Mixed ligand palladium(ii) complexes of 6-methoxy-2-oxo-1,2- dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazones with triphenylphosphine co-ligand: Synthesis, crystal structure and biological properties

Article abstract of DOI:10.1039/c2dt31079a

A series of new 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazone ligands (H₂L1-H₂L5) and their corresponding palladium(ii) complexes [Pd(L1)(PPh₃)] (1), [Pd(L2)(PPh₃)] (2), [Pd(HL3)(PPh₃)]Cl (3), [Pd(L4)(PPh₃)] (4) and [Pd(L5)(PPh₃)] (5), have been synthesized in order to evaluate the effect of terminal N-substitution in thiosemicarbazone moiety on coordination behaviour and biological activity. The new ligands and their corresponding complexes were characterized by analytical and various spectral techniques. The molecular structure of the complexes 2-5 were characterized by single crystal X-ray diffraction studies which revealed that the ligands H₂L2, H₂L4 and H₂L5 are coordinated to palladium(ii) as binegative tridentate (ONS^2-) by forming six and five member rings whereas, the ligand H₂L3 coordinated to Pd(ii) as uninegative tridentate (ONS^-). The interactions of the new complexes with calf thymus DNA (CT-DNA) have been evaluated by absorption and ethidium bromide (EB) competitive studies which revealed that complexes 1-5 could interact with CT-DNA through intercalation. Further, the interactions of the complexes with bovine serum albumin (BSA) were also investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic methods, which showed that the new complexes could bind strongly with BSA. Antioxidant studies showed that all the complexes have a strong antioxidant activity against 2-2′-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azino-3-ethylbenzthiazoline-6-sulfonic acid diammonium salt (ABTS) cation radical. In addition, in vitro cytotoxicity of the complexes against human lung cancer (A549) cell line was assayed which showed that 4 has higher cytotoxic activity than the rest of the complexes and cisplatin.

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PAPER
Mixed ligand palladium(II) complexes of 6-methoxy-2-oxo-1,2-
dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarbazones with
triphenylphosphine co-ligand: Synthesis, crystal structure and biological
properties†
Eswaran Ramachandran,a Duraisamy Senthil Raja,a Nattamai S. P. Bhuvaneshb and
Karuppannan Natarajan*a
Received 18th May 2012, Accepted 26th June 2012
DOI: 10.1039/c2dt31079a
A series of new 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted
thiosemicarbazone ligands (H2L1–H2L5) and their corresponding palladium(II) complexes [Pd(L1)-
(PPh3)] (1), [Pd(L2)(PPh3)] (2), [Pd(HL3)(PPh3)]Cl (3), [Pd(L4)(PPh3)] (4) and [Pd(L5)(PPh3)] (5), have
been synthesized in order to evaluate the effect of terminal N-substitution in thiosemicarbazone moiety on
coordination behaviour and biological activity. The new ligands and their corresponding complexes were
characterized by analytical and various spectral techniques. The molecular structure of the complexes 2–5
were characterized by single crystal X-ray diffraction studies which revealed that the ligands H2L2, H2L4
and H2L5 are coordinated to palladium(II) as binegative tridentate (ONS2−) by forming six and five
member rings whereas, the ligand H2L3 coordinated to Pd(II) as uninegative tridentate (ONS−). The
interactions of the new complexes with calf thymus DNA (CT-DNA) have been evaluated by absorption
and ethidium bromide (EB) competitive studies which revealed that complexes 1–5 could interact with
CT-DNA through intercalation. Further, the interactions of the complexes with bovine serum albumin
(BSA) were also investigated using UV-visible, fluorescence and synchronous fluorescence spectroscopic
methods, which showed that the new complexes could bind strongly with BSA. Antioxidant studies
showed that all the complexes have a strong antioxidant activity against 2-2′-diphenyl-1-picrylhydrazyl
(DPPH) radical and 2,2′-azino-3-ethylbenzthiazoline-6-sulfonic acid diammonium salt (ABTS) cation
radical. In addition, in vitro cytotoxicity of the complexes against human lung cancer (A549) cell line was
assayed which showed that 4 has higher cytotoxic activity than the rest of the complexes and cisplatin.
complexes are good alternative candidates for metal–organic
Introduction
antitumor drugs due to their structural and thermodynamic simi-
larities to platinum(II) complexes,6,7 a number of palladium(II)
The platinum containing antitumor drugs like cisplatin, carbo-
platin, and oxaliplatin have widely been used in clinics,1 but
thiosemicarbazone complexes have been synthesised and exami-
ned for their potential as antitumor agents.8–15 In this area,
there are limitations due to the drug resistance over a period of
time, severe side effects in causing nausea and to the failure of
kidney and liver, which are typical of heavy metal toxicity.2–5
Hence, attempts have been made extensively to replace these
drugs with more-efficient, less toxic, and target-specific non-
covalent DNA binding anticancer drugs. Since palladium(II)
palladium(II) complexes of heterocyclic thiosemicarbazones
have attracted considerable attention and many of them showed
remarkable cytotoxic activity.15,16
In the recent past, our group has been actively engaged in
the study on the variable coordination behaviours of
thiosemicarbazones,17–24 and semicarbazones25 with diverse
transition metals and on the biological properties of the resulting
complexes. The results obtained revealed that thiosemicarba-
zones derived from 2-oxo-1,2-dihydroquinoline-3-carbaldehyde
and substituted 2-oxo-1,2-dihydroquinoline-3-carbaldehyde and
their transition metal complexes exhibiting significant cytotoxic
activities. However, the structural and biological properties of
mononuclear palladium complexes of 6-methoxy-2-oxo-1,2-
dihydroquinoline-3-carbaldehyde 4N-substituted thiosemicarba-
zone have not been explored. Therefore, it seemed important for
us to compare the coordination behaviour and the potential
aDepartment of Chemistry, Bharathiar University, Coimbatore, 641 046,
India. E-mail: k_natraj6@yahoo.com; Fax: +91 422 2422387;
Tel: +91 422 2428319
bDepartment of Chemistry, Texas A&M University, College Station,
TX 77843, USA
†Electronic supplementary information (ESI) available: Synchronous
spectra of BSA (1 μM) in the presence of increasing amounts of the
complexes 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E) (0–25 μM) in the wave-
length difference of Δλ = 15 nm and Δλ = 60 nm (Fig. S1 and S2).
CCDC reference numbers 831519, 834540, 880537 and 841993 for the
complexes 2, 3, 4 and 5. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c2dt31079a
13308 | Dalton Trans., 2012, 41, 13308–13323
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Scheme 1 The synthetic routes of the ligands and its corresponding palladium(II) complexes.
biological activities of mononuclear palladium(II) complexes of
6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-sub-
stituted thiosemicarbazone.
using a Nicolet Avatar model FT-IR spectrophotometer from
KBr discs. The electronic spectra of the complexes were
recorded with a Jasco V-630 spectrophotometer using DMSO as
the solvent. Emission spectra were measured using a Jasco FP
Herein, we present the synthesis, structure, DNA interaction,
protein binding, antioxidative and cytotoxicity studies of mono-
nuclear palladium complexes of 6-methoxy-2-oxo-1,2-dihydro-
quinoline-3-carbaldehyde 4N-substituted thiosemicarbazones.
The crystal structures of the new complexes have been deter-
mined by X-ray crystallography. The investigation of the biologi-
cal properties of the new complexes have been carried out
focusing on the binding properties with calf thymus DNA
(CT-DNA) and competitive binding studies with ethidium
bromide (EB). The synthetic routes of the ligands and their
corresponding palladium complexes are shown in Scheme 1.
1
6600 spectrofluorometer. H NMR spectra were recorded on a
Bruker AMX 500 at 500 MHz NMR spectrometer using TMS as
an internal reference. The melting points were recorded with a
Lab India Melting point apparatus.
Synthesis of the ligands
6-Methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde thio-
semicarbazone (H2L1). Thiosemicarbazide (0.911 g, 0.01 mol)
dissolved in warm methanol (50 mL) was added to a methanol
solution (50 mL) containing 6-methoxy-2-oxo-1,2-dihydro-
quinoline-3-carbaldehyde (2.032 g, 0.01 mol). The mixture was
refluxed for an hour during which period a yellow colour precipi-
tate was formed. The reaction mixture was then cooled to room
temperature and the solid compound was filtered. It was then
washed with methanol and dried under vacuum. Yield: 86%.
MP: 258–260 °C, Elemental Analysis calculated for:
C12H12N4O2S (%): C, 52.16; H, 4.38; N, 20.28; S, 11.61. Found
(%): C, 52.09; H, 4.24; N, 20.31; S,11.56 IR (KBr, cm−1): 3392
(ms) ν(NH); 3160(ms) ν(NH2); 1651(s) ν(CvO); 1611, 1530(s)
ν(CvN) + ν(CvC); 840(m) ν(CvS). UV-vis (DMSO), λmax
Experimental section
Materials and methods
All the reagents used were of analytical or chemically pure
grade. Solvents were purified and dried according to standard
procedures.26 Doubly distilled water was used to prepare buffers.
Ethidium bromide (EB), bovine serum albumin (BSA), calf
thymus DNA (CT-DNA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased from
Sigma-Aldrich and used as received. 6-Methoxy-2-oxo-1,2-
dihydroquinoline-3-carbaldehyde27 and the starting complex,
[PdCl2(PPh3)2]28 were prepared by literature methods. Elemental
analyses (C, H, N, S) were performed on a Vario EL III Elemen-
tar analyzer instrument. Infrared spectra of the ligand and the
metal complexes were recorded in the range of 4000–400 cm−1
1
(nm): 263, 656, 409 (π → π*, n → π*). H NMR (DMSO-d6);
δ 11.88 (s, N(3)H); 11.62 (s, N(2)H); 8.71 (s, 1H, C(1)H);
8.26–8.29 (d, 2H, N(4)H2); 8.03 (s, 1H, C(6)H); 7.62 (s, 1H,
C(10)H); 7.19 (d, 1H, C(7)H); 7.11 (d, 1H, C(8)H); 3.78 (s, 3H,
C(11)H).
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6-Methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-4N-
methylthiosemicarbazone (H2L2). It was prepared using the
same procedure as described for H2L1 with 4-methyl-3-thio-
semicarbazide (1.052 g, 0.01 mol) and 6-methoxy-2-oxo-1,2-
dihydroquinoline-3-carbaldehyde (2.032 g, 0.01 mol). A yellow
coloured product was obtained. Yield: 83%. MP: 271–273 °C,
Elemental Analysis calculated for: C13H14N4O2S (%): C, 53.79;
H, 4.86; N, 13.30; S, 11.04. Found (%): C, 53.71; H, 4.79;
N, 13.43; S, 11.17. IR (KBr, cm−1): 3372(ms) ν(NH); 1658(s)
ν(CvO); 1551(s) ν(CvN) + ν(CvC); 856 (m) ν(CvS). UV-
vis (DMSO), λmax (nm): 264, 352, 409 (π → π*, n → π*).
1H NMR (DMSO-d6); δ 11.90 (s, N(3)H); 11.68 (s, N(2)H);
8.63 (s, 1H, C(1)H); 8.55 (s, 1H, N(4)H); 8.27 (s, 1H, C(6)H);
7.27 (s, 1H, C(10)H); 7.19 (d, 1H, C(7)H); 7.12 (d, 1H, C(8)H);
3.79 (s, 3H, C(11)H); 3.05 (s, 3H, C(13)H).
8.46 (s, 1H, C(1)H); 8.30(s, 1H, C(6)H); 7.37 (d, 1H, C(7)H);
7.26 (s, C(10)H). 7.16–7.17 (d, 1H, C(8)H); 3.78 (s, 3H, C(11)
H); 2.49 (s, 6H, C(13, 14)H).
Synthesis of complexes
[Pd(L1)(PPh3)] (1). To a solution of [PdCl2(PPh3)2] (0.100 g;
0.143 mmol) in toluene (20 cm3), the ligand, H2L1 (0.040 g;
0.143 mmol) and two drops of triethylamine were added. The
mixture was heated under reflux for 5 h. The resulting solution
on slow evaporation yielded an orange coloured crystalline
powder. It was washed with toluene and dried under vacuum.
The crystals obtained were found to be not suitable for X-ray dif-
fraction. Yield: 79%, MP: 275–277 °C. Anal. calcd for
C30H25N4O2PPdS (%): C, 56.04; H, 3.92; N, 8.71; S, 4.99;
Found (%): C, 56.19; H, 3.86; N, 8.64; S, 4.81. IR (KBr disks,
cm−1): 3244(m) ν(NH2); 1606, 1547 (s) ν(CvN) + ν(CvC);
1354 (s) ν(C–O); 746 (m) ν(C–S); 1445, 1096, 695 (for PPh3),
UV-vis (DMSO), λmax (nm): 268 (intra-ligand transition); 385
(LMCT s/d); 433 (MLCT). 1H NMR (DMSO-d6); δ 8.52 (s, 1H,
C(1)H); 8.49 (s, 1H, C(6)H); 7.15–7.71 (m, 18H, aromatic); 6.98
(s, 2H, N(4)H2); 3.81 (s, 3H, C(11)H).
6-Methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-4N-
ethylthiosemicarbazone (H2L3). It was prepared using the same
procedure as described for H2L1 with 4-ethyl-3-thiosemi-
carbazide (1.192 g, 0.01 mol) and 6-methoxy-2-oxo-1,2-dihy-
droquinoline-3-carbaldehyde (2.032 g, 0.01 mol). An yellow
colour product was obtained. Yield: 89%. MP: 275–277 °C,
Elemental Analysis calculated for C14H16N4O2S (%): C, 55.25;
H, 5.30; N, 18.41; S, 10.54. Found (%): C, 55.37; H, 5.21;
N, 18.38; S, 10.61. IR (KBr, cm−1): 3158 (ms) ν(NH); 1649 (s)
ν(CvO); 1621, 1536 (s) ν(CvN) + ν(CvC); 825 (m) ν(CvS).
UV-vis (DMSO), λmax (nm): 263, 354, 410 (π → π*, n → π*).
1H NMR (DMSO-d6); δ 11.90 (s, N(3)H); 11.61 (s, N(2)H);
8.62 (s, 1H, C(1)H); 8.57 (s, 1H, N(4)H); 8.28 (s, 1H, C(6)H);
7.27 (s, 1H, C(10)H); 7.18 (d, 1H, C(7)H); 7.15 (d, 1H, C(8)H);
3.80 (s, 3H, C(11)H); 3.63 (q, 2H, C(13)H); 1.19 (t, 3H,
C(14)H).
[Pd(L2)(PPh3)] (2). It was prepared using the same procedure
as described for 1 by the reaction of [PdCl2(PPh3)2] (0.100 g;
0.127 mmol) with ligand, H2L2 (0.042 g; 0.143 mmol). Dark
orange coloured crystals obtained were found to be suitable for
X-ray diffraction. Yield: 75%, MP: 254–256 °C, Elemental
Analysis calculated for C31H27N4O2PPdS (%): C, 56.61; H,
4.25; N, 8.52; S, 4.87; Found (%): C, 56.75; H, 4.32; N, 8.45; S,
4.73. IR (KBr disks, cm−1): 3179 (m) ν(NH); 1598, 1550 (s)
ν(CvN) + ν(CvC); 1344 (s) ν(C–O); 747 (m) ν(C–S); 1434,
1098, 693 (for PPh3), UV-vis (DMSO), λmax (nm): 274(intra-
ligand transition); 401 (LMCT s/d); 459 (MLCT). 1H NMR
(DMSO-d6); δ 8.67 (s, 1H, C(1)H); 8.62 (s, 1H, N(4)H); 8.38 (s,
1H, C(6)H); 7.22–7.73 (m, 18H, aromatic); 3.82 (s, 3H, C(11)
H); 2.75 (s, 3H, C(13)H).
6-Methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-4N-
phenylthiosemicarbazone (H2L4). It was prepared using the
same procedure as described for H2L1 with 4-phenyl-3-thiosemi-
carbazide (1.672 g, 0.01 mol) and 6-methoxy-2-oxo-1,2-dihy-
droquinoline-3-carbaldehyde (2.032 g, 0.01 mol). A yellow
coloured product was obtained. Yield: 92%. MP: 284–286 °C,
Elemental Analysis calculated for: C18H16N4O2S (%): C, 61.35;
H, 4.58; N, 15.90; S, 9.10. Found (%): C, 61.21; H, 4.49; N,
15.79; S, 9.27. IR (KBr, cm−1): 3301(ms) ν(NH); 1656 (s)
ν(CvO); 1619, 1539 (s) ν(CvN) + ν(CvC); 837(m) ν(CvS).
UV-vis (DMSO), λmax (nm): 264, 354, 413 (π → π*, n → π*).
1H NMR (DMSO-d6); δ 12.06 (s, N(3)H); 11.97 (s, N(2)H);
10.15 (s, 1H, C(1)H); 8.84 (s, 1H, N(4)H); 8.42 (s, 1H, C(6)H);
7.27 (m, 8H, aromatic); 3.78 (s, 3H, C(11)H).
[Pd(HL3)(PPh3)]Cl (3). It was prepared as described for 1 by
the reaction of [PdCl2(PPh3)2] (0.100 g; 0.143 mmol) with
ligand, H2L3 (0.044 g; 0.143 mmol). Dark orange coloured pre-
cipitate formed was filtered and crystals were grown from
DMF, found to be suitable for X-ray diffraction. Yield: 81%,
MP: 263–265 °C, Elemental Analysis calculated for
C32H30ClN4O2PPdS (%): C, 54.32; H, 4.27; N, 7.92; S, 4.53;
Found (%): C, 54.39; H, 4.39; N, 7.65; S, 4.47. IR (KBr disks,
cm−1): 3300(m) ν(NH2); 1646 (s) ν(CvO); 1608, 1551 (s)
ν(CvN) + ν(CvC); 746 (m) ν(C–S); 1434, 1097, 692 (for
PPh3), UV-vis (DMSO), λmax (nm): 276(intra-ligand transition);
6-Methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde-4(N,N)-
dimethylthiosemicarbazone (H2L5). It was prepared using the
same procedure as described for H2L1 with 4,4-dimethyl-3-thio-
semicarbazide (1.192 g, 0.01 mol) and 6-methoxy-2-oxo-1,2-
dihydroquinoline-3-carbaldehyde (2.032 g, 0.01 mol). A yellow
coloured product was obtained. Yield: 86%. MP: 294–296 °C,
Elemental Analysis calculated for C14H16N4O2S (%): C, 55.25;
H, 5.30; N, 18.41; S, 10.54. Found (%): C, 55.31; H, 5.24;
N, 18.37; S,10.68. IR (KBr, cm−1): 3427(ms) ν(NH); 1648(s)
ν(CvO); 1601, 1510(s) ν(CvN) + ν(CvC); 865(m) ν(CvS).
UV-vis (DMSO), λmax (nm): 261, 301, 411 (π → π*, n →
π*).1H NMR (DMSO-d6); δ 11.87 (s, N(3)H); 11.10 (s, N(2)H);
1
415 (LMCT s/d); 437 (MLCT). H NMR (DMSO-d6); δ 12.40
(s, N(3)H); 8.73 (s, 1H, C(1)H); 8.54 (s, 1H, N(4)H); 8.44 (s,
1H, C(6)H); 7.63–7.99 (m, 18H, aromatic); 3.79 (s, 3H, C(11)
H); 3.57 (q, 2H, C(13)H); 1.17 (t, 3H, C(14)H).
[Pd(L4)(PPh3)] (4). It was prepared as described for 1 by the
reaction of [PdCl2(PPh3)2] (0.100 g; 0.143 mmol) with ligand,
H2L4 (0.050 g; 0.143 mmol). Dark orange coloured precipitate
formed was filtered and the crystals were grown from acetone–
DMF solution, found to be suitable for X-ray diffraction. Yield:
84%, MP: 218–220 °C, Elemental Analysis calculated for
13310 | Dalton Trans., 2012, 41, 13308–13323
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Table 1 Experimental data for crystallographic analyses
Complex 2
Complex 3
Complex 4
Complex 5
CCDC deposit number
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
α(°)
β(°)
831519
C31H27N4O2PPdS
657.00
834540
C35H38.11ClN5O3.56PPdS
790.62
880537
C42H41N4O4PPdS
835.22
841993
C32H29N4O2PPdS
671.02
110(2) K
110(2) K
110(2) K
110(2) K
0.71073 Å
Monoclinic
P2(1)/c
1.54178 Å
Monoclinic
P2(1)/c
1.54178 Å
Triclinic
ˉ
P1
1.54178 Å
Triclinic
ˉ
P1
14.2896(12)
11.4236(10)
18.1864(15)
90
13.110(2)
12.080(2)
24.071(4)
90
12.2382(5)
12.4005(5)
13.5466(5)
67.059(2)
09.8863(3)
11.3723(4)
13.9140(5)
101.755(2)
105.532(2)
103.784(2)
1402.71(8) Å3
2
109.9050(10)
90
113.173(11)
90
80.237(2)
γ(°)
Volume
Z
81.519(2)
2791.4(4) Å3
4
3504.5(10) Å3
4
1858.29(13) Å3
2
Density (calculated)
Absorption coefficient
F(000)
1.563 Mg m−3
0.834 mm−1
1336
1.498 Mg m−3
6.324 mm−1
1622
1.493 Mg m−3
5.356 mm−1
860
1.589 Mg m−3
6.879 mm−1
684
Crystal size (mm3)
Theta range for data
collection
0.17 × 0.13 × 0.02
2.14 to 27.49°
0.50 × 0.04 × 0.04
4.00 to 59.99°
0.10 × 0.09 × 0.02
3.68 to 59.99°
0.05 × 0.04 × 0.03
3.44 to 59.99°
Index ranges
−18 ≤ h ≤ 18
−14 ≤ k ≤ 14
−23 ≤ l ≤ 23
31 688
6377 [R(int) = 0.0375]
99.4%
−14 ≤ h ≤ 14
−13 ≤ k ≤ 13
−26 ≤ l ≤ 26
46 209
5163 [R(int) = 0.0590]
99.3%
−13 ≤ h ≤ 13
−13 ≤ k ≤ 13
−15 ≤ l ≤ 15
41 891
5379 [R(int) = 0.0571]
97.7%
−11 ≤ h ≤ 11
−12 ≤ k ≤ 12
−15 ≤ l ≤ 15
31 583
4078 [R(int) = 0.0459]
98.0%
Reflections collected
Independent reflections
Completeness to
θ = 27.49°
Absorption correction
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Semi-empirical from
equivalents
Max. and min.
0.9868 and 0.8712
0.7860 and 0.1441
0.9004 and 0.6165
0.8202 and 0.7248
transmission
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2σ(I)]
6377/0/363
1.037
R1 = 0.0262
wR2 = 0.0613
R1 = 0.0350
wR2 = 0.0657
5163/0/437
1.014
5379/18/471
1.099
4078/0/373
1.054
R1 = 0.0355
wR2 = 0.0895
R1 = 0.0452
wR2 = 0.0915
1.043 and −0.831
R1 = 0.0355
wR2 = 0.0969
R1 = 0.0397
wR2 = 0.0987
0.830 and −0.728
R1 = 0.0224
wR2 = 0.0544
R1 = 0.0250
wR2 = 0.0550
0.302 and −0.543
R indices (all data)
Largest diff. peak and hole 0.586 and −0.475
(e Å−3
)
C36H29N4O2PPdS (%): C, 60.13; H, 4.06; N, 7.79; S, 4.46;
Found (%): C, 60.07; H, 4.11; N, 7.83; S, 4.51. IR (KBr disks,
cm−1): 3260 (m) ν(NH); 1614, 1561 (s) ν(CvN) + ν(CvC);
1315 (s) ν(C–O); 746 (m) ν(C–S); 1436, 1095, 691 (for PPh3),
UV-vis (DMSO): λmax (nm): 270 (intra-ligand transition); 483
(LMCT s/d); 465 (MLCT). 1H NMR (DMSO-d6); δ 8.92 (s, 1H,
C(1)H); 8.67 (s, 1H, N(4)H); 8.38 (s, 1H, C(6)H); 7.27–7.78
(m, 23H, aromatic); 3.81 (s, 3H, C(11)H).
C(1)H); 8.41(s, 1H, C(6)H); 7.12–7.72 (m, 18H, aromatic); 3.84
(s, 3H, C(11)H); 3.05 (s, 6H, C(13, 14)H).
Single crystal X-ray diffraction studies
Single-crystal X-ray diffraction data of complex 2 was collected
on a BRUKER APEX 2 X-ray (three-circle) diffractometer,
whereas the data for the complexes 3, 4 and 5 were collected on
a BRUKER GADDS X-ray (three-circle) diffractometer at
110 K. Integrated intensity information for each reflection was
obtained by reduction of the data frames with APEX2.29 The
integration method employed a three dimensional profiling algor-
ithm and all data were corrected for Lorentz and polarization
factors, as well as for crystal decay effects with the program
SADABS.30 The structure of complexes, 2, 3, 4 and 5 were
solved by direct methods using the program SHELXTL.31 The
refinement and all further calculations were carried out using
SHELXTL. The hydrogen atoms bound to carbon were placed in
idealized positions and were set riding on the parent atom.
Hydrogen atoms attached to N and O were located from the
Fourier difference maps and were set riding on the respective
[Pd(L5)(PPh3)] (5). It was prepared as described for 1 by the
reaction of [PdCl2(PPh3)2] (0.100 g; 0.143 mmol) with ligand,
H2L5 (0.044 g; 0.143 mmol). Dark orange coloured crystals
obtained were found to be suitable for X-ray diffraction. Yield:
78%, MP: 281–283 °C, Elemental Analysis calculated for
C32H29N4O2PPdS (%): C, 57.27; H, 4.36; N, 8.35; S, 4.78;
Found (%): C, 57.14; H, 4.28; N, 8.25; S, 4.63. IR (KBr disks,
cm−1): 3147 (m) ν(NH); 1593, 1550 (s) ν(CvN) + ν(CvC);
1354 (s) ν(C–O); 747 (m) ν(C–S); 1436, 1094, 693 (for PPh3),
UV-vis (DMSO), λmax (nm): 262 (intra-ligand transition); 385
(LMCT s/d); 457 (MLCT). 1H NMR (DMSO-d6); δ 8.63 (s, 1H,
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Fig. 1 An ORTEP view of the complex 2 with the atom numbering scheme.
parent atom. All non-hydrogen atoms were refined with anisotro-
pic thermal parameters. A molecule of DMF and water were
found solvated in complex 3. The thermal parameters of O90
(water) indicated that it is partially occupied and the occupancy
was refined (0.56). For DMF, the thermal ellipsoids were
elongated. A trial to refine the disorder has only increased the
number of refinement parameters along with the restraints and
did not improve the reliability factors. In complex 4, two acetone
molecules, each disordered between two positions, were found
solvated. The structures were refined (weighted least squares
refinement on F2) to convergence.31 Crystal data and details of
structure refinements for complexes are listed in Table 1.
Further support for the binding of the complexes to DNA via
intercalation was obtained from emission quenching experi-
ments. For all the experiments, the DNA was pre-treated with
ethidium bromide for 30 min. Then the test solutions were added
to this mixture of EB-DNA, and the change in the fluorescence
intensity was measured. The excitation and the emission wave-
length were 515 nm and 613–617 nm, respectively.
Protein binding studies
The excitation wavelength of BSA at 280 nm and the emission
at 345 nm were monitored for the protein binding studies. The
excitation and emission slit widths and scan rates were main-
tained constant for all the experiments. Samples were carefully
degassed using pure nitrogen gas for 15 min. Quartz cells
(4 × 1 × 1 cm) with high vacuum Teflon stopcocks were used for
degassing. Stock solution of BSA was prepared in 50 mM phos-
phate buffer (pH = 7.2) and stored in the dark at 4 °C for further
use. Concentrated stock solution of the compounds were pre-
pared as mentioned for the DNA binding experiments except
that the phosphate buffer was used instead of Tris-HCl buffer for
all the experiments. Titrations were manually done by using a
micropipette for the addition of the compounds. For synchronous
fluorescence spectra also, the same concentration of BSA and
the compounds were used and the spectra were measured at two
different Δλ (difference between the excitation and emission
wavelengths of BSA) values such as 15 and 60 nm.
DNA binding experiments
The UV-vis absorption spectroscopic studies and the DNA
binding experiments were performed at room temperature. The
purity of the CT-DNA was verified by taking the ratio of the
absorbance values at 260 and 280 nm in the respective buffer,
which was found to be 1.8 : 1, indicating that the DNAwas suffi-
ciently free of protein. The DNA concentration per nucleotide
was determined by absorption spectroscopy using the molar
extinction coefficient value of 6600 dm3 mol−1 cm−1 at 260 nm.
The complexes were dissolved in a mixed solvent of 5% DMSO
and 95% Tris-HCl buffer for all the experiments. Absorption
titration experiments were performed with a fixed concentration
of the compounds (25 μM) while gradually increasing the con-
centration of DNA (10–50 μM). While measuring the absorption
spectra, an equal amount of DNA was added to both the test
solution and the reference solution to eliminate the absorbance
of DNA itself.
Antioxidant assays
The DPPH radical scavenging activities of the complexes were
measured according to the method of Blois.32 The complexes at
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Fig. 2 An ORTEP view of the complex 3 with the atom numbering scheme. The DMF and water molecules are omitted for clarity.
Table 2 Selected bond lengths [Å] and angles [°]
Complex 2 Complex 3 Complex 4 Complex 5
The total antioxidant activity of the samples was measured
by ABTS radical cation decolorization assay according to the
method of Re et al.33 ABTS˙+ (2,2′-Azino-3-ethylbenzothiazo-
line-6-sulfonic acid diammonium salt) was produced by reacting
7 mM ABTS aqueous solution with 2.4 mM potassium persul-
fate in the dark for 12–16 h at room temperature. Prior to assay,
this solution was diluted in ethanol (about 1 : 89 v/v) and equili-
brated at 30 °C to give an absorbance of 0.700 0.02 at 734 nm.
After the addition of 1 mL of diluted ABTS solution to various
concentrations of the complexes in ethanol, the absorbance was
measured at 30 °C exactly 30 min after the initial mixing.
Pd(1)–N(1)
Pd(1)–O(1)
Pd(1)–S(1)
Pd(1)–P(1)
2.0262(16) 2.031(3)
2.0332(14) 2.063(2)
2.040(3)
2.053(3)
2.024(2)
2.0393(17)
2.2786(5)
2.2347(6)
2.2352(10) 2.2167(10) 2.2385(6)
2.2775(10) 2.2838(10) 2.2843(7)
N(1)–Pd(1)–O(1) 93.10(6)
N(1)–Pd(1)–S(1) 84.31(5)
O(1)–Pd(1)–S(1) 177.09(4)
N(1)–Pd(1)–P(1) 176.97(5)
O(1)–Pd(1)–P(1) 89.84(4)
92.25(11)
84.22(9)
175.88(7)
175.52(9)
91.93(7)
92.43(11)
84.07(9)
176.16(7)
175.23(9)
91.11(7)
92.30(4)
93.18(7)
83.95(6)
171.79(5)
166.81(6)
92.01(5)
92.45(2)
S(1)–Pd(1)–P(1) 92.765(19) 91.54(4)
Cytotoxicity assays
various concentrations were taken and the volume was adjusted
to 100 μL with methanol. About 5 mL of a 0.1 mM methanolic
solution of DPPH was added to the aliquots of compound and
standards (BHA, BHT) and shaken vigorously. Negative control
was prepared by adding 100 μL of methanol in 5 mL of 0.1 mM
methanolic solution DPPH. The tubes were allowed to stand for
20 min at 27 °C. The absorbance of the sample was measured at
517 nm against the blank (methanol). Radical scavenging
activity of the samples was expressed as IC50 which is the con-
centration of the compound required to inhibit 50% of DPPH
concentration.
MTT assay. Cytotoxic effect of the compounds on human
lung cancer cells (A549) were assayed by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.34 The cells were seeded at a density of 10 000 cells per
well in 200 μL and then the compounds were added to the cells
at a final concentration of 1, 10, 25 and 50 μM in the cell culture
media. After 48 h, the wells were treated with 20 μL MTT
(5 mg mL−1 PBS) and incubated at 37 °C for 4 h. The purple
formazan crystals formed were dissolved in 200 μL DMSO and
read at 570 nm in a micro plate reader.
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Fig. 3 An ORTEP view of complex 4 with the atom numbering scheme. The distorted DMF molecules are omitted for clarity.
formulae of the complexes 2, 4, 5 and 3 are [Pd(L)PPh3] and
[Pd(HL)PPh3]Cl respectively. The crystal data, data collection
and refinement parameters for the new Pd(II) complexes were
summarized in Table 1.
Results and discussion
Synthesis and characterization
The synthetic routes of the ligands and their Pd(II) complexes, 1,
2, 3, 4 and 5 are shown in Scheme 1. The complexes 1–5 were
prepared by direct reaction of the corresponding ligand with pre-
cursor complex, [PdCl2(PPh3)2] in 1 : 1 mole ratio. While all
other ligands coordinated as dibasic tridentate in 1, 2, 4 and 5,
the H2L3 alone coordinated as a monobasic tridentate in 3. All
the new ligands and their mononuclear palladium complexes
were characterised by elemental analysis, IR, UV-vis and
1H-NMR spectroscopy and the data are given in the Experimen-
tal section. The IR peak shift in ν(CvN), ν(CvO) and ν(CvS)
of the ligands in the complexes gave evidence for the coordi-
Crystal structures of the complexes 2–5
The molecular structure of the complexes 2 and 3 along with the
atomic numbering scheme are given in Fig. 1 and 2. The selected
bond lengths and bond angles are summarised in Table 2. The
single crystal X-ray diffraction study reveals that complexes 2
and 3 are crystallized in a monoclinic crystal system with the
space group P2(1)/c. In complex 2 the coordination geometry
around Pd(II) can be described as a distorted square planar, the
palladium atom being bonded to binegative tridentate ONS
donor ligand molecules in such a way that five and six mem-
bered chelate rings are formed and the remaining site is occupied
by triphenylphosphine. In complex 3, the Pd(II) ions adopt a dis-
torted square planar geometry, the palladium atom being bonded
to monobasic tridentate ONS donor ligand molecules and the
fourth vacant site is occupied by triphenylphosphine ligand and
the complex neutralized by one chlorine atom which is present
in lattice. The trans angles of O1–Pd1–S1; 177.09(4)° and
N–Pd1–P; 166.81(6)° (for 2) and O(1)–Pd(1)–S(1); 175.88(7)°,
1
nation of the ligand to palladium ion. In the H-NMR spectra of
the new complexes the absence of the signal for N(2)H indicated
that sulphur coordinated to the palladium ion in the complexes
1–5 in the thiolate form. The absence of the peak for N(3)H in
the complexes 1, 2, 4 and 5 indicated that the oxygen coordi-
nated to Pd(II) ion as the phenolic form and in complex 3, the
presence of the N(3)H peak clearly indicated that the oxygen
coordinated to the palladium in the oxo form. These obser-
vations have been confirmed by X-ray single crystal structure
analysis. The single crystal X-ray analysis showed that the
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Fig. 4 An ORTEP view of complex 5 with the atom numbering scheme.
N(1)–Pd(1)–P(1); 175.52(9)° (for 3) indicated a slight deviation
from the expected linear trans geometry, suggesting distortion in
the square planar coordination geometry. The dihedral angle
between the mean planes of the coordinating five-member chela-
tion ring and the six member one is 2.18° and 3.97° for 2 and 3
respectively.
6.92° for 4 and 5 respectively. The analysis of the bond lengths
and angles (Table 2) of the complexes 2–5 gives further support
to the belief that the oxo group oxygen of complex 3 bonded to
the metal ion in neutral mode and in the rest of the complexes
the oxo group oxygen is bonded to the metal ion as O−.
The ORTEP view of complexes 4 and 5 along with the coordi-
nating atomic numbering scheme are depicted in Fig. 3 and 4,
and selected bond lengths and bond angles are listed in Table 2.
The complexes 4 and 5 crystallized in a triclinic system with the
DNA binding studies
DNA binding is the predominant property looked for in pharma-
cology when evaluating the potential of new antitumor com-
plexes, and hence, the interaction between DNA and the
synthesized complexes needs to be investigated. The mode and
tendency for the binding of the complexes 1–5 to CT-DNA were
studied with different methods.
ˉ
space group P1. In both 4 and 5, the coordination geometry
around Pd(II) is a distorted square planar, the palladium atom
being bonded to binegative tridentate ONS donor ligand mo-
lecules in such a way that five and six membered chelate rings
are formed. The remaining site is occupied by triphenylpho-
sphine. The trans angles of complex 4 O1–Pd1–S1; 176.16(7)°
and N–Pd1–P; 175.23(9)° and complex 5 are O(1)–Pd(1)–S(1);
171.79(5)° and N(1)–Pd(1)–Cl(1); 166.81(6)° which showed a
slight deviation from the expected linear trans geometry,
suggesting distortion in the square planar coordination geometry.
The dihedral angle between the mean planes of the coordinating
five-member chelation ring and the six member one is 6.38° and
Electronic absorption titration
Electronic absorption spectroscopy is one of the most useful
techniques for the investigation of the mode of interaction of
metal complexes with DNA.35,36 Complexes binding to DNA
through intercalation usually results in hypochromism with or
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Fig. 5 Electronic spectra of complexes 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E) in Tris-HCl buffer upon addition of CT-DNA. [Complex] = 25 μM,
[DNA] = 0–50 μM. Arrow shows the absorption intensities decrease upon increasing DNA concentration.
without a small red or blue shift, since the intercalative mode
involves a strong stacking interaction between the planar aro-
matic chromophore and the base pairs of DNA.21,37 The results
of absorption spectra of complexes in the absence and presence
of CT-DNA is given in Fig. 5. Upon increasing the concentration
of DNA to the test complexes, the absorption bands of the com-
plexes 1 and 2 exhibited hypochromism of 57.49% and 62.79%
and 25.41% with blue shifts of 3 and 4 and 2 nm at 382 and 384
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1 < 2 < 3 < 5 < 4, though it has been found that complexes 1–5
can bind to DNA by intercalation, the binding mode needs to be
proven through further experiments.
Ethidium bromide (EB) displacement studies
The absorption titration results indicate that the complexes effec-
tively bind to DNA. In order to confirm the binding mode and
compare their binding affinities, ethidium bromide displacement
experiments were carried out. EB is a planar cationic dye which
is widely used as a sensitive fluorescence probe for native DNA.
EB emits intense fluorescent light in the presence of DNA due to
its strong intercalation between the adjacent DNA base
pairs.39,40 Hence, EB displacement technique can provide indir-
ect evidence for the DNA binding mode. The displacement tech-
nique is based on the decrease of fluorescence resulting from the
displacement of EB from a DNA sequence by a quencher and
the quenching is due to the reduction of the number of binding
sites on the DNA that is available to EB. Fig. 7 shows the fluor-
escence quenching spectra of DNA-bound EB by complexes and
illustrates that as the concentration of the complexes increases,
the emission band at 613 nm exhibited hypochromism up to
14.88, 16.07, 18.15, 20.20 and 21.11% with blue shifts of 3, 2,
4, 4 and 3 nm of the initial fluorescence intensity for 1–5
respectively. The observed decrease in the fluorescence intensity
clearly indicates that the EB molecules are displaced from their
DNA binding sites and are replaced by the complexes under
investigation.41 The quenching parameter can be analyzed
according to the Stern–Volmer equation,
Fig. 6 Plots of [DNA]/(εa − εf) versus [DNA] for the complexes 1–5
with CT-DNA.
and 427 nm whereas the absorption bands of complex 3 at 384
and 432 nm exhibited a hypochromism of about 70.30 and
64.08% with blue shifts of 3 and 1 nm. However, complex 4 at
391 nm and 470 nm exhibited a hypochromism of about 80.48%
and 68.05% with blue shifts of 4 and 3 nm and complex 5 at
392 and 463 nm exhibited a hypochromism of about 73.03%
and 65.49% with blue shifts of 5 and 2 nm, respectively. These
results revealed that all the new palladium complexes bind to the
DNA helix via intercalation, due to stacking interaction between
the planar aromatic chromophore and the base pairs of DNA.
Complex 4 showed more hypochromicity than the other com-
plexes, indicating that the binding strength of complex 4 is much
stronger than the other complexes. In order to further compare
the binding strength of the palladium complexes, their intrinsic
binding constants (Kb) were determined from the following
equation.38
F0=F ¼ Kq ½Qꢀ þ 1
where F0 is the emission intensity in the absence of compound,
F is the emission intensity in the presence of compound, Kq is
the quenching constant, and [Q] is the concentration of the com-
pound. The Kq value is obtained as a slope from the plot of F0/F
versus [Q]. From the Stern–Volmer plot (Fig. 8) of F0/F versus
[Q], the quenching constants (Kq) were obtained from the slope
which were 7.21 × 103 M−1, 7.29 × 103 M−1, 8.46 × 103 M−1
,
½DNAꢀ=ðεa ꢁ εf Þ ¼ ½DNAꢀ=ðεb ꢁ εf Þ þ 1=Kbðεb ꢁ εf Þ
10.35 × 103 M−1 and 9.75 × 103 M−1 respectively for 1–5.
Further, the apparent DNA binding constant (Kapp) were calcu-
lated using the following equation,
where, [DNA] is the concentration of DNA in the base pairs, εa
is the apparent absorption coefficient corresponding to Aobs
/
[compound], εf is the extinction coefficient of the free compound
and εb is the extinction coefficient of the compound when fully
bound to DNA. From the plot of [DNA]/(εa − εf) versus [DNA]
(Fig. 6), the intrinsic binding constant Kb was calculated from
the ratio of the slope and the intercept. The intrinsic binding con-
stant (Kb) values were 2.31( 0.04) × 104 M−1, 3.26( 0.02) ×
104 M−1, 3.78( 0.07) × 104 M−1, 5.46 ( 0.05) × 105 M−1 and
6.18( 0.06) × 104 M−1 for complexes 1, 2, 3, 4 and 5 respect-
ively. The experimental values of Kb revealed that complexes
1–5 bind to DNA via the intercalative mode. From the results
obtained, it has been found that complex 4 strongly binds with
CT-DNA compared to the remaining complexes, which may be
due to the presence of the phenyl ring in the terminal nitrogen of
the respective ligand, preferring interaction of the complex with
the DNA bases through π–π interaction. From the above experi-
mental studies the order of binding affinity of the complexes is
KEB½EBꢀ ¼ Kapp½complexꢀ
where the compound concentration is the value at a 50%
reduction in the fluorescence intensity of EB, KEB (1.0 ×
107 M−1) is the DNA binding constant of EB, [EB] is the con-
centration of EB = 12 μM), and they were found to be 8.65 ×
105 M−1, 8.74 × 105 M−1, 10.15 × 105 M−1, 12.42 × 105 M−1
and 11.70 × 105 M−1 respectively for 1–5. From these exper-
imental data, it is seen that the order of binding affinities of the
complexes is in the order 4 > 5 > 3 > 2 > 1, which is in agree-
ment with the results observed from the electronic absorption
spectra. Furthermore, the observed quenching constants and
binding constants of the palladium(II) complexes suggested that
the interaction of the complexes 1–5 with DNA should be of
intercalation.23
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Fig. 7 Fluorescence quenching curves of ethidium bromide bound to DNA: 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). [DNA] = 12 μM, [EB] = 12 μM,
and [complex] = 0–25 μM. Arrow shows the emission intensity changes upon increasing complex concentration.
Protein binding studies
involved in the transport of metal ions and metal complexes with
drugs through the blood stream. Usually, the binding of drugs to
these proteins may lead to either a loss or enhancement of the
biological properties of the original drug. Fluorescence spectra
are useful in the qualitative analysis of the binding of chemical
Fluorescence quenching of BSA by mononuclear palladium(II)
complexes. It has been proved that the interaction of drugs with
blood plasma proteins particularly with serum albumin is
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existence of just a single binding site in BSA for all of the com-
pounds. From the values of Kq and Kbin, it is inferred that
complex 4 interacts with BSA more strongly than the rest of the
complexes. Here again, complex 4 shows better activity due to
the presence of a phenyl group at the terminal N atom of the
coordinated ligand which can interact with the active site by
making it more hydrophobic. On decreasing the bulkiness in
substitution, the binding constant value (K) also decreases.
Quenching usually occurs either by dynamic or static quench-
ing. Dynamic quenching is a process in which the fluorophore
and the quencher come into contact during the transient exist-
ence of the excited state. On the other hand, static quenching
refers to the formation of a fluorophore–quencher complex in the
ground state. A simple method to determine the type of quench-
ing is UV-visible absorption spectroscopy. UV-visible spectra of
BSA in the absence and presence of the complexes (Fig. 12)
showed that the absorption intensity of BSAwas enhanced as the
complexes were added, and there was a little blue shift of about
1 nm for all the complexes. It showed that there exists a static
interaction between BSA and the added compounds due to the
formation of the ground state complex of the type of BSA-
compound reported earlier.41
Fig. 8 Stern–Volmer plots of the fluorescence titrations of the com-
plexes 1, 2, 3, 4 and 5.
compounds to BSA. Generally, the fluorescence of BSA is
caused by two intrinsic characteristics of the protein, namely
tryptophan and tyrosine. Changes in the emission spectra of
tryptophan are observed in response to protein conformational
transitions, subunit associations, substrate binding, or denatura-
tion. The intrinsic fluorescence of BSA will provide considerable
information on their structure and dynamics and is often utilized
in the study of protein folding and association reactions. The
interaction of BSA with the complexes was studied by fluor-
escence measurement at room temperature. A solution of BSA
(1 μM) was titrated with various concentrations of the complexes
1–5 (0–25 μM). Fluorescence spectra were recorded in the range
of 290–450 nm upon excitation at 280 nm. The effects of the
compound on the fluorescence emission spectrum of BSA are
given in Fig. 9. Addition of the new complexes to the solution of
BSA resulted in a significant decrease of the fluorescence inten-
sity of BSA at 346 nm, up to 79.45, 85.52, 87.49, 89.51 and
87.94% from the initial fluorescence intensity of BSA
accompanied by a hypsochromic shift of 1–3 nm for the com-
plexes 1–5 respectively. The observed blue shift is mainly due to
the fact that the active site in protein is buried in a hydrophobic
environment. These results indicated a definite interaction of all
of the complexes with the BSA protein.22,24
Characteristics of synchronous fluorescence spectra
In order to investigate in detail the structural changes which
occurred to BSA upon the addition of new compounds, synchro-
nous fluorescence spectra of BSA were measured before and
after the addition of test compounds. The results provide reason-
able information on the molecular microenvironment, particu-
larly in the vicinity of the fluorophore functional groups.41 It is a
well-known fact that the fluorescence of BSA is normally due to
the presence of tyrosine, tryptophan and phenylalanine residues
and hence, spectroscopic methods are usually applied to study
the conformation of serum protein. According to Miller,42 the
difference between the excitation wavelength and emission
wavelength (Δλ = λemi − λexi) indicates the type of chromophore.
A higher Δλ value such as 60 nm is characteristic of the trypto-
phan residue while a lower Δλ value such as 15 nm is character-
istic of the tyrosine residue.39 Fig. S1 and S2† show the
synchronous fluorescence spectra of BSA with various concen-
trations of test complexes recorded at Δλ = 15 nm and Δλ =
60 nm respectively. In the synchronous fluorescence spectra of
BSA at Δλ = 15, addition of the complexes to the solution of
BSA resulted in an increase of the fluorescence intensity of BSA
at 302 nm; 1.09, 1.47, 1.51, 2.35 and 1.69 times of the initial flu-
orescence intensity of BSA for the complexes 1, 2, 3, 4 and 5
respectively. But, in the case of synchronous fluorescence
spectra of BSA at Δλ = 60, addition of the compounds to the
solution of BSA resulted in a significant decrease of the fluore-
scence intensity of BSA at 343 nm, up to 81.14, 82.53, 86.12,
88.53 and 86.56% of the initial fluorescence intensity of BSA
accompanied by a trivial blue shift of 1 and 2 nm for complexes
1–5. The spectral studies clearly suggested that the fluorescence
intensities of both the tryptophan and tyrosine were decreased
but the emission wavelength of the tryptophan residues is blue
shifted with increasing concentration of the complexes. At the
same time, there is no change in the emission wavelength of
Furthermore, fluorescence quenching data were analysed with
the Stern–Volmer equation and Scatchard equation. From the
plot of F0/F versus [Q] the quenching constant (Kq) can be cal-
culated (Fig. 10). If it is assumed that the binding of compounds
with BSA occurs at equilibrium, the equilibrium binding con-
stant can be analysed according to the Scatchard equation:
log½I0 ꢁ I=Iꢀ ¼ log Kbin þ n log½Qꢀ
where Kbin is the binding constant of the compound with DNA
and n is the number of binding sites. The number of binding
sites (n) and the binding constant (Kbin) have been found from
the plot of log(I0 − I)/I versus log [Q] (Fig. 11). The calculated
Kq, Kbin, and n values are given in Table 3. The calculated value
of n is around 1 for all of the compounds, indicating the
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Fig. 9 The emission spectrum of BSA (1 μM; λexi = 280 nm; λemi = 346 nm) in the presence of increasing amounts of the complexes 1 (A), 2 (B), 3
(C), 4 (D) and 5 (E) (0–25 μM). Arrow shows the emission intensity changes upon increasing complex concentration.
tyrosine. It suggested that the interaction of compounds with
BSA affects the conformation of the tryptophan micro-region.21
It further indicated that the hydrophobicity around tryptophan
residues is strengthened. The hydrophobicity observed in
fluorescence and synchronous measurements confirmed the
effective binding of all the complexes with the BSA. Hence, the
strong interaction of these compounds with BSA suggested that
the complexes may be fit for anticancer studies.
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Table 3 Quenching constant (Kq), binding constant (Kbin) and number
of binding sites (n) for the interactions of complexes with BSA
Complexes
Kq (M−1
)
Kbin (M−1
)
n
1
2
3
4
5
1.89(0.05) × 105
2.19(0.17) × 105
2.20(0.17) × 105
2.78(0.23) × 105
2.74(0.45) × 105
03.05(0.04) × 105
02.66(0.12) × 106
04.54(0.05) × 106
12.51(0.09) × 107
02.12(0.17) × 107
1.01
1.25
1.47
1.52
1.30
Fig. 10 Stern–Volmer plots of the fluorescence titration of the com-
plexes 1–5 with BSA.
Fig. 12 UV absorption spectra of BSA (10 μM) in the presence of the
complex (5 μM).
Table 4 The radical scavenging activity of the complexes
IC50 values (μm)
Complexes
DPPH˙
ABTS˙
Complex 1
Complex 2
Complex 3
Complex 4
Complex 5
BHA
08.56
09.45
08.62
08.38
11.95
25.58
45.99
06.95
18.68
08.90
06.79
24.15
21.58
37.99
Fig. 11 Scatchard plots of the fluorescence titration of the complexes
1–5 with BSA.
BHT
Antioxidant activity
ABTS radicals than that of the standard antioxidants BHA and
BHT.
Since the palladium complexes exhibited good DNA and protein
binding affinity, it was considered meaningful to study the anti-
oxidant activity of these complexes. Hence, a systematic study
was carried out on the antioxidant potential of the new com-
plexes along with standards, such as butylated hydroxy anisole
(BHA) and butylated hydroxyl toluene (BHT), against DPPH
and ABTS radicals with respect to different concentrations of the
complexes and the results are shown in Table 4. It is seen from
the results that complexes 1–5 possess potent antioxidant activi-
ties. However, complex 4 showed better activity than the other
four complexes, which may be due to the electron withdrawing
effect of the phenyl group on the terminal nitrogen atom of the
ligand. It is to be noted that the new palladium(II) complexes
showed a better antioxidant activity against the DPPH and
In vitro cytotoxic activity studies by MTT assay
The positive results obtained from the DNA binding, protein
binding and antioxidative studies encouraged us to test the cyto-
toxicity of the complexes against a human lung cancer cell line
(A549). The cytotoxicity assay for the new Pd(II) complexes was
assessed using the method of MTT reduction. Cisplatin was used
as a positive control. The complexes were found to be cytotoxic
to lung cancer cell line (A549). The IC50 value (50% inhibition
of cell growth for 48 h) of palladium complexes 1, 2, 3, 4 and 5
are shown in Fig. 13. The new palladium complexes exhibited
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Dalton Trans., 2012, 41, 13308–13323 | 13321

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financial assistance received, and the University Grants Com-
mission, New Delhi, for the award of a Fellowship in Science
for Meritorious Students (RFSMS) to E.
R under the
UGC-SAP-DRS programme, are gratefully acknowledged.
References
1 L. Kelland, Nat. Rev. Cancer, 2007, 7, 573–584.
2 T. Boulikas and M. Vougiouka, Oncol. Rep., 2003, 10, 1663–1682.
3 E. Wong and C. M. Giandomenico, Chem. Rev., 1999, 99, 2451–2466.
4 M. Galanski, V. B. Arion, M. A. Jakupec and B. K. Keppler, Curr.
Pharm. Des., 2003, 9, 2078–2089.
5 D. Wang and S. Lippard, Nat. Rev. Drug Discovery, 2005, 4, 307–320.
6 J. Ruiz, N. Cutillas, C. Vicente, M. D. Villa and G. Lopez, Inorg. Chem.,
2005, 44, 7365–7376.
7 E. J. Gao, C. Liu, M. C. Zhu, H. K. Lin, Q. Wu and L. Liu, Anti-Cancer
Agents Med. Chem., 2009, 9, 356–368.
Fig. 13 The IC50 values (50% inhibition of cell growth for 48 h) of
complexes 1–5 are depicted.
8 S. Padhye, Z. Afrasiabi, E. Sinn, J. Fok, K. Mehta and N. Rath, Inorg.
Chem., 2005, 44, 1154–1156.
9 S. Halder, S. M. Peng, G. H. Lee, T. Chatterjee, A. Mukherjee, S. Dutta,
U. Sanyal and S. Bhattacharya, New J. Chem., 2008, 32, 105–114.
10 D. K. Demertzi, M. A. Demertzis, J. R. Miller, C. S. Frampton,
J. P. Jasinski and D. X. West, J. Inorg. Biochem., 2002, 92, 137–140.
11 P. I. da, S. Maia, A. G. de, A. Fernandes, J. N. Silva, A. D. Andricopulo,
S. S. Lemos, E. S. Lang, U. Abram and V. M. Deflon, J. Inorg. Biochem.,
2010, 104, 1276–1282.
12 A. I. Matesanz and P. Souza, J. Inorg. Biochem., 2007, 101, 245–253.
13 A. I. Matesanz, C. Hernández, A. Rodriguez and P. Souza, J. Inorg.
Biochem., 2011, 105, 1613–1622.
14 L. Otero, M. Vieites, L. Boiani, A. Denicola, C. Rigol, L. Opazo,
C. O. Azar, J. D. Maya, A. Morello, R. L. K. Siegel, O. E. Piro,
O. E. Castellano, M. Gonzalez, D. Gambino and H. Cerecetto, J. Med.
Chem., 2006, 49, 3322–3331.
15 K. Husain, M. Abid and A. Azam, Eur. J. Med. Chem., 2007, 42, 1300–
1308.
higher cytotoxic effects on lung cancer cells with lower IC50
values indicating its efficiency in killing the cancer cells even at
low concentrations. The cytotoxic effectiveness of complexes 3,
4 and 5 with an IC50 of 18, 7 and 10 μM was higher than that of
cisplatin (25 μM). There are reports in the literature on the cyto-
toxic effects of the complexes with longer incubation time
periods and such a longer incubation period may result in the
development of cellular resistance for that particular complex.
But, the data obtained for our complexes showed a higher cyto-
toxicity with a short incubation period which is highly signifi-
cant when compared to the results of Beckford et al.43
Furthermore, the IC50 values of 7 μM for complex 4 confirmed a
much higher cytotoxic effect than the rest of the complexes indi-
cating the effect of the substitution at the terminal nitrogen of the
complexes for the enhanced antitumor activities.
16 K. Husain, M. Abid and A. Azam, Eur. J. Med. Chem., 2008, 43, 2016–
2028.
17 R. Prabhakaran, P. Kalaivani, R. Jayakumar, M. Zeller, A. D. Hunter,
S. V. Renukadevi, E. Ramachandran and K. Natarajan, Metallomics,
2011, 3, 42–48.
18 P. Kalaivani, R. Prabhakaran, F. Dallemer, P. Poornima, E. Vaishnavi,
E. Ramachandran, V. Vijaya Padma, R. Renganathan and K. Natarajan,
Metallomics, 2012, 4, 101–113.
Conclusion
19 E. Ramachandran, P. Kalaivani, R. Prabhakaran, N. P. Rath, S. Brinda,
P. Poornima, V. V. Padma and K. Natarajan, Metallomics, 2012, 4, 218–
227.
20 P. Kalaivani, R. Prabhakaran, E. Ramachandran, F. Dallemer,
G. Paramaguru, R. Renganathan, P. Poornima, V. Vijaya Padma and
K. Natarajan, Dalton Trans., 2012, 41, 2486–2499.
21 D. Senthil Raja, G. Paramaguru, N. S. P. Bhuvanesh, J. H. Reibenspies,
R. Renganathan and K. Natarajan, Dalton Trans., 2011, 40, 4548–4559.
22 D. Senthil Raja, N. S. P. Bhuvanesh and K. Natarajan, Eur. J. Med.
Chem., 2011, 46, 4584–4594.
23 E. Ramachandran, P. Kalaivani, R. Prabhakaran, M. Zeller, J. H. Bartlett,
P. O. Adero, T. R. Wagner and K. Natarajan, Inorg. Chim. Acta, 2012,
385, 94–99.
24 E. Ramachandran, S. P. Thomas, P. Poornima, P. Kalaivani,
R. Prabhakaran, V. V. Padma and K. Natarajan, Eur. J. Med. Chem.,
2012, 50, 405–415.
25 D. Senthil Raja, N. S. P. Bhuvanesh and K. Natarajan, Inorg. Chem.,
2011, 50, 12852–12866.
26 A. I. Vogel, Text Book of Practical Organic Chemistry, Longman,
London, 5th edn, 1989, p. 268.
27 M. K. Singh, A. Chandra, B. Singh and R. M. Singh, Tetrahedron Lett.,
2007, 48, 5987–5990.
Mononuclear palladium(II) complexes containing 6-methoxy-2-
oxo-1,2-dihydroquinoline-3-carbaldehyde 4N-substituted thio-
semicarbazones and triphenylphosphine have been synthesised and
characterized with a view to study the effect of substitution at the
terminal N of the ligand on the coordination behaviour and their
biological activities. The DNA interaction and protein binding
properties of the new complexes were evaluated by absorption
and fluorescence spectroscopies and the results suggested that
the binding affinity of the complexes increases with the increase
in size of substitution at the terminal nitrogen of the thiosemi-
carbazone moiety. The free radical scavenging assay results
showed that all the complexes possess significant activity. In
addition, the in vitro cytotoxicity of the new complexes 1–5 was
assayed against A549 cancer cell line and all the complexes
exhibited good cytotoxic activity. The IC50 cytotoxicity values
indicted that complex 4 is more cytotoxic when compared to the
rest of the complexes.
28 J. L. Burmeester and F. Basolo, Inorg. Chem., 1964, 3, 1587–1593.
29 APEX2, Program for Data Collection and Integration on Area Detectors,
BRUKER AXS Inc., 5465 East Cheryl Parkway, Madison, WI 53711-
5373, USA.
Acknowledgements
30 G. M. Sheldrick, SADABS (version 2008/1): Program for Absorption
Correction for Data from Area Detector Frames, University of Gottingen,
2008.
Council of Scientific and Industrial Research, New Delhi, India
[Grant No. 01(2216)/08/EMR-II & 21(0745)/09/EMR-II] for the
13322 | Dalton Trans., 2012, 41, 13308–13323
This journal is © The Royal Society of Chemistry 2012

View Article Online
31 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr., 2007,
64, 112–122.
38 A. Wolf, G. H. Shimer and T. Meehan, Biochemistry, 1987, 26, 6392–
6396.
32 M. S. Blois, Nature, 1958, 181, 1199–1200.
39 F. J. Meyer-Almes and D. Porschke, Biochemistry, 1993, 32, 4246–
33 R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C. Rice-
Evans, Free Radical Biol. Med., 1999, 26, 1231–1237.
34 T. Mossman, J. Immunol. Methods, 1983, 65, 55–63.
35 A. F. Tanious, D. Y. Ding, D. A. Patrick, C. Bailly, R. R. Tidwell and
W. D. Wilson, Biochemistry, 2000, 39, 12091–12101.
36 C. Y. Zhong, J. Zhao, Y. B. Wu, C. X. Yin and P. Yang, J. Inorg.
Biochem., 2007, 101, 10–18.
4253.
40 G. M. Howe, K. C. Wu and W. R. Bauer, Biochemistry, 1976, 19, 339–
347.
41 D. Senthil Raja, N. S. P. Bhuvanesh and K. Natarajan, Dalton Trans.,
2012, 41, 4365–4377.
42 J. N. Miller, Proc. Anal. Div. Chem. Soc., 1979, 16, 203–208.
43 F. A. Beckford, M. Shaloski, G. Leblanc, J. Thessing, L. C. L. Alleyne,
A. A. Holder, L. Li and N. P. Seeram, Dalton Trans., 2009, 10757–
10764.
37 Z. C. Liu, B. D. Wang, B. Li, Q. Wang, Z. Y. Yang, T. R. Li and Y. Li,
Eur. J. Med. Chem., 2010, 45, 5353–5361.
This journal is © The Royal Society of Chemistry 2012
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