Nickel(II) complexes having different configurations controlled by N,N,O-donor Schiff-base ligands in presence of isothiocyanate as co-ligand: Synthesis, structures, comparative biological activity and DFT study

Article abstract of DOI:10.1016/j.poly.2015.07.055

Abstract Four mononuclear nickel(II) complexes, viz. [NiL¹(OAc)(H₂O)₂]·2H₂O (1), [NiL¹(NCS)(H₂O)₂]·H₂O (1a), [NiL²(OAc)(H₂O)] (2), and [NiL²

Full text of DOI:10.1016/j.poly.2015.07.055

Polyhedron 101 (2015) 93–102
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Nickel(II) complexes having different configurations controlled
by N,N,O-donor Schiff-base ligands in presence of isothiocyanate
as co-ligand: Synthesis, structures, comparative biological activity
and DFT study
Jaydeep Adhikary a, Priyanka Kundu a, Sanchari Dasgupta a, Sudeshna Mukherjee b, Sreya Chattopadhyay b,
Gabriel Aullón c, Debasis Das a,
⇑
a Department of Chemistry, University of Calcutta, 92 A. P. C. Road, Kolkata 700 009, India
b Department of Physiology, University of Calcutta, 92 A. P. C. Road, Kolkata 700 009, India
c
`
`
`
Departament de Química Inorganica and Institut de Química Teorica i Computacional, Universitat de Barcelona, Martí i Franques 1-11, 08028 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Four mononuclear nickel(II) complexes, viz. [NiL1(OAc)(H2O)2]ꢀ2H2O (1), [NiL1(NCS)(H2O)2]ꢀH2O (1a),
[NiL2(OAc)(H2O)] (2), and [NiL2(NCS)] (2a) (where HL1 = 2-[(2-Morpholin-4-yl-ethylimino) -methyl]-phe
nol and HL2 = 2-[(2-Pyrrolidin-1-yl-ethylimino)-methyl]-phenol) have been synthesized and structurally
characterized. Single crystal X-ray analysis reveals the presence of square planar coordination geometry
about nickel for 2a, whereas others have distorted octahedral geometry. DFT calculations have done to
explore the origin of different geometries of 1a and 2a although their preparative procedure is same.
The influence of different geometries i.e., octahedral and square-planar on the anticancer activity of
Ni(II) have been investigated on Erhlich’s ascities carcinoma (EAC) cells and the order of anticancer activ-
ity is 2a > 1a > 2 > 1. The biological results are further compared to the activity of cis-platin.
Ó 2015 Elsevier Ltd. All rights reserved.
Article history:
Received 5 June 2015
Accepted 20 July 2015
Available online 26 July 2015
Keywords:
Schiff base Ni(II) complex
Square planar and octahedral geometry
DFT study
Bioactivity
1. Introduction
(Scheme 1). We have tried to explain the origin of such transforma-
tion from octahedral 2 to square planar 2a by DFT calculation.
Nickel(II) has very rich coordination chemistry owing to its
inherent ability to espouse various geometries [1–7]. This versatile
coordination chemistry of nickel has an area of considerable
importance in inorganic chemistry with implications in other areas
of chemistry and biology [8–10]. Schiff base complexes present
suitable biometric properties that can mimic the structural fea-
tures of the active sites, and they have been extensively used in
various fields such as disease treatment, biochemical reaction
and biological regulator [11–14]. Some articles about the interac-
tion of DNA with macrocyclic Schiff base nickel(II) complex have
been reported [15–18]. In our lab octahedral Ni(II) Schiff base com-
plexes have been widely studied in various biological field [19]. But
the activity of square planar Ni(II) complex is yet to explore in bio-
logical study. For this purpose, we have prepared octahedral Ni(II)
complexes (1 and 2) of two closely related Schiff base ligands (HL1
and HL2). Further treatment of 1 and 2 with sodium thoicyanate
produce octahedral 1a and square planar 2a, respectively
Anticancer activity of all the complexes has been studied on
Erhlich’s ascities carcinoma cell. All the four complexes show
excellent anticancer activity. ROS generation is responsible for
such activity. DNA fragmentation activity of the complexes has
been proved by fluorescence imaging. Experimental outcome sug-
gest that square planar complex (2a) is the most efficient among
the all. This can be explained as the high penetration property
due to planarity as reported previously [20–22]. Cytotoxicity result
of the complexes corroborates that toxicity level is lesser towards
normal cell in compare to cancer cell.
2. Experimental
2.1. Physical methods and materials
Elemental analyses (carbon, hydrogen and nitrogen) were per-
formed using a Perkin–Elmer 240C elemental analyzer. Infrared
spectra (4000–500 cmꢁ1) were recorded at 27 °C using a Perkin–
Elmer RXI FT-IR spectrophotometer with KBr pellets. Electronic
spectra (1400–200 nm) were obtained at 27 °C using a Shimadzu
⇑
Corresponding author.
E-mail address: dasdebasis2001@yahoo.com (D. Das).
http://dx.doi.org/10.1016/j.poly.2015.07.055
0277-5387/Ó 2015 Elsevier Ltd. All rights reserved.

94
J. Adhikary et al. / Polyhedron 101 (2015) 93–102
Scheme 1. Schematic route of complexes.
UV-3101PC with methanol as solvent and reference. Thermal anal-
yses (TG–DTA) were carried out on Mettler Toledo
solution (10 mL) of salicylaldehyde (0.244 g, 2 mmol) and the
resulting solution was refluxed for 30 min. Then, methanolic solu-
tion (5 mL) of Nickel acetate (0.497 g, 2 mmol) was added and
resulting solution was stirred for 1 h. The green solution was fil-
tered, kept in a CaCl2 desiccator in dark and after a few days crys-
tals of complex 1, suitable for X-ray data analysis were obtained.
(Yield 73%). Anal. Calc. for C30H54N4Ni2O15: C, 43.47; H, 6.52; N,
6.67. Found: C, 43.41; H, 6.49; N, 6.62%. UV–Vis–NIR (methanol,
nm) kmax = 621, 758, 949.
a
(TGA/SDTA851) thermal analyzer in flowing dinitrogen (flow rate:
30 cm3 min1). Ambient temperature magnetic susceptibility mea-
surements were performed with Magway MSB Mk1 magnetic sus-
ceptibility balance. All chemicals were obtained from commercial
sources and used as received. Solvents were dried according to
standard procedure and distilled prior to use. Salicylaldehyde,
N-(2-aminoethyl) morpholine, N-(2-aminoethyl) pyrolidine,
Nickel(II) acetate hexahydrate and Sodium thiocyanate were pur-
chased from Aldrich.
2.2.2. Synthesis of Complex [NiL1(NCS)(H2O)2]ꢀH2O (1a)
After following the same procedure as for 1 aqueous solution of
Sodium thiocyanate (0.162 g, 2 mmol) was added to it and the
solution was stirred for more 15 min. Resulting green solution
was filtered, kept in a CaCl2 desiccator. Few days later green single
crystals of 1a were obtained, suitable for X-ray data collection.
(Yield 79%). Anal. Calc. for C14H23N3NiO5S: C, 41.58; H, 5.69; N,
2.2. Synthesis of complexes
2.2.1. Synthesis of Complex [NiL1(OAc)(H2O)2]ꢀ2H2O (1)
A methanolic solution (5 mL) of N-(2-aminoethyl) morpholine
(0.260 g, 2 mmol) was added dropwise to
a hot methanolic

J. Adhikary et al. / Polyhedron 101 (2015) 93–102
95
Table 1
Crystallographic data and details of structure refinement for the complexes.
1
1a
2
2a
Empirical formula
Formula mass
Crystal system
Space group
a (Å)
2(C15H24N2NiO6), 3(H2O)
828.15
C14H21N3NiO4S, H2O
404.11
C15H22N2NiO4
353.04
C14H17N3NiOS
334.07
monoclinic
C2/c (No. 15)
25.2737(16)
9.6509(6)
16.6677(11)
90
110.885(2)
90
3798.4(4)
4
triclinic
triclinic
monoclinic
P21/n (No.14)
10.182(5)
18.687(5)
16.093(5)
90
105.660(5)
90
2948.4(19)
8
ꢀ
ꢀ
P1 (No. 2)
P1 (No. 2)
8.7824(3)
9.6530(3)
11.5092(4)
95.749(1)
90.179(2)
111.469(1)
902.61(5)
2
9.5340(3)
11.1002(3)
16.7590(5)
74.277(1)
75.438(1)
70.363(1)
1582.59(8)
4
b (Å)
c (Å)
a
(°)
b (°)
c
(°)
V (Å3)
Z
T (K)
293
1.062
1.448
1752
293
1.218
1.487
424
293
1.482
1.245
744
293
1.456
1.505
1392
l
(Mo K
a
) (mmꢁ1
)
)
Dcalc (g cmꢁ3
F(000)
h max (°)
26.9
34.0
32.9
29.1
Total, Unique Data (Rint
)
21880, 4066 (0.029)
3480
13312, 6819 (0.018)
5831
28951, 11098 (0.023)
8190
44749, 7823 (0.038)
5813
Observed (I > 2r(I))
Nref, Npar
4066, 259
0.0335, 0.0920, 1.04
ꢁ0.35, 0.75
6819, 241
0.0327, 0.0916, 0.98
ꢁ0.37, 0.54
11098, 413
0.0393, 0.1810, 0.70
ꢁ0.37, 0.65
7823, 361
0.0365, 0.1024, 0.97
ꢁ0.77, 0.41
R, wR2, S
Residual extrema (e Åꢁ3
)
10.39. Found: C, 41.56; H, 5.64; N, 10.35%. UV–Vis–NIR (methanol,
2.4. Computational details
nm) kmax = 612, 755, 916.
Unrestricted calculations were carried out using the Gaussian09
package [29]. The hybrid density function method known as B3LYP
was applied [30]. Effective core potentials (ECP) were used to rep-
resent the innermost electrons of the nickel and the basis set of
valence double-f quality for associated with the pseudopotentials
known as LANL2DZ [31]. The basis set for the main group elements
was 6-31G⁄ (S, C, N, O and H) [32]. Solvent effects of methanol and
water were taken into account by PCM calculations [33], keeping
the geometry optimized for gas phase (single-point calculations).
2.2.3. Synthesis of Complex [NiL2(OAc)(H2O)] (2)
Complex 2 was synthesized by following exact procedure as
mentioned for complex 1. Here N-(2-aminoethyl)pyrolidine
(0.228 g, 2 mmol) was used in place of N-(2-aminoethyl) morpho-
line in time of Schiff base preparation. X ray diffractable green
crystals of complex 2 were obtained from the final solution after
one week (Yield 76%). Anal. Calc. for C15H22N2NiO4: C, 50.99; H,
6.23; N, 7.93. Found: C, 50.91; H, 6.17; N, 7.91%. UV–Vis–NIR
(methanol, nm) kmax = 609, 753, 900.
2.5. Bio-activity
2.2.4. Synthesis of Complex [NiL2(NCS)] (2a)
2.5.1. Isolation and culture of Erhlich’s ascities carcinoma (EAC) cells
EAC cells were isolated aseptically from the peritoneal cavity of
tumor-bearing mouse after injecting 2–3 ml of PBS into the peri-
toneal cavity. The collected cells were placed in sterile petriplates
and incubated at 37 °C for 2 h to separate the tumor cells from cells
of macrophage lineage. The non-adherent tumor cells were
After adopting the same procedure as for 2, aqueous solution of
sodium thiocyanate (0.162 g, 2 mmol) was added. The solution
turned reddish brown immediately from green. Final solution
was kept for crystallisation. X ray suitable red crystals of complex
2a were obtained from the resulting solution after 2 days. (Yield
82%). Anal. Calc. for C14H17N3NiOS: C, 50.29.47; H, 5.08; N, 12.57.
Found: C, 50.23; H, 5.06; N, 12.53%. UV–Vis–NIR (methanol, nm)
kmax = 473, 753, 873.
2.3. X-ray data collection and structure determination
Diffraction data for 1–4 were collected at room temperature
(293 K) on a Bruker Smart CCD diffractometer equipped with gra-
phite–monochromated Mo
Ka radiation (k = 0.71073 Å). Cell
refinement, indexing and scaling of the data set were carried out
using Bruker SMART APEX and Bruker SAINT package [23]. The struc-
ture were solved by direct methods and subsequent Fourier analy-
ses [24] and refined by the full-matrix least-squares method based
on F2 with all observed reflections using SIR-92 and SHELX-97 [25],
software. For the complexes, all non-hydrogen atoms were refined
with anisotropic thermal parameters and the hydrogen atoms
were fixed at their respective positions riding on their carrier
atoms and refined anisotropically. All the calculations were per-
formed using the WinGX System, Ver 1.80.05 [26], PLATON99 [27],
Fig. 1. UV–Vis spectra of 10ꢁ2 (M) solution of complex 1, 1a, 2, 2a in methanol
medium.
ORTEP3 [28] programs. Selected crystallographic data and refine-
ment details are displayed in Table 1.

96
J. Adhikary et al. / Polyhedron 101 (2015) 93–102
Fig. 5. Molecular structure of complex 2a with the atom numbering scheme.
Fig. 2. Molecular structure of complex 1 with the atom numbering scheme.
The cells were serum-starved for 24 h. After 24 h the cells were
cultured with increasing doses (0–150 M) of the compounds
l
(1–4) and also in presence of a standard free radical scavenger
10 nM N-acetyl cysteine (NAC). At the end of the culture, media
along with the cells were aspirated and were aspirated and were
centrifuged at 3000 rpm for 5 min. The cells from each group were
collected and with them different experiments were performed
[34,35].
2.5.2. Isolation and culture of hepatocytes
Liver was perfused in situ with collagenase (0.05% p/v) and then
the liver was removed. A portion of the liver was chopped with a
blunt forceps and single cell suspensions were made in
RPMI-1640. The cell population was passed through a nylon mesh
with 70 lM pore size. Cell viability was determined using Trypan
blue exclusion test and then the cells were cultured in the same
way the EAC cells were cultured [35,36].
2.5.3. Cytotoxicity assay
Fig. 3. Molecular structure of complex 1a with the atom numbering scheme.
Change in percentage of viability of EAC cells and hepatocytes
after treating them with the increasing doses of the compounds
was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
te-trazolium bromide (MTT) assay. The cultured cells were resus-
pended in PBS containing MTT (0.05 mg/ml) and were incubated
for 4 h at 37 °C. The media containing MTT was aspirated out and
200 ll of dimethyl sulfoxide was added to dissolve the formed
insoluble formazan salt. The absorbance was read spectrophoto-
metrically at 540 nm and the number of viable cells is proportional
to the amount of formazan salt formed and was measured as the
percent of the control [37].
2.5.4. Detection of intracellular ROS content
EAC cells, after treatment with the compounds, were lysed in
ice-cold lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA,
1 mM EDTA, 20 mM NaF, 100 mM Na3VO4, 0.5% NP-40, 1% Triton
X-100, 1 mM PMSF, pH 7.4) with freshly added protease inhibitor
cocktail. It was centrifuged at 14,000g for 25 min at 4 °C and the
supernatant (total cell lysate) was collected, aliquoted and was
used for estimation of intracellular ROS content. The protein con-
tent in the lysates was measured by Micro Lowry method for esti-
mation of protein. ROS content of the EAC cells was measured
Fig. 4. Molecular structure of complex 2 with the atom numbering scheme.
according to Toban-Velasco et al. [40]. 5 ll of whole cell lysate of
each group were incubated in presence of 5 mM dihyroethidium
at 37 °C for 60 min. Fluorescent signals were recorded at the end
of the incubation at an excitation wavelength of 480 nm and an
emission wavelength of 525 nm in a spectrofluorimeter (JASCO).
aspirated out gently and washed repeatedly with PBS and then cul-
tured in RPMI-1640 medium in presence of 10% FBS, Streptomycin
(100 U/ml) and gentamycin (50 U/ml) at 37 °C in a CO2 incubator.

J. Adhikary et al. / Polyhedron 101 (2015) 93–102
97
Table 2
Selected bond distances for complexes 1–2a.
1
1a
2
2a
Ni01–O1
Ni01–O2
Ni01–O3
Ni01–O4
Ni01–N1
Ni01–N2
2.0377(15)
2.110(2)
2.0384(18)
2.1182(15)
2.0024(18)
2.2307(17)
Ni1–O1
Ni1–O3
Ni1–O4
Ni1–N1
Ni1–N2
Ni1–N3
2.0351(11)
2.0544(11)
2.1711(12)
2.0056(11)
2.2771(12)
2.0486(14)
Ni1–O1
Ni1–O2
Ni1–O3
Ni1–O4
Ni1–N1
Ni1–N2
2.0187(17)
2.0908(16)
2.1142(16)
2.1633(19)
1.999(2)
Ni1–O1
Ni1–N1
Ni1–N2
Ni1–N3
1.8343(18)
1.8450(19)
1.959(2)
1.863(2)
2.1843(19)
Table 3
Selected bond distances for complexes 1–2a.
1
1a
2
2a
O1–Ni01–O2
O1–Ni01–O3
O1–Ni01–O4
O1–Ni01–N1
O1–Ni01–N2
O2–Ni01–O3
O2–Ni01–O4
O2–Ni01–N1
O2–Ni01–N2
O3–Ni01–O4
O3–Ni01–N1
O3–Ni01–N2
O4–Ni01–N1
O4–Ni01–N2
N1–Ni01–N2
90.18(7)
90.68(7)
86.58(6)
89.77(7)
170.83(6)
87.05(7)
174.38(7)
94.80(8)
86.67(7)
88.40(6)
178.10(7)
97.75(7)
89.78(7)
97.22(6)
81.93(7)
O1–Ni1–O3
O1–Ni1–O4
O1–Ni1–N1
O1–Ni1–N2
O1–Ni1–N3
O3–Ni1–O4
O3–Ni1–N1
O3–Ni1–N2
O3–Ni1–N3
O4–Ni1–N1
O4–Ni1–N2
O4–Ni1–N3
N1–Ni1–N2
N1–Ni1–N3
N2–Ni1–N3
87.14(5)
87.72(4)
90.75(5)
169.30(4)
91.72(5)
82.51(5)
176.13(4)
99.88(5)
89.46(5)
94.17(5)
85.21(4)
171.97(5)
81.77(5)
93.85(5
O1–Ni1–N2
O1–Ni1–N1
O2–Ni1–O3
O2–Ni1–O4
O2–Ni1–N1
O2–Ni1–N2
O2–Ni1–C14
O3–Ni1–O4
O3–Ni1-N1
O3–Ni1–N2
O3–Ni1–C14
O4–Ni1–N1
O4–Ni1–N2
O4–Ni1–C14
N1–Ni1–N2
N1 -Ni1 –C14
N2 –Ni1 -C14
172.21(7)
90.13(7)
97.28(7)
158.95(7)
95.30(7)
87.40(7)
128.20(7)
61.71(7)
167.31(7)
99.10(7)
31.13(6)
105.67(7)
96.79(7)
30.75(7)
83.19(7)
136.19(7)
101.60(7)
O1–Ni1–N1
O1–Ni1–N2
O1–Ni1–N3
N1–Ni1–N2
N1–Ni1–N3
N2–Ni1–N3
94.71(7)
176.37(7)
86.44(8)
85.70(8)
175.41(8)
93.43(8)
96.39(5)
A standard curve was prepared using increasing concentration of
DHE incubated in parallel and results were expressed as moles of
DHE produced per mg of protein [38–40].
complex with the complexes and the decrease in fluorescence
was measured. 3.9 M DNA (genomic DNA isolated from EAC cells
using the method of Laird et al., 1991) was incubated with 1.1
l
lM
ethidium bromide and various concentrations of the complexes.
2.5.5. Detection of DNA fragmentation by fluorescence microscopy
For detection of DNA fragmentation, which is hallmark of apop-
tosis, in EAC cells treated with or without increasing doses of the
compounds, the EAC cells were fixed and nuclear DNA stained with
DAPI (0.2 mg/ml for 15 min at room temperature). A fluorescent
microscope was used to detect the signal from DAPI. Digital images
were captured and were controlled with Dewinter Caliper Pro soft-
ware [34].
The reaction mixtures were excited at 552 nm and the fluorescence
intensities were recorded at 624 nm using
troflourimeter [20,41].
a JASCO spec-
3. Result and discussion
3.1. Syntheses, FT-IR spectra and UV–Vis spectra of the complexes
Two Schiff base ligands HL1 and HL2 are prepared through the
classical method where salicylaldehyde is refluxed with
N-(2-aminoethyl)-morpholine and N-(2-aminoethyl)-pyrrolidine,
respectively in methanol medium for half an hour. HL1 and HL2
are further treated with nickel(II) acetate in situ separately to pre-
pare complexes 1 and 2 respectively. Whereas, separate treatment
of HL1 and HL2 with nickel(II) acetate and followed by sodium thio-
cyanate give complexes 1a and 2a respectively. All the four com-
plexes are characterized by routine physicochemical techniques
as well as by X-ray single crystal structure analyses. All the com-
plexes show IR bands due to C@N stretch in the range 1618–
2.5.6. DNA binding studies of the compounds by competitive
fluorescence displacement assay
Interactions of the prepared compounds with double stranded
DNA were studied by incubating the DNA–ethidium bromide
1649 cmꢁ1 and skeleton vibrations in the range 1536–1560 cmꢁ1
.
1a and 2a have one sharp peak around 2100 cmꢁ1 is due to C@N
stretching of SCN ligand. A broad band in the range 3300–
3500 cmꢁ1 is due to hydrogen bonded O–H group of coordinated
water molecule present in complexes 1, 1a and 2. (Supporting
Information, Figs. S1–S4). But such peak is absent in case of 2a
which implies the absence of hydrogen bonded O–H group.
Magnetic susceptibility measurements for 1, 1a and 2 (leff ꢂ 3.2
B.M. at 298 K) suggest that nickel(II) possesses octahedral configu-
ration whereas diamagnetic 2a nickel(II) acquire square planar
geometry. Electronic spectra (Fig. 1) recorded in methanol also
reveal that coordination environment around nickel(II) in 1, 1a
Fig. 6. Intermolecular H-bonding (blue dots) and intramolecular H-bonding (yellow
dots) present in 1. (Color online.)

98
J. Adhikary et al. / Polyhedron 101 (2015) 93–102
Fig. 7. H-bonded (blue dots) polymeric structure of 1a. (Color online.)
(present in axial position). Here acetate anion is bridged via mono
dentate mode. In 1a, NiII is coordinated via three donor sites of HL1,
N of one isothicyanato ligand and two water molecules. In complex
2, one acetate anion coordinate Ni centre through two oxygen
atoms in bidentate fashion. Sixth position is occupied by one metal
coordinated water molecule. Complex 2a, NiII is coordinated via
three donor site of HL2 and Nitrogen of one isothicyanato ligand.
Selected bond lengths are reported in Table 2. The NiꢁN1,
NiꢁN2, NiꢁO1 bond distances vary in the ranges 1.8450(19)–
2.0056(11), 1.959(2) –2.2307(17), and 1.8343(18)–2.0377(15) Å,
respectively. Selected bond angles are shown in Table 3. The mean
N1ꢁNiꢁO1, N1ꢁNiꢁN2, and O1ꢁNiꢁN2 bond angles are 91.34°,
83.15°, and 172.18°, respectively. Packing diagram for all the com-
plexes are shown in Fig. S5–S8 (See Supporting Information). The
adjacent mononuclear molecules of 1 are linked by O–H(water)
ꢀ ꢀ ꢀO(phenoxyl) intermolecular hydrogen bonds. Each molecule of
1 has an intramolecular hydrogen bond O–H(water)ꢀ ꢀ ꢀO(acetate)
also as depicted in Fig. 6. Hydrogen bonded 1D polymeric structure
of 1a is represented in Fig. 7. Dimerization of single molecule of 2
via hydrogen bonding has been illustrated in Fig. 8.
Fig. 8. Dimerization of complex 2 via intermolecular H-bonding (blue dotts). (Color
online.)
3.3. DFT Study
and 2 is roughly octahedral and square planar in 2a [42,43].
Complex 1, 1a, 2 display three weak absorption bands at ꢃ615,
In an attempt to understand the formation of nickel complexes,
we have performed a theoretical study based in DFT calculations to
evaluate the thermodynamic feasibility of these compounds. In
addition to the energetic profile and relative stability of the species
involved, our study provides an insight into structural features of
involved complexes. In our calculation, solvent effects have been
considered in all reactions by an implicit method to obtain Gibbs
free energies. Both methanol and water are taken as solvent, but
only former will be discussed in this section. We have defined
two main pathways in our study: (a) substitution of the monoden-
tade acetate by a isothiocyanate one, via a pentacoordinated inter-
mediate; and, (b) acetate isomerization to bidentate coordination
mode replacing a water molecule, following by the introduction
of a isothiocyanate to exchange it, generating a square planar com-
plex. In all cases, the pathways are evaluated from the octahedral
ꢃ755 and ꢃ900 nm
(e , 3–10) assigned to
/dm3 molꢁ1 cmꢁ1
spin-allowed 3A2g ? 3T1g(P), 3A2g ? 3T1g(F) and 3A2g ? 3T2g(F)
transitions respectively expected for octahedral d8 ions. In case of
complex 2a, bands around ꢃ755 and ꢃ920 are slightly blue shifted.
Disappearance of peak around ꢃ615 nm along with the generation
of one new intense bands for 2a at the lower wavelength region
[ꢃ471 nm (
e
/dm3 molꢁ1 cmꢁ1)] indicates a large crystal-field split-
ting and is consistent with the square planar geometry of nickel(II)
as reported previously [44].
3.2. Crysallographic description of complexes 1–4
The structure of complexes have been depicted in Figs. 2–5. All
the four complexes are discrete mononuclear in nature. Complex 1
& 2 are made using tridentate ligand HL1 whereas 3 & 4 are made
with another tridentate ligand HL2. Both the ligands have N, N, O
donor site, where HL1 and HL2 contain morpholine and pyrrolidine
moiety respectively. Complexes 1, 1a and 2 have one octahedrally
disordered NiII centre. On the other hand in complex 2a, NiII centre
have perfectly square planar geometry. Complex 1 contains two
coordinating water molecules (one in axial and another in equato-
rial position with respect to ligand plane) and one acetate anion
compound [Ni(
j
1-OAc)(H2O)2(L)] (A), having
L
an O,N,N0-
tridentate ligand.
3.3.1. Substitution of an octahedral complex
Since the most favored reaction for ligands substitution of hex-
acoordinated complexes proceed via a pentacoordinated interme-
diates in two steps, this pathway is studied (Scheme 2). The
pentacoordinated intermediates (AB) have been characterized for
both LMorph and LPyrr ligands, having similar stability from the

J. Adhikary et al. / Polyhedron 101 (2015) 93–102
99
100
80
60
40
20
0
A
2a
1a
2
1
5
M
25
M
50
M
75
M
100
M
125
M
150
M
Drug+NAC
Fig. 10. Fluorescence emission spectra of EB–DNA fragmentation in absence
(Control) and presence of complex 1a and 2a.
60
40
20
0
B
100
2a
1a
80
2
1
60
2a
1a
2
40
20
0
1
0 M
5
M
25
M
50
M
75
M
100 M 150 M
5
M
25
M
50
M
75
M
100
M
125
M
150
M
Fig. 11. Effect of the complexes on survival of normal primary mouse hepatocytes.
Isolated hepatic cells were incubated with increasing doses of the complexes (0–
150 lM) for 24 h. After 24 h, cells were collected and the percentage of dead cells
was assayed. For determination of cell death, harvested cells were incubated with
MTT for 4 h and the amount of formation of formazan salt was measured at 540 nm
using a spectrophotometer.
300
250
200
150
100
C
2a
1a
2
reactants, about 28 kcal/mol. Besides the charges of reactants and
products are not the same, the high ability of acetate and isothio-
cyanate to generate hydrogen bonds will affect dramatically on the
thermodynamics. As example, when one solvent molecule is
explicitly included by forming a hydrogen bonding, they are stabi-
lized only 4 kcal/mol from insolated models. Nevertheless, we have
decided to generate pentacoordinated intermediates by lengthen-
ing of sixth ligand and full reoptimizaton of molecular structure.
When distances are sufficiently large to not find energetic changes,
similar energy from the reactants about 15 kcal/mol has been
obtained. And now, the final substituted octahedral complex (B)
is only +5 kcal/mol from the reactants. Moreover, the influence of
solvent, methanol or water, is very scarce in this pathway, and
all energetic differences are less than 1 kcal/mol.
All three involved complexes present same structural trends.
Hexacoordinate complexes presents particularly identical dis-
tances for three donors (OAr,NIm,NAm) arount the nickel, Ni–
OAr ꢂ 2.04, Ni–NIm ꢂ 2.01, but small diferences of 0.03 Å are found
for Ni–NAm, being larger for LMorph than LPyrr complexes (2.23 and
2.20 Å, respectively). The fac disposition of tridentate ligand is
completed by one water in equatorial, which Ni–OEq distances
(2.11 and 2.22 Å in A and B, respectively) change by the presence
of intramolecular hydrogen bonding in acetate complexes but
1
0 M
5
M
25
M
50
M
75
M
100 M 150 M
Fig. 9. Effect of the complexes 1, 1a, 2 and 2a on tumor cell survival. EAC cells were
incubated with increasing doses of the complexes (0–150 M) in presence of
l
10 mM N-acetyl cysteine (NAC) for 24 h. After 24 h, cells were collected and assayed
for the effect of the compounds on: (A) Cell death – For determination of cell death,
harvested cells were incubated with MTT for 4 h and the amount of formation of
formazan salt was measured at 540 nm using a spectrophotometer. (B) Intracellular
ROS content was estimated by incubating the cell lysates from each groups with
DHE (25 lg/ml) for 1 h using a spectrophotometer using excitation and emission
wavelengths of 495 nm and 525 nm respectively. (C) DNA fragmentation – For
detection of DNA fragmentation, treated EAC cells were fixed, permeabilised and
stained with DAPI and visualized under a fluorescence microscope. The figures
shown here are representative of six different figures with similar results. All values
represent mean S.E. of six independent experiments in each case.

100
J. Adhikary et al. / Polyhedron 101 (2015) 93–102
Fig. 12. TGA plot of complex (a) 1 and 1a; (b) 2 and 2a.
Table 4
Half maximal inhibitory concentration (IC50) in
lM of the complex.
Complex Half maximal inhibitory concentration (IC50) in
lM on tumor cells
2a
1a
2
1
cis-
19.634
22.32
24.08
26.08
24.1
Platin
Table 5
Scheme 2. Substitution of acetate ligand by isothiocyanate in an octahedral
Half maximal inhibitory concentration (IC50) in
cell.
lM of the complex towards normal
complex.
Complex Half maximal inhibitory concentration (IC50) in
mouse hepatocytes
lM on primary
2a
1a
2
1
cis-
41.96
61.39
79.92
98.42
24.1
Platin
tridentate ligand are situated into basal plane. The distances Ni–
OAr and Ni–NIm are 1.93 and 1.98 Å, and differences of 0.02 Å for
Ni–NAm are newly found in LMorph (2.13) and LPyrr (2.11) complexes.
The environment is completed with two water molecules in the
fourth basal and the aplical positions at 2.16 and 2.11 Å, respec-
tively, and other structural changes are less important.
Scheme 3. Genaration of square planar complex from octahedral one.
Another interesting point for these nickel compounds are
its electronic structure. Since nickel(II) is a d8 ion, it should present
two unpaired electrons in pseudo-octahedral or square-pyramidal
environments. Our calculations show spin densities in the metal
atoms between 1.58 and 1.63, revealing that 80% of those two elec-
trons are locating in the nickel atom. More than 10% are delocal-
ized in the tridentate ligand, specially in the aromatic and iminic
framework. Additionally, frequency calculations display character-
istics in the infrared spectra: one band about 1633 cmꢁ1 for termi-
nal carbonyl group in acetate complex (A), and two about 2107 and
811 cmꢁ1 for thiocynate one (B). An imine stretching band changes
Scheme 4. Interchanging of two lignads.
from 1710 (A) to 1707 (B) cmꢁ1
.
absence in thiocyanate one. The axial positions with leaving
ligands are identical depending of the nature of this ligand (Ni–
3.3.2. Generating a square-planar complex
O
Ac/NNCS are 2.04 and 2.08 Å), and we would remark the angular
A second pathway to obtain a thiocyanate complex from acetate
geometry of coordinated N-thiocyanate (121°). Finally, small
changes are found for axial water molecule induced by trans influ-
ence (2.29 and 2.26 Å in A and B, respectively).
The pentacoordinated species (AB) are clearly decribed as
square pyramidal, having distances shorter than those found in
hexacoordinate complexes. The three donors (OAr,NIm,NAm) of
one was studied. It proceeds from [Ni(
j
1-OAc)(H2O)2(L)] complex (A)
that isomerizes to [Ni(
j
2-OAc)(H2O)(L)] (C) changing the coordina-
tion mode for acetate ligand, and after thiocyanoate replaces the car-
boxylato generating a square-planar complex D (Scheme 2). The first
stepcorrespondsto an isomerization of theacetate anion in tripletas
ground state for the complex (and ꢃ80% of the spin densities

J. Adhikary et al. / Polyhedron 101 (2015) 93–102
101
remains in nickel atoms). Our calculations reveal that chelate com-
plex (C) is only 0.8 (LMorph) and 1.1 (LPyrr) kcal/mol higher in energy
than monodentate one, but they become the more stable if the leav-
ing water molecules remain bound to coordinated water, as it is
found in crystal structures (-2.4 ans ꢁ1.7 kcal/mol for LMorph and
LPyrr derivatives, respectively). Moreover, an accessible transition
state for this isomerization are localizated having low energy for
the two systems (ꢃ+7 kcal/mol). For this reason, we can propose
an easy accessibility to the interconversion between coordination
modes of acetate ligand.
compounds and maximum cytotoxicity is observed at the highest
dose i.e., 150 M. Table 4 shows the IC50 values of complexes 2a,
1a, 2, and standard chemotherapeutic drug cis-platin
towards cancer cells. Our results strongly suggest that the Ni
complexes especially 2a, 1a and 2 are effective than cis-platin
on EAC cells.
l
1
a
To explore the nature of the cell death by compounds we inves-
tigate the level of ROS generation in the EAC cells. We have found
that treatment of the EAC cells with the compounds increased ROS
generation by more than 2fold. Researchers have established that
cancer cells have higher basal level of ROS and any agent that
increases ROS generation is termed chemotherapeutic agent
(Manna et al., 2012) [45]. In the present study we have shown that
our compounds increased the level of ROS in EAC cells which ulti-
mately led to EAC cell death (Fig. 9B). To confirm that ROS is the
main player in orchestrating EAC cell death, we co-treated the cells
with a standard ROS scavenger N-acetyl cysteine (NAC). We found
that co-treatment of the cells with 10 mM NAC protected the EAC
cells from ROS induced cell death by the compounds as evidenced
by increase in cell viability in the NAC co-treated groups
(Fig. 9A).Induction of apoptosis by the compounds 2a and 1a are
confirmed by observing DNA fragmentation (hallmark of apoptotic
cell death) in the EAC cells following treatment of EAC cells by the
compounds (Fig. 9C).
We have studied fluorescence quench of ethidium bromide
bound DNA by the compounds as a function of interaction between
the complexes and the DNA. Fig. 10 shows increasing concentra-
tion of four complexes results in decrease in fluorescence intensity
of the ethidium–DNA complex. This indicates that all the four com-
plexes are able to kick out ethidium bromide from DNA. Our data is
in accordance with the reports of other investigators which states
that Ni complexes exhibit groove binding interactions with DNA
(Zhu et al., 2010)[20].
Optimized geometries (C) reveal minor changes in the distances
between nickel and tridentate ligand, in comparison with related
terminal complexes (A), whereas Ni–O distances are about 2.10
and 2.12 Å for equatorial and axial bonds, larger than monodentate
one (2.04 Å). However, an important distortion about ideal octahe-
dral geometry is observed by chelate requirements of
four-membered rings. Carboxylate frequencies in the would
appear at 1612 and 1488 cmꢁ1 (C) according with the chelate
mode (
monodentate complexes (A) due the intramolecular hydrogen
D
= 124 cmꢁ1), and they pattern are similar to those found
bonding (
D
= 171 cmꢁ1). However, the imine stretching is moved
to 1698 cmꢁ1 (C).
The following step for the second path is the substitution of
both acetate and water by thiocianate. This reaction leads a change
in the spin ground state, generating a diamagnetic ion, induced by
square-planar geometry around the nickel atoms, being null spin
density and diamagnetic behavior. The evaluated energies for this
reaction are unfavored by more than +18 kcal/mol. When water
molecule is added to the leaving acetate generating hydrogen bon-
dins, they decrease to +14 kcal/mol, being always LPyrr derivative
more preferred than LMorph one. All metal–ligand distances in the
square-planar complexes (D) are dramatically shorter that those
found in octahedral complexes (more than 0.1 Å). A special case
is found for the Ni-NCS distance (1.88 vs. 2.08), probably by major
The most common problem with most chemotherapeutic drugs
is that the drugs are toxic towards both cancer cells as well as nor-
mal cells. Our data shows that the Ni complexes are comparatively
less toxic towards normal primary hepatocytes than EAC cells. The
IC50 of the compounds towards hepatocytes is much higher when
compared to IC50 of cis-platin. So it can be said that the complexes
are highly toxic to cancer cells but less toxic towards normal cells
(Fig. 11, Table 5).
contribution of the p-backbonding in the square planar geometry.
The stereochemical modification of the nickel atom produces
important changes in the infrared spectra: (a) the imine
stretching decrease to 1682 cmꢁ1, (b) C-S band of thiocynate
increase to 888 cmꢁ1, and (c) C–N band of thiocyanate is slight
moved to 2117 and 2129 cmꢁ1, for LMorph and LPyrr complexes,
respectively.
3.3.3. Interchanging ligands
Finally, we have studied the interchanging reactions for nickel
complexes, as it is shown in Scheme 3. In all cases, we have oba-
tined that complexes containing LPyrr are more favored than
LMorph ones by 1.3–3.2 kcal/mol. It suggests than LPyrr acts as better
ligand than LMorph, according to largest Brönsted basicity of the
pyrrolidine than the morpholine (pKb are 2.7 and 5.5, respectively).
In the neutral form of tridentate ligand (as phenol derivative),
identical energies have been found in both metahnol and water
solvents, indicating that it has not influence of the media.
However, when uncoordinated ligand is considered in its anion
form of (as phenolate salt), water stabilizes slightly LPyrr complexes
3.5. Thermogravimetric analysis
Thermal studies of all the four complexes show stepwise decom-
position. Fig. 12(a) and Fig. 12(b) depict the TGA diagrams of the
complexes of ligands HL1 and HL2, respectively. Decomposition pat-
tern clearly suggest that 2a is the most stable among the four. 2a
starts to disintegrate over 250 °C whereas other complexes start to
lose weight below 100 °C. That further tells 1, 1a and 2 contain water
molecules in crystal structure but 2a doesn’t bear any water mole-
cules. The loss of four water molecules present in complex 1 takes
place in single steps whereas 1a loss three water molecules in two
successive steps on heating. First step weight loss for 1 and 1a are
14.7% (Calcd. 17.07%) and 16.3% (Calcd 13.4%), receptively between
70 °C and 160 °C. Complex 2 shows first step weight loss of 7.5%
(Calcd. 5.0%) corresponds to the elimination of one water molecules
in the temperature range of 40–170 °C. All four species on further
heating generate NiO as the thermally stable end product (for com-
plex 1, Expt. wt loss = 86.7% at 730 °C, theo. Wt. loss = 82.4%; for
complex 1a Expt. wt loss = 86.5% at 640 °C, theo. Wt. loss = 81.6%;
for complex 2 Expt. wt loss = 83.23% at 800 °C, theo. Wt. loss = 78.9%
and for complex 2a Expt. wt loss = 76.4% at 630 °C, theo. Wt.
loss = 77.6%).
than LMorph
, by only 0.3 kcal/mol. The theoretical data has
been tabulated in Table S1–S3 (Supporting Information). (See
Scheme 4)
3.4. Biological activity of the complexes
The effect of the treatment of the compounds on Erhlich’s
ascites carcinoma cells on cell survivality is determined by MTT
assay. It is observed that among the four compounds, square pla-
nar 2a is most efficient in killing the tumor cells and least cyto-
toxicity is observed when the cells are treated with 1(Fig. 9A). In
all the cases, cytotoxicity increased with increasing dose of the

102
J. Adhikary et al. / Polyhedron 101 (2015) 93–102
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4. Conclusions
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Two N,N,O-donor Schiff-base ligands namely 2-[(2-Morpholin-
4-yl-ethylimino)-methyl]-phenol and 2-[(2-Pyrrolidin-1-yl-ethylimi
no)-methyl]-phenol have been designed and synthesized with the
view to explore the influence of non-coordinating heterocyclic parts
of the ligands on the structural variation of nickel complexes in pres-
ence of acetate and isothiocyanate as co-ligands. In presence of acet-
ate as co-ligand, both Schiff-base ligands yield octahedral complexes,
whereas, isothiocyanate helps to generate nickel complexes with
two different configurations, octahedral with HL1 and
square-planar with HL2. Investigation reveals that the nature of the
heterocyclic ring part present in N,N,O donor Schiff-bases i.e. ‘‘mor-
pholin’’ moiety in ligand HL1 and ‘‘pyrrolidine’’ moiety in ligand
HL2 is likely to be instrumental in generating Ni(II) complexes having
different configuration in presence of SCN as co-ligand. DFT calcula-
tions optimized correctly the molecular geometries of involved spe-
cies. However, relative energies of final isothiocyanoate complexes
do not agree with obtained compounds. The presence of the
intramolecular hydrogen bonding interactions modifies relative sta-
bility of calculated species, indicating that not only dielectric contin-
uum has to be taken into account, and these interactions with protic
solvents should be considered. Anticancer activity of the complexes
on Erhlich’s ascities carcinoma (EAC) reveals that Ni(II) having
square-planar geometry exhibits better activity over analogous
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Appendix A. Supplementary data
CCDC 971485–971488 contains the supplementary crystallo-
graphic data for the four complexes. These data can be obtained
free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.
html, or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or
e-mail: deposit@ccdc.cam.ac.uk. Supplementary data associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.poly.2015.07.055.
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