Indian Journal of Chemistry
Vol. 50A, March-April 2011, pp. 465-473
Copper(I) complexes of modified nucleobases and vitamin B3 as potential
chemotherapeutic agents: In vitro and in vivo studies
N J M Sanghamitraa, M K Adwankarb, A S Juvekarb, V Khurajjamb & C Wycliff a, A G Samuelsona
a
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560 012, India
Email: ashoka@ipc.iisc.ernet.in
b
Division of Chemotherapy, Advanced Centre for Treatment, Research and Education in
Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai 410 208, India
Received 27 October 2010; accepted 3 January 2011
Three new complexes of Cu(I) have been synthesized using ancillary ligands like thiopyrimidine (tp) a modified
nucleobase, and nicotinamide (nic) or vitamin B3, and characterized by spectroscopy and X-ray crystallography. In vitro
cytotoxicity studies of the complexes on various human cancer cell lines such as Colo295, H226, HOP62, K562, MCF7 and
T24 show that [Cu(PPh3)2(tp)Cl] (1) and [Cu(PPh3)2(tp)]ClO4 (2) have in vitro cytotoxicity comparable to cisplatin.
Complex [Cu(nic)3PPh3]ClO4 (3) is non-toxic and increases the life span by about 55 % in spontaneous breast tumor model.
DNA binding and cleavage studies show that complex (3) binds to calf thymus DNA with an apparent binding constant of
5.9 × 105 M and completely cleaves super-coiled DNA at a concentration of 400 µM, whereas complexes (1) and (2) do not
bind DNA and do not show any cleavage even at 1200 µM. Thus, complex (3) may exhibit cytotoxicity via DNA cleavage
whereas the mechanism of cytotoxicity of (1) and (2) probably involves a different pathway.
Keywords: Bioinorganic chemistry, Metallodrugs, Copper, Antitumor activity, Thiopyrimidine, Nicotinamide, Cytotoxicity,
Lipophilicity
The FDA approved gold phosphine based antiarthritic
drug, auranofin, and antitumor drug, cisplatin, have
created immense interest in inorganic chemists to
develop new metallodrugs.1,2 Soft metal centers like
Cu(I) and Au(I) are known to possess biological
activity3-5 but need to be stabilized in biological media
with sulfur or phosphorous containing ligands. In
many instances the biological properties of the ligands
are enhanced on complexation with metals, as
reported
for
1,2-bis(diphenylphosphinoethane)
(DPPE) where the anticancer activity of DPPE
increases on coordination to Cu(I) and Au(I).5 DPPE
based Cu(I) complexes have shown promising
antitumor activity in a range of cell lines like PA1,
CHO and human ovarian carcinoma cell lines.3 The
thioglucose derivative auranofin is also an effective
cytotoxic agent against p388 leukemia.2 The Au(I)
trialkylphosphine complexes of 2-thiopyridine and
2-thiopyrimidine are reported to have carcinogenic
and antitumor properties and have been used as
drugs.6-8 Nicotinamide (vitamin B3) is known for its
cytostatic activity9,10 achieved by maintaining
mitochondrial membrane potential, but metal
complexes of nicotinamide do not appear to have
been used as drugs.
In this report, we have described the synthesis of
three Cu(I) complexes of PPh3 with heterocyclic
thione thiopyrimidine and nicotinamide. The in vitro
antitumor activity in different cell lines such as colon
carcinoma (Colo205), human lung carcinoma (H226
and HOP62), human erythroid leukemia (K562),
human breast carcinoma (MCF7) and human bladder
carcinoma (T24) was studied by semi-automated
sulforhodamine-B assay (SRB assay)11 while in vivo
activity was carried out in murine tumor models, viz.,
spontaneous mouse-mammary tumor, L1210-murine
leukemia and P388 leukemia. Previous study on the
detailed mechanism of action of a Cu(I) complex of
DPPE reveals that the complexes bind to calf thymus
(CT) DNA and cleave DNA in vitro and in vivo.12
Hence, we have studied the DNA binding ability of
these complexes with CT DNA and in vitro cleavage
of supercoiled (SC) DNA. The lipophilicity of the
complexes was studied by UV-visible spectroscopic
technique to understand their activity profiles.
Materials and Methods
The solvents, acetonitrile, dichloromethane and
petroleum ether, were dried and distilled over calcium
hydride, P2O5 and sodium ketyl radical respectively.
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INDIAN J CHEM, SEC A, MARCH-APRIL 2011
Thiopyrimidine and calcium hydride were obtained
from Aldrich, USA. PPh3, nicotinamide and acetic
acid were obtained from SD Fine Chem. Ltd., India.
Cu(CH3CN)4ClO4 and CuCl were synthesized by
literature methods.13,14 The calf thymus DNA, agarose
(molecular biology grade), carboxymethyl cellulose
(CMC) and ethidium bromide, were purchased from
Sigma, USA. Supercoiled pUC19 (cesium chloride
purified) DNA was from Bangalore Genei, India.
RPMI 1640 and fetal bovine serum (FBS) were
procured from GIBCO-BRL, Invitrogen Life
Technologies, USA. Cisplatin was purchased from
SD Fine Chem. Ltd., India. Sterile plasticware were
purchased from Nunc, Denmark. Tris-HCl buffer
solution was prepared by using milli-Q water.
1
H-NMR spectra were recorded either on a Bruker
ACF 200 MHz or AMX 400 MHz spectrometer with
tetramethylsilane (TMS) as the internal reference.
31
P{1H} NMR spectra were recorded either on a
Bruker AMX 400 MHz spectrometer operating at
162.2 MHz or a Bruker ACF 200 MHz operating at
81.1 MHz with H3PO4 (85 %) as the external
reference. IR spectra were recorded in the solid state
as KBr pellets on a Bruker Equinox 55 spectrometer.
UV-vis spectra were recorded with Perkin Elmer
(Lambda 55) spectrometer. Emission spectra were
recorded with Perkin Elmer (LS 50B) fluorescence
spectrometer. ELISA plates of SRB assay were read
on the Sunrise model of Tecan, Austria.
yellow solution was formed. The solvent was
removed under vacuum and the volume of the
solution was reduced to half and layered with diethyl
ether, to give orange yellow crystals, suitable for
single crystal XRD. The complex was obtained in
nearly quantitative yield. 31P and 1H NMR was
recorded in CDCl3. 1H NMR (CDCl3): 6.6(t, 1H, tp),
8.07(s, 2H, tp), 7.04 – 7.57(m, 30H, PPh3). 31P NMR
gives a single peak at 0.9ppm. IR (KBr pellet): 1095
(ν ClO4).
Cu(nic)3PPh3ClO4 (3)
Cu(CH3CN)4ClO4 (0.327 g, 0.001 mole) was
reacted with PPh3 (0.262 g, 0.001 mole) in
dichloromethane (20 mL) for 1 hr, and then
nicotinamide (0. 366 g, 0.003 mole) was added. After
30 min, an off-white precipitate was formed. This was
stirred for another 24 h to ensure the completion of
reaction, which was checked by the absence of PPh3
in the supernatant solution by TLC. The precipitate
was dissolved in hot ethanol, which on cooling gave
colorless crystals and a blue solution. These crystals
were found to contain three molecules of
nicotinamide, one PPh3 coordinated to Cu(I) when
analyzed by XRD. (Yield 88 %). 31P and 1H NMR
was recorded in acetone d6. 6.95(s, 3H, nic), 7.4-7.47
(m, 15 H PPh3 and 3 H nic), 7.7 (s, 3H nic) 8.4 (s, 3H
nic). 31P NMR (0.04). IR (KBr pellet): 1090 (ν ClO4).
X-ray crystallography
Synthesis and characterization of the complexes
Cu(thiopyrimidine)(PPh3)2Cl (1)
CuCl (0.099 g, 0.001 mole) was reacted with PPh3
(0.524 g, 0.002 mole) in acetonitrile (30 mL) for
10 min before adding thiopyrimidine (0. 112 g,
0.001 mole). A bright orange yellow solution was
formed, which gave a deep yellow precipitate after
1 hour. The precipitate was filtered and re-dissolved
in dichloromethane and layered with petroleum ether
to give orange yellow crystals suitable for single
crystal XRD. The complex was obtained in nearly
quantitative yield. 31P and 1H NMR was recorded in
CDCl3. 6.6 (t, 1H, tp), 8.1(s, 2H, tp), 7.18 – 7.46
(m, 30H, PPh3). 31P NMR gives a single peak
at –3.41ppm.
Single crystals of complexes (1), (2) and (3) were
separately glued to the tip of glass fibers along the
largest dimension. Data were collected on a Bruker
AXS single crystal diffractometer equipped with
Smart Apex CCD detector and a sealed Mo-Kα source
working at 2.2 KW and 50/35 (kv/mA). Intensity data
were collected at room temperature. Crystallographic
computations were performed using the WINGX
(1.63.02) package.15 Data was corrected for Lorentz
and polarization effects. The structures were solved
by the combination of Patterson and Fourier
techniques and refined by full-matrix least-squares
method using the SHELX program.16 The hydrogen
atoms of the complex (1) were geometrically fixed
and the hydrogen atoms for complex (2) were located
from the difference Fourier map and refined.
Cu(thiopyrimidine)(PPh3)2ClO4 (2)
Cu(CH3CN)4ClO4 (0.327 g, 0.001 mole) was
reacted with PPh3 (0.524 g, 0.002 mole) in acetonitrile
(30 mL) for 10 min. To this solution thiopyrimidine
(0.112 g, 0.001 mole) was added. A bright orange
Description of biological assays
SRB assay17
Human tumor cell lines were cultured in RPMI1640 medium supplemented with (FBS) (10 %) at
SANGHAMITRA et al.: ANTICANCER Cu(I) COMPLEXES OF MODIFIED NUCLEOBASES & VITAMIN B3
37 °C and were maintained in a CO2 incubator in an
atmosphere of 5 % CO2 in tissue culture flasks. The
confluent cultures (70 %) were used to determine the
cytotoxic effects of the test compounds. Single cell
suspension of these tumor cells was made and cell
count was adjusted to 1 × 105 to 5 × 105 cells/mL. Cell
number for seeding was derived from a calibration
curve set up with known number of cells, for each cell
line. 96-well plate was seeded with this cell
suspension, each well receiving 90 µl of it. The plate
was then incubated at 37 °C temperature in CO2
incubator for 24 h to ensure adequate cell-growth
prior to determination of cell growth inhibition. The
drugs (10 µl) were then added at appropriate
concentrations, followed by further incubation for
48 h. Experiment was terminated by gently layering
the cells in the wells with 30 % chilled TCA. The
plates were kept in refrigerator for 1 h, following
which they were washed thoroughly with tap water,
dried and stained with 0.4 % SRB in 1 % acetic acid.
Excess SRB dye was removed by washing the plates,
3 to 4 times, with 1 % acetic acid. The bound SRB
was eluted with Tris (10 mM, pH 10.5). Absorbance
was read at 540 nm with 690 nm reference
wavelength, in the ELISA-plate reader. Optical
density of drug-treated cells was compared with that
of control cells and growth inhibition was calculated
as percent values. Each compound was tested at four
different concentrations (10, 20, 40 & 80 µg/mL), in
triplicate, on the human malignant cell lines.
Concentration for 50 % growth-inhibition (IC50)
≤ 10 µg/mL was considered to indicate activity. For
each of the experiments, a known anticancer drug
cisplatin was used as a positive control.
In vivo xenograft studies
Anticancer activities of complexes (1) and (2) were
evaluated in the P388 leukemia, L1210 and Lewis
lung carcinoma xenograft models. Male BDF-1 mice
was used in all the carcinoma models except in P388
leukemia, where female BDF-1 mice was used. All of
the studies using laboratory animals were approved by
the Institutional Ethics Committee for ‘Animal Care
and Use’ of the Advanced Centre for Treatment,
Research and Education, Navi Mumbai, India and all
the applicable institutional and governmental
guidelines for the human care and use of laboratory
animals were adhered to.
In vivo anticancer activity of complexes (1) and (2)
was evaluated in lewis lung carcinoma, P388 murine
leukemia and L1210 mouse model. In the case of
467
lewis lung carcinoma model, on day 0 of each
experiment, the tumor was removed and minced.
Normal saline was added and 0.2 mL was
administered to each animal by intramuscular
injection. On day 1, all mice were randomized and
then divided into three groups each group contain six
mice. Mice weights were taken on day 1 and 5.
Treatment schedule for the mice groups was from day
7 to day 15 by 1 trough 9 schedule via intraperitonial
(IP) injections. Drugs were prepared in 10 % DMSO
(first weight of drug was taken and dissolved in
100 % DMSO and then distilled water was added to
make final concentration of DMSO to 10 %). The
mice received IP injections of 0.1 mL of the
compounds (100 mg/kg) at morning from day 1 to day
9. On day 7, day 11, day 15 and day 19 tumor volume
was measured in cc.
In the case of P388 murine leukemia model, 1×106
cells/mouse were injected via IP into BDF1 female
mice on day 0. On day 1, all mice were randomised
and then divided into five groups, each group
containing six mice. Weights of mice were taken on
day 1 and day 5. Treatment schedule for the mice
groups was from day 1 through day 9. Drugs were
prepared in 10 % DMSO in one experiment and CMC
in a second experiment. The mice received IP
injections of 0.2 ml of the compounds (100 mg/kg) at
morning from day 1 to day 9. On day 7 and day 9
tumor volume was measured in cc. For L1210 model,
1 × 105 cells/mouse were injected via IP into the
BDF1 male mice on day 0. On day 1, all mice were
randomised and then divided into seven groups, each
group containing six mice. Weights of mice were
taken on 1st and 5th days. Treatment schedule for the
mice groups was from day 1, day 5 and day 9. Drugs
were prepared in CMC and the mice received IP
injections of 0.2 mL of the compounds (75, 50 and
25 mg/kg) at morning from day 1, day 5 and day 9.
On day 1, day 5, day 7, day 11, day 15 and day 19,
tumor volume was measured in cc.
Anticancer activity of complex (3) was evaluated
in spontaneous breast carcinoma and L1210 mouse
model. In both the models, cells were injected
intraperitonially into BDF1 female mice on day 0. On
day 1, all mice were randomised and then divided into
five groups, each group containing six mice.
Treatment schedule for the mice groups was from day
1 through day 9. The drug was prepared in water. The
mice received IP injections of 0.2 mL of the
compounds (30 mg/kg) for L1210 model and
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INDIAN J CHEM, SEC A, MARCH-APRIL 2011
50 mg/kg for spontaneous breast carcinoma model at
morning on day 1, day 5 and day 9. From day 5
through day 35, tumor volume was measured in cc.
Tumor volume (Tvol) was calculated in accordance
with the equation; Tvol = L × W2 × 0.5, where L is the
maximum length of the tumor and W is the minimum
length. At the end of study the mice were sacrificed
by cervical dislocation and the tumors were excised
and fixed in 10 % phosphate buffered formalin until
further evaluation. The tumor volumes are expressed
as mean ± standard error for three mice in each group.
One-way analysis of variance (ANOVA) was used for
multiple comparisons followed by Student’s t-test to
find out difference between individual treatments.
Body weights and water consumption were measured
in all the experiments to assess toxicity.
Lipophilicity measurements
The lipophilicity of the complexes were measured
by standard shake flask technique.18 A chloroform
solution of each of the complexes (1 mg/mL) was
separately prepared. This solution 40 µL was added to
6 mL of CHCl3 and divided into two parts. The
absorbance of one 3 mL part was recorded to get A0.
To another part, 3 mL of milli-Q water was added and
stirred for 4 h. The CHCl3 layer was separated by
separatory funnel and the solution was centrifuged at
16000 rpm for 15 min and water particles were
removed. Absorbance of the CHCl3 solution was
recorded to obtain A1. A0 is ACHCl3 and A0 – A1 gives
AH2O. Since the complexes were not soluble in water,
the partition coefficient in CHCl3-water system, i. e.,
log PCHCl3 was measured and then log Poct was
calculated using the following regression equation18,
log Poct = (1.343 + log PCHCl3)/1.126.
DNA binding and cleavage
The concentration of CT DNA was determined
from the absorption intensity19, assuming an ε value
of 6600 M-1 cm –1 at 260 nm. Binding of the
complexes were studied by ethidium bromide (EtBr)
displacement method by monitoring the fluorescence
change of CT DNA-bound-EtBr in Tris-HCl
(5 mM)/NaCl (5mM). Excitation wavelength was set
to 510 nm and emission was monitored at 600 nm.
Binding constant of complex (3) with CT DNA was
calculated using the reported procedure.20 Supercoiled
pUC19 DNA (5 µL, ∼500 ng) in 50 mM Tris-HCl
buffer (pH 7.2) was treated with the metal complex
(5 µL of respective concentration). The total volume
of solution loaded on gel was 10 µL. After incubation
for 1 h at 37 °C, the samples were added to the loading
buffer containing 25 % bromophenol blue, 0.25 %
xylene cyanol, and 30 % glycerol (3 µL), and the
solution was finally loaded on 0.8 % agarose gel
containing 1.0 µg/mL EtBr. Electrophoresis was
carried out for 2 h at 100 V in TBE buffer. Bands were
visualized by UV light and photographed using Kodak
Gel Documentation system.
Results and Discussion
The reactions of CuCl with thiopyrimidine (tp) and
PPh3 in the ratio of 1:1:2 gives complex (1) in which tp
coordinates in monodentate fashion through S (Scheme
1). The complex obtained with CuCl in the presence of
NaH or pyridine by deprotonating tp also resulted in
the same complex. This suggests that the tp is more
stable in its protonated form in the complex. The
chloride ion coordinated to the copper center is
involved in strong hydrogen bonding with the
pyrimidine N-H. The reaction of Cu(CH3CN)4ClO4
with PPh3 and thiopyrimidine in 1:2:1 ratio in
acetonitrile gives complex (2), which was found to be
coordinated to tp through both N and S with a ClO4ion sitting outside the coordination sphere (Scheme 2).
Figure 1 shows the ORTEP view of complexes (1), (2)
and (3) (50 % probability thermal ellipsoids).
Hydrogen atoms were omitted for clarity. The bond
distances (angstrom) of complexes (1), (2) and (3) are
given in Table 1 and the corresponding bond angles
(degrees) are given in Table 2. The crystallographic
data for complexes (1), (2) and (3) are given in Table 3.
Thus, copper (I) interacts with thiopyrimidine in
different ways and might be responsible for bringing
about anticancer activity in a unique way.
Lipophilicity is an important physicochemical
parameter that contributes to the toxicity and effectiveness
of a drug. The in vivo distribution of a drug involves
partitioning between the extracellular aqueous medium
and the cell membrane mostly made up of the lipid
molecules. Hence, lipophilicity of a drug directly
correlates with its affinity for the cell membrane. Since
the best mimic of a cell membrane is
n-octanol, according to Hansch and Fujita convention,
lipophilicity of a drug is expressed as the logarithm
of its octanol-water partition coefficient21,22,
log Poctanol = log (Coctanol/ Cwater), where Coctanol is
concentration of drug in octanol and Cwater is
concentration of drug in water. Lipophilicity of these
complexes was studied by shake flask technique.23
Since complexes (1) and (2) were not soluble in
SANGHAMITRA et al.: ANTICANCER Cu(I) COMPLEXES OF MODIFIED NUCLEOBASES & VITAMIN B3
Fig. 1Molecular structure of complexes (1), (2) and (3). [50 % probability thermal ellipsoid].
469
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INDIAN J CHEM, SEC A, MARCH-APRIL 2011
water, the partition coefficient was first measured in
CHCl3 and log Poctanol was calculated indirectly as
described in the experimental section. The concentrations
Table 1Bond distances for complexes (1), (2) and (3)
Bond
Cu(1)-P(1)
Cu(1)-P(2)
Cu(1)-Cl(1)
Cu(1)-S(1)
Cu(1)-N(1)
Cu(1)-S(2)
(1)
2.2848(11)
2.2947(12)
2.3633(12)
2.3800(13)
Bond distance (Å)
(2)
2.2137(13)
2.2607(13)
(3)
2.186(3)
Cytotoxicity of the copper(I) complexes
2.121(4)
2.4913(15)
2.105(8)
Table 2Bond angles for complexes (1), (2) and (3)
Bond angle (o)
Angles
P(1)-Cu(1)-P(2)
P(1)-Cu(1)-Cl(1)
P(2)-Cu(1)-Cl(1)
P(1)-Cu(1)-S(1)
P(2)-Cu(1)-S(1)
Cl(1)-Cu(1)-S(1)
N(1)-Cu(1)-P(1)
N(1)-Cu(1)-P(2)
N(1)-Cu(1)-S(2)
P(1)-Cu(1)-S(2)
P(2)-Cu(1)-S(2)
N(1)-Cu(1)-N(1)
of the complexes were determined by UV-visible
absorption spectroscopy by monitoring the absorbance at
260 nm. The absorption of complex (1) and (3) was
monitored at 260 nm and for complex (2) at 290 nm. The
lipophilicities of the complexes were found to be 3.02,
2.76 and 0.75 for the complexes (1), (2) and (3)
respectively. The high toxicity shown by complexes (1)
and (2) in vivo could be attributed to the high lipophilicity.
(1)
(2)
(3)
122.41(4)
111.81(4)
98.65(4)
102.18(5)
112.87(4)
108.69(4)
128.95(5)
126.28(10)
99.72(10)
68.21(11)
112.58(5)
103.58(5)
118.87(5)
98.64(7)
Thio derivatives of DNA bases are known to induce
cellular sensitization to ultraviolet A (UVA) and
combination of non lethal doses of UVA and thiobases
show cooperative cytotoxicity to cultured human cells.24
Thiopurines have been used as antileukemic agents.24
Niacin deficiency is also known to impair cell cycle
arrest and DNA damage induced apoptosis, leading to
the survival of cells with leukemogenic potential.9
Previously Ru(II) complexes of thiopurines and
thiopyrimidines have shown potent activity against
ovarian cancer cells.25 Hence, it was of interest to
evaluate the anticancer activities of the complexes by
studying the potential inhibitory effects of the complexes
on the growth of cancer cells in various cancer cell lines.
All three complexes showed consistent in vitro
cytotoxicity in human malignant cell lines. The
complexes were found to inhibit more than 80 % growth
of the cells at 20 µg /mL concentration after 48 h similar
Table 3Crystallographic data for complexes (1), (2) and (3)
(1)
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (deg.)
β (deg.)
γ (deg.)
Volume (Å3)
Z
Density (calc.) (mg/m3)
Crystal size (mm3)
Reflections collected
Independent reflections
(Goodness-of-fit on F2)c
(2)
(3)
C40H34ClCuN2P2S
735.68
Monoclinic
P21/c
14.427(5)
10.127(3)
24.357(8)
90
94.491(5)
90
3547.7(19)
4
1.377
C40H34ClCuN2O4P2S
799.68
Monoclinic
P21/n
10.0774(15)
15.400(2)
25.138(4)
90.000(3)
78.655(2)
90.000(3)
3825.0(10)
4
1.389
C36H33ClCuN6O4P
791.66
Tetragonal
R -3
15.28(4)
15.28(4)
37.78(12)
90.00
90.00
120.00
7639(37)
9
1.549
0.12 × 0.13 × 0.1
27478
7231 [R(int) = 0.0644]
1.113
0.22 × 0.07 × 0.16
32759
9004 [R(int) = 0.0766]
1.101
0.21 × 0.16 × 0.26
1663
[R(int) = 0.1436]
1.306
R1 = 0.0635, wR2 = 0.1245
R1 = 0.0880, wR2 = 0.1775
Final R1a , wR2b [I > 2σ(I)]
Final R1a, wR2b (all data)
R1 = 0.0953, wR2 = 0.1366
R1 = 0.1456, wR2 = 0.2026
a
b
2
2 2
2 2 1/2 c
R1 = (Σ||Fo| - |Fc||) / (Σ|Fo|); wR2 = [Σ(w|Fo| -|Fc| ) / Σw|Fo| ) ] ; GOF = [w(Fo2-Fc2) 2]/(n-p)1/2.
R1 = 0.0675, wR2 = 0.1589
R1 = 0.1026, wR2 = 0.1762
SANGHAMITRA et al.: ANTICANCER Cu(I) COMPLEXES OF MODIFIED NUCLEOBASES & VITAMIN B3
to cisplatin, except in K562 cell line in which these
complexes show better inhibition than cisplatin. The IC50
values of the complexes are given in Table 4 which
shows that complexes (1) and (2) are more potent
in vitro in the cell lines Colo205, H226, HOP62 and
K562 than complex (3), whereas the activity is similar
for MCF7 and T24. Complexes (1) and (2) are found to
be equivalent to or more active than cisplatin in all the
cell lines. Previously Ru(II) complexes of
thiopyrimidines and thiopurines have been shown to
have an IC50 of 18 µM in ovarian cancer cell lines25,
whereas in this study in a different set of cell lines we
have observed IC50 values of ~ 5-7 µM for Cu(I)
complexes of thiopyrimidines (1) and (2).
Since these three complexes showed consistent
activity in a range of cell lines their in vivo activity
was studied. Unlike the in vitro behavior, the in vivo
activity of the complexes was different. The in vivo
results and % ILS of the complexes are given in
Table 5. These results show that complex (3) is active
and non-toxic in spontaneous breast carcinoma and
L1210 whereas complexes (1) and (2) are active and
non-toxic in P388 leukemia. However, complexes (1)
and (2) were very toxic in Lewis lung carcinoma and
Table 4In vitro IC50 values for the complexes for the
different cell lines
Complex
(1)
(2)
(3)
Cisplatin
IC50 (µg/mL)a
Colo205 H226 HOP62 K562 MCF7
5
5
7
10
5
5
5
5
10
5
15
16
18
18
16
9
7
8
20
7
T24
5
5
8
5
a
The IC50 values are derived from graphs plotted using the average
% inhibition values obtained from 3 different sets of experiments.
471
P388 when the vehicle was 10 % DMSO, and so further
experimentation was not carried out. On the other hand,
with carboxymethyl cellulose (CMC) as a vehicle, these
two complexes were active and non-toxic, although the
% ILS was only 50 % and 37.5 % for complexes (1) and
(2) respectively at 75 mg/kg concentration. Unlike
complexes (1) and (2), complex (3) was non-toxic in
both spontaneous breast carcinoma model and murine
leukemia L1210. Complex (3) did not show any activity
in L1210 but showed activity at 50 mg/kg on the
35th day with 55 % ILS in spontaneous breast carcinoma
upon intravenous injection. Higher toxicity of
complexes (1) and (2) could be partly due to the
observed higher log Poct value of more than 2.6. As we
have discussed before the non-toxicity of complex (3)
could be ascribed to its higher water solubility having a
log Poct value of 0.75.
Metal complex-DNA interactions
Since it is reported that the key step in the mechanism
of the anticancer activity of thiopurine based drugs is the
incorporation of thiobases into the nucleic acids,24,26, 27 we
have studied the DNA binding and DNA cleavage
activity of the complexes. Intercalation of EtBr in CT
DNA enhances its fluorescence intensity as the
fluorescence quenching by solvent molecules is
prevented. Binding of small molecules to CT DNA can be
conveniently studied by monitoring the fluorescence of
EtBr, since it is displaced from DNA and the fluorescence
intensity is reduced. A plot of fluorescence intensity
versus concentration of drug/small molecule permits
evaluation of the apparent binding constant. The apparent
binding constant of complex (3) with calf thymus DNA is
5.9 × 105 M. Complexes (1) and (2) also reduce the EtBr
fluorescence intensity but Kapp could not be obtained as
Table 5The % ILS of the complexes in different mouse models
Cell lines
P388
L 1210
Dose/route
100mg/kg IP
75 mg/kg IP
50 mg/kg IP
25 mg/kg IP
P388
75 mg/kg IP
25 mg/kg IP
P388
75 mg/kg IP
50 mg/kg IP
25 mg/kg IP
Spontaneous breast tumor 50 mg/kg, IV
Lewis lung carcinoma
100 mg/kg. IP
100 mg/kg IP
a
Schedule
1–9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
1, 5, 9
% ILSa/ % tumor growth inhibitionb
Comments
(1)a
(2)a
(3)b
10 % DMSO
10
00
NDc
Inactive & toxic
00
14.3
NDc
CMC
14.3
7.1
Both inactive
7.1
7.1
10 % DMSO
23.5
17.6
NDc
Both inactive
5.8
5.8
CMC
37.5
50
NDc
1 and 2 active and
CMC
non toxic at all these
25.0
37.5
CMC
doses.
25.0
25.0
Water
55.0
Active on day 35
10 % DMSO
Toxic
Toxic
NDc
Expt. terminated
Vehicle
1–9
% increase in lifespan (% ILS); b % tumor growth inhibition; c Not done.
472
INDIAN J CHEM, SEC A, MARCH-APRIL 2011
50 % reduction in fluorescence intensity of EtBr-DNA
complex was not achieved. Due to the high
hydrophobicity of the complexes a precipitate was
formed on further addition of the complexes to the
EtBr-DNA solutions. However, before the onset of
precipitate formation, the EtBr fluorescence was
decreased to 13.2 % and 22.5 % after initial addition of
1200 µM of complexes (1) and (2) respectively. The
fluorescence quenching of the EtBr-DNA solution on
addition of these complexes is shown in Fig. 2.
Interestingly, complexes (1) and (2) are weakly
fluorescent when excited at 250 nm and emit at 364 nm
in 5 mM tris-HCl and 5 mM NaCl. As the emission
spectra are not affected by the addition of CTDNA, this
emission could not be used for binding studies.
Since a previous study12 showed that the Cu(I)
DPPE complexes cleave DNA in vivo, SC DNA
cleavage study was carried out. Figure 3 shows the
cleavage of SC DNA by the complexes (1), (2) and (3).
Complex (3) shows complete cleavage at 400 µM but
in the case of complexes (1) and (2) no cleavage was
observed even at 1200 µM as shown in Fig. 3.
However, these two complexes showed retardation in
movement, which might be due to the reduction in the
super helical density of the complex bound DNA.
Potent anticancer drugs, adriamycin and cisplatin,
behave in a similar fashion and do not cleave SC
DNA.28 It has also been observed that adriamycin and
actinomycin D result in elongation of DNA and
reduction in DNA synthesis respectively.29,30 So,
in vitro DNA cleavage does not always translate to
cytotoxicity. Complexes (1) and (2) show potent in vitro
cytotoxicity at < 10 µg/mL concentration, whereas
complex (3) shows cytotoxicity only at < 20 µg/mL dose
level. These complexes probably follow a different
pathway for the observed antitumor activity.
Interaction with biologically relevant molecules
While elucidation of the mechanism of action of the
metallodrugs, it is also crucial to ascertain the active
species in vivo, after intravenous injection of the metal
complexes. Especially since copper complexes readily
undergo ligand substitution and redox reactions, it is
difficult to pinpoint the nature of the active species
[Cu(I) or Cu(II)] with certainty. Once intravenously
administered, metallodrugs can react with human
serum albumin (HSA) and intracellularly abundant
thiols like glutathiones and undergo redox reactions.
Hence, in order to understand the structural integrity
under physiological conditions, it is important to study
the binding of the metal complexes with plasma
Fig. 2Fluorescence quenching of EtBr-DNA solution upon addition
of different concentrations of the complexes in 5 mM Tris-HCl and
5 mM NaCl. [■, (1); ●, (2); ▲, (3). λex = 510 nm; λem = 600 nm].
Fig. 3Gel electrophoresis diagrams for the cleavage of
supercoiled (SC) pUC19 DNA with (A) complexes (1) and (2) and
(B) complex (3). [Cleavage of supercoiled pUC19 DNA (0.5 µg) by
400 µM of complex (3) in a 5 mM tris HCl/NaCl buffer (pH 7.2) at
37 °C containing 1 % DMF. Forms I and II are SC and nicked
circular (NC) DNA. 1200 µM of complexes (1) and (2) were used].
proteins, such as human serum albumin (HSA) and
intracellularly abundant thiols like glutathiones. The
interaction of the most active complex (3) with bovine
serum albumin (BSA), reduced glutathione (GSH),
oxidized glutathione (GSSG) and methionine (meth)
was studied by 31P NMR.
In the case of complex (3), there was no ligand
exchange reaction even in the presence of 5 equivalents
of GSH as observed from the unchanged peak position
of PPh3 in the 31P NMR spectrum. As a control
experiment, similar reactivity studies were done with
the complex [Cu(PPh3)4]ClO4. It was observed that
31
P NMR value was completely shifted to –5 ppm (free
PPh3 value) from –0.7 ppm (complex), and most
SANGHAMITRA et al.: ANTICANCER Cu(I) COMPLEXES OF MODIFIED NUCLEOBASES & VITAMIN B3
importantly, the intensity of the peak at –0.7 ppm was
completely diminished while the intensity of the peak
at –5 ppm (free PPh3) and +30 ppm (phosphine oxide)
increased with increasing concentrations of GSSG and
GSH. These results point to the fact that complex (3)
retains its structural integrity in the presence of HSA
under physiological conditions, and suggests that the
activity of complex (3) could be due to the Cu(I) species.
Conclusions
In the present work, we have shown potent anticancer
activities of thionucleobase and nicotinamide containing
copper(I) phosphine complexes. By using the
differences in the fluorescence intensities of bound and
free EtBr as a probe, we have monitored DNA binding.
In the presence of these complexes, there was decrease
in the fluorescence intensity showing release of EtBr
from the DNA-EtBr complex. The % quenching of
EtBr-DNA fluorescence was highest about 55 % for
complex (3) whereas the quenching is only 13 % and
22 % for complex (1) and (2) respectively. So
complex (3) binds to CT DNA more efficiently than
complexes (1) and (2). Similarly in an in vitro DNA
cleavage studies with SC DNA we have also showed
that complex (3) cleaves about 90 % of SC DNA at
0.4 mM concentration whereas complex (1) and (2)
showed only retardation in the mobility of bound
SC-DNA. Since complexes (1) and (2) showed potent
in vitro cytotoxicity but they do not exhibit DNA
binding and DNA cleavage activity, the results indicate
that complexes (1) and (2) exert cytotoxicity by some
other mechanism which may not involve direct cleavage
of DNA. However, their in vivo activity was not
encouraging owing to the toxicity observed. Hence, our
experimental results suggest that complex (3) holds forth
promise as a drug candidate, with potent in vitro and
in vivo antitumor activity, having optimum lipophilicity,
leading to reduced toxicity.
Supplementary Data
The X ray crystallographic files (in CIF format) for
the structure determination of the complexes have been
deposited with the Cambridge Crystallographic
Data Centre under CCDC 767500 (complex 1),
CCDC 767499 (complex 2), CCDC 767501
(complex 3). These can be obtained free of charge from
the CCDC via www.ccdc.cam.ac.uk/data_request/cif.
Acknowledgement
Generous financial support from DBT, New Delhi,
India, is gratefully acknowledged.
473
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