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
3.3.1. Results and Discussion ..................................................................................................... 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
3.4.1. Results and Discussion ..................................................................................................... 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
Representative NMR and ESI-mass spectra of scheme 2 ............................................................... 157
Representative NMR and ESI-mass spectra of scheme 3 ............................................................... 160
Representative NMR and ESI-mass spectra of scheme 4 ............................................................... 165
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 20. Chemical structure of (51). ................................................................................................. 41
Figure 21. Chemical structure of (52). ................................................................................................. 42
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 28. Chemical structure of (68). ................................................................................................. 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
Figure 49. The docked configuration of 40E2 in the binding site of HDAC6 (PDB ID 1t69).
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-
carbothioamide)ruthenium(II)]chloride 13 in MeOD-d4. ................................................................... 154
Figure 52. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 11 in MeOD-d4. ................................................................... 155
Figure 53. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 13 in MeOD-d4. ................................................................... 155
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-
carbothioamide)ruthenium(II)]chloride 13 in CH2Cl2. ....................................................................... 156
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 72. ESI-MS of [chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 27 in CH2Cl2. ....................................................................... 165
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
Figure 76. 1HNMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
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
Figure 78. 1HNMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-
(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
Figure 81. 13C{H}NMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
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
Figure 83. 13C{H}NMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-
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
Figure 86. ESI-MS of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
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
Figure 88. ESI-MS of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-
(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
Table 20. H bonds and lipophilic contacts formed between HDAC6 and 31 and the individual
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.
Figure 20. Chemical structure of (51).
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.
Figure 21. Chemical structure of (52).
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
Figure 28. Chemical structure of (68).
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-
carbothioamide)ruthenium(II)] chloride (11)
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-
carbothioamide)ruthenium(II)] chloride (13)
Compound 13 was synthesized following the general synthetic procedure C using N-
(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,
3J= 6 Hz, 1H, H-15), 5.65 (d, 3J= 6 Hz, 1H, H-17), 5.59 (d, 3J= 6 Hz, 1H, H-18), 5.42
(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-
carbothioamide)ruthenium(II)] chloride (14)
Compound 14 was synthesized following the general synthetic procedure C using N-
(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-
carbothioamide)ruthenium(II)] chloride (15)
Compound 15 was synthesized following the general synthetic procedure C using N-
(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-
carbothioamide)ruthenium(II)] chloride (16)
Compound 16 was synthesized following the general synthetic procedure C using N-
(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),
8.20 (t, 3J= 8 Hz, 1H, H-3), 7.76 (t, 3J= 6 Hz, 1H, H-2), 7.37 (d, 3J= 9 Hz, 2H, H-8/H-
12), 6.93 (d, 3J= 8 Hz, 2H, H-9/H-11), 5.97 (d, 3J= 6 Hz, 1H, H-15), 5.88 (d, 3J= 6
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)
The compound 18 was synthesized following the general complexation procedure C
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,
H-1), 8.42 (d, 3J= 9 Hz, 1H, H-4), 8.26 (td, 3J= 8 Hz, 4J= 1 Hz, 1H, H-3), 7.76 (td, 3J=
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),
1.21 (d, 3J= 7 Hz, 3H, H-20), 1.17 (d, 3J= 7 Hz, 3H, H-22) ppm. 13C{1H} NMR
(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)
The compound 20 was synthesized following the general complexation procedure C
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)
The compound 21 was synthesized following the general complexation procedure C
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)
The compound 22 was synthesized following the general complexation procedure C
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)
The synthesis of compound 25 was performed following the general complexation
procedure C, using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (100 mg, 0.34
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)
The synthesis of compound 26 was performed following the general complexation
procedure C, using N-(4-sulfamoylphenyl)pyridine-2-carbothioamide (80 mg, 0.27
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.
13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 159.7
(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,
3J= 6 Hz, 1H, H-15), 6.06 (d, 3J= 6 Hz, 1H, H-17), 6.02 (d, 3J= 6 Hz, 1H, H-18), 5.75
(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,
H-1), 8.43 (d, 3J= 8 Hz, 1H, H-4), 8.30 (td, 3J= 8 Hz, 4J= 1 Hz, 1H, H-3), 7.85 (td, 3J=
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
[Chlorido(η6-p-cymene)(4-oxo-4-((4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)amino)butanoic acid)osmium(II)]chloride (33)
Compound 33 was synthesized following the general complexation procedure using
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
[Chlorido(η6-p-cymene)(8-oxo-8-((4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)amino)octanoic acid)ruthenium(II)]chloride (34)
Compound 34 was synthesized following the general complexation procedure using
29 (140 mg, 0.36 mmol, 2 eq.) and [Ru(η6-p-cymene)Cl2]2 (111 mg, 0.18 mmol, 1
eq.). Yield: 186 mg (74%), red solid. Elemental analysis found: C, 52.22; H, 5.28; N,
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),
8.29 (t, 3J= 8 Hz, 1H, H-3), 7.85 (t, 3J= 6 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), 6.05 (d, 3J= 6 Hz, 1H, H-23), 5.94 (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= 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
[Chlorido(η6-p-cymene)(8-oxo-8-((4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)amino) octanoic acid)osmium(II)]chloride (35)
Compound 35 was synthesized following the general complexation procedure using
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)
Compound 36 was synthesized following the general complexation procedure using
30 (120 mg, 0.35 mmol, 2 eq.) and [Ru(η6-p-cymene)Cl2]2 (106 mg, 0.17 mmol, 1
eq.). Yield: 143 mg (63%), red solid. Elemental analysis found: C, 44.11; H, 4.33; N,
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),
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 = 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
(C-23) ppm. MS (ESI+): m/z 579.1004 [36–2Cl–H]+ (mex = 579.1008).
87
[Chlorido(η6-p-cymene)(N1-hydroxy-N4-(4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)succinamide)osmium(II)]chloride (37)
Compound 37 was synthesized following the general complexation procedure using
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,
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), 6.14
(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),
2.67 (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
[Chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)octanediamide)ruthenium(II)]chloride (38)
Compound 38 was synthesized following the general complexation procedure using
31 (80 mg, 0.20 mmol) and [Ru(η6-p-cymene)Cl2]2 (61 mg, 0.10 mmol). Yield: 77 mg
(54%), red solid. Elemental analysis found: C, 49.56; H, 5.37; N, 7.59, calculated for
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
[Chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-carbothioamido-
κ2N,S)phenyl)octanediamide)osmium(II)]chloride (39)
Compound 39 was synthesized following the general complexation procedure using
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,
H-2/H-9/H-11), 7.60 (d, 3J= 9 Hz, 2H, H-8/H-12), 6.23 (d, 3J= 6 Hz, 1H, H-23), 6.14
(d, 3J= 6 Hz, 1H, H-25), 6.08 (d, 3J= 6 Hz, 1H, H-26), 5.82 (d, 3J= 6 Hz, 1H, H-22),
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
(C-27) ppm. MS (ESI+): m/z 725.2201 [39–2Cl–H]+ (mex = 725.2244).
90
[Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-(pyridine-2-
carbothioamido-κ2N,S)phenyl)octanediamide)rhodium(III)]chloride (40)
Compound 40 was synthesized following the general complexation procedure using
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)
Compound 41 was synthesized following the general complexation procedure using
31 (120 mg, 0.30 mmol) and [Ir(Cp*)Cl2]2 (119 mg, 0.15 mmol, 1 eq.). Yield: 130 mg
(54%), red solid. Elemental analysis found: C, 43.21; H, 4.86; N, 6.85, calculated for
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.
13C{1H} NMR (100.61 MHz, CDCl3 (0.3 mL)/MeOD-d4 (0.1 mL), 25 °C): δ = 173.9
(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
Molecular weight (g mol-1) 580.28 638.62
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
Temperature (K) 372(2) 372(2)
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
Goodness-of-fit on F2 1.049 1.016
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
3.3.1. Results and Discussion
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
Molecular weight (g mol-1) 293.36 652.21
Crystal size (mm) 0.32×0.10×0.08 0.26 × 0.10 × 0.08
Wavelength (Å) 0.71073 0.71073
Temperature (K) 100(2) 100(2)
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
Goodness-of-fit on F2 1.050 1.116
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
3.4.1. Results and Discussion
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.
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.
139
Figure 49. The docked configuration of 40E2 in the binding site of HDAC6 (PDB ID 1t69).
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.
Table 19. H bonds and lipophilic contacts formed between HDAC8 and 31 and the individual
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
Table 20. H bonds and lipophilic contacts formed between HDAC6 and 31 and the individual
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-
carbothioamide)ruthenium(II)]chloride 13 in MeOD-d4.
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Figure 52. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-bromophenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 11 in MeOD-d4.
Figure 53. 13C{H}NMR Spectrum of [chloro(η6-p-cymene)(N-(4-methoxyphenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 13 in MeOD-d4.
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.
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Representative NMR and ESI-mass spectra of scheme 2
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.
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Figure 58. 13C{H}NMR Spectrum of [bromido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-
carbothioamide)ruthenium(II)]bromide 17 in MeOD-d4.
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
Representative NMR and ESI-mass spectra of scheme 3
Figure 62. 1HNMR Spectrum of [chlorido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-
carbothioamide)ruthenium(II)]chloride 24 in MeOD-d4.
Figure 63. 1HNMR Spectrum of [bromido(η6-p-cymene)( N-(4-sulfamoylphenyl)pyridine-2-
carbothioamide)ruthenium(II)]bromide 25 in MeOD-d4.
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.
Figure 71. ESI-MS of [chlorido(η6-p-cymene)(N-(4-sulfamoylphenyl)pyridine-2-
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.
Representative NMR and ESI-mass spectra of scheme 4
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.
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.
167
Figure 76. 1HNMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4.
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.
168
Figure 78. 1HNMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-
(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
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.
Figure 81. 13C{H}NMR spectrum of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in MeOD-d4.
170
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.
Figure 83. 13C{H}NMR spectrum of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-
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.
Figure 85. ESI-MS of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
carbothioamido)phenyl)octanediamide)ruthenium(II)]chloride 38 in CH3OH.
172
Figure 86. ESI-MS of [chlorido(η6-p-cymene)(N1-hydroxy-N8-(4-(pyridine-2-
carbothioamido)phenyl)octanediamide)osmium(II)]chloride 39 in CH2Cl2.
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.
173
Figure 88. ESI-MS of [Chlorido(η5-pentamethylcyclopentadienyl)(N1-hydroxy-N8-(4-
(pyridine-2-carbothioamido-κ2N,S)phenyl)octanediamide)iridium(III)]chloride 41 in
CH3COCH3.