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Complexes of Ruthenium and Osmium Based on Oxicam

Scaffold as Potential Anticancer Agents

A thesis submitted as partial fulfillment for the degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

BY

Adnan Ashraf

2015

Department of Chemistry

University of Sargodha

Sargodha, Pakistan

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O Lord!

Advance me in Pure knowledge & Lead me to the Straight Path (Ameen)

iii

Dedication

Every challenging work needs self effort as well as

guidance of many elders especially of those who are

very close to your heart

I would like to dedicate this humble effort to all

those people whose affection, encouragement and

prays of day and night made me able to get success

and honour

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DECLARATION

It is certified that the research work mentioned in this thesis entitled “Complexes of

Ruthenium and Osmium Based on Oxicam Scaffold as Potential Anticancer

Agents” is original and nothing has been stolen /copied/ plagiarized from any other

source.

___________

(Supervisor 1)

Dr. Waseeq Ahmad Siddiqui

Assistant Professor,

Department of Chemistry,

University of Sargodha, Sargodha

___________

(Supervisor 2)

Dr. Muhammad Hanif

Assistant Professor,


COMSATS IIT, Abbotabad

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DECLARATION

It is certified that the research mentioned in this thesis entitled “Complexes of


Agents” by Adnan Ashraf is original and nothing has been stolen/copied/plagiarized

from any source.

___________

Adnan Ashraf

Ph.D Scholar


University of Sargodha, Sargodha

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Certificate

It is certified that the research contained in the thesis entitled “Complexes of


Agents” by Adnan Ashraf is original and the research work reported in this thesis has

been completed according to the requirements of HEC & UoS.

Signature of Student Signature of Supervisor with stamp

Name Adnan Ashraf Name Dr. Waseeq Ahmad Siddiqui

Signature of Supervisor with stamp

Name Dr. Muhammad Hanif

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APPROVAL CERTIFICATE

It is certified that research work reported in this thesis entitled “Complexes of


Agents” submitted by Adnan Ashraf is accepted in its present form by Department of

Chemistry, University of Sargodha, Sargodha, Pakistan, as satisfying the partial

requirement for the degree of Doctor of Philosophy in Chemistry.

Dr. Waseeq Ahmad Siddiqui Dr. Muhammad Hanif Supervisor 1 Supervisor 2

Assistant Professor Assistant Professor

Department of Chemistry Department of Chemistry

University of Sargodha COMSATS IIT.

Sargodha Abbotabad

Dr. Muhammad Sher Chairman,

Department of Chemistry

University of Sargodha

Sargodha

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ACKNOWLEDGEMENT

All praises to Almighty Allah, the Creator and Sustainer of universe, who is the Origin of all knowledge

and wisdom. Without Him nothing could be happened. He enabled me to successfully complete my research

work. All regards to Holy Prophet Muhammad (S. A. W.) who paved us to right path with essence of faith

in Allah.

I would like to express my gratitude and appreciation to my supervisor, Dr.Waseeq Ahmad

Siddiqui, Assistant Professor, Department of Chemistry, University of Sargodha, Sargodha for his

sympathetic attitude and providing the available facilities throughout the course of foregoing investigation.

I am greatly indebted to my respectable supervisor Dr. Muhammad Hanif Assistant Professor, COMSATS,

Abottabad / Research Fellow, School of Chemical Sciences, University of Auckland, New Zealand for his

valuable and scholarly guidance and continuous encouragement, sincere cooperation, meticulous criticism,

indefatigable zeal and patronizing concrns.

My sincerest thanks are to the Chairman, Dr. Muhammad Sher, Department of Chemistry,

University of Sargodha, Sargodha for providing facilities during the conduct of this research work.

It is immense pleasure to express my profound indebtness and gratitude to Dr. Christian

Hartinger, Associate Professor and Deputy Head (Research) of School of Chemical Sciences, University of

Auckland, New Zealand for providing me an opportunity to work in his lab as Visiting Researcher. I

would also like to express my thanks and respect Dr.Nawaz Tahir, Department of Physics, University of

Sargodha and Dr. Nadeem Arshad Qadri, King Abdul Aziz University, Saudi Arabia, Tanya Groutso,

School of Chemical Sciences, University of Auckland, New Zealand for X-ray Crystallographic studies.

I would like to express my gratitude to Higher Education Commission (HEC), Islamabad,

Pakistan for providing me necessary funds for carrying out research project and sponsoring my research

fellowship both within country and abroad. I am also thankful to Hi-Tech. Laboratory staff, University of

Sargodha for spectroscopic studies and for their cooperation.

I also extend admiration and appreciation to my lab colleagues Dr. Farhana Aman and Ms.

Shahana Zainab for their cooperation and supportive attitude. I acknowledge my heartily thanks to my

Family for their loving and convivial behaviors and for their prayers which always sooth & calm me. I

would like to thank my friends who were always available on the spot for sharing joys and sorrows during

this duration. I am highly indebted for their support, cooperation and nice attitude.

A cordial thank to all who directly or indirectly helped me during my lab work. May Allah bless

all above mentioned personalities long, happy and peaceful life. (Ameen)

Adnan Ashraf

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Abbreviations

2D two dimensional NMR m multiplet (NMR)

DCM dichloromethane mg milligram

d doublet (NMR) min minute

q Quartet(NMR) ovlp.m Overlapped multiplet (NMR)

δ chemical shift µM micromolar

d6- DMSO deuterated dimethyl sulfoxide mL milliliter

DNA 2’-deoxyribonucleic acid mM millimolar

D2O deuterated water mmol millimol

e.g. exempli gratia m.p. melting point

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

etc. et cetera (and other things NMR nuclear magnetic resonance

(spectroscopy)

ESI electrospray ionization pH pondus Hydrogen

(power of hydrogen)

g gram pKa logKa (acid dissociation constant)

h hour ppm parts per million

DCM dichloromethane s singlet (NMR)

GMP guanosine 5’monophosphate t triplet (NMR)

Hz hertz °C degree Celsius

TLC Thin Layer Chromatography DMF Dimethyl Formamide

IR Infrared SAR Structure Activity Relationship

KBr Potassium bromide NSAIDs Non-steroidal anti-inflammatory

drugs

CDCl3 deuterated chloroform MeOD-d4 deuterated methanol

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List of Figures

Fig. 1.1: X-ray crystal structure ruthenium(II)-enzyme complex 2

Fig. 1.2: Platinum based drugs used in clinics 4

Fig. 1.3: A prodrug active only in acidic environment 5

Fig. 1.4: Platinum drug-HPMA conjugates 5

Fig. 1.5: Structure of trans-[PtCl2L1L2] complexes 5

Fig. 1.6: Platinum (IV) complexes in clinical trials 6

Fig. 1.7: Platinum (IV) complexes of bioactive ligands 7

Fig. 1.8: Non platinum anticancer activemetal complexes 8

Fig. 1.9: Ruthenium based anticancer complexes in clinical trials 9

Fig. 1.10: Piano stool configuration of Ru(II)-arene complexes 10

Fig. 1.11: Structures of different arene ligands used for bonding to Ru(II) 10

Fig. 1.12: Ru(II) arene complexes as anticancer agents 11

Fig. 1.13: Structures of some RAPTA Complexes 11

Fig. 1.14: Examples of dinuclear Ru(II)-arene complexs 13

Fig. 1.15: Examples of osmium-arene anticancer complexes 14

Fig. 1.16: General aqueous reactivity of osmium(II)–arenes 15

Fig. 1.17: Some representative Oxicams 16

Fig. 1.18: Coordination behaviour of piroxicam with different metal ions 17

Fig. 1.19: Piroxicamato ligand containing arene ruthenium complex 18

Fig. 1: Comparison of 1H NMR of compounds 7a-g 79

Fig. 2: Comparison of 13

C NMR of compounds 7a-g 79

Fig. 3: Ortep diagram of Compound 6a, 7a and 7b 80

Fig. 4: 1H NMR of complexes 10a-g 84

Fig. 5: 13

C NMR of complexes 10a-g 85

Fig. 6: Ortep diagrams of compound 10a and 10d 87

Fig. 7: 1H NMR of the ligand 11, 12 and complexes 13a-d, 14a-d 90

Fig. 8: 13

C NMR of the ligand 11, 12 and complexes 13a-d, 14a-d 91

Fig. 9: Ortep diagram of complexes 13a and14d 92

Fig. 10: Ortep diagram of compounds 15a, 18 and 18c 97

Fig. 11: Comparison of 1H NMR of compound 20d, 20g, 20i and 20l 101

Fig. 12: Ortep diagrams of chalcones 103

Fig. 13: Aromatic region in 1H NMR of compounds 21a-g 106

Fig. 14: Ortep diagrams of compound 21d, 21d, 21g and 21k 107

Fig. 15: Ortep diagram of pyrazole based Ru(II) monodentate complexes 110

Fig. 16: 1H NMR of compounds 23-23g 112

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List of Tables

Table-1: Crystal structure data for the compound 6a, 7a and 7b 81

Table-2: Anti-cancer Activity of complexes 7a-g 82

Table-3: Crystal structure data for the complexes 10a and 10d 86

Table-4: Selected bond lengths for complexes 10a and 10d 87


Table-6: Crystal structure data for the complexes 13a and 14d 93

Table-7: Some selected bond lengths of compound 13a and 4d 93

Table-8: Crystal structure data for the compounds 15a, 18 and 18c 97

Table-9: Selected bond lengths for compound 15a, 18 and 18c 98

Table-10: Anti-cancer Activity of complexes 18, 18a-c 98

Table- 11: Selected bond lengths for compounds 20d-f, 20h, 20i, 20k-n 103

Table 12a: Crystal structure data for the compounds 20d, 20e, 20f and 20h 103

Table 12b: Crystal structure data for the compounds 20i, 20k, 20l and 20m 104

Table-13: Crystal structure data for the compound 21a, 21d, 21g and 21k 107


Table-15: Crystal structure data for the compounds 22d, 22h, 22i and 22j 110

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List of Publications as co-auther during Ph.D

Research Papers:

F. Aman, M. Hanif, W. A. Siddiqui, A. Ashraf, L. Filak, J. Reynisson, T.

Soehnel, S. Jamieson, C. Hartinger, Anticancer Ruthenium(η6-p-cymene)

Complexes of Non-steroidal Anti-inflammatory Drug Derivatives.,

Organometallics, 2014, 33, 5546-5553.

F. Aman, M. Asiri, W. A. Siddiqui, M. N. Arshad, A. Ashraf, V. A. Blatov, N. S. Zakharov Multilevel topological description of molecular packings of

1,2-benzothiazines, Cryst. Eng. Comm., 2014, 16, 1963-70.

F. Aman, M. Hanif, A. Ashraf, W. A. Siddiqui, S. Jamieson, C. Hartinger,

“Oxicams as bioactive ligand systems in anticancer RuII(p-cymene)

complexes” in 12th

European Biological Inorganic Chemistry Conference

Zurich Switzerland 24-28 Aug 2014, 19 (Suppl 2): S779–S814.

International Crystal Structure Reports:

1. Ashraf, A., Tahir, M. N., Siddiqui, W. A., Perveen, N. (2012) 2-(1H-

Benzimidazol-2-yl)-N-[(E)-(dimethylamino)methylidene]benzenesulfonamide

Acta. Cryst. E68, o2069.

2. Aman, F., Siddiqui, W. A., Ashraf, A., Siddiqui, H. L., Parvez, M., (2012) N-

Benzyl-4-hydroxy-2-methyl-1,1-dioxo-2H-16,2-benzothiazine-3-

carboxamide Acta. Cryst. E68, o1921.

3. Ali, U. S., Siddiqui, W. A., Ashraf, A., Tahir, M. N., (2012) 2,6-Dibromo-4-

chloroaniline Acta. Cryst. E68, o1904.

4. Aman, F., Siddiqui, W. A., Ashraf, A., Siddiqui, H. L., Parvez, M., (2012) 4-

Hydroxy-2-methyl-1,1-dioxo-N-phenyl-2H-16,2-benzothiazine-3-

carboxamide Acta. Cryst. E68, o1790.

5. Aman, F., Siddiqui, W. A., Ashraf, A., Tahir, M. N., (2012) 2,3-Dihydro-16,2-benzothiazine-1,1,4-trione Acta. Cryst. E68, o1306.

6. Akhtar, T., Siddiqui, W. A., Ashraf, A., Tahir, M. N., (2012) 2,2-(4-Methyl-

4H-1,2,4-triazole-3,5-diyl)dibenzenesulfonamide, Acta. Cryst. E68, o754.

7. Aman, F., Siddiqui, W. A., Ashraf, A., Tahir, M. N., (2012) 4-Hydroxy-2-

methyl-1,1-dioxo-2H-1λ6,2-benzothiazine-3-carboxylic acid hemihydrates,

Acta. Cryst. E68, o621-o622.

8. Siddiqui, W. A., Ashraf, A., Siddiqui, H. L., Akram, M., Parvez, M. (2012)2-

(N-Cyclohexylcarbamoyl)benzenesulfonamide, Acta. Cryst. E68, o370.

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Table of Contents

ACKNOWLEDGEMENT ............................................................................................................................. viii

Abbreviations ............................................................................................................................................ ix

List of Figures .............................................................................................................................................. x

List of Tables .............................................................................................................................................. xi

List of Publications as co-auther during Ph.D ........................................................................................... xii

Abstract ................................................................................................................................................xxviii

1. Introduction ................................................................................................................................................1

1.1 Cancer ...................................................................................................................................................1

1.2 Available therapeutic options for cancer treatment ............................................................................1

1.3 Chemotherapy ......................................................................................................................................2

1.4 Classes of chemotherapeutics ..............................................................................................................3

1.4.1 Antimetabolites .............................................................................................................................3

1.4.2 Alkylating agents ...........................................................................................................................3

1.4.3 Topoisomerase inhibitors ..............................................................................................................3

1.5 Metal based anticancer agents ............................................................................................................4

1.6 Platinum based anticancer agents .......................................................................................................4

1.7 New trends in platinum(II) based drugs ...............................................................................................5

1.8 Platinum(IV) complexes in clinical studies ...........................................................................................6

1.9 Platinum(IV) complexes with biological relevant ligands .....................................................................6

1.10 Non-Platinum Anticancer Agents .......................................................................................................7

1.11 Ruthenium based anticancer agents ..................................................................................................9

1.12 Activation by reduction hypothesis ....................................................................................................9

1.13 Ru(II)-arene complexes ................................................................................................................... 10

1.14 Dinuclear Ru(II)-arene complexes ................................................................................................... 12

1.15 Osmium based anticancer agents ................................................................................................... 13

1.16 Osmium based organometallic complexes ..................................................................................... 14

1.17 Metal complexes of bioactive ligands ............................................................................................. 15

1.18 Oxicam: An eminent class of NSAIDs ............................................................................................... 15

1.19 Applications of oxicam derivatives .................................................................................................. 16

1.20 Metal complexes of oxicam derivatives .......................................................................................... 17

1.21 Aims and objectives of research work ............................................................................................ 19

1.22 Scope of work .................................................................................................................................. 19

2 Experimental work ................................................................................................................................... 20

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2.1 Materials ............................................................................................................................................ 20

2.2 Instrumental Methods and Techniques ............................................................................................ 20

2.3 Biological Studies ............................................................................................................................... 21

2.4 Synthesis of pro-oxicam primary amides 6a-g and their Ru(II) Complexes 7a-g .............................. 22

2.4 General procedure for synthesis of 6a-g ........................................................................................... 22

2.4.1 2-Methyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid amide

(6a)....................................................................................................................................................... 22

2.4.2 2-Ethyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid amide

(6b) ...................................................................................................................................................... 23

2.4.3 2-Propyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid amide

(6c) ....................................................................................................................................................... 23

2.4.4 2-Butyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid amide

(6d) ...................................................................................................................................................... 24

2.4.5 2-Cyanomethyl-4-hydroxy-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid

amide (6e) ........................................................................................................................................... 24

2.4.6 3-Carbamoyl-4-hydroxy-1,1-dioxo-1H-1λ6-benzo[e][1,2]thiazin-2-yl)-acetic acid methyl ester

(6f) ....................................................................................................................................................... 24

2.4.7 2-Benzyl-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-dioxide (6g) ....................... 25

2.5 General procedure for the synthesis of 7a-g .................................................................................... 25

2.5.1 Chlorido(2-methyl-4-oxido-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid

amide)(η6-p-cymene)ruthenium(II) (7a) .............................................................................................. 26

2.5.2 Chlorido(2-ethyl-4-oxido-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid

amide)(η6-p-cymene)ruthenium(II) (7b) ............................................................................................. 26

2.5.3 Chlorido(2-propyl-4-oxido-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid

amide)(η6-p-cymene)ruthenium(II) (7c) .............................................................................................. 27

2.5.4 Chlorido(2-butyl-4-oxido -1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2]thiazine-3-carboxylic acid

amide)(η6-p-cymene)ruthenium(II) (7d) ............................................................................................. 28

2.5.5 Chlorido(2-cyanomethyl-4-oxido-1,1-dioxo-1,2-dihydro-1λ6-benzo[e][1,2] thiazine-3-carboxylic

acid amide(η6-p-cymene)ruthenium (II) (7e) ...................................................................................... 28

2.5.6 Chlorido(3-carbamoyl-4-oxido-1,1-dioxo-1H-1λ6-benzo[e][1,2]thiazin-2-yl)-acetic acid methyl

ester)(η6-p-cymene)ruthenium(II) (7f) ................................................................................................ 29

2.5.7 Chlorido(2-benzyl-4-oxido-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide)(η6-p-

cymene)ruthenium(II) (7g) .................................................................................................................. 30

2.6 Synthesis of pro-oxicam secondary amides 9a-g and their Ru(II) complexes 10a-g ......................... 31

2.6 General procedure for synthesis of 9a-g ........................................................................................... 31

2.6.1 2-Benzyl-N-phenyl-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide (9a) ....... 31

2.6.2 2-Benzyl-N-(2-chlorophenyl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide

(9b) ...................................................................................................................................................... 32

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(9c) ....................................................................................................................................................... 32


(9d) ...................................................................................................................................................... 33

2.6.5 2-Benzyl-N-cyclohexyl-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide (9e) .. 33

2.6.6 2-Benzyl-N-benzyl-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide (9f) ......... 34

2.6.7 2-benzyl-N-(2,6-dimethylphenyl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-

dioxide (9g) .......................................................................................................................................... 34

2.7 General procedure for synthesis of 10a-g ......................................................................................... 35

2.7.1 Chlorido(2-benzyl-N-phenyl-4-oxido-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide)(η6-

p-cymene)ruthenium(II) (10a) ............................................................................................................. 35

2.7.2 Chlorido(2-benzyl-N-(2-chlorophenyl)-4-oxido-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-

dioxide)(η6-p-cymene)ruthenium(II) (10b) .......................................................................................... 36


dioxide)(η6-p-cymene)ruthenium(II) (10c) .......................................................................................... 37


dioxide)(η6-p-cymene)ruthenium(II) (10d) .......................................................................................... 37

2.7.5 Chlorido(2-benzyl-N-cyclohexyl-4-oxido-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-

dioxide)(η6-p-cymene)ruthenium(II) (10e) .......................................................................................... 38

2.7.6 Chlorido(2-benzyl-N-benzyl-4-oxido-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide)(η6-

p-cymene)ruthenium(II) (10f) ............................................................................................................. 39

2.7.7 Chlorido(2-benzyl-N-(2,6-dimethylphenyl)-4-oxido-2H-benzo[e][1,2] thia-zine-3-carboxamide

1,1-dioxide)(η6-p-cymene)ruthenium(II) (10g) .................................................................................... 40

2.8 Synthesis of N-benzyl analogues of Piroxicam/Isoxicam related ligand 11, 12 and their Ru(II)/Os(II)

complexes 13a-d, 14a-d .......................................................................................................................... 41

2.8 General procedure for the synthesis of 11, 12 .................................................................................. 41

2.8.1 2-Benzyl-N-(pyridin-2-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbox-amide-1,1-dioxide (11)

............................................................................................................................................................. 41

2.8.2 2-Benzyl-N-(5-methyl-isoxazol-3-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carbox-amide-1,1-

dioxide (12) .......................................................................................................................................... 42

2.9 General procedure for the synthesis of 13a,b, 14a,b ........................................................................ 43

2.9.1 Dichlorido-(2-benzyl-N-(pyridin-2-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide)(η6-p-cymene)ruthenium(II) (13a) .......................................................................................... 43

2.9.2 Dichlorido-(2-benzyl-N-(pyridin-2-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide)(η6-p-cymene)osmium(II) (13b) .............................................................................................. 44

2.9.3 Dichlorido-(2-Benzyl-N-(5-methyl-isoxazol-3-yl)-4-hydroxy-2H-benzo[e] [1,2]thiazine-3-carbox-

amide-1,1-dioxide)(η6-p-cymene) ruthenium(II) (14a) ....................................................................... 44

2.9.4 Dichlorido-(2-Benzyl-N-(5-methyl-isoxazol-3-yl)-4-hydroxy-2H-benzo[e] [1,2]thiazine-3-carbox-

amide-1,1-dioxide)(η6-p-cymene)Osmium(II) (14b) ............................................................................ 45

xxiii

2.10 General synthetic procedure for the synthesis of 13c,d, 14c,d ...................................................... 46

2.10.1 Chlorido(2-benzyl-4-oxido-N-(pyridin-2-yl)-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide)(η6-p-cymene)ruthenium(II) (13c) .......................................................................................... 46

2.10.2 Chlorido(2-benzyl-4-oxido-N-(pyridin-2-yl)-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide)(η6-p-cymene)osmium(II) (13d) .............................................................................................. 47

2.10.3 Chlorido(2-Benzyl-N-(5-methyl-isoxazol-3-yl)-4-oxido-2H-benzo[e][1,2] thiazine-3-carbox-

amide-1,1-dioxide)(η6-p-cymene)ruthenium(II) (14c) ......................................................................... 47

2.10.4 Chlorido(2-Benzyl-N-(5-methyl-isoxazol-3-yl)-4-oxido-2H-benzo[e][1,2] thiazine-3-carbox-

amide-1,1-dioxide)(η6-p-cymene)osmium(II) (14d) ............................................................................ 48

2.11 Oxicam derivatives functionalized at position 2 and 3 and their Ru(II)/Os(II) complexes 15a, 16a,

17a,b, 18a-c ............................................................................................................................................. 49

2.11 General procedure for the synthsis of 15-18 .................................................................................. 50

2.11.1 2-benzyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide (15) .......................................................................................................................................... 50

2.11.2 2-benzyl-N-(1H-indazol-6-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-dioxide

(16) ...................................................................................................................................................... 50

2.11.4 2-methyl-N-(1H-indazol-6-yl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide 1,1-dioxide

(17) ...................................................................................................................................................... 51

2.11.4 2-methyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxamide-1,1-

dioxide (18) .......................................................................................................................................... 51

2.12 General procedure for the synthesis of 15a-18a, 16b, 18b ............................................................ 52

2.12.1 Dichlorido(2-benzyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-

carboxamide-1,1-dioxide)(η6-p-cymene) ruthenium(II) (15a) ............................................................. 52

2.12.2 Dibromido-(2-benzyl-N-(1H-indazol-6-yl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-


2.12.3 Diiodido-(2-benzyl-N-(1H-indazol-6-yl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-carboxamide-

1,1-dioxide)(η6-p-cymene) ruthenium(II) (16b) ................................................................................... 53

2.12.4 Dichlorido-(2-methyl-N-(1H-indazol-6-yl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-


2.12.5 Dichlorido[2-methyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-

carboxamide-1,1-dioxide](η6-p-cymene)Ruthenium(II) (18a) ............................................................. 54

2.12.6 Dichlorido(2-methyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-

carboxamide-1,1-dioxide)(η6-p-cymene)Osmium(II) (18b) ................................................................. 55

2.12.7 Oxalato(2-methyl-N-(pyridin-4-ylmethyl)-4-hydroxy-2H-benzo[e][1,2] thiazine-3-

carboxamide-1,1-dioxide)(η6-p-cymene)Osmium(II) (18c) ................................................................. 56

2.13 Synthesis of 1,2-benzothiazine based chalcones 20a-o .................................................................. 57

2.13 General synthetic procedure for the synthesis of 20a-o................................................................. 57

2.13.1 3-Benzylidene-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-one (20a) ..................... 57

xxiv

2.13.2 3-(2-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20b)58

2.13.3 3-(3-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20c) 58

2.13.4 3-(4-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20d)58

2.13.5 3-(2-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20e) .. 59

2.13.6 3-(3-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20f) ... 59

2.13.7 3-(4-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20g) .. 60

2.13.8 3-(3-Chloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20h) .. 60

2.13.9 3-(4-Chloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20i) ... 60

2.13.10 3-(2,4-Dichloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20j)

............................................................................................................................................................. 61

2.13.11 3-(3-Fluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20k) . 61

2.13.12 3-(4-Fluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20l) .. 62

2.13.13 3-(2,6-Difluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one

(20m) ................................................................................................................................................... 62

2.13.14 3-(2-Methyl-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20n) 62

2.13.15 3-(3-Hydroxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2] thiazin-4-one (20o)

............................................................................................................................................................. 63

2.14 Synthesis of pyrazole based benzenesulfonamides as ligands 21a-i .............................................. 63

2.14 General procedure for synthesis of 21a-i ........................................................................................ 64

2.14.1 2-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamide (21a) ......................................................... 64

2.14.2 2-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21b) ............................... 64

2.14.3 2-(5-(3-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21c) ............................... 65

2.14.4 2-(5-(4-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21d) ............................... 65

2.14.5 2-(5-(3-florophenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21e) ...................................... 65

2.14.6 2-(5-(3-chlorophenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21f) .................................... 66

2.14.7 2-(5-(3-bromophenyl)-1H-pyrazol-3-yl)benzenesulfonamide (21g) ................................... 66

2.14.8 2-[5-(3-Ethoxy-2-hydroxy-phenyl)-1H-pyrazol-3-yl]-benzenesulfonamide (21h) .................... 66

2.14.9 2-[5-(2,4-Dichloro)-1H-pyrazol-3-yl]-benzenesulfonamide (21i) ............................................. 67

2.15 Synthesis of Ru(II) complexes of pyrazole based benzenesulfonamide as a monodentate donor

system 22a-j ............................................................................................................................................ 67

2.15 General porocedure for the synthesis of 22a-j ........................................................................... 67

2.15.1 Dichlorido(2-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-cymene)ruthenium(II)

(22a)..................................................................................................................................................... 68

2.15.2 Dichlorido(2-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfon-amido)(η6-p-

cymene)ruthenium(II) (22b) ................................................................................................................ 68

xxv

2.15.3 Dichlorido(2-(5-(3-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfon-amido)(η6-p-

cymene)ruthenium(II) (22c) ................................................................................................................ 68

2.15.4 Dichlorido(2-(5-(4-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfon-amido) (η6-p-

cymene)ruthenium(II) (22d) ................................................................................................................ 68

2.15.5 Dichlorideo(2-(5-(3-fluoroyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (22e) ................................................................................................................ 69

2.15.6 Dichlorido(2-(5-(3-chloroyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (22f) ................................................................................................................. 69

2.15.7 Dichlorido(2-(5-(3-bromoyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (22g) ................................................................................................................ 69

2.15.8 Dichlorido(2-(5-(2-hydroxy-4-ethoxyphenyl)-1H-pyrazol-3-yl)benzene-sulfonamido) (η6-p-

cymene)ruthenium(II) (22h) ................................................................................................................ 70

2.15.10 Oxalato(2-(5-(4-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfon-amido) (η6-p-

cymene)ruthenium(II) (22j) ................................................................................................................. 70

2.16 Synthesis of Ru(II) complexes of pyrazole based benzenesulfonamide as a bidentate donor ligands

23a-g ........................................................................................................................................................ 71

2.16 General procedure for the synthesis of 23a-g ................................................................................ 71

2.16.1 Chlorido(2-(5-phenyl-1H-pyrazol-3-yl)benzenesulfonamido)(η6-p-cymene) ruthenium(II) (23a)

............................................................................................................................................................. 71

2.16.2 Chlorido(2-(5-(2-methoxyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (23b) ................................................................................................................ 72


cymene)ruthenium(II) (23c) ................................................................................................................ 73


cymene)ruthenium(II) (23d) ................................................................................................................ 73

2.16.5 Chlorido(2-(5-(3-fluoroyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (23e) ................................................................................................................ 74

2.16.6 Chlorido(2-(5-(3-chloroyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (23f) ................................................................................................................. 75

2.16.7 Chlorido(2-(5-(3-bromoyphenyl)-1H-pyrazol-3-yl)benzenesulfonamido) (η6-p-

cymene)ruthenium(II) (23g) ................................................................................................................ 75

3 RESULTS AND DISCUSSION ....................................................................................................................... 77

3.1 Synthesis of pro-oxicam primary amides 6a-g and their Ru(II) complexes 7a-g ............................... 77

3.1.2 FT-IR ............................................................................................................................................ 78

3.1.3 1H NMR ....................................................................................................................................... 78

3.1.4 13C NMR of metal complexes ...................................................................................................... 79

3.1.5 ESI-MS ......................................................................................................................................... 80

3.1.6 X-ray Crystallography ................................................................................................................. 80

xxvi

3.1.7 Anticancer Activity...................................................................................................................... 81

3.2 Synthesis of pro-oxicam based secondary amides 9a-g and their Ru(II) complexes 10a-g .............. 82

3.2.1 Synthesis and Spectral Characterization .................................................................................... 82

3.2.2 Synthesis ..................................................................................................................................... 82

3.2.3 FT-IR Studies ............................................................................................................................... 83

3.2.4 1H NMR ....................................................................................................................................... 83

3.2.5 13C NMR ...................................................................................................................................... 84

3.2.6 ESI-MS ......................................................................................................................................... 85



3.3 Synthesis of N-benzyl analogues of Piroxicam/Isoxicam related ligand 11, 12 and their Ru(II)/Os(II)

complexes 13a-d, 14a-d .......................................................................................................................... 88

3.3.1 Synthesis ..................................................................................................................................... 88

3.3.2 FT-IR ............................................................................................................................................ 89

3.3.3 1H NMR ....................................................................................................................................... 89

3.3.4 13C NMR ...................................................................................................................................... 91

3.3.5 ESI-MS ......................................................................................................................................... 91


3.4 Oxicam derivatives functionalized at position 2 and 3 and their Ru(II)/Os(II) complexes 15a, 16a,

17a,b, 18a-c ............................................................................................................................................. 94

3.4.1 Synthesis ..................................................................................................................................... 94

3.4.2 FT-IR ............................................................................................................................................ 95

3.4.3 1H NMR ....................................................................................................................................... 95

3.4.4 13C NMR ...................................................................................................................................... 96

3.4.5 MS Analysis ................................................................................................................................. 97



3.5 Synthesis of 1,2-benzothiazine based Chalcones 20a-o .................................................................... 99

3.5.1 Synthesis ..................................................................................................................................... 99

3.5.2 FT-IR ............................................................................................................................................ 99

3.5.3 1HNMR ...................................................................................................................................... 100

3.5.4 13C NMR .................................................................................................................................... 101

3.5.5 ESI-MS ....................................................................................................................................... 102

3.5.6 X-rays Crystallography .............................................................................................................. 102

3.6 Synthesis of pyrazole based benzenesulfonamide as ligands 21a-i ................................................ 104

xxvii

3.6.1 Synthesis ................................................................................................................................... 104

3.6.2 Characterization ....................................................................................................................... 105

3.6.3 FT-IR .......................................................................................................................................... 105

3.6.4 1H NMR ..................................................................................................................................... 105

3.6.5 ESI-MS ....................................................................................................................................... 106

3.6.6 Molecular structures ................................................................................................................ 106

3.6.7 Anticancer Activity.................................................................................................................... 107

3.7 Synthesis of Ru(II) complexes of pyrazole based benzenesulfonamide as a monodentate donor

system 22a-j .......................................................................................................................................... 108

3.7.1 Synthesis ................................................................................................................................... 108

3.7.2 FT-IR .......................................................................................................................................... 109

3.7.3 MS Analysis ............................................................................................................................... 109

3.8 Synthesis of Ru(II) complexes of pyrazole based benzenesulfonamide as bidentate donor ligands

23a-g ...................................................................................................................................................... 111

3.8.2 FT-IR .......................................................................................................................................... 111

3.8.3 1H NMR ..................................................................................................................................... 112

3.8.4 MS Analysis ............................................................................................................................... 112

4. Future Plan .................................................................................................................................... 113

Conclusion ............................................................................................................................................. 114

xxviii

Abstract

Metal based drugs have been used for medicinal purposes since ages but their potential was

realized after the discovery of the first metal based chemotherapeutic agent, i.e., cisplatin, which

became one of the most successful anticancer drugs espcecially for the treatment of testicular

cancer. Other members of this class include oxaliplatin, carboplatin and nedaplatin. The use of

platinum based drugs is limited due to their adverse side effects (e.g., nephrotoxicity,

neurotoxicity, nausea, vomiting etc.) and intrinsic or acquired resistance. These limitations

prompted bioinorganic chemists to develop new strategies to treat cancer with other metal based

anticancer agents with higher efficacy and lesser undesired effects. Therefore, different metal

complexes of titanium, iron, cobalt, gallium, ruthenium and osmium etc. were investigated.

Among these NAMI-A (imidazolium [tetrachlorido(dimethylsulfoxide)(1H-

imidazole)ruthenate(III)]), KP46 (trismaltolate gallium) and KP1019 (indazolium trans-

[tetrachloridobis(1H-indazole)ruthenate(III)]) have entered clinical trials. On the other hand,

Ru(II)/Os(II) half sandwich organometallic complexes increase the lipophilic character of

complexes and facilitate their uptake into cells. RAPTA type complexes are among the most

popular examples of half sandwich organometallics. Furthermore, the coordination of bioactive

ligands with these established organometallic pharmacophores may enhance the efficacy of these

biologically active compounds by altering their physicochemical and pharmacological properties.

In particular, the use of bioactive ligands such as hydroxypyrones, quinolones and non-steroidal

anti-inflammatory drugs (NSAIDs) often resulted in promising bioactivity of the compounds. In

this thesis, the use of oxicam based NSAIDS as ligands for Ru(II) and Os(II) was investigated.

For this purpose different series of ligands based on the oxicam scaffold were prepared. These

include 1,2-benzothiazine based primary amides, secondary amides, indazole and methyl pyridyl

based secondary amides, piroxicam as well as isoxicam analogues, 1,2-benzothiazine based α,β-

unsatuarated ketones and pyrazole based benzenesulfonamides. Furthermore, these ligands were

reacted with Ru(II) and Os(II) cymene dimer to obtain organometallic complexes. All the ligands

and complexes were characterized with different spectroscopic techniques including FT-IR, 1H,

13C NMR, elemental analysis, high resolution mass spectrometry and twenty seven compounds

were analyzed by single crystal X-ray diffraction analysis. The cytotoxic activity of the

complexes towards human colorectal carcinoma HCT116, non-small cell lung carcinoma NCI-

H460 and cervical carcinoma SiHa cells was investigated by using the sulforhodamine B (SRB)

assay.

The prepared ligands behaved as monodentate (N donor) or bidentate chelators (O,O-, N,O- and

N,N-donor systems) depending upon the ligand structure as well as reaction conditions such as

xxix

nature of solvent used for reaction. The 1,2-benzothiazine based primary amides were

synthesized by reacting compound 5 with different alkylating agents in basic conditions to isolate

ligands 6a-g. These O,O-coordinating ligands were used to synthesize the organometallic

ruthenium complexes 7a-g (Scheme-1) with piano-stool configuration. These complexes were

evaluated for their anticancer activity and results indicate that only 7f and 7g were active against

three different human cancer cell lines. On the other hand, 1,2-benzothiazine based secondary

amides were synthesized by reacting compound 8 with different aniline derivatives to obtain

O,O-chelating ligands 9a-g. When these ligands were reacted with [Ru(cym)Cl2]2, the same O,O-

coordination behavior was observed to stabilize metal complexes 10a-g by forming six-

membered rings and giving rise to piano stool type geometry (Scheme-2). All these complexes

were found active against different anticancer cell lines and the most lipophilic compound was

found the most active with an IC50 value of 13.58 µM. The N-benzyl analogues of piroxicam and

isoxicam 11 and 12 were also prepared and reacted with MII(η

6-p-cymene). Compounds 11 and

12 can act as monodentate ligands through their pyridyl/isoxazolyl nitrogen atom and as bidentate

chelators to Ru(II) and Os(II) metal ions by forming six membered rings through

pyridyl/isoxazolyl nitrogen and the amide oxygen atoms (Scheme-3). In compounds 15-18

functionalization at position 3 was carried out to get indazolyl/pyridyl goup-containing oxicam

analogues which act as monodentate ligands and coordinate to ruthenium/osmium centres

through pyridyl/indazolyl nitrogens. The results of anticancer activity studies revealed that

organo-Ru(II) and -Os(II) complexes 18a-c are more active than the free ligand 18 (Scheme-4).

The 1,2-benzothiazine based chalcones (Scheme-5) were obtained as intermediates which were

reacted with hydrazine to isolate 1,2-benzothiazine based pyrazole containing sulphonamide

ligands 21a-j. The results of anticancer activity assays show that halogen containing derivatives

are more active in this series (Scheme-6). These pyrazole containing sulphonamides were reacted

with [Ru(cym)Cl2]2 to isolate complexes in which these sulfonamides acted either as

monodentate or bidentate ligands. In complexes 22a-k ligands coordinated mondentately through

the pyrazole nitrogen (Scheme-7) while in complexes 23a-g they coordinated bidentately via

pyrazole and sulphonamide nitrogen atoms by forming rather stable seven membered rings

(Scheme-8).

The biological investigations indicate moderate to high IC50 values for these complexes and it

was also observed that within the series of compounds, the most lipophilic complex was the most

active.

INTRODUCTION

Chapter 1 Introduction

1

1. Introduction

1.1 Cancer

Cancer has become the second deadliest disease after cardiovascular diseases world

wide. More than 100 different types or forms of cancer are known which are severely

affecting the human health [1]. This disease has created such an alarming situation

that in the year 2012, 14 million new cases of cancer were reported and it is expected

that in the next 20 years these cancer cases may reach up to 22 million. Cancer

incidence and mortality rate due to this disease are continuously growing and it is

expected that mortality rate may reach up to 13 million per year in the next few

decades due to cancer alone which was 8.2 million in 2012. Cancer incidence is so

common in Central America, South America, Asia, as well as in Africa that more than

60 % cancer cases through out the world are registered from these regions. Cancer has

become the cause of more than 70% deaths in these regions of the world [2]. It was

found that cancer burden around the global has increased to such an extent that more

than half million people died only in united states in the year 2008 while 1.5 million

were registered as cancer patients in USA [3].

1.2 Available therapeutic options for cancer treatment

Several therapeutic options are available which can be employed in order to eradicate

or minimize the fatal effects of this deadly disease. Some commonly employed

approaches include surgery, radiotherapy, targeted therapy and chemotherapy [4,5].

Surgery: It is applicable for the treatment of primary and localized tumors.

Radiation Therapy: Specifically, it is targeting for primary solid/localized

tumors and mainly dependent on diagnostic imaging, radiology and

radiography. It destroys cancer cells locally but unfortunately this technique is

not uselful when tumor metastasize in the body. Moreover, healthy cells are

also damaged in this technique.

Chemotherapy: Chemotherapy is found to be more suitable approach for

reducing the tumor size before surgery and also after surgery when tumor is

metastasized. Chemotherapy is regarded as the most efficacious method for

the treatment of disseminated cancer cells. In these days several

chemotherapeutic agents are being used in clinics for the cancer therapy [6-9].


2

Targeted therapy: Drugs or substances that block growth and metastasis of

cancer by interfering selectively with specific target molecules [10-15].

1.3 Chemotherapy

Chemotherapy involves the administration of a therapeutic agent against any disease,

while in case of cancer it involves the utilization of single or combination of drugs in

order to mere down the effect of this horrendous disease. These chemotherapeutic

agents interfere with the DNA replication processes through interaction with protein

such as topoisomerase I and II which can act as an adept component in replication

process. These agents create covalent binding with DNA and also interact with

nitrogenous bases of nucleic acid through intercalation. Furhtermore, these

chemotharputic agents may interact with enzymes, protein and sulfur containing

amino acids, so all these sites act as target sites for chemotherapeutic agents.

Therefore, interaction of metal based anticancer agents and proteins, antioxidants and

cellular components are important to understand the bioactivity both in vitro and in

vivo. These interactions may cause drug inactivation (related to resistance) or

activation (in case of prodrugs) and also responsible for drug delivery. The interaction

of metallodrugs with DNA has been extensively investigated but interaction with

protein is not well characterized. The combination of advanced mass spectrometric

methods with NMR and single crystal X-ray diffraction boosted the research to map

the interactions of metallodrugs with the proteome and identify key protein as

target [16]. In case of ruthenium based chemotherapeutic agents, it selectively bound

to N of the imdazole ring of histidine residue (His 15) in eggwhite lysozome (a single

chain protein). This selective binding of the Ru-fragment places the complex in

asymmetric environment which may become the bases of ruthenium-catalyzed

enantioselective synthesis [17].

Fig. 1.1: X-ray crystal structure ruthenium(II)-enzyme complex [17].


3

The interaction of metallodrugs with sulphur-containing amino acids L-methionine

and L-cysteine play a major role in chemistry of both Pt(II), Pt(IV) and Ru(III)

anticancer agents and also making platform for the interactions with Ru(II)-arene as

well [18]. Therefore, amino acid and protein binding results might account for the

low toxic side effects of this class of anticancer agents [19]. On the other hand, the

relatively weak binding to amino acids and proteins could perhaps aid in transport and

delivery of active species to cancer cells prior to binding to DNA or RNA [20].

1.4 Classes of chemotherapeutics

Several classes of compounds have been used in clinics for treatment of cancer. Some

prominent chemotherapeutic agents include:

1.4.1 Antimetabolites

These are structurally related to nitrogenous bases of nucleic acids such as purines

and pyrimidines and disturb the normal cell cycle.

Purine analogues e.g., Tioguanine, Fludarabine

Pyrimidine analogues e.g., 5-fluorouracil, floxuridine, gemcitabine

1.4.2 Alkylating agents

Several alkylating agents are employed as chemotherapeutic agents. Some common

examples are:

1. Triazines e.g., Dacarbazine

2. Alkyl sulfonates e.g., Busulfan

3. Nitrogen mustards e.g., Cyclophosphamide; Chlorambucil

4. Nitrosoureas e.g., lomustine, carmustine, streptozocine

5. Platinum (II) or (IV) complexes e.g., Cisplatin, carboplatin, oxaliplatin

1.4.3 Topoisomerase inhibitors

These inhibitors hinder the process of DNA replication as well as transcription. Other

approaches that can be employed for cancer treatment include hormone inhibitors,

signal transduction and enzyme inhibitors, mitosis inhibitors, antibodies and cytotoxic

antibiotics [21].


4

1.5 Metal based anticancer agents

Metallopharmaceuticlas have been used as therapeutic and diagnostic agents since

ages and these are used for the treatment of different diseases like arthritis,

inflammation, diabetes and cancer [22-24]. Therefore, the development of metal

based drugs has brought a revolution in medicinal chemistry. Now a days, there is a

growing interest in metal-based drugs design as anticancer agents. The activity can be

further enhanced by using bioactive ligands possibly due to synergistic and/or

additive effect. These new bioactive compounds may result in different mode of

action and can overcome the problems associated with previously known metal based

drugs [25].

1.6 Platinum based anticancer agents

A remarkable work on design and development of platinum based compounds has

been established after the discovery of cisplatin. This worldwide best-selling anti-

cancer drug is still in clinical use for about 50-70% cancer patients, besides its toxic

side effects and drug resistance against some specific tumors [26]. It is currently

successfully used for the treatment of a range of solid tumors such as colorectal, non-

small cell lungs and genitourinary ovarian, bladder, carcinoma tumors as well as

bronchogenic tumors of head or neck and gastric cancer. But numerous side effects

associated with the administration of this chemotherapeutically active drug such as

vomiting, nausea, neurotoxicity, nephrotoxicity and ototoxicity as well as drug

resistance have mere down its importance in treatment of cancer [27,28]. Most of the

changes have been based upon the modification of the leaving group and coordination

behaviour of the ligands attached to platinum metal centre. Later on, other cisplatin

analogues with similar structural features and mode of action got international

approval. The strategies for the synthesis of cisplatin analogues include the selection

of some interesting bioactive ligands merged with Pt(II) metal ion and got

international approval for the treatment of different cancers [29].

Pt

Cl

H3N

H3N

Cl

PtPt

H3N

H3N

O

O

O

O

O

OH2N

NH2

O

O

Cisplatin Carboplatin Oxaliplatin Nedaplatin

Pt

O

OH3N

H3N

O

Fig. 1.2: Platinum based drugs used in clinics


5

1.7 New trends in platinum(II) based drugs

Initial studies suggest that cisplatin is the active while its trans-isomers is inactive

against cancer. Also the presence of ethylene diamine ligand and leaving group such

as halido or oxalato groups was attributated for anticancer activity of cisplatin.

However, later it was shown that some trans isomers of Pt(II) are as active as

cisplatin. In addition, several new innovative approaches have been employed to

synthesize new platinum (II) complexes having outstanding chemotherapeutical

potential and some examples are:

I. Synthesis of platinum based prodrugs for their specific targeted activity such as

thioplatin and cis-dichlorobis(2-hydroxyethylamine)platinum(II) [9,28,30,31].

Fig. 1.3: A prodrug active only in acidic environment

II. Synthesis of new platinum based polymer drugs such as AP5280 and AP5286 by

coupling drug with hydroxylpropyl mathacrylamide (HPMA) copolymer for their

better accumulation into tumor cells [32].

OPt

O

NH2

H2N

O

O

OPt

O

NH3

NH3

O

O

R =

H2C

H2C

CH3

HN

O

HC OH

CH3

CH3

HN

O

Gly

Phe

Leu

Gly

NH

R

95 5

Fig. 1.4: Platinum drug-HPMA conjugates

III. Synthesis of some highly cytotoxic trans complexes [28]

N

Pt

H3N Cl

Cl

N

Pt

N

Cl

Cl

SN

Pt

N

S

Cl

Cl

Fig. 1.5: Structure of trans-[PtCl2L1L2] complexes


6

1.8 Platinum(IV) complexes in clinical studies

Furthermore, efforts have been made to develop platinum-based anticancer agents

including prodrug Pt(IV) complexes with least side effects and higher

efficacy [24,31,33]. The use of platinum(IV) pro-drugs shows several advantages over

platinum(II) analougs i.e., they are stable during oral administration, their stability

reduce the side effects and their structural modifications via axial ligands can be used

to improve pharmacological profile [34]. Furthermore, different platinum (IV)

complexes e.g., tetraplatin (also known as ormaplatin) [35], iproplatin, satraplatin and

LA-12 have been entered in clinical trials [36]. Tetraplatin, was abandoned due to

severe and unpredictable cumulative neurotoxicity whereas the activity of iproplatin

was not better than those of cisplatin or carboplatin [37,38]. While, satraplatin and

LA-12, are currently undergoing clinical trials. Satraplatin was investigated for

treatment of hormone refractory prostate cancer in combination with prednisone [39].

Furthermore, these compounds can be activated from Pt(IV) to Pt(II) by using

glutathione and other reducing agents [40,41] while reduction process may also be

achieved by irradiation with the potential for development of photoactivated

drugs [42]. Tumor-specific cellular recognition motifs may also used to target cancer

cells without effecting healthy tissue and activated only upon delivery to the tumor

only [43].

H2N

NH2

Pt

Cl

Cl

Cl

Cl

H2N

H2N

Pt

OH

OH

Cl

Cl

H3N

NH2

Pt

O

O

Cl

Cl

O

O

H3N

NH2

Pt

O

O

Cl

Cl

O

O

Tetraplatin Satraplatin Iproplatin LA-12

Fig. 1.6: Platinum (IV) complexes in clinical trials

1.9 Platinum(IV) complexes with biological relevant ligands

With the passage of time, more sophisticated pro-drugs were synthesized by

introducting bioactive ligands or targeting agents in the detachable axial positions

because it can be relatively easily modified. These positions provide opportunities to

increase the selectivity against cancerous tissues or even intracellular targeting.

Furthermore, lipophilicity profile effects the antiproliferative potency and cellular


7

accumulation of the complexes but reduction potentials of Pt(IV) to Pt(II) have not

effect the antiproliferative properties. Keppler and Galanski varied the lipophilicity of

the axial ligands by preparing esters of succinic anhydride. They prepared

bis(carboxylato) dichlorido(ethane-1,2-diamine)platinum(IV) compounds, some of

which displayed impressive IC50 values in the low nM range [44,45].

Pt

O

O

ClH3N

ClH3N

O

O

Pt

O

O

ClHN

ClNH

O O

O O

Pt

O

O

ClH3N

ClH3N

O

O

Cl Cl

O

O

Cl Cl

Fig. 1.7: Platinum(IV) complexes of bioactive ligands

1.10 Non-Platinum Anticancer Agents

After the success of platinum based anticancer agents, the chemotherapeutic field has

flourished gradually but these agents can be employed only in treatment of a limited

number of cancers. Furthermore their use causes significant side effects and acquired

resistance [46]. These problems prompted chemists to develop alternative strategies to

explore new bioactive ligands and their transition metal complexes with promising

pharmacological properties [47]. Therefore, considerable efforts have been made to

develop non-platinum anticancer agents which can impact hugely on future cancer

chemotherapy. The variation in metal can affect the chemical behaviour, mode of

action, cellular distribution as well as cellular uptake, ligand exchange kinetics,

activity spectrum and toxicological profile of metallodrugs. Several other metals were

employed to explore new chemotherapeutic agents. Budotitane and titinocene was the

first titanium based non platinum compound whose anticancer activity was

evaluated [48-52]. The anticancer activity of some gallium salts was also tested such

as gallium nitrate as well as gallium chloride and they were found to be efficient

against bladder cancer but because of lower intestinal absorption and toxicity, their

use was limited [53]. It was observed that formation of insoluble hydrolytic products

leads to lower drug uptake by tumor cells. In order to improve the bioavailability and


8

stability of gallium based drugs, some lipophilic ligands should be introduced.

[Tris(8-quinolinolato)gallium(III)] commonly known as KP46 and gallium maltolate

complexes are some of the representatives of gallium based antitumor drugs having

reasonable bioavaibilty and promising antitumor potential [54]. Another innovative

approach was to carry out the coordination of gallium metal with those ligands that

were already exhibiting anticancer potential such as the coordination of gallium metal

centre with some carbazones [55].

O

O

O

Co2(CO)6Ti

Cl

ClN

ON

O

N

O

Ga

Titanocene KP46 Co-AAS Ferrocifen derivatives

H3CH2C

OH

O(CH2)nNMe2Fe

n = 2, 3, 5, 8

Fig. 1.8: Non-platinum anticancer active metal complexes.

The biological properties of alkyne hexacarbonyldicobalt (Co2(CO)6) species [56] and

therir cytotoxic properties were initially investigated in 1987 [57] and then studied in

more detail again since 1997 [58]. The subsequent structure-activity studies and

biochemical properties of a acetylsalicylic acid cobalt complex Co-ASS were

investigated and it become a leading compound for this class of drugs [59]. Clinical

studies on aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) have

indicated a correlation between the long-term intake of NSAIDs and positive effects

for cancer patients thereby making NSAIDs interesting candidates for

chemoprevention and combination chemotherapy [60]. Furthermore, the NSAID-like

properties of Co-ASS and other related complexes was studied and found that this

lead compound have potential to inhibit the NASID main target COX-1 and COX-

2 [59]. Co-ASS strongly inhibits the NSAID main target enzymes COX-1 and COX-

2 [59] and on the basis of its good stability, it could be concluded that the active

species was indeed the intact organometallic complex [61].

In search of new anticancer agents, multiple coordination ligands have been used to

prepare iron based complexes with different heterocyclic bonding sites. Different

thiosemicarbazone based iron complexes have been found active against different cell

lines [62]. Testing of anticancer potential of iron complexes was also carried out and

it was observed that [Fe(C5H5)2]X type complexes show promising in vivo anticancer


9

activity [63]. Several ferrocene derivatives of some highly efficient chemotherapeutic

agents were also prepared such as ferrocifen derivatives [63].

1.11 Ruthenium based anticancer agents

Ruthenium compounds are the promising anticancer agents particularly against

metastasized tumors and have less toxicity than cisplatin. Ruthenium based

complexes are famous due to their stability and diversity towards different ligand

systems. These aspects make ruthenium based complexes readily available for

different practical applications [64,65]. Two drugs namely imidazolium trans-

[tetrachlorido(dimethylsulfoxide)(1H-imidazole)ruthenate(III)] (NAMI-A),

indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) and sodium

trans-[tetrachloridobis(1H-indazole)ruthenate(III)](KP1339) have been tested under

clinical trials. These compounds are structurally similar to each other but their

cytotoxicity profile is different. KP1019/KP1339 is active against colon carcinomas

while NAMI-A is effective against metastasized cancers. These compounds have

shown promising results but induced damage to the DNA in a different manner than

that of platinum containing compounds [66,67].

HN

N HN

HNRu

Cl

Cl

Cl

Cl

S

O CH3

CH3

+ RuCl

Cl

Cl

Cl

NNH

NHN

HN

HN+

_

_

NAMI-A KP1019

Fig. 1.9: Ruthenium based anticancer complexes in clinical trials.

1.12 Activation by reduction hypothesis

The mode of action of ruthenium(III) complexes is not fully clarified but Ru(III)

complexes are thought to serve as prodrugs. There are mainly two important

procedures for the activation of these compounds i.e., activation-by-reduction and

aquation. The electrochemical potential inside a solid tumor become the bases of

activation-by-reduction hypothesis which is lower than in the surrounding of normal

tissues because of hypoxic environment of the tumor which facilitate the reduction of


10

Ru(III) to Ru(II) [68-70]. Glutathione in the cell and ascorbic acid in the blood may

act as reducing agents for ruthenium complexes. Detoxification from drugs and

xenobiotic is initiated by tripeptide glutathione in the cells [71] which is ubiquitous

molecule, produced by all organs especially in the liver [72]. In most of aerobic

organisms, ascorbic acid (AsH) is an important antioxidant and cell protector and it

plays a central role in the processes like corticosteroid synthesis, tyrosine oxidation

and aromatic hydroxylation [73].

1.13 Ru(II)-arene complexes

On the other hand half sandwich organoruthenium complexes open the new horizons

for drug design. The structure activity relationship demonstrated that the binding of

arene ligand to Ru(II) increases the lipophilic character of complex and facilitate their

uptake inside the cells. It is found that Ru(II)-arene complexes usually exhibit half

sandwich structures and piano stool configuration where the arene ligands form the

seat while other ligands form the legs of this piano stool configuration [74].

arene

Ru

XY

Z

n

n = 0, +1, +2

Fig. 1.10: Piano stool configuration of Ru(II)-arene complexes.

It is observed that mostly Ru(II) arene complexes are activated by hydrolysis before

reaching their target sites [75,76]. A variety of different sized arenes were introduced

for π bonding to metal centre to enhance stabilization.

benzene p-cymene indan tetralin dibenzosuberane fluorene

5,6-dihydrophenathrene biphenyl dihydroanthracene tetrahydroanthracenecyclophane

Fig. 1.11: Structures of different arene ligands used for bonding to Ru(II).


11

Furthermore, the size of arene ligand greatly enhances the cytotoxic effect of some of

the ruthenium complexes. The cytotoxic activity of [(η6-arene)Ru(II)Cl(en)]

complexes increases in the following order: benzene < p-cymene < biphenyl <

dihydroanthracene < tetrahydroanthracene (IC50 = 17 < 10 < 5 < 2 < 0.5 μM [77].

Some O,O- and N,N- containing half-sandwich Ru(II) arene complexes which target

guanine N7 in DNA and display promising in vivo and in vitro anticancer

activity [42].

NN

HN

HO

OO

Ru

OC

Ru

H2N

NH2Cl

RuCl

O

O

DW1 [arene)Ru(II)Cl(acac)] [arene)Ru(II)Cl(en)]

+

Fig. 1.12: Ru(II) arene complexes as anticancer agents

The RAPTA family, [Ru(arene)PTACl2], PTA = 1,3,5-triaza-7-phosphaadamantane

and DW-1 are other the most important examples of these type of complexes [67,78].

DW-1 is the structural mimic of the natural product staurosporine, a potential

inhibitor of protein kinase and this compound presents a metal chirality. The other

enantiomer DW-2 has displayed a better selectivity for one particular kinase Pim-1

and its efficacy is 100 times higher than staurosporine [79].

In early reports on potentially interesting biological activity of RAPTA-C led to

development of RAPTA class of compounds of general formula [(η6-

arene)Ru(II)Cl2(PTA)] complexes (Figure 1.13). [80-82]

RuCl

Cl

P N

NN

RuCl

Cl

P N

NN

RuCl

Cl

P N

NN

RuCl

Cl

P N

NN

NNH3C

CH3

RAPTA-C RAPTA-B RAPTA-T RAPTA-Im

BF4

Fig. 1.13: Structures of some RAPTA Complexes

Intially, in vitro anticancer activity of RAPTA compounds was tested against TS/A

adenocarcinoma and HBL-100 epithelial (non-cancerous) cell lines by using MTT

assy. These compounds were found inactive aginst non-tumorigenic HBL-100 cell


12

line but only mild or no cytotoxicity towards TS/A cell line (66>300 μM) [83]. Two

compounds, RAPTA-T and RAPTA-Im have shown good cytotoxic selectivity

towards the TS/A cell line over the HBL-100 cell line. An in vivo anticancer activity

of RAPTA-B and RAPTA-C was performed in mice bearing the MCa mammary

carcinoma [83] but none of both were found active against the primary tumors.

Furthermore, both were effective in reducing the number and weight of solid lung

metastases that originate from the primary tumor. These promising in vivo results

established RAPTA compounds as potential antimetastatic agents [83]. The advantage

of these complexes was specific inhibition of the growth of purine adenocarcinoma

cells TS/A without damaging non-tumorgenic human mammary cells HBL-100 but it

was determined that protonation of phosphamine ligand leads to loss of selectivity but

higher cytotoxicity. The higher cytotoxicity can be explained by less liphophilic

character exhibited by positively charged species which leads to better intracellular

entrapment [15].

When RAPTA-C was incubated with supercoiled pBR322 DNA then pH-dependent

DNA damage was observed i.e., it damage DNA at pH 7.0 and below but not in

physiological pH, indicating the potential of RAPTA-C to damage DNA selectively in

diseased hypoxic cells [82]. The DNA-damaging ability of RAPTA complex did not

correlate with the observed antimicrobial activity, indicating a non-DNA-based

mechanism of cytotoxicity in vitro. Furthermore, the interaction of RAPTA-C with

intracellular proteins was observed by laser ablation inductively coupled mass

spectrometry (LA-ICP-MS) suggesting that protein-binding may be a major factor in

the activity of these compounds [82].

1.14 Dinuclear Ru(II)-arene complexes

The concept of ruthenium arene based dinuclear complexes was recently flourished

because of their promising biological potential and unique anticancer properties.

Hence, different linker chains including alkyl chains, ethylenediamine, 1,2-

diphenylethylenediamine, pyridine derive linkers etc have been developed to attach

two Ru(II)-arene units in a single moiety. These dinuclear ruthenium arene complexes

contain both monodentate (through nitrogen) or bidentate type of coordination having

O,O-, N,O- and N,N- donar ligands [84-86].


13

N (CH2)nO

O

CH3

Ru

Cl

N O

O

H3C

Ru

Cl

Ru Cl

ClPN

NN

RuCl

Cl PN

N N

NNH

Ru Cl

NHN

RuCl

N

HN N N

Ru

RuCl

ClCl

O

+

O

HN

O

HN

Linker

2+

Fig. 1.14: Examples of dinuclear Ru(II)-arene complexs

Alkyl chains containing dinuclear Ru(II)-arene complexes are involved to cause

inhibition of RNA synthesis as well as unwinding of DNA [87] and it was found that

antiproliferative activity was increased due to the presence of different coordinating

ligands [86]. In case of RAPTA type of dinuclear complexes the relative

conformation of the ruthenium is controlled by stereochemical configuration of

diamine linker moieties. It was also found that significant cytoxicity increased when

isomers are with a closed conformation against different cell lines [84].

Later on dinuclear Ru(II)-arene complexes were synthesised in which numerous

disulfoxide linkers such as BESE (1,2-bis(ethylsulfinyl)ethane) and BESP (1,3-

bis(ethylsulfinyl)propane) were employed to join two unit together in a single

molecule. These dinuclear complexes have exhibited promising chemotherapeutic

activity against mammary cancer cell lines in vivo [88].

1.15 Osmium based anticancer agents

In last two decades, extensive research has been devoted to iron and ruthenium

complexes where studies on bioactive osmium complexes are inadequate. Osmium

behavior is distinct from ruthenium because of several features including slower

ligand exchange kinetics, preference for higher oxidation states, stronger spin-orbit

coupling and π-back-donation from lower oxidation states. Therefore, osmium

complexes are relatively inert, stable in physiological conditions and may become

interesting alternatives of ruthenium based complexes. Different synthetic approaches

have been adopted to design structural analogs of ruthenium based anticancer


14

compounds. Therefore, a range of osmium complexes including, mono and

multinuclear compounds have been isolated with interesting chemical and biological

properties [89-92]. Furthermore, osmium based complexes has been investigated for

their anticancer properties and reasonable results have been found which form the

basis of recent development in Os(II) organometallic chemistry [93-98].

1.16 Osmium based organometallic complexes

Ruthenium as well as osmium have comparable sizes but both exhibit quite different

chemistries. Therefore, they show different hydrolysis rates, solubility, lipophilicity

and ligand exchange kinetics, etc [99-102]. All above mentioned factors also depend

upon the ligands attached to the metal centre. Introduction of arene ligands increases

the stability of these organometallic complexes and prevent their oxidation to Os(III).

Several investigations were carried out to determine the effect of ligands and these

investigations helped scientists to develop highly efficient chemotherapeutic

agents [77,103-105].

NN

HN

HO

OO

Os

OC

Os

H2N

NH2ClN

NH

SFOs

Cl

N N

NOs

Cl

PF6

Cl NPF6

OsCl

Cl

P N

NN

OOs

Cl

N

OOs

Cl

ON

Fig. 1.15: Examples of osmium-arene anticancer complexes

Some osmium analogues of RAPTA complexes were synthesized and their anticancer

activity determined against several cell lines like A549 lung carcinoma, HT29 colon

carcinoma and T47D breast cancer cell lines. It was revealed that osmium complexes

exhibit almost similar activities against colon carcinoma compared to RAPTA

complexes [106,107].


15

The synthesis of a new [(η6-biphenyl)Os(II)(en)Cl]PF6 complex was also reported by

Sadler research group. The antiproliferative profile of the complex indicate low

micromolar range IC50 of the complex against human lung A549 and ovarian A2780

cancer cells [101]. Furthermore, the replacement of neutral N,N- coordinating ligand

with O,O- anionic ligand produce low cytotoxicity and faster hydrolysis rate. The low

activi

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