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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Div. Chem. Sci. (Engl. Transl.) 35 (1985), p. 2125 Woodward, R. B., Wintner, C., Tetrahedron Lett. (1969), p. 2689 Marchenko, G. A., Mukhametzyanov, A. S., Tselinskii, I. V., Zh. Org. Khim. 21 (1985), p. 1426; J. Org. Chem. USSR (Engl. Transl.) 21 (1985), p. 1297 Marchenko, G. A., Mukhametzyanov, A. S., Tselinskii, I. V., Ermoshkin, A. S., Zh. Org. Khim. 21 (1985), p. 1429; J. Org. Chem. USSR (Engl. Transl.) 21 (1985), p. 1300 Zyusin, I. N., Izv. Akad. Nauk, Ser. Khim. 47 (1998), p. 1263; Russ. Chem. Bull. (Engl. Transl.) 47 (1998), p. 1231 Hou, Y., Chen, Y., Amro, N. A., Wahu-Mesthrige, K., Andreana, P. R., Liu, G.-Y., Wang, P. G., Chem. Commun. 19 (2000), p. 1831 Bhat, J. I., Clegg, W., Maskill, H., Elsegood, M. R. J., Menneer, I. D., Miatt, P. C., J. Chem. Soc., Perkin Trans. 2 (2000), p. 1435 Kubrina, L. N., Yakubovich, L. M., Vanin, A. F., Izv. Akad. Nauk SSSR, Ser. Biol. (1988), p. 844; Biol. Bull. Acad. Sci. USSR (Engl. Transl.) (1988), p. 533 Vanin, A. F., Vedernikov, Y. P., Galagan, M. E., Ignatov, S. M., Kubrina, L. N., Malenkova, I. V., Mordvintsev, P. I., Kostyanovskii, R. G., Izv. Akad. Nauk SSSR, Ser. Biol. (1991), p. 136; Chem. Abs. 114 (1991), p. 223269t Challis, B. C., Challis, J. A., N-Nitrosimines and N-Nitrosoimines Vol. 2, Ed. S. Patai, S., John Wiley and Sons, New York 1982, p. 1151 Olszewska, T., Milewska, M. J., Gdaniec, M., Malusznska, H., Polonski, T., J. Org. Chem. 66 (2001), p. 510 Rehse, K., Birkhofer, G., Arch. Pharm. 328 (1995), p. 77 Rehse, K., Ludtke, E. Arch. Pharm. 328 (1995), p. 17 Rehse, K., Ciborski, T., Ludtke, E., Arch. Pharm. 327 (1994), p. 771 Rehse, K., Ludtke, E., Arch. Pharm. 327 (1994), p. 581

183 Rehse, K., Schleifer, K. J., Martens,

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A., Kampfe, M., Arch. Pharm. 327 (1994), p. 393 Rehse, K., Schleifer, K. J., Arch. Pharm. 326 (1993), p. 929 Rehse, K., Kampfe, M., Schleifer, K. J., Arch. Pharm. 326 (1993), p. 483 Rehse, K., Brummer, U., Unsold, E., Pharmacology, 53 (1998), p. 820 Rehse, K., Schleifer, K. J., Ludtke, E., Bohme, E., Arch. Pharm. 327 (1994), p. 359 Rehse, K., Schleifer, K. J., Ciborski, T., Bohn, H., Arch. Pharm. 326 (1993), p. 791 Zhu, X.-Q., He, J.-Q., Li, Q., Xian, M., Lu, J., Cheng, J. P., J. Org. Chem. 65 (2000), p. 6729 Clark, R. D., Helmkamp, J. Org. Chem. 29 (1964), p. 1316 Besthorn, E., Chem. Ber. 43 (1910), p. 1519 Thoman, C. J., Hunsberger, S. J., Hunsberger, I. M., J. Org. Chem. 33 (1968), p. 2852 Bartsch, R. A., Chae, Y. M., Ham, S., Birney, D. M., J. Am. Chem. Soc. 123 (2001), p. 7479 Woodward, R. B., Hoffmann, R., The Conservation of Orbital Symmetry, VCH, Weinheim 1970 Yu, H., Chen, W.-T., Goddard, J. D., J. Am. Chem. Soc. 112 (1990), p. 7529 Akiba, K., Fukawa, I., Mashita, K., Inamoto, N., Tetrahedron Lett. (1968), p. 2859 Jappy, J., Preston, P. N., Tetrahedron Lett. (1970), p. 1157 Thoman, C. J., Hunsberger, M., J. Org. Chem. 33 (1968), p. 2852 Fairful, A. E. S., Peak, D. A., J. Chem. Soc. (1955), p. 796 Tsuda, K., Fukushima, S., J. Pharm. Soc., Jpn. 62 (1942), p. 64 Paskovich, D. H., University of Ottawa, personal communication (1963) Zimmerman, H., Paskovioh, D. H., J. Am. Chem. Soc., 86 (1984), p. 2149 Hrabie, J. A., Klose, J. R., Wink, D. A., Keefer, L. K., J. Org. Chem. 58 (1993), p. 1472

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K. W., Wink, D. K., Methods Enzymol. 268 (1996), p. 281 Maragos, C. M., Morley, D., Wink, D. A., Dunams, T. M., Saavedra, J. E., Hoffman, A., Bove, A. A., Isaac, L., Harbie, J. A., Keefer. L. K., J. Med. Chem. 34 (1991), p. 3242 Drago, R. S., Paulik, F. E., J. Am. Chem. Soc. 82 (1960), p. 96 Keefer, L. K. CHEMTECH 28 (1998), p. 30 Keefer, L. K., Annu. Rev. Pharmacol. Toxicol. 43 (2003), p. 585 Davies, K. M., Wink, D. A., Saavedra, J. E., Keefer, L. K., J. Am. Chem. Soc. 123 (2001), p. 5473 Gow, A. J., Thom, S. R., Brass, C., Ischiropoulos, H., Microchem. J. 56 (1997), p. 146 Drago, R. S., Karstetter, B. R., J. Am. Chem. Soc. 83 (1960), p. 1819 Longhi, R., Ragsdale, R. O., Drago, R. S., Inorg. Chem. 1 (1962), p. 768 Saavedra, J. E., Booth, M. N., Hrabie, J. A., Davies, K. M., Keefer, L. K., J. Org. Chem. 64 (1999), p. 5124 Smith, D. J., Chakravarthy, D., Pulfer, S., Simmons, M. L., Hrabie, J. A., Citro, M. L., Saavedra, J. E., Davies, K. M., Hutsell, T. C., Mooradian, D. L., Hanson, S. R., Keefer, L. K., J. Med. Chem. 39 (1996), p. 1148 Pulfer, S. K., Ott, D., Smith, D. J., J. Biomed. Mater. Res. 37 (1997), p. 182 Hrabie, J. A., Saavedra, J. E., Roller, P. P., Southan, G. J., Keefer, L. K., Bioconjugate Chem. 10 (1999), p. 838 Saavedra, J. E., Billiar, T. R., Williams, D. L., Kim, Y.-M., Watkins, S. C., Keefer, L. K., J. Med. Chem. 40 (1997), p. 1947 Liu, J., Saavedra, J. E., Lu, T., Song, J-G., Clark J., et al., J. Pharmacol. Exp. Ther. 300 (2002), p. 18 Foley, R. D. P., Quarfordt, S. H., Saavedra, J. E., Keefer, L. K., et al., Transplantation 71 (2001), p. 193 Saavedra, J. E., Dunams, T. M., Flippen-Anderson, J. L., Keefer, L. K., J. Org. Chem. 57 (1992), p. 6134

221 Saavedra, J. E., Srinivasan, A.,

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Bonifant, C. L., Chu, J., Shanklin, A. P., Flippen-Anderson, J. L., Rice, W. G., Turpin, J. A., Davies, K. M., Keefer, L. K., J. Org. Chem. 66 (2001), p. 3090 Saavedra, J. E., Shami, P. J., Wang, L. Y., Davies, K. M., Booth, M. N., Citro, M. L., Keefer, L. K., J. Med. Chem. 43 (2000), p. 261 Schneider, J. L., Halfen, J. A. Jr., Young, V. G., Tolman, W. B., New J. Chem. 22 (1998), p. 459 Srinivasan, A., Kebede, N., Saavedra, J. E., Nikolaitchik, A. V., Brady, D. A., Yourd, E., Davies, K. M., Keefer, L. K., Toscano, J. P., J. Am. Chem. Soc. 123 (2001), p. 5465 Magson, I. L., Drugs of the Future 25 (2000), p. 701; Pluta, R. M., Oldfield, E. H., Boock, R. J., J. Neurosurg. 87 (1997), p. 746 Qureshi A. I., Suarez, J. I., Bhardwaj, A., Yahia, A. M., Tamargo, R. J., Ulatowski, J. A., Crit. Care Med. 28 (2000), p. 824 Wolf, E. W., Banerjee, A., Soble-Smith, J. Jr., Dohan, F. C., White, R. P., Robertson, J. T., J. Neurosurg. 89 (1998), p. 279 Tierney, T. S., Clatterbuck, R. E., Lawson, C., Thai, Q.-A., Rhines, L. D., Tamargo, R. J., Neurosurgery 49 2001, p. 945 Hellstrom, W. J. G., Wang, R., Champion, H. C., Sikka, S. C., Keefer, L. K., Doherty, P., J. Androl. P-33 (1997) Wang, R., Domer, F. R., Sikka, S. C., Kadowitz, P. J., Hellstrom, W. J. G., J. Urol. Baltimore 151 (1994), p. 234 Djerassi, C., NO, University of Georgia Press, Athens 1998 Hanson, S. R., Hutsell, T. C., Keefer, L. K., Mooradian, D. L., Smith, D., J. Adv. Pharmacol. 34 (1995), p. 383 Espadas-Torre, C., Oklejas, V., Mowery, K., Meyerhoff, M. E., J. Am. Chem. Soc. 119 (1997), p. 2321 Zapol, W. M., Hurford, W. E., New Horiz. 1 (1993), p. 638

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4

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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4 The Role of S-Nitrosothiols in the Biological Milieu

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).

93

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4 The Role of S-Nitrosothiols in the Biological Milieu

•

[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 in the large molecular weight (>3K) protein fraction and were very stable to denitrosation (t1/2 ∼3h). These authors also showed that the molecular specie(s) responsible for S-nitrosation is freely diffusible and has to be transported to the cell surface before internal S-nitrosation could take place. In a recent comprehensive study, Feelish and coworkers (Bryan et al., 2004) determined the concentrations of RSNOs, N-nitrosamines (RNNOs) NO2 − , NO3 − , heme nitrosyl (NO-heme) in plasma, RBCs, as well as brain, heart, liver, kidney, lung, and aortic tissues of rats. Furthermore, the levels of these analytes were monitored under conditions of eNOS inhibition, hypoxia and redox state. The emerging picture was that RSNOs were detected in all of the tissues examined in comparable levels to NO-

4.1 Structure and Cellular Reactivity of RSNOs

heme species which are known to turn on guanylate cyclase and turn off cytochrome c oxidase and cytochrome P450 activities. The RBCs contained the most RSNOs (∼250nM). The next largest RSNO concentration was in aorta (∼100nM) followed by liver and kidney (∼35nM), heart and lung (∼8–10nM) and plasma (∼1nM). In all of these tissues, the RNSO were associated with the protein fraction. Upon eNOS inhibition, [RSNOs], [RNNOs], and [NO3 − ], underwent an initial transient increase followed by depletion. The transient increase was explained as follows: as NO levels fall subsequent to eNOS inhibition and approach [O2 •− ], ONOO− will be formed under diffusion controlled rates (Scheme 4.1 [i]) which can lead to rapid RSNO formation (Scheme 4.1 [k]) or decompose to yield NO2 • plus OH• or be converted to NO3 − . NO2 • can yield RS• (Scheme 4.1 [e]) which in turn can react with NO to generate RSNOs (Scheme 4.1 [ f ]). The depletion of most of the NO-related metabolites indicated that these species are dependent on eNOS-generated NO. Another fascinating finding of this study was the rapid potentiation of RSNO formation accompanying hypoxia. The concentration of RSNOs in the brain increased by ∼250% 10min after a hypoxic episode. The conclusion of the authors was that these responses were too rapid to occur via the N2 O3 route (third-order process) but instead could only take place via the peroxynitrite- NO2 • route that generates RS• (Scheme 4.1 [e]) which can directly react with NO to form RSNOs. This study is the first to demonstrate that S-nitrosation takes place in all tissues and cells and is a dynamic process that can occur in vivo at similar rates to heme nitrosation. In addition, RSNOs were shown to be as labile as heme-nitrosyl species in that they were rapidly formed and destroyed in response stimuli such as hypoxia and tissue redox status. 4.1.2.4

Metalloprotein Mediated

The multi-copper carrying enzyme ceruloplasmin (CP), found in large amounts in liver and nervous tissues, has been shown to convert NO to RSNOs. The proposed mechanism involves the binding of NO to the CP type I Cu-sites. The NO is then oxidized to NO+ and transferred to RS− giving rise to RSNO (Innoue et al., 1999). Perhaps the most physiologically important metalloprotein to be S-nitrosated by NO is hemoglobin (Hb). The S-nitrosation of Hb-Cys 93 by NO was first demonstrated by Stamler and coworkers in 1996 (Jia et al., 1996; Stamler et al., 1997). To this day HbSNO remains controversial with respect to its mechanism of formation and physiological relevance. The wealth of data on HbSNO formation has given rise to two broad interpretations that either support the hypothesis that the S-nitrosation of Hb-Cys 93 is redox catalyzed by the heme and is under allosteric control or that it is N2 O3 or NO2 • -mediated without the involvement of the hemes. The main point of argument with the formation of HbSNO is that in vitro the exposure of NO to oxyhemoglobin (HbFe(II)-O2 ) results in the production of methemoglobin (HbFe(III)) plus nitrate (Eq. (2)). NO + HbFe(II)-O2

→

HbFe(III) + NO3 −

(2)

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4 The Role of S-Nitrosothiols in the Biological Milieu

Since the rate constant for this reaction is estimated to be ∼9×107 M−1 s−1 (Eich et al., 1996; Herold et al., 2001) the contention is that this reaction would predominate in vivo, thus minimizing the formation of HbSNO. In the following section the evidence for and against a role of the heme in HbSNO formation is discussed. In their initial report, Stamler and co-workers (Jia et al., 1996) showed that exposure of Hb to NO resulted in the S-nitrosation of C93 in the â-chains. The reported yield upon exposure of Hb to 10-fold molar excess of NO, was 1mol S–NO per tetramer. Given that there are 2 C 93/tetramer this is a 50% yield. Hb-SNO formation was accompanied by the conversion of oxyHb (HbFe(II)O2 ) to metHb (HbFe(III)). Hbâ C93 S-nitrosation could also be accomplished by exposure to RSNOs (GSNO or CysNO). The rates RSNO-dependent Hb-S-nitrosation was ∼10-fold larger in oxyHb than in deoxy-Hb. Conversely, the rate of spontaneous decay of deoxy-Hb-SNO was ∼20-fold larger than oxy-Hb-SNO. An explanation for this differential reactivity was presented in a subsequent study (Stamler et al., 1997) where protein modeling data based on the X-ray structures of Hb in T and R states indicated that in OxyHb the SNO of Cys â 93 is protected from solvent. In contrast, in deoxyHb the SNO is highly exposed to solvent. The implication was that the NO+ on Cys â93-S–NO could be transferred to thiols in RBC and eventually effluxed to induce vasodilation under conditions of low O2 saturation. Gow and Stamler (Gow and Stamler, 1998) looked more carefully at Hb-SNO chemistry under more physiological conditions i.e. at NO to Hb ratios ranging from 1:100 to 10:1. They reported that at NO:Hb ratios of ∼1:20 NO is bound to the hemes. As O2 is slowly introduced the NO is transferred from the Heme to the âC93 thiol and O2 Hb-SNO is converted to the R state. They also presented spectral evidence that in the absence of oxygen, at low NO:Hb ratios (<1:20) the major product is nitrosyl Hb, whereas metHb is produced at intermediate NO:Hb ratios (1:20 to 1:2) and nitrosylHb is once again produced at ratios of >1:2. The authors proposed that when Heme-Fe(II)-NO is in close proximity to âC93-SH, NO is directly transferred to yield SNO• H. They further proposed that O2 would then act as an electron acceptor to yield RSNO plus superoxide. Heme-Fe(II)-NO + C93 SH •

C93 -SNO H + O2 +

⇔ →

C93 -SNO• H HONS-C93 + O2

(3) •−

(4)

Alternatively they suggested that NO might be transferred from the heme to the C93SH. Interestingly, the maximum yield of Hb-SNO formed in this study was <17% of the total â-subunit thiols. The Stamler group (Gow et al., 1999) then proposed that the estimated ∼1% of the O2 -vacant hemes in the R-state could be the sites of S–NO catalysis. The implication of this was that NO would not be consumed (via Eq. (2)) since oxygen is absent from these sites. The evidence presented to support this hypothesis was mainly from EPR data on heme-Fe(II)-NO formation yields as a function of NO added to Hb that had varying saturations of O2 . The EPR data indicated that in the T-state heme nitrosylation was competitive with heme oxidation (via Eq. (2)). However, in the R-state, at low O2 saturation (<20%), heme nitrosylation displayed cooperativity.

4.1 Structure and Cellular Reactivity of RSNOs

EPR data also showed that in the R-State metHb formation reached a saturating value of ∼40% of total at large O2 -saturation (40–80%). When ∼1ìM NO was introduced to oxy Hb (∼50ìM) ∼400nM HbSNO was detected. This corresponds to 0.5% of the Hb â C93 being S-nitrosated. The authors then treated RBC under normoxic conditions (∼99% saturation) with 1ìM NO. The [HbSNO] isolated under these conditions was ∼100nM i.e. ∼10% of the NO added. Given that the mean [Hb] in RBCs is ∼25ìM (Gow et al., 1999) or 50ìM in free thiol this corresponds to 0.02% of Hb â C93 being S-nitrosated. The NO binding to Hb oxygenated to various degrees, was also studied with EPR and UV/Vis spectroscopy by Huang and coworkers (2001). Contrary to the results of Gow et al. (1999), NO binding did not show cooperativity under conditions stabilizing either the R-state (0.01M phosphate) or the T state (0.1M phosphate), both with Hb or with whole blood. Liao’s group (Han et al., 2002) compared the reaction of bolus NO with free Hb and RBCs. Upon exposure of NO donors (DEA-NO) to cell-free oxyHb only MetHb was detected. However, with bolus NO both MetHb and nitrosylHb were detected. The amount of nitrosylHb decreased with increasing O2 saturation. These authors proposed that NO first forms MetHb at the NO–oxyHb interface, converting the latter to MetHb. MetHb is then reduced to deoxyHb and further reacts with another NO to yield nitrosylHb (Eq. (2), (5) and (6). NO + Hb-Fe(III)

→

Hb-Fe(II) + NO+

(5)

NO + Hb-Fe(II)

→

Hb-Fe(II)-NO

(6)

The proof of this hypothesis was that CN, which binds tightly to MetHb, resulted in no detectable nitrosylHb. The fact that HbSNO was still formed even with the CN− -blocked Hb suggested that HbSNO could be formed in a heme-independent manner. In RBC suspensions, under the same experimental conditions, a ∼2-fold larger amount of nitrosylHb was detected in comparison to free oxyHb but no HbSNO was detected. Again the nitrosylHb-formation was attenuated with CN− . In the experiments of Gow et al. (1999) NO was added in bolus. Lancaster and colleagues (Joshi et al., 2002) argued that substantial oxidative reactions leading to the production of nitrosating agents such as N2 O3 or NO2 • , may occur before the bolus NO solution is dispersed. They suggested that the HbSNO were likely formed from N2 O3 or NO2 • rather than by a heme-catalyzed mechanism. To test this they repeated the experiments of Gow et al. (1999) but NO was introduced either by bolus NO addition or by NO-donors which liberate NO uniformly throughout the solution. The results obtained were that when NO donors were employed the only product observed was methemoglobin; whereas bolus addition to Hb produced several unidentifiable species as well as nitrite, suggesting that the N2 O3 route was operative. Interestingly, both methods of NO-introduction produced ∼50nM per 50ìM Hb (or 100ìM in reactive free thiol) which corresponds to ∼0.05% of Hb â C93 being S-nitrosated, 10-fold lower yield than reported by Gow et al. (1999). In early 2003 Stamler and Singel and coworkers (Luchsinger et al., 2003) proposed a MetHb intermediate route for HbSNO formation. This mechanism was very similar to that proposed by Han et al. (2002). In this study, they presented EPR evidence that

97

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that at low NO/heme ratios (0.05–0.75) the predominating reactions at the â subunits were HbSNO (Eq. (7)]) and nitrosylHb, with ∼88% of the nitrosylHb (Eq. (8)) ending up on the â subunits. NO + Hb-Fe(III)

Hb-Fe(II) + NO

→

Hb-Fe(III)NO

↔

Hb-Fe(II)NO+ + â C93 S−

→

Hb-Fe(II) + â C93 SNO

(7)

→

Hb-Fe(II)NO

(8)

At larger NO/heme ratios (>2) the predominant reaction was nitrite plus nitrosylHb (Eq. (9)) which was equally distributed amongst the á and â subunits NO + Hb-Fe(III)

→

Hb-Fe(III)NO

↔

Hb-Fe(II)NO+ + OH−

→

Hb-Fe(II) + NO2 −

(9)

So what should we make of the published data? It is clear that free Hb gets nitrosated at â C93 residues. As Herold and Rock (2003) found out in their comprehensive study, the â C93 S-NO yield is dependent on: the volumes of the NO and Hb solutions, the rates of mixing of these solutions; and the concentrations of phosphate ion and NO. As they point out, their data indicate that â C93 S-NO can be formed by one of three chemical pathways: the N2 O3 route at large [NO]; the NO2 • route at low [NO]; and the Hb-Fe(III) route (Eqs. (5) and (7)). In addition to these routes English and coworkers (Romeo et al., 2003) have demonstrated that commercial preparations of human erythrocyte Hb contained complexed superoxide dismutase (SOD) monomer. They also showed that only the Hb:SODmonomer complex was able to transfer NO from GSNO to â C93 without having it react with the oxyheme via the postulated mechanism in Eqs. (10)–(12). oxyHb-SH + Cu2+ ZnSOD 1+

↔

oxyHb-S• + Cu1+ ZnSOD + H+

(10)

+

↔

GSH + NO + Cu ZnSOD

(11)

•

↔

oxyHb-SNO

(12)

GSNO + Cu ZnSOD + H

NO + oxyHb-S

2+

The implication of these observations was that these reactions take place in isolation “within an oxyHb-SOD encounter complex” channeling the released NO directly to the â C93 -thiyl radical. In this manner, NO will not be consumed by reacting with oxyHb and producing NO3 − (Eq. (2)). 4.1.2.5

Transnitrosation

Transnitrosation is defined as the transfer of NO (NO+ ) from one thiol to another without the generation of NO (Eq. (13)). RSH + R′SNO

↔

RSNO + R′SH

(13)

4.2 Postulated Physiological roles of RSNOs

Based on computational studies and mass spectral evidence Houk and colleagues (2003) have proposed that transnitrosation proceeds through a nitroxyldisufide intermediate (Scheme 4.2 b)

S

O

O

N

N

-S

S

a

O S

S-

N

b

c

S Scheme 4.2

They make the very interesting assertion that, depending on the lifetime and molecular environment of an appropriate electron acceptor (O2 , or Cu2+ ), may form the corresponding radical which can readily yield disulfide and NO via the postulated mechanism in Scheme 4.3. O N

O Cu2+

O S

N

S

Cu+

O S

N

N S

S

S

S

S

Scheme 4.3

4.2

Postulated Physiological roles of RSNOs 4.2.1

Regulation of Blood Flow by HbSNO

The HbSNO-hypothesis of Stamler and coworkers can be summarized as follows: at the lungs Hb is in its R-state and 99% of its hemes are oxygenated the remaining 1% vacant hemes (mainly in the â subunits) react with NO yielding nitrosyHb. The NO is then transferred to â Cys93 forming â Cys93 NO. In the R-state â Cys93 NO is protected from solvent and is therefore stable. In hypoxic tissues Hb is converted to the T-State where â Cys93 NO is exposed to solvent and the â Cys93 -bound NO is transferred, via transnitrosation, to thiols on the RBC membrane anion exchange protein AE1 and transported out of the RBCs (Pawloski et al., 2001). The exported NO equivalents then dilate peripheral blood vessels, thus recruiting more RBCs to the site and thus improving blood flow and oxygenation. As supporting evidence, Stamler and coworkers showed that arterial RBC from rats and humans contained ∼100nm nitrosoHb and ∼300nm HbSNO. In contrast, venous RBCs contained ∼70nM nitrosoHb and ∼70nM HbSNO (Jia et al., 1996; McMahon et al., 2002). Feelish and colleagues were able to confirm the levels obtained in the rat but no detectable nitrosoHb or HbSNO could be detected in humans or monkeys. These authors attributed this to the difference in the demonstrated differences in reactivity between â C125 of rodents and

99

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4 The Role of S-Nitrosothiols in the Biological Milieu

â C93 of primates (Rassaf et al., 2003). In other studies, Gladwin’s group (Gladwin et al., 2002; Reiter et al., 2002) failed to detect any change in HbSNO levels in human forearm during A-V transit. Further adding to the puzzle, Han et al. (2002) failed to detect any changes in plasma Nitrosyl/NitrosoHb levels subsequent to inhalation of NO. The oft-touted argument against a physiological role for HbSNO is that even if it does form in RBCs, the Hb-bound NO has to make quite a journey to get to smooth muscle cells: it must first cross the RBC plasma membrane, then the RBC free zone (Liao et al., 1999), then cross the endothelial cell membranes twice and finally go through the smooth muscle membrane. Why would nature adopt such a convoluted route when the endothelial cells next to the smooth muscle cells are producing membrane-diffusable NO? We and others have demonstrated that an endothelial, cell surface protein-disulfide isomerase-mediated mechanism, does exist for the rapid influx of RSNO bound-NO (Zai et al. 1999; Ramachandran et al., 2001). Whether the csPDI route plays a role in the transfer of NO-equivalents from RBCs remains to be answered. 4.2.2

Regulation of Ventilatory Response in the Brain by RSNOs

Gaston and colleagues (Lipton et al., 2001) demonstrated that injection of GSNO into the brain respiratory centre (nucleus tractus solitarus, NTS) subsequent to a short period of hypoxia, increased ventilation. A closer examination revealed that the CysGly NO was better than GSNO in effecting this response. The authors then showed that this response was receptor mediated as l-Cys-NO yielded a better response then its d-analogue. They further postulated that ã-glutamyltranspeptidase, (ã-GT), the enzyme that converts GSH to CysGly, might regulate this process. As proof of this hypothesis, the injection of GSNO into the NTS of ã-GT-deficient mice (-/-) resulted in a ∼75% attenuation over the wild type, of the ventilatory response. 4.2.3

Role of RSNOs in Platelet Function

NO has long been identified as an inhibitor of platelet aggregation. This effect has principally been attributed to the NO-dependent stimulation of platelet guanylate cyclase (GC), leading to elevated cGMP which, through the stimulation of G-kinases, inhibits Ca2+ mobilization. Many reports on RSNO-dependent platelet inhibition have appeared where anti-platelet effects of RSNOs have generally been attributed to their NO-storage properties i.e. NO once released acts through the GC route. However, several lines of evidence point to RSNOs affecting platelet function through GC-independent routes at the platelet surface (many references). One of the proteins implicated in RSNO-mediated platelet inhibition is protein-disulfide isomerase (PDI). PDI is a dimeric protein composed of identical monomers (∼55KDa), initially found in the endoplasmic reticulum (ER) (Goldberg et al. 1963). PDI is widely distributed in eukaryotic tissues and makes up ∼1% of the total protein content of cells.

4.2 Postulated Physiological roles of RSNOs

One of the most studied functions of PDI is its ability to catalyze isomerization and rearrangement of disulfide bonds (Eq. (14)) in the ER contributing to a proper folding of nascent proteins (Novia et al., 1992). PDI R –S–S–R + R –SH ↔ 1

1

2

R1 –S–S–R2 + R1 –SH

(14)

PDI contains a C-terminal tetrapeptide sequence known as the endoplasmic retention signal, KDEL. This anchor mediates the interaction between plasma membrane and membranes of the Golgi apparatus via a KDEL receptor. The PDI KDEL receptor complex is recycled back into the endoplasmic reticulum (Xiao et al., 1999). It is thought that a saturation of the retention mechanism results in the secretion of PDI which is deposited on the cell membrane and stabilized by electrostatic interactions (Terada et al., 1995). The secreted PDI is termed cell surface PDI (csPDI) Detwiler and co-workers were the first to demonstrate a role for csPDI in platelet physiology (Chen et al., 1995; Essex et al., 1995). Essex and co-workers (1999, 2001) and Hogg and co-workers (Burgess et al., 2000) showed that the inactivation of csPDI with anti-PDI antibodies and thiol alkylating agents inhibited platelet activation and aggregation. Recent studies have identified two distinct activities for csPDI. The first activity elucidated by Lahav and colleagues (Lahav et al., 2000, 2002, 2003), is the catalysis of disulfide bond formation between integrins á2bâ3, the fibrinogen receptor, and á2â1 the collagen receptor and their ligands, thus promoting covalently linked, irreversible, adhesion of platelets to other platelets and to other vascular cells. The second activity, termed denitrosation (Eq. (15)), is catalyzed by the reduced form of the same active site thiols that are involved in the thiol-disulfide exchange activity of the enzyme (Zai et al., 1999; Ramachandran et al. 2001; Raturi and Mutus, 2004). In this process csPDI catalyzes the release of NO from RSNOs. csPDI R–S–N=O →

1/2R–S–SR + NO•

(15)

Therefore, RSNOs appear to regulate PDI-dependent adhesion by competitively inhibiting integrin-ligand disulfide bond formation and, in the process, producing NO which prevents further activation of recruited platelets via the GC/G-kinase route. Thiols, protein or small molecular weight, can be S-nitrosated either by reaction with NO-oxidation products (Scheme 4.1) in hydrophobic domains of plasma proteins or transnitrosated at the cell–plasma interface of NO-producing cells (endothelial cells) or NO-storing cells (RBCs). Efforts to determine the true levels in plasma and in the various cellular compartments are complicated owing to thermal sensitivity and photosensitivity (Sexton et al., 1994; Alpert et al., 1997; Mutus et al., 1999) of the RS–NO bond. Techniques currently employed for the detection and quantification of RSNOs have been summarized in recent reviews (Martinez Riuz and Lamas, 2004; Rasaf et al., 2004). However, it must be made clear that these detected [RSNO] represent steady-state snapshots of a dynamic endocrine-modulated system. It could be that our present

101

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4 The Role of S-Nitrosothiols in the Biological Milieu

techniques are missing most of the “NO-action potential” and are reporting resting values. The number of enzymes and functional proteins that are reportedly regulated by S-nitrosation is on the rise. For example, a search of PUBMED with the key word S-nitrosation revealed some 70 reports of in vitro regulation of enzymes, proteins and cellular processes that are affected by S-nitrosation. Some of these processes that have been well characterized include, nuclear regulatory proteins the NMDA receptor and the ertrocyte anion exchange protein 1 (AE1) (see review by Gaston, 2003). A natural extension of these studies would be to identify the pools of proteins in cells and plasma that are nitrosylated (the nitrosylome) via proteomics. Although some advances have been made in this direction with the biotin switch method, where the S–NO is switched to a biotin label (Jaffrey et al., 2001) the technique is plagued by low sensitivity. The identification of the nitrosylome with techniques that allow temporal resolution shorter than signaling frequencies, under physiological and pathological conditions, will be the key to elucidation of the nitrosylation pathway.

4.3

Conclusion

As this brief survey indicates, RSNOs can be rapidly and specifically formed and degraded in a similar time frame to that observed in NO-guanylate cyclase signaling. An extrapolation of the in vitro reports of RSNO-modulated processes leads the observer to the conclusion that a vast array of pivotal events could potentially be regulated by S-nitrosation. Therefore the great task before us is to sort out the underlying biochemical and physiological mechanisms of RSNO-mediated signal transduction.

103

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5

Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms Tara P. Dasgupta, Danielle V. Aquart

Nitric oxide, NO, has emerged as an important biological messenger [1–3] during the last 15 years and is also known to be an extremely powerful ligand to metal ions [4, 5]. Its superior binding constants compared to CO and O2 and its flexibility in forming varieties of coordination geometries such as linear, bent and bridged, allows it to dominate the coordination sphere of the metal [6, 7]. NO has a singly occupied ð∗ orbital, but is neither a strong one-electron oxidant nor a strong one-electron reductant [8]. It reacts readily with substitution-labile redox-active metal ions and the metal complexes show a range of NO character from nitrosyl, NO+ (linear, sp hybridized and isoelectronic with CO) to nitroxyl, NO− (bent, sp2 hybridized and isoelectronic with O2 ). In the former case considerable charge transfer from NO to metal takes place, whereas the reverse charge transfer, metal to NO, occurs in the latter [8–13]. The most classic examples of metal nitrosyl complexes [4] are the “brown-ring” complex, [Fe(H2 O)5 NO]2+ formed during the qualitative test for nitrate, Roussin’s red and black salts formulated as K2 [Fe2 (NO)4 S2 ] and K[Fe4 (NO)7 S3 ] respectively, and sodium nitroprusside (SNP), Na2 [Fe(CN)5 NO]. It is interesting to note that the nitrosyl moieties of SNP and Roussin’s salts exhibit significant NO+ character and they are also excellent NO donors. In particular, SNP is used widely to induce hypotension during surgery [14, 15]. It significantly reduces the cerebral infarct size [16, 17] and also inhibits platelet aggregation [18]. Transurethral administration of SNP can also induce an erectile response in cats without much side effects [19]. The syntheses, structures and properties of wide varieties of metal nitrosyl complexes have been well documented [4, 5, 20–23]. However, the bulk of the complexes reviewed previously are of academic interest and only a few of these metal nitrosyl complexes have been considered as biologically effective NO donors. It was observed that the metal nitrosyls with significant NO+ character are subject to attack from a variety of nucleophiles and have hypertensive properties. This could be due to the strong trans- labilizing effect of NO. In contrast, the metal nitrosyl compounds with the general formula [M(CN)5 NO]n− , where the NO ligand was either neutral (for M = Co) or anionic (for M = Cr) showed no vasodilatory effect [24]. 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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5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

Metal centers, primarily iron proteins, become the principal target for NO [25–27] under bioregulatory conditions. It is generally agreed that the trans-labilizing effect plays a major role in the activation of certain enzymes. The best example is the ferroheme enzyme soluble guanylyl cyclase [28, 29]. NO attacks the metal center and the resulting nitroxyl complex exerts a trans-effect, labilizing the axial ligand trans to NO. This triggers the conformational change which activates the enzyme for the catalytic formation of cyclic guanylyl monophosphate from the corresponding triphosphate. It has been reported that NO plays a vital role as an inhibitor of metalloenzymes such as cytochrome P450 [30], cytochrome oxidase [31], nitrile hydratase [32–35] and catalase [36]. It has also been implicated in the vasodilatory properties of a salivary ferriheme protein of blood sucking insects [37]. The mechanism proposed for the biosynthesis of NO involves the interaction of arginine with the ferric iron of the nitric oxide synthase (NOS) enzyme’s heme prosphetic group followed by an electron transfer process [1, 38, 39].

5.1

Iron Complexes 5.1.1

Nitroprusside

Sodium nitroprusside (SNP), which is also known as Nipruss or Nipride to medical practitioners, was the first iron nitrosyl complex, prepared as far back as 1850 by Playfair [40]. The hypotensive property of SNP was first demonstrated by Johnson [41] in 1929. It was shown that application of a moderate dose of SNP reduces the blood pressure of a severely hypertensive patient without any side effect [42]. Since that time considerable research has been carried out to understand the mode of action of nitroprusside and its metabolic fate. SNP is now regarded as a potent vasodilator that causes muscle relaxation by releasing NO which activates the cytosolic isozyme of guanylyl cyclase [43–46]. The ruby-red compound, SNP, is prepared by the reaction of nitrite ion with hexacyanoferrate ion, [Fe(CN)6 ]4− [47, 48], as shown in Eq. (1) [Fe(CN)6 ]4− + NO− + H2 O

→

[Fe(CN)5 NO]2− + CN− + 2OH−

(1)

The complex anion, [Fe(CN)5 NO]2− , is diamagnetic and its infrared spectrum shows an N–O stretching vibration at 1939cm−1 , higher than for neutral NO, signifying a net positive charge on the NO [49, 50]. Hence, the positively charged nitrogen of NO in SNP is readily attacked by nucleophiles such as OH− , RS− , and S2− to give as products, [Fe(CN)5 NO2 ]4− , [Fe(CN)5 (NOSR)]3− and [Fe(CN)5 (NOS)]4− respectively [49–51]. The reactions of SNP with thiols and thiolate ions are complicated [42]. The nitroprusside ion is reduced to [Fe(CN)5 NO]3− which rapidly loses the cyanide ion trans to NO (trans-effect) to form the paramagnetic complex ion, [Fe(CN)4 (NO)]2− (Fig. 5.1). The equilibrium constant for the formation of [Fe(CN)4 (NO)]2− from nitroprusside ion is 6.8×10−5 M and the rate constant for cyanide release is 280s−1 at 25 ˚C [52]. The

5.1 Iron Complexes O

N

102.3

C

102.7

Fe

C N

88.9

N C N

177.1

C 176.1

N

Fig. 5.1 Structure of

[Fe(CN)4 NO]2− .

five-coordinated species was isolated as (Ph4 P)2 [Fe(CN)4 (NO)]. The X-ray analysis of the compound revealed that the anion has a square pyramidal structure with an Fe– NO distance of 156.5 pm which is shorter than the Fe–NO distance (166.6 pm) in the nitroprusside ion [53]. The X-ray structure [54, 55] along with the EPR spectra [56] and visible spectra (570–630 nm) in both aqueous and non-aqueous solvents (57, 58) support the resonance structure FeI –NO+ ↔ FeII –NO, which explains the substitution lability of this five-coordinated species [59, 60]. SNP spontaneously releases NO both thermally and photochemically [61–65], but is quite stable in the dark and in aqueous in vitro physiological media [66]. This implies that absorption of heat and light energy induces electron transfer from the Fe2+ center to the NO+ ligand, resulting in weakening of the Fe–NO bond and subsequent release of NO [65]. SNP also decomposes in an aqueous environment in the presence of biological reductants [65, 66] and some transition metal ions to produce nitric oxide. As indicated before, thiols are well known biological reductants and can improve the potency of SNP as a hypotensive agent by releasing NO [62, 66–68]. The detailed mechanism of reduction of SNP by l-cysteine to produce NO has been reported [69]. It was observed that NO release is favored at lower pH values and cysteine concentration whereas at higher pH and cysteine concentration formation of NO− occurs. The mechanism of the reaction (Scheme 5.1) involves three clear stages. The first two stages resemble the stepwise reversible two-electron reduction of NO+ to NO− and are similar in rates. However, the third stage is the slow substitution of NO− by cysteine in the reduced nitroprusside ion [69]. The release of cyanide from nitroprusside ion seems to be a prior requirement for NO release [42, 50, 60, 61, 70, 71] and the rate of the reaction is retarded by the addition of NaCN, indicating reversible loss of CN− from the nitroprusside ion. The reaction is also catalysed by

111

5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

112

H+

CySSCy

O N -SCy

2-

O N

+ CyS

Fe

-

K1

NO 2

.

CyS

k1

Fe

3-

O N

HCN

CN kCN

Fe

O N

2-

at lower [Cy S H] NO

Fe

k-1

fast +

1st STAGE

2OH -, KOH

Fe

3-

CySat higher [Cy S H]

4-

2nd STAGE

+ H2O

K2 O N-SCy

3-

Fe

k2

. CySSCy

CyS

3rd STAGE NO - + other Fe(II) product(s)

+

[FeII(CN) 4SCy]3-

H+

k3 r. d. s .

O N CyS-

+

3-

Fe

HNO 1) 2HNO

N2O + H2O

3) HNO + CySH

NO2- + N2O + H+ .. CySH CyS-NH-OH NH 2OH + CySSCy

4) NO + 0.5 O2

NO2

2) HNO + 2NO .

5) 2NO 2 + H2O

NO2- + NO3- + 2H+

Scheme 5.1 Proposed overall mechanism for the reaction between nitroprusside and L-cysteine.

Cu2+ , which has been the common feature for the decomposition of S-nitrosothiols [72–77]. The reduction of SNP by another well known biological reductant, vitamin C or l-ascorbic acid [78] which exists in cellular systems at millimolar concentration [79] has also been investigated thoroughly and the mechanism of NO release has been proposed [80] (Scheme 5.2). The rate of NO release increases up to about pH 7 and then decreases at higher pH due to the reaction between nitroprusside ion and OH− . The two ionized form of ascorbate present in aqueous solution reduce SNP in the order A2− >>HA− >>H2 A (H2 A = ascorbic acid) to release NO. Similar results were obtained by Wanat et al. [81] for the complex ion [Fe(CN)5 (NO2 )]3− . The outer sphere reduction of SNP by ascorbate involves three clear stages with NO being released in the last stage. The reaction is catalyzed by alkali metal ions which act as a glue between two negatively charged ions, nitroprusside and ascorbate. The formation of this ion triplet [82] seems to be a prior requirement for outer sphere electron transfer between two negatively charged ions [83, 84]. Since the catalysis by metal ions increases with cationic size, the partial deaquation mechanism explains the observed catalytic efficacy sequence for M+ (M+ =Li+
5.1 Iron Complexes

K1

H2A

K2

-

HA

2-

A

-5 K1 = 5.62 x 10 -12 K2 = 1.62 x 10

st 1 STAGE

[Fe(CN)5NO]

2-

+

+ Mn+

2-

A

KIT

[Fe(CN)5NO]

2-

.Mn+.A2-

330, 394 nm

k1 -

2OH , KOH 345, 430 nm

[Fe(CN)5NO]

3-

n+

+ M

+

A.-

4-

[Fe(CN)5(NO2)] + H2O fast

does not produce NO

kCN , KCN

[Fe(CN)4NO]

2-

-

+ CN

350, 615 nm

nd

2

STAGE -

HA [Fe(CN)4NO] rd

3

2-

k3 A2-

STAGE

k'2 [Fe(CN)4NO]

K2 2-

A

k''2

3-

.

A-. / HA

+

A2- + 3H+

4-

(very slow)

NO nitric oxide

[Fe(CN)4(NH2OH]

[Fe(CN)4(A)] +

+

.

2-

th 4 STAGE

+ A

Scheme 5.2 Overall reaction mechanism for the reaction between SNP and L-ascorbic acid.

In general, the primary reaction step in the reaction of the nitroprusside ion with thiols and thiolates [59, 85, 86] is the formation of [Fe(CN)5 –N(O)SR]3− followed by the loss of RS∗ to form [Fe(CN)5 NO]3− . The radical RS∗ annihilates itself to form insoluble R2 S2 [59, 85] and [Fe(CN)5 NO]3− produces [Fe(CN)6 ]4− and NO. The second step only occurs in the pH range 6.5–8.5 with the loss of RSNO which in turn produces NO and RSSR [87, 88]. A large number of reactions involving SNP and oxygen, nitrogen and carbon centered nucleophiles have been reported [50, 51, 89] and these reactions are of immense importance from both synthetic and mechanistic standpoints, but are of very little interest in terms of biological significance. Simple primary and secondary amines [90–94] undergo nitrosation reactions with SNP to produce [Fe(CN)5 NH2 R]3− and [Fe(CN)5 NHR2 ]3− along with some [Fe(CN)5 H2 O]3− . A dissociative mechanism (rate = k1 K[nitroprusside][amine]) has been proposed for

113

114

5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

the reaction with most simple primary amines [95, 96] and a dissociative interchange pathway (rate = k2 K[nitroprusside][amine]2 ) is proposed for secondary amines [97, 98], where K is the equilibrium constant for adduct formation and k1 and k2 are the rate constants. SNP can produce NO both enzymatically and nonenzymatically in the dark provided that vascular tissue is present [61]. This is possible because vascular tissue, including cell membranes and smooth muscle cell membranes always contains biological reductants such as thiols, hemoproteins, and ascorbate. It is also suggested that the presence of NADH or NADPH as cofactor in the biological tissue may also be a requirement for NO release [61, 71, 99, 100]. 5.1.2

Iron Porphyrin Nitrosyls

The physiological effects of NO are mediated through the enzyme receptor, soluble guanylyl cyclase (sGC) [101,102]. The affinity of NO for sGC is approximately 1010 , which is about six orders of magnitude greater than that of CO [103, 104]. Two types of sGC have been identified. The first type, sGC1, isolated from the bovine lung enzyme [104,105] has a soret band at 426 nm and á, â bands at 558 and 528 nm respectively [104–108]. sGC1 contains six-coordinate heme with two histidines bound to heme iron, one of which is photolabile [105]. The second type, sGC2, has also been isolated from bovine lung with stoichiometric amounts of ferrous heme and exhibits a soret band at 431 nm and a single á/â band at 555 nm [108–110]. This observation is consistent with a high-spin five-coordinated square pyramidal heme containing a single histidine ligand. NO interacts with both sGC1 and sGC2 producing a fivecoordinated nitrosyl heme species [108, 110–112] (Scheme 5.3). It is quite evident that the ferrous complexes of porphyrins, both natural and synthetic, have extremely high affinities towards NO. A series of iron (II) porphyrin nitrosyls have been synthesized and their structural data [11, 27] revealed non-axial symmetry and the bent form of the Fe–N=O moiety [112–116]. It has been found that the structure of the Fe–N–O unit in model porphyrin complexes is different from those observed in heme proteins [117]. The heme prosthetic group is chemically very similar, hence the conformational diversity was thought to arise from the steric and electronic interaction of NO with the protein residue. In order to resolve this issue femtosecond infrared polarization spectroscopy was used [118]. The results also provided evidence for the first time that a significant fraction (35%) of NO recombines with the heme-Fe(II) within the first 5 ps after the photolysis, making myoglobin an efficient NO scavenger. High-spin Fe(II) porphyrin complexes are considerably more labile than the Fe(III) analogs. The bimolecular rate for the nitrosylation [119] of model compound Fe(TPP), where TPP is tetraphenyl porphyrin, determined by flash photolysis in toluene, has been reported to be quite fast (kobs =5.2×109 M−1 s−1 ). The activation parameters, ÄH≠ and ÄS≠ values are 2.6kJmol−1 and −515Jmol−1 K−1 respectively. On the other hand for a water soluble Fe(II)TPP compound the NO uptake rate (kON =4.5×105 M− s−1 ) is about three orders of magnitude larger [120] than for the Fe(III) analogs (5×102 M−1 s−1 ).

5.1 Iron Complexes

NH

N N

II

N

Fe N

N N NO

HN

sGC1

N

N Fe N

N

N

N

NO

Fe N

N N

Scheme 5.3 Nitrosylation of soluble

HN

guanylyl gyclase1 and soluble guanylyl cyclase2.

sGC2

Fe(III) porphyrins are known to undergo reductive nitrosylation in the presence of excess NO. For example, FeIII (TPP)Cl reacts with NO in toluene containing a small amount of methanol to give FeII (TPP)(NO) [121, 122] as shown in Scheme 5.4. Similarly, when an aqueous ferri-hemoglobin, metHb, is exposed to NO, the ferrohemoglobin NO adduct, Hb(NO), is produced [123]. Recently, it was shown [124] by EPR, electrochemical and spectroelectrochemical techniques, that the water soluble FeIII TMPyP (TMPyP = meso-tetrakis(N-methylpyridinium-4-yl)porphyrin) undergoes reductive nytrosylation at all pHs to form FeII (NO)TMPyP. The reaction of CytII with NO to form the adduct CytII –(NO) is quite slow (kNO =8.3M−1 s−1 ) and is a function of [NO] and [OH− ]. The formation of nitrosohemoglobin (HbFeII (NO)), both in vitro and in vivo, by the transfer of NO from nitroaspirin, the lead compound of a new class of nitric oxide releasing nonsteroidal anti-inflammatory drugs has been investigated by EPR spectroscopy [125]. This study indicates the potential of EPR spectroscopy as a tool to monitor the transfer and distribution of NO from nitrovasodilators to hemoglobin. Fe(TPP)Cl + NO

Fe(TPP)(Cl)(NO) CH3OH

Fe(TPP)NO

+ NO Fe(TPP) + CH ONO + HCl Scheme 5.4 Reaction of Fe(TPP)Cl with nitric 3 oxide.

115

116

5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

5.1.3

Dinitrosyl Complexes (DNICs)

Dinitrosyl-iron complexes [126–129] are formed during the reaction of Fe(II) with NO in the presence of low molecular weight thiols, aminoacids, peptides or proteins (mainly through the cysteine and histidine moieties) and were first detected by EPR signals in biological tissues [130–132]. Further investigations revealed that DNICs assist in antiplatelet [133] and vasorelaxation [134] activities, enhance cardiac resistance to ischemia, reperfusion and blood pressure lowering [135–139]. They also show inhibition to glutathione reductase and glutathione S-transferase (GST) [140, 141] and induce accumulation of heat shock protein HSP70 [142, 143]. These evidences clearly suggest that DNICs function as transport and storage systems for NO in vivo [134, 144–146]. DNICs, on interacting with low molecular weight thiols, can interconvert between a paramagnetic and diamagnetic state, provided the correct ratios of Fe2+ : thiol exist [147]. These complexes take the form shown in Fig. 5.2, and may contain a range of oxygen, nitrogen, sulfur and even phosphorus ligands [148–154]. + L

ON Fe ON

L

L = -SR, -NR 2, -OR

DNIC

Fig. 5.2 General structure of DNIC.

DNICs are relatively stable and can be stored at −80°C for weeks without much loss of NO [155], however, their stabilities decrease dramatically at room temperature to render unbound NO. NO bound to thiols may account for the low yield of free NO sometimes detected in solutions involving DNICs in the presence of high concentrations of thiols [126]. The decomposition is acid catalyzed to free NO and S-nitrosothiol (RSNO). Of course, protein-bound DNICs are more stable and serve as potential ‘sinks’ for low molecular weight DNICs. DNICs are spontaneously [128] formed in aqueous media using a simple Fe(II) salt, S-nitrosothiol and thiol, with a ratio of Fe2+ /RSH of 1:20. NO is transferred quantitatively from the sulfur atom in the RSNO to the iron. The complete mechanism is yet to be fully determined. A 1:2 ratio results in the formation of an EPR silent yellow dinuclear iron complex ([Fe2 (RS)2 (NO)4 ]. At the higherer ratio, the green paramagnetic, mononuclear dinitrosyl predominates. The reaction is very straightforward at pH 7.8, under an inert atmosphere and in water. Under anaerobic conditions the stability of this compound is enhanced, however, in the presence of air and hydrogen peroxide, it readily decomposes to give the dinuclear complex [126] which is similar in structure to the Roussin red salt, as shown in Scheme 5.5. DNICs continue to be a relevant aspect of nitric oxide research and indicate the high affinity of NO for metal centers, especially ferrous ions, when compared with sulfur bound NO complexes, such as S-nitrosothiols.

5.1 Iron Complexes Fe 2+ RSNO RSH

RSNO ON RSH ON

SR Fe SR

RSH O2, H2O2

R S

ON

NO Fe

Fe S

ON

NO

Scheme 5.5 Interconversion of DNICs with an iron-sulfur nitrosyl.

R

5.1.4

Iron–Sulfur Cluster Nitrosyls

The interactions of NO with the iron–sulfur cluster moieties of several enzymes generate iron–sulfur–nitrosyl cluster compounds [156]. However, synthetic nitrosyl clusters such as Roussins black salt (RBS), Roussins red salt (RRS), Roussins red ester (RRE) and [FeNOS]4 (Fig. 5.3) are well known [129, 157] and can be synthesized easily [158–164]. For example, the RBS can be prepared by mixing FeSO4 with NaNO3 and (NH4 )2 S in aqueous solution [158]. RRE salts are generally insoluble in water, but recently the water soluble sulfonated derivative, Na2 [Fe2 (SCH2 CH2 SO3 )2 (NO)4 ], has been prepared [165]. The crystal structures of tetraphenylarsonium [166] and tetraethylammonium [167] salts have been determined and the cluster anion has been found to possess C3v symmetry. Electrochemical studies of stable RBS show the existence of four reduction

NO

S

ON

Fe

Fe ON

S

S

Roussin's Red Salt

S

Fe

NO

NO

Roussin's Red Ester

Fe

S

NO

R

Fe

S

Fe S

Fe(NO)2 Fe (NO)2

ON

S Fe

S

Roussin's Black Salt

R

ON

Fe S

NO (ON)2Fe

ON

-

O N

2-

Fe

S

ON

(FeNOS) 4

Fig. 5.3 Structures of some well-known iron-sulfur cluster nitrosyls.

NO

117

118

5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

steps producing corresponding negatively charged (–1 to –4) species. The ammonium salt of the electrogenerated species, [Fe2 S3 (NO)7 ]2− , has been isolated and the crystal structure of the compound determined, confirming that the anion is a considerably distorted form of the parent anion RBS− [168]. Detailed photochemical studies of RBS and RRS have revealed that the photolysis of RRS produces RBS and NO quantitatively (Scheme 5.6) and the RBS produced undergoes further photodecomposition to generate NO and iron (III) [165, 168]. RBS has been tested as an NO delivery drug to the vascular and brain tissues by thermal as well as photochemical means [169, 170]. Due to the high solubilities in aprotic solvents, Roussin’s salts are able to penetrate the endothelial cell membrane easily and deliver NO for hours [169]. RBS has been found to inhibit ADP-induced platelet aggregation [171] and Roussin’s salts in general show a bacteriostatic effect, presumably due to the interaction of released NO and iron–sulfur proteins [172]. Fe2S2(NO)42-

hv

oxidation Fe4S3(NO)72- + S2-

Fe2S2(NO)32- + NO RRS Fe4S4(NO)74-

Scheme 5.6 Photochemical release of

nitric oxide from Roussin’s red salt.

5.2

Ruthenium Complexes

The high affinity of ruthenium (III) for nitric oxide [173–175] has initiated an explosion in the synthesis of ruthenium nitrosyl complexes. Ruthenium (III) readily scavenges for NO to form rather stable mononitrosyls [176–180]; in fact, it has been shown [181] that ruthenium (III) can easily abstract NO from l-arginine. Initial approaches toward appropriate synthetic routes dealt with accomplishing high water solubility for ruthenium complexes [182]. This led to the chelation of polycarboxylate ligands to ruthenium (III) prior to NO binding. Ruthenium (III) polyaminocarboxylates K[Ru(Hedta)Cl] [183] (Fig. 5.4) rapidly bind NO at physiological pH in a 1:1 stoichiometry at 7°C, via the aqua substituted compound [182] [Ru(Hedta)(H2 O)], to give the stable mononitrosyl complex, [RuII (Hedta)(NO)] through electron transfer between NO and the central metal. Infrared analysis [176] at 1897cm−1 , confirmed the presence of a linear Ru(II) –NO bond. Different forms of the precursor of Ru(Hedta)(NO) lead to other ruthenium nitrosyls [183]. The ability of these polycarboxylate [182–185] compounds to scavenge NO is ideal for their application to various biological systems, as alleviators of some of the diseases which are the consequences of high NO toxicity [176, 186, 187]. Some of these compounds have been shown to preferentially inhibit iNOS [178, 183, 188] rather than cNOS. Employing an NO scavenger rather than an NOS inhibitor eliminates enzyme specificity [183]. The Ru–NO bond is extremely stable and remains intact

5.2 Ruthenium Complexes O O

COOH

COOH

Cl

O

N

N

N

O Ru

Ru

Cl

N

N

N

O

CO2H

Cl

COOH

O

COOH

Fig. 5.4 Structures of Ru(Hedta)(NO), A, and

B

A

trans-[RuCl2 (nic)4 ], B - NO scavengers.

throughout redox and substitution reactions [183]. However, some ruthenium porphyrins [189–193] react readily with NO to form the thermally stable corresponding nitrosyl complexes which release NO on photolysis. Recently, some thermally stable Ru (II) nitrosyl complexes have been synthesized [194, 195]. These compounds include the salen- [194, 195] and salophen-type [194] complexes represented by RuII (R-salen)(X)(NO) and RuII (R-salophen)(X)(NO), where X = Cl− , ONO− , salen = N,N′-ethylenebis(salicylideneiminato) dianion, salophen = N,N′-1,2-phenylenebis(salicylideneiminato), and R = alkyl group. The Ru-salophen complexes are prepared following the synthetic route depicted in Scheme 5.7. The corresponding salen complexes have also been prepared in a similar fashion using 1,2diaminoethane rather than phenylenediamine. The structures of [Ru(salen)(NO)(Cl)], [Ru(salen)(ONO)(NO)] and [Ru(t Bu2 salophen)(Cl)(NO)] were determined by single crystal X-Ray diffraction [194, 195]. All these complexes are found to release NO on photoexcitation in both organic and aqueous solvents, as shown in Fig. 5.5. The kinetic studies of the subsequent reverse reaction to reform the nitrosyl complexes show that the rates of NO uptake are dramatically dependent on the type of solvent, with second-order rate constants ranging from 5×10−4 M−1 s−1 in acetonitrile to 4×107 M−1 s−1 in toluene [194]. O

R'

OH

H2N

refluxing ethanol

+

N

N

H2N R

R'

OH

HO

R

R' R

X

C N

1. 2NaH 2. Ru(NO)Cl 3

N C Ru

R'

O R

O NO

R' R

Scheme 5.7 Synthesis of ruthenium salophen complexes. R = t Bu, R′ = H, X = Cl, or R = R′ = t Bu.

119

120

5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms NO O C=N

Ru

Sol

O N=C

hv

O C=N

kNO

Ru

O

+ NO

N=C

Cl

Cl

Ru(salen)(Cl)(NO)

Fig. 5.5 Photolysis of a Ru–NO complex.

Photorelease of NO [196–203] is not confined to these salen and salophen complexes, but is also observed in other ruthenium nitrosyl species [155], namely trans[RuCl(NO)(cyclam)]2+ , where cyclam is 1,4,8,11-tetraazacyclotetradecane, and trans[Ru(NH3 )4 L(NO)]3+ , Ru(NO)Cl5 , and Ru(NO)Cl3 (Fig. 5.6). These species are able to effect greater hypotensive responses [204–206] even when compared with the more common NO-releasing agents, such as SNP. In fact, the hypotensive effects of cyclamNO [204] become completely diminished in the presence of an NO scavenger, indicating the NO-releasing function of these compounds. The binding power and the reduction potential of the ruthenium complex can be modified by varying the ligand, L; and thus altering the trans-effect capability of NO [207, 208]. This in turn affects the dissociation of NO from the complex. Such a study of the trans-labilization was performed [207], and showed (Scheme 5.8) that NO readily dissociates from trans[Ru(L)(NH3 )4 NO]Cl3 (L = imidazole, pyridine, or sulfite) upon irradiation [209, 210], due to the strong nitrosonium (NO+ ) character of the bound NO. When L is imidazole the NO release is further facilitated since it coordinates to Ru(II) through a carbon atom of the imidazole, which facilitates the formation of the reduced state of NO [211]. The increase in NO production has been achieved in the ruthenium complex trans[NO(P(OEt)3 )(NH3 )4 Ru]3+ , by the introduction of strong trans-labilizing phosphito ligands and achieving biologically accessible reduction potentials [212, 213].

Cl

Ru

Cl

Ru(NO)Cl 3

2-

NO

NO

Cl

Cl Cl

NO

Ru Cl Cl

3+

H3N Ru NH3 H3N NH3

Cl

L

trans-[Ru(NH3)4L(NO)] 3+

Ru(NO)Cl 5

2+ NO HN Ru NH HN NH Cl

trans-[RuCl(NO)(cyclam)]2+ Fig. 5.6 Structures of some typical ruthenium nitrosyl complexes.

5.3 Other Metal Nitrosyls

(a) trans-[Ru(L)(NO)(NH3)4]3+ -

e

trans-[Ru(L)(NO)(NH3) 4]2+ H2O

trans-[Ru(L)(H2O)(NH3)4]2+ + NO

(b) trans-[RuCl(NO)(cyclam)]2+

etrans-[RuCl(NO)(cyclam) ] + Cl- H2O

trans-[Ru(NO)(H2O)(cyclam)]2+ H2O

trans-[Ru(H2O)2(cyclam)]2+ + NO Scheme 5.8 Dissociation of NO from different Ru–NO complexes.

NO release is considerably slowed down when trans-[Ru(Cl)(NO)(cyclam)] is reduced (Scheme 5.8(b)). It has also been observed [204] that the reduction in blood pressure in hypertensive Wistar rats is more prolonged by the administration of cyclam-NO than when SNP is administered. This suggests that the metal cyclam NO complex could be used as a long lasting vasodilator [214]. The dual role of these ruthenium complexes, as both an NO donor and scavenger, appears to lie strongly in the binding constant of NO to Ru(II) and the ligands present, which can facilitate NO dissociation. Clearly the investigative approach into these systems has just touched the surface and the future appears very promising in these potential NO source/scavenger systems.

5.3

Other Metal Nitrosyls

The occurrence of transition metal nitrosyls is more common among Fe and Ru metals, as previously discussed, and these compounds show greater biological applications. However, ongoing investigations have included Mn, Cu, Os, Ir and, to a greater extent, Co. What remains to be thoroughly looked into is the ease with which the NO ligand is removed or lost by these compounds. Manganese nitrosyl porphyrins [215] are considered good models for the iron-nitric oxide analogs, which are relatively unstable but very vital to many biological operations. A six-coordinate manganese nitrosyl porphyrin of the form (por)Mn(NO)(L), where por can be TTP (TTP = tetra(4-methylphenyl)porphine) and L = piperidine, methanol, 1-methylimidazole, has been prepared [216] in moderate yields by the reductive nitrosylation of the (por)MnCl complex with NO in piperidine. The crystal structures of these compounds give indication of a linear Mn–NO bond [215]. Examples of the osmium and iridium complexes are Os(PPh3 )2 Cl(NO) and Ir(PPh3 )3 (NO), respectively [216]. The osmium compound gave, on reaction with HCl, the first characterized complex with the feature of an N-coordinated HNO, Os(PPh3 )2 Cl2 (HNO), which was confirmed by X-ray crystallography. On the other hand, the nitrosylated iridium compound gave the hydroxylamine complex [216]. The nitric oxide reduction of Cu(dmp)2 (H2 O)2+ in aqueous media gives a Cu(II)– NO complex via an inner-sphere mechanism [216] (dmp = 2,9-dimethyl-1,10-phen-

121

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5 Metal–NO complexes: Structures, Syntheses, Properties and NO-releasing Mechanisms

anthroline). However, this complex is only transient in nature and readily undergoes nucleophilic attack by ROH. Due to this behavior there is no known recorded spectral evidence for its existence. The more commonly met cobalt nitrosyls [217, 218] originate from the reaction of porphyrins or â-halogenated porphyrins of cobalt with a nitrosating agent such as NOBF3 under argon. The reactions occur via electron transfer from the central metal. Such compounds include, (TPPBr4 )Co(NO), where TPPBr4 is 7,8,17,18-tetrabromo5,10,15,20-tetraphenylporphyrin; also [(NO− )Co(III)TSPc]4− Co(Pc(NO) and [Co(2-TMpyP)(NO)], (Pc = phthalocyaninato, TSPc = 3,10,17,24-tetrasulfophthalocyanine, 2-TMpyP = tetra(1-methyl-2-pyridyl)porphine).

5.4

Conclusion

Since the discovery of NO as an EDRF (endothelium derived relaxing factor) and as a molecule which plays important roles in mammalian bioregulation and immunology, there has been an upsurge of research interest in developing suitable NO donors. Indeed, hundreds of NO donors have been developed but not many of them are metal–NO complexes. This is surprising since numerous thermally stable metal nitrosyl complexes have been synthesized and characterized since the discovery of coordination compounds. In fact, among the metal nitrosyl complexes, SNP, which was synthesized as early as the mid-eighties, is still regarded as the only established NO donor drug used as a hypotensive anaesthetic during surgery. There have been developments of nitrosyl complexes including DNICs, and iron–sulfur cluster nitrosyls, but still much more research work by chemists is needed. The present chapter summarizes some of the recent findings in the area of metal nitrosyl complexes, specifically, their functions as NO donors, and their biological applications. The mechanistic details of NO release from these metal nitrosyl complexes have also been reviewed. Hopefully the chapter will inspire and motivate chemists to concentrate their research interest towards the development of new metal nitrosyl complexes with biological activities superior to those of the existing NO donors.

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186 187 188

189

190

Fricker, S. P., Biochem. Biophys. Res. Commun. 292 (2002), p. 519 Beirith, A., Creczynski-Pasa, T. B., Bonetti, V. R., Konzen, M., Seifriz, I., Paula, M. S., Franco, C. V., Calixto, J. B., Eur. J., Pharmacol. 369 (1999), p. 289 Pieper, G. M., Roza, A. M., Adams, M. B., Hilton, G., Johnson, M., Felix, C. C., Kampalath, B., Darkes, M., Wanggui, Y., Cameron, B., Fricker, S. P., J. Cardiovasc. Pharmacol. 39 (2002), p. 441 Morioka, Y., Ishikawa, A., Tomizawa, H., Miki, E., J. Chem. Soc., Dalton Trans. (2000), p. 781 Marmion, C. J., Murphy, T., Nolan, K. B., Chem. Commun. (2001), p. 1870 Davies, N. A., Wilson, M. T., Slade, E., Fricker, S. P., Murrer, B. A., Powell, N. A. Henderson, G. R., Chem. Commun. (1997), p. 47 Cameron, B. R., Darkes, M. C., Yee, H., Olsen, M., Fricker, S. P., Skerlj, R. T., Bridger, G. J., Davies, N. A., Wilson, M. T., Rose, D. J., Zubieta, J., Inorg. Chem. 42 (2003), p. 1868 Morbidelli, L., Donnini, S., Filippi, S., Messori, L., Piccioli, F., Orioli, P., Sava, G., Ziche, M., Br. J., Cancer 88 (2003), p. 1484 Pieper, G. M., Roza, A. M., Adams, M. B., Hilton, G., Johnson, M., Felix, G. C., Kampalath, B., Darkes, M., Wanaggui, Y., Cameron, B., Fricker S. P., J. Cardiovasc. Pharmacol. 39 (2002), p. 441 Moncada, S., Higgs, A., New Engl. J., Med. 329 (1993), p. 2002 Parratt, J. R., J. Physiol. Pharmacol. 48 (1997), p. 493 Knowles, R. G., Merrett, M., Slater, M., Moncada, S., Biochem. J. 270 (1990), p. 883 Miranda, K. M., Bu, X., Lorkovic, I., Ford, P. C., Inorg. Chem. 36 (1997), p. 4838 Kadish, K. M., Adamian, V. A., Caemelbecke, E. V., Tan, Z., Tagliatesta, P., Bianco, P., Boschi, T., Yi, G.-B. Khan, M. A., Ritcher-Addo, G. B., Inorg. Chem. 35 (1996), p. 1343

191 Bohle, D. S., Goodson, P. A., Smith,

B. D., Polyhedron 15 (1996), p. 3147 192 Yi, G.-B. Khan, M. A., Ritcher-Addo,

G. B., Inorg. Chem. 35 (1996), p. 3453 193 Bohle, D. S., Hung, C.-H. Smith,

B. D., Inorg. Chem. 37 (1998), p. 5798 194 Works, C. F., Jocher, C. J., Bart,

195

196 197

198 199

200

201

202 203 204

205

206

207 208

209

G. D., Bu, X., Ford; P. C., Inorg. Chem. 41 (2002), p. 3728 Bordini, J., Hughes, D. L., Da Motta Neto, J. D., da Cunha, C. J., Inorg. Chem. 41 (2002), p. 5410 Carter, T. D., Bettache, N., Ogden, D., Br. J. Pharmacol. 122 (1997), p. 971 Bettache, N., Carter, T. D., Corrie, J. E., Ogden, D., Trentham, D. R., Methodol. Enzymol. 268 (1996), p. 266 Makings, L. R., Tsien, R. Y., J. Biol. Chem. 269 (1994), p. 6282 Carther, T. D., Bettache, N., Ogden, D., Trentham, D. R., J. Physiol (London) 467 (1993), p. 165 Williams, J. H., Bettache, N., Trentham, D. R., Bliss, T. V. P., J. Physiol (London) 467 (1993), p. 166 Slocik, J. M., Ward, M. S., Somayajula, K. V., Shepherd, R. E., Transition Met. Chem. 26 (2001), p. 351 Gorelsky, S. I., Lever, A. B. P., Int. J., Quantum Chem. 80 (2000), p. 636 Works, C. F., Ford, P. C., J. Am. Chem. Soc. 122 (2000), p. 7592 Marcondes, F. G., Ferro, A. A., Souza-Torsoni, A., Sumitani, M., Clarke, M. J., Franco, D. W., Tfouni, E., Krieger, M. H., Life Sci. 70 (2002), p. 2735 de Barros, B. F., Toledo, J. C., Franco, D. W., Tfouni, E., Krieger, M. H., Nitric Oxide 7 (2002), p. 50 Torsoni, A. S., de Barros, B. F., Toledo, J. C. Jr., Haun, M., Krieger, M. H., Tfouni, E., Franco, D. W., Nitric Oxide 6 (2002), p. 247 Clarke, M. J., Gaul, J. B., Struct. Bonding 81 (1993), p. 147 Dodsworth, E. S., Vleck, A. A., Lever, A. B. P., Inorg. Chem. 33 (1994), p. 1045 Borges, S., Davanzo, C. U., Castellano, E. E., Z-Schpector, J., Silva, S. C., Franco; D. W., Inorg. Chem. 37 (1998), p. 2670

References 210 Gomes, M. G., Davanzo, C. U., Silva,

211

212

213

214

S. C., Lopes, L. G. F., Santos, P. S., Franco, D. W., J. Chem. Soc., Dalton Trans. (1998), p. 601 Lopes, L. G. F., Wieraszko, A., El-Sherif, Y., Clarke, M. J., Inorg. Chim. Acta 312 (2001), p. 15 Wieraszko, A., Clarke, M. J., Lang, D. R., Lopes, L. G. F., Franco, D. W., Life Sci. 68 (2001), p. 1535 Lorkovic, I. M., Miranda, K. M., Lee, B., Bernhard, S., Schoonover, J. R., Ford, P. C., J. Am. Chem. Soc. 120 (1998), p. 11674 Lang, D. R., Davis, J. A., Lopes, L. G. F., Ferro, A. A., Vasconcellos,

215

216 217 218

L. C. G., Franco, D. W., Tfouni, E., Wieraszko, A., Clarke, M. J., Inorg. Chem. 39 (2000), p. 2294 Zahran, Z. N., Lee, J., Alguindigue, S. S., Khan, M. A., Ritcher-Addo, G. B., J. Chem. Soc., Dalton Trans. 1 (2004), p. 44 Ford, P. C., Lorkovic, I. M., Chem. Rev. 102 (2002), p. 993 Vilakazi, S., Nyokong, T., Polyhedron 19 (2000), p. 229 Kadish, K. M., Ou, Z., Tan, X., Boschi, T., Monti, D., Fares, V., Tagliatesta, P., J. Chem. Soc., Dalton Trans. (1999), p. 1595

129

131

6

The NO-releasing Heterocycles Alberto Gasco, Karl Schoenafinger

A number of heterocyclic systems release NO under physiological conditions and thus mimic NO actions; they may be subdivided into three classes: – heterocyclic N-oxides – mesoionic heterocycles – other heterocyclic systems The subject has been treated as part of a general discussion of NO-donors in a number of reviews [1–4]. This chapter will briefly introduce the general properties of these systems and the chief methods to synthesise them, after which it will concentrate on their NO-donor properties and NO-dependent biological activities. The term NO will be used here as a family name, embracing not only nitric oxide (NO• ) but also its two redox forms, nitroxyl (HNO) and nitrosonium ion (NO+ ), which play important roles in the complex signalling system connected with NO• [5]. The specific redox form involved in the NO-release will be indicated, if known, when necessary for the discussion.

6.1

Heterocyclic N-oxides 6.1.1

Furoxans

Furoxan is an old heterocyclic system, known to chemists thanks to an argument over its structure and its intriguing chemistry. NMR spectroscopy and X-ray crystallography resolved the problem of its structure and showed that it is the 1,2,5-oxadiazole 2-oxide (1). Furoxan derivatives can consequently exist as a pair of position isomers when the substituents at the ring are different. Conventionally it is numbered assigning 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

132

6 The NO-releasing Heterocycles

H

H

4

3

N

+ N O

5

2

O

1

1 Structure 6.1

the 2-position to the "pentavalent" nitrogen atom. The controversy over its structure, its chemistry, and the reactivity of its side-chain functional groups, have been exhaustively reviewed [6–9] and are also the subject of a very complete two-volume monograph [10,11]. The readers are referred to this bibliography for complete information, not only on the very extensive chemistry of furoxans, but also for references to additional reviews, including those published before the true structure of the furoxan system was known. The pharmacological properties of furoxans have also recently been reviewed [12]. 6.1.1.1

General Properties

The simple unsubstituted furoxan system has recently been synthesised [13]. X-ray analysis indicates that the ring is planar and shows the typical structural features observed for many other furoxans, namely an unusually short N(2)–O(exo) bond (1.240Å), in keeping with the strong IR absorption í=1620s(cm−1 ) (N+ –O− ), and a rather long O(1)–N(2) bond (1.44Å). It is a colorless stable liquid, as are furoxans substituted with simple alkyl groups, while the diaryl and the alkyl-aryl substituted derivatives are crystalline white solids; all these substances are insoluble in water. ClogP algorithm analysis of a number of octanol–water partition coefficients of differently substituted products has extended the possibility of calculating log P values to the whole class of furoxan derivatives [14]. Furoxans are very feeble bases, even when an amino group is attached to the ring. 4-Amino-3-methylfuroxan behaves as a Hammett base [15], as does benzofuroxan (2, R=H) [7]. By contrast, the two methylfuroxan carboxylic acid isomers show equal high dissociation constants [9]. The electronic constants for the 3-methylfuroxanyl and 4-methylfuroxanyl moieties (3 and 4) have been measured; both appear to be strong electron-attracting groups 4

H3C

3

5

N

6

O N+ 1 O

2

R 7

2 Structures 6.2–6.4

H3C

- +N N O O 3

+ N

N O 4

O

-

6.1 Heterocyclic N-oxides

by inductive effect, but only a weak mesomeric electron-withdrawing effect has been found [16]. In general, furoxans are fairly stable compounds in acid solution but are sensitive to bases [6, 9]. This is true in particular for the parent ring and for the 4- and 3-monosubstituted compounds. The former undergoes extensive decomposition, while the latter two produce á-hydroxyimino substituted nitrile oxides 5 and á-nitro substituted nitriles 6, respectively. 4-Aryl-3-methylfuroxans 7, unlike their 4-methyl isomers, give Angeli’s rearrangement, namely they are converted to the corresponding 3-arylisoxazolin-4-one oximes 8, by the action of concentrated alkali hydroxides or alkoxides (Scheme 6.1). R

H + N

N O

R O

+ C N O

C

-

N

R

H R CH C N

+ N N O O

OH

NO2 6

5 Ar N

CH3

NOH

Ar

+ N O O

N O

7

8

Scheme 6.1 Decomposition in alkali solution of 4- and

3-monosubstituted furoxans and 4-aryl-3-methylfuroxans.

The asymmetrically substituted furoxans 9a and 9c undergo interconversion under the action of heat [6, 10]. The dinitrosoethylene structure 9b has been proposed as an intermediate (Scheme 6.2). This equilibrium has been studied in depth, in particular in 1,1,2,2-tetrachloroethane solution. Electron-releasing groups consistently favor the 4-position, while a similar preference for the 3-position is not in general observed for electron-withdrawing groups. The energy barriers for interconversion are lower R1

+ N

N O

O

-

C

ON

NO 9b

(R1)

(R1)

5

N

R

-

+ O

R

N

N O 9c

(R1) NO

7

R 6

O N+ O10a

R1

R C

9a

4

R

R1

R

NO 10b

10c

Scheme 6.2 Interconversion between the furoxan isomers 9a, 9c and between the benzofuroxan isomers 10a, 10c.

O N+ O N

-

133

134

6 The NO-releasing Heterocycles

when the ring bears an electron-releasing substituent, and thus great care must be paid to selecting and controlling the temperature in reactions with furoxans, in order to avoid thermal isomerisation. In general this process is a very important route to obtain the other asymmetrically substituted derivative when only one is available. NMR spectroscopy is the principal tool to determine the structure of a pair of furoxan isomers [6, 10]: the N-oxide group exerts a shielding effect on the C and H nuclei linked to the 3-position, compared to the same nuclei linked to the 4-position in the other isomer. This is true also for the hydrogen nuclei of C(H)n groups. This shielding influence is exerted also on more distant protons, but the effect is attenuated. Another useful probe for structural assignments in furoxan isomers is the big difference in chemical shifts of the ring carbon atoms. In fact the 3-carbon atom shows a strong upfield shift compared to the 4-carbon (e.g. 113.8ppm and 156.1ppm respectively in dimethylfuroxan). The fusion of the benzene ring at the 3–4 bond of the furoxan system dramatically decreases the energy barrier of thermal interconversion, with the consequence that, in benzofuroxan derivatives 10a and 10c, the equilibrium is active at room temperature [6, 7, 10] (Scheme 6.2). In 5(6)-substituted benzofuroxans, there are a number of reasons for the preference for a tautomer at equilibrium. Nevertheless, the presence of an electron-withdrawing group in 5(6)-substituted benzofuroxans frequently favors the 6- tautomer over the 5- tautomer. In 4(7)-substituted benzofuroxans the steric interactions between the substituent and the N+ –O− moiety play important roles, and thus the 4-isomer is frequently favored at equilibrium. Light also induces furoxan isomerisation [6, 10]. Photo-equilibration is different from thermal-equilibration, and is strongly influenced by the wavelength of the light. For example, irradiation of both isomers of amino-phenylfuroxans in ether with 254nm light produces a mixture in which the two products are present in similar amounts. By contrast, the 3-amino derivative is quantitatively converted to the 4-isomer on irradiation with light >300nm. 6.1.1.2

Synthesis

A very large number of furoxan derivatives have been synthesised, taking advantage of the reactivity of side-chain functional groups in the ring [6, 9, 11]. Here, the principal procedures used to synthesise the furoxan system will only be discussed briefly [6–8, 10] (Scheme 6.3). 6.1.1.2.1

Dimerisation of nitrile oxides This is the principal route to obtain symmetrically substituted furoxans. The reaction normally occurs by heating the isolated nitrile oxide 11 or by keeping it in a neutral medium. This reaction is fast and is the main reason why it is difficult to conserve isolated nitrile oxides.

6.1 Heterocyclic N-oxides M + (CNO)

-

16

M = Ag Hg/2

+ R C N O

Halogen = (R)2

R - HCl

11 R

O

- H2O

NO2

C N

R CH2 NO2 NX OY

OH 12

OH

+ N O-

- HNO2

R

N 13

R

N

Cl

C

14

+ R CH N2 15

Scheme 6.3 Synthesis of symmetrically substituted furoxan derivatives.

Owing to the instability of nitrile oxides, they are frequently generated from stable precursors. A common procedure starts from nitrolic acids 12 which, on heating or by the action of sodium bicarbonate, spontaneously lose nitrous acid giving the corresponding furoxans. Nitrolic acid is sometimes generated in situ. A typical example is the treatment of both aliphatic and aromatic methyl ketones with N2 O4 to give diacylfuroxans. Other precursors of nitrile oxides are hydroxamic acid halides 13. Under the action of bases they easily produce nitrile oxides. Primary nitro compounds 14 also produce nitrile oxides on dehydration. Diazocompounds stabilised by an electron-withdrawing group 15 and fulminates 16, treated with nitrosating agents and halogens respectively, are also used as precursors of nitrile oxides. 6.1.1.2.2

Dehydrogenation of á-dioximes (glyoximes) The dehydrogenation of dioximes symmetrically substituted (17, R=R1 , Scheme 6.4) with a variety of oxidants, including potassium ferricyanide, halogens, hypohalides, nitric acid, nitrogen dioxides, and lead tetracetate, is a common route to symmetrically substituted furoxans. In the case of dioximes bearing different substituents (R≠R1 ) the furoxan isomer obtained can depend on the configuration of the starting dioxime. The classic example is the oxidation of p-methoxybenzil dioxime with ferricyanide (Scheme 6.5); this reaction affords the 4-phenyl furoxan isomer 18 in a regiospecific manner if the dioxime amphi-form 19 is used, the 3-phenyl isomer 20 is produced if the amphi-

135

6 The NO-releasing Heterocycles

136

R C

C

N

N

R1

R1

R Ox

N

N O

OH OH

+ O

-

17 Scheme 6.4 Dehydrogenation of substituted dioximes. C6H5

C

C6H4OCH3

C

N

C6H5

+ N N O O

N OH

C6H4OCH3

OH

C6H5

C6H4OCH3

- +N N O O

C6H5 HO

(Amphi form )

C N

C HO

C6H4OCH3

N

(Amphi form )

18

19

20

21

HO C6H5 C C HO

N N

C6H4OCH3

N

C6H5 C C

C6H4OCH3

N

OH

OH (Anti form )

(Syn form )

22

23

18 + 20

Scheme 6.5 Dehydrogenation of p-methoxybenzil dioxime.

form 21 is used, and a mixture of 18 and 20 when the oxidation is conducted on either the anti-form 22 or the syn-form 23 [17]. This stereospecific oxidation does not occur for all dioximes, probably due to isomerisation of the dioxime during the reaction or to different reaction mechanisms involved in the use of different oxidants. When the lipophilic–hydrophilic balance of the two furoxan isomers is appropriate, they are easily separated by chromatography or fractional crystallisation. For example, the synthesis of 4-hydroxymethyl3-furoxancarboxamide (CAS 1609), one of the most promising furoxancarboxamide vasodilators (see later), passes through the intermediate formation of a mixture of the two isomeric methyl hydroxymethylfuroxancarboxylic esters, which can easily be separated by recrystallisation from isopropyl acetate [18]. 6.1.1.2.3

Action of nitrogen oxides on olefins Addition of N2 O3 to alkenes is another efficient method to produce furoxans. The reaction goes through the intermediate formation of “pseudonitrosites” 24 which are converted into á-nitrooximes 25 (Scheme 6.6) which, in turn, by dehydration afford furoxans.

6.1 Heterocyclic N-oxides R1

R R CH CH R1

HC CH ON NO2

HON

R1

R

R1

R

C CH NO2

+ N

N O

O

25

24

Dimer Scheme 6.6 Production of furoxans from olefins under the action of N2 O3 .

Pseudonitrosites can sometime be isolated and converted into furoxans by heating in alcohol or water. In some cases the reaction is carried out with a mixture of nitrogen oxides; for example, 4-nitro-3-phenylfuroxan is obtained by the action on cinnamaldehyde of nitrogen oxides generated by a mixture of nitric and arsenious oxides [9]. 6.1.1.2.4

Other methods Other methods can be used to prepare the furoxan system; the most common are spontaneous decomposition of azidonitroolefins 26, dehydration of á-nitrooximes 25, thermolysis of o-nitro phenylazides 27 and oxidation of o-nitrosubstituted aromatic amines 28 (Scheme 6.7). All these procedures have been discussed in detail in Ref. [10]. The latter two methods are of paramount importance in the synthesis of benzofuroxan derivatives. R

R1 C C

N3

NO2 26

- N2

R

R1

R

+ N

N O

N3

- H2O

O

- N2

Ox

R

O2N

2

25 NO2 27

H2N 28

Scheme 6.7 Other methods to prepare the furoxan and benzofuroxan system.

6.1.1.3

NO-release

The capacity of furoxan derivatives to behave as NO-donors was first demonstrated by Feelisch et al. [19], who showed that furoxan derivatives produce nitric oxide when dissolved in physiological solution in the presence of thiols. Among the reaction products, they isolated nitrite and, in lesser amounts, nitrate, which are the final oxidation products of nitric oxide in aerobic water solution, as well as dioxime derivatives, which are the reduction products of the furoxans. They also evidenced a marked pH-dependent production of S-nitrosothiols. Working with N,N′-diisopropylfuroxan3,4-dicarboxamide (29, Ipramidil) and an excess of glutathione (GSH), the amount of S-nitrosoglutathione formed increased with increasing pH until pH9, above which it

137

138

6 The NO-releasing Heterocycles H3C

CH3

CH

NH HN CH CH3 C C O O + N N O O

H3C

29

Structure 6.29 R1 R2

R2S

R1 N

R2SH

R1

R

N

N

OH

OH

R1

R

S

R2

N - N+ O O-

R

R1

R

S

S N

N O

O

-NO -

R2

N O

Ox

-

R2SH

R R2SNO

+ N O O R2S

-

NO

R2SH

R

R1

R1

R2

N

N O

O

NO2

[NXOY]

-

+ NO3

Ox

R

C CO N S

S

.

-NO -

R2

O N

R

R1 N

S

R2

O

Scheme 6.8 NO-release from furoxan derivatives in the presence

of thiols. Adapted from Ref. [19] with permission.

decreased, probably due to the increasing decomposition of furoxan under the action of the basic pH. The rate of NO formation in the reaction between l-Cysteine (l-Cys) and a fixed amount of Ipramidil increased as the l-Cys/furoxan ratio increased and became constant for ratios above 50:1. A reasonable mechanism to justify their findings has been proposed (Scheme 6.8). This mechanism implies the attack of the thiolate anion on the 3- or/and the 4position of the ring, with formation of intermediates that produce nitroxyl anion (NO− ). This NO-redox form may be oxidised by oxygen to NO• that, in its turn, is oxidised to nitrite, and in lesser amounts, to nitrate, through the intermediate formation of oxygenated nitrogen species. These are strong nitrosating species that can interact with the thiols present in the solution giving nitrosothiols. These last products can decompose giving nitric oxide. In the case of the attack of the thiolate anion at the 3-position, nitrosothiols may also be formed by direct interaction between thiols and the intermediate derived from the opening of the furoxan ring. The reaction between 4-phenyl-3-furoxancarbonitrile and an excess of thiophenol in water solution at physiological pH has been closely studied [20]. Nitrite and the 5-amino-3-phenyl-4phenylthioisoxazole (30) were isolated in similar yield among the reaction products.

6.1 Heterocyclic N-oxides

139

This suggests that the attack of the thiolate anion, at least with this product, occurs principally on the 3-position of the furoxan ring. An alternative mechanism to that discussed above was proposed to explain NO-donation by this product. It implies the preliminary cleavage of the 1–2 bond of the furoxan ring, rather than of the 2–3 bond as suggested by Feelisch, to give a tertiary nitroso intermediate. Reasonable mechanisms may be put forward to explain the production of different NO-redox forms from this intermediate [20] (Scheme 6.9). Interestingly, some furoxans, such as 31 and related compounds, produce NO, detected as nitrite, spontaneously without the assistance of thiols [21].

S

C N + N

N O

-

C N

C N O

N

N O

S

S

S

O

N

-

+

N

- NO O

O 30

Scheme 6.9 Reaction between 4-phenyl-3-furoxancarbonitrile and an excess of thiophenol.

In conclusion, either thiol-induced or spontaneous NO-release from furoxans, depending on the substituent at the ring, can occur at physiological pH values through complex mechanisms that may involve more than one redox form of NO. Additional careful investigation is required to clarify these processes. NO-release from furoxans in cells and tissues may be thiol induced, but could also occur through enzymatic activation; this aspect of the pharmacochemistry of furoxan has been very little investigated to date. 4-Phenyl-3-furoxancarbonitrile and its 3-phenyl isomer have been found to increase the concentration of the soluble guanylate cyclase (sGS) in cultured rat lung fibroblasts (RFL-6-cells) in a manner that is dependent on the intracellular concentration of thiol groups [22]. Enzymatic activation to release NO cannot be excluded for furoxans such as the furoxancarboxamide 32. This product was found to dilate segments of rabbit femoral artery precontracted with phenylephrine, in a dose-dependent manner [23]. When the experiments were repeated with segments preincubated with Proadifen, an inhibitor of cytochrome P450, a significant rightwards shift of the concentration-response curve was observed, indicating possible involvement of this enzyme in NO-release. CH3 SO2

+ N

N O

CO

S CH2 CH2 N CH3 O

-

31

Structures 6.31, 6.32

+ N

N O 32

NH CH2 N

O

-

NH2

NO2

140

6 The NO-releasing Heterocycles

6.1.1.4

Biological Actions

Many furoxan derivatives display typical NO-donor dependent biological activities [12]. In particular they stimulate the sGC and trigger both antiaggregatory and vasodilating action. Ipramidil (29) and the phenylsulfonyl substituted furoxan 33 are among the first furoxans that were reported to activate sGC from rat liver, in the presence of l-Cys, and in human platelets, respectively, in both cases in a dose-dependent manner [19, 24]. Their potency as stimulators was much decreased by the presence of oxyhemoglobin (HbO2 ), a potent nitric oxide scavenger. As a consequence of these findings, a large series of phenylfuroxans 34 substituted with diverse electron-withdrawing and electronreleasing groups and the two 3,4-bis(phenylsulfonyl) and 3,4-bis(cyano) derivatives 35 and 36, were studied for their capacity to activate partially purified rat lung soluble guanylate cyclase in the presence of l-Cys [25]. Broadly speaking, the presence at the ring, in particular at the 3-position (derivatives 34a), of electron-withdrawing substituents such as NO2 , CN and SO2 C6 H5 induced potent enzyme activating action. This is in keeping with the hypothesis that the thiol induced NO-release from furoxans in physiological solution is due to an initial nucleophilic attack on the ring by the thiolate anion. These products were also found to trigger strong vasodilating activity on rabbit aorta rings precontracted with noradrenaline, and potent inhibitory effects on collagen-induced human platelet aggregation.

R1

SO2

H3C

+ N

N O

O

-

+ N

N O

O

R1 -

-

a 33

O

+ N

N O

O2S

SO2 + N

N O

O

N C -

C N + N

N O

O

-

b 34

35

36

Structures 6.33–6.36

There is no doubt that the classes of furoxan derivatives most extensively studied for their NO-dependent activities are the furoxans condensed to the benzene ring and to heterocyclic systems, the furoxan sulfones, the cyano substituted furoxans and the furoxancarboxamides. 6.1.1.4.1

Condensed furoxans Benzofuroxans (2), benzodifuroxan (37), and benzotrifuroxan (38) are systems that have long been known [6–8,10,11]. Early studies showed that 37 is a potent in vivo and in vitro vasodilator, as are its furazanobenzofuroxan and thiazolobenzofuroxan analogues [26]. Structural comparison between 37 and the extended glyceryl trinitrate (GTN) conformation has shown a close similarity in the relative position between 1 and 3 positioned NO2 moieties in GTN and the O–N+ –O− substructures in the furoxan

6.1 Heterocyclic N-oxides

rings. More recently the product was found to be a potent activator of the sGC in C6 cells and in human platelets and a potent in vitro vasodilator [27, 28]. Both these activities decreased markedly when the experiments were conduced in the presence of either 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a well known inhibitor of the sGC, or HbO2 . By contrast, the stimulating effect on platelet sGC increased in the presence of dithiothreitol [28]. The benzotrifuroxan (38) is an increasingly potent activator of sGC in C6 cells, and an in vitro vasodilator, but 2, and its 4(7) and 5(6) CH3 and CN substituted derivatives, have shown feeble activity. This behavior seems to be correlated with the NO-donor properties of the products. In fact 2 (R=H) and its substituted derivatives did not produce nitrite when incubated with l-Cys in water solution at physiological pH and temperature, while 37 and 38 gave large amounts of nitrites (45% and 96% respectively). Both NO• and HNO were detected in the incubates of the two products by chemiluminescence and gas chromatography. The trimer furoxans 39 and 40, in which R is alkyl, aryl, alkoxy, and CH2 –X (X=Cl, OH, OMe, NH2 ) groups can be formally considered open models of 38. These substances dilate rat aorta strips precontracted with noradrenaline in a dose-dependent manner, in some cases more effectively than GTN [29]. The products 39, in which a 4,3′:4′,4′′-connection is present between the three furoxan rings, are more potent than the corresponding analogs 40 that have a 3,3′:4′,3′′-connection. The activity of all these products is decreased by the presence of HbO2 or of methylene blue (MB), another inhibitor of sGC, and it parallels the capacity of the substances to produce nitrite in the presence of thiols. The products 39 and 40 (R=CH3 , CH2 NH2 ) were also tested for their in vitro antiplatelet activity, and in vivo for their vasodilating behavior [30]. Again the derivatives 39 were the most potent antiplatelet agents. All products showed hemodynamic profiles similar to those of other known NO-donors when administered intraduodenally (i.d.) to anesthetised pigs. They lowered the heart preload and afterload, with minimal or no effect on contractility and heart rate. 4,7-Dimethyl-1,2,5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (41) is also an old product [7, 11, 31] that has recently been found to react with GSH to give S-nitrosoglutathione, NO• and HNO [32]. It stimulates partially purified rat lung soluble guanylate cyclase, but not the heme-deficient enzyme. The activation is inhibited by ODQ. The product also displays significant vasodilator activity on rat thoracic aorta rings at nanomolar concentrations. Finally, [1,2,5]oxadiazolo[3,4-d]pyrimidine-5,7-dione 1oxide derivatives (42, R,R1 =CH3 ,H) release NO, detected as nitrite, in the presence of thiols. A mechanism for this release has been proposed [33]. 6.1.1.4.2

Furoxan sulfones and carbonitriles A number of arylsulfonylfuroxan derivatives 43 (R = alkyl, aryl, alkoxy), have been studied, principally for their in vitro vasodilating and antiaggregatory properties [34, 35]. The involvement of NO in these actions has, in general, been evidenced through decreased activity in the presence of ODQ and/or HbO2 . The derivatives bearing the arylsulfonyl groups at the 3- position of the ring proved to be more potent than the 4-arylsulfonyl substituted isomers, and the pair of isomeric phenyl-phenylsulfonylfuroxans (43a,b R=C6 H5 ) was more potent than the cor-

141

142

6 The NO-releasing Heterocycles O N - +N O

+ O O N N

N O

-

+ O O N O N+ O-

O

N+ Oa

b

- + O N

N+ O-

37

O +N

O N+

3"

3 4

N O

R R N O

O

4"

+N

N

3' 4'

+ N

O N 38

R R

O

-

N

N

N

-

N O

O

N

4

4"

3

3" 3' 4'

- +N N O O

O N+ O-

40

39 Structures 6.37–6.40

-

CH3

O

O + N

R1

N

N

O

-O N +

N+ O-

CH3

N

O

ON+ O N

R

41

42

Structures 6.41, 6.42 R

SO2Ar N+

N O

O

SO2Ar

R

- +N N O O

-

a

b 43

Structure 6.43

responding methyl analogs (43a,b R=CH3 ).The most potent product was the 3,4bis(phenylsulfonyl) substituted furoxan 35. The pharmacological profile of CHF 2206 (43a Ar=R=C6 H5 ) [36] and of CHF 2363 (43a Ar=C6 H5 , R=OC2 H5 ) [37] have been closely examined by the Chiesi Company. CHF 2363 did not show in vitro cross tolerance with GTN and, when administered to anesthetised rats by intravenous infusion, was a more potent vasodilator than isosorbide-5-mononitrate (IS-5-MN), which was taken as reference (Fig. 6.1). A number of water soluble sulfonyl derivatives (44, X=SO2 , n=2,3) and some related compounds (44, X=O,S, n=2,3) bearing a chain with a basic function at the ring, have

6.1 Heterocyclic N-oxides

(a)

(b)

% Relaxation

100 µmol kg-1 min-1

% change in MAP

50

0 -10.0

-7.5

-5.0

-2.5

log (conc. (M)) Fig. 6.1 (a) in vitro GTN tolerance and CHF 2363 cross-tolerance: concentration–response curves for relaxation of noradrenaline-contracted rat aortic strips by GTN ({) and CHF 2363 (Ä) before (open symbols) and after (solid symbols) 90min exposure to 550ìM GTN. (b) Effect of

CHF 2363 on mean arterial pressure: peak effect induced on mean arterial pressure (MAP) by intravenous infusion of CHF 2363 (open columns) and IS-5-MN (cross-hatched columns) in the anaesthetised rat. Adapted from Ref. [37] with permission.

been studied for their ability to relax rat aorta strips precontracted with noradrenaline [21]. R1 R

X

N

N O

+

CH2 n

N R1

O

a

R1 R

X

- +N N O O

CH2 n

N R1

b 44

Structure 6.44

The vasodilating potencies were, to a great extent, modulated within the series. A number of these products have been studied in vivo at the Cassella-Hoechst laboratories [38]. The best product was found to be derivative G95-6527 (44a, R=C6 H5 , X=S, n=2, R1 =CH3 ); when administered i.d. to anesthetised pigs it induced a significant decrease in both systolic and diastolic blood pressures (Fig. 6.2). The onset of the action was smooth and the maximum effect was reached after 30min and lasted for over 2h. The high potency in stimulating rat lung soluble guanylate cyclase sGC, and the potent in vitro vasodilating and antiaggregatory activities shown by 4-phenyl3-furoxancarbonitrile [20], prompted the synthesis of other furoxancarbonitriles 45 in which R is an alkyl, aryl, aminoalkyl, and alkoxy groups. The whole series of products was tested on rat aorta strips precontracted with noradrenaline [39]. Most of the 3-CN members of the series (45a) were more potent than the 4-CN isomers

143

6 The NO-releasing Heterocycles

Haemodynamic profile (anaesthetized pig - 3 mg/kg i.d.) 10 0

∆ BP(mm Hg)

144

-10 -20 -30 -40

Fig. 6.2 Hemodynamic profile of G95-6527:

-50 0

30

60

90

120

min

150

180

systolic blood pressure BPs ( ) and diastolic blood pressure BPd ( ) (P. Martorana, personal communication).

(45b). The hemodynamic profiles for a number of these compounds were evaluated on anesthetised pigs after i.d. administration, and were consistent with that of CAS 1609 (see below), suggesting similar in vivo NO-release characteristics. R1

O R

C N N

N

+

O

O

R

C N

- +N N O O

-

a

b 45

R

O

C N N

N O

R R2

+ O

+ -O N

-

a

R1

C N N

R2

O b

46

Structures 6.45, 6.46

6.1.1.4.3

Furoxancarboxamides Perhaps the most studied furoxan derivatives are the furoxancarboxamides 46 in which R, R1 , R2 are represented by a variety of groups. The Cassella-Hoechst laboratories have studied hundreds of these products. A number of patents cover their synthesis and vasodilating activities [40]. During this research, a number of structure–activity relationships emerged. The most active compounds appear to be the 3-furoxancarboxamide isomers, and the possibility of an internal hydrogen bond interaction between the exocyclic oxygen and the carboxamide function appears likely [18]. CAS 1609 (46a, R=CH2 OH, R1 =R2 =H) emerged as a suitable candidate for clinical studies [41]; administered i.d. or intravenously (i.v.) to anesthetised pigs, it decreased heart preload and afterload, cardiac output, left ventricular work and myocardial oxygen consumption, all in a dose-dependent manner (Fig. 6.3). The onset of the effects was smooth and the duration long. Long-term administration in conscious dogs did not develop tolerance, unlike the IS-5-MN taken as a reference (Fig. 6.4). The lack of tolerance appears to be one of the most important determinants of furoxans as vasodilators [23, 37].

6.1 Heterocyclic N-oxides

Fig. 6.3 Hemodynamic profile of CAS 1609 on

anesthetised dog (0.3mgkg−1 i.v.): systolic blood pressure (BPs), diastolic blood pressure (BPd), left ventricular end diastolic pressure (LVEDP), diastolic pulmonary artery pressure (PAPd), heart rate (HR), left ventricular

contractility (dp/dt), cardiac output (CO), total peripheral resistance (TPR), stroke volume (SV), left ventricular stroke work (LVSW), and myocardial oxygen consumption (MVO2 ). Adapted from Ref. [41] with permission.

C AS 1 6 09 (2 × 0 .5 m g /k g)

IS MN ( 3 × 10 mg /k g )

∆ B Ps (mm H g)

d ay 1

da y 5

d ay 3

10

10

10

0

0

0

-1 0

-1 0

-1 0

-2 0

-2 0

-2 0

-3 0

-3 0

-3 0

-4 0

-4 0

-4 0

0

6

12

0

6

12

0

6

12

h ou r s

Fig. 6.4 In vivo tolerance study of CAS 1609 and IS-5-MN (H. Bohn, personal communication).

6.1.1.5

NO-donor Hybrid Furoxans

A very recent development in the field of NO-donors is the design of drug/NO-donor hybrids [42]. These products are obtained by joining a drug, or an appropriate part of it, to an NO-donor moiety, either directly or through an appropriate spacer. The link is generally stable but may sometimes be vulnerable to metabolic cleavage. The resulting product may retain the pharmacological properties of the parent drug while also displaying new activities due to its new ability to release NO. These hybrids are

145

6 The NO-releasing Heterocycles

146

O H3C

n = 2, 3, 4

R1 = CH3 , CONH2 , CN

CF3 O2N

SO2Ph

CO2(CH2)nO N

H3C

N

O

CH3

N

O

+ O

O

N H

O CH2

N

R1

N

H

+ N

N

47

48

CH3

CH3

R = SO2C6H5, C6H5

O C O

-

O (CH2)3

O

O

R

N

N

C

+

O

O

O

N

O

N

+

1 + O

b

SO2Ph

N

R

O

(CH2)3 O N

CH2 N

49

H3C S CH2

O

O

-

a

N H

O

R1 = CH3 , CONH2 , CN

O C

O C

O

NO2

O

N

CH2 CH2 X

N

N

CH3

O

-

50 R1

+

O

R = C6H5, SO2C6H5 R = CH3, CONH2, CN

R

+ N

N O

O

X=O X = NHCH2

-

51

N O

O CH3OOC H3C

CH2

N

COOCH3 N

CH3

H

52

Structures 6.47–6.52

absorbed, distributed and excreted as a single unit, which provides an advantage over the simultaneous administration of the two components of the hybrid. Many drugs have been hybridised with furoxan moieties and the resulting products studied for their dual activity. They include á1 -antagonists, â1 -antagonists, Ca2+ -channel blockers, K+ -channel activators, nonsteroidal anti-inflammatory drugs (NSAIDs), H2 and H3 -antagonists. All these products have been reviewed [4, 12]. More recently, Ca2+ -channel activators [43], REC 15/2739, a uroselective á1 -antagonist [44], aspirin [45], rabeprazole, a potent inhibitor of H+ /K+ -ATP-ase enzyme [46], were linked to furoxan moieties to give hybrids 47–50 respectively. A number of reports, both old and recent, describe antiviral, antimicrobial, antiparasitic, and antitumoral properties of a number of furoxan derivatives [7, 11, 12]. Since it is known that nitric oxide displays potent cytotoxic actions in the immune

6.1 Heterocyclic N-oxides

system [47], it is possible that these actions are at least partially dependent on the product’s NO-donor properties. No demonstration has yet been reported of the involvement of NO in such activities; however, a series of hybrid furoxan derivatives linked to metronidazole have recently been proposed on this basis [48]. The products 51 displayed potent anti-Helicobacter pylori activity, in particular against metronidazole resistant strains. However, the potential contribution to this activity of the redox properties and/or of the NO-release associated to the furoxan system did not emerge clearly. In fact, the furazan (1,2,5-oxadiazole) analogs, which have different redox behavior and do not release NO, behave similarly. A problem in hybrid design is its “balance”; in fact, when two pharmacophoric groups are joined to produce a hybrid, the biological properties of one group must not prevail over those of the other, since otherwise the hybrid is unbalanced. The furoxan system appears to be a flexible tool to reach this objective. In fact the NOreleasing properties of the furoxan system can be modulated by changing the substituent at the ring. Examples of substituted furoxan system used to balance a hybrid are compounds 52. Here, differently substituted furoxan moieties were joined with the 4-phenyl-1,4-dihydropyridine pharmacofore group, which blocks the voltage dependent l-type calcium channels. All these products were found to dilate rat aorta strips precontracted with a K+ -ion solution in a dose-dependent manner [49]. Analysis of the dose-response curves obtained in the presence of ODQ showed that product 52 where R=CH3 was principally a Ca2+ -blocker, product 52 where R=CN was principally a NO-dependent vasodilator, and product 52 where R=CONH2 was a well balanced hybrid with vasodilating properties, due to its capacity both to block the Ca2+ -channels and to release NO. 6.1.2

3,4-Dihydro-1,2-diazete 1,2-dioxides (1,2-diazetine 1,2-dioxides) 6.1.2.1

Generalities

The simple 3,4-dihydro-1,2-diazete 1,2-dioxide (1,2-diazetine 1,2-dioxide, DD) system 53 is unknown. 3,3,4,4-Tetramethyl derivative 54 was the first compound of the class to be synthesised [50]. It has an unusually low triplet energy and is a useful triple quencer [51, 52]. Many other derivatives of 53 have recently been prepared in view of their NO-donor properties. H H

+ O N N + O

H H

-

-

53

Structures 6.53, 6.54

H3C CH3 + O N N H3C CH + O 3 54

147

148

6 The NO-releasing Heterocycles

6.1.2.2

Synthesis

Oxidation of the 1,2-bishydroxylamines 55 produces the corresponding DDs (Scheme 6.10). When in 55 both hydroxylamino groups are at tertiary carbon atoms, the reaction can be conducted using sodium periodate or bromine as oxidant, to give the DDs 57 [50, 53]. The oxidation is thought to produce vicinal dinitroso compounds 56, which as a consequence of the intramolecular interaction of the two nitroso groups afford the final DDs [54, 55]. Sodium hypobromite is used when one hydroxylamino group is at a tertiary and the other at a secondary carbon atom, as well as with 1,2bishydroxylamino derivative of cycloheptane, to give DDs 58 [54, 56]. Oxidation of 55 (R1 , R2 , R3 , R4 =CH3 ) with an excess of NaNO2 in acid media yields 54, through a different mechanism [57]: the 1,2-bis(nitrosohydroxylamine) 59 is formed as a stable intermediate. This product can be isolated using less than one equivalent of NaNO2 , further reaction with an excess of NaNO2 or MnO2 leads to 54. á-Hydroxylamino substituted oximes 60 (Scheme 6.11) are oxidized with sodium hypohalogenites to produce 3-halogeno substituted DDs 62 [58–60]. Again the reaction is thought to produce the intermediate vicinal dinitroso compounds 61. The dimer of á-nitrosooxime 63 was isolated from the reaction mixture, together with the expected DD 62 (R1 =R2 =CH3 , X=Br) when 60 (R1 =R2 =CH3 ) was treated with sodium hypobromite [59]. Finally, oxidation of 63 with HNO3 or N2 O4 gave the nitroderivative 64 [54]. R3

R2

R4

R3

C

NHOH

Br2

C

NHOH

or NaIO4

R4 C

R2

NO

C

R1

R1

R4

R2 R1

NaOBr

56

55

NO

R3

+ O N

-

N + O-

R1, R2, R3, R4 = Alkyl

57

NaNO2/H +

R3 H3C

H3C

CH3

NO

C

N

OH

C

N

OH

CH3

H

R2 R1

NO

59 NaNO2/H + or MnO2

54

Scheme 6.10 Synthesis of DDs.

+ O N

-

N + O58

R1 = R2 = CH3; R3 = CH3, C2H5, C6H5 R1 = CH3; R2 - R3 = (CH2)4 R1 = H; R2 - R3 = (CH2)5

6.1 Heterocyclic N-oxides R1 X

R1

R2

C

N

OH

C

NHOH

NaOX

R2

CH3 60

R1 X

C

NO

C

NO

+ O N

-

CH3

N O R2 CH3 + -

61

62

R1 = Alkyl, C6H5; R2 = Alkyl R1 - R2 = (CH2)5, (CH2)4 X = Br, Cl

Dimer

H3C H3C

H3C NO2 + O N

N OH C C

HNO3

NO

or N2O4

CH3

N H3C CH + O 3

63

-

64

Scheme 6.11 Synthesis of 3-halogeno and 3-nitro substituted DDs.

6.1.2.3

NO-release

Early studies showed that tetramethyl substituted DD 54 produces NO• either by photolysis in acetonitrile with 2537Å light, or by vapor phase pyrolysis at 250°C (0.01mmHg) [50], and that a number of DDs decompose into olefins and nitrogen oxides when refluxed in benzene [59]. More recently, DDs have been found to release nitric oxide spontaneously following Eq. (1) when gently heated in a neutral water solution [61]. Release over 10min was evaluated by detecting nitrites, the major oxidation products of NO• in aerobic water solution, by a polarographic method. R3

R4

R2 R1

+ O N

-

N + O-

R1

R4

+

C C R2

2 NO

R3

(1)

Nitric oxide release was confirmed by EPR spectroscopy using imidazoline nitroxide and imidazoline N-oxide nitroxide derivatives as spin traps for NO• [56]. Through this technique the NO-release rate was evaluated for a number of DDs, both in buffered water (pH7.5) and in dimethyl sulfoxide (DMSO). In the case of 3-halogeno derivatives 62, the NO-release was found to be strongly influenced by the presence of thiols [60]. This interaction was monitored by HPLC and chemiluminescence. The decomposition was shown to be dependent on pH and thiol concentration, but was only slightly influenced by the nature of the thiol. At a fixed thiol concentration, the rate was proportional to the DD concentration. In contrast, at fixed DD concentration, there was a complex relationship between decomposition rate and thiol concentration. For example, working with a 150ìM pH7.5 buffer solution of 62 (R1 -R2 =(CH2 )5 , X=Br), an increase in the NO-release rate was found on increasing the l-Cys concen-

149

150

6 The NO-releasing Heterocycles

tration from 10−5 M to 10−3 M. A further increase in the l-Cys concentration induced a decrease in NO-release rate. No NO-release was observed when the concentration of the thiol exceeded 50mM. Similar parabolic profiles were observed with other thiols. The mechanism in Scheme 6.12 was proposed for thiol induced NO-release from 3-halogeno DDs [60]. O

R1 X

+ O N

R1 X

N R2

N+ O CH3

R2

CH3

N + O -

R1 X

R1 X

+ O N

CH3

N R2

O-

O-

CH3

62

R2

OH C

N

C

OH

R1 H2O

CH3

R2

C C

R2

N CH3

O-

R2

NO

C

- ( RS)2

CH3

- RS

69

O SR

R1 RS -

C R2

RSH

-

-

N

C

RSH

CH3

O

R1

NO

- NO

70

-X - ( RS)2

-

65 -X

R1

+ O N

RS -

N

N R2

R1

O SR

RS -

C

.

R1

N

OH C

NO CH3

N NHOH

C R2

CH3

.

68

67

66

Scheme 6.12 NO-release from DD derivatives in the presence of thiols. Adapted from Ref. [60] with

permission.

The attack by the thiolate anion on the N-oxide oxygen of 62 produces the intermediate sulfenic acid derivative 65, which, in the presence of thiols, further reacts with the thiolate anion, to give the oxime 66, which has been isolated among the reaction products. By contrast, spontaneous loss of the halide anion from 65 affords the nitroso intermediate 67 that, by losing NO• and the thiyl radical directly, or through 68, produces the á-nitrosoolefin 69. By a Michael type reaction with water this last product immediately yields the final oxime 70, which has been isolated among the reaction products. 6.1.2.4

Biological Properties

DDs display typical NO-dependent activities. They have been found to activate sGC in human platelets, to inhibit platelet aggregation and to induce acceleration of platelet disaggregation [61–64]. On isolated rings of male and female rat thoracic aorta, they trigger spasmolitic effects that, in the case of 62 (R1 -R2 =(CH2 )5 , X=Br, Fig. 6.5) are comparable to those of GTN [61–63]. They trigger vasodilating effects on perfused rat tail arteries contracted with noradrenaline and on rat mesenteral blood vessels precontracted with PGF2a [56, 60, 65]. Hereditary hypertensive rats showed a significant decrease in arterial blood pressure following administration of DDs by the

6.1 Heterocyclic N-oxides 100% 80% 60% 40% 20% 0% 58a

58b

62a

Fig. 6.5 Comparison of the effect of 58a

(R1 =R4 =H; R2 -R3 =(CH2 )5 ), 58b (R1 =R2 =R3 =CH3 ; R4 =H), 62a (R1 =C2 H5 , R2 =CH3 , X=Br) and 62b (R1 -R2 =(CH2 )4 , X=Br) on the human

62b

platelet soluble guanylate cyclase activity ( ), amount of nitric oxide formed ( ), spasmolytic activity ( ), and hypotensive effect ( ). Adapted from Ref. [61] with permission.

intraperitoneal route (i.p.) or through a catheter into the left jugular vein [56, 61, 62, 65]. The ability of the products to activate sGC frequently parallels their capacity to release NO• , with few exceptions [61–64]. Derivatives bearing a bulk substituent pattern at the 3,4 positions appear to be the most active, but the limited number of products considered makes this structure–activity relationship uncertain. In a small series of products, a rather good agreement was found between the product’s ability to release NO• and its in vitro spasmolytic activity, its in vivo antihypertensive activity, and its capacity to activate sGC [61]. However this is not a general trend. In particular, the 3-halogen derivatives 62 display strong vasorelaxing activities that are not always correlated with their spontaneous NO-release rates [60]. The effect of thiols on NO-release of these products, discussed above, undoubtedly plays a role in this behavior. 6.1.3

Other Heterocyclic N-oxides

Other heterocyclic N-oxides have been found to display biological activities that may depend on their proven or hypothesised ability to release NO; these include 4H-pyrazol-4-one 1,2-dioxides 71, 2H-1,2,3-triazole 1-oxides 72, benzotetrazine 1,3dioxides 73 and 1,2,3-benzotriazine 3-oxides 74. These systems have been less extensively studied than the furoxan and 1,2-diazetine dioxide systems discussed above. O R

R1

R2

R1

+N N + O O-

+ N

N N

O

O

-

N+

-

N

N

N+ ON

R

R

N R

71

Structures 6.71–6.74

72

73

N

74

+ O-

151

152

6 The NO-releasing Heterocycles

6.1.3.1

4H-pyrazol-4-one 1,2-dioxides (pyrazolone N,N-dioxides)

The parent heterocyclic system 71 (R=R1 =H) is unknown. X-ray analysis of the dimethyl derivative shows a remarkably long N–N bond (1.659Å) [66]. This unusual length had already been predicted by a perturbation molecular orbital (PMO) study [67]; it may be explained by the dipole–dipole repulsion between the N+ –O− groups, enhanced by the antiaromatic character of the ring system. Pyrazolone N,N-dioxides have also been studied for their ability to give 1,3-dipolar cycloadditions with various unsaturated compound [68]. A number of pyrazolone N,N-dioxides, bearing alkyl, aryl, arylalkyl and substituted-phenyl groups at the ring, have been synthesised in view of their potential capacity to release NO. The products were obtained in poor yields, by the action of NaNO2 /HNO3 on the corresponding á,â-unsaturated oximes 75 [69–71]. OH N R CH CH C R1 N OH 75

NaNO2/HNO3

71 + N N+ O-

-O

76

Structures 6.75, 6.76

These compounds have been claimed to release NO [69] and this possibility has been studied in detail for the derivative 71 (R=R1 =C6 H5 ) [70]. The product was found to release NO• spontaneously (chemiluminescence detection) when dissolved in pH7.4 buffered water solution at 37°C. Nitric oxide release was increased by the presence of an excess of either l-Cys or GSH. A number of compounds 71 display typical NO-dependent activities, such as activation of the sGC [70, 72], vasodilation [70] and antiplatelet activities [69, 71, 72] as well as antithrombotic properties in an in vivo thrombosis model [69, 72]. Some structure–activity relationships have emerged from these studies. The inhibition potencies of the products on platelet aggregation induced by collagen appears chiefly to be influenced by a combination of the electron, lipophilicity and membrane affinity properties of the substituents [69]. The corresponding oximes 76 are generally less potent than the parent oxoderivatives 71. The in vivo antithrombotic potency of the diphenyl derivative 71 (R=R1 =C6 H5 ) was found to be higher than that of the water-soluble derivative 71 (R=CH3 , R1 =C6 H4 OCH2 COOH) [69, 70]. This was explained by its better absorption from the gastrointestinal tract.

6.1 Heterocyclic N-oxides

6.1.3.2

2H-1,2,3-triazole 1-oxides

A number of 2H-1,2,3-triazole 1-oxides 72 were prepared by chemists at the Cassella Company as potential NO-donors in view of their formal structural similarity with furoxan derivatives [18]. Derivative 72a was studied in depth. It was obtained by cupric sulfate oxidation of intermediate 79, derived from the action of the substituted phenylhydrazine 78 on the oximino acetoacetic acid amide 77 (Scheme 6.13). The product was found to display weak vasodilating activity on isolated guinea pig pulmonary arteries, and possible involvement of nitric oxide in this action was suggested. CH3

CH3 H3C O

C

HO

HN C

C

O

CH CH3 O

+

O

-

N+

-

NH2

O

NH

N+

O

H N

H3C

HN

C

C

N

N

HO 77

78

C

CH CH3 O

N

79 O2/CuSO4

CH3 O CH CH3 C N H3C H + N N O N

+ O N O 72a

Scheme 6.13 Synthesis of 2H-1,2,3-triazole 1-oxide derivative 72a.

6.1.3.3

1,2,3,4-Benzotetrazine 1,3-dioxides and 1,2,3-Benzotriazine 3-oxides

A recent report shows that 1,2,3,4-benzotetrazine 1,3-dioxide nitroderivatives 73 (R=NO2 ) are thiol dependent NO-donors and potent activators of the sGC [73]. Activation of the sGC was inhibited in the presence either of ODQ or of the NO• scavenger 2(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO). All products inhibited ADP-induced aggregation of human platelets, with the potency sequence 7-nitro >5,7-dinitro >5-nitro. A patent claims both in vitro vasodilating and sGC stimulatory NO-dependent activities for some 1,2,3-benzotriazine 3-oxide derivatives 74 [74]. Compound 74

153

154

6 The NO-releasing Heterocycles

(R=CH3 ) proved to release small amounts of nitric oxide spontaneously, this release being increased by the presence of l-Cys and GSH.

6.2

Mesoionic Heterocycles

The mesoionic compounds are represented by structures that cannot be properly described by Lewis forms not involving charge separation. Typical examples are the sydnones 80; the first example was obtained at the University of Sydney by the action of acetic anhydride on N-nitrosophenylglycine [75]. They were consequently named after the Australian town [76]. These structures are best approximated as resonance hybrids. They can be represented by any contributing mesomeric structure a, b, c or by the general structure d. R + N N O

R + N O

-

a

R + N -N O

-

N

O

O b

R

O

N + N O

O

d

c 80

Structure 6.80

Some mesoionic heterocycles, structurally correlated to the sydnones and represented by a general 5-membered ring molecule that contains the NO-moiety, behave as NO-donors. They include sydnonimines 81 and mesoionic 1,2,3,4-oxatriazolium derivatives 82 and 83. The latter will be named 3-R-1,2,3,4-oxatriazolium-5-olates (82) and 3-R-1,2,3,4-oxatriazolium-5-R1 -amenates (83) [77]. R + N N O

R + N N NR1

81

N O 82

R + N N O

N O

NR1

83

Structures 6.81–6.83

For NO-release from these compounds it is essential that they decompose easily, either spontaneously or after enzymatic cleavage of stabilizing substituents at the imino group, to nitrosohydrazine intermediates that represent the real NO-precursors.

6.2 Mesoionic Heterocycles

6.2.1

Sydnonimines 6.2.1.1

General Properties

Among the NO-donor mesoinoic heterocycles, the sydnonimines have attracted by far the most interest. They were first obtained by Brookes et al. [78] and Kato et al. [79] by the action of nitrous fumes on á-(N-methyl-N-nitrosoamino)-nitriles followed by the action of acetic anhydride. Whereas sydnones represent a family of stable heterocycles, sydnonimines, with the exception of 3-hydroxysydnonimine (81, R=OH, R1 =H) that forms an inner salt [80], are unstable if not protonated to a salt or acylated to stable N-acylimino derivatives. In this case they can be stored at room temperature O

O N + N N O

- C O N

H Cl

N + N N O

O CH2CH3

Molsidomine 84

- H N

Linsidomine (SIN-1) 85 CH3

O N + N

O

N

- C N

O

N + N N O

H3C

O CH3

Ciclosidomine

Pirsidomine (CAS 936)

86

87 O

OH

CH3 H3C

O - C N

N + N N O

O

O

OH

S

HO

O

N

H3C

HO

H Marsidomine (CAS 754) 88

CH3

N + N

H Cl

N

N H

O

C4144 90

Structures 6.84–6.90

N + N N

O

C89-4095 89 S

H3C

CH3

H Cl - H N

155

156

6 The NO-releasing Heterocycles

for a long time if protected from light. At neutral conditions sydnonimines that are not substituted at the exocyclic nitrogen undergo a ring opening reaction. This instability is the reason why sydnonimines and sydnones differ in their capability to release NO, since formation of nitric oxide from the intact heterocycles may occur under photolytic conditions or under thermic stress but is unlikely to occur physiologically. The most prominent examples of sydnonimines studied as NO-donors bear a substituted amino group at the 3-position, among them Molsidomine (N-ethoxycarbonyl3-morpholinosydnonimine) 84, SIN-1 (Linsidomine, 3-morpholinosydnonimine hydrochloride) 85, Ciclosidomine (N-cyclohexylcarbonyl-3-morpholinosydnonimine hydrochloride) 86, Pirsidomine (CAS 936, N-(4-methoxybenzoyl)-3(cis-2,6-dimethylpiperidino)sydnonimine 87, Marsidomine (CAS 754, 3-(cis-2,6-dimethylpiperidino)sydnonimine tartrate) 88, C89-4095 (3-(3,3-dimethyl-1,1-dioxo-1,4-thiazane-4-yl)sydnonimine hydrochloride) 89 and C4144 (3-(3,3-dimethyl-1,4-thiazane-4-yl)sydnonimine hydrochloride) 90. The chemistry of sydnonimines has been extensively reviewed [81] and only the main methods to synthesise the biologically most interesting derivatives will be discussed here. 6.2.1.2

Synthesis

The general synthesis of 3-dialkylaminosydnonimines [82, 83] is outlined in Scheme 6.14. R R

N

N Reduction

O

R

91 R

R R

N H

C N

R

N

N

R1CHO

H

NaCN

R

H

O Hofmanndegradation

N

O

R1

93

CN

94

H HCl

O - C R 2 N

R N R + NaHCO3 N ClCOR2

N O

H

N

Cl

-

HCl

R

N O

NaNO2

R1

R

R1

H 96

CH

N H

92 R R N + N

N

R1

N

CH CN

N

95

Scheme 6.14 General synthesis of 3-dialkylamino sydnonimines.

N,N-dialkylhydrazines 93, prepared either by reduction of nitrosamines 91 or by Hofmann degradation of ureas 92 [84] are cyanomethylated with aldehydes and

6.2 Mesoionic Heterocycles

sodium cyanide to hydrazinoacetonitrile derivatives 94, that, after nitrosation and ring closure with strong acids, like hydrochloric acid, yield the N-unsubstituted sydnonimines in their protonated form 95. Treatment of these compounds with acid chlorides or other acylating agents in the presence of bases, like sodium bicarbonate or pyridine, gives the acylated 96 [85, 86]. According to this procedure 3-alkyl substituted sydnonimines have been prepared as well, starting from alkylamines instead of N,Ndialkylhydrazines [87, 88]. On the other hand N-nitroso and N-nitrosydnonimines can be prepared by nitrosation and nitration of sydnoniminium salts 95 [78, 79]. 3-Monoalkylaminosydnonimines are rarely described in the literature. The 3benzylamino-4-benzoyl derivative 98 was obtained by a reaction of 3-(N-chloracetylN-benzylamino)-4-benzoylsydnone (97, R=C6 H5 CH2 ) with liquid ammonia [89] and a small group of other 3-monoalkylaminosydnonimines 100 was obtained by the action of strong acids on the nitroso compounds 99 (Scheme 6.15) [90]. Cl R

H2C

R

O

C N + N O N O

C

NH3

O

-

O 98

N

-

H

R

H3C

99

C

N

97 R CN N H3C N CH 2 CH3 N O

O

H N+ N

HCl

H N + N N

O

H Cl

N

-

H 100

Scheme 6.15 Synthetic routes to 3-monoalkylaminosydnonimines.

Annelated sydnonimines 103, 104 have also been prepared starting from phthalazine and dihydrophthalazine derivatives 101, 102 and proved to be highly active NO donors (Scheme 6.16) [91]. 6.2.1.3

NO-release

The stability of Molsidomine 84 and SIN-1 85 in aqueous systems under acidic, basic, thermic and photolytic conditions was investigated by Asahi et al. [92] (Scheme 6.17). In alkaline solutions the carbamate group is hydrolysed and decarboxylated to 85. This product immediately opens to SIN-1A 105 that, in turn, is transformed into SIN-1C 106. The authors postulated that elimination of HNO underlies this last transformation. The stability of solutions of Ciclosidomine 86 has been studied by Carney, who found that the decomposition of Ciclosidomine is quite similar to that of Molsidomine,

157

158

6 The NO-releasing Heterocycles N

1. COCl2

N

N

N

2. TMSCN

CN 101

NaNO2

C

N + N N

O

O N

Cl

-

H 103

102

RCOCl

Base

N + N N

O

O N C R 104 Scheme 6.16 Synthesis of condensed sydnonimines.

O

O N + N N O

O - C O N

CH2CH3

N + N

OH -

N

N

O

-

H

SIN-1 84

85 spontaneously

O O N

N

CHCN

- HNO

N O

N

CH2CN

N

SIN-1C

SIN-1A

106

105

Scheme 6.17 Decomposition of Molsidomine 84 in alkaline solution.

being essentially a sequential hydrolysis followed by ring opening and cleavage [93]. He also found a rapid degradation of this compound under the influence of light. Under physiological conditions Molsidomine is rapidly converted to SIN-1, mainly by the action of liver esterases [94]. In hepatectomized rats the drug did not show any hypotensive effect, suggesting that the parent compound is inactive and the pharmacological effects are mediated by its metabolites.

6.2 Mesoionic Heterocycles

Compared with Molsidomine, both SIN-1 and SIN-1A are reported to induce similar, but more rapid hypotensive action [95, 96]. SIN-1A, after undergoing oxidation in the presence of oxygen or, in vivo, possibly by redox-active enzymes such as cytochrome C [97, 98] or by reaction with ferric myoglobin formed during reperfusion injury [99], releases NO through an intermediate radical cation. No decomposition of SIN-1A was observed in the absence of oxidants and light. Irradiation with visible light remarkably enhances the oxidative NO-release as well as the oxygen consumption from SIN-1 in buffered solutions [100]. In vitro, this reaction produces stoichiometric amounts of superoxide anion and consumes oxygen. Thus, a close correlation exists between the rate of NO-release and oxygen consumption [101]. Based on these observations an oxidative mechanism of NO-release from sydnonimines was proposed. It is exemplified for SIN-1A in Scheme 6.18. This product is attacked by molecular oxygen and transformed into the radical cation 107 while the oxygen is converted to superoxide anion. The unstable radical cation loses NO• giving the hydrazinium cation 108 which immediately is deprotonated to the final SIN-1C. Since NO• reacts with superoxide anion at a diffusion-controlled rate to give peroxynitrite anion (OONO− ) [102], the production of this species is inevitable during NO-release from sydnonimines in in vitro systems. Peroxynitrite anion is a source of hydroxyl (OH• ) and nitrogen dioxide (NO• 2 ) radicals. These reactive species, when produced in high concentrations, can induce oxidative stress, and thus can be the source of toxicity [103, 104]. O

O2

N O

N

.

O 2-

O

. N+

CH2CN

N

CH2CN

N

O

105

N

107

NO

O

O N

N 106

CHCN H+

+ N N

.

CH2CN

108

Scheme 6.18 Oxidative mechanism of NO-release from SIN-1A 105.

The hypothesis that HNO is not involved during NO-release from sydnonimines was confirmed by the study of NO-release from C78-0652 109, the dimethyl derivative of SIN-1A (Scheme 6.19). This product closely resembles SIN-1A in its biological and pharmacological behavior, showing a clear NO-dependent vasodilating effect on guinea pig pulmonary arteries and hypotensive action in anesthetized and conscious dog models [105].

159

160

6 The NO-releasing Heterocycles N O O

Ox

N N -

C CN H3C

+ N N

O

NO.

C CN H3C CH 3

CH3

109 -H

O

HNO rearrangement

+

+ N N C CN H3C CH3

CN O

CN

N N C CH3 CH3

H2O

H O

N N H

- acetone

110

Scheme 6.19 NO-release from the dimethyl derivative of SIN-1A 109.

Its chemical structure does not allow elimination of HNO, thus supporting the oxidative pathway of activation to NO• , a mechanism still possible in this blocked SIN-1A derivative. The final product of the NO-release was found to be 1-amino-2cyanomorpholine (110) [106]. Its formation can be rationalized assuming that, after the oxidative NO-release, deprotonation occurs at the á-position of the morpholine, followed by migration of the cyano group and hydrolytic cleavage of the hydrazone moiety. Sterically hindered amino substituents or amino substituents with electron-withdrawing groups in the 3-position of the sydnonimine ring, as in Marsidomine 88, C89-4095 89 or C4144 90, have little impact on the stability of the heterocyclic system toward ring opening, but are able to slow down the process of oxidative NO-release from the nitroso intermediates [107]. According to this mechanism 3-alkylsydnonimines turned out to be less interesting as NO-releasing compounds because the corresponding ring-opened nitroso derivatives are much more resistant to oxidants. Also, introducing aminocarbonyl or sulfonyl substiuents at the exocyclic nitrogen resulted in a higher chemical and metabolic stability of the sydnonimine nucleus, and thus in a much lower or no NO-mediated vasodilating and antiplatelet activity in vivo. In conclusion, sydnonimines represent a class of NO-donors that, with the exception of N-acylated derivatives that need chemical hydrolysis or enzymatic activation by esterases, release NO• spontaneously in the presence of oxidants without requiring further activation with enzymes or thiols.

6.2 Mesoionic Heterocycles

6.2.1.4

Biological Properties

The pharmacological effects of sydnones and sydnonimines have been reviewed by Ackermann [108] and Boes et al. [109], and those of sydnonimines have been reported by Granik et al. [2, 3] and Wang et al. as a part of a general discussion on NO-donors [4]. Sydnonimines, and particularly 3-dialkylaminosydnonimines, were discovered as possible drug candidates due to their vasodilating and antihypertensive action [95, 110, 111]. Molsidomine has been on the market for more than 25years as an antianginal, nitrate-like drug for the treatment of coronary heart disease that lowers myocardial oxygen consumption, cardiac output, left ventricular work and arterial blood pressure [112–115]. The mechanism of action is a direct activation of soluble guanylate cyclase leading to elevated levels of cGMP [99] mediated by the release of NO• , that can occur from its metabolite SIN-1A [97, 107, 116]. Compared with other NO-releasers like nitrates, Molsidomine and other sydnonimines like Pirsidomine 87 [117] and C89-4095 89 [118] show diminished or no development of tolerance and a higher antiaggregatory activity in platelets [119]. The higher antiaggregatory and antithrombotic activity can be explained by the fact that the NO-release from sydnonimines can occur in the blood plasma independently of the presence of activating cellular cofactors. The antithrombotic activity of C894095 has been reported to be inhibited by oxyhemoglobine, thus demonstrating a clear sGC- and cGMP-mediated mechanism for platelet inhibition. C4144 90 showed a Suntest 750 W m-² Molsidomine

Pirsidomine

Linsidomine

C-4144

100 90 80

Content (%)

70 60 50 40 30 20 10 0 0

5

10

15

20

25

30

35

40

45

50

55

60

Time (min)

Fig. 6.6 Comparison of the photostability of different sydnonimines in the Suntest [106].

65

161

6 The NO-releasing Heterocycles

promising pharmacological profile due to its long-lasting antianginal and vasodilating activity and better thermal and photolytic stability than other sydnonimines (Fig. 6.6) [119, 120]. Pirsidomine showed a prolonged vasodilating activity in vitro and a long duration of action in vivo in a dog model. Compared with isosorbite-5-mononitrate (IS-5-MN) this sydnonimine developed no tolerance in a conscious dog model at doses of both drugs that produced similar pharmacological effects (Fig. 6.7) [121–123].

Tolerance study in conscious renal hypertensive dogs Oral treatment for 5 days (3 times daily) Afterload

Preload Pirsidomine 2 mg/kg p.o.

10 2 ∆ LVEDP (mmHg)

∆ BPs (mmHg)

0 -10 -20 -30 -40

0 -2 -4 -6 -8 -10

0

1

2

3

4

5

6

time (h)

0

1

2

3

4

5

6

1

2

3

4

5

6

IS-5-MN 10 mg/kg p.o. 10

2 ∆ LVEDP (mm Hg)

∆ BPs (mmHg)

162

0 -10 -20 -30 -40

0 -2 -4 -6 -8 -10

0

1

2

3

4

1st

5

6

3rd

time (h)

5th

0

day

Fig. 6.7 Comparison of the tolerance development to Pirsidomine and IS-5-MN.

The lack of tolerance has been explained by the fact that the NO-release from these compounds is spontaneous and independent of the presence of thiols [124], that, by contrast, may be an essential cofactor in the action of the nitrate. Martorana et al. [125] showed a marked antiischemic effect of Pirsidomine in a dog model of myocardial infarction. In a placebo controlled, double-blind, multicenter study with 47 chronic stable angina pectoris patients Pirsidomine showed significant hypotensive, anti-ischemic and antianginal effects [126]. SIN-1, the first metabolite of Molsidomine, is the sydnonimine that attracted the highest interest for in vitro as well as in vivo studies. In vitro this molecule has been

6.2 Mesoionic Heterocycles

shown to act as an NO-releaser as well as a superoxide and peroxinitrite generator. Most of its effects are reported to be NO-mediated. In human platelets SIN-1 activated guanylate cyclase and elevated cGMP contents, leading to the inhibition of thrombininduced platelet aggregation [127]. Groves et al. [128] demonstrated inhibition of platelet adhesion following balloon angioplasty in a pig model and a frog model. Chesnais et al. [129] found a cGMP-mediated negative inotropic effect that was accentuated by the addition of the superoxide scavenging enzyme SOD. Xiao et al. [130] found a cGMP-mediated down-regulation of human natural killer cells by this sydnonimine. In endothelial cells SIN-1 has been demonstrated to produce a long term antioxidant and cytoprotective effect via induction of the ferritin gene [131] to reduce re-oxygenation injury in cardiomyocytes and to protect against endothelial dysfunction during myocardial ischemia reperfusion [132, 133]. In contrast to this, pro-oxidant and cytotoxic effects have also been reported to result in the decreased viability of different cell types [134, 135]. 6.2.2

Mesoionic Oxatriazoles

Mesoionic 1,2,3,4-oxatriazolium-5-olates 82 and the corresponding 5-amenate derivatives 83 are structurally related to sydnones 80 and to sydnonimines 81, respectively. As with sydnonimines, the 5-amenate derivatives are stable only as salts or in their N-acylated forms. An extensive investigation of the NO-releasing and of the biological properties of this class started about 10 years ago, when researchers at the GEA Farmaceutisk Fabrik, Copenhagen, synthesized a large number of these compounds, among them GEA-3162 (3-(3,4-dichlorophenyl)-1,2,3,4-oxatriazolium-5-amenate hydrochloride) 111, GEA-3175 (3-(3-chloro-2-methylphenyl)-1,2,3,4-oxatriazolium-5-(4Cl Cl

H Cl + N N N

O

- H N

H3C

+ N N N

O

O - S N

O

GEA-3162

GEA-3175

111

112

Cl

H Cl H3C

Cl

+ N N N

O

- H N

Cl H3C

+ N N N

O

HN

CH2 C

- C O N

GEA-5024

GEA-5624

113

114

Structures 6.111–6.114

CH3

CH

163

164

6 The NO-releasing Heterocycles

methylphenylsulfonyl)amenate) 112, GEA-5024 (3-(3-chloro-2-methylphenyl)-1,2,3,4oxatriazolium-5-amenate hydrochloride) 113, and GEA-5624 (3-(3-chloro-2-methylphenyl)-1,2,3,4-oxatriazolium-5- (2-propinylaminocarbonyl)amenate) 114. 6.2.2.1

Synthesis

The first 1,2,3,4-oxatriazolium-5-olate derivative (82, R=C6 H5 ) was synthesised by Ponzio [136] in 1933 by the action of trinitromethane (nitroform) on a phenyldiazonium salt 115 and it was called “pseudooxatriazol” (Scheme 6.20). Later the same product was obtained using potassium diazomethanedisulfonate followed by treatment with nitrous acid [137]. The most general route to these derivatives implies the action of nitrous acid on 1-substituted semicarbazides 116 [138]. The reaction goes through the corresponding 1-nitrososemicarbazides 117, that can be isolated. In this case, they can undergo cyclisation to the final products in acid media [139]. Using this procedure a number of products endowed with hypotensive [139–141] or antianginal and antithrombotic properties [142] were obtained. Another very simple way to prepare products 82 (R=alkyl) is the action of phosgene on 1-alkyl-1-nitrosohydrazines 118, in the presence of a base [143]. The first synthesis of a 1,2,3,4-oxatriazolium-5-amenate derivative (83,R=R1 =C6 H5 ) was reported by Busch et al. as early as 1896 [144]. The product was obtained by the nitrosation of the diphenylthiosemicarbazide precursor 119. The real structure remained unclear until 1971, when Christophersen et al. [145] assigned the correct mesoionic structure to the compound. These authors prepared a series of mesoionic +

C6H5N2 115

CH(NO2)3

CH2(SO3K)2

O SO2K C6H5 HN N

C SO2K

HNO2

R + N N

H+

O

N O

R

H

N N N

H 117

COCl2

R N N

H O

HNO2

R

H C N H

N N H

H

O

82

H

O

H C N

N

118 Scheme 6.20 Synthetic routes to mesoionic 1,2,3,4-oxatriazolium-5-olates.

116

6.2 Mesoionic Heterocycles H

S R

C N N N

H

R1 H 119 HNO2 or C2H5ONO

H

R N N

N HN CN

H

R + N N

R

120

ON

CN 121

N O

HCl

NR1

HN C HN N C6H11 H2N

83

NO 122

Scheme 6.21 Synthetic routes to mesoionic 1,2,3,4-oxatriazolium-5-amenates.

oxatriazolium-5-amenate derivatives by action of nitrous acid on 1-substituted and 1,4-disubstituted thiosemicarbazides (119, R=alky, aryl; R1 =H, alkyl, aryl, Scheme 6.21). Alternatively Karup et al. [146, 147] developed a synthetic route for 3-aryl substituted derivatives similar to that reported by Busch et al. [144] starting from arylhydrazines and potassium thiocyanate leading to thiosemicarbazides that, on nitrosation with ethyl nitrite, gave good yields of 3-substituted mesoionic 1,2,3,4oxatriazolium-5-amenate hydrochlorides. 3-Aryl derivatives have also been prepared from 2-aryl-1-cyanohydrazines 120 and nitrous acid through the intermediate formation of the corresponding nitroso-cyanohydrazines 121 [148]. Finally the product 83 (R=C6 H11 , R1 =H) was obtained by Finnegan et al. [149] on treatment of 1-cyclohexyl2-guanyl-1-nitrosohydrazine (122) with HCl. Similarly to the sydnonimines, unsubstituted oxatriazolium-5-amenates have been acylated with various acylating systems to N-acyl-, N-carbamoyl-, N-alkoxycarbonyland N-sulfonyl-derivatives [148] that represent chemically stable compounds (Scheme 6.22). Nitrosation of 5-amenate derivatives has been reported to lead to unstable nitroso derivatives 123 that eliminate nitrogen spontaneously, forming the corresponding 5-olate analogues 124 [150]. Only in special cases the free bases of mesoionic 1,2,3,4oxatriazolium-5-amenates, unsubstituted at the imine nitrogen, are stable enough to allow isolation and chemical characterization at room temperature [148]. 6.2.2.2

NO-release

As for sydnonimines, the ability of mesoionic derivatives 82 and 83 to produce NO is strictly connected to their capacity to decompose to nitrosohydrazine intermediates. Mesoionic 1,2,3,4-oxatriazolium-5-olates represent a thermally stable class of compounds, but they undergo a relatively facile nucleophilic ring-opening reac-

165

166

6 The NO-releasing Heterocycles

R + N N R1COCl

R + N N N O

- H N

R2NCO

N

O

R + N N N

R3SO2Cl

N

O

- H N

NaNO2/HCl

O - C NR 2 N

O

R + N N N

R + N N

O - C R 1 N

O

O S N R3

O

R + N N N

N

O

N

R + N N

O - N2

123

N

O

O

-

124

Scheme 6.22 Acylation and nitrosation of 5-amenate derivatives of mesoionic 1,2,3,4-oxatriazoles.

tion with water under basic conditions. In addition they eliminate carbon dioxide to yield nitrosohydrazines 125 or react with amines and other nucleophiles to give nitrososemicarbazides 126 [145] as shown in Scheme 6.23. H

R

N

O

N N

NaOH

R + N N

O O

H

N 125

HNR2R3

H

R

R2

N N O

C N

N O 126

R + N N N O

- H N

OH

-

R

CN N N O N H 127

Scheme 6.23 Ring-opening reactions of mesoionic

5-olate and 5-amenate 1,2,3,4-oxatriazoles to nitrosohydrazine derivatives.

R3

6.2 Mesoionic Heterocycles

5-Amenate derivatives that do not carry substitutents at the exocyclic nitrogen atom undergo a ring-opening reaction to nitroso cyanohydrazines 127 upon treatment with bases in aqueous solutions [151]. Noncyclic nitrosohydrazines have been reported to function as NO-sources and to exhibit antithrombotic and antihypertensive effects in animal models that were similar to their corresponding cyclized compounds [139, 150]. 5-Amenates acylated at the exocyclic nitrogen are stable as solid compounds but decompose in aqueous solutions releasing NO• . This decomposition depends on the pH and most importantly on their chemical structure [147, 152]. The proposed mechanism of NO-release is shown in Scheme 6.24. It is related to that postulated for sydnonimines. The main difference is that here 5-substituted amenates 128 are able to react with water to form acyclic nitroso semicarbazides 129 directly without needing enzymatic cleavage, and these intermediates release NO• by an oxidative or thiol mediated mechanism that is not fully understood [153]. Due to the electron-demanding carbamoyl substructure on the nitroso hydrazine intermediates 129 the oxidative process that initiates the NO-release is much slower than with the active sydnonimine metabolites. The elimination of HNO from the nitroso intermediates and the subsequent oxidation to NO• cannot be completely ruled out for this type of compounds. In vivo, an alternative, possibly thiol mediated route for the NO• formation plays a role in the activity [147]. In this reaction the formation of nitrosothiols as unstable NO• precursor intermediates is the most likely process. R

-HNO

R

O2

R R

+

N N N O

-

X

N

H2O

H R N N X N C N O O H

+

.

RSH

R

X = CO, SO2 128

.

- NO , - H , - O2-

R N N X C N O H

-RSNO

129

NO

.

H R N N X C N H O H

Scheme 6.24 Proposed mechanism of NO release from

mesoionic 1,2,3,4-oxatriazolium-5-amenates.

6.2.2.3

Biological Properties

Derivatives 82 (R=cycloalkyl) proved to be central nervous system stimulants, whereas 3-alkyl analogs were claimed as vasodepressant or hypotensive agents with some residual central nervous system activity [139, 154]. Mesoionic oxatriazolium-5-amenates 83 exhibit the whole range of biological properties typical of NO-releasing compounds that stimulate soluble guanylate cyclase; namely antihypertensive activity in animals following the relaxation of the vascular

167

168

6 The NO-releasing Heterocycles

smooth muscles, inhibition of platelet aggregation and adhesion to vessel walls, fibrinolytic and broncholytic activity, both in vitro and in vivo, relaxing effects on the trachea and inhibition of leukocyte function [146, 155–157]. The vasorelaxing action on rabbit aorta strips, the tracheal relaxation on guinea pig trachea, the antiaggregatory action on human platelets as well as the thrombolytic properties of a large series of 5-amenates bearing a number of different substituents both on the 3-position and the 5-imino group were closely investigated. A comparison with the corresponding rates of NO-release evaluated in phosphate buffer pH7.4 indicates that, broadly speaking, fast NO-donors should be the most promising as vasorelaxant drugs, while slow NO-releasers seem to be most promising as antiplatelet and antithrombotic agents [152]. Antibacterial potency for these products has been shown in an Escherichia coli model by Virta et al. [158], and the inhibition of oxidation of low density lipoproteins was demonstrated by Malo-Ranta et al. [159]. Inhibition of serum induced DNA synthesis and proliferation of vascular smooth muscle cells have been shown for GEA-3162. The urea derivative GEA-5624 inhibited only the DNA synthesis but not the proliferation by a NO mediated cGMP independent mechanism. In fact this activity was not abolished by the presence of either oxyhemoglobin or ODQ [160]. Tumor cell growth and lymphocyte proliferation have also been reported by Vilpo et al. and Kosonen et al. to be inhibited by this NO-donors [161, 162]. GEA-3162 exhibited a protective effect on isolated rat heart during ischemia and reperfusion [163] and has been reported to inhibit gastric ulceration induced by ethanol when administrated intragastrically [164]. GEA-3175 induced relaxation of bovine bronchioles through a cGMP-mediated opening of large conductance Ca2+ -activated K+ -channels [165, 166]. GEA-5024 was reported to cause dose-dependent apoptotic cell death in metastatic murine melanoma cells [167] and showed antimalaria potency [168].

6.3

Other Heterocyclic Systems

In vivo, some other heterocyclic systems behave as NO-donors. Typical examples are the 3-amino-4-alkyl or arylalkyl derivatives of 1,2,4-oxadiazol-5(4H)one 133 [169, 170]. These products can be synthesised following Scheme 6.25. O CH3CH2O

C

O

O RBr

NH CN

CH3CH2O

C

NH2OH

N CN R

130

131

CH3CH2O

C

NOH N R

132

- C2H5OH

O

O

N

C

N NH2

R

NH2 133

Scheme 6.25 Synthesis of 1,2,4-oxadiazol-5(4H)one derivatives.

Action of the appropriate alkyl bromide on N-cyanourethane 130 affords the Nalkyl cyanourethanes 131 that, under the action of the hydroxylamine, through the intermediate formation of unstable hydroxyguanidines 132, yield the final products 133. These products have been found to display antithrombotic properties after

6.3 Other Heterocyclic Systems

oral administration to rats. The strongest activity was shown by the 4-pentyl and 4-benzyl derivatives. Substitution and hydrogenation of the benzyl group decreased the antithrombotic effect. These products are probably transformed in vivo into the corresponding hydroxyguanidines 132 that, in their turn, might produce nitric oxide by enzymatic activation, as has been found to occur for other hydroxyguanidines [171]. In effect, the 4-benzyl derivative (133, R=C6 H4 CH2 ) released NO• (detected by chemiluminescence) when incubated with rat liver microsomes [169]. Since arylazoamidoximes release nitric oxide when incubated in rat liver microsomial fraction [172], 3-arylazo-1,2,4-oxadiazol-5-ones 136 have been prepared from the corresponding arylazoamidoximes 134 as their potential pro-drugs [172]. Reaction with chloroformate afforded compounds 135 which underwent cyclisation to 136 in alkaline medium (Scheme 6.26). O C O Ph O N H

C NH2

C6H5OCOCl

N O

OH

R N N C

N N

-

O

O

N H

N

NH2

N N

Ar

Ar 134

135

136

Scheme 6.26 Synthesis of 3-arylazo-1,2,4-oxadiazol-5-ones.

NOH

OH

R1 C

- HNO

N N C R HON 137

R1

N

R

N N

138

- H2O

NH

NH

NOH

O

R1

R

139

Scheme 6.27 Synthesis of a series of 1,3,4-triazol-1-oles.

In an in vivo thrombosis model, these products displayed antithrombotic effects that were generally lower than those of the corresponding azoamidoximes. This is probably due to a rather slow cleavage of the prodrugs 136 to the drugs 134. The most potent oxadiazol-5-one proved to be the phenyl derivative (136, Ar=C6 H5 ). Azaurolic acid 137 is a compound that displays antithrombotic activity and that, both in solution and in the solid state, releases N2 O, which is the product of dimerization/dehydration of HNO, giving the stable compound 138 (R=R1 =CH3 , Scheme 6.27). This latter product also displays anthitrombotic activity [173]. These findings prompted the synthesis of a series of 1,3,4-triazol-1-oles 138 which were obtained by cyclisation of the parent oximes 139 in aqueous alkali. All products showed antithrombotic activity; the most potent was found to be the dimethyl derivative, which also significantly decreased blood pressure when administered orally to spontaneously hypertensive rats. Possible involvement of NO in this action was suggested but not demonstrated.

169

170

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Corell, T. N., Lissau, B. G., Petersen, F. P., Alhede, B. I. F., WO Patent (1992) 92/13847 Karup, G. L., Preikschat, H. F., Wilhelmsen, E. S., Pedersen, S. B., Marcinkiewicz, E., Cieslik, K., Gryglewski, R. J., Pol. J. Pharmacol. 46 (1994), p. 541 Masuda, K., Kamiya, T., Kashiwa, K., Chem. Pharm. Bull. 19 (1971), p. 559 Finnegan, W. G., Henry, R. A., J. Org. Chem. 30 (1965), p. 567 Rehse, K., König, P., Arch. Pharm. (Weinheim) 328 (1995), p. 137 Christophersen, C., Treppendahl, S., Acta Chem. Scand. 26 (1972), p. 858 Gryglewski, R. J., Marcinkiewicz, E., Robak, J., Michalska, Z., Madej, J., Curr. Pharm. Des. 8 (2002), p. 167 Holm, P., Kankaanranta, H., Metsa-Ketela, T., Moilanen, E., Eur. J. Pharmacol. 346 (1998), p. 97 Wallace & Tiernan Inc. GB Patent (1964) 1065684 Karup, G. L., Preikschat, H. F., Wilhelmsen, E. S., Pedersen, S. B., Corell, T. N., Alhede, B. I. F., WO Patent (1994) 94/03442 Corell, T., Pederson, S-B., Lissau, B., Moilanen, E., Metsa-Ketela, T., Kankaanranta, H., Vuorinen, P., Vapaatalo, H., Rydell, E., Andersson, R., Marcinkiewicz, E., Corbut, R., Gryglewski, R. J., Pol. J. Pharmacol. 46 (1994), p. 553 Karup, G. L., Preikschat, H. F., Pedersen, S. B., Corell, T. N., Wilhelmsen, E. S., WO Patent (1996) 962/11191 Virta, M., Karp, M., Vuorinen P., Antimicrob. Agents Chemother. 38 (1994), p. 2775 Malo-Ranta, U., Yla-Herttuala S., Metsa-Ketela T., Jaakkola O., Moilanen E., Vuorinen P., Nikkari T., FEBS Lett. 337 (1994), p. 179 Laehteenmaeki, T., Sievi E., Vapaatalo H., Br. J. Pharmacol. 125 (1998), p. 402 Vilpo, J. A., Vilpo, L. M., Vuorinen, P., Moilanen, E., Metsa-Ketela, T.,

References

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164

165

166

167

The Biology of Nitric Oxide 4 (1994), p. 286 Kosonen, O., Kankaanranta, H., Vuorinen, P., Moilanen, E., Eur. J. Pharmacol. 337 (1997), p. 55 Szekeres, M., Dezsi, L., Monos, E., Metsa-Ketela, T., Acta Physiol. Scand. 161 (1997), p. 55 Asmawi, M. Z., Moilanen, E., Annala, K., Rahkonen, P., Kankaanranta, H., Eur. J. Pharmacol. 378 (1999), p. 123 Kosonen, O., Kankaanranta, H., Moilanen, E., Eur. J. Pharmacol. 394 (2000), p. 149 Kosonen, O., Kankaanranta, H., Malo-Ranta, U., Ristimaki, A., Moilanen, E., Br. J. Pharmacol. 125 (1998), p. 247 Xie, K., Wang, Y., Huang, S., Xu, L., Bielenberg, D., Salas, T., McConkey,

168 169 170 171

172

173

D. J., Jiang, W., Fidler, I. J., Oncogene 15 (1997), p. 771 Taylor-Robinson, A. W., Biochem. Soc. Trans. 25 (1997), p. 262S Rehse, K., Bade, S., Arch. Pharm. Pharm. Med. Chem. 329 (1996), p. 535 Rehse, K., Bade, S., Eur. J. Pharm. Sci. 4 (1996), S121 Jousserandot, A. Boucher, J.-L., Desseaux, C., Delaforge, M., Mansuy, D., Bioorg. Med. Chem. Lett. 5 (1995), p. 423 Rehse, K., Bade, S., Harsdorf, A., Clement, B., Arch. Pharm. Pharm. Med. Chem. 330 (1997), p. 392 Rehse, K., Piechocki, D., Schober, M., Scheffler, H., Reitner, N., Unsöld, E., Arch. Pharm. Pharm. Med. Chem. 329 (1996), p. 511

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7

C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas S. Bruce King 7.1

Introduction

C-Nitroso compounds, oximes, N-hydroxyguanidines and N-hydroxyureas each contain an N–O bond and release nitric oxide (NO) or one of its redox forms under some conditions. The nitrogen atom of a C-nitroso compound formally exists in the +1 oxidation state, the same oxidation state as nitroxyl (HNO), the one-electron reduced form of NO. The nitrogen atoms of oximes, N-hydroxyguanidines, and Nhydroxyureas each formally exist in the –1 oxidation state, the same oxidation state as hydroxylamine. Consequently, the direct formation of NO (formal oxidation state = +2) from any of these species requires oxidation, one electron for a C-nitroso compound and three electrons for an oxime, N-hydroxyguanidine or N-hydroxyurea. This chapter summarizes the syntheses and properties, NO-releasing mechanisms and the known structure–activity relationships of these compounds.

7.2

C-Nitroso Compounds 7.2.1

Alkyl and Aryl C-Nitroso Compounds 7.2.1.1

Syntheses and Properties

Alkyl and aryl C-nitroso compounds contain a nitroso group (–N=O) directly attached to an aliphatic or aromatic carbon. As compounds with a nitroso group attached to a primary or secondary carbon exist primarily as the oxime tautomer, the stable examples of C-nitroso compounds contain nitroso groups attached to tertiary carbons, such as 2-methyl-2-nitroso propane (1, Fig. 7.1) or nitroso groups attached to carbons bearing an electron-withdrawing group (–CN, –NO2 , –COR, –Cl, –OAc, Fig. 7.1). Oxidation of alkyl and aryl hydroxylamines provides the most direct route to alkyl and 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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7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas R1 N O 1

R2 R3

N O

R1 and R2 = alkyl, aryl Fig. 7.1 Basic Structures of C-alkyl and R3 =-CN, -NO2, -COR, -Cl, OAc aryl nitroso compounds.

aryl C-nitroso compounds [1, 2]. A number of synthetic routes provide access to compounds with a nitroso group attached to a carbon bearing an electron-withdrawing group. These have recently been reviewed [3]. Aliphatic and aromatic C-nitroso compounds may exist as monomers, dimers or adopt the tautomeric oxime structure. As noted, stable examples of monomeric Cnitroso compounds contain nitroso groups attached to tertiary carbons or nitroso groups attached to carbons bearing an electron-withdrawing group. These monomeric C-nitroso compounds have a deep blue or green color (for 1, ëmax = 300, 665 nm, å = 100, 20) and an infrared stretch for the N=O bond between 1680 and1450 cm−1 [4]. C-Nitroso dimers are colorless and show an infrared stretch for the N=N bond between 1410 and 1175 cm−1 [4]. Dimerization of C-nitroso compounds requires 6– 10 kcal mol−1 and dissociation of C-nitroso dimers requires 20–30 kcal mol−1 of energy [3]. The bond dissociation energy of the C–N bond of aliphatic C-nitroso compounds ranges between 36 and 40 kcal mol−1 making homolytic C–N bond cleavage of these compounds a reasonable pathway to NO formation [5]. 7.2.1.2

NO-releasing Mechanisms

2-Methyl-2-nitroso propane (1, Scheme 7.1) undergoes both thermal and photochemical homolytic C–N bond cleavage to yield NO and the tert-butyl radical [6]. 2-Methyl2-nitroso propane also acts as a spin-trap and reacts with the tert-butyl radical to give di-tert-butyl nitroxide (2) as shown by electron paramagnetic resonance (EPR) spectroscopy (Scheme 7.1) [7]. Photochemical irradiation of 1 results in NO release, activation of soluble guanylate cyclase and vascular relaxation [8]. C-Nitroso compounds also inhibit yeast aldehyde dehydrogenase, indicating their potential to mimic the effects of nitroxyl (HNO) [9].

N O 1

hv or heat

+ NO

N O

N O 2

Scheme 7.1 Thermolysis or photolysis of C-nitroso compounds to nitric oxide.

7.2 C-Nitroso Compounds

7.2.2

Acyl C-Nitroso Compounds 7.2.2.1

Syntheses and Properties

Acyl nitroso compounds (3, Scheme 7.2) contain a nitroso group (–N=O) directly attached to a carbonyl carbon. Oxidation of an N-acyl hydroxylamine derivative provides the most direct method for the preparation of acyl C-nitroso compounds [10]. Treatment of hydroxamic acids, N-hydroxy carbamates or N-hydroxyureas with sodium periodate or tetra-alkyl ammonium periodate salts results in the formation of the corresponding acyl nitroso species (Scheme 7.2) [11–14]. Other oxidants including the Dess–Martin periodinane and both ruthenium (II) and iridium (I) based species efficiently convert N-acyl hydroxylamines to the corresponding acyl nitroso compounds [15–18]. The Swern oxidation also provides a useful alternative procedure for the oxidative preparation of acyl nitroso species [19]. Horseradish peroxidase (HRP) catalyzed oxidation of N-hydroxyurea with hydrogen peroxide forms an acyl nitroso species, which can be trapped with 1, 3-cyclohexanone, giving evidence of the formation of these species with enzymatic oxidants [20]. O

O R

oxidation NH OH

R = -alkyl, aryl, -OR, -NHR

R

N O 3

Scheme 7.2 Oxidation of N-acyl hydroxyl-

amines to C-acyl nitroso compounds.

Acyl nitroso compounds react with 1, 3-dienes as N–O heterodienophiles to produce cycloadducts, which have found use in the total synthesis of a number of nitrogen-containing natural products [21]. The cycloadducts of acyl nitroso compounds and 9, 10-dimethylanthracene (4, Scheme 7.3) undergo thermal decomposition through retro-Diels–Alder reactions to produce acyl nitroso compounds under non-oxidative conditions and at relatively mild temperatures (40–100°C) [11–14]. Decomposition of these compounds provides a particularly clean method for the formation of acyl nitroso compounds. Photolysis or thermolysis of 3, 5-diphenyl-1, 2, 4-oxadiazole-4-oxide (5) generates the aromatic acyl nitroso compound (6) and benzonitrile (Scheme 7.3) [22, 23]. Other reactions that generate acyl nitroso compounds include the treatment of 5 with a nitrile oxide [24], the addition of N-methyl morpholine N-oxide to nitrile oxides and the decomposition of N, O-diacylated or alkylated N-hydroxyarylsulfonamides [25–29]. C-Acyl nitroso compounds are highly reactive species and no examples of stable and isolable C-acyl nitroso compounds have been reported. Charge-reversal and neutralization–reionization mass spectrometry experiments provided the first spectroscopic evidence for the existence of acyl nitroso compounds in the gas phase [30, 31]. Recently, time-resolved infrared spectroscopic measurements revealed evidence for the first direct observation of an acyl nitroso compound in solution [22]. Pho-

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7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas

O N

retroDiels Alder

R

O

O R

3 acyl nitroso compound

4 R = -alkyl, aryl, -OR, -NHR Ph

O

N

O

O

hv

N

Ph

Ph

N O

6

5

N O

+

Ph

N Scheme 7.3 Alternative methods of

C-acyl nitroso compound formation.

tolysis of 5 generates the aromatic acyl nitroso species (6, Scheme 7.3), which has infrared resonances at 1735, 1590, 1560, and 1235 cm−1 that correlate very well with calculated values [22]. 7.2.2.2

NO-releasing Mechanisms

C-Acyl nitroso compounds are reactive species that react with 1, 3-dienes as N–O heterodienophiles and alkenes bearing allylic hydrogens as enophiles [12]. Triphenylphosphine deoxygenates acyl nitroso compounds to give isocyanates [32]. Generation of C-acyl nitroso compounds in a non-nucleophilic solvent and in the absence of these reactive partners leads to dimerization and the ultimate formation of the corresponding anhydride [12]. Generation of C-acyl nitroso compounds in the absence of 1, 3-dienes but in the presence of nucleophiles results in the formation of nitrous oxide [33], the dimerization and dehydration product of nitroxyl (HNO), the one-electron reduced form of NO [34]. The identification of nitrous oxide from these reactions provides good evidence for the intermediacy of HNO. The periodate oxidation of hydroxamic acids in the presence of amines yields amides and a similar amount of nitrous oxide (Scheme 7.4) [33]. Periodate oxidation of benzohydroxamic acid yields the acyl nitroso species (6) that reacts as an electrophile toward the amine to form a tetrahedral intermediate (Scheme 7.4). Decomposition of this intermediate O Ph

O

NaIO4

NH OH

Ph

RNH2

HO Ph

N 6 O

NHR N O

O Ph

NHR + HNO

HNO

Scheme 7.4 Nucleophilic substitution of a C-acyl

nitroso compound with nitroxyl formation.

N2O + H2O

7.2 C-Nitroso Compounds

gives the observed amide and HNO, which dimerizes and dehydrates to nitrous oxide (Scheme 7.4). Generation of acyl nitroso compounds through the thermal decomposition of acyl-nitroso compound-cyclopentadiene cycloadducts in the presence of amines also yields amides and nitrous oxide [33]. Time-resolved infrared spectroscopic studies reveal a second-order rate constant of 1.3 ± 0.5 × 105 M−1 s−1 for the addition of diethyl amine to 6 and estimate the lifetime of an acyl nitroso species in organic solution at infinite dilution as 1 ms [22]. The observation of a weak broad band at 2650 cm−1 (N–H stretch) that grows in intensity during this reaction provides evidence of the direct formation of HNO during this substitution [22]. The observation of nitrous oxide formation during the periodate oxidation of hydroxamic acids in water, the periodate or HRP catalyzed oxidation of N-hydroxyurea and the thermal decomposition of N-hydroxyurea derived acyl nitroso-9, 10-dimethylanthracne cycloadducts in water all suggest the initial formation of HNO following hydrolysis of a C-acyl nitroso species [20, 35–37]. N, O-Diacylated or O-alkylated N-hydroxysulfonamides release nitroxyl (HNO) upon hydrolysis or metabolic dealkylation, as determined by gas chromatographic identification of nitrous oxide in the reaction headspace [27–29, 38]. Scheme 7.5 depicts the decomposition of a representative compound (7) to a C-acyl nitroso species that hydrolyzes to yield HNO. Either hydrolysis or metabolism removes the O-acyl or O-alkyl group to give an N-hydroxy species that rapidly decomposes to give a sulfinic acid and an acyl nitroso species. This C-acyl nitroso species (8) hydrolyzes to the carboxylic acid and HNO (Scheme 7.5). These compounds demonstrate the ability to relax smooth muscle preparations in vitro and also inhibit aldehyde dehydrogenase, similar to other HNO donors [27, 29].

Cl

O O R1 S N R2 O O 7

O-dealkylation or O-deacylation Cl

O O H S N R2 O O 8

R1 = alkyl or acyl R2 = alkyl or aryl O Cl

SO2H + R 2

H2O N O

PhCO2H + HNO

Scheme 7.5 Nitroxyl formation from a diacyl N,O–N-hydroxysulfonamide.

7.2.2.3

Structure–Activity Relationships

Extensive structure–activity relationships for the oxidative formation of C-acyl nitroso compounds or the release of NO or HNO from C-acyl nitroso compounds do not exist. However, the –R group of the cycloadducts of acyl nitroso compounds and 9, 10dimethylanthracene (4, Scheme 7.3) appears to strongly influence the rate that these compounds undergo retro-Diels–Alder reactions to produce acyl nitroso compounds.

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7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas

In general, C-acyl nitroso compounds-9, 10-dimethylanthracene cycloadducts derived from hydroxamic acids (–R = alkyl, aryl, t1/2 = 4.1 h for −R = −Ph at 60°C) decompose more slowly than those derived from N-hydroxycarbamates or N-hydroxyureas [11, 13, 14]. Further addition of alkyl groups to the N atom of N-hydroxyurea-derived cycloadducts produces a further increase in the rate of the retro-Diels–Alder reaction of these cycloadducts [36]. These general trends suggest the possibility of the development of acyl nitroso compound-9, 10-dimethylanthracene cycloadducts as a general class of HNO or NO donors with varied release profiles.

7.3

Oximes 7.3.1

Syntheses and Properties

The two most common methods for the preparation of simple oximes are the direct condensation of hydroxylamine with a ketone and the nitrosation of a carbon bearing an acidic proton (Scheme 7.6) [39]. Other methods for the preparation of simple oximes include the addition of NOCl to olefins, the addition of Grignard reagents to the conjugate bases of nitro compounds, the photolysis of organic nitrites (the Barton reaction), the alcoholic cleavage of cyclic ketones with NOCl, the oxidation of primary amines and the reduction of nitro compounds [2]. Flavin-containing monoxygenases also convert some aliphatic primary amines to their corresponding oximes through the intermediacy of the hydroxylamine in the presence of liver microsomes [40]. O R2 HO

NaNO2 R1

H+

HO

O R2

N

NH2OH R1

-H2O

R2

N R1

Scheme 7.6 General oxime synthesis.

The structurally unique nitro-containing oximes, such as FK409, a semi-synthetic metabolite of Streptomyces griseosporeus [41, 42], (9, Scheme 7.7) comprise the most widely studied group of NO-releasing oximes. These compounds spontaneously release NO in aqueous solution at pH 7.4 as determined by EPR spectroscopy, and chemiluminescence NO detection [43, 44]. The synthetic preparation of these compounds relies on the addition of acidic sodium nitrite to a 1,3-diene conjugated with an amide group [45, 46]. This sequence directly and efficiently introduces the nitro and oxime group into the 1, 3-diene in a single synthetic operation. Oximes generally demonstrate good stability in the solid state when stored at low temperature. Simple oximes show reasonable stability in neutral aqueous solution but hydrolyze to hydroxylamine and the parent ketone under acidic or basic catalysis [2]. As noted, nitro-containing oximes, such as FK409 (9), spontaneously decompose

7.3 Oximes

R1

R1

NH2 HCl/NaNO2 R2

R2 O

NOH NH2

NO2

O

O2N NOH CONH2 9, FK409

Scheme 7.7 Synthesis of nitro-containing oximes from dienes.

at pH 7.4 to NO and the corresponding diketone (10, Scheme 7.8) as determined by mass spectrometry [43, 44]. The rate of decomposition of FK409 increases with increasing pH and solutions of 9 below pH 4 demonstrate good stability [44, 46]. Further oxidation of oximes results in the formation of nitroso or nitro compounds or the oxidative cleavage of the carbon–nitrogen double bond [2].

O2N NOH

pH = 7.4

O O

CONH2 9, FK409

10

+ NO

CONH2

Scheme 7.8 Decomposition of FK409 to nitric oxide.

The pKa value for dissociation of the –NOH proton in oximes ranges between 10 and 13 in water and 15 and 28 in dimethyl sulfoxide (DMSO) [47]. Calculations show that the bond dissociation energies for the O–H bond in oximes is around 90 kcal mol−1 [47]. Oxime anions have a weak ultraviolet absorbance at 265 nm (å= 200) and these anions have oxidation potentials between –0.15 and –0.75 V as measured in acetonitrile and compared to the ferrocene/ferrocenium couple [4, 47]. Oximes display two strong resonances in the infrared, one between 3600 and 2700 cm−1 for the O– H stretch and another between 1685 and1520 cm−1 for the carbon–nitrogen double bond stretch [4]. The carbon atom of the carbon–nitrogen double bond group typically resonates between 155 and160 ppm (relative to tetramethyl silane) in the 13 C nuclear magnetic resonance (NMR) spectrum and the –NOH proton typically resonates between 6.5 and 8 ppm (relative to tetramethylsilane) in the 1 H NMR spectrum [4].

183

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7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas

7.3.2

NO-releasing Mechanisms

Two general mechanisms exist for NO release from oximes: 1. hydrolysis to hydroxylamine followed by hydroxylamine oxidation and 2. direct oxidation and decomposition of the oxime. A number of simple alkyl and aryl oximes show vasoactive properties upon conversion to NO [48]. EPR studies show the in vivo formation of iron nitrosyl hemoglobin (HbNO) following the administration of cyclohexanone oxime, a known hemotoxin [49–51] to rats [52]. Studies with 15 N cyclohexanone oxime reveal that the oxime group acts as the source of the nitric oxide group found in the iron nitrosyl hemoglobin [52]. In vitro incubation of cyclohexanone oxime with rat blood also produces HbNO, suggesting that the metabolism of cyclohexanone oxime to NO occurs in blood [52]. The initial hydrolysis of cyclohexanone oxime to hydroxylamine, which further reacts with hemoglobin to yield HbNO [53], has been proposed as a reasonable mechanism of in vivo NO production from cyclohexanone oxime (Scheme 7.9) [52]. HO

N

O hydrolysis

+ NH2OH

oxyHb HbNO

Scheme 7.9 Hydrolysis of cyclohexanone oxime

to hydroxylamine with nitric oxide formation.

Liver microsomes oxidize 4-(chlorophenyl)methyl ketone oxime to produce the corresponding ketone, nitroalkane as well as NO, nitrite and nitrate (Scheme 7.10) [54]. This cytochrome P450 catalyzed conversion requires NADPH and oxygen, and control studies with superoxide dismutase (SOD) show that ketone formation depends on superoxide while nitroalkane formation does not [54]. Similarly, microsomal oxidation of acetoxime yields nitrite/nitrate but purified inducible nitric oxide synthase (iNOS) fails to convert acetoxime to these species [55]. Mechanistically, these cytochrome P450 catalyzed conversions of oximes to NO appear to occur through two pathways: 1. oxidation of the oxime by a cytochrome P450-derived reactive oxygen species, such as superoxide and 2. direct oxidation of the oxime by an enzyme-bound oxidant such as an iron (III) peroxide or a high-valent iron oxo complex [54, 55]. A ferric heme porphyrin model of nitric oxide synthase converts fluorenone oxime to NO in the presence of oxygen [56]. Mechanistic studies show the initial formation of a high-spin ferric heme-oxime complex that undergoes homolysis to form an iminoxyl Cl

Cl

Cl

P450

+

+ NO

O2, NADPH NOH

O

Scheme 7.10 P450 Cytochrome-mediated

nitric oxide formation from oximes.

NO2

7.3 Oximes

NOH

R

N R

Ar

-1 e-

R N OH

R

N O

R R

OH/H2O N O

11a NOH N

R HO R

O N O

R

-1 eR

+ HNO

NO

Ar 11b Scheme 7.11 Oxidative decomposition of oximes to nitric oxide.

radical [56]. Oxygen insertion yields an iron peroxy-nitroso species that decomposes through O–O bond homolysis to NO and fluoreneone [56]. Some aromatic substituted oximes of quiniculidin-3-ones (11a, b Scheme 7.11) also act as NO donors under mild biomimetic oxidative conditions, as determined by electrochemistry [57, 58]. Under basic conditions, these oximes exist primarily as the anion and single-electron oxidation of the anion produces an iminoxyl radical (Scheme 7.11) [58]. Further reaction of this radical with either the hydroxyl radical or water yields a C-nitroso-C-hydroxy intermediate that decomposes to HNO and the corresponding aldehyde or ketone (Scheme 7.11) [58]. Further single-electron oxidation of HNO would yield NO. The addition of a 2-OH group to the aromatic ring proximal to the oxime group yields the best NO donor of this series, which also activates soluble guanylate cyclase [57, 58]. Despite their use in a number of pharmacological experiments and commercial availability, the mechanism of NO release from nitro-containing oximes, such as FK409, remains poorly understood. Structural studies indicate the relationship of acidity of the protons á to the nitro group to the ability of these compounds to release NO [59]. These results led to the suggestion that FK409 decomposes to NO through a Nef reaction, which produces HNO as a by-product [44]. The oxidant for conversion of HNO to NO during FK409 decomposition remains to be identified and FK409 decomposition to NO does not depend on oxygen [43, 44]. Any proposed mechanism of FK409 to NO must account for the following observations: 1. NO formation depends on the presence of both the nitro and oxime groups, 2. the hydrolysis of both groups to ketones and 3. the rate of decomposition increases with increasing pH [44]. 7.3.3

Structure–Activity Relationships

Extensive structure–activity relationships for NO release from oximes do not currently exist. Structural changes do influence the rate of NO release from the nitro-containing oximes, such as FK409 (9, t1/2 = 46 min, Fig. 7.2) [46, 59]. Changing the alkyl group attached to the carbon of the nitro group can increase (12, -CH2 OCH3 , t1/2 = 2.6 min, Fig. 7.2) or decrease (13, -tertBu, t1/2 = 2900 min, Fig. 7.2) the rate of NO release from

185

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7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas

H3CO

O2N NOH

O2N

O2N

O2N

NOH

NOH

NOH

CONH2

CONH2

CONH2

12

13

9, FK409

NH O 14

N

Fig. 7.2 FK409 and other nitric oxide-releasing nitro-oximes.

these compounds [46]. Replacement of the amide carbonyl group with a methylene group also reduces the rate of NO release, as demonstrated by the pyridyl derivative (14, t1/2 = 107 min, Fig. 7.2), a relatively slow release NO donor [46, 59, 60]. The observed changes in the rate of NO release in these compounds upon structural alteration appears related to changes in the acidity of the proton á to the nitro group [59], which provides further support for a Nef reaction-like mechanism of NO release [44].

7.4

N-Hydroxyguanidines 7.4.1

Syntheses and Properties

A number of methods for the preparation of N-hydroxyguanidines exist. Addition of hydroxylamine to a cyanamide, prepared by the condensation of an amine with cyanogen bromide, represents the most straightforward preparation of N-hydroxyguanidines (Scheme 7.12) [61–63]. Alternatively, the condensation of hydroxylamine with S-alkyl thioureas (often with Hg+2 ion catalysis) yields N-hydroxyguanidines (Scheme 7.12) [64]. This approach successfully produces synthetic N-hydroxy-larginine (15, Fig. 7.3), the biochemical intermediate in the nitric oxide synthase mediated conversion of l-arginine to l-citrulline and nitric oxide [64]. The condensation of amines with 1-benzyloxy-3-benzyloxycarbonylthiourea, prepared from benzyl chloroformate, provides another alternative approach to N-hydroxyguanidine synthesis [65]. RNH2

BrCN

SR RHN

NH

RNCN

RHN

NH2

NHOH

NH2OH Hg+2

NHOH

NH2OH

RHN

NH2

Scheme 7.12 General N-hydroxyguanidine synthesis.

7.4 N-Hydroxyguanidines NHOH H2N

O

N H

O

NHOH OH

NH2 15

H2N

OH

N H

NH2 16

Fig. 7.3 Structure of N-hydroxy-L-arginine and its protonated form.

N-Hydroxyguanidines demonstrate good stability if stored at low temperature (– 20°C) and under an inert atmosphere [66]. N-Hydroxyguanidines decompose under basic conditions but demonstrate relative stability as mineral acid salts [66]. Hydroxylation of the guanidine group decreases the basicity of this group from approximately 13.6 (pKa of l-arginine) to 8.1 (pKa of l-N-hydroxy-l-arginine) [66]. NHydroxyguanidines, including, N-hydroxy-l-arginine, undergo one- and two-electron oxidation with oxidation potentials of ∼ 0.5 and 1.0–1.5 V, respectively [67, 68]. While a number of tautomeric forms of N-hydroxy-guanidines exist, both experimental and theoretical studies indicate that the protonated species (16, Fig. 7.3) represents the most likely physiologically relevant form of N-hydroxy-l-arginine [69, 70]. 7.4.2

NO-releasing Mechanisms

Chemical oxidation of N-hydroxyguanidines produces either NO or nitroxyl (HNO), depending upon the oxidant. Oxidation of model N-hydroxyguanidines with lead tetraacetate or a potassium ferricyanide/hydrogen peroxide mixture produce NO, while oxidation with lead oxide, silver carbonate and organic peracids yield HNO [71, 72]. Oxidation of model N-hydroxyguanidines with copper(II) gives small amounts of HNO, presumably through the corresponding aminoxyl radical [73]. Oxidation of N-hydroxyguanidines with singlet oxygen produces nitrite, evidence for the intermediacy of NO [74]. The corresponding ureas or cyanamides usually comprise the organic products of these oxidations [71–74]. Nitric oxide synthase catalyzes the oxygen and NADPH-dependent conversion of N-hydroxy-l-arginine to l-citrulline and NO [75]. Despite the physiological importance of this reaction, a complete mechanism for the nitric oxide synthase catalyzed conversion of N-hydroxy-l-arginine to NO does not exist. In general, single-electron reduction of the resting ferric heme yields a ferrous heme that binds oxygen (Scheme 7.13) [76, 77]. Further reduction of this species yields a peroxy-iron species that reacts with either N-hydroxy-l-arginine or an N-hydroxy-l-arginine-derived radical to give a tetrahedral intermediate that decomposes to l-citrulline and NO (Scheme 7.13, shown with a nitrogen-derived radical) [76, 77]. A number of potential mechanisms, featuring activated iron heme-based oxidants and both oxygen and nitrogen centered radicals, exist and have been summarized [77, 78]. Incubation of synthetic N-tert-butyloxy and N-(3-methyl-2-butenyl)oxy-l-arginine (17, 18, Scheme 7.13) with neuronal nitric oxide synthase produces NO and provides support for radical formation through hydrogen atom abstraction from an N–H bond [78]. However, the recent demonstration of tetrahydrobiopterin radical formation during nitric oxide synthase

187

188

7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas NHR

Fe(III)

1 e-, O2

O O Fe(III)

1 e-

Fe(II)O2

15 H2N Fe(III)

NO + L-citrulline + Fe(III)

N

O

O

O

R = -(CH2)3CH(NH3+)CO2-

O

N

H2N

O

O N H

OH

H2N

NH2

17

O

N

OH

N H 18

NH2

Scheme 7.13 Nitric oxide synthase-mediated conversion of N-hydroxy-L-arginine to nitric oxide.

catalyzed oxidation of N-hydroxy-l-arginine, the natural substrate, may eliminate the requirement for an N-hydroxy-l-arginine-derived radical in NO formation during this reaction [79]. Cytochrome P450 enzymes and peroxidases also catalyze the oxidative formation of NO from N-hydroxyguanidines and these reactions have also been recently reviewed [68, 80]. 7.4.3

Structure–Activity Relationships

Nitric oxide synthase demonstrates relatively strict structural requirements for the catalysis of NO formation from N-hydroxyguanidines other than N-hydroxy-l-arginine [81]. For example, N-hydroxy-l-homoarginine (19) acts as an NO-producing substrate of NOS while N-hydroxy-l-nor-arginine (20) does not (Fig. 7.4) [81]. This difference in reactivity has been rationalized by comparing the possible positioning of these substrates in relation to the reactive oxygen species generated by nitric oxide NH2

NHOH H2N

Cl

OH

N H

NOH

O

21

19 NH2

NHOH H2N

NH

NHOH OH

N H

H2N

H2N O 20

N H 22

NHOH H2N

N H 23

Fig. 7.4 Other N-hydroxyguanidine nitric oxide

producing substrates of nitric oxide synthase.

7.5 N-Hydroxyureas

synthase. In addition to these l-amino acid-derived N-hydroxyguanidines, N-arylN′-hydroxyguanidines, such as N-(4-chlorophenyl)-N′hydroxyguanidine (21), act as NO-producing substrates of the inducible form of nitric oxide synthase (Fig. 7.4) [82]. The incubation of 21 with inducible nitric oxide synthase in the presence of tetrahydrobiopterin results in the formation of NO, as measured by the oxyhemoglobin assay and the corresponding urea [82]. While 21 binds the enzyme weaker than Nhydroxy-l-arginine, the rate of NO production is only four times lower and classic nitric oxide synthase inhibitors block nitric oxide production [82]. The assay of a number of other N-aryl-N′hydroxyguanidines with inducible nitric oxide synthase reveals the presence of a mono-substituted N-hydroxyguanidine group and an N-phenyl ring bearing a small non-electronegative para-substitutent as important structural features for NO formation in these compounds [77]. In addition to these aromatic substituted N-hydroxyguanidines, simple alkyl substituted N-hydroxyguanidines, such as N-butyl-N′-hydroxyguanidine (22) act as efficient NO-producing substrates of nitric oxide synthase (Fig. 7.4) [83]. An important group of subsequent studies shows the nitric oxide synthase isoform selectivity for a structurally diverse group of non-amino acid N-hydroxyguanidines [84]. In general, the N-aryl-N′-hydroxyguanidines act as selective NO-producing substrates of the inducible isoform of nitric oxide synthase [84]. However, N-cyclopropylN′-hydroxyguanidine (23) demonstrates high selectivity as a NO-producing substrate for the neuronal isoform of nitric oxide synthase (Fig. 7.4) [85]. Such results clearly highlight the potential of isoform selective substrates of nitric oxide synthase as selective NO delivery agents. Interestingly, X-ray crystallographic studies show that some of these non-amino acid-derived N-hydroxyguanidines bind to the nitric oxide synthase active site in a different orientation from N-hydroxy-l-arginine, suggesting that that isoform selectivity of these substrates may arise from differential binding [86].

7.5

N-Hydroxyureas 7.5.1

Syntheses and Properties

The first reported synthesis of hydroxyurea (24) consists of the condensation of hydroxylamine with potassium cyanate (Scheme 7.14) [87]. Condensation of hydroxylamine with ethyl carbamate also gives pure hydroxyurea in good yield after recrystallization (Scheme 7.14) [88]. Nitrogen-15 labeled hydroxyurea provides a useful tool for studying the NO-producing reactions of hydroxyurea and can be prepared by the condensation of N-15 labeled hydroxylamine with either potassium cyanate or trimethylsilyl isocyanate followed by silyl group removal (Scheme 7.14) [89, 90]. Addition of hydroxylamine to alkyl or aryl isocyanates yields alkyl or aryl N-hydroxyureas (Scheme 7.14) [91, 92]. The condensation of amines with aromatic N-hydroxy carbamates also produces N-substituted N-hydroxyureas (Scheme 7.14) [93].

189

190

7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas O base

KOCN + NH2OH.HCl

H2N 24

O H2N

+ NH2OH.HCl

OEt

NH OH

O

NaOH H2N

24

NH OH

O 1) 15NH2OH TMS N C O

H2N

15

NH OH

2) CH3OH 15

N-24 O

NH2OH

R N C O

RHN

O

O RNH2

+

HN O OH

NH OH

RHN

NH OH

Scheme 7.14 General N-hydroxyurea synthesis.

Hydroxyurea (24) exists in three tautomeric forms that include a keto form and two imino forms. For each of these structures the hydroxy group may orient itself either syn or anti to the carbonyl group. A number of single crystal X-ray crystallographic studies show that in the solid state all of the atoms of hydroxyurea except the hydroxy hydrogen reside in a single plane [94–96]. These studies show that hydroxyurea primarily adopts the keto form in which the hydroxy group orients itself anti to the carbonyl group and forms a hydrogen bond to the carbonyl group of a neighboring molecule. A group of recent theoretical studies also indicates that the keto form is significantly more stable than the imino forms (> 10 kcal mol−1 ) with the anti keto form being more stable than the syn by 3.7 kcal mol−1 [97–100]. Hydroxyurea possesses three ionizable protons and potentiometric titration experiments indicate that it behaves as a weak acid with a pKa of 10.6 [92, 101]. Alkyl and aryl substituted hydroxyureas demonstrate similar first pKa s [92]. Nuclear magnetic resonance (NMR) and theoretical experiments reveal that the hydroxy proton is the most acidic in aqueous solution [97, 102]. Cyclic voltammetric experiments show that hydroxyurea demonstrates irreversible behavior with a mid-point redox potential of 1070 mV [103]. Alkyl or aryl substituted N-hydroxyureas generally possess lower mid-point redox potentials [92]. Hydroxyurea demonstrates good stability in the solid state and pure samples can be stored for several weeks before any decomposition occurs [104]. Less than 10% of a

7.5 N-Hydroxyureas

0.15 mM hydroxyurea solution decomposes after 9 months at 4°C in neutral aqueous solution [105]. Hydroxyurea also does not decompose in alkaline solutions (pH 14, 30 min, 25°C) or in acidic conditions (pH 1–1.5, 3 h, 25°C) but hydrolyzes rapidly when refluxed in either acidic or alkaline aqueous solution [105,106]. Hydroxyurea decomposes upon oxidation and treatment of a hydroxyurea solution at 25°C (pH 7) with hydrogen peroxide results in 85% decomposition within an hour [105]. Exposure of hydroxyurea to iron(III) chloride yields a gas, identified by infrared spectroscopy as a mixture of nitrous oxide and carbon dioxide [107]. 7.5.2

NO-releasing Mechanisms

The emergence of hydroxyurea (24) as a new therapy for sickle cell disease and the discovery of both the therapeutic and pathophysiological effects of NO in sickle cell disease focuses attention on hydroxyurea as an NO donor [108, 109]. Growing evidence indicates the in vivo conversion of hydroxyurea to nitric oxide in sickle cell disease patients undergoing hydroxyurea therapy. EPR spectroscopic experiments reveal the dose-dependent formation of HbNO in the blood of rats following intragastric administration of hydroxyurea [89]. Experiments using 15 N-labeled hydroxyurea clearly indicate that the NO of HbNO derives from hydroxyurea [89]. Similar EPR experiments show the production of HbNO in the venous blood of a human taking hydroxyurea [110]. Further studies firmly establish the in vivo formation of NO as patients undergoing chronic hydroxyurea therapy for sickle cell disease demonstrate significant increases in HbNO and plasma nitrite, nitrate and cyclic guanylate monophosphate levels [111–113]. Together, these studies clearly show a measurable increase in NO metabolites in patients following administration of hydroxyurea and strongly suggest in vivo NO formation from hydroxyurea. However at this time, the site and mechanism of metabolic in vivo NO formation from hydroxyurea remains to be identified. Nitric oxide formation from hydroxyurea requires a three-electron oxidation (Scheme 7.15) [114]. Treatment of hydroxyurea with a variety of chemical oxidants produces NO or “NO-related species”, including nitroxyl (HNO), and these reactions have recently been extensively reviewed [114]. Many of these reactions proceed either through the nitroxide radical (25) or a C-nitroso intermediate (26, Scheme 7.15) [114]. The remainder of the hydroxyurea molecule may decompose into formamide or carbon dioxide and ammonia, depending on the conditions and type of oxidant (one-electron vs. two electron) employed. Hydroxyurea reacts with oxy, deoxy and metHb in vitro to form iron nitrosyl hemoglobin (HbNO) and transfers NO to 2–6% of the iron heme groups [115]. Trapping studies using cyanide and carbon monoxide indicate that hydroxyurea oxidizes both oxy and deoxyHb to metHb and reduces metHb to deoxyHb specifically identifying the reaction of hydroxyurea and metHb as the critical reaction in the formation of HbNO from hydroxyurea [115]. Scheme 7.16 depicts the proposed mechanisms of NO and HbNO formation during the reaction of deoxy and metHb with hydroxyurea. Oxidation of hydroxyurea by metHb produces deoxyHb and the nitroxide radical (25,

191

192

7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas O

oxidation

H2N

NH OH

NO

-3e-

24 O H2N 25

O H2N

NH O

N O 26

Scheme 7.15 Oxidative nitric oxide formation from N-hydroxyureas.

Scheme 7.16), which may decompose to NO and bind with deoxyHb to form HbNO (Scheme 7.16) [115, 116]. Reduction of the N–O bond of hydroxyurea by two equivalents of deoxyHb gives metHb and urea [115]. Formation of metHb during the reaction of hydroxyurea with oxyHb, as previously noted [117], provides a pathway for HbNO formation from the reaction of hydroxyurea with oxyHb (Scheme 7.16). O 2 H2N

NH OH 24

O 2 HbFe

+3

2 HbFe

+2

+ NH + 2H O

+ 2 H2N 25

urea O H2N

NH 24 OH

O H2N

NH O

HbFe+2 NO

HbNO

25 O

O HbFe(+2)-O2 oxyHb

+

H2N

NH OH 24

HbFe(+3) + H2O2 + H N 2 metHb

Scheme 7.16 Mechanism of nitric oxide formation from

the reaction of hydroxyurea and hemoglobin.

NH O 25

7.5 N-Hydroxyureas

While the reaction of hydroxyurea with hemoglobin clearly forms NO, calculations based on the observed rate constants derived from these studies reveal that only a small amount of hemoglobin would react under physiological conditions [118–120]. Compared to the reported rapid ìM increase in NO metabolites observed in patients taking hydroxyurea [111], these results suggest that other mechanisms besides the direct reaction of hydroxyurea and hemoglobin probably yield the observed increase of NO metabolites. A potential alternative mechanism for NO production from hydroxyurea includes oxidation of hydroxyurea by superior oxidants to hemoglobin. Both peroxidases and copper-containing proteins, including copper–zinc superoxide dismutase and ceruloplasmin react with hydroxyurea to produce NO rapidly relative to the reaction of hydroxyurea with hemoglobin [20, 121]. These reactions have also been recently reviewed [114]. Given that hydroxylamine reacts rapidly with heme proteins and other oxidants to produce NO [53], the hydrolysis of hydroxyurea to hydroxylamine also provides an alternative mechanism of NO formation from hydroxyurea, potentially compatible with the observed clinical increases in NO metabolites during hydroxyurea therapy. Incubation of hydroxyurea with human blood in the presence of urease results in the formation of HbNO [122]. This reaction also produces metHb and the NO metabolites nitrite and nitrate and time course studies show that the HbNO forms quickly and reaches a peak after 15 min [122]. Consistent with earlier reports, the incubation of hydroxyurea (10 mM) and blood in the absence of urease or with heat-denatured urease fails to produce HbNO over 2 h and suggests that HbNO formation occurs through the reactions of hemoglobin and hydroxylamine, formed by the urease-mediated hydrolysis of hydroxyurea [122]. Significantly, these results confirm that the kinetics of HbNO formation from the direct reactions of hydroxyurea with any blood component occur too slowly to account for the observed in vivo increase in HbNO and focus future work on the hydrolytic metabolism of hydroxyurea. 7.5.3

Structure–Activity Relationships

Experiments using synthetic hydroxyurea derivatives provide information regarding the mechanism and the structural features required for NO release. O-Methylhydroxyurea (27, Fig. 7.5) fails to react with any type of hemoglobin [115]. NO H2N 27

O

O NH OCH3

CH3

H2N

N H

N OH

29

28 O

O HN 30

NH OH

N

NH OH 31

Fig. 7.5 Structure–activity relationships of N-hydroxyureas.

NH OH

193

194

7 C-Nitroso Compounds, Oximes, N-Hydroxyguanidines and N-Hydroxyureas

Methylhydroxyurea (28, Fig. 7.5) oxidizes oxyHb to metHb and reduces metHb to deoxyHb but neither of these reactions produces HbNO, further supporting the mechanism depicted in Scheme 7.16 for the formation of NO and HbNO from the reactions of hydroxyurea and hemoglobin [115]. The O-methyl group of 27 prevents the association and further reaction of 27 with the heme iron [115]. Scheme 7.16 predicts the redox chemistry observed during the reaction of 28 with hemoglobin and the failure to detect HbNO shows the inability of 28 or any derivative radicals to transfer NO during these reactions [115]. These results indicate that nitric oxide transfer in these reactions of hydroxyurea requires an unsubstituted acylhydroxylamine (–NHOH) group. The hydroxyurea derivatives (29-31, Fig. 7.5), which contain an unsubstituted – NHOH group, each react with oxyHb to form metHb and variable amounts of nitrite and nitrate [92]. The rate of reaction of these hydroxyurea derivatives correlates well with that compound’s oxidation potential [92]. These experiments show that aromatic hydroxyureas, such as 30, react with oxyHb 25–80 times faster than hydroxyurea, suggesting that other hydroxyurea derivatives may be superior nitric oxide donors [92]. While compound 31 reacts with oxyHb to form metHb, this reaction does not produce HbNO and low temperature EPR studies indicate the failure of 31 to form a low-spin metHb complex [92]. These results reveal the importance of the formation of a low-spin hydroxyurea-metHb complex to NO and HbNO formation during these reactions. In addition to an unsubstituted –NHOH group, these studies show that HbNO formation from the reaction of hydroxyureas and hemoglobin also requires that the nitrogen atom of the non-N-hydroxy group bears at least a single hydrogen atom.

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H. Y., Xian, M., Poulos, T. L., Wang, P. G., J. Med. Chem. 46 (2003), p. 2271–2274 Li, H. Y., Shimizu, H., Flinspach, M., Jamal, J., Yang, W. P., Xian, M., Cai, T. W., Wen, E. Z., Jia, Q. A., Wang, P. G., Poulos, T. L., Biochemistry 41 (2002), p. 13868–13875 Dresler, W. F. C., Stein, J., Justus Liebig’s Ann. Chem. Pharm. 150 (1869), p. 242 Deghenghi, R., Organic Synth. 5 (1973), p. 645–649 Jiang, J., Jordan, S. J., Barr, D. P., Gunther, M. R., Maeda, H., Mason, R. P., Mol. Pharmacol. 52 (1997), p. 1081–1086 Yasaki, G., Xu, Y. P., King, S. B., Synth. Commun. 30 (2000), p. 2041–2047 Ichimori, K., Stuehr, D. J., Atkinson, R. N., King, S. B., J. Med. Chem. 42 (1999), p. 1842–1848 Huang, J. M., Zou, Z., Kim-Shapiro, D. B., Ballas, S. K., King, S. B. J. Med. Chem. 46 (2003), p. 3748–3753 Higgin, J. J., Yakovlev, G. I., Mitkevich, V. A., Makarov, A. A., Raines, R. T., Bioorg. Med. Chem. Lett. 13 (2003), p. 409–412 Armagan, N., Richards, J. P. G., Uraz, A. A., Acta Crystallogr., Sect. B 32 (1976), p. 1042–1047 Berman, H., Kim, S. H., Acta Crystallogr. 23 (1967), p. 180–181 Kjoller-Larsen, I., Jerslev, B., Acta Chem. Scand. 20 (1966), p. 983–991 Remko, M., Lyne, P. D., Richards, W. G., Phys. Chem. Chem. Phys. 1 (1999), p. 5353–5357 Jabalameli, A., Venkatraman, R., Nowek, A., Sullivan, R. H., J. Chem. Phys. 113 (2000), p. 5784–5790 Jabalameli, A., Zhanpeisov, N. U., Nowek, A., Sullivan, R. H., Leszczynski, J., J. Phys. Chem. A 101 (1997), p. 3619–3625 LaManna, G., Barone, G., Int. J. Quantum Chem. 57 (1996), p. 971–974 Kofod, H., Huang, T. Y., Acta Chem. Scand. 8 (1954), p. 494–502 Bagno, A., Comuzzi, C., Eur. J. Org. Chem. (1999), p. 287–295

103 Swarts, J. C., Aquino, M. A. S., Han,

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J. Y., Lam, K. Y., Sykes, A. G., Biochim. Biophys. Acta-Gen. Subj. 1247 (1995), p. 215–224 Kofod, H., Huang, T. Y., Acta Chem. Scand. 8 (1954), p. 485–493 Fishbein, W. N., Winter, T. S., Davidson, J. D., J. Biol. Chem. 240 (1965), p. 2402–2406 Kofod, H., Acta Chem. Scand. 9 (1955), p. 1575–1586 Harmon, R. E., Dabrowiak, J. C., Brown, D. J., Gupta, S. K., Herbert, M., Chitharanjan, D., J. Med. Chem. 13 (1970), p. 577 Halsey, C., Roberts, I. A. G., Br. J. Haematol. 120 (2003), p. 177–186 Reiter, C. D., Gladwin, M. T., Curr. Opin. Hematol. 10 (2003), p. 99–107 Glover, R. E., Ivy, E. D., Orringer, E. P., Maeda, H., Mason, R. P., Mol. Pharmacol. 55 (1999), p. 1006–1010 Gladwin, M. T., Shelhamer, J. H., Ognibene, F. P., Pease-Fye, M. E., Nichols, J. S., Link, B., Patel, D. B., Jankowski, M. A., Pannell, L. K., Schechter, A. N., Rodgers, G. P., Br. J. Haematol. 116 (2002), p. 436–444 Morris, C. R., Vichinsky, E. P., Van Warmerdam, J., Machado, L., Kepka-Lenhart, D., Morris Jr, S. M., Kuypers, F. A., J. Pediatr. Hematol. Oncol. 25 (2003), p. 629–634 Nahavandi, M., Tavakkoli, F., Wyche, M. Q., Perlin, E., Winter, W. P., Castro, O., Br. J. Haematol. 119 (2002), p. 855–857 King, S. B., Current Med. Chem. 10 (2003), p. 437–452 Huang, J., Hadimani, S. B., Rupon, J. W., Ballas, S. K., Kim-Shapiro, D. B., King, S. B., Biochemistry 41 (2002), p. 2466–2474 Pacelli, R., Taira, J., Cook, J. A., Wink, D. A.m Krishna, M. C., Lancet 347 (1996), p. 900 Stolze, K., Nohl, H., Biochem. Pharmacol. 40 (1990), p. 799–802 Kim-Shapiro, D. B., King, S. B., Bonifant, C. L., Kolibash, C. P., Ballas, S. K., Biochim. Biophys. Acta-Gen. Subj. 1380 (1998), p. 64–74 Kim-Shapiro, D. B., King, S. B., Shields, H., Kolibash, C. P.,

References Gravatt, W. L., Ballas, S. K., Biochim Biophys Acta 1428 (1999), p. 381–387 120 Rupon, J. W., Domingo, S. R., Smith, S. V., Gummadi, B. K., Shields, H., Ballas, S. K., King, S. B., Kim-Shapiro, D. B., Biophys. Chem. 84 (2000), p. 1–11 121 Sato, K., Akaike, T., Sawa, T., Miyamoto, Y., Suga, M., Ando, M.,

Maeda, H., Jpn. J. Cancer Res. 88 (1997), p. 1199–1204 122 Lockamy, V. L., Huang, J. M., Shields, H., Ballas, S. K., King, S. B., Kim-Shapiro, D. B., Biochim. Biophys. Acta-Gen. Subj. 1622 (2003), p. 109–116

199

Part 2 NO Donors’ Applications in Biological Research

203

8

Vasodilators for Biological Research Anthony Robert Butler, Russell James Pearson 8.1

NO-donor Drugs for Biological Research

With the discovery that NO plays a role in vasodilation it has become important that nitrovasodilators (i.e. substances that act as vasodilators by the release of NO) are well-characterised. This includes a clear understanding of the mechanism of NO release. The use of an inappropriate agent may give non-reproducible or false data. At least three crucial factors must be considered when an NO-donor drug is selected: • Is the substance water and/or lipid soluble? • What chemical or biochemical process effects NO release? • Are any toxic or biologically active species produced as a result of NO release? Substances regularly used in biological and physiological research will be examined in the light of these three criteria with the aim of providing researchers with the relevant information to aid in the selection of an NO-donor suitable for the experiments being undertaken.

8.2

Sodium Nitrite (NaNO2 )

Undoubtedly sodium nitrite is a vasodilator [1]. This is seen from anecdotal evidence: when nitrite is used as an antidote to cyanide poisoning hypotension is a major hazard. However, in ex vivo experiments the effect of nitrite is small but the situation in vivo is more difficult to assess, for reasons that will be clear shortly. It is now generally assumed that nitrite acts as a vasodilator because it can undergo a spontaneous reaction to give NO. The termolecular equation (Eq. (1)) sometimes given for this process is certainly incorrect as termoleculer reactions very rarely occur. 3HNO2

→

H2 O + 2NO + HNO3

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

(1)

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8 Vasodilators for Biological Research

In fact the process occurs in two steps and the correct equations are: 2HNO2 N2 O3





H2 O + N2 O3

(2)

NO + NO2

(3)

The rate at which loss of nitrous acid occurs depends upon whether or not N2 O3 , NO and NO2 are lost from the solution as all are gaseous at room temperature and both steps are equilibria. Disregard of these matters has led to some misleading data in the literature [2]. If the concentrations are such that the solubilities of N2 O3 , NO and NO2 are not exceeded and all species stay in solution, the rate at which NO is generated from nitrite is very slow and, consequently, sodium nitrite is a poor source of NO [3]. In addition, because the protonated form (HNO2 ) is what undergoes reaction, production of NO occurs only in an acid medium; a pH as low as 5 is required. This is unrealistic when considering biological situations. Nitrite does occur in blood plasma and there is some debate about its in vivo role [4, 5]. Chemical conversion of nitrous acid into sensible concentrations of NO at blood pH does not take place. In vivo there is, of course, the possibility of enzymatic or biological conversion of nitrite into NO. It is unlikely that xanthine oxidoreductase, although effective, is responsible for this because of high Km values [6–8] but oxyhemoglobin may well act to effect conversion [9]. In view of these facts, the use of NaNO2 as a vasodilator in biological experimentation is highly suspect. It has the virtue of high water solubility but all else is uncertain. One further matter is relevant, a second product of reaction is NO2 (see Eq. (3)) which is highly toxic. There are far better nitrovasodilators for biological experimentation than sodium nitrite. Nitrates have no vasodilator effect at all. However there are enzymes, particularly in the mouth, which convert nitrate into nitrite [10] and so there is the possibility that ingestion of nitrate could result in vasodilation.

8.3

S-Nitrosothiols

Compounds of this structure were first described many years ago by a Scottish chemist named MacBeth. He noted that addition of nitrite to an acid solution of a thiol produced a transient red color, which he credited to formation of an S-nitrosothiol or thionitrite. The chemical properties of these compounds have been described elsewhere in this volume by Mutus (Chapter 4). There are also reviews by Williams [11] and by Wang et al. [12]. All known compounds of this class decompose in what appears to be a spontaneous manner as follows: 2RSNO

→

RS–SR + 2NO

(4)

If the nitrosothiol is made from a naturally-occurring thiol-containing amino acid the S-nitrosothiol should be the perfect agent for the study of nitrovasodilation. The product, other than NO (i.e. the disulfide), is also a naturally occurring substance, and S-nitrosothiols themselves occur naturally [13]. Release of NO occurs at room

8.3 S-Nitrosothiols

temperature. Cysteine is a naturally-occurring S-containing amino acid but nitrosocysteine decomposes so rapidly, even when stored in ice, that results obtained by its use cannot be relied upon. If partial decomposition has occurred before the drug is utilised, it is impossible to know if the observed effect is due to unreacted nitrosocysteine or to NO. Also, NO in aqueous solution is rapidly lost to the atmosphere unless very careful precautions are in place and a solution of decomposed nitrosocysteine may contain little or no NO. It was suggested at one time that the endothelium-derived relaxing factor (EDRF) was nitrosocysteine [14] but that suggestion has been refuted on a number of grounds, including its instability [15]. Glutathione is a tripeptide containing cysteine and nitrosoglutathione (GSNO) is a different matter as it can be isolated and stored (Fig. 8.1). Also it is biologically active, suggesting that it does release NO when used. What prompts it to start releasing NO in solution when it is completely stable as a solid? This question dogged the early use of all nitrosothiols in biological research. Mathews and Kerr [16] produced evidence to suggest there is no correlation between the rate of in vitro NO release from a number of nitrosothiols and their vasodilator action. They concluded that NO release was not the source of their biological activity. Some light was shed on the situation when Williams et al. [17] showed that copper ions are very powerful catalysts for NO release from nitrosothiols. There is enough adventitious copper in normal distilled water to effect fairly rapid NO release from an otherwise stable nitrosothiol, S-nitrosothio-N-acetyl-penicillamine (SNAP). SNO O

O

O H N

GSNO

N H

HO

OH

NH2

O

O SNAP

HO

SNO NHAc

Fig. 8.1 Chemical structure of GSNO and SNAP.

The rate of release of NO from SNAP in the absence of any copper ions (the spontaneous or thermal reaction) is very slow indeed. The reason for the very low stability of nitrosocysteine is that there is a ready complexation of copper ions to this compound, a process that is the precursor of NO release [18]. Further studies by Butler and Williams [19] showed that it is the cuprous ion, Cu(I), which is the active species in effecting NO release from a nitrosothiol, whatever copper salt is present in solution. Cuprous ions are readily generated from Cu(II) by reaction with a thiol: CU++ + RS−

→

CU+ + RS•

(5)

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8 Vasodilators for Biological Research

Thus NO release from a nitrosothiol will not occur unless there is thiol present, as Cu(II) is the more stable oxidation state under most conditions. So, for SNAP, a very conveniently prepared nitrosothiol [20], to act as a source of NO both copper ions and thiols must be present. All, or most, tissue contains thiols so this is no problem but the presence of copper is more doubtful. There is not much copper in the human body but what there is, is concentrated in certain regions, giving concentrations quite high enough to act as a catalyst for NO release. The need for copper ions in NO-release must cast doubt on some quantitative data obtained by the use of SNAP. The amount of NO delivered during ex vivo experimentation may depend on the amount of copper present in the tissue rather than the concentration of SNAP. This makes comparisons between the effect of NO in different tissues unreliable unless the experimenter is sure that the tissues being compared contain similar amounts of copper. There is direct experimental evidence that copper is involved in the release of NO from nitrosothiols in ex vivo tissue [21]. On the other hand, the amount of NO required for physiological effect is so small, picamole quantities, that thermal decomposition of a nitrosothiol may be sufficient, but the presence of copper will inevitably give more NO and it is now accepted that the biological effect of NO depends critically upon the amount delivered; too much may reverse the effect observed when the amount is optimal. The use of GSNO in vasodilator research is particularly unsafe as the effect of copper ions, as determined by in vitro studies, is much more complex than with SNAP. Briefly, Cu(I) ions complex not only with GSNO but also with glutathione disulfide (a reaction product) and this means that the concentration of Cu(I) ions falls as the reaction proceeds, killing the reaction when all the Cu(I) ions have been complexed [22]. The absence of any observed effect when GSNO is used as a vasodilator may be due to removal of copper ions by complexation with glutathione disulfide. In summary nitrosothiols are valuable vasodilators as they are water soluble and produce no toxic by-products. However there is considerable doubt about what prompts the release of NO from what, in vitro, are stable compounds. Release may be thermal and therefore a property of the compound and independent of tissue. On the other hand, Cu(I) ions are such powerful catalysts for NO release that their presence in tissue will greatly enhance the amount of NO formed. NO-delivery then becomes a property of the tissue as well as the compound, complicating interpretation of the results. The situation is further complicated by the suggestion that there is an enzyme responsible for NO-release from nitrosothiols [23]. In spite of all the reservations given above, much reliable and reproducible data has been obtained by the use of nitrosothiols. A few examples from the many possible are now given to illustrate this point. Experiments using GSNO established the role of NO in effecting relaxation of bronchial muscle in guinea pigs [24]. GSNO has proved valuable in the cold preservation of liver because, as an NO-donor, it prevents hepatic injury [25]. After a pulmonary bypass in dogs there is an increase in the activity of nitric oxide synthase but the increase can be prevented by a dose of GSNO. Clearly GSNO, as an NO-donor, obviates the need for increased endogenous synthesis of NO [26].

8.3 S-Nitrosothiols

Most nitrosothiols are soluble enough to be used conveniently for biological experiments. However, in flow systems they produce only transient effects. Thus a bolus injection of SNAP solution into a perfusate passing through an isolated segment of artery produces transient dilation. The solubility of the nitrosothiol prevents retention of the drug at the site of action. However this is countered if the lipophilic nature of the nitrosothiol is enhanced. For example, S-nitroso-N-valerylpenicillamine (SNVP) has sustained vasodilator effect when delivered in perfusion experiments (Fig. 8.2). This characteristic could be of value to physiologists. O HO

SNO Fig. 8.2 S-nitroso-N-valerylpenicillamine

HN

as a sustained vasodilator.

Similarly sugar-SNAPs (Fig. 8.3) developed by Wang and his group [27–30] have interesting pharmocokinetic properties. OH HO HO

O

NHAc

H N

OH

Glucose-1-SNAP SNO

O OH HO HO

O

OH NHAc

Glucose-2-SNAP

HN SNO O

Fig. 8.3 Sugar-SNAPs.

These compounds were designed to permit improved transport of nitrosothiols in mammalian cells using glucose transporters. The aglycone moiety provides the pharmacological activity, whereas the carbohydrate unit enhances water solubility, cell penetration and drug–receptor interaction. The sugar-SNAPs are more stable than SNAP itself and release NO more slowly. An acetylated version was made by Butler et al. [31] (RIG200) (Fig. 8.4) and, in perfusion experiments induced prolonged vasodilation in endothelium-denuded, isolated rat femoral arteries. Butler et al. [32] also developed a series of O-acylated-S-nitrosated thiosugars (Fig. 8.5). Partial de-acylation in tissue should give a mixture of hydrophilic and hydrophobic groups. These compounds can be delivered transdermally and result in greatly enhanced subcutaneous blood flow [33]. Unfortunately, these compounds are not very stable and cannot be isolated. The alcoholic solution, in which they are stored and administered, must be kept on ice during use.

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8 Vasodilators for Biological Research

OAc O

AcO AcO

OAc O

NH SNO

AcHN

Fig. 8.4 RIG200. OAc O

AcO AcO

SNO AcO

D-SNAG OCOC2H5 C2H5OCO C2H5OCO

O SNO

OCOC3H7 O

C3H7OCO C3H7OCO

SNO

C2H5OCO

C3H7OCO

D-SNO-PROP

D-SNO-BUT

OCOC4H9 C4H9OCO C4H9OCO

O SNO

OCOC5H11 O

C5H11OCO C5H11OCO

C4H9OCO

D-SNO-VAL

SNO C5H11OCO

D-SNO-HEX

Fig. 8.5 S-Nitroso-1-thio-2,3,4,6-O-acylated-

glucopyranose compounds of increasing lipophilicity.

S-Nitrosodipeptides are resistant to copper ion catalysed NO release [34], presumably because of poorer complexing ability with Cu(I) ions, but the low solubility of these compounds diminishes their use as vasodilators. A very common reaction, of nitrosothiols is, as discussed in Chapter 4, transnitrosation (Eq. (6)) in which the NO group is transferred from a nitrosothiol to a thiol as NO+ . RSNO + R′S−
(6)  RS− + R′SNO As there are fairly high concentrations of thiols in many tissues this reaction may occur whenever a nitrosothiol is used in ex vivo or in vivo experiments. Thus, the nitrosothiol used may be merely a storage vehicle for NO+ and NO+ passes in a heterolytic reaction to a tissue thiol, probably cysteine or glutathione. It is this nitrosothiol that cleaves homolytically to give the radical species NO. Thus the reaction

8.4 Metallic Nitrosyls

generating NO is independent of the nitrosothiol used. Another possibility is that NO passes directly from the delivered nitrosothiol to the iron of the heme component of guanylate cyclase without ever becoming free. A model reaction for such a process has been developed by Butler and Williams [35]. More detailed research is required to understand exactly how nitrosothiols effect vasodilation. In the opinion of these authors transnitrosation to cellular thiols is the most likely mechanism and this is consistent with their reliability as vasodilators. For initial experimentation SNAP is probably the most convenient compound to use as it is cheap and can be stored. For more sophisticated experiments it is important to choose a drug that has the right solubility or lipophilic properties. The sugar-SNAPs show great promise here.

8.4

Metallic Nitrosyls

Nitric oxide is a very common ligand and can be present in the complex formally as NO, NO+ or NO− . Some such complexes are a source of NO but, with the exception of sodium nitroprusside, few have been used as vasodilators. Because of the importance of sodium nitroprusside it will be considered separately. Of other complexes which can release NO one of the most intriguing is a nitrosylated iron-sulfur complex known as Roussin’s Black salt, NH4 [Fe4 S3 (NO)7 ] [36]. This species releases NO on oxidation, the other products of the reaction are rust and sulfur. Although a convenient source of NO the ionic nature of Roussin’s Black salt might suggest that it would not cross cell members and would have limited value. However the anion [Fe4 S3 (NO)7 ]− is soluble in organic solvents and can readily penetrate muscle tissue. For this reason it is an effective nitrovasodilator in perfusion experiments [37]. It is also an effective disinfectant of contaminated drinking water. The disadvantages of Roussin’s Black salt as an NO-donor drug are the difficulties experienced in its synthesis and poor shelf stability. The by-products of reaction are innocuous but the accumulation of rust in tissue may have adverse effects. A number of other metallic nitrosyl complexes have been examined as NO-donor drugs but none have proven to be of value [38].

8.5

Sodium Nitroprusside (Na2 [Fe(CN)5 NO] ⋅ 2H2 O)

Although its mode is uncertain, sodium nitroprusside (SNP) is one of the most valuable vasodilators. Its use in clinical practice is suspect as the cyano-ligands render cyanide poisoning a possibility. However for ex vivo experiments this consideration is less important but the possibility of some biological action due to these ions remains. The mechanism by which SNP acts as a vasodilator is not fully understood. With the discovery of a physiological role for NO there has been renewed interest in mechanistic studies of reactions involving SNP and a re-examination of studies of SNP undertaken before 1987. So far, only one simple reaction leading to the release

209

210

8 Vasodilators for Biological Research

of NO from SNP in solution has been discovered (Eq. (7)) and that is a photochemical one [37]. hí [Fe(CN)5 NO]2− (7) [Fe(CN)5 H2 O]2− + NO → H2 O It is not unreasonable to suggest that this is the reaction responsible for the vasodilator action of nitroprusside (NP) because, in most experiments, the apparatus is not protected from light. When SNP solution is used during vascular surgery by infusion into a blood vessel it is subject to high levels of illumination and, although protective aluminum foil is provided, it is rarely used. Against this view, the fact that SNP is such a reliable and reproducible vasodilator mitigates against adventitious lighting as the genesis of its vasodilator action. Also, the simple reaction given above is only the first reaction step in a complex series of photochemically produced products. A study [39] has confirmed the photochemical formation of NO but also detected HCN and cyanogen as gaseous products. Duchstein and Rioderen [40] confirmed the ‘dark’ stability of SNP but found that illumination caused immediate formation of NO. The quantum yield for the production of NO is low, 0.20 and 0.32 at 420 and 320 nm, respectively [41]. There is direct evidence from the work of Flitney et al. [42] for a photochemical source of NO in physiological experimentation. Ambient light was found to be sufficient to activate SNP to dilate isolated frog heart preparations and these preparations, when shielded from light, failed to respond to SNP. Furthermore Hirst et al. [43], in a study of the regulation of cell respiration by NO, showed that intense light was necessary to produce an inhibitory effect with SNP, whereas another NO-donor (Roussin‘s Black salt) had an effect with ordinary laboratory lighting. In view of these observations, and the known photochemistry of nitroprusside, it is sensible to shield from direct light all experimental set-ups where SNP is to be used. If direct illumination changes the results obtained, then photochemical release of NO must be considered. The situation, however, is more complicated than has been suggested so far. With mammalian tissues the use of SNP gives highly reproducible results, irrespective of lighting conditions. Clearly lighting is something to be considered, particularly when using amphibian tissue, but other mechanisms of NO release must normally dominate the release of NO from SNP in mammalian tissue. For example SNP is a dilator of mammalian artery even in the complete absence of light [44]. Also the quantum yield for the release of NO from SNP is low and ordinary lighting is unlikely to result in production of enough NO to effect biological action, so there must be a ‘dark’ reaction resulting, eventually, in NO release. SNP reacts readily with hydroxide, amines and carbanions but it is unlikely that any of these reactions is involved in NO release as they are all relatively slow. However, the reaction with thiols is likely to be involved. The reaction is very fast [45] and there are thiols present in most tissues. The overall reaction is thought to be as shown in Scheme 8.1. The adduct formed in the first step (an equilibrium) is highly colored and decomposes either back to reactants or to a reduced species [Fe(CN)5 NO]3− and the thiyl radical RS• . In the presence of oxygen the reduced species is oxidised back to NP, giving a cyclised oxidation of RS− to RS• (and disulfide) but, under anaerobic conditions, NP reacts with eventual release of NO [46]. However, in spite of the desired

8.5 Sodium Nitroprusside [Fe(CN)5NO]2-

+

RS-

[Fe(CN)5N(SR)O]3-

[Fe(CN)5NO]3-

+

RS.

1/2 RS-SR

[Fe(CN)4NO]2-

+

CN-

[Fe(CN)4]2-

+

NO

6[Fe(CN)4]2-

+

6CN-

O2

5[Fe(CN)6]4-

+

Fe2+

Scheme 8.1 The overall reaction of SNP in tissue.

release of NO, this is not a complete account of the ‘dark reaction’ as it occurs only under anaerobic conditions which rarely apply in biological experimentation. It would appear that mammalian tissue effects some other chemical or enzymatic reaction. It has been claimed [47] that when SNP is mixed with whole blood the ion breaks down completely to give Fe2+ , cyanide and NO but NMR studies using 13 C-labelled NP, which could be assayed non-intrusively, did not confirm this result [48]. The cyano ligands of NP are non-labile but in some complexes readily formed from NP (e.g. [Fe(CN)5 H2 O]2− and [Fe(CN)5 NOSR]3− the ligands are labile and this may explain why certain analytical procedures indicate the presence of free cyanide when, in fact, it is still complexed. The exact nature of the NO-releasing reaction and the other products of reaction in mammalian tissue are still unclear. The matter has been discussed by a number of authors and a reductive mechanism in rat hepatocytes and human erythrocytes has been suggested in the presence of NADH and NADPH. Nitroprusside can pass through cell membranes and so there is no intrinsic difficulty with this suggestion. There is direct evidence from spin echo NMR studies to show the conversion, by nitroprusside, of glutathione into glutathione disulfide within erythrocytes [49]. However, the reaction of NP with thiols may be a necessary but not sufficient cause for the release of NO from the ion as there are many thiols in frog heart tissue and NP is a vasodilator only under illumination. Furthermore Sogo et al. [50] could not detect NO generation from NP in human plasma containing cysteine, glutathione, homocysteine and reduced cysteine residues. Therefore, there must be a unique component of mammalian tissues which is involved in the release of NO from NP, and this reaction comes after reaction with thiol. Kowaluk et al. [51] report that NP is readily metabolised to NO in subcellular fractions of bovine coronary arterial smooth muscle and that the dominant site of metabolism is in the membrane fraction. This led to the isolation of a small membrane-bound protein or enzyme that can convert NP into NO. The mechanism shown in Scheme 8.2 combines the thiol reaction and that with an enzyme.

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8 Vasodilators for Biological Research

[Fe(CN)5NO]2-

+

RS-

[Fe(CN)5N(SR)O]3-

+

RS.

1/2 RS-SR

O2 [Fe(CN)5NO]3-

enzyme and protein [Fe(CN)5H2O]2-

+

NO

Scheme 8.2 NP mechanism with a thiol and enzyme.

Although incomplete this scheme gives the most satisfactory account of the release of NO from NP. In spite of an incomplete understanding of the mechanism of action, NP is an excellent vasodilator. For example pulmonary arterial infusion of SNP can reduce lung injury reperfusion following cardiopulmonary bypass in dogs [52]. SNP proved a valuable vasodilator in a study of the effect of experimental diabetes on vascular function in isolated perfused kidneys [53]. In a study of the dilation of the rabbit basilar artery [54] SNP was found to be more effective than NONOates, an effect unchanged by removing the endothelial cells. Such is the success of NP that it is often used as a measure against which all other nitrovasodilators are measured. So, in spite of the lack of clarity in understanding its mode of action, SNP is a commonly used, secure vasodilator. It acts quickly and has a powerful effect. It is water-soluble and a solution, if protected from light, is stable at room temperature. In flow systems the dilator effect is transient. A positive result with SNP does suggest that the effect under scrutiny is an NO-mediated one. However, for complete confidence in interpreting the result, testing the effect of lighting is a wise precaution. If there is a photochemical response then the effect might be difficult to reproduce in other laboratories.

8.6

Organic Nitrates

A number of organic nitrates are potent vasodilators and have been used clinically for over 100 years, particularly for the relief of the symptoms of angina (Fig. 8.6). The most widely used is glyceryl trinitrate (GTN) but the group includes pentaerythrityl tetranitrate (PETN), isosorbide mononitrate (ISMO) and isosorbide dinitrate (ISDN). All are explosive but require detonation and their explosive nature has not restricted their use in medicine. GTN was used in the early experiments to identify the EDRF as NO. Also, it was probably the vasodilator action of GTN which first suggested that NO could be the endogenous EDRF [55]. As GTN requires metabolism to convert it into NO it was fortunate that this occurred within the tissue used for the seminal, ex vivo experiments.

8.6 Organic Nitrates ONO2

O2NO

ONO2

ONO2 ONO2

ONO2

O2NO

GTN

PETN

HO

O2NO O

O O

O ISMN

ONO2

ONO2

ISDN

Fig. 8.6 Four commonly used organic nitrates.

An alcoholic solution of GTN, if protected from light, is stable over many months. Most organic nitrates are only slightly soluble in water and under alkaline conditions [56] hydrolyse to give an alcohol and nitrate (Eq. (8)) or undergo á-H elimination to give nitrite (Eq. (9)). RCH2 ONO2 + OH− −

RCH2 ONO2 + OH

→ →

ROH + NO3 − RCHO + H2 O + NO2

(8) −

(9)

Because of the high pH required for this process and the poor vasodilator properties of nitrite, this process cannot explain the biological action of organic nitrates. There are two problems in the use of organic nitrates as vasodilators: 1. the conditions necessary for the biotransformation that converts an organic nitrate into NO, and 2. the phenomenon of tolerance. The two may be linked. There are probably multiple pathways in the conversion of an organic nitrate into NO, some chemical and some enzymatic. For the former the presence of thiol groups appears to be necessary [57, 58]. The overall reaction is shown in Eq. (10). This is not a balanced equation and does not reflect fully the course of the reaction. A number of studies have shown that thiols, both in vivo and in vitro, can affect this process. In most cases it appears that thiols act as a reducing agent, being converted to disulfide, while nitrite is released (Eq. (11)). RONO2 + R′SH RONO2 + 2R′SH

→ H2 O →

ROH + 1/2 R′SSR′ + NO

(10)

ROH + R′SSR′ + NO2 − + HO−

(11)

However, a few thiols (cysteine, N-acetylcysteine and thiosalicylic acid) also react with organic nitrates to release NO by another process that is difficult to discern. It could be that the nitrite reacts with a thiol to give an S-nitrosothiol, a ready source of NO, but nitrosation is unlikely to occur at biological pHs. Another possible route to NO involves formation of a thionitrate by trans-esterification [59] (Eq. (12)). This species could then decompose to give NO via an intermediate sulfenyl compound [60] (Eq. (13)). R′SNO2

→

R′SONO → R′S(=O)NO

(12)

2R′S(=O)NO

→

R′S(=O)S(=O)R′ + 2NO

(13)

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However, experiments show that non-enzymatic release of NO from an organic nitrate is very slow, far too slow to explain its rapid and dramatic vasodilator effect. Therefore during in vivo and ex vivo experiments it appears that an enzymatic process occurs. The site of this biological process has not been identified. Both an NADPHdependent cytochrome P450 pathway [61–63] and a pathway involving enzymes of the glutathione-S-transferase family [64] have been proposed. Another possibility is that the organic nitrate reacts with a ferrous-heme moiety of hemoglobin and myoglobin to release NO [65]. Also, GTN is yet another substrate for the enzyme xanthine oxidoreductase and the reaction releases NO [66]. A rather different route, involving formation of an organic nitrite by a process requiring reduced flavin, has been proposed [67]. The organic nitrite then nitrosates a cellular thiol and NO is formed from the nitrosothiol (Eqs. (14) and (15)). R′ONO2

[H+ ] →

2RSNO

→

R′ONO

RS− →

R′O− + RSNO

RSSR + 2NO

(14) (15)

In view of the use of organic nitrites in the synthesis of S-nitrosothiol this appears to be a very reasonable suggestion. As there is uncertainty of the metabolic pathway for the release of NO from organic nitrates there must be some doubt about their use in physiological experimentation. They can be a ready source of NO but certain conditions, as yet not fully delineated, have to be in place before NO release occurs. These conditions may not always apply and the result of an experiment using an organic nitrate, if negative, remains slightly doubtful. On the other hand, many significant results have been obtained by the use of organic nitrates, particularly GTN. The other factor affecting the use of organic nitrates is nitrate tolerance, the mechanism of which is unclear. An early explanation of tolerance was thiol depletion [68] but that now seems unlikely as their is an abundance of thiol in most tissue [69]. A more likely explanation is down regulation of the enzymes involved in the biotransformation but few details are available. An interesting suggestion is that GTN induces increased production of superoxide from the vascular wall and tolerance is caused by reaction of NO, produced enzymatically from GTN, with superoxide to give peroxynitrite and then nitrate [70] (Eq. (16)). NO + O2 •−

→

ONOO− → NO3 −

(16)

Ascorbic acid [71] and vitamin E [72] are powerful scavengers of reactive oxygen species and are known to enhance the sensitivity of vascular tissue to organic nitrates. Also the local concentration of NO due to administration of GTN is greatly enhanced by hypoxia [73]. Clinical tolerance is a major problem with the use of GTN in the management of the symptoms of angina. If GTN is delivered transdermally, from a patch, tolerance starts after about 16 h and recovery takes about another 8 h. In view of the time scale it is unlikely that tolerance is a problem in most physiological experimentation but it should always be considered. Rather curiously pentaerythritol tetranitrate (PETN) is resistant to tolerance [74, 75].

8.7 Organic Nitrites

Some new organic nitrates are of interest. Sininitrodil [76] (Fig. 8.7), SPM-4744 and SMP-5185 [77] selectively dilate large microvessels and SPM-5185 is resistant to cross-tolerance with GTN in human blood vessels in vitro. O O

O N O

O

Fig. 8.7 The chemical structure of Sininitrodil.

GTN is a very convenient organic nitrate to use, although during handling its explosive nature should be remembered. If the experimental results are suspect in any way it might be wise to try one of the organic nitrates, which do not display tolerance. In research, as in other areas of human life, it is wise to be nimble.

8.7

Organic Nitrites

Compounds of this family (such as tert-butyl nitrite and isoamyl nitrite) are powerful vasodilators and have been used clinically for the relief of the symptoms of angina for over 100 years (Fig. 8.8). ONO ONO

Fig. 8.8 The chemical structure of tert-butyl nitrite (TBN) TBN

IAMN

and isoamyl nitrite (IAMN).

They are more potent than nitrates and there is no development of tolerance [78]. Their drawbacks are that they are very volatile (and therefore difficult to deliver quantitatively) and photochemically and thermally unstable. Organic nitrites are excellent nitrosating agents, particularly for thiols (Eq. (17)). RONO + RS−

→

RO− + RSNO

(17)

This could be an in vivo route to NO. There are enzymes, which will catalyse this process (e.g. glutathione S-transferase) [79]. In aqueous solution they hydrolyse to alcohol and nitrite (Eq. (18)). RONO + H2 O

→

ROH + NO2 − + H+

(18)

Nitrite can be reduced in vivo to NO by oxyhemeoglobin [9]. At the same time organic nitrites are substrates for xanthine oxidoreductase and direct reduction to NO occurs under anaerobic conditions [8]. For some clinical purposes the volatility of organic nitrites is an advantage as it allows inhalation, but for physiological experimentation the volatility is a serious

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drawback. Also, nitrites have very low solubility in water. Although useful, an organic nitrite is not the nitrovasodilator of first choice.

8.8

NONOates

A very popular category of NO donor, at least in biological research, is the NONOates, first synthesized in the 1960s [80]. They have several nicknames to accompany the array of chemical entities associated with them. Described rather loosely by some as a class of sulfur-free compounds capable of releasing NO [81], these NO/nucleophile adducts are more correctly named diazeniumdiolates. From their generic structure of X[N(O)NO]− , in which X is any suitable secondary amine, these compounds are relatively simple to synthesize, though obviously the choice of X is instrumental in determining the chemical properties and ultimately the overall potency and applicability of the drug molecule. Generally speaking NONOates are stable in the solid form [82] and thus have a good shelf life whilst being highly water soluble, releasing two moles of NO per mole of parent compound once in an aqueous environment [83] (Scheme 8.3). However, in further work, it was found that at neutral pH the actual amount of NO measured is slightly lower than what would be predicted from Scheme 8.3 and so a ratio slightly less than 2:1 is perhaps a more adequate representation [84]. The rate at which NONOates release NO is chemically predictable and can vary in accordance with X; thus half-lives can range from 1 min to 1 day [85]. One of the special characteristics of NONOates, aside from their favorable solubility in water, is that their potency as vasodilators correlates so well with the first order rate with which they spontaneously release NO in aqueous buffer [86]. R N R'

+ N

O N

H2O ONa

R NH

+

2NO

+

NaOH

R'

Scheme 8.3 The release of NO from a NONOate in an aqueous

A non-enzymatic release of NO that is not catalysed by exogeneous thiol or albumin has led to these compounds being used extensively in biological research [86]. In experiments other than the direct correlation (between the rate constant for NO release, from NONOates, and the EC(50) for activating sGC) there is a clear correlation between the rate of NO donation and the observed biological potency. Diethylamine/NO (DEA/NO), spermine/NO (SPER/NO) and diethylenetriamine/NO (DETA/NO) are three commonly used NONOates (Figure 8.9). Of the three NO donors, DEA/NO releases NO the fastest whilst DETA/NO is the slowest at supplying NO. It is therefore completely consistent that, when administered to the cerebrovascular beds of isolated rabbit basilar arteries, the degree of relaxation mirrors the same trend, with DEA/NO and DETA/NO being most and least active [87].

8.8 NONOates O

Diethylamine/NO (DEA/NO)

N

O

N

O

N N

Spermine/NO (SPER/NO)

O N N

H2N

NH2

N H

Diethylenetriamine/NO (DETA/NO)

N

O

O

N H2N

N

NH2

Fig. 8.9 The common NONOates: DEA/NO, SPER/NO

and DETA/NO.

In other studies relating to cerebral ischemia, SPER/NO has shown neuroprotective potential by improving rat brain perfusion [88]. Elsewhere all three NONOates, shown in Fig. 8.9, induced relaxation of the middle cerebral artery (MCA) in a goat model [89]. Whilst these two examples highlight a beneficial role for NONOates in controlling brain ischemic damage, the potential of NONOates and other NO donors, such as SNP, on large cerebral arteries is reduced in conditions of global cerebral ischemia. From a toxicity point of view, NONOates, whilst good sources of NO, have been shown in cases of high NO release, to increase the blood brain barrier’s permeability to unwanted chemical entities, resulting in neuro-degenerative disorders [90]. Whilst this is clearly non-ideal, the observations are not limited to NONOates since the predicted redox state of the “NO-culprits” is far from being well understood or rationalised. What appears to be obvious is the therapeutic potential of NONOates in the management of pulmonary hypertension. Since this symptom is commonplace in sepsis, acute respiratory distress syndrome (ARDS), congenital heart disease and following cardiopulmonary bypass surgery, the application of NONOates in this area holds both promise and scope [91]. Using rat preparations of main and intralobar pulmonary arteries [92], full relaxation was observed using two very different NONOates. MAHMA/NO (Fig. 8.10) and SPER/NO have half-lives of 1.3 min and 73 min, respectively, at 37 °C and pH 7.3. This was reflected in potency and sustainability, with MAHMA/NO showing up to 40 times the potency of SPER/NO but the latter showing activation for over 1 h, compared with several minutes for MAHMA/NO. Varying the X component of the NONOate (X[N(O)NO]− ) can clearly change its biological activity to a large extent, though the mechanism of action once NO is released is undoubtedly the same, since introduction of the guanylate cyclase inhibitor, 1H-[1,2,4]oxa diazolo[4,3-a]quinoxalin-1-one (ODQ), abolished all dilatory activity of

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O

N

O

N N N H2

Fig. 8.10 The chemical structure of MAHMA NONOate.

MAHMA/NO

MAHMA/NO and SPER/NO. On the basis of these results it is clear that synthetic changes to the nucleophile, alter only the chemical kinetics of the drug. This, in turn, alters the biological activity, due to the parallels, already mentioned, between rate of NO release and biological responsiveness. In the discussion so far, the chemical modifications to X have centered around the variability in releasing NO. Brilli et al. [91, 93] highlight an excellent example of how the rationale behind the selection of X can go beyond the confines of how readily it releases NO and instead focus on how the structure may influence distribution in vivo, resulting in compartmental selectivity. DMAEP/NO and ethylputreanine NONOate (Fig. 8.11) are chemically quite distinct to an organic chemist, with the former having a polar-charged tertiary amine as a noticeable difference. Such polarity limits transmucosal flux which, in effect, forms the anchor as to its movement in vivo. Thus when introduced into the trachea of Yorkshire pigs, only free NO can diffuse across transmucosal membranes. Using this approach, DMAEP/NO was shown to selectively reduce the pulmonary vascular resistance index (PVRI) whereas ethylputreanine NONOate and SNP reduced both PVRI and systemic vascular resistance. O

N

O

N O

N

NH

NH2 O

DMAEP/NO 2-(dimethylamino)-ethylputreanine NONOate

O

N

O

N O

N NH2 O ethylputreanine NONOate

Fig. 8.11 Comparing the chemical structure of DMAEP/NO and ethylputreanine NONOate.

Selectivity of this kind erases any fears of systemic hypotension and hypoxemia, which is usually associated with other NO donors applied to such model systems. Similar attempts at selective vasodilation have been reported by other groups [94, 95] and further work by Brilli et al. [96] in this field shows how aerosolized NONOates, in combination with surfactant, improve both oxygenation and PVRI in porcine lung

8.9 NO Inhalation; NO Gas as an NO Donor

injury. More recent work reinforces the use of aerosolized NO adducts to ensure that the NONOates act as completely selective pulmonary vasodilators (SPVs) [85]. In summary, the NONOate family, in addition to being readily water soluble and highly stable in the solid state, can provide continuous release of NO, for periods up to and over 24 h, under physiological conditions. Collectively they show great promise in reversing pulmonary hypertension, with particular value in the treatment of common ailments such as asthma as they can be delivered simply to the lung using a small volume nebulizer. In contrast to neat NO gas, levels of NONOates are gradually reduced in tissue preparations, preventing the likelihood of rebound pulmonary hypertension during biological or clinical studies. The versatility of NONOates makes them ideal research tools [86] for studying NO in many different scenarios. For instance, using NONOates the activation of skeletal and cardiac ryanodine receptors (RyRs) by NO has indicated a new mechanism for regulating force in striated muscle [81]. By way of playing devils advocate, the major drawback of NONOates, concerning toxicity, must be addressed [97]. As in earlier discussion, such toxicity problems are out-with the biological experimentation frame, but are of major relevance to clinical studies. Nitrosation of secondary amines can give rise to carcinogenic agents (Scheme 8.4). R

R NH

R'

+

N2O3

N

NO

+

R'

HNO2 

Scheme 8.4 Nitrosation of secondary amines from NONOates.

The toxicity risk associated with a possible by-product, following the release of NO, from the NONOates, ultimately limits their use to within the boundaries of biological research to serve primarily as vehicles for dispatching NO. The concerns, although not observed in the short term, seriously perturb their use in human studies where ethical permission is sought and, more importantly, jeopardise their role as viable medicines. Despite this, there are examples of their use [98] and potential [99] in human studies. In defence of any reported toxicity, Lam et al. showed that administration of DETA/NO via aerosol in both single and multi dose animal studies did not highlight any evidence of pulmonary or hematological toxicity [100]. In the light of all of the current findings and in the context of this chapter, these compounds remain amongst the best for understanding the role and possibilities for NO in biological systems.

8.9

NO Inhalation; NO Gas as an NO Donor

In its raw form, exogenous NO donation as pure NO gas must be considered as a possible therapeutic route. Unlike all previous subdivisions, administration of this kind is obviously independent of any chemical or enzymatic process prior to the activation of soluble guanylate cyclase. This route lacks sophistication, and can

219

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produce both NO and toxic by-products, such as nitrogen dioxide (NO2 ) [101]. Since NO is given as part of an inhalation cocktail, containing oxygen, it is vital that it reaches its target quickly and is therefore administered locally. This makes the lungs the obvious target organs, since, aside from being highly accessible, they are known to respond to NO as evidenced by their plentiful supply of eNOS, iNOS and nNOS [102]. One of the major drawbacks with the inhalation route is the necessity for specialised delivery apparatus equipped with environmental monitoring systems to ensure the safety of both the patient and the healthcare workers respectively. Local administration of NO to the lungs has been shown to reverse pulmonary hypertension in animal models [103], importantly with no systemic side effects. This is likely to be as a result of surplus NO being removed as nitrosyl-hemoglobin [104]. Such advantages of gaseous NO were first reported in 1991 [105, 106]. In 1999 and 2001 NO gas was approved as a drug in the USA and European Union, for treating hypoxemic respiratory failure in infants [107]. The use of NO as a therapeutic aerosol holds much promise. Alleviating pulmonary hypertension provides medical applications across a broad spectrum of cardiopulmonary disease states. Without doubt one of the hottest areas in which NO gas has been utilised over the last 10 years, is in lung transplantation procedures. From an array of animal models including rats [108], mice [109], rabbits [110], dogs [111] and pigs [112], the beneficial use of NO inhalation to reverse ischemia-reperfusion injury (I-R) and to aid in organ storage, during transplantation, has been substantial. In attempts to swell the pulmonary donor pool, studies have been performed using lungs from non-heart beating donors (NHBDs) [112]. Over the years the major problem with such donors has been early postoperative graft dysfunction resulting from the warm ischemic time period. Using a dog model, with donors sacrificed and then left for 3 h at room temperature, recipient dogs showed reduced pulmonary vascular resistance (PVR), enhanced oxygenation and improved survival when administered NO (40 ppm) during reperfusion. This was seen to be especially vital during the first hour of reperfusion [113]. In addition to aiding blood supply to the donor organ there is substantial evidence to suggest that NO is providing a protective role in tandem [114]. In fact, such observations are attributed to an improvement in survival rate from 57% to 89%. The idea of a protective mechanism originates from lower myeloperoxidase activity in NO treated models [108, 111]. Myeloperoxidase activity, or polymorphonuclear neutrophil (PMN) lung sequestration is central to I-R injury. To test the protective ability of inhaled NO (30 ppm) using pigs, PMN lung sequestration was measured in vivo, while measurements for adhesion of PMNs to cultured pulmonary artery endothelial cells (PAECs) was conducted in vitro. Both were reduced by NO [115]. Similar work, in a dog model [116], showed consistent results, with pulmonary vascular resistance and neutrophil sequestration both being reduced. In addition, thiobarbituric acid-reactive materials, which, usually accumulate during organ storage, were reportedly reduced when taken from animals treated with NO at the time of harvest. As a result of this, it is believed that NO pre-treated lungs exhibited reduced oxidant injury during storage. Implications to how this may reshape organ handling following removal from the donor, described as ‘preconditioning’ [110] are clearly

8.9 NO Inhalation; NO Gas as an NO Donor

huge. Other theories relating to the importance of NO in lung injury show that high concentrations of NO in a mouse model (40 ppm) increase lung expression of tissue factor on alveolar epithelial cells [109]. With all three isoforms of NOS found in the lungs it is perhaps not that surprising to observe NO as such a diverse homeostatic molecule. Stamler et al. [102] reinforce this idea, reporting NO as a mediator, not only in bronchial tone but also in the permeability to sodium/water, ciliary motility, mucus secretion and protection to pollutants and pathogens. This latter role is substantiated by work using NO inhalation in male Sprague-Dawley rats in vivo, ex vivo and in vitro, to show bacterial clearance of Pseudomonas aeruginosa [117]. The results suggest such clearance from direct bactericidal effects and through the influx of alveolar neutrophils with preserved function. In other protective roles, inhalation of the NO-precursor, l-arginine, in guinea pigs, prevented the parafluenza type 3 virus, from inducing airway hyper-responsiveness to histamine [118]. This suggests therapeutic potential in viral respiratory infections. Whilst inhaled NO has shown much potential in lung transplantation, not to mention its benefits during cardiac transplantation [119] and in treating severe pancreatitis [120], it is not recommended in all cases. Hewitt et al. [121] report NO inhalation, in rats, before and during double lung transplantation to actually have detrimental effects. Elsewhere, a large multi-centered trial [122] showed ventilator free survival to be reduced following NO usage in acute lung disorders. Observations of this kind have been attributed to the so-called rebound [102] in pulmonary artery pressure upon termination of NO gas therapy. Rebound is the major drawback associated with NO inhalation since it results ultimately in cardiovascular collapse. Attempts at combating the rebound response have centered around sustaining an NO supply. Schutte et al. [123] used SNP in the lung to prolong NO availability, whilst Stamler et al. [102] suggested channelling a bulk supply of NO into endogenous thiols to give S-nitrosothiols (SNOs) and thus provide a slow sustained release of NO without toxicity worries. O-nitrosoethanol (ENO) was administered via aerosol to neonatal pigs. Such a compound fulfils the criteria of being volatile, stable in oxygen yet capable of heterolytic cleavage in the presence of glutathione. The reaction involving the nucleophilic attack by glutathione upon free NO is far less favorable than that involving ENO. In vivo results correlate with this line of thought where rebound is only observed in animals treated with free NO. Such findings suggest a major limitation with NO inhalation. Although this may indeed be the case, there can be no argument with regard to its experimental use uncovering a reliable local route for introducing selective vasodilation to the cardiopulmonary system. Administration by inhalation has been explored by Brilli [124], mentioned previously for his work with NONOates. Here he uses one of these same NONOates, DMAEP/NO (see Fig. 8.11), in aerosol form. When administered in an aerosolized state, DMAEP/NO again shows selective pulmonary vasodilation in a porcine model. This is achieved without affecting the systemic vascular resistance index (SVRI) or the cardiac index (CI). Work from the same year by Adrie et al. [125] compared aerosolized DEA/NO with aerosolized SNP and inhaled NO, in sheep. As the NONOate has a short half-life (2.1 min), it was predicted that this would be a selective pulmonary vasodilator. However, compared with inhaled NO this was not observed, though SNP

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did selectively reduced PVR by 42% at low doses. Such observations are perhaps not that surprising if one is to revisit the rationale used by Brilli in earlier work [91, 93] (described in Section 8.8) where polarity was used as a handle for selectivity. It is worth mentioning that work some three years earlier [126] than that of Adrie, is inconsistent and indicates that inhaled SNP does not provoke selective pulmonary vasodilation. It would appear that their low doses of SNP were perhaps too low to see any beneficial responses in the light of those used by Adrie (< 10−2 M). In summary, NO inhalation’s major strength is that of rapid, local administration which selectively increases trans-pulmonary efficiency. Its main drawback is clearly with regard to rebound; administration in combination with an NO donor that can remain in the lungs long enough to release NO spontaneously over a prolonged period would be the ideal scenario to circumvent this. The use of NO inhalation in lung related surgery and the management of transplants would appear to be the avenue currently under most exploration. Though, on the basis of current literature information, it will continue to remain so for this particular class of NO donor.

8.10

Sydnonimines

Staying on the theme of sustained nitric oxide inhalation, or at least delivery as alveolar-accessible aerosol particles, the sydnonimine, molsidomine (Fig. 8.12) was compared with SNP and GTN [123]. All three reduced pulmonary hypertension in a dose dependent fashion with order of efficiency being, SNP > SIN−1 >> Molsidomine and GTN. O N N O

N O

N OC2H5 Fig. 8.12 Chemical structure of molsidomine.

Whilst this result suggests that the sydnonimines are in amongst the leading NO donors their favoritism appears to have dwindled in recent years. Most biological studies involving their use were reported back in the early 1990s with a large percentage of the work being conducted in German laboratories. Initially these compounds were used as antihypertensives [127, 128] but due to their ability to reduce venous return, cardiac output, ventricular work and myocardial oxygen consumption, they are more commonly studied as antianginal agents [129–133]. Perhaps the most intriguing characteristic of this family of NO donors is the wellunderstood mechanism by which they release NO [134–136]. The decomposition contains both enzymatic and non-enzymatic steps. Unfortunately the presence of this enzymatic step appears to have resulted in the sydnonimines being categorised

8.10 Sydnonimines

and considered in a similar light to that of GTN. However, as we shall illustrate, sydnonimines do not show tolerance with repeated administration, highlighting a distinct advantage over the organic nitrate collection. The lack of consistency in the nomenclature, resulting from the use of generic, chemical, numerical code and metabolite naming does complicate the general picture and has led to incorrect labelling in the literature. In an attempt to prevent this, molsidomine and pirsidomine, the two main compounds in this class, will now be introduced together with their identical degradative pathways to allow all terminology to be displayed in one scheme (see Scheme 8.5). Molsidomine and pirsidomine, are both stable as solids at room temperature in the absence of light [137]. However the ring opened metabolites SIN-1A and C87-3786 are both photolabile and sensitive to an oxidising environment resulting ultimately in the release of superoxide and NO in stoichiometric quantities [138]. Generation of these two species is an obvious problem due to the resulting formation of peroxynitrite and the generation of • OH, which may initiate lipid peroxidation [139–141] (see Eq. (19)). Such concerns over the formation of peroxynitrite from SIN-1A or C87-3786 are warranted since their cytotoxic effects show close consistency with cellular studies doped with neat peroxynitrite [142, 143]. NO + O2 −

→

ONOO−
 ONOOH
 NO2 • + •OH

(19)

The suitability of SIN-1 as a photoactivatable NO donor was illustrated by Schröder et al. [136]. In the presence of polychromatic visible light, nitrite concentrations increased by 61% whilst oxygen concentrations dropped to 2% of control values. This in vitro example highlights how the oxygen-dependent NO release from SIN-1A is promoted by an external energy source. In a biological setting, Schröder et al. postulate that this energy barrier may be controlled enzymatically. Whilst there are reports [144] that CAS 936 (pirsidomine) has vasodilating activity of its own, there can be no argument over metabolites C87-3754 and C87-3786 being the crucial species with regard to the NO directed pathway. If confirmation of this was at all needed, in vitro and in vivo studies on pirsidomine and metabolites C87-3754 and C87-3786 were performed in which NO accumulation was measured by electron paramagnetic resonance (EPR) [145]. Whilst NO accumulation was observed in all studies involving metabolites, detection of NO from pirsidomine was only seen in the presence of isolated rabbit liver, clearly illustrating its role as a stable prodrug requiring hepatic biotransformation. In other work, C87-3754 has been compared with SIN-1, the equivalent metabolite from molsidomine, and from the organic nitrate class, isosorbide-5-mononitrate (IS5-MN) [146]. In this latter study, which involved long term oral administration in a dog model, IS-5-MN showed tolerance whilst the sydnonimines showed no self- or cross-tolerance. Elsewhere, tolerance studies in dogs by oral or continuous infusion of C87-3754 have shown benefits in the treatment of a failing heart, without tolerance [147] and with better results than those observed by nitroglycerin [148]. The antianginal activity of pirsidomine in dogs is seen following oral (p.o.) (10 mg kg−1 ) [148], intravenous (i.v.) [147] or intraduodenal (i.d.) (1 mg kg−1 ) [149] administration. Using this same latter dose similar effects are observed in pigs [150],

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8 Vasodilators for Biological Research CAS 936 Pirsidomine (3-(cis-2,6-dimethylpiperidino)-N(4-methoxybenzoyl)-sydnonimine)

Molsidomine (N-ethoxycarbonyl-3morpholinosydnonimine)

O N

N N

O

N

N

N NCOOC2H5

O

N

O

OMe Esterase

Esterase

O SIN-1 Linsidomine (3-morpholinosydnonimine)

N

C87-3754 (3-(cis-2,6-dimethylpiperidino)sydnonimine)

N

N

N

N NH.HCl

O

N NH.HCl

O

OH

OH

O N-morpholino-N-nitroso-2aminoacetonitrile

N N

CH2CN

SIN-A

N-(cis-2,6-dimethylpiperidino)-Nnitroso-2-aminoacetonitrile C87-3786

N N

N O

CH2CN

N O

O

O2

O2

O2

O2

NO

NO

H+

H+

SIN-1C (stable metabolite)

N N

(stable metabolite)

N N

CHCN

CHCN



Scheme 8.5 The degradative pathways for molsidomine and pirsidomine.

with a reduction of ischemia, showing a good interspecies relationship in the presence of this vasodilating agent.

8.11 Conclusion

Not surprisingly, with so many comparative studies taking place, molsidomine was directly compared with pirsidomine. Using cytosolic mouse macrophage NO synthase extracts SIN-1, but not C87-3786, reduced enzymatic [3H] arginine turnover to [3H] citrulline [151]. This may highlight a subtle difference in tolerance levels between these two anti-anginal prodrugs. Clinical studies using pirsidomine [152] and SIN-1 [153] have both reinforced the idea of these sydnonimines as anti-ischemic and anti-anginal agents that do not show tolerance. In the latter cited work, SIN-1 also showed sustained vasodilation in the presence of nitroglycerin tolerance, showing that sydnonimines act by a different mechanism to GTN. There is evidence to reinforce this idea as SIN-1 has been shown to operate via formation of S-nitrosoglutathione [154], illustrating how endogenous thiols may be responsible for the sustainability of sydnonimines by storing NO locally. Any difference between the sydnonimines and GTN is likely to center around the unknown enzymatic metabolism required in the latter NO donor. In this section we have highlighted how the sydnonimines represent a credible therapeutic agent for patients showing tolerance to long-term nitroglycerin therapy. In the light of these observations, sydnonimines clearly have a place in the pecking order of NO donors and despite the opening remarks to this section suggesting they were “yesterday’s NO donors” their use in more recent biological studies [155] continues to strengthen their case as worthy drug candidates.

8.11

Conclusion

The choice of nitrovasodilator used in vasodilator research is important when interpreting the results. Undoubtedly the one with the simplest and best-understood chemistry is a NONOate. It is also possible, by selecting the right NONOate, to deliver NO at a predetermined rate. The few disadvantages in the use of NONOates are outlined in that section of this chapter. To promote NONOates is not to denigrate other nitrovasodilators. Surprisingly sodium nitroprusside has proved invaluable in investigating the physiological roles of NO and will continue to do so. However, the reservations expressed earlier should be borne in mind. S-Nitrosothiols are very simple compounds to use and, when made lipid soluble may have great value in certain circumstances. Even if S-nitrosothiols are not intended directly as the source of NO donor, mechanisms involving the intervention of endogenous thiols are rife, highlighting their unique ability to except, store and release NO in a biological setting without any apparent toxicity or tolerance concerns.

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NO Donors as Antiplatelet Agents Anna Kobsar, Martin Eigenthaler 9.1

Introduction

Organic nitrates have been used successfully for more than a century in the treatment of cardiovascular diseases. However, it was not before the late seventies that nitric oxide (NO) was identified as the active component of organic nitrates. It took some more years to recognize the endogenous vasodilator endothelium-derived relaxing factor (EDRF), synthesized in and released from the vascular endothelium, as NO. Besides EDRF, the endothelium secretes another important vasodilator, prostacyclin (PG-I2 ). Both EDRF and PG-I2 not only relax vascular smooth muscle cells, but also strongly inhibit platelet activation. The endogenous vasodilators mediate their effects mainly through elevation of cyclic nucleotides, and the synergistic action of 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) and the crosstalk between these two major signaling pathways is certainly the most potent endogenous mechanism of platelet inhibition. Chronic damage of the vascular endothelium and subsequently decreased release of endothelial factors is now an established key event in the pathogenensis of cardiovascular diseases. In a very recent study, acute inhibition of endogenous NO production in humans caused rapid platelet activation in vivo, which was reversed by sublingual administration of the NO donor glycerol trinitrate, demonstrating that platelet function in vivo is rapidly regulated by NO bioavailability [1]. New drugs that interfere with cyclic nucleotide generation and metabolism have become an essential part in the clinical treatment of cardiovascular diseases. This chapter will focus on EDRF/NO-regulated signaling pathways, however, essential aspects of cAMP signaling in platelets will also be included.

9.2

Molecular Mechanisms of NO-mediated Platelet Inhibition

Inhibition of platelet activation by NO is not exclusively cGMP-dependent but involves mechanisms independent of cGMP like ribosylation or nitrosylation of proteins. For 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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several years it has been hypothesized that NO generated inside the platelet (intracellular NO) acts primarily via cGMP-dependent pathways, whereas extracellular NO prefers cGMP-independent mechanisms. However, until now this has not been confirmed convincingly. Fig. 9.1 summarizes the potential mechanisms of NO action in human platelets.

Fig. 9.1 Nitric oxide mediated inhibition of

platelet activation. Abbreviations used: NO, nitric oxide; EDRF, endothelium-derived relaxing factor; GC, guanylyl cyclase; PDE, phosphodiesterase; cGMP-PK, GMP-dependent protein kinase; Rap1b, small GTPase Rap1b;

VASP, vasodilator stimulated phosphoprotein; hsp27, heat shock protein hsp27; LASP, LIM and SH3 domain containing protein; Tx, thromboxane; IP3 , inositol triphosphate; ADP, adenosine 5′-diphosphate. Adapted from Refs. [44] and [116].

9.2.1

cGMP-dependent NO Signaling Mechanisms

The classical NO-regulated pathways in platelets involve the activation of soluble guanylyl cyclase (GC) with subsequent increase in cGMP which regulates effector molecules such as cGMP-regulated phosphodiesterases (PDEs) and cyclic GMPdependent protein kinases (cGMP-PKs). The latter then phosphorylate specific signaling molecules. To our present knowledge, these signaling cascades represent the major NO-regulated mechanisms of platelet inhibition.

9.2 Molecular Mechanisms of NO-mediated Platelet Inhibition

9.2.1.1

Regulation of cGMP Levels

cGMP levels are up-regulated by synthesis through GCs. In mammalian cells, both membrane-bound and soluble forms of GCs are known. However, until now, only the soluble form of GC, which is located in the cytoplasm, has been found in platelets and there is no evidence that platelets also contain a membrane-bound GC. Soluble GC contains heme as a prosthetic group and is activated by the endogenous EDRF (NO) released from endothelial cells and pharmacological NO-generating agents. Down-regulation of cGMP levels occurs by degradation through PDEs. PDEs are a large group of enzymes consisting of several isozyme families that hydrolyze the 3′-phosphoester bond on cGMP and/or cAMP, thus converting them into biologically inactive 5′-nucleotide metabolites. Platelets contain at least three different types of PDEs (Fig. 9.1 and Table 9.1): the cGMP-stimulated PDE2, the cGMP-inhibited PDE3, and the cGMP-binding cGMP-specific PDE5 [2]. All of them are important targets of cGMP, but not of cAMP. Tab. 9.1: Phosphodiesterases in human platelets.

Isozyme PDE2 cGMP-stimulated PDE PDE3 cGMP-inhibited PDE

Substrate cGMP, cAMP cAMP >> cGMP

PDE5 cGMP-binding cGMP-specific PDE

cGMP

Inhibitors EHNA cGMP, milrinone, amrinone, anagrelide, cilostazol, lixazinone, NSP-513 Dipyridamol, zaprinast, sildenalfil, DMPPO, E4021

PDE2 hydrolyzes both cAMP and cGMP, with similar affinities (Km values of 50 and 35 ìM, respectively), and is stimulated by the binding of cGMP to two allosteric regulatory sites. PDE3 has similar Km values for cAMP and cGMP (0.2 and 0.02 ìM), but PDE3 preferentially hydrolyzes cAMP due to a Vmax that is 10-fold higher for cAMP than for cGMP. PDE3 is inhibited by the binding of cGMP. Therefore, cGMP can potentially decrease (via PDE2) or enhance (via PDE3) a cAMP response. In contrast, PDE3 activity is increased by a direct cAMP-PK-catalyzed phosphorylation as a negative feedback loop, which rapidly returns elevated cAMP levels back to basal [3]. PDE5 is highly specific for cGMP hydrolysis (Km of 1 ìM for cGMP and 150 ìM for cAMP). It contains a cyclic nucleotide-dependent protein kinase phosphorylation site and two allosteric cGMP binding sites on the regulatory domain of each subunit. Binding of cGMP to the catalytic site facilitates cGMP binding to the allosteric sites, which causes exposure of the phosphorylation site [4]. Recent in vitro studies with purified enzymes showed that PDE5 can be phosphorylated by cGMP-PK and by cAMP-PK, leading to increased catalytic activity of PDE5 [5]. This might provide a physiological negative feedback regulation of intracellular cGMP elevation.

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Inhibitors of PDEs regulate platelet cGMP and cAMP levels via decreased degradation of cyclic nucleotides. PDE3 inhibitors have been described as useful anti-platelet and anti-thrombotic tools that also inhibit vascular smooth muscle cell proliferation and arteria intima thickening in animal models [6, 7]. Inhibition of platelet PDEs in combination with low doses of cyclic nucleotide elevating vasodilators could become a very useful tool in clinical situations where specific inhibition of platelet function is desired without the generation of side-effects on vascular smooth muscle cells. The presence of high concentrations of PDE5 in platelets and vascular smooth muscle cells has led to the development of a variety of selective compounds, some of them should prove useful for platelet-specific therapy since they reveal relatively poor vasodilatory effects [8]. Recently, dipyridamole has been demonstrated, under therapeutically relevant conditions, to enhance platelet inhibition by amplifying the signaling of the NO donor SNP [9]. 9.2.1.2

Effector Sites of cGMP

The major target enzymes of cGMP are cGMP-PKs, which mediate their effects through phosphorylation of specific substrate proteins. In mammalian cells, two distinct forms of cGMP-PKs are known: cGMP-PK I that regulates vascular tone and platelet function, and cGMP-PK II that is involved in intestinal ion secretion and growth of bones. Two isoforms of cGMP-PK I are known, cGMP-PK Iá and cGMP-PK Iâ, which differ only in their N-terminal region. Platelets contain high concentrations of the cGMP-PK Iá isoform. In contrast to the cAMP/cAMP-PK system with a basal cAMP concentration (4.4 ìM) close to the concentration of cAMP binding sites (6.2 ìM), the situation for cGMP/cGMP-PK is very different. The basal cGMP concentration in platelets (0.4 ìM) is less than one-tenth of the concentration of cGMP binding sites on the cGMP-PKs (14.6 ìM), suggesting that even severalfold increases in intracellular cGMP levels are capable of stimulating only a small fraction of cGMP-PKs, however, locally higher concentrations of cGMP in certain cell compartments might occur [10]. Nevertheless, activation of cGMP-PKs kinases is the most important mechanism of cGMP action in human platelets, as demonstrated by studies with membranepermeable cyclic nucleotide analogs that selectively activate cGMP-PK and do not affect PDEs. Inhibition of platelet aggregation by these analogs correlated well with the activation of cGMP-PK in intact platelets [11, 12]. Furthermore, in contrast to platelets from healthy persons, cGMP-PK-deficient platelets from patients with chronic myelocytic leukemia showed no inhibition of agonist-induced Ca2+ mobilization from intracellular stores by a specific activator of cGMP-PK, 8-para-chlorophenylthioguanosine3′,5′-cyclic guanosine monophosphate (8-pCPT-cGMP) [13]. In accordance with this finding is the observation that in cGMP-PK-deficient mice, collagen-induced platelet shape change, aggregation, and serotonin release could not be inhibited by 8-pCPTcGMP [14]. The important in vivo role of platelet cGMP-PK I for the inhibition of platelet adhesion to vascular endothelium has been further demonstrated by recent studies

9.2 Molecular Mechanisms of NO-mediated Platelet Inhibition

using a microcirculatory ischemia/reperfusion mouse model with quantification of platelet attachment by an intravital microscopy technique. The comparison of wild type versus cGMP-PK I-deficient mice demonstrated that platelet cGMP-PK I, but not cGMP-PK I present in endothelium or smooth muscle cells, is the key molecule in the prevention of pathological intravascular platelet adhesion. 9.2.1.3

cGMP-PK I Substrates in Platelets

Platelet activation is a complex interplay of platelet agonist receptors, agonist-generated inside-out signals, regulation of signaling molecules, induction of cytoskeletal rearrangements and finally the conformational change and clustering of platelet integrin receptors [15, 16]. Essentially all of these activation steps are effectively inhibited by cGMP-PK I that mediates its effects through phosphorylation of specific substrate proteins. Table 9.2 summarizes the current knowledge of such proteins and the effects of cGMP-PK-mediated phosphorylation on their proposed function in platelets. Tab. 9.2: cGMP-PK I substrates in platelets.

Substrate IP3 receptor (IRAG) RAPIb VASP hsp 27 LASP PDE 5 Thromboxane receptor

Proposed Function of Phosphorylation Downregulation of Ca2+ release from intraceullular stores Inhibits Rap1b activation probably regulation of cell adhesion and secretion Inhibits VASP binding to F-actin inhibits VASP localization to focal contacts and integrins Reduces stimulation of actin polymerization in vitro Inhibits LASP binding to F-actin inhibits LASP localization to focal contacts Enhances cGMP degradation Inhibits TxA2 receptor-associated G protein activation

9.2.1.3.1

Inositol triphosphate (IP3 ) receptor Platelet activation by all stimulatory agonists induces the elevation of cytosolic Ca2+ followed by the activation of multiple Ca2+ -dependent enzymes, like the Ca2+ -dependent protein kinase-C (PKC) isoforms, the Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), cytosolic phospholipase A2 (cPLA2), and the small GTPase Rap1b. Most agonists activate phospholipase C (PLC) and elevate cytosolic Ca2+ by an IP3-dependent release of Ca2+ from intracellular stores, as well as by stimulation of the entry of extracellular Ca2+ . The platelet agonist adenosine 5′-diphosphate (ADP) activates an additional immediate influx of extracellular Ca2+ through an ADP receptor-operated cation channel. Increased levels of cGMP antagonize the activatorevoked Ca2+ release from intracellular stores and the secondary store-mediated Ca2+ influx, but not the ADP receptor-operated cation channel [17]. This effect is certainly mediated by several mechanisms, and one probable mechanism of cGMP action is

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inhibition of PLC activation, however, until now no clear evidence has been found for direct inhibition of PLC by cGMP-PK [18]. Better characterized is the inhibitory action of cGMP on the IP3 receptor. PLC catalyzes the conversion of PIP2 into diacylglycerol (DAG) and IP3. IP3 receptors, which mediate the release of Ca2+ from the dense tubular system and probably also the secondary store-related influx, are directly phosphorylated by cGMP-PK in human platelets [19, 20]. However, the role of this IP3 receptor phosphorylation in the inhibition of intracellular calcium elevation is still not clear. In smooth muscle cells, an additional IP3 receptor-associated protein (IRAG) needs to be phosphorylated by cGMP-PK, since phosphorylation of the IP3 receptor alone is not sufficient to inhibit Ca2+ release [21]. Whether IRAG phosphorylation by cGMP-PK also plays a role in the down-regulation of agonist-induced Ca2+ elevation in platelets needs to be investigated. 9.2.1.3.2

Rap 1b Rap1b, a small GTPase of the Ras family, is highly expressed in human platelets. The functional role of Rap1 in platelets is unclear, but it may be involved in the regulation of platelet adhesion, since in leukocytes Rap1 activation induces cell adhesion. In platelets, Rap1b is subjected to a complex sequential regulation [22]. During platelet activation, Rap1b is activated within seconds by PKC-independent and Ca2+ -mediated (initial activation), as well as PKC-dependent (second wave of activation), mechanisms. Rap1b activation is sustained via a GPIIb/IIIa (integrin áIIbâ3)-dependent mechanism. However, platelet aggregation also induces the inactivation of Rap1b, which correlates with translocation of Rap1b from the plasma membrane to the cytoskeleton. Treatment of platelets with NO leads to Rap1b phosphorylation and inhibition of agonist-induced Rap1b activation [23–25]. However, it is likely that inhibition of Rap1b activation is mediated by phosphorylation-independent mechanisms, e.g. by inhibition of intracellular Ca2+ elevation, which is both necessary and sufficient for Rap1b activation [26]. 9.2.1.3.3

Vasodilator stimulated phosphoprotein (VASP) One of the major substrates of both cGMP-PK and cAMP-PK is the cytoskeletonassociated vasodilator-stimulated phosphoprotein (VASP) that is found in various cell types to be located in focal adhesions, stress fibers, cell–cell contacts, and highly dynamic membrane regions [27]. In platelets, VASP is present in particularly high concentrations [10]. VASP contains three phosphorylation sites: Serine157 , Serine239 , and Threonine278 ; in intact human platelets Serine239 is the initially preferred site of activated cGMP-PK followed by phosphorylation at Serine157 [28, 29]. Depending upon its phosphorylation status and the system analyzed (purified proteins, listeria, or mammalian cells), VASP is able to affect and regulate actin polymerization and actin filament bundling [27]. It is suggested that VASP phosphorylation reduces its binding to F-actin and down-regulates its enhancing functions on actin polymerization, however, the precise molecular mechanism of VASP action in platelets still needs to be characterized. Phosphorylation at Serine157 has been shown to closely

9.2 Molecular Mechanisms of NO-mediated Platelet Inhibition

correlate with inhibition of the fibrinogen receptor GP IIb/IIIa [30]. Studies using VASP-deficient mice showed that inhibition of platelet aggregation by low doses of cyclic nucleotides was impaired in the absence of VASP [31, 32]. Moreover, platelets from VASP-deficient mice also showed enhanced aggregation in response to collagen, increased thrombin-induced activation of GP IIb/IIIa, and enhanced surface expression of P-selectin [31, 32]. Studying in vivo platelet–vessel wall interactions in this mouse model demonstrated that platelet adhesion to endothelial cells was significantly reduced in platelets from VASP-deficient mice compared to wild type mice. Under pathophysiological conditions in an ischemia-reperfusion mouse model, the loss of VASP led to increased platelet adhesion to atherosclerotic endothelium and the subendothelial matrix, and platelet adhesion in VASP-deficient mice was unresponsive to NO. Taken together, these observations indicate that VASP phosphorylation is required to mediate at least part of the inhibitory effect of cGMP on platelet adhesion and GP IIb/IIIa activation under both physiological and pathophysiological conditions. While it is clear that VASP is required for platelet inhibition, the role of VASP phosphorylation in cytoskeletal reorganization and integrin activation is not completely understood. In human endothelial cells, VASP phosphorylation disrupted its binding to focal adhesions and induced a progressive reduction of the actin microfilament system, whereas cells transfected with a VASP mutant, where all three known phosphorylation sites were changed into alanine, were resistant to these effects [33]. Therefore, the role of VASP and VASP phosphorylation still needs to be characterized in the context of the other known cGMP-PK substrate proteins that are involved in microfilament organization. 9.2.1.3.4

Heat shock protein hsp27 In platelets, hsp27 is phosphorylated by the p38 mitogen-activated protein kinase (MAPK)-activated MAPKAPK-2 resulting in stimulation of actin polymerization. In human platelets, hsp27 is additionally phosphorylated by cGMP-PK (and probably also by cAMP-PK) at one site that is different from the MAPKAPK-2 phosphorylation sites [34]. In vitro experiments showed that cGMP-PK-phosphorylated hsp27 reduced the MAPKAPK-2-induced stimulation of actin polymerization [34], however, the exact role of hsp27 in the regulation of platelet activation still needs to be investigated. 9.2.1.3.5

LASP LASP is an actin-binding protein that consists of the LIM-domain, a cysteine-rich zincfinger motif, and the SH3-domain, the Src homology domains recognizing prolinerich motifs of interacting partners. LASP was recently shown to be a cGMP-PK substrate in human platelets [35]. Phosphorylation of LASP occurs at Serine 146 and a ,,constitutively-phosphorylated“ S146D LASP mutant showed reduced binding affinity for F-actin in vitro. Unlike wild type LASP, overexpressed S146D mutant in PtK2 cells was not localized at the cytoskeleton and the leading edge of the cell but was found exclusively in the cytosol, and the transfected cells showed reduced migration

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[35]. However, the exact function of LASP and the role of its phosphorylation in cytoskeletal reorganization and platelet activation remains to be investigated. 9.2.1.3.6

Thromboxane A2 (TxA2 ) receptor Recently, it was shown that the thromboxane receptor itself is a substrate of cGMP-PK and cAMP-PK in HEK293 cells, HEL cells, or with purified enzymes in vitro. Phosphorylation of its cytoplasmic carboxyterminal domain prevented the thromboxane receptor from coupling to and activating G-proteins [36, 37]. For intact platelets, TxA2 receptor phosphorylation has not yet been shown, however, it would provide another explanation for the inhibition of PLC activation and subsequent intracellular Ca2+ elevation and granule secretion in response to cyclic nucleotides. 9.2.1.3.7

Phosphodiesterase PDE5 As discussed above, PDE5 is highly specific for cGMP hydrolysis and its activity is in part regulated by cGMP-PK phosphorylation. Phosphorylation of PDE5 in vitro increased its catalytic activity and PDE5 probably decreases intracellular cGMP levels by this mechanism. 9.2.2

cGMP-independent NO Signaling Mechanisms

NO-mediated platelet inhibition is still thought to be mainly mediated by activation of soluble GS and generation of cGMP. However, with the increasing number of NO-donors and components that inhibit NO-/cGMP-regulated signaling molecules, like guanylyl cyclase inhibitors, it became evident that besides the classical activation cascade, effects of NO can be mediated by cGMP-independent mechanisms which are summarized in Fig. 9.1. Some years ago, NO-donors were shown to increase a cytosolic ADP-ribosyltransferase that ADP-ribosylates a soluble 39 kDa protein in platelets identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [38, 39]. This effect was not related to the stimulation of soluble GS or cGMP-increase, and ADP-ribosylation was further enhanced by the presence of NADPH as an essential cofactor. Also, inhibition of platelet aggregation through interference with platelet thromboxane A2 formation has been suggested for certain NO-donors (S-nitroso cysteine) as a cGMP-independent mechanism [40]. Another possible cGMP-independent mechanism is through peroxynitrite, a reactive oxygen species derived from the interaction of NO with superoxide, that was reported to inhibit ADP-, collagen-, thrombin- and thromboxane-induced platelet activation [41]. It was suggested that the inhibitory action of peroxynitrite might be, at least in part, due to nitrosylation of inhibitory signaling proteins, however, these mechanisms still need to be characterized. Furthermore, NO-donors were shown to regulate the Ca2+ uptake into platelet membrane vesicles in a way independent of cGMP or nitrite ions, the metabolic product of NO. The NO effects were biphasic with an inhibition above 10 nM and a stimulation

9.3 Effects of Different Groups of NO Donors on Platelets

below this concentration. These data suggested that NO is functionally coupled to sarco-endoplasmic reticulum calcium-ATPase (SERCA) of the platelet dense tubular system [42]. However, unlike in other cell types where direct regulation of ion channels by cGMP without involvement of protein kinases has been demonstrated, such mechanisms have not yet been clearly demonstrated for platelets. In summary, the contribution of cGMP-independent mechanisms in NO-donormediated platelet inhibition is still not clear. Another unsolved question is, why cGMP-independent mechanisms of platelet inhibition are not common to all NO donors. In the following, the specific differences between various NO donors with regard to platelet effects will be discussed.

9.3

Effects of Different Groups of NO Donors on Platelets 9.3.1

Diazeniumdiolates 9.3.1.1

DEA/NO (Sodium 2-(N,N-diethylamino)-diazenolate-2-oxide)

DEA/NO is a short-acting NO-donor drug with a half-life of about 2 min in pH 7.4 phosphate buffer at 37 °C, releasing two molecules of NO and one molecule of diethylamine. Preincubation of platelet-rich plasma and washed platelets with DEA/NO inhibited platelet aggregation [43, 44] and activation of the fibrinogen receptor on the platelet surface [45]. The anti-platelet effect of DEA/NO could be abolished by the NO-scavenger hemoglobin, however, not all DEA/NO effects on platelets were cGMP– dependent. A selective inhibitor of sGS, ODQ only slightly decreased the inhibitory effect of DEA/NO on ADP-induced platelet aggregation [44]. In vitro, DEA/NO increased the cGMP level and stimulated VASP phosphorylation, and combination of DEA/NO with YC-1 (activator of soluble GS) induced a synergistic effect on cGMP increase and VASP-phosphorylation in platelets [46, 47]. 9.3.1.2

DETA NONOate ((Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2diolate)

DETA/NO is a stable NO-donor with the longest NO generation half-life of approximately 20 h. Thrombelastography performed on rabbit blood showed that DETA NONOate-derived NO significantly decreased coagulation activity and platelet activation [48]. Monitoring by intravital microscopy showed that DETA/NO attenuated the platelets/endothelial cells adhesion response to endotoxins (e.g. lipopolysaccharides) in murine intestinal venules [49]. The main mechanism of the antiadhesive action of DETA/NO on platelets was activation of soluble guanylate cyclase [49].

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9.3.1.3

MAHMA NONOate ((Z-1-[N-Methyl-N-[6-(N-methylammoniohexyl) amino]]diazen-1- ium-1,2-diolate)

MAHMA NONOate spontaneously dissociates in a pH-dependent, first-order process with a half-life of 1 min at 37 °C. In vivo, examination in anesthetised rats showed, that MAHMA NONOate had both platelet inhibitory and vasodepressor effects [50]. Like other NONOates, MAHMA NONOate inhibited collagen-induced and ADPinduced rat platelet aggregation in a concentration-dependent manner [51]. ODQ, a soluble GS inhibitor, caused only small influence on the concentration–response curve to MAHMA NONOate, indicating that cGMP-independent mechanisms play a crucial role. A potential target of MAHMA NONOate induced inhibition was the sarco-endoplasmic reticulum calcium-ATPase of the platelet dense tubular system [51]. 9.3.2

Sodium Nitroprusside (SNP)

SNP spontaneously releases NO and causes direct relaxation of blood vessels and inhibition of platelet aggregation by activating soluble GS and cGMP-PK [44, 47, 52]. Its effect on platelets could be completely blocked with the soluble GS inhibitor ODQ, indicating that cGMP-independent mechanisms play no, or only a minor, role for SNP action [44, 52]. SNP induced cGMP-PK activation resulted in phosphorylation of specific target molecules like VASP [30, 47, 53, 54]. SNP inhibited the initial platelet adhesion to collagen fibers, collagen-induced intracellular Ca2+ mobilization and actin polymerization, but not long-term adhesion [55]. Combination of SNP with YC-1, an NO-independent activator of soluble GS, induced a synergistic effect in cGMP increase and VASP-phosphorylation [46]. Furthermore, SNP and cAMP elevating agents synergistically inhibited the aggregation of PRP induced by collagen as well as platelet adhesion/aggregation in blood [56]. SNP inhibited agonist-induced Ca2+ -mobilization, but not the basal Ca2+ level in platelets [57]. SNP strongly inhibited p-42- and p38 MAPK activation, fibrinogen binding and surface expression of CD40L and CD62P in response on platelet activating agents [58, 59]. Furthermore, SNP prevented hydroxyl radicals- and reactive oxygen species-mediated membrane damage by inhibiting dioxygenase activity of lipoxygenase in human platelets [60]. 9.3.3

Molsidomine (3-Morpholino-sidnonimine; SIN-1)

SIN-1 spontaneously releases NO and superoxide under physiological conditions thereby stimulating cGMP production. SIN-1 significantly decreased expression of P-selectin and both total and activated GP IIb/IIIa and also promoted reversal of activated GP IIb/IIIa complex in platelets stimulated with thrombin [61]. However, in rats SIN-1 could only partially reduce the degree of platelet activation [62]. SIN-1 stimulated VASP Ser157 phosphorylation and inhibited GP IIb/IIIa activation. Threshold

9.3 Effects of Different Groups of NO Donors on Platelets

concentrations of SIN-1 potentiated the effects of cAMP with respect to inhibition of platelet adhesion and aggregation, VASP phosphorylation, Rap1b phosphorylation, and glycoprotein IIb/IIIa inhibition [25, 30, 56, 63, 64]. In rabbits, SIN-1 activated the plasma fibrinolytic system through inhibition of the release of plasminogen activator inhibitor from platelets in a concentration-dependent manner [65]. Synergism between SIN-1 and aspirin was observed in the inhibition of platelet activation in flowing blood [56]. Furthermore, a combination of SIN-1 and ilopost, a stable prostacyclin analog, reduced infarct size and had synergistic cardioprotective effects on myocardial ischemia in rabbits [66]. 9.3.4

S-Nitrosothiols 9.3.4.1

SNAP (S-Nitroso-N-acetyl-d,1-penicillamine)

SNAP is a synthetic S-nitrosothiol that inhibits ADP- and collagen-induced platelet aggregation and increased cGMP-levels in platelets [47, 67]. SNAP inhibited both thrombin-induced surface expression and release of platelet P-selectin [68]. Not all effects of SNAP on platelets were cGMP-dependent. Matrix metalloproteinase inhibitor and SNAP synergistically inhibited platelet adhesion to fibrinogen through cyclic GMP-independent mechanisms, since inhibitors of soluble GS did not reverse the SNAP effects [69]. Like SNP, SNAP also inhibited dioxygenase activity of lipoxygenase in human platelets thereby preventing membrane damage through hydroxyl radicals and reactive oxygen species [60]. 9.3.4.2

SNVP (S-Nitroso-N-valerylpenicillamine)

SNVP is a lipophilic N-substituted valeryl analog of SNAP. It caused prolonged nitric oxide mediated vasodilatation in rats [70, 71]. In vivo, SNVP prevented the hyperaggregability of circulating platelets caused by angioplasty of rabbit carotid arteries. Here, only minor effects on blood pressure were observed, whereas platelet adhesion was strongly inhibited [71]. 9.3.4.3

GSNO (S-nitroso-glutatione)

GSNO only weakly stimulates soluble GS and exerts its anti-platelet action most likely via enzymatic, rather than spontaneous release of NO [72, 73]. GSNO significantly diminished collagen-induced platelet aggregation. NO release and GSNO ability to stimulate the intracellular cGMP level in platelets was diminished by copper chelating agent [47]. In vitro, GSNO inhibited the adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers [74] as well as the ADP-, collagen- and thrombin-induced platelet aggregation [74–76]. Treatment of platelets with ODQ, a

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selective inhibitor of soluble guanylate cyclase, resulted in decreased cGMP accumulation in response to GSNO, but GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action [47, 75]. GSNO did not affect solid-phase vWF-tumor cell-stimulated platelet surface expression of GPIIb/IIIa, but inhibited GPIIb/IIIa activation [45, 77]. Furthermore, GSNO facilitated production of functional platelets from megakaryocytoid cell line Meg-01 in a cGMP-independent way [78]. 9.3.4.4

CysNO (S-Nitrosocysteine)

CysNO is a potent anti-platelet agent. Like other nitrosothiols, it increased the intracellular GMP level in platelets in vitro and inhibited collagen-induced platelet aggregation and ADP-induced activation of GP IIb/IIIa on the platelet surface [45, 47]. However its potency to release NO, stimulate platelet soluble GS and elevate intracellular platelet cGMP was higher than of GSNO [47, 72]. 9.3.4.5

SNAC (S-Nitroso-N-acetyl-cysteine)

SNAC is the most potent inhibitor of collagen-induced platelet aggregation in vitro compared to other S-nitrosothiols (SNAP, GSNO, CysNO and HomocysNo). Although it increased cGMP-levels in platelets [47], SNAC only partially inhibited thrombin-induced P-selectin expression on a platelet surface [79]. 9.3.4.6

HomocysNO (S-Nitrosohomocysteine)

Like all S-nitrosothiols, HomocysNO inhibited collagen-induced aggregation and stimulated intracellular cGMP-levels in platelets in vitro [47]. 9.3.4.7

RIG200 (N-(S-Nitroso-N-acetylpenicillamine)-2-amino-2-deoxy1,3,4,6, tetra-O-acetyl-beta-D-glucopyranose)

RIG200, a new S-nitrosothiol, consists of SNAP linked to acetylated glucosamine [80] and causes prolonged vasodilatation in isolated rat arteries with damaged endothelium [81]. This effect lasts for more than 4 h in endothelium-denuded vessels but is not seen in intact vessels. Although shown to be NO-mediated, the precise mechanism by which RIG-200 causes sustained vasodilatation remains unknown [80]. RIG200 inhibited ADP- and collagen-induced platelet aggregation in PRP, and its effects were abolished by the NO scavanger hemoglobin [44, 75]. Soluble guanylate cyclase inhibitor ODQ could not inhibit all the effects of RIG200 on platelets. The cGMP-independent effects of RIG200 were different from those of DEA/NO. ODQ only partially diminished RIG-200 effects on collagen-induced, but not on ADP-

9.3 Effects of Different Groups of NO Donors on Platelets

induced platelet aggregation. In contrast, ODQ had no effect on DEA/NO-mediated inhibition of collagen-induced platelet aggregation and slightly reduced the inhibitory effect of DEA/NO on ADP-induced platelet aggregation [44]. 9.3.5

Organic Nitrates 9.3.5.1

GTN (Glyceryl Trinitrate, Nitroglycerin, NTG)

GTN is a stable organic nitrate that does not spontaneously release NO but requires metabolisation [80]. The probable mechanism of organic nitrate action implicates catalyzed release of NO from nitrates by glutathione-S-transferases [82, 83], cytochrome P450 reductase [84], an unidentified 200 kDa microsomal protein in smooth muscle cells, or a combination of enzymatic and nonenzymatic processes [85]. In vitro, GTN exerted anti-aggregatory effects on platelets in PRP and induced cGMP accumulation and could partially inhibit thrombin-induced Ca2+ -mobilization in Fura-2-loaded gel-filtered platelets [44, 57, 86–88]. GTN directly decreased the platelet response to collagen in whole blood and increased the intraplatelet levels of both cGMP [89, 90] and cAMP [89]. GTN potentiated the inhibitory effects of forskolin on platelet aggregation in PRP and whole blood [89]. Whole blood flow cytometry showed that GTN inhibited fibrinogen binding and expression of P-selectin in response to ADP and thrombin ex vivo [91]. Concurrently, GTN was found to exert an inhibitory effect on platelet NOS activity [88]. N-Acetylcysteine induced a threefold potentiation of the anti-aggregatory effect of GTN [86]. In an in vivo rabbit model, GTN prevented the hyper-aggregability of circulating platelets caused by angioplasty. However, bolus GTN failed to inhibit adhesion of platelets after angioplasty, despite inducing a transient reduction in systemic blood pressure [71]. The transdermal administration of GTN caused a significant decrease in systolic blood pressure, however it did not influence platelet cGMP level, platelet aggregation or adhesion molecule expression [90, 92]. 9.3.6

Mesoionic Oxatriazole Derivatives 9.3.6.1

GEA-3162 (1,2,3,4-Oxatriazolium, 5-amino-3-(3,4-dichlorphenyl)-, cloride)

GEA-3162 spontaneously generates NO when dissolved [93, 94]. GEA-3163 inhibited ADP-induced platelet aggregation and induced a dose-dependent increase in cyclic GMP in platelets. The increase in cGMP was potentiated by phosphodiesterase-5 inhibitor, zaprinast, and inhibited by oxyhemoglobin [94].

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9.3.6.2

GEA-3175 (1,2,3, 4-Oxatriazolium, -3-(3-chloro-2-methylphenyl)-5[[(4-methylphenyl) sulfonyl]amino]-, Hydroxide Inner Salt)

GEA-3175 is more stable than GEA-3162 in vitro but still retains its biological activity [95]. The release of NO and NO2 by GEA 3175 was increased 140-fold in the presence of human plasma, as analyzed by ozone chemiluminescence [94]. GEA 3175 inhibited agonist-induced platelet aggregation and induced a more than 4-fold increase in platelet cGMP without affecting cAMP levels [94]. Thrombin-stimulated rises in the cytosolic free Ca2+ concentration and secretion were dose-dependently inhibited by GEA 3175. GEA 3175 showed a reduced capacity to inhibit platelet aggregation of uremic platelets compared to controls [96]. 9.3.7

Other NO Donors 9.3.7.1

OXINO (Sodium trioxdinitrate or Angel’s Salt)

OXINO partially inhibited ADP-induced platelet aggregation in vitro. Flow cytometry analysis of PRP showed that OXINO decreased ADP-induced CD62P expression and fibrinogen binding on platelets [45]. 9.3.7.2

B-NOD (2-Hydroxy-benzoid acid 3-nitrooxymethyl-phenyl ester)

B-NOD is a new NO donor compound. It has a chemical NO-releasing group similar to that of NTG, however, it releases NO in vitro and in vivo. After administration of B-NOD in vivo its activity persists for more than 7 h. In vitro, the release of NO from B-NOD was augmented by the presence of living cells (blood platelets). B-NOD increased cGMP levels and prevented thrombin-induced platelet aggregation in vitro in the same manner as SNP. In vivo, administration of B-NOD in rabbits did not cause a fall in blood pressure or an increase in heart rate [97]. 9.3.8 L-Arginine (L-Arg)

The amino acid l-Arg is a source of endogenous NO, which is synthesized by NO synthase (NOS). l-Arg potentiates smooth muscle cell- and neutropil-mediated antiaggregatory effects on platelets [98–100]. Platelets produce NO from l-Arg in response to collagen. This NO production from l-Arg might be functionally important when platelets are exposed to collagen fibrils of the subendothelium [101]. In vitro, l-Arg enhanced platelet cGMP levels by increasing NO production and reduced platelet aggregation and TxB2 production [99, 102, 103]. In vivo, l-Arg induced hypotension and vasodilatation in humans [102].

9.4 Activators of Soluble Guanylyl Cyclase

9.4

Activators of Soluble Guanylyl Cyclase

These substances directly stimulate soluble GC and elevate cGMP levels in platelets. 9.4.1

YC-1 (3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole)

YC-1, a derivative of benzylindazole, is a novel NO-independent activator of soluble GC. In human washed platelets, YC-1 inhibited platelet aggregation and ATP release induced by U46619, a stable analog of TXA2 , collagen and thrombin in a concentration-dependent manner [95]. YC-1 potentiates the stimulatory action of submaximally effective NO and carbon monoxide concentrations [104, 105]. In vitro, YC-1 increased both cGMP levels and VASP phosphorylation in platelets [46, 95]. In vivo, a significant increase in cGMP and a distinct effect on VASP phosphorylation in rat platelets were observed 1 h after oral administration of YC-1. These biochemical alterations are supported by a significant prolongation in rat- and mice-tail bleeding time [46, 106]. Monitoring of adhesive interactions between platelets and endothelial cells by intravital microscopy showed that YC-1 reduced lipopolysaccharide-induced platelet/endothelial cells adhesion [49]. It also inhibited both initial and long-term platelet adhesion to collagen, collagen-induced intracellular Ca2+ mobilization and actin polymerization [55]. 9.4.2

BAY 41-2272 (3-(4-Amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)1H-pyrazolo [3,4-b]pyridine)

BAY 41-2272 is a novel non-NO-based soluble GC activator. It produced a marked inhibition of platelet aggregation in washed platelets and PRP, however, with much lower potency in PRP. Both NO and prostacyclin exhibited synergistic activity with BAY 41-2272 to inhibit platelet aggregation. In vivo, BAY 41-2272 significantly reduced blood pressure in rats, but it had only a small effect on FeCl3 -induced thrombosis [107].

9.5

cGMP Analogs

Cell-permeable analogs for cGMP have been used on a variety of cell types, including platelets, for many years. These substances are supposed to diffuse across the cell membrane and directly stimulate cGMP-PKs, thereby activating NO signaling cascades downstream of cGC in platelets. Among the variety of substances available, the specificity for potential target enzymes has to be judged critically for each substance. In platelets it was shown recently that nearly all these substances had unpredictable side-effects and inhibited platelet activation by mechanisms independent of cGMP-

247

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9 NO Donors as Antiplatelet Agents

PK [108]. Therefore the use of such substances for platelets requires exact monitoring of their effects, e.g. by controlling phosphorylation of cGMP-PK substrate proteins.

9.6

Inhaled NO and Platelet Inhibition

Effects of inhaled NO on human platelet function are controversial. Using various concentrations (10 to 80 ppm NO) and set-ups of gaseous NO in humans and a variety of animal models the results of many published studies are unclear or even contradictory. Effects observed are 1. a severalfold elevation of plasma cGMP levels [109–111]; 2. no increase in intracellular platelet cGMP [111]; 3. up to 75% reduced platelet aggregation [109, 111–113]; 4. prolonged bleeding time [109, 110, 113]; 5. no effect on bleeding time and/or platelet function [110, 114]. Clearly, changes in bleeding time and platelet function may also be due to effects of NO at the interface of blood cells, endothelium and the vessel wall [110]. Furthermore, the model system used, species differences and accompanying diseases of the cardiovascular or lung system may alter NO action [115]. Therefore, with regard to platelet function no clear effect of inhaled NO can be defined at present and the involvement of cGMPindependent mechanisms as well as effects of NO on other vascular cells may explain the changes observed in hemostasis.

9.7

Conclusion

NO donors have been used for more than a century in the treatment of cardiovascular diseases. Clearly, the NO/cGMP system plays a major role in platelet inhibition in vivo and in vitro, however, the complex regulation of cGMP levels, as well as the crosstalk to the cAMP system, makes it a signaling network that is not yet fully understood. The contribution of cGMP-independent mechanisms in NO signaling in platelets is far from clear. Careful use of the crucial genetically altered mouse models, the variety of NO donors with clear differences in biochemistry and functional platelet effects as well as the many so-called specific activatory or inhibitory research tools will certainly help to elucidate the still unknown areas of NO signaling in platelets in the near future.

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Control of NO Production Noriko Fujiwara, Keiichiro Suzuki, Naoyuki Taniguchi 10.1

Introduction

Nitric oxide (NO) was discovered to be an endothelium derived relaxing factor (EDRF) and plays an important role in a variety of physiological and pathological processes. NO activates guanylate cyclase, resulting in an increase in the concentration of cGMP, which is involved in cardiovascular functions [1]. The biochemical production of NO is catalyzed by NO synthase (NOS) in two steps. l-arginine is first oxidized to produce NG -hydroxy-l-arginine (NHA), which contains a –C(NH2 )=NOH group. In the second step, the oxidative cleavage of the C=N bond of NHA gives NO and l-citrulline (Fig. 10.1) [2]. Three distinct isoforms of NOS have been cloned from a variety of tissues. Endothelial NOS (eNOS, NOS III), which is expressed mainly in vascular endotherial cells, regulates vascular tone and smooth muscle tension [3, 4]. It is generally thought that the NO produced by neuronal NOS (nNOS, NOS I) functions as a neurotransmitter [5, 6]. Both eNOS and nNOS are constitutive subtypes, and their enzyme activities are Ca2+ - and calmodulin-dependent. The inducible subtype (iNOS, NOS II) expressed in macrophages, vascular smooth muscle cells and hepatocytes is Ca2+ - and calmodulin-independent. The NO produced in macrophages functions directly in antibacterial, antiviral and anti-tumor actions [7–9]. To date, +

H 2N

NH2+

H 2N

N H

OH

NH

NH NADPH

H 3N

COO-

L -arginine

O NH

NADPH

O2 +

H2N

+

.NO

O2 +

H 3N

COO-

N G-hydroxy-L-arginine (NHA)

First oxidation

+

H3N

COO-

L -citrulline

nitric oxide

Second oxidation

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

Fig. 10.1 NO formation by the NOS enzyme.

256

10 Control of NO Production

some splice variants of these isoforms have also been identified [10]. Among three NOS isoforms, iNOS in macrophages and smooth muscle cells, activated by cytokines and lipopolysaccaride (LPS), produces a large amount of NO, which plays a key role in the pathology of a variety of diseases, including sepsis and inflammation. Therefore, compounds that specifically inhibit iNOS but do not affect other isoforms are in demand, and a variety of iNOS selective inhibitors have been developed to date. Because excess NO production by nNOS has been found to be associated with pathological conditions, including Alzheimer’s disease [11], neuroimmune diseases and the pathogenesis of AIDS dementia [12, 13], the development of nNOS selective inhibitors is also an ongoing process. In contrast, when an increase in NO production is desired in a variety of clinical situations, isoform-specific substrates or NO donors will be required. Because of the double-edged nature of NO in both basic physiological functions and various pathological conditions, the development of both inhibitors and substrates for each NOS isoform represents a highly desirable goal [14]. We will concentrate here on various compounds that function as NOS inhibitors and NOS substrates with emphasis on the structure–activity relationships. These compounds are important, not only for NO up/down-regulation but also for use as an isoformspecific probe in biomedical studies designed to develop a better understanding of the mechanism of action of NOS.

10.2

Structure of Nitric Oxide Synthase

The reaction leading to NO formation in mammalian cells is catalyzed by the enzyme nitric oxide synthase (NOS) [15–17]. NOS monomers are very large (∼133–161 kD per monomer) modular enzymes that are produced by a series of gene fusion events during evolution. Although the known NOS enzymes are usually referred to as ‘homodimeric’ in their active form, recently Alderton et al. emphasized that they are tetramers of two NOS monomers associated with two calmodulins (CaMs) [10]. NOSs exhibit two domain structures, an N-terminal oxygenase domain and a C-terminal reductase domain. The reductase region is homologous to P450 reductase, and contains FMN, FAD and NADPH binding regions. The oxygenase region contains the l-arginine, tetrahydrobiopterin (BH4 ), and heme-binding sites. The subunits in NOS are aligned in a head to head orientation with the oxygenase domains in contact and the reductase domains in contact only with the oxygenase domains [18–21]. The reductase unit of one monomer supplies electrons from NADPH to the oxygenase unit of the other [22]. This assignment of domain alignment is similar to sulfite reductase [23] and nitrite reductase [24] where the catalytic domain is dimeric [19]. Cloning of the isoforms of NOS has revealed that they share only a 50% primary sequence homology, suggesting that they may differ from each other in regulatory aspects [25]. The crystal structures of the oxygenase domains of murine iNOS monomer [26], murine and human iNOS dimer [26–29] and human and bovine eNOS dimer [29, 30] indicate a high degree of structural similarity within the critical catalytic center among NOS isoforms. However, a novel approach to probe the active site topologies

10.3 NO Formation

of the three NOSs using various aryldiazenes showed that the active site topologies of the three NOS isoforms are different. The differences in the ceiling heights, in the rates of reaction of phenyldiazene with the three isoforms, and the ability of the isoforms to react with naphthyldiazene, suggest that the size of the active site decreases in the order nNOS > iNOS > eNOS [31]. Despite an overall structural similarity, such topological differences and some subtle structural differences among the substrate binding sites of the NOS isoforms can be used to develop isoform-specific inhibitors and substrates. Moreover, X-ray crystal structure analyses have clarified an unexpected fact, that a single structural zinc per dimer is located at the dimer interface and is coordinated by four cysteines, two from each monomer. The zinc and four cysteins play a crucial role in the stability of the NOS homodimer [29].

10.3

NO Formation

All NOS isoforms function as double monooxygenases, generating NO and citrulline from l-argiinine, 2 mol of O2 , and 1.5 mol of NADPH [2, 32, 33]. The electrons from the NADPH are transferred to the heme domain of another subunit via FAD and FMN [15]. The electrons are used in the oxidation of l-arginine as described below. The heme–flavin domain interaction and hence, electron transfer, is regulated by calmodulin [34]. Tetrahydrobiopterin (BH4 ) is also required for NO synthesis and promotes dimerization [35–37]. The l-arginine guanidinium group at the bottom of the heme pocket first donates a proton to the peroxo-iron, facilitating O–O bond cleavage and conversion to a proposed oxo-iron (IV) p-cation radical species, which then rapidly hydroxylates the neutral guanidinium group to give Nù -hydroxy-l-arginine (NHA) [15, 27]. The reaction is similar to those catalyzed by cytochrome P-450 with a proposed oxo-iron intermediate [P-Fe(IV)=O] [38]. NHA is then converted to l-citrulline and NO by an unusual mechanism involving a one-electron oxidation intermediate [P-Fe(III)-OO2− ] [39]. Another unique feature of NOS catalysis is the requirement for BH4 in both steps of the reaction. BH4 is thought to play an essential redox role by providing an electron required to activate the Fe(II)-oxy complex to the active hydroxylating species, Fe(IV)-O or Fe(III)-hydroxy [40]. When BH4 is absent in the oxygenase domain, the rate of the oxidative reaction is markedly decreased, as described in Section 10.8.1.

10.4 L-Arginine and L-Arginine Derivatives

It is generally thought that analogs of the native substrate of NOS isoforms, l-arginine, bind to the active site as an inhibitor or a substrate. Many analogs of l-arginine have been synthesized, and the functions of these compounds have been analyzed.

257

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10 Control of NO Production

10.4.1

Inhibitors

Many analogues of l-arginine are inhibitors of NOS. Typically, they are substituted at one or both of the terminal guanidine (amidino or omega-) nitrogens (Fig. 10.2) [41, 42]. Table 10.1 shows IC50 values and/or Ki values for these inhibitors against three or two NOS isoforms, although the IC50 values are difficult to compare because they depend on the experimental conditions employed. Komori et al. calculated the Ki R3

NH2

(CH2)n NH C R1

R2

n

R1

R2

R3

3

NH

NH2

COOH

L -arginine (substrate)

3

NOH

NH2

COOH

L -NG-hydroxy-arginine (L-NHA)

3

NCH 3

NH2

COOH

L -NG-methyl-arginine

3

NNO2

NH2

COOH

L -NG-nitro-arginine

3

NNH 2

NH2

COOH

L -NG-amino-arginine

3

NCH 2CH 3

NH2

COOH

L -NG-ethyl-arginine(4)

3

NCH 2CHCH3

NH2

COOH

L -NG-allyl-arginine

3

NOCH3

NH2

COOH

L -NG-methoxy-arginine

3

O

NH2

COOH

L -citrulline (product)

3

NH

CH3

COOH

L -iminoethyl-ornithine

3

NH

SCH3

COOH

S-methyl- L-thiocitrulline (L-SMTC) (8)

3

NH

SCH2 CH3

COOH

S-ethyl-L -thiocitrulline (L-SETC) (9)

4

NH

CH3

COOH

L -iminoethyl-lysine

4

NH

CH3

CH(OH)CH(OH)

(intermediate)

(L-NMA) (1)

(L-NNA) (2) (3)

(5) (6)

(L-NIO) (7)

(L-NIL) (10)

N-[5(S)-amino-6,7dihydroxyhepnyl]ethanimidamide (11)

Fig. 10.2 Structure of L-arginine analogs and amino acid-based NOS inhibitors.

10.4 L-Arginine and L-Arginine Derivatives Tab. 10.1: Inhibitory activities of L-arginine derivatives for NOSs.

Compound (No.) iNOS 1 14 2 7.6 3 4 5 6 7 2.2 8 9 10 5 11 12

IC50 (ìM) nNOS eNOS iNOS 10 5.9 2.5 0.52 0.5 8.7 1.7 ∼ 81 8.5 20.5 3.9 34 17 61 138 150 8420

Ki (ìM) nNOS 7.5 0.2 1.2 ∼ 66 0.85 6.0 1.2 0.5

eNOS

11 24

values for some l-arginine analogs towards iNOS and nNOS. From the results, the rank order of potency for the inhibitors for iNOS was found to be l-NG -amino-arginine (3) > l-NG -methyl-arginine (1) > l-NG -hydroxy-arginine (NHA) > l-NG -allyl-arginine (5) = l-NG -nitro-arginine (2) >> l-NG -methoxy-arginine (6) > l-NG -ethyl-arginine (4), whereas for nNOS the order was l-NG -nitro-arginine (2) > l-NG -methyl-arginine (1) > l-NG -allyl-arginine (5) > l-NG -amino-arginine (3) > l-NG -methoxy-arginine (6) >> l-NG -hydroxy-arginine (NHA) > l-NG -ethyl-arginine (4) [43]. These inhibitors were more potent against nNOS than against iNOS with the l-NG -nitro-arginine (2) having the greatest specificity for the nNOS. However, most of these classical l-arginine analogs showed poor selectivity within the isoforms. In other amino acid-based inhibitors, l-N-(1-iminoethyl)-lysine (l-NIL) (10) can be considered as the first amino acid-based inhibitor showing iNOS (IC50 = 5 ìM) vs. eNOS (IC50 = 138 ìM) [44]. Replacing the carboxyl moiety of NIL with a vicinal glycol yielded N-(5(S)-amino6,7-dihydroxyhepnyl)ethanimidamide dihydrochloride (11), having IC50 values for iNOS (12 ìM) and eNOS (8420 ìM). This compound is 700-fold more selective for iNOS than for eNOS [45]. Recently, 5-fluorinated-l-lysine, an l-NIL analog, was also prepared and found to be a selective iNOS inhibitor [46]. In contrast, S-methyl-lthiocitrulline (SMTC) (8) and S-ethyl-l-thiocitrulline (SETC) (9) were more potent against nNOS than iNOS or eNOS (Table 10.1) [47]. 10.4.2

Substrates

NOS isoforms have a narrow specificity with respect to substrates for the production of NO. In addition to the natural substrate, l-arginine, some l-arginine analogs have been reported as substrates for the NOS enzyme. l-methyl-arginine (l-NMA) (1), a widely used NOS inhibitor (see Fig. 10.2 and Table 10.1), is also capable of the NOS-dependent generation of NO, although the proposed mechanisms suggest either l-arginine or NHA as intermediates [48]. l-arginine derivatives, such as homol-arginine and N-hydroxy-homo-l-arginine, have also been reported to be oxidized to

259

260

10 Control of NO Production Tab. 10.2: Kinetic constants for the substrate and inhibitor activity of L-arginine and compounds

12–16. iNOS nNOS eNOS Compound Km (ìM) Ki (ìM) Km (ìM) Ki (ìM) Km (ìM) Ki (ìM) l-arginine 9.5 2.7 1.1 12 35 45 1.5 25 1.0 50 13 20000 13000 5000 65000 15000 10000 14 20000 8 40000 2 25000 50 15 900 770 3000 16 9000 640 2500

the corresponding ureas and NO by NOS enzymes. However, most other arginine derivatives such as d-arginine, l-arginine methylester, N-acetyl-l-arginine and agmatine were found not to be substrates. Lee et al. synthesized some conformationallyrestricted arginine analogs (Fig. 10.3) and reported that they serve as alternative substrates or inhibitors of the three isozymes of NOS [49]. (E)-(12) and (Z)-3,4didehydro-d,l-arginine (13), m-guanidino-d,l-phenylglycine (14) are both substrates NH HN

NH NH2

HN

NH2

COOH

COOH

NH2

NH2 12 (E)-3,4-Didehydro-D,L -arginine

13 (Z)-3,4-Didehydro-D,L -arginine

NH

NH HN

NH2

HN

NH2

O COOH

COOH

NH2

NH2 14 m-Guanidino-D, L-phenylglycine

15 5-Keto-L-arginine

NH HN

N H COOH NH2

16 (E)-propyl-3,4-didehydro-D,L-arginine

Fig. 10.3 Structure of conformationally-restricted L-arginine analogs.

10.5 Non-amino Acid Inhibitors and Non-amino Acid Substrates

and inhibitors of all three NOS isoforms (Table 10.2). The (Z)-isomer (13) was found to be a much poorer substrate for all three NOS than the (E)-isomer (12), having Km values 3–4 orders of magnitude greater than the (E)-isomer (Table 10.2) and kcat /Km values 3–5 orders of magnitude lower than those for the (E)-isomer [49]. The (E)-isomer also exhibits a much lower inhibitory activity than the (Z)-isomer. The m-guanidinod,l-phenylglycine (14) is a much poorer substrate than (E)-3,4-didehydro-d,l-arginine (12), but shows a similar potent inhibitory function. In contrast, 5-keto-d,l-arginine (15) and (E)-N-propyl-3,4-didehydro-d,l-arginine (16) act only as inhibitors.

10.5

Non-amino Acid Inhibitors and Non-amino Acid Substrates

Arginine and its analogs can be regarded as derivatives of guanidine. The guanidium moiety of the arginine is critical for binding between the substrate and enzyme, with smaller contributions from carboxylic acid and peptide nitrogen groups [50]. Therefore, amidine derivatives, which have a guanidium moiety, as shown in Fig. 10.4, have been synthesized and their activities have been assessed as inhibitors and substrates. R

R R

NH

S

C

C

C

H2N

NH

a) amidines

H2N

NH

b) guanidines

H2N

NH

c) isothiourea (ITU)

R R

NH

O

C

C

C

R'

NOH

H2N

NOH

H2N

NH OH

d) oxime

e) hydroxyguanidines

Fig. 10.4 Structure of guanidine derivatives and compounds structurally similar to guanidine.

f) hydroxyurea

261

262

10 Control of NO Production

10.5.1

Guanidine 10.5.1.1

Inhibitor

Guanidine (Fig. 10.4b, R = H) itself is neither an inhibitor nor a substrate, but methylguanidine (Fig. 10.4b, R = CH3 ) and ethylguanidine (Fig. 10.4b, R = CH2 CH3 ) inhibit NOS [51]. In particular, aminoguanidine (Fig. 10.4b, R = NH2 ) has received considerable attention as an inhibitor of NOS due to the early recognition of its selectivity towards iNOS [52]. Although aminoguanidine is much less potent than L-NMA (Ki = 2.5 ìM), or other compounds toward iNOS in vitro (Ki = 39.9 ìM, [53]), many studies have reported that aminoguanidine is beneficial in various experimental models of inflammation and septic shock. The intravenous administration of aminoguanidine at a dose of 1 mg kg−1 min−1 caused the recovery of blood pressure in LPS-treated rabbits that exhibited a lowered blood pressure. However, L-NMA at the same dose increased blood pressure but decreased the heart rate in normal rabbits, while aminoguanidine affected neither cardiovascular function [54]. Aminoguanidine has also been studied extensively in diabetes where it appears to exert NO-dependent and NO-independent beneficial effects [55]. Indeed, aminoguanidine has many other pharmacological functions including inhibition of histamine metabolism [56], polyamine catabolism [57], the formation of advanced glycation end products [58], and catalase activity [59]. Moreover, aminoguanidine prevented the gene expression of iNOS by an unknown mechanism [60]. 10.5.1.2

Substrates

It has been reported that N-hydroxyguanidine (Fig. 10.4e)-containing drugs could be oxidatively cleaved by cytochrome P450 and horseradish peroxidase with the formation of the corresponding ureas and nitrogen oxides including NO [61–63], and even by NOS [64]. Xian et al. synthesized a series of compounds that contain an N-hydroxyguanidine functional group [65]. The results, shown in Fig. 10.5, indicate that the activity of these compounds as an NOS substrate is related to the structure of the substituent group. Although N-hydroxyguanidine (R = H) itself is not a substrate, compounds with small alkyl group substituents function as substrates for all NOS isoforms. When an ethyl group (R = CH2 CH3 ) is attached to hydroxyguanidine, only a low reactivity is observed, but one additional methylene group, n-propyl substitution (R = CH2 CH2 CH3 ), significantly enhances the activity. The presence of an additional methylene group, n-butyl (R = CH2 CH2 CH2 CH3 ) substituent, and an isopropyl (R = CH(CH3 )2 ) substituent also produce good substrates for all NOS isoforms. The Km values of n-butyl-hydroxyguanidine were 33 ìM for iNOS and 67 ìM for nNOS, whereas the Km values of isopropyl-hydroxyguanidine were 77 ìM for iNOS and 56 ìM for nNOS. However, when one more methylene group was attached (n-pentyl, R = CH2 CH2 CH2 CH2 CH3 ), the activity as a substrate decreased,

10.5 Non-amino Acid Inhibitors and Non-amino Acid Substrates

compounds L -arginine

-H -CH3

nNOS eNOS iNOS

-CH2 CH3 -CH2 CH2 CH3 -CH2 CH2 CH2 CH3 -CH2 CH2 CH2 CH2 CH3 -CH(CH3)2

Fig. 10.5 NO formation from the oxidation of N-alkyl-N′-hydroxyguanidines -CH2 CH(CH3)2 in the presence of NOS. The reaction rates were assessed by 0 20 40 60 80 100 a hemoglobin assay and are Relative amounts of NO production from expressed as a percentage of N-alkyl-hydroxyguanidine (% NHA) those of NHA. -C(CH3 )3

and further long alkyl substituents such as n-hexyl (R = CH2 CH2 CH2 CH2 CH2 CH3 ), showed significantly lower activities. Moreover, when the alkyl substituents become too bulky, the activity also decreased. For example, N-iso-butyl-N-hydroxyguanidine (R = CH2 CH(CH3 )2 ), containing one more methyl group attachment at the terminal carbon of N-n-propyl-N′-hydroxyguanidine diminished the activity. Similar effects were also found for N-isopropyl-N′-hydroxyguanidine substituents and N-tert-butylN′-hydroxyguanidine (R = C(CH3 )3 ), which is not a substrate for any of the three NOSs (Fig. 10.5). These data indicate that, when the alkyl substituents are too small, too large or too bulky, the ability as a NOS substrate decreases significantly. This suggests that an interaction between the alkyl group and the hydrophobic cavity in the NOS active site contributes to the relative activity of the synthesized compounds as a substrate [65]. Moreover, N-aryl-N′-hydroxyguanidines have been found to be selective substrates for iNOS [65, 66]. N-phenyl-N′-hydroxyguanidine [65] and N-(4-fluorophenyl)-N′-hydroxyguanidine [66] were found to be the best substrates for iNOS. When the hydroxyguanidine group was changed to a guanidine group (Fig. 10.4b), both N-alkyl and N-aryl-substituted compounds had no activity as a substrate [65], although some of them exhibit an inhibitory function as described above. iNOS utilizes large amounts of l-arginine, and this may result in the depletion of intracellular l-arginine pools and/or an increased uptake of l-arginine from extracellular sources. The induction of iNOS is associated with the induction of arginosuccinate synthase, an enzyme that catalyzes the recycling of l-citrulline to l-arginine. This may supply iNOS with its substrate from endogenous sources [67]. The membrane transport system for l-arginine is also activated with the iNOS induction [68]. In fact, the transport of l-arginine into astrocytes is necessary for maximal iNOS activity and

263

264

10 Control of NO Production

NO generation [69]. However, the question of why extracellular arginine is required for iNOS activity, even though the intracellular concentrations of arginine are well above the Km level and should be sufficient to saturate iNOS, remains unclear. This phenomenon, termed the "arginine paradox," has yet to be adequately resolved [70]. It has also been observed that NO formation in RAW264.7 cells stimulated by LPS is dependent on extracellular l-arginine concentrations in the medium (Kazuma et al., unpublished data). Thus the amount of NO formed from the synthesized N-alkyland N-phenyl-N′-hydroxyguanidines in the medium has been measured, in order to determine whether the synthesized N-alkyl and N-phenyl-N′-hydroxyguanidines function as iNOS substrates in vivo. RAW264.7 cells were seeded on 96 well plates, cultured for 8 h in DMEM (without l-arginine) containing LPS (10 ng ml−1 ) and then washed twice with Krebs–Ringer phosphate buffer. The cells were incubated in Krebs–Ringer phosphate buffer containing 10 ìM DAF2-DA (Calbiochem, La Jolla, CA) and the synthesized NOS substrates for 2 h at 37 °C. NO production from the cells in the medium was measured by NO-specific fluorescence (DAF2-DA). Fig. 10.6 shows that all of these N-alkyl- and N-phenyl-N′-hydroxyguanidines, except N-tertbutyl-N′-hydroxyguanidine, which was not a substrate in vitro (Fig. 10.5), function as substrates for iNOS in vivo as well (Kazuma et al., unpublished data). The potency of the compounds with respect to NO production was similar to the activity assessed by an in vitro hemoglobin assay (Fig. 10.5), although N-isopropyl-N′-hydroxyguanidine was not a good substrate in the cells (Fig. 10.6). Interestingly, although NHA showed Relative amounts of NO production from N-alkyl-hydroxyguanidine (% NHA)

compounds 0

100

200

300

NHA L -arginine

-CH2 CH3 -CH2 CH2 CH3 -CH2 CH2 CH2 CH3 -CH2 CH2 CH2 CH2 CH3 -CH(CH3)2 -C(CH3 )3

Fig. 10.6 NO production from the oxidation of N-alkyl- and N-phenyl-hydroxyguanidines in RAW264.7 cells.

400

500

10.5 Non-amino Acid Inhibitors and Non-amino Acid Substrates

a higher activity than l-arginine by the in vitro hemoglobin assay (Fig. 10.5), the ability of NHA with respect to NO production in the cells was 4-fold lower than that of l-arginine (Fig. 10.6). 10.5.2

Isothiourea (ITU)

Garvey et al. reported that simple alkyl isothioureas (ITUs) (Fig. 10.4c) are potent inhibitors of the three human NOS isoforms [71]. Substitution on the sulfur atom but no substitution on the nitrogen atoms was essential for the inhibitory function because thiourea itself is not an inhibitor. Small alkyl substitutions on the sulfur atom resulted in the most potent inhibitors. The inhibitory potency of the alkyl-ITUs was represented as 1/Ki values (Fig. 10.7). S-methyl-, S-ethyl-, and S-isopropyl-ITU were found to be the most potent inhibitors of all NOS isoforms. If the alkyl groups were too small (hydrogen, R = H), too large (n-butyl, R = CH2 CH2 CH2 CH3 ) or too bulky (tert-butyl, R = C(CH3 )3 ), the inhibitory activity decreased significantly. As observed for N-alkyl-N′-hydroxyguanidines as NOS substrates, there is an optimal length of the hydrophobic chain for an inhibitory function. A comparison between the ability of the N-alkyl-N′-hydroxyguanidines to produce NO (Fig. 10.5) and the inhibitory potency of the S-alkyl-ITUs (Fig. 10.7) will provide a better understanding of structure–activity relationships (SAR). The optimal length of the carbon chain for compounds nNOS eNOS iNOS

L -NMA

-H -CH3 -CH2 CH3 -CH2 CH2 CH3 -CH2 CH2 CH2 CH3 -CH2 CH2 CH2 CH2 CH3

N.T.

-CH(CH3)2 -C(CH3 )3 -CH2 CH(CH3)2

1 10 100 Inhibitory potency of N-alkyl-ITU (1/K i) Fig. 10.7 Inhibitory potencies of N-alkyl-S-isothioureas towards

three NOS isoforms. The inhibitory potencies are expressed as 1/Ki . N.T. means not tested.

265

266

10 Control of NO Production

the N-alkyl-N-hydroxyguanidines is between propyl (C3) and pentyl (C5), whereas that for the S-alkyl-ITUs is between methyl (C1) and propyl (C3). Furthermore, isopropyl appears to be the most suitable alkyl group for both the N-alkyl-N′-hydroxyguanidine as an NOS substrate and the S-alkyl-ITU as an NOS inhibitor. However, the structure of the isopropyl group is quite different from the hydrophobic chain of the native substrate, l-arginine or NHA. Li et al. demonstrated that the binding mode towards nNOS is different for the N-isopropyl-N′-hydroxyguanidine vis-a-vis the N-butyl-N′hydroxyguanidine although the binding mode of the N-butyl-N′-hydroxyguanidine is consistent with that observed for the substrate NHA bound to the nNOS active site [72]. Variations in the interactions between the alkyl group and the hydrophobic cavity in the NOS active site will provide mechanistic insights into NO biosynthesis and a new viewpoint for the development of isoform selective inhibitors and substrates. There are some published reports on in vivo experiments designed to investigate the effects of ITUs in various models associated with NO overproduction. S-methyl ITU reversed the circulatory failure caused by endotoxin in the rat. This beneficial effect of the S-methyl-ITU was associated with an attenuation of liver injury and hepatocellular dysfunction [73]. Similarly, the administration of aminoethyl-ITU (1 mg kg−1 h−1 commencing 2 h after the injection of endotoxin) resulted in beneficial hemodynamic effects and attenuated the degree of liver injury in the rat [74]. Aminoethyl-ITU also prevented iNOS expression in cultured macrophages and in the rat [60]. Seo et al. reported that S-ethyl-ITU suppressed intranucleosomal DNA cleavage in pancreatic â-cells induced by NO produced by iNOS, and that the intravenous administration of S-ethyl-ITU at 0.1 mg kg−1 min−1 caused an increase in blood pressure in both normal and LPS-treated rabbits [54]. Bis-ITUs were selective inhibitors of iNOS compared to eNOS, showing 15–20fold selectivity. S,S′-(1,3-phenylenebis(1,2-ethanediyl))-bis-ITU (Fig. 10.8) inhibits human iNOS with a Ki of 47 nM and has a 190-fold selectivity for human iNOS compared to human eNOS [71]. However, the IC50 values of S-ethyl-ITU, and S,S′(1,3-phenylenebis(1,2-ethanediyl))-bis-ITU for iNOS produced in DLD-1 cells that had been stimulated by a combination of cytokines were 10 and 150 ìM, respectively, despite the quite low Ki values for human iNOS. Therefore the clinical use of these isothiourea-based compounds is limited by their poor cellular penetration [71] and systemic toxicity [75]. From this S,S′-(1,3-phenylenebis(1,2-ethanediyl))-bis-ITU, N-(3-(aminomethyl)benzyl)acetamidine (denoted as 1400W) was derived (Fig. 10.8). The 1400W was found to be a slow, tight binding inhibitor of human iNOS with a Kd value of 7 nM. In contrast, the inhibition of human nNOS and eNOS was weaker, rapidly reversible and competitive with l-arginine, with Ki values of 2 ìM and 50 ìM, respectively. Selectivity was observed in rat aortic rings in which 1400W was more than 1000-fold more potent against iNOS than eNOS and also in a rat model of endotoxin-induced vascular injury [76]. Thus, 1400W is biologically active, unlike the ITUs, and will be useful as a novel therapeutic agent as a potent iNOS selective inhibitor.

10.5 Non-amino Acid Inhibitors and Non-amino Acid Substrates

S

S NH2

HN

H2 N

N

S,S -[1,3-phenylenebis(1,2-ethanediyl)]bisisothiourea

HN

N H

NH2

N -[3-(aminomethyl)benzyl]acetamidine (1400W)

Fig. 10.8 Structure of 1400W.

10.5.3

Amidine

Guanidines and isothiourea can be classified as derivatives of amidines (Fig. 10.4) of which formamidine (Fig. 10.4a, R = H) is the lowest member of the homologous series. Although formamidine and acetamidine (Fig. 10.4a, R = CH3 ) are not NOS inhibitors, propionamidine (Fig. 10.4a, R = CH2 CH3 ) and longer chain amidines inhibit NOS activity [77]. In addition, simple alkyl amidines, butylamidine (Fig. 10.4a, R = CH2 CH2 CH2 CH3 ), inhibit nitrite formation in immunostimulated J774 macrophages (EC50 = 60 ìM) [77]. The optimal length of the carbon chain for the alkyl amidine is between ethyl (C2) and butyl (C4), which is similar to that for the N-alkyl-N′-hydroxyguanidines and the S-alkyl-ITUs. These amidine derivatives

Tab. 10.3: Inhibitory activities of selected substituted 2-iminopiperidines for NOSs.

Compound IC50 (ìM) Selectivity (No.) iNOS nNOS eNOS eNOS/iNOS 17 1.0 1.1 4.7 4.7 18 0.1 0.2 1.1 9 19 0.5 0.4 0.6 1 20 0.08 0.06 0.3 4 21 5.9 28 375 64

267

268

10 Control of NO Production

R1

NH

R2

N H Cyclic amidine

R1

R2

H

H

2-iminopiperidine (17)

CH3

H

4-methyl-2-iminopiperidine (18)

H

CH3

6-methyl-2-iminopiperidine (19)

CH3

CH3

4,6-dimethyl-2-iminopiperidine (20)

H

CH2 C6H 11

Fig. 10.9 Structure of cyclic cyclohexymilmethyl-2-iminopiperidine (21) amidines.

were more selective towards iNOS than the commonly used l-arginine based NOS inhibitors [77]. 10.5.4

Cyclic Amidines are Potent iNOS Selective Inhibitors

Cyclic amidines such as 2-iminopiperidine (17), are more potent inhibitors of iNOS than l-NMA. Substitution on the 2-iminopiperidine ring resulted in more potent and selective inhibitors towards human iNOS (Fig. 10.9 and Table 10.3, reviewed by Salerno [78]). Among them, 4- (18) and 6- (19) methyl derivatives as well as the 4,6-dimethyl compounds (20) were the most potent inhibitors with IC50 values for iNOS of 0.1, 0.5 and 0.08 ìM, respectively. Although the cyclohexylmethyl derivative (21) was less potent, it was the most selective among this six ring series. Recently, a novel cyclic amidine analogue (1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2azabicyclo[4.1.0]heptane hydrochloride, denoted as ONO-1714 (Fig. 10.10), has been Me H H • HCl Cl H

N H ONO-1714

NH

Fig. 10.10 Structure of ONO-1714.

10.5 Non-amino Acid Inhibitors and Non-amino Acid Substrates

developed. It inhibits human iNOS with a Ki of 1.88 nM and inhibits the rodent enzyme with similar potency in vitro [53]. In terms of selectivity, ONO-1714 was found to be 10-fold more selective for human iNOS over human eNOS. ONO-1714 inhibited the LPS-induced elevation of plasma NO3 /NO2 in mice with an ID50 value of 0.01 mg (kg s.c.)−1 , which is 2600-fold more potent than l-NMA with an ID50 value of 26 mg (kg s.c.)−1 . It has been reported that ONO-1714 improves glomerulonephritis [79], intestinal ischemia-reperfusion [80], cardiac dysfunction [81], septic shock [82], pancreatitis [83] and colitis [84] in animal models. Therefore, ONO-1714 has considerable therapeutic potential as a potent iNOS inhibitor. 10.5.5

Indazole

The indazole compounds also inhibit NOS activity. Based on the SAR of indazole compounds, it has been concluded that the inhibition of nNOS is a characteristic property of the indazole nucleus (Fig. 10.11, Table 10.4, [78]). Nitration at the 5-, 6-, or 7-position resulted in a graded increase in inhibitory potency, while the presence of an amino-group at the 5- and 6-position gave less active compounds (Fig. 10.11, Table 10.4). The 7-nitro indazole (7-NI) is a strong NOS inhibitor with IC50 values of 0.9, 5.8 and 0.71 ìM for nNOS, iNOS and eNOS, respectively. Despite the absence of in vitro selectivity in enzymatic or functional assays, 7NI may be considered as the first selective inhibitor of nNOS in vivo. Moore et al. reported that 7NI showed antinociceptive activity in mice without increasing blood pressure and that the antinociceptive activity of the 7-NI was correlated with the inhibition of nNOS activity [85, 86]. The inhibition of nNOS by 7-NI protected against MPTP-induced [87] and NMDA induced [88] neurotoxicity in mice. 7-NI also reduced neurogenic edema formation, presumably by blocking the nNOS present in peripheral nerves [89]. N

R N H

Fig. 10.11 Structure of indazol.

Tab. 10.4: Inhibitory activities of Indazol derivatives for NOSs.

Compound R iNOS H 5-NO2 6-NO2 7-NO2 5.8 5-NH2 6-NH2

IC50 (ìM) nNOS 177.8 47.3 31.7 0.9 > 1000 > 1000

eNOS

0.71

269

270

10 Control of NO Production

10.6

Inhibition of NOS Function Targeted towards Cofactors

Since NOS isoforms require a number of cofactors and/or prosthetic groups, such as FAD, FMN, NADPH, BH4 and calmodulin, when the cofactors cannot function or cannot bind, NOS enzymes are inactive. The flaboprotein inhibitors, diphenyleneiodonium and several of its analogs, inhibit NOS activity by their irreversible effects on the FAD binding site [90]. Calmodulin binding and activation may be an interesting target for the selective inhibition of different NOS isoforms. It was found that a calmodulin inhibitor, such as calmidazolium, W-7 and fendiline inhibit the calmodulin-dependent isoforms, nNOS and eNOS but not iNOS [91]. Kondo et al. reported that a point mutated plant calmodulin M144V, SCaM-1, acts as an antagonist for nNOS activation [92]. Since BH4 analogues bind to the BH4 binding site, pteridine-based compounds have been reported as NOS inhibitors. Among a series of tested compounds, the 4-amino analogue of BH4 , 5,6,7,8-tetrahydro-6(d-thero-1,2-dihydroxypropyl)pterine, was a potent inhibitor of the recombinant rat nNOS both in vitro (Ki = 13 nM) and in vivo [93]. The heme prosthetic group is also required for the catalytic activity of NOSs and is likely responsible for oxygen activation in a manner similar to that of other heme-containing monooxygenases such as cytochrome P450 [94]. Carbon monoxide (CO) is an extremely good ligand for ferrous (Fe2+ ) hemes and is a potent inhibitor of NOS activity [95, 96]. Other heme ligands such as KCN, miconozole [97] and even NO itself [98] were also reported to be capable of NOS inhibition. Since it is well known that imidazoles act as inhibitors of various heme-containing proteins by binding to the heme group [99], imidazole derivatives also inhibit all NOS isoforms [78].

10.7

Regulators of NOS Gene Expression

Aminoguanidine and aminoethyl-ITU, which are weaker iNOS selective inhibitors, inhibited iNOS expression in the macrophage cell line J774.2 cells stimulated by LPS [60]. N-Acetyl-5-hydroxytryptamine (N-acetylserotonin), an inhibitor of sepiapterin reductase in BH4 synthesis, inhibited the expression of iNOS, but not iNOS activity, both in a cultured macrophage cell line RAW264.7 stimulated by LPS and in vascular smooth muscle cells stimulated by interleukin-1â [100]. Moreover, the novel quinazoline derivatives (Fig. 10.12) inhibited the induction of iNOS mRNA in RAW264.7 cells stimulated by LPS (Fig. 10.13A). Surprisingly, however, they enhanced the induction of iNOS mRNA in vascular smooth muscle cells stimulated by interleukin-1â in an opposite manner (Fig. 10.13B) [101]. Although the mechanism by which these agents inhibit or enhance iNOS gene expression remains unclear, the regulation of NO production at the gene expression level may provide new insights in the development of selective inhibitors and selective enhancers of iNOS in various tissues.

10.8 NO Formation by an NOS-independent Pathway

DIQ

H 3C

4-(1,1-dimethyl-1,2-methoxyethylamino) -2-(imidazol-1-yl)-quinazoline dihydrochloride

CH3

O CH3

NH N

N

N

N

IMT 2-(imidazol-1-yl)-4-(2-methoxyethylamino) -5,6,7,8-tetrahydroquinazoline dihydrochloride

O CH3

NH N

N

N

N

IPE 2-{2-[2-(imidazol-1-yl)-5-methylthieno [2,3-d]pyrimidine-4-ylamino]ethoxy} CH3 ethanol dihydrochloride

O NH

OH N

S

N

N

N

Fig. 10.12 Structure of

quinazoline derivatives.

A RAW264.7 cells (-) (-)

(-)

LPS (10 ng ml-1) DIQ IMT IPE 5 50 5 50 5 50

iNOS

B VSMC

IL-1β (10 ng ml-1)

(-) (-)

(-)

DIQ 5 50

5

IMT 50

5

IPE 50

Fig. 10.13 Effects of the quinazoline

iNOS

derivatives on iNOS gene expression in RAW264.7 cells (A) and vascular smooth muscle cells (B).

10.8

NO Formation by an NOS-independent Pathway

It has been found that oxime (Fig. 10.4d) and hydroxyurea (Fig. 10.4f) as well as Nhydroxyguanidine (Fig. 10.4e) also release NO via an NOS-independent pathway as described below.

271

272

10 Control of NO Production

10.8.1

Oxime

Cytochrome P450s have been found to catalyze the oxidative cleavage of C=N bonds of compounds containing a –C=NOH (oxime) function, such as NHA, N-hydroxyguanidine, amidoxime, ketoximes, and aldoximes, producing the corresponding derivatives and NO in vitro [62, 102]. The oxidase function of cytochrome P450s is similar to that of NOSs, both of which utilise the same prosthetic groups (NADPH, FAD, FMN and heme-thiolate). However, cytochrome P450s do not bind BH4 , which is only present in NOSs, and the reactions are not selective in terms of substrates. In addition, the oxidation of N-hydroxyguanidine, amidoximes, and ketoximes by microsomal cytochrome P450s, but not NOSs, is strongly inhibited by superoxide dismutase (SOD), indicating that cytochrome P450s require O2 •− for the oxidation [62]. It has been reported that O2 •− directly oxidizes N-hydroxy-l-arginine [103, 104], and that O2 •− efficiently oxidizes amidoximes with formation of the corresponding amides and nitriles, in addition to nitrogen oxides [102]. The BH4 free-iNOS-catalyzed oxidation of N-hydroxyguanidines is also strongly inhibited by SOD. In addition, the oxidation of N-p-chlorophenyl-N′-hydroxyguanidine by NADPH and O2 in the presence of BH4 free-iNOS led to the formation of the corresponding urea and cyanamide, in addition to nitrite and nitrate. The oxidation of NHA by BH4 -free-iNOS in the presence of NADPH and O2 also leads to the formation of N-cyanoornithine and citrulline [105]. Thus, the oxidation of oximes by cytochrome P450 and BH4 -free-iNOS show similar characteristics (reviewed by Mansuy and Boucher [106]). However, the rate of the reactions was markedly lower (in the 1–10 min−1 range) than iNOS in the presence of BH4 (in the 50–500 min−1 range). 10.8.2

Hydroxyurea

Hydroxyurea (Fig. 10.4f) was first synthesized in 1869 and has been used in the treatment of a variety of cancers and sickle cell disease for a long time. However, the mechanism of the beneficial functions of this agent has remained unclear. Hydroxyurea inhibits ribonucleotide reductase by quenching the catalytically essential tyrosyl free radical of the enzyme [107], and increases the levels of fetal hemoglobin [108, 109]. A variety of experiments have shown an increase in iron nitrosyl hemoglobin, nitrite, and nitrate as a result of the administration of hydroxyurea. These results and the molecular structure of hydroxyurea, which contains an N–O bond, suggest that the release of NO during the oxidative metabolism of hydroxyurea may explain its effects. An EPR study demonstrated that chemical oxidation of hydroxyurea with hydrogen peroxide or copper(II) sulfate in aqueous dimethyl sulfoxide produces NO and nitroxyl, the electron reduced form of NO [110]. Biological oxidants, such as iron- or copper-containing enzymes and proteins also convert hydroxyurea to NO [111]. Moreover, horseradish peroxidase catalyzes the formation of NO from hydroxyurea in the presence of hydrogen peroxide [112]. Huang et al. synthesized a variety of hydroxyurea derivatives and demonstrated that the structure of the hydroxyurea

10.9 Summary

derivative directly controls both the ability of the compound to generate NO and iron nitrosyl hemoglobin (HbNO) and the rate of NO release [113]. The development of new hydroxyurea-based NO donors may be a benefit in the treatment of sickle cell disease and cancers.

10.9

Summary

Nitric oxide (NO), a free radical, is a ubiquitous signaling molecule that is involved in both basic physiological functions and various pathological conditions. NO is produced by three distinct NO synthase (NOS) isoforms, which play distinct roles in a variety of tissues. The up/down regulation of NO release in pathological conditions is a target for drug development. Because of the complexity of the actions of NO from the various isoforms of NOS, the development of isoform-specific or cellselective inhibitors and substrates would be desirable. In the early days, l-arginine analogues such as l-NMA and l-NNA were studied and used as NOS inhibitors. Since l-arginine is regarded as a derivative of guanidine, various guanidine derivatives, such as aminoguanidine, N-alkyl-N′-hydroxyguanidines and N-alkyl-S-isothioureas have been developed as NOS inhibitors and NOS substrates. The focus of this chapter is on the length and structure of the alkyl chains of N-alkyl-N′-hydroxyguanidines and N-alkyl-S-isothioureas suitable for binding to the active site in NOS isoforms. Several reports indicate that when the alkyl substituents are too small, too large or too bulky, the ability as an NOS substrate and as an NOS inhibitor decreases significantly. X-ray crystal structure analyses of the active site in NOS isoforms have made it easy to develop new drugs for targeting the active site. In addition, compounds targeting the specific co-factor requirement of NOS isoforms, and compounds regulating the NOS gene expression have also been developed for the up/down control of NO formation. NO production by a NOS-independent pathway, including the oxidation of hydroxyurea and oxime by cytochrome P450 and peroxidase, or super oxide anion radical, represents a new approach for the regulation of NO formation. The control of NO formation at will by inhibitors or substrates specific for each NOS isoform promises to contribute to a further understanding of the NOS mechanism and therapy for various diseases that are associated with NO.

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Jamal, J., Yang, W., Xian, M, Cai, T., Wen, E. Z., Jia, Q., Wang, P. G., Poulos, T. L., The novel binding mode of N-alkyl-N’-hydroxyguanidine to neuronal nitric oxide synthase provides mechanistic insights into NO biosynthesis, Biochemistry 41 (2002), 13868–13875 Szabo, C., Southan, G. J., Thiemermann, C., Beneficial effects and improved survival in rodent models of septic shock with S-methylisothiourea sulfate, a potent and selective inhibitor of inducible nitric oxide synthase, Proc. Natl. Acad. Sci. USA 91 (1994), p. 12472–12476 Thiemermann, C., Ruetten, H., Wu, C. C., Vane, J. R., The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase, Br. J. Pharmacol. 116 (1995), p. 2845–2851 Tracey, W. R., Nakane, M., Basha, F., Carter, G., In vivo pharmacological evaluation of two novel type II (inducible) nitric oxide synthase inhibitors, Can. J. Physiol. Pharmacol. 73 (1995), p. 665–669 Garvey, E. P., Oplinger, J. A., Furfine, E. S., Kiff, R. J., Laszlo, F., Whittle, B. J., Knowles, R. G., 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo, J. Biol. Chem. 272 (1997), p. 4959–4963 Southan, G. J., Szabo, C., Connor, M. P., Salzman, A. L., Thiemermann, C., Amidines are potent inhibitors of nitric oxide synthases: preferential inhibition of the inducible isoform, Eur. J. Pharmacol. 291 (1995), p. 311–318 Salerno, L., Sorrenti, V., Di Giacomo, C., Romeo, G., Siracusa, M. A., Progress in the development of selective nitric oxide synthase (NOS) inhibitors, Curr. Pharm. Des. 8 (2002), p. 177–200 Ogawa, D., Shikata, K., Matsuda, M., Okada, S., Usui, H., Wada, J., Taniguchi, N., Makino, H.,

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Particular ability of liver P450s3A to catalyze the oxidation of N-omega-hydroxyarginine to citrulline and nitrogen oxides and occurrence in NO synthases of a sequence very similar to the heme-binding sequence in P450s, Biochem. Biophys. Res. Commun. 192 (1993), p. 53–60 White, K. A., Marletta, M. A., Nitric oxide synthase is a cytochrome P-450 type hemoprotein, Biochemistry 31 (1992), p. 6627–6631 McMillan, K., Bredt, D. S., Hirsch, D. J., Snyder, S. H., Clark, J. E., Masters, B. S., Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide, Proc. Natl. Acad. Sci. USA 89 (1992), p. 11141–11145 Klatt, P., Schmidt, K., Mayer, B., Brain nitric oxide synthase is a haemoprotein, Biochem. J. 288 (1992), p. 15–17 Rogers, N. E., Ignarro, L. J., Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from l-arginine, Biochem. Biophys. Res. Commun. 189 (1992), p. 242–249 Rogerson, T. D., Wilkinson, C. F., Hetarski, K., Steric factors in the inhibitory interaction of imidazoles with microsomal enzymes, Biochem. Pharmacol. 26 (1977), p. 1039–1042 Klemm, P., Hecker, M., Stockhausen, H., Wu, C. C., Thiemermann, C., Inhibition by N-acetyl-5-hydroxytryptamine of nitric oxide synthase expression in cultured cells and in the anaesthetized rat, Br. J. Pharmacol. 115 (1995), p. 1175–1181 Fujiwara, N., Okado, A., Seo, H. G., Fujii, J., Kondo, K., Taniguchi, N., Quinazoline derivatives suppress nitric oxide production by macrophages through inhibition of NOS II gene expression, FEBS Lett. 395 (1996), p. 299–303 Andronik-Lion, V., Boucher, J. L., Delaforge, M., Henry, Y., Mansuy, D., Formation of nitric oxide by cytochrome P450-catalyzed oxidation of aromatic amidoximes, Biochem.

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Part 3 Clinical Applications of NO Donors

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Nitric Oxide Donors in Cardiovascular Disease Martin Feelisch, Joseph Loscalzo 11.1

Introduction

Nitric oxide donors comprise a heterogeneous group of different chemical classes of compounds that either decompose spontaneously or are metabolized in cells and tissues to generate nitric oxide (NO). As diverse as the chemistries of the individual agents and the pathways that lead to NO formation from them are, so are the differences in their pharmacodynamic, pharmacokinetic, and toxicological properties. Several extensive reviews on this topic are available in the literature [1], and some of these aspects are dealt with in other chapters of this volume. A common feature of all of these compounds is that they can relax isolated blood vessels in vitro (hence, the older designation, “nitrovasodilators”) and, depending on their mode and rate of biotransformation, are principally capable of enhancing blood flow and lowering blood pressure in vivo. Only a select few compounds are in clinical use today, however, and all these drugs had been introduced into medical practice long before the discovery of NO as a biological signaling molecule. The present chapter is designed to give a brief overview of the history as well as the current use of NO donors in cardiovascular disease, including a brief account of the pharmacological profile and pathways of biotransformation of the major NO-generating drugs currently in use.

11.2

Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present

Nitroglycerin (NTG) as the prototypic NO donor and longer-acting organic nitrates, such as isosorbide dinitrate (ISDN) and isosorbide-5-mononitrate (IS-5N), have been used for the treatment of coronary atherothrombotic disease and its complications (principally, heart failure) for many decades. These agents are primarily used for the prophylaxis and treatment of myocardial ischemia in patients with coronary artery disease, and are highly effective in relieving acute attacks of angina pectoris. Nitroglycerin itself was first proposed for the treatment of angina pectoris by William

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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Murrell, who reported in 1879 that a one percent solution of the drug administered sublingually relieved angina and prevented attacks thereafter [2]. Nitroglycerin has an interesting history and is one of the few medications in current use that was discovered before the twentieth century. It is distinguished from other drugs by the fact that it was adopted by allopathic physicians from homeopathic physicians [3, 4]. Shortly after its first synthesis by the Italian scientist, Ascanio Sobrero, in 1846, Constantine Hering, a leading homeopathic physician in Germany, began exploring the possible therapeutic value of this powerful explosive by systematically investigating its effects in healthy individuals. These so-called homeopathic “provings” were precursors of present-day approaches to screening compounds for toxic and potential therapeutic effects, gradually replacing empiricism. Hering’s interest in nitroglycerin originated from Sobrero’s earlier observation that the substance produced a throbbing headache when placed on the tongue. After confirmation of this effect in volunteers, which in some individuals was achieved with less than 1/300th of a drop, and consistent with the homeopathy doctrine, simila similibus curantur (like cures like), nitroglycerin was advocated for the treatment of headache. Although Hering also shared with the medical community his observation that nitroglycerin affected the pulse, even when administered in very small quantities (suggesting a direct effect on the heart), and the compound was further characterized by a number of physiologists, it neither attracted much interest by allopathic physicians nor was it proposed for use as an antianginal agent until a related compound, amyl nitrite, was introduced in 1867 for the treatment of angina pectoris by the British physician, Thomas Lauder Brunton [5]. Brunton himself suffered from frequent attacks of angina and tested numerous compounds until he found, apparently without knowledge of nitroglycerin, that inhalation of amyl nitrite was effective in relieving his symptoms. His motivation to test this particular compound arose from investigations by Guthrie showing that amyl nitrite lowered blood pressure, and from the contemporary concept that angina was due to high arterial tension secondary to increased vasomotor tone. Interestingly, Brunton went on to demonstrate in animal experiments that nitroglycerin also lowered blood pressure, but never proposed it as a remedy for angina. The similarity of the pharmacodynamic profile of NTG with that of amyl nitrite led Murrell to investigate the former in his patients, and provided the basis for nitrovasodilator therapy of angina pectoris, the treatment of which at the time included venesection, digitalis, opium, and brandy. In parallel with these developments in medicine, the explosive properties of undiluted nitroglycerin made it a compound of prime interest for military use, as well as for the mining industry. The spontaneous explosion hazard posed a serious danger for its production, transport, and use, until the Swedish entrepreneur, Alfred Nobel, found a solution to this problem by adsorbing nitroglycerin onto porous silica, creating an easily-handled solid explosive he called dynamite, that could be produced comparatively safely in factories. By an ironic twist of fate, Nobel developed angina towards the end of his life and refused to be treated with the very substance that had brought him to so much wealth. Although control of the symptoms of acute angina pectoris had become possible with amyl nitrite and NTG, their short duration of action did not allow prophylactic

11.2 Clinical Cardiovascular Applications of NO Donor Therapy – Past and Present

treatment. This problem was addressed with the discovery of long-acting nitrates and the development of slow-release formulations and transdermal therapeutic systems. Pentaerythrityl tetranitrate (PETN) and related nitrate esters were synthesized in the 1930s and pharmacologically characterized in the following years [6], but did not gain much interest until recently (vide infra). In the early 1930s, 10 years after the discovery of insulin, several research groups intensively searched for carbohydrate analogs that were metabolized independent of insulin for use as a substitute diet for diabetics. At that time, John Krantz at the University of Maryland School of Medicine started a large-scale research program on sugar alcohols and their anhydrides, which fulfilled this criterion. One of the compounds he discovered was isosorbide, which was used as an osmotic diuretic initially. As an extension of this work, many of these polyalcohols were converted to their nitrate esters and investigated for their vasodilating properties. In the course of these studies, Krantz and colleagues discovered the long-lasting vasodilator properties of the dinitrated derivative of isosorbide and published their findings in 1939 [7]. Subsequent Swedish studies in the late 1940s demonstrated the prophylactic use of ISDN as an antianginal drug and its prolonged duration of action after oral administration; it was first marketed in Sweden in 1947 for this purpose. Independently, and unaware of these achievements, chemists of an American company resumed nitrate research work on an improved synthesis of ISDN based on Krantz’s data of the 1930s, and introduced the drug to the US market in 1959. They were dismayed to discover that ISDN had already been on the market in Scandinavia for 12 years. Soon thereafter, ISDN became available worldwide as the first long-acting, prophylactic treatment of angina pectoris. The popularity of organic nitrates on both sides of the Atlantic abruptly decreased after reports by Needleman’s group in the late 1960s showing that these compounds undergo rapid degradation in the liver, and that the parent drug could not be identified in the blood even shortly after ingestion, suggesting that nitrates were likely to be ineffective when given orally. This hypothesis clearly contradicted a large body of clinical evidence that nitrates were effective. Some years later, using more sensitive and reliable analytical methods, this interpretation was shown to be incorrect, and the clinical utility of long-acting, oral organic nitrate derivatives affirmed. In 1967, one of the pharmacodynamically active metabolites of ISDN, IS-5N, was shown to be formed in vivo, and shortly thereafter it was introduced as a novel long-acting NO donor with improved bioavailability. In addition to the treatment of angina pectoris, organic nitrates are also quite useful in patients with acute coronary syndromes. In unstable angina, intravenous nitroglycerin can relieve ischemic pain effectively, while in patients with acute myocardial infarction, it is often effective. The benefit of organic nitrates in acute coronary syndromes is not only a result of their hemodynamic benefits, but also, in part, a consequence of their antiplatelet effects [8]. This important antithrombotic action of organic nitrates is mediated by the NO-dependent activation of platelet guanylyl cyclase and impairment of intraplatelet calcium flux [9]. The limiting determinant of efficacy, especially in patients with acute myocardial infarction, is hypotension, which can complicate the hemodynamic consequences of the infarction itself, thereby offsetting the potential benefits of the improved coronary perfusion.

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More recently, some controversy over the use of NO-donors in patients with acute myocardial infarction has arisen based on two clinical trials, GISSI-3 [10] and ISIS-4 [11]. These trials investigated the adjunctive use of nitrate therapy with reperfusion therapy by thrombolysis in patients with acute myocardial infarction. The design of these trials was predicated on a meta-analysis of several small trials suggesting that intravenous nitroglycerin or oral nitrates reduced short-term mortality in infarct patients [12]. Both trials showed trends in favor of nitrate therapy; however, neither reached statistical significance. Of note, approximately 60% of patients in both placebo groups received open label nitrates at the discretion of the treating physician, rendering the final results significantly confounded by this therapeutic bias. Two other important groups of patients with cardiovascular diseases often benefit from NO donor therapies: those with hypertension and those with heart failure. Patients with hypertension, especially those with hypertensive emergencies, can often derive acute control of blood pressure from the judicious intravenous administration of the NO donor sodium nitroprusside (SNP). This compound, first reported by the Scottish chemist, Lyon Playfair, in 1846, is an inorganic complex comprising five cyanide anions and one NO molecule attached to a central iron atom. Its in vivo activity is very short-lived, ending a few seconds after the infusion is terminated, which makes it an ideal compound for controlled hypotension in hypertensive crises or in patients undergoing neurovascular surgery. Although the blood pressure-lowering effects of ISDN in hypertensive patients had been demonstrated as early as 1946, chronic administration of currently available long-acting oral NO donors to patients with chronic essential hypertension is not very effective for blood pressure control, except in cases of myocardial ischemia complicated by hypertension. The use of NO donors for the treatment of heart failure, a use originally suggested by Brunton at the end of the 19th century, has been widely recognized, beginning with the work of Johnson and Hale in the 1950s followed by the elegant work of Cohn and Franciosa in the 1970s in patients with acute severe congestive heart failure or frank pulmonary edema [13]. As a result of this work, nitroglycerin administered in combination with dopamine became an accepted treatment for these patients, substituted only many years later by more selective agents, including dobutamine and, more recently, nesiritide. These early observations in acutely ill heart failure patients were followed by the VHEFT-1 study, which compared ISDN and hydralazine with prazosin or with placebo in patients with chronic congestive heart failure [14]. In this prospective study, the ISDN-containing arm showed a clear benefit over treatment with prazosin or placebo.

11.3

Pharmacological Cardiovascular Mechanism of Action of NO Donors

The pharmacological mechanisms of action of NO donors that contribute to their benefit in coronary artery disease, congestive heart failure, and hypertension are listed in Table 11.1. These actions can be grouped into five categories: vasodilation, decrease in myocardial oxygen consumption, improvement in hemodynamic performance,

11.3 Pharmacological Cardiovascular Mechanism of Action of NO Donors Tab. 11.1: Mechanisms of action of NO donors in cardiovascular disease.

Disorder Stable angina pectoris

Unstable angina pectoris Acute myocardial infarction Congestive heart failure Systolic dysfunction

Diastolic dysfunction

Hypertension

Mechanism Decreased myocardial oxygen consumption –decreased LV end-diastolic dimension –decreased LV filling pressure –decreased LV systolic pressure –decreased PVR Increased coronary blood flow –epicardial coronary artery dilation –stenotic segment dilation –coronary collateral vessel dilation –increased subendocardial perfusion As above Antithrombotic effects As above Antithrombotic effects Improved hemodynamic performance –decreased end-diastolic dimension –decrease filling pressure –decreased systolic pressure –decreased SVR –decreased mitral regurgitation Arterial vasodilation Improved hemodynamic performance –as above –improved lusitropy Arterial vasodilation Altered hemodynamic performance –decreased filling pressure –decreased systolic pressure –decreased SVR

increase in myocardial blood flow, and antithrombotic effects [9, 15, 16]. Some of the elements within these categories overlap; however, they are detailed in the table for completeness. The hemodynamic and antianginal activities of organic nitrates are predominantly mediated by vasodilation of capacitance veins and conductance arteries, i.e., by a peripheral effect rather than by direct coronary dilator action. The reason for the preferential venodilator effect of nitrates is unclear, but may include differences in smooth muscle sensitivity and/or efficacy of biotransformation to NO. Alternatively, metabolically competent enzymes in arterial and venous smooth muscle cells may be inhibited to a different degree by endogenously produced NO. Since venous endothelial cells produce less NO than their arterial counterparts, nitrates may be more effectively metabolized to NO in the venous vascular wall. By dilating capacitance veins, preload is reduced, leading to a reduction in end-diastolic ventricular volume. These changes lead to a lowering of myocardial oxygen requirements and a favorable redistribution of blood flow in the heart with an increase in subendocardial

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myocardial perfusion. Another possible explanation for the blood flow redistribution across the left ventricular wall is that organic nitrates, unlike other NO donors, preferentially dilate large coronary arteries. To what extent this effect contributes to the effectiveness of these drugs in stable angina is unclear, but it is undoubtedly relevant to the relief of coronary vasospasm in patients with Prinzmetal angina. By dilating systemic conductance arteries, together with reducing left ventricular enddiastolic volume, afterload (the resistance against which the heart has to pump blood into the periphery) is decreased, which is another key determinant of myocardial oxygen consumption. Another important effect of organic nitrates is dilation of coronary collateral vessels, which results in improved perfusion of ischemic myocardium. Importantly, organic nitrates do not affect coronary resistance vessels (presumably owing to a lack of biotransformation to NO at this site), thereby minimizing the risk of myocardial ischemia from a coronary “steal” phenomenon (“luxury” perfusion of adequately perfused myocardium at the expense of hypoperfused segments). Amyl nitrite and molsidomine produce hemodynamic effects similar to those of organic nitrates, although headaches are less frequent. Sodium nitroprusside has a more balanced effect on the arterial and venous circulation. In contrast to organic nitrates, sodium nitroprusside also dilates small resistance vessels, which accounts for its potent hypotensive effects and for the potential for coronary steal in patients with active myocardial ischemia.

11.4

Clinically Available NO Donors: Structures and Mechanism of Action

Clinically available NO donors approved for use in the U.S. in patients with cardiovascular disease include nitroglycerin, ISDN, IS-5N, amyl nitrite, and SNP. Pentaerythrityltetranitrate (PETN) has been approved for use in the U.S. for many years, but has been largely replaced by ISDN and IS-5N. Nicorandil and molsidomine [(which is converted to the active moiety, 3-morpholinosydnonimine (linsidomine, SIN-1), in vivo)] are not approved for use in the U.S., but, like PETN, are available abroad. The chemical structures of these agents are given in Figure 11.1. The formulations of each of these vary, and include oral, sublingual, topical (ointment), transdermal (patch), buccal, and intravenous preparations. While the hemodynamic profiles of different organic nitrates are very similar, there are marked differences in the pharmacokinetic properties of the individual compounds. All nitrate esters are prodrugs that must be biodegraded to achieve the desired therapeutic effect. Biotransformation essentially requires enzymatically catalyzed denitration and reduction, with the consequent generation of nitric oxide. The partially denitrated metabolites remain pharmacologically active, albeit of lower potency compared to the parent molecules; and together with the fully denitrated alcohols, are excreted either unchanged or as glucuronide or sulfate conjugates. Despite intense research carried out for more than a century, the precise biochemical and molecular mechanisms by which organic nitrates are metabolized remain controversial. Sulfhydryl groups are probably required, either in the active center of the

11.4 Clinically Available NO Donors: Structures and Mechanism of Action CH2

O

NO2

CH

O

NO2

CH2

O

NO2

ONO2

O

ONO2

O

O

O OH

ONO2

Nitroglycerin

Isosorbide dinitrate

Isosorbide-5-mononitrate

_

CN CH3

2Na

CN Fe CN

+ CN NO

CH CH2 CH2 ONO2 CH3

2

CN

Amyl nitrite

Sodium nitroprusside

NO2 O CH2 NO2 O CH2

C

CH2 O NO2

CH2 O NO2 Pentaerythrityl tetranitrate O O ONO2 NH

N N O

N N N

N

O

N 1

Nicorandil

Mosidomine

Fig. 11.1 Chemical structures of common NO donors.

nitrate metabolizing enzyme(s), as reducing equivalents or as cofactors [17, 18]. Interestingly, virtually all thiol compounds can facilitate conversion of organic nitrates to nitrite, but only a select few concomitantly generate NO [19, 20]. The reason for this selectivity is not understood, but may offer important mechanistic clues as to the enzymatic pathway of NO formation. Thionitrates (S-nitrothiols; RSNO2 ) are thought to be the principal reaction intermediates that give rise to either nitrite or

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nitrite and NO [21]. Since Hay’s original observations on the metabolism of nitroglycerin in blood at the end of the 19th century [22], we know that organic nitrates are converted to nitrite. Inorganic nitrite and nitrate are, however, unlikely mediators of vasorelaxation, as equimolar amounts of nitrite or the fully hydrolyzed organic nitrate injected into blood do not elicit a comparable degree of vasodilation. It is, thus, generally believed that it is the NO produced during organic nitrate biotransformation that accounts for the vasorelaxant effect, although this view has recently been questioned [23, 24]. Very recent work suggests that (in ischemic tissue) nitrite can react with deoxyhemoglobin to generate NO [25]. Regardless of the nature of the active metabolite involved, biotransformation occurs in both vascular smooth muscle and endothelial cells, but is not limited to the vascular wall. In fact, metabolism of organic nitrates occurs in all tissues, with particularly high prevalence in the liver and the intestine [26]. Such extravascular metabolic events are generally believed to contribute only to inactivation (the so-called “first-pass” metabolism) of organic nitrates, but this view may not be entirely correct as the metabolism at these sites can conceivably generate longer-lived NO-adducts that may re-enter the systemic circulation to exert vascular effects distal to their site of generation. Many different enzyme systems have been proposed to be involved in organic nitrate metabolism, including certain cytochrome P450 and glutathione S-transferase isoforms, and other glutathione and NAD(P)H-dependent but poorly characterized enzymatic activities [27–29]. Nitrates may also be metabolized by xanthine oxidoreductase, although this pathway is likely to be limited to hypoxic situations as enzyme activity is inhibited by oxygen [30]. In addition to other nonspecific esterase activities, several bacterial enzymes with amino acid sequence homology to the “Old Yellow Enzyme” (the first flavin-dependent enzyme identified in yeast), as well as fungal and plant enzymes, are capable of nitrate ester degradation, which plays an important role in the removal of contaminant organic nitrates from wastewater in the industrial production process. In addition to these enzymatic pathways, organic nitrates may undergo non-enzymatic metabolism by reacting with thiol-containing biomolecules, including cysteine, glutathione, and sulfhydryl-bearing proteins, such as albumin. None of these pathways, however, has unequivocally been demonstrated to play a key role in the bioactivation of organic nitrates in the vasculature. Many of the earlier investigations in this area suffer from a lack of distinction between organic nitrate breakdown (to nitrite/nitrate and the denitrated metabolites) and activation (to NO) in that only a decrease in concentration of the parent compound or an increase in nitrite/nitrate or denitrated metabolites was measured, without direct assessment of NO and/or cGMP formation. The significance of those early studies for the bioactivation of organic nitrates, therefore, remains unclear. Other studies carried out in cultured cells or isolated tissues of non-vascular origin are equally difficult to interpret. For example, recent evidence suggests that mitochondrial aldehyde dehydrogenase may play a role in the biotransformation of nitroglycerin [24] and in the development of tolerance to organic nitrates [31], although this hypothesis remains controversial at the current time [32]. Thus, the identity of the enzyme(s) involved in the bioactivation of organic nitrates remains an unresolved issue [29].

11.5 Nitrate Tolerance

Although amyl nitrite was the first chemical entity that was specifically advocated for the treatment of angina pectoris, 135 years later we still know little about its mode of action at the molecular level. Considering the relatively high chemical reactivity and nitrosating potential of nitrite esters, which form the basis for their use in preparative organic chemistry, and their rapid reaction with nucleophiles, such as thiols, it is likely that S-nitrosothiols are active intermediates involved in blood vessel relaxation [18, 33]. In view of the high intracellular concentrations of reduced glutathione in mammalian tissues, a substantial portion of amyl nitrite that enters the cell may undergo non-enzymatic reactions. Whether or not their interaction with reactive amino moieties also produces potentially harmful N-nitrosamines is unknown. Although a full characterization of the enzymatic pathways involved in generating NO from organic nitrites and nitrates is lacking, it appears that their metabolic routes in the vasculature are distinct [34]. Whereas the majority of enzymatic breakdown of organic nitrites seems to occur in the cytosol, that of organic nitrates is associated with the membrane fraction. Owing to the high volatility of most simple alkyl nitrites, these compounds have been increasingly used as recreational drugs. In addition to prolonged hypotension, severe methemoglobinemia is a common problem observed after misuse of amyl nitrite, which can lead to acute hemolytic anemia. Despite intense study of the chemical reactivity of the inorganic NO donor SNP with a number of electrophiles and nucleophiles (in particular thiols), the mechanism of NO release from this drug also remains incompletely understood. In biological systems, both enzymatic and non-enzymatic pathways appear to be involved [28]. Nitric oxide release is thought to be preceded by a one-electron reduction step followed by release of cyanide, and an inner-sphere charge transfer reaction between the nitrosonium ion (NO+ ) and the ferrous iron (Fe2+ ). Upon addition of SNP to tissues, formation of iron nitrosyl complexes, which are in equilibrium with S-nitrosothiols, has been observed. A membrane-bound enzyme may be involved in the generation of NO from SNP in vascular tissue [35], but the exact nature of this reducing activity is unknown.

11.5

Nitrate Tolerance

Tolerance to nitrates is defined as the reduction in hemodynamic effect or the requirement for higher doses to achieve a persistent effect with continuous use in the face of constant plasma concentrations [15]. Nitrate tolerance was first described for nitroglycerin in 1888 [36]; it occurs with all organic nitrates, albeit to different extents. For reasons that are not understood, PETN appears to be the least susceptible to the development of tolerance. No, or much less, tolerance is observed with nitrite esters, such as amyl nitrite [37], molsidomine, and sodium nitroprusside. Earlier investigations suggested that a depletion of intracellular thiols is involved in tolerance development [17], but this has not been substantiated in later studies [38, 39]. As with organic nitrate bioactivation, the precise mechanism(s) involved in nitrate tolerance remain(s) unknown, but it is likely to be complex and multifactorial. Two principal

293

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mechanisms have been proposed: impaired biotransformation to NO and increased endothelial generation of reactive oxygen species such as superoxide anions. The sources of the latter likely include NAD(P)H oxidase, nitric oxide synthases, xanthine oxidases, and enzymes of the mitochondrial respiratory chain [40]. Interestingly, hydralazine may potentiate the activity of NO donors by virtue of its ability to inhibit NAD(P)H oxidases [41], the most important source of which, from the standpoint of tolerance, is the endothelial cell [42]. The increased formation of superoxide is thought to be secondary to an increase in vascular angiotensin II following activation of the renin-angiotensin system. Superoxide anion can, in turn, oxidize tetrahydrobiopterin, an essential cofactor for NO generation by nitric oxide synthases; a deficiency of this cofactor leads to so-called “uncoupling” of the enzyme, converting it from a source of NO to a source of superoxide anion. Other potential mechanisms include intracellular l-arginine depletion, also leading to an uncoupling of endothelial nitric oxide synthase, upregulation of phosphodiesterase activity, degradation of certain cytochrome P450 isoforms, plasma volume expansion, and neurohormonal activation leading to loss of the tolerance-dependent sympatho-inhibitory mechanism, blunting the response to the NO donor. Some of the confusion in this area arises from an unfortunate lack of clear distinction between the different circumstances under which a reduction in nitrate efficacy is observed. In this respect it is worth noting that the conditions induced by the bathing of isolated tissue with suprapharmacological concentrations of an organic nitrate have little to do with the clinical situation. As pointed out above, in vivo tolerance is multifactorial and, therefore, cannot be readily mimicked by biochemical manipulations of isolated vascular tissue. It is possible that the contribution of each of the individual pathways involved in nitrate tolerance differs, depending on the hemodynamic situation and the particular dose used, which would explain the high degree of variability observed with combination therapies aimed at preventing or overcoming nitrate tolerance. The success of the measures taken to counteract the development of tolerance depends critically on our understanding of the mechanism(s) involved. Of note, nitrate tolerance is often assessed by measurement of surrogate parameters, such as pulmonary capillary wedge pressure, blood pressure, or finger pulse plethysmographic parameters, rather than hard, clinically relevant endpoints, and the relevance of those surrogates for the overall antiischemic effects is not very clear. Another issue is that the extent and rate of development of tolerance may differ substantially between tissues/target organs. This point is exemplified by earlier observations in munitions workers exposed to high concentrations of nitroglycerin vapors, as well as clinical results from patients on nitrate therapy in whom the nitrate headache typically disappears over the course of 2–3 days, while hypotensive, antianginal, and antiplatelet effects persist in most cases. This issue raises a general question as to the clinical significance of nitrate tolerance, which, in general, appears to be more of an issue in Europe than in the U.S. Attempts to counteract tolerance development include the use of thiols such as N-acetylcysteine, antioxidants such as vitamin C and vitamin E, and angiotensinconverting enzyme inhibitors or angiotensin II receptor antagonists. Other approaches to decreasing the development of tolerance include intermittent therapy

11.6 Is Nitrate Therapy Associated with Adverse Vascular Effects?

with the NO donor (using a nitrate-free period every 24 hours), the use of supplemental l-arginine [43], combination therapy with hydralazine [41], and the use of folate [42, 44]. Nitrate-free intervals, while helpful in some cases, run the risk of creating the problem of inadequate antianginal protection of the patient. Simply switching to another nitrate does not resolve the problem because of marked cross-tolerance between different nitrates.

11.6

Is Nitrate Therapy Associated with Adverse Vascular Effects?

Organic nitrates have a low incidence of unwanted side-effects. Those effects known to occur more frequently, such as headache and orthostatic hypotension, are consequences of the vascular actions of NO and, for the most part, minor. Often, tolerance to those unwanted effects develops faster than that to the principal pharmacodynamic effect, and appropriate dose adjustments may alleviate the problem. Despite an impeccable safety record in hundreds of millions of patients over the course of nearly 130 years of therapeutic use, nitrate therapy has recently been suspected to affect endothelial function adversely [45], and to be associated with increased oxidative stress and impaired mitochondrial function [46]. In view of the potent antioxidant properties of NO, this conclusion at first appears counterintuitive, but may be a consequence of enhanced angiotensin II levels and is likely to occur with any vasodilator. The principal difference with NO donors is that the interaction of the NO with superoxide can lead to the formation of the potent oxidant peroxynitrite; however, neither tissue markers of peroxynitrite nor cardiac mechanical function appear to be affected during long-term nitroglycerin treatment, suggesting that organic nitrate therapy does not result in oxidative damage of the heart [47]. Moreover, recent investigations from our own laboratory demonstrate that nitrate metabolism is associated with a marked increase in the level of S-nitrosothiols, compounds known to act as storage forms of NO, conceivably conferring an additional level of protection.

11.7

Conclusions

Similar to other popular drugs such as aspirin, organic nitrates would probably not have overcome today’s regulatory hurdles to enter the clinical arena considering, for example, their mutagenic potential, the development of tolerance, and their almost instantaneous side-effects (hypotension, severe headache). These potential problems are direct consequences of the production of the endogenous messenger molecule NO, but either do not represent a concern in vivo or can be controlled by careful dose titration and choice of the optimal formulation. Sodium nitroprusside suffers from the problem of cyanide accumulation and associated toxicity during prolonged infusion. Molsidomine has been off the market in Europe for some time owing to toxicological concerns (nasopharyngeal carcinoma in rats) that arose from an effect

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later shown to be specific to rodents. Most NO donors in clinical use today were introduced into medicine in an era well before governmental regulation became very restrictive. In fact, nitroglycerin and amyl nitrite were introduced to medicine based on empiric observation without scientific proof of either mode of action or efficacy. Today, the contributions of Hering and his followers, demonstrating that a powerful explosive can be safely administered to humans, have been largely forgotten. Were it not for the peculiar doctrines of homeopathy, nitroglycerin would probably never have been proposed as a remedy for anything. In the last 15 years, safety concerns about NO-generating compounds in general, and the complexity of NO action and metabolism in particular, limited the pharmaceutical industry’s interest in developing new NO-donors. Nevertheless, some progress has been made in the last decade, in particular with “NO-enhanced medicines”, i.e., combination molecules of established drugs with an NO-releasing moiety (either an S-nitrosothiol or an organic nitrate). Several of these compounds show promising pharmacological profiles with clearly reduced untoward side-effects, but none of the lead compounds under development has been approved for clinical use at the time of this writing. An improved understanding of the biological chemistry of NO in recent years and the availability of specific biomarkers for NO in tissues are likely to have a positive impact on the development of selective and effective NO donors for novel cardiovascular applications in the near future.

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Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders: Clinical Status and Therapeutic Prognosis David R. Janero, David S. Garvey

12.1

Introduction

The notion that inhibitors of human platelet activity might be useful therapeutics rests upon century-old observations [1]. The eminent pathologist Virchow, in 1860, theorized that damage to the blood vessel wall triggers aggregation of blood elements and risks occlusion of the vessel lumen. Bizzozero soon thereafter linked the adhesive properties of the small, colorless corpuscles in human blood to coagulation and clot formation (thrombosis). By 1883, the colorless corpuscles were called platelets, and their defensive physiological role in restoring vascular integrity (hemostasis) became sufficiently established such that, in 1910, hemorrhagic tendency (“bleeding time”) was proposed as a clinical index of platelet number/activity. Subsequent work has provided great insight into the life cycle, physiology, and regulation of the human platelet, lately at molecular resolution. Evolving knowledge about the human platelet has accompanied an increased appreciation of the clinical significance of thromboembolic disorders, whose pathogenesis at least partly reflects an occlusive thrombus, either attached to the blood vessel wall or circulating in the vasculature (embolus). Their efficacy and safety limitations notwithstanding, anti-platelet drugs have clear clinical outcome benefits against morbidity and mortality: they reduce the incidence of nonfatal stroke or myocardial infarction in patients at risk of occlusive vascular events [2]. Recent reviews are available for background and primary references on platelet biogenesis and function in human health and disease [3, 4] and on current plateletinhibitor drugs [2, 5]. To preface consideration of the existing clinical data regarding NO donors as anti-platelet therapy, the following section relates human platelet physiology to pathological thrombus formation and the influence of nitric oxide (NO) therein.

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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12.2

Human Platelets, Thromboembolic Disorders, and NO

Human platelets are anucleate, subcellular fragments derived from megakaryocyte cytoplasm within bone marrow. Formed and released into the blood during thrombopoiesis, platelets circulate in the body for about 7–10 days as discrete entities and are replenished at the rate of ≈1011 per day. The high blood concentration (2–3×108 ml−1 ), small size (≈2–3 ìm diameter), and discoid shape of platelets ensure their efficient dissemination throughout the vasculature of the healthy human. These features and the hydrodynamic tendency of flowing blood to push platelets toward the blood vessel wall make circulating platelets highly effective monitors of the integrity of the body’s entire vascular tree [3, 6]. As a critical component of primary hemostasis, the resulting patrol system is well-engineered to fulfill its fundamental, life-saving physiological role: arrest bleeding from wounds. Since severe inherited platelet disorders in humans are very rare [7, 8], the platelet phenotype usually reflects a delicate balance between intrinsic platelet properties defined at thrombopoiesis and a stringently controlled interplay among pro- and anti-thrombotic mediators from the platelet itself, blood cells, and the blood vessel wall (particularly its endothelial lining) [3, 4, 9]. The vascular endothelium is an interface biocompatible with flowing blood. The healthy endothelial lining responds to physical and biochemical stimuli by balancing its production of smooth-muscle relaxing and contracting substances such that vascular tone and homeostasis are maintained. Likewise, the vascular endothelium contributes dynamically to the molecular control of platelet activity [10]. Healthy endothelium is inherently anti-thrombotic due to its regulated production of, predominantly, two potent, synergistic platelet inhibitors: the lipid prostacyclin (PGI2 ), an eicosanoid product of the enzymatic oxygenation of arachidonic acid, and the radical nitric oxide (NO), the gaseous product of enzymatic l-arginine (l-arg) oxidation by the constitutive endothelial nitric oxide synthase (eNOS) isoform [11]. Human platelets contain a constitutive NOS plus a NOS isoform (iNOS) induced rapidly upon platelet stimulation [12]. Although the precise in vivo contributions of various NO sources often remain elusive from clinical studies, NO from both the vascular endothelium and the platelet helps prevent thrombotic episodes and is critical to platelet quiescence and vessel patency [13, 14]. In contrast to agents that inhibit enzymatic production of platelet activators (e.g., aspirin) or bind to platelet surface receptors for agonists/adhesion molecules (e.g., GPIIb/IIIa antagonists) [2, 15], NO is a commonpathway platelet inhibitor that directly stimulates guanylate cyclase production of guanosine 3′,5′-cyclic monophosphate (cGMP), which suppresses platelet function regardless of the particular receptor-mediated agonist(s) activating the platelet [16] (Fig. 12.1). NO (and NO-derived nitrogen oxides) may also inhibit platelet function independently from the conventional, cGMP-mediated mechanism [16]. Normally, quiescent platelets freely circulate through the vasculature, reflective of the hemocompatible character of the vascular endothelium and the anti-thrombotic nature of healthy human blood vessels. Traumatic vascular damage incites a spatially and temporally coordinated platelet transformation encompassing several major, sequential phenotypic changes: platelet adhesion to subendothelial matrix components

12.2 Human Platelets, Thromboembolic Disorders, and NO

PGI2 G

PAF

R

G

ADP

R

G

R

G

Agonists / Activators

Catecholamines Thrombin Collagen

R

PI ATP

R

G

AC

+ PLP IP3 TxA2

AMP Activation protein expression (e.g., P-selectin)

+ +

[Ca++]cytosolic

storage

release

+

Ca++ stored

-

R TK

Shear

GTP

GC

+ NO

cGMP NOS

PDE

GMP

Fibrinogen binding to GPIIb/IIIa Aggregation

Thrombus formation

NO/NO donor

L-Arg

>

Ca++

Fig. 12.1 Schematic representation of human platelet activation. Platelet activators/agonists act generally through receptors R coupled to G-proteins G or kinases, such as tyrosine kinase TK. Receptor-mediated signal transduction activates phospholipases (PLP), some of which catabolize phosphoinositol (PI) to generate inositol trisphosphate (IP3 ), and others which liberate arachidonic acid substrate for eicosanoid [including thromboxane A2 (TXA2 )] production. IP3 is a positive effector, stimulating calcium (Ca++ ) release from intra-platelet stores, whereas prostayclin [prostaglandin I2 , (PGI2 )], stimulates calcium storage by binding to a specific receptor and activating adenylate cyclase (AC) to produce cAMP as second messenger. Calcium mobilization from internal platelet stores stimulates calcium entry into the platelet from the external milieu down a concentration gradient. Elevated cytosolic free calcium

Secretion/ degranulation (ADP, TxA2)

L-Arg

>

>

PDE

cAMP

stimulates the platelet, whereas a reduction in platelet cytosolic calcium decreases the level of platelet activation. Nitric oxide (NO), generated from either an exogenous NO donor or from platelet nitric oxide synthase (NOS) acting upon L-arginine (L-arg) substrate taken up from the plasma, stimulates guanylate cyclase (GC) conversion of GTP to the second messenger cGMP, which inhibits stored calcium release and external calcium entry and thus suppresses platelet activation, regardless of the agonist(s) activating the platelet. Both cAMP and cGMP are actively broken down by phosphodiesterase (PDE) enzymes to AMP and GMP, respectively. Increased platelet activity may encompass the key activation responses listed and culminate in thrombus formation. The symbols + and − denote stimulation or inhibition, respectively. Solid arrows ( ) signify enzymatic reactions; broken arrows ( ) signify effector pathways or responses.

exposed to flowing blood by the vascular insult; platelet shape change, activation, and secretion (degranulation); further platelet recruitment into the wound site; and elaboration of matrix molecules to consolidate the aggregated platelets [4, 17] (Fig. 12.2).

Key Activation Responses

R

Thromboxane

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

Fig. 12.2 Schematic depiction of the role of platelets in thrombus formation. (a) Circulating platelets are kept inactive by prostaglandin (i.e., prostacyclin, PGI2 ) and nitric oxide (NO) released by vascular endothelial cells. Endothelial cells also express CD39 on their surface, which inhibits platelet activation by converting ADP – a potent platelet agonist – to AMP. (b, c) At sites of blood vessel wall injury, platelets adhere to the exposed subendothelium through interactions between collagen, von Willebrand factor, and fibronectin and their receptors on the platelet, integrin á2â1, glycoprotein Ib-IX (GP Ib-IX), and integrin á5â1, respectively. Both thrombin and ADP induce the platelet into an active conformation. (d) The activated platelet secretes ADP, platelet-derived growth factor (PDGF), and fibrinogen from

internal storage granules (“degranulation”) and releases newly-synthesized thromboxane A2 (TXA2 ). ADP and TXA2 signal circulating platelets to become activated and change shape, starting the process of platelet recruitment into the vascular injury site. (e) Glycoprotein IIb/IIIa (GP IIb/IIIa) receptors expressed on the activated platelet surface bind fibrinogen, leading to the formation of fibrinogen bridges between the platelets (“platelet aggregation”). Aggregation and the simultaneous generation of a fibrin meshwork lead to platelet thrombus or clot formation. (f) Clot retraction then leads to formation of a stable thrombus. Figure reproduced with permission from Ref. [2]. Copyright 2002 Nature Publishing Group (http://www.nature.com).

The balance of opposing pro- and anti-platelet forces determines the overall hemostatic response. Successful hemostasis is achieved when assorted signal-transduction systems, mediators, white blood cells, and platelet receptors for agonists and adhesion molecules overcome the local resistance against platelet activation to generate

12.2 Human Platelets, Thromboembolic Disorders, and NO

a stable thrombus that stops the bleeding at the injury site and allows healing to commence [18]. Platelets cannot inherently distinguish between a wounded, hemorrhagic blood vessel that has lost its integrity and an intact, but diseased, vessel predisposed toward local platelet adhesion and activation. Not surprisingly, therefore, hemostatic platelet plug formation and pathological thrombosis share many mediators and platelet responses. The platelet regulatory functions of the human vascular endothelium are impaired by pro-inflammatory and conventional cardiovascular risk factors (including hypercholesterolemia, male gender, family history, age, obesity, smoking, diabetes, hyperhomocysteinemia) and during the course of disorders including atherosclerosis, coronary artery disease, essential hypertension, the hypertension of preganacy (preeclampsia), hemolytic uremia syndrome, and thrombotic thrombocytopenic purura [19, 20, 21]. A vasoconstrictive, pro-thrombotic environment promoting occlusive platelet aggregate formation also contributes to atrial fibrillation and the failure of synthetic vessel grafts and revascularization techniques such as endarterectomy, angioplasty, and stenting [22, 23, 24]. Perhaps the most compelling illustration of the clinical significance of pathological thrombosis is the role of platelets in precipitating most acute coronary syndromes [25, 26]. Occlusive atherosclerotic plaque in coronary arteries, while eliciting stable angina, seldom causes acute unstable angina or acute myocardial necrosis (infarction). Plaque fissure, erosion, or rupture (particularly, rupture of the plaque’s protective fibrous cap) abruptly transitions the chronic, stable state into an acute coronary syndrome. Plaque disruption exposes flowing blood to several subendothelial components adhesive to platelets. At the injury site, hemodynamic forces and a sequence of platelet {adhesion →activation →secretion →recruitment →aggregation}, reminiscent of the hemostatic response, support local formation of a thrombus composed mostly of platelet aggregates in a fibrin reticulum (Fig. 12.2). When coronary arteries are narrowed or occluded by this mechanism over a sufficient period of time, nutritive blood flow to working heart muscle becomes critically compromised (ischemia), leading to acute coronary syndromes (including unstable angina and myocardial infarction) having the potential for devastating clinical manifestations (e.g., cardiac electrical instability, sudden death). Indeed, continuing platelet activation in patients with acute ischemia/infarction is associated with adverse prognosis [27]. A similar acute thrombotic scenario dominated by the platelet in other large- and medium-sized arteries (including peripheral and carotid arteries) has been implicated in the pathogenesis of debilitating peripheral vascular disease and ischemic stroke [28, 29]. Since acute cardiovascular syndromes are the main reasons for admission to coronary care units in the United States and much of the Western world, thrombosis represents a leading cause of morbidity and mortality [30]. Dysregulation of the vascular endothelium has emerged as a critical component of most thrombotic disorders [10, 21]. Often without any anatomical sign of atherosclerosis, many cardiovascular diseases express a vasomotor abnormality termed endothelial dysfunction, indexed clinically as impaired endothelium-dependent vasodilation [31]. Although its mechanism is multifactorial, endothelial dysfunction is characterized by diminished vascular NO production and/or bioavailability [32]. The

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

resultant NO-deficient state creates an imbalance in an important platelet inhibitor relative to other endothelium-derived vasoactive factors. Attenuated platelet NO production/responsiveness and abnormal circulating and urinary biomarkers for NO and platelets are also associated with many cardiovascular diseases and often correlate with coronary risk factors [10, 31, 32]. This scenario implies that endothelial dysfunction and platelet NO resistance pathologically compromise the NO-dependent, anti-thrombotic properties of the vessel wall. Thus, the principal causes of hospitalization and death in the Western world, ranging from childhood stroke to acute coronary syndromes, share a NO-related, pro-thrombotic vasodilator dysfunction. Given the fundamental role of the human platelet in the etiology of atherothrombosis, the proven efficacy of platelet-inhibitor drugs in the prevention and treatment of cardiovascular disease is unremarkable. First-generation anti-platelet agents (aspirin, ticlopidine, and clopidogrel) and the anticoagulants heparin and warfarin reduce the risk of vascular death by about 20% and of non-fatal myocardial infarction and stroke by about 30% in patients with angina, suspected acute myocardial ischemia, or a past history of myocardial ischemia, stroke, or transient ischemic attacks [2, 27, 33, 34]. In a wide range of patients at high risk of occlusive vascular disease, chronic plateletinhibitor therapy (commonly, low-dose aspirin) offers primary protection against myocardial ischemia, stroke, and death [35, 36]. But some patients with coronary artery disease, diabetes mellitus, hypercholesterolemia, and hypertension exhibit increased platelet aggregability that is not readily inhibited by, or may even be refractory to, aspirin [34, 37]. Despite aspirin treatment in patients with acute coronary syndromes or carotid artery stenosis, persistent platelet activation is associated with adverse prognosis [37]. Second-generation anti-platelet agents, such as GPIIb/IIIa antagonists, have yet to prove their general value and safety and may require intravenous administration [2, 15]. Risks, safety issues, and efficacy limitations associated with current anti-platelet/anti-thrombotic clinical strategies complicate patient management [2, 15, 34, 37]. The inability of current drugs to stem the persistence of thrombosis as a leading cause of morbidity and mortality and ameliorate the high incidence of major ischemic cardiovascular events invites new approaches toward improved anti-platelet therapy. One of these, the use of NO donors, has great intrinsic appeal from the crucial importance of NO as a physiological anti-thrombotic agent and the increasing recognition that vascular NO insufficiency is a component of various atherothrombotic diseases (vide supra). The clinical benefits of organic nitrates are attributed mainly to their vasodilator property, effected through their denitration (bioactivation) to yield NO [38]. Since 1967, nitrovasodilators of diverse chemical structures have been known to have anti-platelet effects in vitro [39], suggesting that at least some of their therapeutic benefit in diseases associated with a heightened thrombotic risk may reflect in vivo platelet inhibition. Furthermore, the anti-platelet actions of NO are uniquely diverse: NO inhibits platelet activation, aggregation, degranulation, and recruitment and promotes platelet disaggregation [13, 40]. The following sections will summarize the salient clinical data on NO donors as anti-platelet agents, organized according to the specific NO donor studied (Table 12.1). The discussion will consider only those clinical trials in which a NO donor

12.2 Human Platelets, Thromboembolic Disorders, and NO Tab. 12.1: NO donors examined in clinical trials for anti-platelet/anti- thrombotic activity.*

NO donor

Structure O

Nitroglycerine

NO2

O

O2N

O

O2N O

NO2

H O

O H

Isosorbide dinitrate O2N O

O NO2

H O

O H

Isosorbide 2-mononitrate HO

OH

H O

O H

Isosorbide 5-mononitrate

O NO2

NO CN Fe NC CN CN

2-

NC

2Na+ .2H2O

Sodium nitroprusside O

+ N N N O

3-Morpholinosydnonimine (SIN-1) O

N

Molsidomine

* References

NH2

O O

O

HO

S-Nitroso-glutathione

N

+ N N O

O

NH

N H

S N H N

O O OH

O

are cited in the text.

was administered to human subjects and at least one purported index of platelet activity was assessed. This presentation will also encompass clinical studies that have attempted to document a prognostic outcome benefit from a known platelet inhibitory NO donor, whether or not platelet activation status was assessed. By these

305

306

12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders Tab. 12.1: (continued)

NO donor

Structure O

NH

HO

N H

NH2

l-Arginine

NH2

O O

O O

O

NO2

NCX-4016

criteria, although isolated human platelets are a convenient, homogeneous model, NO donors identified solely as in vitro inhibitors of isolated human platelets will not be dealt with e.g., [41–48] (Table 12.2). Likewise, the very limited, far from consensus, clinical data on the anti-platelet effect of NO gas e.g., [49], ingested inorganic nitrate (NO3 − ) e.g., [50], or a nitrate-rich diet [51] are not considered. It should also be noted that some NO donors have been studied in the clinic e.g., [52, 53] and are prescribed as cardiovascular medicines (e.g., pentaerithrityltetranitrate [54]) without published documentation of their potential platelet effects in a human outcome trial.

Tab. 12.2: Examples of NO donors shown to inhibit isolated human platelet activity in vitro.

NO Donor

Structure -

O

N N

DEA/NO

Reference

O N

[41] -

O

+ H 3N

O N

N N

N H

SPER/NO

O

S

[42]

CO2H

O N

NH2

N

[43]

S-NO-Captopril O HO

S

N

O

HN

S-NO-Acetyl-cysteine

O

[44]

12.3 Nitrovasodilators Tab. 12.2: (continued)

NO Donor

Structure

Reference

O HO

S

N

O

HN

S-NO-N-acetyl-penicillamine

[45]

O O

O O

HO

O NO 2

H H

21-NO-Prednisolone (NCX-1015)

H

O

[46] CN + O N

4-Phenyl-3-furoxancarbonitrile

GEA 3175

[47]

N O

Cl

+ N N N O

N O

S O

[48]

O2N O O2N O O NO2

Pentaerythrityltetranitrate

O NO2

[54]

12.3

Nitrovasodilators 12.3.1

Glyceryl Trinitrate, Nitroglycerin (GTN)

Over a century ago, empirical observation was made that organic nitrates, including glyceryl trinitrate (GTN), alleviate angina. Since then, GTN has been a mainstay therapy for angina and cardiac failure, even with the possible loss of effectiveness (tolerance) over extended dosing [55] and the risk of platelet hyperactivity in GTNtolerant patients [54]. Despite this venerable therapeutic history, the mechanism of GTN bioactivation to NO is speculative at best [38]. In 1967, some 15 years before identification of NO as a biological entity, GTN was the first nitrovasodilator shown

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

capable of inhibiting human platelet activity (i.e., aggregation) in vitro [39]. As subsequently demonstrated, the suprapharmacological concentrations required reflect the fact that (human) platelets lack a robust, intrinsic capacity for organic nitrate bioactivation to NO [56]. The 1980s saw the earliest clinical investigation of GTN’s potential to inhibit human platelet function in vivo [57–60]. Sublingual and intravenous GTN, at therapeutic doses or greater, was generally found to increase hemorrhagic tendency (i.e., prolong bleeding time) in healthy subjects and in coronary disease patients. Negligible, if any, effect of GTN administration on more specific platelet endpoints (e.g., number of circulating platelet aggregates or agonist-induced platelet aggregation in plateletrich plasma in vitro) could be demonstrated. The sole subject in these four studies who did show a marked inhibition of ex vivo platelet aggregation after one hour of high-dose GTN infusion also had the greatest hemodynamic response (i.e., bloodpressure drop). Accordingly, the prolongation of bleeding time observed in these early, uncontrolled studies likely reflected vasodilation and increased venous capacitance rather than pharmacological inhibition of platelet function [60]. Reagrdless of mechanism, the effect of therapeutic GTN on bleeding time in healthy volunteers appeared slight at best in a subsequent, controlled trial [61]. Under some conditions of blood collection and in vitro aggregometry, the inhibitory effect of GTN on agonist-induced platelet aggregation was not expressed unless exogenous thiol (e.g., N-acetyl-cysteine) was added prior to aggregometry [62]. Yet in a double-blind, randomized, controlled, cross-over trial with healthy male volunteers, transdermal GTN at doses 2–4-fold above therapeutic levels had no effect on ADP-induced platelet aggregation in whole blood, whether or not N-acetyl-cysteine had been co-administered [63]. This trial may have been confounded by the development of nitrate tolerance in its subjects. Although not conducted in a double-blind, placebo-controlled, cross-over fashion, the study of Diodati et al. represents a methodological and conceptual turning-point in the clinical investigation of GTN as an anti-platelet agent [64]. These investigators demonstrated that inhibition of human platelet aggregation by GTN is so transient and reversible that in vitro aggregometry must be conducted without delay after GTN administration. Consequently, Diodati et al. employed bedside aggregometry in whole blood, which eliminated the need for platelet-rich plasma preparation and allowed assessment of the potential anti-platelet effect of therapeutic GTN infusion in coronary artery disease patients within 30 s of phlebotomy. Under these assay conditions, GTN infusions incrementally titrated to produce no more than a 10% decrease in mean arterial blood pressure were shown to inhibit in vitro platelet aggregation by at least 50% in 8 out of 10 total subjects, regardless of platelet agonist. Notably, the anti-platelet effect was lost within 15 min post-infusion. GTN’s anti-aggregatory and hemodynamic (i.e., hypotensive) effects did not correlate, suggestive of direct platelet inhibition by GTN in vivo. Subsequent clinical studies provided additional evidence that GTN exerts antiplatelet activity in vivo and offered insight into the nature of GTN’s anti-aggregatory pharmacology. A dose–effect relationship between intravenous GTN and inhibition of platelet aggregation was uncovered in healthy male subjects, in whom plasma

12.3 Nitrovasodilators

concentrations of GTN and its metabolites correlated with the anti-aggregatory efficacy [65]. Sublingual GTN administered at a therapeutic dose to healthy subjects and to patients with stable angina and hyperaggregable platelets inhibited ADP-induced platelet aggregation by ≈35%, as evaluated within three minutes post-dosing by in vitro aggregometry in platelet-rich plasma [66]. An increase in platelet cGMP was not observed and was perhaps lost during plasma preparation. Two studies with transdermal GTN were likewise positive. In healthy subjects, an anti-platelet effect could be demonstrated by whole-blood aggregometry up to 2 h after GTN patch application, provided that a phosphodiesterase inhibitor was introduced into the blood at the time of sampling and aggregometry was carried out within 1 min of phlebotomy [67]. The lack of effect of transdermal GTN on heart rate or blood pressure in this study suggested that GTN had exerted a direct anti-platelet effect in vivo. The same dose of transdermal GTN was administered in a subsequent randomized, doubleblind, controlled trial to stable angina patients, in whom GTN was vasoactive [68]. Significant platelet inhibition was observed: in vitro whole-blood aggregation to ADP decreased by ≈25% in 70% of the patients administered GTN and in only 27% taking a placebo. In contrast, a hemodynamically effective infusion of GTN into coronary disease patients, either before or after bypass surgery, did not influence three perioperative indices of hemostasis: bleeding time, in vitro platelet adherence to glass, and in vitro clot formation [69]. More direct platelet function tests (e.g., aggregation) were not conducted. The significance of this omission is highlighted by a report that, in healthy volunteers given transdermal GTN at a hemodynamically active dose, fibrinolytic capacity was unaffected, yet ADP-induced platelet aggregation in vitro was inhibited [70]. Three subsequent GTN infusion studies were predicated on the importance of activated platelets in precipitating most acute coronary syndromes [25–27]. The first exploited the observation that rapid atrial pacing in patients with stable coronary artery disease causes platelet hyperaggregability across the coronary bed, similar to the platelet hyperresponsiveness that helps precipitate unstable angina [71]. To this intent, Diodati et al. sampled both coronary sinus and arterial blood from stable coronary disease patients at rest and up to 10 min after inception of atrial pacing, with or without a therapeutic GTN infusion causing hypotension [72]. Pacing-induced platelet activation (i.e., platelet hyperaggregability) was observed in coronary-sinus blood and was abrogated by GTN pretreatment. In another study [73], patients premedicated with aspirin (300 mg, p.o.) were administered intravenously a vasoactive GTN dose within 48 h of the onset of either acute myocardial infarction or unstable angina. Platelet activation was determined prior to GTN infusion and after GTN administration once blood pressure returned to post-infusion values as activation antigen/adhesion protein (P-selectin, GPIIb/IIIa) expression on the platelet surface. According to this virtually immediate readout, GTN infusion inhibited the platelet activation that persisted in acute coronary syndrome patients treated with aspirin. Similar acute inhibitory effects of therapeutic GTN infusion on basal and stimulated platelet adhesion-protein expression have also been documented in healthy male volunteers [74].

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Two somewhat paradoxical GTN trials remain to be considered. In one, patients within 5 days of an ischemic or hemorrhagic stroke were randomized to receive either placebo patches or a therapeutic, vasoactive dose of transdermal GTN over the ensuing 12 days [75]. Neither in vitro platelet aggregation nor platelet activation antigen expression was affected on the first or the eighth day of GTN treatment. The nitrate tolerance that developed in these subjects and their relatively late treatment after clinical presentation may help explain the negative result. In patients with non-insulin-dependent diabetes, free of cardiovascular complications, sublingual or transdermal GTN did not inhibit ADP-induced platelet aggregation in platelet-rich plasma [76]. Yet up to 12 h after dosing, GTN significantly inhibited platelet aggregation in control nondiabetics. Since antioxidants (glutathione, vitamin E) normalized the hyperaggregability of the diabetic patients’ platelets, underlying oxidative stress may have confounded GTN’s potential anti-platelet action. Can the anti-platelet properties of GTN be linked to significant prognostic impact in a positive clinical outcome study? Aside from a small, randomized, placebocontrolled trial demonstrating that sublingual GTN increased maximum walking distance in patients with peripheral vascular disease [77], two major investigations have dealt with this question. The first is a meta-analysis of the collective mortality results from seven randomized, controlled, intravenous GTN trials in patients at high mortality risk from acute myocardial infarction [78]. This analysis is quite controversial, since virtually all of the individual trials considered were insufficiently powered to detect clinical benefit, and none was designed to assess GTN’s mortality effect. Nevertheless, the meta-analysis suggested that intravenous GTN elicited an early mortality reduction of around 35% in acute myocardial infarction. The greatest impact on mortality seemed to occur during the first week of follow-up. In contrast, GISSI-3, a multi-center, randomized, placebo-controlled trial, was specifically designed and powered to assess whether early intravenous plus subsequent transdermal GTN improved cardiac function and six-week survival after acute myocardial infarction [79]. Alone or together with the ACE inhibitor lisinopril, GTN did not show any independent effect on severe ventricular dysfunction or mortality, whereas lisinopril had positive effects on both outcome measures. The negative GISSI-3 trial argues against a substantive, clinically beneficial anti-platelet effect of GTN in acute myocardial infarction, at least of the tenor that had been demonstrated for aspirin [2, 27, 33, 34]. However, the additive effect of lisinopril plus GTN in reducing mortality in the GISSI-3 trial could reflect a component of in vivo platelet inhibition by GTN. Several factors may have conspired to obscure any difference between GISSI-3 control vs. GTN-treated patients, including: a heterogeneity of response with varying infarct size; the intensive subject pre-exposure to thrombolytic therapy and aspirin; and the administration of nitrovasodilators (including intravenous GTN) to over 50% of the control subjects.

12.3 Nitrovasodilators

12.3.2

Isosorbide Dinitrate (ISDN) and Isosorbide Mononitrate (ISMN)

Along with GTN, ISDN is the organic nitrovasodilator most widely used in clinical practice for the treatment of angina, although tolerance may develop with extended dosing [55, 80]. ISDN has a longer duration of action than GTN, since its in vivo metabolites, IS-2-MN and IS-5-MN, are cleared relatively slowly [81]. By the late 1980s, it was well established that ISDN and its mononitrate metabolites, at suprapharmacological concentrations, inhibited isolated human platelet aggregation in vitro, IS-2-MN being the most potent anti-aggregatory agent of the three, and IS-5-MN, the weakest [82]. Reminiscent of the initial clinical experience on the anti-thrombotic effects of GTN, early reports constitute essentially observational studies on ISDN or ISMN infused into either healthy subjects or stable angina patients. In vitro aggregation in platelet-rich plasma was commonly used to index an in vivo platelet effect post-dosing. Despite the very weak response of isolated human platelets to ISDN or ISMN, four uncontrolled studies by DeCaterina et al. in stable angina patients demonstrated that therapeutic levels of infused ISDN, IS-5-MN, or IS-2-MN markedly inhibited subsequent adenosine diphosphate- and adrenaline-induced platelet aggregation in vitro and acutely reduced the number of circulating platelet aggregates [83–86]. An antiplatelet effect persisted up to 30 min after discontinuation of mononitrate infusion and up to 60 min after ISDN infusion. Although platelet inhibition by ISDN could be detected at hemodynamically neutral doses, the anti-platelet effects of ISDN and its mononitrate metabolites were generally dose-dependent and correlated well with the degree of blood pressure drop. At the highest doses, the platelet-inhibitory effect diminshed, presumably due to excessive vasodilation that incited a compensatory discharge of pro-aggregatory catecholamines. Indeed, catecholamine discharge may help account for the reported lack of an anti-aggregatory effect of oral IS-5-MN in stable angina patients [87]. Yet platelet sensitivity to PGI2 increased, a finding reminiscent of the correlation between PGI2 level and in vitro inhibition of platelet aggregation noted in aortic blood of ischemic heart disease patients after bolus oral ISDN administration [88]. In patients with peripheral (femoral) and coronary artery diseases, oral ISDN did not alter radiolabeled platelet deposition over the femoral atherosclerotic lesion site, but did synergize the inhibition of platelet deposition by PGE1 , a platelet-inhibitory prostaglandin [89]. These results [88, 89] implicate eicosanoid mediators in the anti-platelet activity of ISDN in vivo. Not every platelet endpoint need be sensitive to ISDN/ISMN: oral IS-5-MN administered to healthy volunteers inhibited platelet activating factor (PAF)-induced aggregation in vitro without affecting platelet secretion, plasma cGMP level, or bleeding time [90, 91]. Virtually all of the above cited studies were uncontrolled and involved acute oral or intravenous ISDN/ISMN administration followed within hours by in vitro platelet activity assessment. In contrast, Sinzinger et al. showed in an uncontrolled trial that oral ISDN (100 mg daily) administered to coronary artery disease patients over four weeks inhibited ADP-induced aggregation in platelet-rich plasma in vitro and reduced the number of circulating platelet aggregates and platelet production of thromboxane

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

B2 (TxB2 ), a vasoconstrictive platelet activator [92]. Wallen et al. reported the results of a randomized, double-blind, placebo-controlled cross-over study of 20 mg ISDN given twice daily for two weeks to ischemic heart-disease patients either at rest or after platelet aggregation induced in vivo by dynamic exercise [93]. A number of operational factors were controlled in this study to obviate potential complications from nonstandard patient compliance, diurnal variations in platelet function, and changes in subject posture that could increase plasma catecholamines and spuriously activate platelets. Although exercise potentiated platelet aggregability and secretion and ISDN had clinical effects (e.g., blood pressure lowering, headache induction), no inhibition of platelet function was seen. Three randomized, double-blind, placebo-controlled studies, one of infused ISDN in patients undergoing cardiopulmonary bypass [69], the others of ISDN [70, 94] and IS-5-MN [70] in healthy volunteers, likewise failed to show any effect of these organic nitrates on platelet function, despite significant blood pressure drops in the nitrate-treated subjects. These data make it tempting to conclude that the acute anti-platelet effects of ISDN/ISMN observed in uncontrolled studies are not readily verifiable in comparing active treatment and placebo in controlled clinical trials. Whether this difference in study design alone fully explains the contrasting efficacy findings is questionable: an uncontrolled trial documented only marginal immediate platelet inhibition by graded ISDN infusions into stable angina patients [95], whereas a randomized, placebo-controlled study involving aspirin-treated men with acute myocardial infarction demonstrated that infused ISDN reduced both in vitro platelet aggregation and adhesion [96]. An outcome-based clinical study showed that up to 16 days of transdermal ISDN treatment in four pre-eclamptic women elicited a virtually immediate and profound improvement in fetoplacental circulation [97]. The improvement might have reflected inhibition of microthrombus formation, but platelet activity was not assessed. One large, multi-center, randomized, placebo-controlled clinical trial of IS-5-MN in a wide range of low-to high-risk patients with suspected or documented acute myocardial infarction has been conducted [98]. In this so-called ISIS-4 trial, the treatment group received daily oral doses of controlled-release IS-5-MN for one month. Reminiscent of the GISSI-3 trial with GTN [79], the ISIS-4 trial offered no evidence that IS-5-MN started early in acute myocardial infarction improved survival, even in patients free of non-study nitrates, although there were clear signs (hypotension, headache) that IS-5-MN was indeed active. As with GISSI-3, most patients in the ISIS-4 trial had undergone thrombolysis. The possibility thus remains that nitrate therapy might exert a decisive anti-platelet effect in, for example, patients not eligible for thrombolysis. The ISIS-4 trial has been criticized for employing IS-5-MN, a weak NO donor [73, 82]. 12.3.3

Sodium Nitroprusside (SNP)

The nitrovasodilator sodium nitroprusside (SNP) has been used for decades to manage acute hypertensive crises and congestive heart failure complicating myocardial ischemia [99]. However, prolonged SNP administration is limited by tolerance, the

12.3 Nitrovasodilators

need for parenteral administration, and the potential toxicity of the cyanide generated upon NO release [100]. The anti-aggregatory effect of therapeutic levels of SNP on isolated human platelets was initially documented in 1974 [101]. Soon thereafter, the much greater in vitro potency of SNP as inhibitor of human platelet aggregation relative to the organic nitrovasodilators GTN and ISDN was noted [102]. This difference could most readily be rationalized by the fact that organic nitrates require bioactivation to generate NO, whereas SNP is a coordination complex that releases NO both spontaneously at physiological pH and as a consequence of tissue catabolism [103]. In uncontrolled clinical studies, Mehta and Mehta demonstrated that infused SNP titrated to produce a hemodynamic response normalized the characteristically high number of circulating platelet aggregates in heart-failure patients and dosedependently decreased (by 20–40% pre-infusion responses) ADP- or epinephrineinduced platelet aggregation in platelet-rich plasma in vitro [57, 104]. The first prospective, controlled human study of clinical doses of infused SNP on platelet function was also positive: SNP infused for blood pressure control during anesthesia prior to coronary bypass surgery acutely and dose-dependently inhibited (by up to 50%) platelet aggregation to ADP and epinephrine and concomitantly prolonged bleeding time [105]. As measured by whole-blood aggregometry in vitro, the platelet hyperaggregability induced by rapid atrial pacing in stable coronary artery disease patients was blunted acutely by therapeutic SNP infusion, whereas SNP did not affect these subjects’ platelet aggregability to ADP or thrombin prior to pacing [72]. In subjects with normal left-ventricular function undergoing elective coronary bypass surgery, a 60 min therapeutic SNP infusion prevented the up-regulation of platelet adhesion molecules up to 35–60 min post-infusion without hemodynamic effect, as documented in two randomized, prospective, placebo-controlled studies [106, 107]. Three essentially negative SNP trials must be acknowledged. In an uncontrolled trial with healthy volunteers, clinical doses of infused SNP inhibited neither platelet Pselectin expression nor ADP-induced aggregation in vitro, but did suppress epinephrine-induced aggregation up to 4 min after discontinuation of the SNP infusion [108]. This study is complicated by the fact that SNP, by markedly increasing heart rate, likely induced catecholamine release that would have primed platelet aggregation. Another study found no acute effect of therapeutic and vasoactive SNP infusion on platelet aggregation to collagen or ADP in whole blood from patients with angina, either with or without angiographic evidence of atherosclerosis [109]. A randomized, doubleblind, placebo-controlled, two-way cross-over study demonstrated that the platelet activation observed within 5 min after the start of hemodialysis was not affected by a 15 min SNP infusion delivered via the inlet of the hemodialysis device [110]. Since there was no blood-pressure effect of SNP in this study, the negative platelet result may reflect sub-optimal dosing and/or scavenging of SNP-derived NO by the erythrocyte hemoglobin in the whole-blood dialysate. Perhaps the in vivo anti-platelet effects of SNP are most likely seen when platelets are pre-activated in vivo (e.g., by plaque rupture in unstable angina patients). A meta-analysis of the collective outcome results from three randomized, controlled intravenous SNP trials suggested that intravenous SNP reduces early mortality by ≈35% in acute myocardial infarction [78]. Since each of the component trials was

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12 Nitric Oxide Donors as Anti-platelet Agents for Thromboembolic Disorders

not designed to assess SNP’s efficacy in reducing mortality, the conclusion of clinical benefit is controversial. Suggestion that SNP could have a platelet-related effect sufficient to improve clinical outcome comes from the demonstration that SNP infused during coronary bypass attenuated the associated systemic inflammatory response, an important determinant of the success of open-heart surgery [111].

12.4

Oxatriazolium NO Donors 12.4.1

Sydnonimines

The sydnonimine class of NO donors is typified by 3-morpholinosydnonimine (SIN1), which is generated from its precursor, molsidomine, mainly in the liver [112]. Molsidomine has a slower onset and longer duration of action than conventional nitrovasodilators due to its relatively slow conversion to SIN-1, whereas SIN-1 itself has a rapid onset and short duration of action [113]. NO release from SIN-1 occurs spontaneously in blood with concomitant generation of superoxide anion radical (O2 •− ) [114]. Both molsidomine and SIN-1 are vasodilators, and this effect was first exploited clinically in 1978 to alleviate angina [115]. Since molsidomine and SIN-1 do not induce tolerance or cross-tolerance with conventional nitrates, both sydnonimines received considerable attention in the 1980s for their potential to serve as tolerancefree alternatives to classic nitrovasodilators in stable angina and heart failure [113]. The overall therapeutic appeal of molsidomine and SIN-1 has since been weakened by the potential generation of the potent oxidizer peroxynitrite (ONOO− ) from SIN1 decomposition products NO and O2 •− , the frequent dosing necessitated by SIN1’s short duration of action, and concerns about the ill-understood sydnonimine mechanism of vasodilation [116]. Both molsidomine and SIN-1 have been evaluated clinically for anti-platelet effects. In a double-blind, placebo-controlled study with 12 healthy volunteers, oral administration of a 4 mg dose of molsidomine markedly attenuated platelet-activating factor (PAF)-induced platelet aggregation in vitro up to 30 min post-dosing. 24 h thereafter, molsidomine’s anti-aggregatory effect was lost [88]. In a subsequent uncontrolled study by the same group, six male and six female healthy volunteers were administered molsidomine intravenously at a dose of 60 ìg kg−1 . In eight of the ten patients evaluated, in vitro platelet activation by PAF was significantly attenuated 40 min postinfusion. Plasma levels of molsidomine, its active metabolite (SIN-1), and cGMP were measured up to 1 h following molsidomine infusion. Plasma concentrations of both molsidomine and SIN-1 dropped off rapidly after molsidomine dosing, and plasma cGMP was unchanged from pre-treatment level. The lack of a SIN-1 affect upon plasma cGMP was attributed to the target of NO activation. NO stimulates cGMP production from the intracellular (cytosolic) form of guanylate cyclase, as opposed to the membrane-bound isoform. Plasma cGMP may be a marker for the activity of the membrane-bound guanylate cyclase isoform [117].

12.4 Oxatriazolium NO Donors

A double-blind, placebo-controlled study in which a 4 mg dose of molsidomine was orally administered confirmed the anti-aggregatory effects of the drug when administered intravenously. Although the anti-platelet effects were maintained 1 h after molsidomine administration, bleeding time was unaffected [89]. In an uncontrolled study in patients with obliterating atherosclerosis of the lower extremities, molsidomine and PGI2 were evaluated for potential synergistic actions on platelet and fibrinolytic activities. At the doses given, neither molsidomine nor PGI2 as a monotherapy affected either function. However, the two drugs combined enhanced the overall anti-platelet and fibrinolytic activity without concomitant potentiation of the hypotensive effects of the two compounds. The synergism observed was attributed to the complimentary mechanisms by which each drug exerts its effects. The anti-platelet activity was attributed to a NO-mediated potentiation by cGMP of the anti-aggregatory action of PGI2 -mediated cAMP production. The fibrinolytic synergism was characterized as arising from an inhibition of plasminogen inhibitor release from platelets by NO and a PGI2 mediated release of tissue plasminogen activator from endothelial cells [118]. In an uncontrolled study with stable angina patients, the vasodilator and in vitro anti-platelet effects of graded doses of infused SIN-1 were assessed. Platelet responses to either ADP or the thromboxane B2 mimetic, U-46619, indicated that SIN-1 treatment only marginally affected platelet aggregation to either agonist [92]. Another uncontrolled study of SIN-1 in the same patient population examined the effects of the drug on platelet Ca+2 handling. SIN-1 reduced cytosolic Ca+2 in unstimulated platelets by decreasing Ca+2 influx, while in stimulated platelets it attenuated Ca+2 mobilization from internal stores. Superoxide dismutase (SOD), but not catalase, potentiated these responses. Thus, the effects of SIN-1 on Ca+2 handling resembled those of NO as modulated by the simultaneous release of O2 •− . By virtue of catalase’s lack of effect in this in vitro aggregation system, the influence of SIN-1 on Ca+2 handling was shown to be independent of O2 •− conversion to H2 O2 . Since SOD scavenges O2 − and thus limits ONOO− formation, it was felt unlikely that ONOO− contributed to the platelet Ca+2 handling response to SIN-1; rather, it was concluded that SOD increases NO bioavailability [119]. Molsidomine and its NO-donor active metabolite, linsidomine, have been studied in one large-scale, placebo-controlled, double-blind outcome study involving over 4,000 subjects [120]. In this so-called ESPRIM trial, patients with acute myocardial infarction and no signs of overt heart failure were randomly assigned to receive within 24 h of symptom onset either placebo or linsidomine (1 mg h−1 intravenously for 48 h) followed by 16 mg oral molsidomine daily for 12 days. All other standard treatments,but not vasodilators, were allowed at physicians’ discretion. With the exception of increased headache frequency in the treatment group, sydnonimine treatment did not reduce 35 day or long-term mortality or affect the incidence of major or minor adverse events. Similar to the GISSI-3 trial [79] with GTN and the ISIS-4 trial with IS-5-MN [98], the sydnonimine ESPRIM trial failed to demonstrate an independent survival benefit of an anti-platelet NO donor.

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12.5

Nitrosothiol NO Donors 12.5.1

S-Nitroso-glutathione (GSNO)

S-Nitroso-glutathione (GSNO) is a naturally-occurring NO donor that can act as a physiological NO surrogate or congener in vivo [121]. Cells and platelets synthesize GSNO by as-yet ill-defined mechanisms probably involving reaction between glutathione and nitrosating nitrogen oxides (e.g., N2 O3 , N3 O4 ). Generation of NO from GSNO likely occurs through mechanisms compatible with physiological pH, oxygen tension, and redox poise: e.g., transition metal (particularly copper)-catalyzed decomposition and/or transnitrosylation (transfer of a NO+ -equivalent from GSNO to an acceptor thiol) [116]. Plasma glutathione peroxidase augments NO liberation from GSNO [122]. Another mammalian enzyme, glutathione-dependent formaldehyde dehydrogenase, may also facilitate platelet GSNO turnover, although the enzyme’s presence in platelets is unconfirmed [123]. Whether synthesized endogenously or sequestered from plasma, platelet GSNO can be mobilized during platelet activation to dampen platelet activity [124]. For over a decade, GSNO has been known to inhibit isolated human platelet activation and aggregation [125]. The mechanism of this inhibition is complex. The prevalent signal-transduction pathway for platelet inhibition by NO (liberation of NO from GSNO to activate platelet guanylate cyclase and increase platelet cGMP content) may operate along with cGMP-independent mechanisms involving platelet protein nitration and NO metabolites such as ONOO− [126, 127]. Erythrocytes efficiently scavenge GSNO-derived NO, raising doubt about GSNO’s potential to exert an anti-thrombotic effect in vivo [128, 129]. Yet de Belder and colleagues demonstrated in healthy male volunteers that GSNO infusions up to 1.5 ìg min−1 for 5 min markedly inhibited ADP-induced platelet aggregation in platelet-rich plasma in vitro at doses that only marginally influenced vascular tone [130]. These investigators also showed that a 30 min GSNO infusion into healthy female volunteers (2.5 mg maximum total dose) inhibited in vitro platelet aggregation by up to 66% without altering systemic blood pressure or pulse [131]. Subsequent studies in stable angina patients undergoing coronary angioplasty [132] and in females with severe preeclampsia [133] documented that infused GSNO (16–22 mg total dose) reduced the platelet activation-protein expression associated with these maladies. Particularly noteworthy was the finding that hemodynamically neutral GSNO doses exerted a platelet inhibitory effect in angioplasty patients pre-medicated with aspirin and GTN [132]. These studies gave rise to the concept that GSNO is a “platelet-selective” NO donor preferentially affecting platelet activation over vasodilation. In contrast, the ready bioactivation of the organic nitrates GTN, ISDN, and ISMN by the vascular endothelium, but not by the (human) platelet, predisposes these drugs toward smooth-muscle relaxation and vasodilation. Further support for this concept was provided by Langford et al.’s demonstration that both infused GTN and GSNO inhibited the systemic platelet activation in unstable angina patients af-

12.5 Nitrosothiol NO Donors

ter aspirin treatment, but GSNO was much better tolerated [73]. Likewise, infused GSNO exerted a significant, acute anti-platelet effect (reduction of P-selectin surface expression) in bypass patients without hemodynamic influence when infused after the subjects had been weaned off of cardiopulmonary bypass, though all subjects had received GTN and anticoagulant (heparin) [134]. A negative report that GSNO infused into stable angina patients during coronary bypass surgery did not alter platelet P-selectin and GPIIb/IIIa expression may have been confounded by GSNO photodegradation within the cardiopulmonary bypass circuit [135]. In a series of four small trials, Markus and colleagues studied whether intravenous GSNO affected thromboembolus formation in patients undergoing either carotid endarterectomy or angioplasty and stenting for symptomatic carotid artery stenosis. Despite routine administration of aspirin and heparin to these patients, procedurallyinduced vascular endothelial denudation incites circulating cerebral thromboemboli, the frequency of which can be quantified as a surrogate endpoint of platelet activity by noninvasive ultrasonography. Initial data demonstrated that GSNO infusion (to a maximum total dose of ≈500 nmol kg−1 or until a threshold blood-pressure drop was attained) reduced the mean embolization frequency after carotid endarterectomy by 80% [136]. The effect was rapid in onset and sustained for 6 h, although the GSNO infusion had been terminated 2 h post-endarterectomy. A follow-up randomized, double-blind, placebo-controlled trial with a larger number of patients showed that a comparable, 90 min GSNO infusion elicited a 98% reduction in the frequency of embolic signals (vs. saline-treated controls) during the first 4 h after carotid endarterectomy [137]. A significantly diminished embolization frequency was maintained up to 24 h post-surgery relative to the saline group. Consonant with these results, short-term GSNO infusion markedly and rapidly reduced the frequency of embolic signals in patients with actively embolizing symptomatic carotid stenosis who had not undergone endarterectomy [138]. No side-effects (e.g., hypotension, hemorrhage) were observed in these patients, despite the fact that the suppression of embolization by GSNO persisted over 24 h. That the anti-thromboembolic effect of GSNO was not restricted to the setting of endarterectomy could also be demonstrated by a randomized, double-blind, placebo-controlled study in which a 90 min GSNO infusion virtually eliminated embolic signals within the first 6 h after angioplasty and stenting for treatment of symptomatic, high-grade carotid stenosis [139]. Although the frequency of thromboembolism correlates with perioperative stroke risk in the clinical setting of carotid endarterectomy or angioplasty, the consistently positive results of Markus and colleagues [136–139] do not imply significant clinical benefit, for the studies were not powered for an effect on morbidity or mortality. Suggestion from this work that GSNO might have the potential for clinical outcome benefit in patients with carotid artery stenosis comes from the reduction in recurrent throboembolic events after GSNO treatment vs. placebo group noted in one study [138] and the trend toward improved overall clinical status in another [139]. A case report demonstrating that a total GSNO infusion of 7.5 mg over 90 min rapidly improved the condition of one subject with a rare pre-eclampsia variant characterized by thrombotic microangiopathy is similarly suggestive of GSNO’s potential to exert a prognostic clinical effect that is platelet-related [140].

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12.6 L-Arginine {S(+)-2-Amino-5-[(aminoiminomethyl)amino]pentanoic acid} (L-arg)

The conditionally essential amino acid l-arginine (l-arg) has varied roles in mammalian metabolism [141]. Some of l-arg’s bioactivity could modulate platelet function: l-arg is a secretagogue for insulin, an anti-thrombotic humoral factor, and serves as the physiological substrate for catalytic NO production by NOS. Human platelets internalize circulating plasma l-arg through high- and low-affinity transport systems that supply platelet NOS with substrate for NO biosynthesis, and platelet function is influenced by endogenously generated NO [142, 143]. Consequently, a substratecontrol mechanism exists by which l-arg supplementation could potentiate directly platelet NO production and inhibit platelet activity. Less direct means have also been proposed to explain how supplemental l-arg might increase bioactive NO in the vasculature and/or elicit an anti-platelet effect advantageous to vascular health [144]. Regardless of the mechanisms through which exogenous l-arg influences platelet activity, the association between impaired platelet-derived NO production and acute coronary syndromes suggests that platelet NO deficiency contributes to thromboembolic disorders [13]. Although not without side-effects at very high chronic doses, l-arg supplementation is appealing from the standpoint of safety, since the finite catalytic capacity of the NOS system and the hydrolytic activity of (vascular) arginase would tend to obviate NO overload and its adverse sequellae [141]. As shown in double-blind, placebo-controlled, randomized studies with healthy subjects, both infused [145] and oral [146] l-arg significantly inhibited (by ≈40%) ADP-induced platelet aggregation in vitro and potentiated platelet cGMP content. The effect, though, was weak: the plasma concentration of l-arg required to produce an anti-platelet effect was some 2-fold above normal, steady-state levels, and the oral anti-aggregatory l-arg dose was 4-fold greater than the usual daily l-arg intake in humans. The infused l-arg dose that effectively inhibited platelet activity (30 g total) was hypotensive and increased heart rate, whereas the oral anti-platelet dose (7 g per day over 3 days) did not affect blood pressure, suggestive of oral l-arg platelet selectivity. A comprehensive, randomized, placebo-controlled trial of infused bolus l-arg and its enantiomer (d-arg) included healthy subjects, non-insulin dependent diabetics, hypertensive subjects, and normotensives with primary hypercholesterolemia [147]. A blood-pressure drop and an acute inhibition of ADP-induced aggregation in plateletrich plasma were observed in all subjects after l-arg administration (≤5 g). Both responses to l-arg infusion closely correlated in magnitude, were weaker in noninsulin dependent diabetics and hypercholesterolemics, and declined with increasing age. Notably, d-arg did not elicit any of the l-arg effects, which were reduced by some 70% when superimposed upon ongoing, nonselective NOS inhibition with infused l-N-monomethyl-arginine (L-NMMA). Since d-arg is not a NOS substrate, and L-NMMA is a substrate-competitive NOS inhibitor, the l-arg effects observed in this study were theorized to reflect a rise in vascular NO production by eNOS. In contrast, the inhibition of platelet aggregation observed in vitro after a 5 min l-arg infusion (160 mg total dose) into healthy subjects and patients with angiographic

12.7 NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester]

atherosclerosis paralleled the anti-aggregatory effect of l-arg when added to whole human blood, suggestive of a direct effect on platelet NOS [109]. Hypercholesterolemic subjects without clinically evident cardiovascular disease and free of vasoactive or anti-platelet medications were the focus of a double-blind, randomized, placebo-controlled trial of long-term dietary l-arg supplementation (8.4 g daily for 2 weeks), which was well tolerated with no side-effects and markedly elevated circulating l-arg levels [148]. But l-arg only modestly and variably (by ≈20% on average) attenuated the platelet hyperreactivity characteristic of hypercholesterolemia, an effect that persisted 2 weeks after l-arg discontinuation in some subjects. The rigorous inclusion criteria for entry into this positive trial appear key to the modest l-arg effect observed, for a randomized, double-blind, placebo-controlled study of dietary l-arg (3 g, 3 times per day for 1 month) in coronary disease patients on standard therapy did not alter platelet activation-protein (P-selectin) expression [149]. Whether the negative result reflects the vasoactive and anti-platelet medicines (aspirin, nitrovasodilators) that had been administered as standard therapy for coronary artery disease or a suboptimal l-arg dose remains open to question. In nonmedicated, healthy male volunteers, l-arg infused at doses that normalized a NOS-inhibitor-induced increase in blood pressure did not affect platelet activation status (P-selectin expression, number of circulating platelet-leukocyte aggregates) [150]. Standard pre- and peri-operative therapy with aspirin and heparin did not appear to have a confounding effect in a randomized, double-blind, placebo-controlled l-arg trial in patients undergoing carotid endarterectomy [137]: a 90 min l-arg infusion (30 g total dose) reduced the frequency of embolic signals by 79% vs. placebo-treated group during the first 4 h after the procedure. A significant reduction in embolization was maintained up to 24 h post-surgery. Two small trials suggest that l-arg infusion could have a therapeutic effect related to in vivo platelet inhibition. Intravenous l-arg treatment (10.5 g daily for 7 days) of patients with peripheral vascular disease decreased ADP- and collagen-induced platelet aggregation in vitro, increased plasma cGMP, elongated the pain-free walking period, and improved absolute and pain-free walking distances [151]. A higher l-arg dose (16 g daily for 21 days) increased the duration and distance of pain-free walking for up to 6 weeks after treatment in patients with peripheral vascular disease [152].

12.7

NCX-4016 [2-Acetoxybenzoate 2-(1-nitroxy-methyl)-phenyl ester]

A recent trend in the pharmaceutical industry has been to harness the intrinsic tissueprotective properties of NO for improving the gastric tolerance of nonsteroidal antiinflammatory drugs (NSAIDs). This trend has led to the synthesis of hybrid, chimeric molecules containing an NSAID or aspirin moiety and a NO-donor functionality [153, 154]. One such hybrid is a NO-releasing derivative of aspirin, NCX-4016. In a doubleblind, randomized, placebo-controlled gastrointestinal safety assessment in healthy subjects, NCX-4016 (400 or 800 mg twice daily for 7 days) acted like aspirin as an inhibitor of arachidonic acid-induced platelet aggregation in vitro [155]. Whether

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this anti-platelet effect reflected NO-donor activity at all could not be determined from this study, since, for example, the des-NO-donor analog of NCX-4016 was not investigated.

12.8

Conclusion and Future Prospects

Pharmacological platelet inhibition has therapeutic value in preventing major cardiovascular events [34]. Aspirin prophylaxis reduces morbidity and mortality in patients at risk of clinical complications from occlusive vascular disease [35, 36]. Anti-platelet therapy also helps ease, but nowhere near eliminates, the growing, worldwide healthcare burden from patients with coronary risk factors, acute coronary syndromes, atherosclerosis, failed vessel grafts, and unsuccessful percutaneous revascularization attempts, with or without stenting [2, 27, 33, 34]. These and other conditions are generally characterized by a pro-thrombotic state, expressed clinically as hyperactive platelets and deficiencies in platelet- and endothelium-derived NO [10, 31, 32, 156]. The routine clinical use of NO-donor, anti-anginal nitrovasodilators in the management of ischemic syndromes, myocardial infarction, and cardiac failure [55, 80] and the pathogenic involvement of platelets in these same situations [25–27] invite the possibility that the salutary effects of nitrovasodilator drugs partly reflects an NO-based anti-platelet action over and above vasodilation. If so, then a clear therapeutic niche would exist in several high-impact cardiovascular disorders for platelet-inhibitory NO donors, especially since current anti-platelet drugs have not solved the medical problem of thrombosis. In vivo, platelet activation is inherently complex, involving many interactive, often iterative factors [3, 4, 9, 17, 18] (Figs. 12.1 and 12.2). The physiological chemistry of NO and NO-derived metabolites is profoundly influenced by their environment [157]. These factors conspire to limit severely the translational significance of laboratory demonstrations that NO donors in vitro inhibit the agonist-induced activation of isolated platelets. In vitro platelet systems might provide clinically useful data in, for example, targeting compounds or identifying anti-platelet drugs acting within the NO signal-transduction pathway (e.g., NO-dependent protein kinase modulators, guanylate cyclase activators) [158–160]. Likewise, although laboratory results with animal models of thrombosis generally support the notion that NO donors have anti-platelet effects in vivo, even the best thrombosis models can only approximate evolving thromboembolic disease in humans, during which vascular redox tone and steady-state NO background may flux dramatically [161, 162]. Reminiscent of the trend with laboratory studies, most (33 out of 43 cited above) uncontrolled clinical trials with either healthy volunteers or cardiovascular patients suggest that oral and intravenous NO donors at therapeutic doses acutely inhibit platelet activation in vivo (vide supra). Aside from their lack of long-term dosing and a placebo control group, several considerations restrict the predictive clinical value of these uncontrolled clinical studies: limited numbers of subjects; nonuniform criteria for subject entry and treatment outside of the trial; induction of adrenaline or

12.8 Conclusion and Future Prospects

catecholamine discharge in response to NO-donor induced vasorelaxation; lack of independent dosing optimization; and undocumented patient compliance. In vitro measurements of platelet activation status (e.g., agonist-induced aggregation; activation protein expression) or integrated evaluation of overall hemostasis (e.g., bleeding time) invite further complication, since the diagnostic utility of these parameters for monitoring anti-platelet strategies and predicting clinical outcomes is far from established [163]. Methodological artifacts may be compounded by the transient and rapidly reversible nature of platelet inhibition by NO, which prohibits extensive platelet studies post-dosing. Time constraints obviate examination of multiple endpoints of platelet activation status or even aggregation alone over a range of agonists and agonist concentrations [164]. The sensitivities of platelet endpoints (aggregation, adhesion protein expression, etc.) to a given exogenous NO exposure can differ markedly [165, 166], and the chemical form of NO delivery per se may modify the subsequent platelet response [167]. These factors lend an air of uncertainty as to what degree uncontrolled clinical studies can represent the effectiveness of an NO donor as an anti-thrombotic agent. Nonetheless, the aggregate study-design and methodological considerations would tend to underestimate potential platelet inhibition by an NO donor in vivo, as would the likelihood that some trial subjects with cardiovascular disease had NO-resistant platelets [168]. Many of these same factors impinge upon placebo-controlled clinical trials of NO donors and platelet activation in vivo. As opposed to open-label, uncontrolled trials, it has been somewhat more difficult to show a convincing, clear-cut difference on platelet activity between placebo and active NO-donor treatment (14 positive controlled trials out of 25 cited above). Perhaps this is because distinctions between placebo and treatment groups tend to be reduced, if not masked, by the considerable intrinsic genetic variability of the responsiveness of individuals to anti-platelet agents [169]. Drugs inconsistently administered outside of the trials may also have had an obfuscating effect: appropriate heart disease therapy includes aspirin and nitrovasodilators, which have anti-platelet effects themselves (vide supra). Nitrates administered outside of two large, randomized, multi-center, placebo-controlled trials (GISSI-3 [79], ISIS-4 [98]) may have contributed to their failure to show an independent effect of nitrovasodilator therapy on clinical outcome (i.e., survival advantage) that would at least be suggestive of in vivo platelet inhibition by an NO donor. Although in vitro platelet inhibition by a NO donor was reported as early as 1967 [39], there is no extant, clear-cut demonstration from a well-powered, randomized, controlled and blinded trial that a NO donor at therapeutic doses has in vivo antiplatelet activity sufficient to exert an independent effect upon clinical outcome. Since there is a greater likelihood for false negative than false positive results in the three such trials (GISSI-3 [79], ISIS-4 [98], ESPRIM [120]) reported so far, the possibility that NO-donor drugs could exert an independent anti-platelet effect for prognostic benefit in cardiovascular disease must still stand. The rational design of small-molecule, platelet-targeted NO donors compatible with vascular homeostasis and function that release bioactive NO only in the presence of activating platelets might yield pharmacologically attractive, safe, and tolerance-free compounds with which to verify this possibility. A site- and event-specific NO donor would be expected to prevent

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platelet aggregation and thrombus formation regardless of the agonist(s) operative and should efficiently counteract the pro-thrombotic and vasoconstrictor mediators produced by activated platelets themselves without exerting undue hemodynamic side-effects (e.g., concomitant hypotension or hemorrhage). Testing of such an agent in a properly designed and controlled clinical study would seem to be a worthwhile goal toward demonstrating an independent, anti-platelet effect of an NO donor on classical outcome measures, provided that patient entry criteria were well-defined. Potential criteria could include: standardization as to anti-platelet drug response; compromised endogenous NO bioactivity; platelets not refractory to NO and activated by a pre-existing condition such as unstable angina or post-bypass grafting; no pre-medication with platelet-active drugs. With regard to vascular devices, biocompatible, anti-thrombotic NO-donor coatings might offer ways to improve the success rate of revascularization procedures and extend their applicability [170, 171]. In the light of experimental and clinical demonstration that sustained organic nitrate use alters vascular biochemistry and function [172, 173], the paucity of data regarding the effects of long-term NO-donor administration on the human platelet is particularly noteworthy. Improved technology for monitoring platelet activity in the clinical setting would be most welcome. As for now, demonstrations that NO donors with various chemical structures acutely inhibit human platelet activity under certain clinical conditions in both healthy subjects and patients with cardiovascular disease await confirmation of their true significance by “gold-standard” validation from positive human outcome trials.

Acknowledgement

We thank Deborah Farnham for reprint assistance, Dawn Spooner for illustration assistance and Ginny Braman for technical comments.

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NO and Gene Regulation Jie Zhou, Bernhard Brüne 13.1

Formation of NO and RNI-signaling

With the discovery of the EDRF (endothelium derived relaxing factor), its identification as nitric oxide (NO) and the notion that NO is a versatile molecule beyond the vascular system, it came as some surprise that this small molecule powers signal transmission in nearly all areas of life [1, 2]. NO taught us to revise traditional thinking and to appreciate that formation of a radical stirs efficient pathophysiological signaling in biology/medicine. Signal transmission of NO is often elicited by “reactive nitrogen intermediates” (RNI) rather than the NO radical itself. The RNI comprise oxidation states and adducts of the products of nitric oxide synthase (NOS), including NO-radical (• NO), NO− and NO+ , as well as the subsequent adducts of these species such as NO2 , NO2 − , NO3 − , N2 O3 , N2 O4 , S-nitrosothiols, peroxynitrite and nitrosyl-metal complexes [3, 4]. Determination of RNI involvement in biological responses is often based on the use of compounds that mimic an endogenous response by administration of chemically diverse NO donors, by blocking RNI formation with NOS-inhibitors, or by using knockout mice that lack isotype specific NOS [5]. NO is produced by NOS that converts l-arginine to citrulline and NO [6]. Three isoenzymes known as neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) are named after the cell type from which they were first isolated and cloned. Isoenzymes show variations in terms of a high (iNOS) versus low (eNOS, nNOS) output capacity and differ with respect to their basic mechanisms of activation. The major distinction is regulation of nNOS as well as eNOS by a cytosolic calcium increase resulting in a pulsative enzyme activation versus the cytokine-inducible iNOS that produces RNI until the enzyme is degraded [7]. Biological signaling attributed to RNI is, in a first and simple approach, distinguished as either being cGMP-dependent or cGMP-independent [2]. Binding of RNI to the heme moiety of soluble guanylyl cyclase and concomitant cGMP formation constitutes the classical RNI response with broad implications for vascular medicine, at the same time acknowledging the landmark discovery of EDRF [8]. Besides the cGMP-signaling cascade that is mimicked by lipophilic cGMP analogs, alternative signaling pathways are activated by RNI via redox and additive chemistry. This may 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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promote covalent modification of proteins or oxidation events that do not require attachment of the NO group [3, 9]. Among these modifications, S-nitrosylation/Snitrosation [10], protein nitration, oxidation and cGMP-independent phosphorylation have increasingly attracted scientific attention as (ir)reversible post-translational protein alterations. These cGMP-independent protein modifications often elicit signal transmission that culminates in activation or suppression of genes [11–13]. Most, if not all, gene regulatory activities evoked by RNI are indirect. Up to now there is no evidence for the existence of DNA elements within promoter regions of eukaryotic genes that directly respond to RNI. Thus, to understand the signaling qualities of RNI in regulating genes we need to consider modification of transcription factors, their compartmentalization, their action as transcriptional activators or inhibitors, the stability of target mRNAs as well as transcription factor protein stability regulation. Numerous studies have analyzed the regulatory effects of RNI on transcription factors and transcriptional regulation. Although outside the main focus of this article, to summarize these primary observations we provide a table showing several transcription factors and their regulation under the impact of RNI (Table 13.1). For extensive coverage of the primary literature we refer to a number of review articles [11–14]. Often, RNI exert contradictory effects with regard to transcription factor activation. This may reflect the use of RNI with different signaling properties, different concentrations of RNI, cell-free versus intact cell systems, different types of cells, as well as the fact that activation of transcription factors is a result of complex upstream signaling cascades that themselves are targeted by RNI. For example, in resting cells RNI increase NF-êB- or AP-1-dependent gene transcription while RNI attenuate these responses in activated cells [13]. Thus, the signaling qualities of RNI depend on the biological milieu, i.e. the presence or absence of modulatory co-signals, often considered to be superoxide [15]. Along that line, the primary target of RNI as well as specific molecular modification(s) often remain unknown and different parallel versus interfering signals may be subjected to different redox control mechanisms. Considering thiol modification, an important post-translational protein alteration, and taking into account that many transcription factors share redox sensitivity based on thiol residues found in their DNA-binding domain, it is not surprising that their activity is under the control of RNI. However, a simple prediction on activation versus inhibition of gene activation as a result of RNI is still lacking. Any conditions of stress are potentially harmful to cells and require appropriate defence responses. Among other systems, the hypoxia inducible factor-1á (HIF-1á) [16–19] and the tumor suppressor protein p53 [20–22] are important transcriptional regulators that act in response to stress signals to coordinate a cellular reply by inducing growth arrest, apoptosis or adaptation [23]. Ultimately, the nature and intensity of stress signals, the cell type, and the cellular context will dictate the final outcome. Multiple levels of regulation must ensure that p53 and HIF-1á are fine-tuned to guarantee appropriate transcriptional regulation of target genes to cope with the stress situation. Interestingly, among multiple signals, hypoxia and RNI emerged as activators of both, p53 and HIF-1á.

13.2 p53 Regulation under the Impact of RNI Tab. 13.1: Transcription factors under the control of RNI. Selected examples for the regulatory

impact of RNI on prokaryotic and eukaryotic transcription factors. In a very simplistic way activation versus inhibition by RNI are indicated. Prokaryotic factors Transcription factor (TF) SoxR, OxyR Fur LAC9 Ace1

Modulation by RNI −rather uniform results showing activation −de-repression of genes that are under the control of Fur −attenuated DNA-binding in vitro −inhibition of DNA-binding in yeast

Eukaryotic notably mammalian factors NF-êB −activation in resting cells, low level RNI −inhibition in stimulated cells, high level RNI AP-1 −activation in unstimulated cells, low level RNI −inhibition in activated cells Sp1, Egr-1 (zinc finger TFs) −rather uniform inhibition −activation if Sp1 de-represses the e.g. TNFá promoter VDR, RXR (nuclear hormone R) −inhibition of DNA-binding and reporter activity PPARã −activation at low level RNI −inhibition at high level RNI NFAT −inhibition in activated NK cells HSFs −activation (HSP70 expression) p53 −activation (for details see below) HIF-1 −activation under normoxia (for details see below) −inhibition under hypoxia TF: transcription factor, R: receptor, Fur: ferric uptake regulation protein, NF-êB: nuclear factorêB, AP-1: activator protein-1, Egr-1: early growth response-1, VDR: 1á,25-dihydroxy-vitamin D3 receptor, RXR: retinoid X receptor, PPARã: peroxisome proliferator-activated receptor ã NFAT: nuclear factor of activated T-cells, HSF: heat shock factor, p53: tumor suppressor p53, HIF-1: hypoxia inducible factor-1.

13.2

p53 Regulation under the Impact of RNI 13.2.1

Basic Considerations: p53 Phosphorylation and Mdm2 Binding

p53, a prototype tumor suppressor, controls cell cycle progression and apoptosis with the notion that these functions are altered in many tumors, thus contributing to malfunction. In unstressed cells p53 exhibits an extremely short half-life and the protein amount is maintained at a low, often undetectable, level by efficient proteasomal degradation. Cellular stress, such as DNA damage, oncogene activation, or hypoxia, stabilizes p53 predominantly by post-translational modification, with the notion that a common denominator in all p53-inducing stresses may be nucleolar disruption [24]. As a transcriptional activator p53 promotes transcription not only of cell cycle regulating genes such as p21WAF1/CIP1 or murine double minute 2 (mdm2) but also apoptotic ones, exemplified by Bax or Fas, [20–22]. In addition, transcription-

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independent actions contribute to the activation of proapoptotic pathways. However, these effects are not exerted indiscriminately. Quantitative and qualitative changes endow p53 with improved capability to alter the cell phenotype, either to take care of the damage or to eliminate affected cells from the replicative pool to prevent its expansion into malignant progenitor cells [25]. It appears that the choice between induction of growth arrest versus apoptosis is defined by the balance of survival signals, i.e. the cellular context as well as the particular genotype of the cell with genetic alterations affecting (in)directly the functional status of p53 [22]. Stability regulation of p53 is regulated by Mdm2, which functions as a p53-specific E3 ubiquitin-ligase. The mdm2 gene is transcribed under the control of p53 and the Mdm2 protein binds p53 at the N-terminus to facilitate its proteasomal degradation [26–28]. Ubiquitination of p53 requires a conserved Mdm2 binding region of eight cysteine and one histidine amino acids that form two zinc-binding sites, known as a RING-finger motif. The RING-finger domain additionally facilitates nuclear export of p53 [29, 30]. Blocking p53 nuclear export attenuates Mdm2-mediated p53 degradation and concomitantly stabilizes and actives p53, [31–33]. These considerations are relevant for p53 stabilization. When UV attenuates the p53/Mdm2 interaction, ubiquitination of p53 is reduced, culminating in protein stabilization [34, 35]. Although under debate, phosphorylation of p53 at serine 15 may be needed to dissociate p53 and Mdm2 [36, 37] and phosphorylation of p53 at several sites, among others serine 15, is facilitated by RNI [38] as well as other stress conditions such as UV, IR or CdCl2 treatment [39, 40]. Besides phosphorylation, an altered compartmentalization of p53 achieved by leptomycin B, a chromosome region maintenance 1 (CRM1)/exportin1mediated nuclear export inhibitor, activated p53 [41, 42]. It is predicted that serine 15 phosphorylation of p53 following UV-treatment reduced nuclear export of p53 [43]. 13.2.2

Molecular Mechanisms of RNI-evoked p53 Stabilization

For some time it has been appreciated that iNOS- or NO donor-derived RNI stabilize p53 [44, 45]. Supporting evidence came from experiments in iNOS-deficient macrophages that failed to localize p53 to the nucleus after in vivo bleomycin exposure [46]. Upregulation of p53 targets such as p21(Waf1/Cip1) or Bax in response to RNI, supported the idea that p53 was transcriptionally active [47, 48]. Experiments in thymocytes from p53 null mice or in mutant p53 human lymphoblastoid cells revealed that these cells were less vulnerable to RNI, implying that p53 may transmit a proapoptotic RNI-response [49, 50]. RNI-accumulated p53 revealed a distinct phosphorylation pattern, predominantly at serine 15 [38, 51]. This goes along with current thinking that post-translational modification of p53 at the N- and/or C-terminus contributes to protein stability regulation. Reports on the involvement of the ataxia telangiectasia-mutated (ATM) kinase in facilitating RNI-evoked phosphorylation are controversially discussed. Wang et al. ruled out a contribution from ATM or the alternate reading frame (ARF) tumor suppressor protein in p53 accumulation [52]. Based on studies in isogenic human cell lines and MEFs from gene knockout (ATM-/- ) mice these observations were challenged by demonstrating that

13.3 HIF-1á Regulation under the Impact of RNI

serine 15 phosphorylation is ATM- and ATM- and Rad3-related (ATR)-dependent but p38- and DNA-PK-independent, although mechanisms of ATM/ATR activation by RNI remained obscure [53]. Cell fractioning and heterokaryon analysis suggested that RNI, in some analogy to leptomycin B, prevented nuclear-cytoplasmic shuttling of p53 which causes nuclear protein stabilization/activation [54]. In addition to phosphorylation, reversible down-regulation of Mdm2 by RNI may contribute to activation of p53 [52]. An initial but transient drop in Mdm2 may account for early accumulation of p53 in response to RNI, whereas in a second phase p53 remains stabilized although Mdm2 increases above controls due to transactivation of the mdm2 gene [52]. Under conditions of elevated Mdm2 expression, nuclear trapping of p53 appears as a rational explanation for p53 accumulation [55]. Fig. 13.1 schematically depicts concepts to rationalize RNI actions in accumulating p53. Concepts on attenuated nuclear-cytoplasmic shuttling of serine 15-phosphorylated p53 to account for RNI action came from heterokaryon analysis, although causation between an altered compartmentalization and serine 15-phosphorylation awaits clarification. Leptomycin B, an established inhibitor of p53 nuclear export, targets an active cysteine residue in CRM1 to attenuate nuclear export. This may apply for RNI as well, considering that RNI may nitrosylate thiol residues. Observations on the nuclear trapping of p53 were corroborated when RNI increased nuclear import and retention of p53 in neuroblastoma cells in which p53 is primarily cytoplasmatic [56]. Transactivation of p53 and serine 15 phosphorylation were noticed in cells co-cultured with RNI-releasing macrophages and were correlated to iNOS expression in ulcerative colitis. This highlights RNI generation and activation of a p53 response pathway during chronic inflammation [53]. Activation of divers signaling pathways by RNI, culminating in gene expression, has been proven by the use of DNA microarrays [57] or differential analysis of library expression [58]. Among different group of genes activated by RNI was a subpopulation of genes that specifically required p53. These data support the basic observation that RNI stabilize and activate p53 under pathophysiological conditions. However, at present it cannot be ruled out that nitrosative stress during chronic inflammation might lead to mutations within the p53 gene, thus contributing to carcinogenesis [59, 60].

13.3

HIF-1á Regulation under the Impact of RNI 13.3.1

Lessons from Hypoxia: Basic Considerations of HIF-1á Stability Regulation

Intracellular recognition of decreased oxygen tension, i.e. hypoxia, and an appropriate response to meet these alterations is predominantly facilitated via the oxygendependent transcription factor HIF-1 as a consequence of stability regulation and/or protein synthesis of the HIF-1á subunit. Pioneering work on erythropoietin, a classical hypoxia-responsive target gene, led to the discovery of HIF-1 [61] while more recently an integrated picture of oxygen sensing emerged [16, 19, 62–64]. HIF-1 is a

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Fig. 13.1 Stability regulation of p53 by early vs. late RNI actions. RNI by (in)directly activating ATR/ATM provoke phosphorylation of p53. In turn tetramerization of p53 and binding of the coactivator p300/CBP will provoke gene activation. This may go along with RNI-evoked nuclear retention of ubiquitinated p53 although

expression of Mdm2, a p53-downstream target gene, is increased. An early action of RNI may involve transient downregulation of Mdm2 which would be consistent with p53 stabilization due to reduced poly-ubiquitination. For details see text.

heterodimer composed of one of the three alpha subunits (HIF-1á, HIF-2á or HIF3á) and one HIF-1â subunit [65]. HIF-1â is constitutively expressed and is identical to the aryl hydrocarbon receptor (AhR), known as AhR nuclear translocator (ARNT). HIF-1, as implied by its name, is predominantly active under hypoxic conditions as a result of stabilization of the alpha subunit, i.e. HIF-1á. In the presence of oxygen the alpha subunit is unstable and is generally undetectable due to polyubiquitination by an E3-ubiquitin ligase complex that is built among other proteins by the von Hippel–Lindau protein (pVHL), followed by 26S proteasomal degradation [66, 67]. An oxygen-dependent prolyl-4-hydroxylase, similar to proline hydroxylation of collagens, covalently modifies a domain of HIF-1á known as the oxygen-dependent degradation domain (ODD) by hydroxylating proline residues 402 and 564 [68, 69]. Hydroxylases

13.3 HIF-1á Regulation under the Impact of RNI

are known as orthologs of C. elegans Egl-9, designated as PH domain-containing enzymes (PHD), i.e. prolyl hydroxylases (PHD1, PHD2, PHD3 and PHD4) [70–72]. Hydroxylated HIF-1á form hydrogen bonds with pVHL side chains which promote polyubiquitination of HIF-1á, followed by proteasomal degradation [66, 67, 73]. Enzymes require 2-oxoglutarate and iron as cofactors, thus implying why “hypoxicmimetics”, e.g. the iron chelator desferrioxamine, accumulates HIF-1á by attenuating PHD activity. Besides the transactivation domain residing in the ODD another one is found in the extreme C-terminus of HIF-1á, known as the C-terminal transactivation domain (CTAD). Hydroxylation of asparagine 803 by FHI (factor inhibiting HIF-1) within the CTAD [68, 74] renders HIF-1á unable to bind to the p300/CBP coactivator, thus preventing transactivation capabilities of HIF-1. Hypoxia attenuates Pro564/402 and Asn803 hydroxylation, which in turn provokes HIF-1á protein stabilization, HIF-1â association, coactivator recruitment, subsequent activation of HIF-1 which results in expression of those targets that contain HRE (hypoxia responsive element) sites with the core DNA sequence 5′-ACGTG-3′ [64]. Target genes are categorized according to their signaling qualities with involvements in erythropoiesis, iron homeostasis, glucose/energy metabolism, viability decisions, or vascular development/remodeling [63, 75]. HIF-1 targets are related to physiology as well as pathology and therefore it is predictable that gene products contribute not only to cell protection but also to cell pathologies, in close association with several major disease states such as ischemic cardiovascular disorders, pulmonary hypertension, stroke, pregnancy disorders or cancer [63, 75]. 13.3.2

Stability Regulation of HIF-1á by NO/RNI in Normoxia versus Hypoxia

There are several lines of independent research showing HIF-1á stabilization, HIF-1 DNA binding and HIF-1 transactivation under normoxia by RNI [18]. Experiments in a variety of human, pig or bovine cells ruled out species specific or cell type restricted effects. The use of chemically distinct NO donors such as S-nitrosoglutathione (GSNO, considered the most physiological NO donor), NOC-18 (Z-1-1[2-aminoethylamino]diazen-1ium-1,2-diolate), NOC-5 (3-(hydroxy-1-(1-methylethyl)-2-nitrosohydazino)-1-propanamine, SNAP (S-nitroso-N-acetyl-d,l-penicillamine), or others indeed suggest RNI involvement [18]. Along that line the time- and concentrationdependent effects of RNI on HIF-1á accumulation have been established by using NO donors with different half-lives [76]. RNI-evoked activation of the human vascular endothelial growth factor (VEGF) promoter under normoxia in combination with deletion and mutation analysis of the VEGF promoter indicated that the RNI-responsive cis-elements were the HIF-1 binding site and an adjacent ancillary sequence that is located immediately downstream within the HRE [77, 78]. Experiments with GSNO, a nitrosonium donor, and observations that GSNO effects are reversed by dithiothreitol, lead to the proposal that S-nitrosylation stabilizes HIF-1á [79]. Although S-nitrosation of HIF-1á is confirmed in vitro, a biological significance and any (in)direct role in HIF-1á stability regulation by RNI awaits clarification [80]. The use of NO donors often raises questions on the pathophysiological importance of

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RNI signaling with respect to relevant concentrations. This potential drawback was overcome by overexpression human iNOS, thereby accumulating HIF-1á. This supports the notion that autocrine or paracrine produced RNI are capable of stabilizing HIF-1á under normoxia [76]. Supporting evidence came from a transwell co-culture set-up of lipopolysaccharide/interferon-ã activated, and thus iNOS-derived RNI producing macrophages and tubular LLC-PK1 detector cells, showing that activated, but not resting, macrophages elicited HIF-1á accumulation in LLC-PK1 cells [81]. However, considering various intracellular targets of RNI it should not be extrapolated from these results that this is a uniform reaction, especially if the intracellular redox milieu changes, i.e. by the formation of superoxide. Interference of O2 − with RNI signaling, and vice versa, is established and understandable by the diffusion-controlled radical interaction which may redirect signaling qualities of RNI towards other species, i.e. ONOO− . Experiments with the redox cycler DMNQ (2,3-dimethoxy-1,4-naphthoquinone) to generate O2 − and/or H2 O2 (derived from superoxide dismutase-triggered conversion of O2 − to H2 O2 ) attenuated RNI-elicited HIF-1á accumulation [82], which favors the assumption that agents increasing reactive oxygen intermediates (ROI), including ONOO− , destabilize HIF-1á [83]. These observations predict that the ability of RNI to stabilize HIF-1á depends to some extent on the intracellular redox milieu and is subject to modulation by cosignals. Analogous observations are shown by seminal observations in 1998/1999 stating that carbon monoxide (CO) and RNI inhibit hypoxia-induced HIF-1á accumulation [84–86]. As mechanistic insights start to become clear it appears that HIF-1á suppressing actions under low RNI concentrations depend on the inhibition of mitochondrial respiration, since it is absent in p0 -cells and is mimicked by inhibitors of mitochondrial respiration [87]. The authors propose that destabilization of HIF-1á by RNI under hypoxia is unlikely to result from oxidative stress, i.e. ROS formation, rather correlating with inhibition of mitochondrial respiration by NO which leaves more oxygen available for PHDs, thus allowing PHD activity to be regained although oxygen tension is low [87, 88]. Alternatively, the level of free iron may change under the conditions of RNI formation and hypoxia, which again may contribute to the regulation of PHD activity. Determination of free iron and PHD activity under conditions of O2 − and RNI formation, as well as under conditions of cotreatment, will help to clarify various proposals. RNI signaling and HIF-1á accumulation is supported in several experimental systems. Cell density-induced HRE activation demands the production of RNI, which are generated by densely cultured cells as a diffusible paracrine factor [89]. In human prostate cells RNI use Ras, mitogen activated protein kinase (MAPK) and HIF-1á signaling to activate HRE which connects this pathway with survival or growth advantages of tumor cells, because inhibition of iNOS blocked production of an angiogenic activity in thioglycolate-induced peritoneal and murine RAW264.7 macrophages [90]. It can be concluded that VEGF contributes to macrophage-dependent angiogenic activity and modulation of VEGF mRNA levels in macrophages is, at least in part, under the control of the iNOS pathway. Attenuating iNOS provokes formation of antiangiogenic factors which makes RNI likely players in the regulation of macrophage-

13.4 RNI, p53 and HIF-1 in Tumor Biology

dependent angiogenic activity in vivo, in wound repair and possibly in tumor development [90]. The idea that NO donors or an active iNOS promote HIF-1á accumulation demanded mechanistic explanations considering the existing details proposed for HIF1á stability regulation. In close analogy to hypoxia, RNI have been shown to decrease ubiquitination of HIF-1á and to dissociate pVHL from HIF-1á [91]. Considering that the HIF-1á-pVHL interaction requires prolyl hydroxylation of HIF-1á raises the possibility that RNI blocks HIF-1á prolyl hydroxylation. An in vitro HIF-1á-pVHL capture assay implied dose-dependent inhibition of PHD activity by GSNO, while the association of a synthetic peptide resembling the hydroxylated ODD domain of HIF-1á with pVHL remained intact. It can be concluded that hypoxia and RNI use overlapping signaling pathways and/or modifications to evoke HIF-1á stabilization. As proposed schematically in Fig. 13.2, RNI may block PHD-activity, attenuate proline hydroxylation of HIF-1á, dissociate HIF-1á from pVHL with the consequence of protein stabilization based on decreased proteasomal degradation. It is known that RNI interact with iron (II) in heme- or non-heme-containing proteins [4], exemplified by spectroscopic studies when NO directly binds to the ferrous iron in protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase [92] or to isopenicillin N synthase [93]. These enzymes coordinate Fe2+ in their catalytic domain in a 2-histidine-1-carboxylate facial triad which is the defining structural motif of mononuclear non-heme iron(II) enzymes [94]. HIF-1á prolyl hydroxylases (PHDs) belong to a non-heme Fe2+ -containing family of enzymes. Thus, it is rational to assume Fe2+ -coordination by RNI in the catalytic site of PHD and thus competition of RNI with oxygen. The concept of direct PDH inhibition by RNI as an explanation of HIF-1á accumulation was recently challenged by the observation that the NO donor NOC18 did not inhibit HIF-1á hydroxylation, ubiquitination, and degradation [95]. Instead, based on pulse labeling studies the authors proposed increased HIF-1á synthesis. Unfortunately, major conclusions in this study are derived from overexpression experiments of FLAG-HIF-1á and HA-pVHL and it remains open whether overexpressed proteins share regulatory features of endogenous proteins. This becomes a concern, considering that expression of FLAG-tagged HIF-1á remained constant under the impact of NOC18, and thus a difference in the HIF-1á/pVHL interaction would not be expected. However, considering the proposed role of PI3K in HIF-1á translational regulation it may turn out as an exception to the rule that in some cells PI3K is stimulated by RNI which in turn provoke HIF-1á translation regulation. This scenario has also been noticed for hypoxia [96]. Under conditions where PI3K/Akt stimulation by hypoxia and/or RNI occurs, translation control mechanisms may overlap with blocked degradation pathways in accumulating HIF-1á.

13.4

RNI, p53 and HIF-1 in Tumor Biology

The critical role that HIF-1 plays in cancer biology can be deduced from immunohistochemistry data showing elevated levels of HIF-1á in a variety of primary malignant

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Fig. 13.2 Stability regulation of HIF-1á and

activation of HIF-1 by RNI. HIF-1á is subjected to hydroxylation by PHDs and FIH that allows recruitment of pVHL, subsequent polyubiquitination and concomitant 26S proteasomal degradation. RNI attenuate PHD

as well as FIH activity under normoxia, thus abrogating HIF-1á hydroxylation. Binding of HIF-1â constitutes the active HIF-1 dimer. Subsequent binding of the coactivator p300/CBP promotes gene activation. For details see text.

tumors and/or tumor metastases with normal levels of HIF-1á in benign tumors [97]. The interior of a growing tumor becomes progressively hypoxic as its size increases, based on the notion that oxygen only diffuses around 150–200 microns from capillaries. Thus stabilization of HIF-1á is in part because of its induction through the ubiquitous pathway of oxygen sensing and signaling. In addition, tumor-specific genetic alterations, i.e. mutations involving oncogenes and tumor suppressor genes, often enhance HIF-1 expression [19, 75]. For example, loss of pVHL, PTEN or p53 tumor suppressor genes is associated with HIF-1á expression as well as the transforming potential of the v-Src oncogenes. The striking upregulation of HIF-1á in many dif-

13.5 Conclusions

ferent tumors by both physiologic and epigenetic mechanisms raises the question how HIF-1 impacts tumor biology. Apparently, HIF-1 allows metabolic adaptation to hypoxia, promotes angiogenesis, enhances survival and stimulates proliferation. Therefore, aberrant HIF-1á overexpression in brain, breast, cervical, esophageal or ovarian cancers is correlated with treatment failure and mortality. Moreover, RNI have been shown to play an important role in tumor growth and progression [98]. Expression of NOS has been demonstrated in a variety of tumors including breast, head, neck, prostate, bladder, colon, and CNS tumors such as glioblastomas [99]. RNI promote tumor growth by multiple actions such as regulating blood flow, maintaining the vasodilatory tone, promoting metastasis by increasing vascular permeability as well as affecting matrix metalloproteinases and stimulating angiogenesis. The observation that RNI share with hypoxia the ability to stabilize HIF-1á may be relevant for various aspects of tumor biology. Conversely, several reports have documented that increased production of RNI reduced tumor cell survival and induced tumor cell death [100]. At least in part, mechanisms may point to a proapoptotic mechanism being compatible with RNI-evoked p53 stabilization [44]. However, as the role of RNI in affecting apoptosis can be pro- as well as anti-apoptotic, the impact of RNI is linked to pro- as well as anti-tumor activities [101]. It appears that high levels of RNI formation, as generated via iNOS, may be cytostatic or cytotoxic, whereas low level RNI generation via constitutively active NO-synthases can have the opposite effect and promote tumor growth [102, 103]. The regulation of tumor growth by RNI represents an important new dimension in cancer research. Multiple facets of RNI signaling such as HIF-1á or p53 accumulation as well as the regulatory impact of RNI on apoptosis need consideration to determine the precise role of RNI in tumor biology and to understand contrasting observations of RNI in promoting or inhibiting the etiology of cancer.

13.5

Conclusions

Altered gene expression by RNI constitutes an integral component to explain their signal transmission capabilities. This appears relevant for RNI in coordinating inflammation, affecting proliferation, differentiation and regulating cell survival decisions. Transcriptional regulation is in the sphere of RNI actions and, among multiple transcription factors, HIF-1á and p53 are emerging regulatory targets that link RNI signaling with medical related problems of tumor biology and/or cell viability decisions. Mechanistically, we are beginning to understand how RNI mimic a hypoxic response by attenuating prolyl hydroxylase activity under normoxia and how RNI stabilize p53 by affecting protein phosphorylation and/or an altered compartmentalization of p53. Fig. 13.3 summarizes these aspects and implicates pathophysiological consequences ranging from cellular signaling, i.e. adaptation, to death and tumor biology. RNI have not been considered classical, i.e. originally identified activators of p53 or HIF-1á. This provokes the question of the relevance of RNI in activating gene

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Fig. 13.3 HIF-1á and p53 as mediators of RNI

signaling. RNI affect HIF-1á stabilization to promote its interaction with HIF-1â. Recruitment of the coactivator p300/CBP causes gene activation to elicit pathophysiological responses. RNI activate

p53, e.g. causing tetramerization and recruitment of the coactivator p300/CBP provokes gene activation to suppress cell cycle progression and/or to elicit apoptosis. HIF-1, p53 as well as direct RNI actions contribute to affect tumor pathogenesis. For details see text.

expression via p53 or HIF-1. Gene expression profiling may help to answer this question in the future as well as our search for medical symptoms associated with RNI formation and transcriptional regulation via p53 and/or HIF-1. Assuming numerous genes to be under the control of RNI, using multiple transcriptional regulators, we need to establish a hierarchy of gene activation processes that determines and allows one to predict the signaling qualities of RNI.

Acknowledgement

We apologize to researchers whose primary observations that form the basis for our current knowledge in this active field could not be cited due to space limitations, or have been acknowledged indirectly by citing review articles, only. Our work was supported by grants from Deutsche Forschungsgemeinschaft (Br 999), Deutsche Krebshilfe (10-2008-Br2) and the Sander Foundation (2002.088.1).

Abbreviations

Abbreviations

ATM ATR AhR ARNT cGMP CBP CTAD GSNO HIF-1 HRE (i)NOS MAPK Mdm2 ODD p53 PHD pVHL RNI ROI VEGF

ataxia telangiectasia-mutated ATM- and Rad3-related Aryl hydrocarbon receptor AhR nuclear translocator cyclic guanosine monophosphate CREB (cAMP-response element binding protein)-binding protein C-terminal transactivation domain S-nitrosoglutathione hypoxia inducible factor-1 hypoxia-response element (inducible) nitric oxide synthase mitogen activated protein kinase murine double minute oxygen-dependent degradation domain tumor suppressor p53 prolyl hydroxylase domain-containing protein von Hippel–Lindau protein reactive nitrogen intermediates reactive oxygen intermediates vascular endothelial growth factor

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Nitric Oxide and Central Nervous System Diseases Elizabeth Mazzio, Karam F. A. Soliman 14.1

General Overview – Gaining Control over Various NOS Enzymes that Concurrently Contribute to Degenerative CNS Diseases

Nitric oxide (NO) is an unstable gaseous radical that plays a central role in human neurological and cardiovascular function, as well as central nervous system (CNS) disease. NO is synthesized from the electron oxidation of l-arginine to l-citrulline through various isoforms of nitric oxide synthase (NOS). The NOS enzyme is an oxidoreductase that requires oxygen, heme iron, tetrahydrobiopterin, â-nicotinamide adenine dinucleotide phosphate, flavin mononucleotide and flavin adenine dinucleotide [1–4]. And, the three primary isoforms of NOS are neuronal NOS (NOSI:nNOS), inducible NOS (NOS-2:iNOS) and endothelial NOS (NOS-3:eNOS). NOS-1 and NOS-3 have distinct similarities in that both isoforms are activated by intracellular Ca2+ /calmodulin [3, 5], and the NO generated is a signaling molecule that regulates 3′,5′-cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG) phosphorylation events that control autonomic, central cardiovascular and neurological functions [1, 6–9]. Nitric oxide generated by NOS-2 is markedly different from NOS 1 and 3, where its primary role resides in monocyte/macrophages assisting in destruction of infection, invasive pathogens or necrotic debris. While NO molecules generated by NOS 1 and 3 are primarily transient signaling molecules, NO synthesized by NOS-2 plays a central role in immune response to injury, where it can also contribute to the destruction of healthy tissue during chronic or acute inflammatory diseases [10, 11]. In the CNS, an inflammatory response triggered by injury, infection or neuronal necrotic debris is carried out in part by glia (microglia and astrocytes). Astrocytes respond to cytokines via signaling through stress-activated mitogen-activated protein kinases (MAPKs) that phosphorylate and activate transcription factor/DNA binding elements initiating iNOS mRNA transcription [12, 13]. Since iNOS protein expression is highly up-regulated by pro-inflammatory cytokines, CNS inflammation can yield dangerously high quantities of NO over extended periods of time, possibly posing an irreversible threat to the health and viability of post-mitotic neurons [14]. All three isoforms can diverge from their functional regulatory roles and during disease states contribute to NO mediated pathologies of the CNS. Excessive accuNitric 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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mulation of NO by nNOS or iNOS can prompt its reactivity with molecular oxygen (O2 ) or other reactive oxygen species (ROS) to produce a number of reactive nitrogenated species (RNS), such as dinitrogen trioxide [N2 03 ] nitrogen dioxide [NO2 ], peroxynitrite [ONOO− ], peroxynitrous acid [ONOOH], nitroxyl ions [NO− ], nitrosonium ions [NO+ ], and dinitrosyl iron complexes [15]. The accumulation of ONOO− / ONOOH can induce both nitration/nitrosylation of proteins and oxidation of lipid membrane structures, that contribute to neuronal apoptosis, loss of mitochondrial electron transport (ETC) function and degradation of nuclear DNA [16–21]. The accumulation and concentration of iNOS, nitrosylated proteins and lipid peroxidation markers in the CNS juxtaposes both aging [22] and neurodegenerative injury associated with Alzheimer’s disease (AD) [23, 24], inflammation [25], autoimmune encephalomyelitis [26], ischemia, head trauma [27, 28], spinal cord injury, [29], stroke [30], multiple sclerosis (MS) [31], amyotrophic lateral sclerosis (ALS) [32], cerebrovascular damage, traumatic brain injury [33], HIV1-related encephalitis and dementia [34], demyelinating diseases [35], cerebral malaria [36] and Parkinson’s Disease (PD) [37]. Pharmaceutical manipulation as a means to effectively combat and minimize the detrimental effects of NO can be achieved through integration of drugs that either indirectly or directly target diverse effects on 1. NOS mRNA, 2. NOS enzyme activity, 3. the conversion of NO to neurotoxic species or 4. block downstream cell signaling systems involved with NO-mediated apoptosis and necrosis. While nNOS and iNOS contribute to toxic events through overproduction of NO, conversely, it is the loss of eNOS activity that can create injurious effects to the brain. NO generated by eNOS (located in the vascular endothelium), evokes vasodilation and a deficit can initiate hypertension/vasospasms, exacerbating stroke or other CNS diseases that require abundant delivery of blood, nutrients and oxygen to damaged neurons [38, 39]. And, for this reason, existing research has generally concluded that a therapeutic approach to combat the toxicity of NO in CNS disease involves employing agents that simultaneous down-regulate iNOS and nNOS, and augment eNOS [40, 41]. This can be achieved with either selective NOS enzyme inhibitors, or by manipulating equivalent cell signaling controls that perpetuate beneficial diverse effects on various isoforms of CNS NOS. For example, a number of promising agents can concomitantly antagonize pro-inflammatory transcription of glial iNOS mRNA, increase vascular eNOS, and block cell death associated with nNOS and glutamate. Some of these include poly(ADP-ribose)-polymerase-1 (PARP-1) inhibitors [42–45], thiazolidinediones (TDs)/peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists [46–50] phosphodiesterase (PDE) inhibitors (PDE-1C, PDE4, PDE-5) [51–55] and stress-activated mitogen-activated protein kinase of 38 kDa (p38/SAPK)/c-Jun-NH2 -terminal kinase (JNK/SAPK)-inhibitors [56–60]. Another promising class of drugs to treat NO-related pathologies are superoxide dismutase (SOD) mimetics [61–64], which can rapidly dissipate superoxide (O2 − ) and prevent the formation of ONOO− which, if present, appears to be the single most deleterious RNS in the CNS. ONOO− can trigger hypertension through attenuating eNOS, induce apoptosis through opening of the mitochondrial permeability transition pore complex (PTPC), and impart global oxidative stress throughout the brain. Removing O2 − or increasing the ratio of NO/ONOO− appears to switch NO from being a neurotoxic to a

14.2 Signaling Controls – Neuronal NOS: TYPE-I

neuroprotective /vasodilatory molecule. Other important cell signaling controls that can be targeted to concurrently regulate various isoforms of NOS to provide a simultaneous anti-flammatory, vasodilatory effect and/or block the downstream toxic effects of ONOO− include: adenylate cyclase, 3′,5′-cyclic adenosine monophosphate (cAMP), cGMP, protein kinase A (PKA), tyrosine kinase, protein phosphatase, Janus tyrosine kinase, angiotensin converting enzyme (ACE), extra-cellular signal-regulated protein kinase 1/2 (ERK/1/2), ERK[1/2], p38/SAPK, JNK/SAPK, PPAR-gamma, PARP-1, bcl2 family proteins, glutathione, nicotinic acid and antagonizing the effects of inositol 1,4,5-triphosphate (IP3 ) or blocking Ca2+ voltage/ligand activated receptors. Each of these will be briefly discussed in terms of mechanism, impact and control over lethal forms of NO that contribute to CNS disease.

14.2

Signaling Controls – Neuronal NOS: TYPE-I 14.2.1

Neurotransmission

In order to gain understanding as to how to combat the detrimental effects of NO in the CNS, it is worthwhile to briefly delineate the normal function of each NOS isoform, its upstream regulatory controls and the downstream target of molecular NO. In neurons, NO produced by nNOS plays a central role in cell signaling through activation of soluble guanylyl cyclase (sGC) [65, 66]. Soluble GC is variably dispersed in CNS neurons and activated by NO through its interaction with the catalytic heme binding domain of á1 and â1 subunits, prompting a conformational change that initiates conversion of guanosine triphosphate (GTP) to cGMP [67–70]. In neurons, the accumulation of intracellular cGMP can directly control cyclic nucleotide-gated membrane ion channels [71, 72] or coordinate a multitude of diverse signaling effects through activation of PKG [73]. In general, protein kinases serve to add a negative phosphate group onto protein structures, thereby altering electrostatic charges which result in the modification of structural/functional characteristics that control open/closed or on/off signaling systems [74]. While there are many types of neuronal protein kinases, PKG specifically regulates neurotransmission and feed-back responses required for adaptation and sensitization to environmental stimuli. For this reason, the activity of nNOS in the CNS is integral to neurophysiological sensory function such as vision, learning, nocioception, antinocioception, hearing, digestion, circadian rhythms, sleeping, olfaction, memory, reproduction, neuro-endocrine function and cognition [75–82]. One of the primary functions of CNS nNOS in the regulation of sGC is control over neurotransmitter (NT) release, which coordinates communication through relaying synapses. NO can either act as a direct neuromodulator [83, 84] or its role lies in carrying out Ca2+ initiated nNOS/ NO/sGC/cGMP/PKG signaling, that regulates synaptic NT release [85–90]. The presence of NO near and or around neurons is known to induce rapid neuronal firing and augment NT release, and this is consistently

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demonstrated in studies of diverse neuronal cell type such as glutaminergic, nicotinic, gamma aminobutyric acid (GABA)-ergic, cholinergic and adrenergic neurons [86–89, 91–94]. Depending upon the type of NT released and postsynaptic modulation of permeability to select ions, NO can augment both excitatory [92, 95] or inhibitory postsynaptic potentials [96]. Although a specific mechanism defining the role for NO in NT release has not been elucidated, studies consistently demonstrate that excitatory NT receptor activation can lead to Ca2+ influx, activation of nNOS and NT release, effects that are blocked by tetrodotoxin, Ca2+ -deficient buffers, intracellular Ca2+[I] buffering agents, hemoglobin (NO trapping agent) or nNOS inhibitors such as N(g)-nitro-l-arginine methyl ester (l-NAME) or N(g)-monomethyl-l-arginine (l-NMMA) [94, 97]. While NO produced endogenously through nNOS can carry out these effects, an exogenous supply of larginine or NO donors such as S-nitrosoacetylpenicillamine and S-nitrosoglutathione can also elicit NT release, effects that are blocked by conotoxin, N-type Ca2+ channel blockers, hemoglobin, inhibitors of NOS: l-NMMDA, 7-nitroindazole, sGC: 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (ODQ), PKG, and are exacerbated by permeable analogues of cGMP, such as 8-bromo-cGMP and dibutyl-cGMP [92, 94, 96–99]. These findings support a clear role for Ca2+ -activated nNOS, and regulation of NT release through PKG. While there is a wealth of information suggesting a relationship between Ca2+ / NO and NT release, there is little to no information defining a specific role for PKG in regulating synaptic events at the axonal nerve terminal. Further compelling, are reports that suggest that the NO molecule itself, independent of signaling systems can potentiate Ca2+ -evoked exocytosis of NT. Calcium has a well known role in NT release through its activation of Ca2+ kinases [Ca2+ /calmodulin-dependent protein kinase CAM kinase I, II] [100] that phosphorylate the synaptic vesicle protein synapsin, reducing affinity to actin and thereby initiating release of vesicles from the active zone of nerve terminals [101, 102]. However, a fairly high concentration of nNOS is also located at the nerve terminal and 3-nitrotyrosine (3-NT) has been detected in synaptic vesicles proteins such as synaptobrevin, synaptophysin, munc-18, SNAP25, indicating that NO may play a role in both docking and priming of vesicles prior to release [103–105]. Further, 3-NT is a protein oxidative marker for ONOO− [106, 107], indicating that ONOO− is being produced in the nerve terminal apparently contributing to an unknown but powerful role in synaptic vesicle destabilization. While an exact mechanism is not known, ONOO− may augment vesicle release by irreversibly inhibiting synaptosomal, plasma and SERCA/reticulum membrane Ca2+ ATP-ase pumps, which may potentiate cytosolic Ca2+[I] , creating a lower threshold for NT receptor activation and exocytosis of synaptic vesicles [108, 109]. Moreover, in the presence of NO donors, the evoked NT release is abolished in the presence of Cu2+ /Zn2 +-SOD, O2 -scavengers, Ca2+ chelator: EGTA and hemoglobin [94, 110, 111]. These findings indicate a synergistic role between ONOO− , Ca2+ and NT release, and further suggest a pre-eminent role for O2 − in this process, although posing a questionable role for PKG. While there are many aspects to the neurotoxic effects of nNOS, which are discussed in greater detail in Section 14.5, the lethal effects appear to be directly due to

14.2 Signaling Controls – Neuronal NOS: TYPE-I

accumulation of the NO molecule itself, independent of sGC activity, cGMP or PKG [112]. In disease states, the amassing of NO produced during glutamate induced – Ca2+ – overload is lethal to neurons, where both cGMP and PKG exert a non-toxic regulatory and potentially neuroprotective role [65, 113]. Therefore, a targeted endpoint for antagonizing the direct effects of nNOS, would be upstream to the function of the enzyme, achieved by controlling Ca2+[I] . 14.2.2

Neuronal Calcium Homeostasis

The primary upstream control over nNOS is the neuronal concentration of intracellular Ca2+[I] . Typically, the control of Ca2+[I] is regulated by homeostatic efflux/influx cell membrane transport systems, such as Ca2+ ATP-ase pumps, ion channels, uniporters and H+ /Ca2+ :Na2+ exchangers. In neurons, the initial rise in Ca2+[I] can occur through the influx of extracellular Ca2+ , triggered by activation of plasma membrane voltage dependent or ligand-gated Ca2+ channels and/or the loss of Ca2+ uptake from internal storage compartments, such as the endoplasmic reticulum (ER) and mitochondria. Typically, a potent spike in Ca2+ i triggers an all or none-response in excitable type cells. However, in neurons, this can also contribute to toxicity where either depolarization or overactivation of glutaminergic ionotropic Ca2+ activated channels: N-methyl-d-aspartate (NMDA), á-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) and kainite (KA), and G protein-coupled metabotropic receptors, linked to phosphospholipase C (PLC) [114] contribute to the rapid influx of Ca2+ , the activation of nNOS and neurotoxicity. The rapid influx of Ca2+ , and phospholipase C (PLC) release of IP3 , can collectively escalate the rise in Ca2+[I] , by inducing Ca2+ -induced Ca2+ release (CICR) through activation of ER-IP3 and ryanodine sensitive Ca2+ release channels, and opening of the mitochondrial permeability transition pore complex (PTPC) initiating massive Ca2+ efflux through a reverse Na2+ /Ca2+ antiporter in the mitochondria [115, 116, 117]. Preventing the initial rise of Ca2+[I] , in a superfluous environment of glutamate is neuroprotective, and can be achieved by blocking Ca2+ entry at the NMDA receptor: MK-801, N-alkylglycines, the AMPA/kainite receptor: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitroquinoxaline-2,3dione (DNQX), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo-(f)-quinoxaline (NBQX) or the metabotropic glutamate receptor: 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) [92, 118–123]. Further, blunting the rise in intracellular Ca2+[I] , can be mediated by blocking ryanodine sensitive Ca2+ release channels (dantrolene) or with PLC inhibitors, internal ER IP3 receptor antagonists, Ca2+ chelators or calmodulin antagonists [112, 123–127]. The resting voltage of the plasma membrane also plays a critical role in establishing the propensity of glutamate to open voltage-activated Ca2+ channels. Depolarization or leakage of Na2+ ions can prompt a lower threshold for action potential, creating a hyper-excitable membrane. Depolarization reduces the affinity of Mg2+ to create a voltage-dependent block of the NMDA channel, leading to opening of the pore and a greater ligand-receptor affinity for glutamate [128]. In contrast, hyperpolarizing the membrane or increasing extracellular Mg2+ concentration can establish a tighter Mg2+ voltage-dependent block of the

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NMDA receptor, with less susceptibility to Ca2+ mediated entry by glutamate [129, 130]. In total, antagonizing direct upstream control over the nNOS isoform can be achieved in part by application of internal and external Ca2+ channel blockers, Ca2+ buffering agents, hyperpolarizing agents or selective nNOS inhibitors such as 6 or 7nitroindazole, 1-(2-trifluoromethylphenyl) imidazole or 6-phenyl-2-aminopyridines [131–133].

14.3

Signaling Controls, Endothelial NOS: Type-3 14.3.1

EDRF/Vascular Tone

While nNOS is critical to the function of neurotransmission, endothelial NOS presents itself in vascular endothelium and platelets, serving a crucial role in cardiovascular function and CNS blood flow. NO generated by eNOS induces robust vasodilation, endothelial smooth muscle relaxation, and antagonizes thrombosis/platelet aggregation [8, 9, 134]. Both NO and prostacyclin are referred to as endothelium derived relaxing factors (EDRF) and a chronic deficit of one or both can lead to hypertension, atherosclerosis, stroke and congestive heart failure [135–138]. Endothelial NOS is constitutive, augmented in the presence of l-arginine [139], regulated by Ca2+ /calmodulin and controls sGC/cGMP-dependent activation of PKG which regulates phosphorylation of proteins that initiate a vasodilatory response [8, 140]. In the cardiovascular system, the eNOS/NO/sGC/cGMP/PKG vasodilatory response is triggered by prostacyclin, bradykinin, potassium chloride, low blood pressure, low oxygen tension, acetylcholine, histamine and vasoactive intestinal polypeptide (VIP) [141–145]. Bradykinin is a key regulator and potent activator of eNOS in the circulatory system. Agents that can prevent the degradation of bradykinin such as ACE inhibitors, also evoke vascular relaxation and vasodilation [146]. ACE inhibitors provide a dual protective mechanism, in that they augment a vasodilatory response to bradykinin and attenuate formation of angiotensin II (Ang II) [147, 148], that if present yields vasoconstrictor effects by acting on endothelial AT-type 1 receptors [149]. Moreover, a reduction in Ang II can also prevent its potent role in the up-regulation of NADPH oxidase activity which generates O2 − and in the presence of NO forms a potent oxidizing and vasoconstrictor molecule, ONOO− [150, 151]. In the cardiovascular system, ONOO− is detrimental because it can directly evoke hypertension [152] and induce oxidative injury to the vessel walls, effects that are attenuated by removing O2 − [153] or exacerbated in SOD-/- knockout mice [154]. Superoxide scavengers, that are one and the same with ACE inhibitors such as captopril, can have powerful therapeutic effects with ample capacity to potentiate bradykinin and even further increase NO/ONOO− [155–158]. Increasing the ratio of NO/ONOO− in the cardiovascular system has a number of beneficial effects, and for this reason ACE inhibitors can ef-

14.3 Signaling Controls, Endothelial NOS: Type-3

fectively improve circulatory function, reduce risk of heart attack, stroke, myocardial infarction, hypertension, congestive heart failure and general mortality from cardiovascular related events [159, 160]. Alternatively, EDRF/NO can also be potentiated by administration of l-arginine or nitrates such as nitroglycerin, glycerol trinitrate, amyl nitrite and isosorbide dinitrate, that can also effectively treat cardiovascular disease, myocardial ischemia, angina pectoris and hypertensive crisis [138, 161]. 14.3.2

eNOS, Cyclic AMP/GMP Regulation

Unlike nNOS, where Ca2+ channel blockers provide neuroprotective effects, agents that increase the activity of eNOS are beneficial. While eNOS is regulated in part by Ca2+ /calmodulin in a similar fashion to nNOS, it is also highly regulated by phosphorylation at serine residues through activity of phosphatidylinositol-3-OH-kinase (PI3K/AK2) and PKA [162, 163]. The effects of PKA in the potentiation of eNOS are quite robust and independent of changes in Ca2+[I] [164]. Agents that can augment intracellular cAMP (which activate PKA), such as forskolin, 8-bromo-cAMP, beta-adrenergic receptor agonist isoproterenol and adrenomedullin also heighten eNOS activity and induce a vasodilatory response [165–167]. And, primary vasoactive substances such as bradykinin and VIP also activate eNOS through a mechanism involving phosphorylation of serine residues, effects which are blocked by inhibitors of both PKA and PI3K [163, 168, 169]. These findings suggest an upstream control over eNOS via cAMP/PKA, whereas downstream effects of NO generated by eNOS involve controlling cGMP/PKG-mediated vasodilation. These findings indicate that an elevation of both cyclic nucleotides by PDE inhibitors should synergistically potentiate vasodilation of EDRF/NO and instill beneficial effects to cardiovascular function. Likewise, sildenafil or PDE-5 inhibitors are considered promising in the treatment of coronary heart disease, where they can prevent the degradation of cGMP, evoke endothelial vasodilation and attenuate platelet activation/adhesion in patients with heart disease [54, 55, 170, 171]. Moreover, the application of PDE-1 selective inhibitor, 8-methoxymethyl-1-methyl-3-(2methylpropyl)-xanthine thwarts the degradation of cGMP and induces vasodilation, effects that are blocked by sGC inhibitor ODQ and restored by NO donors, defining a specific role for NO in regulating GS/cGMP and PKG [172]. Similar effects are observed with cAMP specific-PDE inhibitors such as the type 3 inhibitors: CI 930, cilostazol milrinone, 94120 and LY 195115, which can effectively heighten NO/EDRF in vascular tissue, thoracic aorta and arterioles effects that are blocked by NOS inhibitors. These findings corroborate a role for cAMP in regulating eNOS/NO/GS/cGMP and PKG [53, 173, 174]. The application of PDE4 inhibitors (rolipram and denbufylline), also prevents degradation of cAMP and can induce endothelial relaxation, effects that are reversed by addition of l-NMMA and restored by addition of l-arginine [174]. While these studies demonstrate that PDE inhibitors augment the function of eNOS in cardiovascular tissue, the effects of blocking degradation of AMP can also downregulate the iNOS isoform in immunocompetant cells during CNS inflammation. For example, an increase of cAMP in astrocytes, microglia and other cell types can suppress NO production, and block

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induction of NOS-2 mRNA [51,175–178]. Together, cAMP selective PDE-4 /PDE-1C inhibitors may provide powerful anti-inflammatory, vasodilatory effects and thereby antagonize NOS imbalances that contribute to CNS disease. Further, since the effects of cGMP in neurons do not appear to contribute to toxicity [65, 112, 113], PDE-5 inhibitors may yield dual benefit although there is little research on this topic. Other known factors that can evoke EDRF/NO, include the statin drugs, green tea catechins [139, 155, 162, 179], lysophospholipids [180] sauna bath therapy [181], physical exercise [182], indomethacin [183], estrogen [184, 185], glutathione [186], and vitamin C [187]. In total, increasing eNOS activity can be achieved by administration of ACE inhibitors, organic nitrites, phosphodiesterase inhibitors, antioxidants, larginine, angiotensin II receptor (AT1 ) blockers, and prostacyclin analogs such as isocarbacyclin.

14.4

Signaling Controls, Inducible NOS: Type-2 14.4.1

Inflammation, Microglia and Astrocytes

During CNS inflammation, the largest contributor to the production of NO is cells of monocyte/ macrophage lineage, including microglia and astrocytes. Under normal conditions, astrocytes provide a protective functional and regulatory role in the brain by sustaining a nourishing interstitial milieu and platform for neurons to thrive. Astrocytes aid in regulating electrolyte balance, nutrient/metabolic transport, H+ ion concentrations, and assist with neuronal repair [188]. Because astrocytes are basically the CNS housekeeper, they are equipped with robust neuroprotective apparatus. Some of these include a high intracellular concentration of reduced glutathione (GSH), vigorous antioxidant enzymes such as catalase and glutathione peroxidase (GSH-Px), the ability to synthesize glycogen as a storage fuel, to release trophic factors: nerve growth factor, brain-derived neurotrophic factor, transforming growth factor-â, fibroblast growth factor, to produce anti-inflammatory cytokines, and to provide a rapid disposal route for extra-cellular glutamate via high capacity Na+ -coupled glutamate uptake transporters (GLT1 and GLAST1) with metabolic detoxification through glutamine synthetase [188–192]. During trauma or CNS disease, astrocytes can diverge from their normal defensive function, and respond to necrotic debris by creating a toxic inflammatory response. Markers of inflammation in astrocytes consist of morphological changes evident by cell proliferation, swelling, hypertrophy, increased expression of glial fibrillary acidic protein (GFAP), iNOS, cycloxygenase (COX) and expression of immunological proteins inherent to antigen-presenting cells such as class I/II major histocompatibility complex (MHC) antigens, vascular adhesion molecules (VLA-1, VLA-2, and VLA-6), lymphocyte function-associated molecule-3 and release of pro-inflammatory mediators such as tumor necrosis factoralpha (TNF-á), interleukin 1â (IL-1â), interferon–gamma (IFN-ã), and prostaglandins [193, 194]. CNS inflammatory disease often accompanies reduced expression or func-

14.4 Signaling Controls, Inducible NOS: Type-2

tion of astrocytic GLT1 and GLAST1, leading to the accumulation of extracellular glutamate [188, 195, 196]. While there are many contributors to neurological damage during inflammation, the primary vulnerabilities posed by astrocytes with regards to NO are: the extensive production of a highly diffusible NO molecule, depletion of intracellular GSH and reduced ability to handle glutamate, which accumulates in extracellular synaptic spaces resulting in excitotoxic neuronal cell death [14, 129, 191, 197, 198]. Microglia cells (resident macrophages of the brain) also become lethal participants, demonstrating ample facility to proliferate, migrate, infiltrate to various regions of the brain and participate in active phagocytosis. Active microglia also display similar characteristics to antigen-presenting cells typical to the humoral immune response, including the ability to express complement receptors, MHC class I and II antigens, Fc gamma receptors, immunoglobulin E receptors, intercellular adhesion molecule1 (ICAM-1), VCAM-1, iNOS, COX-2, and to produce and release pro-inflammatory cytokines: TNF-á and IL-1â [199-204]. Although experimentally induced CNS inflammation in rodents is associated with expression of iNOS in both microglia and astrocytes, in the human brain, astrocytes are believed to be a primary generator of NO. Studies consistently demonstrate that adult and fetal astrocytes produce abundant NO and express iNOS mRNA; however there are discrepancies amongst findings regarding iNOS in activated human microglia [194, 205, 206]. Microglia cells can however, release pro-inflammatory cytokines such as TNF-á and IL-1â that in turn can induce expression of iNOS in astrocytes. Therefore, both cell types are lethal participants in neurodegenerative CNS disease. 14.4.2

Stress Activated and Extra-cellular Kinases

Expression of glia NOS-2 mRNA is primarily mediated by kinase signaling pathways that are initiated in response to surface contact with pro-inflammatory cytokines such as TNF-á, IL-1â, IFN-ã, S100B, or pathogenic antigenic substrates derived from bacterial, parasitic or viral agents [207–211]. While there are a number of kinase signaling cascades that control induction of iNOS in glia [212–214], primarily proinflammatory agents trigger one or more of the three central mixed lineage kinase (MLK) pathways: ERK/MAPK/1/2, p38/SAPK and JNK/SAPK [58, 209, 215–217]. MLK signaling cascades are highly complex, interconnected and classified under mitogen-activated protein kinase phosphorelay modules that control diverse signal transduction pathways within mammalian cells, in particular in response to stress [218]. Ultimately, in astrocytes, SAPK/ERKs control the pro-inflammatory response, by directly phosphorylating and activating DNA binding elements and transcription factors such as activator protein-1 (AP-1), cAMP response element binding protein (CREB), Jun-c and nuclear factor kappaâ (NF-kappaâ), that permit binding to the DNA promotor region of iNOS, initiating mRNA transcription through RNA polymerase II [12, 13, 215]. The activity of p38/SAPK is central to the genetic changes that occur in response to cytokine activated pro-inflammatory signaling in astrocytes and microglia. p38/SAPK

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enzyme inhibitors such as 4-(4-fluorophenyl)-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole or SB203580, exert powerful anti-inflammatory effects, blocking expression of iNOS, TNF-á and IL-1â [12, 13, 58, 219]. Moreover, SB203580 is effective in blocking iNOS nuclear transcription in several models of inflammation and in response to various types of pro-inflammatory stimuli such as bacterial/viral pathogenic antigens [209, 220]. While a substantial role for p38/SAPK and JNK/SAPK in regulating cytokine regulation of iNOS is consistently reported [12, 217, 221, 222], results have been variable in defining a role for ERK/MAPK/1/2, which is typically involved with cell growth and proliferation signaling. Use of inhibitor PD98059 has been reportedly both ineffective and effective in blocking LPS/cytokine induction of iNOS in primary astrocytes, glioma and microglia cell lines [12, 58, 223]. The activities of p38/SAPK, JNK/SAPK or ERK/MAPK/1/2 are essentially involved with phosphorylation of CREB, AP-1 and NF-kappaâ, guiding conformational changes in these proteins, that allow binding to the DNA promoter region of the iNOS gene [215, 221, 224]. While protein kinases turn on expression of genes, protein phosphatase 1 (PP1) and PP2A typically cleave a phosphate group off at serine residue 133/142 of CREB [225]. In astrocytes, both PP1 and PP2A control iNOS mRNA and NF-kappaâ DNA binding, events that are reportedly upregulated in the presence of inhibitors such as okadaic acid [226]. These findings suggest that PP1 and PP2A antagonize the function of SAPK enzymes, either directly or in competition between phosphorylation/dephosphorylation of transcription factor/binding elements in the nucleus that regulate iNOS transcription. There are also other counteracting controls to the activation of NF-kappaâ in astrocytes, such as expression and accumulation of Ikappaâ-alpha (IKA) and Ikappaâ-beta (IKB) proteins [227]. IKA and IKB are inhibitory proteins that are degraded upon phosphorylation by IKA and IKB kinases, which correspond to the upregulation of NF-kappaâ. Increased activation of IKA can inactivate NF-kappaâ translocation and antagonize association with the CREB-binding protein, which is critical in initiating iNOS transcription [215, 221, 224, 228]. It appears that some of the anti-inflammatory cytokines such as IL-4 or IL-10 mediate effects via this signaling, where they up-regulate IKA mRNA, thereby reducing NOS-2 [211]. 14.4.3

Cyclic AMP/Protein Kinase a

Other protein kinases may indirectly influence the activation of NF-kappaâ. For example, in contrast to the pro-inflammatory effects typically observed with activation of kinases, the elevation of cAMP activates PKA and blocks transcription of iNOS mRNA [51, 178, 229, 230]. Astrocytes contain a variety of NT receptors that are coupled to Gsadenylate cyclase [231] and, either activation of â-adrenergic/dopamine receptors or employing agents that increase cAMP, such as forskolin (adenylate cyclase activator), PDE inhibitors [i.e. pentoxifylline], dibutyrl cAMP, or 8-bromo cAMP can attenuate lipopolysaccharide (LPS)/cytokine activated iNOS mRNA in microglia, astrocytes and a number of other cell types [51, 176, 177, 178, 232–237]. In contrast, agents that suppress the intracellular concentration of cAMP such as H-89 and Rp-cAMP are pro-

14.4 Signaling Controls, Inducible NOS: Type-2

inflammatory and augment iNOS expression in astrocytes [51]. Although there is an abundance of evidence to support cAMP/PKA activation and suppression of iNOS mRNA, in theory, this is the opposite to what would be expected by cAMP/PKA which typically phosphorylates CREB, a regulatory promotor of transcription [238]. However, this is not the case, and studies consistently demonstrate that cAMP suppresses the transcription of pro-inflammatory proteins in diverse immuno-competent cells including astrocytes and microglia [178, 229, 230, 237, 239]. Although the mechanism of action for cAMP in mediating effects on iNOS in astrocytes has not been clearly elucidated, there are preliminary studies that indicate plausible controls. The antiinflammatory effects of cAMP may involve activation of cAMP-responsive element that initiates transcription of suppressor of cytokine signaling proteins (SOCS-1,3) [240]. In immuno-competent cells, SOCS proteins counteract cell signaling directed by pro-inflammatory cytokines such as TNF-á, IL-1â, IFN-ã, by directly blocking phosphorylation and activation of janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT)–(JAK/STAT) pathway [212]. In astrocytes, tyrosine phoshorylation of JAK2 and STAT1 alpha/beta are required for LPS/IFN-ã induction of iNOS mRNA [213]. Moreover, cytokine induction of iNOS in other type of cells are blocked by JAK2 inhibitor tyrphostin B42 [241], which also prevents degradation of IKA and blocks the translocation of the NF-kappa B p65 into the nucleus [242]. Agents that can increase intracellular cAMP, such as forskolin and dibutyryl cAMP also induce SOCS-3 mRNA protein expression in immuno-competent cells [243]. Overexpression of SOCS-3, can antagonize the pro-inflammatory effects of IL-1â, IFN-ã and block cytokine induction of iNOS [244, 245]. Ultimately, it is possible that the means by which tyrosine kinase inhibitors such as genestein [214] and adenylate cyclase activators [229], attenuate iNOS transcription in astrocytes, may merge at SOSC-3/enzyme inactivation of JAK/STAT, both preventing phosphorylation and activation of JAK [246]. There is also evidence to link cAMP to both JAK/STAT and the function of ERK/MAPK/1/2, a regulatory control of iNOS in astrocytes [58, 223]. In macrophages, ERK/MAPK/1/2, can phosphorylate STAT1alpha and thereby activate JAK2/STAT1alpha which is integral for the cytokine induction of iNOS [247]. In glia, both stress and inflammation can provoke activation of the ERK/MAPK/1/2 signaling [248, 223, 249], that is blocked by cell permeable analogues of cAMP, such as 8-CPT cAMP or other cAMP potentiating agents such as forskolin/isobutylmethylxanthine or isoproterenol [248]. These data suggest that cAMP potentiation of SOSC-3 [243] may prevent the activation of JAK/STAT [240] and block ERK1/2 activation of NFkappaâ [58, 223]. In glia, elevation in cAMP juxtaposes the elevation of IKA, and inactivation of NF-kappaâ [250]. Future research will be required to delineate a more precise mechanism for cAMP in control of iNOS. However, these findings suggest therapeutic application of selective cAMP-specific PDE inhibitors, which negatively control cytokine activation of iNOS.

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14.4.4

Cyclic AMP–Phosphodiesterase Inhibitors

Both astrocytes and microglia contain PDE-4 and PDE-1C, and both show highspecificity for cyclic AMP [178, 251–253]. As mentioned previously, PDE-1C and PDE- 4 inhibitors also have beneficial effects on cardiovascular function by augmenting endothelial NO/EDRF. While there is very little research investigating the potential use of PDE-4 inhibitors in CNS disease, its primary pharmaceutical use is well known in the treatment of chronic inflammatory diseases in the periphery, such as arthritis, chronic obstructive pulmonary disease and asthma. A large number of studies consistently show that PDE-4 inhibitors modulate anti-inflammatory effects in basophils, eosinophils, macrophages, lymphocytes and neutrophils [239, 254]. Although few studies have examined the efficacy of PDE-4 inhibitors in CNS disease, rolipram (PDE-4 I) and non-specific PDE inhibitors such as ibudilast, can suppress production and release of TNF-á, IL-1â, NO and O2 − in glial cells [52, 178, 239, 255]. In vivo, non-specific PDE inhibitors are under investigation, showing capacity to reduce neurodegeneration associated with cerebral ischemia, vascular dementia, AD, and stroke [256–259]. 14.4.5

Peroxisome Proliferator-activated Receptor-gamma

There is also evidence to support another pathway for cAMP, involving the peroxisome proliferator-activated receptor gamma (PPAR-gamma). PPAR-gamma is a nuclear hormone ligand receptor that, if activated, can induce a conformational change directly to DNA coactivator complexes (i.e. CREB-binding protein), preventing activation of NF-kappaâ, AP-1 and binding to the iNOS promoter, thereby suppressing iNOS mRNA transcription [47, 48, 49, 260–262]. In the CNS, PPAR-gamma receptors are expressed in astrocytes and throughout the brain and spinal cord [263]. Administration of lipophilic PPAR-gamma activators such as non-steroidal anti-inflammatory agents (NSAIDS) (indomethacin, fenoprofen, ibuprofen, flufenamic acid and diclofenac), the TDs (ciglitizone, rosiglitazone, troglitazone) or prostaglandin 15-deoxydelta12,14-prostaglandin J2 can gain direct access to the nucleus and exert powerful anti-inflammatory effects by directly blocking transcription of pro-inflammatory proteins [264–266]. The activation of PPAR-gamma is directly associated with the reduction of Ikappaâ kinase, elevated expression of IKA mRNA and inactivation of NF-kappaâ/ AP-1, which also corresponds to the suppression of a host of proinflammatory proteins such as iNOS, MHC-II antigen, ICAM-1, P-selectin, COX-2 and TNF-á [153, 261, 267–272]. Cyclic AMP may yield immunosuppressive effects through altering the PPAR-gamma receptor. In astrocytes, norepinephrin (NE) can induce upregulation of PPAR-gamma mRNA and protein, effects that are blocked by the â-adrenergic antagonist propranolol [273]. Similarly, NE can act on â-adrenergic receptors triggering activation of adenylate cyclase and cAMP [274], which are associated with inactivation of NF-kappaâ [51]. Future research will be required to

14.5 The Neurotoxicity of NO

delineate whether cAMP or PKA can directly upregulate genetic transcription of the PPAR-gamma receptor. While the use of PPAR-gamma agonists are widely known to treat other diseases such as cancer [275], diabetes [276, 277], arthritis and microbial sepsis [153, 269, 278], there are a growing number of studies demonstrating that these agents may protect against NO-mediated injury to the CNS [279]. In experimental models of PD, administration of pioglitazone can attenuate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced dopaminergic degeneration and loss of nigrostriatal function, effects that also correspond to reduction of iNOS, NO, inactivation of NF-kappaâ and heightened concentration of IKA [268, 280]. Moreover, PPAR-gamma ligands such as troglitazone and ciglitizone attenuate microglial activation in the presence of betaamyloid (Aâ), also corresponding to reduction of astrocyte proliferation, neurotoxicity and expression of pro-inflammatory modulators such as COX and MAC-1 [281]. PPAR-gamma agonists such as pioglitazone, also provide therapeutic advantage in animal models of experimental autoimmune encephalomyelitis, where reduction of neurological dysfunction and preserved myelin is associated with reduction of TNFá, IFN-ã, reduction in T cell proliferation and elevated concentration of IKA [282, 283]. While PPAR-gamma agonists appear to prevent neurodegeneration associated with CNS inflammatory conditions, they also augment the function of eNOS. Administration of TDs and rosiglitazone reduce blood pressure [284] and attenuate cardiac damage associated with ischemic/reperfusion-injury [50]. Interestingly, there is recent evidence to suggest that PPAR-gamma agonists also prevent excitotoxicity in neurons, independent of an inflammatory response [46]. These findings indicate significant promise for this class of drugs, having combined effects to antagonize global inflammation, iNOS, protect against excitotoxicity, while potentiating the function of eNOS. In summary, controlling pro-inflammatory cytokine signaling associated with iNOS protein expression in glia, may be achieved with NSAIDS (indomethacin, ibuprofen), cAMP-specific phosphodiesterase inhibitors, â-adrenergic agonists, PPARgamma ligands, ERK/MAPK/1/2, p38/SAPK and JNK/SAPK inhibitors, select SAPK inhibitors such as quercetin [285], tyrosine kinase inhibitors, or compounds that augment IKA, inhibit NF-kappaâ translocation, activation or DNA binding.

14.5

The Neurotoxicity of NO 14.5.1

Oxidative Stress

In the CNS, accumulation of NO generated by either iNOS or nNOS, in the presence of O2 , can produce a number of nitrogen oxide species [17, 286]. However, the formation of peroxynitrite/peroxynitrous acid (ONOO− /ONOOH) is thought to be the most critical in terms of contributing to neurodegenerative injury, where singlet NO− is innocuous and even neuroprotective [287, 288, 298, 290]. Peroxynitrite is generated by

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the chemical reactivity of superoxide (O2 − ), and NO, and once formed, can protonate to yield ONOOH that can spontaneously decompose into • NO2 and OH• [85,107]. In the CNS, low concentration of ONOO− is not toxic [291], however, during disease states the accumulation of ONOO− /ONOOH, without adequate handling capacity can be dangerous. These are potent oxidizing agents with ample capacity to destroy the integrity of lipid membrane structures and protein amino acid resides such as cysteine, glutathione, methionine, tyrosine, guanine and tryptophan [16, 18, 292]. In neurons and other biological systems, lipid/protein oxidation and nitrosylation reactions can render structural and conformational changes that lead to inactivation or malfunction of cellular constituents, contributing to nerve cell death. Some of these include: ONOO− -mediated inactivation of glyceraldehyde-3-phosphate dehydrogenase, creatine kinase and aconitase [16, 286, 293], inactivation of ETC-complex I, II, and V (F1, FO-ATPase), displacement of oxygen at cytochrome oxidase, loss of oxidative phosphorylation (OXPHOS) [20, 21, 293–295], oxidation of adenine nucleotide translocase resulting in PTPC opening and apoptosis [296, 297], hyper-activation of PARP-1 nuclear enzyme leading to depletion of energy substrates [43], loss of aerobic/anaerobic ATP production, inhibition of Ca2+ and Na2+ -K+ ATP-ase pumps [16, 108, 109], inactivation of antioxidant enzymes such as GSH-Px, catalase and SOD [16, 293, 298, 299, 300], depletion of endogenous GSH, which serves as an ONOO− detoxification system [197], and lipid membrane degradation yielding 4-hydroxynonenal (4-HNE) and nitrosoperoxo–lipid oxidation adducts [16, 292]. While the presence of ONOO− /ONOOH can trigger a surplus of deleterious events that contribute to cell death, ultimately, these center around a heightened synergy between oxidative stress created by NRS and ROS. In vitro, or in vivo, the oxidation, nitrosylation and inactivation of proteins mediated by ONOO− , NO2 , and ONOOH can lead to the accumulation of the protein oxidation marker 3-NT, easily detected by immuno-histochemical analysis [18, 301, 302]. Accumulation of 3-NT in the CNS is observed during aging, inflammation and chronic degenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) where its presence precedes, and accumulates in proximity to neurodegenerative lesions and corresponds to the loss of neurological function [303–308]. The accumulation of 3-NT is also observed tantamount to formation of protein carbonyls (a marker of ROS mediated damage) [303, 307], indicating a co-operative degenerative synergy by a number of radical species potentially including the hydroxyl radical (OH• ), hydrogen peroxide (H2 02 ), O2 − and ONOO− /ONOOH. There is ample evidence to suggest that the presence of ONOO− in the CNS, may render near complete exhaustion of normal endogenous antioxidant defenses such as alpha-tocopherol, ascorbic acid and GSH [309, 310]. Peroxynitrite can inhibit the catalytic activity of nearly all antioxidant enzymes, such as GSH-Px, glutathione reductase, catalase, glutathione S-transferase [298–300] Mn2+ -SOD and Cu2+ /Zn2+ SOD [311–313]. NO/ONOO− can also initiate the release of free Fe2+ from iron-sulfur heme centers and ferritin [286], oxidize transition metal complexes [16], and, with a deficit of H2 O2 metabolizing enzymes, escalate production of the destructive OH• radical. In total, the presence of ONOO− in the CNS creates an incredible vulnerability and platform for ROS-mediated damage, by rendering near complete annihilation

14.5 The Neurotoxicity of NO

of major endogenous cytosolic and mitochondrial antioxidant defenses. This is further substantiated by evidence demonstrating accumulation of thiobarbituric acid reactive substances (TBARS, a biomarker of lipid peroxidation) and toxic lipid peroxidative aldehyde products such as 4-HNE observed in the presence of 3-NT and protein carbonyls in CNS diseases, indicating massive oxidative stress [292, 305, 314, 315]. The effects of ONOO− in the CNS are pre-eminently dangerous because this molecule may initiate a progressive cycle that perpetuates itself. The overproduction of ONOO− can lead to the inactivation of SOD, which is required for endogenous dissolution of superoxide (O2 −• ) [313]. The molecular availability of O2 −• in biological systems is the rate-limiting factor for production of ONOO− , which is the most damaging NRS species [288–290, 318]. Moreover, during inflammatory diseases, elevated production of O2 −• can occur through mitochondrial dysfunction, NADPH oxidase or Ca2+ -activated xanthine oxidase, which propels reactivity with NO to form even more ONOO− , that can then further exacerbate the inactivation of mitochondrial and cytosolic SOD [309, 316, 317]. The inability to remove O2 −• can severely impact the lethality of the NO molecule, whereas scavenging O2 −• is extremely neuroprotective. This is consistently demonstrated in studies that show a reduction of neurological injury involving 3-NT, NO, ONOO− and 4-HNE, with administration of SOD mimetics, or in cell models and transgenic mice overexpressing Mn2+ -SOD, Cu2+ /Zn2+ -SOD or nNOS/iNOS (-/-) knock out [316–321]. In contrast, NO-mediated pathologies are exacerbated in SOD deficient (-/-) mice [322]. Therapetic value SOD/catalase mimetics such as manganese-salen complexes [62] are currently under investigation and show significant promise to reduce neurological injury associated PD [323], AD [324] aging [325], stroke [326] ALS [63] and autoimmune encelphalomyelitis [327]. While SOD mimetics can prevent the formation of ONOO− , once formed, GSH is the major detoxification system and intracellular sink for removing ONOO− [197]. Once again, the presence of ONOO− in the CNS can be very dangerous, because this molecule can also create a cyclic and progressive depletion of thiols and GSH. ONOO− can oxidize thiols by a two-electron oxidation reaction, and ONOOH can directly and irreversibly deplete intracellular GSH by a one-electron oxidation formation of thiyl radicals that can initiate a chain reaction [19, 328]. Agents that augment intracellular GSH, such as 3H-1,2-dithiole-3-thione or gamma-glutamylcysteine ethyl ester (GGCE) [329–331] can attenuate the neurotoxic effects of ONOO− . Conversely, preventing the synthesis of GSH by inhibiting GSH synthetase with buthionine sulfoximine can exacerbate cellular damage by ONOO− [329, 332]. These findings establish equally important roles for GSH and SOD, in blocking the formation of / and detoxifying ONOO− to reduce neurological injury. 14.5.2

Mitochondrial Impairment

The production of NO within or around neurons, can impart irreversible adverse affects on mitochondrial O2 -dependent cellular respiration [21, 333, 334]. Degenerative diseases that involve NO correspond to toxicity that parallels the loss of mito-

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chondrial OXPHOS, impaired ETC function and a deficit of ATP [335]. Conversely, administration of nNOS inhibitors can initiate a robust elevation in whole body O2 oxygen utilization [21], suggesting a powerful adverse influence of NO on the function of the mitochondria. The effects of NO in the CNS could be catastrophic, in particular because post-mitototic neurons must sustain aerobic production of ATP to fulfill ongoing requirements for a plethora of anabolic processes, such as protein synthesis, DNA repair, membrane voltage, neuronal trafficking, Ca2+ homeostasis, axonal/dendritic outgrowth, NT packaging and synaptic release. NO can either gain direct access to neurons through diffusion, or it can be produced intracellularly via Ca2+ activated cytosolic nNOS [336, 337]. A rise in cytosolic Ca2+ juxtaposes the activation of a Ca2+ uniporter / and sequestering of Ca2+ into the mitochondria, where it can further activate constitutive mitochondrial NOS (mtNOS), thus generating NO in close proximity to the ETC [338–340]. While defining the sequence homology of mtNOS, using cross-reactivity to selective monoclonal antibodies has not been consistently demonstrated [334, 341], the enzymatic function of mtNOS is known to require l-arginine, Ca2+ , NADPH, calmodulin, tetrahydrobiopterin and its activity is inhibited by NOS inhibitors- l-NMMA and (S)-2-amino-(1-iminoethylamino)-5- thiopentanoic acid [334, 342]. In isolated mitochondria, the presence of l-arginine can produce a dose-dependent production of NO and l-citrulline, that juxtaposes heightened production of OH• , H2 02 , O2 and ONOO− /ONOOH and a loss in OXPHOS, ATP, cell respiration, effects that are reversed in the presence of NOS inhibitors [309, 333, 334, 342–344]. These findings suggest that the detrimental effects of mtNOS on cell respiration, appear to be in the main, due to the formation of NO or ONOO− /ONOOH. The production of ONOO− in the mitochondria of neurons is highly likely, due to mtNOS being located in the same organelle that accommodates the enormous aerobic metabolic requirement for O2 . The human brain consumes approximately 20% of whole body O2 , and in the mitochondria 1–2% of the O2 consumed is converted to O2 − through autoxidation of semiquinones of ubiquinone and flavin NADHdehydrogenase [308, 345]. In the mitochondria, O2 − can react rapidly with NO to form ONOO− , or ONOO− can be formed through the reactivity of O2 with nitroxyl ions [NO− ], that are emitted upon contact of NO with ferrocytochrome c or ubiquinol [18, 293, 346]. Once formed, ONOO− , can directly produce a concentration-dependent loss of mitochondrial respiration and OXPHOS [309]. In mitochondrial isolates, addition of l-arginine or Ca2+ can also lead to the accumulation of ONOO− and ROS, concomitant to the loss of cell respiration, effects that are reversed also in the presence of SOD, uric acid, oxymyoglobin, GGCE, reduced thiols and nNOS inhibitors: NMMA or l-NAME [331, 344, 347–349]. Cultured cells that overexpress mitochondria Mn2+ -SOD appear to be more resistant to NO-mediated cell death [320]. These findings clearly suggest that the presence of O2 − is responsible for the detrimental inhibitory effects of NO on the mitochondria, through production of ONOO− . In the mitochondria, ONOO− can mediate damage to OXPHOS by nitrosylating/oxidizing tyrosine or thiol functional groups, rendering catalytic inactivation of complex I [NADH: ubiquinone oxidoreductase], complex II [succinate: ubiquinone oxidoreductase] and complex V (F1, FO-ATPase), thereby impeding ETC/ OXPHOS

14.5 The Neurotoxicity of NO

and aerobic production of ATP [15, 20, 21, 293]. HNO is equally as toxic as ONOO− , contributing to the inactivation of complex II [350]. Moreover, the NO molecule itself, is known to be a reversible inhibitor of complex IV [20, 351]. Nitric oxide has a high affinity for heme, much like its role in sGC, however in the mitochondria this can be detrimental. NO is a suffocating agent, and its mechanism is similar to carbon monoxide, where its presence competitively and reversibly inhibits cytochrome oxidase by displacing O2 at cytochromes a+a3, CuA, and CuB, thereby blocking the active site with Fe-nitrosyl adducts [a32+NO] or nitrite bound adducts [a33+CuB2 +NO2 -] [352] and raising the Km for O2 [294, 295, 353]. Interestingly, SOD, can reverse the effects of NO on the mitochondria and block the toxic effects of NO, indicating that the effects of NO on complex IV do not contribute to the lethality or collapse of cell function. Further, these findings suggest a detrimental lethal role for ONOO− that far exceeds its ability to inhibit mitochondrial respiration. 14.5.3

Permeability Transition Pore Complex, Apoptosis

The damaging effects of ONOO− on the mitochondria also initiate mitochondrialdirected apoptosis [354]. The accumulation of Ca2+ in the mitochondria can lead to activation of mtNOS and production of ONOO− which can directly trigger opening of the mitochondrial permeability transition pore complex (PTPC), and thereby initiate the controlling event in a sequel of apoptotic cell death [296, 297, 345, 348, 355]. While Ca2+ is thought to initiate opening of the PTPC, ONOO− appears to play a more pre-eminent role, because opening of the PTPC is blocked by NOS inhibitors, SOD mimetics and ONOO− scavengers during Ca2+ mediated apoptosis [348, 356, 357]. The effects of ONOO− on PTPC are catastrophic and in essence define a death sentence. Peroxynitrite can maintain the PTPC in its open state, creating a pore of permeability that allows passage of high molecular weight solutes (<1500 Da), such as cytochrome c (Cyt-C) to traverse between the mitochondrial and the cytosolic compartment [297, 358]. In the presence of ONOO− the release of Cyt-C into the cytosol, can lead to its binding to apoptotic protease activating factor (APAF-1), ATP and procaspase-9, initiating auto-activation of caspase-9, and activation of caspase-3, resulting in cell death by degradation of DNA and cytoskeletal proteins [345, 358, 354, 359]. NO toxicity can be reduced in the presence of substances that block the PTPC such as cyclosporin A (CsA), N-methyl-valine-4-cyclosporin A [357] or agents that scavenge O2 − such as SOD and uric acid [348]. The fact that CsA can prevent the toxic effects of ONOO− suggests that PTPC-directed apoptosis, rather than inhibition of mitochondrial respiration or oxidation of cellular proteins, is by far the most important means by which NO imparts its lethal effects on neurons. There are several hypotheses for a specific mechanism by which ONOO− can control the open state of the PTPC. Briefly the PTPC is regulated by primary constituents of the pore, including the inner membrane adenine nucleotide translocase (ANT) and the outer membrane protein voltage-dependent anion channel (VDAC or porin). The VDAC–ANT complex can bind to signaling proteins that modulate permeability transition, such as pro-apoptotic Bax (which opens the pore) and anti-apoptotic Bcl-2

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(which closes the pore) [358]. Anti-apoptotic proteins such as Bcl-2, Bcl-w, Bcl-xl directly interact with the VDAC channel, thereby closing the pore and preventing Cyt-C release, APAF-1/Cyt-C-procaspase-9 activation and activation of caspase-3 [360–363]. In contrast, Bax or Bak pro-apoptotic proteins undergo conformational changes to allow translocation to the mitochondrial membrane, where Bax interacts with VDAC forming a Bax–VDAC pore allowing passage of Cyt-C [364, 365]. ONOO− is believed to adversely affect several of these events. First, the ability of ONOO− to decouple the ETC creates a loss of mitochondrial membrane voltage, directly associated with increased vulnerability of PTPC opening [348]. Second, ONOO− /NO can carbonylate or oxidize thiols, potentially Cys-56 and Cys-159 on the ANT, that may initiate a change in conformation to allow for sustained interaction with cyclophilin (CyP-D) to facilitate opening of the pore [293, 366–369]. Moreover, ONOO− may accelerate CyP-D, translocation from the matrix to the PTPC, that typically occurs during conditions of Ca2+ -overload and oxidative stress [358]. Thirdly, and likely most important, NO may control the PTPC indirectly by activating p38/SAPK and JNK/SAPK, that in turn inactivates/phosphorylates Bcl-2 at ser 70 [370, 371]. The phosphorylation of Bcl-2 can cause a loss of anti-apoptotic properties, rendering its inability to maintain the PTPC in a closed position allowing pro-apoptotic events to govern, including activation of Bax, translocation of Bax from the cytosol and opening of the PTPC [286, 297, 358, 370]. Further, NO-mediated activation of SAPKs can also activate proapopototic proteins (Bax Bad, Bcl-Xs), concomitant to inactivation and loss of Bcl-2 mRNA and protein expression [286, 372–374]. Many studies consistently corroborate this relationship between NO and apoptosis, where cell death corresponds to elevated activation and translocation of pro-apoptotic Bax/Bak [309, 331, 376, 377]. In contrast, over expression of Bcl-2, Bcl-xL, or use of Bcl-2:20-34 can block neurotoxicity by NO donors, ONOO− [378], AMPA, NMDA [379] and Ca+ overload, all of which initiate toxicity through NO [371]. Further, studies also confirm that neuronal death by glutamate or NO in various models of injury are spared in animal or cell genetic knockout of (-/-) bak(-/-) JNK or with administration of p38/SAPK and JNK/SAPK inhibitors [380–386]. In total, prevention of the neurotoxic effects of NO/NO-donors can be achieved either by targeting upstream events of mtNOS/nNOS using NOS inhibitors, SOD mimetics or ONOO− scavengers [348, 356, 357] or downstream via overexpression of Bcl-2 /Bcl-xL or use of p38/SAPK and JNK/SAPK inhibitors, which prevent the inactivation of Bcl-2 and the activation of Bax [57, 59, 370, 387–389]. As discussed in Section 14.4.2, the transcription of iNOS mRNA in glia is also controlled by p38/SAPK and JNK/SAPK via regulating phosphorylation of NF-kappaâ. Therefore, inhibitors of stress activated MLKs, may have powerful diverse effects in antagonizing both the production of NO in CNS glia, and preventing neuronal apoptosis in the presence of ONOO− . Phosphorylated/activated p38/SAPK-P and JNK/SAPK-P play a lethal role in human CNS disease [224] and in experimental models of CNS injury, the accumulation of phosphorylated (p38/SAPK-P) corresponds to neurological damage, where its presence materializes in neurons, astrocytes and reactive microglia as inflammation progresses [390]. In human Parkinson’s disease, a loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) corresponds to upregulation of astrocytic iNOS, NO [391] as well as elevation in

14.5 The Neurotoxicity of NO

p38/SAPK-P and JNK/SAPK-P [392]. In animal models of PD, administration of MPTP can selectively destroy dopaminergic neurons of the SNzc, concomitant to the accumulation of astrocytic iNOS, NO, ONOO− [393–395] elevation of Bax and reduction in Bcl-2 [396], effects that are blocked by the p38/SAPK and JNK/SAPK inhibitors, SB203580 /SP60012 [217, 397]. Similarly, the loss of Bcl-2 in knockout mice exacerbates the toxic effects of MPTP [398], where protection is observed in transgenic mice overexpressing Bcl-2, or in Bax [396, 399] nNOS and iNOS knock out mice [316]. Therapeutic approaches with agents such as ginsenoside, which enhance Bcl-2/ Bcl-xl or reduce Bax/iNOS [400] and nNOS inhibitors such as 7-nitroindazole, all show protection against MPTP [131, 401, 402]. The close relationship between NO and p38/SAPK-P is also observed in the pathology of AD. In humans, there is a concomitant rise in p38/SAPK-P in neurons displaying neurofibrillary pathologies [403] and increased expression of iNOS occurs in response to mutation of presenilin-1, SB100, advanced glycation end products or beta-amyloid (Aâ) generated by proteolytic cleavage of amyloid precursor protein (APP) [404-408]. In experimental models of AD, elevation of glia iNOS is co-localized with p38/SAPK-P in response to the presence of Aâ [409]. Likewise, transgenic models useful for the study of AD, such as non-specific enolase-controlled APP mice, display an increase in APP and Aâ, that corresponds to p38/SAPK-P and JNK/SAPK-P [410]. Further, CNS models of AD or aging display a reduction of bcl-2, in association with rapid senescence, elevation of 3-NT, NO, Aâ and neuronal apoptosis, suggesting that the loss of bcl-2 control over the MPT in neurons is intricately involved with NO-mediated pathologies [411, 412]. 14.5.4

Excitotoxicity, Poly(ADP-ribose)-polymerase-1

While the accumulation of NO during CNS inflammation is in part due to glial iNOS, the intraneuronal production of NO occurs in response to excitatory NT receptor activation and prompted by Ca2+ influx. Glutamate regulates excitatory neurotransmission in the brain, however during inflammation or CNS disease, its accumulation can contribute to excitotoxic neuronal cell death via a NO mediated pathway. Excitotoxic nerve cell death is involved with the pathogenesis of many CNS diseases including acute trauma, PD, AD, ALS, ischemia, stroke and Huntington’s disease [413]. Astrocytes play a critical upstream role in protecting neurons from glutamate by contributing to the routine clearance of glutamate through uptake transport systems, human EAAT-1 (rat GLAST) and human EAAT-2 (rat GLT-1), and detoxification by glutamine synthetase [191, 192]. During CNS disease, a loss of astrocytic GLT-1/GLAST expression or function can lead to accumulation of glutamate in extracellular-presynaptic junctions, where it can initiate nerve cell death via Ca2+ overload and activation of nNOS. The loss of astrocytic GLT/GLAST mRNA accompanies many human CNSNO-mediated pathologies, corresponding to increased susceptibility to seizure, excitotoxicity and nNOS- mediated nerve cell death [118, 414–417]. Experimental models that employ chemical block of GLT/GLAST or GLT/GLAST knock out mice report induce increased susceptibility to seizure [418, 419] epilepsy [420], excitotoxicity [421] and loss of neurological function [191, 192, 422]. In experimental models of injury,

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abnormal GLT-1/GLAST function is observed in response to ischemia [422, 423] experimental autoimmune encephalomyelitis [424] human T-lymphotropic virus [425] induced thiamin deficiency [426] and oxidative insult by H2 02 /Fe+3 [420, 427, 428]. These findings suggest that GLT-1/GLAST may be damaged during CNS inflammation, possibly through oxidative stress. As discussed in Section 14.4.3, cAMP is a powerful anti-inflammatory mediator in astrocytes, where it can serve to both downregulate iNOS as well as augment GLT-1/GLAST mRNA [429–431]. Therapeutic agents such as PDE4, 1C inhibitors that can both attenuate iNOS and augment glutamate uptake and dissolution in astrocytes may provide a powerful front against NO mediated pathologies. During CNS disease, the accumulation of glutamate can initiate hyperexcitability of neuronal membranes, creating a vulnerability of Ca2+ entry via further activation of ionotrophic glutamate receptors. Subsequently, Ca2+ overload can lead to the activation of both nNOS/ mtNOS, and formation of ONOO− which appears to be exceedingly more important in triggering apoptosis [336, 348,356, 357, 432] than the rise in Ca2+ [433]. Membrane depolarization by glutamate can occur through overstimulation of metabotropic glutamate (mGluR) receptors, which mediate signaling through GTP and antagonize hyperpolarizing currents through K+ channels and GABA receptor-linked channels [434]. Glutamate can further depolarize the membrane by stimulating ionotropic AMPA/KA receptors initiating influx of Ca2+ and Na2+ , contributing to both hyper-osmotic stress and cell death [336, 434]. The entry of Na2+ through the AMPA/KA channel can exacerbate the loss of voltage, leading to release of the Mg2+ voltage dependent block of the NMDA linked Ca+ channel [436], allowing for greater sensitivity to receptor activation by glutamate [114]. Electrolytes that can hyperpolarize the membrane, can render glutamate less likely to initiate Ca2+ influx. A high concentration of K+ , can reduce neuronal excitotoxicity initiated by KA [437] and Mg2+ can primarily prevent the toxic effects of NMDA receptor activation [129]. Ultimately, glutamate can act on all three receptor types, NMDA, Kainate/AMPA and mGluR to synergistically contribute to Ca2+ overload. The rise in Ca2+ i can further perpetuate itself, by triggering Ca2+ -induced Ca2+ release (CICR), which is a reverse mobilization of Ca2+ from endogenous storage organelles, such as the mitochondria through PTPC opening and the ER via IP3 and ryanodine Ca2+ activated-release channels [116, 117, 438, 439]. Subsequently, the rise in Ca2+[I] can activate nNOS/ mtNOS and generate NO, which directly contributes to toxicity, independent of sGC or catalytic production of cGMP [432]. In contrast, increasing the intracellular concentration of cGMP, either by using cell permeable analogues such as 8-bromo-cGMP, or by preventing the breakdown of cGMP via selective PDE5 inhibitors, dipyridamole/zaprinast or nonselective PDE inhibitor, aminophylline can reduce the toxicity of glutamate, effects that are blocked by the KT5823, a PKG inhibitor [113]. The neuroprotective effects of cGMP are also observed in ROS and NO-mediated toxicity [440, 441]. These findings clearly point to the amassing of NO as the central deleterious event. NMDA/AMPA/KA receptor activation evokes a rise in cytosolic Ca2+ , activating a high capacity ruthenium red sensitive Ca2+ uniporter that transports Ca2+ into the mitochondria, which then activates mtNOS [338–340, 442]. Activation of nNOS/mtNOS,

14.5 The Neurotoxicity of NO

can evoke a rise in NO/ ONOO− [356, 441, 443], which can oxidize PTPC-ANT, activate p38/SAPK and JNK/SAPK leading to phosphorylation/deactivation of bcl-2, activation of Bax [297, 358], opening of the PTPC [368, 444], Cyt-C release, activation of caspase-3 and cell death [293, 297, 389, 445–447] [see Section 14.5.3]. Opening of the PTPC also corresponds to reverse transport of Ca2+ back into the cytosol, through a Na2+ /Ca2+ exchanger anti-port system [116, 296, 357]. This contributes to swelling of the mitochondria, a sharp rise in cytosolic Ca2+[I] and activation of Ca2+ -dependent proteases, nucleases, and lipases that digest cellular material. Further, Ca2+ activation of phospholipase A2 (PLA2 ) can catalyze the downstream degradation of membrane phospholipids, resulting in release of free fatty acids, lysophospholipids, arachidonic (AA) and docosahexaenoic acid [438], which also contribute to the opening of the PTPC [358]. AA is also further metabolized into prostaglandins, epoxides, thromboxanes and lipid peroxides, and formation of neurotoxic peroxidative products such as 4-HNE [313, 438, 448]. Together, the detrimental effects of Ca2+ [I] become indistinguishably merged with those of ONOO− . Glutamate toxicity is also thought to involve activation of poly (ADP-ribose) polymerase-1 (PARP-1) [449]. PARP-1 is a nuclear repair enzyme, and its hyper-activation in the presence of ROS or ONOO− is thought to play a primary role in cell death [42, 449, 450]. The activity of PARP-1 appears to be downstream to PTPC opening, and is subject to cleavage by caspase-3 [449]. In the presence of NO/glutamate, indirectly blocking the PTPC with JNK/ p38 SAPK inhibitors, which increase bcl-2/bax or inhibiting caspase-3 and PARP-1 can protect against cell death [43, 112, 443, 451, 452]. Although it is not clear exactly how PARP-1 controls cell death, previous literature has described PARP-1 activation as an attempt to repair excessive damage to the DNA in order to preserve the genomic structure. Because the substrate for PARP-1 is nicotinamide adenine dinucleotide (NAD+ ) [453], the rapid utilization of NAD+ can lead to the loss of its regular function as a reducing equivalent to drive production of ATP, thereby rendering collapse of glycolysis, pentose phosphate shunt, tricarboxylic acid cycle, and inability to produce cellular energy [452, 453]. In neurons, PARP-1 inhibitors such as 3-aminobenzamide, 5-iodo-6-amino-1,2-benzopyrone, 3,4-dihydro5-[4-1(1-piperidynil) butoxy]-1(2H)-isoquinolinone and benzamide [453] prevent neuronal cell death by NMDA and NO [43, 452] and PARP (–/–) mice are resistant to glutamate, NMDA and NO toxicities [453]. In glia, PARP-1 inhibitors may also downregulate iNOS mRNA [45], and selected PARP-1 inhibitors such as 3-aminobenzamide and 1,5-dihydroxyisoquinoline are also powerful OH• scavengers, giving these agents dual neuroprotective properties [454]. PARP-1 inhibitors have shown considerable promise against NO-mediated CNS pathologies, both preventing nerve damage and eliciting improved neurological function after traumatic brain injury, hypoglycemia [42, 455, 456], septic shock, stroke, ischemia-reperfusion, diabetes mellitus [453, 457, 458] and cardiovascular disease [44]. While all of these events are interconnected, the two most critical are neuronal rapid influx of Ca2+ and activation of nNOS, both which trigger all downstream events. Therefore, directly blocking the toxic effects of glutamate/NO, can be achieved through many means, including selective nNOS inhibitors, Ca2+ channel blockers [NMDA, Kainate/AMPA] [ryanodine-sensitive, IP3 ], calmodulin antagonists, Ca2+

367

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385

Index 1400W 266 94120 353 a ACE inhibitor 7, 310, 352 acetylcholine 352 acute coronary syndrome 309 acute hemolytic anemia 293 acute myeloid leukemia (AML) 16 acute myocardial infarction 288, 313, 315 acute respiratory distress syndrome (ARDS) 217 adenine nucleotide translocase 363 adenylate cyclase activator 357 aging 348 AhR nuclear translocator (ARNT) 334 AIDS 19 AIDS dementia 256 alanosine 64 aliphatic N-nitrosourea 60 Alzheimer’s disease (AD) 256, 348, 360 amidine derivative 261 aminoethyl-ITU 270 aminoguanidine 262, 270 aminophylline 366 amlodipine 7 amyl nitrite 290 amyloid precursor protein (APP) 365 amyotrophic lateral sclerosis (ALS) 348, 360 Angel’s salt 246 angina 286, 304 angina pectoris 285–287 angiogenesis 339 angioplasty 317 angiotensin-converting enzyme (ACE) inhibitor 7, 310, 352 anti-platelet drugs 299, 304–306, 308, 315, 319–322 apoptosis 339, 366

arachidonic acid (AA) 367 arthritis 359 aryl hydrocarbon receptor (AhR) 334 arylazoamidoxime 169 arylsulfonylfuroxan 141 ascorbic acid 56 aspirin 10, 18, 309 astrocyte 354 ataxia telangiectasia-mutated kinase 332 atherosclerosis 15, 303 autoimmune encephalomyelitis 348, 366 azaurolic acid 169 3′-azido-3′-deoxythymisylate (AZT) 22 azidonitroolefins 137 b B-NOD 246 BAY 41-2272 247 1,2,3,4-benzotetrazine 1,3-dioxide 153 benzofuroxan 137 BHT 68 bond dissociation energy (BDE) 60 bradykinin 352 c C4144 156 C89-4095 156 C-acyl nitroso compound 180 calcium channel blocker 7 calmodulin 4, 256 calmodulin antagonist 351 cAMP 353, 357 cAMP response element binding protein (CREB) 355 cancer 15, 273, 359 Captropril 7 carbon monoxide 270 carcinogen 72 carcinogenesis 57, 333 carcinogenic agent 219

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

386

Index carcinogenicity 62 carcinogenisis 60 carotid artery stenosis 317 carotid endarterectomy 317 caspase 24 C-diazeniumdiolate 63 cell death 360 central nervous system (CNS) disease 347–349, 354, 355, 358–361, 365–367 cerebral malaria 348 cGMP 235, 236, 300, 349 cGMP analogs 247 cGMP-independent phosphorylation 330 cGMP-PKs kinase 236 chronic obstructive pulmonary disease (COPD) 12 CI 930 353 ciclosidomine 156, 157 Cilazapril 7 cilostazol milrinone 353 cisplatin 17 C-nitroso compound 177–179 CNS inflammation 354 cobalt nitrosyl 122 colon cancer 18 colorectal cancer 18 colorimetric technique 56 condensed furoxan 140 coronary artery disease 304 coxsackievirus 21 cupferron 65, 66 cyclic amidine 268 cyclic AMP–phosphodiesterase inhibitor 358 cycloxygenase (COX) 354 CysNO (S-nitrosocysteine) 244 cysteine protease 21, 24 cytochrome oxidase 360 cytochrome P450 37, 184, 272, 292 cytosine arabinoside (Ara-C) 18 d daunorubicin 16 DEA/NO 241 deazetization 74 dementia 348 demyelinating disease 348 denbufylline 353 dephostatin 63 derivative of amidine 267 Dess–Martin periodinane 179

DETA/NO 241 diabetes 359 diabetes mellitus 304 diazeniumdiolate 24, 68–71, 216 diazeniumdiolate (NONOate) anticancer drugs 15 diazonium ion 58 diazotate ion 61 3,4-dihydro-1,2-diazete 1,2-dioxide (1,2diazetine 1,2-dioxide, DD) 147 dinitrosyl-iron complex 116 dioxime 135 dipyridamole 43 dipyridamole/zaprinast 366 docosahexaenoic acid 367 dopastin 64 Drago complex 76 e Enalapril 7 endoplasmic reticulum 351 endothelial nitric oxide synthase (eNOS) 300 endothelial NOS (eNOS or NOS III) 4 endothelium-derived relaxing factor (EDRF) 205, 352 eNOS 352, 353 EPR spectroscopy 115 Epstein-Barr virus 21 f familial adenomatous polyposis (FAP) 18 fatty acid 367 FK409 182, 183, 185 flavoprotein 44 5-fluorouracil 17 Fremy’s salt 58 furoxan 131, 132, 134–141, 144, 146, 151 furoxancarbonitrile 143 furoxancarboxamide 144 g gas-liquid chromatography GEA-3162 245 GEA-3175 246 gene regulation 329, 330 genestein 357 glioblastomas 339 glutamate 366

56

Index glutamate toxicity 367 glutathione (GSH) 16, 36, 137, 205, 354 glutathione S-transferase 292 glutathione peroxidase 316 glutathione-S-transferase 36 glyceryl trinitrate 15, 33, 307–310, 312, 315, 316 anti-platelet effect 308 Grignard reagent 182 GSH 361 GSNO 243 GTN–NO conversion 43 guanidine 262 guanylate cyclase (GC) 100 h H2 -antagonist 13 HbSNO (hemoglobin nitrosothiol) 95– 97, 99, 100 regulation of blood flow 99 head trauma 348 headache 295 heart failure 288 hemoglobin 95, 193, 350 hemoglobin assay 264 hemostasis 300, 302 heparin 304 herpes simplex virus type 1 21 heterocyclic N-oxide 131 HIF-1 335, 337 HIF-1á 333 histamine 352 histocompatibility complex (MHC) 354 HIV 19 HIV-1 replication 20 HMG-CoA reductase inhibitor 7 homocysNO (S-nitrosohomocysteine) 244 horseradish peroxidase (HRP) 179 4H-pyrazol-4-one 1,2-dioxides (pyrazolone N,N-dioxides) 152 hsp27 239 2H-1,2,3-triazole 1-oxide 153 Huntington’s disease 365 hydralazine 295 hydroxamic acid 19, 179 hydroxamic acids 10 hydroxyl radical 360 hydroxylamine 184 hydroxyurea 271, 272 hypercholesterolemia 304, 319 hypertension 15, 304 hypoxemic respiratory failure 220

hypoxia

330, 333, 337, 339

i impotence 15 indazole 269 inducible NOS (iNOS or NOS II) 4 inflammation 348 influenza virus 21 inhaled NO 248 inhibitor of NOS 258 iNOS 364 iNOS inhibitor 259 insulin 23 ionic diazeniumdiolate 79 iridium 121 iron porphyrin nitrosyl 114 IS-5N 290 ischemia 348, 365 ISDN 290 isosorbide dinitrate (ISDN) 285, 290, 311 isosorbide mononitrate (ISMN) 311 isosorbide-5-mononitrate 285 isothioureas (ITU) 265, 273 IUPAC 69 j janus tyrosine kinase (JAK) 357 Japanese encephalitis virus 21 JS-K 16 l l-arginine 3, 246 anti-platelet effect 318 l-arginine derivative 257, 259, 260, 273 l-ascorbic acid 112 l-citrulline 3, 187 l-cysteine 111 LASP 239 lead oxide 187 lead tetraacetate 187 leptomycin B 333 linsidomine 315 lipase 367 lipid peroxide 367 lipid prostacyclin (PGI2 ) 300 lisinopril 310 LY 195115 353 lysophospholipid 367 m macrophage 357 MAHMA NONOate 242 manganese nitrosyl porphyrin

121

387

388

Index marsidomine 156 Meisenheimer complex 16 mesoionic compound 154 metal–NO complex 109, 122 2-methyl-2-nitroso propane 178 metalloprotein 95 microbial sepsis 359 microglia cell 354, 355 mitochondria 351 mitochondria of neuron 362 mitochondrial aldehyde dehydrogenase (mtALDH) 40 mitochondrial-directed apoptosis 363 mitogen-activated protein kinase (MAPK) 17, 239, 336, 347 mixed lineage kinase (MLK) 355 molsidomine 156, 159, 222, 223, 242, 314 mouse hepatitis virus 21 multiple sclerosis (MS) 360 myeloperoxidase 220 myocardial oxygen consumption 288 n N-aryl-N′-hydroxyguanidine 189 N-aryl N-nitrosamine 60 N-diazeniumdiolate 75, 76, 78, 79, 81, 82 mechanism of NO release 76 nonthrombogenic blood-contact surface 81 reaction 79 reversal of cerebral vasospasm 80 synthesis 77 treatment of impotency 81 N-hydroxy-l-arginine 187 N-hydroxyguanidine 186, 262, 271 N-hydroxy-N-nitrosoamine 63, 64, 67, 72 property 68 reactivity 70 synthesis 66 N-hydroxyurea 181, 182, 189, 190 N-nitrosamine 56, 59, 61–63 physical property 59 reaction 59 N-nitrosimine 72 N-nitroso compound 55, 56, 59, 62, 72 N-nitrosoimine property 74 synthesis 75 Nù -hydroxy-l-arginine (NHA) 257 NAD(P)H oxidase 294

NCX-1000 11 NCX-1015 11 NCX-4016 10, 319 Nef reaction 186 neurodegenerative CNS disease 355 neurodegenerative injury 348 neuroimmune disease 256 neuronal apoptosis 364 neuronal calcium homeostasis 351 neuronal NOS (nNOS or NOS I) 4 neuronal protein kinase 349 neurotoxic effects of NO 364 neurotransmitter 349 Nicorandil 14 nicotinamide adenine dinucleotide 367 Nicox 10 nitrate therapy 295 3-nitratomethyl-PROXYL (NMP) 13 nitrate tolerance 293–295 nitric oxide antiviral effects 21 biological actions 6 inhibition of bone resorption 22 inhibition of cysteine proteases 24 reaction 6 nitric oxide synthase 187, 294 nitric oxide synthase (NOSs) 3 nitric-oxide-releasing nonsteroidal antiinflammatory drugs (NONSAIDs) 11 nitroarene 9 nitroglycerin 245, 285, 290 7-nitro indazole (7-NI) 269 nitrolic acid 135 NitroMed 10 nitrosation of oxime 67 nitrosoglutathione (GSNO) 205, 225 nitrosohemoglobin 115 nitrosonium 6 nitrososemicarbazide 166 nitrosoxacin 65 nitrosyl 109 nitrosyl hemoglobin (HbNO) 191, 273 3-nitrotyrosine 350 nitrovasodilator 225 nitroxyl 7, 44, 109, 180, 181 nitroxyl anion 138 nNOS 349 nNOS inhibitor 352 NO-aspirin 11 NOC18 337 NO donor anticancer drugs 15

Index classification 7 new therapeutic applications 14 NO-release mechanism 7 polymeric films 23 treatment of diabetes 23 NO-donor hybrid furoxan 145 NO gas 219–221 NO inhalation 219–222 NO synthase (NOS) 255 NO-releasing heterocycle 131 non-steroidal anti-inflammatory drug (NSAID) 17 NONOate 216 NOS isoform 256, 257 3-NT 360 nuclear magnetic resonance (NMR) 69 nuclease 367 o O-derivatized diazeniumdiolate 79 O-nitrosoethanol (ENO) 221 okadaic acid 356 olefin 136 ONO-1714 268, 269 organic nitrate 24, 212–215, 245, 287, 290, 295 mechanism of action 34 reductive denitration 41 tolerance 34, 42 organic nitrate metabolism 292 organic nitrite 44, 182, 214, 215 orthostatic hypotension 295 osmium 121 1,2,3,4-oxatriazolium-5-olate 163–165 1,2,4-oxadiazol-5(4H)one 168 osteoporosis 22 oxatriazole 163 oxatriazolium-5-amenate 167 oxidative phosphorylation 360 oxidative stress 295, 359, 366 oxime 177, 182–185, 271 OXINO 246 oxyhemeoglobin 215 oxyhemoglobin 140 p p53 331 p53 phosphorylation 331 p53 stabilization 332 Parkinson’s disease (PD) 348, 360, 364 PARP-1 inhibitor 367 PD98059 356

pentaerythrityltetranitrate (PETN) 287, 290 peripheral blood mononuclear cells (PBMC) 22 permeability transition pore complex 363 peroxisome proliferator-activated receptor gamma (PPAR-gamma) 358 peroxynitrite 6, 21, 240, 295, 359, 363 peroxynitrite anion 159 PF9404C 12 phosphodiesterase 234 phosphorylation 237–239 photolysis of nitrosimine 73 pirsidomine 156, 162, 223 platelet 300, 303–306, 308, 310, 311, 313, 318, 322 platelet activation 237, 320 platelet inhibition 233, 234, 236, 239– 243, 247, 248, 320, 321 pneumonia 21 poecillanosine 65 poliovirus 21 potassium ferricyanide 187 Proadifen 139 prolyl hydroxylase 335 propranolol 358 prostaglandin 23, 358, 367 protein phosphatase 1 (PP1) 356 protein-disulfide isomerase (PDI) 100 proteomics 102 protoporphyrin IX (heme) 4 r Rap1b 238 Rapid Access to Intervention Development (RAID) 17 reactive nitrogen intermediate (RNI) 329–333, 336, 337, 339, 340 reactive nitrogen species (RNS) 6 reactive oxygen intermediate (ROI) 336 renin-angiotensin system 42 restenosis 15 Retro-Diels–Alder reaction 179, 181 retroviruse 21 RIG200 244 RNI signaling 336 rolipram 353 Roussins black salt (RBS) 117 Roussins red ester (RRE) 117 Roussins red salt (RRS) 117 6-(R)-tetrahydrobiopterin 4 ruthenium 10 ruthenium nitrosyl complex 118

389

390

Index s S-nitroso-glutathione (GSNO) platelet inhibition 316 S-nitrosothio-N-acetyl-penicillamine (SNAP) 205 S-nitrosothiol (RSNO) 22, 24, 34, 91, 112, 204, 213, 214, 221, 225, 243, 291 enzymatic consumption 92 formation in the biological milieu 93 regulation of ventilatory response in the brain 100 role in platelet function 100 structure and cellular reactivity 91 S-nitrosation 94 S-nitrosylation reaction 24 sarco-endoplasmic reticulum calciumATPase (SERCA) 241 SB203580 356 sclerosis 348 selective pulmonary vasodilator (SPV) 219 sickle cell disease 191, 273 silver carbonate 187 SIN-1 156, 159, 162, 242, 314 SNAC (S-nitroso-N-acetyl-cysteine) 244 SNAP 243 SNVP 243 sodium nitrite 203 sodium nitroprusside (SNP) 18, 110, 209, 225, 242, 288, 290, 312 anti-aggregatory effect 313 soluble guanylyl cyclase (GC) 110, 114, 234, 247, 349 spinal cord injury 348 statin 7 stroke 348, 365 structure–activity relation (SAR) 61 substantia nigra pars compacta (SNpc) 364 substrate for iNOS 263 substrate for NOS 259, 262 sugar-S-nitrosothiol 19 sugar-SNAPs 207 sulfydryl depletion 42 superoxide dismutase (SOD) 42, 98, 361, 363 Swern oxidation 179 sydnone 154, 161 sydnonimine 155, 161, 222, 225 anti-platelet effect 314

t Taxol 17 tea 56 tetrahydrobiopterin (BH4 ) 256 thiazolidinediones 348 thiobarbituric acid reactive substances (TBARS) 361 thiyl radical 210 thrombogenic effect 23 thrombolysis 288, 312 thrombopoiesis 300 thrombosis 303 thromboxane 367 transnitrosation 98 transnitrosation reaction 59 tromboxane A2 receptor 240 tumor biology 339 tumor development 337 tumor metastases 338 tyrosine kinase inhibitor 357 u uric acid

363

v vaccinia virus 21 vascular endothelial growth factor (VEGF) 335 vasoactive intestinal polypeptide 352 vasodilation 288 vasodilator 203 vasodilator-stimulated phosphoprotein (VASP) 238 vesicular stomatitis virus 21 viral protease 21 virus 19 vitamin C 112 von Hippel–Lindau protein (pVHL) 334 w warfarin

304

x xanthine 39, 40 xanthine dehydrogenase 38 xanthine oxidase 38, 294 xanthine oxidoreductase (XOR) 292 y YC-1

247

38, 45,