thesis titas mukherjee.pdf

Coordination Chemistry of Some Platinum

Metals with N, S and O Donor Ligands:

Synthesis, Characterization and Reactivity

THESIS SUBMITTED THESIS SUBMITTED THESIS SUBMITTED THESIS SUBMITTED

FOR FOR FOR FOR

THE DEGREE OFTHE DEGREE OFTHE DEGREE OFTHE DEGREE OF

DOCTOR OF PHILOSOPHY DOCTOR OF PHILOSOPHY DOCTOR OF PHILOSOPHY DOCTOR OF PHILOSOPHY ININININ SCIENCE SCIENCE SCIENCE SCIENCE (CHEMISTRY)(CHEMISTRY)(CHEMISTRY)(CHEMISTRY)

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TITAS MUKHERJEE TITAS MUKHERJEE TITAS MUKHERJEE TITAS MUKHERJEE

Department of ChemistryDepartment of ChemistryDepartment of ChemistryDepartment of Chemistry

The University of BurdwanThe University of BurdwanThe University of BurdwanThe University of Burdwan

BurdwanBurdwanBurdwanBurdwan----713104, India713104, India713104, India713104, India

JuneJuneJuneJune----2014201420142014

“All truths are easy to understand once All truths are easy to understand once All truths are easy to understand once All truths are easy to understand once

they are discoveredthey are discoveredthey are discoveredthey are discovered,,,, the point is to the point is to the point is to the point is to

discover them.discover them.discover them.discover them.”

-----Galileo GalileiGalileo GalileiGalileo GalileiGalileo Galilei

DedicatedDedicatedDedicatedDedicated

totototo

My Beloved FamilyMy Beloved FamilyMy Beloved FamilyMy Beloved Family

THE UNIVERSITY OF BURDWAN

Dr. Pabitra Chattopadhyay

Associate Professor

Department of Chemistry

Golapbag Burdwan –713104, India Ph.: +91-342-2558554 extn. 424 Fax: +91-342-2530452 Mobile: +91-9434473741 e-mail: [email protected]

Dated: 02-06-2014

To whom it may concernTo whom it may concernTo whom it may concernTo whom it may concern

This is to certify that the thesis entitled “Coordination Chemistry of Some Platinum

Metals with N, S and O Donor Ligands : Synthesis, Characterization and Reactivity” is the

result of work done by Mrs. Titas Mukherjee, M. Sc., who started her work in November, 2009

and has registered her name in the University of Burdwan for the award of Doctor of Philosophy

in Science (Chemistry) under my guidance and supervision (vide Regn No.: R-

PhD/Regn./Chem/SC/348 dated 19-12-2011). She has fulfilled all the conditions necessary for

the award of Ph. D. degree in Science (Chemistry) of The University of Burdwan. This work

described in present dissertation has never been submitted in full or part, for the award of any

degree by the candidate or by any other person.

Certified further that Mrs. Mukherjee has completed the Ph.D. Course Work framed by

Department of Chemistry, The University of Burdwan, and delivered one seminar lecture on

January 31, 2014 defending her dissertation and the lecture was highly appreciated by the

audience.

( Pabitra Chattopadhyay)

CONTENTS

ACKNOWLEDGEMENT a-b

PREFACE c-d

CHAPTER-I A partial review of platinum metal complexes with N,S

and O donor ligands

I.1. Platinum Metals

I.2. Ruthenium

I.3. Osmium

I.4. Rhodium

I.5. Iridium

I.6. Palladium

I.7. Platinum

I.8. N, S and O donor Ligands (Literature survey)

I.9. Present Work

I.10. Physical measurements

References

1-65

CHAPTER-II

Palladium(II) Complexes of Dithiocarbamic Acids:

Synthesis, Characterization, Crystal Structure and DNA

Binding Study

II.1. Introduction

II.2. Experimental Section

II.3. Results and Discussions

II.3.1.Synthesis and Characterizations

II.3.2.Structural description of complexes 1 and 4

II.3.3.Spectroscopic Analysis

II.3.4.DNA Binding Experiments

II.4. Epilogue

References

66-95

Chapter-III

Palladium(II) and Platinum(II) Complexes of

Deprotonated Carboxamide Ligands: Synthesis,

Structural Characterization and Binding Interactions

with DNA and BSA

III.1. Introduction

III.2. Experimental Section

III.3. Results and Discussions

III.3.1.Synthesis and Characterizations

III.3.2.Structural description of complexes 1a, 1b and 1c

III.3.3.Spectroscopic Analysis

III.3.4.Computational Details

III.3.5. Binding Experiments with Calf Thymus DNA

III.3.6. Binding Experiments with BSA of 1a and 1b

III.4. Epilogue

References

96-134

CHAPTER-IV

Neutral Cyclometalated Rhodium(III) Complexes

Bearing Dithiocarbamate Derivative: Synthesis,

Structural Characterization, Antibacterial Activity and

DNA Binding Study

IV.1. Introduction

IV.2. Experimental Section

IV.3. Results and Discussions

IV.3.1.Synthesis and Characterizations

IV.3.2.Structural description of complex 1

IV.3.3.Spectroscopic Analysis

IV.3.4.Theoretical Calculation

IV.3.5. DNA Binding Experiments

IV.3.6. Antimicrobial Screening

IV.4. Epilogue

References

135-161

CHAPTER-V

Synthesis, Characterization, Interaction with DNA and

Bovine Serum Albumin(BSA), and Antibacterial Activity

of Cyclometalated Iridium(III) Complexes Containing

Dithiocarbamate Derivative

V.1. Introduction

V.2. Experimental Section

V.3. Results and Discussions

V.3.1.Synthesis and Characterizations

V.3.2.Structural description of complex 2

V.3.3.Spectroscopic Analysis

V.3.4.Theoretical Calculation

V.3.5. DNA Binding Experiments

V.3.6. Antimicrobial Screening

V.3.7. BSA Binding Experiments

V.4. Epilogue

References

162-193

CHAPTER-VI

Synthesis of platinum (II) complexes of some new

pyrrolyl azo ligands: spectroscopic characterization and

studies on DNA intercalation

VI.1. Introduction

VI.2. Experimental Section

VI.3. Results and Discussions

VI.3.1.Synthesis and Characterizations

VI.3.2.Structural description of ligand HL7

VI.3.3.Spectroscopic Analysis

VI.3.4.DNA Binding Experiments

VI.4. Epilogue

References

194-213

SYNOPSIS i - xiii

LIST OF PUBLICATIONS & ATTENDING CONFERENCES A - C

REPRINTS

a

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

I owe a great deal of thanks to a number of people who have supported me during my life

and academic career. Foremost, I wish to express my sincere gratitude for the mentoring of

Professor Dr. P. Chattopadhyay through the years of my thesis research. It’s difficult to put into

words how much I have matured and learned while a member of his group. It is equally difficult

to describe my owe and gratefulness for the endless offerings of knowledge and insightful

suggestions from Prof. Chattopadhyay. Not only has my knowledge of coordination chemistry

been expanded tremendously, but the development of my understanding of how to plan, execute,

and critically evaluate chemical research is something that will no doubt be the foundation for

success in my professional career.

I also wish to express my appreciation of my teachers at The Department of Chemistry,

The University of Burdwan, Dr. A. K. Ghosh, Head, Department of Chemistry, B.U, Prof. S.

Basu, Prof A. K. mukhopadhyay, Prof. B.K. Ghosh, Dr. S. Bhattacharya, Prof. S. Laskar, Prof. P.

Ghosal, Dr. B. Ray, Dr. D. Das, Dr. T. Das, Dr. B. Mondal, Dr. R. Ghosh Dr. A. Sinhababu, Dr.

S. S. Bhattacharya for their worthy suggestions and tremendous support during my research

activity. My friend as well as the faculty member of B. U, Mrs. M. Baskey demands a special

acknowledgement for her valuable cooperation during my research time. I would also like to thank

all of staffs at Department of Chemistry, B. U. for their kind helps in everything that they can.

Besides, I would like to thank the Department of Science and Technology (DST) and

University Grant Commission (UGC), New Delhi, India for financial support and the

Department of Chemistry, University of Burdwan for research facilities. I gratefully

acknowledge the authority of Sadya High school, Sadya, Burdwan, for the cooperation in doing

my research work.

I owe a special thanks to Prof. E. Zangrando, Dipartimento Di Scienze Chimiche, Trieste,

Italy; Jaromir Marek, CEITEC MU, Faculty of Science, Masaryk University, Czech Republic,

Prof. G. Hundal, and Prof. M.S. Hundal, Department of Chemistry, Guru Nanak Dev

University, Amritsar, Punjab, India for their assistance regarding the crystal data collection. I

am grateful to Dr. S. Banerjee, Govt. College of Engineering and Leather Technology, Salt Lake

Sector-III, Kolkata, India and Dr. B. Chattopadhyay, Department of Solid State Physics, Indian

Association for the Cultivation of Science, Jadavpur, Kolkata, India for their help in theoretical

study.

b

Our lab was always an enjoyable place to work, made so by the other people in the group.

I thank them for their welcoming attitude, their passed on knowledge, help and friendship. Life in

the lab would certainly have been much duller without them. I would like to express my heartiest

thanks to the past and present members of the Chattopadhyay’s group Dr. S. Pramanik, Dr. S.

Dhara, Dr. S. Sarkar, Dr. A. Patra, Dr. S. Dey, Mrs. H. Paul,, Mr. B. Mandal, Mr. U. Saha,

Mr. R. Chakraborty, Ms. S. sen, Mr. B. Sen, Ms. M. Mukherjee, Mr. S. Pal and Mr. S. Lohar

and all other research scholars of this department for their instant co-operation and pleasant

association. I am endlessly surprised how many epiphanies and good ideas come from casual

discussions of science with them.

Last, but not the least, my acknowledgement goes out to my family, which has been an

unfaltering source of love and encouragement. It would be hard for anyone to waiver in their

endeavors with the knowledge that they always have such a wonderful family to lift them up in

their difficult times. Anything good in me came from them. I have been blessed with an amazing

family and I could not have done this without them. Most of all I thank my parents for all the

support, belief, and love that they gave me during their life. I would like to convey my gratitude

to all mighty God for giving me such an academic and cooperative family after my marriage also.

I am thankful to my father-in-law and mother-in-law for their unflinching love and constant

encouragement to achieve my goals. My mother-in-law deserves a very special position to me as

her undoubtedly enthusiastic attitude triggered me to complete my research work and thesis

within the stipulated time. I am thankful to my dearest two juniors, my sister and my sister-in-

law for their joyous company throughout my life. Words can merely describe their cooperation

towards my achievement. I am also grateful to my other family members. My maternal uncle,

Mr. A. Chakraborty has also played a very significant role in motivating my research. In

addition, my husband has always made life easy for me, supporting me in my career choice,

cheering me on from the sidelines, and providing companionship during my research activity. I

acknowledge his friendly cooperation and valuable scientific suggestions.

Department of Chemistry,

The University of Burdwan (Titas Mukherjee)

c

PREFACEPREFACEPREFACEPREFACE

The work embodied in this thesis entitled “Coordination Chemistry of Some

Platinum Metals with N, S, O Donor Ligands : Synthesis, Characterization and

Reactivity” was initiated in November 2009, to explore the coordination behaviour of

N,S,O donor sets with some of the platinum metal ions. The characterization of platinum

metal complexes i.e. of Pd(II), Pt(II), Rh(III) and Ir(III) complexes with N,S, O donor

sets are carried out using X-ray crystallographic, spectroscopic and physicochemical

tools. The thesis consists of six chapters on the studies of coordination chemistry of

various platinum metal complexes having N,S,O donor ligands.

Platinum metal complexes having N,S,O donor set has been paid considerable

attention in the coordination chemistry due to the significant role of platinum metal ions

and its complexes in biological systems in several ways. Importance of N,S,O donor sets

and their platinum metal complexes along with the present study are outlined in

Chapter I. The subject matter of Chapter II is the systematic description of syntheses and

structural characterizations of a series of palladium (II) complexes of three

dithiocarbamic acids and their reactivity towards calf thymus DNA. Chapter III includes

syntheses and study of coordination chemistry of three deprotonated carboxmide ligand

systems with palladium (II) and platinum (II) metal ions using different structural

characterization tools along with theoretical study and also the study of the binding

interactions of the complexes with calf thymus DNA and bovine serum albumin.

Chapter IV comprises an account of an interesting coordination behavior of the

cyclometalated rhodium (III) complexes containing three dithiocarbamate derivatives and

their syntheses and structural characterizations . Theoretical study, antibacterial activity

test and calf thymus DNA and bovine serum albumin binding study has also been

integrated in this chapter. Synthesis, characterizations, interactions with calf thymus

DNA and bovine serum albumin, antibacterial study and theoretical study of

cyclometalated iridium (III) complexes containing three dithiocarbamate derivative has

been illustrated in Chapter V. Synthesis, characterization and studies on DNA

d

intercalation of platinum (II) complexes with two pyrrolyl azo ligands are demonstrated

in Chapter VI.

In keeping with the general practice of reporting scientific observation, due

acknowledgement has been made whenever the work described as based on the findings

of other investigators. I must take the responsibility of any unintentional oversights and

errors which might have crept in spite of due precautions.

Department of Chemistry

The University of Burdwan (Titas Mukherjee)

1

CHAPTER-I

A partial review of platinum metal complexes with N,S,O donor ligands

2

I.1 Platinum Metals The members of second and third triads of group VIII (groups 8, 9 and 10) viz.

ruthenium, rhodium, palladium and osmium, iridium, platinum are together known as the

platinum metals because they occur together in platinum bearing ores. These are all rare

elements, their sum total abundance in the earth’s crust is ~2×10-5 %. The electronic configurations of the elements: Ru(4d7 5s1); Rh(4d8 5s1) ; Pd(4d10);

Os(5d66s2); Ir(5d7 6s2); Pt(5d9 6s1). All the six elements have almost the same atomic size.

This is one reason for the platinum metals showing comparable chemical behaviour. The

first ionization potential virtually remains stagnant for Ru(7.36) (710.6) and Rh(7.46)

(720) and then slowly increases to Pt(9.0). General similarities are observed in their

having high ionization potentials and high positive standard electrode potentials for

M2+/M couples. All these elements have a large number of binary compounds namely

oxides, sulphides and halides. Their small atomic size and particularly the very small ionic

size lead expectedly to a dominant complex chemistry. Only in the cases of rhodium,

palladium, iridium, and platinum have the freezing points of the platinum metals been

accurately determined. They are, respectively, 1966, 1554, 2454, and 1773°C. The melting

point is believed to be above 2400°C., and perhaps above 2450°C, and that of osmium is

believed to be about 2700°C. The estimated boiling points of the platinum metals are: Ru,

4900°C. ; Rh, 4500°C. ; Pd, 3000-3980°C. ; Os, 5500°C.; Ir, 5300°C.; Pt, 4530°C.

Scheme 1. Position of platinum metals in periodic table.

3

However, the wide variations in the chemistry of platinum metals arise due to

varying stabilities of the different oxidation states and stereochemistries. The common

oxidation states are: Ru, +3(III); Os, +4(IV), Rh, +3(III); Ir, +4(IV),+3(III); Pd, +4(IV),

+2(II); Pt, +4(IV), +2(II). There is also the inherent tendency of the 4dn and 5dn

configurations to pair their spins. All these lead to formation of diamagnetic low spin

complexes of platinum metals. Besides forming the usual sigma-bonded complexes with

common ligands like H2O, halides, nitrogen and sulphur donor ligands they are also quite

capable of forming an extensive series of π-complexes. With the exception of Pd and Pt

they all form binary carbonyls. All the platinum metals, however, form carbonyl halides,

and other π-bonded complexes such as with isocyanides, mixed complexes with π-bonding

ligands, etc. Finally platinum forms interesting complexes with acetylene, ethylene and

also forms a Pt-C bonded acetylacetonato complex.

Scheme 2. Flow chart for refining of platinum group metals by solvent extraction.

4

I.2 Ruthenium

Ruthenium (Ru) constitutes the group 8 transition metals (Table 1) positioned in

the second row of the periodic table. The name from the latin word Ruthenia (Russia).

Ruthenium is exceedingly rare and is the 74th most abundant metal on earth found in

platinum and other ores. The relative abundance is 7×10-3 %. The ability of ruthenium to

assume a wide range of oxidation states (from -2 to +8) and coordination geometries

provides unique opportunities for catalysis. The most stable oxidation state of this element

is +2 and +3. Both are substitutionally inert, although the latter to a greater extent. This

makes ruthenium unique within the periodic table and is a feature which has proved useful

for a number of single-electron transfer studies. Ruthenium has 26 isotopes whose half-

lives are known, with mass numbers from 90 to 115. Of these 7 are stable: 96Ru, 98Ru, 99Ru, 100Ru, 101Ru, 102Ru, and 104Ru. Naturally, the most common isotope is 102Ru with an

abundance of 31.6%.

Ruthenium is mainly used as a catalyst. Some ruthenium complexes absorb light

throughout the visible spectrum and are being actively researched in various, potential,

solar energy technologies. Ruthenium compounds are encountered relatively rarely by

most people. All ruthenium compounds should be regarded as highly toxic and as

carcinogenic. Compounds of ruthenium stain the skin very strongly. It seems that ingested

ruthenium is retained strongly in bones. Ruthenium oxide, RuO4, is highly toxic and

volatile, and to be avoided.

I.2.1 Properties of the ruthenium

The ability of ruthenium to assume a wide range of oxidation states (from -2 to +8)

and coordination geometries provides unique opportunities for catalysis. The most stable

oxidation state of this element is +2 and +3. Both are substitutionally inert, although the

latter to a greater extent. This makes ruthenium unique within the periodic table and is a

feature which has proved useful for a number of single-electron transfer studies.

5

Table 1. Some parameters of elemental ruthenium.

Parameter

Atomic symbol Ru

Atomic number 44

Atomic weight/g mol-1 101.1

Electronic configuration [Kr] 4d75s1

Density(20°C)/g cm-3 12.2

Melting point/°C 2,250

Boiling point/°C 4,150

ΔHfus/ kJ mol-1 38.59

ΔHvap/ kJ mol-1 619

Electronegativity (Pauling Scale) 2.2

Electrical resistivity (20°C)/μ_ cm 71

Number of stable isotopes 7

Metal radius (12-coordinated) (in pm) 134

Effective ionic radius (6-coordinated) (in pm) 56.5 (Ru5+ ion)

62 (Ru4+ ion)

68 (Ru3+ ion)

71.5 (Ru2+ ion)

I.2.2 Coordination chemistry and biological application of ruthenium

Ruthenium compounds are suitable for use in biological applications due to their

slow ligand exchange rates which are close to those of cellular processes and the

capability of the metal to mimic iron in binding to specific biological molecules. An

additional feature is that the oxidation states Ru(II), Ru(III) and Ru(IV) are accessible

under physiological conditions, with reductants such as glutathione and ascorbate being

capable of reducing Ru(III) and Ru(IV) and the oxidation of Ru(II) readily induced by

molecular oxygen or cytochrome oxidase [1]. The cytological stain, Ruthenium Red,

[(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5]6+, was the first compound of this metal to be used

in biology[2]. Since then, numerous medicinal properties of the metal have been

discovered including antimalarial, antimicrobial and anticancer.

6

Furthermore, the radioactive nucleotide of ruthenium (97Ru) has shown promise as a

radiopharmaceutical, with advantages over the currently widely used technetium

alternative (99mTc) due to its longer half life allowing for more manipulation prior to use;

however there are problems due to its lack of commercial availability [2].

Organometallic complexes of ruthenium is the most common in lower oxidation

states and contain ligands ranging from arenes, allyls and cyclopentadienyls to carbonyl,

nitrido and cyano groups[3]. Since metallo-complexes form the basis of this research their

chemistry has been covered. Ruthenium complexes exist as either mono- or bis-arene

structures, commonly with the metal in either the zero or +2 oxidation state [3].

Ruthenium(II) mono-arene complexes are known as ‘half-sandwich’ compounds with a

pseudo octahedral ‘piano-stool’ structure, as shown in Figure 1 [4].

Figure 1. The general chemical structure of a half-sandwich metal mono-arene complex

(M = Ru). In applications as anticancer agents X is most often a halide and Y,Z is

generally a chelating group with NN, NS, SS, NO or OO donor atoms.

The η6-arene occupies three coordination sites (the ‘seat’), with the other ligands (X,

Y and Z) at the remaining sites (the ‘legs’) [5]. These complexes possess stable metal-

arene bonds, resulting in reduced tendency to oxidise to the +3 oxidation state [6]. Due to

the similarity in size of the two metals, the structures of monoarene complexes of

ruthenium have been found to be very close to those of the osmium analogues with many

bond lengths identical, however their chemistries have been shown to vary considerably

[7]. As a result of the interest in this class of complexes as anticancer drugs, extensive

investigations [8] into the effect of varying the arene [5] and X, Y, and Z [9] on the

properties of the complexes have illustrated the extent to which exchange rates, structures,

redox behavior [10], solution chemistry [11] and bio-chemistry [12] are dependent on the

7

ligands. The knowledge gained has allowed the design of complexes for specific

applications. Although reports of the aqueous chemistry of Ru(III) complexes are much

more limited than for Ru(II) [13], good solubility is generally observed for both metals [4,

6]. This is naturally dependent on the ligands involved and may become important in

terms of tuning complexes for clinical applications.

Ru(II) mono-arene structures with three non-equivalent ligands possess chirality,

with a stereogenic centre at the metal. This gives rise to potential applications as

asymmetric catalysts for a range of reactions, including alkene hydrogenation, Diels-Alder

reactions and alkene metathesis [14]. Bis-arene Ru(II) sandwich complex of the type

shown in Figure 2, on which the work discussed in this thesis is based, may be synthesised

from the appropriate chloride-bridged dimer, [(η6-arene)MCl2]2 (M = Ru), by reaction

with a silver salt in acetone (to remove bound chloride ligands) affording a mono-arene

acetone intermediate. Reaction with a second arene in trifluoroacetic acid (TFA) yields the

bis-arene structure [15]. As for mono-arene complexes, bis-arene complexes are more

highly documented for ruthenium. A search of the Cambridgecrystallographic database

(CCDC) revealed that while there are over 50 X-ray crystal structures of ruthenium bis-

arene complexes. The stark contrast between these numbers and those for mono-arene

complexes of ruthenium of ca. 1600, clearly indicates the limited research to date on

ruthenium complexes.

Figure 2. The general chemical structure of a metal bis-arene complex (M = Ru).

Mono-arene complexes of ruthenium containing biologically-active ligands are

known most frequently for amino acids and peptides where coordination to the metal is

through σ-bonding via functional donor group atoms [16]. Bis-arene complexes are less

common and significantly, there are no reported Ru(III) bisarene complexes of

biologically-active ligands. Nevertheless, there are a number of ruthenium examples

8

including complexes of the essential amino acid phenylalanine [17] and derivatives of

dopamine, a neurotransmitter which is important in the regulation of many internal

pathways [15] (Figure 3). Importantly, the biologically activity of such complexes has not

been investigated to date.

Figure 3. The chemical structures of [(η6-p-cymene)Ru(η6-phenylalanine)]2+ [17] (left)

and [(η6-p-cymene)Ru(η6-H-dopamine)]2+ [12] (right).

I.3 Osmium

Osmium (from Greek osme meaning "smell") is a chemical element (Table 2)

which is hard, brittle, and bluish-white transition metal in the platinum group that is found

as a trace element in alloys, mostly in platinum ores. Osmium is the densest naturally

occurring element. Osmium was discovered 41 years before its second row analogue,

however much less is known about its chemistry. This is most likely due to the higher cost

of the metal and the greater difficulty associated with compound synthesis. Although

osmium is found in a wider range of oxidation states than ruthenium (-2 to +8 compared to

0 to +8), this does not extend to coordination number. Osmium has seven naturally

occurring isotopes, six of which are stable: 184Os, 187Os, 188Os, 189Os, 190Os, and (most

abundant) 192Os.

Third row transition metals are commonly more inert than those in the second and

first rows and consistent with this, osmium is more inert than ruthenium, with ligand

exchange rates around 105 times slower. Because of the volatility and extreme toxicity of

its oxide, osmium is rarely used in its pure state, and is instead often alloyed with other

metals. Those alloys are utilized in high-wear applications. Osmium alloys such

9

as osmiridium are very hard and, along with other platinum group metals, are used in the

tips of fountain pens, instrument pivots, and electrical contacts, as they can resist wear

from frequent operation.

Table 2. Some parameters of elemental osmium.

Parameter

Atomic symbol Os

Atomic number 76


Electronic configuration [Xe]4fJ45d66s2







Electrical resistivity (20°C)/μ_ cm 8.12



Effective ionic radius (6-coordinated) (in pm) 54.5 (Os6+ ion)

57.5 (Os5+ ion)

63 (Os4+ ion)

I.3.1 Properties of the osmium

Although osmium is found in a wider range of oxidation states than ruthenium (-2

to +8 compared to 0 to +8), this does not extend to coordination number. As a result of the

lanthanide contraction, osmium is very similar in size to ruthenium, however due to its

position in the third row, its outer 5d orbitals are highly exposed and hence sensitive to the

electronic nature of coordinating ligands. Apart from the most well-known osmium

10

complex, osmium tetroxide (OsO4), the majority of complexes are low-spin octahedral. In

contrast to both ruthenium and iron, no characteristic cationic aqua ion [M(OH2)6]3+/2+

species has been detected for osmium.

I.3.2 Coordination chemistry and biological application of osmium

The chemistry of transition metal complexes of thiosemicarbazones has been

receiving considerable attention largely because of their pharmacological properties [18].

Thiosemicarbazones usually bind to a metal ion, either in the neutral thione form (1) or in

the anionic thiolate form (2), as bidentate N,S donor ligands forming five-membered

chelate rings [18,19]. However, incorporation of a third donor site (D) into these

thiosemicarbazone ligands, linked to the carbonylic carbon via one or two intervening

atoms, normally results in D,N,S tricoordination (3) [18,20,21]. In this note we report the

chemistry of two ruthenium and osmium complexes of the same ligand, Viz.

salicylaldehyde thiosemicarbazone (Hsaltsc, where H stands for the dissociable proton).

Though free Hsaltsc exists in the thione form (4) [22], it is known to coordinate as a

dianionic tridentate O,N,S donor [21] (see Scheme 3). Reaction of Hsaltsc with

[M(PPh3)3X2] (M ) Ru, Os; X ) Cl, Br) afforded complexes of the type [M(PPh3)2-

(saltsc)2] where the salicylaldehyde thiosemicarbazone ligand is coordinated, in spite of

having the phenolic oxygen as the potential third donor site, as a bidentate N,S-donor

ligand, forming a four-membered chelate ring (5). The steric bulk of the coligand PPh3

appears to be the driving force for this rather unexpected coordination mode of the

salicylaldehyde thiosemicarbazone ligand. The syntheses, characterization, and cyclic

voltammetric properties of these two [M(PPh3)2(saltsc)2] complexes are described here.

11

Scheme 3. N,S donor coordination sites to M = Os.

Basuli et. al. reported the unusual coordination mode of salicylaldehyde

thiosemicarbazone (1, Scheme 4) observed in a group of [M(PPh3)2(saltsc)2] complexes

(where M = Ru, Os and saltsc = anion of salicylaldehyde thiosemicarbazone) [23]. It

appeared that two factors might be responsible for such unusual coordination mode of

salicylaldehyde thiosemicarbazoe: (i) the steric bulk of the two triphenylphosphine ligands

and (ii) intramolecular hydrogen bonding between the phenolic hydrogen and the imine

nitrogen. The present work has originated from their previous attempt to find out the

actual driving force behind such coordination mode of the thiosemicarbazone ligand. To

do that, the triphenylphosphine ligand is kept unchanged while the thiosemicarbazone

ligand has been modified by removing the hydroxy group from the salicylaldehyde

fragment to prevent any intramolecular hydrogen bonding. They have used

thiosemicarbazones of benzaldehyde and two para substituted benzaldehydes (2). The

ligands are abbreviated in general as HL-R, where H stands for the dissociable proton and

R for the substituent. Ligands of this type are known to bind to a metal ion as a

monoanionic bidentate N,S donor forming stable fivemembered chelate ring (3) [24].

However, reaction of these ligands with [M(PPh3)3X2] (where M = Ru, Os and X = Cl, Br)

afforded complexes of type [M(PPh3)2(L-R)2] where the thiosemicarbazone ligand is

12

coordinated as a bidentate N,S donor ligand forming a four-membered chelate ring (4).

The synthesis, structure and cyclic voltammetric properties of the [M(PPh3)2-(L-R)2]

complexes are described here with special reference to the nature of steric interaction

responsible for the observed mode of binding of the thiosemicarbazone ligands.

Scheme 4. Osmium complexes of 1-4.

Medicinal applications of osmium are much less common because it is generally

not considered to be biologically relevant and in some forms can be highly toxic.

However, some osmium complexes have been used for the treatment of rheumatoid

arthritis in animals and anticancer activity has been reported for some Os(III) and Os(IV)

neutral complexes [4]. In addition, trypanocidal activity against T. b. brucei has been

reported [5,6].

13

I.4 Rhodium

Rhodium is a metallic element of atomic number 45 and atomic weight 102.905

which was discovered in 1803 by W.H. Wollaston. The name rhodium naming it after the

Greek word for “rose” because of its rose-color [25], commonly found in aqueous

solutions of its salts. Rhodium is a member of platinum group metals, of which it is found

the widest application due to its several properties. It is unaffected by air and water up to

875 K, and unaffected by acids, but is attached by molten alkalis [26]. Rhodium remains

bright in all atmospheric exposures at room temperatures and is completely resistant to a

variety of corrosives. These properties plus the high and relatively uniform reflectivity

(75-80%) and the ease with which hard, bright electrodeposits can be produced have led to

wide use of rhodium for jewelry, reflectors and electrical contacts. Rhodium has certain

specific uses but the principle application of rhodium is an alloying element for platinum.

The 10% rhodium-platinum alloy is used in the oxidation of ammonia, for spinnerets,

glass-fiber bushings, furnace windings and thermocouples. A trace of rhodium is required

in the liquid bright gold used for decorating glass and porcelain to achieve a very fine-

grained, bright deposit. Rhodium has been used in large quantities as the reduction catalyst

for NOx in catalytic converters. Small amounts of rhodium plus indium or ruthenium are

used in dental alloys to produce a very fine-grained casting.

All the platinum metals are generally associated with each other including rhodium.

However, the relative proportions of the individual metals are by no means constant and

the more important sources of rhodium are the nickel-copper-sulfide ores found in South

Africa and in Sudbury, Canada, which contain about 0.1% rhodium. The production

methods of rhodium are used by the alternative solvent extraction processes and ion

exchange techniques. These methods offer superior efficiency and they are increasingly

replacing the classical process that is not high efficiency and costly recycling [27]. The

diagram for refining rhodium is shown in Scheme 2.

I.4.1 Properties of the rhodium

The oxidation states of rhodium are listed in Table 3. The oxidation state +1 and +3

are the most important, the other oxidation states are rare. The -1 and 0 states occur in

carbonyls including clusters and in nitrosyls, the +2 state principally in the carboxylates,

and the +4, +5 and +6 states mainly as fluorides. The geometry of rhodium(I), (d8) is

14

predominantly square planar, but a few five-coordinate species have been isolated. Many

of the square planar complexes are of importance in catalysis, since the metal atom can

increase its coordinate number by accepting ligands in the apical sites [26]. The geometry

of rhodium (III) (d6) is almost invariable octahedral. The electronic (visible) spectra of

rhodium(III) complexes display two bands toward the blue end of the spectrum, although

in many instances only the first spin allowed ligand-field band (1T1g←1A1g) is observed,

since the second band is often obscured by charge transfer transitions which the [28].

Table 3. Some parameters of elemental rhodium.

Parameter

Atomic symbol Rh

Atomic number 45


Electronic configuration [Kr] 4d85s1






ΔHf (monoatomic gas)/ kJ mol-1 556(±11)



Number of naturally occurring isotopes 1


Effective ionic radius (6-coordinated) (in pm) 55 (Rh5+ ion)

60 (Rh4+ ion)

66.5 (Rh3+ ion)

15

I.4.2 Compounds of rhodium

Heating rhodium metal or the trichloride in oxygen at 600oC, or simply heating the

trinitrate, produces dark-grey Rh2O3; it is the only stable oxide formed by this metal. The

yellow precipitate formed by the addition of alkali to aqueous solutions of rhodium(III) is

actually Rh2O3.5H2O rather than a genuine hydroxide. Electrolytic oxidation of Rh3+

solutions and addition of alkali gives a yellow precipitate of RhO2. H2O, but attempts to

dehydrate this produce Rh2O3. Black anhydrous RhO2 is obtained by heating Rh2O3 in

oxygen under pressure. The octahedral hexafluorides are obtained directly from the

elements and both are volatile, extremely reactive and corrosive solids, RhF6 being the

least stable of the platinum metal hexafluorides. The pentafluorides of rhodium may be

prepared by the thermal dissociation of the hexafluorides. It is also highly reactive and are

respectively dark-red and yellow solids, RhF4 is a purple-red solid, usually prepared by the

reaction of the strong fluorinating agent BrF3 on RhBr3 [29]. The most familiar and most

stable of the halides of rhodium is the trihalides. Those of rhodium range in color from the

red RhF3 to black RhI3. The anhydrous trihalides are generally unreactive and insoluble in

water but, excepting the triiodide which is only known in this form, water-soluble hydrates

can be produced by wet methods. The dark-red RhCl3.3H2O is the most common

compound of rhodium and the usual starting point for the preparation of other rhodium

compounds, and is itself best prepared from the metal sponge. This is heated with KCl in a

stream of Cl2 and the product extracted with water. The solution contains K2[Rh(H2O)Cl5]

and treatment with KOH precipitates the hydrous Rh2O3 which can be dissolved in

hydrochloric acid and the solution evaporated to dryness. RhBr3.2H2O also is formed from

the metal by treating it with hydrochloric acid and bromine.

I.4.3 Coordination chemistry of rhodium

Metal dithiocarbamates are the subject of current research activity due to their

potential applications and their interesting coordination behavior [30]. There are some

examples of dithiocarbamate binuclear compounds, but most complexes are mononuclear

in solution, although in some cases crystallize in polymeric forms [31]. The early

preparation of mononuclear rhodium (I and III) dithiocarbamates was reported by Cotton

and McCleverty [32], while an interesting example of a binuclear complex is the cation

[RhIII2{S2CN(Me)2}5]+, in which the two rhodium atoms achieve an octahedral

16

environment as a consequence of the additional coordination of two bridging sulfur atoms

from two different dithiocarbamate groups (Scheme 5) [33]. Thus, although transition

metal complexes containing dithiocarbamate groups as bridging or, most frequently, as

chelating ligands are well-known, the chemical behavior of dithiocarbamate metal

complexes as metalloligands remains essentially unexplored. Only very recently, the

neutral rhodium and iridium(III) tris(dithiocarbamate) complexes have been described as

metalloligands toward the silver cation Ag+, forming cationic complexes of the type

[Ag{M(S2CNR2)3}2]BF4 (M = Rh, Ir) [34]. It is worth keeping in mind that each of the

two sulfur atoms of a metal-coordinated chelate dithiocarbamate ligand maintains an

additional coordination capability due to the availability of, at least, one pair of potentially

bonding electrons. This potentiality has been contemporarily exploited in the coordination

chemistry of thiolato and pyridine-2-thiolato complexes allowing the tailored synthesis of

homo- and heteronuclear rhodium aggregates [35].

Scheme 5. Preparatory method for dithiocarbamate rhodium complexes.

Octahedral N donor diimine rhodium(III) complexes are of interest because of

their ability to undergo a chemically irreversible two-electron reduction involving ligand

loss to form squareplanar rhodium(I) complexes. This change in coordination number

from six to four opens up sites for binding substrates for catalysis and has been utilized to

carry out the electrocatalytic reduction of CO to methanol [36]. Further, octahedral

17

diimine rhodium(III) complexes are of interest as they have been used in the process of

photochemically reducing H2O to H2 [37]. Tris-diimine [38] and tris-dithiolate [39]

rhodium(III) complexes have been previously reported. The tris-diimine complexes all

exhibit ligand-based π-π* emission in frozen solvent matrixes at 77 K and no emission in

fluid solution at room temperature [40]. By contrast, in frozen solvent matrixes at 77 K,

the tris-dithiolate complexes exhibit metalbased d-d* emission for the

Rh(Me2dtc)3{Me2dtc = dimethyldithiocarbamate} and both d-d* and ligand-tometal

charge transfer (π-d*) for the Rh(SS)3{SS = sulfur analogues of β-diketones} [39]. The

Pt(NN)(SS) complexes exhibit solvatochromic absorption bands and solution

luminescence arising from a metal/dithiolate to a diimine charge transfer excited state

[41]. In addition, they display diiminebased π-π* emission at 77 K. These systems led us

to explore the analogous diimine dithiolate system on a d6 Rh(III) metal center in order to

investigate if unique photophysical and electrochemical properties result and what ligands

are lost during the two electron Rh(III) to-Rh(I) reduction. In mixed ligand systems, the

ligand lost depends on the nature of the ligands present. Mixed ligand systems allow one

to tune the electrochemical potential and affect the reactivity of the rhodium metal center.

We have recently shown that cis-[Rh-(bpy)2(OTf)2][OTf] where bpy = 2,2’-bipyridine and

OTf = trifluoromethanesulfonate acid is a versatile intermediate for synthesizing mixed

diimine ligand complexes of rhodium(III) by a high yield synthetic pathway [42]. Here,

we report on the synthesis of novel [Rh(bpy)2 (SS)][PF6]n complexes, where SS =

dimethyldithiocarbamate (Me2dtc), diethyldithiocarbamate (Et2dtc),

dibenzyldithiocarbamate (Bz2dtc), 1,1-dicyanoethylene-2,2-dithiolate (i-mnt), and 1-

(ethoxycarbonyl)-1-cyanoethylene-2,2-dithiolate (ecda), using the triflate complex as the

starting material.

18

Scheme 6. Synthesis of rhodium complexes.

Since the discovery of a diazo compound by Peter Griess in1858, large numbers of

azo compounds have been reported by several workers [43]. The azo group is among the

most versatile active units, which may be photochromic [44], proton-responsive [45] and

redox active [46], depending on the chemical constitution in the molecule. Diazo

compounds are used as contrast enhanced lithography (CEL) materials and the photoacid

generator (PAG) for printing plates based on thermal crosslinking [47]. Incorporation of

an azo group in N-heterocycles with an inbuilt C=N bond in a conjugated state generates

an azoheterocycle which bears an azoimine function (–N=N–C=N–) [48]. Imidazole is

ubiquitous in chemistry and biology [49]. We have taken a simple strategy to functionalise

imidazole by incorporating an arylazo (Ar-N=N–) group, and arylazoimidazoles have

been used to explore the coordination chemistry with different transition and non-

transition metal ions [50]. We have recently designed 1-alkyl-2-{(o-

thioalkyl)phenylazo}imidazoles [51]. As a ligand, this exhibits both bidentate (N, N0) and

tridentate (N, N’, S) natures depending on the chemical environment of the metal center

19

[52] (see scheme 7). The isoelectronic, d6 Rh(III) and Ir(III) complexes have been less

thoroughly probed compared to the Ru(II) analogs, no doubt in part due to the more

difficult chemistry.

Scheme 7. Different isomeric form of Rhodium complexes with azo ligands(NNS-donor).

I.4.4 Organometallic compounds and biological application of rhodium

Transition-metal-based compounds constitute a class of chemotherapeutics, which

are widely used in the clinic [53]. Especially precious metals, for example, platinum

compounds being used in the treatment of cancer, silver compounds being used for

antimicrobial agents and gold compounds use in the treatment of rheumatoid arthritis [54].

The interaction between DNA molecules and heavy metal compounds has been studied by

several researchers for nanoelectronics, development of antitumor drugs [55] and tracing

biological activity. In recent years, rhodium complexes have attracted in the interaction of

complexes with biomolecules, for example, the binding effect of the antitumor complex

rhodium(II) acetate [Rh2(O2CCH3)4] to the plasmid DNA has been studied under different

molar ratio of [Rh2(O2CCH3)4] compound to base pair of DNA and reaction time. The

structure of [Rh2(O2CCH3)4] is shown in Figure 4.

20

Figure 4. Structure of [Rh2(O2CCH3)4]L, axial ligand; R, carbon chain of carboxylate

groups; R = CH3, rhodium(II) acetate.

For the organometallic compounds, carbonyl hydrides and carbonylate anions are

obtained by reducing neutral carbonyls and in addition to mononuclear metal anions,

anionic species of very high nuclearity have been obtained, often by thermolysis. These

are especially numerous for rhodium and in certain Rh13, Rhl4 and Rh15 anions have

structures conveniently visualized either as polyhedra encapsulating further metal atoms,

or alternatively as arrays of metal atoms forming portions of hexagonal close packed or

body centred cubic lattices stabilized by CO ligands. [Rhl3H3(CO)24]2- (Figure 5a) is

typical. The incorporation of interstitial or encapsulated heteroatoms is a common and

stabilizing feature. Carbon is the most common and may originate from the solvent or

from cleavage of a CO ligand. The carbido C contributes 4 electrons to the cluster bonding

and in the 90-electron species [Rh6C(CO)15]2- features trigonal prismatic coordination of

Rh6 about the central C (Figure 5b). Even more complicated structures are found for the

large rhodium clusters containing 2 carbido C atoms: [Rh12(C)2(CO)25] (Figure 5c); the

cluster also has 14 pendant terminal CO groups, 10 μ-CO groups and one μ3-CO. In

contrast, [Rh15(C)2(CO)28]- has individual 6- coordinate (octahedral) carbide C atoms

symmetrically placed on each side of a central rhodium which itself has 12 rhodium

nearest neighbours in addition to the 2 C atoms. Again, the approach to metal structures is

notable and is one of the main interests in constructing large clusters and studying their

chemical and catalytic activity. The structures of compounds mentioned above are shown

in Figure 5.

21

Figure 5. Schematic representations of the metal cores of some clusters;

(a) [Rhl3H3(CO)24]2-, (b)[Rh6C(CO)15]2- and (c) [Rh12(C)2(CO)25].

H, P, As, S have also been encapsulated in ions such as [Rh13(H)3(CO)24]2-,

[Rh9(P)(CO)21]2-, [Rhl0As(CO)22]3- and [Rh17(S)2(CO)32]3-. More recently N has been

encapsulated in [Rh14(N)2(CO)25]2- and [Rh23(N)4(CO)38]3-. The latter is the largest

rhodium cluster so far characterized. It consists of an irregular polyhedron of 21 rhodium

atoms encapsulating a pair of particularly close (257.1 pm) rhodium atoms as well as 4 N

atoms each of which is located in a semi octahedral site.

Rhodocene, [Rh(5-C5H5)2], is also known but is unstable to oxidation and has a

tendency to form dimeric species. However, the yellow rhodicenium cations are certainly

known and are entirely analogous to the cobalticenium cation in their resistance to

oxidation and susceptibility to nucleophilic attack.

I.5 Iridium

Iridium (group 9), with atomic number 77 (Table 4), is very hard, lustrous, brittle,

silver-coloured and unreactive. Iridium is the second denser element known belong to the

platinum group metals. Iridium is one of the least abundant elements of the earth’s crust,

even less abundant than gold or platinum (0.4 parts per billion by weight, 0.05 parts per

billion by moles). Iridium was discovered along with osmium by Smithson Tennant in

1803, and its name means “prismatic colours” in Greek (“iridios”) due to the many

colours of its compounds. It is the most corrosion-resistant metal known and it resists

attack by any acid. Iridium is attacked by molten salts such as sodium chloride (NaCl) and

sodium cyanide (NaCN). The iridium complexes synthesized in this work also show many

different colours. Iridium is found in natural alloys with platinum and osmium in alluvial

22

deposits. Commercially, iridium is recovered as a by-product from the nickel mining

industry. The main use of iridium is as a hardening agent for platinum alloys. With

osmium, it forms an alloy that is used for tipping pens, and compass bearings. Iridium is

used in making crucibles and other equipment that is used at high temperatures. It is also

used to make heavy-duty electrical contacts. Iridium was used in making the international

standard kilogram, which is an alloy of 90% platinum and 10% iridium. Radioactive

isotopes of iridium are used in radiation therapy for the treatment of cancer.

Table 4. Some parameters of elemental iridium.

Parameter

Atomic symbol Ir

Atomic number 77


Electronic configuration [Xe] 4f14 5d7 6s2



Boiling point/°C 4430



Molar heat capacity J mol-1K-1 25.10





Effective ionic radius (6-coordinated) (in pm) 71 (Ir5+ ion)

76.5(Ir4+ ion)

82 (Ir3+ ion)

I.5.1 Properties of the iridium

The known oxidation states of iridium are from -1 to +6, the most stable are +3 and

+4. The common oxidation states of iridium in organometallic and coordination

23

compounds are +I and +III, whereas compounds with Ir in high oxidation states, i.e. +4

and +5, are very rare. In the synthesized complexes, the oxidation state is +3, resulting in a

[Xe] 5d6 electron configuration for an octahedral metal centre, a d6 electron configuration

for a 2nd or 3rd row metal ion is invariably low-spin, which means the electrons are

paired, and the complexes are kinetically inert with diamagnetic properties. In the case of

Ir, however, some intriguing reactivity is associated with high-valent intermediates. For

example, complexes of IrIV and IrV have been proposed as key intermediates in C–H bond

activation [56], oxygen atom transfer reactions, and water oxidation chemistry [57].

Simple complexes of IrIV are also known as strong one-electron oxidants [58]. The high

reactivity and limited stability of high-valent Ir compounds often precludes their

identification and characterization.

I.5.2 Compounds of iridium

Iridium forms compounds in oxidation states between −3 to +6. Well-

characterized examples of the highest oxidation state are rare, but include IrF6 . In

addition, it was reported in 2009 that iridium(VIII) oxide (IrO4) was prepared under

matrix isolation conditions (6 K in Ar) by UV irradiation of an iridium-peroxo complex.

This species, however, is not expected to be stable as a bulk solid at higher temperatures

[59]. Iridium dioxide, IrO2, a brown powder, is the only well-characterized oxide of

iridium [60]. A sesquioxide, Ir2O3, has been described as a blue-black powder which is

oxidized to IrO2 by HNO3 [57]. The corresponding disulfides, diselenides, sesquisulfides

and sesquiselenides are known and IrS has also been reported. Iridium also forms iridates

with oxidation states +4 and +5, such as K2IrO3 and KIrO3 [61]. No binary hydrides of

iridium, IrxHy are known. No monohalides or dihalides are known, whereas

trihalides, IrX3, are known for all of the halogens [60]. For oxidation states +4 and above,

only the tetrafluoride, pentafluoride and hexafluoride are known. Iridium hexafluoride,

IrF6, is a volatile and highly reactive yellow solid, composed of octahedral molecules.

Iridium pentafluoride has similar properties but it is actually a tetramer.

24

Hexachloroiridic(IV) acid, H2IrCl6, and its ammonium salt are the most important

iridium compounds from an industrial perspective [62]. They are involved in the

purification of iridium and used as precursors for most other iridium compounds, as well

as in the preparation of anode coatings. Iridium trichloride, IrCl3, which can be obtained in

anhydrous form by dissolving Ir2O3 in hydrochloric acid, is often used as a starting

material for the synthesis of other Ir(III) compounds. Another compound used as a starting

material is ammonium hexachloroiridate(III), (NH4)3IrCl6. Iridium(III) complexes

are diamagnetic (low-spin) and generally have anoctahedral molecular geometry. Among

organo-iridium compounds contain iridium–carbon bonds where the metal is usually in

lower oxidation states, Vaska's complex,IrCl(CO)[P(C6H5)3]2 (viz. Figure 6), which has

the unusual property of binding to the dioxygen molecule, O2 [63].

Anotheroneis Crabtree'scatalyst,a homogeneouscatalyst for hydrogenation reactions [64]

These compounds are both square planar, d8 complexes, with a total of 16 valence

electrons, which accounts for their reactivity [65].

Figure 6. Vaska's complex.

I.5.3 Coordination chemistry of iridium

Little is known about the quantitative aspects of the higher oxidation state solution

chemistry of the group 9 metal such as Ir. Thus, in a very recent paper, Wang et al. [66]

noted the paucity of information about the solution chemistry of iridium in its higher

oxidation states, despite the fact that numerous studies on the oxidation of Ir(III)

complexes have been reported. Typically, the products of oxidation of Ir(III) complexes

have not been well characterized and the chemistry is poorly defined., However, those

Ir(IV), Ir(V), and Ir(VI) compounds that have been prepared are noted for their strong

oxidizing powers. The majority of studies on iridium complexes concern the preparation,

structural characterization, and spectroscopic properties of a very limited range of

25

Ir(RRdtc) (where RRdtc = dialkyldithiocarbamate), complexes and electrochemical

studies are limited to reduction [67]. Apparently, there are no previous studies on iridium

diselenocarbamates in any oxidation state, and no information is available on higher

oxidation state iridium dithiocarbamate or diselenocarbamate complexes. Detailed studies

on the electrochemical oxidation of Ir(RRdtc), and Ir(Et,dsc), enable the Eo values for the

[Ir(RRdtc)]+/Ir(RRdtc), and selenium analogue redox couples to be obtained. This

completes the measurement of the triad of potentials for the M(IV)/M(III) couples and

allows them to be compared for the first time in order to ascertain the order of redox

stability. Studies on the reactivities of the electrochemically generated Ir(IV) complexes

and their decomposition pathways also can be compared with those of their Co(IV) and

Rh(IV) counterparts in an endeavor to understand the order of reactivity of the oxidation

state IV species.

Triscyclometalated Ir(III) complexes can be prepared by three different synthetic

routes using either 2-phenylpyridine (ppyH) or 1-phenylpyrazole (ppzH) as the

cyclometalating ligand precursors (Scheme 8). The first method involves treating Ir(acac)3

with 3 equiv of the free ligand in glycerol, at refluxing temperatures (Method A, eq 2)

[68]. The triscyclometalated complexes can also be prepared from the appropriate â-

diketonate derivative [(C^N)2Ir(OÔ), OÔ= 2,2,6,6-tetramethyl-3,5-heptanedione (dpm)]

(Method B) or dichloro-bridged dimer [(C^N)2Ir(-Cl)2Ir(C^N)2] (Method C), by heating

the Ir complex with a 2-3-fold excess of cyclometalating ligand in glycerol. These

syntheses work equally well for other pyridine-type ligands (e.g., tpyH, 46dfppyH), as well

as for phenylpyrazoles (e.g., 46dfppzH, tfmppzH). Methods B and C have several

advantages over Method A. The dichloro-bridged dimmers [69] and (C^N)2Ir(OÔ)8

compounds are easily prepared in high yield from IrCl3.H2O, a starting material less

expensive than Ir(acac)3. In addition, Methods B and C give higher yields than does

Method A. For example reactions using Method C and either ppyH or ppzH give yields

between 80 and 85% (based on the starting dimer, reaction temperature 200 °C) versus 45-

60% using Method A [70]. Previous routes to make tris-cyclometalated Ir(III) complexes

also utilize either IrCl3.H2O or (C^N)2Ir(í-Cl)2Ir(C^N)2 complexes as starting materials

[71]. However, these methods employ a large excess of the cyclometalating precursor

ligands as solvent, making it necessary to prepare the desired HC^N compounds on a

26

relatively large scale. The reaction temperature and nature of the cyclometalating ligand

strongly affect the facial/meridional product ratios of the reactions. For the nonfluorinated

ligands ppyH, tpyH, and ppzH, both Methods B and C give the facial isomer as the

predominant product when reaction temperatures are >200 °C. A small amount of the

meridional isomer (typically 1-3%) is sometimes also present in the crude reaction

mixture. The meridional impurity can be readily removed by recrystallization or column

chromatography. Higher yields of the mer-Ir(C^N)3 complexes (68-80%) are obtained

using Method C at a lower temperature, that is, 140°C. Interestingly, different results are

obtained with the fluorine-substituted ligands, 46dfppzH, tfmppzH, and 46dfppyH. All of

these ligands give the facial isomer as the major product when Methods A or B are used at

high temperature (>200 °C). However, when using Method C, we found that 46dfppzH

and tfmppzH give meridional isomers as principal products, whereas 46dfppyH gives

mixtures of fac/ mer-isomers, along with other unidentified products, even at 140°C.

Therefore, pure samples of mer-Ir(46dfppy)3 were prepared by Method C using 2-

ethoxyethanol as solvent at 120 °C. The coordination geometry of the precursor

complexes used in Method C [(C^N)2Ir(-Cl)2Ir(C^N)2] has the Ir-N bonds in a mutually

trans disposition. This suggests that cyclometalation by the third C^N ligand leads directly

to the formation of the meridional isomer, as observed when the syntheses are carried out

at a lower temperature. When the reaction temperature for Method C is raised to >200 °C,

however, isomerization of either the starting materials or the formed meridional isomers

needs to occur to form the observed facial products (vide infra).

Where, [(C^N) = ppy/ tpy/ 46dfppy; (O^O) = dpm; [(C^NH) = ppz/ 46dfppz/ tfmppz

Scheme 8. Synthetic methodology of cyclometalated Ir(C^N)3 complexes.

27

Chart 1. Cyclometalating ligands used to prepare Ir(C^N)3.

I.5.4 Organometallic Complexes of iridium

Iridium(III) complexes containing cyclometallating ligands, such as fac-Ir(ppy)3,

have found interest in OLED applications as their emission from triplet MLCT states is

found to be very efficient and also this type of complex is robust and features synthetic

versatility and reversible electrochemistry. Between these two extremes are complexes

containing only one or two anionic ligating atoms and five or four neutral ligands

respectively, which may display excited states of intermediate (MLCT/LC) or quite

different (e.g. LLCT) character [72]. All these characteristics have allowed iridium based

complexes to displace complexes based on other metals in various applications [73].

During the first attempts to obtain [Ir(bpy)3]3+, De Simone et al. in 1969 reported the

formation of [Ir(bpy)2Cl2]Cl instead as undesired product by fusing K3IrCl6.3H2O with

bpy at 270°C. Characterisation of the obtained complex by NMR revealed it to be the cis

28

isomer [74]. Similar types of complexes were shortly after synthesised, for instance in

1969 Chiswell et al prepared cis- [Ir(phen)2Cl2]Cl (phen = 1,10-phenanthroline) starting

from K3IrCl6 as well [75] Broomhead presented a different synthetic method to obtain the

same phenanthroline based complex in 1971[76]. The synthesis was carried out following

two steps, in the first [Ir(phen)Cl4]2[phenH]+ was prepared from (NH4)3IrCl6·2H2O and

1,10-phenanthroline by reflux in acidic water for 2 hours. The second step consisted of

heating the phenanthrolinium salt in refluxing glycerol for 1 hour to give cis-

[Ir(phen)2Cl2]Cl as a yellow solid. These early examples of iridium polypyridyl complexes

reflected the synthetic difficulties and the harsh conditions needed when preparing even

simple iridium(III) complexes [77]. A new synthetic method to synthesise this class of

iridium(III) complexes was introduced by Brewer et al. in 1990, when complexes

containing different types of polypyridyl bidentate chelates [IrL2Cl2]+ were obtained from

IrCl3.3H2O upon addition of two equivalents of the chelate L in a mixture of EtOH and

H2O[78].

In 2000, in fact Thompson and coworkers published a paper reporting the first

OLED based on Ir(ppy)3 [79]. Watts et al. also reported the synthesis and photophysical

properties of complexes containing both cyclometallating and polypyridyl ligands (such as

[Ir(ppy)2(bpy)]+. [Ir(ppy)2(bpy)]+ displayed intermediate electrochemical and

photophysical properties between the LC predominant character of [Ir(bpy)3]3+ and the

MLCT character of [Ir(ppy)3] [80].

I.6 Palladium

Palladium (Pd), named after the asteroid Pallas, is arguably the most versatile and

ubiquitous metal in modern organic synthesis [81]. Palladium-mediated processes have

become essential tools, spanning countless applications in the syntheses of natural

products, polymers, agrochemicals, and pharmaceuticals. In part, this far-reaching scope is

due to palladium’s ability to participate in catalytic transformations, as well as its high

functional group tolerance. Nearly every area of organic synthesis has been impacted by

this versatile transition metal, which has fundamentally changed the way retrosynthetic

analysis is approached.

Palladium is a steel-white metal (Table 5), which does not tarnish in air. Palladium

has the lowest melting point and density of the platinum metals. Annealed palladium is

29

soft and ductile, but it becomes much stronger and harder through coldworking. Hydrogen

readily diffuses through heated palladium, so this method is often used to purify the gas.

Finely divided palladium is used as a catalyst for hydrogenation and dehydrogenation

reactions. Palladium is used as an alloying agent and for making jewelry and in dentistry.

White gold is an alloy of gold which has been decolorized by the addition of palladium.

The metal is also used to make surgical instruments, electrical contacts, and watches.

Table 5. Some parameters of elemental palladium.

Parameter

Atomic symbol Pd

Atomic number 46


Electronic configuration [Kr] 4d10


Melting point/°C 1,554.9




Molar heat capacity J mol-1K-1 25.98




Effective ionic radius (6-coordinated) (in pm) 75.5 (Pd4+ ion)

90 (Pd3+ ion)

78 (Pd4+ ion)

Effective ionic radius (4-coordinated) (in pm) 100 (Pd4+ ion)

I.6.1 Properties of the palladium

Although palladium can exist in a number of different oxidation states, useful

organic methods are dominated by the use of Pd(0) and Pd(II) [81], although the utility of

Pd(IV) [82] has been steadily emerging in its own right. There are relatively few known

30

compounds with palladium unambiguously in the +3 oxidation state, though such

compounds have been proposed as intermediates in many palladium-catalyzed cross-

coupling reactions [83]. In 2002, palladium(VI) was first reported [84]. The increased

stability of the even-numbered oxidation states (e.g., 0, +2, +4) can be rationalized by the

low tendency of palladium to undergo one-electron or radical processes; conversely, it

readily participates in two-electron oxidation or reduction. Palladium’s ability to undergo

facile and reversible two-electron operations has contributed to its widespread use as a

catalyst, since each oxidation state can yield different chemistry. Reactions such as cross-

couplings and olefin hydrogenation are common to the Pd(0) platform, while

transformations such as alcohol oxidation and cycloisomerization can be achieved using

Pd(II).

I.6.2 Compounds of palladium

Palladium generally prefers low oxidation states. The first stable organopalladium

compounds with this +4 formal oxidation state, PdIV(C6F5)2Cl2(L–L) (L–L = bidentate

neutral ligands), were isolated in 1975; then, alkylpalladium(IV) species were also isolated

and intensively studied [85]. However, an organopalladium compound with a formal

oxidation state exceeding +4 has never been identified. On the other hand, highly

electronegative fluorine ligands reportedly can produce +5 and +6 oxidation states in

inorganic Pd compounds, although such species are unstable and have not been well

characterized [86]. Electrochemical formation of PdO3 is also suggested [87]. Elemental

palladium reacts with chlorine to give palladium(II) chloride; it dissolves in nitric acid and

precipitates palladium(II) acetate on addition of acetic acid. These two compounds and

the bromide are reactive and relatively inexpensive, making them convenient entry points

to palladium chemistry. All three are not monomeric; the chloride and bromide often must

be refluxed in acetonitrile to obtain the more reactive acetonitrile complex monomers.

Palladium(II) chloride is the principal starting material for many other palladium catalysts.

It is used to prepare heterogeneous palladium catalysts: palladium on barium sulfate,

palladium on carbon, and palladium chloride on carbon. It reacts with triphenylphosphine

in coordinating solvents to give bis(triphenylphosphine)palladium(II) dichloride, a useful

catalyst. The other major palladium(0) complex, (Pd2(dba)3), is prepared by

reducing sodium tetrachloropalladate in the presence of dibenzylidene acetone[88]. The

31

great many reactions in which palladium compounds serve as catalysts are collectively

known as palladium-catalyzed coupling reactions. Prominent examples include the Heck,

Suzuki and Stille reactions. Palladium(II) acetate, (Pd(PPh3)4, Pd2(dba)3) are useful in

this regard, either as catalysts or as starting points to catalysts.

I.6.3 Coordination chemistry of palladium

The reported carboxamide ligands L1–L6 are prepared in moderate yields by the

reactions between appropriate diamines with 6-methyl-2-picolinic acid or picolinic acid in

pyridine in the presence of triphenylphosphite according to the literature method [89]. The

complexes 1–6 were obtained from the reaction of each ligand with palladium(II) acetate

dihydrate in CH3CN in excellent yields (Scheme 9). The complexes were isolated as

yellow solids, which are much soluble in DMSO and DMF except for 3. The Pd(II)

complexes were stable for several weeks in the air. They were characterized by 1HNMR,

FT-IR spectroscopies and elemental analyses. Crystals of the complex 5 for X-ray

structure determination were grown by slowly evaporating their solutions in DMF. Four

nitrogen atoms of the [6-Me2-bpb]2- ligand coordinate to a Pd(II) ion to form a

mononuclear complex unit 5. There are hydrogen bonds between carbonyl oxygen atoms

and hydrogen atoms of water molecules to form one-dimensional zigzag chains. The

molecule unit is distorted much from a square planar because of steric bulkiness of the

methyl groups of pyridyl rings (C1–N1–Pd1–N4 torsion angle of 27.3(2)0). The fact that

Pd(II) complexes with the tetradentate N4-type ligands showed efficient catalytic activity

led us to investigate the polymerization of norbornene with the palladium complexes [90].

When MMAO was used as a co-catalyst, the palladium complexes 1–6 displayed high

catalytic activity for norbornene polymerization. Control experiments showed that absence

of either palladium complex or modified methylaluminoxane (MMAO) did not

polymerize norbornene. This is the first example that neutral Pd(II) complexes with N4-

type tetradentate ligands catalyzed the norbornene polymerization, to our knowledge.

32

Scheme 9. Synthetic pathway for PdN4.

Over the past quarter-century dithiolene-R-diimine (A), dithiolato-R-diimine (B),

and dithiolato-R-diamine (C) complexes of the group VIII metals have attracted the

attention of numerous research groups because of their unique properties, which include

solution luminescence, solvatochromism, large molecular hyperpolarizabilities, and large

excited-state oxidation potentials [91]. In particular, much of this work has focused on the

optoelectronic properties of the platinum(II) complexes of types A and B (Figure 6).

Interestingly, despite the significant structural and electronic variation among the systems

that have been explored thus far, photochemical or chemical oxidation has resulted in

dithiolene/dithiolato-centered rather than metal-centered oxidation [92]. In 1985,

Srivastava and co-workers first suggested that the type A complexes [Pt(bpy)(tdt)] and

[Pd(bpy)(tdt)] (bpy = 2,2’-bipyridine; tdt = 3,4-toluenedithiolato) both exhibited the

ability to act as a photosensitizer for the formation of singlet oxygen. They attributed this

ability to the unique LLCT-based excited state of these complexes [93]; however, they

were unable to fully characterize the photooxidation products of these reactions. Since this

initial report, the chemical and photochemical oxidation of nickel-containing complexes,

principally of type C, has been studied extensively. Depending on the reaction conditions,

a wide variety of products have been isolateds monosulfenates [RS-M-SOR], disulfenates

[M-(SOR)2],monosulfinates [RS-M-SO2R], disulfinates [M-(SO2R)2], mixed sulfinate-

sulfenate [ROS-M-SO2R], and disulfonate [M-(SO3R)2] [94]. The formation of the

disulfonate complexes is particularly noteworthy because they involve the oxidative

33

cleavage of the hypothetically strong metal-sulfur bonds. More recently, attention has

again shifted to the heavier members of the triad, with palladium(II) complexes of type C

and platinum(II) complexes of type B being shown to undergo photoinduced oxidation in

the presence of atmospheric oxygen to yield monosulfenate, disulfenate, mixed

sulfinate/sulfenate, monosulfinate, and disulfenate complexes [95]. Additionally, Connick

and Gray recently reported the complete structural and spectroscopic characterization of

the photooxidation products of [Pt(bpy)(bdt)] (3) (bdt = 1,2-benzenedithiolato),

identifying both a monosulfinate and a disulfinate complex depending on the oxidation

conditions. The widely differing behavior of these two closely related complexes naturally

raises the question of the role that the metal atom plays in the oxidation process. In an

attempt to address this question, it was decided to study the oxidation behavior of the third

member of this family, [Pd(bpy)(bdt)] (2).

Figure 6. General complex structure of MN2S2 chromophore (M = Pd).

Phenylazophenyl ligand and its derivatives form through complexation five

membered ring complexes. These show a planar chelating ring, therefore with the five

torsion angles near zero. We have performed the statistical analysis of Pd–N, Pd–C

distances, C–Pd–N angles, N=N and C–N distances. The Pd–N distance exhibits a narrow

range after removing the data with refcode NEPDIV, [(CxN)Pd(ppp)][ClO4] [96] (CxN =

phenylazophenyl- C,N; ppp = bis(2-(diphenylphosphino)ethylphenylphosphine-P,P’,P”)

which is a highly distorted five-coordinated palladium structure. The Pd–C distance is

shorter than that found for Pd–N because of the negative charge on a deprotonated carbon

atom, both distances (Pd–C and Pd–N) show a narrow range.

Regarding the C–Pd–N angle, it shows a narrow range (bite angle characteristic for

phenylazophenyl ligand), nevertheless structures NEPDIV and AZFAPD11,

[(F6acac)Pd(CxN)] [97] (F6acac = hexafluoroacetylacetonate, CxN = phenylazophenyl-

34

C,N) have values of 71.80 and 74.40, which are considerably different from the mean. The

N=N distance is longer than that observed for the N=N bond in trans-azobenzene. The C–

N distances are different upon complexation, the distance involving the C in the chelating

ring being shorter than that in trans-azobenzene [98]. On complexation, the observed

elongation of the N=N distance and shortening of the N–C distance relative to those in

trans-azobenzene, could arise from the higher contribution of a resonance structure with

charge separation, C6H4–N+–N-–C6H5 and the subsequent delocalization of the positive

and negative formal charges over the phenylene and the N-phenyl rings, respectively, the

latter being partially prevented by the ring rotation.

N

N

Pd

Figure 7. Complex structure of Pd-azo complex.

I.7 Platinum

Platinum was discovered by pre-Columbian South Americans and taken to Europe

about 1750. Platinum is a metallic element of atomic number 78 and atomic weight

195.08. It is found the widest application due to its several properties. Platinum is a

lustrous, silvery-white, malleable and ductile metal. It is unaffected by air and water, and

will only dissolve in aqua regia (HCl/HNO3) and molten alkali. Platinum is used in

jewellery, anti-cancer drugs, catalysts and catalytic convertors. The properties of platinum

are tabulated in Table 6. The production methods of platinum are used by the alternative

solvent extraction processes and ion exchange techniques as already shown in Scheme 2.

I.7.1 Properties of the platinum

Platinum is silvery-white and lustrous, and is both malleable and ductile. It is also

readily obtained in finely divided forms which are catalytically very active. Platinum

black is a velvety-black powder obtained by adding ethanol to a solution of PtCl2 in

aqueous KOH and warming. Another property of platinum which has led to numerous

laboratory applications is its coefficient of expansion which is virtually the same as that of

soda glass into which it can therefore be fused to give a permanent seal. Like rhodium and

iridium, platinum has the fcc structure predicted by band theory calculations for elements

35

with nearly filled d shells. The maximum oxidation state of platinum is +6 in PtF6 and has

no oxidation state below zero. For platinum, however, both +2 and +4 are prolific and

form a vital part of early as well as more recent coordination chemistry. Platinum exhibits

a strong preference for the square planar geometry. The kinetic inertness of platinum(II)

complexes has led to their extensive use in studies of geometrical isomerism and reaction

mechanisms. In the divalent state, platinum shows the class-b characteristic of preferring

CN- and ligands with nitrogen or heavy donor atoms rather than oxygen or fluorine.

Platinum(IV) by contrast is more nearly class-a in character and is frequently reduced to

platinum(II) by P- and As-donor ligands. The organometallic chemistry of these metals is

rich and varied and that involving unsaturated hydrocarbons is the most familiar of its

type.

Table 6. Some parameters of an elemental platinum.

Parameter

Atomic symbol Pt

Atomic number 78


Electronic configuration [Xe] 4f85d96s1




ΔHfus/ kJ mol-1 19.7(±2.1)

ΔHvap/ kJ mol-1 469(±25)

ΔHf (monoatomic gas)/ kJ mol-1 545(±21)



Metal radius (12-coordinated) (in pm) 138.5

Effective ionic radius (6-coordinated) (in pm) 57 (Pt5+ ion)

62.5 (Pt4+ ion)

80 (Pt2+ ion)

36

I.7.2 Compounds of platinum

Platinum forms only one reasonably well-characterized oxide PtO2, although the

existence of many others has been made. The stable oxide of platinum is found, instead, in

the higher oxidation state. Platinum can form a mono and a di-sulfide. Black PtS2 is

obtained when H2S is passed through aqueous solution of platinum(IV) and green PtS are

best obtained by heating PtCl2, Na2CO3 and S. The only hexa- and penta-halides are the

dark-red PtF6 and [PtF5]4 which are both obtained by controlled heating of Pt and F2. It is

one of the strongest oxidizing agents known, oxidizing both O2 (to O2+, [PtF6]-). Platinum

alone forms all 4 tetrahalides and these vary in color. The diamagnetic “trichloride” and

“tribromide” of platinum contain platinum(II) and platinum(IV) and the triiodide probably

does also. Platinum dichlorides are less well known [99].

I.7.3 Coordination chemistry of platinum

The simple coordination compound cis-diamminedichloroplatinum(II), or

cisplatin, is an effective anticancer drug that has been used in the clinic since 1978 [100].

Its success has given rise to the second-generation platinum drugs carboplatin and

oxaliplatin. These three platinum(II) complexes are believed to operate by a similar

mechanism. Aquation of the leaving groups, chloride for cisplatin and carboxylate and

oxalate for carboplatin and oxaliplatin, respectively, generates reactive cis-

diam(m)ineplatinum cations, which react readily with the purine nucleobases in DNA

[101]. Structural distortions in DNA induced by platinum binding [102] trigger multiple

cellular responses that ultimately lead to cell death [103]. Despite the clinical success of

these compounds, the requirement for intravenous administration and associated long-term

toxic side effects diminish the quality of life for patients. Platinum anticancer complexes

in the 4+ oxidation state have shown considerable promise both for oral administration

and for reduction of systematic toxicity [104]. The orally administered platinum(IV)

complex satraplatin progressed as far as phase III in clinical trials [105]. The increased

stability of these complexes, due to their low-spin d6 electronic configuration, aids in their

survival of the acidic environment of the gastrointestinal tract before being absorbed into

the bloodstream. They operate by a mechanism similar to that of the first- and second-

generation platinum(II) analogues. An activation step, namely, reduction from

37

platinum(IV) to platinum(II), must occur before aquation and DNA binding, however

(Scheme 10). In addition to their kinetic stability, another favorable property of

platinum(IV) complexes relative to their platinum(II) counterparts is the presence of two

additional coordination sites that can be modified to alter their pharmacokinetic properties.

By varying the two axial ligands, one can predictably alter the redox potential and

lipophilicity [106] of the platinum(IV) complex while leaving the DNA-binding cis-

diammineplatinum moiety unaltered. Furthermore, the axial coordination positions serve

as binding sites for other biologically active ligands, which may have synergistic effects

with platinum therapy, as demonstrated by us and by others [107]. The ability to tether

platinum(IV) complexes via the axial ligands to various nanodelivery devices for

increased cellular uptake and selectivity is another advantage. The design of new

platinum(IV or II) anticancer complexes, however, is limited by the current synthetic

methodology [108]. Most of the newly tested platinum(IV or II) complexes bear either

chloro, hydroxo, or carboxylato axial ligands. The development of new synthetic

methodologies for accessing the platinum-ion manifold can expand the range of

complexes having novel properties.

Scheme 10. Activation step of cis-platin formation.

The interest in dinuclear platinum(III) complexes is steadily increasing because of

their very interesting chemical properties. They contain a metal-metal single bond which

is generally supported by two or four bridging ligands (the latter generally indicated as

“lantern shaped” complexes) [109] and references therein]. Only few exceptions with

three bridging ligands, or unsupported by covalent bridges [110], have been so far

reported. Usually the bridging ligands form fivemember rings comprising the two

platinum centers and a set of three atoms providing a suitable bite, for example, NCO

(including pyrimidine nucleobases),NCS,NCN, SCS, OXO (X = C, S, P), or PXP (X = O,

C) [111].

38

The organometallic chemistry of platinum has been thoroughly studied; it has great

historical significance and has provided a basis for understanding many catalytic reactions

[112]. Ligand substitution at platinum(II) or platinum(IV) is slower than in analogous

palladium complexes, and this has limited the use of platinum compounds in self-

assembly through dynamic coordination chemistry. However, the relative inertness of

organoplatinum complexes [113] can be an advantage in self-assembly by way of

secondary bonding interactions, such as hydrogen bonding, and there have been

impressive advances in the synthesis of complex organometallic structures using

alkylplatinum complexes as building block molecules. For example, a binuclear

organoplatinum(II) complex cation [O=C{4-C6H4Pt-(PPh3)2{3-C5H4NC(=O)NH2)}2]2+

(Scheme 11) forms a supramolecular polymer through hydrogen bonding between amide

groups, while the diplatinum(IV) complex cation [{PtMe2(4-

CH2C6H4CO2H)(bu2bipy)}2{μ-4-C5H4NCH2NHC(=O)C(=O)NHCH2-4-C5H4N}]2+

(Scheme 10, NN=bu2bipy=4,4’-di-tert-butyl-2,2’-bipyridine) [114] forms a double

stranded polymer through hydrogen bonding between both carboxylic acid and amide

groups.

Scheme 11. Supramolecular Polymer A with Amide Groups and a Double-Stranded

Polymer B with Both Amide and Carboxylic Acid Groups.

39

The π-acidity of the azoimine group, -N=N-C=N-, and its ability to stabilize low

valent metal redox states has encouraged the study of its chemistry and molecular

architecture design with this group [115]. 2-(Arylazo) pyridine (aap, 1) is one such

molecule whose coordination chemistry with transition metals has been extensively

studied and maximum effort has been given to explore the chemical activity of the

platinum group metals [116]. However, platinum remains untouched. Much current

research on platinum(II) with N-donor ligands is targeted to synthesize analogues of

cisplatin. The use of N,N-donor ligands instead of ammonia appeared favorable because of

the thermodynamic stability of the chelate complexes. The π-acidity of the function, -

N=N-C=N-, has manifested itself in [Pd(N,N)(O,O)] (where N,N-2-(arylazo)pyridines;

O,O_catecholates, Scheme 12) in a ligand–ligand charge transfer transition (LLCT) [117].

Scheme 12. General synthetic steps to synthesize [Pt(NN)(OO)] complexes.

I.7.4 Organometallic compounds of platinum

Platinum has played major roles in the development of organometallic chemistry.

The first compound containing an unsaturated hydrocarbon attached to a metal was

[Pt(C2H4)Cl2]2 and platinum methyls were among the first-known transition metal alkyls.

For σ bonded compounds, platinum have been known since the beginning of this century

and commonly involve the stable (PtMe3) group; and compounds of the divalent metals. In

the platinum(II) compounds the metal is always octahedrally coordinated and this is

40

frequently achieved in interesting ways. Moreover, platinum(II) are among the most stable

σ bonded organo-transition metal compounds.

On the basis of the 18-electron rule, the d8s2 configuration is expected to lead to

carbonyls of formula [M(CO)4] and this is found for nickel. On the other hand, carbonyl

complex of platinum is not stable. It may be added that the introduction of halides (which

are σ bonded) can reverse the situation: the colorless of [PtX3(CO)]- are quite stable.

Reductions of [PtC16]2- in an atmosphere of CO provide a series of clusters, [Pt3(CO)6]n 2-

(n = 1-6, 10) consisting of stacks of Pt3 triangles in slightly twisted columns; Pt-Pt = 266

pm in triangles, 303-309 pm between triangular planes.

A feature of these and other platinum clusters is that they mostly have electron

counts lower than predicted by the usual electron counting rules [118]. The

cyclopentadienyls of platinum are less stable than those of nickel, and while the heavier

pair of metals form some mono cyclopentadienyl complexes, neither forms a metallocene.

Alkene and alkyne complexes are important not only for their part in stimulating the

development of bonding theory but also for their catalytic role in some important

industrial processes [119]. These are of the forms [PtC13Alk]-, [PtC12Alk]2 and

[PtC12Alk2] which provide the most stable compound of this type. They are generally

prepared by treating a platinum(II) salt with the hydrocarbon when a less strongly bonded

anion is displaced. A common property of coordinated alkenes is their susceptibility to

attack by nucleophiles such as OH-, OMe- , MeCO2 - and Cl-.

I.7.5 Pyrrolazo ligand

Pyrrolazo dyes are organic compounds easily prepared by the diazotization of

Aniline and its derivatives. The intermediary diazotate form is highly reactive that it has to

be coupled with aromatic substances in acidic solutions at low temperature (-5-0oC) to

yield pyrrolazo dyes. General appearances of pyrrolazo dyes are red, violet or brownish

colors in their crystalline state. Most of these compounds are only partly soluble or water

insoluble. Nevertheless, their solubility can be increased by the addition of organic [120]

solvent such as chloroform, methanol, ethanol, dichloromethane, dimethylformamide,

tetrahydrofuran and acetone. The general structure of pyrrollazo dyes are shown in Figure

8. Azo dyes comprise the largest group of organic reagent used in spectrophotometric

analysis. They are found in a variety of industrial applications because of their color

41

fastness. These dyes are characterized by chromophoric azo group (-N=N-) offering a

wide range spectrum of colors. They also used for coloring consumer goods such as

leather, clothes, food, toys, plastic and cosmetics [121].

Figure 8. General structure of pyrrolazo dyes. G may be H, OH, NH2, SO3H, NO2 halogen

atoms or other groups.

. Pyrrolazo dyes are sensitive chromogenic reagents in addition to being interesting

complexing agents, and have been used as reagents for spectrophotometry, solid phase

extraction and liquid chromatography [122]. The application in spectrophotometry is

based on the colored compounds resulting from their reaction with most metals, especially

some transition metals, usually stable chelate complexes are produced. They have been

employed in separation procedures, because of their limited solubility in aqueous solution

but greater in organic solvent. Some of them have also proved to be particularly useful as

indicators in complexometric titrations.

I.7.6 Biological activity of Pt-complexes

Cisplatin (cis-diamminedichloroplatinum(II), CDDP) was chemically described in

1845 [123] but its antitumor properties were only found accidentally by Rosenberg in

1965 [124,125]. While investigating the influence of an electric field on the growth of the

E.coli bacteria, Rosenberg found that cells stopped dividing and displayed strong

filamentous growth. This phenomenon appeared to be caused by the presence of small

amounts of compounds, like cis-[PtCl2(NH3)2] and cis-[PtCl2(NH3)2], formed by slowly

dissolving Pt electrodes in the ammonium chloride electrolyte [126]. Following this

discovery a large number of platinum complexes were tested for their antiproliferative

effect. The complexes having cis geometry were found to be antitumor active and cisplatin

NH N

N

G

42

the most active. The trans isomer of cisplatin, transplatin, showed no antitumor activity

[127]. The structures of cisplatin and transplatin are shown in Figure 9.

Pt

Cl Cl

NH 3H 3N

Pt

Cl NH 3

ClH 3 N

c is-p la tin tra n s-p la tin Figure 9. Structure of the antitumor drug, cisplatin, and its inactive trans isomer,

transplatin.

Cisplatin successfully entered into clinical trials in 1971 [128] the first clinical test

was performed by Hill et al. and was approved by the United States FDA in 1978.

Cisplatin is routinely used in the clinic, appearing the most effective against testicular and

ovarian cancer [129]. The response rates of other solid malignances, such as head, neck,

small-cell lung, oesophageal, can be improved with cisplatin treatment, although this

effect appears to be temporary [130]. In combination with other antitumor drugs, such as

vinblastine and bleomycin, a synergistic effect has been achieved. With testicular cancer,

when recognized in an early stage, curing rates exceed 90%. Common problems

associated with cisplatin in the clinic include nephrotoxicity, ototoxicity and

myelosuppresion. Procedures such as forced diuresisand pharmacological interventions

with S-containing chemoprotectants [131] have helped to alleviate the dose-limiting

nephrotoxicity. In addition to the serious side effects, inherent or treatment-induced

resistant tumor cell sub-populations also limit the therapeutic efficacy of cisplatin [125].

These toxic side effects of cisplatin limit the dose that can be administrated to patients;

typical doses are 100 mg/m2, which is usually given at a three-weekly schedule.The most

significant advantage in obviating the side effects of cisplatin has come from the process

of analogues development, i.e. the search for structural analogues of cisplatin that fulfill

one or all of the next criterions:

1. Development of new selectivities, including an activity spectrum wider than

cisplatin and, especially, activity in cisplatin-resistant tumors.

2. Modification of the therapeutic index, that is to say, a higher clinic efficacy to

reduce toxicity, with activity at least in the same range as cisplatin.

43

3. Modification of the pharmacological properties, such as solubility, which could

result in improved ways of administration.

The ligand exchange kinetics of Pt compounds is largely determined by the nature

of the leaving groups (i.e. the group that first leaves the Pt under the used reaction

conditions). Complexes with strongly coordinating leaving groups do not have antitumor

activity, also very weakly coordinating leaving groups do not lead to high activity [132].

This inactivity can be caused by either the low reactivity or the very high reactivity of

these compounds. In addition, the trans effect of the strongly coordinating group would

cause the release of the amine ligand. Pt complexes with weakly coordinating ligands are

not active, because they show too much reactivity, the most probable being a rapid

reaction with other intracellular components. The nature of the non-leaving groups also

influences the reactivity of platinum compounds. For drugs of general formula cis-

[PtCl2(amine)2] with several ligands as nonleaving groups have shown antitumor activity.

These can be monodentate ligands (NH3) or didentate ligands, like diethylenediamine (en)

or diaminocyclohexane (dach). The activity of the platinum complexes decreases along

the series NH3>RNH2>R2NH>R3N (R is an alkyl subtituent). Steric hindrance and

hydrogen-bonding ability of the ligands are important factors in determining the reactivity

[133]. The amines can act as hydrogen donor to the O6 atom of a guanine and to a 5’

phosphate group in DNA, thus stabilizing Pt-G binding [134]. These interactions are

important for the thermodynamics (by stabilizing the Pt-d(pGpG) adduct) and for the

kinetics of the reaction (by driving the Pt complex to the N7 of guanine) [135]. So all

these observations resulted in a list of requirements for the structure of platinum

complexes exhibiting antitumor activity, the so-called Structure Activity Relationships

(SARs):

1. A cis geometry is required with the general formula cis-[PtX2(amine)2] for Pt(II),

and for Pt(IV) the formula cis-[PtX2Y2(amine)2]. Monofunctional binding cationic

complexes are inactive.

2. The X ligands (leaving groups) should be of intermediate strength (Cl-, SO42-,

carboxylate ligands). For Pt(IV) complexes the Y ligands should have a trans

orientation and can be Cl-, OH-, or [O(CO)CnH2n+1]-.

44

3. The non-leaving group amine ligands should contain at least one NH moiety,

necessary for hydrogen-bonding interactions with DNA (H-bonding to the O6 of

guanine and to the 5’ phosphate group).

Mechanism of action: Although a variety of known carrier-mediated transport

systems influence cellular accumulation of cisplatin, there is little evidence for an active

carrier-mediated uptake mechanism. Because cisplatin accumulation is neither saturable,

nor competitively inhibited by structural analogues, it has been suggested that the drug

enters in the cell through passive diffusion [136]. Involvement of high-capacity facilitated

diffusion via gated channels has been also proposed [137], but the steric demands of

cisplatin prohibit entrance through most of the well-characterized ion channels. Recently it

has been reported that the copper transporter Ctr1 is involved in the uptake of cisplatin,

since it has been demonstrated that deletion of the yeast homologue of the Ctr1 gene,

which encodes a high-affinity copper transporter, results in increased cisplatin resistance

and reduces the intracellular accumulation of cisplatin [138]. Once cisplatin enters the

cell, where the chloride concentration is much lower than in plasma (~4mM) the drug

undergoes hydrolysis to form positively charged active species for subsequent interaction

with cellular nucleophiles [139]. The ultimate target for cisplatin inside the cell is DNA.

The exact interaction mode between cisplatin and DNA and the cellular distribution are

not known yet. However, inside the cell there are many competitors for DNA binding

present, as well as in the nucleus, such as small molecules and ions which compete for

cisplatin binding [Cl-, (HPO4)2-, OH-, H2O], amino acids, peptides, proteins, and

polyphosphates [138-140]. Pt-protein binding is thought to play an important role in the

toxicity and the mechanism of cisplatin-resistance.

I.7.7 DNA-Binding studies of Pt-complexes

Because platinum belongs to the group of “B”-type metals, its ion reacts

preferentially with N atoms rather than O atoms. In fact, the preferred binding site in DNA

is the N7 atom of purines nucleobases [141]. At physiological pH the N3 atom of thymine

is protonated, the N3 of purines are sterically hindered and aromatic nitrogens without a σ

lone pair are excluded for platinum coordination. The N1 of adenine and the N3 atom of

cytosine are suitable positions for platinum binding. In Figure 10 a scheme with all the

45

potential binding positions is represented. All four positions can be platinated, but the

preferred binding step is the N7 atom of guanine, which shows a strong kinetic preference

[142]. This tendency results from the strong basicity of that position and from hydrogen

bond interactions between ammine protons of cisplatin with O6 in guanine and their

accessibility for the platinum complexes [143]. The reaction of cisplatin with DNA leads

to the formation of six major categories of Pt-DNA adducts, the most important ones

schematically depicted in Figure 11. The major adduct formed by cisplatin was found to

be the 1,2-intrastrand d(GpG) cross-links on adjacent purine bases, followed by 1,2-

intrastrand d(ApG) cross-links between an adjacent adenine and guanine, 1,3-intrastrand

d(GXG) and 1,4-intrastrand d(GXXG) cross-links between purines separated by one or

two intervening bases, respectively. A small percentage of cisplatin was found to be

involved in interstrand cross-links, linking the two strands of the DNA double helix, or in

monofunctional adducts coordinated to a single purine and protein-DNA cross-link, in

which cisplatin coordinates a protein molecule and a nucleobases[144].

Figure 10. Possible platinum binding sites on DNA.

46

The consequences of these cross-links to the cell and how they lead to cell death

are largely unknown. Results to date, obtained in numerous cell lines, suggest that

cisplatin damaged DNA causes cell cycle perturbation, and arrest in the G2-phase to allow

repair of the damage, and in the case of inadequate repair, the cells eventually undergo an

abortive attempt at mitosis that results in cell death via an apoptotic mechanism [145].

Transplatin is not able to form 1,2-intrastrand cross-links, because of the steric hindrance

of the two ammine groups in trans position; instead it may form 1,3-intrastrand cross-links

between two G residues, or between a G and a C residue, separated by at least one base.

The amount of monofunctional adducts and protein-DNA cross-links formed is much

higher for transplatin than for cisplatin. Another difference between the two isomers is

that interstrand cross-links formed by transplatin are between complementary G and C

residues, whereas cisplatin forms only G interstrand cross-links. It was reported that under

physiological conditions the major adducts are the monofunctional adducts, allowing the

formation of protein-DNA cross-links. In vivo transplatin interstrand cross-links might not

form because of the slow rate (t1/2 ≈ 24 hr) of the cross-linking reaction, and the trapping

of the monoadduct species by thiol-containing proteins [146].

Figure 11. Main adducts formed in the interaction of cisplatin with DNA. (a) 1,2-

intrastrand cross link; (b) 1,3-intrastrand cross-link; (c) interstrand cross-link; (d) protein-

DNA cross link.

47

I.8 N, S and O donor ligands

Considering the versatility and importance of platinum metal complexes of the

organic moiety having N, S and O donor dithiocarbamate, carboxamide and azo

containing ligands in different fields like biology, industry, catalysis, various organic

compounds containing N, S and O donor sets have been used to design new coordination

complexes to explore new chemistry for several years. The hard (amidato-N), borderline

(pyridine-N) and soft (amine-N, azo-N, pyrrol-N and dithiocarbamate-S) nature of ligand

donor sites and their flexibility to occupy the different geometric coordination site around

the metal ions ultimately dictate the characteristic properties of the synthetic metal

complexes as well the bio-active site(s) in biological systems. Therefore, with the aid of

the ligands containing such donor sites (and their combination) will provide metal

complexes whose properties might be interesting and sometimes relevant to the

coordination chemistry and an impetus for the present work. The coordination chemistry

as well as the biological activity of rhodium, iridium, palladium and platinum metals to

the N, S and O donor (dithiocarbamate, carboxamide and azo) ligands are still now not too

much explored compared to the ruthenium and osmium analogue. In order to establish the

chemical as well as the biological relevance, the aforesaid four metals are chosen in the

area of our work. Here, some palladium, platinum, rhodium and iridium complexes are

listed in Table 7 as literature survey.

Table 7. N, S and O donor ligands and their rhodium, iridium, palladium, and platinum

metal complexes.

Ligands Comments Ref.

Palladium chemistry

N C

Me

H2C

S

S-

EtOOC

1

[Pd(1)Cl]n

FTIR, NMR, Nephrotoxicity study, Biological study

147

48

S-

S-

2b

Me

S-

S-

2a

[Pd(2a)(PPh3)2],

[Pd(2b)2(PPh3)2]

FTIR, X-Ray Structure, 31PNMR

148

S

S-

3

[Pd(3)2]

FTIR, NMR, ESR-study 149

OMe

S

S-

4

[Pd(4)2]

FTIR, NMR, X-Ray structure 150

N

S

S-Me

EtOOC

5

[Pd(5)(L)(Cl)]

X-Ray Structure, FTIR, TGA,

Cytotoxicity study

150

MeOC S-

S-MeOC

6

[Pd(6)(L1)(L2)], L1 = tBuCN,

L2=CH(NHXy)(NEt2)

X-Ray Structure, FTIR, NMR

151

N

Me S-

S-MeOOC

7

[Pd(7)2]

X-Ray Structure, FTIR, TGA,

Biological study

151

49

HN

OO

NH

NN

X1X2

R1 R2

8

[Pd(8)2]

X-Ray Structure, FTIR, UV-vis, 1HNMR

152

Platinum chemistry

N

HN

O

Me9H

[PtII(9)2], [PtII(9H)(9)Cl]

X-Ray Structure, FTIR, 2D-NMR,

ESMS-study, UV-vis, Biological

(Antitumor) study

153

N

N

OC2H5HN

O

HN

O

OC2H5

10H

[Pt(10H)(Me)2]

X-Ray Structure, FTIR, UV-vis, 1HNMR

154

50

N

NOH

HO

Cl

11

[Pt(11)(tba)]

X-Ray Structure, FTIR, 1HNMR,

Powdered X-Ray structure, DFT

155

OH

N

N

Cl

HO

Cl12

[Pt(12)(tba)]

FTIR, 1HNMR 156

NN

O

Me

O

PPh2

R

13

[Pt(13)(Cl2)]

Study on Heck reaction, FTIR, 1HNMR, DFT

157

Rhodium chemistry

14

N-

[Rh(14)(15)Cl]+

IR,NMR, Electrochemistry study,

Luminescent study

158

51

15

N N

N N

NH2

N

Me

Ph

S

S -1 6

[Rh{(16)}(CO)2]

FTIR, X-Ray Structure, UV-vis 159

17N N

[Rh(14)(17)]+

IR,NMR, Electrochemistry study,

UV-vis

160

18

O

P S

iPr

Me

tBuPh Ph

[Rh(18)(cod)]+

FTIR, X-Ray structure, UV-vis,

Chemical reactivity

161

19

N NtBuBut

H

[Rh(19)(COE)]2, [Rh(19)2H(Cl)],

[Rh(19)2Cl]

X-Ray Structure, DFT study, UV-

vis

162

20

N NMe

H

PPh2

[Rh(20)(cod)]+, [Rh(20)(CO)2]+

[Rh(20)(CO)2]+

X-Ray Structure, IR-Spectra,

NMR , Catalytic activity

163

NN +

21

[Rh(21)(cod)(Cl)],

[Rh(22)(cod)(Cl)], [Rh(23)(CO)2]

X-Ray Structure, FTIR, ESI-Mass,

Chemical reactivity

164

52

NN +

22

NN

23

NN

NN

24

[Rh(24)(CO)2]+

X-Ray Structure, 2DNMR, UV-

vis, Catalytic activity

165

N

N N

R

25

N

N N

R

HN

N

26

[Rh(25)(26)]+

X-Ray Structure, UV-vis, Catalytic

activity

166

53

N

N

Et

N N

H

S

Me27

[Rh(27)Cl3]

X-Ray Structure, DFT study, UV-

vis, ESI-Mass, FTIR

167

Iridium chemistry

Me

Me

S

S -28

[Ir(28)2]

FTIR, Emission lifetime

study

168

29

Et

Et

S

S-

30N N

OMeMe

[Ir(29)(30)2]

FTIR, 1HNMR, 13CNMR,

X-Ray Structure, UV-vis,

OLED-fabrication,

Electrochemistry

169

31

N

NPh

[Ir(29)(31)2]

FTIR, 1HNMR, X-Ray

Structure, UV-vis

170

32N N

OFF

[Ir(29)(32)2]

FTIR, 1HNMR, X-Ray

Structure, UV-vis, OLED-

fabrication

171

54

N N

OOMeMeO

33

[Ir(29)(33)2]

FTIR, 1HNMR, X-Ray

Structure, UV-vis, OLED-

fabrication

172

34

N-

N N

N N

NH2

N

35

[Ir(35)(34)2], [Ir(34)3]


Luminescence spectra, CV

173

N

NNPri

36

[(36)Ir(PPh3)2(H)2+

X-Ray Structure, FTIR,

ESI-Mass, UV-vis

174

37

HN

N

N

N

S

R

[Rh(37)Cl3]


ESI-Mass, FTIR

Electrochemistry

175

I.9 Present work

Considering the above facts and the importance of the platinum metal complexes

of nitrogen-sulphur donor sets, different series of newly designed and previously reported

ligands having S2(dithiocarbamate), N4(carboxamide), N2(Pyrrolazo) and

CN(phenylpyridine) donor sets have been used to synthesize new metal complexes in this

present work. (Scheme 13). Here, in this thesis work many different

platinum/palladium/rhodium/iridium complexes of nitrogen-sulphur donor sets have been

designed by suitably tuning the other donor atom like carbon from phenylpyridine. The

new ligands and all the new complexes have been characterized with the help of

microanalytical, physico-chemical and spectroscopic tools, and the old ligands have also

55

been verified spectroscopically after the synthesis of the organic moieties for this purpose.

In one case, detailed structure of one ligand has been established by single crystal X-ray

crystallography that is one pyrrolazo ligand (HL7).

N

N

Me

S

SH

O

N

S

SH

N

NS

SH

HL2HL1

HL3

NH HN

NN

OO

N

NH HN

NN

OO

H2L4 H2L5

N

NH HN

NN

OO

Br

H2L6

NH

N

N

Cl

NH

N

N

NO2

N-HL7

HL82-C6H5Py

Scheme 13. Structures of the ligands used in this present work.

56

In most cases, the structures of the metal complexes have been confirmed by X-ray

diffraction study. Redox properties of the complexes has also been examined by cyclic

voltammetric measurements study. Biological behavior such as CT-DNA and protein

(BSA) binding study as well as antibacterial study of the suitable complexes have been

performed. Very interestingly, we have shown that the catechol reactivity towards the

palladium metal not as a ligand rather than it acts in the medium as a water scavenger.

I.10 Physical measurements

(i) Elemental analyses: The elemental analyses were performed using a Perkin-

Elmer model 240C and 2400 CHN elemental analyzers.

(ii) Conductance measurements: The solution electrical conductivity was

measured using Systronics 304 digital conductivity meter with a solution concentration of

ca.10-3 moldm-3.

(iii) Infrared spectra: The infrared spectral data were recorded in KBr disc

(4000-200 cm-1) on a JASCO FT-IR model 420 Spectrophotometer and PerkinElmer FTIR

model RX1 spectrometer .

(iv)UV-Vis spectra: The UV-Vis Spectral data were recorded using JASCO UV-

VIS / NIR model V-570 Spectrophotometer.

(v) 1H-NMR spectra: 1H-NMR spectral data were recorded in suitable solvent

using Brucker 300, 500 MHz FT-NMR spectrometer.

(vi) Electrochemical studies: The Electrochemical studies were carried out under a

dinitrogen atmoshphere with a PAR electrochemistry system 250 potentiostat / versastat

and 270 softwere package. All results were collected at 298 K with the saturated calomel

electrode (SCE) as reference. Reported potentials are uncorrected for junction contribution

with tetrabutylammonium perchlorate (TBAP) as supporting electrolyte.

(vii) Fluorescence spectra: Fluorescence spectral data were recorded in suitable

solvent using Hitachi-2000 fluorimeter.

All reference in this thesis are given in the format:

Authors, name, Journals/periodicals/books, Volume (Year) Page number.

57

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66

CHAPTER-II

Palladium(II) Complexes of Dithiocarbamic

Acids: Synthesis, Characterization, Crystal

Structure and DNA Binding Study

67

Abstract

A series of distorted square planar palladium (II) complexes with dithiocarbamic

acids of formulation [Pd(L)2], where L1H = 4-methylpiperazine-l-carbodithioic acid

(4-MePipzcdtH), L2H = morpholine-4-carbodithioic acid (MorphcdtH), and

L3H = 4-benzylpiperidine-l-carbodithioic acid (4-BzPipercdtH) for complex 1, 2, 3 and 4,

respectively, was isolated in pure form. All the complexes were characterized by

physicochemical and spectroscopic methods. The detailed structure of complex 1 and 4

were characterized by single crystal X-ray crystallography. The interaction of these

palladium complexes with CT-DNA was investigated with the help of absorption and

emission spectroscopic tools. The association constant Kb was deduced from the

absorption spectral study, while the number of binding sites (n) and the binding constant

(K) were calculated from relevant fluorescence quenching data, suggesting the

intercalative interaction of the complexes with CT-DNA due to the stacking between the

aromatic chromophore and the base pairs of DNA.

68

II.1 Introduction

Transition metal complexes of various aliphatic, aromatic and heterocyclic

dithiocarbamate ligands have been widely investigated because of their medicinal,

industrial and analytical applications [1–3]. Dithiocarbamate complexes of palladium (II)

and platinum (II), as well as of isoelectronic gold (III), show remarkable antitumour

properties, and in some cases, their cytotoxic activity is superior to that of cisplatin [4–6].

Previous studies have shown that the presence of ligands with sulphur donor atoms

appears to be a prerequisite in conferring antitumour properties on palladium (II)

complexes [5]. Dithiocarbamates, which are used as fungicides and pesticides [7], are of

great interest because they display cytotoxic properties and have also been applied in the

treatment of metal poisoning [2, 8–10]. The nature of the heterocycle attached to the

dithiocarbamate fragment has a crucial effect on the electronic properties of these ligands

and therefore on their potential pharmacological attributes as well as the catalytic

properties of their metal complexes [11].

Taking into account the above facts and our continuous interest on the interaction of

newly designed metal complexes of nitrogen/sulfur ligands with calf thymus-DNA [12],

in this chapter, we report an account of the synthesis, structural characterization and DNA

binding study of distorted square planar palladium (II) complexes with three

dithiocarbamates of general formula [Pd(L)2], , where L1 = 4-Methylpiperazine-l-

carbodithioic acid anion, L2 = Morpholine-4-carbodithioic acid anion and L3 = 4-

Benzylpiperidine-l-carbodithioic acid anion for complexes 1, 2 and 3, respectively. The

complexes were characterized by physicochemical and spectroscopic methods; in

addition, the structures of complexes 1 and 4 were characterized by single crystal X-ray

crystallography, UV-vis, IR-spectroscopy, ESI-Mass and 1HNMR. The binding constant

Kb of the three complexes with DNA has been derived from UV–vis study and the

quenching constant Ksv have also been determined from fluorescence displacement

experiments using ethidium bromide as spectral probe to establish the binding mode of

small molecules to double-helical DNA.

69

II.2 Experimental Section

II.2.1 Solvents and Reagents

Solvents and reagents were obtained from commercial sources and used without

further purification unless otherwise stated. Acetonitrile (MeCN) was dried by distillation

over calcium hydride. For electrochemical experiments further purification was achieved

by KMnO4/Li2CO3 treatment [13] followed by distillation over P4O10. Water was

deionized and then distilled from alkaline permanganate. Ethanol and methanol were

distilled from magnesium ethoxide and magnesium methoxide, respectively. Pyridine was

distilled over and kept in presence of solid KOH. N,N’-Dimethylformamide (DMF) was

kept over powdered BaO for 24h, filtered and then distilled in vacuo. Benzene was first

stirred with concentrated sulphuric acid until it was free from thiophene. To remove acid

it was then shaken twice with water, once with 10% sodium carbonate solution, again

with water, and finally dried over anhydrous calcium chloride, and was distilled.

Chloroform and dichloromethane were made acid free by washing first with sodium

bicarbonate solution then five to six times with water followed by keeping over

anhydrous calcium chloride for 24h, and distilled. Tetra-n-butyl ammonium perchlorate

(TBAP) was prepared from tetra-n-butylammonium bromide and 70% aqueous perchloric

acid [14,15]. This was recrystallized from ethanol and dried in vacuo. Dinitrogen was

purified by bubbling through an alkaline dithionite solution. 1-Methyl-piperazine (Fluka)

was dried by refluxing over sodium hydroxide beads and also stored over sodium

hydroxide. Morpholine and 4-benzylpiperidine, purchased from Aldrich, and PdCl2 from

Arora Matthey were used as received. All other chemicals used were of analytical reagent

grade. Solvents used for spectroscopic studies and for synthesis were purified and dried

by standard procedures before used [16].

II.2.2 Physical Measurements

The elemental (C, H, N) analyses were performed on a Perkin Elmer model 2400

elemental analyzer. Electronic absorption and IR spectra were obtained on a JASCO UV–

Vis/NIR spectrophotometer model V-570 and on a Perkin–Elmer FTIR model RX1

spectrometer (KBr discs, 4000–300 cm-1), respectively. 1H NMR spectra were recorded

on a Bruker AC300 spectrometer using TMS as an internal standard in DMSO-d6 solvent.

70

Electrospray ionization (ESI) mass spectra of the complexes were recorded with a

QtofMicro Instrument (Waters, YA263).

II.2.3 X-Ray Crystal Structure Analysis

Single crystals of the complex 1 and 4 were obtained from a solution of 1 in DMF

on slow evaporation. The diffraction data were collected at 120(2) K with a four-circle j-

axis KUMA KM-4 diffractometer (KUMA Diffraction, Wroclaw) equipped with a CCD

detector and an Oxford Cryostream Cooler. The x-scan technique was performed at

different u, h and j offsets for covering the whole independent part of reflections in the h

range 3.5–27.57° with monochromated MoKα radiation (k = 0.71073Å). The data

reduction was carried out using the CrysAlis RED program [17]. An empirical absorption

correction implemented in SCALE3 ABSPACK scaling algorithm was applied [17]. The

structure was determined by direct methods (SHELXS-97), and all non-H atoms were

refined anisotropically on F2 using SHELXL-97 full matrix leastsquares procedure [18].

All H-atoms were found from difference Fourier maps and refined using a riding model,

and the positions of those belonging to the lattice water molecules were refined. The

crystallographic data such as data collection parameters and other crystallographic

information for the complex 1 and 4 are summarized in Table II.1. Crystallographic data

for have been deposited with the Cambridge Crystallographic Data Centre, CCDC No.

833198 for complex 1.

II.2.4 Syntheses of Ligands

A common synthetic method was followed to obtain the acidic form of the anionic

ligands, 4-Methylpiperazine-l-carbodithioic acid anion (L1) [19], Morpholine-4-

carbodithioic acid anion (L2) [20] and 4-Benzylpiperidine-l-carbodithioic acid anion (L3)

[21]. The procedure for the preparation of 4-Benzylpiperidine-l-carbodithioic acid is here

described in detail; the other ligands were obtained as solid products in the same way.

Carbon disulphide (4.00 g, 52.62 mmol) was slowly added to a cold solution of 4-

Benzylpiperidine (8.75 g, 50 mmol) in EtOH (15 ml) with vigorous stirring over 2.0 h.

The yellowish white precipitate was collected and washed with diethyl ether. The crude

product was recrystallized from isopropyl alcohol. All the solid products were

characterized by physico-chemical and spectroscopic methods. Yield: 92–96%.

71

Table II.1. Crystallographic data for the complexes 1 and 4.

Complex 1 4

Empirical Formula C12H22N4PdS4.3H2O C12H22N4PdS4

Formula Weight 511.02 457.02

Crystal system Orthorhombic Triclinic

Space group C mcm P1

a (Å) 11.4373(11) 4.47030(10)

b (Å) 6.7609(5) 8.4501(2)

c (Å) 25.858(2) 12.1978(3)

Volume (Å3) 1999.5(3) 444.437(18)

Temperature (K) 120(2) 120(4)

Z 4 1

ρρρρcalc (g/cm3) 1.702 1.708

µ(mm-1) 1.487 1.512

F(000) 1044 232

θθθθ range (deg) 3.50 - 27.57 1.7 - 34.44

Refl. collected / unique 6618 / 1199 13963/6830

R(int) 0.0386 0.0264

Final R indices [I > 2σσσσ(I)] R1= 0.0648, wR = 0.1293 R1= 0.0316, wR2= 0.0596

R indices (all data) R1= 0.0659, wR2 = 0.1298 R1= 0.0529, wR2= 0.0682

Goodness-of-fit on F2 1.540 1.007

aR = ∑||Fo |- |Fc||/∑|Fo|;

bwR2 = { ∑ [w(Fo

2 –Fc2)2] / ∑w [(Fo

2)2]}1/2

72

II.2.5 Syntheses of Palladium (II) Complexes

The metal complexes were prepared by reacting palladium (II) chloride with the

respective organic compound in equimolar ratio following a common procedure. For

complexes 1, 2, and 3, the organic species L1H (89.4 mg, 0.508 mmol) or L2H (83 mg,

0.509 mmol) or L3H (116 mg, 0.53 mmol), respectively, were mixed with 0.254 mmol of

palladium (II) chloride. The resulting mixture was refluxed for 6 h in ethanol. The solid

yellow colored product of complex 1 was obtained by evaporation of the solvent. While

in case of 2 and 3, a brown and a yellow colored solid product, respectively, precipitated

out from the reaction medium. The solid mass of complexes was filtered, washed with

ethanol and dry diethyl ether, and finally dried under vacuum over P4010. Yield: 70-74%.

Complex 4 was synthesized as follows: To an 15 ml ethanolic solution of

catechol(0.055 g, 0.50 mmol) triethylamine(1.0 mmol, 0.101 g) was added slowly in

stirred condition. Then to it L3H (89.4 mg, 0.508 mmol) and PdCl2 (0.089 g, 0.50 mmol)

were added with vigorous stirring followed by reflux at 70-80°C for 5-6 h. After cooling,

an brownish yellow solid resulted, filtered and washed with ethanol followed by diethyl

ether. Recrystallisation from ethanol gives yellow crystals suitable for X-ray diffraction.

Yield: 0.05 g, 86-90%.

II.3 Results and Discussions

II.3.1 Syntheses and Characterizations

The organic compounds L1H, L2H, and L3H were synthesized by the reaction of

the respective amine with carbon disulphide in ethanol, and later characterized by IR and

1H NMR spectral analyses. These ligands have two sulphur donors and the corresponding

palladium(II) complexes were obtained in good yield from the reaction of PdCl2 with the

respective organic moieties in 1:2 molar ratio in ethanol medium at refluxing condition

(see Scheme 1).

LnH + PdCl2

6 h, Reflux

Ethanol[Pd(Ln)2]

Where Ln = L

1, L

2 and L

3

Scheme 1. Synthetic procedures for the preparation of complexes.

73

When L1 is mixed with PdCl2 in ethanolic medium, complex 1 forms. Again,

when a mixture of catechol and triethylamine mixed with palladium (II) chloride and then

added to the ethanolic solution of L1, complex 4 forms (Path-a). The same complex 4

also forms when ethanolic solutions of L1 is mixed with PdCl2 and then catechol and

triethylamine mixture added (Path-b). If complex 1 again dissolved in ethanol and then to

it added a mixture of triethylamine and catechol dropwisely, then the formation of

complex 4 occurs (Path-c) (see scheme 2). Complex 1 and 4 differs only in crystal

packing architecture and the difference is only in the crystalline water content. Complex 1

has the three molecules of water of crystallization, whereas complex 4 has no such water

of crystallization in packing arrangement. Though palladium-catechol complex formation

is so common from literature survey, here, palladium prefers to ligate with

dithiocarbamate moiety only rather to form mixed ligand palladium-dithiocarbamate-

catechol complex. This suggest that, here catechol is acting as a water scavenger in the

formation of palladium–dithiocarbamate complex.

Scheme 2. Different synthetic pathway of complex 4.

[Pd(L1)2](Catechol+Et3N)+PdCl2+L1PdCl2+L1+(Catechol+Et3N)

Catechol+Et3N EtOH

L1+PdCl2

(4)

[Pd(L1)2].3H2O

(1)

Path-a

Path-b

EtOH EtOH

Path-c

EtOH

74

Table II.2. Palladium (II) complexes with dithiocarbamate ligands.

Microanalytical data (Table II.3) confirm the composition of the complexes

and spectroscopic data of 1, 2, 3 and 4 along with the X-ray crystallographic results of 1

and 4 indicate the organic moieties, 4-Methylpiperazine, Morpholine and 4-

Benzylpiperidine in the reaction medium are converted to 4-Methylpiperazine-l-

carbodithioic acid (L1H), Morpholine-4-carbodithioic acid (L2H) and 4-

Benzylpiperidine-l-carbodithioic acid (L3H) respectively, and their palladium (II)

complexes 1-4.

Table II.3. Microanalyticala data of the palladium (II) complexes (1-4).

Compounds Elemental analyses Found (calcd.)

Conductance

(Λo)a C H N

[Pd(L1)2] .3H2O(1) 31.71 (31.54) 4.77 (4.85) 12.18 (12.26) 53

[[Pd(L2)2](2) 28.03 (27.87) 3.69 (3.74) 6.42 (6.50) 45

[Pd(L3)2](3) 51.46 (51.43) 5.33 (5.31) 4.72 (4.61) 42

[Pd(L1)2] (4) 31.54 (30.58) 4.85 (4.03) 12.26 (13.12) 8

amho.cm2 mol-1 in DMF .

The mononuclear metal complexes 1-4 are soluble in DMF and DMSO, but

sparingly soluble in methanol and ethanol. The conductivity measurement of the

complexes in DMF showed conductance values in the range of 8-53 Λo mol-1 cm-1 at

300K, suggesting that these complexes exist in solution as non-electrolytes [22].

LnH Complex

4-Methylpiperazine-l-carbodithioic acid (4-MePipzcdtH ) (L1H) [Pd(L1)2] .3H2O(1)

[Pd(L1)2] (4)

Morpholine-4-carbodithioic acid ( MorphcdtH ) (L2H) [Pd(L2)2] (2)

4-Benzylpiperidine-l-carbodithioic acid (4-BzPipercdtH ) (L3H) [Pd(L3)2] (3)

75

II.3.2 Structural description of complexes 1 and 4

An ORTEP view of complex 1 and 4 with the atom numbering scheme is illustrated

in Figures II.1and II.3 and a selection of bond distances and angles is listed in Table II.4

and II.5. The structural analysis evidenced that the complex resides on a 2/m site, with

the mirror plane bisecting the S-C-S angles and containing the palladium (II) ion, so that

the crystallographic independent part is only a quarter of the whole complex. Moreover

two residuals, one lying at the intersection of two mirror planes, and consequently on a

two-fold axis (m2m site), and the second located on a mirror plane, were interpreted as

lattice water molecules (O1, O2). According to the site multiplicity, the ratio Pd:O1:O2 is

1:1:2 and the compound is formulated as [Pd(L1)2].3H2O.

The palladium atom has a distorted square planar geometry defined by two

symmetric dithiocarbamate ligands that behave as chelating monobasic. The Pd-S bond

distances, all equal to 2.327(1) Å due to the local symmetry, are within the range of

2.300-2.359 Å found in bis(dithiocarbamate)palladium (II) complexes retrieved in the

CSD (Cambridge Structural Database, Version 5.32.1) [23]. The formation of the four

membered PdS2C chelate ring induces a value of the S-Pd-S angle to 75.57(7)°, with the

carbon atoms C(1) slightly displaced by 0.187(8) Å on opposite directions of the PdS4

mean plane. The chelating S-Pd-S angle is not unusual, being close comparable to the

corresponding average value 75.7(6)° calculated for the cited fragments detected in CSD.

The C-S bond distance, of 1.719(5)Å, shows an intermediate double and single bond

character. The substituted piperazine ring has a chair conformation with the methyl group

occupying an equatorial position. The nitrogen atom N (2) with a flat geometry (the C-N-

C angles amount to 358.2°, C-N =1.321(1)Å), that indicates an sp2 hybridized atom and

an electron delocalization with the CS2 fragment.

All the geometrical values are comparable to those observed in the similar bis

chelated palladium (II) complexes having the piperidine [24] and 4-methylpiperidine-

dithiocarbamate [19] as ligands, indicating that these rings have a negligible effect on the

Pd-S bond distances and the chelating S-Pd-S angles, being in the range of 2.3096(9)-

2.3187(9)Å and 75.40(3)-75.28(3)° for the four independent S of the former complex and

of 2.3189(7)-2.3300(7)Å, 75.52(2)° for the centrosymmetric species of the latter.

Finally, the crystal packing of 1 shows a 1D polymeric zig-zag chain built by H-

bonds running along axis c (Figure II.5). In fact, the water molecule O1 connects through

76

H-bonds two metal complexes through N…H-OH interactions and acts as H-bond

acceptor with respect to two symmetry related solvent molecules O2, as reported in Table

II.6.

Figure II.1. ORTEP view of the complex [Pd(4-MePipzcdt)2].3H2O (1) with atom

labeling scheme of the crystallographic independent part. (Symmetry codes: (i) -x,y,z;

(ii)x,-y,-z; (iii) -x,-y,-z).

Figure II.2. The 1D polymeric zig-zag chain of the complex 1 built by H-bonds running

along axis c.

77

Figure II.3. ORTEP drawing (35% ellipsoid probability) of complex [Pd(4-MePipzcdt)2

(4).

Table II.4. Selected bond distances (Å) and bond angles (°) for 1.

Bond distances (Å) Bond angles (°)

Pd-S(1) 2.327(1) S(1)-Pd-S(1)#3 75.57(7)

S(1)-C(1) 1.719(5) S(1)-Pd-S(1)#1 180.0

C(1)-N(2) 1.321(1) S(1)-Pd-S(1)#2 104.43(7)

N(2)-C(3) 1.478(6) S(1)#3-C(1)-S(1) 112.1(4)

C(3)-C(4) 1.513(8) N(2)-C(1)-S(1) 123.9(2)

C(4)-N(5) 1.468(7) C(1)-S(1)-Pd 85.7(2)

N(5)-C(6) 1.488(10)

Symmetry codes: #1 -x,-y,-z+1 #2 x,-y,-z+1 #3 -x,y,z

78

Table II.5. Selected bond distances (Å) and bond angles (°) for 4.

Bond distances (Å) Bond angles (°)

Pd-S(2) 2.330(3) S(1)-Pd-S(2) 74.96(10)

S(2)-C(1) 1.662(8) S(2)-Pd-S(3) 104.83(9)

C(1)-N(1) 1.278(10) S(2)-Pd-S(4) 179.73(16)

N(1)-C(2) 1.480(10) S(1)-C(1)-S(2) 113.10(4)

C(2)-C(3) 1.522(11) N(1)-C(1)-S(2) 125.80(6)

C(3)-N(2) 1.429(14) C(1)-S(2)-Pd 86.70(3)

N(2)-C(6) 1.452(10)

Symmetry codes: #1 -x,-y,-z+1 #2 x,-y,-z+1 #3 -x,y,z

Table II.6 Intermolecular hydrogen bond parameters (Å/deg) of 1.

D-H…A d(D-H) d(H...A) d(D...A) <DHA

O(1)-H(11)...N(5) 0.82(13) 1.96(13) 2.788(8) 176(3)

O(2)-H(21)...O(1)a 0.85(5) 2.38(8) 2.952(10) 125(9)

O(2)-H(22)...O(2)b 0.86(2) 2.56(3) 3.402(13) 168(10)

Symmetry codes: (a) x–1/2, y+1/2, z; (b) –x+1/2–1, y+1/2, z.

II.3.3 Spectroscopic analysis

II.3.3.1 IR Spectra

The IR spectra of the complexes are compared with the free ligands (Figures II.4-

II.6) in order to confirm the ligand coordination to the metal, and for this there are some

reference peaks that are of good help for achieving this goal. A prominent band, due to

the C-H stretching of the N-CH3 group [25] was identified around 2850 cm-1 in the IR

spectrum of the free acid 4-MePipzcdtH, while it was found to be shifted at higher

frequency (2924 cm-1) in the palladium complex. The band near 1500 cm-l indicates a

considerable double bond character of the C---N bond, as confirmed by the X-ray structure.

This fact could be ascribed to the electron releasing ability of the heterocyclic group

79

towards the sulphur atoms, a feature that induces an electron delocalization over the

carbon-nitrogen bond and the CS2 fragment. This is shown by the νC=N shift to higher

energies (ca. 1505-1450 cm-l) with respect to the free acids (ca. 1445-1430cm-1), and

these bands lie in between the stretching frequencies expected for a double C=N (1640-

1690 cm-l) and single C-N bond (1250-1350 cm-1). The blue-shift of the C=N stretching

frequency on going from the free acids to their metal complexes gives support to the

typical bidentate character [26] of the carbodithioic acid ligands. Two bands in the region

of 1030-950 cm-l (separated by less than 20 cm-l) assignable to the anti-symmetric νa(SCS),

and one band for the symmetric vs(SCS) stretch in the region 695-670 cm-l of the complexes

suggest the unsymmetrical chelating bidentate mode of coordination to the metal ion [27].

The stretchings due to νCOC (asym. and sym.), νN-CH3 and νCCC (asym. and sym.) remain

unchanged in the spectra of the complexes and in the free ligands. This observation helps

to exclude any coordination to the metals via nitrogen and oxygen donors. Finally a band

in the spectra of the complexes in the range 423-426 cm-1 is attributable to the νPd-S

stretching frequency [28]. The blue-shift of the C=N stretching frequency on going from

L3H to complex 4 gives support to the typical bidentate character [26] of the carbodithioic

acid ligand. Two bands in the region of 1034-997 cm-l (separated by less than 20 cm-l)

assignable to the anti-symmetric νa(SCS), and one band for the symmetric vs(SCS) stretch at

698cm-l of the complex 3 suggest the unsymmetrical chelating bidentate mode of the

metal ion coordination. The stretchings due to νN-CH3 and νCCC (asym. and sym.) remain

unchanged in the spectrum of the complex and in the free ligand (Table II.7). This

observation helps to exclude any coordination to the metals via nitrogen donors. Finally a

band in the spectrum of the complex at 436 cm-1 is attributable to the νPd-S stretching

frequency [28]. Very characteristic signal for 4 is absent at around 3400-3650cm-1 for O-

H stretch for water which was highly focused in complex 1(see Figures II.7 and II.8).

80

Figure II.4. FTIR spectrum of L1H using KBr disk.

Figure II.5. FTIR spectrum of L2H using KBr disk.

81

Figure II.6. FTIR spectrum of L3H in KBr disk.

Figure II.7. FTIR spectrum of complex 1 in KBr disk.

82

Figure II.8. FTIR spectrum of complex 4 in KBr disk.

Table II.7 IR spectral data of the ligands and palladium (II) complexes

Compound IR data (cm-1)

νSCS(s) νSCS(as) νC=N νPd-S

4-Methylpiperazine-l-carbodithioic

acid (L1H)

684 1023 1451 -

Morpholine-4-carbodithioic acid

(L2H)

689 1029 1468 -

4-Benzylpiperidine-l-carbodithioic

acid (L3H)

699 1072 1496 -

[Pd(L1)2] .3H2O (1) 992 1000 1497 438

[Pd(L2)2] (2) 1007 1025 1492 440

[Pd(L3)2] (3) 1010 1027 1508 436

[Pd(L1)2] (4) 997 1034 1491 436

83

II.3.3.2 1HNMR Spectra

The 1HNMR spectra of the [Pd(L)2] complexes (in dmso-d6) put in evidence the

complexes formation with a general downfield shift of signals with respect to the free

organic moieties. Three distinct peaks in the 1HNMR spectrum of 1 indicates the presence

of three types of hydrogens in the complex in accord with the crystal structure symmetry.

The signal at ca. 3.79 ppm of the eight H atoms of piperidine S2C-N(CH2)2 groups in the

coordinated ligand ring is downfield shifted compared to the value at ca. 3.15 ppm in the

free ligand, and the signal at ca. 4.35 ppm is assigned to the six H of N-CH3 (see

representative spectrum of 1) (Table II.8). The spectrum of 2 shows only two multiplet

signals, one at ca. 3.78-3.86 ppm accounts for the eight H of S2C-N(CH2)2 and another at

ca. 3.65-3.67 ppm for the eight H of O(CH2)2 of the morpholine ring. In the spectrum of

3, a characteristic triplet signal at ca. 3.18 ppm of four H of the two benzyl -CH2- groups

attached to the piperidine ring is observed in addition to other multiplets.

Figure II.9. 1HNMR spectrum of complex 1 in DMSO d6 with respect to TMS.

84

Table II.8. 1HNMR spectral data of the complexes (1-3).

Scheme 3. Alphabetic labeling of the hydrogens in 1-3 for 1HNMR spectral

characterization.

II.3.3.3 Electronic Spectra

The electronic absorption spectra of the complexes 1-3 were recorded at room

temperature using DMSO as solvent and the data are tabulated in Table II.9. The spectra

exhibit a sharp band around 305 nm along with a shoulder at ca. 340 nm assignable to the

intramolecular π→π* and n→π* transitions, respectively. The availability of the electrons

on the donor atoms for the electronic transition increases in the order 2 < 1 < 3, thereby

increasing the molar extinction coefficient values. This is due to the presence of a highly

electronegative oxygen atom in the ligand of complex 2, and of a N-CH3 and of a CH2-

benzyl substituent in 1 and 3, respectively.

Compound Ha Hb Hc Hd

1 3.79

(m, 8H)

3.15

(m, 8H)

4.35

(m, 6H)

_

2 3.65

(m, 8H)

3.80

(m, 8H)

_ _

3 3.91

(m, 8H)

1.75

(m, 8H)

3.18

(t, 4H)

7.21

(m, 10H)

N NH

c3C

S

S

NNCH3

S

S

Pd

Hb

Ha

Ha

Hb

1O

N

S

S

O

N

S

S

Pd

HaHb

HbH

a2

N

N

S

S

N

N

S

S

Pd

Ha

Hb

Hb

Ha

Hc

HdH

d

Hd

Hd

Hd

3

85

Table II.9. Electronic absorption spectral data of the complexes.

Compound λmax(nm) (10-5, ε/dm3 mol-1cm-1)a

1 300(5821), 339(942), 686(828)

2 297(4497), 335(745), 678(1122)

3 304 (9877), 340(1412), 681(913)

4 295(5450), 335(820), 680(790)

a in DMSO solvent

II.3.4 DNA binding experiments

The interaction of the palladium (II) complexes with calf thymus DNA (CT-DNA)

has been investigated using absorption and emission spectra. To examine the binding

mode of metal complexes with DNA, electronic absorption spectroscopy is used as a

distinctive characterization tool. In general, binding of the metal complex to the DNA

helix is testified by an increase of the n → π* band of palladium(II) complex due to the

involvement of strong intercalative interactions between the effective chromophore of the

complexes and the base pairs of DNA [29-31]. The absorption spectra of the free metal

complexes and of their adducts with CT-DNA are given in Figures II.10 and II.11. The

extent of the hyperchromism in the absorption band is generally consistent with the

strength of intercalative interaction [32-35] and the observed spectral changes indicate a

strong interaction of the palladium (II) complexes with CT-DNA.

In order to establish the binding strength of the metal complexes with CT-DNA, the

apparent association constant Kb was determined from the spectral titration data using the

following equation [36]:

[DNA]/(εa-εf)= [DNA]/(εb-εf) + 1/[Kb(εb-εf)]

where [DNA] is the concentration of DNA, εf, εa and εb correspond to the

extinction coefficient, respectively, for the free palladium(II) complex, for each addition

of DNA to the palladium(II) complex and for the palladium (II) complex in the fully

bound form. Plots of [DNA]/(εa-εf) vs [DNA] (Figure II.12) gave the apparent association

constant Kb as the ratio of slope to the intercept. The values of Kb for the complexes were

estimated to be 0.89 × 105 M-1 (R = 0.99763, n = 4 points), 0.32 × 105 M-1 (R = 0.99438, n

86

= 5 points) 1.54 × 105 M-1 (R = 0.9974, n = 5 points) and 0.86 × 105 M-1 (R = 0.99986, n =

5 points) for 1, 2, 3 and 4 respectively.

Figure II.10. Electronic spectral titration of complex 1 with CT-DNA at 300 nm in Tris

HCl buffer; [complex] = 2.52×10-5; [DNA]: (a) 0.0, (b) 1.25×10-6, (c) 2.5×10-6,

(d) 3.75×10-6, (e) 5.0×10-6 mol L-1. The arrow denotes the gradual increase of

DNA concentration.

(a) (b) Figure II.11. (a) Electronic spectral titration of complex 2 with CT-DNA at 297 nm in

Tris-HCl buffer; (b) Electronic spectral titration of complex 3 with CT DNA at

304 nm in Tris-HCl buffer; [complex] = 2.75×10-5; [DNA]: (a) 0.0,(b) 1.25×10-6

(c) 2.5×10-6, (d) 3.75×10-6, (e) 5.0×10-6, (f) 6.25×10-6 mol L-1. The arrow

denotes the gradual increase of DNA concentration.

280 300 320 340

0.00

0.05

0.10

0.15

e

aA

bso

rb

an

ce

Wavelength(nm)

280 300 320 340 360

0.00

0.02

0.04

0.06

0.08

0.10

f

a

Ab

so

rban

ce

Wavelength(nm)

270 300 330 360 390

0.00

0.15

0.30

0.45

0.60

0.75

0.90

f

a

Ab

so

rb

an

ce

Wavelength(nm)

87

1 2 3 4 5 6 70

20

40

60

80

100 1c

1b

1a[D

NA

]/(ε

a-ε

f) x

10

10

[DNA] x 106

Figure II.12. Plot of [DNA]/(εa-εf) vs [DNA] for the absorption titration of CT-DNA

with the Pd(II) complexes in Tris-HCl buffer;association constant Kb:0.89 x10M-1 for 1

(R = 0.99763, n = 4 points); 0.32 x 105 M-1 for 2 (R = 0.99438, n = 5); 1.54 x 105 M-1 for

3 (R = 0.9974, n = 5).Where 1a indicates 1, 1b indicates 2, 1c indicates 3.

The binding propensity of the complex to CT-DNA has been analyzed by the

fluorescence spectral technique using the emission intensity of ethidium bromide (EB).

Although EB does not show any emission in the buffer medium due to fluorescence

quenching by solvent molecules, it shows an emission band in the presence of CT-DNA

due to intercalative binding to the helix. The decrease in fluorescence intensity of the

DNA-bound EB (with excitation wavelength of 522 nm) decreases with the increasing

concentration of the complexes (see Figures II.13, II.14, II.15) because the binding of the

palladium complexes to DNA promotes the release of the EB molecules from the DNA

and concomitant decrease in the fluorescence emission [36]. This quenching due to the

addition of the palladium (II) complexes is in agreement with the linear Stern–Volmer

equation [37]:

I0/I = 1+ Ksv [Q]

where I0 and I represent the fluorescence intensities in the absence and presence of

quencher, respectively. Ksv is a linear Stern–Volmer quenching constant, Q is the

concentration of the quencher. In the quenching plot (insets of Figures II.13-II.15) of I0/I

88

vs. [complex], Ksv value is given by the slope of the regression line. The Ksv values are

0.39 × 104, 0.87 × 104, 0.82 × 104 and 0.68 × 104 for complexes 1, 2, 3 and 4 respectively)

suggesting a strong affinity of each palladium (II) complex to CT-DNA. The titration data

obtained from the fluorescence experiment can be helpful also for calculating the number

of binding sites and the apparent binding constant. In the following equation [38]:

log[(I0 - I)/I] = logK + n log[Q]

where, K and n represent the binding constant and number of binding sites of

palladium complex to CT-DNA, respectively. The calculated K values were found to be

6.7 × 104, 1.6 × 104, 12.5 × 104 and 8.3 × 104 for 1, 2, 3 and 4 respectively, with a trend

similar to the apparent association constant values of the complexes.

Figure II.13. Emission spectra of the CT-DNA-EB system in Tris-HCl buffer upon titration

with complex 1. λex = 522 nm; [EB] = 9.6×10-5 mol L-1, [DNA] = 1.25×10-5; [Complex]: (a)

0.0, (b) 1.36×10-5, (c) 2.72×10-5, (d) 4.08×10-5, (e) 5.44×10-5, mol L-1. The arrow denotes

the gradual increase of complex concentration.

580 600 620 640 660 680 700

200

400

600

800

1000 a

e

Inte

nsit

y

Wavelength(nm)

89

1.5 3.0 4.5 6.0

1.00

1.05

1.10

1.15

1.20

I 0/ I

[Complex] x 105

Figure II.13a. Plot of I0/I vs. [complex] of 1; Ksv = 0.39 × 104 (R = 0.99746, n = 4

points) in tris-HCl buffer upon titration of complex 1 with CT-DNA-EB system.

Figure II.14. Emission spectra of the CT-DNA-EB system in tris-HCl buffer upon

titration with complex 2. λex = 522 nm; [EB] = 9.6×10-5 mol L-1, [DNA] = 1.25×10-5;

[Complex]: (a) 0.0, (b) 1.38×10-5, (c) 2.75×10-5 ,(d) 4.12×10-5, (e) 5.50×10-5, (f) 8.25×10-

5 mol L-1. The arrow denotes the gradual increase of complex concentration.

570 600 630 660 6900

200

400

600

800

1000

f

a

Inte

nsit

y

Wavelength (nm)

90

1 2 3 4 5 6 7 8 9

0.90

1.05

1.20

1.35

1.50

1.65

I 0 / I

[Complex]x105

Figure II.14a. Plot of I0/I vs. [complex] of 2; Ksv = 0.87 × 104 (R = 0.98873, n = 5 points)

in tris-HCl buffer upon titration of complex 2 with CT-DNA-EB system.

Figure II.15. Emission spectra of the CT-DNA-EB system in Tris-HCl buffer upon

titration with complex 3. λex = 522 nm; [EB] = 9.6×10-5 mol L-1,[DNA] = 1.25×10-5;

[Complex]: (a) 0.0, (b) 1.25×10-5, (c) 2.5×10-5 ,(d) 3.75×10-5, (e) 5.00×10-5, mol L-1.The

arrow denotes the gradual increase of complex concentration.

570 600 630 660 690 720 7500

200

400

600

800

1000

e

a

Inte

nsi

ty

Wavelength (nm)

91

1 2 3 4 5 6

0.96

1.04

1.12

1.20

1.28

1.36

I o/ I

[Complex] x 105

Figure II.15a. Plot of I0/I vs. [complex] of 3; Ksv = 0.82 × 104 (R = 0.99965, n = 5 points)

in tris-HCl buffer upon titration of complex 3 with CT-DNA-EB system

The number of binding sites n, determined from the intercept of log[(I0 – I)/I] vs log[Q]

(see Figures II.16-II.18), are 1.19, 1.10 1.25 and 1.18 for complexes 1, 2, 3 and 4

respectively, indicating the existence of about a single binding site in DNA.

Figure II.16. Plot of log[I0-I/I] vs. log[complex] for the titration of CT-DNA-EB

system with complex 1 in Tris-HCl buffer.

-4.8 -4.6 -4.4 -4.2 -4.0

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log

[(I o

-I)/

I]

log[Complex]

92

Figure II.17. Plot of log[I0-I/I] vs. log[complex] for the titration of CT-DNA-EB system

with complex 2 in Tris-HCl buffer.

Figure II.18. Plot of log[I0-I/I] vs. log[complex] for the titration of CT-DNA-EB system

with complex 3 in Tris-HCl buffer.

-4.80 -4.65 -4.50 -4.35 -4.20

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log

[(I o

-I/I

]

log[Complex]

-4.95 -4.80 -4.65 -4.50 -4.35

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log

[(I o

-I)/

I]

log[Complex]

93

II.4 Epilogue

We reported here the synthesis and characterization of three palladium (II)

complexes with dithiocarbamates, and the solid state structure of one of these has been

established using single crystal X-ray crystallography. The electronic spectral titration of

all the complexes with CT-DNA in tris-HCl buffer showed a significant intercalative

interaction due to the stacking between the aromatic chromophore and the base pairs of

DNA with the apparent estimated association constant Kb in the range 6.7-12.5 × 104 M-1.

The linear Stern–Volmer quenching constant Ksv and the binding sites n of the complexes

to CT-DNA have been also determined from the ethidium bromide fluorescence

displacement experiments, suggesting a good affinity of the complexes to CT-DNA.

Detail of CT-DNA binding experiment is tabulated in Table II.10.

Table II.10. Complete analytical data for the DNA binding study of 1, 2, 3 and 4.

Compound Kb (M-1) KSV No. of Binding sites

(n)

1 0.89 x105 0.39 x104 1.19

2 0.32 x105 0.87 x104 1.10

3 1.54 x105 0.82 x104 1.25

4 0.86 x105 0.68 x104 1.18

94

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[30] V.A. Bloomfield, D.M. Crothers, I. Tinoco, Physical Chemistry of Nucleic Acids,

Harper and Row, New York (1974) 432.

[31] R. Vijayalakshmi, M. Kanthimathi, V. Subramanian, B. U. Nair, Biochem Biophys

Acta 1475 (2000) 157.

[32] S. A. Tysoe, R. J. Morgan, A. D. Baker, T.C. Strekas, J Phys Chem 97 (1993) 1707.

[33] J. K. Barton, A. T. Danishefsky, J. Goldberg, J Am Chem Soc 106 (1984) 2172.

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[37] O. Stern, M. Volmer, Z Phys 20 (1919) 183.

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.

96

CHAPTER-III

Palladium(II) and Platinum(II) Complexes of

Deprotonated Carboxamide Ligands: Synthesis,

Structural Characterization and Binding

Interactions with DNA and BSA

97

Abstract

Four neutral complexes [ML] (where M = Pd (1a, 1c, 1d) and Pt (1b), N,N'-bis(2-

pyridinecarboxamide)-1,2-benzene ligand = H2L4, N,N

'-bis(2-pyridinecarboxamide)-2,3-

pyridine ligand = H2L5 , N,N

'-bis(2-pyridinecarboxamide)-5-bromo-2,3-pyridine ligand =

H2L6) have been synthesized and characterized by physico-chemical and spectroscopic

tools along with the detailed structural analysis by single crystal X-ray crystallography

and theoretical (DFT) study. In solid state, the compounds are isomorphous and

isostructural showing the formation of trimeric species [ML]3. 3H2O. Electrochemical

study of 1a showed a quasi-reversible reductive response at E1/2 = –1.148 V assignable to

the Pd(II)/Pd(I) couple, while a metal centered irreversible oxidative peak centred at

+0.977 V was observed in the voltammogram of 1b. The interaction of the four

complexes with CT-DNA has been investigated using spectroscopic tools and viscosity

measurement. In each case the association constant (Kb) was deduced from the absorption

spectral study and the number of binding sites (n) and the binding constants (K) were

calculated from relevant fluorescence quenching data, suggesting a non-covalent

interaction between the metal complex and DNA, which could be assigned to an

intercalative binding. In addition, the interaction of 1a and 1b was ventured with bovine

serum albumin (BSA) with the help of absorption and fluorescence spectroscopy

measurements. Through these techniques, the apparent association constant (Kapp) and the

binding constant (K) could be calculated for each complex.

98

III.1 Introduction

The last few decades have witnessed a remarkable interest in pyridine–N

containing carboxamide complexes in various fields of biological relevance like

asymmetric catalysis [1,2], dendrimer [3], molecular receptor synthesis [4] and also in the

synthesis of compounds with possible antitumour properties [5]. The carboxamide [–

C(O)NH–] group of the primary structure of protein represents an important ligand

construction unit in coordination chemistry, since its chelating rigid nature imparts a

unique balance of stability versus reactivity, and has allowed for impressive

developments in a variety of catalytic transformations.

Deprotonated amide groups readily coordinate metal ions through the amide-N

and/or -O atom [6] forming a stable delocalized electronic system. The anticancer

properties of cis-platin and palladium (II) complexes stem from the ability of the cis-

MCl2 fragment to bind to DNA bases. However, cis-platin also interacts with non-cancer

cells, mainly binding molecules containing -SH groups, resulting in nephrotoxicity [7,

11]. This has aroused interest in the design of novel palladium (II) [8, 10] and platinum

(II) complexes with better efficacy and lower toxicity. Serum albumins are the major

soluble protein constituents of the circulatory system and serve as a depot and as a

transfer protein along with several physiological functions. BSA has been one of the most

extensively studied of these proteins, particularly because of its structural homology with

human serum albumin [9].

In the present chapter we have turned our attention to explore the amide

functionality of the N,N'-bis(2-pyridinecarboxamide)-1,2-benzene ligand (H2L

4), N,N'-

bis(2-pyridinecarboxamide)-2,3-pyridine ligand (H2L5), N,N

'-bis(2-pyridine-

carboxamide)-5-bromo-2,3-pyridine ligand (H2L6) towards palladium (II) and platinum

(II) ion. We report herein three novel palladium (II) (1a, 1c, 1d) and one platinum (II)

(1b) complexes with the tetradentate ligand having two deprotonated amide-N and two

pyridinic-N donors. These species were structurally characterized by X-ray diffraction

and also by means of different physico-chemical, spectroscopic and computational

studies. In addition, interaction of complexes 1a to 1d with CT-DNA and also of

complexes 1a and 1b with bovine serum albumin (BSA) have been studied. In order to

establish the association mode of these small molecules to DNA, the binding constant

(Kb) and the quenching constant (Kq) of the complexes with the double-helix has been

99

determined from absorption study and fluorescence displacement experiments using

ethidium bromide as spectral probe. Beside this, the strong binding activity of complexes

1a and 1b with bovine serum albumin (BSA) were examined using absorption-

fluorescence spectroscopy, further supported by viscosity measurements.

III.2 Experimental Section

III.2.1 Solvents and Reagents

All chemicals and reagents were obtained from commercial sources and used as

received, unless otherwise stated. 2-Pyridinecarboxylic acid, 1,2-diaminobenzene and

triphenyl phosphite were purchased from Aldrich and K2PtCl4 and PdCl2 procured from

Across, were used as such. The solvents used were purified following the standard

procedures; all other chemicals used were of analytical reagent grade. For detailed

purification and drying of solvents follow chapter II under section II.2.1.

III.2.2 Physical Measurements

The elemental (C, H, N) analyses were performed on a Perkin Elmer model 2400

elemental analyzer. Electronic absorption spectra and IR spectra were obtained on a

JASCO UV–Vis/NIR spectrophotometer model V-570 and on a Perkin-Elmer FTIR

model RX1 spectrometer (KBr discs, 4000-300 cm-1), respectively. 1H NMR spectra were

recorded on a Bruker AC300 spectrometer using TMS as an internal standard in DMSO-

d6 solvent. Electro spray ionization (ESI) mass spectra of complexes 1a and 1b were

recorded with a Qtof Micro Instrument (Waters, YA263). The fluorescence spectra

complex bound to DNA were obtained at an excitation wavelength of 522 nm in the

Fluorimeter (Hitachi-4500). Viscosity experiments were conducted on an Ostwald’s

viscometer, immersed in a thermostated water-bath maintained at 25°C. Redox potentials

were measured in CHI620D potentiometer in DMF using TBAP as supporting electrolyte

at room temperature. Electrochemical setup was a three-electrode cell with glassy carbon,

Ag/AgCl and a platinum wire as a working, reference and counter electrode, respectively.

Molar conductances (ΛM) were measured in a systronics conductivity meter 304 model in

dimethylformamide at complex concentration of ~10-3 mol L-1.

100

III.2.3 X-Ray Crystal Structure Analysis

Data collections of compounds 1a, 1b and 1c were carried at room temperature on

a Bruker Smart Apex diffractometer equipped with CCD. (λ = 0.71073 Å). Cell

refinement, indexing and scaling of the data sets were done by using programs Bruker

Smart Apex and Bruker Saint packages [12]. The structures were solved by direct

methods and subsequent Fourier analyses and refined by the full-matrix least-squares

method based on F2 with all observed reflections [13]. Hydrogen atoms were placed at

calculated positions, those of lattice water molecules of compound 1b were located on

the ∆Fourier map and analogously for molecule Ow1 of 1a. All the calculations were

performed using the WinGX System, Ver 1.80.05 [14]. Crystal data and details of

refinements are given in Table III.1. Crystallographic data for have been deposited with

the Cambridge Crystallographic Data Centre, CCDC No. 856198 and 873080 for 1a and

1b, respectively.

III.2.4 Syntheses of Ligands

III.2.4.1 Synthesis of H2L4: The ligand H2L

4 was synthesized following reported method

[15] with slight modification. Pyridinic solution (10 ml) of 2-pyridinecarboxylic acid

(1.23 g, 10 mmol) and 1,2-diaminobenzene (0.54 g, 5 mmol) was mixed under stirring

condition followed by the dropwise addition of triphenyl phosphite (3.1 g, 10 mmol) at

80°C for 4 h and settled overnight. A pale brown crystalline solid resulted, was washed

with methanol to give long white needles. Yield: 96%.

III.2.4.2 Synthesis of H2L5: 2-pyridinecarboxylic acid (1.23 g, 10 mmol) and 2, 3-

diaminopyridine (0.545 g, 5 mmol) were added together to a total of 10 mL pyridine and

the mixture was stirred at warm condition for 10 mins. Then triphenyl phosphite (3.1 g,

10 mmol) was added slowly to the mixture. The mixture was stirred and heated at 80°C

under reflux for 5 h, after which it was cooled to room temperature and settled overnight

and the white solid appeared. It was washed with methanol to give long white needles. It

was further dissolved in methanol and the white crystals, suitable for X-ray diffraction,

were obtained by slow evaporation. Yield: 93%.

III.2.4.3 Synthesis of H2L6: H2L

6 was synthesized in an identical manner to that

described for H2L5 with 5-Bromo-2, 3-diaminopyridine in place of 2, 3-diaminopyridine.

Yield: 95%.

101

Table III.1. Crystallographic data for complexes 1a, 1b and 1c.

Complex (1a).1/2H20 (1b).1/2H20 (1c)

Empirical Formula C54H42N12O9Pd3 C54H42N12O9Pt3 C51H41N15O10Pd3

Fw 1322.20 1588.27 1343.19

Crystal System Monoclinic Monoclinic Monoclinic

Space group C 2/c C 2/c C 2/c

a, Å 14.5231(17) 14.5777(5) 4.47030(10)

b, Å 20.2671(17) 20.2335(6) 8.4501(2)

c, Å 16.4470(17) 16.4016(5) 12.1978(3)

β, deg 97.365(3) 97.237(2) 91.193(2)

V, Å3 4801.1(9) 4799.2(3) 444.437(18)

Z 4 4 1

Dcalcd, g cm–3 1.829 2.198 1.708

µ (Mo-Kα) mm–1 1.188 8.799 1.512

F(000) 2640 3024 232

θ range, deg 1.73 - 28.22 1.73 - 31.00 1.7 - 34.44

no. of reflns collcd 26979 36287 13963

no. of indep reflns 5836 7530 6830

Rint 0.0513 0.0325 0.0264

no. of reflns (I>2σ(I)) 3951 5593 5188

no. of refined params 357 362 190

Goodness-of-fit (F2) 1.025 1.010 1.007

R1, wR2 (I >2σ(I)) [a] 0.0344, 0.0767 0.0231, 0.0487 0.0316, 0.0596

R indices (all data) 0.0623, 0.0890 0.0428, 0.0554 0.0529, 0.0682

aR = ∑||Fo |- |Fc||/∑|Fo|;

bwR2 = { ∑ [w(Fo

2 –Fc2)2] / ∑w [(Fo

2)2]}1/2

102

III.2.5 Syntheses of Palladium (II) and Platinum (II)-Complexes

III.2.5.1 Synthesis of [Pd(L4)].1/2H20 (1a)

To a solution of H2L4 (0.636 g, 0.50 mmol) in dry DMF (10 mL) was added

NaH (0.0237g, 1.00 mmol) and the resulting suspension was stirred for 30 min. To the

resulting light yellow solution, PdCl2 (0.089 g, 0.50 mmol) dissolved in DMF was added

in portions with continuous stirring for a period of 3 h. The yellow precipitate resulted

was filtered and washed with diethyl ether and vacuum dried. The residue was further

dissolved in DMF (6.0 mL), filtered, and the volume of the filtrate was reduced to 3.0

mL. Yellow crystals, suitable for X-ray diffraction, were obtained by slow evaporation.

Yield: 0.46 g, 82-85%.

III.2.5.2 Synthesis of [Pt(L4)].1/2H20 (1b)

To a solution of H2L4 (0.636 g, 0.50 mmol) in dry DMF (10.0 mL), NaH (0.0237

g, 1.00 mmol) was added and the resulting suspension was stirred for 30 min. To the

resulting light yellow solution, aqueous K2PtCl4 (0.208 g, 0.50 mmol) was added in

portions with vigorous stirring under nitrogen atmosphere and stirring has continued for

further 10 h. The resulting orange-red solution was allowed to evaporate slowly,

obtaining orange micro crystals suitable for X-ray diffraction studies. Yield: 0.38 g, 79-

80%.

III.2.5.3 Synthesis of [Pd(L5)] (1c)

NaH (0.0237 g, 1.00 mmol) was added to a dimethylformamide solution (10 ml)

of H2L5 (0.159 g, 0.50 mmol). After the mixture had been stirred at room temperature for

a short time, a yellow solution resulted, to which PdCl2 (0.089 g, 0.50 mmol) dissolved in

DMF added and the mixture was stirred for 4 h. The yellow precipitate resulted was

filtered and washed with diethyl ether and vacuum dried resulting an analytically pure

solid. It was further dissolved in DMF and the yellow crystals, suitable for X-ray

diffraction, were obtained by slow evaporation. Yield: 0.05 g, 85-90%.

III.2.5.4 Synthesis of [Pd(L6)] (1d)

Complex 1d was synthesized in an identical manner to that described for L5 with

5-Bromo-2, 3-diaminopyridine in place of 2, 3-diaminopyridine Yield: 0.046 g, 80-82%.

103

III.3 Results and Discussions

III.3.1 Syntheses and Characterizations

The organic amide ligand H2Ln (n = 4, 5, 6) were synthesized by the reaction of 1,

2-diaminobenzene, 2, 3-diaminopyridine and 5-Bromo-2, 3-diaminopyridine respectively

with 2-pyridinecarboxylic acid stirring at 80-85°C in pyridine medium, and have been

characterized by IR and 1H NMR spectral analyses. The palladium (II) complexes 1a, 1c

and 1d and platinum (II) complex 1b were obtained in good yield from the reaction of

palladium (II) chloride and potassium tetrachloro palatinate (II), respectively with the

tetradentate ligand L in 1:1 molar ratio in the DMF medium with prolonged stirring at

room temperature (viz Scheme 1 and Table III.2). The complexes were isolated as yellow

solids, which are much soluble in DMSO and DMF. The palladium (II) complexes were

stable for several weeks in the air.

Scheme 1. Synthetic routes of the complexes.

X

N NO O

N N

Pd

N NO O

N N

Pt

PdCl2 in DMF

K2PtCl4

DMF-H2O, N2

X

NH HNO O

N N

H2L4

Stir, 3h

Stir, 12h

1b.1/2H2O

Y Y

NH HNO O

N N

When X=CH, Y=H; H2L4

When X=N, Y=H; H2L5

When X=N, Y=Br; H2L6

When X=CH, Y=H;1aWhen X=N,Y=H; 1cWhen X=N, Y=Br; 1d

104

Table III.2. Palladium (II) and platinum (II) complexes with amide ligands.

Microanalytical data (Table III.3) confirm the composition of the complexes

and spectroscopic data of 1a, 1b, 1c and 1d along with the X-ray crystallographic results

of 1a, 1b and 1c indicate the organic moieties, 1, 2-diaminobenzene, 2, 3-

diaminopyridine and 5-Bromo-2, 3-diaminopyridine in the reaction medium are converted

to N,N'-bis(2-pyridinecarboxamide)-1,2-benzene (H2L4), N,N'-bis(2-pyridine-

carboxamide)-2,3-pyridine (H2L5) and N,N'-bis(2-pyridinecarboxamide)-5-bromo-2,3-

pyridine (H2L6 ) respectively, and their palladium (II) complexes 1a, 1c, 1d and platinum

(II) complex 1b.

Table III.3. Microanalytical data of palladium (II) and platinum (II) complexes (1a - 1d).

Compounds Elemental analyses Found (calcd.) Conductance

(Λo)a C H N

[Pd(L4)].1/2H20 (1a) 49.05 (48.26) 3.20 (3.94) 12.71 (12.18) 51

[Pt(L4)].1/2H20 (1b) 40.83 (40.67) 2.66 (2.64) 10.58 (10.54) 48

[Pd(L5)] (1c) 48.19 (46.02) 2.62 (1.98) 16.53 (14.26) 7

[Pd(L6)] (1d) 40.62 (41.85) 2.01 (3.46) 12.71 (13.93) 6

amho.cm2 mol-1 in DMF.

The monomeric complexes 1a – 1d are soluble in DMF and DMSO but insoluble in

methanol and ethanol. The conductivity measurement of complexes in DMF showed the

conductance values in the range of 6-51 Λo mol-1 cm-1 at 300 K. These values suggest that

the complexes exist as non-electrolytes in solution [16].

LnH Complex

N,N'-bis(2-pyridinecarboxamide)-1,2-benzene ( H2L4 )

[Pd(L4)].1/2H20 (1a)

[Pt(L4)].1/2H20 (1b)

N,N'-bis(2-pyridinecarboxamide)-2,3-pyridine ( H2L5 ) [Pd(L5)] (1c)

N,N'-bis(2-pyridinecarboxamide)-5-bromo-2,3-pyridine ( H2L6 ) [Pd(L6)] (1d)

105

III.3.2 Structural description of the complexes 1a, 1b and 1c

The X-ray structural analysis show that complexes 1a and 1b are isomorphous and

isostructural showing the formation of [ML]3.3H2O aggregates (M = Pd and Pt,

respectively L = bis(pyridine-2-carboxamide)benzene dianion. The independent

crystallographic unit comprises of one and half neutral metal complex, being one located

on a two-fold axis passing through the metal and bisecting the Namide-M-Namide bond

angle, as shown in Figures III.1 and III.2 for the palladium and platinum derivative 1a

and 1b respectively. The crystals contain also some disordered water lattice molecules.

As expected, in 1a and 1b the metal ion is chelated by the tetradentate dianionic

ligand L in a square planar coordination geometry and due to the nature of the ligand, all

the atoms in each complex are coplanar. The bond lengths reported in Table III.4 indicate

that the values relative to the pyridine N donors are significantly longer by ca. 0.1 Å than

those of the amide nitrogen atoms, either in the Pd and Pt complexes, and the Pd-N bond

values are in agreement with those reported in analogous complexes [17]. On the other

hand the Pd-N bond lengths appear slightly longer with respect to the correspondent Pt-N

values in agreement with the metal ionic radius.

It is worth noting of the supramolecular arrangement observed in these compounds,

being the complexes arranged as trimeric entities where complex Pd1(Figure III.1) and

Pt1(Figure III.2) of C2 symmetry; stacking is observed in between a pair of two

symmetry related Pt1 and Pt2 units (Figure III.3), similar packing pattern has been found

in between Pd1 and Pd2 units. Within this trimer the platinum metal ions in 1b are

separated by 3.2897(1) Å, in an almost collinear arrangement forming a Pt(1)-Pt(2)-Pt(1)

angle of 178.64(1)°. The correspondent figures in 1a are close comparable being of

3.2903(4)Å and 179.33(1)° , respectively. This packing feature is not unusual for square

planar complexes having aromatic ligands and the aggregation of complexes are

stabilized by a combination of π-π stacking interactions between the pyridine and phenyl

rings of the ligands and d8-d8 metallophilic contacts [18-20]. A structural indication of the

latter interaction between the metals is the slight displacement from the N4 donor set

plane of atom M(1) by ca. 0.03 Å towards M(2) (M = Pt or Pd) inside each trimer. A

rotation of ca. 140° is requested to complex Pt1 in order to be superimposed to Pt2 that

occupies the center (Figure III.3), and similarly for the palldium complex.

106

Since the number of water molecules and crystal packing are closely comparable for

1a and 1b, we limit here the description for the former compound. The lattice water

molecules reside sideways to the trimer entities and are connected through H-bonds. In

fact O2w, which is located on a two-fold axis, is weakly bound to O1w (O…O = 3.16Å),

which in turn connects the carbonyl oxygens O(1) and O(3) of symmetry related

complexes (O….O = 2.80, 3.01Å, respectively) forming a 2D packing arrangement

parallel to the crystallographic ab plane.

1a

Figure III.1. ORTEP drawing (35% ellipsoid probability) of complex 1a located

on a crystallographic two-fold axis. (primed atoms at -x, y, -z+1/2).

1b

Figure III.2. ORTEP drawing (35% ellipsoid probability) of complex 1b located

on a crystallographic two-fold axis. (primed atoms at -x, y, -z+1/2).

107

Figure III.3. Complex trimers in the crystal packing of 1b connected through H-bonds

occurring among carbonyl groups and lattice water molecules. A similar packing is

observed in 1a.

The complex of formulation [PdL']3.4H2O where the metal is chelated by the ligand

L' (= bis(pyridine-2-carboxamide)pyridine dianion) [21]. The independent crystallo-

graphic unit comprises of one neutral metal complex and a half one located on a two-fold

axis passing through the metal and bisecting the Namide-M-Namide bond angle. The complex

co-crystallize with disordered water lattice molecules and the water content is detected

likely for the presence in the ligand of an uncoordinated N that interacts with the

disordered water molecules. The metal ion is chelated by the tetradentate dianionic ligand

L5 (1c) in a square planar coordination geometry and due to the nature of the ligand, all

the atoms in the complex are coplanar. The bond lengths reported in Table III.4 indicate

that the values relative to the pyridine N donors are significantly longer by ca. 0.1 Å than

those of the amide nitrogen atoms in the palldium complex, and the Pd-N bond values are

in agreement with those reported in analogous complexes with similar ligand halogen-

and methyl-substituted [22]. It is worth noting the supramolecular arrangement observed

in this compound (Figure III.4), being the complex arranged as trimeric entity (Figure

III.5).

108

Figure III.4. ORTEP drawing (35% ellipsoid probability) of complex 1c. Due to the

imposed crystallographic C2 symmetry, the atom N(8) is to be considered

interchangable with group C(26)-H (the independent position was refined as a

mixed C/N species).

Figure III.5. Packing of complex trimers in 1c connected through H-bonds occurring

among carbonyl groups and disordered lattice water molecules.

109

Table III.4. Coordination bond lengths (Å) and angles (°) for compounds 1a, 1b and 1c.

1a, M= Pd 1b, M= Pt 1c, M=Pd M(1)-N(1) 2.054(3) 2.040(3) 2.053(2)

M(1)-N(4) 2.055(3) 2.044(2) 2.056(2)

M(1)-N(2) 1.939(3) 1.953(2) 1.962(2)

M(1)-N(3) 1.951(3) 1.944(3) 1.940(2)

M(2)-N(5) 2.053(3) 2.043(3) 2.051(2)

M(2)-N(6) 1.946(3) 1.958(2) 1.952(2)

M(1)-M(2) 3.290(4) 3.289(1) 3.3099(2)

N(1)-M(1)-N(2) 81.33(10) 81.28(10) 81.95(9)

N(1)-M(1)-N(3) 165.28(11) 165.79(10) 165.92(9)

N(1)-M(1)-N(4) 112.51(10) 112.64(10) 112.22(9)

N(2)-M(1)-N(3) 84.22(11) 84.81(10) 84.26(10)

N(2)-M(1)-N(4) 166.13(11) 166.01(10) 165.79(9)

N(3)-M(1)-N(4) 81.91(11) 81.22(10) 81.54(9)

N(5)-M(2)-N(6) 81.85(11) 81.37(11) 81.74(9)

N(5)-M(2)-N(6') 165.73(11) 165.90(10) 166.08(9)

N(5)-M(2)-N(5') 112.41(15) 112.72(14) 112.17(13)

N(6)-M(2)-N(6') 83.90(15) 84.55(15) 84.36(13)

M(1)-M(2)-M(1') 179.33(1) 178.64(1) 177.42(1)

Primed atoms at -x, y, -z+1/2.

Within this trimer the Pd metal ions in 1c are separated by 3.3099(2) Å, in an

almost collinear arrangement forming a Pd(1)-Pd(2)-Pd(1) angle of 177.42(1)°. This

packing feature is not unusual for square planar complexes having aromatic ligands and

the aggregation of complexes accounts for π-π interactions between the pyridine and

phenyl rings of the ligands and dz2-dz

2 orbital interactions between the metals [23]. A

structural indication of the latter interaction between the metals is the slight displacement

from the N4 donor set plane of atom M(1) by ca. 0.03 Å towards M(2) (M = Pd) in the

complex. A rotation of ca 140° is requested to complex Pd1 in order to be superimposed

to Pd2 that occupies the center. The single crystal X-ray structure of 1c shows two

110

independent water molecules and thus four per complex trimer. Both molecules are

disordered over two positions which account for the requisite to interact through H-bond

with the uncoordinated N of the pyridine ring.

III.3.3 Spectroscopic analysis

III.3.3.1 IR Spectra

The characteristic IR frequency data observed in the IR spectra of the compounds

have been tabulated in Table III.5. For carboxamide ligands (H2L4-H2L

6) an IR band at

around 1640-1645 cm-1 is assigned due to C=O stretching and a split band at around

3317-3320 and 3292-3294 cm-1 is assigned due to N-H vibrations. In the IR spectrum of

1a-1d the absence of νN-H stretching clearly indicates that the ligand H2L4-H2L

6 is

coordinated to Pd(II) or Pt(II) ion in the deprotonated form. In the complexes the νC=O

vibration has been shifted to lower frequency at around 1600-1635 cm-1 due to metal

ligand chelating effect. For the complex 1a and 1b, νO-H stretching at 3400-3500cm-1

shows due to water molecules as a solvent of crystallization.

Table III.5. IR spectral data of the ligands , palladium (II) and platinum (II) complexes.

Compound νC=O ννννN-H ννννM-N

N,N'-bis(2-pyridinecarboxamide)-

1,2-benzene ( H2L4 )

1640 3318, 3293 -

N,N'-bis(2-pyridinecarboxamide)-

2,3-pyridine ( H2L5 )

1642 3317, 3294 -

N,N'-bis(2-pyridinecarboxamide)-5-

bromo-2,3-pyridine ( H2L6 )

1645 3320, 3292 -

[Pd(L4)].1/2H20 (1a) 1629 - 419

[Pt(L4)].1/2H20 (1b) 1635

- 415

[Pd(L5)] (1c) 1600 - 435

[Pd(L6)] (1d) 1601 - 440

111

III.3.3.2 Electronic Spectra

The electronic absorption spectra of complexes 1a to 1d were recorded at room

temperature using DMF as solvent. The spectra exhibit a sharp band around 280 nm

assignable to the intramolecular π → π* transition. Another relative high intensity band

around 310 nm is due to a charge transfer from amide ligand core to metal, i.e. LMCT

(Table III.6). The low energy tail of the charge transfer band that appears in the visible

region of the spectrum is responsible for the yellow and orange-yellow color of the

solution containing 1a, 1b, 1c and 1d respectively. Here, it is observed that the transition

for the palladium (II) complex shifted to lower energy compared to that of the platinum

(II) derivative, and this study is in accordance with the theoretical calculation of the

energy of HOMO and LUMO for 1a and 1b, being LUMO for complex 1a lower lying

compared to 1b (see computational part).

Table III.6. Electronic absorption spectral data of 1a to 1d.

Compound λmax(nm) (10-5, ε/dm3 mol-1cm-1)a

1a 280 (5010), 315( 7015)

1b 282 (4950), 317 (6900)

1c 280 (4,900), 308 (5,510)

1d 284 (5,415), 311 (6,980)

a Solvent used for every experiment is DMF

III.3.3.3 Electrochemistry

The electrochemical properties for 1a and 1b have been studied by cyclic

voltammetry (CV) at a platinum working electrode in dimethylformamide (0.1M TBAP

as supporting electrolyte) at room temperature. The cyclic voltammograms of 1a and 1b

are displayed in Figures III.6 and III.7, respectively. The CV scan of 1a revealed a one-

electron quasi-reversible reductive response at E1/2 = –1.148 V (Epc = –1.245 V and Epa =

–1.051 V; ∆E = 194 mV) assignable to Pd(II)/Pd(I) couple. On the contrary, platinum (II)

in 1b is irreversibly oxidized to platinum (IV) by two electron stoichiometry [24, 25]

centred at +0.977V.

112

Figure III.6. Cyclic voltammogram (scan rate 100 mV/s) of 1a in DMF solution of 0.1 M

TBAP, using platinum working electrode.

Figure III.7. Cyclic voltammogram (scan rate 100 mV/s) of 1b in DMF solution

containing 0.1 M TBAP, using platinum working electrode.

From the theoretical calculation of HOMO and LUMO energy of both

complexes, it may be derived that the comparatively lower energy of LUMO of 1a may

be responsible for the reduction of palladium (II)/palladium (I) and accordingly the higher

energy of HOMO of 1b for the irreversible oxidation of platinum (II) to platinum (IV)

(see computational part).

113

III.3.4 Computational details

The DFT calculations for the isolated complexes 1a and 1b were performed using

Dmol3 code [26] in the framework of a generalized-gradient approximation (GGA) [27].

The geometry of the molecules were fully optimized using the hybrid exchange-

correlation functional BLYP [28] and a double numeric plus polarization (DNP) basis set

(Figures III.8-III.10). The electronic structures were also calculated at the same level. No

constraints on bond lengths, bond angles or dihedral angles were applied in the

calculations, and all atoms were free to optimize. Convergence was assumed to be

reached when the total energy change between two consecutive self-consistent field

(SCF) cycles was less than 1x10-5a.u.

Figure III.8. Surface plots of frontier orbitals of 1a.

Figure III.9. Surface plots of frontier orbitals of 1b.

H0M0 LUM0

H0M0 LUM0

114

Figure III.10. Molecular orbital diagrams.

III.3.5 Binding experiments with calf thymus-DNA

The mode of interaction of the complexes 1a to 1d with calf thymus DNA (CT-

DNA) has been investigated using absorption and emission spectroscopic tools as well as

by viscosity and cyclic voltammetry measurements.

The binding experiments with calf thymus DNA for complexes 1a to 1d were

monitored following the same procedure previously reported [29] with UV–Vis and

fluorescence spectroscopic tools and also by viscosity and cyclic voltammetry

measurements. All the experiments involving the interaction of the complexes with CT-

DNA were carried out in MilliQ water containing tris–HCl buffer (pH 8.04). The solution

of CT-DNA in the buffer gave a ratio of UV absorbance of ca. 1.8–1.9:1 at 260 and 280

nm, indicating that the CT-DNA was sufficiently free of protein [30]. Stock solution of

DNA was always stored at 40C in the dark and used within four days. The CT-DNA

concentration per nucleotide was determined spectrophotometrically by employing an

1b 1a

115

extinction coefficient of 6600 M-1 cm-1 at 260 nm [31]. The complexes were dissolved in

a solvent mixture of 1% DMSO and 99% tris–HCl buffer at 1.0 x 10-4 M-1 concentration.

Absorption spectral titration experiment was performed by keeping constant the

concentration of the complex (10 µM) and varying the CT-DNA concentration. While

measuring the absorption spectra, an equal amount of CT-DNA was added to both the

complex solutions and the reference solution to take into account the absorbance of DNA

itself. In the emission quenching experiment, ethidium bromide (EB) was used as a

common fluorescent probe for the DNA in order to examine the mode and process of

metal complex binding to the double-helix [32]. A 5.0 µL of the EB tris–HCl buffer

solution(1.0 mmol L-1) was added to 1.0 mL of DNA solution (at saturated binding levels)

[33], stored in the dark for 2 h. Then the solution of each of the Pd(II) and Pt(II)

complexes was titrated into the DNA/EB mixture and diluted in tris–HCl buffer to 5.0 mL

to get the solution with the appropriate complex/ CT-DNA mole ratio. After the

incubation at room temperature for 30 min, the fluorescence spectra of EB bound to DNA

were recorded (λex = 522 nm) in the Hitachi 4500 Fluorimeter. All measurements were

performed at ambient temperature. The binding interaction of the metal complexes with

DNA was studied by the well known method employing the Ostwald’s viscometer. The

CT-DNA solution (5 µM) was titrated with platinum (II) and palladium (II) complexes

(0.5–3.5 µM), following the change of the viscosity in each case. Data are presented as

(η/η0)1/3 versus the ratio of concentration of the compound and CT-DNA, where η is the

viscosity of CT-DNA in presence of the compound and ηo is the viscosity of CT-DNA

alone. Viscosity values were calculated from the observed flow time of CT-DNA-

containing solution corrected from the flow time of buffer alone (t0), η = t–t0 [34].

III.3.5.1 Absorption spectroscopy

Electronic absorption spectroscopy is used as a distinctive characterization tool

for examining the binding mode of metal complexes with DNA [33, 35]. In intercalative

binding mode, the π* orbital of the intercalated ligand can couple with the π orbital of the

base pairs, thus decreasing the π→π* transition energy and resulting in bathochromism.

On the other hand, the coupling π orbital was partially filled by electrons, thus decreasing

the transition probabilities and concomitantly resulting in hypochromism [36]. The

absorption spectra of the free metal complexes and of their adducts with CT-DNA (at a

116

constant concentration of the compounds) are given in Figures III.11 to III.14 for

complexes 1a to 1d, respectively. The extent of hyperchromism in the absorption band is

generally consistent with the strength of intercalative interaction [28, 36]. As the

concentration of CT-DNA is increased, it was found that the Pd and Pt complexes at 270

nm and 268 nm exhibit hyperchromicity of 3.5/19.76% and 3.7/24.02%, respectively.

This feature might be ascribed to the fact that both of the co-complexes could uncoil the

helix structure of DNA and made more bases embedding in DNA exposed [37-39]. In

order to establish the binding strength of the metal complexes with CT-DNA, the

apparent association constant Kb was determined from the spectral titration data using the

following equation [40].

1/∆εap = 1/ (∆εKb D) + 1/∆ε

where, ∆εap= |εa-εf|, ∆ε= |εb-εf|, D = [DNA], and εa, εb and εf are respectively the

apparent, bound and free extinctions coefficient of each of the compound in respective

cases. Kb, expressed as M–1, is derived from the slope of the graph obtained by plotting

the [DNA]/(εa-εf) vs [DNA] (Figures III.15-III.16). The Kb values for complexes for 1a

to 1d were estimated to be 0.36 × 104 M–1 (R = 0.99999, n = 5 points), 0.93× 104 M–1 (R =

0.99885, n = 5 points), 0.80 × 105 M–1 (R = 0.99889, n = 5 points) and 0.78× 105 M–1 (R =

0.99894, n = 5 points) respectively.

In order to corroborate the binding mode of intercalation of the palladium (II) and

platinum (II) complexes with CT-DNA, we employed ethidium bromide (EB) that

interacting with DNA, represents a characteristic indicator of intercalation. The maximal

absorption of EB at 479 nm decreased and shifted to 499 nm in presence of DNA (Figure

III.17), typically indicating insertion between the base pairs [41]. The absorption spectra

of the mixture solution of EB, palladium(II) complex 1a and DNA and similarly for

platinum(II) complex 1b are reported (Figures III.17(a) and III.17(b), respectively). The

observed behavior could be indicative of (1) being EB strongly bound to complex 1a (or

1b), the result is a decrease amount of EB intercalated into DNA; or (2) there exists a

competition between the palladium (II) (or platinum (II)) complex and EB towards DNA

intercalation, so releasing some free EB from DNA–EB complex. However, here the

former account could be irrelevant because of the appearance of a new absorption band.

117

Figure III.11. Electronic spectral titration of complex 1a with CT-DNA in tris-HCl

buffer; [1a] = 2.62×10-5; [DNA]: (a) 0.0, (b) 1.25×10-6, (c) 2.5×10-6, (d) 3.75×106,

(e) 5.0×10-6 , (f) 6.25×10-6 mol L-1. The arrow denotes the gradual increase of DNA

concentration.

Figure III.12. Electronic spectral titration of complex 1b with CT-DNA in tris-HCl

buffer; [1b] = 2.64×10-5; [DNA]: (a) 0.0, (b) 1.25×10-6, (c) 2.5×10-6, (d) 3.75×10-6, (e)

5.0×10-6, (f) 6.25×10-6 mol L-1. The arrow denotes the gradual increase of DNA

concentration.

300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

0.12 f

a

Ab

so

rb

an

ce

Wavelength(nm)

300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

0.12

f

a

Ab

sorb

an

ce

Wave length (nm)

118

Figure III.13. Electronic spectral titration of complex 1c with CT-DNA at 267nm in tris-

HCl buffer;[1c]: 3.42×10-5; [DNA]: (a) 0.0, (b) 1.25×10-6, (c) 2.5×10-6, (d) 3.75×106, (e)

5.0×10-6, (f) 6.25×10-6 mol L-1. The arrow denotes the gradual increase of DNA

concentration.

Figure III.14. Electronic spectral titration of complex 1d with CT-DNA at 269 nm in

tris- HCl buffer; [1d] = 2.62×10-5; [DNA]: (a) 0.0, (b) 1.25×10-6, (c) 2.5×10-6, (d)

3.75×106, (e) 5.0×10-6 , (f) 6.25×10-6 mol L-1. The arrow denotes the gradual increase of

DNA concentration.

300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

0.12 f

a

Ab

so

rb

an

ce

Wavelength(nm)

300 400 500 600 700 800

0.00

0.02

0.04

0.06

0.08

0.10

0.12

f

a

Ab

sorb

an

ce

Wave length (nm)

119

Figure III.15. Comparative plot of [DNA]/(εa-εf) vs [DNA] for the absorption titration of

CT-DNA with complexes 1a and 1b in tris-HCl buffer. Association constant Kb:

0.36 × 104 M-1 (R = 0.99999, n = 5 points) for 1a; 0.93× 104 M-1 (R = 0.99885, n =

5 points) for 1b.

Figure III.16. Comparative plot of [DNA]/(εa-εf) vs [DNA] for the absorption titration of

CT-DNA with complexes 1c-1d in tris-HCl buffer. Association constant Kb: 0.80× 105M-

1 (R = 0.99999, n = 5 points) for 1c; 0.78× 105 M-1 (R = 0.99885, n = 5 points) for 1d.

1 2 3 4 5 6 7

40

50

60

70

80

90

1c

1d

[DNA] x 106

[DN

A]/

( εε εεa-

εε εε f) x 1

010

120

(a) (b)

Figure III.17. (a) The electronic spectra of 1.0 ×10-5 M EB (A); (A) + 2.5×10-5 M DNA

(B); (B) + 2.5×10-5 M Pd(II) complex (C) in tris–HCl buffer. (b) The electronic spectra of

1.0 ×10-5 M EB (A); (A) + 2.5×10-5 M DNA (B); (B) + 2.5×10-5 M platinum (II) complex

(C) in tris–HCl buffer.

III.3.5.2 Fluorescence quenching analysis

The binding propensity of palladium and platinum complexes to CT-DNA has

been analyzed by the steady-state emission quenching experiments using the emission

intensity of ethidium bromide (EB). It is well known that EB can intercalate

nonspecifically with DNA, causing a strong fluorescence. Other compounds competing

with EB to intercalation in DNA will induce displacement of bound EB and a decrease in

the fluorescence intensity. This fluorescence-based competition can provide indirect

evidence for the DNA-binding mode. The fluorescence intensity of the EB/DNA system

(with excitation wavelength of 522 nm) is reduced by the increasing concentration of the

complexes (Figures III.18, III.20, III.22, and III.24), and caused by EB migration from a

hydrophobic to an aqueous environment [42]. The quenching of EB bound to DNA by 1a

to 1d is in agreement with the linear Stern–Volmer equation [43]:

I0/I = 1+ Kq [Q]

where I0 and I represent the fluorescence intensities in the absence and presence

of quencher, respectively. Kq is a linear Stern–Volmer quenching constant, Q is the

concentration of the quencher. In the quenching plot ( Figures III.19, III.21, III.23, and

450 480 510 540 570 600

0.0

0.2

0.4

0.6

0.8

1.0

B

C

A

Ab

sorb

ance

Wave length(nm)450 480 510 540 570 600 630

0.0

0.2

0.4

0.6

0.8

1.0

B

C

A

Ab

sorb

an

ce

Wave length (nm)

121

III.25) of I0 /I vs. [complex], Kq is given by the ratio of the slope to the intercept. The Kq

values are 0. 14 × 104 , 0.44× 104 , 0.42× 104 (R = 0.99746, n = 4 points) and 0.19× 104

(R = 0.99876, n = 5 points)for complexes 1a to 1d respectively implies that all the

complexes can insert between DNA base pairs and the platinum (II) complex can bind to

DNA more strongly than the palladium(II) complex which is consistent with the

absorption data. The titration data obtained from the fluorescence experiment can be

helpful also to calculate the number of binding sites and the apparent binding constant. In

the following equation [44]:

log[(I0 - I)/I] = logK + n log[Q]

K and n represent the binding constant and number of binding sites of palladium

complex to CT-DNA, respectively. The number of binding sites n, determined from the

intercept of log[(I0 – I)/I] vs log[Q] (Figures III.26-III.27), are 1.07, 1.18, 0.96 and 1.03

for 1a to 1d, respectively, indicating the existence of about a single binding site in DNA

and a weaker association for the complexes. The K values were calculated to be 0.32 ×

104, 0.77 × 104, 0.36 × 104 and 0.28× 104 for 1a to 1d, respectively, with a trend similar to

the apparent association constant values of the complexes.

Figure III.18. Emission spectra of the CT-DNA-EB system in tris-HCl buffer upon

titration with complex 1a. λex = 522 nm; [EB] = 9.6×10-5, [DNA] = 1.25×10-5;

[1a]: (a) 0.0, (b) 1.31×10-5, (c) 2.62×10-5, (d) 3.93×10-5, (e) 5.24×10-5, (f) 6.55×10- 5 mol

L-1. The arrow denotes the gradual increase of complex concentration.

575 600 625 650 675 700 725 750 775

0

150

300

450

600

750

900

1050

1200

1350

Inte

nsit

y

Wavelength(nm)

f

a

122

Figure III.19. Plot of I0/I vs. [complex] of 1a; Kq = 0.16× 104 (R = 0.99876, n = 5 points) in

tris-HCl buffer upon titration of complex 1 with CT-DNA-EB system.


titration with complex 1b. λex = 522 nm; [EB] = 9.6×10-5, [DNA] = 1.25×10-5;

[1b]: (a) 0.0, (b) 1.32×10-5, (c) 2.64×10-5, (d) 3.96×10-5, (e) 5.28×10-5, (f) 6.60×10-5

mol L-1.

1 2 3 4 5 6 7

1.00

1.02

1.04

1.06

1.08

1.10

1.12

I o/I

[Complex]X105

575 600 625 650 675 700 725 750 775

0

150

300

450

600

750

900

1050

1200

1350

Inte

nsit

y

Wavelength(nm)

f

a

123

Figure III.21. Plot of I0/I vs. [complex] of 1b; Kq = 0.14× 104 (R = 0.99932, n = 5

points) in tris-HCl buffer upon titration of complex 1 with CT-DNA-EB system.


titration with complex 1c. λex = 522 nm; ; [EB] =0.96 x 10-4 molL-1; [DNA] = 9.9 x 10-6

molL-1; [1c]: (a) 0.0, (b) 1.36 x 10-5 , (c) 2.72 x 10-5 , (d) 4.08 x 10-5 , (e) 5.44 x 10-5 molL-

1.The arrow indicates the increase of compound concentration.

1 2 3 4 5 6 7

1.00

1.02

1.04

1.06

1.08

1.10

I o/I

[Complex]X105

550 600 650 700 750

0

200

400

600

800

1000

e

a

Inte

nsit

y

Wavelength(nm)

124

Figure III.23. Plot of I0/I vs. [complex] of 1c; Kq = 0.42× 104 (R = 0.99746, n = 4 points)

in tris-HCl buffer upon titration of complex 1 with CT-DNA-EB system.


titration with complex 1d. λex = 522 nm; [EB] = 0.96 x 10-4 molL-1, [DNA] =

1.25×10-5 molL-1; [1d]: (a) 0.0, (b) 1.31×10-5, (c) 2.62×10-5, (d) 3.93×10-5, (e)

5.24×10-5, (f) 6.55×10-5 mol L-1. The arrow denotes the gradual increase of

complex concentration.

1 2 3 4 5 6 7 8 90.90

1.05

1.20

1.35

1.50

1.65

I 0 / I

[Complex]x105

575 600 625 650 675 700 725 750 775

0

150

300

450

600

750

900

1050

1200

1350

Inte

nsit

y

Wavelength(nm)

f

a

125

Figure III.25. Plot of I0/I vs. [complex] of 1d; Kq = 0.19×104 (R = 0.99876, n = 5

points)in tris-HCl buffer upon titration of complex 1 with CT-DNA-EB system.

Figure III.26. Comparative plot of log[I0-I/I] vs. log[complex] for the titration of CT-

DNA-EB system with complexes 1a and 1b in tris-HCl buffer medium.

-4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

-4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1

-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

log

[(I o

-I)/

I]

1a

1b

log

[(I o

-I)/

I]

log[Complex]

1 2 3 4 5 6 7

1.00

1.02

1.04

1.06

1.08

1.10

I o/I

[Complex]X105

126

Figure III.27. Comparative plot of log[I0-I/I] vs. log[complex] for the titration of CT-

DNA-EB system with complexes 1c and 1d in tris-HCl buffer medium.

III.3.5.3 Viscosity measurements

Since optical photophysical probes generally provide necessary, but insufficient

clues to further clarify the interactions between the complex and DNA, viscosity

measurements were carried out. Hydrodynamic measurements, sensitive to length change

(i.e. viscosity and sedimentation), are regarded as the least ambiguous and the most

critical tests of binding in solution in the absence of crystallographic structural data. A

classical intercalation model demands that the DNA helix lengthens as base pairs are

separated in order to accommodate the binding ligand, leading to an increase in DNA

viscosity. In contrast, a partial, non-classical intercalation of compound could bend (or

kink) the DNA helix, reducing its effective length and, concomitantly, its viscosity [34].

The results obtained in these viscosity measurement studies suggest that all the

compounds 1a to 1d can intercalate between adjacent DNA base pairs, causing an

extension of the helix with a concomitant increase of the DNA viscosity. The effects of

both compounds on the viscosity of DNA are shown in Figures III.28-III.29.

-4.6 -4.5 -4.4 -4.3 -4.2

-1.6

-1.5

-1.4

-1.3

-1.2

-1.1

-1.0

-0.9

1c

1d

log[Complex]

log

[(I o

-I)/

I]

127

Figure III.28. Effect of increasing amounts of palladium (II) and platinum (II) complexes

on the relative viscosity of CT-DNA at 25°C.

Figure III.29. Effect of increasing amounts of palladium (II) complexes on the relative

viscosity of CT-DNA at 250C.

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.30

0.35

0.40

0.45

0.50

0.55

0.60

1a

1b

(( (( η−

ηη

−η

η−

ηη

−η

00 00/η/η /η/η

00 00)) ))(1

/3)

(1/3

)(1

/3)

(1/3

)

[M]/DNA

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

(n-n

o/n

o)1

/3

[M]/DNA

1d

1c

128

III.3.6 Binding experiments with bovine serum albumin (BSA) of 1a and 1b

III.3.6.1 Absorption spectral characterization

The binding mode of complexes 1a and 1b with BSA were examined by

electronic absorption titration with BSA. The absorption spectra of the free metal

complexes and of their adducts with BSA are given in Figures III.30 and III.31 for

complex 1a and 1b, respectively. The spectra indicate a significant increase in the

absorbance of BSA by increasing the concentration of the complex and are indicative of

the fact that BSA adsorbs strongly the complex on its surface [45]. From these titration

data the apparent association constant (Kapp) of the complexes with BSA has been

determined using the following equation [36]:

1/(Aobs – A0) = 1/(Ac – A0) + 1/ Kapp(Ac – A0) [comp]

where, Aobs is the observed absorbance of the solution containing different concentrations

of the complex, A0 and Ac are the absorbance of BSA and of the complex at 280 nm,

respectively. The enhancement of the absorbance at 280 nm was attributable to the

complex absorption at BSA surface. Based on the linear relationship between 1/(Aobs - A0)

vs the reciprocal concentration of the complex with a slope of 1/Kapp(Ac - A0) and an

intercept equal to 1/(Ac - A0) (Figure III.32), the value of Kapp was determined to be

1.262× 105 M-1 (R = 0.99967, n = 5 points) and 1.402× 105 M-1 (R = 0.99991, n = 5

points), for 1a and 1b, respectively.

Figure III.30. Absorption titration spectra of BSA in presence of complex 1a.

Concentration range of the complex is 0-6.25× 10-6 M-1.

129

Figure III.31. Absorption titration spectra of BSA in presence of complex 1b

Concentration range of the complex is 0-6.25× 10-6 M-1 .

Figure III.32. The linear dependence of 1/(A _ A0) on the reciprocal concentration of 1a

and 1b.

III.3.6.2 Fluorescence quenching analysis

In the fluorescence quenching experiment, the fluorescence emission spectrum

of BSA was studied increasing the concentration of the quencher. The fluorescence

130

quenching is described by the Stern–Volmer relation [45], similarly as described above

for CT-DNA binding experiments (Figures III.33 and III.35). From the slope of the

regression line in the derived plot of I0/I vs [complex] (Figures III.34 and III.36) the Kq

values for the complexes were determined to be 4.29× 104 for 1a (R = 0.99825 for five

points) and 4.62 × 104 for 1b (R = 0.99948 for five points), indicating a strong affinity of

both of the complexes to BSA.

Figure III.33. Fluorescence quenching titration of BSA varying the concentration of 1a,

[complex] = 0, 1, 2, 3, 4 and 5 × 6.35 ×10-6M.

Figure III.34. Plot of I0/I vs. [complex] of 1a; 4.29× 104 for 1a (R = 0.99825 for five points).

1 2 3 4 5 6 7

1.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

I O/I

[Complex]x106

300 320 340 360 380 400

0

500

1000

1500

2000

2500

f

a

Inte

nsit

y

Wavelength(nm)

131

Figure III.35. Fluorescence quenching titration of BSA varying the concentration of 1b,

[complex] = 0, 1, 2, 3, 4 and 5 × 6.35 ×10-6M.

Figure III.36. Plot of I0/I vs. [complex] of 1b; 4.62 × 104 for 1b (R = 0.99948 for five points).

III.4 Epilogue

Three novel square planar palladium (II) and one platinum (II) complexes 1a to

1d of deprotonated tetradentate ligands N,N'-bis(2-pyridinecarboxamide)-1,2-benzene;

N,N'-bis(2-pyridinecarboxamide)-2,3-pyridine; N,N'-bis(2-pyridinecarboxamide)-5-bromo-

2,3-pyridine have been synthesized and characterized using various spectroscopic

300 320 340 360 380 400 420

0

500

1000

1500

2000

2500

f

a

Inte

nsit

y

Wavelength(nm)

1 2 3 4 5 6 7

1.10

1.15

1.20

1.25

1.30

1.35

I O/I

[Complex]x106

132

measurements. The X-ray structural characterization revealed that the palladium (II) and

platinum (II) derivatives are isomorphous and are packed to form a trimeric motif with

complexes connected by π-π interactions between the aromatic rings of the ligands and

metallophilic bonding. The complexes have been found to interact with CT-DNA through

an intercalative mode, which was investigated by absorption, fluorescence and viscosity

measurement tools. The quenching rate constant, binding constant and number of binding

sites were calculated according to the relevant fluorescence data. The binding constants

indicate that the DNA-binding affinity, as well as the binding trend with BSA, increases

from palladium (II) to platinum (II), in accordance with the relevant viscosity

measurement study. The information obtained from the present work is indicative of the

development of potential probes of DNA structure in future applications.

133

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135

CHAPTER-IV

Neutral Cyclometalated Rhodium(III) Complexes

Bearing Dithiocarbamate Derivative: Synthesis,

Structural Characterization, Antibacterial

Activity and DNA Binding Study

136

Abstract Reaction of three different dithiocarbamates (4-MePipzcdtH, L1H; MorphcdtH,

L2H and 4-BzPipercdtH, L3H) with [Rh(2-C6H4py)2Cl]2.1/4CH2Cl2 afforded a class of

rhodium (III) complexes of the type [RhIII(2-C6H4py)2(L)]. The complexes were fully

characterized by several spectroscopic tools along with a detailed structural

characterization of [Rh(2-C6H4py)2(L1)] (1) by single crystal X-ray diffraction.

Structural analysis of 1 showed a distorted octahedron in which both of the 2-

phenylpyridyl nitrogens are in axial positions, trans to one another and the sulfur atoms

are opposite to the phenyl rings. Electrochemical analysis by cyclic voltammetry reveals

irreversible redox behavior of the rhodium centre in 1, 2 and 3. DNA binding ability of

complex 1 has also been evaluated from the absorption spectral study as well as

fluorescence quenching properties, suggesting the intercalative interaction of the complex

with CT-DNA due to the stacking between the aromatic chromophore and the base pairs

of DNA. Antibacterial activity of complexes has also been studied by agar disc diffusion

method against some species of pathogenic bacteria (Escherichia coli, Vibrio cholerae,

Streptococcus pneumonia and Bacillus cereus).

137

IV.1 Introduction

Rhodium (III) complexes are the subject of current research activity in the

interaction of complexes with biomolecules as well as the rhodium catalyst is able to

fulfill its role over the other conventional catalysts due to the capability of the metal to

change its coordination number from six to four and also the oxidation state from

rhodium (III) to rhodium (I). This change appears as a chemically irreversible two-

electron reduction involving ligand loss from octahedral rhodium (III) to form square

planar rhodium (I) complexes. Loss of the ligand depends on the nature of the ligands

present in mixed ligand systems which allow one to tune the electrochemical potential

and affect the reactivity of the rhodium metal center [1]. The discovery of the catalytic

properties of Wilkinson’s catalyst, viz. [RhCl(PPh3)3] naturally brought about a

widespread search for other rhodium phosphines with catalytic activity [2,3]. Further,

octahedral diimine rhodium (III) complexes are of interest as they have been used in the

process of photochemical reduction of H2O to H2 [4].

The dithiocarbamates (R2NCS2-) have been considered as versatile ligands for

bonding to transition as well as main group metal ions [5-17], and got an enormous

attention because of their importance in several fields such as the chemical industry,

biology and biochemistry [18-21]. The nature of the heterocycle attached to

dithiocarbamate fragment appears crucial so as to vary the electron properties of these

ligands and thus to control the potential pharmacological attributes as well as the catalytic

efficiency of the metal complexes [22]. Coordination complexes of platinoids with

dithiocarbamato ligands are known in the literature [9-13] and also palladium (II) and

platinum (II) complexes of dithiocarbamato groups together with mono- or diamine

ligands [14-17]. But to the best of our knowledge so far, report of rhodium (III)

cyclometalated complexes bearing dithiocarbamate derivative is still unexplored.

The binding interactions of these complexes with DNA have also been studied

systematically to explore the biological activity of the new complexes as we know the

fact of the activity of cisplatin by coordination to DNA [23, 24]. And from through

pharmacological mechanistic studies, it is also known that small molecules interact with

DNA via electrostatic forces, groove binding, or intercalation [25], and their effectiveness

depends on the mode and affinity of the binding [26]. Intercalation is one of the most

important among these interactions. Therefore, the search for drugs that show

138

intercalative binding to DNA has been an active research area for the past several decades

[27].

Encouraged by the advantages of the facts stated above, we isolated a new series of

cyclometalated rhodium (III) complexes bearing dithiocarbamate derivatives by a high

yield synthetic pathway under mild reaction conditions. The present chapter deals with

the chemistry of these [RhIII(2-C6H4py)2(L)] complexes, where L1 = 4-Methylpiperazine-

l-carbodithioic acid anion, L2 = Morpholine-4-carbodithioic acid anion and L3 = 4-

Benzylpiperidine-l-carbodithioic acid anion (see scheme 2) for complexes 1, 2 and 3,

respectively. The complexes have been synthesized and characterized by physicochemical

and spectroscopic methods; in addition, the structure of complex 1 was characterized by

single crystal X-ray crystallography, UV-vis, IR-spectroscopy, and electrochemistry. The

binding interactions of the complex 1 with calf thymus-DNA (CT-DNA) have also been

studied systematically to explore the mode of biological activity as part of our continuing

interest [12, 28, 29] using absorption spectral study as well as fluorescence quenching

properties, suggesting the intercalative interaction of the complex with CT-DNA due to

the stacking between the aromatic chromophore and the base pairs of DNA. In addition,

antibacterial activity of the complexes (1, 2 and 3) against some pathogenic bacteria,

namely Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and Bacillus cereus

has also been studied by agar disc diffusion method.

IV.2 Experimental Section

IV.2.1 Solvents and Reagents

Rhodium trichloride, 2-phenylpyridine, morpholine and 4-benzylpiperidine

(Aldrich) were purchased and used without further purification. [Rh(2-

C6H4py)2Cl]2.1/4CH2Cl2 was prepared following the reported procedure [30]. 4-

Methylpiperazine (Aldrich) has been dried by refluxing over NaOH beads, the colorless

liquid obtained after distillation and stored over NaOH beads. Solvents used for

spectroscopic studies and for synthesis were purified and dried by standard procedures

before used. The organic moieties, 4-methylpiperazine-l-carbodithioic acid (4-

MePipzcdtH, L1H), morpholine-4-carbodithioic acid (MorphcdtH, L2H) and 4-benzyl-

piperidine-l-carbodithioic acid (4-BzPipercdtH, L3H) were obtained as solid products

following the reported procedure [12].

139

IV.2.2 Physical Measurements

The Fourier transform infrared spectra of the ligand and the complexes were

recorded on a Perkin-Elmer FTIR model RX1 spectrometer using KBr pellet in the range

4000 - 300 cm-1. The solution phase electronic spectra were recorded on a JASCO UV–

Vis/NIR spectrophotometer model V-570 in the range 200-1100 nm. Elemental analyses

were carried out on a Perkin-Elmer 2400 series-II CHNS Analyzer. The fluorescence

spectra complex bound to DNA were obtained at an excitation wavelength of 522 nm in

the Fluorimeter (Hitachi-2000). Mass spectra of 1, 2 and 3 were recorded on Micromass

Q-Tof microTM. NMR spectrum of the ligands and complexes has been recorded on

Bruker DPX-300. Solution conductivity and redox potentials were measured using

Systronics Conductivity Meter 304 model and CHI620D potentiometer in DMF at

complex concentration of ~10-3 mol L-1. Viscosity experiments were conducted on an

Ostwald’s viscometer, immersed in a thermostated water-bath maintained to 25oC.

IV.2.3 X-Ray Crystal Structure Analysis

X-ray data of the suitable crystal of complex 1 were collected on a Bruker’s Apex-

II CCD diffractometer using MoKα (λ = 0.71069). The data were corrected for Lorentz

and polarization effects and empirical absorption corrections were applied using

SADABS from Bruker. A total of 13691 reflections were measured out of which 4012

were independent and 2299 were observed [I > 2σ(I)] for theta (θ) 32°. The structure was

solved by direct methods using SIR-92 [31] and refined by full-matrix least squares

refinement methods based on F2, using SHELX-97 [32]. The two fold axis passes through

the metal ion, nitrogens of the piprazine ring and their substituent carbon atoms.

Therefore the asymmetric unit contains half the molecule. All non-hydrogen atoms were

refined anisotropically. The refinement showed rotational disorder in the piprazine ring

which could be resolved by splitting the two unique carbon atoms into two components

and refining their sof and thermal parameters as free variables with restraints over the

bond distances. All hydrogen atoms were fixed geometrically with their µ iso values 1.2

times of the phenylene and methylene carbons and 1.5 times of the methyl carbons. All

calculations were performed using Wingx package [33, 34]. Important crystallographic

parameters are given in Table IV.1. Crystallographic data for have been deposited with

the Cambridge Crystallographic Data Centre, CCDC No. 932588 for 1.

140

Table IV.1. Crystallographic data for the complex 1.

Complex 1

Empirical Formula C28H27N4RhS2

Formula Weight 586.59

Crystal system Monoclinic

Space group C 2/c

a (Å) 16.683(5)

b (Å) 17.840(4)

c (Å) 10.251(5)

β, deg 126.223(5)

Volume (Å3) 1999.5(3)

Temperature (K) 2461.3(15)

Z 4

ρcalc (g/cm3) 1.583

µ(mm-1) 0.889

F(000) 1200

θ range (deg) 1.90 -31.84

Refl. collected / unique 13691 / 4012

R(int) 0.0544

No. of reflns (I > 2σ(I)) 2299

No. of refined paramaters 357

Goodness-of-fit (F2) 1.034

R1, wR2 (I >2σ(I)) [a] 0.0473, 0.1041

R indices (all data) R1 = 0.1053, .1342

aR = ∑||Fo |- |Fc||/∑|Fo|;

bwR2 = { ∑ [w(Fo

2 –Fc2)2] / ∑w [(Fo

2)2]}1/2

141

IV.2.4 Syntheses of Ligands


ligands, 4-Methylpiperazine-l-carbodithioic acid anion (L1) [19], Morpholine-4-

carbodithioic acid anion (L2) [20] and 4-Benzylpiperidine-lcarbodithioic acid anion (L3)

[21]. The procedure for the preparation and spectroscopic evidences of dithiocarbamate

ligands has already described in detail in chapter II under section II.2.4.

IV.2.5 Syntheses of Rhodium (III) Complexes

The complexes have been synthesized following a common procedure stated as

below. The ligand, L1H (177 mg, 1.0 mmol) for complex 1 or L2H (164 mg, 1.0 mmol)

for complex 2 or L3H (253 mg, 1.0 mmol) for complex 3 was dissolved in DMSO-MeCN

(v/v 1:1) solvent mixture and to this ligand solution dropwise MeCN solution of [Rh(2-

C6H4py)2Cl]2.1/4CH2Cl2 (468mg, 0.5mmol) was added. The mixture was then refluxed in

nitrogen atmosphere for 12 h and the color changed from faded yellow to orange. On

slow evaporation of this solution, orange coloured microcrystalline solid appeared, which

was subjected to purification by column chromatography using MeCN as an eluant.

Needle shaped crystals of [Rh(2-C6H4py)2(L1)] suitable for X-ray diffraction study were

grown from this solution on evaporation at ambient temperature.

IV.3 Results and Discussions

IV.3.1 Syntheses and Characterization

The bidentate sulphur ligands (L1H, L2H, and L3H) were synthesized by the

reaction between carbon disulfide with different amines in ethanol, and later characterized

by FTIR and 1H NMR. Treatment of these ligands with [Rh(2-C6H4py)2Cl]2.1/4CH2Cl2 at

refluxing condition in DMSO-MeCN (v/v 1:1) solvent mixture having nitrogen

atmosphere resulted in cleavage of the chloro bridge and led to formation of mononuclear

rhodium(III) complexes of general formula of [RhIII(2-C6H4py)2(L)] which were obtained

from the column chromatography using acetonitrile as orange colored microcrystalline

solid on evaporation. Here, the dithiocarbamates behaves as bidentate monobasic ligands

(see Scheme 1 and Table IV.2). The formulations of the complexes have been confirmed

by spectroscopic methods and elemental analyses.

142

Scheme 1. Synthetic method for the preparation of rhodium (III) complexes.

Table IV.2. Rhodium (III) complexes with dithiocarbamate ligand.

Microanalytical data (Table IV.3) confirm the composition of the complexes

and spectroscopic data of 1, 2 and 3 along with the X-ray crystallographic results of 1

indicate the organic moieties, 4-Methylpiperazine, Morpholine and 4-Benzylpiperidine in

the reaction medium are converted to 4-Methylpiperazine-l-carbodithioic acid (L1H),

Morpholine-4-carbodithioic acid (L2H) and 4-Benzylpiperidine-l-carbodithioic acid

(L3H) respectively, and their rhodium (III) complexes 1-3. The complexes (1-3) are

sparingly soluble in common organic solvents except hexane but fairly soluble in DMF

and DMSO, and are stable in both the solid state and solution state in air. The complexes

are diamagnetic in nature.

LnH Complex

4-Methylpiperazine-l-carbodithioic acid (4-MePipzcdtH ) (L1H) [RhIII(2-C6H4py)2(L1)] (1)

Morpholine-4-carbodithioic acid ( MorphcdtH ) (L2H) [RhIII(2-C6H4py)2(L2)] (2)

4-Benzylpiperidine-l-carbodithioic acid (4-BzPipercdtH ) (L3H) [RhIII(2-C6H4py)2(L3)] (3)

DMSO-MeCN

X

N SH

S

LnH + [Rh(2-C6H4py)2Cl2]2

Reflux,12 h[Rh(2-C6H4py)2(Ln)]

n = 1, Complex 1n = 2, Complex 2n = 3, Complex 3

X= -N-CH3 , 4-MePipzcdtH (L1H)

X= -O, MorphcdtH (L2H)

X= -CH-CH2Ph , 4-BzPipercdtH(L3H) 2-C6H4py ppy

N

143

Table IV.3. Microanalytical data of the rhodium (III) complexes (1-3).


IV.3.2 Structural description of complex 1

An ORTEP view of the complex [Rh(2-C6H4py)2(L1)] (1) with atom labeling

scheme is illustrated in Figure IV.1, and a selection of bond distances and angles is listed

in Table IV.4. The structural analysis evidenced that the complex resides on a C 2/c site

in the monoclinic crystal system. The crystal structure of 1 shows a distorted octahedron

in which both of the 2-phenylpyridyl nitrogens are in axial positions, trans to one another

and the sulfur atoms are opposite to the phenyl rings. As would be expected, both Rh-C

σ-bonds are equal in length (1.994(4)Å) and significantly shorter than the Rh-N dative

bond lengths of 2.039(3)Å due to the Rh-C σ-bonds increasing electron density on the

metal center. The bond distance of Rh-C in 1 is comparable with the previously reported

Rh-C bonds in cyclometalated complex (1.996(9)Å) but the bond Rh-N is slightly longer

than those (1.987(7)Å) [36], but both are comparable with the reported values [37]. Both

Rh–S distances are also equal in length (2.4854(11)Å), and these are longer than those in

[Rh(Et2NCS2)3] (2.364(3)Å) [38] due to trans influence of the strong σ-donating carbon

atoms of the phenyl groups and shorter than those in similar type complex [{Rh(Bu2-

C6H4py)2}2{S2P(OMe)2}] (2.548(2)Å) due to attachment of sulphur atoms to carbon to of

more electronegativity than phosphorous, for this reason, the S-Rh-S angle of 71.00(5)o is

smaller compared to the observed value of 79.40(1)o in the previously report [37].


(Λo)a C H N

[RhIII(2-C6H4py)2(L1)] (1) 57.33(57.21) 4.64(4.58) 9.55(9.32) 130

[RhIII(2-C6H4py)2(L2)] (2) 56.54(56.52) 4.22(4.06) 7.33(7.29) 127

[RhIII(2-C6H4py)2(L3)] (3) 63.53(63.44) 4.87(4.79) 6.35(6.02) 132

144

Figure IV.1. ORTEP view of the complex [Rh(2-C6H4py)2(L1)] (1) with atom labeling

scheme (excluded H for clarity). (Symmetry codes: (i) -x,y,z; (ii) x,-y,-z; (iii) -x,-y,-z).

Table IV.4. Selected bond distances (Å) and bond angles (°) for 1.

Bond length (Å)

Rh(1) - C(11) 1.994(4) Rh(1) - C(11)#1 1.994(4)

Rh(1) - N(1) 2.039(3) Rh(1) - N(1)#1 2.039(3)

Rh(1) - S(1) 2.4854(11) Rh(1) - S(1)#1 2.4855(11)

Bond angle (o)

C(11)-Rh(1)-N(1) 80.30(13) C(11)#1-Rh(1)-N(1)#1 80.29(13)

C(11)-Rh(1)-N(1)#1 93.30(13) C(11)#1-Rh(1)-N(1) 93.30(13)

N(1)-Rh(1)-S(1) 170.86(11) N(1)-Rh(1)-N(1)#1 171.09(17)

C(11)-Rh(1)-S(1) 100.14(11) C(11)#1-Rh(1)-S(1)#1 100.15(11)

C (11)#1-Rh(1)-S(1) 90.15(9) N(1)#1-Rh(1)-S(1)#1 90.15(9)

N(1)#1-Rh(1)-S(1) 97.12(9) N(1)-Rh(1)-S(1)#1 97.11(9)

S(1)-Rh(1)-S(1)#1 71.00(5) C(11)-Rh(1)-C(11)#1 88.8(2)

Symmetry transformations used to generate equivalent atoms: #1 -x+1, y, -z+3/2

145

IV.3.3 Spectroscopic analysis

IV.3.3.1 IR Spectra

The IR spectra of the ligands display an intense stretch at 2850 cm-1 and 1445-1430

cm-1 correspond to γC-H of N-Me and γC=N respectively. The γC-H of N-Me of L1H for 1

(2910cm-1) is blue shifted than the γC-H of N-Me of the free ligand (2850 cm-1) indicating

metal ligand coordination. The band around 1590 cm-l indicates a double bond character

of C-N bond in the ligand frame, which is confirmed from the bond length of the X-ray

structure. This fact could be attributed to the electron releasing ability of the heterocyclic

group towards the sulphur atoms, a feature that induces an electron delocalization over

the carbon-nitrogen bond and the CS2 fragment. This is shown by the νC=N shift to higher

energies (ca. 1510-1465 cm-l) with respect to the free acids (ca. 1445-1430 cm-1) (see

Table IV.5), and these bands lie in between the stretching frequencies expected for a

double C=N (1610-1690 cm-l) and single C-N bond (1250-1350 cm-1). The blue-shift of

the C=N stretching frequency on going from the free acids to their metal complexes gives

support to the typical bidentate character [42] of the carbodithioic acid ligands. Two

bands in the region of 1040-965 cm-l (separated by less than 20 cm-l) assignable to the

νa(SCS) and one band for the vs(SCS) stretch in the region 705-675cm-l of the complexes

suggest the unsymmetrical chelating bidentate mode of coordination to rhodium(III) ion

[43]. The stretchings due to νCOC (asym and sym), νN-Me and νCCC (asym and sym) remain


to exclude any coordination to the metals via nitrogen and oxygen donors.

Table IV.5. IR spectral data of the ligands and rhodium (III) complexes.

Compound ννννSCS(s) ννννSCS(as) ννννC=N

4-Methylpiperazine-l-carbodithioic acid(L1H) 684 1023 1451

Morpholine-4-carbodithioic acid (L2H) 689 1029 1468

4-Benzylpiperidine-l-carbodithioic acid (L3H) 699 1072 1496

[RhIII(2-C6H4py)2(L1)] (1) 695 1025 1492

[RhIII(2-C6H4py)2(L2)] (2) 698 1036 1485

[RhIII(2-C6H4py)2(L3)] (3) 704 1082 1505

146

IV.3.3.2 Electronic Spectra

The electronic spectra of 1, 2 and 3 in DMF were shown in Figure IV.2. The

spectral data have been tabulated in Table IV.6. Complexes 1, 2 and 3 display a lower

energy band at 675nm, 672nm and 675nm, respectively with low extinction coefficient

values that correspond to the d–d transition. The higher energy band at 360 nm for all

three complexes with high extinction coefficient values are due to the coordinated carbon

atom from pipyridine moiety, C(σ)-Rh(III) charge transfer (LMCT) transition. The other

higher energy intense transitions at 374 nm and 360 nm are due to the n→π* and π→π*

charge transfer transitions.

Figure IV.2. Electronic absorption spectra of 1, 2 and 3 in DMF.

Table IV.6. Electronic absorption spectral data of the complexes.

Complex λmax(nm) (10-5, ε/dm3 mol-1cm-1)a

1 360(7845), 374sh(6780), 385(6840), 675(160)

2 360(7900), 375sh(6680), 390(6700), 672(210)

3 360(7800), 382sh(6600), 390(6640), 675(200)

a in DMF solvent

350 400 450 500

0

1

2

3

4

-----(3)

-----(1)

-----(2)

Ab

so

rb

an

ce

Wavelength(nm)

147

IV.3.3.3 Redox studies

The cyclic voltammograms (CV) of the complexes 1, 2 and 3 were recorded in

DMF solvent at room temperature. Three electrode cell set up such as platinum, Ag/Ag+

(non-aquous) and a platinum wire as a working, reference and auxiliary electrode

respectively have been used for measurements. The cyclic voltammograms of all the three

complexes 1, 2 and 3 have been shown in Figure IV.3 and the electrochemical data have

been tabulated in Table IV.7. The complexes exhibit an irreversible reductive response at

E1/2 value ≈ -0.697 V to -0.767 V versus Ag/Ag+ (non-aqueous) corresponds to Rh3+/Rh+

couple. Small differences in the ∆Ep values 652 mV, 658mV, 654 mV for 1, 2 and

respectively) have been observed that increases in the order 2> 3>1. This indicates that

the ease of reduction from rhodium (III) to rhodium (I) with respect to ligand electronic

environment is supposed to be much more in case of complex 1 and least in complex 2.

Figure IV.3. Cyclic voltammograms (scan rate 50 mV/s) of 1, 2 and 3 in DMF solution

with 0.1 M TBAP, using platinum working electrode.

Table IV.7. Electrochemical dataa for the complexes 1, 2 and 3.

Complex Epc(V) Epa(V) ∆∆∆∆Ep(mV) E1/2(V)

1 -1.023 -0.371 652 -0.697

2 -1.096 -0.438 658 -0.767

3 -1.077 -0.413 654 -0.744

a Potentials versus non-aqueous Ag/Ag+ reference electrode, scan rate 50 mV/s,

supporting electrolyte: tetra-N-butylammonium perchlorate (0.1 M).

-1.5 -1.2 -0.9 -0.6 -0.3 0.0

-2

0

2

4

6

8

10

I ( µµ µµ

A)

E (V)

231

148

The trend of reduction potential values followed can be explained by the

availability of the electrons on the donor atoms of the dithiocarbamate ligands. The

electron donating capacity through σ bond of the six membered heterocyclic ring

increases in the order 2< 3<1 owing to the presence of different substituents at the

heteroatom i.e. highly electronegative O atom (2), -R effect of benzylic group (3), +I

effect of Me group (1) and so the trend of reduction potential followed as such, which is

further supported by theoretical calculation obtained from DFT study.

IV.3.4 Theoretical calculation

To clarify the configurations and energy level of the complexes 1, 2 and 3, DFT

calculations were carried out in G09W program using B3LYP/6-31G(d) calculation and

correlation function as implemented in the Gaussian program package Gaussian 09.

Thermal contribution to the energetic properties was considered at 298.15 K and one

atmosphere pressure (Figures IV.4-IV.6).

Figure IV.4. Optimized structure of complex 1.

HOMO(136) -0.1913 eV LUMO(137) -0.0469

eV

149


HOMO(132) - 0.1946eV LUMO(133) - 0.0500eV

150


IV.3.5 DNA binding experiments

All the experiments involving CT-DNA were studied by spectroelectronic titration

and fluorescence quenching technique by using ethidium bromide (EB) as a DNA

scavenger and performed the experiment as our previously standardized method [41].

Tris–HCl buffer solution was used in all the experiments involving CT-DNA. This tris–

HCl buffer (pH 7.9) was prepared using deionized and sonicated HPLC grade water

(Merck). The CT-DNA used in the experiments was sufficiently free from protein as the

ratio of UV absorbance of the solutions of DNA in tris–HCl at 260 and 280 nm

(A260/A280) was almost ~1.9. The concentration of DNA was determined with the help of

the extinction coefficient of DNA solution at 260 nm (ε260 of 6600 L mol-1 cm-1) [41].

Stock solution of DNA was always stored at 4oC and used within four days. Concentrated

stock solution of the complex 1 was prepared by dissolving the compound in DMSO and

suitably diluted with tris–HCl buffer to the required concentration for all the experiments.

Absorption spectral titration experiment was performed by keeping constant the

HOMO(156) - 0.1916eV LUMO(157) - 0.0472eV

151

concentration of the complex 1 and varying the CT-DNA concentration. To eliminate the

absorbance of DNA itself, equal solution of CT-DNA was added both to the complex 1

solution and to the reference solution.

In the ethidium bromide (EB) fluorescence displacement experiment, 5 µL of the

EB tris–HCl solution (1.0 mmol.L-1) was added to 1.0 mL of DNA solution (at saturated

binding levels), stored in the dark for 2.0 h. Then the solution of the compound was

titrated into the DNA/EB mixture and diluted in tris–HCl buffer to 5.0 mL to get the

solution with the appropriate complex 1/CT-DNA mole ratio. Before measurements, the

mixture was shaken up and incubated at room temperature for 30 min. The fluorescence

spectra of EB bound to DNA were obtained at an excited wavelength of 522 nm in the

Fluorimeter (Hitachi-2000). The interaction of the complex 1 with calf thymus DNA (CT-

DNA) has been investigated by using absorption and emission spectra.

IV.3.5.1 Absorption spectral study

Electronic absorption spectroscopy is an effective method to examine the binding

modes of complex 1 with DNA. In general, binding of the compound to the DNA helix is

testified by an increase of the CT band complex 1 due to the involvement of strong

intercalative interactions between an aromatic chromophore of compound and the base

pairs of DNA [42-44]. The absorption spectra of complex 1 in the absence and presence

of CT-DNA is given in Figure IV.7. The extent of the hyperchromism in the absorption

band is generally consistent with the strength of intercalative binding/interaction [45, 46].

Figure IV.8 indicates that the complex 1 interacts strongly with CT-DNA (Kb = 1.54 x105

M-1), and the observed spectral changes may be rationalized in terms of intercalative

binding [47]. In order to further illustrate the binding strength of the complex 1 with CT-

DNA, the intrinsic binding constant Kb was determined from the spectral titration data

using the following equation [48]:

[DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/[Kb (εb–εf)] (1)

where [DNA] is the concentration of DNA, εf, εa and εb correspond to the extinction

coefficient, respectively, for the free complex 1, for each addition of DNA to the complex

1 and for the complex 1 in the fully bound form. A plot of [DNA]/(εa–εf) versus [DNA],

gives Kb, the intrinsic binding constant as the ratio of slope to the intercept.

152

Figure IV.7. Electronic spectral titration of complex 1 with CT-DNA at 267nm in tris-

HCl buffer; [Compound] = 1.09 x 10-4; [DNA]: (a) 0.0, (b) 1.25 x 10-6 , (c) 2.50 x 10-6 ,

(d) 3.75 x 10-6, (e) 5.00 x 10-6 , (f) 6.25 x 10-6mol.L-1. Arrow indicates the increase of

DNA concentration.

Figure IV.8. Plot of [DNA]/(εa–εf) versus [DNA] for the absorption of CT-DNA with

complex 1 in tris-HCl buffer.

From this [DNA]/( εa–εf) versus [DNA] plot (Figure IV.8), the binding constant

Kb for complex 1 was estimated to be 1.54 x 105 M-1 (R = 0.99746 for five points),

indicating a strong binding of the complex 1 with CT-DNA.

153

IV.3.5.2 Fluorescence quenching technique

Fluorescence intensity of EB bound to DNA at 612 nm shows a decreasing trend

with the increasing concentration of the compound. The quenching of EB bound to DNA

by the compound is in agreement with the linear Stern–Volmer equation [49]:

I0/I = 1 + Ksv [Q] (2)



concentration of quencher. In the quenching plot (Figure IV.9-IV.10) of I0/I versus

complex 1, Ksv value is given by the ratio of the slope to intercept. The Ksv value for the

complex 1 is 0.87 x 104 (R = 0.98873 for five points), suggesting a strong affinity of 1 to

CT-DNA.

Figure IV.9. Emission spectra of the CT-DNA-EB system in tris–HCl buffer upon the

titration of the compound complex 1. Kex = 522 nm; [EB] =0.96 x 10-4 molL-1; [DNA]

= 9.9 x 10-6 molL-1; [Compound]: (a) 0.0, (b) 1.36 x 10-5 , (c) 2.72 x 10-5 (d) 4.08 x 10-5 ,

(e) 5.44 x 10-5 , (f) 6.80 x 10-5molL-1. Arrow indicates the increase of compound

concentration.

560 600 640 680 720 760

200

400

600

800

1000

1200

Flu

re

sc

en

ce

in

ten

sit

y (

a.u

.)

λλλλ (nm)

f

a

154

Figure IV.10. Plot of I0/I versus complex 1 for the titration of CT-DNA–EB system with

complex 1 using spectrofluorimeter; linear Stern–Volmer quenching constant (Ksv for

complex 1 = 0.87 x 104 ; (R = 0.98873 for five points).

IV.3.5.3 Number of binding site calculation

Flurorescence quenching data were used to determine the binding sites (n) for

the compound 1 with CT-DNA. Figure IV. 9 shows the fluroscence spectra of EB-DNA

in the presence of different concentrations of compound 1. It can be seen that the

fluroscence intensity at 612 nm was used to estimate Ksv and n.

If it is assumed that there are similar and independent binding sites in EB-DNA, the

relationship between the fluroscence intensity and the quencher medium can be deduced

from the following Eq. (3):

nQ + B → Qn….B (3)

where B is the flurophore, Q is the quencher, [nQ + B] is the postulated complex between

the flurophore and n molecules of the quencher [42]. The constant K is given by Eq. (4):

K = [Qn….B]/[Q]n.[B] (4)

If the overall amount of biomolecules ( bound or unbound with the quencher) is Bo, then

[Bo] = [Qn…B]+ [B], where [B] is the concentration of unbound biomolecules, and the

relationship between the fluorescence intensity and the unbound biomolecule as [B]/[Bo]

= I/Io , that is:

log[(Io-I)/I] = logK + nlog[Q] (5)

155

Where (n) is the number of binding site of compound complex 1 with CT-DNA, which

can be determined from the slope of log[(Io-I)/I] versus log[Q],as shown in the Figure

IV.11. The calculated value of the number of binding sites (n) is 1.10 (R= 0.99869 for

five points). The value of (n) approximately equals 1, and thus indicates the existence of

one binding site in DNA for compound 1.

Figure IV.11. The linear plot shows log[(Io-I)/I] versus log[Q],where R = 0.99869 for

five points.

IV.3.5.4 Viscosity Measurement

To further clarify the nature of interaction between complex 1 and CT DNA,

viscosity measurements were carried out. Upon binding, a DNA intercalator causes an

increase in the viscosity of the DNA double helix due to its insertion between the DNA

base pairs and consequently to the lengthening of the DNA double helix. In contrast, a

partial and/or nonclassical intercalation could bend (or kink) the DNA helix, reducing the

effective length and its viscosity [50]. The method is generally considered the least

unambiguous to probe the mode of binding of a compound to DNA. The effect of 1 on

viscosity of CT DNA is shown in Figure IV.12. The viscosity of DNA increased

dramatically upon addition of complex 1 and is nearly linear (R2 = 0.99621 for nine

points). These results strongly indicate that the complex 1 deeply into the DNA base pairs

in intercalative fashion.

156

Figure IV.12. Effect of increasing amount of 1 on the relative viscosity of CT DNA in

tris-HCl, 50 mM NaCl buffer (R2 = 0.99621 for nine points).

IV.3.6 Antimicrobial screening

The biological activities of free dithiocarbamic acids and its rhodium (III)

derivatives (1, 2 and 3) have been studied for their antibacterial activities by agar well

disc diffusion method [51-53]. The antibacterial activities were done at 100 µg/mL

concentration of different compounds in DMF solvent by using three pathogenic gram

negative bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumoniae) and one

gram positive pathogenic bacteria (Bacillus cereus). DMF was used as a negative control.

The petri dishes were incubated at 37°C for 24h. After incubation plates were observed

for the growth of inhibition zones. The diameter of the zone of inhibition was measured in

mm.

IV.3.6.1 Antibacterial activity

Antibacterial activity of the dithicarbamic acids (HL) and the corresponding

complexes are tabulated in Table IV.8 and also shown by bar diagram in Figure IV.13.

Comparisons of the biological activity of the dithicarbamates and their rhodium (III)

derivatives with the standard antibiotic, chloramphenicol at different concentrations have

been carried out taking usual precautions. From this study, it is inferred that all the

rhodium (III) complexes have higher activity than the ligand only, but little less efficient

than the antibiotics.

0 2 4 6 8 10

1.00

1.04

1.08

1.12

1.16

(n/n

o)1

/3

[Complex 1]/[DNA]

157

Table IV.8. Antibacterial data of free dithiocarbamic acids (LnH) and rhodium (III)

complexes (1, 2 and 3) (100 µg/ ml).

Compound for

Treatment

Inhibition zone in mm

E. coli V.cholerae S.pneumoniae B. cereus

L1H 05 05 04 03

L2H 05 04 08 04

L3H 04 07 06 03

1 11 14 17 06

2 16 17 20 08

3 12 14 18 07

Chloramphenicol 22 29 24 09

DMF 0 0 0 0

Figure IV.13. Zone inhibition bar diagram of the LnH ligands and their corresponding

rhodium (III) complexes.

0

5

10

15

20

25

30

E.C V.C S.P B.C

L1H

L2H

L3H

1

2

3

Chlorampheni

colDMF

158

The increased activity may be due to the increase of the delocalization of π-

electrons over the whole chelate ring imparts the increased lipophilic character to the

metal complexes. This higher lipophilicity of the complexes facilitates the penetration

ability with a greater extent into the bacterial cell membranes, and as result it perturbs the

respiration process of the bacteria and diminish the further growth of the microorganisms.

IV.4 Epilogue

Three complexes of diimine dithiocarbamate mixed ligand framework Rh(2-

C6H4py)2(L1)](1), Rh(2-C6H4py)2(L

2)](2), Rh(2-C6H4py)2(L3)] (3) have been synthesized

and characterized by means of solid and solution phase spectroscopic studies including

the X-ray structure of 1. With the knowledge gained from the present study, attempts are

now underway to bind these ligands in the C, N, S-coordination fashion to iridium and

other metal ions having octahedral geometry. The present study of interaction with CT-

DNA shows that these cyclometalated rhodium (III) complexes having dithiocarbamate

moieties are good intercalative binding to with CT-DNA with an adequate number of

coordination sites and this strongly binding ability of the complexes as intercalator

encourage to develop these materials as good anticancer candidates. From the

antibacterial studies it is found that all the metal complexes have higher activities than the

free dithiocarbamic acids (LnH) against four pathogenic bacteria (Escherichia coli, Vibrio

cholerae, Streptococcus pneumonia and Bacillus cereus, among these three complex 2

has more antibacterial effect.

159

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162

CHAPTER-V

Synthesis, Characterization, Interaction with

DNA and Bovine Serum Albumin (BSA), and

Antibacterial Activity of Cyclometalated

Iridium(III) Complexes Containing

Dithiocarbamate Derivative

163

Abstract Three new mono-nuclear cyclometalated iridium (III) complexes having

dithiocarbamate ligand of the type [IrIII(2-C6H4py)2(L)], (where 2-C6H4py = 2-

phenylpyridine; and L1H = 4-MePipzcdtH, L2H = MorphcdtH and L3H = 4-BzPipercdtH

for 1, 2 and 3 respectively) were synthesized from [Ir(2-C6H4py)2Cl]2.1/4CH2Cl2 by

displacing the two bridged chlorides with one dithiocarbamate ligand. The complexes

were characterized by using physico-chemical and spectroscopic tools along with the

detailed structural analysis of [Ir(2-C6H4py)2(L2)] (2) by single crystal X-ray diffraction.

Structural analysis of 2 showed a distorted octahedron in which the nitrogen donor of one

2-phenylpyridine and the carbon donor of another 2-phenylpyridyl ligand are in axial

positions and trans to one another. Electrochemical analysis by cyclic voltammetry

showed the irreversible two-electron equivalent reduction voltammograms of 1, 2 and 3

attributable to iridium (III) to iridium (I). The electronic characterizations of these

complexes are consistent with significant delocalization of the sulfur electron density

onto the empty metal d-orbital. The intercalative interaction of the complexes with CT-

DNA was evaluated using absorption, fluorescence quenching and viscosity measurement

techniques. The binding affinity of these complexes with bovine serum albumin (BSA)

was estimated in terms of quenching constants using the Stern-Volmer equation. Study of

antibacterial activity of complexes by agar disc diffusion method against some species of

pathogenic bacteria was also performed.

164

V.1 Introduction

The chemistry of iridium (III) complexes has been receiving considerable current

attention in the field of development of organic light-emitting diodes, catalysts and

anticancer agents. In recent years, iridium (III) complexes, particularly, bis- and tris-

cyclometalated (C^N) complexes of iridium(III) based on 2-phenylquinoline and its

derivatives have been widely used as one of the promising electroluminescent materials

for the OLED technology because cyclometalized iridium(III) complexes having a d6-

electron configuration show strong spin-orbit coupling, efficient intersystem crossing

from the singlet excited state to the triplet manifold, as well as an enhancement of the T1-

S0 transition, thereby displaying efficient phosphorescent emission at room temperature

[1-10]. In this regard, several reports on the evolution of selective recognition for Hg2+

with multisignaling optical–electrochemical response by introducing the sulfur atoms to

these cyclometalated ligands are also too many in the literature [11-17] compared to the

reports of biological applications of iridium(III) complexes [18-28]. Among these

biological applications, DNA binding studies have been one of focused studies to explore

the intercalation characteristics of the iridium(III) complexes into DNA in quest of

relatively inert iridium-based anticancer complexes of higher potency, higher cancer cell

selectivity, lower resistance and reduced side effects [25-28]. But to the best of our

knowledge so far, study of interaction with DNA and bovine serum albumin (BSA), and

antibacterial activity of cyclometalated iridium (III) complexes bearing dithiocarbamate

derivative is still unexplored.

The dithiocarbamate (R2NCS2-) type of ligands are significantly commendable as

each of the two sulfur atoms of a metal-coordinated chelate dithiocarbamate ligand

maintains an additional coordination capability due to the availability of, at least, one pair

of potentially bonding electrons. This potentiality has been contemporarily exploited to

explore the important applications of this type of ligands and metal dithiocarbamates in

several fields such as the chemical bonding and industry, biology and biochemistry [29-

35]. It is also noteworthy that the nature of the heterocycle and the metal ion attached to

dithiocarbamate fragment control the potentiality of pharmacological as well as catalytic

efficiency of the metal complexes [36].

Keeping the facts of the advantages stated above, in this chapter, an account of

syntheses, characterizations, electrochemical behavior and biological properties of a new

165

series of three mononuclear cyclometalated iridium (III) complexes bearing

dithiocarbamate derivatives has been described. The report deals with the chemistry of

the type of [IrIII(2-C6H4py)2(L)] complexes, where 2-C6H4py = 2-phenylpyridine; and L1

= 4-Methylpiperazine-l-carbodithioic acid anion, L2 = Morpholine-4-carbodithioic acid

anion and L3 = 4-Benzylpiperidine-l-carbodithioic acid anion (see scheme 2) anion for

complexes 1, 2 and 3, respectively. The complexes were characterized by

physicochemical and spectroscopic methods; in addition, the structure of complex 2 was

characterized by single crystal X-ray crystallography, UV-vis, IR-spectroscopy.

Electrochemical analysis by cyclic voltammetry showed the irreversible two-electron

equivalent reduction voltammograms of 1, 2 and 3 attributable to iridium (III) to iridium

(I). The electronic characterizations of these complexes are consistent with significant

delocalization of the sulfur electron density onto the empty metal d-orbital. The

intercalative mode of binding interactions of these complexes with calf thymus-DNA

(CT-DNA) have been investigated systematically to explore the biological activity of the

new complexes as we know through pharmacological mechanistic studies that the

effectiveness depends on the intercalative binding to DNA [37-40] using absorption,

fluorescence quenching and viscosity measurement techniques. The association of

complex 2 with bovine serum albumin (BSA) with through spectroscopic tools has also

been examined and the binding affinity of the complex with bovine serum albumin (BSA)

was estimated in terms of quenching constants using the Stern-Volmer equation. In

addition, antibacterial activity of the complexes (1, 2 and 3) against some pathogenic

bacteria, namely Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and

Bacillus cereus has also been studied by agar disc diffusion method.

V.2 Experimental Section

V.2.1 Solvents and Reagents

Iridium trichloride, 2-phenylpyridine, morpholine and 4-benzylpiperidine

(Aldrich) were purchased and used without further purification. [Ir(2-

C6H4py)2Cl]2.1/4CH2Cl2 was prepared following the reported procedure [42]. Calf

thymus-DNA was obtained from Bangalore Genie, India. Solvents used for spectroscopic

studies and for synthesis were purified and dried by standard procedures before used. The

organic moieties, 4-methylpiperazine-l-carbodithioic acid (4-MePipzcdtH, L1H),

166

morpholine-4-carbodithioic acid (MorphcdtH, L2H) and 4-benzylpiperidine-l-

carbodithioic acid (4-BzPipercdtH, L3H) were obtained as solid products following our

earlier report [43].

V.2.2 Physical Measurements

The Fourier transform infrared spectra of the ligand and the complexes were

recorded on a Perkin-Elmer FTIR model RX1 spectrometer using KBr pellet in the range

4000 - 300 cm-1. The solution phase electronic spectra were recorded on a JASCO UV–

Vis/NIR spectrophotometer model V-570 in the range 200-1100 nm. Elemental analyses

were carried out on a Perkin-Elmer 2400 series-II CHNS Analyzer. Steady-state

fluorescence emission and excitation spectra were recorded with a Hitachi F-4500 FL

spectrophotometer. Mass spectra of 1, 2 and 3 were recorded on Micromass Q-Tof

microTM. NMR spectrum of the ligands and complexes has been recorded on Bruker

DPX-300. Solution conductivities were measured using Systronics Conductivity Meter

304 model and redox potentials by CHI620D potentiometer in DMF at complex

concentration of ~10-3 mol L-1. The stock solutions of protein (1.00 × 10−4 mol L−1) was

prepared by dissolving BSA in 0.05 M phosphate buffer at pH 7.4 and stored at 0-4 °C in

the dark. The concentration of BSA was determined from optical density measurements,

using the values of molar absorptivity of ε280 = 44,720 M−1 cm−1 [44].

V.2.3 X-Ray Crystal Structure Analysis

X-ray data of the suitable crystal of complex 2 were collected on a Bruker’s Apex-

II CCD diffractometer using MoKα (λ = 0.71069). The data were corrected for Lorentz

and polarization effects and empirical absorption corrections were applied using

SADABS from Bruker. A total of 13691 reflections were measured out of which 4012

were independent and 2299 were observed [I > 2σ(I)] for theta (θ) 32°. The structure was

solved by direct methods using SIR-92 and refined by full-matrix least squares refinement

methods based on F2, using SHELX-97 [45]. The two fold axis passes through the metal

ion, nitrogens of the piprazine ring and their substituent carbon atoms. Therefore the

asymmetric unit contains half the molecule. All non-hydrogen atoms were refined

anisotropically. The refinement showed rotational disorder in the piprazine ring which

could be resolved by splitting the two unique carbon atoms into two components and

167

refining their sof and thermal parameters as free variables with restraints over the bond

distances. All hydrogen atoms were fixed geometrically with their Uiso values 1.2 times of

the phenylene and methylene carbons and 1.5 times of the methyl carbons. All

calculations were performed using Wingx package [46,47]. Important crystallographic

parameters are given in Table V.1. Crystallographic data for have been deposited with the

Cambridge Crystallographic Data Centre, CCDC No. 932587 for 2.

V.2.4 Syntheses of Ligands


ligands, 4-Methylpiperazine-lcarbodithioic acid anion (L1), Morpholine-4-carbodithioic

acid anion (L2) and 4-Benzylpiperidine-lcarbodithioic acid anion (L3). The procedure for

the preparation and spectroscopic evidences of dithiocarbamate ligands has already

described in detail in chapter II under section II.2.4.

V.2.5 Syntheses of Iridium (III) Complexes

The complexes have been synthesized with the help of a common procedure stated

as below. To the solution of the ligand, L1H (177 mg, 1.0 mmol) for complex 1 or L2H

(164 mg, 1.0 mmol) for complex 2 or L3H (253 mg, 1.0 mmol) for complex 3 in DMSO-

MeCN (v/v 1:1) solvent mixture, the solution of [Ir(2-C6H4py)2Cl]2.1/4CH2Cl2 (557 mg,

0.5 mmol) in acetonitrile was added. The resulting mixed solution was then refluxed in

nitrogen atmosphere for 12 h and the color changed from faded yellow to orange. On

slow evaporation, microcrystalline solid appeared, which was then subjected to

purification by TLC on a silica plate with C6H6–MeCN (v/v 3:1) as an eluant, an orange

band separated, which was extracted with MeCN. Needle shaped crystals of [Ir(2-

C6H4py)2(L2)] suitable for X-ray diffraction study were grown from this solution on

evaporation at ambient temperature.

168

Table V.1. Crystallographic data for the complex 2.

Complex 2

Empirical Formula C27H24IrN3O2S2, 0.5H2O


Crystal system Triclinic

Space group P -1

a (Å) 9.7183(4)

b (Å) 11.4054(5)

c (Å) 12.3144(6)

β, deg 100.737(2)

Volume (Å3) 1274.16(10)

Temperature (K) 2

Z 1.769


µ(mm-1) 664

F(000) 1.77 -27.00

θ range (deg) 19902

Refl. collected / unique 5488

R(int) 0.0224




R1, wR2 (I >2σ(I)) [a] 0.0193 , 0.0491

R indices (all data) R1 = 0.0211, 0.0500

aR = ∑||Fo |- |Fc||/∑|Fo|;

bwR2 = { ∑ [w(Fo

2 –Fc2)2] / ∑w [(Fo

2)2]}1/2

169

V.3 Results and Discussions

V.3.1 Syntheses and Characterization

The ligands L1H, L2H, and L3H were synthesized by the reaction between carbon

disulfide with different amines in ethanol, and later characterized by FTIR and 1H NMR.

Treatment of these ligands with [Ir(2-C6H4py)2Cl]2.1/4CH2Cl2 at refluxing condition in

DMSO-MeCN (v/v 1:1) solvent mixture having nitrogen atmosphere resulted in cleavage

of the chloro bridge and led to formation of mononuclear iridium(III) complexes of

general formula of [IrIII(2-C6H4py)2(L)] which were obtained from the column

chromatography using acetonitrile as organge colored microcrystalline solid on

evaporation. Here, the dithiocarbamates behaves as bidentate monobasic ligands

(viz.Scheme 1 and Table V.2).

Scheme 1. Synthetic method of iridium (III) complexes.

Table V.2. Iridium (III) complexes with dithiocarbamate ligand.

LnH Complex

4-Methylpiperazine-l-carbodithioic acid (4-MePipzcdtH ) (L1H) [(2-C6H4py)2 IrIII (L1)] (1)

Morpholine-4-carbodithioic acid ( MorphcdtH ) (L2H) [(2-C6H4py)2 IrIII (L2)] (2)

4-Benzylpiperidine-l-carbodithioic acid (4-BzPipercdtH ) (L3H) [(2-C6H4py)2 IrIII (L3)] (3)

MeOH-MeCN

X

NSH

S

N

LnH + [Ir(2-C6H4py)2Cl2]2Reflux / 10 h

[(2-C6H4py)2IrLn]


2-C6H4pyX= -N-CH3 , 4-MePipzcdtH (L1H)


X= -CH-CH2Ph , 4-BzPipercdtH (L3H)

1

2

3456

7

8

170

Microanalytical data (Table V.3) confirm the composition of the complexes

and spectroscopic data of 1, 2 and 3 along with the X-ray crystallographic results of 2

indicate the organic moieties, 4-Methylpiperazine, Morpholine and 4-Benzylpiperidine in

the reaction medium are converted to 4-Methylpiperazine-l-carbodithioic acid (L1H),

Morpholine-4-carbodithioic acid (L2H) and 4-Benzylpiperidine-l-carbodithioic acid (L3H)

respectively, and their iridium (III) complexes 1-3.

Table V.3. Microanalytical data of the iridium (III) complexes (1-3).


The complexes (1-3) are sparingly soluble in common organic solvents except

hexane but fairly soluble in DMF and DMSO, and are stable in both the solid state and

solution in air. The spectroscopic and elemental analyses confirm the formulations of the

complexes. The molar conductivity of freshly prepared solution (~1 x 10-3 M

concentration) of the complexes in DMF are fairly consistent with a non-electrolytes. The

magnetic susceptibility measurements are in agreement with the diamagnetic nature of the

complexes.

V.3.2 Structural description of complex 2

An ORTEP view of the complex [Ir(2-C6H4py)2(L2)] (2) with atom labeling

scheme is illustrated in Figure V.1, and the selected bond distances and bond angles are

listed in Table V.4. The structural analysis evidenced that the complex resides on a P-1

site in the triclinic crystal system. The crystal structure of 2 is a distorted octahedron in

which the nitrogen donor of one 2-phenylpyridine and the carbon donor of another 2-

phenylpyridyl ligand are in axial positions and trans to one another. As would be

expected, both Ir-C σ-bonds are equal in length (1.994(4)Å) and significantly shorter than


(Λo)a C H N

[(2-C6H4py)2 IrIII (L1)] (1) 49.76(49.03) 4.03(3.89) 8.29(8.76) 42

[(2-C6H4py)2 IrIII (L2)] (2) 48.92(48.01) 3.65(3.41) 6.34(6.97) 35

[(2-C6H4py)2 IrIII (L3)] (3) 55.98(55.22) 4.29(4.11) 5.60(6.10) 45

171

the Ir-N dative bond lengths of 2.039(3)Å. The bond distance of Ir-C in 2 is comparable

with the previously reported Ir-C bonds in cyclometalated complex (1.996(9)Å) but the

bond Ir-N is slightly longer than those (1.987(7)Å) [48], but both are comparable with the

reported values [49]. Both Ir–S distances are also equal in length (2.4854(11)Å), and

these are longer than those found in [Ir(Et2NCS2)3] (2.364(3)Å) [49] due to the

attachment of sulphur atoms to carbon of more electronegativity than phosphorous, for

this reason, the S-Ir-S angle of 71.00(5)o is smaller compared to the observed value of

79.40(1)o in the previous report [49]. The structural analysis showed a highly disordered

water molecule O1W near the inversion centre, it was refined with 0.5 sof. The hydrogens

attached to it were located from the difference Fourier and were initially refined with 0.5

sofs with their isotropic thermal parameters fixed as 1.2 times that of the oxygen and O-H

distance at 0.84(2)Å.

Figure V.1. ORTEP view of [Ir(2-C6H4py)2(L2)] (2) with atom labeling scheme of the

crystallographic independent part. (Symmetry codes: (i) -x,y,z; (ii) x,-y,-z; (iii) x,-y,-z).

172

Table V.4. Selected bond distances (Å) and bond angles (°) for 2.

Symmetry transformations used to generate equivalent atoms: #1 -x+1, y, -z+3/2

V.3.3 Spectroscopic analysis

V.3.3.1 IR Spectra

The IR spectra of the ligands display an intense stretch at 2850 cm-1 and 1445-1430

cm-1 correspond to γC-H of N-Me and γC=N respectively. The γC-H of N-Me of L1H for 1

(2915cm-1) is blue shifted than the γC-H of N-Me of the free ligand (2850 cm-1) indicating

metal ligand coordination. The band around 1500 cm-l indicates a double bond character

of C-N bond in the ligand frame, which is confirmed from the bond length of the X-ray

structure. This fact could be attributed to the electron releasing ability of the heterocyclic

group towards the sulphur atoms, a feature that induces an electron delocalization over

the carbon-nitrogen bond and the CS2 fragment. This is shown by the νC=N shift to higher

energies (ca. 1480-1496 cm-l) with respect to the free acids (ca. 1445-1430 cm-1) (see

Table V.5), and these bands lie in between the stretching frequencies expected for a

double C=N (1610-1690 cm-l) and single C-N bond (1250-1350 cm-1). The blue-shift of

the C=N stretching frequency on going from the free acids to their metal complexes gives

support to the typical bidentate character [50] of the carbodithioic acid ligands. Two

bands in the region of 1042-996 cm-l (separated by less than 20 cm-l) assignable to the

Bond length (Å)

Ir(1)-S(1) 2.490(8) Ir(1)-N(3) 2.011(3)

Ir(1)-S(2) 2.459(7) Ir(1)-C(16) 2.006(3)

Ir(1)-N(2) 2.047(2) Ir(1)-C(27) 2.040(2)

Bond angle (o)

C(27)-Ir(1)-N(3) 80.38(11) N(2)-Ir(1)-S(1) 97.17(8)

C(27)-Ir(1)-S(1) 88.85(7) N(3)-Ir(1)-S(1) 99.59(9)

S(1)-Ir(1)-S(2) 71.16(2) C(27)-Ir(1)-S(2) 95.28(7)

S(2)-Ir(1)-N(2) 100.14(11) C(27)-Ir(1)-N(2) 172.51(9)

C(16)-Ir(1)-N(2) 80.27(11) C(16)-Ir(1)-S(1) 169.99(8)

C(16)-Ir(1)-N(3) 90.27(11) C(16)-Ir(1)-C(27) 94.54(11)

N(3)-Ir(1)-N(2) 94.18(11) N(3)-Ir(1)-N(2) 94.18(11)

173

νa(SCS) and one band for the vs(SCS) stretch in the region 708-678cm-l of the complexes

suggest the unsymmetrical chelating bidentate mode of coordination to iridium(III) ion

[51]. The stretchings due to νCOC (asym and sym), νN-Me and νCCC (asym and sym) remain


to exclude any coordination to the metals via nitrogen and oxygen donors.

Table V.5. IR spectral data of the ligands and iridium (III) complexes.

V.3.3.2 Electronic Spectra

The electronic spectral data of 1, 2 and 3 in DMSO solution have been tabulated

in Table V.6. Complexes 1, 2 and 3 display a lower energy band at 678 nm, 674 nm and

676 nm, respectively with low extinction coefficient values that correspond to the d–d

transition. The higher energy band around 385-390 nm for all three complexes with high

extinction coefficient values are due to the coordinated carbon atom from pipyridine

moiety, C(σ)-Ir(III) charge transfer (LMCT) transition. The other higher energy intense

transitions at 352 nm and 359 nm are due to the n→π* and π→π* charge transfer

transitions. In deoxygenated solution, 1-3 exhibit green fluorescence with no

phosphorescence in solution state (Figure V.2). Here, the emission intensity remained

almost unaffected in presence of Zn2+, Hg2+, Cd2+ and Pb2+ metal ions and oxygen also.

Compound νSCS(s) νSCS(as) νC=N νIr-S

4-Methylpiperazine-l-carbodithioic acid (L1H) 684 1023 1451 -

Morpholine-4-carbodithioic acid (L2H) 689 1029 1468 -

4-Benzylpiperidine-l-carbodithioic acid (L3H) 699 1072 1496 -

[(2-C6H4py)2 IrIII (L1)] (1) 695 998 1490 433

[(2-C6H4py)2 IrIII (L2)] (2) 699 1015 1474 436

[(2-C6H4py)2 IrIII (L3)] (3) 707 1035 1499 434

174

Figure V.2. Emission spectra of the complexes at 10-5 M in deoxygenated acetonitrile

solution at 298 K (λex = 364 nm, 340 nm and 313 nm for 1, 2 and 3 respectively).

Table V.6. Electronic absorption spectral data of the complexes.

Complex λmax(nm) (logε)a

1 435 (2.50), 390 (3.35), 359sh (3.95), 335 (3.92), 313 (3.97)

2 438 (2.60), 386 (3.35), 353sh (3.94), 314 (3.92)

3 442 (2.65), 382 (3.39), 352sh (3.90), 319 (3.96)

a in DMSO solvent

V.3.3.3 Redox studies

The cyclic voltammograms (CV) of the complexes 1, 2 and 3 were recorded in

DMF solvent at room temperature. Three electrode cell set up such as platinum, Ag/AgCl

and a platinum wire as a working, reference and auxiliary electrode respectively have

been used for measurements. The cyclic voltammograms of all the three complexes 1, 2

and 3 have been shown in Figure V.4 and the electrochemical data have been tabulated in

Table V.7. The complexes exhibit an irreversible reductive response at E1/2 value ≈ -0.766

V to -0.810 V (versus Ag/AgCl) corresponds to Ir3+/Ir+ couple. Small differences in the

∆Ep values 665 mV, 593 mV and 630 mV for 1, 2 and 3 respectively) have been observed

175

that increases in the order 1 > 3 > 2. The trend of reduction potential values can be

explained by the energy level diagram for the frontier π MOs of complexes obtained from

the DFT calculation (viz. supporting information). This study supports the least cathodic

potential value for 2 (-0.766 V) in accordance with the calculated LUMO of complex 2 is

of the least energy (LUMO (133) = -0.0473 eV) where as the LUMO of the highest

energy (LUMO (137) = -0.0443 eV) is in agreement with the highest cathodic potential (-

0.810 V) for 1.

Figure V.3. Cyclic voltammograms (scan rate 50 mV/s) of 1, 2 and 3 in DMF solution of

M TBAP, using platinum working electrode. Potentials are vs. non-aqueous Ag/AgCl.

Table V.7. Electrochemical dataa for the complexes 1, 2 and 3.

Complex Epc(V) Epa(V) ∆∆∆∆Ep(mV) E1/2(V)

1 -1.094 -0.464 665 -0.810

2 -1.143 -0.478 593 -0.766

3 -1.063 -0.470 630 -0.779

aPotentials versus non-aqueous Ag/Ag+ reference electrode, scan rate 50 mV/s,

supporting electrolyte: tetra-N-butylammonium perchlorate (0.1 M).

-1.4 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0

-2

-1

0

1

2

3

4

5Black = 3, Red = 2, Blue = 1

I ( µµ µµ

A)

E (V)

176

V.3.4 Theoretical calculation

To clarify the configurations and energy levels of the complexes 1, 2 and 3, DFT

calculations were carried out in G09W program using B3LYP/6-31G(d) calculation and

correlation function as implemented in the Gaussian program package Gaussian 09.

Thermal contribution to the energetic properties was considered at 298.15 K and one

atmosphere pressure (Figures V.4 -V.6).

Figure V.4. Optimized structure of complex 1.

HOMO(136) = -0.1791 eV LUMO(137) = -0.0443 eV

177


HOMO(132) = -0.1828eV LUMO(133)= -0.0473eV

178


V.3.5 DNA binding experiments

All the experiments involving CT-DNA were studied by spectrophotometric

titration and fluorescence quenching technique by using ethidium bromide (EB) as a

DNA scavenger in tris-buffer solution and performed the experiment as our previously

standardized method [52].

Fig.s6. Energy level

HOMO(156) = -0.1794 eV

LUMO(157) = -0.0445 eV

179

Concentrated stock solution of the complex 2 was prepared by dissolving the

compound in DMSO and suitably diluted with tris–HCl buffer to the required

concentration for all the experiments. Absorption spectral titration experiment was

performed by keeping constant the concentration of the complex 2 and varying the CT-

DNA concentration. To eliminate the absorbance of DNA itself, equal solution of CT-

DNA was added both to the complex 2 solution and to the reference solution.

In the ethidium bromide (EB) fluorescence displacement experiment, 5 µL of the

EB tris–HCl solution (1.0 mmol.L-1) was added to 1.0 mL of DNA solution (at saturated

binding levels), stored in the dark for 2.0 h. Then the solution of the compound was

titrated into the DNA/EB mixture and diluted in tris–HCl buffer to 5.0 mL to get the

solution with the appropriate complex 1/CT-DNA mole ratio. Before measurements, the

mixture was shaken up and incubated at room temperature for 30 min. The interaction of

the complex 2 with calf thymus DNA (CT-DNA) has been investigated by using

absorption and emission spectra along with the viscometric measurements to discern

between DNA intercalation and groove binding modes.

V.3.5.1 Absorption spectral study

Electronic absorption spectroscopy is an effective method to examine the binding

modes of complex 2 with DNA. In general, binding of the compound to the DNA helix is

testified by an increase of the CT band complex 2 due to the involvement of strong

intercalative interactions between an aromatic chromophore of compound and the base

pairs of DNA [53,54]. The absorption spectra of complex 2 in the absence and presence

of CT-DNA is given in Figure V.7. The extent of the hyperchromism in the absorption

band is generally consistent with the strength of intercalative binding/interaction [55,56].

Figure V.7 indicates that the complex 2 interacts strongly with CT-DNA (Kb = 0.31 x105

M-1), and the observed spectral changes may be rationalized in terms of intercalative

binding [57]. In order to further illustrate the binding strength of the complex 2 with CT-

DNA, the intrinsic binding constant Kb was determined from the spectral titration data

using the following equation [58] :

[DNA]/(εa–εf) = [DNA]/(εb–εf) + 1/[Kb (εb–εf)] (1)

where [DNA] is the concentration of DNA, εf, εa and εb correspond to the extinction

coefficient, respectively, for the free complex 2, for each addition of DNA to the complex

180

1 2 3 4 5 6 7

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

[DN

A]/

( εε εεa- εε εε

f) x

10

10

[DNA] x 106

2 and for the complex 2 in the fully bound form. A plot of [DNA]/(εa–εf) versus [DNA],

gives Kb, the intrinsic binding constant as the ratio of slope to the intercept. From the

[DNA]/( εa–εf) versus [DNA] plot (Figure V.8), the binding constant Kb for complex 2

was estimated to be 0.31 x 105 M-1 (R = 0.99478 for five points).

Figure V.7. Electronic spectral titration of complex 2 with CT-DNA at 269 nm in tris-

HCl buffer; [Complex 2] = 1.09 x 10-4; [DNA]: (a) 0.0, (b) 1.25 x 10-6, (c) 2.50 x10-6, (d)

3.75 x 10-6, (e) 5.00 x 10-6, (f) 6.25 x 10-6 molL-1. The arrow indicates the increase of

DNA concentration.

Figure V.8. Plot of [DNA]/(εa–εf) versus [DNA] for the absorption of CT-DNA with the

complex 2 in tris-HCl buffer.

300 400 500 600 7000.0

0.1

0.2

0.3

0.4 f

a

Ab

so

rb

an

ce

Wavelength (nm)

181

V.3.5.2 Fluorescence quenching technique

Fluorescence intensity of EB bound to DNA at 612 nm shows a decreasing trend

with the increasing concentration of the compound (Figure V.9). The quenching of EB

bound to DNA by the compound is in agreement with the linear Stern–Volmer equation

[59]:

I0/I = 1 + Ksv [Q] (2)



concentration of quencher. In the quenching plot in Figure V.10 of I0/I versus complex 2

Ksv value is given by the ratio of the slope to intercept. The Ksv value for the complex 2 is

0.39 x 104 (R = 0.99746 for five points), suggesting a strong affinity of the compound to

CT-DNA.

Figure V.9. Emission spectra of the CT-DNA-EB system in tris-HCl buffer upon the

titration of complex 2. λex = 522 nm; [EB] = 9.6×10-5 mol L-1, [DNA] =

1.25×10- 5; [Complex]: (a) 0.0, (b) 1.36×10-5, (c) 2.72×10-5, (d) 4.08×10-5,

(e) 5.44×10-5 mol L-1. The arrow denotes the gradual increase of the

concentration of complex.

V.3.5.3 Number of binding site calculation

Flurorescence quenching data were used to determine the binding sites (n) for the

complex 2 with CT-DNA. Figure V.10 shows the fluroscence spectra of EB-DNA in the

600 650 700 7500

200

400

600

800

1000a

e

Inte

ns

ity

Wavelength(nm)

182

presence of different concentrations of compound 2. It can be seen that the fluroscence

intensity at 612 nm was used to estimate Ksv and n.




nQ + B → Qn….B (3)



K = [Qn….B]/[Q]n.[B] (4)

If the overall amount of biomolecules ( bound or unbound with the quencher) is Bo, then

[Bo] = [Qn…B]+ [B], where [B] is the concentration of unbound biomolecules, and the

relationship between the fluorescence intensity and the unbound biomolecule as [B]/[Bo]

= I/Io , that is:


Where (n) is the number of binding site of compound complex 2 with CT-DNA, which


V.11. The calculated value of the number of binding sites (n) is 1.19 (R= 0.99862 for five

points). The value of (n) approximately equals to 1, and thus indicates the existence of

just a single binding site in DNA for compound 2.

Figure V.10. Plot of I0/I vs. complex 2 for the titration of CT-DNA–EB system with

complex 2 using spectrofluorimeter; linear Stern–Volmer quenching constant (Ksv) for

complex 2 = 0.39 x 104 ; (R = 0.99746 for five points).

2 4 6 8

1.05

1.20

1.35

1.50

1.65

0

[Complex] x 105

183

Figure V.11. Plot of log[(Io-I)/I] versus log [Complex 2] for the titration of complex 2

with CT-DNA-EB system in tris-HCl buffer.

V.3.5.4 Viscosity Measurement

To obtain direct evidence of DNA interaction with complex 2 and specifically to

discriminate between DNA intercalation and groove binding modes, viscosity

measurements were carried out. Upon binding, a DNA intercalator causes an increase in

the viscosity of the DNA double helix due to its insertion between the DNA base pairs

and consequently to the lengthening of the DNA double helix. In contrast, a partial and/or

nonclassical intercalation could bend (or kink) the DNA helix, reducing the effective

length and its viscosity [60].

The effect of 2 on viscosity of CT DNA is shown in Figure V.12. The viscosity of

DNA increased dramatically upon addition of complex 2 and is nearly linear at low

concentration region. These results strongly indicate that the imine dithiocarbamate

compound intercalated deeply into the DNA base pairs. According to the theory of Cohen

and Eisenberg [61] the slopes value of this line in this plots correlate well with the DNA-

ligand binding modes. Thus, groove binding compounds usually display a slope close to

0.0, whereas slope values reported in the literature typically lie in the range 0.8–1.5 for

DNA monointercalants and 1.3–2.3 for DNA bisintercalants [62-66]. Here, it is evident

that complex 2 interacts with CT-DNA as a bifunctional intercalant agent since the slope

value was calculated to be 1.999 ± 0.004.

-4.8 -4.6 -4.4 -4.2 -4.0

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log

[(I o

-I)/

I]

log[Complex 2]

184

0.0 2.0 4.0 6.0 8.0 10.0

0.99

1.02

1.05

1.08

1.11

1.14

1.17

(n/n

o)1

/3

[Complex 2]/[DNA] x 10-2

Figure V.12. Viscometric measurement experiments of calf thymus DNA and complex

2 at 25 oC (50 mM tris- buffer); assays were performed in triplicate. Slope: 1.999 ±

0.004; R2 =0.99621 for 9 points.

V.3.6 Antimicrobial screening

The biological activities of free dithiocarbamic acids and the iridium(III)

derivatives of dithiocarbamates (1, 2 and 3) have been studied for their antibacterial

activities by agar well diffusion method [67-69]. The antibacterial activities were done at

100 µg/mL concentration of different compounds in DMF solvent by using three

pathogenic gram negative bacteria (Escherichia coli, Vibrio cholerae, Streptococcus

pneumoniae) and one gram positive pathogenic bacteria (Bacillus cereus). DMF was used

as a negative control. The Petri dishes were incubated at 37°C for 24h. After incubation

plates were observed for the growth of inhibition zones. The diameter of the zone of

inhibition was measured in mm.

V.3.6.1 Antibacterial activity

Antibacterial activity of the dithicarbamic acids (HL) and the corresponding

complexes are tabulated in Table V.8 and bar diagram Figure V.13. Comparisons of the

biological activity of the dithicarbamates and their iridium(III) derivatives with the

standard antibiotics, chloramphenicol at different concentrations have been carried out

185

taking usual precautions. From this study, it may be concluded that inferred that all the

iridium(III) complexes have higher activity than the ligand only, but little less efficient

than the antibiotics. The increased activity may be due to the increase of the

delocalization of π-electrons over the whole chelate ring imparts the increased lipophilic

character to the metal complexes. This higher lipophilicity of the complexes facilitates the

penetration ability with a greater extent into the bacterial cell membranes, and as result it

perturbs the respiration process of the bacteria and slowdowns the further growth of the

microorganisms.

Table V.8. Antibacterial data of free dithiocarbamic acids (LnH) and iridium (III) complexes (1, 2 and 3) (100 µg/ ml).

Compound for

Treatment


E. coli V.cholerae S.pneumoniae B. cereus

L1H 03 03 04 02

L2H 04 03 07 03

L3H 04 06 05 03

1 07 06 05 05

2 13 15 14 13

3 08 09 09 07

Chloramphenicol 19 28 30 20

DMF 0 0 0 0

186

Figure V.13. Zone inhibition bar diagram of the LnH ligands and their corresponding

iridium (III) complexes.

V.3.7 BSA-binding experiments

The binding study with bovine serum albumin (BSA) for complex 2 was done

dissolving BSA in MilliQ water (1.0×10-5 M-1) and the stock solution of the complex was

prepared in DMSO-H2O (1:99 v/v) mixture at 1.0×10-5 M-1 concentration. Both the

absorption and fluorescence quenching experiments (λex = 280 nm) were performed by

gradually increasing the complex concentration, keeping fixed the concentration of BSA.

All the experimental sets were carefully degassed purging pure nitrogen gas for 5 min.

V.3.7.1 Absorption spectral technique

The absorption spectra of BSA in the absence and presence of iridium (III)

complex 2 at different concentrations were recorded in DMSO-H2O medium (Figure

V.14) The spectra indicate a significant increase in the absorbance of BSA by increasing

the concentration of the complex and are indicative of the fact that BSA adsorbs strongly

the complex on its surface. From these data the apparent association constant (Kapp) of the

complex with BSA has been determined using the following equation [70]:

1/(Aobs – A0) = 1/(Ac – A0) + 1/ Kapp(Ac – A0)[complex]

Where, Aobs is the observed absorbance of the solution containing different

concentrations of the complex at 280 nm, A0 and Ac are the absorbances of BSA and the

complex at 280 nm, respectively, with a concentration of C0, and Kapp represents the

0

5

10

15

20

25

30

E.C V.C S.P B.C

L1H

L2H

L3H

1

2

3

Chloramphenicol

DMF

187

apparent association constant. The enhancement of absorbance at 280 nm was due to

absorption of the surface complex, based on the linear relationship between 1/(Aobs - A0)

vs reciprocal concentration of the complex with a slope equal to 1/Kapp(Ac - A0) and an

intercept equal to 1/(Ac - A0) (Figure V.15). The value of the apparent association

constant (Kapp) determined from this plot is 8.4388× 104 M-1(R = 0.99462 for five points).

V.3.7.2 Fluorescence quenching

The effect of increasing the concentration of the complex 2 on the fluorescence

emission spectrum of BSA were studied and represented in Figure V.16. With the

addition of complex BSA fluorescence emission is quenched. The fluorescence

quenching is described by the Stern–Volmer relation [59]:

I0/I =1+ KSV [complex]

Where, I0 and I represent the fluorescence intensities of BSA in the absence and

presence of quencher, respectively. KSV is the linear Stern–Volmer quenching constant

and [complex] the molar concentration of the quencher. A linear plot (Figure V.17)

between I0/I against [complex] was obtained and from the slope we calculated the KSV as

5.31 × 105(R = 0.98942 for five points).

Figure V.14. Absorption titration spectra of BSA in presence of complex 2.

Concentration range of complex is 0 - 6.25× 10-6 M-1.

260 280 300 320

0.0

0.5

1.0

1.5

2.0

2.5 f

a

Ab

so

rb

an

ce

Wavelength(nm)

188

Figure V.15. Linear dependence of 1/A _ A0 on the reciprocal concentration of 2.

Figure V.16. Fluorescence quenching titration of BSA varying the concentrations of

complex 2, [Complex]: (a) 0.0, (b) 1×10-5, (c) 2×10-5, (d) 3×10-5 and (e) 4×10-5 mol L-1;

Inset shows the Stern–Volmer plot.

2 4 6 8 10

2

4

6

8

1/(

AO

bs-A

O)

(1/[Complex])x105

300 350 400 450

0

1000

2000

3000

4000

5000

6000

e

a

Inte

ns

ity

Wavelength(nm)

189

Figure V.17. Plot of I0/I vs. [complex] 2 (R = 0.99948 for five points).

V.4 Epilogue

Three neutral cyclometalated iridium (III) complexes bearing dithiocarbamate

derivatives formulated as [Ir(2-C6H4py)2(L1)](1), [Ir(2-C6H4py)2(L

2)](2), [Ir(2-

C6H4py)2(L3)] (3) have been synthesized and characterized by means of solid and solution

phase spectroscopic studies including the detailed structural analysis of complex 2 by

single crystal X-ray crystallographic study. The present study of interaction with CT-

DNA shows that these cyclometalated iridium (III) complexes are good intercalator to

CT-DNA with an adequate number of coordination sites. The detailed study on

viscometric measurement shows that complex 2 interacts with CT DNA as bifunctional

intercalants. These results is in support of the cytotoxicity behaviour of the complexes.

However, from the antibacterial studies it is found that all the complexes have higher

activities than the free dithiocarbamic acids (LH) against four pathogenic bacteria

(Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and Bacillus cereus, among

these three complexes, complex 2 has more antibacterial effect.

1 2 3 4 5

1.0

1.5

2.0

2.5

3.0

I O/I

[Complex]x106

190

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194

CHAPTER-VI

Synthesis of Platinum (II) Complexes of Some

New Pyrrolyl Azo Ligands: Spectroscopic

Characterization and Studies on DNA

Intercalation

195

Abstract

Reaction of K2[PtCl4] with the pyrrolyl azo ligands (4-Chloro-phenyl)-(1H-pyrrol-

2-yl)-diazene(HL7) and (4-Nitro-phenyl)-(1H-pyrrol-2-yl)-diazene(HL8) afford the

complexes [Pt(Ln)(MeCN)2]ClO4 (1a and 1b) in good yield. The products have all been

fully characterized by elemental analysis, IR, and 1HNMR spectroscopy. The detailed

structure of ligand HL7 is characterized by single crystal X-ray crystallography.

Furthermore, we present a method to determine the mode of interaction of 1a with calf

thymus DNA (CT-DNA) using absorption, emission spectral techniques and also to find

out the binding constant Kb and the linear Stern–Volmer quenching constant KSV. The

results indicate that 1a strongly interacts with CT-DNA in intercalative binding mode.

196

VI.1 Introduction

Metal chelates of conjugated ligands are of abiding interest in inorganic research,

especially those bearing π-acidic ligands to promote properties like successive electron

transfer, intense color, biologically active and oxidation state of ambivalence. The

number of heteroatoms, the ring size, and the substituent in the heterocycle ring

significantly modify the π-acidity and regulate the physical, chemical and biological

properties of the complexes [1,2]. The coordination of a ligand to a positively charged

metal centre might enhance the reactions of nucleophiles with the ligand and thus form a

fascinating area in metal-assisted organic molecule synthesis [3-6]. Metal complexes with

aryl-azo ligands undergo different organic transformations at the pendant aryl ring such as

hydroxylation [4,7], thiolation [3] and C-N coupling reaction with aromatic amines [5,6].

Not only in case of chemical transformations, but also it can provide an intrinsic step to

synthesize bio-active molecules.

The anticancer properties of cis-Pt(NH3)2Cl2 [8–16] gave an impetus to the

research in the field of platinum metal chemistry. This activity is due to the cis-PtCl2

fragment that binds with DNA bases. However, the drug also interacts with non-cancer

cells, mainly with molecules having thiol groups. This causes nephrotoxicity. The

anticancer properties of biologically important platinum (II) complex has aroused interest

in the design of bio-active complexes of platinum (II) of better activity and lower toxicity.

Mono-dentate ligands can bind in both cis- and trans arrangements around the metal and

the stability of the isomers depend on several factors. Consequently, bidentate ligands are

more reliable for the preparation of cis-Pt(II)-complexes as bi-dentate ligand can lock the

cis-positions and other sites can occupy by solvent molecules or halide ions, in particular

with platinum(II) [17–24]. The reaction of DNA bases with platinum (II) complexes of

chelating N,N’–donors having cis-MCl2 configuration constitutes a model system which

may allow for exploration of the mechanism of the anti-tumor activity of cis-platin. We

are intending to incorporate higher steric crowding around the target metal centre by

using different ligands with azoamine chelating mode (–N=N–C-NH–), which will open

an avenue to find out mechanistic aspect of nucleophilic interaction with the metal centre

under different local environments.

197

In the present work, we introduce the azo-amine system in the form of N,N’-donor

bis chelates of platinum. The synthetic method is described along with X-ray structure of

one pyrrol-azo ligands such as (4-chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7) and (4-

nitro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL8) (Scheme 1). Herein we report two novel

distorted square planar platinum (II) complexes with the bidentate ligand having one

deprotonated amine-N, one azo-N donors and two MeCN solvent molecules with general

molecular formula [Pt(Ln)(MeCN)2](ClO4) where n=7, 8. The synthesis and spectral

characterization (FTIR, UV-vis, ESI-Mass and 1HNMR) of the complexes has been

described. The intercalative mode of binding interactions of these complexes with calf

thymus-DNA (CT-DNA) have been investigated systematically to explore the biological

activity of the new complexes as we know through pharmacological mechanistic studies

that the effectiveness depends on the intercalative binding to DNA.

VI.2 Experimental Section

VI.2.1 Solvents and Reagents

K2PtCl4 and PdCl2 were purchased from Arrora Mathey, Calcutta, India. Pyrrol

azo ligands were synthesized according to the published procedure[25]. Pyrrol , 4-chloro-

aniline and 4-nitro-aniline were obtained from Aldrich and recrystallized further from the

suitable solvent. Sodium perchlorate (NaClO4) was prepared and recrystallized by a

previously reported method [26]. Nitrogen gas was purified by successive bubbling

through alkaline pyrogallol solution and concentrated sulfuric acid. Commercial grade

SRL silica gel (60–120 mesh) was used for column chromatography. All other chemicals

and solvents used in preparative work were of reagent grade and were used as received.

VI.2.2 Physical Measurements

Microanalytical data (C, H, N) were collected using a Perkin–Elmer 2400

elemental analyzer. Spectroscopic data were obtained using the following instruments:

UV–Vis spectra, JASCO UV–Vis/NIR spectrophotometer model V-570; IR spectra (KBr

disk, 4000–200 cm-1), Perkin-Elmer FTIR model RX1 spectrometer; Bruker AC300

spectrometer using TMS as an internal standard in DMSO-d6 solvent. The fluorescence

spectra of complex bound to DNA were obtained at an excitation wavelength of 522 nm

in the Fluorimeter (Hitachi-4500). Electrochemical setup was a three-electrode cell with

198

glassy carbon, Ag/AgCl and a platinum wire as a working, reference and counter

electrode, respectively. Molar conductances (ΛM) were measured in a systronics

conductivity meter 304 model in dimethylformamide at complex concentration of ~10-3

mol L-1.

VI.2.3 X-Ray Crystal Structure Analysis

Single crystals suitable for X-ray diffraction study of HL7 were grown by slow

evaporation of a methanolic solution at ambient temperature. Diffraction data collection

of HL7 was carried out at 150(2) on an Oxford Diffraction Xcalibur CCD system using

MoKα radiation(λ = 0.71073 Å). Cell refinement, indexing, and scaling of the data-set

were performed using CrysAlis [27], Denzo, and Scalepack [28]. The structure was

solved by direct methods and refined by full-matrix least-squares based on F2 with all

observed reflections [29]. Hydrogens were fixed at geometrical positions. All calculations

were performed using the WinGX system, Ver 1.80.05 [30].Crystal data and details of

refinement are reported in Table VI.1. Crystallographic data for have been deposited with

the Cambridge Crystallographic Data Centre, CCDC No. 885460 for HL7.

VI.2.4 Syntheses of Ligands

The syntheses of the ligands were carried out following the common procedure of

coupling pyrrole with the diazotized different para-substituted aniline (Scheme 1). The

procedure for the preparation of (4-chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7) is here

described in detail, being the other ligand obtained as solid products in the same way.

VI.2.4.1 Synthesis of (4-Chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7)

A diazotized solution of para-chloro aniline (2.55 g, 20 mmol) was added

dropwise to an alkaline solution of pyrrol (1.4g, 20 mmol) with continuous stirring at

0°C-5°C. The temperature was controlled at ice-cold conditions. pH of the solution was

maintained in moderate alkaline (7–9) range by adding Na2CO3 solution dropwise if

necessary. The red colored compound so precipitated was filtered. Finally the compound

was purified from mixture of solvent dichloromethane and n-hexane (1:1). A brown

crystalline compound of HL7 was obtained from the red colored solution through slow

199

evaporation of the solvent. Single crystals of HL7 suitable for X-ray crystallography were

obtained from a hexane-toluene solvent mixture of the compound on slow evaporation at

room temperature. Synthesis of (4-nitro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL8) was

carried out following the similar method described above taking 4-nitro aniline in place

of 4-chloro aniline.

VI.2.5 Syntheses of Platinum (II) Complexes

VI.2.5.1 Synthesis of [Pt(L7)(MeCN)2]ClO4 (1a)

To a nitrogen flushed solution of K2PtCl4 (0.415 g, 1 mmol) in water (10 ml)

under refluxing condition was added HL7 (0.206 g, 1.0 mmol) and triethylamine mixture

in MeCN (15 ml). The reaction was continued for 6 h and cooled to room temperature

(r.t.). The brown–red solution was evaporated in a steam bath and the solution was

reduced to half of its original volume. Then the solution was added to aqueous

NaClO4(0.140 g, 1mmol). The dark precipitate was filtered and washed with hot water

and finally with cold MeCN–H2O (1:1; v:v). A deep orange–red band eluted using C6H6–

MeCN (3:1; v:v). Microcrystals were obtained by complete evaporation of the eluted

solution at room temperature, which were not suitable for X-ray diffraction.

VI.2.5.2 Synthesis of [Pt(L8)(MeCN)2]ClO4 (1b)

To the acetonitrilic solution of organic moiety HL8 (0.216g, 1.0 mmol) followed

by triethylamine, a solution of K2PtCl4 (0.415 g, 1 mmol) in MeCN-H2O (20 mL)

previously purged with N2 was added dropwise and the resulting solution was refluxed

for ∼ 6 h under N2. The solvent of the resulting solution was reduced to 1/3rd of the total

volume, and water solution of NaClO4 (0.140 g, 1mmol) was added dropwise in stirring

condition. The resulting mixture was stirred for ∼ 2 h and then by filtering a red solid

mass was obtained. The product was dissolved in the minimum amount of

dichloromethane and pure platinum complex was obtained by chromatographic separation

over a silica gel column using C6H6–MeCN (3:2; v:v) as eluant. Microcrystals were

obtained by complete evaporation of the eluted solution at room temperature, which were

not suitable for X-ray diffraction.

200

Table VI.1. Crystallographic data for the ligand HL7.

Empirical Formula C10H8N3Cl


Crystal system Triclinic

Space group P 21/c

a (Å) 7.6867(4)

b (Å) 9.9521(6)

c (Å) 12.7722(6)

β, deg 89.479(4)

Volume (Å3) 947.51(9)

Temperature (K) 2461.3(15)

Z 4


F(000) 424

θ range (deg) 3.07 - 25.00

Refl. collected / unique 20544 / 6672

R(int) 0.0544




R1, wR2 (I >2σ(I)) [a] R1 = 0.0330, wR2 =0.0592

R indices (all data) R1 = 0.0614, wR2 = 0.0630

aR = ∑||Fo |- |Fc||/∑|Fo|;

bwR2 = { ∑ [w(Fo

2 –Fc2)2] / ∑w [(Fo

2)2]}1/2

201

VI.3 Results and Discussions

VI.3.1 Syntheses and Characterizations

The organic moieties LnH (n=1, 2) were prepared by coupling pyrrol with the

diazonium ion obtained from para-chloroaniline and para-nitroaniline respectively.

These organic moieties act as bidentate N,N donor ligands which are interesting chelators

due to the presence of hard donor azo-N and borderline base pyrrol-N centres. The ligand

has been characterized by spectroscopic tools and the X-ray diffraction studies.

The complexes 1a and 1b were obtained in good yield from the reaction of the

K2PtCl4 with equimolar amount of the organic moiety (HL7, HL8) followed by

trithylamine in acetonitrile medium in refluxing condition under dinitrogen (N2)

atmosphere followed by the addition of water solution of sodium perchlorate to the

reaction mixture at cold condition (viz. Scheme 1 and Table VI.2).

Scheme 1. Synthetic procedures of complexes 1a and 1b.

NH

N

N

R

K2MCl4

N N

N

R

MNCMe

MeCN

ClO4

HL7, R = Cl

HL8, R = NO2

+

NEt3, MeCN,

Reflux

Under N2, 4hFiltrate

M = Pt, R = Cl, 1a

M = Pt, R = NO2, 1b

NaClO4

Stir for 30mins

202

Table VI.2. Platinum (II) complexes with azo ligands.

Microanalytical data (Table VI.3) confirm the composition of the complexes and

spectroscopic data of 1a and 1b indicate the organic moieties, para-chloro aniline, para-

nitro aniline in the reaction medium are converted to (4-Chloro-phenyl)-(1H-pyrrol-2-yl)-

diazene(HL7) and (4-Nitro-phenyl)-(1H-pyrrol-2-yl)-diazene(HL8) respectively, and their

platinum (II) complexes 1a and 1b.

Table VI.3. Microanalytical data of the platinum (II) complexes (1a and 1b).


The complexes 1a and 1b are soluble in DCM, acetonitrile, methanol, and DMF.

The conductivity measurements indicate that both the platinum(II) complex species are

1:1 electrolytes in methanolic solution.

VI.3.2 Structural description of ligand HL7

Single crystals of HL7 were obtained from a hexane–toluene solvent mixture of

the compound on slow evaporation at room temperature. The crystal structure of the

ligand is shown in Figure VI.1 together with the atom labeling scheme for all non-H

atoms, and a selection of bond lengths and angles appear in Table VI.4. The

HLn Complex

(4-Chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7) [Pt(L7)(MeCN)2]ClO4 (1a)

(4-Nitro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL8) [Pt(L8)(MeCN)2]ClO4 (1b)


(Λo)a C H N

[Pt(L7)(MeCN)2]ClO4 (1a) 28.93(28.90) 2.25(2.23) 12.05(12.00) 92

[Pt(L8)(MeCN)2]ClO4 (1b) 28.41(28.37) 2.21(2.18) 14.20(14.18) 105

203

crystallographic independent unit comprises molecules A and B, which are comparable

with respect to bond distances and angles, as indicated in Table VI.4 where values of A

and B agree within their esd’s. The molecules are arranged about a pseudo inversion

center, but oriented so that their mean planes form a dihedral angle of 53.1°. This

disposition allows the double H-bond interactions N–H…N of 3.012 and 3.074Å. Both

molecules present coplanar atoms, indicating an electron delocalization inside each

molecule. The crystal packing (Figure VI.2) shows A parallel aligned in a head-tail

arrangement (being referred by a crystallographic center of symmetry) to form weak π–π

interactions between the phenyl and pyrrole ring of the adjacent symmetry related

molecule.

.

Figure VI.1. ORTEP drawing (50% ellipsoid probability) of the two independent

molecules of HL7.

Figure VI.2. Crystal packing viewed down axis c (dark spheres indicates molecules B).

204

Table VI.4. Selected bond lengths (Ǻ) and bond angles (°) of HL7.

Bond length (Å)

C(1) - N(7) 1.435(2) C(9) - N(10) 1.377(2)

N(7) - N(8) 1.281(2) C(9) - C (13) 1.388(3)

N(8) - C(9) 1.382(2) N(10) - C(11) 1.356(2)

Bond angle (o)

C(2)-C(1)-N(7) 125.51(17) N(10)-C(11)-C(12) 109.10(18)

C(6)-C(1)-N(7) 115.54(17) C(11)-C(12)-C(13) 107.27(17)

N(8)-N(7)-C(1) 113.61(16) N(10)-C(9)-C(13) 107.48(17)

N(7)-N(8)-C(9) 113.86(17) N(8)-C(9)-C(13) 125.92(18)

N(10)-C(9)-N(8) 126.60(18) C(11)-N(10)-C(9) 108.79(17)

Monoazopyrrole HL7 has three forms within a unit cell: one shows no hydrogen

bonding while A and B are both intermolecularly hydrogen bonded to each other between

the nitrogen of pyrrole and the nitrogen of an azo bond with N-H…N bond distances of

3.020Å (N10a-N7) and 3.087Å (N10-N7a). Intramolecular hydrogen bonding between N-

10 of pyrrole and a nitrogen of the azo bond for the ligand as the distances between the

N10 proton of pyrrole and the nitrogen of the azo bond [HL7 : 2.592 Å] are slightly

smaller than the sum of the van derWaals radii of hydrogen (1.20Å) and nitrogen (1.50Å)

atoms[31]. Surprisingly, the N-10 proton of the ligand is rotated away from the nitrogen

of the azo bond, since the H-N10a-C9a angle (127.7Å) is larger than the H-N10a-C11a

angle (122.8Å). The structure of the ligand is nearly planar, where the dihedral angle

between the pyrrole and phenyl planes is only 5.36Å. As shown by side-view drawings in

Figure VI.1. Significantly, complexation improves the planarity of azopyrrole molecules.

The mean deviation of carbon and nitrogen atoms from the π-delocalized skeletons

containing azo, phenyl, and pyrrole groups is 0.047Å in the ligand. All configurations for

N=N-C(pyrrole) are in a trans-cis form. HL7 exhibits a N=N bond distance of 1.28Å,

which is longer than those of simple monoazo dyes with thiophene (1.226Å)[32] and

longer than expected due to lack of π-resonance effects (1.271Å)[33] whereas

monoazothiophenes with electron-withdrawing groups for N=N (1.297-1.314Å)[32].

205

VI.3.3 Spectroscopic analysis

VI.3.3.1 IR Spectra

The IR spectra of the complexes are compared with the free ligands in order to

confirm the ligand coordination to the metal, and for this there are some reference peaks

that are of good help for achieving this goal. The IR spectrum of the free ligand, HL7

showed a characteristic band at ∼ 3198 cm-1 for νN-H, which is absent in the spectra of the

platinum (II) complex 1a, and that for HL8 at ∼ 3200 cm-1 is also absent in the spectra of

1b. The νN=N band in the free ligand, HL7 is 1483 cm-1 and it is shifted to lower wave

numbers in the complex 1a (1477cm-1) (viz. Figure VI.3), suggesting coordination to the

metal ion.

Figure VI.3. FTIR spectrum of complex 1a in KBr disk.

The similar result obtained for 1b. The characteristic bands at 1,380–1,385 cm-1 is

assigned to ν(N=N) of the coordinated azo ligands (HL7/HL8) in the complexes. As we

know, in going from –N=N–(σ2π2) to (σ2π2 π*1), the bond order decreases from 2 to 1.5;

therefore, it was observed that the vibrational frequency of ν(N=N) decreases

significantly from the free ligand value (1,420–1,425cm-1). Here the back-bonding from

206

Pt(dπ) to azo(π*) orbitals is expected to become unimportant upon azo anion radical

formation. The complexes [Pt(Ln)(MeCN)2]ClO4 show sharp absorption bands around

1090 cm-1 and 626 cm-1 assignable to ν(ClO4-) which are absent for the organic moieties.

Table VI.5. IR spectral data of the ligands and platinum (II) complexes.

VI.3.3.2 1HNMR spectra

The 1HNMR spectra of the organic moieties and the [Pt(Ln)(MeCN)2]ClO4

complexes were recorded in CDCl3. The characteristic signals for the protons of HL7 (viz.

Figure VI.4) and HL8 appeared in the spectra as usual fashion in support of the proposed

structural formulae. In the 1HNMR spectra of the complexes the protons appeared at

characteristic δ values and these values are in accordance with the proposed structure. It

was observed that characteristic signals for the protons of the complexes appeared at

higher δ values with respect to the free organic moieties. The NH-pyrrole signal appeared

at δ = ~9.25 ppm which is absent in the 1HNMR spectra of the complexes.

Compound ννννN-H ννννN=N ν(ClO4-)

(4-Chloro-phenyl)-(1H-pyrrol-2-yl)-

diazene (HL7) 3198 1380,1483 _

(4-Nitro-phenyl)-(1H-pyrrol-2-yl)-

diazene (HL8) 3195 1382,1485 _

[Pt(L7)(MeCN)2]ClO4 (1a) - 1477 1089,623

[Pt(L8)(MeCN)2]ClO4 (1b) - 1472 1091,626

207

Figure VI.4. 1HNMR spectrum of HL7 in CDCl3 with respect to TMS.

VI.3.4 DNA binding experiments

VI.3.4.1 Absorption spectral study

The binding mode of platinum (II) complex 1a with calf thymus DNA was

examined by electronic absorption titration with CT-DNA. As shown in the Figure VI.5,

the spectra indicate a significant hyperchromism effect centered around 298 nm

absorption maximum, suggesting that there is a strong interaction between the platinum

(II) complex and DNA. The spectral change might be interpreted as due to the

intercalative binding nature of the adducts, [34] since platinum (II) complexes containing

azo-pyrrole systems (organic ligand), which likely facilitates the formation of van der

Waals contacts or hydrogen bonds during interaction with DNA helix. From these

titration data the intrinsic binding constants (Kb) of the complexes with CT-DNA has

been determined using the following equation, [35,36]

[DNA]/(εa-εf) = [DNA]/(εb-εf) + 1/[kb(εb-εf )]

Where [DNA] represents the DNA concentration, εf and εb are the extinction

coefficients for the free and fully bound platinum (II) complex, respectively, and εa the

metal complex extinction coefficient during each addition of DNA. The [DNA]/(εa - εf)

plot against [DNA] gave a linear relationship (Figure VI.6). The intrinsic binding

208

300 450 600

0.25

0.50

0.75

f

a

Ab

so

rb

an

ce

Wavelength(nm)

constants (Kb) for the complex 1a was calculated from the slope to intercept ratio (Kb =

0.79 × 105 M-1,R = 0. 0.9679 for five points). The value is in close agreement with those

of the well-established intercalative binding rather than groove binding agent [37].

Figure VI.5. Electronic spectral titration of complex 1a with CT-DNA at 267 nm

in Tris-HCl buffer; [Compound] = 1.12 x 10-4; [DNA]: (a) 0.0, (b) 1.25 x 10-6 , (c) 2.50

x 10-6 ,(d) 3.75 x 10-6, (e) 5.00 x 10-6 , (f) 6.25 x 10-6mol.L-1. Arrow indicates the

increase of DNA concentration.

Figure VI.6. Plot of [DNA]/(εa–εf) versus [DNA] for the absorption of CT-DNA

with complex 1a in tris-HCl buffer.

VI.3.4.2 Fluorescence displacement experiments

Ethidium bromide (EB) fluorescence displacement experiments were also

performed in order to investigate the interaction mode of the complex with CT-DNA. In

209

fact the EB fluorescence intensity (at λex = 522 nm) will be enhanced in presence of DNA

because of its intercalation into the helix, and it was quenched by the addition of another

molecule that displaces EB from DNA [38]. Here, the significant decreases of the

fluorescence intensity of EB bound to DNA at around 610 nm were recorded by

increasing the concentration of the complex 1a as shown in Figure VI.7. The observation

of EB fluorescence quenching due to the releasing of some EB molecules from the EB-

DNA system is supportive to the interaction of the platinum (II) complex with CT-DNA

through the intercalative binding mode. Here the quenching of EB bound to DNA by the

platinum (II) complexes is in agreement with the linear stern–Volmer equation [39]:

I0/I =1+ KSV [complex]


quencher, respectively. KSV is the linear stern–Volmer quenching constant and [complex]

the molar concentration of the quencher. From the slope of the regression line in the

derived plot of I0/I vs [complex] (Figure VI.8), the KSV values for the complex was

determined. This was found to be 0.64 × 104 for 1a (R = 0.99983 for five points),

indicating a strong affinity of platinum (II) complexes to CT-DNA.

Figure VI.7. Emission spectra of the CT-DNA-EB system in tris–HCl buffer upon the

titration of the compound complex 1a. Kex = 522 nm; [EB] =0.96 x 10-4 molL-1;

[DNA] = 9.9 x 10-6 molL-1; [Compound]: (a) 0.0, (b) 1.36 x 10-5, (c) 2.72 x 10-5,

(d) 4.08 x 10-5, (e) 5.44 x 10-5, (f) 6.80 x 10-5 mol.L-1. Arrow indicates the

increase of compound concentration.

560 580 600 620 640 660 680 7000

250

500

750

1000

f

a

Flu

orescen

ceIn

ten

sit

y(a

.u.)

Wave length(nm)

210

Io/I

Figure VI.8. Plot of I0/I versus complex 1a for the titration of CT-DNA–EB system

with complex 1a using spectrofluorimeter; linear Stern–Volmer quenching constant

(Ksv) for complex 1a = 0.64 x 104; (R = 0.98987 for five points).

VI.3.4.3 Number of binding site calculation

Flurorescence quenching data were used to determine the binding sites (n) for

the complex 1a with CT-DNA. Figure VI.7 shows the fluroscence spectra of EB-DNA in

the presence of different concentrations of complex 1a. It can be seen that the fluroscence

intensity at 612 nm was used to estimate Ksv and n.




nQ + B → Qn….B (3)



K = [Qn….B]/[Q]n.[B] (4)

If the overall amount of biomolecules (bound or unbound with the quencher) is

Bo, then [Bo] = [Qn…B]+ [B], where [B] is the concentration of unbound biomolecules,

and the relationship between the fluorescence intensity and the unbound biomolecule as

[B]/[Bo] = I/Io , that is:


Where (n) is the number of binding site of compound complex 1a with CT-DNA, which


211

VI.9. The calculated value of the number of binding sites (n) is 1.08 (R= 0.98763 for five

points). The value of (n) approximately equals 1, and thus indicates the existence of one

binding site in DNA for complex 1a.

Figure VI.9. The linear plot shows log[(Io-I)/I] versus log[Q], where R = 0.98763 for

five points.

VI.4 Epilogue

Two new square planar platinum(II) complexes of two pyrrolyl azo dyes (4-

Chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7) and (4-Nitro-phenyl)-(1H-pyrrol-2-yl)-

diazene (HL8) have been synthesized and characterized by means of solid and solution

phase spectroscopic studies including the X-ray structure of ligand HL7. Further, binding

interaction study of complex 1a with calf-thymus DNA clearly indicates that the mode of

interaction is intercalative binding with a significant value of the binding constant. The

present work is a viable model study to understand the conditions which can be optimized

to carefully analyze the interaction of the pyrrolyl azo compounds with DNA for later

application in therapeutic drug.

212

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i

Synopsis

ii

SYNOPSISSYNOPSISSYNOPSISSYNOPSIS

During the last decade, medicinal applications of metal complexes have

experienced rapid growth throughout the world with the advent of science. Among all,

the platinum metal complexes containing N, S and O donor sets are of great importance

for their activity in biological as well as chemical aspect. Considering the increasing

demand of this field, a program has been taken to study the reactivity of some platinum

metals with some newly designed or already known organic moieties having N,S, O

donor sites. Herein, the new complexes of platinum metals having the hard (amidato-N),

borderline (pyridine-N) and soft (amine-N, azo-N, pyrrol-N and dithiocarbamate-S)

donor ligands have been synthesized in facile route of high yield. These newly designed

complexes are described here in detail. The complexes have been characterized using

physicochemical (viz. elemental analyses, conductance measurement) and spectroscopic

(viz. IR, 1HNMR spectra, UV-Vis) tools along with the detailed structural analyses using

single crystal X-ray difractometer in most of the cases. The systematic spectroscopic and

physico-chemico study of the interaction of some selective complexes with calf thymus-

DNA (CT-DNA) and bovine serum albumin (BSA) in tris–HCl buffer medium of

biological pH have been carried out to explore the biological acitivity of the resulting

complexes. The electrochemical study using cyclic voltammetry (CV) has also been

performed in search of the chemical reactivity of the complexes.

The thesis entitled “Coordination Chemistry of Some Platinum Metals with N, S, O

Donor Ligands : Synthesis, Characterization and Reactivity”, divided into six chapters.

Chapter I (Introduction) highlights brief overview of the platinum metals (Ru, Os, Rh, Ir,

Pd and Pt), i.e., occurrence, general properties, coordination behavior of their complexes,

characteristic feature of different oxidation states along with known chelate chemistry of

platinum metals having dithiocarbamate, carboxamide and azo ligands i.e. N, S, O donor

chelators. It includes also the scope of the present work which stems from the following

facts: (i) the coordination chemistry of platinum metal complexes involving N, S, O

donation sites has been the cynosure among the chemists due to the application of these

chelates in the field of chemical and biological reactivity and also in medicine, (ii) the

potential role played by platinum metal ions being present in the active sites of a large

iii

number of metalloproteins having the MN2S2 chromophore, has stimulated efforts to

design of new ligand frames having N, S donor sets and characterize platinum metal

complexes as models for providing a better understanding of the biological system, (iii)

from literature it is revealed that it’s a challenge for the researchers to design and

synthesize of appropriate ligand frame having N, S, O donor sets and to explore their

depth in complexation behavior with platinum metal ion. Each of these goals has been

dealt with in the subsequent chapters.

Chapter II describes the synthesis, spectral, structural characterization of a family

of four-coordinated palladium(II) complexes of 4-methylpiperazine-l-carbodithioic acid

(4-MePipzcdtH=L1H), morpholine-4-carbodithioic acid (MorphcdtH=L2H), and 4-

benzylpiperidine-l-carbodithioic acid (4-BzPipercdtH=L3H) along with the reactivity of

complexes. Single-crystal X-ray structure analysis of one of the complexes, [Pd(L1)2]

.3H2O(1) (Figure 1a) reveals that the Pd(II) complexes (1-4) are square planar and

monomeric in nature, and shows a 1D polymeric zig-zag chain built by H-bonds running

along axis c (Figure 2). On reaction with catechol as water scavenger at room temperature

in ethanol, the complex 1 was successfully converted to the Pd(II) complex formulated as

[Pd(L1)2] (4) (Figure 1b) viz. Scheme 1.

Scheme 1. Synthetic pathway of complexes 1 and 4.

These complexes were characterized by FT-IR, 1H NMR and X-ray diffraction.

Here monomeric 1 complex is soluble in DMF and DMSO, but sparingly soluble in

[Pd(L1)2](Catechol+Et3N)+PdCl2+L1PdCl2+L1+(Catechol+Et3N)

Catechol+Et3N EtOH

L1+PdCl2

(4)

[Pd(L1)2].3H2O

(1)

Path-a

Path-b

EtOH EtOH

Path-c

EtOH

iv

acetonitrile and methanol, while 4 is interestingly soluble in polar organic solvents like

acetonitrile, methanol, DMF and DMSO. The conductivity measurement of complexes 1-

4 in DMF show such conductance values suggesting that all the four complexes exist as

non electrolytes in solution.

(a) (b)

Figure 1. An ORTEP drawing (35 % probability ellipsoid) of [Pd(4-MePipzcdt)2].3H2O

(1) and [Pd(4-MePipzcdt)2] (4) with atom labeling scheme.

Figure 2. The 1D polymeric zig-zag chain of the complex 1 built by H-bonds running

along axis c.

Infrared spectra of all the complexes 1-4 exhibit an intense band at ca. 1491-1508

cm-1 assignable to νC=N (1497 cm-1, 1492 cm-1, 1508 cm-1 and 1491 cm-1 for 1, 2, 3 and 4

respectively). In IR spectra of all the complexes, the bands at 992-1010 cm-1 (992 cm-1,

1007 cm-1, 1010 cm-1 and 997 cm-1 for 1, 2, 3 and 4 respectively) attributable to νSCS(s)

were observed. On the other hand, the bands at 1000-1034 cm-1 assignable to νSCS(as)

(1000 cm-1, 1025 cm-1, 1027 cm-1 and 1034 cm-1 for 1, 2, 3 and 4 respectively) suggest

v

the unsymmetrical chelating bidentate mode of the metal ion coordination. In addition to

these, the characteristic band at 436-440 cm-1 in the four complexes corresponds to the

νPd-S stretching frequency.

In the electronic spectra of complexes, a sharp band around 305 nm along with a

shoulder at ca. 340 nm assignable to the intramolecular π → π* and n → π* transitions,

respectively. The availability of the electrons on the donor atoms for the electronic

transition increases in the order 2 < 1 < 3, thereby increasing the molar extinction

coefficient values.

The interaction of the four palladium (II) complexes with calf-thymus DNA (CT-

DNA) have been investigated by using UV-visible absorption and fluorescence emission

spectroscopy (viz. Table 1). In recent years platiunum metals containing complexes have

received considerable interest in nucleic acid chemistry. Since such systems are relevant

to the models for both protein-DNA and antitumor agent-DNA interactions. Therefore, it

is important to rationalize how such compounds with peptides affect the DNA binding.

The binding constant Kb in the range 0.32-1.54 × 105M-1. and the Stern-Volmer

quenching constant Ksv in the range 0.39-0.87 × 104 M-1 determined from the DNA

binding experiments of the compounds strongly corroborate their intercalation to the

DNA helix. The linear Stern–Volmer quenching constant Ksv and the binding sites n of

the complexes to CT-DNA have been also determined from the ethidium bromide

fluorescence displacement experiments, suggesting a good affinity of the complexes to

CT-DNA.

Table 1. Complete analytical data for the DNA binding study of 1, 2, 3 and 4.

Compound Kb (M-1) KSV No. of Binding sites

(n)

1 0.89 x105 0.39 x104 1.19

2 0.32 x105 0.87 x104 1.10

3 1.54 x105 0.82 x104 1.25

4 0.86 x105 0.68 x104 1.18

vi

This work is a viable model study to understand the conditions which can be

optimized to carefully analyze the interaction of the said compounds with DNA for later

application in therapeutic drug.

Chapter III concerns the synthesis of the square planar palladium(II) and

platinum(II) complexes of deprotonated carboxamide organic moieties and

characterization. The present contribution aims to study to explore the amide functionality

of the N,N'-bis(2-pyridinecarboxamide)-1,2-benzene ligand (H2L

4), N,N'-bis(2-

pyridinecarboxamide)-2,3-pyridine ligand (H2L5), N,N

'-bis(2-pyridinecarboxamide)-5-

bromo-2,3-pyridine ligand (H2L6) towards palladium (II) and platinum (II) ion.

Three palladium(II) and one platinum(II) complexes were obtained in good yield

from the reaction of PdCl2 and K2PtCl4, with equimolar amounts of the organic moiety in

the DMF medium (Scheme 2). The organic moiety (L) acts as a tetradentate neutral

ligand with N4 donor centers in these complexes. All the complexes obtained in this

study have been characterized by several physico-chemical and spectroscopic tools along

with single crystal X-ray crystallographic studies. The X-ray structural characterization

revealed that the palladium (II) and platinum (II) derivatives are isomorphous (Figure 3)

and are packed to form a trimeric motif with complexes connected by π-π interactions

between the aromatic rings of the ligands and metallophilic bonding.

Infrared spectra of all the complexes 5-8 exhibit an intense band at ca. 1600-1635

cm-1 assignable to νC=O, has shifted to lower frequency than the free ligand due to metal

ligand chelating effect. In the IR spectrum of 5-8 the absence of N-H strech absorption

clearly indicates that the ligand H2L4-H2L

6 is coordinated to Pd(II) or Pt(II) ion in the

deprotonated form. In addition, the νM-N band is observed in the range of 415- 440 cm-1

for all the complexes suggesting metal ligand coordination.

In the electronic spectra of complexes, a sharp band around 280-284 nm

assignable to the intramolecular π → π* transition was observed. The spectra exhibit a

relative high intensity band around 308-317nm assignable to the charge transfer from

amide ligand core to metal, i.e. LMCT. The low energy tail of the charge transfer band

that appears in the visible region of the spectrum is responsible for the yellow and orange-

yellow color of the solution containing the complexes.

vii

In solution, the complex 5 displayed a quasi-reversible reductive voltammogram

having at E1/2 = –1.148 V (Epc = –1.245 V and Epa = –1.051 V; ∆E = 194 mV)

assignable to Pd(II)/Pd(I) couple. On the contrary, platinum (II) in 6 is irreversibly

oxidized to platinum (IV) by two electron stoichiometry centred at +0.977V. The

theoretical calculation using DFT of HOMO and LUMO energy of both complexes

supports the experimental findings towards the electrochemical properties.

Scheme 2. Synthetic pathway of complexes 5-8.

5 6

Figure 3. ORTEP diagram of complex 5 and 6.

The interaction of all the palladium (II) and platinum (II) complexes 5-8 with calf

thymus DNA (CT-DNA) has been investigated by using absorption, emission spectra and

X

N NO O

N N

Pd

N NO O

N N

Pt

PdCl2 in DMF

K2PtCl4

DMF-H2O, N2

X

NH HNO O

N N

H2L4

Stir, 3h

Stir, 12h

6.1/2H2O

Y Y

NH HNO O

N N

When X=CH, Y=H; H2L4

When X=N, Y=H; H2L5

When X=N, Y=Br; H2L6

When X=CH, Y=H; 5When X=N,Y=H; 7When X=N, Y=Br; 8

viii

also with viscosity measurement techniques. In the electronic absorption spectroscopy,

the binding modes of metal complexes with DNA are found to be strong intercalative

interactions between an aromatic chromophore of complexes and the base pairs of DNA.

The extent of the hypochromism in the charge transfer band is generally consistent with

the strength of intercalative binding/interaction. All the four complexes interact with

DNA strongly (for complexes 5-8: Kb = 0.36 x104 - 0.93 × 104 M–1). The Ksv value for all

the four complexes are in the range 0.14x104 - 0.44 x104, suggesting a strong affinity of

the complexes to CT-DNA. The results obtained in the viscosity measurement studies

suggest that all the compounds 5 to 8 can intercalate between adjacent DNA base pairs,

causing an extension of the helix with a concomitant increase of the DNA viscosity. The

binding mode of complexes 5 and 6 with bovine serum albumin (BSA) were examined by

electronic absorption titration and fluorescence quenching experiment with BSA. The

Kapp values obtained from the absorption study (1.262× 105 M-1 for 5 and 1.402× 105 M-1

for 6) and the Kq values resulted from fluorescence study(4.29× 104 for 5 and 4.62 × 104

for 6) are indicative of a strong affinity of both of the complexes to BSA.

Chapter IV and Chapter V deal with the cyclometalated rhodium (III) and

iridium(III) complexes of the dithiocarbamate ligands (Scheme 3) respectively and their

characterizations using various physicochemical and spectroscopic tools along with single

crystal X-ray studies. In addition, antibacterial activities of all the rhodium and iridium

complexes against some pathogenic bacteria were investigated along with their binding

interactions with DNA and BSA.

Scheme 3. Synthetic pathway of Rhodium and Iridium complexes.

DMSO-MeCN

X

N SH

S

LnH + [M(2-C6H4py)2Cl2]2

Reflux,10-12 h[M(2-C6H4py)2(Ln)]

n = 1, Complex 9/12n = 2, Complex 10/13n = 3, Complex 11/14

X= -N-CH3 , 4-MePipzcdtH (L1H)


X= -CH-CH2Ph , 4-BzPipercdtH(L3H) 2-C6H4py ppy

N

M=Rh/Ir

ix

From the X-ray structural analysis, the crystal structure of rhodium complex

[Rh(2-C6H4py)2(L1)] (9) (Figure 4) exhibits a distorted octahedron in which both of the 2-

phenylpyridyl nitrogens are in axial positions, trans to one another and the sulfur atoms

are opposite to the phenyl rings. Whereas, the crystal structure of iridium complex [Ir(2-

C6H4py)2(L2)] (13) (Figure 5) shows a distorted octahedron in which the nitrogen donor

of one 2-phenylpyridine and the carbon donor of another 2-phenylpyridine ligand are in

axial positions and trans to one another. Apparently all the rhodium and iridium

complexes are similar in crystalline motif, though there are some remarkable differences

in their X-ray bonding pattern.

Figure 4. ORTEP diagram of [Rh(2-C6H4py)2(L1)] (9)

Figure 5. ORTEP diagram of [Ir(2C6H4py)2(L2)].

x

The characteristic IR bands assignable to νC=N around 1485–1505 cm-1 for Rh

(1492 cm-1, 1485 cm-1 and 1505 cm-1 for 9, 10 and 11 respectively) and for Ir (1490 cm-1,

1474 cm-1 and 1499 cm-1 for 12, 13 and 14 respectively) shift to higher energies (ca.

1510-1465 cm-l) with respect to the free acids and these bands lie in between the

stretching frequencies expected for a double C=N (1610-1690 cm-l) and single C-N bond

(1250-1350 cm-1) and this gives support to the typical bidentate character [42] of the

carbodithioic acid ligands. The bands in the range of 695–704 cm-1 assignable to the

νSCS(s) (695 cm-1, 698 cm-1 and 704 cm-1 for 9, 10 and 11 respectively) and 1025–1082

cm-1 to νSCS(as) (1025 cm-1, 1036 cm-1 and 1082 cm-1 for 9, 10 and 11 respectively) for Rh

and at around 695–707 cm-1 (νSCS(s)) (695 cm-1, 699 cm-1 and 707 cm-1 for 12, 13 and 14

respectively) and 998–1035 cm-1(νSCS(as) (998cm-1, 1015 cm-1 and 1035 cm-1 for 12, 13

and 14 respectively) for Ir. The observed bands for all the suggest the unsymmetrical

chelating bidentate mode of coordination to both rhodium(III) and iridium (III) ions.

The electronic absorption spectra of the rhodium(III) and iridium (III) complexes

were recorded at room temperature using DMF as the solvent. The higher energy band at

360 nm for three rhodium(III) complexes and at 385-390 nm for three iridium(III)

complexes with high extinction coefficient values are due to the coordinated carbon atom

from pipyridine moiety, C(σ)-M(III) charge transfer (LMCT) transition. The other higher

energy intense transitions at 374 nm and 360 nm for rhodium and at 352 nm and 359 nm

for iridium are due to the n→π* and π→π* charge transfer transitions respectively.The

rhodium (III) and iridium (III) complexes exhibit the characteristic broad d-d absorption

band around 715nm in their solution electronic spectra.

Electrochemical activity of the complexes are examined by cyclic voltammetry

using a Pt-disk working electrode, Ag/Ag+ (non-aquous) reference electrode and a Pt-wire

auxiliary electrode in dry DMF and in presence of [n-Bu4N]ClO4 as supporting

electrolyte. In solution, all the rhodium (III) and also the iridium (III) complexes exhibit

an irreversible reductive response at E1/2 value ≈ -0.697 V to -0.767 V versus Ag/Ag+

(non-aqueous) corresponds to Rh3+/Rh+ couple and at E1/2 value ≈ -0.766 V to -0.810 V

(versus Ag/AgCl) corresponds to Ir3+/Ir+ couple. This observation suggests that the

electron donating capacity through σ bond of the six membered heterocyclic ring of the

dithiocarbamate ligands depends upon the availability of the electrons on the donor atoms

xi

of the ring. The trend of reduction potential followed as such, is further supported by

theoretical calculation obtained from DFT study.

The study of interaction of rhodium (III) complex [Rh(2-C6H4py)2(L1)] (9) and

iridium (III) complex [Ir(2-C6H4py)2(L2)] (13) with CT-DNA were investigated using

absorbance study, fluorescence quenching study and also viscosity measurement

technique. Kb for rhodium complex and iridium complex were estimated to be 1.54 x 105

M-1 and 0.31 x 105 M-1 respectively. The Ksv values for the rhodium complex and iridium

complex were obtained is 0.87 x 104 and 0.39 x 104 respectively. These shows that these

cyclometalated complexes having dithiocarbamate moieties are good intercalative binding

to with CT-DNA with an adequate number of coordination sites and this strongly binding

ability of the complexes as intercalator encourage to develop these materials as good

anticancer candidates. The binding study with bovine serum albumin (BSA) of the

iridium (III) complex [Ir(2-C6H4py)2(L2)] (13) was also indicative of the fact that, the

cyclometalated iridium (III) complex have a good affinity towards BSA and (Kapp)

determined from this study is 8.4388× 104 M-1 and KSV obtained as 5.31 × 105 .

The antibacterial activity of the dithicarbamic acids and their rhodium (III) (9-11)

and iridium (III) complexes (12-14) were investigated and against four pathogenic

bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and Bacillus

cereus) and compared with standard antibiotic, chloramphenicol at different

concentrations (viz. Figure 6).

Figure 6. Zone inhibition bar diagram of the LnH ligands and their corresponding

iridium(III) complexes.

0

5

10

15

20

25

30

E.C V.C S.P B.C

L1H

L2H

L3H

1

2

3

Chloramphenicol

DMF

xii

From this study (viz. Figure 6), it may be inferred that all the rhodium (III) and

iridium(III) complexes have higher activity than the ligand only, but little less efficient

than the antibiotics.

Chapter VI shows the synthesis and isolation of a new family of platinum(II)

complexes incorporating pyrrol azo ligands (viz. Scheme 4). This series of pyrrol azo

ligands (4-Chloro-phenyl)-(1H-pyrrol-2-yl)-diazene (HL7) and (4-Nitro-phenyl)-(1H-

pyrrol-2-yl)-diazene (HL8) containing nitrogen donor centers and their platinum (II)

complexes have been characterized by elemental analyses, spectroscopic and other

physico-chemical tools. One ligand (HL7) has been subjected to single-crystal X-ray

analysis (Figure 7). In the structure of the ligand, the crystal packing shows a parallel

alignment in a head-tail arrangement (being referred by a crystallographic center of

symmetry) to form weak π–π interactions between the phenyl and pyrrole ring of the

adjacent symmetry related molecule. The conductivity measurement of the complexes in

methanolic solution shows the conductance value in such a range revealing that the

complexes exist as the 1:1 electrolytic nature in the solution state.

Scheme 4. Synthetic procedures of azo complexes.

NH

N

N

R

K2MCl4

N N

N

R

MNCMe

MeCN

ClO4

HL7, R = Cl

HL8 , R = NO2

+

NEt3, MeCN,

Reflux

Under N2, 4hFiltrate

M = Pt, R = Cl, 15

M = Pt, R = NO2, 16

NaClO4

Stir for 30mins

xiii

Figure 7. ORTEP drawing (50% ellipsoid probability) of the two independent molecules

of HL7.

The infrared spectra of complexes 15 and 16 show characteristic sharp peaks at

1477 cm-1 and 1472 cm-1 respectively are assigned to νN=N of the coordinated azo ligands

(HL7/HL8) in the complexes. This band shifted to lower wave numbers in case of

complexes than the free ligand suggesting coordination to the metal ion. The

characteristic peaks at around 3200 cm-1 for νN-H in the ligand spectra is absent complexes

inferred the metal-ligand coordination. The complexes [Pt(Ln)(MeCN)2]ClO4 show sharp

absorption bands around 1090 cm-1 and 626 cm-1 assignable to ν(ClO4-) which are absent

for the organic moieties. The well-resolved 1H NMR spectra of the complexes clearly

indicate that the NH-pyrrole signal appeared at δ = ~9.25 ppm which is absent in the

1HNMR spectra of the complexes. The characteristic signals for the protons of the

complexes appeared at higher δ values with respect to the free organic moieties in support

of the proposed structural formulae.

The binding mode of platinum (II) complex 15 with calf thymus DNA was

examined by electronic absorption titration and fluorescence displacement experiments

with CT-DNA. The intrinsic binding constants (Kb) for the complex 15 was calculated

from the slope to intercept ratio (Kb = 0.79 × 105 M-1,R = 0. 0.9679 for five points) and

from the slope of the regression line in the derived plot of I0/I vs [complex, the KSV values

for the complex obtained was found to be 0.64 × 104 for 15 (R = 0.99983 for five points).

The calculated binding site also appeared as single binding. These values are in close

agreement with those of the well-established intercalative binding rather than groove

binding agent.

A

List of PublicationsList of PublicationsList of PublicationsList of Publications

A. Related to thesis work

1. Cyclometalated rhodium(III) complexes bearing dithiocarbamate derivative: Synthesis,

characterization, interaction with DNA and biological study.

T. Mukherjee, B. Sen, A. Patra, S. Banerjee, G. Hundal, P. Chattopadhyay,

Polyhedron, 2014, 69, 127–134.

2. Palladium(II) and platinum(II) complexes of deprotonated N,N’-bis(2-pyridinecarboxamide)-

1,2-benzene: Synthesis, structural characterization and binding interactions with DNA and BSA.

T. Mukherjee, B. Sen, E. Zangrando, G. Hundal, B. Chattopadhyay, P. Chattopadhyay,

Inorg. Chim. Acta, 2013, 406,176–183.

3. Palladium(II) complexes of dithiocarbamic acids: synthesis, characterization, crystal structure

and DNA binding study.

T. Mukherjee , S. Sarkar , J. Marek , E. Zangrando, P. Chattopadhyay,

Trans. Met. Chem, 2012, 37, 155–161.

4. Synthesis, characterization, interaction with DNA and bovine serum albumin (BSA), and

antibacterial activity of cyclometalated iridium(III) complexes containing dithiocarbamate

derivative.

T. Mukherjee, M. Mukherjee, S. Banerjee, G. Hundal, P. Chattopadhyay,

(Communicated to J. Coord. Chem, 2014)

5. N4 and S4 coordination to the palladium(II) centre: synthesis, structural characterization and

biological activity.

T. Mukherjee, G. Hundal, E. Zangrando, P. Chattopadhyay

(Under Preparation)

6. Synthesis of new pyrrolyl containing azo compounds of Pd(II)/Pt(II) : Spectroscopic

characterization and studies on DNA intercalation.

T. Mukherjee, E. Zangrando, P. Chattopadhyay

(Under Preparation)

B

B. Other works

7. Synthesis, characterization, crystal structure, and DNA-binding of ruthenium(II) complexes of

heterocyclic nitrogen ligands resulting from a benzimidazole-based quinazoline derivative.

H. Paul, T. Mukherjee, M.G.B. Drew, P. Chattopadhyay,

J. Coord. Chem, 2012, 65,1289–1302.

8. A water soluble Al3+ selective colorimetric and fluorescent turn-on chemosensor and its

application in living cell imaging.

S Sen, T. Mukherjee, B. Chattopadhyay, A.Moirangthem, A.Basu, J. Marek, P. Chattopadhyay,

Analyst, 2012, 137, 3975–3981.

9. Zinc(II) complexes of 1,3-bis(2-pyridylmethylthio)propane: Anion dependency, crystal

structure and DNA binding study.

A. Patra, S. Sarkar, T. Mukherjee, E. Zangrando , P. Chattopadhyay,

Polyhedron, 2011, 30, 2783–2789.

10. 5-Nitro-1,10-phenanthroline bis(N,N-dimethylformamide- K’O)-bis(perchlorato) copper(II):

synthesis, structural characterization,and DNA-binding study.

S. Dey, T. Mukherjee, S. Sarkar, H.S. Evans, P. Chattopadhyay,

Trans. Met. Chem., 2011, 36, 631–636.

11. Development of a highly selective cell-permeable ratiometric fluorescent chemosensor for

oxorhenium(V) ion.

S. Sen, T. Mukherjee, S. Sarkar, S. K. Mukhopadhyay, P. Chattopadhyay,

Analyst, 2011, 136, 4839-4845.

12. An oxamato bridged trinuclear copper(II) complex: Synthesis, crystal structure, reactivity,

DNA binding study and magnetic properties.

S. Dey, S. Sarkar, T. Mukherjee, B. Mondal, E. Zangrando, J.P. Sutter, P. Chattopadhyay,

Inorg. Chim. Acta, 2011, 376, 129–135.

13. Coordination behavior of pyridylmethylthioether system with cupric chloride and cupric

bromide: C-S bond cleavage and crystal structures.

S. Sarkar, S. Dey, T. Mukherjee, E. Zangrando, M. G.B. Drew, P. Chattopadhyay,

J. Mol. Struct, 2010, 980, 94–100.

C

14. Copper(II) complex of in situ formed 5-(2-pyridyl)-1,3,4-triazole through C–S bond cleavage

in 1,2-bis(2-pyridylmethylthio)-bis-ethylsulphide: Synthesis, structural characterization and

DNA binding study.

S. Sarkar, T. Mukherjee, S. Sen, E. Zangrando, P. Chattopadhyay,

J. Mol. Struct., 2010, 980, 117–123.

Attending Conferences

National

1.) International Year of Chemistry-2011 (IYC-2011), March 15-17, 2011.

(Participation) The University of Burdwan, Burdwan, India.

2.) Recent Advances in Chemical Science (RACS-2012), March 15-17, 2012.

(Participation) The University of Burdwan, Burdwan, India.

International

1.) Royal Society of Chemistry, Roadshow (RSC-Roadshow-2013), 5th

February, 2013.

(Participation) Indian Association for the Cultivation of Science (IACS), Kolkata, India.

2.) Poster entitled :

“Neutral Cyclometalated Rhodium(III) Complexes bearing Dithiocarbamate Derivative

:Synthesis, Structural Characterization and DNA Binding Study”

presented in Modern Trend in Inorganic Chemistry (MTIC-XV), December 13-16, 2013

organised by IIT-Roorkee, Uttarakhand, India.

Reprints

Palladium(II) complexes of dithiocarbamic acids: synthesis,characterization, crystal structure and DNA binding study

Titas Mukherjee • Sandipan Sarkar •

Jaromir Marek • Ennio Zangrando •

Pabitra Chattopadhyay

Received: 14 October 2011 /Accepted: 21 November 2011 / Published online: 16 December 2011

� Springer Science+Business Media B.V. 2011

Abstract A series of distorted square planar palla-

dium(II) complexes with dithiocarbamic acids of general

formula [Pd(L)2], where L = 4-methylpiperazine-l-carbo-

dithioic acid anion, morpholine-4-carbodithioic acid anion

or 4-benzylpiperidine-l-carbodithioic acid anion for com-

plexes 1a, 1b and 1c, respectively, have been synthesized.

The complexes were characterized by physicochemical and

spectroscopic methods; in addition, the structure of com-

plex 1a was characterized by single crystal X-ray crystal-

lography. The interaction of these palladium complexes

with CT-DNA was investigated with the help of absorption

and emission spectroscopy. The association constant Kb

was deduced from the absorption spectra, while the number

of binding sites and the binding constant were calculated

from the fluorescence quenching data. The results suggest

an intercalative interaction of the complexes with CT-

DNA.

Introduction

Transition metal complexes of various aliphatic, aromatic

and heterocyclic dithiocarbamate ligands have been widely

investigated because of their medicinal, industrial and

analytical applications [1–3]. Dithiocarbamate complexes

of platinum(II) and palladium(II), as well as of isoelec-

tronic gold(III), show remarkable antitumour properties,

and in some cases, their cytotoxic activity is superior to

that of cisplatin [4–6]. Previous studies have shown that

the presence of ligands with sulphur donor atoms appears

to be a prerequisite in conferring antitumour properties on

palladium(II) complexes [5]. Dithiocarbamates, which are

used as fungicides and pesticides [7], are of great interest

because they display cytotoxic properties and have also

been applied in the treatment of metal poisoning [2, 8–10].

The nature of the heterocycle attached to the dithiocarba-

mate fragment has a crucial effect on the electronic prop-

erties of these ligands and therefore on their potential

pharmacological attributes as well as the catalytic proper-

ties of their metal complexes [11].

Taking into account the above facts and our continuous

interest on the interaction of metal complexes of nitrogen/

sulphur ligands with calf thymus-DNA(CT-DNA) [12–14],

herein we report an account of the synthesis, structural

characterization and DNA binding properties of palla-

dium(II) complexes with three dithiocarbamates, namely

4-methylpiperazine-l-carbodithioic acid (4-MePipzcdtH),

morpholine-4-carbodithioic acid (MorphcdtH) and 4-ben-

zyl-piperidine-l-carbodithioic acid (4-BzPipercdtH). The

binding constants Kb of each of the three complexes with

DNA have been derived from UV–vis spectroscopy, and

the quenching constants Ksv have been determined from

fluorescence displacement experiments using ethidium

bromide.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11243-011-9569-9) contains supplementarymaterial, which is available to authorized users.

T. Mukherjee � S. Sarkar � P. Chattopadhyay (&)

Department of Chemistry, Burdwan University,

Golapbag, Burdwan 713104, India

e-mail: [email protected]

J. Marek

CEITEC MU, Faculty of Science, Masaryk University,

Kamenice 5/A2, 625 00 Brno, Czech Republic

E. Zangrando

Dipartimento di Scienze Chimiche e Farmaceutiche,

Via Licio Giorgieri 1, 34127 Trieste, Italy

123

Transition Met Chem (2012) 37:155–161

DOI 10.1007/s11243-011-9569-9

personal copy

Experimental

1-Methyl-piperazine (Fluka) was dried by refluxing over

sodium hydroxide beads and also stored over sodium

hydroxide. Morpholine and 4-benzylpiperidine, purchased

from Aldrich, and PdCl2 from Arora Matthey were used as

received. All other chemicals used were of analytical

reagent grade.

The elemental (C, H, N) analyses were performed on a

Perkin Elmer model 2400 elemental analyzer. Electronic

absorption and IR spectra were obtained on a JASCO

UV–Vis/NIR spectrophotometer model V-570 and on a

Perkin–Elmer FTIR model RX1 spectrometer (KBr discs,

4,000–300 cm-1), respectively. 1H NMR spectra were

recorded on a Bruker AC300 spectrometer using TMS as

an internal standard in DMSO-d6 solvent. Electrospray

ionization (ESI) mass spectra of the complexes were

recorded with a QtofMicro Instrument (Waters, YA263).

The fluorescence spectra of the adducts with DNA were

obtained at an excitation wavelength of 522 nm with a

Hitachi-2000 fluorimeter.

Preparation of the ligands

A common synthetic method was followed to obtain the

acidic form of the anionic ligands, 4-methylpiperazine-l-

carbodithioic acid anion (mpca) [15], morpholine-4-car-

bodithioic acid anion (mca) [11] and 4-benzylpiperidine-l-

carbodithioic acid anion (bpca) [16]. The procedure for the

preparation of 4-benzylpiperidine-l-carbodithioic acid (bpcaH)

is here described in detail; the other ligands were obtained

as solid products in the same way.

CS2 (4.00 g, 52.62 mmol) was slowly added to a cold

solution of 4-benzylpiperidine (8.75 g, 50 mmol) in EtOH

(15 ml) with vigorous stirring over 2.0 h. The yellowish

white precipitate was collected and washed with diethyl

ether. The crude product was recrystallized from isopropyl

alcohol. All the solid products were characterized by

physico-chemical and spectroscopic methods. Yield:

92–96%.

Preparation of the palladium(II) complexes

The metal complexes were prepared by reacting PdCl2 with

the respective ligand in equimolar ratio following a com-

mon procedure. For complexes 1a, 1b and 1c, the dith-

iocarbamic acids, mpcaH (89.4 mg, 0.508 mmol), mcaH

(83 mg, 0.509 mmol) or bpcaH (116 mg, 0.53 mmol),

respectively, were mixed with PdCl2 (45 mg, 0.254 mmol)

in EtOH (10 ml). The resulting mixture was refluxed for

6 h. The solid yellow product of complex 1a was obtained

by evaporation of the solvent. For 1b and 1c, a brown and a

yellow solid product, respectively, precipitated out from

the reaction medium. The complexes were filtered off,

washed with EtOH and dry Et2O and dried under vacuum

over P4010. Yield: 70–74%.

[Pd(L1)2] (1a): C12H22N4PdS4, Anal. Found: C, 31.7; H,

4.8; N, 12.2; Pd, 23.2; S, 27.9; Calc.: C, 31.5; H, 4.9; N,

12.3; Pd, 23.3; S, 28.1. IR (cm-1): mC=N, 1,497; ma(SCS),1,000, 992; mPd–S, 438. ESI–MS (m/z): [M ? Na?],

480.0099 (15% abundance); [M ? H?] 458.0179 (75%

abundance). Conductivity (Ko, ohm-1 cm2 mol-1) in

DMF: 53. 1H NMR (d, ppm in dmso-d6): 4.35 (br, 6H of

N–CH3); 3.79 (q, 8H of S2C–N(CH2)2); 3.15 (m, 8H of

–N(CH2)2). Yield: 75–77%.

[Pd(L2)2] (1b): C10H16N2O2PdS4; Anal. Found: C, 28.0;

H, 3.7; N, 6.4; Pd, 24.9; S, 29.9; Calc.: C, 27.9; H, 3.7; N, 6.5;

Pd, 24.7; S, 29.8. IR (cm-1): mC=N, 1,492; ma(SCS), 1,025,1,007; mPd–S, 440; ESI–MS (m/z): [M ? Na?], 453.9262

(20% abundance); [M ? H?], 431.9342 (64% abundance).

Conductivity (Ko, ohm-1 cm2 mol-1) in DMF: 45. 1H NMR

(d, ppm in dmso-d6): 3.78–3.86 (m, 8H of S2C–N(CH2)2);

3.65–3.67 (q, 8H of O(CH2)2). Yield: 80–82%.

[Pd(L3)2] (1c): C26H32N2PdS4; Anal. Found: C, 51.5; H,

5.3; N, 4.7; Pd, 17.4; S, 21.3; Calc. C, 51.4; H, 5.3; N, 4.6;

Pd, 17.5; S, 21.1. IR (cm-1): mC=N, 1,508; ma(SCS),1,027,1,010; mPd–S, 436. ESI–MS (m/z): [M ? Na?], 630.2257

(12% abundance); [M ? H?], 608.2337 (35% abundance).

Conductivity (Ko, ohm-1 cm2 mol-1) in DMF: 42. 1H

NMR (d, ppm in dmso-d6): 7.19–7.29 (m, 10H of C6H5);

3.91 (m, 8H of S2C–N(CH2)2); 3.18 (t, 4H of CH2,

j = 6.2); 2.21 (m, 2H of CH); 1.73–1.77 (m, 8H of

(CH2)2). Yield: 75–78%.

X-ray crystal structure analysis

Single crystals of the complex 1a were obtained from a

solution of 1a in DMF on slow evaporation. The diffraction

data were collected at 120(2) K with a four-circle j-axisKUMA KM-4 diffractometer (KUMA Diffraction, Wro-

claw) equipped with a CCD detector and an Oxford

Cryostream Cooler. The x-scan technique was performed

at different u, h and j offsets for covering the whole

independent part of reflections in the h range 3.5–27.57�with monochromated MoKa radiation (k = 0.71073A).

The data reduction was carried out using the CrysAlis RED

program [17]. An empirical absorption correction imple-

mented in SCALE3 ABSPACK scaling algorithm was

applied [17]. The structure was determined by direct

methods (SHELXS-97), and all non-H atoms were refined

anisotropically on F2 using SHELXL-97 full matrix least-

squares procedure [18]. All H-atoms were found from

difference Fourier maps and refined using a riding model,

and the positions of those belonging to the lattice water

molecules were refined. Details of data collection and

refinement are given in Table 1.

156 Transition Met Chem (2012) 37:155–161

123

personal copy

DNA binding experiments

All the experiments involving CT-DNA were studied by

UV absorbance or fluorescence displacement of ethidium

bromide (EB) and performed following our previously

reported methods [12–14].

Results and Discussion

The organic compounds mpcaH, mcaH and bpcaH (Fig. 1)

were synthesized by reaction of the respective amine with

carbon disulphide in ethanol and characterized by IR and1H NMR spectra. These ligands have two sulphur donors,

and the corresponding palladium(II) complexes were

obtained in good yield from the reaction of PdCl2 with the

respective ligands in 1:2 molar ratio in refluxing ethanol.

These mononuclear metal complexes are soluble in

DMF and DMSO, but sparingly soluble in methanol and

ethanol. Conductivity measurements in DMF showed

conductance values in the range of 42–53 Ko mol-1 cm-1

at 300 K, suggesting that these complexes exist in solution

as non-electrolytes [19].

Structural description of complex 1a

An ORTEP view of complex 1a with the atom numbering

scheme is illustrated in Fig. 2, and a selection of bond

distances and angles is listed in Table 2. The structural

analysis showed that the complex resides on a 2/m site,

with the mirror plane bisecting the S–C–S angles and

containing the palladium(II) atom, such that the crystallo-

graphically independent moiety is only a quarter of the

whole complex. Moreover, two residuals, one lying at the

intersection of two mirror planes, and consequently on a

twofold axis (m2 m site), and the second located on a

mirror plane, were interpreted as lattice water molecules

(O1, O2). According to the site multiplicity, the ratio Pd/

O1/O2 is 1:1:2, and the compound is formulated as

[Pd(mpca)2]�3H2O.

The palladium atom has a distorted square planar

geometry defined by two symmetric dithiocarbamates that

behave as chelating monobasic ligands. The Pd–S bond

distances, all equal to 2.327(1) A due to the local sym-

metry, are within the range of 2.300–2.359 A found for

25 bis(dithiocarbamate)palladium(II) complexes retrieved

from the CSD (Cambridge Structural Database, Version

5.32.1) [20]. The formation of the four-membered PdS2C

chelate ring induces a value of the S–Pd–S angle of

75.57(7)�, with the carbon atoms C(1) slightly displaced by

0.187(8) A on opposite directions of the PdS4 mean plane.

The chelating S–Pd–S angle is not unusual, being closely

comparable to the corresponding average value (75.7(6)�)calculated for the cited complexes retrieved from the CSD.

The C–S bond distance, of 1.719(5) A, is intermediate

between double- and single-bond character. The substituted

piperazine ring has a chair conformation with the methyl

group occupying an equatorial position. The nitrogen atom

N(2) with a flat geometry (the C–N–C angles amount to

Table 1 Crystal data and details of refinements for 1a�3H2O

Empirical formula C12H22N4PdS4�3H2O

Formula weight 511.02

Crystal system Orthorhombic

Space group C mcm

a (A) 11.4373(11)

b (A) 6.7609(5)

c (A) 25.858(2)

Volume (A3) 1,999.5(3)

Temperature (K) 120(2)

Z 4

qcalc (g/cm3) 1.702

l(mm-1) 1.487

F(000) 1,044

Crystal size (mm3) 0.30 9 0.25 9 0.10

h range (deg) 3.50–27.57

Reflections collected/unique 6,618/1,199

R(int) 0.0386

Final R indices [I[ 2r(I)] R1 = 0.0648, wR2 = 0.1293

R indices (all data) R1 = 0.0659, wR2 = 0.1298

Goodness-of-fit on F2 1.540

Residuals, (e.A-3) 1.201, –1.250

X

N

S

S

(-)

Fig. 1 Structures of the anions of dithiocarbamic acids. X = N–CH3,

mpca; X = O, mca; X = CH2CH2Ph, bpca

Fig. 2 An ORTEP view of the complex [Pd(4-MePipzcdt)2]�3H2O

(1a) with atom labelling scheme of the crystallographic independent

part. (Symmetry codes: (i) -x, y, z; (ii) x, -y, -z; (iii) -x, -y, -z)

Transition Met Chem (2012) 37:155–161 157

123

Author's personal copy

358.2�, C–N = 1.321(1) A) indicates an sp2 hybridized

atom and electron delocalization within the CS2 fragment.

All the geometrical values are comparable to those

observed in similar bischelated Pd(II) complexes having

piperidine [21] and 4-methylpiperidine-dithiocarbamate

[22] ligands, indicating that these rings have a negligible

effect on the Pd–S bond distances and the chelating S–Pd–

S angles, being in the range of 2.3096(9)–2.3187(9) A and

75.40(3)–75.28(3)� for the four independent sulphur atoms

of the former complex and 2.3189(7)–2.3300(7) A and

75.52(2)� for the centrosymmetric species of the latter.

Finally, the crystal packing of 1a shows a 1D polymeric

zigzag chain built by H-bonds running along the c axis

(Fig. s1). In fact, the water molecule O1 connects two

metal complexes through N…H–OH H-bonds interactions

and acts as an H-bond acceptor with respect to two sym-

metry-related solvent molecules O2, as reported in Table 3.

Spectroscopic analysis

The IR spectra of the complexes were compared with those

of the free ligands in order to deduce the mode of ligand

coordination to the metal. A prominent band, due to the C–H

stretching of the N–CH3 group [23], was identified around

2,850 cm-1 in the IR spectrum of the free acid 4-Me-

PipzcdtH, but was shifted to higher frequency (2,924 cm-1)

for the palladium complex. A band near 1,500 cm-l indi-

cates a considerable degree of double-bond character of the

C=N bond, as confirmed by the X-ray structure. This can be

ascribed to the electron-releasing ability of the heterocyclic

group towards the sulphur atoms, a feature that induces

electron delocalization over the carbon–nitrogen bond and

the CS2 fragment. This is shown by the mC=N shift to higher

energies for the complexes (ca. 1,505–1,450 cm-l) with

respect to the free acids (ca. 1,445–1,430 cm-1), and these

bands lie between the stretching frequencies expected for a

double C=N (1,640–1,690 cm-l) and single C–N bond

(1,250–1,350 cm-1). The blueshift of the C=N stretching

frequency on going from the free acids to their metal com-

plexes provides evidence for the bidentate character [24] of

the carbodithioic acid ligands. Two bands in the region of

1,030–950 cm-l (separated by\20 cm-l) assignable to the

anti-symmetric ma(SCS), and one band for the symmetric

vs(SCS) stretch in the region 695–670 cm-l for the complexes,

suggest an unsymmetrical chelating bidentate coordination

mode [25]. The stretchings due to mCOC (asym. and sym.),

mN–CH3 and mCCC (asym. and sym.) remain unchanged in the

spectra of the complexes and the free ligands. This obser-

vation helps to exclude any coordination to the metals via

nitrogen and oxygen donors. Finally, a band in the spectra of

the complexes in the range 423–426 cm-1 is attributable to

the mPd–S stretching frequency [21].

The 1HNMR spectra of the [Pd(L)2] complexes (in

dmso-d6) show a general downfield shift of signals for the

complexes with respect to the free ligands. Three distinct

peaks in the 1H NMR spectrum of complex 1a indicate the

presence of three types of hydrogen in the complex, in

accord with the symmetric crystal structure. The multiplet

at ca. 3.79 ppm assigned to the eight H atoms of the

coordinated piperidine S2C–N(CH2)2 groups is shifted

downfield compared to the value of ca. 3.15 ppm for the

free ligand, while the signal at ca. 4.35 ppm is assigned to

the six protons of the N–CH3 groups. A multiplet at ca.

3.65–3.67 ppm is assigned to the eight O(CH2)2 protons of

the morpholine ring. In the spectrum of complex 1c, a

characteristic triplet at ca. 3.18 ppm is assigned to four

Table 2 Selected bond

distances (A) and bond angles

(�) for 1a

Symmetry codes: #1 -x, -y,

-z?1 #2 x, -y, -z?1

#3 -x, y, z

Bond distances (A) Bond angles (�)

Pd–S(1) 2.327(1) S(1)–Pd–S(1)#3 75.57(7)

S(1)–C(1) 1.719(5) S(1)–Pd–S(1)#1 180.0

C(1)–N(2) 1.321(1) S(1)–Pd–S(1)#2 104.43(7)

N(2)–C(3) 1.478(6) S(1)#3–C(1)–S(1) 112.1(4)

C(3)–C(4) 1.513(8) N(2)–C(1)–S(1) 123.9(2)

C(4)–N(5) 1.468(7) C(1)–S(1)–Pd 85.7(2)

N(5)–C(6) 1.488(10)

Table 3 Intermolecular hydrogen bond parameters (A/deg) of 1a

D–H…A d(D–H) d(H…A) d(D…A) \DHA

O(1)–H(11)…N(5) 0.82(13) 1.96(13) 2.788(8) 176(3)

O(2)–H(21)…O(1)a 0.85(5) 2.38(8) 2.952(10) 125(9)

O(2)–H(22)…O(2)b 0.86(2) 2.56(3) 3.402(13) 168(10)

Symmetry codes: (a) x - 1/2, y ? 1/2, z; (b) -x?1/2 - 1, y ? 1/2, z


123


protons of the two benzyl –CH2– groups attached to the

piperidine ring.

The electronic absorption spectra of the three complexes

were recorded at room temperature using DMSO as sol-

vent, and the data are tabulated in Table 4. The spectra

exhibit a sharp band around 305 nm along with a shoulder

at ca. 340 nm assignable to intramolecular p ? p* and

n ? p* transitions, respectively. The availability of the

electrons on the donor atoms for the electronic transition

increases in the order 1b\ 1a\ 1c, thereby increasing

the molar extinction coefficient values. This is due to the

presence of a highly electronegative oxygen atom in the

ligand of complex 1b, compared to N–CH3 and CH2-ben-

zyl substituents in 1a and 1c, respectively.

DNA binding experiments

The interaction of the palladium(II) complexes with calf

thymus DNA (CT-DNA) has been investigated using

absorption and emission spectroscopy. In general, binding

of a palladium(II) complex to DNA is accompanied by an

increase in the n ? p* band, due to strong intercalative

interactions between the effective chromophore of the

complexes and the base pairs of DNA [26, 27]. The

absorption spectra of the free metal complexes and of

their adducts with CT-DNA are given in Figs. 3 and 4.

The extent of hyperchromism in the absorption band is

generally consistent with the strength of the intercalative

interaction [28–30], and the observed spectral changes

indicate a strong interaction of the palladium(II) complexes

with CT-DNA.

In order to establish the binding strength of the metal

complexes with CT-DNA, the apparent association con-

stants Kb were determined from the spectral titration data

using the following equation [31]:

½DNA�=ðea � ef Þ ¼ [DNA]=ðeb � ef Þ þ 1=½Kbðeb � ef Þ�where [DNA] is the concentration of DNA and ef, ea and ebcorrespond to the extinction coefficients, respectively, for

the free palladium(II) complex, for each addition of DNA

to the palladium(II) complex and for the palladium(II)

Table 4 Electronic absorption spectral data

Compound kmax(nm) (10-5, e/dm3 mol-1cm-1)a

1a 300(5821), 339(942), 686(828)

1b 297(4497), 335(745), 678(1122)

1c 304(9877), 340(1412), 681(913)

a In DMSO solvent

280 300 320 3400.00

0.05

0.10

0.15 e

a

Ab

sorb

ance

Wavelength(nm)

Fig. 3 Electronic spectral titration of complex 1a with CT-DNA at

300 nm in Tris–HCl buffer; [complex] = 2.52 9 10-5; [DNA]:

(a) 0.0, (b) 1.25 9 10-6, (c) 2.5 9 10-6, (d) 3.75 9 106,

(e) 5.0 9 10-6 mol L-1. The arrow denotes the gradual increase in

DNA concentration

(b)

0.00

0.02

0.04

0.06

0.08

0.10

f

a

Ab

sorb

ance

Wavelength(nm)280 300 320 340 360 270 300 330 360 390

0.00

0.15

0.30

0.45

0.60

0.75

0.90

f

a

Ab

sorb

ance

Wavelength(nm)

(a)

Fig. 4 a Electronic spectral titration of complex 1b with CT-DNA

at 297 nm in Tris–HCl buffer; b electronic spectral titration of complex

1c with CT-DNA at 304 nm in Tris–HCl buffer; [complex] =

2.75 9 10-5; [DNA]: (a) 0.0, (b) 1.25 9 10-6, (c) 2.5 9 10-6,

(d) 3.75 9 10-6, (e) 5.0 9 10-6, (f) 6.25 9 10-6 mol L-1. The arrowdenotes the gradual increase in DNA concentration


123


complex in the fully bound form. Plots of [DNA]/(ea–ef)versus [DNA] (Fig. 5) gave the apparent association con-

stant Kb as the ratio of slope to the intercept. The values of

Kb were estimated as 0.89 9 105 M-1 (R = 0.99763,

n = 4 points), 0.32 9 105 M-1 (R = 0.99438, n = 5 points)

and 1.54 9 105 M-1 (R = 0.9974, n = 5 points) for

complexes 1a, 1b and 1c, respectively.

The binding propensity of the complexes to CT-DNA

has been analysed by fluorescence spectroscopy using the

emission intensity of ethidium bromide (EB). The fluo-

rescence intensity of the DNA-bound EB (with excitation

wavelength of 522 nm) decreases with increasing concen-

trations of the complexes (Figs. 6, 7, 8), indicating that

binding of the complexes to DNA releases EB molecules

[32]. This quenching on addition of the palladium(II)

complexes is in agreement with the linear Stern–Volmer

equation [33]:

I0=I ¼ 1þ Ksv½Q�where I0 and I represent the fluorescence intensities in the

absence and presence of quencher, respectively. Ksv is the

1 2 3 4 5 6 70

20

40

60

80

100 1c

1b

1a

[DN

A]/(

ε a-ε f)

x 10

10

[DNA] x 106

Fig. 5 Plot of [DNA]/(ea–ef) versus [DNA] for the absorption

titration of CT-DNA with the Pd(II) complexes in Tris–HCl buffer;

association constant Kb: 8.89 9 104 M-1 for 1a (R = 0.99763, n = 4

points); 3.18 9 104 M-1 for 1b (R = 0.99438, n = 5); 15.39 9 104

M-1 for 1c (R = 0.9974, n = 5)

580 600 620 640 660 680 700

200

400

600

800

1000 a

e

Inte

nsi

ty

Wavelength(nm)

1.5 3.0 4.5 6.0

1.00

1.05

1.10

1.15

1.20

I 0/ I

[Complex] x 105

Fig. 6 Emission spectra of the CT-DNA–EB system in Tris–HCl

buffer upon titration with complex 1a. kex = 522 nm; [EB] = 9.6 9

10-5 mol L-1, [DNA] = 1.25 9 10-5; [Complex]: (a) 0.0, (b) 1.369 10-5, (c) 2.72 9 10-5, (d) 4.08 9 10-5, (e) 5.44 9 10-5, mol L-1.

The arrow denotes the increasing complex concentration. Inset: plotof I0/I versus [complex] of 1a; Ksv = 0.39 9 104 (R = 0.99746,

n = 4 points)

580 600 620 640 660 680 7000

200

400

600

800

1000

f

a

Inte

nsi

ty

Wavelength(nm)

1 2 3 4 5 6 7 8 90.90

1.05

1.20

1.35

1.50

1.65

I 0 / I

[Complex] x105


buffer upon titration with complex 1b. kex = 522 nm; [EB] = 9.6 9

10-5 mol L-1, [DNA] = 1.25 9 10-5; [Complex]: (a) 0.0, (b)1.38 9 10-5, (c) 2.75 9 10-5,(d) 4.12 9 10-5, (e) 5.50 9 10-5,

(f) 8.25 9 10-5 mol L-1. The arrow denotes the increasing complex

concentration. Inset: plot of I0/I versus [complex] of 1b; Ksv = 0.87

9 104 (R = 0.98873, n = 5 points)

570 600 630 660 690 720 7500

200

400

600

800

1000

e

a

Inte

nsi

ty

Wavelength(nm)

1 2 3 4 5 60.96

1.04

1.12

1.20

1.28

1.36

I o/ I

[Complex] x 105


buffer upon titration with complex 1c. kex = 522 nm; [EB] = 9.6 9

10-5 mol L-1, [DNA] = 1.25 9 10-5; [Complex]: (a) 0.0, (b) 1.25 9

10-5, (c) 2.5 9 10-5,(d) 3.75 9 10-5, (e) 5.00 9 10-5, mol L-1.

The arrow denotes the increasing complex concentration. Inset:plot of I0/I versus [complex] of 1c; Ksv = 0.82 9 104 (R = 0.99965,

n = 4 points)


123


linear Stern–Volmer quenching constant, and Q is the

concentration of the quencher. In the quenching plot (insets

of Figs. 6, 7, 8) of I0/I versus [complex], Ksv is given by the

slope of the regression line as 0.39 9 104, 0.87 9 104 and

0.82 9 104 for complexes 1a, 1b and 1c, respectively,

suggesting a strong affinity of each complex for CT-DNA.

The titration data obtained from the fluorescence

experiments can be helpful also for calculating the number

of binding sites and the apparent binding constant, by means

of the following equation [34]:

log½ðI0 � IÞ=I� ¼ logK þ n log½Q�where K and n represent the binding constant and number

of binding sites of the complex, respectively. The values of

n, determined from the intercept of plots of log[(I0 - I)/I]

versus log[Q] (see Figs. s2, s3 and s4), are 1.19, 1.10 and

1.25 for complexes 1a, 1b and 1c, respectively, suggesting

a single binding site in the DNA. The calculated K values

were 0.67 9 105, 0.16 9 105 and 1.25 9 105 for 1a, 1b

and 1c, respectively.

Conclusion

We reported here the synthesis and characterization of

three palladium(II) complexes of dithiocarbamate ligands,

and the solid-state structure of one of these has been

established using single crystal X-ray crystallography. The

electronic spectral titration of all three complexes with CT-

DNA in Tris–HCl buffer showed a significant intercalative

interaction, with the apparent estimated association con-

stant Kb in the range 0.32–1.54 9 105 M-1, which is

comparable with the reported values for other palladium

complexes of dithiocarbamate derivatives [35] The Stern–

Volmer quenching constants suggest a good affinity of the

complexes for CT-DNA.

Supporting material

Crystallographic data for compound 1a have been deposited

with the Cambridge Crystallographic Data Centre, CCDC

No. 833198. Copies of this information are available on

request at free of charge from CCDC, 12 Union Road,

Cambridge, CB21EZ, UK (fax:?44-1223-336-033; e-mail:

[email protected] or http://www.ccdc.cam.ac.uk).

Acknowledgments Financial support from Department of Science

and Technology (DST), New Delhi, India, is gratefully acknowl-

edged. T. Mukherjee is grateful to University Grants Commission

(UGC), New Delhi, India, for the fellowship. The authors are grateful

to Dr. Dipankar Koley of CDRI, Lucknow, for 1H NMR spectral

assistance.

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123



Palladium(II) and platinum(II) complexes of deprotonated N,N0-bis(2-p-yridinecarboxamide)-1,2-benzene: Synthesis, structural characterizationand binding interactions with DNA and BSA

Titas Mukherjee a, Buddhadeb Sen a, Ennio Zangrando b, Geeta Hundal c, Basab Chattopadhyay d,Pabitra Chattopadhyay a,⇑aDepartment of Chemistry, Burdwan University, Golapbag, Burdwan 713104, IndiabDipartimento di Scienze Chimiche e Farmaceutiche, Via Licio Giorgieri 1, 34127 Trieste, ItalycDepartment of Chemistry, Guru Nanak Dev University, Amritsar 143005, IndiadDepartment of Solid State Physics, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India

a r t i c l e i n f o

Article history:Received 18 December 2012Received in revised form 20 March 2013Accepted 23 April 2013Available online 2 May 2013

Keywords:Palladium(II) complexPlatinum(II) complexCrystal structureDNA and BSA binding study

a b s t r a c t

Two neutral complexes [ML] (where M = Pd (1a) and Pt (1b), L = bis(pyridine-2-carboxamide)benzenedianion) have been synthesized and characterized by physico-chemical and spectroscopic tools alongwith the detailed structural analysis by single crystal X-ray crystallography and theoretical (DFT) study.In solid state the compounds are isomorphous and isostructural showing the formation of [ML]3�3H2Otrimeric species. Electrochemical study of 1a showed a quasi-reversible reductive response atE1/2 = �1.148 V assignable to the Pd(II)/Pd(I) couple, while a metal centered irreversible oxidative peakcentred at +0.977 V was observed in the voltammogram of 1b. The interaction of the complexes withCT-DNA has been investigated using spectroscopic tools and viscosity measurement. In each case theassociation constant (Kb) was deduced from the absorption spectral study and the number of bindingsites (n) and the binding constant (K) were calculated from relevant fluorescence quenching data, suggest-ing a non-covalent interaction between the metal complex and DNA, which could be assigned to an inter-calative binding. In addition, the interaction of 1a and 1b was ventured with bovine serum albumin (BSA)with the help of absorption and fluorescence spectroscopy measurements. Through these techniques, theapparent association constant (Kapp) and the binding constant (K) could be calculated for each complex.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

The last few decades have witnessed a remarkable interest inpyridine carboxamide complexes in various fields of biological rel-evance like asymmetric catalysis, [1,2] dendrimer [3] and molecu-lar receptor synthesis, [4] and also in the synthesis of compoundswith possible antitumour properties [5]. The carboxamide[–C(O)NH–] group of the primary structure of proteins representsan important ligand construction unit in coordination chemistry,since its chelating rigid nature imparts a unique balance of stabilityversus reactivity, and has allowed for impressive developments ina variety of catalytic transformations.

Deprotonated amide groups readily coordinate metal ionsthrough the amide–N and/or –O atom [6] forming a stable delocal-ized electronic system. The anticancer properties of cisplatin andpalladium(II) complexes stem from the ability of the cis-MCl2 frag-ment to bind to DNA bases. However, cisplatin also interacts with

non-cancer cells, mainly binding molecules containing –SH groups,resulting in nephrotoxicity [7]. This has aroused interest in thedesign of novel palladium(II) [8] and platinum(II) complexes withbetter efficacy and lower toxicity. Serum albumins are the majorsoluble protein constituents of the circulatory system and serveas a depot- and as a transfer protein along with several physiolog-ical functions. BSA has been one of the most extensively studied ofthese proteins, particularly because of its structural homology withhuman serum albumin [9].

In the present work we have turned our attention to explore theamide functionality of the N,N

0-bis(2-pyridinecarboxamide)-1,2-

benzene ligand (H2L) towards palladium(II) and platinum(II) ion.We report herein two novel Pd(II) (1a) and Pt(II) (1b) complexeswith the tetradentate ligand having two deprotonated amide-Nand two pyridinic-N donors. These species were structural charac-terized by X-ray diffraction and also by means of different physico-chemical, spectroscopic and computational studies. In addition,interaction of complexes 1a and 1b with CT-DNA and also withbovine serum albumin (BSA) have been studied. In order to estab-lish the association mode of these small molecules to DNA, the

0020-1693/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ica.2013.04.033

⇑ Corresponding author.E-mail address: [email protected] (P. Chattopadhyay).

Inorganica Chimica Acta 406 (2013) 176–183

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binding (Kb) and the quenching constant (Kq) of the complexeswith the double-helix has been determined from UV–Vis studyand fluorescence displacement experiments using ethidium bro-mide as spectral probe. Beside this, the strong binding activity ofcomplexes 1a and 1b with BSA was examined using absorption-fluorescence spectroscopy, further supported by viscositymeasurements.

2. Experimental

2.1. Materials and instrumentation

All chemicals and reagents were obtained from commercialsources and used as received, unless otherwise stated. 2-Pyridine-carboxylic acid, 1,2-diaminobenzene and triphenyl phosphite werepurchased from Aldrich and K2PtCl4 and PdCl2 procured fromAcross, were used as such. The solvents used were purified follow-ing the standard procedures, all other chemicals used were of ana-lytical reagent grade.

The elemental (C, H, N) analyses were performed on a PerkinElmer model 2400 elemental analyzer. Electronic absorption spec-tra and IR spectra were obtained on a JASCO UV–Vis/NIR spectro-photometer model V-570 and on a Perkin–Elmer FTIR model RX1spectrometer (KBr discs, 4000–300 cm�1), respectively. 1H NMRspectra were recorded on a Bruker AC300 spectrometer usingTMS as an internal standard in DMSO-d6 solvent. Electrospray ion-ization (ESI) mass spectra of complexes 1a and 1b were recordedwith a QtofMicro Instrument (Waters, YA263). The fluorescencespectra complex bound to DNA were obtained at an excitationwavelength of 522 nm in the Fluorimeter (Hitachi-2000). Viscosityexperiments were conducted on an Ostwald’s viscometer, im-mersed in a thermostated water-bath maintained at 25 �C. Redoxpotentials were measured in CHI620D potentiometer in DMF usingTBAP as supporting electrolyte at room temperature. Electrochem-ical setup was a three-electrode cell with glassy carbon, Ag/AgCland a platinum wire as a working, reference and counter electrode,respectively. Molar conductances (KM) were measured in a sys-tronics conductivity meter 304 model in dimethylformamide atcomplex concentration of �10�3 mol L�1.

2.2. Preparation of the ligand

The ligand H2L was synthesized following reported method [10]with slight modification. Pyridinic solution (10 ml) of 2-pyridine-carboxylic acid (1.23 g, 10 mmol) and 1,2-diaminobenzene(0.54 g, 5 mmol) was mixed under stirring condition followed bythe dropwise addition of triphenyl phosphite (3.1 g, 10 mmol) at80 �C for 4 h. and settled overnight. A pale brown crystalline solidresulted, was washed with methanol to give long white needles.Yield; 96%. Anal. Calc. for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60.Found: C, 68.16; H, 3.94; N, 1762.18%. Anal. Calc.: IR (KBr, cm�1):mC@O, 1677; mNH, 3317.9. ESI-MS (m/z): parent 318.11 (100% abun-dance); [M+H+] 319.12 (19.7% abundance).

2.3. Preparation of the complexes

2.3.1. Synthesis of Pd(L) (1a)To a solution of H2L (0.636 g, 0.50 mmol) in dry DMF (10 mL)

was added NaH (0.0237 g, 1.00 mmol) and the resulting suspen-sion was stirred for 30 min. To the resulting light yellow solution,PdCl2 (0.089 g, 0.50 mmol) dissolved in DMF was added in portionswith continuous stirring for a period of 3 h. The yellow precipitateresulted was filtered and washed with diethyl ether and vacuumdried. The residue was further dissolved in DMF (6.0 mL), filtered,and the volume of the filtrate was reduced to 3.0 mL. Yellow

crystals, suitable for X-ray diffraction, were obtained by slowevaporation.

(1a).H2O: C18H14N4PdO3: Anal. Calc.: C, 49.05; H, 3.20; N, 12.71.Found: C, 48.26; H, 3.94; N, 12.18%. IR (KBr,cm�1): mC@O, 1630;mPd–N, 415. ESI-MS (m/z): [M+Na+], 463.01 (16% abundance);[M+H+] 442.01 (96.2% abundance). Conductivity (Ko, ohm�1 cm2 -mol�1) in DMF: 51. UV–Vis in DMF, knm (e) (e, dm3 mol�1 cm�1):281 (8,264), 305 (6,960). Yield: 65–68%.

2.3.2. Synthesis of Pt(L) (1b)To a solution of H2L (0.636 g, 0.50 mmol) in dry DMF (10.0 mL),

NaH (0.0237 g, 1.00 mmol) was added and the resulting suspen-sion was stirred for 30 min. To the resulting light yellow solution,aqueous K2PtCl4(0.2075 g, 0.50 mmol) was added in portions withvigorous stirring under nitrogen atmosphere and stirring has con-tinued for further 10 h. The resulting orange-red solution was al-lowed to evaporate slowly, obtaining orange micro crystalssuitable for X-ray diffraction studies.

(1b).H2O: C18H14N4PtO3: Anal.: C, 40.83; H, 2.66; N, 10.58Found: C, 40.67; H, 2.64; N, 10.54. IR (KBr, cm�1): mC@O, 1637;mPt–N, 435. ESI-MS (m/z): [M+Na+], 554.22 (20% abundance);[M+H+], 532.22 (81% abundance). Conductivity (Ko, ohm�1 cm2 -mol�1) in DMF: 48. UV–Vis in DMF, knm (e) (e, dm3 mol�1 cm�1):278 (5,142), 313 (5,564). Yield: 50–52%.

2.4. X-ray data collection and structural determination

Data collections of compounds 1a and 1b were carried at roomtemperature on a Bruker Smart Apex diffractometer equipped withCCD. (k = 0.71073 Å). Cell refinement, indexing and scaling of thedata sets were done by using programs Bruker Smart Apex andBruker Saint packages. [11] The structures were solved by directmethods and subsequent Fourier analyses and refined by thefull-matrix least-squares method based on F2 with all observedreflections [12]. Hydrogen atoms were placed at calculated posi-tions, those of lattice water molecules of compound 1b werelocated on the DFourier map and analogously for molecule Ow1of 1a. All the calculations were performed using the WinGX Sys-tem, Ver 1.80.05. [13] Crystal data and details of refinements aregiven in Table 1.

Table 1Crystallographic data for compounds 1a and 1b.

1a�1.5H20 1b�1.5H20

Empirical formula C54H42N12O9Pd3 C54H42N12O9Pt3Fw 1322.20 1588.27Crystal system monoclinicSpace group C 2/ca (Å) 14.5231(17) 14.5777(5)b (Å) 20.2671(17) 20.2335(6)c (Å) 16.4470(17) 16.4016(5)b (�) 97.365(3) 97.237(2)V (Å3) 4801.1(9) 4799.2(3)Z 4Dcalc (g cm�3) 1.829 2.198l (Mo Ka) (mm�1) 1.188 8.799F(000) 2640 3024h (�) 1.73–28.22 1.73–31.00No. of reflections collected 26979 36287No. of independent reflection 5836 7530Rint 0.0513 0.0325No. of reflection (I > 2r(I)) 3951 5593No. of refined parameter 357 362Goodness-of-fit (GOF) on F2 1.025 1.010R1, wR2 (I > 2r(I))a 0.0344, 0.0767 0.0231, 0.0487R indices (all data) 0.0623, 0.0890 0.0428, 0.0554

a R1 =P

||Fo| � |Fc||/P

|Fo|, wR2 = [P

w(Fo2 � Fc2)2/P

w(Fo2)2]½.

T. Mukherjee et al. / Inorganica Chimica Acta 406 (2013) 176–183 177


2.5. DNA binding studies of palladium(II) and platinum(II) complexes

The binding experiments with calf thymus DNA for complexes1a and 1b were monitored following the same procedure previ-ously reported by us [14] with UV–Vis and fluorescence spectro-scopic tools and also by viscosity and cyclic voltammetrymeasurements.

All the experiments involving the interaction of the complexeswith CT-DNA were carried out in MilliQ water containing tris–HCl buffer (pH 8.04). The solution of CT-DNA in the buffer gave aratio of UV absorbance of ca. 1.8–1.9:1 at 260 and 280 nm, indicat-ing that the CT-DNA was sufficiently free of protein [15]. Stocksolution of DNA was always stored at 4 �C in the dark and usedwithin four days. The CT-DNA concentration per nucleotide wasdetermined spectrophotometrically by employing an extinctioncoefficient of 6600 M�1 cm�1 at 260 nm [16]. The complexes weredissolved in a solvent mixture of 1% DMSO and 99% tris–HCl bufferat 1.0 � 10�4 M�1 concentration. Absorption spectral titrationexperiment was performed by keeping constant the concentrationof the complex (10 lM) and varying the CT-DNA concentration.While measuring the absorption spectra, an equal amount ofCT-DNA was added to both the complex solutions and the refer-ence solution to take into account the absorbance of DNA itself.

In the emission quenching experiment, ethidium bromide (EB)was used as a common fluorescent probe for the DNA in order toexamine the mode and process of metal complex binding to thedouble-helix [17]. A 5.0 lL of the EB tris–HCl buffer solution(1.0 mmol L�1) was added to 1.0 mL of DNA solution (at saturatedbinding levels) [18], stored in the dark for 2 h. Then the solution ofeach of the Pd(II) and Pt(II) complexes was titrated into the DNA/EB mixture and diluted in tris–HCl buffer to 5.0 mL to get the solu-tion with the appropriate complex/ CT-DNA mole ratio. After theincubation at room temperature for 30 min, the fluorescence spec-tra of EB bound to DNA were recorded (kex = 522 nm) in the Hitachi4500 Fluorimeter. All measurements were performed at ambienttemperature.

The binding interaction of the metal complexes with DNA wasstudied by the well known method employing the Ostwald’s vis-cometer. The CT-DNA solution (5 lM) was titrated with Pt(II) andPd(II) complexes (0.5–3.5 lM), following the change of the viscos-ity in each case. Data are presented as (g/g0)1/3 versus the ratio ofthe concentration of the compound and CT-DNA, where g is theviscosity of CT-DNA in presence of the compound and g0 is the vis-cosity of CT-DNA alone. Viscosity values were calculated from theobserved flow time of CT-DNA-containing solution corrected fromthe flow time of buffer alone (t0), g = t–t0 [19].

2.6. Protein (BSA) binding experiments of palladium(II) andplatinum(II) complexes

The binding study with bovine serum albumin (BSA) for com-plexes 1a and 1b were done dissolving the BSA in MilliQ water(1.0 � 10�5 M�1) and the stock solutions of each of the complexeswere prepared in DMSO–H2O (1:99 v/v) mixture at 1.0 � 10�5 M�1

concentration. Both the absorption and fluorescence quenchingexperiments (kex = 280 nm) were performed by gradually increas-ing the complex concentration, keeping fixed the concentrationof BSA. All the experimental sets were carefully degassed purgingpure nitrogen gas for 5 min.

2.7. Computational details

The DFT calculations for the isolated complexes 1a and 1b wereperformed using Dmol3 code [20] in the framework of a general-ized-gradient approximation (GGA) [21]. The geometry of the mol-ecules were fully optimized using the hybrid exchange–correlation

functional BLYP [22] and a double numeric plus polarization (DNP)basis set (Supplementary material, Figs. s1–s3). The electronicstructures were also calculated at the same level. No constraintson bond lengths, bond angles or dihedral angles were applied inthe calculations, and all atoms were free to optimize. Convergencewas assumed to be reached when the total energy change betweentwo consecutive self-consistent field (SCF) cycles was less than1 � 10�5 a.u.

3. Results and discussion

3.1. Synthesis and characterization

The organic ligand L was synthesized by the reaction of 1,2-diaminobenzene with 2-pyridinecarboxylic acid stirring at80–85 �C in pyridine medium, and it has been characterized byIR and 1H NMR spectral analyses. The palladium(II) and plati-num(II) complexes 1a and 1b were obtained in good yield fromthe reaction of palladium(II) chloride and potassium tetrachloroplatinate(II), respectively with the tetradentate ligand L in 1:1 Mratio in the DMF medium with prolonged stirring at room temper-ature (viz Scheme 1).

The monomeric complexes 1a and 1b are soluble in DMF andDMSO but insoluble in methanol and ethanol. The conductivitymeasurement of complexes in DMF showed the conductance val-ues in the range of 42–53Ko mol�1 cm�1 at 300 K. These valuessuggest that the complexes exist as non-electrolytes in solution[23].

3.2. Structural description of 1a and 1b

The X-ray structural analysis show that complexes 1a and 1bare isomorphous and isostructural showing the formation of[ML]3�3H2O aggregates (M = Pd and Pt, respectively L = bis(pyridine-2-carboxamide)benzene dianion. The independent crystallographicunit comprises of one and half neutral metal complex, being onelocated on a twofold axis passing through the metal and bisectingthe Namide–M–Namide bond angle, as shown in Figs. 1 and 2 for thePt derivative 1b. The crystals contain also some disordered waterlattice molecules.

As expected, in 1a and 1b the metal ion is chelated by the tet-radentate dianionic ligand L in a square planar coordination geom-etry and due to the nature of the ligand all the atoms in eachcomplex are coplanar. The bond lengths reported in Table 2 indi-cate that the values relative to the pyridine N donors are signifi-cantly longer by ca. 0.1 Å than those of the amide nitrogenatoms, either in the Pd and Pt complexes, and the Pd–N bond val-ues are in agreement with those reported in analogous complexes

N NO O

N NPd

N NO O

N NPt

1a

1b

H2L

NH HNO O

N N

Scheme 1. Synthetic routes of the complexes.

178 T. Mukherjee et al. / Inorganica Chimica Acta 406 (2013) 176–183


[24]. On the other hand the Pd–N bond lengths appear slightlylonger with respect to the correspondent Pt–N values in agreementwith the metal ionic radius.

It is worth noting of the supramolecular arrangement observedin these compounds, being the complexes arranged as trimericentities where complex Pt2 (or Pd2) of C2 symmetry (Figs. 1 ands4) is stacking in between a pair of two symmetry related Pt1 (orPd1) units (Figs. 3 and s5). Within this trimer the Pt metal ionsin 1b are separated by 3.2897(1) Å, in an almost collinear arrange-ment forming a Pt(1)–Pt(2)–Pt(1) angle of 178.64(1)�. The corre-spondent Figures in 1a are close comparable being of 3.2903(4) Åand 179.33(1)�, respectively. This packing feature is not unusualfor square planar complexes having aromatic ligands and theaggregation of complexes are stabilized by a combination of p–pstacking interactions between the pyridine and phenyl rings ofthe ligands and d8–d8 metallophilic contacts. [25–27]. A structuralindication of the latter interaction between the metals is the slightdisplacement from the N4 donor set plane of atom M(1) by ca.0.03 Å towards M(2) (M = Pt or Pd) inside each trimer. A rotationof ca. 140� is requested to complex Pt1 in order to be superimposedto Pt2 that occupies the center (Fig. 3), and similarly for the Pdcomplex.

Since the number of water molecules and crystal packing areclose comparable for 1a and 1b, we limit here the description forthe former compound. The lattice water molecules reside sidewaysto the trimer entities and are connected through H-bonds. In factO2w, which is located on a twofold axis, is weakly bound to O1w

(O� � �O = 3.16 Å), which in turn connects the carbonyl oxygensO(1) and O(3) of symmetry related complexes (O� � �O = 2.80,3.01 Å, respectively) forming a 2D packing arrangement parallelto the crystallographic ab plane.

3.3. Spectral properties

3.3.1. Electronic absorption spectral studiesThe electronic absorption spectra of complexes 1a and 1b were

recorded at room temperature using DMF as solvent. The spectraexhibit a sharp band around 280 nm assignable to the intramolec-ular p? p⁄ transition. Another relative high intensity band around310 nm is due to a charge transfer from amide ligand core to metal,i.e. LMCT. The low energy tail of the charge transfer band that ap-pears in the visible region of the spectrum is responsible for theyellow and orange-yellow color of the solution containing 1a and1b, respectively. Here, it is observed that the transition for the pal-ladium(II) complex shifted to lower energy compared to that of the

Fig. 1. ORTEP drawing (35% ellipsoid probability) of complex B of 1b located on acrystallographic twofold axis. (The same label scheme applies also to 1a; primedatoms at �x, y, �z + 1/2).

Fig. 2. ORTEP drawing (35% ellipsoid probability) of complex A of 1b. (The samelabel scheme applies also to 1a).

Table 2Coordination bond lengths (Å) and angles (�) for compounds 1a and 1b.

1a, M = Pd 1b, M = Pt

M(1)–N(1) 2.054(3) 2.040(3)M(1)–N(4) 2.055(3) 2.044(2)M(1)–N(2) 1.939(3) 1.953(2)M(1)–N(3) 1.951(3) 1.944(3)M(2)–N(5) 2.053(3) 2.043(3)M(2)–N(6) 1.946(3) 1.958(2)M(1)–M(2) 3.2903(4) 3.2897(1)N(1)–M(1)–N(2) 81.33(10) 81.28(10)N(1)–M(1)–N(3) 165.28(11) 165.79(10)N(1)–M(1)–N(4) 112.51(10) 112.64(10)N(2)–M(1)–N(3) 84.22(11) 84.81(10)N(2)–M(1)–N(4) 166.13(11) 166.01(10)N(3)–M(1)–N(4) 81.91(11) 81.22(10)N(5)–M(2)–N(6) 81.85(11) 81.37(11)N(5)–M(2)–N(60) 165.73(11) 165.90(10)N(5)–M(2)–N(50) 112.41(15) 112.72(14)N(6)–M(2)–N(60) 83.90(15) 84.55(15)M(1)–M(2)–M(10) 179.33(1) 178.64(1)

Primed atoms at �x, y, �z + 1/2.

Fig. 3. Complex trimers in the crystal packing of 1b connected through H-bondsoccurring among carbonyl groups and lattice water molecules. A similar packing isobserved in 1a.



platinum(II) derivative, and this study is in accordance with thetheoretical calculation of the energy of HOMO and LUMO for 1aand 1b, being LUMO for complex 1a lower lying compared to 1b(Fig. s3).

3.3.2. ElectrochemistryThe electrochemical properties for 1a and 1b have been studied

by cyclic voltammetry (CV) at a platinum working electrode indimethylformamide (0.1 M TBAP as supporting electrolyte) atroom temperature. The cyclic voltammograms of 1a and 1b aredisplayed in Figs. 4 and 5, respectively. The CV scan of 1arevealed a one-electron quasi-reversible reductive response atE1/2 = �1.148 V (Epc = �1.245 V and Epa = �1.051 V; DE = 194 mV)assignable to Pd(II)/Pd(I) couple. On the contrary, platinum(II) in1b is irreversibly oxidized to platinum(IV) by 2e� stoichiometry[28,29] centred at +0.977 V. From the theoretical calculation ofHOMO and LUMO energy of both complexes, it may be derived thatthe comparatively lower energy of LUMO of 1a may be responsiblefor the reduction of Pd(II)/Pd(I) and accordingly the higher energyof HOMO of 1b for the irreversible oxidation of Pt(II) to Pt(IV)(Fig. s3).

3.4. Binding experiments with calf thymus-DNA

The mode of interaction of the complexes 1a and 1b with calfthymus DNA (CT-DNA) has been investigated using absorptionand emission spectroscopic tools as well as by viscosity and cyclicvoltammetry measurements.

3.4.1. Absorption spectroscopyElectronic absorption spectroscopy is used as a distinctive char-

acterization tool for examining the binding mode of metal com-plexes with DNA [18,30]. In intercalative binding mode, the p⁄

orbital of the intercalated ligand can couple with the p orbital ofthe base pairs, thus decreasing the p? p⁄ transition energy andresulting in bathochromism. On the other hand, the coupling porbital was partially filled by electrons, thus decreasing the transi-tion probabilities and concomitantly resulting in hypochromism[31]. The absorption spectra of the free metal complexes and oftheir adducts with CT-DNA (at a constant concentration of thecompounds) are given in Fig. 6 and s6 for complexes 1a and 1b,respectively. The extent of hyperchromism in the absorption bandis generally consistent with the strength of intercalative interac-tion [28–31]. As the concentration of CT-DNA is increased, it wasfound that the Pd and Pt complexes at 270 nm and 268 nm exhibit

hyperchromicity of 3.5/19.76% and 3.7/24.02%, respectively. Thisfeature might be ascribed to the fact that both of the co-complexescould uncoil the helix structure of DNA and made more basesembedding in DNA exposed [32–34]. In order to establish the bind-ing strength of the metal complexes with CT-DNA, the apparentassociation constant Kb was determined from the spectral titrationdata using the following equation [35]

1=D�ap ¼ 1=ðD�KbDÞ þ 1=D�

where, Deap = |ea � ef|, De = |eb � ef|, D = [DNA], and ea, eb and ef arerespectively the apparent, bound and free extinctions coefficient ofeach of the compound in respective cases. Kb, expressed as M�1, isderived from the slope of the graph obtained by plotting the[DNA]/(ea�ef) versus [DNA] (Fig. 7). The Kb values for complexesfor 1a and 1b were estimated to be 0.36 � 104 M�1 (R = 0.99999,n = 5 points) and 0.93 � 104 M�1 (R = 0.99885, n = 5 points),respectively.

In order to corroborate the binding mode of intercalation of thePd(II) and Pt(II) complexes with CT-DNA, we employed ethidiumbromide (EB) that, interacting with DNA, represents a characteris-tic indicator of intercalation. The maximal absorption of EB at479 nm decreased and shifted to 499 nm in presence of DNA(Fig. s7), typically indicating insertion between the base pairs[36]. The absorption spectra of the mixture solution of EB, palla-dium(II) complex 1a and DNA and similarly for platinum(II) com-plex 1b are reported as Supplementary (Figs. s7(a) and s7(b),respectively). The observed behavior could be indicative of: (1)

Fig. 4. Cyclic voltammogram (scan rate 100 mV/s) of 1a in DMF solution of 0.1 MTBAP, using platinum working electrode.

Fig. 5. Cyclic voltammogram (scan rate 100 mV/s) of 1b in DMF solution containing0.1 M TBAP, using platinum working electrode.

300 400 500 600 700 8000.00

0.02

0.04

0.06

0.08

0.10

0.12 f

a

Abs

orba

nce

Wavelength(nm)

Fig. 6. Electronic spectral titration of complex 1a with CT-DNA at 271 nm in tris–HCl buffer; [1a] = 2.62 � 10�5; [DNA]: (a) 0.0, (b) 1.25 � 10�6, (c) 2.5 � 10�6, (d)3.75 � 106, (e) 5.0 � 10�6, (f) 6.25 � 10�6 mol L�1. The arrow denotes the gradualincrease of DNA concentration.



being EB strongly bound to complex 1a (or 1b), the result is adecrease amount of EB intercalated into DNA; or (2) there existsa competition between the palladium(II) (or platinum(II)) complexand EB towards DNA intercalation, so releasing some free EB fromDNA–EB complex. However, here the former account could beirrelevant because of the appearance of a new absorption band.

3.4.2. Fluorescence quenching analysisThe binding propensity of palladium and platinum complexes

to CT-DNA has been analyzed by the steady-state emissionquenching experiments using the emission intensity of ethidiumbromide (EB). It is well known that EB can intercalate nonspecifi-cally with DNA, causing a strong fluorescence. Other compoundscompeting with EB to intercalation in DNA will induce displace-ment of bound EB and a decrease in the fluorescence intensity. Thisfluorescence-based competition can provide indirect evidence forthe DNA-binding mode. The fluorescence intensity of the EB/DNAsystem (with excitation wavelength of 522 nm) is reduced by theincreasing concentration of the complexes (Figs. 8 and s8), andcaused by EBmigration from a hydrophobic to an aqueous environ-ment [37]. The quenching of EB bound to DNA by 1a and 1b is inagreement with the linear Stern–Volmer equation [38]:

I0=I ¼ 1þ Kq½Q �

where I0 and I represent the fluorescence intensities in the absenceand presence of quencher, respectively. Kq is a linear Stern–Volmerquenching constant, Q is the concentration of the quencher. In thequenching plot (insets of Figs. 8 and s8) of I0/I versus [complex],Kq is given by the ratio of the slope to the intercept. The Kq valuesare 0. 14 � 104 and 0.44 � 104 for complexes 1a and 1b respectivelyimplies that both complexes can insert between DNA base pairs andthat the platinum(II) complex can bind to DNA more strongly thanthe palladium(II) complex which is consistent with the absorptiondata.

The titration data obtained from the fluorescence experimentcan be helpful also to calculate the number of binding sites andthe apparent binding constant. In the following equation [39]:

log½ðI0 � IÞ=I� ¼ log K þ n log ½Q �K and n represent the binding constant and number of binding

sites of palladium complex to CT-DNA, respectively. The number ofbinding sites n, determined from the intercept of log[(I0 � I)/I] ver-sus log[Q] (Fig. 9), are 1.07 and 1.18 for 1a and 1b, respectively,indicating the existence of about a single binding site in DNA anda weaker association for the complexes. The K values were calcu-lated to be 0.32 � 104 and 0.77 � 104 for 1a and 1b, respectively,with a trend similar to the apparent association constant valuesof the complexes.

3.4.3. Viscosity measurementsSince optical photophysical probes generally provide necessary,

but insufficient clues to further clarify the interactions between thecomplex and DNA, viscosity measurements were carried out.Hydrodynamic measurements, sensitive to length change (i.e. vis-cosity and sedimentation), are regarded as the least ambiguous andthe most critical tests of binding in solution in the absence of crys-tallographic structural data. A classical intercalation model de-mands that the DNA helix lengthens as base pairs are separatedin order to accommodate the binding ligand, leading to an increasein DNA viscosity. In contrast, a partial, non-classical intercalationof compound could bend (or kink) the DNA helix, reducing itseffective length and, concomitantly, its viscosity [19]. The resultsobtained in these viscosity measurement studies suggest that boththe compounds 1a and 1b can intercalate between adjacent DNAbase pairs, causing an extension of the helix with a concomitant in-crease of the DNA viscosity. The effects of both compounds on theviscosity of DNA are shown in Fig. 10.

3.5. Binding experiments with bovine serum albumin (BSA)

3.5.1. Absorption spectral characterizationThe binding mode of complexes 1a and 1b with BSA were

examined by electronic absorption titration with BSA. The

1 2 3 4 5 61.0

1.5

2.0

2.5

3.0

3.5

4.0

1a

[DN

A]/(

ε a-ε

f)X10

10

[DNA]X106

1b

Fig. 7. Comparative plot of [DNA]/(ea � ef) vs. [DNA] for the absorption titration ofCT-DNA with complexes 1a and 1b in tris–HCl buffer; association constant Kb:0.36 � 104 M�1 (R = 0.99999, n = 5 points) for 1a; 0.93 � 104 M�1 (R = 0.99885, n = 5points) for 1b.

575 600 625 650 675 700 725 750 7750

150

300

450

600

750

900

1050

1200

1350

Inte

nsity

W avelength(nm )

f

a

1 2 3 4 5 6 7

1.00

1.02

1.04

1.06

1.08

1.10

1.12

I o/I

[Com plex]X105

Fig. 8. Emission spectra of the CT-DNA-EB system in tris–HCl buffer upon titrationwith complex 1a. kex = 522 nm; [EB] = 9.6 � 10�5; [DNA] = 1.25 � 10�5; [1a]: (a) 0.0,(b) 1.31 � 10�5, (c) 2.62 � 10�5, (d) 3.93 � 10�5, (e) 5.24 � 10�5, (f) 6.55 �10�5 mol L�1. The arrow denotes the gradual increase of complex concentration.Inset shows the plot of I0/I vs. [1a]; Kq = 0.16 � 104 (R = 0.99876, n = 5 points).

-4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1-2.0

-1.8

-1.6

-1.4

-1.2

-1.0

1a

1blog[

(I o-I)/I

]

log[Complex]

Fig. 9. Comparative plot of log[I0-I/I] vs. log[complex] for the titration of CT-DNA-EB system with complexes 1a and 1b in tris–HCl buffer medium.



absorption spectra of the free metal complexes and of their ad-ducts with BSA are given in Figs. 11 and s9 for complex 1a and1b, respectively. The spectra indicate a significant increase in theabsorbance of BSA by increasing the concentration of the complexand are indicative of the fact that BSA adsorbs strongly the com-plex on its surface [40]. From these titration data the apparentassociation constant (Kapp) of the complexes with BSA has beendetermined using the following equation [31]:

1=ðAobs � A0Þ ¼ 1=ðAc � A0Þ þ 1=KappðAc � A0Þ½comp�where, Aobs is the observed absorbance of the solution containingdifferent concentrations of the complex, A0 and Ac are the absor-bance of BSA and of the complex at 280 nm, respectively. Theenhancement of the absorbance at 280 nm was attributable to thecomplex absorption at BSA surface. Based on the linear relationshipbetween 1/(Aobs � A0) versus the reciprocal concentration of thecomplex with a slope of 1/Kapp(Ac � A0) and an intercept equal to1/(Ac � A0) (Fig. 12), the value of Kapp was determined to be1.262 � 105 M�1 (R = 0.99967, n = 5 points) and 1.402 � 105 M�1

(R = 0.99991, n = 5 points), for 1a and 1b, respectively.

3.5.2. Fluorescence quenching analysisIn the fluorescence quenching experiment, the fluorescence

emission spectrum of BSA was studied increasing the concentra-tion of the quencher (Figs. 13 and s10). The fluorescence quenching

is described by the Stern–Volmer relation, [40] similarly asdescribed above for CT-DNA binding experiments. From the slopeof the regression line in the derived plot of I0/I versus [complex](insets of Figs. 13 and s10) the Kq values for the complexes weredetermined to be 4.29 � 104 for 1a (R = 0.99825 for five points)and 4.62 � 104 for 1b (R = 0.99948 for five points), indicating astrong affinity of both of the complexes to BSA.

4. Conclusions

Two novel square planar palladium(II) and platinum(II) com-plexes 1a and 1b of deprotonated tetradentate ligand N,N

0-bis(2-

pyridinecarboxamide)-1,2-benzene have been synthesized andcharacterized using various spectroscopic measurements. TheX-ray structural characterization revealed that the palladiumd(II)and platinum(II) derivatives are isomorphous and are packed toform a trimeric motif with complexes connected by p–p interac-tions between the aromatic rings of the ligands and metallophilicbonding. The complexes have been found to interact with CT-DNA through an intercalative mode, which was investigated byabsorption, fluorescence and viscosity measurement tools. Thequenching rate constant, binding constant and number of bindingsites were calculated according to the relevant fluorescence data.The binding constants indicate that the DNA-binding affinity, as

0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.30

0.35

0.40

0.45

0.50

0.55

0.60

1a

1b

(η− η

0/η0)(1

/3)

[M]/DNA

Fig. 10. Effect of increasing amounts of Pd(II) and Pt(II) complexes on the relativeviscosity of CT-DNA at 25 �C.

280 350 420 490 560 6300.0

0.2

0.4

0.6

0.8

1.0

1.2

f

a

Abs

orba

nce

Wave length (nm)

Fig. 11. Absorption titration spectra of BSA in presence of complex 1a. Concentra-tion range of complex is 0–6.25 � 10�6 M�1.

2 4 6 8 102

4

6

8

10

12

14

16

(1 / [Complex]) x 105

1a

1b

1/(A

Obs

-AO)

Fig. 12. The linear dependence of 1/A � A0 on the reciprocal concentration ofcomplexes 1a and 1b.

1 2 3 4 5 6 71.00

1.05

1.10

1.15

1.20

1.25

1.30

1.35

I O/I

[Complex]x106

300 320 340 360 380 4000

500

1000

1500

2000

2500

f

aIn

ten

sity

Wavelength(nm)

Fig. 13. Fluorescence quenching titration of BSA varying the concentrations ofcomplex 1a, [complex] = 0, 1, 2, 3, 4 and 5 � 6.35 � 10�6 M. Inset shows the Stern–Volmer plot.



well as the binding trend with BSA, increases from Pd(II) to Pt(II), inaccordance with the relevant viscosity measurement study. Theinformation obtained from the present work is indicative of thedevelopment of potential probes of DNA structure in futureapplications.

Acknowledgements

Financial support from Council of Scientific and Industrial Re-search (CSIR), New Delhi, India is gratefully acknowledged. E.Zangrando thanks MIUR-Rome, PRIN 2007HMTJWP_002 for finan-cial support.

Appendix A. Supplementary material

CCDC 856198 and 873080 contains the supplementary crystal-lographic data for 1a and 1b. These data can be obtained free ofcharge from The Cambridge Crystallographic Data Centre viawww.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-ated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ica.2013.04.033.

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Cyclometalated rhodium(III) complexes bearing dithiocarbamatederivative: Synthesis, characterization, interaction with DNAand biological study

Titas Mukherjee a, Buddhadeb Sen a, Animesh Patra a, Snehasis Banerjee b, Geeta Hundal c,Pabitra Chattopadhyay a,⇑aDepartment of Chemistry, Burdwan University, Golapbag, Burdwan 713104, IndiabGovt. College Of Engineering and Leather Technology, Salt Lake Sector-III, Kolkata 98, IndiacDepartment of Chemistry, Guru Nanak Dev University, Amritsar 143005, India

a r t i c l e i n f o

Article history:Received 31 August 2013Accepted 22 November 2013Available online 1 December 2013

Keywords:Cyclometalated rhodium(III) complexDithiocarbamatesCrystal structureDFTDNA bindingAntimicrobial study

a b s t r a c t

Reaction of three different dithiocarbamates (4-MePipzcdtH, L1H; MorphcdtH, L2H and 4-BzPipercdtH,L3H) with [Rh(2-C6H4py)2Cl]2.1/4CH2Cl2 afforded a class of rhodium(III) complexes of the type[RhIII(2-C6H4py)2(L)]. The complexes were fully characterized by several spectroscopic tools along with adetailed structural characterization of [Rh(2-C6H4py)2(L1)] (1) by single crystal X-ray diffraction. Struc-tural analysis of 1 showed a distorted octahedron in which both of the 2-phenylpyridyl nitrogens are inaxial positions, trans to one another and the sulfur atoms are opposite to the phenyl rings. Electrochemicalanalysis by cyclic voltammetry reveals irreversible redox behavior of the rhodium centre in 1, 2 and 3. TheirDNA binding ability have been also evaluated from the absorption spectral study as well as fluorescencequenching properties, suggesting the intercalative interaction of the complexes with CT-DNA due to thestacking between the aromatic chromophore and the base pairs of DNA. Antibacterial activity of complexeshas also been studied by agar disc diffusion method against some species of pathogenic bacteria(Escherichia coli, Vibrio cholerae, Streptococcus pneumonia and Bacillus cereus).

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Rhodium(III) complexes are the subject of current researchactivity in the interaction of complexes with biomolecules as wellas the rhodium catalyst is able to fulfill its role over the other con-ventional catalysts due to the capability of the metal to change itscoordination number from six to four and also the oxidation statefrom Rh(III) to Rh(I). This change appears as a chemically irrevers-ible two-electron reduction involving ligand loss from octahedralRh(III) to form square planar Rh(I) complexes. Loss of the liganddepends on the nature of the ligands present in mixed ligand sys-tems which allow one to tune the electrochemical potential and af-fect the reactivity of the rhodiummetal center [1]. The discovery ofthe catalytic properties of Wilkinson’s catalyst, viz. [RhCl(PPh3)3]naturally brought about a widespread search for other rhodiumphosphines with catalytic activity [2,3]. Further, octahedral dii-mine rhodium(III) complexes are of interest as they have been usedin the process of photochemical reduction of H2O to H2 [4].

The dithiocarbamates (R2NCS2�) have been considered as versa-tile ligands for bonding to transition as well as main group metal

ions [5–17], and got an enormous attention because of their impor-tance in several fields such as the chemical industry, biology andbiochemistry [18–21]. The nature of the heterocycle attached todithiocarbamate fragment appears crucial so as to vary the elec-tron properties of these ligands and thus to control the potentialpharmacological attributes as well as the catalytic efficiency ofthe metal complexes [22]. Coordination complexes of platinoidswith dithiocarbamato ligands are known in the literature [9–13]and also palladium(II) and platinum(II) complexes of dithiocarb-amato groups together with mono- or diamine ligands [14–17].But to the best of our knowledge so far, report of rhodium(III)cyclometalated complexes bearing dithiocarbamate derivative isstill unexplored.

The binding interactions of these complexes with DNA havealso been studied systematically to explore the biological activityof the new complexes as we know the fact of the activity ofcisplatin by coordination to DNA [23,24]. And from thorough phar-macological mechanistic studies it is also known that small mole-cules interact with DNA via electrostatic forces, groove binding, orintercalation [25], and their effectiveness depends on the modeand affinity of the binding [26]. Intercalation is one of the mostimportant among these interactions. Therefore, the search fordrugs that show intercalative binding to DNA has been an active

0277-5387/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.poly.2013.11.028

⇑ Corresponding author. Tel.: +91 342 2558339; fax: +91 342 2530452.E-mail address: [email protected] (P. Chattopadhyay).

Polyhedron 69 (2014) 127–134

Contents lists available at ScienceDirect

Polyhedron

journal homepage: www.elsevier .com/locate /poly


research area for the past several decades [27]. Moreover, althoughrhodium metal is not bio-essential element but its compoundshave useful applications in the biological field [28–31] and havesignificant pharmacological effects through the interaction withDNA [32].

Encouraged by the advantages of the facts stated above, we iso-lated a new series of cyclometalated rhodium(III) complexes bear-ing dithiocarbamate derivatives by a high yield synthetic pathwayunder mild reaction conditions. The present report deals with thechemistry of these [RhIII(2-C6H4py)2(SS)] complexes, whereSS = 4-MePipzcdtH, MorphcdtH, 4-BzPipercdtH with special refer-ence to their formation, structural characterization and electro-chemical behavior. The binding interactions of these complexeswith calf thymus-DNA (CT-DNA) have also been studied systemat-ically to explore the mode of biological activity as part of ourcontinuing interest [12,33]. In addition, antibacterial activity ofthe complexes (1, 2 and 3) against some pathogenic bacteria,namely Escherichia coli, Vibrio cholerae, Streptococcus pneumoniaand Bacillus cereus has also been studied by agar disc diffusionmethod.

2. Experimental

2.1. Materials and physical measurements

Rhodium trichloride, 2-phenylpyridine (2-C6H4py), morpholineand 4-benzylpiperidine (Aldrich) were purchased and used with-out further purification. [Rh(2-C6H4py)2Cl]2�1/4CH2Cl2 was pre-pared following the reported procedure [34]. 4-Methylpiperazine(Aldrich) has been dried by refluxing over NaOH beads, the color-less liquid obtained after distillation and stored over NaOH beads.Solvents used for spectroscopic studies and for synthesis werepurified and dried by standard procedures before used. The organicmoieties, 4-methylpiperazine-l-carbodithioic acid (4-MePipzcdtH,L1H), morpholine-4-carbodithioic acid (MorphcdtH, L2H) and 4-ben-zyl- piperidine-l-carbodithioic acid (4-BzPipercdtH, L3H) were ob-tained as solid products following the reported procedure [12].

The Fourier transform infrared spectra of the ligand and thecomplexes were recorded on a Perkin-Elmer FTIR model RX1 spec-trometer using KBr pellet in the range 4000–300 cm�1. The solu-tion phase electronic spectra were recorded on an JASCO UV–Vis/NIRspectrophotometer model V-570 in the range 200–1100 nm.Elemental analyses were carried out on a Perkin–Elmer 2400 ser-ies-II CHNS Analyzer. The fluorescence spectra complex bound toDNA were obtained at an excitation wavelength of 522 nm in theFluorimeter (Hitachi-2000). Mass spectra of 1, 2 and 3 were re-corded on Micromass Q-Tof micro™. NMR spectrum of the ligandsand complexes has been recorded on Bruker DPX-300. Solutionconductivity and redox potentials were measured using SystronicsConductivity Meter 304 model and CHI620D potentiometer in DMFat complex concentration of �10�3 mol L�1. Viscosity experimentswere conducted on an Ostwald’s viscometer, immersed in athermostated water-bath maintained to 25 �C.

2.2. Syntheses of [Rh(2-C6H4py)2(L1)] (1) [Rh(2-C6H4py)2(L

2)] (2) and[Rh(2-C6H4py)2(L

3)] (3)

The complexes have been synthesized following a common pro-cedure stated as below. The ligand, L1H (89.4 mg, 0.508 mmol) forcomplex 1, L2H (83.8 mg, 0.508 mmol) for complex 2 or L3H(127.0 mg, 0.508 mmol) for complex 3 was dissolved in DMSO–MeCN (v/v 1:1) solvent mixture and to this ligand solution drop-wise MeCN solution of [Rh(2-C6H4py)2Cl]2�1/4CH2Cl2 (468 mg,0.5 mmol) was added. The mixture was then refluxed in nitrogenatmosphere for 12 h and the color changed from faded yellow to

orange. On slow evaporation of this solution orange colouredmicrocrystalline solid appeared, which was purified by extractingthe orange band in column chromatography using MeCN as aneluant. Needle shaped crystals of [Rh(2-C6H4py)2(L1)] suitable forX-ray diffraction study were grown from this solution onevaporation at ambient temperature.

Rh(2-C6H4py)2(L1)] (1): [C28H27N4RhS2]; Yield: 85%. Anal. Calc. C,

57.33; H, 4.64; N, 9.55. Found: C, 57.21; H, 4.58; N, 9.32%. IR(cm�1): mC@N, 1495; ma(SCS), 1005, 996; ESI-MS (m/z): [M+Na+],609.576(25% abundance); [M+H+] 587.588 (69% abundance). Con-ductivity (Ko, M�1 cm�1) in DMF: 30; 1H NMR (d, ppm in dmso-d6): 4.36 (m, 3H of N–CH3); 3.82 (m, 4H of S2C-N(CH2)2); 3.20(m, 4H of –N(CH2)2); protons of 2-C6H4py: C1-H (8.83, d, 2H),C5-H (8.02, d, 2H), C4-H (7.70, d, 2H), C3-H (7.69, m, 2H), C6-H,C7-H and C8-H (7.43, m, 6H), C2-H (7.28, m, 2H).

[Rh(2-C6H4py)2(L2)] (2): C27H24N3ORhS2; Yield: 80%. Anal. Calc.

C, 56.54; H, 4.22; N, 7.33. Found: C, 56.52; H, 4.06; N, 7.29%. IR(cm�1): mC@N, 1485; ma(SCS), 1030, 1014; ESI-MS (m/z): [M+Na+],596.528 (20% abundance); [M+H+], 574.538 (64% abundance). Con-ductivity (Ko, M�1 cm�1) in DMF: 27. 1H NMR (d, ppm in dmso-d6):3.86 (m, 4H of S2C-N(CH2)2); 3.66 (m, 4H of O(CH2)2); protons of2-C6H4py: C1-H (8.80, d, 2H), C5-H (7.99, d, 2H), C4-H (7.69, d,2H), C3-H (7.67, m, 2H), C6-H, C7-H and C8-H (7.40, m, 6H), C2-H(7.24, m, 2H).

[Rh(2-C6H4py)2(L3)] (3): C35H32N3RhS2; Yield: 73%. Anal. Calc. C,

63.53; H, 4.87; N, 6.35. Found: C, 63.44; H, 4.79; N, 6.02%. IR(cm�1): mC@N, 1505; ma(SCS),1035, 1020; ESI-MS (m/z): [M+Na+],684.682 (18% abundance); [M+H+], 662.688 (42% abundance). Con-ductivity (Ko, M�1 cm�1) in DMF: 32. 1H NMR (d, ppm in dmso-d6):7.29–7.32 (m, 5H of C6H5); 3.94 (m, 4H of S2C-N(CH2)2); 3.21 (t, 2Hof CH2); 2.23 (m, 4H of CH2); protons of 2-C6H4py: C1-H (8.86, d,2H), C5-H (8.02, d, 2H), C4-H (7.70, d, 2H), C3-H (7.68, m, 2H),C6-H, C7-H and C8-H (7.38, m, 6H), C2-H (7.21, m, 2H).

2.3. X-ray crystallography

X-ray data of the suitable crystal of complex 1were collected ona Bruker’s Apex-II CCD diffractometer using Mo Ka (k = 0.71069).The data were corrected for Lorentz and polarization effects andempirical absorption corrections were applied using SADABS fromBruker. A total of 13691 reflections were measured out of which4012 were independent and 2299 were observed [I > 2r(I)] for the-ta (h) 32�. The structure was solved by direct methods using SIR-92and refined by full-matrix least squares refinement methods basedon F2, using SHELX-97 [35]. The two fold axis passes through the me-tal ion, nitrogens of the piprazine ring and their substituent carbonatoms. Therefore the asymmetric unit contains half the molecule.All non-hydrogen atoms were refined anisotropically. The refine-ment showed rotational disorder in the piprazine ring which couldbe resolved by splitting the two unique carbon atoms into twocomponents and refining their sof and thermal parameters as freevariables with restraints over the bond distances. All hydrogenatoms were fixed geometrically with their Uiso values 1.2 timesof the phenylene and methylene carbons and 1.5 times of themethyl carbons. All calculations were performed using Wingxpackage [36,37]. Important crystallographic parameters are givenin Table 1.

2.4. Theoretical calculation

To clarify the configurations and energy level of the complexes1, 2 and 3, DFT calculations were carried out in G09W programusing B3LYP/6-31G(d) calculation and correlation function asimplemented in the Gaussian program package GAUSSIAN 09. Ther-mal contribution to the energetic properties was considered at298.15 K and one atmosphere pressure.

128 T. Mukherjee et al. / Polyhedron 69 (2014) 127–134


2.5. DNA binding experiments

All the experiments involving CT-DNA were studied by spectro-electronic titration and fluorescence quenching technique by usingethidium bromide (EB) as a DNA scavenger and performed theexperiment as our previously standardized method [38].

Tris–HCl buffer solution was used in all the experiments involv-ing CT-DNA. This tris–HCl buffer (pH 7.9) was prepared usingdeionized and sonicated HPLC grade water (Merck). The CT-DNAused in the experiments was sufficiently free from protein as theratio of UV absorbance of the solutions of DNA in tris–HCl at 260and 280 nm (A260/A280) was almost �1.9. The concentration ofDNA was determined with the help of the extinction coefficientof DNA solution at 260 nm (e260 of 6600 L mol�1 cm�1) [38]. Stocksolution of DNA was always stored at 4 �C and used within fourdays. Concentrated stock solution of the complex 1 was preparedby dissolving the compound in DMSO and suitably diluted withtris–HCl buffer to the required concentration for all the experi-ments. Absorption spectral titration experiment was performedby keeping constant the concentration of the complex 1 and vary-ing the CT-DNA concentration. To eliminate the absorbance of DNAitself, equal solution of CT-DNA was added both to the complex 1solution and to the reference solution.

In the ethidium bromide (EB) fluorescence displacement exper-iment, 5 lL of the EB tris–HCl solution (1.0 mmol.L�1) was added to1.0 mL of DNA solution (at saturated binding levels), stored in thedark for 2.0 h. Then the solution of the compound was titrated intothe DNA/EB mixture and diluted in tris–HCl buffer to 5.0 mL to getthe solution with the appropriate complex 1/CT-DNA mole ratio.Before measurements, the mixture was shaken up and incubatedat room temperature for 30 min. The fluorescence spectra of EBbound to DNA were obtained at an excited wavelength of522 nm in the Fluorimeter (Hitachi-2000). The interaction of thecomplex 1 with calf thymus DNA (CT-DNA) has been investigatedby using absorption and emission spectra.

2.6. Antimicrobial screening

The biological activities of free dithiocarbamic acids and therhodium(III) derivatives of dithiocarbamates (1, 2 and 3) have beenstudied for their antibacterial activities by agar well diffusionmethod [39–41]. The antibacterial activities were done at

100 lg/mL concentration of different compounds in DMF solventby using three pathogenic gram negative bacteria (E. coli,V. cholerae, S. pneumoniae) and one gram positive pathogenicbacteria (B. cereus). DMF was used as a negative control. The Petridishes were incubated at 37 �C for 24 h. After incubation plateswere observed for the growth of inhibition zones. The diameterof the zone of inhibition was measured in mm.

3. Results and discussion

3.1. Synthesis and characterization of complexes

The bidentate sulfur ligands (L1H, L2H, and L3H) were synthe-sized by the reaction between carbon disulfide with differentamines in ethanol, and later characterized by FTIR and 1H NMR.Treatment of these ligands with [Rh(2-C6H4py)2Cl]2�1/4CH2Cl2 atrefluxing condition in DMSO–MeCN (v/v 1:1) solvent mixture hav-ing nitrogen atmosphere resulted in cleavage of the chloro bridgeand led to formation of mononuclear rhodium(III) complexes ofgeneral formula of [RhIII(2-C6H4py)2(L)] which were obtained fromthe column chromatography using acetonitrile as orange coloredmicrocrystalline solid on evaporation. Here, the dithiocarbamatesbehaves as bidentate monobasic ligands (see Scheme 1). The com-plexes (1–3) are sparingly soluble in common organic solvents ex-cept hexane but fairly soluble in DMF and DMSO, and are stable inboth the solid state and solution in air. The molar conductivity offreshly prepared solution (�1 � 10�3 M concentration) of 1(KM = 130 M�1 cm�1), 2 (KM = 127 M�1 cm�1) and 3 (KM = 132 -M�1 cm�1) in DMF are fairly consistent with a non-electrolyte,respectively. The complexes are diamagnetic in nature. The formu-lations of the complexes have been confirmed by spectroscopicmethods and elemental analyses.

3.2. Structural description of complex 1

An ORTEP view of the complex [Rh(2-C6H4py)2(L1)] (1) withatom labeling scheme is illustrated in Fig. 1, and a selection of bonddistances and angles is listed in Table 2. The structural analysis evi-denced that the complex resides on a C 2/c site in the monocliniccrystal system. The crystal structure of 1 shows a distorted octahe-dron in which both of the 2-phenylpyridyl nitrogens are in axialpositions, trans to one another and the sulfur atoms are oppositeto the phenyl rings. As would be expected, both Rh-C r-bondsare equal in length (1.994(4) Å) and significantly shorter than theRh-N dative bond lengths of 2.039(3) Å due to the Rh-C r-bondsincreasing electron density on the metal center. The bond distanceof Rh-C in 1 is comparable with the previously reported Rh-Cbonds in cyclometalated complex (1.996(9) Å) but the bond Rh-Nis slightly longer than those (1.987(7) Å) [42], but both are compa-rable with the reported values [43]. Both Rh–S distances are also

Table 1Crystallographic data for complex 1.

Empirical formula C28H27N4RhS2Identification code shelxlFormula weight 586.59Crystal system monoclinicSpace group C 2/ca (Å) 16.683(5)b (Å) 17.840(4)c (Å) 10.251(5)b (�) 126.223(5)V (Å3) 2461.3(15)Z 4Dcalc. (g cm�3) 1.583l (Mo Ka) (mm�1) 0.889F(000) 1200h (�) 1.90–31.84Number of reflections collected 13691Number of independent reflections 4012Rint 0.0544Number of reflections (I > 2r(I)) 2299Number of refined paramaters 357Goodness-of-fit (GOF) on (F2) 1.034R1, wR2 (I > 2r(I)) 0.0473, 0.1041R indices (all data) R1 = 0.1053, 0.1342

DMSO-MeCN

X

N SH

S

LnH + [Rh(2-C6H4py)2Cl2]2Reflux / 12 h

[Rh(2-C6H4py)2Ln]


2-C6H4pyX= -N-CH3 , 4-MePipzcdtH (L1H)X= -O, MorphcdtH (L2H)X= -CH-CH2Ph , 4-BzPipercdtH (L

3H)

N 1

2

3456

7

8

Scheme 1. Synthetic method of rhodium(III) complexes.

T. Mukherjee et al. / Polyhedron 69 (2014) 127–134 129


equal in length (2.4854(11) Å), and these are longer than those in[Rh(Et2NCS2)3] (2.364(3) Å) [44] due to trans influence of thestrong r-donating carbon atoms of the phenyl groups andshorter than those in similar type complex [{Rh(Bu2-C6H4py)2}2{S2P(OMe)2}] (2.548(2) Å) due to attachment of sulfur atoms tocarbon to of more electronegativity than phosphorous, for this rea-son, the S–Rh–S angle of 71.00(5)� is smaller compared to the ob-served value of 79.40(1)� in the previous report [43].

3.3. IR spectra

The IR spectrum of the ligands display an intense stretch at2850 cm�1 and 1445–1430 cm�1 correspond to cC–H of N-Me andcC=N respectively. The cC–H of N–Me of L1H for 1 (2910 cm�1) is blueshifted than the cC–H of N–Me of the free ligand (2850 cm�1) indicat-ing metal ligand coordination. The band around 1590 cm�l indi-cates a double bond character of C–N bond in the ligand frame,which is confirmed from the bond length of the X-ray structure.This fact could be attributed to the electron releasing ability ofthe heterocyclic group towards the sulfur atoms, a feature that in-duces an electron delocalization over the carbon–nitrogen bondand the CS2 fragment. This is shown by the mC=N shift to higherenergies (ca. 1510–1465 cm�l) with respect to the free acids (ca.1445–1430 cm�1), and these bands lie in between the stretching

frequencies expected for a double C@N (1610–1690 cm�l) and sin-gle C–N bond (1250–1350 cm�1). The blue-shift of the C@Nstretching frequency on going from the free acids to their metalcomplexes gives support to the typical bidentate character [45]of the carbodithioic acid ligands. Two bands in the region of1040–965 cm�l (separated by less than 20 cm�l) assignable to thema(SCS) and one band for the vs(SCS) stretch in the region 705–675 cm�l of the complexes suggest the unsymmetrical chelatingbidentate mode of coordination to rhodium(III) ion [46]. Thestretchings due to mCOC (asym and sym), mN–Me and mCCC (asymand sym) remain unchanged in the spectra of the complexes andin the free ligands. This observation helps to exclude any coordina-tion to the metals via nitrogen and oxygen donors.

3.4. Electronic spectra

The electronic spectra of 1, 2 and 3 in DMF were shown in Fig 2.The spectral data have been tabulated in Table 3. Complexes 1, 2and 3 display a lower energy band at 675, 672 and 675 nm,

Fig. 1. ORTEP view of the complex [Rh(2-C6H4py)2(L1)] (1) with atom labeling scheme (excluded H for clarity). (Symmetry codes: (i) �x, y, z; (ii) x, �y, �z; (iii) �x, �y, �z).

Table 2Coordination bond lengths [Å] and angles [�] for complex 1.

Bond length (Å)Rh(1)–C(11) 1.994(4) Rh(1)–C(11)#1 1.994(4)Rh(1)–N(1) 2.039(3) Rh(1)–N(1)#1 2.039(3)Rh(1)–S(1) 2.4854(11) Rh(1)–S(1)#1 2.4855(11)

Bond angle (�)C(11)–Rh(1)–N(1) 80.30(13) C(11)#1–Rh(1)–N(1)#1 80.29(13)C(11)–Rh(1)–N(1)#1 93.30(13) C(11)#1–Rh(1)–N(1) 93.30(13)N(1)–Rh(1)–S(1) 170.86(11) N(1)–Rh(1)–N(1)#1 171.09(17)C(11)–Rh(1)–S(1) 100.14(11) C(11)#1–Rh(1)–S(1)#1 100.15(11)C(11)#1–Rh(1)–S(1) 90.15(9) N(1)#1–Rh(1)–S(1)#1 90.15(9)N(1)#1–Rh(1)–S(1) 97.12(9) N(1)–Rh(1)–S(1)#1 97.11(9)S(1)–Rh(1)–S(1)#1 71.00(5) C(11)–Rh(1)–C(11)#1 88.8(2)

Symmetry transformations used to generate equivalent atoms: #1 �x + 1, y, �z +3/2.

350 400 450 5000

1

2

3

4

-----(3)

-----(1)-----(2)

Abs

orba

nce

Wavelength(nm)

Fig. 2. Electronic absorption spectra of 1, 2 and 3 in DMF.



respectively with low extinction coefficient values that correspondto the d–d transition. The higher energy band at 360 nm for allthree complexes with high extinction coefficient values are dueto the coordinated carbon atom from pipyridine moiety, C(r)–Rh(III) charge transfer (LMCT) transition. The other higher energyintense transitions at 374 nm and 360 nm are due to the n? p⁄

and p? p⁄ charge transfer transitions.

3.5. Redox studies

The cyclic voltammograms (CV) of the complexes 1, 2 and 3were recorded in DMF solvent at room temperature. Three elec-trode cell set up such as platinum, Ag/Ag+ (non-aquous) and aplatinum wire as a working, reference and auxiliary electroderespectively have been used for measurements. The cyclic voltam-mograms of all the three complexes 1, 2 and 3 have been shown inFig. 3 and the electrochemical data have been tabulated in Table 4.The complexes exhibit an irreversible reductive response at E1/2value � �0.697 to �0.767 V versus Ag/Ag+ (non-aqueous) corre-sponds to Rh3+/Rh+ couple. Small differences in the DEp values652, 658, 654 mV for 1, 2 and 3 respectively) have been observedthat increases in the order 2 > 3 > 1. This indicates that the easeof reduction from Rh(III) to Rh(I) with respect to ligand electronicenvironment is supposed to be much more in case of complex 1and least in complex 2. The trend of reduction potential values fol-lowed can be explained by the availability of the electrons on thedonor atoms of the dithiocarbamate ligands. The electron donatingcapacity through r bond of the six membered heterocyclic ring in-creases in the order 2 < 3 < 1 owing to the presence of differentsubstituents at the heteroatom i.e. highly electronegative O atom(2), �R effect of benzylic group (3), +I effect of Me group (1) andso the trend of reduction potential followed as such, which is fur-ther supported by theoretical calculation obtained from DFT study(viz. supporting information).

3.6. DNA binding study of [Rh(2-C6H4py)2(L1)]

3.6.1. Absorption spectral studyElectronic absorption spectroscopy is an effective method to

examine the binding modes of complex 1 with DNA. In general,binding of the compound to the DNA helix is testified by an in-crease of the CT band complex 1 due to the involvement of strongintercalative interactions between an aromatic chromophore ofcompound and the base pairs of DNA [26,47,48]. The absorptionspectra of complex 1 in the absence and presence of CT-DNA is gi-ven in Fig. 4. The extent of the hyperchromism in the absorptionband is generally consistent with the strength of intercalativebinding/interaction [49,50]. Fig. 5 indicates that the complex 1interacts strongly with CT-DNA (Kb = 1.54 � 105 M�1), and the ob-served spectral changes may be rationalized in terms of intercala-tive binding [51]. In order to further illustrate the binding strengthof the complex 1 with CT-DNA, the intrinsic binding constant Kb

was determined from the spectral titration data using the follow-ing equation [52]:

½DNA�=ðea � efÞ ¼ ½DNA�=ðeb � efÞ þ 1=½Kbðeb � ef Þ� ð1Þ

where [DNA] is the concentration of DNA, ef, ea and eb correspond tothe extinction coefficient, respectively, for the free complex 1, foreach addition of DNA to the complex 1 and for the complex 1 inthe fully bound form. A plot of [DNA]/(ea–ef) versus [DNA], givesKb, the intrinsic binding constant as the ratio of slope to the inter-cept. From the [DNA]/(ea–ef) versus [DNA] plot (Fig. 5), the bindingconstant Kb for complex 1 was estimated to be 1.54 � 105 M�1

(R = 0.99746 for five points), indicating a strong binding of the com-plex 1 with CT-DNA.

Table 3Electronic absorption spectral data.

Compound kmax (nm) (10�5, e/dm3 mol�1cm�1)a

1 360(7845), 374sh(6780), 385(6840), 675(160)2 360(7900), 375sh(6680), 390(6700), 672(210)3 360(7800), 382sh(6600), 390(6640), 675(200)

a In DMF solvent.

-1.5 -1.2 -0.9 -0.6 -0.3 0.0

-2

0

2

4

6

8

10

I(µ A

)

E (V)

231

Fig. 3. Cyclic voltammograms (scan rate 50 mV/s) of 1, 2 and 3 in DMF solution of0.1 M TBAP, using platinum working electrode.

Table 4Electrochemical dataa for the complexes 1, 2 and 3.

Complex Epc (V) Epa (V) DEp (mV) E1/2 (V)

1 �1.023 �0.371 652 �0.6972 �1.096 �0.438 658 �0.7673 �1.077 �0.413 654 �0.744

a Potentials versus non-aqueous Ag/Ag + reference electrode, scan rate 50 mV/s,supporting electrolyte: tetra-N-butylammonium perchlorate (0.1 M).

300 400 500 600 7000.0

0.1

0.2

0.3

0.4 f

a

Abs

.

λ(nm)

Fig. 4. Electronic spectral titration of complex 1with CT-DNA at 267 nm in tris–HClbuffer; [Compound] = 1.09 � 10�4; [DNA]: (a) 0.0, (b) 1.25 � 10�6, (c) 2.50 � 10�6,(d) 3.75 � 10�6, (e) 5.00 � 10�6, (f) 6.25 � 10�6mol L�1. Arrow indicates theincrease of DNA concentration.



3.6.2. Fluorescence quenching techniqueFluorescence intensity of EB bound to DNA at 612 nm shows a

decreasing trend with the increasing concentration of the com-pound. The quenching of EB bound to DNA by the compound isin agreement with the linear Stern–Volmer equation [53]:

I0=I ¼ 1þ Ksv½Q � ð2Þ

where I0 and I represent the fluorescence intensities in the absenceand presence of quencher, respectively. Ksv is a linear Stern–Volmerquenching constant, Q is the concentration of quencher. In thequenching plot in Fig. 7 of I0/I versus complex 1 Ksv value is givenby the ratio of the slope to intercept. The Ksv value for the complex1 is 0.87 � 104 (R = 0.98873 for five points), suggesting a strongaffinity of 1 to CT-DNA.

3.6.3. Number of binding sitesFlurorescence quenching data were used to determine the bind-

ing sites (n) for the compound 1 with CT-DNA. Fig. 6 shows thefluroscence spectra of EB-DNA in the presence of different concen-trations of compound 1. It can be seen that the fluroscence inten-sity at 612 nm was used to estimate Ksv and n.

If it is assumed that there are similar and independent bindingsites in EB-DNA, the relationship between the fluroscence intensityand the quencher medium can be deduced from the following Eq.(3):

nQ þ B ! Qn . . .B ð3Þwhere B is the flurophore, Q is the quencher, [nQ + B] is the postu-lated complex between the flurophore and n molecules of thequencher [26]. The constant K is given by Eq. (4):

K ¼ ½Qn . . .B�= Q½ �n:½B� ð4ÞIf the overall amount of biomolecules (bound or unbound with thequencher) is Bo, then [Bo] = [Qn� � �B]+ [B], where [B] is the concentra-tion of unbound biomolecules, and the relationship between thefluorescence intensity and the unbound biomolecule as [B]/[Bo] = I/Io, that is:

log½ðIo � IÞ=I� ¼ logKþ n log½Q � ð5Þwhere (n) is the number of binding site of compound complex 1with CT-DNA, which can be determined from the slope oflog[(Io�I)/I] versus log[Q], as shown in the Fig. 8. The calculatedvalue of the number of binding sites (n) is 1.10 (R = 0.99869 for fivepoints). The value of (n) approximately equals 1, and thus indicatesthe existence of one binding SITE in DNA for compound 1.

1 2 3 4 5 6 7

5.05.56.06.57.07.58.08.59.0

[DN

A]/(

ε a-ε

f) x

1010

Fig. 5. Plot of [DNA]/(ea–ef) vs. [DNA] for the absorption of CT-DNA with thecomplex 1 in tris–HCl buffer.

560 600 640 680 720 760

200

400

600

800

1000

1200

Flur

esce

nce

inte

nsity

(a.u

.)

λ (nm)

f

a

Fig. 6. Emission spectra of the CT-DNA-EB system in tris–HCl buffer upon thetitration of the compound complex 1. Kex = 522 nm; [EB] = 0.96 � 10�4 mol L�1;[DNA] = 9.9 � 10�6 mol L�1; [Compound]: (a) 0.0, (b) 1.36 � 10�5, (c) 2.72 � 10�5,(d) 4.08 � 10�5, (e) 5.44 � 10�5, (f) 6.80 � 10�5 mol L�1. Arrow indicates theincrease of compound concentration.

1 2 3 4 5 6 7 8 91.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

I / I o

[Complex 1] x 105

Fig. 7. Plot of I0/I vs. complex 1 for the titration of CT-DNA–EB systemwith complex1 using spectrofluorimeter; linear Stern–Volmer quenching constant (Ksv) forcomplex 1 = 0.87 � 104; (R = 0.98873 for five points).

-4.0 -4.2 -4.4 -4.6 -4.8

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

log[

(I o-I)

/I]

log[Complex 1]

Fig. 8. The linear plot shows log[(Io � I)/I] vs. log[Q], where R = 0.99869 for fivepoints.



3.6.4. Viscosity MeasurementTo further clarify the nature of interaction between complex 1

and CT DNA, viscosity measurements were carried out. Upon bind-ing, a DNA intercalator causes an increase in the viscosity of theDNA double helix due to its insertion between the DNA base pairsand consequently to the lengthening of the DNA double helix. Incontrast, a partial and/or nonclassical intercalation could bend(or kink) the DNA helix, reducing the effective length and its vis-cosity [54]. The method is generally considered the least unambig-uous to probe the mode of binding of a compound to DNA. Theeffect of 1 on viscosity of CT DNA is shown in Fig. 9. The viscosityof DNA increased dramatically upon addition of complex 1 and isnearly linear (R2 = 0.99621 for nine points). These results stronglyindicate that the complex 1 deeply into the DNA base pairs in inter-calative fashion.

3.7. Antibacterial activity

Antibacterial activity of the dithicarbamic acids (HL) and thecorresponding complexes are tabulated in Table 5. Comparisonsof the biological activity of the dithicarbamates and their rho-dium(III) derivatives with the standard antibiotics, chloramphenicolat different concentrations have been carried out taking usual pre-cautions. From this study, it is inferred that all the rhodium(III)complexes have higher activity than the ligand only, but little lessefficient than the antibiotics. The increased activity may be due tothe increase of the delocalization of p-electrons over the wholechelate ring imparts the increased lipophilic character to the metalcomplexes. This higher lipophilicity of the complexes facilitates thepenetration ability with a greater extent into the bacterial cell

membranes, and as result it perturbs the respiration process ofthe bacteria and diminish the further growth of themicroorganisms.

4. Conclusion

Three complexes of diimine dithiocarbamate mixed ligandframework Rh(2-C6H4py)2(L1)](1), Rh(2-C6H4py)2(L2)](2), Rh(2-C6H4py)2(L3)] (3) have been synthesized and characterized bymeans of solid and solution phase spectroscopic studies includingthe X-ray structure of 1. With the knowledge gained from the pres-ent study, attempts are now underway to bind these ligands in theC,N,S-coordination fashion to iridium and other metal ions havingoctahedral geometry. The present study of interaction withCT-DNA shows that these cyclometalated rhodium(III) complexeshaving dithiocarbamate moieties are good intercalative binding towith CT-DNA with an adequate number of coordination sites andthis strongly binding ability of the complexes as intercalator encour-age to develop these materials as good anticancer candidates. Fromthe antibacterial studies it is found that all the metal complexeshave higher activities than the free dithiocarbamic acids (LH)against four pathogenic bacteria (E. coli, V. cholerae, S. pneumoniaand B. cereus, among these three complex 2 has more antibacterialeffect.

Acknowledgements

Financial support from the Council of Scientific and IndustrialResearch (CSIR), New Delhi, India is gratefully acknowledged.

Appendix A. Supplementary data

CCDC 932588 contains the supplementary crystallographic datafor 1. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the CambridgeCrystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,UK; fax: (+44) 1223-336-033; or e-mail: [email protected] data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.poly.2013.11.028.

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0 2 4 6 8 10

1.00

1.04

1.08

1.12

1.16

(n/n

o)1/

3

[Complex 1]/[DNA]

Fig. 9. Effect of increase of amount of 1 on the relative viscosity of CT DNA in tris–HCl having 50 mM NaCl buffer. (R2 = 0.99621 for nine points).

Table 5Antibacterial data of free dithiocarbamic acids (LH) and rho-dium(III) complexes (1, 2 and 3) (100 lg/ml).

Compound fortreatment


E. coli V.cholerae

S.pneumoniae

B.cereus

L1H 05 05 04 03L2H 05 04 08 04L3 04 07 06 031 11 14 17 062 16 17 20 083 12 14 18 07Chloramphenicol 22 29 24 09DMF 0 0 0 0



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