Syntheses, structures, optical properties and biological activities of bimetallic complexes

Article abstract of DOI:10.1039/c4ra11991f

A bimetallic Schiff base-metal complex [Ni(L)Na(CH₃CN)(ClO₄)] (1) and a coordination polymer [Zn₂(L)(N₃)₂]_n (2) (H₂L = N,N′-bis(3-methoxysalicylidehydene)cyclohexane-1,2-diamine) have been synthesized and characterised by IR, elemental analyses and single crystal X-ray diffraction. The solid state structure reveals that complex 1 is a heterometallic Ni(ii)-Na(i) compound and complex 2 is a homometallic coordination polymer of Zn(ii) bridged by azide anions. The ligand coordinates to the Ni(ii) ion or one of the Zn(ii) ions through two imine nitrogen atoms and two phenolic oxygen atoms in 1 and 2, respectively. The Na(i) ion is coordinated by four oxygen atoms of the phenoxo and methoxy groups of the ligand. This is further bonded to perchlorate in 1. The optical properties of 1 and 2 have been investigated. The cellular toxicity, cellular uptake and DNA binding of complex 1 have been explored.

Full text of DOI:10.1039/c4ra11991f

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Syntheses, structures, optical properties and
biological activities of bimetallic complexes†
Cite this: RSC Adv., 2014, 4, 64725
Mamata Mahato,a Dhriman Dey,b Souvik Pal,a Sudeshna Saha,a Abhrajyoti Ghosh,c
d
a
*
Klaus Harms and Hari Pada Nayek
A
bimetallic Schiff base-metal complex [Ni(L)Na(CH3CN)(ClO4)] (1) and
a
coordination polymer
[Zn2(L)(N3)2]n (2) (H2L
¼
N,N0-bis(3-methoxysalicylidehydene)cyclohexane-1,2-diamine) have been
synthesized and characterised by IR, elemental analyses and single crystal X-ray diffraction. The solid
state structure reveals that complex 1 is a heterometallic Ni(II)–Na(I) compound and complex 2 is a
homometallic coordination polymer of Zn(II) bridged by azide anions. The ligand coordinates to the Ni(II)
ion or one of the Zn(II) ions through two imine nitrogen atoms and two phenolic oxygen atoms in 1 and
2, respectively. The Na(I) ion is coordinated by four oxygen atoms of the phenoxo and methoxy groups
of the ligand. This is further bonded to perchlorate in 1. The optical properties of 1 and 2 have been
investigated. The cellular toxicity, cellular uptake and DNA binding of complex 1 have been explored.
Received 8th October 2014
Accepted 19th November 2014
DOI: 10.1039/c4ra11991f
www.rsc.org/advances
with different polydentate organic ligands.11,12 One of these
ligands is Schiff base ligands which have been utilized to prepare
Introduction
several biologically important metal complexes. In this context,
metal complexes of ‘salen-type’ Schiff bases, which are formed
by the condensation of salicylaldehyde or various salicylalde-
hyde derivatives with diamines have been recently investigated
for their biological activities. For instance, Mn(III)–salen and
salophen complexes have been used to investigate their
apoptosis and anti-tumour activities towards cancer cells.13,14
Several Fe(III)–salen complexes have also shown to be effective in
DNA damage in human cells.15 Salen and salophen ligands form
complexes with metal with d10 electronic conguration (for
example Zn2+) and such complexes have been used as uores-
cence probes and uorescent marker in cells.16–18 Cytotoxicity
and DNA binding ability of several bimetallic homo- or hetero-
nuclear complexes have also been investigated.10,19–21
One of our recent research interests is to synthesize poly-
nuclear metal complexes using Schiff base ligands and inves-
tigate their catalytic, magnetic, optical properties as well as
biological activities. In this work, we have chosen a salen-type
ligand H2L, synthesized from the condensation of 1,2-dia-
minocyclohexane with o-vanillin.22 Various coordination modes
of H2L are shown in Scheme 1.
The design and synthesis of effective metal-based pharmaceu-
ticals has been an active research area since the landmark
discovery of cisplatin and its usefulness for the treatment of
cancer.1–3 Presently, cisplatin, carboplatin and oxaliplatin are
most widely used chemotherapeutic agents. However, their
numerous side effects and ineffectiveness because of frequent
development of resistance limit their applications as chemo-
therapeutic agents.4–6 Therefore, a great attention has been
drawn to design and synthesis of novel metal-based drugs with
enhanced efficiency and minimal side effects. These anti-cancer
drugs interact with DNA of cancer cells and block the division of
cancer cells, thus causing death of the cells. The mode of
interactions are of these drugs with DNA is classied as covalent
binding and non-covalent interactions.7–9 Of course, the mode of
interactions and pharmacological activities can be regulated by
the nature of metal ion and the organic ligands encapsulating
the metal ion.10 Therefore, recent research works have been
focused to investigate anticancer agents based on bio-essential
metal ions such as nickel(II), zinc(II), copper(II) and iron(III)
aDepartment of Applied Chemistry, Indian School of Mines, Dhanbad-826004,
Jharkhand, India. E-mail: hpnayek@yahoo.com
bDepartment of Biochemistry, University of Calcutta, Kolkata-700019, West Bengal,
India
cDepartment of Biochemistry, Bose Institute, Centenary Campus, Kolkata-700054,
West Bengal, India
dFachbereich Chemie, Philipps Universitat Marburg, Hans Meer-wein Strasse,
¨
Marburg, Germany
† Electronic supplementary information (ESI) available. CCDC 1026191 and
1026192. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c4ra11991f
Scheme 1 Different coordination modes of H2L.
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Fig. 1 Molecular structure of 1 and 2. Hydrogen atoms are omitted for
clarity. Selected bond lengths [A˚] and bond angles [ꢁ]; 1: Ni1–O1
1.843(3), Ni1–O2 1.844(3), Ni1–N1 1.858(4), Ni1–N2 1.850(4), Na1–O1
2.345(4), Na1–O2 2.340(4), Na1–O3 2.476(4), Na1–O4 2.561(5), Na1–
O6 2.357(5), Na1–N3 2.470(5), O1–Ni1–O2 83.10(14), N1–Ni1–N2
86.69(18), O1–Na1–O2 62.95(12), O1–Na1–O3 64.67(14), O2–Na1–
O4 63.61(12), N3–Na1–O6 92.37(18); 2: Zn1–O1 2.006(5), Zn1–O2
2.006(5), Zn1–N1 2.039(6), Zn1–N2 2.027(6), Zn1–N3 2.027(7), Zn2–
O1 2.069(6), Zn2–O2 2.080(5), Zn2–N3 2.026(7), Zn2–N6 1.942(8),
O1–Zn1–O2 78.8(2), N1–Zn1–N2 81.9(2), O1–Zn2–O2 75.75(19), N3–
Zn2–N6 109.3(3).
Scheme 2 Syntheses of complex 1 and 2.
We report that a discrete bimetallic complex [Ni(L)Na(CH3-
CN)(ClO4)] (1) and a coordination polymer [Zn2(L)(N3)2]n (2)
were synthesized by the reaction of H2L with nickel(II) acetate
tetrahydrate or zinc(II) nitrate hexahydrate respectively (Scheme
2). Herein we describe the synthesis, solid-state structure,
optical properties of complexes 1 and 2. The cellular toxicity,
cellular uptake and DNA binding studies of complex 1 have also
been reported.
and two oxygen atoms (O3 and O4) of the –OMe groups. The
sodium ion (Na1) is further coordinated by a nitrogen atom (N3)
of an acetonitrile solvent molecule and an oxygen atom (O6) of
the perchlorate anions and thus results a six coordinated
geometry environment around Na1 atom. The perchlorate ion
[ClO4]ꢀ is bonded to Na1 with mono-dentate fashion leaving the
other oxygen atoms inert. The Ni–O and Ni–N bond lengths are
Results and discussion
A Schiff base ligand (H2L) has been synthesized by reacting 1,2-
diaminocyclohexane with o-vanillin in ethanol according to
literature procedure.22 Complexes 1 and 2 were obtained by
reacting H2L with nickel acetate tetrahydrate and sodium
perchlorate (1) or zinc nitrate hexahydrate and sodium azide (2)
in acetonitrile or methanol respectively. Detailed syntheses
procedures are provided in the Experimental section.
˚
˚
in the range of 1.843(3)–1.844(3) A and 1.850(4)–1.858(4) A
respectively, which are comparable with the average Ni–O (1.845
˚
˚
A) and Ni–N (1.849 A) bond lengths obtained from crystal
structure database (CSD) and full agreement with the recent
reported values.23–25 The Ni1/Na1 non bonded distance is
˚
3.3709 A. The solid-state structure of 2 reveals that there are two
All the compounds are characterized by standard analytical
techniques. A strong band at due to the azomethine (C]N)
group at 1620 and 1628 cmꢀ1 are observed in 1 and 2 respec-
tively. The bands in the range of 2928–2937 cmꢀ1 are due to
alkyl C–H bond stretching of the methoxy (–OMe) groups for the
complexes 1 and 2. The characteristics bands for perchlorate
ion are appeared at 1080 and 1101 cmꢀ1 in 1. The splitting of
the band is indicative of the presence of coordinated perchlo-
rate ion in 1, as also supported by the solid-state structure. The
solid-state structures of all the compounds were established by
single crystal X-ray diffraction. Complex 1 and 2 crystallize in
the monoclinic space group Cc and P21/c with four formula
units per unit cell respectively. Fig. 1 and 2 shows the molecular
structure of 1 and 2 respectively. Table 1 provides the details of
crystallographic measurement. Single crystal structure deter-
mination reveals that Ni1 is occupying the N2O2 cavity of the
ligand and coordinated by two nitrogen atoms (N1 and N2) and
two phenoxo oxygen atoms (O1 and O2) of the ligand. This
results in square planer geometry around Ni1. A sodium ion
crystallographically independent zinc(II) ions (Zn1 and Zn2).
Zn1 possesses a distorted square pyramidal geometry and
coordinated by two nitrogen atoms (N1 and N2), two oxygen
atoms (O1 and O2) of N2O2 cavity of the Schiff base ligand and
one nitrogen atom (N3) of azide anion (N3ꢀ).
Fig. 2 Fragment of coordination polymer of 2. Hydrogen atoms are
(Na1) is coordinated by two phenolic oxygen atoms (O1 and O2) omitted for clarity.
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Table 1 Single crystal data and structure refinement parameters for
complexes 1 and 2
Compound
1
2
Formula
Formula mass
T/K
C
24H27Cl1N3O8Ni1Na1 C22H24N8O4Zn2
602.63
100(2)
0.71073
595.23
100(2)
˚
l/A
0.71073
0.34 ꢂ 0.06 ꢂ 0.06
Monoclinic
P21/c
11.307(2)
17.304(2)
11.837(2)
103.41(2)
2252.9(6)
4
Crystal dimensions/mm 0.41 ꢂ 0.34 ꢂ 0.21
Crystal system
Space group
Monoclinic
Cc
˚
a/A
11.394(13)
29.490(4)
9.154(10)
123.07(4)
2577.8(5)
4
˚
b/A
˚
c/A
b/ꢁ
3
˚
Fig. 3 UV-vis spectra of complexes 1 and 2.
V/A
Z
Dc/g cmꢀ3
1.553
0.927
1.755
2.179
m/mmꢀ1
broblast cell (Vero 76) cells, it was necessary to assess the
toxicity of the compounds. Cytotoxicity was evaluated by MTT
experiments following a protocol detailed in the Experimental
section using Vero 76 and HeLa cell lines. Incubated cells were
seeded in 96-well plates at a density of 1 ꢂ 104 cells per well
density for 24 h. The cytotoxicity has been evaluated with
respect to negative control DMSO (1%) using SDS as a positive
control (with a growth-inhibition value above 90%). At lower
concentration (0–10 mM) complex 1 showed no cytotoxic effect
on the cells. Further increment of the concentration resulted in
gradual increase in cytotoxic effect on the cells (data not
shown). However, above 25 mM concentration of complex 1, it
became very difficult to perform the assay mainly due to
precipitation of the compound over and around the cells
resulting into rapid cell death. It is most likely that above a 25
mM concentration the cytotoxicity does not originate exclusively
from the interaction between the metal-containing compounds
and the DNA but also from the excess of complex 1 that covers
the cells (outside the cell membrane), which does not allow for
proper growth. In case of complex 2, due to its little solubility
cytotoxicity assay couldn't be performed. The growth-inhibition
percentages obtained at low concentrations (0.05–50 mM) of
complex 1 didn't allow the calculation of the IC50 values.
Cytotoxicity analysis revealed similar scenario in both the tested
cell lines.
For in vitro DNA interaction analysis, increasing concentra-
tion of complex 1 was incubated with 1 mg of supercoiled pUC19
plasmid DNA. It has been observed that for complex 1, inter-
action with DNA has increased with increasing concentrations
with highest interaction observed at 1.5 mM concentration of
complex 1 (Fig. 4). In the control lanes (lane 1 and 12) DNA is
visible even without addition of compound, probably due to the
diffusion of the compound resulting in a low range visualization
of control DNA. Interestingly, we observed that the migration
pattern for the supercoiled plasmid showed a slight change with
an increase in complex 1 concentration. In order to identify
localization of complex 1 in or near the cell nucleus, studies
were undertaken using an Epi-uorescence microscope. The
experimental results indicate clear localization of complex 1
F(000)
1248
2.428–27.098
15 794
1216
2.535–27.000
35 459
q Range/ꢁ
Measured reections
Independent
reections/Rint
Parameters
R1 (I > 2s(I))
wR2 (all data)
Goodness-of-t on F2
5056/0.0415
4773/0.0910
343
325
0.0377
0.0926
1.034
0.649, ꢀ0.347
0.0895
0.1910
1.057
1.238, ꢀ1.579
ꢀ3
˚
Drmax,min/e A
Zn2 is six-coordinated by four oxygen atoms (O1, O2, O3 and
O4) of the ligand and two nitrogen atoms (N3 and N6) of two
unique azide anions (N3ꢀ). Zn2 and Zn1 of adjacent units are
bridged by an azide anion in end-on fashion, thus forming a 1D
polymeric structure (Fig. 2). The intramolecular Zn–Zn non-
bonded distance is 3.1077 A. The Zn–O and Zn–N bond
lengths are 2.006(5)–2.080(5) A and 1.942(8)–2.039(6) A respec-
tively, which are in good agreement with the literature values
˚
˚
˚
˚
˚
(average Zn–O and Zn–N bond lengths are 1.914 A and 2.002 A
respectively).26
UV-vis absorption study
The UV-vis spectrum of the ligand was previously reported. The
bands because of p–p* transition and n–p* transition were
found at 221, 259 and 332 nm respectively.22 The absorption
spectra of complexes 1 and 2 were recorded in acetonitrile.
Fig. 3 shows the UV-vis spectra. The spectrum of 1 shows two
ligand-centred bands at 249 and 343 nm which are red-shied
due to coordination to metal ions.
A band at 404 nm was observed due to d–d transition of Ni(II)
ion. Three absorption bands can be found in the spectrum of 2
at 226, 268 and 349 nm. All these bands are due to intra-ligand
transitions and are also red-shied because of the coordination
of the zinc(II) ions as compared to free ligands.
Cellular cytotoxicity, DNA binding and cellular uptake studies
Before studying the in vitro interaction and internalization of mostly inside the nucleus (green uorescence) probably due to
the complex 1 and 2 using a noncancerous monkey kidney its interaction with the cellular DNA. We used DAPI [40,6-
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range and the binding was found maximum at 25 mM concen-
tration of complex 1. However, at higher concentration of
complex 1, the uorescence intensity gradually fell down
probably because of lower solubility of complex 1 at concen-
trations higher than 25 mM. To this end, we believe that complex
1 is a useful DNA binding molecule as evident from present
analysis. However, further in depth research is necessary to
explore the possibilities of this group of small molecules as DNA
binding agents.
Conclusions
A discrete heterometallic complex, [Ni(L)Na(CH3CN)(ClO4)] (1)
and a 1 D polymer, [Zn2(L)(N3)2]n (2) were synthesized from a
Schiff-base
ligand,
N,N0-bis(3-methoxysali-
Fig. 4 1% Agarose gel electrophoresis. (A) 1 mg of pUC19 DNA was
preincubated with increasing concentration of complex 1 and then
loaded on a 1% agarose gel. Lane contents are as follows: 1-pUC19 in
DMSO; 2-11-pUC19 with 0.5 mM, 0.1 mM, 0.2 mM, 0.5 mM, 0.8 mM, 1
mM, 1.5 mM, 2.0 mM, 2.5 mM, and 5.0 mM of complex 1, respectively in
DMSO; and 12-pUC19 in 10 mM Tris–HCl, pH-80 buffer. (B) Densi-
tometric analysis of the obtained gel bands showing differential fluo-
rescence intensity of the complex 1 bound DNA. Highest intensity was
recorded for 1.5 mM complex 1.
cylidehydene)cyclohexane-1,2-diamine (H2L). The solid state
structures of 1–2 were established by single crystal X-ray
diffractions. Absorption spectra of both complexes showed
ligand-centred electronic transitions. Cellular cytotoxicity,
cellular uptake and DNA binding ability of 2 were investigated.
The synthesis of small metal complexes suitable for DNA
binding in process and will be communicated in future.
Experimental
diamidino-2-phenylindole (DAPI)], which binds to the A–T rich
regions of DNA (produce blue uorescence upon binding) as a
control uorescent stain to show that indeed complex 1 was
localized in the nucleus of the cells (Fig. 5). We observed that
the uorescence intensity of complex 1 upon DNA binding
increases with increasing complex 1 at lower concentration
All synthetic work was performed under aerobic atmosphere.
Solvents were distilled prior to use. Commercially available
reagents were used as received. The ligand, N,N0-bis(3-
methoxysalicylidehydene)cyclohexane-1,2-diamine (H2L) was
prepared according to a literature procedure.22
Synthesis of [Ni(H2L)Na(ClO4)(CH3CN)] (1)
A solution of nickel(II) acetate tetrahydrate (0.25 mmol, 0.0622
g) in 5 mL of acetonitrile was added to a solution of the ligand
(0.25 mmol, 0.096 g) in 5 mL of acetonitrile. The mixture was
reuxed for 2 h. Aer cooling to room temperature, a solution of
sodium perchlorate (0.25 mmol, 0.035 g) in 5 mL of acetonitrile
was added and continued reuxing for additional 1 h. The
brown coloured solution was allowed to evaporate in room
temperature. Dark reddish brown crystals were obtained aer
one week. Yield: 0.061 g (63.8%). Anal. Calc. for C24H27N3O8-
Ni1Cl1Na1: C 47.83; H 4.52; N 6.97. Found: C 47.62; H 4.48; N
6.83. IR (KBr, cmꢀ1): 2929 (m), 1620 (s), 1606 (m), 1548 (4), 1475
(s), 1451 (m), 1326 (m), 1246 (s), 1225 (m), 1169 (w), 1101 (s),
1080 (s), 979 (w), 867 (w), 753(m), 624 (w), 572 (w).
Synthesis of [Zn2(L)(N3)2]n (2)
A solution of zinc nitrate hexahydrate (0.5 mmol, 0.130 g) in 5
mL of methanol was added to a 5 mL solution of the ligand (0.5
mmol, 0.191 g) and reuxed for 1 h. A 10 mL of methanol–water
Fig. 5 Localization of complex 1 in Vero cells: the fluorescence solution (4 : 1) containing sodium azide (1 mmol, 0.065 g) was
microscopic image shows that most of the complex 1 get incorpo-
rated into the nucleus. (I) represents the nucleus of individual cells
stained with DAPI (blue fluoresence) while (II) represents localization of
complex 1 at the nucleus (green fluorescence); (III), and (IV) represent
added and the mixture was reuxed for additional 2 h. It was
ltered in hot condition and the ltrate kept for slow evapora-
tion. Single crystals suitable for X-ray diffraction were appeared
aer three weeks. Yield: 0.126 g (65.9%). Anal. Calc. for
the enlarged nucleus of a two neighboring cell one stained with DAPI
(III) and the other stained with complex 1 (IV), respectively.
C
22H24N8O4Zn2: C 44.39; H 4.06; N 18.82. Found: C 43.83; H
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3.97; N 18.46; IR (KBr, cmꢀ1): 2928 (m), 2106 (s), 2067 (s), 1628 were permeabilized with 0.1% Triton X 100 in PBS at room
(s), 1550 (w), 1475 (m), 1439 (m), 1379 (m), 1280 (m), 1253 (m), temperature for 20 minutes. Cells were then incubated with
1218 (s), 1082 (w), 1012 (w), 967 (w), 847 (w), 742 (s).
various concentration of complex 1 for two hours. Cells were
then washed three times with PBS. Finally, the coverslips were
mounted with a DAPI containing mounting media. The cells
were then evaluated using a Zeiss Scope.A1 Epiuorescence
microscope. Fluorescence intensity of complex 1 was evaluated
at 515–550 nm while DAPI was visualised at 440–470 nm.
Analytical techniques
Elemental analyses were performed on a VARIO MICRO cube
analyser by Elemental analyser system GMBH. IR spectra in KBr
(4500–500 cmꢀ1) were recorded using a PerkinElmer RXI FT-IR
spectrometer. The electronic spectra of 1–2 were recorded with a
Shimadzu UV-1800 spectrometer.
Acknowledgements
X-ray crystallography
This work was nancially supported by DST, India under FAST
Track Scheme (Order no. SR/FT/CS-96/2011, dated 15/06/2012).
A.G. was supported by Ramanujan Fellowship from Department
of Science and Technology, India (SR/S2/RJN-106/2012).
The data of 1–2 were collected on a Bruker D8 Quest diffrac-
tometer equipped with a Photon 100 CMOS area detector
system, using MoKa radiation with graphite monochromator
˚
(l ¼ 0.71073 A) at T ¼ 100(2) K. The structure was solved by
direct methods (SIR92)27 and rened on F2 by full-matrix least-
squares methods using SHELXL-97.28,29 Non-hydrogen atoms
were anisotropically rened. H-atoms were included in the
renement on calculated positions riding on their carrier
atoms. Hydrogen atoms of the coordinated acetonitrile in 1
Notes and references
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are added to the molecular formula. The function minimized
P
2
2 2
was [ w(Fo ꢀ Fc ) ] (w ¼ 1/[s2(Fo2) + (aP)2 + bP]), where
´
P ¼ (Max(Fo2,0) + 2Fc )/3 with s2(Fo2) from counting statistics.
2
The function R1 and wR2 were (s||Fo| ꢀ |Fc||)/s|Fo| and
5 E. Wong and C. M. Giandomenico, Chem. Rev., 1999, 99,
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2 2
[sw(Fo ꢀ Fc ) /s(wFo4)]1/2, respectively.†
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Cellular cytotoxicity assay (MTT assay)
7 G. Barone, A. Terenzi, A. Lauria, A. M. Almerico, J. M. Leal,
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previously.30 Briey, Vero 76 cell line (monkey epithelial cell
line) and HeLa cell lines were maintained in DMEM containing
10% FBS and 1% penicillin and streptomycin antibiotic
concentration. The cells were kept in a humidied incubator
with 5% CO2 at 37 ꢁC. 1 ꢂ 104 cells were seeded per well in a 96
well plate. When the cells were 80% conuent they were incu-
bated with the varied concentrations of complex 1 for 24 h. Aer
incubation, cells were washed with PBS and incubated with
´
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740 ng of plasmid DNA was incubated with the indicated
concentration of complex 1 solubilised in DMSO at a nal
volume of 20 ml for a period of one hour at 37 ꢁC. Aer this the
samples were run in a 1% agarose gel. The gel was then stained
with ethidium bromide (10 mg mLꢀ1) solution and visualised on
a UV transilluminator.
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Preliminary cellular uptake and localization studies
0.2 ꢂ 106 Vero 76 cells were seeded on a cover slip in 35 mm
17 Y. Hai, J.-J. Chen, P. Zhao, H. Lv, Y. Yu, P. Xu and J.-L. Zhang,
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