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ErnstMoritzArndtUniversität Greifswald


Synthesis, characterization,
antitumor and antimicrobial activities
of heterocyclic transition metal complexes



Inauguraldissertation

zur

Erlangung des akademischen Grades

doctor rerum naturalium (Dr. rer. nat.)

an der MathematischNaturwissenschaftlichen Fakultät

der

ErnstMoritzArndtUniversität Greifswald




vorgelegt von

Magdalena Karolina Buczkowska

geboren am 30.03.1982

in Starogard Gdański (Polen)





Greifswald, Mai 2011
Die vorliegende Arbeit entstand in der Zeit von April 2007 bis Dezember 2010 am Ernst
MoritzArndt Universität Greifswald.





































Dekan.........................Prof. Dr. Klaus Fesser

1. Gutachter................Prof. Dr. Patrick Bednarski

2. Gutachter...............Prof. Dr. Franciszek Sączewski

Tag der Promotion.....05.10.2011..........




































Dedicated to my husband Arek
and my Parents
Abstract
__________________________________________________________________________________________

“Synthesis, characterization, antitumor and antimicrobial activities
of heterocyclic transition metal complexes ”

Magdalena Buczkowska, working group Prof. P. J. Bednarski
Department of Pharmaceutical and Medicinal Chemistry, University of Greifswald

Transition metal complexes play a crucial role in antitumor therapy. Complexes of
platinum, ruthenium as well as lanthanum and gallium have been investigated in preclinical as
well as in clinical studies. The best known platinum(II) agents approved worldwide, cisplatin
or carboplatin, are used in nearly 50% of all cancer therapies. Due to their severe toxicity,
their high activity is not always satisfactory and side effects are frequently encountered.
Therefore, ongoing efforts wordwide are aiming to identify novel metal.based substances
with less toxicity and new mechanisms of action.
This work focused on the development of new metal.based drugs that could act against
human cancer cells. It was motivated in part by a previous study with Cu(II) complexes,
which reported new coordination compounds with SOD mimicking and cytotoxic activities.
On the basis of this previous work we chose several commercially available heterocyclic
carboxylic acids to synthesize new metal ion complexes in search of interesting biological
activity.
New as well as previously reported Cu(II), Co(II), Pt(II) and Zn(II) complexes were
synthesized using ligands 1.6. The chelating 2:1 ligand.metal complexes were obtained
generally in water at room temperature in the reaction of metal(II) chloride with
corresponding aromatic nitrogen ligands. The precipitates were washed with diethyl ether and
dried in desiccator.

The synthesized chelating complexes were characterized by the use of usual spectroscopic
methods, elemental analyses and HPLC chromatography and some by X.ray crystallography. Abstract
__________________________________________________________________________________________

Such coordination compounds are easily formed by transition metals with free orbitals d that
can accept the donor electron pairs. The coordination is through the heterocyclic nitrogen and
carboxylate oxygen donor atoms, which was shown by analysis of the characteristic
functional groups in the IR spectra. The d.d transitions and absorption of visible light in
Cu(II) and Co(II) complexes make them highly colored, blue, green or green.blue,
respectively.
The configuration at the coordination center was established in some cases by X.ray
crystallography. Most of the already published structures are trans coordinated. This led to the
assumption that other uncrystallized complexes were also trans coordinated. However, X.ray
data of the Cu(II) complex of 5 showed quite unexpectedly the cis configuration. On the other
hand, the LC/MS experiments with the Pt(II) complex of 5 indicated that this complex exists
in two isomeric forms, i.e., cis and trans at the Pt(II) center.
Through the use of density functional calculations we optimized the structures and
calculated the energies and dipole moments. The differences in energy for all complexes were
about 6. to 15.fold lower when compared to cis and transplatin. The DFT calculations
confirmed that the trans.isomers are more stable than their cis.isomers.
UV.Vis stability studies with most of the synthesized complexes as well as some other
Cu(II) complexes were performed to study the spectral changes over 24 h at pH 7.4, 37 °C in
the absence and presence of glutathione, a tripeptide present at millimolar concentrations in
the cancer cells, as well as ascorbate. Time.dependent changes in the UV.vis spectra were
observed in almost all cases, indicating instability of the complexes in the cells and their
possible decomposition to lose the ligand and release the metal ion. In the case of Cu(II)
complexes, reduction of Cu(II) to Cu(I) may take place.
The synthesized coordination metal(II) complexes were tested for their potential
antiproliferative activities by using the crystal violet staining method in a panel of human
cancer cell lines, LCLC.103H, 5637 and A.427. Out o f all complexes, three Pt(II) complexes
of 2, 5 and 6 showed satisfactory activity and for these complexes the IC values were 50
additionally determined in the RT.4, DAN.G and MCF. 7 cancer cell lines. Interestingly, the
active Pt(II) complexes were the chelating trans complexes, which is quite unexpected, based
on the dramatic difference in activities between cisplatin and its inactive trans.isomer,
transplatin.
All of the complexes were also tested for their potential antimicrobial activities in
comparison to standard antibiotics on such bacterial strains as Staphylococcus aureus,
Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and yeast Candida maltosa. Abstract
__________________________________________________________________________________________

Co(II) complexes have been especially known to act against bacterial strains. The activity of
the Co(II) complexes was indeed the highest of all metal(II) complexes. The ligand 2 (a
nicotinic acid isomer) was also found active. This fact could explain why some antibacterial
activity was found in the MIC assay.
In addition to the complexes synthesized in this work, several novel heterocyclic metal(II)
complexes of copper, ruthenium, platinum, gallium, osmium and lanthanum from other
research groups were screened for their antiproliferative activity, some of which exhibited
very potent activity in the cancer cell lines.
In conclusion, Pt(II) complexes with bis.chelating heterocyclic carboxylate ligands
represent a particularly interesting new class of coordination complexes from the view point
of their structural and biological properties.

1 Table of contents
__________________________________________________________________________________________
Table of contents


Table of contents 1

Abbreviations 6
1. Introduction 7

1.1. Cancer 7
1.1.1. Cancer chemotherapy 7
1.2. Metal ion complexes as anticancer agents 9
1.2.1. Development of a new anticancer agent 11
1.2.2. Classes of metal based pharmaceuticals 12
1.3. Antiinfective metal ion complexes 13
1.4. Biological properties of copper 15
1.4.1. Copper complexes with anti.inflammatory activity 16
1.4.2. Copper complexes with antimicrobial and antifungal activity 17
1.4.3. Copper complexes with antitumor activity 18
1.5. Biological properties of cobalt 22
1.5.1. Cobalt complexes with antimicrobial activity 22
1.5.2. Cobalt complexes with antitumor activity 24
1.6. Biological properties of zinc 26
1.6.1. Zinc complexes with antimicrobial activity 28
1.6.2. Zinc complexes with antitumor activity 29
1.7. Platinum complexes 30
1.7.1. Platinum complexes with antimicrobial activity 30
1.7.2. Platinum complexes with antitumor activity 31
1.8. Ruthenium complexes 38
1.8.1. Ruthenium complexes with antitumor activity 39
1.9. Gallium complexes with antitumor activity 40
1.10. Lanthanum complexes with antitumor activity 42
1.11. Osmium complexes with antitumor activity 42
1.12. Metal coordination chemistry 43
1.12.1. Copper 44
1.12.1.1. Copper(I) complexes 45
1.12.1.2. Copper(II) complexes 46 2 Table of contents
__________________________________________________________________________________________

1.12.1.3. Copper(III) complexes 47
1.12.2. Cobalt 47
1.12.2.1. Cobalt(II) complexes 48
1.12.2.2. Cobalt(III) complexes 48
1.12.2.3. Cobalt(IV) and cobalt(V) complexes 49
1.12.3. Platinum 49
1.12.3.1. Platinum(0) complexes 50
1.12.3.2. Platinum(I) complexes 50
1.12.3.3. Platinum(II) complexes 50
1.12.3.4. Platinum(III) complexes 51
1.12.3.5. Platinum(IV) complexes 51
1.12.4. Zinc 52

2. Aims of the thesis 54

56 3. Results

3.1. Synthesis and structural characterization of copper(II), cobalt(II), zinc(II) 56
and platinum(II) complexes
3.1.1. Metal(II) complexes of 2.pyrazine.carboxylic acid 59
3.1.1.1. Copper(II) complex of 2.pyrazine.carboxyli c acid (1a) 59
3.1.1.2. Hydrated copper(II) complex of 2.pyrazine. carboxylic acid (1b) 60
3.1.1.3. Hydrated cobalt(II) complex of 2.pyrazine. carboxylic acid (1c) 61
3.1.1.4. Platinum(II) complex of 2.pyrazine.carboxy lic acid (1d) 61
3.1.1.5. Zinc(II) complex of 2.pyrazine.carboxylic acid (1e) 62
3.1.2. Metal(II) complexes of 2.picolinic acid 63
3.1.2.1. Hydrated copper(II) complex of 2.picolinic acid (2a) 63
3.1.2.2. Hydrated cobalt(II) complex of 2.picolinic acid (2b) 64
3.1.2.3. Platinum(II) complex of 2.picolinic acid (2c) 65
3.1.2.4. Zinc(II) complex of 2.picolinic acid (2d) 66
3.1.3. Metal(II) complexes of 1H.imidazole.2.carbox ylic acid 66
3.1.3.1. Copper(II) complex of 1H.imidazole.2.carbo xylic acid (3a) 67
3.1.3.2. Cobalt(II) complex of 1H.imidazole.2.carbo xylic acid (3b) 68
3.1.3.3. Platinum(II) complex of 1H.imidazole.2.car boxylic acid (3c) 68
3.1.4. Metal(II) complex of benzimidazole.2.carboxy lic acid 69
3.1.4.1. Cobalt(II) complex of benzimidazole.2.carb oxylic acid (4a) 69 3 Table of contents
__________________________________________________________________________________________
3.1.5. Metal(II) complexes of 1.methylimidazole.2.c arboxylic acid 70
3.1.5.1. Copper(II) complex of 1.methylimidazole.2. carboxylic acid (5a) 70
3.1.5.2. Cobalt(II) complex of 1.methylimidazole.2. carboxylic acid (5b) 71
3.1.5.3. Cobalt(II) complex of 1.methylimidazole (5c) 72
3.1.5.4. Platinum(II) complex of 1H.methylimidazole.2.carboxylic acid (5d) 73
3.1.6. Platinum(II) complex of 2.methyl.pyrazinecar boxylate (6a) 74
3.2. Crystal structures 76
3.2.1. Crystal structure of the complex 5a 77
3.2.2. Crystal structure of the complex 5b 78
3.3. HPLC studies 79
3.3.1. Chromatograms of the ligands and products 79
3.4. LC/MS studies 82
3.4.1. Copper(II) complex (1a) 82
3.4.2. Copper(II) complex (1b) 83
3.4.3. Cobalt(II) complex (1c) 83
3.4.4. Copper(II) complex (5a) 84
3.4.5. Cobalt(II) complex (5b) 85
3.4.6. Platinum(II) complex (5d) 87
3.5. UV.Vis stability studies 88
3.5.1. Metal(II) complexes investigated for stability 89
3.5.2. UV.Vis studies in aqueous buffer 90
3.5.3. UV.Vis studies in the presence of glutathione 91
3.5.4. UV.Vis stability studies with the presence of ascorbic acid 93
3.6. Computational studies of the metal(II) complexes of 1.methylimidazole.2. 95
carboxylate
3.6.1. The theoretical background 95
3.6.2. Determination of the energy 96
3.7. Screening for antiproliferative activity 100
3.7.1. Introduction to the antiproliferative studies 100
3.7.2. Copper(II) complexes 102
3.7.3. Cobalt(II) complexes 106
3.7.4. Zinc(II) complexes 107
3.7.5. Platinum(II) complexes 108
3.7.5.1. Platinum(II) complexes bearing carboxylate ligands 108
3.7.5.2. Platinum(II) phenanthrolines from Prof. Aldrich.Wright’s group 111
3.7.5.3. Platinum(II) complexes from Prof. Keppler’s group 116 4 Table of contents
__________________________________________________________________________________________

3.7.6. Ruthenium(II) complexes 119
3.7.7. Gallium(III) complex 130
3.7.8. Lanthanum(III) complex 134
3.7.9. Osmium(II) complex 137
3.8. The antimicrobial screening 138
3.8.1. The modified agar diffusion method 138
3.8.2. The microbouillon serial dilution method 141

4. Discussion 144

4.1. Synthesis of the metal(II) complexes and their structural confirmation 144
4.2. Stability studies 147
4.3. Computational studies 149
4.4. Antiproliferative activity 150
4.5. Antimicrobial activity 152

5. Conclusions 154

6. Experimental section 155

6.1. General information 155
6.2. Synthesis of the coordination compounds 157
16.2.1. Bis(Pyrazine.2.carboxylato.N ,O).copper (II) (1a) 157
16.2.2. Diaqua.bis(Pyrazine.2.carboxylato.N ,O).copper (II) (1b) 158
16.2.3. Diaqua.bis(Pyrazine.2.carboxylato.N ,O).cobalt (II) (1c) 158
16.2.4. Dichloro.bis(Pyrazine.N ,N).platinum (II) (1d) 158
16.2.5. Bis(Pyrazine.2.carboxylato.N ,O).zinc (II) * 2.5 H O (1e) 158 2
16.2.6. Diaqua.bis(2.picolinate.N ,O).copper (II) (2a) 159
16.2.7. Bis(2.picolinate.N ,O).cobalt (II) * 2.25 H O (2b) 159 2
16.2.8. Bis(2.picolinate.N ,O).platinum * 0.5 H O (II) (2c) 159 2
16.2.9. Diaqua.bis(2.picolinate.N ,O).zinc * 2 H O (II) (2d) 159 2
16.2.10. Bis.(4.imidazolecarboxylato.N ,O).copper (II) (3a) 160
16.2.11. Diaqua.bis.(4.imidazolecarboxylato.N ,O).cobalt (II) (3b) 160
16.2.12. Diaqua.bis.(4.imidazolecarboxylato.N ,O).platinum (II) (3c) 160
16.2.13. Bis(benzimidazole.2.carboxylato.N ,O).cobalt (II) * 4.5 H O (4a) 160 2
6.2.14. Bis.(1.Methylimidazole.2.carboxylato.N´,O). copper (II) (5a) 161

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