Facile solid state ball milling as a green strategy to prepare 2-(2,4-dichlorophenoxy)-N′-(2-hydroxybenzylidene)acetohydrazide complexes

Article abstract of DOI:10.1016/j.saa.2014.05.062

2-(2,4-Dichlorophenoxy)-N′-(2-hydroxybenzylidene)acetohydrazide (H₂L) complexes were prepared by ball milling involving the reaction of ligand with Ni(II), Co(II), Cu(II) and VO(II) salts (mechanochemical syntheses). The compounds were elucidat

Full text of DOI:10.1016/j.saa.2014.05.062

Accepted Manuscript
Facile Solid state ball milling as a green strategy to prepare 2-(2,4-dichlorophe-
noxy)-N’-(2-hydroxybenzylidene)acetohydrazide complexes
Ahmed Fekri, Rania Zaky
PII:
S1386-1425(14)00853-1
http://dx.doi.org/10.1016/j.saa.2014.05.062
SAA 12224
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomo-
lecular Spectroscopy
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 April 2014
10 May 2014
22 May 2014
Please cite this article as: A. Fekri, R. Zaky, Facile Solid state ball milling as a green strategy to prepare 2-(2,4-
dichlorophenoxy)-N’-(2-hydroxybenzylidene)acetohydrazide complexes, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.05.062
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Facile Solid state ball milling as a green strategy to prepare 2-(2,4-
dichlorophenoxy)-N'-(2-hydroxybenzylidene)acetohydrazide complexes
Ahmed Fekri*, Rania Zaky
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura, Egypt
ABSTRACT
2-(2,4-dichlorophenoxy)-N'-(2-hydroxybenzylidene)acetohydrazide
(H2L)
complexes were prepared by ball milling involving the reaction of ligand with Ni(II),
Co(II), Cu(II) and VO(II) salts (mechanochemical syntheses). The compounds were
elucidated by elemental analysis, spectroscopy (1H NMR, IR, UV-visible, MS
spectra), and physical measurements (magnetic susceptibility and molar conductance).
IR spectra suggested that the H2L behaved as a monodentate and/or bidentate ligand
coordinating via azomethine nitrogen and/or deprotonated enolized carbonyl oxygen.
The electronic spectra of the complexes and their magnetic moments provided
information about geometries. The antimicrobial activities of the ligand and its
complexes were studied against gram positive bacteria; Staphylococcus aureus, gram-
negative bacteria; Escherichia coli and pathogenic fungi; Candida albicans by using
minimum inhibition concentrations method (MIC). Also, the antioxidant (ABTS-
derived free radical method) and cytotoxic (in vitro Ehrlich Ascites) activities of the
isolated compounds were evaluated.
Keywords:
Ball milling
Hydrazone complexes
Antimicrobial
Cytotoxic
Antioxidant
1. Introduction
Ball milling is an effective methodology in organic as well as inorganic synthesis
[1]. From the view point of green chemistry, it was of interest to investigate the
complexation reaction using ligand and metal salts in the solid state. This method was
environmentally friendly and easy to handle, while requires short reaction times and
solventless conditions [2,3].
1

As reported in the literature, Indole-3 acetic acid, naphthalene acetic acid, and 2,4-
dichlorophenoxyacetic acid were the well-known plant growth regulating hormones,
widely used in agricultural field [4,5]. The derivatives of these hormones were also
found to have plant growth regulating and other biological activities [6].
The growing interest in Schiff base compounds is essentially because of their
raised biological activity and also they make possible development of unique
materials. Schiff base are used as chelating ligands in coordination chemistry and
their metal complexes were of great interest for many years [7]. Wherever, the
existence of metal ions bonded to biologically active compounds may enhance their
activities [8,9]. Transition metal ions with different oxidation state have a strong role
in bioinorganic, redox enzyme systems, electrochemistry, environmental chemistry,
metallic deactivators and separation technique [10-17].
The present work aims to synthesize of 2-(2,4-dichlorophenoxy)-N'-(2-
hydroxybenzylidene)acetohydrazide (H2L) and its metal complexes with Co(II),
Ni(II), Cu(II), and VO(II) ions by ball-milling. Moreover, one-pot two-step
mechanochemical synthesis used to prepare a complex without isolating
intermediates. The possible modes of chelations were discussed based on various
spectroscopic methods (IR, UV–visible, MS). The antioxidant, antimicrobial and
cytotoxic activities of the isolated compounds were evaluated.
2. Experimental
2.1. Materials and reagents
All chemicals used were of analytical reagent grade or of the highest purity
available.
Salicylaldehyde,
2-(2,4-dichlorophenoxy)acetic
acid,
[Co(CH3COO)2].4H2O, [Ni(CH3COO)2].4H2O, [Cu(CH3COO)2].H2O and VOSO4
were bought from Sigma-Aldrich. Absolute ethyl alcohol (EtOH), diethyl ether,
dimethyl formamide (DMF) and dimethylsulfoxid (DMSO) were spectroscopic pure
from BDH. Thin-layer chromatography (TLC) was performed on silica gel GF254
precoated plates (Fluka). The chromatograms were visualized under UV light (at 254
and 366 nm).
2

2.2. Instrumentations
The ball-mill was a Retsch MM 2000 swing mill with a 10 cm3 stainless steel,
double-walled beaker with fittings for circulating coolants. Two stainless steel balls of
12 mm diameter were used. Ball-milling was performed at 20225 Hz frequency,
usually at room temperature (without circulating liquid and the temperature did not
rise above 30°C). Infrared spectra of the ligand and its complexes were recorded on a
FTIR spectrophotometer (Mattson 5000, Madison, USA) in the range 4,000–400 cm-1
1
in KBr disks. H NMR spectrum of the ligand was recorded in DMSO on an EM-390
(200 MHz) on a Varian Mercury-300 instrument (Switzerland). Mass spectra were
recorded on a Mattson 5000 FTIR spectrophotometer. Magnetic moment values were
evaluated at room temperature (25 1°C) using a magnetic susceptibility balance
(Johnson Matthey Wayne, Pennsylvania, USA) with Hg[Co(SCN)4] as calibrant. The
electronic spectra of the complexes were recorded in DMSO solution using a
Shimadzu UV 240 (P/N 204-58000) spectrophotometer (USA) in the range 200–900
nm. Elemental analyses (C, H and N) were performed on a Perkin-Elmer 2400 Series
II Analyzer. The determination of metal contents in the complexes was carried out
according to standard methods [18]. Molar conductance of the complexes were
determined by preparing 10-3 mol L-1 solutions in DMSO and measured on an YSI
Model 32 conductivity bridge.
2.3. Synthesis of 2-(2,4-dichlorophenoxy)-N'-(2-hydroxybenzylidene)acetohydrazide
(H2L)
2.3.1. Synthesis of H2L by ball milling
The 2,4-dichlorophenoxyacetic acid hydrazide was prepared according to
literature procedure [19]. The hydrazide (2.35 g; 10.0 mmol) and salicylaldehyde
(1.22 g; 10.0 mmol) were carefully weighed into a Retsch MM 2000 stainless steel
ball-mill vessel equipped with steel balls (10.0 g), the vessel was closed and the
milling was carried out at room temperature [20]. After every 5 min milling cycles,
the progress of the reaction was monitored by TLC. The milling continued for 15 min
overall, till no traces of reactants were found (Scheme 1).
3

2.3.2. Synthesis of H2L by using solvent
The 2,4-dichlorophenoxyacetic acid hydrazide (2.35 g; 10.0 mmol) was refluxed
with salicylaldehyde (1.22 g; 10.0 mmol) in 30 ml absolute ethanol for 1h. White
crystals were formed, filtered off, washed with EtOH and diethyl ether, and
recrystallized from EtOH. The purity of the compounds was checked by TLC.
2.4. Synthesis of metal complexes
2.4.1. Synthesis of metal complexes by ball-milling
A clean and dry ball-milling vessel equipped with 2 stainless steel balls was filled
with 2-(2,4-dichlorophenoxy)-N'-(2-hydroxybenzylidene)acetohydrazide (H2L) (0.339
gm; 1.00 mmol) and 1.00 mmol of acetate salts of Ni(II), Co(II), Cu(II), or sulphate
salt of VO(II) [21]. The mixtures were ball-milled at room temperature for 2 min. The
progress of the reaction was tested by TLC. This milling cycle was repeated, if
necessary, until the reaction was completed. Further purification was recrystallized
from ethanol, if necessary.
2.4.2. Synthesis of metal complexes by using solvent
The complexes were prepared according to a traditional method by reflux
equimolar amounts of H2L (3.39; 10.0 mmol) and 10.0 mmol of Ni(II), Co(II), Cu(II)
and VO(II) salts. The reaction mixture was refluxed on a water bath for 1–3 h. The
precipitate was filtered off, washed with hot EtOH and distilled water and finally
preserved in a vacuum desiccators over anhydrous CaCl2. The complexes are powder-
like, stable in laboratory atmosphere, and soluble in DMF or DMSO. The complexes
were characterized by physical and spectroscopic techniques as follows.
2.5. One-pot two-step mechanochemical synthesis
The all-at-once synthesis of complex was also explored by milling hydrazide,
salicylaldehyde and [Cu(CH3COO)2].H2O together in the appropriate mole ratio for
17 min. In particular, after in situ formation of the ligand by ball milling, metal salt
was added directly to the jar and milling continued for a further 2 min. This approach
also gave the target complex selectively with no solvent needing to be added. Overall,
4

these syntheses have some potential advantages in terms of time and to avoid the use
of solvents for the purification of intermediates.
2.6. Antimicrobial activity
The compounds, antibiotic ampicillin and antifungal colitrimazole were dissolved
in DMSO at concentrations of 1.00 µg ml-1. Twofold dilutions for each one were
prepared separately. The suspensions of microorganisms at concentrations of 10.0
CFU ml-1 (colony forming unit ml-1) were inoculated to the corresponding wells [22].
The plates were incubated at 36 ºC for 24 and 48 h for gram positive bacteria;
Staphylococcus aureus, gram-negative bacteria; Escherichia coli and pathogenic
fungi; Candida albicans by using MIC. The MIC values were determined as the
lowest concentration that completely inhibited visible growth of the microorganism as
detected by unaided eye.
2.6.1. ABTS free radical and scavenging activity.
ABTS-derived free radical method was preferred than other methods that the
produced color remains stable more than 1 h. For each of the tested compounds
2.00 ml of ABTS solution (60.0 mM) was added to 3.00 ml MnO2 solution (25.0
mg/ml), all prepared in 5.00 ml aqueous phosphate buffer solution (pH 7, 0.1 M). The
mixture was shaken, centrifuged, filtered and the absorbance of the resulting green-
blue solution (ABTS radical solution) at λ734 nm was adjusted to approx. 0.50. Then,
50.0 ml of (2.00 mM) solution of the tested compound in spectroscopic grade
MeOH/phosphate buffer (1:1) was added. The absorbance was measured, and the
reduction in color intensity was expressed as inhibition percentage. L-ascorbic acid
was used as a standard antioxidant (positive control). Blank sample was run without
ABTS and using MeOH/phosphate buffer (1:1) instead of tested compounds.
Negative control was run with ABTS and MeOH/phosphate buffer (1:1) only [23-26].
2.6.2. Cell proliferation assay
HePG2 cells were seeds in a 96-well plate at a density of 1.0x104 cells/well [27] at
37 ºC for 24 h under 5% CO2. Different concentration of drugs were added to each
well and cultured for 48 h. The treated cells were washed with PBS and 100 µl of
MTT solution (5.00 mg/ml MTT stock in PBS diluted to 1.00 mg/ml with 10%
5

RPMI-1640 medium) was added to each well and incubated for 4 h at 37 ºC. Finally,
100 µl of DMSO was added and optical densities at 570 nm were measured using a
plate reader (EXL 800). The relative cell viability in percentage was calculated as
(A570 of treated samples/A570 of untreated sample) X 100.
3. Results and Discussion
The isolated solid complexes were stable in air and easily soluble in DMF and
DMSO. The complexes were decomposed on heating above 300 ºC. The molar
conductance values in DMSO were 3-9 Ohm-1cm2mol-1 indicating that all complexes
were non-electrolytes. The colors, yield and elemental analyses of the isolated
complexes were listed in tables 1, 2.
3.1. IR and 1H NMR spectra
In the IR spectrum of H2L, the bands observed at 1676, 1619, 3443 and 3262
cm-1 assigned to ν(C=O) [28], ν(C=N)azo [29], ν(OH) [30] and ν(NH) [31] vibrations,
respectively (Table 3).
1
The H NMR spectrum of H2L in DMSO (Scheme 2) revealed that this
compound is formed as mixtures of its anti and syn isomers (geometrical isomers).
The signals indicated the presence of some additional peaks in the compound. The
1
formation of both isomers was reflected in H NMR spectrum as difference in
1
intensities of proton signals corresponding to both isomers. The H NMR spectrum
showed signals at (11.82; 10.95) and (11.57; 9.99) ppm assignable to the protons of
(NH) and (OH), respectively. The multiple signals observed in the 6.83-7.71 ppm
region are assigned to the aromatic protons. Also, the signals observed at (5.27; 4.82)
are assigned to active methylene protons (–CH2). Moreover, the two signals showed at
(8.49; 8.31) ppm are attributed to (–N=C–H) protons.
The shifts of the characteristic absorption bands of the ligand on complexation
as well as the appearance of new vibrational bands indicated that H2L considered as
monodentate and/or bidentate depending on the metal salt used and the reaction
conditions (Scheme 3).
In [Ni(H2L)2(OAc)(H2O)2] and [VO(H2L)2(SO4)] complexes, H2L acted as a
neutral monodentate ligand coordinating via azomethine nitrogen (C=N)azo. This
mode of complexation is supported by the shift of ν(C=N)azo to lower wave numbers.
6

The ν(C=O), ν(OH) and ν(NH) vibrations remain more or less at the same position.
The IR spectra of these complexes showed new bands at 459 and 468 cm-1 which may
be attributed to ν(Ni–N) and ν(V–N), respectively. Also, the appearance of bands at
1051 and 975 cm-1 assignable to the SO stretching vibrations suggested bidentate
sulfate.
On the other hand, the IR spectra of 2, 3, 4, 5, 6 and 8 complexes (Table 3)
showed that the ligand worked as a mononegative bidentate coordinating via the
azomethine nitrogen (C=N)azo and the deprotonated enolized carbonyl group (=C-O-).
This mode of complexation was supported by the disappearance of both ν(C=O) and
ν(NH) with simultaneous appearance of new bands in 1620-1637 and 1196-1202 cm-1
region assignable to ν(C=N*) and ν(C–O)enolic [33]; the negative shift of ν(C=N)azo
and the appearance of new bands in (558-570) cm-1 and (445-475) cm-1 region
assigned to ν(M–O) and ν(M–N), respectively [33]. Also, the complexes involved
acetate ion in coordination sphere showed two bands in (1371-1387) cm-1 and (1464-
1477) cm-1 region attributed to the νas(O–C–O) and νs(O–C–O) of the acetate group.
The difference rang about 90 cm-1 between those two bands indicating the bidentate
bonding for the acetate group [34].
3.2. Magnetic moments and electronic spectra
The
electronic
spectra
of
[Ni(HL)(OAc)(H2O)2].2H2O
and
[Ni(H2L)2(OAc)(H2O)2] complexes showed two bands at (18182, 16949) and (29585,
3
3
27933) assigned to A2g → 3T1g(F) and A2g → 3T1g(P) transitions, respectively, in an
octahedral geometry [35]. The calculated values of Dq, B, β and υ2/υ1 values lie in the
range reported for an octahedral structure (Table 4). The position of υ1 (11293 &
10527 cm-1) was calculated theoretically. Also, the value of the magnetic moment (3.2
& 3.4 BM) could be taken as an additional evidence for an octahedral geometry.
The electronic spectra of [Co(HL)(OAc)(H2O)2] and [Co(HL)2(H2O)2]
complexes showed two bands at 14286 and (17241, 17857) cm-1 attributed to
4
4T1g →4A2g(F) and T1g
→
4T1g(P) transitions, respectively, in an octahedral
configuration [35]. The calculated values of Dq, B, β and υ2/υ1 were in good
agreement with those reported for octahedral Co(II) complexes. The position of υ1
7

(6645 cm-1) was calculated theoretically. Also, the magnetic moment value (4.9 & 5.1
BM) was consistent with octahedral geometry around the Co(II) ion.
The Cu(II) complexes have magnetic moment value (1.9 & 2.1 BM),
indicating the presence of Cu(II) ion. The electronic spectra of [Cu(HL)2] and
[Cu(HL)(OAc)] complexes (Figure 1) exhibited a broad band with a maximum at
14925 and 14815 cm-1, respectively due to the 2B1g → 2A1g transition, as reported for
square planar Cu(II) complex [35].
The electronic spectra of [VO(HL)2] and [VO(H2L)2(SO4)] complexes showed
2
a band at 17391 and 17544 cm-1, respectively assigned to B2 → 2E(υ2) in a square-
pyramidal configuration [35].
3.3. Biological activity
3.3.1. Antimicrobial activity
The Diseases caused by microbial infections were a serious menace to the
health of human being and often have connection to some other diseases when the
body system gets debilitated. Developing antimicrobial drugs and maintaining their
potency in opposition to resistance by different types of microorganisms as well as a
broad spectrum of antibacterial and antifungal activities were some of the major
concerns of research in this area.
All of the tested compounds showed a remarkable biological activity against
different types of gram positive bacteria; Staphylococcus aureus, gram-negative
bacteria; Escherichia coli and pathogenic fungi; Candida albicans by using MIC as
shown in table 5 [36,37]. Among all the tested compounds, [VO(H2L)2(SO4)]
complex has the most potent antibacterial and antifungal activity (93-100%). In
general, all the metal complexes have higher antimicrobial activity than the ligand and
this may be due to the change in structure caused by coordination tends to make metal
complexes act as more potent bactereostatic agents, thus inhibiting the growth of the
microorganisms. Moreover, coordination reduces the polarity of the metal ion mainly
because of the partial sharing of its positive charge with the donor groups within the
chelate ring system formed during the coordination. This process, successively,
increases the lipophilic nature of the central metal atom, which favors its permeation
more efficiently through the lipid layer of the microorganism, so destroying them
8

more destructively. Increased activity of the metal chelates is due to the lipophilic
nature of the metal ion in complexes. Furthermore, the mode of action of Schiff base
compounds may involve the formation of a hydrogen bond through the azomethine
nitrogen atom with the active center of the cell constituents, resulting in interference
with normal cell process [38].
3.3.2. Antioxidant activity for ABTS
By screening antioxidant activity using ABTS inhibition method for isolated
compounds [39], it was observed that all complexes have lower antioxidant activity in
comparison with the ligand (87.88%) except [VO(H2L)2(SO4)] (89.23%) and
[VO(HL)2] (88.85%) complexes showed high antioxidant activity comparable to
ascorbic acid (Figure 2). In general, a range of vanadium compounds have various
biological activities as anti-amoebicagents and anti-diabeticagents [40,41].
3.3.3. Cell proliferation assay
In view of the biological activity of Schiff bases, we firstly were evaluated the
ability of hydrazone derivatives to inhibit cancer cell growth against Hepatocellular
carcinoma liver (HCC) [42]. In our experiments, IC50 values (compound
concentration that produces 50% of cell death) were calculated. For comparison
purposes the cytotoxicity of Fluorouracil (5-FU) and the free ligand as well as the
metal complexes was evaluated under the same experimental conditions. It is clearly
observed that complexation with metal has a synergistic effect on the cytotoxicity
(Figure 3). The comparison of cytotoxicity indicated that [VO(H2L)2(SO4)] and
[VO(HL)2] complexes showed much lower IC50 values (2.414 and 2.485 µg/ml) for
HePG2, respectively. Therefore, the chelation of the free ligand with metal ion is
essential for anticancer activities of these complexes. These results were consistent
with the cases of many other analogues of hydrazones. Importantly, it should be
emphasized that VO(II) complexes exhibits considerable cell growth inhibition
activity against human liver hepatocellular carcinoma HepG2 cells similar to that of
5-FU (2.355 µg/ml) for HePG2. These gratifying results are encouraging its further
screening in vitro. Therefore, its further biological evaluation in vivo as well as
studies of mechanism of action is necessary.
9

4. Conclusions
In this work, Ni(II), Co(II), Cu(II), and VO(II) complexes of 2-(2,4-
dichlorophenoxy)-N'-(2 -hydroxybenzylidene)acetohydrazide (H2L) were synthesized
by two different methods, the first was by ball milling as a green strategy and the
second was by refluxing as the traditional method. The structures of the isolated
complexes were interpretive by different spectroscopic techniques. The results of
most complexes showed that the ligand act as mononegative bidentate except in the
[Ni(H2L)2(OAc)(H2O)2] and [VO(H2L)2(SO4)] complexes, the ligand act as neutral
monodentate. Also, the ligand and its complexes were screened for antimicrobial,
antioxidant activity (using ABTS method), and in vitro Ehrlich as cites assay. The
results revealed that the ligand and VO(II) complexes showed the highest inhibitory
antioxidant activity by comparable to ascorbic acid. Moreover, VO(II) complexes
have excellent activity similar to that of 5-FU for inhibiting the tumour cells
compared to the free ligand.
REFRENCES
[1] F.K.V. Moreira, J.M. Marconcini, L.H. Mattoso, Cellulose 19 (2012) 2049–2056.
[2] A.C. Tella, U.B. Eke, A.Y. Isaac, C.A. Ojekanmi, Orbital Elec. J. Chem. 3 (2011)
94–103.
[3] M. Ferguson, N. Giri, X. Huang, D. Apperley, S.L. James, Green Chem. 2014,
doi: 10.1039/C3GC42141D
[4] T. Gaspar, C. Keveks, C. Penel, H. Greppin, D.M. Reid, T.A. Thorpe, In Vitro
Cell. Dev. Biol. Plant 32 (1996) 272–289.
[5] P.H. Moura-Costa, L. Lundoh, J. Trop. For. Sci. 7 (1994) 338–340.
[6] G.N. Naik, R.P. Bakale, A.H. Pathan, S.G. Ligade, S.A. Desai, K.B. Gudasi, J.
Chemistry 2013 (2013) 1–13.
[7] S. Ershad, L.A. Sagathforoush, G. Karim-nezhad, S. Kangari, Int. J. Electrochem.
Sci. 4 (2009) 846–854.
10

[8] O.A. El-Gammal, E.A. Elmorsy, Y.E. Sherif, Spectrochim. Acta A 120 (2014)
332–339.
[9] E.F.F. da Cunha, T.C. Ramalho, D.T. Mancini, E.M.B. Fonseca, A.A. Oliveira, J.
Braz. Chem. Soc. 21 (2010) 1787–1806.
[10] C. Nuñez, R. Bastida, A. Macias, C. Lodeiro, L. Valencia, Inorg. Chim. Acta
362 (2009) 3454–3460.
[11] M.S. Ureta-Zanartu, P. Bustos, M.C. Diez, M.L. Mora, C. Gutierrez,
Electrochim. Acta. 46 (2001) 2443–2592.
[12] A. Ciszewski, G. Milczarek, J. Electroanal. Chem. 469 (1999) 18–26.
[13] S. Trevin, F. Bedioui, M.C Gomez Villegas, C. Bied-Charreton, J. Mater. Chem.
7 (1997) 923–928.
[14] M.V. Angelusiu, S.F. Barbuceanu, C. Draghici, G.L. Almajan, Eur. J. Med.
Chem. 45 (2010) 2055–2062.
[15] G. Tamasi, L. Chiasserini, L. Savini, A. Sega, R. Cini, J. Inorg. Biochem. 99
(2005) 1347–1359.
[16] N. Filipovic, H. Borrmann, T. Todorovic, M. Borna, V. Spasojevic, D. Sladic, I.
Novakovic, K. Andjelkovic, Inorg. Chim. Act 362 (2009) 1996–2000.
[17] Y. Zhang, L. Zhang, L. Liu, J. Guo, D. Wu, G. Xu, X. Wang, D. Jia, Inorg.
Chim. Acta 363 (2010) 289–293.
[18] A.I. Vogel, Quantitative Inorganic Analysis, Longmans, London, 1989.
[19] P. Nun, C. Martin, J. Martinez, F. Lamaty, Tetrahedron 67 (42) (2011) 8187–
8194.
[20] G.N. Naik, R.P. Bakale, A.H. Pathan, S.G. Ligade, S.A. Desai, K.B. Gudasi, J.
11

Chem., 2013 (2013) 1–13.
[21] A.C. Tella, U.B. Eke, A.Y. Isaac, C.A. Ojekanmi, Orbital Elec. J. Chem. 3
(2011) 94–103.
[22] I. Stylianakis, A. Kolocouris, N. Kolocouris, G. Fytas, G.B. Foscolos, E.
Padalko, J. Neyts, D. Clerq, E. Bioorg, Med. Chem. Lett. 13 (2003) 1699–1703.
[23] E. Lissi, B. Modak, R. Torres, J. Escobar, A. Urza, Free Radical Res. 30 (1999)
471–477.
[24] A.B.A. El-Gazzar, A.M.S. Youssef, M.M. Youssef, A.A. Abu-Hashem, F.A.
Badria, Eur. J. Med. Chem. 44 (2009) 609–624.
[25] R. Aeschlach, J. Loliger, C.B. Scott, A. Murcia, J. Butler, B. Halliwel, I.O.
Aruoma, Food Chem. Toxicol. 32 (1994) 31–36.
[26] H.J. Mauceri, N.N. Hanna, M.A. Beckett, D.H. Gorski, M.J. Staba, K.A. Stellato,
K. Bigelow, R. Heimann, S. Gately, M. Dhanabal, G.A. Soff, V.P. Sukhatme,
D.W. Kufe, R.R. Weichselbaum, Nature, 394 (1998) 287–291.
[27] Y. Morimoto, K. Tanaka, Y. Iwakiri, S. Tokuhiro, S. Fukushima, Y. Takeuchi,
Biol. Pharm. Bull. 18 (1995) 1417–1422.
[28] R.R. Zaky, T.A. Yousef, J. Mol. Struct. 1002 (2011) 76–85.
[29] K.M. Ibrahim, I.M. Gabr, G.M. Abu El-Reash, R.R. Zaky, Monatsh. Fur.
Chem.140 (2009) 625–632.
[30] K.M. Ibrahim, I.M. Gabr, R.R. Zaky, J. Cood. Chem. 62 (2009) 1100–1116.
[31] K.M. Ibrahim, R.R. Zaky, E.A.Gomaa, M.N. El-Hady, Res. J. Pharm. Biol.
Chem. 2 (2011) 391–404.
[32] R.R. Zaky, K.M. Ibrahim, I.M. Gabr, Spectrochim. Acta. 81 (2011) 28–34.
12

[33] T.A. Yousef, T.H. Rakha, U. El-Ayaan, G.M. Abu El-Reash, J. Mol. Struct. 1007
(2012) 146–157.
[34] R.R. Zaky, Phosphorus, Sulfur, and Silicon and the Relat. Elem. 186 (2011)
365–380.
[35] F.A. Cotton, G. Wilkinson, C.A. Murillo, M. Bochmann, Advanced Inorganic
Chemistry, 6th ed., John Wiley & Sons Inc., 2003.
[36] P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, R.H. Yolken, in: G.L.
Wood, J.A. Washington (Eds.), Manual of Clinical Microbiology, Am. Soc.
Microbiol., Washington, DC, 1995.
[37] R.N. Jones, A.L. Barry, T.L. Gavan, I.I.J.A. Washington, in: E. H. Lennette, A.
Ballows, W.J. Hausler Jr, H.J. Shadomy (Eds.), Manual of Clinical Microbiology,
fourth edn, Am. Soc. Microbiol. (1972), Washington DC 985.
[38] N. Raman, V. Muthuraj, S. Ravichandran, A. Kulandaisamy, Proc. Indian Acad.
Sci. 115 (2003) 161–167.
[39] J.E.N. Dolatabadi, A. Mokhtarzadeh, S.M. Ghareghoran, G. Dehghan, Adv.
Pharm. Bull. 4 (2014) 101–104.
[40] D.C. Crans, B. Zhang, C.R. Roberts, Inorg. Chem. 49 (2010) 4245–4256.
[41] B.A. Reul, S.S Amin, S.M. Brichard, Br. J. Pharmocol. 126 (1999) 467–477.
[42] G. Kismali, F.M. Emen, T. Yesilkaynak, O. Meral, D. Demirkiran, T. Sel, N.
Kulcu, Eur. Rev. Med. Pharmacol. Sci. 16 (2012) 1001–1012.
13

Scheme1. The outline of the synthesis of ligand and its metal complexes.

Scheme 2. 1H NMR spectrum of 2-(2,4-dichlorophenoxy)-N'-(2-hydroxybenzylidene)acetohydrazide
exist in tautomer in DMSO.
Scheme 3. IR spectra of (A) H2L, (B) [Cu(HL)2] and (C) [Cu(HL)(OAc)] complexes.

Fig. 1. Electronic Spectra of (A) [Cu(HL)2] and (B) [Cu(HL)(OAc)] complexes in DMSO.
100
80
60
40
20
0
C0 C1 C2 C3 C4 C5 C6 C7 C8 C9
Fig. 2. Anti-oxidant assays by ABTS method of ligand and its metal complexes.
6
5
4
3
2
1
0
C0 C1 C2 C3 C4 C5 C6 C7 C8 C9
Fig. 3. The cytotoxicity (IC50) of tested compounds on HepG2 cell line.

Table 1
Elemental analysis and physical data of H2L and its metal complexes prepared by ball milling
Compound
Empirical Formula
F.W. Found (Calculate)
% Found (Calculate)
Yield
(%)
Colour
White
C
H
N
Cl
M
-
H2L
53.26
3.60
8.31
20.97
C15H12Cl2N2O3
339.231 (339.170)
95
98
97
(53.12) (3.57) (8.26) 20.93
[Cu(HL)(OAc)]
CuC17H14Cl2N2O5
460.718 (460.712)
44.33 3.09 6.10 15.41
(44.32) (3.06) (6.08) (15.41) (13.79)
13.82
Green
[Co(HL)2(H2O)2]
CoC30H26Cl4N4O8
771.290 (771.284)
46.74
3.38
7.30
18.43
7.64
Yellow
(46.72) (3.39) (7.26) (18.41) (7.64)
[Ni(H2L)2(OAc)(H2O)2]
NiC32H31Cl4N4O12
832.182 (832.301)
46.19
3.75
6.73
17.06
7.05
Pale-green 95
Dark-green 99
(46.23) (3.90) (6.84) (17.11) (7.11)
[VO(H2L)2(SO4)]
VC30H24Cl4N4SO11
845.462 (845.490)
42.62
2.86
6.63
8.39
6.02
(6.23)
(42.68) (2.99) (6.67) (8.41)
Table 2
Elemental analysis and physical data of H2L and its metal complexes prepared by using solvent
Compound
Empirical Formula
F.W. Found (Calculate)
% Found (Calculate)
Yield
(%)
Colour
White
Λm*
C
H
N
Cl
M
-
H2L
53.26
3.60
8.31
20.97
C15H12Cl2N2O3
339.231 (339.170)
90
78
85
-
(53.12) (3.57) (8.26) 20.93
[Cu(HL)2]
CuC30H22Cl4N4O6
739.881 (739.874)
48.79
3.34
7.62
19.22
8.61
Green
3
5
(48.70) (3.00) (7.57) (19.19) (8.59)
[Co(HL)(OAc)(H2O)2]
CoC17H18Cl2N2O7
492.312 (492.122)
41.54
3.71
5.71
14.44
12.06
Yellow
(41.49) (3.68) (5.69) (14.42) (11.97)
[Ni(HL)(OAc)(H2O)2].2H2O
NiC17H22Cl2N2O9
528.097 (527.912)
38.71
4.24
5.40
13.47
11.24
Pale-Green 83
6
3
(38.68) (4.20) (5.31) (13.45) (11.12)
[VO(HL)2]
48.55
3.05
7.61
19.21
6.93
VC30H22Cl4N4O7
743.661 (743.264)
Yellow
80
(48.48) (2.98) (7.54) (19.10) (6.85)
* in DMSO (ohm-1cm2 mol-1)

Table 3
Most important IR spectral bands of H2L, its metal complexes in cm-1
No Compound
ν(NH) ν(OH)phenolic ν(C=O) ν(C=N)azo (CH2) ν(C=N *) ν(C-O)enolic
1
2
3
4
H2L
[Cu(HL)2]
[Cu(HL)(OAc)]
[Co(HL)(OAc)(H2O)2]
3262
3443
3443
3472
3444
1676
1619
1601
1603
1604
2988
3042
3024
3014
-
-
-
-
-
-
-
-
1626
1620
1635
1202
1200
1196
5
6
7
8
9
[Co(HL)2(H2O)2]
-
-
3492
3444
3475
3445
3465
-
-
1605
1606
1606
1603
1608
3014
3015
2927
3043
3060
1635
1637
-
1623
-
1196
1196
-
1201
-
[Ni(HL)(OAc)(H2O)2].2H2O
[Ni(H2L)2(OAc)(H2O)2]
[VO(HL)2]
3137
-
1678
-
[VO(H2L)2(SO4)]
3264
1678
Table 4
Magnetic moments, electronic spectra and ligand field parameters of metal complexes of H2L
Compound μeff (BM) Band position (cm-1) Dq (cm-1) B (cm-1)
β
υ2/υ1
2
3
4
5
6
7
8
9
2.1
1.9
5.1
4.9
3.2
3.4
1.8
1.8
14925
14815
-
-
-
-
-
-
-
-
14286; 17241
14286; 17857
18182; 29585
16949; 27933
17391
767
809
1120
1057
-
767
809
933
881
-
0.79 2.15
0.83 2.15
0.89 1.61
0.85 1.61
-
-
-
-
17544
-
-
Table 5
Antimicrobial activity in terms of MIC (mg/mL) after 48 h
S. Aureus (mg/mL)
E. Coli (mg/mL)
C. Albicans (mg/mL)
Compound
DIZ
(mm)
24
-
11
17
13
9
12
11
10
18
24
DIZ
(mm)
22
-
DIZ
(mm)
-
28
5
19
5
5
14
8
6
% Activity index
% Activity index
% Activity index
100
-
100
-
-
Ampicillin
Colitrimazole
100
18
68
18
18
50
29
21
75
93
1
2
3
4
5
6
7
8
9
46
71
54
38
50
46
42
75
100
6
27
64
55
27
55
41
50
73
82
14
12
6
12
9
11
16
18
21
26

 Mechanochemical synthesis
One-pot two-step reaction

 Spectroscopic characterization
 Studying the antimicrobial, cytotoxicity and antioxidant activities