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)
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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
List of Publications ................................................................................................................................. 4
List of Schemes ....................................................................................................................................... 5
List of Figures ......................................................................................................................................... 8
List of Tables ........................................................................................................................................ 13
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
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. 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
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
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
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
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)
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
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
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.
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-