1

SYNTHESIS AND ANTIMICROBIAL STUDIES OF

IRON (III) COMPLEXES OF CIPROFLOXACIN,

PENICILLINS AND ISATIN DERRIVATIVE

BY

EZE FABIAN IFEANYI

PG/ M.SC/07/43054

DEPARTMENT OF PHARMACEUTICAL AND

MEDICINAL CHEMISTRY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA

MARCH, 2011

2

Synthesis and Antimicrobial Studies of Iron (III) Complexes

of Ciprofloxacin, Penicillins and Isatin Derivative

By

Eze Fabian Ifeanyi

PG/ M.Sc/07/43054

Department of Pharmaceutical and Medicinal Chemistry

Faculty of Pharmaceutical Sciences

University of Nigeria, Nsukka

March, 2011

3

Synthesis and Antimicrobial Studies of Iron (III) Complexes of

Ciprofloxacin, Penicillins and Isatin Derivative

By

Eze Fabian Ifeanyi

PG/ M.Sc/07/43054

A research project report submitted to the Department of Pharmaceutical and

Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Nigeria,

Nsukka in partial fulfillment of the requirements for the award of Master of Science

degree (M.Sc) in Medicinal Chemistry.

Project Supervisor: Dr. U. Ajali

March, 2011

DEDICATION

To the sick and suffering

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ACKNOWLEDGEMENT

I am grateful to the almighty God for seeing me through this tedious

research work. A lot of people had been God’s instrument in this work. This work

was supervised by Dr. U. Ajali. His friendly advices created the enabling

environment for the work. I thank him immensely. Dr. P.O. Ukoha of the Dept. of

Pure and Industrial Chemistry provided a lot of technical and material support. I

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appreciate him a lot. I am indebted to Juhel (Pharm) Nig. Ltd. Enugu for donating

pure samples of Ciprofloxacin and Penicillins. Dr. C.J. Mbah, my H.O.D,

recommended me to Juhel for assistance. I thank him.

This work was sponsored by my mother, Mrs. V. Eze, and my sister,

Uchenna, may God bless them abundantly. Mr. G.C. Ebi, Dr. F.B.C. Okoye and

Pharm. E.O. Omeje made a lot of useful suggestions. My sister, Nkechi was a

source of motivation. She inspired me into the P.G. programme. I thank my

brothers, Emeka and Ikechukwu, my sisters; Chinonyelum and Chinedu for their

encouragement and prayers, and my colleagues; Sule, Amaka, Ifeyinwa, and

Rosy for keeping me company.

CERTIFICATION

The work embodied in this project report is an original investigation and

has not been submitted in part or full for any other diploma or degree of this or

any other university.

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………………………………… ………………………….....

Dr. (Mrs.) N. J. Nwodo Dr. U. Ajali

Head of Department Supervisor

………………………………………………

External Examiner

ABSTRACT

Complexation is very relevant in pharmacy as a means of modifying the

pharmacological, toxicological and physico- chemical properties of drugs.In this

study, iron (III) complexes of ciprofloxacin, ampicillin, amoxicillin, and

cloxacillin were synthesized through systematic choice of solvents and reaction

conditions. A Schiff base derived from Isatin (Indole-1,2-dione) and

ethylenediamine, and its iron (III) complex were also synthesized. The aqueous

solubility profiles of the complexes were determined. The complexes showed

improved aqueous solubility when compared to those of the corresponding

ligands. Relative thermal and acid stabilities were determined

spectrophotometrically. Results showed that the complexes have enhanced

thermal and acid stabilities with respect to the pure ligands. Antimicrobial studies

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carried out on the complexes showed that the complexes have decreased

antimicrobial activities against most of the microorganisms used. Ciprofloxacin

complex, however, showed almost the same activity as the corresponding ligand.

The iron (III) complex of the Schiff base derived from isatin and ethylenediamine

showed enhanced antibacterial activity against B.subtilis and S. aureus, but less

activity against P. aeruginosa, E. coli, and Salmonella typhi. Job’s method of

continuous variation and the approximate molecular weight of the complexes,

determined by measurement of boiling point elevation (ebullioscopy), suggested a

1:2 metal to ligand stoichiometry for most of the complexes). The structures of

the complexes were proposed based on the stoichiometry, infrared and

ebullioscopic data and assuming a coordination number of 6 or 4 for iron.

TABLE OF CONTENTS

Title page i

Dedication ii

Acknowledgement iii

Certification iv

Abstract v

CHAPTER ONE: GENERAL INTRODUCTION 1

1.1 Introduction 1

1.2 Complexes 3

1.2.1 Definition of Complex 3

1.2.2 Types of Complexes 5

1.2.3 Chemistry of complexes 8

1.2.3.1 Structure and bonding in metal ion (Coordination) Complexes 8

1.2.3.2 Methods of Studying the structure of Complexes 10

1.2.3.3 Theories and bonding in Transition metal complexes 11

1.2.3.4 IUPAC Nomenclature of Coordination Compounds 12

1.2.3.5 Isomerism in Transition metal Complexes 14

1.2.4 Reaction Conditions Favourable for Complex Formation 15

8

1.2.5 Complex Stability 17

1.2.5.1 Measurement of Complex Stability 17

1.2.6 Methods of Analysis and Determination of Complexes 20

1.3 Antibiotics 20

1.3.1 Definition of Antibiotics 21

1.3.2 Classes of Antibiotics 21

1.3.2.1β-lactam Antibiotics 21

1.3.2.2 Non- lactam Antibiotics 25

1.4 Medicinal Chemistry of the Essential Trace Elements 29

1.4.1 Iron 29

1.5 Aim of Study 30

CHAPTER TWO: MATERIALS AND METHODS 31

2.1 Equipment 31

2.2 Reagents and Solvents 31

2.3 Microorganisms 31

2.4 Synthesis of the Complexes 31

2.5 Ebullioscopic Determination of Molecular Weight of the Complexes 33

2.6 Job’s Method of Continuous Variation 33

2.7 Determination of some Physicochemical Parameters 34

2.7.1 Aqueous Solubility 34

2.7.2 Thermal and Acid Stabilities 34

2.8 Determination of melting Point

2.9 Spectral. Analysis

2.9.1 UV- Visible Spectral Analysis

2.9.2 Infrared Spectral Analysis

2.10 Antimicrobial Studies on the Complexes

2.10.1 Preparation of the Culture Media 2.10.2

Maintenance and Activation of the Microorganisms 2.10.3

Microbiological Sensitivity Screening Test

2.10.4 Determination of Minimum Inhibitory Concentration (MIC)

CHAPTER THREE: RESULTS 37

3.1

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CHAPTER FOUR: DISCUSSION AND CONCLUSION

4.1 Possible Synthetic Reaction 54

4.2 Antimicrobial Results 57

4.3 Thermal and Acid Stability 57

4.4 Infrared Spectrophotometrical Data 58

4.5 Conclusion 60

References 61

LIST OF TABLES AND FIGURES

Table 1.1 Pharmaceutical Properties affected by complexation 1

Table 1.2 Drug-Metal Complexes and their Properties 2

Table 1.3 Classification of Molecular Complexes 6

Table 1.4 PH Ranges for Metal –EDTA Titration 16

Table 1.5 Some semi-synthetic Penicillins 22

Table 3.1 Percentage Yield of the Complexes 37

Table 3.2 approximate Molecular weight of the Complexes 37

Table 3.3 Aqueous Solubility 38

Table 3.4 Relative Thermal Stability of the Complexes 38

Table 3.5 Relative Acid Stability of the Complexes 38

Table 3.6 Result of sensitivity Screening 39

Table 3.7 Minimum Inhibitory Concentrations 39

Table 3.8 Result of Job’s Method of Continuous Variation for Ciprofloxacin Complex 40

Table 3.9 Result of Job’s Method of Continuous Variation for Cloxacillin Complex 40

Fig. 3.1 Job’s Plot for Ciprofloxacin Complex 41

Fig. 3.2 Job’s Plot for Cloxacillin Complex 42

Fig 3.3 UV- Visible Spectrum of Ciprofloxacin 43

Fig 3.4 UV-Visible Spectrum of Fe3+ complex of Ciprofloxacin 44

Fig 3.5 UV-Visible Spectrum of Amoxicillin Complex 45

Fig 3.6 UV-Visible Spectrum of Fe3+ Complex of Cloxacillin 46

Fig 3.7 UV-Visible Spectrum of Fe+ Complex of Isatin Schiff base 47 Fig

3.8 UV-Visible Spectrum of Ferric Chloride 48

Fig 3.9 Infrared Spectrum of Ciprofloxacin 49

Fig 3.10 Infrared Spectrum of Fe3+ Complex of Ciprofloxacin 50

Fig 3.11 Infrared Spectrum of Fe3+ Complex of Cloxacillin 51

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Fig 3.12 Infrared Spectrum of Fe3+ Complex of Amoxicillin 52

Fig 3.13 infrared Spectrum of Fe3+ Complex of Isatin Schiff base 53

CHAPTER ONE

GENERAL INTRODUCTION

1.1 INTRODUCTION

Many drugs possess modified pharmacological, toxicological and physicochemical

properties when administered in the form of metal complexes. (Guangguo et al, 2003). It is

obvious that these complexes must possess some properties that are different from those of

their constituents; otherwise there would be no evidence for their existence. Among the

properties that may be altered upon complex formation are solubility, energy absorption,

conductance, partitioning behaviour and chemical reactivity. (Guangguo et al, 2003).

Some of the properties of drugs are so pertinent to dosage forms and drug delivery that

they are identified as pharmaceutical or biopharmaceutical properties. Complex formation may

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affect these properties adversely or sometimes favourably. Many of these pharmaceutical

properties, affected by complexation, with corresponding examples of drug complexes are

given in table 1.1.

Table 1.1 Pharmaceutical Properties Affected by Complexation

Property Example (Connors, 2000)

1. Physical state Nitroglycerin-cyclodextrin

2. Volatility Iodine-PVP

3. Solid-state stability Vitamin A-cyclodextrin

4. Chemical stability Benzocaine-caffeine

5. Solubility Aspirin-caffeine

6. Dissolution rate Phenobarbital-cyclodextrin

7. Partition coefficient Benzoic acid-caffeine

8. Permeability Prednisone-dialkylamides

9. Absorption rate Salicylamide-caffeine

10. Bioavailability Digoxin-cyclodextrin

11. Biological activity Indomethacin-cyclodextrin

Table 1.2 Drug – metal Complexes and their Properties

Properties Drug-metal Complexes

Improved antibacterial activity, water Co(II), Ni(II), Cu(II) and Zn(II)

solubility and solid state stability complexes of 3-salicylidenehydrazono-2-

indolinone

Solubility, stability and light absorption Cu2+ complex of ciprofloxacin

(Guangguo, 2003)

Antifungal activity Co2 +-bisisatinthiocarbohydrazone (Satisha, 1999)

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For many systems, it has been shown that the complex provides faster dissolution and greater

bioavailability than does the physical mixture. The processing characteristics (physical state,

stability, flowability, etc.) of the complex also may be better than those of the free drug (Connors,

2000).

Not all complexation is intentional or desirable, and some dosage form incompatibilities may

be the result of unwanted complexation reactions. For examples, some widely used polyethers

(Tweens, Carbowaxes or PEGS) can form precipitates with H- bond donors such as phenols and

carboxylic acids. A substance used widely in liquid dosage forms as a complexer of metal ions is

EDTA. The purpose of this application of complexation is to improve drug stability by inhibiting

reactions (usually oxidations) that are catalyzed by metal ions, the complexed form of the metal ion

being catalytically inactive. Citric acid (in the form of citrate anion) also is used for this purpose.

(Connors, 2000).

Drug complexation experiments could help medicinal chemists to predict some dosage

form incompatibilities, explain the mode of action of some drugs as well as devise new methods of

drug analysis. Formation of coordination complexes provides the basis for many analytical

methods for determination of metals in food, drug and biological systems (e.g. complexiometric

titration). Very low concentration of metal ions can also be determined spectrometrically by

complexation with a ligand that produces spectral change, (e.g. colorimetric analysis).The ferric

hydroxamate method for the detection and determination of carboxylic acid derivatives is another

important application of complexation in pharmaceutical analysis. Here, a carboxylic acid

derivative is reacted with hydroxyl amine to form the corresponding hydroxamic acid. An excess

of Fe (III) is added, and this forms a red-violent coordination complex with the hydroxamic acid

and the concentration of the complex determined spectrometrically (Sandell E.B, 1989).

Complexation is a useful means of studying the protein-binding of drugs. Some of the

constituents of the blood (e.g. HSA are capable of forming complexes with drugs and this has

important practical implication. Drug distribution and clearance are affected by its binding

characteristics. Complexes occur widely in biological systems, so the application of complex

formation processes in therapy is a reasonable approach to drug design. Among the most important

biological manifestations of complexation are many metal-ion coordination complexes whose

study constitutes a large part of bioinorganic chemistry. Examples of these complexes include:

haemoglobin (iron), cytochrome (iron), vitamin B12 (cobalt), carboxypeptidaseA (zinc) etc.

Molecular complexation in biological systems also occurs, as in DNA base pairing and stacking

interactions. Numerous antimicrobial and anti-neoplastic agents are believed to exert their action

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by means of complex formation with DNA base pair (Intercalation), e.g. daunorubicin and

actinomycin D.

Many toxic effects of excessive metal-ion concentration can be treated by agents that

form strong coordination complexes, via chelation, thus aiding the excretion of the metal. Among

the metals whose toxicity can be treated by chelation therapy are lead, copper, cobalt, mercury,

nickel, iron and zinc. Coordination compounds could also release valuable trace elements needed

for maintenance of life when they are administered as drugs.

Complexation should, therefore, be given adequate attention in pharmaceutical research.

1.2 COMPLEXES

1.2.1 DEFINITION OF A COMPLEX

A complex is a chemical specie formed by the association of two or more interacting

molecules or ions (Connors, 2000). The term complex is also used to describe a substance

composed of two or more substances, each of which is capable of an independent existence,

bound by forces weaker than the regular chemical bonds. (Cotton et al, 1999)

A complex is also a species of definite substrate to ligand stoichiometry that can be formed in

equilibrium process in solution, and also may exist in the solid state. (Connors, 2000)

It should be noted that complexes are not formed with classic covalent bonds.

Some Basic Terms Associated with Complexes

Substrate: This is the reactant, usually an electron pair acceptor (Lewis acid) whose physical or

chemical properties are observed experimentally.

Ligand: The second interactant, usually an electron pair donor (Lewis base) which donates the pair of

electron used for bonding.

Chelate: (from chelos; Greek word for Crab) is a ring structure formed when the ligand has more than

one binding site and occupies more than one coordination position.

Chelated complexes are more stable than similar complexes with unidentate ligands as

dissociation of the complex involves breaking many bonds rather than one. The more rings that

are formed, the more stable the complex is. (Lee, 1996). Chelating agents with three, four and

six donor atoms are known and are termed tridentate, tetradentate and hexadentate ligands

respectively.

Some common polydentate ligands (chelating agents) are listed below.

HOO.CH2

HOO.CH2 N –CH2 –CH2 – N

CH2.COOH

CH2.COOH

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Ethylenediamine tetracetic acid (EDTA)

O-

O

H

C

Several chelate complexes are of biological importance. Haemoglobin in the red blood

cell contains an iron-porphyrin complex. Chlorophyl in green plants contains a magnesium-

porphyrin complex. Vitamin B12 is a cobalt complex and the cytochrome oxidase enzymes

contain iron and copper. The body contains several materials which could form chelate

compounds with metals, for example adrenaline, citric acid and cortisone. Metal poisoning by

lead, copper, iron, chromium and nickel results from these metals forming weak unwanted

complexes, thus preventing normal metabolism. For this reason, dermatitis from chromium or

nickel salts is treated with EDTA cream. Lead and copper poisoning are treated by drinking an

aqueous solution of EDTA which complexes with the unwanted lead or copper ions at

appropriate pH. (Lee, 1996).

1.2.2 TYPES OF COMPLEXES

Complexes are classified into two groups based on the type of chemical boding between the

interactants. These are coordination complexes and molecular complexes.

Coordination Compounds (Metal ion Complexes)

Coordination complexes consist of a central transition metal atom or cation, which acts as a

lewis acid, surrounded by one or more ligands which act as Lewis base (electron pair donor).

The ligands can be monodentate, ‘one tooth’ or polydentate (contain more than one binding site

capable of coordinating with the metal) some of which can act as chelating agents.

(Cotton et al, 1999)

Salicylaldehyde anion

CH2 NH2

NH2 CH2

Ethylenediamine

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The ligands could be uncharged molecules e.g. H2O, NH3, CO, EDTA, ethylenediamine,

dipyridyl, O-phenanthroline, Ciprofloxacin, Penicillins, Isatin e.t.c or negatively charged

radical e.g. I–, Cl–, OH–, S2-, NO2–, NO2

-, CN– e.t.c. The number of coordinate bonds between

the metal ion and the ligand(s) is called coordination number. The ligands are bound to the

metal by coordinate covalent bond between the ligand electron pairs and the empty d-orbital of

the metal. These d-orbitals, according to ligand field theory, split into high and low energy

groups under the influence of the asymmetric (typically octahedral) electric field created by the

array of ligands. This accounts for the highly coloured nature of the complexes when (as

frequently happens) the d-orbital splitting energy difference corresponds to visible-light

wavelengths (Cotton et al, 1999).

Coordination compounds could arbitrarily be classified according to the nature of the

ligands into:

Classical (or Werner Complexes): Ligands bind to metal, almost exclusively, via their lone

pairs of electrons residing on the main group atoms of the ligand. Typical of such ligands are

H2O, NH3, Cl–, CN–, en–. Examples of such complexes are:

[Co (NH3)6]Cl3, (Fe (C2O4)3]K3, [Co (EDTA)]– e.t.c.

Organometallic Compounds: Ligands are organic (e.g. alkenes, alkynes, alkyls) as well as

organic-like ligand such as phosphines, hydride and CO. Example: (C5H5)Fe (CO)2 CH3,

Ferrocene e.t.c.

Bioinorganic compounds: Ligands are those provided by nature such as porphyrins. Example:

haemoglobin, cyanocobolamin (Cotton et al, 1999)

Molecular Complexes

Molecular complexes consist of an association of molecules held by weak, non-covalent

intermolecular forces. The non-covalent forces are of three broad types:

(a) The electrostatic forces among ions and molecules possessing permanent dipole moments.

(b) The induction (or polarization) forces between an ion and a non-polar molecule or a polar

molecule and a non-polar molecule.

(c) The dispersion (London) forces, which operate between all molecules.

There is no systematic classification of molecular complexes, nor has a system of

nomenclature been developed to describe them. Particular types, however, may be classified

in terms of the kinds of interactions involved in their formation, the kinds of interactants

involved or the kinds of complex formed, (Connors et al, 2000).

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Table 1.3: Classification of Molecular Complexes

Type of bonding or interaction Example

Charge transfer complexes Solution of iodine in organic solvent

Hydrogen bonding Adenine-thymine

Hydrophobic interaction Solvophobic effect

Stacking interaction In adjacent DNA pairs

Based on type or structure of interactants

Small molecule-small molecule complex

Small molecule-macro molecule binding

Drug-protein complex

Enzyme-substrate complex

Drug-receptor complex

Antigen-antibody complex

Type of structure of complex

. Self –associated aggregate, e.g. acetic acid, caffeine

. Micelle e.g. Soap molecules

. Inclusion complex e.g. α-cyclodextrin

. Clathrate e.gUrea-long chain molecules

Charge-transfer (CT) complexes, also called electron donor-acceptor (EDA) complexes,

may be formed when one interactant can act as the electron donor and the other as the electron

acceptor. The appearance of a new electronic absorption band, not attributable to either the

donor or the acceptor, is often taken as evidence for change transfer complexation. Example,

when iodine is dissolved in aliphatic hydrocarbons or cabontetrachloride, the solution has a

violet colour, characteristic of iodine, but solutions in aromatic hydrocarbons, alcohols, or

ethers are brown. It is inferred that, in the later solvents, a complex is formed and, because of

the colour (spectral) change, charge-transfer is implicated. The solvent is the electron donor

and iodine the electron acceptor.

Hydrogen bonded complexes are observed readily in solvents that do not compete as H-

bond donors or acceptors. The complex between phenol and pyridine in inert solvents is an H-

bond complex. Perhaps the most famous hydrogen bonded complexes are those of adenine-to-

17

thymine of guanine–to-cytosine, which, as constituents of deoxyribonucleic acid, are

responsible for the double-helix structure of the DNA molecule.

When two planar molecules undergo a primarily hydrophobic association, the total

surface area of the complex exposed to the solvent can be minimized if molecules are in plane-

to-plane contact. This plane-to-plane orientation is called stacking interaction. The purine-

pyrimidine H-bonded base pairs in DNA are planar assemblies that undergo stacking

interactions with adjacent pairs.

Most complexes probably involve a combination of interactions.

Self association is a type of complexation in which a molecule forms complexes with others of

its own species. If S represents a molecule capable of self association, then S2 is its dimmer, S3

its trimer e.t.c. Benzene and caffeine form dimers in aqueous solution.

A micelle is a special form of self-aggregated complex in which the interactant is a surfactant, a

molecule possessing both a polar and non-polar portion.

Inclusion complexes are formed when a macrocyclic compound, possessing an intra-

molecular cavity of molecular dimensions, interacts with a small molecule that can enter the

cavity. The macrocyclic molecule is called the host and the small included molecule the guest.

Crown ethers present a non-polar external molecular surface, but the interior of the cavity is

relatively polar. As a consequence, polar guests such as ions can enter the cavity and, because

their polarity is masked by the surrounding host, exhibit unusual chemistry. For example,

KMnO4, which is not soluble in non-polar solvents, can be extracted into organic solvents from

water in the presence of crown ethers.

The cyclodextrins are macrocyclic hosts that are formed by the action of certain bacterial

enzymes on starch. They consist of α-D-glucose units joined with glycosidic (ether) linkages.

The interior of the cavity is lined with these glycosidic bonds and, therefore, is relatively non-

polar (i.e., relative to water), whereas the exterior of the molecule is quite polar because of the

large number of hydroxyl groups. The diameters of the cavities of the cyclodextrins are

approximately 5 Ǻ (for α –cyclodextrin), 6 to 7 Ǻ (for β) and 8 to 9 Ǻ (for ). Thus, small

guest molecules, or parts of molecules, may enter the host cavity to form inclusion complexes,

whose stabilities are in part the result of the hydrophobic effect. Many properties of the guest

molecule may be altered by inclusion in a cyclodextrin; these include volatility, solubility and

chemical stability. Numerous practical applications have been suggested (Mulliken, 1969).

The literature on molecular complexes frequently now uses the term molecular

recognition, which can be taken to mean a non-covalent interaction in which complementary

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features of the two interactants result in significant specificity in the complex formation process

(Mulliken, 1969).

1.2.3 CHEMISTRY OF COMPLEXES

1.2.3.1 STRUCTURE AND BONDING IN METAL-ION COORDINATION

COMPLEXES

Alfred Werner’s coordination theory in 1893 was the first attempt to explain the

bonding in coordination complexes (Lee, 1996). He concluded that in complexes the metal

shows two different sorts of valency:

(a) Primary Valency: This is the number of charges on the complex ion. It is non-directional.

In compound, this charge is matched by the same number of charges from negative ions.

Example, the complex [Co (NH3)6]Cl3 actually exists as [Co(NH3)6]3+ and 3Cl–. There are three

ionic bonds, thus the primary valency is 3.

(b) Secondary Valency: The number of secondary valencies equals the number of ligand

atoms coordinated to the metal. This is called coordination number. It is directional. Each metal

has a characteristic number of secondary valencies. Thus in [Co (NH3)6]Cl3 the three Cl– are

hold by primary valencies and the six NH3 by secondary valencies.

Since secondary valencies are directional, a complex ion has a particular shape; e.g. the

complex ion [Co (NH3)6]3+ is octahedral. The most common coordination number in transition

metal complexes is 6 and the shape is usually octahedral. The coordination number of four is

also common and this gives rise to either tetrahedral or square planar complexes.

In the complexes [Co(NH3)6]Cl3, [Co(NH3)5Cl]Cl2, and [Co(NH3)4Cl2]Cl, Werner

established that the coordination number is 6 each and the primary valencies are three, two and

one respectively. The possible arrangements of six groups round one atom are a planar

hexagon, a trigonal prism and an octahedron.

Werner then compared the number of isomeric forms he had obtained

Planar hexagon Trigonal prism Octahedron

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with the theoretical number for each of the possible shapes. His results strongly suggested that

these complexes have an octahedral shape. More recently the X-ray structures have been

determined and these establish that the shape is octahedral. With a bidentate ligand such as

ethylenediamine, two optically active isomers have been found.

In a similar way, Werner studied a range of complexes which included [Pt(NH3)2 Cl2]2+ and

[Pd (NH3)2 Cl2]. The coordination number is 4 and the shape could be either tetrahedral or

square planar. Werner was able to prepare two different isomers for these complexes. A

tetrahedral complex can only exist in one form, but a square planar complex can exist in two

isomeric forms. This proved that these complexes are square planar rather than octahedral

.2.3.2 METHODS OF STUDYING THE STRUCTURES OF COMPLEXES

X –ray crystallography: This is the most powerful method for determining crystal structures. It

provides details of the exact shape, the bond length and bond angles of the atoms in the

structure.

Spectroscopy: Electronic spectra (UV and visible) provide valuable information on the energy

of the orbitals and the shape of the complex. By this means it is possible to distinguish between

tetrahedral and octahedral complexes and whether the shape is distorted or regular.

Cryoscopy: This involves the measurement of the extent of freezing point depression of a

liquid when a chemical substance is dissolved in it. This measurement is based on Raoult’s

law: “For dilute solutions, the freezing point depression is proportional to the number of

particles present’’. (Ibemesi, 1994).

i.e. ∆ Tf = Kf M

Or ∆ Tf = Kf W2 x 1000

M2 W1

Where

∆Tf = Difference between the F. P of solvent and solution

M = molaility of the solution

Cl Pt

NH3

Cl NH3 Cis

Cl Pt

NH3

NH3 Cl

Trans

20

W2 = weight of solute dissolved

W1 = weight of solvent

M2 = molecular weight of the solute

Kf = molal depression constant (unique for each solvent). (Ibemesi, 1994).

Cryoscopic measurements can be used to find if a molecule dissociates and how many ions

are formed. If a molecule dissociates into two ions it will give twice the expected depression

for a single particle, and so on. Information on the number of particles formed and that from the

total number of charges can be used together to establish the structure of a complex. Cryoscopy

is also used to determine molecular weight of a substance.

Electrical Conductivity: The electrical conductivity of a solution of an ionic material

depends on the concentration of the solute as well as the number of charges on the species

which are formed on dissolution. The total number of charges on the species formed when the

complex dissolves can be deduced by comparison of its molar conductivity with that of known

simple ionic materials. Conductivity is directly proportional to number of charges.

Measurement of Magnetic Moment: This provides information about the member of

unpaired electron spin present in a complex. From this, it is possible to decide how the

electrons are arranged and which orbital are occupied. Sometimes the structure of the complex

can be deduced from this. For example, the compound Ni (NH3)4 (NO3)2 2H2O might contain

four NH3 molecules coordinated to Ni2+ in a square planar [Ni (NH3)4]2+ ion and two molecules

of water of crystallization and have no unpaired electrons. Alternatively the water might be

coordinated to the metal, giving an octahedral [Ni(H2O)2(NH3)4]2+ complex with two unpaired

electrons. Both these complex ions exist and their structures can be deduced from magnetic

measurements.

Measurement of dipole Moments: This could be used to distinguish the cis and trans isomers

of a complex. For example, the complex ,[Pt (NH3)2 Cl2] is square planar and can exist as cis or

trans forms. The dipole moments from the various metal-ligand bonds cancel out in the trans

configuration. However, a finite dipole moment is obtained for the cis arrangement. This

method applies to non-ionic complexes only. (Lee, 1996).

1.2.3.3 THEORIES OF BONDING IN TRANSITION METAL COMPLEXES

Valence bond theory (developed by Pauling):

The theory holds that coordination compounds contain complex ions, in which ligands form

coordinate covalent bonds to the metal. Thus the ligands must have a lone pair of electrons, and

21

the metal must have an empty orbital of suitable energy available for bonding. The theory

considers which atomic orbitals on the metal are used for bonding from which the shape and

stability of the complex are predicted. The theory, however, fails to explain why these

complexes are coloured as well as why their magnetic properties vary with temperature.

Crystal field theory (Proposed by Bethe and Van Vleck).

This theory assumes that the attraction between the central metal and the ligands in a complex

is purely electrostatic. The following assumptions are made.

(i) Ligands are treated as point charges

(ii) There is no interaction between metal orbitals and ligand orbitals

(iii) The d-orbitals on the metal all have the same energy (degenerate) in the free

atom.However, when a complex is formed the ligands destroy the degeneracy.

The d-orbital is subdivided into the eg orbitals (dx2–y2 and dz2) and t2g orbitals (dxy, dxz and dyz).

In an octahedral complex, the metal is at the centre of the octahedron and the ligands at the six

corners. The lobes of the two eg orbitals point along the axes x, y and z while those of the three

t2g orbitals point in between the axes. It follows that the approach of six ligands along the axes

will increase the energy of the dx2 –y2 and dz2 orbitals much more than it increases the energy

of dxy, dxz and dyz orbitals. Thus, under the influence of an octahedral ligand filed, the d-orbitals

spilt into two groups of different energies. The energy difference is called d-orbital splitting

energy, ∆o. This energy difference most often corresponds to visible-light wavelengths and this

accounts for the coloured nature of the transition metal coordination complexes. An improved

and more complete model of the crystal filed theory, which incorporates molecular orbital

theory and make some allowance for covalency, is known as ligand field theory.

Molecular Orbital Theory

This theory fully allows for both covalent and ionic contributions to bonding. It has a

great advantage that it is easily extended to cover bonding. - Bonding helps to explain how

metals in low oxidation states [eg [Nio (CO)4] can form complexes even though there is no

change on the metal. The theory holds that the atomic orbitals of the ligands overlap with the

metal d-orbitals of right energy to form molecular orbitals.

1.2.3.4 IUPAC NOMENCLATURE OF COORDINATION COMPOUNDS

Rules:

1. The positive ion (cation, not central metal) is named first followed by the negative ion.

22

2. When naming a complex ion, the ligands are named before the central metal; they are named

alphabetically (numerical prefixes do not affect the order)

3. When writing the formular of complexes, the complex ion is enclosed in square bracket. The

metal is written first and the coordinated groups are listed in the order: Negative ligands,

neutral ligands, positive ligands.

4.Multiple occurring monodentate ligands receive a prefix according to the number of

occurrences: di-, tri-, tetra, penta-, e.t.c. Polydentate ligands, or ligands preceded by numerical

prefix eg ethylenediamine, dipyridyl e.t.c) receive bis-, tris-, tetrakis, pentakis -, e.t.c.

5. (a)Names of negative ligands end in –O. this replaces the final ‘e’ when the anion ends with-

‘ate’, eg. Sulphate –sulphato. It replaces ‘ide’: cyanide becomes cyano.

Some negative ligands:

CH3COO– acetato CN– cyano

-NO2 nitro OH– hydroxo

HS– mercapto –SCN thiocyanato

–ONO nitrito –NCS isothiocyanato

(b) Neutral ligands usually retain their names, with some of these exceptions:

NH3 ammine NO nitrosyl

H2O aqua or aquo CO carbonyl

(c) Positive ligands end in -ium e.g.

NH2NH2 - hydrazinuim

6. The oxidation state of the central metal is shown by Roman numeral in bracket immediately

following its name. If the complex is an anion, the central atom’s name will end in-

‘ate’ and its Latin name used if available (except Mercury). (Cotton et al, 1999).

7. If the complex contains two or more metal atoms, it is termed polynuclear. The bridging

ligands which link the two metal atoms together are linked by the prefix μ- . If there are two or

more bridging groups of the same kind, this is indicated by di - , tri- e.t.c. Bridging groups

are listed alphabetically with the other groups unless the symmetry of the molecule allows a

simpler name. If a bridging group bridges more than two metals, it is shown as 3 , 4 , 5 ,

or6

, to indicate how many atoms it is bonded to.

Examples:

[Co (NH3)5 Cl]SO4 Pentaamminechloro cobalt (III) sulphate.

[Cd (en)2 (CN)2 ] Dicyano bis (ethylenediamine) cadimium(II).

[CU NH3 Cl5]3– Aminepentachlorocuprate (II) ion

23

[Zn (NCS)4]2+ Tetrathiocyanato-N-zinc (II)

Na3 [Ag (S2O3)3]2 Sodium bis (thiosulphato) argentate (I)

Fe (C5H5)2 Bis (Cyclopentadienyl) iron (II)

[(NH3)5 Co.NH2. Co (NH3)5] (NO3)5 -amido bis [pentaamminecobalt (III)] nitrate.

[(Co)3 Fe (CO)3 Fe (CO)3] Tri -carbonyl-bis (tricarbonyl iron (O).

1.2.3.5 ISOMERISM IN TRANSITION METAL COMPLEXES

Isomerism is a phenomenon whereby two or more compounds have the same chemical

formular but different chemical structures. Because of the complicated formular of many

coordination compounds, the variety of bond types and the number of shapes possible, many

different types of isomerism occur.

Ionization Isomerism: This type of isomerism is due to the exchange of groups between the

complex ion and the ions outside it. Eg [Co (NH3)5 Br] SO4 and [Co(NH3)5SO4]Br. The former

is red-violet and gives a white precipitate with BaCl2 solution, while the latter is red in colour

and gives no precipitate with BaCl2, instead, it gives cream-coloured precipitate of Ag Br with

AgNO3, confirming the presence of free Br– ions.

Linkage Isomerism: Linkage isomerism occurs when the ligands contain more than one atom

which could donate an electron pair, e.g. –NO2 or –ONO, SCN- or –SCN e.t.c. The nitrito and

nitro complexes of cobalt constitute isomers.

Geometric

Isomerism:

Square planar complexes such as [Pt (NH3)2Cl2] can be prepared in two forms: cis and

trans. If the complex is prepared by adding NH4OH to a solution of [PtCl4]2– ions, the complex

has a finite dipole moment and must therefore be cis. The complex prepared by treating

[Pt(NH3)4 ]2+ with HCl, however, has no dipole moment and must, therefore, be trans.

H2N Co

ONO

H2N NH3 NH3

NH3

H3N Co

NO2

H3N NH3 NH3

NH3

and

2+ 2+

Pentaammine nitrito cobalt (III) ion (red in colour, easily decomposed by acid

Pentaammine nitrito cobalt (III) yellow, stable to acid)

24

Optical Isomerism

Optical isomerism is common in octahedral complexes involving bidentate groups. For

e.g. [Co (en)2 Cl2]+ shows cis and trans forms (geometric isomerism). In addition the cis form

is optically active and exist in d and l forms, making a total of three isomers. Optical isomerism

also occurs in polynuclear complexes.

1.2.4 REACTION CONDITIONS FAVOURABLE FOR COMPLEX FORMATION

The reaction conditions, such as temperature and pH, favourable for complex formation

depend largely on the type of ligand, the charge on the metal ion and the type of metal (whether

the metal is in the first, second or third row of transition elements).

Ligands which cause only a small degree of crystal field splitting are termed weak field ligands

while those that cause large splitting are called strong field ligands. The spectrochemical series

of ligands is given below.

← Weak field ligands

I – <Br– < S2– < Cl– < NO3– < F– < OH– < EtOH < Oxalate <H2O <EDTA (NH3 and pyridine) <

ethylenediamine <dipyridyl < O– phenanthroline <NO2– < CN– < CO.

→ Strong field ligands.

The series is experimentally determined. The halides are in the order expected form

electrostatic effect. In other cases, a pattern of increasing donation is followed:

Halide donors < O donors < N donors < C donors. (Lee, 1996)

The magnitude of the crystal field stabilization energy of complexes increases as the charge on

the metal ion increases. It also increases by about 30% between adjacent members down a

group of transition elements. The thermodynamic stability, and hence ease of formation,

increases in that order. Complexes where the metal is in the (+ 3) oxidation states are generally

more stable than those where the metal is in the (+2) state:

trans

Pt

NH3

NH3

Cl

Cl Pt

NH3

Cl

Cl

NH3 cis

25

For a given ligand and metal the factors that influence complex formation include pH,

temperature, solvent, duration of reaction and continuous stirring. EDTA, for example, forms

stable complex with most metal ions in solution. The stability of the complex formed, however,

depends on the pH of the solution. Most divalent metals form stable EDTA complexes at pH >

7 while most trivalent metals form stable complexes at low pH, say 3. The application of this is

that by adjusting pH, one metal may be determined in the presence of the other in

complexiometric titration. The pH ranges for metal titration with EDTA is given below.

Table 1.4 pH ranges for Metal-EDTA titration

Fe3+ 1.5

Cu2+ 3.0

Zn2+ 4.0

Fe2+ 5.0

Ca2+ 8.0

Mg2+ 10.0

Temperature is another important factor that affects complex formation. If

complexation is exothermic, it would be favoured by low temperature and vice versa. Many

complexation reactions occur readily at room temperature, e.g. ciprofloxacin –Cu2+ (Guangguo,

et at, 2003), EDTA –Metals, Fe3+ – K2 C2 O4 . H2O (Szafran Z. et al, 1991) e.t.c. Schiff base

ligands require higher temperatures with reflux. e.g. in the synthesis of binuclear iron (III) –

iron (III) complexes with the tetradentate Schiff base, N, NI–bis (Salicylidene),

ethylenediamine and dicarboxylic acid bridging ligands, the reaction mixture is heated to

45–50oC under reflux with continuous stirring for 30 mins. (Zdenek et al, 1996). Similarly, in

the synthesis of metal complexes of 3 – salicylidenhydrazono-2-indolinone Schiff base, the

temperature is maintained between 60 – 65oC under reflux with continuous stirring for 2hrs.

(Sandra et al, 2003)

Solvent effects on complex-formation vary and can be complicated sometimes, but their

study may offer insight into the nature of the intermolecular interactions responsible for the

formation of the complex. A quantitative theory of solvent effects on complex formation has

been developed (Connors K. A. et al, 1992). The solvation contribution to the total free energy

of complex formation can be either stabilizing (if the complex is solvated more extensively

than the reactants) or destabilizing (if the reverse is applicable).

26

1.2.5 COMPLEX STABILITY

For the general complex formation equilibrium:

mS +nL Sm Ln

the overall binding constant, βmn is defined as βmn = [Sm Ln] where

[S]m[L]n

S = substrate, L = ligand, brackets denote molar concentrations. The binding constant is also

known as stability constant, formation constant or association constant. The reciprocal quality

is the dissociation constant. These constants depend on the identities of the substrate and

ligand. They also depend on the solvent and temperature (Connors et al, 2000). The binding

constant is an important measure of complex stability, and is related to the standard free energy

of complex formation by

∆GoII = – RT ln KII

where R = gas constant, T = absolute temperature, KII = binding constant for formation of

complex with I: I stoichiometry.

1.2.5.1 MEASUREMENT OF COMPLEX STABILITY

If a property of the substrate is altered upon its complexation with a ligand,

measurement of the property as a function of ligand concentration provides a means for

estimating the binding constant. The following methods have been devised.

Spectrometry: This method is based on the change in light absorption. Suppose the absorption

spectrum of the substrate is changed significantly upon binding, a wavelength at which a

substantial change in absorption occurs could be selected and, assuming that Beer’s law is

obeyed by the species, a particular chrornophore could be targeted and absorbance measured at

interval as a function of concentration.

Absorbance is additive. At a total substrate concentration absorbance is given as

Ao = s b St

In the presence of the ligand the absorbance is

AL = s b [S] + l b [L] + ll b [SL] where ll is the molar absorptivity of the complex (I: I

stoichiometry assumed).

This spectrometric method is applicable in the ultraviolet, visible and infrared regions.

Nuclear magnetic resonance spectrometry can as also be applied where a change in the

chemical shift is measured (Connors KA, 1987).

27

Chemical Reactivity: If the rate of a chemical reaction (such as hydrolysis) undergone by the

substrate is either increased or decreased by binding to the ligand, the stability constant can be

measured.

Consider the kinetic scheme:

Here R is a reagent that reacts with the substrate S, and complex SL, but does not form

complexes, P is the product and Ks, Kll are second order rate constants. The result of the

mathematical development is

Where qll = 1 – Kll/Kls and kls is the measured second order rate constant in a solution having

ligand concentration, [L].

Potentiometry: If the activity of an ion is changed upon complex formation, it may be

possible to make use of the measurement of electrical potential, E, according to Nernst

equation:

E= constant + (RT/nF) ln a

Where a is the ion activity, n is the no. of electrons in the redox process and F is the Faraday.

Potentiometry is the most widely used method for the study of metal ion coordination

complexes, for which the activity of the metal ion, ligand, or the hydrogen ion may be

measured (Hartley et al, 1980).

Solubility: Here, the total apparent solubility, St, of the substrate is measured as a function of

total ligand concentration, Lt. Because the system is prepared to contain excess (solid)

substrate, the free-substrate concentration is maintained constant at its intrinsic molar

solubility, So. Therefore the mass balance on substrate can be written thus:

St = So + [SL]

Combination of the above equation with Kll = [SL]

[S][L]

and Lt = [L] + SL

SL + R

KS

P

P Kll

S+R

1 + Kll [L] KS –– Kl

s qll Ku [L] = KS

28

yields St = So + Kll So Lt ... *

1 + KllSo

Equation * predicts that St is a linear function of Lt. The binding constant is obtained with

Kll = Slope

So (1 –slope)

There are other methods that, like the solubility method, involve a distribution between two

phases. The apparent partition coefficient of solutes between two immiscible solvent can be a

measure of complex formation. Several chromatographic methods are based on a similar

principle, the retention volume, or time, of a substrate being measured as a function of ligand

concentration.

Dialysis: This technique is applicable when one interactant, such as the substrate, is a very

large molecule and the other; the ligand is a small molecule. It is, therefore, used widely to

study the binding of drugs to proteins.

In dialysis, two compartments containing solvents are separated by a semi permeable

membrane. In one compartment (No1) the non-diffusible substrate is placed, and in the other

(No 2) the diffusible ligand is placed. The system then is allowed to come to equilibrium. At

equilibrium the free ligand concentration [L] is equal in the two compartments. The solutions in

the two compartments are analyzed for their total ligand concentration. Hence

[Lt]1 = [L]1 + [bound L]1

[Lt)2 = [L]2

and the equilibrium condition is [L]1 = [L]2.

Therefore i can be calculated for compartment No 1.

Using i = Lt – [L] (Connors et al, 2000)

St

i = average no of drug molecules bound per protein molecule at free-drug concentration [L]

Lt = total drug concentration

St = total protein concentration.

The experiment is repeated at different ligand concentrations to obtain i as a function of [L].

The data then are analyzed in terms of the model equation (Connors, 1987).

1.2.6 METHODS OF ANALYSIS AND DETERMINATION OF COMPLEXES

Most complex ions absorb light in the UV – visible region of the electromagnetic

spectrum. This property is usually exploited in both qualitative and quantitative analyses of

29

complexes. The various methods of characterization of complexes have been discussed in

section 1.2.3.2

Spectrometrical determination of the concentration of complexes in solution is based on

Beer-Lambert’s law: “When a monochromatic light is incident on dilute solutions, the

amount of light absorbed is proportional to the concentration of the absorbing species’’.

A = b c

Where A = absorbance, b= path length of the container

= molar absorptivity (dm3 mol–1 cm–1)

C = concentration (mol dm–3)

If the complex absorbs in the visible region of the spectrum, this is called colorimetric analysis.

Colorimetric analysis is based on colour matching with standard solutions. It is a valuable

method for complex analysis since most transition metal complexes are coloured.

1.3 ANTIBIOTICS

1.3.1 DEFINITION OF ANTIBIOTICS

An Antibiotic is any natural substance secreted by microorganism to ward off other

microorganisms. (Brown, 1996). Bacteria or moulds might secrete chemicals that interfere with

attacking microorganisms to harm, kill or slow them down.

Antibiotic is a chemical compound derived from or produced by a living organism or semi-

synthesized or produced as synthetic small analogue of naturally occurring compound, which is

capable, in small concentration, of inhibiting the life processes of microorganisms (Olaniyi A.

A., 2005). This is a more general definition.

In another view, an antibiotic is a drug that kills or slows the growth of bacteria. Antibiotics

constitute one class of antimicrobials. Antimicrobial is a larger group which also includes anti-

viral, anti-fungal, and anti-parasitic drugs. (Samuel U., 2002)

The first antibiotic was discovered by Alexander Fleming in 1928 in a significant

breakthrough for medical science. Fleming saw that the mould, penicillium, was inhibiting

bacteria growth.

1.3.2 CLASSES OF ANTIBIOTICS

Although there are several classification schemes for antibiotics; based on bacterial

spectrum (broad verses narrow), route of administration (injectable versus oral versus topical),

30

or type of activity (bactericidal vs. bacteriostatic), the most useful basis of classification is

chemical structure, Antibiotics within a structural class will generally have similar patterns of

effectiveness, toxicity and allergic potentials.

1.3.2.1 BETA LACTAM ANTIBIOTICS

Chemically, these antibiotics contain a β-lactam ring, i.e. a 4-membered cyclic amide.

HNO

They are divided into penicillins, cephalosporins, penems (eg clavulanic acid), carbapenems

(eg thienamycin) and monobactams.

THE PENICILLINS

The natural or biosynthetic penicillins are obtained by fermentation of the moulds,

penicillium notatum and penicillium chrysogenum. They are not effective against gram-

negative bacteria (rods). Chemical modifications have been carried out on 6-Aminopenicillanic

acid (6-APA), which is regarded as the parent compound of all penicillins, to produce a large

number of semi-synthetic penicillins with more desirable properties such as

(a) Improved acid stability, eg phenethicillin

(b) Stability to penicillinase (β-lactamase) enzyme, eg. Ampicillin;

(c) Broadened spectrum of activity, eg Ampicillin;

(d) Improved metabolic or pharmacological efficiency such as slow excretion, better tissue

diffusion and better oral absorption, eg. Cloxacillin, amoxicillin.

(e) Decreased allergenicity

Penicillins are characterized by three fundamental structural features:

i. Fused β –lactam ring;

ii. A free carboxyl group;

iii. One or more substituted amide side chain.

The basic structure consists of thiazolidine ring connected to a β-lactam ring, to which is

attached a side chain R.

31

C O OH

R-C-NH-

CH3N

S

O

CH3

O 1

34

567

Basic structure of penicillin

Natural penicillins

When R is CH3 CH2CH = CH CH2 Penicillin F or I (2-Pentenyl Penicillin)

R = C6 H5 CH2– Penicillin G or II (Benzyl Penicillin)

R = HO C6 H4 CH2– Penicillin X or III (P-Hydroxy benzyl Penicillin)

R = CH3(CH2)6– Penicillin K or IV (n– heptyl penicillin)

R = C6H5OCH2– Penicillin V (Phenoxymethyl Penicillin)

Table 1.5

Some Semi-synthetic Penicillins

R Name Characteristics

C6H5CH

NH2

Ampicillin (–) – 6(amino-2- phenyl

acetamido) -3,3-dimethyl -7- Oxo-thia-

1-aza bicyclo [3,2,0] heptane-2-

carboxylic acid

Acid-stable, penicillinase

sensitive, broad spectrum

of activity, very little

bound to protein.

32

HO-C6H4-CH

NH2

Amoxicillin Acid-stable, penicillinase

sensitive.

C6H5CH

COOH

Carbenicillin Acid-sensitive,

penicillinase-resistant,

broad spectrum of activity.

NO

CH3

Cl

Cloxacillin Acid-stable, penicillinase-

resistant, oral and

parenteral.

C6H5-O-CH

CH3

Phenethicillin Acid-stable, penicillinase-

sensitive,

CH2CH3

C6H5-O-CH

Propicillin Acid-stable penicillinase-

resistant.

CLOXACILLIN

Cloxacillin is a semi-synthetic antibiotic in the same class as penicillin. It was discovered

and developed by Beecham and sold under a number of trade names, including Cloxapen,

Cloxacap and Orbenin. It is used to treat a wide variety of infections, including sinusitis, strep

throat, pneumonia, respiratory tract infection, ear infections, dental abscesses, skin infections

and STDs like gonorrhea and syphilis, and infections of the genital and urinary tracts.

Mode of Action: Cloxacillin kills bacteria by destroying the cell wall of the invading

microorganisms. It is, however, not effective against viruses and most fungi. There are also a a

number of bacteria, the so called “super bugs” that are resistant to penicillin antibiotics

including cloxacillin.

Chemistry: The systematic IUPAC name for cloxacillin is (2S,5R,6R)-6-{[3-(2-Chlorophenyl)-

5-oxazole-4-carbonyl] amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-

33

carboxylic acid. It has amolecular formular of C19H18ClN3O5S and molecular weight of 435.88

g mol-1.

CH3

O

CH3

ON

S

OCH

ON

Cl

CH3

H

O

NC

Cloxacillin

Pharmacokinetics: Cloxacillin is administered either orally or through intra-muscular route. It

has a bioavailability of 37- 90 % and protein binding of 95%. Its elimination half life is 30

minutes to 1 hour and excretion is renal and biliary.

AMOXICILLIN

Amoxicillin is one of the semi-synthetic penicillins discovered by Beecham scientists. It is

a moderate spectrum, bacteriolytic β-lactam antibiotic used to treat bacterial infections. It is

usually the drug of choice within the class because it is better absorbed, following oral

administration, than other β-lactam antibiotics. It is also a treatment for cystic acne.

Amoxicillin acts by inhibiting the synthesis of bacterial cell wall. It inhibits cross-linkage

between the linear peptidoglycan polymer chains that make up a major component of the cell

walls of both Gram-positive and Gram-negative bacteria.

Chemistry: The systematic IUPAC name for amoxicillin is (2S, 5R, 6R)-2-{[(2R)-2-amino-2-

(4-hydroxyphenyl)-acetyl] amino}-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo [3, 2, 0] heptane-2-

carboxylic acid. It has a formular of C16H19N3O5S and molecular weight of 365.4 g mol-1

CH3

CH3

CH-C-NH

NH2

ON

S

OO

CH

HO

O

Amoxicillin

34

Pharmacokinetics: Amoxicillin is well absorbed following oral administration with

bioavailability of 95%. Less than 30% of the drug is biotransformed in the liver. It has an

elimination half life of 61.3 minutes and its excretion is renal.

Properties of Penicillins

The solubility and other physical and chemical properties of penicillins are affected by

the nature of the acyl side chain (Olaniyi A, 2005). They are incompatible with, and inactivated

by heat, moisture, oxidizing agents, metals, (Cu, Zn), acids, alkalis, penicillinase, (β-

lactamase), alcohol and glycerine. They are unstable in aqueous solutions; they are, therefore,

buffered to pH 6 – 6.8 using citrate or phosphate buffers. They are relatively strong acids, pKa

between 2.5 and 3.0. However, those with basic groups in the side chain exist as zwitterions, eg

Ampicillin.

Penicillins are very reactive owing to the strained amide bond in the fused β-lactam

ring. They are susceptible to attack by nucleophilas, eg NaOH, penicillinase, alkoxides e.t.c, as

well as by electrophiles such as Cu2+, Zn2+, dil HCl etc. The ease of hydrolysis of the β-lactam

ring by water, hydroxide ion, or alkoxide ion, is the main cause of deterioration of penicillins

forming penicilloic acid. (Olaniyi A, 2005).

1.3.2.2 NON-LACTAM ANTIBIOTICS

The Quinolones

These are a new class of highly potent, orally active, broad spectrum antibiotics

developed from the original 1,8-naphthyridine urinary antibacterial agent, nalidixic acid.

Several new compounds in this class, notably the fluoroquinolones are more potent with

broader spectrum of activity than nalidixic acid.

The fluoroquinolones possess both a 6-fluoro substituent and a 7-piperazinyl group on

the quinolone pharmacophore which increased the potency, expanded the spectrum and appears

to have prevented the development of plasma-mediated resistance.

35

COOH

C2H5

Me N N

O

Nalidixic acid

COOH

N

O

NN

R RR

F

Fluoroquinolone12

3

45

6

21

R1 =R3 = H, R2 = Et ; Norfloracin

R1 = R3 = H, R2= ; Ciprofloxacin

R1 = Me ,R2 = Et, R3= H; Perfloxacin.

R1 =Me, R2 = NHMe, R3 = H; Amifloxacin.

Mode of Action of quinolones:

They act by inhibiting the bacterial DNA synthesis, initially by inhibiting ATP-

dependent DNA super coiling by binding to subunit of DNA-gyrase; secondarily, they also

inhibit the relaxation of super coiled DNA and finally, they also block the DNA nicking –

closing enzyme that is ultimately for DNA elongation.

Structure – Activity Relationship:

(a) The 4-keto and the 3- COOH groups are required for activity. Modification to the –

COOH results in an inactive compound. However, replacement of –COOH group with

isothiazolo ring increases potency.

36

(b) A C-6 fluorine atom increases potency as it increases lipophiilcity thus facilitating

penetration into tissues and cells.

(c) Substitution with a piperazinyl group at C -7 leads to potent antibiotic with broad

spectrum of activity.

Ciprofloxacin Ciprofloxacin is a member of the second generation fluoroquinolone class of synthetic

antimicrobial agent s that has broad spectrum of activity against both Gram-positive and Gram-

negative organisms. It was first patented in 1983 by A.G. Bayer and subsequently approved by

the United States Food and Drug Administration (FDA) in 1987. Intravenous formulation was

later introduced in 1991.

Chemistry: Ciprofloxacin is 1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl quinoline-3-

carboxylic acid. It has a molecular formular of C17H18FN3O3 and molecular weight of 331.4

g mol-1 .It is a faintly yellowish to white crystalline substance.

N

OF COOH

NN

H

Ciprofloxacin

Mode of Action: Ciprofloxacin, like most fluoroquinolones, functions by inhibiting DNA

synthesis, initially by inhibiting ATP-dependent super coiling by binding to subunit A of DNA-

gyrase. Secondarily, they also inhibit the relaxation of the super coiled DNA, a reaction not

dependent on ATP. Finally, they also block the DNA nicking-closing enzyme that, in the

absence of drug interference, is ultimately responsible for DNA elongation. (Olaniyi, 2005)

Pharmacokinetics: The effects of 200 – 400 mg of ciprofloxacin given intravenously are linear;

drug accumulation does not occur when administered at 12 hour intervals. Bioavailability is

approximately 70-80 % with no significant first pass effect. Intravenous administration

produces a similar serum level as those achieved with 500 mg administered orally. I.V

administration over 60 minutes given every 8 hours produces similar serum level of the drug as

37

750 mg administered orally every 12 hours. Metabolism is hepatic including CYP1A2. The

elimination half life is 4 hours and excretion is renal.

Indications: The licensed uses of ciprofloxacin in adult population are as follows: typhoid fever

(enteric fever) caused by Salmonella typhi, acute uncomplicated cystitis in females, chronic

bacterial prostatitis, lower respiratory infections, acute sinusitis, skin and skin structure

infections, infectious diarrhea e.t.c. Ciprofloxacin is not recommended for the treatment of

tuberculosis.

Oral and I.V fluoroquinolones are not licensed by the United states F.D.A. for use in

children due to the risk of permanent injury to the musculoskeletal system.

ISATIN

Isatin or 1H-indoline-2, 3-dione is an indole derivative obtained by Erdman and Laurent in

1841 as a product from oxidation of indigo dye by nitric acid and chromic acid. The compound

is found in many plants. Isatin and its shiff bases are investigated for their antimicrobial and

other pharmaceutical properties.

Synthesis: Isatin is commercially available. It may be prepared by cyclizing the condensation

product of cloral hydrate, aniline and hydroxylamine in sulphuric acid.

H2SO4

HO-NH2

OH

NHCl3C

NH2

OH

OH

N

O

NHO

O Indolin-2,3-dione This reaction is called Sandmeyer isonitrosoacetanilide isatin synthesis after

Traugott Sandmeyer who discovered it in 1919.

Pure isatin is an orange red solid with a melting point of 200º C.

38

1.4 MEDICINAL CHEMISTRY OF THE ESSENTIAL TRACE ELEMENTS

1.4.1 IRON

Biologically iron is the most important transition element. It is involved in

several different processes:

(a) As an oxygen carrier in the blood (haemoglobin)

(b) Oxygen storage in muscle tissue (myoglobin)

(c) As an electron carrier in animals, plants and bacteria (cytochromes) and for

electron transfer in plants and bacteria (ferredoxins).

(d) Storage and scavenging of iron in animals (ferretin and transferrin)

(e) As a content of enzymes like aldehyde oxidase, peroxidase and succinic

dehydrogenase (the aerobic oxidation of carbohydrates). (Lee, 1996).

Numerous iron (II) and iron (III) compounds, complexes and solutions

have been used as haematinics in the past. However because of their greater

gastrointestinal irritation and poor absorption, iron (III) compounds and their

preparations are used rarely today. Ferrous fumarate, ferrous gluconate, ferrous

sulphate (oral solution, syrup, and tablets) and dried ferrous sulphate are official

in the USP.

Iron dextran injection, a colloidal iron (III) hydroxide with partially

hydrolyzed dextran, and iron sorbitex injection, a complex of iron with sorbitol

and citric acid, are cited in the USP as injectable forms for patients with poor

gastrointestinal tolerance or poor absorption of iron.

A study carried out in Finland has cast doubt on the advisability of the

routine use of haematinics because men with higher levels of ferritin were found

to be more prone to heart attack. Interpretation of the results included speculation

about iron’s ability to give rise to free radicals after reaction with oxygen. The use

of haematinics without substantiated need is not advisable. (Clarence et al, 2000).

1.5 AIM OF STUDY

39

The aims of the current study are the following:

1. To prepare new Fe3+ complexes of ampicillin, amoxicillin, cloxacillin,

ciprofloxacin and a Schiff base derived from isatin and ethylenediamine, with

a view to improving the aqueous solubility, gastric acid stability and solid

dosage form stability of the drugs.

2. To investigate the antimicrobial properties of the iron (III) complexes of

these drugs and compare them with those of the free drugs (ligands).

3. To investigate the aqueous solubility, thermal and acid stability of these

complexes.

4. To carryout preliminary characterization of the iron (III) complexes of these

drugs and hence propose a structure for each complex.

CHAPTER TWO

MATERIALS AND METHODS

2.1 EQUIPMENT

Magnetic stirrer (Gallenkamp, England), Ultraviolet- visible spectrophotometer (Jenway

6305, Barlowood Sci. ltd, Dunmow), Infrared spectrophotometer (Shimadzu, ® Japan), pH

40

meter (Jenway, Dunmou), electronic weighing balance (Adventurer, OHAUS corp. China),

melting point apparatus (Electrothermal, ® England) were used.

2.2 REAGENTS AND SOLVENTS

Pure ciprofloxacin hydrochloride (Juhel Pharm. ltd, Enugu),amoxicillin powder

(Becham pham. Pakistan) cloxacillin (Neuchem pharm. Lagos) and ampicillin trihydrate (c/o

Neuchem pharm. Lagos) were obtained from Juhel pharm. Nig. ltd, Enugu & Neuchem (F&P)

ltd Lagos. Pure isatin powder and ethylenediamine were obtained from Dr. P.O. Ukoha’s

research lab. Dept. of Pure & Ind. Chem., UNN. Ferric chloride (Merck. Germany), analytical

grades of the following solvents (manufactured by Aldrich Chem. Ltd): absolute ethanol,

methanol and dioxan, and distilled water (Lion Table water Ltd UNN) were used as obtained.

2.3 MICROORGANISMS

The following microorganisms (clinical isolates): Staphylococcus aureus, Pseudomonas

aeruginosa, Bacillus subtilis, Escherichia coli, Salmonella typhi, Shigella spp, Aspergillus

niger and Candida albicans were used. They were maintained in the Department of

Pharmaceutics, University of Nigeria Nsukka.

2.4 SYNTHESIS OF THE COMPLEXES

Synthesis of Fe3+ Complex of Ciprofloxacin

Ciprofloxacin HCl (368 mg) was dissolved in a minimum quantity of distilled water. To this

solution was then added FeCl3 (81.25 mg) dissolved in absolute ethanol. The mixture was

stirred continuously with magnetic stirrer at room temperature for 3 hrs. The pH was adjusted

to 7.0-8.0 with dil. NaOH. The resulting red solution was transferred into an evaporating dish

and allowed to evaporate slowly at room temperature for two weeks. The reaction was

monitored spectrometrically. The red crystals formed were purified by recrystallizing in a

minimum quantity of ethanol, and weighed. (Guangguo et al, 2003)

Synthesis of Cloxacillin-Fe3+ Complex

Cloxacillin powder (435 mg) was dissolved in dioxane (100 ml). To this solution was added

an alcoholic FeCl3 solution containing 162.5 mg of FeCl3 in 100 ml of ethanol. The mixture

was stirred continuously for 4 hours at room temperature, at the end of which clay-brown

crystals separated from the solution. The mixture was filtered and the crystals washed

thoroughly with dioxane, dried in a desiccator and weighed. (Guangguo et al, 2003)

41

Synthesis of Amoxicillin-Fe3+ Complex

Amoxicillin powder (365 mg) was dissolved in methanol (100 ml) in a beaker. FeCl3 (162.5

mg) was put in another beaker containing ethanol (10 ml). The two solutions were mixed

together and stirred with a magnetic stirrer under reflux, maintained at 40oC for 4 hrs. The

resulting green solution, which was foaming, was transferred into an open beaker and allowed

to stand for 1 week. The green crystals obtained were washed thoroughly with small quantity of

ethanol, dried and weighed.

Synthesis of Ampicillin –Fe3+ Complex

Ampicillin trihidrate (403 mg) was dissolved in methanol (100 ml) in a beaker. FeCl3

(81.25 mg) was put in another beaker containing ethanol (10 ml). The two solutions were

mixed together and stirred with a magnetic stirrer under reflux, maintained at 40◦C for 4 hrs.

The resulting brown solution was transferred into an open beaker and allowed to stand for 1

week. The brown crystals obtained were washed thoroughly with small quantity of ethanol,

dried and weighed. (Guangguo et al, 2003)

Synthesis of Isatin-ethylenediamine Schiff base Ligand.

Isatin (147 mg) was dissolved in minimum quantity of absolute ethanol placed in an ice

bath. To this solution, 100% ethylenediamine (60 mg) maintained at 4oC was added and the

mixture stirred continuously for 2 hrs. The mixture was acidified with CH3COOH to pH 4 -

4.5. The temperature was maintained at 0-5oC throughout. Rusty brown product, separated after

two days, was filterd and washed with ethanol. The solid product was weighed after drying in a

desiccator.

Synthesis of Fe3+ Complex of the Isatin Schiff base

The Schiff base derived from isatin and ethylenediamine (190 mg) was dissolved in

minimum amount of absolute ethanol. To this refluxing solution, FeCl3 (82 mg) dissolved in

ethanol was added. The refluxing was continued for 2 hrs, at 60oC. The reddish brown product

was filtered, washed with ethanol and dried in a desiccator over CaCl2 .

These reactions were monitored spectrometrically and chromatographically.

2.5 EBULLIOSCOPIC DETERMINATION OF MOLECULAR WEIGHT OF THE

COMPLEXES

42

Each of the complexes (10 mg) was dissolved in absolute ethanol (50 ml) in a small

beaker. 2 ml of each of the solutions was put in a narrow fusion tube connected to a

thermometer by a thread, and heated in paraffin bath. The boiling point elevation in each case,

i.e. the difference between the boiling point of the solution and that of pure ethanol, was

recorded. The approximate molecular weight M2 of each solute was calculated using the

Raoult’s law:

M2 = Kb . W2 . 1000

∆Tb W1

Where Kb = Ebullioscopic constant (1.22 molal-1 for ethanol)

∆Tb = Boiling point elevation

W2 /W1 = concentration of the solute

2.6 JOB’S METHODS OF CONTINUOUS VARIATION

Standard solution (l mmole) each of ferric chloride and the ligands was prepared.

Different volumes (1ml, 2ml … 9ml) of the ferric chloride solution were delivered into nine

appropriately labeled test tubes using a clean burette. Different volumes of the ligands solution

were added to the test tube containing the ferric chloride solution such that the total volume of

each mixture is 10.00 ml. This keeps the total number of mole of reactants constant throughout

the series of mixtures but varies the mole fraction of each reactant from mixture to mixture.

Each of the solution mixture was shaken thoroughly, stirred in a small beaker with magnetic

stirrer for 30 mins and allowed to stand for 24 hrs to equilibrate. Samples were removed from

each test tube and placed in the cuvette of a UV-visible spectrophotometer. The absorbances

were read at the λ max of each complex. A graph of mole fraction of ferric ion versus

absorbance was plotted and analyzed carefully to determine the mole fraction of ferric ion

when there is the most significant change in absorbance.

The maximum change will occur when the mole fraction of the reactants is closest to the

actual stoichiometric ratio. Both the formular of the complex and stoichiometry were

determined using this approach.

2.7 DETERMINATION OF SOME PHYSICIOCHEMICAL PARAMETERS OF THE

COMPLEXES AND LIGANDS

2.7.1 AQUEOUS SOLUBILITY

43

Saturated solution (10 ml) of each of the complexes and ligands at ambient temperature was

evaporated to dryness in an evaporating dish. The mass of the solid left in each case was

determined. The solubilities were calculated using the relation:

S =mass

volume1000X

The solubilities of the complexes were compared with those of the ligands.

2.7.2 THERMAL AND ACID STABLITIES

The relative thermal and acid stabilities of the complexes were determined

spectrophotometrically. Dilute solutions of the complexes (0.1 mg/ml) were prepared, their

absorption spectra generated and the wavelength at maximum absorption band (λ max) in each

case noted. Seven 0.1 mg/ml solutions of each of the complexes were prepared and the

temperature regulated to 20oC, 30oC, 40oC, 50oC, 60o, 70oC and 80oC respectively. The

solutions were allowed to stand for 24hrs and the absorbance in each case measured. Similarly,

the same concentration of these solutions was prepared at the pH range of 1-6 and the changes

in absorbance measured. These changes in absorbance with pH and temperature, assuming

Beer Lambert’s law is obeyed, is a measure of the stability of the complexes.

2.8 DETERMINATION OF MELTING POINT

Small quantities of the complexes and the isatin Schiff base were placed in a narrow

capillary tube sealed at one end and placed into the melting point apparatus. The apparatus was

switched on and the temperature allowed to increase gradually. The tube was observed through

the magnifying glass and the temperature at which each of the compounds just started flowing

was noted as the melting point

2.9 SPECTRAL ANALYSIS

2.9.1 UV-VISIBLE SPECTRAL ANALYSIS

The UV-Visible spectra of the Fe3+ complexes of ciprofloxacin, cloxacillin, amoxicillin and

the isatin Schiff base (in distilled water) and the ligands (in distilled water, dioxan, methanol

44

and ethanol respectively) were recorded in UV 6305 PC spectrophotometer (Jenway) using

1cm quartz cuvettes.

2.9.2 INFRARED SPECTRAL ANALYSIS

The IR spectra of the complexes and the ligands were determined at Sheda Science and

Tech. Complex, Abuja. The compounds were prepared in KBr disc and the spectra recorded in

FTIR (Shimadzu, ® Japan).

2.10 ANTIMICROBIAL STUDIES ON THE COMPLEXES

The ligands and complexes were assayed for antimicrobial activity by the agar diffusion

method. Solutions of the complexes and pure ciprofloxacin were made in distilled water and

those of the other ligands in DMSO.

2.10.1 PREPARATION OF THE CULTURE MEDIA

The culture media employed for the anti-microbial investigation were nutrient agar, for

bacteria, and Saboraud’s dextrose agar for fungi and yeast.

Nutrient agar formular

Beef extract 1.0 g NaCl 5.0 g

Peptone 5.0 g Distilled water 1000 ml

Agar 15.0 g Yeast extracts 2.0 g

Approximate pH 7.4

Saboraud’s Agar Formular

• Mycological peptone 10.0 g

• Dextrose 40.0 g

• Agar 15.0 g

• Distilled water 1000 ml

• Approx. pH 5.2

The various quantities of the needed ingredients were mixed together and dissolved in 1

dm3 of distilled water and allowed to stand for about 15 mins. The mixture was distributed in

bijou bottles and sterilized by autoclaving at 121oC for 15 minutes. The sterilized media were

maintained in a molten state until used.

2.10.2 MAINTENANCE AND ACTIVATION OF THE MICROORGANISMS

45

The microorganisms had been maintained by weekly sub culturing and incubation at 37 oC

(for bacteria) and 25 oC (for fungi and yeast). A 24 hour old culture of test organism was

always employed. A constant cell population size of 106 Cfu /ml was used throughout the

studies. The cell size was established by dilution and standardization through comparison with

MacFalan optical density.

2.10.3 MICROBIOLOGICAL SENSITIVITY SCREENING TEST

Sterile Petri dishes (sterilized at 170 oC for 1 hr. in hot air oven) were prepared and

labeled. Exactly 0.1 ml of 106 cfu/ ml suspension of microorganisms was placed aseptically at

the centre of the Petri dishes using a sterile pipette and 30 ml of molten nutrient agar or

Saboraud’s dextrose agar poured into them. The seeded dishes were rotated to ensure even

spread of seeded organisms and then allowed to solidify.

When set, wells were bored in the seeded dishes with a cork borer (8 mm in diameter). The

agar disc diffusion method (Lovian, 1980) was followed. Each sample was tested at the same

concentration level of 5 mg/ml. Two drops of each of the sample solution were dropped in the

wells using a pipette. The plates were incubated at 32 oC for 24 hours (for bacteria) and at 25oC

for 48 hrs. (for fungi and yeast). For each test, double determination was made and mean values

of the clear inhibition zone diameter (IZD) measured and recorded after incubation.

2.10.4 DETERMINATION OF MINIMUM INHIBITORY CONCENTRATION

(MIC)

The preparation of seeded plates followed the same procedure as described above. To

determine the MIC, each of the plates was divided into six sectors and wells of 8 mm diameter

bored on each sector using sterile cork borer. Six dilutions of each solution of the ligands and

complexes were made (5, 2.5, 1.25, 0.625, 0.3125 and 0.175 mg/ml) and two drops of each

solution placed on appropriately labeled cup on each dish in increasing order of concentrations.

The plates were incubated as above. Triplicate plates were prepared for each determination and

zones of inhibition measured and recorded as mean IZD± SEM. A linear plot of square of

inhibition zone diameter (IZD2) against logarithm of concentration was made for each

organism and test sample. The antilog of the intercept at the log conc. axis (i.e. when IZD2 = 0)

represents the MIC.

46

CHAPTER THREE

RESULTS

3.1 YIELD OF THE SYNTHESIS

The percentage yield, colour and the melting point of the products are listed in table 3.1

Table 3.1 PERCENTAGE YIELD OF THE COMPLEXES

47

Cpf-

Fe3+

Cloxa- Fe3+ Amoxil--

Fe3+

Ampi-

Fe3+

Isat-eth-

Fe3+

Isat-eth Schiff

base

Duration of reaction

3 hrs 4 hrs 4 hrs 4 hrs 2 hrs 2 days

Solvent Ethanol/ water

Dioxane Methanol Methanol Ethanol Ethanol

Initial Colour

Brown Rusty brown

Brown Brown Brown Orange

Final colour

Red Clay-brown

Green Brown Brown Brown

Melting point (°C)

172

150

142

46

225

210

Percentage Yield (%)

80

95

63.5

32

65

77

3.2 MOLECULAR WEIGHT AND STOICHIOMETRY OF THE COMPLEXES

The molecular weight and stoichiometry of the complexes are given in table 3.2. They were

determined through boiling point elevation and Job’s method of continuous variation

respectively.

Table 3.2 APPROXIMATE MOLECULAR WEIGHT OF THE COMPLEXES

Approx. Mol. Weight (g mol-1) Expected Metal- ligand

Stoichiometry

CPF-Fe3+ 891 1:2

Cloxa- Fe3+ 586 1:1

Amoxil- Fe3+ 532 1:1

Amp- Fe3+ 917 1:2

Isatin-eth. ligand 182 1:1 (isatin:ethylenediamine ratio)

Isat-eth.- Fe3+ complex 543 1:2

3.3 PHYSICOCHEMICAL PROPERTIES OF THE COMPLEXES AND LIGANDS

3.3.1 AQUEOUS STABILITY

The solubilities of the complexes and the ligands in water at room temperature are displayed

in table 3.3.

Table 3.3 AQUEOUS SOLUBILITY

Pure Ligand Fe3+ Complex

48

Compound Solubility (g dm-3) Solubility (g dm-3)

Ciprofloxacin < 1 >5

Cloxacillin negligible 1.8

Amoxicillin negligible 2.0

Ampicillin negligible 2.0

Isatin 0.5

Isatin-eth. Schiff base 0.8 2.2

3.3.2 RELATIVE THERMAL STABILITY

The absorbance of 0.1 mg/ml solutions of each of the ligands and their iron (III) complexes at

different temperatures are given in tables 3.4 and 3.5 respectively. These changes in absorbance

with temperature are a measure of the thermal stability of the compounds.

Table 3.4 ABSORBANCES OF THE COMPLEXES AT DIFFERENT TEMPERATURES

Complexes 20°C 30°C 40°C 50°C 60°C 70°C 80°C λmax

Cpf-Fe3+ 0.609 0.686 0.689 0.686 0.665 0.635 0.629 443 nm

Cloxa-Fe3+ 0.114 0.122 0.122 0.120 0.109 0.102 0.083 385 nm

Amoxil-Fe3+ 0.095 0.109 0.128 0.133 0.126 0.122 0.105 440 nm

Isat-eth-Fe3+ 0.525 0.516 0.522 0.514 0.511 0.407 0.223 495 nm

Table 3.5 ABSORBANCES OF THE LIGANDS AT DIFFERENT TEMPERATURES

Ligands 20°C 30°C 40°C 50°C 60°C 70°C 80°C λmax

Ciprofloxacin 0.348 0.362 0.311 0.240 0.170 0.104 0.081 315

Cloxacillin 0.721 0.727 0.683 0.455 0.337 0.190 0.184 290

Amoxicillin 0.661 0.664 0.643 0.428 0.369 0.312 0.126 254

Isat. Schff base 0.304 0.306 0.296 0.217 0.212 0.105 0.101 300

3.3.3 RELATIVE ACID STABILITY

The absorbance of 0.1 mg/ml solution of each of the ligands and their iron (III) complexes

at different pHs are given in tables 3.6 and 3.7 respectively. These changes in absorbance with

pHs are a measure of the acid stability of the compounds.

Table 3.6 ABSORBANCES OF THE COMPLEXES AT DIFFERENT pHs

49

Complexes pH 1 pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 λmax

Cpf-Fe3+ 0.216 0.246 0.256 0.291 0.360 0.362 0.368 443 nm

Cloxa-Fe3+ 0.008 0.013 0.053 0.055 0.072 0.110 0.122 385 nm

Amoxil-Fe3+ 0.645 0.511 0.452 0.360 0.124 0.118 0.109 440 nm

Isat-eth-Fe3+ 0.417 0.424 0.443 0.448 0.502 0.514 0.516 495 nm

Table 3.7 ABSORBANCES OF THE LIGANDS AT DIFFERENT pHs

Ligands pH 1 pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 λmax

Ciprofloxacin 0.150 0.152 0.171 0.220 0.300 0.315 0.360 315

Cloxacillin 0.437 0.449 0.550 0.554 0.696 0.704 0.722 290

Amoxicillin 0.387 0.404 0.499 0.511 0.603 0.620 0.664 254

Isat. Schff base 0.123 0.178 0.283 0.283 0.291 0.306 0.308 300

ANTIMICROBIAL RESULTS

The inhibitory zone diameter (IZD), in mm, and the minimum inhibitory concentration

(MIC) of the ligands and complexes at a concentration of 5 mg/ml are given in tables 3.8 and

3.9 respectively

Table 3.8 RESULT OF SENSITIVITY SCREENING

Ciprofloxacin Cloxacillin Amoxicillin Ampicillin Isatin

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Pure

Isatin

Schif

base

Fe3

Cplx

50

S.aureus 44.0 48.0 31.5 - 31.5 - 30.0 - 18.0 20.5 24.0

B.subtilis 45.0 46.0 32.0 - 32.0 - 30.0 - 18.5 20.0 24.0

P.aeruginosa 48.5 48.0 20.0 - 29.5 - 28.0 - 14.0 12.5 9

E.coli 40.0 45.0 28.0 - 34.5 - 33.5 - 17.5 15.0 9

S. typhi 48.5 11.0 27.0 - 36.0 - 35.5 - 14.0 13.0 9

Shigella spp. 30.0 27.0 30.0 - 35.0 - 30.0 -

Asp. Niger - - 30.0 - 28.0 - 35.0 -

C.albicans - - - - - - - -

Table 3.9 MINIMUM INHIBITORY CONCENTRATION (MIC) OF THE DRUGS IN µg/ml

TABLE 3.10 RESULT OF JOB’S METHOD OF CONTINOUS VARIATION FOR

CIPROFLOXACIN COMPLEX

Metal- ligand ratio Mole fraction of Fe3+ Absorbance

1:9 0.1 0.337

Ciprofloxacin Cloxacillin Amoxicillin Ampicillin Isatin

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Ligand Fe3+

Cplx

Pure

Isatin

Schif

base

Fe3

Cplx

S.aureus 12.0 7.9 12.5 - 12.5 - 12.5 - 125.0 125 25.0

B.subtilis 6.3 5.0 12.5 - 12.5 - 12.5 - 125.0 125 25.0

P.aeruginosa 3.2 3.2 22.0 - 14.0 - 15.0 - 177.0 177 >200

E.coli 2.8 2.8 14.5 - 12.5 - 12.5 - 125.0 125 >200

S. typhi 0.18 177 14.5 - 11.0 - 12.5 - 177.0 177 >200

Shigella spp. 14 12.5 12.5 - 11.0 - 12.5 -

Asp. Niger - - 14.5 - 15.0 - 15.0 -

C.albicans - - - - - - - -

51

2:8 0.2 0.692

3:7 0.3 0.943

4:6 0.4 0.882

5:5 0.5 0.742

6:4 0.6 0.600

7:3 0.7 0.453

8:2 0.8 0.280

9:1 0.9 0.157

Table 3.11 RESULT OF JOB’S METHOD OF CONTINOUS VARIATION FOR

CLOXACILLIN COMPLEX

Metal- ligand ratio Mole fraction of Fe3+ Absorbance

1:9 0.1 0.150

2:8 0.2 0.224

3:7 0.3 0.283

4:6 0.4 0.360

5:5 0.5 0.411

6:4 0.6 0.288

7:3 0.7 0.215

8:2 0.8 0.158

9:1 0.9 0.092

Fig 3.1 Job’s Plot for Ciprofloxacin Complex

52

The mole fraction of Iron (III) at the intersection of the two straight lines is 0.32. The mole ratio is 3.2: 6.8 which corresponds to 1: 2metal – ligand stoichiometry.

0

0.1

2.0.

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 0.2 0.4 0.6 0.8 1Mole Fraction of Fe3+

Absorbance

53

Fig 4.2 Job's Plot for Cloxacillin Complex

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

Mole Fraction of Iron (III)

Abs

orba

nce

Series1

The mole fraction of Iron (III) at the intersection of the two straight lines is

0.48.The mole ratio is 4.8 : 5.2 which corresponds to 1 : 1 metal – ligand

stoichiometry.

3.5 SPECTRAL DATA

The UV-Visible and infrared spectra of the drugs are displayed in figs. 3.3 – 3.13

54

Fig. 3.3 UV-VISIBLE SPECTRUM OF CIPROFLOXACIN

55

Fig.3.4 UV-Visible Spectrum of Fe3+ Complex of Ciprofloxacin

56

Fig.3.5 UV-Visible Spectrum of Amoxicillin Complex

57

Fig. 3.6 UV-Visible Spectrum of Fe3+ Complex of Cloxacillin

58

Fig. 3.7 UV-Visible Spectrum of Fe3+ Complex of Isatin Schiff Base

59

Fig. 3.8 UV-Visible Spectrum of Ferric Chloride

60

Fig. 3.9 Infrared Spectrum of Ciprofloxacin

61

Fig. 3.10 Infrared Spectrum of Fe3+ Complex of Ciprofloxacin

62

Fig. 3.11 Infrared Spectrum of Fe3+ Complex of Cloxacillin

63

Fig. 3.12 Infrared Spectrum of Fe3+ Complex of Amoxicillin

64

Fig. 3.13 Infrared Spectrum of Fe3+ Complex of Isatin Schiff Base

65

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 POSSIBLE EQUATIONS OF THE REACTIONS

Iron, just like most transition elements mainly exhibits coordination number of 6 giving

an octahedral structure, though a few tetrahedral complexes, where it assumes coordination

number of 4, are formed. (Lee, 1996). From the infrared spectra, approximate molecular

weight and stoichiometric ratio and assuming coordination number of 6 or 4, the following

structures have been proposed for the complexes

Formation of Fe3+ - Ciprofloxacin Complex

N

OFC

O

O

N

O

N N

F C

O

O

Fe N NN

OF COOH

NN

H

FeCl3

Cl

Cl

H H2

Ciprofloxacin is considered the best of the second generation quinolones family. There

have been several reports about the synthesis and crystal structure of metal complexes with

ciprofloxacin. Recently, it has been found that during the synthesis of a metal complex

containing the fluoroquinolone ligand, the piperazinyl group detaches and is substituted by a

chloro group. (Guangguo et al, 2003)

66

Proposed Structure for Fe3+ Complex of Amoxicillin

Cl

CH3

CH3

CH-C-NH

NH2O

ON

S

O

OC

Fe

2+O

The green colour of the complex suggests reduction of the iron (III) ions to iron (II).

Participation of a univalent negative ligand such as the chloro group in coordinate covalent

bonding with the metal (reductive substitution) has been suggested. This could account for

this observation. Participation of the phenolic oxygen is also suggested since the absorption

band for the OH group is lacking in the infrared spectrum of the complex.

Proposed Structure for Ampicillin Complex

CH3

CH3

CH-C-NH

NH2

N

S

OO

C

OO

Fe

3+

Steric crowding of bonds in the molecule and the use of the amide nitrogens for

coordination are suggested to have caused the poor yield of this complex

67

Formation of Isatin- ethylenediamine (Schiff base) Ligand

This is a Shiff reaction; reaction between a carbonyl group of the indolin-1, 2-dione

and the amino group of the ethylenediamine to form indolin-2- imine (Schiff base)

N

H

H2NCH2CH2NH2

O

O N

H

O

NCH2CH2NH2

+

Proposed Structure for Fe3+ Complex of the Isatin Schiff Base

N

H

O

NCH2CH2NH2

N

H

O

H2NH2CH2CN

Fe

68

Proposed Structure for the Fe3+ Complex of Cloxacillin

CH3

OFe

Cl C-NH

NO

CH3

ON

S

OC

OCH3

3+

In this complex a coordination number of 4 has been proposed for iron giving a

tetrahedral or square planar complex. This is because of the bulky yield of the complex

during synthesis. The use of amide nitrogen for coordination is possible but not often

favoured energetically due to low availability of electrons brought about by inductive and

mesomeric effects. In addition, Fe3+ shows a preference for forming complexes with ligands

which coordinate through O as opposed to N. (Lee, 1996)

4.2 ANTIMICROBIAL RESULTS

Ciprofloxacin complex shows improved antibacterial activity against S. aureus and

B.subtilis when compared to that of the ligand. It had almost the same activity against

P.aeruginosa, E.coli and Shigella as the ligand and lower activity against Salmonella typhi

when compared with that of the ligand as shown in the MIC result. Fe3+ complexes of

amoxicillin, ampicillin and cloxacillin showed decreased antibacterial and antifungal activity

when compared to those of the corresponding ligands. This could be attributed to loss of some

essential pharmacophoric moities due to coordination with the metal ion. The sites used for

dative bonding with the central metal ion are no longer available for binding with the biological

receptors in the microorganisms. This can also be explained on the basis of the mode of action

of the drugs. Several antibiotics have the property of forming chelates with metal ions needed

for normal functioning of enzyme system in the microorganisms and consequently destroy

them. The complexed form of the drugs is not capable of such chelation since their binding

69

sites are already occupied. A deviation from the optimal lipophilicity due to increased aqueous

solubility can also account for the decreased activity. Fe3+ complex of Schiff base derived from

isatin and ethylene diamine shows enhanced activity against B. subtilis and S. aureus, but less

activity against P. aeruginasa, E. coli and samonella. The influence of the central metal ion on

the antimicrobial activity of isatin complexes is evident.

4.3 THERMAL AND ACID STABILITIES

Complexation offers a useful means of manipulating the redox potentials of drug

molecules, which determines reactivity and hence the stability of the molecules. The higher the

redox potential the more the reactivity and hence, lower stability and vice versa.

From the results of the absorbance of the complexes and ligands at different temperatures

and pH, the differences in absorbance were very significant in the ligands but not very

significant in the complexes even at harsh conditions: 80oC and at low pH of 1. This shows that

the concentration of these complexes were still high at such adverse conditions. This is as a

result of the greater ability of the complexes to withstand acid medium (less ease of acid

hydrolysis). Pure Ciprofloxacin and penicillins are not appreciably stable in aqueous and acid

media. One can, therefore conclude that these complexes are more stable than the pure ligands

and hence have advantage over the ligands as drugs.

4.4 INFRARED SPECTROPHOTOMETRICAL DATA Ciprofloxacin Complex:

I.R Absorption Band (cm-1) Inference (functional group)

2730 C-H Stretching (-CH2 -of the cycloprophyl

ring)

1616.06 Sp2 C=C of aromatic ring or C=O group

coordinated to metal

3392 (weak and broad) -OH group coordinated to metal

1454 Sp2 C-C of the aromatic ring

Pure Ciprofloxacin

3528 OH group of the carboxyl group

1707 C=O group of ketone

1627 C=C of aromatic

70

The disappearance of the strong absorption peak at 3528cm-1 (found in the

ligand but not in the complex) indicates the loss of the OH group of carboxyl

group to coordination with the metal. The band at 1707.6cm-1 due to the

ketone group, was not detected in the spectrum of the complex, indicating

that this moiety participates in coordinate bonding with the metal ion. The

corresponding band was recorded at 1616cm-1. The shift towards lower wave

number is consistent with this participation.

Isat- eth-Fe3+ Complex

Infrared Data (cm-1) Inference (Functional Group)

2923.57 C-H aliphatic

3399.90 (weak and broad) Bound –NH of ethylenediamine

1608 C=C of aromatic ring

The absence of any strong absorption band in the spectrum of this complex is an indication

that all the functional groups present are bonded to the metal ion. The ligand shows strong

absorption bands at about 1720cm-1 and above 3400cm-1 due to the carbonyl and amino groups

respectively.

Cloxacillin Complex

Infrared Data (cm-1) Inference (Functional Group)

1737.55 C=O group of acid or amide

1658.49 Sp2 C=C of aromatic

3000-2724 (very prominent peak) C-H aliphatic

1610.28 Sp2 C-C of aromatic

71

Amoxicillin –Fe3+ Complex

Infrared Data (cm-1) Inference (Functional Group)

2850 C-H stretching (-CH3)

1747 C=O of acid

1454 Sp2 C-C of aromatic ring

1376 Geminal dimethyl group on the thiazoline ring

840 Para disubstituted benzene ring

The absence of any strong absorption band beyond 3000 cm -1 indicates participation of the

amino group of the side chain and the -OH group of the phenolic group in dative bonding

with the metal.

72

4.5 CONCLUSION

Complexation improves the aqueous solubility, thermal and acid stabilities of ciprofloxacin,

penicillins and Isatin. This improvement in solubility can be exploited in parenteral

administration of these drugs. Loss of these drugs to deterioration during storage would be

minimized if they are stored in the form of their coordination complexes. Complexes of these

drugs, therefore, have advantage over the pure drugs due to this inherent stability. Oral

administration of drugs in their complex forms may reduce absorption in the lipid layers. This

is because the improved aqueous solubility would result in lower partition coefficient. Co-

administration of these drugs with iron tablets is therefore discouraged because complexation

may occur in vivo.

The structure – activity relationship of drugs could be predicted by complexation reactions.

If a particular biological activity of a drug is lost or diminished on complexation with metal

ions, it would be reasonable to suggest that one or more of the groups bonded to the metal is

necessary for activity. Further tests can be conducted to confirm the pharmacophoric moieties.

Most coordination compounds of Isatin (indolindione) are more effective antibacterial

drugs than isatin itself. The activity depends on the central metal ion of the complexes.

73

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