Current Topics in Medicinal Chemistry - Volume 5, Issue 7, 2005

Despite the fact that certain nitrogen oxide species (e.g. nitroglycerin) have been used to treat vascular ailments such as angina for over 100 years, the discovery of endogenous generation of nitric oxide (NO) and its role in the regulation of vascular function took many by surprise since it represented a unique and novel signaling paradigm in mammalian biology. Since this discovery, there has been a virtual explosion of research activity on the physiology, pathophysiology, biochemistry, chemistry and pharmacology of NO and related nitrogen oxides. The therapeutic potential associated with pharmacological manipulation of NO (and related/derived nitrogen oxides) has advanced beyond the mere treatment of angina. It is becoming increasingly clear that an unbalance in nitrogen oxide homeostasis is involved in a variety of illnesses and disorders. Nitrogen oxide related therapies, involving either increases or decreases in nitrogen oxides, can be envisioned (or are currently in use) for the treatment of a variety of vascular disorders including pulmonary hypertension, atherosclerosis, male erectile dysfunction, smooth muscle cell restenosis and shock as well as for the prevention and/or treatment of ischemia reperfusion injury, neurodegenerative disease, heart failure, and cancer, among many others. This list will most assuredly grow as the intense research on the physiology and pathophysiology of NO, and related nitrogen oxides, continues. Like many biological signaling/effector species, NO has numerous and diverse activities ranging from its role as an important vascular agent to its function as a neurotransmitter and immune response agent. Although the important biological actions of nitrogen oxides makes their manipulation an important therapeutic strategy for a number of illnesses, the diversity of activity also presents some difficulties with respect to pharmacological/physiological specificity. Thus, the development and characterization of specific inhibitors or activators of the various isoforms of the nitric oxide synthases, as well as the generation of NO-donors with varied pharmacokinetic and/or distribution profiles are vitally important to the future of NO-based therapies. A single class or family of NO-donor or NOS regulator agents is insufficient in addressing all the possibilities associated with NO-related therapy. Moreover, it is now established that redox congeners of the nitrogen oxides may have distinct biological actions. One of the best examples of this is the difference between the activities of NO and HNO, which are related to each other by a single electron but have, for the most part, "orthogonal" biological properties. Indeed, the therapeutic potential for NO and HNO is quite distinct. Thus, the development of a variety of donors of specific nitrogen oxides (e.g. NO versus HNO) is also vital to the further growth of this field. This issue of "Current Topics in Medicinal Chemistry" is focused on work regarding the development and utility of nitrogen oxide-based therapies. This issue serves to, among other things, illustrate the diversity of strategies for the manipulation of biological nitrogen oxides as well as bring to light the importance of doing so. Clearly, the realization of the tremendous potential of nitrogen oxide-based therapy requires the development of diverse chemistry as illustrated herein. Finally, a further demand for expanding the "chemical repertoire" for manipulating nitrogen oxide biology will come as discoveries of new roles for nitrogen oxides in biology are made.

Investigational NO-related therapeutic agents span the range from prodrugs that elevate NO levels, to scavengers of NO, and inhibitors of endogenous NO synthesis. Related agents that influence nitrogen oxides, in addition to NO itself, are also to be considered. Organic nitrates have been used for over 130 years in cardiovascular therapy and hybrid nitrates continue to lead the way in clinical development for an increasing range of disease states. Selectivity for inhibition of NO synthase (NOS) isoforms is a continuing goal. Conversely, N-hydroxyarginine derivatives are substrates for NOS and represent a new NO donor class. Diazeniumdiolate (NONOate) NO donors have been essential for understanding of NO biology and are being developed as NO donor and HNO donor prodrugs including as photolabile sources. N-Hydroxyurea has been used as a cancer drug for decades and is approved for sickle cell disease treatment, but also provides a lead compound for design of NO and HNO donors. Nitroaliphatics and nitrosoaliphatics also represent chemical classes that include NO donors and exhibit biological activity that mimics that of NO. Nitroprusside has been in use since the 1920's and metal ion complexes continue to be explored, including as caged NO donors. Clear and exciting opportunities exist for new therapies based upon NO-related drugs. Despite the extensive clinical use of NO donor drugs and the role of NOS activation in the action of several prescription medications, challenges remain to the development of new NO-related therapeutics, but these are surmountable and are outweighed by the opportunities.

This review includes the non-patent literature up to October 2004 that deals with selective neuronal nitric oxide synthase inhibitors (highest potency is for the neuronal isozyme). Some non-selective inhibitors or selective inducible nitric oxide synthase inhibitors are mentioned if they are related to compounds that are discussed; structures of these compounds generally are not given. In vitro inhibition constants are given either as IC50 values or as Ki values. An IC50 value, the inhibitor concentration that produces 50% inhibition in the presence of a constant concentration of substrate, is obtained by extrapolation of several rate data points to 50% inhibition. Ki values are derived from several types of plots that relate the concentration of inhibitor with enzyme velocity in the presence of a variety of substrate concentrations [1]. The Ki value can be estimated from the IC50 value [2]. Although the two inhibition constants are related, they are not the same; generally, the reported Ki values tend to be lower than the IC50 values. If specifics are desired about how the data were collected, then the reader will have to look in the literature cited. No attempt was made to be exhaustive in citing all references related to specific inhibitors; rather, examples of literature references are given for each inhibitor described.

Diazeniumdiolate ions are convenient and, for a variety of applications, uniquely advantageous nitric oxide (NO) dosage forms. Ionic diazeniumdiolates generate bioactive NO in physiological fluids truly spontaneously (i. e., without metabolism or redox activation), with reliable half-lives ranging from 2 seconds to 20 hours depending on the ion's structure. They are generally simple-to-prepare solids with excellent shelf life and high NO content - up to 40% by weight. Very importantly from the pharmaceutical point of view, the ionic diazeniumdiolates can be easily derivatized to prodrug forms that can be activated for NO release enzymatically, photolytically, or by slowed hydrolysis, allowing for rational design of strategies for targeting pharmacological delivery of NO to sites of need without unwanted collateral exposure of other NO-sensitive compartments. In addition to their world-wide sale for use in probing the chemical biology of NO, published proof-of-concept studies with diazeniumdiolates suggest several more lucrative applications. These include: converting existing drugs and biologicals to NO-releasing form to improve performance and/or extend patent life; diazeniumdiolating medical devices for improved biocompatibility; anticancer drug discovery; use as surgical aids and for wound repair; field generation of NO gas; and nonmedical uses such as extending the post-harvest life of cut flowers. Future work aimed at exploiting the full clinical potential of diazeniumdiolate technology will be pursued in this laboratory and strongly encouraged in others, with a concurrent fundamental research effort to broaden the knowledge base from which further opportunities can be inferred [e. g., exploiting the very recent finding that some diazeniumdiolates appear to offer a versatile platform from which nitroxyl (HNO)-generating prodrugs can be developed].

The photochemistry and use of recently developed photosensitive precursors to nitric oxide (NO) are reviewed. As "caged NO" donors these precursors are able to deliver NO in a spatially and temporally controllable manner. These properties have made such precursors useful in applications in biology and medicine, especially in elucidating neurophysiological roles of NO and in new cancer therapies.

Recent comparisons of the pharmacological effects of nitric oxide (NO) and nitroxyl (HNO) donors have demonstrated that the responses to these redox-related nitrogen oxides are nearly universally dissimilar. These analyses have suggested the existence of mutually exclusive signaling pathways as a result of discrete chemical interactions of HNO and NO with a variety of critical biomolecules. Although the mechanisms of action are currently unresolved, the pharmacological responses to HNO are promising for clinical treatment of cardiovascular diseases such as heart failure, myocardial infarction and stroke. This review provides a detailed discussion of the most commonly utilized donors of HNO as well as a guideline for the characterization of novel donors.

Hydroxyurea has emerged as a new therapy for sickle cell disease but a complete mechanistic description of its beneficial actions does not exist. Patients taking hydroxyurea show evidence for the in vivo conversion of hydroxyurea to nitric oxide (NO), which also has drawn interest as a sickle cell disease treatment. While the chemical oxidation of hydroxyurea produces NO or NO-related products, NO formation from the reactions of hydroxyurea and hemoglobin do not occur fast enough to account for the observed increases in patients taking hydroxyurea. Both horseradish peroxidase and catalase catalyze the rapid formation of nitric oxide and nitroxyl (HNO) from hydroxyurea. In these reactions, hydroxyurea is converted to an acyl nitroso species that hydrolyzes to form HNO. The ferric heme protein then oxidizes HNO to NO that combines with the heme iron to form a ferrous-NO complex that may act as an NO donor. In general, acyl nitroso compounds, regardless of the method of their preparation, hydrolyze to form HNO and the corresponding carboxylic acid derivative. Similarly, the incubation of blood and hydroxyurea with urease rapidly form NO-related species suggesting the initial urease-mediated hydrolysis of hydroxyurea to hydroxylamine, which then reacts quickly with hemoglobin to form these products. These studies present two NO releasing mechanisms from hydroxyurea that are kinetically competent with clinical observations.

FK409 was discovered during a screening program for prostaglandin like compounds from microbial products by measuring inhibition of platelet aggregation and vasodilation. FK409, a semisynthetic compound derived from acidic nitrosation of microbial broth, was shown to be a potent vasodilator with a similar pharmacological profile to organic nitrates such as nitroglycerin (NTG). FK409 dilated the larger diameter coronary vascular vessels more potently than those with a smaller diameter in vitro, and was more potent than NTG in a dog angina pectoris model. Clinical development of FK409 for angina pectoris included a preliminary open efficacy study in about twenty patients with angina pectoris showing an improvement in 60-70 % of patients. Anemia proved a drug related adverse event. A new study was carried out on around 20 patients with ischemic heart disease, but in the longer term the anemia remained. It was concluded that FK409 had a comparable efficacy to organic nitrates, but an undesirable adverse effect, and development was terminated. Back-up compounds for FK409, explored improvement of the pharmacokinetic profile: an increase in duration of action and a reduction of the risk of anemia. The pharmacological action of FK409 was associated with increased cGMP levels in aortic smooth muscle; and release of NO was observed by physicochemical methods. Synthesis of chemically more stable derivatives of FK409 with slower NO release was aimed at longer pharmacokinetic profiles and a lower incidence of anemia. FR144220 and FR146881 were identified as chemically more stable compounds with a longer duration of pharmacological action.

Because of the chemical and physical properties of nitric oxide, its effective use and delivery for therapeutic application represents a significant challenge. Accordingly, current understanding of nitric oxide biology largely stems from the use of nitric oxide prodrugs and adducts whose biological activities are based on their ability to release nitric oxide or a redox-related species. Among the structurally diverse ensemble of nitric oxide donor compounds reported to date are the C-nitroso compounds. These compounds have only recently been investigated with respect to their potential as nitric oxide donors, although they have been known and studied for over 120 years. Here, we consider the synthesis and physico-chemical properties of the C-nitroso compounds and the available data regarding their biological activities. Synthetic methods reviewed include direct substitution of H by NO, oxidative approaches, and the addition of various oxides of nitrogen across multiple bonds. The electronic spectra of C-nitroso compounds and the mechanism and thermodynamics of monomer-dimer equilibration are described. The physico-chemical and biological properties of two related classes of compounds, the diazetine dioxides and the furoxans, are also described.

Organic nitrates, such as nitroglycerin, have been used in clinical practice for more than one century for the treatment of angina, even before the identification of Nitric Oxide (NO) as the so-called Endothelium Derived Relaxing Factor (EDRF). Recently, multiple functions of this molecule in biology and pathophysiology have been discovered and alterations in the NO signalling pathway have often been associated with disease progression in mammals, providing a strong rationale for the use of NO as a potential drug. To have a therapeutic benefit from NO properties, an elegant approach has been designed coupling well-known existing drugs with moieties able to slowly release NO following enzymatic metabolism. "Hybrid nitrates", in which activities of both the native drug and NO are present, have been obtained with the aim of originating safer and more active drugs. The technology consists in the choice of the appropriate chemical spacer arm carrying the nitric ester in order to obtain the best pharmacodynamic and pharmacokinetic profile. The connecting linkers already explored are of different chemical structure, ranging from aliphatic chains to heteroaromatic rings. The molecules so far obtained have already demonstrated their potential therapeutic interest in both pharmacological tests and clinical trials. In this review, we describe the approach and the possibility of generating new chemical entities, combining well-known drugs with an NO-donating moiety in order to increase activity and safety, along with examples of their activity and potential therapeutic application in different pathologies. A few significant examples of molecules in the early preclinical stage, as well as in advanced clinical development will be described.

Nitric oxide (NO) has been implicated in a wide variety of disease states. Both inhibitors and substrates of nitric oxide synthase (NOS) could have great therapeutic potential in the treatment of these diseases. There is considerable pharmacological interest in developing inhibitors of NOS, and hundreds of inhibitors have been identified. In contrast, the study on NOS substrates is less active. The advances in the identification, design and development of NOS substrates are discussed in this review. The focus is the chemistry and biochemistry of N-hydroxyguanidines. The crystal structures of substrates bound to NOSs and the mechanisms of NOS catalyzed NO generation from substrates are also discussed.