Synthesis, Spectral Characterization and Cytotoxic Activity of 3d-
Transition Metal complexes with o-vanillin
benzoylhydrazone Ligand
Husna Sarfina Bt Husain
(23617)
Bachelor of Science with Honours
(Resource Chemistry)
Faculty of Resource Science and Technology
Synthesis, Spectral Characterization and Cytotoxic Activity of 3d-Transition Metal
complexes with o-vanillin benzoylhydrazone Ligand
Husna Sarfina Bt Husain (23617)
A Dissertation Submitted in Partial Fulfillment of the
Final Year Project 2 (STF 3015)
Supervisor: Assoc. Prof. Dr. Md. Abu Affan
Resource Chemistry
Department of Chemistry
Faculty of Resource Science and Technology
University Malaysia Sarawak
4 July 2012
I
Acknowledgement
I would like to express the deepest gratitude to my dearest supervisor, Assoc. Prof. Dr. Abu
Affan, Chemistry Department, UNIMAS, for his constant encouragement, guidance and support
from the initial to the final of the project. This thesis would not have been possible to complete
without his help. Besides, I would like to express my deepest appreciation to Mr. Salam, PhD
student of Chemistry Department, UNIMAS, for his occasional helps to develop my
understanding. Furthermore, I would like to give special thank to Miss Norihan, M. Sc. student
of Chemistry Department, UNIMAS, for her cooperation with me to do the study on FT-IR and
UV-Visible spectroscopy. Besides, I would like to express sincere thank to all academic staff of
Department of Chemistry for their inspiration and valuable advice to accomplish this work. I
would like to grab this opportunity to thanks my family and my friends who involved directly or
indirectly by giving their encouragement to me in completing this project. Finally, I would like
to offer my regards and blessing to all of those who supported me in any respect during the
completion of the project.
II
Declaration
No portion of the work referred to this thesis has been submitted in support of an application for
another degree qualification of this or any other university or institution of higher learning.
_______________________________
Husna Sarfina Bt Husain (23617)
Chemistry Department
Faculty of Resources Science and Technology
University Malaysia Sarawak
III
Table of Contents
Acknowledgement …………………………………………………………………………. I
Declaration …………………………………………………………………………………. II
Table of contents …………………………………………………………………………… III
List of abbreviations ………………………………………………………………………... V
List of figures ………………………………………………………………………………. VII
List of schemes …………………………………………………………………………….. VIII
List of tables ………………………………………………………………………………... IX
Abstract …………………………………………………………………………………….. 1
CHAPTER 1
1.0 Introduction
1.1 Hydrazone ligands and transition metal complexes …………………………….
1.2 Objectives ……………………………………………………………………….
2
3
CHAPTER 2
2.0 Literature review
2.1 Synthesis of Schiff base hydrazone ligands and their transition transition
metal complexes ………………………………………………………………...
2.2 Cytotoxic activity of transitional metal complexes of hydrazone ligand ………
4
9
CHAPTER 3
3.0 Material and methods
3.1 Spectral characterization of complexes …………………………………………
3.2 Synthesis of hydrazone ligand (1) and its 3d-transition metal complexes (2-7)
3.2.1 Synthesis of o-vanillin benzoylhydrazone (C15H14N2O3) (1) ………
3.2.2 Synthesis of Mn(II) complex (2) with ligand (1) …………………..
3.2.3 Synthesis of Fe(II) complex (3) with ligand (1) ……………………
3.2.4 Synthesis of Co(II) complex (4) with ligand (1) ……………………
3.2.5 Synthesis of Ni(II) complex (5) with ligand (1) ……………………
10
11
12
13
14
15
IV
3.2.6 Synthesis of Cu(II) complex (6) with ligand (1) ……………………
3.2.7 Synthesis of Zn(II) complex (7) with ligand (1) ……………………
3.3 Cytotoxicity activity …………………………………………………………….
16
17
18
CHAPTER 4
4.0 Result and discussion …………………………………………………………………..
4.1 Physical and analytical data …………………………………………………….
4.2 UV-Visible analysis data ……………………………………………………….
4.3 FT-IR spectroscopy …………………………………………………………….
4.4 1H NMR spectrum ………………………………………………………………
4.5 Cytotoxicity test of ligand (1) and its transition metal(II) complexes (3-7) …...
19
19
22
25
31
34
CHAPTER 5
5.0 Conclusion ……………………………………………………………………………...
36
CHAPTER 6
6.0 Suggestion for further research …………………………………………………………
37
References …………………………………………………………………………………. 38
Appendixes …………………………………………………………………………………. 41
V
List of abbreviations
Ar-H Aromatic ring
br broad
CHN Carbon-Hydrogen-Nitrogen
DMSO Dimethyl Sulfoxide
FT-IR Fourier transform infrared spectroscopy
1H NMR Proton Nuclear Magnetic Resonance Spectroscopy
KBr Potassium Bromide
LC50 Lethal concentration 50
MHz Megahertz
m Medium
m.p Melting point
mL Millilitre
nm Nanometre
n Nonbonding
ppm Part per million (µg/mL)
s Strong
UV-Visible Ultraviolet-Visible spectroscopy
v Wave number
w Weak
X-ray X-radiation
% Percentage
π* Pi anti-bonding
π Pi bonding
cm-1
Wave number or reciprocal wavelength
oC Degree Celsius
δ Delta
λmax Lambda max wavelength
Ω-1
cm2mol
-1 Molar conductivity
VI
List of figures
Figure 1: Tautomeric form of 4-methylphenylamino acetoacetylacetone hydrazone
Figure 2: Keto and enol form of aroylhydrazone
Figure 3: Formation of hydrazone derivatives
Figure 4: Synthesize of 3-carbaldehyde chrome-(benzoyl) hydrazone
Figure 5: N´-(5-chloro-2-hydroxybenzylidene)-4-Dimethylaminobenzohydrazide and N´(2,4-
Dichlorobenzylidene)-4-Dimethylaminobenzohydrazide
Figure 6: UV-Visible spectrum of [C15H14N2O3] (1) in MeOH (1×10-4
M)
Figure 7: UV-Visible spectrum of [Fe(C15H12N2O3)H2O] (3) in MeOH (1×10-4
M)
Figure 8: UV-Visible spectrum of [Zn(C15H12N2O3)H2O] (7) in MeOH (1×10-4 M)
Figure 9: IR spectrum of [C15H14N2O3] (1) (As KBr disc)
Figure 10: IR spectrum of [Fe(C15H12N2O2)H2O] (3) (As KBr disc)
Figure 11: IR spectrum of [Zn(C15H12N2O2)H2O] (7) (As KBr disc)
Figure 12: 1H-NMR signal of [C15H14N2O3] (1) in DMSO-d6
Figure 13: 1H-NMR of [Zn(C15H12N2O3)H2O] (7) in DMSO-d6
Figure 14: Toxicity test of [Cu(C15H12N2O3)H2O] (6)
Figure 15 UV-Visible spectrum of [Co(C15H12N2O3)H2O] (4) in MeOH (1×10-4
M)
Figure 16: UV-Visible spectrum of [Ni(C15H12N2O3)H2O] (5) in MeOH (1×10-4
M)
VII
List of schemes
Scheme 1: Synthesis o-vanillin benzoylhydrazone (1)
Scheme 2: Synthesis of [Mn(C15H12N2O3)H2O] (2)
Scheme 3: Synthesis of [Fe(C15H12N2O3)H2O] (3)
Scheme 4: Synthesis of [Co(C15H12N2O3)H2O] (4)
Scheme 5: Synthesis of [Ni(C15H12N2O3)H2O] (5)
Scheme 6: Synthesis of [Cu(C15H12N2O3)H2O] (6)
Scheme 7: Synthesis of [Zn(C15H12N2O3)H2O] (7)
VIII
List of tables
Table 1: The physical data and theoretical elemental analysis of ligand and its complexes
Table 2: Molar conductivity values of complexes (2-7)
Table 3: The λmax (nm) peaks of hydrazone ligand (1) and its transition metal complexes (2-7)
Table 4: IR spectra of hydrazone ligand (1) and its metal complexes (2-7) (cm-1
)a
Table 5: 1H NMR signals for ligand (1) and complex (7)
Table 6: The LC50 of ligand (1) and its complexes (7)
1
Synthesis, Spectral Characterization and Cytotoxic Activity of 3d-Transitional Metal
Complexes with o-vanillin benzoylhydrazone Ligand
Husna Sarfina Bt Husain
Chemistry Department
Faculty of Resources Science and Technology
University Malaysia Sarawak
ABSTRACT
Six new Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes (2-7) of o-vanillin
benzoylhydrazone ligand (1) have been synthesized and characterized. The ligand (1) and its 3d-
metal complexes (2-7) were characterized by elemental analysis, molar conductivity, UV-
Visible, FT-IR and 1H NMR spectroscopic studies. The spectroscopic data of the complexes (2-
7) suggested that the ligand (1) is acted as a dinegative tridentate chelating system and is
coordinated to central metal(II) atom through phenoxide-O, atomethine-N and carboxylate-O
atoms. Four coordinated molecular structure has been proposed for the synthesized 3d-transition
metal complexes. The ligand (1) and its metal complexes (2-7) have also been tested for their
cytotoxicity and found to be less active against Artemia salina.
Keywords: Hydrazone ligand; Transition metal salts; spectral analysis; cytotoxicity.
ABSTRAK
Enam komplek (2-7) baru iaitu Mn(II), Fe(II), Ni(II), Cu(II) and Zn(II) bagi o-vanilin
benzohidrazon ligan (1) telah disintesis dan dianalis. Ligan (1) dan komplek (2-7) telah
dianalisis mengunakan analisis elemen, molar konduktiviti, UV-Visible, FT-IR dan 1H NMR
spectra. Data spectroskopi bagi komplek (2-7) menyatakan ligan (1) bertindak sebagai
dinegative tridentate chelating system dan dikordinate kepada metal(II) utama melalui atom
phenoxide-O, atomethine-N dan carboxylate-O. Empat koordinat molekul telah dicadangkan
bagi sintesis komplek 3d-logam peralihan. Ligan (1) dan metal komplek (2-7) juga telah uji
citotoxiciti dan dijumpai rendah aktif terhadap Artemia salina.
Kata kunci: Hidrazon ligan; logam peralihan; analisis spectra; citotoxiciti.
2
CHAPTER 1
1.0 INTRODUCTION
1.1 Hydrazone ligands and transition metal complexes
Hydrazone ligands have similarities in their donor properties with unsymmetrical salen which is
condensation product of salicylaldehyde and 1,2-diaminoethane then, unsymmetrical salen can
act as effective catalyst toward alkene epoxidation (Das et al., 2011). The chemistry of
hydrazones plays an important role in coordination chemistry. Hydrazone ligands and their metal
complexes have great attention in coordination chemistry (Raman et al., 2004). The metal
complexes have been widely used in medical like antibiotic, antibacterial, antiviral, antiparasitic,
radio-sensitizing agent and also anticancer agents (Kurdekar et al., 2011). The condensation
reaction of primary amines with a carbonyl compound yields Schiff bases which are offer
opportunities for inducing substrate chirality, tuning the metal centred electronic factor,
enhancing the solubility and stability either homogenous or heterogeneous catalyst (Aysegul et
al, 2004). Then, the presence of metals ion bonded to biologically active compound will enhance
their activities because of present N, S, and O donor atom in ligands (Saghatforoush et al., 2008).
Hydrazones have special characteristics which can form stable metal complexes with most
transition metals ions and also showed in antimicrobial, anti-tuberculosis, anti-tumor activity
(Al-sha’alan, 2007). In hydrazone ligands which have hydroxyl radical and superoxide anion
have contribute in DNA-binding abilities and pharmacological activities (Qian et al., 2009).
3
On the other hand, the azo-hydrozane tautomerism has ability in colour tone and photostabilty of
azo dye and also in design compound having required colour properties (Shamns et al., 2011).
Recently, heterocyclic hydrazones have ability in pharmacological properties because of iron
scavenging and anti-tubercular activities and can also acts as chelating agent and versatile modes
bonding (Saleem & Mousa, 2011).
In view of the above studies and applications of hydrazone ligands and their metal complexes,
the author has synthesized several 3d-transition complexes of hydrazone ligand (1). These
complexes have been characterized and also studied their cytotoxicity against Artemia salina.
1.2 Objectives
The main objectives in this research are
i. to synthesis o-vanillin benzoylhydrazone and it’s Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and
Zn(II) complexes.
ii. to characterize hydrazone ligand (1) and it’s 3d transitional metal complexes (2-7) by
CHN analyses, UV-visible, FT-IR and 1H NMR spectral analyses.
iii. to determine the molar conductance value of the synthesized metal(II) complexes (2-7).
iv. to evaluate cytotoxicity of hydrazone ligand and it’s metal(II) complexes against brine
shrimp (Artemia salina).
4
CHAPTER 2
2.0 LITERATURE REVIEW
2.1 Synthesis of Schiff base hydrazone ligands and their transition metal complexes
Schiff base with hydrazone-based functional groups have their versatility in coordinating metal,
pharmacological, biological activities and also established some drugs (Aza et al., 2007). Besides
that, heterocyclic azo dyes have important applications in textile colorants, photo-responsive
biomaterial, optical sensing of metal ions and non-linear optics (Shams et al., 2011). In
preparation functionalized acylic and heterocyclic hydrazone dye based on 2-N-Acylamino-
4,5,6,7-tetrahydrobenzo[b]thiophene system (Shams et al., 2011).
The bis-tridentate aroylhydrazone with binicotinate conformation to isolate 3-hydroxy-5-
(hydroxymethyl)-2-methyl-4-pyridinecarbonylhydrazone is prepared from the condensation
reaction of salicylaldehyde and acid hydrazide (Hermes-Lima et al., 2001). They have found that
the iron chelator pyridoxal isonicotinoyl hydrazone had potential in antioxidant against OH. This
can be induced by Fe(III)-EDTA, ascorbate and O2.
The tetraaldehyde phenylhydrazone can act as binucleating tetraphenylhydrazone and give
organic ligand which mimicking protein-metal binding sites in biological system (Karabӧcek,
2005). Based on Karabӧcek (2005), the tetraaldehyde phenylhydrazone can also be used as
therapeutic agent and catalytic agent.
4-methylphenylamino acetoacetylacetone hydrazone can be formed by condensation reaction of
4-methylphenylamino acetohydrazide with acetylacetone in EtOH 1:1 ratio (EL-Tabl et al.,
2007).
5
Based on EL-Tabl et al., (2007), the dinuclear copper and iron complexes of 4-
methylphenylamino acetoacetylacetone hydrazone were able to mimic bimetallic sites in various
enzymes and tautomeric formed (Figure 1).
CH3
HO
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Figure 1: Tautomeric form of 4-methylphenylamino acetoacetylacetone hydrazone
Arolhydrazone was bonded to metal ion in keto or enolic form (Boaming et al., 2009), (Figure
2). Aroylhydrazone has also ability to form chelating to metal ion and containing pyridine in the
aroylhydrazone make it become more attracted in analytical reagent because of their sensitivity
(Das et al., 2011). Pyridyl-containing hydrazone was displayed intriguing structure like helicate,
molecular box, molecular cleft and square (Boaming et al., 2009).
HO
OH
Figure 2: Keto and enol form of arolhydrazone
6
Hydrazide/hydrazone derivatives can be formed coumarine, pyridine, thiozole and thiophene
derivative which are suitable in pharmaceutical application (Mohera et al., 2010). Then,
Moherab et al., (2010) have synthesized hydrazide/hydrazone derivatives from
cynoacetylhydrazine with 3-acetylpyridine in 1,4-dioxane shown in Figure 3.
NHNH2
CN
COCH3
NC
CH3
Figure 3: Formation of hydrazone derivatives
Besides that, 3-carbaldehyde chrome-(benzoyl) hydrazone is synthesized ethanolic solution of
benzoyl hydrazide to ethanol containing 3-carbaldehyde chrome-(benzoyl)hydrazone dropwise
(Yong & Zheng-yin, 2010), (Figure 4). The reaction mixture was refluxed and washed several
time with ethanol and recrystallized from DMF and water (Yong & Zheng-yin, 2010).
7
OH
(CH3CO2)2O
98% H2SO4
OCOOCH3
AlCl3
heat
OH
COCH3 PdCl3
DMF
CHO
NH2
Figure 4: Synthesize of 3-carbaldehyde chrome-(benzoyl) hydrazone
Calix[n]arene molecule have donor group like oxygen and nitrogen atom which can give ability
to coordinate with hard and soft transition metal (Podyachev et al., 2011). The example of
calix[n]arene is 25,26,27,28-tetrakis[(2-pyridinylmethylidene)hydrazinocarbonylmethyloxy]-
2,8,14,20-tetrahiacalix[4]arene (Podyachev et al., 2011). In preparation of 25,26,27,28-
tetrakis[(2pyridinylmethylidene) hydrazinocarbonylmethyloxy]-2,8,14,20-tetrathiacalix[4]arene
by suspension tetrahydrazide in EtOH and DMF the picolinaldehyde then, hexane is added after
the solvent was removed from reaction mixture in distillation under vacuum (Podyachev et al.,
2011).
For synthesis of N´-(5-chloro-2-hydroxybenzylidene)-4-Dimethylaminobenzohydrazide using 5-
chlorosalicylaldehyde and 4-dimethylaminobenzohydrazide then, dissolved in ethanol under
room temperature for one hour (De-Suo, 2011). Besides that, De-Suo (2011) have reported that,
in synthesis N´-(2,4-Dichlorobenzylidene)-4-Dimethylaminobenzohydrazide is using 2,4-
dichlorobenzaldehyde and 4-dimethylaminobenzohydrazide (Figure 5). These two compounds
are air stable colourless block-shaped crystal and crystal form are soluble in methanol, ethanol,
acetonitrile and chloroform (De-Suo, 2011).
8
OH
Figure 5: N´-(5-chloro-2-hydroxybenzylidene)-4-Dimethylaminobenzohydrazide and N´-(2,4-
Dichlorobenzylidene)-4-Dimethylaminobenzohydrazide
In addition, to antitumor activities, metal complexes with Schiff bases have interesting structural
possibilities. From the literature survey, studies on transition metal complexes derived from o-
vanillin benzolhydrazone ligand containing ONO-donors are still lacking. For this reason, the
author has taken decision for the synthesis and characterization of 3d-transition metal complexes
with o-vanillin benzoylhydrazone and also to study their biological activity.
9
2.2 Cytotoxicity activity of transitional metal complexes of hydrazone ligand
Cytotoxicity is degree of agent being destructive to cell or being toxic. The toxicity is degree of
cell being poisonous. According to Agarwala et al., (2005), phenylglyoxal-bis(thiosemi-
carbazone) and anthracene-9-carboxaldehyde thiosemicarbazone inhibit the bacterial growth of B
Subtilis and E Coli. According to Mohareb et al., (2011), hydrazide-hydrazone derivatives were
able to inhibit the growth of the tested human tumor cell lines in dose manner. The human cell
lines representing different types name which are breast adenocarcinoma, non-small cell lung
cancer and cancer by using in vitro system. The ligand of [C19H15N3O2] derived from 2,6-
diaminopyridine and salicylaldehyde which is non-toxic to neuroblastoma cell but when formed
complex with Zn(II) ion complex give more toxic than in Cd(II) ion complex (Fadzel et al.,
2010). The toxicity can be study by used LD50 of rat and showed CuL give more value and L is
salicylidenesulfamethoxazole (Iqbal et al., 2008). Yon and Zheng-yin (2010) found that, 3-
carbaldehyde chrome-(benzoyl) hydrazone and its rare earth metal complexes exhibit good
antioxidant activity. The antioxidant can give anti-aging, anti-cancer and anti-cardiovascular
diseasese and antioxidant activities of 3-carbaldehyde chrome-(benzoyl) hydrazone and its
complexes were determined by superoxide and hydroxyl radical scavenging methods in vitro
(Yon and Zheng-yin, 2010). The cytotoxicity activity has also reported by Abbas and Farghlay
(2010) against human breast cancer (MCF-7) in vitro by using colon cancer cell line HCT-116,
liver carcinoma cell line HEPG2-1 and human breast cell line MCF-7.
10
CHAPTER 3
3.0 MATERIAL AND METHODS
3.1 Spectral characterization of complexes
This research was conducted in Inorganic Research Laboratory at UNIMAS. All chemicals were
purchased from Fluka, Aidrich or J.T. Baker. All solvents were purified according to standard
procedure (Armarego et al., 1996). CHN analyses, UV-visible, FT-IR and 1H NMR were used
for characterization of hydrazone ligand and its transition metal complexes. The CHN analysis
was recorded with Flash EA 1112 Series CHN elemental analyzer and electronic spectra were
recorded with methanol and dimethyl sulfoxide on a Perkin Elmer Lambda 25 UV-Visible
spectrometer. FT-IR spectra were recorded on KBr disc using Perkin Elmer Spectrum GX
Fourier-Transform (4000-400 cm-1
). 1H NMR spectra were recorded on JEOL 500 MHz-NMR
spectrophotometer with dimethyl sulfoxide. The molar conductance values were measured with
methanol solvent at room temperature using Jenway 4510 conductivity meter.
11
3.2 Synthesis of hydrazone ligand (1) and its 3d-transition metal complexes (2-7)
3.2.1 Synthesis of o-vanillin benzoylhydrazone (C15H14N2O3) (1)
Benzhydrazide (1.36 g, 0.01 mol) in 20 mL of absolute ethanol was added drop wise into 20 mL
of ethanolic solution of 2-hydroxy-3-methoxybenzaldehyde (1.52 g, 0.01 mol). The mixture was
stirred and refluxed for 4 hour (Scheme 1). The yellow microcrystalline solids formed were
filtered off and dried in vacuo over anhydrous silica gel. The yellow microcrystals were
recrystallized from ethanol and dried in vacuo over anhydrous silica gel. Yield: 2.14 g, 74 %,
m.p 192-194 0C.
CHO
OH
OCH3
NH2
Absolute ethanol
Refluxed 4 hoursOH
OCH3
Scheme 1: Synthesis o-vanillin benzoylhydrazone (1)
12
3.2.2 Synthesis of Mn(II) complex (2) with ligand (1)
The ligand (1) (1.35 g, 0.005 mol) was dissolved in absolute methanol (20 mL) into the reaction
flask. Then, a methanolic solution of manganese(II) acetate tetrahydrate (1.23 g, 0.005 mol) was
added drop wise into the reaction flask to produce green-yellow solution. The resulting reaction
mixture was refluxed 5 hours (Scheme 2) and cooled to room temperature. Yellow green
microcrystals were formed, filtered off and washed with cold heptane and dried in vacuo over
anhydrous silica gel. Yield 1.90 g, 74%, m.p 289-291 0C
OH
OCH3
Mn(CH3COO)2.4H2O
Absolute MeOHrefluxed 5 hour
OCH3
Mn
H2O
2CH3COOH
Scheme 2: Synthesis of [Mn(C15H12N2O3)H2O] (2)
13
3.2.3 Synthesis of Fe(II) complex (3) with ligand (1)
The ligand (1) (1.35 g, 0.005 mol) was dissolved in absolute methanol (20 mL) into reaction
flask. Then, a methanolic solution of iron(II) chloride.xH2O (0.63 g, 0.005 mol) was added drop
wise into the reaction flask to produce black solution. The resulting reaction mixture was
refluxed 5 hours (Scheme 3) and cooled to room temperature. Black microcrystals were formed,
filtered off and washed with cold heptane and dried in vacuo over anhydrous silica gel. Yield:
1.49 g, 75%, m.p 282-284 0C.
OH
OCH3
Absolute MeOHrefluxed 5 hour
OCH3
Fe
H2O
2HCl
FeCl2.xH2O
Scheme 3: Synthesis of [Fe(C15H12N2O3)H2O] (3)
14
3.2.4 Synthesis of Co(II) complex (4) with ligand (1)
The ligand (1) (1.35 g, 0.005 mol) was dissolved in absolute methanol (20 mL) into the reaction
flask. Then, a methanolic solution of Cobalt(II) chloride hexahydrate (1.19 g, 0.005 mol) was
added drop wise into reaction flask to produce chocolate solution. The resulting reaction mixture
was refluxed 5 hours (Scheme 4) and cooled to room temperature. Chocolate microcrystals were
formed, filtered off and washed with cold heptane and dried in vacuo over anhydrous silica gel.
Yield: 1.68 g, 66%, m.p. 216-218 0C.
OH
OCH3
Absolute MeOHrefluxed 5 hour
OCH3
Co
H2O
2HCl
CoCl2.6H2O
Scheme 4: Synthesis of [Co(C15H12N2O3)H2O] (4)