Embed Size (px)
Citation preview
SYNTHESIS OF METALLATED MACROCYCLES AND THEIR INTERACTION WITH DNA/NUCLEOTIDES
,-ir',-
ABSTRACT THESIS / ' " " ^
SUBMITTED FOR THE AWARD OF THE DEGREE OF
Bottor of ^tjiloieiDplip
I i
IN
A ^ \ \ ' -^.y-'
CHEMISTRY ^ J^l
> *
.A BY J^y:
MUBASHIRA AZIZ
^ ^
s^s
^ ^ ^
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2 0 0 8
^^'f^rBfliP
Abstract
Abstract
Medicinal Inorganic Chemistry is an area of growing interest owing to the fact that
inorganic pharmaceuticals play a key role in clinical therapy (e.g; cisplatin and second
generation alternatives carboplatin and oxaliplatin - chemotherapeutic agents for solid
malignancies) and diagnostics (e.g; MR! contrast agents). For these inorganic
compounds, the metal serves as structural center for organizing the organic ligands in the
biologically relevant chemical space. Transition metals appear more appealing for this
purpose because they can support a multitude of coordination numbers and geometries
that go far beyond sp, sp^ and sp^ hybridization of carbon. The other key aspects for using
metal containing compounds as structural scaffolds are; the kinetic stability of the
coordination sphere in the biological environment, the stablization of certain or unusual
ligand geometries, acquired redox activity, enhanced cellular uptake, different modes of
action and synergistic effect from metal ion and ligands.
Ligands can modify the deleterious effects of metal ion overload, inhibit selected
metalloenzymes or facilitate systemic bioavailability of metal ions and can assist in
targeting specific tissues or enzymes. Some important ligand modifying effects include
modifying the reactivity, lipophilicity, stabilizing specific oxidation states and
contributing to substitution inertness. Biological systems themselves provide numerous
examples of designer ligands such as metalloprotein haemoglobin, manganese superoxide
dismutase (MnSOD) and catalase.
11
Since DNA has been identified as the primary molecular target of metal based anticancer
drugs, interaction of well tailored metal complexes with DNA ascertains the extent and
mode of DNA binding and the potential of these complexes to act as chemotherapeutic
agents. There are various modes of DNA interaction—covalent, non-covalent,
intercalation etc. DNA targeted metal based drugs which involve covalent binding to
nucleobase moieties have shown low degree of selectivity. Recently, non-covalent DNA
binding metal complexes particularly metallointercalators have received attention in the
development of efficient anticancer drugs. Intercalation is well known to strongly
influence the properties of the DNA and has been reported to as preliminary step in
mutagenesis. The interaction of metal complexes with nucleobases and/or nucleotides is
very well established, and is now known to be the basis of antitumor drug action. The
metal complexes, which are favoured as effective drug candidates are molecules that;
(i) damage DNA
(ii) block DNA synthesis indirectly through inhibition of nucleic acid precursor
biosynthesis
(iii) disrupt hormonal stimulation of cell growth
(iv) must recognize nucleic acid particularly in sequence specific fashion and then bind to
DNA in a way that alters their function.
Recent advances in ligand design for metal-based drugs reveal that the ligand
modifications in geometry, size, hydrophobicity, planarity, charge and hydrogen bonding
I l l
of the complexes, lead to spectacular changes in binding modes, location, affinity and to
a different cleavage effect.
Macrocycles form a major class of ligand molecules that encompasses a wide range of
structure and functional groups. Their structures can be easily manipulated allowing the
preparation of a variety of ring sizes as well as the addition of different side chains
producing different types of macrocyclic molecules suitable for specific uses. With these
perspectives, we have synthesized and characterized a variety of macrocyclic transition
metal complexes. The interaction of these complexes with CT DNA/5'GMP have been
studied by various biophysical techniques.
New water soluble Co(II), Ni(II) and Cu(II) complexes of 4,15-bis(2-hydroxyethyl)-
2,4,6,13,15,17- hexaazatricyclodocosane were synthesized and characterized by various
techniques, viz. elemental analysis, conductivity measurements, infrared, electronic, ESI-
MS, ' H and ' C NMR spectroscopy. Molar conductance measurements in aqueous
solution showed that complexes are ionic in nature. On the basis of spectroscopic data, a
square planar geometry was assigned to the complexes involving four N-atoms of the two
cyclohexane moieties. Interaction studies of [C2oH42N602Co]Cl2 and [C20H42N6O2CU ]Cl2
with CT DNA were carried using UV/Visible absorption spectroscopy, fluorescence
spectrophotometry, cyclic voltammetry and viscosity measurements. Absorption spectral
traces reveal 27.7% and 23.3 % hyperchromism for complexes [C2oH42N602Co]Cl2 and
[C2oH42N602Cu]Cl2, respectively indicative of strong binding to CT DNA. These results
were authenticated by fluorescence quenching experimer\ts and viscosity measurements.
IV
The intrinsic binding constants Kb of [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 are
2.94.10'* M"' and 2.71.10'* M"', respectively. Early transition metals show preference for
06 position while later ones copper and cobalt prefer N7 position of DNA base guanine.
To validate this hypothesis, interaction studies of copper (11) and cobalt (II) complexes
were carried out with 5'GMP, which revealed electrostatic interactions are more favored
alongwith hydrogen bonding than coordinate covalent interaction to N7 position of
guanine. The stability of the complexes were investigated by UV/Vis and cyclic
voltammetry. The UV/Vis spectra and the voltammograms of the complexes exhibited no
change over a wide pH range, which suggested the robust nature of these complexes in
solution. The electrochemical behaviour of complexes was studied in Tris-HCI/KCl
buffer and displayed a quasireversible M'VM' redox couples. In presence of DNA the
complexes exhibited a shift in the formal potential with a decrease in anodic and cathodic
peak currents indicating the binding of complexes to CT DNA.
Benzimidazoles are very useful subunits for the development of molecules of
pharmaceutical or biological interest. The biological relevance of these heterocyclic
aromatic building blocks is due to their structural similarity to purine nucleobases. New
benzimidazole derived homodinuclear macrocyclic complexes of Cobalt(Il), Copper(II)
and Zinc(ll) were isolated from the newly synthesized ligand 2,2,2',2'-S,S[bis(bis-N,N-2-
thiobenzimidazolyloxalato-l,2-ethane)]. The structures of the complexes were elucidated
by elemental analysis, molar conductance measurements, IR, ' H N M R , '^C NMR,
electronic and ESI MS spectroscopic techniques. In complex [C36H24N804S4Co2]Cl4,
Co(ll) ions possess a tetrahedral coordination environment composed of O2S2 donor
atoms while its Cu(II) and Zn(II) counterparts [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] respectively reveal a six coordinate octahedral structure, defined
by the O2S2 donors from the macrocyclic ring and two chloride ions. Molar conductance
and spectroscopic data also support the proposed geometry of the complexes. DNA
binding properties of complexes [C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] Were investigated using electronic absorption spectroscopy,
fluorescence spectroscopy, viscosity measurements and cyclic voltammetry. The
absorption spectra of the complexes [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2]
with calf thymus DNA showed hypochromism, while complex [C36H24N804S4Co2]Cl4
showed hyperchromism attributed to a partial intercalation and electrostatic binding
modes respectively. The intrinsic binding constant Kb of complexes
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] were
determined as 16.6 . 10'* M"', 4.25 . 10'* M"' and 3.0 . 10'* M"', respectively. The decrease
in the relative specific viscosity of calf thymus DNA with increasing concentration of the
complexes authenticates the partial intercalation binding mode. Gel electrophoresis of
complex [C36H24N8O4S4CI4CU2] with pBR322 plasmid DNA demonstrated that complex
exhibits excellent "artificial" nuclease activity via an oxidative cleavage pathway.
Homonuclear bis-macrocyclic complexes where the two metal ions act in unison may
bind much more tightly than the corresponding monometallic complexes through a
concerted two point DNA binding mechanism. A new series of bis-macrocyclic
VI
complexes of Co(II), Ni(II) and Cu(II) containing pyridyl bridges between 13-membered
macrocyclic subunits have been synthesized via in situ one pot template condensation
reaction (lOPTCR). The proposed structures of these new dinuclear complexes are
consistent with the data obtained from elemental analysis, molar conductance, IR, EPR,
UV/Vis, ESI-MS, ' H and ' C spectroscopy. The complexes have square planar geometry
with four secondary nitrogen atoms coordinated to the metal ion. DNA binding properties
of the complexes [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 were investigated
by absorption and emission titrations; cyclic voltammetry and viscosity measurements.
Complexes [C2iH43NiiCo2](C104)4 and [C2iH43Ni|Cu2](C104)4 are avid DNA binders
with binding constants K 1.64 x 10 and 2.05 x 10 , respectively. Hyperchromism,
decrease in emission intensity of DNA bound EthBr and changes observed in the
viscosity and cyclic voltammograms in presence of added metal complexes reveals that
the complexes bind to DNA predominantly by electrostatic attraction, substantiated by
absorption titration with 5'GMP. No shifts in the visible absorption bands were observed
which implies that there is no change of the coordination environment of the metal ions.
These results reveal that the complexes interact with 5'GMP exclusively through
hydrogen bonding of-NH- groups to base nitrogen atom or phosphate oxygen atom or by
electrostatic interactions of the metal ions utilizing the phosphate oxygen atoms of the
nucleotide. The quenching of EthBr bound to DNA by the metal complex is in good
agreement with the linear Stern-Volmer equation.
Vll
Functionalized pyran-2-ones are key structural motifs found in a large number of
biologically important natural products. In this context, another series of new
macrocyclic Co(II), Ni(II), Cu(II) and Zn(II) complexes were synthesized by schiff base
condensation reaction of 1,2-diaminocyclohexane and the P-diketonate functionalized
coumarin ligand 3-acetoacetyl-7-methyl -pyrano-(4,3-b)-pyran-2,5-dione. The formation
of the ligand and the complexes was ascertained from elemental analysis, IR, 'H N M R ,
C NMR, UV/Vis and mass spectroscopies. Molar conductance and spectroscopic data
reveal an octahedral geometry of the complexes around the metal ions. In vitro DNA
binding studies of the ligand and the C3gH44N40i8Cl2 complex were investigated by
electronic absorption and fluorescence titrations. The ligand exhibited hyperchromism
due to the aggregation of the planar ligand molecule on the DNA surface. The complex
shows hypochromism in the UV region and the hyperchromism at the d-d absorption
bands, which suggests that the complex has intimate association with CT DNA and binds
to helix via partial intercalation and simultaneously increases the probability for
electrostatic association due to the strong Lewis acid copper center. The binding
constants K for the ligand and the complexes are 3.2.10^ M"' and 2.18 .10^ M"
respectively. To identify the specific binding mode, absorption titration of
C38H44N40,8Cl2 complcx with 5'GMP were performed. Hyperchromism at the UV
absorption bands and no change at the d-d absorption bands supported a non-covalent
binding mode. This was also confirmed by NMR studies of the complex with 5'GMP.
The intrinsic binding constants were also estimated from emission titrations using
VIII
Scatchard equation and were found to be 3.0 .10'* M''and 2.0 .lO"* M"' for the ligand and
the complex, respectively. The metal complex also cleaved pBR322 plasmid DNA by an
oxidative cleavage mechanism in presence of ascorbic acid as the reducing agent. In vitro
cytotoxicity of the ligand was determined against different human cancer cell lines using
standard protocols. The highest growth inhibitory effect was observed for CNS cancer
SNB-75 cell lines.
SYNTHESIS OF METALLATED MACROCYCLES AND THEIR INTERACTION WITH DNA/NUCLEOTIDES
THESIS SUBMITTED FOR THE AWARD OF THE DEGREE OF
Bottor of ^I)tlQ£iDpI)p
Yi IN
CHEMISTRY i i ^ j l
47.. . ; .
MUBASHIRA AZIZ
DEPARTMENT OF CHEMISTRY ALIGARH MUSLIM UNIVERSITY
ALIGARH (INDIA)
2008
- . .r>*
T6866
To !My Ramify
(Dr.(Mrs.) Taru^^rjmand Department of Chemistry AUgarh Muslim University
Aligarh-202002, India.
Phone No. 0571-2703893 E-mail: [email protected]
Certificate
The work embodied in this thesis entitled "Synthesis of Metallated Macrocycles and
their Interaction with DNA/Nucleotides" is the result of original researches carried out
by Miss. Mubashira Aziz under my supervision and is suitable for the award of Ph.D
degree.
Dr. (Mrs.) Farukh Arjmand
CONTENTS
Page No.
Acknowledgements
Publications
Abstract i-viii
Abbreviations
CHAPTER I: Introduction 1-37
CHAPTER H: Experimental 38-53
CHAPTER HI: Synthesis, Spectroscopic Studies of New 54-81
Water-Soluble Co(II) and Cu(II) Macrocyclic
Complexes of 4,15-bis(2-hydroxyethyl)
-2,4,6,13,15,17- hexaazatricyclodocosane:
Their Interaction Studies with Calf Thymus
DNA and Guanosine 5' Monophosphate
CHAPTER IV: Synthesis and Characterization of Dinuclear
Macrocyclic Cobalt(II), Copper(II) and Zinc(II)
Complexes derived from 2,2,2,2 -S,S
[bis(bis-N,N-2-thiobenzimidazolyloxalato-l,2-ethane)]:
DNA Binding and Cleavage Studies
82-110
CHAPTER V: Synthesis and Biophysical Studies of
Bis-macrocyclic cobalt/copper (II)
Complexes having Pyridine Spacer with
CT DNA and 5' GMP
111-135
CHAPTER VI: Cytotoxic Activity of a Potent Coumarin ligand
3-acetoacetyl-7-methyl-pyrano-(4,3-b)-pyran-2,5-dione
against Different Cell Lines: Design of Metal based
Chemotherapeutic Agents derived from this Ligand
and their DNA Binding Profile
136-161
REFERENCES: 162-185
Acknowledgements
Acknowledgements
In the name of "Allah, Most Gracious, Most Merciful"
All praise and thanks to Almighty Allah for letting me to accomplish this research
endeavor. During this long and eventful journey I have had a vigorous training to
excellence in research, to move forward with investigations in depth and in particular, I
have learned a lot about myself I found weakness where I thought I was strong and
strengths where I felt vulnerable. To say it has been a learning experience is a vast
understatement, I will remember everything I have learned here and use it to guide me
through the rest of my days both inside and outside of the lab.
I am eternally grateful to my able supervisor Dr. (Mrs.) Farukh Arjmand for her
proficient guidance, incessant encouragement, incredible patience and constructive
critiques. Her skills as an innovative and an accomplished researcher, ebullient scientific
attitude, good organizing ability, enthusiasm, inspiration and her great efforts to explain
things clearly and simply, served to give me a sense of direction in research and life in
general. Beyond research, she is an exemplary advocate, psychologist, editor, motivator
or to put it simply a complete institution in itself
Much gratitude is extended to Prof. Sartaj Tabassum for so generously sharing his
knowledge and expertise. His thoughtful advice and deep insight into the work are
extremely valuable in getting through the difficult times, whenever the problems got
tough.
My sincere thanks to Chairman, Department of Chemistry, AMU, Aligarh for providing
me the necessary research facilities during the period of my research work.
Thanks are due to Regional Sophisticated Instrumentation Center, Central Drug Research
Institute and Indian Institute of Technology, Bombay for providing access to their CHN,
IR, NMR, mass spectra and EPR facilities, respectively.
My incredible thanks to University Grants Commission (Project Fellow) and Council of
Scientific and Industrial Research (Senior Research Fellow), New Delhi for their kind
financial support through scheme No. 12-21/2003 (SR) and 01 (1982)/05-EMR-II),
respectively.
I would like to thank all the past and present lab colleagues for their cooperation
throughout my research work. The biggest thanks belong to Irshad, Mala and Suvigya
for their sound advice, good company, and fruitful discussions and of course to the
wonderful moments we had together.
My thanks also go to my friends and roommates for being so nice and considerate to me.
I cannot end without thanking my loving and supportive family without whom I could
have never made it this far. I wish to thank my parents, who raised me, supported me,
taught me and encouraged me to do my best in all matters of life. My heartfelt thanks to
my special aunt for her understanding and unflinching courage. You have contributed
your entire life to our family. You always did everything you could and gave everything
you had and just knowing you were always there, has made all the difference.
And last but not the least many thanks to my elder brother and sisters for their love, care,
continuous support and interest in what 1 do. It is to my family that I dedicate this work.
Mubasnira Aziz
Publications
Publications
1. Synthesis, Spectroscopic Studies of New Water-Soluble Co(II) and Cu(II) Macrocyclic
Complexes of 4,15-bis(2-hydroxyethyl)-2,4,6,13,15,17- hexaazatricyclodocosane: Their
Interaction Studies with Calf Thymus DNA and Guanosine 5' Monophosphate.
Farukh Arjmand, Mubashira Aziz and Mala Chauhan, J. Inclusion Phenom. Macrocyclic Chem., 61,265 (2008).
2. Synthesis and Characterization of Dinuclear Macrocyclic Cobalt(II), Copper(II) and
Zinc(II) Complexes derived from 2,2,2 ,2 -S,S[bis(bis-N,N-2-thiobenzimidazolyloxa!ato-
1,2-ethane)]: DNA Binding and Cleavage Studies.
Farukh Arjmand and Mubashira Aziz, Eur. J. Inorg. Chem., 2008 (doi: 10.1016/j.ejmech.2008.05.006).
3. Synthesis and Biophysical Studies of Bis-macrocyclic cobalt/copper (II) Complexes
having Pyridine Spacer with CT DNA and 5' GMP.
Faruidi Arjmand and Mubashira Aziz, Chemistry: An Asian Journal (Communicated) 2008.
4. Cytotoxic Activity of a Potent Coumarin ligand 3-acetoacetyl-7-methyl-pyrano-(4,3-
b)-pyran-2,5-dione against Different Cell Lines: Design of Metal based
Chemotherapeutic Agents derived from this Ligand and their DNA Binding Profile.
Farukh Arjmand and Mubashira Aziz, Bioorg. Med. Chem., (Communicated) 2008.
Abstract
Abstract
Medicinal Inorganic Chemistry is an area of growing interest owing to the fact that
inorganic pharmaceuticals play a key role in clinical therapy (e.g; cisplatin and second
generation alternatives carboplatin and oxaliplatin - chemotherapeutic agents for solid
malignancies) and diagnostics (e.g; MRI contrast agents). For these inorganic
compounds, the metal serves as structural center for organizing the organic ligands in the
biologically relevant chemical space. Transition metals appear more appealing for this
purpose because they can support a multitude of coordination numbers and geometries
that go far beyond sp, sp^ and sp^ hybridization of carbon. The other key aspects for using
metal containing compounds as structural scaffolds are; the kinetic stability of the
coordination sphere in the biological environment, the stablization of certain or unusual
ligand geometries, acquired redox activity, enhanced cellular uptake, different modes of
action and synergistic effect from metal ion and ligands.
Ligands can modify the deleterious effects of metal ion overload, inhibit selected
metalloenzymes or facilitate systemic bioavailability of metal ions and can assist in
targeting specific tissues or enzymes. Some important ligand modifying effects include
modifying the reactivity, lipophilicity, stabilizing specific oxidation states and
contributing to substitution inertness. Biological systems themselves provide numerous
examples of designer ligands such as metal loprotein haemoglobin, manganese superoxide
dismutase (MnSOD) and catalase.
Since DNA has been identified as the primary molecular target of metal based anticancer
drugs, interaction of well tailored metal complexes with DNA ascertains the extent and
mode of DNA binding and the potential of these complexes to act as chemotherapeutic
agents. There are various modes of DNA interaction—covalent, non-covalent,
intercalation etc. DNA targeted metal based drugs which involve covalent binding to
nucleobase moieties have shown low degree of selectivity. Recently, non-covalent DNA
binding metal complexes particularly metallointercalators have received attention in the
development of efficient anticancer drugs. Intercalation is well known to strongly
influence the properties of the DNA and has been reported to as preliminary step in
mutagenesis. The interaction of metal complexes with nucleobases and/or nucleotides is
very well established, and is now known to be the basis of antitumor drug action. The
metal complexes, which are favoured as effective drug candidates are molecules that;
(i) damage DNA
(ii) block DNA synthesis indirectly through inhibition of nucleic acid precursor
biosynthesis
(iii) disrupt hormonal stimulation of cell growth
(iv) must recognize nucleic acid particularly in sequence specific fashion and then bind to
DNA in a way that alters their function.
Recent advances in ligand design for metal-based drugs reveal that the ligand
modifications in geometry, size, hydrophobicity, planarity, charge and hydrogen bonding
Ill
of the complexes, lead to spectacular changes in binding modes, location, affinity and to
a different cleavage effect.
Macrocycles form a major class of ligand molecules that encompasses a wide range of
structure and functional groups. Their structures can be easily manipulated allowing the
preparation of a variety of ring sizes as well as the addition of different side chains
producing different types of macrocyclic molecules suitable for specific uses. With these
perspectives, we have synthesized and characterized a variety of macrocyclic transition
metal complexes. The interaction of these complexes with CT DNA/5'GMP have been
studied by various biophysical techniques.
New water soluble Co(ll), Ni(II) and Cu(ll) complexes of 4,15-bis(2-hydroxyethyl)-
2,4,6,13,15,17- hexaazatricyclodocosane were synthesized and characterized by various
techniques, viz. elemental analysis, conductivity measurements, infrared, electronic, ESI-
MS, H and C NMR spectroscopy. Molar conductance measurements in aqueous
solution showed that complexes are ionic in nature. On the basis of spectroscopic data, a
square planar geometry was assigned to the complexes involving four N-atoms of the two
cyclohexane moieties. Interaction studies of [C2oH42N602Co]Cl2 and [C20H42N6O2CU ]Cl2
with CT DNA were carried using UV/Visible absorption spectroscopy, fluorescence
spectrophotometry, cyclic voltammetry and viscosity measurements. Absorption spectral
traces reveal 27.7% and 23.3 % hyperchromism for complexes [C2oH42N602Co]Cl2 and
[C2oH42N602Cu]Cl2, respectively indicative of strong binding to CT DNA. These results
were authenticated by fluorescence quenching experiments and viscosity measurements.
IV
The intrinsic binding constants Kb of [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 are
2.94.10'' M"' and 2.71.10'* M'', respectively. Early transition metals show preference for
06 position while later ones copper and cobalt prefer N7 position of DNA base guanine.
To validate this hypothesis, interaction studies of copper (II) and cobalt (II) complexes
were carried out with 5'GMP, which revealed electrostatic interactions are more favored
alongwith hydrogen bonding than coordinate covalent interaction to N7 position of
guanine. The stability of the complexes were investigated by UV/Vis and cyclic
voltammetry. The UV/Vis spectra and the voltammograms of the complexes exhibited no
change over a wide pH range, which suggested the robust nature of these complexes in
solution. The electrochemical behaviour of complexes was studied in Tris-HCl/KCl
buffer and displayed a quasireversible M'VM' redox couples. In presence of DNA the
complexes exhibited a shift in the formal potential with a decrease in anodic and cathodic
peak currents indicating the binding of complexes to CT DNA.
Benzimidazoles are very useful subunits for the development of molecules of
pharmaceutical or biological interest. The biological relevance of these heterocyclic
aromatic building blocks is due to their structural similarity to purine nucleobases. New
benzimidazole derived homodinuclear macrocyclic complexes of Cobalt(II), Copper(II)
and Zinc(II) were isolated from the newly synthesized ligand 2,2,2',2'-S,S[bis(bis-N,N-2-
thiobenzimidazolyloxalato-l,2-ethane)]. The structures of the complexes were elucidated
by elemental analysis, molar conductance measurements, IR, 'H N M R , '^C NMR,
electronic and ESI MS spectroscopic techniques. In complex [C36H24N804S4Co2]Cl4,
Co(II) ions possess a tetrahedral coordination environment composed of O2S2 donor
atoms while its Cu(II) and Zn(II) counterparts [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] respectively reveal a six coordinate octahedral structure, defined
by the O2S2 donors from the macrocyclic ring and two chloride ions. Molar conductance
and spectroscopic data also support the proposed geometry of the complexes. DNA
binding properties of complexes [C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] wcrc investigated using electronic absorption spectroscopy,
fluorescence spectroscopy, viscosity measurements and cyclic voltammetry. The
absorption spectra of the complexes [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2]
with calf thymus DNA showed hypochromism, while complex [C36H24N804S4Co2]Cl4
showed hyperchromism attributed to a partial intercalation and electrostatic binding
modes respectively. The intrinsic binding constant Kb of complexes
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] were
determined as 16.6 . 10'*M"', 4.25 . lO'^M'' and 3.0 . lO' M"', respectively. The decrease
in the relative specific viscosity of calf thymus DNA with increasing concentration of the
complexes authenticates the partial intercalation binding mode. Gel electrophoresis of
complex [C36H24N8O4S4CI4CU2] with pBR322 plasmid DNA demonstrated that complex
exhibits excellent "artificial" nuclease activity via an oxidative cleavage pathway.
Homonuclear bis-macrocyclic complexes where the two metal ions act in unison may
bind much more tightly than the corresponding monometallic complexes through a
concerted two point DNA binding mechanism. A new series of bis-macrocyclic
VI
complexes of Co(Il), Ni(II) and Cu(II) containing pyridyl bridges between 13-membered
macrocyclic subunits have been synthesized via in situ one pot template condensation
reaction (lOPTCR). The proposed structures of these new dinuclear complexes are
consistent with the data obtained from elemental analysis, molar conductance, IR, EPR,
UV/Vis, ESI-MS, 'H and ' C spectroscopy. The complexes have square planar geometry
with four secondary nitrogen atoms coordinated to the metal ion. DNA binding properties
of the complexes [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 were investigated
by absorption and emission titrations; cyclic voltammetry and viscosity measurements.
Complexes [C2iH43N|iCo2](C104)4 and [C2iH43NiiCu2](C104)4 are avid DNA binders
with binding constants K/, 1.64 x 10 and 2.05 x 10 , respectively. Hyperchromism,
decrease in emission intensity of DNA bound EthBr and changes observed in the
viscosity and cyclic voltammograms in presence of added metal complexes reveals that
the complexes bind to DNA predominantly by electrostatic attraction, substantiated by
absorption titration with 5'GMP. No shifts in the visible absorption bands were observed
which implies that there is no change of the coordination environment of the metal ions.
These results reveal that the complexes interact with 5'GMP exclusively through
hydrogen bonding of-NH- groups to base nitrogen atom or phosphate oxygen atom or by
electrostatic interactions of the metal ions utilizing the phosphate oxygen atoms of the
nucleotide. The quenching of EthBr bound to DNA by the metal complex is in good
agreement with the linear Stern-Volmer equation.
vu
Functionalized pyran-2-ones are key structural motifs found in a large number of
biologically important natural products. In this context, another series of new
macrocyclic Co(II), Ni(II), Cu(II) and Zn(II) complexes were synthesized by schiff base
condensation reaction of 1,2-diaminocyclohexane and the P-diketonate functionalized
coumarin ligand 3-acetoacetyl-7-methyl -pyrano-(4,3-b)-pyran-2,5-dione. The formation
of the ligand and the complexes was ascertained from elemental analysis, IR, 'H N M R ,
' C NMR, UV/Vis and mass spectroscopies. Molar conductance and spectroscopic data
reveal an octahedral geometry of the complexes around the metal ions. In vitro DNA
binding studies of the ligand and the C38H44N4O18CI2 complex were investigated by
electronic absorption and fluorescence titrations. The ligand exhibited hyperchromism
due to the aggregation of the planar ligand molecule on the DNA surface. The complex
shows hypochromism in the UV region and the hyperchromism at the d-d absorption
bands, which suggests that the complex has intimate association with CT DNA and binds
to helix via partial intercalation and simultaneously increases the probability for
electrostatic association due to the strong Lewis acid copper center. The binding
constants K* for the ligand and the complexes are 3.2.10^ M"' and 2.18 .10^ M"'
respectively. To identify the specific binding mode, absorption titration of
C38H44N4O18CI2 complex with 5'GMP were performed. Hyperchromism at the UV
absorption bands and no change at the d-d absorption bands supported a non-covalent
binding mode. This was also confirmed by NMR studies of the complex with 5'GMP.
The intrinsic binding constants were also estimated from emission titrations using
Vlll
Scatchard equation and were found to be 3.0 .10'* M''and 2.0 .lO"* M"' for the ligand and
the complex, respectively. The metal complex also cleaved pBR322 plasmid DNA by an
oxidative cleavage mechanism in presence of ascorbic acid as the reducing agent. In vitro
cytotoxicity of the ligand was determined against different human cancer cell lines using
standard protocols. The highest growth inhibitory effect was observed for CNS cancer
SNB-75 cell lines.
bzim
CTDNA
CV
dach
DMF
DMSO
EthBr
EtOH
GMP
IL
LMCT
LNT
MeOH
OD
PTS
SRB
tris
TCNE
UV
Abbreviations
benzimidazole
calf thymus DNA
cyclic voltammetry
diaminocyclohexane
dimethylformamide
dimethylsulfoxide
ethidium bromide
ethanol
guanosine monophosphate
intraligand
ligand to metal charge transfer
liquid nitrogen temperature
methanol
optical density
para toluene sulphonic acid
Sulphorhodamine B
tris(hydroxymethyl)aminomethane
tetracyanoethylene
ultra-violet
CHAPTER I
Introduction
Introduction
Medicinal Inorganic Chemistry is an interdisciplinary area of growing interest, which
offers exciting possibilities for the design of novel metal-based therapeutic
agents/diagnostic agents with unique mechanisms of action [1-8]. Undoubtedly, the well
recognized success of Bamett Rosenberg in 1960 with the discovery of cisplatin [cis-
(PtCl2(NH3)2)] as the first archetypical inorganic anticancer drug opened new vistas in the
frontier areas of research in chemical and biological sciences [9-12]. The success of
cisplatin has aroused great interest in the development of new innovative metal
complexes - 'classic' platinum based, non-classic platinum-based (as they did not mimic
cisplatin in their modes of action) and non-platinum metal complexes [13,14]. Hitherto,
thousands of complexes in last four decades have been synthesized and investigated in
vitro and in vivo for active therapeutic profile to treat diseases including Aids HIV,
Alzheimer's, cancer and diabetes [15,16]. Amongst the noteworthy are: AMD3100,
Mozobil- a potent anti HIV agent [17], NAMI-A and KP1019-rutheniimi anficancer
agents [18-20], BMOV- Bis(maltolatooxovanadium IV) the first purpose designed
vanadium based insulin enhancing antidiabetic agent [21-23] (Figure 1). Non-platinum
class of metal-based chemotherapeutics possess preconditions viz; differences in
coordination geometry, binding preferences according to the HSAB, hard and soft acids
and bases principle (late transition metal divalent ions show preference to N7 and 06
atoms of guanine, which were considered as borderline Pearson bases; hard acids prefer
hard bases and so on), electrovalency and redox activity and icinetics of ligand exchange
reactions or even the replacement of essential metals [24].
(b)
(c) NH '
e
"N Cl>^ I ^ C l
ci" I \i
O ^ V '"CH3 CH3
© NH
N H
(d)
Figure 1. Structures of chemotherapeutic metal-based agents (a) AMD3100 (b) BMOV (c) NAMI-A and (d) KP1019.
These preconditions form the chemical basis for a diversity of pharmacologically relevant
interactions with biomolecules viz; molecular DNA and nucleotides. This has opened up
a new field of nucleic acid—metal ion interaction that is contributing beyond traditional
sciences, biotechnology, biochemistry, genetics, medicinal chemistry and others. Metal
ions are essential for charge compensation of the negatively charged phosphate sugar
backbone [25-27]. They are instrumental for proper folding, and are crucial cofactors for
ribozyme catalysis [28,29]. The design of strategy for synthesis of the metal-based drugs
employs well-tailored multifunctional biologically active ligands [30-32]. The reactivity
of the metal is subordinate to the activity of the ligands; the metal ions rather serve to
modulate/tune this activity and achieve specific molecular recognition with a defined
target thereby limiting drug toxicity. Metal complexation alters the solubility and
lipophilicity of the drug resulting in changes in its pharmacokinetics, biodistribution and
biotransformation [33]. Transition metals possess different oxidation states which not
only allow for modification of the 3-dimensional space into which the molecule can fit
but significantly allows them to participate in biological redox chemistry [34]. In
addition, the ability to undergo ligand exchange reactions offer opportunities for metals
to interact and coordinate with nucleobases of molecular target DNA [35]. By optimizing
choice of ligands and oxidation state, kinetics and thermodynamic properties of
metallopharmoceuticals can be exploited for specific therapeutic requirements [36].
To design a metal- based applicable anticancer drug, macrocyclic ligands hold promise as
they are rigid enough to provide metal binding sites and orient functional groups
stereoselectively, yet flexible enough to accommodate structural changes required for
induced fit recognition of biological targets [37]. Metallomacrocycles are of interest in
fields as diverse as catalysis, selective metal recovery, sensors and therapy and diagnosis
[38-43]. Saturated tetraazamacrocycles-l,4,7,10-tetraazacyclododecane (cyclam) and
1,4,8,11- tetraazacyclododecane (cyclen) and their derivatives have been extensively
studied for their antitumour and anti-HIV activity [44-46]. There is current medical
interest in the drug AMDS 100, a bicyclam containing cyclam units connected by a p-
phenylene bis(methylene) (p-xylyl) linker. Mozobil AMD3100 is in phase (II) clinical
trials for stem cell transplantation used in the treatment of patient who have cancers
involving the blood and immune system [47]. It is also most potent anti-HIV agent
known [48]. These cyclams are strong chelating agents. Complexation of AMDS 100 to
Zn(II) enhances co-receptor binding strength and anti-HIV activity, whereas Pd(II)
binding inactivates the drug. The order of activity is Zn(II)2 > AMDS 100 > Ni(ir) 2 >
Cu(II) 2 » Co(II) 2 > » Pd(II) 2 [49].
BiTPC, a Bi(m) complex of TPC (1,4,7,10-tetrakis(2-pyridylmethyl)-1,4,7,10-
tetraazacyclododecane exhibited a very high cytotoxic activity against melanoma B16-
BLC cells [50]. The IC50 of BiTPC is 4.1x10* M, which was 100 times more potent than
the cisplatin. Stable Au(III) complex of 1,4,7-tetrazacyclododecane is known to induce
distortions of DNA double helix [51] and was foimd to be more cytotoxic than cisplatin
against A-549 and HCT-116 cell lines (Figure 2).
XL S J\ ^ N ^ N ^ CI. /CI I
N ^ ^ ^ HNC ^^«
H
Figure 2. Structures of TPC (left) andAuTACN (right).
A characteristic feature of many malignant cancers is the rapid, and unregulated growth
of cell populations. These cells also typically possess genetic instability, predisposing
them to develop resistance to chemotherapeutic agents [52,53]. During cancer
chemotherapy, after single agent treatment, a resistance subpopulation of cells is often
seen to survive. To circumvent this resistance for effective clinical treatment, a co
administration of a combination of at least two drugs is administered simultaneously or
sequentially to inhibit tumour growth [54]. This approach although successful leads to
toxicity problems and therefore to reduce non-tumour cell toxicity chemotherapeutic
agents with specific target location are being developed [55-57]. Towards this approach,
two biologically significant moieties are tethered in combination and thereby referred as
'conjugates' to yield a more efficacious, less toxic chemotherapeutic drug. Darren Magda
et al have synthesized a conjugate of motexafm gadolinium MGd and an experimental
drug demonstrating significant tumours targeting with a porphyrin methotrexate
(Matrex® trade name) MTX N-[4-[[(2,4-diamino-6-
pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid -a DNA synthesis inhibitor
[58] (Figure 3). The resulting conjugates exhibited greater activity than the corresponding
amide conjugate or methotrexate alone. In vivo tumour localization of the conjugates is
often correlated with higher molecular weight and more lipophilic molecules which
extent circulation times and improves uptake of the conjugates [59,60]. This approach has
facilitated the development of anticancer drugs with improved selectivity and
bioavailablity where the macrocyclic core behaves as a 'pro-drug' that cleaves under
physiological conditions. Currently, MGd is undergoing clinical trials for a range of
cancer indications [61,62]. MGd, which is MRI detectable, has been extensively studied
in combination with X-ray radiation therapy (XRT) or as a direct chemotherapeutic agent
either alone or in conjunction with other anticancer agents [63,64].
MGdl MTX2
ir,N—Gi—*ct
0 p:xi O OR NHi Ri
3-a-isomer 3ir-i»onief
Figure 3. Synthesis of ester conjugate from MGd and MTX.
Polyaza macrocyclic ligands and complexes bearing additional functional pendant arms,
such as aminoalkyl, hydroxyalkyl, carboxylic acid, amide and carboxylic esters have
received much attention owing to their interesting chemical properties and potential
applications in various fields [65-72]. It has been demonstrated that introduction of
additional functional groups into polyaza macrocylic compounds causes a considerable
change into chemical and biological properties [73,74]. Since the properties of the
macrocyclic compounds are correlated with structural characteristic of the ligands, many
efforts have been made to obtain new types of macrocyclic compounds, which are
expected to exhibit specific properties [75]. Structural parameters, which can be varied,
include the metal ions viz; size, degree of unsaturation and types and numbers of
substituents on the macrocyclic rings. Shin-Goel Kang et al have synthesized a new
macrocyclic ligand 3,14-dimethyl-2,6,13,17-tetraazatricyclo [16,4,0,0] docosa-2,12-diene
that contains two cyclohexane rings at the macrocyclic backbone, by the non-template
reaction of methyl vinyl ketone with 1,2-diaminocyclohexane [76]. Although a majority
of macrocyclic ligands have beep prepared by metal ion template condensation of ketones
and multiamines, some of them have also been obtained by non-template reactions
[77,78].
Template directed synthesis is the organization of an assembly of atoms with respect to
one or more geometric loci to achieve a particular linking of atoms [79,80]. It utilizes
metal ions as the "anchor" of template complex. Lawrance et al used a Cu " template to
produce ethylene side bridged 15-membered macrobicycles from piperazine derivatives
[81]. The advantages of this template synthesis are the ease and high yield of the reaction
as well as the addition of a pendant donor through reduction of a nitro group to amine
(Figure 4).
Ligands similar to those of Lawrance et al were produced by template reactions involving
N,N'-bis[(CH2)n-NH2]-l,4-diazacycloheptanes as the starting materials in place of
piperazine [82,83]. Besides, this topology is useful for designing therapeutic drugs since
they provide unsaturated coordination environment and possess vacant coordination sites
for binding/activation with substrate/drug target [84,85].
^2* n ^ N ,NH2
N NH, HCHO
EtNO,
2*
Figure 4. Template closure of a side-bridged macrocycle.
A highly efficient synthesis of carbamate polyazamacrocycles derived from chloroethyl
pendant armed macrocycles was achieved by A. Sutherland et al [86]. These
polyazamacrocycles were as cytotoxic as the well-known anticancer alkylating agents
chlorambucil and melphalan and were known to exhibit significant biological acUvity
(antiparasitic activity) owing to their pheirmacokinetic tunable substituents and reactive
side chains [87] (Figure 5). Tuning the nature of these arms allows the creation of smart
molecules with sp>ecific physical and chemical properties, which can be utilized for many
applications in medicinal and analytical chemistry and NMR imaging. Polyamine
mustards (derivatives of 1,4,7-triazacyclononane, 1,4,7,10-tetraazacyclododecane and
1,4,8,11-tetraazacyclotetradecane) are candidates for conversion into hypoxia-selective
prodrugs (an inactive form, which is metabolized in vivo to the active species) via
complexation with metals [88]. The novel Cu(II) complexes of tacn, cyclen and cyclam
were assessed in vitro as hypoxia-selective cytotoxins. Bioreductive drugs are prodrugs,
which are selectively activated by intracellular reductases under hypoxic conditions. This
is a consequence of an insufficient formation of new blood vessels in rapidly growing
tumours and it is a major physiological difference between tumour and normal tissues
[89]. The cylen mustard complex showed 24-fold selectivity as hypoxia selective
bioreductive prodrug with an IC50 value of 2 (iM against lung tumour cell line A549
(Figure 6).
• " - ' o N N ° r D
Cl \ 1 ^-^ ^r\ u H
Figure 5. Structures ofN-functionalized macrocyclic ligands.
10
cu
2+ 2+
,N. N [ cu—j-ci a-
CI JC^J OH PF6-
cr ^7 \-^ci CI
Figure 6. Structures of Cu(II) complexes of N-Mustard derivatives of (a) Cyclen and (b)TACN
A wide variety of linked macrocyclic ligands including species covalently linked by
secondary species have been reported in an attempt to investigate metal ion recognition
by such systems, including metal ion binding of mono-ring and xylyl-Iinked bis-ring
systems [90,91]. Majority of these have involved polyamine rings although a number of
mixed donor macrocyclic systems have also been investigated. Shim Sung Lee and
Lindoy et al have synthesized NO2S2 donor macrocycle from the ring closure reaction
between Boc-N-protected 2,2'-iminobis(ethanethiol) and 2,2'-(ethylenedioxy)bis(benzyl
chloride) followed by deprotection of the Boc-group. a,a'-Dibromo-p-xylene was
employed as a dialkylating agent to bridge two macrocyles to yield the corresponding N-
linked product [92]. Binuclear complexes of this N-linked macrocyle with heavy metals
Hg(ll) and Cd(II) were also isolated (Figure 7). There is a growing interest in Cu(II)
complexes of tetradentate bis(benzimidazol-2-yl)dithiother ligands as they offer N2S2
chromophore in blue proteins [93]. Addison et al have studied Cu(ll) complexes of linear
N2S3 ligands containing pyridyl, quinolyl and benzimidazolyl termini [94]. The structure
11
and properties of Cu(II) complexes of trithioether ligands containing substituted
pyrazolyl [l,9-bis(3,5-dimethyl-l-pyrazolyl)-2,5,8-trithianonane,pzttn], benzimidazolyl
[l,9-bis(3,5-benzimidazol-2-yl)-2,5,8-trithianonane, bzttn] and pyridyl [(l,9-bis(pyrid-2-
yl)-2,5,8-trithianonane, pttn] moieties were described [95,96]. S. Usha and M.
Palanaindavar have described the effect of incorporating increasing number of sulphur
donors and varying chelate ring size on the structure and spectra of Cu(II) complexes of
bis(benzimidazol-2-yl)tetra- and penta-thioether ligands [97].
Figure 7. Xylyl-bridged ditopic NO2S2 donor macrocyle
The study of these ligands provides an opportunity to gain insight into the relative
importance of contributions from the coordination of thioethers and the benzimidazole
which are readily accessible to the positive M(II)/M(I) redox potentials. In addition,
imidazoles are common component of pharmacologically active molecules [98].
Benzimidazole group plays important role in several minor groove DNA binders such
Hoechst 33258 and Hoechst 33342 and display wide range of biological activities
including inhibition of DNA topoisomerase 1, antitumour and antiparasitic activities [99-
102]. These facts make imidazole and its derivatives important target analytes.
12
Deoxyribonucleic acid (DNA) is the primary cellular target molecule for most anticancer
therapies [103]. Binding studies of small molecules with DNA are very important in the
development of new therapeutic reagents and DNA molecular probes [104]. Nucleic
acids under physiological conditions are polyanions composed of heterocyclic bases
linked to a sugar-phosphate backbone. Thus, they are quite amenable to probe with
positively charged transition metal ions.
DNA is a flexible, dynamic and polymorphic in structure that can assume a variety of
interconverting forms [105]. Each form has its own biological role in the regulation of the
life of cell. Selective stabilization of one of these forms can help to discover and better
imderstand their biological roles. Genetic information is provided by the DNA in at least
two different ways. First, the sequence of its nucleotides determine the primary structure
of the protein. Second, DNA can regulate gene expression through its shape [106].
Coordination compoimds offer many binding modes to polynucleotides, including outer
sphere non-covalent binding, metal coordination to nucleobases and phosphate backbone
sites, as well as strand cleavage induced by oxidation using redox-active metal centres
(Figure 8). In covalent binding, the labile ligand of the complexes is replaced by a
nitrogenous base of DNA such as guanine N7. On the other hand, the non-covalent DNA
interactions include intercalation, electrostatic interaction, hydrogen bonding and groove
(surface) binding of cationic metal complexes along outside of DNA helix, along major
or minor groove. Intercalation involves the partial insertion of aromatic heterocyclic rings
of ligands between the DNA base pairs [107].
13
Figure 8. External binding (left), intercalation (middle), groove (right) binding
Considerable progress was made in past few years on the identification of metal binding
sites in large DNA molecules; however, to obtain information and to determine the
coordination of a metal ion to a specific site, interaction with low-molecular building
blocks of large nucleic acids viz mononuleotides and dinucleotides has become
mandatory (Figure 9). Purine and pyrimidine nucleoside bases have been focus of many
such studies. An excellent review article by Lippert has appeared a few years ago
summarizing the multiplicity of behaviours known for most relevant purines (adenine,
guanine and other 6-oxo purines) and pyrimidines [108]. It presents a series of general
rules about different metal-nucleobase coordination modes with different metal ions.
Similarly, Gutierrez et al have described structures of related copper(lI) complexes with
purine nucleobase as a valuable source of information on molecular recognition patterns.
The presence of amino or oxo group in C6 atom of adenine or guanine and its derivatives,
respectively significantly increases their possibilities for developing a suitable
14
intramolecular inter ligand interactions that reinforces the corresponding Cu-N
(nucleobase) coordination bond [109].
0 1 II S' • I J
0-P-O—1 Q
"OH H 3- ^
7
1'
J'
0
fl NH
N NH2 3
0 I II 5 '
"OH W
NH, 7 61 '1
1
3
r
Figure 9. Chemical structures of guanosine-5'monophosphate (5'-GMP^') and adenosine-5-monophosphate (5 '-AMP^') with atomic numbering.
Relatively few studies have been focused on the potential of the DNA-binding ability of
the macrocyclic complexes. However, Kimura and colleagues have described a series of
macrocyclic complexes linked to intercalators and have shown that these interact with
DNA in a sequence-selective manner [110-112]. On the basis of structure-activity
correlation, sequence-specific DNA binding properties can be invoked by varying the
substituents on the macrocyclic skeleton. Macrocycles are known to exhibit anticancer
activity and a few- namely bryostatin have entered phase II human clinical trials for the
treatment of melanoma, non-hodgkins lymphoma and renal cancers [113]. Bryostatins are
a unique family of emerging cancer chemotherapeutic candidates isolated from marine
bryozoa (Figure 10). An understanding of the structural basis for these bryostatin
activities like high affinities for protein kinase PKC signal transduction is important for
the development of simpler and clinically superior chemotherapeutic agents. Structure-
15
activity relationships and related computer niodeling of bryostatins suggest that
bryostatin binding is a cooperative event between two functional domains: one, the
recognition domain containing elements that contact the receptor, and a second, a spacer
which serves to properly orient and constraint the recognition domain.
Figure 10. Structure of Bryostatin
The numerous side effects of cisplatin as a chemotherapeutic agent leave room for the
selection of other metals for the synthesis of bioactive molecules. Among these, Cu(II)
ion is especially attractive due to its occurrence in biological systems and participation as
an integral part of the active site of different types of metalloproteins, which recognize its
coordination with the human body fimctions [114-116]. The exploration of copper
complexes as chemical nucleases is well documented [117-123] because they possess
biological accessible redox potential and relatively high nucleobase affinity [124,125]
and are therefore selective at the molecular target site. Besides this, the tuning and
prediction of redox potentials by structure modification provides a rational strategy for
the development of tumour selective bioreductive drugs.
16
The macrocyclic copper (II) complexes of the formula [Cu(L')]Cl2, [Cu(L^)]Cl2,
[Cu(L^)]Cl2, [Cu(L^)]Cl2 and [Cu(L^)](C 104)2 where (L ' = 3,10-bis(2-methylpyridine)-
1,3,5,8,10,12-hexaazacyclotetradecane, L = 3,10-bis(2-propionitrile)-l,3,5,8,10,12
hexaazacyclotetra- decane, L = 3,10-bis(2-hydroxyethyl)-l,3,5,8,10,12
hexaazacyclotetradecane, L'' = 3,10-bis(2-benzyl)-l,3,5,8,10,13-hexaazacyclotetradecane
and L = 2,6,9,13-tetraparacyclophane) (Figure 11) interacted with DNA in different
binding modes and exhibited effective nuclease activities [126,127]. The functional
groups on the side chain of macrocycles play a key role in deciding the mode and extent
of binding of Cu complexes to DNA. Therefore, extensive studies using different
structural macrocycles to evaluate and understand the factors that determine mode and
mechanism of the binding interaction of macrocyclic transition metal complexes with
DNA are necessary. Absorption and fluorescence spectroscopic studies, cyclic
voltammetry and viscosity measurements were used for the interaction of these
macrocycles with CT DNA.
17
^ / ^ > — C H 2 — N ^ C u ^ 5^CH2
[CULV^
H / \ H
[CULV*
^
'CN
H<y
'OH
ICuL 3,2+
[CuL 4,2+
^
Cu
A-\
[CuL^
Figure 11. Chemical structures of the macrocyclic copper (II) complexes
18
The complex [Cu(L )]Cl2 exhibits an increase in molar absorptivity (hyperchromism)
with no shift. On addition of CT DNA, all the macrocyclic complexes except [Cu(L )]
CI2 show decrease in molar absorptivity (hypochromism) of the LMCT absorption band
as well as red shift (Figure 12).
200 220 240 260 280 300 320 340
Wavelength (nm)
(b)
200 220 240 260 280 300 320 340 Waveiwigth (nm)
220 240 260 2to 300 320 340
Wavetength (nm)
Figure 12. Absorption spectra of (a) fCul'jCh (b) fCuL^JCh (c) fCuL^JCh in the absence (...) and presence (- ) of increasing amounts of DNA
19
These observations suggest that copper complexes of L ' , L"* and L* ligands bind to DNA
through partial intercalation by providing an aromatic moiety present in the side chain
attached to macrocycle or within the macrocycle to overlap with the stacking base pairs
of DNA helix. On the other hand, complexes [Cu(L^)]Cl2 and [Cu(L^)]Cl2 lack any fused
aromatic ring to facilitate intercalation rather they possess -NH and -OH groups, which
form hydrogen bonding with the base pairs of DNA.
Furthermore, binding constants (Kb) were also determined for copper complexes of L ' , L^
and L as 2.1 X 10 M ' , 4 X 10 M"' and 7.8 x 10 M ', respectively. The difference in
binding constants of the three complexes to DNA further support that the functional
groups on the side chains of macrocycle play a key role in deciding the mode and extent
of binding of complexes to DNA.
Competitive ethidium bromide binding study was employed to understand the mode of
DNA interaction of [Cu(L')]Cl2, [Cu(L^)]Cl2 and [Cu(L^)]Cl2 complexes. The addition of
the complexes to DNA pretreated with EthBr causes appreciable reduction in emission
intensity, indicating that the complexes compete with EthBr in binding to DNA (Figure
13). The quenching constants Ksv were determined by Stem-Volmer equation. The values
for the copper complexes L , L and L were 1.34, 0.74 and 0.39, respectively.
20
500 600 700 Waveler)gtb(nm)
(b)
500 600 700
Wavelengtti(nm)
900-1
600-c
i§ 300-
U'' 500
1
R f\ i\ 600
i " ;
^ 700
(c) Wavelength(nm)
Figure 13. Emmision spectra ofEB bound to DNA in the absence (...) and presence (- ) of the (a) fCuL'jCh (b) fCuL^JCh (c) [CuL^JCh
Cyclic voltammetry is the most effective and versatile electroanalytical technique
available for the mechanistic study of redox systems [128]. Complexes [Cu(L')]Cl2,
[Cu(L^)]Cl2 show irreversible redox behaviours with low Ipc/Ipa values while [Cu(L^)]Cl2
exhibits reversible CV. Redox potential of the complex follow the trend [Cu(L')]Cl2
>[Cu(L )]Cl2 >[Cu(L )]Cl2 illustrating the ability of the hexaazamacrocycles to stabilize
the Cu(I) state. The more decrease of the peak current for complex [Cu(L^)]Cl2 than that
21
of [Cu(L^)]Cl2 suggest the stronger binding affinity complex [Cu(L^)]Cl2 to DNA (Figure
14).
Figure 14. Cyclic voltammogram offCuLJCh in the absence (-) and in presence (inner line) of DNA
Viscosity measurements were also carried out to understand the binding mode of these
complexes [Cu(L')]Cl2, [Cu(L^)]Cl2 and [Cu(L^)]Cl2 with CT DNA. In presence of
[Cu(L^)]Cl2, there was no obvious effect on the relative specific viscosity CT DNA
whereas complexes [Cu(L')]Cl2 and [Cu(L^)]Cl2 decreased the viscosity of DNA. The
decrease in viscosity may be explained by a binding mode, which produced bends or
kinks in DNA (Figure 15).
22
0.0 0.1 0.2 0.3 0.4 i 2 «
[CuLl"/IDNAJ
Figure 15. Effect of increasing amounts offCul'jCh (? ) fCuL^JCh (?) [CuL^JCh (*)on the relative specific viscosity ofCTDNA
A series of macrocyclic /intercalator adducts have been prepared from cyclam and the 1-
and 2-substituted anthraquinones (l-[(2-aminoethyl)amino]anthracene-9,10-dione
(lC2mac), l-[(3-aminopropyl)amino]anthracene-9,10-dione (lC3mac), 2-[(3-
aminopropyl)amino] anthracene-9,10-dione (2C3mac) (Figure 16). These adducts and
their Cu(II) complexes depict excellent DNA binding properties. UV spectrophotometric
titrations were carried out to compare the affinities of (lC2mac) and its copper complex
with salmon sperm DNA [129].
2
1-position n=2; lC2mac
C' ''Q^' ^ n=3;lC3mac
H I I H 2-position n= 3; 2C3mac
Figure 16. Cyclam/anthraquinone macrocyclic addiict.
23
With increasing concentration of the DNA, a steady decrease in absorbance was observed
(Figure 17). The apparent binding constants obtained from the half-reciprocal plots were
found to be 4.7.10^ M'' and 6.2.10^ M ' for (lC2mac) and (Cu-lC2mac), respectively
suggesting that the metal plays an important role in determining the affinity of the
complex for DNA.
9a» »4» wavdength (nm) •knffh (nm)
Figure 17. UV spectrophotometric titration curves for (a) lC2mac and (b) Cu-lC2mac.
The DNA binding of the cyclam/anthraquinone adducts and their metal complexes were
compared by reacting them with plasmid DNA. The binding assays revealed that addition
of the macrocycle had a strong influence on the binding of the intercalator moiety. This
was attributed to the hydrogen bonding association between the macrocycle and the
DNA. Complexation of the copper to the (lC2mac) adduct substantially modifies the
binding of the complex as indicated by the lower concentrations required to fully unwind
24
the supercoiled DNA. The dipositive charge of copper within the macrocycle and also the
availability of the nucleophilic sites in the axial positions of the Cu(II) results in the
increased DNA binding and/or unwinding (Figure 18).
(a) (b)
Figure 18. Gel electrophoresis pattern of pBR322 plasmidDNA treated with (a)lC2mac and (b) Cu-lC2mac.
Macrocyclic SchifF base dinuclear Cu(II) complex of the formula [Cu2LCl2]Cl2.3H20,
where L = bis-p-xyxlyl BISDIEN was synthesized and characterized by L. N. Ji et al
[130], The interaction of the complex with calf thymus DNA was investigated by
electronic absorption spectra, fluorescence spectroscopy, cyclic voltammetry and
viscosity measurements. The addition of CT DNA to the metal complex results in an
appreciable decrease in absorption, intensity (hypochromism) indicating interaction
between the dinuclear complex and CT DNA. Structurally, the ligand provides the
aromatic moiety extending from the metal centre to overlap with the DNA base pairs by
an intercalative binding mode (Figure 19,20).
25
Figure 19. Chemical structure of the bis-p-xylylBISDIEN ligand.
Wavelength (nm)
Figure 20. Absorption spectra of [Cu2LCl2]Cl2.3H20, in the absence (...) and presence (-) of increasing amounts ofDNA.
Emission titrations of the complex with the EB boimd DNA revealed that the complex
competes with EB in binding to DNA as depicted in Figure 21 by the reduction in
emission intensity with increasing concentrations of the complex. The fluorescence
quenching curve illustrates that the emission quenching of EB bound to DNA by the
complex was in good agreement with the linear Stem-Volmer equation.
26
1 '
0 - • I ' 1 • 1 ' 1
(a) V\fewelength(nm)
(b) [Cu,LCLf*/tDNA]
Figure 21. (a) Emmision spectra ofEB bound to DNA in the absence (...) and presence (- ) of[Cu2LCl2]Cl2.3H20, (b) Stem -Volmer quenching plot.
Cobalt complexes have received less attention in comparison to other transition metal
complexes [131-134] although they exhibit interesting metallointercalation and DNA
cleavage properties [135,136]. Besides this, cobalt containing complexes offer an
exciting possibility for oral delivery of a wide variety of peptide based drugs- the most
efficient chemotherapeutics. Co(Ill) is a deactivating carrier system for highly cytotoxic
organic drugs. These can be released from the metal centre upon reduction to Co(II) and
exert their activity in hypoxic tissue. Co (III) with its high charge density is a potent
candidate for hydrolyzing phosphodiester bonds of DNA. E. L. Chang et al [137] have
developed a kinetically inert Co(III)-cylen based complex (Co(III)-cycmmb) for
inhibition of the translation of RNA into proteins.
27
The interaction was mediated through the kinetic inertness of Co(III) for the phosphate
groups of the nucleotides, as well as coordination of Co(III) to the nitrogenous bases. The
second pathway occurred at longer contact times and was mediated by the hydrolysis of
the phosphodiester backbone. This was first report that demonstrated use of a metal-
chelate complex to achieve the inhibition of the translation of RNA into proteins (Figiire
22).
CI
. i N—Co ST-—Co \
^OCHj
o
Figure 22. Structure of the Co(III)-cycmmb complex
Dinuclear copper(II) and Zinc (II) complexes containing Bis-TACN macrocycle bridged
by bis-4-methylphenol were synthesized and evaluated for their DNA binding ability
[138]. The extent of binding of two complexes to CT DNA was determined by measuring
their effects on the UV spectroscopy and fluorescence spectra of DNA (Figure 23). Upon
addition of an increasing amount of DNA to the complexes, hypochromism and slight red
shift were observed indicating a moderate binding of the complexes with DNA (Figure
24). The relative binding ability of the complexes was also studied by measuring the
change in the fluorescence intensity of EB bound DNA upon increasing the complex
concentration. The emission intensity showed a reduction upon the addition of complexes
28
suggesting an intercalative binding mode. The apparent binding constants for the Cu(II)
and Zn(II) complexes were found to be 1.0.10^ M ' and 1.1.10^ M"', respectively. The
similar values result from the intercalation of the 4-methyl phenol group and the
hydrophobic property of the rigid macrocyclic ligand, which facilitates DNA binding.
The complexes effectively promote the cleavage of plasmid DNA via the synergistic
mechanism of the metal ions with the rigid ligand framework.
Figure 23. Structure of bridged bis-TACN macrocyclic ligand.
Figure 24. Absorption spectra of dimiclear (a) Cu(II) and (b) Zn (II) complexes of bis-TACN macrocycle.
29
Nickel plays versatile and sometimes controversial role in living systems [139]. The
molecular basis of carcinogenic nickel compounds is quite elusive because they are
weakly mutagenic in most assay systems and only induce rather weak oxidative stress
[140,141]. In fact, several nickel complexes have been found to inhibit proliferation of
diverse cancer cells [142,143]. Nickel (II) affects the DNA repair mechanisms by
inhibiting the base excision repair (BER) and nucleotide excision repair (NER) processes
[144,145], which may become a potential merit in the design of antitumour agents. It is
known that cisplatin-DNA adducts are repaired in cells primarily through the NER
pathway [146]. As a platinum group metal, nickel(II) may share some similarity with
platinum(II). Therefore, if DNA could be one of the cellular targets for nickel
compounds, their effects on DNA function could be enhanced by the inhibition of DNA
repair process.
Macrocyclic complex [Ni(CR)] (€104)2 where CR = [2,12-dimethyl-3,7,ll,17-
tetraazabicyclo-[11.3.1]heptadeca-l(17),2,ll,13,15-petaenato] was prepared by the
template condensation reaction using 2,5 diacetyl pyridine and bis-(3-aminopropylamine)
while complex [Ni(cyclam)](C104)2 (where cyclam= 1,4,8,11 tetraazacyclotetradecane)
was obtained by the reaction between Ni(C104)2.H20 and cylcam at ratio of 1:1 in
aqueous EtOH solution (Figure 25). Interaction of these complexes with nucleotides
GMP, AMP and ApG in aqueous solution has been monitored by 'H, '^N N M R and UV
30
spectroscopy. The complex NiCR oxidized GMP gradually but do not affect AMP
significantly [147].
H,C. ^ *V^CHj ,NHHN
NH u HN ^NHHN
Figure 25. Structure of CR (CR=[2.12-dimethyl-3,7,11.17-tetraazabicyclo-[11.3.1]heptadeca-l(17),2,ll,13,15-petaenato]) and cyclam (cyclam=1,4,8,11 tetraazacyclotetradecane).
Chemical nucleases have become an invaluable research tool in the fields of biology,
bioinorganic chemistry, therapy and molecular biology [148,149]. Chemical nucleases
are compoimds that mimic the action of enzymatic nucleases and are often dependent on
their active sites including metal ions particularly Zn(II), Mg(II) and Fe(II), owing to
their ability to act as Lewis acids [150]. Chemical nucleases are smaller than enzymes
and capable to attack macromolecular regions that are inaccessible to enzymes and also
their specificity and efficiency may be modulated [151]. In recent years, many artificial
metalloenzymes have been suggested as substitutes for natural enzymes to cleave DNA.
Many complexes of transition metal ions have been designed and studied as artificial
cleavage agents. Among the commonly used ligands 1,4,7,10-tetraazacyclododecane
(cyclen) has a special structure and can coordinate with most metal ions (transition metal
31
ions and lanthanide ions) [152-154]. Therefore, metal cyclen complexes were widely
used in DNA recognition, cleavage and enzyme mimics [155-157]. Many nucleases carry
one or more metal centres. In most cases synergistic effects exist between the metal
centres. Polyaza macrocyclic ligand 3,6,9,17,20,23-hexaa2atricyclo[23.3.1.1] triaconta-
l(29),ll(30),12,14,25,27-hexaene (BMXD) was first synthesized in 1990 [158]. The
ligand consists of a macrocycle formed by two diethylenetriamine moieties separated by
two m-xylyl bridges, and is capable of coordinating one or two Cu(II) centers via its
diethylene triamine moieties (Figure 26). The Cu2-BMXD complex was capable of
undergoing DNA cleavage reaction via an oxidative pathway with reasonably high
efficiency [159].
N Cu ,Cu---N
Figure 26. Chemical structure of the [CU2BMXD] complex.
Although there are compounds that are capable of performing remarkable hydrolytic
cleavage of DNA, oxidative agents that cleave DNA are far more superior. Similarly, a
series of multinuclear polyamine metal complexes containing chiral dipeptide linkage
32
were synthesized and used as artificial nuclease enzyme models [160]. Burstyn et al have
studied a series of macrocyclic Cu(II) complexes and they have proposed a mechanism of
DNA hydrolysis and shown the importance of macrocyclic ring size and substituents on
the ring for reactivity [161-163]. The reactivity of DNA cleaving agents was strongly
enhanced when their structures comprised groups having high affinity for the DNA. The
binding subunits increase the local concentration of the catalyst at the binding site leading
to the acceleration of the reaction. M. Chikira et al appended an acridine moiety to 1,4,7-
triazacyclononane ([9]aneN3) complex. The conjugation of the metal complex with the
intercalating group resulted in a higher cleavage activity both in the oxidative and
hydrolytic cleavage mechanisms [164] (Figure 27).
NH HN .7CF3COOH
H,0
Figure 27. Macrocyclic artificial DNA cleavage agents.
Zinc is the most abundant trace intracellular element and plays an important role in both
genetic stability and function [165]. Mechanistically, zinc has a significant impact on
DNA as a component of chromatin structure, DNA replication and transcription and
33
DNA repair [166]. Due to its good Lewis acidity, redox inactivity, rapid ligand exchange
and less toxicity Zn(II) is frequently encountered in both natural and artificial nucleases
[167]. Xiao-Qi Yu have designed and synthesized the conjugate ligand of cyclen-uracil
and correlated macrocyclic polyamine Zn(II)complexes having DNA cleaving activity
(Figure 28). These complexes have special properties; i) uracil as a nucleobase was
favourable to recognize DNA at low concentrations and ii) the metal ion in the complex
was bound to imide of uracil group through an intermolecular mode to form unique and
stable supramolecular complexes, which benefits the DNA cleavage process [168,169].
Kimura and co-workers have also demonstrated that Zn(II) complexes of 12-membered
macrocyclic tetraamine selectively interacts with uridine and thymidine nucleotides
through specific Zn ^ imide N-bonding [170].
•R—N ; = 0
Figure 28. Uracil conjugated Zn(II) macrocyclic complex.
A series of bimetallic systems have been synthesized and investigated for their catalytic
cleavage activity of DNA models and DNA itself, as the catalytic efficiency of many
natural metallonucleases often relies on the cooperative action of two or more metal ions.
34
The two metal ions not only increase the effect of Lewis acid catalysis but also make
possible other activation modes, such as leaving group activation [171]. R. Breslow
prepared a bimetallic macrocyclic complex by connecting two tridentate macrocyclic
ligand with a rigid 4,4'-biphenyl linker (Figure 29). The reactivity towards several
phosphate esters increased 5 times than the corresponding monometallic system [172].
'.^.. rf *»"? ? » • *
\ /r\ //
2+ Figure 29. Structure of the ditopic bimetallic Zn complex.
35
Present work
The interaction of metal complexes with nucleic acids and their constituents is of central
importance to many aspects of their structure and fimction. These complexes offer
opportunity to explore the effects of central metal atom, the ligand and the coordination
geometries on the binding events. Tailored multifunctional ligands for metal-based
medicinal drugs play an integral role in modulating the potential toxicity of a
metallodrug. In metal- based drug candidates, the metal ion organizes the organic ligands
in the three dimensional space permitting the site-specific interaction by matching the
shape, symmetry and functionality of metal complexes to that of DNA target. Such an
approach complements the molecular diversity in the quest for the discovery of
therajjeutic compounds with superior biological activity.
Macrocycles are important and ubiquitous ligands in transition metal coordination
chemistry, imparting thermodynamic and kinetic stabilities to their complexes uncommon
or non-existent with other acyclic ligands. The molecular topology of the ligand
framework assists these metal ions to achieve specificity for the target site DNA and
enhances their DNA binding affinity. On account of the unique coordination and
structural properties of pendant armed macrocyclic complexes and their roles in
bioinorganic chemistry, new macrocyclic complexes of 4,15-bis(2-hydroxyethyl)-
2,4,6,13,15,17-hexaazatricyclodocosane were synthesized from diaminocyclohexane,
formaldehyde and 2-aminoethanol. Complexes bearing cyclohexylamine moiety, a non-
planar amine ligand are known to affect the kinetics and cytotoxicity. The molecular
36
planar amine ligand are known to affect the kinetics and cytotoxicity. The molecular
architecture of these complexes bearing -NH and -OH groups of appended arms with
two cyclohexane rings, provides a rationale to explore their DNA binding properties.
The binding of these complexes with CT DNA was studied by UV/Vis, fluorescence
spectroscopy, viscosity and cyclic voltammetry. These complexes exhibit efficient DNA
binding activity, which was ascertained by their interaction with CT DNA; furthermore
specific binding behaviour was studied with 5'GMP employing UV/Vis and NMR
spectroscopy.
Benzimidazoles are very useful subunits for the development of molecules of
pharmaceutical or biological interest. A new series of benzimidazole-derived dinuclear
O2S2 donor macrocyclic complexes were synthesized. Their DNA binding abilities were
investigated by UV/Vis and fluorescence spectroscopy, cyclic voltammetry and viscosity
measurements. The results suggest that the complexes bind to DNA by a partial
intercalative binding mode. The cleavage of supercoiled pBR322 plasmid DNA by the
Cu(II) complex in absence or presence of reducing agent was observed using gel
electrophoresis. The complex exhibited an efficient nuclease like activity via an oxidative
cleavage mechanism.
Complexes featuring two or more metal centres held at an optimum distance can
modulate the sequence selective recognition of the biological target. Therefore, an
attempt was made to synthesize bis-macrocyclic complexes via in situ one pot template
condensation reaction of pyridynyl, an aromatic nitrogen-nitrogen linker, formaldehyde
37
of these complexes with DNA. Interaction with 5'GMP was studied by absorption
spectroscopy.
Coumarin derivatives have been proven to fimction as chemotherapeutic agents with a
variety of biological activities including the anticancer activity. A new series of metal
complexes were synthesized from a coumarin ligand 3-acetoacetyl-7-methyl-pyrano-(4,3-
b)-pyran-2,5-dione and diaminocyclohexane via a template reaction to give a schiff base
type macromolecule. The DNA binding affinity of the ligand and the copper complex
was evaluated by electronic absorption spectroscopy and flourescence spectroscopy.
Interaction with 5'GMP was elucidated by absorption and NMR spectroscopy. In vitro
antitumour activity of the ligand against different human cancer cell lines were
determined using standard protocols for cell viability. The ligand exhibited highest
growth inhibiting effect against CNS cancer SNB-75 cell lines. It is inferred that the
metal complexes will possess greater cytotoxic effect then the free ligand since chelation
enhances the inhibitory power of the ligands.
CHAPTER II
Experimental
38
CHAPTER II
EXPERIMENTAL METHODS
The following techniques were employed.
1. Characterization techniques
1.1 Infrared spectroscopy
1.2 Ultra-violet and visible spectroscopy
1.3 Nuclear magnetic resonance spectroscopy
1.4 Electron paramagnetic resonance spectroscopy
1.5 Mass spectroscopy
1.6 Molar conductance measurements
2. DNA binding studies
2.1 Cyclic voltammetry
2.2 Absorption spectral studies
2.3 Fluorescence spectral studies
2.4 Gel electrophoresis
2.5 Viscometry studies
1. Characterization techniques
1.1 Infrared spectroscopy
The infrared spectroscopy is a useful technique to characterize a compound. It results
from transition between vibrational and rotational energy levels. IR region of the
39
electromagnetic spectrum covers a wide range of wavelength from 200 to 4000 cm' . It
has been found that in IR absorption, some of the vibrational frequencies are associated
with specific groups of atoms and remain same irrespective of the molecules in which the
group is present. These are called characteristic frequencies [173] and their constancy
results from the constancy of bond force constants from molecule to molecule. The
important observation that the IR spectrum of a complex molecule consists of
characteristic group frequencies makes IR spectroscopy, unique and powerful tool in
structural analysis.
1.2 Ultraviolet and visible spectroscopy
When a molecule absorbs radiation, its energy is increased. This increased energy is
equal to the energy of the incident photon expressed by the relation
E = hv
E =hc/X
where h is Planck's constant, v is the frequency, X is the wavelength of the radiation and
c is the velocity of light. Most of the compoimds absorb light in the spectral region
between 200 and 800 nm resulting in the excitation of electrons of the molecules from
ground state to higher electronic states. In transition metals, all the five'd' orbitals viz.
9 7 7
dxy, dyz, dxz, dz and dx -y are degenerate. However, in coordination compounds due to the
presence of ligands, this degeneracy is destroyed and d orbitals split into two groups t. g
40
(dxy, dyz and dxz) and Cg (d ^ and dx .y ) in an octahedral complex and t and e in a
tetrahedral complex. The set of tjg orbitals goes below the original level of degenerate
orbitals in octahedral complexes and the case is reversed in tetrahedral complexes (Figxire
30a-b).
Free ion
/<^ti<ixW • V
Hypothetical ion in a spherically symmetric field
''jy <r: *(«
Octahedral field
mq
'k
4P?
Figure 30a. Splitting of the d energy levels in an octahedral complex.
Ion in spherically symmetric Illy syc
field Tetrahedral field
. -^ a.vv dxz "vz
N X
\ \
4Dq Bari cvntvr
6Dq
J.2 dr2-y2
Figure 30b. Ligand field splitting of a tetrahedral complex, 'g' subscript is omitted in Tj symmetry.
41
At energy higher than the ligand field absorption bands, we commonly observe one or
more very intense bands that go off scale unless log e is plotted. These are the charge
transfer bands, corresponding to electron transfer processes that might be either ligand —•
metal (L —>• M) or metal -* ligand (M -* L). In case of octahedral complexes M —» L
transitions occur for metal ion complexes that have filled, or nearly filled, t2g orbitals with
ligands that have low lying empty orbitals. The L -> M charge-transfer (C-T) spectra
have been studied more thoroughly. The electron density is transferred from the ligand to
the metal. This is favoured when the central metal has a high oxidation state, and is 'short
of electrons'. The ligand n orbitals are lower in energy than the metal t2g orbitals. No net
oxidation-reduction usually occurs, because of the short lifetime of the excited state
[174].
1.3 Nuclear magnetic resonance spectroscopy
The nuclei of certain isotopes possess a mechanical spin or angular momentum. The
NMR spectroscopy is concerned with nuclei having nuclear spin quantum number
1 = 1/2, example of which include 'H , '^C, ^ 'P , '"Sn and '^F.
For a nucleus with 1 = 1/2, there are two values for the nuclear spin angular momentum
quantum number mi = ±1/2 which are degenerate in the absence of a magnetic field.
However, in the presence of the magnetic field, this degeneracy is destroyed such that the
42
positive value of mi corresponds to the lower energy state and negative value to higher
energy state separated by AE.
In an NMR experiment, one applies strong homogeneous magnetic field causing the
nuclei to presses. Radiation of energy comparable to AE is then imposed with radio
frequency transmitter equal to precision or Larmor fi-equency and the two are said to be
in resonance. The energy can be transferred from the source to the sample. The NMR
signal is obtained when a nucleus is excited from low energy to high energy state.
1.4 Electron paramagnetic resonance spectroscopy
EPR spectroscopy [175] is the branch of absorption spectroscopy in which radiation
having frequency in the microwave region is absorbed by molecules possessing electrons
with unpaired spins. The unpaired electrons in radicals or in complexes of transition
metal centers with only partially filled d orbitals features a spin, the orientation of which
in a magnetic field can give rise to two energetically different states. For an electron of
spin S = 1/2, the spin angular momentum quantum number will have values of ms = ±1/2.
In absence of magnefic field, the two values of ms i.e. +1/2 and -1/2 will give rise to a
doubly degenerate spin energy state. If a magnetic field is applied, this degeneracy is
lifted and leads to the non-degenerate energy levels. The low energy level will have the
spin magnetic moment aligned with the field and correspond to the quantum number ms =
-1/2. On the other hand, the high energy state will have the spin magnetic moment
43
opposed to the field and correspond to the quantum number m^ = +1/2. The energy E of
the transition is given by;
E = hv = gPHo
Where h is Plank's constant, v the frequency of radiation, p the Bohr magneton, Ho the
field strength and g the spectroscopic splitting factor.
The magnitude of g is characteristic for the paramagnetic system and depends upon the
orientation of the molecule with respect to the magnetic field. From the observed value of
g it is possible to determine oxidation and spin states or even details in the coordination
sphere of the metal centers. If the paramagnetic radical or ion has g value independent of
the orientation of the crystal it is said to be isotropic and if the value of g depends on the
orientation of the crystal and is said to be anisotropic.
1.5 Mass spectroscopy
At electron beam energy of about 9 to 15 electron volts, depending on the molecule
involved, a molecular ion is formed by interaction with the beam electrons. Recognition
of the parent ion (actually a radical ion) is of great importance because it gives the
molecular weight of the sample [176]. At this point, the molecular weight is an exact
numerical molecular weight not the approximation obtained by the all other molecular
weight procedures. Mass spectra is usually obtained at an electron beam energy of 70
electron volts and under these conditions numerous fragment ions (including the parent
44
ions) versus their relative concentrations constitutes the mass spectrum of the samples.
The fragmentations occur on the basis of mass/charge (m/z). The largest peak in the
spectrum is called base peak and assigned a value of 100 %. The other peaks are reported
as percentage of the base peak.
1.6 Conductance measurements
The conductivity measurement is one of the simplest and easily available techniques used
to study the nature of the complexes. It gives direct information regarding whether a
given compound is ionic or covalent. For this purpose, the measurement of molar
conductance (Am) which is related to the conductance value in the following manner is
made.
cell constant x conductance Am =
concentration of solute expressed in mol cm"
Conventionally, solutions of 1x10" M strength are used for the conductance
measurements. Molar conductance values of different types of electrolytes in a few
solvents are given as, 1:1 electrolyte has a value of 80-115 ohm"' cm^ mol"' in MeOH,
65-90 ohm"' cm^ mol"' in DMF, 78-80, 50-70 ohm"' cm^ mol"' DMSO and 35-45 ohm"'
cm^ mol' in EtOH [177,178]. Similarly a solution of 2:1 electrolyte has a value of 160-
45
220 ohm"' cm^ mol' in MeOH, 130-170 ohm"' cm^ mol"' in DMF and 70-90 ohm"' cm^
mol"' in EtOH.
2. DNA binding studies
Calf thymus DNA was procured from Sigma Chemical Company and Tris-base was
obtained from E. Merck. All the experiments involving interaction of the complexes with
CT DNA were conducted in buffer containing Tris (0.01 M) adjusted to pH 7.2 with
hydrochloric acid. The CT DNA was dissolved in Tris HCl buffer and was dialyzed
against the same buffer overnight. Solutions of CT DNA gave ratios of UV absorbance at
260 and 280 nm above 1.8, indicating that the DNA was sufficiendy free of protein [179].
DNA concentration per nucleotide was determined by absorption specfroscopy using the
molar absorption coefficient 6600 dm^ mol"' cm"' at 260 nm [180]. The stock solution
was stored at 4°C.
2.1 Cyclic voltammetry
Cyclic voltammetry involves the measurement of current-voltage curves under diffusion
controlled, rnass transfer conditions at a stationary elecfrode, utilizing synmietrical
triangular scan rates ranging from a few millivolts per second to hundred volts per
second. The triangle returns at the same speed and permits the display of a complete
polarogram with cathodic (reduction) and anodic (oxidation) waveforms one above the
other. Two seconds or less is required to record a complete polarogram [181].
46
Consider the reaction
O + ne • R (i)
Assuming semi-infinite linear diffusion and a solution containing initially only species O.
With the electron held at a potential Ei where no electrode reaction occurs. The potential
is swept linearly at v v/sec so that the potential at any time is
E(t) = Ei-vt
or Epeak = E°-0.0285
The rate of electron transfer is so rapid at the electrode surface that species O and R
immediately adjust to the ratio according to the Nemst equation, which is as follows.,
Co (0,t) = Co* - [nFA(7rDo)' ]-' J I(7c)(t-T)-"^ dx (ii)
I = nFACo*(7iDoCT)' x(CTt) (iii)
Redox (electron-transfer) reactions of metal complexes can be investigated by cyclic
voltammetry. An electrode is immersed in a solution of the complex and voltage is swept
while current flow is monitored. No current flows until oxidation or reduction occurs.
After the voltage is swept over a set range in one direction, the direction is reversed and
swept back to the original potential. The cycle may be repeated as often as desired.
Figure 31 shows the cyclic voltammograms (CV) for a reversible one-electron redox
reaction such as,
CpFe(CO)LMe • CpFe(CO)LMe^ + e"
47
Sweeping the potential in an increasing direction oxidize the complex as the anodic
current la flows; reversible reduction of CpFe(CO)LMe* generates cathodic current Ic on
the reverse sweep. The magnitude of the current is proportional to the concentration of
the species being oxidized or reduced.
The measured parameters of interest on these cyclic voltammograms are Ipa/Ipc the ratio
of peak currents, Epa - Epc the separation of peak potentials and the formal electrode
potential E°. For a Nemstian wave with stable product, the ratio Ipa/Ipc - 1 regardless of
scan rate, E° and diffusion coefficient, when Ipa is measured from the decaying cixrrent as
a base line. The difference between Epa and Epc (AEp) is a useful diagnostic test of a
Nemstian reaction. Although AEp is slightly a function of E°, it is always close to
2.3RT/ nF.
0 Switching time, X
(a) (b) Figure 31. (a) Cyclic potential sweep (b) Resulting cyclic voltammogram
The technique yields information about reaction reversibilities and also offers a rapid
means of analysis for suitable systems. The method is particularly valuable to study
interaction of metal ions to DNA as it provides a usefiil compliment to other methods of
48
investigation, such as UVA^is spectroscopy. Cyclic voltammetric studies were
accomplished on a CH Instrument Electrochemical analyzer using a three-electrode
configuration comprised of a Pt wire as the auxiliary electrode, a platinum micro-cylinder
as the working electrode and Ag/AgCl as the reference electrode. Electrochemical
measurements were made imder nitrogen atmosphere. All electrochemical data were
collected at 298 K and are uncorrected for junction potentials.
2.2 Absorption spectral studies
In a closed constant volume system, the rate of a chemical reaction is defined as the rate
of change of the concentration of any of the reactants and products with time. The
concentration can be expressed in any units of quantity per imit volume e.g. moles per
liter, moles per cubic centimeter. The rate will be defined as positive quantity regardless
of the component whose concentration change is measured.
The rate of a chemical reaction is not measured directly instead the concentration of one
of the reactants or products is determined as a fiincfion of time. A common procedure for
determining the reaction order is to compare the experimental results with integrated rate
equations for reactions of different orders. For a first order rate equation, integrating by
separate variables using integration limits such that at t = 0, c = Co and at t = t, c = c.
[182].
-dc/dt = kc
49
or In (CQ/C) = kt
The intrinsic binding constant Kb of the complex to CT DNA was determined from
equation (1), through a plot of [DNA]/ e^-Cf vs [DNA], where [DNA] represents the
concentration of DNA, and Ea, Cf, and Sb the apparent extinction coefficient (Aobs /[M]),
the extinction coefficient for free metal complex (M), and the extinction coefficient for
the free metal complex (M) in the fully bound form, respectively. In plots of [DNA]/ea-Ef
vs. [DNA], Kb is given by the ratio of slope to intercq)t [183,184]. These absorption
spectral studies were performed on USB 2000 Ocean Optics and Shimadzu UV-1700
PharmaSpec UVA^is spectrophotometers.
[DNA] / |8a-efl = [DNA]/|8a-£d +l/Kb|ea-8f| (1)
Absorption spectral titration experiments were performed by maintaining a constant
concentration of the complex and varying the nucleic acid/nucleotide concenfration. This
was achieved by diluting an appropriate amount of the metal complex solutions and CT
DNA/5'GMP stock solutions while maintaining the total volume constant. This results in
a series of solutions with varying concentrations of CT DNA/5'GMP but a constant
concentration of the complex. The absorbance (A) was recorded after successive
additions of CT DNA/5'GMP. While measuring the absorption spectra an equal amount
of CT DNA/5'GMP was added to both the compound solution and the reference solution
to eliminate the absorbance of the CT DNA/5'GMP itself
50
2.3 Fluorescence spectral studies
When molecules that have absorbed light are in a higher electronic state, they must lose
their excess energy to return back to the ground state. If the excited molecule returns to
the ground state by emitting light, it exhibits fluorescence and spectrum thus obtained is
called emission spectrum. This phenomenon is generally seen at moderate temperature in
liquid solution. The emission spectrum is obtained by setting the excitation
monochromator at the maximum excitation wavelength and scanning with emission
monochromator. Often an excitation spectrum is first made in order to confirm the
identity of the substance and to select the optimum excitation wavelength. Further
experiments were carried out to gain support for the mode of binding of complexes with
CT DNA. Non-fluorescent or weakly fluorescent compounds can often be reacted with
strong fluorophores enabling them to be determined quantitatively. On this basis
molecular fluorophore EthBr was used which emits fluorescence in presence of CT DNA
due to its strong intercalation. Quenching of the fluorescence of EthBr bound to DNA
were measured with increasing amount of metal complexes as a second molecule and
Stem-Volmer quenching constant K was obtained from the following equation 2 [185].
I o / I = l + K r (2)
where r is the ratio of total concentration of complex to that of DNA and IQ and I are the
fluorescence intensities of EthBr in the absence and presence of complex. Binding
51
constant K of the metal complexes was also determined from equations 3 and 4
(Scatchard equations) by emission titration [186,187].
CF = C T ( I / I O - P ) ( 1 - P ) (3)
r/CF = K(n-r) (4)
Where Cp is the free probe concentration, Cj is the total concenfration of the probe added
I and lo are fluorescence intensities in presence and absence of CT DNA, respectively and
P is the ratio of the observed fluorescence quantimi yield of the bound probe to that of the
free probe. The value P was obtained as the intercept by extrapolating from a plot of I/Io
vs 1/ [DNA], r denotes ratio of CB (=CT-CF) to the DNA concentration i.e., the bound
probe concentration to the DNA concentration, K is the binding constant and CF, is the
free metal complex concentration and "n" is the binding site nimiber. Emission intensity
measurements were carried out using Hitachi F-2500 spectrofluorometer at room
temperature.
2.4 Gel electrophoresis
Gel electrophoresis is a technique widely used for separation and analysis of charged
biomolecules like nucleic acids [188,189]. Any charged biomolecule migrates when
placed in an electric field. The ratio of migration of a molecule depends on its net charge,
size, shape and the applied current. This can be represented as follows
V = E.q / f
52
Where V = velocity of migration of the molecule, E = electric field in volts/cm, q = net
charge on the molecule, f = frictional coefficient which is function of mass and shape of
molecule. The movement of a charged molecule in an electric field is often expressed in
terms of electrophoretic mobility (|i), which is defined as the velocity per unit of electric
field.
H = V / E = E .q / f .E
ji = q / f
For molecules with similar conformation, "f " varies with size but not with shape. Thus
electrophoretic mobility (ji) of a molecule is directly proportional to the charge density
(charge/mass ratio). Molecules with different charge/mass ratio migrate under the electric
field at different rates and hence get separated. This is the basic principal for all the
electrophoretic techniques. Depending upon the nature of support medium,
electrophoresis is of different types such as paper, starch, polyacrylamide and agarose gel
electrophoresis. We have opted agarose gel electrophoresis, because agarose gels are
more porous as compared to polyacrylamide gels and are, therefore, used to fi^ctionate
large macromolecules such as DNA that caimot be readily penetrate into and move
through other types of supporting materials. Agarose is a linear polymer of D-galactose
and 3,6-anhydro-L-galactose. When an electric field is applied across agarose gel, DNA
molecules that are negatively charged at neutral pH, migrates towards oppositely charged
electrode at rates determined by their molecular size and conformation. DNA molecules
of the same size but with different conformation travel at different rates. The order of
53
migration velocity in the increasing order of various forms of DNA is: supercoiled DNA
> linear double strand DNA > open circular DNA.
2.5 Viscometry studies
The hydrodynamic changes are the consequence of the change in length of the molecule,
the diminished bending between layers and the diminished length-specific mass.
Viscosity measurements were carried out using Ostwald's viscometer at 29 ± 0.01 °C.
Flow time was measured with a digital stopwatch. Each sample was measured three times
and an average flow time was calculated. Data were presented as (r|/r|o) versus binding
ratio ([M]/[DNA]), [190,191] where r| is a viscosity of DNA in the presence of complex
and r|o is the viscosity of DNA alone. Viscosity values were calculated from the
observed flow time of DNA containing solution (t >100s) corrected for the flow time of
buffer alone (to), ti = t- to.
CHAPTER m
Synthesis, Spectroscopic Studies of New Water-Soluble Co(II) and Cu(II) Macrocyclic Complexes of 4,15-bis(2-hydroxyethyl)-
2,4,6,13,15,17- hexaazatricyclodocosane: Their Interaction Studies with Calf Thymus DNA and Guanosine 5' Monophosphate
54
CHAPTER III
Experimental
Cobalt chloride hexahydrate (C0CI2. 6H2O), nickel chloride hexahydrate (NiCli. 6H2O),
copper chloride dihydrate (CUCI2.2H2O), Tris-base, formaldehyde, ethanolamine (E.
Merck) and 1, 2-dianiinocyclohexane (Fluka) were used as received. Calf thymus DNA
(CT DNA) and guanosine 5'monophosphate disodiimi salt (5'GMP) were purchased from
Sigma chemical Co. and Fluka, respectively. All reagent grade compounds were used
without further purification. Carbon, hydrogen and nitrogen contents were determined
using Carlo Erba analyzer Model 1108. Molar conductances were measured at room
temperature on a Digisun Electronic conductivity Bridge. Fourier-transform ER (FTIR)
spectra were recorded on an Interspec 2020 FTIR spectrometer. UVA^is spectra were
recorded on a UV-1700 Pharma Spec Shimadzu spectrophotometer in H2O and the data
were reported as A-max/nm. The EPR spectrum of the copper complex was acquired on a
Varian E 112 spectrometer using X-band frequency (9.1GHz) at liquid nitrogen
temperature in solid state. The ' H and * C NMR spectra were obtained on a Bruker DRX-
300 spectrometer operating at room temperature. Chemical shifts were reported in ppm.
Electrospray mass spectra were recorded on Micromass Quattro II triple quadrupol mass
spectrometer. Molecular modeling of the complex was carried out by using CS Chem
Draw 3D Pro 5.0.
\ » ^ / ,0 KV\ 55
All the experiments involving interaction of the complex with CT DNA were performed
in twice distilled buffer containing tris (hydroxymethyl)-aminomethane (Tris, 0.01 M)
and adjusted to pH 7.5 with hydrochloric acid. Solution of 5'GMP was prepared in
double distilled water. NMR experiments with 5'GMP were carried out in D2O on a
Bruker Avance II 400 NMR spectrometer at 25 ^C. Cyclic voltammetric studies were
performed on a CH Instrument Electrochemical analyzer in a single compartmental cell
with 5 mM Tris-HCl/50 mM KCl buffer as supporting electrolyte. A three-electrode
configuration was used comprising of a Ft wire as auxiliary electrode, platinum micro
cylinder as working electrode and Ag/AgCl as the reference electrode. Electrochemical
measurements were made under a dinitrogen atmosphere.
Synthesis of 4,15-bis(2-hydroxyethyl)-2,4,6,13,15,17- hexaazatricyclodocosane
cobalt(II) Dichloride IC20H42N6O2C0 ]Cl2
To a stirred methanol solution (50 mL) of C0CI2. 6H2O (5.94 g, 25 mmol) were slowly
added 1,2-diaminocyclohexane (6.08 mL, 50 mmol) formaldehyde (2.8 mL, 100 mmol)
and ethanolamine (3.07 mL, 50 mmol). The resulting mixture was refluxed for ca 24 h
until a dark orange colored solution appeared. The volume of the solution was reduced to
15 mL on a rotary evaporator and then kept in refrigerator overnight. The dark orange
crystals were separated and washed thoroughly with hexane and then dried in vacuo.
56
Synthesis of 4,15-bis(2-hydroxyethyl)-2,4,6,13,15,17- hexaazatricyclodocosane
nickel(II) Bichloride [C2oH42N602Ni]Cl2
The [C2oH42N602Ni]Cl2 complex was prepared by using NiCl2.6H20 (5.92 g, 25 mmol)
according to the method outlined for complex [C20H42N6O2C0 ]Cl2. The brown coloured
product obtained was washed with methanol and dried in vacuo.
Synthesis of 4,15-bis(2-hydroxyethyl)-2,4,6,13,1547- hexaazatricyclodocosane
copper(II) Dichloride IC2oH42N602Cu]Cl2
The complex [C2oH42N602Cu]Cl2 was prepared by adopting the procedure given for
[C2oH42N602Co]Cl2 using CUCI2.2H2O (4.26 gm, 25 mmol). The dark blue coloured
complex obtained was filtered, washed with hexane and dried in vacuo.
Results and discussion
The one step template reaction of 1,2-diaminocyclohexane, formaldehyde and
ethanolamine in presence of metal chloride salts yielded the Cobalt(II), Nickel(II) and
Copper(II) macrocyclic complexes of 4,15-bis(2-hydroxyethyl)-2,4,6,13,15,17-
hexaazatricyclodocosane in which 2-hydroxyethyl groups are appended as shown in
Scheme 1. These complexes are extremely stable in solid state; readily dissolve in H2O,
MeOH, EtOH, DMF and DMSO. Molar conductivity data for 10' M solutions in H2O
observed in the range -150-200 O'cm^mol"' are in accordance with those expected for
1 -.2 electrolytes, implying the non-participation of chloride anions in coordination to the
MCl2 + + HCHO + HO V
^ NH,
57
2+
,2CI
M = Co(II), Ni(II), Cu(II)
Scheme 1. Proposed structure of complexes [C2oH42N602Co]Cl2, [C2oH42N602Ni]Cl2 and [C20H42N6O2CUJCI2.
metal ion. The important properties and physical data of the complexes are given in Table
1. The stretching mode v(O-H) in the IR spectra of all the complexes confirm to the
existence of free hydroxyl groups. Thus, the potentially hexadentate ligand behaves as a
tetradentate ligand with the central metal ion assuming a square planar coordination
geometry as also authenticated by the UV/Vis absorption spectroscopy. The molecular
model of the complex [C2oH42N602Co]Cl2 as depicted in Figure 32 indicates that there is
58
no strain on any bond and angle. The interaction studies of complex [C2oH42N602Co]Cl2
and [C2oH42N602Cu]Cl2 with CT DNA and guanosine 5'monophosphate in aqueous
solution were examined by various techniques while [C2oH42N602Ni]Cl2 was synthesized
only for NMR studies. Molecular architecture of the macrocyclic complexes which,
contain —NH— and —OH groups of appended arms with two cyclohexane rings, provides
a rationale to explore their DNA binding propensity. An additional feature is their
solubility in water — a highly significant physical property of pharmacological
relevance, as all biochemical reactions are based on small molecules that dissolve in an
aqueous phase [192].
Figure 32. Three dimensional molecular model of the [C2ot'U2N602Co]Cl2 complex.Colour Scheme Co" Purple; N blue; O red; C gray; H light blue; lone pairs pink.
59
I R spectra
The IR spectra of the complexes [C2oH42N602Co]Cl2, [C2oH42N602Ni]Cl2 and
[C2oH42N602Co]Cl2 exhibit an intense band in 3100-3130 cm' region due to v(N-H)
[193] of the coordinated secondary amino group, which was further confirmed by the
appearance of bands in the 534-550 cm"' range corresponding to v(M-N) vibrations [194].
Absorption bands due to v(CH2) vibrations of the cyclohexyl ring and the ethanolamine
moiety were observed at 2860 - 2931 cm'' and 2367- 2380 cm"' region, respectively
while a moderate peak at ~ 1600 cm"' was assigned to 5(N-H) vibrations [195]. The
strong characteristic bands at 1441 - 1452 cm"' and 1043 - 1049 cm"' were associated
with the C-N and C-0 vibrations of the ethanolamine fragment. The stretching mode of
the 0-H group observed at ca. 3200 cm' suggests that the oxygen atom does not interact
with the metal ion [196] (Table 2). Together with these observations, the absence of the
free -NH2 group stretching modes at ~ 3300 - 3400 cm"' confirmed the condensation and
the subsequent cyclization resulting in the formation of new macrocyclic complexes.
NMR Spectra
The 'H and '^C NMR spectra of the diamagnetic [C2oH42N602Ni]Cl2 complex were
obtained in D2O solution. The 'H NMR spectrum revealed an intense signal at 4.79 ppm
assigned to the presence of free -OH protons of the ethanolamine pendant arm as
observed for the analogous ethanolamine metal complexes [197]. Signature due to the
two -CH2 groups of the ethylene chain appeared at ~ 3.67 ppm (-CH2OH) and at 3.36
60
ppm (-CH2N), respectively [198,199]. The cyclohexane ring protons were observed in
the 2.56- 2.86 ppm and 1.44-1.74 ppm range as doublets [200,201] (Table 3).
The '^C NMR spectrum confirms the ' H N M R data. The '^C NMR spectrum features
various resonances due to the cyclohexyl ring carbons at 19.00, 20.29, 23.94, 26.70,
39.13 ppm [202, 203] (Table 4). A doublet appearing at 58.58- 58.85 ppm has been
assigned to the -CH2OH carbons of ethanolamine moiety [204]. Additional signals at
54.64 ppm and at 68.62 ppm were observed due to -N-CH2-GHr-0H carbons and - N -
C-N-C-N- carbons of the macrocyclic framework, respectively [205].
Electronic absorption spectra
The electronic spectra of the complexes [C2oH42N6C)2Co]Cl2, [C2oH42N602Ni]Cl2 and
[C2oH42N602Cu]Cl2 wcrc characterized by strong ligand absorptions at around 224-240
nm. The visible region of the [C2oH42N602Cu]Cl2 complex exhibited a very broad and
intense band centered at 570 nm, which was assigned to Big • Aig transition [206,
207], a characteristic of CUN4 chromophore with the copper ion in the square planar
environment. The electronic spectrum of the [C2oH42N602Co]Cl2 complex displays two
main absorption bands; an intense band at 340 nm attributed to the charge transfer (CT)
transitions; and a broad band at 490 nm ascribed to the d-d transition indicative of the
square planar Co(II) ion [208]. Similarly, the [C2oH42N6C)2Ni]Cl2 complex exhibits an
intense charge transfer band at 340 nm. The d-d band due to the Ni(Il) ion of the complex
[C2oH42N602Ni]Cl2 was not fully resolved, and seemed to be obscured under the charge
transfer band, which was tailed to near IR region. However, a discernible shoulder
61
appeared at 510 nm, which may be assigned to 'Aig(F) • 'Big(G) transition, typical
of a low spin d* electronic configuration of square planar Ni(II) ion [209,210].
EPR Spectrum
The solid state X-band EPR spectrum of the [C2oH42N602Cu]Cl2 complex was recorded
at 77K (liq-N2 temperature) using TCNE as a field marker (2.00277). The spectrum
exhibits a broad gj. component with the splitting of the g y component, reflecting the
coupling with the Cu(II) nucleus (I = 3/2). The g n value at 2.19 and gi at 2.05 are quite
similar to the values reported for other related square planar copper (II) complexes and
are typical for axially symmetrical Cu(II) complexes with the unpaired electron in the
dxl ^ orbital with ^Big ground state [211]. The order g n >gj. > gc (2.0023) further confirm
that the ground state of the Cu(II) is predominantly dx - ^.
Solution stability studies
To confirm the stability of complexes [C2oH42N602Co]Cl2 and [C2oH42N6C)2Cu]Cl2 in
buffered solution at various pH values, UVA/ is and cyclic voltammetric studies were
performed under conditions similar to those used for DNA binding studies. UVA^is
spectra of complex [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 exhibited no change in
the position of the intraligand band over a pH range 2-12 with only minor change in
intensity. The complexes also display similar spectra over a period of 24 h and no
precipitation was observed (Figure 33a- b). The non-precipitation after a 24 h time period
and over a broad pH range suggests the robust nature of these complexes in solution. The
solution stability of complex [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 were ftirther
62
studied by means of cyclic voltanunetery in the pH range 2-10. The voltammetric
behaviour of the complexes was found to be reproducible and they exhibited
quasireversibility [212] typical for one electron transfer process corresponding to
M(II)/M(I) redox couple (Figure 34a -b)
30O4I0
Wavelength KM
Fig. 33a. UV/Vis absorption spectra of the [C20H42N6O2CUJ Ch complex as a function of pH. Conditions: Tris-HCl buffer, 25 C; The curves from top to bottom correspond to pH 12, 10, 8, 6, 4, 2 respectively.
Wavelength (nm)
Figure 33b. UV/Vis absorption spectra of the [C2oH42N602Cu]Cl2 complex after 24 h as a function of pH. Conditions: Tris HCl buffer, 25 "C; The curves from top to bottom correspond to pH 12, 10, 8, 6, 4, 2 respectively.
63
(i) (ii) l U
7J
U
U
i i l
I f
u M
• - U
.£• j i j
y—v,^^ y^ \
/ \^„^ / ^^^,„-^
—-—*-^ y ^^ ' \ , / ' ^
y^ '-•^...^^_^^
•111 4L2 -03 «S -M - U
rv;
Figure 34a. Cyc//c voltammetric data for [C2oH42N(,02Co]Cl2 complex at pH i) 2, ii) 4. Hi) 6, iv) 8, v) 10. Conditions: 5 mM Tris-HCl/50 mM KCl buffer. 25 "C; scan rate 0 1 VS'.
64
(i) (ii)
1J
u 14
1 ^
1«
0«
0«
04
i l
0
-OJ
4 4
• - - • • - - • 1 I 1 f • • • ' • • ' •
i /
/I y"^ \
y^ 1 y/ , j / ^
/-""^'"'^l--^"* ' ^ ( ^y ^ • • • 1 ' 1 1 1 • • 1 1 —
0.« 04
(iii)
D% 0 -OJ A t
I
iv;
Figure 34b. Cyclic voltammetric data for [C2oH42N602Cu]Cl2 complex at pH i) 2, ii) 4, iii) 6, iv) 8, v) 10. Conditions: 5 mM Tris-HCl/50 mM KCl buffer. 25 "C; scan rate 0.1
vs-'.
65
Mass spectra
Electrospray mass spectra in the positive ion mode were recorded for the complexes
[C2oH42N602Co]Cl2, [C2oH42N602Ni]Cl2 and [C2oH42N602Cu]Cl2. The ESI MS spectrum
of the [C2oH42N602Cu]Cl2 complex shows the molecular cation peak, [M-Cl]"^ at m/z =
496 corresponding to a mass loss of 35.45 due to the one chloride ion from the parent
macrocycle. The spectrum shows some prominent peaks corresponding to the various
fragments of the complex. The molecular cation during the fragmentation process yields
the species such as; (C16H35N5OCICU + 2H^ 411; (C,6H32N4ClCu) 378; (C,2H24N4ClCu
+ 2H^ 326 and [C,2H24N4Cu + 2H^f^ 145; from the loss of C4H9NO, C4H,oN202,
C8H18N2O2CI and C8H18N2O2CI2 fragments, respectively, which confirm the structure of
the [C2oH42N602Cu]Cl2 complex. The molecular cation peaks relating to the complexes
[C2oH42N602Co]Cl2 and [C2oH42N602Ni]Cl2 were not observed. Nevertheless, many
fragments were observed in each of the specfra to characterize obvious compositions of
the complexes. Peaks defining [M-2Cr + 2ltf* and (Ci2H24N4ClNi + 6H^ fragments
were recorded at m/z 23 land 326,respectively for [C2oH42N602Ni]Cl2 complex, while for
[C2oH42N602Co]Cl2 complcx (C12H24N4CIC0 + 3H^ fragment was observed at m/z 321.
Other peaks at m/z 143 were ascribed to [C12H24N4C0 + lltf^ and [Ci2H24N4Ni +
2B^f* fragments for [C2oH42N602Co]Cl2 and [C2oH42N602Ni]Cl2 complex, respectively.
DNA binding studies
DNA is the primary intracellular target of antitumour drugs. Interaction of the complexes
with DNA can induce DNA damage, which leads to blockage of cell division and
66
eventually cell death. Interaction with 5'GMP provides supportive evidence in favor of
the DNA binding studies.
Absorption Titration
The absorption spectral titration of the [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2
complexes with CT DNA was followed by monitoring the absorbance of intraligand
bands (Figure 35a-b). Any interaction between the complex and the DNA is expected to
perturb the ligand centered spectral transitions of the complexes. Intensity of the spectral
band of the complexes at 224 nm and 240 nm were found to increase with the increasing
concentration of the DNA. The complexes [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2
exhibited hyperchromism of 27.7% and 23.3% when saturated around [DNA]/[complex]
= 0.75 and 0.58, respectively. The hyperchromic and hypochromic effect are the spectral
features of DNA concerning its double helix structure. The "hyperchromic effect" which
results from the structural damage of DNA observed in these complexes is indicative of
strong binding of the complexes to CT DNA. However, no red shift was observed in the
absorption traces, which ruled out coordinate covalent binding with N7 base moieties of
DNA. Complex-interaction occurs with exterior phosphates of DNA primarily via
electrostatic attraction [213]. Nevertheless, DNA double helix possesses many hydrogen
bonding sites positioned on the edges of the DNA bases, it is quite probable that the
coordinated -NH- groups and the dangling -OH groups could form hydrogen bonds with
the DNA base pairs [214, 215], contributing to the overall hyperchromism. No significant
change was observed in the absorbance of LF transition of the complexes on the addition
67
of CT DNA revealing that the binding of metal complexes via N7 of guanine is quite
unlikely. This was evidenced by the UVA'is absorption spectral studies of the complexes
with 5'GMP. The present ligand framework lacks extended n- systems; the intercalative
binding mode is therefore, overruled. In order to further compare quantitatively the
affinity of complexes bound to CT DNA the intrinsic binding constants Kb of the
complexes were also determined (Figure 35a-b, inset). The binding constants obtained
for [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 using equation (1) are 2.94.10'' M'' and
2.71.10'* M ' , respectively, revealing that both the complexes bind strongly to CT DNA
with almost same affinity.
0.000 200.00 250.00 300.00 350.00 AOO.OO
Wavelength (nm)
Figure 35a. Absorption spectral traces of [C2oH42N602Co]Cl2 complex in Tris-HCl buffer upon addition ofCTDNA. Arrows show the absorbance changes upon increasing concentration of the CT DNA. Inset: Plots of [DNAJ/Sa-e/vs [DNA] for the titration of CT DNA with [C2oH42N602Co]Ch complex; (*) experimental data points; full lines, linear fitting of the data. [C2oH42N602Co]Cl2=1.23. Iff'' M.
68
2.000
0.000 200.00 220.00 240.00 260-00 280.00 300.00
Wovelength (nm)
Figure 35b. Absorption spectral traces of [C2oH42^602Cu]Cl2 complex in Tris-HCl buffer upon addition ofCTDNA. Arrows show the absorbance changes upon increasing concentration of the CT DNA. Inset: Plots of [DNAJ/Sa-S/vs fDNAJ for the titration of CT DNA with [C2oH42N602Cu]Cl2 complex; (^) experimental data points; full lines, linear fitting of the data. [C2oH42N602Cu]Cl2=1.33.1(r^ M.
Interaction studies with 5'GMP
Interaction of metal complexes with nucleotides is notably important to illustrate the
binding sequence with DNA at the molecular level. Previously, it has been observed that
interaction of 5'GMP with Cu " ion occurs mainly via N7 site [216, 217] while 06
position is favoured in other early transition metal ions [218]. The absorption spectra of
complexes [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 on interaction with 5'GMP show
69
hyperchromism in the intraligand bands, with a red shift (bathochromic) of 5 nm for the
complex [C2oH42N602Cu]Cl2 (Figure 36a-b). However, no distinct spectral changes were
observed in the LF bands, obscuring the possibility of coordination of the metal ions
either to N7 or 06 of GMP. The hyperchromicity implies that the interaction between the
complexes and the GMP is a combination of electrostatic and hydrogen bonding with the
oxygen of the negatively charged phosphate group to the - NH- of the complex [219] or
possibly via 06 atom of nucleobase with pendant - OH of the complex. The
fimctionalized - OH appendage facilitates in carrying the metal ion to the site in the major
groove.
0.000^ 190.00 250.00 300 .00 350-00 400-00
Wave leng th ( n m )
Figure 36a. Absorption spectral traces of [C2()H42N602Co]Cl2 complex in H2O upon addition of 5 'GMP.
70
1.5001-
0)
c o L.
o l/t
Xi
<
1.000
0.500
0.000 195.00 300.00 400.00
Wavelength (nm)
500.00
Figure 36b. Absorption spectral traces of [C2oH42N602Cu]Cl2 complex in H2O upon addition of 5'GMP.
' H and *P NMR spectroscopic studies with 5'GMP
The conclusive evidence for the interaction of complexes [C2oH42N602Co]Cl2 and
[C2oH42N602Cu]Cl2 with 5'GMP was further obtained by ' H NMR and ^'P NMR
spectroscopy, being most sensitive and a reliable technique (Fig 37a- b). The ' H NMR of
5'GMP in D2O solvent records the proton resonance of guanine Hg at 8.07 ppm and
ribose Hi'-Hs' at 3.8-5.8 ppm, respectively. The resonance of 2-NH2 was obscured due to
the exchange of protons with deuterium solvent. For paramagnetic complexes, the
71
chemical shift of protons adjacent to the metal center will be significantly perturbed and
there is an apparent line broadening. On interaction of complex with 5'GMP, there is no
line broadening however, H2' resonance of ribose records an increase in intensity while
the other peaks of ribose show downfield shift. The G-Hs signal has diminished but does
not display significant downfield shift (8.07 ppm in free 5'GMP to 8.1 ppm in
[C2oH42N602Co]Cl2 + 5'GMP bound), which shows non-involvement of N7 position of
guanine in coordination [220]. These results are also consistent with UVA^is interaction
studies of complex [C2oH42N602Co]Cl2 with DNA and 5'GMP. ^'P WAR of 5'GMP
records a signal at 3.65 ppm, which is completely quenched on addition of complex
[C2oH42N6C)2Co]Cl2 suggcstive of involvement of phosphate moiety via electrostatic
binding mode.
Compared with the ' H N M R spectra of 5'GMP and 5'GMP + [C2oH42N6C)2Co]Cl2, the ' H
NMR spectra of complex [C2oH42N602Cu]Cl2 + 5'GMP displayed remarkable changes.
The Hg signal due to 5'GMP at 8.07 ppm disappears, while peaks due to Hi'-Hs' showed
significant line broadening with downfield shifts. These observations depict that the
complex [C2oH42N6C)2Cu]Cl2 shows tendency for coordination to the N7 position of the
guanine compared to complex [C2oH42N602Co]Cl2. However, phosphate binding of
complex [C2oH42N602Cu]Cl2 to 5'GMP cannot be overruled owing to the absence of any
signal in the ^'P NMR of [C2oH42N602Cu]Cl2 + 5'GMP. These NMR results strongly
support the phosphate binding of complexes [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2
72
(Hi)
J . I . ' I V » - " • • • « » "
(ii)
(i)
4 u.
1
i#j
ill A L
- r • • "
5 -T- ppm
Figure 37a. 'HNMR spectra ofi) 5 'GMP and the reaction ofii) [C20H42N6O2C0] Ch and [C2oH42N602Cu]Cl2 complexcs (2.5 mM) with 5 'GMP (5 mM) in D2O at 25 V .
73
' \^H^^\*h^t^JrW^^ It I • T < •1 -I -t -1
Figure 37b. ^'PNMR spectrum of 5 'GMP in D2O at 25 "C
with 5'GMP, and simultaneous coordination of complex [C2oH42N602Cu]Cl2 to the N7
atom of nucleobase.
Fluorescence spectroscopic studies
To exclude the possibility of the intercalative binding mode an ethidium bromide assay
was carried out. Ethidium bromide is a conjugate planar intercalating molecule emitting
intense fluorescence when bound to DNA. Decrease in emission intensity results when a
second DNA binding molecule either replaces EthBr or accepts the excited state electron
from EthBr [221]. Complexes [C2oH42N602Co]Cl2 and [C20H42N6O2CUICI2 do not display
74
luminescence either alone or in tris buffer. The addition of complex [C2oH42N6C)2Co]Cl2
and [C2oH42N602Cu]Cl2 to the EthBr-DNA system resulted in the reduction in the
emission intensity (Figure 38a-b). As complexes [C2oH42N6C)2Co]Cl2 and
[C2oH42N602Cu]Cl2 bind to DNA primarily via surface binding, they cannot displace the
strongly DNA bound EthBr. So the observed quenching occurs through the photoelectron
transfer mechanism [222]. The larger quenching extent of complex [C2oH42N602Cu]Cl2
than the complex [C2oH42N602Co]Cl2 is expected, as the reduction of the DNA-bound
complex [C2oH42N602Co]Cl2 (E1/2 = -0.285 V) is more difficult than the complex
[C2oH42N602Cu]Cl2 (E1/2 = -0.180 V). The relative fluorescence intensity, plotted as a
function of CT DNA concentration (in terms of [M]/[DNA]) (Insets) is in good
agreement with the linear Stem-Volmer equation (2), which also proves that the
complexes bind to DNA. The value of the fluorescence quenching constant Kgv obtained
as the slope of IQ/I VS r (=[complex]/[DNA]) for complexes [C2oH42N602Co]Cl2 and
[C2oH42N602Cu]Cl2 are found to be 0.3 and 0.5, respectively.
Viscosity measurements
To obtain further support for the binding modes of the complexes with DNA, viscosity
measurements were carried out as hydrodynamic measurements sensitive to length
changes are regarded as the most critical tests of a binding model in solution in the
absence of crystallographic structural data [223]. For DNA binding of a complex, a
partial or a non-classical mode of binding could bend or kink the DNA helix, reduce its
effective length and concomitantly its viscosity [224].
75
a ^^M is.67 21.N
|Coiipki]/|DNA|.)0-'
27J
400 ' I ' 900
' I ' M (nm) (00 TOO
I ' 1 1 ' I
no
Figure 38a. Emission spectra ofEB bound to DNA in the presence of complex in Tris-HCl buffer. Arrows show the intensity changes upon increasing concentration of the [C2oH42N602Co]Cl2 complcx. Inset: Fluorescence quenching curves of DNA bound EB [C20H42N6O2C0] CI2 6.6. la^ M-33.0. lOr^ M, fCTDNAJl.ZlOr^M; (^). experimental data points; full lines, linear fitting data.
ar
• 7 -
JJ I I M 1t.t7 2t.S« |C(»pla|/|DNA|.lir<
J7j
100
Figure 38b. Emission spectra of EB bound to DNA in the presence of complex in Tris-HCl buffer. Arrows show the intensity changes upon increasing concentration of the C20H42N6O2CI2CU complex. Inset: Fluorescence quenching curves of DNA bound EB [C20H42N6O2CI2CU] 6.6. W'^M-SS.O. 10'^ H [CT-DNA] 1.2.10-^M; (^). experimental data points; full lines, linear fitting data.
76
The effect of the complexes [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 on the viscosity
of the CT DNA is shown in Figure 39. The relative specific viscosity decreases steadily,
which imply complexes bind to CT DNA via electrostatic mode [130]. Moreover, the
decreasing pattern of DNA viscosity occurs probably due to the insertion of the flexible -
OH pendant arm into the DNA helix permitting the hydrogen bond interaction with the
DNA base pairs [225]. These results are consistent with observed hyperchromic effect of
[C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 bound to CT DNA.
;? 1.2 n
T -
0.65 1.3 1.95 1/R {=IM1/[DNAJ )
2.63
Figure 39. Effects of increasing amount of [C2oH42N60^o]Cl2 complex (•),fC2oH42N602CuJ Ch complex (u) on the relative viscosity ofCTDNA at 29 ±0.1 "C. [M]= complex concentration
Cyclic voltammetry
The cyclic voltammograms of complexes [C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2
were studied in aqueous solution containing 5 mM Tris-HCl/50 mM KCl buffer obtained
at 0.1 VS" scan rate in the absence and presence of CT DNA. Both the complexes
display quasi-reversible electrochemical waves for M(II)/M(I) couple. The CV response
77
of the complex [C2oH42N602Co]Cl2 in the absence of DNA (Figure 40a) shows reduction
of Co(II) to Co(I) at a cathodic peak potential of -0.400 V and the corresponding
oxidation wave at -0.110 V. The separation of anodic and cathodic peak potentials (AEp)
is -0.01 V and the ratio of anodic to cathodic peak currents Ipa/Ipc is 0.133, indicating a
quasi-reversible redox process. The formal potential Em estimated, as the average of Epa
and Epc is -0.405 V. Complex [C2oH42N602Cu]Cl2 exhibited one quasi-reversible redox
couple corresponding to the Cu(II)/Cu(I) redox state with Ipa/Ipc =0.322 and the Epc and
Epa values of -0.440 V and 0.020 V respectively (Figure 40b) For this couple, AEp and
the formal potential Emare 0.460 V and -0.210 V, respectively.
Keeping all the parameters constant, quasi-reversibility of the electron transfer was
maintained in the presence of DNA. Addition of DNA to complex [C2oH42N602Co]Cl2
and [C2oH42N602Cu]Cl2 resulted in reduction in anodic and cathodic peak currents
coupled with shifts in Epc, Epa and E1/2 values. However, for complex [C2oH42N602Co]Cl2
the anodic peak appeared weakly. The apparent reduction in the peak currents of
[C2oH42N602Co]Cl2 and [C2oH42N602Cu]Cl2 was attributed to the diffusion of the metal
complexes bound to the large slowly diffusing DNA molecule [126]. The shift in the Em
values indicates the binding of the complexes [C2oH42N602Co]Cl2 (-0.285 V) and
[C2oH42N602Cu]Cl2 (-0.180 V) to the DNA surface. These electrochemical results are in
agreement with the above spectral results supporting that the complexes bind to DNA
surface involving electrostatic interaction.
78
-1.2 "0.2 0 -0.2 -0.4 -0.6 -0.8 -1.0 -1.2
Potential/V
Figure 40a. Cyclic voltammograms (5 mM Tris-HCl/50 mM KCl buffer, 2f) of [C2oH42N602Co]Cl2 complex and complex in absence (Curve a) and presence (curve b) ofCTDNA.
100
0.3 a2 0.1 0 -0.1-0.2-0.3-0.4-0.5-0.6-0.7-0.8
Potential/V
Figure 40b. Cyclic voltammograms (5 mM Tris-HCl/50 mM KCl buffer. 25^) of [C2oH42N602Cu]Cl2 complcx and complex in absence (Curve a) and presence (curve b) ofCTDNA.
79
o
z
o
u
o
c 3 O
S
U
o S s u
I a
0 0 ^ 00 ^
/--
o o 00 00
ON CO
00 00 >r\
s 00
^ uS • ^
(?N
, ^
o 00
in s
OS 0\
o o
o q
o o 00 •<4-
o <n oi
o
u (0
4>
o
2
3
"o
CN ^
(U 00
g O
as vo
_ p 2 m
fN
3 X>
.
Q
o
73 u o
o r O)
o OS (N
O • *
tN
R u
a d
a
13 U
So
c/)
X
• f t
s o U
U U u 'o' U «
o
u
o
o
u
u u
f-l
o
IS
o
80
U 3 u o "^
o
u
u
o
^
s "^
o I
o >
^ y, ^
o i n u-i
Tiro >n
(N • *
>r»
o o
00
00
o
3: o >
o 00 ro <N
fO
u U
o vo ^; «s •<»
o
u
d 3C O >
5. O
o vo oo
CO
00 OS 00
CM
O en m
w V X V O
o o
CN
o
o
-a
-i e
E o U
o U
M
u fS
o vo
7^ (N
:£ o <N
U
Z <N
u es
o >o
^ ^
o
o
3
u (N
o fS
o ;z; cs
£ o
o
81
S
a >^
"a, a o
U
cs ffi u
1
^ 1
1 ffi u
1
<s ffi u
1
w o t s
ffi u
1
ffi o
1
X 0)
t o U
1
5 *—H
VO 0 0
r4 1
*r\ <S
VO rn rn
r-vq r<S
OS r-• ^
M
U • 1—t
z fS
o
^ o (S
y
s a 3
a o u «s
U
r^^ n
ffi 1
z 1
u "
<s
X u
M
K u ^
/ ^ w o <s
u 1 ^ -
>> o 1
X u ^
"& s
U
<s vq 00
o
•<t
o rr <o
>r) 0 0 0 0 >o
1 00
00 <r>
m *-*4
0\ m o o 0^ f - M
fS
u • »-«
CM
6 ^"
r o <N
u '—'
CHAPTER IV
Synthesis and Characterization of Dinuclear Macrocyclic Cobalt(II), Copper(II) and Zinc(II) Complexes derived from 2,2,2,2 -S,S[bis(bis-
N,N-2-thiobenzimidazolyloxalato-l,2-ethane)]: DNA Binding and Cleavage Studies
82
CHAPTER IV
Experimental
Reagent grade chemicals were used without further purification for all syntheses.
C0CI2.6H2O, CUCI2.2H2O, ZnCh. K2CO3, KI, 1,2-dibromoethane, diethyloxalate, Tris-
base (Merck), 2-mercaptobenzimidazole (Sigma-Aldrich Chemie), 6X loading dye
(Fermental Life Science) and supercoiled pBR322 plasmid DNA (Genei) were used as
received and calf thymus DNA (CT DNA) was obtained from Sigma. Microanalysis
(%CHN) was performed on a Carlo Erba analyzer model 1108. Molar conductances were
measured at room temperature with a Digisun electronic conductivity bridge. Infrared
spectra were collected by using KBr pellets on an Interspec 2020 FT-IR spectrometer.
Shimadzu UV-1700 PharmaSpec UVA^is specfrophotometer was used to record the
elecfronic spectra. Data were reported in nm. ' H and '^C spectra were recorded on a
Bruker DRX-300 specfrometer at 300 MHz. Chemical shifts were reported on the 6 scale
in parts per million (ppm). The EPR spectrum of the copper complex was acquired on a
Varian E 112 spectrometer using X-band frequency (9.1GHz) at liquid nifrogen
temperature in solid state. The electrospray mass spectra were recorded on a Micromass
Quattro II triple quadrupol mass spectrometer. Emission spectra were determined with a
Hitachi F-2500 fluorescence spectrophotometer. Cyclic voltammetry was performed on
CH-instrument electrochemical analyzer. The supporting electrolyte was 0.4 M KNO3 in
water. All samples were purged with nitrogen prior to measurements at room
83
temperature. A three-electrcxie configuration was used comprised of a Pt micro cylinder
as a working electrode, Pt wire as auxiliary electrode and an Ag/AgCl electrode as a
reference electrode. All the experiments involving the interaction of the complexes with
DNA were conducted in aerated Tris-HCl buffer (0.01 M, pH 7.2).
The cleavage of supercoiled pBR322 plasmid DNA in absence of activating agents was
observed using gel electrophoresis. In reactions using supercoiled pBR322 plasmid DNA
300 ng in Tris-HCl buffer (10 mmol) at pH 7.4 was treated with complex
[C36H24N8O4S4CI4CU2] (50-250 \iM). The samples were incubated for 1 h at 37 °C. A
loading buffer containing 25 % bromophenol blue, 0.25 % xylene cyanol, 30 % glycerol
was added and electrophoresis was carried out at 100 V for 2 h in Tris-HCl buffer using 1
% agarose gel containing 1.0 ^g/mL ethidium bromide
The DNA cleavage with added reductant ascorbic acid was monitored as in case of
cleavage experiment without added reductant using agarose gel electrophoresis.
Reactions using pBR322 plasmid DNA in Tris-HCl buffer at pH 7.4 was treated with
complex [C36H24N8O4S4CI4CU2] (50-250 ^mol) and ascorbic acid (10 )imol). The samples
were incubated for 1 h at 37 °C. The gels were documented and analyzed by UVP gel.
Synthesis of N^-bis(2-mercaptobenzimidazolyl)oxalate hydrochloride
C,9H25N405S2Cl3
The ligand C19H25N4O5S2CI3 was synthesized according to the reported procedure [226]
as described. To a methanolic solution of 2-mercaptobenzimidazole (5.00 g, 33 mmol),
diethyl oxalate (2.26 mL, 16 mmol) was added in a 2:1 molar ratio. The solution was
84
heated under reflux for ca. 1 h. After cooling, concentrated. HCl (6 mL) was added
dropwise with stirring. The cream crystalline product formed, was filtered off, washed
thoroughly with hexane and dried in vacuo.
Synthesis of 2,2,2',2'-S,S[ bis(bis-N,N-2-thiobenziinidazolyI oxalato-1,2- ethane)]
C38H29N9O5S4
A mixture of C19H25N4O5S2CI3, (4.62 g, 10 mmol) 1,2-dibromoethane (0.86 mL, 10
mmol), anhydrous K2CO3 (1.38 g, 10 mmol) and KI in acetonitrile was stirred for ca. 24
h at 35±5 °C. The reaction mixture was poured into water, made alkaline with NaOH
solution. The TLC pure compound was isolated, washed with H2O and dried in vacuo.
Synthesis of 2,2,2', 2'-S,S[bis(bis-N,N-2-thiobenzimidazoIyloxalato-l,2-
ethane)Cobalt(II)lchloride[C36H24N804S4Co2]Cl4
The ligand C38H29N9O5S4 (1.9 g, 25 mmol) and C0CI2.6H2O (1.36 g, 50 mmol) were
dissolved in 20 mL MeOH. The solution was heated to reflux for 24 h, resulting in a clear
blue solution. Slow evaporation of the solution afforded a deep blue TLC pure crystalline
compound.
Synthesis of Dichloro-2,2,2%2'-S,S[bis(bis-N,N-2-thiobenziniidazolyloxaIato-l,2-
ethane)Copper(II)] [C36H24N8O4S4CI4CU2]
To a methanolic solution of ligand C3gH29N905S4 (1.9 g, 25 mmol), CUCI2.2H2O (0.85 g,
50 mmol) in 50 mL was added and heated to reflux for 24 h. On slow evaporation of the
reaction mixture a green compound obtained was filtered and dried in vacuo.
85
Synthesis of Dichloro-2^^',2'-S,S[bis(bis-N;M-2-thiobenzimidazolyloxalato-U-
ethane)Ziiic(II)] [C36H24N804S4Cl4Zn2].
This complex was synthesized by a similar method as described for
[C36H24N804S4Co2]Cl4 using anhydrous ZnCh (0.68 g, 50 mmol). The cream coloured
complex obtained was isolated and dried in vacuo.
Results and discussion
The ligand C38H29N9O5S4 and its metal complexes [C36H24N804S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] were synthesized by a procedure as
illustrated in Scheme 2. Formulations of the ligand and the complexes were verified by
elemental analysis, molar conductivity as well as by UVA^is, mass and NMR
spectroscopy. Molar conductance measurement of the complexes in DMSO corresponds
to the 1:4 electrolytic nature for complex [C36H24N804S4Co2]Cl4 while complexes
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] behave as non-electrolytes. Evidently,
in complexes [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] the metal ions are
hexacoordinated surrounded by O2S2CI2 environment, adopting an octahedral geometry,
while in complex [C36H24N804S4Co2]Cl4 the Co(II) ions are in tetrahedral environment
with O2S2 donor set, confirmed by UVA^is and mass spectral data. Complexes
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] and ligands
C19H25N4O5S2CI3 and C38H29N9O5S4 were soluble in DMF, DMSO and MeOH,
respectively.
86
N. I T >—SH + C2H5OOCCOOC2H5
N / \ N
N ^ ^ N
MeOH
KjCOj
KI
Cone. HCl
•N
N ^ O
N ^ O />—SH
N
CH3CN 1,2-Dibromoethane
CI N
M = Cu(II). Zn(II)
Scheme 2. Synthetic route and proposed structures of the ligands C19H25N4OSS2CIS and C3SH29N9OSS4 and complexes fC3^24^x0484002] CI4, [C36H24NHO4S4CI4CU2] and fC36H24N,04S4Cl4Zn2j.
87
The important properties and physical data of the ligands and the metal complexes are
given in Table 5. Energy minimized structvire using CS Chem Draw 3D Pro 5.0 as shown
in Figure 41 validated the proposed structures. DNA binding studies were performed with
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] complexes.
Figure 41. Three dimensional molecular model of [C36H24M804S4Co2]Cl4 complex; Colour Scheme Co" dark blue; N light blue; S yellow; O red; C gray.
Infrared Spectra
The IR spectrum of the ligand C19H25N4O5S2CI3 exhibited a characteristic band at 2368
cm'' attributed to the u(S-H) vibrations [226], indicating that the thiol proton remains
attached to the sulphur atom. Howeve?, the absence: of u(N-H) stretching band at ~
3400cm" [227] unplies its deprotonation and subsequent ligand formation with
88
diethyloxalate. The presence of an intense o(C=0) stretching absorption detected at 1625
cm' confirms the bridging of the two imidazole rings [228]. Two medium intensity bands
at 1523 cm' and 1353 cm"' were assigned to the o(C=N) and u(C-N), respectively [229].
The IR spectrum of the ligand C38H29N9O5S4 was quite similar to C19H25N4O5S2CI3
except for the missing peak at 2368 cm"' due to the -SH group. The deprotonation of
thiol groups was also inferred from the presence of a broad band at ca. 2853 cm'
belonging to the u(CH2) of the aliphatic chain. Additionally, the pure characteristic u(C-
H) modes of the ring residues in C19H25N4O5S2CI3 and C38H29N9O5S4 were observed in
the wave regions at 3100 cm"'. The IR spectra of the complexes [C36H24N804S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] reveal slight but specific differences
from the spectra of free ligands. On complexation, the shifting of the ligand u(C=0) and
u(C-S) bands towards the lower side suggests the involvement of oxygen and sulphur
atoms in coordination. Of particular interest is the lower frequency region, characteristic
for the metal-chloride and metal-oxygen stretching vibrations. Complexes
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] display metal-
oxygen bands at 493-509 cm"'. The appearance of a band at ~ 455 cm"' region in
complexes [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] supports the formation of
metal-chloride bonds while its absence in the IR spectrum of [C36H24N804S4Co2]Cl4
indicates that the chloride ions do not participate in coordination and behave as
counterions. This was also supported by the ESI MS and conductivity measurements
(Table 6).
89
NMR Spectra
The formation of new macrocyclic complexes were also ascertained by ' H and ' C NMR
spectra of the ligand C19H25N4O5S2CI3, C38H29N90$S4 and complex
[C36H24N804S4Cl4Zn2]. The signal due to the - SH functionality appearing at 2.95 ppm in
the free ligand spectrum of C19H25N4O5S2CI3 was missing both in the ligand
C38H29N9O5S4 and complex [C36H24N804S4Cl4Zn2] [230]. Absence of the characteristic -
NH proton signal of the imidazole ring at 12.00— 14.00 ppm corresponds to the
deprotonation of the -iSfH group. The observation of a singlet for methylene protons at
2.75-2.80 ppm [231] and a multiplet for the benzimidazole protons in the range 7.15-7.69
ppm indicated the equivalence of the two halves of the ligand C38H29N9O5S4. Compared
with the free ligand, complex [C36H24Ng04S4Cl4Zn2] showed proton signals at nearly
identical positions (Table 7). The '^C NMR spectra of the ligands C19H25N4O5S2CI3 and
C38H29N9O5S4 exhibited the resonances due to the carbonyl -C=0 and ring carbon atoms
of the imidazole moiety at 151.24—164.27 ppm and at 112.96-139.25 ppm, respectively.
Signals at 39.5 ppm were ascribed to the S-CH2-CH2-S [232] carbons of the ligand
C38H29N9O5S4. Complex [C36H24N804S4Cl4Zn2] displayed spectral pattern similar to that
of the ligands except downfield chemical shifts of carbon signals. The carbonyl signal
was desheilded in the spectra of the metal complex, which appeared at 164.27 ppm in
comparison to 151.24 ppm in C38H29N9O5S4, inferring coordination through the oxygen
atom of the ligand. Significant chemical shift was also observed for -S-CH2- resonances,
90
which implies coordination via sulphur atoms. These observed spectral features
conformed to the proposed macrocyclic structure (Table 8).
Mass spectra
The ESI MS spectra of the ligand C19H25N4O5S2CI3, C38H29N9O5S4 and the complex
[C36H24N804S4Co2]Cl4 wcrc obtained in the acetonitrile solutions. The spectrum of the
ligand C19H25N4O5S2CI3 shows peaks at m/z 429 and 494 were assigned to the
(Ci6H,2N402S2Cl2+2ir) and (C17H17N4O3S2CI3 +H^ compounds, respectively. For the
ligand C36H24N8O4S4 peaks centered at m/z 817 and 775 corresponded to the
(C38H29N905S4-2H^ and (C36H26N805S4.H20-3H^ species, respectively. The ESI MS
spectrum of the complex [C36H24N804S4Co2]Cl4 shows molecular ion peak at m/z 1019.
Peaks at m/z 981, 478, 222 and 224 correspond to the ion peaks resulting from the
successive expulsion of the chloride ions from the complex entity. This implies that the
chloride ions were not coordinated to the Cobalt ion.
EPR spectrum
The solid state X-band EPR spectrum of the complex [C36H24N8O4S4CI4CU2] acquired at
LNT imder the magnetic field strength 3000±1000 guass with tetracyanoethylene
(TCNE) as field marker (g = 2.0027) showed an anisotropic spectrum, exhibiting both
parallel and perpendicular g values. The EPR spectrum of complex
[C36H24N8O4S4CI4CU2] consists of a very broad axial symmetrical line shape with g|| =
2.16, gi = 2.08 values and gav - 2.106 computed from the formula gav = g\\' + 2 gi^ / 3,
suggesting an octahedral geometry. The trend g||> gx>ge (2.0023) revealed that the
91
unpaired electron was located in the dx .y' orbital of the Cu" ions characteristic of axial
symmetry [233]. The parameter G = ig[\-2)/ (gi-2), which measure the exchange
interaction between the metal center in a polycrystalline solid. According to Hathaway
and Billing [234] G>4 indicate negligible exchange interaction in the solid complex. For
the Cu(II) complex G <4 indicates considerable exchange interaction in the complex.
Electronic spectra
The electronic spectra of the ligands C19H25N4O5S2CI3, C38H29N9O5S4 and complexes
[C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] were recorded
in DMSO at room temperature. The UV region of the electronic spectra of the ligands,
both free and in the coordinated form was dominated by a pair of bands, one intense and
a narrow band at 270 - 290 nm and a second less intense band around 242 - 247 nm. The
low energy UV band has been assigned to an intraligand n —• n* transition of the
benzimidazolyl groups and the high energy UV band was ascribed to intraligand charge
transfer transitions. In the visible region, complex [C36H24N804S4Co2]CU displays two
bands, one low at 617 nm and a high at 680 nm assigned to ''A2 • ' 'T] transition,
typical for tetrahedral Co(II) complexes [235]. The high value of the molar extinction
coefficient and the blue colour of the Co(II) complex, also authenticate tetrahedral
stereochemistry. On the other hand complex [C36H24N8O4S4CI4CU2] showed a band with
low extinction coefficient at 800 nm due to ^Big ^^Aig transition [236] supporting an
octahedral geometry around Cu(II) metal ion.
92
DNA binding studies
Absorption spectral studies
The binding of the metal complexes to DNA helix is often characterized through
absorption spectral titration, followed by the changes in the absorbance and shift in the
wavelength. The absorption spectra of [Cj6H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2]
and [C36H24N804S4Cl4Zn2] in the absence and presence of CT DNA are illustrated in
Figure 42a-c, respectively. Upon the addition of CT DNA to [C36H24N804S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2], interesting changes in the
absorbances of the intraligand absorption bands of the complexes were observed —
hyperchromism for [C36H24N804S4Co2]Cl4 and hypochromism for [C36H24N8O4S4CI4CU2]
and [C36H24N804S4Cl4Zn2]. Hyperchromism and hypochromism are the spectral changes
typical of a metal complex association with the DNA helix [237, 238]. The
hypochromicity, characteristic of intercalation [239] has been usually attributed to the
interaction between the electronic states of the compound chromophores and those of the
DNA bases [240], while the red shift has been associated with the decrease in the energy
gap between the highest and the lowest molecular orbitals (HUMO and LUMO) after
binding of the complex to DNA [241]. The observed hyporchromism for
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] unambiguously revealed the active
participation of bzim moieties in associating with the DNA [242]. However, the lack of
red shift suggests that the binding mode of both [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] was not classical intercalation. Because of the bulky structure of
93
the complexes, the bzim rings cannot completely intercalate. When one of the four bzim
rings inserts into the helix, the other rings extends away from the plane due to the
stereochemistry effect and hence decreasing the effective area of overlap. Interestingly,
the complex [C36H24N804S4Co2]Cl4, inspite of containing bzim rings exhibits a
hyperchromism, suggestive of electrostatic binding. Due to electrostatic binding on the
DNA surface, it appears to imdergo distortion in the coordination sphere resulting in the
enhanced absorption intensity of the intraligand bands. Similar reports were observed for
DNA binding studies of ruthenium complexes derived from tetradentate
bis(benzimidazol-2-yl)dithioether ligands [243]. Therefore, the observed spectral changes
were rationalized in terms of partial intercalation and electrostatic binding. To further
illustrate the DNA binding sfrength, the intrinsic binding constant Kb were determined by
equation 1 for complexes [C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and
' [C36H24N804S4Cl4Zn2] which were found to be 16.6 . lO'* M"', 4,25. 10'* M"' and 3.0 . 10*
M"', respectively. The binding constants of these complexes were lower in comparison to
those observed for typical classical intercalators [244]. The diminution of the intrinsic
binding constants could be explained by the steric constraints imposed by the ligand
framework and thus encouraging a partial intercalation binding mode for the complexes.
94
XOJCO
Wtvckoflk (on)
Figure 42a. Absorption spectral traces of complex fC36H24Ns04S4CoJCl4 in Tris-HCl buffer upon addition ofCTDNA. Inset: Plots of[DNA]/ Sa-e/vs [DNA] for the titration of CT DNA with complex fC36H24N804S4Co2]Cl4 (m), experimental data points; full line, linear fit of the data. [CompIex]= 0.33 • 10^ M, [DNA] = 0.86 - 4.30- 10" M.
ieiw 2NM Wiveleiiftk(iim)
200X0
Figure 42b. Absorption spectral traces of complex [€3^24^804S^l/Zu2} in Tris-HCl buffer upon addition ofCTDNA. Inset: Plots of [DNA]/ Sa-C/vs [DNA] for the titration of CT DNA with complex [C36H24N8O4S4CI4CU2] (m), experimental data points; full line, linear fit of the data. [Complex]= 0.33 • Iff" M, [DNA] = 0.86 — 4.30- Iff^ M.
95
Wavtln(lk(ui)
Figure 42c. Absorption spectral traces of complex fC36H24Ns04S4Cl4Zn2] in Tris-HCl buffer upon addition ofCTDNA. Inset: Plots of[DNA]/ Sa-S/vs [DNA] for the titration of CT DNA with complex [Ci^24Ns,OS4^U'Z.n2j (m), experimental data points; full line, linear fit of the data. [Complex] = 0.66- ICT^M, [DNA] = 0.44-2.21- W^M.
Fluorescence studies
Upon excitation at % —• n* transitions either in DMSO or in presence of CT DNA,
complexes [C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2]
cannot emit luminescence. Hence, steady state competitive binding studies of the three
complexes were monitored by a fluorescent EthBr displacement assay. EthBr, a planar
aromatic heterocyclic dye intercalates non-specifically into the DNA, which causes it to
fluoresce strongly [245].
EthBr(weakfluorescent)+DNA(non-fluorescent) '-* ^ EthBr-DNA(strong fluorescent)
Competitive binding of second DNA binding macromolecule results in the displacement
of bound EthBr and a decrease in the fluorescence intensity, furnishing indirect evidence
for the DNA binding mode. The emission spectra of EthBr bound DNA in the absence
96
and presence of increasing amounts of complex [C36H24Ng04S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] are given in Figure 43a-c. The
emission intensity of DNA-EthBr system (Kem =590 nm) decreases appreciably, whicli
indicated tiiat the compounds could replace EthBr from the DNA-EthBr system. Such a
characteristic change is often observed in intercalative binding modes [246]. The
quenching plots (inset) followed the linear Stem-Volmer relationship (equation 2).
The Stem -Volmer quenching constant Ksv values for complexes C36H24Ng04S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] obtained from equation 2 were found
to be 0.04,0.26 and 0.06, respectively.
i
**Wav*l«ngth (•m)'*'
Figure 43a. Emission spectra of EthBr bound to DNA in the presence of complex [C36H24N8O4S4C0JCI4 in Tris-HCl buffer. Arrows indicate the intensity changes upon increasing concentration of the complex. Inset: Fluorescence quenching curve of DNA bound EthBr with complex (*), experimental data points; full lines, linear fit of the data. [Complex] = 0.33 -1.66- lO'^M, [DNA] = 0.66 • Kf^M; A„ = 510 nm.
97
1503
600 Wivtbaftk (M) 650
Figure 43b. Emission spectra of EthBr bound to DMA in the presence of complex [C36H24N8O4S4CI4CU2J in Tris-HCl buffer. Arrows indicate the intensity changes upon increasing concentration of the complexes. Inset: Fluorescence quenching curve ofDNA bound EthBr with complex (^), experimental data points; full lines, linear fit of the data. [Complex] ^0.33- 1.66- 10'^M, [DMA] = 0.66- KT^M; Xex = 510 nm.
Wavelength (nm)
Figure 43c. Emission spectra of EthBr bound to DNA in the presence of complex [C36H24N804S4Cl4Zn2] in Tris-HCl buffer. Arrows indicate the intensity changes upon increasing concentration of the complexes. Inset: Fluorescence quenching curve of DNA bound EthBr with complex (^), experimental data points; full lines, linear fit of the data. [Complex] = 0.66-2.66- 10"'M, [DNA] = 0.33 • Iff^M; k^ = 510 nm.
98
The effect of the complexes C36H24N804S4Co2]Cl4, [C36H24N8O4S4CI4CU2] and
[C36H24N804S4Cl4Zn2] on the fluorescence intensity of EthBr was also investigated in
presence of increasing amounts of DNA. The fluorescence intensity of EthBr has a
gradual enhancement with the addition of DNA in the absence and presence of metal
complexes. Fluorescence Scatchard plots [247] for the binding of EthBr to DNA in
presence of metal complexes (Figure 44) were obtained from equation;
rEB/CEB,f=(n-rEB)[KEB/l+KM] (5)
where res is the ratio of bound EthBr to total nucleotide concentration; CEB,ft
concentration of free EthBr; n, maximum value of TEB, KEB, and KM, intrinsic binding
constant for EthBr and metal complexes to DNA, respectively, and CM,f, concentration of
free metal complex. Using fluorescence to obtain rEB, binding isotherms were
constructed. With the addition of the complexes, the slope that is Kobs decreases with the
increase in the concentration of the complex indicating inhibition of EthBr binding
(Figure. 45). From the above equation (5) we obtain the relationship;
Kobs = [KEB/l+KM] (6)
The reciprocal of equation (6) produces a straight line equation;
1/Kobs = (KM /KEB) CM,f, + 1/ KEB- (7)
The concentrations of free complexes were approximated from the following equation;
rM = n - rEB - TEB/ KEB CEB (8)
CM,f = CM - TM CDNA (9)
99
(a) (b)
* tt-f" ^ riJ> d** 0 * #* ^ ^ # * «!^
(c)
I I I I I I I I
0.038 0.04S 0.054 0.062 0.07 0.078 0.086 0.094
Figure 44. Scatchard plots for the binding ofEthBr to DMA in presence of a) 0.3 • 10'''M
b) 1.33 • / O ' V c) 3.30 • l(fM of the representative [C36H24N8O4S4C0ZICU complex; [EthBr] "= 0.66 • lO^M; (^), experimental data points; full lines, linear fit of the data.
Where TM is the ratio of bound complex to total DNA concentration, CM,f and CM are
concentrations of free and total metal complex, respectively. Replots of Kobs gave the
approximate binding constants of the complexes [C36H24N804S4Co2]Cl4
[C36H24Ng04S4CUCu2] and [C36H24Ng04S4 CI4 Zn2], which were calculated to be 2.49 .
10\ 1.04. 10" and 1.85 . 10\ respectively.
100
(a) (b)
02S
0.2
<^0.1S
x. 0.1 •
•^0.05
0 ^
J.1 6.2
C^,/10^M
9.3 — I
0.0S1
(c)
0.001 0.088
C M . , / 1 0 - ' M
0.115
Figure 45. Replot of observed binding constant of EthBr to DMA for a) fC36ff24N804S4Co2jCl4 complex b) [C36H24N8O4S4CI4CU2] complex and c) [C36H24N8O4S4 CI4 ZnJ complex: (^,m), experimental data points; full lines, linear fit of the data.
101
DNA Cleavage activity
The chemical nuclease like activity of the [C36H24N8O4S4CI4CU2] complex, has been
investigated by agarose gel electrophoresis using supercoiled pBR322 plasmid DNA as a
substrate. The pBR322 DNA was mixed with different concentrations of
[C36H24N8O4S4CI4CU2] in Tris HCl buffer in the absence and presence of reductant
ascorbic acid and mixtures were incubated at 37 °C for Ih. Control experiments using
only complex or ascorbic acid failed to show any apparent cleavage activity, hi the
absence of ascorbic acid, pBR322 plasmid DNA was converted from SC (form I) to NC
(form II), (Figure 46a) revealing the efficient cleavage like activity and at higher
concentrations of complex [C36H24N8O4S4CI4CU2], increased band intensities were
observed. Since Cu(II) complexes are catalytically active in the oxidation of ascorbic acid
by dioxygen involving Cu(I) intermediate species, we have examined the cleavage like
activity of [C36H24Ng04S4Cl4Cu2] in presence of ascorbic acid at increasing concentration
of the complex. There was complete conversion of SC form I to nicked DNA form (II)
with almost disappearance of form (III). The bands are more intensified in presence of
ascorbic acid than in the absence of ascorbic acid as seen from Figure 46b. The intense
nuclease activity was apparently caused by enhanced stabilization of Cu(I) species
formed upon its reduction by ascorbic acid. This supports oxidative cleavage mechanism
of [C36H24N8O4S4CI4CU2] where Cu(II) complex is first reduced by ascorbic acid to form
the Cu(I) species, which then binds to DNA forming a Cu(I)complex-DNA adduct. This
102
adduct then reacts with dioxygen to form a peroxodicopper (II) derivative which could
generate active oxygen species required for cleavage [248].
Viscosity studies
To further explore the interaction properties between the metal complexes and the DNA,
the relative specific viscosity of DNA was examined by varying the concentration of the
added metal complexes. Measuring the viscosity of DNA is a classical technique used to
analyze the DNA binding mode in solution [249]. Hydrodynamic measurements i.e.
viscosity, sedimentation, rotational diffusion etc sensitive to molecular lengths provide
strong evidences for the DNA-binding modes, especially in absence of crystallographic
and NMR structural data [250]. A classical intercalation model results in the lengthening
of the DNA helix as the base pairs are separated to accommodate the binding molecule,
leading to an increase in the DNA viscosity. In contrast, partial intercalators as well as
NC
sc
Figure 46a. Gel electrophoresis diagram showing cleavage ofpBR322 supercoiled DNA (300 ng) by [C36H24N8O4S4CI4CU2] complex, in absence of ascorbic acid; 250 fiM [C36H24N8O4S4CI4CU2] + DNA, (lane 1), 200 juM [C36H24N8O4S4CI4CU2] + DNA (lane 2), 150 fiM [C36H24N804S4Cl4Cu2]+ DNA (lane 3), 100 fiM [C36H24N8O4S4CI4CU2] + DNA (lane 4) and 50 fiM [C36H24N8O4S4CI4CU2] + DNA (lane 5), control DNA (lane 6).
lUJ
1 2
NC
sc
Figure 46b. Gel electrophoresis diagram showing cleavage ofpBR322 supercoiled DNA (300 ng) by [C36H24N8O4S4CI4CU2] complex, in presence of ascorbic acid (10 fxM) 250 liM by [C36H24N8O4S4CI4CUZJ + DNA, (lane 1), 200 juM by [C36H24N8O4S4CI4CU2J + DNA (lane 2), 150 fiM by [C36H24N804S4Cl4Cu2]+DNA (lane 3), 100 fiM by [C36H24N8O4S4CI4CU2] + DNA (lane 4) and 50 /iM by fC36H24N804S4Cl4Cu2j+DNA (lane 5), control DNA (lane 6).
covalent binders could bend or kink DNA helix, reduce its effective length and in turn its
viscosity [251, 252]. Figure 47 depicts the effect of complexes C36H24N804S4Co2]Cl4,
[C36H24N8O4S4CI4CU2] and [C36H24N804S4Cl4Zn2] on the DNA viscosity. The plot
reveals that the relative solution viscosity decreased with increasing concentration of the
complexes, indicating that the DNA becomes more compact, resulting in DNA
aggregation. The aggregation reduces the number of independently moving DNA
molecules in solution and hence leads to a decrease in viscosity. These results support
that complexes bind to CT-DNA by partial intercalation [253]. Further, due to the bulky
structure of the complexes, they would act as a wedge to pry apart one side of a base pair
stack but not fully separate the stack as required by the classical intercalation model.
.^ 0.4
0.2
104
Complex I
complei 3
I < I
0.2 0.4 0.6 1/R = IComplex | / |DNA|
0.8
Figure 47. Effect of increasing amount of [C36H24N804S4Co2]Cl4 complex (m),[C36H24N804S4Cl4CuJ complex (A) and [CiJiiitNsOSACUZnz} complex (^) on the relative viscosity ofCT-DNA at 29 ±0.1 °C. [DMA] = 0.25 • la" M, [M] = 0.05 - 0.25-
Cyclic voltammetry
CV is an electroanalytical technique used to study the mechanistic behaviour of redox
systems of new metal complexes. Due to the resemblance between the electrochemical
and biological reactions, CV provides a useful complement to study the binding of metal
complexes to DNA, in addition to other spectroscopic methods of investigation. The
cyclic voltammograms (Figure 48,49) of the complexes [C36H24Ng04S4Co2]Cl4 and
[C36H24N8O4S4CI4CU2] show cathodic and anodic peaks due to the reduction of M " and
its subsequent oxidation. In absence of CT DNA, complex [C36H24N804S4Co2]CU
exhibits a quasireversible redox wave for a one-electron transfer process corresponding to
the Co'VCo' redox couple with an anodic peak potential Epa of-0.27V and a cathodic
peak potential of-0.35V, respectively. The formal potential Em taken as an average of
anodic and cathodic peak values is -0.310 V and the corresponding AEp value was 0.08V.
105
No appreciable change was observed at different scan rates. In presence of CT DNA,
there is a shift in AEp value (0.16V) as well as in the E\n value (-0.28V), indicative of
strong binding of the complex to CT DNA. The cyclic voltammogram of complex
[C36H24N8O4S4CI4CU2] also reveals a one electron quasireversible behaviour with E1/2 = -
0.33V and AEp= 0.16V (Epa= -0.25V and Epc= -0.41V) [254]. In presence of DNA
reversibility of the electron transfer was maintained with a significant shift in AEp value
(0.15V) as well as in E1/2 value (-0.375V). In addition to the changes in the formal
potential, the peak currents in the cyclic voltammograms of both the complexes
decreased than those in absence of DNA, indicating a strong binding of the complexes
[C36H24N804S4Co2]Cl4 and [C36H24N8O4S4CI4CU2] with DNA.
Figure 48. Cyclic voltammograms at a scan rate 0.2 Vs'' in DMSO/H2O (5:95) a) free [C36H24Ns04S4Co2]Cl4 compicx b) [C36H24N8O4S4C0JCI4 complex in presence of CT DNA.
106
Figure 49. Cyclic voltammograms at a scan rate 0.2 Vs'' in DMSO/H2O (5:95) a) free [C36H24N8O4S4CI4CU2J complex b) [C36H24N8O4S4CI4CU2] complex in presence ofCT DNA.
From reversible redox reactions of the free and bound species, the corresponding
equilibrium constants for binding of each oxidation state to DNA can be obtained from
the Nemst equation.
Eb°-Ef°=0.0591og(KK/K2+)
Where Eb° and Ef° are the formal potentials of M"/ M ' couple, in the free and bound
forms respectively and Ki+and K2+ are the corresponding binding constants for +1 and
+2 species to DNA. K1+/K2+ has been determined using the above equation and found to
be 3.22 and 5.76 for complexes [C36H24N804S4Co2]Cl4 and [C36H24N8O4S4CI4CU2],
whose potential shifts are 0.03V and 0.045V, respectively.
107
a o o -o a
CO
O
u
u . < ^ r — I
O N
so u 5 « » - ^
ia t3 s «^ U « J es U
S z
• § - ^ «» ,9 o U >>c«
' ^ 90
«/) ^
o
GO
I I—^ 00
2 13
U
(X
ffi
U
N
u o
rrj rn
'— ^ ^
r^ )S. 5 ^ ^ ^
:2S ^ s
Ti-as "t
(N vd r-~
•<t
1
>o rn
t~-* - H
0 0
<N ft trt
.- VO
«o >—•
^^ SO vq
m • ^
tN
o o
o <o v^
o
/--*\ t~-rn
d ^^
(N
^~~*
• O m CN
CN
,
o (N VO
oo 00 o
, ^ •<t
rn
- oo
^ o
/—*s
r- 3: ^ f*^
<N c i , ^
00 00
1
O O r
U
o o
CO
d "& o
U =^ u DO
m
u C/5
O Z
X ^m
u
I o s
o ON
o
00
00
u
J-u
u • •
00
d
U
«N
3 u J-u 00
PC so
u
o 00 (N
O •*
00
d 00
NO
y
108
-J u
r» O U
•w C/3 •>*
o 00
^; • » r«
s& rft
u M V H Ji "D-S o u
T3 a R TT
w f>
o . ^ ;2: o\ r*
« ao
u r« fO
u «s
CZ) <n
o
^ H
u M
13 a A .Sf ^ 0) .o '^rf
o A •*-a T3
B •
^ V
2
• 1
s vSi.
<s a N •*
U
O £
• ^
EC ve
r T >
U ^ • • r i
'O s e:
F ^
<>< s U -? (J
• V
c» T T
o 00 12; I t r<
ffl
o I
1/5 I
u >
o II u
? o
u
en
m
(N
m
00
X V
o ; ) ^ is
m
u 00
O •«r
z v%
u
m <n 00 <N
c^ CN Os (N
O
0 ^ f - M
VO
f O OS <n
OS O m
•* o «/
00
o
00
CO OS
0 0
OS
cs
00
00
C4 VO cn
r-o f o
OS
«o f o
oo so r*^
r*i ( N *o
t ^ *--H « 0
VO 0 \ •<t
f«^ OS •«t
^ M
OS •<t
C/3 m
o 0^ ^ <S
0 0 f *1
u
o 6* u t on o
0 0
^, n
^ ro
U
3
u -5 u n in 6
X
u
c ^ u " J -
tn O
0 0
^ . n
SO
m
u
109
,_ <s a Kl
J
•* O
0 0
• ^
u H « "B. a o u -o sa A
• ^
o OK
0 0 «n
u »»r
U M
CZ5 •/> O • *
z,
o ot ^ 9t
z,
H 3 L
<s
3 u C/3
d oo
^ ^
^ 0 CO
U '—'
•>f (/J
O ON
0 0
CO
n 00 O
a o\
u
§•
1 1
r-
' 1
"D
1
r-
in vo
^ 00 rn r~
X ^ ^ ^
o oo (S
r j
1
1
U 1
U 1
1
10
a N
CO
o:
»s G
5/3
d £ «»
N
^
t^ <N
S ""
t^ Tf
r« <s
"" 5s
u
I/]
o
u
u C/3
6
u
^
S
5 5 0\ <N
tN
o
OS
o ON
00 .
H 3
s o
9 V
u I
I C/3
CHAPTER V
Synthesis and Biophysical Studies of Bis-macrocyclic cobalt/copper (II) Complexes having Pyridine Spacer with CT DNA and 5' GMP
I l l
CHAPTER V
Experimental
Reagent grade Chemicals and solvents were used without further purification.
C0CI2.2H2O, NiCl2.6H20, CUCI2.2H2O, Tris-base (Merck), l,8-diamino-3,6-diazaoctane
(Fluka), formaldehyde, perchloric acid (BDH) and 2,6-diaminopyridine (Lancaster) were
used as received. Calf thymus DNA (CT DNA) and guanosine-5'-monophosphate
disodium salt (5'GMP) were purchased from Sigma chemical Co. and Fluka,
respectively.
Molar conductances X^ (in il'cm^ mol"'): at 25 °C, Digisun electronic conductivity
bridge. UVA'is Spectra: USB 2000 Ocean Optics spectrometer; H2O solns. Anax in nm.
IR Spectra: KBr pellets, Interspec 2020 FT-IR spectrometer; in cm*'. ' C and ' H N M R
Spectra: at 75 MHz and 300 MHz respectively; Bruker-DRX-300 spectrometer; 5 in ppm.
EPR Spectra: Varian-E-112 spectrometer; at the X-band frequency (9.1GHz) at liq. N2
temp. Elemental analyses: Carlo Erba analyzer model 1108. Electrospray mass spectra;
Micromass Quattro II triple quadrupol mass spectrometer.
All the experiments involving interaction of the complex with CT DNA were performed
in twice distilled buffer containing tris(hydroxymethyl)-aminomethane (Tris, 0.01 M) and
adjusted to pH 7.5 with hydrochloric acid. Solution of 5'GMP was prepared in double
distilled water. Absorption spectral titration experiments at constant concentration of the
complexes [6.6 x 10' M] while varying the CT DNA concentration and 5'GMP were
112
performed on USB 2000 Ocean Optics spectrometer and UV-1700 PharmaSpec UV-Vis
spectrophotometer (Shimadzu) respectively. Cyclic voltammetric studies were paformed
on a CH Instrument Electrochemical analyzer in a single compartmental cell with 0.4 M
KNO3 as a supporting electrolyte. A three-electrode configuration was used comprising
of a Pt wire as auxiliary electrode, platinum micro cylinder as working electrode and
Ag/AgCl as the reference electrode. Electrochemical measurements were made under a
dinitrogen atmosphere. All electrochemical data were collected at 25°C
and are uncorrected for junction potentials.
Synthesis of 2,6-bis[l,3,6,9,12-pentaazacyclotridecane pyridine cobalt(II)]
perchlorate [C2iH43N„Co2l (€104)4
To a stirred methanol solution of C0CI2.2H2O (2.37 g, 10 mmol) were slowly added 1,8-
diamino-3,6-diazaoctane (1.49 mL, 10 mmol), 37% formaldehyde (3.03 mL, 20 mmol)
and 2,6- diaminopyridine (0.54 g, 5 mmol). The resulting mixture was refluxed for ca. 24
h until a dark reddish brown solution appeared. This solution was cooled and filtered
under vacuum. Excess perchloric acid in methanol was added to the filtrate and left to
stand over night. Reddish brown solid product was filtered off, thoroughly washed with
methanol and dried in vacuo over fused CaCl2.
113
Synthesis of 2,6-bis[l,3,6,9,12-pentaazacyclotridecane pyridine nickel(II)]
perchlorate [CiiajaNnNiz] (€104)4
This dark brown coloured complex was synthesized by a procedure similar to that
described for [C2iH43N,iCo2] (004)4 using NiCh.eHzO (2.37 g, 10 mmol).
Synthesis of 2,6-bis[l,3,6,9,12-pentaazacyclotridecane pyridine copper(II)]
perchlorate [C21H43N11CU2I (€104)4
This light brownish complex was obtained by the method analogous to that for
[C2iH43NnCo2] (C104)4 using CUCI2.2H2O (1.70 g, 10 mmol).
Results and discussion
The new bis-macrocyclic complexes [C2iH43NnCo2](C104)4, [C2iH43NiiNi2](C104)4 and
[C2iH43NiiCu2](C104)4 have been synthesized by the template condensation reaction of
l,8-diamino-3,6-diazaoctane, formaldehyde and 2,6-diaminopyridine with Co(II), Ni(II)
and Cu(II) chlorides, respectively. These complexes were isolated by the addition of
perchloric acid in methanol as depicted in Scheme 3. These complexes are stable at room
temperature and are soluble in polar solvents such as wato", DMF and DMSO but
insoluble in methanol and diethylether. The values of molar conductance measured in
water indicate that the complexes are 1:4 electrolytes. Analytical data were consistent
with the proposed formulation of the bis-macrocyclic complexes. DNA binding studies
were performed with [C2iH43NiiCo2](C104)4 and [C21H43N11CU2](€104)4 complexes. The
analogous nickel complex was synthesized only for NMR studies. The important
properties and physical data of the complexes are given in Table 9.
114
% ^ ^
NH, H,N
HCIO4
NH,
+ MCI2 + HCHO + \ //^
NH,
.4CIO4
M = Co(Il), Ni(II) &Cu(II)
Scheme 3. Synthetic route for the bis-macrocyclic complexes [C2iH^ [C2,H43N„Co2] (004)4 and fC2iH43N„Cu2](C104)4.
'104)4.
Infrared spectra
The solid-state IR spectra of the bis-macrocyclic complexes [C2iH43NiiCo2](CI04)4,
[C2iH43N|iNi2](C104)4 and [C2iH43NiiCu2](CI04)4 exhibit peak at 1558-1650 cm' and a
sharp peak at 1462-1505 cm"' attributed to the azomethine u(C=N) and u(C>=C)
stretching modes, [255,256] respectively indicating the presence of pyridine spacer
moiety in the complexes. The absence of characteristic D(NH2) vibration [257] at 3400
115
cm"' supports the deprotonation of the amine groups and the formation of the macrocyclic
framework via condensation with formaldehyde. The bands observed in the range 1538-
1550 cm"' corresponding to \)(C—N) confirm the macrocyclic structure of complexes.
Additionally, broad bands at 3200-3250 cm' were ascribed to the stretching vibration of
v(NH) of the (NH-CH2-CH2-NH) linkage [258]. Other diagnostic bands of medium
intensity at 2769-2781 cm"' and 1300-1400 cm"' were assigned to C—H stretching and
bending vibrational modes, respectively [259]. The bands in the region 480—513 cm"'
were ascribed to the u(M—N) stretching vibrations. The spectra also display strong
absorption bands near 1000 cm"' (antisymmetric stretch) and sharp bands at 625—633 cm"
' (antisymmetric bend) due to the uncoordinated C104~ anions [260-262] (Table 10).
NMR spectral studies
To further elucidate the structure of the bis-macrocyclic complexes, the diamagnetic
complex [C2iH43NiiNi2](C104)4 was characterized by ' H and ' C NMR spectroscopy,
showing characteristic aliphatic and aromatic signals with chemical shifts in accordance
with the proposed structure (Table 11). The aromatic region of the spectrum of
[C2iH43NiiNi2](C104)4 was consistent with the presence of a doublet at 7.69-8.02 ppm
and a well-resolved triplet at 6.97-7.31 ppm due to equivalent and non-equivalent
hydrogens from the pyridine spacer [263]. Comparison of ' H NMR spectra for free 2,6-
diaminopyridine with [C2|H43NiiNi2](C104)4 also evidences that condensation of 2,6-
diaminopyridine has occurred and confirmed the formation of the bis-macrocycle. The 'H
NMR spectrum of the free 2,6-diaminopyridine displays primary amine proton signals at
116
5.29 and 5.59-5.61 ppm which disappear in [C2iH43NiiNi2](C104)4 with simultaneous
emergence of a new prominent peak at 2.58 ppm corresponding to the -CH2-N-CH2-
linkage protons [264]. A series of non-overl^ping multiplets in the high field region at
3.12-3.88 ppm and 4.86 ppm were attributed to the -(CH2)2-NH-(CH2)2-NH- chain of the
macrocyclic framework [265,266].
The '^C NMR spectrum reveals signals in the region 32.6-39.04, 45.58-48.77 and 51.31-
55.73 ppm representing - CH2- carbons of macrocyclic backbone [267]. Peaks at 134.23
and 120.12 ppm appear due to the aromatic ring carbons. This pattern of ' C NMR
resonances was consistent with ' H NMR data and agrees well with the proposed bis -
architecture of the complexes (Table 12).
Mass spectroscopy
ESI MS mass spectroscopy provides the key evidence for the formation of the
macrocyclic complexes. The ESI MS spectrum of the [C2iH43NiiNi2](C104)4 complex
shows peaks at m/z 868, 385, 223 and 141 corresponding to the molecular ions of the
formulations [M-C104' + SH't, [M-2C104- + 2ltf^, [M-3C104' + H^]^^ and [M-
4CIO4' ] ^ respectively, resulting from the stepwise loss of the perchlorate anions (Table
13). The mass spectrum also shows some prominent peaks representing successive
fragments of the complex molecule. The appearance of peaks at m/z 522, 161, 121, 305
and 100 are ascribed to the species [Ci3H23N5Cl208Ni], [Ci3H23N5Cl208Ni - 2CIO4" f^,
[C8H2oN5Cl208Ni - 2 C 1 0 4 ] ^ [C6H,6N4Cl208Ni - C104] '^ and [C6H,6N4Cl208Ni -
2C104"]^^ respectively. The peaks occurring at m/z 149 and 77 corresponding to 1,8-
117
diamino-3,6-diazaoctane and pyridine group, respectively, further confirm this
Segmentation pattern.
Electronic absorption Spectra
The UVA^is absorption spectra of [C2iH43NiiCo2](C104)4, [C2iH43NiiNi2](C104)4 and
[C2iH43NiiCu2](C104)4 complexes (1x10'^ M) were recorded at 25 °C in H2O. In the UV
region the complexes display two main transitions i) an intense band at 325-340 nm,
attributed to LMCT transitions and ii) a strong band at 230-250 nm ascribed to
intraligand charge transfer transitions [268]. The visible absorption spectra of complex
[C2iH43NiiCo2](C104)4 was characterized by an absorption band at 425 nm due to the
metal d-d electronic transition attributed to 'Aig • 'Big transition [269] consistent with
a square planar geometry around the cobalt metal ion. Similarly, complex
[C2iH43NiiNi2](C104)4 displayed a shoulder at 415 nm [229] assigned to the 'Aig—• 'A2g
transition of low spin Ni(II) in a square planar environment. Complex
[C2iH43NiiCu2](C104)4 shows a distinct absorption band at 585 nm due to ligand field d-d
transitions assigned to Big—• A2g transitions, again in agreement with the square planar
geometry of Cu(II) ion [270].
EPR spectral studies
The solid state X-band EPR spectra of the complex [C2iH43NiiCu2](C104)4 was acquired
at a frequency of 9.1 GHz under the magnetic field strength 3,000±1,000 guass using
tetracyanoethylene (TCNE) as field marker at LNT. The complex shows an anisotropic
spectrum with gy = 2.049 and gi = 2.01 and gav = 2.02 computed fi-om the expression gav
118
= (g|| + 2gi^)/3. These parameters are in good agreement to the values reported for other
related square planar Cu(II) systems [271] and are typical of axially synmietrical d'
Cu(II) complexes. The trend gn > gj. > 2 reveals that the unpaired electron is present in
the dx -y orbital [272]. For a Cu(II) complex, g\\ is a parameter sensitive enough to
indicate covalence. For a covalent complex, g|| < 2.3 and for an ionic environment, g\\ =
2.3 or more. In the present complex gn < 2.3 indicates an appreciable metal-ligand
covalent character [273]. The G factor defined as G = (gy - 2)/(gi- 2) indicative of
exchange interactions between the Cu(n) sites equal to four suggests negligible exchange
interactions.
Biophysical studies with CT DNA and 5'G]VIP
DNA binding is the critical step for many cytotoxic compounds as it is the primary
pharmacological target of antitumor drugs. Metal complexes are particularly attractive
systems to study as the ligating organic molecules are held firmly in place by the metal
ions, giving specific shape and surface features to the complex that can be exploited to
affect the DNA-metallocomplex interaction. The mode and propensity of binding of
[C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 complexes to CT DNA has been
evaluated by the absorption titration, luminescence titration, cyclic voltammetry and
viscosity measurements.
Absorption titration
The potential CT DNA binding ability of complexes [C2iH43NiiCo2](C104)4 and
[C2iH43NiiCu2](C104)4 were studied by UVA^is spectroscopy by following the intensity
119
changes of the intraligand transition bands. Figure 50a-b displays the absorption spectra
of [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 in the absence and presence of CT
DNA respectively. On addition of CT DNA to the aqueous solution of
[C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 there is an incremental increase in
absorbance of IL transitions. A moderate bathochromic shift (4-5 nm) in UV region was
also observed for [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4. Hyperchromicity
with bathochromic shift is a feature of DNA binding mode either through covalent
coordinate linkage to N7 nucleobase of DNA [116, 274] or electrostatic interaction to the
major/minor groove of DNA helix [275].
100 «00 Wovatangth (nm)
500
Figure 50a. UV spectral traces of [C2iH43N,,Co2j(Cl04)4 complex in Tris-HCl buffer (0.01 M, pH 7.2) upon addition ofCTDNA. Inset: Plots of [DNA]/ Ea-Sf versus [DNA]; (m), experimental data points; full lines, linear fitting of the data, [complex] = 6.6 x 10'^ M.
120
I
•a
<
OJO
0.86
300 400 Wavelength |nm)
500
Figure 50b. UV spectral traces of [C2iH43NnCuJ(€104)4 complex in Tris HCl buffer (O.OIM, pH 7.2) upon addition ofCTDNA. Inset: Plots of [DNAJ/ Ca-e/versus [DNAJ; (m), experimental data points; full lines, linear fitting of the data; [complex] = 6.6 x ICF^
M.
There is every possibility for both binding modes due to the molecular structure of the
complexes-strong Lewis acid metal centres separated at an optimum distance, which
favours coordination to the N7 of guanine nucleobase while electrostatic interaction
could be due to interaction of positively charged metal ions with negatively charged
oxygens of the phosphate backbone of the DNA helix. Since no significant changes were
observed in the LF bands covalent/coordinate linkage to the N7 of nucleobase is strongly
ruled out. This is also evidenced by the absorption titration with 5'GMP. Intercalation
which leads to hypochromic spectral feature [276] is again ruled out in complexes
[C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C 104)4. Therefore it is reasonable to assume
that the complexes bind electrostatically to DNA. The cationic core of
121
[C2iH43NiiCo2](CI04)4 and [C2iH43NiiCu2](C104)4 could exert an enhanced electrostatic
attraction due to an increase in the positive charge by the incorporation of more than one
metal centers in a single molecule [277]. The higher the positive charge a dinuclear
species possesses, the more cellular uptake is observed [278]. This feature along with
high binding affinity of [C2iH43NuCo2](C104)4 and [C2iH43NiiCu2](CI04)4 may facilitate
access to DNA target in a tumor cell. Besides this, divalent cations can repel and displace
other counterions, leading to strong attraction with the proximal phosphate groups. The
result is DNA bending by electrostatic collapse around a divalent cation [279]. Such
general affinity would induce structurally ill-defined DNA aggregates [280]. Recently
Farrell et al. [281] have reported a phosphate backbone-binding mode for a polynuclear
Platinum (II) complex that has planar arrays of hydrogen bond donors leading to
association with the DNA phosphate backbone. It is obvious that the coordinated -NH-
groups of the present macrocyclic complexes may also sterically clash with the DNA
surface and involve in the hydrogen bonding with the phosphate oxygens. Additionally
hydrogen bonding, in which the -NH- of the ligand serves as a hydrogen bond donor
would be expected to create partial negative charge on the ligand nitrogen atoms. This
charge effect would make the ligand a better o-donor to the metal, rendering the
coordination of metal ion to the nucleobase difficult. In order to compare quantitatively
the binding strength the intrinsic binding constant Kb for [C2iH43NiiCo2](CI04)4 and
[C2iH43N)iCu2](C104)4 were calculated to be 1.64x10* M"' and 2.05x10* M'' respectively.
These values are significantly lower than typical intercalators (EthBr-DNA ~ 10 M"').
122
Interaction with 5' GMP
To corroborate our results of UVA^is titrations with CT DNA, interaction studies of
[C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](Cl04)4 with 5'GMP in double distilled water
were carried out (Figure 51a-b). The interaction between the metal complex and 5'GMP
is expected to perturb the ligand field transitions of the metal complex due to
coordinate/covalent linkage either to N7 or 06 of guanine base [282]. However upon
addition of 5'GMP, there is substantial increase in the absorption of IL bands in the UV
region while small changes were observed at the absorption maxima due to LMCT bands.
No shifts in the visible absorption bands were observed which implies that there is no
change of the coordination environment of the metal ions [283]. Coordination to the N7
and 06 positions of 5'GMP are thus ruled out, in view of the steric hindrance by the
macrocyclic rings. These results reveal that the complexes interact with 5'GMP
exclusively through hydrogen bonding of -NH- groups to base nitrogen atom or
phosphate oxygen atom or by electrostatic interactions of the metal ions utilizing the
phosphate oxygen atoms of the nucleotide.
Fluorescence spectroscopic studies
As the complexes [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 are non-emissive
both in presence and absence of DNA, competitive DNA binding experiments with the
proven DNA intercalator, ethidium bromide provided additional information about the
DNA binding properties of the bis- macrocyclic complexes [C2iH43N|iCo2](C104)4 and
123
wt Wavelength (nm)
am w«
Figure 51a- UVspectral traces [C2iH4}NnCo2](004)4 complex in double distilled water upon the addition of 5 'GMP.
0.100
A
• o r b k
30000 40000 Wavalangth (nm)
Figure 51b. UV spectral traces [C21H43N1 /CuJ (€104)4 complex in double distilled water upon the addition of 5 'GMP.
124
[C2iH43NiiCu2](C104)4. EthBr shows reduced fluorescence intensity in a buffer medium
due to the quenching by solvent molecules and an increase in fluorescence intensity when
bound to DNA, because of its strong intercalation between the DNA base pairs and
stabilization of its excited state [284]. Addition of Complexes [C2iH43NiiCo2](C104)4 and
[C2iH43NiiCu2](C104)4to EthBr-DNA system decreases the emission intensity at 590 nm
indicating that complexes have a good affinity for DNA (Figure 52a-b). The extent of
fluorescence quenching reflects the extent of binding of complexes to DNA [285]. Two
mechanisms have been proposed to account for the quenching viz; the displacement of
ethidium bromide from DNA and electron transfer from excited ethidium to an acceptor
(e.g; cupric ion, Cu " ) [286]. As complex [C2iH43NiiCo2](C104)4 and
[C2iH43N||Cu2](C104)4 bind to DNA via surface binding, displacement of strongly bound
EthBr cannot take place. Instead the observed quenching is due to the facile
intramolecular photoinduced electron transfer from the excited EthBr to complex bound
to DNA [287]. The quenching extent by [C2]H43NiiCu2](CI04)4 is larger than
[C2iH43N|iCo2](C104)4 This is expected, as the reduction of DNA-bound complex
[C2iH43N,,Cu2](CI04)4 is easier than [C2iH43NnCo2](C104)4, (E° = ^ 4 5 mV and ^ 1 5
mV for [C2iH43NiiCo2](C104)4and [C2iH43NiiCu2](CI04)4, respectively). Moreover, it is
possible that the low energy ligand based TC* orbitals could facilitate the photoinduced
electron transfer.
125
600 WavtUngth (nm)
Figure 52a. Emission spectra of EthBr bound to CT DNA in the presence of [C2iH43NiiCo2](Cl04)4 and in Tris-HCl buffer. Inset: Plots ofljl vs [complex]/[DNA]; [DNA] = 3.3xia^M; X,^^ SlOnm.
600 Wavelength (ntn)
700
Figure 52b. Emission spectra of EthBr bound to CT DNA in the presence of [C2iH43NiiO,6Cu2Cl4] and in Tris-HCl buffer. Inset: Plots of UI vs [complex]/[DNA]; [DNA] = 3.2xW^M; Xex= SlOnm.
126
The emission spectra of the EthBr-bound DNA in the absence and presence of complex
[C2iH43NiiCo2](C104)4 and [C2iH43NnCu2](C104)4 alongwith the titration plots (inset)
are given in Figure 52a-b. The plots depict that the quenching of EthBr bound to DNA by
the metal complex is in good agreement with the linear Stem-Volmer equation, implying
that the complex competes with EthBr in binding to DNA.
In the plot of IQ/I VS r, the Stem-Volmer quenching constant K v is given by the ratio of
the slope to the intercept. The Kgv value for the complex [C2iH43NnCo2](C104)4 and
[C2iH43NiiCu2](C104)4 as estimated by using equation 2 were found to be 0.07 and 0.44
respectively.
Viscosity studies
To explore further the mode of binding to DNA, the viscosity of the CT DNA solution
were measured by varying the concentration of the added metal complexes.
Hydrodynamic method, such as determination of viscosity, which is exquisitely sensitive
to the change in length of DNA, is the most effective means for studying the binding
mode of complexes to DNA in solution especially in absence of crystallographic data
[288]. Electrostatic interactions typically cause less pronoimced or no change in the DNA
solution viscosity, while partial intercalation induces static bends in DNA double helix,
reducing its effective length and concomitantly its viscosity [126].
The variation of the relative specific viscosity with addition of the increasing
concentration of the complex [C2iH43NiiCo2](C104)4 and [C2iH43NiiCu2](C104)4 is given
in the Figure 53a-b. The viscosity of DNA decreases steadily with increasing
127
concentration of the metal complex, indicating that the DNA becomes more compact
because of its interaction with metal complex. These experimental results support the
idea that the complex [C2iH43NuCo2](C104)4 and [C2iH43NuCu2](C104)4 bind to DNA
by simple electrostatic interactions. In addition to this hydrogen bonding interactions of
the coordinated -NH- groups with the base pairs leads to the bending of the DNA chain
resulting decrease in the DNA viscosity [221]. These observations are consistent with the
results from UVA^is and luminescence titrations.
Cyclic Voltammetry
Cyclic Voltammetry is employed to study the interaction of metal complexes with DNA
owing to the resemblance between electrochemical and biological reactions. Based on the
shift of the formal potentials in the cyclic voltammograms, the relative binding affinities
and binding modes of the metal complexes with DNA can be deduced. The redox
behavior of the bis-macrocyclic complex [C2iH43NiiCo2](C104)4 and
[C2iH43NiiCu2](C104)4 in absence and presence of CT DNA has been studied by means
of cyclic voltammetry in H2O/DMSO (95:5) over a sweep range of 1.6 to -0.8 V at a scan
rate of 0.3 VS' . The CV of bis-macrocyclic complex [C2iH43NiiCo2](C104)4 (Figure 54)
in the absence of CT DNA features a non-Nemstian one quasi-reversible redox wave at
cathodic peak potential Epc of -0.430 V and and anodic peak potential of -0.401 V,
attributed to the Co(Il)/(I) couple. The formal electrode potential E1/2 estimated as the
128
033 0£6 1 1/R=IM]/IDNA]
Figure 53a. Effect of increasing amount of [C2iH4iNuCo2] (004)4 complex on the relative viscosity ofCTDNA at 29 ±0.1 "C. [DNA] =4x1 a'M.
0.04 0.06 1/R«[M]/[DNA]
Figure 53b. Effect of increasing amount of [C21H43N11CU2](004)4 complex on the relative viscosity ofCTDNA at 29 ±0.1 "C. [DNA] =4xia^M.
129
average of the anodic and cathodic peak potentials is -0.415 V (-415mV). The ratio of
anodic and cathodic peak currents, Ipa/Ipc ~0.5 and AEp =0.30 V (30 mV) (Nemstian value
= 59 mV) imply a one electron transfer process of Co(II)/Co(I). The voltammograms
obtained at various scan rates does not show any marked change. In presence of CT DNA
at the same concentration of metal complex, there is a considerable shift in the formal
potential E1/2 = -0.445 V. The cathodic peak potential shifts to negative side (-0.490 V),
while the anodic peak potential shifts to the positive side (-0.400 V). The ratio Ipa/Ipc
decreases (0.43) for the CT DNA bound complex [C2iH43NiiCo2](C104)4 suggesting that
the complex binds strongly to CT DNA by electrostatic association [289]. Cyclic
Voltammogram of the complex [C2iH43N|iCu2](C104)4 (Figure 55) shows a well-defined
redox process corresponding to the formation of the Cu(II)/Cu(I) couple with formal
Figure 54. Cyclic voltammograms at a scan rate 0.2 Vs'' in H2O; curve a [C2iH43NnCo2](€104)4 complex alone; curve b [C21H43N11C02](004)4 complex in presence ofCTDNA.
130
Figure 55. Cyclic voltammograms at a scan rate 0.2 Vs'' in H2O; curve a [C2iH43NiiCu2j(Cl04)4 complex alone; curve b [C2iH43NiiCu2](Cl0^4 complex in presence ofCTDNA.
potential E1/2 = -0.375 V. The couple is found to be quasi-reversible with AEp =70 mV
and the ratio of the anodic to cathodic peak currents (Ipa/Ipc = 0.23) correspond to the
simple one electron process. The large peak width for the one electron couple
Cu(II)/Cu(I) indicates the structural reorganization of the coordination sphere during the
electron transfer [290]. Upon the addition of CT DNA the formal electrode potential E°
value (-0.415 V) shifts towards more negative side.
The apparent reduction in peak currents of [C2iH43N|iCo2](C104)4 and
[C2iH43NiiCu2](C104)4 upon the addition of CT DNA is attributed to the diffusion of the
complexes bound to large, slowly diffusing DNA molecules. These results are
comparable with the behaviour of other reported mononuclear and their corresponding
131
polynuclear complexes [291]. The occurrence of a single redox wave implies that the two
copper (II) centers in [C2iH43NiiCu2](C104)4 simultaneously exchange electrons with the
electrode and that the coulumbic interactions between the metal centers are negligible
[292]. Further the shifts in E\a to more negative values support the binding of the
complexes to DNA surface with significant contributions from electrostatic forces of
attraction [293].
o
o U
en
o
M
ct
'O
^^ ct u
•••*
ct a ct
'O a
, , R
V) b JS
&H
T t
' ^
o u
n 9 U »H
;5 < I S
u R
• *
^
o u > ^
r<
o\
T3 JJ
U
c s o
2
3 o U
X
B o U
O
• o^
u o
<N 00
<n '"'
OS
Tt
CN m vo <N
VO 0\
v^ ^
ON
S .
CN T - ^
O
r<
,_ <N VO
ON
• *
(N (N VO (N
^ ON
u-i >*-•
2 /—s • *
^ vo (N
<N r-u-i
OS (N
«o
O oo <n s_^
f 2;
00 00
rsi
vo
4=
'•B -a u
o (N
<N c< r«
t^ o CO
vo
c
2
•I Q
o
o vo
I
o ON
132
d u
CN O u z f»l
:£ <N
u
d u
Z z :S
<N
u
o u n 3
u z m a?
fS
u
S
O U
I o 00 • *
«n o w
133
c o
en
o 00
o O
o o 00
>r> o w-1
fN VO • ^
o U
(3 u p
00 o vo
PC
u 00
ON VO r~ tN
u-> r r tN
o
4i
oi O
h-i s
U
r~ •* (N rr^
O »r> {N en
o o <N CT)
X u "E, B o U
O U
IN
o q
u
d
u
d
3
134
a a
"a a o
U
H
u I
U
U
U
I o O
0 0
oo 00 rn
I
0 0
U CM
u
a a a ^^
"a a o
O U
H
X ^
OH
1 CM
X
u ts
ffi u 1
1
u z 1
«N
K U 1
>< u
t o U
CN '—' C> <N
^
• ^
r'l
m t^ r-; r-; >o oo' >r> T t ^H 00 ro U-) ^ vS >r> Tf
O 0 \
1
^ (N r<^
• « •
r—V • •
o u s_x
C4
>-•
z CO
^ ts
u b i . » J
135
• N 1 9i
a o u •V
o mm
u * P 4
Z w^ v H
M
u <4-l
o et
mm 03 U
V a
«« 03 s c« s ^ o ^
• 1-H
V
2 C4 H
+
o u t S + p — 1
tc + d u c^
s +
S' +
d o
1
+ r—1
+ , d u 1
2 U—J
t o U
^ • *
r4
0 0
0 0
0 0
• < l -
* o U
^ <N
U^
CHAPTER VI
Cytotoxic Activity of a Potent Coumarin ligand 3-acetoacetyI-7-methyi-pyrano-(4,3-b)-pyran-2,5-dione against Different Cell Lines: Design of Metal based Chemotherapeutic Agents derived from this Ligand and
their DNA Binding ProHIe
136
CHAPTER VI
Experimental
Reagent grade chemicals were used without further purification for all syntheses.
Co(C104)2.6H20, Ni(C104)2.6H20, Cu(C104)2.6H20 and Zn(C104)2.6H20. rac-1,2-
diaminocyclohexane, triethylorthoformate, 4-hydroxy-6-methyl-2H-pyran-2-one (Fluka)
and Tris-base (Merck) were used as received. Calf thymus DNA (CT DNA) was obtained
from Sigma. 6X loading dye and supercoiled pBR322 plasmid DNA were procured from
Fermental Life Science and Genei respectively. Microanalysis (%CHN) was performed
on a Perkin Elmer elemental analyzer model 2400. Molar conductances were measured at
room temperature with a Eutech Instruments CON 510 digital conductometer. Infrared
spectra were collected by using KBr pellets on a Perkin Elmer spectrum RX-1 FT-IR
spectrometer. ' H and ' C spectra were recorded on a Bruker Avance-II spectrometer at
400 MHz and 100 MHz respectively. Chemical shifts were reported on the 5 scale in
parts per million (ppm). The EPR spectrum of the copper complex was acquired on a
Varian E 112 spectrometer using X-band frequency (9.1GHz) at liquid nitrogen
temperature in solid state. The electrospray mass spectra were recorded on a Micromass
Quattro II triple quadrupol mass spectrometer. Emission spectra were recorded with a
Hitachi F-2500 fluorescence spectrophotometer.
All the experiments involving the interaction of the complexes with DNA were
conducted in aerated Tris-HCI buffer (0.0 IM, pH 7.2).
137
The cleavage of supercoiled pBR322 plasmid DNA in absence of activating agents was
observed using gel electrophoresis. In reactions using supercoiled pBR322 plasmid DNA
(300 ng) in Tris-HCI (10 mmol) buffer at pH 7.4 was treated with complex
C38H44N4O18CI2CU (50-250 ^mol). The samples were incubated for 1 h at 37° C. A
loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol
was added and electrophoresis was carried out at 100 V for 2 h in Tris-HCI buffer using
1% agarose gel containing 1.0 ^g/mL ethidium bromide. The DNA cleavage with added
reductant ascorbic acid was monitored as in case of cleavage experiment without added
reductant using agarose gel electrophoresis. Reactions using supercoiled pBR322 plasmid
DNA in Tris-HCI buffer at pH 7.4 was treated with complex C38H44N4O18CI2CU (50-250
^mol) and ascorbic acid (10 jimol). The samples were incubated for 1 h at 37 °C. All the
gels were viewed by UVP gel doc system and photographed.
The anticancer activities against different human cancer cell lines were carried out using
SRB assay. The human tumour cell lines of the cancer-screening panel were grown in
RPMI-1640 medium containing 5% foetal bovine serum and 2 mM L-glutamine. For a
typical screening experiment, cells were inoculated into 96-well microtitre plates in 100
^L at plating densities ranging from 5000 to 40,000 cells per well depending on the
doubling time of individual cell lines. After cell inoculation, the microtitre plates were
incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to
addition of ligand C13H10O6. Following compound addition, the plates were incubated for
an additional 48 h at 37 °C, 5% CO2, 95% air and 100% relative humidity. For adherent
138
cells, the assay was terminated by the addition of cold trichloroacetic acid.
Sulforhodamine B solution (100 ^L) at 0.4% (w/v) in 1% acetic acid was added to each
well, and plates were incubated for 10 min at room temperature. After staining, unbound
dye was removed by washing five times with 1% acetic acid and the plates were air-
dried. Bound stain was subsequently solublized with 10 mM trizma base, and the
absorbance was read on an automated plate reader. The growth inhibitory rate of treated
cells was calculated by (ODcontroi - ODtestVODcontroi x 100%.
Synthesis of [3-acetoacetyI-7-inethyl-pyrano-(4,3-b)-pyran-2,5-dione] CnHioOg
The ligand was synthesized by a procedure as follows [294].
4-hydroxy-6-methyl-2H-pyran-2-one (0.01 mol, 1.00 g) was added in portions to a
preheated mixture of moist ethylorthoformate (0.01 mol, 1.66 mL) containing traces of p-
toluene sulphonic acid. The reaction mixture was heated for 15 minutes and cooled to 10
C when an orange coloured crystalline solid precipitated. It was recrystallized from
chloroform.
General Procedure for the synthesis of macrocyclic complexes
C38H44N40,8Cl2Co, C38H44N40i8Cl2Ni, C38H44N40,8Cl2Cu and C38H44N40l8Cl2Zn
To a solution of an appropriate metal perchlorate (5.00 mmol) in 20 mL of methanol,
1.22 mL (10.00 mmol) of rac-l,2-diaminocyclohexane was added dropwise. The reaction
mixture was stirred at 35 °C for 30 minutes and then 2.62 g (10.00 mmol) of ligand
C13H10O6 dissolved in 30 mL of methanol was added, stirred for additional 1 h and heated
under reflux for 6 h until peculiar colour changes were observed. The reaction mixture
139
was filtered and the filtrate was evaporated under vacuum to remove the solvent. The
product obtained was purified by repeated recrystallazation from MeOH/CHCb and air-
dried.
Results and discussion
The complexes having a general formula [ML2(dach)2(H20)2](C104)2 where M = Co(II),
Ni(II), Cu(II) and Zn(II) and L = 3-acetoacetyl-7-methyl-pyrano-(4,3-b)-pyran-2,5-dione
and dach = diaminocyclohexane were prepared in situ by mixing a methanolic solution of
metal perchlorates to the solution of diaminocyclohexane and the 3-acetoacetyl-7-methyl-
pyrano-(4,3-b)-pyran-2,5-dione ligand on reflux in 1:2:2 molar ratio (Scheme 4). The
ligand was synthesized by the method described earlier [294] by the reaction of 4-
hydroxy-6-methyl-2H-pyran-2-one with preheated mixture of moist ethylorthoformate
with traces of PTS. The reaction mixture yielded yellow crystalline solid on heating for
15 minutes and cooling at 10 °C. The ligand was recrystallized from a mixture of
chloroform. The complexes [ML2(dach)2(H20)2](C104)2 were also recrystallized from
chloroform/methanol mixture and pure complexes were obtained by separating the
compound by preparatory TLC (CHCI3/CH3OH, 95:5). The yield of the complexes was <
50%. The complexes were air stable and insoluble in water, sparingly soluble in MeOH,
good soluble in DMSO, DMF and CHCI3. Molar conductance measurements showed that
complexes are ionic in nature. The formation of complexes was ascertained by elemental
analysis, which serves as a basis for the determination of their empirical formulae,
confirmed by IR spectroscopy, 'H , '^C N M R spectroscopy and mass spectral data. All
140
these spectroscopic data supported the presented formulae and the proposed structure of
the complexes. The important properties and physical data of the complexes are given in
Table 14.
II n* II / ^ M ( C I O 4 ) 2 . 6 H j 0 /-V^> i '^^^-r-^ °
HjC'^^'^^O^O H,N NH, I HN ,N ' CH3
where M =Co(II), Ni(Il), Cu(n) and Zn(II)
Scheme 4
IR spectroscopy
IR spectra of the ligand CuHioOe and the complexes [ML2(dach)2(H20)2](C104)2 were
recorded in solid state as KBr discs in the range 500-3800 cm'". The ligand CnHioOe
exhibits the characteristic stretching vibrations of the carbonyl groups of lactone rings at
1760 cm' and 1724 cm"' and low frequency sharp band observed at 1626 cm"' was
assigned to stretching vibration of the v(C=0) of the P-diketonate residue, which was in
resonance with the lactone ring [295-297]. A broad band at 3431 cm"' characteristic of
v(OH) vibration was attributed to the enolic OH vibrations implying that the ligand
141
exhibits keto-enol tautomerisation [298,299]. This was confirmed by the presence of a
vinylic v(=CH) stretch at 3073 cm"'. Other medium intensity bands in the range 1300-
1400 cm"' due to the in plane bending modes of v(CH3) and v(=CH) groups were also
observed. Absorption maxima found at 1554 cm' and 1197 cm"' were attributed to the -
v(C=C) and v(C-O) stretching modes of the pyran rings, respectively. The IR spectra of
the macrocyclic complexes [ML2(dach)2(H20)2](C104)2 revealed some notable features.
Disappearance of v(C=0) at 1724 cm"' and 1626 cm"' and the appearance of a strong
band in the range 1641-1653 cm"' assigned to v(C=N) provided strong evidence for the
conversion of ketonic groups into the azomethine groups of the Schiff base residues
[300]. The macrocycle formation through Schiff base condensation was supported by the
appearance of new bands in the range 2920-2935 cm' and 2857-2861 cm"' due to
symmetric and asymmetric v(CH2) vibrations of the dach moieties. Additionally the IR
spectra of the complexes also display a wide band at 3329-3487 cm"' associated with the
stretching vibrations of the water molecules and the coordinated v(NH) groups. This
substantiates the fact that both the ligand and the metal complexes show tautomerism.
However, bands belonging to v(OH) bending vibrations could not be assigned, owing to
the overlay with very intense v(C=N). The conclusive evidence for the coordination mode
of metal complexes was deduced by the observation of new v(M-N) bands around 513-
527 cm' in the far IR region [301,302]. Similarly, the presence of uncoordinated
perchlorate anions was depicted by the lack of splitting of v(Cl-O) bands observed in the
range 1055-1114 cm"' and 624-649 cm"' [303] (Table 15).
142
NMR Spectra
The identification of ligand CisHjoOe and the metal complexes was further obtained from
NMR spectroscopy. The ' H NMR spectrum of the ligand C13H10O6 displayed a broad
signal at 15.75 ppm, characteristic of enolic proton [304]. Peaks with chemical shifts near
6.25-8.76 ppm were ascribed to -C=CH and -CH=C-C ring protons. The signals for the
alkyl -CH3 protons and the vinylic =CH protons were detected at 2.09-2.31 ppm, 2.41-
2.45 ppm and 4.09-4.12 ppm. Similar chemical shifts were observed for the
corresponding protons in ' H NMR spectrum of the complex C38H44N40i8Cl2Zn. The -
OH resonance disappears while a new peak at 5.61-5.70 ppm emerges due to the
coordinated -NH proton. Additional peaks in the range 1.88-1.92 and 1.96-2.18 ppm
absent in the spectrum of the ligand CnHioOe were attributed to different cyclohexyl ring
protons (Table 16).
The ' C NMR spectra of the ligand CnHioOe and the complex C38H44N40i8Cl2Zn
corroborated well with the proposed structures. In the spectrum of CuHioOe, the ' C
NMR signals for the two lactonic carbonyl carbons appear at 156.13 and 159.31 ppm
[305]. Resonances due to -C=0 and =CH-OH of the diketone residue appear at 199 ppm
and 168 ppm, respectively [306]. However, significant changes were observed in the
spectrum of complexes C38H44N40i8Cl2Zn. A new signal appeared at 175.33 ppm due to
C=N, confirming the cyclization of the ligand with the dach moieties. Resonances at
20.09 and 24.38-28.94 were ascribed to the cyclohexyl ring carbons and the CH3 groups.
143
respectively. Other peaks remained almost undisturbed in the spectrum of the complex
C38H44N40i8Cl2Zn (Table 17).
Electronic spectra
The electronic spectrum of the ligand CuHioOe in CHCI3 show intense absorption bands
at 220 nm, 279 nm and 437 nm. The strong bands appearing at the low energy side were
assigned to n-+ TC* associated with the C=0 group. The bands at the higher energy arise
from 7t-* 7t* transitions within the lactonic rings or exocyclic carbonyl groups [307].
These absorption bands in complexes C38H44N4O18CI2C0, C38H44N40i8Cl2Ni,
C38H44N4O18CI2CU and C38H44N40i8Cl2Zn are shifted to longer wavenumbers compared
to those of ligands. A prominent band at 309-350 nm was ascribed to n —* 7t* transition of
the azomethine chromophore [308]. In the visible region, complexes C38H44N4O18CI2C0,
C38H44N40i8Cl2Ni and C38H44N4O18CI2CU revealed bands at 520 nm, 670 nm and 588 nm
ascribed to d-d transitions of the Co(II), Ni(II) and Cu(II) ions, respectively in an
octahedral coordination geometry [309-311].
Mass spectroscopy
Electrospray ionization mass spectra of the ligand CnHioOg and the metal complexes
C38H44N4O18CI2C0, C38H44N40i8Cl2Ni, C38H44N4O18CI2CU and C38H44N40i8Cl2Zn were
obtained to confirm their proposed molecular formulae. The ligand C13H10O6 displayed
the molecular ion peak at m/z 262.3. The mass spectra of complexes C38H44N4O18CI2C0,
C38H44N40i8Cl2Ni, C38H44N4O18CI2CU and C38H44N40i8Cl2Zn recorded molecular ion
peaks at m/z 973.7, 973.0, 979.0, 981.1 respectively. Fragments obtained by the
144
successive expulsion of the coordinated water molecules were also depicted in the mass
spectra of the complexes C38H44N4O18CI2C0, C38H44N40i8Cl2Ni, C38H44N4O18CI2CU and
C38H44N40i8Cl2Zn, supporting the proposed molecular formulae (Table 18).
EPR spectrum
Solid state X-band EPR spectrum of the C38H44N4O18CI2CU complex obtained at LNT at
9.1 GHz, recorded an isotropic signal at 2.04 with the unpaired electron located in dz
orbital having an octahedral geometry [312].
In vitro DNA binding studies
Absorption titration witli CT DNA
Electronic absorption spectroscopy is one of the most useful techniques for DNA binding
studies of metal complexes. Absorption spectral titrations were performed by maintaining
a constant concentration of the ligand CuHioOe (0.066.10'^ M) and the complex
C38H44N4O18CI2CU (0.016.10"^ M) and varying the nucleic acid concentration (0.120.10'^
M - 0.301.10-' M for ligand CnHioOe and O.OSS.IO" M - 0.277.10-^ M complex
C38H44N4O18CI2CU. The absorption spectra of the ligand CnHioOe and complex
C38H40N4O16CI2CU in the absence or presence of CT DNA are given in Figure 56. The
ligand exhibits the absorption maxima at 279 nm and 437 nm attributed to n-*n* and
n—*n* transitions respectively. In presence of CT DNA, both the absorption bands
exhibited hyperchromism and the band at 279 nm also undergoes a red shift of 5 nm. The
hyperchromicity implies that some interaction other than intercalation has occurred
between ligand and DNA, because intercalation would lead to hypochromism and
145
bathochromic shift in UV absorption spectra [313]. The intrinsic binding constant Kb for
ligand to CT DNA was calculated to be 3.2.10^ M"', a value lower than those obtained for
typical intercalators (EB-DNA ~10^). It appears that the hyperchromism occurs due to the
aggregation of the planar ligand molecules on the DNA surface and/or intimate surface
binding of the lactone rings along the DNA backbone [314]. The complex
C38H44N4O18CI2CU exhibits three intraligand transitions at 215 nm, 279 nm and 309 nm
attributed to 7t—»7t* and n ^ n* transitions, respectively and a d-d transition maximum at
588 nm. In presence of CT DNA, interesting changes in absorption spectra were
observed. Unlike the ligand C13H10O6, the complex exhibited hypochromism at 309 nm
absorption band attributed to n—» n*, while as the d-d transition at 588 nm undergoes
hyperchromism with no red shift (Figure 57, 58).
Mavelength (nir.)
Figure 56. Absorption spectral traces of CuH/oOc ligand in Tris HCl buffer upon addition ofCTDNA. Arrow shows the absorbance change upon increasing concentration of the CT DNA. Inset: Plots of [DNA]/ Ea-Sf vs [DNA] for the titration of CnHwOe ligand with CT DNA; (m) experimental data points; full lines, linear fitting of the data.
146
This pattern reveals that while the UV absorption band undergoes hypochromism
suggesting that the complex has intimate association with CT DNA and binds to the helix
via partial intercalation [315, 316], at the same time electrostatic interaction due to the
presence of strong Lewis acid copper centre of the complex to the negatively charged
phosphate of the DNA helix probability increases which is clearly implied by
hyperchromism of d-d absorption maxima [274,317]. The observed hypo and
hyperchromic effects reveal that the complex C38H44N4O18CI2CU possesses more than one
DNA binding mode. A good quality data was not obtained from titrations at metal d-d
bands, making the determination of intrinsic binding constant inaccurate. Hence, binding
constant was calculated at the intraligand band and was found to be 2.18. I O ^ M ' .
Figure 57. Absorption spectral traces ofCi8H44N40i^l^u complex in Tris-HCl buffer upon addition of CT DNA Inset: Plots of [DNA]/ Sa-Sf vs [DNA] for the titration of C3SH44N4O18CI2CU complex with CT DNA; (u) experimental data points; full lines, linear fitting of the data. Arrow shows the absorbance change upon increasing concentration of the CT DNA.
147
ma
Wiveleiigth(iim)
Figure 58. Absorption spectral traces ofC38H44N40isCl^u complex in Tris-HCl buffer upon addition ofCTDNA at d-d absorption band. Arrow shows the absorbance change upon increasing concentration of the CTDNA.
Absorption titration with 5'GMP
To identify the specific binding site of the metal complex C38H44N40igCl2Cu to DNA,
interaction studies with the nucleotide 5'GMP were examined by absorption titrations.
On increasing the concentration of the nucleotide there was a sharp increase in intensity
of the UV absorption band as shown in Figure 59. No significant changes were observed
at the d-d absorption maxima. Therefore, coordination towards the N7 atom of guanine of
the copper complex has been precluded. Hyperchromism at the UV absorption bands thus
implies that the complex binds to DNA non-covalently, most probably through
electrostatic interaction or by partial intercalation.
148
Wavelength (nm)
Figure 59. Absorption spectral traces of C38H44N4O18CI2CU complex in Tris-HCl buffer upon addition of 5'GMP Arrows show the absorbance changes upon increasing concentration of the 5 'GMP.
'H and 'P NMR studies with 5'GMP
The interaction of complex C38H44N4O18CI2CU with guanosine 5'monophosphate was
evaluated by using ' H and ^'P NMR technique. The 'H NMR of 5'GMP in D2O solvent
recorded the proton resonance of guanine H8 at 8.10 ppm and ribose Hl'-HS' at 3.8-5.8
ppm respectively. For paramagnetic complexes, the chemical shift of proton adjacent to
the metal centre will be perturbed resulting in line broadening. On interaction of complex
C38H44N40igCl2Cu with 5'GMP the H8 signal of guanine does not alter significantly
(8.12 ppm) (Figure 60a-b). At different time intervals the spectra remains unaltered,
revealing the non-participation of N7 of guanine in covalent binding [318]. Furthermore,
there is line broadening of H8 and Hr-H5' signals of ribose sugar which indicate the
presence of paramagnetic Cu(II) center in the proximity of these protons. ^'P NMR of
149
5'GMP reveals a signal at 3.67 ppm. On interaction of complex C38H44N4O18CI2CU with
5'GMP, there is upfield shift of 3.60 ppm (0.07 ppm) indicative of electrostatic binding
mode of the complex C38H44N4O18CI2CU to the phosphate group of 5'GMP. Since there is
no large upfield shift, it implies that the complex has weaker electrostatic tendency with
the phosphate group and involves other non-covalent DNA binding mode. These
observations were further corroborated by UV and fluorescence data.
Fluorescence titrations
The ligand C13H10O6 and the complex C38H44N4O18CI2CU emit luminescence in Tris-
buffer with a maximum appearing at 567 nm and 525 nm respectively. Upon the addition
of increasing concentration of DNA (0.033.10"^M -0.166.10"^M), the emission intensity
of both the ligand CnHioO^ and the complex C38H44N4O18CI2CU enhances gradually
(Figure 61a-b). The emission enhancement is usually correlated with the extent to which
the compounds penetrate into the hydrophobic pockets of DNA, thereby avoiding the
quenching effects of solvent water molecules [319]. Due to the reduced solvent mobility
at the binding site by the hydrophobic DNA macromolecule a decrease in vibrational
modes of relaxation results and hence quenching efficiency, especially in case of
intercalation [320, 321]. The emission titration results thus support the idea that both the
ligand as well as the complex is protected from the solvent water molecules and can
insert deeply into the DNA helix [322].
150
(a)
-o-?-o ' 6- 4'
7 O
re SSsJssssssHs:
V^W;^ NH
"OH H" 3' 2'
• • 7 C S 1 3 1 1
(b) : n
J} ^/W^>N--,^.JLi^:----w.
Figure 60. 'H NMR spectra of (a) 5'GMP and (b) the reaction of C3sli44N40i^l2Cu complex (2.5 mM) with 5 'GMP (5 mM) in D2O at 25 "C.
151
To compare the binding affinity of the ligand and the complex C38H44N40igCl2Cu the
binding data obtained from the emission spectra were fitted in the Scatchard equation to
acquire the binding parameters. A plot of r/Cp vs r gave the binding constants 3.0 .10* M'
'and 2.0 .lO'' M"' for the ligand CnHioOe and the complex C38H44N4O18CI2CU
respectively. The calculated binding constants are different from those obtained by
absorption titration due to the different spectroscopic and different calculation method.
DNA Cleavage
The interaction of the complex C38H44N4O18CI2CU with supercoiled pBR322 plasmid
DNA was studied both in absence and presence of reductant, ascorbic acid employing gel
electrophoresis. The binding of the complex with pBR322 plasmid DNA was determined
by its ability to change its conformation from supercoiled (SC) to nicked open circular
(NC) form. Different concentrations of complex C38H44N4O18CI2CU were mixed with the
pBR322 plasmid DNA in Tris-HCI buffer in the absence and presence of the reductant
ascorbic acid and mixtures were incubated at 37 °C for Ih. In the absence of ascorbic
acid, pBR322 plasmid DNA was converted from SC (form I) to NC (form II). In presence
of ascorbic acid at increasing concentration of the complex there was complete
conversion of SC form I to nicked DNA form (II). The bands are more intensified in
presence of ascorbic acid as depicted in Figure 62a-b. Control experiments using only
complex or ascorbic acid failed to show any apparent cleavage activity. An oxidative
152
(a)
«n too Wtveleiiglh(ii
(b)
' ' I mo
Wivelenglh (nm)
Figure 61. Emission enhancement spectra of (a) CnHioOe ligand and (b) C3SH44N4OisChCu complex in presence ofCT DNA in Tris-HCl buffer. Arrows show the intensity changes upon increasing concentration of the CT DNA C/jH/oOc and C3SH44N4O/SCI2CU (0.033.10' M); Inset: Scatchard plots of the fluorescence titration data; (m) experimental data points; full lines, linear fitting of the data.
153
cleavage mechanism has been proposed where Cu (II) is first reduced to the Cu (1) [323]
by the ascorbic acid which then binds to DNA forming a Cu(I)complex-DNA adduct.
NC
sc
NC
- • SC
Figure 62. Gel electrophoresis diagram showing cleavage of supercoiled pBR322 plasmid DNA (300 ng) by CS8H44N4O18CI2CU complex, (a) in absence of ascorbic acid; 250fiMC38H44N4O,8Cl2Cu + DNA, (lane 1), 200/uMC38H44N40,sCl2Cu + DNA (lane 2), I5OMMC38H44N4O/8CI2CU + DNA (lane 3), 100 ^M C38H44N4O/8CI2CU + DNA (lane 4) and 50 fiM C38H44N4O18CI2CU +DNA (lane 5), control DNA (lane 6); (b) in presence of ascorbic acid (10 ^M) 250 nM C38H44N4O18CI2CU + DNA, (lane 1), 200 fiM C38H44N4O18CI2CU + DNA (lane 2), 150 fiM C38H44N40,8Cl2Cu + DNA (lane 3), 100 nM C38H44N4O18CI2CU + DNA (lane 4) and 50 nM C38H44N40,8Cl2Cu +DNA (lane 5), control DNA (lane 6).
154
In Vitro Cytotoxicity
The cytotoxic and/or the growth inhibitory effects of the ligand CnHioOe against the
different human tumor cell lines derived from leukemia, non-small cell lung cancers,
colon cancers; CNS cancer and melanoma were determined, using standard protocol for
cell viability. The dose response curves (Figure 63) were evaluated and the optical data
retrieved from logio concentration summarized in Table 19.
The ligand CuHioOe exhibited inhibitory effect of almost same order with logio GI50 >-
4.43 for all cell lines except CNS cancer (SNB-75), which exhibited highest inhibition
factor, logio GI50 = -5.56 at a concentration of 3.98 ^M. These experiments support that
the compound exhibits a strong growth inhibitory effect selectively against CNS cancer
SNB-75 cell line.
Since chelation of metal ion with ligand enhances the inhibitory power of the compounds,
therefore it is inferred that the complexes will possess greater cytotoxic effect than the
free ligand itself
155
(a) (b)
r I'
L 1 1 1 _
U«««»«-i»fcO- • «>M»
r . . . A . . . NCI-KW
(c) (d)
{•
n'-l |4 . _ _ ^ MCT-IJ ,
tr.-=.-=.-=.i.-«i:^zr!r!rx * ,.-• '—•
nlkkhr)
(e)
J r
MilsMM*
fcjPA'i;t-4="i=ji--^-i««.r«="55isr-:S
r 1 ' •
UUim.m , MM ^ _ K-MU.> . . ^ . . MC-MU^ » IMOC-m . . . 4 UACC4X . . . 4 . .
Figure 63. Dose response activity curves ofCijHioOe ligand against a) leukemia (b) Non-Small cell lung cancer (c) Colon cancer (d) CNS cancer (e) Melanoma cell lines.
156
o U
<s
u oe
o •* iz; ^
00 rr>
u M
0) k< o a & o u -o a R t e
o o
<*>
u 'O a ct .SP a> JS
• r f
« IH
o A •^ 73
"« u
A S
e R f"-^ A u
• P 4
w
8 N
ao
d • V
;z; ^ !S
0 0 n
u -o c R
s
u 00 ^ N
o T
z 00 f»)
u r.
^; «s
h* j a Ck
- * 1-H
00
o
00
H U
o
C 3 O
b
3
2
3 O
"o U
U
U o
U X
o. B o
c a
o •* ON 0 0 CO J ^
o m 0 \ ON »r) i n
rJ t ^
bO c
o o
X
u
t^ r 0 0
0\
T3 6
o 00 r-1
o U
<s
u 00
d
u
o <r»
ON • * vo 00 vd vd
o\ d 00
o
A
00 vq >r5
O 00
d z
u
ON fO
«n vo vd vd
<n
o OS
& 3
OH
o
3
u u
00
d z!
u
00 (N
0 0 <N
vd >«
09
ro
o
r •^
2 PQ
O CN)
c N
o 00
d z i i i i CJ O
CQ
u 00
^ 00 r^
u o U M
u 0 0
o T f
z ^
so n
u en O H V
a S o . U rA P H
" 'c a B « ^
10 C
61)-T s U 00
VO i-i
oo * • ^
f»l
«u -M -a «M B O 03 eo s « V. >i5 _£*
.o
s^l R »*) H U
c N U
00
d
I 00
u
3
o u
00
d
i oo
u
u 00
d •t I 00
u
o u u
oo
d
I 00
u
o o en
d
ex 3 o
o
in so
OS
O
00
O VO
ON VO
Ov
3 00 VO
m m
o VO r>. ^
VO <N VO »—'
t—(
fO r f m
o
c«
o o
00 o m
o
r< >r> VO • — I
0 0
»o m 0 \ (N
t--tN •T)
VO VO <n
O
o fo
<r> s
00 00
o 0 \ <N
Ov W-)
VO u-
s
•n 00
CM
>
!
u >
157
0 \
0 \ •^ VO *-
VO 00
• > : 1 -r<) Ov CN
O <n w->
Ov OO «r>
£ u II >
II u y
u o I 2
158
c N u
oo
6
I oo m
u
vO
o o
X
u
ir> t-> m 1—<
1—H
m rvi o\ o (N
W-) Ti
cs • *
<N
CS • — ( 1-0\ o rl-
ro 0 0 O
VO r 0 0
ex 3 O la
o
(N <N
00
I VO 0\
1 1
00
1-00 1-CO 00 •^
VO ^ t^
Tiro 00
-^ 0 0 00 ^ a
o
X O
a: u X u
i
vq
U II u
X u U
H
s
159
a SI
u C
u oo
6
I 00
u
o o
X
u
00 n- 22 Tt 00 <^
00
<N 2 2
<N
0 0 ON OO
s 2 ^ ^ T t >n
o »n
^r —' f<^ ^ 00 t^ vo -^ r<^ t-«; - - O N vo 0\ (N O ON <n « 0 • ^
ON as 0 0
CO
s o X
u
u II u
X u I I .
o II u
•8
o II u
o II u
X
u I o o u
X u o u I
u
S II 9 o X u
•Si JO a H
160
o U
r«
u 00 o
^ ;
r l 00
u M
9i
a,
•
15 S c N
00 ^
o z ^
00
su o
o
-o C3
U
en V) W
s
l-H U) M 00
u 2 0}
T3
a a U
u 00
o rr
z, •* :S 00 f»l
u
u 00
o •V
:S fO
H U
+
o IN
X
X
B o
i
o o
u
00
ON
O Ov
ON
CN ON
o U IN
o
ON <N
ON ON
ON
00 '-«r NO ON ON
ON
^
o
ON
3
o
o
NO
ON
CNI ^
NO ON
O <N ON ^
00 ON
C N
U U U U O
i i i ^ 00 00 00 00
u u u u
161
Table 19.% Mean optical densities Growth Inhibition for selected cancer cell lines by CisHioOeligand
Panel/ Cell Line
Leukemia CCRF-CEM HL-60 (TB) K-562 MOLT-4 RPM1-8226 SR Lung cancer A549/ATCC EKVX HOP-62 NCI-H226 NCI-H23 NC1-H322M NCI-H460 NCI-H522 Colon Cancer COLO-205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 CNS Cancer SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 Melanoma MALME3M M14 SK-MEL-2 SK-MEL-28 SK-MEL-05 UACC-257 UACC-62
Time Zero
0.322 0.273 0.175 0.420 0.244 0.421
0.275 0.697 0.688 1.213 0.418 0.502 0.169 0.408
0.399 0.473 0.146 0.121 0.229 0.375 0.227
0.474 0.385 0.481 0.650 0.489 0.242
0.642 0.243 0.479 0.417 0.306 0.607 0.335
Ctrl
1.083 0.812 1.108 1.480 0.546 0.936
1.205 1.241 1.972 2.057 1.448 1.342 1.356 1.515
2.296 1.203 1.125 0.847 0.983 1.497 1.252
1.349 0.692 1.327 1.891 1.070 1.180
1.210 1.046 0.976 0.912 1.514 1.604 1.629
Logio
Mean Optical Densities
-8.4
0.946 0.784 1.194 1.457 0.502 0.899
1.194 1.311 2.018 2.049 1.487 1.333 1.311 1.478
2.300 1.025 1.064 0.782 0.977 1.485 1.291
1.330 0.639 1.303 1.934 0.883 0.970
1.225 0.988 0.960 0.928 1.265 1.552 1.495
-7.4
0.923 0.705 1.054 1.363 0.508 0.974
1.215 1.302 2.047 2.257 1.503 1.321 1.371 1.528
2.506 1.140 1.075 0.774 0.968 1.412 1.194
1.304 0.632 1.314 1.920 0.883 0.965
1.199 0.978 0.893 0.930 1.199 1.594 1.483
-6.4
0.961 0.700 0.931 1.236 0.448 0.994
1.130 1.202 1.999 1.984 1.396 1.296 1.155 1.515
2.222 1.029 0.992 0.804 0.929 1.439 1.172
1.211 0.609 1.282 1.854 0.847 0.850
1.236 0.963 0.862 0.945 1.388 1.483 1.352
concentration
-5.4
0.821 0.648 0.957 1263 0.450 0.873
1.125 1.191 1.946 2.010 1.531 1.295 1.221 1.546
2.204 1.170 1.058 0.743 0.950 1.466 1.104
1.209 0.595 1.250 1.858 0.769 0.895
1.218 0.969 0.864 1.046 0.948 1.474 1.340
-4.4
1.023 0.697 1.067 1.479 0.530 0.857
1.067 1.155 1.974 2.024 1.583 1.340 1.084 1.472
2.105 1.108 0.990 0.679 0.854 1.509 1.293
1.179 0.558 1.277 1.811 0.755 1.144
1.074 0.961 0.886 0.919 1.012 1.424 1.297
-8.4
82 95
109 98 86 93
99 113 104 99
104 99 96 96
99 76 94 91 99 99
104
99 83 97
104 68 76
103 93 96
103 76 93 90
Percent Growth
-7.4
79 80 94 89 88
107
101 111 106 124 105 97
101 101
111 91 95 90 98 92 94
95 81 98
102 68 77
98 92 S3
104 74 99 89
-6.4
84 79 81 77 68
111
92 93
102 91 95 95 83
100
96 76 86 94 93 95 92
84 73 95 97 62 65
105 90 77
107 90 88 79
-5.4
66 70 84 80 68 88
92 91 98 94
108 94 89
103
95 95 93 86 96 97 86
84 69 91 97 48 70
101 91 78
127 53 87 78
-4.4
92 79 96
100 95 85
85 84
100 96
113 100 77 96
93 87 86 77 83
101 104
81 56 94 94 46 96
76 89 82
102 58 82 74
References
162
References
[I] S. M. Cohen, Curr. Opin. Chem. Biol., 11, 115 (2007).
[2] K. H. Thompson, C. Orvig, Dalton Trans., 761 (2006).
[3] P. J. Dyson, G. Sava, Dalton Trans., 1929 (2006).
[4] N. Farrell, Coord. Chem. Rev., 232, 1 (2002).
[5] C. Orvig, M. J. Abrams, Chem. Rev., 99, 2201 (1999).
[6] K. H. Thompson, C. Orvig, Science, 300, 936 (2003).
[7] N. Farrell, Bioorganometallics, ed. G. Jaouen, Wiley-VCH, Weinheim, 2005.
[8] Z. J. Guo, P. J. Sadler, Angew. Chem. Int. Ed, 38, 1513 (1999).
[9] B. Lippert (Ed.), Cisplatin, Chemistry and Biochemistry of a leading Anticancer
Drug VCHA & Wiley-VCH, Zurich, (1999).
[10] B. Rosenberg, L. vanCamp, T. Krigas, Nature, 205, 698 (1965).
[II] B. Rosenberg, Interdiscip. Sci. Rev., 3, 134 (1978).
[12] T. Storr, K. H. Thompson, C. Orvig, Chem. Soc. Rev., 35, 534 (2005).
[13] C. X. Zhang, S. J. Lippard, Curr. Opin. Chem. Biol, 7, 481 (2003).
[14] X. Wang, Z. Guo, Dalton Trans., 1521 (2008).
[15] R J. P. Williams, J. J. R. Frausto da Silva, Biological Chemistry of the elements,
Oxford University Press, Oxford, (2001).
[16] R. W. Y. Sun, D. L. Ma, E, L. M. Wong, C. M. Che, Dalton Trans., 4884 (2007).
[17] L. Ronconi, P. J. Sadler, Coord Chem. Rev., 251,1633 (2007).
163
[18] C. G. Hartinger, S. Zorbas-Seifried, M. A Jakupec, B. Kynast, H. Zorbas, B. K.
Keppler,y. Inorg. Biochem., 100, 891 (2006).
[19] A. Bergamo, G. Sava, Dalton Trans., 1267 (2007).
[20] G. Sava, S. Zorzet, C-Turrin, F. Vita, M.R. Soranzo, G. Zabucchi, M. Cocchietto, A.
Bergamo, S. Digiovone, G. Pezzoni, L. Sartor, S. Garbisa, Clin. Cancer Res., 9,1898
(2003).
[21] K. H. Thompson, J. H. McNeill, C. Orvig, Chem. Rev.. 99,2561 (1999).
[22] Y. Shechter, G. Eldberg, A. Shisheva, D. Gefel, N. Sekar, S. Qian, R. Bruck, E.
Gershonov, D.C. Crans, Y. Goldwasser, M. Fridkin, J. Li, in Vanadium Compounds:
Chemistry, Biochemistry and Therapeutic Applications, ed. A.S. Tracey and D.C.
Crans, ACS Symposium Series 711, Washington D.C, 308 (1998).
[23] P. Caravan, L. Gelmini, N. Glover, F.G. Herring, H. Li, J. H. McNeill, S. J. Rettig, I.
A. Setyawati, E. Shuler, Y. Sun, A. S. Tracey, V.G. Yuen, C. Orvig, 7. Am. Chem.
Soc, 117,12759(1995).
[24] M. A. Jakupec, M. Galanski, V. B. Arion, C. G. Hartinger, B. K. Keppler, Dalton
Trans., 183(2008).
[25] R. K. O. Sigel, A. M. Pyle, Chem. Rev., 107, 97 (2007).
[26] R. K.O. Sigel, Eur. J. Inorg. Chem., 12, 2281 (2005).
[27] D. E. Draper, D. Grilley, A. M. Soto, Annu. Rev. Biophys. Biomol. Struct., 34, 221
(2005)
[28] E. Freisinger, R. K. O. Sigel, Coord Chem. Rev., 251, 1834 (2007).
164
[29] B. T. Wimberly, D. E. Brodersen, W. M. demons. Jr., R. J. Morgan-Warren, A. P.
Carter, C. Vonrhein, T. Hartsch, V. Ramakrishnan, Nature, 407,327 (2000).
[30] M. J. Clarke, Coord. Chem. Rev., 236, 209 (2003).
[31] J. E. Debreczenni, A. N. Bullock, G. E. Atilla, D. S. Williams, H. Bregman, S.
Knapp, E. Meggers,/Ingew. Chem. Int. Ed., 45, 1580 (2006).
[32] V. Barve, F. Ahmad, S. Adsule, S. Banerjee, S. Kulkami, P. Katiyar, C. E. Anson,
A. K. Powell, S. Padhye, F. H. Sarkar, J. Med Chem., 49, 3800 (2006).
[33] T. W. Hambley, Dalton Trans., 4929 (2007).
[34] E. Meggers, Curr. Opin. Chem. Biol, 11, 287 (2007).
[35] S. M. Fricker, Dalton Tram., 4903 (2007).
[36] P. C. A. Bruijnincx, P. J. Sadler, Curr. Opin. Chem. Biol, 12, 197 (2008).
[37] X. Liang, J. A. Parkinson, M. Weishaupl, R. O. Gould, S. J. Paisey, H. -S. Park, T.
M. Hunter, C. A. Blindauer, S. Parsons, P. J. Sadler, J. Am. Chem. Soc, 124, 9105
(2002).
[38] M. Shionoya, E. Kimura, Y. litaka, J. Am. Chem. Soc, 112,9237 (1990).
[39] E. Kimura, X. H. Bu, M. Shionoya, S. J. Wada, S. Maruyama, Inorg. Chem., 31,
4542(1994).
[40] E. Kimura, Prog. Inorg Chem., 41, 443 (1994).
[41] Y. Inouye, T. Kanamori, M. Sugiyama, T. Yoshida, T. Koike, M. Shionoya, K.
Enomoto, K. Suchiro, E. Kimura, Antiviral Chem. Chemother., 6, 337 (1995).
[42] L. Fabbrizzi, M. Liccheili, P. Pallavicini, P. Sacchi, Supramol Chem., 13, 569
165
(2001).
[43] S. Muregesan, S. J. Shetty, T. S. Srivastava, O. P. D. Noronha, A. M. Samuel, Appl.
Radial. Isot., 55, 641 (2001).
[44] F. Liang, C. T. Wu, H. K. Lin, T. Li, D. Z. Gao, Z. Y. Li, Z. Y. Li, H. K. Lin, D.
Z. Gao, J. Wei, C. Y. Zheng, M. X. Sun, Bioorg. Med Chem. Lett., 13, 2469 (2003).
[45] F. Liang, P. Wang, X. Zhou, T. Li, Z. Y. Li, H. K. Lin, D. Z. Gao, C. Y. Zheng, C.
T. Wu, Bioorg. Med Chem. Lett.. 14, 1901 (2004).
[46] E. de Clercq, Nat. Rev. Drug Disc, 2, 581 (2003).
[47] E. de Clercq, N. Yamamoto, R. Pauweb, M. Baba, D. Schols, H. Nakashima, J.
Balzarini, Z. Debyser, A. Murrer, D. Schwartz, D. Thomthon, G. Bridger, S. Fricker,
G. Henson, M. Abrams, D. Dicker, Proc. Natl. Acad. Sci., USA, 89, 5286 (1992).
[48] W. Zhan, Z. Liang, A. Zhu, S. Kurtkaya, H. Shim, J. P. Snyder, D. C. Liotta, J. Med
Chem., 50, 5655 (2007).
[49] J. A. Este, C. Cabrera, E. de Clercq, S. Struyf, J. van Dannme, G. Bridger, R. T.
Skerlj, M. J. Abrams, G. Henson, A. Gutierrez, B. Clotet, D. Schols, Mol.
Pharmacol.. 55, 1667 (1997).
[50] X. Wang, X. Zhang, J. Lin, J. Chen, Q. Xu, Z. Guo, Dalton Trans.. 2379 (2003).
[51] P. F. Shi, Q. Jiang, J. Liu, Y. M.Zhao, L. P. Lin, Z. J. Guo, J. Inorg. Biochem.. 100,
939 (2006).
[52] N. M. Liang, K. D. Tew, in Encyclopedia of Cancer, ed. J. R. Bertino Academic
Press, San Diego, 1, 560 (1997).
166
[53] S. A. Chowdhary, A. L. List, in Encyclopedia of Cancer, ed. J. R. Bertino Academic
Press, San Diego, 1, 610 (1997).
[54] J. H. Goldie, A. J. Goldman, Drug resistance in Cancer: Mechanisms and Models,
Cambridge University Press, Cambridge, UK, (1998).
[55] R. V. J. Chari, Adv. Drug Delivery Rev., 31, 89 (1998).
[56] A. Wunder, G. Stehle, H. H. Schrenk, G. Hartung, D. L. Heene, W. M. Borst, H.
Sinn, Int. J. Cancer, 76, 884 (1998).
[57] C. R. Gardener, J. Alexander, in Drug Targeting, ed, P. Buri and A. Gumma,
Elsevier science Publishers, Amsterdam, 145 (1985).
[58] W. H. Wei, M. Fountain, D. Magda, Z. Wang, P. Lecane, M. Mesfin, D. Miles, J. L.
Sessler, Org. Biomol. Chem., 3, 3290 (2005).
[59] R. Pignatello, S. Guccione, S. Forte, C. Di Giacoma, V. Sorrenti, L. Vicari, G.
Uccello Barretta, F. Balzano, G. Puglisi, Bioorg. Med. Chem., 12, 2951 (2004).
[60] A. N. Vis, A. van der Gaast, B. W. G. van Rhijn, T. K. Catsburg, C. Schmidt, G. H.
J. Mickisch, Cancer Chemother. Pharmacol, 49, 342 (2002).
[61] Drugs, R& D, 5, 52 (2004).
[62] M. P. Mehta, W. R. Shapiro, M. J. Glantz, R. A. Patchell, M. A. Weitzner, C. A.
Meyers, C. J. Schultz, W. H. Rao, M. Leibenhout, J. Ford, W. Curran, S. Phan, J. A.
Smith, R. A. Miller, M. F. Renschler, J. Clin. Oncol., 20, 3445 (2002).
[63] R. Shimanovich, S. Hannah, V. Lynch, N. Gerasimchuk, T. D. Mody, D. Magda, J.
L. Sessler, J. T. Groves, J. Am. Chem. Soc, 123,3613 (2001).
167
[64] S. M. Young, F. Qing, A. Harriman, J. L. Sessler, W. C. Dow, T. D. Mody, G. W.
Hemmi, Y. Hao, R. A. Miller, Proc. Natl. Acad ScL. USA, 96,2569 (1999).
[65] M. Meyer, V. Dahaoul-Gingrey, C. Lecomte, R. Guilard, Coord. Chem. Rev., ITS-
ISO, 1313 (1998).
[66] J. Costamagna, G. Ferraudi, B. Matsuhiro, M. Campos-Vallette, J. Canales, M.
Villagran, J. Vergas, M. J. Aguirre, Coord Chem. Rev., 196 125 (2000).
[67] D. Parker, Chem. Soc. Rev., 19,271 (1990).
[68] V. Alexander, Chem. Rev., 95, 273 (1995).
[69] K. P. Wainwright,y<c/v. Inorg. Chem., 52,293 (2001).
[70] M. Vicente, R. Bastida, A. Macias, L. Valencia, C. F. G. C. Geralds, C. D. Brondino,
Inorg. Chim. Acta, 35S, 1141 (2005).
[71] V. Artero, M. Fontecava, Coord Chem. Rev., 249, 1518 (2005).
[72] L. Siegfried, A. Comparone, M. Neuburger, T. A. Kaden,y. Chem. Soc, Dalton
Trans., 30 (2005).
[73] Z. Zhang, S. Mikkola, H. Lonnberg, Org Biomol. Chem., 1, 854 (2003).
[74] P. V. Bernhardt, G. A. Lawrance, Coord Chem. Rev., 104, 297 (1990).
[75] A. A. Khandar, S. A. H. Yazdi, M. Khatamian, P. McArdle, S. A. Zarei,
Polyhedron, 26, 33 (2007).
[76] S. G. Kang, J. K. Kweon, S. K. Jung, Bull. Korean Chem. Soc, 12, 483 (1991).
[77] R. Bembi, T. G. Roy, A. K. Jhanji, Inorg Chem., 27, 496 (1998).
[78] W. Hay, M. A. Ali, B. Jeeragh,y. Chem. Soc, Dalton Trans., 2763 (1998).
168
[79] D. H. Busch, J. Inclusion Phenom. Macrocyclic Chem., 11, 389 (1992).
[80] D. H. Busch, N. A. Stephenson, Coord. Chem. Rev.. 100, 119 (1990).
[81] L. M. Engelhardt, G. A. Lawrance, T. M. Manning, A. H. \^\\\Xt, Aust. J. Chem., 42,
1859(1989).
[82] R. D. Hancock, M. P. Ngwenya, P. W. Wade, J. C. A. Boeyens, S. M. Dobson,
Inorg. Chim. Acta, 164, 73 (1989).
[83] G. Pattrick, R. D. Hancock, Inorg. Chem., 30, 1419 (1991).
[84] E. L. Hegg, K. A. Deal, L. L. Kiessling, J. N. Burstyn, Inorg. Chem., 36,1715
(1997).
[85] E. L. Hegg, S. H. Mortimore, C. L. Cheung, J. E. Huyett, D. R. Powell, J. N.
Burstyn, Inorg Chem.. 38, 2961 (1999).
[86] J. M. Wilson, F. Giordani, L. J. Farrugia, M. P. Barrett, D. J. Robins, A. Sutherland,
Org Biomol. Chem., 5,3651 (2007).
[87] L. L. Parker, F. M. Anderson, C. C. O'Hare, S. M. Lacy, Bioorg. Med. Chem., 13,
2389 (2005).
[88] L. L. Parker, S. M. Lacy, L. J. Farrugia, C. Evans, D. J. Robins, C. C. O'Hare, J. A.
Hartley, M. Jaffar, I. J. Stratford, J. Med Chem., 47, 5683 (2004).
[89] E. Reisner, V. B. Arion, B. K. Keppler, A. J. L. Pombeiro, Inorg. Chim. Acta, 361,
1569(2008).
[90] M. Fainerman- Melnikova, A. Nezhadahi, G. Rounaghi, J. C, Mc-Murtrie, J. Kim,
K. Gloe, M. Langer, S. S. Lee, L. F. Lindoy, T. Nishimura, K. M. Park, J. Seo, Dalton
169
Trans., 122 (2004).
[91] J. Kim, T. H. Ahn, M. Lee, A. J. Leong, L. F. Lindoy, B. R. Rumbel, B. W. Skelton,
T. Strixner, G. Wei, A. H. White, J. Chem. Soc, Dalton Trans.. 3993 (2002).
[92] Y. Jin, II Yoon, J. Seo, J. E. Lee, S. T. Moon, J. Kim, S. W. Han, K. M. Park, L.
F. Lindoy, S. S. Lee, Dalton Trans., 788 (2005).
[93] E. Bouwman, W. L. Driessen, J. Reedijk, Coord. Chem. Rev., 104, 143 (1990).
[94] R. P. F. Kanters, R.Yu, A. W. Addison, Inorg. Chim. Acta, 196, 97 (1992).
[95] B. Adhikary, C. R. Lucas, Inorg. Chem.. 33, 1376 (1994).
[96] S. Liu, C. R. Lucas, R. C. Hynes, J. P. Charland, Can. J. Chem., 70, 1773 (1992).
[97] S. Usha, M. Palanaindavar, J. Chem. Soc. Dalton Trans., 2277(1994).
[98] Y. Zhang, R. Yang, F. Liu, K. Li, Anal. Chem.. 76,7336 (2004).
[99] N. Spink, D.G. Brown, J.V. Skelly, S. Neidle, Nucleic Acids Res., 11, 1607 (1994).
[100] D. S. Pilch, Z. Xu, Q. Sun, E. J. LaVoie, L.F. Liu, K.J. Breslauer, Proc. Natl. Acad.
5c/., USA, 94, 13565(1997).
[101] J. Mann. A. Barren, Y. Opoku-Baohen, E. Johansson, G. Parkinson, L. R. Kelland,
S. Neidle, J. Med Chem.. 44,138 (2001).
[102] F. A. Tanious, D. Hamelberg, C. Baily, A. Czamy, D. W. Boykin, W. D. Wilson, J.
Am. Chem. Soc, 126, 143 (2004).
[103] M. F. Brana, M. Cacho, A. Gradillas, B. de Pascual-Teresa, A. Ramos, Curr.
Pharma. Des., 7, 1745 (2001).
[104] Z. S. Yang, Y. L. Wang, G. C. Zhao, Anal. Scl, 20,1127 (2004).
170
[105] B. Spingler, C. D. Pieve, Dalton Trans., 1637 (2005).
[106] A. Herbert, A. Rich, J. Biol. Chem., 271, 11595 (1996).
[107] A.D. Richards, A. Rodger, Chem. Soc. Rev., 36,471 (2007).
[108] B. Lippert, Coord Chem. Rev., 200-202,487 (2000).
[109] D. C. Lazarte, M. del Pilar. B. Blanco, J. G. Santos, J. M. G. Perez, A.
Castineiras, J. N. Guiterrez, Coord Chem. Rev., 252, 1241 (2008).
[110] S. Aoki, E. Kimura, J. Am. Chem. Soc, 122, 4542 (2000).
[111] E. Kimura, H. Kitamura, K. Ohtani, T. Loike, J. Am. Chem. Soc, 122,4668 (2000).
[112] E. Kikuta, R. Matsubura, N. Katsube, T. Koike, E. Kimura, J. Inorg. Biochem., 82,
239 (2000).
[113] P. A. Wender, J. DeBraBander, P.G. Harran, J. M. Jimenez, M. F .T. Koehler, B.
Lippa, C. M. Park, C. Siedenbiedel, G. R. Pettit, Proc Natl. Acad. Sci., USA, 95,
6624(1998).
[114] K. J. Humphreys, K. D. Karlin, S. E. Rokita, J. Am. Chem. Soc, 124, 8055 (2002).
[115] S. T. Frey, H. H. J. Sun, N. N. Murthy, K. D. Karlin, Inorg. Chim. Acta, 242, 329
(1996).
[116] T. Ito, S. Thyagarajan, K. D. Karlin, S. E. Rokita, Chem. Commun., 4812 (2005).
[117] D. Jayaraju, A. K. Kondapi, Curr. Sci., 81, 787 (2001).
[118] T. Biver, F. Secco, M.R. Tine, M. Venturini, J. Inorg Biochem., 98, 33 (2004).
[119] A. R. Chakravarty, P. A. N. Reddy, B. K. Santra, A. M. Thomas, Proc Indian
Acad Sci., 114,391(2002).
171
[120] N. K. Singh, S. B. Singh, N. Singh, A. Shrivastav, Biometals, 16, 471 (2003).
[121] Z. S. Yang, Y. L. Wang, Y. C. Liu, G. C. Zhao, Anal. ScL. 21,1355 (2005).
[122] C. Sissi, F. Mancin, M. Gatos, M. Palumbo, P. Tecilla, U. Toneliato, Inorg. Chem.,
44,2310(2005).
[123] R. Cejudo, G. Alzuet, M. G. Alvarez, J. L. G. Gimenez, J. Borras, M. L.
Gonzalez, J. Inorg. Biochem., 100, 70 (2006).
[124] W. K. Pogozelski, T.D. Tullius, Chem. Rev., 98,1089 (1998).
[125] C. J. Burrows, J. G. Muller, Chem. Rev.. 98,1109 (1998).
[126] J. Liu, T. Zhang, T. Lu, L. Qu, H. Zhou, Q. Zhang, L. Ji, J. Inorg. Biochem.,
91, 269 (2002).
[127] J. Liu, T. B. Lu, H. Deng, L. N. Ji, Transition Met. Chem., 28, 116 (2003).
[128] D. H. Johnston, H. H. Thorp, J. Am. Chem. Soc., 117, 8933 (1995).
[129] L.T. Ellis, D. F. Perkins, P. Turner, T. W. Hambley, Dalton Trans., 2728 (2003).
[130] J. Liu, T.-B. Lu, H. Li, Q.- L. Zhang, L. -N. Ji, T. -X. Zhang, L. -H. Qu, H. Zhou,
Transition Met. Chem., 27, 686 (2002).
[131] A. M. Thomas, M. Nethaji, A. R. Chakravarty, J. Inorg. Biochem., 98,1087 (2004).
[132] P. A. N. Reddy, B. K. Santra, M. Nethaji, A. R. Chakravarty, J. Inorg. Biochem.,
98, 377 (2004).
[133] C. W. Jiang, J. Inorg Biochem., 98, 497 (2004).
[134] F. Liu, K. Wang, G. Bai, Y. Zhang, L. Gao, Inorg Chem., 43, 1799 (2004).
[135] C. V. Sastri, D. Eswaramoorthy, L. Giribabu, B. G. Maiya, J. Inorg. Biochem., 94,
172
138(2003).
[136] Q. L. Zhang, J. G. Liu, J. Z. Liu, H. Li, Y. Yang, H. Xu, H. Chau, L. N. Ji, Inorg.
Chim. Acta, 339,34 (2002).
[137] J. B. Delehanty, T. C. Stuart, D. A. Knight, E. R. Goldman, D. C. Thach, J. E.
Bongard, E.L. Chang, RNA, 11, 831 (2005).
[138] J. Qian, W. Gu, H. Liu, F. Gao, L. Feng, S. Yan, D. Liao, P. Cheng, Dalton Trans.,
1060(2007).
[139] J. P. Lancaster (Ed.), The Bioinorganic Chemistry of Nickel, VCH, New York,
(1998).
[140] C. S. Huang, J. X. Li, M. Costa, Z. Zhang, S. S. Leonard, V. Castranova, V.
Vallyathan, G. Ju, X. L. Shi, Cancer Res., 61, 8051 (2001).
[141] C. Mayer, R. G. Klein, H. Wesch, P. Schmezer, Mutat. Res., 420, 85 (1998).
[142] M.B. Ferrari, F. Bisceglie, G. Pelosi, M. Sassi, P. Tarasconi, M. Comia, S.
Capacchi, R. Albertini, S. Pineli, J. Inorg Biochem., 90, 113 (2002).
[143] S. S. Matkar, L. A. Wrischnik, P. R. Jones, U. Hellmann- Blumberg, Biochem.
Biophys. Res. Commun., 343, 754 (2006).
[144] A. Hartwig, T. Schwerdtle, Toxicol. Lett., Ill, 47 (2002).
[145] K. S Kasprzak, F. W. Sunderman Jr., K. Salnikow, Mutat. Res.. 533,67 (2003).
[146] G. Chu, J. Biol Chem., 269, 787 (1994).
[147] Y. Liu, E. Sletten, J. Inorg Biochem., 93, 190 (2003).
[148] Q. Yang, J. -Q. Xu, Y. -S. Sun, Z. -G. Li, Y. -G. Li, X. -H. Qian, Bioorg. Med.
173
Chem. Lett., 16, 803 (2006).
[149] Q. X. Xiang, J. Zhang, P. -Y. Liu, C. -Q. Xia, Z. -Y. Zhou, R. -G. Xie, X.-Q. Yu,
J. Inorg. Biochem., 99, 1661 (2005).
[150] A. Blasko, T. C. Bruice, y4cc. Chem. Res.. 32,475 (1999).
[151] W. S. Bowen, W. E. Hill, J. S. Lodmell, Methods. 25,344 (2001).
[152] P. Caravan, J. J. Ellison, T. J. McMurry, R. B. Lauffer, Chem. Rev.. 99,2993
(1999).
[153] B. Aoki, E. Kimura, Chem. Rev.. 104, 769 (2004).
[154] L. Luker, J. Kotek, P. Vojtisek, P. Hermann, Coord Chem. Rev.. 216-217,287
(2001).
[155] M. Shionoya, E. Kimura, M. Shiro,y. Am. Chem. Soc. 116, 3848 (1993).
[156] D. M. Epstein, L. L. Chappell, H. Khalili, R. M. Supkowski, W. D. Horrocks, J. R.
Morrow, Inorg. Chem.. 39, 2130 (2000).
[157] A. Schrodt, A. Neubrand, R. van Eldik, Inorg Chem., 36,4579 (1997).
[158] R. Menif, A. E. Martell, P. J. Squattrito, A. Clearfield, Inorg. Chem.. 29,4723
(1990).
[159] M. C. B. Oliveira, M. S. R. Couto, P. C. Severino, T. Foppa, G. T. S. Martins, B.
Szpoganicz, R. A. Peralta, A. Neves, H. Terenzi, Polyhedron. 24, 495 (2005).
[160] Y. G. Fang, J. Zhang, S. Y. Chen, N. Jiang, H. H. Lin, Y. Zhang, X. Q. Yu,
Bioorg. Med Chem.. 15, 696 (2007).
[161] E. L. Hegg, J. N. Burstyn, Inorg Chem.. 35, 7474 (1996).
174
[162] K. A. Deal, J. N. Burstyn, Inorg. Chem.. 35, 2792 (1996).
[163] K. A. Deal, A. C. Hengge, J. N. Burstyn, J. Am. Chem. Soc, 118, 1713 (1996).
[164] T. Hirohama, H. Arii, M. Chikira, J. Inorg. Biochem., 98, 1778 (2004).
[165] I. E. Dreosti, Mutat. Res., 475, 161 (2001).
[166] K. H. Falchuk, Mol. Cell Biochem.. 188,41 (1998)
[167] F. Mancin, P. Tecilla, New. J. Chem., 31, 800 (2007).
[168] C. Q. Xia, N. Jiang, J. Zhang, S. Y. Chen. H. H. Liu, X. Y. Tan, Y. Yue, X. Q.
Yu, Bioorg Med. Chem., 14, 5756 (2006).
[169] X. Y. Wang, J. Zhang, K. Li, N. Jiang, S. Y. Chen, H. H. Lin, Y. Huang, L. J.
Ma, X. Q. Yu, Bioorg. Med. Chem., 14, 6745 (2006).
[170] M. Shionoya, T. Ikeda, E. Kimura, M. Shiro, J. Am. Chem. Soc, 116, 3848 (1994).
[171] N. H. Williams, B. Takasaki, M. Wall, J. Chin, cc. Chem. Res.. 32,485 (1999).
[172] W. H. Chapman Jr, R. Breslow,/. Am. Chem. Soc, 117,5462 (1995).
[ 173] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds, Wiley, New York, (1986).
[174] B. E. Douglas, D. H. McDaniel, J. J. Alexander, Concept and Models of
Inorganic Chemistry, J, Wiley and Sons, Inc., New York, (1993).
[175] R. S. Drago, Physical Methods in Inorganic Chemistry, Reinhold Publishing
Corporation, New York, (1968).
[176] R. M. Silverstein, G.C. Bassler, Spectroscopic identification of organic
compounds, John Wiley and sons. Inc. New York. London (1964).
175
[177] B. J. Hathway, D. G. Holah, J. D. Postlethwaite, 7. Chem. Soc, 3215 (1961).
[178] W. J. Geary, Coord Chem. Rev., 7, 81 (1971).
[179] J. Marmur, J. Mol. Biol.. 3,208 (1961).
[180] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am. Chem. Soc., 76,
3047(1954).
[181] A. J. Bard, L. R. Faulkner, Electrochemical Methods, Wiley, New York,
219(1980).
[182] K. J. Laidler, Chemical Kinetics, Tata McGraw Hill Inc., New York, (1973).
[183] A. Wolfe, G. H. Shimer, T. Meehan, Biochemistry, 26,6392 (1987).
[184] D. E. V. Scgemechel, D. M. Crother, Biopolymers, 10, 465 (1971).
[185] J. R. Lakowiez, G. Webber, Biochemistry, 12,4161 (1973).
[186] G. D. Liu, J. P. Liao, Y. Z. Fang, S. S. Huang, G. L. Sheng, R. Q. Yu, Anal. Sci.,
18,391(2002).
[187] G. D. Liu, J. P. Liao, S. S. Huang, G. L. Shen, R. Q. Yu, Anal. Sci., 17,1031
(2001).
[188] A. Kornberg, T. A. Baker, DNA replication, second edition university Science
Books United States of America.
[189] R. E. Farell. Jr., RNA Methodologies, A laboratory guide for isolation and
characterization. Third edition Elsevier Academic press united states of America
[190] G. Cohen and H. Eisenberg, Biopolymers, 8, 45 (1969).
[191] M. Eriksson, M. Leijon, C. Hiort, B. Norden, A. Graslund, Biochemistry, 33,
176
5031 (1994).
[192] T. Nogrady, in Medicinal Chemistry, A biochemical approach, Oxford
University press, New York, Oxford, 6, (1985).
[193] P.Comba, S. M. Luther, O. Maas, H. Pritzkow, A. Vielfort, Inorg. Chem., 40, 2335
(2001).
[194] S. M. Annigeri, M. P. Sathisha, V. K. Revankar, Transition Met. Chem., 32, 81
(2007).
[195] G. Du, A. Ellem, L. K. Woo, Inorg. Chem., 42, 873 (2003).
[196] Y.Chen, Q. Liu, Y. Deng, H. Zhu, C. Chen, H. Fan, D. Liao, E. Gao, Inorg Chem.,
40, 3725 (2001).
[197] A. Channa, J. W. Steed, Dalton Trans.. 2455 (2005).
[198] Y. Xie, W. Bu, A. S. C. Chen, X. Xu, Q. Liu, Z. Zhang, Inorg Chim. Acta, 30,
257 (2000).
[199] S. Tabassum, S. Parveen, F. Arjmand, Acta Biomat.. 1, 677 (2005).
[200] J. Gao, J. H. Reibenspies, R. A. Zingaro, F. R. Woolley, A. E. Martell, A.
Clearfield, Inorg Chem., 44, 232 (2005).
[201] J. Gregolinski, J. Lisowski, T. Lis, Org Biomol. Chem., 3, 3161 (2005).
[202] N. Kuhnert, A. L. Periago, G. M. Rossignolo, Org. Biomol. Chem., 3, 524 (2005).
[203] J. Gao, A. E. Martell, Org Biomol. Chem., 1, 2801 (2003).
[204] S. A. Li, J. Xia, D. X. Yang, Y. Xu, D. F. Li, M. F. Wu, W. X. Tang, Inorg
Chem., 41, 1807(2002).
177
[205] M. P. Suh, S. G. Kang, Inorg. Chem.. 27, 2544 (1988).
[206] H. Aneetha, Y. H. Lai, S. C. Lin, K. Panneersellvam, T. H. Lu, C. S. Chang, J.
Chem. Soc, Dalton Trans., 2885 (1999).
[207] R. N. Prasad, M. Agrawal, S. Sharma, Indian J. Chem., 43A, 337 (2004).
[208] A. D. Naik, S. M. Annigeri, U. B. Gangadarmath, V. K. Revankar, V. B. Mahale,
Transition Met. Chem., 27, 333 (2002).
[209] M. Chauhan, F. Arjmand, Transition Met. Chem., 30, 481 (2005).
[210] K. J. Inoue, M. Ohba, H. Okawa, Bull. Chem. Soc. Jpn., 75, 99 (2002).
[211] S. Chandra, Sangeetika, S. Thakur, Transition Met. Chem., 29, 925 (2004).
[212] G. M. Escander, L. F. Sala, Can. J. Chem., 70, 2053 (1992).
[213] J. Chen, X. Wang, Y. Shao, J. Zhu, Y. Li, Q. Xu, Z. Guo, Inorg. Chem., 46, 3306
(2007).
[214] M. Baldini, M. B. Ferrari, F. Bisceglie, G. Pelosi, S. Pinelli, P. Tarasconi, Inorg.
Chem., 42, 2049 (2003).
[215] M. Chauhan, F. Arjmand, Chem. Biodiv., 3, 660 (2006).
[216] P. G. Daniele, E. Prenesti, S. Berto, V. Zelano, R. Aruga, Ann. Chim., 229 (2004).
[217] Y. G. Gao, M. Sriram, A. H. J. Wang, Nucleic Acids Res., 21,4093 (1993).
[218] S. Moradell, J. Lorenzo, A. Rovira, M. S. Robillard, F. X. Aviles, V. Moreno, R. de
Llorens, M. A. Martinez, J. Reedijk, A. Llobet, J. Inorg. Biochem., 96, 493 (2003).
[219] A. Robertazzi, J. A. Platts, J. Biol. Inorg. Chem., 10, 854 (2005).
[220] G. S. Manning, Q. Rev. Biophys., 11, 179 (1978).
178
[221] B. Selvakumar, V. Rajendiran, P. V. Maheswari, H. S. -Evans, M. Palanaindavar,
J. Inorg. Biochem.. 100, 316 (2006).
[222] R. B. Lopez, B. L. Leon, T. Boussie, T. J. Meyer, Tetrahedron Lett.,. 37, 5437
(1996).
[223] S. Satyanarayana, J. C. Dabrowiak, J. B. Chaires, Biochemistry, 32,2573 (1993).
[224] V. G. Vaidyanathan, B. U. Nair, Dalton Trans., 2842 (2005).
[225] J. Kang, H. Wu, X, Lu, Y. Wang, L. Zhou, Spectrochim. Acta A, 61, 2041
(2005).
[226] F. Arjmand, B. Mohani, S. Ahmad, Eur. J. Med. Chem., 40,1103 (2005).
[227] S. M. Sondhi, M. Johar, R. Shukla, R. Raghubir, N. Bharti, A. Azam, Aust. J.
CAe/«., 54,461(2001).
[228] S. M. Annigeri, A. D. Naik, U. B. Gangadharmath, V. K. Revankar, V. B. Mahale,
Transition Met. Chem., 27, 316 (2002).
[229] X. Ottenwaelder, A. Aukauloo, Y. Joumaux, R. Carrasco, J. Cano, B. Cervera, L
Castro, S. Curreli, M. C. Munoz, A. L. Rosello, B. Soto, R. R. Garcia, Dalton
Trans., 25\6 {2005).
[230] R. M. Silverstein, G. C. Bassler, T. C. Mori!!, Spectrometric Identification of
Organic Compounds, John Wiley & Sons, Inc., 179 (1974).
[231] M. Vaidyanathan, R. Balamurugan, U. Sivagnanam, M. Palanaindavar, 7. Chem.
Soc, Dalton Trans.. 3498 (2001).
[232] G. Rajsekhar, C. P. Rao, P. Saarenketo, K.Nattinen, K. Rissanen, New. J. Chem..
179
28, 75 (2004).
[233] J. Monocol, M. Mudra, P. Lonnecke, M. Hewitt, M. Valko, H. Morris, J. Scorec,
M. Melnik, M. Mazur, M. Koman, Inorg. Chim. Acta, 360, 3213 (2007).
[234] B. J. Hathaway, D.E. Billing, Coord. Chem. Rev., 5,143 (1970).
[235] A.Romerosa, C. S.Bello, M. S.Ruiz, A. Caneschi, V. Mckee, M. Peruzzini, L.
Sorace, F. Zanibini, Dalton Trans., 3233 (2003).
[236] A. B. P. Lever, Inorganic Electronic Spectroscopy, Elsevier Publishing
Company, Amsterdam, London NY, 61 (1968).
[237] J. K. Barton, A. T. Danishefsky, J. M. Goldberg, ^m. Chem. Soc, 106,2172
(1984).
[238] C. S. Chow, J. K. Barton, Methods Enzymol, 111, 219 (1992).
[239] J. Z. Wu, L. Yuan, J. F. Wu, J. Inorg Biochem., 99,2211 (2005).
[240] B. D. Wang, Z. Y. Yang, P. Crewdson, D. Q. Wang, 7! Inorg. Biochem., 101, 1492
(2007).
[241] J. H. Tan, Y. Lu, Z. S. Huang, L. Q. Gu, J. Y. Wu, Eur. J. Inorg. Chem., 42, 1169
(2007).
[242] A. K. Patra, S. Dhar, M. Nethaji, A. R. Chakrvarty, Dalton Trans., 896 (2005).
[243] V. Rajendiran, M. Surali, E. Suresh, S. Sinha, K. Somasundaram, M.
Palanaindavar, Dalton Trans., 148 (2008).
[244] R. Ren, P. Yang, W. Zheng, Z. Hua, Inorg Chem., 39, 5454 (2000).
[245].Y. Wang, Z. N. Yang, Z. N. Chen, Bioorg Med Chem. Lett., 18,298 (2008).
180
[246] Z. Y. Yang, B. D. Wang, Y. H. Li, J. Organomet.Chem., 691,4156 (2006).
[247] H. Li, X. Y. Le, D. W. Pang, H. Deng, Z. H. Xu, Z. H. Lin, J. Inorg. Biochem., 99,
2240 (2005).
[248] L. M. Mirica, X. Ottenwealder, T. D. P. Stack, Chem. Rev., 104,1013 (2004).
[249] S. Satyanarayana, J. C. Dabrowiak, J. B. Chaires, Biochemistry, 31,9319 (1992).
[250] T. M. Kelly, A. B. Tossi, D. J. McConnell, T. C. Strekas, Nucleic Acids Res., 13,
6017(1985).
[251] C. R. Brodie, J. Collins, J. R. A. Wright, Dalton Trans., 1145 (2004).
[252] C. S. Allardyce, P. J. Dyson, D. J. Ellis, S. L. Heath, Chem. Commm., 1396 (2001).
[253] H. Chao, W.J. Mei, Q. W. Huang, L. N. Ji,y. Inorg. Biochem., 91, 165 (2002).
[254] X. Lu, K. Zhu, M. Zhang, H. Liu, J. Kang, J. Biochem. Biophys. Methods., 52, 189
(2002).
[255] S. Gonzalez, L. Valencia, R. Bastida, D.E. Fenton, A. Macias, A. Rodriguez, J.
Chem. Soc, Dalton Trans., 3551 (2002).
[256] C. Jubert, A. Mohamadou, C. Gerard, S. Brandes, A. Tabard, J. P. Barbier, J.
Chem. Soc. Dalton Trans., 2660 (2002).
[257] L. Tei, A.J. Blake, A. Bencini, B. Valtancoli, C. Wilson, M. Schroder, J. Chem.
Soc, Dalton Trans., AMI (2000).
[258] M. Vicente, R. Bastida, C. Lodeiro, A. Macias, A.J. Parola, L. Valencia, S.E. Spey,
Inorg Chem., 42, 6768 (2003).
[259] M. Meyer, L. Fremond, A. Tabard, E. Espinosa, G.Y. Vollmer, R. Guilard, Y. Dory
181
New J. Chem., 29, 99 (2005).
[260] S. Gou, M. Qian, Z. Yu, C. Duan, X. Sun, W. Huang, J. Chem. Soc, Dalton
Tran^., 3232(2001).
[261] H. Keypour, S. Salehzadeh, R. G. Pritchard, R. V. Parish, Inorg. Chem., 39, 5787
(2000).
[262] E. Bertolo, R. Bastida, D.E. Fenton, C. Lodeiro, A.J. Macias, A. Rodroguez, J.
Inclusion Phenom. Macrocydic Chem., 45, 155 (2003).
[263] N.W. Alcock, G. Clarkson, P. B. Glower, G. A. Lawrance, P. Moore, M.
Napitupulu, Dalton Trans., 518 (2005).
[264] M. Soibinet, D. Gusmeroli, L. Siegfried, T.A. Kaden, C. Palivan, A. Schweiger,
Dalton Trans., 2138 (2005).
[265] H.L. Chan, H.Q. Liu, B.C. Tzeng, Y.S. You, S. M. Peng, M. Yang, C. M Che,
Inorg. Chem., 41,3161 (2002).
[266] D.A. Knight, J.B. Delehanty, E.R. Goldman, J. Bongard, F. Streich, L.W. Edwards,
E.L. Chang. Dalton Trans., 2006 (2004).
[267] B. Graham, M. J. Grannas, M.T.W. Heam, C. M. Kepert, L. Spiccia, B.J. Skelton,
A. H. White, Inorg Chem., 39, 1092 (2000).
[268] B. Cervera, J.L. Sanz, M.J. Ibanez, G. Villa, F. Lloret, M. Julve, R. Ruiz, X.
Ottenwaelder, A. Aaukauloo, S. Poussereau, Y. Joumaux, M.C. Munoz, J. Chem.
Soc, Dalton Trans., 781 (1998).
[269] B.S. Garg, R.K. Sharma, E. Kundra, Transition Met. Chem., 30, 552 (2005).
182
[270] N. Raman, A. Kulandaisamy, K. Jayasubramanian, Indian J. Chem., 41A, 942
(2002).
[271] Y. Dong, G.A. Lawrance, L.F. Lindoy, P. Turner, Dalton Trans., 1567 (2003).
[272] A. M. Herrera, R. J. Staples, S. V. Kryatov, A.Y. Nazarenko, E. V. Akimova,
Dalton Trans.. 846 (2003).
[273] S. Srinivasan, P. Athappan, G. Rajagopal, Transition Met. Chem., 26, 588 (2001).
[274] M. Chauhan, K. Banerjee, F. Arjmand, Inorg Chem., 46, 3072 (2007).
[275] R. S. Kumar, S. Arunachalam, Polyhedron, 25, 3113 (2006).
[276] J. -Z. Wu, L. Yuan, J. -F. Wu, J. Inorg. Biochem., 99, 2211 (2005).
[277] K.J. Humphreys, A.E. Johnson, K.D. Karlin, S.E. Rokita, J. Biol. Inorg. Chem., 7,
835 (2002).
[278] A.L. Harris, X. Yang, A. Hegmans, L. Povirk, J. J. Ran, L. Kelland, N. P. Farrell,
Inorg Chem.. 44, 9598 (2005).
[279] 1. Rouzina, V.A. Bloomfield, Biophys. J., 74, 3152 (1998).
[280] A.D. Richards, A. Rodger, Chem. Soc. Rev., 36,471 (2007).
[281] S. Komeda, T. Moulaei, K. K. Woods, M. Chikuma, N. P. Farrell, L.D. Williams,
J. Am. Chem. Soc, 128, 16092 (2006).
[282] M. Chauhan, F. Arjmand, J. Organomet. Chem., 692, 5156 (2007).
[283] T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M. Chikira, P.T. Selvi,
M. Palaniandavar,y. Inorg. Biochem., 99, 1205 (2005).
[284] J.B. Lepecq, C. Paoletti,^ Mol. Biol., 11, 87 (1967).
183
[285] B.C. Baguley, M. LeBret, Biochemistry, 23, 937 (1984).
[286] A. Raja, V. Rajendiran, P. U. Maheswari, R. Balamurugan, C.A. Kilner, M. A.
Halcrow, M. Palaniandavar, J. Inorg. Biochem., 99, 1717 (2005).
[287] P.T. Selvi, H.S. Evans, M. Palaniandavar, J. Inorg. Biochem., 99, 2110 (2005).
[288] P.U. Maheswari, M. Palaniandavar, 7. Inorg Biochem., 98, 219 (2004).
[289] S. Mahadevan, M. Palanaindavar, Inorg Chem., 37, 693 (1998).
[290] M. Palanaindavar, T. Pandiyan, M. Laximinarayan, H. Manohar, J. Chem. Soc,
Dalton Trans., 455 (1995).
[291] Y. Dong, L.F. Lindoy, P. Turner, G. Wei, Dalton Trans., 1264 (2004).
[292] C. De Alwis, J. A. Crayston, T. Cromie, T. Eisenblatter, R.W. Hay, D. Lampeka
Ya, L.V. Tsymbal, Electrochim. Acta, 45, 2061 (2000).
[293] P. T. Selvi, M. Palanaindavar, Inorg Chim. Acta, 337,420 (2002).
[294] K. S. Siddiqi, F. Arjmand, M. Rehman, S. Tabassum, S. A. A. Zaidi, J. Chem.
Research (S), 84, (1994). J. Chem. Research (M), 0383-0393 (1994).
[295] B. S. Creavan, D. A. Egan, K. Kavanagh, M. McCann, A. Noble, B. Thati, M.
Walsh, Inorg Chim. Acta. 359, 3976 (2006).
[296] Y. S. Kim, S. Y. Park, H. J. Lee, M. E. Suh, D. Schollmeyer, C. O. Lee, Bioorg
Med. Chem., 11, 1709(2003).
[297] V. Fargeas, M. Balouch, E. Metay, J. Bafreau, D. Menard, P. Gosselin, J. P.
Berge, C. Barthomeuf, J. Lebreton, Tetrahedron, 60, 10359 (2004).
[298] X. S. Tai, M. Y. Tan, Spectrochim. Acta A., 61, 1767 (2005).
184
[299] M. Cindric, T. K. Novak, K. Uzarevic.J. Mol. Struct., 750, 135 (2005).
[300] H. Khanmohammadi, R. Arabahmadi, M. H. Abnosl, H. R. Khavasi, Polyhedron,
26, 4963 (2007).
[301] I. Yilmaz, H. Temel, H. Alp, Polyhedron, 27, 125 (2008).
[302] S. llhan, H. Temel, 1. Yilmaz, M. Sekerci, J. Organomet. Chem., 692, 3855 (2007).
[303] M. Fernandez, R. Bastida, A. Macias, P. Lourido, L. Valencia, Polyhedron, 26,
5317(2007).
[304] R. M. Silverstein, F. X. Webster, Spectrometric Identification of Organic
Compounds, 6 Ed, John Wiley India Edition, 173.
[305] S. A. Kotharkar, D. B. Shinde, Bioorg. Med. Chem. Lett., 16, 6181 (2006).
[306] 1. Yavari, M. Bayat, Tetrahedron, 59, 2001(2003).
[307] K. Abe, K. Atsufuji, M. Ohba, H. Okawa, Inorg Chem., 41,4461 (2002).
[308] Q. Shi, R. Cao, X. Li, J. Luo, M. Hong, Z. Chen, New. J. Chem.. 26,1397 (2002).
[309] M. Scarpellini, A. Neves, R. Homer, A. J. Bortoluzzi, B. Szpoganics, C. Zucco,
R.A. Nome Silva, V. Drago, A. S. Mangrich, W. A. Ortiz, W. A. C. Passos, M. C.
B. de Oliveira, H. Terenzi, Inorg Chem., 42, 8353 (2003).
[310] J. Pons, A. Chadghan, A. A. Larena, J. F. Piniella, J. Ros, Inorg. Chim. Acta, 324,
342 (2001).
[311] J. M. Moratal, M. J. M. Ferrer, A. Donaire, J. Castells, J. Salgado, H. R.
Jimenez, Dalton Trans., 3393 (1991).
[312] H. Kurosaki, R. K. Sharma, S. Aoki, T. Inoue, Y. Okamoto, Y. Suguira, M. Doi, T.
185
Ishida, M. Otssuka, M. Goto, J. Chem. Soc, Dalton Trans., 441 (2001).
[313] M. Baldini, M. B. -Ferrari, F. Bisceglie, P. P. Dall'Aglio, G. Pelosi, S. Pinelli, P.
Tarasconi, Inorg. Chem.. 43, 7170 (2004).
[314] F. M. O'Reilly, J. M. Kelly, J. Phys. Chem. B., 104, 7206 (2000).
[315] S. M. Hecht, J. Nat. Prod., 63, 158 (2000).
[316] Z. M. Wang, H. K. Lin, Z. F. Zhou, M. Xu, T. F. Liu, S. R. Zhu, Y. T.
Chen, Bioorg. Med. Chem., 9, 2849 (2001).
[317] F. Zhang, Q. Zhang, W. Wang, X. Wang, J. Photochem. Photobiol. A: Chemistry,
184,241(2006).
[318] S, van Rijt, S. van Zutphen, H. den Dulk, J. Brouwer, J. Reedijk, Inorg. Chim.
Acta, 359, 4125 (2006).
[319] Y. Wang, Z. Y. Yang, Q. Wang, Q. K. Cai, K. B. Yu, J. Organomet. Chem., 690,
4557 (2005).
[320] X. Xu, K. C. Zheng, L. J. Lin, H. Li, Y. Gao, L. N. Ji, J. Inorg. Biochem.. 98, 87
(2004).
[321] J. Liu, H. Chao, Y. X. Yaan, H. -J. Yu, L. -N. Ji, Inorg. Chim. Acta, 359, 3807
(2006).
[322] B. Wang, Z. Y. Yang, T. Li, Bioorg. Med Chem., 14, 6012 (2006).
[323] A. M. Thomas, G. C. Mandai, S. K. Tiwary, R. K. Rath, A. R. Chakravarthy, 7.
Chem. Soc, Dalton Trans., 1395 (2000).
![[Bis(2-pyridyl-[kappa]N)amine]chlorido([eta]6 … · 2017. 3. 23. · [Bis(2-pyridyl-jN)amine]chlorido-(g6-hexamethylbenzene)ruthenium(II) hexafluoridophosphate dichloromethane solvate](https://img.pdfslide.us/doc/110x75/60ee35de6a41681269719553/bis2-pyridyl-kappanaminechloridoeta6-2017-3-23-bis2-pyridyl-jnaminechlorido-g6-hexamethylbenzenerutheniumii.jpg)
![methoxy-pyridyl)]-benzimidazole derivatives Supporting ... · Novel bright blue emissions IIB group complexes constructed with various polyhedron-induced 2-[2′-(6-methoxy-pyridyl)]-benzimidazole](https://img.pdfslide.us/doc/110x75/611dc45d3b745e14fc5b42aa/methoxy-pyridyl-benzimidazole-derivatives-supporting-novel-bright-blue-emissions.jpg)
![Plate 1: 1H NMR spectrum of pyridyl[1,5-a]-4](https://img.pdfslide.us/doc/110x75/620a6bc27d3ba434de7c84da/plate-1-1h-nmr-spectrum-of-pyridyl15-a-4-.jpg)
