In situ oxidation triggered heteroleptically deprotonated cobalt(III) and homoleptic nickel(II) complexes of diacetyl monoxime derived tri-nitrogen chelators; Synthesis, molecular structures and biological assay

Article abstract of DOI:10.1016/j.ica.2017.07.036

Two tri-nitrogen chelating unsymmetrical Schiff base ligands, 3-(hydroxyimino)-2-butanone-2-(1H-benzothiazol-2-yl)hydrazone and 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone are synthesized by the condensation reaction of diacetyl monoxime with 2-hydrazino-benzothiazole/benzimidazole. Ligands have shown ML₂ type octahedral coordination towards Ni(II) and in situ generated Co(III) ions. Ligands and complexes are characterized by various spectro-analytical techniques. Both the ligands have shown a similar mode of ligation, but a different mode of deprotonation towards the cobalt and nickel ions. Ligands are left neutral in the case of nickel complexes while anionic in the case of cobalt complexes. Further, in situ oxidation of Co(II) to Co(III) has triggered a different mode of deprotonation between the two ligands of the same cobalt complex. Out of the four complexes synthesized, three complexes are characterized by single crystal X-ray diffraction technique, to evidence the structural facts. A comparative account of bond lengths of two complexes of the benzothiazole based ligand is presented. Structures of all the four complexes have been checked preliminarily for their NCI-60 Human Cancer Cell Line anticancer screening. Further, one among the four, the nickel complex of benzothiazole core is used for one dose growth inhibition screening against 60 different human cancer cell lines. The tested complex has shown highest growth inhibition over a Non-Small Cell Lung Cancer cell line EKVX. In addition, compounds are screened for their antibacterial and antifungal potencies against few microorganisms. The complexes have shown promising antimicrobial potencies.

Full text of DOI:10.1016/j.ica.2017.07.036

Accepted Manuscript
Research paper
In situ oxidation triggered heteroleptically deprotonated Cobalt(III) and homo-
leptic Nickel(II) complexes of diacetyl monoxime derived tri-nitrogen chelators;
Synthesis, molecular structures and Biological assay.
Vinayak Kamat, Avinash Kotian, Anupama Nevarekar, Krishna Naik, Dhoolesh
Kokare, Vidyanand K. Revankar
PII:
S0020-1693(17)30525-X
http://dx.doi.org/10.1016/j.ica.2017.07.036
ICA 17759
DOI:
Reference:
Inorganica Chimica Acta
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Received Date:
Revised Date:
Accepted Date:
10 April 2017
13 July 2017
14 July 2017
Please cite this article as: V. Kamat, A. Kotian, A. Nevarekar, K. Naik, D. Kokare, V.K. Revankar, In situ oxidation
triggered heteroleptically deprotonated Cobalt(III) and homoleptic Nickel(II) complexes of diacetyl monoxime
derived tri-nitrogen chelators; Synthesis, molecular structures and Biological assay., Inorganica Chimica Acta
(2017), doi: http://dx.doi.org/10.1016/j.ica.2017.07.036
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In situ oxidation triggered heteroleptically deprotonated Cobalt(III) and homoleptic
Nickel(II) complexes of diacetyl monoxime derived tri-nitrogen chelators; Synthesis,
molecular structures and Biological assay.
Vinayak Kamata, Avinash Kotiana, Anupama Nevarekara, Krishna Naika, Dhoolesh
Kokarea and Vidyanand K. Revankara*
a Department of Chemistry, Karnatak University, Dharwad-580003, India.
* Corresponding author
Abstract: Two tri-nitrogen chelating unsymmetrical Schiff base ligands, 3-
(hydroxyimino)-2-butanone-2-(1H-benzothiazol-2-yl)hydrazone and 3-(hydroxyimino)-2-
butanone-2-(1H-benzimidazol-2-yl)hydrazone are synthesized by the condensation
reaction of diacetyl monoxime with 2-hydrazino-benzothiazole/benzimidazole. Ligands
have shown ML2 type octahedral coordination towards Ni(II) and in situ generated Co(III)
ions. Ligands and complexes are characterized by various spectro-analytical techniques.
Both the ligands have shown a similar mode of ligation, but a different mode of
deprotonation towards the cobalt and nickel ions. Ligands are left neutral in the case of
nickel complexes while anionic in the case of cobalt complexes. Further, in situ oxidation
of Co(II) to Co(III) has triggered a different mode of deprotonation between the two
ligands of the same cobalt complex. Out of the four complexes synthesized, three
complexes are characterized by single crystal X-ray diffraction technique, to evidence the
structural facts. A comparative account of bond lengths of two complexes of the
bezothiazole based ligand is presented. Structures of all the four complexes have been
checked preliminarily for their NCI-60 Human Cancer Cell Line anticancer screening.
Further, one among the four, the nickel complex of benzothiazole core is used for one
dose growth inhibition screening against 60 different human cancer cell lines. The tested
complex has shown highest growth inhibition over a Non-Small Cell Lung Cancer cell
line EKVX. In addition, compounds are screened for their antibacterial and antifungal
potencies against few microorganisms. The complexes have shown promising
antimicrobial potencies.
Keywords: Diacetyl monoxime, Benzothiazole Schiff base, 2-hydrazinobenzimidazole,
Heteroleptic deprotonation, Co(III) complexes.
1

Highlights :
 Two new tridentate ligands 3-(hydroxyimino)-2-butanone-2-(1H-benzothiazol-2-
yl)hydrazone and 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)
hydrazone are synthesized and characterized.
 Ni(II) and in situ generated Co(III) complexes of the ligands are synthesized and
characterized by the single crystal X-ray diffraction method.
 Ligands are left neutral in the case of nickel complexes while anionic in the case
of cobalt complexes.
 One dose growth inhibition screening against 60 different human cancer cell lines
is carried out for Nickel complex of benzothiazole core.
 Ligands and complexes are tested for their antibacterial and antifungal potencies.
1. Introduction
Transition metal complexes of hydrazone based Schiff base ligands have attracted
the coordination chemists for a long time. Substituted organic heterocycles with donor
atoms in tuned positions have readily found their use in designing such ligand systems [1-
4]. Benzothiazoles and benzimidazoles are the class of organic heterocycles, which have
imparted biological applications to their transition metal complexes, antitumor and
antimicrobial assessments being predominant. [5-8].
Due to their structure, stability and reactivity, oximes have kindled the interest in
designing ligand systems for transition metal ions [9, 10]. The oxime functionality is
amphiprotic with a mildly acidic hydroxyl group and slightly basic nitrogen atom [11].
Hence it is widely recognized to produce different modes of coordination [12]. The
structural diversity of oxime ligands varies from mononuclear to higher nuclearity via
oximato bridges [13]. But mononuclear octahedral complexes are preferred if the ligand
provides a stable tridentate mode of coordination [11, 14].
ML2 type cobalt complexes of tridentate hydrazones, often show in situ oxidation
of Co(II) to Co(III) [15]. In such complexes with uninegative tridentate Schiff bases,
electric neutrality is maintained by the presence of a counter anion [16]. In contrast,
2

Co(III) complexes with dinegative tridentate ligands have received less attention. In
addition, transition metal complexes, particularly Co(III) complexes derived from
unsymmetrical and symmetrical Schiff bases have drawn considerable attention in the
past for their important biological applications [17]. These facts have motivated the
present work, wherein synthesis, structural characterization and biological evaluation of
four novel transition metal complexes are undertaken.
2. Experimental
2.1. Materials and physical measurements
Diacetyl monoxime obtained from Thomas-Baker and other reagents including 2-
mercaptobenzimidazole/benzothiazole obtained from SD-Fine Chemicals were used as
supplied. Solvents were purified and dried according to the standard procedures. The
metal salts used were in their hydrated form, i.e., CoCl2.6H2O, NiCl2.6H2O. Reaction
progress was maintained through silica gel thin layer chromatographic technique.
The CHN analysis was performed on a Thermo quest elemental analyzer.
Molecular weights and formulae were calculated without solvent molecules unless
1
explicitly stated. The H NMR spectra were recorded on AGILENT VNMRS-400/JEOL
ECX-500 spectrometer, in a DMSO-d6 solvent. Infrared spectra were recorded in KBr
discs in the region 4000–400 cm−1 on a Nicolet-6700 FT-IR spectrometer. The solution
state UV–Vis spectra of all the compounds in methanol were recorded on a JASCO V-
670 UV-Vis spectrophotometer. The EI mass spectrum of the ligand was obtained with a
Shimadzu GCMS-QP2010S spectrometer. The ESI mass spectral data for all the
complexes were obtained using a Waters UPLCTQD mass spectrometer. Magnetic
susceptibility measurements were carried out on polycrystalline samples, using a Faraday
balance at room temperature with Hg[Co(SCN)4] as reference.
2.2. Synthesis of Ligands
2.2.1. 3-(hydroxyimino)-2-butanone-2-(1H-benzothiazol-2-yl)hydrazone (L1H2)
The schematic representation of the synthesis of L1H2 is given in Scheme 1. First,
2-hydrazinobenzothiazole was obtained from 2-mercaptobenzothiazole by the reported
method [18]. In the next step, 2-hydrazinobenzothiazole (1.65 g, 0.01 mol) was refluxed
3

with diacetyl monoxime (1.01g, 0.01 mol) in 20 mL of methanol for 1 h. On cooling, the
separated white slimy product was filtered and washed with cold methanol. The solid
product was dried in vacuo for 4 h.
4
H
S
N
3
N
5
10
2
11
7
N
S
N
OH
S
N
Diacetyl monoxime
Methanol, Reflux 4h
Reflux 10h
SH
9
N
1
NH
6
NH2-NH2.H2O (80%)
NH2
8
Scheme 1 Synthesis of L1H2.
Yield: 83%. M.P.: 280°C. Color: white; Anal. Calc. for C11H12N4OS (%): C, 53.21; H,
1
4.87; N, 22.56. Found for L1H2 (%): C, 53.53; H, 5.01; N, 22.32. H NMR (400 MHz,
DMSO- d6): δ (ppm) 11.66 (s, 1H, OH), 11.46 (s, 1H, NH), 7.70-7.05 (m, 4H, C7 - C10H),
2.11 (s, 3H, C1H), 2.01 (s, 3H, C4H). EI-MS (m/z): 248 (M+). IR (KBr, v, cm−1): 3442,
1612, 1571, 1545, 750. λmax (nm): 231 (π→π∗), 313 (n→π∗ ).
2.2.2. 3-(hydroxyimino)-2-butanone-2-(1H-benzimidazol-2-yl)hydrazone (L2H2)
The synthesis of L1H2 was carried out according to the previously developed
strategy [19].
Yield: 55%. M.P.: 240°C. Color: buff. Anal. Calc. for C13H17N5O3 (%): C, 53.60; H, 5.88;
N, 24.04. Found for L2H2 (%): C, 53.91; H, 5.43; N, 23.72. 1H NMR (400 MHz, DMSO-
d6): δ (ppm) 11.36 (b, 2H, OH and NH), 7.23 - 7.21 (m, 2H, C7H and C10H ), 6.96 - 6.94
(m, 2H, C8H and C9H ), 3.15 (s, 2H, ring NH), 2.12 (s, 3H, C1H), 2.09 (s, 3H, C4H), 1.88
(s, 3H, C13H). EI-MS (m/z): 231 (M+). IR (KBr, v, cm−1): 3516, 3168, 1673, 1016, 748.
λmax (nm): 218 (π→π∗), 305(n→π∗ ).
2.3. Syntheses of metal complexes
The complexes were obtained by refluxing the respective hexa hydrated metal
chlorides (0.119 g, 0.500 mmol) with the methanolic solution of ligands (L1H2 0.248 g or
L1H2 0.231 g, 1.00 mmol) for 4 h. The crystalline product was obtained by evaporating
the solvent under reduced pressure. Single crystals were obtained by evaporating the
methanolic solution of respective complexes.
4

2.3.1. [Co(L1H)(L)]
Yield: 84%. Color: red. Anal. Calc. for C22H21N8O2S2Co (%): C, 47.82; H, 3.83; N, 20.28.
1
Found for [Co(L1H)(L)] (%): C, 47.53; H, 3.48; N, 19.92. H NMR (500 MHz, DMSO-
d6): δ (ppm) 10.65 (s, 1H, OH), 7.43-6.82 (m, 8H, C7 - C10H), 2.64 (s, 6H, C1H), IR (KBr,
v, cm−1): 3380, 1586, 1437, 1016, 747, 489. λmax (nm): 219 (π→π∗), 287(n→π∗ ),
487(LMCT). ESI-MS (positive mode m/z): 553.
2.3.2. [Co(L2H)(L)]
Yield: 69%. Color: red. Anal. Calc. for C22H23N10O2Co (%): C, 50.97; H, 4.47; N, 27.02.
1
Found for [Co(L2H)(L)] (%): C, 51.33; H, 4.15; N, 27.42. H NMR (500 MHz, DMSO-
d6): δ (ppm) 12.25 (s, 2H, ring NH), 11.80 (s, 1H, OH), 6.96 - 6.56 (m, 8H, C7H - C10H ),
2.68 (s, 6H, C1H), 2.19 (s, 6H, C4H). IR (KBr, v, cm−1): 3373, 1657, 1627, 1591, 1456,
751. λmax (nm): 213 (π→π∗), 296(n→π∗ ), 476(LMCT). ESI-MS (positive mode m/z): 519.
2.3.3. [Ni(L1H2)2]Cl2
Yield: 88%. Color: yellow. Anal. Calc. for C22H24N8O2S2NiCl2 (%): C, 42.20; H, 3.86; N,
17.89. Found for [Ni(L1H2)2]Cl2 (%): C, 42.54; H, 3.56; N, 18.22. IR (KBr, v, cm−1):
3394, 1578, 1519, 1073, 752, 464. λmax (nm): 220 (π→π∗), 313(n→π∗ ), 828(d-d
transtion). ESI-MS (positive mode m/z): 553, 249.
2.3.4. [Ni(L2H2)2]Cl2
Yield: 54%. Color: yellow. Anal. Calc. for C22H26N10O2NiCl2 (%): C, 44.63; H, 4.43; N,
23.66. Found for [Ni(L2H2)2]Cl2 (%): C, 44.44; H, 4.18; N, 23.28. IR (KBr, v, cm−1):
3383, 1650, 1578, 747. λmax (nm): 211 (π→π∗), 300(n→π∗ ), 818(d-d transtion). ESI-MS
(positive mode m/z): 519, 232.
2.4. Crystallographic data collection and refinement
The single crystal X-ray diffraction data were obtained at 296 K on a Bruker
SMART APEX2 CCD area detector diffractometer using a graphite monochromated Mo-
Kα (λ = 0.71073 Å) radiation source. Bruker SAINT Software package [20] was used to
integrate the frames using a narrow-frame algorithm. The structure was solved on Olex2
[21] with ShelXT [22] structure solution program using Intrinsic Phasing and refined
5

with the XL [23] refinement package using Least Squares minimization. All non-
hydrogen atoms were refined anisotropically. A summary of the crystallographic data and
structure refinement for [Co(L1H)(L1)] and [Ni(L2H2)2]Cl2 are tabulated in table 1, while
the data for [Ni(L2H2)2]Cl2 is provided in the supplementary information (SI) along with
other geometric tables (SI-T1 to SI-T5). The mercury-3.8 package was used to generate
molecular graphics [24].
Table 1. Crystallographic data and structure refinement
[Co(L1H)(L1)]
[Ni(L1H2)2]Cl2
Crystal data
Chemical formula
Mr
C22H21CoN8O2S2
552.52
C22H24N8NiO2S2·2(Cl)·2(H2O).O
678.25
Crystal system, space group
Temperature (K)
a, b, c (Å)
Monoclinic, P21/c
296
Triclinic, P-1
296
10.2921 (2), 13.8972 (3), 10.6157 (2), 11.3691 (2),
17.0674 (3)
13.4250 (2)
100.160 (1), 99.940 (1), 101.713(1)
 = 93.979 (1)
2435.29 (8)
4

V (Å3)
1524.71 (5)
2
Z
Radiation type
Mo K
Mo K
 (mm-1)
0.91
0.99
Crystal size (mm)
Data collection
Diffractometer
0.25 × 0.15 × 0.1
0.3 × 0.15 × 0.1
Bruker APEX-II CCD
Bruker APEX-II CCD
No. of measured, independent and
39089, 10467, 7875
19485, 5352, 4456
observed [I > 2(I)] reflections
Rint
0.026
0.029
6

(sin /)max (Å-1)
Refinement
0.802
0.595
R[F2 > 2(F2)], wR(F2), S
No. of reflections
No. of parameters
H-atom treatment
max, min (e Å-3)
0.037, 0.107, 1.03
10467
0.039, 0.112, 1.05
5352
356
419
Mixed
Mixed
0.48, -0.41
0.77, -0.67
2.5. NCI-60 Human Cancer Cell Line Screening Methodology
The compound [Ni(L1H2)2]Cl2 was tested against a panel of 60 human cancer cell
lines at the National Cancer Institute, Rockville, MD[25]. The cytotoxicity studies were
conducted using a 48 h exposure protocol using the sulforhodamine B assay [26, 27].
The One-dose data was reported as the percent growth inhibition (GI %) of treated
cells. The number reported is growth relative to the no-drug control and relative to the
time zero number of cells. This has allowed detection of both growth inhibition (values
between 0 and 100) and lethality (values less than 0).
2.6. Growth inhibitory activity of microbial strains[28]
The MIC values of ligands and complexes for the growth inhibition of six
different pathogenic microbial genomes S. epidermidis ,E. faecalis, K. pneumoniae, E.
coli, A. fumigatus and C. albicans were determined in HIMEDIA M210 Brain heart
infusion (BHI) broth. This is used for the propagation of pathogenic cocci and other
fastidious organisms associated with allied pathological investigations. BHI broth had the
following composition.
Ingredients in g/L. Calf brain, infusion from, 200.00; Beef heart infusion from, 250.00;
Proteose peptone, 10.00; Dextrose, 2.00; Sodium chloride, 5.00; Disodium phosphate,
2.50. And the final pH (at 250C) was 7.4+/-0.2.
7

Nine dilutions of each sample were done with BHI for MIC determination. In the
initial tube, 20 μL of the drug was added to the 380 μL of BHI broth. Then, 200 μL of
BHI broth was added into the next 9 tubes separately for dilution. From the initial tube,
200μL was transferred to the first tube containing 200 μL of BHI broth. This was
considered as 10-1 dilution. From 10-1 diluted tube, 200 μL was transferred to the second
tube to make 10-2 dilution. The serial dilution was repeated up to 10-9 dilution for each
drug. From the maintained stock cultures of required organisms, 5 μL was taken and
added into 2 mL of BHI broth. In each serially diluted tube, 200 μL of above culture
suspension was added. The tubes were incubated for 24 hours and observed for turbidity.
3. Results and Discussion
The primary aim of the work was to synthesize Co(II) and Ni(II) complexes of
newly designed, structurally interesting ligand systems. Accordingly, the ligands L1H2
and L2H2 were synthesized by condensing diacetyl monoxime with 2-hydrazino-
benzothiazole/benzimidazole. These ligands are of structural interest, due to their
different possible modes of deprotonation and coordination. But in the present case, a
stable ML2 type of tri-nitrogen chelation is observed in all the four complexes. Ionizable
protons in the ligands being moderately acidic, deprotonation has depended itself on the
metal salt tuned pH. Hence the ligands behaved neutrally with nickel centers, while
anionic with the cobalt centers. Further, in situ oxidation of Co(II) to Co(III) during
complexation has made the ligands of cobalt complexes heteroleptically deprotonated.
All the complexes are soluble in methanol, ethanol, acetone and DMSO. The
analytical and spectroscopic data for ligands and complexes are presented in the
experimental section. The analytical data and spectral characterization results of all the
complexes are consistent with the proposed structures. Cobalt complexes are diamagnetic,
as expected for low spin octahedral Co(III) complexes. This observation is further
supported by sharp signals in the diamagnetic region of 1H NMR of these complexes. The
observed magnetic moments of 3.10 and 3.18 BM for [Ni(L1H2)2]Cl2 and [Ni(L2H2)2]Cl2
are in accordance with 2 unpaired electrons, as expected for octahedral nickel(II)
complexes.
3.1. Crystallographic structures
8

Single crystals of [Co(L1H)(L)], [Ni(L1H2)2]Cl2 and [Ni(L2H2)2]Cl2 suitable for X-
ray diffraction studies were grown by slow evaporation of the methanolic solution of
these complexes. Mercury rendered ortep projections of [Co(L1H)(L1)] and [Ni(L1H2)2]2+
showing 50% displacement ellipsoids are given in Fig.1, while that of [Ni(L2H2)2]Cl2 in
SI-F1.
Fig.1. Ortep projections of [Co(L1H)(L1)] and [Ni(L1H2)2]Cl2 showing 50%
displacement ellipsoids (Counter anions and crystallization solvents are omitted for
clarity).
[Co(L1H)(L)]has crystallized in the monoclinic crystal system with P21/c space
group while [Ni(L1H2)2]Cl2 in P-1. The asymmetric unit of [Co(L1H)(L)] contains a
neutral molecule of the complex without any counter anions. But the asymmetric unit of
[Ni(L1H2)2]Cl2 has [Ni(L1H2)2]2+cation, 2 chloride counter ions along with 3 water
molecules. The metal ion is coordinated to two tridentate N, N, N chelators (termed as
ligand A and ligand B) in both complexes. Both the ligands have maintained s-cis
configuration at the diacetyl fragment. Ligand L2H2, which had s-trans configuration [19]
has changed its configuration due to the free rotation of C2-C3 single bond during
complexation. In each coordination sphere, both the ligands are practically planar and are
almost perpendicular to each other. The mean planes of the two ligands in cobalt and
nickel complexes have maintained a dihedral angle of 85.760 and 89.040 respectively.
The ligating atoms of ligands being identical in all the cases, the central metal ion is
9

coordinated in a core of 6 nitrogens. Two tridentate ligands have coordinated in
meridional fashion with the ring-N (N4), imine-N (N2) and oxime-N (N1) as donor
atoms.
In each complex, azomethine nitrogens of the two ligands (N2A-N2B) reside trans
to each other whereas the other two donor sites (N1A-N1B and N4A-N4B) have
remained mutually cis to each other. Though the ligating pattern is same in these
complexes, the mutual orientation of two ligands in a complex is found to be different for
[Co(L1H)(L)] and [Ni(L1H2)2]Cl2. This is schematically represented in Fig.2. If this kind
of difference in mutual orientation of unsymmetrical tridentate ligands is observed within
the two complexes of the same metal ion, that would eventually lead to stereoisomerism
[29]. Further, the two nickel complexes, [Ni(L1H2)2]Cl2 and [Ni(L2H2)2]Cl2 have shown
similar structural features and same spatial orientation of ligands.
Fig.2. Schematic representation of the mutual orientation of ligands in [Co(L1H)(L)] and
[Ni(L1H2)2]Cl2.
Ligands have formed two five-membered chelate rings with the metal center. The
N-M-N bite angles in all the cases have significantly deviated from ideal octahedral bite
angle of 900, indicating significant deviations from ideal octahedral geometry in the
complexes.
3.2. Mode of deprotonation through bond length analysis
The ligands used in this investigation has essentially two ionizable protons viz
hydrazide-NH and hydroxyl-OH. Hence the ligands can provide N, N, N ligation either in
the neutral form or in the anionic form. If the ligand is behaving anionic, it can do so by
the deprotonation of either of the 2 ionizable hydrogens or by both. This opens up 4
10

different modes of deprotonation for the ligand to give the same mode of ligation. These
modes are depicted in the Fig.3 for the complexes of benzothiazole based ligand.
H
H
N
S
N
S
N
N
N
N
N
N
O
OH
M
M
(B)
(A)
N
S
N
N
S
N
N
N
N
N
O
OH
M
M
(D)
(C)
Fig.3. Different modes of deprotonation for the complexes benzothiazole based ligand.
It is very difficult to assign the correct mode of deprotonation of the ligand with
spectral techniques. Even in the X-ray structure, exactly locating all the hydrogen atoms
from the difference Fourier map is cumbersome. Hence additionally, bond lengths in the
structures of [Co(L1H)(L)] and [Ni(L1H2)2]2+ can be compared to establish the correct
mode of deprotonation of the ligands. Bond lengths of these two structures are compared
in Fig. 4.
11

N
1.35
1.44
S
1.74
1.34
1.30
1.74
1.33
N
1.30
1.88
N
N
1.38
1.96
Ligand A
1.39
OH
1.93
Co
1.98
O
1.28
1.39
1.93
Ligand B
N
N
1.88
1.34
1.31
1.75
1.75
N
1.38
1.45
S
1.33
1.30
N
H
N
1.36
1.48
S
1.74
1.28
1.75
1.35
N
1.30
1.28
2.00
N
N
1.38
2.11
Ligand A
1.40
OH
2.11
Ni
2.11
OH
1.38
1.40
2.14
Ligand B
N
N
2.00
1.28
1.30
1.73
1.75
N
1.36
1.48
S
1.36
1.28
N
H
Fig.4. Comparison of bond lengths for [Co(L1H)(L1)] and [Ni(L1H2)2]2+ (in Å).
In the crystal structure of [Ni(L1H2)2]Cl2, the presence of two chloride counter
anions directly confirms the neutral form (mode A) of both the ligands. But in the
structure of [Co(L1H)(L)], no counter anions are observed suggesting an anionic mode of
coordination for both the ligands. Comparison of C5-N4 and C5-N3 bond lengths in the
structures, suggest the deprotonation of hydrazide-NH and delocalization of charge, in the
ligands of the cobalt complex. In addition, in the ligand B of the cobalt complex, the N1-
O1 bond length has reduced to 1.28 Å, indicating a double bond character. This suggests
the deprotonation of a hydroxyl group and delocalization of charge acquired on the
12

oxygen via the conjugated -N=C- groups [30]. Conclusively, in the cobalt complex, the
structure of ligand A is close to mode C while the structure of ligand B is close to mode D
making the complex heteroleptic. According to the electric neutrality principle, the
presence mono-negative and di-negative anions in the cobalt complex eventually confirm
the in-situ oxidation of cobalt from 2+ to 3+ oxidation state.
The Co-N bond lengths are in the range of 1.88-1.98 Å while the Ni-N bond
lengths are in the order of 2.00-2.11 Å. The shorter bond lengths and stronger
coordination which is due to the smaller size of Co(III) ion, is in accordance with the
earlier reports for similar Co(III) complexes [12]. Bond length analysis even shows
stronger coordination of azomethine nitrogen atoms in both the complexes.
3.3. Mass spectral analysis
The EI mass spectrum of ligands L1H2 (SI-F2) and L2H2 has given molecular ion
peaks [M]+ at m/z 248 and 231 respectively, corresponding to the exact mass of organic
motif. Both the ligands have shown similar fragmentation pattern (SI-F3).
In the positive mode ESI-MS of the cobalt complexes (SI-F4 and SI-F5), base
peaks are observed at m/z 553 and 519. This exactly corresponds to the mass of
[Co(L1H)(L1)+H]+ and [Co(L2H)(L2)+H]+species. This assignment is in good agreement
with the ascribed +3 oxidation state for cobalt, as cobalt in +2 state would have given
these peaks at an m/z of 554 and 520. In the mass spectra (SI-F6 and SI-F7) of nickel
complexes, peaks observed at m/z 553 and 519 are assigned to [Ni(L1H2)2-H]+ and
[Ni(L2H2)2-H]+ species. The base peaks observed at m/z of 249 and 232 in these
complexes are corresponding to [M+H]+ species of the of the corresponding ligands.
3.4. Electronic spectral analysis
The free ligand L1H2 exhibited broad bands in the UV region at a λmax of 231 and
313 nm respectively. These bands are assigned to the intraligand π→π∗ and n→π∗
transitions respectively. L2H2 has shown these transitions at 218 and 305 nm. These bands
have appeared in all the complexes with a slight shift in their λmax. This shift is attributed
to the change in π conjugation and coordination of azomethine nitrogens upon
complexation [31, 32].
13

6
In strong field octahedral cobalt(III) complexes, t2g configuration transforms as
3
3
1
1
1A1g ground term. Excitation of a electron to eg level spans T1g + T2g + T1g + T2g with
low lying spin-triplet states. Hence there are two principal spin-allowed
transitions, 1A1g→1T1g and 1A1g→1T2g [33, 34]. Cobalt complexes of L1H2 and L2H2 have
shown a new non-ligand band around 487 and 476 nm respectively. The ε values and the
positions of these peaks fall in the normal range expected for octahedral Co(III)
complexes [35, 36]. Due to its high intensity, it may be easily attributed to ligand to
Co(III) charge transfer (LMCT) [32, 37]. Further, octahedral Co(III) complexes are
expected to show a weak low energy 1A1g→1T2g transition [38]. In the present case, these
transitions are obscured by the broad and intense charge transfer bands [39]. Nickel
complexes have shown a very broad band centered at 828 and 818 nm respectively for
3
[Ni(L1H2)2]Cl2 and [Ni(L2H2)2]Cl2. This is assignable to A2g→3T2g transition for an
octahedral Ni(II) complex derived from diimine type ligands [40].
3.5. NCI-60 Human Cancer Cell Line Screening
The NCI-60 Human Tumour Cell Lines Screen utilizes 60 different human tumor
cell lines, representing leukemia, melanoma and cancers of the lung, colon, brain, ovary,
breast, prostate, and kidney cancers to identify and characterize novel compounds with
growth inhibition or killing of tumor cell lines. Among all the complexes, the structure of
[Ni(L1H2)2]Cl2 was selected by NCI for one dose screening based on its ability to add
diversity to the NCI small molecule compound collection.
Growth inhibition percent in single dose assay (10−5 M) of [Ni(L1H2)2]Cl2 against
60 human cancer cell lines is summarized in SI-T6. Among the 60 human cancer cell
lines tested, the nickel complex derived from benzothiazole core has very effectively
inhibited the growth of Non-Small Cell Lung Cancer cell line EKVX (GI %: 73.34) and
Breast Cancer cell line MDA-MB-468(GI %: 69.95). In addition, the compound has even
shown considerable activity against Leukaemia cell lines MOLT-4 (GI %: 64.78) and SR
(GI %: 65.20). Remaining tested cell lines have shown moderate to less sensitivity
towards the compound.
Further to obtain the IC50 value of [Ni(L1H2)2]Cl2 against the lung cancer cell line,
different concentrations of the drug (300, 150, 75, 37.5, 18.75, 9.375 µg/mL) of samples
14

was treated. The obtained cell viability (in%) are 56.94, 61.80, 68.18, 71.79, 75.50 and
83.04 respectively (Negative control : 100%). These results are plotted in Fig.5. The
results suggest that the compound exhibit good cytotoxic activities in a concentration
dependent manner. The IC50 value obtained from the graph is found to be 207.7 µg/ml.
Fig.5. Cytotoxicity of [Ni(L1H2)2]Cl2 against lung cancer cell line.
3.6. Antibacterial and antifungal assay
Benzothiazole and benzimidazole derivatives are well known antimicrobial agents
with chemotherapeutic importance [41]. The antimicrobial potency of newly synthesized
ligands and complexes were evaluated against gram-positive bacteria (S. epidermidis,E.
faecalis), gram-negative bacteria (K. pneumoniae, E. coli) and fungi (A. fumigatus and C.
albicans. Ciprofloxacin and fluconazole were used as standards for antibacterial and
antifungal assays respectively. The results are tabulated in table 2.
Table 2. In vitro antimicrobial activity (MIC in μg/mL)
Gram-positive
bacteria
Gram-negative
bacteria
Fungi
Microbial
Strains
S.
E.
K.
E.
A.
C.
epidermidis faecalis pneumoniae
coli fumigatus albicans
L1H2
0.4
50
3.12
50
0.4
0.4
15

L2H2[19]
[Co(L1H)(L1)]
[Co(L2H)(L2)]
[Ni(L1H2)2]Cl2
[Ni(L2H2)2]Cl2
Ciprofloxacin
Flucanazole
0.8
0.8
0.4
0.4
1.6
1.6
-
12.5
3.12
12.5
50
0.4
0.4
0.8
6.25
0.4
0.8
-
3.12
12.5
6.25
12.5
12.5
1.6
0.4
0.4
0.4
0.4
0.8
-
0.4
0.8
0.4
0.4
0.4
-
3.12
1.6
-
-
16
8
Between the two gram positive bacterial strains, synthesized compounds have
shown excellent growth inhibition activity against S. epidermidis. Hence the cobalt
complexes are 2-4 times more active than the standard., ciprofloxacin. K. pneumoniae is
found to be sensitive in gram-negative strains. Upon complexation, cobalt complex of
L1H2 has shown 2-16 fold activity enhancement against gram-negative strains.
According to Overtone's concept of cell permeability, the lipid membrane of the
cell favours the passage of lipid-soluble materials. Hence lipophilicity plays an important
role in antimicrobial activity. In the case of chelation of a bulky ligand to a metal cation,
due to the overlap of the ligand orbitals and partial sharing of positive charge of the metal
ion with donor groups, the polarity of the metal ion is significantly reduced[42]. Due to
the increased delocalization of the π-electrons over the whole chelate ring, lipophilicity of
the complex increases. This enhances the penetration of the complexes into the lipid
membrane, which in turn blocks the metal binding sites on enzymes of
microorganisms[43]. Hence the metal complexes seem to block the synthesis of proteins
and disturb the process of respiration of the cell restricting the further growth. Hence the
enhanced activity of cobalt complexes in comparison with nickel complexes could be
related to reduced overall polarity of the molecule, which increases the lipophilic nature
of the complex, favoring the efficient permeation through lipid layer of the
microorganism[44].
Compounds are inhibiting the growth of A. fumigatus and C. albicans even at a
very low concentration of 0.4 μg/mL. This makes the complexes very effective against
fungal strains, over the tested standard-fluconazole.
16

4. Conclusions
In the present work two new Schiff base ligands, 3-(hydroxyimino)-2-butanone-2-
(1H-benzothiazol-2-yl)hydrazone and 3-(hydroxyimino)-2-butanone-2-(1H-
benzimidazol-2-yl)hydrazone are synthesized and characterized. The ligands bind the
metal centers in tridentate mode through ring-N, azomethine-N and oxime-N atoms
respectively. Analytical and spectroscopic data for the complexes indicate that the ligands
have shown ML2 type octahedral coordination towards Ni(II) and in situ generated Co(III)
ions. [Co(L1H)(L)]has crystallized in the monoclinic crystal system with P 21/c space
group and [Ni(L1H2)2]Cl2 in P -1. X-ray structures have shown meridional fashion of 2
tridentate ligands to give distorted octahedral geometry to the complexes. From the
crystal structures and comparison of bond lengths, it is clear that the ligands have shown
the similar mode of ligation, but a different mode of deprotonation with the metal ions.
Ligands are left neutral in the case of nickel complexes while anionic in the case of cobalt
complexes. Further, in situ oxidation of Co(II) to Co(III) has triggered a different mode of
deprotonation between the two ligands of the same cobalt complex. Among the 60 human
cancer cell lines tested, [Ni(L1H2)2]Cl2 has very effectively inhibited the growth of Non-
Small Cell Lung Cancer cell line EKVX (GI %: 73.34) and Breast Cancer cell line MDA-
MB-468(GI %: 69.95). Further, synthesized compounds have shown excellent growth
inhibition activity against S. epidermidis. Complexes are even found to be very effective
against fungal strains, over the tested standard-fluconazole.
Appendix A. Supplementary data
CCDC 1491779, 1491782 and CCDC 1524740 contains the supplementary
crystallographic data for [Co(L1H)(L)], [Ni(L1H2)2]Cl2 and [Ni(L2H2)2]Cl2. 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.
Acknowledgements
The authors thank USIC, Karnatak University, Dharwad, for providing spectral
and SC-XRD facilities. Recording of ESI-mass spectra from IIT Kanpur and CDRI-
Lucknow is greatly acknowledged. NCI-USA is acknowledged for NCI-60 Human
17

Tumor Cell Lines Screen under Developmental Therapeutics Program. One of the authors
(Vinayak Kamat) is thankful to UGC-New Delhi for providing financial assistance under
UGC-SRF (101864/June 2013).
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Graphical abstract
21