UNIVERSITI PUTRA MALAYSIA

SYNTHESIS, AND PHYSICO-CHEMICAL AND BIOLOGICAL CHARATERIZATION OF NEW SCHIFF BASES DERIVED FROM

THIOPHENE AND THEIR TRANSITION METAL COMPLEXES

MD.UWAISULQARNI OSMAN

FS 2007 8

SYNTHESIS, AND PHYSICO-CHEMICAL AND BIOLOGICAL CHARATERIZATION OF NEW SCHIFF BASES DERIVED FROM THIOPHENE AND THEIR TRANSITION METAL COMPLEXES

By

MD.UWAISULQARNI OSMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Master of Science

April 2007

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

SYNTHESIS, AND PHYSICO-CHEMICAL AND BIOLOGICAL

CHARATERIZATION OF NEW SCHIFF BASES DERIVED FROM THIOPHENE AND THEIR TRANSITION METAL COMPLEXES

by

MD.UWAISULQARNI BIN OSMAN

April 2007

Chairman: Mohamed Ibrahim Mohamed Tahir, PhD Faculty: Science Several new Schiff bases formed from S-benzyldithiocarbazate (SBDTC) and S-

methyldithiocarbazate (SMDTC) with selected aldehyde/ketones containing a thiophene

ring namely, 1-(2-thienyl)-1-propanone and thiophene-2-carbaldehyde in 95 % of ethanol

have been synthesized. Complexes of Cobalt(III), Nickel(II), Copper(II), Zinc(II) and

Cadmium(II) with these Schiff bases were prepared. These compounds were

characterized by elemental analyses and various physico-chemical techniques. Their

thermal behaviour was also investigated. Single crystal X-ray analysis was carried out on

nine single crystals. The Co(III) complexes are six-coordinated whereas, Ni(II), Zn(II)

and Cd(II) complexes are four-coordinated. Unfortunately, none of the Cu(II) complexes

produced single crystals but, it is proposed, that they are also four-coordinated. The

Schiff bases and their metal complexes were evaluated for their cytotoxic and

antimicrobial activities. Cytotoxic screening was carried out against Human ovarian

cancer cells (CaOV-3), Human breast carcinoma cells with negative estrogen receptor

(MDA-MB-231) and Human liver carcinoma cells (HEP-G2). Antimicrobial screening

II

was carried out against four bacteria and three fungi. All compounds were found to have

low or no activity except for Cu(SBT2P)2. H2O which is active against Bacillus subtilis-

wild type (B29).

III

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

SINTESIS, DAN FIZIKAL-KIMIA SERTA KAJIAN SECARA BIOLOGI TERHADAP BES SCHIFF TERBITAN DARIPADA THIOPHENE DAN

KOMPLEK LOGAM PERALIHAN MASING-MASING

Oleh

MD.UWAISULQARNI BIN OSMAN

April 2007

Pengerusi: Mohamed Ibrahim Mohamed Tahir, PhD Fakulti: Sains Beberapa bes Schiff baru telah berjaya disintesiskan daripada S-benzildithiokarbazat

(SBDTC) dan S-metildithiokarbazat (SMDTC) yang bertindak balas dengan

aldehid/keton terpilih yang ia mengandungi gelang thiophen iaitu, 1-(2-thienil)-1-

propanon and thiophen-2-carbaldehid dengan menggunakan pelarut etanol (95 %).

Pelbagai komplex Cobalt(III), Nikel(II), Kuprum(II), Zink(II) dan Cadmium(II) juga

berjaya disediakankan dengan menggunakan bes Schiff tersebut. Kesemua sampel telah

dicirikan dengan menggunakan pelbagai teknik kimia-fizik. Kajian terhadap sifat

termanya juga telah dijalankan. Analisis sinar-X telah dijalankan terhadap sembilan

hablur tunggal. Didapati bahawa komplek Co(II) berkoordinasi enam manakala, Ni(II),

Zn(II) dan Cd(II) adalah berkoordinasi empat. Malangnya, tiada hablur tunggal dapat

dihasilkan daripada Cu(II) tetapi adalah dicadangkan ia mempunyai struktur empat

koordinasi berdasarkan maklumat yang diperolehi. Bes Shiff dan komplek logamnya

telah diuji untuk menilai aktiviti antimikrob dan sitotoksik. Saringan sitotoksik telah

dijalankan dengan menggunakan CaOV-3 (Sel kanser ovari manusia), MDA-MB-231

IV

(Sel barah payu dara dengan penerima estrogen negatif) and HEP-G2 (Sel barah hati

Manusia). Saringan antimikrob telah dijalankan terhadap empat bakteria dan fungi

terpilih. Ujian biologi telah menunjukkan bahawa kesemua sampel yang disintesiskan

tidak memberi sebarang aktiviti kecuali Cu(SBT2P)2. H2O, di mana ia aktif menentang

Bacillus subtilis-wild type (B29).

V

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to my supervisors Dr. Mohamed Ibrahim

Mohammed Tahir and Professor Dr. Karen A. Crouse for guidance, comment,

encouragement and unlimited help throughout the duration of my studies.

I would also like to express my appreciation to Prof. Bohari M. Yamin, Universiti

Kebangsaan Malaysia and Dr. Andrew R. Cowley, University of Oxford for their

contributions to the single crystal X-ray analyses. Also, my thanks are extended to Dr.

Rozita Rosli and Mr. Nasir Umar Tsafe from the Faculty of Medicine and Health Science,

UPM for their help in the cytotoxicity studies. My appreciation is also forwarded to Kak

Nora from the Molecular and Cell Biology Laboratory, Institute of Bioscience, Universiti

Putra Malaysia for running the antimicrobial tests.

I would like to thank the Ministry of Science, Tecnology and Innovation of Malaysia for

the funding of this project under the Intensification of Research in Priority Areas Scheme

(IRPA grant no: 09-02-04-0755-EA001). Many thanks to Universiti Putra Malaysia for

rewarding the Pasca Scholarship.

VI

I would like to thank to all my labmates, Khoo Teng Jin, Chew Kar Beng, Eddy Chan

Mun Hoe , Fiona How Ni Foong, Thahira Begum (my grammar lady ☺), Norazlinaliza,

Tan Sang Loon and Abdul Fatah. Without Khoo Teng Jin, three crystals would not have

been analyzed. There were done at Oxford during his attachment programme. Without

Thahira, I would not have been able to submit this thesis to my supervisor and co-

supervisor without many grammaritical errors. Furthermore, without my other labmates I

would have been unables to have had life full of laugher through my stay here.

I would also like to express my gratitude and appreciation to my parents, Osman bin

Napiah and Bariah binti Marpan and all my brothers and sisters Nasir, Nasri, Nazri,

Risnawaty, Siti Aishah and Siti Norazizah. Last but not least , I would like to express my

love to Asmaa Azyaati binti Jamaluddin for her patience and support.

VII

This thesis submitted to the Senate of Univesiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Mohamed Ibrahim Mohamed Tahir, D.Phil Lecturer Faculty of Science Universiti Putra Malaysia (Chairman) Karen Badri Anne Crouse, PhD Professor Faculty of Science Universiti Putra Malaysia (Member) ______________________

AINI IDERIS, PhD Profesor/Dean School of Graduate Studies,

Universiti Putra Malysia

Date: 14 JUNE 2007

VIII

IX

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: Mohamed Ibrahim Mohamed Tahir, D. Phil. Lecturer Faculty of Science Universiti Putra Malaysia (Chairperson) Karen Anne Crouse, Ph.D Professor Faculty of Science Universiti Putra Malaysia (Member)

_______________________ AINI IDERIS, PhD Profesor/Dean School of Graduate Studies

Universiti Putra Malysia

Date:14 JUNE 2007

X

DECLARATION

I hereby declare that the thesis is based on my original work except for the quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

__________________________ MD.UWAISULQARNI BIN OSMAN Date: 4 MAY 2007

XI

TABLE OF CONTENTS

Page

ABSTRACT II ABSTRAK IV ACKNOWLEDGEMENTS VI APPROVAL VIII DECLARATION XI LIST OF TABLES XIV LIST OF FIGURES XV LIST OF REACTION SCHEMES XVII LIST OF ABBREVIATIONS XVII

CHAPTER

I INTRODUCTION 1 Research Problems 10

Objectives 10

II LITERATURE REVIEW 11 III METHODOLOGY 27

Preparation of Substituted Dithiocarbazate Compounds 28 S-benzyldithiocarbazate (SBDTC) 28 S-methyldithiocarbazate (SMDTC) 29

Preparation of Schiff Bases 29 N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic

acid phenyl ester (SBT2P) 29 N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid

phenyl ester (SBT2C) 29 N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic

acid methyl ester (SMT2P) 30 N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid methyl ester (SMT2C) 30

Preparation of Metal Complexes 31 Preparation of Metal Complexes of SBT2P, SBT2C, SMT2P and SMT2C 31

Physico-Chemical Analysis 32 Melting Point Determination 32 Carbon, Hydrogen, Nitrogen and Sulfur (CHNS) Analyses 32 Inductively Coupled Plasma-Atomic Emission

Spectroscopic Analyses (ICP-AES) 32 Mass Spectrometric Analyses 33

XII

Nuclear Magnetic Resonance (1H and 13C NMR) Spectroscopic Analyses 33 Fourier Transform-Infrared (FT-IR) Spectroscopic Analyses 33 Molar Conductivity Measurements 33 Magnetic Susceptibility Measurements 34 Ultraviolet/Visible (UV-Vis) Spectroscopic Analyses 34 Single Crystal X-Ray Diffraction 34 Thermogravimetric Analysis (TGA) and Derivative Thermogravimetric (DTG) Analyses 36 Differential Thermal Analysis (DSC) 36

Biological Activity Studies 36 Cytotoxic Assays 36 Qualitative Antimicrobial Assays 37 Quantitative Antimicrobial Assays 38

IV RESULTS AND DISCUSSION 39 Spectroscopic Characterization of the Ligands (SBT2P, SBT2C, SMT2C and SMT2P) and Their Metal Complexes 39

V CONCLUSION 102 REFERENCES 105 APPENDICES 116 BIODATA OF THE AUTHOR 210

XIII

LIST OF TABLES

Table Page

1 Analytical data and physical properties of the Schiff bases and their metal complexes. 41

2 ICP-AES data of the metal complexes 43

3 1H NMR data of the Schiff bases 50

4 13C NMR data of the Schiff bases 51

5 Infrared data of the ligands and metal complexes 55

6 Magnetic susceptibility and molar conductivity measurements of the metal complexes 58

7 Electronic Spectral data of the Schiff bases and their metal complexes 61

8 Comparison of selected bond lengths in the dithiocarbazate Schiff bases derived from thiophene. 67

9 Comparison of selected bond lengths in metal complexes of dithiocarbazate Schiff bases derived from thiophene 77

10 Thermal data of Schiff bases and their metal complexes based on TGA ,DTG and DSC curves 86

11 Qualitative antimicrobial assay results 97

12 Quantitative antimicrobial assay results 98

13 Cytotoxic activity 100

XIV

LIST OF FIGURES

Figure Page

1 Bond lengths (Å) and angles ( ° ) in of thiophene ring 1

2 Structure of bidendate ligand 5

3 Structure of multidendate ligand 5

4 Structure of S-benzyldithiocarbazate (SBDTC) 5

5 Structure of S-methyldithiocarbazate (SMDTC) 5

6 Structure of Cisplatin 8

7 Structure of square-based pyramidal complex metal with carbine cluster 12

8 Structure of octahedral complex 12

9 Structure of double butterfly cluster 12

10 Structure of [Cu(Lm)2] 13

11 Structure of [Ni(Lm)2] 13

12 Structure of SnPh2Cl(tctsc) 14

13 Structure of SnCl2(tctsc)2 14

14 The packing of the molecules of SBDTC viewed down the b axis 17

15 Different conformers exist in structure of SMDTC 17

16 Conformation of S-methyl dithiocarbamate 18

17 Tautomeric form of Schiff base derived from dithiocarbazate 18

18 Possible ligand to metal coordination modes 19

19 Structure of [Cd(dpksbz)NCS]2 21

20 Structure of Sn(SNNNS)I2 22

21 Structure of Zn2(SNNNS)2 22

XV

22 Different sizes of the cyclic rings for the thiosemicarbazone 24

23 General structure of new dithiocarbazate Schiff bases derived from thiophene 39

24 Structure of N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic acid phenyl ester (SBT2P) 44

25 N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid phenyl ester (SBT2C) 44

26 Structure of N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic acid methyl ester (SMT2P) 44

27 Structure of N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid methyl ester (SMT2C) 45

28 Schiff bases of 2,6-diacetylpyridine with S-benzyldithiocarbazate and S-methyldithiocarbazate 46

29 Tautomeric forms of thiophene containing Schiff bases derived from dithiocarbazate 47

30 Coordination sites of NS Schiff bases 52

31 ORTEP diagram of SBT2P with 50% probability displacement ellipsoids and the atomic numbering scheme 63

32 Packing arrangement of SBT2P 63

33 ORTEP diagram of SBT2C with 50% probability displacement ellipsoids and the atomic numbering scheme 65 34 ORTEP diagram of SMT2P with 50% probability displacement

ellipsoids and the atomic numbering scheme 67

35 ORTEP diagram of Zn(SBT2P)2 with 50% probability displacement ellipsoids and the atomic numbering scheme 69

36 ORTEP diagram of Ni(SBT2P)2 with 50% probability displacement ellipsoids and the atomic numbering scheme 69

37 ORTEP diagram of Ni(SMT2P)2 with 50% probability displacement ellipsoids and the atomic numbering scheme 72

XVI

38 ORTEP diagram of Cd(SMT2P)2 with 50% probability displacement ellipsoids and the atomic numbering scheme 72

39 ORTEP diagram of Co(SMT2C)3 with 50% probability displacement ellipsoids and the atomic numbering scheme 74

40 ORTEP diagram of Zn(SMT2C)2 with 50% probability displacement ellipsoids and the atomic numbering scheme 76

LIST OF REACTION SCHEMES

Scheme Page

1 Formation of thiophene-S-oxide as a primary reactive metabolite 3

2 Preparation of Ru (II) and Pd(II) complexes of 5-nitrothiophene-2-carboxaldehyde thiosemicarbazones 14

3 General equations for the reduction process of Cu(I) from thiosemicarbazonecopper(II) 26

4 General equation for reaction of the Dithiocarbazate Schiff bases 48

5 A mechanism for the formation of Dithiocarbazate Schiff bases 49

6 Postulated for the first decomposition of the metal complexes 85

LIST OF ABBREVIATIONS

A.ochraceous Aspergillus ochraceous (398)

B.Subtilis Bacillus subtilis- wild type (B29)

C.albicans Candida albicans (C.A)

CaOV-3 Human Ovarian Cancer cell

CD50 Cytotoxic dose at 50%

CEM-SS Human cell T-lymphoblastic leukemia

XVII

CGCs cerebella granule cells

CHNS Carbon, Hydrogen, Nitrogen and Sulfur analyses

DMF Dimethylformamide

DMSO-d6 Deuterited -dimethylsulphoxide

DNA Deoxyribonucleic acid

dsDNA doubly stranded Deoxyribonucleic acid

DTG Derivative Thermogravimetric

FT-IR Fourier Transform-Infrared

GC-MS Gas Chromatography- Mass Spectrometer

HEP-G2 Human Liver carcinoma cells

HL60 Human Tlymphoblastic leukemic cells

HT-29 Human colon adenocarcinoma cells

ICP-AES Inductively coupled plasma-atomic emission spectroscopy

MDA-MB-231 Human breast carcinoma cells with negative estrogen receptor

MIC Minimum inhibitory concentration

MRSA Methicillin resistant Staphylococcus Aureus

NMR Nuclear magnetic resonance

ORTEP Oak Ridge Thermal Ellipsoid Program

P.aeruginosa Pseudomonas aeruginosa (60690)

S.cerivisiae Saccharomyces cerivisiae (20341)

S.typhimurium Salmonella typhimurium

SBDTC S-benzyldithiocarbazate

SBT2C N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid phenyl ester

XVIII

XIX

SBT2P N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic acid phenyl ester

SMDTC S-methyldithiocarbazate

SMT2C N’-Thiophen-2-ylmethyl-hydrazinecarbodithioic acid methyl ester

SMT2P N’-(1-Thiophen-2-yl-propyl)-hydrazine carbodithioic acid methyl ester

TGA Thermogravimetric analysis

UV-Vis Ultraviolet/Visible

CHAPTER I

INTRODUCTION

In recent years, considerable attention has been paid to the metal complexes due to the

importance in understanding the effect of metal complexes in biological activity.

Modification of the structure of the metal complexes by coordinating with different

organic compounds has been carried out to enhance the potential use as drugs. However,

studies related to drug design are limited and knowledge of the physico-chemical

characteristics of potentially active compounds are required. Considering the wide scope

of the subject, present research is restricted to the thiophene derivatives of

dithiocarbazate with five transition metals, namely, cobalt(III), nickel(II), copper(II),

zinc(II) and cadmium(II) and their biological activities.

Structure of the Thiophene ring

The structure of thiophene ring itself has been determined by previous reports [1] and is

as shown in the figure below:

Figure 1: Bond lengths (Å) and angles ( ° ) in of thiophene ring.

1

History of thiophene compounds

The history of thiophene has been documented [2] and is summarized as follows:

In 1844, Laurent synthesized thionessal (tetraphenylthiophene) but the structure of that

compound was not able to be determined until Victor Meyer understood the properties of

the ‘unknown’ compound.

Victor Meyer used 250L of benzene with 25L of concentrated sulfuric acid. The “black-

acid” produced by this process was converted into the lead salt and dry distillation of this

salt with ammonium chloride gave a “crude thiophene”. The thiophene formed from this

treatment, 140g from a 2660g portion of the lead salt, was found to boil at 84oC and

contain about 28% sulphur (the sample was about 70% thiophene and 30% benzene). A

portion, 70g was brominated and a dibromo derivative, which was found to be C4H2Br2S,

was obtained. Victor Meyer then could surmise that the original material, before

bromination, was C4H4S. The dibromothiophene resisted dehydrobromination in boiling

alcoholic potassium hydroxide and the bromo groups could not be removed by reduction.

Its properties were similar to those of the bromobenzenes and Victor Meyer quickly

grasped its aromatic nature.

The history of the naming the C4H4S compound was described by Thorpe. Firstly, Meyer

suggested the name thianthren, then thiophan, next thiol and finally thiophene, meaning

that it was a sulphur-containing compound giving derivatives to those of the phenyl series.

2

Biological studies of the thiophene ring system

As certain physical and chemical properties of benzene and thiophene were similar,

researchers studied biological activity of the thiophene ring, based on the activity of the

benzene ring. Some research was also carried out on furan rings, analogues of the

thiophene ring, the only difference being the substitution of the sulphur by the oxygen

atom [3, 4].

In 1992 [5], the initial research on biological activity of the thiophene ring was reported

and that thiophene-S-oxides were formed intermediately in vivo and acted as reactive

metabolites. A mechanism was proposed as follows:

S

S

O

O

SSCH2CH

COOH

NHCOCH3

S SG

O

SSCH2CH

COOH

NHCOCH3

rats

GSH

1

2

Heat

GSH = glutathione

1 = dihydrothiophene sulfoxide mercapturate

2 = N-acetyl-S-(2-thienyl)-L-cysteine

Scheme 1: Formation of thiophene-S-oxide as a primary reactive metabolite

3

From the scheme above, it was found that the metabolite 1 was present in urine of rats

after treatment with thiophene. It was also suggested that metabolite 1 was derived from

the reactive thiophene sulfoxide intermediate and reacted with glutathione. Finally,

transformation of 2 from 1 was through a dehydration reaction, a reaction well known for

hydrothiophene sulfoxide.

Remarkably, research has revealed that thiophene itself can cause death of cerebella

granule cells (CGCs) in presence of rat liver postmitochondrial (S9) fraction as a source

of biotransformation enzyme [6]. It was believed that the metabolism of thiophene was

the same as reported previously, where thiophene was converted to reactive thiophene-S-

oxide through oxidation [5]. However, damage due to interaction between doubly

stranded deoxyribonucleic acid (dsDNA) with thiophene-S-oxide can be detected by

using an electrochemical DNA-biosensor [7].

Dithiocarbazate Derivatives

In metal complexes containing Schiff bases, the environment at the coordination center

can be modified by attaching different substituents to the ligand for the fine-tuning of

structure and reactivity [8, 9, 10, 11, 12]. Research has shown that different ligands show

different biological properties even with slight modifications in their molecular structure.

Dithiocarbazates, NH2NHCS2-, react with aldehydes or ketones via a condensation

reaction to produce a dithiocarbazate Schiff base. The Schiff bases are reacted with

4

transition metal salts, bonding through the mercaptide sulphur and azomethine nitrogen

atoms to form a bidentate ligand (Figure 2) [13], although in some cases they can behave

as multidentate ligands (Figure 3), bonding through the sulphur and two nitrogen atoms,

depending on the nature of the aldehyde or ketone used [9].

Figure 2: Structure of Figure 3: Structure of bidendate ligand multidendate ligand

Dithiocarbazates, such as S-benzyldithiocarbazate, SBDTC (Figure 4) [14], S-

methyldithiocarbazate, SMDTC (Figure 5) [15] and their Schiff bases have potential as

antimicrobial and anticancer agents. Furthermore, the activity of these compounds is

sometimes enhanced by the presence of coordinated transition metal ions.

H2N

HN S

S

H2N

HN S

CH3

S

Figure 4: Structure of Figure 5: Structure of S-benzyldithiocarbazate S-methyldithiocarbazate (SBDTC) (SMDTC)

5