Synthesis and characterization of mixed-ligand diimine-piperonal thiosemicarbazone complexes of ruthenium(II): Biophysical investigations and biological evaluation as anticancer and antibacterial agents

Article abstract of DOI:10.1016/j.molstruc.2011.02.029

We have used a novel microwave-assisted method developed in our laboratories to synthesize a series of ruthenium-thiosemicarbazone complexes. The new thiosemicarbazone ligands are derived from benzo[d][1,3]dioxole-5- carbaldehyde (piperonal) and the complexes are formulated as [(diimine) ₂Ru(TSC)](PF₆)₂ (where the TSC is the bidentate thiosemicarbazone ligand). The diimine in the complexes is either 2,2′-bipyridine or 1,10-phenanthroline. The complexes have been characterized by spectroscopic means (NMR, IR and UV-Vis) as well as by elemental analysis. We have studied the biophysical characteristics of the complexes by investigating their anti-oxidant ability as well as their ability to disrupt the function of the human topoisomerase II enzyme. The complexes are moderately strong binders of DNA with binding constants of 10⁴ M ^-1. They are also strong binders of human serum albumin having binding constants on the order of 10⁴ M^-1. The complexes show good in vitro anticancer activity against human colon cancer cells, Caco-2 and HCT-116 and indeed show some cytotoxic selectivity for cancer cells. The IC₅₀ values range from 7 to 159 μM (after 72 h drug incubation). They also have antibacterial activity against Gram-positive strains of pathogenic bacteria with IC₅₀ values as low as 10 μM; little activity was seen against Gram-negative strains. It has been established that all the compounds are catalytic inhibitors of human topoisomerase II.

Full text of DOI:10.1016/j.molstruc.2011.02.029

Journal of Molecular Structure 992 (2011) 39–47
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis and characterization of mixed-ligand diimine-piperonal
thiosemicarbazone complexes of ruthenium(II): Biophysical investigations
and biological evaluation as anticancer and antibacterial agents
a
a
a
a
Floyd A. Beckford a, , Jeffrey Thessing , Michael Shaloski Jr. , P. Canisius Mbarushimana , Alyssa Brock ,
⇑
Jacob Didion a, Jason Woods a, Antonio Gonzalez-Sarrías b, Navindra P. Seeram b
a Science Division, Lyon College, Batesville, AR 72501, USA
b Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 January 2011
Received in revised form 15 February 2011
Accepted 16 February 2011
Available online 19 February 2011
We have used a novel microwave-assisted method developed in our laboratories to synthesize a series of
ruthenium-thiosemicarbazone complexes. The new thiosemicarbazone ligands are derived from
benzo[d][1,3]dioxole-5-carbaldehyde (piperonal) and the complexes are formulated as [(diimine)2
Ru(TSC)](PF6)2 (where the TSC is the bidentate thiosemicarbazone ligand). The diimine in the complexes
is either 2,20-bipyridine or 1,10-phenanthroline. The complexes have been characterized by spectroscopic
means (NMR, IR and UV–Vis) as well as by elemental analysis. We have studied the biophysical charac-
teristics of the complexes by investigating their anti-oxidant ability as well as their ability to disrupt the
function of the human topoisomerase II enzyme. The complexes are moderately strong binders of DNA
with binding constants of 104 Mꢀ1. They are also strong binders of human serum albumin having binding
constants on the order of 104 Mꢀ1. The complexes show good in vitro anticancer activity against human
colon cancer cells, Caco-2 and HCT-116 and indeed show some cytotoxic selectivity for cancer cells. The
Keywords:
Thiosemicarbazone
Diimine
DNA
Anticancer
Human serum albumin
Topoisomerase II
IC50 values range from 7 to 159
l
M (after 72 h drug incubation). They also have antibacterial activity
M; little activity
against Gram-positive strains of pathogenic bacteria with IC50 values as low as 10
l
was seen against Gram-negative strains. It has been established that all the compounds are catalytic
inhibitors of human topoisomerase II.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
Ruthenium complexes of diimine ligands such as 2,20-bipyridine
(bpy) and 1,10-phenanthroline (phen) are widely used in
Schiff bases are an important set of chelating ligands in main
group and transition metal coordination chemistry and have
become an important class of compounds in medicinal and phar-
maceutical fields as well [1–3]. Metal complexes of Schiff bases
have been designed and synthesized to explore their pharmacolog-
ical activity, for instance finding applications as model analogs of
certain metallo-enzymes. One particular set of Schiff bases that
has been studied very aggressively over the past decade is the thio-
semicarbazones. Thiosemicarbazones are of considerable pharma-
cological interest since a number of derivatives have shown a
broad spectrum of chemotherapeutic properties. The wide range
of biological activities possessed by substituted thiosemicarba-
zones includes cytotoxic, antitumor [4], antibacterial [5], and anti-
viral [6] properties. The biological properties of the ligands can be
modified and enhanced by linkage to metal ions [7–9].
bioinorganic chemistry particularly as probes for DNA. Some of these
compoundsalsopossessinteresting anticancerproperties andmay be
candidates for drugs [10–15]. While it is believed that DNA is a pri-
mary target for such complexes, since DNA replication is integral to
the progression of these diseases, there is also the recognition that
the observed biological activity is not always related to their DNA-
binding ability. Consequently studies that seek to investigate other
possible targets such as enzymes and other proteins are being under-
taken [16–18]. Thebindingofdrugstoplasmaproteinsisafundamen-
tal factor, important in determining the overall pharmacological
activity of the drug. Among the human serum proteins, albumin
(HSA)acts as a reservoir for a long duration of action, and binding ulti-
mately affects drug absorption, metabolism, distribution and excre-
tion, properties that are of key importance to drug development.
Since HSA serves as a transport carrier for drugs, it is important to
study the interactions of potential drugs with this protein.
In this paper we report on a study of a family of mixed-ligand
diimine ruthenium complexes of the type [(bipy)2Ru(TSC)](PF6)2
⇑
Corresponding author. Tel.: +1 870 307 7212; fax: +1 870 307 7496.
E-mail address: floyd.beckford@lyon.edu (F.A. Beckford).
and [(phen)2Ru(TSC)](PF6)2 where TSC is
a
chelating
0022-2860/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2011.02.029

40
F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
thiosemicarbazone ligand derived from piperonal. We report on
their biophysical reactivity (-interaction with DNA and human ser-
um albumin), their cytotoxicity towards a number of human can-
cer cell lines and their antibacterial behavior towards a number
of pathogenic and facultative bacteria. We also report their
capability as anti-oxidants by investigating their reactivity with
2,2-diphenyl-1-picrylhydrazyl (dpph) radicals.
N, 16.58. m.p. 169 °C. IR (cmꢀ1):
1589;
(C@S) 1255, 832. 1H NMR (300.08 MHz, DMSO-d6):
m (NH2, NH) 3375, 3143; m (C@N)
m
d = 6.07 (2H, s, H1), 7.66 (1H, s, H3), 7.07 (1H, d J = 8.1 Hz, H5),
6.92 (1H, d J = 7.8 Hz, H6), 8.56 (1H, s, H8), 7.94 (1H, s, NbH), 11.32
(1H, s, NaH), 3.58 (2H, q, NbACH2), 1.14 (3H, t, NbACH2ACH3). 13C
NMR (75.463 MHz, DMSO-d6); d = 14.72 (NbACH2ACH3), 38.22
(NbACH2ACH3), 101.47 (C1), 105.23 (C6), 108.19 (C3), 123.78 (C5),
128.84 (C4), 141.50 (C8), 148.83 (C2), 148.05 (C7), 176.46 (C9).
pPhTSC. Yield 94% of a light-yellow solid. Analysis – Calc. for
2. Experimental
C
15H13N3O2S: C, 60.18; H, 4.38; N, 14.04. Found: C, 60.21; H, 4.50;
N, 13.95. m.p. 189 °C. IR (cmꢀ1):
(NH2, NH) 3353, 3134; (C@N)
1597;
(C@S) 1253, 869. 1H NMR (300.08 MHz, DMSO-d6): d = 6.08
2.1. Materials and methods
m
m
m
Analytical or reagent grade chemicals were used throughout. All
the chemicals including solvents were obtained from Sigma–
Aldrich (St. Louis, MO, USA) or other commercial vendors and used
as received. The metal complexes were synthesized using a
Discover S-Class microwave reactor (CEM, Matthews, USA). Micro-
analyses (C, H, N) were performed by Desert Analytics, Tucson,
USA. Proton and carbon nuclear magnetic resonance (NMR) spectra
(2H, s, H1), 7.85 (1H, s, H3), 7.55 (1H, d J = 14 Hz, H5), 7.38 (1H, d
J = 15 Hz, H6), 8.07 (1H, s, H8), 10.11 (1H, s, NbH), 11.74 (1H, s,
NaH), 6.93–7.25 (5H, m, NbAC6H5). 13C NMR (75.463 MHz, DMSO-
d6); d = 101.50 (C1), 105.67 (C6), 108.18 (C3), 142.67 (C8), 149.09
(C2), 148.10 (C7), 175.78 (C9). The phenyl carbons were located in
a closely-spaced cluster between 120 and 130 ppm that overlapped
with the signals from the aromatic ring of the benzodioxole moiety.
were recorded in dimethylsulfoxide-d6 on
a Varian Mercury
300 MHz spectrometer operating at room temperature. The resid-
ual 1H and 13C present in DMSO-d6 (2.50 and 39.51 ppm respec-
tively) were used as internal references. Infrared (IR) spectra in
the range 4000–500 cmꢀ1 were obtained using the ATR accessory
on a Nicolet 6700 FTIR spectrophotometer. The electronic spectra
were recorded using quartz cuvettes on an Agilent 8453 spectro-
photometer in the range 190–1100 nm using samples dissolved
in DMSO. Fluorescence spectra were recorded on a Varian Cary
Eclipse spectrophotometer.
2.1.1.2. Metal complexes. The starting ruthenium complexes,
[(phen)2RuCl2]ꢁH2O and [(bpy)2RuCl2], were synthesized as de-
scribed in the literature [19]. The target complexes were synthe-
sized by the following general method: Equimolar amounts of
[(phen)2RuCl2]ꢁH2O or [(bpy)2RuCl2] and the appropriate ligand
was suspended in 8–10 mL of ethylene glycol in a 35-mL reaction
vessel. The vessel was capped and the reaction mixture saturated
with argon for 15 min. The reaction vessel was then placed in the
microwave reactor and heated at 150 °C for 5 min (using a dynamic
method). The dark brown suspension became a dark red solution.
This solution was poured onto 5–10 mL of a saturated aqueous
solution of KPF6 which resulted in the immediate precipitation of
a red solid. The solid was collected by vacuum filtration, washed
with water followed by ether and then dried at the vacuum pump.
The product was recrystallized from dichloromethane/ether (1 and
2), ethanol/hexanes (3 and 4) or ethanol/ether (5).
2.1.1. Synthesis of compounds
2.1.1.1. Ligands. The ligands, 2-(benzo[d][1,3]dioxol-5-ylmethyl-
ene)hydrazinecarbothioamide, (HpTSC), 2-(benzo[d][1,3]dioxol-
5-ylmethylene)-N-methylhydrazinecarbothioamide,
(MepTSC),
2-(benzo[d][1,3]dioxol-5-ylmethylene)-N-ethylhydrazinecarbo
thioamide, (EtpTSC) and 2-(benzo[d][1,3]dioxol-5-ylmethyl-
ene)-N-phenylhydrazinecarbothioamide (PhpTSC), were syn-
thesized as follows: Equimolar amounts piperonal (benzo[d]
[1,3]dioxole-5-carbaldehyde) and the appropriate N(4) alkyl-
substituted thiosemicarbazide were suspended in 80 mL of
absolute anhydrous ethanol containing a few drops of glacial
acetic acid. The reaction mixture was heated at reflux for 3–
4 h and a pale yellow suspension resulted. The reaction mixture
was cooled and filtered through a glass-sintered crucible. The
pale-yellow solid which was obtained was thoroughly washed
with ethanol followed by ether and dried by suction.
[(bpy)2Ru(HpTSC)](PF6)2. 1. Red solid. Yield: 180 mg (47%). Analy-
sis; Calc. for C29H25F12N7O2P2Ru S: C, 37.59; H, 2.72; N, 10.58. Found:
C, 37.84: H, 2.73; N, 10.75. IR (cmꢀ1):
m
(NH2, NH) 3424(w), 3373,
(NAN) 1036; (C@S) 1255, 830; kmax
); 294 nm (3.8); 430 nm (broad).
3152(w);
(log
m
(C@N) 1620;
m
m
e
[(bpy)2Ru(EtpTSC)](PF6)2. 2. Red solid. Yield: 259 mg (66%). Anal-
ysis; Calc. for C31H29F12N7O2P2Ru S: C, 39.00; H, 3.06; N, 10.26.
Found: C, 39.29; H, 3.18; N, 10.18. IR (cmꢀ1):
m
(NH2, NH) 3421(w),
3113(w, b);
(log ); 303 nm (3.8); 276 nm (broad).
m (C@N) 1586; m (NAN) 1034; m (C@S) 1247, 827; kmax
e
pHTSC. Yield 80% of an off-white solid. Analysis – Calc. for
C9H9N3O2S: C, 48.42; H, 4.06; N, 18.82. Found: C, 48.40; H, 4.02;
[(phen)2Ru(HpTSC)](PF6)2ꢁ0.25C4H10O. 3. Red solid. Yield: 166 mg
(47%). Analysis; Calc. for C34H27.5F12N7O2.5P2Ru S: C, 41.12; H, 2.79;
N, 18.67. m.p.189 °C. IR (cmꢀ1):
m
(NH2, NH) 3427, 3252, 3153;
m
N, 9.87. Found: C, 41.84; H, 2.42; N, 10.33. IR (cmꢀ1):
m
(NH2, NH)
(NAN) 1036; (C@S)
e); 300 nm (sh); 430 nm (broad).
(C@N) 1592;
m
(C@S) 1260, 838. 1H NMR (300.08 MHz, DMSO-
3424, 3373(w), 3152(w);
1251, 830; kmax (log
m
(C@N) 1620;
m
m
d6): d = 6.04 (2H, s, H1), 7.63 (1H, s, H3), 7.04 (1H, d J = 8.4 Hz,
H5), 6.89 (1H, d J = 7.8 Hz, H6), 7.92 (1H, s, H8), 8.01 (1H, s, NbH),
8.04 (1H, s, NbH), 11.3 (1H, s, NaH). 13C NMR (75.463 MHz,
DMSO-d6); d = 102.01 (C1), 105.97 (C6), 108.81 (C3), 124.55 (C5),
129.42 (C4), 142.68 (C8), 148.70 (C2), 149.54 (C7), 178.30 (C9).
pMeTSC. Yield 96% of a pale-yellow solid. Analysis – Calc. for
[(phen)2Ru(MepTSC)](PF6)2. 4. Dark-red solid. Yield: 256 mg
(71%). Analysis; Calc. for C34H27F12N7 O2P2Ru S: C, 41.30; H, 2.75;
N, 9.92. Found: C, 42.20; H, 2.62; N, 10.19. IR (cmꢀ1):
m
(NH2,
NH) 3424(w), 3337(w);
m
(C@N) 1620;
m
(NAN) 1036;
m
(C@S)
1256, 833; kmax (log ); 360 nm (sh); 440 nm (broad).
e
C
10H11N3O2S: C, 50.62; H, 4.67; N, 17.71. Found: C, 50.62; H, 4.62;
N, 17.50. m.p. 209 °C. IR (cmꢀ1):
(NH2, NH) 3344, 3152; (C@N)
1590;
(C@S) 1283, 828. 1H NMR (300.08 MHz, DMSO-d6): d =
[(phen)2Ru(EtpTSC)](PF6)2. 5. Dark-red solid. Yield: 286 mg
m
m
(71%). Analysis; Calc. for C35H29F12N7 O2P2Ru S: C, 41.92; H, 2.92;
m
N, 9.78. Found: C, 41.80; H, 2.68; N, 9.88. IR (cmꢀ1):
m
(NH2, NH)
6.07 (2H, s, H1), 7.66 (1H, s, H3), 7.07 (1H, d J = 8.1 Hz, H5), 6.91
(1H, d J = 7.8 Hz, H6), 8.51 (1H, s, H8), 7.94 (1H, s, NbH), 11.38 (1H,
s, NaH), 3.00 (3H, t, NbACH3). 13C NMR (75.463 MHz, DMSO-d6);
d = 30.78 (NbACH3), 101.47 (C1), 105.15 (C6), 108.19 (C3), 123.76
(C5), 128.88 (C4), 141.38 (C8), 148.82 (C2), 148.06 (C7), 177.51 (C9).
pEtTSC. Yield 84% of a pale-yellow solid. Analysis – Calc. for
3420, 3371(w);
(log ); 292 nm (3.8); 400 nm (broad).
m (C@N) 1574; m (NAN) 1036; m (C@S) 826; kmax
e
2.2. DNA-interaction studies
All experiments involving interaction of the complexes with
DNA were carried out in Tris buffer (5 mM Tris, 50 mM NaCl, pH
C11H13N3O2S: C, 52.57; H, 5.21; N, 16.72. Found: C, 52.66; H, 5.06;

F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
41
7.20). Stock solutions of ct-DNA were prepared by dissolving com-
mercial nucleic acids in buffer and stored at 4 °C for more than 24 h
to achieve homogeneity. After dilutions DNA concentration per
nucleotide phosphate was determined spectrophotometrically
using the molar absorption coefficient of 6600 Mꢀ1 cmꢀ1 at
260 nm [20]. A solution of ct-DNA in the buffer gave a ratio of
UV absorbance at 260 and 280 nm of P1.8 indicating that the
DNA did not require purification [21]. The DNA stock solutions
were stored at ꢀ20 °C and used within 1 week after their prepara-
tion. The doubly purified water used in all experiment was from a
Milli-Q system.
1.50 M NaCl, 100 mM MgCl2, 5 mM dithiothreitol and 300
l
g BSA/
mL) to the 10x ATP Buffer B (20 mM ATP in water) and was used at
1/5 volume. The metal complexes were added from a 3 mM stock
solution made fresh in a 1:1 solution of DMSO and Tris buffer
to yield varying concentrations (20
lM, 50 lM, 100 lM, and
300 M). Etoposide used as a control poison was added from a
l
10 mM stock solution in DMSO to create a 1 mM concentration
in the reaction. Also a control lane, in which DMSO was added to
DNA at the highest concentration it appears in a single reaction,
was included. DNA (pHOT-1) was used at a concentration of
12.5 ng/lL. Human topoisomerase IIa (3 units, 1.5 lL) was added
and the reactions were immediately transferred from the ice bath
to a 37 °C hot water bath for 30 min. The reactions were termi-
nated by adding 1/10 volume of 10% sodium dodecyl sulfate. Pro-
2.2.1. Viscosity measurements
Viscosity studies were carried out using a Cannon-Manning
semi micro-dilution viscometer (type 75, Cannon Instruments
Co., State College, PA, USA) immersed vertically in a thermostatted
water bath maintained at 31 1 °C. The viscosity of DNA solutions
was measured in the presence and absence of the metal complexes.
teinase
K was added at a concentration of 50 lg/mL. The
reactions were incubated in the hot water bath for an additional
15 min. 1/10 volume of 10ꢂ gel loading buffer was added and
the reactions removed from the water bath. They were cleaned
up via a CIA extraction. An equal volume of CIA (chloroform:iso-
amyl alcohol 24:1) was added, the mixture vortex and then briefly
centrifuged. The upper blue layer was split in half and placed into
corresponding wells of two 1% agarose gels in Tris–Borate–EDTA
buffer (TBE). The gels were run at 70 V for 2 h in TBE. One gel con-
The DNA concentration was maintained at 10
plex concentration varied from 0 to 60 M. Data are presented as
vs. 1/R, where R = [DNA]/[complex] and is the viscosity
of DNA in the presence of the complex and 0 is the relative viscos-
lM, while the com-
l
1/3
(g/g0)
g
g
ity of DNA alone. Relative viscosity values were calculated from the
observed flow times of DNA solution (t) corrected for the flow time
tained 0.2
lg/mL ethidium bromide. This EB gel was run with TBE
of buffer alone (t0), using the expression
was measured with a digital stopwatch and each sample was mea-
sured three times and the average flow time was used.
g
0 = (t ꢀ t0)/t0. Flow time
containing 0.5 l
g/mL ethidium bromide. This gel was then de-
stained in DNA-free water for 30 min while the other gel was
stained in an ethidium bromide/buffer solution for 30 min. It was
then de-stained as mentioned before. The bands on the gel were
imaged under UV light using a GEL Logic 440 Imaging System with
Kodak Molecular Imaging Software.
2.2.2. Ethidium bromide displacement experiments
In the ethidium bromide (EB) displacement experiment, a 3-mL
sample of a solution that was 10 lM DNA and 0.33 lM EB (satu-
rated binding levels [22]), in Tris buffer was titrated with aliquots
of a concentrated solution of a complex producing reaction mix-
tures with varied mole ratio of complex to ct-DNA. After each addi-
tion the solution was vortex for 30 s and allowed to mix at the
appropriate temperature for 5 min before measurement. The fluo-
rescence spectrum of the solution was obtained by exciting at
520 nm and measuring the emission spectra from 540 to 700 nm
using 5 nm slits.
2.4. DPPH anti-oxidant assay
The assay was conducted in a 96-well plate with a total volume
of 250
lL in each well. To various concentrations of the metal com-
plex DPPH was added (to a final concentration of 150
lM) and the
reactions incubated at 25 °C for 30 min in the dark (shielded from
light) before the absorbances were read at 520 nm. Ascorbic acid
(AA) was used as a comparison and a reagent blank along with a
control were included on the plate. The control is a solution of
DPPH and the reagent blank is a solution of the complex at the
appropriate concentrations.
2.2.3. Chemical nuclease activity
The DNA unwinding and cleavage ability of the complexes were
evaluated by agarose gel electrophoresis of supercoiled pBR322
DNA. The experiments were done in the dark or after exposure to
ꢀ
ꢁ
Acontrol ꢀ Asample
SC% ¼
ꢂ 100%
long-radiation UV light. Samples of pBR322 DNA (0.1
incubated with the complexes (in concentrations ranging from 10
to 300 M) in Tris buffer (50 mM Tris, 18 mM NaCl, pH 8.2) at
room temperature for 1 h in the dark or subjected to 365 nm light.
The reactions were quenched by addition of 3 L of loading buffer
lg/lL) were
Acontrol
l
SC% is the scavenging capacity. IC50 values denote the concentration
of metal complex required to scavenge 50% of the DPPH radicals and
were obtained from a plot of SC vs. [metal complex]. Acontrol is the
absorbance of the control and Asample is the absorbance of the
sample.
l
(0.25% bromophenol blue and 15% Ficoll in water). Samples of the
reaction mixtures were loaded onto a 1% agarose gel in TBE buffer
(89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.2). The gels were
subjected to electrophoresis for 1 h at 70 V, followed by staining
with 0.5
l
g/mL ethidium bromide for 30 min. The bands on the
2.5. Reaction with human serum albumin (HSA)
gel were visualized under UV light and photographed using a
GEL Logic 440 Imaging System with a Kodak Molecular Imaging
Software.
For the fluorescence titration, a similar procedure to the EB dis-
placement experiments was done. Solutions of HSA (fatty acid-
free) were prepared in Tris buffer (50 mM Tris, pH 7.40, 100 mM
NaCl) and stored in the dark at 4 °C. The protein concentration
was determined spectrophotometrically using the molar absorp-
tivity of 36,000 Mꢀ1 cmꢀ1 at 280 nm [23]. In the experiments, a
2.3. Activity against topoisomerase II
The capability of the ruthenium complexes to affect human
topoisomerase II activity was analyzed using gel electrophoresis.
3.0-mL solution of HSA (5 lM) was placed in a quartz cuvette
The reaction mixture was a total of 20
lL and Tris buffer (50 mM
and titrated with various amounts of a concentrated solution of
Tris, 18 mM NaCl, pH 7.2) was used as the reaction buffer for the
complexes. The ‘‘complete buffer’’ was made fresh daily by adding
equal volumes of 10x Topo II Assay Buffer A (0.5 M Tris–HCl (pH 8),
the complex producing solutions with varied mole ratios of com-
plex to HSA. The complex concentration ranged from 3 to 30
lM.
The fluorescence spectra of the solutions were obtained by exciting

42
F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
at 295 nm and measuring the emission spectra from 300 to
500 nm.
bacteria were maintained on nutrient agar and cultured in Mueller
Hinton Broth and compounds were dissolved in DMSO. The MIC
was determined from a micro-dilution method. Approximately
30 mL of bacteria culture were incubated overnight at 37.0 °C to
produce exponentially growing cells. This was then diluted to yield
a suspension containing 1.5 ꢂ 108 CFU/mL (based on comparison
of the turbidity to a 0.5 McFarland standard). Subsequently,
250 lL of this bacteria mixture were inoculated into a sterile 96-
well microplate. Various amounts of the compounds were added
to wells to give predetermined concentrations ranging from 2 to
50 lM. The well absorbances (at 600 nm) were recorded on a Bio-
Tek Synergy HT microplate reader immediately after inoculation.
The microplates were incubated for 24 h at 37.0 °C, and then the
absorbance recorded again after the 24 h period. The MIC was de-
fined as the lowest concentration of compound that did not pro-
duce any visible cell-growth or change in absorbance after
incubation. Solvent, media and positive growth controls were in-
cluded on each plate. Ampicillin or streptomycin was used as a
standard comparison. Each plate contained three replicates of each
concentration and two separate experiments were done.
2.6. Cell culture
Cell lines included two human colon cancer cells: HCT-116
(human colon carcinoma) and Caco-2 (human epithelial colorectal
adenocarcinoma). In addition, normal human colon cells CCD-18Co
(human colon fibroblasts) were included. All cell lines were ob-
tained from the American Type Culture Collection (ATCC, Rockville,
MD, USA) and maintained at the University of Rhode Island. Caco-2
cells were grown in EMEM medium supplemented with 10% v/v fe-
tal bovine serum, 1% v/v nonessential amino acids, 1% v/v L-gluta-
mine and 1% v/v antibiotic solution (Sigma). HCT-116 cells were
grown in McCoy’s 5a medium supplemented with 10% v/v fetal bo-
vine serum, 1% v/v nonessential amino acids, 2% v/v HEPES and 1%
v/v antibiotic solution. CCD-18Co cells were grown in EMEM med-
ium supplemented with 10% v/v fetal bovine serum, 1% v/v nones-
sential amino acids, 1% v/v L-glutamine and 1% v/v antibiotic
solution and were used from PDL = 26 to PDL = 35 for all experi-
ments. Cells were maintained at 37 °C in an incubator under a 5%
CO2/95% air atmosphere at constant humidity and maintained in
the linear phase of growth. The pH of the culture medium was
determined using pH indicator paper (pHydrion™ Brilliant, pH
5.5–9.0, Micro Essential Laboratory, NY, USA) inside the incubator.
All of the test samples were solubilized in DMSO (<0.5% in the cul-
3. Results and discussion
The ligands were synthesized using a general method involving
the acid-catalyzed condensation of piperonal with the correspond-
ing N-alkyl-substituted thiosemicarbazide in ethanol. The com-
plexes were made using a microwave-assisted thermal reaction
of the dichlorobis(diimine)ruthenium(II) starting material with
the ligands. [Ru(diimine)2Cl2] and ligand were suspended in the
ethylene glycol solvent and the reaction mixture saturated with ar-
gon. The mixture was then heated using a dynamic method that
was developed as follows: time = 5 min; temperature = 150 °C;
power = variable; stirring = max; cooling = on. The reaction pro-
duced red solids that are insoluble in alcohols and water but are
very soluble in acetone, CH2Cl2 and DMSO. From microanalytical
and spectroscopic data we propose that the complexes can be for-
mulated as [(diimine)2Ru(TSC)](PF6)2 (Fig. 1).
ture medium) by sonication and were filter sterilized (0.2 lm)
prior to addition to the culture media. Control cells were also run
in parallel and subjected to the same changes in medium with a
0.5% DMSO.
2.6.1. Cytotoxicity assay
The assay was carried out as described previously [24] to mea-
sure the IC50 values for samples. Briefly, the in vitro cytotoxicity of
samples were assessed in tumor cells by a tetrazolium-based color-
imetric assay, which takes advantage of the metabolic conversion
of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe-
nyl)-2-(4-sulfenyl)-2H-tetrazolium, inner salt] to a reduced form
that absorbs light at 490 nm. Cells were counted using a hemacy-
tometer and were plated at 2000–5000 cells per well, depending
on the cell line, in a 96-well format for 24 h prior to drug addition.
Test samples and a positive control, etoposide (4 mg/mL), were sol-
ubilized in DMSO by sonication. All samples were diluted with
media to the desired treatment concentration and the final DMSO
concentration per well did not exceed 0.5%. Control wells were also
included on all plates. Following a 24 h, 48 h or 72 h drug-
incubation period at 37 °C with serially diluted test compounds,
MTS, in combination with the electron coupling agent, phenazine
methosulfate, was added to the wells and cells were incubated at
37 °C in a humidified incubator for 3 h. Absorbance at 490 nm
(OD490) was monitored with a spectrophotometer (SpectraMax
M2, Molecular Devices Corp., operated by SoftmaxPro v.4.6 soft-
ware, Sunnyvale, CA, USA) to obtain the number of surviving cells
relative to control populations. The results are expressed as the
median cytotoxic concentrations (IC50 values) and were calculated
from six-point dose response curves using 4-fold serial dilutions.
Each point on the curve was tested in triplicate. Data are expressed
as mean SE for three replicates on each cell line.
3.1. Nuclear magnetic resonance
A simple comparison of the NMR spectra of the ligands and the
metal complexes reveals the presence of the azomethinic proton
signal in the spectra of the complexes which is indicative of the
non-deprotonation of the ligand confirming the neutrality of the
2.7. Antimicrobial assay
The in vitro antimicrobial activity of the complexes was investi-
gated as the minimum inhibitory concentration (MIC) against a
number of Gram-positive (Staphylococcus aureus, Bacillus cereus,
Enterococcus faecalis) and Gram-negative (Escherichia coli, Pseudo-
monas aeruginosa, Salmonella typhimurium) bacteria strains. The
Fig. 1. Structure of the ligands and complexes synthesized in the study.

F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
43
coordinated thiosemicarbazone. In all the complexes there is a
general upfield shift of nearly 1–1.5 ppm, which reflects coordina-
tion through the azomethine nitrogen. We have previously noted
this behavior for similar complexes [25]. The signal ascribed to
the aminic proton in the free ligands along with that for the alde-
hydic proton is buried under the multiplet of peaks between 6 and
9 ppm that is associated with the diimine group but we have seen
for similar complexes that the aminic proton generally move
downfield by as much as 0.5 ppm. This is indicative of the binding
of the thiocarbonyl group and is the result of a decrease in the elec-
tron density caused by electron withdrawal by the metal ions from
the thione sulfur. The protons associated with the 1,3-dioxole frag-
ment do not shift and show up at approximately 6.00 ppm for all
the complexes. The other proton signals show up at their expected
positions.
3.2. Infrared spectra
Thiosemicarbazones exhibit characteristic bands correspond-
ing to various functional groups in specific energy regions. Thio-
semicarbazone ligands can coordinate in a number of different
ways. Most commonly they bind as either of two tautomeric
forms – a neutral thione form or the anion from the thiol form.
Infrared spectrophotometry can be used to identify the coordi-
nated form and it was observed that the characteristic absorption
Fig. 2. Fluorescence spectra of the EB-DNA complex in the absence and presence of
increasing amounts of 2. kex = 520 nm, [EB] = 0.33
lM, [DNA] = 10
lM, [2] (mM):
0–60 in 5 lM increments. Temperature = 303 K.
F0
F
¼ 1 þ KSV½Ruꢃ ¼ 1 þ Kqs0½Ruꢃ
ð1Þ
peaks of all complexes are similar. The absence of a
m (SAH)
absorption in the region 2600–2500 cmꢀ1 is considered as evi-
dence that the thione form of the ligands exist in the solid state
In this equation F0 and F are the fluorescence intensities of the reac-
tion solution in the absence and presence of the metal compound.
KSV is the Stern–Volmer quenching constant which is the measure
of the effectiveness of the complex as a quencher. Fig. 3 shows
the Stern–Volmer plots and the excellent linearity of the plots con-
firms the quenching behavior. The values of the quenching con-
stant, seen in Table 1, range from 1.6 ꢂ 104 to 4 ꢂ 104 Mꢀ1
indicating that these are only moderate quenchers. In general, the
complexes with the phen unit are stronger quenchers than the
bpy complexes. This might implicate an intercalative mode (albeit
weak) of binding. To assess the strength of the binding, Eq. (2)
was employed to calculate the apparent binding constant.
[26,27]. There are two or three weak and broad bands in the
m
(NAH) region (3450–3150 cmꢀ1) and these signals play an impor-
tant role in evaluating the nature of the bonding in thiosemicar-
bazone complexes. The presence of these bands supports the
thione formulation of the ligand in the complexes and they do
not shift significantly on complexation. The coordination sites
can also be inferred from the spectral bands attributed to the
C@N iminic and C@S thioamide IV groups. The ligands show a
medium intensity band at 1570–1620 cmꢀ1 that we ascribe to
C@N and these are shifted slightly to higher or lower energy
upon complexation. The bands in the free ligand attributed to
the C@S group shifts to lower frequencies by 4–27 cmꢀ1. The size
of the shifts suggest that the ligand coordinates as a neutral,
bidentate (through the azomethine nitrogen and thiocarbonyl
sulfur) ligand in all the complexes. The presence of the azomethi-
nic hydrogen in all the complexes indicates the lack of thiolate
formation.
KEB½EBꢃ
Kapp
¼
ð2Þ
½Ruꢃ50%
In this equation KEB is the binding constant for ethidium bromide,
taken as 1.2 ꢂ 106 Mꢀ1 [28], and [Ru]50% is the concentration of
the complex that causes a 50% reduction of the initial fluorescence.
These values are shown in Table 1 and are on the order of 104 Mꢀ1
which again suggest that the complexes are moderate binders.
The quenching of the fluorescence can occur by two common
mechanisms – dynamic and/or static quenching. We propose that
in the reactions here the quenching is predominantly static. In such
a scenario a quencher-fluorophore complex is formed. This is in-
ferred from the bimolecular quenching constant (Kq in Eq. (1)) cal-
3.3. Interaction with DNA
3.3.1. Ethidium bromide competition experiment
The complexes do not have
a peak with strong enough
absorption to enable reproducible investigations by absorption
spectrophotometry. So we have investigated the reaction of
the complexes with calf thymus DNA via a fluorescence compe-
tition experiment. Ethidium bromide (EB) is a well-known dye
that is commonly used as a marker for nucleic acids by interca-
lating between the base pairs and generating a fluorescent ad-
duct. The fluorescence from the adduct may be quenched by
addition of a compound that can displace the EB from the bind-
ing sites on the DNA [25]. This quenching may be taken as evi-
dence that the compound can bind to DNA likely in a similar
fashion to EB. Using 2 as a typical example, (also see Supple-
mental Information – Sl), we can see from Fig. 2 that the com-
plex can indeed reduce the fluorescence of the EB-DNA solution.
culated by using s0 = 22 ns [29] for the EB-DNA complex. Kq for the
reactions are on the order of 1012 Mꢀ1 sꢀ1 which is two orders of
magnitude larger than the limiting value of 1010 Mꢀ1 sꢀ1 [30] con-
sidered the largest possible value in aqueous solution.
3.3.2. Viscometric studies
We have studied the interaction of the complexes with DNA by
viscometry. This method is the most definitive way to verify if a
small molecule can bind to DNA via an intercalative mechanism.
The classical DNA intercalators will lengthen the strands resulting
in an increase in the viscosity of the DNA solutions. On the other
hand, complexes that bind exclusively in the DNA grooves by a par-
tial or non-classical intercalation of the compound, (under the
same conditions), can reduce its effective length, and consequently,
A
quantitative estimate of this quenching behavior may be
obtained by treating the data according to the Stern–Volmer
equation (1):

44
F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
Fig. 5. Agarose gel electrophoresis diagram for the cleavage of pBR322 DNA by 1 at
ambient temperature in the dark (A) and upon irradiation with 365 nm light (B)
under aerobic conditions. Irradiation time was 1 h and incubation time was 1 h.
Lane DNA, DNA alone, Lanes 1–8, DNA + 10, 20, 30, 40, 50, 100, 200 and 300 mM;
Lane 10, DNA ladder.
Fig. 3. Stern–Volmer plots for the reaction of the complexes with EB-DNA at 308 K.
3.3.3. Chemical nuclease activity
The chemical nuclease activity of complexes has been assessed
by their ability to convert supercoiled pBR322 DNA from Form I to
Form II by agarose gel electrophoresis in the dark as well as with
UV irradiation under aerobic conditions. When circular plasmid
DNA is probed by electrophoresis, relatively fast migration is nor-
mally observed for the intact supercoil form (Form I). If scission oc-
curs on one strand (nicking), the supercoil will relax to generate a
slower-moving open circular form (Form II). Fig. 5, (see also Sup-
plemental Information – S2), shows the electrophoretic separation
of the DNA after incubation with 1 in the dark and after irradiation.
In general it is clear that the complexes show some cleavage of the
DNA and there is also a slight increase in this activity as the con-
Table 1
Binding constants for interaction of the complexes with the EB-DNA complex at
303 K.
1
2
3
4
5
104 KSV (Mꢀ1
103 Kapp (Mꢀ1
1011 Kq (Mꢀ1 sꢀ1
)
1.59 0.04 2.20 0.08 4.04 0.15 3.33 0.08 2.99
6.34
7.22
)
7.77
10.0
11.0
18.2
10.6
14.2
9.38
13.6
)
At 293 K, KSV
,
Kapp and Kq are (2.32 0.11) ꢂ 104 Mꢀ1
,
7.94 ꢂ 103 Mꢀ1 and
1.05 ꢂ 1012 Mꢀ1 sꢀ1 respectively for 2.
centration of the complexes is increased (up to 300 lM). The ex-
its viscosity by bending the DNA helix [31]. The viscometric data,
1/3
tent of the cleavage is more pronounced when the reaction
mixtures are irradiated with UV light (365 nm). There are no
apparent differences in the cleavage ability of the complexes. This
would suggest that the mechanism of cleavage is similar among
the complexes.
presented as a plot of (
g
/g0)
vs. the binding ratio [Ru]/[DNA], is
shown in Fig. 4. It was observed for the complexes that the viscos-
ity of the DNA solutions did not change significantly over the stud-
ied range of metal concentrations. There is an overall slight
decrease in viscosity (with the exception of 3 which showed incon-
sistent changes). This would suggest that the complexes can possi-
bly interact with DNA via partial intercalation or groove binding. It
is well known that [Ru(bpy)3]2+ interacts with DNA strictly via
electrostatic interactions and since our complexes are charged, this
mode of interaction is also a possibility. Given the electronic nature
of the piperonal sub-structure, major intercalation into DNA by
this moiety seems improbable. So the viscometry results support
the idea that if the complexes intercalate into DNA, the interaction
is weak.
3.3.4. Anti-topoisomerase activity
Though DNA is implicated as the main target for anticancer
ruthenium compounds, affecting the activity of topo II may also
be an effective mode of anti-neoplastic activity. Type II topoisome-
rases (topo II) are a class of ubiquitous enzymes that modulate the
topological problems associated with DNA replication, transcrip-
tion, and other nuclear processes by introducing temporary single-
or double-strand breaks in the DNA. Topo II is a major enzyme in
neoplastic cells. Consequently topoisomerases have been one of
the major molecular targets in anticancer drug development. The
ability of the complexes to affect the catalytic activity of human
topoisomerases II was studied by a DNA relaxation assay. Super-
coiled plasmid DNA was treated with topo II in the presence of
varying concentrations of the complex and the products were ana-
lyzed by gel electrophoresis on agarose gel. Depending on the nat-
ure of the products one can propose if the complex is a poison or an
inhibitor. Topo II-targeting agents control the topo II activity either
by poisoning the enzyme or by inhibiting the enzyme. Topo II poi-
sons act by increasing the concentration of covalent enzyme-
cleaved DNA complexes. Catalytic inhibitors prevent topo II from
carrying out its required physiological functions. In our experi-
ments (see Supplemental Information – S3) enzyme activity is
characterized by conversion of pHOT1 DNA from the supercoiled
conformation (SC, Form I, Lane 1) to the fully relaxed conformation
(R, Form II; see Lanes 2 and 3). The species migrating between
the two forms are the various topoisomers. Careful examination
of the electrophorograms suggests that the complexes can inhibit
Fig. 4. Effect of increasing concentrations of complexes 1, 2, 4 and 5 on the relative
viscosity of ct-DNA solutions at 304 K = 1 K.

F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
45
the relaxation of the enzyme at high concentrations as evidenced
by the reduction in the number of the topoisomers but a fully re-
laxed conformation is not observed. So we propose that the com-
plexes can act as catalytic inhibitors of topo II.
3.4. Anti-oxidant behavior
Based on the data from experiments that show that the com-
plexes can bind to DNA and further that there is strong activity
regarding the cleavage of pBR322 plasmid DNA, we investigated
the anti-oxidant properties of the complexes by using the 2,2-di-
phenyl-1-picrylhydrazyl (dpph) radical method. The model of the
scavenging of dpph radicals is a simple, rapid and convenient
method that is extensively used to evaluate the anti-oxidant prop-
erties of compounds. The dpph radicals are stable but in the pres-
ence of compounds capable of donating hydrogen atoms the
radical property is destroyed resulting in a color change from pur-
ple to yellow. In our experiments we used ascorbic acid as a stan-
dard comparison. It was observed that the complexes can indeed
reduce the concentration of the initial dpph radicals in solution
and this is taken as evidence of their anti-oxidant capabilities. All
Fig. 6. Emission spectra of HSA in the absence and presence of increasing amounts
of 2. kex = 295 nm, [HSA] = 5.0
lM, and [2] (lM); 0–35 in 2.5 lM increments.
Temperature = 303 K.
the compounds have similar IC50 values of 232
221 M and 265 M for 2, 3, 4 and 5 respectively. Under our reac-
tion conditions ascorbic acid has an IC50 of 82 M suggesting that
lM, 235 lM,
l
l
l
our compounds are about three times less effective as the well-
known anti-oxidant under the reaction conditions. We can specu-
late that the cleavage of plasmid DNA by the complexes may not
involve the generation of radical species.
3.5. Reaction of the complexes with HSA
Human serum albumin (HSA) is the most abundant blood ser-
um protein, occurring to the extent of 0.63 mM. The protein serves
as a transport unit for a wide variety of endogenous substances
including drugs. In this study we investigated the binding of the
complexes to HSA using fluorescence spectroscopy. HSA has a
well-known structure that contains a single tryptophan residue
that is responsible for the majority of the intrinsic fluorescence
of the protein. On excitation at 295 nm HSA has strong fluores-
cence emission at 350 nm. This emission can be attenuated by a
small molecule binding at or near the tryptophan as this amino
acid unit is quite susceptible to changes in its environment. As a
representative example, Fig. 6 shows that addition of 2 to HSA
can very efficiently reduce the fluorescence so we can conclude
that the complexes in general can bind to HSA. We can obtain a
quantitative estimate of the strength of this binding by treating
the data in an analogous manner to the EB-DNA competition
experiments. That is, we can use the Stern–Volmer equation
(Eq. (1)). Treatment of the data in this manner resulted in the plots
shown in Fig. 7. It was observed that the plots were not linear as
predicted from the equation but showed significant positive devi-
ations from linearity at higher concentration of added metal com-
plexes. There are two common explanations for this deviation. First
the HSA fluorescence can be quenched via both of the common
quenching mechanisms operating simultaneously. For proteins
the quenching constants are approximately the same [32,33].
Alternatively, there may be more than one independent binding
site on the HSA and they are not all equivalently accessible to
the complexes. Under these circumstances, the binding constant
for the reaction can be obtained from a modified Stern–Volmer
(MSV) analysis [34]. This involves treating the data using Eq. (3):
Fig. 7. Stern–Volmer plots for the reaction of the complexes with HSA at 308 K
the protein. K is the effective quenching constant for the fluoro-
phore which can be taken as a binding constant assuming the de-
crease in fluorescence comes from the interaction of the HSA with
the complexes. Fig. 8 shows the plot of F0/F0 ꢀ F vs. 1/[Ru] from
which we obtain K from the ratio of the intercept to the slope.
The derived binding constants are between 104 and 105 Mꢀ1 (Table
2) indicating that they are strong binders. The average number of
binding sites as measured by f was determined to be about 1.3. This
number does not conclusively settle the question as to whether
there is a single binding site on the protein.
3.6. Cytotoxicity assay
The in vitro cytotoxicity of the complexes 1–5 against two hu-
man cancer cell lines, HCT-116 (colon carcinoma) and Caco-2 (epi-
thelial colorectal adenocarcinoma) and a non-cancerous cell line,
CCD-18Co (colon fibroblasts) was investigated using a tetrazo-
lium-based (MTS) colorimetric assay. Etoposide, a potent anti-
neoplastic drug, was used as a standard comparison treatment.
The IC50 values (Table 3), the median cytotoxic concentrations,
were determined after 24, 48 and 72 h of drug exposure. Generally,
the longer the exposure time the more cytotoxic is the complexes
with the 72 h exposure being between 1.2 and 2 times more effec-
tive compared to the 24 h exposure. As another generality, the
Caco-2 cell line is slightly more sensitive to the complexes than
F0
1
1
f
¼
þ
ð3Þ
F0 ꢀ F fK½Ruꢃ
Here f = fraction of the fluorophore that is initially accessible to the
complex. This may be interpreted as the number of binding sites on

46
F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
have on the cytotoxic behavior. An interesting point to note is that
all the complexes are less cytotoxic to the normal cells CCD18-Co
indicating a definite cytotoxic selectivity towards cancer cells. In
the best case for 4 for example, the compound is 17 times less cyto-
toxic to the normal cells. This effect is even more pronounced than
for the standard drug treatment with etoposide being only about
ꢄ2.5 times more lethal to the cancer cells vs. the non-cancerous
cells.
The IC50 values for the complexes (Table 3) are in the range
7–159 lM after incubation for 72 h, indicating the good to poor
cytotoxicity profiles of the complexes. Under the experimental
conditions 1 and 4 show comparable or better activity to etoposide
against both HCT-116 and Caco-2. In general however the com-
plexes do not reach the activity of the standard against the cancer
cell lines. This is in itself not an unusual issue. It is known that
some ruthenium complexes have low in vitro toxicity but show
good in vivo characteristics. For example, complexes of the type
[(arene)Ru(PTA)Cl]+ exhibited low activity against cancer cells
but had very good anti-metastatic activity in vivo [34].
3.7. Antibacterial assay
The in vitro antimicrobial efficiency of the complexes against a
number of Gram-positive and Gram-negative bacteria has been
determined as the minimum inhibitory concentrations. The com-
pounds were tested at low concentrations due to solubility issues
and to avoid excessive concentrations of the solvent DMSO in the
wells. The complexes generally were more active against the
Gram-positive bacteria than the Gram-negative species. Complex
1 showed no inhibition against the four bacteria strains used: B.
cereus, S. aureus, S. typhimurium or E. coli. In addition 4 and 5
showed no inhibition against B. cereus and S. aureus. Complex 2
showed very good activity against both B. cereus and S. aureus with
Fig. 8. Plots of the modified Stern–Volmer equation, F0/(F0 ꢀ F) vs. 1/[Ru], for the
reaction of the complexes with HSA.
the HCT-116 cell line. For instance at 72 h exposure, 1 is 1.3 times
as cytotoxic against the Caco-2 line. We can speculate that the al-
kyl substituent on the thiosemicarbazone ligand is probably not
very critical. 4, which contain a methyl substituent on N3, is the
most cytotoxic with IC50 of 8.6 and 6.6 lM at 72 h exposure against
a MIC of 10
or E. coli 3 had an MIC of 20
aureus and 50 M against E. coli. It did not show any activity
against S. typhimurium. It should be pointed out that no inhibition
is relative only to the highest concentration (50 M) of complex
lM but it showed no inhibition against S. typhimurium
HCT-116 and Caco-2 respectively. These values are 2–2.5 times as
cytotoxic, under our assay conditions, as the comparison drug eto-
poside. With the bpy complexes, the un-substituted (H) complex
(1) is more active than the ethyl-substituted analog. On the other
hand, for the phen complexes the ethyl-substituted complex is
more active. So we cannot draw any definitive conclusions as to
any effect that the diimine or the thiosemicarbazone substituent
l
M against B. cereus, 30 M against S.
l
l
l
that we used. We have studied the antibacterial activity of the free
ligands as well and in general the complexes were more active
than the free ligands which showed no measureable activity at
Table 2
Binding constants for the interaction of the complexes with HSA.
Temperature (K)
104 K (Mꢀ1) (R2 beneath)
1
2
3
4
5
293
298
303
308
1.96 0.49 (0.981)
3.82 0.11 (0.999)
–
–
–
3.61 0.21 (0.993)
3.35 0.31 (0.995)
–
1.10 0.21 (0.996)
6.60 0.35 (0.992)
–
1.45 0.35 (0.992)
–
–
3.40 0.06 (0.992)
9.10 0.84 (0.986)
9.35 0.35 (0.992)
5.10 0.12 (0.999)
8.10 0.76 (0.977)
4.72 0.05 (0.999)
Table 3
IC50 values (
l
M) representing the anti-proliferative activity of all the complexes in a panel of three human cell lines – 2 tumorigenic (HCT-116 and Caco-2) and one non-cancerous
(CCD-18Co).
Comp.
HCT-116
24 h
Caco-2
24 h
CCD-18Co
24 h
48 h
72 h
48 h
72 h
48 h
72 h
IC50
1
2
3
4
36.9 4.9
32.4 3.3
27.5 3.3
39.2 4.0
31.0 4.4
22.2 4.7
220
nd
221
212
181
6
153
321
179
136
136
4
5
5
3
2
145
304
167
114
127
3
4
4
2
2
198
101
4
6
174
6
159
4
148
3
138
3
130
3
67.4 4.1
13.4 3.1
56.1 2.0
22.9 1.5
55.6 3.6
8.6 2.1
42.6 3.7
18.3 1.4
97.0 6.4
19.5 2.8
65.9 3.8
23.1 1.2
76.4 4.0
15.2 3.7
56.2 1.9
17.9 1.3
58.9 4.1
6.6 2.4
36.8 2.8
16.5 2.0
6
6
4
17.8 2.5
65.9 2.9
27.2 1.2
5
Etoposide
50.1 1.5
45.2 1.1
42.2 1.0

F.A. Beckford et al. / Journal of Molecular Structure 992 (2011) 39–47
47
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The project described was supported by Award Number
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