Transition metal complexes of N′-(2-(hydroxyimino)propanoyl)isonicotinohydrazide: Synthesis, characterization, DNA interaction and anticancer evaluation

Article abstract of DOI:10.1016/j.ica.2017.02.034

A new tetradentate chelating ligand, N′-(2-(hydroxyimino)propanoyl)isonicotinohydrazide and its selective transition metal complexes have been synthesized and evaluated for anticancer behavior. The ligand structure was determined by single-crystal X-ray crystallography. All complexes were investigated by elemental, thermogravimetric analysis, IR, NMR, ESI Mass and electronic spectroscopic studies. The ligand exhibited monoclinic lattice structure with Cc space group, having 4 ligand molecules in each unit cell. The geometrical isomerism (E and Z) was observed in ligand due to the restricted rotation along the >C[dbnd]N– functional group. The interaction of ligand and complexes with calf-thymus DNA (CT-DNA) has been extensively studied using absorption, emission, viscosity and thermal denaturation studies with E. coli DNA. The DNA cleavage properties were screened using plasmid pBR322 DNA by gel electrophoresis method. Complexes have been evaluated for their in vitro antiproliferative activity against human cancer cells of different origin such as MCF-7, Mia-Pa-Ca-2 and DU-145 by using SRB (sulforhodamine B) assay, in which iron complex have shown better antiproliferative activity compared to remaining complexes.

Full text of DOI:10.1016/j.ica.2017.02.034

Accepted Manuscript
Research paper
Transition Metal Complexes of N'-(2-(hydroxyimino)propanoyl)isonicotinohy-
drazide: Synthesis, Characterization, DNA Interaction and Anticancer Evalua-
tion
Divya Hegde, Ganesh N. Naik, Ramesh S. Vadavi, V. Shyam Kumar, Delicia
A. Barretto, Kalagouda B. Gudasi
PII:
S0020-1693(16)31043-X
http://dx.doi.org/10.1016/j.ica.2017.02.034
ICA 17462
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
21 December 2016
8 February 2017
22 February 2017
Please cite this article as: D. Hegde, G.N. Naik, R.S. Vadavi, V.S. Kumar, D.A. Barretto, K.B. Gudasi, Transition
Metal Complexes of N'-(2-(hydroxyimino)propanoyl)isonicotinohydrazide: Synthesis, Characterization, DNA
Interaction and Anticancer Evaluation, Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.
2
017.02.034
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Transition Metal Complexes of N'-(2-(hydroxyimino)propanoyl)isonicotinohydrazide:
Synthesis, Characterization, DNA Interaction and Anticancer Evaluation
1
1
2
3
3
Divya Hegde , Ganesh N. Naik , Ramesh S. Vadavi , Shyam Kumar V. , Delicia A. Barretto
1
and Kalagouda B. Gudasi *
1
Department of Chemistry, Karnatak University, Pavate Nagar, Dharwad-580 003,
Karnataka, India.
2
Department of Chemistry, B.K. College, Belgaum-590 001, Karnataka, India.
3
Department of Biotechnology and Microbiology, Karnatak University, Pavate Nagar,
Dharwad-580003, Karnataka, India.
Abstract
A new tetradentate chelating ligand, N'-(2-(hydroxyimino)propanoyl)isonicotinohydrazide
and its selective transition metal complexes have been synthesized and evaluated for
anticancer behavior. The ligand structure was determined by single-crystal X-ray
crystallography. All complexes were investigated by elemental, thermogravimetric analysis,
IR, NMR, ESI Mass and electronic spectroscopic studies. The ligand exhibited monoclinic
lattice structure with Cc space group, having 4 ligand molecules in each unit cell. The
geometrical isomerism (E and Z) was observed in ligand due to the restricted rotation along
the >C=N- functional group. The interaction of ligand and complexes with calf-thymus DNA
(CT-DNA) has been extensively studied using absorption, emission, viscosity and thermal
denaturation studies with E. coli DNA. The DNA cleavage properties were screened using
plasmid pBR322 DNA by gel electrophoresis method. Complexes have been evaluated for
their in vitro antiproliferative activity against human cancer cells of different origin such as
MCF-7, Mia-Pa-Ca-2 and DU-145 by using SRB (sulforhodamineB) assay, in which iron
complex have shown better antiproliferative activity compared to remaining complexes.
1

*
Corresponding author: Prof. K. B. Gudasi
Tel: +91-836-2215286 (Extn-28); Fax: +91-836-2771275, E-Mail: drkbgudasi@kud.ac.in
Key words: Ketoxime; Ethyl pyruvate; Transition Metal Complexes; DNA Binding and
Cleavage Studies; Antiproliferative studies.
1
. Introduction
An oxime is a chemical compound belongs to hydroxylamine family, with the general
1
2
1
2
formula R R C=N-OH, where R is organic side chain and R is either hydrogen, forming an
aldoxime, or another organic group, forming ketoxime [1-2]. These molecules are appear to
be amenable to biotransformations such as reduction, oxidation, hydrolysis and conjugations
with organic and inorganic molecules [3]. Several oximes/ N-OH containing compounds
were reported as effective antimicrobial, anti-inflammatory, to serve as efficient drugs in non-
absorbed iron chelating therapy and to be potential agents for treatment of osteoarthritis [4].
Numerous aliphatic and aromatic oxime ligands have been shown to possess fungicidal [5-6],
bactericides [7], analgesic [8], antioxidant [9-10], antitumor [11-13], insecticidal [14],
antiviral [15], antimalarial [16], antilepral [17] properties. Oximes are also known to initiate
DNA cleavage and ability to relax blood vessels [18-20]. Thus, both the increasing synthetic
effort and growing body of scientific information with respect to N-OH compounds hold
great promise for the development of novel therapeutic drugs in various fields of medicine
[
21-22].
Another versatile class of organic compounds called pyruvic acids, known to possess
anti-inflammatory activities by suppressing the generation of tumor necrosis factor-α (TNF-
α) and interleukin-6 (IL-6), and inhibiting the expression of pro-inflammatory agents such as
inducible nitric oxidase synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-
inflammatory mediators [23-30]. The anti-inflammatory properties and the ability to
2

scavenger ROS (reactive oxygen species) confirmed in numerous studies of pyruvate and its
-
analogues in animal models of human disease [31-36]. Pyruvate (CH COCOO ) the anionic
3
form of a pyruvic acid, involves in the intermediary metabolism process as a product of
glycolysis and the starting substrate for the tricarboxylic acid (TCA) cycle [37]. Under
aerobic conditions, pyruvate enters into mitochondria, where it undergoes oxidative
decarboxylation in a reaction catalyzed by the enzyme complex, pyruvate dehydrogenase to
form acetyl coenzyme A and carbon dioxide. Therefore, with the view of making good use of
pyruvic acids, we used pyruvic acid derived side chain for the construction of novel possible
drug molecule.
The pharmacological behavior of any drug molecules are mainly depends upon their
ability to reachtarget in vivo. Most of the synthesized drugs are usually fails either in late
development or early stage of clinical trials due poor ADME (absorption, distribution,
metabolism and excretion) properties [38]. Several researchers proved that, transition metal
complexes are the better than corresponding simple ligands due to their better ADME
properties [39-41]. On coordination, ligands might improve their bioactivity profiles, while
some inactive ligands may acquire pharmacological properties and consequently they have
become an important class of selective binding agents for nucleic acids [42]. Totally, three
platinum (II) compounds, cisplatin, carboplatin and oxaliplatin have been approved by the
FDA (FDA approvals in 1978, 1989, and 2002, respectively). Nonetheless, side effects
associated with these complexes and the development of tumor resistance, have led to the
search for new generations of metal based anticancer agents [43]. Substituted hydrazones of
pyruvate derivatives and their transition metal associates are capable of interacting with
specific conformations of DNA double helix or with specific DNA base sequences, and
eventually to induce apoptosis. Redox activeness of first row transition metal ions in the
3

ligand periphery reflects their ability to cleave DNA in the oxidative process. Hence
functions of DNA will alter and may reduce the rapid cell growth in cancer infected cells.
Considering with these literature support, we have synthesized a novel ketoxime
based compound composed of isoniazid as parental moiety and pyruvic acid derivative as
side chain and terminal hydroxyl amine as functional group. The intension of the present
ligand is, to obtain synergistic behaviour of isoniazid, pyruvate and hydroxyl amine
functional groups. To get better anticancer behaviour, we have further synthesized various
transition metal complexes of current ligand and tested for anticancer activity.
2
2
. Experimental
.1. Materials
Isonicotinic acid, ethyl pyruvate, tris-base and tris-HCl, ethidiumbromide (EtBr) and
Calf thymus DNA were obtained from Sigma Aldrich and were of analytical grade.
Hydrazine hydrate, potassium hydroxide, hydroxylamine hydrochloride, acetic acid and metal
chlorides/nitrates of AR grade obtained from Spectrochem, India. Plasmid DNA-pBR322 was
purchased from Chromous Biotech, India. Agarose was obtained from Lonza (India). All the
solvents were of either HPLC or AR grade and obtained from S.D. Fine chemicals.
2
.2. Methods
Metal content of Fe(III), Co(II), Ni(II) and Cu(II) complexes was determined
volumetrically and that of Zn(II) complex, gravimetrically. The chloride content was
-3
determined in the form of AgCl. Conductance measurements were recorded in DMF (10 M)
using ELICO-CM-82 conductivity bridge with cell type CC-01 and cell constant 0.53. The IR
spectra were recorded on a Nicolet 170SX FT-IR spectrometer with the fixed range of 4000-
-1
4
00 cm using KBr discs. Carbon, hydrogen and nitrogen content were determined using
1
13
Thermo quest elemental analyzer. H and C NMR spectra of ligand and zinc complex were
recorded in DMSO-d6 on a Bruker 500 MHz spectrometer. Electronic spectra were recorded
4

-
on a Hitachi-U-3310 UV-visible spectrophotometer in the 200-900 nm range using DMF (10
3
M) solvent. Thermogravimetric study was carried out over a temperature range of 25-
-1
1
2
2
000°C using TGAQ500 analyzer with a heating rate of 10°C min .
.3. Synthesis
.3.1. Preparation of the Ligand
The ligand was synthesized in four simple steps as presented in Scheme 1. All the
1
13
intermediates are characterised by H and C NMR and GC mass spectral studies. Isoniazid
was synthesised as per the literature report [44]. Synthesis of the ligand includes following
steps.
2
.3.1.1. Synthesis of N'-(2-oxopropanoyl)isonicotinohydrazide (IV)
To a methanolic solution of isoniazid (5.00g, 36.46 mM) anequimolar quantity of
ethyl pyruvate (4.23g, 36.46 mM) was added and stirred for an hour. The solid separated was
filtered, washed with cold methanol and dried in air. The product was recrystallized from hot
ethanol. The purity of the compound was checked by TLC on precoated silica gel plates
(Rf:0.45, Hexane:Ethyl acetate, 7:3 ratio).
2
.3.1.2. Synthesis of N'-(2-(hydroxyimino)propanoyl)isonicotinohydrazide (V)
The methanolic solution of KOH (2.86g, 51.01mM) was added to methanolic solution
of hydroxylamine hydrochloride (2.36g, 34.00 mM) under stirring. The reaction mixture was
kept in ice bath for 5 to 10 min. During the reaction, KCl generates and starts precipitating
out from the solution. Once the precipitation of KCl was complete, N'-(2-
(oxopropanoyl)isonicotinohydrazide (4.00 g, 17.00 mM) was added with stirring and the
mixture was filtered immediately under vacuum. The solvent was removed from the filtrate
under vacuum to get solid product of potassium salt. This salt was acidified with 1.25N acetic
acid to get free ketoxime product [45]. The purity of the compound was checked by TLC on
precoated silica gel plates (Rf:0.45 in Hexane: Ethyl acetate, 1:9).
5

Yield 75%; m.p. 240ºC; colorless solid. Anal. Calcd for C H N O (%): C, 48.65; H, 4.54;
9
10
4
3
-1
N, 25.21. Found (%): C, 48.47; H, 4.32; N, 25.14. IR (cm ): amide carbonyl ν(C6=O1) 1667,
carbonyl ν(C7=O2) 1634, azomethine ν(C=N) 1602, amide ν(N2H/N3H) 3044/3112,
1
hydroxyl ν(OH) 3400. H NMR of major product (7E, 9Z isomer) (500 MHz, DMSO-d ,
6
ppm): 11.92 (s, O3H, 1H), 10.64 (s, N3H, 1H), 9.98(s, N2H, 1H), 8.76 (dd, Jmeta=2.5Hz,
Jortho=5.5Hz, 1H, C1H and C5H), 7.76 (dd, Jmeta=2.0Hz, Jortho=5.5Hz, 1H, C2H and C4H),1.92
1
3
(
s, C8H, 3H). C NMR major product (7E, 9Z isomer) (500 MHz, DMSO-d , ppm): 163.31
6
(C6), 163.25 (C7), 150.57 (C9), 149.31(C1 and C5), 139.52(C3), 121.32 (C2 and C4),
1
9
.72(C8). H NMR of minor product (7Z, 9E isomer) (500 MHz, DMSO-d , ppm): 11.83 (s,
6
O3H', 1H), 10.93 (s, N3H', 1H), 9.68(s, N2H', 1H), 8.79 (dd, Jmeta=2.5Hz, Jortho=5.5Hz, 1H,
C1H' and C5H'), 7.80 (dd, Jmeta=2.0Hz, Jortho=5.5Hz, 1H, C2H' and C4H'), 1.88 (s, C8H', 3H).
1
3
C NMR minor product (7Z, 9E isomer) (500 MHz, DMSO-d , ppm): 164.08 (C6'), 163.21
6
(C7'), 150.47 (C9'), 149.32 (C1' and C5'), 139.52(C3'), 121.34 (C2' and C4'), 9.72 (C8').
2
.3.2. Synthesis of Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) complexes
The ligand (V) (0.5g, 1 mM.) dissolved in methanol was added drop wise to an
aqueous methanolic solution of Co(II), Ni(II), Cu(II), Zn(II) nitrates and FeCl (1mM)
3
andstirred for 6-7 hrs at room temperature to obtain respective complexes. After completion
of the reaction, the precipitate obtained was filtered, washed with methanol followed by ether
and air dried.
CoL: Yield 65%; claret red solid. Anal. Calcd for K [Co (C H N O )].H O (%): C, 21.67;
2
3
18 20 12 20
2
-1
H, 2.22; N, 16.85; Co, 17.73. Found(%): C, 21.69; H, 2.28; N, 16.75; Co, 17.8. IR (cm ):
carbonyl ν(C6=O1) 1659, new ν(C=N) 1617.
NiL: Yield 60%; vert green solid. Anal. Calcd for K [Ni (C H N O )].H O (%):C, 21.69;
2
3
18 20 12 20
2
-1
H, 2.22; N, 16.86; Ni, 17.67. Found (%): C, 21.62; H, 2.18; N, 16.79; Ni, 17.52. IR (cm ):
carbonyl ν(C6=O1) 1655, new ν(C=N) 1610.
6

CuL: Yield 62%; brown solid. Anal. Calcd for [Cu (C H N O )].2H O(%): C, 20.17; H,
2
9
16
6
13
2
-1
4
.06; N, 14.11; Cu, 21.34. Found (%): C, 20.12; H, 4.01; N, 14.08; Cu, 21.29. IR (cm ):
carbonyl ν(C7=O2) 1624, new ν(C=N) 1615.
ZnL: Yield- 71%; white solid. Anal. Calcd for [Zn (C H N O )].2H O (%): C, 18.54; H,
2
9
16
6
13
2
1
3
.46; N, 14.41; Zn, 22.43. Found (%): C, 18.68; H, 3.87; N, 14.35; Zn, 22.68. H NMR (500
MHz, DMSO-d , δ, ppm): 10.92 (s, N3H, 1H), 8.79 (d, J=7.0 Hz, 1H, C1H), 8.64(d, J=7.5
6
Hz, 1H, C5H), 7.81 (d, J=7.0 Hz, 1H, C4H), 6.57 (d, J=6.5 Hz, 1H, C2H), 1.22 (s, C8H, 3H).
1
3
C NMR (500 MHz, DMSO-d , δ, ppm): 165.32 (C6), 153.05 (C7), 150.35 (C9), 149.30 (C1
6
-1
&
C5), 139.18 (C3), 121.16 (C2 & C4), 10.66 (C8).IR (cm ): carbonyl ν(C7=O2) 1620, new
ν(C=N) 1603.
FeL: Yield 69%; dark red solid. Anal. Calcd for [Fe (C H Cl N O )].H O(%): C, 19.17; H,
2
9
16
4
4
7
2
3
1
.22; N, 9.94; Cl, 25.15; Fe, 19.81. Found (%): C, 19.19; H, 3.19; N, 9.91; Cl, 25.12; Fe,
-1
9.78. IR (cm ): carbonyl ν(C7=O2) 1626, new ν(C=N) 1618.
2
.4. X-ray Crystallography
Single
crystal
X-ray
diffraction
study
of
the
ligand
N'-(2-
(hydroxyimino)propanoyl)isonicotinohydrazide was carried at 296 K using BRUKER
SMART APEX-CCD diffractometer with MoK\α radiation (λ =0.71073). All the non-
hydrogen atoms were refined with anisotropic temperature factors. The structures were
2
solved using SHELXL-97 and refined using Full-matrix least squares F . All the calculations
were performed using WinGX crystallographic software. Molecular geometry calculations
were performed using PARST and the structure drawing was obtained with ORTEP-3 for
windows program [46-48].
2
.5. DNA Binding studies: An extensive study of interaction of the prepared compounds
with DNA was undertaken.
7

2
.6. UV Absorption Studies
The concentration of CT-DNA was calculated from its known extinction coefficient at
-1
-1
2
60 nm (6600 M cm ). Absorption titration experiments were performed by maintaining a
constant ligand and metal complex concentration (20 μM) and varying nucleotide
concentration (0-60 μM) in buffer. After addition of DNA to the ligand and metal complex,
the resulting solution was allowed to equilibrate at 25ºC for 20 min, after which absorption
readings were noted. The data were then fit to following equation (1) to obtain intrinsic
binding constant Kb [49-50].
[
DNA]/[ε –ε ]=[DNA]/[ε –ε ]+1/Kb[ε –ε ]
(1)
a
f
b
f
b
f
Where, [DNA] = concentration of DNA in base pairs,
εa= is the extinction coefficient observed for the MLCT absorption band at the given DNA
concentration,
εf= is the extinction coefficient of the complex without DNA, and
εb= is the extinction coefficient of the complex when fully bound to DNA.
A plot of [DNA]/[ε -ε ] versus [DNA] gave a slope 1/[ε - ε ] and intercept equal to (1/Kb)[ε
b
a
f
a
f
-
εf]. The intrinsic binding constant Kb is the ratio of slope to the intercept.
2
.7. Thermal Denaturation Study
DNA melting experiments were carried out in phosphate buffer (pH 7.2) on a
HITACHI U-3310 spectrophotometer equipped with a temperature-controlling programmer
ETC-717 (5ºC). Solutions of E. coli DNA (Escherichia coli DNA) in phosphate buffer gave a
ratio of UV absorbance at 260 and 280nm (A260/A280) as 1.8-1.9, indicating that the DNA
was pure and sufficiently free of contaminants like the proteins and RNA. UV melting
profiles were obtained by scanning A260 absorbance monitored at a heating rate of 5ºC /min
for solutions of E. coli DNA (100 μM) in the absence and presence of ligand and metal
complexes (20 μM) from 25 to 85ºC. The melting temperature T , which is defined as the
m
8

temperature where half of the total base pairs are unbound, was determined from the
midpoint of the melting curves [51].
2
.8. Competitive Binding Fluorescence Measurements
The apparent binding constants (Kapp) of the complexes were determined by
fluorescence spectral technique using ethidiumbromide (EtBr) bound CT-DNA solution in
phosphate buffer. The changes in fluorescence intensities at 596nm (546 nm excitation) of
EtBr bound to DNA were recorded with an increasing amount of ligand and metal complex
concentration. EtBr was non-emissive in phosphate buffer due to fluorescence quenching of
the free EtBr by the solvent molecules [52-53].
2
.9. Viscosity Measurements
Viscosity experiments were carried out using Ostwald micro-viscometer maintained
at 26ºC in a thermostatic water bath. Flow time of solutions in phosphate buffer (pH=7.2)
was recorded in triplicate and an average flow time was calculated. Data were presented as
0
1/3
(
η/η ) versus binding ratio, where η is the viscosity of DNA in the presence of complex and
0
η is the viscosity of DNA alone [52-53].
2
.10. DNA Cleavage Studies
The DNA cleavage study of the synthesized compounds were determined by agarose
gel electrophoresis using the E. coli pBR322 plasmid as a target. The synthesized compounds
100μM) were dissolved in 6 % DMSO (dimethylsulfoxide) solvent and mixed with the
(
target plasmid (1:1). The mixture was then incubated at 37ºC for 2 h. After the incubation
period, the plasmid and the compound mixture was mixed with the tracking dye bromo-
phenol blue (1:1). It was then loaded into 1 % agarose gel (containing 0.5 μg/mL ethidium
bromide) wells along with two control wells, the first one with the untreated plasmid and the
second one with the plasmid treated with DMSO solvent. Finally it was electrophoresed at 50
V constant voltage for about 30 mins using Tris-EDTA (TAE) buffer [52-53]. The bands
9

were visualized by UV light and photographed for analysis. The extent of cleavage of the
supercoiled DNA was determined by measuring intensities of the bands using Molecular
Imager Geldoc gel-XR imaging system (BIORAD).
2
.11. Experimental procedure for SRB Assay
The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum
and 2 mM L-glutamine. For the present screening experiment, cells were inoculated into 96
well microtiter plates in 90 µL at 5000 cells per well. After cell inoculation, the microtiter
plates were incubated at 37 °C, 5 %CO , 95 % air and 100 % relative humidity for 24 hr prior
2
to addition of experimental drugs. Experimental drugs were solubilized in DMSO to prepare
-2
a stock of 10 M concentration. At the time of experiment, four 10-fold serial dilutions were
made using complete medium. Aliquots of 10 µl of these different drug dilutions were added
-4
to the appropriate micro-titer wells already containing 90 µl of medium, resulting in10 M,
-5
-6
-7
1
0 M, 10 M and 10 M drug concentrations.
After addition of the compound, plates were incubated at standard conditions for 48
hours and assay was terminated by the addition of cold TCA (trichloroacetic acid). Cells were
fixed in situ by the gentle addition of 50 µl of cold 30 % (w/v) TCA (final concentration,
1
0% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates
were washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (50
µl) at 0.4% (w/v) in 1 % acetic acid was added to each of the wells, and plates were incubated
for 20 minutes at room temperature. After staining, unbound dye was recovered and the
residual dye was removed by washing five times with 1 % acetic acid. The plates were air
dried. Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was
read on an Elisa plate reader at a wavelength of 540 nm with 690 nm reference wavelength.
Percent growth was calculated on a plate-by-plate basis for test wells relative to
control wells. Percent Growth was expressed as the ratio of average absorbance of the test
1
0

well to the average absorbance of the control wells×100. Using the six absorbance
measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at
the four concentration levels (Ti)], the percentage growth was calculated at each of the drug
concentration levels. The dose response parameters were calculated for each test article.
Growth inhibition of 50% (GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is
the drug concentration resulting in a 50% reduction in the net protein increase (as measured
by SRB staining) in control cells during the drug incubation. The drug concentration resulting
in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of
drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as
compared to that at the beginning) indicating a net loss of cells following treatment is
calculated from [(Ti-Tz)/Tz] x 100 = -50 [54-55].
3
. Results and Discussion
The ligand is soluble in chloroform, hot methanol, DMF and DMSO, whereas
complexes are soluble in DMF and DMSO. The complexes are non-hygroscopic and stable in
both solid and solution phases. The analytical data for the complexes is in good agreement
with their formulae. The molar conductance values are too low for FeL, CuL and ZnL to
account for any dissociation of the complexes in DMF, suggesting their non-electrolytic
nature. The conductance of Co(II) and Ni(II) complexes clearly indicate their ionic
behaviour. White crystals suitable for XRD studies were isolated by slow evaporation of
methanolic solution of ligand. Attempts to grow single crystals of metal complexes were
unsuccessful. However, the complexes are comprehensively characterized by various
established physico-chemical techniques. The compound code and corresponding electronic
spectral details are presented in Table 1, supplementary information.
3
.1. Crystal structure description of ligand
1
1

The crystal structure refinement details, selective bond lengths and selective bond
angles are presented in Tables 1 to 3 respectively. The ORTEP diagram, molecular packing
diagram, molecular planes are presented in the Figures 1 to 3 respectively. The frames were
integrated with the Bruker SAINT Software package using a narrow-frame algorithm.
The new molecule has exhibited monoclinic structure having the space group Cc with
a = 14.32(4) Å, b = 8.91(3) Å, c = 9.44(3) Å, α = 90°, β = 114.65°, γ = 90°, V=1095.55(11)
3
Å and Z=4 for the formula C H N O . There are two molecular planes in the molecule
9
10
4
3
where the pyridine ring moiety forms one plane and hydroxyl amine part forms another plane
with an angle 85.13°. The amide carbonyl oxygen O1 is involved in intermolecular
interaction with nitrogen N2. The hydroxyl oxygen O3 is involved in intermolecular
interaction with amide carbonyl oxygen O2 and amide nitrogen N2 and hydrogen H7 bonded
to it forms intermolecular hydrogen bonding with O2. The hydrogen, H6 bonded to N3and
H5 bonded to N2 are involved in intermolecular hydrogen bonding with N1 and O1
respectively as shown in Figure 4. The nitrogen N1 in the pyridine ring shows intermolecular
interaction with amide nitrogen N3.
3
.2. Infrared spectral studies
The diagnostic IR bands of ligand and its complexes are compiled in Table 4. Ligand
has shown tetra dentate mode of coordination towards all metal ion [56]. The strong signal
-1
observed at 1667 cm in the IR spectrum of the ligand was assigned to amide carbonyl
(>C6=O1) [57]. This signal was disappeared in FeL, CuL and ZnL due to its involvement in
coordination with corresponding metal ions via enolisation with adjacent N2H followed by
deprotonation. This mode of coordination was further supported by the appearance of new
-1
signal in the region of 1614 to 1618 cm corresponds to ν(-C6=N2). This coordination
1
behavior was also confirmed by the aid of H NMR spectral studies, where proton signal
observed for N2H in the ligand spectrum was disappeared in its corresponding zinc complex.
1
2

In remaining two complexes (CoL and NiL) (>C6=O1) band was shifted to lower frequencies
-1
by 8-12 cm , on formation of the complexes indicates its coordination behavior towards
metal ions.
-1
The strong band observed at 1634 cm in the spectrum of the ligand was assigned to
-1
(
>C7=O2) has shifted to lower wave number by 8-14 cm , in FeL, CuL and ZnL suggesting
its involvement in coordination. In case of Co and Ni complexes, the signal due to (>C7=O2)
was disappeared. This indicates that, (>C7=O2) group was undergone enolisation with
adjacent N3H group, and deprotonated and finally coordinated to metal ion. This
coordination behavior was supported by the appearance of new signal in the region of 1617 -
-1
1
610 cm corresponding to new ν (-C7=N3) in Co and Ni complexes.
-1
The band due to N3H group observed at 3112 cm in the ligand spectrum was
disappeared in Co and Ni complexes and shifted to lower frequencies in remaining
complexes (CuL, ZnL and FeL). The disappearance of signal is due to the enolisation of N3H
with adjacent (C7=O2) functional group. The negative shift suffered by ν(N3H) in CuL, ZnL
and FeL compared to ligand suggests its coordination towards metal center [58-59]. The band
-1
due to N2H was observed at 3044 cm in the ligand spectrum was shifted to lower
frequencies in Co and Ni complexes and disappeared in remaining complexes. The
disappearance of N2H signal in CuL, ZnL and FeL indicates, its involvement in enolisation
with adjacent (C6=O1) functional group. The coordination of N2H and N3H were confirmed
1
by H NMR spectral studies.
-1
The bands observed at 1602 and 1556 cm in the ligand spectrum assigned to
azomethine ν(>C9=N4) and pyridine ν(C1=N1) respectively, have remain unaltered even on
complex formation, suggesting their non-involvement in coordination [57]. In the ligand
-1
spectrum the broad bands at 3400 cm was assigned to ν(O3H) was absent in the spectrum of
zinc complex, suggesting its involvement in coordination via deprotonation. This is further
1
3

1
supported by the absence of the signal corresponding to OH in the H NMR spectrum of zinc
-1
complex. The broad band observed in the region of 3432-3421 cm in all complexes was
assigned to ν(OH) of coordinated/lattice held water molecule. This is further confirmed by
-1
the appearance of a weak non-ligand band at 856, 839, 853, 888 cm assignable to rocking
mode of coordinated water molecule in cobalt, nickel, copper and iron complexes
respectively [57]. The presence of lattice held and/or coordinated water molecules are further
confirmed by thermo-gravimetric analysis. All the complexes except FeL, have shown two
-1
-1
new bands in the region of 1384 cm and 1060-1064 cm and is assigned to ν (NO ) and
1
3
ν (NO ) respectively. This confirms the presence of coordinated nitrate ion in chelating
5
3
fashion [60].
.3. NMR studies.
3
1
13
The detailed H and C NMR spectral assignments of ligand and its Zn(II) complex
are presented in experimental section. The numbering scheme followed for the NMR
1
13
assignment of carbons and protons is presented in Scheme 1. The H and C NMR spectra of
ligand are depicted in Figures 5-6, and those of Zn (II) complex in Figures 7-8 respectively.
1
13
Both H and C NMR spectra of ligand have shown two set of signals indicates the presence
of ligand in E and Z isomeric forms, arising due to restricted rotation across N3=C7 and
N4=C9 bonds [61-64] as presented in Scheme 1.
The three well separated singlets at 11.92/11.83, 9.98/9.68 ppm and 10.64/10.93 ppm
are assigned to O3H, N2H and N3H protons respectively [65]. In Zinc (II) complex, the
signal due to O3H proton was absent, indicates its deprotonation followed by coordination.
The signal at 9.98/9.68 ppm, assigned to N2H in the ligand spectrum was absent in the
spectrum of Zn complex indicates its involvement in enolisation with adjacent carbonyl
1
3
group. This is further confirmed by C NMR spectral studies. The signal due to C6 appeared
at 163.31 ppm in the ligand spectrum has shifted downfield to 165.32 ppm on coordination
1
4

indicating the involvement of oxygen in coordination. The singlet observed at 10.64/10.93
ppm assigned to N3H in the ligand spectrum, was shifted to 10.92 ppm on complex formation
indicates its involvement in coordination. Aromatic protons of ligand have not shown any
significant shift after complex formation indicates the noninvolvement of pyridine nitrogen in
coordination.
The signal corresponds to C7 carbon at 163.25 ppm in ligand spectrum was shifted to
1
53.05 ppm on complex formation indicates the participation of carbonyl oxygen (C7=O) in
coordination. The signal due to C9 carbon appeared at 150.57 ppm in the ligand spectrum has
not shown any significant shift on complex formation. The aromatic carbons appeared in the
region of 139.31 to 149.31 ppm in the ligand spectrum and these signals do not shown any
considerable shifts on complex formation. This fact supports the noninvolvement of pyridine
nitrogen in coordination.
1
13
It is evident from IR, H NMR and C NMR spectra of zinc complex that the ligand
coordinates through amide carbonyl oxygen and NH nitrogen towards one zinc(II) ion and
second amide carbonyl oxygen and OH oxygen towards another zinc(II) ion in tetra-dentate
fashion.
3
.4. Electronic spectral studies
Electronic spectra of ligand and their complexes are recorded in order to assign the
plausible geometry around the metal ions. The electronic spectrum of the ligand shows a
band at 269 nm assigned to π→ π* transition. Electronic spectrum of cobalt complex shows a
4
4
d–d transition around 521 nm assigned to T (F) → T (P) transition, which is consistent
1
g
1g
with an octahedral geometry. The bands at 393and 601 nm with the nickel complex are
3
3
3
3
assigned to A (F) → T (P) and A (F) → T (F) respectively and are suggestive of an
2
g
1g
2g
1g
octahedral geometry. The copper complex shows an absorption around 575 nm assigned to
3
3
E → T transition, suggesting an octahedral geometry [66].
g
2g
1
5

3
.5. Mass spectral studies
The mass spectrum of the ligand has a molecular ion peak at 222 which corresponds
to its molecular weight. The ESI mass spectra of cobalt, nickel, copper, zinc and iron
+
+
+
+
complexes show molecular ion peaks at m/z 900(M ), 897(M+1) , 542(M ), 548(M+1) and
+
5
42(M ) which correspond to their respective molecular weights.
.6. Thermal studies
3
In order to understand the thermal stability and decomposition pattern, we have
undertaken the thermogravimetric studies of all synthesized complexes in the temperature
range 25-1000ºC. The decomposition details of various metal complexes are presented Table
S2 of supplementary information. The graphs represent the decomposition along with
derivative graphs are depicted in Figure S9 to S11 of supplementary information.
All the complexes exhibit a weight loss around 100ºC indicating the presence of
lattice held water. In the thermograms of CoL, NiL and FeL complexes, a weight loss in the
range of 1.78- 3.4% was observed between 68-110ºC, 75-106ºC and 68-102ºC respectively
suggesting presence of one lattice held water molecule in each metal complexes. In case of
CuL and ZnL, weight loss of 6.56 to 6.58%, is observed in the range of 62-130 ºC and
correspond to the loss of two lattice held water molecules [67].
The loss of lattice held water molecules is followed by the loss of coordinated water
molecules in all the complexes. The weight loss of 3.67- 4.1% in the temperature range of 99
to 150ºC, in CoL and NiL was assigned to loss of two coordinated water molecules. In case
of Zn complex, weight loss of 12.21% is observed in the temperature range of 110 to 180ºC,
assignable to the loss of four coordinated water molecules. The combined weight loss of four
coordinated water molecules along with two nitrates/four chlorides is observed in case of
CuL and FeL complexes in the temperature range of 130-258ºC [68]. The loss of coordinated
nitrate ions is observed in the temperature range of 150 -257ºC in CoL, NiL, and ZnL. The
1
6

loss of one ligand in case of CuL, ZnL and FeL and two ligands in CoL and NiL is observed
between the temperature range of 250 to 809ºC and the weight loss is in the range of 38.11 to
4
3
3
5.2%.
.7. DNA Binding Studies
.7.1. Absorption Spectral Studies
Examining the changes in absorption spectrum of the metal complexes upon addition
of increasing amounts of DNA is one of the most widely used methods to know the
interaction of metal complexes with DNA and for determining overall binding constants. The
absorption spectra of ligand and complexes in the presence and absence of CT-DNA is shown
in the Figure 9. Compound bound to DNA through intercalation usually results in
hypochromic effect and red shift (bathochromic effect).The extent of the hypochromism is
commonly consistent with the strength of intercalative interaction. Upon addition of calf-
thymus DNA to ligand and its complexes, there is a decrease in molar absorptivity
(hypochromism) of the intense intra ligand absorption bands (240-400 nm) of complexes.
The hypochromismin case of ligand (260 nm), cobalt complex (266 nm), nickel complex
(
266 nm and 326 nm), copper complex (318 nm), zinc complex (260 nm) and iron complex
278 nm) indicate strong binding with DNA. In order to compare the DNA-binding affinities
(
of these complexes quantitatively, their intrinsic binding constants with CT-DNA is obtained
by monitoring the changes in absorption at intra ligand band with increasing concentrations
4
-1
4
-1
4
-1
of DNA using equation (1) and is found to be 1.46 x 10 M , 2.95 x 10 M , 0.953 x 10 M ,
4
-1
4
-1
4
-1
3
.63 x 10 M , 0.802 x 10 M and 3.89x 10 M for ligand, cobalt, nickel, copper, zinc and
iron complexes respectively, indicating their strong binding to DNA except nickel and zinc
complexes which shows weaker interaction.
3
.7.2. DNA Thermal Denaturation
1
7

Intercalation of small molecules into the double helix is known to increase the helix
melting temperature(Tm), the temperature at which the double helix denatures into single-
stranded DNA. The extinction coefficient of DNA bases at 260 nm in the double strand is
much less than that in the single-stranded form. Hence, melting of the helix leads to an
increase in the absorption at this wavelength. E. coli DNA was seen to melt at 55-60ºC
(phosphate buffer) in the absence of complex. The melting temperature of DNA on addition
of ligand, nickel and zinc complexes is found to be in the region 60-65ºC and for cobalt,
copper and iron complexes it is found in the range 65-70ºC which indicates that cobalt,
copper and iron complexes binds strongly compared to the ligand, nickel and zinc complexes
(Figure 10). This behavior is comparable to classical intercalations.
3
.7.3. Ethidium Bromide Displacement Assay
The competitive DNA binding of complexes has been studied by examining changes
in emission intensity of ethidium bromide (EtBr) bound to CT-DNA as a function of the
concentration of complexes added. Though the emission intensity of EtBr in buffer medium
is quenched by the solvent molecules, it is enhanced by its stacking interaction between
adjacent DNA base pairs. Ligand and complexes when added to DNA pretreated with EtBr
{
[DNA]/[EtBr]=1:1}, the DNA-induced emission intensity of EtBr has decreased as shown in
Figure 11. Addition of a second DNA binding molecule would quench the EtBr emission by
either replacing the DNA-bound EtBr (if it binds to DNA more strongly than EtBr) or
accepting an excited state electron from EtBr. The apparent binding constant (Kapp) has been
calculated from the equation (2) [69].
KEtBr[EtBr]=K [Compound]
(2)
app
7
-1
where, K[EtBr] is 1 x10 M and the concentration of EtBr is 20 μM; [compound] is the
concentration of the compound causing 50% reduction in the emission intensity of EtBr. The
6
-1
Kapp values for ligand and iron complex are 5.0x10 M , for cobalt and copper complexes
1
8

6
-1
6
-1
are 6.6x10 M and for nickel and zinc complexes are 4.0x10 M . The higher values of Kapp
indicate that these compounds bind to DNA by intercalation.
3
.7.4. Viscosity Measurements
Absorption and emission studies generally provide necessary, but not sufficient clues
to support intercalative binding mode. To further clarify the nature of interaction between the
complexes and DNA, viscosity measurements have been carried out. Lengthening of DNA
helix occurs on intercalation as base pairs are separated to accommodate the binding ligand
leading to increase in DNA viscosity [70]. Partial or on classical intercalation of ligand may
bend or kink the DNA helix, thereby decreasing its effective length and subsequently
viscosity. The values of relative specific viscosities of DNA in the absence and presence of
complexes are plotted against [complex]/[DNA].The relative viscosities of CT-DNA bound
to ligand and its complexes, increased with increasing concentration (Figure 12), indicative
of a classical intercalation [71]. The increased degree of viscosity which may depend on the
binding affinity of compounds to CT-DNA follows the order CuL>FeL>CoL>LH>NiL>ZnL.
3
.8. DNA Cleavage Studies
The extent to which the newly synthesized ligands and their metal complexes could
function as DNA cleavage agents is examined using supercoiled (SC) plasmid pBR322 DNA
as a target. The agarose gel electrophoresis method employed to study the efficiency of
cleavage by the synthesized compounds. The photograph representing the cleavage of DNA
by various samples is presented in Figure 13.
The characterization of DNA recognition by transition metal complex has been aided
by the DNA cleavage chemistry that is associated with redox-active or photoactivated metal
complexes. The electrophoretic analysis clearly reveals that the new molecule and their metal
complexes have acted on DNA as there has been a difference in molecular weight between
the control and the treated DNA samples. Usually the pBR 322 plasmid shows nicked/open
1
9

circular, linear and supercoiled DNA bands [72-73]. It is observed that at a given
concentration, there is diminishing of these bands with a prominent streak when treated with
the complexes compared to standard. The data clearly suggest that, DNA cleavage activity
for CoL and FeL is more compared to CuL and other compounds have not shown any
significant cleavage. The average activity is observed for NiL and ZnL. Out of all cleavage
experimental results, the best activity is observed for iron complex and in which, DNA was
cleaved at 100 μM concentration. The experiment was extended to various concentrations
like 75μM, 50μM, 25μM and 12.5μM to evaluate the lowest activity limit. The results are
well within the acceptable limit up to 25μM concentration.
3
.9. Anticancer Activity Studies
The anticancer activities of ligand, and its metal complexes such as CoL, CuL and
FeL are tested against human MCF-7 (Human Breast Cancer Cell Line), Mia-Pa-Ca-
(Human Pancreatic Cancer Cell Line) and DU145 (Human Prostate Cancer Cell Line)
2
cancer cell lines. The results are compared with the results obtained by analyzing adriamycin
under identical conditions using SRB assay. The LC50, TGI and GI50 values are compared
between LH, CoL, CuL and FeL for their action on DU-145, MCF-7 and Mia-Pa-Ca-2 cell
lines and results are compiled in Table 5 and represented graphically in Figure 14.
The complexes exhibited enhanced cytotoxic activities in all the three cell lines
compared to ligand alone. In this case, the enhancement of antiproliferative activity, with
respect to ligand, might be related to their different structural characteristics and mainly
attributed to redox active metal ions. The results show that the cell viability of all the tested
cell lines has decreased with increasing the concentration of the compounds and has exhibited
moderate cytotoxic activities in a concentration dependent manner. All the tested compounds
have shown better result for DU-145 cell lines and shown good to moderate result for MCF-7
and Mia-Pa-Ca-2 cell lines. The iron complex has shown better antiproliferative results
2
0

compared to other compounds tested. However the activity is less compared to the standard.
The photography representing the cell lines tested for various metal complexes along with
ligand are presented in Figure 15 and comparative graphs are presented in Figure 16.
4
. Conclusion
A novel ligand N'-(2-(hydroxyimino)propanoyl)isonicotinohydrazide was synthesized
and characterized with various spectroscopic, single crystal X-ray diffraction and analytical
techniques. The ligand has exhibited amido-imidol tautomerism and presence of (7E, 9Z) and
(7Z, 9E) isomers were confirmed by NMR spectral studies. The metal:ligand stoichiometry
arrived at, is 3:2 for cobalt and nickel complexes while it is 2:1 for other complexes. Their
molar conductance values suggest that Co(II) and Ni(II) complexes are electrolytes but the
remaining complexes are non-electrolytes. The ligand has exhibited its tetradentate
coordinating property. In the present studies, we have obtained three different type complexes
and all are assigned based on characterization results and along with literature support [74-
8
2]. The M3L2 and M2L type of structures are commonly available in literature for oxime
type ligands [74-82]. The tentative structures for the complexes synthesized are depicted in
Figure 17. The complexes have shown moderate anticancer activity against DU-145 cancer
cell lines. Among the complexes tested but less than the standard, iron complex has shown
better activity. The intercalative mode of binding to DNA is confirmed by absorption,
emission, thermal-denaturation and viscosity studies.
Acknowledgement
The authors thank the USIC, Karnatak University, Dharwad for providing the spectral
facilities and thankful to UGC for providing UGC-UPE fellowship and UPE-FAR-I program.
Authors are thankful to Dr. M. Netaji for his help in solving the crystal data.
2
1

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2
6

H
O
O
OH
O
O
H
O
III
N
O
N
NH2
Ethyl pyruvate
N
Hydrazine hydrate
Stirring
H
EtOH/H SO
2
4
O
Reflux
Stirring
N
I
N
II
N
N
IV
NH OH.HCl
2
Acetic acid
4
KOH
N
OH
8
3
N
9
H
(Z)
N(E) OH
OH
O
N
H
N
2
1
3 7
O
2
N
O
6
H
3
(7E,9Z)
4
5
2
N
1
N
1
OH
Z)
N
H
(
O
N
V
(
E)
Amido form
N
OH
(7Z,9E)
N
Imidol form
isomers)
(
Scheme 1.Synthetic route, Numbering pattern, Amido-Imidol tautomer forms, and E & Z isomerism for ligand
2
7

Figure 1. ORTEP diagram for ligand
2
8

Figure 2. Packing diagram for ligand representing the presence of four ligand molecule in each unit cell
2
9