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)