Nickel(ii) and copper(ii) complexes constructed with N2S2 hybrid benzamidine-thiosemicarbazone ligand: Synthesis, X-ray crystal structure, DFT, kinetico-catalytic and in vitro biological applications

Article abstract of DOI:10.1039/c5ra18568h

With the aim of assessing whether transition metal complexes might be utilized as efficient biocatalysts and biological drugs, new monofunctional nickel(ii) and copper(ii) complexes of types NiL (1) and CuL (2) [H₂L = N-(N′,N′-diethylaminothioc

Full text of DOI:10.1039/c5ra18568h

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P. Viswanathamurthi, V. Krishnaswamy, N. Raju, B. K. Manickam Dakshinamoorthi, K. PT and J. Malecki,
RSC Adv., 2015, DOI: 10.1039/C5RA18568H.
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Nickel(II) and copper(II) complexes constructed with N2S2 hybrid
benzamidine-thiosemicarbazone ligand: Synthesis, X-ray crystal structure,
DFT, kinetico-catalytic and in vitro biological applications
a
a
b
Paranthaman Vijayan, Periasamy Viswanathamurthi,* Krishnaswamy Velmurugan,
b
c
Raju Nandhakumar, Manickam Dakshinamoorthi Balakumaran, Pudupalayam
c
d
Thangavelu Kalaichelvan, Jan Grzegorz Malecki
a
Department of Chemistry, Periyar University, Salem-636 011, India.
b
c
Department of Chemistry, Karunya University, Karunya Nagar, Coimbatore - 641 114, India.
Centre for Advanced Studies in Botany, School of Life Sciences, University of Madras, Guindy
Campus, Chennai - 600 025, Tamil Nadu, India.
d
Department of Crystallography, Silsian University, Szkolna 9, 40-006 Katowice, Poland
*To whom correspondence should be addressed, eꢀmail: viswanathamurthi72@gmail.com
Fax: +91 427 2345124
1

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Abstract
With the aim of assessing whether transition metal complexes might be utilized as efficient
biocatalysts and biological drugs, new monofunctional nickel(II) and copper(II) complexes of
the types NiL (1) and CuL (2) [H L = Nꢀ(N’,N’ꢀdiethylaminothiocarbonyl)benzimidoyl
2
chlorideꢀ2ꢀaminoacetophenoneꢀNꢀmethylthiosemicarbazone] were synthesized. The compounds
are remarkably stable and were obtained in good yields. The structural elucidation was carried
out thoroughly in both solid and solutionꢀstate by elemental analyses and various spectroscopic
1
techniques (IR, UVꢀvis, H NMR, EPR) as well as ESI mass spectrometry. The molecular
structure of the compounds was investigated by singleꢀcrystal Xꢀray diffraction analyses. The
analyses showed tetradentate coordination of ligand in nickel(II) and copper(II) complexes, in
which the metal atom exhibits a square planar geometry with N S donor fashion. Structural
2
2
optimization, HOMOꢀLUMO energy calculation, Natural Bond Orbital (NBO) analysis of H L
2
and its complexes 1, 2 were investigated by Density Functional Theory (DFT). The trend in
binding affinities of the compounds with biomolecules such as calf thymus DNA (CTꢀDNA) and
bovine serum albumin (BSA) protein were investigated by different spectrophotometric methods
revealed an intercalative mode of interaction. Furthermore, enzyme kinetic studies reflected that
the square planar complexes (1, 2) are also effective in mimicking catecholase (3,5ꢀDTBC) and
phosphatase (4ꢀNPP) like activities over parent H L. The highest k values suggested that the
2
cat
selected compounds displayed higher rate of catalytic efficiency. In vitro cytotoxicity of the
complexes on human skin cancer melanoma (A375), human cervical cancer (HeLa) and human
hepatocellular carcinoma (Hep3B) cancer cell lines demonstrated that the complexes had broadꢀ
spectrum of antiꢀcancer activity with low IC50 values. The morphological assessment data
obtained by acridine orange/ethidium bromide (AO/EB) and diamidinoꢀ2ꢀphenylindole (DAPI)
staining revealed that the complex 2 induces apoptosis much more effectively than 1.
Key words: Nickel(II) and copper(II) complex, X- ray structure, DFT, DNA/BSA binding,
Kinetics, MTT, Apoptosis
2

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Introduction
Ligand design is becoming an increasingly important part of the synthetic activity in
chemistry. This is of course because of the subtle control that ligands exert on the metal center to
1
,2
which they are coordinated. Notably, the choice of the hybrid ligands is particularly crucial in
the stabilization of highly reactive species, unusual oxidation states in promoting original
3
,4
5,6
reactivity and implementing catalytic as well as biological properties. In previous, many
article have shown that monoꢀ and bidentate ligands system may suffer from insufficient stability
for in vitro and in vivo studies due to rapid ligand exchange reactions with plasma components.
In common many metal(II) cores containing medium and soft donor atoms ligands were
particularly recommended for in vitro and in vivo studies. Thus, chelators with a mixed sulfur
and nitrogen donor sphere might be very suitable for formation of metal complexes and some of
7
them have found in medicinal applications. With above fact in mind, tetradentate N, S donor
Schiffꢀbase ligands have extensively been used as supporting ligands in dꢀblock chemistry
8
,9
because of their ability to stabilize metals in different oxidation states.
Thioureas are potentially very versatile ligands, as they are able to coordinate to a range
of metal centers as either neutral ligands, monoanions, or dianions. The effectiveness of thiourea
is believed to be due to the presence of lone pair of electrons in atom such as nitrogen, sulphur
1
0
and oxygen and πꢀ electron cloud that are capable of forming dative covalent bond with metals.
The derivatives of thiourea such as Nꢀ(dialkylaminothiocarbonyl)benzimidoyl chlorides were
first synthesized and incorporated into the synthesis of corresponding benzamidines, which can
readily be obtained by reactions of the benzimidoylchlorides and primary amines, with numerous
1
1
metals have been broadly studied. The benzamidines can be functionalized with additional
donor group to modify the properties depending on the requirements. The thiosemicarbazones
have been chosen to functionalize benzamidoyl chlorides due to their considerable significance
with respect to their versatile coordination behavior and promising biological and pharmaceutical
1
2ꢀ15
properties.
The new tetradentate chelator, has been synthesized from benzimidoyl chloride
moiety and Nꢀmethylthiosemicarbazide (Scheme 1).
3

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N
N
NH2
NH
N
S
Stirring
EtOH
N
N
N
S
S
HN
Cl
S
HN
HN
HN
H L
2
Scheme 1 Synthetic route of H L in this study
2
The discovery of cisplatin in 1965 was a major milestone in cancer chemotherapy, which
forms a PtꢀDNA adducts and ultimately inhibits cellular DNA transcription and replication.
However, usage of cisplatin suffers from unprepossessing side effects such as drug resistance
1
6ꢀ18
and severe toxicity such as nephrotoxicity and neurotoxicity.
So, the new metal based drugs
with novel mechanisms of action are being developed as an alternative to platinum based drugs.
Numerous Co(III), Ni(II), Cu(I/II), Zn(II) and Ru(II) complexes have been probed in quest of
1
9aꢀc
new developed inorganic drugs.
Among the various metal(II) complexes, nickel and copper
ions are considered as the best promising alternatives to cisplatin as anticancer drugs because its
1
9d
play energetic role in biological functions such as DNA, BSA, HSA and RNA interactions.
To find highly effective, target specific and less toxic drugs, much effort has been
devoted to the development of metalꢀbased anticancer agents binding to DNA through a
noncovalent interaction. Three binding modes of metal complexes with DNA exist in a
noncovalent way, electrostatic, groove, and intercalation. Many important applications of these
complexes require that they can bind to DNA via an intercalative mode that could induce cellular
degradation. The planarity of the ligand and the presence of aromatic systems are factors that
2
1a
favor the intercalation. Moreover, most of the recent research on clinical agents has shown that
drugs (metal complexes) not only bind to the prime molecular target DNA, but also strongly
2
1b
interact with serum albumin proteins such as BSA and HSA. It is essential to explore drugꢀ
protein interactions as most of the drugs bound to serum albumin are usually transported as a
protein complex. Attention has also been focused on the proteins that drive and control cell cycle
progression.
4

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On the other hand, the catecholase/phosphataseꢀlike activities of some model
coordination complexes have been a topic of recent interest for the development of new
2
3
bioinspired catalysts. Catechol oxidase is a copperꢀcontaining typeꢀIII activeꢀsite protein
Scheme 2) that catalyzes for twoꢀelectronꢀtransfer reaction during the oxidation of a wide range
(
of catechols known as catecholase activity, while hydrolase belong to a class of metalloenzymes
that catalyzes the hydrolysis of several type of substrates including phosphate esters (4ꢀNPP),
2
4a
peptides and nuclei acids. Most of the researchers have focused on dinuclear systems so far to
match the original enzyme structurally where there exists a dimericꢀcopper active center. Recent
investigations have also shown that some manganese(II/III), nickel(II), zinc(II) and cobalt(II/III)
24b,c
species can also mediate such catechol oxidation.
Therefore, much attention has been focused
on the catecholase/phosphatase like properties of benzamidineꢀthiosemicarbazone containing
nickel(II) and copper(II) complexes.
N
N
N
N
N
O
O
N
CuA
CuB
N
N
N
N
N
N
Scheme 2 Bio active dimeric copperꢀcontaining typeꢀIII protein
Therefore, through this above contribution, we reported the ligand [Nꢀ
diethylaminothiocarbonyl)]benzimidoylchlorideꢀ2ꢀaminoacetophenoneꢀNꢀ methylthiosemicarba
(
ꢀ
zone] (H L) to synthesize new nickel(II) and copper(II) complexes (1, 2) as shown in Scheme 3.
2
Furthermore, the investigation of binding properties with calf thymus DNA (CTꢀDNA) and BSA
protein on the new complexes have been carried out. Moreover H L and its complexes 1, 2 were
2
also investigated for possible catecholase and phosphatase like activities. In addition, cytotoxic
activity of the new compounds towards normal Vero cell line, A375 (human melanoma skin
cancer) human cervical cancer (HeLa) and human hepatocellular carcinoma (Hep3B) cell lines
were evaluated using MTT assay along with cell death analysis by AO/EB and DAPI staining.
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Results and discussion
Synthesis and characterization
The benzamidineꢀthiosemicarbazone ligand H L was synthesized by the reaction of
2
1
0
Nꢀ(diethylaminothiocarbonyl)benzimidoylchloride
with
oꢀaminoacetophenoneꢀ
2
5
thiosemicarbazone in presence of supporting base Et N. The synthesis of nickel(II) and
3
copper(II) complexes 1, 2 were achieved in good yields by the reaction of H L with
2
Ni(OAc) .4H O and Cu(OAc) .H O respectively (Scheme 3). In this reaction, the H L acted as a
2
2
2
2
2
dibasic tetradentate ligand with N S fashion. The new compounds under investigation are
2
2
crystalline and nonhygroscopic solids, airꢀstable in solution and in the solid state at room
temperature, soluble in common organic solvents such as methanol, ethanol, benzene,
chloroform, dichloromethane, acetone, dimethylsulfoxide, dimethylformamide and insoluble in
hexane, petroleum ether and diethyl ether. The complexes have been characterized satisfactorily
1
by elemental analyses, IR, UVꢀvis, HꢀNMR and ESIꢀMS spectral studies. In addition, the
molecular structure of the ligand H L and its complexes 1, 2 was confirmed by single crystal Xꢀ
2
ray crystallography.
N
N
N
N
N
N
N
N
S
NH
N
S
N
N
S
[
Ni(OAc] . 4H O
2 2
[Cu(OAc] . H O
2
2
Ni
Cu
EtOH/CH Cl
EtOH/CH Cl2
2
2
2
S
S
S
N
HN
N
HN
HN
HN
H L
2
1
2
Scheme 3 Synthesis of nickel(II) (1) and copper(II) (2) complexes
IR spectroscopy has been used to confirm the deprotonation and exact binding mode of
the ligand. In the IR spectrum of the ligand, there are three νNH bands were observed in the range
ꢀ1 26
of 3236ꢀ3302 cm . On complexation, two νNH peaks disappeared from the spectra of
complexes indicate the deprotonation of these groups in the ligand and a sharp band at 3390ꢀ
ꢀ1
3
417 cm was assigned to the νNH stretch of the H CꢀNHꢀCꢀS group. A strong vibration
3
6

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ꢀ1
observed at 1668ꢀ1717 cm in the spectrum of ligand corresponding to νC=N groups which were
ꢀ1
shifted to lower frequency in the complexes. A sharp band at 838 cm was attributed to νC=S
group of diethylaminothiocarbonyl moiety in the H L. This was shifted to lower frequencies
2
ꢀ
1
region at 784ꢀ790 cm in the complexes indicates the participation of sulphur in the
ꢀ1
coordination. Furthermore, the one more band at 781 cm was assigned to νC=S group present in
the thiosemicarbazone part of H L, which was completely disappeared in the spectra of
2
ꢀ1
complexes and new band appeared around 761ꢀ764 cm was assigned to νCꢀS indicates the
thioneꢀthiol tautomerism of –NH–C=S group and subsequent coordination of thiolate sulfur after
deprotonation. These clearly confirm the coordination occurs through nitrogen and sulphur
atoms.
1
The clearest characterization of the synthesized compounds that can be seen from the Hꢀ
NMR spectra of ligand H L and its complex 1. The datas are summarized in the experimental
2
1
section and the resulting spectra are depicted as Fig. S1, S2. In the H NMR spectra, the signal
due to the –NH hydrazinic group of ligand appeared at 12.72 ppm which was absent in complex
2
7
supporting the ketoenolization and coordination of thiolate sulphur. The signal due to the C ꢀ
ar
NH group appeared at 8.47 ppm in the spectrum of H L has completely disappeared in the
2
spectrum of complex indicates deprotonation upon complex formation. The signal appeared at
8.10 ppm was attributed to NHꢀCH group of H L, which was shifted to a high field region at
3 2
2
8
4
.85 ppm even after complexation. However, the proton signals of the two methylene groups in
H L and its complex 1, which should consequently be two quartets, appear as four well
2
6
separated multiplet resonances at 3.74ꢀ4.12 ppm. This results in magnetic nonequivalence with
respect to their axial and equatorial positions of four methylene protons as indicated by four
2
9
separated signals with ABX splitting patterns as previously reported. Furthermore two triplet
3
signals region at 1.22ꢀ1.33 ppm for the methyl groups in N(CH CH ) are observed in the spectra
2
3 2
7
of H L and its complex 1. In addition, the spectra of H L and its complex 1 showed a series of
2
2
overlapping multiplets for aromatic protons at 6.40ꢀ7.54 ppm.
The UV–vis spectra of the complexes have been recorded in DMSO solution (0ꢀ48 h)
(Figure S3) are similar, suggesting that the complexes retain their structure in solution within the
timeframe used for the biological experiments. Electronic spectra of the compounds displayed
intense absorptions in the visible and ultraviolet regions. The absorptions in the ultraviolet region
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are assignable to transitions within the ligand orbitals. The bands around 236ꢀ298 nm were
*
*
15a
assigned to ligandꢀcentred (LC) π→π and n→π transitions. The lowest energy absorption
2
+
2+
maxima located in the 367ꢀ374 nm range may be assigned to an S(pπ)→M(dπ) (M = Ni , Cu )
LMCT transition caused by the promotion of the electron from the full HOMO of the ligand of
primarily sulfur pπ character to the empty LUMO of metal ion dπ character. Furthermore, the
complexes showed a broad bands at 431ꢀ491 nm region, which is attributed to the dꢀd transitions
8
9
bands of spinꢀpaired d (complex 1) and d (complex 2) species with a squareꢀplanar structure
Fig. S3). The geometry of the complexes further confirmed by single crystal Xꢀray diffraction
analyses.
The EPR spectrum measured at 9.45 GHz (Xꢀband) of the complex 2 correspond to an
axial gꢀtensor showing broad signals (Fig. S4). The gꢀparameters, g and g , were determined for
(
||
┴
the complex 2, but no coupling parameters could be extracted. The averaged gꢀvalues (g ) were
av
obtained through the relationship between the anisotropic parameter of the gꢀtensor, from the
following expression g = (g + 2×g )/3. The complex 2 exhibits an anisotropic spectra with g =
av
||
┴
||
2
.078, g = 2.031 and g = 2.046 which is consistent with the presence of a square planer
┴ av
geometry as evident from the crystal structure of 2. These parameters are in good agreement with
2
+
9
that of the related square planar Cu systems and are typical of axially symmetrical d copper(II)
15b
complexes (Fig. S4).
The ESIꢀMS analyses of H L and its complexes 1, 2 shows the most abundant peaks at
2
+
m/z, 441.55, 498.42, 503.52 respectively which were assigned to [M + H] ions. The obtained
molecular masses are in good agreement with that of the calculated molecular masses (Fig. S5ꢀ
S7).
Single crystal X-ray diffraction studies
Singleꢀcrystal Xꢀray diffraction studies of compounds (H L, 1 and 2) confirm the
2
conclusions drawn from the spectroscopic studies. To gain insight into the coordination
chemistry and structural parameters of these compounds, single crystals were isolated as good
quality by slow evaporation of a concentrated ethanol/dichloromethane solution and
characterized by Xꢀray diffraction. Details of the data collection, solution and refinement are
gathered in the experimental section and Table 1. The ORTEP view of the molecular structure of
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compounds along with a partial atom numbering scheme is shown in Figure 1ꢀ3 and important
bond lengths and angles for the compounds are summarized in Table 2.
Fig. 1 Perspective view (25% probability ellipsoids) of H L with the atom numbering scheme.
2
Figure 1 depicts the molecular structure of H L. The H L crystallized in the triclinic Pі
2
2
space group. Selected bond lengths and angles are summarized in Table 2. The protonation of the
nitrogen atoms N3 and N5 is experimentally justified by the detection of peaks of electron
density in the final Fourier map, which can be assigned to corresponding hydrogen atoms are
involved intermolecular hydrogen bonding. The N(3)ꢀH(3)ꢀS(1) hydrogen bond forms the
1
1
intramolecular ring of S (6) type. The C–N and C=N bond distances (see Table 2) found in H L
2
agree well with that reported for similar other ligands containing C–N/C=N bonds. The thione
form is confirmed by the bond length of C(1)ꢀS(1) (1.704 Å), C(17)ꢀS(2) (1.674 Å) which are
very close to a formal C=S bond length (1.60 Å). Examination of packing diagram of H L
2
revealed that in the structure exhibit intermolecular πꢀπ stacking interaction between the parallel
C(9)ꢀC(14) ring and its equivalent obtained by (ꢀx, 1ꢀy, ꢀz) symmetry transformation although
the geometrical parameters with distance of 3.575 Å and shift equal to 1.440 Å indicate rather
weak interaction as shown in Fig S8.
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Table 1 Crystal data and structure refinement for ligand H L and its complexes 1, 2
2
H L
2
1
2
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell
C H N S
440.62
100(2) K
0.71073 Å
Triclinic
Pī
C H N Ni S
497.32
295(2) K
0.71073 Å
orthorhombic
Pbca
C H Cu N S
502.15
295(2)
0.71073 Å
orthorhombic
Pbca
22
28
6
2
22 26
6
2
22 26
6
2
dimensions
a (Å)
b (Å)
c (Å)
α (°)
7.831(6) Å
10.956(8) Å
14.357(18) Å
75.594(18)°
89.60(3)°
13.2007(7) Å
17.5298(9) Å
20.1722(11) Å
90°
13.3637(4)
17.6802(7)
19.9696(6)
90°
β (°)
90°
90°
γ (°)
Volume (Å )
72.16(2)°
90°
90°
3
3
3
3
1
2
132.47(5) Å
4668.0(4)Å
8
4718.3(3)Å
8
Z
Density (calcd)
MgM
Absorption
coefficient
F(000)
3
3
3
1
.292 Mg/m
ꢀ1
1.415 Mg/m
1.414 Mg/m
ꢀ3
-1
-1
1.032 mm
1.124 mm
2
.291 mm
468
2080
2088
3
0.12 x 0.07 x 0.03
0.37 x 0.17 x 0.03
3.44 to 25.04°
0.25 x 0.22 x 0.17
Crystal size ( mm )
Theta range for
data collection
Index ranges
3.18 to 73.12°
3.608 to 25.07°
ꢀ9<=h<=8,
ꢀ15<=h<=15
ꢀ20<=k<=20
ꢀ19<=l<=24
16086
ꢀ15<=h<=11
ꢀ21<=k<=14
ꢀ23<=l<=19
13366
ꢀ
ꢀ
13<=k<=12
17<=l<=17
Reflections
collected
12690
Independent
reflections
Data/restraints/para 4299 / 0 / 275
meters
4299 [R(int) =
0.0182]
4122 [R(int) = 0.0666]
4122 / 0 / 284
1.059
4158 [R(int) = 0.0314]
4158 / 0 / 284
1.049
G2oodness-of-fit on 1.132
F
Final R indices
I>2sigma(I)]
R indices (all data) R1= 0.0364,
wR2 = 0.1011
R1= 0.0360,
wR2= 0.1007
R1= 0.0440,
wR2= 0.1096
R1= 0.0607,
wR2 = 0.1180
R1= 0.0372,
wR2= 0.0910
R1= 0.0511,
wR2= 0.0968
[
1
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Fig. 2 Perspective view (25% probability ellipsoids) of complex 1 with the atom numbering
scheme.
1
1

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Table 2 Selected bond lengths (Å) and bond angles (°) for H L and its complexes 1, 2 with the
2
optimized geometrical values
H L
2
Xꢀray DFT
1
Xꢀray
DFT
2
Xꢀray
DFT
C(1)-N(1)
C(1)-S(1)
C(1)-N(2)
C(2)-N(2)
C(2)-N(3)
C(9)-N(3)
C(15)-N(4)
C(17)-N(5)
C(17)-N(6)
C(17)-S(2)
1.339(2) 1.358
1.704(2) 1.784
1.362(2) 1.371
1.309(2) 1.325
1.333(3) 1.356
1.418(2) 1.428
1.274(2) 1.301
1.354(2) 1.386
1.323(2) 1.338
1.674(2) 1.730
N(3)-Ni(1)
S(1)-Ni(1)
N(4)-Ni(1)
S(2)-Ni(1)
N(3)-C(6)
N(3)-C(13)
N(4)ꢀC(19)
S(2)ꢀC(21)
S(1)ꢀC(1)
1.872(2)
2.149(9)
1.900(2)
2.157(9)
1.353(3)
1.409(3)
1.319(4)
1.729(3)
1.717(3)
1.306(4)
1.890 N(3)-Cu(1)
2.262 S(1)- Cu(1)
1.911 N(4)- Cu(1)
2.276 S(2)- Cu(1)
1.354 N(3)-C(2)
1.419 N(3)-C(9)
1.315 N(4)ꢀC(15)
1.805 S(2)ꢀC(17)
1.789 S(1)ꢀC(1)
1.339 N(2)ꢀC(2)
1.932(2)
2.244(7)
1.981(2)
2.230(9)
1.353(3)
1.400(3)
1.308(4)
1.739(3)
1.720(3)
1.308(3)
1.954
2.365
1.997
2.361
1.367
1.412
1.314
1.808
1.789
1.335
N(2)ꢀC(6)
N(1)-C(1)-S(1)
C(2)-N(3)-H(3)
C(15)-N(4)-N(5) 118.5(1) 119.6
N(6)-C(17)-S(2) 122.8(1) 125.7
N(5)-C(17)-S(2) 120.6(1) 118.5
120.5(1) 120.1
116.3(1) 113.2
N(3)ꢀNi(1)ꢀN(4)
N(4)ꢀNi(1)ꢀS(2)
S(2)ꢀNi(1)ꢀS(1)
N(3)ꢀNi(1)ꢀS(1)
N(3)ꢀNi(1)ꢀS(2)
N(4)ꢀNi(1)ꢀS(1)
93.5(1)
92.0
86.7
88.9
92.4
N(3)ꢀCu(1)ꢀN(4) 92.22(9)
N(4)ꢀCu(1)ꢀS(2) 86.67(7)
91.9
85.7
90.5
92.4
169.8
174.2
87.48(2)
85.65(3)
95.32(7)
S(2)ꢀCu(1)ꢀS(1)
89.58(3)
N(3)ꢀCu(1)ꢀS(1) 94.83(7)
168.47(8) 173.0 N(3)ꢀCu(1)ꢀS(2) 166.17(7)
167.35(8) 175.3 N(4)ꢀCu(1)ꢀS(1) 164.80(7)
C(9)-N(3)-H(3)
116.3(1) 116.6
Table 3 Hydrogen bonds for the H L and its complexes 1, 2 [Å and °]
2
Compound
H L
2
D–H….A
N(3)ꢀH(3)ꢀS(1)
d(D–H)
0.86
d(H…A)
2.28
d(D….A)
2.968(2)
<(DHA)
137.2
N(5)ꢀH(5)ꢀS(2)
N(6)ꢀH(6)ꢀN(4)
C(2)ꢀH(2A)ꢀS(1)
C(20)ꢀH(20A)ꢀN(5) 0.96
C(16)ꢀH(16B)ꢀN(5) 0.96
C(19)ꢀH(19B)ꢀS(1) 0.97
0.86
0.86
0.97
2.61
2.19
2.57
2.24
2.21
2.56
3.351(2)
2.577(2)
2.963(3)
2.600(5)
2.611(4)
2.949(3)
145.2
106.9
104.3
101.2
103.9
104.2
1
2
Symmetry operation: H L: : 'x, y, z'; 'ꢀx, ꢀy, ꢀz', Complex 1: 'x, y, z''ꢀx+1/2, ꢀy, z+1/2''x+1/2,ꢀ
2
y+1/2,ꢀz''ꢀx, y+1/2, ꢀz+1/2''ꢀx, ꢀy, ꢀz''xꢀ1/2, y, ꢀzꢀ1/2''ꢀxꢀ1/2, yꢀ1/2,z''x, ꢀyꢀ1/2, zꢀ1/2', Complex 2:
x, y, z''ꢀx+1/2, ꢀy, z+1/2''x+1/2,ꢀy+1/2,ꢀz''ꢀx, y+1/2, ꢀz+1/2''ꢀx, ꢀy, ꢀz''xꢀ1/2, y, ꢀzꢀ1/2''ꢀxꢀ1/2, yꢀ
'
1/2,z''x, ꢀyꢀ1/2, zꢀ1/2'. [D= donator, A=acceptor]
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Fig. 3 Perspective view (25% probability ellipsoids) of complex 2 with the atom numbering
scheme.
A perspective view of the complexes 1 and 2 with the atomic numbering scheme is
depicted in Fig. 2, 3, while selected bond lengths and angles are given in Table 2. The complexes
1
, 2 crystallized in the triclinic belonging to the Pbca space group. The metal ion adopted a
square planer geometry with bonding of the H L as dibasic tetradentate (SNNS) donors. The
2
o
o
trans angles are N(3)ꢀNi(1)ꢀS(2), 168.47(8) and N(4)ꢀNi(1)ꢀS(1), 167.35(8) (for 1) and N(3)ꢀ
o
o
Cu(1)ꢀS(2), 166.17(7) and N(4)ꢀCu(1)ꢀS(1), 164.80(7) (for 2) which showed a deviation from
the expected linear trans geometry, suggesting distortion in the squareꢀplanar coordination
geometry. The metal bond distances NiꢀN, NiꢀS in complex 1 and CuꢀN, CuꢀS in complex 2
31a,b
(Table 2) are well agree with reported nickel(II) and copper(II) complexes.
Furthermore,
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there exists intermolecular hydrogen bonding between the the atom (C–H⋯N/S contact) of the
H L and its complexes 1, 2 showed in Table 3. The unit cell packing diagram of compounds
2
along with hydrogen bonding is given in Fig. S9.
Theoretical calculations
HOMO and LUMO are referred to as frontier orbitals. The energy difference between the
HOMO and LUMO is termed as HOMO–LUMO energy gap. The difference in energy between
two frontier orbitals can be used to predict the strength and stability of transition metal
complexes as well as the colors they produce in solution. The calculations using the density
functional theory (DFT) method of GAUSSIANꢀ09 were carried out to electronic structure of
3
2
compounds. A molecule with a small gap is more polarized and is known as soft molecule. The
lowering of the HOMOꢀLUMO band gap is essentially a consequence of the large stabilization
of the LUMO due to the strong electronꢀaccepting ability of the electronꢀacceptor group.
The molecular structure of the H L and its complexes 1, 2 the deprotonated forms were
2
characterized through computational studies in a solid phase. Contour plots of frontier molecular
orbitals of the complexes and the energy gap was also calculated (Fig. 4). Positive and negative
regions are shown in red and blue colors respectively, and the geometrical parameters have
already been given in Table 1. In general, there is a good correlation between the optimized
structures and Xꢀray crystal structures of H L and its complexes 1, 2. The perfect square planer
2
geometries of the complexes are also reflected in their calculated structures. It is seen that the
complexes, the electron density of the HOMO is localized largely on the metal ion surrounding
(N, S atom) only and to a very smaller extent from the ligand, while in the LUMO major
contributions from ligand and to a lesser extent from the metal ion surrounding without relevant
contributions. So the electron transfer occurs from the highest occupied molecular orbital
(HOMO) to the lowest unoccupied molecular orbital (LUMO). It can be inferred that the electron
transfer in complexes is related to LMCT. The calculated HOMO energies of the complexes vary
as H L (ꢀ5.68 eV) < 1 (ꢀ5.25 eV) < 2 (ꢀ5.31 eV) and those of LUMO exhibit a similar trend: H L
2
2
(
ꢀ2.04 eV) < 1 (ꢀ2.15 eV) < 2 (ꢀ2.05 eV). The HOMO–LUMO energy gaps in compound H L
2
(3.64 eV), complex 1 (ꢀ3.12 eV) and 2 (ꢀ3.20 eV) are almost the small different, which is
consistent with the experimentally observed order of biological efficiencies of complexes. The
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4

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calculated atomic charge, electronic configuration of donor atoms and metal centered atoms are
listed in Table 4.
H L
2
1
2
HOMO = E = ꢀ5.68 eV
E= ꢀ3.64 eV
HOMO = E = ꢀ5.27 eV
E= ꢀ3.12 eV
HOMO = E = ꢀ5.25 eV
E= ꢀ3.20 eV
LUMO = E = ꢀ2.04 eV
LUMO = E = ꢀ2.15 eV
LUMO = E = ꢀ2.05 eV
Fig. 4 Frontier molecular orbitals of compounds and their HOMOꢀLUMO energy gap and
B3LYP/6ꢀ311G ground state optimized geometry of compounds (top).
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Table 4 Charges (a.u.) and electron configurations of atoms for complexes 1, 2
Complex 1
Complex 2
Charge
eV)
Charge
(eV)
Atom
Electronic configuration
Electronic configuration
(
[
5
[
4
[
3
[
4
core]4S(0.41)3d(8.60)4p(0.02)
S( 0.02)
core]2S( 1.32)2p(
.31)3p(0.01)
core]2S( 1.35)2p( 4.00)
p( 0.02)
core]3S(1.80)3p(4.47)4S(0.01)
p(0.01)
[core]4S(0.43)3d(9.35)4p(0.01)5S
(0.01)
[core]2S( 1.33)2p(4.37)
3p(0.01)
[core]2S( 1.36)2p( 4.04)
3p(0.02)
[core]3S(1.81)3p(4.52)4S(0.01) 4p
(0.01)
Ni/Cu 0.95397
N1(6) ꢀ0.65053
N2(7) ꢀ0.37386
S1(2) ꢀ0.28649
1.19451
ꢀ0.72124
ꢀ0.42138
ꢀ0.34276
[
4
core]3S(1.82)3p(4.58)4S(0.01)
p( 0.01)
C1(11) 0.34264 [core]2S( 0.86)2p(2.77)3p(0.02) 0.34451 [core]2S( 0.86)2p( 2.77)3p(0.02)
S2(3) ꢀ0.36154 [core]3S( 1.81)3p(4.53)4p(0.01) ꢀ0.42362
[
3
core]2S(0.86)2p(2.79)3S(0.01)
p(0.03)
[core]2S(0.87)2p(2.79)3S(0.01)3p
(0.03)
C2(39) 0.31027
0.30966
From the assumption of the Table 4, the electron populations on s and p orbitals of
nitrogen and sulfur donor atoms in both complexes are less than the expected values of valence
orbitals, while the computed electron population in the central ion in both complex 1, 2 is more
2
+
8
9
33
than the expected value in M (Ni, Cu) with d , d electronic configuration respectively. The
calculated formal charge on the metal ion (1 = 0.954 eV; 2 = 1.195 eV) in the complexes was
lower than the formal charge +2, confirming a significant charge donation from the ligand H L.
2
This confirms the electron transmission of donor atoms toward the central metal. The calculated
electronic configurations (Table 4) of the donor atoms with reference to s and p orbitals are
consistent with electron donation towards the metal ion.
DNA interaction studies
Stability of compounds
Before to carry out DNA binding studies, the stability of the complex is paramount
for its application. The stability of the selected compounds (H L, 1 and 2) has been detected
2
in TrisꢀHCl buffer (5 mM TrisꢀHClꢀ50 mM NaCl buffer, pH = 7.2) at four different time
points (0 min, 1 hr, 6 hr and 12 hr) through the UV absorption method. The UVꢀvis spectral
results did not reveal any appreciable change in either the intensity or the position of the
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6

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absorption bands in TrisꢀHCl buffer solution (Fig. S3). This demonstrates that the
compounds keep a stable structure under physiological conditions.
Fluorescence emission titration
DNA is often a vital target to mediate the targeted apoptosis or necrosis to the cells
replication. So the binding affinity of the metal complexes to CTꢀDNA was used as primary
studies for chemotherapeutic agents. These studies were carried out different spectral
methods. Considered as a basic testing method, fluorescence spectroscopy is one of the most
3
4
commonly used techniques for exploring the interaction of any drug with CTꢀDNA. This
technique is commonly used to study the binding mode (and their likely nature) between DNA
and metal complexes. The emissive titration studies have been performed by monitoring the
changes in emission intensity by aliquot addition of DNA. Usually, intercalation mode of
interaction between the metal complexes and DNA results in hypochromism with or without
red/blue shift; on the other hand, nonꢀintercalative/electrostatic interaction causes
hyperchromism. Therefore, typical emissive titration curves for the selected compounds in
the absence and presence of CTꢀDNA at different increasing concentrations were
implemented.
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Fig. 5 Fluorescence titrations of H L, 1 and 2 (25 ꢁM) with CTꢀDNA (0ꢀ50 ꢁM) (left).
2
Scatchard plots of r/Cf vs r for selected compounds (right).
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8

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Table 5 Fluorescence spectral parameters for compounds H L, 1 and 2 bound to CTꢀDNA
2
Compound
Kb
Ksv
Kapp
3
4
4
3
3
5
5
H L
4.30×10
9.3x10
8.01x10
2
4
4
1
2
8.50×10
4.49×10
1.82x10
2.17x10
3.21x10
3.45x10
From the emissive titration spectra (Fig. 5), it is apparent that upon the addition of
increasing concentration of CTꢀDNA (0ꢀ50 ꢁM) to a solution of H L displayed
2
hypochromism (2.41%) at 304 nm along with small red shift (∼2 nm). Complexes 1, 2 shows
different behavior relative to those of the ligand and exhibits the band at 406 and 528 nm
showed hypochromism of about 36.36 and 53.27% with a small red shift respectively (∼4
nm). The observed hypochromism is due to an intercalative mode of binding involving a
strong stacking interaction between extending aromaticity of the ligand and the base pairs of
3
5
DNA. The binding of compounds to DNA either leads to hypochromism, which provides a
measure of the strength of the intercalative. Based on the emission enhancement, the intrinsic
binding constant was obtained according to the Scatchard equation:
C = C (F – F ) / F – F0)
b
t
0
max
here, C is the total compound concentration, F being the observed fluorescence emission
t
intensity at given DNA concentration, F being the intensity in the absence of DNA and Fmax is
0
the fluorescence of the totally bound compound. The fluorescence spectra of the tested
compounds with increasing concentration of CTꢀDNA are depicted in Fig. 5. Intrinsic binding
constant (K ) were cast in the form of a Scatchard plot of r/C versus r, where r is the binding
b
f
ratio of C / [DNA] and C is the free ligand concentration. The intrinsic binding constant (K )
b
f
b
values for the interaction of H L, 1 and 2 with CTꢀDNA are listed in Table 5. The binding
2
3
4
−1
constant (K ) of the compounds was in the range of 4.30 × 10 to 8.50 × 10 M . However, the
b
complexes interact with CTꢀDNA more strongly than the ligand. The observed values of Kb
revealed that the compounds bind to DNA via intercalative mode, where the order of DNAꢀ
binding affinity is 1 > 2 > H L. However, the binding mode needs to be proved through some
2
more experiments.
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Ethidium bromide displacement study
Fluorescenceꢀquenching experiments with EBꢀbound DNA was undertaken to understand
the mode of DNA interaction with the metal complexes. The molecular fluorophore EB emits
intense fluorescence at about 600 nm in the presence of DNA due to its strong intercalation
3
6
between adjacent DNA base pairs. Addition of a second molecule, which bound to DNA more
strongly than EB, (actually the fluorescence intensity of EB increases by almost 20ꢀfold after
binding to DNA) would displace bound EB and quenched the DNAꢀinduced EB emission. The
extent of quenching of the fluorescence of EB bound to DNA would reflect the extent of DNA
3
7
binding of the second molecule. From the above fact in mind, as the concentration of the H L
2
and its complexes 1, 2 increases, the emission band at 604 nm (Fig. 6) for EB exhibited
hypochromism up to 26.97, 47.95 and 31.41% with a slight red/blue shift from the initial
fluorescence intensity respectively. The quenching of emission intensity of ethidium bromide
upon addition of compounds shows that the complexes probably compete with EB for the
binding with DNA. Further quantitative assessment of the magnitude of interaction was
ascertained by the classical Stern−Volmer equation:
I /I = 1 + K [Q]
0
sv
where I and I are the emission intensities of EB bound CTꢀDNA in the absence and presence of
0
the quencher (complexes) concentration [Q], respectively, which gave the SternꢀVolmer
quenching constant (K ). The K value is obtained with a slope from the plot of I /I versus [Q]
sv
sv
0
were shown in Fig. 6. The quenching constant (K ) values were obtained from the slope, thus
sv
indicating that the complexes strongly bind to DNA. The quenching constant (K ) values were
sv
obtained from the slope, which were listed in Table 5.
2
0

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Fig. 6 Fluorescence titrations of H L, 1 and 2 (0ꢀ50 µM) with EB bound CTꢀDNA (7.5 µM)
2
(
left). Scatchard plots of I /I vs Q for selected compounds (right).
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Further the apparent DNA binding constant (K ) values were also calculated using the
app
following equation:
K [EB] = K [M50%
]
EB
app
ꢀ7
ꢀ1
where KEB = 1.0 × 10 M is the DNA binding constant of EB, [EB] is the concentration of EB
7.5 ꢁM) and [M50%] is the concentration of the compound used to obtain a 50% reduction in
(
fluorescence intensity of DNA pretreated with EB. The Kapp value for the ligand and their
complexes 1 and 2 were given in Table 5. The DNA binding ability of the compounds follows
the order 1 ~ 2 > H L, which is consistent with the results obtained from the above fluorescence
2
spectral studies.
Viscosity measurement
This technique give us useful information on how the conformation of DNA is influenced
by the binding mode of the metal complex to DNA. In general, the viscosity of CTꢀDNA
increases when a compound binds with DNA in intercalating mode but remains unchanged when
3
8
a compound binds with DNA in electrostatic mode. When the H L and its complexes 1, 2 were
2
treated with CTꢀDNA (200 ꢁM), the concentrations of compounds (0ꢀ120 ꢁM) increased from a
ratio of R = 0ꢀ0.1(1/R = [compound]/[DNA]), the relative viscosity of DNA enlarged steadily in
the order 2 > 1 > H L. The relative viscosity of DNA solution exhibits an increase upon addition
2
of all compounds and it is more pronounced in the case of the complexes (Fig. 7). The observed
behavior of the DNA viscosity upon addition of the compounds may be considered evidence of
the existence of an intercalative binding mode to DNA, a conclusion which clarifies the
preliminary indications derived from fluorescence spectroscopic studies.
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2

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1
/3
Fig. 7 Relative viscosity (η/η0) of CTꢀDNA in TrisꢀHCl buffer solution in the presence of
tested compounds at increasing amounts (r = 0ꢀ0.1).
BSA interaction studies
Fluorescence spectral study
Interaction between the most abundant blood protein, that is, serum albumin, and
complexes have attracted immense current interest because of their structural homology with
3
9a
human serum albumin.
To understand the mechanism of interaction between selected
compounds and BSA, fluorescence quenching experiments have been carried out. The
fluorescence spectra of BSA showed a broad band with a maximum at ~340 nm. The relative
3
3
ratio of fluorescence intensity for three amino acids and the functions were reported earlier.
Therefore, the fluorescence behavior of BSA can provide significant information about the
structure, dynamics, and protein folding. A solution of BSA (1 µM) was titrated with various
increasing concentrations of the compounds H L, 1 and 2 (0ꢀ50 µM) in the range of wave length
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2
90ꢀ430 nm (λ = 280 nm). Fig. 8 showed that the effect of increasing concentration of
ex
compounds on the fluorescence emission of BSA.
Fig. 8 Fluorescence titrations of H L, 1 and 2 (0ꢀ50 ꢁM) with BSA (1 ꢁM) (left). SternꢀVolmer
2
plots of I /I vs Q and Scatchard plots of Log[IoꢀI/I] vs Log[Q] for selected compounds (right).
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4

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Table 6 Quenching parameters of BSA for compounds H L, 1 and 2
2
Compound
Ksv
Kq
Kbin
‘n’
1.5
5
5
5
13
13
13
7
9
9
H L
2
1.83x10
1.83x10
6.05x10
1
2
1.19x10
1.02x10
1.19x10
1.02x10
1.34x10
1.44x10
1.9
1.8
The extent of quenching of fluorescence intensity, as expressed by the value of Stern–
Volmer constant (K ), is a measure of protein binding affinity of the complexes. To have a deep
sv
insight into the quenching progression, quenching constant (K ) was evaluated from following
q
SternꢀVolmer equation:
I /I = 1+ K [Q] = 1+ K τo [Q]
0
sv
q
where I and I are the fluorescence intensity in the absence and presence of the quencher,
0
respectively. k is the quenching rate constant, τo is the fluorescence life time of biopolymer
q
ꢀ8
39b
BSA ( o
τ = 10 s) , K and [Q] are the SternꢀVolmer quenching constant and the concentration
sv
of the quencher respectively. Quenching constant (K ) have been found from the plot of log (I -I)
q
0
1
2
/
I versus log [Q] (Fig. 8). It can be seen that the K value of BSA is higher than 1.0 × 10 ,
q
which is much higher than the molecular fluorescence diffusing constant as shown in Table 6. So
the foregoing fluorescence quenching mechanism of the compoundsꢀBSA system cannot be
dynamic quenching. It may be static quenching. Nevertheless, the hypothesis needs to be proved
through some more experiments.
UV-Visible spectral study
Furthermore it has been proved that the fluorescence quenching mechanism of the
compound is static quenching by UVꢀvis absorption spectral studies. The fluorescence quenching
mechanisms are usually classified as either static or dynamic quenching. Static quenching
usually results from the formation of a complex between the quencher and the fluorophore in the
ground state, whereas in dynamic quenching the fluorophore and the quencher get in touch with
4
0
each other during the transient existence of the excited state. An effective method to identify
the type of quenching was UVꢀvisible absorption spectroscopy. UVꢀvisible spectra of BSA in the
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absence and presence of the compounds (Fig. 9) showed that the absorption intensity of BSA
was enhanced as the complexes were added, and there was a little blue shift. It revealed that
there existed a static interaction between BSA and the added complexes due to the formation of
the ground state complex of the type of BSAꢀcompound.
Fig. 9 Absorbance spectra of the compounds H L, 1 and 2 with BSA.
2
Binding constant and binding site number
The binding constant K was calculated using the Scatchard equation
b
Log [I -I / I] = log K + n log [Q]
0
bin
where, Kbin is the binding constant of the compound with BSA and ‘n’ is the number of binding
sites. The number of binding sites (n) and the binding constant (K ) have been found from the
bin
4
1
plot of log (I -I) / I versus log [Q] (Fig. 8). The calculated K , and n values are given in Table
0
bin
6. The calculated value of ‘n’ is around 2 indicating the existence of two binding site available in
BSA for all of the compounds. The higher values of K and K indicates a strong interaction
q
bin
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between the BSA protein and the complexes over the ligand used in this study. It is noticed that
the values of binding constant of the compounds follows the order 2 > 1 > H L, which is
2
consistent with the results obtained from above binding affinity studies.
Catechol oxidase activity
3
,5ꢀdiꢀtertbutylcatechol (3,5ꢀDTBC) was employed as a substrate to study the catalytic
performance of the H L and its complexes 1, 2 mainly because its low reduction potential makes
2
it easy to oxidize and the bulky tertꢀbutyl groups prevent further over oxidation such as ring
4
2
opening and it is shown a low quinoneꢀcatechol reduction potential (Scheme 4). The reactions
were carried out at room temperature in aerobic conditions and were monitored by the
fluorescence spectroscopic technique. Moreover, the oxidation product of 3,5ꢀdiꢀ
tertbutylbenzoquinone (3,5ꢀDTBQ) is highly stable and exhibited a characteristic emission
ꢀ4
intensity at λemis = 437 nm (λ = 401) in pure DMSO. To monitor the reaction, a 1 × 10 M
ex
solution of selected compounds were treated with 100 equivalents of 3,5ꢀDTBC. Upon the
addition of the catecholic substrate, a new band (Fig. 10) was gradually appeared at about 437
nm in time due to the formation of the oxidized product 3,5ꢀDTBQ. Thus, the experiment clearly
proves that the oxidation of 3,5ꢀDTBC to 3,5ꢀDTBQ which is catalyzed by the synthesized
compounds, as it is well established that 3,5ꢀDTBQ shows a maximum at λemis = 437 nm in pure
DMSO.
OH
OH
O
O
Catalyst
DMSO, O2.
Scheme 4 Catalytic oxidation of 3, 5ꢀDTBC to 3, 5ꢀDTBQ in DMSO in the presence of oxygen.
Kinetic study of the catecholase activity
To understand the kinetic aspects of catalysis for tested compounds, the rate constant for
compounds as catalyst was determined by traditional initial rate methods (detailed description
provided in the experimental section). The catalytic behavior shows saturation kinetics, and a
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treatment based on the Michaelis–Menten model seemed to be appropriate under excess substrate
43a
conditions. The observed rate constants (k ) were extracted by the initial rate method. Plots of
i
ki vs. [3,5ꢀDTBC] gave nonꢀlinear curve of decreasing slope (Fig. S10) which are best described
by the following equations.
k1
k
2
CS
C + P
C + S
k-1
k
-2
Vmax[S]
KM + [S]
V =
1
K
V
M
1
1
=
+
max [S]
Vmax
V
A plausible mechanism for 3,5ꢀDTBC oxidation promoted by metal(II) complexes is
schematically depicted in Scheme 5. Unfortunately we were unable to characterize the
intermediates. However, when a mixture of starchꢀpotassium iodide solution was added to a
mixture of complex and 3, 5ꢀDTBC, blue coloration developed, which indicates that hydrogen
peroxide was produced during the course of reaction. It is interesting to note that no blue
coloration was observed in absence of 3,5ꢀDTBC. A believable mechanistic path of the
formation of H O as byꢀproduct during the oxidation of 3,5ꢀDTBC to 3,5ꢀDTBQ catalyzed by
2
2
4
3b
metal(II) compounds was suggested by Chyn and Urbach.
2
8

Page 29 of 53
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DOI: 10.1039/C5RA18568H
tBu
+
N
N
2
N
S
tBu
HO
ꢀH+
M
1
/2 H O
OH
2
2
1e
N
N
S
HN
N
N
N
N
t
Bu
N
N
S
HO
ꢀO
N
S
M
M
S
But
tBu
tBu
N
S
N
O
O
O
N
1
/2 O + H+
1e
2
HN
HN
1
/2 H O
2 2
ꢀO
But
But
ꢀ
H+
N
N
N
N
tBu
+H+
tBu
ꢀ
H+
N
N
S
O
N
N
S
O
M
M
S
ꢀO
tBu
SH
ꢀO
tBu
N
N
HN
HN
Scheme 5 Plausible mechanism for oxidation of 3,5ꢀDTBC by complexes 1, 2
2
9