Nickel(II) complexes of polyhydroxybenzaldehyde N4-thiosemicarbazones: Synthesis, structural characterization and antimicrobial activities

Article abstract of DOI:10.1007/s11243-013-9777-6

Nickel(II) complexes with 2,3-dihydroxybenzaldehyde N4-substituted thiosemicarbazone ligands (H₃L¹-H₃L ⁴) have been synthesized and characterized with the aim of evaluating the effect of N4 substitution in the thiosemicarbazone moiety on their coordination behavior and biological activities. Two series of nickel(II) complexes with the general formulae [Ni(H₃L)(H₂L)]ClO ₄ and [Ni₂(HL)₂] were characterized by analytical and spectral techniques. The molecular structure of one of the complexes, namely, [Ni(H₃L⁴)(H₂L ⁴)]ClO₄ was established by single crystal X-ray diffraction studies. The crystal structure of this complex revealed that two H₃L⁴ ligands are coordinated to nickel(II) in different modes; one as a neutral tridentate ONS ligand and the other is as a monoanionic tridentate (ONS^-) ligand. The antimicrobial activities of the compounds were tested against 25 bacterial strains via the disc diffusion method, and their minimum inhibitory concentration (MIC) and minimum microbicidal concentration were evaluated using microdilution methods. With a few exceptions, most of the compounds exhibited low-to-moderate inhibitory activities against the tested bacterial strains. However, the complexes [Ni ₂(HL³)₂] (7) and [Ni₂(HL ⁴)₂] (8) indicated higher inhibitory activity against Salmonella enterica ATCC 9068 (MIC values 15.7 and <15.7 μg/ml, respectively), compared with gentamicin as the positive control (MIC 25 μg/ml). Complex (7) also inhibited Streptococcus pneumoniae more efficiently (MIC 31.2 μg/ml), compared with gentamicin (MIC > 50 μg/ml). The toxicities of the compounds were tested on brine shrimp (Artemia salina), where no meaningful toxicity level was noted for both the free ligands and the complexes. The cytotoxicities of the compounds on cell viability were determined on MCF7, PC3, A375, and H413 cancer cells in terms of IC₅₀; complexes [Ni(H₃L³)(H₂L³)]ClO ₄ (3), [Ni₂(HL³)₂] (7) and [Ni ₂(HL⁴)₂] (8) exhibited significant cytotoxicity on the tested cell lines.

Full text of DOI:10.1007/s11243-013-9777-6

Transition Met Chem (2014) 39:81–94
DOI 10.1007/s11243-013-9777-6
Nickel(II) complexes of polyhydroxybenzaldehyde N4-
thiosemicarbazones: synthesis, structural characterization
and antimicrobial activities
•
Hana Bashir Shawish Mohammadjavad Paydar Chung Yeng Looi
•
•
•
Yi Li Wong Elaheh Movahed Siti Nadiah Abdul Halim Won Fen Wong
•
•
•
•
Mohd-Rais Mustafa Mohd Jamil Maah
Received: 15 July 2013 / Accepted: 25 October 2013 / Published online: 8 November 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract Nickel(II) complexes with 2,3-dihydroxybenz-
aldehyde N4-substituted thiosemicarbazone ligands (H3L1–
H3L4) have been synthesized and characterized with the aim
of evaluating the effect of N4 substitution in the thiosemi-
carbazone moiety on their coordination behavior and bio-
logical activities. Two series of nickel(II) complexes with
the general formulae [Ni(H3L)(H2L)]ClO4 and [Ni2(HL)2]
were characterized by analytical and spectral techniques.
The molecular structure of one of the complexes, namely,
[Ni(H3L4)(H2L4)]ClO4 was established by single crystal
X-ray diffraction studies. The crystal structure of this com-
plex revealed that two H3L4 ligands are coordinated to
nickel(II) in different modes; one as a neutral tridentate ONS
ligand and the other is as a monoanionic tridentate (ONS-)
ligand. The antimicrobial activities of the compounds were
tested against 25 bacterial strains via the disc diffusion
method, and their minimum inhibitory concentration (MIC)
and minimum microbicidal concentration were evaluated
using microdilution methods. With a few exceptions, most of
the compounds exhibited low-to-moderate inhibitory activ-
ities against the tested bacterial strains. However, the com-
plexes [Ni2(HL3)2] (7) and [Ni2(HL4)2] (8) indicated higher
inhibitory activity against Salmonella enterica ATCC 9068
(MIC values 15.7 and\15.7 lg/ml, respectively), compared
with gentamicin as the positive control (MIC 25 lg/ml).
Complex (7) also inhibited Streptococcus pneumoniae more
efficiently (MIC 31.2 lg/ml), compared with gentamicin
(MIC [ 50 lg/ml). The toxicities of the compounds were
tested on brine shrimp (Artemia salina), where no mean-
ingful toxicity level was noted for both the free ligands and
the complexes. The cytotoxicities of the compounds on cell
viability were determined on MCF7, PC3, A375, and H413
cancer cells in terms of IC50; complexes [Ni(H3L3)
(H2L3)]ClO4 (3), [Ni2(HL3)2] (7) and [Ni2(HL4)2] (8)
exhibited significant cytotoxicity on the tested cell lines.
Introduction
The synthesis and characterization of coordination com-
pounds with ligands containing nitrogen and sulfur donors
have evolved during the last few years as one of the main
research areas in coordination chemistry as well as in bio-
chemistry [1, 2]. Among the nitrogen/sulfur compounds,
thiosemicarbazones are one of the most studied ligands, not
only because of their flexibility to bind metal ions in various
modes, but also because of the wide range of applications
that they possess [3, 4]. The capability of thiosemicarba-
zones to coordinate to metals is due to the delocalization of
electron density over the NHC(S)NHN system, which is
improved by substitution at the N(4) position. Consequently,
thiosemicarbazones can coordinate to metal ions as mono-
dentate ligands through the thiocarbonyl sulfur or as biden-
tate through the thiocarbonyl sulfur and one of the hydrazinic
nitrogen atoms, leading to the formation of either a five or a
four membered chelate ring (Scheme 1a) [5, 6]. Further-
more, the presence of an additional coordinating group in the
carbonylic part opens up many more coordination
H. B. Shawish Á S. N. A. Halim Á M. J. Maah (&)
Department of Chemistry, Faculty of Sciences, University of
Malaya, 50603 Kuala Lumpur, Malaysia
e-mail: mjamil@um.edu.my
M. Paydar Á C. Y. Looi Á Y. L. Wong Á M.-R. Mustafa
Department of Pharmacology, Faculty of Medicine, University
of Malaya, 50603 Kuala Lumpur, Malaysia
E. Movahed Á W. F. Wong
Department of Medical Microbiology, Faculty of Medicine,
University of Malaya, 50603 Kuala Lumpur, Malaysia
123

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Transition Met Chem (2014) 39:81–94
Scheme 1 Coordination
possibilities of
thiosemicarbazones
X
R
M
R
M
M
N
N
H
S
N
R1
N
H
S
N
R1
N
S
H
N
R1
N
H
N
H
R2
R2
R2
b
a
OH
possibilities, increasing the flexibility in coordination to both
hard and soft metal ions, and resulting in the formation of
complexes having five- and six-membered chelate rings
(Scheme 1b) [7]. The biological activities of thiosemicar-
bazones have gained considerable interest due to their good
activities as anticancer [8, 9], antimalarial [10], antiepileptic
[11] and antifungal [12] agents. It has been shown that
complexation of thiosemicarbazones with metal ions can
increase their biological activities. In order to find new
compounds with higher activity, variation of the thiosemi-
carbazone frame has been extensively studied. Relationships
between the structures of thiosemicarbazones, their transi-
tion metal complexes and their interactions with DNA are
evident in a number of cases [13, 14].
OH
1
N
S
2
H
N
3
4
H
N
H
R
Scheme 2 Chemical structure of 2,3-dihydroxybenzaldehyde –N4-
substituted thiosemicarbazone. H3L1; R=H, H3L2; R=–CH3, H3L3;
R=–C6H5, H3L4; R=–C2H5
A number of thiosemicarbazone metal complexes with
ONS-donor ligands, especially salicylaldehyde thiosemi-
carbazone ligands, have been synthesized and character-
ized by means of X-ray diffraction and spectroscopic
techniques [15, 16]. The ONS-donor set can lead to unu-
sual stereochemical, electrochemical, and electronic prop-
erties in many of their metal complexes [17].
Experimental
Materials and measurements
The bioinorganic chemistry of nickel has been rapidly
expanded since the discovery of nickel in the active site of
several metalloenzymes [18]. Although certain nickel
compounds are considered to have environmental toxicity
and carcinogenic nature, many structurally characterized
nickel complexes have shown antibacterial, antifungal, and
anticancer/antiproliferative activity. The key target of
anticancer metallodrugs is DNA; thus, the interaction of
Ni(II) complexes with DNA is mainly dependent on the
structure of the ligand and can involve intercalative
behavior and/or DNA cleavage ability [19].
2,3-Dihydroxybenzaldehyde, thiosemicarbazide, 4-methyl-
3-thiosemicarbazide, 4-ethyl-3-thiosemicarbazide, 4-phe-
nyl-3-thiosemicarbazide, nickel(II) perchlorate hexahy-
drate, and nickel(II) acetate tetrahydrate were obtained
from Sigma-Aldrich. All the solvents used in the reaction
were from Merck and used as received.
FT-IR spectra were recorded on a Perkin-Elmer Spec-
trum RX-1 spectrophotometer as KBr pellets in the fre-
quency range of 4,000–400 cm-1. NMR spectra were
recorded in deuterated DMSO-d6 on an ECA 400 MHz
instrument. Elemental analyses were performed on a
Thermo Finnigan Eager 300 CHNS elemental analyzer.
Although metal complexes of salicylaldehyde thiosemi-
carbazone ligands have received considerable attention in
view of their chemistry and biological properties, fewer
studies have focused on the complexation behavior and
biological activities of polyhydroxybenzaldehyde thiosem-
icarbazone derivatives [20–22]. In this paper, we report the
preparation, structural characterization and antimicrobial
studies of nickel complexes of 2,3-dihydroxybenzaldehyde
N4-substituted thiosemicarbazone.
Synthesis of the ligands
The thiosemicarbazone ligands H3L (Scheme 2) were
prepared following the published procedure [20] by treat-
ment of 2,3-dihydroxybenzaldehyde with N4-substituted
thiosemicarbazide in a 1:1 molar ratio, under standard
reflux conditions.
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Transition Met Chem (2014) 39:81–94
83
Synthesis of complexes [Ni(H3L)(H2L)]ClO4 (1–4)
alginolyticus, V. cholerae, V. fischeri, V. harveyi, V.
parahaemolyticus, V. vulnificus and Yersinia pestis.
To a colorless solution of 2,3-dihydroxybenzaldehyde thi-
osemicarbazone (1 mmol) (H3L1; 0.21 g, H3L2; 0.23 g,
H3L3; 0.29 g, H3L4; 0.24 g) in ethanol (15 ml) was added
an ethanolic solution of Ni(ClO4)2Á6H2O (0.36 g, 1 mmol).
In each case, the dark green solution was stirred for about
4 h under reflux. The clear solution after filtration was left
for slow evaporation at room temperature.
The medium used in the assay for the antibacterial test
was Difco yeast extract–peptone–dextrose (YPD) broth and
agar medium (BD, NJ, USA). Bacterial initial suspensions
were prepared by the direct colony method. The colonies
were taken from the plate, suspended in 5 ml of sterile
Difco YPD broth and allowed to grow in a rotary shaker
(200 rpm) at 37 °C. The bacterial culture was then diluted
using sterile 0.85 % saline, and its turbidity was adjusted
by comparing with 0.5 McFarland’s standard to obtain
108 CFU/ml bacterial suspension [28].
Synthesis of complexes [Ni2(HL)2] (5–8)
2,3-Dihydroxybenzaldehyde thiosemicarbazone (2 mmol)
(H3L1; 0.42 g, H3L2; 0.46 g, H3L3; 0.58 g, H3L4; 0.48 g)
was dissolved in 20 ml ethanol. To this, a solution of
Ni(OAc)2Á4H2O (0.248 g, 1 mmol) in ethanol (10 ml) was
added. The mixture was refluxed for 2 h. The red solid
which precipitated out was filtered off and washed thor-
oughly with water and ethanol followed by ether, dried in
air and kept in a desiccator over silica gel.
Kirby–Bauer disc–diffusion experiments were carried
out as follows. An aliquot of 65 ll of each microorganism
was swabbed uniformly across a Difco YPD agar plate
using cotton swabs. Then, a sterile filter paper disc (diam-
eter 6 mm) impregnated with 20 ll of the compound
solution was placed on the surface of the agar. After treat-
ment, the plate was inverted (to avoid moisture on the agar
surface) and incubated for 16–18 h in air at 35 °C. At the
end of the incubation period, the plates were examined for
the inhibition of bacterial growth by measuring the diameter
(in mm) of the inhibition zone surrounding each filter paper
disc. In general, larger zones of inhibition indicate a more
active compound against the organism [29]. A bacterial
strain was considered resistant (R) to the drug tested when
the zone of inhibition was B12 mm; intermediate (I) when
the inhibition zone was between 12 and 15 mm, or sus-
ceptible (S) if the inhibition zone was C15 mm [30].
The microdilution plate method with resazurin was
applied to determine the minimum inhibitory concentra-
tions (MIC) and minimum microbicidal concentrations
(MMC) of the test compounds [31]. The 96-well plates
were prepared by dispensing 180 ll of nutrient broth to the
first row and 100 ll of the broth to the rest of the plate. A
20 ll aliquot from the stock solution (with a concentration
of 10 mg/ml) of the test compound was added into the first
row of the plate. Then, twofold serial dilutions were carried
out to obtain a range of concentrations from 1,000 to
7.81 lg/ml. A 10 ll aliquot of diluted bacterial suspension
was added to each well to give a final concentration of
5 9 105 CFU/ml. Finally, 10 ll resazurin solution was
added to each well inoculated with bacteria, and the plates
were incubated at 37 °C for 24 h. The MIC was defined as
the lowest concentration of the test compound that pre-
vented the resazurin color change from blue to pink. The
experiment was performed in triplicate using gentamicin as
positive control. In a solvent control test, the effect of 10 %
DMSO was studied on the growth of microorganisms, and
no inhibitory activity was observed [32].
X-ray crystallography
Green crystals of complex 4 were grown by slow evapo-
ration of the mother liquor and were analyzed by single
crystal diffraction. The data were collected at 273 K on a
Bruker Apex 2 diffractometer with Mo–Ka radiation
˚
(k = 0.71073 A), and the data reduction was performed
using Bruker SAINT [23]. The structure was solved using
direct methods, which yielded the positions of all non-
hydrogen atoms. These were refined first isotropically and
then anisotropically. All hydrogen atoms were placed in
calculated positions with fixed isotropic thermal parame-
ters and included structure factor calculations in the final
stage of full-matrix least-squares refinement. All calcula-
tions were performed using the SHELXTL suite of pro-
grams [24]. Molecular graphics were drawn using ORTEP
[25] and PLATON [26]. Material for publication was
prepared using publCIF [27].
In vitro antimicrobial assays
Stock solutions of the test compounds were prepared in
10 % v/v aqueous DMSO solution (Fisher chemicals) at
concentrations of 10 mg/ml. Antimicrobial activities of the
compounds was tested against 38 microorganisms,
including 25 pathogenic bacteria (standard and clinical
strains): Bacillus subtilis, Escherichia coli, Enterococcus
faecalis, Listeria innocua, L. monocytogenes, L. welshi-
meri, Proteus mirabilis, Pseudomonas aeruginosa, Sal-
monella enterica, S. enteritidis, S. typhi, S. typhimurium, S.
dysenteriae, S. flexneri, S. sonnei, Staphylococcus aureus,
S. epidermidis, Streptococcus pneumoniae, Vibrio
The MMC of each compound was evaluated by plating
10 ll of the samples from the wells with no indicator color
change, on YPD agar plates, followed by 16-h incubation
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Transition Met Chem (2014) 39:81–94
at 37 °C. The lowest concentration with no colony was
defined as the MMC of the compound against the micro-
organism [33].
the next day, the cells were treated with a twofold dilution
series of six concentrations of the solvents, and then
incubated at 37 °C in 5 % CO2 for 48 h. MTT solution
(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide)
was added at 2 mg/ml and after 2 h of incubation at 37 °C
in 5 % CO2, DMSO was added to dissolve the formazan
crystals. The plates were then read with a Chameleon
multitechnology microplate reader (Hidex, Turku, Finland)
at 570 nm absorbance. The cell viability percentage after
exposure to the solvents for 48 h was calculated by the
previously described method [36]. The ratio of the absor-
bance of treated cells to the absorbance of DMSO-treated
control cells was determined as cell viability (%). The
concentration of the solvent required to reduce the absor-
bance of treated cells to 50 % of the DMSO-treated control
cells was defined as the IC50.
Brine shrimp lethality assays
Brine shrimp lethality assays (BSLA) were carried out as
follows. Sea salt (38 g) was dissolved in distilled water (1 l)
to prepare artificial sea water as brine shrimp growth med-
ium. After filtration, brine shrimp (Artemia salina) eggs were
added to the medium and allowed to hatch by incubation at
room temperature in the dark for 48 h. The brine shrimps
were then seeded in 6-well plates (10 shrimps and 3 ml of
artificial sea water per well) and treated with 6 concentra-
tions of each compound (200, 100, 50, 25, 12.5, 6.25 lg/ml).
Control experiments were simultaneously conducted. After
24 h, the number of surviving shrimps was counted and data
were analyzed using PROBIT analysis software to determine
the median lethal concentration (LC50). The experiments
were done in triplicate [34].
Results and discussion
Synthesis and general properties
In vitro anticancer activity
Four 2,3-dihydroxybenzaldehyde N4-substituted thiosemi-
carbazones (H3L1–4) have been used in this study, differing
in the substituent on nitrogen N4 of the thiosemicarbazone
moiety (R = H; H3L1, CH3; H3L2, C6H5; H3L3 and C2H5;
H3L4), in order to observe their influence on the biological
activities of the complexes. Reaction of these thiosemi-
carbazone ligands with Ni(ClO4)2Á6H2O in a molar ratio
1:1 afforded mononuclear complexes 1–4 of the general
formula [Ni(H3L)(H2L)]ClO4 (Scheme 3a), while reactions
of these thiosemicarbazones with Ni(OAC)2Á4H2O in a
molar ratio 2:1 (ligand:metal) yielded complexes (5–8) of
the general formula [Ni2(HL)2] (Scheme 3b). The stoichi-
ometries, partial elemental analyses and colors of the
complexes are shown in Table 1. The crystals of the
nickel(II) complexes (1–4), prepared by slow evaporation
of the solution remaining after filtration of the reaction
mixtures, were green colored and soluble in common
organic solvents such as methanol, ethanol, acetone, and
acetonitrile. They were stable to air and moisture and could
be manipulated in air without noticeable decomposition,
although the brightness of the crystals decreased when they
were taken out of solution. Complexes (5–8), were isolated
as brown solids, insoluble in common organic solvents
such as methanol, dichloromethane, and acetonitrile, but
soluble in DMF and DMSO.
Human prostate cancer cell line (PC3) and human malig-
nant melanoma cell line (A375) were purchased from the
American Type Culture Collection (ATCC, Manassas, VA,
USA). Human breast cancer cell line (MCF-7) was
acquired from Cell Lines Service (300273; Eppelheim,
Germany) and Carcinoma-derived human oral keratinocyte
cells (H413) were received from Professor Ian Charles
Paterson. MCF-7 and A375 cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM, Life Technologies, Inc.,
Rockville, MD, USA) supplemented with 10 % heat-
inactivated fetal bovine serum (FBS, Sigma-Aldrich, St.
Louis, MO, USA), 1 % penicillin and streptomycin. PC3
cells were grown in Roswell Park Memorial Institute
medium (RPMI) supplemented with 10 % heat-inactivated
fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO,
USA), 1 % penicillin and streptomycin. H413 cells were
grown in Dulbecco’s Modified Eagle Medium/Ham’s F-12
(DMEM/F12) supplemented with 10 % heat-inactivated
fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO,
USA), 1 % penicillin and streptomycin. Cells were cul-
tured in tissue culture flasks (Corning, USA) and were kept
in an incubator at 37 °C under a humidified atmosphere
with 5 % CO2. For experimental purposes, cells in expo-
nential growth phase (approximately 70–80 % confluency)
were used.
In the mononuclear complexes (1–4), the two thiosemi-
carbazone ligands bound to nickel(II) behave as tridentate
ONS chelating ligands, but in different coordination modes as
evidenced by the IR spectra and X-ray analysis. One ligand is
coordinated in its neutral form through the thione sulfur,
azomethine nitrogen and the phenol oxygen, while the other is
The influence of the ionic solvents was determined by
MTT assay [35]. MCF-7, A375, PC3 and H413 cells were
treated for 48 h. On the first day, 1.0 9 104 cells were
seeded into a 96-well plate for 24-h incubation assay. The
cells were incubated overnight at 37 °C in 5 % CO2. On
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Transition Met Chem (2014) 39:81–94
85
R
N
H
H
N
H
R
H
N
N
H
O
N
H
N
S
H
O
S
O
Ni
O
H
O
O
Ni
Ni
H
O
S
N
N
ClO4
S
H
H
O
H
N
N
N
H
H
R
N
H
a
R
b
Scheme 3 Proposed structures of the prepared complexes
Table 1 Stoichiometries, color and partial elemental analysis of nickel(II) thiosemicarbazone complexes
Complex
Stoichiometries
Yield %
Color
Anal. calc. (found) %
C
H
N
[Ni(H3L1)(H2L1)]ClO4Á2H2O (1)
[Ni(H3L2)(H2L2)]ClO4Á2H2O (2)
[Ni(H3L3)(H2L3)]ClO4Á(3)
[Ni(H3L4)(H2L4)] ClO4ÁH2O (4)
[Ni2(HL1)2] (5)
C16H17N6O8S2ClNiÁ2H2O
C18H21N6O8S2ClNiÁH2O
C28H25N6O8S2ClNi
68
72
64
72
62
70
73
76
Green
31.21 (31.30)
34.55 (34.47)
45.95 (46.01)
36.74 (36.81)
35.86 (35.84)
37.15 (37.12)
48.88 (48.79)
40.58 (40.64)
3.44 (3.32)
3.71 (4.64)
3.44 (3.49)
4.16 (4.05)
2.63 (2.53)
3.46 (3.43)
3.22 (3.20)
3.75 (3.82)
13.65 (13.68)
13.43 (13.49)
11.49 (11.40)
12.86 (12.84)
15.68 (15.66)
14.44 (14.38)
12.21 (12.18)
14.20 (14.23)
Green
Green
C20H25N6O8S2NiClÁH2O
C16H14N6O4S2Ni2
Green
Brownish red
Brown
Brown
Brown
[Ni2(HL2)2]ÁH2O(6)
C16H18N6O4S2Ni2ÁH2O
C16H18N6O4S2Ni2
[Ni2(HL3)2] (7)
[Ni2(HL4)2] (8)
C20H22N6O4S2Ni2
coordinated in its monodeprotonated form through the thione
sulfur, azomethine nitrogen and phenolate oxygen. Magnetic
moment measurements indicate the paramagnetic nature of
these nickel(II) complexes with room temperature magnetic
moments in the range of 2.92–3.08 B.M, consistent with
octahedral nickel(II) complexes [37]. The thiosemicarbazone
ligand in complexes (5–8) is bound in its doubly deprotonated
formasa tridentateONS donorina bridgingmodethrough the
thiolate sulfur, azomethine nitrogen and phenolic oxygen.
Magnetic moment measurements indicate the diamagnetic
natureofthesenickel(II)complexes.Duetothepoorsolubility
of these complexes, attempts to grow crystals suitable for
X-ray studies were not successful.
4 are shown in Table 2. Figure 1 shows the molecular
structure, and the most important bond lengths and angles
are shown in Table 3. This compound crystallizes in the
monoclinic space group P2(1)/c with two molecular weight
units per cell. The asymmetric unit of complex 4 (Fig. 2)
consists of two distorted octahedral [Ni(H3L4)(H2L4)]?
cations and two perchlorate counteranions, as well as two
water molecules of solvation within the crystal lattice. In
each cation, the nickel center is coordinated to two dif-
ferent 2,3-dihydroxybenzaldehyde-N4-ethylthiosemicarba-
zone ligands, which are placed approximately
perpendicular to each other. One neutral ligand is coordi-
nated through the azomethine nitrogen, thione sulfur and
phenol oxygen, while the other is monoanionic and coor-
dinated through the azomethine nitrogen, thione sulfur and
phenolate oxygen. This arrangement gives rise to an
octahedral environment in such a way that four bicyclic
chelate systems are formed.
Crystal structure of complex 4
The crystallographic data and structure refinement param-
eters obtained from X-ray diffraction analysis for complex
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Transition Met Chem (2014) 39:81–94
˚
Table 2 Crystal data and structure refinement parameters for com-
plex 4
Table 3 Selected bond lengths (A) and bond angles (°) of complex 4
Bond lengths
Bond angels
Parameters
[Ni(H3L4)(H2L4)]2(ClO4)2Á2H2O
Ni1–N4
Ni1–N1
Ni2–N7
Ni2–N10
Ni1–O3
Ni1–O1
Ni2–O7
Ni2–O5
Ni1–S2
Ni1–S1
Ni2–S3
Ni2–S4
S2–C18
S1–C8
2.015(3)
2.025(3)
2.032(3)
2.031(3)
2.054(3)
2.108(3)
2.048(3)
2.114(3)
2.3972(1)
2.4073(1)
2.3958(1)
2.4116(1)
1.682(5)
1.691(4)
1.683(4)
1.680(4)
1.282(4)
1.286(5)
1.344(5)
1.274(5)
1.351(5)
1.347(5)
1.283(5)
1.349(5)
1.360(5)
1.375(5)
1.371(4)
1.336(4)
1.358(4)
1.366(5)
1.333(4)
1.365(5)
N4–Ni1–N1
N4–Ni1–O3
N1–Ni1–O3
N4–Ni1–O1
N1–Ni1–O1
O3–Ni1–O1
N4–Ni1–S1
N1–Ni1–S1
O3–Ni1–S1
O1–Ni1–S1
N4–Ni1–S2
N1–Ni1–S2
O3–Ni1–S2
O1–Ni1–S2
S1–Ni1–S2
N10–Ni2–N7
N10–Ni2–O7
N7–Ni2–O7
N10–Ni2–O5
N7–Ni2–O5
O7–Ni2–O5
N10–Ni2–S3
N7–Ni2–S3
O7–Ni2–S3
O5–Ni2–S3
N10–Ni2–S4
N7–Ni2–S4
O7–Ni2–S4
O5–Ni2–S4
S3–Ni2–S4
178.47(14)
88.70(11)
92.51(11)
92.28(13)
86.92(13)
84.25(12)
96.60(9)
Empirical formula
Formula weight, M
Temperature, T (K)
C40 H52 Cl2 N12 Ni2 O18 S4
1,305.50
273
˚
Wavelength, Mo Ka (A)
Crystal system
k = 0.71073
Monoclinic
P2(1)/c
Space group
Unit cell dimensions
84.35(10)
89.14(8)
˚
a (A)
12.0076(2)
26.8576(4)
17.2286(3)
90.00
˚
b (A)
168.81(10)
83.59(10)
95.09(10)
169.71(8)
89.25(10)
98.48(5)
˚
c (A)
a (°)
b (°)
c (°)
101.3100(10)
90.00
3
˚
Volume V (A )
5,448.24(15)
4
S4–C38
S3–C28
N7–C27
N10–C37
N11–C38
N4–C17
N8–C28
N2–C8
Z
176.47(13)
87.59(11)
95.28(11)
96.60(11)
85.75(11)
83.99(10)
94.61(9)
Absorption coefficient, l (cm-1
)
1.023
Reflections collected
Density (calculated) (mg m-3
F(0 0 0)
48,139
)
1.592
2,696
Data/parameters/restraint
S
11,141/703/0
1.009
Independent reflections
R[F2 [ 2r(F2)]
wR(F2)
6,858 [R(int) = 0.0503]
0.1151
N1–C7
83.30(9)
N5–C18
C2–O1
91.31(8)
0.1241
167.64(8)
82.65(10)
94.85(9)
Largest difference peak and hole 0.6618 and 0.7454
O2–C3
3
˚
(e A )
O4–C13
O3–C12
O5–C22
O6–C23
O7–C32
O8–C33
166.38(8)
87.71(8)
98.90(4)
Because of the different character of the atoms forming
the environment around the Ni(II) ion, the coordination
octahedron is distorted, where the Ni atoms are in a trans-
N(1)N(4)-cis-O(1)O(3)-cis-S(1)S(2) configuration and
trans-N(7)N(10)-cis-O(5)O(7)-cis-S(3)S(4) configuration,
respectively. The trans angles involving the two imine
nitrogens, N(1)–Ni1–N(4) of 178.46(15)° and N(7)–Ni2–
N(10) of 176.44(13)°, are \180°. The O(1)–Ni1(1)–O(3)
angle is 83.83(11)° and O(5)–Ni2(1)–O(7) angle is
83.78(10)°, while the S(1)–Ni1–S(2) angle of 98.47(5)°
and S(3)–Ni2–S(4) of 98.89(5)° are [90°, revealing that
the two thiosemicarbazone ligands exert significant steric
effects on each other. The bond lengths between each
nickel(II) ion and the donor atoms are very unequal,
Fig. 1 Molecular structure of complex [Ni(H3L4)(H2L4)]ClO4Á2H2O,
displacement ellipsoids are drawn at 70 % probability level, perchlo-
rate ion and water molecule are omitted for clarity
123

Transition Met Chem (2014) 39:81–94
87
increasing in the order Ni–N \ Ni–O \ Ni–S as shown in
Table 2. However, the average Ni–N bond distance of
˚
2.025(3) A is in the normal range for Ni–N bond distances
of octahedral nickel(II) complexes with thiosemicarba-
zones [38]. The coordination modes of the oxygens of the
2,3-dihydroxybenzaldehyde moieties in the two ligands are
different; the average value of bond distances O(1)–C(2),
˚
1.360(5) A, is only 0.015 A shorter than the average value
˚
˚
of bond O(2)–C(3), 1.375(5) A, while the average value of
˚
bond distances O(3)–C(12), 1.336(4) A, is 0.038 A shorter
˚
˚
than the average value of O(2)–C(3), 1.375(5) A. Conse-
quently, the Ni–O bond distances are different and lay in
˚
the range 2.049–2.120 A. These values are close to the
Fig. 3 Unit cell packing of complex 4 along a axis
bond distances observed in other nickel salicylaldehyde
complexes. The other values are very similar to those
reported for other nickel complexes with ONS-donor
thiosemicarbazones [39].
Spectroscopic studies
The most characteristic vibrational frequencies and their
tentative assignments for the 2,3-dihydroxybenzaldehyde-
N4-substituted thiosemicarbazone ligands H3L, and their
nickel(II) complexes are listed in Table 5. In the solid state,
all the thiosemicarbazones remain in the thione form, as
indicated by the bands found at around 1,390–1,340 cm-1
attributable to m(C=S). Furthermore, the asymmetric
stretching frequency of the thioamide group is observed in
the region of 855–800 cm-1. Moreover, the m(S–H) band
that would appear at around 2,500–2,800 cm-1 cannot be
seen in the spectra of all these ligands, while a sharp band
due to the vibration of N2–H is observed at
3,200–3,150 cm-1 in the spectra of all the ligands. These
results support the existence of the thione tautomer in the
solid state [40, 41]. The bands around 3,300–3,420 cm-1
are attributed to m(N4–H), while the bands at the region of
3,500–3,370 cm-1 are assigned to OH stretching of
the polyhydroxybenzaldehyde thiosemicarbazone. The
The bicyclic chelate systems Ni(1), S(2), C(18), N(5),
N(4), C(17), C(11), C(12), O(3) and Ni(1), S(1), C(8),
N(2), N(1), C(7), C(1), C(2), O(1) are considerably devi-
ated from planarity, as evidenced by the maximum devia-
˚
˚
tion of 0.345(2) A for O(3) and 0.24(3) A for O(1),
respectively, from their respective mean planes.
The packing of the molecules in the unit cell is shown in
Fig. 3. The molecules are held together by a system of
hydrogen bonds in which both [Ni(H3L4)(H2L4)]? cations
act as donors through the nitrogens N(2), N(3), N(5), N(6),
N(8), N(9), N(11) and N(12) and the oxygens O(2), O(4),
O(6), and O(8), and as acceptors through the oxygen atoms
O(2), O(3), O(6) and O(7) (Table 4). The two perchlorate
anions and water molecules also act as acceptors. The
molecular structures of the non-crystallized complexes 1, 2
and 3 are expected to be similar to that of complex 4.
Fig. 2 Asymmetric unit cell for
complex 4
123

88
Transition Met Chem (2014) 39:81–94
˚
electron density of the azomethine group is reduced due to
coordination of nitrogen to the metal atom. However, this
band is not a pure vibrational band of C=N stretching, but
it is a combination band of [m(C–N) ? d(NH)] stretching
vibrations [42]. Meanwhile, the shift to higher frequency
of the m(N–N) band, observed in the spectra of the com-
plexes, has also been related to the electronic delocaliza-
tion produced as a consequence of coordination through
the azomethine nitrogen atom. Further evidence for the
coordination of the azomethine nitrogen is obtained from
the presence of a new band 520–580 cm-1, assignable to
m(Ni–N), for all the nickel complexes. The interaction of
thiosemicarbazone ligands with nickel(II) through sulfur in
all of these complexes is confirmed by the shift in the
thioamide band to lower energies, together with a decrease
in intensity when compared with the corresponding bands
of the free ligands. In complexes 1–4, the bands assigned
to the C=S stretching at 1,390–1,340 and 855–800 cm-1 in
the free ligands have shifted to 1,342–1,330 and
780–775 cm-1, respectively, while the thioamide stretch-
ing in complexes 5–8 was observed at 1,330–1,315 cm-1
Table 4 Hydrogen-bond geometry (A, °) for complex (4)
Donor–HÁÁÁacceptor
D–H
HÁÁÁA
DÁÁÁA
D–HÁÁÁA
N(2)–H(2A)ÁÁÁO(10)
N(2)–H(2A)ÁÁÁO(9)
O(2)–H(2B)ÁÁÁO(17)
N(3)–H(3A)ÁÁÁO(9)
N(3)–H(3A)ÁÁÁO(10)
O(4)–H(4A)ÁÁÁO(3)
O(4)–H(4A)ÁÁÁO(6)
N(5)–H(5A)ÁÁÁO(18)
O(6)–H(6A)ÁÁÁO(11)
N(6)–H(6B)ÁÁÁO(18)
N(8)–H(8A)ÁÁÁO(16)
O(8)–H(8B)ÁÁÁO(2)
O(8)–H(8B)ÁÁÁO(7)
N(9)–H(9A)ÁÁÁO(14)
N(11)–H(11A)ÁÁÁO(13)
N(12)–H(12A)ÁÁÁO(13)
0.86
0.86
0.82
0.86
0.86
0.82
0.82
0.86
0.82
0.86
0.86
0.82
0.82
0.86
0.86
0.86
2.32
2.55
1.87
2.30
2.52
2.31
1.95
2.03
2.69
2.53
2.26
1.91
2.33
2.26
2.16
2.44
3.149(6)
3.043(5)
2.673(3)
3.122(5)
3.053(6)
2.754(4)
2.720(4)
2.842(5)
2.903(5)
3.256(6)
3.057(6)
2.685(4)
2.742(4)
3.118(6)
2.977(6)
3.209(6)
163
118
164
160
121
114
155
158
173
143
154
157
112
171
157
149
azomethine stretching frequency is observed at and around 735 cm-1. This last band is attributed to
1,611–1,542 cm-1
while strong to medium bands C–Sbridging stretching. The wavenumber shift of the thio-
observed in the range of 1,210–1,060 cm-1 are assigned to amide band of complexes 5–8 (thiol coordination) would
the hydrazinic (N–N) bonds. be expected to be larger than in the complexes 1–4 (thione
,
The assignments of the vibrational frequencies of the coordination), due to the lowering of the C–S bond order
complexes were made by comparing with those of the free owing to deprotonation in the case of thiol coordination.
ligands. The IR spectra of the nickel complexes suggest The presence of a new band at around 1,525–1,559 cm-1
that the coordination of the thiosemicarbazone to nickel(II) in the IR spectra of complexes 5–8 due to the newly
occurs through the azomethine nitrogen, phenolate oxygen formed m(N=C) supports the above observation. The loss
and thione sulfur. The azomethine m(C=N) vibration in the of the O–H proton upon coordination is difficult to assign
range of 1,611–1,542 cm-1 in the free ligands shows a in polyhydroxybenzaldehyde thiosemicarbazone com-
positive shift to higher frequency 1,617–1,581 cm-1 after plexes, due to the presence of more than one OH which
complexation, indicating participation of the azomethine can engage in both inter- and intra-molecular hydrogen
group in the coordination to nickel(II), such that the bonds [21].
Table 5 IR Spectral assignments for the thiosemicarbazone ligands and their nickel(II) complexes
Compound
m(C=N)
m(C=S)/m(C–S)
m(C–O)
m(N–N)
m(Ni–O)
m(Ni–N)
H3L1
H3L2
H3L3
H3L4
1,611
1,543
1,542
1,542
1,617
1,581
1,607
1,606
1,601
1,591
1,597
1,595
1,342, 824
1,389, 853
1,389, 850
1,384, 807
1,335, 779
1,342, 789
1,324, 773
1,340, 780
1,320, 737
1,333, 736
1,335, 737
1,316, 734
1,279
1,290
1,289
1,307
1,270
1,272
1,264
1,280
1,227
1,260
1,263
1,260
1,068
1,043
1,064
1,051
1,087
1,086
1,061
1,088
1,094
1,090
1,095
1,089
–
–
–
–
–
–
–
–
[Ni(H3L1)(H2L1)](ClO4)2Á2H2O (1)
[Ni(H3L2)(H2L2)] (ClO4)2ÁH2O (2)
[Ni(H3L3)(H2L3)](ClO4)2 (3)
[Ni(H3L4)(H2L4)](ClO4)2Á2H2O (4)
[Ni2(HL1)2] (5)
467
474
455
464
486
447
424
445
523
581
569
569
545
521
520
521
[Ni2(HL2)2]ÁH2O (6)
[Ni2(HL3)2] (7)
[Ni2(HL4)2] (8)
123

Transition Met Chem (2014) 39:81–94
89
However, the coordination of phenolate oxygen is
indicated by the lowering in the m(C–O) band by
10–30 cm-1 when compared with the free ligand. These
observations are supported by the observation of a Ni–O
benzene shielding cone. Unfortunately, the 1H NMR
spectra of complexes [Ni2(HL1)2] (5) and [Ni2(HL4)2] (8)
could not be assigned due to spectral broadening which
may be due to the presence of paramagnetic species formed
with solvent in the axial positions [44]. The aromatic ring
protons are observed as multiplets between 7.59 and
6.61 ppm, which are practically unaffected by metal
complexation.
band at 480–424 cm-1
.
1H NMR is a helpful tool for identification of the
coordination sites in diamagnetic nickel(II) complexes
when taken in conjunction with other spectroscopic infor-
mation. The chemical shifts of the thiosemicarbazone
ligands and their diamagnetic nickel complexes
[Ni2(HL2)2] (6) and [Ni2(HL3)2] (7) are presented in
Table 6. A singlet observed at 11.41 and 11.78 ppm for the
free ligands H3L2 and H3L3, respectively, is assigned to the
thioamidic proton N(2)H. This signal is absent from the
spectra of complexes 6 and 7, showing that coordination of
the ligands H3L2 and H3L3 to nickel(II) is in the thiolate
In vitro antimicrobial assays
The disc diffusion method, developed by Bauer et al., is a
well-standardized and highly reproducible qualitative
method of antimicrobial susceptibility testing that allows
the simultaneous testing of a number of compounds in a
relatively easy and inexpensive manner. In this study, the
disc diffusion method was applied to test the activity of the
compounds against 25 bacterial strains. The diameter of the
inhibition zone was correlated with the susceptibility level
of each microorganism against the test compound
(Table 7). The bacterial strains indicated diverse suscepti-
bilities to these compounds, but a significant percentage of
the tested bacterial strains demonstrated high resistance.
In order to gain more quantitative data, microdilution
plate analysis was also performed in the present study
(Table 8). Overall, most of the compounds demonstrated
low-to-moderate antibacterial activities.
1
form [43]. In the H NMR spectra of both complexes, the
azomethine proton signal is observed as two singlets; one is
shifted upfield, while the other is shifted downfield when
compared with the free ligands. This pattern of shifts is
probably due to different withdrawal of electron density
from the thiosemicarbazone moiety. The appearance of
CH=N as two signals suggests the coordination of two
inequivalent azomethine nitrogens in these complexes.
Furthermore, two signals are observed at 9.50, 8.90 ppm
and 9.21, 9.54 ppm for complexes 6 and 7, respectively;
each integrates as one hydrogen corresponding to the
m-OH of the thiosemicarbazone moiety.
The complexes indicated higher inhibitory activities
than the corresponding free ligands, and the susceptibility
levels of the Gram-negative bacteria to the complexes were
higher than those of the Gram-positive bacteria. Complexes
(7) and (8) exhibited significant antimicrobial activity
In the 1H NMR spectra of complexes 6 and 7, the N(4)H
appears at 6.30 and 6.38 ppm, respectively, and has shifted
upfield compared with the free H3L2 and H3L3 ligands,
perhaps due to the presence of this proton within the
Table 6 1H NMR spectral assignments for the thiosemicarbazone ligands and their nickel(II) complexes 6 and 7 in DMSO-d6
Compound
Chemical shifts, d (ppm)
N(2)H
OH
C=N
N(4)H
Aromatic due to Tsc
Aliphatic due to Tsc
H3L2 (2)
11.41(s, 1H)
9.26(s, 2H)
8.38(s, 1H)
8.39(s, 1H)
7.38(d, 1H)
6.41(dd, 1H)
6.66(t, 1H)
3.01(d, 3H)
[Ni2(HL2)2]ÁH2O (6)
–
9.50(s, 1H)
8.90(s, 1H)
8.33(s, 1H)
8.42(s, 1H)
6.30(s, 1H)
7.33(d, 2H)
6.75(d, 2H)
6.61(t, 2H)
2.96(d, 3H)
H3L3 (3)
11.78(s, 1H)
9.55(s, 1H)
9.04(s, 1H)
8.52(s, 1H)
10.03(s, 1H)
7.59(d, 1H)
7.50(d, 2H)
7.36(t, 2H)
–
7.19(t, 1H)
6.71(dd, 1H)
6.69(t, 1H)
[Ni2(HL3)2] (7)
–
9.54(s, 1H)
9.21(s, 1H)
8.99(s, 1H)
8.47(s, 1H)
6.38(s, 1H)
7.58–6.63(m, 13H)
–
123

90
Transition Met Chem (2014) 39:81–94
Table 7 Inhibitory activity of the compounds against different bacterial species using Kirby–Bauer disc diffusion method
Tested microorganism
Inhibitory activity of compounds based on the inhibition zones (IZ)a
H3L1
H3L2
H3L3
H3L4
1
2
3
4
5
6
7
8
B. subtilis
I
I
R
R
R
I
I
I
I
I
I
I
I
I
I
E. faecalis
I
S
R
I
S
I
I
R
I
R
I
R
I
I
I
I
S
I
E. coli ATCC 25922
L. innocua
I
I
I
R
I
I
R
R
R
R
R
R
R
R
R
R
I
I
R
I
I
I
R
I
R
R
I
S
S
S
I
R
I
L. monocytogenes
L. welshimeri
P. mirabilis
P. aeruginosa
S. enterica ATCC 9068
S. enteritidis
S. typhi
I
R
R
R
R
R
R
R
R
R
R
R
R
I
R
I
I
I
S
R
I
R
R
I
I
R
I
S
R
S
R
I
I
I
R
I
R
S
S
I
I
R
I
S
I
S
S
S
I
S
S
S
R
I
S
S
I
S
S
S
S
I
R
R
I
R
I
S
I
S
S
I
I
I
I
I
R
R
S
I
S. typhimurium
S. dysenteriae
S. flexneri
R
R
I
R
I
I
I
I
I
R
S
I
I
I
I
I
I
I
I
S
I
S
I
I
S
I
I
I
S. sonnei
R
I
R
I
I
I
I
I
I
S
I
S. aureus
I
I
I
I
I
I
I
I
S. epidermidis
S. pneumoniae
V. alginolyticus
V. cholerae
I
R
R
I
I
I
R
R
I
S
I
I
I
I
I
I
R
I
R
R
R
R
R
I
R
I
I
I
R
I
R
I
S
I
I
I
I
I
I
I
I
R
S
I
I
I
I
I
I
I
I
I
V. fischeri
S
I
I
S
S
R
I
S
S
R
I
I
S
S
R
I
S
S
I
S
S
I
I
S
S
I
V. harveyi
I
S
I
S
S
S
S
V. parahaemolyticus
V. vulnificus
Yersinia pestis
a
R
R
I
I
I
R
R
R
R
R
R
I
I
I
S
R
I
I
I
R
I
I
R resistant (IZ = 0 mm), I intermediate (0 \ IZ B 1 mm), S susceptible (IZ [ 1 mm)
Table 8 In vitro antimicrobial activity of the compounds using microdilution method
Tested microorganisms Minimum inhibitory concentration (MIC)/minimum microbicidal concentration (MMC) (lg/ml)
H3L1
H3L2
H3L3
H3L4
1
2
MIC
MMC
500
MIC
MMC
MIC
MMC MIC
MMC
MIC
MMC
500
MIC
MMC
500
B. subtilis
250
250
250
500
500
500
250
250
500
250
1,000
500
500
500
125
500
250
250
1,000 250
250 62.5
1,000 250
500 1,000
[1,000 250
250 500
500
500
125
250
250
E. faecalis
500
500
125
500
125
500
250
500
250
125
250
250
250
250
[1,000
250
[1,000
250
E. coli ATCC 25922
L. innocua
[1,000 125
250 500
1,000
1,000
500
62.5
125
1,000
500
500
L. monocytogenes
L. welshimeri
P. mirabilis
500 500
1,000
250
[1,000 250
500 250
500
1,000 1,000 [1,000 125
500 1,000
[1,000
500
500
500
250
250
500
500 250
500 125
500 250
500
250 500
1,000
250
125
P. aeruginosa
S. enterica ATCC 9068
S. enteritidis
250
250 125
250
125
250
250
500
500
125
1,000
500
250
500
62.5
62.5
62.5
250
250
62.5
125
500 [1,000 125
250 500 250
250
500 125
500 125
[1,000 250
[1,000 250
125
S. typhi
[1,000
250
500
S. typhimurium
S. dysenteriae
1,000 1,000 [1,000 250
[1,000 500 500 250
500 1,000
500 500
500
125
123

Transition Met Chem (2014) 39:81–94
91
Table 8 continued
Tested microorganisms Minimum inhibitory concentration (MIC)/minimum microbicidal concentration (MMC) (lg/ml)
H3L1
H3L2
H3L3
H3L4
1
2
MIC
MMC
500
MIC
MMC
MIC
MMC MIC
MMC
250
MIC
MMC
125
MIC
MMC
S. flexneri
250
500
250
250
500
250
500
125
250
250
250 250
500
500
500
125
250
500
250
250
250
500
500
500
125
500
125
125
250
125
500
62.5
31.2
62.5
250
S. sonnei
1,000
500
500 500
250 250
[1,000 250
250 125
250
250
500
125
125
500
S. aureus
125
500
S. epidermidis
S. pneumoniae
V. alginolyticus
V. cholerae
V. fischeri
500
500 [1,000
62.5
250 250
[1,000 1,000 [1,000 125
500 500
500 1,000
[1,000
250
500
1,000
125
250
250
125
125
250
250 250
250 125
250 250
125 125
500 250
250 125
250
500
125
125
500
250
250
125
125
[1,000 250
250
62.5
62.5
125
500
31.2
62.5
62.5
62.5
500
62.5
V. harveyi
125
500
500
125
250
250
250
500
500
125
1,000
250
V. parahaemolyticus
V. vulnificus
Y. pestis
1,000
250 250
[1,000 250
1,000 125
1,000 1,000 [1,000 250
250 1,000 [1,000 250
125
250
250
Tested microorganisms
Minimum inhibitory concentration (MIC)/minimum microbicidal concentration (MMC) (lg/ml)
3
4
5
6
MIC
MMC
MIC
MMC
250
MIC
MMC
MIC
MMC
B. subtilis
250
250
250
125
250
125
250
62.5
62.5
62.5
125
125
250
125
250
125
31.2
125
125
62.5
125
125
250
125
250
500
500
500
250
500
250
500
125
62.5
62.5
250
250
250
250
500
250
62.5
250
250
125
250
250
500
125
500
250
125
250
500
500
250
250
62.5
31.2
62.5
250
250
125
62.5
62.5
250
125
250
250
250
62.5
31.2
500
250
500
500
250
[1,000
500
500
500
250
500
125
500
250
250
62.5
125
125
500
62.5
125
125
125
250
500
250
125
31.2
62.5
125
250
250
500
1,000
500
E. faecalis
500
500
E. coli ATCC 25922
L. innocua
125
250
[1,000
1,000
500
500
[1,000
[1,000
1,000
[1,000
500
1,000
250
L. monocytogenes
L. welshimeri
P. mirabilis
1,000
500
1,000
500
500
1,000
250
P. aeruginosa
S. enterica ATCC 9068
S. enteritidis
S. typhi
125
500
62.5
62.5
62.5
125
125
500
500
250
125
125
250
125
250
250
250
250
250
250
500
250
250
500
250
500
500
500
500
500
500
500
125
125
250
250
125
250
250
S. typhimurium
S. dysenteriae
S. flexneri
1,000
62.5
62.5
250
250
S. sonnei
125
500
250
250
500
500
125
S. aureus
250
S. epidermidis
S. pneumoniae
V. alginolyticus
V. cholerae
250
1,000
250
250
V. fischeri
62.5
62.5
V. harveyi
62.5
125
250
125
125
125
250
500
500
V. parahaemolyticus
V. vulnificus
Y. pestis
[1,000
500
1,000
123

92
Transition Met Chem (2014) 39:81–94
Table 8 continued
Tested microorganisms
Minimum inhibitory concentration (MIC)/minimum microbicidal concentration (MMC) (lg/ml)
7
8
Gentamicin
MIC
MIC
MMC
MIC
MMC
MMC
B. subtilis
250
125
250
500
250
500
125
250
125
500
62.5
15.7
62.5
125
250
250
250
250
125
125
62.5
250
250
500
125
125
62.5
125
500
125
500
250
250
250
125
62.5
15.7
62.5
250
500
62.5
250
31.2
62.5
125
250
125
125
125
31.2
125
62.5
500
[1,000
250
0.8
[50
1.6
1.6
[50
3.1
E. faecalis
E. coli ATCC 25922
L. innocua
[1,000
500
62.5
125
62.5
250
31.2
0.8
1.6
L. monocytogenes
L. welshimeri
P. mirabilis
500
1.6
3.1
500
1.6
3.1
250
0.8
3.1
P. aeruginosa
S. enterica ATCC 9068
S. enteritidis
S. typhi
125
3.1
6.25
50
6.25
\15.7
31.2
62.5
125
31.2
25
125
500
3.1
1.6
6.25
6.25
S. typhimurium
S. dysenteriae
S. flexneri
[1,000
125
3.1
125
6.25
12.5
6.25
0.8
12.5
125
250
12.5
12.5
0.8
S. sonnei
125
62.5
S. aureus
125
125
250
250
250
125
125
S. epidermidis
S. pneumoniae
V. alginolyticus
V. cholerae
62.5
31.2
125
3.1
6.25
[50
25
[50
12.5
6.25
6.25
12.5
6.25
12.5
1.6
125
12.5
12.5
25
V. fischeri
250
V. harveyi
125
62.5
V. parahaemolyticus
V. vulnificus
Y. pestis
62.5
31.2
62.5
125
125
500
12.5
25
3.1
against S. enterica ATCC 9068. The MIC values of the two
compounds (15.7 and \15.7 lg/ml) toward S. enterica
ATCC 9068 were lower than that of gentamicin, the
positive control (25 lg/ml). Complex (7) also demon-
strated higher inhibitory power against S. pneumoniae
Table 9 Brine shrimp lethality assay (BSLA)
Compound
LC50 value (lg/ml) SD
H3L1
H3L2
H3L3
H3L4
97.79 1.09
105.17 1.46
9,361.07 0.68
77.35 0.93
88.32 1.73
96.38 3.84
63.56 1.27
59.29 0.78
92.41 1.45
87.61 1.19
72.43 1.05
58.76 0.92
(MIC
31.2 lg/ml),
compared
with
gentamicin
(MIC [ 50 lg/ml). Two of the ligands, H3L1 and H3L4
indicated significant antibacterial activity against V. fisc-
heri, with MIC values \100 (MIC = 62.5 lg/ml), which
is, however, higher than gentamicin (MIC = 6.25 lg/ml).
The ligand H3L3 inhibited E. faecalis, L. innocua and S.
epidermidis with MIC value of 62.5 lg/ml. However, the
inhibitory level of the ligand was comparable with genta-
micin only against E. faecalis (MIC [ 50 lg/ml), and for
the other two was much higher than the positive control.
[Ni(H3L1)(H2L1)](ClO4)2Á2H2O (1)
[Ni(H3L2)(H2L2)] (ClO4)2ÁH2O (2)
[Ni(H3L3)(H2L3)](ClO4)2 (3)
[Ni(H3L4)(H2L4)](ClO4)2Á2H2O (4)
[Ni2(HL1)2] (5)
[Ni2(HL2)2]ÁH2O (6)
[Ni2(HL3)2] (7)
[Ni2(HL4)2] (8)
Brine shrimp lethality assays
values (Table 9). Most of the ligands indicated higher LC50
values than their corresponding complexes. In general,
none of the compounds indicated high cytotoxicity against
The toxicities of the compounds were tested on brine
shrimp (A. salina), and the results are expressed in LC50
123

Transition Met Chem (2014) 39:81–94
93
Table 10 Cytotoxic activity of the compounds on different cancer cells in terms of IC50
Compound
IC50 value (lg/ml) SD
MCF7
PC3
78.22 4.86
A375
H413
H3L1
H3L2
H3L3
H3L4
50.43 4.77
43.75 5.21
97.52 6.19
26.08 1.86
58.36 3.97
31.40 2.04
17.43 1.27
27.61 0.96
46.21 5.30
39.91 2.49
14.22 0.72
27.55 1.61
52.78 3.81
57.22 4.29
78.31 8.17
59.03 6.08
37.16 2.53
34.28 3.11
24.61 2.98
56.73 5.27
41.83 5.10
57.29 7.26
18.30 2.01
25.18 1.49
43.19 5.12
48.97 3.84
127.08 13.54
53.22 4.26
47.21 2.15
41.74 3.76
18.08 0.38
32.81 2.53
48.15 5.21
51.32 3.70
14.45 0.62
26.11 1.05
73.41 6.29
109.17 8.45
89.43 5.93
36.51 2.18
45.90 3.92
27.63 0.67
52.11 2.87
51.89 6.02
68.55 4.73
19.27 0.93
37.43 1.81
[Ni(H3L1)(H2L1)](ClO4)2Á2H2O (1)
[Ni(H3L2)(H2L2)] (ClO4)2ÁH2O (2)
[Ni(H3L3)(H2L3)](ClO4)2 (3)
[Ni(H3L4)(H2L4)](ClO4)2Á2H2O (4)
[Ni2(HL1)2] (5)
[Ni2(HL2)2]ÁH2O (6)
[Ni2(HL3)2] (7)
[Ni2(HL4)2] (8)
A. salina, and the lowest LC50 value was obtained for
complex (7) (LC50 = 58.76 0.92 lg/ml).
based on the spectroscopic results and other studies.
Attempts to get good crystals of these complexes were
unsuccessful, preventing X-ray diffraction studies. The
substituents at nitrogen N4 did not have an effect on the
geometry of the nickel(II) complexes, but did influence the
biological activity. The results of disc diffusion and mic-
rodilution tests indicated various levels of inhibition, con-
ducted by the complexes and their corresponding ligands,
against the tested bacterial strains. In general, the antimi-
crobial activities of the ligands were lower than their cor-
responding complexes. However, only a few combinations
of complexes with the tested bacterial strains gave signif-
icant inhibition.
MTT cell viability assays
The effect of the compounds on cell viability was deter-
mined by MTT assay on MCF7, PC3, A375, and H413
cells. Table 10 shows the in vitro anticancer IC50 values
recorded on the studied cell lines. Complex (7) induced the
highest cell growth inhibition activity on the tested cell
lines; among the rest, complexes (3) and (8) also exhibited
moderately high cytotoxicities against the cancer cells.
Other compounds showed low-to-moderate cytotoxicities.
The good antimicrobial and inhibition activity for
complex (7) could be due to the existence of a phenyl
substituent on the thiosemicarbazone moiety, which is
more hydrophobic compared with the other substituents.
Moreover, the inhibitory effect of complex (7) is larger
than of complex (3) (complex with N4–phenyl substituent).
This may be related to the coordinatively unsaturated
square planar geometry in complex (7), giving the oppor-
tunity for easy binding of DNA to the metal [45].
Supplementary material
CCDC 941774 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via htt:www.ccdc.cam.ac.uk/conts/ retrieving html, or from
the Cambridge crystallographic data centre, 12 Union Road,
Cambridge CB21E2, UK; fax: (?44) 1223-336033; or
e-mail:deposit@ccdc.cam.ac.uk. Supplementary data asso-
ciated with this article can be found in the online version.
Conclusion
Acknowledgments The authors acknowledge the support from PPP
(PS 484/2010B) and UMRG (RG206/11AFR) from University of
Malaya (UM). The authors also would like to thank the Department of
Chemistry, University of Malaya for facilities to carry out the
research work.
In this study, reaction of thiosemicarbazones with nickel
perchlorate gave mononuclear nickel(II) complexes of the
type [Ni(H3L)(H2L)]ClO4 in which one thiosemicarbazone
acts as uninegative tridentate and the other as neutral tri-
dentate ligand forming bis-octahedral complexes. The
reaction with nickel acetate gave nickel(II) complexes of
the type [Ni2(HL)2] (L = ONS-donor ligand) with the
thiosemicarbazone coordinated as a binegative tridentate
ligand. The [Ni2(HL)2] series are most probably binuclear,
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