Nitrogen-Based Diazeniumdiolates: Versatile Nitric Oxide-Releasing

Chemistry for Everyone edited by

Products of Chemistry

George B. Kauffman California State University Fresno, CA 93740

Nitrogen-Based Diazeniumdiolates: Versatile Nitric Oxide-Releasing Compounds in Biomedical Research and Potential Clinical Applications

Joseph E. Saavedra* Intramural Research Support Program, SAIC, National Cancer Institute, Frederick, MD 21702; *[email protected] Larry K. Keefer The Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, MD 21702

Nitric oxide (NO) has been known to chemists for more than two centuries. As recently as 1987 this diatomic free radical was widely considered to be just a toxic gas, one of the constituents of acid rain, smog, and tobacco smoke as well as a precursor of other harmful oxides of nitrogen responsible for nitrosamine formation. Our own laboratory spent many years studying NO indirectly as a precursor and metabolite of carcinogenic nitrosamines (1, 2). However, by 1988 the evidence was overwhelming that NO was an integral part of normal physiological function (3–7), giving way to a major revolution in biomedical research. NO functions as a protective, regulatory, and signaling agent involved in regulation of blood pressure and clotting, neurotransmission, and immune response (Figure 1). These crucial bioregulatory functions and the awareness that our bodies produce from one to nine mmol of NO every day (8) have made this compound one of the most studied molecules in biomedical science. A testimony to this fact is clearly seen in the 1998 award of the Nobel Prize in Physiology or Medicine to three American scientists, Robert Furchgott, Louis Ignarro, and Ferid Murad, for their discoveries of the roles of NO as a signaling molecule (9).

are known at the present time. These range in molecular weight from about 130 kDa to 160 kDa. Two of the isoforms, nNOS and eNOS, are constitutive enzymes that are regulated by Ca2+ influxes. The third isoform, inducible NO synthase, iNOS, is produced in many cell types, with especially high levels being formed as a response to inflammatory stimuli. The biosynthesis of NO occurs as L-arginine is oxidized to N G-hydroxy-L-arginine followed by further oxidation to L-citrulline (10) as shown in Scheme I. The NO synthases normally take care of all the NO production needs to ensure our well-being as long as a proper balance is maintained. However, excessive NO formation can dangerously lower blood pressure and may contribute to tissue damage in chronic diseases like rheumatoid arthritis. NO reacts with superoxide (O2᎑), a by-product of mitochondrial respiration, giving reactive nitrogen species that can effectively damage proteins and DNA (11). To counteract the negative consequences of physiological overproduction of NO, drug

O

O

H2N H C OH C

NO and Drug Design

H2C

Three different isoforms of the enzyme family (the NO synthases) that catalyzes the production of NO in mammals

HN

H2N H C OH C

O2

CH2

H2C

CH2

NADPH

C H2N

NH

CH2

C H2N

NOH

N G-hydroxy-L-arginine

L-arginine

O2

cytostasis penile erection uterine relaxation

gastrointestinal motility

HN H2O

CH2

HO C

NADPH

O

H2N CH H2O

CH2

NO

bronchodilation

vasorelaxation

H2C CH2

+

NO

HN

immune function

neurotransmission antiplatelet action

Figure 1. Bioeffector functions of NO.

C

O

H2N L-citrulline

Scheme I. Biosynthesis of NO from L-arginine.

JChemEd.chem.wisc.edu • Vol. 79 No. 12 December 2002 • Journal of Chemical Education

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Chemistry for Everyone

development efforts in NO synthase inhibitors are abundant in the literature (12). On the other hand, when the flux of NO is insufficient, serious health problems such as respiratory distress, impotence, unwanted clot formation, and collapsed blood vessels can arise as well. We will limit our treatise to this area of research, which is the development of compounds that allow for controlled release and targeted delivery of NO in deficient areas. Furthermore, we will focus on chemical agents that were developed in our laboratory. Drugs that provide extra NO at sites of need have been available for some time and some are listed in Figure 2. These include nitrate esters like nitroglycerin (GTN) and isosorbide dinitrate, compounds that are dispensed orally or through a patch, to relieve angina by dilating microvessels as NO is metabolically generated from them. The production of the NO radical from nitrate esters requires a three-electron reduction, but this metabolism decreases in efficiency on continued use of the drugs, contributing to “nitrate tolerance” (13). Isoamyl nitrite is a nitrite ester that is administered by inhalation, generating NO by direct reduction or via nitrosation of thiols to form S-nitrosothiols that are in turn metabolized to NO. Although nitrite esters in general seem to be good sources of NO, they are easily hydrolyzed to nitrite ion and are potent nitrosating agents that lead to undesirable, potentially carcinogenic products such as N-nitrosamines. The fourth compound shown in Figure 2 is sodium nitroprusside (SNP), a drug used in emergency room situations when the patient’s blood pressure fluctuates at dangerously high levels. This agent, saving the lives of many patients, is infused directly into the circulatory system where a one-electron reduction rapidly produces NO, lowering blood pressure systemically. On the downside, however, this one-electron reduction also leads to the formation of a free cyanide ion and an iron(II) center that in turn catalyzes the production of highly toxic nitrogen species (14). Molecular NO has been introduced into wide clinical practice; inhalation of low concentrations of the gas (e.g., 20 ppm) mixed in air produces selective dilation of blood vessels in the lung, and thus it is used in intensive care units to CH2ONO2

H3C

CHONO2 CH2ONO2 nitroglycerin (GTN)

ONO2 O O

CHCH2CH2ONO H3C isoamyl nitrite

2− NO CN NC Fe 2Na+ CN NC CN

ONO2 isosorbide dinitrate

sodium nitroprusside (SNP)

Figure 2. Some commonly used clinical nitrovasodilators.

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treat disorders such as adult respiratory distress syndrome and persistent pulmonary hypertension of the newborn (15). However, because NO’s biological half-life is short, continuous delivery of the gas is necessary, requiring the patient to remain in an inhalation chamber or on a respirator. Furthermore, abrupt removal of NO from the breathing air can cause blood pressure in the lung to rebound to levels higher than those seen before treatment was begun, posing some serious limitations in inhalation therapy. Several other types of NO donors exist, some of which require an oxidative metabolism rather than the three- or one-electron reductions discussed earlier. These are compounds such as cupferron and alanosine, which are N-nitrosohydroxylamine derivatives, and sydnonimines. S-Nitrosothiols, like S-nitrosoglutathione and S-nitrosocysteine, also generate NO; moreover, their possible role in storage and transport of nitric oxide intensifies their importance in drug discovery (16). Physical and Chemical Properties of Nitric Oxide In order to effectively introduce our nitrogen-based NO donors and the strategies for site-directed administration of these drugs, it is important to first present some of the basic physical and chemical properties of this remarkable bioeffector molecule. Electrical storms produce small amounts of NO by the direct combination of nitrogen and oxygen. Oxidation of ammonia is a chief commercial process for its manufacture, while the reduction of nitric acid to NO with mercury or copper is a well-known laboratory procedure. The reduction of sodium nitrite in the presence of ferrous ion is also a convenient preparative method. NO is a free radical, the simplest known thermally stable paramagnetic molecule, with a solubility in water of about 2 mM at room temperature and 1 mM at 37 ⬚C. The boiling point of this compound is ᎑151.8 ⬚C, while its freezing point is ᎑163.6 ⬚C. In the gas phase nitric oxide is colorless; however, the liquid and the solid are blue in color. This free radical contains 11 valence electrons and has a bond length of 1.15 Å. The eleventh electron is placed in a π* orbital and it is responsible for NO’s rich chemistry, inducing direct and indirect biological effects. Although NO is a quite stable chemical entity in the pure state, it is nonetheless reactive in biological systems. In aqueous media the oxidation of NO by O2 produces nitrite (NO2᎑). Reduction of NO gives NO᎑, which in aqueous medium protonates and dimerizes, then loses water to form N2O. Superoxide anion radical (O2᎑) and NO couple to form peroxynitrite, ONOO ᎑ , which upon protonation to ONOOH rapidly rearranges to nitrate and hydrogen ions (11). NO forms complexes with transition metals and binds readily to heme-containing proteins, leading to the formation of metal nitrosyl species that can in turn mediate important physiological functions (17). NO is so rapidly inactivated by blood that the controlled delivery of this molecule to the organ or cell type where it is needed and without affecting other NO-sensitive parts of the anatomy remains a challenge. Our objective has been to develop strategies to sequester the NO functionality into a chemically stable carrier that can be directed to the deficient site and carry out its effector pathway without systemwide consequences.

Journal of Chemical Education • Vol. 79 No. 12 December 2002 • JChemEd.chem.wisc.edu


Reactions with Lewis Bases: Formation of NO-Releasing Diazeniumdiolates We have thus far introduced most of the properties of NO in relation to its free radical nature and have looked briefly into its redox chemistry. However, another important characteristic of NO, at least for the purpose of preparing stable biologically active agents, is its Lewis acid property (18). The adducts of nucleophiles, or electron donors, with nitric oxide have been known for many years; however, their biological properties were not investigated until recently (19). It is the electron acceptor quality of NO that gives us the rationale for the preparation of nonclassical NO-releasing compounds—that is, agents that dissociate back to the nucleophile and NO on acid-catalyzed hydrolysis without oxidation (the sydnonimines) or reduction (required by the nitrate esters and nitroprusside ion). A process that clearly depicts NO’s Lewis acid chemistry is the reaction of sulfite ion (SO32᎑) with two molecules of NO to give O3SN2O22᎑ (18). This compound does not revert to NO when dissolved in water but actually disproportionates to N2O and sulfate (20). Angeli’s salt (Na2N2O3) is prepared, not by reaction with NO, but by the nitration of hydroxylamine; however, structurally speaking, this compound is an oxygen adduct of NO represented by the structure [ON2O2]2᎑ (21). Like the sulfite analog, this material disproportionates to N2O, but it also generates significant amounts of NO at low concentration in neutral buffers (19). This spontaneous release of NO under physiological conditions was confirmed by Vanin et al. (22) as they discovered that administration of the compound dilated the blood vessels of a living animal. Active methylene groups react with NO to give Nnitrosohydroxylamines, many with the general structure [᎑O2N2–CR1R2–N2O2᎑]; these are known as Traube compounds (23, 24). Complexes of these structures may be considered carbon/NO adducts and are generally very stable, but have not been reported to release a significant amount of NO upon nonredox dissociation at physiological pH. Analogous adducts in which the N2O2᎑ group is attached to carbon may be obtained from oximes (25) and enamines (26).

ations as general reaction types between NO and amine nucleophiles. Two preparative techniques were used to carry out these reactions. One method simply required bubbling NO into an ether solution of the desired substrate at ᎑78 ⬚C, while the second procedure required high pressure in an autoclave. Both methods gave the same quaternary ammonium salt of the diazeniumdiolate; however, the latter provided higher yields. The ammonium salts were conveniently converted to more stable sodium salts with sodium ethoxide and only symmetrical, unhindered amines, as large as di-n-hexylamine, were successfully converted to the corresponding diazeniumdiolate. In our laboratory, we have found that medium-pressure exposure of only five atm NO is adequate for these reactions in organic solvents even at room temperature. In many instances, we have directly isolated the sodium salts of the diazeniumdiolates by reacting the amine with NO in methanol:ether solutions in the presence of one equivalent of sodium methoxide. We have also extended the availability of these complexes to those derived from unsymmetrical secondary amines. Many of the diazeniumdiolates derived from amine nucleophiles, when dissolved in buffers, blood, or cell culture medium, dissociate to regenerate NO and the nucleophile as the by-product. All of these compounds have a strong chromophore at approximately 250 nm in the ultraviolet (UV) region, a convenient marker for their detection, quantification, and quality control. Their dissociation rates in aqueous media can be measured by following either the disappearance of this UV peak or the appearance of NO as a product (e.g., by chemiluminescence; ref 29). The dissociation of many of these anions to NO at physiological pH is induced by protonation of the amino nitrogen (30) and proceeds with simple pseudo-first order kinetics at pH 7.4 with half-lives

+

R2NH

H R2N

2 NO

+

N N

Reaction of NO with Nucleophilic Amines Leading to the Formation of NO-Releasing Diazeniumdiolate Species Complex formation between primary or secondary amines and NO was originally reported by Drago and Paulik (27). We now refer to these adducts as 1-amino-substituted diazen-1-ium-1,2-diolates or simply diazeniumdiolates. The reaction proceeds according to Scheme II, where one equivalent of amine reacts with two equivalents of NO. The second equivalent of base is protonated, keeping the newly formed [N(O)NO]᎑ group in its stable anionic form. Drago and Paulik introduced the reaction of diethylamine with NO to obtain (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, DEA/NO (see Figure 3), as the diethylammonium salt. The thrust of their work was to obtain additional evidence on the Lewis acid or electron pair acceptor property of NO. This work was later extended to other primary and secondary amines (28), and thus they came to establish these complex-

O−

+

−

O

R2NH

+

R2N

O− −

N

+

R2N

O−

N N

+

H2 NR2

N

O

+

H2 NR2

+

−

NaOH

O

R2NH

+

R2N

+

O−

N N

Na+

+

H2O

−

O

Scheme II. Reaction of amines with NO to form NO-releasing diazeniumdiolates.


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ranging from 2 s to 20 h at 37 ⬚C (31). We have as yet no convincing explanation for, or ability to predict, the extreme differences in dissociation rate among these compounds, but they are nevertheless reliable sources of NO and because of the vast differences in their half-lives, they may be tailored to different biomedical applications. We have synthesized a wide variety of these adducts, diversifying the nucleophile residues that may encompass simple primary or secondary amines, polyamines, or secondary amino acids. Some selected examples of our diverse diazeniumdiolate library are shown in Figure 3. DEA/NO, for example, at 37 ⬚C and pH 7.4 has a halflife of 2 min, and the half-life of the five-membered heterocyclic pyrrolidine derivative, PYRRO/NO, is only 3 s at 37 ⬚C (32). Proline in methanolic sodium methoxide reacted with NO to give PROLI/NO, an adduct that turned out to be the fastest NO releaser that we have encountered so far, with a half-life of 1.8 s in physiological conditions (33). IPA/NO is an example of a diazeniumdiolate of a primary amine (19). Numerous zwitterionic diamine, triamine and tetramine diazeniumdiolates, as illustrated by MAHMA/NO, DETA/NO and SPER/NO in Figure 3, were synthesized by exposing the free bases to NO in various organic solvents (34, 35). These diazeniumdiolated zwitterions are not very soluble in organic solvents, in contrast to their parent amines with multiple nitrogens; therefore, the reaction with NO can be limited to only one nitrogen site as the solid products precipitate from solution. These powders are collected by filtration and are usually stable indefinitely in closed containers at freezer temperatures. The isolated materials may contain as much as 40% NO by weight and at physiological pH most of them release two full equivalents of the NO free radical. Spermine, an important biomolecule, reacts with NO to give the single complex SPER/NO (Figure 3) in spite of

NH2

NH

+

−

H2N

O

N +

NH2+

N −

+

N

+

O Na IPA/NO

O−

NH2

N

O

H2N

N O

N +

O−

N

N − O MAHMA/NO

−

SPER/NO

−

+

N

N

NO

N −

+

O Na

NH3+

N −

O

N +

N

O− DETA/NO

DEA/NO

H N O N

N −

O

N +

−

O

N O−Na+

PIPERAZI/NO

O

N +

+

O−Na+ N O−Na+

N −

N

N

PROLI/NO

O−Na+ PYRRO/NO

Figure 3. Anionic and zwitterionic diazeniumdiolates discussed in this review.

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having four amine groupings in the molecule. Studies of the fundamental chemistry of these zwitterions have provided some important information on the polyamine-NO interactions and the structural features of the adducts. One crucial finding to surface from this research was the fact that NO reacts with secondary amino groups even when more than one primary amino nitrogen is present in the same molecule. This work has provided diazeniumdiolates of varied half-life, thus facilitating research on NO’s bioregulatory effects. Spontaneous NO Releasers in Biomedical Research The ability of anionic or zwitterionic diazeniumdiolates to generate NO in physiological media with a wide range of rates makes them ideal agents for probing many aspects of cardiovascular and other biological functions (36), where the biological effects of these compounds correlated well with the NO release rates seen in parallel work using phosphate buffers. Thus experiments with rabbits demonstrated that diazeniumdiolates had blood pressure-lowering activity that persisted only as long as there was NO release (37). For these purposes, a fast NO releaser such as DEA/NO (t1/2 = 2 min) was more potent than one with a longer half-life. The blood pressure reached a minimum within one min of injecting a tiny amount intravenously (1 nmol/kg of body weight was plenty for inducing a significant drop in blood pressure), followed by a rapid recovery. The longer acting SPER/NO (t1/2 > 30 min) was less potent at the one-min time point, but lowered blood pressure was still observable 30 min after injection (37). To inhibit overproliferation of cells in the blood vessel wall after arterial injury, on the other hand, long-acting diazeniumdiolates are preferable. Exposing cultured rat aorta vascular smooth muscle cells to 0.5 mM DETA/NO (t1/2 = 20 h, structure shown in Figure 3) completely suppressed the growth of these cells for 48 h without any observed toxicity while similar treatment with DEA/NO (t1/2 = 2 min) had no effect (38). Cerebral vasospasm, a life-threatening disorder caused by the rupture of a blood vessel in the brain, strikes thousands of people each year in the United States alone. Some research suggests that these spastic blood vessel collapses are triggered when hemoglobin in a hemorrhage-produced clot scavenges endogenous nitric oxide from nearby cells that otherwise would be used to keep the affected vessel open. Although neurosurgeons can identify those with high risk factors and assess the severity of an on-going spasm, they are often unable to curtail neurological damage or to prevent the deaths that often follow a spasm. However, two research groups have approached this problem with some success in animal models. Pluta et al., at the US National Institute of Neurological Diseases and Stroke (NINDS), hypothesized that infusing a fast-acting NO-generating agent such as PROLI/NO (Figure 3) upstream from the affected artery might relieve the spasm without dilating blood vessels further downstream, since PROLI/NO has a half-life of only 1.8 s at body pH and temperature. When the NINDS group infused this diazeniumdiolate into the carotid arteries of monkeys, the spasms were reversed while the blood pressure remained unaffected (39).



The second group (Wolf and colleagues of the University of Tennessee at Memphis) achieved parallel results with an opposite approach. Rather than a short-lived NO donor, this group chose a long-lived NO generator, DETA/NO (t1/2 = 20 h, Figure 3), which they injected into the cerebrospinal fluid bathing the brain of their subject dogs to dilate the spastic vessels, counting on the blood-brain barrier to contain the drug and keep it from affecting the general circulation. Intermittent injections of DETA/NO prevented the spasm throughout the animals’ vulnerable period and eliminated the need for continuous infusion (40). As we mentioned earlier, inhaled NO gas is used in emergency situations to treat acute respiratory distress syndrome or pulmonary hypertension and is a successful way of reducing blood pressure in the lungs of children with persistent pulmonary hypertension of the newborn. However, because the short half-life of NO requires continuous delivery of the gas, Hampl et al. (41) applied intermittent dosages of DETA/ NO in the form of aerosols to rats with induced pulmonary hypertension. Their treatment with nebulized DETA/NO once a day for four consecutive days produced a lasting reduction of pulmonary vascular resistance without observable toxicity or other adverse effects. NO is also a prime mediator of relaxation in the corpus cavernosum. This is a muscle whose relaxation produces penile erection by allowing increased arterial inflow and restricted venous outflow in the penis. Once again, because of its short half-life in aerobic media, NO gas is not clinically functional in the treatment of impotence. Because diazeniumdiolates allow for the controlled pharmacological delivery of NO, Champion et al. (42) induced erections in cats by injecting PROLI/NO, DEA/NO, PIPERAZI/NO (Figure 3) and other NO donors of this class. Although the duration of the maximum erection was less than that of the control (a combination of phentolamine, papaverine, and prostaglandin E1), their data support further clinical investigations with these compounds for treatment of erectile dysfunction. Physical and Chemical Modifications of Diazeniumdiolates for Pharmacological Targeting The anionic complexes discussed above decompose spontaneously at physiological pH, some of them at a very rapid rate. Unfortunately, a compound that generates NO spontaneously also produces, in addition to the desired effect at the localized site, many unwanted side effects at NO-sensitive sites throughout the organism. The researcher’s challenge, then, is to target NO to a specific site at a specified rate so that it will affect only that target. This selectivity can sometimes be achieved through local administration. In treating impotence, for example, one can avoid systemic effects by injecting a fast-acting NO generator directly into the penis to produce an erection. However, more often, physicians must deliver NO to an internal organ for which direct injection is not a clinically acceptable option. By taking advantage of the chemical versatility of the diazeniumdiolate functionality, we have devised general strategies to prepare both tissue-selective NO donor drugs and materials containing NO delivery agents that can be physically placed near the target organs. The synthetic strategies comprise: the incorporation of the

[N(O)NO]᎑ functionality into polymeric matrices of different structural types; prodrug design involving protection of the terminal oxygen; and the binding of a diazeniumdiolate prodrug to a biomolecule or biocompatible molecule.

Polymer-Bound Diazeniumdiolates We have incorporated the diazeniumdiolate [N(O)NO]᎑ functionality of compounds comprising the structure X–N2O 2᎑, where X is an amino group, into polymeric matrices of different structural types (43). Figure 4 depicts the generalized polymer types considered in this investigation. In Figure 4, left, a low molecular weight diazeniumdiolate is simply incorporated into a polymer matrix such as polyethylene glycol, polyurethane, or polycaprolactone giving a blend of the general structure 1. In Figure 4, center, the diazeniumdiolate functionality [N(O)NO]᎑ is covalently attached to a polymer containing pendent amino groups (structure 2). In Figure 4, right, the [N(O)NO]᎑ group is directly attached to the polymer backbone, 3. Polymer blends (structure 1 of Figure 4) can drastically alter the time course of NO release and may provide a prolonged shelf life for the NO-containing compound by shielding the diazeniumdiolate group from proton sources that could otherwise initiate NO release. This technique has been used in the development of biocompatible coatings for artificial materials that come into contact with the blood stream. By applying a coating of polyvinyl chloride (PVC) blended with MAHMA/NO (structure 1 in Figure 3), Espadas-Torre et al. were able to fabricate a biosensor electrode allowing unrestricted blood flow past the sensor tip (44). The diazeniumdiolate stopped blood platelets from adhering, but it created a new problem by leaching out of the polymer, a feature which could potentially create systemic effects such as hypotension. Our laboratory recently described an example of 2 (see Figure 4) in which PIPERAZI/NO was linked to heparin through nucleophilic displacements of primary sulfate groups on the polysaccharide chain (Scheme III). The diazeniumdiolated heparin, 4, exhibited significant heparinlike anticoagulant activity; however, unlike the native heparin, this NO donor had the ability to inhibit and even reverse platelet aggregation in experiments conducted with plateletrich human plasma, making it a dual-mechanism anticoagulant (45). A cross-linked polyethylenimine exposed to NO, represented by structure 3 of Figure 4, showed potent anticoagulant activity when coated on artificial blood vessels installed in a baboon’s circulatory system. In this case, the nucleophile

X

[N2O2]−

X

[N2O2]−

1

X

− [N2O2]

X 2

[N2O2]− [N2O2] −

X 3

Figure 4. Incorporation of the diazeniumdiolate functional group, [N(O)NO]᎑, into polymers: structure 1, as polymer blends; structure 2, via covalent attachment to groups pendent to the polymer chain; structure 3, by covalent attachment to the polymer backbone. [Figure adapted from Smith et al. (43) with permission.]


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carrier residue was anchored to the polymer, preventing it from leaching into the circulatory system and providing completely localized NO release. This work demonstrated that incorporation of the diazeniumdiolate functionality into a stationary solid can control the rate of NO release and limit its exposure to the tissues in close proximity to where the polymer is physically placed (46).

Prodrug Formation via O 2 Functionalization Chemistry While the localization strategies of the polymers above are promising for a wide variety of applications, it seemed to us that converting ionic diazeniumdiolates to more stable derivatives through covalently binding various protecting groups to the terminal oxygen of the [N(O)NO]᎑ group (47) might provide a more general targeting strategy. Selectivity could then be achieved by employing protecting groups that can only become detached for subsequent NO release under specific conditions of pH or enzymatic activities prevailing at the target sites. This “prodrug” approach provides a strategy that allows the diazeniumdiolate to move freely throughout the circulatory system but prevents it from spontaneously dissociating. This can be effected by covalently attaching a protective group at the terminal oxygen, as in 6, according to Scheme IV. The ideal prodrug will not release NO until it is metabolically reconverted to the anionic diazeniumdiolate, 5. Timothy Billiar, a University of Pittsburgh trauma surgeon who had investigated important aspects of NO biosynthesis in the liver, believes that the antioxidant, vasodilatory, and antiadhesive properties of NO generated locally by such a drug might improve his patients’ health without organ trans-

plantation. With the collaboration of this group we were able to test our prodrug hypothesis. The ideal starting diazeniumdiolate was PYRRO/NO, an ion that releases two molecules of NO with a half-life of 3 s at body temperature and pH. Because the liver contains an abundance of enzymes (the cytochromes P450) that catalyze the epoxidation of vinyl ethers among their many drug metabolizing activities, Billiar’s group hypothesized that vinylated PYRRO/NO would undergo epoxidation and subsequently hydrolyze to NO (as in Scheme V) rapidly enough to provide the required liver selectivity. The prodrug V-PYRRO/NO, 7, was synthesized and the Pittsburgh group tested it in rats. As expected, the compound raised the levels of cyclic guanosine-3´,5´monophosphate (a marker of NO generation) in the liver without dramatically lowering the animals’ blood pressure. Billiar’s group then generated an experimental model of liver failure in rats and showed that diazeniumdiolate 7 dramatically reduced the extent of programmed cell death in the rats’ livers (32). +

R2NH2

+ 2 NO

pH 7.4

O R2N

−

O

R´X

+

N

N

O

N+

R2N

−

− −

OR´

N

5

X

+

6

OSO3−

Metabolism

CH2 Scheme IV. Generic description of prodrug formation via O2 functionalization chemistry and subsequent metabolic regeneration of NO.

O CO2−

OSO3− OO

O

NHSO3−

OH PIPERAZI/NO

O OH

O

O− −

heparin

O

N

N

N

+

−

N+

O−

Cytochrome P450

N

O

N

N V-PYRRO/NO 7

N

O−

N N

CH2

N

O CO2−

OSO3− OO

N

+

N

+

O O

Epoxide Hyrdolase

pH 7.4, 37 °C

O−

O− N

N

+

N

O

CHOHCH2OH

PYRRO/NO

O

NHSO3−

3-second half-life

OH O

NH + 2 NO

OH 4 Scheme III. Converting a polysaccharide to NO-releasing form with PIPERAZI/NO.

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Scheme V. V-PYRRO/NO: Proposed mechanism of metabolic activation for a liver-selective prodrug. [Adapted from Saavedra et al. (32) with permission.]



Attachment of O 2 Substituted Polyfunctional Diazeniumdiolates to Biocompatible Molecules In order to introduce an NO-releasing group into biomedically important substrates, we have explored the utility of MOM-PIPERAZI/NO, compound 8 of Figure 5 (48). This compound has three very important features. One is the nonionic nature of the diazeniumdiolate. Secondly, it hydrolyzes very slowly under physiological conditions with a half-life of 17 days. Finally, the nucleophilic character of the second nitrogen offers a reactive site for tandem functionalization. We have been able to develop procedures for conjugating this NO-releaser to a wide variety of other functional molecules, including amino acids, drugs, nucleosides, phospholipids (48), synthetic polymers (49), polysaccharides (45), and proteins (50). Some examples are shown in Figure 5. Hrabie et al. (50) prepared the diazeniumdiolated albumin 9 shown in Figure 5. The protein conjugate contained 20 [N(O)NO]᎑ groups per molecule and released all 40 molecules of NO with a half-life of about three weeks under physiological conditions. A cardiology group at Indiana Uni-

O

O N O serum albumin

N N

N

−

+

N OCH2OCH3 O

N H

20 lysine residue

linker

NO-releasing moiety

diazeniumdiolated protein 9

1. linker reagents 2. albumin −

O N N+ N OCH2OCH3

HN

MOM-PIPERAZI/NO 8 heparin

−

O

polyvinyl chloride (PVC)

OCH2OCH3 +

N

−

N

O

N

OCH2OCH3 +

N

N

N Cl

N CH2

CH2 CH CH2

Cl

N CH

CH2

CH

O −

CO2

OO OH O

−

OSO3

diazeniumdiolated PVC

O −

NHSO3

versity, led by Keith March, devised experiments to see if this albumin derivative could be useful in controlling healing of blood vessels subjected to balloon angioplasty. This procedure involves the insertion of a balloon into a narrowed artery, stretching the vessel, and restoring normal blood flow. The downside of this procedure is that abnormal healing of the injury associated with the required overexpansion often occurs, with renewed thickening of the arterial wall (restenosis) again interfering with blood flow; this life-threatening complication occurs in hundreds of thousands of patients worldwide each year. The cardiology group subjected pigs to angioplasty in their coronary arteries; however, immediately before the procedure they injected the diazeniumdiolated albumin into the pericardium, a sac enveloping the animal’s heart whose contents bathe the outside half of the coronary arteries. Because of the high molecular weight of the protein they expected that it would remain concentrated in the pericardial fluid and provide prolonged exposure of the artery’s outer wall to the NO it released. Two weeks after treatment the animals were examined, and it was found that this procedure inhibited the excessive proliferation of arterial tissue, reducing thickening in the vessel wall by 50% compared to control experiments in which pigs received underivatized albumin (51). The diazeniumdiolated polysaccharide 10 described also in Figure 5 is the methoxymethyl (MOM)-protected version of 4 (mentioned earlier; see Scheme III). This heparin derivative also exhibits anticoagulant activity (49). The third example in Figure 5 relates to work by Mark Meyerhoff ’s group at the University of Michigan. As mentioned in a previous section, they used blends of PVC and MAHMA/NO (see structure 1, Figure 4) to coat electrodes before placing them in the bloodstream to measure blood gases and other important analytes. In the original experiments, use of untreated PVC coatings on the electrode resulted in platelets being deposited on the surface of the probe at a rapid rate. This diminished contact between the biosensor and the ions being analyzed. The blended coatings containing the NO releaser inhibited clot adhesion to the sensor’s surface while allowing unrestricted blood flow at the site of the implant. However, a portion of the low molecular weight diazeniumdiolate leached out of the polymer, creating the potential problem of systemic effects such as hypotension. To circumvent this limitation, we covalently attached the nucleophilic MOM-PIPERAZI/NO to the PVC polymer backbone, as in Figure 5. When the Meyerhoff group placed the diazeniumdiolated PVC in contact with platelet-rich plasma, the methoxymethyl group slowly hydrolyzed, freeing the diazeniumdiolate ion, and dissociating two NO molecules; this reduced platelet adhesion relative to controls using underivatized PVC (49). Analogous materials are being devised for coating the many medical devices that come into contact with whole blood; these include vascular grafts, dialysis shunts, and biosensors.

OH

Conclusions diazeniumdiolated heparin 10

Figure 5. Converting polymers to NO-releasing form with MOMPIPERAZI/NO. [Figure adapted from Saavedra and Keefer (52) with permission.]

It is well established that NO is a key bioeffector molecule in the cardiovascular, nervous, and immune systems. A great deal of research has focused on the areas in which reduced bioavailability of NO is responsible for, or at least


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implicated in, a range of diseases and where the delivery of exogenous NO is an attractive therapeutic option. A vigorous basic research program in organic chemistry is key to any progress we may make in our drug discovery efforts. In seeking a next generation of products, we will continue preparing diazeniumdiolates with the goal of applying our growing knowledge about the fundamental biological, physical, and chemical properties of this class of compounds into a host of clinical benefits. Acknowledgment This project has been funded in part through the National Cancer Institute contract No. NO1-CO-56000. Literature Cited 1. Keefer, L. K. CHEMTECH 1998, 28 (8), 30. 2. Hansen, T. J.; Croisy, A. F.; Keefer, L. K. IARC Sci. Publ. 1982, 41, 21. 3. Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 9265. 4. Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524. 5. Marletta, M. A.; Yoon, P. S.; Iyengar, R.; Leaf, C. D.; Wishnok, J. S. Biochemistry 1988, 27, 8706. 6. Moncada, S.; Radomski, M. W.; Palmer, R. M. J. Biochem. Pharmacol. 1988, 37, 2495. 7. Garthwaite, J.; Charles, S. L.; Chess-Williams, R. Nature 1988, 336, 385. 8. Tannenbaum, S. R.; Wishnok, J. S.; Leaf, C. D. Am. J. Clin. Nutr. 1991, 53, 247S. 9. Bradbury, J. Lancet 1998, 352, 1287. 10. Marletta, M. A.; Tayeh, M. A.; Hevel, J. M. BioFactors 1990, 2, 219. 11. Beckman, J. S.; Koppenol, W. H. Am. J. Physiol. 1996, 271, C1424. 12. Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. J. Med. Chem. 1995, 38, 4343. 13. Fung, H.-L.; Bauer, J. A. Cardiovasc. Drugs Ther. 1994, 8, 489. 14. Wink, D. A.; Cook, J. A.; Pacelli, R.; DeGraff, W.; Gamson, J.; Liebmann, J.; Krishna, M. C.; Mitchell, J. B. Arch. Biochem. Biophys. 1996, 331, 241. 15. Roberts, J. D., Jr.; Zapol, W. M. Semin. Perinatol. 2000, 24, 55. 16. Stamler, J. S.; Simon, D. I.; Osborne, J. A.; Mullins, M. E.; Jaraki, O.; Michel, T.; Singel, D. J.; Loscalzo, J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 444. 17. Burstyn, J. N.; Yu, A. E.; Dierks, E. A.; Hawkins, B. K.; Dawson, J. H. Biochemistry 1995, 34, 5896. 18. Drago, R. S. Adv. Chem. Ser. 1962, 36, 143. 19. Maragos, C. M.; Morley, D.; Wink, D. A.; Dunams, T. M.; Saavedra, J. E.; Hoffman, A.; Bove, A. A.; Isaac, L.; Hrabie, J. A.; Keefer, L. K. J. Med. Chem. 1991, 34, 3242. 20. Switkes, E. G.; Dasch, G. A.; Ackermann, M. N. Inorg. Chem. 1973, 12, 1120. 21. Hope, H.; Sequeira, M. R. Inorg. Chem. 1973, 12, 286. 22. Vanin, A. F.; Vedernikov, Y. I.; Galagan, M. E.; Kubrina, L. N.; Kuzmanis, Y. A.; Kalvin’sh, I. Y.; Mordvintsev, P. I. Biochemistry (Moscow) 1991, 55, 1048. 23. Traube, W. Liebigs Ann. Chem. 1898, 300, 81. 24. Woodward, R. B.; Wintner, C. Tetrahedron Lett. 1969, 10, 2689.

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Nitrogen-Based Diazeniumdiolates: Versatile Nitric Oxide-Releasing

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