design of protein-targeted organometallic complexes as

Design of Protein-Targeted Organometallic

Complexes as Anticancer Agents

A Thesis Submitted to Department of Chemistry,

Quaid-i-Azam University, Islamabad, in part fulfillment of the

requirement for the degree of

Doctor of Philosophy

In

Inorganic/Analytical Chemistry

by

Jahan Zaib Arshad

Department of Chemistry Quaid-i-Azam University

Islamabad, Pakistan (2019)

Dedicated

To

My Parents and My Wife

1

Acknowledgements

First of all, I am grateful to Almighty Allah for every good thing which I have in my

life.

I wish to express fervent sense of thankfulness to my supervisor A/Prof. Dr. Amir

Waseem for providing me the opportunity to work in his research group. His

inspiring guidance, valuable suggestions, good manners and generous support make

me possible to accomplish this task. I am highly privileged to have a nice and friendly

mentor in shape of you.

With deep sense of gratitude and appreciation, I would like to express my sincere

thanks to my foreign supervisor Prof. Dr. Christian Hartinger for giving me

acceptance to complete most of my PhD work in his lab at the University of

Auckland, New Zealand. That one year stay was definitely a fascinating and enriching

experience of my life. Thank you Christian for providing excellent lab facilities, for

your inspiring guidance, advice, support and encouragement throughout the project,

for proofreading and correcting of all my manuscripts. Thank you for giving me a

warm welcome party at your home on Christmas. Thank you for being a great mentor

and I feel proud to be a part of Hartinger group.

I must say big thank to a person who really helped me to complete my PhD. My co-

supervisor Dr. Muhammad Hanif. I highly appreciate your support as supervisor for

throughout my PhD both at during your stay at the COMSATS University of

Islamabad, Abbottabad Campus and at the University of Auckland, New Zealand. I

am highly grateful for introducing me to such an exciting field of medicinal inorganic

chemistry (anticancer compounds development) with complete guidance and support

during these years and also providing me up-to date knowledge in this field. Special

thanks for picking me up from the airport when I first arrived Auckland then showing

me around the beautiful places in the city and also helping me in visa extension

Besides the great support outside the University, I like to thank you for your guidance,

encouragement and the time you spent to share your expert knowledge to discuss

problems of synthetic or analytical nature. Thank you for developing me inside the

proper sense of research. Furthermore I would like to thank you for proofreading and

correcting all my written work, including all papers, presentation and this dissertation.

In short, I found a spectacular mentor and a gentle big brother in shape of you.

2

I am extremely thankful to Prof. Dr. M. Siddique (Chairman, Department of

Chemistry, Quaid-i-Azam University, Islamabad) and Prof. Dr. Amin Badshah

(Dean of Natural Sciences, Quaid-i-Azam University, Islamabad) for providing me

the opportunity to enroll in the university for the completion of my PhD.

I am highly grateful to Sanam Movassaggi for teaching me all my lab skills and also

helping me in NMR measurement training. Thank you for such a nice and cooperative

support during my stay in Auckland.

I am extremely thankful to Dr. Adnan Ashraf for helping me in various aspects both

inside and outside of the lab.

I am very grateful to Mrs. Shahida Perveen for her kind support in the lab.

I am highly indebted to many people for their cooperation in the completion of my

dissertation: Dr. Mario Kubanik for elemental analysis; Sanam Movassaggi for

cancer cell line studies; Jóhannes Reynisson and Ayesha Zafar HDAC inhibition

and molecular modelling studies; Kelvin Tong for DFT calculations, Tanya Groutso

for measuring my single X-ray crystal structures; Dr. Tilo Söhnel for crystal structure

refinement; Tony Chen for ESI-MS measurements; Dr. Michael Schmitz for NMR

training and Radesh Singh and Tasdeeq Mohammed for keeping the labs running

and ordering everything we need to work.

I am highly grateful to all colleagues and members of the Hartinger groups,

especially Betty, Kelvin, Adnan, Shahida, Mathew, Mario, Dianna and Hannah, for

their help and nice cooperation.

I highly acknowledge the support of Higher Education commission (HEC) for

providing funding to me for my IRSIP stay at the University of Auckland, New

Zealand.

I am thankful to my teachers at the Department of Chemistry, Quaid-i-Azam

University, Islamabad for their support and guidance.

I would like to say thank you to all my friends (both in Pakistan and New Zealand) for

their much appreciated support and encouragement.

I would like to express my sincere thanks to my wife Fozia Jahanzaib for her kind

and moral support during my whole PhD and professional career as well. Thank you

for being always there for me.

3

Finally, I would like to say that no acknowledgement would ever adequately express

my gratitude to my whole family for their years of love, care and emotional support.

Special thanks to my parents for their endless supports and love. I can’t pay their

share what they have invested in my character and career build up. So, hats off to

both of you my sweet Mom and Dad. Nothing is more beautiful in the world than my

lovely parents.

Jahan Zaib Arshad

4

List of Publications

1. Arshad, J.; Hanif, M.; Zafar, A.; Movassaghi, S.; Tong, K.; Reynisson, J.;

Kubanik, M.; Waseem, A.; Söhnel, T.; Jamieson, S., Organoruthenium

and‐Osmium Complexes of 2‐Pyridinecarbothioamides Functionalized with a

Sulfonamide Motif: Synthesis, Cytotoxicity and Biomolecule Interaction.

ChemPlusChem 2018, 83, 612-619.

2. Arshad, J.; Hanif, M.; Movassaghi, S.; Kubanik, M.; Waseem, A.; Söhnel, T.;

Jamieson, S. M.; Hartinger, C. G., Anticancer Ru(η6-p-cymene)Complexes of

2-Pyridinecarbothioamides: A Structure–Activity Relationship Study. Journal

of Inorganic Biochemistry 2017, 177, 395-401.

3. Waseem, A.; Arshad, J., A Review of Human Biomonitoring Studies of Trace

Elements in Pakistan. Chemosphere 2016, 163, 153-176.

4. Waseem, A.; Arshad, J.; Iqbal, F.; Sajjad, A.; Mehmood, Z.; Murtaza, G.,

Pollution Status of Pakistan: A Retrospective Review on Heavy Metal

Contamination of Water, Soil, And Vegetables. BioMed research

international 2014, http://dx.doi.org/10.1155/2014/813206.

5

List of Schemes

This PhD thesis is based on following published or unpublished schemes:

1. Anticancer Ru (η6-p-cymene) Complexes of 2-Pyridinecarbothioamides: A

Structure–Activity Relationship Study. (published in Journal of Inorganic

Biochemistry 2017, 177, 395-401)

2. Impact of Metal Ions and Halide Leaving Groups on the Biological Activity of

Organometallic N-(4-fluorophenyl)pyridine-2-carbothioamide Anticancer

Agents. (manuscript in preparation)

3. Organoruthenium and‐Osmium Complexes of 2‐Pyridinecarbothioamides

Functionalized with a Sulfonamide Motif:Synthesis, Cytotoxicity and

Biomolecule Interaction. (published in ChemPlusChem, 2018, 83, 612-619)

4. Targeting Epigenetic Changes: Multitargeted Vorinostat (SAHA)-derived

Metal Complexes with Potent Anticancer and Histone Deacetylase Inhibitory

Activity. (manuscript in preparation)

6

TableofContentsAcknowledgement .................................................................................................................................. 1

List of Publications ................................................................................................................................. 4

List of Schemes ....................................................................................................................................... 5

List of Figures ......................................................................................................................................... 8

List of Tables ........................................................................................................................................ 13

Abbreviations ........................................................................................................................................ 15

Abstract ................................................................................................................................................. 16

CHAPTER 1: INTRODUCTION ...................................................................................................... 19

1.1. DNA targeting agents ................................................................................................................ 20

1.1.1. Platinum-based anticancer drugs ........................................................................................ 20

1.1.1. Mechanism of action of cisplatin and analogous drugs ...................................................... 21

1.2. Non-platinum complexes as anticancer agents .......................................................................... 22

1.3 Protein targeted anticancer agents............................................................................................... 24

1.3.1. Thioredoxin reductase inhibitors ........................................................................................ 24

1.3.2. Transferrin and albumins for transport and/or delivery of Ru drugs in clinical trials ......... 26

1.3.3. Kinase Inhibitors ................................................................................................................. 28

1.3.4. Cathepsin B Inhibitors ........................................................................................................ 29

1.3.5. Histone Protein Targeting ................................................................................................... 30

1.3.6. Plectin Inhibitors ................................................................................................................. 31

1.3.7. Histone deacetylases inhibitors (HDACis) ......................................................................... 32

1.3.8. Carbonic anhydrase inhibitors ............................................................................................ 46

CHAPTER 2: EXPERIMENTAL ..................................................................................................... 51

2.1. Chemicals ................................................................................................................................... 52

2.2. Instrumentation .......................................................................................................................... 53

2.3. Bioanalytical Assays .................................................................................................................. 53

2.3.1 Sulforhodamine B Cytotoxicity Assay ................................................................................ 53

2.3.2. Stability of complexes 9 and 10 in aqueous solution .......................................................... 53

2.3.3. Stability of complexes 24–27 in aqueous solution and reactivity with amino acids ........... 53

2.3.5. Calculated logarithmic octanol/water partition coefficient (clogP) .................................... 54

2.3.6. Molecular Modelling of complexes 24 and 27 against CA II ............................................. 54

2.3.7. Stability of complexes 38–41 in aqueous solution and reactivity with amino acids .......... 54

2.3.8. HDAC inhibition of compounds 29, 31, 38–41 .................................................................. 54

2.3.9. Dynamic simulation of ligand 31 and its complexes 38–41 against HDAC6 and HDAC8 ......................................................................................................................................... 55

2.4. General procedures for the synthesis of PCAs ligands .............................................................. 57

2.5. General procedures for the syntheses of metal complexes of PCAs .......................................... 58

2.6. Synthesis of PCA based succinic/suberanilic carboxylic acid ligands ...................................... 78

2.7. Synthesis of PCA based succinic/suberic hydroxamic acid ligands .......................................... 79

7

2.8. Synthesis of metal complexes of PCA based carboxylic acid and hydroxamic acid derivatives ......................................................................................................................................... 81

CHAPTER 3: RESULTS & DISCUSSION ...................................................................................... 92

Scheme 3.1. Anticancer Ru(η6-p-cymene)complexes of 2-pyridinecarbothioamides: A structure–activity relationship study ............................................................................................ 94

3.1.1. Results and discussion ........................................................................................................ 94

3.1.2. Stability in aqueous solution ............................................................................................. 102

3.1.3. In vitro antiproliferative activity and lipophilicity ............................................................ 103

3.1.4. Quantitative estimate of drug-likeness of ligands ............................................................. 106

Scheme 3.2. Impact of metal ions and leaving halido groups on the biological activity of organometallic N-(4-fluorophenyl)pyridine-2-carbothioamide anticancer agents ............. 109

3.2.1. Introduction ....................................................................................................................... 109

3.2.1. Results and Discussion ..................................................................................................... 109

3.2.2. In vitro antiproliferative activity ....................................................................................... 115

Scheme 3.3. Organoruthenium and -osmium complexes of 2-pyridinecarbothioamides functionalized with a sulfonamide motif: Synthesis, cytotoxicity and biomolecule interaction ...................................................................................................................................... 117


3.3.2. Stability in aqueous solution and reactivity toward amino acids ...................................... 121

3.3.3. In vitro anticancer activity ................................................................................................ 122

3.3.4. Molecular Modelling ........................................................................................................ 123

Scheme 3.4. Targeting epigenetic changes: multitargeted vorinostat (SAHA)-derived metal complexes with potent anticancer and histone deacetylase inhibitory activity ............. 126


3.4.2. Stability in aqueous solution and reactivity with amino acids .......................................... 132

3.4.3. In vitro anticancer activity ............................................................................................... 134

3.4.4. HDAC inhibition ............................................................................................................... 135

3.4.5. Molecular dynamic simulations ........................................................................................ 137

Conclusion .......................................................................................................................................... 141

References ........................................................................................................................................... 143

Appendix A ......................................................................................................................................... 154

Representative NMR and ESI-mass spectra of scheme 1 ............................................................... 154




8

List of Figures

Figure 1. Chemical structure of platinum drugs approved by FDA (1-3) and drugs used

locally in Japan (4), Korea (5) and China (6). ...................................................................................... 21

Figure 2. Cisplatin forming adducts with DNA [Reprinted with permission from ref.12b.

Copyright 2005 Nature Reviews Drug Discovery]. .............................................................................. 22

Figure 3. The chemical structures of tris(8-quinolinolato)gallium(III) (7), (KP46) gallium

maltolate (8), Butotitane (9) and titanocene dichloride (10). ................................................................ 23

Figure 4. The chemical structures of Auranofin (11) and gold phosphole complex GoPI (12). .......... 25

Figure 5. The chemical structures of carbo-RAPTA-C (13) and Ru(II) arene complexes of

benzimidazol-2-ylidene (14a–14d). ...................................................................................................... 26

Figure 6. The chemical structures of KP1019 (15) and NKP-1339 (16). ............................................ 28

Figure 7. Chemical structures of (17) and (18). .................................................................................. 29

Figure 8. Chemical structures of RAPTA-T (19) and (20) as Cathepsin B inhibitors. ........................ 30

Figure 9. Chemical structure of RAPTA-C (21) and RAED-C (22). ................................................... 30

Figure 10. Chemical structure of RuII/OsII(cymene) complexes of PCAs (23A–28A) and

(23B–28B). ........................................................................................................................................... 31

Figure 11. Histone deacetylase inhibitors (HDACis) (29–31) approved by FDA. .............................. 33

Figure 12. cis-Platinum(II) complex conjugated with SAHA (32) and Belinostat drug (33). ............. 34

Figure 13. Chemical structures of (34), (35) and (36). ........................................................................ 36

Figure 14. Chemical structure of (37). ................................................................................................. 36

Figure 15. Chemical Structure of (38). ................................................................................................ 38

Figure 16. Chemical structures of JAHA (39) and its analogues (40–43). .......................................... 39

Figure 17. Chemical structures of triazole based JAHA analogues (44a-44b), (45), (46a-46b). ........ 40

Figure 18. Chemical structure of pojamide (47). ................................................................................. 40

Figure 19. Chemical structure of (48–50). ........................................................................................... 41



Figure 22. Chemical structures of (53–55). ......................................................................................... 43

Figure 23. Chemical structures of (56–59). ......................................................................................... 44

Figure 24. Chemical structures of Fc-SAHA (60) and p-Fc-SAHA (61). ........................................... 45

Figure 25. Chemical structures of (62) and (63). ................................................................................. 46

Figure 26. The chemical structures of Indisulam (64) and SLC-0111 (65). ....................................... 47

Figure 27. Chemical structure of (66) and (67). ................................................................................... 48


Figure 29. Chemical Structure of metallocene (69). ............................................................................ 49

9

Figure 30. Chemical structures of (70a–70d). ..................................................................................... 50

Figure 31. The molecular structures of 3 (top) and 6 (bottom) drawn at 50% probability

level. ...................................................................................................................................................... 96

Figure 32. Comparison of the 1H NMR spectra in MeOD-d4 recorded for ligand 3 and after

complexation with [Ru(cym)Cl2]2. The protons of the PCA ligand were shifted after

coordination to Ru and the most significant change was observed for H1 after complexation

as indicated by a shift from 8.67 ppm in 3 to 9.66 ppm in 11. .............................................................. 99

Figure 33. The molecular structures and atom numbering for metal complexes 12 and 13 at

50% probability level. Solvent molecules and counterions were omitted for clarity. ........................ 100

Figure 34. Molecular structure of 13 with the π interaction between the pyridine rings of two

molecules indicated. Co-crystallized solvents and counterions were omitted for clarity. .................. 101

Figure 35.1H NMR spectra of 9 in D2O recorded after 0.5, 2 and 24 h, showing the

chlorido/aqua ligand exchange reaction to occur very rapidly. The dashed grey lines indicate

the positions of the protons of the chlorido complex 9. ...................................................................... 102

Figure 36. ESI-mass spectrum of 9 after 7 days of incubation in water (bottom) or 60 mM

HCl (top). The mass spectrum in HCl shows the partial exchange of the thiocarbamide sulfur

atom of 9 with O (9O). ......................................................................................................................... 103

Figure 37. Molecular structures for metal complexes 17, 18, and 20 with 50% thermal

ellipsoid probability level. Hydrogen atoms, solvents and counter ions are omitted for clarity. ........ 113

Figure 38. Molecular structure of N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 23

drawn at 50% probability level. .......................................................................................................... 118

Figure 39. Molecular structure of 27neutral drawn at 50% probability level. ...................................... 119

Figure 40. 1H NMR spectroscopic study of the reaction between 24 and His in D2O,

monitored for 72 h. The peaks of His are highlighted in grey boxes. ................................................. 122

Figure 41. The modelled configuration of 24E2 in the catalytic site of carbonic anhydrase II

(PDB ID 3PYK). a) Hydrogen bonds are depicted as green dotted lines between the metal

complex and the amino acids Thr199, and Thr200. Lipophilic interactions are represented as

purple dotted lines with Val121, Leu60 and Leu198. b) The enantiomer 24E2 is shown in the

binding pocket with the protein surface rendered. Red depicts a negative partial charge on the

surface, blue depicts a positive partial charge and grey shows neutral/lipophilic areas. .................... 124

Figure 42. Molecular structure of 28 drawn at 50% probability level. The intermolecular

hydrogen bonding are shown between the carboxylic acid and amide groups. .................................. 127

Figure 43. Molecular structure of one of the enantiomers of 33 drawn at 50% probability

level. The counter ion and residual MeOH were removed for clarity. ............................................... 129

Figure 44. Molecular structure of 33 drawn at 50% probability level. Two enantiomeric

molecules of 33 are connected by two chloride counter ions through H-bonds with the amide

protons of two molecules. ................................................................................................................... 129

10

Figure 45. 1H NMR spectra of 40 in D2O (bottom), after addition of AgNO3 (2 eq.), and in the

presence of NaCl (104 mM) and HCl (60 mM). ................................................................................. 132

Figure 46. 1H NMR spectra of 39 in D2O (bottom) recorded 0.5, 2 and 6 h after dissolution. ......... 133

Figure 47. 1H NMR spectra of 41 in D2O (bottom), and 24 hours after the addition of Cys (1

eq., middle; 2 eq.,top). ........................................................................................................................ 133

Figure 48. The docked configuration of 39E2 in the binding site of HDAC8 (PDB ID 1t69).

(a) Hydrogen bonds are depicted as green dotted lines between ligand and the amino acids

Asp101and His180. The Zn interaction is shown with solid lines. (b) 39E2 is shown in the

binding pocket with the protein surface rendered. Blue depicts a positive partial charge on the

surface, red negative and grey neutral/lipophilic. ............................................................................... 138


The complex is shown in the binding pocket with the protein surface rendered. Blue depicts a

positive partial charge on the surface, red negative and grey neutral/lipophilic. ................................ 139

Figure 50. 1HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 11 in MeOD-d4. ................................................................... 154

Figure 51. 1HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-


Figure 52. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-


Figure 53. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-


Figure 54. ESI-MS of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 11 in CH2Cl2. ....................................................................... 156

Figure 55. ESI-MS of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-


Figure 56. 1H NMR Spectrum of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 17 in MeOD-d4. ................................................................... 157

Figure 57. 1H NMR Spectrum of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-

fluorophenyl) pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CDCl3. ................................ 157

Figure 58. 13C{H}NMR Spectrum of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 17 in MeOD-d4. ................................................................... 158

Figure 59. 13C{H}NMR Spectrum of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-

fluorophenyl) pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CDCl3. ................................ 158

Figure 60. ESI-MS of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 17 in CH2Cl2. ....................................................................... 159

Figure 61. ESI-MS of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)

pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CH2Cl2. ....................................................... 159

11

Figure 62. 1HNMR Spectrum of [chlorido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 24 in MeOD-d4. .................................................................. 160

Figure 63. 1HNMR Spectrum of [bromido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 25 in MeOD-d4. .................................................................. 160

Figure 64. 1HNMR Spectrum of [iodo(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]iodide 26 in MeOD-d4. ...................................................................... 161

Figure 65. 1HNMR Spectrum of [chloro(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)osmium(II)]chloride 27 in MeOD-d4. ...................................................................... 161

Figure 66. 1H NMR spectrum of 24 and 27 in DMSO-d6 recorded after 15 min of dissolution.

The spectra showed peaks assigned to the NH protons as well as minor products, presumably

due to DMSO/Cl ligand exchange reactions. ...................................................................................... 162

Figure 67. 13C{H}HNMR Spectrum of [chlorido(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]chloride 24 in MeOD-d4. ..................... 162

Figure 68. 13C{H}HNMR Spectrum of [bromido(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]bromide 25 in MeOD-d4. ..................... 163

Figure 69. 13C{H}HNMR Spectrum of [iodo(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-

2-carbothioamide)ruthenium(II)]iodide 26 in MeOD-d4. ................................................................... 163

Figure 70. 13C{H}HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)osmium(II)]chloride 27 in MeOD-d4. ......................... 164

Figure 71. ESI-MS of [chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 24 in CH3OH. ....................................................................... 164



Figure 73. Comparison of 1HNMR spectrum of ligand 8-oxo-8-((4-(pyridine-2-

carbothioamido)phenyl)amino)octanoic acid 29 and its complex [chlorido(η6-p-cymene)(8-

oxo-8-((4-(pyridine-2-carbothioamido)phenyl)amino) octanoic acid)ruthenium(II)]chloride 34

in MeOD-d4. ........................................................................................................................................ 165

Figure 74. 1HNMR spectrum of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)

octanediamide 31 in DMSO-d6. ......................................................................................................... 166

Figure 75. 1HNMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-

carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in MeOD-d4............................... 166


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4. ................................. 167

Figure 77. 1HNMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-

(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride 40 in

MeOD-d4. ............................................................................................................................................ 167

12


(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride 41 in

MeOD-d4. ............................................................................................................................................ 168

Figure 79. 13C{H}NMR spectrum of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)

octanediamide 31 in DMSO-d6. ......................................................................................................... 168

Figure 80. 13C{H}NMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-

carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in MeOD-d4............................... 169


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4. .................................. 169

Figure 82. 13C{H}NMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-

hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]

chloride 40 in MeOD-d4. ..................................................................................................................... 170


hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]

chloride 41 in MeOD-d4. ..................................................................................................................... 170

Figure 84. ESI-MS of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide

31 in CH3OH. ...................................................................................................................................... 171

Figure 85. ESI-MS of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-

carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in CH3OH. ................................. 171


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in CH2Cl2. ..................................... 172

Figure 87. ESI-MS of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-

(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride 40 in

CH3COCH3. ........................................................................................................................................ 172


(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride 41 in

CH3COCH3. ........................................................................................................................................ 173

13

List of Tables

Table 1. X-ray diffraction measurement parameters for single crystals of ligands 3 and 6. ................ 96

Table 2. Selected bond lengths (Å) and angles (°) for ligands 3 and 6 and complexes 12 and

13. ......................................................................................................................................................... 97

Table 3. X-ray diffraction measurement parameters for single crystals of 12 and 13. ...................... 101

Table 4. In vitro anticancer activity (mean IC50 values ± standard deviations) of PCA ligands

1–8 and their respective Ru(cym) complexes 9–16 in human colorectal (HCT116), non-small

cell lung (NCI-H460) cervical (SiHa) carcinoma cell lines and colon carcinoma (SW480)

cells (exposure time 72 h). .................................................................................................................. 105

Table 5. clogP values for ligands 1–8 calculated with ChemDraw 12.0,

Molinspiration(www.molinspiration.com) and ALOGPS 2.1.113 ....................................................... 106

Table 6. The calculated molecular properties used for the calculation of the quantitative

estimate of druglikeness (QED). MW (molecular weight), clogP for the ligands using the

average logP of seven different programs via the ALOGPS 2.1 applet at

http://www.vcclab.org. HBA (hydrogen bond acceptor), HBD (hydrogen bond donor), PSA

(polar surface area) calculated viawww.molinspiration.com or ChemBio3D 12.0 software,

ROTB (rotatable bonds), AROM (number of aromatic rings) and Alerts (number of structural

alerts). Calculation of the weighted QED for maximum information content (QEDwmo) was

carried out according to ref.114 ............................................................................................................ 107

Table 7. X-ray diffraction parameters for the measurement of single crystals of 17, 18, and

20. ....................................................................................................................................................... 114

Table 8. Selected Bond Lengths (Å) and Angles (°) for 17, 18 and 20. where M = Ru, Os and

X = Cl, Br, I. ....................................................................................................................................... 114

Table 9. IC50 (μM) for ligand 1 and their respective RuII, OsII, RhIII and IrIII complexes (9, 17–

22) in human colorectal (HCT116), non-small cell lung (NCI-H460) cervical (SiHa)

carcinoma cell lines and colon carcinoma (SW480) cell lines. .......................................................... 115

Table 10. X-ray diffraction measurement parameters for 23 and 27neutral. ......................................... 120

Table 11. Conductivity measurements of ligand 23 and complexes 24–27 in acetonitrile

(0.1 mM). ............................................................................................................................................ 121

Table 12. In vitro anticancer activity (IC50 values) of ligand 23, its respective Ru/Os(cym)

complexes 24, 25, 26 and 27 and related compounds F-SN 1 and plecstatin-1 in human

colorectal (HCT116), non-small cell lung (NCI-H460) cervical (SiHa) and colon carcinoma

(SW480) cells(exposure time 72 h). The clogP values for the PCAs 23 and F-SN 1 are also

given. ................................................................................................................................................... 123

14

Table 13. The H bonds and lipophilic interactions of the modelled compounds with amino

acid residues of carbonic anhydrase II. ............................................................................................... 124

Table 14. X-ray diffraction parameters for the measurement of single crystals of ligand 28

and its Os(cym) complex 33. .............................................................................................................. 130

Table 15. Comparison of selected bond lengths (Å), angles (°), and torsion angles (°) of 28

and its Os(cym) complex 33. .............................................................................................................. 131

Table 16. In vitro cytotoxic activity (mean IC50 values ± standard deviations) of PCA-

carboxylic acid and their organometallic complexes (28, 29, 32, 33, 34 and 35) as well as

PCA-hydroxamic acids and their organometallic complexes (30, 31, 36, 37, and 38–41) in the

human cancer cell lines HCT116 (colon), NCI-H460 (non-small cell lung), SiHa (cervix), and

SW480 (colon) given in μM as determined by the SRB assay (exposure time 72h). ......................... 135

Table 17. Single dose mean values for the residual activity of HDAC8 after treatment with

29–31, and 38–41 at 10 μM. The numbers in brackets are the two recorded data points (n = 2). ...... 136

Table 18. Inhibitory activity (IC50 in nM) of PCA-hydroxamic acid 31 and its organometallic

complexes 38–41 against HDAC1, HDAC6, and HDAC8 in comparison to SAHA. ........................ 137

Table 19. H bonds and lipophilic contacts formed between HDAC8 and 31 and the individual

enantiomers of its metal complexes. ................................................................................................... 139


enantiomers of its metal complexes. ................................................................................................... 140

15

Abbreviations

DNA 2’-deoxyribonucleic acid mM millimolar

DMSO-d6 deuterated dimethyl sulfoxide μM micromolar

MeOD-d4 deuterated methanol nM nanomolar

CDCl3 deuterated chloroform mL milliliter

D2O deuterated water mg milligram

CA carbonic anhydrase δ chemical shift

J coupling constant (NMR) eq. equivalent

e.g. exempli gratia (for example) ppm parts per million

brs broad singlet (NMR) K Kelvin

d doublet (NMR) Å angstrom

t triplet h hour

m multiplet (NMR) °C degree Celsius

td triplet of doublet min minute

ESI electrospray ionization TEA triethylamine

Hz hertz nm nanometer

2D two dimensional DMSO dimethyl sulfoxide

3D three dimensional THF tetrahydrofuran

m/z mass by charge ratio DCM dichloromethane

HDAC histone deacetylases MeOH methanol

IC50 half maximal inhibitory SAHA suberoylanilide

concentration hydroxamic acid

et al. et alii (and others) MS mass spectrometry

etc. et cetera (and other things) MHz mega hertz

PCA pyridne-2-carbothioamide p-cymene para-cymene

µS/cm micro siemens per centimeter η6 eta-6-coordination

pH pondus hydrogenii NMR nuclear magnetic power of hydrogen

16

Abstract

DNA is considered as the ultimate target of platinum based anticancer drugs which

are widely used in clinics but the toxicity and resistance induced by these compounds

have halted their success. In recent past, proteins or enzymes have been explored as

alternate targets for metal-based anticancer agents. These enzymes or proteins are

involved in metabolic pathways associated with cancer development. These include

transferrin, albumin, kinase, cathepsin B, thioredoxin reductase, plectin, carbonic

anhydrase and histone deacetylase etc. Many compound classes of metal

complexes have been investigated against such targets. The ruthenium and osmium

complexes of pyridine-2-carbothioamides (PCAs) stabilized by η6-arene ring were

introduced as orally administrable anticancer agents with potential to bind with the

histone proteins to interrupt the chromatin activity (Chemical Science., 2013, 4,

1837–1846). Recently, in vivo examination of these compounds revealed selective

binding to plectin and they termed as plecstatin (Angewandte Chemie International

Edition., 2017, 56, 8267-8271). In this doctoral thesis, PCA ligands were

functionalized with groups which can bind to specific enzymes or proteins such

as carbonic anhydrase and histone deacetylase. The new PCA ligands were then

converted to their respective organometallic compounds of Ru(II), Os(II),

Rh(III) and Ir(III). All novel PCAs and their corresponding complexes were

evaluated for their cytotoxic potential against different cancer cell. The

organometallic compounds were studied for their hydrolytic stability as well as

their interactions with biomolecules such as amino acids and proteins by using

a range of biophysical methods.

For structure activity relationships study, a series of N-phenyl substituted pyridine-2-

carbothiamides (PCAs) were obtained by systematically varying the substituents at

the phenyl ring. The PCAs were then converted to their corresponding RuII(η6-p-

cymene) complexes. In preliminary examination, these metal based compounds were

studied for their acidic and hydrolytic stability. In cytotoxic assay, the lipophilic

PCAs 1–4 showed cytotoxicity in the low micromolar range and 6 was the most

potent compound of the series with an IC50 value of 1.1 μM against HCT116 colon

cancer cells. These observations were correlated with calculated octanol/water

partition coefficient (clogP) data and quantitative estimated druglikeness. A similar

17

trend as for the PCAs was found in their Ru complexes, where the complexes with

more lipophilic ligands proved to be more cytotoxic in all tested cell lines. In general,

the PCAs and their organoruthenium derivatives demonstrated excellent drug-likeness

and cytotoxicity with IC50 values in the low micromolar range, making them

interesting candidates for further development as orally active anticancer agents.

In order to investigate the impact of metal centres on anticancer activity, Rh and Ir

analogues of the most promising and orally active compound plecstatin (9) were

prepared. Within the same group, the lighter metal fragments ruthenium and rhodium

complexes showed increased cytotoxicity as compared to their respective heavier

congener i.e. osmium and iridium. However, changing the halido leaving group

resulted in slight decrease in activity with exception of ruthenium-bromido 17 and

osmium-iodido 20 complexes in H460 cancer cell line.

To further explore the carbonic anhydrase as another potential target for these

compounds, PCA was functionalized with sulfonamide group and convert into RuII

and OsII(η6-p-cymene) complexes. The presence of the sulfonamide motif in many

organic drugs and metal complexes endowed these agents with interesting biological

properties and may result in the latter case in multitargeting agents. The compounds

were characterized with standard methods and the in vitro anticancer activity data was

compared with studies on the hydrolytic stability of the complexes and their reactivity

to small biomolecules. A molecular modelling study against carbonic anhydrase II

revealed plausible binding modes of the complexes in the catalytic pocket.

In a multitargeting approach, by incorporating several bioactive components – a metal

centre, a pyridinecarbothioamide and a hydroxamic acid – in a novel pharmacophore,

highly cytotoxic functionalized PCAs and their organometallic compounds were

obtained. The PCA ligand 31 bearing the vorinostat (SAHA) pharmacophore and their

respective organoruthenium, osmium, rhodium and iridium complexes 38–41

displayed potent cytotoxicity but these results showed slight correlation towards

HDACi studies. In HDAC inhibition assay against HDAC1, HDAC6 and HDAC8, the

PCA-SAHA derivative 31 and its organometallic compounds 38–41 showed

inhibitory activity in nanomolar range and some derivatives were more potent

inhibitors than the approved drug SAHA. The HDACi mechanism further confirmed

by dynamic simulation where compound 31 and its enantiomeric complexes 39 and

18

40 chelated with Zn2+ ion of HDAC8 and HDAC6 and formed several interactions

within their binding pocket.

Overall, this doctoral thesis comprises of seven new ligands (6, 7, 23, 28–31) and

twenty six novel organometallic complexes(10–18, 20–22, 24–27, 32–41), while

single crystals of four ligands (3, 6, 23, 28) and seven complexes (12, 13, 17, 18, 20,

27neutral, 33) are reported.

19

CHAPTER 1: INTRODUCTION

20

INTRODUCTION

1.1. DNA targeting agents

1.1.1. Platinum-based anticancer drugs

The serendipitous discovery of cisplatin was a landmark towards development of

metal-based drugs. It prompted the investigation of metal-based compounds with

biological activity. In combination, the extensive research in the field of molecular

biology, more specifically the information at genetic and cellular level has not only

helped in revealing the perceptive mode of action of existing inorganic drugs but also

established the roots towards rational development of metal based chemotherapeutics.

Cisplatin, 1 (Figure 1) was previously known as Peyrone’s chloride named after

Michele Peyrone, who synthesized it in 1844.1 In 1960ies, the cytotoxic properties of

cisplatin was revealed by Barnett Rosenberg when he observed the filamentous

growth of Escherichia coli bacteria under the influence of electric impulses that lead

to inhibition of their cell division. He observed the filamentous growth of bacteria in

electric field and their cell division was ceased. Rosenberg and colleagues identified

cisplatin as a key compound responsible for the antiproliferative effect.2 Clinical trials

were initiated in 1971, and after circumventing number of obstacles, cisplatin was

finally approved in 1978 by the Food and Drug Administration (FDA) as anticancer

drug, with exemplary success in treating testicular and ovarian cancer.3 Nowadays,

the approach of combined chemotherapy also broadened the spectrum of cisplatin

drug towards treatment of various malignant cancers.4

To overcome the multidrug resistance and side effects offered by cisplatin in cancer

treatment triggered intensive research to design new platinum-based

chemotherapeutic agent. From thousand of synthesized platinum complexes in last 40

years, only carboplatin 2 and oxaliplatin 3 have received worldwide clinical approval

so far (Figure 1).5 Carboplatin approved by FDA in 1989 equipped with features of

slow ligand substitution kinetics and with minimal side effects showed better

compatibility as compared to cisplatin in cancer chemotherapy.6 However, as the

active form of carboplatin form the adduct with the DNA similar to cisplatin and

therefore the problem of chemoresistance remain the same.7 In line, the third

generation platinum drug oxaliplatin came into clinics in 2002 and found very

effective in treatment of cisplatin-resistant tumors.8 Oxaliplatin in combination with

21

5-fluoruracil and folic acid is currently employed for the treatment of colorectal9 and

lung cancer.10 In addition, three other platinum-based drugs like nedaplatin 4,

lobaplatin 5, and heptaplatin 6 are also in clinical practice for the treatment of several

tumors in Japan, Korea and China, respectively (Figure 1).3c, 11

Figure 1. Chemical structure of platinum drugs approved by FDA (1-3) and drugs used

locally in Japan (4), Korea (5) and China (6).

1.1.1. Mechanism of action of cisplatin and analogous drugs

After the intravenous administration of cisplatin into blood stream the execution of its

anticancer property within the body occurs in multi-step process that includes cellular

uptake, activation by hydrolysis, formation of DNA adduct and cell death induced by

apoptotic mechanism.12 The cellular uptake of cisplatin primarily contributed by

passive diffusion or mediated by active transport via membrane proteins such as the

copper transporter Ctr1.3b, 13 Once inside the cell, cisplatin is hydrolyzed that

accounted the formation of single or double positively charged aqua species

[Pt(NH3)2Cl(H2O)]+ and [Pt(NH3)2(H2O)2]2+.3b After activation these positively

charged aquated species move into nucleus and bind with nitrogen atoms of DNA

bases i.e. with guanine and adenine and forming main intrastrand crosslinks of type

1,2-d (GpG) (around 65%), 1,2-d (ApG) (around 25%) (Figure 2).14 These cisplatin

adducts induce a conformational change in the DNA, resulting in the inhibition of

DNA replication and transcription which in turn lead towards the programmed cell

death in cancer tissues.4b, 15

22

Figure 2. Cisplatin forming adducts with DNA [Reprinted with permission from ref.12b.

Copyright 2005 Nature Reviews Drug Discovery].

1.2. Non-platinum complexes as anticancer agents

Platinum drugs are very successful in clinics, however, the non-specific mechanism of

action of these drugs to target the DNA via covalent interactions also caused damage

to normal cells resulted in producing the severe side effects, such as ototoxicity,

neurotoxicity, nausea, nephrotoxicity and vomiting. Another problem associated with

platinum based drugs, are acquired or intrinsic resistance in cancer chemotherapy.16

This has stimulated the development of the other types of novel metal

chemotherapeutics that act via alternate mode of action contributing that higher

selectivity towards tumor cells is essential for the improvement of cancer

chemotherapy. Due to these facts, research on non-platinum based drugs increased in

the past few years. Among non-platinum metal drug design metals such as gold,

gallium, titanium, rhodium, iridium, iron, ruthenium and osmium are under extensive

investigation due to their promising potential in cancer chemotherapeutics. These

non-platinum metals offers the advantage of having different chemical behavior

including different oxidation states, redox potential, coordination geometry, additional

binding sites preferably to biomolecules according to HSAB principle etc and also

offers a chance to modulate the rate of hydrolysis or ligand exchange kinetics.17

Therefore, all these features reflected that non-platinum compounds may have

different mode of actions, bioavailability and biological activity.9 The arsenic based

compound TrisenoxR (As2O3)18 approved in 2000 by FDA is used in clinic for the

treatment of acute promyelocytic leukemia. It’s involved in multiple intracellular

transduction pathways and these actions include stimulation of apoptosis, inhibition of

23

multiplication of cancer cells, and inhibition of growth of new blood vessels. Further

clinical examination of arsenic trioxide also revealed the great potential of this

compound in the treatment of malignant diseases.19

Gallium(III) nitrate17 the first of its generation has used as chemotherapeutic agent

against cancer associated hypocalcaemia20 (Figure 3). This has stimulated the clinical

development of gallium(III) maltolate 7 (Figure 4) has been found effective in

inhibiting the hepatocellular carcinoma cell growth and also induces apoptosis in

lymphoma cell lines.21 In 2003, a related compound tris(8-quinolinolato) gallium(III)

(KP46) 8 (Figure 3) has entered in clinical trials and showed promising preclinical

efficacy in primary explanted melanoma22 and renal cancer cells.23

Butotitane (cis-diethoxybis(1-phenylbutane-1,3-dionato-κ2O1,O2)titanium(IV) 9

(Figure 3), was the first titanium based anti-cancer agent in clinical trials but later its

clinical development was halted due to formulation issues. Afterwards, another

titanium base compound titanocene dichloride 10 (Figure 3) reached in clinical trials

but it was abandoned in phase II as no advantages over other treatment regimens were

observed.24

Figure 3. The chemical structures of tris(8-quinolinolato)gallium(III) (7), (KP46) gallium

maltolate (8), Butotitane (9) and titanocene dichloride (10).

24

1.3 Protein targeted anticancer agents

In cancer chemotherapeutics, the DNA binding mechanism caused damage to healthy

cells along with severe side effects including resistance to these DNA binding drugs.25

In recent past, the research in the field of genomic and proteomics identified the

potential involvement of various proteins or enzymes in cancer cells survival or its

progression, therefore identification of protein targets has inspired the rational design

of protein-targeted anticancer drugs.12b The proteins such as transferrin, albumin,

kinase, cathepsin B, plectin, carbonic anhydrase and histone deacetylase etc are

proved as widely studied proteomics targets in cancer chemotherapy.

As metal complexes offer the advantages of peculiar features that include facile

construction of 3D structure that can tightly fit in to the enzyme’s active sites

increasing both selectivity and opportunity to bind with various protein’s residues.

Further, in metal complexes a labile metal-ligand bond e.g. halides also offers the

opportunity to strongly bind with nucleophiles of amino acid side chains upon

hydrolysis. All these features made metal complexes as potential cancer

chemotherapeutics towards proteins or enzymes inhibition.20, 26

1.3.1. Thioredoxin reductase inhibitors

Thioredoxin reductase TrxR belongs to the family of glutathione reductase catalyzes

the reduction of thioredoxin and in combination with thioredoxin and NADPH this

system triggers the reduction of disulfide reductase and involve in several metabolic

pathways (antioxidative network, nucleotide synthesis). In TrxR the selenocysteine

being part of its active site responsible for the catalytic mode of action of the

enzyme.27 The overexpression of TrxR has been found in carcinogenesis, cancer

progression and resistant to chemotherapy that made it an important therapeutic target

in cancer chemotherapy.28

The gold(I) complexes containing the electrophilic gold center can easily bind to the

nucleophilic sulfur and selenium containing residues through covalent interaction.

The selenoprotein inhibition by gold(1+);(2S,3R,4S,5R,6R)-3,4,5-triacetyloxy-6-

(acetyloxymethyl)oxane-2-thiolate;triethylphosphane(Auranofin), 11 (Figure 4)

indicates its direct involvement in TrxR inhibition along with perturbation of its

biochemistry that is responsible for protein expression.29 In a crystallographic adduct

experiment another gold phosphole complex {1-phenyl-2,5-di(2-

25

pyridyl)phosphole}AuCl (GoPI) 12 (Figure 4) formed covalent interaction with

cysteine residue of active site of glutathione reductase by losing its phosphole ligand

(Figure 4). This “undressing” of gold complex suggested general mechanism of action

of gold agents with cysteine and selenocysteine containing enzymes.30

Figure 4. The chemical structures of Auranofin (11) and gold phosphole complex GoPI (12).

Arsenic trioxide As2O3 has been found as an effective chemotherapeutic agent

towards acute promyelocytic leukemia (APL).31 Although, the mechanism of action

of this metallodrug was poorly understood but in 2007 Lu et al., reported the

importance of As2O3 as thioredoxin reductase inhibitors as it’s potentially block the

active site of the selenoenzyme thioredoxin reductase.32

Ruthenium complexes are famous for their protein binding potential can inhibit the

TrxR activity due to “soft” character of Ru metal centre. Two of the famous

ruthenium compounds NAMI-A imidazolium trans-

imidazoledimethylsulfoxidetetrachloro-ruthenate(III)and KP1019 (indazolium trans-

[tetrachloridobis(1H-indazole)ruthenate(III)]) compared to gold complexes were less

potent in inhibiting the TrxR1 and ineffective against TrxR2.33 Among them, the most

efficacious ruthenium derivative1,1-cyclobutanedicarboxylateruthenium-arene 1,3,5-

triaza-7-phosphatricyclo[3.3.1.1]-decane(carbo-RAPTA-C) 13 (Figure 5) inhibited

50% of TrxR activity at very low submicromolar concentration.34 In another study,

the interaction of Ru(II) arene complexes of benzimidazol-2-ylidene 14a–14b (Figure

5) with thiols and selenol induced the inhibition of enzymes such as TrxR and

cathepsin B. These compounds showed more selectivity for the inhibition of

selenoenzyme TrxR compared with IC50values 50-times lowered than calculated for

cysteine rich cathepsin B. Furthermore, strong antiproliferative effect of these

complexes accounted for efficient cellular accumulation and also affected the tumor

cell’s metabolic pathways (e.g. cell morphology, cell respiration and glycolysis).35

26

Figure 5. The chemical structures of carbo-RAPTA-C (13) and Ru(II) arene complexes of

benzimidazol-2-ylidene (14a–14d).

1.3.2. Transferrin and albumins for transport and/or delivery of Ru drugs in

clinical trials

Ruthenium-based drugs being non-toxic in nature and capable to overthrown the

multidrug resistance induced by platinum drugs that have made them as possible

alternate to platinum-based drugs.36 Ruthenium-based drugs such as indazolium trans-

[tetrachloridobis(1H-indazole)ruthenate(III)](KP1019) 15 (Figure 6)37 and the sodium

salt analogue of KP1019 sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)])

(NKP-1339) 16 (Figure 6)38 and now recently in clinical trials they named it IT-139

equipped with high tumor inhibiting potential because of their strong affinity towards

biological molecules such as human serum albumin and transferrin39 and in addition

it’s activation in to more reactive specie in the reductive tumor milieu as compared to

healthy tissues.40 It has been suggested that both human serum albumin and

transferrin proteins played the role of transport and delivery system for ruthenium-

based compounds which is also found important for inducing their chemotherapeutic

activity.41 The interest in iron protein transferrin has been developed because tumor

cells requires more iron than healthy cells and therefore transferrin receptors (CD7)

got overexpressed and that caused the metallodrug to bound to transferrin in cancer

cells in a selective accumulative manner.42 Beside this in tumor tissues blood vessels

leakage and ineffective lymphatic drainage lead to enhanced accumulation in

inflamed and cancer tissues , an effect also known as “enhanced permeability and

retention effect” (EPR).41, 43

In a comparative study analysis, it is revealed that KP1019 preferentially 15-times

more binds to albumin in the blood stream as compared to transferrin. 37b, 44

27

The clear picture of mechanism of action of KP1019/NKP-1339 is not completely

resolved yet but redox chemistry suggested that it undergoes activation-by-reduction

mechanism. Under physiological conditions, there are two oxidation states of

ruthenium i.e. Ru(III) and Ru(II) but ruthenium compounds in oxidation state (II)

easily undergoes the ligand substitution reaction (replacement of chloro ligands by

aqua ligands) than Ru(III) and hence readily forms the aquated specie that has more

reactivity towards biological molecules in the cells. Hence, the mechanism of

activation of Ru(III) complexes in reductive milieu of solid tumors clearly proved

their prodrug nature, while keeping the healthy cells away from their toxic effects.45

Besides the DNA intercalation and proteins binding mechanisms, the activated Ru(II)

specie also accompanied by different intracellular features such as generation of

reactive oxygen species (ROS)46 that can stimulate apoptosis via mitochondrial

pathway47 and upregulation of the p38 mitogen-activated protein kinase (MAPK)

stress response pathway.48 Unlike platinum drugs, NKP-1339 does not seem likely to

target DNA but they are accompanied by multiple features of targeting cancer cells

that hold promising for the activity of this compound.38

Furthermore, the IT-139 exhibited a distinct protein-binding pattern involve the

blockade of important cellular protection mechanism. As in a recent study, IT-139

inhibited the stress induced upregulation of glucose-regulated protein of 70 kDa

(GRP78).49 The endoplasmic reticulum (ER) chaperone GRP78 upregulated in

various malignant tumors and its inhibition renders tumor cells vulnerable to

endogenous metabolic and radical stress, hypoxia and the effects of cytotoxic

compounds.50 In addition to GRP78, other important chaperones including major heat

shock proteins potentially inhibited by IT-139 exposure of cancer cells in vitro.51

28

Figure 6. The chemical structures of KP1019 (15) and NKP-1339 (16).

1.3.3. Kinase Inhibitors

Kinases are considered as important therapeutic target for novel anticancer agents.

They potentially catalyze the shift of phosphate groups from ATP to biomolecules and

involved in important cellular functions including cell cycle regulation.26a In this

regard, Meggers’ ruthenium arene complexes26a, 52, such as the racemic carbonyl(η5-

2,4-cyclopentadien-1-yl)(9-hydroxypyrido[2,3-a]pyrrolo[3,4-c]carbazole-5,7(1H,6H)-

dionato- ĸN1,ĸN12)ruthenium(DW 1/2) 18 (Figure 7) potently inhibited series of cyclic

dependent kinases. In line, DW1/2 found strong inhibitor of glycogen synthase kinase

3 (GSK-3) and especially of the proto-oncogene serine/threonine-protein kinase Pim-

1, where the S enantiomer showed remarkable inhibition capacity in picomolar

range.53

Analogous to staurosporine 17 (an organic kinase inhibitor) (Figure 7), several

ruthenium complexes albeit with cyclopentadienyl ligand rather than arene rings have

also been developed as selective kinase inhibitors. Unlike staurosporine, ruthenium

(II)-cyclopentadienyl analogues exhibited high selectivity for specific kinases.53-54

This high selectivity may be attributed to 3D shape of the pseudo octahedral

ruthenium centre that affords unique structures that match with different binding

pockets of different kinases. The crystal adduct formation of these complexes with

kinases also confirmed their binding to ATP binding sites while the metal atom only

defined the geometry of molecule and did not involve in any binding interaction to the

residues in the active site. 52b, 53, 54b

29

Figure 7. Chemical structures of (17) and (18).

1.3.4. Cathepsin B Inhibitors

In past few years cathepsin B (Cat B) has turned out to be plausible target in cancer

chemotherapy55 as its overexpression has found to be associated with development

and progression several tumors.56 In a study conducted by Casini and coworkers the

docking experiment of RAPTA compounds particularly the (methyl-)(1,3,5-triaza-7-

phosphatricyclo[3.3.1.1]-decane)ruthenium(II)]dichloride (RAPTA-T) 19 (Figure 8)

with cathepsin B indicated that ruthenium (II) ion binds to cysteine (Cys-29) in the

catalytic pocket of the enzyme and the alkyl and aryl portion found favorable

interaction with hydrophobic sites of protein imparting further stability. Further, the

compound 19 also potently inhibited cat B with IC50 value in low micromolar range

i.e. 1.5 µM. Hence, Cat B inhibitory potency of these two RAPTA compounds along

with formation of RAPTA-cat B adduct clearly reflects the potential of these

compounds as potent Cat B inhibitors.57 In another study, sugar-derived phosphite

based ruthenium complexes also bearing various arene co-ligands were evaluated for

their anticancer potential58 and the most lipophilic compound [dichlorido(η6-

biphenyl)(3,5,6-bicyclophosphite-1,2-O-cyclohexylidene-α-

Dglucofuranose)ruthenium(II)] 20 (Figure 8) exhibited excellent cytotoxic potential in

low micromolar range.. The compound 20 showed reactivity to the DNA model

nucleobase 9-ethylguanine and did not show any DNA binding interaction. In further

examination, the compound 20 potently inhibited the cat B similar to that of RAPTA-

T that clearly showed the possible mode of action of this compound.

30

Figure 8. Chemical structures of RAPTA-T (19) and (20) as Cathepsin B inhibitors.

1.3.5. Histone Protein Targeting

In search of other possible mechanism of action of ruthenium based compounds, one

of the emerging non-cytotoxic antimetastatic compound [(ƞ6-p-cymene)Ru(1,3,5-

triaza-7-phosphaadamantane)Cl2] RAPTA-C 21 bound in central proteinaceous part

of histone proteins59 while the relatively cytotoxic anti-primary tumor compound[(η6-

p-cymene)Ru(ethylenediamine)Cl]PF6(RAED-C) 22 (Figure 9) is bound in outer ring

of double helix of DNA of chromatin.60 . DNA foot printing analysis with both naked

DNA and nucleosome core particles (NCPs) showed that RAED-C formed the adduct

with GG nucleotide sites of the DNA, whereas weak or no interaction is observed in

case of RAPTA-C. The crystallographic studies revealed that RAPTA-C formed the

adduct at three sites of NCPs in bivalent mode of coordination with histidine, lysine

and glutamate residues of the histone proteins. On the other hand, the RAED-C

formed the adduct at two different histone sites i.e. the one at the histone glutamate

and second involve the adducts with DNA. The computational investigation of above

mentioned two complexes with NCPs indicated that actually bulky

phosphaadamantane ligand of RAPTA-C that prefers to selectively bind with histone

sites of NCPs rather than the DNA and hence interfering with the chromatin activity

reflecting another possible mode of action of ruthenium based compound.

Figure 9. Chemical structure of RAPTA-C (21) and RAED-C (22).

31

Similarly, metal complexes of 2-pyridinecarbothioamides (PCAs) 23A, 25A, 23B,

and 25B were crystallized out with NCPs (Figure 10).61 This crystallographic

measurement revealed that RuII(cymene) complexes 23A and 25A form adducts at

two sites H2B His-79 and H2B´ His-79, while the OsII(cymene)complexes 23B and

25B form adducts at three sites H2B His-106, H2B His-79 and H2B´ His-79 of

histone proteins and hence influences the dynamics of chromatin structure. The

complexes 25A and 25B formed adducts with His-106 in a similar fashion to

RAPTA-C. In contrast to RAPTA-C these RuII and OsII complexes of PCAs 23A–28A

and 23B–28B exhibited remarkable acidic stability with strong cytotoxic potential

against human colon carcinoma SW480, human ovarian cancer CH1 cell lines while

moderate cytotoxicity against human lung cancer A549 cell line. Moreover,

complexes containing the N-phenyl, N-4-fluorophenyl and N-mesityl ligands also

displayed the strongest cytotoxicity towards CH1 and SW480 cells with IC50 values

lower than 8 μM, but also moderate cytotoxic effect in A549 cells. Furthermore, the

quantitative estimates of drug-likeness (QEDwmo) of Ru(cymene) complexes 23A

(0.54), 25A (0.53) and 26A (0.56) was found higher than clinically approved

anticancer drugs erlotinib (0.41), imatinib (0.41), tamoxifen (0.43), dasatinib (0.46)

and sorafenib (0.51) and all these features make them an interesting candidate for

development of orally active anticancer agents.

Figure 10. Chemical structure of RuII/OsII(cymene) complexes of PCAs (23A–28A) and

(23B–28B).

1.3.6. Plectin Inhibitors

In other proteomics targets, targeting plectin has been found to a promising anticancer

strategy. As plectin targeting cause the non-mitotic tubule (MT) network to undergo

G0/G1 arrest and hence affect the motility of cancer cells. In a recent study, by Meier

32

and coworkers reported the in vivo activity of these Ru-PCAs complexes after oral

administration showed the potential of these compounds to selectively bind to plectin

and RuII(cymene) complex of N-4-fluorophenyl pyridine-2-carbothioamide 25A

(Figure 10) named as Plecstatin-I successfully targeted the plectin and exhibited

excellent anticancer activity against primary tumors in CT-26 colon cancer cells and

more so in the invasive B16 melanoma tumor model after oral administration. Hence,

this mentioned study clearly reflects the strong protein binding nature of these

compounds along with tendency to develop as orally active metallodrug for the

treatment of solid tumors .62

1.3.7. Histone deacetylases inhibitors (HDACis)

Histone deacetylases (HDACs) are one of the most important therapeutic targets that

have been thoroughly studied for anticancer activity. Histone acetylases (HATs)

together with HDACs acetylate and deacetylate lysine residues on histones,

respectively. Histone acetylases (HATs) responsible for relaxed chromatin structure

that is associated with the up-regulation of gene transcription. In contrast, HDACs

associated with condensed chromatin structure that lead to transcriptional suppression

of genes.63 The overexpression of HDACs, found in cancer cells lead to histone

hypoacetylation which silence the tumor suppression genes and cancer cell survival is

promoted.64 Therefore, HDACs contributed towards development of histone

deacetylase inhibitors (HDACi) by promoting acetylation of histones and induce

inhibition of cancer cell growth and ultimate cell death under the reprogrammed

processes.64b, 65 In this perspective, hydroxamic acid has been proved as effective

HDACis and uptill now three hydroxamic acid derivatives namely Vorinostat

(SAHA) 29, Belinostat (PXD-101) 30 and Panobinostat (LBH-589) 31 (Figure 11)

approved by FDA for the treatment of different cancers.66

33

Figure 11. Histone deacetylase inhibitors (HDACis) (29–31) approved by FDA.

In crystal adduct formation of clinically approved HDAC inhibitors with human

HDACs, the hydroxamic acid moiety coordinates with the active-site zinc ion. Several

HDAC inhibitors have thus been designed with a pharmacological model consist of a

zinc binding group (ZBG), a chain linker and surface recognition cap group which

interacts with amino acid residues on the enzyme surface.

1.3.7.1 Platinum–HDACis Conjugates

The strategy of combining the HDACis with platinum drugs rendered the nuclear

DNA with cytotoxic DNA targeting agents, has been utilized to improve enhance

efficiency of platinum drugs in cancer chemotherapeutic. Marmion and co-workers

combined cis-[Pt(NH3)2(H2O)2](NO3)2] with the malonate derivatives of SAHA and

belinostat and synthesized cis-[PtII(NH3)2(malSAHAH-2)] 32 and cis-[Pt(NH3)2(mal-

p-Bel-2H)] 33 (Figure 12) in an attempt to capitalize the potential synergistic effect of

combining platinum complexes with HDACis.67 Unfortunately, these Pt–HDACis

conjugates upon reaching the nucleus did not undergo hydrolysis and failure of

desired mechanism of intracellular aquation of PtII complexes to discharge the

HDACi resulted in reduced potency of these compounds.

34

Figure 12. cis-Platinum(II) complex conjugated with SAHA (32) and Belinostat drug (33).

The sluggish kinetics of Pt(II)–HDACi conjugates towards intracellular aquation,

prompted the use of octahedral Pt(IV) complexes to achieve the desired chemical

properties. The square planar Pt(II) complexes containing two axial ligands afforded

the synthesis of Pt(IV) complexes. The Pt(IV) complexes being kinetically inert

doesn’t make any undesired interaction with nucleophile prior to reaching the tumor

cells and hence devoid of other problems associated with cisplatin and its analogues.

The intracellular reduction of Pt(IV) complexes followed by simultaneous release of

original cytotoxic Pt(II) drugs as well as two axial ligands may proved as the possible

mechanism of action of these compounds.68

Satraplatin, an analogue of cisplatin that contains two acetate ligands in the axial

positions, has entered Phase III clinical trials for hormone refractory prostate cancer

(HRPC).69 Similarly, Valproic acid (VA), being famous for use as antiepileptic and

anticonvulsant drug, has recently been acknowledged as one of the short-chain fatty

acid class of HDAC inhibitor.70 Like other HDACis, VA triggered its antiproliferative

effect through cell cycle arrest, cell apoptosis, metastasis, angiogenesis,

differentiation, and senescence.71 Shen and co-workers coupled Valproic acid (VA)

with cis,cis,trans-diaminedichlorodihydroxy-platinum(IV) [Pt(NH3)2Cl2(OH)2] 34 to

synthesize a satraplatin-like Pt(IV)–Valproic acid complex

[Pt(NH3)2Cl2(COOCH(CH2CH2CH3)2)] 35 prodrug (Figure 13). The conjugate 35

first treated with reducing agent ascorbic acid that resulted in reduction into

platinum(II) complex accompanied by liberation of axial VA and hence exhibited the

HDAC inhibitory activity similar to VA.72 Further, the complex 35 exhibited

remarkable potency against four cancer cell lines i.e. human lung carcinoma A549,

human breast cancer BCap37, human ovarian carcinoma SKOV-3 and human

35

hepatocellular carcinoma HepG2 cell lines with IC50 value low micromolar range i.e.

0.14–0.20 μM. In a comparative study analysis, the potency of 35 was higher than 34,

VA or a mixture of 34 and VA, indicating that the conjugation of Pt(IV) complex with

VA accounted for enhanced cytotoxicity. In cellular distribution studies increased

binding capacity of 34 to cell membrane was observed due to its increased

lipophilicity that accounted for its availability in cytosol. In the nucleus, increase of

histone acetylation by VA and relax of chromatin structure allow binding of Pt(II)

complex i.e. cisplatin to DNA. Hence, this synergistic effect of Pt(II) complex and

VA accounted for high cytotoxicity of 35. In vivo examination also revealed that 35

significantly inhibited the tumor growth in an A549 tumor xenografts model of mice

with minimal side effects as compared to 34. Besides all this, this study lacks in direct

comparison between the cytotoxicity of 35 and mixture of 35 reduction products i.e.

cisplatin and VA, so actual extent of synergism within the molecule remained unclear.

Further, there was no conclusive evidence of 35 reduction in intracellular

environment. As a fact, 35 showed less HDAC inhibitory activity as compared to

ascorbic acid pretreated-35 showing that reduction process may represents a rate

limiting step in the activation of this compound in the cells.

In a similar study, biological activity of Pt(IV) conjugate 35 has been compared with

that of its isomer cis,cis,trans-diamminedichloridobis(n-octanoato)platinum(IV) 36

(Figure 14). Both complexes 35 and 36 exhibited remarkable cytotoxicity in micro- or

submicromolar range against various human cancer cell lines and most prominent

effect was observed on cells derived from malignant pleural mesothelioma. Osella and

co-workers concluded that this excellent anticancer was only due to cisplatin moiety

released by the intracellular Pt(IV) to Pt(II) reduction and the highly lipophilic nature

of Pt(IV) complexes contributed towards pronounced efficacy as compared to

cisplatin. Further, the absence of synergism between Valproic acid and cisplatin was

also revealed because of the too low concentration (~µM levels) of Valproic acid in

the cells released from ctc-[Pt(NH3)2(VA)2Cl2]complex to inhibit the histone

deacetylase, since the IC50 value of Valproic acid was in the mM range. Moreover,

Pt(IV) conjugate complex 36 despite deprived of any HDAC inhibitory activity

contributed towards more cytotoxicity than 35. 73

36

Figure 13. Chemical structures of (34), (35) and (36).

Kasparkova and co-workers conjugated platinum(IV)-diazido with suberoyl-bis-

hydroxamic acid (SubH) as axial ligands to synthesize a photoactivatable complex to

target genomic DNA and HDACs.74 The complex cis,trans-[Pt(N3)2(Sub)2(tBu2bpy)]

(where tBu2bpy = 4,4′-di-tert-butyl-2,2′-bipyridine) 37 (Figure 14) when irradiated

with ultraviolet or visible light resulted in simultaneous release of cytotoxic Pt(II)

specie and HDAC inhibitor i.e. SubH and displayed 6–11 times more potency against

cisplatin-resistant A2780cisR and cisplatin-sensitive A2780 cell lines than cisplatin.

This remarkable activity of 37 was attributed due to its inhibit HDAC inhibitory

activity via SubH, which increases histone acetylation levels and also makes

chromatin DNA more venerable to damage induced by Pt(II) moiety. Besides this

dual functionality, the compound 37 also effectively block the RNA polymerization

and formed interstrand cross links with the DNA to block transcription and replication

for favorable antitumor effects.

Figure 14. Chemical structure of (37).

37

To fill out the gaps left by Shen and Osella and their respective co-workers and to

reveal the mechanism of action of Pt(IV)–HDACis conjugates, Pt(IV) derivatives of

cisplatin, oxaliplatin and trans-[Pt(n-butylamine)(piperidino-piperidine)Cl2]+ were

synthesized by Gibson his co-workers and their biological activities were compared

with two different HDAC inhibitors valproate (VPA) and 4-phenylbutyrate (PhB).75

The Pt(IV) compound of cisplatin, containing two axial Phenylbutyrate ligand, ctc-

[Pt(NH3)2(PhB)2Cl2] 38 (Figure15) proved as an excellent cytotoxic agent with

potency 100 times more than cisplatin. The compound 38 also revealed significant

potency more than the other of Pt(IV) compounds of cisplatin with either two

hydroxido, two acetato or two valproato ligands. Data also suggested the pronounced

potency of Pt(IV) derivatives of cisplatin containing the two axial HDAC inhibitors

i.e. phenylbutyrate or valproate ligands than their oxaliplatin analogs.

Further examination of Pt(IV) derivative of bis-PhB 38 revealed enhanced cellular

accumulation than its bis-PhB counterpart. In mechanistical studies, DNA platination

confirmed for 38 signifying the importance of axial HDAC inhibitors facilitates the

Pt(II) moiety towards DNA intercalation. Moreover, the complex 38 also blocked 60–

70% HDAC activity inside the neoplastic cells and hence proved the effect of

synergism between Pt(II) specie and PhB. With remarkable potency compound 38

displayed nuclear DNA fragmentations, apoptosome complex formation and also

induce induces activation of caspase (3 and 9) that are typical apoptotic

characteristics. Although the revealed mechanism is HDAC inhibition that can lead to

increased DNA platination but 4-phenylbutyrate or valproate once inside the cell can

also affect many cellular processes. Therefore, Gibson demonstrated that the

increased cytotoxicity cannot be attributed to one particular cellular event. Hence,

these “dual-targeted” metal complexes may prompt as “multi-targeted” pro-drugs that

once inside the cells can provoke different cellular events that can kill the cancer

cells.

38

Figure 15. Chemical Structure of (38).

1.3.7.2. Ferrocene-capped HDACis

In one study, Spencer and his coworkers modified clinically used HDAC inhibitor

suberoylanilide hydroxamic acid (SAHA) by replacement of the terminal phenyl ring

with ferrocene moiety and has been synthesized a ferrocene-capped HDAC inhibitor

named as Jay Amin hydroxamic acid (JAHA) 39 (Figure 16) because in molecular

docking examination it binds in a similar fashion to SAHA 29. In docking

experiment with HDAC8, JAHA formed classical interaction between the

hydroxamate moiety and the catalytic zinc ion and the ferrocenyl group situated at the

entrance area of the pocket that is surrounded by amino acid residues such as Tyr100,

Phe152, and Tyr306.

In this study, JAHA and its analogues were also tested against class I and II HDACs.

JAHA displayed HDAC inhibitory profile similar to SAHA with IC50 vale ranges

from 0.0008 µM to 1.36 µM. Compound 40 (Figure 16) exhibited potential similar to

39 against HDAC 1, 2 and 3, but it is 10 fold less potent towards HDAC6 and four

times more potent towards HDAC8. Compound 42 (Figure 16) containing the original

SAHA derivative and ferrocene moiety displayed excellent inhibitory potential

towards HDACs 1 and 2 and notably, against the HDAC6 with IC50value 90 pm,

whereas 41 (Figure 16) showed the lowest IC50 value toward HDAC8 with a IC50 = 2

nM. On the other hand, complex 43 (Figure 17) with a shorter alkyl chain length

exhibited poor HDAC inhibition. None of the complexes 39–43 showed any

significant inhibition of class IIa HDACs (4, 5, 7 and 9). However the low cytotoxic

potential of these compounds against human breast cancer cells MCF-7 was attributed

to their lower cellular permeability as a result of the ferrocene group. In further

examination, the compounds 41 and 42 also promoted chromatin acetylation and

acetylation of α-tubulin with same potency as that of SAHA drug. This study reflected

39

that modification of the aryl “cap” of SAHA accounted for HDAC inhibitory potential

almost similar to SAHA drug.

Figure 16. Chemical structures of JAHA (39) and its analogues (40–43).

In another attempt, a small library of triazole based JAHA analogues (Figure 17) were

synthesized through click chemistry by Spencer and his coworkers. In molecular

docking studies, the compound 44b (Figure 17) in which the triazole moiety attached

with ferrocene cap showed excellent binding potential with zinc moiety of HDAC8.

However, the compound 46a (Figure 17) in which triazole moiety adjacent to

hydroxamic acid group leads to steric clash with HDAC8. Similarly, in HDAC

inhibition assay the complex 44b showed excellent HDAC inhibition potential

comparable to SAHA drug but weak potential exhibited by its shorter chain length

derivative 44a (Figure 17), again highlighting the importance of chain length for

HDAC inhibition. Following previous HDAC binding trend, the compound 45 (Figure

17) and 46a in which triazole is directly attached with hydroxamic acid group were

devoid of any HDAC inhibition activity. However, the complex 46b (Figure 18)

displayed HDAC inhibitory towards HDACs 1–3 and submicromolar potency towards

HDAC8. The lead compound of this study, 44b also engaged in α-tubulin acetylation

(HDAC6 substrate) and lysine acetylation (in chromatin) similar to SAHA and also

induced the cell cycle arrest.

40

Figure 17. Chemical structures of triazole based JAHA analogues (44a-44b), (45), (46a-46b).

In further extension to JAHAs chemistry, Spencer and his co-workers synthesized a

ferrocene containing o-aminoanilide HDAC inhibitor named as pojamide, 4776

displayed both greater selectivity and significant inhibition potency against HDAC3

with IC50 value of 0.09 µM. In docking experiment against HDAC3, pojamide formed

characteristics benzamide-zinc interaction along with other plausible interactions with

amino acid residues. On further examination, 47 inhibited 90% of invasion in

HCT116 colorectal cancer cells leading to a conclusion that HDAC3 inhibition is

effective in blocking cellular invasion. On treating HCT116 cells with pojamide,

sodium nitroprusside and glutathione its cytotoxicity is remarkably enhanced due to

its facile conversion into ferrocenium salt (FeIII-pojamide) that revealed the dual mode

of action of pojamide with advantageous compatibility as compared to similar potent

HDACis.76

Figure 18. Chemical structure of pojamide (47).

1.3.7.3. Rhenium–HDACis Conjugates

In recent approach, Alberto and coworkers incorporated cyclopentadienyl rhenium

tricarbonyl CpReCO3 in to SAHA backbone to synthesize HDAC inhibitors 48–50

41

(Figure 19). The in vitro examination revealed that complexes 48–50 were slightly

less active than SAHA suggesting that incorporation of CpReCO3 moiety might

hindered the favorable enzyme-inhibitor interaction. Further examination also

revealed the low cellular uptake of complexes 48–50 as compared to SAHA.

Moreover, changing the position of amide linker on Cp ring did not produce any

appreciable effect on cytotoxic potential.

Figure 19. Chemical structure of (48–50).

Recently, Mao and co-workers synthesized a Re(I)CO3–HDAC inhibitor conjugate 51

(Figure 20) containing a SAHA derivative and also bearing 4,7-diphenyl-1,-10-

phenanthroline N^N ligand to generate a phosphorescent HDAC inhibitor. In

cytotoxic assay the complex 51 was found to 2.5 fold more potent than SAHA in

HeLa cells and exhibited the HDAC inhibitory potential comparable to SAHA along

with significant acetylation of histone H3 in a dose dependent manner. Moreover,

different mechanistic studies revealed that 51 can induce caspase-independent

paraptosis through mitochondria-related events including mitochondrial membrane

permeabilization and reactive oxygen species (ROS) generation.


42

1.3.7.4. Gold Complex as HDACi

The anticancer potential of gold(III) complexes have been restricted due to their low

solubility under physiological condition.64a, 77 With unprecedented solubility, Yang,

Che and co-workers synthesized a novel gold(III) porphyrin analogue [5-

hydroxyphenyl-10,15,20-triphenylpor-phyrinato gold(III) chloride)] 52 (Figure 21)

with cytotoxic potential 100–3,000 time higher than that of cisplatin against human

breast cancer cell line MDA-MB-231.78. In vivo examination of 52 revealed the

suppression of mammary MDA-MB-231 tumor growth in nude mice. These affects

are attributed to inactivation of Wnt/β-catenin signaling along with inhibition of all

Class I HDACs (HDAC1, HDAC2, HDAC3 and HDAC8). Further in molecular

modeling study the favorable interaction of complex 52 with binding pocket of

HDAC8 was observed with excellent binding energy of −9.67 kcal/mol. Hence, this

data all together suggested that compound 52 may develop as promising anticancer

HDAC inhibitor.


1.3.7.5. Fluorescent Ruthenium/Iridium Complexes Conjugated with HDACis

Fluorescent-HDAC inhibitors can be used as an efficient tool for analyzing the HDAC

activities also combine with their therapeutic capabilities or considered as novel

theranostic agents having both diagnostic and therapeutic potential. Taking into

account to this concept Mao and coworkers reported fluorescent ruthenium(II)

polypyridyl complexes containing N1-hydroxy-N8-(1,10-phenanthrolin-5-

yl)octanediamide, a SAHA derivative, to synthesize RuII–HDACi hybrid complexes

53–55 (Figure 22) with dual optical and inhibitory activities.79 The HDAC inhibitory

effect revealed that complexes 53–55 exhibited strong to moderate inhibitory activity

43

with IC50 values ranges from 6.66 µM to 85.52 µM. In molecular docking studies the

complex 55 bind to the active site zinc ion of HDAC8 through hydroxamic acid

moiety in a similar fashion to that of SAHA while RuII–polypyridyl groups are well

accommodated in the shallow pocket of HDAC8 that is surrounded by hydrophobic

residues Ile34, Phe152, and Leu308. Furthermore the complex 55 increased the

acetylation of histone H3 in HeLa cells. In line, the most lipophilic complex 55

successfully penetrated into HeLa cells and being localized in cytoplasmic region that

minimized its chances to bind to DNA. In vitro examination of complexes 53–55

revealed that the complex 55 exhibited a higher cytotoxic potency than those of the

widely used clinical chemotherapeutic agents i.e. cisplatin and SAHA. Further,

mechanistic studies revealed that complex 55 can induce apoptosis via mitochondrial

dysfunction and reactive oxygen species (ROS) generation. The mitochondrial

membrane potential MMP and reactive oxygen species ROS generated by complex 55

was far more than those of SAHA, that may also accounted for its higher

antiproliferative activity. So, this study clearly showed the potential of fluorescent

RuII–HDACi conjugates to develop as promising anticancer agents with dual

characteristics of imaging and HDAC inhibition.

Figure 22. Chemical structures of (53–55).

Photodynamic therapy (PDT) have emerged as alternative tool in cancer

chemotherapy due to its strong therapeutic efficacy and minimal side effects as

compared to radio- and chemotherapy.80 In PDT, photosensitizer mostly localized in

tumor specified area and upon irradiating these substance causes oxidative damage to

tissues due to development of ROS. More recently, cyclometalated Ir(III) complexes

are reported to work as efficient photosensitizer for PDT.81

44

In this study, Mao and coworkers conjugated N1-hydroxy-N8-(1,10-phenanthrolin-5-

yl)octanediamide, a phenanthroline modified SAHA derivative ligand , to

cyclometalated Ir(III)complexes to synthesize Ir(III)–HDACi complexes 56–59

(Figure 23) to study synergistic inhibition effects on cancer cells due to their HDAC

inhibitory potency and therapeutic imaging activity.82 The photophysical properties

of these respective complexes revealed that complexes 56–59 effectively taken up by

HeLa cells and mostly retained in the cytoplasm. The compounds 56–59 were also

screened against various human cancer cell lines. Under dark condition, respective

complexes exhibited moderate cytotoxicity but after irradiated at 325 nm and 425 nm

wavelengths a marked increase in cytotoxic potential was observed (IC50 = 3.1 – 21.9

µM) and also displayed very low phototoxicity against human normal liver cells LO2.

The complexes 56–59 showed the potent HDAC inhibitory effect and treatment of

HeLa cells with 56 increased the acetylation of histone H3. Moreover, irradiated cells

displayed higher histone H3 acetylation levels than in the dark. However different

mechanistic studies showed that complex 56 induced the apoptotic cell death through

inhibition of HDACs, increase of Caspase 3/7 pathway, ROS productions and

mitochondrial dysfunction. Upon exposure to UV light radiation, all those previously

describe biological effects of complex 56 are significantly enhanced. So, this study

supported the fact the methodology of combining phosphorescent Ir(III) complexes

with protein targeted drug design may prove as effective strategy for the development

of multidimensional metallodrug.

Figure 23. Chemical structures of (56–59).

In order to prevent the side effects of HDAC inhibitors and for targeted drug delivery

approach, Gasser and coworkers synthesized a photoactivatable organometallic

45

HDAC inhibitor (p-Fc-SAHA) 61 (Figure 24) by photocaging ferrocene based SAHA

(Fc-SAHA) 60 (Figure 24) with a photolabile protecting group, 1-(bromomethyl)-4,5-

dimethoxy-2-nitrobenzene.63c The organometallic complex (Fc-SAHA) 60 was

successfully released from photoactivatable complex 61 upon irradiating the longer

UV wavelength (350 nm). HDAC inhibitory profile has suggested that p-Fc-SAHA 61

was 30 to 600 times less active than Fc-SAHA against HDAC1, HDAC2 and

HDAC6. As expected, this suggested that upon irradiating Fc-SAHA 60 maintained

its inhibitory activities. So, this study clearly demonstrates the possibility of

developing an effective light-controlled organometallic HDAC inhibitor.

Figure 24. Chemical structures of Fc-SAHA (60) and p-Fc-SAHA (61).

1.3.7.6. Ruthenium/Rhodium Piano Stool Complexes Conjugated with HDACis

RuII and RhIII piano stool complexes have emerged as potential anticancer

metallodrugs. 61, 83 More recently, Walton and coworkers synthesize RuII and RhIII

piano stool complexes 62 and 63 (Figure 25) that can act as a histone deacetylase

inhibitors (HDACi)84. The RuII and RhIII piano stool complexes were coupled with

previously reported N1-hydroxy-N8-(1,10-phenanthrolin-5-yl)octanediamide, a

phenanthroline substituted SAHA derived ligand. The compounds 62 and 63 showed

effective micromolar antiproliferative activity against non-small cell lung carcinoma

cells (H460). However, the cytotoxicity of RhIII complex 63 (IC50 = 4.1µM) was five

folds higher than RuII complex 61 (IC50 = 21µM) and also comparable to clinically

approved inhibitor SAHA (IC50 =1.4µM). However, these two complexes 62 and 63

showed comparable HDAC inhibition activity that was also somewhat closer to

SAHA drug suggesting their potential as HDAC inhibitors. Moreover, RuII and RhIII

piano stool complexes 62 and 63 didn’t show any sign of DNA intercalation or

covalent binding unfolding that their anticancer activity is due to HDAC inhibition.

46

Figure 25. Chemical structures of (62) and (63).

1.3.8. Carbonic anhydrase inhibitors

Carbonic anhydrases the most widely studied zinc containing metalloenzyme

catalyses the reversible hydration of carbon dioxide to bicarbonate and a proton and

also involved in various physiological processes. In past few years carbonic

anhydrases has got attention due to overexpression of CA isozymes IX and XII in

cancer cells of many hypoxic tumors where they provide a pH-regulating system that

exploited by cancer cells for their survival and progression. Hence, targeting

specifically the tumor associated CA IX and XII has found to be promising strategy in

developing anticancer drugs with minimum side effects.85

To inhibit the biochemical features of tumor associated CA IX and XII that assist in

cell survival of hypoxic tumor, the sulfonamides(R-SO2NH2) proved as excellent

inhibitors of carbonic anhydrase (CAs). Sulfonamides constitute an important class of

pharmacological agent which form adducts with Zn2+ ion of active site of CAs

enzyme by nitrogen atom of the sulfamoyl moiety and disrupt the catalytic process.

The remaining R group or scaffold of drug molecule involves in various interactions

with protein residues that further stabilize the enzyme-inhibitors adduct.86 This was

demonstrated by X-ray crystallographic analysis of the adduct formation between

various CAs and many representatives of sulfonamide-based inhibitors.86c, 87 In line,

two of the sulfonamide derivatives (N-(3-chloro-7-indolyl)-1, 4-

benzenedisulfonamide (Indisulam) 64 (Figure 26) and (4-(4-fluorophenylureido)-

benzenesulfonamide (SLC-0111) 65 (Figure 26) are in clinical phase II and I,

respectively for the treatment of solid metastatic tumor overexpressing CA IX and

XII. 88

47

Figure 26. The chemical structures of Indisulam (64) and SLC-0111 (65).

In order to achieve the desired selectivity towards CA IX and XII, metal based

compounds bearing sulfonamide ligands have been developed as novel approach to

deliver organometallic drug-like compounds as future therapies.

1.3.8.1 Re/99mTc labeled benzenesulfonamides as CAIs

Various studies have reported that CA IX used as hypoxic tumor markers and this

may lead to have novel therapeutic and diagnostic applications towards management

of metastatic tumor. Studies from Dubois et al. demonstrated that the CA IX active

site is accessible for sulfonamides only under hypoxic conditions89. Therefore,

radiolabelled sulfonamides may serve as powerful tool to visualize hypoxic tumors

also equipped with therapeutic application towards functional inhibition of hypoxic

tumors89. In this regard Akurathi and coworkers describe the synthesis of

99mTc(CO)3labeled 4-(2-aminoethyl)benzene-sulfonamide (conjugated with an N-(2-

picolyl-amine)-N-acetic acid moiety 66 (Figure 27) with reliable (radio)chemical

yield and purity. Its rhenium analogue 67 (Figure 27) was also prepared and showed a

KIs 58 nM for CA IX and significantly reduces the extracellular acidification induced

by CA IX. On the other hand, in vivo studies revealed that 99mTc-radiolabelled

conjugate 65 has been localized very low in tumor tissues that restricted its

application in visualization of CA IX expressing tumor tissues90. Hence, it appeared

that [99mTc]–66 is not a promising tracer for visualization of CA IX expressing

tumors.

48

Figure 27. Chemical structure of (66) and (67).

In a recent study Lu et al. synthesized a series of novel benzenesulfonamide CA IX

inhibitors containing tridentate coordinating sites complexed with Re or 99mTc labeled

tricarbonyl core 68 (Figure 28).91 In hypoxic CA IX expressing HeLa cells the

rhenium analogues exhibited strong to moderate binding affinity with IC50 values

ranging from 3–116 nM. One of the radiolabeled technetium tricarbonyl 99mTc(CO)3+

complex displayed high potency with IC50 value 9 nM against CA IX expressing

HeLa cells and found potentially significant in development of diagnostic and

therapeutic agent for the treatment of hypoxic solid state tumor .91


49

1.3.8.2. Metallocene as CAIs

Metallocenes such as ferrocene and ruthenocene are sandwich compounds have

potential as promising therapeutic agents against a large number of cancers. Poulsen

and co-workers have evaluated the potential of metallocenes 69 as CA inhibitors

separated from sulfonamide moiety (ZBG) by either a 1,4or a 1,5-1,2,3-triazolelinkers

(Figure 29).92 In crystallographic adduct formation of metallocenes with human CA

II, the sulfonamide moiety formed the interaction with catalytic zinc, while the

hydrophobic ferrocene or ruthenocene better adjusted in the hydrophobic pocket

within the enzyme active site.92 More important, these complexes gifted with more

potency than analogues containing a simple phenyl ring and also displayed strong

selectivity towards cancer associated CA IX or XII. Although both organic and metal

based compounds displayed similar biopharmaceutical properties (LogP, LogD,

solubility etc) revealed that remarkable activity and isoform selectivity is due to better

3-dimensional arrangement of metallocene in the active site that is inaccessible by 2-

dimensional organic groups.93

Figure 29. Chemical Structure of metallocene (69).

1.3.8.3. Piano stool complexes as CAIs

Monnard et al. synthesized d6-piano-stool complexes 70a–70d (Figure 30) bearing an

arylsulfonamide anchor as hCA II inhibitor. Interestingly, ruthenium biphenyl

complex 70d [(η6-biphenyl)Ru(bispy)Cl]+ displayed the highest affinity towards hCA

II, with inhibition constant 145.3 nM. Carbonic Anhydrase II (hCA II) served as a

model host for these complexes. As the co-crystal structure of hCA II, with 70c

revealed that complex coordinated with the protein in a conventional manner i.e. the

sulfonamide group bound to the catalytic zinc site while the aryl spacer forms close

contacts with the hydrophobic residues V121, F131, V135, L141, L198, P202, L204.

Ruthenium arene scaffold is located at the entrance of the cavity. The ruthenium

50

metal centre did not make any interaction with protein residues and maintained its

coordination sphere despite the presence of a chloride leaving group94. The evidences

of binding ability and high affinity of these piano stool complexes suggests that their

isoform selective towards tumor associated CA IX and XII can be achieved by

making slight changes to arene ring that significantly enhances the complex-enzyme

interaction.

Figure 30. Chemical structures of (70a–70d).

51

CHAPTER 2: EXPERIMENTAL

52

EXPERIMENTAL

2.1. Chemicals

All air- and moisture-sensitive reactions were carried out under nitrogen atmosphere

using standard Schlenk techniques. Chemicals obtained from commercial suppliers

were used as received and were of analytical grade. Tetrahydrofuran (THF),

dichloromethane (DCM), diethyl ether (Et2O), acetonitrile and triethylamine (TEA)

were first dried through a solvent purification system (LC Technology Solutions Inc.,

SP-1 solvent purifier), degassed under a N2 flow, and the stored in a Schlenk flask.

Methanol (MeOH) was dried using standard procedures and stored over activated

molecular sieves (3Å). Ethanol (EtOH) and methanol (MeOH) were dried over

activated molecular sieves (3 Å) in Erlenmeyer flasks for two days prior to use.

4-Fluoroaniline, α-terpinene, 2-picoline, and Na2S·9H2O were purchased from Merck,

4-chloroaniline, 4-bromoaniline, p-toluidine, p-anisidine, 4-aminoacetophenone, N,N-

dimethyl-p-phenylenediamine, p-phenylenediamine (98%), 4-

aminobenzenesulfonamide, sulfur succinic anhydride (≥99%), suberic acid, acetic

anhydride, ethylchloroformate, NaOH, conc. HCl, NH2OH·HCl (98%),

NaOCH3(≥97.0%), and osmium tetroxide (98%) were purchased from Sigma-

Aldrich.L-cysteine (Cys), L-methionine (Met), and L-histidine (His) were obtained

from Ak Scientific. Ruthenium(III) chloride hydrate (99%), iridium(III) chloride

hydrate and rhodium(III) chloride hydrate were from Precious Metals Online.

The ligands N-(4-fluorophenyl)pyridine-2-carbothioamide 1,61 N-(4-

chlorophenyl)pyridine-2-carbothioamide 2,95 N-(4-bromophenyl)pyridine-2-

carbothioamide 3, N-(p-tolyl)pyridine-2-carbothioamide 4, N-(4-

methoxyphenyl)pyridine-2-carbothioamide 5,96 N-(4-aminophenyl)pyridine-2-

carbothioamide 8,97 and complexes [chlorido(η6-p-cymene)(N-(4-

fluorophenyl)pyridine-2-carbothioamide)ruthenium(II)] chloride 961, [chlorido(η6-p-

cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)osmium(II)] chloride 1961

were synthesized by adopting standard procedures. The dimers bis[dichlorido(η6-p-

cymene)ruthenium(II)],98 bis[dichlorido(η6-p-cymene)osmium(II)],99

bis[dichlorido(η5-pentamethylcyclopentadienyl)rhodium(III)]100 and

bis[dichlorido(η5-pentamethylcyclopentadienyl)iridium(III)],101 were synthesized by

following reported procedures.

53

2.2. Instrumentation

1H and 13C{1H} and 2D (COSY, HSQC, HMBC) NMR spectra were recorded on

Bruker Avance AVIII400 MHz NMR spectrometer at ambient temperature at 400.13

MHz (1H) or 100.61 MHz(13C{1H}). For NMR experiments DMSO-d6, MeOH-d4,

CDCl3, D2O were used as solvents. Chemical shifts are reported versus SiMe4 and

were determined by reference to the residual solvent peaks.

High resolution mass spectra were recorded on a Bruker micrOTOF-QII mass

spectrometer in positive electrospray ionization (ESI) mode. Elemental analyses were

carried out on an Exeter Analytical Inc-CE-440 Elemental Analyser. X-ray diffraction

measurements of single crystals were carried out on a Bruker SMART APEX2

diffractometer with a CCD area detector using graphite monochromated Mo-Kα

radiation (λ = 0.71073 Å). The molecular structures were solved and refined with the

SHELXL-2016102 and Olex2103 program packages. The molecular structures were

visualized using Mercury 3.9.

2.3. Bioanalytical Assays

2.3.1 Sulforhodamine B Cytotoxicity Assay

Sulforhodamine B assay was used to determine the anticancer activity of compounds

against HCT116, NCI-H460 and SiHa cells by using the reported method. Similar

protocol was also used against SW480 cells (from ATCC).104 The cells were seeded at

5000 cells/well in 96-well microculture plates and allowed to settle for 24 h.

2.3.2. Stability of complexes 9 and 10 in aqueous solution

Hydrolytic stability of 9 and 10 was carried out by dissolving the compounds (1–2

mg/mL) in D2O and 1HNMR spectra were recorded after 0.5, 2, 24, 48,72 h and 7d

and ESI-mass spectra after 0.5, 24, 72 h and 7 days. To determine the stability in

acidic medium, 9 was dissolved in 60 mMHCl and the incubation mixture was

analyzed by ESI-MS after 0.5, 24, 72 h and 7 days.

2.3.3. Stability of complexes 24–27 in aqueous solution and reactivity with amino

acids

The hydrolytic stability of 24–27 was studied by dissolving the compounds (1–2

mg/mL) in D2O.1H NMR spectra were recorded after 0.5, 2, 24, 48, 72, 96 and 120h

and ESI-mass spectra after 0.5, 24,96 h and 7 d. To determine the reactivity with the

54

amino acids Cys, His and Met, 24 or 27 (1–2 mg/mL) was dissolved in D2O and 2

equivalents of the respective amino acids were added. The incubation mixture was

analyzed by 1H NMR spectroscopy and ESI-MS after 0.5, 2, 24, 48, 72, and 96 h.

2.3.4. Conductivity measurements of ligand 23 and its complexes 24–27

The conductivity in acetonitrile was determined for ligand 23 and complexes 24–27

(0.1 mM) on an Oakton CON 700 Conductivity/°C/°F Benchtop Meter at room

temperature.98

2.3.5. Calculated logarithmic octanol/water partition coefficient (clogP)

ChemBioDrawUltra 15.0 was used to determine the calculated logarithmic octanol-

water partition coefficient (clogP) of ligands 1–8 and 23.

2.3.6. Molecular Modelling of complexes 24 and 27 against CA II

Scigress Ultra version F.J 2.6105 was used for the modelling of the ligands into the

crystal structure of human carbonic anhydrase II (PDB ID 3PYK).94 Hydrogen atoms

were added to the structures and the ligands were built into the binding pocket based

on co-crystallized [chlorido{N-[di(pyridin-2-yl-κN)methyl]-4-

sulfamoylbenzamide}{(1,2,3,4,5,6-η)-(1R,2R,3R,4S,5S,6S)-1,2,3,4,5,6-

hexamethylcyclohexane-1,2,3,4,5,6-hexayl}ruthenium(II)]. The ligands were first

structurally optimized followed by short 1 ps molecular dynamics simulations using

the MM2 force field .106

2.3.7. Stability of complexes 38–41 in aqueous solution and reactivity with amino

acids

The hydrolytic stability of the complexes was studied by dissolving 1–2 mg/mL in

D2O/d4MeOD (38 and 39) or D2O (40 and 41). 1H NMR spectra were recorded after

0.5, 2, 24, 48, 72, 96 and 120 h. To determine the reactivity with the amino acids Cys,

His and Met, the compounds (1–2 mg/mL) were dissolved in D2O/d4-MeOD (38 and

39) or D2O (40 and 41) and 2 equivalents of the respective amino acid were added.

The incubation mixtures were analyzed by 1H NMR spectroscopy after 0.5, 2, 24, 48,

72, 96 and 120 h and ESI-MS after 0.5 h, 1and 7 d.

2.3.8. HDAC inhibition of compounds 29, 31, 38–41

HDAC1, HDAC6 and HDAC8 inhibition assays were performed using a fluorescent

HDAC activity assay kit (Reaction Biology CORP., USA).The substrates were the

55

fluorogenic peptides RHKAcKAcAMC (for HDAC8; residues 379–382from p53), and

RHKKAcAMC (for HDAC1 and 6; residues 379–382 from p53).

Initially, the inhibition of HDAC8 by 29, 31, and 38–41 was determined at a

concentration of 10 μM (n = 2). Ligand 29 was found to be inactive (83% residual

activity) at this concentration and was not further evaluated. Compounds 31 and 38–

41 were found to inhibit HDAC8 at 10 μM and were therefore further studied to

determine their IC50values against HDAC1, HDAC6 and HDAC8in 10 point mode (n

= 1). The highest concentrations used were 10 μM for 38–41 and 100μM for 31. The

IC50 values were calculated using GraphPad Prism 4 based on a sigmoidal dose-

response equation.

2.3.9. Dynamic simulation of ligand 31 and its complexes 38–41 against HDAC6

and HDAC8

The Scigress Ultra version F.J 2.6 program105 was used for the modelling of 31 and its

complexes in both enantiomeric forms in the crystal structures of HDAC6 (PDB ID:

5eei,resolution 1.32 Å)107 and HDAC8 (PDB ID: 1t69,resolution 2.91 Å).108

Hydrogen atoms were added to the structures and the ligands were built into the

binding pocket based on SAHA, the co-crystallised ligand found in the crystal

structures. The ligands were first structurally optimised followed by a short 1 ps

molecular dynamics (MD) simulation using the MM2106 force field. The crystal

structure was locked and served only as a scaffold during the optimisations and

molecular dynamics simulations.

56

Scheme 1

Anticancer Ru(η6-p-cymene)Complexes of 2-

Pyridinecarbothioamides: A Structure–Activity

Relationship Study

57

2.4. General procedures for the synthesis of PCAs ligands

Method A: For the synthesis of carbothioamide ligands 6 and 7, a mixture of N-

substituted aniline (25 mmol), sulfur (75 mmol), Na2S·9H2O (0.5 mol %) and 2-

picoline (15 mL) was refluxed at 150 °C for 72 h.61 After cooling, the solvent was

evaporated under reduced pressure. The dark solid residue dissolved in

dichloromethane and twice filtered through a bed of silica gel. Rotary evaporator used

to evaporate the solvent. Pure product was obtained after recrystallization from

methanol.

Method B: For the synthesis of carbothioamides ligand 9, a mixture of N-substituted

aniline (50 mmol), sulfur (120 mmol), sodium sulfide nonahydrate (5 mol %) and 2-

picoline (100 mmol) was refluxed at 150 oC for 18 h. The 2M aqueous solution of

sodium hydroxide (100 mL) was transferred into above reaction mixture and filtered.

The conc. hydrochloric acid added dropwise to the filtrate solution to acidify its pH

upto 5 and resulting yellow precipitates filtered off and washed with 100 mL of

water.97 These precipitates was dissolved in dichloromethane and filtered through a

bed of silica gel. Pure orange yellow crystalline product was obtained by

recrystallization from acetonitrile and dried.

N-(4-Acetylphenyl)pyridine-2-carbothioamide (6)

Compound 6 was prepared following general procedure A using 4-acetylaniline (3.37

g, 25 mmol), sulfur (2.40 g, 75 mmol), Na2S·9H2O (0.12 g, 0.5 mol%) and 2-picoline

(15 mL).Yield: 4.93g, (77%), yellow-orange solid. Elemental analysis found: C,

64.77; H, 4.67; N, 10.81, calculated for C14H12N2OS·0.2H2O: C, 64.92; H, 4.81; N,

10.78. 1H NMR (400.13 MHz, DMSO-d6, 25 °C): δ = 12.47 (s, 1H, NH), 8.70 (d, 3J=

6 Hz, 1H, H-4), 8.52 (d, 3J= 8 Hz, 1H, H-1), 8.19 (d, 3J= 8 Hz, 2H, H-9/H-11), 8.05

(m, 3H, H-3/H-8/H-12), 7.68 (ddd, 3J= 7 Hz, 3J= 5 Hz, 4J= 1 Hz, 1H, H-2), 2.59 (s,

3H, COCH3) ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ =

196.8(COCH3), 190.7 (C-6), 152.6 (C-5), 147.4(C-1), 143.1(C-7), 137.8 (C-3), 134.3

58

(C-10), 128.7 (C-9/C-11), 126.6 (C-8/C-12), 124.7 (C-2), 123.4 (C-4), 26.7(Car-

COCH3) ppm. MS (ESI+): m/z 279.0568 [6 + Na]+ (mex = 279.0563).

N-(4-(Dimethylamino)phenyl)pyridine-2-carbothioamide (7)

Compound 7 was prepared following general procedure A using N,N-dimethyl-p-

phenylenediamine (3.40 g, 25 mmol), sulfur (2.40 g, 75 mmol), Na2S·9H2O(0.12 g,

0.5 mol%) and 2-picoline (15 mL). Yield: 5.34 g, (83%), red needles. Elemental

analysis found: C, 64.33; H, 5.71; N, 15.64, calculated for C14H15N3S·0.25H2O: C,

64.22; H, 5.97; N, 16.05. 1H NMR (400.13 MHz, DMSO-d6, 25 °C) δ = 12.09 (s, 1H,

NH), 8.65 (d, 3J= 7 Hz, 1H, H-4), 8.53 (d, 3J= 8 Hz, 1H, H-1), 8.02 (td, 3J= 7 Hz, 4J=

1 Hz, 1H, H-3), 7.90 (m, 2H, H-8/H-12), 7.62 (ddd, 3J= 7 Hz, 4J= 1 Hz, 1H, H-2),

6.76 (d, 3J= 9 Hz, 2H, H-9/H-11), 2.93 (s, 6H, N(CH3)2) ppm. 13C{1H}NMR (100.61

MHz, DMSO-d6, 25 °C): δ = 186.5 (C-6), 152.8 (C-5), 148.7 (C-10), 147.2 (C-1),

137.7 (C-3), 128.4 (C-7), 126.0 (C-8/C-12), 124.4 (C-2), 124.2 (C-4), 111.5 (C-9/C-

11), 40.1 (Car-N(CH3)2) ppm. MS (ESI+): m/z 280.0884 [7 + Na]+ (mex = 280.0879).

2.5. General procedures for the syntheses of metal complexes of PCAs

Method C. A solution of [M(L)X2]2 (M = Ru, Os, Rh, Ir; L = η6-p-cymene, η5-

pentamethylcyclopentadienyl; X = Cl, Br, I) (1 equiv.) in dry DCM (20 mL) was

added to a stirred solution of carbothioamide ligand (2 equiv.) in dry THF (20 mL).

The reaction mixture was stirred for 4 h at 40 °C under nitrogen atmosphere. A

change in color from brown to deep red was observed immediately after the addition

of dimer. Rotavap was used to evaporate the solvent and the residue was dissolved in

a minimal volume of DCM, followed by addition of n-hexane that resulted in

immediate precipitation. After placing it in the fridge overnight, the precipitate was

filtered, and dried under vacuum.

59

Method D. The respective carbothioamide (2 equiv.) was dissolved in absolute DCM

(20 mL) and a solution of [Ru(cym)Cl2]2 (1 equiv.) in absolute DCM (20 mL) was

added. The reaction mixture was stirred for 4 h at room temperature under nitrogen

atmosphere. The solvent was concentrated in vacuo to ca. 5 mL and n-hexane was

added for precipitation in the fridge. The solvent was decanted and subsequent drying

in vacuo yielded analytically pure solid product.

Method E. The carbothioamide ligand (2 equiv.) was dissolved in dry MeOH (30

mL) followed by addition of 1 mL acetic acid. [Ru(cym)Cl2]2 (1 equiv.) was added to

the stirred solution of the ligand and stirred for another 4 h under nitrogen

atmosphere. Rotavap was used to evaporate solvent. The solid residue was washed

with ethyl acetate (2 × 10 mL) followed by with diethyl ether (2 × 10 mL) and dried

under vacuum to isolate the desired product.

[Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2-

carbothioamide)ruthenium(II)] chloride (10)

Compound 10 was synthesized following the general synthetic procedure C using N-

(4-clorophenyl)pyridine-2-carbothioamide (100 mg, 0.40 mmol) and [Ru(η6-p-

cymene)Cl2]2 (122 mg, 0.20 mmol).Yield: 171 mg, (77%), red solid. Elemental

analysis found: C, 48.63; H, 4.24, N, 4.97, calculated for C22H23Cl3N2RuS·0.15C6H14:

C, 48.44; H, 4.46; N, 4.93. 1HNMR (400.13 MHz, MeOD-d4, 25 °C): δ = 9.63 (d, 3J=

6 Hz, 1H, H-1), 8.40 (d, 3J= 8 Hz, 1H, H-4), 8.25 (t, 3J= 8 Hz, 1H, H-3), 7.81 (t, 3J= 7

Hz, 1H, H-2), 7.56 (m, 4H, H-9/H-11/H8/12), 6.02 (d, 3J= 6 Hz, 1H, H-15), 5.92 (d,

3J= 6 Hz, 1H, H-17), 5.87 (d, 3J= 6 Hz, 1H, H-18), 5.61 (d, 3J= 6 Hz, 1H, H-14), 2.73

(sept, 3J= 6 Hz, 1H, H-21), 2.20 (s, 3H, H-19), 1.20 (d, 3J= 6 Hz, 3H, H-20), 1.13 (d,

3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, MeOD-d4, 25 °C): δ = 159.9

(C-1), 155.5 (C-5), 140.9 (C-3), 139.8 (C-7), 134.9 (C-10), 130.8 (C-9/C-11), 130.5

(C-2), 127.6 (C-8/C-12), 125.1 (C-4), 107.1 (C-16), 105.2 (C-13), 89.2 (C-15), 89.1

60

(C-17), 86.5 (C-18), 84.8 (C-14), 32.4 (C-21), 22.9 (C-20), 21.9 (C-22), 18.8 (C-19)

ppm. MS (ESI+): m/z 483.0236 [10–2Cl–H]+ (mex = 483.0231).

[Chlorido(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-


Compound 11 was synthesized following the general synthetic procedure D using N-

(4-bromophenyl)pyridine-2-carbothioamide (100 mg, 0.34 mmol) and [Ru(η6-p-

cymene)Cl2]2 (104 mg, 0.17 mmol).Yield: 143 mg,(70%), dark red solid. Elemental

analysis found: C, 44.39; H, 3.90; N, 4.63, calculated for C22H23BrCl2N2RuS: C,

44.09; H, 3.87; N, 4.67. 1HNMR (400.13 MHz, CDCl3, 25 °C): δ = 9.34 (d, 3J= 6 Hz,

2H, H-1/H-4), 8.06 (t, 3J= 8 Hz, 1H, H-3), 7.64 (d, 3J= 8 Hz, 2H, H-8/H-12), 7.57

(m,3H, H-2/H-9/H-11), 5.69 (d, 3J= 6 Hz, 1H, H-15), 5.59 (d, 3J= 6 Hz, 1H, H-17),

5.52 (d, 3J= 6 Hz, 1H, H-18), 5.37 (d, 3J= 6 Hz, 1H, H-14), 2.76 (sept, 3J= 6 Hz, 1H,

H-21), 2.20 (s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H, H-20), 1.14 (d, 3J= 7 Hz, 3H, H-22)

ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C): δ = 157.1 (C-1), 139.7 (C-3),

136.1 (C-10), 132.4 (C-8/C-12), 128.6 (C-2), 127.0 (C-9/11),126.4 (C-4), 106.1 (C-

16), 102.8 (C-13), 87.6 (C-15), 87.2 (C-17), 84.6 (C-18), 83.8 (C-14), 31.1 (C-21),

22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z 528.9731 [11–2Cl–H]+

(mex = 528.9723).

61

[Chlorido(η6-p-cymene)(N-(p-tolyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride (12)

Compound 12 was synthesized following the general synthetic procedure E using N-

(p-tolyl)pyridine-2-carbothioamide (100 mg, 0.44mmol) and [Ru(η6-p-cymene)Cl2]2

(134 mg, 0.22mmol). Yield: 111mg, (47%), dark red solid. Elemental analysis found:

C, 52.04; H, 5.08; N, 5.00, calculated for C23H26Cl2N2RuS·0.1C6H14: C, 52.19; H,

5.09; N, 5.16. 1HNMR (400.13 MHz, MeOD-d4, 25 oC): δ = 9.67 (d, 3J= 6 Hz, 1H, H-

1), 8.44 (d, 3J= 8 Hz, 1H, H-4), 8.30 (td, 3J=8 Hz, 4J= 1.5 Hz, 1H, H-3), 7.85 (td, 3J=

7 Hz, 4J= 1 Hz, 1H, H-2), 7.51 (d, 3J= 8 Hz, 2H, H-9/H-11), 7.41 (d, 3J= 8 Hz, 2H, H-

8/H-12), 6.05 (d, 3J= 6 Hz, 1H, H-15), 5.94 (d, 3J= 6 Hz, 1H, H-17), 5.91 (d, 3J= 6

Hz, 1H, H-18), 5.65 (d, 3J= 6 Hz, 1H, H-14), 2.74 (sept, 3J= 6 Hz, 1H, H-21), 2.24 (s,

3H, -CH3), 2.21 (s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H, H-20), 1.13 (d, 3J= 7 Hz, 3H, H-

22) ppm. 13C{1H} NMR (100.61 MHz, MeOD-d4, 25 °C): δ = 193.7 (C-6), 160.2 (C-

1), 154.7 (C-5), 141.1 (C-3), 140.8 (C-10), 136.4 (C-7), 131.4 (C-9/C-11), 130.8 (C-

2), 126.1 (C-4), 125.0 (C-8/C-12), 107.3 (C-16), 105.5 (C-13), 89.3 (C-15), 89.2 (C-

17), 86.7 (C-18), 85.0 (C-14), 32.4 (C-21), 22.9 (C-20), 21.9 (C-22), 21.3 (C-19), 18.8

(Car-CH3) ppm. MS (ESI+): m/z 463.0782 [12–2Cl–H]+ (mex = 463.0777).

62

[Chlorido(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-



(4-methoxyphenyl)pyridine-2-carbothioamide (90 mg, 0.37mmol) and [Ru(η6-p-

cymene)Cl2]2 (113 mg, 0.18 mmol). Yield: 183 mg, (87%), dark red solid. Elemental

analysis found: C, 49.86; H, 4.53; N, 5.24; calculated for C23H26Cl2N2ORuS: C,

50.18; H, 4.76; N, 5.09. 1HNMR (400.13 MHz, CDCl3, 25 °C): δ = 9.59 (d, 3J= 9 Hz,

1H, H-1), 9.43 (d, 3J= 5 Hz, 1H, H-4), 8.04 (t, 3J= 9 Hz, 1H, H-3), 7.83 (d, 3J= 8 Hz,

2H, H-8/H-12), 7.57 (t, 3J= 6 Hz, 1H, H-2), 6.98 (d, 3J= 9 Hz, 2H, H-9/H-11), 5.72 (d,


(d, 3J= 6 Hz, 1H, H-14), 3.84 (s, 3H, -OCH3), 2.76 (sept, 3J= 6 Hz, 1H, H-21), 2.20 (s,

3H, H-19), 1.20 (d, 3J= 7 Hz, 3H, H-20), 1.14 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H}

NMR (100.61 MHz, CDCl3, 25 °C): δ = 159.6 (C-5), 157.7 (C-1), 154.0 (C-10),

140.0 (C-3), 130.9 (C-7), 129.0 (C-8/C-12), 127.3 (C-2), 126.8 (C-4), 114.5 (C-9/C-

11), 106.4 (C-16), 103.0 (C-13), 87.7 (C-15), 87.3 (C-17), 84.8 (C-18), 84.0 (C-14),

55.7 (-OCH3), 31.1 (C-21), 22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+):

m/z 479.0731 [13–2Cl–H]+ (mex = 479.0732).

63

[Chlorido(η6-p-cymene)(N-(4-acetylphenyl)pyridine-2-



(4-acetylphenyl)pyridine-2-carbothioamide (100 mg, 0.39 mmol) and [Ru(η6-p-

cymene)Cl2]2 (116 mg, 0.19 mmol). Yield: 197 mg, (84%), red solid. Elemental

analysis found: C, 51.21; H, 4.68; N, 4.91, calculated for C24H26Cl2N2ORuS: C,

51.25; H, 4.66; N, 4.98. 1HNMR (400.13 MHz, MeOD-d4, 25 °C): δ = 9.66 (d, 3J= 6

Hz, 1H, H-1), 8.44 (d, 3J= 8 Hz, 1H, H-4), 8.29 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3),

8.19 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.84 (td, 3J= 6 Hz, 4J= 1 Hz, 1H, H-2), 7.74 (d, 3J=

9 Hz, 2H, H-8/H-12), 6.05 (d, 3J= 6 Hz, 1H, H-15), 5.94 (d, 3J= 6 Hz, 1H, H-17), 5.90

(d, 3J= 6 Hz, 1H, H-18), 5.65 (d, 3J= 6 Hz, 1H, H-14), 3.77 (s, 3H, OCH3), 2.75 (sept,

3J=6 Hz, 1H, H-21), 2.66 (s, 3H, COCH3), 2.21 (s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H,

H-20), 1.13 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25

°C): δ = 197.2 (CO), 159.2 (C-5), 157.3 (C-1), 139.9 (C-3), 136.0 (C-7),134.4 (C-10),

129.5 (C-9/C-11), 128.8 (C-8/C-12), 127.4 (C-2), 124.8 (C-4), 106.3 (C-16), 103.0

(C-13), 87.7 (C-15), 87.3 (C-17), 84.7 (C-18), 83.9 (C-14), 31.1 (C-21),

26.8(COCH3), 22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z 491.0731

[14–2Cl–H]+ (mex = 491.0721).

64

[Chlorido(η6-p-cymene)(N-(4-(dimethylamino)phenyl)pyridine-2-



(4-(dimethylamino)phenyl)pyridine-2-carbothioamide (100 mg, 0.39 mmol) and

[Ru(η6-p-cymene)Cl2]2 (116 mg, 0.19 mmol). Yield: 182 mg, (83%), red solid.

Elemental analysis found: C, 49.47; H, 5.28; N, 6.36, calculated for

C24H29Cl2N3RuS·0.33C6H14·0.66CH2Cl2: C, 49.36; H, 5.44; N, 6.48. 1HNMR (400.13

MHz, MeOD-d4, 25 °C): δ = 9.63 (d, 3J= 5 Hz, 1H, H-1), 8.39 (d, 3J= 8 Hz, 1H, H-4),

8.25 (t, 3J= 7 Hz, 1H, H-3), 7.80 (t, 3J= 6 Hz, 1H, H-2), 7.56 (d, 3J= 9 Hz, 2H, H-8/H-

12), 6.94 (d, 3J= 8 Hz, 2H, H-9/H-11), 6.01 (d, 3J= 6 Hz, 1H, H-15), 5.92 (d, 3J= 6

Hz, 1H, H-17), 5.87 (d, 3J= 6 Hz, 1H, H-18), 5.61 (d, 3J= 6 Hz, 1H, H-14), 3.07 (s,

6H, N(CH3)2), 2.74 (sept, 3J= 6 Hz, 1H, H-21), 2.21 (s, 3H, H-19), 1.20 (d, 3J= 7 Hz,

3H, H-20), 1.12 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3,

25 °C): δ = 197.2 (C-6), 159.6 (C-5), 157.2 (C-1), 152.3 (C-10), 140.0 (C-3), 135.8

(C-7), 129.5 (C-8/C-12), 128.8 (C-2), 127.3 (C-4), 124.8 (C-9/C-11), 106.2 (C-16),

102.9 (C-13), 87.7 (C-15), 87.3 (C-17), 84.7 (C-18), 83.9 (C-14), 31.1 (N(CH3)2),

26.8 (C-21), 22.8 (C-20), 22.0 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z 492.1047

[15–2Cl–H]+ (mex = 492.1041).

65

[Chlorido(η6-p-cymene)(N-(4-aminophenyl)pyridine-2-



(4-aminophenyl)pyridine-2-carbothioamide (50 mg, 0.22 mmol) and [Ru(cym)Cl2]2

(67 mg, 0.11mmol).Yield: 73 mg, (62%), black/dark red solid. Elemental analysis

found: C, 45.94; H, 5.15; N, 6.75, calculated for

C22H25Cl2N3RuS·0.33CH2Cl2·1.33H2O: C, 45.63; H, 4.86; N, 7.15. 1HNMR (400.13

MHz, MeOD-d4, 25 °C): δ = 9.60 (d, 3J= 6 Hz, 1H, H-4), 8.32 (d, 3J= 8Hz, 1H, H-1),



Hz, 1H, H-17), 5.82 (d, 3J= 6 Hz, 1H, H-18), 5.56 (d, 3J= 6 Hz, 1H, H-14), 2.73 (sept,

3J=6 Hz, 1H, H-21), 2.20 (s, 3H, H-19), 1.20 (d, 3J= 7 Hz, 3H, H-20), 1.13 (d, 3J= 7

Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1

mL), 25 °C): δ = 158.8 (C-1), 148.7 (C-10), 140.2 (C-3), 136.6 (C-7), 129.5 (C-8/C-

12), 126.0 (C-2), 124.4 (C-4), 117.1 (C-9/C-11) 106.1 (C-16), 104.1 (C-13), 88.3 (C-

15), 88.2(C-17), 85.4 (C-18), 83.9 (C-14), 31.7 (C-21), 22.9 (C-20), 21.9 (C-22), 18.9

(C-19) ppm. MS (ESI+): m/z 464.0734 [16–2Cl–H]+ (mex = 464.0768).

66

Scheme 2

Impact of Metal Ions and Halide Leaving Groups on

the Biological Activity of Organometallic N-(4-

fluorophenyl)pyridine-2-carbothioamide Anticancer

Agents

67

[bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide (17)

The compound 17 was synthesized following the general complexation procedure C

using N-(4-fluorophenyl)-2-pyridinecarbothioamide (75 mg, 0.32 mmol) and [Ru(η6-

p-cymene)Br2]2 (127.5 mg, 0.13 mmol). Yield: 121 mg, (60%), Red solid. Elemental

analysis found: C, 42.34; H, 3.88; N, 4.54, calculated for C22H23Br2FN2RuS: C, 42.12;

H, 3.70; N, 4.47. 1H NMR (400.13 MHz, MeOD-d4, 25 oC): δ = 9.67 (d, 3J= 6 Hz, 1H,

H-1), 8.44 (d, 3J= 8 Hz, 1H, H-4), 8.29 (td, 3J= 8 Hz, 4J= 1 Hz, 1H, H-3), 7.83 (td, 3J=

7 Hz, 4J= 1 Hz, 1H, H-2), 7.63 (m, 2H, H-9/H-11), 7.35 (t, 3J= 8 Hz, 2H, H-8/H-12),

6.04 (d, 3J= 6 Hz, 1H, H-15), 5.92 (d, 3J= 6 Hz, 1H, H-17), 5.89 (d, 3J= 6 Hz, 1H, H-

18), 5.68 (d, 3J= 6 Hz, 1H, H-14), 2.80 (sept, 3J= 6 Hz, 1H, H-21), 2.28 (s, 3H, H-19),

1.21 (d, 3J= 6 Hz, 3H, H-20), 1.15 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR

(100.61 MHz, MeOD-d4, 25 °C): δ = 194.1 (C-6), 165.0 (C-10), 162.5 (C-5), 160.8

(C-1), 154.7 (C-7), 141.0 (C-3), 130.7 (C-2), 128.9 (C-9), 125.8 (C-11), 125.0 (C-4),

117.9 (C-8), 117.7 (C-12), 108.2 (C-16), 105.0 (C-13), 89.3 (C-15), 89.2 (C-17), 86.7

(C-18), 85.8 (C-14), 32.6 (C-21), 22.9 (C-20), 21.9 (C-22), 19.3 (C-19) ppm. MS

(ESI+): m/z 467.0531 [17–2Br–H]+ (mex = 467.0526).

68

[iodo(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]iodide (18)


using N-(4-fluorophenyl)pyridine-2-carbothioamide (65 mg, 0.28 mmol) and [Ru(η6-

p-cymene)I2]2 (136.8 mg, 0.14 mmol). Yield: 179 mg, (87%), Red solid. Elemental

analysis found: C, 36.87; H, 3.20; N, 3.66, calculated for C22H23FI2N2RuS: C, 36.63;

H, 3.21; N, 3.88. 1H NMR (400.13 MHz, MeOD-d4, 25 oC): δ = 9.64 (d, 3J= 6 Hz, 1H,


7 Hz, 4J= 1 Hz, 1H, H-2), 7.65 (m, 2H, H-9/H-11), 7.35 (t, 3J= 8 Hz, 2H, H-8/H-12),

6.03 (d, 3J= 6 Hz, 1H, H-15), 5.89 (d, 3J= 6 Hz, 1H, H-17), 5.86 (d, 3J= 6 Hz, 1H, H-

18), 5.71 (d, 3J= 6 Hz, 1H, H-14), 2.89 (sept, 3J=6 Hz, 1H, H-21), 2.37 (s, 3H, H-19),


(100.61 MHz, MeOD-d4, 25 °C): δ = 194.0 (C-6), 164.9 (C-10), 162.4 (C-5), 161.7

(C-1), 154.9 (C-7), 140.7 (C-3), 130.2 (C-2), 128.9 (C-9), 125.8 (C-11), 125.1 (C-4),

117.9 (C-8), 117.7 (C-12), 109.3 (C-16), 104.7 (C-13), 89.5 (C-15), 89.1 (C-17), 86.9

(C-18), 86.8 (C-14), 33.0 (C-21), 23.0 (C-20), 22.1 (C-22), 20.1 (C-19) ppm. MS

(ESI+): m/z 467.0531 [18–2I–H]+ (mex = 467.0538).

69

[iodo(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)osmium(II)]iodide (20)


using N-(4-fluorophenyl)pyridine-2-carbothioamide (55 mg, 0.24 mmol) and [Os(η6-

p-Cymene)I2]2 (137 mg, 0.12 mmol). After completion of reaction, the solid product

was filtered followed by washing with dichloromethane (2 × 10 mL) and

tetrahydrofuran (1 × 10 mL) and afterwards dried in vacuum. Yield: 130 mg, (66%),

Black solid. Elemental analysis found: C, 32.82, H, 2.86, N, 3.37, S, 3.96, calculated

for C22H23FI2N2OsS: C, 32.60; H, 2.86; N, 3.46 S, 3.96. 1H NMR (400.13 MHz,

MeOD-d4, 25 oC): δ = 9.60 (d, 3J= 6 Hz, 1H, H-1), 8.47 (d, 3J= 9 Hz, 1H, H-4), 8.22

(td, 3J= 7 Hz, 4J= 2 Hz, 1H, H-3), 7.71 (td, 3J= 7 Hz, 4J= 1 Hz, 1H, H-2), 7.64 (m, 2H,

H-9/H-11), 7.35 (t, 3J= 8 Hz, 2H, H-8/H-12), 6.18 (d, 3J= 6 Hz, 1H, H-15), 6.06 (d, 3J= 6 Hz, 1H, H-17), 6.03 (d, 3J= 6 Hz, 1H, H-18), 5.87 (d, 3J= 6 Hz, 1H, H-14), 2.78

(sept, 3J=6 Hz, 1H, H-21), 2.43 (s, 3H, H-19), 1.20 (d, 3J= 7 Hz, 3H, H-20), 1.13 (d,

3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, MeOD-d4, 25 °C): δ = 164.8

(C-10), 162.8 (C-1), 162.6 (C-5), 155.0 (C-7), 140.6 (C-3), 131.2 (C-2), 128.9 (C-9),

128.8 (C-11), 125.4 (C-4), 117.9 (C-8), 117.7 (C-12), 100.5 (C-16), 97.1 (C-13), 81.5

(C-15), 81.5 (C-17), 78.9 (C-18), 78.1 (C-14), 32.9 (C-21), 23.2 (C-20), 22.1 (C-22),

20.0 (C-19) ppm. MS (ESI+): m/z 557.1103 [20–2I–H]+ (mex = 557.1115).

70

[chlorido(η5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)rhodium(III)]chloride (21)


using N-(4-fluorophenyl)pyridine-2-carbothioamide (130 mg, 0.56 mmol) and

[(C5(CH3)5RhCl2)]2 (173 mg, 0.28 mmol). Yield: 268 mg, (88%), orange solid.

Elemental analysis found: C, 50.80; H, 4.70; N, 5.35, calculated for

C22H24Cl2FN2RhS.0.3C6H14 C, 50.40; H, 5.01; N, 4.94. 1H NMR 400.13 MHz,

CDCl3, 25 oC): δ = 9.64 (brd, 3J= 7 Hz, 1H, H-1), 8.77 (d, 3J= 5 Hz, 1H, H-4), 8.21 (t,

3J= 8 Hz, 1H, H-3), 7.94 (t, 2H, H-9/H-11), 7.67 (t, 3J= 7 Hz, 1H, H-2), 7.18 (t, 3J= 9

Hz, 2H, H-8/H-12), 1.70 (s, 15H, CH3-Cp*) ppm. 13C{1H} NMR (100.61 MHz,

CDCl3, 25 °C): δ = 162.8 (C-10), 160.4 (C-5),153.8 (C-1), 140.1 (C-3), 128.8 (C-9/C-

11), 127.1 (C-2), 126.6 (C-4), 116.2 (C-8), 116.0 (C-12), 97.5 (Cp*-C), 9.1 (Cp*-

CH3) ppm. MS (ESI+): m/z 469.0621 [21–2Cl–H]+ (mex = 469.0614).

[chlorido(η5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)pyridine-2-

carbothioamide) iridium(III)]chloride (22)


using N-(4-clorophenyl)pyridine-2-carbothioamide (100 mg, 0.40 mmol) and

[(C5(CH3)5RhCl2)]2(123 mg, 0.20 mmol). Yield: 176 mg (81%), Red solid. Elemental

analysis found: C, 42.71; H, 3.80; N, 4.31, calculated for

C22H24Cl2FIrN2S.0.1C4H8O.0.1C6H14: C, 42.73; H, 4.09; N, 4.33. 1H NMR (400.13

71

MHz, CDCl3, 25 oC): δ = 9.30 (d, 3J= 6 Hz, 1H, H-1), 8.74 (d, 3J= 5 Hz, 1H, H-4),

8.08 (t, 3J= 7 Hz, 1H, H-3), 7.74 (t, 2H, H-9/H-11), 7.56 (t, 3J= 6 Hz, 1H, H-2), 7.15

(t, 3J= 9 Hz, 2H, H-8/H-12), 1.70 (s, 15H, CH3-Cp*) ppm. 13C{1H} NMR (100.61

MHz, CDCl3, 25 °C): δ = 163.9 (C-10), 160.0 (C-5), 154.5 (C-1), 139.8 (C-3), 129.2

(C-9/C-11), , 126.3 (C-2), 116.2 (C-4), 115.9 (C-8/C-12), 90.1 (Cp*-C), 8.8 (Cp*-

CH3) ppm. MS (ESI+): m/z 559.1195 [22–2Cl–H]+ (mex = 559.1200).

72

Scheme 3

Organoruthenium and -osmium Complexes of 2-

Pyridinecarbothioamides Functionalized with a

Sulfonamide motif: Synthesis and Cytotoxicity

73

N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (23)

Ligand 23 was prepared following the general procedure B using sulfanilamide

(2.153g, 12.5 mmol), sulfur (0.96 g, 30 mmol), sodium sulfide nonahydrate (0.15 g, 5

mol %) and 2-picoline (2.46 mL, 25 mmol). Yield: 2.45 g, (67%), yellow solid.

Elemental analysis found C, 49.73; H, 4.00; N, 14.88 calculated for

C12H11N3O2S2·0.2CH3CN: C, 49.39; H, 3.88; N, 14.86. 1H NMR (400.13 MHz,

DMSO-d6, 25 oC) δ = 12.48 (s, 1H, -NH), 8.70 (d, 3J= 5 Hz, 1H, H-4), 8.53 (d, 3J= 8

Hz, 1H, H-1), 8.13 (d, 3J= 9 Hz 2H, H-9/H-11), 8.06 (td, 3J= 8 Hz, 4J= 1 Hz, 1H, H-

3), 7.89 (d, 3J= 9 Hz, 2H, H-8/H-12), 7.68 (ddd, 3J= 8 Hz, 3J= 5 Hz, 4J= 1 Hz, 1H, H-

2), 7.41 (s, 2H, -SO2NH2) ppm. 13C{1H}NMR (100.61 MHz, DMSO-d6, 25 °C): δ =

191.1 (C-6), 152.5 (C-5), 147.4 (C-1), 141.9 (C-7), 141.5 (C-10), 137.9 (C-3), 126.7

(C-9/C-11), 126.1 (C-2), 124.8 (C-4), 124.3 (C-8/C-12) ppm. MS (ESI+): m/z

316.0190 [23+Na]+ (mex = 316.0157).

[chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride (24)

The synthesis of compound 24 was performed following the general complexation

procedure C, using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (120 mg, 0.41

mmol) and [Ru(η6-p-cymene)Cl2]2 (124mg, 0.20 mmol). After completion of reaction,

the solvent was concentrated in vacuum up to 5 mL and n-hexane was added for

further precipitation in the fridge. The solid product was filtered followed by washing

74

with dichloromethane (2 × 10 mL) and dried in vacuum. Yield: 130 mg, (53%), red

solid. Elemental analysis found: C, 39.84; H, 3.89; N, 5.81, calculated for

C22H25Cl2N3O2RuS2·0.7CH2Cl2·1.25H2O: C, 40.00; H, 4.27; N, 6.17.1HNMR (400.13

MHz, MeOD-d4, 25 oC): δ = 9.66 (d, 3J= 6 Hz, 1H, H-1), 8.43 (d, 3J= 8Hz, 1H, H-4),

8.29 (td, 3J= 8 Hz, 4J(H3,H1)= 2 Hz, 1H, H-3), 8.09 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.85

(t, 3J= 8 Hz, 1H, H-2), 7.76 (d, 3J= 9 Hz, 2H, H-8/H-12), 6.05 (d, 3J= 6 Hz, 1H, H-

15), 5.94 (d, 3J= 6 Hz, 1H, H-17), 5.91 (d, 3J= 6 Hz, 1H, H-18), 5.65 (d, 3J= 6 Hz, 1H,

H-14), 2.74 (sept, 3J= 7 Hz, 1H, H-21), 2.21 (s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H, H-

20), 1.13 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz, CDCl3, 25 °C):

δ = 191.1 (C-6), 152.5 (C-5), 158.0 (C-1), 153.8 (C-7), 142.3 (C-10), 139.81 (C-3),

129.3 (C-9/C-11), 127.4 (C-2), 125.3 (C-4), 125.1 (C-8/C-12) 106.3 (C-16), 103.6 (C-

13), 87.7 (C-15), 87.4 (C-17), 84.9 (C-18), 83.9 (C-14), 31.0 (C-21), 22.3 (C-20), 21.4

(C-22), 18.3 (C-19) ppm. MS (ESI+): m/z 528.0353 [24–2Cl–H]+ (mex = 528.0356).

[bromido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide (25)



mmol) and [Ru(η6-p-cymene)Br2]2 (125 mg, 0.17 mmol). After completion of

reaction, the solvent was concentrated in vacuum up to 5 mL and n-hexane was added

for further precipitation in the fridge. The solid product was filtered followed by

washing with dichloromethane (2 × 10 mL) and dried in vacuum. Yield: 145 mg,

(62%), red solid. Elemental analysis found: C, 39.31; H, 3.71; N, 5.75, calculated for

C22H25Br2N3O2RuS2·0.2C4H8O: C, 38.96; H, 3.81; N, 5.98.1HNMR (400.13 MHz,

MeOD-d4, 25 oC): δ = 9.66 (d, 3J= 6 Hz, 1H, H-1), 8.45 (d, 3J= 8Hz, 1H, H-4), 8.30

(td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 8.11 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.83 (m, 3H, H-

2/H-8/H-12), 6.05 (d, 3J= 6 Hz, 1H, H-15), 5.94 (d, 3J= 7 Hz, 1H, H-17), 5.90 (d, 3J=

6 Hz, 1H, H-18), 5.69 (d, 3J= 6 Hz, 1H, H-14), 2.81 (sept, 3J= 7 Hz, 1H, H-21), 2.28

75

(s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H, H-20), 1.15 (d, 3J= 7 Hz, 3H, H-22) ppm.

13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 158.8

(C-1), 153.3 (C-7), 142.9 (C-10), 140.0 (C-3), 129.5 (C-9/C-11), 127.5 (C-2), 125.9

(C-4), 125.7 (C-8/C-12) 107.6 (C-16), 103.4 (C-13), 87.8 (C-15), 87.4 (C-17/C-18),

85.1 (C-14), 31.3 (C-21), 22.5 (C-20), 21.6 (C-22), 18.9 (C-19) ppm. MS (ESI+): m/z

528.0353 [25–2Cl–H]+ (mex = 528.0340).

[iodido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]iodide (26)



mmol) and [Ru(η6-p-cymene)I2]2 (133 mg, 0.14 mmol). After completion of reaction,

the solid product was filtered followed by washing with dichloromethane (2 × 10 mL)

and tetrahydrofuran (1 × 10 mL) and afterwards dried in vacuum. Yield: 187 mg,

(88%), Red solid. Elemental analysis found: C, 35.99; H, 3.72; N, 4.72, calculated for

C22H25I2N3O2RuS2·0.75C4H8O: C, 35.89; H, 3.74; N, 5.02. 1HNMR (400.13 MHz,

MeOD-d4, 25 oC): δ = 9.63 (d, 3J= 6 Hz, 1H, H-1), 8.42 (d, 3J= 8Hz, 1H, H-4), 8.25

(td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 8.09 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.77 (m, 3H, H-

2/H-8/H-12), 6.03 (d, 3J= 6 Hz, 1H, H-15), 5.88 (d, 3J= 7 Hz, 1H, H-17), 5.85 (d, 3J=

7 Hz, 1H, H-18), 5.70 (d, 3J= 6 Hz, 1H, H-14), 2.89 (sept, 3J=7 Hz, 1H, H-21), 2.37

(s, 3H, H-19), 1.21 (d, 3J= 7 Hz, 3H, H-20), 1.17 (d, 3J= 7 Hz, 3H, H-22) ppm.


(C-1), 139.2 (C-3), 128.7 (C-9/C-11), 128.2 (C-2), 127.5 (C-4), 124.9 (C-8), 124.6 (C-

12), 103.1 (C-13), 87.7 (C-15), 87.4 (C-17), 85.7 (C-18), 85.2 (C-14), 31.5 (C-21),

22.4 (C-20), 21.6 (C-22), 19.4 (C-19) ppm. MS (ESI+): m/z 528.0353 [26–2Cl–H]+

(mex = 528.0354).

76

[chloride(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)osmium(II)]chloride (27)

The synthesis of compound 27 was performed following general complexation

procedure C, using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (90 mg,

0.31mmol) and [Os(η6-p-cymene)Cl2]2 (121 mg, 0.15 mmol). After complete work up

the solid product was washed with dichloromethane (2 × 10 mL) and dried using

rotavap. Yield: 168 mg, (80%), black solid. Elemental analysis found: C, 39.28; H,

3.94; N, 5.87; S, 8.96, calculated for C22H25Cl2N3O2OsS2·0.1C6H14: C, 38.93; H, 3.82;

N, 6.03; S, 9.20. 1HNMR (400.13 MHz, MeOD-d4, 25 oC): δ = 9.50 (d, 3J= 6 Hz, 1H,

H-1), 8.43 (d, 3J= 9 Hz, 1H, H-4), 8.21 (t, 3J= 8 Hz, 1H, H-3), 8.04 (d, 3J= 9 Hz, 2H,

H-9/H-11), 7.73 (d, 3J= 8 Hz, 1H, H-2), 7.63 (d, 3J)= 9 Hz, 2H, H-8/H-12), 6.14 (d,


(d, 3J= 6 Hz, 1H, H-14), 2.64 (sept, 3J=7 Hz, 1H, H-21), 2.27 (s, 3H, H-19), 1.19 (d,

3J= 7 Hz, 3H, H-20), 1.08 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR (100.61 MHz,

CDCl3, 25 °C): δ = 158.4 (C-1), 139.5 (C-3), 129.5 (C-9/C-11), 127.3 (C-2), 125.2

(C-4), 124.2 (C-8/C-12), 96.3 (C-13), 79.4 (C-15), 78.9 (C-17), 76.3 (C-18), 73.7 (C-

14), 31.0 (C-21), 22.6 (C-20), 21.5 (C-22), 18.1 (C-19) ppm. MS (ESI+): m/z

618.0918 [27–2Cl–H]+ (mex = 618.0925).

77

Scheme 4

Targeting Epigenetic Changes: Multitargeted

Vorinostat (SAHA)-derived Metal Complexes with

Potent Anticancer and Histone Deacetylase Inhibitory

Activity

78

2.6. Synthesis of PCA based succinic/suberanilic carboxylic acid ligands

To a stirred solution of succinic anhydride or suberic anhydride (1.3 equiv.) in

chloroform (90 mL), N-(4-aminophenyl)-pyridine-2-carbothioamide (1 equiv.)

dissolved in chloroform (90 mL) was added dropwise slowly at 0 °C under an

atmosphere of nitrogen. After complete addition, the mixture stirred for 45 minutes at

room temperature.109 The yellow precipitates was filtered, washed with hot water (100

mL) followed by recrystallization from hot methanol and dried under vacuo to yield

analytically pure yellow solid product. Moreover, the reaction mixture filtrate was

also dried using rotavap followed by washing with hot water, recrystallization from

hot methanol and dried under vacuum to afford second crop of pure product.

4-oxo-4-((4-(pyridine-2-carbothioamido)phenyl)amino)butanoic acid (28)

Compound 28 was prepared following the general procedure using succinic anhydride

(284 mg, 2.83 mmol, 1.3 eq.) and N-(4-aminophenyl)-pyridine-2-carbothioamide(500

mg, 2.18 mmol, 1.0 eq.). Single crystals suitable for X-ray diffraction analysis of 28

were obtained by slow evaporation of methanol. Yield: 438 mg, (61%), yellow

powder. Elemental analysis found: C, 58.03; H, 4.59; N, 12.39, calculated for

C16H15N3O3S: C, 58.34; H, 4.59; N, 12.76. 1H NMR (400.13 MHz, DMSO-d6, 25 °C)

δ = 12.21 (s, 1H, -CSNH), 12.12 (brs, 1H, -OH), 10.09 (s, 1H, -CONH ), 8.67 (d, 3J=

5 Hz, 1H, H-4), 8.52 (d, 3J= 8 Hz, 1H, H-1), 8.03 (t, 3J= 8 Hz, 1H, H-3), 7.90 (d, 3J=

8 Hz, 2H, H-9/H-11), 7.64 (d, 3J= 8 Hz, 3H, H-2/H-8/H-12), 2.57 (m, 2H, H-15), 2.54

(m, 2H, H-14) ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ = 188.8 (C-6),

173.8 (C-13), 170.1 (C-16), 152.7 (C-5), 147.3 (C-1), 137.8 (C-3), 137.5 (C-7), 134.0

(C-10), 126.3 (C-8/C-12), 124.6 (C-2), 124.3 (C-4), 118.7 (C-9/C-11), 31.0 (C-14),

28.8 (C-15) ppm. MS (ESI+): m/z 352.0732 [28+Na]+ (mex = 352.0720); MS (ESI-):

m/z 328.0756 [28–H]+ (mex = 328.0750).

79

8-Oxo-8-((4-(pyridine-2-carbothioamido)phenyl)amino)octanoic acid (29)

Compound 29 was prepared following the general procedure using suberic anhydride

(1.062 g, 6.80 mmol, 1.3 eq.) and N-(4-aminophenyl)-pyridine-2-

carbothioamide(1.200 g, 5.23 mmol, 1.0 eq.). Yield: 0.840 g, (41%), yellow powder.

Elemental analysis found: 62.60; H, 6.10; N, 11.23, calculated for C20H23N3O3S: C,

62.32; H, 6.01; N, 10.90. 1H NMR (400.13 MHz, DMSO-d6, 25 °C) δ = 12.20 (s, 1H,

-CSNH), 11.97 (brs, 1H, -OH), 9.89 (s, 1H, -CONH), 8.67 (d, 3J= 5 Hz, 1H, H-4),

8.52 (d, 3J= 8 Hz, 1H, H-1), 8.03 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.89 (d, 3J= 8 Hz,

2H, H-9/H-11), 7.65 (m, 3H, H-2/H-8/H-12), 2.31 (t, 3J= 7 Hz, 2H, H-14), 2.20 (t, 3J=

7 Hz, 2H, H-19), 1.59 (m, 2H, H-15), 1.50 (m, 2H, H-18), 1.30 (m, 4H, H-16/H-17)

ppm. MS (ESI+): m/z408.1358 [29+Na]+ (mex = 408.1340); MS (ESI-): m/z 384.1382

[29 – H]+ (mex = 384.1389)

2.7. Synthesis of PCA based succinic/suberic hydroxamic acid ligands

NH2OH was generated from NH2OH·HCl (4 eq.) and NaOCH3 (4 eq.) in methanol

(50 mL) and this mixture was stirred for 1 h. The solvent was evaporated under

reduced pressure and the residue was dried under vacuum overnight. Anhydrous

ethylchloroformate (2 eq.) and anhydrous TEA (3 eq.) were added to a solution of 28

or 29 (1 eq.) in anhydrous THF (200 mL) under nitrogen atmosphere. The reaction

mixture was stirred for 1 h at room temperature, while NH2OH (4 equiv.) was

dissolved in dry methanol (40 mL). It was added to the reaction mixture under

nitrogen atmosphere and stirred for another 24 h at room temperature. The reaction

mixture was diluted with deionized water (100 mL) and concentrated on a rotary

evaporator. A yellow precipitate formed which was filtered and subsequently washed

with deionized water (2 × 10 mL) and dried. The crude product was suspended in

dichloromethane (20 mL), filtered and washed with DCM (2 × 10 mL) to obtain a

yellow solid.

80

N1-hydroxy-N4-(4-(pyridine-2-carbothioamido)phenyl)succinamide (30)

Compound 30 was prepared following the general procedure using NH2OH·HCl (422

mg, 6.07 mmol, 4 eq.), NaOCH3 (328 mg, 6.07 mmol, 4 eq.) in methanol (50 mL), 4-

oxo-4-((4-(pyridine-2-carbothioamido)phenyl)amino)butanoic acid 28 (500 mg, 1.52

mmol, 1 eq.), anhydrous ethyl chloroformate (289 µL, 3.04 mmol, 2 eq.) and

anhydrous TEA (635 µL, 4.55 mmol, 3 eq.). Yield: 128 mg, (29%), yellow powder.

Elemental analysis found: 54.34; H, 4.58; N, 14.43, calculated for

C16H16N4O3S·0.25C4H8O·0.25CH2Cl2: C, 54.00; H, 4.86; N, 14.60. 1H NMR (400.13

MHz, DMSO-d6, 25 °C) δ = 12.20 (s, 1H, -CSNH), 10.44 (s, 1H, -NHOH), 10.13 (s,

1H, -CONH), 8.71 (brs, 1H, -OH), 8.66 (d, 3J= 5 Hz, 1H, H-4), 8.52 (d, 3J= 8 Hz, 1H,

H-1), 8.03 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.90 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.65

(m, 3H, H-2/H-8/H-12), 2.58 (t, 3J= 7 Hz, 2H, H-15), 2.30 (t, 3J= 7 Hz, 2H, H-14)

ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ = 188.9 (C-6), 170.3 (C-13),

168.3 (C-16), 152.7 (C-5), 147.3 (C-1), 137.8 (C-3), 137.5 (C-7), 134.0 (C-10), 126.3

(C-8/C-12), 124.6 (C-2), 124.3 (C-4), 118.7 (C-9/C-11), 31.5 (C-14), 27.4 (C-15)

ppm. MS (ESI+): m/z 367.0841 [30+Na]+ (mex = 367.0805); MS (ESI-): m/z 343.0865

[30–H]+ (mex = 343.0868).

N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide (31)

Compound 31 was prepared following the general procedure using NH2OH·HCl (361

mg, 5.19 mmol, 4 eq.), NaOCH3 (280 mg, 5.19 mmol, 4 eq.), 8-oxo-8-((4-(pyridine-2-

carbothioamido)phenyl)amino)octanoic acid 29 (500mg, 1.30mmol, 1 eq.), anhydrous

ethyl chloroformate (247 µL, 2.59 mmol, 2 eq.) and anhydrous TEA (543 µL, 3.89

81

mmol, 3 eq.). Yield: 168 mg, (32%), yellow powder. Elemental analysis found: 59.37;

H, 6.06; N, 13.91, calculated for C20H24N4O3S·0.1H2O: C, 59.71; H, 6.06; N, 13.93.

1H NMR (400.13 MHz, DMSO-d6, 25 °C) δ = 12.20 (s, 1H, -CSNH), 10.32 (s, 1H, -

NHOH), 10.01(s, 1H, -CONH), 8.67 (d, 3J= 5 Hz, 1H, H-4), 8.64 (brs, 1H, -OH), 8.52

(d, 3J= 8 Hz, 1H, H-1), 8.03 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.89 (d, 3J= 9 Hz, 2H,

H-9/H-11), 7.65 (m, 3H, H-2/H-8/H-12), 2.31 (t, 3J= 7 Hz, 2H, H-14), 1.94 (t, 3J= 7

Hz, 2H, H-19), 1.59 (m, 2H, H-15), 1.49 (m, 2H, H-18), 1.28 (m, 4H, H-16/H-17)

ppm. 13C{1H} NMR (100.61 MHz, DMSO-d6, 25 °C): δ = 188.9 (C-6), 171.3 (C-13),

169.1 (C-20), 152.7 (C-5), 147.3 (C-1), 137.8 (C-3), 137.5 (C-7), 134.0 (C-10), 126.3

(C-8/C-12), 124.6 (C-2), 124.3 (C-4), 118.8 (C-9/C-11), 36.4 (C-14), 32.2 (C-19),

28.4 (C-16/C-17), 25.0 (C-15/C-18) ppm. MS (ESI+): m/z 423.1467 [31+Na]+ (mex =

423.1446); MS (ESI+): m/z 399.1491 [31–H]+ (mex = 399.1503).

2.8. Synthesis of metal complexes of PCA based carboxylic acid and hydroxamic acid derivatives

A solution of dimeric [M(L)Cl2]2 (M = Ru, Os, Rh, Ir; L = η6-p-cymene, η5-

pentamethylcyclopentadienyl) precursor (1 eq.) in dry DCM (20 mL) was added to a

stirred solution of a pyridine-2-carbothioamide carboxylic or hydroxamic acid ligand

(2 equiv.) in dry THF (20 mL).Orange or dark red precipitates were formed and the

reaction mixture was stirred for 4 h at 40 °C under nitrogen atmosphere. After cooling

the reaction mixture to room temperature, it was placed in the fridge overnight. The

precipitates were filtered and washed with DCM (2 × 5 mL), followed by drying

using rotavap to yield the pure product. Moreover, the filtrate also concentrated in

vacuo upto 10 ml and n-hexane was added for precipitation in the fridge. The solvent

was decanted and the solid residue was washed with DCM (2 × 5 mL) and THF (2 × 5

mL), followed by drying using rotary evaporator to afford another batch of product.

82

[Chlorido(η6-p-cymene)(4-oxo-4-((4-(pyridine-2-carbothioamido-

κ2N,S)phenyl)amino)butanoic acid)ruthenium(II)]chloride (32)

Compound 32 was synthesized following the general complexation procedure using

28 (130 mg, 0.40 mmol, 2 eq.) and [Ru(η6-p-cymene)Cl2]2 (121 mg, 0.20 mmol, 1

eq.). Yield: 189 mg (75%), red solid. Elemental analysis found: C, 47.23; H, 4.55; N,

5.27, calculated for C26H29Cl2N3O3RuS·0.9CH2Cl2·0.4C6H14: C, 47.14; H, 4.91; N,

5.63. 1H NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.67 (dd, 3J= 6 Hz, 4J= 1 Hz, 1H,


6 Hz, 4J= 1 Hz, 1H, H-2), 7.80 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.59 (d, 3J= 9 Hz, 2H, H-

8/H-12), 6.06 (d, 3J= 6 Hz, 1H, H-19), 5.95 (d, 3J= 6 Hz, 1H, H-21), 5.91 (d, 3J= 6

Hz, 1H, H-22), 5.65 (d, 3J= 6 Hz, 1H, H-18), 2.71 (m, 5H, H-14/H-15/H-25), 2.21 (s,

3H, H-23), 1.21 (d, 3J= 7 Hz, 3H, H-24), 1.13 (d, 3J= 7 Hz, 3H, H-26) ppm.

13C{1H}NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 171.9

(C-16), 159.0 (C-1), 153.8 (C-5), 140.4 (C-3), 139.8 (C-7), 130.0 (C-8/C-12), 125.9

(C-2), 124.9 (C-4), 120.8 (C-9/C-11), 106.8 (C-20), 104.4 (C-17), 88.2 (C-19/C-21),

85.6 (C-22), 84.4 (C-18), 31.8 (C-15), 31.6 (C-25), 29.4 (C-14), 22.8 (C-24), 21.8 (C-

26), 18.8 (C-23) ppm. MS (ESI+): m/z 564.0895 [32–2Cl–H]+ (mex = 564.0886).

83


κ2N,S)phenyl)amino)butanoic acid)osmium(II)]chloride (33)


28 (100 mg, 0.30 mmol, 2 eq.) and Os(η6-p-cymene)Cl2]2 (120 mg, 0.15 mmol, 1 eq.).

Single crystals suitable for X-ray diffraction analysis were obtained by slow diffusion

of diethyl ether into a concentrated solution of 33 in methanol. Yield: 82 mg (37%),

dark red solid. Elemental analysis found: C, 41.42; H, 4.19; N, 5.51; S, 4.12,

calculated for C26H29Cl2N3O3OsS·1.30H2O: C, 41.74; H, 4.26; N, 5.62; S, 4.29. 1H

NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.58 (d, 3J= 6 Hz, 1H, H-1), 8.49 (d, 3J= 8

Hz, 1H, H-4), 8.28 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.80 (m, 2H, H-2/H-9/H-11),

7.59 (d, 3J= 9 Hz, 2H, H-8/H-12), 6.23 (d, 3J= 6 Hz, 1H, H-19), 6.13 (d, 3J= 6 Hz, 1H,

H-21), 6.08 (d, 3J= 6 Hz, 1H, H-22), 5.82 (d, 3J= 6 Hz, 1H, H-18), 2.70 (m, 5H, H-

14/H-15/H-25), 2.29 (s, 3H, H-23), 1.19 (d, 3J= 7 Hz, 3H, H-24), 1.09 (d, 3J= 7 Hz,

3H, H-26) ppm. 13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25

°C): δ = 194.0 (C-6), 175.4 (C-13), 171.7 (C-16), 159.7 (C-1), 153.8 (C-5), 140.5 (C-

3), 139.4 (C-7), 132.9 (C-10), 130.7 (C-8/C-12), 125.8 (C-2), 125.5 (C-4), 120.6 (C-

9/C-11), 98.1 (C-20), 97.3 (C-17), 80.0 (C-19), 79.7 (C-21), 77.1 (C-22), 74.8 (C-18),

31.7 (C-15), 31.5 (C-25), 29.3 (C-14), 23.1 (C-24), 22.0 (C-26), 18.6 (C-23) ppm. MS

(ESI+): m/z 654.1466 [33–2Cl–H]+ (mex = 654.1502).

84


κ2N,S)phenyl)amino)octanoic acid)ruthenium(II)]chloride (34)




5.97, calculated for C30H37Cl2N3O3RuS: C, 52.09; H, 5.39 N, 6.08. 1H NMR (400.13

MHz, MeOD-d4, 25 °C) δ = 9.67 (d, 3J= 6 Hz, 1H, H-1), 8.43 (d, 3J= 8 Hz, 1H, H-4),



Hz, 1H, H-25), 5.91 (d, 3J= 6 Hz, 1H, H-26), 5.65 (d, 3J= 6 Hz, 1H, H-22), 2.74 (sept,

3J= 7 Hz, 1H, H-29),2.42 (t, 3J= 8 Hz, 2H, H-14), 2.31 (t, 3J= 7 Hz, 2H, H-19), 2.21

(s, 3H, H-27), 1.74 (m, 2H, H-15), 1.64 (m, 2H, H-18), 1.43 (m,4H, H-16/H-17), 1.21

(d, 3J= 7 Hz, 3H, H-28), 1.13 (d, 3J= 7 Hz, 3H, H-30) ppm. 13C{1H} NMR (100.61

MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 173.8 (C-13), 171.8 (C-20),

158.6 (C-1), 154.2 (C-5), 140.2 (C-3), 139.4 (C-7), 134.9 (C-10), 129.6 (C-8/C-12),

125.5 (C-2), 124.9 (C-4), 120.8 (C-9/C-11), 106.5 (C-24), 104.1 (C-21), 88.1 (C-23)

88.0 (C-25), 85.3 (C-26), 84.1 (C-22), 37.2 (C-14), 33.0 (C-19), 31.4 (C-29), 28.8 (C-

16), 28.7 (C-17), 25.7 (C-15), 25.5 (C-18), 22.8 (C-28), 21.8 (C-30), 18.8 (C-27) ppm.

MS (ESI+): m/z 620.1521 [34–2Cl–H]+ (mex = 620.1529).

85


κ2N,S)phenyl)amino) octanoic acid)osmium(II)]chloride (35)


29 (120 mg, 0.31 mmol, 2 eq.) and [Os(η6-p-cymene)Cl2]2 (123 mg, 0.16 mmol, 1

eq.). Yield: 132 mg (54%), dark red solid. Elemental analysis found: C, 46.21; H,

5.11; N, 4.86; S, 3.61, calculated for C30H37Cl2N3O3OsS·0.5H2O 0.2C6H14: C, 46.43;

H, 5.10; N, 5.21; S, 3.97. 1H NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.57 (d, 3J= 6

Hz, 1H, H-1), 8.48 (d, 3J= 8 Hz, 1H, H-4), 8.27 (t, 3J= 8 Hz, 1H, H-3), 7.80 (m, 3H,

H-2/H-9/H-11), 7.58 (d, 3J= 9 Hz, 2H, H-8/H-12), 6.22 (d, 3J= 6 Hz, 1H, H-23), 6.12

(d, 3J= 6 Hz, 1H, H-25), 6.07 (d, 3J= 6 Hz, 1H, H-26), 5.81 (d, 3J= 6 Hz, 1H, H-22),

2.65 (sept, 3J=6 Hz, 1H, H-29), 2.42 (t, 3J= 7 Hz, 2H, H-14), 2.30 (t, 3J= 7 Hz, 2H, H-

19), 2.28 (s, 3H, H-27), 1.73 (m, 2H, H-15), 1.64 (m, 2H, H-18), 1.42 (m,4H, H-16/H-

17), 1.19 (d, 3J= 7 Hz, 3H, H-28), 1.09 (d, 3J= 7 Hz, 3H, H-30) ppm. 13C{1H} NMR

(100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 194.4 (C-6), 173.8 (C-

13), 171.8 (C-20), 160.7 (C-1), 153.9 (C-5), 141.0 (C-3), 139.7 (C-7), 132.8 (C-10),

131.5 (C-8/C-12), 126.4 (C-2), 126.2 (C-4), 120.9 (C-9/C-11), 98.2 (C-24), 97.5 (C-

21), 80.4 (C-23/C-25), 77.8 (C-26), 75.2 (C-22), 37.3 (C-14), 33.0 (C-19), 31.6 (C-

29), 28.8 (C-16), 28.7 (C-17), 25.6 (C-15), 25.5 (C-18), 23.2 (C-28), 22.1 (C-30), 18.9

(C-27) ppm.MS (ESI+): m/z 710.2092 [35–2Cl–H]+ (mex = 710.2113).

86

[Chlorido(η6-p-cymene)(N1-hydroxy-N4-(4-(pyridine-2-carbothioamido-

κ2N,S)phenyl)succinamide)ruthenium(II)]chloride (36)




7.49, calculated for C26H30Cl2N4O3RuS·1.1H2O.0.6CH2Cl2: C, 44.29; H, 4.67; N,

7.77. 1H NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.66 (d, 3J= 5 Hz, 1H, H-1), 8.44

(d, 3J= 8 Hz, 1H, H-4), 8.29 (t, 3J= 8 Hz, 1H, H-3), 7.85 (t, 3J= 6 Hz, 1H, H-2), 7.79

(d, 3J= 8 Hz, 2H, H-9/H-11), 7.59 (d, 3J= 9 Hz, 2H, H-8/H-12), 6.05 (d, 3J= 6 Hz, 1H,

H-19), 5.94 (d, 3J= 6 Hz, 1H, H-21), 5.90 (d, 3J= 6 Hz, 1H, H-22), 5.65 (d, 3J= 6 Hz,

1H, H-18), 2.74 (m, 3H, H-15/H-25), 2.49 (t,3J= 7 Hz, 1H, H-14),2.21 (s, 3H, H-23),


(100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 158.5 (C-1), 153.8 (C-

5), 140.2 (C-3), 139.2 (C-7), 132.5 (C-10), 129.6 (C-8/C-12), 125.7 (C-2), 125.2 (C-

4), 120.5 (C-9/C-11), 106.6 (C-20), 104.0 (C-17), 87.9 (C-19), 87.8 (C-21), 85.2 (C-

22), 84.2 (C-18), 32.2 (C-15), 31.3 (C-25), 28.2 (C-14), 22.7 (C-24), 21.8 (C-26), 18.7


87


κ2N,S)phenyl)succinamide)osmium(II)]chloride (37)


30 (100 mg, 0.29 mmol) and [Os(η6-p-cymene)Cl2]2 (115 mg, 0.15 mmol). Yield: 163

mg (73%),dark red solid. Elemental analysis found: C, 39.49; H, 4.02; N, 6.49; S,

3.72, calculated for C26H30Cl2N4O3OsS·1.1H2O.0.68CH2Cl2: C, 39.21; H, 4.14; N,

6.86; S, 3.92. 1H NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.58 (d, 3J= 6 Hz, 1H, H-

1), 8.50 (d, 3J= 8 Hz, 1H, H-4), 8.28 (td, 3J= 8 Hz, 4J= 1 Hz, 1H, H-3), 7.80 (m, 3H,



2.67 (m, 5H, H-14/H-15/H-25), 2.29 (s, 3H, H-23), 1.19 (d, 3J= 7 Hz, 3H, H-24), 1.09

(d, 3J= 7 Hz, 3H, H-26) ppm. 13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4

(0.1 mL), 25 °C): δ = 196.7 (C-6), 173.3 (C-13), 171.7 (C-16), 161.2 (C-1), 154.9 (C-

5), 141.2 (C-3), 140.8 (C-7), 134.1 (C-10), 131.7 (C-8/C-12), 126.7 (C-2), 125.3 (C-

4), 121.6 (C-9/C-11), 98.9 (C-20), 98.8 (C-17), 81.4 (C-19) 80.9 (C-21), 78.5 (C-22),

75.5 (C-18), 32.5 (C-25), 31.0 (C-15), 28.6 (C-14), 23.3 (C-24), 22.1 (C-26), 18.6 (C-

23) ppm. MS (ESI+): m/z 669.1575 [37–2Cl–H]+ (mex = 669.1594).

88


κ2N,S)phenyl)octanediamide)ruthenium(II)]chloride (38)


31 (80 mg, 0.20 mmol) and [Ru(η6-p-cymene)Cl2]2 (61 mg, 0.10 mmol). Yield: 77 mg


C30H38Cl2N4O3RuS·H2O: C, 49.72; H, 5.56; N, 7.73. 1H NMR (400.13 MHz, MeOD-

d4, 25 °C) δ = 9.67 (d, 3J= 6 Hz, 1H, H-1), 8.44 (d, 3J= 8 Hz, 1H, H-4), 8.29 (t, 3J= 8

Hz, 1H, H-3), 7.85 (t, 3J= 7 Hz, 1H, H-2), 7.80 (d, 3J= 9 Hz, 2H, H-9/H-11), 7.59 (d,

3J= 9 Hz, 2H, H-8/H-12), 6.06 (d, 3J= 6 Hz, 1H, H-23), 5.95 (d, 3J= 6 Hz, 1H, H-25),

5.91 (d, 3J= 6 Hz, 1H, H-26), 5.65 (d, 3J= 6 Hz, 1H, H-22), 2.74 (sept, 3J= 7 Hz, 1H,

H-29), 2.42 (t, 3J= 7 Hz, 2H, H-14), 2.31 (s, 3H, H-27), 2.11 (t, 3J= 8 Hz, 2H, H-19),

1.73 (m, 2H, H-15), 1.64 (m, 2H, H-18), 1.42 (m, 4H, H-16/H-17), 1.21 (d, 3J= 7 Hz,

3H, H-28), 1.13 (d, 3J= 7 Hz, 3H, H-30) ppm. 13C{1H} NMR (100.61 MHz, CDCl3

(0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 173.8 (C-13), 158.9 (C-1), 153.8 (C-5),

140.4 (C-3), 139.8 (C-7), 132.9 (C-10), 129.9 (C-8/C-12), 125.8 (C-2), 125.0 (C-4),

120.8 (C-9/C-11), 106.8 (C-24), 104.3 (C-22), 88.1 (C-23) 88.0 (C-25), 85.4 (C-26),

84.3 (C-21), 37.4 (C-14), 34.3 (C-19), 31.5 (C-29), 29.1 (C-16), 29.1 (C-17), 25.8 (C-

15), 25.1 (C-18), 22.8 (C-28), 21.8 (C-30), 18.8 (C-27) ppm. MS (ESI+): m/z

635.1630 [38–2Cl–H]+ (mex = 635.1657).

89


κ2N,S)phenyl)octanediamide)osmium(II)]chloride (39)


31 (120 mg, 0.30 mmol) and [Ru(η6-p-cymene)Cl2]2 (118.4 mg, 0.15 mmol). Yield:

141 mg (59%), dark red solid. Elemental analysis found: C, 43.23; H, 4.92; N, 6.48;

S, 3.59, calculated for C30H38Cl2N4O3OsS·H2O.0.3CH2Cl2: C, 43.36; H, 4.88; N,

6.68; S, 3.82. 1H NMR (400.13 MHz, MeOD-d4, 25 °C) δ = 9.58 (d, 3J= 6 Hz, 1H, H-

1), 8.50 (d, 3J= 8 Hz, 1H, H-4), 8.28 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.81 (m, 3H,



2.65 (sept, 3J=7 Hz, 1H, H-29), 2.42 (t, 3J= 8 Hz, 2H, H-14), 2.29 (s, 3H, H-27), 2.11

(t, 3J= 7 Hz, 2H, H-19), 1.73 (m, 2H, H-15), 1.65 (m, 2H, H-18), 1.42 (m, 4H, H-

16/H-17), 1.17 (d, 3J= 7 Hz, 3H, H-28), 1.09 (d, 3J= 7 Hz, 3H, H-30) ppm. 13C{1H}

NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 176.9 (C-13),

173.8 (C-20), 159.8 (C-1), 154.1 (C-5), 140.5 (C-3), 139.7 (C-7), 132.7 (C-10), 130.8

(C-8/C-12), 125.7 (C-2), 125.1 (C-4), 120.9 (C-9/C-11), 98.1 (C-24), 97.5 (C-21),

80.2 (C-23), 79.8 (C-25), 77.3 (C-26), 74.7 (C-22), 37.4 (C-14), 34.4 (C-19), 31.6 (C-

29), 29.2 (C-16), 29.1 (C-17), 25.8 (C-15), 25.1 (C-18), 23.2 (C-28), 22.1 (C-30), 18.7


90

[Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-

carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride (40)


31 (130 mg, 0.32 mmol) and [Rh(Cp*)Cl2]2 (100 mg, 0.16 mmol). Yield: 158 mg

(69%), orange solid. Elemental analysis found: C, 49.06; H, 5.58; N, 7.87, calculated

for C30H39Cl2N4O3RhS·0.4CH2Cl2: C, 49.11; H, 5.40; N, 7.54. 1H NMR (400.13

MHz, MeOD-d4, 25 °C) δ = 10.07 (s, 1H, -CONH), 9.07 (d, 3J= 6 Hz, 1H, H-1), 8.45

(d, 3J= 8 Hz, 1H, H-4), 8.34 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.94 (td, 3J= 6 Hz, 4J=

2 Hz, 1H, H-2), 7.82 (dd, 3J= 8 Hz, 4J= 2 Hz, 2H, H-9/H-11), 7.61 (d, 3J= 9 Hz, 2H,

H-8/H-12), 2.42 (t, 3J= 7 Hz, 2H, H-14), 2.10 (t, 3J= 7 Hz, 2H, H-19), 1.74 (s, 15H,

Cp*-CH3), 1.65 (m, 4H, H-15/H-18), 1.42 (m, 4H, H-16/H-17) ppm. 13C{1H}NMR

(100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 173.6 (C-13), 171.5

(C-20), 154.9 (C-1), 154.6 (C-5), 140.8 (C-3), 139.5 (C-7), 130.0 (C-8/C-12), 125.7

(C-2), 125.6 (C-4), 120.5 (C-9/C-11), 98.5 (Cp*-C), 37.0 (C-14), 32.8 (C-19), 28.6

(C-16), 28.5 (C-17), 25.4 (C-15), 25.3 (C-18), 9.1 (Cp*-CH3) ppm. MS (ESI+): m/z

637.1720 [40–2Cl–H]+ (mex = 637.1727).

91

[Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-

carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride (41)


31 (120 mg, 0.30 mmol) and [Ir(Cp*)Cl2]2 (119 mg, 0.15 mmol, 1 eq.). Yield: 130 mg


C30H39Cl2IrN4O3S·0.5CH2Cl2: C, 43.54; H, 4.79; N, 6.66. 1H NMR (400.13 MHz,

MeOD-d4, 25 °C) δ = 10.06 (s, 1H, -CONH), 9.06 (d, 3J= 6 Hz, 1H, H-1), 8.49 (d, 3J=

8 Hz, 1H, H-4), 8.32 (td, 3J= 8 Hz, 4J= 2 Hz, 1H, H-3), 7.91 (td, 3J= 6 Hz, 4J= 1 Hz,

1H, H-2), 7.82 (dd, 3J= 9 Hz, 4J= 2 Hz, 2H, H-9/H-11), 7.62 (d, 3J= 9 Hz, 2H, H-8/H-

12), 2.42 (t, 3J= 7 Hz, 2H, H-14), 2.10 (t, 3J= 7 Hz, 2H, H-19), 1.76 (s, 15H, Cp*-

CH3), 1.71 (m, 2H, H-15), 1.65 (m, 2H, H-18), 1.41 (m, 4H, H-16/H-17) ppm.


(C-13), 171.8 (C-20), 156.1 (C-1), 154.9 (C-5), 140.8 (C-3), 139.8 (C-7), 132.8 (C-

10), 131.1 (C-8/C-12), 125.8 (C-2), 125.7 (C-4), 120.8 (C-9/C-11), 91.6 (Cp*-C),

37.2 (C-14), 33.0 (C-19), 28.9 (C-16), 28.8 (C-17), 25.7 (C-15), 25.6 (C-18), 8.9

(Cp*-CH3) ppm. MS (ESI+): m/z 727.2294 [41–2Cl–H]+ (mex = 727.2315).

92

CHAPTER 3: RESULTS & DISCUSSION

93

Scheme 1

Anticancer Ru(η6-p-cymene)Complexes of 2-

Pyridinecarbothioamides: A Structure–Activity

Relationship Study

94

Scheme 3.1. Anticancer Ru(η6-p-cymene)complexes of 2-pyridinecarbothioamides: A structure–activity relationship study

3.1.1. Results and discussion

With the aim to establish structure activity relationship and to inspect the influence of

the lipophilicity of the coordinated ligand with regard to biological activity, a series of

pyridine-2-carbothioamide complexes substituted at the phenyl ring by varying the

substituents in terms of electron-withdrawing and -donating properties as well as

considering the protonation potential of the substituents was prepared. Their

biological activity against a panel of cell lines while attempting to rationalize their

cytotoxicity with regards to the physicochemical properties.

The PCA ligands 6 and 7 were synthesized by adopting a literature procedure used

before for the preparation of 1–5 and 8.61, 95, 97 Briefly, the N-substituted aniline was

refluxed for 48–72 h with an excess of sulfur and 2-picoline in the presence of

catalytic amounts of sodium sulfide (Scheme 1). After work up, the ligands were

purified by recrystallization from methanol/acetonitrile, to yield the PCAs from 77 to

83% yield, which is in a similar range as reported previously for related

compounds.61, 97

Scheme 1. Synthesis of the PCA ligands 1–8 and the respective Ru(cym)Cl complexes 9–16.

95

The PCA ligands were characterized by NMR spectroscopy, ESI-MS, elemental and

single crystal X-ray diffraction analysis, if crystals were obtained. The 1HNMR

spectra of PCAs in deuterated solvents (CDCl3/DMSO-d6) featured the thioamide

proton resonance at ca. 12 ppm. Comparison of the chemical shifts found for

equivalent 2-picolinamides shows that the amide protons of 6 and 7 were more

deshielded which caused a downfield shift of ca 2.5 ppm.110 The chemical shifts of

the individual pyridine proton and carbon atoms were observed in the range 7.65–8.70

ppm and 124.2–157.4 ppm, respectively, and both were practically unaffected by the

nature of N-phenyl substituents which however impacted the proton and carbon atom

shifts observed for the phenyl ring. For example, the H-9/H-12 protons as well as

H8/H12 protons of ligands 3 and 4, bearing electron-withdrawing chloro and electron-

donating methyl substituents, respectively, were shifted by ~1 ppm. A similar trend

was observed for the C9/C11 and C8/C12 carbon atoms with chemical shifts of ~3

ppm in the 13C{1H} NMR spectra.

Single crystals of the ligands N-(4-bromophenyl)pyridine-2-carbothioamide 3 and N-

(4-acetylphenyl)pyridine-2-carbothioamide 6, suitable for X-ray diffraction analysis,

were obtained by slow evaporation from methanol and they crystallized in the triclinic

and monoclinic space groups P-1 and P21/c, respectively. Selected bond lengths and

angles are listed in Table 2 and the crystallographic data are shown in Table 1. In the

molecular structures of both 3 and 6 (Figure 31), the pyridine and phenyl ring are co-

planar. In general, the structures of both compounds are very similar. The C–S bond

lengths are approximately the same, as were the torsion angles for S–C6–C5–N1 at -

179.7(1) and -172.1(1)°. Both 3 and 6 showed an offset π-stacking interaction

between the phenyl substituents of adjacent molecules.

96

Figure 31. The molecular structures of 3 (top) and 6 (bottom) drawn at 50% probability level.

Table 1. X-ray diffraction measurement parameters for single crystals of ligands 3 and 6.

3 6

CCDC 1540259 1540262

Formula C12H9BrN2S C14H12N2OS

Molecular weight (g mol-1) 293.19 256.32

Crystal description yellow needles orange needles

Crystal size (mm) 0.32 × 0.32 × 0.28 0.32×0.12×0.10

Wavelength (Å) 0.71073 0.71073

Temperature (K) 372(2) 372(2)

Crystal system triclinic monoclinic

Space group P-1 P21/c

a (Å) 7.7193(3) 11.379(5)

b (Å) 8.5870(3) 5.684(5)

c (Å) 9.6210(4) 18.815(5)

α (°) 76.991(2) 90.000(5)

β (°) 80.006(2) 92.780(5)

γ (°) 65.540(2) 90.000(5)

Volume (Å3) 563.27(4) 1215.5(12)

Z 2 4

Final R indices [I>2σ(I)] 0.0294 0.0686

R indices (all data) 0.0775 0.1181

Goodness-of-fit on F2 1.038 1.014

97

Table 2. Selected bond lengths (Å) and angles (°) for ligands 3 and 6 and complexes 12 and

13.

3 6 12 13

Ru–S - - 2.3469(7) 2.3483(16)

Ru–Cl1 - - 2.4001(7) 2.4059(17)

Ru–N1 - - 2.102(2) 2.106(5)

C6–S 1.662(18) 1.656(19) 1.695(3) 1.699(6)

C6–N2 1.341(2) 1.347(2) 1.319(4) 1.318(7)

C6–C5 1.515(2) 1.504(3) 1.484(4) 1.477(8)

C5–N1 1.345(2) 1.341(2) 1.353(3) 1.375(8)

C1–N1 1.331(2) 1.338(2) 1.350(3) 1.342(7)

C7–N2 1.405(2) 1.403(2) 1.433(3) 1.433(7)

N1–Ru–S 81.36(6) 81.53(14)

N1–Ru–Cl1 83.68(6) 83.17(14)

S–Ru–Cl1 89.44(3) 90.40(6)

The N-phenyl-substituted pyridine-2-carbothioamides (PCAs) 1–8 were used to

prepare a series of new Ru(cym) complexes 10–16 and for comparison 9 61(Scheme 1)

by adding the dimeric precursor [Ru(cym)Cl]2 in absolute dichloromethane to a

solution of the respective PCA ligand in absolute tetrahydrofuran. After stirring the

reaction mixture for 4 h at 40 °C and workup, the mononuclear complexes were

obtained in 62–87% yield.

Surprisingly, conducting this complexation reaction under the same conditions in

methanolic solution resulted in the appearance of two species in the 1HNMR spectra.

In this protic solvent, the thioamide group was deprotonated which resulted in N,N'-

coordination (10–20%) of the mono-anionic PCA rather than N,S-coordination as in

case of neutral PCA.110-111 This switch in coordination mode in protic solvents was

found to be dependent on time, temperature and the pH value. In an attempt to avoid

formation of a mixture of coordination isomers, we aimed to shift the equilibrium to

maintain the thioamide in its protonated state. For this purpose, the PCAs were

dissolved in 3.3% acetic acid/methanol and Ru(cym) was added. This procedure

yielded only one species with PCA acting as a neutral N,S-chelating ligand. However,

this method resulted in low yield (40–54%) which could be improved to 80–90%

when absolute THF and DCM were used. Furthermore, 11 was also obtained by using

absolute DCM as the solvent and stirring the reaction mixture for 4 h at room

98

temperature, following a literature procedure.112 Unfortunately, the latter method

cannot be applied for all ligands because of their low solubility in DCM, which

therefore requires the use of the solvent combinations as mentioned before.

The 1HNMR spectra (Figure 50–51; Appendix A) of the organometallic compounds

were recorded in MeOD-d4/CDCl3.The H4 and H1 proton of the pyridine ring were

most deshielded, which confirms N,S-bidentate coordination of the pyridine nitrogen

and thioamide moiety to the Ru. The most drastic shift compared to the ligand was

observed for H1 at ca.1 ppm (compare Figure 31 for 3 and 11). The methyl protons

H19 of p-cymene appeared as singlets while the isopropyl protons H20 and H22

coupled to H21 and therefore were detected as two doublets in the range of 2.10–2.43

ppm and 1.02–1.21 ppm, respectively. The p-cymene aromatic protons H14, H15,

H17 and H18 were observed in the range of 5.54–6.94 ppm as four doublets (Figure

32). Signal for the thioamide proton were not observed in all complexes, possibly due

to fast exchange of the NH proton in deuterated solvents. In the 13C{1H}NMR spectra

(Figure 52–53; Appendix A) of the Ru complexes, the quaternary carbon atom of the

thioamide functionality appeared in the range of ~192–197 ppm for complexes 12 and

15, however, this carbon atom was not detectable for the other complexes. Similarly,

C5 and C7 were not visible in 11. The pyridine carbon atoms C5 and C1 next to the

pyridine nitrogen coordinated to the Ru center were detected most downfield and

appeared in the range of 155–160 ppm and 157–160 ppm, respectively. The remaining

carbon atoms C2, C3 and C4 of the pyridine ring appeared in the range of 123.4–

140.2 ppm.

99

Figure 32. Comparison of the 1H NMR spectra in MeOD-d4 recorded for ligand 3 and after

complexation with [Ru(cym)Cl2]2. The protons of the PCA ligand were shifted after

coordination to Ru and the most significant change was observed for H1 after complexation

as indicated by a shift from 8.67 ppm in 3 to 9.66 ppm in 11.

The complexes were also characterized by electrospray ionization mass spectrometry

(ESI-MS). The ESI-mass spectra (Figure 54–55; Appendix A) of all complexes

featured a peak at a m/z value assigned to [M–Cl]+ ions but the most abundant peak

was from a [M–2Cl–H]+ species in dichloromethane solutions.

The molecular structures of 12 and 13 were determined by single crystal X-ray

crystallography. Crystallographic parameters including bond lengths and bond angles

are given in Tables 2 and 3. Single crystals of 12 were grown by slow diffusion of

diethyl ether into a methanol solution and crystallized in the space group C2/c. A

single crystal of 13 with a space group of P21/n was obtained by slow evaporation of a

saturated solution of the complex in methanol and ethyl acetate. The complexes

crystallized in monoclinic crystal systems with the Ru center adopting pseudo

octahedral coordination geometry.

In contrast to organometallic complexes of N-phenyl-picolinamide where an N,N’

coordination mode was found,110 the molecular structures of 12 and 13 showed an

N,S-coordination mode of the PCA ligands towards ruthenium (Figure 33). The

charge of these cationic complexes was balanced by chloride as the counterion. The

bite angles between adjacent atoms in the coordination sphere of ruthenium were

around 85°. The Ru–S bond lengths at ca. 2.347 Å were very similar in the complexes

100

and the C6–S bond was elongated as compared to the ligands, indicating more single

bond character (Table 2). In line, the C6–N2 distance was shorter than in 3 and 6,

indicating increased double bond character upon coordination of the Ru center to the

S atom. The Ru–Cl1 bond lengths observed were2.4001(7)and 2.4059(17)Å,

respectively for 12 and 13 (Table 2).The torsion angle S–C6–C5–N1 for a

structurally-related osmium complex was 4.1(4)°,61 while it was 17.63° and 19.14° for

12 and 13, and analogous Ru–PCAmaleimide derivative.112 In contrast the analogous

torsion angles C6–N2–C7–C12 for the Ru complexes 12 and 13 were smaller than in

the Os derivative but similar to the Ru–PCAmaleimide derivative.112

In the structures of 12 and 13, two enantiomers were present. In case of 13 they were

linked through π stacking of the pyridine moieties of the PCA ligand (3.958 Å;

Figure 34). In addition, the chloride counterions Cl2 were found in both structures to

be involved in H bonds with the amide NH and the N2–H···Cl2 distances were 3.078

Å and 3.071 Å for 12 and 13.

Figure 33. The molecular structures and atom numbering for metal complexes 12 and 13 at

50% probability level. Solvent molecules and counterions were omitted for clarity.

101

Figure 34. Molecular structure of 13 with the π interaction between the pyridine rings of two

molecules indicated. Co-crystallized solvents and counterions were omitted for clarity.

Table 3. X-ray diffraction measurement parameters for single crystals of 12 and 13.

12·CH3OH·H2O 13·C4H8O2

CCDC 1540260 1540261

Formula C24H26Cl2N2RuS O2 C27H34Cl2N2O3RuS


Crystal description red block red block

Crystal size (mm) 0.2 × 0.2 × 0.05 0.38 × 0.14 × 0.08

Wavelength (Å) 0.71073 0.71073


Crystal system monoclinic monoclinic

Space group C2/c P21/n

a (Å) 31.976(5) 14.1171(5)

b (Å) 8.665(5) 8.4673(3)

c (Å) 24.103(5) 25.211(10)

α (°) 90 90

β (°) 124.757(5) 101.823(3)

γ (°) 90 90

Volume (Å3) 5486.o(3) 2949.63(19)

Z 8 4

Final R indices [I>2σ(I)] R1 = 0.0356, wR2 = 0.0968

R1 = 0.0563, wR2 = 0.1053

R indices (all data) R1 =0.0405, wR2 = 0.1001

R1 = 0.1100, wR2 = 0.1250


102

3.1.2. Stability in aqueous solution

The parent compounds to this series of PCA–Ru(cym) derivatives were shown to be

very stable under acidic conditions,61 while they undergo a chlorido/aqua ligand

exchange reaction in water. To determine the aqueous stability of complexes 9 and

10, they were dissolved in D2O and 1H NMR spectra were recorded over a time

course of 0.5, 3, 24, 48 and 72 h (Figure 35). The compounds hydrolyzed very quickly

to form an aqua complex and even after 30 mins of incubation in D2O, more than 60%

of the complex was already hydrolyzed. While after 2 h two sets of peaks for the

chlorido and aqua complexes can be detected, the 1H NMR spectrum recorded after

24 h shows the conversion to the aqua complex to be complete, as indicated by a well-

resolved spectrum. The formed aqua species were stable for more than a week as

demonstrated by 1H NMR spectroscopy.

Figure 35.1H NMR spectra of 9 in D2O recorded after 0.5, 2 and 24 h, showing the

chlorido/aqua ligand exchange reaction to occur very rapidly. The dashed grey lines indicate

the positions of the protons of the chlorido complex 9.

The NMR experiments were complemented by ESI-MS studies with a special focus

on the stability in presence of 60 mMHCl, and compared to that in aqueous solutions.

The former environment was chosen to resemble stomach conditions, and estimate

stability in acidic media as one of the beneficial conditions for potential oral

administration. The incubation mixtures were analyzed after 0.5, 24, 72 h and 7 days.

The spectrum of 9 dissolved in water featured a peak at m/z 467.0556 as the base peak

which was assigned to [9–H–2Cl]+ (m/zcalc 467.0531; Figure 36). The spectrum hardly

changed over the time course of a week and the latter peak was still the most

103

abundant. Incubation of 9 in 60 mMHCl on the other hand gave a mass spectrum in

which the peak assigned to the [9–H–2Cl]+ was still the most abundant, but in

addition a peak at m/z 503.0302 was detected and assigned to [9–Cl]+ (m/zcalc

503.0295). In HCl solution an exchange of the thiocarbamide S with an O atom was

observed with peaks at m/z 451.0778 and 487.0541 for [9O–H–2Cl]+ and [9O– Cl]+

respectively (Figure 36).

Figure 36. ESI-mass spectrum of 9 after 7 days of incubation in water (bottom) or 60 mM

HCl (top). The mass spectrum in HCl shows the partial exchange of the thiocarbamide sulfur

atom of 9 with O (9O).

3.1.3. In vitro antiproliferative activity and lipophilicity

Carbothioamides are potent gastric mucosal protectants.113 The fluoro-substituted

PCA 1 and structurally-related N-(2,6-difluorophenyl)-pyridine-2-carbothioamide

exhibited very low acute toxicities in mouse models, indicating high tolerability in

vivo.113 We reported earlier that the coordination of Ru or Os centers to PCAs results

in potent antiproliferative agents in human ovarian teratocarcinoma (CH1), colon

carcinoma (SW480) and non-small cell lung cancer (A549) cells after 96 h exposure

with the p-fluoro derivative 9 being the most potent Ru compound in the MTT

assay.61 This derivative was included in this study as a benchmark and compared to its

ligand 1 and the analogous 2–8 as well as their respective complexes 10–16 in terms

of their antiproliferative activity in sulforhodamine B (SRB) assays with human

colorectal carcinoma (HCT116), non-small cell lung carcinoma (H460), cervical

104

carcinoma (SiHa) and colon carcinoma (SW480) cells. The potential of ligands and

metal complexes to inhibit the growth of cancer cells is summarized in Table 4.

The Ru(cym) complexes 9–13 and 15 exhibited potent cytotoxic activity in HCT116,

NCI-H460 and SiHa cells with IC50 values in the low micromolar range, which is

clearly associated with the cytotoxic activity of their respective PCA ligands and gave

similar IC50 values as the complexes in these cell lines. However, in case of 14 and

16, complexation reduced the cytotoxic potency of the ligands, with 16 being the least

active derivative. The SW480 human colon carcinoma cells were the most chemo-

resistant cells included in this assay. However, with the exception of 16, complexation

significantly enhanced the cytotoxicity of ligands 1–7 and the complexes 9–15 gave

IC50 values in the range 7.8–15 μM in this cell line. Surprisingly, the ruthenium

complex 14 bearing the most active ligand 6 was less cytotoxic than its uncoordinated

ligand. It should be noted that the chloride ions present in the cell culture medium

should prevent chlorido/aqua ligand exchange reactions to occur.

105

Table 4. In vitro anticancer activity (mean IC50 values ± standard deviations) of PCA ligands

1–8 and their respective Ru(cym) complexes 9–16 in human colorectal (HCT116), non-small

cell lung (NCI-H460) cervical (SiHa) carcinoma cell lines and colon carcinoma (SW480)

cells (exposure time 72 h).

Compound IC50 value (µM)

HCT116 NCI-H460 SiHa SW480

1 5.7± 0.7 7.8 ± 1.8 16± 6 33± 2

2 4.3 ±1.3 3.8 ± 0.3 10 ± 1 23±2

3 5.2 ± 1.3 5.0 ± 0.2 11 ± 1 23± 6

4 9.2 ± 2.3 9.5 ± 0.5 28 ± 3 149 ± 69

5 9.8 ± 3.4 11 ± 1 35 ± 6 77 ± 20

6 1.1 ± 0.2 1.1 ± 0.1 5.9 ± 2.1 25 ± 12

7 13 ± 3 12 ± 1 38 ± 5 96 ± 15

8 59 ± 7 52 ± 1 97 ± 0.2 >300

9 6.5 ± 0.3 10 ± 2 8.3± 0.7 9.9± 0.7

10 5.5 ± 0.4 6.2± 0.5 13 ± 1 7.8 ± 0.7

11 7.1 ± 1.2 8.2± 0.8 15 ± 1 9.9 ± 1.3

12 8.7 ± 2.5 9.4 ± 1.0 19 ± 1 8.8± 1.5

13 12 ± 1 15± 2 35 ± 4 11 ± 1

14 17 ±2 23± 4 50 ± 3 15 ± 1

15 10 ± 0.4 15 ± 1 33 ± 2 12 ± 1

16 146 ± 19 >300 >300 >300

As the cytotoxicity of anticancer agents is often linked to their ability to accumulate in

cells, the lipophilicity of 1–8 was calculated. Higher lipophilicity allows compounds

to pass through membranes more efficiently and is often given as octanol/water

partition coefficient (logP). The octanol/water partition coefficient was calculated

(clogP) using Chemdraw 12.0, molinspiration (www.molinspiration.com) and

ALOGPS 2.1 (Table 5). As the Ru(cym)Cl moiety is present in all the

organoruthenium complexes 9–16, the clogP values should depend on ligands 1–8

only. In general, the most lipophilic ligands 1–4 were the most potent cytotoxins

when coordinated to a Ru moiety. The least lipophilic ligand 8 resulted in the least

106

active anticancer agent 16, signifying the major role of lipophilicity in the bioactivity

of these compounds.

Table 5. clogP values for ligands 1–8 calculated with ChemDraw 12.0,

Molinspiration(www.molinspiration.com) and ALOGPS 2.1.114

Compound clogP

ChemDraw Molinspiration ALOGPS 2.1

1 2.87 2.80 2.59

2 3.44 3.32 3.05

3 3.59 3.45 3.18

4 3.12 3.09 2.79

5 2.61 2.69 2.36

6 2.25 2.54 2.29

7 2.79 2.74 2.54

8 1.40 1.71 1.64

3.1.4. Quantitative estimate of drug-likeness of ligands

As the compounds were developed with the aim to achieve oral application, the

quantitative estimate of drug-likeness was calculated to predict their potential as

orally active compounds. The weighted quantitative estimate of drug-likeness of the

ligands based on maximum information content (QEDwmo) was determined for ligands

1–8 (Table 6). The PCAs 1–8 showed excellent drug-likeness with QEDwmo values

around 0.8–0.9. Interestingly, ligand 6 has highest QEDwmo value of 0.91 and was

also the most potent compound. However, its complex 14 was only moderately active

in the cytotoxicity assay. 1–4 were found to have fairly similar QEDwmo and IC50

values in all cell lines. Furthermore, their respective complexes also shared the same

trend in cytotoxic studies.

107

Table 6. The calculated molecular properties used for the calculation of the quantitative estimate of druglikeness (QED). MW (molecular weight), clogP for

the ligands using the average logP of seven different programs via the ALOGPS 2.1 applet at http://www.vcclab.org. HBA (hydrogen bond acceptor), HBD

(hydrogen bond donor), PSA (polar surface area) calculated viawww.molinspiration.com or ChemBio3D 12.0 software, ROTB (rotatable bonds), AROM

(number of aromatic rings) and Alerts (number of structural alerts). Calculation of the weighted QED for maximum information content (QEDwmo) was

carried out according to ref.115

Compound MW ALOGPS HBA HBD PSA ROTB AROM Alerts Unweighted QEDwn Weighted QEDw

mo

1 232.27 2.59 2 1 24.92 3 2 1 0.79 0.82

2 248.72 3.05 1 1 24.92 3 2 1 0.72 0.83

3 293.18 3.18 1 1 24.92 3 2 1 0.75 0.86

4 228.31 2.79 1 1 24.92 3 2 0 0.73 0.86

5 244.31 2.36 2 1 34.15 4 2 0 0.91 0.90

6 256.32 2.29 2 1 41.99 4 2 0 0.93 0.91

7 257.35 2.54 2 1 28.16 4 2 0 0.91 0.91

8 229.30 1.64 2 2 50.94 3 2 1 0.84 0.79

108

Scheme 2

Impact of Metal Ions and Halide Leaving Groups on

the Biological Activity of Organometallic N-(4-

fluorophenyl)pyridine-2-carbothioamide Anticancer

Agents

109

Scheme 3.2. Impact of metal ions and leaving halido groups on the biological activity of organometallic N-(4-fluorophenyl)pyridine-2-carbothioamide anticancer agents

3.2.1. Introduction

3.2.1. Results and Discussion

The careful modification at phenyl ring of PCAs and their conversion in to

Ru(cymene) and Os(cymene) complexes61, 112, 116 led to the identification of potent

antiproliferative agents. In this regard, the effect of different metal ions ( RhIII and

IrIII) and leaving group (Cl, Br and I) on the biological properties of the most

cytotoxic Ru(cymene)Cl complex of fluorinated–PCA 961, 116, has been elucidated.

N-4-fluorophenyl pyridine-2-carbothioamide (PCA-F) 161 was prepared according to

reported method using 4-fluoroaniline (25 mmol), sulfur (75 mmol), sodium sulfide

nonahydrate (0.5 mol %) and 2-picoline (15 mL) as a reagent (Scheme 2). After

recrystallization from hot methanol the PCA-F 1 obtained with 79% yield. . The

1HNMR spectra of PCA-F 1 has been recorded in CDCl3/MeOD-d4. In CDCl3, the

thioamide proton (-CSNH) at ca 12 being more deshielded as compared to amide

proton (-CONH) of picolinamide accounted for downfield shift of ca ~2.5 ppm.110

The chemical shifts of individual pyridine protons and carbon atoms of compound 1

observed in the range 7.55–8.67 ppm and 125.8–160.7 ppm, respectively while the

individual protons and carbon atoms of phenyl ring were chemically observed in the

range of 7.16–8.00 ppm and 116.1–126.7 ppm, respectively.

The ligand 1 was converted into different metal arene derivatives [M(arene)(PCA-

F)X2]2 where M = RuII, OsII RhIII IrIII; arene = p-cymene,

pentamethylcyclopentadienyl (Cp*); and X = Cl, Br, I (Scheme 2). For the synthesis

of RuII, OsII, RhIII, IrIII complexes of PCA-F a solution of dimeric precursor

[RuII/OsII(η6-p-cymene)X2]2 or [RhIII/IrIII(Cp*)X2]2 in absolute dichloromethane

(DCM) was transferred in to the solution of compound 1 in absolute tehtrahydrofuran

(THF) and stirred the reaction for 4 h at 40 oC.116 After the complete work up and

precipitation, the respective mononuclear complexes obtained in the 60–88% yield.

110

Scheme 2. Synthetic route to N-fluorophenyl pyridine-2-carbothioamide 1 and its

organometallic RuII, OsII, RhIII and IrIII complexes (9, 17–22) along with the NMR

spectroscopy numbering scheme.

The organometallic compounds were characterized by NMR spectroscopy, ESI–MS,

elemental and single crystal XRD analysis. The 1HNMR spectra (Figure 56–57;

Appendix A) of these metal complexes have been recorded in MeOD-d4/CDCl3. In

metal complexes the H-4 and H-1 protons are most deshielded that is in accord with

electron donating effect of pyridine nitrogen and thioamide moiety to the metal atom.

In comparison to ligand in all complexes the most substantial change observed for H1

at ca.1 ppm. In p-cymene, the methyl protons H-19 appeared as singlet and isopropyl

protons H20 and H22 appeared as two doublets in the range of 2.21–2.43 ppm and

1.13–1.21 ppm, respectively. The isopropyl CH proton H21 appeared as a septet in

the range of 2.65–2.89 ppm and the p-cymene ring protons H14, H15, H17 and H18

were observed as four doublets in the range of 5.64–6.18 ppm. However in complexes

21 and 22, the -CH3 protons of Cp* ring appeared as a singlet at 1.70 ppm (Figure 57;

Appendix A). In 13C{1H}NMR spectrum (Figure 58–59; Appendix A) of both the

quaternary carbon atoms of thioamide (C6) and pyridine ring (C5) RuII/OsII(η6-p-

cymene) complexes, deshielded by ca. 3 ppm and 2 ppm, respectively, that also

supports the bidentate N,S-coordination mode of the ligand towards metal atom. In

RuII/OsII(η6-p-cymene)and RhIII/IrIII(Cp*) complexes the remaining carbon atoms of

111

pyridine ring (C1–C4) in appeared in the range of 125.0–162.4 ppm and 116.2–154.5

ppm, respectively. Moreover, in RuII/OsII and RhIII/IrIII complexes the chemical shifts

values of carbons atoms of phenyl ring (C7–C12) were observed in the range of

116.3–165.0 ppm and 115.9–163.0 ppm, respectively. Furthermore, the η6-p-cymene

ring carbon atoms of ruthenium complexes 9, 17 and 18 significantly shifted

downfield by ca. 8 ppm as compared to its osmium analogue 19 and 20, respectively.

Similarly, the quaternary carbons of Cp* ring in rhodium complex 21 observed with a

downfield shift of ca.7 ppm as compared to its iridium analogue 22. Moreover, in

RhIII/IrIII complexes the –CH3 carbon atoms of Cp* ring appeared at approximately ~9

ppm. The nature of complexes was confirmed by ESI-mass spectrum (Figure 60–61;

Appendix A) in positive ion mode featured a peak at a m/z value assigned to [M – X]+

ion but the most abundant peak was from [M– 2X– H]+ specie. The similar ionization

behavior in ESI-MS was observed for other metal complexes of PCAs. 61, 112, 116-117

The experimental m/z values were close to the calculated values.

The formation of complexes was further confirmed by elemental analysis. The

elemental analysis data of the complexes were close to the theoretical values. In line

with appearance of signals of n-hexane and THF in 1H NMR, these respective

solvents were also used to calculate the elemental analysis values of compounds 21

and 22.

The solid state molecular structure of the complexes 17, 18, and 20 were determined

by single crystal X-ray diffraction analysis. Crystallographic data and structural

refinement parameters are given in Table 7, whereas selected bond lengths and bond

angles are given in Table 8. Crystals of 18 and 20 were obtained by slow diffusion of

diethyl ether into solutions of complexes in methanol and crystallized out in the space

groups P2(1)/n and P2(1)/c, respectively . While, the crystal of 17 was obtained by

slow evaporation of saturated solution in methanol and ethyl acetate, with a space

group of P2(1)/n. These three crystals adopted monoclinic crystal system similar to

previously reported Ru(cym) and Os(cym) complexes of pyridine-2-carbothioamide.61

The complexes displayed the common piano stool geometry, where η6-p-cymene ring

forms the seat of the piano-stool, while an N,S chelating N-(4-fluorophenyl)pyridine-

2-carbothioamide ligand 1 and halide ligand form the three legs of the stool. In

molecular structure of complexes 17, 18 and 20 (Figure 37) the PCA-F ligand 1 binds

to ruthenium/osmium metal via pyridine nitrogen and sulfur atom, forming five

112

membered ring with bite angle N(1)–Ru–S of 81.54(9)o, 81.6(1)o and 80.46(9)o,

respectively. The M–S bond lengths observed for complexes 17, 18 and 20 were

2.3429(14) (Å), 2.3666(10) (Å) and 2.3519(9) (Å), respectively. However, the

determined M–S bond distance was significantly longer than M–N bond in all

complexes. Among crystallized complexes, the largest M–X bond lengths i.e.

2.7334(4) Å and 2.7165(3) Å observed for 18 and 20 than 2.5465(10) Å for 17 due to

more polarized nature of M–I bond as compared to M–Br bond. The torsion angle

N1–C5–C6–S for 20 is -4.3(4)ᴼ which is similar to previously reported osmium

complex61 and implies that osmium PCA complexes form delocalization system

within the carbothioamide and pyridine ring. On the other hand, the larger torsion

angles C6–N2–C7–C12/C6–N2–C7–C8 for these complexes >57ᴼ implied that the

delocalized system does not extend to the Namide-substituted ring. Moreover, there is

an offset π-stacking interaction is observed between pyridyl–pyridyl, cymene–phenyl

and pyridyl–phenyl of the adjacent molecules in 17, 18 and 20 respectively.

113

Figure 37. Molecular structures for metal complexes 17, 18, and 20 with 50% thermal

ellipsoid probability level. Hydrogen atoms, solvents and counter ions are omitted for clarity.

114

Table 7. X-ray diffraction parameters for the measurement of single crystals of 17, 18, and

20.

17.C4H8O2 18 20

Formula

C26H31Br2FN2

O2RuS C22H23FIN2

RuS C22H23FI2N2

OsS

Molecular Weight (g mol-1) 715.48 594.48 810.48 Crystal Description Red Block Black Block Black Block Crystal Size (mm × mm × mm) 0.38x0.10x0.05 0.28x0.28x0.22 0.35x0.34x0.10Wavelength (Å) 0.71073 0.71073 0.71073 Temperature (K) 372(2) 372(2) 372(2) Crystal System Monoclinic Monoclinic Monoclinic Space Group P2(1)/n P2(1)/n P2(1)/c a (Å) 14.100(5) 6.4580(2) 15.2088(12) b (Å) 8.944(5) 17.6353(6) 11.3216(9) c (Å) 22.700(5) 19.1159(7) 13.9552(11) α (°) 90.000(5) 90 90 β (°) 91.891(5) 96.899(2) 94.179(4) γ (°) 90.000(5) 90 90 Volume (A^3) 2861(2) 2161.32(13) 2396.5(3) Z 4 4 4 Final R indices [I>2ơ(I)] 0.0825 0.0537 0.0302 R indices (all data) 0.0954 0.1132 0.0801 Goodness-of-fit on F2) 1.002 0.881 1.179

Table 8. Selected Bond Lengths (Å) and Angles (°) for 17, 18 and 20. where M = Ru, Os and

X = Cl, Br, I.

Bond Lengths (Å) 17.C4H8O2 18 20

M–S 2.3429(14) 2.3666(10) 2.3519(9) N(1)–M 2.103(3) 2.094(3) 2.098(3) M–X(1) 2.5465(10) 2.7334(4) 2.7165(3) Bond Angles (°) 17.C4H8O2 18 20

N(1)–M–S 81.54(10) 81.54(9) 80.48(9) N(1)–M–X(1) 82.38(10) 84.11(9) 83.57(8) S–M–X(1) 89.49(4) 89.76(3) 89.74(3) Torsion Angles (°) 17.C4H8O2 18 20

C6–N2–C7–C12 57.0(6) 75.2(5) – C6–N2–C7–C8 – – 96.6(5) N1–C5–C6–S – 16.7(5) 4.3(4) N1–C1–C6–S 16.7(5) – –

115

3.2.2. In vitro antiproliferative activity

The fluoro substituted PCAs being gastric mucosal protectant are known to have low

toxicity in mouse suggesting better in vivo tolerability.113 Based on established

anticancer activity, the impact of metal ions (Ru, Os, Rh, Ir) and leaving groups (Cl,

Br, I) has been evaluated on the most active Ru(cymene)complex of p-fluoro

substituted PCA 9 61, 116 against four human cancer cell lines i.e. HCT116, H460,

SiHA and SW480 (Table 9). In cytotoxic assay, complexes demonstrated anticancer

activity with IC50 values from low micromolar to micromolar range. The IC50 value

of fluoro substituted PCA ligand was lower or almost comparable to its respective

complexes. Among synthesized metal complexes, the ruthenium complex with

chloride leaving group 9 showed highest antiproliferative activity with IC50 values of

6.5 µM, 8.3 µM and 4.3 µM against HCT116, SiHa and SW480 cell lines

respectively while in H460 cell line its analogue ruthenium-bromido complex 17 was

most cytotoxic with IC50 value of 6.8 µM. In general, within the same group the

lighter metal fragments ruthenium and rhodium complexes (9, 17, 18 and 21)

exhibited pronounced cytotoxicity in all four cell lines as compared to their heavier

congener i.e. osmium and rhodium (19, 20 and 22), respectively. On the other hand,

changing the halido leaving group with the more lipophilic one (Cl < Br < I) resulted

in slight decrease in anticancer activity except for ruthenium-bromido 17 and

osmium-iodo complexes 20 in H460 cell line.

Table 9. IC50 (μM) for ligand 1 and their respective RuII, OsII, RhIII and IrIII complexes (9, 17–

22) in human colorectal (HCT116), non-small cell lung (NCI-H460) cervical (SiHa)

carcinoma cell lines and colon carcinoma (SW480) cell lines.

Compound IC50 value (µM)

HCT116 H460 SiHA SW480 1 5.7 ± 0.7 7.8 ± 1.8 16 ± 6 9.9 ± 0.7

9 6.5 ± 0.3 10.3 ± 1.8 8.3 ± 0.7 4.3 ± 1.2

17 7.7 ± 0.5 6.8 ± 1.0 17 ± 2 7.6 ± 0.7

18 7.5 ± 0.3 7.1 ± 0.9 17 ± 1 7.5 ± 0.8

19 18 ± 1 24 ± 2 21 ± 3 10 ± 2

20 19 ± 1 18 ± 1 31 ± 2 24 ± 1

21 11 ± 2 12 ± 2 22 ± 5 8.9 ± 1.1

22 15 ± 2 18 ± 4 46 ± 6 24 ± 6

116

Scheme 3

Organoruthenium and -osmium Complexes of 2-

Pyridinecarbothioamides Functionalized with a Sulfonamide

motif: Synthesis, Cytotoxicity and Biomolecule Interaction

117

Scheme 3.3. Organoruthenium and -osmium complexes of 2-pyridinecarbothioamides functionalized with a sulfonamide motif: Synthesis, cytotoxicity and biomolecule interaction


Bioactive PCAs can act as S,N-bidentate ligands to metal ions to access a library of

organometallic and coordination compounds.61, 113, 118We functionalized a PCA ligand

with a sulfonamide, a motif found in many drugs and involved in interactions with the

active sites of CAs. The sulfonamide-substituted PCA 23 was prepared in a one-pot

synthesis by refluxing sulfanilamide and elemental sulfur in 2-picoline for 18 h with a

catalytic amount of sodium sulfide (Scheme 3). After work up and recrystallization

from acetonitrile, 23 was obtained in a good yield of 67%. The ligand was

characterized by NMR spectroscopy, ESI-MS, elemental analysis and single crystal

X-ray diffraction. In the1HNMR spectrum of 23, the thioamide proton was detected at

12.48 ppm. This corresponds to a downfield shift of ca. 2 ppm as compared to the

amide proton of picolinamide ligands.110 The protons of the pyridine ring were

observed in the range of 7.6–8.7 ppm, while the signals assigned to the aromatic

phenyl protons were detected in the range of 7.8–8.2 ppm. In the 13C{H}NMR

spectrum the pyridine ring carbon atoms were detected in the range of 124–153 ppm

while the carbons of the aromatic ring resonated between 124.3 and 141.5 ppm. The

ESI-mass spectrum of the ligand featured the pseudo molecular ion [23 + Na]+ at m/z

316.0157 which is in close agreement with the calculated value.

Scheme 3. Synthetic route to N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 23 and its

organometallic RuII and OsII complexes 24–27 with the numbering scheme used to assign the

signals in the NMR spectra.

118

The molecular structure of N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 23 was

determined by single crystal X-ray diffraction analysis (Figure 38). Crystals were

grown by slow evaporation from a methanol-dichloromethane mixture at room

temperature. PCA 23 crystallized in the monoclinic space group Cc (compare Table

10 for the crystallographic parameters). The hydrogen and oxygen atoms of the

sulfonamide group were involved in intermolecular H bonds with other molecules of

23. The pyridine and benzene rings were found to be disordered indicating a strong

displacement along the S2-C10-C7-N2 and C6-C5-C2 axes in the molecule.

Figure 38. Molecular structure of N-(4-sulfamoylphenyl)pyridine-2-carbothioamide 23

drawn at 50% probability level.

Compound 23 was converted into the corresponding RuII(cym) and OsII(cym)

complexes 24–27 in good yields (53–88%). The reactions were performed under

nitrogen atmosphere by reacting 23 (2 eq.) with [Ru/Os(cym)X2]2 (1 eq.) in a mixture

of tetrahydrofuran and dichloromethane at 40 °C for 4 h (Scheme 3). The red to dark

red/black products were obtained after filtration61 and were characterized by 1D and

2D NMR spectroscopy, ESI-MS and elemental analysis. The 1HNMR spectra of all

complexes were recorded in MeOD-d4 (Figures 62–65; Appendix A). Due to the fast

H/D exchange in protic deuterated solvents, the thioamide proton was not detected

while the spectra recorded for 24 and 27 in DMSO-d6 featured peaks at around 7.3

ppm (Figure 66; Appendix A). The H4 and H1 protons of the pyridine ring were

deshielded due to coordination of the pyridine nitrogen atom causing a shift by

approximately 1 ppm. The nature of the metal ion had only a slight effect on the 1H

and 13C{1H} NMR chemical shifts of the PCA ligand. The 13C{1H} spectra (Figures

67–70; Appendix A) contained most of the expected peaks but some of the quaternary

carbon atoms were not detected, presumably because of too low concentration of the

119

samples. Importantly, the spectra showed significant differences for the aromatic p-

cymene C–H atoms for the Ru complex 24 as compared to its Os counterpart 27.

These carbon atoms resonated about 10 ppm downfield in case of 24 as compared 27.

Similar shifts have been observed for related compounds while in other cases the

shifts were less pronounced.83b, 119

The molecular structure of a single crystal formed from slow diffusion of diethyl ether

into methanol solution of 27 was determined by single crystal X-ray diffraction

analysis (Figure 39; compare Table 10 for the crystallographic parameters). The Os

center adopted a pseudooctahedral coordination geometry and 23 coordinated to the

metal ion as an anionic N,S-bidentate ligand after deprotonation of the amide group.

Therefore, we label this compound as 27neutral. This is in contrast to all other

molecular structures of related Ru and Os complexes where the PCA ligand was

neutral and a complex cation was formed.61, 112, 116 The Os–cymcentroid and Os–Cl

distances were 1.671 Å and 2.442(4) Å and therefore similar to those reported for

related complexes.61, 112, 116 The Os–S1 and Os–N1 bond lengths were 2.355(4) and

2.133(1) Å. The C6–S1 bond (1.754(15) Å in 27neutral) was elongated as compared to

1.655(5) Å for 23, indicating a higher single bond character. The C6–N2 distance of

1.251(19) Å in 27neutral was slightly shorter compared to a bond length of 1.345(6) Å

in 9, demonstrating increased double bond character upon coordination of the Os

center to the S atom and deprotonation of the amide group. The latter bond is hardly

modified when PCA coordinates as a neutral ligand to a metal center.120

Figure 39. Molecular structure of 27neutral drawn at 50% probability level.

120

Table 10. X-ray diffraction measurement parameters for 23 and 27neutral.

23 27neutral

CCDC 1829882 1829883

Formula C12H11O2N2S2 C22H24ClN3O2OsS2


Crystal size (mm) 0.32×0.10×0.08 0.26 × 0.10 × 0.08

Wavelength (Å) 0.71073 0.71073


Crystal system monoclinic monoclinic

Space group Cc P-1

a (Å) 4.8844(6) 6.9829(7)

b (Å) 28.476(3) 12.2144(10)

c (Å) 8.8935(9) 13.5379(12)

α (°) 90 79.167(5)

β (°) 94.869(7) 83.956(6)

γ (°) 90 82.303(6)

Volume (Å3) 1232.5(2) 1120.06(18)

Z 4 2

Calculated Density (mg/mm3) 1.581 1.934

Absorption coefficient (mm-1) 0.433 6.024

F(000) 608 636

Theta range (°) 25.233 24.403

Number of Parameters / Reflections (all) 204 / 2214 289 / 3613

Final R indices [I > 2σ(I)] R1=0.0412

wR2= 0.0741

R1= 0.0844

wR2 = 0.1594

R indices (all data) R1= 0.0514

wR2 = 0.0774

R1= 0.1071

wR2 = 0.1668


121

To confirm the ionic nature of the complexes, conductivity measurements were

performed for 23 and its complexes 24–27 in acetonitrile. All the complexes showed

higher conductivity than the neutral ligand (Table 11), indicating their ionic nature.

However, it should be noted that the conversion of the cationic form into the neutral

form may be accompanied by the release of HCl.

Table 11. Conductivity measurements of ligand 23 and complexes 24–27 in acetonitrile

(0.1 mM).

Compound Conductivity

(µS/cm)

Temperature

(°C)

23 5 21.6

24 32 21.9

25 68 21.9

26 73 22.8

27 21 23.1

ESI-MS was also used to confirm the formation of the metal complexes (Figure 71–

72; Appendix A). In light of the molecular structure of 27neutral, which features the

PCA ligand in its deprotonated form coordinated to Os, it is interesting to note that the

mass spectrum of 27 recorded in positive ion mode featured a peak at a m/z value

assigned to [M–Cl]+ ions but the most abundant peak was from a [M–2Cl–H]+

species, which was the only peak found for the Ru complexes. The elemental analysis

data of the complexes were in close agreement with the theoretical values for the

protonated complexes 24–27 with a chlorido counterion.

3.3.2. Stability in aqueous solution and reactivity toward amino acids

The aqueous stability of complexes 24–27 was determined by NMR spectroscopy and

ESI-MS. The compounds were dissolved in D2O and 1H NMR spectra were recorded

after 0.25, 1, 3, 24, 48, 72, 96 and 120 h. The compounds underwent chlorido/aqua

ligand exchange reactions within 15 min of incubation in D2O. There was no change

in the spectrum over a period of 120 h, indicating the high stability of the formed aqua

species.

Depending on the nature of metal ion and co-ligands, metal complexes are prone to

undergo ligand exchange when encountered with biomolecules such as proteins. In

order to understand the nature of such interactions, reactions of 24 and 27 with the

122

amino acids L-cysteine (Cys), L-methionine (Met), and L-histidine (His)were

monitored by 1H NMR spectroscopy in D2O. Despite that both 24 and 27, undergo

immediate hydrolysis, they did not react with amino acids within 24 h of incubation at

1 : 1 and 1 : 2 (complex : amino acid) molar ratio (Figure 40 for His), after which

another equivalent of amino acid was added and the reaction was followed for another

96 h. The 1H NMR spectra however remained largely unchanged with only a minor

amount of another species (< 5%) forming, possibly due to adduct formation with the

amino acids. This low reactivity was further confirmed by ESI-MS, where no adduct

formation was observed with amino acids. The relative high stability of the aqua

species of these complexes is unique compared to that of analogous Ru PCA

complexes.

Figure 40. 1H NMR spectroscopic study of the reaction between 24 and His in D2O,

monitored for 72 h. The peaks of His are highlighted in grey boxes.

3.3.3. In vitro anticancer activity

The cytotoxicity of ligand 23 and its respective complexes 24–27 was determined in

human HCT116 colorectal, H460 non-small cell lung, SiHa cervical, and SW480

colon carcinoma cells (Table 12). The sulfonamide-substituted PCA ligand 23 was

moderately active only in the HCT116 cancer cell line with an IC50 value of 105 μM.

The Ru(cym) and Os(cym)complexes were inactive in all tested cancer cell lines. This

is surprising given the fact that plecstatin-1 and other related derivatives were highly

cytotoxic (Table 12).62, 112, 116 The low potency may be related to the comparatively

low lipophilicity of ligand 23 (clogP = 0.92) as compared to N-(4-

123

fluorophenyl)pyridine-2-carbothioamide in F-SN 1 (clogP = 2.88),116possibly

interfering with efficient accumulation in cancer cells. Another explanation may be

that the sulfonamide substituent hinders the interaction of the complex with plectin,

which was identified as the target for plecstatin-1.62

Table 12. In vitro anticancer activity (IC50 values) of ligands 23, its respective Ru/Os(cym)

complexes 24, 25, 26 and 27, and related compounds F-SN 1 and plecstatin-1 in human

colorectal (HCT116), non-small cell lung (NCI-H460) cervical (SiHa) and colon carcinoma

(SW480) cells(exposure time 72 h). The clogP values for the PCAs 23 and F-SN 1 are also

given.

Compound IC50 value (µM) clogP

HCT116 NCI-H460 SiHA SW480

23 105 ± 3 >300 >300 >300 0.92

24 >211 >300 >300 >300 -

25 >300 >300 >300 >300 -

26 >300 >300 >300 >300 -

27 >300 >300 >300 >300 -

F-SN 1116 5.7± 0.7 7.8 ± 1.8 16 ± 6 33 ± 2 2.88

plecstatin-1116 6.5 ± 0.3 10 ± 2 8.3 ± 0.7 9.9 ± 0.7 -

3.3.4. Molecular Modelling

As crystal structure of h-CA II with a co-crystallized Ru complex (SRX) featuring a

sulfonamide functional group has been reported (PDB ID: 3PYK),94 we modelled

ligand 23 and both possible enantiomers of its chiral Ru and Os complexes 24(24E1

and 24E2) and 27(27E1 and 27E2), respectively, into the catalytic pocket using a

molecular dynamics approach. The results were compared to that of a co-crystallized

Ru complex (SRX) with a sulfonamide functional group. All the compounds were

found to interact through H bonds with Thr residues in close proximity to the Zn ion

in the active site, to which the sulfonamide moieties bound (Table 13). In addition,

they formed lipophilic interactions with Val121, Leu60, and Leu198, as did SRX (in

addition to Pro202). The ligand and its complexes practically adopted the same

conformation, independent of the chirality at the metal center. The predicted pose of

24E2 is shown in Figure 41a with its hydrogen bonds with Thr199 and Thr200 via the

124

oxygen atom of the sulfonamide group. Complex 24E2 is residing deep in the catalytic

site of the enzyme showing an excellent fit (Figure 41b), as did all the other

complexes, and blocks access to the Zn ion coordinated to His94, His96, and His119.

This demonstrates that the enzyme is a viable target, which however would have to be

verified experimentally.

Figure 41. The modelled configuration of 24E2 in the catalytic site of carbonic anhydrase II

(PDB ID 3PYK). a) Hydrogen bonds are depicted as green dotted lines between the metal

complex and the amino acids Thr199, and Thr200. Lipophilic interactions are represented as

purple dotted lines with Val121, Leu60 and Leu198. b) The enantiomer 24E2 is shown in the

binding pocket with the protein surface rendered. Red depicts a negative partial charge on the

surface, blue depicts a positive partial charge and grey shows neutral/lipophilic areas.

Table 13. The H bonds and lipophilic interactions of the modelled compounds with amino

acid residues of carbonic anhydrase II.

Compound H bonds Lipophilic interactions

SRX Thr199 Val121, Leu198, Pro202

23 Thr200 Val121, Leu198

24E1 Thr199, Thr200 Val121, Leu198

24E2 Thr199, Thr200 Val121, Leu198, Leu60

27E1 Thr199, Thr200 Val121, Leu198

27E2 Thr200 Val121, Leu198, Leu60

125

Scheme 4

Targeting Epigenetic Changes: Multitargeted Vorinostat

(SAHA)-derived Metal Complexes with Potent Anticancer

and Histone Deacetylase Inhibitory Activity

126

Scheme 3.4. Targeting epigenetic changes: multitargeted vorinostat (SAHA)-derived metal complexes with potent anticancer and histone deacetylase inhibitory activity


In our efforts to design multitargeted anticancer agents, i.e. a drug contains

more than one pharmacophore in a single molecule,121 the design concept of the

organometallic HDACi presented here is based on a bioactive metal centre, that

can undergo ligand exchange reactions and form covalent bonds to target donor

atoms; a SAHA-inspired hydroxamic acid moiety as the Zn-binding group; and

a pyridine-2-carbothioamide (PCA) ligand. PCA-based organometallics were

shown to interact selectively with plectin62 and to have a preference for amino

acid side chains over DNA, as shown in nucleosome core particle binding

studies.61 The high stability of PCA–metal bonds even under acidic conditions

provides a structural scaffold to the pharmacophore making it suitable for oral

administration.61, 122

The PCA-based hydroxamic acid ligands 30 and 31 were prepared in two steps

(Scheme 4). Succinic or suberic anhydride were reacted with N-(4-

aminophenyl)pyridine-2-carbothioamide to afford pyridine-2-carbothioamide

succinic acid 28 and pyridine-2-carbothioamidesuberic acid 29 in yields of 61

and 41%, respectively. PCAs 28 and 29 were converted into the respective

hydroxamic acids 30(29%) and 31(32%) with NH2OH, ethylchloroformate, and

Et3N. This conversion was characterised by an upfield shift of the broad COOH

singlet in the 1H NMR spectra from ca. 12 ppm in 28 and 29 to ca.8.70 ppm for

the hydroxyl proton in 30 and 31. X-ray diffraction analysis of single crystals of

28 showed that it crystallised in the orthorhombic space group Pbca (Table 14,

Figure 42). The C6=S bond length of 1.665(3) Å was significantly longer than

found for the carbonyls C13=O1 and C15=O2 at 1.232(3) and 1.232(3) Å,

respectively (Table 15). The aromatic rings are co-planar stabilised by an

intramolecular H bond between N1 of the pyridine ring and the amide HN2

(2.107 Å). The molecules form an expansive network of intermolecular H

bonds that involve the carboxylic acid HO3 of one molecule and the carbonyl

O2 of another (HO3···O2 distance of 2.669 Å; Figure 42). In addition, the amide

127

proton HN3 and carbonyl oxygen form another set of intermolecular hydrogen

bonds with an HN3···O1 distance of 2.085 Å.

Scheme 4. Synthesis of the pyridine-2-carbothioamide carboxylic (28 and 29) and

hydroxamic acids (30 and 31) and their respective organometallic RuII , OsII , RhIII and IrIII

complexes (32–41).

Figure 42. Molecular structure of 28 drawn at 50% probability level. The intermolecular

hydrogen bonding are shown between the carboxylic acid and amide groups.

128

Both the carboxylic (28 and 29) and hydroxamic (30 and 31) acid derivatives were

converted to organometallics by reaction with the dimeric precursors [M(cym)Cl2]2

(M = Ru, Os; cym = η6-p-cymene) or [M(Cp*)Cl2]2 (M = Rh, Ir; Cp* =

pentamethylcyclopentadienyl) in 37–75% yield (Scheme 4). In the 1H NMR spectra,

coordination of 28–31 to the metal ions caused deshielding of the pyridine proton H1

accompanied by downfield shifts of ca.1 ppm (Figure 73; Appendix A) depending on

the metal centre (δ = 9.1–9.7 ppm). In contrast, H4, which is involved in a hydrogen

bond with S1 in the crystal structure of 28, becomes more shielded and shifts upfield

by ca. 0.2 ppm to around 8.5 ppm (Figure 73; Appendix A). The compounds were

also characterised by 13C{1H} NMR spectroscopy, elemental analysis and

electrospray ionisation mass spectrometry (ESI–MS), all of which supported the

identity of the compounds. The ESI-mass spectra (Figure 85–88; Appendix A) of all

complexes featured [M–2Cl–H]+ ions as base peaks with the experimental m/z values

and isotope distributions in close agreement to the calculated values. This shows the

ease of deprotonation of the thioamide proton while in the solid state the amide

remains protonated. This was confirmed by single crystal X-ray diffraction analysis of

33 (Table 14). Complex 33 crystallised in the monoclinic space group P21/c as two

enantiomers and the structure featured the characteristic piano-stool configuration

where the Os is coordinated to cym, the S,N-chelating PCA 28, and a chlorido ligand

(Figure 43). The chloride counterion formed intermolecular H bonds with the amide

proton HN3 and the thioamide proton HN2 to bridge two enantiomeric molecules of 33

(Figure 44). The Os−cymcentroid and the Os–Cl distances of 1.679(1) Å and 2.417 (2)

Å, respectively, were in a similar range as observed for structurally related

[Os(cym)(PCA)Cl] complexes.61 For coordination of the Os centre to S1 and N1, the

H bond N1···HN2 for in 28 was broken. This resulted in the PCA ligand to lose its

planarity (torsion angle C5–C6–N2–C7 135.08°) seen in the molecular structure of

28. Upon metal coordination, the bond C6–S1 was elongated and C6–N2 was

contracted as compared to 28, as observed for related compounds.61, 122 This indicates

higher single bond character for C6–S1 and higher double bond character for the C6–

N2 bond (Table 15).

129

Figure 43. Molecular structure of one of the enantiomers of 33 drawn at 50% probability

level. The counter ion and residual MeOH were removed for clarity.

Figure 44. Molecular structure of 33 drawn at 50% probability level. Two enantiomeric

molecules of 33 are connected by two chloride counter ions through H-bonds with the amide

protons of two molecules.

130

Table 14. X-ray diffraction parameters for the measurement of single crystals of ligand 28

and its Os(cym) complex 33.

28 33·CH3OH

CCDC 1831913 1831914

Formula C16H15N3O3S C27H33Cl2N3O4OsS

Molecular Weight (g mol-1) 329.37 756.72

Crystal Description yellow needle red block

Crystal Size (mm × mm × mm) 0.38 × 0.12 × 0.12 0.38 × 0.10 × 0.10

Wavelength (Å) 0.71073 0.71073

Temperature (K) 100 100

Crystal System orthorhombic monoclinic

Space Group Pbca P21/c

a (Å) 9.8887(3) 14.6784(6)

b (Å) 16.6029(5) 18.1415(7)

c (Å) 18.0686(5) 11.2070(5)

α (°) 90 90

β (°) 90 107.259(2)

γ (°) 90 90

Volume (A3) 2966.52 2849.92

Z 8 4

Calculated Density (mg/mm3) 1.475 1.764

Absorption coefficient (mm-1) 0.238 4.773

F(000) 1376 1496

Theta range (°) 25.252 26.370

h range 11 18

k range 19 22

l range 21 14

Number of Reflections 3489 6600

R(int) 0.0610 0.0612

Goodness-of-fit on F^2 1.046 1.125

Final R indices [I>2σ(I)] R1 = 0.0528

wR2 = 0.1509

R1 = 0.0495

wR2 = 0.1028

R indices (all data) R1 = 0.0683

wR2 = 0.1648

R1 = 0.0692

wR2 = 0.1088

131

Table 15. Comparison of selected bond lengths (Å), angles (°), and torsion angles (°) of 28

and its Os(cym) complex 33.

Bonds (Å) 28 33·CH3OH

Os–cymcentroid – 1.679

Os–S1 – 2.336(2)

Os–N1 – 2.103(4)

Os–Cl1 – 2.416(1)

C1–N1 1.336(2) 1.348(8)

C5–N1 1.337(2) 1.361(9)

C5–C6 1.512(2) 1.475(9)

C6–S1 1.661(2) 1.691(5)

C6–N2 1.337(2) 1.331(8)

C13–N3 1.356(2) 1.347(8)

C13–O1 1.229(2) 1.218(8)

C16–O2 1.235(2) 1.260(2)

C16–O3 1.301(2) 1.330(2)

Bond Angles (°) 28 33·CH3OH

N1–Os–S1 – 80.9(1)

N1–Os–Cl1 – 81.4(1)

S1–Os–Cl1 – 86.97(5)

C2–C1–N1 123.4(2) 122.8(5)

C4–C5–N1 123.0(2) 120.3(5)

N1–C5–C6 116.0(1) 115.7(5)

N2–C6–C5 111.9(1) 119.2(5)

N2–C6–S1 127.5(1) 122.4(4)

C5–C6–S1 120.6(1) 118.4(4)

O2–C16–O3 123.7(2) 118.0(1)

O2–C16–C15 122.2(2) 124.0(1)

O3–C16–C15 114.1(2) 117.0(1)

Torsion Angles (°) 28 33·CH3OH

N1–C5–C6–S1 172.4(1) -5.2(7)

C6–N2–C7–C8 -179.9(2) -49.5(9)

132

3.4.2. Stability in aqueous solution and reactivity with amino acids

The stability of compounds 38–41 in d4-MeOD/D2O (38 and 39) or D2O (40

and 41) was studied by 1H NMR spectroscopy (Figures 45 and 46). Compound

40 was remarkably soluble in water (47 mM), especially compared to 39 (0.6

mM). All complexes underwent chlorido/aqua ligand exchange, which was

complete within 15 min for 38, 40 and 41 and too fast to determine reaction

kinetics for by NMR spectroscopy, while 39 reacted more slowly and the

process took about 6 h. The formation of the aqua complexes was confirmed by

addition of 2 eq. of AgNO3, which gave identical spectra (Figure 45). The aqua

species were stable for at least 5 d. The ligand exchange appeared to be at least

partly reversible in the presence of 104 mM NaCl or 60 mM HCl, however,

precipitation of probably the chlorido complex complicated data interpretation.

The reversibility of the ligand exchange reaction was indicated, for example, in

case of 40 by a shift of the signal assigned to H1 from 8.41 ppm for the aqua

species to 9.08 ppm for the chlorido complex in the 1H NMR spectra (Figure

45).

Figure 45. 1H NMR spectra of 40 in D2O (bottom), after addition of AgNO3 (2 eq.), and in the

presence of NaCl (104 mM) and HCl (60 mM).

133

Figure 46. 1H NMR spectra of 39 in D2O (bottom) recorded 0.5, 2 and 6 h after dissolution.

Furthermore, 38–41 were studied for their reactions with L-cysteine (Cys), L-

methionine (Met), L-histidine (His), in 5% D4-MeOD/D2O (38 and 39) or D2O (40

and 41) by 1H NMR spectroscopy (Figure 47). These experiments supported the

results found in the stability studies. The compounds were highly stable and no adduct

formation with His, Met and Cys was observed over 5 d. This level of stability of the

compounds is remarkable, especially in presence of Cys, which has been reported to

induce decomposition of Ru(arene) complexes.123

Figure 47. 1H NMR spectra of 41 in D2O (bottom), and 24 hours after the addition of Cys (1

eq., middle; 2 eq.,top).

134

3.4.3. In vitro anticancer activity

The cytotoxicity of the PCAs 28–31 and their organometallic complexes was

determined in human colorectal (HCT116), non-small cell lung (H460), cervical

(SiHa) and colon carcinoma (SW480) cells (Table 16). From the carboxylic acid

derivatives and their complexes, only PCA 29 was moderately cytotoxic while for

none of the complexes of 28 and 29 the IC50 concentration was reached. The

hydroxamic acid derivatives 30 and 31 showed biologically activity, with especially

the close SAHA derivative 31 displaying excellent antiproliferative potency with IC50

values in the high nanomolar range (Table 16). It was therefore at least 2 orders of

magnitude more potent than its carboxylic acid analogue 29. This demonstrates the

essential role of the hydroxamic acid functional group in the biological activity of

many HDACi. Only the organometallic compounds formed with 31 showed

cytotoxicity and their potency depended strongly on the metal fragment. Ru(cym)38

and Os(cym)39 were low to moderately cytotoxic, while the Cp* complexes of Rh 40

and Ir 41 showed the highest antiproliferative activity, with 40 being similarly

cytotoxic as 31.

135

Table 16. In vitro cytotoxic activity (mean IC50 values ± standard deviations) of PCA-

carboxylic acid and their organometallic complexes (28, 29, 32, 33, 34 and 35) as well as

PCA-hydroxamic acids and their organometallic complexes (30, 31, 36, 37, and 38–41) in the

human cancer cell lines HCT116 (colon), NCI-H460 (non-small cell lung), SiHa (cervix), and

SW480 (colon) given in μM as determined by the SRB assay (exposure time 72h).

Compound IC50 values (μM)a HCT116 NCI-H460 SiHa SW480

SAHA 0.46 ± 0.09 0.57±0.01 1.6±0.1 1.3 ±0.07 28 > 200 > 200 > 200 > 200 32 > 200 > 200 > 200 > 200 33 > 200 > 200 > 200 >200 29 127 ± 9 119 ± 26 161 ± 20 191 ± 13 34 > 200 > 200 > 200 > 200 35 > 200 > 200 > 200 > 200 30 90 ± 7 71 ± 48 178 ± 2 176 ± 2 36 > 200 > 200 > 200 > 200 37 > 200 > 200 > 200 > 200 31 0.30 ± 0.14 0.98 ± 0.30 1.6 ± 0.6 1.5 ± 0.3 38 30 ± 3 120 ± 24 126 ± 15 124 ± 8 39 42 ± 5 136 ± 71 73 ± 5 170 ± 124 40 0.97 ± 0.10 3.5 ± 0.3 3.3 ± 0.1 3.3 ± 0.2 41 3.4 ± 0.5 11.4 ± 0.6 12 ± 0.3 11 ± 1 a200 μM was the highest concentration used in the assay.

3.4.4. HDAC inhibition

Based on the cytotoxic data, 29–31 and 38–41 were selected for screening of HDAC8

inhibition at a concentration of10 µM. The carboxylic acid 29 and the hydroxamic

acid 30 showed very low activity at this concentration with residual HDAC8 activity

of 100 and 83%, respectively (Table 17). The presence of the hydroxamic acid in 30

proofed beneficial with a slight inhibition of HDAC8 and this was confirmed for the

SAHA derivative 31 with only 9% residual HDAC8 activity. This fact also

demonstrates the role of the length of the aliphatic chain which is required for the

hydroxamic acid group to reach the Zn ion deep in the active site of the enzyme.

Notably, all complexes of 31 were more potent than the ligand at this concentration

and they were therefore included in a study to determine their IC50 values against

HDAC1, HDAC6 and HDAC8 (Table 18). PCA 31 and its organometallic compounds

38–41 exhibited excellent HDAC inhibitory potential with IC50 values in the nM

range. They were more potent inhibitors of HDAC1 and HDAC8 than the clinically

approved drug SAHA and equally potent against HDAC6. In particular, 31 and its

136

Rh(Cp*) complex 40 were strong inhibitors of HDAC6 compared to SAHA. The

lower activity of 31 against HDAC1 and HDAC8 was enhanced when it was

coordinated to organometallic moieties. In general, the organometallic compounds

showed a slight selectivity for HDAC6, as would be expected given their structural

similarity with SAHA, which was about an order of magnitude more potent against

HDAC6 than HDAC1 and HDAC8 in this assay. The influence of the metal centre

may be explained by two effects. The metal centre can undergo ligand exchange

reactions and despite not seeing adduct formation with isolated amino acids, the

protein microenviroment may support covalent bond formation or electrostatic

interaction of the aquated complex cation within the binding site.61 Moreover, the

metal fragment can be considered as a bulky group that may form hydrophobic

interactions or hydrogen bonds with aromatic amino acid side chains.124 Comparison

of the HDAC inhibitory and cytotoxicity data shows limited correlation, which may

be a result of a contribution of the PCA ligand and the metal centre to the mode of

action through an alternative pathway.

Table 17.Single dose mean values for the residual activity of HDAC8 after treatment with

29–31, and 38–41 at 10 μM. The numbers in brackets are the two recorded data points (n = 2).

Compound HDAC activity / %

29 100 [96,103]

30 83 [82,84]

31 9 [9,9]

38 -2.8 [-2.7,-2.9]

39 -0.1 [-0.2,0.1]

40 -3.8 [-3.8,-3.8]

41 -3.9 [-3.6,-4.1]

137

Table 18. Inhibitory activity (IC50 in nM) of PCA-hydroxamic acid 31 and its organometallic

complexes 38–41 against HDAC1, HDAC6, and HDAC8 in comparison to SAHA.

Compound IC50 values (nM)

HDAC1 HDAC6 HDAC8

SAHA 306 20 306

31 474 5 901

38 27 14 45

39 34 25 87

40 195 6 34

41 54 12 31

3.4.5. Molecular dynamic simulations

To understand the HDAC inhibitory activity of 31 and the two enantiomers of

its complexes 38–41 in comparison to SAHA, a molecular modelling approach

was used in combination with molecular dynamics simulations. The active site

of HDAC8 consists of a long, narrow channel leading to a cavity that contains

the catalytic machinery. The walls of the channel are formed by Tyr100,

Tyr306, His180, Phe152, Gly151 and Met 274 and are primarily

hydrophobic.108, 125Studies with SAHA confirmed that the Zn2+ ion and also

Tyr306 are the important active site components (Table 19).125-126 Upon

modelling, 31 and its enantiomeric metal complexes showed a good fit in the

binding pocket as they superimposed over SAHA and interacted with Zn

through the hydroxamate motif (Figure 48 for 39E2). In all cases, the metal

fragments were sitting above the protein surface. With exception of one of the

enantiomers of 40, the complexes formed H bonds with His180 (and the

majority also with Asp101), while all but one of the enantiomers of 39 and 31

showed lipophilic contacts with Tyr100 through the ligand backbone (Table

19). The latter fact may be of relevance when interpreting the HDAC inhibition

data for 31 which was the by far least active HDAC8 inhibitor.

Modelling the same compounds into HDAC6 resulted in similar observations as for

HDAC8 with the compounds interacting with the Zn ion but the metal complexes

were found lying in a nearby second channel as compared with SAHA and 31. This

positioning supports additional interactions of the metal moiety with the protein

through functionally important active site residues such as Tyr745, Pro464, Phe583,

138

His463 and Gly473 (Figure 49 for 40E2).107 Notably, the enantiomeric structures offer

different binding options with amino acid side chains, most significantly His463 with

its imidazole moiety, which may well undergo a ligand exchange reaction with one

enantiomer, while the other has the labile chlorido ligand pointing away from it.


(a) Hydrogen bonds are depicted as green dotted lines between ligand and the amino acids

Asp101and His180. The Zn interaction is shown with solid lines. (b) 39E2 is shown in the

binding pocket with the protein surface rendered. Blue depicts a positive partial charge on the

surface, red negative and grey neutral/lipophilic.

139


The complex is shown in the binding pocket with the protein surface rendered. Blue depicts a

positive partial charge on the surface, red negative and grey neutral/lipophilic.


enantiomers of its metal complexes.

Compound H bonds Lipophilic contacts

SAHA Tyr306 -

31 - Tyr100

38E1 His180, Asp101 Tyr100

38E2 His180, Asp101 Tyr100

39E1 His180, Asp101 -

39E2 His180, Asp101 Tyr100

40E1 His180, Asp101 Tyr100

40E2 - Tyr100

41E1 His180, Asp101 Tyr100

41E2 His180 Tyr100

140


enantiomers of its metal complexes.

Compound H bonds Lipophilic contacts

SAHA Tyr745, His573,

His574 -

31 Gly473 -

38E1 Tyr745 Pro464, His463

38E2 Tyr745 Pro464, His463, Phe583

39E1 Tyr745 Pro464, His463

39E2 Tyr745 Pro464, Phe583

40E1 Tyr745 His463

40E2 Tyr745 His463

41E1 Tyr745 His463

41E2 Tyr745 His463

141

Conclusions

The central theme of this research is to develop the novel metal-based anticancer

agents with non-classical mode of action. The “proof of concept” has reflected in

most of the synthesized compounds based on the specific design hypotheses.

Pyridine-2-carbothioamides are the bioactive S,N-bidentate ligands and their

complexation to biologically active metal centre can result in synergistic effects,

different modes of action as well as increased solubility. In structure-activity

relationship study, a series of N-phenyl substituted pyridine-2-carbothioamides and

their organometallic RuII(cym) complexes were prepared. The new derivatives were

modified at the phenyl ring and three procedures were optimized for the synthesis of

the complexes to ruled out the formation of coordination isomers and to obtain pure

complexes in the desired N,S-coordination mode, as was demonstrated by X-ray

diffraction analysis as well as spectroscopic studies. Representative compounds

exhibited remarkable stability in aqueous and acidic medium of 60 mMHCl. Most of

the PCAs and their organoruthenium compounds were shown to be potent anticancer

agents in human cancer cell lines. The cytotoxicity in cancer cell lines was correlated

with the clogP values calculated for the PCAs. Based on established anticancer

activity the most cytotoxic Ru(η6-p-cymene)complex of N-fluorophenyl substituted

PCA 9 has been taken into account to study the impact of metal ions (Ru, Os, Rh, Ir)

and leaving groups (Cl, Br, I). Within the group, the complexes of the lighter metals

(Ru and Rh) exhibited greater anticancer activity than their heavier congener (Os and

Ir) in cytotoxic assay, while the influence of leaving group only observed in H460

cancer cell line.

Another series of compounds involving functionalization of PCA pharmacophore

with a sulfonamide and the preparation of its half sandwich complexes to target the

enzyme carbonic anhydrase. The Ru(cym) and Os(cym) complexes were synthesized

and thoroughly characterized. The molecular structure of 27 suggests deprotonation of

the carbothioamide moiety, while structures of several other PCA complexes

crystallized in the protonated form. We evaluated the compounds for their stability in

aqueous solution and reactivity with biomolecules. The compounds undergo a quick

chlorido/aqua ligand exchange but are surprisingly unreactive to amino acids. The

antiproliferative activity could only be determined for ligand 23 in HCT116 cells.

142

While binding to CA II, as determined by molecular dynamic simulations studies,

may not result in anticancer activity, this shows that the compounds are still capable

of interacting with the Zn ion in the catalytic site of CA II.

In a more rational approach, we have combined different pharmacophores in a single

molecule. The bioactive PCA moiety was functionalised with hydroxamic and

carboxylic acid residues and both the linker and metal fragment were varied. The

PCA 31 is structurally related to SAHA and its Rh complex 40 was a potent

cytotoxin. HDAC1, HDAC6 and HDAC8 inhibition studies revealed minor

correlation with the cytotoxic activity and suggest an impact of the other bioactive

moieties beyond the SAHA-derived fragment on the biological activity. Ligand 31

and the metal complexes still show a similar HDAC inhibition pattern as SAHA in

these isoforms. The ability to act as Zn chelators in HDACs was demonstrated by

computational methods, which suggest at least in case of HDAC6 an impact of

chirality on the binding to the protein.

This work demonstrates that the M(arene)-PCA system (where M = RuII, OsII,

RhIII, IrIII) offers the opportunity to design anticancer metallodrugs with novel

mode of actions. In future, further research efforts will be concentrated on

evaluation of PCAs and their half sandwich complexes in other cancer cell lines

to find out the broader spectrum of their cytotoxicity. The interaction of

complexes of PCAs can be evaluated against other cellular proteins such as

cathepsin B, thioredoxin reductase, matrix metalloproteinase to determine the

multitargeted nature of these compounds. The cellular accumulation studies of

histone deacetylase targeted half sandwich complexes can be evaluated along

with determination of HDAC activity inside the cells. Further, in vivo studies

can provide better picture about general toxicity and potential of these

compounds as anticancer agents.

In conclusion, metal(arene)complexes with PCA-type ligands are the promising

candidates towards development of protein-targeted anticancer drugs.

143

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Appendix A

Representative NMR and ESI-mass spectra of scheme 1

Figure 50. 1HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 11 in MeOD-d4.

Figure 51. 1HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-


155

Figure 52. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-


Figure 53. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-


156

Figure 54. ESI-MS of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 11 in CH2Cl2.

Figure 55. ESI-MS of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-

carbothioamide)ruthenium(II)]chloride 13 in CH2Cl2.

157


Figure 56. 1H NMR Spectrum of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 17 in MeOD-d4.

Figure 57. 1H NMR Spectrum of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-

fluorophenyl) pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CDCl3.

158

Figure 58. 13C{H}NMR Spectrum of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-


Figure 59. 13C{H}NMR Spectrum of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-

fluorophenyl) pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CDCl3.

159

Figure 60. ESI-MS of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-

carbothioamide)ruthenium(II)]bromide 17 in CH2Cl2.

Figure 61. ESI-MS of [chlorido(η5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)

pyridine-2-carbothioamide)rhodium(III)]chloride 21 in CH2Cl2.

160


Figure 62. 1HNMR Spectrum of [chlorido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-


Figure 63. 1HNMR Spectrum of [bromido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-


161

Figure 64. 1HNMR Spectrum of [iodo(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)ruthenium(II)]iodide 26 in MeOD-d4.

Figure 65. 1HNMR Spectrum of [chloro(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-

carbothioamide)osmium(II)]chloride 27 in MeOD-d4.

162

Figure 66. 1H NMR spectrum of 24 and 27 in DMSO-d6 recorded after 15 min of dissolution.

The spectra showed peaks assigned to the NH protons as well as minor products, presumably

due to DMSO/Cl ligand exchange reactions.

Figure 67. 13C{H}HNMR Spectrum of [chlorido(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]chloride 24 in MeOD-d4.

163

Figure 68. 13C{H}HNMR Spectrum of [bromido(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]bromide 25 in MeOD-d4.

Figure 69. 13C{H}HNMR Spectrum of [iodo(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-

2-carbothioamide)ruthenium(II)]iodide 26 in MeOD-d4.

164

Figure 70. 13C{H}HNMR Spectrum of [chloro(η6-p-cymene)(N-(4-

sulfamoylphenyl)pyridine-2-carbothioamide)osmium(II)]chloride 27 in MeOD-d4.


carbothioamide)ruthenium(II)]chloride 24 in CH3OH.

165

Figure 72. ESI-MS of [chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-carbothioamide)ruthenium(II)]chloride 27 in CH2Cl2.


Figure 73. Comparison of 1HNMR spectrum of ligand 8-oxo-8-((4-(pyridine-2-carbothioamido)phenyl)amino)octanoic acid 29 and its complex [chlorido(η6-p-cymene)(8-oxo-8-((4-(pyridine-2-carbothioamido)phenyl)amino) octanoic acid)ruthenium(II)]chloride 34 in MeOD-d4.

166

Figure 74. 1HNMR spectrum of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)

octanediamide 31 in DMSO-d6.


carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in MeOD-d4.

167


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4.


(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride 40 in

MeOD-d4.

168


(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride 41 in

MeOD-d4.

Figure 79. 13C{H}NMR spectrum of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)

octanediamide 31 in DMSO-d6.

169


carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in MeOD-d4.


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4.

170


hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]

chloride 40 in MeOD-d4.


hydroxy-N8-(4-(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]

chloride 41 in MeOD-d4.

171

Figure 84. ESI-MS of N1-hydroxy-N8-(4-(pyridine-2-carbothioamido)phenyl)octanediamide

31 in CH3OH.


carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in CH3OH.

172


carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in CH2Cl2.


(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride 40 in

CH3COCH3.

173


(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride 41 in

CH3COCH3.

design of protein-targeted organometallic complexes as

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