Synthesis, in vitro cytotoxic and antiviral activity of cis-[Pt(R(-) and S(+)-2-α-hydroxybenzylbenzimidazole)2Cl2] complexes

Article abstract of DOI:10.1016/j.ejmech.2004.09.017

A pair of enantiomeric platinum(II) complexes of cis-[Pt(R(-) and S(+)-HBB)₂Cl₂] (HBB = 2-α- hydroxybenzylbenzimidazole) was synthesized and evaluated for its preliminary in vitro cytotoxic activity on the human MCF-7 breast cancer and HeLa cervix cancer cell lines and antiherpes virus activity against bovine herpesvirus type 1 (BHV-1). In general, it was found that Pt(II) complexes were less cytotoxic on both cell lines than cisplatin and were comparable to carboplatin. There was no significant difference in cytotoxicity between two enantiomers, and the antiviral test results showed that the Pt(II) complexes and their carrier ligands R(-) and S(+) HBB had no effects inhibiting replication of BHV-1.

Full text of DOI:10.1016/j.ejmech.2004.09.017

European Journal of Medicinal Chemistry 40 (2005) 135–141
www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro cytotoxic and antiviral activity of cis-[Pt(R(–)
and S(+)-2-a-hydroxybenzylbenzimidazole)2Cl2] complexes
M. Gökçe a, S. Utku a, S. Gür b, A. Özkul c, F. Gümüs¸ a,*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Gazi, 06330 Etiler-Ankara, Turkey
b Department of Microbiology, Faculty of Veterinary Medicine, University of Kocatepe, Afyon, Turkey
c Department of Virology, Faculty of Veterinary Medicine, University of Ankara, Dıs¸kapı-Ankara, Turkey
Received 21 June 2004; received in revised form 6 September 2004; accepted 10 September 2004
Available online 24 November 2004
Abstract
A pair of enantiomeric platinum(II) complexes of cis-[Pt(R(–) and S(+)-HBB)2Cl2] (HBB = 2-a-hydroxybenzylbenzimidazole) was syn-
thesized and evaluated for its preliminary in vitro cytotoxic activity on the human MCF-7 breast cancer and HeLa cervix cancer cell lines and
antiherpes virus activity against bovine herpesvirus type 1 (BHV-1). In general, it was found that Pt(II) complexes were less cytotoxic on both
cell lines than cisplatin and were comparable to carboplatin. There was no significant difference in cytotoxicity between two enantiomers, and
the antiviral test results showed that the Pt(II) complexes and their carrier ligands R(–) and S(+) HBB had no effects inhibiting replication of
BHV-1.
© 2004 Elsevier SAS. All rights reserved.
Keywords: 2-a-Hydroxybenzylbenzimidazole; Pt(II) complexes; Cytotoxic activity; Antiviral activity
1. Introduction
ing diamines as carrier-ligands as an alternative compound to
cisplatin can slow or block repair enzymes [6].
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]. Despite the success of cispl-
atin, problems regarding intrinsic or acquired resistance and
side effects have encouraged the development of new plati-
num drugs. Acquired resistance, as well as the intrinsic resis-
tance observed in some patients, is multifactoral in nature
and includes contributions from differential drug uptake, cel-
lular detoxification systems, and DNA repair mechanisms
[2,3]. Structural modification of the carrier-ligand in cispl-
atin may alter the spectrum of antitumor activity and over-
come resistance [4].
The studies on the search for new platinum drugs revealed
that not only the chemical constitution but also the configu-
ration of the carrier-ligand determines the extent of the anti-
tumor effect [7]. Since the chirality of the carrier-ligands deter-
mines the tendency of a complex to be transported through
the cell membrane [8] and DNA has a chiral structure and its
reaction with stereoisomeric diamineplatinum(II) complexes
yields adducts which are diastereomeric to each other and
thus hinder the DNA function to different extents, the use of
optically pure chiral ligands might be an alternative means in
order to get better characteristics for platinum compounds.
In a previous paper, we reported the synthesis and cha-
racterization of the complexes of the structure, cis-
[Pt(L2)Cl2]·H2O where L is 5(6)-non/or-chlorosubstituted-2-
hydroxymethylbenzimidazole and the determination of their
preliminary in vitro cytotoxic effects by “Rec-Assay” test
[9]. The DNA-binding properties of these two Pt(II) com-
plexes were also examined and it was determined that the
DNA platinated with these compounds was specifically rec-
ognized by high mobility group (HMG) domain protein,
The carrier-ligand of the platinum DNA-damaging agents
may affect biodistribution; rates and type of DNA adduct for-
mation, recognition of damaged DNA by repair enzymes or
regulatory/binding proteins [5]. The use of sterically demand-
* Corresponding author. Tel./fax: +90 312 212 0236.
E-mail address: fgumus@gazi.edu.tr (F. Gümüs¸).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.09.017

136
M. Gökçe et al. / European Journal of Medicinal Chemistry 40 (2005) 135–141
HMG1 [10]. It was also determined that some of the new
2-substituted benzimidazole Pt(II) complexes we synthe-
sized have in vitro cytotoxic activities on the human RD
Rhabdomyosarcoma [11] and MCF-7 breast cancer [12] cell
lines.
molecular hydrogen bounded imidazole N–H. The com-
plexes exhibited N–H stretching bands centered at 3170 cm–1
sharper than those of the ligands due to breaking of tautomer-
izm, indicating that the N–H group was not involved in the
coordination [16,17]. The complexes showed a broad band
centered at 3422 cm–1 assigned to t (OH) which is not
appreciably shifted from the free ligand values, suggesting
that coordination does not occur through the oxygen.
In the present study to determine whether enantiomorphic
properties of the platinum complexes influence their cyto-
toxic activities, two enantiomerically pure Pt(II) complexes
bearing R(–) or S(+)-2-a-hydroxybenzylbenzimidazole
(HBB) as carrier-ligands were synthesized and evaluated for
their preliminary in vitro cytotoxic activities on the human
MCF-7 breast cancer and HeLa cervix cancer cell lines. With
the consideration that interference of platinum complexes
with viral nucleic acids could be important also in the treat-
ment of viral disease, antiherpesvirus (bovine herpesvirus
type 1, BHV-1) activities of the platinum complexes, cis-
[Pt(R(–)-HBB)2Cl2] and cis-[Pt(S(+)-HBB)2Cl2], were also
tested.
The Pt–N and Pt–Cl vibrations are considered to be char-
acteristic for dichloro-diamine platinum complexes. But the
metal–nitrogen stretching bands could not be distinguished
from other ring skeleton vibrations present in the spectra.
According to the kinetic trans effect [18], the synthesis
method used is expected to yield complexes with cis geom-
etry. In the far IR region of the complexes’s spectra, a new
broad band appeared assigned to t (Pt–Cl) centered at
327 cm–1. As 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 [19]. In some cases, cis-dichloro complexes
show only one band due to low resolutions independent from
cis or trans-configurations [20]. Although no shoulder was
apparently observable at the Pt–Cl stretching band of the
complexes 3 and 4, the broad nature of the band suggested
the presence of the bands overlapped in this domain. In
addition the position of the t (Pt–Cl) band of the complexes
was at 327 cm–1 and the geometry can be assigned as cis. The
other bands in the spectrum of the complexes were similar to
those in the ligands spectrum except for slight shifts in their
positions and changes in their intensities due to coordination.
The insolubility of the complexes in the other organic
solvents made it necessary to record the 1H-NMR spectra in
dimethylsulfoxide-d6 (DMSO-d6). All measurements were
recorded immediately in order to avoid the ligand exchange
reactions between the Pt(II) complexes and DMSO-d6.
The 1H-NMR spectrum of the Pt(II) complexes in
DMSO-d6 is indicative of complex formation. The 1H-NMR
spectra of R(–)- and S(+)-HBB-platinum(II)complexes 3 and
4 are identical. The spectra of the complexes compared to
those of the free ligands showed considerable difference. The
large downfield shifts in the imidazole N–H signal in the
spectra of the complexes with respect to their ligands are a
result of an increase in the N–H acid character after platinum
binding [21]. The chemical shift variation upon coordination
(Dd = d complex – d free ligand) observed, show that some of
the values are positive, indicating a decrease in electronic
density on the 2-substituted benzimidazole ligands with co-
ordination to the platinum. The 1H-NMR signals of the plati-
num complexes tend to broaden, this is consistent with the
literature [22].
2. Results and discussion
2.1. Synthesis and characterization of the complexes
The carrier-ligands R(–)-HBB and S(+)-HBB (1 and 2) in
the Pt(II) complexes (3 and 4) were prepared starting from
commercially available R(–) and S(+) mandelic acid, accord-
ing to Phillips method [13] as shown in Scheme 1. Their
melting points and optical rotation values were identical to
the data reported in the literature [14,15]. The Pt(II) com-
plexes 3 and 4 were synthesized by the reaction of enantio-
merically pure ligands with K2PtCl4 in 0.5 N HCl as de-
scribed in Section 4. The melting points of the complexes
were above 300 °C.
The complexes were characterized by elemental analysis,
1
fast atom bombardment (FAB) mass, infrared (IR) and H-
NMR spectra. It was not possible to obtain suitable crystals
for structural determinations. However, from the results of
the techniques employed it is possible to purpose possible
structures (Scheme 1). Elemental analysis suggested a 1:2
(metal/ligand) stoichiometry for the complexes.
The IR spectra of the complexes have shown characteristic
changes when compared to free ligands. The ligands show
broad bands in the region 3530–2300 cm–1 due to the inter-
In the FAB MS spectrum of platinum complex 3, the peak
with very low intensity corresponding to its molecular ion,
M+, was shown at m/z 713.9 (calc. 714.51) while in the
spectrum of complex 4 this peak did not appear. Peaks at m/z
609.6 and 609.5 assignable to fragments corresponding to the
loss of Cl– and OH– ions of platinum complexes 3 and 4,
Scheme 1
K2PtCl4, 0.5 N HCl, 50 °C.
. (a) R(–) or S(+)-mandelic acid, 4 N HCl, reflux 1–2 days, (b)

M. Gökçe et al. / European Journal of Medicinal Chemistry 40 (2005) 135–141
137
respectively, were observed. The loss of Cl– ions has been
well documented for similar structure of complexes [23].
The test results for the platinum(II) complexes on the
human HeLa cervix cancer cell line showed that the com-
plexes were less active than cisplatin. Considerable T/Ccorr.
values of the complexes 3 and 4 on HeLa cell line were in
between 40 and 20 µM concentration. (Table 2).
2.2. Biological activities
In general, it was found that Pt(II) complexes were less
cytotoxic on both cell lines than cisplatin and were compa-
rable to carboplatin. Although the complexes bearing R(–)-
HBB ligands were found to be slightly more active than the
complexes bearing S(+)-HBB ligands on the MCF-7 cell
line, no significant differences between the enantiomeric
forms of platinum(II) complexes were observable. These
results are consistent with previous reported data that the
differences in biological activity of enantiomeric forms of
platinum(II) compounds with chiral amine ligands are not so
pronounced unless the rational freedom of the asymmetric
center is reduced by placing, for instance, the chiral center(s)
on the carbon chain linking two nitrogen atoms of a chelate
diamine [7].
Although there is some evidence to suggest that other
biological targets may be important in the cisplatin mecha-
nism, it is generally accepted that DNA is the primary target
[24]. DNA-binding reactions of platinum complexes are usu-
ally viewed as kinetically controlled processes [25,26].
Steric hindrance of the carrier-ligands can slow reaction
kinetics [27]. The presence of bulky, planar amine ligands in
[Pt(anion)2] complexes and their orientation with respect to
the coordination plane, as well as their substituents, can
reduce the rates of DNA-binding compared to aliphatic am-
mine and amine complexes [6]. The ligands used in this study
are not flate due to the a-hydroxybenzyl moiety. This may
result in severe steric constraints around the Pt atom, making
the complexes less reactive than cisplatin. For the same
reason Pt–Cl hydrolysis of the platinum complexes 3, 4 may
be relatively slow compared to cisplatin. A detailed study
2.2.1. Preliminary cytotoxicity test
The preliminary antiproliferative activity of the platinum
complexes bearing enantiomeric R(–) or S(+)-HBB groups
as carrier-ligands were determined on the human MCF-
7 breast cancer and HeLa cervix cancer cell lines. MCF-7 and
HeLa cells were incubated for 72 h with 40, 20, 10, 1, 5,
0.5 µM of the Pt(II) complexes and cisplatin and carboplatin
used as reference compounds. The antiproliferative activity
values of the complexes and the reference compounds ex-
pressed as T/Ccorr. are presented in Table 1.
In the test on the human MCF-7 cancer cell line at 0.5, 1,
5 µM concentration T/Ccorr. values of the [Pt(R(–)/and S(+)-
HBB)Cl2] (3 and 4) except for complex 3 at 5 µM concentra-
tion were >100. At these concentrations, the reference cispl-
atin and carboplatin had T/Ccorr. values of ca. 82.89%,
66.69%, 10.52% and >100%, >100%, 81.64%, respectively.
At 10 µM concentration, cytotoxic effects of the complexes 3
and 4 amounted to T/Ccorr. values of ca. 57.91% and 80.85%,
respectively. While at the same concentration T/Ccorr. values
of the reference compounds cisplatin and carboplatin were
found to be –22.44% (cytocidal effect) and 87.79%, respec-
tively.
A clear antiproliferative effect was observed for both
complexes synthesized by increasing the concentration to
20 µM. At these dosage T/Ccorr. values of complexes 3 and 4
were around 12.63% and 16.99%, respectively. At this con-
centration cytocidal effects were observed for cisplatin and
carboplatin (–42.80% and –8.88%, respectively). At the con-
centration of 40 µM, cytocidal effects were observed for the
complexes synthesized and for the reference compounds.
Table 1
Effect of the Pt(II) complexes on the proliferation of MCF-7 cell line
[T/C]corr.
(%) (S.D.) a
Compound
40 µM
20 µM
10 µM
5 µM
1 µM
0.5 µM
>100
Cis-[Pt(R(–)HBB)2Cl2]
Cis-[Pt(S(+)HBB)2Cl2]
Cisplatin
–51.55 1.12 b
–20.86 4.92 b
–49.91 6.26 b
–9.88 0.25 b
12.63 4.72
16.99 1.24
–42.80 20.47 b
–8.88 3.51 b
57.91 26.89
80.85 18.24
–22.44 3.00 b
87.79 20.17
94.26 2.83
>100
>100
>100
>100
10.52 5.14
81.64 1.31
66.69 20.15
>100
82.89 4.90
>100
Carboplatin
a S.D., standard deviation.
b Cytocidal effect.
Table 2
Effect of the Pt(II) complexes on the proliferation of HeLa cell line
[T/C]corr.
(%) (S.D.) a
Compound
40 µM
20 µM
10 µM
5 µM
1 µM
>100
>100
0.5 µM
>100
Cis-[Pt(R(–)HBB)2Cl2]
Cis-[Pt(S(+)HBB)2Cl2]
Cisplatin
–5.37 1.01 b
8.12 0.50
–48.60 12.85 b
55.69 9.95
36.18 8.15
–53.69
50.40 4.72
66.22 5.16
84.66 9.38
71.07 6.89
>100
–58.81 15.68 –54.43 15.00 13.21 4.85
44.03 4.40
–14.90 b
b
Carboplatin
16.13 2.43
59.37 20.43
76.78 19.22
89.72 11.03
>100
>100
a S.D., standard deviation.
b Cytocidal effect.

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M. Gökçe et al. / European Journal of Medicinal Chemistry 40 (2005) 135–141
determining the complex binding and hydrolysis rate con-
stants of the complexes will be necessary to investigate these
hypotheses. The insolubility of the Pt(II) complexes in water
compared to cisplatin may also be related to their lower
cytotoxicity.
Plaque assay revealed no significant difference in plaque
numbers and plaque sizes which were detected between the
viruses incubated with the platinum complexes and R(–)-
HBB ligand (Table 3). Based on the data obtained from
plaque assay, one step growth curves of the BHV-1 showed
that there are no obvious changes between growth kinetics of
the viruses. These results indicated that platinum complexes
3, 4 and R(–)-HBB ligand have no effects inhibiting replica-
tion of BHV-1.
2.2.2. Antiherpes virus effect tests
Although it is well known that HBB selectively inhibits
only certain viruses belonging to the Picornavidae family,
while it is completely inactive against the main viruses of
other families [15,28], in this study with the aim of evaluat-
ing to what extent complexation affects the antiherpesvirus
activity of the R(–) and S(+)-HBB ligands, the antiviral
activity of the ligands and the complexes against BHV-
1 were tested.
In all series of BHV-1 infected Madin-Darby bovine kid-
ney (MDBK) cells incubated with 150, 50 and 20 µM of
platinum complexes 3, 4 and also 10 µM of R(–)-HBB ligand
1, high toxicity resulted in massive cellular death were found
as early as at 8–12 h postinoculation (pi). S(+)-HBB 2 was
found extremely toxic for MDBK cells in its all concentra-
tions used in research. The rest of the cells completed their
incubation period in parallel to non-treated MDBK cells
showing BHV-1 indicative cytopathic changes until 84 h pi
(Fig. 1).
3. Conclusion
The main purpose of this work was to investigate whether
differences in cytotoxicity on MCF-7 and HeLa cell lines
exist between a pair of enantiomeric complexes, [Pt(R(–)-
HBB)2Cl2] and [Pt(S(+)-HBB)2Cl2] and to test their antiher-
pesvirus type 1 (BHV-1) activities. The test results showed
that no significant differences in cytotoxicity between enan-
tiomers were observable and the platinum complexes and
their carrier-ligands, R(–) and S(+)-HBB, had no effects
inhibiting replication of BHV-1. In general, the complexes,
which were found to be less cytotoxic than cisplatin, exhib-
ited moderate cytotoxicity comparable to carboplatin in both
MCF-7 and HeLa cell lines. In conclusion, complexes 3 and
Fig. 1. One step growth curve of BHV-1 in MDBK cells in the presence of Pt(II) complexes (3) and (4) and ligand R(–)-HBB (1).
Table 3
Average plaque numbers of BHV-1 dilutions treated and untreated with Pt(II) complexes (3) and (4) and ligand R(–)-HBB (1)
Log
Plaque numbers of BHV-1 after incubation with
10 dilutions
3
4
R(–)-HBB (1)
Plain
DMEM
UC
UC
UC
UC
86
10 µM
UC
UC
UC
UC
79
5 µM
UC
UC
UC
UC
83
1 µM
UC
UC
UC
UC
82
10 µM
UC
UC
UC
UC
78
5 µM
UC
UC
UC
UC
81
1 µM
UC
UC
UC
UC
81
5 µM
UC
UC
UC
UC
80
1 µM
UC
UC
UC
UC
91
1
2
3
4
5
6
7
11
11
12
10
10
11
11
10
11
1
2
2
1
1
1
0
0
2
UC, uncountable.

M. Gökçe et al. / European Journal of Medicinal Chemistry 40 (2005) 135–141
139
4 should be considered for further antitumor activity studies
within an extended series of structural derivatives and a
broader range of cell lines.
[␣]D20: R(–)-HBB hydrochloride: –82.3° (c = 1, water)
(–83.6° (water) [14], –82.7° (water) [15]), S(+)-HBB hydro-
chloride: +83.7° (c = 1, water) (+84.5° (water) [14], +83.2°
(water) [15]), m.p. 210 °C (isopropyl alcohol/ether (1:1))
(210–211 °C [15]).
4. Experimental
4.1.3. Synthesis of cis-[Pt(R(–)-HBB)2Cl2] (3)
and cis-[Pt(S(+)-HBB)2Cl2] (4)
4.1. Chemistry
To a stirred solution of R(–) or S(+)-HBB (0.45 g, 2 mmol)
in 0.5 N HCl (10 ml) was added a solution of K2PtCl4
(0.415 g, 1 mmol) in 0.5 N HCl (10 ml) drop wise over 2 h at
room temperature. The reaction mixture, protected from
light, was heated at 50 °C for 1 or 2 days for R(–) or S(+)
enantiomer, respectively. A pink solution and reddish oil was
formed during this period. The solution was discarded and
the reddish oil was solidified to give a pink precipitate by
treating with ethanol/ether mixture (1:1 volume) and stirring
for 1 h, which was then collected by filtration, washed with
ethanol followed by ether. This procedure was repeated for
three times. The product was dried in vacuo.
4.1.1. Materials
Melting points were measured on an Electro thermal
9200 melting point apparatus and are uncorrected. Elemental
˙
analyses were performed by TÜBITAK Laboratory (Ankara,
Turkey). 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, the samples
were prepared as Nujol mulls on CsI windows. Proton Mag-
netic Resonance (1H-NMR) spectra were recorded in
DMSO-d6 (Merck) on a Bruker 400 MHz spectrometer. All
chemicals and solvents used were of reagent grade (Merck,
Aldrich, Sigma), and were used without further purification.
Thin layer chromatography (TLC) was performed on pre-
coated aluminum plates (Silicagel 60 F254, Merck). Plates
were visualized by UV light, Dragendorff reagent, and iodine
vapor. Specific rotations were measured with a Rudolph
Research Analytical Autoral (IV) polarimeter, at room tem-
perature (20 °C); concentrations are expressed as g per
100 ml. Positive-ion FAB+ mass spectra were recorded on a
ZapSpec spectrometer using 3-nitrobenzyl alcohol (NBA) as
the matrix solvent.
1H-NMR (DMSO-d6): d 13.65 (broad s, 2H, 2 × N–H,
exchangeable with D2O), 7.98–7.54 (m, 4H, Ar–H), 7.39–
7.12 (m, 8H, Ar–H), 6.88–6.32 (m, 6H, Ar–H), 6.25 (s, 2H, 2
× O–H, exchangeable with D2O), 5.71 (s, 2H, 2 × CH), IR
(KBr): t 3170 (N–H), 1036 (C–O), 327 (Pt–Cl) cm–1. Cis-
[Pt(R(–)-HBB)2Cl2] 3: yield: 44.8%, 0.320 g pure. FAB-
MS(+): m/z 713.9 [M]+, calc. 714.51, 609.6 [M – 2Cl– –
2OH–]+, calc. 609.58. Anal. calc. for C28H24Cl2N4O2Pt: C,
47.06; H, 3.38; N, 7.84%. Found: C, 46.52; H, 3.78; N,
7.09%. Cis-[Pt(S(+)-HBB)2Cl2] 4: yield: 39.9%, 0.285 g
pure. FAB-MS(+): m/z 609.5 [M – 2Cl– – 2OH–]+, calc.
609.58. Anal. calc. for C28H24Cl2N4O2Pt: C, 47.06; H, 3.38;
N, 7.84%. Found: C, 47.22; H, 3.32; N, 7.39%.
4.1.2. Synthesis of R(–) and S(+)-2-␣-hydroxybenzyl-
benzimidazoles. [R(–) and S(+) HBB] (1 and 2)
R(–) or S(+)-mandelic acid 2.28 g (15 mmol) and 1,2-
phenylendiamine 1.08 g (10 mmol) were dissolved in 10 ml
4 N hydrochloric acid and refluxed for 3 h. The reaction
mixture was then allowed to cool to room temperature and
placed in a refrigerator (5 °C) for 1 h. A white precipitate,
formed, was filtered off and then dissolved in 25 ml water.
The clear solution was neutralized with a solution of 20%
aqueous solution of K2CO3 and a white precipitate which
formed, was filtered off, washed with water and recrystal-
lized several times from ethanol/water (1:1). R(–)-HBB 1
yield: 45.86%, 1.03 g pure, S(+)-HBB 2 yield: 43.63%,
0.98 g pure, m.p. 204 °C (ethanol/water (1:1)) (lit. 205–
4.2. Biological test
4.2.1. Preliminary cytotoxicity test
4.2.1.1. Cell lines and growth conditions. The human MCF-
7 breast and HeLa cervix cancer cell lines used in this study
was obtained from the Cell Culture Collection (HUKUK No:
00092502 and 90061901, respectively) of Institute for Foot
and Mouth Disease (IFMD) (Turkey).
The cells were grown in Dulbecco’s (Seromed, Germany)
minimal essential medium (DMEM) enriched with 10% fetal
calf serum (FCS) (Biochrom, Germany), 100 mg ml–1 strep-
tomycin and 100 IU ml–1 penicillin in a humidified atmo-
sphere of 5% CO2 at 37 °C. The cells were harvested using
1
207 °C [29]). H-NMR (DMSO-d6): d 12.24 (broad s, 1H,
N–H), 7.52–7.49 (m, 4H, Ar–H), 7.37–7.34 (m, 2H, Ar–H),
7.29–7.25 (m, 1H, Ar–H), 7.19–7.15 (m, 2H, Ar–H), 6.57 (s,
1H, OH), 5.97 (s, 1H, CH). IR (KBr): t 3425 (O–H), 3200–
3100 (N–H), 1045 (C–O) cm–1.
Trypsin
(Bibco
Life
Technologies,
UK)/Versen
For evaluation of the specific rotations of the enantiomers,
the hydrochlorides of R(–) and S(+) HBB were prepared,
since the optical rotation values [␣]D for the base form of the
enantiomers in absolute ethanol and acetone were very low
[14]. R(–) or S(+) HBB were dissolved in absolute ether and
saturated with a stream of dry HCl. The precipitate was
filtered off and recrystallized from isopropyl alcohol/ether
(1:1).
(0.05%:0.02%) solution. Mycoplasma contamination was
routinely monitored and only mycoplasma-free cultures
were used.
4.2.1.2. In vitro chemosensitivity assay on the human
MCF-7 breast and HeLa cervix cancer cell lines. The
preliminary in vitro testing of the platinum complexes on

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M. Gökçe et al. / European Journal of Medicinal Chemistry 40 (2005) 135–141
antitumor activity was carried out on human MCF-7 breast
and HeLa cervix cancer cells according to a previously pub-
lished microtiter test [30]. Briefly, the cells were seeded in to
96-well plates (Greiner GmbH, Germany) in a volume of
100 µl as to be 18–22 cells per microscopic area. After
attachment to the culture surface, the cells were incubated in
an atmosphere containing 5% CO2 at 37 °C for 24 h. After
48 h, the growth medium was carefully removed by suction
and 200 µl of fresh medium were added into each well. The
medium used contained an adequate volume of a stock solu-
tion of the respective compound in order to obtain the desired
test concentration (0.5, 1, 5, 10, 20 and 40 µM solvent:
DMSO, the complexes tested were added to the culture
medium such that the final DMSO was 0.1% (v/v)). Sixteen
wells were used for each compound (complex 3, 4, cisplatin
and carboplatin) tested for individual concentrations while
16 wells were reserved for the cell culture control, which
contained the corresponding amount of DMSO. After the
72 h incubation time, the medium was removed and the cells
were fixed with 100 µl 1% glutardialdehyde in phosphate-
buffered saline (PBS) per well for 25 min. The fixative was
replaced by 150 µl PBS/well and the plates were stored in the
refrigerator (4 °C). Cell biomass was determined by a crystal
violet staining technique [31].
BHV-1 Cooper strain at a multiplicity of infection (MOI) of
0.1 plaque morphing unit (PFU) per cell. Cells were incu-
bated at 37 °C for 1 h for providing viral attachment onto cell
membrane. After intensive washing of cell surface with pre-
warmed PBS, DMEM containing the platinum compounds
and ligands (dissolved in DMSO) in final concentrations of
150, 50, 20, 10, 5 and 1 µM were added to culture flasks. One
series of cell were incubated with plain DMEM serving as
non-treated virus control. The cells infected were further
incubated at 37 °C for several days until cytopathic effect
(cpe) in cells infected with non-treated BHV-1 has appeared
during daily microscopic evaluations. The cultures were fro-
zen and thawed twice after cpe reached 95% of cells. The
yields of the virus were aliquoted in small volumes and
stored at –80 °C until use.
4.2.2.3. Detection of infectivity titer. To detect in vitro infec-
tivity of the viruses after incubation with the compounds
tested, the plaque assay was used. Confluent MDBK cells
grown in 24-well plates were infected with 10-fold serial
dilutions (0.2 ml) of viruses harvested from cells incubated
with/without the compounds. After 1 h adsorption at 37 °C,
cells were overlaid with 1.6% carboxymethylcellulose in
DMEM. The cells were incubated for further 48 h and then
were fixed with 10% formaldehyde for 1 h at room tempera-
ture. After removal, fixative cells were stained with crystal
violet (0.35%) in ethanol. Plaques formed by per dilution
were measured (or counted) and represented as the average
of duplicate inoculation wells.
The effects of the platinum complexes were expressed as
corrected T/C values according to the following equations:
T/Ccorr. % = T - Co / C - Co × 100
͔ ͓
͓
͑
͒ ͑
͔͒
where T is the mean absorbance of the treated cells, C is the
mean absorbance of the controls, and Co is the mean absor-
bance of the cells at the time (t = 0) when the drug was added.
When the absorbance of treated cells was less than that of
the culture at t = 0 (Co), the extent of cell killing was
calculated as
4.2.2.4. One step growth curve. The growth dynamics of the
virus after incubation with and without the compounds tested
were performed as described by Chowdhury et al. [32].
Briefly, confluent MDBK cells were infected with viruses at
a MOI of 5 PFU per cell. After 1 h of adsorption at 4 °C,
residual input viruses were removed. The cultures were
washed three times with PBS, and 5 ml of medium was added
to each flask before further incubation (37 °C). At indicated
time intervals, replicate cultures were frozen. Virus yields
were determined by titration on MDBK cells. Each data point
represented the average of duplicate samples obtained from
separate infections.
Cytocidal effect % = Co - T /Co × 100
͔ ͓
͓
͑
͒
͔
Absorbance was measured at 578 nm using a Titertek
Multiscan Plus MKII Autoreader.
All platinum (II) complexes were tested in two indepen-
dent duplicates.
4.2.2. In vitro antiherpes virus activity tests
4.2.2.1. Virus and cell culture. Cooper strain of BHV-1 used
in the research was propagated in MDBK cells with routine
procedure. The virus was stored at –80 °C in small aliquots
after purification and infectivity titer of the virus was also
calculated.
Acknowledgements
˙
We would like to thank Professor Dr. Ibrahim Burgu
(Faculty of Veterinary Medicine, Ankara University) for his
valuable support. Financial support of part of this work (EF
02/2000-20) by the Research Foundation of Gazi University
is gratefully acknowledged.
4.2.2.2. Test for antiviral effect. Serial concentrations of
R(–) and S(+)-HBB ligands and their platinum complexes 3
or 4 were tested on the BHV-1 infected MDBK cells and the
results were compared in terms of plaque size and number
detected pi in parallel to non-treated BHV-1. MDBK cells
monolayered in tissue culture flasks were infected with
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