Synthesis and structure-activity relationship of benzylamine supported platinum(iv) complexes

Article abstract of DOI:10.1039/c3nj41141a

A series of benzylamine derivative (BAD) supported platinum(iv) complexes (PtCl₄(BADs)₂) have been synthesized. The complexes were tested in vitro against the MCF-7 cell line, and the 4-fluoro and 4-chloro containing complexes expressed impressive anticancer activities. Their DNA binding nature for a structure-activity relationship (SAR) study was investigated with physicochemical-indicators (PCI) which categorized them as good intercalators for host-guest chemistry. A mechanism for drug efficacy is proposed by analysis of the resultant viscosity and surface tension of PtCl ₄(BADs)₂-DNA solutions named as the drug-friccohesity interaction (DFI). The complexes have shown significant antioxidant activity, determined on the basis of free radical scavenging effects due to their terminating action against the reactive species.

Full text of DOI:10.1039/c3nj41141a

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Synthesis and structure–activity relationship of
benzylamine supported platinum(IV) complexes†
Rakesh Kumar Ameta,a Man Singh*a and Raosahab Kathalupant Kaleab
Cite this: NewJ.Chem., 2013,
37, 1501
A series of benzylamine derivative (BAD) supported platinum(IV) complexes (PtCl4(BADs)2) have been
synthesized. The complexes were tested in vitro against the MCF-7 cell line, and the 4-fluoro and
4-chloro containing complexes expressed impressive anticancer activities. Their DNA binding nature for
a structure–activity relationship (SAR) study was investigated with physicochemical-indicators (PCI)
which categorized them as good intercalators for host–guest chemistry. A mechanism for drug efficacy
is proposed by analysis of the resultant viscosity and surface tension of PtCl4(BADs)2–DNA solutions
named as the drug-friccohesity interaction (DFI). The complexes have shown significant antioxidant
activity, determined on the basis of free radical scavenging effects due to their terminating action
against the reactive species.
Received (in Montpellier, France)
16th December 2012,
Accepted 20th February 2013
DOI: 10.1039/c3nj41141a
www.rsc.org/njc
be accepted that Pt(IV) complexes act as prodrugs via reductive
Introduction
activation to their reactive Pt(II) complexes.12,13 Pt(IV) based
In the recent past, bioinorganic complexes of platinum have
been a fascinating area of research. Advancements in coordina-
tion chemistry have emerged from contemporary interests in
the fields of agriculture, nanomedicine, MEMS (microelectro-
mechanical systems) and NEMS (nanoelectromechanical
systems).1 Due to increasing interest, cisplatin, a platinum
complex (PC), has been used in the treatment of various kinds
of human cancer, although side-effects, such as nephrotoxicity
and drug resistance, have been a great constraint for its wider
uses.2 Many Pt(II) and Pt(IV) complexes have been synthesized
for their anticancer chemistry.3–6 Pt(II) complexes show several
restrictions compared with those of Pt(IV), however, Pt(IV) com-
plexes are also cytotoxic in nature, but have some advantages in
comparison to Pt(II) complexes.7,8 In the higher oxidation state,
two extra ligands and the change from planar to octahedral
geometry, together with higher kinetic inertness compared to
the Pt(II) complexes, make Pt(IV) complexes interesting candi-
dates for the design of new platinum-based anticancer drugs.9
However, whilst Pt(IV) complexes such as tetraplatin, iproplatin
and satraplatin have been in clinical trials, unfortunately, they
have not gained clinical approval up to now3,4 due to high
general toxicity, disputed benefit and other factors.9–11 It could
drugs would have better activity due to extracellular reduction
which would lead to deactivation and general toxicity when they
are reduced primarily in the cell.14 Currently, several anticancer
drugs have been successfully used to cure cancer diseases,15
however, such drugs are not effective on solid tumors, for
example, breast cancer, whose occurrence has been increasing
over the past few years.16,17 Therefore, cancer chemotherapy
against solid tumors is a research thrust area where effective
drugs are required. Thus, the selection of benzylamine deriva-
tives in this work as ligands to prepare Pt(IV) complexes and
their anticancer screening against the MCF-7 cell line are an
endeavour towards obtaining efficient anticancer drugs which
may be probed for anticancer activities in order to inhibit
cytotoxicity such as is found with other platinum complexes.3
Also, platinum complexes with benzylamine derivatives as
ligands are used for their catalytic or biological applications.4
In general, the synthesis of Pt(IV) complexes is a difficult task
but their DNA binding activity (DBA) is helpful in the identifi-
cation of their anticancer nature8 and is considered a great
incentive for their synthesis. DBA has been extensively investi-
gated over the past several decades, due to the extraordinary
potentiality in anticancer activities, DNA structural and depen-
dent electron transfer probes, DNA foot-printing, sequence-
specific cleaving agents and many others.18,19 DNA is a primary
molecular target of PC based anticancer drugs due to their
molecular mechanics and dynamics. Therefore, the PC–DNA
interaction ascertains the extent and mode of a drug’s chemo-
therapeutic potential. The DNA targeted PCs act as intercalators
a School of Chemical Sciences, Central University of Gujarat, Gandhinagar-382030,
India. E-mail: ametarakesh40@gmail.com, mansingh50@hotmail.com;
Fax: +91 079-23260076; Tel: +91 079-23260210
b School of life Sciences, JNU, New Delhi-110067, India
† Electronic supplementary information (ESI) available: Details of absorption
titration of complex–DNA interaction. See DOI: 10.1039/c3nj41141a
c
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that can lead to the development of efficient anticancer drugs due 446.94 (Pt–N), 346 (Pt–Cl). 1H NMR dH (500 MHz; DMSO-d6;
to non-covalent binding.20 Their anticancer activity with DBA is Me4Si) 5.373 (2H, s, PhCH2NH2), 3.890 (2H, s, PhCH2N),
studied with the aim of designing new platinum antitumor 7.629–7.615 (1H, d, PhH, J = 7 MHz), 7.466–7.451 (1H, d,
agents with low toxicity in the blood stream and non-tumoral PhH, J = 7.5 MHz), 7.408–7.354 (2H, m, PhH). 13C NMR dC
tissues.19,20 Apart from their anticancer activities, transition (500 MHz; DMSO-d6; Me4Si) 47.77 (C1), 135.72 (C2), 132.91 (C3),
metal complexes are being widely used as preliminary materials 131.05 (C4), 129.77 (C5), 127.82 (C6) and 130.14 (C7). +ve
in the development of therapeutic interventions due to their ESI-MS: 622.0361 [M + 2] (calc. for [C14H16N2Cl6Pt] = 620).
antioxidant potential.21 Their antioxidant activities are found to UV/Vis = lmax (DMSO)/nm 265 (e/dm3 molꢁ1 cmꢁ1 1775).
prevent ageing and different diseases associated with oxidative
Synthesis of bis(((3-chlorophenyl)methanamine)tetrachloro-
damage, due to their terminating action against reactive species platinum) [M3CBA]. Reaction of K2PtCl4 (0.602 mmol) and
such as free radicals.22 Thus, new anticancer platinum(IV) com- 3-chlorobenzylamine (1.204 mmol) produced a yellow precipi-
plexes have been synthesized whose antitumor, DBA and anti- tate. Yield: 0.3015 g, 80%. Elemental analysis, found: C, 27.00;
oxidant studies would be useful in the medicinal field.
H, 2.36; N, 4.20%. Calcd for C14H16N2Cl6Pt: C, 27.12; H, 2.6; N,
4.52%. IR (KBr): nmax/cmꢁ1 3238.8 and 3193.9 (NH2), 1475 and
1430 (Ph, CQC), 798.88 (mono substituted Ph), 1083.7 (C–N),
708.16 to 683.67 (C–Cl), 438.78 (Pt–N), 348 (Pt–Cl). 1H NMR dH
(500 MHz; DMSO-d6; Me4Si) 5.363 (2H, s, PhCH2NH2), 3.653
Experimental section
Materials and methods
Potassium tetrachloroplatinate (K2PtCl4, 99.99%) and BADs (2H, s, PhCH2N), 7.588–7.393 (4H, m, PhH). 13C NMR dC (500
(49.5%) were used (Sigma Aldrich) as received. Structures of MHz; DMSO-d6; Me4Si) 49.66 (C1), 141.11 (C2), 130.70 (C3),
the complexes were determined with FTIR (Perkin Elmer) in a 133.41 (C4), 129.07 (C5), 128.01 (C6) and 127.98 (C7), +ve ESI-
KBr palate with polystyrene thin film as a calibration standard. MS: m/z 622.0364 [M + 2] (calc. for [C14H16N2Cl6Pt] = 620). UV/
1H and 13C NMR spectra were recorded in (CD3)2SO Vis = lmax (DMSO)/nm 270 (e/dm3 molꢁ1 cmꢁ1 1987).
(NMR, 99.99%) with a Bruker-Biospin Avance-III 500 MHz
Synthesis of bis(((4-chlorophenyl)methanamine)tetrachloro-
FT-NMRspectrometer using TMS as an internal standard for platinum) [M4CBA]. M4CBA was prepared with K2PtCl4
chemical shifts. Mass spectra were obtained with an Agilent (0.602 mmol) and 4-chlorobenzylamine (1.204 mmol) to give a
Q-TOF LC/MS with ESI+ mode with acetonitrile and water in a yellow precipitate. Yield: 0.3088 g, 82%. Elemental analysis,
3 : 7 ratio as the mobile phase. Elemental analysis was per- found: C, 26.98; H, 2.41; N, 4.40%. Calcd for C14H16N2Cl6Pt: C,
formed with a Euro vector instrument. UV/Vis spectra were 27.12; H, 2.6; N, 4.52%. IR (KBr): nmax/cmꢁ1 3230.6 and 3193.9
recorded with a Spectro 2060 plus model UV/Vis spectrophot- (NH2), 1491 and 1447 (Ph, CQC), 802.04 (mono substituted Ph),
ometer over 200–600 nm using a 1 cm path length cuvette in 1096–1018.4 (C–N), 842.46 (C–Cl), 495.92 (Pt–N), 352 (Pt–Cl).
DMSO at 2 ꢀ 10ꢁ3 M (molar).
1H NMR dH (500 MHz; DMSO-d6; Me4Si) 5.313 (2H, s,
PhCH2NH2), 3.623 (2H, s, PhCH2N), 7.518–7.505 (2H, d, PhH,
J = 6.5 MHz), 7.452–7.433 (2H, d, PhH, J = 9.5 MHz). 13C NMR dC
Synthesis of platinum complexes
Synthesis of bis(phenylmethanamine)tetrachloroplatinum (500 MHz; DMSO-d6; Me4Si) 49.42 (C1), 137.44 (C2), 132.81 (C5),
[MBA]. MBA was prepared by the General procedure given in 131.16 (C3 and C7), 128.81 (C4 and C6). +ve ESI-MS: m/z
ESI† with K2PtCl4 (0.602 mmol) and benzylamine (1.204 mmol) 622.0362 [M + 2] (calc. for [C14H16N2Cl6Pt] = 620). UV/Vis = lmax
to give a yellow precipitate. Yield: 0.2676 g, 80%. Elemental (DMSO)/nm 265 (e/dm3 molꢁ1 cmꢁ1 1836).
analysis, found: C, 30.01; H, 3.0; N, 4.88%. Calcd for
14H18N2Cl4Pt: C, 30.51; H, 3.29; N, 5.08%. IR (KBr): platinum) [M4FBA]. K2PtCl4 (0.602 mmol) and 4-fluorobenzyl-
max/cmꢁ1 3259 and 3202 (NH2), 1496 and 1455 (Ph, CQC), amine (1.204 mmol) gave a yellow precipitate. Yield: 0.2571 g,
Synthesis of bis(((4-fluorophenyl)methanamine)tetrachloro-
C
n
757.14 (mono substituted Ph), 1079 (C–N), 479.6 (Pt–N), 338 72%. Elemental analysis, found: C, 28.54; H, 2.55; N, 4.56%.
1
(Pt–Cl). H NMR dH (500 MHz; DMSO-d6; Me4Si) 5.273 (2H, s, Calcd for C14H16N2Cl4F2Pt: C, 28.64; H, 2.75; N, 4.77%. IR (KBr):
PhCH2NH2), 3.577 (2H, s, PhCH2NH2), 7.383–7.354 (3H, m,
n
max/cmꢁ1 3259 and 3202 (NH2), 1496 and 1455 (Ph, CQC),
PhH), 7.325–7.312 (2H, d, PhH, J = 6.5 MHz). 13C NMR dC 757.14 (mono substituted Ph), 1079.6 (C–N), 834.1 (C–F), 475.51
(500 MHz; DMSO-d6; Me4Si) 50.18 (C1), 138.49 (C2), 129.23 (Pt–N), 349 (Pt–Cl). 1H NMR dH (500 MHz; DMSO-d6; Me4Si)
(C4 and C6), 128.95 (C3 and C7) and 128.21 (C5), +ve ESI-MS: 5.301 (2H, s, PhCH2NH2), 3.625 (2H, s, PhCH2N), 7.449–7.438
m/z 552.1544 [M + 1] (calc. for [C14H18N2Cl4Pt] = 551). UV/Vis = (2H, d, PhH, J = 5.5 MHz), 7.23–7.195 (2H, t, PhH, J = 17.5 MHz).
lmax (DMSO)/nm 270 (e/dm3 molꢁ1 cmꢁ1 2432).
13C NMR dC (500 MHz; DMSO-d6; Me4Si) 49.41 (C1), 134.81 (C2),
Synthesis of bis(((2-chlorophenyl)methanamine)tetrachloro- 131.39 (C3 and C7), 115.53 (C4 and C6), 161.09 (C5). +ve ESI-MS:
platinum) [M2CBA]. M2CBA was prepared with K2PtCl4 m/z 590.097 [M + 3] (calc. for [C14H16N2Cl4F2Pt] = 587.183). UV/
(0.602 mmol) and 2-chlorobenzylamine (1.204 mmol) which Vis = lmax (DMSO)/nm 265 (e/dm3 molꢁ1 cmꢁ1 2229).
gave a yellow precipitate. Yield: 0.2636 g, 70%. Elemental
In vitro anticancer activity
analysis, found: C, 26.95; H, 2.4; N, 4.12%. Calcd for
C
14H16N2Cl6Pt: C, 27.12; H, 2.6; N, 4.52%. IR (KBr): nmax/cmꢁ1 Cell viability was estimated colorimetrically using 2-(3-diethyl-
238.8 and 3198.0 (NH2), 1475 and 1447 (Ph, CQC), 753 amino-6-diethylazaniumylidene-xanthen-9-yl)-5-sulfobenzene-
(mono substituted Ph), 1055 (C–N), 679.6 to 630 (C–Cl), sulfonate, SRB assay as standard.23
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Cell lines and culture conditions
estimation of their relevance in inducing structural changes in
medium, the temperature was controlled at 298.15 K with
ꢃ0.01 1C by an auto temperature control LAUDA ALPHA RA 8
thermostat. About 15 to 20 measurements for each composition
were made to assure high precision. Flow time and pendent
drop were measured with a digital stopwatch and drop counter,
respectively, and were repeated five times to obtain average flow
times and numbers of pendent drops. The data are presented
as (Z/Z0)1/3 versus binding ratio; Z is the dynamic viscosity of
DNA with complexes while the Z0 is the viscosity of the DNA
mixture in buffer.33
Conductance and zeta potentials of DNA solutions with and
without complexes were measured with a LABINDIA, PICO+
conductivity and Microtrac Zetatrac, U2771, DLS, respectively at
250 1C. 0.1, 0.01 and 0.001 M aqueous KCl solutions having
conductances 12.88, 1.413 and 147 mS cmꢁ1 respectively, were
used for calibration of the conductivity meter. An auto sus-
pended solution of alumina suspension (400-206-100) was used
as a zeta potential standard. Initially, for DMSO + Tris buffer a
set-zero was made for the zeta potential. For both the measure-
ments, the DNA concentration was kept constant while the
concentration of the PtCl4(BADs)2 was varied from 50 to 400 mM
with 50 mM intervals.
Human breast cancer cell lines (MCF-7) were obtained from
NCI, USA, and grown in minimal essential medium (MEM).
Eagles media were supplemented with 10% heat inactivated
fetal bovine serum (FBS, Sigma-Aldrich), 2 mM L-glutamine
and 1 mM sodium pyruvate (Hyclone) in humidified CO2
incubators.
Assay of cytotoxicity in cancer cell lines
The cytotoxicities of platinum complexes were determined by
SRB assay where E5000 cells were seeded into each well of a 96
well clear flat bottom polystyrene tissue culture plate and
incubated for 2 h in MEM. An additional 190 mL cell suspension
was added in each well containing 10 mL test sample in 10%
DMSO with 10 mL Adriamycin (doxorubicin) as a positive drug
control. Each experiment was carried out in 3 replicate wells.
After an incubation of 48 h, 100 mL of 0.057% SRB solution (w/v)
was added into each well. Then 200 mL of 10 mM Tris base
solution (pH 10.5) was added into each well and the wells
shaken smoothly. The cell viability was assayed by absorption
at 510 nm with a microplate reader. The experiments were
repeated thrice with 5 replicates each time and 99% reprodu-
cibility was obtained.
DPPH free radical antioxidant activity
DNA binding
Antioxidant or scavenging activities were determined on the
basis of a free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH)
scavenging effect.34 Stock solutions of 1000 mM of the tested
samples and DPPHꢄ (0.002%) were prepared with DMSO–water
(1 : 1). To prepare samples, 1 mL of 0.002% DPPHꢄ solution was
added into 1 mL of complex of 50, 100, 150, 200, 250, 300, 350
and 400 mM separately. The reaction mixtures were thoroughly
mixed by shaking the test tubes vigorously and incubated at
25 1C for 60 min keeping in a water bath in the dark.
Absorbance was measured at 517 nm in a spectro 2060 plus
modeled UV/Vis spectrophotometer. The radical scavenging
activity was determined as a result of the decrease in absor-
bance of DPPH,35 and calculated with the following formula:
CT-DNA (Sigma) was used as received (analytical grade).
Tris–HCl buffer (10 M, pH = 7.2) was prepared in Milli-Q water,
and used for DNA stock solution preparation, absorption titra-
tion, viscosity, surface tension and zeta potential measurements.
The DNA concentration was determined using an absorption
spectrophotometer as a molar absorptivity (6600 Mꢁ1 cmꢁ1) at
260 nm.24,25 The CT-DNA in buffer gave a ratio of UV absorbance
at 260 and 280 nm of 1.8–1.9, indicating the DNA was free of
protein.26–29 Absorption titrations in Tris-buffer were performed
at 10, 30, 50, 70 and 90 mM, to which DNA stock solutions
(50 mM) were added, (ri = [complex]/[DNA] = 0.2, 0.6, 1, 1.4
and 1.8). The PtCl4(BADs)2–DNA solutions were incubated at
room temperature for 15 min before recording the absorption
spectra. To elucidate their binding strength, an intrinsic binding
constant (Kb) with CT-DNA was obtained by monitoring a change
in absorbance of the DNA with increasing amounts of
Scavenging activity (%) = (A0 ꢁ AS/A0) ꢀ 100
AS is the absorbance of DPPH with a tested compound and A0 is
the absorbance of DPPH without a tested compound (control).
Radical scavenging potential was expressed as an EC50
value and represents the test compound concentration at which
50% of the DPPH radicals are scavenged. The data calculated
for antioxidation are presented as means ꢃSD of three
30
PtCl4(BADs)2, and calculated with the following equation:
½DNAꢂ ½DNAꢂ
1
¼
þ
ea ꢁ eb
eb ꢁ ef Kbðeb ꢁ ef Þ
ea, ef and eb are the apparent, free and bound complex extinction determinations.
coefficients, respectively. ef was determined using a calibration
curve of an isolated metal complex, with the Beer–Lambert law.
Results and discussion
Synthesis
ea was determined as the ratio between the measured absor-
bance and PtCl4(BADs)2 concentration such as Aobs/[Pt]. A plot
of [DNA]/(ea ꢁ ef) vs. [complex] produced a slope of 1/(eb ꢁ ef), The synthesis of PtCl4(BADs)2 was achieved by reaction of
and a Y intercept equal to 1/Kb(eb ꢁ ef); Kb is the ratio of the K2PtCl4 with different BADs (Fig. 1a–e) in ethanol–aqueous
slope to the Y intercept.30
solution in a 1 : 2 ratio over 24 h (reaction, Scheme 1).
For their synthesis, K2PtCl4 and BADs were reacted in a ratio
Viscosity and surface tension measurements were con-
ducted with a Borosil Mansingh survismeter, BMS.31,32 For an of 0.602 mmol to 1.204 mmol respectively and the complexes
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Fig. 2 Growth curves for a human breast cancer cell line (MCF-7).
We estimated the GI50, TGI and LC50 in mg mLꢁ1 as a
response parameter (Table 1) which inferred a 50% growth
inhibition, resultant total growth inhibition and a net loss of
50% cells after treatment respectively.
Fig. 1 (a–e) Structures of the ligands (L = BADs).
The less than 10 mg mLꢁ1 of GI50 depicts anticancer activity
with respect to ADR and cisplatin.38 Data analysis for M4CBA
and M4FBA (GI50 o 10) showed impressive anticancer activity
as compared to standard ADR and cisplatin which have GI50
values o10 (Table 1). The GI50 = 13.4 and 11.9 values of M2CBA
and M3CBA respectively, were near to standard against the
Scheme 1 Synthesis of Pt(IV) complexes where L = BADs (Fig. 1a–e).
were formed as yellow powders. The reaction of tetrachloro- MCF cell line while MBA, having GI50 = 21.9, is far from
platinate (a compound of platinum +2) with a simple primary anticancer activity. This data analysis led us to infer that the
amine, to give a compound of platinum +4, is an oxidation anticancer nature of PtCl4(BADs)2 is associated with the o-, m-
process which is carried out in the presence of an oxidant, but and p-halide substitution of the benzylamine ligands as MBA o
many coloured complexes of Pt(IV) have been synthesized in M2CBA o M3CBA o M4CBA = M4FBA. Several platinum
aqueous organic solvent without adding any oxidant.5 For complexes have been synthesized for the curing of the cancer
structure determination, elemental analysis, FTIR, 1H and but our focus was to investigate the anticancer activity of
13C NMR, LC-MS and UV/Vis were used and data are given in synthesized platinum(IV) complexes with benzylamine deriva-
experimental section. The platinum nitrogen coordinate bond tives as ligands. However, cytotoxicity studies of the ligands
vibrations were from 495.92 to 438.78 cmꢁ1 36,37
The ESI +ve only are being pursued in our laboratory and will soon be
.
mass spectra had peaks of M + 1 for MBA, M + 2 for M2CBA, communicated in the next paper of the series on benzylamine
M3CBA, M4CBA and M + 3 for M4FBA, confirming their derivatives. Thus, the complexes most probably will have bright
molecular mass. The UV/Vis absorption from 265 to 270 nm prospects as effective anticancer drugs, especially against solid
and 1H NMR coupling constant within 5 to 9 MHz confirmed tumors like other Pt(IV) complexes.4,9
their trans and octahedral geometry.5,6,38 The elemental analysis,
1
FTIR, H and 13C NMR, LC-MS and UV/Vis data for structural
DNA binding activities
illustration were in close agreement.
DBA is a recent mechanism for SAR studies of metallic com-
pounds, depicted in absorption spectra. Thus, the PtCl4(BADs)2
were mixed with CT-DNA which resulted in changes in absor-
Anticancer activity
The complexes were tested in vitro against the MCF-7 human bance. Generally, the extent of hypochromism reveals the
breast tumor cell line in comparison to Adriamycin (ADR) by intercalative binding strength of complexes can be attributed
colorimetric micro culture 2-(3-diethylamino-6-diethylazaniumyl- to the interaction with DNA bases, as a characteristic for
idene-xanthen-9-yl)-5-sulfo-benzenesulfonate (SRB) assay.23 intercalation.40,41 Similarly, a hyperchromic effect might be
The data were also compared with cisplatin whose data were ascribed to an external contact or to a partial uncoiling of
taken from the literature.38 This assay has several advantages DNA structure, exposing more bases of the DNA due to electro-
over the tetrazolium assays, being able to distinguish between static binding.42,43 We observed a significant hypochromic
cytostatic effects because the drug decreases the rate of cell effect in absorption titrations of interacting DNA with MBA,
proliferation and a cytotoxic effect is a true decrease in the M2CBA, M3CBA, M4CBA and M4FBA which exposes their
number of viable cells.39 The MCF-7 interaction activity for 10, intercalation40,41 with the base pairs of DNA (Fig. S1–S5, ESI†).
20, 40 and 80 mg mLꢁ1 PtCl4(BADs)2 was plotted (Fig. 2).
With UV spectrophotometric titration, the PtCl4(BADs)2–DNA
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Table 1 LC50, TGI and GI50 in mg mLꢁ1 against the MCF-7 cell line and
antioxidant EC50 values for PtCl4(BADs)2
Entry
Complex
LC50
TGI
57.3
45.5
46.7
42.7
40.8
30.6
GI50
21.9
13.4
11.9
EC50 (mM)
1
2
3
4
5
6
7
MBA
480
77.6
480
480
480
62.4
430
80
—
—
150
195
M2CBA
M3CBA
M4CBA
M4FBA
ADR
o10
o10
o10
o10
Cisplatin38
430
binding constants (Kb) were 1 ꢀ 104, 1.5 ꢀ 104, 1 ꢀ 104, 1 ꢀ 104
and 1.33 ꢀ 104 M2 cm for MBA, M2CBA, M3CBA, M4CBA and
M4FBA respectively. The Kb values predict the stronger inter-
actions caused by M2CBA and M4FBA whose intercalating
strengths depend on the size and electron densities of inter-
acting aromatic rings including an amine group, due to a
combined effect of hydrophobic and hydrophilic inter-
actions.40–43 The DBA of a ligand such as benzylamine was
investigated (Fig. S6, ESI†) and the absorption attributed inter-
action with DNA was lower in comparison to its complex
(Fig. S1, ESI†). Thus the complex is more strongly intercalated
with DNA than the free ligand. Table S1 (ESI†) presents a
comparative study of lmax for MBA, M2CBA, M3CBA and
M4CBA which is 240 nm and M4FBA which is 245 nm without
DNA, while with DNA they showed an almost negligible absor-
bance at these wavelengths. Thus, their nature is changed
entirely on binding with DNA for the development of a concept
of SAR for the understanding of their anticancer mechanism.
Fig. 3 Effect of increasing amounts of PtCl4(BADs)2 on viscosity and surface
tension of CT-DNA (5 ꢀ 10ꢁ5 M), a drug efficacy study.
respectively) were plotted against 1/R (R = [DNA]/[complex] =
0.2, 0.6, 1.0, 1.4, 1.8) (Fig. 3).
An increase in concentration of PtCl4(BADs)2 increased the
viscosity of DNA (Fig. 3) which is explained in terms of
the behaviour of the PtCl4(BADs)2 as DNA intercalators. For
ethidium bromide, the relative viscosity48 of DNA is increased
with a slope varying from 0 to 0.9448 whereas with 0.0351,
0.0317, 0.0312 and 0.0327 and 0.0318 slopes, the relative
viscosity of DNA increased for MBA, M2CBA, M3CBA, M4CBA
and M4FBA respectively. A decrease in slope value, maybe due
to an interaction of PtCl4(BADs)2 with DNA, made DNA longer,
which is reasonably explained as due to another interaction
occurring between DNA and PtCl4(BADs)2. The measured DNA
binding constant for PtCl4(BADs)2 is lower than that of ethi-
dium bromide. The viscosity of free ligand (benzylamine) with
DNA was measured and found to be lower than that of the
complex, from which it was inferred that interaction of DNA
with free ligand is weaker as compared to that with complex.
Therefore, a higher increase in viscosity of PtCl4(BADs)2–DNA
as compared to ethidium bromide and free ligand could have
produced a lower binding constant which clearly ascertained
intercalation.
Viscosity
For detailed DNA binding patterns, spectrophotometric studies
are supported by fluid dynamics in the form of viscosity data
(Fig. 3) as the most critical test for a classical intercalation
model. The viscosity is a classical intercalative mode in the case
of a DNA solution which is increased due to an increase in
overall DNA length44 with an expression of their molecular
mechanics and dynamics expressed as:
Ecovalent = Ebond + Eangle + Edihedral
Surface tension
Apart from the viscosity, the surface tension is an interaction
probing thermodynamic indicator to depict the disruption of
intramolecular forces existing in DNA, and acts as evidence for
stronger interactions with higher viscosity. This force disrup-
tion is measured as a decrease in surface tension, and therefore
the surface tension values of a DNA solution with increasing
amounts of PtCl4(BADs)2 (1/R = 0.2, 0.6, 1.0, 1.4, 1.8) (Fig. 3)
have been determined. The resultant surface tension of
PtCl4(BADs)2–DNA decreased which confirmed that cohesivity
or intramolecular interaction of DNA is lost on interaction with
the PtCl4(BADs)2.
The DNA and complexes have their specific covalent bond
lengths, bond angles and dihedral energies which develop a
critical DFI model as is given in the above equation and
discussed below. Thus, a partial non-classical intercalation
produces a bend or kink in the DNA helix that reduces its
effective length concomitantly. It is well known that with
cisplatin, a decrease in relative viscosity is explained due to
covalent binding which causes shortening in an axial length of
the double helix DNA.45 A partial intercalator decreases the
relative viscosity by reducing an axial length, whereas a classical
organic intercalator such as ethidium bromide increases the
relative viscosity by increasing an axial length of the DNA.46,47
To observe covalent binding or classical organic interaction,
Conductivity and zeta potential
their relative specific viscosities (Z/Z0)1/3 (Z0 and Z are specific The conductivity and zeta potential analyses for PtCl4(BADs)2–
viscosity contributions of DNA with and without PtCl4(BADs)2, DNA interactive complexes were key factors for the conformational
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Table 2 Conductivities (mS) of interacting and pure DNAa
adenine and thymine or guanine and cytosine are co-ordinately
or covalently bonded with platinum through intrastrand or
interstrand binding,49–52 producing higher viscosities. Thus,
the drug efficacy or intrinsic activity is dependent on its
disruptive activity as well as fluidity, and both should be in
an inverse manner because at first, the drug breaks DNA
cohesivity and then it interacts. Such behaviour of the
PtCl4(BADs)2–DNA interaction has shown the higher viscosities
and lower surface tension of a PtCl4(BADs)2–DNA solution
(Fig. 3). Thus, disruption of cohesivity, and the development
of interaction bonding or intercalation can be retrieved from
the DNA–drug interacting mechanism49–52 leading to the devel-
opment of a new model, referred to as DFI. DFI has inferred a
decrease in cohesivity as well as an increase in interaction
(Fig. S6, ESI†) which is supported by the anticancer activities of
PtCl4(BADs)2 (Table 1). To our best understanding, in DNA–
complex interactions, such a comparative study of surface
tension and viscosity as DFI has never been reported before;
this could be beneficial and useful in identifying the anticancer
nature of such metal complexes.
Entry Conc. (mM) MBA M2CBA M3CBA M4CBA M4FBA
1
2
3
4
5
6
7
8
50
772
648
601
547
510
471
446
404
689
654
594
547
523
469
434
399
678
605
555
522
480
446
413
366
586
555
518
480
411
420
392
355
617
594
569
525
487
444
406
354
100
150
200
250
300
350
400
a
DNA solution of 50 mM with 878 mS cmꢁ1 conductance.
Antioxidant activities
Antioxidant activities were analysed by the scavenging effect of
a stable free radical di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium
(DPPH) as per standard procedure,34,35 with a slight modifica-
tion. The percentage scavenging activity of PtCl4(BADs)2 was
determined in a concentration-dependent mode with a com-
parison with the DPPH free radical’s absorption at 517 nm.34,35
The DPPH free radical’s absorption at 517 nm in DMSO–water
was 0.440 to 0.445, a maximum. The complexes from 50 to
400 mM at an interval of 50 mM, showed a decrease in absorp-
tion, from which their antioxidant activities could be inferred34
(Fig. 5).
Fig. 4 Zeta potential of DNA (5 ꢀ 10ꢁ5 M) in the absence and presence of
increasing amounts of PtCl4(BADs)2.
behaviour of an isolated DNA chain with the complexes.
A DNA solution of 50 mM had an 878 mS cmꢁ1 conductivity
while the addition of increasing amounts of PtCl4(BADs)2
decreased the conductivities (Table 2). DNA molecules are
negatively charged due to phosphate groups, but by interaction
with PtCl4(BADs)2, the negative charge density is decreased
due to the positively charged metal which balances the charge
density.
The EC50 values for MBA, M4CBA and M4FBA (Table 1) and
M2CBA and M3CBA showed a 35.31 and 29.61% maximum
scavenging effect at 50 and 150 mM respectively.
With complexes, the zeta potential of the resultant
PtCl4(BADs)2–DNA also decreased (Fig. 4), from which it could
be inferred that an increase in Coulombic interaction and the
disassociation of counterions in DNA was limited.
The decrease in conductivity and zeta potential confirmed
strong interactions where the DNA could structurally be mod-
ified as a result of this interaction.
Drug efficacy studies or DFI with DNA binding
The intrinsic structural domain of an activity of a drug is
related to its therapeutic effect noted as the relative ability of
a drug–receptor complex with a maximum functional response.
Viscosity data in monitoring drug efficacy are gaining momen-
tum for a drug intake for trajectory areas because of their
functional domains. Initially, an effective anticancer drug breaks
cohesivity of DNA (distortion of intermolecular forces) and
decreases surface tension; secondly, the nitrogen atoms of
Fig. 5 Free radical scavenging activities of PtCl4(BADs)2.
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