Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes

Article abstract of DOI:10.1016/j.ica.2017.09.006

In search of a new antimicrobial and antioxidant with improved potency, we designed and synthesized a pair of chromone hydrazones and their transition metal complexes. The characterization and elucidation of the structure of the prepared compounds were performed by elemental analysis, IR, electronic, EPR and thermo gravimetric analyzes, as well as conductivity and magnetic susceptibility measurements. The EPR spin Hamiltonian parameters of copper complexes were calculated. The spectroscopic data showed that the ligands act as a monobasic tridentate. The coordination sites with the metal (II) ion are pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. Hydrazones and their metal complexes have shown antimicrobial activity against Gram-positive bacteria (S. aureus and B. subtilis); Gram-negative bacteria (P. aeruginosa and E. coli) and fungus (C. albicans) and also the synthesized ligands were tested for their antioxidant activity in vitro by DPPH scanning and the ABTS radical method. The substitution requirements for favorable antioxidant activity were investigated. Compounds containing a phenolic hydroxyl group at the imine (azomethine) position as well as an additional electron donor group exhibited lower IC₅₀ values and the result shows that all hydrazones have a significant antioxidant activity compared to the standard antioxidants Trolox.

Full text of DOI:10.1016/j.ica.2017.09.006

Accepted Manuscript
Research paper
Design, synthesis, antimicrobial and antioxidant activity of 3-formyl chromone
hydrazone and their metal (II) complexes
Jessica Elizabeth Philip, Shanty Angamaly Antony, Sneha Jose
Eeettinilkunnathil, M.R. Prathapachandra Kurup, Mohanan Puzhavoorparambil
Velayudhan
PII:
S0020-1693(17)30649-7
http://dx.doi.org/10.1016/j.ica.2017.09.006
ICA 17862
DOI:
Reference:
Inorganica Chimica Acta
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Received Date:
Revised Date:
Accepted Date:
13 June 2017
1 September 2017
2 September 2017
Please cite this article as: J.E. Philip, S.A. Antony, S.J. Eeettinilkunnathil, M.R.P. Kurup, M.P. Velayudhan, Design,
synthesis, antimicrobial and antioxidant activity of 3-formyl chromone hydrazone and their metal (II) complexes,
Inorganica Chimica Acta (2017), doi: http://dx.doi.org/10.1016/j.ica.2017.09.006
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Design, synthesis, antimicrobial and antioxidant activity of 3-formyl
chromone hydrazone and their metal (II) complexes
Jessica Elizabeth Philip, Shanty Angamaly Antony, Sneha Jose Eeettinilkunnathil, M. R.
Prathapachandra Kurup and Mohanan Puzhavoorparambil Velayudhan
Department of Applied Chemistry, Cochin University of Science and Technology, Kochi-22
E-mail: mohan@cusat.ac.in,
Corresponding author
Mohanan P. V., Ph.D.
Department of Applied Chemistry, Cochin University of Science and Technology, Cochin-682022,
Kerala, India.
Tel: +91 9447184874
Email: mohan@cusat.ac.in,
Abstract: In search of a new antimicrobial and antioxidant with improved potency, we
designed and synthesized a pair of chromone hydrazones and their transition metal
complexes. The characterization and elucidation of the structure of the prepared compounds
were performed by elemental analysis, IR, electronic, EPR and thermo gravimetric analyzes,
as well as conductivity and magnetic susceptibility measurements. The EPR spin Hamiltonian
parameters of copper complexes were calculated. The spectroscopic data showed that the
ligands act as a monobasic tridentate. The coordination sites with the metal (II) ion are
pyrone oxygen, azomethine nitrogen and hydrazonic oxygen. Hydrazones and their metal
complexes have shown antimicrobial activity against Gram-positive bacteria (S. aureus and
B. subtilis); Gram-negative bacteria (P. aeruginosa and E. coli) and fungus (C. albicans) and
also the synthesized ligands were tested for their antioxidant activity in vitro by DPPH
scanning and the ABTS radical method. The substitution requirements for favorable
antioxidant activity were investigated. Compounds containing a phenolic hydroxyl group at
the imine (azomethine) position as well as an additional electron donor group exhibited
lower IC50 values and the result shows that all hydrazones have a significant antioxidant
activity compared to the standard antioxidants Trolox.
1

Keywords: 3-formyl chromone, tridentate ligands, EPR spectra, antioxidant activity,
antimicrobial activity.
1. Introduction
The current escalation of knowledge of free radicals and reactive oxygen species (ROS) in
biology produces a medical revolt that promises a fresh era of health and disease management
[1]. It is ironic that oxygen, a crucial element for life, in certain situations has deleterious
-•
effects on humans [2-5]. They consist of a superoxide anion radical (O2 ), a peroxy radical
•
(RO2 ), an alkoxy radical (RO•), a hydrogen peroxide (H2O2) and singlet oxygen (oxygen
molecule in the electronically excited state) [6]. The hazardous effects of ROS include
oxidative damage to biological molecules such as lipids, proteins, nucleic acids and sugars
and also affect the balance between ROS production, cellular defense mechanisms are
transferred to more oxidative conditions (the state is called oxidative stress). It has been
established that oxidative stress is implicated in a wide range of diseases, such as
inflammation, neurodegenerative disorders (Alzheimer's disease, Parkinson's disease),
atherosclerosis, diabetes and cancer [7-13].
An antioxidant is a stable molecule appropriate to give an electron to a free radical
rampaging and neutralize it, thereby dropping its capability to damage [14]. These
antioxidants sustain or inhibit cell damage primarily through their free radical scavenging
property [15]. These low molecular weight antioxidants can safely interact with free radicals
and terminate the chain reaction before the crucial molecules are damaged. Some of these
antioxidants, including glutathione, ubiquinol and uric acid, occur during normal metabolism
in the body [16-17].
2

The Chromones are oxygen-containing heterocyclic compounds having a
benzoannelated γ-pyrone ring (4H-chromen-4-one, 4H-1-benzopyran-4-one) as the parent
compound, which is a common and integral constituent of a variety of medicinal agents
[6,18-19]. These compounds show the variety of pharmacological activities and the change
in their structure offer a high degree of diversity that has been found useful for the search for
novel therapeutic agents [6,20-21]. Several natural and synthetic benzopyrone derivatives
have been reported to exert remarkably antimicrobial, anti-tubercular and antifungal activity
[22-24]. The development of new defensive and potentially beneficial agents based on
chromone is an important research point in medicinal chemistry, with the core of chromone
being a key element in the pharmacophores of many biologically active molecules with
various medicinal applications [25].
Taking into account the biological importance and remarkable structural behavior of
these compounds, in this paper we wish to report some of our findings in the preparation of a
pair of hydrazone that were obtained by the condensation of the respective 3-formyl
chromone with different aromatic hydrazides. The synthesized compounds were subjected to
in-vitro antioxidant screening against DPPH and ABTS radical and their antimicrobial
activities were screened against selected bacteria and fungi.
2. Experimental
2.1.
Materials
Reagents used for the synthesis are 3-formyl chromone, 4-hydroxy benzhydrazide,
benzhydrazide, (Sigma Aldrich). All other reagents were of analytical grade and the Organic
solvents (ethanol, absolute ethanol, methanol, diethylether, dimethylformamide (DMF) and
dimethylsulfoxide (DMSO)) employed were either 99% purity or purified by known
laboratory procedures[26].
2.2. Physical measurements
3

Elemental analyses were performed on a Vario EL III elemental analyzer. Melting point of
the ligands and their complexes were measured. IR spectra (KBr cm-1) were recorded on a
JASCO FT- IR spectrometer in the range of 4000-400 cm-1 .1H/13C NMR spectra were
recorded on a Bruker Advance III 400MHz FT-NMR spectrometer using CHCl3/DMSO-d6 as
solvent and TMS as an internal standard (chemical shifts in δ ppm). Electronic spectra were
recorded at room temperature on a Thermo Scientific™ Evolution 201 UV-Visible
spectrophotometer. Thermo gravimetric Analysis (TG/DTG) were carried out from room
temperature up to 800 oC at a heating rate of 10 oC/min on Perkin Elmer Diamond TG/DTA
analyzer. The molar conductivities of the complexes in DMF solutions (10-3 M) at room
temperature were measured using a Systronic model 303 direct reading conductivity meter.
Mass spectra of the hydrazones were recorded by direct injection on WATERS 3100 Mass
Detector using Electron Spray Ionization (ESI) technique designed for routine HPLC-MS
analyses. Magnetic susceptibility measurements of the complexes were carried out on a
Vibrating Sample Magnetometer using Hg [Co(SCN)4] as a calibrant. The EPR spectra of the
complexes in the solid state at 298 K and in DMF at 77 K were recorded on a JEOL,
JapanModel: JES-FA200 ESR Spectrometer using TCNE as the standard, with 8.75-9.65
GHz microwave frequency.
2.3. Synthesis of chromone hydrazones
The synthesis of chromone hydrazones are shown in scheme 1. Aromatic hydrazides (1.00
mmol), 3-formylchromone (1.00 mmol), and a few drops of acetic acid were dissolved in 25
mL of ethanol, and the mixture was stirred and refluxed for 6 h. The precipitate was
collected, washed with ethanol, and dried in vacuo. All compounds were prepared similarly
shown in scheme 1 and characterized as below.
4

2.3.1. N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide - Yield 78% as yellow
powder; m.p. 171-173oC; IR (KBr, cm−1): 3206 (N-H), 1680 (C=O, pyrone), 1655 (C=O,
hydrazonic), 1571 (C=N), 1238(C–O), 1035 (N–N); 1H NMR (DMSO-d6, δ, ppm): 11.72 (s,
1H, OH); 8.78 (s, 1H, CH=N); 8.59 (s, 1H, oxo ring); 7.92- 6.84 ( m, chromone ring and
phenyl ring). 13C NMR (DMSO-d6, δ, ppm): 177.5(C=O), 163.77 (-N-N=CH-), 162.70,
157.20, and 152.80 (non protonated carbon), ESIMS [M+1]: 293.1. Anal. Calcd for
C17H12N2O3 (292.08): C, 68.45; H, 4.28; N, 9.39; Found: C, 68.40; H, 4.19; N, 9.36%.
2.3.2. 4-hydroxy-N'-((4-oxo-4H-chromen-3-yl)methylene)benzohydrazide -Yield 76% as
yellow powder; m.p. 169–170oC; IR (KBr, cm−1): 3400-3100 (broad O-H & N-H), 1680
(C=O, pyrone), 1651 (C=O, hydrazonic), 1580 (C=N), 1232 (C–O), 1028 (N–N); 1H NMR
(CHCl3, δ, ppm): 11.73 (s, 1H, OH); 8.60 (s, 1H, CH=N); 8.8 (s, 1H, oxo ring);8.15- 6.87 (
m, chromone ring and phenyl ring); 13C NMR (CHCl3, δ, ppm): 176.00 (C=O),163.71 (-N-
N=CH-),162.72, 160.83, 157.21and 152.83 (non protonated carbon), ESIMS [M+1]: 309.2.
Anal. Calcd for C17H12N2O4 (308.18): C, 66.25; H, 3.92; N, 9.09; Found: C, 66.30; H, 3.88;
N, 9.13%.
2.4. Synthesis of complexes
About 1 mmol of metal (II) acetate dissolved in 30mL methanol was added gradually to 1
mmol of the ligand, dissolved in DMF. The reaction mixture was heated under reflux for 8h.
The resulting precipitates were filtered, washed with methanol and hexane then diethyl ether
5

and finally air-dried. The complexes were kept in desiccators over anhydrous calcium
chloride. The yield was noted. All complexes were prepared similarly.
Table 1 Analytical and physical data of the chromone hydrazones (HL1 & HL2) and their metal (II)
complexes.
Elemental Analysis, % Found (calac.)
Yield
%
M.P.
oC
No
Reaction
HL1
Complex M.F. [F.W.]
Colour
C
H
N
M
-
68.40
(68.45)
4.19
(4.28)
9.36
(9.39)
1
2
C17H12N2O3 [292.08]
Yellow
Brown
78
62
ꢀꢁꢂ
HL1+
Ni(OAc)2
49.33
(49.28)
4.47
(4.35)
6.15
(6.05)
12.53
(12.68)
C19H20N2NiO8 [463.06]
>250
HL1+
Cu(OAc)2
53.03
(52.84)
3.38
(3.73)
6.40
(6.49)
14.65
(14.71)
3
C19H16N2CuO6[431.88]
Green
65
>250
HL1+
Zn(OAc)2
49.47
(49.53)
4.06
(4.16)
6.13
(6.08)
14.11
(14.20)
4
5
6
7
8
C19H19N2ZnO7.5 [459.04]
C18H14N2O4 [322.31]
Yellow
Yellow
Brown
Green
Yellow
67
76
72
70
66
>250
ꢀꢁꢃ
66.30
(66.23)
3.88
(3.92)
9.13
(9.09)
HL2
-
HL2+
Ni(OAc)2
47.58
(47.64)
4.29
(4.21)
5.72
(5.85)
12.07
(12.25)
C19H20N2NiO9 [479.06]
C19H18N2CuO8[465.90]
C19H14N2ZnO6 [431.73]
>250
>250
>250
HL2+
48.89
3.93
6.12
13.51
Cu(OAc)2
HL2+
Zn(OAc)2
(48.98)
52.79
(52.86)
(3.89)
3.20
(3.27)
(6.01)
6.41
(6.49)
(13.64)
15.09
(15.15)
2.5.Antioxidant activity determination
2.5.1. DPPH radical scavenging assay
The DPPH (2-2'-diphenyl-1-picrylhydrazyl) radical scavenging action of the compounds was
evaluated according to the method of Blios.45. The DPPH radical is a stable free radical
having λmax at 517 nm and showed characteristic deep violet colour. The loss of violet colour
of the DPPH solution by the abstraction of hydrogen from the substrate serves as a marker of
the scavenging of DPPH radicals. A variable concentration of the test compounds was added
to a solution and the final volume was made up to 3 mL with solvent. The solution was
incubated at 37 °C for 20 min. The decrease in absorbance of DPPH was measured at 517
nm. The same experiment carried out without the test compounds serve as a control.
Percentage inhibition can be calculated by using the equation.
ꢌꢍꢎꢏꢊꢆꢉꢐꢊꢑ − ꢌꢍꢎꢒꢓꢔꢕꢑꢖ
%ꢄꢅꢆℎꢇꢈꢇꢉꢇꢊꢆꢄ =ꢄꢋ
ꢗ × 100ꢄ
ꢌꢍꢎꢏꢊꢆꢉꢐꢊꢑ
6

A graph was plotted with concentration (µM) against % inhibition. The concentration
at which there is 50% fall in absorbance of DPPH solution was determined from the graph
(IC50). The IC50 value in µM was calculated from the above obtained concentration value. All
experiments were carried out in triplicate. The 6-hydroxyl-2, 5, 7, 8-tetramethylchroman-2-
carboxylic acid (Trolox) was used as antioxidant reference.
2.5.2. (ABTS˙+) radical cation assay
It is based on the ability of antiradical molecules to quench the ABTS radical. It is a blue
green coloured chromophore shows characteristic absorption at 734 nm. By the addition of
antioxidants to the radical cation reduces it to ABTS and loss its characteristic colour
(decolourization). In this method, an antioxidant was added to a pre-formed ABTS radical
solution, and after 20 min period, the remaining ABTS˙+ is quantified spectrophotometrically
at 734 nm. The ABTS˙+ was produced by reacting 7 mM ABTS in H2O with 2.45 mM
potassium persulfate (K2S2O8), stored in the dark at room temperature for 24 hrs before use
(the radical was stable for more than two days under these conditions). The ABTS˙+ solution
was further diluted in methanol until the initial absorbance value of 0.80 0.02 at 734 nm at
30oC. Then, in a 3 mL solution of ABTS˙+, was added a methanolic solution of the compound
under study so that their final concentrations vary between 0 and 15 µM. In each case, a 20-
80% decrease in the initial absorbance of the reaction solution was achieved. The absorbance
was recorded at 0, 1, 4 and 6 min. The scavenging capacity of test compounds was calculated
using the following equation:
ꢌꢍꢎꢏꢊꢆꢉꢐꢊꢑ − ꢌꢍꢎꢒꢓꢔꢕꢑꢖ
ꢙ
ꢝ ꢞ
ꢋ
ꢗ
× 100
ꢌꢍꢘꢎ˙ ꢒꢏꢓꢚꢖꢆꢛꢇꢆꢛꢄꢖꢜꢜꢖꢏꢉꢄ % ꢄ= ꢄ
ꢌꢍꢎꢏꢊꢆꢉꢐꢊꢑ
Where Acontrol is absorbance of a control lacking any radical scavenger and Asample is
absorbance of the remaining ABTS˙⁺ in the presence of scavenger. Graphs of antioxidant
concentration versus percent absorbance reduction were then plotted. The concentration of
7

Trolox giving the same percentage reduction of absorbance at 734 nm as the 1 mM
antioxidant solution was calculated from the three point graphs. The results were thus
expressed as Trolox equivalent antioxidant capacity (TEAC) values. For each hydrazone and
each concentration, measurements were performed in triplicate. Absorbance values were
corrected for radical decay using blank solutions.
2.6. Antimicrobial activity
The synthesized chromone hydrazones and metal complexes were tested for their in vitro
antibacterial and antifungal activity against the sensitive organisms Staphylococcus aureus
(ATCC 25923) and Bacillus subtilis (ATCC 6635) as Gram positive bacteria, Pseudomonas
aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) as Gram negative bacteria
and Candida albicans (ATCC 10231) as fungus strain using the disc diffusion method
[Kirby-Bauer Test ][27](for the qualitative determination) and the serial dilutions in liquid
broth method for determination of MIC values. The antibiotic chloramphenicol was used as
reference in the case of bacteria, and nystatin in the case of fungi.
The chromone hydrazones and metal complexes were dissolved in DMSO. The test
was carried on medium potato dextrose agars (PDA) which have a mixture of 200 g potatoes,
6 g dextrose and 15 g agar [28] .Uniform dimension filter paper discs were impregnated with
equal volume (10 µL) from the exact concentration of dissolved compounds and carefully
located on the inoculated agar surface. After incubation for 36 h at 27 oC in the case of
bacteria and for 48 h at 24 oC in the case of fungi, inhibition of the organisms was measured
and used to calculate mean of inhibition zones and all experiments were carried out in
parallel sets of triplicate. Data were analyzed by the analysis of variance (ANOVA) using the
Origin pro 8.5 statistical software and the mean differences were separated using Tukey's
studentized test at the 1% level of probability. Time kill kinetic studies were conducted for
the compounds against one Gram positive, one Gram negative bacteria and against fungus.
8

In the experiment, an overnight culture of the isolates was used up. 1mL of 106 CFU mL-1 of
each culture was inoculated in sterilized nutrient broth media containing 25 mg mL-1 of the
compounds. The experiment was conducted for 13 h in a shaker at 30°C. Likewise, control
was prepared for each microorganism without having the test compound. The CFU count was
taken at regular 1 h interval. For that, 1mL of each culture was spread on nutrient agar plates
from 0 h to 13 h and each plate was incubated for 24 h at 30°C. The CFU (colony forming
unit) was calculated and plotted in graph.
2.6.1. Minimum Inhibitory Concentration (MIC)
All the compounds showed substantial inhibition of both bacteria and fungi, were tested
further for the Minimum Inhibitory Concentration (MIC). Standard pathogenic bacterial
strains S. aureus, B. subtilis, P. aeruginosa, E. coli and C. albicans as fungus. The resazurin
reduction method was used for finding out the MIC in 96 well microplates [29]. All the
compounds were dissolved in DMSO having a final stock concentration of 10 mg mL-1. Fifty
microlitres of this stock solution was serially diluted to eight times and 50 μL of each serially
diluted compound was added to microplate wells. All the microbial cultures were grown in
nutrient agar to reach 0.5 McFarland concentrations and 50 mL of this culture was added to
each well. Microplate contents were mixed well and incubated at room temperature for 12
hours. After 12 h of incubation, 10 μL of mixture from each well was spread on the agar plate
and checked for the Colony Forming Units (CFU). Further 30 μL of 0.1% resazurin solution
was added to each well and incubated up to another 24 hours. Microplate well contents were
observed in the change in colour from blue to pink. Those wells that have microbes growing
will change the blue resazurin into pink color. The well, which remains blue after 24 hours of
incubation indicates there are no microorganisms survived in the well, the minimum
concentration where no microbial growth found are considered as MIC value.
9

3. Result and Discussion
3.1.Characterization of ligands and complexes
The structure of the synthesized chromone hydrazones and their metal complexes were
elucidated by elemental analysis, electronic, FTIR, 1H/13C NMR and ESI-MS spectra. The
results of elemental analysis (C, H, N) with molecular formula (Table 1) are good agreement
with those calculated for the suggested formula and the melting points (Table 1) are sharp
signifying the purity of the prepared chromone hydrazones. The metal (II) complexes were
isolated pure in very good yields and they are of various colours. The ligands and the
complexes are very much soluble in DMF and DMSO, moderately soluble in methanol and
acetonitrile and practically insoluble in water, carbon tetrachloride, chloroform and benzene.
They are very air stable solids at room temperature without decomposition for a long time.
Moreover, the structure of the ligands (HL1 & HL2) was deduced from mass spectral data
which showed the [M+1] peak at 293.1 & 309.2 a.m.u. respectively, confirming its formula
weight (F.W. 292.08 and 308.18 respectively; Figs. C: 1-2 (supplementary data)) it supported
the suggested structure of the ligands.
3.1.1. FTIR spectra
The characteristic IR bands of the chromone hydrazones (Table 3) give important information
about the various functional groups present in it. Strong bands due to the ν (N-H) and ν
(C=O) modes at 3220 and 1641 cm-1 are observed in the spectrum of HL1 which suggests that
the hydrazone exists in the amido form in the solid state [30]. A band at 1678 cm-1 is due to
the presence of (C=O, pyrone). A prominent band at 1587 cm-1 due to azomethine ν (C=N)
formation of ligand HL1. It further confirms the ν (C–O), ν (N–N) bands at 1238, 1035 cm-1
respectively. Similarly in the case of ligand HL2 a strong broad band around 3394 cm-1 is due
to ν (N-H & O-H). A strong band at 1650 cm-1 observed in the spectrum which suggests that
the HL2 exists in the amido form in solid state. Band at 1675 cm-1 is due to the presence of
10

(C=O, pyrone). A prominent band at 1605 cm-1 due to azomethine ν (C=N) formation of
ligand HL2. It further confirms the ν (C–O), ν (N–N) bands at 1235, 1048 cm-1 respectively.
The comparison of the complexes with respective chromone hydrazone shown that
all complexes had broad band in the range of 3690-3025 cm-1 assigned to ν (OH) of the
coordinated water molecules linked with the complexes [31] and also the OH of the
hydrazine moiety which are confirmed by elemental, AAS, and thermal analyses. All the
metal (II) complexes displayed distinct sharp band in 1571-1531 cm-1 region corresponds to
the characteristic -C=N- group stretching vibration indicating the coordination of azomethine
nitrogen to metal (II) centre as observed in the literature [32]. This coordination of
azomethine nitrogen is also supported by band at 1029-1013 cm-1 corresponding to ν (N-N)
stretching vibration. On comparison with literature reports, ν(N-N) vibrations was found to
be in higher frequencies in the complexes due to the increase in double bond character, off-
setting the loss of electron density via donation to the metal[33].
The bands at 1580 cm-1 assigned to ν(C=N), in the chromone hydrazones were shifted
to lower wave number region in all complexes. The band at 1655 cm-1 assigned to ν(C=O)
hydrazonic in the free ligand was shifted to (1647-1598) cm-1, [34] which indicate that the
azomethine nitrogen and hydrazonic (C=O) are in chelation. In complexes 1 & 4 the
chelating bidentate acetate (CH3COO-) group was present due to the bands around 1424-1414
and 1337-1325cm-1. These two bands are due to νas (COO-) and νs (COO-), respectively. The
separation of the two bands, ∆ ν = (νas- νs) = 88cm-1, is comparable to the values cited for the
bidentate character of the acetate group ∆ ν = 75-90 cm-1[35]. On the other hand, complexes
2, 3, 5 & 6 showed new bands characteristic for νas (COO-) and νs (COO-) of acetate ion in
the ranges 1549-1531 and 1413-1389cm-1. The higher difference between the two bands
indicates the monodentate nature of the acetate group, in which one ‘O’ atom of acetate was
coordinated to the metal center, while the second was hydrogen bonded to, a geminal H2O
11

[36]. The appearance of the non - ligand bands at 557-510 and 473-403 cm-1 were assigned to
ν (M-O) and ν (M-N), respectively.
Table 3 Characteristic IR spectral data of the ligand (HL1 & HL2) and its complexes.
IR spectra (cm)-1
N
o
Compounds
Mono/
Bidantate
acetate
ν( C=O)
pyrone
ν( C=O)
hydrazonic
ν(OH, NH)
ν( C=N)
ν( C-O)
ν( N-N)
ν( M-O)
ν( M-N)
1
2
3
4
6
7
8
9
HL1
-, 3220
1678
1645
1656
1641
1675
1639
1634
1654
1641
1617
1605
1597
1650
1617
1598
1593
1587
1545
1531
1560
1580
1571
1555
1550
1238
1220
1228
1224
1232
1219
1229
1196
1035
1029
1019
1027
1028
1015
1021
1013
-
-
-
[Ni(L1)(OAc)(H2O)]
.2H2O
3666-2919
(broad)
ν
as 1431,
νs 1328
as 1531,
νs 1323
as 1529,
510
526
533
-
436
403
460
-
ν
[Cu(L1)(OAc)(H2O)]
3445
[Zn(L1)(OAc)]
.2.5H2O
ν
3618-3088
νs 1397
3394
HL2
-
(broad)
[Ni(L2)(OAc)(H2O)]
.2H2O
3690-3043
(broad)
νas 1451,
520
512
557
449
423
473
νs 1364
[Cu(L2)(OAc)
(H2O)].H2O
νas 1511,
νs 1374
3416-3025
3630-2997
ν
as 1536,
[Zn(L2)(OAc)]
νs 1388
3.1.2. 1HNMR spectra
Proton Nuclear Magnetic Resonance (1 H NMR) Spectroscopy is a powerful tool used for the
determination of the structure of compounds. The 1H NMR spectra of the chromone
hydrazones and Zn (II) (because of their diamagnetic nature) have been recorded with DMSO
as solvent. 1H and 13C NMR spectral data (δ ppm) of the ligand relative to TMS (0 ppm) in
DMSO-d6, Tables 2; Figs. A: 1-2 & B: 1-2 (supplementary data), give further support of the
suggested structure of the ligand.
Table 2
1H & 13C NMR Spectral data of ligands HL1 & HL2
1HNMR
13CNMR
δ ppm (DMSO)
δ ppm
(DMSO)
Assignments
Assignments
HL1
11.72
8.78
(s,1H,OH)
177.5
-C=O
(s,1H,CH=N-)
163.77
-N-N=CH-
8.59
(s,1H,oxo ring)
162.70,157.20,152.80
Non protonated carbon
7.92 -6.84
(m, chromone & phenyl ring)
12

HL2
11.73
10.12
8.80
(s,1H,OH)
176.00
C=O
(s,1H,OH)
(s,1H,oxo ring)
163.06
-N-N=CH-
162.72,160.83,
157.21,152.83
8.60
(s,1H,CH=N-)
Non protonated carbon
7.88-6.84
(m, chromone & phenyl ring)
The comparison of the NMR spectra of the complexes with the spectra of ligands
gave valuable information regarding the coordination mode of ligands during complexation.
The 1H NMR spectrum obtained for complexes 3 and 6 was not good due to the poor
solubility of the compound. In the spectra of the free hydrazones there are sharp singlets in
the range of 11-12 ppm showing the existence of iminol form in solution. They also gave
singlets in the range of 10-11 ppm with an area integral of one which is due to phenolic OH
protons. Syntheses and characterization of Zn (II) complexes derived from ONO donor
chromone hydrazones the intensity of these signals significantly decreases, which suggests
that these protons are easily exchangeable and confirm the assignment. Peaks for aromatic
protons were found in the region 6.8-8 ppm. Peaks are in the region 11-12 ppm corresponds
to iminol protons found in the spectra of free hydrazones were absent in the spectra of
complexes indicating the coordination of iminol oxygen to zinc. In the spectrum of
complexes, a singlet with an integral area of three is found at 2.0-1.9 ppm and it is assigned
to the methyl group present in acetate which is coordinated to zinc. Also a singlet with area
integral one is appeared at 8.27 & 8.7 ppm may indicate the presence of -CH- proton. All
other peaks observed in the spectra of free hydrazones are slightly shifted. The 1 H NMR
spectra of complexes 3 and 6 are shown in the Figs. A: 3-4 (supplementary data).
3.1.3. Solution studies
UV -Vis spectra and Molar conductivity measurements
13

The electronic spectral data of the ligand in DMF (Table 3) showed two bands λmax (DMF)
nm (ε in 103 dm3 mol-1cm-1): 237 (13.7), 341 (8.6) for HL1 and 241(14.1), 347 (7.9) for HL2.
The higher energy band may be assigned to π-π* transitions of the azomethine linkage and
the aromatic rings. The medium energy band may be assigned to n-π* transitions of the C=O
and C=N groups. The metal complexes in DMF solutions present absorption maxima
attributable to the hydrazone ligand together with the absorptions, around 419-697 nm, due to
ligand to metal charge transfer and d-d transitions of the metals in the complexes [37]. The
→π* and to
bands in the range 340 and 241 nm in the electronic spectra of hydrazones due n
π→π* transitions suffered marginal shifts (350 and 252 nm) upon complexation. This may be
due to the weakening of the C=O bond and the extension of conjugation upon complexation.
The shift occurs also due to the coordination of the metal (II) ion are pyrone oxygen,
azomethine nitrogen and hydrazonic oxygen. The electronic spectral data of the metal (II)
complexes are given in Table 4.
The molar conductance values of the synthesized Ni (II), Cu (II) and Zn (II)
complexes in DMF (10-3) were measured at room temperature and the results were shown in
Table 4. The values showed that all complexes have non-electrolytic nature [38].
Table 4
Electronic spectra, magnetic moments and molar conductivity data of the ligands (HL1&HL2) and its
metal complexes.
Electronic spectral
bandsa (nm)
b
µ eff
.
conductancea
No
1
Compounds
HL1
λmaxa (nm)/(εmax L cm-1
mol-1)
B.M.
(Ω-1 cm2 mol-1)
237 (1.37) - π→π*
→π*
361 (8.6) - n
-
-
260 (4.57) - π→π*
316 (4.42) - π→π*
366 (4.79) - n→π*
429 (2.45) - CT
589 – dd
2
3
3.45
1.65
12.5
[Ni(L1)(OAc)(H2O)].2H2O
254 (4.46) - π→π*
310(1.88) - π→π*
→π*
407(5.91) - n
428 (6.03) - CT
9.4
[Cu(L1)(OAc)(H2O)]
642 (1.14) – dd
272 (4.77) - π→π*
358 (2.85) - n→π*
410 (2.52) - CT
4
6
D
-
8.6
-
[Zn(L1)(OAc)].2.5H2O
HL2
241 (1.41) - π→π*
→π*
359 (7.9) - n
14

257 (3.83) - π→π*
316 (5.19) - π→π*
353 (6.12) - n→π*
611 - dd
7
3.32
15.4
[Ni(L2)(OAc)(H2O)].2H2O
257 (5.20) - π→π*
377 (5.70) - π→π*
→π*
437(5.31) - n
462 (2.85) - CT
8
9
1.72
D
13.7
9.5
[Cu(L2)(OAc)(H2O)].H2O
[Zn(L2)(OAc)]
629 (1.24) - dd
260 (4.26) - π→π*
349 (3.30) - n→π*
430(1.12) - CT
a Solutions in DMF (10-3 M), values of εmax are in parentheses and multiplied by 104 (L mol-1 cm-1).
b μ eff. is the magnetic moment of the complex.
D - diamagnetic
3.1.4. TG/DTG
The thermal (thermo gravimetric) analysis was mainly used to confirmation of the associated
water or solvent molecule to be in the coordination sphere or in the outer sphere of the
complex [39] and information on their properties, nature of intermediate and final products of
thermal decomposition can be obtained. From TGA curves, the weight loss was calculated for
the different steps and compared with theoretically calculated weight for the suggested
formulae based on the results obtained from elemental analyses as well as molar conductance
and AAS measurements. TGA indicated the formation of metal oxide as the end product from
which the metal content could be calculated and compared with that obtained from analytical
determination. Thermo grams of metal complexes indicate decompositions around 100oC is
due to hydrated water (except complex 2 & 6) and also (complex 1, 2, 4 & 5) around 200 °C
is due to coordinated water molecules. The results of thermal analysis (Table - 5, Figs. E: 1-6
(supplementary data)) of these complexes are in agreement with elemental analyses.
Table 5
TG/DTG of metal (II)complexes
Temperature
range(oC)
Mass loss (%)
Obsd. (Cald.)
Complexes
Decomposition product
Loss of 2H2O (hyd)
Loss of H2O (coordinated) &
acetate
80-105
297-354
7.62 (7.77)
17.47 (18.03)
[Ni(L1)(OAc)(H2O)].2H2O
15

229
258-306
307-369
4.55 (4.16)
13.93 (14.25)
22.35(22.07)
Loss of H2O (coordinated)
Acetate
C6H6
[Cu(L1)(OAc)(H2O)]
50-120
450-477
9.96 (9.80)
14.44(14.89)
Loss of 2.5 H2O (hyd)
C2H4O2
[Zn(L1)(OAc)].2.5H2O
[Ni(L2)(OAc)(H2O)].2H2O
50-130
173-248
277-368
7.16 (7.51)
4.33 (4.06)
22.14(22.05)
Loss of 2H2O (hyd)
Loss of H2O (coordinated)
C6H6O
60-114
200-248
290-429
4.05 (3.86)
4.26 (4.01)
32.33 (33.08)
Loss of H2O (hyd)
Loss of H2O (coordinated)
C7H9NO2
[Cu(L2)(OAc)(H2O)].H2O
[Zn(L2)(OAc)]
150-280
419-484
43.66 (43.01)
6.58 (6.97)
C10H8N2O2
CH4
3.1.5. Magnetic measurements
Due to the Jahn–Teller distortion of CuII-ion (d9) it lower the symmetry, complete
interpretations of the spectra and magnetic properties are somewhat difficult [40]. The
magnetic moment values of the copper complexes are 1.68, 1.72 B.M., which is consistent
with the presence of one unpaired electron. In nickel complexes the magnetic moment values
are 3.45 and 3.32 B.M; indicate that complexes follows octahedral geometry.
3.1.6. EPR studies
The EPR spectra of the copper (II) complexes in DMF at 77 K and in a polycrystalline state
at 298 K were recorded in the X-band, using 100-kHz modulation frequency and 9.5 GHz
microwave frequency. All the EPR spectra are simulated using Easy Spin 5.0.20 package[41]
and the experimental (red) and simulated (blue) best fits are included. EPR parameters of the
copper (II) complexes are presented in Table 6 and Figs. 1- 4.
Copper (II) complex (2 & 5) displayed Figs. 1- 4 axial spectra in the polycrystalline
state at 298 K with gǁ and g⊥ values. The variation in the gǁ and g⊥ values indicates that in
the solid state, the geometry of the compounds is affected by the nature of coordinating
ligands.
Fig 1: EPR spectrum of Complex 2 at 298 K; Fig 2: EPR spectrum of Complex 2 in DMF at 77K;
Fig 3: EPR spectrum of Complex 5 at 298 K; Fig 4: EPR spectrum of Complex 5 in DMF at 77K
16

1
2
3
4
For complex (2 & 5), displayed axial features; since it is magnetically concentrated,
hyperfine splitting was not clear in both parallel and perpendicular regions. The geometric
parameter G, represents the exchange interaction between copper canters in the
polycrystalline compound and is calculated for each complexes using the equation, G = (gǁ -
2.0023)/(g⊥ - 2.0023) for axial spectra [42]. If G > 4.4, exchange interaction is negligible and
if it less than 4.4, considerable exchange interaction is indicated in the solid complex. The
calculated G values are greater than 4.4 for complexes which indicate that there are no
copper-copper exchange interaction is present in the polycrystalline state of the complexes.
2
As gǁ > g
⊥
> 2.0023, a square pyramidal geometry consistent with dx -y2 ground state can be
assigned to the copper (II) complexes (2 & 5), thereby ruling out the possibility of a trigonal
bipyramidal structure (where gǁ < g ).
At 77 K in DMF solution, complexes 2 & 5 (Figs.1-4) exhibits axial spectrum. The
EPR parameters gǁ and g incorporated with the energies of d-d transition were used to
⊥
⊥
evaluate the bonding parameters.
The following simplified expression were used to calculate the bonding parameters
K2 (gǁ - 2.0023) Ed-d/8λ0
ǁ =
K2
⊥ = (g⊥ - 2.0023) Ed-d/2λ0
17

Where Kǁ and K⊥ are orbital reduction factors and λ0 represents the one electron spin orbit
coupling constant which equals -828 cm-1.
Hathaway [42] has pointed out that for pure sigma bonding Kǁ ≈ K⊥ ≈ 0.77, for in
plane π-bonding Kǁ < K⊥ and for out-of-plane π-bonding, K⊥< Kǁ. For complexes (2 & 5), it
is observed Kǁ < K⊥ indicates significant in-plane π-bonding is present in the complex.
The gǁ values also provide information regarding the nature of metal-ligand bond
[43]. The gǁ value is normally 2.3 or larger for ionic and less than 2.3 for covalent metal-
ligand bonds. The gǁ values obtained for complexes (2) and (5) indicate a significant degree
of covalency in the metal-ligand bonds [44].
Table 6 Spin Hamiltonian and bonding parameters of copper (II) chromone hydrazones
Polycrystalline state
DMF solution (77 K)
(298 K)
Complex
gǁ
g⊥
gav
G
gǁ
g⊥
Kǁ
K⊥
[Cu(L1)(OAc)(H2O)] (2)
2.190
2.19
2.066
2.059
2.1073
2.1027
2.9466
3.3104
2.190
2.20
2.066
2.057
0.6609
0.6829
0.7700
0.7185
[Cu(L2)(OAc)(H2O)].H2O(2)
Fig 5: Proposed structure of complex 1, 2, 3, 4, 5 & 6
O
O
.2 H2O
O
N
O
H2O
O
N
.H2O
O
N
O
N
Ni
Cu
O
O
O
18

O
O
O
N
O
O
N
O
. 2.5 H2O
. 2 H2O
N
N
H2O
Ni
Zn
O
O
O
OH
O
O
O
O
O
.H2O
H2O
N
N
O
N
N
Zn
Cu
O
O
O
O
O
OH
OH
3.2. Antioxidant activity
The antioxidant activity of chromones and their derivatives has attracted increasing interests
and been extensively investigated, mainly in- vitro system. It has been reported that over
production of free radicals may induce some oxidative damages to bio molecules such as
carbohydrates, proteins, lipids and DNA, thus accelerating aging, cancer, cardiovascular
diseases, and inflammation and so on.
3.2.1. Scavenging radical activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
In this method the decrease in absorbance (stable free radical DPPH) at 517 nm, in presence
of chromone hydrazones were tested. All tested chromone hydrazones shows UV absorbance
between 230 and 350 nm ranges. These values do not interfere with the kinetic measurement
of DPPH absorbance at 517 nm. The absorbance of DPPH radical decrease in presence of
antioxidants is due to hydrogen transfer from the antioxidant, thus forming the DPPH-H
stable compound. Methanol solutions of DPPH radical at different concentrations of
19

hydrazones were checked over a 20 min period. The chromone hydrazones were studied to
investigate their antiradical ability by comparing their scavenging effects with standard
Trolox. We find that the inhibitory effect of the compounds tested on DPPH radical is
concentration dependent and the suppression ratio increases with increasing sample
concentrations in the range tested. The IC50 values of the chromone hydrazones were
determined (HL1 & HL2 = 3.71 & 1.54 mg/mL respectively) and standard Trolox is
6.25mg/mL. Compounds having a hydroxyl group at para position show the lowest IC50
values. A lower IC50 value means higher DPPH free radical scavenging activity.
Hydrogen atom abstraction from phenolic ring under the examined conditions is quite
easier for compounds HL2 as shown in Scheme 2. As a consequence the reaction of DPPH
radicals may create nitrogen radical on the antioxidant considered. This is in good agreement
with Dolenc et al. [24] concerning the auto oxidation process of hydrazones. Studying
structurally different hydrazones, we proposed for those compounds having at least one
hydrogen atom on nitrogen, the formation of nitrogen cantered radical (hydrazonyl radical)
which is stabilized through conjugation forming delocalised carbon cantered radicals. The in
vitro antioxidant activities of synthesized chromone hydrazones may be due to the relative
ability for the N-H hydrogen atom abstraction from the compound and also substitutions on
aromatic or hetero aromatic hydrazides.
Scheme 2: Proposed reaction of DPPH with p-hydroxybenzhydrazide derivative
20

3.3.2. Scavenging radical activity on 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS˙+) radical cation
The antioxidant activity of chromone hydrazones were evaluated by using the Trolox
equivalent antioxidant capacity values (TEAC). In this method we measure the relative
ability of antioxidant compound to scavenge the radical cation [2, 2′-azinobis (3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS˙+)] and compared with a standard amount of
synthetic antioxidant Trolox [6-hydroxy- 2, 5, 7, 8-tetramethylchroman-2-carboxylic acid],
it’s a vitamin E analogue. The TEAC value means the concentration of standard Trolox with
the same antioxidant capacity as 1 mM concentration of the antioxidant compounds here
chromone hydrazones under investigation.
This method based on the generation and detection of coloured long-lived radical cation
ABTS is chemically oxidized by potassium persulphate to give the relatively stable (over 24
h) ABTS radical. The concentration of ABTS˙+ is measured at 734 nm [25]. Thus the
antioxidant-induced reduction of the ABTS˙+ concentration is directly related to the
antioxidant capacity of the compound being tested. ABTS radicals are involved in an electron
transfer process. All screening compounds exhibited at various levels radical cation
scavenging activity. There were no differences between the TEAC values for the chromone
hydrazones at 1 min, 4 min and 6 min (data not shown). As seen in Fig. 6, compounds HL2
were the most effective ABTS˙+ radical scavengers depending on concentration range (0-15
μM). ABTS˙+ absorbance decreases significantly with all chromone hydrazone
concentrations, the effect being less pronounced for hydrazone HL1 at concentrations above
12 µM. The scavenging effects on ABTS˙+ decreased in the order Trolox>HL1>HL2. Table 7
summarised the results obtained in terms of Trolox equivalents capacity. All chromone
hydrazones possess TEAC values equivalent or inferior values. This might be due to a better
21

transfer of radical and better resonance stabilisation of the radical species for these
compounds (Table 7).
Fig.6 ABTS radical scavenging activity of chromone hydrazones compared with standard Trolox. Values
are average of 3 independent measurements of each compound.
HL1
HL2
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2
4
6
8
10
12
14
16
Conc mg/mL
Table 7: Antioxidant activity of chromone hydrazones by ABTS radical. Values are average of 3
independent measurements of each compound.
Compounds TEAC
3a
3.23
1.48
3.09
3b
Trolox
3.3.Antimicrobial activity
The antibacterial activity of all compounds against Mycobacterium were plotted in a bar
diagram and represented in Fig. 7. Fig. 7 represents the zone of inhibition for bacteria and
fungi at three different concentrations (1, 0.5 and 0.25 mg mL-1) with a standard deviation of
the triplicate values (Table 8). Antifungal zone of inhibition showed by the synthetic
compounds represented in Fig. 7. Figure 8 and table 9 represent the MIC of the compounds
synthesized against the microorganisms. Chloramphenicol was used as a reference in the
22

case of bacteria and nystatin in the case of fungi were tested at a concentration of 10 mg mL-1.
Most of the compounds showed good microbial inhibition for all tested microorganisms such
as S. aureus and B. subtilis, two Gram negative bacteria P. aeruginosa and E. coli and the
pathogenic fungus C. albicans in the disc diffusion method. Complexes 2 and 5 showed a
good zone against S. aureus, in the case of B. subtilis complex 2, 3, 5 and 6; for P.
aeruginosa complex 2, 5 and 6; for E. coli the complex 5 showed good inhibition zone
diameter. All the other compounds with an inhibition zone of up to 35 mm. But in the case of
C. albicans complex 5 shows good inhibition zone diameter. The ratio of structural activity of
the synthesized compounds was explained based on the microorganisms tested. Among all
compounds, complex 5 has shown that the best inhibition against all tested microorganisms
may be due to the copper (II) complex. The presence of the p-hydroxyl group has increased
the activity of complex 5 against all microorganisms tested. Complex 2 has shown a
significant inhibition against Gram-positive bacteria, which is due to the presence of copper
(II). The compound HL2 showed greater inhibition of bacteria and less for fungi indicating
that the compound may be a better antibacterial than antifungal activity. This indicates that
the synthesized compounds are biologically active as antimicrobials. B. subtilis was inhibited
to the maximum extent by most compounds.
Fig 7: Comparison of zone diameters of compounds
23

Table 8: Inhibitory activity of the compounds expressed as zone of inhibition (mm)a
Mean of zone diameter, nearest wholea (mm)
Gram-positive bacteria
Gram-negative bacteria
Yeasts
Organism
Staphylococcus
aureus
(ATCC 25923)
Pseudomonas
aeruginosa
(ATCC 27853)
Bacillus subtilis
(ATCC 6635)
Escherichia coli
(ATCC 25922)
Candida albicans
(ATCC 10231)
HL1
22.4±0.2
27.3±0.4
31.7±0.2
29.5±0.1
23.5±0.2
28.2±0.4
31.2±0.1
29.4±0.3
00
26.4±0.3
30.5±0.1
34.9±0.2
33.6±0.2
25.3±0.3
31.5±0.4
36.2±0.1
32.6±0.3
00
21.2 ±0.2
27.6±0.1
31.0±0.3
28.2±0.2
22.4±0.2
29.3±0.1
35.7±0.4
31.3±0.1
00
22.1±0.3
23.5±0.1
28.8±0.2
24.3±0.2
22.3±0.1
24.3±0.2
30.4±0.2
26.8±0.2
00
21.2±0.2
22.5±0.1
28.7±0.2
24.8±0.1
20.8±0.3
25.6±0.2
30.7±0.2
26.3±0.4
00
(1)
(2)
(3)
HL2
(4)
(5)
(6)
Control
Chloramphenicol
Nystatin
34.0±0.1
-
35.1 0.2
-
35.8±0.1
-
32.1±0.3
-
-
31.5±0.1
aThe zone diameters have been calculated in mm by digital vernier caliper. Each value represents a mean standard deviation (SD) of three
replications. Values followed by the same letter(s) in each column are not statistically different according to Tukey's test (P < 0.01).
Fig 8: The Minimum Inhibitory Concentration (MIC) for the compounds
24

Table 9: The Minimum Inhibitory Concentration (MIC) for the compounds
Minimum Inhibitory Concentration (MIC) in mg mL-1
Gram-positive bacteria
Gram-negative bacteria
Fungi
Organism
Staphylococcus
aureus
Pseudomonas
aeruginosa
Bacillus subtilis
Escherichia coli
Candida albicans
HL1
125.00
31.25
15.60
31.25
62.50
31.25
15.60
31.25
12.00
62.50
15.60
15.60
31.25
62.50
31.25
15.60
15.60
12.00
125
250
125
(1)
31.25
15.6
31.25
62.50
31.25
15.6
15.6
12.00
-
62.50
31.25
62.50
125
62.50
31.25
62.50
62.50
31.25
15.6
(2)
(3)
HL2
(4)
62.50
15.6
31.25
12.00
-
(5)
(6)
31.25
-
Chloramphenicol
Nystatin
-
-
15.00
–: not detected inhibition; control; dimethylsulfoxide.
After screening for the antimicrobial properties of compounds, the minimum
inhibitory concentration (MIC) of compounds were tested and compared with the marketed
drugs as shown in Table 9. The MIC of complex 5 showed that, they are most active against
bacteria S. aureus, B. subtilis, P. aeruginosa and E. coli and fungi C. albicans with value of
15.6 mg mL-1. The complex 2 were shown moderate MIC value of 31.25 mg mL-1 against
and E. coli and fungi C.albicans. It was revealed that compound showed a prominent
25

bacterial zone of inhibition and found to vary statistically insignificantly up to 1% level with
chloramphenicol of the same concentration. Similarly, compounds show a prominent fungal
zone of inhibition and comparable to the marketed antifungal drug nystatin.
Further, to understand the time for actual inhibition of test microorganisms with the
application of the compounds, time-kill kinetic study was performed. Time-kill kinetic study
exhibits basic pharmacodynamic information on the relationship between the synthesized
compound and the growth of microorganisms. This test thereby contributes to a better
understanding of current and future application of the compound against the diseases caused
by the respective bacteria or fungi. Time kill kinetics study for complex 5 against one Gram
positive, one Gram negative bacteria and on one fungus is shown in Fig. 9.
As shown in Fig. 9, the untreated controls in each case represented the normal growth
curve of P. aeruginosa, S. aureus and C. albicans, where lag period remained for 1 h. After
that, the exponential growth or the log phase occurred followed by a stationary phase.
Whereas, in case of complex (5) for both the microorganisms, a very short exponential
growth phase was observed in compare to the untreated control. The growth inhibition of
Pseudomonas sp. and Staphylococcus sp. was observed at 3-4 h of incubation period in case
of complex (5). At 4th hour of incubation the bacterial CFU enters in the declining phase i.e.
death phase. When compound complex (5) was applied to the Candida sp. a negligible
growth phase was seen to occur. Consequently, the growth inhibition was found at 2nd hour of
incubation. After an hour of growth inhibition, the cells entered in the death phase. Thus this
observation revealed that the complexes show promising bactericidal and fungicidal activities
respectively.
Fig 9 : Time kill kinetics study for complex 5 against one Gram positive (a. Staphylococcus sp.),
one Gram negative (b. Pseudomonas sp. ) bacteria and one fungus(c. Candida sp).
26

b
c
a
3.4. Conclusion
The condensation reaction of 3-formyl chromone with benzhydrazide and 4-
hydoxybenzhydrazide, afforded the tridentate chromone hydrazones, HL1 and HL2, ligands.
The reactions of the ligands with nickel (II), copper (II) and zinc (II) salts gave neutral
mononuclear complexes. The spectroscopic data showed that the ligand acts as monobasic
tridentate ligand through the γ - pyrone oxygen, azomethine nitrogen and hydrazonic oxygen.
The ligand and complexes showed antimicrobial activity. From the time-kill kinetic study
revealed that the complexes show promising bactericidal and fungicidal activities. In the case
of ligands shows better antioxidant activity than standard Trolox.
Acknowledgements
One of the authors (Jessica Elizabeth Philip) is grateful to the University Grand Commission
(UGC), Government of India for financial assistance in the form of Senior Research
Fellowship and also authors are thankful to the Department of Applied Chemistry, Cochin
University of Science and Technology, Cochin-22, India; Central Institute of Fisheries
Technology, Indian Council of Agricultural Research, Kochi-29,India; SAIF, IIT Bombay,
India and SAIF CUSAT, India for providing necessary facilities to carry out this work.
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