Nitric Oxide Donors: For Pharmaceutical and Biological Applications

Nitric Oxide Donors Edited by Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi

Further Titles of Interest

O. Kayser, R. H. Müller (Eds.)

C.-H. Wong (Ed.)

Pharmaceutical Biotechnology

Carbohydrate-based Drug Discovery

Drug Discovery & Clinical Applications

2 Volumes

2004 ISBN: 3-527-30554-8

J.-C. Sanchez, G. L. Corthals, D. F. Hochstrasser (Eds.)

Biomedical Application of Proteomics 2004 ISBN: 3-527-30807-5

2003 ISBN: 3-527-30632-3

H. Buschmann, T. Christoph, E. Friderichs, C. Maul, B. Sundermann (Eds.)

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G. Müller, S. Petry (Eds.)

Lipases and Phospholipases in Drug Development From Biochemistry to Pharmacology 2004 ISBN: 3-527-30677-3

Nitric Oxide Donors For Pharmaceutical and Biological Applications

Edited by Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi

Editors Professor Dr. Peng George Wang 876 Biological Sciences Building 484 W. 12th Avenue The Ohio State University Columbus, OH 43210, USA Dr. Tingwei Bill Cai 876 Biological Sciences Building 484 W. 12th Avenue The Ohio State University Columbus, OH 43210, USA Professor Dr. Naoyuki Taniguchi Department of Biochemistry Graduate School of Medicine Osaka University Medical School 2-2 Yamadoka, Suita Osaka 565-0871, Japan

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library. Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the Internet at http://dnb.ddb.de c 2005 WILEY-VCH Verlag GmbH & Co KGaA,
Weinheim Printed on acid-free and chlorine-free paper All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form – by photoprinting, microfilm, or any other means – nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition Steingraeber Satztechnik GmbH, Dossenheim Printing Strauss GmbH, Mörlenbach Bookbinding Litges & Dopf Buchbinderei GmbH, Heppenheim Printed in the Federal Republic of Germany ISBN-13 978-3-527-31015-9 ISBN-10 3-527-31015-0

V

Contents

Part 1 Chemistry of NO Donors 1

1

1.1 1.1.1 1.1.2 1.2 1.3 1.3.1 1.3.2 1.4 1.4.1 1.4.2 1.5 1.5.1 1.5.1.1 1.5.1.2 1.5.1.3 1.5.1.4 1.5.2 1.5.2.1 1.5.2.2 1.5.3 1.5.4 1.5.5 1.5.6 1.6

NO and NO Donors 3 Tingwei Bill Cai, Peng George Wang, and Alvin A. Holder Introduction to NO Biosynthesis and NO donors 3 Nitric Oxide Synthases 3 Chemistry of Reactive Nitrogen Species 6 Classification of NO Donors 7 New Classes of NO Donors under Development 9 Nitroarene 9 Hydroxamic Acids 10 Development of NO-Drug Hybrid Molecules 10 Nitrate Hybrid Molecules 11 Furoxan Hybrid Molecules 13 New Therapeutic Applications of NO Donors 14 NO Donors against Cancer 15

Diazeniumdiolates (NONOates) as Promising Anticancer Drugs 15 The Synergistic Effect of NO and Anticancer Drugs 17 NO-NSAIDs as a New Generation of Anti-tumoral Agents 17 Other NO Donors with Anticancer Activity 18 NO against Virus 19 HIV-1 Induces NO Production 19 Antiviral and Proviral Activity of NO 21 Inhibition of Bone Resorption 22 Treatment of Diabetes 23 Thromboresistant Polymeric Films 23 Inhibition of Cysteine Proteases 24 Conclusion 24 References 26

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

VI

Contents

2

2.1 2.1.1 2.1.2 2.1.3 2.1.4 2.1.5 2.1.6 2.1.7 2.2 2.3

3

3.1 3.2 3.2.1 3.2.2 3.2.3 3.2.4 3.3 3.3.1 3.3.2 3.3.3 3.3.4 3.4 3.4.1 3.4.2 3.4.3 3.5 3.5.1 3.5.2 3.5.2.1 3.5.2.2 3.5.3 3.5.4 3.5.4.1 3.5.4.2 3.5.4.3 3.5.5

Organic Nitrates and Nitrites 33 Roger Harrison Organic Nitrates 34

Direct Chemical Reaction between Organic Nitrates and Thiols 35 Glutathione-S-transferase 36 Cytochrome P-450-dependent Systems 37 Membrane-bound Enzyme of Vascular Smooth Muscle Cells 38 Xanthine Oxidoreductase 38 Mitochondrial Aldehyde Dehydrogenase 40 Tolerance 42 Organic Nitrites 44 Conclusions 45 References 47

N-Nitroso Compounds 55 Arindam Talukdar, Peng George Wang Introduction 55 N-Nitrosamines 56 Synthesis of Nitrosamines 58

Physical Properties and Reactions of N-Nitrosamines 59 Structure–Activity Relationship of N-Nitrosamines 61 Application of N-Nitrosamines 62 N-Hydroxy-N-nitrosoamines 63 Biologically Active N-Hydroxy-N-nitrosamine Compounds 64 Synthesis of N-Hydroxy-N-nitrosamines 66 Properties of N-Hydroxy-N-nitrosamines 68 Reactivity of N-Hydroxo-N-nitrosamines 70 N-Nitrosimines 72 Mechanism of Thermal Reaction of N-Nitrosoimine 73 Properties of N-Nitrosoimines 74 Synthesis of N-Nitrosoimines 75 N-Diazeniumdiolates 75 Mechanism of NO Release 76 Synthesis of N-Diazeniumdiolates 77 Ionic Diazeniumdiolates 79 O-derivatized Diazeniumdiolates 79 Reactions of N-Diazeniumdiolates 79 Clinical Applications 80 Reversal of Cerebral Vasospasm 80 Treatment of Impotency 81 Nonthrombogenic Blood-contact Surfaces 81 Future Directions 81 References 83

Contents

4

4.1 4.1.1 4.1.1.1 4.1.2 4.1.2.1 4.1.2.2 4.1.2.3 4.1.2.4 4.1.2.5 4.2 4.2.1 4.2.2 4.2.3 4.3

5

5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.2 5.3 5.4

6

6.1 6.1.1 6.1.1.1 6.1.1.2 6.1.1.2.1 6.1.1.2.2 6.1.1.2.3 6.1.1.2.4 6.1.1.3 6.1.1.4

The Role of S-Nitrosothiols in the Biological Milieu 91 Bulent Mutus Structure and Cellular Reactivity of RSNOs 91 RSNO Structure 91 Enzymatic Consumption of RSNOs 92 Formation of RSNOs in the Biological Milieu 93 Nitrite Mediated 93 NO Mediated 93 NO Oxidation Products Mediated 93 Metalloprotein Mediated 95 Transnitrosation 98 Postulated Physiological roles of RSNOs 99 Regulation of Blood Flow by HbSNO 99

Regulation of Ventilatory Response in the Brain by RSNOs 100 Role of RSNOs in Platelet Function 100 Conclusion 102 References 103

Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms 109 Tara P. Dasgupta, Danielle V. Aquart Iron Complexes 110 Nitroprusside 110 Iron Porphyrin Nitrosyls 114 Dinitrosyl Complexes (DNICs) 116 Iron–Sulfur Cluster Nitrosyls 117 Ruthenium Complexes 118 Other Metal Nitrosyls 121 Conclusion 122 References 123

The NO-releasing Heterocycles 131 Alberto Gasco, Karl Schoenafinger Heterocyclic N-oxides 131 Furoxans 131 General Properties 132 Synthesis 134 Dimerisation of nitrile oxides 134

Dehydrogenation of á-dioximes (glyoximes) 135 Action of nitrogen oxides on olefins 136 Other methods 137 NO-release 137 Biological Actions 140

VII

VIII

Contents

6.1.1.4.1 6.1.1.4.2 6.1.1.4.3 6.1.1.5 6.1.2 6.1.2.1 6.1.2.2 6.1.2.3 6.1.2.4 6.1.3 6.1.3.1 6.1.3.2 6.1.3.3 6.2 6.2.1 6.2.1.1 6.2.1.2 6.2.1.3 6.2.1.4 6.2.2 6.2.2.1 6.2.2.2 6.2.2.3 6.3

7

7.1 7.2 7.2.1 7.2.1.1 7.2.1.2 7.2.2 7.2.2.1 7.2.2.2 7.2.2.3 7.3 7.3.1 7.3.2 7.3.3 7.4

Condensed furoxans 140 Furoxan sulfones and carbonitriles 141 Furoxancarboxamides 144 NO-donor Hybrid Furoxans 145 3,4-Dihydro-1,2-diazete 1,2-dioxides (1,2-diazetine 1,2-dioxides) 147 Generalities 147 Synthesis 148 NO-release 149 Biological Properties 150 Other Heterocyclic N-oxides 151 4H-pyrazol-4-one 1,2-dioxides (pyrazolone N,N-dioxides) 152 2H-1,2,3-triazole 1-oxides 153 1,2,3,4-Benzotetrazine 1,3-dioxides and 1,2,3-Benzotriazine 3-oxides 153 Mesoionic Heterocycles 154 Sydnonimines 155 General Properties 155 Synthesis 156 NO-release 157 Biological Properties 161 Mesoionic Oxatriazoles 163 Synthesis 164 NO-release 165 Biological Properties 167 Other Heterocyclic Systems 168 References 170

C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas 177 S. Bruce King Introduction 177 C-Nitroso Compounds 177 Alkyl and Aryl C-Nitroso Compounds 177 Syntheses and Properties 177 NO-releasing Mechanisms 178 Acyl C-Nitroso Compounds 179 Syntheses and Properties 179 NO-releasing Mechanisms 180 Structure–Activity Relationships 181 Oximes 182 Syntheses and Properties 182 NO-releasing Mechanisms 184 Structure–Activity Relationships 185 N-Hydroxyguanidines 186

Contents

7.4.1 7.4.2 7.4.3 7.5 7.5.1 7.5.2 7.5.3

Syntheses and Properties 186 NO-releasing Mechanisms 187 Structure–Activity Relationships 188 N-Hydroxyureas 189 Syntheses and Properties 189 NO-releasing Mechanisms 191 Structure–Activity Relationships 193 References 195

Part 2 NO Donors’ Applications in Biological Research 201

8

Vasodilators for Biological Research 203 Anthony Robert Butler, Russell James Pearson

8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 8.10 8.11

NO-donor Drugs for Biological Research 203 Sodium Nitrite (NaNO2 ) 203 S-Nitrosothiols 204 Metallic Nitrosyls 209 Sodium Nitroprusside (Na2 [Fe(CN)5 NO] ⋅ 2H2 O) 209 Organic Nitrates 212 Organic Nitrites 215 NONOates 216 NO Inhalation; NO Gas as an NO Donor 219 Sydnonimines 222 Conclusion 225 References 226

9

NO Donors as Antiplatelet Agents 233 Anna Kobsar, Martin Eigenthaler Introduction 233

9.1 9.2 9.2.1 9.2.1.1 9.2.1.2 9.2.1.3 9.2.1.3.1 9.2.1.3.2 9.2.1.3.3 9.2.1.3.4 9.2.1.3.5 9.2.1.3.6 9.2.1.3.7 9.2.2

Molecular Mechanisms of NO-mediated Platelet Inhibition 233 cGMP-dependent NO Signaling Mechanisms 234 Regulation of cGMP Levels 235 Effector Sites of cGMP 236 cGMP-PK I Substrates in Platelets 237 Inositol triphosphate (IP3 ) receptor 237 Rap 1b 238 Vasodilator stimulated phosphoprotein (VASP) 238 Heat shock protein hsp27 239 LASP 239 Thromboxane A2 (TxA2 ) receptor 240 Phosphodiesterase PDE5 240 cGMP-independent NO Signaling Mechanisms 240

IX

X

Contents

9.3 9.3.1 9.3.1.1 9.3.1.2 9.3.1.3

9.3.2 9.3.3 9.3.4 9.3.4.1 9.3.4.2 9.3.4.3 9.3.4.4 9.3.4.5 9.3.4.6 9.3.4.7 9.3.5 9.3.5.1 9.3.6 9.3.6.1 9.3.6.2 9.3.7 9.3.7.1 9.3.7.2 9.3.8 9.4 9.4.1 9.4.2

9.5 9.6 9.7

10

10.1 10.2

Effects of Different Groups of NO Donors on Platelets 241 Diazeniumdiolates 241 DEA/NO (Sodium 2-(N,N-diethylamino)-diazenolate-2-oxide) 241 DETA NONOate ((Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate) 241 MAHMA NONOate ((Z-1-[N-Methyl-N-[6-(N-methylammoniohexyl) amino]]diazen-1ium-1,2-diolate) 242 Sodium Nitroprusside (SNP) 242 Molsidomine (3-Morpholino-sidnonimine; SIN-1) 242 S-Nitrosothiols 243 SNAP (S-Nitroso-N-acetyl-d,1-penicillamine) 243 SNVP (S-Nitroso-N-valerylpenicillamine) 243 GSNO (S-nitroso-glutatione) 243 CysNO (S-Nitrosocysteine) 244 SNAC (S-Nitroso-N-acetyl-cysteine) 244 HomocysNO (S-Nitrosohomocysteine) 244 RIG200 (N-(S-Nitroso-N-acetylpenicillamine)-2-amino-2-deoxy1,3,4,6, tetra-O-acetyl-beta-d-glucopyranose) 244 Organic Nitrates 245 GTN (Glyceryl Trinitrate, Nitroglycerin, NTG) 245 Mesoionic Oxatriazole Derivatives 245 GEA-3162 (1,2,3,4-Oxatriazolium, 5-amino-3-(3,4-dichlorphenyl)-, cloride) 245 GEA-3175 (1,2,3, 4-Oxatriazolium, -3-(3-chloro-2-methylphenyl)-5[[(4-methylphenyl) sulfonyl]amino]-, Hydroxide Inner Salt) 246 Other NO Donors 246 OXINO (Sodium trioxdinitrate or Angel’s Salt) 246 B-NOD (2-Hydroxy-benzoid acid 3-nitrooxymethyl-phenyl ester) 246 l-Arginine (l-Arg) 246 Activators of Soluble Guanylyl Cyclase 247 YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole) 247 BAY 41-2272 (3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)1H-pyrazolo [3,4-b]pyridine) 247 cGMP Analogs 247 Inhaled NO and Platelet Inhibition 248 Conclusion 248 References 249

Control of NO Production 255 Noriko Fujiwara, Keiichiro Suzuki, Naoyuki Taniguchi Introduction 255 Structure of Nitric Oxide Synthase 256

Contents

10.3 10.4 10.4.1 10.4.2 10.5 10.5.1 10.5.1.1 10.5.1.2 10.5.2 10.5.3 10.5.4 10.5.5 10.6 10.7 10.8 10.8.1 10.8.2 10.9

NO Formation 257 l-Arginine and l-Arginine Derivatives 257 Inhibitors 258 Substrates 259 Non-amino Acid Inhibitors and Non-amino Acid Substrates 261 Guanidine 262 Inhibitor 262 Substrates 262 Isothiourea (ITU) 265 Amidine 267 Cyclic Amidines are Potent iNOS Selective Inhibitors 268 Indazole 269 Inhibition of NOS Function Targeted towards Cofactors 270 Regulators of NOS Gene Expression 270 NO Formation by an NOS-independent Pathway 271 Oxime 272 Hydroxyurea 272 Summary 273 References 274

Part 3 Clinical Applications of NO Donors 283

11

11.1 11.2 11.3 11.4 11.5 11.6 11.7

12

12.1 12.2 12.3

Nitric Oxide Donors in Cardiovascular Disease 285 Martin Feelisch, Joseph Loscalzo Introduction 285

Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present 285 Pharmacological Cardiovascular Mechanism of Action of NO Donors 288 Clinically Available NO Donors: Structures and Mechanism of Action 290 Nitrate Tolerance 293 Is Nitrate Therapy Associated with Adverse Vascular Effects? 295 Conclusions 295 References 297

Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders: Clinical Status and Therapeutic Prognosis 299 David R. Janero, David S. Garvey Introduction 299 Human Platelets, Thromboembolic Disorders, and NO 300 Nitrovasodilators 307

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Contents

12.3.1 12.3.2 12.3.3 12.4 12.4.1 12.5 12.5.1 12.6 12.7 12.8

Glyceryl Trinitrate, Nitroglycerin (GTN) 307 Isosorbide Dinitrate (ISDN) and Isosorbide Mononitrate (ISMN) 311 Sodium Nitroprusside (SNP) 312 Oxatriazolium NO Donors 314 Sydnonimines 314 Nitrosothiol NO Donors 316 S-Nitroso-glutathione (GSNO) 316 l-Arginine {S(+)-2-Amino-5-[(aminoiminomethyl)amino]pentanoic acid} (l-arg) 318 NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester] 319 Conclusion and Future Prospects 320 References 323

13

NO and Gene Regulation 329 Jie Zhou, Bernhard Brüne

13.1 13.2 13.2.1 13.2.2 13.3 13.3.1

Formation of NO and RNI-signaling 329 p53 Regulation under the Impact of RNI 331 Basic Considerations: p53 Phosphorylation and Mdm2 Binding 331 Molecular Mechanisms of RNI-evoked p53 Stabilization 332 HIF-1á Regulation under the Impact of RNI 333 Lessons from Hypoxia: Basic Considerations of HIF-1á Stability Regulation 333 Stability Regulation of HIF-1á by NO/RNI in Normoxia versus Hypoxia 335 RNI, p53 and HIF-1 in Tumor Biology 337 Conclusions 339 Abbreviations 341 References 342

13.3.2 13.4 13.5

14

Nitric Oxide and Central Nervous System Diseases 347 Elizabeth Mazzio, Karam F. A. Soliman

14.1

General Overview – Gaining Control over Various NOS Enzymes that Concurrently Contribute to Degenerative CNS Diseases 347 Signaling Controls – Neuronal NOS: TYPE-I 349 Neurotransmission 349 Neuronal Calcium Homeostasis 351 Signaling Controls, Endothelial NOS: Type-3 352 EDRF/Vascular Tone 352 eNOS, Cyclic AMP/GMP Regulation 353 Signaling Controls, Inducible NOS: Type-2 354 Inflammation, Microglia and Astrocytes 354 Stress Activated and Extra-cellular Kinases 355 Cyclic AMP/Protein Kinase a 356

14.2 14.2.1 14.2.2 14.3 14.3.1 14.3.2 14.4 14.4.1 14.4.2 14.4.3

Contents

14.4.4 14.4.5 14.5 14.5.1 14.5.2 14.5.3 14.5.4

Cyclic AMP–Phosphodiesterase Inhibitors 358 Peroxisome Proliferator-activated Receptor-gamma 358 The Neurotoxicity of NO 359 Oxidative Stress 359 Mitochondrial Impairment 361 Permeability Transition Pore Complex, Apoptosis 363 Excitotoxicity, Poly(ADP-ribose)-polymerase-1 365 References 369

Index 385

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XV

Preface The discovery of the physiological and pathophysiological roles of nitric oxide (NO) during the 1980s was one of the most surprising and exciting developments in biological research. NO exhibits a broad range of biological activities. Thus, it comes as no surprise that, as far back as 1992, the editors of the journal Science called NO the molecule of the year, and in 1998, three scientists, R.F. Furchgott, L.J. Ignarro, and F. Murad, were awarded the Nobel Prize in physiology and medicine for their contribution to elucidating the role of nitric oxide in the functions of living organisms. As a simple diatomic free radical, NO is generally considered to represent the biologically important form of the endothelium-derived relaxing factor (EDRF). Cellular NO is almost exclusively generated via the oxidation of L-arginine, which is catalyzed by nitric oxide synthetases (NOS). Under physiological conditions, NO directly activates soluble guanylate cyclase (sGC) to transform guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP), followed by kinase-mediated signal transduction. The endogenous formation of NO plays a key role in many bioregulatory systems, including smooth muscle relaxation, platelet inhibition, neurotransmission, and immune stimulation. Due to the instability and inconvenient handling of aqueous solutions of authentic NO, there is increasing interest in using compounds capable of generating NO in situ. These compounds are called NO donors, or NO releasing agents. Glyceryl trinitrate (GTN) may be the most well known NO donor. Although the use of GTN for medicinal purposes dates back more than 150 years, little had been revealed about its physiological mechanism of action before the 1980s. It is well known that the epoch-making invention realized by Alfred Nobel in 1863 paved the way for controlled detonation of GTN. Therefore, when Nobel’s physician recommended GTN as a treatment of his angina pectoris, Nobel wrote: “Isn’t it the irony of fate that I have been prescribed N/G 1 [nitroglycerine] to be taken internally! They called it Trinitrin, so as not to scare the chemist and the public.” Nobel would not have found it ironic if he had known that it was NO, released from GTN in vivo, that helps relieve angina. In addition to organic nitrates, many other chemicals can be transformed into NO in vitro or in vivo. Due to the diversity of NO donor structures, the pathway for each class of compounds to generate NO could differ significantly, e.g., enzymatical vs. Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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Preface

non-enzymatical, reductive vs. oxidative, etc. As each class of compounds offers distinct biochemical properties, this allows us to choose a compound that best meets the demands of specific investigations. Insufficient NO production causes serious medical problems. Many diseases such as hypertension, atherosclerosis and restenosis involve the deficiency of NO production. Therefore, a compound that can release NO under specific conditions can be used therapeutically to palliate NO underproduction. In fact, the best known NO donor, glyceryl trinitrate, has been used for over a century to relieve acute attacks of angina pectoris. In 1998, Carl Djerassi published a book entitled “NO”, where he plotted the success of a biotech company producing NO donor compounds to treat male impotence. In reality, NO donor compounds have a variety of biomedical applications. Our latest search using the keyword “nitric oxide donor” at ScienceFinder revealed that there are 2,880 published research papers on NO donors. More importantly, there have been 105 US and world patents on the applications of NO donors in the treatment of cardiovascular diseases, central nervous systems diseases, diseases related to immunity, physiological disorders and many other medical situations. Besides supplementation of NO in a situation where a NO insufficiency may underlie the pathology, NO donors can also regulate NO-based physiological pathways, i.e., male erectile dysfunction, and improve drug safety and efficacy, such as gastrointestinal toxicity of non-steroidal anti-inflammatory drugs. Since the mid-1980s, the development of new NO donors has offered several advantages over the previous NO donors, such as spontaneous releasing NO, donating NO under controlled rates, and even targeting NO to certain tissues. The current trends in NO donor development include discovery of new NO donors, finding novel applications of old NO donors, development of NO-drug hybrids and site-specific delivery of NO. Although a number of reviews and books on NO have been published, we felt that there was a need to publish a comprehensive text addressing the basic principles of all aspects of NO donors. This book is not only an informative resource for basic scientists in the NO field, but also for all clinicians and biologists interested in the applications of NO donors. This 14-chapter book is divided into three sections ranging from the basic chemistry of NO donors to clinically applied science. The first seven chapters present a review of medicinal chemistry of all classes of NO donors. The next three chapters continue to discuss the application of NO donors and NO inhibition in biological research. The final four chapters of the book address other important issues on biological functions of NO donors. Integrating internationally recognized authors for each chapter was not an easy job. We really appreciate the help from all these hard-working authors. We are also grateful to the editors at Wiley-VCH – without their continuous support this project would never have been possible. We would like to sincerely thank faculty members, postdoctoral fellows, graduate and undergraduate students who have contributed so much in Wang’s and Taniguchi’s laboratories on nitric oxide research. These people are Libing Yu, Zhengmao Guo, Andrea McGill, Johnny Ramirez, Jun Li, Ming Xian, Adam Janczuk, Yongchun Hou, Vladislav Telyatnikov, Yingxin Zhang, Xuejun Wu, Alvin A. Holder, Qiang Jia, Zhong Wen, Xiaoping Tang, Xinchao Chen, Jaime Martin Franco, Mingchuan Huang, Dongning Lu, Arindam Talukdar, Noriko

Preface

Fujiwara, Satoshi Kazuma, and Yasuhide Miyamoto. P. George Wang acknowledges the continuing funding support (NIH 54074) over the past ten years from the National Institute of Health on the development of nitric oxide donors. Naoyuki Taniguchi was supported by the 21st Center of Excellence Program funded by the Ministry of Education, Culture, Sports, Science and Technology, Japan. December 2004

Peng George Wang Tingwei Bill Cai Naoyuki Taniguchi

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List of Contributors

Danielle V. Aquart Department of Chemistry University of the West Indies Mona, Kingston 6 Jamaica

Tara P. Dasgupta Chemistry Department University of the West Indies Mona Campus, Kingston 7 Jamaica

Bernhard Brüne University of Kaiserslautern Faculty of Biology Department of Cell Biology Erwin-Schroedinger-Straße 13/4 67663 Kaiserslautern Germany

Martin Eigenthaler Universität Würzburg Medizinische Poliklinik Klinikstr. 6–8 97070 Würzburg Germany

Anthony Robert Butler University of St Andrews Bute Medical School The Bute Building St Andrews Fife KY16 9TS Scotland United Kingdom Tingwei Bill Cai The Ohio State University Department of Chemistry 100 W 18th Ave Columbus, OH 43210 U.S.A.

Martin Feelisch Boston University School of Medicine 650 Albany, X304 Boston, MA 02118 U.S.A. Noriko Fujiwara Department of Biochemistry Hyogo College of Medicine 1-1 Mukogawa-cho Nishinomiya Hyogo 663-8501 Japan David S. Garvey NitroMed Inc. 125 Spring St. Lexington, MA 02421 U.S.A.

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

XX

List of Contributors

Alberto Gasco Faculty of Pharmacy University of Turin Via Pietro Giuria 9 10125 Turin Italy Roger Harrison Department of Biology and Biochemistry University of Bath Claverton Down Bath, BA2 7AY United Kingdom Alvin A. Holder Colorado State University Department of Chemistry Fort Collins, CO 80523 U.S.A. David R. Janero NitroMed Inc. 125 Spring St. Lexington, MA 02421 U.S.A. Kiyomi Kikugawa School of Pharmacy Tokyo University of Pharmacy and Life Science 1432-1 Horinouchi Tokyo 192-0392 Japan S. Bruce King Wake Forest University Department of Chemistry Salem Hall Winston-Salem, NC 27109 U.S.A.

Anna Kobsar Universität Würzburg Institut für Klinische Biochemie und Pathobiochemie Josef-Schneider-Str. 2 97080 Würzburg Germany Joseph Loscalzo Boston University School of Medicine 715 Albany Street, E113 (Dept. Medicine) W507 (Whitaker CVI) Boston, MA 02118 U.S.A. Elisabeth Mazzio College of Pharmacy and Pharmaceutical Sciences Florida A & M University Tallahassee FL 32307 U.S.A. Bulent Mutus Chemistry and Biochemistry University of Windsor Essex Hall, 401 Sunset Avenue Windsor, Ontario N9B 3P4 Canada Russell James Pearson University of St Andrews School of Chemistry Purdie Building St Andrews Fife KY16 9ST Scotland United Kingdom Karl Schoenafinger Synthetic Medicinal Chemistry Aventis Pharma Deutschland Industriepark Hoechst 65926 Frankfurt am Main Germany

List of Contributors

Karam F. A. Soliman College of Pharmacy and Pharmaceutical Sciences Florida A & M University Tallahassee FL 32307 U.S.A.

Naoyuki Taniguchi Department of Biochemistry Graduate School of Medicine Osaka University Medical School, 2-2 Yamadoka, Room B-1, Suita Osaka 565-0871 Japan

Keiichiro Suzuki Department of Biochemistry Hyogo College of Medicine 1-1 Mukogawa-cho Nishinomiya Hyogo 663-8501 Japan

Peng George Wang The Ohio State University Faculty Departments of Chemistry and Biochemistry 484 W 12th Avenue, Columbus, OH 43210 U.S.A.

Arindam Talukdar The Ohio State University Department of Chemistry and Biochemistry 484 W 12th Avenue Columbus, OH 43210 U.S.A.

Jie Zhou University of Kaiserslautern Faculty of Biology Department of Cell Biology Erwin Schroedinger Straße 13/4 67663 Kaiserslautern Germany

XXI

Part 1 Chemistry of NO Donors

3

1

NO and NO Donors Tingwei Bill Cai, Peng George Wang, and Alvin A. Holder

Nitric oxide (NO), a magic free radical gas molecule, has been shown to be involved in numerous physiological and pathophysiological processes. Among its diverse functions, NO has been implicated in the relaxation of vascular smooth muscle, the inhibition of platelet aggregation, neurotransmission (Viagra reverses impotence by enhancing an NO-stimulated pathway), and immune regulation [1]. It was named the molecule of the year in 1992 by Science and was the subject of the Nobel Prize in 1998. NO has limited solubility in water (2–3 mM), and it is unstable in the presence of various oxidants. This makes it difficult to introduce as such into biological systems in a controlled or specific fashion. Consequently, the development of chemical agents that release NO is important if we are to target its bioeffector roles to specific cell types for biological and pharmacological applications. Based on our comprehensive review of NO donors [2], this chapter focuses on recent progress and current trends in NO donor development and novel applications which are not covered by the following chapters.

1.1

Introduction to NO Biosynthesis and NO donors 1.1.1

Nitric Oxide Synthases

Endogenous NO is produced almost exclusively by l-arginine catabolism to l-citrulline in a reaction catalyzed by a family of nitric oxide synthases (NOSs) [3]. In the first step, Arg is hydroxylated to an enzyme-bound intermediate Nù -hydroxy-l-arginine (NHA), and 1 mol of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and O2 are consumed. In the second step, NHA is oxidized to citrulline and NO, with consumption of 0.5 mol of NADPH and 1 mol of O2 (Scheme 1.1). Oxygen activation in both steps is carried out by the enzyme-bound heme, which derives electrons from NADPH. Mammalian NOS consists of an N-terminal oxyNitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

4

1 NO and NO Donors

H2N

NH2 NH

H2N

NH

1 NADPH

O

N OH

COO

Arginine

NH

0.5 NADPH

N O

O2 H2O

O2 H2O H3N

NH2

H3N

COO

H2N

N w-Hydroxyarginine

COO

Citrulline

Nitric Oxide

Scheme 1.1 Endogenous synthesis of nitric oxide.

genase domain that binds iron protoporphyrin IX (heme), 6-(R)-tetrahydrobiopterin (H4 B) and Arg, and a C-terminal reductase domain that binds FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide), and NADPH, with a calmodulin binding motif located between the two domains. To be active, two NOS polypeptides must form a homodimer. The reductase domains each transfer NADPH-derived electrons, through FAD and FMN, to the heme located in the adjacent subunit. Three distinct isoforms of NOS have been identified – neuronal, macrophage and endothelial types, and each is associated with a particular physiological process (Table 1.1). Constitutive endothelial NOS (eNOS or NOS III) regulates smooth muscle relaxation and blood pressure; constitutive neuronal NOS (nNOS or NOS I) is involved in neurotransmission and long-term potentiation; the NO produced from inducible NOS (iNOS or NOS II) in activated macrophage cells acts as a cytotoxic agent in normal immune defense against microorganisms and tumor cells. The constitutive isoforms (nNOS and eNOS) require added Ca2+ and calmidulin for activity and produce a relatively small amount of NO, while the inducible isoform (iNOS) has tightly bound Ca2+ and calmodulin, and produces a relatively large amount of NO. Tab. 1.1: Properties of NOS isoforms.

NOS nNOS (NOS-I) iNOS (NOS-II) eNOS (NOS-III)

Locations Brain, spinal cord, peripheral Macrophages, other tissues Endothelium

Characteristics Constitutive, Ca2+ dependent Inducible, Ca2+ independent Constitutive, Ca2+ dependent

Major Biological Functions Neuromediator Host defender, cytotoxic Vasodilator tone modulator

The first step of an NOS catalyzed reaction is a “classical” P450-dependent Nhydroxylation of a guanidine, except for the involvement of H4 B. As shown in Scheme 1.2, Fe(III)heme 1 first accepts one electron to give Fe(II)heme 2, which binds O2 to produce ferrous-dioxy heme 3. The second electron from H4 B reduces 3 to peroxyiron 4. Arg donates a proton to 4 to facilitate O–O bond cleavage to generate an oxo-iron (IV) cation radical species 5, which then rapidly hydroxylates the neutral guanidinium to NHA [4]. The second step of NOS oxidation is a greater challenge to enzymologists since there is no direct analogy in other systems. A variety of proposed reaction steps can be

1.1 Introduction to NO Biosynthesis and NO donors

FMNH2

FMNH .

H4B

O2

O2

FeIII

FeII

S

S

S

1

2

3

NH2 R . + H4B N + N H H H _ O O

FeII

NH2 R

NH2 R

N H N H _ OH O

4

OH

N

N H

FeIII

FeIV +. S 5

FeII S

5

S

1

Scheme 1.2 The first step of NOS reaction.

roughly summarized in three mechanisms (Scheme 1.3). The popular Mechanism I was proposed by Marletta and modified by Ingold and others [5, 6], a superoxoiron(III)heme intermediate 6 abstracts the hydrogen atom of the NHA to furnish an iminoxy radical 8, which upon nucleophilic attack by the hydroperoxoiron(III)heme 7 on its carbon generates NO and citrulline. This mechanism, however, appears not to be supported by the crystal structure analysis of the NOS-NHA complex [7–9] or by a recent spectral study [10]. The second mechanism was proposed by Groves (Mechanism II), where the NOS-catalyzed aerobic oxidation of NHA occurs via a radical-type auto-oxidation process [11, 12], i.e., NHA is oxidized by the Fe(III) heme to generate an iminoxyl radical 8, which tautomerizes to the á-nitroso radical 12. Insertion of a dioxygen molecule between 12 and Fe(II) heme forms an energetic á-nitrosoperoxy Fe(III) heme intermediate that decomposes to generate NO [13, 14]. However, direct ligation of NHA to heme iron has been precluded by the X-ray crystallographic data [7–9]. The third mechanism, proposed by Silverman and others [15–18], mainly inMechanism I NH2 R

N H

NH2 8

NH2

N OH

R

O 2 , e-

N H

R

O

N O

H

O FeIII

H2N R

N O O H

N H

N H

N H

O

O

FeIII

6

R

H

O

O

FeIII

NH2

N O

OH

9

FeIII

7

+ NO

10

FeIII

Mechanism II NH2

NH2 R

N H

R

N OH -H+

N H

N O

FeIII

N H

N H III

Fe

R

OH O2 , e

-

N H O

N O

NH2 OH

R

N H O

Fe

O2

R

H2N

N O

12

6

Fe

Scheme 1.3 The second step of NOS reaction.

NH2

III

R +

-H

16

R

Fe

III

18

+

NO

FeIV 14

13

N N H O

NH2 O-

FeIII

R

N H

O

+ NO

O-

O

O

O O

H2 N

17

-

O

NH2 N H

O

N O

N H

R

N H O FeIII

15 H

N O H O

O III

N O

N H

8

11

N H

NH2

R

FeII

NH2

NH2 R

N H

FeIII

Mechanism III

NH2

R

19

FeIII

20

6

1 NO and NO Donors

volves the oxidation of the nitrogen on the protonated N-hydroxyguanidino moiety (Mechanism III). It was suggested that the initial N–H bond cleavage by superoxoiron(III)heme 6 generates a radical cation intermediate 15, which, upon heterolysis of the O–H bond, gives the iminoxy radical 17. The nucleophilic attack of peroxoiron(III)heme 18 on 17 gives an intermediate similar to 13, which decomposes to NO and citrulline. More recently, Stuehr has emphasized the involvement of H4 B in the second step of the NOS reaction [19–21]. 1.1.2

Chemistry of Reactive Nitrogen Species

One of NO’s major biological actions is to activate guanylate cyclase directly to generate cyclic guanosine monophosphate (cGMP) as an intracellular second messenger, followed by kinase-mediated signal transduction. In another pathway, NO undergoes oxidation or reduction in biological systems to convert to many different reactive nitrogen species (RNS). It can react with molecular oxygen (O2 ), superoxide anion (O2 −• ) or transition metals (M) to produce RNS such as N2 O3 , NO2 , NO2 − , NO3 − , peroxynitrite (OONO− ), and metal-nitrosyl adducts (Route A, Scheme 1.4) [22, 23]. Among these RNS, peroxynitrite stands out as an important species [24, 25]. The reaction between NO and O2 −• produces peroxynitrite at a diffusion controlled rate [26–28]. Peroxynitrite is a strong oxidizing and nitrating species that causes molecular damage leading to disease-causing cellular dysfunction [29, 30]. NO can also be rapidly oxidized by oxygen, superoxide or transition metals to nitrosonium (NO+ ) which reacts with nucleophilic centers such as ROH, RSH and RR′NH to produce RO–NO, RS–NO or RR′N–NO, respectively (Route B, Scheme 1.4) [31, 32]. These products subsequently undergo other reactions to exhibit their biological effects. In addition, NO also undergoes a one-electron reduction to produce nitroxyl (NO− ) (Route C, Scheme 1.4). The reducing potential of this reduction is approximately +0.25 V [33]. Nitroxyl converts rapidly to N2 O under physiological conditions. Other competing reactions RONO ROH

RSNO RSH

NO+

L-Arg

RR'-NO RR'NH oxidation

NH2OH

NOS

RSH

reduction

NO

B H2O NO2-

H2O2 ONOO-

M M-NO

NO-

N2O

C M

A O2-

M-NO

ONOO-

M NO2-

O2

NO2 / N2O3

O2-

NO3( NO2- )

O2

NO NO2-

ONOO-

O2

H2O

RS-NO

NO2- / NO3-

Scheme 1.4 Oxidation and reduction of reactive nitrogen species.

M M-NO

1.2 Classification of NO Donors

of nitroxyl include addition to thiol groups (singlet NO− ) to generate NH2 OH, and reaction with oxygen (triplet NO− ) to form peroxynitrite (OONO− ). Nitroxyl has also been proven to exhibit many biological functions [34], such as vasodilatation [35–37] and cytotoxicity [38–40].

1.2

Classification of NO Donors

Intensive research on the biological functions of NO and other reactive nitrogen oxide species demands exogenous sources of NO donors as research tools and pharmaceuticals. Since the mid-1980s, the development of new NO donors has offered several advantages over the previous NO donors, such as spontaneous release of NO, donation of NO under controlled rates, and even the targeting of NO to certain tissues. The structural dissimilarities of the diverse NO donors have led to remarkably varied chemical reactivities and NO-release mechanisms. Generally NO donors release NO through three kinds of mechanisms. The first route is that donating NO spontaneously, which releases NO through thermal or photochemical self-decomposition of e.g. S-nitrosothiols, diazeniumdiolates, oximes. The second route is that releasing NO by chemical reactions with acid, alkali, metal and thiol. Organic nitrates, nitrites and syndnonimines give NO though this mechanism. The third route is enzymatic oxidation where NO donors, for example, N-hydroxyguanidines, need metabolic activation by NO synthases or oxidases for NO release. Some NO donors release NO by more than one route, e.g. organic nitrates can also generate NO by enzymatic catalysis. Classification of all NO donors could be confusing, since all nitrogen– oxygen-bonded compounds have the potential to decompose, be oxidized, or be reduced to produce reactive nitrogen species. However, similar chemical structures usually have a similar NO-releasing mechanism, so all current NO donors and their pathways of NO generation are summarized in Table 1.2 according to their chemical classification. Many medicines may work by an NO-dependent mechanism. Recent studies have shown that angiotensin-converting enzyme (ACE) inhibitors (i.e. Enalapril, Captropril, Cilazapril) improve endothelium-dependent vasodilator responsiveness [41–43]. ACE inhibitors inhibit the degradation of bradykinin, thereby augmenting NO production. Another calcium channel blocker, amlodipine, also releases NO from blood vessels, and kinins mediate the generation of NO [44]. These new findings give a good explanation for the cardioprotective effects of these drugs. Furthermore, estrogen, statins (HMG-CoA reductase inhibitor) and essential fatty acids have the ability to augment NO synthesis [45, 46]. All of the above molecules do not have structural moieties which can release NO directly, so they can be called NO stimulators, and they are not discussed in this book. Currently used NO donors will be introduced in the following chapters.

7

8

1 NO and NO Donors Tab. 1.2: Current major classes of NO donors.

Chemical Class

Representative Compounds

Pathway of NO Generation Non-enzymatic

Enzymatic

Thiols

Cyt-P450, GST, etc

Hydrolysis, transnitrosation, thiols, light, heat Light, thiols, reductants, nucleophiles

Xanthine oxidase, etc

OH− , light

Cyt-P450 related enzyme

Light, heat

Peroxidases

Thiols, light

?

Spontaneous, enhanced by thiols, light, metal ions

Unknown enzymes

Light, heat

?

Spontaneous, thiols

?

Thiols

Unknown enzyme

Thiols

?

Spontaneous, enhanced by light, oxidants, pH>5

Prodrugs require enzymatic hydrolysis

Spontaneous, O2 /FeIII -porphyrin

Cyt-P450

ONO2

Organic nitrate

O2NO ONO2 H3C O

Organic nitrite

H 3C

Metal-NO complex

Na2 [Fe(CN)5 (NO)]•2H2 O

NO

H3C

HO NO

N-Nitrosamine

N Me OH

N-Hydroxyl nitrosamine

O- NH4+

N

A membranebound Enzyme

N O Me

+N _

Nitrosimine

N

N

Nitrosothiol

O

N

O

N

AcHN S

O

CO2H

C-Nitroso compound Diazetine dioxide

O2N

N

R1 R2

N

R3

N

+ _O

R4

R

+ _

N

N

O

O Ar

+

N

Oxatriazole5-imine

O-

+

R

Furoxan & benzofuroxan

O

N

_

N

NH.HCl

O

NH

Syndonimine

O

N

R2

Oxime

N

+

_N

O

R1 NOH

O2 N

CONH2

1.3 New Classes of NO Donors under Development Tab. 1.2 (continued)

Chemical Class

Representative Compounds

Pathway of NO Generation Non-enzymatic

Enzymatic

Anto-oxidation enhanced by metal ions

Catalase/H2 O2

Oxidants

NOS, Cyt-P450

H2 O2 /CuZn-SOD or ceruloplasimin, H2 O2 /Cu2+ , heme-containing proteins

Peroxidase

?

Guanylate cyclase

H

Hydroxyamine

N

OH

H

NH

N-Hydroxyguanidine & guanidine

HO N H

CO2H

N H

NH2 O

Hydroxyurea

H2N

NH OH

O OH

Hydroxamic acid

N H

1.3

New Classes of NO Donors under Development

Different types of NO donors will be discussed in the other chapters except for the following two classes. 1.3.1

Nitroarene

6-Nitrobenzo[á]pyrene (6-nitroBaP) was found to release NO under visible-light irradiation, while no such photodegradation was observed with other nitrated BaPs, such as 1- and 3-nitroBaPs [47]. It can induce DNA strand breaks upon photoirradiation. NO is generated from 6-nitroBaP via 6-nitriteBaP, which is produced from 6-nitroBaP by an intramolecular rearrangement mechanism (Scheme 1.5). This finding may be useful for the development of a new type of photochemically triggered NO donors. NO hν

NO2

NO2

6-nitroBaP

6-nitriteBaP

Scheme 1.5 Photochemical reaction of 6-nitroBap.

O 6-Oxy-BaP radical

9

10

1 NO and NO Donors

1.3.2

Hydroxamic Acids

Hydroxamic acids [general formula RC(O)N(R′)OH] have been used as inhibitors of peroxidases [48], ureases [49] and matrix metalloproteinases, and as anti-hypertensive, anti-cancer, anti-tuberculous and antifungal agents [50, 51]. Although some of these bioactivities are attributed to the chelating ability of the hydroxamate group, the hypotensive effects are due to their ability to release NO [52]. Experiments have shown that hydroxamic acids can transfer NO to ruthenium(III) and cause vascular relaxation in rat aorta by activation of the iron-containing guanylate cyclase enzyme. Of the hydroxamic acids investigated, benzohydroxamic acid (Fig. 1.1) showed higher NO releasing ability than aceto-, salicyl-, and anthranilic hydroxamic acids.

O N H

OH Fig. 1.1 Benzohydroxamic acid.

1.4

Development of NO-Drug Hybrid Molecules

An innovative approach to harnessing the beneficial properties of NO is to attach an NO-releasing moiety to an existing drug (Fig. 1.2). Different hybrid compounds can offer various drug actions with synergistic effects, with reduced toxicity and side effects. Several pharmaceutical companies are actively engaged in this research area. A series of compounds are currently in the Phase-I or Phase-II clinical study. For example, Nicox in France (www.nicox.com) has developed the NO-releasing derivative of acetylsalicyclic acid, NCX-4016, which is claimed to be able to overcome the major drawback associated with the use of aspirin as a pain reliever [53]. NCX-4016 also shows a broader mechanism than aspirin and can inhibit additional inflammatory mediators [54]. NitroMed in Boston (www.nitromed.com) has reported that nitrosylated á-adrenoreceptor antagonists moxisylate (S-NO-moxisylate) had lower toxicity and fewer adverse side effects in the treatment of erectile dysfunction [55].

General:

Drug

NO CH3

OAc O O NCX-4016

Fig. 1.2 NO-drug conjugates.

CH3

O

CH3

ONO2

N

O

H3C H3C

CH3

S-NO-Moxisylyte

SNO CH3

1.4 Development of NO-Drug Hybrid Molecules

1.4.1

Nitrate Hybrid Molecules

Recently, the combination of a nitrate moiety with another bioactive substructure in a single molecule has received particular attention. Nitric-oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) are chemical entities obtained by adding a nitroxyalkyl moiety via an ether linkage to a conventional NSAID, such as NO-aspirin (NCX-4016), a prototype NO-NSAID. Because the use of NSAIDs is associated with significant gastro-intestinal toxicity, the development of safer NSAIDs, NO-NSAIDs, has been demonstrated to be rational and successful [56]. NO-NSAIDs inhibit inflammation via cyclo-oxygenase (COX)-dependent and independent NO-related effects. The mechanism can be explained by a general feature of NO, its capability to modify proteins that contain cysteine residues by causing the S-nitrosation of thiol groups in the enzyme catalytic center [57]. It has been proved that NCX-4016 causes the S-nitrosation and inhibition of interleukin(IL)-1â converting enzyme(ICE)-like cysteine proteases (caspases) involved in pro-IL-1â and pro-IL-18 processing, which are pivotal in the pro-inflammatory cytokine hierarchy. The NO-NSAIDs are not only devoid of gastro-intestinal toxicity, but are also more effective anti-inflammatory drugs than their parent compounds (Scheme 1.6). A similar compound, B-NOD, showed a similar activity, and does not affect blood pressure [58]. NCX-4215 and NCX-456 are designed according to the same principle. NO-NSAIDs are currently undergoing clinical Phase II trials. NO releasing

O O

NO

Inhibition of caspase

O O

Anti-inflammatory

ONO2

effect

NCX-4016 Hydrolysis Acetyl salicyclic acid

Inhibition of prostaglandin-forming COXs

Scheme 1.6 Anti-inflammatory mechanism of NO-NSAIDs.

NCX-1000 (Fig. 1.3) is another prodrug obtained by adding a nitroxybutyl moiety to ursodeoxycholic acid (UDCA), a steroid that is selectively metabolised by hepatocytes [59]. Because NCX-1000 is almost exclusively metabolised in the liver, NO is delivered directly to hepatic cells. It has been demonstrated that NCX-1000 protects against liver damage induced by Con A in mice by modulating the live-resident immune system, and reduces portal pressure in cirrhotic rats. This drug may provide a novel therapy for the treatment of patients with portal hypertension or immunomediated liver injury [60]. Using a similar approach, NCX-1015, a nitro-prednisolone, was designed and showed NO-releasing ability in biological fluids [61]. It was more

11

12

1 NO and NO Donors

O OH O O

O

ONO2

OH O

4

O

O

ONO2

O ONO2

NH2

NCX-4215

NCX-456

B-NOD

O O

O

4

ONO2

O O

O H

OMe

HO

H

H

ONO2

H H

HO

O OH

H

OH

H

O NCX-1000

NCX-1015

Fig. 1.3 Structures of NO-NSAIDs.

potent than prednisolone in acute and chronic inflammation. NO not only synergizes with the glucocorticoid moiety to produce the anti-inflammatory effect, but also counteracts the osteoclast activity of prednisolone, which causes a major side-effect of glucocorticoid drugs. Moreover, NCX-1015 showed significant bronchodilating activity [62]. As inhaled NO has been suggested as a useful therapy to induce airway dilation [63–65], NO-linked steroids may be a very effective therapy for airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). The potential synergism between NO and steroids locally released in a damaged tissue, may reduce the therapeutically effective doses of steroids and prevent the side-effects of steroids. The combination of beta-blockers with nitrovasodilators is an efficient therapeutic approach in coronary heart disease. Therefore, nitration of beta-blockers could produce an NO donor, while keeping its beta adrenergic receptor blocking effects. The S-S enantiomer of a metoprolol derivative named PF9404C (Fig. 1.4) was synthesized accordingly [66]. Pharmacological and biochemical experiments showed that when in contact with living cells, PF9404C can generate substantial amounts of NO, leading to cyclic GMP formation and vasorelaxation. Unlike rapid NO donors, PF9404C produces a slowly developing and sustained relaxation of the vessel. Its beta-blocking potency is close to that of S(-)-propranolol, four-fold higher than metoprolol. When beta-blockers alone are administered to hypertensive patients, the total peripheral resistance remains higher than in normotensives. If PF9404C is used in hypertensive patients, the NO actions, including potent vasodilating actions, inhibition of leukocyte–endothelial cell interaction, as well as platelet adherence and aggregation and vascular smooth muscle cell proliferation, may exert beneficial effects in hypercholesterolemia and ischaemic heart injury. Thus, PF9404C exhibits antihypertensive

1.4 Development of NO-Drug Hybrid Molecules

O OH

O

N H

O OH

ONO2

N H

O

ONO2 Fig. 1.4 Structures of â-blocker NO hybrid.

and cardioprotective actions through a double mechanism, NO donation and betablockade. Several reports indicate the involvement of superoxide in the mediation of tolerance [67–69]. Based on these reports, a bifunctional superoxide dismutase-mimic NO donor was designed by Haj-Yehia’s group [70]. The nitrate ester was incorporated into a nitroxide such as 3-hydroxymethyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (HMP) by its conversion into 3-nitratomethyl-PROXYL (NMP) (Scheme 1.7). HMP is a stable, metal-independent, low-molecular weight SOD-mimic with excellent cellpermeability. So NMP is the first compound that can simultaneously generate NO and destroy superoxide. This may lead to novel nontolerance-inducing nitrovasodilators. OH Nitric / sulfuric acid N O

-5 °C, 20 min

HMP

ONO2 N O NMP

Scheme 1.7 Preparation of NMP.

1.4.2

Furoxan Hybrid Molecules

H2 -antagonists are a class of drugs used in the management of peptic ulcer and gastric-acid-related disorders. Since they fail to trigger protection against gastric damage induced by NSAIDs, it would be useful to combine the antisecretory activity of H2 -receptor antagonists with the NO-dependent gastroprotective effect in the same molecule. Some new H2 -antagonist-containing NO-donor moieties have been synthesized [71]. The H2 -antagonistic substructures were derived from lamtidine and tiotidine, respectively. NO-releasing moieties were chosen from phenysulfonyl furoxan, nitrates and nitrosothio functions. The experimental results showed that only the hybrid compounds were able both to antagonize histamine effects on guinea pig papillary muscle and to display in vivo antisecretory and gastroprotective action. The best results were obtained with the lamtidine/furoxan hybrid structure, such as

13

14

1 NO and NO Donors

H2-Antagonistic Substructure

NO-Donor Moiety

Spacer

CN N N

O

3N

N H

H

O 2

SO2Ph N

21

O

N

O

Fig. 1.5 General structure of H2 -antagonist/NO hybrids and example compound.

21 (Fig. 1.5), while others hybrids showed ambiguous results. These compounds could be the prototypes of a new class of drugs, which may be useful in the therapy of gastric hypersecretion combined with inflammatory disorders. Nicorandil is an antianginal drug, which has the properties of both K+ channel openers and NO donors [72]. Structurally, it is a nicotinamide derivative with a nitrate group in its chemical structure (Fig. 1.6). The hybrid molecules of furoxan and nicorandil derivatives may achieve an ideal cardiovascular drug with good selectivity, efficiency and low toxicity [73]. A series of hybrid drugs designed by linking the furoxan ring to nicorandil analogues was investigated. Several of these compounds had good vasodilatory activity [74]. Compound 22 was further tested for its hypotensive effects in anaesthesized rats, and was able to significantly reduce blood pressure 3 h after administration. Its hypotensive effects could prevail for a further 3 h. These preliminary results indicate that the furoxan-nicorandil derivatives are a useful lead in the design of NO-donor compounds for hypertension. O

O N H

2 ONO2

N

O N

N N

H3CO Nicorandil

CH3 O

N

O

22

Fig. 1.6 Structures of Nicorandil and its hybrid.

1.5

New Therapeutic Applications of NO Donors

Due to the numerous possible reactions and related biological consequences, inappropriate overproduction of NO can cause a series of disease states such as a variety of neurodegeneration diseases including inflammation, rheumatic disease, septic shock, diabetes mellitus, and cerebral ischemia. Therefore development of isoformspecific NOS inhibitors to regulate NO synthesis has been an active research area.

1.5 New Therapeutic Applications of NO Donors

On the other hand, insufficient NO production also causes serious medical problems. Many diseases such as hypertension, atherosclerosis and restenosis involve a deficiency of NO production. Therefore, a compound that can release NO under specific conditions can be used therapeutically to palliate NO underproduction. In fact, the best known NO donor, glyceryl trinitrate, has been used for over a century to relieve acute attacks of angina pectoris. In 1998, Carl Djerassi published a book titled “NO”, where he plotted the success of a biotech company producing NO donor compounds to treat male impotence [75]. Currently, NO donor compounds have a variety of biomedical applications. Though current understanding of NO physiology and pathology seems incomplete, the largely indirect, correlative information suggests that both NO excess and insufficiency can elicit tissue injury and diseases. So far the most purported NO-insufficiency diseases are cardiovascular. The oldest NO donor drug, glyceryl trinitrate, has been used as a vasodilator since 1879. Besides supplementation of NO in a situation where an NO insufficiency may underlie the pathology, NO donors can also regulate an NObased physiological pathway, i.e., male erectile dysfunction, and improve drug safety and efficacy, i.e., gastrointestinal toxicity of nonsteroidal anti-inflammatory drugs. In addition to cardiovascular disorders [76], nerve system diseases [77] and inflammation [78], the benefit of NO in many other diseases has now been recognized. 1.5.1

NO Donors against Cancer

NO produced by activated macrophages plays an important role in modulating the host defense mechanism against tumor cells [79, 80]. Several in vitro studies have also shown that NO donors are cytotoxic to tumor cells leading to apoptosis, mainly involving changes in mitochondrial permeability transition and release of cytochrome c from the mitochondria [81]. NO released from inducible NO synthase inhibits metastasis at a higher level, but at a lower concentration, NO may cause induction of NO resistance and permit the growth of tumor cells [82]. Although there are reports indicating the genotoxicity of NO, exposure of whole cells to NO donors resulted in no appreciable mutations as compared to alkylating agents [83]. The reason is that large amounts of NO are required to generate DNA alterations and the formation of other reactive species and nitrosated DNA from NO is limited. There are also numerous defense mechanisms, such as ascorbate and glutathione, as well as intraand intercellular consumption of NO, which limit the mutation of DNA [84]. The dual role of NO in carcinogenesis is very confusing, so further studies are still needed to clarify it [85]. 1.5.1.1

Diazeniumdiolates (NONOates) as Promising Anticancer Drugs

Diazeniumdiolate compounds have already shown anti-leukemia activity [86]. However, these NO donors release NO systemically and cause severe side effects on the vascular system, so their therapeutical use has been limited. Upon modulation at the

15

1 NO and NO Donors

16 OO N O2N

N+

F

N

NO2

CH3

N

N

O

O

O

N

HN O

O JS-K

S

N

N N O

HOOC

5-FU/NONOate

NO

O

O

OH O

HO HO HO

H N

HN O

S-Nitrosocaptopril

S

CH3 CH3 CH3 NO

Man-1-SNAP

Fig. 1.7 Anticancer NO donors.

oxygen with nitro-aromatic substituents, these derivatives can release NO in target cells after a hydrolytic or enzymatic action. JS-K (Fig. 1.7), an example of O-protected diazeniumdiolate developed by the US National Cancer Institute (NCI), has attracted much attention [87]. JS-K can be attacked by the nucleophilic thiol group of glutathione (GSH), with the formation of the Meisenheimer complex; then the NONOate moiety leaves, and at physiological condition, the NONOate decomposes to release NO (Scheme 1.8). The aryl moiety of JS-K is bonded to the thiol group of GSH to give S-(2,4-dinitrophenyl)glutathione (DNP-SG). The drug kills, or slows the growth, of cancer cells without harming healthy cells [88]. For example, acute myeloid leukemia (AML) is the most common and deadly form of leukemia. Tests with the drug showed it triggered the destruction of AML cells grown in vitro. In the HL-60 human myeloid leukemia assay system, the IC-50 of JS-K is 0.5 ìM, while the IC-50 of a chemotherapeutic agent, daunorubicin, is 0.01 ìM. It also slowed the growth of AML cells in mice. In other tests on cell cultures, JS-K did the same, but to a lesser extent, in prostate, colon, and breast cancer cells. It also inhibited the growth of prostate cancer cells in mice. JS-K is found to react with glutathione S-transferases (GST), which help pump foreign substances out of certain cells. GSTs help the liver get rid of toxic substances in blood, but they also help cancer cells resist chemotherapy drugs. When GSTs in cancer cells interact with JS-K, there are two anticancer effects: GST activity is inhibited, making the cells less resistant to chemotherapy drugs, and NO is released. O HO

N H

O

S

JS-K + GSH -O

NH2

H N O

O-

O

N+

N N+ O-

O OH

NO2

HO

O

N H

O

NH2

H N S

OH O

O NO2

N N

O O

NO2

JS-K Meisenheimer complex -O

N

ON+

DNP-SG

N N

O 2 NO

+

HN

N O

Scheme 1.8 Mechanism of JS-K.

O

pH 7.4 O 4-Carbethoxy-PIPERAZI/NO

1.5 New Therapeutic Applications of NO Donors

It has been shown that JS-K inhibited cell growth with concomitant activation of mitogen-activated protein kinase (MAPK) members, ERK, JNK, p38 and their downstream effectors, c-Jun and AP-1 [89]. Inhibitors of these MAPK pathways abrogated the growth inhibitory actions of JS-K. In addition to the actions of JNK as a kinase for c-Jun, it was shown that c-Jun is also an ERK target. Furthermore, JS-K generated NO in culture and NO inhibitors antagonized both MAPK induction and the growth inhibitory effects of JS-K. These results suggest two possible mechanisms for the mediation of JS-K growth inhibitory actions, namely, NO-induction of MAPK pathway constituents as well as possible arylation reactions. The data support the idea that prolonged MAPK activation by JS-K action is important in mediating its growthinhibitory actions. In 2003, NCI accepted JS-K into its Rapid Access to Intervention Development (RAID) program, which tries to speed the development of new cancer therapies. Work done so far by the University of Utah within the RAID program has shown that JS-K is active against a broad spectrum of cancer cells. JS-K thus represents a promising platform for novel growth inhibitory analog synthesis. 1.5.1.2

The Synergistic Effect of NO and Anticancer Drugs

The 5-fluorouracil (5-FU) and NONOate conjugates (Fig. 1.7) were prepared and their cytotoxicity was tested [90]. The median effect doses of the conjugates for DU145 and HeLa cancer cell lines were 2–4-fold lower than that of 5-FU. In another study by Wink et al., the cytotoxicity of cisplatin was enhanced about 60-fold after NONOate pretreatment for 30 min [91]. The enhancement of cytotoxicity of 5-FU/NONOate conjugates and cisplatin-NONOate combination has shown that there is a synergistic effect between anticancer drugs and NO. Another study by Jia et al. demonstrated that the cytotoxicity of Taxol was enhanced by S-nitrosocaptopril (Fig. 1.7) [92]. This effect is primarily mediated via the increased influx of Taxol by NO into intracellular compartments, while NO-induced cytotoxicity cannot be excluded. In another separate study, researchers found organic nitrates can also increase the efficiency of cytostatic therapy and retard the development of drug resistance [93]. The combined therapy results in significant increase in life span and number of survivors among mice bearing leukemia P388 and L-1210. Comparative studies of organic nitrates and a similar compound in which ONO2 moieties were replaced by OH groups demonstrated that the presence of NO2 is required for adjuvant activity of compounds and confirmed that NO modifies the antitumor effects of cytostatics. It was also shown that the NO donor retards the development of drug resistance to cyclophosphamide. 1.5.1.3

NO-NSAIDs as a New Generation of Anti-tumoral Agents

Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin, were utilized primarily to protect from inflammation. Other biological effects were also found gradually, for example, induction of apoptosis [94, 95], stimulation of immune activity

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[96], and inhibition of angiogenesis [97]. Studies of patients with familial adenomatous polyposis (FAP) have demonstrated a reduction by approximately 50% of colonic adenomas or colorectal cancer among patients while using aspirin [98]. The growing evidence suggests that tumor inhibition may be mediated by at least two major cellular events. These involve the ability of NSAIDs to maintain the equilibrium between proliferation and apoptosis rates in colonocyte, and their inhibitory effect on angiogenesis [99, 100]. The main problem with the regular use of NSAIDs is the occurrence of side effects such as the increased risk of gastrointestinal bleeding and the developments of ulcers [101]. In recent years, NO-NSAIDs, such as NO-aspirin (NCX 4016) (Fig. 1.2), have been developed as “safe” NSAIDs [102]. NO-aspirin is 2500–5000-fold more potent than traditional aspirin in inhibiting the growth of colon cancer cells in vitro [103]. The corresponding test in mice also confirmed the strong inhibitory effect of NO-aspirin in intestinal carcinogenesis and suggests that NONSAIDs merit further evaluation as chemopreventive agents against colon cancer [104]. A detailed study showed that NO-aspirin inhibits â-catenin/T cell factor (TCF) signaling in colon cancer cells by disrupting the nuclear â-catenin/TCF association, whereas aspirin has no affect [105]. Two clinical trials of aspirin for the prevention of cancer were published in March 2003 [106, 107]. In one study, 635 patients with a recent history of adenomas received either the placebo or 325 mg of aspirin per day. A colonoscopy was performed on 81% of the patients after at least one year. One or more adenomas were found in 17% of patients in the aspirin group compared to 27% in the placebo group. Another study gave a similar result. These two studies indicate that daily use of aspirin is associated with a significant reduction in the incidence of colorectal adenomas in patients with previous colorectal cancer. If aspirin therapy is stopped, the reduction in the risk of adenomas dissipates. Also, NSAIDs may decrease the incidence of carcinomas of the esophagus, stomach, breast, lung, prostate, urinary bladder and ovary [108]. The clinical use of these agents is limited to patients with FAP. Due to the clear protective effect of aspirin, NO-NSAIDs can be a good alternative, which can give the beneficial effects of both NO and NSAIDs. In March 2003, NCI awarded a grant to the University of Michigan to conduct a clinical trail of NO-releasing aspirin (NCX-4016). This placebo-controlled study will assess the pharmacokinetics of different doses of NCX 4016 in patients at risk of colon cancer. 1.5.1.4

Other NO Donors with Anticancer Activity

As well as NONOates, other NO donors also showed anticancer activity independently. Sodium nitroprusside (SNP), a metal-NO complex, showed cytotoxic effects on the cells of some patients with malignant lymphoma (ML), acute myelocytic leukemia (AML) or chronic myelomonocytic leukemia (CMMoL), but not with multiple myeloma [109]. SNP and cytosine arabinoside (Ara-C) did not share the drug resistance. Interestingly, SNP had no effect on lymphocytes of healthy volunteers. These results suggest that SNP has an anti-tumor effect on human hematological malignant cells.

1.5 New Therapeutic Applications of NO Donors

A series of sugar-S-nitrosothiols (sugar-SNAPs), for example, glucose-1-SNAP, have shown promising pharmacokinetic properties [110]. These compounds were designed based on the observation that facilitated transport of monosacharrides in mammalian cells was accomplished by the glucose transporter family of transmembrane properties. They were constructed from an aglycone unit conjugated with a mono- or oligosaccharide. Compared to SNAP, sugar-SNAPs had higher stability and slower NO-releasing properties in aqueous solution. Glucose-SNAPs were more cytotoxic than SNAP. The enhanced cytotoxicity of glucose-1-SNAP and glucose-2SNAP may be related to their affinity for glucose transporters present on plasma membranes, but relative experiments have not yet been done. Another possible explanation is that glucose-SNAP binds to glucose transporters and decomposes to release NO, then NO causes the apoptosis of cancer cells. Recently, mannose-SNAPs were also developed, for example Man-1-SNAP (Fig. 1.7). The cytotoxicity of Man-1SNAP was just as potent as that of glucose-SNAP [111]. Hydroxamic acid derivatives, which belong to a new class of NO donors, have been shown to inhibit the matrix metalloproteinases (MMPs) [112]. MMPs are a family of zinc-dependent endopeptidases, which play a critical role in multiple steps in the metastatic cascade and facilitate neoangiogenesis. Numerous hydroxamic acids, such as marimastat, have been developed, that bind the zinc atom in the active catalytic domain of MMPs. During a randomized Phase III trial, comparing marimastat with placebo in patients with metastatic breast cancer, marimastat was not associated with an improvement in progression-free survival or overall survival. Other studies also indicated no benefit for MMP inhibitors when used either in combination with chemotherapy or sequentially after first-line chemotherapy in a variety of cancers [113]. Currently, many pharmaceutical companies have suspended clinical development of this kind of agent. 1.5.2

NO against Virus 1.5.2.1

HIV-1 Induces NO Production

It was found that the HIV envelope glycoprotein in vitro increases the production of NO by human monocyte-derived macrophages [114]. NO production is increased in patients who have AIDS [115], and the increased concentrations of nitrite in AIDS patients with opportunistic infections is caused by T gondii, Pneumocystis carinii, Mycobacterium tuberculosis, and Mycobacterium avium, whereas nitrite concentrations are normal in symptom-free patients. It was also confirmed that there was increased production of NO in the sera of children with HIV-1 infection, and of circulating cytokines, such as interleukin 1â, tumor necrosis factor á, and interferon ã. It is postulated that rises in the concentrations of these cytokines may represent a substantial stimulation of NO production [116]. In contrast, it has been shown that there was no altered endogenous nitrate formation in eight patients with AIDS, most of whom had opportunistic infections [117]. It has also been noted that there were high

19

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1 NO and NO Donors

nitrite and nitrate concentrations in 39 patients with AIDS, especially in those with lower CD4 cell counts, whereas in symptom-free patients no such increase was seen [118]. However, AIDS patients with opportunistic infections were not selected for assessment of NO production. Groeneveld and colleagues [119] have shown that serum nitrate concentrations are higher in symptom-free HIV-1-infected patients than in healthy individuals. NO production was measured in vitro from peripheral blood leucocytes of HIV-1-infected patients after measuring nitrite concentrations from peripheral blood mononuclear cells and polymorphonuclear leucocytes [120, 121]. An increase in nitrite production in AIDS patients, especially in those with opportunistic infections, was also seen. There is substantial induction of the iNOS gene in primary cultures of human monocyte-derived macrophages, concomitant with the peak of virus replication, and exposure to low concentrations of NO donors results in a significant increase in HIV-1 replication [122]. Acute infection of macaques with a pathogenic strain of the simian immunodeficiency virus increased gene expression of iNOS in mononuclear cells obtained from bronchoalveolar lavage [123]. At the time of systemic viral load peak, NO production was greatly raised in the monkeys [123]. The activated lung macrophages of neonatal rats produced significantly more NO than did those of infant and adult rats [124]. Since HIV-1 infection in neonates progresses to AIDS more rapidly than does infection in later life in human beings, these investigators speculate that excessive NO may explain the rapid progression of HIV-1 infection to AIDS during infancy. The concentrations of nitrite or nitrate in the sera of patients infected with HIV-1 are substantially raised, especially in those with low CD4 cell counts [118]. However, during HIV-1 infection, it is difficult to find out whether the NO production is attributable to virus replication or to opportunistic infections, or both. In vitro there is a substantial rise in nitrite concentrations from blood mononuclear cells and polymorphonuclear leucocytes from patients with AIDS, especially in those with neurological disorders and pulmonary disease caused by intracellular opportunistic pathogens [121]. Interestingly, the serum concentrations of nitrate are positively correlated with plasma and cell-associated viral loads, which suggests that HIV-1 may induce NO synthesis in vivo [119]. However, the results clearly show that there is a close relation between viral replication and iNOS expression or peaks of plasma nitrate in the absence of any opportunistic infections, in either in macaques or infected patients [119, 122, 123]. NO acts as an autocrine factor that mediates HIV-1 replication; as at the molecular level, NO seems to stimulate long-terminal repeat-mediated transcription [125]. It was noted that exogenous NO increases replication of HIV-1 T-tropic isolates in primary T cells or T-cell lines, and inhibitors of iNOS partly block HIV-1 replication, especially that induced by tumor necrosis factor á [125]. The contrasting effects of exogenous NO, particularly NO donors, may depend on the type of NO donors, their releasing kinetics, and the dose used in the study design.

1.5 New Therapeutic Applications of NO Donors

1.5.2.2

Antiviral and Proviral Activity of NO

Antiviral effects of NO are known for several viruses, including murine poxvirus, herpes simplex virus, Epstein-Barr virus, coxsackievirus, and influenza virus [126, 127]. Virus infection induces directly or indirectly (through interferon ã production) overproduction of NO because of localized iNOS expression in the area of infection [128]. Many pathological effects of NO are thought to be produced via its interaction with oxygen radicals, producing peroxynitrite [129]. Since the antiviral effects of NO do not require immune recognition of infected cells, and since NO can pass readily into cells, it provides a useful early defence against viral infections before the development of a specific immune response; thus NO may be a host response modulator rather than a simple antiviral agent. Viral infections against which NO and its derivatives are thought to have inhibitory effects include DNA and RNA viruses, such as poliovirus, Japanese encephalitis virus, mouse hepatitis virus, vesicular stomatitis virus, herpes simplex virus type 1, vaccinia virus, and Epstein-Barr virus [126]. NO may inhibit an early stage in viral replication, and thus prevent viral spread, promoting viral clearance and recovery of the host. The earliest host response to viral infections is non-specific and involves induction of cytokines, especially tumor necrosis factor á and interferonã. These cytokines are potent inducers of iNOS, which generates large amounts of endogenous NO [130]. Thus, NO could be a vital factor in inducing the host’s innate immunity to control the initial stages of viral infections. Despite the protective effect of NO against various viral infections, workers in several studies have shown a harmful role of NO in many systems. NO seems to play a part in the development of pneumonia caused by influenza virus [128], in the pathogenesis in mice of tick-borne encephalitis flavivirus infection [131], and in worsening the course of the murine myocarditis caused by coxsackievirus B3 [132]. In addition, pneumonia in mice induced by herpes simplex virus type 1 could be suppressed by the inhibitor of iNOS [133]. The issue of whether NO acts as an inhibitor of viral replication or as a harmful agent, therefore, remains unanswered. This issue is particularly evident in HIV-1 infection, since NO seems to act as a double-edged sword in the pathogenesis of HIV-1. The antiretrovirus properties of NO were shown in mice infected with Friend leukemia virus, a murine retrovirus. NO produced by NO-generating compounds or activated macrophages inhibits viral replication in fibroblast cultures, and is involved in defens against this murine retrovirus in vivo [134]. It was also reported that NO donors can inhibit HIV-1 replication in human monocytes through induction of iNOS [135]. The life cycle of many viruses, including retroviruses, depends on viral proteases that cleave viral glycoproteins into individual polypeptides, and these enzymes are necessary for viral replication. NO can inactivate coxsackievirus [136]. Since cysteine proteases are critical for the virulence and replication of many viruses, nitrosation of viral cysteine proteases may be a mechanism of antiviral host defense. NO mediates nitrosation of cysteine and aspartyl proteases of HIV-1, and it was suggested that this

21

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1 NO and NO Donors

mechanism may have a role in the inhibition of HIV-1 replication [137]. Later it was shown that NO donors inhibit the HIV-1 reverse transcriptase activity, through the modulation of the catalytic activity of cysteine enzymes [138]. The modulation mechanism of the HIV-1 reverse transcriptase activity may be relevant for the development of new strategies for inhibition of HIV-1 replication. NO has complex roles in immunological host responses against viruses, and especially against HIV-1 infection. In HIV-1 infection, NO cannot be rigidly classified as an anti-inflammatory or proinflammatory molecule, but it can be deemed a true inflammatory mediator. Many studies support a proviral effect of NO in HIV-1 infection, mainly based on stimulation of viral replication, and on toxic effects on various cells, including central nervous system cells, via oxidative injury that may cause cellular and organ dysfunction, and immunosuppression and immunopathology, especially in the central nervous system. In several studies on the antiviral effects of NO on HIV-1 infection, the proviral or antiviral effects of NO seem to be strictly related to the active production of NO during HIV-1 infection. The universal speculative interpretation of the dichotomous effect of NO is that overproduction of this substance, especially in the primary infection and in late stages of the disease, leads to active viral replication with consequent harmful effects on the course of the disease. Conversely, low production of NO may cause a reduction in or inhibition of HIV-1 replication, especially during the symptomless stage of the disease, or during treatment with highly active combined antiretroviral drugs. Recently, it has been found that NO donors inhibit HIV-1 replication in acutely infected human peripheral blood mononuclear cells (PBMCs), and have an additive inhibitory effect on HIV-1 replication in combination with 3′-azido-3′-deoxythymisylate (AZT) [139, 140]. S-nitrosothiols (RSNOs) inhibit HIV-1 replication at a step in the viral replicative cycle after reverse transcription, but before or during viral protein expression through a cGMP-independent mechanism. In the latently infected U1 cell line, NO donors and intracellular NO production stimulate HIV-1 reactivation. These studies suggest that NO both inhibits HIV-1 replication in acutely infected cells and stimulates HIV-1 reactivation in chronically infected cells. Thus, NO donors may be useful in the treatment of HIV-1 disease by inhibiting acute infection, or reactivating a latent virus. 1.5.3

Inhibition of Bone Resorption

NO is recognized as a mediator of bone cell metabolism, where it regulates osteoblast and osteoclast activity [141–143]. Osteoporosis, which frequently occurs in postmenopausal women, is a systemic skeletal disease associated with abnormal bone resorption. Addition of NO or NO donors to osteoclasts in vitro results in a reduction in bone resorption, whereas NO synthase inhibitors increase bone resorption, both in vitro and in vivo. Further research has shown that NO reduces bone resorption, via inhibition of the cysteine protease cathepsin K, which is believed to be a key protease in bone resorption. Most of the NO donors, i.e., nitroglycerin, 3-

1.5 New Therapeutic Applications of NO Donors

morpholinosydnonimine (SIN-1), S-nitrosothiols, sodium nitroprusside (SNP), have an IC50 value for cathepsin K from 0.01 ìM to about 1000 ìM. So NO donors may be a new generation of therapeutic agents for inhibiting the bone resorption activity of osteoclasts. There is also evidence suggesting that prostaglandin plays an important role as a regulator of bone remodelling in response to various stimuli, such as cytokines, sex hormones, and mechanical loading [144, 145]. Moreover, both cytokine-induced activation of NO and prostaglandin E2 (PGE2 ) pathways may act in a concerted manner to influence bone cell activity and bone turnover [146, 147]. So the combination of cyclooxygenase (COX) inhibitors and NO donors would exert more potent modulatory effects on bone turnover and bone mass. A report from NiCox indicated that flurbiprofen nitroxybutylester (HCT1026) was significantly more efficacious than the parent compound, flurbiprofen, at inhibiting osteoclast formation and bone resorption in vitro and prevented ovariectomy-induced bone loss in vivo [148]. HCT1026 may be of clinical value in the prevention and treatment of inflammatory diseases such as rheumatoid arthritis, which are characterized by joint inflammation as well as periarticular and systemic bone loss. 1.5.4

Treatment of Diabetes

SIN-1, a non-enzymatic NO donor, has been reported to inhibit insulin release in isolated pancreatic islets [149]. However, another report showed that l-arginine, an NO donor, could stimulate glucose-induced insulin secretion from the pancreas of diabetic rats [150]. Further studies showed that non-enzymatic NO donors such as SIN-1, sodium nitrite, sodium nitropusside, and S-nitroso-N-acetyl-dl-penicillamine (SNAP), increased insulin sensitivity through stimulation of NO production in the liver [151]. It was found that, besides the known vascular effect, enzymatic NO donors, such as organic nitrates, also have a hypoglycaemic/antihyperglycaemic effect. A pharmaceutical combination for the treatment and prevention of diabetes mellitus was invented, comprising at least one enzymatic NO donor and optional antidiabetic active ingredients [152]. The basis of the invention is the recognition of a new insulinsensitizing effect and synergism using NO donors and conventional antidiabetic drugs. 1.5.5

Thromboresistant Polymeric Films

Hydrophobic polymer materials that slowly release NO can be used on the surface of medical devices. Many medical devices suffer from the surface adhesion of blood platelets. To minimize this thrombogenic effect, blood thinners such as heparin, coumarin, and aspirin are often used. However, systemic administration of antiplatelet agents could increase the risk of uncontrolled bleeding elsewhere in the body. In contrast, biocompatible polymer films would solve this problem [153]. It is possible to create polymeric surfaces that mimic the inner surface of a blood vessel by

23

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1 NO and NO Donors

locally releasing NO. Such in situ NO generation would inhibit platelet adhesion and activation on the polymer surface. Because of its short-life in blood (<1 s), NO is potentially an ideal species for improving the thromboresistivity of polymeric materials for biomedical applications [154]. Various diazeniumdiolate NO donors have been incorporated into polymer matrices [155, 156]. These films showed good NO-releasing ability and improved blood compatibility. 1.5.6

Inhibition of Cysteine Proteases

S-nitrosylation reactions, which transfer NO from a NO donor to a protein sulfhydryl group, can affect protein functions in biological systems [157]. If S-nitrosylation occurs in the active sites of the enzymes it may inhibit the catalytic activity of the enzymes. Cysteine proteases comprise a large class of enzymes from plant, animal, and bacterial sources. They play important roles in various biological processes [158]. NO donors inhibit cysteine proteases by modification of the cysteine catalytic residue of the enzymes, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain [159]. Since cysteine proteases are critical for the replication of many viruses, bacteria, fungi, and parasites, cysteine proteases appear as promising targets for anti-parasite chemotherapy [160]. NO-releasing drugs could have an enhancing role in the therapeutic treatment of parasitic diseases, such as malaria [161]. Caspases, a family of cysteine proteases activated during apoptosis, are also therapeutic targets for modulating inflammation, as ICE/caspase-1 activation is the limiting step in the process of maturation of the cytokines IL-1â and IL-18, which are pivotal in the pro-inflammatory cytokine hierarchy [162]. Specific inhibitors are being developed by various pharmaceutical companies [163]. NO-NSAIDs are promising anti-inflammatory drugs as caspase inhibitors.

1.6

Conclusion

The synthesis of compounds which can release NO is relatively easy but, for therapeutic uses, they must have tissue selectivity, evenly controlled manner, and remain in a subtoxic range. Notwithstanding the many new classes of NO donors that have been reported, organic nitrates, diazeniumdiolate and S-nitrosothiols are still the three most important NO donors. They have the obvious advantages of well-proven NO donors, decomposing rapidly in solution, and mimicking the endogenous nitrosothiols. In clinical use, only organic nitrates and sodium nitroprusside show on prescriptions. However, patients taking long-term nitrates often develop tolerance, and prolonged sodium nitroprusside administration can give rise to cyanide accumulation in the body. S-nitrosothiols do not share these drawbacks. Perhaps it will take several years for new NO donor drugs to be used extensively. For targeted therapy, protected diazeniumdiolates as prodrugs may be more suitable than other

1.6 Conclusion

classes. By selecting a protecting group that can be metabolically removed by enzymes unique to the target tissue, NO release should be concentrated at the target site. The liver selective NO donor, V-PYRRO/NO, is a successful example[164]. The development of hybrid NO donors, such as NO-NSAIDs, which are formed by linking an NO-releasing moiety to a well-established bioactive molecule, seems a promising trend. These hybrid compounds can either abolish detrimental side effects, or reduce toxicity, or produce synergistic effects. Upon understanding the complex chemistry, biochemistry, and molecular biology of NO, NO donors should have more therapeutic applications.

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2

Organic Nitrates and Nitrites Roger Harrison

Glyceryl trinitrate (GTN), already known as nitroglycerin and the basis of dynamite, was first employed therapeutically, in the relief of anginal pain, by Murell in 1879 [1]. Now, over 120 years later, GTN and related organic nitrates (Fig. 2.1) are widely used as vasodepressors in the clinical treatment of a range of cardiovascular diseases, including angina, acute myocardial infarction and congestive heart failure [2, 3]. In fact, even before Murell’s report, Brunton had, in 1867, described using the organic nitrite, isoamyl nitrite (IAN) in the treatment of angina [4]. IAN is now seldom used for this purpose. It does, however, find minor application in the diagnostic evaluation CH2ONO2 GTN

CH ONO2 CH2 ONO2 H

O2NO

O

2

ISDN

O

IAN

CH3

5

H

CH

ONO2

CH2

CH2ONO

CH3 Fig. 2.1 Commonly used organic nitrates and

IBN

CH3

CH CH3

CH2 ONO

nitrites. GTN, glyceryl trinitrate; ISDN, isosorbide dinitrate; IAN, isoamyl nitrite; IBN, isobutyl nitrite.

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

34

2 Organic Nitrates and Nitrites

of cardiac murmurs [5] and for the treatment of cyanide poisoning [6]. It is also widely used as a recreational drug, as is its homologue, isobutyl nitrite [IBN] [7–9] (Fig. 2.1).

2.1

Organic Nitrates

The generally accepted mechanism of action of organic nitrates involves their entry into vascular smooth muscle cells, wherein they are converted to nitric oxide (NO). NO activates soluble guanylate cyclase (sGC), which in turn leads to increased levels of cyclic guanosine-3-5-monophosphate (cGMP), activation of cGMP-dependent protein kinase (cGK-I) and vasorelaxation [2, 10, 11]. There are, however, many uncertainties in this overall picture. In particular, the precise mechanism whereby GTN is converted to NO has been vigorously debated. Any hypothesis concerning the mechanism of action of organic nitrates must take into account the phenomenon of tolerance [12, 13]. With the introduction of GTN patches, in the early 1980s, it became clear that continuous exposure led, within 24–48 hours, to dramatic loss of effect. Responsiveness could, however, be restored within 48 hours of discontinuation of the drug. Early in vitro studies indicated that tolerance to GTN was accompanied by decrease in the levels of tissue thiols; moreover tolerance could be reversed by dithiothreitol. This led Needleman and colleagues [14–17] to propose the existence of a nitrate receptor containing essential thiol groups at the active site. According to their proposal, interaction of SH groups with organic nitrates leads to oxidation of the former to disulfide and denitration of the latter to nitrite. Reversal of tolerance by dithiothreitol was explained in terms of reduction of the disulfide back to active SH groups. The hypothesis was subsequently developed by Ignarro et al. [11, 18], who proposed that nitrite is transformed to NO and thence to S-nitrosothiol. The latter then activates sGC, so raising the levels of cGMP and effecting vascular relaxation (Scheme 2.1). Subsequent publications have queried aspects of this scheme, particularly the essential intermediacy of S-nitrosothiol. While S-nitrosothiols undoubtedly activate the sGC/cGMP system and cause vasodilation [11, 19, 20], the chemistry of their formation from NO and thiols at physiological pH is problematic [21]. Moreover, evidence that they, rather than NO itself [10, 22], directly initiate the vasodilatory cascade has been questioned in detail [23]. The involvement of nitrite poses similar difficulties. Ignarro proposed that nitrite is protonated to nitrous acid which then, under acid conditions, decomposes to NO (Scheme 2.1). While this non-enzymic conversion is feasible [24], the extent of formation and decomposition of nitrous acid at physiological pH is debatable and extensive pharmacological arguments have been advanced against the role of nitrite as an intermediate in this scheme [23, 25]. These will be considered in more detail later. Despite these objections, the essential mechanism of vasorelaxation, that requires metabolism of organic nitrates to NO, is still generally accepted and conversion of GTN to NO has been experimentally demonstrated in both smooth muscle [26–28] and endothelial cells [27, 28]. Possible mechanisms

2.1 Organic Nitrates

2 R1

SH

R1 S

S

R1

NO2- + ROH

RONO2 H+

HONO H+ NO GTP

R11 SH R11 SNO

sGC cGMP

Scheme 2.1 Simplified mechanism whereby organic nitrates (RONO2 ) effect vasorelaxation. Thiols (R1 –SH) interact with organic nitrates to give nitrite (NO2 − ), which is converted successively to nitrous acid (HONO) and NO. NO then reacts with a thiol (R11 –SH) to give a nitrosothiol

Relaxation

(R11 –SNO),

which upregulates sGC, leading to increase in cGMP and relaxation. In the course of their interaction with organic nitrate, thiols (R1 –SH) are oxidised to disulfides (R1 –S–S–R1 ). Adapted from Ignarro et al. [11].

of GTN–NO conversion, both enzymic and non-enzymic will be considered in what follows. 2.1.1

Direct Chemical Reaction between Organic Nitrates and Thiols

In view of the evidence implicating thiols in the vasodilatory activity of organic nitrates, early consideration was given to the extent to which NO can be generated by direct chemical interaction between organic nitrates and SH groups. Feelisch and Noack reported that chemical interaction of GTN with several thiols, in phosphate buffer, led to generation of NO, as determined by oxyhaemoglobin [29] or chemiluminescence [30] assay. However, the specificity of the former assay has been criticised [31] by Thatcher and colleagues, who themselves failed to detect generation of NO, by millimolar mixtures of GTN and cysteine, by means of a sensitive NO-specific electrode [32]. NO was detected by other workers, albeit in amounts too low to account for sGC activation [33, 34]. In one of these cases [33], rates of NO production were some 20-fold higher in the presence of plasma, but were significantly reduced following heating, indicating the presence, in plasma, of a heat-labile component involved in NO production. More recently, Artz et al. [35] have provided spectroscopic evidence that, while GTN/cysteine can activate sGC, this is not via transformation of GTN to NO. These findings are not only contrary to the idea that interaction of GTN and

35

36

2 Organic Nitrates and Nitrites

cysteines can mediate non-enzymic production of NO, but also challenge the concept of the intermediacy of NO in GTN-induced activation of sGC (see Section 2.3). Nevertheless, considerable research effort has been expended on studies of mechanisms of GTN–NO bioconversion. Heat treatment of smooth muscle or endothelial cells is known to abrogate their ability to release NO from GTN [27, 28, 36], and the present consensus is firmly in favour of enzymic transformation of GTN to NO in vivo. 2.1.2

Glutathione-S-transferase

In their early studies of the influence of thiols on the phamacology of organic nitrates, Needleman and colleagues demonstrated that GTN is metabolised to glyceryl dinitrate (GDN) in perfused rat liver with accompanying depletion of glutathione (GSH) [37]. GSH is a substrate for glutathione-S-transferase and several reports have supported the involvement of this enzyme in the activation of GTN in vivo. Thus, Jakoby and colleagues [38, 39] showed that glutathione-S-transferase catalyses the conversion of GTN to GDN and nitrite, with the concomitant oxidation of GSH to GSSG. More recently, Lau and Benet [40], working with subcellular fractions of rabbit liver, demonstrated that GTN is metabolised preferentially to 1,2-GDN in the cytosol and to 1,3-GDN in the microsomes. They provided evidence for the involvement of glutathione-S-transferase in this process and concluded that different isoenzymes generated specific GDN isomers. Several such isoenzymes have, in fact, been purified from human aorta [41]. Despite these positive findings, results from inhibitor studies are controversial. Thus, sulphobromophthalein, a known inhibitor of glutathione-S-transferase [42] has been reported both to inhibit [43] and not to inhibit [44–47] vasorelaxation by GTN, while other findings [48] favored involvement of the enzyme in GTN metabolism of cultured smooth muscle cells. With regard to the actual determination of GTN-induced NO, Harrison and colleagues [49] followed the generation of both nitrite and NO by homogenates of dog carotid artery in the presence of GTN. Addition of an inhibitor of glutathione-Stransferase led to a 78% decrease in nitrite production but had no effect on NO release. Moreover, whereas a linear relationship existed between glutathione-S-transferase activity and nitrite production, enzyme activity and NO production were unrelated. Additionally, the authors demonstrated that purified preparations of those glutathioneS-transferase isoforms that occur in dog carotid smooth muscle metabolised GTN to nitrite but not to NO. On the basis of these studies it was concluded that glutathioneS-transferase is not involved in the bioconversion of GTN to NO. The extent to which nitrite can serve as a physiological source of NO has been extensively discussed. As noted above, Ignarro and colleagues proposed that nitrite is an intermediate in the bioconversion of GTN to NO (Scheme 2.1). However, nitrite has been shown to be 1000-fold less effective than GTN in the stimulation of sGC [50]. While this difference could be attributed to the inability of hydrophilic nitrite to cross cell membranes, levels of intracellular and plasma concentrations of nitrite have been

2.1 Organic Nitrates

shown to be similar [51]. Bennett and Marks [25] have argued that, if endogenous intracellular concentrations of nitrite are (like those in plasma) micromolar, then it is hard to envisage how extra nitrite generated from nanomolar concentrations of GTN could affect cell function. The present consensus, therefore, is that, in the absence of unequivocal evidence that glutathione-S-transferase plays a major role in either bioconversion of GTN to NO or in GTN-induced vasorelaxation, it acts simply to catalyse generation of nitrite; in this context, a ‘non-productive’ competing metabolic route. 2.1.3

Cytochrome P-450-dependent Systems

Servent and colleagues [52] reported that GTN is metabolised in rat liver microsomes by an NADPH-dependent cytochrome P-450 system, yielding GDN, glyceryl mononitrate (GMN) and NO. Moreover, Schroeder and Schroer [53] showed that inhibitors of cytochrome P-450 reduce cGMP stimulation by GTN in kidney epithelial cells. Cytochrome P-450 systems are also present in both smooth muscle [54, 55] and endothelial [56] cells. In studies of hepatic [57] and aortic [58] microsomes, Bennett and colleagues showed that bioconversion of GTN to GDN was NADPH dependent and was inhibited by the cytochrome P-450 inhibitor, SKF 525A. In hepatic microsomes, moreover, conversion of GTN led to activation of sGC [59]. The involvement of cytochrome P-450 in vascular bioactivation of GTN, however, is controversial. Using cultured rat lung fibroblast cells as a model for vascular smooth muscle cells [60], Schroeder [61] was able to demonstrate that proadifen, an inhibitor of cytochrome P-450, decreased GTN-induced stimulation of cyclic GMP by up to 81%; the glutathione-S-transferase inhibitor, sulfobromophthalein, was without effect under similar conditions. In further support, Bennett et al. [62] demonstrated that 7-ethoxyresorufin, a cytochrome P-450 substrate, inhibited GTN-induced relaxation, cGMP accumulation and aortic biotransformation of GTN. On the other hand, Salvemini and colleagues [46] were unable to block aortic relaxation or bioconversion of GTN to NO by either SKF-525A or by another inhibitor, metyrapone. In further inhibitor studies, SKF 525A [51, 56, 60], metapyrone [63] and cimetidine [59] all failed to affect GTN-induced vasodilation. It is worth noting here that Ahlner and colleagues [2] warn against facile interpretation of data derived by using inhibitors of cytochrome P-450 systems; several different isoenzymes exist with different sensitivities to ‘classical’ inhibitors. In fact, one of these isoenzymes, CYP3A4-NADPH-cytochrome P-450 reductase, has been shown to be particularly effective in the generation of NO from isosorbide dinitrate [64]. Similar concerns about specificity apply to diphenyleneiodonium (DPI), which has been shown to inhibit biotransformation of GTN in rat aortic microsomes and GTN-induced lowering of blood pressure in rats [65]. DPI was shown to inhibit GTN transformation by purified NADPH-cytochrome P-450 reductase, but, as the authors acknowledge, DPI inhibits several flavin-containing enzymes, including xanthine oxidoreductase (see later).

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2.1.4

Membrane-bound Enzyme of Vascular Smooth Muscle Cells

Fung and colleagues examined the metabolic conversion of organic nitrates in subcellular fractions of bovine coronary artery smooth muscle cells [66, 67]. They found NO-generating capacity to be present in membrane fractions and, with the use of marker enzymes, identified plasma membrane as the primary location. The enzyme involved in bioconversion was not glutathione-S-transferase [68] and differed from those that catalyse activation of organic nitrites [69]. Partial purification [70] established that the molecular sizes of the native enzyme and subunits were approximately 200 kDa and 58 kDa respectively, and that enzymic activity depends on the presence of a free thiol group. Evidence was presented that the enzyme did not involve cytochrome P-450. 2.1.5

Xanthine Oxidoreductase

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, that, because of its ready availability from cows’ milk, has been studied for over 100 years [71]. Its enzymology is, accordingly, well characterized [72, 73]. XOR is a complex molybdoflavoprotein, comprising two identical 147 kDa subunits, each of which contains one Mo, one FAD and two Fe2 S2 redox centres. Long known to be involved in the later stages of purine catabolism, it catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. It has, however, a wide range of reducing substrates, including not only hypoxanthine and xanthine, but also a broad spectrum of N-heterocyles and aldehydes [74]. Electrons are donated from such substrates to the Mo site of XOR and are rapidly equilibrated between the redox centres before being passed to NAD+ or to molecular oxygen at the FAD site. Reduction of NAD+ gives NADH, while that of oxygen generates hydrogen peroxide and superoxide. XOR can also act as an NADH oxidase, in which NADH donates electrons to the FAD site. These interrelationships are outlined in Fig. 2.2. The enzyme occurs in two forms, xanthine dehydrogenase (XDH) and xanthine oxidase (XO). XDH and XO can be interconverted by thiolmodifying reagents, but proteases convert XDH irreversibly to XO. Both XDH and XO can reduce molecular oxygen, although XO is more efficient in this respect [73]; only XDH can reduce NAD+ . It is worth noting that the term, ‘xanthine oxidase’ is commonly used for the enzyme in general. In this chapter, XOR will be used in this context and ‘xanthine oxidase’ (XO) will refer specifically to the form that cannot reduce NAD+ . It has long been known that XOR is capable of reducing inorganic nitrate [76–79]. However, this reaction was largely ignored until relatively recently. In 1998, Miller et al. [80] reported that purified bovine milk XOR catalyses the reduction of GTN, as well as of inorganic nitrate and nitrite, to NO under hypoxic conditions. The electron donor in this work was NADH. In continuing studies, Doel et al. [81] demonstrated the XOR-catalysed generation of NO, not only from GTN, but also from isosorbide dinitrate (ISDN) (Fig. 2.1) and from the isomeric mononitrates (Figs. 2.1 and 2.3). In

2.1 Organic Nitrates

Hypoxanthine

Xanthine

Xanthine

Urate

Mo Fe2S2 FAD

NADH

NAD+ O2 NADH

NAD+

O2-• H2O2

Fig. 2.2 Schematic diagram showing

XOR-catalysed oxidation of hypoxanthine and xanthine (also most reducing substrates) at the molybdenum (Mo) site, and of NADH at the FAD site. Reduction of NAD+ or of molecular oxygen takes place at FAD. Adapted from Harrison [73].

the presence of xanthine (rather than NADH) as reducing substrate, NO could not be detected, even by the sensitive chemiluminescence assay used for this purpose. In fact, GTN was shown to be reduced to inorganic nitrite in essentially stoichiometric proportions; an observation that was initially surprising in view of the recently demonstrated ability of XOR to catalyse reduction of nitrite to NO [82–85]. The relative slowness of the further reduction of nitrite to NO, in this experimental system was, however, explained [81] in terms of inhibition by GTN and, more especially, by xanthine; NADH shows no such substrate inhibition [85].

O6 5 2

O2NO

3

OH

4

1

O

O2NO

O

1 2

5 6

4

OH

3

O

and endo- respectively to the fused isosorbide-5-mononitrate, showing that pyran ring system. Reproduced with permission from Doel et al. [81]. orientation of the nitro groups is exo-

Fig. 2.3 Isosorbide-2-mononitrate and

Reduction of organic nitrates was shown to be very much faster with xanthine than with NADH as electron donor [81]. The relative inefficiency of NADH as a reducing substrate has also been noted in the cases of XOR-catalysed reduction of organic nitrites [86] (see later) and of molecular oxygen [87]. It is interesting to note that isosorbide-2-mononitrate was reduced with 7-fold greater efficiency than was isosorbide-5-mononitrate [81]. This can be rationalised on the basis of the structures of the two isomers in which the nitrate groups of the 2-isomer and the 5-isomer are located exo and endo respectively to the fused bis-pyran ring system (Fig. 2.3).

39

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2 Organic Nitrates and Nitrites

In contrast to inorganic nitrate [88] or nitrite [85], which are reduced at the Mo site of XOR, organic nitrates were shown [81] to be reduced initially at the FAD site of XOR (Fig. 2.4). As discussed above, cytochrome P-450 can catalyse the same reduction and other flavoproteins, as yet unidentified, may well do the same. Indeed organic nitrates have been shown to be reduced by flavins alone [89]. NO3-

Xanthine

NO2Urate

Mo

NO2NO•

Fe2S2 Fig. 2.4 Schematic diagram showing

NADH

FAD

XOR-catalysed reduction of nitrates and nitrites. Organic nitrates are reduced at the FAD site to inorganic nitrite; this can be further reduced to NO at the molybdenum (Mo) site. Organic nitrites are reduced at the FAD site, in this case, Organic nitrates directly to NO. Inorganic nitrates are reduced at Organic nitrites Mo. Adapted from Harrison [73]. NO2NO•

NAD+

Although generation of NO, catalysed by purified XOR in the presence of GTN, is relatively slow [81], O’Byrne et al. [90] were able to demonstrate NO-induced inhibition of platelet aggregation, in platelet-rich human plasma, in the presence of GTN and XOR. The anti-aggregation effect was dose-dependently inhibited by allopurinol, a classic inhibitor of XOR. Bennett and colleagues [91] observed that, in the presence of xanthine, purified XOR catalysed the conversion of GTN to GDN and that this conversion was inhibited by DPI. Although, as noted above, DPI inhibits several flavin-containing enzymes, including XOR, the authors argued against involvement of the latter in GTN metabolism, citing the inablity of a high speed supernatant of rat aorta to biotransform organic nitrates. This argument is based on the premise that XOR is primarily cytosolic and may be questioned in the light of the enzyme’s membrane association [92, 93]. They did, however, also show that organic nitrate-induced aortic relaxation was not inhibited by allopurinol. It is worth noting that S-nitrosothiols, originally implicated by Ignarro and colleagues in the bioactivation of organic nitrates (Scheme 2.1), have been shown to be reduced to NO in the presence of XOR [94]. 2.1.6

Mitochondrial Aldehyde Dehydrogenase

A recent publication from Stamler and colleagues [95, 96] identified mitochondrial aldehyde dehydrogenase (mtALDH) as a key enzyme in GTN metabolism. The au-

2.1 Organic Nitrates

thors focused on consistent evidence that the major dinitrate, arising from GTN bioconversion in smooth muscle, is 1,2-GDN, rather than the 1,3-isomer [11, 97, 98]. A mouse macrophage cell line, displaying this regioselectivity, was used as a source of the relevant enzyme, which proved to be present predominantly in the 100,000 g supernatant fraction; purification led to its identification as mtALDH. GTN-induced generation of 1,2-GDN was confirmed to be localized in the mitochondrial fraction of the macrophage cells and shown to be blocked by known ALDH inhibitors. Purified mtALDH catalysed the stoichiometric formation of 1,2-GDN and nitrite from GTN, in a reaction that was shown to be dependent on the presence of thiols. It was argued that reductive denitration of GTN involves interaction of one of two adjacent active site thiols with GTN to yield an –SNO2 group, which then reacts with the other thiol to yield nitrite and a disulfide. In a final, essential step the inactive disulfide is converted back to two thiols by external thiol compounds (Scheme 2.2). It is interesting to note that this mechanism is essentially that foreseen by Needleman and colleagues in their proposed nitrate receptor (see earlier). SH SH Thi o ls

RONO2

S

+

NO2-

S ROH

Scheme 2.2 Mechanism of reductive

SNO2 SH

denitration of organic nitrates (RONO2 ) to nitrite, catalysed by mtALDH. Adapted from Chen et al. [95].

Regarding the vasculature, treatment of aortic rings with mtALDH inhibitors attenuated GTN-dependent relaxation, formation of 1,2-GDN and accumulation of cGMP. Moreover, when tolerance was induced by pre-treatment with GTN, generation of 1,2-GDN and cGMP were again decreased, as was the activity of mtALDH. In whole animals, ALDH inhibitors were shown to attenuate the hypotensive effects of GTN in rabbits and rats. While the above evidence supports the bioconversion of GTN to nitrite, the mechanism of NO generation is less clear. As noted above, intermediacy of inorganic nitrite in GTN-induced NO production was postulated both by the Ignarro hypothesis (Scheme 2.1) and by studies on glutathione-S-transferase. Arguments against such involvement are largely based on the pharmacological ineffectiveness of nitrite, compared with that of GTN [25, 50, 51]. In addressing this issue, Chen et al. [95] propose that mtALDH-catalysed generation of nitrite directly in the mitochondria would allow further reduction to NO in situ, either by components of the electron transport chain or by disproportionation of nitrous acid (HNO2 ), formed by protonation of nitrite in the intermembrane space. However, detailed arguments against both these possibilities have been advanced by Bennett and coworkers [99]. The latter

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authors also make the point that, in rat and rabbit aortae, the majority of mtALDH is present in the cytosol, rather than in the mitochondria. Moreover, they question the specificity of some of the mtALDH inhibitors used by Chen and colleagues and provide tolerance data (see next section) that are inconsistent with a major role for mtALDH in the vascular biotransformation. 2.1.7

Tolerance

As noted earlier, tolerance toward organic nitrates is an established clinical phenomenon, that can be mimicked in vitro. Early explanations of tolerance focused on depletion of thiol levels, that could be restored by external addition or simply with time. Such explanations fitted neatly with Needleman’s ‘nitrate receptor’ hypothesis [14–17, 100], whereby critical reduced thiol groups are oxidised to inactive disulfides in the course of biotransforming nitrates. Addition of sulfydryl donors, such as dithiothreitol, would then lead to restored activity by reducing the disulfide groups back to active thiols. Very similar arguments apply to mtALDH [95] (see above), in which unknown thiols are seen as performing the same task [101]. The importance of sulfydryl depletion in tolerance has, however, been questioned by Boesgaard et al. [102], who failed to show diminution of sulfydryl levels in tolerant rats. Moreover, Fung and colleagues [13, 103] argue that reversal of tolerance by exogenous thiols does not necessarily support the sulfydryl depletion hypothesis, on the premise that added thiols can react chemically with nitrates, extracellularly, to generate S-nitrosothiols, which are vasodilators in their own right. More recently, Munzel, Harrison and coworkers have proposed the involvement of the renin-angiotensin system in nitrate tolerance [104]. They envisage activation of this system, in response to chronic exposure to GTN, whereby increased levels of circulating angiotensin II lead to protein kinase C-mediated activation of a vascular NAD(P)H oxidase and elevated superoxide levels. The increased superoxide is seen as reacting with GTN-induced NO and so inhibiting vasodilation. This hypothesis is based on data derived from a rabbit model of in vivo tolerance involving transdermal GTN patches. Reduction in vasodilator responses of tolerant aortae was associated with increased vascular superoxide generation that could be inhibited by DPI. This was true of responses to either GTN or acetylcholine and, in both cases, the responses could be restored by a superoxide dismutase analogue [105]. GTN-induced vasodilation in tolerant aorta was increased by removal of the endothelium, a source of superoxide, [105] or by addition of Tiron, a scavenger of superoxide [106]. In contrast, Ratz et al. [91], working with a similar model in rats, failed to reproduce several of the above findings and concluded that, overall, their data were not consistent with increased superoxide production as a causative factor in the reduced relaxation responses of tolerance. On the other hand, Gori and Parker [107] endorse the importance of superoxide in tolerance and go on to propose a ‘unifying hypothesis’, whereby superoxide is seen not only as directly affecting NO levels but also as contributing to other tolerance mechanisms, including sulfydryl depletion (see above)

2.1 Organic Nitrates

and inactivation of enzymes involved in cGMP generation or GTN-NO conversion (see below). Involvement of sGC in tolerance has been favored by other workers. Thus, Axelsson and Anderson [109] found that nitrate tolerance was correlated with reduced cGMP response and an alteration in cGMP turnover. Tolerance-induced changes at this level should affect responses from both organic nitrates and more direct sources of NO. However, evidence both for [109] and against [110–113] cross-tolerance has been reported in different experimental systems. Levels of cGMP can be affected not only by sGC, but also by cGMP phosphodiesterase, and the activity of the latter enzyme has been implicated in tolerance. In this case also, experimental data are contradictory. An inhibitor of the enzyme, dipyridamole, has been shown to improve [114] and not to improve [115] the relaxant effects of GTN in nitrate-tolerant human systems. In this context, it is worth noting that, in their studies of the effects of GTN/cysteine on sGC, Artz et al. [35] observed that GTN alone oxidises and inactivates sGC, suggesting yet another possible contribution to tolerance. In studies of tolerance overall, most experimental effort has focused on the role of down-regulation of enzymic GTN–NO conversion. Hinz and Schroeder [113] examined the phenomenon in cultured cells, which have the advantage, over isolated organs, of maintaining biochemical function over relatively long periods, so allowing recovery from tolerance to be studied. Exposure of the cells to therapeutically relevant levels of GTN led to abolition of GTN-induced cGMP after 5 hours. Sensitivity was restored after 30 hours, but recovery could be blocked by cycloheximide. These findings implicate de novo synthesis or up-regulation of proteins in recovery from tolerance and would be consistent with more than one mechanistic hypothesis. Thus, inactivation of sGC, of anti-oxidant enzymes (such as superoxide dismutase) or of GTN-converting enzymes would all be consistent with the data. However, the authors argued against two of these possibilities. They compared results obtained with either GTN or spermidine NONOate, which spontaneously releases NO without the need for enzymic catalysis. The cGMP response to spermidine NONOate in GTN-tolerant cells was the same as in nontolerant cells. This shows that the effectiveness of NO is unaffected in GTN-tolerant cells, suggesting that neither the activity of sGC nor the levels of superoxide are changed in the tolerant state. The conclusion of these studies was, accordingly, that induction of tolerance involves down-regulation of enzymes catalyzing NO release from organic nitrates. Concerning possible mechanisms of down-regulation of GTN-converting enzymes, Hasegawa et al. [116] addressed this issue in studies of endothelium-denuded rat aortic strips. They found that bioactivation of GTN was impaired by pre-exposure to endogenous NO and proposed that GTN-induced tolerance results from inhibition of the GTN-converting step by NO itself. In view of the possible involvement of XOR in GTN metabolism (see above), it is of interest that this enzyme has been shown to be inhibited by NO [117]. In fact, XOR was progressively inactivated in the course of catalyzing the reduction of GTN [81]. Similar inactivation occurs during the reduction of inorganic nitrite to NO [85]. This latter inactivation was shown to involve a ‘suicide’ reaction within the enzyme-substrate complex, rather than interaction between XOR and newly formed free NO [88]. It is not clear whether inactivation of XOR

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in the course of GTN reduction occurs at the GTN–nitrite or at the nitrite–NO step. A further possibility has recently been introduced by Saleem and Ohshima [118], who report that, under anaerobic conditions, XOR reduces NO to nitroxyl (HNO), which inactivates the enzyme. Whatever the mechanism, the very fact that XOR is inactivated in the course of catalyzing the reduction of organic nitrates would be consistent with its involvement in their bioactivation and in the associated phenomenon of tolerance. It should also be noted that cytochrome P-450 is also inactivated by NO [64, 119] and similar considerations regarding its possible involvement in tolerance apply. In their studies of the flavoprotein inhibitor, DPI, Bennett and colleagues [91, 120] found that DPI inhibited GTN-induced accumulation of cGMP to the same extent in aortae from naïve and from GTN-tolerant animals. They argued that, if inactivation of flavoproteins is a major cause of tolerance, then there should be less scope for further, DPI-induced inactivation in tolerant animals, compared with naïve ones. Their conclusion, accordingly, was that, while flavoproteins may participate in biotransformation of organic nitrates and associated vasodilation, their alteration cannot be the basis for nitrate tolerance. The same group drew similar conclusions from their findings with mtALDH [99]. Cyanamide and propionaldehyde, respectively an inhibitor and a substrate of ALDH, were shown to diminish GTN-induced relaxation to the same extent in aortic tissue from tolerant and non-tolerant animals. They reasoned that inhibition of relaxation caused by cyanamide or propionaldehyde in tolerant aortae could not be via further inhibition of mtALDH and was more likely to result from non-specific inhibition of GTN-induced relaxation. Finally, in this context, it should be noted that Laursen et al. [121] dispute the whole concept that nitrate tolerance is associated with reduced bioconversion of GTN to NO. Using in vivo spin-trapping combined with ex vivo cryogenic EPR spectroscopy, they showed that in vivo bioconversion of GTN to NO was dose-dependently increased during prolonged infusion in GTN-tolerant rats and that GTN-induced levels of NO were similar in tolerant and nontolerant tissues. They confirmed the previous findings of Munzel et al. [105] that removal of the endothelium from isolated aortae of nitrate-tolerant rats improves GTN-mediated vasorelaxation and concluded by supporting the superoxide-based tolerance hypothesis of the latter workers (see above).

2.2

Organic Nitrites

As mentioned earlier, the therapeutic use of organic nitrites [4] actually predates that of organic nitrates [1]. Clinical utilisation of nitrites has, however, been very much less and this is reflected in the relatively sparse attention given to their mechanisms of action. Alkyl nitrites react readily with thiols to form S-nitrosothiols [122], which show biological effects similar to those of NO [11]. Nevertheless, glutathione-Stransferase has been implicated in the metabolism of organic nitrites, via intermediate

2.3 Conclusions

S-nitrosoglutathione [123–125]. Fung and colleagues [126] studied the metabolism of IAN and IBN to NO in vascular smooth muscle cells, and identified major and minor enzymic involvement, associated respectively with the cytosolic and microsomal fractions. The molecular weights of the NO-generating enzymes, determined by radiation inactivation target size analysis, were 263 kDa for the cytosolic enzyme and 79 kDa for the microsomal enzyme. Largely on this basis, it was concluded that the former enzyme was distinct from glutathione-S-transferase; the relationship of the microsomal enzyme with that previously identified as metabolising GTN (see earlier) was deemed to be uncertain. However, the authors went on to describe differences between organic nitrites and organic nitrates in their vascular actions, hemodynamic effects and tolerance [127]. More recently, purified xanthine oxidoreductase (XOR) has been shown to catalyse the reduction of IAN and IBN to NO, in the presence of xanthine under anaerobic conditions [86]. The stoichiometric ratio of NO production to xanthine oxidation (to urate) was 2:1; a result that is predicted on the basis of a two-electron reduction of xanthine to urate and one-electron reduction of organic nitrite to NO. Kinetic analysis showed that NO generation in this system was considerably faster than that from inorganic nitrite [85, 86] or from organic nitrates [81] and that inorganic nitrite is unlikely to be a reaction intermediate. On the basis of these data, XOR would seem to be a strong candidate for involvement in the bioactivation of organic nitrites in vivo. It is tempting to equate XOR, which occurs in both the cytosol and in membranes [73], with the organic nitrite-metabolising cytosolic enzyme identified by Kowaluk and Fung [126]; the molecular weight of XOR (294 kDa) falls within the 95% confidence limits (236–298 kDa) established for their enzyme.

2.3

Conclusions

The hypothesis formulated by Needleman, Ignarro and colleagues (Scheme 2.1) has served as the basis for most subsequent research into the mechanisms by which organic nitrates induce vasorelaxation. In particular, attention has been directed towards enzymes involved in the bioconversion of organic nitrates to NO. In fact, no unequivocal major candidate has emerged. While a number of enzymes, including glutathione-S-transferase, XOR and mtALDH, have been shown to catalyse the conversion of organic nitrates to nitrite, the significance of this is unclear. Not only is the involvement of nitrite as an intermediate in NO generation debatable, but also the means by which nitrite is converted to NO in vivo are unknown. Thus, although Chen and colleagues [95] proposed mechanisms for this conversion within the mitochondria, this was not backed by experimental evidence and has been disputed by others [97]. Purified XOR, on the other hand, is certainly capable of catalyzing the reduction of nitrite to NO [85] but its involvement in the physiological conversion of organic nitrates has not been unequivocally demonstrated. However, much of the above has very recently been thrown into question by Kleschyov et al. [128], who make the case that GTN induces vasorelaxation without

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the intermediacy of NO. They found that pharmacological (nanomolar) concentrations of GTN induced almost complete vasorelaxation in precontracted aortae without increasing basal NO production; significant increases in NO production were observed only at suprapharmacological (micromolar) concentrations. The authors relate these findings to earlier work, in which biphasic concentration–relaxation curves were seen as reflecting the involvement of two different enzyme systems [2]. These comprised a high affinity, low capacity system operating at picomolar/nanomolar concentrations of GTN and a low affinity, high capacity system functioning in the micromolar/millimolar range. Kleschyov and colleagues identified the latter with that involved in NO production from suprapharmacological concentrations of GTN (and, incidentally, also from ISDN, which only has a monophasic concentration–effect curve, and is some two orders of magnitude less potent than GTN as a vasorelaxant). They proposed that pharmacologically relevant concentrations of GTN activate the sGC/dGMP/cGK-1 pathway and induce vasorelaxation without involving NO. Further support for this idea was provided by the observation that production of NO from micromolar concentrations of GTN was the same in GTN-tolerant and nontolerant rats; a finding consistent with that of Laursen et al. [121] (see above). Evidence was provided that mtALDH could be involved in the high affinity pathway, but not in the other. Clearly, many questions remain. If NO is an intermediate in GTN-mediated relaxation, then the enzyme(s) involved in GTN–NO conversion are not identified with certainty. Moreover, several hypotheses, not necessarily mutually exclusive, compete to explain the phenomenon of tolerance. Finally, it is plausibly argued that pharmacologically relevant concentrations of GTN activate the sGC/GK-1 system by some yet unidentified mechanism that does not involve NO. There is still much scope for study.

47

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3

N-Nitroso Compounds Arindam Talukdar, Peng George Wang

3.1

Introduction

N-Nitroso compounds have been known since the preparation of dimethylnitroamine. Ever since the tumorigenic property of this compound was described in 1956 by Magee and Barnes [1], a large number of N-nitroso compounds have been synthesized and tested for carcinogenic activity [2, 3]. Systematic studies on the natural occurrence of N-nitroso compounds are lacking but a few studies have shown that these compounds may occur in certain microorganisms [4, 5] and in a variety of mushrooms [6]. At least one of these compounds, strephozotrin, is a potent carcinogen [7, 8]. N-Nitroso compounds have been found in a large variety of foods and consumer products such as meat [9], beer [10], cosmetics [11], infant pacifiers [12], and drug formulations [13]. Their widespread prevalence can be attributed to the relative ease of formation and to the abundance of their amine precursors in the environment. The occurrence of N-nitroso compounds in these products is of great concern, owing to the carcinogenic properties that many of them exhibit [14]. N-Nitrosodimethylamine (DMN) has been detected in urban air samples [15] and the presence of N-nitroso compounds, tentatively identified as N-nitroso derivatives of some pesticides [16, 17], has been reported in samples from water treatment plants and river water. Nitrosonornicotine has been found in unburned smoking tobacco, chewing tobacco, and snuff [18]. N-Nitroso compounds occur in many operations in the rubber industry. Some nitrosamines (nitrosodiphenylamine, N-N-dinitrosopentamethylenetetramine, polymerized N-nitroso 2,2,4-trimethyl-1,2-dihydroquinoline and N-methyl-N-4-dinitroso aniline) are used as organic accelerators and antioxidants in the production of rubber and often the products are found to be contaminated with such compounds [19]. The majority of the analytical methods for detection of N-nitroso compounds have employed gas chromatography (GC) or liquid chromatography (LC) in conjunction with a thermal energy analyzer (TEA) [20], which relies on the pyrolytic breakdown of N–NO moieties to release the nitrosyl radical. Despite the isolation techniques used, the quantitative determination of N-nitroso compounds requires a concomitant posiNitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

56

3 N-Nitroso Compounds

tive identification of the molecule. For this reason, the preferred method of analysis, gas-liquid chromatography allied to a nitrogen-sensitive detector, must be linked to high-resolution mass spectrometry to confirm the presence of N-nitroso compounds. Results should be considered positive only when or if mass-spectroscopic techniques have confirmed their unequivocal presence. Eisenbrand and Preussman [21] have described a colorimetric technique in which nitrosamines are cleaved to nitrosyl bromide and secondary amines, and the liberated NO+ ion is measured colorimetrically after reacting with N-1-naphthalenyl-1,2ethanediamine. Fine and Rufeh have reported [22] an instrument which is specific to the N-nitroso functional group and is capable of detecting N-nitroso compounds in foodstuffs at the ìg kg−1 level with little or no concentration or purification. Ascorbic acid has been found to be the most effective and useful inhibitor of amine nitrosation [23]. Ascorbic acid inhibits the formation of DMN from oxytetracycline and nitrite, and also from aminophenazone (aminopyrine) and nitrite. Tannins are present in a variety of foods, competing with secondary amines for nitrite and thus leading to a reduction in the amount of nitrosamine formed [24]. Tea exerts a relatively strong inhibitory potency for N-nitroso compound formation both in vitro and in humans. The active constituents may be related to their polyphenols content, especially the tea catechin derivatives [25]. This chapter summarizes the synthesis, physical properties and reactions of Nnitroso compounds [26]. They may be subdivided into four classes: – N-Nitrosamines – N-Hydroxy-N-nitrosoamines – N-Nitrosoimines – N-Diazeniumdiolates

3.2

N-Nitrosamines

Increasing attention is being paid to the chemistry of N-nitrosamines owing to the toxicity, carcinogenic, mutagenic, and teratogenic properties of these compounds [27–29]. In general, N-nitroso compounds can be divided into two different types (Fig. 3.1). Type I includes dialkyl, alkyl aryl and diaryl nitrosamines, while Type II are nitrosamines with an electron-withdrawing group attached to the nitrogen bearing the NO group, e.g. N-nitrosamides, N-nitrosoureas, N-nitrosoguanidines, Nnitrosocarbamates and other N-acyl-substituted N-nitroso compounds. N-Nitroso compounds have been found to exist as syn and anti rotamers [30, 31] due to restricted rotation of the N–N bond resulting from nitrogen lone-pair delocalization (Fig. 3.2). This delocalization causes the hydrogens at the á-carbons to become acidic as evident by their base-catalyzed reactions, such as exchange with deuterium

3.2 N-Nitrosamines

H3C

N

H3C

N O H3C

HO

N

N O

NO N N N O

N N O

H3C

N NO

N H

OH

R

Dephostatin

NO N

NO N ON

O2 N NO2

5

R

N ON N

N

4

3

2

1

NDMA

N

NO

N NO N NO

N

6

8

7

Type I O

O (PhCH2)2N

N

Ph N NO

NO

HO HO

10

9

O S N O NO 12

HN 11

O2 N

O O

OH O

N NO

O 13

H2N

OH N O

NO

N N NO 14

Type II Fig. 3.1 Structure of some N-nitrosoamines amines.

R'

R

O

R'

N

N O R R, R' = alkyl, aryl, COR, CONHR

Fig. 3.2 N–N restricted rotation in N-nitrosamines.

[32], stereoselective á-alkylation with alkyl iodide, and isomerization at the á-position [33]. The á-carbons of nitrosamines undergo enzymatic hydroxylation followed by oxidative cleavage leading to the formation of alkyldiazo hydroxides, alkyldiazonium ions and alkyl cations [34]. These cations are postulated to initiate the process of carcinogenesis in some cases by alkylating the base units of DNA [35, 36].

57

58

3 N-Nitroso Compounds

3.2.1

Synthesis of Nitrosamines

N-Nitrosation of amines is an important and well-established reaction in organic synthesis [2, 3]. N-Nitrosoamines were first reported by Geuther in 1863 [37]. The most general reagent is nitrous acid, generated from sodium nitrite and mineral acid in water or in mixed alchohol–water solvents [38, 39]. Other nitrosating agents: a source of the nitrosonium ion (NO+ ) such as dinitrogen tetraoxide (N2 O4 ), nitrosonium tetrafluoroborate (NO+ BF4 − ) [40], Fremy’s salt, bis(triphenylphosphine)nitrogen(1+) nitrite [41], N-haloamides and sodium nitrite under phase-transfer conditions [42], [NO+ ,Crown,H(NO3 )2− ] [43], oxyhyponitrite [44], dinitrogen tetroxide [45], solid acids (i.e., oxalic acid dehydrate [46], inorganic acidic salts [47] and hydrolysable chloride salts [48]), and sodium nitrite, have also been used. Endogenous nitrosamines are formed by nitrosation of amines in the body, via their acid or bacterial-catalyzed reaction, with nitrite or with oxidative products of NO generated during inflammation or infection [49, 50]. Primary amines react readily with nitrosating agents (Scheme 3.1) to provide deamination products. The intermediates, primary nitrosamines (RNHNO), are not stable; therefore after a series of rapid reactions, they give rise to the diazonium ion (RN2 + ), and then decompose to the final products. The reactions of secondary amines can stop at the nitrosamine stage, since no á-hydrogen atoms are available for the necessary proton transfer reactions, which lead to diazonium ion formation. + Na O N O

HCl

H H O N O +

HCl H O N O

+ N O

+ N O

Nitrosonium ion

NO+ R1 N

R2

NO R1 N R2

(1)

H H .. NH2

R

R

H

H

O H

H

.. +N O

.. N N O + H

R

R

.. .. N N O H H

H O H

.. .. N N O H

O +

H

N-nitrosamine

H+

R

.. N

.. N

(2)

+ OH2

R

+ N

.. N

Scheme 3.1 Formation of nitronium ion and its reaction with primary and secondary amines.

3.2 N-Nitrosamines

Nitrosation of primary amides results in deamination to produce carboxylic acid and nitrogen as products. Secondary amides, when nitrosated, give the corresponding nitrosamides in a reversible process [51]. In order to obtain good yields of the nitrosamides, it is best to add a base to remove the acid formed (Scheme 3.2). This reaction also occurs with ureas and carbamates. NO+

RCONH2

NO+ RCONHR'

RCO2H + N2 + RCONHR' NO

Base

(1)

RCONR'

(2)

NO

Scheme 3.2 Nitrosation of primary and

secondary amides.

3.2.2

Physical Properties and Reactions of N-Nitrosamines

Most N-nitrosoamines are liquids with limited water solubility, except for those with three carbon atoms or less. However, they are soluble in common organic solvents, such as alcohols, ketones, esters and halogenated hydrocarbons. These compounds are relatively stable in aqueous solution at physiological pH, but are light-sensitive. All N-nitroso compounds exhibit ultraviolet absorption in the 230–240 nm and 330– 350 nm regions. In the infrared region N-nitroso compounds show a characteristic but weak band at 1445–1490 cm−1 (N=O stretching) which is different from the analogous band at 1605–1620 cm−1 in C-nitroso compounds and nitrite esters [52, 53]. The N–N bond of N-nitrosamine can undergo two possible modes of cleavage, homolytic and heterolytic cleavage (Scheme 3.3), producing fragments capable of affecting nitrosation [54–56]. The alternative possibility is that direct transfer of the NO group can occur without the intermediacy of a free nitrosating agent formed from the above fragments. This is the basis of the so-called transnitrosation reactions [57] of nitrosamines where a direct transfer of the nitroso group occurs, by attacking a nucleophile at the N-atom. Aromatic N-nitrosamines [54] and aromatic N-nitrosoureas have been demonstrated to undergo homolytic cleavage of the N–NO bond to give NO [58, 59]. Aromatic N-nitrosoureas and N-nitrososulfonamides have also been shown to be heterolytically cleaved to give nitrosonium ion (NO+ ) in solution. Thus, some aromatic N-nitroso compounds can act as donors of NO or NO+ . On the other hand, O

homolysis

N N

O N N

heterolysis

.N O

N. +

.

N. -

Scheme 3.3 Two possible modes of N–NO bond cleavages of N-nitrosamine.

+

+N

O

59

60

3 N-Nitroso Compounds

aliphatic N-nitrosoureas do not release NO, and there has been no report of aliphatic N-nitrosamines that readily undergo N–NO bond cleavage. The most critical parameter to determine their NO-releasing potential is the N–NO bond energy. Studies have shown that the homolytic cleavage of N–NO bonds generating a NO radical, is thermodynamically more favorable than the heterolytic cleavage, generating a pair of ions, by 23.3–44.8 kcal mol−1 [60, 61]. The mechanism for carcinogenisis by N-nitrosamines at the molecular level is probably by á-hydroxylation catalyzed by a variety of oxidases and oxygenases, for example cytochrome P450-related enzymes. Decomposition of á-hydroxy-N-nitroso compounds produces powerful alkylating agents which damage DNA. N-Nitrosamines also have the potential to decompose spontaneously in vivo leading to the formation of alkylating agents as well (Scheme 3.4, Eq. (1)). Aliphatic N-nitroso compounds, such as N-nitrosodimethylamine (NDMA), produce little NO, this in part may be because their N–NO bond dissociation energies (BDE) are relatively high, and therefore there is a preference for dealkylation. Another event, denitrosation, which accounts for 10–20% of total nitrosamine metabolism, is carried out by the same cytochrome P450-related enzyme. After one-electron oxidation of the á-carbon atom, it generates the highly unstable á-nitrosamino radical. This unstable radical intermediate readily releases NO (Scheme 3.4, Eq. (2)) [62]. For example, compounds 2, 3 release NO in the liver and blood with yields of 1% and 0.01% respectively [63]. The nitrosation products of hexamethylenetetramine, 1,3,5-trinitrosoheuahydro-1,3,5-triazine (6) and 3,7-dinitroso-1,3,5,7-tetrazabicyclo[3.3.1] nonane (7), have been shown to form NO at yields of 3.1% or 1.3% in vitro at 37 °C (1 h, pH 7.4), respectively [64]. R' R P450

R' R

R'

OH N NO

+ + R N2

O (1)

N NO

DNA spont.

R'

+ + R N2

DNA Alkylated DNA

OH

R' R

N NO

R' P450

R

N H NO

H R

+ NO N R'

(2)

Scheme 3.4 Mechanism of carcinogenesis by N-nitrosamine.

N-Aryl-N-nitrosamines have higher NO-releasing potential than N-alkyl-N-nitrosamines. This is because the resonance effect between the aromatic ring and neighboring nitrogen increases the NO-generating ability. Electron withdrawing groups on the aromatic ring can weaken the N–NO bond, and enhance the NO-releasing ability. Some derivatives of 2 generate NO spontaneously and are therefore good nitrosating agents [65]. When the resonance effect is weakened by ortho-substituted

3.2 N-Nitrosamines

groups, or when N-nitrosamines have a bulky N-substituted group, such as tert-butyl, the NO-releasing ability diminishes [66]. Type II nitrosamines have two reaction pathways. One pathway involves nucleophilic attack at the carbon of C=O to generate a tetrahedral intermediate which decomposes to an active diazotate ion (R–N=N–O− ). The other pathway involves the nucleophililc attack on the nitrogen of the nitroso group resulting in denitrosation (Scheme 3.5). The nucleophile can be a biologically prevalent thiol, therefore type II compounds are often used as NO donors for the formation of S-nitrosothiols [67, 68].

R Nu ..

a

R' O N n inatio R C Nu + N O deam C R' Oa N b O R' Nu N n i O trosa R C NH + tion b N

Alkylating

O

Scheme 3.5 Two reaction pathways followed by Type II nitrosamines.

In general, type II compounds show greater NO-releasing ability than type I Nnitrosamines. This can be explained by the electronic repulsion between the carbonyl oxygen and nitroso oxygen, or the attraction of the lone-pair electrons at nitrogen, by the carbonyl group; both features weaken the N–NO bond. 3.2.3

Structure–Activity Relationship of N-Nitrosamines

The attempt to rationalize the connection between the molecular structures of organic compounds and their biological activities comprises the field of structure–activity relations (SAR) studies. Correlations between structure and activity are important for the development of pharmacological agents, herbicides, pesticides, and chemical communicants (olfactory and gustatory stimulants) and the investigation of chemical toxicity and mutagenic and carcinogenic potential. Practical importance attaches to these studies because the results can be used to predict the activity of untested compounds. In addition, SAR studies can direct the researcher’s attention to molecular features that correlate highly with biological activity, thus suggesting mechanisms or further experiments. SAR studies have been used to some extent in the pharmaceutical and agricultural industries. The methods are beginning to be applied to the important problems of chemical toxicity and chemical mutagenesis and carcinogenesis. The early structure–activity studies [69, 70] were limited in the number of compounds studied but showed that reasonable correlations could be drawn between the structure of compounds and their biological activity without a complete understanding of the underlying mechanisms involved. Chou and Jurs [71] expanded the approach to structure–activity relationships by applying computer-assisted mathematical and statistical methods to a large set of N-nitroso compounds. These methods

61

62

3 N-Nitroso Compounds

come under the broad heading of pattern-recognition techniques. The software used was developed by Jurs and his co-workers and is referred to as ADAPT [72]. Since physicochemical measurements were not available for such a large set of compounds, computer-generated parameters were used. A recent study of N-nitroso compounds has been presented by Dunn and Wold [73, 74] using a pattern-recognition technique called SIMCA. This method performs principal components analysis for each class of compounds using physicochemical measurements as descriptors. It has been found that a large number of molecular structural descriptors are essential to predict the structure–activity relationship. They are steric, lipophilic, electronic properties of a molecule, molecular connectivity, counts of atoms, bonds, rings etc. have been shown to be highly correlated with a variety of biological activity including mutagenicity. Wishnok and Archerz reported [75], with some degree of confidence, an estimate of carcinogenic activity for 51 N-nitrosamines through structure–activity relationships by correlating the number of carbon atoms with the carcinogenic activity. A year later, Singer, Taylor and Lijinskyzl [76] linked liposolubility with nitrosamine carcinogenicity through quantitative SAR. Wishnok and co-workers later reported [77] a quantitative Hansch–Taft SAR for N-nitrosamine carcinogenicity which demonstrated that variation in carcinogenicity could be correlated with a number of molecular properties. They determined water–hexane partition coefficients and electronic inductive effects of substituents at the á-carbon to be the most important features, which suggested that transport properties were important in carcinogenicity. Wishnok and coworkers [78] have predicted organ specificity using physicochemical properties of N-nitrosodialkyl amines. Partition coefficients, electronic factors, and a measure of steric hindrance gave a near perfect prediction. The carcinogenicity of cyclic N-nitroso compounds varies greatly, depending upon the number and nature of á-substituents [79]. These á-substituents are, in general, forced to occupy axial orientations [80]. As a result, the spatial distance between the nitroso oxygen and the á-hydrogen is expected to increase due to twisting of the ring in order to relax the axial steric strain. This increase in distance would make the intramolecular proton abstraction process difficult. N-nitrosoamines cannot be dissected into active and inactive regions, but they must be considered in their entirety. Therefore, one cannot simply look at the compounds and predict their carcinogenicity because no single region of the structure is responsible for the biological activity [81]. 3.2.4

Application of N-Nitrosamines

Although N-methyl-N-nitrosourea can induce cancer in human beings, its derivatives were found to be potent antitumor agents. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1-(2-chloroethyl)3-(2,6-dioxo-3-piperidyl-1-nitrosourea (PCNU) 1-(2-Choroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea showed antitumor activity by alkylating with DNA [82–84]. N-Nitrosourea-based prodrugs designed to become activated by tumor-associated proteases were found to provide enhanced

3.3 N-Hydroxy-N-nitrosoamines

antitumor activity and reduced systemic toxicity. Tripeptides representing substrates for plasmin were linked by an amide bond to N′-(2-aminoethyl)-N-(2-chloroethyl)-Nnitrosourea and the corresponding N′-Me derivative. Cancer cells expressing high plasminogen activator activity are highly sensitive to these prodrugs in the presence of plasminogen, but not in its absence [85]. The N-methyl-N-nitrosourea derivative streptozotocin is an antibiotic with diabetogenic, carcinogenic and antitumor activity thought to act via alkylation of DNA and proteins. Evidence points to a release of bioactive NO from streptozotocin, which appears to contribute significantly to streptozotocin-induced cytotoxicity. Apparently both the antineoplastic and carcinogenic activities of N-nitrosoureas result from modification of DNA [86]. A stable nitrosoguanidine-induced mutant strain of Streptomyces griseoverticillatus produced a new antibiotic designated tuberactinomycin-N [87]. This compound contained capreomycidine as the guanidine moiety and had stronger antitubercular activity and lower general toxicity in mice and guinea pigs than did tuberactinomycinA. A novel compound designated nitrosostromelin [88], which is represented by the formula EtMeC(OH)(CH2 )8 CH(OH)CH[N(OH)NO] CH2 OH, functional derivatives, and pharmaceutically acceptable salts thereof. Nitrosostromelin inhibits stromelysin and may be useful for the treatment and prevention of rheumatoid arthritis and osteoarthritis. A hybrid drug N-2-chloroethylnitrosoureidodaunorubicin [89] that combines structural and functional features of both anthracyclines and nitrosoureas showed increased antitumor activity with less toxicity than doxorubicin, and its bifunctional properties provide the opportunity for simultaneous treatment of individual cancer cells with two cytotoxic modalities as well as treatment of heterogeneous populations typical of bladder cancers. N-Nitrosamines have been shown to be inhibitors of cysteine-containing enzymes. For example, dephostatin and other N-methyl-N-nitrosoanilines (1) were found to be inhibitors of the protein tyrosin phosphatases, papain and caspase [90, 91]. Inhibition results from the S-nitrosation of the critical cysteine residues in the active sites of the enzymes by the nitrosamines. Compounds 6 and 7 have been found to inhibit thrombus formation in arterioles and venules of rats [92], while N-nitrosamide 9 exhibited vasodilation and mutagenicity as a result of NO release [93]. N-Nitroso-N-cyclohexylhydroxylamine is a synergistic agent for insecticides [94]; it increases the insecticidal activity of chlordane. Certain N-alkylphenylnitrosamines [95] and alkyl N-alkyl-N-nitrosocarbamates [96] are insecticides and fungicides, and may also be used for the impregnation of fabrics, etc. They have larvicidal action against Drosophila melanogaster and can also kill all moth larvae and weevils.

3.3

N-Hydroxy-N-nitrosoamines

N-Hydroxy-N-nitrosoamines also known as ‘nitrosoalkoxyamine’ belong to the class of C-diazeniumdiolate compounds. Several protocols have been used in the past to name compounds containing the diazeniumdiolate functional group. This has not

63

3 N-Nitroso Compounds

64

only hidden their functional group status but has made it extremely difficult to find and interpret literature on the subject. 3.3.1

Biologically Active N-Hydroxy-N-nitrosamine Compounds

The N-hydroxy-N-nitrosamino functionality (Fig. 3.3) occurs naturally in the antibiotics alanosine (17) [97, 98] and dopastin (15) [99]. Alanosine (l-2-amino-3(N-nitrosohydroxylamino)propionic acid), an antibiotic isolated from Streptomyces alanosinicus possesses antiviral and antitumor activity. It is an investigational antineoplastic drug which, as a close analog of aspartic acid, interferes with the aspartate utilizing enzymes of purine and pyrimidine biosynthesis. Enzymatic oxidation of the á-amino group of alanosine to the carbonyl level permits decomposition of the hydroxynitrosamine moiety into nitric oxide (Scheme 3.6, Eq. (1)) in a reaction that resembles the â-decarboxylation of oxalacetate. The nitric oxide free radical is highly reactive and exhibits potent cytocidal, mutagenic, and vasodilatory activities. O N

N

+

H 4 N- O

O-

N

N O O

N

N

O- NH3+ N+ O-

Cupferron (16)

Dopastin (15)

HO

O N

N OH

n-C7H15

+ N H

.

O N

N OH

O + N N OH H3C(H2C)14H2C

22

23

OH O + N

-

N OH CH2OH 25

n-C15H31

O + N

HOOC

N OH

21

20

O + N N OH i-PrH2C(H2C)11H2C

18

NH2

19

(CH2)8

N-O O N N

Alanosine (17)

O

OH

H2 N

O

O

+

+

OAc O + N

O + N N OH i-PrH2C(H2C)9H2C

24

N OH

26

Fig. 3.3 Important N-hydroxy-N-nitrosoamines.

Dopastin is an experimental antihypertensive agent which has been noted to potently inhibit copper-dependent dopamine â hydroxylase [100]. The pharmacological

3.3 N-Hydroxy-N-nitrosoamines 2 e-

O N .. -O

O NH3+

N

COO

N -

.. -O

enzymatic

N

O COO(1)

spontaneous

O

O H2 C

COO-

+ O

N

2O

N.

N

Scheme 3.6 Generation of NO from alanosine.

properties of N-hydroxy-N-nitrosamines have been ascribed to their ability to serve as structural analogs of carboxylic acids [101, 102] and to their ability to chelate metal ions [103]. These features do not involve covalent modification of the hydroxynitrosamino group, which is generally stable under physiological conditions in the absence of enzyme action [104]. Apart from dopastin and alanosine, some other naturally occurring N-oxy-N-nitrosamines have also been isolated. Compound 19 was isolated independently in a soil screen for antibiotics (nitrosofungin) [105] and in a search for a treatment against apple canker (propanosine) [106]. Fragin, 20, was originally isolated as a plant growth inhibitor [107] and has since been found in numerous bacterial cultures [108]. Homoalanosine, 21, was found have herbicidal activity [109]. Nitrosoxacins A, B, C (structures 22–24) were isolated from the soil microbe fermentation and found to be lipoxygenase inhibitors [110], and stromelysin inhibitor nitrosostromelin (25) has been isolated from a related Streptomyces culture [111]. Poecillanosine, 26, isolated from a marine sponge [112], represents a possible new source of naturally occurring diazeniumdiolates. Further work is clearly warranted since these materials show a variety of interesting biological activities including, for example, the ability of alanosine and synthetic analogs to inhibit reproduction of the common house fly [113]. Keefer’s group has disclosed a series of structurally innovative N-oxy-N-nitrosamines such as 17 [114]. These compounds are heat-stable in solution and are slow NO releasers. More importantly, the decomposition to carcinogenic nitrosamines is excluded. The best known member of the synthetic N-nitroso-N-oxyarylamine family is N-nitroso-N-oxybenzeneamine ammonium salt (cupferron, 16) [115], which is commonly used as a metal chelator and as a polymerization inhibitor. Cupferron undergoes thermal dissociation to generate NO. At room temperature, both in the solid state and in solution, it is relatively stable, generating very little NO. However, it can be thermally or photochemically decomposed to an azoxy compound and NO [116]. It was also found that cupferron could release NO under enzymatic [117], electrochemical [118], and chemical oxidation [119] (Scheme 3.7). During the one-electron oxidation step, cupferron is oxidized to the unstable oxy radical, which spontaneously decomposes to nitrosobenzene and NO. Cupferron and its derivatives have a NONO moiety attached directly to carbon instead of an oxygen or nitrogen atom. The advantage of this type of NO donor is that, after NO release, the byproducts can be selected

65

66

3 N-Nitroso Compounds electrochemical O

O N N

O -NH4+

N N

-1e-

O

O N N

.

. . O + NO

oxy radical

enzymatic

Scheme 3.7 Electrochemical or enzymatic generation of NO from cupferron.

to be noncarcinogenic [120]. Fine tuning of the NO donor structure allows preparation of novel donors with specific targeting effects. Ortho-substituted derivatives of cupferron are good donors both in vitro and in vivo. These compounds show faster decomposition rates than cupferron because the ortho-substitution prevents the NONO moiety from becoming planar [121]. Para-substituted cupferrons constitute a set of redox-sensitive NO donors. These compounds release NO via spontaneous dissociation during one-electron oxidation. Electron-withdrawing groups can increase the oxidation potential and make NO release easier. The instability of cupferron and its ortho- and para-substituted derivatives can be a liability in the pharmaceutical realm where targeted delivery is crucial to the success of NO donor drug efforts. In general, the preferred substituent groups attached to –N(O− )NO are á-naphthyl, ortho-substituted phenyl or heterocyclic aromatic rings. These groups can also be biologically active moieties, such as progesterone, estrogen, epinephrine or other catecholamines, which can be designed to target the NO-releasing agent to a specific organ or tissue [122]. 3.3.2

Synthesis of N-Hydroxy-N-nitrosamines

The syntheses of N-hydroxy-N-nitrosamines are usually carried out by the nitrosation of the corresponding N-hydroxyamines (Scheme 3.8) [123, 124]. N-Hydroxyamines are readily obtained by the reduction of the corresponding nitro-compounds. The most efficient methods are neutral or basic reactions. Recent applications of this method have resulted in the preparation of a variety of cupferron derivatives (Scheme 3.8) via nitrosation of phenylhydroxylamine with amyl nitrite/ammonia [125] or methyl nitrite/ammonia [126]. Behrend and Konig have shown that the organic

NO2 R

O- NH4+ N NO

NHOH EtONO

Zn NH4Cl

NH3

R

O

NHOH

+ NaNO2

R

-

N N OH

HCl

Scheme 3.8 Synthesis of cupferron derivative.

3.3 N-Hydroxy-N-nitrosoamines

nitrites can also be used under acidic conditions to produce the “free acid” (O2 protonated) form of the diazeniumdiolate directly [127], although this is usually accomplished via reaction with acidified inorganic nitrite and demonstrated by the preparation of cupferron [128]. Acidified nitrite can successfully produce diazeniumdiolates in the presence of free hydroxyls [129] and primary amines [130]. Nitrosyl chloride and nitrosylsulfuric acid can also serve as the nitrosating agents [131]. The nitrosation of oximes can also serve to produce N-hydroxy-N-nitrosamines (Scheme 3.9). The acidified nitrite procedure can be applied to oximes derived from terpenes [132] and hydroxyguanidine [133] for the synthesis of N-hydroxy-Nnitrosamines. However, some á,â-unsaturated oximes were converted into pyrazole1,2-dioxides [134]. The alkyl nitrites under basic conditions have produced interesting results. Quinone dioximes yielded only monodiazeniumdiolates [135], while simple aliphatic oximes gave products resulting from addition to the imine double bond [136]. O-

H2N H 2N

HONO

+

+

NOH

N N O-

RHN

RHN

-

OH

O

N O- Na+

+N

N i-PrONO NaOMe

N

N O

OH

Scheme 3.9 Nitrosation of oxime and dioxamines.

The reaction of NO with a wide variety of enamines, derived from the respective aldehydes and ketones can produce N-hydroxy-N-nitrosamine (Scheme 3.10) [137]. These compounds are relatively stable to thermal decomposition and do not release copious volumes of NO upon adding to concentrated mineral acids. However, on dissolution in aqueous buffer at pH 7.4 and 37 °C, a slow release of NO could be detected by the chemiluminescence method. O N

O NO

N N

N

O

OScheme 3.10 Reaction of enamine with NO.

The reaction of NO with a variety of compounds containing acidic protons in the presence of strong base was described long ago (Scheme 3.11, Eq. (1)) [138]. This work was largely ignored as a preparative method until a Russian group extended the methodology for the preparation of diazeniumdiolates of malonate esters (Scheme 3.11, Eq. (2)) [139, 140] and a variety of ketones [141, 142].

67

68

3 N-Nitroso Compounds

O

NO

+Na -O

NaOMe

O

ON

N

+

-

N

C

N

O- Na+

O NaOMe +

Na -O

N

O N

O

-

+

-

(1)

N

O- Na+

N

C H2

+ CH3COOMe

31

MeOOC MeOOC

CH2

NO

MeOOC

NaOMe

MeOOC

O CH + N

N

O- Na+

(2)

32

Scheme 3.11 Reaction of nitric oxide with carbanion.

BHT (2,6-di-tert-butyl-4-methylphenol), a phenolic antioxidant, on reaction with NO under neutral conditions, results in scavenging of the potentially harmful NO via radical reactions [143]. Sodium phenolate under basic conditions undergoes a Traube-type reaction at the ortho-position to produce a cupferron derivative [144]. When the ortho-positions are sterically blocked and the para-position does not bear a proton, cyclohexadienone diazeniumdiolates may be formed (Scheme 3.12) [145]. An alternative method includes the reaction of Grignard reagents with NO followed by treatment with ammonia or other bases. ONa t-Bu

O t-Bu

NO

t-Bu

t-Bu Scheme 3.12 Synthesis of

N2O2- Na+ cyclohexadienone C-diazeniumdiolate.

3.3.3

Properties of N-Hydroxy-N-nitrosamines

The diazeniumdiolate functional group is a monobasic acid (Scheme 3.13), which is also unstable in acidic solutions. This has created some difficulties in measuring its properties. The aromatic compounds (cupferron analogs) have pKa values between 3.5 and 4.4 in water solutions [146], while the pKa values of aliphatic derivatives range from 5.1 for nitrosofungin [147] to 6.4 for fragin (20) [148]. N-Hydroxy-NO N R

-

O

NaOH

+

N N

OH

R

+ N

Scheme 3.13 Monobasic nature of

O- Na+ C-diazeniumdiolate functional group.

3.3 N-Hydroxy-N-nitrosoamines N X

O

N+ O

N N X

X

O

28

27

N

O

OR

N + O

N N X

O OR Fig. 3.4 Resonance forms and tautomers of the

29

30

diazeniumdiolate functional group.

nitrosamines are relatively stable to thermal decomposition and do not release copious volumes of NO upon adding to concentrated mineral acids. The anionic form can exist as a hybrid of resonance forms 27 and 28 (Fig. 3.4) [137]. Many X-ray crystallographic studies support structure 27 as the predominant determinant of its physicochemical properties. The X-ray crystal structure study has shown that the –N2 O2 − functional group is essentially planar and that the oxygens are in the syn configuration (i.e., on the same side) with respect to the diazene double bond, an arrangement designated the Z isomer by IUPAC rules [149]. This is the bonding which gives rise to the group’s name “diazen” representing the N=N linkage and “ium” the formal positive charge, and “diolate” includes the two negatively charged oxygens. The point of attachment to the additional substituent (X) is considered as position1, so the full name becomes “diazen-1-ium-1,2-diolate” for the anions. Substitution on the oxygens gives rise to either the O2 -substituted diazeniumdiolates (29) or the O1 -substituted diazeniumdiolates (30). There has been an upsurge in the synthesis of anions with the X[N(O)NO] (X = O− , CR , SO− , and NR2 ) functional group as a probe for studying the biology of nitric oxide (NO) and nitroxyl (NO− /HNO). Nuclear magnetic resonance (NMR) spectroscopy of diazeniumdiolates has rarely received attention. Aliphatic protons on the carbon to which the N2 O2 group is attached are shifted downfield by 2–3 ppm, as might be expected by analogy with the carboxylates [150] and the fact that carbon generally appears at 58–62 ppm in carbon NMR spectra [151] if no other influences are present. Most useful is a study of the 15 N NMR spectra of several diazeniumdiolates including C-, N-, O-, and S-bound compounds [152]. 14 N NMR spectroscopy may also be useful since the N-oxide nitrogen signal of O2 -alkylated diazeniumdiolates has been reported to be relatively sharp (ca. –65 to –70 ppm relative to nitromethane) [153]. The 15 N chemical shifts of the N–O1 nitrogens of several selectively labeled analogs are reported to be 320– 360 ppm relative to ammonia [154]. Additional analytical studies of great interest but limited scope include photoelectron spectroscopy [155], EPR [156], polarography [157], and cyclic voltammetry [124].

69

70

3 N-Nitroso Compounds

3.3.4

Reactivity of N-Hydroxo-N-nitrosamines

N-Hydroxy-N-nitrosamines with an aliphatic group at O2 produce a compound stable to aqueous acid and base (Fig. 3.4, 29) [158], whereas all other N-hydroxy-Nnitrosamines are susceptible to hydrolysis and appropriate O2 -derivatives also render these materials vulnerable. The hydrolysis endpoint is the formation of nitroxyl (HNO) [which dimerizes to form nitrous oxide (N2 O)] and a C-nitroso compound. These products are formed from aryl [159] and alkyl bound unsubstituted diazeniumdiolates as well as O1 -alkylated derivatives [160]. Studies of the solvolysis of O1 -alkyl derivatives are complicated by their tendency to decompose via competing radical pathways [161], but the O1 -benzyl derivatives are unique in that they hydrolyze back to the original synthetic precursors (Scheme 3.14) [162]. p-MePh

N

OCH2Ph

p-MePhCH2NHOCH2Ph Scheme 3.14 Reaction

H+

of O1 -benzyl derivative in presence of aqueous acid.

+

N O

HONO

The mode of decomposition of O1 -alkylated diazeniumdiolates bound to carbonyls is also exceptional because they can undergo rearrangement to hyponitrites before decomposition (Scheme 3.15) [163]. Scheme 3.15

O

R

O t Bu

O t Bu

N

O

N R

O

O

N

H2O

N

-N2

Decomposition of O1 -alkylated RCOO tBu diazeniumdiolate bound to carbonyl group. O

O2 -Tosyl diazeniumdiolates are quite stable and are the most studied derivatives [164]. Solvolysis in sodium methoxide solution generates the alkylated C-diazeniumdiolate (Scheme 3.16), while under more neutral conditions a reaction analogous to that of the acyl compounds occurs. The added stability of the tosylates has enabled labeling studies, which have shown that these reactions are probably initiated by migration of the acyl and tosyl groups to O1 . O N R

+ N

OTs

O

MeO-

N R

+ N

OCH3 Scheme 3.16 Alkylation of N-hydroxy-N-nitrosamine.

Stevens [164] standardised the preparation of azoxy compounds from the O2 tosylated acyl diazeniumdiolates. The reaction of Grignard reagents with O2 -alkyl diazeniumdiolates was found to produce azoxy compounds (Scheme 3.17), although radical side reactions can sometimes interfere in some solvents [165, 166]. The carbon-bearing diazeniumdiolate functional group is also a potential nucleophilic site since both the unsubstituted and alkylated versions of this functional

3.3 N-Hydroxy-N-nitrosoamines

O N

-

O

+

OTs

N

RMgX

N

+

R

N

+

TsOMgX

Scheme 3.17 Reaction of Grignard reagent with O2 -alkylatedcompound.

group stabilize a negative charge on an adjacent carbon. Thus, protons on carbons adjacent to diazeniumdiolates are acidic and in the presence of base these sites can be alkylated [139, 140], halogenated [167, 168], and cyanated [168] (Scheme 3.18).

+

-

Na O

N

ON

O C H2

NaH OMe

+

Na -O

N

ON

O OMe

ClCN CN

Scheme 3.18 Reaction of C-diazeniumdiolate as a nucleophile.

Carbanions derived from diazeniumdiolates can also undergo the Knoevenagel [169] (Scheme 3.19, Eq. (1)) and Michael reactions [170] (Scheme 3.19, Eq. (2)). Under suitable conditions, â-substituted diazeniumdiolates can undergo elimination to yield products derived from the olefin [171] and, in a related reaction, bis(diazeniumdiolates) can undergo elimination of one N2 O2 R group to produce olefins [172]. NaOMe

PhCHO + CH2(N2O2Me)2

CH2(N2O2Me)2

PhCHOHCH(N2O2Me)2

CH2=CHCN

(NCCH2CH2)2C(N2O2Me)2

KOH

(1)

(2)

Scheme 3.19 Knoevenagel and Michael reactions.

Hou et al. developed a method that controlled the generation of a nanomolar amount of NO [173]. A self-assembled monolayer of N-nitroso-N-oxy-p-thiomethylbenzeamine ammonium salt bound to a gold electrode via a thiol linkage was used for the reaction. When an electric potential was applied, one-electron electrochemical oxidation led to the release of NO (Scheme 3.20). There was a linear relationship between the amount of NO generated and the area of the electrode, indicating that the amount of NO release could be controlled by selecting an appropriately sized H4 N+ O -

N

NO

H4 N+O -

O

NO

N e-

Au

HS

N

S Au

+ S

Au

NO

Scheme 3.20 Generation of

nanomolar amounts of NO.

71

72

3 N-Nitroso Compounds

electrode surface area. This approach may be used in microelectrode arrays for biochemical applications. Most of the C-diazeniumdiolates are not NO donors since they hydrolyze to produce nitrous oxide directly [174]. However, it has been found that carefully selected compounds can serve as NO donors under physiological conditions via alternative reaction pathways. Many cupferron analogs release NO via enzymatic oxidation [175] as do O1 -alkylated diazeniumdiolates [176]. Several novel types of NO-releasing Nhydroxy-N-nitrosamines have been prepared. These new preparative methods have been described in earlier sections. The precursors are enamines (Scheme 3.10), phenolates (Scheme 3.12), nitriles, and N-hydroxyguanidines (Scheme 3.9).

3.4

N-Nitrosimines

An abundance of research has been reported on the carcinogenic potential of Nnitroso compounds in recent years. Historically, nitrosimines were studied because they were potential carcinogens [177, 178]. Most nitrosimine type NO donors are heterocyclic compounds. They are the N-nitrosation products of their corresponding imines. Several types of nitrosoimines have been reported in the literature, such as 1,3-disubstituted nitrosiminobenzimidazoles (33) [179], 1,3,4-thiadiazole2-nitrosimines (34) [180], benzothiazole-2(3H)-nitrosimines (35) [181], thiazole-2nitrosimines (36) [182], oligonitroso sydnonimines (37) [183], 3-alkyl-N-nitroso-sydnonimines (38) [184, 185] and 2H-1,3,4-thiadiazine nitrosimines (39) [186] (Fig. 3.5). These nitrosimines have the ability to inhibit platelet aggregation in vitro. Some of them also exhibit antithrombotic and blood pressure lowering abilities in vivo [187, 188]. R N

R2 N NO

N

N

N N R

R

33

O

S

N _ +

NR RN

ON N

N _ +

O N NO

37

N NO

N

N

N R

1

34

S

R3

S

NO

R

1

R

NO

2

36

35

S

R N _+ N O 38

R1 N NO

N NO N N R

2

39

Fig. 3.5 N-Nitrosoimines.

Among all the reactions of nitroso compounds the most widely studied has been homolysis to generate NO [189]. However, other reactions are known; for example, N-nitrosoaziridenes lose N2 O to form alkenes in a cheletropic fragmentation [190]. In contrast, N-nitrosimines (Scheme 3.21) spontaneously lose nitrogen rather than

3.4 N-Nitrosimines

NO [177], resulting in the corresponding ketone. Loss of nitrogen has been shown to be quantitative. It has been documented that nitrogen evolution was shown to proceed via first-order kinetics (t1/2 =4.75 h) for 40 with R1 =4-MePh and R2 =2-MePh. Steric bulk and conjugation stabilize nitrosimines. For example, 40 with R1 =tertBu and R2 =2-MePh is stable for weeks, but no dialkylnitrosimines are known. The 3-substituted 2-nitrosiminobenzothiazonines (e.g., 35a) are among the more stable derivatives known and require heating, for example in refluxing methanol, for deazetization. R1 R2

N

N O

-N2

R1 R2

40

O Scheme 3.21 Loss of nitrogen resulting in corresponding ketone.

Nitrosimines should be stored at lower temperature and protected from light. They are relatively stable when dissolved in aqueous buffers. They release both NO and N2 O, the latter via a nitroxyl intermediate (HNO). In the presence of thiols, formation of N2 O increases at the expense of NO production, which suggests that NO release involves an oxidative mechanism. However, nitrososydnonimines can release up to two moles of NO per molecule, one from the nitrosimine and another from the resulting sydnonimine. 3.4.1

Mechanism of Thermal Reaction of N-Nitrosoimine

Nitrosoimines can undergo thermal reaction, a unimolecular, two-step mechanism has been proposed, as shown in Scheme 3.22 [193]. In this mechanism, a concerted electrocyclization is envisioned to form the strained four-membered ring in 41, followed by a presumably forbidden, but highly exothermic, deazetization to give 41. The electrocyclic ring closure is, at first glance, a 4-electron process, analogous to the cyclization of butadiene [194] or acrolein [194, 195]. This would be expected to involve rotation around the C=N bond coupled with C–O bond formation. N O

S N N R 35

-N2

S N N O R

N

S O N R

41

a R = Me c R = Pr e R = Bn b R = Et d R = Ph

Scheme 3.22 Thermal reaction of benzothiazole-2(3H)-nitrosimines.

Photolysis of the nitrosimines, such as 35a, gives rise to a variety of products that appear to have come from loss of NO and subsequent radical reactions (Scheme 3.22) [196]. Similar products, indicative of radical reactions, are also observed in the thermolysis of sterically hindered nitrosimines (e.g., 40, with R1 = tert-Bu and R2 = 2-MePh) [197]. Steric constraints were proposed to disfavor the cyclic pathway

73

74

3 N-Nitroso Compounds

(Scheme 3.22), thus diverting the reaction to the radical pathway. The differences in the products observed in Scheme 3.22 as compared to Scheme 3.23 require that the two mechanisms be different and are consistent with a concerted reaction for the thermal deazetization of unhindered nitrosimines. N O

S

hν

S N

N N R

MeOH

N R

.

S

.

N R

CN

35

S NH N R

SH CN N R

Scheme 3.23 Thermal

S N R

CN 2

deazetization of unhindered nitrosimines.

Nitrososydnonimines and thiazole-2-nitrosimines are also susceptible to photolytic cleavage of the C=N–NO bond. The corresponding sydnonimine salts are formed at 37 °C in aqueous solution in 90% yield. At higher temp (70 °C), ring opening is observed. In methanol solution about 25% of sydnones are obtained. It is interesting to note that the formation of N2 O from nitrososydnonimine is increased up to 11-fold by the addition of glutathione, while the amount of NO is decreased. In the presence of light and thiols, soluble guanylate cyclase (sGC) is activated. These observations suggest that the nitroxylate anion NO− might play an important role in the stimulation of sGC [187]. 3.4.2

Properties of N-Nitrosoimines

The tendency of the N-nitrosoketimines to decompose to the corresponding ketones parallels the propensity of the parent ketimines toward hydrolysis, which in some cases is effected merely by atmospheric moisture. In each case, resistance to decomposition is enhanced by the presence of bulky groups. It was surprising, then, to discover that a given N-nitrosoketimine is transformed to the corresponding ketone as readily in the absence of, as in the presence of, moisture (e.g., under nitrogen, in the solid state and in a dry solvent; at reduced pressure; and on a column of silica gel during attempted chromatography). Furthermore, when highly stable N-nitrosimine was allowed to stand at room temperature in water for several days, it was recovered virtually unchanged, while heating a carbon tetrachloride solution of the same compound on a steam bath caused total decomposition to the ketone within 30 min. This is not to deny that hydrolysis may be a competing reaction, especially for the less stable N-nitrosimines. That the above decomposition is not hydrolysis was indicated by a kinetic study; appropriate plots of nitrogen evolution vs. time indicated that the reaction is first order [198]. The fact that nitrogen gas is the other product of this decomposition was proved by infrared and mass spectrometry. Numerous attempts to react N-nitrosoimines with a variety of halides produced either unchanged starting materials or the ketone, the latter being formed by loss of nitrogen from the nitroso

3.5 N-Diazeniumdiolates

compound. However, reaction of N-nitrosoimine with triethyloxonium fluoroborate produced an epoxide in some cases, presumably by combination of the intermediate diazomethane (or the carbene derived from it) with transiently formed acetaldehyde. Three of the possible structures contributing to the resonance of N-nitrosoketimines are shown in Fig. 3.6. None of the physical measurements essential for the determination of their true structure has yet been made. Still, some evidence is provided by their chemical properties and physical characteristics. The individual N-nitrosoketimines vary widely in stability [198]. R

R R,

NN

O

R,

I

R +

+ N NO

R II

,

N NO III

- Fig. 3.6 Three of the possible structures contributing to the resonance of N-nitrosoketimines.

3.4.3

Synthesis of N-Nitrosoimines

A series of 3-substituted 2-nitrosiminobenzothiazolines (35a–e) as well as the disubstituted analog 35f were prepared (Scheme 3.24) [198]. The reaction of an arylamine hydrochloride with potassium thiocyanate gave the corresponding unsymmetrical thiourea [199]. Oxidative cyclizations with bromine provide the iminobenzothiazolines [191, 200], which on treatment with sodium nitrite in acetic acid afforded the nitrosiminobenzothiazoline (35a–f). The nitrosiminobenzoselenazoline (42) was similarly prepared. 1. Et2O R1CN + R2MgBr

2. MeOH

R1 R2

NOCl NH CCl4

R1

N N O

R2

Scheme 3.24 Synthesis of N-Nitrosimines by nitrosyl chloride.

Ketimines were successfully nitrosated by treatment with nitrosyl chloride in cold carbon tetrachloride resulting in the formation of N-nitrosoimines (Scheme 3.25) [201]. In an alternative method Zimmermang [202] independently synthesized (Scheme 3.26) three very stable, highly hindered N-nitrosoketimines by the action of dinitrogentetroxide on the ketimines.

3.5

N-Diazeniumdiolates

N-diazeniumdiolates are an interesting class of compounds, presently under development, which can deliver NO specifically to a target site. Diazeniumdiolates are NO nucleophile complexes capable of releasing NO in an aqueous environment or in

75

76

3 N-Nitroso Compounds

S Ph

H

N

SCN-

Ph

HCl

R

MeO

N

NH2

Br2

S NH N R

R

N O

S

Se

N N CH3

NaNO2

S

HOAc

N R

N O N

N O N

N R 42

Scheme 3.25 Synthesis of nitrosoiminobenzothiazoline.

N2O4 Mes2C

NH

NaOAc, CCl4

Mes2C

N N O Scheme 3.26 Synthesis of N-nitrosodimesitylimine with dinitrogen tetroxide.

response to a shift in the local pH [203–205]. The first reported N-bound diazeniumdiolate was the NO adduct of diethylamine (DEA/NO, Scheme 3.27) commonly known as ‘Drago complex’ [206] after Russell Drago, who synthesized it back in 1960. Since these materials are thus over 100 years “younger” than their C-bound analogs, there is less chemistry reported. Furthermore, they were virtually ignored until this past decade when people realized the potential of N-diazeniumdiolates for dependable and site-specific NO donor features [207, 208]. 2

NH +

2NO

+ N N

OScheme 3.27 NO adduct of

+ diethylamine, commonly called N O Et2NH2 ‘Drago Complex’.

N-diazeniumdiolates spontaneously dissociate at physiological pH to release nitric oxide (NO) by stable first order kinetics with half-lives ranging from 2 s to 20 h [209, 210]. They are blessed with many attributes that make them an especially attractive starting point for designing solutions to important clinical problems, namely they are stable as solids, have structural diversity, a controlled rate of release of NO on hydrolysis, and a rich derivatization chemistry that facilitates targeting of NO to specific sites of need, a critical goal for therapeutic uses of a molecule with natural bioeffector roles in virtually every organ [208]. 3.5.1

Mechanism of NO Release

The mechanism of NO release from N-diazeniumdiolates is depicted in Fig. 3.7. If R3 of the generic structure shown at the top is a cation, NO is generated spontaneously on protonation of the anionic portion along with the formation of dialkylamine. If R3 is covalently bound, it must be removed first to free the anion before spontaneous

3.5 N-Diazeniumdiolates

R1

+ N N

R2

N OR3 If R3 is covalently bonded

If R3 is ionically bonded

metabolism and/or hydrolysis

+ H R1 N H +

O-

2NO

+ H

R2

R1

+ N N

R2

OFig. 3.7 Mechanism of NO

- release for the N O N-diazeniumdiolates.

NO generation can begin. Some examples of masking the diazeniumdiolate ion in this way for pharmacological advantage are listed in Fig. 3.9 below [208]. 3.5.2

Synthesis of N-Diazeniumdiolates

The N-diazeniumdiolate class of compounds with the general structure R1 R2 N– [N(O)NOR3 ] have been prepared by exposing primary, secondary, and polyamines to nitric oxide (NO). The Drago complex can be synthesized by the reaction of diethylamine (DEA) with NO (Scheme 3.28). They are in general less stable than their carbon analogs, and so have never been isolated in their protonated forms. Soon after their initial synthesis, it was discovered that the ammonium salts are quite hygroscopic and that the sodium salts, which were prepared either by metathesis [211] or directly by the use of added base [212] in the reaction mixture, were more stable.

H2 N

H N

NO

NH2 H 2N

O O + N N

N

-

Scheme 3.28 Reaction of NO with amine to synthesize NH2 diazeniumdiolate.

The enormous diversity of isolable materials that can be synthesized by varying R1 and R2 of Fig. 3.7 can be extended even further by covalently binding different R3 groups to the terminal oxygen of the ionic diazeniumdiolate moiety. All three R groups of Fig. 3.7 can be varied over a wide range to produce isolable materials. Salts in which R3 is ionically bound to the diazeniumdiolate oxygen have proven especially useful for generating controlled fluxes of NO spontaneously in aqueous media. This can lead to a variety of well-defined outcomes. For example, when R3 is a methoxymethyl (MOM) group and R1 R2 N is a piperazine ring, the diazeniumdiolate (MOM-PIPERAZI/NO; Fig. 3.9) is still a spontaneous NO releaser at physiological pH, but the rate-limiting step is hydrolytic cleavage of the MOM group;

77

78

3 N-Nitroso Compounds

in this case, the MOM derivative’s half-life for NO generation is 17 days [213]. Most other diazeniumdiolates with covalently bound R3 groups have proven effectively stable toward spontaneous hydrolysis, but by choosing R3 groups that are vulnerable to cleavage by specific enzymes, agents capable of cell- or organ-selective NO generation after system-wide administration can be prepared. Structures of some Ndiazeniumdiolates are shown in Figs. 3.8 and 3.9. Based on this N-diazeniumdiolates can be divided into two groups: ionic and O-derivatized N-diazeniumdiolates. H2 N +

H2 N

N O

H2 N

NH2

+N -

N O

+ NH3

N +N O

O

t1/2 = 5-46 min

-

N

-

-

O

+N O

N

-

O

t1/2 = 20 h

-

t1/2 = 1 min

H N

- + COO Na N +N

+ NH2 Me

N

H -

N

N +N O

-

N

-

O Na+

N +N O

-

O Na+

t1/2 = 2 s

-

N

N +N O

N O Na+

-

O Na+

t1/2 = 2 min

t1/2 = 5 min

t1/2 = 3 s

Fig. 3.8 Structure and half-life of ionic N-diazeniumdiolates.

NO2

-O

N +N

N

N +N O

N

O

NO2 O

O

N +N N O

O O

(liver selective NO donor; activated by cytochrome p450)

(can be activated by nucleophilic aromatic substitution reaction)

(Cell-permeanent NO donor; activated by esterase)

H N

OH

N -

N +N O

-O N

O

O

(spontaneously hydrolyzes; half-life 17 days at pH 7.4 and 37 0C)

Fig. 3.9 Structure of O2-substituted N-diazeniumdiolates.

O

+N

OH

N O

OH OH

(Generates NO after activation by glycosidases at pH 5.6)

3.5 N-Diazeniumdiolates

3.5.2.1

Ionic Diazeniumdiolates

These are the most common diazeniumdiolates, formed by the reaction of secondary amines and polyamines with nitric oxide in basic media [214, 215]. They are stable solids, capable of regenerating two equivalents of nitric oxide along with the starting amine in neutral or acidic buffers. The half-life of NO generation varies from a few seconds to many hours, depending on the amine. The decomposition to NO is a spontaneous, first-order reaction at constant pH. 3.5.2.2

O-derivatized Diazeniumdiolates

The diazeniumdiolate anions react with electrophiles to produce stable covalent compounds (Fig. 3.9) [213, 216]. These compounds have the ability to act as prodrugs, releasing nitric oxide only when metabolically or enzymatically converted to the diazeniumdiolate anion [217–219]. Several compounds of this class have been synthesized by reaction of alkyl or aryl halides, sulfate esters, epoxides, etc. with the ionic diazeniumdiolates [220, 221]. 3.5.3

Reactions of N-Diazeniumdiolates

Unlike the C-diazeniumdiolates, the N-diazeniumdiolates have not been induced to form stable O1 -derivatives. The O2 alkylation of the N-diazeniumdiolates may be accomplished using simple alkyl halides (Scheme 3.29), epoxides, alkyl sulfates, and aryl halides [220, 221]. The compounds are generally stable, and many have been prepared with the goal of using the alkyl group as a protecting group that can be removed selectively to regenerate the N-diazeniumdiolate [213]. R1 R2

+ N N

ON O

R 3X

R1 R2

+ N N

OScheme 3.29 Alkylation of

N OR3 N-diazeniumdiolates.

While direct alkylation has been used to introduce a variety of substituents at O2 to modify the NO-releasing properties of molecules containing the diazeniumdiolate functional group, it has also proven possible to perform synthetic manipulations on several already existing O2 -substituted compounds without disturbing the diazeniumdiolate group (Scheme 3.30). Halogens on side chains can undergo nucleophilic displacement by amines [220] or thiolates [222]and can be dehydrohalogenated [220]. N-Diazeniumdiolates can also undergo some reactions other than those directly associated with NO release. They act as bidentate chelators and form complexes with a variety of transition metal ions. Suitable choice of the ligands can lead to the formation of complexes in which the diazeniumdiolates act as a monodentate ligand coordinated to Cu(II) at O2 only [223].

79

80

3 N-Nitroso Compounds

O N

+ N

Et2N pyr O N

O

-

N

Et2N

N

OCH2CH2Br

MeNH2 O N

Br

+ N

OCH CH2

N

OCH2CH2NHMe

N

OCH2CH2SAc

+

Et2N

Et3N

+ -

N

-

Et2N

NaOH

+

O

MeC(O)SH O N Et2N

+

Scheme 3.30 Reaction at O2 without disturbing the diazeniumdiolate group.

The N-diazeniumdiolates are quite photosensitive. Studies of various O2 -substituted compounds, both alkyl and aryl, revealed a primary photochemical reaction involving cleavage of the N=N bond to yield a nitrosoamine and an O-substituted nitrene which rearranges to a C-nitroso compound (Scheme 3.31), the latter is often isolated as the oxime [224]. + N N

O-

hν

N OR

Et2NNO

+

RNO Scheme 3.31 Photochemical reaction involving cleavage of the N=N bond.

3.5.4

Clinical Applications 3.5.4.1

Reversal of Cerebral Vasospasm

Subarachnoid hemorrhage is a life-threatening disorder caused by the rupture of an intracranial aneurysm present in the brain. Unfortunately, during surgery it is difficult to remove all of the excess blood from the cranium before closing the surgical opening. Any residues of blood can predispose the affected artery to undergo uncontrolled, spastic contractions. The resulting perturbations in cerebral blood flow can produce serious neurological deficits, even death if not stopped immediately. It was thought that the hemoglobin in the clot surrounding an artery induces these spasms by robbing the muscle controlling the vessel diameter of the endogenous NO (called endothelium-derived relaxing factor) that it needs to remain properly relaxed,

3.5 N-Diazeniumdiolates

thereby permitting contracting factors to run amok. On administration of various Ndiazeniumdiolates into the carotid artery of an animal subject, a complete reversal of the spastic condition was immediately observed. If clinical trials, now in the planning stages, are successful, a considerably improved prognosis could become available for the several thousand individuals who are affected by cerebral vasospasm each year [225–228]. 3.5.4.2

Treatment of Impotency

Millions of men worldwide suffer from an inability to achieve and maintain an erection sufficient for the completion of sexual intercourse. A few years ago, it was discovered that NO is a critical physiological effector of penile erection; by inducing relaxation of the corpus cavernosum, NO allows blood to engorge the penis and maintain its tumescent state. Hellstrom and colleagues [229] found that the diazeniumdiolates both increased the length of the penis and raised the pressure within the corpus cavernosum when administered transurethrally under experimental conditions [230]. It is not often that current research is featured in a popular novel. Carl Djerassi [231] wrote in 1998 a ‘science-in-fiction’ book, NO, where the principal character, Renu Krishnan, a molecular biologist, discovers that salts formed by reacting certain nucleophiles with the bioregulatory agent nitric oxide (NO) can regenerate pharmacologically active NO on contact with body fluids and can treat erectile dysfunction. 3.5.4.3

Nonthrombogenic Blood-contact Surfaces

Many devices (e.g., biosensors, extracorporeal membrane oxygenation equipment, heart-lung bypass machines, and renal dialysis systems) when coming into contact with blood can cause unwanted clot formation. To minimize this thrombogenic foreign-body response, antiplatelet agents such as heparin, coumadin, and aspirin are often used. However, systemic administration of such agents concomitantly increases the risk of uncontrolled bleeding elsewhere in the body. One way to prevent this potentially life-threatening side effect is to coat the foreign body with an immobilized drug that limits antiplatelet action to the contact surface between the blood and the device. NO technology offers an attractive way to accomplish this because NO is a potent inhibitor of platelet adhesion, aggregation, and activation [232, 233]. The chemically versatile N- diazeniumdiolates can incorporated into blood-insoluble polymers that generate molecular NO with very short half life. 3.5.5

Future Directions

Targeted delivery is critical to the success of future NO/donor drug discovery efforts. The goal must be to provide quantities of NO, through local administration

81

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3 N-Nitroso Compounds

to the specific site(s) of need, that are adequate to achieve the desired physiological effect without disturbing the many other NO-sensitive portions of the anatomy [234, 207]. However, many organs, tissues, and cell types are difficult or impossible to access by local administration, in such cases research efforts are aimed at the development of prodrugs. Many N-diazeniumdiolates are being examined as candidate prodrugs for chemotherapeutic treatment for drug-resistant tumors, reducing the risk of restenosis after coronary angioplasty, killing intracellular parasites, and inhibiting metastasis.

83

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The Role of S-Nitrosothiols in the Biological Milieu Bulent Mutus

Some 30 years after the discovery of NO as a signaling molecule we are still in the dark about many aspects of its biochemistry, cell biology and physiology. The following is the mythical fate of the enzymatically generated NO, subscribed to by the author of this chapter: while some fraction of NO remains unchanged in blood most of it is converted rapidly to S-nitrosothiols (RSNOs) or to higher oxides of nitrogen in hydrophobic cellular components. These “NO-equivalents” are transported throughout the body by mechanisms that are largely unknown. At selective interfaces, the NO-equivalents transfer their “NO” to protein thiols giving rise to RSNOs and affecting protein structure function in a similar manner to phosphorylation-mediated signal transduction. The S-nitrosated proteins are then denitrosated by chemical or enzymatic means to resting levels, to await the next wave of nitrosation-mediated signaling. This chapter summarizes the current state of knowledge on the role of S-nitrosothiols in mammalian systems under the following headings: Structure and cellular reactivity of RSNOs; formation of RSNOs in the biological milieu; and physiological role of RSNOs.

4.1

Structure and Cellular Reactivity of RSNOs 4.1.1

RSNO Structure

The S–N=O bond is labile and as a result energetic parameters related to the S–N bond have relied on computational investigations (Barberger et al., 2000; 2001; Fou et al., 2002; de Oliveira et al., 2002; Lu et al., 2001). The predicted S–N and N–O bond lengths from one such investigation (Baciu and Gauld, 2003) are presented in Fig. 4.1.

Nitric Oxide Donors Peng George Wang, Tingwei Bill Cai, Naoyuki Taniguchi (Eds.) c 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Copyright
ISBN: 3-527-31015-0

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H

H

H

1.843 S

N 1.168

C

H

S N 1.793

O Fig. 4.1 Schematic illustration

with selected bond lengths, of the lowest energy conformations of the S-nitrosothiols trans-HSNO

O 1.181

and cis-CH3 SNO obtained at the B3P86/6-311+G(2df,p) level of theory (Baciu and Gauld, 2003).

The in vitro RSNO stabilities appear to depend on the structure of the sulfur substituent. In general, reported RSNO stabilities follow the trend R=3°C>R=2°C>R =1°C. Substituent-mediated homolytic mechanisms for RSNO decomposition have been implicated. However, a recent computational study (Barberger et al., 2001) has discounted the homolysis-route for S–NO decomposition since it was determined that bond dissociation enthalpies (BDEs) activation parameters were essentially independent of substituent structure (31–32kcalmol−1 ) and predicted to be too high to occur at physiologically relevant temperatures. For example, the predicted half-life with respect to homolytic S–N cleavage was 2.1years at 37°C. The decomposition of these compounds was then tested experimentally. The RSNO decomposition was rapid and followed zero-order kinetics and, contrary to other reports, (Gow et al., 1997; Singh et al., 1996) thiols stabilized RSNO decomposition and changed the decomposition kinetics to first order, with NO and disulfide as the only products. The authors concluded that bimolecular, processes such as RSNO-thiol adducts, RSNO dimerization and catalytic processes involving metal ions, various oxygen species, and enzymes could be responsible for RSNO-decomposition and reactivity in vivo. 4.1.1.1

Enzymatic Consumption of RSNOs

The enzymes superoxide dismutase (Jourd’heuil et al., 1999; Johnson et al. 2001; Romeo et al., 2003), xanthine oxidase (Trujillo et al., 1998) protein disulfide isomerase (Ramachandran et al., 2001; Rautri and Mutus, 2004), thioredoxin reductase (Nikitovic and Holmgren, 1966; Jensen et al., 2001) ã-glutamyl transpeptidase (Askew et al., 1995; Lipton et al., 2001) glutathione-dependent formaldehyde dehydrogenase (GDFDH) (Liu et al., 2001) have been shown to metabolize RSNOs in vitro. A potential in vivo role for RSNO catabolism has only been demonstrated with GDFDH in yeast, mice and E. coli. In the wild type of these organisms RSNOs are found in the protein fractions with trace amounts in the cytosol. However, in knock-out organisms made deficient in GDFDH cytosolic both protein-RSNO and cytosolic-RSNO levels were elevated (Liu et al., 2001).

4.1 Structure and Cellular Reactivity of RSNOs

4.1.2

Formation of RSNOs in the Biological Milieu 4.1.2.1

Nitrite Mediated

The principal in vitro route to the formation of RSNOs is through the reaction of nitrous acid or protonated nitrite (HNO2 ) with thiols (Eq. (1)). Since the pKa of HNO2 is 3.37, this reaction is unlikely to occur in cells and tissues where the pH is maintained at 7.4. (1) HONO + RSH → RSNO + H2 O However, this reaction can and has led to errors in the measurement of RSNOs in biological fluids when the samples are improperly buffered to avoid the HNO2 -route to S-nitrosation (Tsikas, 2003). Enzymatic conversion of nitrite to RSNOs has been reported. Glutathione-Stransferase catalyzed generation of RSNOs from organic nitrites was initially demonstrated in rat liver microsomes (Ji et al., 1996). Subsequently, this activity has been identified in the rat heart and lung GSTs (Akerboom et al., 1997). 4.1.2.2

NO Mediated

NO reacts directly with thiols in vitro to yield RSNOs only in the absence of oxygen (Gow et al., 1997). Therefore, this reaction, Scheme 4.1, is unlikely to occur in biological systems. 4.1.2.3

NO Oxidation Products Mediated

The in vivo RSNO-producing reactions resulting from NO oxidation and related products are summarized in Scheme 4.1. The reaction between NO and O2 giving rise to N2 O4 [a] is third order and, as a result, highly concentration dependent. NO and O2 are low polarity compounds that are more soluble in hydrophobic compartments of cells, such as membranes and protein hydrophobic domains, than in the aqueous phase (Wink et al., 1993; Lui et al., 1998; Nedosparov et al., 2000). NO2 • formed from the homolysis of N2 O4 [b], reacts with NO giving rise to N2 O3 [c] a powerful S-nitrosating agent. Hydrophobic phase, N2 O3 could then selectively S-nitrosate thiols in the proximity of the hydrophobic–aqueous interface [d]. N2 O3 in aqueous environments is rapidly hydrolyzed to nitrite. N2 O3 -dependent S-nitrosation has been demonstrated in the case of serum albumin (Nedospasov et al., 2000; Rafikova et al., 2002), in membranes (Liu et al., 1998) and in the protein-disulfide isomerase dependent transfer of NO-equivalents from extracellular RSNOs to the cytosol (Ramachandran et al., 2001).

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•

[b’]

N=O + O=O

Scheme 4.1

Other postulated routes (Jourd’heuil et al., 2003) to RSNO formation include the reaction between NO and O2 to yield NO2 • via a second-order reaction. NO2 • and thiolate anion, RS− , react giving rise to thiyl radical, (RS• ) [e]. RS• then reacts with NO to yield RSNO [ f ]. The reaction between RS• and RS− can also be the source of non-enzymatic generation of superoxide anion (O2 •− ) [g], [h]. O2 •− reacts with NO to produce peroxynitrite (ONOO− ) [i] (Szabo, 2003). Thiols react with ONOOH to form RSNOs [k] (van der Vliet et al.,1998). Intracellular and extracellular RSNO metabolism has been studied in LPS activated macrophages (Zhang and Hogg, 2003). This study showed that ∼0.02% of the NO produced in response to LPS, (detected as NO2 − ) was converted to cytosolic RSNOs and that all of the RSNOs detected were