Synthesis, characterization and anticancer activity of mono- and dinuclear Ni(II) and Co(II) complexes of a Schiff base derived from o-vanillin

Article abstract of DOI:10.1016/j.poly.2018.12.055

A Schiff base ligand, 6,6′-dimethoxy-2,2′-[o-phenylenebis(nitrilomethylidyne)]diphenolato (LA), and its mononuclear and dinuclear Ni(II) and Co(II) complexes were synthesized. Structural elucidation was carried out via elemental analysis, molar conductivi

Full text of DOI:10.1016/j.poly.2018.12.055

Accepted Manuscript
Synthesis, characterization and anticancer activity of mono- and binuclear Ni(II)
and Co(II) complexes of a Schiff base derived from o-vanillin
Hadariah Bahron, Siti Solihah Khaidir, Amalina Mohd Tajuddin, Kalavathy
Ramasamy, Bohari M. Yamin
PII:
S0277-5387(19)30015-4
https://doi.org/10.1016/j.poly.2018.12.055
POLY 13673
DOI:
Reference:
To appear in:
Polyhedron
Received Date:
Revised Date:
Accepted Date:
29 September 2018
21 December 2018
21 December 2018
Please cite this article as: H. Bahron, S.S. Khaidir, A.M. Tajuddin, K. Ramasamy, B.M. Yamin, Synthesis,
characterization and anticancer activity of mono- and binuclear Ni(II) and Co(II) complexes of a Schiff base derived
from o-vanillin, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2018.12.055
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Synthesis, characterization and anticancer activity of mono- and binuclear
Ni(II) and Co(II) complexes of a Schiff base derived from o-vanillin
,
a
a
a,e
b,c
Hadariah Bahron,* Siti Solihah Khaidir, Amalina Mohd Tajuddin, Kalavathy Ramasamy
and Bohari M. Yamind
aFaculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam,
Selangor, Malaysia.
bFaculty of Pharmacy, Universiti Teknologi MARA (UiTM), 42300 Bandar Puncak Alam,
Selangor Darul Ehsan, Malaysia
cCollaborative Drug Discovery Research (CDDR) Group, Pharmaceutical and Life Sciences
Community of Research, Universiti Teknologi MARA (UiTM), 40450 Shah Alam, Selangor Darul
Ehsan, Malaysia
dFaculty of Science and Technology, Universiti Sains Islam Malaysia, Bandar Baru Nilai, 71800
Nilai, Negeri Sembilan, Malaysia.
eAtta-ur-Rahman Institute for Natural Product Discovery (AuRIns), UiTM Puncak Alam,
Selangor, Malaysia.
*
Corresponding author: hadariah@salam.uitm.edu.my
ABSTRACT
A Schiff base ligand, 6,6’-dimethoxy-2,2’-[o-phenylenebis(nitrilomethylidyne)]diphenolato (LA),
and its mononuclear and dinuclear Ni(II) and Co(II) complexes were synthesized. Structural
elucidation was carried out via elemental analysis, molar conductivity, IR, NMR, UV-Visible
spectroscopy, TGA and magnetic susceptibility data. Single crystal X-ray diffraction of the Ni(LA)
complex revealed a square planar geometry of the Ni(II) centre, with the ligand acting as a
tetradentate ONNO chelate. Upon coordination to the metal centers, the (C=N) and (C-O) peaks
shifted to lower frequencies, with appearance of new peaks assignable to (M-N) and (M-O) in
-1
the ranges 535-560 and 421-460 cm , respectively. Ni(LA) and Ni (LA) complexes are
2
diamagnetic, whereas the Co(LA) and Co (LA) complexes are paramagnetic with 3 unpaired
2
electrons each. All the complexes display low molar conductivities in acetonitrile indicating non-
electrolytic behavior. Anticancer screening against human colorectal cancer HCT116 cell lines
revealed that both dinuclear complexes were more active than their mononuclear counterparts.

Ni (LA) showed the highest potency with an IC value of 0.81 M.
2
50
Keywords: mononuclear, dinuclear, complexes, Schiff base, HCT116, anticancer, o-vanillin.
1
. Introduction
Since the introduction by Hugo Schiff in 1864, Schiff bases, also known as imines or azomethines,
have gained phenomenal popularity mainly due to their ease of synthesis, air stability and relatively
cheap starting materials. They are good chelating agents, especially those with potentially
coordinating functional groups such as S-H or O-H near the site of condensation [1]. Their ability
to chelate metal cations is greatly enhanced by introducing donor atoms at strategic positions in
the molecule, allowing a huge array of metal complexes with interesting structural architectures
[2-4] such as binuclear and polynuclear.
The presence of nitrogen and oxygen donor atoms renders these compounds effective and
stereospecific as catalysts for bond formation, oxidation, reduction, hydrolysis and other
transformation of inorganic and organic compounds [5-8]. Fine tuning of molecular electronic
properties can be achieved by placing electron withdrawing or donating substituents in the
molecules, making them extremely attractive for investigation for various applications such as
antimicrobial agents [9], sensor materials [10], pharmacological and electrochemical agents [11].
Schiff bases and their complexes have been reported to show significant bioactivity [12] owing to
the azomethine linkage and/or hetero atoms and metals present in their structures. The assessment
of novel synthetic Schiff bases as cytotoxic and cytostatic agents has irrefutably been growing
exponentially because of their proven effectiveness as attractive frontrunner structures [13].
Anticancer properties are commonly observed to increase upon coordination of these ligands with
transition metals, such as zinc, copper, cobalt and nickel [14]. This enhanced bioactivity may be
explained by Overton’s concept and chelation theory [15] that are based on lipophilicity and
penetration of the complexes through the lipid membrane. However, there were isolated reports of
ligands displaying higher bioactivity than their metal complexes [16], indicating the potency of
the organic compounds by themselves.

st
Cancer has been ranked among the most fatal diseases of the 21 century. A wide range of
cytotoxic drugs with different mechanisms of action are used to treat this disease, either alone or
in combination [17]. The main drawback of these drugs is the side effects that are caused mainly
by their indiscriminate action against both cancerous and healthy cells. There is an urgent need to
find new and improved anticancer agents to fight this disease.
This paper describes the synthesis, structure elucidation and anticancer screening of a Schiff base
ligand, LA, derived from ortho-vanillin and ortho-phenylenediamine, together with its
mononuclear and binuclear nickel(II) and cobalt(II) complexes, annotated as Ni(LA), Ni (LA),
2
Co(LA) and Co (LA), respectively. The ligand and complexes were characterized via their
2
physicochemical and spectral properties, and X-ray crystallography for Ni(LA). The activity of
the compounds against human colorectal cancer HCT116 is also reported.
2
. Experimental part
2
.1. Synthesis
The chemicals used in this investigation were of analytical grade. Ortho-vanillin (OVan), ortho-
phenylenediamine (OPD), nickel(II) acetate tetrahydrate and cobalt(II) acetate tetrahydrate were
purchased from Sigma Aldrich (St Louis, US). All chemicals and solvents were used as purchased
without prior purification.
2
.1.1. Synthesis of the ligand LA
The ligand synthesis is depicted in Scheme 1. OVan (30 mmol, 4.56 g) and OPD (15 mmol, 1.62
g) were dissolved in ethanol (20 mL), separately. Both solutions were mixed in a round-bottomed
flask and refluxed for an hour, after which an orange precipitate appeared. The mixture was left to
cool at room temperature before the precipitate was filtered off, washed with cold ethanol and
dried in a desiccator overnight.

Elemental analysis for LA, analysed as C H N O , calc (found): C 70.20 (69.90), H 5.33 (5.36),
2
2
20
2
4
-1
1
N 7.44 (7.20)%. IR bands (KBr pellet, cm ): 1612, 1248, 1205. H NMR (500 MHz, CDCl ) δ
3
(ppm): 11.20 (s, 1H), 9.93 (s, 1H), 7.29 (1H), 7.20 (dd, J = 7.8, 1.4 Hz, 1H), 7.13 (dd, J= 8.0, 1.4
1
3
Hz, 1H), 6.98 (t, J= 7.90 Hz, 1H), 3.93 (s, 3H). C NMR δ (ppm): 164.10 (C-OH), 161.90 (C=N),
1
42.00-115.00 (Ar-C), 56.00 (-OCH3)
2
.1.2. Synthesis of the mononuclear complexes
The mononuclear complex synthesis is depicted in Scheme 2. The orange LA ligand (0.6 mmol,
.2511 g) and nickel(II) acetate tetrahydrate (0.6 mmol) were mixed in a round-bottomed flask
0
with ethanol (15 mL), then refluxed for one hour, upon which a change of color to red was
observed. After slow cooling, a brick red precipitate was formed. It was filtered off, washed with
cold ethanol and air dried. The solid Ni(LA) precipitate was recrystallized in a mixture of
chloroform and dimethylsulfoxide (DMSO). The Co(LA) complex was prepared using the same
protocol. The reaction of the ligand LA (0.4592 g, 1.22 mmol) with cobalt(II) acetate tetrahydrate
(0.3039g; 1.22 mmol) afforded a brown precipitate.
Elemental analysis for Ni(LA), analysed as C H N NiO (H O), calc (found): C 58.58 (58.52), H
2
2
18
2
4
2
-1
1
4
.47 (4.27), N 6.21(6.31)%. IR (KBr pellet, cm ): 1610, 1242, 1195, 550, 437. H NMR (500
MHz, CDCl ) δ (ppm): 8.27 (s, 1H), 7.72 (dd, J = 6.1, 3.3 Hz, 1H), 7.21-7.15 (m, 1H), 6.95 (d, J
3
1
3
=
7.6 Hz, 1H), 6.72 (d, J = 7.3 Hz, 1H), 6.57 (t, J = 7.8 Hz, 1H), 3.87 (s, 3H), C NMR δ (ppm):
1
54.00 (C-OH), 142.00 (C=N), 127.00 -112.00 (Ar-C), 55.00 (-OCH ).  = 0 BM
3
eff
Elemental analysis for Co(LA) analysed as C H CoN O (H O) , calc (found): C 56.30 (56.50),
2
2
18
2
4
2
2
-1
H 4.72 (4.37), N 5.97 (5.97)%. IR (KBr pellet, cm ): 1605(vs), 1244, 1193, 542, 456.  = 3.96
eff
BM
2
.1.3. Synthesis of the dinuclear complexes
The dinuclear complex synthesis is depicted in Scheme 3. The Ni (LA) complex was obtained by
2
reacting the ligand LA (0.4514 g, 1.2 mmol) with nickel(II) acetate tetrahydrate (0.5972 g, 2.4

mmol). The ligand and metal salt were dissolved in 25 mL of a 4:1 ratio of absolute EtOH:CHCl3
and refluxed for three hours. The mixture was left to cool to room temperature and the brick red
precipitate obtained was filtered off, washed several times with cold EtOH, air dried and stored in
a desiccator over blue silica gel. The Co (LA) complex was prepared using the same protocol
2
where the ligand LA (0.4514 g, 1.2 mmol) and cobalt(II) acetate tetrahydrate (0.5978 g, 2.4 mmol)
were reacted, affording a brown precipitate.
Elemental analysis for Ni (LA) analysed as C H N Ni O (H O)(CH CO ) , calc (found): C
2
22 18
2
2
4
2
3
2 2
-1
4
9.74 (49.47), H 4.17 (4.18), N 4.46 (4.64)%. IR (KBr pellet, cm ): 1609, 1244, 1193, 526, 458.
1
H NMR (500 MHz, CDCl ) δ (ppm): 8.30 (s, 1H), 7.74 (dd, J = 6.0, 3.3 Hz, 1H), 7.22 (dd, , J =
3
6
.1, 3.2 Hz, 1H), 6.95 (d, J = 7.9 Hz,1H), 6.73 (d, J = 7.1 Hz, 1H), 6.59 (t, J = 7.8 Hz, 1H), 3.88
1
3
(
s, 3H). C NMR δ (ppm): 154.00 (C-OH), 142.00 (C=N), 127.00-112.00 (Ar-C), 55.00 (-OCH ).
3
eff = 0 BM
Elemental analysis for Co (LA) analysed as C H Co N O (H O) , calc (found): C 48.37 (49.03),
2
22 18
2
2
4
2
3
-1
H 4.43 (4.57), N 5.13 (4.88)%. IR (KBr pellet, cm ): 1605, 1243, 1194, 543, 458.  = 3.56 BM
eff
2
.2. Physical measurements
Elemental analyses (carbon, hydrogen and nitrogen) were performed on a Thermo Finnigan Flash
EA 110 Elemental Analyzer. Infrared spectra were recorded as KBr discs in the range 4000-400
-1
cm on a Perkin-Elmer FT-IR 1600 spectrometer. Molar conductivities were measured on a
Mettler Toledo 730 Series conductivity meter using a conductivity standard of 1413 S/cm.
Mgnetic susceptibilities were measured on a Sherwood Auto balance. UV-Visible absorption
-5
spectra were recorded as 1 x 10 M acetonitrile solutions on a Perkin Elmer Lambda 35 UV-Vis
spectrometer. Thermal analyses were performed in a nitrogen atmosphere using a TA Instruments
1
13
Q50 TGA analyser. H and C NMR spectra were recorded in deuterated chloroform on a Bruker
Avance 500 MHz spectrometer. All measurements were carried out at room temperature and
pressure.
2
.3. X-ray crystallographic analysis

Red crystals of Ni(LA) were grown from a 1:3 mixture of CHCl :DMSO. A single crystal was
3
selected, mounted on a glass fiber with epoxy cement and placed in the goniometer prior to data
collection. A Bruker D8 QUEST with a photon CCD area-detector diffractometer (Bruker, AXS
Inc., Madison, WI, USA) and graphite-monochromated Mo-Kα radiation (λ =0.71073 Å) was used
for data collection. The data collected were reduced using the program SAINT [18] and an
empirical absorption correction was applied using SADABS [19]. The structure was solved by
direct methods and refined by using the full- matrix least-squares method using the SHELXTL
[20] software package. All non-H atoms were anisotropically refined. The hydrogen atoms bonded
to water oxygen atom were located by difference syntheses and refined isotropically. The
molecular graphics were created using SHELXTL and MERCURY software. The PLATON
program was used for the molecular structure calculation [21]. Atomic scattering factors and
anomalous dispersion corrections were taken from the International Tables for X-ray
crystallography.
2
2
.4 Cytotoxicity assay
.4.1 Cell Culture
The human colorectal carcinoma cell line, HCT116 (ATCC® CCL-247™), was cultured in the
Roswell Park Memorial Institute, RPMI 1640 Medium w/25mM HEPES & L-Glutamine, Biowest,
supplemented with 10% heat inactivated fetal bovine serum (FBS)(PAA Laboratories) and 1%
penicillin/streptomycin, Sigma Aldrich, (St Louis, US). The cultures were maintained in a
humidified incubator at 37 °C in an atmosphere of 5% CO2.
2
.4.2 MTT assay

HCT116 cells were plated at 7,000 cells per well and allowed to incubate at 37 C for 24 hours.
The Schiff base LA and its metal complexes were dissolved in DMSO separately as stock solutions
with a concentration of 1000 M. Serial dilution with DMSO was carried out before being added
to each well. DMSO is a commonly used solvent in bioactivity investigations for metal complexes
[
22] and being an aprotic solvent, DMSO has small tendency to interact with metal complexes
23]. The cells were treated with the compounds at concentrations ranging between 0.01 and 100
[
M and incubated at 37 C for 72 hours. The cytotoxicity of the compounds was assessed using
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, from Sigma Aldrich, as described
by [24], with minor modifications. Briefly, 50 L of 0.06 mol/L MTT solution (Sigma) was added
to each well and the plates were incubated at 37 C for 4 hours. All solutions were aspirated and
the formazan crystals were dissolved in DMSO (Merck). The plate was read at 450 nm. Data
generated were used to plot a dose-response curve from which the concentration of compounds
required to kill 50% of the cell population (the IC value) was determined. Neat DMSO was used
5
0
as a negative control.
3
. Results and discussion
The physical properties and elemental analyses data for the Schiff base LA and its complexes are
presented in Table 1. The experimental percentages of C, H and N were in close accord with the
theoretical values, indicating formation of the intended compounds. The melting points of the
complexes were higher than that of the ligand (Table 1), a trait commonly reported [25], likely due
to increased molecular sizes and to a certain extent, the presence of stronger dative covalent and
ionic bonds. The molar conductivity values of the complexes in acetonitrile were very low, in the
-1
2
-1
range 0.03-0.22  cm mol , indicative of the non-electrolytic nature of the complexes.
3
.1. Infrared spectroscopy
The significant infrared bands of the LA ligand and its metal complexes are listed in Table 2. The
spectra, as shown in Fig. 1, provide valuable information on the functional groups present in the
compounds [26]. The spectrum of the free Schiff base exhibited a high intensity band at 1612
-1
cm , attributed to (C=N) stretching in accordance with the report by Ghani et al. [27]. This band
-1
was observed to be shifted slightly to lower frequencies of 1605-1610 cm in the complexes, likely

due to coordination of the metal atom to the imine nitrogen atom through sharing of the N lone
pair of electrons in a dative manner. The lowering of the frequency indicated a weakening of the
C=N bond upon coordination to the metal center, caused by a decrease in the -electron density
due to an inductive effect.
The involvement of deprotonated phenolic oxygen atom in chelation was indicated by the shifting
to lower frequencies of the (C-O)phenolic peak. The medium intensity (C-O)phenolic band that was
-1
-1
observed at 1248 cm in LA shifted slightly to lower wavenumbers of 1243 cm in Co (LA) and
2
-1
-1
1
244 cm in Ni(LA), Ni (LA) and Co(LA). There are two bands between 1540 and 1490 cm
2
that are not present in the ligand spectra that can be assigned to coupled (C=N) + (C=C) + (C-
C) modes [28-29].
The coordination of the imine nitrogen and phenolic oxygen atoms to the metal centers was
-1
supported by the appearance of weak (M-N) and (M-O) bands at 526-543 and 456-460 cm ,
respectively. This observation is in good agreement with previous assignments [30-31].
3
.2. NMR spectroscopy
1
13
The H and C NMR data for LA and Ni(LA) are listed in the Tables 3 and 4, respectively. The
1H NMR spectra (Fig. 2) showed integrations and multiplicities consistent with the proposed
structures. The peak at δ 9.94 ppm for LA represented the signal for the -OH hydrogen atom. In
the spectrum of Ni(LA), deprotonation of OH upon complexation was observed by the
disappearance of the -OH hydrogen atom signal.
The chemical shift of the azomethine hydrogen atom (-HC=N-) was shielded in the spectrum of
Ni(LA) at δ 8.27 ppm compared to the free Schiff base at δ 8.66 ppm, inferring coordination of
the Ni(II) ion to the LA ligand through the azomethine nitrogen atom [32]. The spectra of the
ligand and Ni(LA) show aromatic hydrogen atom chemical shifts in the ranges of δ 6.87-7.29 and
6
.56-7.73 ppm, respectively. The non-equivalent aromatic hydrogen atoms interact with each other
to produce spin-spin splitting patterns indicative of meta and ortho interactions, with J coupling
values of 1.4-3.3 Hz and 6.1-8.0 Hz, respectively. The methoxy signals appeared unsplit and stand
out as tall sharp singlets in the upfield region of δ 3.87 ppm in LA and Ni(LA).

1
3
The C NMR spectra recorded signals of sharp peaks representing the chemically non-equivalent
1
3
carbon atoms in the compound (Table 4). The C peak for C-O was observed at δ 154 ppm, C=N
at δ 142 ppm, aromatic C atoms in the range δ 112-127 ppm and –OCH at δ 55 ppm.
3
3
.3. UV-Visible spectroscopy
The UV-Visible data and band assignments are listed in Table 5. The spectra of the free
ligand and its complexes exhibited a high intensity band in the region 200-300 nm, assignable to
π–π* of the aromatic benzene and π–π*(C=N) of the azomethine groups. Upon complexation, the
(C=C)
metal complexes showed a hypsochromic shift (blue shift) to lower wavelengths for both electronic
transition bands, likely due to the possible occurrence of a significant amount of -backbonding
from the metal to the ligand, enriching the C=N bond with electrons and strengthening it.
*
The peaks attributed to n–π were observed in the range 330-377 nm. In the electronic
spectra of the metal complexes, except for that of Co(LA), the bands showed hypsochromic
shifting to lower wavelengths, indicating strengthening of the C=N bond. All the complexes
exhibited a weak broad band in the region 432-494 nm due to a ligand to metal charge transfer
(LMCT) from the  of the ligand to the hybrid orbitals of the metal atoms.
3
.4. Thermogravimetric analysis
The elemental microanalysis of the compounds, shown in Table 1, indicate that Ni(LA), Ni (LA),
2
Co(LA) and Co (LA) contained one, one, two and three molecules of water, respectively. TGA
2
was performed to investigate the nature of the bonding of the water molecules in these complexes.
The compounds were analysed in a platinum pan under nitrogen over the temperature range 30-
4
50 C with 10 C/minute increments. The temperature intervals and the percentage mass loss are
listed in Table 6, presenting the summary of the thermal decomposition of the water molecules
throughout the analysis. Ni(LA), Ni (LA) and Co(LA) showed a mass degradation at temperatures
2
below 160 C, indicating that water moieties are present as solvation molecules.

The thermogram of Co (LA) showed two stages of thermal degradation below 250 C. An
2
equivalence of two water molecules were lost between 36 and 125 C, and one at a higher
temperature of 125-210 C. This observation indicates that there are two lattice and one
coordinated water molecule in Co (LA). It is therefore noted that one of the Co(II) centres might
2
have a coordination number of 5 to account for this coordinated H O molecule, which is likely to
2
be the one that is bonded to the –OCH moieties, taking steric factors into account.
3
3
.5. Magnetic susceptibility
The magnetic moment values for the metal complexes furnish information on the geometry of the
central metals. There are two possible geometries for four-coordinate complexes, namely square
planar and tetrahedral [33]. Both Ni(LA) and Ni (LA) showed diamagnetism, indicating that the
2
8
compounds assume a square planar geometry, as expected for a Ni(II) 3d species [34]. The Co(II)
complexes revealed a  value of 3.88 B.M., very close to the spin only magnetic moment,  ,
eff
so
7
of 3.87 B.M. for 3 unpaired electrons. It is therefore suggested that the Co(II) d centre in Co(LA)
assumes a tetrahedral geometry. For Co (LA), one of the Co(II) centres is proposed to have
2
tetrahedral geometry whereas the other Co(II) ion, bonded to the fifth ligand of H O, takes a
2
distorted square pyramidal geometry.
3
.6. Structure description of Ni(LA)
The nickel complex crystallized in the monoclinic system with the space group P 21/n, a =
3
8
.8525(4) Å, b = 19.061(8) Å, c = 11.8339(5) Å, β = 99.546(2)°, Z = 4 and V = 1969.91(15) Å .
The crystal system and refinement parameters are shown in Table 7. The ORTEP diagram of the
complex, along with the atomic labelling scheme, is shown in Fig. 3. The Ni(II) atom is
coordinated within an ONNO square planar donor set, provided by the tetradentate Schiff base
ligand, LA. Selected bond lengths and bond angles are listed in Table 8. The Ni1—O2 and Ni1—
O3 distances are 1.8385(14) and 1.8419(14) Å, respectively, slightly shorter than the Ni1—N2
(1.8532(16) Å) and Ni1—N1 (1.8564(16) Å) bond lengths, as expected. Both phenolic hydroxyl
groups in the ligand are deprotonated upon complexation. The square planar geometry of the
complex is distorted as evidenced by the O2—Ni1—O3 and O2—Ni1—N2 bond angles of
8
4.18(6) and 175.35(7), respectively. The presence of a water molecule of crystallization in the

crystal structure is consistent with the results obtained from the micro-elemental analysis data
(
Table 1) and the TGA investigations (Table 6). A similar crystal structure of the analogous
Ni(C H N O )].H O, without the presence of methoxy groups, has been reported by Ghaemi et
[
2
0
14
2
2
2
al. [35].
In the crystal structure, the complex molecules are linked by a C7--H7A…O3 intermolecular
hydrogen bond, the symmetry code of which is given in Table 9, to form a zigzag chain along the
c axis (Figure 4). In addition, the solvated water molecule also forms hydrogen bonds with the
oxygen atoms O1, O2, O3 and O4 of the ligand, as listed in Table 9.
3
.7. Cytotoxicity assay against HCT 116 cell line
Fig. 5 shows the dose-response curves of LA and its complexes, from which their respective IC50
was derived as illustrated in Fig. 6 and listed in Table 10. The relative cytotoxicity of LA and its
complexes against HCT116 is in the order of Ni (LA) > Ni(LA) > LA > Co (LA) > Co(LA). The
2
2
ligand, LA, yielded IC at 29.40  5.90 µM indicating that even without the presence of any metal,
5
0
the ligand itself has the ability to interact and alter the DNA present in HCT116, hence retarding
its growth. Ni (LA) complex, on the other hand, exhibited the most potent anticancer activity with
2
IC recorded at 0.81  0.22 µM. It is interesting to note that contrary to the nickel(II) complexes,
5
0
both the cobalt(II) complexes have lower anticancer activities than their parent ligand.
While nickel(II) complexes exhibited much higher anticancer activities than their analogous
cobalt(II) complexes, both dinuclear complexes out-performed their mononuclear counterparts.
This observation indicates that to a certain extent, the anticancer potency of these compounds is
also metal-dependent.
4
. Conclusion
The Schiff base LA acts as a tetradendate ligand and coordinates to metal centers through its
phenolic O and azomethine N donor atoms. The appearance of new M-N and M-O peak in IR
spectra evidenced the involvement of azomethine N and phenolic O in complexation and the
1
disappearance of phenolic OH peak in H NMR spectra supported the coordination of the metal

ion via phenolic O. Ni(LA) single crystal x-ray diffraction data shows a distorted square planar
geometry with one solvated water molecule. With regard to cytotoxicity against HCT116,
dinuclear Ni (LA) was the most potent bioactive agent, followed by it mononuclear counterpart,
2
the parent ligand LA and the cobalt(II) compounds.
Appendix A. Supplementary data
CCDC 1533932 contains the supplementary crystallographic data for Ni(LA). 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)
1
223-336-033; or e-mail: deposit@ccdc.cam.ac.uk.
6
. ACKNOWLEDGEMENTS
The authors wish to thank Universiti Teknologi MARA for the research funding (600-
IRMI/PERDANA5/3 BESTARI (046/2018)) and the MyBrainSc scholarship by the Ministry of
Higher Education of Malaysia. Universiti Teknologi MARA is gratefully acknowledged for the
research facilities provided.
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Table 1
The physical properties and elemental analyses data for Schiff base ligand, LA and it complexes
series
Molar
Melting
Point
Elemental Percentages
Found (Calculated)
Molecular Formula
(RMM)
Yield
(%)
Conductivity
Compound
LA
Color
-1
2
-1
(
 cm mol )
(°C)
C (%)
H (%)
N (%)
C
25
H
26
N
2
O
4
69.90
(70.20)
5.36
(5.33)
7.20
(7.44)
Orange
89.8
88.5
170
N/A
(
418.49)
Brick
red
C
22
H
18
N
2
Ni O
4
(H
2
O)
58.52
(58.58)
4.27
(4.47)
6.31
(6.21)
Ni(LA)
>300
0.03
0.04
(451.10)
Ni
(CH CO
627.89)
(H
469.36)
C
22
H
18
N
2
2
O
4
(H
2
O)
Brick
red
49.47
(49.74)
4.18
(4.17)
4.64
(4.46)
Ni
2
(LA)
3
2
)
2
83.1
>300
(
C
22
H
18CoN
2
O
4
2
O)
2
56.50
(56.48)
4.37
(4.54)
5.97
(5.49)
Co(LA)
Co (LA)
Brown
Brown
94.57
98.44
>300
>300
0.22
0.15
(
C
22
H
18Co
2
N
2
O
4
(H
2
O)
3
49.03
(48.37)
4.57
(4.43)
4.88
(5.13)
2
(
546.31)
Table 2
Selected IR data of LA ligand and metal complexes in cm-1

(C-O)
Phenolic
(C-O)
Methoxy
Compound
(C=N)
(M-N)
(M-O)
LA
1612
1609
1609
1605
1605
1248
1205
1195
1193
1193
1194
-
-
Ni(LA)
1244
1244
1244
1243
535
526
542
543
460
458
456
458
Ni (LA)
2
Co(LA)
Co (LA)
2
Table 3
1
H chemical shifts for LA, Ni(LA) and Ni (LA)
2
Chemical shift, (ppm)
3
4
5
10
11
C2-
OCH
3
Assign.
C -H
Ar.)
C -H
C -H
(Ar.)
C -H
(Ar.)
C -H
1
8
C -OH
N=C -H
(
(Ar.)
(Ar.)

1
1.20
(s)
1H)
9.93
(s)
(1H)
7.29
(s)
(1H)
7.20
(dd;7.8,1.4)
(1H)
7.21-7.15
(m)
7.13
(dd; 8.0,1.4)
(1H)
6.95
(d;7.6)
(1H)
6.95
(d; 7.9)
(1H)
6.98
(t;7.9)
(1H)
6.72
(d;7.3)
(1H)
6.72
(d;7.1)
(1H)
3.93
(s)
(3H)
3.87
(s)
(3H)
3.87
(s)
LA
-
(
8
.27
7.72
6.57
(t;7.8)
(1H)
6.59
(t;7.8)
(1H)
Ni(LA)
-
-
(s)
(dd; 6.1,3.3)
(1H,)
(1H)
(1H)
7.22
8
.30
7.74
Ni
2
(LA)
(s)
(dd; 6.1,3.3) (dd; 6.1, 3.2)
(1H) (1H)
(1H)
(3H)
Note: Ar.=Aromatic, s=singlet, d=doublet, dd=doublet of doublet, t=triplet, m=multiplet
Table 4
1
3
C NMR chemical shifts for LA, Ni(LA) and Ni (LA)
2
Chemical shift, (ppm)
Assign.
N=C8-
H
C -H
2
C -H
6
C -H
3
C -H
5
C -H
4
C -H
11
C -H
10
C -H
9
OC H
3
7
1
C -OH
(Ar.)
(Ar.)
(Ar.)
(Ar.)
(Ar.)
(Ar.)
(Ar.)
(Ar.)
5
6.30
LA
196.61 151.65 148.30 127.69 124.53 121.96 121.89 120.81 119.58
157.85 154.19 142.79 127.75 127.27 124.16 119.41 115.10 114.85
186.62 179.65 166.37 139.46 130.53 127.31 124.08 119.42 115.16
117.99
Ni(LA)
112.80
112.82
55.29
55.30
2
Ni (LA)
Note: Ar.=Aromatic
Table 5
The UV-Visible data
Transition assignment
Wavelength (nm)
Molar absorptivity, ()
*
π–π benzene
250
306
337
215
283
330
432
254
300
369
480
219
269
358
489
256
290
377
494
26094
19668
5237
4764
2564
2636
1392
5058
1957
2977
755
34240
15634
21150
5667
12739
5309
6012
1708
*
LA
π–π
(C=N)
n–π*
π–π* benzene
*
π–π (C=N)
Ni(LA)
n–π*
LMCT
*
π–π benzene
*
π –π (C=N)
2
Ni (LA)
n –π*
LMCT
*
π–π benzene
*
π–π (C=N)
Co(LA)
n–π*
LMCT
*
π–π benzene
*
π–π (C=N)
2
Co (LA)
n–π*
LMCT
Table 6
The temperature intervals and the percentage of water loss
Decomposition
temperature
Weight loss (%)
Compound
Molecular formula
Lost species
Found Calc.
(C)

Ni(LA)
Ni (LA)
Co(LA)
Co (LA)
C
22
H
18
N
2
NiO
Ni
CH CO
4
(H
2
O)
O)
70-120
70-100
36-175
3.90
3.34
7.80
3.99
2.87
One lattice water molecule
One lattice water molecule
C
22
H
18
N
2
2
O
4
(H
2
2
(
3
2 2
)
C
22
H
18CoN
2
O
4
(H
2
O)
2
7.67
6.5
Two lattice water molecule
Two lattice water molecule
3
6-125
7.6
3.8
2
C
22
H
18Co
2
N
2
O
4
(H
2
O)
3
1
25-210
3.29 One coordinated water molecule

Table 7
Summary of crystal data and structure refinement parameters for Ni(LA) complex
Compound
Ni(LA)
CCDC
1533932
Identification code
Empirical formula
Formula weight
Temperature
Ni(LA)
C22 H20 N2 Ni O5
451.11
0(2) K
Wavelength
0.71073 Å
Monoclinic
P 21/n
Crystal system
Space group
Unit cell dimensions
a = 8.8525(4) Å

= 90°.
b = 19.0681(8) Å
c = 11.8339(5) Å


= 99.546(2)°.
= 90°.
3
Volume
1969.91(15) Å
4
Z
3
Density (calculated)
Absorption coefficient
F(000)
1.521 Mg/m
-1
1.023 mm
936
3
Crystal size
0.500 x 0.320 x 0.270 mm
2.759 to 28.417°.
Theta range for data collection
Index ranges
-11<=h<=11, -25<=k<=25, -15<=l<=15
Reflections collected
67732
Independent reflections
Completeness to theta = 25.242°
Refinement method
4928 [R(int) = 0.0525]
99.9 %
2
Full-matrix least-squares on F
Data / restraints / parameters
4928 / 3 / 279
2
Goodness-of-fit on F
1.066
Final R indices [I>2sigma(I)]
R indices (all data)
R1 = 0.0356, wR2 = 0.0766
R1 = 0.0544, wR2 = 0.0869
n/a
Extinction coefficient

-3
Largest diff. peak and hole
0.424 and -0.354 e.Å
Table 8
Selected bond lengths (Å) and bond angles ()
Bond lengths (Å)
Ni1 O2
Ni1 O3
Ni1 N2
Ni1 N1
1.8385 (14)
1.8419 (14)
1.8532 (16)
1.8564 (16)
Bond angles ()
O2 Ni1 O3
O2 Ni1 N2
O3 Ni1 N2
O2 Ni1 N1
O3 Ni1 N1
N2 Ni1 N1
84.18 (6)
175.35 (7)
95.32 (7)
94.88 (7)
173.34 (7)
86.14 (7)
Table 9
Intermolecular hydrogen bonds (Å, )
D --H ..A
D --H (Å)
H ..A (Å)
D--A (Å) D --H ..A ()
i
i
O1W --H1WA ..O3
O1W --H1WA ..O4
O1W --H1WB ..O1
O1W --H1WB ..O2
0.83 (3)
0.83 (3)
0.83 (2)
0.83 (2)
0.95
2.28 (3)
2.39 (3)
2.20 (19)
2.47 (3)
2.47
2.990 (2) 145(3)
3.114 (3) 147(3)
3.001 (3) 163(3)
3.034 (3) 127(3)
3.201 (3) 134
i
i
ii
C9
--H9A ..O2
(symmetry codes; i= -x,2-y,1-z , ii= -1/2+x,3/2-y,-1/2+z)
Table 10
IC of the investigated compounds against HCT116
5
0
Compound
Ni (LA)
0.81  0.22
Ni(LA)
1.46  0.56
LA
29.40  5.90
Co (LA)
45.50  11.39
Co(LA)
60.25  5.50
2
2
IC , M
5
0
Each data represents mean + standard deviation of quadruplicates.

H
O
N
N
Ethanol
hr
2
OH
+
OH
HO
1
H N
NH2
2
OCH3
OCH3
H CO
3
OVan
OPD
Scheme 1. Synthesis of LA ligand
LA
N
N
N
O
N
O
2
+
M
M , Ethanol
hr
M = Ni(II) / Co(II)
OH
HO
1
OCH3
H CO
3
OCH3
H CO
3
LA
M(LA)
Scheme 2. Synthesis of mononuclear metal complex M(LA)
N
N
N
O
N
O
2
M2+ , Ethanol: Chloroform
hr
M
M
3
OH HO
H CO
OCH3
3
O
O
CH3
H3C
M2(LA)
M = Ni(II) / Co(II)
LA
Scheme 3. Synthesis of dinuclear metal complex M (LA)
2
CAPTION TO THE FIGURES
Fig. 1. IR spectra of LA, Ni(LA), Ni (LA), Co(LA) and Co (LA)
2
2
1
Fig. 2. H NMR spectra of LA, Ni(LA)and Ni (LA)
2

Fig. 3. ORTEP diagram of the Ni(LA) complex with the atom labelling scheme, drawn at 50%
probability ellipsoids
Fig. 4. The crystal packing of the Ni(LA) viewed along a axis. All non-hydrogen bonded hydrogen
atoms are omitted for clarity. The hydrogen bonds are indicated by dashed lines.
Fig. 5. IC dose-response curves for LA and its complexes
5
0
Fig. 6 IC for LA and its complexes
5
0
FIGURE 1
C=N C-OOH C-
M-N M-O
Co2(LA)
Co(LA)
Ni2(LA)
Ni(LA)
LA
4
000
3600
3200
2800
2400
2000
1600
1200
800
400
Wavenumber, cm-1

FIGURE 2
FIGURE 3

FIGURE 4
FIGURE 5
IC50
1
1
50
00
Co(LA)
Co2(LA)
LA
Ni(LA)
5
0
0
Ni2(LA)
0
.005
0.05
0.5
5
50
Concentration, µM

FIGURE 6
IC50
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
6
0.25
4
5.5
2
9.4
4
.6
1
.46
0
.81
Standard 5-FU
LA
Ni(LA)
Ni2(LA)
Co(LA)
Co2(LA)
Synopsis of the graphical abstract
The Schiff base ligand LA was reacted with nickel(II) acetate and cobalt(II) acetate in different
L:M ratios and under different reaction conditions, resulting in mononuclear and dinuclear
complexes of both metals. Characterization was carried out via physicochemical and spectral
techniques, including single crystal X-ray crystallography for Ni(LA). The Ni(II) centre has a
distorted square planar geometry, with the ligand acting as a tetradentate ONNO chelate. There are
4
asymmetric units, each containing one water molecule of crystallization and one Ni(LA)
complex, in a unit cell. All the compounds were screened for anticancer activity against HCT116
cell lines and Ni(LA) emerged as the most potent compound with the lowest IC value of 0.81
5
0
mM. The order of anticancer potency was Ni (LA) > Ni(LA) > LA > Co (LA) > Co(LA).
2
2