast and HeLa cervix cancer cell lines (for experimental
procedure, see Supporting Information). The antiproliferative
activity values of the compounds tested expressed as T/Ccorr are
presented in Figures 3 and 4. IC50 values of the platinum
Recently, in a study on the reaction of Zn7 metallothionein
with cis- and trans-[Pt(N-donor)2Cl2] anticancer complexes,
which bear less voluminous carrier ligands than those of the
carrier ligands of our complexes, it has been reported that
N-donor ligands of cis-[Pt(N-donor)2Cl2] compounds were
replaced by cysteine thiolates but that the majority of trans-
Pt(II) compounds retain their N-donor ligands.35 In the study it
has been determined that steric hindrance represents the major
factor responsible for the different reactivities of Pt(II) drugs
with Zn7 metallothionein. It has also been reported that a lower
reactivity of drugs could also be accomplished by modulating
the electrophilicity of the Pt(II) center. It is expected that the
steric hindrance and the electronic effect of the carrier ligands
may influence the stability of the benzimidazole-platinum(II)
complexes. Therefore, we have been particularly interested in
examining whether the carrier ligands of cis-[PtL2Cl2] complexes
1-3, 5, and 6 are replaced by nucleophiles. For the reactivity
studies we used iodide as nucleophile. In the literature, it was
reported that from an experimental point of view, application
of I- as nucleophile instead of bionucleophiles like nucleotides,
oligonucleotides, and nucleophilic plasma components such as
S-containing amino acids, peptides, or proteins was of consider-
able advantage, since it allows a fast and convenient quantitative
determination of the products by HPLC.36–42 It was also reported
that in the stability studies of some antitumor active Pt(II)
complexes, the use of iodide instead of the main plasma
component albumin as nucleophile gave the same order of
reactivity and allowed the assessment of the in vivo stability.38
In this study, the reactivity of compounds 1-3, 5, and 6
against attack of strong nucleophile I- was monitored by ESI-
LC/MS spectroscopy (for experimental details, see Supporting
Information). The reactions of the compounds with KI were
carried out under pseudo-first-order conditions using excess
potassium iodide. The starting and the reaction products were
well separable and identified by ESI-LC/MS. Platinum contain-
ing ions were distinguishable by the m/z cluster value resulting
from the presence of isotopes. The Pt content of the collected
fractions was determined by inductively coupled plasma mass
spectrometry (ICP-MS) under the conditions presented in
Supporting Information.

Cytotoxicity of Substituted Benzimidazole-Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1349
Figure 3. Antiproliferative effects of the benzimidazole-platinum(II) complexes (1-3, 5, 6), cisplatin, and carboplatin on the MCF-7 cell line
(-, 0.5 µM; b, 1 µM; 2, 5 µM; 9, 10 µM; [, 20 µM; ×, 40 µM).
complexes tested, calculated (using Prism4, GraphPad Software)
from the dose-survival curves for the growth inhibition of both
cell lines used for each incubation time measured, are presented
in Table 2. For comparison, in vitro antiproliferative activities
of the ligands L1-L3, L5, and L6 against both cell lines used
were also performed. IC50 values of the ligands are the averages
of two independent determinants and are presented in Table 3.
In the test on the human MCF-7 cell line at 0.5, 1, 5, and 10
µM concentrations (except for compound 2 and carboplatin at
10 µM concentration) T/Ccorr values of all the compounds
synthesized and the reference compound carboplatin were >100
or not a considerable amount. Cisplatin possessed a considerably
higher activity and almost completely blocked the growth of
the MCF-7 cells at a concentration of 5 µM at the last measuring
time point, 144 h.
Although compound 2 was moderately cytotoxic at 10 µM
concentration at the last measuring time point (T/Ccorr ) 45.15%
at 10 µM, t ) 144 h), there seemed to be no great difference
between the antiproliferative activity values of compounds 1,
2, and 6 at 20 µM concentration (T/Ccorr ) 27.20%, 34.03%,
and 23.61%, respectively, at 20 µM, t ) 144 h). For compounds
3 and 5 no significant cytotoxicity was observed over a period
of 144 h at 20 µM. At 40 µM, they showed a significant
reduction in cell growth (T/Ccorr ) 15.97% and 18.63%,
respectively, t ) 144 h).
In contrast to cisplatin and carboplatin, which reached their
maximum effects at the last measuring time point (T/Ccorr
)
3.32% at 5 µM, t ) 144 h, and T/Ccorr ) 13.20% at 20 µM, t
) 144 h, respectively) compounds 1, 2, 3, and 5 reached their
maximum effects at an incubation time of 72 h (T/Ccorr
)
25.13%, 30.59%, 53.57%, and 51.79%, respectively, at 20 µM,
t ) 72 h). Compound 6 required up to 144 h to reach its
maximum effect.
At a concentration of 40 µM, cytocidal effects were observed
for compounds 1, 2, and 6 at all the time points measured against
MCF-7 cell line (data not shown except for 6).
In the test on the human HeLa cell line at dosages of 0.5, 1,
5, and 10 µM, T/Ccorr values of all the compounds synthesized
(except for compound 2 at 10 µM concentration (T/Ccorr
49.31%, t ) 96 h)) were >100 or did not amount to considerable
values.
)
Compound 2 was found to be more active than carboplatin
and caused a significant reduction of cell growth at 20 µM
concentration (T/Ccorr ) 44.07%, 23.16%, 4.98%, and 2.43%; t
) 48, 72, 96, and 120 h, respectively). At 20 µM concentration,
compound 6 resulted in an almost 2-fold increase in cytotoxicity
(T/Ccorr ) 11.32%, 19.76%, 25.04%, and 24.74%; t ) 48, 72,
96, and 120 h, respectively) relative to compounds 1 and 5 in
the HeLa cell line.

1350 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Gu¨mu¨s¸ et al.
Figure 4. Antiproliferative effects of the benzimidazole-platinum(II) complexes (1-3, 5, 6), cisplatin, and carboplatin on the HeLa cell line (-,
0.5 µM; b, 1 µM; 2, 5 µM; 9, 10 µM; [, 20 µM; ×, 40 µM).
Table 2. IC50 Values Obtained for Compounds 1-3, 5, 6, Cisplatin, and Carboplatin
IC50 (µM)
MCF-7
96 h
HeLa
compd
48 h
72 h
120 h
144 h
48 h
72 h
96 h
120 h
1
2
3
5
6
13.73 ( 2.20 12.96 ( 1.49 15.84 ( 4.15 19.88 ( 7.98 11.43 ( 4.79 29.39 ( 1.32 29.16 ( 3.09 18.90 ( 1.03 16.04 ( 1.22
16.68 ( 2.05 13.49 ( 3.42 19.25 ( 9.76 14.04 ( 6.49 7.10 ( 1.13 17.31 ( 2.27 11.29 ( 3.23 13.10 ( 1.29 11.25 ( 3.72
26.38 ( 11.37 20.27 ( 6.13 24.48 ( 9.84 36.36 ( 12.07 20.98 ( 9.60 27.32 ( 4.99 21.54 ( 1.38 10.04 ( 3.75 36.52 ( 12.64
20.21 ( 8.97 20.98 ( 6.18 23.52 ( 3.54 33.95 ( 9.85 34.14 ( 12.37 31.67 ( 9.23 20.76 ( 5.76 11.69 ( 1.25 18.68 ( 10.87
23.12 ( 6.13 23.71 ( 4.78 16.11 ( 3.79 19.84 ( 2.40 16.96 ( 3.23 11.42 ( 1.78 15.89 ( 6.79 12.54 ( 4.81 16.23 ( 4.53
cisplatin
5.06 ( 2.83
3.75 ( 1.23 1.47 ( 0.69 0.96 ( 0.34
1.06 ( 0.56
1.25 ( 0.54
0.75 ( 0.31 1.00 ( 0.31 1.22 ( 0.47
carboplatin 33.57 ( 8.17 22.53 ( 6.23 23.62 ( 6.73 12.39 ( 5.06
9.36 ( 3.15 33.57 ( 12.35 15.67 ( 4.47 10.39 ( 2.13 6.20 ( 1.98
Table 3. IC50 Values Obtained for Ligands L1-L3, L5, and L6 after
72 h of Incubation
incubation time of 96 h) and cisplatin > 2 > 6, carboplatin >
1, 3, 5 (for the incubation time of 72 h), respectively.
IC50 (µM)
At this stage of the study it is not possible to explain clearly
why compound 3 was less cytotoxic in both cell lines than the
other compounds tested. But it might be thought that hydroxyl
groups of this compound may interact strongly with the
membrane and/or intracellular proteins, causing the compound
to be less active. Its higher reactivity against I- compared to
the other compounds tested except for 1 is in agreement with
its less antiproliferative activity.
MCF-7
HeLa
L1
L2
L3
L5
L6
>40
>40
32.27
25.60
>40
35.13
38.42
>40
37.21
>40
The result of the preliminary antiproliferative activity studies
indicates that, in general, on the basis of calculated IC50 values,
the following order of relative in vitro antiproliferative activity
of the compounds tested with MCF-7 and HeLa cell lines could
be considered: cisplatin > 1, 2, 6 > carboplatin, 3, 5 (for the
For the interpretation of the cytotoxic properties, if the relative
order of reactivity (against KI, 1 . 3 > 6 > 2 > 5) and
antiproliferative activity (1, 2, 6 > 3, 5 and 2, 6 > 1, 3, 5 against
MCF-7 and HeLa cell lines, respectively) of the compounds is

Cytotoxicity of Substituted Benzimidazole-Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1351
Table 4. In Vitro Drug Uptake for Compounds 1-3, 5, 6, and Cisplatin
Pt uptake (µmol of Pt per 1 × 106 cells)a
decrease in mobility for the form I bands was observed (Figure
5). These compounds accelerated the mobility of the form II
band of plasmid DNA similarly as did cisplatin, whose
bifunctional binding shortens and condenses the DNA helix.43
These compounds significantly modified electrophoretic mobil-
ity; a coalescence of the two forms I and II bands was observed,
similar to cisplatin. The concentration required to achieve total
unwinding of the pBR322 plasmid DNA for the compounds 2,
3, 6, and cisplatin were 50, 25, 12.5, and 6.25 µM, respectively
(for compounds 2, 3, 6, and cisplatin, lines 11, 10, 9, and 8,
respectively in Figure 5). The presence of a coalescence point
indicates a strong unwinding of the supercoiled DNA.44 Figure
5 also suggests that compounds 3, 6, and cisplatin changed
conformations of plasmid DNA from negative supercoil to
positive supercoil at higher concentrations than that of their
coalescence points.
compd
MCF-7
HeLa
log Pb
1
2
3
5
0.300
0.228
3.877
0.253
0.823
0.057
0.198
0.099
1.245
0.103
0.572
0.061
2.02 ( 0.25
1.56 ( 0.28
0.87 ( 0.23
1.86 ( 0.59
1.13 ( 0.57
6
cisplatin
a Cellular uptake of platinum in MCF-7 and HeLa cells treated with 30
µM concentrations of compounds 1-3, 5, 6, and cisplatin for 4 h. Data
represent the mean values of two independent experiments. b The partition
coefficient of the carrier ligands of the corresponding complexes obtained
by using the software ACD/LogP.
compared, although there is not a clear correlation, there seem
to be an inverse correlation between the reactivity and antipro-
liferative activity of compounds 2, 3, and 6.
For Pt(II) complexes 1 and 5, no comigration of the forms I
and II bands of plasmid DNA was observed at the concentration
range tested. However, in the range of concentrations from 12.5
to 100 µM the electrophoretic mobility of form I DNA decreased
slightly and form II DNA increased slightly. The slight increase
in the mobilities of form II and particularly form I plasmid DNA
treated with compounds 1 and 5 with respect to untreated DNA
was observed in a range of concentrations from 0.097 to 12.5
µM (Figure 5). In the literature, it has been reported that
bifunctional DNA-cisplatin adducts, both intra- and interstrand
cross-links, unwind DNA with a higher efficiency than monoad-
ducts.44 Small changes in the mobility of the form I band of
pBR322 plasmid DNA treated with some of the Pt(II) and Pd(II)
complexes containing sterically demanding carrier ligands were
attributed to monofunctional binding.45 Thus, the slight increase
in the mobilities of form II and particularly form I plasmid DNA
treated with compounds 1 and 5 may be interpreted that binding
of these compounds to plasmid DNA below 12.5 µM is a
monofunctional manner. The reason for the lower reactivity of
compounds 1 and 5 to plasmid DNA compared with that of the
other compounds tested is not clear. But it may be thought that
the greater electron density on the chlorine atoms and acetoxy
groups of compounds 1 and 5, respectively, might influence
their interactions with DNA negatively. The potential for the
formation of hydrogen bonds with DNA by these compounds
also seems to be very limited.
In vitro antiproliferative activity studies of the ligands against
MCF-7 and HeLa cell lines for an incubation time of 72 h
demonstrated that ligands L1, L5, and L6 (except for L6 against
the MCF-7 cell line) were inactive in the range of concentrations
tested. Although their cytotoxicity was less than those of their
corresponding Pt(II) complexes, L2 and L3 were found to be
cytotoxic (Table 3). It might be thought that they may play a
role in cytotoxicity of their corresponding Pt(II) complexes 2
and 3.
Cellular Uptake Studies. To determine drug accumulation
in MCF-7 and HeLa cells, the cells were incubated with
compounds 1-3, 5, 6, and cisplatin for 4 h. The intracellular
platinum content was then measured using ICP-MS (for
experimental details, see Supporting Information) and calculated
as µmol of Pt per 1 × 106 cells (Table 4). The cellular uptake
experiments used in this study cannot distinguish between
cytosolic platinum and membrane bound platinum. For this
reason, the results do not necessarily reflect the actual cytosolic
concentration of the platinum(II) complexes. In general, the
benzimidazole Pt(II) complexes tested showed higher cellular
accumulation in both cell lines used than cisplatin. Comparison
of the cellular concentrations of the compounds tested in both
cell lines showed that the order of the platinum accumulation
was 3 . 6 . 1 > 5 > 2 > cisplatin, while their relative
cytotoxicities against MCF-7 and HeLa cell lines were cisplatin
> 1, 2, 6 > 3, 5, and cisplatin > 2 > 6 > 1, 3, 5. For compounds
3 and 6, cellular accumulation was found to be about 68- and
14-fold (for MCF-7 cells) and 20- and 9-fold (for HeLa cells)
higher than that of cisplatin. Interestingly, platinum accumulation
of the benzimidazole Pt(II) complexes in MCF-7 cells, which
is estrogen-receptor positive breast cancer cell type, was found
to be higher than that in HeLa cells. This result must be taken
into consideration and investigated in detail in future studies.
Interaction with pBR322 Plasmid DNA. In order to detect
whether synthesized compounds 1-3, 5, and 6 induce confor-
mational changes in the DNA helix and whether there is a
relationship between the plasmid-DNA binding affinity and
the cytotoxicity of the compounds, we investigated their capacity
to remove and reverse the supercoiling of closed circular
pBR322 plasmid DNA as assessed by electrophoretic mobility
measurements on agarose gels (see Supporting Information).
In the electrophoretograms the untreated pBR322 plasmid DNA,
which is a mixture of mainly covalently closed circular form I
and a small amount of open circular form II bands, was used
as the control (Figures 5 and 6).
The increase in intensity of the form II band (compared to
that in the untreated DNA) with the increase in concentrations
of the compounds tested may be due to partial nicking of form
I DNA to produce form II DNA as a result of covalent binding
of the compounds.
To analyze the ability of the compounds to nick DNA, the
gels were also run in the presence of ethidium bromide (data
not shown except for representative compound 2) (Figure 6).
In the presence of ethidium bromide, the decrease in the
electrophoretic migration of the form I bands of the pBR322
plasmid DNA-Pt(II) complexes adducts was eliminated. How-
ever, the increase in the electrophoretic migration of the form
II bands was observed as it has been observed in the case of
the gels that had been run without ethidium bromide. The data
obtained suggest that after the nicking event the platinum
complexes would remain attached to DNA, ensuring a three-
dimensional shape change in form II that is not reverted in the
presence of ethidium bromide.
As has been previously reported for cisplatin, the unwinding
of the DNA produced by the Pt(II) complexes 2, 3, and 6, which
have the potential to form a hydrogen bond with DNA, may
support the formation of DNA intrastrand cross-links.46 Al-
When pBR322 plasmid DNA was interacted with increasing
concentrations of compounds 2, 3, and 6 and cisplatin, a

1352 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Gu¨mu¨s¸ et al.
Figure 5. Modification of gel electrophoretic mobility of pBR322 plasmid DNA when incubated with various concentrations of compounds 1-3,
5, 6, and cisplatin. Concentrations (in µM) are as follows: (lines 1 and 13) untreated pBR322 plasmid DNA; (lines 2) 0.097; (line 3) 0.195; (line
4) 0.39; (line 5) 0.78; (line 6) 1.56; (line 7) 3.125; (line 8) 6.25; (line 9) 12.5; (line 10) 25; (line 11) 50; (line 12) 100. The top and the bottom bands
correspond to form II (open circular) and form I (covalently closed circular) plasmids, respectively.
and/or adenine-adenine (AA) regions, we carried out restriction
endonuclease analysis of the compound-pBR322 plasmid DNA
adducts digested by BamHI and HindIII enzymes (see Support-
ing Information). BamHI and HindIII enzymes bind at the
recognition sequence 5′-G/GATCC-3′ and 5′-A/AGCTT-3′ and
cleaves these sequences just after 5′-guanine and 5′-adenine sites,
respectively, and, as a result, convert form I and form II DNA
to linear form III DNA.48
Figure 6. Electrophoresis in agarose gel containing ethidium bromide
As the concentration of the compounds tested was in-
following incubation of pBR322 plasmid DNA with various concentra-
creased, it was seen that BamHI and HindIII (except for
tions of representative compound 2. Concentrations (in µM) are as
compound 2) digestion was increasingly prevented (Figures
7 and 8). This is probably due to conformational change in
the DNA brought about by the covalent binding of the
compounds with the plasmid DNA. Arranged in the order of
decreasing prevention of BamHI and HindIII digestion, the
compounds were cisplatin > 3 > 1, 6 > 2 > 5 and 5 > 1,
6 > cisplatin > 3 > 2, respectively.
follows: (lines 1 and 11) untreated pBR322 plasmid DNA; (line 2)
100; (line 3) 50; (line 4) 25; (line 5) 12.5; (line 6) 6.25; (line 7) 3.125;
(line 8) 1.56; (line 9) 0.78; (line 10) 0.39. The top and the bottom
bands correspond to form II (open circular) and form I (covalently
closed circular) plasmids, respectively.
though it is not possible to conclude that the cytotoxic activity
of these compounds is directly correlated to their interactions
with DNA, strong unwinding of the supercoiled DNA caused
by compounds 2 and 6 and cisplatin was found to be in
accordance with their in vitro antiproliferative activities.
Compounds 3 and 6 were accumulated in both cell lines used
and/or interacted strongly with the membrane proteins of these
cells in significantly higher amounts than cisplatin (Table 4).
The interaction of these compounds, particularly 6 with the
pBR322 plasmid DNA, was observed at an approximately
similar effective concentration as with cisplatin. But these
compounds were found to be less antiproliferative active than
cisplatin against the cell lines used. This gives support to the
idea that DNA platination is a necessary condition for cytotoxic
activity of platinum complexes but may not be the sole
mechanism that determines their cytotoxicity.47 It is well-known
that the role of additional hydrogen-bonding interactions both
in the kinetics of the DNA binding process and in the
stabilization of the adduct structure is very important.11 Struc-
tural factors, i.e., steric and electronic constraints imposed by
the 2′-hydroxyethyl moiety, may further stabilize the platinated-
plasmid DNA adduct and may be responsible for the decreased
antiproliferative activity and the increased reactivity presumably
to bionucleophiles of the compound 3. It is also very possible
that in the test conditions the hydroxyl group could form an
intramolecular hydrogen bond with the N1-H moiety of the
benzimidazole ring, and this interaction may be responsible for
the poor antiproliferative activity, since compound 6, the N1-
CH3 analogue of compound 3, was found to be more active
than compound 3 in both cell lines used.
Among the benzimidazole Pt(II) complexes tested, com-
pounds 2 and 3, with the ligands having a free N1-H moiety,
inhibit BamHI restriction enzyme activity to a greater extent
than HindIII restriction enzyme activity, thus demonstrating their
affinity for GG regions of the plasmid DNA. On the other hand,
it is interesting to note the greater inhibition of HindIII over
BamHI restriction enzyme activity by compounds 5 and 6
(especially 5) (those with N1-methylated ligands). It can be
speculated that in modification of pBR322 plasmid DNA,
MAcOMBim ligands in the structure of compound 5 may play
a specific role, since compound 5 is not recognized by HindIII
enzyme even at its 0.39 µM concentration. It is interesting to
note that besides its low antiproliferative activity and reactivity
(against KI), compound 5 is also the least reactive compound
to plasmid DNA and nearly inactive for binding to GG regions.
It is also noteworthy that compound 5 is highly effective at
targeting adenine-rich sequence on DNA (Figures 7 and 8). The
different cellular processing of DNA modified by this compound
might be relevant to the low antiproliferative activity of this
compound especially against MCF-7 cells. The formation of
monofunctional adducts assumed may be sufficient to cause
enough conformational change in pBR322 plasmid DNA treated
with 1 and 5 such that HindIII digestion is prevented below
their concentrations, 12.5 µM.
The results of the plasmid DNA interaction and the restriction
studies suggest that changing the chemical structure of the
benzimidazole ligands may modulate DNA binding mode and
the sequence selectivity, and the presence of a hydrogen-bond
donor, such as a hydroxyl group or N1-H moiety on the
benzimidazole carrier ligands of the Pt(II) complexes, seems
to have a significant effect on the DNA unwinding.
BamHI and HindIII Digestion of Drug-pBR322 Plas-
mid DNA. In order to assess whether the benzimidazole-
Pt(II) complexes show affinity toward guanine-guanine (GG)

Cytotoxicity of Substituted Benzimidazole-Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1353
Figure 7. Electrophoretograms for the BamHI digested mixtures of pBR322 plasmid DNA after their treatment with various concentrations of
compounds 1-3, 5, 6, and cisplatin. Concentrations (in µM) are as follows: (lines 1 and 2) untreated pBR322 plasmid DNA and pBR322 DNA
linearized by BamHI, respectively. For compounds 1, 2, 5, and 6: (line 3) 6.25; (line 4) 12.5; (line 5) 25; (line 6) 50; (line 7) 100. For compound
3: (line 3) 1.56; (line 4) 3.125; (line 5) 6.25; (line 6) 12.5; (line 7) 25; (line 8) 50; (line 9) 100. For cisplatin: (line 3) 0.39; (line 4) 0.78; (line 5)
1.56; (line 6) 3.125; (line 7) 6.25; (line 8) 12.5; (line 9) 25. Roman numerals I, II, and III indicate form I (covalently closed circular), form II (open
circular), and form III (linear) plasmids, respectively.
Figure 8. Electrophoretograms for the HindIII digested mixtures of pBR322 plasmid DNA after their treatment with various concentrations of
compounds 1-3, 5, 6, and cisplatin. Concentrations (in µM) are as follows: (first and the last lines) untreated pBR322 plasmid DNA and pBR322
DNA linearized by HindIII, respectively. For compound 1: (line 2) 6.25; (line 3) 3.125; (line 4) 1.56; (line 5) 0.78; (line 6) 0.39; (line 7) 0.195. For
compound 2 and 3: (line 2) 100; (line 3) 50; (line 4) 25; (line 5) 12.5; (line 6) 6.25; (line 7) 3.125. For compound 5: (line 2) 1.56; (line 3) 0.78;
(line 4) 0.39. For compound 6: (line 2) 3.125; (line 3) 1.56; (line 4) 0.78; (line 5) 0.39. For cisplatin: (line 2) 50; (line 3) 25; (line 4) 12.5; (line
5) 6.25; (line 6) 0.39. Roman numerals I, II, and III indicate form I (covalently closed circular), form II (open circular), and form III (linear)
plasmids, respectively.
Cell Cycle Studies. The effects of the representative com-
pounds 1 and 2, which were found to be the most active
compounds against the cell line tested, for initial studies on the
cell cycle profile of the MCF-7 and HeLa cells, respectively,
were examined by propidium iodide (PI) staining and flow
cytometry (see Supporting Information). Cisplatin was used
under the same experimental conditions as the control and for
comparison. After treatment of MCF-7 and HeLa cells for 72 h
with compounds 1 and 2, respectively, at 20 µM concentrations
equivalent to their T/Ccorr values of ∼20% (drug concentration
required to inhibit cell growth by ∼80%), it was observed that
compounds 1 and 2 had no significant effect on the cell cycle
profile of MCF-7 and HeLa cells, respectively, compared to
untreated controls (Figure 9). On the other hand, compound 2
induced a significant increase in the SubG1 cell population
(which is representative of cells with fragmented DNA) with
no arrest in G2/M and G0/G1 phases under the test conditions
applied in HeLa cells with respect to untreated control and
cisplatin. This indicates that compound 2 induces sufficient
levels of DNA damage. The results of the cytotoxicity and the
plasmid DNA helix unwinding and the cell cycle modification
studies on compound 2, one of the two most stable compounds
against model nucleophile I- among the compounds tested, seem
to be in accordance with each other. The result obtained in this
study is in agreement with the results obtained in our ongoing
studies using annexin V and PI double staining by flow
cytometry in which compound 2 induced “early apoptotic” and
“late apoptotic and necrotic” cells in HeLa cells by ∼2- and
∼5-fold, respectively, compared to untreated control (data not
shown). However, the effect of antiproliferative compounds on
apoptosis inductions and cell cycle modifications may depend
on cell type and concentration of the test compounds; the
treatment of MCF-7 cells over 72 h with compound 1 did not
appear to produce any significant SubG1 population increase
compared to the untreated control. This is consistent with its
much weaker modification on pBR322 plasmid DNA compared
to cisplatin.
Treatment of both MCF-7 and HeLa cells for 72 h with
cisplatin at 20 and 1 µM concentrations, respectively, equivalent
to T/Ccorr values of ∼20%, resulted in a small increase in the
SubG1 cell population and a significant increase in G2/M cell
fractions and a reduction in G0/G1 cell fractions. These findings
are consistent with previous reports that cisplatin, whose mode
of cell death is concentration dependent,49 induces G2/M arrest
in MCF-750 and HeLa51 cell lines at the same concentrations
used in this study.
Most of the antineoplastic drugs in current use block the cell
cycle in the S or G2/M phases. Many tumor cells are resistant
to classical chemotherapeutic agents, since they are endowed
with mutations that inactivate apoptotic machinery or activate
antiapoptotic pathways. Therefore, the characterization of novel,
apoptosis-independent anticancer drugs appears to be signifi-
cantly relevant for possible therapeutic applications.52 The
different effects of compounds 1 and 2 on the cell cycle profiles
of the MCF-7 and HeLa cells, respectively, can be due to
multiple causes that may be related to DNA and/or non-DNA
targets interactions of these compounds. Especially for com-
pound 1, which was found to be the most reactive compound

1354 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Gu¨mu¨s¸ et al.
Figure 9. Cell cycle distribution and SubG1 peak demonstration. a: HeLa cell control consists of medium. b: HeLa cells were exposed to cisplatin.
c: HeLa cells were exposed to compound 2. d: MCF-7 cell control consists of medium. e: MCF-7 cells were exposed to cisplatin. f: MCF-7 cells
were exposed to compound 1. Data are representative of three separate experiments with similar results. The horizontal axis of each histogram
represents DNA content; the vertical axis denotes cell number. ((light-blue) SubG1; (light-purple) G0/G1; (pink) S; (dark-purple) G2/M).
in “iodide assay” and less reactive compound against pBR322
plasmid DNA, non-DNA targets interaction may be important
for its antiproliferative activity against MCF-7 cell line. To
confirm all the assumptions, it is necessary to study the
interaction of the Pt(II) complexes with DNA and the specific
proteins in detail.
may be concluded that the mechanism of antiproliferative
activity of compounds 1 and 2 against MCF-7 and HeLa cell
lines, respectively, is probably different from that of cisplatin.
These differences reflect the distinct nature of the carrier ligands
and might be promising agents, and further studies on com-
pounds 1 and 2 are warranted.
The biochemical mechanism of cisplatin cytotoxicity involves
the binding of the drug to DNA and non-DNA targets and the
subsequent induction of cell death through apoptosis, necrosis,
or both.46 It is known that most cisplatin molecules that enter
the cell are bound to proteins rather than DNA, and there is
experimental evidence showing that the former type of damage
also plays a role in the initiation of apoptotic pathways.53 For
example, oxaliplatin, the R,R isomer of [Pt(oxalate)(diaminocy-
clohexan)], is currently in clinical use against metastatic
colorectal cancer in which cisplatin is inactive54 and shows a
level of DNA binding lower than ciplatin. Although the overall
conformational alterations induced in DNA by the major
intrastrand adducts of cisplatin and oxaliplatin are similar,55
oxaliplatin induces higher levels of apoptosis in cell lines
endowed with resistance to cisplatin. The structures of these
two compounds are distinct and differently recognized by
mismatch-repair proteins and some damage-recognition proteins.
It has been reported that in cancer cells the rate of protein
binding is high for oxaliplatin.54 It has also been postulated that
the hydrophobic diaminocyclohexan moiety in oxaliplatin may
direct drug reactivity toward cellular proteins with sulfydryl
groups within hydrophobic pockets, which may be poorly
reactive with cisplatin. Considering the data reported in the
literature and the preliminary results obtained in this study, it
To determine whether the hydrophobicity of the Pt(II)
complexes influences their cytotoxicities and accumulations on
the cell lines tested and their plasmid DNA interactions,
theoretical calculations of hydrophobicity of the carrier ligands
L1-L3, L5, L6 as log P were performed using the software ACD/
LogP. It has been reported in a systematic study of the hydro-
phobicity of cis-diam(m)ineplatinum(II) complexes that when
the leaving groups are unchanged, the hydrophobicity of a
platinum complex is linearly related to that of the am(m)ine
ligand.56 It was also shown that the calculated log Poct correlates
well with observed cellular uptake for the complexes. The log P
values of the ligands L1-L3, L5, L6 are 2.02 ( 0.25, 1.56 (
0.28, 0.87 ( 0.23, 1.86 ( 0.59, 1.13 ( 0.57, respectively. If
compounds 1-3, 5, and 6 in order of decreasing cytotoxicity
against MCF-7 and HeLa cell lines (1, 2, 6 > 3, 5 and 2 > 6
> 1, 3, 5, respectively) are compared with the order of the log P
values of the corresponding ligands (L1 > L5 > L2 > L6 >
L3), although it is not clear, there seems to be a positive
correlation (except for compound 5) between log P and anti-
proliferative activity of the compounds against MCF-7 cell line.
However, in the case of the cytotoxicity against HeLa cell line,
it is more difficult to establish a correlation. In addition, it is
very possible to conclude that there seems to be an inverse
correlation (except for compound 3) between the log P (L1 >

Cytotoxicity of Substituted Benzimidazole-Pt(II) Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1355
to the published procedures as shown in the Scheme 1. Melting
points and 1H NMR data of the ligands are presented in the
Supporting Information.
L5 > L2 > L6 > L3) and the plasmid DNA binding affinity (6
> 3 > 2 > 5, 1) of the compounds.
In summary, while an inverse correlation between the cellular
accumulation and the log P values of the compounds 3, 5, and
6 was observed, there seems to be a positive correlation between
platinum uptake and the antiproliferative activities of compounds
1, 5, and 6 for both cell lines used.
Synthesis of the Platinum(II) Complexes. ESI-LC/MS, IR, 1H
NMR, and FAB-MS data of compounds 1-6 are presented in the
Supporting Information.
[Pt(L1)2Cl2]·3H2O (1). To a stirred solution of L1 (0.180 g, 1.08
mmol) in 0.5 N HCl (15 mL) was added a solution of K2PtCl4
(0.250 g, 0.60 mmol) in 0.5 N HCl (5 mL) dropwise over 30 min
at room temperature. The reaction mixture protected from light was
heated at 60 °C for 5 days. After that time the mixture was kept in
the refrigerator at 4 °C for 12 h. The resulting crude precipitate
was filtered off and purified by repeated washing with small portions
of water, ethanol, and diethyl ether and dried in vacuo to yield
0.150 g (41.66%) of 1. Anal. (C16H14Cl4N4Pt·3H2O) C, H, N.
[Pt(L2)2Cl2]·2DMF (2). To a stirred solution of L2 (0.125 g,
1.69 mmol) in an ethanol-water mixture (5:10 mL) was added an
aqueous solution of K2PtCl4 (0.162 g, 0.39 mmol in 5 mL of water)
dropwise over 30 min at room temperature. The pH was adjusted
to ∼7 and kept constant with the addition of 0.1 M KOH. The
reaction mixture protected from light was stirred at room temper-
ature for 7 days. After that time the mixture was kept in the
refrigerator at 4 °C for 12 h. The resulting crude precipitate was
filtered off and purified by repeated washing with small portions
of water, ethanol, and diethyl ether and dried in vacuo and
recrystallized from DMF by slow evaporation. Yield: 0.082 g
(32.86%) of 2. Anal. (C20H20Cl2N4O4Pt·2DMF) C, H, N.
[Pt(L3)2Cl2]·H2O (3). A similar procedure was carried out as
described for compound 2 using L3 (0.250 g, 1.50 mmol) and
K2PtCl4 (0.366 g, 0.88 mmol) at 60 °C for 5 days. Yield: 21.19%,
0.110 g (21.19%). Anal. (C18H20Cl2N4O2Pt·H2O) C, H, N.
[Pt(L4)2Cl2] (4). L4 (0.122 g, 0.68 mmol) and K2PtCl4 (0.160 g,
0.39 mmol) was dissolved in 7 mL of DMF. The reaction mixture
protected from light was heated at 50 °C for 7 days. Precipitated
KCl was removed by filtration. A solution of 5% aqueous KCl (3
mL) was then added to the filtered solution, and the mixture was
stirred for 2 h. The resulting crude precipitate was filtered off and
purified by repeated washing with small portions of water, ethanol,
and diethyl ether and dried in vacuo to yield 0.050 g (20.00%) of
4. Anal. (C18H18Cl4N4Pt) C, H, N.
[Pt(L5)2Cl2]·6H2O (5). A similar procedure was carried out as
described for compound 2 using L5 (0.200 g, 0.98 mmol) and
K2PtCl4 (0.240 g, 0.58 mmol) at room temperature for 10 days.
Yield: 0.184 g (24.02%). Anal. (C22H24Cl2N4O4Pt·6H2O) C, H, N.
[Pt(L6)2Cl2]·H2O (6). A similar procudure was carried out as
described for compound 2 using L6 (0.070 g, 0.40 mmol) and
K2PtCl4 (0.094 g, 0.23 mmol) at 60 °C for 3 days. Yield: 0.083 g
(32.59%). Anal. (C20H26Cl2N4O3Pt·H2O) C, H, N.
Determination of the Reactivity of the Compounds against
Model Nucleophile I-, Preliminary Cytotoxicity Test, Cellular
Uptake Studies, Interaction with pBR322 Plasmid DNA,
BamH1 and HindIII Restriction Enzyme Digestion and Cell
Cycle Analysis, and SubG1 Detection by Flow Cytometry.
Experimental details are presented in the Supporting Information.
Conclusion
The preliminary data obtained in this study lead us to
conclude that (i) compounds 1, 2, and 6, which were found to
be in vitro antiproliferative active higher or equal to carboplatin
against the human MCF-7 and HeLa cell lines, may be regarded
as having potential antitumor activity. (ii) The results of the
plasmid DNA interaction and the restriction studies suggest that
changing the chemical structure of the benzimidazole ligands
may modulate DNA binding mode and the sequence selectivity.
Although it is not possible to make a clear conclusion about
the necessity of the free N1-H moiety on the cytotoxic activities
of the compounds tested against the cell lines used, the presence
of a hydrogen-bond donor, such as a hydroxyl group or N1-H
moiety on the benzimidazole carrier ligands of the Pt(II)
complexes, seems to have a significant effect on the DNA
unwinding. (iii) The antiproliferative activity mechanism of
compounds 1 and 2 against MCF-7 and HeLa cells, respectively,
is probably different from that of cisplatin. These differences
reflect the distinct nature of the carrier ligands and might make
them promising agents, and further studies on compounds 1 and
2 are warranted. (iv) In general, there is no clear correlation
between the plasmid DNA binding affinity and the stability and
the antiproliferative activity of the compounds tested. (v) In
addition, it also has to be examined whether antiproliferative
activity and the mode of action of the compounds including
cell cycle modification and apoptosis induction are cell-type
dependent.
Experimental Section
Synthesis. All chemicals and solvents used were reagent grade
(Merck or Aldrich or Sigma) and were used without further
purification. Analytical thin-layer chromatography (TLC) was
performed on 60 F254 precoated silica gel plates (Merck). Plates
were visualized by UV light, Dragendorff reagent, or iodine vapor.
Instruments. Melting points were determined with an Electro-
thermal 9200 melting point apparatus and are uncorrected. Elemen-
¨
˙
tal analyses for C, H, N were performed by TUBITAK Laboratory
(Ankara, Turkey) on a CHNS analyzer, model Leco-932, and were
within (0.4% of theoretical values. IR spectra were recorded on
KBr pellets and Nujol mulls on a Mattson 1000 FTIR spectrometer
in the range of 4000-200 cm-1. For the region 400-200 cm-1
,
1
the samples were prepared as Nujol mulls on CsI windows. H
NMR spectra were recorded in DMSO-d6 (Merck) on a Bruker 400
MHz spectrometer. FAB+ mass spectra were recorded on a ZapSpec
spectrometer using 3-nitrobenzyl alcohol (NBA) as the matrix
solvent. The ESI-LC/MS analyses for the identification of the
benzimidazole-Pt(II) complexes synthesized and the determination
of the reactivity of the compounds against model nucleophile I-
were performed with an Agilent 1100 HPLC system (Waldbronn,
Germany). ICP-MS analyses for the determination of platinum
levels of samples for the reactivity of the compounds against I-
and cellular uptake studies were performed by using a standard
Agilent 7500a benchtop ICP/MS fitted with a cross-flow nebulizer
and a quartz spray chamber (experimental details of the spectral
analyses are presented in the Supporting Information). The purity
of the synthesized compounds was established as >95% by
˙
Acknowledgment. We are grateful to Prof. Dr. Ibrahim
Burgu (Faculty of Veterinary Medicine, Ankara University),
Prof. Dr. Ali S¸engül (Department of Immunology, Gülhane
Military Medical Academia, Ankara) and Dr. Recep Ergül (Foot
and Mouth Disease Institute, Ankara, Turkey) for their valuable
support. For helpful assistance we thank Research Assistant
Humay Akın (Faculty of Veterinary Medicine, Ankara Univer-
sity). We thank the Research Foundation of Gazi University
(EF-02/2002-06, EF-02/2004-28 and 02/2007-24) for financial
support.
Supporting Information Available: Experimental details of the
1
1
elemental analysis and H NMR.
Synthesis of the Ligands. Ligands L1-L6, used as carrier ligands
in the structures of the Pt(II) complexes, were prepared according
spectral analyses, melting points, and H NMR data of ligands
1
L1-L6, elemental analysis, IR, H NMR, FAB-MS, and ESI-LC/
MS data of compounds 1-6, crystallographic data for compound

1356 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
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