Synthesis, characterization and tumor inhibitory activity of a novel Pd(ii) complex derived from methanethiol-bridged (2-((1H-benzo[d]imidazol-2-yl)methylthio)-1H-benzo[d]imidazol-6-yl)(phenyl)methanone

Article abstract of DOI:10.1039/C8NJ03057J

In this study, we designed a therapeutic active Pd(ii) complex with the new (2-((1H-benzo[d]imidazol-2-yl)methylthio)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone ligand in good yield. The structure of the ligand and its Pd(ii) complex was characterized via IR, UV-visible, ¹H-NMR, ¹³C-NMR, mass spectroscopy, TGA and powder XRD techniques. The spectral data of the Pd(ii) complex indicated the bidentate bonding mode for bis-benzimidazole and suggested a tetrahedral geometry for the metal complex. The in vitro antiproliferative effect of the BIPM ligand and Pd(ii) complex were tested against the MCF7, A549, Ehrlich ascites carcinoma (EAC) and Daltons lymphoma ascites (DLA) carcinoma cell lines. The metal complex exhibited excellent antiproliferative potency with a significant IC₅₀ value of ～10 μm against the EAC cell line compared to the ligand alone with a value of ～17 μM. Further, the in vivo antitumor effect study on the Pd(ii) complex against a murine EAC tumor model system showed obvious extended survivability. The tumor inhibitory mechanism of the Pd(ii) complex is due to its antiangiogenic effect and promotion of apoptosis, as verified by DNA condensation and FACS analysis. The potential photo-induced binding mode on double-stranded calf thymus DNA and protein cleavage activity study on pBR322 DNA of the complex confirmed its apoptotic characteristics. The significant hypochromic shift due to the strong π-π stacking interaction between the metal complex and the base pairs of DNA was clearly shown by the intrinsic DNA binding constant, k_b. The molecular docking study on the Pd(ii) complex interaction with DNA further confirmed its inhibition ability. The experimental results and drug-likeness properties of the Pd(ii) complex suggest its potential applications, which can be developed as a potent anticancer drug in the near future.

Full text of DOI:10.1039/C8NJ03057J

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Synthesis, characterization and tumor inhibitory
activity of a novel Pd(II) complex derived from
methanethiol-bridged (2-((1H-benzo[d]imidazol-
Cite this: New J. Chem., 2019,
43, 790
2-yl)methylthio)-1H-benzo[d]imidazol-
6-yl)(phenyl)methanone
a
a
b
c
Sunil Kumar N., Krishnamurthy G., * Yadav D. Bodke, Vikas H. Malojirao,
d
e
c
Ravikumar Naik T. R., Shivananda Kandagalla and Prabhakar B. T.
In this study, we designed a therapeutic active Pd(II) complex with the new (2-((1H-benzo[d]imidazol-
-yl)methylthio)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone ligand in good yield. The structure of the
2
1
13
ligand and its Pd(II) complex was characterized via IR, UV-visible, H-NMR, C-NMR, mass spectroscopy,
TGA and powder XRD techniques. The spectral data of the Pd(II) complex indicated the bidentate
bonding mode for bis-benzimidazole and suggested a tetrahedral geometry for the metal complex. The
in vitro antiproliferative effect of the BIPM ligand and Pd(II) complex were tested against the MCF7, A549,
Ehrlich ascites carcinoma (EAC) and Daltons lymphoma ascites (DLA) carcinoma cell lines. The metal
complex exhibited excellent antiproliferative potency with a significant IC50 value of B10 mm against the
EAC cell line compared to the ligand alone with a value of B17 mM. Further, the in vivo antitumor effect
study on the Pd(II) complex against a murine EAC tumor model system showed obvious extended
survivability. The tumor inhibitory mechanism of the Pd(II) complex is due to its antiangiogenic effect
and promotion of apoptosis, as verified by DNA condensation and FACS analysis. The potential photo-
induced binding mode on double-stranded calf thymus DNA and protein cleavage activity study on
pBR322 DNA of the complex confirmed its apoptotic characteristics. The significant hypochromic shift
due to the strong p–p stacking interaction between the metal complex and the base pairs of DNA was
Received 20th June 2018,
Accepted 16th November 2018
b
clearly shown by the intrinsic DNA binding constant, k . The molecular docking study on the Pd(II)
complex interaction with DNA further confirmed its inhibition ability. The experimental results and
drug-likeness properties of the Pd(II) complex suggest its potential applications, which can be developed
as a potent anticancer drug in the near future.
DOI: 10.1039/c8nj03057j
rsc.li/njc
2
second leading cause of death after heart diseases. Molecular
events leading to the establishment of cancer is classified in to
1
. Introduction
Cancer is the uncontrolled, rapid and pathological proliferation 6 major hallmarks (Hanahan 2011). The events are more or less
of abnormal cells, which is one of the most formidable afflictions interdependent, which regulates the progression of the disease.
1
worldwide. Cancer causes about 550 000 deaths a year and is the Evading angiogenesis and resistance to apoptosis are considered
as prime hallmarks. Neoangiogenesis contributes the progressive
a
Department of Chemistry, Sahyadri Science College, Kuvempu University,
destruction through enhanced microvessel density (MVD), which is
Shimoga, Karnataka, 577203, India. E-mail: gkmnaiksahyadri@gmail.com;
Fax: +91 08182 241793; Tel: +91-9448774649
3
an important surrogate marker. Tumor tissue displays an enormous
b
c
neovasculature, which is the recruitment of new blood vessels from
the existing vasculature. Moreover, it is a key factor in the develop-
ment of metastatic carcinoma and a requisite for the invasive nature
Department of Industrial Chemistry, Kuvempu University, Shankarghatta,
Karnataka, India
Molecular Biomedicine Laboratory, Postgraduate Department of Studies and
Research in Biotechnology, Sahyadri Science College, Kuvempu University,
Shimoga, Karnataka, India
4
of tumors. Simultaneously, tumor cells become more resistant to
cell death, which is a normal phenomenon in eliminating malfunc-
tional cells, leading to establishment of the tumor.
Current therapies suffer from the major limitation of side
effects and drug resistance; therefore, the continuous search for
d
e
Centre for Nano Science and Engineering (CeNSE), Indian Institute of Science,
Bangalore, Karnataka, India
Department of Biotechnology and Bioinformatics, Kuvempu University,
Shankarghatta, Karnataka, India
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newer and safer anticancer drugs remains critical in medicinal docking studies of the metal complex. The results demonstrate
chemistry. Compounds that bear heteroatoms such as nitrogen, the drug-likeness properties of the metal complex, which can be
sulfur and oxygen increase the strength of the complex by forming further developed into a potential therapeutic agent in the future.
5
hydrogen bonds with DNA. In recent years, the interactions
between transition metal complexes and DNA and protein
molecules have been an active research area in bioinorganic 2. Materials and methods
chemistry, which have been shown to play a very important role
in medicine, pharmacy and diagnostics. It is well known that
2
.1. General experiments
6
3,4-Diaminobenzophenone, o-phenylenediamine, potassium
DNA is the primary pharmacological target of many anticancer
7
hydroxide, chloroacetic acid, sodium metal, carbon disulphide,
LR grade methanol, LR grade hydrochloric acid and palladium
acetate were procured from Sigma-Aldrich (INDIA), Himedia
compounds. The interaction of metal complexes with specific
DNA sequences has been studied in the hope of understanding
the mechanism of their tumor inhibition for the design of new
drugs and recognizing specific sites or conformations targeted
(INDIA), and Labo Chemicals (INDIA) (commercially available
8
,9
from local sources) and were used as received without further
purification. Freshly distilled methanol was employed for
all synthetic purposes. Spectroscopic grade solvents were
employed for spectroscopic determinations. All other chemicals
were of AR grade. The progress of each coupling reaction was
monitored by TLC. Yields refer to isolated yields after column
chromatographic purification of compounds with a purity of
Z97%.
on DNA.
The benzimidazole moiety is part of the chemical structure of
10
vitamin B12, which is a biologically relevant natural compound.
Benzimidazole and its derivatives have attracted continuous
interest due to their diverse biological activities. As a typical
heterocyclic ligand, the large benzimidazole rings not provide
potential supramolecular recognition sites for p - p stacking
interactions, but also act as hydrogen bond acceptors and donors
11
The products of these reactions were authenticated by
matching their spectroscopic data with that reported in the
to assemble multiple coordination geometries. Metal complexes
are important in the development of antitumour drugs due to
their interaction with biomolecules in the human body and the
high stability of their final products under various conditions.
Metal complexes have been employed in antitumour therapy since
1
13
literature. H and C NMR spectra were recorded on a Bruker
2,13 400 MHz spectrometer and the thermal gravimetric analysis of
the metal complex was performed on a Diamond TG/DT
Analyzer (TG/DTA) from room temperature to 850 1C and at a
1
the discovery of the cytotoxicity of cis-platin (cis-Pt(NH ) Cl ) by
3
2
2
À1
14,15
heating rate of 20 1C min at the Sophisticated Analytical
Rosenberg and coworkers in the late 1960s.
The discovery
Instruments Facility, Cochin University, Cochin, Kerala, India
and Centralized Instrumentation Facility, Mysore University,
Karnataka, India. The chemical shifts were reported in d (ppm)
with tetramethylsilane (TMS) as the internal standard. The
signals were designated as follows: s, singlet; d, doublet;
t, triplet and m, multiplet. Elemental analyses were carried
out with a Perkin-Elmer 2400 Series II C, H, N analyzer. The
molecular weights of the unknown compounds were characterized
via LC-MS spectroscopy at the Centralized Instrumentation
Facility, Mysore University, Karnataka, India. UV-vis spectra were
recorded on a Varian, Cary 5000. Fourier transform infrared
(FT-IR) spectra of the compounds were with KBr pellets (100 mg)
on a Shimadzu Fourier transform infrared (FT-IR) spectrometer.
Melting points were determined in an electrically heated apparatus
by placing the sample in a glass capillary sealed at one end.
Reactions were monitored by thin layer chromatography on
of cis-platin in the field of antitumour therapy has initiated the
design and synthesis of novel metal complexes including palla-
dium to improve the therapeutic activity of antitumour drugs.
Owing to the strong binding ability of Pt(II) metal ion with sulfur,
after Pt(II)-based drugs are taken in the human body, there is high
potential for binding with sulfur-donor biomolecules, which are
16
found in peptides, proteins and enzymes, leading to strong
17,18
cytotoxicity.
The antitumour activity of Pt(II)-based drugs may
be due to the interaction between the metal complex and genomic
17,19
DNA.
Owing to the structural similarities and significant
overlap of the coordination chemistry of platinum and palladium,
20
these two metals are closely related. Thus, palladium-based
complexes have also been investigated intensively in the field of
21,22
medicinal chemistry.
Focus on this aspect, our research group attempted to synthe-
size, structurally characterization and evaluate the biological
properties of a new pharmacologically active [Pd(BIPM)(OAc)2]
complex derived from the (2-((1H-benzo[d]imidazol-2-yl)-
methylthio)-1H-benzo[d]imidazol-5-yl)(phenyl)methanone [BIPM]
ligand. The study on biological properties of the ligand and metal
0.25 mm silica gel plates, which were visualized under UV light
and KMnO staining.
4
2.2. Synthesis of BIPM ligand
complex was focused on evaluating their cytotoxicity against The synthesis of the BIPM ligand involved three steps. The
cancer of multiple origin, specifically, MCF7, A549, Ehrlich ascites intermediate a and b required for the synthesis of BIPM were
2
3–25
carcinoma (EAC) and Daltons lymphoma ascites (DLA). The Pd(II) prepared by following the literature methods (Scheme 1).
complex showed remarkable activity against the Ehrlich ascites 2.2.1. Synthesis of (2-mercapto-1H-benzo[d]imidazol-6-yl)-
carcinoma (EAC) cell line. The in vivo studies further confirmed (phenyl)methanone (a). Potassium hydroxide (0.52 g, 9.21 mmol)
its tumor regressing activity by inducing antiangiogenesis and was dissolved in 15 mL of anhydrous methanol in a 250 mL flask,
apoptosis. Its mechanism of cell death is due to DNA damage, and the mixture was stirred for approximately 10 min. Carbon
as evidenced by the DNA binding, DNA cleavage and molecular disulfide (0.82 g, 9.21 mmol) was dissolved in anhydrous
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(
calculated) for C14
H
10
N
2
OS: C-68.45 (68.73), H-03.99 (04.19),
À1
N-14.45 (14.57). IR (KBr, cm ): 3138 (n –NH), 1642 (n CQO),
1601 (n Ar–CQN), 1524 (n Ar–CQC), 1278 (n Ar–C–N), 749
(
n S–CH ). LC-MS mass (C H N OS) = 385.12 (M + 1).
2
22 16 4
1
H NMR (DMSO-d , 400 MHz): d = 8.26 (1H, s, H ), 7.86 (1H,
6
1
s, H13), 7.73 (1H, s, H
6
), 7.71–7.69 (2H, d, JH24,H28 = 7.6 Hz),
7
9
4
.66–7.65 (1H, d, JH8 = 3.6 Hz), 7.61–7.59 (2H, d, JH17,H20
.6 Hz), 7.56–7.55 (1H, d, JH9 = 2.4 Hz), 7.53–7.52 (1H, t, JH26
=
=
.4 Hz), 7.19–7.18 (2H, d, JH18,H19 = 4.4 Hz), 7.17–7.16 (2H, t,
1
3
J
H25,H27 = 6 Hz), 4.83 (2H, s, H11). C NMR (DMSO-d
6
, 100.62
MHz): d = 195.75 (C ), 150.26 (C ), 144.39 (C ), 137.99 (C ),
2
1
2
12
23
1
32.10 (C , C ), 130.53 (C ), 129.35 (C , C , C , C ), 128.41
28 24 26 15 14 5 4
(
C
C , C , C ), 123.95 (C ), 121.99 (C , C , C , C ), 120.52 (C ,
18), 28.70 (C11). UV-vis: lmax in (nm): D 27 027 cm (370 nm).
27 25 8 7 20 17 9 6 19
À1
2.3. Synthesis of metal complex
Scheme 1 Reagents and conditions: (1) KOH/MeOH – 75–80 1C, (2) 4 N
HCl/100 1C, (3) Na metal/MeOH, and (4) Pd(OAc) /MeOH – 75–80 1C.
2
2.3.1. Synthesis of [Pd(BIPM)(OAc)
An methanolic solution of [Pd(CH COO)
3 2
] (Pd(II) complex) (d).
] (0.17 g, 0.78 mmol)
was mixed with a hot stirring methanolic solution of BIPM
2
2
6
(
0.31 g, 0.78 mmol). The mixture was stirred under reflex for
methanol (5 mL) and added dropwise to the above stirring approximately 6 h. A pale green-coloured solid precipitate was
solution followed by heating. Then a solution of 3,4-diamino- formed, which was filtered. The solid product was recrystallized
benzophenone (1.95 g, 9.21 mmol) in methanol (20 mL) was from methanol and kept in a vacuum desiccator. The melting
added to the above reaction mixture. The reaction mixture was point of the product was 229–237 1C. Yield: 65%. Elemental
22 4 5
thoroughly stirred and refluxed for 5 h. It was initially yellow analysis (%) found (calculated) for C26H N O PdS: C-51.19
and then turned brown as the reaction progressed, and the (51.28), H-03.53 (03.64), N-09.06 (09.20), Pd-17.43 (17.48). IR
À1
evolution of hydrogen sulfide gas was observed. The reaction (KBr, cm ): 3195 (n –NH), 1665 (n CQO), 1619 (n Ar–CQN),
was monitored by TLC (ethyl acetate : pet ether). After comple- 1524 (n Ar–CQC), 1271 (n Ar–C–N), 742 (n S-CH ), 572 (n S - Pd),
2
tion of the reaction, the mixture was poured into a beaker with 480 (n O - Pd), 465 (n N - Pd). LC-MS mass (C H N O PdS) =
2
6 22 4 5
1
ice-water and acidified with 4 N hydrochloric acid to pH 4–5 to 608.00 (M + 1). H NMR (DMSO-d
6 1
, 400 MHz): d = 8.57 (1H, s, H ),
obtain a white precipitate. The precipitate was then filtered and 8.48 (1H, s, H13), 8.33–7.72 (11H, Ar–H), 7.71 (2H, s, H11), 2.47
2
3
À1
3
recrystallized from methanol. White solid: yield 69%. LC-MS (6H, s, COCH ). UV-vis: lmax (nm): D 16 474 cm (607 nm),
À1
mass (C14
.2.2. Synthesis of 2-(chloromethyl)-1H-benzo[d]imidazole
b). Chloroacetic acid (3.51 g, 37.12 mmol) was dissolved in
H
10
N
2
OS) = 254.02 m/z (M + 1).
D 23 980 cm (417 nm).
2
2.4. Pharmacology
(
40 mL of 4 N hydrochloric acid and stirred for nearly 20 min. The synthesized BIPM and Pd(II) complex were initially tested
o-Phenylenediamine (1.92 g, 17.12 mmol) was added to the for their cytotoxic and antiproliferative activity against the
above solution and the mixture was left stirring with reflex for MCF7, A549, Ehrlich ascites carcinoma (EAC) and Daltons
nearly 4 h. The product was confirmed by TLC (ethyl acetate: pet lymphoma ascites (DLA) cell lines in vitro. The metal complex
ether). Then the above hot solution was poured into ice cold showed considerable IC50 values for the Ehrlich ascites carci-
3
water with stirring and conc. NH was added dropwise. The noma (EAC) cell line; hence, it was further investigated to study
yellow precipitate obtained was filtered. The precipitate was then its antiangiogenic activity on murine ascites using the standard
2
4,25
27,28
recrystallized from methanol.
Yellow solid: yield 74%. LC-MS procedure.
results.
.2.3. Synthesis of (2-((1H-benzo[d]imidazol-2-yl)methylthio)- 2.4.1. Cell culture and in vitro treatment. Cell lines including
In general the Pd(II) complex exhibited excellent
mass (C H ClN ) = 167.21 m/z (M + 1).
8
7
2
2
1H-benzo[d]imidazol-6-yl)(phenyl)methanone [BIPM] (c). The MCF7, A549, Ehrlich ascites carcinoma (EAC) and Daltons
0.08 g of sodium metal (3.73 mmol) was added to a solution lymphoma ascites (DLA) were cultured under standard laboratory
of 2-mercaptobenzimidazole (0.84 g, 3.32 mmol) in 20 mL conditions. The cells were grown in DMEM medium (Gibco-
anhydrous methanol and stirred vigorously for 30 min. Then Invitrogen, USA), supplemented with 10% heat-inactivated Foetal
2-chloromethyl benzimidazole (0.55 g, 3.32 mmol) was added to Bovine Serum (FBS) (Invitrogen, USA), Pen Strep (Sigma Aldrich,
the above reaction mixture in portions and it was left stirring USA) and NaHCO (0.37%) and incubated at 37 1C in an atmo-
3
for nearly 3 h. The yellowish-white compound obtained was sphere of 95% air and 5% CO with 98% humidity. The synthe-
2
filtered, washed with methanol and dried. The product was sized BIPM ligand and Pd(II) complex were initially screened for
confirmed by TLC (ethyl acetate : pet ether). The precipitate was their cytotoxic and antiproliferative activity in vitro using the
then filtered and recrystallized from the methanol. White solid; Trypan blue, MTT assay and LDH release assay, as described
27,29
yield 82%: m.p. 196–199 1C. Elemental analysis (%) found earlier,
at varying concentrations (0, 5, 10, 25 and 50 mM in
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DMSO solvent) in an increasing order with time intervals ranging
The study included 30 mice and each group comprised n = 6
between 0–48 h, keeping DMSO solvent as the only control. The and divided into 5 groups. Group I served as the tumor negative
appropriate vehicle control and 5-fluorouracil were used as the control and received DMSO (i.p). Group II was treated with
2
7
À1
positive controls. Each experiment was repeated a minimum of 5-fluorouracil (5 mg kg ) and served as the positive control.
3
À1
independent times.
Group III was administered with BIPM ligand (20 mg kg ).
2
.4.2. MTT assay. The MTT assay was initially performed Groups IV and V animals were administered with Pd(II) complex
À1
to determine the extent of preliminary proliferation against (25 and 50 mg kg ) by intraperitoneal injection (i.p). The
various cell lines such as MCF7, A549, Ehrlich ascites carcinoma treatment group received 3 doses on alternating days after
(
EAC) and Daltons lymphoma ascites (DLA). The cell lines were the onset of tumor. All the tumor parameters were recorded,
3
2
treated with BIPM, Pd(II) complex and 5-fluorouracil, as described and the survivability of the animals was also monitored.
previously. The cells treated with compound and that without
were incubated for 48 h. Then the MTT reagent (5 mg mL ) was in the control and Pd(II) complex-treated nuclei of the ascitic cells
added and the color change due to the proliferating cells was in vivo was measured by acridine orange and Giemsa staining.
30
2.4.8. Acridine orange and Giemsa stain assay. The differences
À1
32,33
estimated. The concentration of Pd(II) complex resulting in a 50% In brief, the EAC cells either treated with Pd(II) complex (25 and
À1
reduction in cell viability (IC50) was then estimated.
50 mg kg body weight in vivo) or untreated were harvested
2.4.3. Trypan blue dye exclusion assay. The effect of BIPM, and smeared on a glass slide, and fixed with methanol and
Pd(II) complex and 5-fluorouracil on the cell viability of MCF7, acetic acid (3 : 1). Then the cells were hydrated with PBS and
A549, Ehrlich ascites carcinoma (EAC) and Daltons lymphoma stained using acridine orange or Giemsa solution (0.1%). The
ascites (DLA) cells was examined using the trypan blue dye cells were washed with PBS and viewed under a Lawrence and
3
0
exclusion assay, as described previously. The cells treated Mayo Lynx Reg microscope.
with or without Pd(II) complex were harvested and resuspended
2.4.9. Peritoneal angiogenesis assay. The restraint of
in 0.4% trypan blue and the viable cells were counted using a neovessel formation in the peritoneum of the mice bearing
hemocytometer. The inhibitory concentration (IC50) value was EAC treated with or without Pd(II) complex were photographed
estimated after 48 h of treatment.
using a Lawrence and Mayo Lynx Reg microscope, as reported
3
4
2.4.4. LDH leak assay. The lactate dehydrogenase (LDH) earlier.
assay was performed to assess the LDH release following
the treatment of MCF7, A549, Ehrlich ascites carcinoma (EAC)
2.5. DNA binding studies
and Daltons lymphoma ascites (DLA) cells with BIPM, Pd(II) As is known, DNA is the primary pharmacological target
complex and 5-fluorouracil (0, 10, 20, and 50 mM) after 48 h of of antitumor drugs; therefore, it is essential to explore the
3
0
incubation, as described previously. Briefly, the cells were interactions of small molecules with DNA. Particularly, metal
lysed using 0.1% Triton X-100 in PBS. The amount of LDH complexes in a well-tailored ligand framework are responsible
released in both the culture media and cell lysate was measured for the specific shape and surface features that complement the
3
5
at 490 nm using an ELISA reader (Robotronics, India). The molecular target site. Hence, understanding the mechanisms
percentage of LDH release was calculated as LDH release in by which drugs interact with nucleic acids and their correlation
media (LDH release in media + intracellular LDH release) Â 100. with biological effects has been a significant focus in medicinal
2.4.5. FACS analysis. The percentage of proliferating/ chemistry. The binding mode and tendency of the Pd(II)
apoptotic cells and mode of cell death were measured through complex to interact with CT DNA determine its potential as a
FACS analysis in the presence or absence of the Pd(II) complex chemotherapeutic agent. Thus, the interaction of the metal
(0, 5 and 10 mM) against EAC cells. The cells were either stained complex with DNA was studied via various analytical techniques.
with propidium iodide and the results were analyzed by using In the present work the interactions of DNA with the complex was
3
1
the WinMDI version 2.9 software.
.4.6. Animal models and ethics. Swiss albino male mice
studied by electronic absorption titration.
2
Electronic absorption spectroscopy is widely employed
weighing 25–28 g were housed under standard laboratory to determine the binding characteristics of metal complexes
conditions with food and water ad libitum. All procedures for with DNA. All the experiments linking the interaction of the
animal experimentation used were approved by the Institu- complex with CT DNA were carried out in Tris–HCl buffer
tional Animal Ethics Committee, National College of Pharmacy, (5 mM Tris–HCl/10 mM NaCl at pH 7.2) containing 5% DMSO
Shimoga, India, in accordance with the CPCSEA guidelines for at room temperature. A solution of CT DNA in buffer gave a UV
the laboratory animal facility (NCP/IAEC/CL/101/05/2012-13).
absorbance at 275 and 325 nm, signifying the CT DNA was
3
6
2.4.7. Animal tumor models and treatment. The antitumor adequately free from protein. DNA binding experiments were
efficacy of the Pd(II) complex was tested against EAC cells performed at room temperature. The disodium salt of CT DNA
in vivo. A fixed number of Ehrlich Ascites carcinoma cells was stored at 4 1C. The concentration of DNA was measured
6
À1
À1 37
(
1 Â 10 cells per animal) were injected in the peritoneal cavity using its extinction coefficient at 260 nm (6600 M cm ).
of the donor mouse and allowed to multiply. The cells were Stock solutions were stored at 4 1C and used within 4 days.
withdrawn from the donor mouse, diluted in 1X PBS, counted Doubly distilled water was used to prepare solutions. Concen-
6
and re-injected to the experimental animals (1 Â 10 cells per trated stock solutions of the complex were prepared by dissolving
animal) for developing EAC liquid tumor.
the complex in DMSO and diluting suitably with the corresponding
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3
8
buffer to the required concentration for all the experiments.
The Lamarckian genetic algorithm (LGA) was implemented
Absorption titration experiments were performed by varying the with default parameters. Finally based on the AutoDock scoring
concentration of CT DNA (0–50 mM) while keeping the metal function, the lowest energy docked conformation was selected
complex concentration (10 mM) constant. The absorbance as the binding mode and post-docking analysis was rendered
4
5
was recorded after each addition of CT DNA. The absorbance using the UCSF Chimera software.
data was then fitted to eqn (1) to obtain the intrinsic binding
constant (k
b
) values for the interaction of the complex 2.8. Drug-likeness prediction
with DNA.
The similarity of the synthesized compound with known drugs
based on its molecular and structural features was determined
using the Molinspiration Cheminformatics server (http://
www.molinspiration.com/). The important properties of the
[DNA]/(e
a
À e
f
) = [DNA]/(e
b
À e
f
) + 1/[k
b
(e
b
À e
f
)]
(1)
where, e
a
, e
f
and e
b
are the apparent, free and bound metal drug-likeness features such as molecular size, hydrogen bonding,
complex extinction coefficients, respectively. The plot of [DNA]/ and hydrophobicity influence in the ADMET properties of the drug
(
e
b
À e
f
) versus [DNA] gave a slope of 1/(e
b
À e
) and y-intercept molecules. The Molinspiration server calculates the molecular
f
À1
equal to [k
y-intercept.
b
(e
b
À e
f
)] , where, k
b
is the ratio of the slope to the property based on Lipinski’s rule of five or Pfizer’s rule of five or
46
simply rule of five.
2.6. DNA cleavage studies
3
. Results and discussion
Since metal complexes have great ability to bind with the
double helical structure of DNA, they also have the capability 3.1. Chemistry
to denature DNA. This ability of metal complexes extends the
The preparation of intermediate compounds a and b was per-
focus to study carcinoma, where the denaturing of affected
DNA cells may stop the spreading of the abnormal duplication
of healthy cells. In this context, the DNA cleavage property
of the Pd(II) complex was analyzed via the concentration-
dependent DNA cleavage agarose gel electrophoresis method
using super coiled pBR322 plasmid DNA as the target in the
absence of reductant. The ability of Pd(II) complex on DNA
cleavage was reflected by the relaxation of the super coiled (SC)
circular form of pBR322 DNA, resulting in the linear circular
formed according to the standard procedures in ref. 23–25,
respectively. The final compound BIPM is the fused product
of compounds a and b, which was achieved by stirring of
compounds a and b in strong sodium metal base in dry
methanol in a suitable molar ratio. The bis-benzimidazole
ligand [BIPM] was characterized via elemental analysis, IR,
3
9
1
13
H NMR, C NMR and mass spectral data.
The Pd(II) complex was prepared by the treating of hot
methanolic solution of the ligand (BIPM) with the methanolic
solution of the palladium acetate. The complex was stable
under atmospheric conditions and soluble in polar organic
solvents, such as DMF, DMSO, and MeCN, and partially soluble
in common organic solvents. The elemental analysis of the
Pd(II) complex was consistent with the calculated results from
(LC) and/or nicked circular (NC) form. The DNA cleavage study
of the complex was performed on 0.3 mg of pBR322 DNA
incubated at 37 1C for 1 h with an increasing concentration
of metal complex (10–20 mM) in aqueous buffer solution
(50 mM Tris–HCl/50 mM NaCl, pH 7.2), and after electrophoresis
it was photographed under UV light.
the empirical formula, as presented in Table 1. The molar
À3
conductivities of 10
M of the complex in DMF at room
2.7. Molecular docking studies
temperature was measured and the result obtained was
À1
À1
2
A molecular docking study of Pd(II) complex with DNA was 03.29 O mol cm . The complex was characterized based
4
0
performed using the Autodock 4.2 software. The crystal on the following studies.
structure of three different DNA regions was selected for docking, The infrared spectral data of BIPM and its Pd(II) complex are
that is, the DNA dodecamer (PDB ID: 423D), GC-rich (PDB ID: summarized in Table 2 and presented in Fig. 1. The IR spectra
41
42
43
1
QC1) and AT-rich (PDB ID: 226D) regions. All the crystal of the ligand and complex were recorded in the wavelength
À1
structures were retrieved from the Protein Data Bank and region of 4000–400 cm . The uncoordinated BIPM displayed a
the receptor (DNA) and compound preprocessed using the characteristic (nN–H) band at 3138 cm and medium intensity
À1
À1
AutoDock tools. The grid was placed in a cubic box (9 Â 7 Â bands at 1601 and 1278 cm assigned to the (nCQN) and (nC–N),
3
3
3
5
nm ), (5 Â 9 Â 5 nm ) and (9 Â 7 Â 4 nm ) in size for the respectively, of the imidazole moiety. The sharp band at
4
4
À1
DNA dodecamer, GC-rich and AT-rich regions, respectively.
1642 cm is due to nCQO and the aromatic nCQC stretching
Table 1 Analytical and physical properties of BIPM and Pd(II) complex
C, H, N and metal (%) analysis found (calculated)
À2
À1
Entry
Compound
Mol. wt
Yield (%)
M
C
H
N
Ohm cm mol
1
2
BIPM (light yellow)
384.12
608.00
82
65
—
17.43
68.45
51.19
03.99
03.53
14.45
09.96
—
03.29
[Pd(BIPM)(OAc)
2
] (pale green)
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Table 2 FTIR spectral data (cm ) of BIPM and Pd(II) complex
NJC
À1
Entry
Compound
N–H
CQC (Ar)
CQN (Ar)
CQO
C–N
S–C
Others
1
2
Ligand
[Pd(BIPM)(OAc)
3138
3180
1524
1545
1601
1619
1642
1665 & 1720
1278
1285
749
730
—
2
]
M–N = 465
M–O = 480
M–S = 572
Fig. 1 FT-IR spectrum of BIPM and its Pd(II) complex.
À1
appeared at 1524 cm . The band due to the methanethiol- spectrum of BIPM showed a signal at 195.7 ppm for the CQO
À1
bridged nS–CH2 appeared at 749 cm . In the spectrum of the group and the two signals at 150.2 and 144.3 ppm are assigned
Pd(II) complex, the band due to nN–H remained unaltered except to the (N–CQN) of the imidazole ring of the C2 and C12
for a slight shift in its position, indicating the non-participation carbons, respectively. The peaks in the region of 137.9–
À1
of the N–H group. The band observed at 1458 cm is assigned 120.5 ppm are due to the aromatic carbons and the signal
to the ring stretching, ring bending and ring out of plane at 28.7 ppm corresponds to the methanethiol-bridged carbon
4
7–52
deformation.
2
These bands were shifted towards a higher (–S–CH ). The above results confirm the structure of the ligand,
1
frequency upon complexation with the metal ion. The slightly as shown in Fig. 3. The H NMR spectrum of the Pd(II) complex
higher frequency is due to the nCQN of the metal complex at is presented in Fig. 4. The aromatic protons in the range
À1
À1
1
619 cm and the new stretching bands observed at 465 cm
of 8.33–7.72 ppm for the imidazole and benzene rings are
in the Pd(II) complex are due to the coordination through imine shifted to slightly upfield due to the increased conjugation
5
3
nitrogen atom of benzimidazole. In the metal complex, upon coordination. The existence of two singlets due to the
À1
À1
the stretching bands observed at 1665 cm and 1720 cm
correspond to the position of the (nCQO) of the ketotic CO band respectively, indicating the –NH group does not participate in
and acetate CO band, respectively. The shift in the position the coordination. The S–CH group proton resonance appeared
1
–NH of the imidazole H and H13 appear at 8.57 and 8.48 ppm,
2
of the CO band of the ketotic group towards a higher frequency as a singlet at 4.71 ppm, which shifted slight upfield due to the
in the spectrum of the metal complex indicates the extended coordination of the sulphur with Pd(II) metal. The other singlet
conjugation in the complex. The coordination occurs through at 2.47 ppm corresponds to the protons of the acetate group.
the sulphur atom of the methanethiol bridge of BIPM to the
Pd(II) metal ion, which is evidenced by the band at 572 cm
The LC-MS spectrum of BIPM is in good agreement with its
calculated value, with its molecular ion peak obtained at m/z
À1
5
4
due to nS–CH2
.
In the metal complex, the small absorption 385, as presented in Fig. 5. The molecular ion peak of the Pd(II)
À1
band at 480 cm corresponds to the nM–O mode of bonding of complex was obtained at m/z 608 with 93% area of absorption.
acetate. In addition, the mass spectrum of the complex also showed
The H NMR spectra of the BIPM ligand and Pd(II) complex an ionic peak at m/z 610 with a considerably lower area of
were obtained in DMSO-d at room temperature. The spectrum absorption (4.42%), which is the other stable characteristic
1
6
of BIPM showed two singlets, which are attributed to the proton isotopic form of the palladium metal ion. This indicates that
of the imidazole –NH, with H and H at 8.26 and 7.86 ppm, metal : ligand ratio is 1 : 1, as shown in Fig. 6.
1
13
respectively. The multiplets in the range of 7.73–7.16 ppm
correspond to both the imidazole and aromatic benzene ring complex, it is important to know the electronic structure of the
protons. The singlet at 4.83 ppm represents the bridged S–CH ligand and metal complex. Thus, the electronic absorption spectra
of the BIPM ligand, which is shown in Fig. 2. The C NMR of the ligand and metal complex were recorded in DMSO in
To understand the physicochemical properties of a metal
2
1
3
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1
Fig. 2 H NMR spectrum of BIPM.
13
Fig. 3
C NMR spectrum of BIPM.
the wavelength range of 200–900 nm (Table 3 and Fig. 7). The
Owing to the solubility of the metal complex, it probably will
spectrum of BIPM displayed a broad absorption band at D = not be crystalline in nature. The powder XRD analysis data
À1
2
7 027 cm (370 nm), which can be assigned to the inter- indicate the amorphous nature of the Pd(II) complex, which is
molecular charge transfer interaction from the aromatic nucleus summarized in Table 4 and Fig. 8. All the peaks of the Pd(II)
51
to the neighboring nitro acceptor center. The electronic absorp- complex were observed in the diffractogram in the 2y range of
tion spectrum of the Pd(II) complex showed two absorption bands 0–901, indicating the slightly crystalline nature of the Pd(II)
À1
at D
1
= 16129 and D
2
= 23300 cm , which are assigned to the complex. The identification of the metal complex was done
3
3
3
3
56
T (F) - A (F) (n ) and T (F) - T (P) (n ) transitions, respectively. using known methods. The high degree of crystallinity of
1
2
1
1
1
2
The broad intense d–d band observed in the region of 430 nm is the BIMP was exhibited by the corresponding 2y low-angle diffrac-
5
7,58
characteristic band due to the presence of the Pd(II) ion, which is tion peaks at 7.311, 20.041 and 30.511
in the Pd(II) complex.
(F) - The X-ray reflections indexed to the Pd(II) complex with diffrac-
) not observed in the spectrum since the frequency of tion peaks at 2y values of 39.601, 47.011, 68.261 and 81.221
the absorption of this band may fall in the IR region; hence, the matched with the literature JCPDS Standard Card (No. 05-0681).
complex is expected to have a distorted tetrahedral geometry. The analysis of the obtained powder XRD pattern of the
55
3
coordinated to the BIPM. The other expected band for T
1
3
2 3
T (F) (n
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1
Fig. 4 H NMR spectrum of Pd(II) complex.
Fig. 5 LCMS spectra of BIPM.
complex suggests that the complex probably had a mixed form energy (DE*), enthalpy (DH*), entropy (DS*) and Gibbs free
of squarer planar and hexagonal close packed geometry. energy change (DG*) of the decomposition, were evaluated
5
9
In the present investigation, the heating rate was suitably graphically by employing the Coats–Redfern relation. The
À1
controlled at 20 1C min under a nitrogen atmosphere and the data is summarized in Table 6. The activation energy values
weight loss was measured from ambient temperature to 850 1C. reflect the thermal stability of the complex. The activation
The data is presented in Table 5 and Fig. 9. The thermogram energy of decomposition was found to be in the range of
À1
of Pd(II) complex showed three decomposition steps in the 1.437 kJ mol . The DE* was found to be positive for the
temperature range of 30–850 1C. The first step of decomposi- complex, indicating that the decomposition reactions proceed
tion within the temperature range of 30–120 1C correspond to at a normal rate.
the loss of two acetate groups with a mass loss of 19.393% and
activation energy of 1.437 kJ mol . The subsequent step in the
À1
3.2. Biological activity studies
range of 290–380 1C corresponds to the complete dissociation
of the ligand with the mass loss of 63.13%, leaving PdO as the
residue.
3.2.1. Pharmacology
3.2.1.1. Pd(II) complex as a lead compound and its SAR. The
synthesized Pd(II) complex containing the bis-benzimidazole
The thermodynamic activation parameters of the decompo- ligand (BIPM) was initially screened for its cytotoxicity and
sition processes of the dehydrated complex, namely, activation antiproliferative activity on the MCF7, A549, Ehrlich ascites
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Fig. 6 LCMS spectra of Pd(II) complex.
Table 3 Electronic absorption spectral bands of the ligand BIPM and Pd(II) carcinoma (EAC) and Daltons lymphoma ascites (DLA) cell lines
complex
in vitro together with the BIPM ligand and 5-fluorouracil
À1
(Table 7). The BIPM ligand alone exhibited cytotoxicity primar-
ily against Ehrlich Ascites Carcinoma cells with an IC50 value of
B17 mM. Whereas, after forming a complex with the palladium
metal salt, i.e., the Pd(II) complex, its potency increased,
exhibiting promising antiproliferative efficacy against Ehrlich
Ascites Carcinoma cells with an IC50 value of B10 mM in three
different cytotoxic assay systems, i.e., Trypan blue, MTT and
LDH release assays. The vehicle control alone did not alter the
cell viability. Obviously, the benzimidazole itself is a more
potent biologically active moiety; however, the conjugation of
palladium metal with BIPM ligand enhanced its potentiality.
The presence of Pd(II) at the center of the complex and the
geometrical arrangement of the complex are the main criteria
for its potential as a lead compound.
Compound
BIPM
Electronic transition n (cm
)
meff (BM)
27 027
16 417 and 23 980
—
03.29
[
Pd(BIPM)(OAc)
2
]
3.2.1.1.1. Pd(II) complex induces cell death in EAC cells.
Further to study the mechanism of the cytotoxicity of the
selected lead compound Pd(II) complex, the cells were subjected
to FACS analysis. For this analysis, EAC cells were treated with
an increasing concentrations of Pd(II) complex (0, 5 and 10 mM,
Fig. 7 Electronic absorption spectra of BIPM and Pd(II) complex.
Table 4 Powder XRD spectral data for the Pd(II) complex
d
Peak no.
2y
y in degrees
y in radians
sin y
h k l
Cal.
Obs.
a in Å
1
2
3
4
.
.
.
.
39.87
46.94
68.08
81.70
19.93
23.47
34.04
40.85
0.347
0.409
0.594
0.713
0.340
0.398
0.557
0.654
1 1 1
2 0 0
2 2 0
3 1 1
2.259
1.934
1.376
1.177
2.265
1.952
1.381
1.184
3.91
3.86
3.89
3.90
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6
0
outcome parameters. Ehrlich ascites tumor implantation
induces a set of local inflammatory reactions, with increasing
vascular permeability, which result in intense edema formation,
61
cellular migration and progressive ascite fluid formation. Ascite
fluid is the direct nutritional source for tumor cells and a rapid
increase in ascite fluid with tumor growth meets the nutritional
62
requirement of growing tumor cells. Hence, a decrease in ascite
fluids accounts for the suppression of tumor growth.
In the present study, the lead molecule Pd(II) complex was
À1
administered at the concentration of 25 and 50 mg kg body-
À1
weight together with BIPM (20 mg kg ) and 5-fluorouracil
À1
th
th
Fig. 8 Powder XRD spectrum of Pd(II) complex.
(5 mg kg ) i.p., as determined by the LD50 studies on the 4 ,
6
th
and 8 day after tumor implantation. Upon treatment with
BIPM and Pd(II) complex on the murine ascite tumor, the ligand
exhibited anti-tumor activity against the EAC tumor model;
whereas, the tumor inhibitory potential increased with the
Pd(II) complex treatment, where was a dose-dependent decrease
in body weight (Fig. 11a and b). The ascite secretion and tumor
cell growth responsible for the establishment of the tumor was
also reduced compared to the control (Fig. 11c). The treatment
further expanded the mice survivability, which was two-fold
higher from 10 days to 30 days (Fig. 11d). The treatment did not
induce any toxicity to the animals (data not shown) and the
physical morphology of the Pd(II) complex-treated tumor mice
4
8 h) and the cell death was analysed using flow cytometry,
following staining with propidium iodide (PI). The results
showed a significant increase in the Pd(II) complex-induced
cell death in the EAC cells with an increase in concentration
(B21.9% cell death at 5 mM and B26.8% cell death at 10 mM)
(Fig. 10). This observation suggests that the Pd(II) complex
could effectively induce cell death in
dependent manner.
a concentration-
3.2.1.2. In vivo carcinoma studies on EAC
3
.2.1.2.1. The Pd(II) complex regresses ascite tumor growth. (Fig. 11e) suggests that the Pd(II) complex can be a potent target
Murine ascites are a suitable model system for preliminary specific antitumor agent. Additionally, all the results were
screening, which play a critical role in drug development. This compared with the positive control, 5-fluorouracil.
is a rational and a hierarchical approach beginning with
toxicology and pharmacology studies, progressing to primary
3.2.1.2.2. Pd(II) complex induces apoptosis and antiangio-
tumors to identify therapeutic targets and models of metastatic genesis. Resistance to apoptosis and angiogenesis are the two
disease to compare drugs using rigorous, clinically relevant important characteristics that promote the establishment of
Table 5 Thermoanalytical results (TGA and DTA) for the Pd(II) complex
Mass
Total mass
Metallic
residue
Complex
Pd(BIPM)(OAc)
TG range (1C) DTA (1C)
30–120
95(+), 200(+), 220(À), 335(À),
n* loss (%) loss (%)
Assignment
[
2
]
1
2
19.39
61.31
Loss of two acetate groups
Loss of organic part of the ligand
80.70
PdO
2
90–380 350(À), 445(À), 470(À), 580(À)
n* = number of decomposition steps.
Fig. 9 Thermoanalytical curves (TGA and DTA) of Pd(II) complex.
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Table 6 Thermodynamic data for the thermal decomposition of the Pd(II) complex
À1
À1
À1
À1
À1
Complex
Pd(BIPM)(OAc)
Decomp. temp. (1C)
E* (kJ mol
)
ln A (min
)
DH* (kJ mol
)
DS* (kJ mol
)
DG* (kJ mol
)
[
2
]
247
1.437
2.584
2.780
2.913
À0.617
À1.643
À2.266
À39.681
À40.682
À40.933
09.19
13.43
15.97
3
4
70
45
Table 7 IC50 values of Pd(II) complex calculated based on the Trypan blue, MTT and LDH release assays at 48 h in MCF-7, A549, EAC and DLA cells
Cancer cell line
MCF7
DMSO
5-Fluorouracil
BIPM
Pd(II) complex
Trypan blue-IC50 value (mM)
MTT-IC50 value (mM)
LDH release assay IC50 values (mM)
Z100
Z100
Z100
3.1 Æ 0.3
3.18 Æ 0.5
3.23 Æ 1.2
54.3 Æ 0.2
56.3 Æ 0.2
69.6 Æ 0.2
48.8 Æ 0.7
48.3 Æ 0.9
60.3 Æ 0.4
A549 cells
EAC cells
DLA cells
Trypan blue-IC50 value (mM)
MTT-IC50 value (mM)
LDH release assay IC50 values (mM)
Z100
Z100
Z100
4.21 Æ 0.2
4.9 Æ 0.2
4.34 Æ 0.6
49.3 Æ 1.1
52.7 Æ 0.3
55.3 Æ 0.5
45.1 Æ 1.2
45.7 Æ 1.8
49.8 Æ 0.9
Trypan blue-IC50 value (mM)
MTT-IC50 value (mM)
LDH release assay IC50 values (mM)
Z100
Z100
Z100
3.9 Æ 0.3
3.45 Æ 0.4
3.12 Æ 0.7
17.3 Æ 0.4
17.9 Æ 0.2
18.1 Æ 0.8
9.7 Æ 0.6
10.1 Æ 0.6
10.2 Æ 0.2
Trypan blue-IC50 value (mM)
MTT-IC50 value (mM)
LDH release assay IC50 values (mM)
Z100
Z100
Z100
3.21 Æ 0.7
3.72 Æ 0.6
3.97 Æ 0.1
42.3 Æ 0.2
43.5 Æ 0.3
54.2 Æ 0.1
37.1 Æ 0.5
37.2 Æ 0.9
47.7 Æ 2.0
Values indicate the mean Æ SEM and statistically significant values are expressed as *p o 0.05 and **p o 0.01.
tumors and there is a direct correlation between these two metastatic potential of tumors. Measurement of neovasculari-
6
3,64
characteristics.
The inhibition of angiogenesis may lead to zation or microvessel density (MVD) is a widely used surrogate
the promotion of apoptosis resulting in cell death, and thereby marker in pathological specimens and tumor models to assess
3
4,66
tumor inhibition. Several anticancer drugs with these dual the prognosis of the disease.
The current study revealed
effects are used in current cancer therapy and many are in that the Pd(II) complex has potent antitumor efficacy and
6
4
clinical trials. When we verified the possible proapoptotic activation of antiangiogenesis may be one of the possible
effect of the selected lead compound Pd(II) complex on EAC underlying mechanisms of tumor inhibition. This is because
cells using the Giemsa staining assay and Acridine orange stain angiogenesis was evident in the inner lining of the peritoneum
assay, it was found that the morphological changes in the EAC of the EAC tumor-bearing mice and it is a reliable model to
66
cells treated with the Pd(II) complex indicated typical apoptotic study the angiogenesis-dependent tumor growth. Subsequently,
structures, including cell shrinkage, condensed nuclei and we verified the effect of the Pd(II) complex for the angioprevention
formation of apoptotic bodies (Fig. 12a and b).
effect. Surprisingly, the Pd(II) complex showed decreased angio-
Tumor growth and metastasis are dependent on angiogenesis, genesis compared to the immense angiogenesis in the peritoneum
6
5
as demonstrated in many in vivo experiments. Increased lining of the untreated tumor-bearing mice (Fig. 12c).
neovasculature may not only increase tumor growth, but also 3.2.2. DNA binding and DNA cleavage studies. One of
enhance hematogenous tumor embolization. Thus, inhibiting the important cellular hallmarks of apoptosis is DNA damage
tumor angiogenesis may arrest tumor growth and decrease the resulting in nuclear fragmentation. Our results revealed that
Fig. 10 Pd(II) complex induces cell death. EAC cells were treated with the Pd(II) complex at different concentrations (0, 5 and 10 mM, 48 h). Promotion of
apoptosis demonstrated through FACS analysis.
8
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Fig. 11 Effect of Pd(II) complex on Ehrlich Ascites Carcinoma (EAC) in mice. Ascite tumor was induced in 6–7 week-old Swiss albino mice by injecting
À1
À1
EAC cells intraperitoneally. Two doses of Pd(II) complex (25 mg kg and 50 mg kg ) were each administered to the tumor-bearing mice on alternate
days after three days of tumor growth. (a) Reduction in percentage of tumor growth. (b) Decreased number of cells. (c) Decrease in ascite secretion.
À1
À1
À1
(
d) Kaplan–Meier survival curve. (e) Physical morphology of the control, 5-fluorouracil – 5 mg kg , BIPM – 20 mg kg , Pd(II) complex – 25 mg kg and
À1
Pd(II) complex – 50 mg kg treated tumor mice.
the cytotoxicity effect induced by the Pd(II) complex is mediated hypochromism and red-shifting due to the strong stacking p–p
by apoptosis. Thus, verify the possible DNA damage induced by interaction between the aromatic chromophore ligand of the metal
67–69
the Pd(II) complex, we further conducted DNA binding and complex and the base pairs of DNA.
cleavage studies. In general, the absorption spectrum of a metal When the BIPM of the Pd(II) complex intercalated with the
complex bound to DNA through intercalation exhibits significant base-pairs of CT DNA, the p* orbital of the complex coupled
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Fig. 12 Tumour inhibition effect of Pd(II) complex on EAC-bearing mice. EAC cells were cultured in vivo and administered with or without Pd(II) complex
À1 À1
(
25 mg kg and 50 mg kg body weight, i.p.). (a) Acridine orange stain photograph. (b) Giemsa stain photograph. (c) Pd(II) complex-induced reduction in
peritoneal angiogenesis.
with the p orbital of the CT DNA base-pairs, which led to a The concentration of CT DNA directly affected the absorption
decrease in the p–p* transition energy of BIPM in the Pd(II) bands of the metal complex, as a result the hypochromic
complex. This caused a regular decrease in lmax, and the intra- phenomenon was detected upon the successive addition of
ligand transition of BIPM shifted to a longer wavelength (red CT DNA with an increase in concentration. The slight shift in
shift). The coupled p* orbital of the complex also participated the absorption towards a higher wavelength indicated that the
as a partially occupied electron orbital due to the overlapping Pd(II) complex containing four N could interact with CT DNA.
with the p orbital of the DNA base pairs (effect of charge The results showed that upon the successive addition of CT
transfer) to decrease the transition probabilities of the complex. DNA to the complex, greater binding activity was observed. The
As a result, the transition intensity of the complex was weakened, [DNA]/(e
resulting in a hypochromic effect, which is mainly ascribed to constant, k
a
À e
f
) ratio was plotted against [DNA] and the intrinsic
, was obtained from the ratio of the slope to the
7
0
b
the interaction between the electronic states of the complex
7
1
chromophores and that of the DNA bases. The observed
hypochromic change without changing the wavelength in the
UV spectrum of the complex indicated that the complex binds
7
2
with DNA through the groove binding mode. The extent of
hypochromism is normally found to correlate with the groove
binding strength. The binding constant of the complex with
CT DNA (k
absorbance using eqn (1).
b
) was determined by monitoring the changes in
7
3
The binding of Pd(II) complex to the CT DNA helix is shown
in Fig. 13. The absorption phenomenon of the Pd(II) complex
(at a constant concentration) was studied in both the presence
and absence of CT DNA. As a result, the absorption spectrum
of the complex exhibited two intense absorption bands at
Fig. 13 Electronic absorption spectra of Pd(II) complex in buffer solution
5 mM Tris–HCl/10 mM NaCl at pH 7.2) in the presence of increasing
amounts of CT-DNA. The arrow shows the intensity changes upon
and ligand-to-metal charge transfer transitions, respectively. increasing the DNA concentration. Plot of [DNA]/(e – e ) vs. [DNA].
279 nm and 320 nm in the UV region, corresponding with the
(
intra-ligand (p–p*) transition of the aromatic chromophore
a
f
8
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the SC form (lane 2). This phenomenon indicates that the Pd(II)
75
complex is capable of performing direct double-strand scission.
The DNA fragmentation is clear evidence and provides addi-
tional proof for the induction of apoptosis due to the cytotoxic
effect of the Pd(II) complex. As a result, this complex is
well-suited for therapeutic applications, particularly in cancer
chemotherapeutics.
3.2.3. Molecular docking study of synthesized Pd(II) complex
with DNA. To further supports the in vitro results, computer-
aided molecular docking studies of the Pd(II) complex with the
minor grooves of DNA mixed nucleotide sequences (sequences:
Fig. 14 Gel electrophoresis diagram showing the cleavage of pBR322
plasmid DNA in aqueous buffer 50 mM Tris–HCl/50 mM NaCl, pH 7.2 and
in the presence of metal complex: lane 1, DNA + control; lane 2, DNA + d(ACCGACGTCGGT) dodecamer (PDB ID: 423D), GC-rich (PDB
2
1
0 mM of Pd(II) complex; lane 3, DNA + 15 mM of Pd(II) complex; and lane 4,
ID: 1QC1) and AT-rich (PDB ID: 226D)) were carried out to
elucidate its mode of interaction and binding affinity. Targeting
the minor grooves of DNA with small molecules is an important
tool in molecular recognition of a specific DNA-sequence. The
molecular docking analysis shows that the Pd(II) complex binds
to the minor grooves for the mixed nucleotide sequence,
GC-rich DNA and AT-rich DNA (Fig. 15). The relative binding
energies of the docked structure of the Pd(II) complex with
the DNA sequence d(ACCGACGTCGGT)2 dodecamer, GC-rich
DNA and AT-rich DNA were found to be À9.38, À5.80 and
DNA + 20 mM of Pd(II) complex.
À1
À7.93 kcal mol , respectively. These interactions were stabi-
lized by hydrogen bonds, where three hydrogen bonds were
formed between the ligand and mixed nucleotide sequence, two
hydrogen bonds formed between the ligand and AT-rich DNA
Fig. 15 Molecular docking of the Pd(II) complex with DNA. (a) DNA–Pd(II) and three hydrogen bonds between the ligand and GT-rich
complex binding pose in the minor groove of the mixed sequence (PDB DNA. Together these results show the high binding affinity of
ID: 423D). (b) In the minor groove of the AT-rich DNA (PDB ID: 226D).
the Pd(II) complex with DNA.
(c) GC-rich region (PDB ID: 1QC1).
3.2.4. Drug-likeness properties. The analysis showed
that the molecular weight of the compound is slightly higher
than the acceptable range (MWT r 500) and the remaining
intercept. The k
2
b
value obtained for the Pd(II) complex was features are within the limit of Lipinski’s rule of five (Table 8),
5
.78 Â 10 M. The significant difference in the DNA binding even though some commercially available compounds with
76
affinity of the Pd(II) complex mainly depends on the metal a molecular weight 4500 are used as potential drugs. The
chelates, and therefore an increase in metal chelates resulted in number of hydrogen bond donors (OH and NH atoms) and
stronger binding affinity with DNA. Our results are consistent acceptors (O and N atoms) in the synthesized compound were
with earlier reports on the preferential binding to CT DNA with found to be 9 and 2, respectively and its milogP value (octanol/
7
4
metal complexes.
water partition coefficient) was within the acceptable range
7
7
The cleavage study of the Pd(II) complex was performed via (o5 Lipinski’s rule). Another important physicochemical
agarose gel electrophoresis using supercoiled pBR322 plasmid property, TPSA (topological polar surface area), was measured,
DNA as a substrate without the addition of a reductant, as which is the sum of all polar atoms such as oxygen, nitrogen
shown in Fig. 14. The cleavage studies were carried out by and attached hydrogen. TPSA is a good descriptor, illustrating
varying the concentration of the Pd(II) complex. With a succes- intestinal absorption, hydrogen bonding potential, bioavail-
sive increase in the metal complex concentration, the super- ability, blood–brain barrier penetration (BBB) and Caco-2 cell
coiled form (SC) of DNA gradually converted in to the nicked permeability. The TPSA value r140 Å and the number of
7
8–80
circular (NC) form and then to the linear form (LC). The most rotatable bonds (r10) indicates good bioavailability.
impressive cleavage feature observed for the Pd(II) complex was Together these results indicate the characteristics of the synthe-
that the LC form of DNA appeared before the disappearance of sized compound as a drug.
Table 8 Molecular properties of Pd(II) complex
Molecular properties
Compound
No.
(ChemSpider ID)
WM
milog P
TPSA
No. of ON
9
No. of OH and NH
2
No. of rotatable bonds
7
Volume
453.29
1
.
Pd(II) complex
608.97
À1.95
118.04
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Paper
4
. Conclusion
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3
4
5
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1H-benzo[d]imidazol-6-yl)(phenyl)methanone [BIPM] and its
[Pd(BIPM)(OAc) ] (Pd(II) complex). The ligand and its complex
2
were characterized using various analytical techniques, and the
obtained results are in good agreement with the proposed
structure of the BIPM ligand and Pd(II) complex.
Furthermore, the pharmacology of the metal complex was
studied via in vitro growth inhibition assay, in vivo growth
inhibition assay, DNA binding, DNA cleavage, molecular docking
on DNA and drug-likeness property studies. As an in vitro
approach, we screened the BIPM ligand and Pd(II) complex for
cytotoxic and antiproliferative effects against the MCF7, A549,
Ehrlich ascites carcinoma (EAC) and Daltons lymphoma ascites
6
K. Jeyalakshmi, Y. Arun, N. S. P. Bhuvanesh, P. T. Perumal,
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7
80–798, DOI: 10.1039/C4QI00234B.
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7
8
2
R. Esteghamat-Panah, H. Farrokhpour, H. Hadadzadeh,
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(DLA) cell lines. The Pd(II) complex emerged as a lead compound
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with potent cytotoxic and antitumor efficacy by inhibiting
neovascularisation and promoting apoptosis against EAC cell
lines, which were further confirmed by the FACS analysis. This
study highlighted that the Pd(II) complex is a promising anti-
cancer molecule with multiple modes of actions, such as
antiproliferative and angiopreventive effects together with its
remarkable apoptosis inducing action. One of the important
cellular hallmarks of apoptosis is DNA damage, resulting in nuclear
fragmentation. Our results revealed that the cytotoxicity effect
induced by the Pd(II) complex is mediated by apoptosis. DNA
binding, DNA cleavage and molecular docking studies were per-
formed to verify the possible DNA damage induced by the Pd(II)
complex. The results indicated that the metal complex exhibits a
remarkable ability to bind and denature the double helical struc-
ture of DNA. The studies on its drug-likeness properties provided
positive review on the therapeutic nature of the metal complex.
Considering the obtained results, further studies should be con-
ducted on the therapeutic applications of the Pd(II) complex,
particularly cancer chemotherapeutics.
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Conflicts of interest
16 Q. W. Wang, P. L. Lam, R. S. M. Wong, G. Y. M. Cheng,
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06 | New J. Chem., 2019, 43, 790--806
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