BACKGROUND OF THE INVENTION [0002] Diazeniumdiolates are useful for a host of clinical applications because of their ability to generate bioactive nitric oxide (NO) under physiological conditions.

Many of the known diazeniumdiolates and their clinical applications are disclosed in recently issued patents. For example, U. S. Patent Nos. 5,155,137 (Keefer et al.) and 5,250,550 (Keefer et al.) disclose complexes of nitric oxide with polyamines. U. S.

Patent No. 5,366,997 (Keefer et al.) discloses oxygen-substituted derivatives of nucleophile-nitric oxide adducts as nitric oxide donor drugs. U. S. Patent No. 5,389,675 (Christodoulou et al.) discloses mixed ligand complexes of nitric oxide-nucleophile adducts. U. S. Patent Nos. 5,405,919 (Keefer), 5,525,357 (Keefer et al.) and 5,718,892 (Keefer et al.) disclose polymer-bound nitric oxide/nucleophile adduct compositions.

U. S. Patent No. 5,632,981 (Saavedra et al.) discloses biopolymer-bound nitric oxide- releasing compositions. U. S. Patent No. 5,691,423 (Smith et al.) discloses polysaccharide-bound nitric oxide-nucleophile adducts. U. S. Patent No. 5,721,365 (Keefer et al.) discloses N-substituted piperazine diazeniumdiolates. U. S. Patent No.

4,954,526 (Keefer et al.) discloses nitric oxide-primary amine complexes useful as cardiovascular agents. U. S. Patent Nos. 5,039,705 (Keefer et al.) and 5,208,233 (Keefer et al.) disclose anti-hypertensive compositions of secondary amine-nitric oxide adducts.

U. S. Patent No. 5,185,376 (Diodati et al.) discloses therapeutic inhibition of platelet aggregation by nucleophile-nitric oxide complexes. U. S. Patent No. 5,650,447 (Keefer et al.) discloses nitric oxide-releasing polymers to treat restenosis and related disorders.

U. S. Patent No. 5,676,963 (Keefer et al.) discloses implants, prostheses, and stents comprising polymer-bound nitric oxide/nucleophile adducts capable of releasing nitric oxide. U. S. Patent No. 5,700,830 (Korthuis et al.) discloses the use of nitric oxide adducts for reducing metastatic risk. U. S. Patent Nos. 5,714,511 (Saavedra et al.) and 5,814,666 (Keefer et al.) disclose selective prevention of organ injury in sepsis and shock using selective release of nitric oxide in vulnerable organs. U. S. Patent No.

5,731,305 (Keefer et al.) discloses anti-hypertension compositions of secondary amine- nitric oxide adducts. U. S. Patent No. 5,910,316 (Keefer et al.) discloses encapsulated and non-encapsulated nitric oxide generators useful as antimicrobial agents.

[0003] Other diazeniumdiolates useful in a host of applications include those described in U. S. Patent Application No. 09/254,301, which discloses O2-arylated and O2-glycosylated diazeniumdiolates, and U. S. Patent No. 6,232,336, which discloses amidine-or enamine-derived diazeniumdiolates.

[0004] In addition to their positive regulatory activities, such as vasodilation and inhibition of platelet aggregation, it is often desirable to exploit nitric oxide's toxic actions such as, for example, its antimicrobial activity. Because nitric oxide is not by itself uniformly toxic to all cells, with many mammalian cell types being especially resistant to NO exposures even at high fluxes, a more general way to effect NO-based toxicity is needed.

[0005] One way to effect NO-based toxicity is by generating the nitric oxide in an environment that contains a superoxide ion. These two radicals can combine to produce peroxynitrite ion (ONOO-), an especially reactive toxicant, as in the following equation: °2-+ NO < ONOO- However, because superoxide ions may be entirely absent from the cells, there exists a need in the art for a compound capable of generating both NO and 02-at once, thereby ensuring the presence of peroxynitrite in the active site.

[0006] One structural class of peroxynitrite-generating NO donor drug that has, in some embodiments, been approved for clinical use is the sydnonimine family.

Oxidative activation of molsidomine produces peroxynitrite when molecular oxygen serves as the oxidant, a phenomenon that explains some of the toxic side effects of therapies in which sydnonimines are administered systemically. This feature poses a substantial limitation to the use of sydnonimines as NO donor drugs in many desired applications. For example, when sydnonimines are administered systemically, the peroxynitrite evolution is not localized, thereby causing undesired and damaging side- effects on non-target cells. In addition, the use of sydnonimines as peroxynitrite generators is limited because the chemical structure does not allow for large and diverse combinatorial libraries to be derived. Furthermore, sydnonimines are capable only of releasing one mole of nitric oxide per molecule, thereby limiting the generation of peroxynitrite to one mole per mole of sydnonimine.

[0007] The invention is a novel class of compounds that have important advantages over the sydnonimine class of compounds. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

SUMMARY OF THE INVENTION [0008] The invention provides a novel class of compounds capable of releasing nitric oxide (NO) and concurrently generating a flux of superoxide (02). When released from the novel compounds of the invention, the NO and 02-are capable of combining to form a peroxynitrite ion (ONOO-). Advantageously, the compounds of the invention are capable of providing peroxynitrite at the target site. Targeting helps to limit or pinpoint the toxic action of the peroxynitrite to the desired cellular location and minimize unwanted side effects to non-target cells. Further, the compounds of the invention are not limited to one mole or less each of superoxide and nitric oxide per mole of compound, as are the sydnonimines, but rather are capable of releasing at least two moles of NO and an unlimited concurrent flux of superoxide from the same molecule, thereby capable of generating up to two moles of peroxynitrite per mole of drug.

[0009] The compounds of the invention are diazeniumdiolates which include a superoxide-generating moiety. The term"diazeniumdiolate"as used herein is meant to include those compounds described herein having the functional group f N (O) NO] (hereinafter abbreviated as N202-) and are capable of releasing nitric oxide either spontaneously or upon cleavage of a protecting group. The superoxide-generating diazeniumdiolate compounds of the invention include the structural moieties XfN2023R- Q or Q-XfN202] or Q-XiN2023R, wherein Q is a superoxide-generating moiety and the [N202] group characterizes the diazeniumdiolate. This superoxide-generating moiety is chemically bonded to the N'nitrogen or to the 02-oxygen of the fNl (Ol) N202] moiety, either directly or optionally through linking groups R and X.

[0010] By"superoxide-generating moiety"is meant that the moiety functions to produce a superoxide ion. For example, the production of a superoxide ion can be accomplished by the release of superoxide from the moiety itself. Further, the moiety can catalyze the reduction of environmental molecular oxygen (02) to a superoxide ion (02').

[0011] The invention also provides a polymer which includes superoxide-and NO-generating functional groups bound to a polymer. By"bound to a polymer,"it is meant that the superoxide-and NO-generating functional groups are associated with, part of, incorporated with or contained within the polymer matrix physically or chemically. Physical association or bonding of the superoxide-and NO-generating functional groups to the polymer may be achieved by coprecipitation of the polymer with superoxide-and NO-generating functional groups as well as by covalent bonding of the superoxide-and NO-generating functional groups to the polymer. Chemical bonding of the superoxide-and NO-generating functional group to the polymer may be achieved by, for example, covalent bonding of the nucleophilic moiety of the NO- generating moiety to the polymer such that the nucleophile residue to which the NO- generating moiety is attached forms part of the polymer itself, i. e., is in the polymeric backbone or is attached to pendant groups on the polymer backbone. The manner in which the superoxide-and NO-generating moieties are associated with, part of, incorporated with or contained within, i. e.,"bound,"to the polymer is inconsequential to the invention and all means of association, incorporation and bonding are contemplated herein.

[0012] The invention also provides a composition which includes a superoxide- generating diazeniumdiolate and a carrier. Polymeric compositions are also provided which include a carrier and a nitric oxide-releasing polymer having superoxide- generating moieties bound to the polymer.

[0013] The invention further provides a method of treating biological disorders in which dosage with nitric oxide/superoxide cogeneration or peroxynitrite evolution would be beneficial comprising administering a superoxide-generating diazeniumdiolate in an amount sufficient to generate a therapeutically effective amount of peroxynitrite.

By"nitric oxide/superoxide cogeneration"is meant that the compounds of the invention are capable of releasing nitric oxide while concurrently generating superoxide. By "peroxynitrite evolution"is meant that the nitric oxide/superoxide cogeneration products combine to form peroxynitrite.

[0014] The invention also provides a method of treating an inanimate object with a superoxide-generating diazeniumdiolate of the invention to reduce the presence of an infectious agent or to prevent the presence of an infectious agent.

DETAILED DESCRIPTION OF THE INVENTION [0015] The invention provides a compound comprising a [N202-] functional group and a superoxide-generating moiety. In particular, the invention provides a compound that is capable of generating nitric oxide and superoxide. Even more particularly, the invention provides a compound of Formula (I) : wherein X and R3 are organic or inorganic moieties, z and z'can be the same or different, with z'being 0 or 1, and z being 0 or an integer of at least 1, and Q3 is a superoxide-generating moiety, with the proviso that when z is 0, then X is a superoxide- generating moiety or it is a linking group substituted with a superoxide-generating moiety.

[0016] In a preferred embodiment of the invention is a compound of Formula (II) : wherein x'and y'are the same or different and are 0 or 1, x and y are the same or different and are 0 or an integer of at least 1, Rl, R2, and R3 are the same or different and are organic or inorganic moieties, and Ql, Q2, and Q3 are the same or different and are superoxide-generating moieties, with the proviso that at least one of x, y or z is at least 1. It will be appreciated by those skilled in the art that although the"mono" diazeniumdiolate compound of the invention is shown in Formula I, compounds containing more than one diazeniumdiolate group, e. g., di, tri, tetra, penta, hexa, hepta, octa, nona, deca, and even higher, are contemplated by the invention, e. g., when any of Qn or Rn is substituted with at least one N202-functional group.

[0017] Advantageously, the compounds of the invention have new and useful properties. Many compounds of the invention are stable at neutral or acidic conditions in the absence of nucleophiles (i. e., will not generate nitric oxide). Another advantageous property of the compounds of the invention is that the linkage between the oxygen and the linking group R3 (i. e., when z'is 1) or between the 02-oxygen and the superoxide-generating moiety Q3 (i. e., when z'is 0 and z is at least 1) is often susceptible to cleavage by nucleophiles, including hydroxide ions. When the compounds of the invention are placed into a basic or nucleophilic environment, the linkage to the 02-oxygen can be broken. When the 02-oxygen linkage is broken, the diazeniumdiolate ion spontaneously degrades via a predictable, first order mechanism, giving rise to NO. The superoxide-generating moiety either produces superoxide itself or catalyzes the reduction of environmental O2 to superoxide. The superoxide- generating moieties are capable of generating a perpetual flux of superoxide in the environment, as long as there is a supply of molecular oxygen in the environment.

[0018] Further, the NO released from the diazeniumdiolate ion and the superoxide generated by the superoxide-generating moiety, e. g., Ql, Q2, and/or Q3, are capable of combining to form peroxynitrite, generally as follows: 02-+ NO- ONOO- [0019] As will be described in further detail hereinafter, compounds of the invention can be comprised of any number of [N202-] functional groups and superoxide-generating moieties. For example, by bonding superoxide-generating moieties covalently to the [N202-] functional group through the 02-oxygen or a linking group R3 covalently attached to the 02-oxygen, as well as covalently bonding a superoxide-generating moiety directly to the nitrogen of the [N202-] functional group or through a linking group X covalently bonded to the nitrogen of the [N202-] functional group, with each such linking group X or R3 having a structure that can be varied over in principle an infinite number of possibilities, exceptionally large and diverse combinatorial libraries can be derived from the versatile diazeniumdiolate structure. Similarly, numerous nitric oxide-releasing functional groups can be incorporated into one compound in accordance with the invention. The chemical versatility of the diazeniumdiolate structure therefore allows for exceptionally diverse compounds capable of releasing a large number of superoxide moieties and a large amount of NO. In addition, such compounds are capable of cogenerating large quantities of nitric oxide and superoxide, which can in turn combine to generate peroxynitrite. Further, this chemical versatility can be used to make the action of inventive compounds localized, i. e., localized release of the NO and superoxide ions. This localization limits the nitric oxide/superoxide exposure and hence the peroxynitrite exposure to desired cellular locations and allows for the pinpointing of the toxic action to the desired target therefore minimizing unwanted side effects to non-target cells.

[0020] Further, upon cleavage of the R3 linking group from the oxygen of the [N202-] functional group, the linking group R3 can be substituted with a nucleophile provided by the environment. If the nucleophile provided by the environment is part of an enzyme, that enzyme can be inactivated. The susceptibility to nucleophilic attack of certain compounds also makes them particularly amenable to designing prodrugs for targeting peroxynitrite to nucleophilic tissue components, body sites and microenvironments in the body.

[0021] Turning to the compounds of Formula I, superoxide-generating moieties such as Q3 envisioned by the invention include, for example, any superoxide-generating moiety capable of releasing superoxide itself or catalyzing the reduction of environmental oxygen to superoxide. In particular, these superoxide-generating moieties include metal surfaces such as those described in Anpo et al., Top. Catal., 8: 189-198 (1999); nitrofurantoin and certain substituted diazapentalenes such as those described in Nagano et al., Kikan Kagaku Sosetsu, 34: 195-205 (1998) and Nagano, T., Yuki Gosei Kagaku Kyokaishi, 47: 843-854 (1989) ; enolizable sugars including dihydroxyacetone (investigated as a prototype for larger analogs), glycated proteins, and fructosyl amines such as those described in references 191-193 of Fridovich, I., Annu.

Rev. Biochem., 64: 97-112 (1995); luminol as described in Faulkner et al., Free Radic.

Biol. Med., 15: 447-451 (1993); thiols, flavins, quinones, catecholamines, and pterins such as those described in Cross et al., Biochem. Biophys. Acta, 1057: 281-298 (1991); peroxyl radicals from carbohydrates, nucleotides, purines, and pyrimidines such as those described in Bielski et al., Int. J. Radiat. Biol., 59: 291-319 (1991); riboflavin and nitroaromatic compounds such as those described in Chauqui et al., Radiat. Phys.

Chem., 30: 365-373 (1987) ; hydrazines, pyridines, and quinones such as those described in Sawyer et al., Biol. Med., Proc. Int. Conf., 4th, 88-91 (1986); flavins, hemoglobin, catecholamines, pteridine derivatives, aromatic nitro compounds and hydroxylamines, redox dyes such as, for example, Alizarine Yellow, Cu (II) and Fe (II) complexes, hydroquinones, melanin, and thiols, such as those described in Niviere et al., Anal. Free Radic. Biol. Systems, 11-19 (1995), all of which are incorporated herein by reference.

[0022] Preferably, the superoxide-generating moiety is selected from the group consisting of an alkylsulfoxide, an Amadori (condensation product) compound, an aminophenol, an anthracycline, a biphenyl, a bispyridinium, a catechol such as a catecholamine or a catecholestrogen, and derivatives thereof, cytochrome P450 reductase, a diazapentalene, a dihydropyridine, a hydroquinone, NAD (P), NAD (P) H, a nitroaromatic, paraquat, a phenol, a phenothiazine, a phenoxazine, a phenylenediamine, a porphyrin, a psoralen, a pterin, a pyridine such as 2,5-dihydroxypyridine, a quinone, a quinoneimine, a quinonediimine, riboflavin, a substituted or unsubstituted diazapentalene, a thiol, a Cu (II) complex, a Fe (II) complex, a 4,4'-bis (dimethylamino)- diphenyl, multi-ring analogues thereof and derivatives thereof. By"derivatives"is meant to include not only structural derivatives, but also various oxidation states and metabolites thereof. Catecholamines is meant to include epinephrine, DOPA, and derivatives thereof. Nitroaromatic is meant to include a nitrofurazone. Porphyrins is meant to include a heme, an oxyheme, a metalloporphyrin such as, for example, and derivatives thereof. Quinone derivatives is meant to include a catechol, a hydroquinone, a heteroaromatic quinone such as, for example, a 1,2-benzoquinone, a 1,4-benzoquinone, a 1,2-aminophenol, a 1,4-aminophenol, a 1,2- diaminobenzene, a 1,4-diaminobenzene, multi-ring analogues thereof and derivatives thereof.

[0023] Other preferred superoxide-generating moieties include aclacinomycin A, cororubicin, daunorubicin, diazaquone, doxorubicin, estrone 3,4-quinone, fredericamycin A, hypocrellin, mansonon D, mitomycin C, nanafrocin, rifampicin, rubomycin, a thespone, and streptonigrin. These moieties can be provided with a N202- functional group or a nitric oxide-releasing functional group, such as those described herein, as illustrated in Example 13.

[0024] Other preferred superoxide-generating moieties include gentisinic acid, pyrocatechuic acid, pyrogallic acid, caffeic acid, ferulic acid, etoposides, 2- hydroxylestradiol, pterins, such as neopterin, pyrazines, such as lamprene, pteridines such as, for example, multi-ring analogues thereof, and derivatives thereof. It will be appreciated by those skilled in the art that these moieties can also be provided with a N202-functional group or a nitric oxide-releasing functional group.

[0025] Radiolytic (X and y) and photolytic generation of superoxide is described in, for example, Chapter 3 of Afanase'ev, I. B.,"Superoxide Ion: Chemistry and Biological Implications,"Vol. I, CRC Press, Boca Raton, 1989). The invention therefore envisions the use of the novel superoxide-generating diazeniumdiolates in radiotherapy and photodynamic therapy applications, especially if ketone, arylamino, phenolic, or formyl groupings capable of sensitizing the process are present in the molecule.

Phthalocyanine, sulfite ion, and solid CdS can also serve this purpose. Rate constants for reaction of numerous 1-electron reductants with 02 are given in Table I of this chapter. For example, an alkyl hydroxylamine reduced 02 (eq. 28 in the chapter), as did cuprous, ferrous, and mercurous complexes. Also, NADH and 5-hydroxytryptophan are said to react with singlet 02 to give superoxide. Decomposition of a-hydroxyperoxyl radicals generated from alcohols, unsaturated fatty acids, amino acids, and sugars is covered in Section V of this chapter.

[0026] Turning now to the linking group R3 of Formula I, R3 is preferably an organic moiety selected from the group consisting of an alkyl, an aryl, and a nonaromatic cyclic.

By"alkyl"is meant acyclic moieties containing carbon and hydrogen and optionally containing nitrogen, oxygen, sulfur, phosphorus, and halogen substituents. Preferably, the alkyl moiety is a Cl 24-containing moiety. By"aryl"is meant a moiety containing at least one aromatic ring including, for example, homocyclic, heterocyclic, and polycyclic aromatic structures and derivatives thereof. Preferably, the aryl moiety is a Cl 30- containing moiety. By"polycyclic"is meant to include a multicyclic structure having either homocyclic or heterocyclic structures or both forming part of the multicyclic structure and, unless otherwise indicated, can include both aromatic and nonaromatic structures. By"nonaromatic cyclic"is meant a moiety containing at least one ring structure and no aromatic groups including, for example, homocyclic, heterocyclic, and polycyclic nonaromatic structures and derivatives thereof. Preferably, the nonaromatic cyclic moiety is a C330-containing moiety.

[0027] It will be appreciated by those skilled in the art that R3 can be freely substituted with at least one superoxide-generating moiety or more than one superoxide- generating moiety, such as those described herein. For example, R3 can be substituted with at least 2 to about 100 superoxide-generating moieties and, more particularly, R3 can be substituted with at least 2 to about 20 superoxide-generating moieties. By way of further example, superoxide-generating moiety substitutions of even greater than 100 are envisioned. For larger molecules, a great number of superoxide-generating moiety substitutions, e. g., of greater than one thousand, are contemplated. It will be appreciated by those skilled in the art that the number of superoxide-generating moieties substituting R3 may be dependent on the desired end-use of the inventive compounds.

[0028] Advantageously, the R3 linking group is an aryl group. 02-arylated diazeniumdiolates suitable for the invention, as well as their chemical synthesis, are disclosed in, for example, WO 98/13358 (Keefer et al.), and U. S. App. No. 09/254,301 (Keefer et al.), and incorporated herein by reference.

[0029] When R3 is an aryl group, as defined herein, it is intended to include all aryl groups that are (or can be made) amenable to reaction with the oxygen atom of a diazeniumdiolate. Illustrative of the aryl groups R3 are acridine, anthracene, benzene, benzoquinone, benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, dinitrophenyl, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine, porphin, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrocoline, pyrrole, quinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole. Preferably, the linking group R3 includes the following structural units: and isomers thereof.

[0030] In keeping with the invention, the linking group R3 can be variably derivatized with numerous substituents well known in the art. For example, the substituents of the linking group R3 can include XfN (O) NO]-, wherein X is as defined hereinafter and is the same X of Formula I, as well as halo, hydroxy, alkylthio, arylthio, alkoxy, aryloxy, amino, mono-or di-substituted amino, a polyamino, ammonio or substituted ammonio, nitroso, cyano, sulfonato, mercapto, nitro, oxo, oximino, a polycyclic, aromatic polycyclic, Cl 24 alkyl, C212 alkenyl, C324 cycloalkyl, C224 heterocyclic, C324 alicyclic, a Cl 30 aryl, benzyl, phenyl, substituted benzyl, substituted phenyl, benzylcarbonyl, benzoyl, saccharides, substituted benzylcarbonyl, substituted benzoyl, and phosphorus derivatives. Illustrative phosphorus derivatives include phosphate and phosphono, (HO) 2P (O) 0- and substituted (HO) 2P (O) 0- moieties, wherein one or more oxygen atoms can be independently replaced by S or NR*, wherein R* is understood to be a Ci. io containing alkyl, cycloalkyl, or aryl group. Illustrative substituents include Cl 24 acyl, and wherein R is hydrogen, substituted or unsubstituted C123 alkyl, substituted or unsubstituted C323 cycloalkyl, substituted or unsubstituted Cl_12 alkenyl, benzyl, phenyl, substituted benzyl or substituted phenyl, and said substituted benzyl or substituted phenyl is substituted with one to five substituents selected from the group consisting of nitro, halo, hydroxy, C, 24 alkyl, C, 24 alkoxy, amino, mono-C, 24 alkylamino, di-C, 24 alkylamino, cyano, phenyl and phenoxy. Preferred saccharides include ribose, glucose, deoxyribose, dextran, starch, glycogen, lactose, fucose, galactose, fructose, glucosamine, galactosamine, heparin, mannose, maltose, sucrose, sialic acid and cellulose. Other preferred saccharides include phosphorylated, 3,5- cyclophosphorylated, and polyphosphorylated hexoses and pentoses.

[0031] Examples of substituted aryl compounds of the invention that can be linked to the diazeniumdiolate group include dinitrophenol (a benzene), hypoxanthine (a purine), uridine (a pyrimidine), vitamin K5 (a naphthalene), ribosyl uridine (a nucleoside), combinations thereof and derivatives thereof.

[0032] In another embodiment of the invention, the linking group R3 is substituted with at least one superoxide-generating moiety.

[0033] In another embodiment of the invention, the linking group R3 is identical to or structurally analogous to molecules, or substituents thereof, normally found in living organisms. These biologically relevant groups can be selected from nucleotides, nucleosides, and nucleic acids, peptides, including peptide hormones, non-peptide hormones, vitamins and other enzyme cofactors such as porphyrins, and others.

Examples of biologically relevant aryl groups are thyroxine, NAD (or NADH), chlorophyll, hypoxanthine, uridine, and vitamin K5.

[0034] In accordance with the invention, any of the compounds in the class of compounds defined as diazeniumdiolates can be subjected to substitution with a superoxide-generating moiety. Thus, for the compounds having Formula I, X can be any organic or inorganic moiety, such as those disclosed in, for example, U. S. Pat. Nos.

5,155,137; 5,250,550; 5,366,997; 5,389,675; 5,405,919; 5,525,357; 5,718,892; 5,632,981; 5,691,423; 5,721,365; 5,039,705; 5,208,233; 5,185,376; 5,650,447; 5,676,963; 5,700,830; 5,714,511; 5,814,666; 5,731,305; 5,910,316 and U. S.

Pat. App. No. 09/254,301, and WO 99/01427, and incorporated herein by reference.

Preferably, X contains atoms in addition to or other than carbon and hydrogen, and is linked to the nitrogen of the [N202-] functional group through an atom other than carbon.

Most preferably, X is an amine, and is linked to the nitrogen of the [N202-] functional group through a nitrogen atom. Suitable moieties of X also include, but are not limited to, C, 24 alkyl, aryl, and nonaromatic cyclic, as defined herein. Preferably, the aryl moiety is a C, 30-containing moiety. Preferably, the nonaromatic cyclic moiety is a C3-30-containing moiety.

[0035] In accordance with the invention, suitable moieties of X may also include superoxide generators. In other words, X may itself be a superoxide-generating moiety.

By"superoxide-generating moiety"is meant that X can be any suitable superoxide- generating moiety as hereinabove described, such as those superoxide-generating moieties defined as Qn, e. g., Ql, Q2, Q3, etc.

[0036] The moiety X of Formula I can be substituted or unsubstituted with additional moieties, such as, for example, fN (O) NO], [N (O) NO3, superoxide-generating moieties such as those defined hereinabove, halo, hydroxy, alkylthio, alkoxy, aryloxy, amino, mono-or di-substituted amino, cyano, sulfonato, mercapto, nitro, substituted or unsubstituted C,, 2 alkyl, substituted or unsubstituted C38 cycloalkyl, substituted or unsubstituted C2-8 heterocycloalkyl, substituted or unsubstituted C2-12 alkenyl, benzyl, phenyl, substituted benzyl, substituted phenyl, benzylcarbonyl, benzoyl, saccharides, substituted benzylcarbonyl, substituted benzoyl, and phosphorus derivatives. Illustrative phosphorus derivatives include phosphato and phosphono moieties. Illustrative phosphato moieties include (HO) 2P (O) 0- and substituted (HO) 2P (O) 0- moieties, wherein one or more oxygen atoms can be independently replaced by S or NR*, wherein R* is understood to be a C, 8-containing alkyl, nonaromatic cyclic, or aryl group.

Preferred substituents include C,, 2 acyl, and wherein R is Ci. jo substituted or unsubstituted alkyl, C3 l l alkenyl, C3 8 substituted or unsubstituted cycloalkyl, benzyl, phenyl, substituted benzyl or substituted phenyl, and the substituted benzyl or substituted phenyl is substituted with one or two substituents selected from the group consisting of halogen, hydroxy, Cl 4 alkyl, Cl 4 alkoxy, amino, mono-CI-4 alkylamino, di-C, 4 alkylamino, phenyl and phenoxy. Preferred saccharides and polysaccharides include ribose, glucose, deoxyribose, dextran, starch, glycogen, lactose, galactose, fructose, glucosamine, galactosamine, heparin, mannose, maltose, sucrose, sialic acid, and cellulose. Other preferred saccharides are phosphorylated, 3,5- cyclophosphorylated, and polyphosphorylated pentoses and hexoses.

[0037] It will be appreciated by those skilled in the art that X can be freely substituted with at least one superoxide-generating moiety or more than one superoxide- generating moiety, such as those described herein. For example, X can be substituted with at least 2 to about 100 superoxide-generating moieties and, more particularly, X can be substituted with at least 2 to about 20 superoxide-generating moieties. By way of further example, superoxide-generating moiety substitutions of even greater than 100 are envisioned. For larger molecules, a great number of superoxide-generating moiety substitutions, e. g., of greater than one thousand, are contemplated. It will be appreciated by those skilled in the art that the number of superoxide-generating moieties substituting X may be dependent on the desired end-use of the inventive compounds.

[0038] In another embodiment of the invention, X of Formula I is: wherein R3 of Formula I is a CI-12 straight chain alkyl, a C3 l2 branched chain alkyl, a C2-12 straight chain or a C3, 2 branched chain alkenyl, a C1-12 acyl, sulfonyl, carboxamido, a glycosyl group, a Cl 30 aryl group or a group of the formula (-CH2-)nON=N(O)NR24R25, wherein n is an integer of 2-8, and R24 and R25 are independently a Cl l2 straight chain alkyl, a C3 l2 branched chain alkyl, or a C2-12 branched chain alkenyl, or R24 and R25, together with the nitrogen atom to which they are bonded, form a heterocyclic group selected from the group consisting of a pyrrolidino, a piperidino, a piperazino and a morpholino group; R21 is hydrogen, hydroxyl, OM, wherein M is a cation, halo, XlR22, wherein Xl is O, NR23 or S, and R22 and R23 are independently selected and are a Cl 24 alkyl, a C3-24 cycloalkyl, a C224 alkenyl, a Ce-so aryl, or a heterocyclic group, and optionally, R2l, R22, R23, R24 and R25 can be a linking group substituted with superoxide- generating moieties.

[0039] In another embodiment of the invention, X is an inorganic moiety as described in U. S. Patent No. 5,212,204. Preferred embodiments of Formula I, in which X is an inorganic moiety, are'038- (sulfite) and-0' (oxide).

[0040] In another embodiment of the invention, the inventive compounds are derived from the compounds disclosed in WO 98/13358, and thus have the Formula (II) : wherein x'and y'are the same or different and are 0 or 1, Q1, Q2 and Q3 are the same or different and are superoxide-generating moieties, x and y are the same or different and are 0 or an integer of at least 1, with the proviso that at least one of x, y or z is at least 1, Rl and R2 are the same or different and are hydrogen, a Cl l2 straight chain alkyl, a Cl l2 alkoxy or acyloxy substituted straight chain alkyl, a C2-12 hydroxy or halo substituted straight chain alkyl, a C3 l2 branched chain alkyl, a C3 l2 hydroxy, halo, alkoxy, or acyloxy substituted branched chain alkyl, a C2-12 straight chain alkenyl, or a C3 l2 branched chain alkenyl, wherein R1 and R2 are optionally substituted with hydroxy, alkoxy, acyloxy, halo, or benzyl; or wherein R1 and R2 together with the nitrogen atom to which they are bonded form a heterocyclic ring selected from the group consisting of : wherein E is (CH2) W or NR6, w is 1 to 12, p is 1 or 2, A and B can be the same or different and are NR7, O or S, m is 1 to 5, and m'is 0 to 4 with the proviso that (m + m') is 1 to 5; R5 is hydrogen, a C-18 straight chain alkyl, a C3-8 branched chain alkyl, a C3-8 cycloalkyl, a C630 aryl, or a carboxyl; R6 and R7 are independently hydrogen, C38 cycloalkyl, C1-12 straight or C3 l2 branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, or t-butoxycarbonyl; and R8 is hydrogen, a C, 6 straight chain alkyl or a C36 branched chain alkyl.

[0041] Preferably, Qn (e. g., Ql, Q2 and Q3, which are the same or different) is a group of formula wherein Rio, Rl l, Rl2, and R13 can be the same or different and are hydrogen, hydroxy, a halo, nitro, an acyl, an acyloxy, an amino, a polyamino, an alkyl, preferably a C324 alkyl, an alkoxy, an alkenyl, preferably a C3-24 alkenyl, an alicyclic, preferably a C324 alicyclic, a heterocyclic, preferably a C2-30 heterocyclic, a carboxy and esters thereof, an oxime, a polycyclic, an aryl, preferably a Cl 30 aryl, or Rio and Ru and/or R12 and R13, together with the carbon atoms to which they are bonded, form a nonaromatic cyclic, polycyclic, or aromatic fused ring. Preferably, the aromatic fused ring system thus formed is the quinone derivitive of an acridine, an anthracene, a benzofuran, a benzothiophene, a benzoxazole, a benzopyrazole, a benzothiazole, a carbazole, a cinnoline, an indole, an isobenzofuran, an isoindole, an isoquinoline, a naphthalene, a phenanthrene, a phenanthridine, a phenothiazine, a phenoxazine, a phthalazine, a phthalocyanine, a pyrrocoline, a quinoline, a quinoxaline, a quinazoline, combinations thereof, or derivatives thereof.

[0042] It will be appreciated by those skilled in the art that Qn can be substituted or unsubstituted. In that regard, Rio, Rn, R12, and R13 can be independently substituted with a hydroxy, a halo, a nitro, an oxo, a thio, a phospho, an acyl, an acyloxy, an amino, a polyamino, an alkyl, preferably a Cl 24 alkyl, an alkoxy, an alkenyl, preferably a C324 alkenyl, an alicyclic, preferably a C324 alicyclic, a nonaromatic cyclic, preferably a C3-30 nonaromatic cyclic, an oxime, a polycyclic, an aromatic polycyclic, an aryl, preferably a C1-30 aryl.

[0043] In a preferred embodiment of the invention, Qn is [0044] Further examples include compounds derived from the polyamine diazeniumdiolates, such as those disclosed in U. S. Patent No. 5,250,550 and WO 98/13358, incorporated herein by reference, and thus have the Formula (III) : wherein b and d can be the same or different and are 0 or 1, R, 4, Rlsn Rl6, Rl7, and Rl8 are the same or different and are selected from the group consisting of hydrogen, C38 cycloalkyl, Cri-12 straight or C3, 2 branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, or 2,2,2-trihalo-t-butoxycarbonyl, and i, j, and k are the same or different and are integers from 2 to 12. Rn of Formula (III) may also be substituted with one or more superoxide-generating moieties, as hereinabove defined.

[0045] In another embodiment of the invention is a compound having the formula: wherein X and R are organic or inorganic moieties which can be the same or different and can be superoxide generating moieties or substituted with superoxide generating moieties, wherein at least one of X or R is a superoxide-generating moiety or substituted with a superoxide-generating moiety.

[0046] In another embodiment of the invention is a compound of Formula (IV): wherein Z is and wherein Rip and R20 are the same or different and are selected from the group consisting of hydrogen, C38 cycloalkyl, C,, 2 straight or C3, 2 branched chain alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl, and 2,2,2,-trichloro-t-butoxycarbonyl, and f is an integer from 0 to 12. Rn of Formula (IV) may also be substituted with superoxide-generating moieties, as hereinabove defined.

[0047] Another particularly useful embodiment of the invention includes compounds of Formula I, wherein X is an amidine-or enamine-derived diazeniumdiolate, such as those disclosed in U. S. Patent No. 6,232,336, and incorporated herein by reference.

[0048] Another particularly useful embodiment of the invention includes compounds of Formula I, wherein X is a polymer, or wherein any compound of the invention is incorporated into a polymeric matrix. Both of these embodiments result in the [N202-] functional group being"bound to the polymer."By"bound to a polymer,"it is meant that the [N202-] functional group is associated with, part of, incorporated with or contained within the polymeric matrix physically or chemically.

[0049] Physical association or bonding of the [N202l functional group to the polymer may be achieved by coprecipitation of the polymer with a nitric oxide/ nucleophile complex as well as by covalent bonding of the [N202-] group to the polymer. Chemical bonding of the [N202-] group to the polymer may be by, for example, covalent bonding of the nucleophile moiety of the nitric oxide/nucleophile adduct to the polymer such that the nucleophile residue to which the [N202] group is attached forms part of the polymer itself, i. e., is in the polymer backbone or is attached to pendant groups on the polymer backbone. The manner in which the nitric oxide- releasing [N202-] functional group is associated with, part of, or incorporated with or contained within, i. e.,"bound"to the polymer is inconsequential to the invention and all means of association, incorporation and bonding are contemplated herein.

[0050] Site-specific application of the polymer-bound adduct composition enhances the selectivity of action of the nitric oxide-releasing [N202-] functional group. If [N202-] functional groups attached to the polymer are localized due to its insolubility, then the effect of their nitric oxide release, and therefore peroxynitrite evolution, will be concentrated in the tissues with which the polymer is in contact. If the polymer is soluble, selectivity of action (e. g., the targeting of specific cell types, tissues, or organs) can still be arranged by, for example, linkage to or derivatization of an antibody specific to the target tissue. Similarly, linkage of [N202-] groups to small peptides that mimic the recognition sequences of ligands for important receptors provides localized nitric oxide/superoxide cogeneration or peroxynitrite evolution, as would linkage to oligonucleotides capable of site-specific interactions with target sequences in a nucleic acid.

[0051] The compounds of the invention can also be derived from the materials disclosed in U. S. Patent Nos. 5,525,357 (Keefer et al.), and 5,405,919 (Keefer et al.), and in U. S. Patent Application Serial No. 08/419,424 (Smith et al.), and are incorporated herein by reference. Any of a wide variety of polymers can be used in the context of the invention. Illustrative of polymers suitable for use in the invention are polyolefins, such as polystyrene, polypropylene, polyethylene, polytetrafluorethylene, polyvinyl chloride, polyvinylidene difluoride, and polyethers such as polyethylene glycol, polysaccharides such as dextran, polyesters such as poly (lactide/glycolide), polyamides such as nylon, polyurethanes, polyethylenimine, biopolymers such as peptides, proteins, oligonucleotides, antibodies and nucleic acids, starburst dendrimers, and the like. In this regard, a polymer containing a diazeniumdiolate can be reacted with a saccharide, such that the saccharide becomes bound to the [N202]-functional group.

[0052] Formation of a diazeniumdiolate from a biopolymer provides a biopolymer- bound diazeniumdiolate composition that can be applied with specificity to a biological site of interest. Site-specific application of the biopolymer-bound diazeniumdiolate enhances the selectivity of action of the nitric oxide-releasing diazeniumdiolate, which occurs following the target-specific cleavage of, for example, the 02-R3 bond or the o2- Q3 linkage of the compounds of Formula (I). As with the other polymers disclosed above, if the superoxide generator attached to the biopolymer is localized because of the inherent properties of the molecule, then the effect of its nitric oxide release will be concentrated in the tissues with which they are in contact. If the biopolymer is soluble, selectivity of action can still be arranged by, for example, attachment to or derivatization of an antibody specific to the target tissue. Similarly, linkage of diazeniumdiolate groups to small peptides that mimic the recognition sequences of ligands for important receptors provides localized nitric oxide/superoxide cogeneration or peroxynitrite evolution, as would linkage to oligonucleotides capable of site-specific interactions with target sequences in a nucleic acid. Other proteins, peptides, polypeptides, nucleic acids and polysaccharides can be similarly utilized. U. S. Patent No. 5,405,919 (Keefer et al.) and U. S. Patent No. 5,632,981 (Saavedra et al.), incorporated herein by reference, disclose similar compounds and processes useful in the preparation of the compounds of the invention.

[0053] It is contemplated that the polymeric compounds of the invention can be used to coat medical devices or can form part of the medical device itself. By"medical device"it is meant to refer to any device that contacts tissue, blood, or other bodily fluids in the course of its use or operation. The bodily fluids are those fluids which are found in or are subsequently used in patients or animals. Preferred medical devices include, for example, prostheses, stents, and medical implants, such as breast implants, prior to surgical connection to the body as a means of reducing the risk of solid state carcinogenesis associated therewith.

[0054] The superoxide-generating diazeniumdiolates of the invention can have a wide range of utilities, in part because of the multifaceted role of nitric oxide, superoxide, and peroxynitrite in bioregulatory processes. Accordingly, the invention also provides a composition, including a pharmaceutical composition, comprising a superoxide-generating diazeniumdiolate, such as those described herein. Preferably, the pharmaceutical composition additionally comprises a pharmaceutically acceptable carrier.

[0055] One skilled in the art will appreciate that suitable methods of administering the superoxide-generating diazeniumdiolate compounds or compositions of the invention to an animal, such as a mammal, are available and, although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and more effective reaction than another route. Pharmaceutically acceptable carriers are also well-known to those who are skilled in the art. The choice of carrier will be determined, in part, both by the particular composition and by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of the invention.

[0056] Formulations suitable for oral administration include (a) liquid solutions, such as an effective amount of the inventive compound dissolved in diluents, such as water or saline, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can include the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

[0057] The compounds of the invention, either alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0058] Formulations suitable for parenteral administration include aqueous and non- aqueous solutions, isotonic sterile injection solutions, which can contain buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e. g., water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0059] The dose administered to an animal, particularly a human, in the context of the invention should be sufficient to effect a therapeutic response in the animal over a reasonable time frame. The dose will be determined by the strength of the particular compositions employed (taking into consideration, at least, the rate of nitric oxide/superoxide cogeneration or peroxynitrite evolution, the extent of nitric oxide/superoxide cogeneration or peroxynitrite evolution, and the bioactivity of the decomposition products derived from the inventive compounds) and the condition of the animal, as well as the body weight of the animal to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side effects that might accompany the administration of a particular composition. A suitable dosage for internal administration is 0.01 to 100 mg/kg per day. A preferred dosage is 0.01 to 35 mg/kg per day. A more preferred dosage is 0.05 to 5 mg/kg per day. A suitable concentration of the inventive compounds in pharmaceutical compositions for topical administration is 0.05 to 15 % (by weight). A preferred concentration is from 0.02 to 5%. A more preferred concentration is from 0.1 to 3%.

[0060] In view of the above, the invention provides methods of using the superoxide-generating diazeniumdiolates of the invention. In one embodiment, a method of treating an animal, such as a mammal, with a biological disorder treatable with nitric oxide/superoxide cogeneration or peroxynitrite evolution, is provided. The method comprises administering to the animal, e. g., the mammal, an amount of a superoxide-generating diazeniumdiolate sufficient to treat the biological disorder in the animal. In this embodiment,"biological disorder"can be any biological disorder, including a biological disorder due to a genetic defect or infection with an infectious agent, such as a virus, bacterium or parasite, as long as the disorder is treatable by nitric oxide/superoxide cogeneration or peroxynitrite evolution.

[0061] In another embodiment, a method is provided for treating an animal, such as a mammal, for infection with, for example, a virus, a bacterium, or a parasite (e. g., leishmania). The method comprises administering to the animal, e. g., the mammal, an amount of a superoxide-generating diazeniumdiolate sufficient to treat the infection in the animal.

[0062] In one aspect of this embodiment of the invention, a method is provided for treating an animal, such as a mammal, for infection with, for example, a virus, such as a retrovirus, in particular HIV, more particularly HIV-1, a bacterium, such as a Gram- positive bacterium, or a parasite, such as Giardia, any one of which comprises a zinc finger protein that can be inactivated by nitric oxide/superoxide cogeneration or peroxynitrite evolution. By"zinc finger protein"is meant a protein comprising a short amino acid domain containing cysteines alone or cysteine and histidine ligands, both of which coordinate with zinc and interact with nucleic acids (South and Summers,"Zinc Fingers,"Chapter 7, In: Adv. Inorg. Biochem. Ser., 8, pp. 199-248 (1990), incorporated herein by reference, including the content of all references cited therein).

By"inactivated"is meant partial or complete loss of activity of the zinc finger protein to be inactivated. The method comprises administering to the animal, e. g., the mammal, an amount of a compound of the invention sufficient to inactivate the zinc finger protein in said infectious agent so as to treat the infection in the animal.

[0063] The above-described method also can be adapted as a means of treating a plant, plant cell, or tissue culture thereof for infection with an infectious agent, such as a virus, e. g., tobacco streak virus (TSV) or alfalfa mosaic virus (AIMV) (South and Summers (1990), supra ; and Sehnke et al., Virology 168: 48 (1989)).

[0064] The methods described herein are useful against zinc fingers comprising the motif C-X2-C-X4-H-X4-C (see, e. g., Wain-Hobson et al., Cell 40 (i): 9-17 (1985)), in which"C"represents cysteine,"H"represents histidine,"X"represents any amino acid, and the numbers"2"and"4"represent the number of"X"amino acids. Such a motif is characteristic of retroviruses, in particular the gag protein of retroviruses. Accordingly, the methods herein are useful against retroviruses, such as HIV, and, in particular, HIV- 1 (Rice et al., Nature Medicine 3 (3): 341-345 (1997); and Rice et al., Reviews in Medical Virology 6: 187-199 (1986)), which includes nucleocapsid p7 proteins (NCp7 proteins) that have two zinc binding domains. Actual and/or potential zinc fingers also have been identified in, among others, the gene products of the EIA genomic region of adenoviruses, the large T antigens from simian virus 40 (SV40) and polyoma viruses, the UvrA protein in E. coli (Culp et al., PNAS USA 85: 6450 (1988)), murine leukemia virus (MuLV-F; Green et al., PNAS USA 86: 4047 (1989)), and bacteriophage proteins (Berg, Science 232: 484 (1986)), such as gene 32 protein (G32P) from bacteriophage T4 (Giedroc et al., Biochemistry 28: 2410 (1989)). Such proteins can be isolated in accordance with methods known in the art (see references cited in South and Summers (1990), supra), and the 02-aryl diazeniumdiolates, which can inactivate such zinc finger proteins, can be identified in accordance with, for example, the zinc finger assay described herein and in Rice et al., J. Med. Chem. 39: 3606-3616 (1996).

[0065] In yet another embodiment of the invention, a method for treating an animal, such as a mammal, for cancer and metastasis thereof is provided. The method comprises administering to the animal an amount of a superoxide-generating diazeniumdiolate sufficient to prevent the growth or metastasis of the cancer in the animal.

[0066] In one aspect of this embodiment, a method for treating an animal, such as a mammal, for cancer is provided, wherein the cancer is due, at least in part, directly or indirectly, to the activity of a zinc finger protein that can be inactivated by a nitric oxide/superoxide cogeneration or peroxynitrite evolution. The method comprises administering to the animal an amount of a compound of the invention sufficient to inactivate the zinc finger protein so as to treat the cancer in the animal (Rice et al., PNAS 89: 7703-7707 (1992)), i. e., prevent the growth or metastasis of the cancer in the animal.

[0067] In still yet another embodiment, a method is provided for treating an animal, such as a mammal, for cancer, wherein the cancer is resistant to treatment with a chemotherapeutic agent (see, e. g., Kelley et al., Biochem. J., 304: 843-848 (1994)), in particular a DNA damaging agent, such as an alkylating agent or an oxidizing agent, due, for example, to the action of an enzyme that adversely affects the activity of the chemotherapeutic agent. The method comprises administering to the animal an amount of a superoxide-generating diazeniumdiolate sufficient to render the cancer in the animal susceptible to treatment with the chemotherapeutic agent. Accordingly, such a method can be used as an adjunct therapy to chemotherapy as needed.

[0068] For example, certain compounds of Formula I of the invention can be synthesized to fit into the active site of glutathione S-transferase (GST), specifically isoenzyme ac (see, e. g., Ji et al., Biochemistry 32 (49): 12949-12954 (1993); and Ji et al., Biochemistry 36: 9690-9702 (1997)). Accordingly, irreversible consumption of glutathione from the active site of glutathione S-transferase-Xe with a specifically tailored superoxide-generating diazeniumdiolate can prevent the enzyme from detoxifying a variety of xenobiotic compounds, such as chemotherapeutic drugs, especially alkylating agents, such as chlorambucil, melphalan and hepsulfam, and other DNA-damaging agents, such as agents that induce electrophilic attack or oxidation, by enzymatic conjugation of the compound with glutathione (see, e. g., Morgan et al., Cancer Chemother. Pharmacol. 37: 363-370 (1996)). This method also has applicability to screening drug-resistant cancer cell lines in vit7-o.

[0069] In another embodiment, a method is provided for treating an inanimate object for the presence of a potentially infectious virus, bacterium, or parasite. The method comprises contacting the inanimate object with an amount of a superoxide-generating diazeniumdiolate sufficient to reduce the presence of the potentially infectious virus, bacterium or parasite. By"potentially infectious"is meant the capability of infecting an animal, such as a mammal, directly or indirectly.

[0070] In one aspect of this embodiment, a method is provided for reducing on an inanimate object the presence of a potentially infectious agent, such as a virus, a bacterium, or a parasite, any one of which comprises a zinc finger protein that can be inactivated by nitric oxide/superoxide cogeneration. The method comprises contacting the inanimate object with an amount of a compound of the invention sufficient to inactivate the zinc finger protein so as to reduce the presence of the potentially infectious agent, e. g., virus, bacterium, or parasite, on the inanimate object.

EXAMPLES [0071] The following examples further illustrate the invention and should not be construed as in any way limiting its scope. With respect to the following examples, NO was obtained from Matheson Gas Products (Montgomeryville, PA). Proton NMR spectra were recorded with a 300 MHz Varian Unity Plus or a Varian XL-200 NMR spectrometer. Chemical shifts are reported in parts per million (ppm) downfield from TMS. Ultraviolet (UV) spectra were run on a Hewlett Packard 8451A Diode Array spectrophotometer. Elemental analyses were performed by Atlantic Microlab Inc.

Example 1: [0072] This example illustrates the preparation of a compound of the invention, 1-B, according to the following reaction: Preparation of 1-A [0073] To a solution of 1.53 g (0.009 mol) of juglone in 100 mL of acetone was added 2 g (0.015 mol) of anhydrous potassium carbonate followed by 2.45 g (0.012 mol) of 1, 5-difluoro-2,4-dinitrobenzene as a solid. The reaction mixture was stirred at room temperature for 72 h and concentrated to dryness under vacuum, then the residue was extracted with dichloromethane. The mixture was filtered through a layer of magnesium sulfate and evaporated to give 2.7 g of glass. The crude material was recrystallized from ethanol to give 2. 31 g of compound 1-A. mp: 164-5°C ; NMR (CDCl3) 6 6.51 (d, 1H), 7.65 (m, 1H), 7.96 (t, H), 8.21 (m, 1H), 8.99 (d, 1H). Anal.

C, H, N. Calcd. for C16H7FN207 : C, 53.64; H, 1.97; N, 7.82. Found: C, 53.60; H, 2.03; N, 7.74.

Preparation of 1-B [0074] A solution of 1 g (0.0028 mol) of compound 1-A in 20 mL of acetone was cooled to 0°C. Solid DMA/NO (398 mg; 0.0031 mol) was added in small lots over a 1 minute period. The ice bath was removed and the reaction mixture was stirred for 72 h, filtered, treated with 5 g of silica gel and placed on a rotary evaporator. The solvent was allowed to evaporate while the silica gel was evenly coated with the crude product. The silica gel containing the crude material was placed in a sample injection module (SIM) for low solubility samples. This was connected to a Kp-Sil column, then attached to a Flash 40 system and chromatographed using dichloromethane as the eluting solvent to give 271 mg of pure compound 1-B: mp 145-6°C ; UV (ethanol) max (s) 244 nm (10.73 mM-'cm-'), 270 nm (7.16 mM~Icm-l), and 310 nm (4.13 mNT'cm-1) ; NMR (CDC13) 6 3.06 (s, 6H), 6.70 (s, 1H), 6.94 (q, 2H), 7.63 (d, 1H), 7.93 (t, 1H), 8. 17 (d, 1H), 8.97 (s, 1H). Anal. C, H, N. Calcd. for C18Hl4N509 : C, 48.77; H, 2.96; N, 15.80. Found: C, 49.17; H, 3.05; N, 15.45.

Example 2: [0075] This example confirms that the cosubstrate glutathione (GSH) can serve as the nucleophile needed to initiate NO generation from 1-B. NO generation was monitored by a chemiluminescence method as previously described (Keefer et al., Methods Enzymol, 268: 281-293 (1996)). Briefly, 2 mL of pH 7.4 phosphate buffer was purged with inert gas into an NO-specific detector (TEA Model 502 A, Thermo Electron Corp., Waltham, MA) for 2.5 min. No signal was detected, even when compound 1-B was added at a final concentration of 1 uM at the 2.5-minute time point. Abundant NO generation was recorded following addition of GSH at 8 mM. The results indicate that NO is produced in neutral aqueous medium only when a strong nucleophile (in this case GSH) is present.

Example 3: [0076] This example provides evidence that the cosubstrate GSH can serve as the reductant that drives concurrent superoxide production.

Part A: Release of superoxide from compound 1-B [0077] NADPH, glutathione (reduced), DMSO, xanthine oxidase, hypoxanthine, and horse ferricytochrome c were purchased from Sigma Chemical Co. (St. Louis, MO).

Compound 1-B was dissolved in DMSO as a 4-5 mM stock solution. The compound was then diluted to working stocks of no more than 500 uM in a potassium phosphate buffer, pH 7.4.

[0078] Superoxide generation was measured by following the reduction of horse ferricytochrome c (a protein easily reduced by 02-that is used in a standard assay for this reactive intermediate) spectrophotometrically at 550 nm (£550nm = 21 mM-'cm-'). A Hewlett Packard 8451A UV-Visible Diode Array Spectrophotometer was used to follow the reaction, and Hewlett Packard 845x UV-Visible ChemStation software was used to display and analyze the results.

[0079] Various concentrations of ferricytochrome c, compound 1-B, glutathione (GSH), and/or NADPH were incubated at 37°C or room temperature and the reduction of cytochrome c was followed kinetically in quartz cuvettes which were blanked against the buffer or the buffer plus cytochrome c.

[0080] Cytochrome c at a concentration of 100 uM in buffer showed little or no change in absorbance, even after adding 1 mM GSH. Reduction began immediately on adding 20 uM of compound 1-B, however, providing evidence for superoxide generation.

Part B: Inhibition of GST activity by compound 1-B [0081] Compound 1-B was dissolved in DMSO to provide a 4-5 mM stock. The compound was then diluted to working stocks of no more than 500 tM in potassium phosphate buffer. The pH of this buffer was 6.5 for glutathione S-transferase (GST) enzymatic inhibition assays using the reaction of GSH with 1-chloro-2, 4-dinitrobenzene (CDNB) as a standard for measuring the catalytic activity of GST under various conditions.

[0082] The extent of enzymatic conversion of the substrate CDNB by isoforms of GST at 37°C was monitored by following increasing absorbance at 340 nm (s 340 nm = 9.6 mM-lcmi'). CDNB was dissolved in ethanol at 24 mM.

[0083] Reactions were conducted in 1 mL of buffer (50 mM, pH 6.5) containing 10 pg of GST--IV enzyme. Glutathione (1 mM) and CDNB (1.2 mM) were added after 2 min and the reaction was followed for 3 min. In an identical scenario, 10 tM of compound 1-B was incubated with the GST enzyme for 2 min prior to the addition of GSH and CDNB to determine the rate at which compound 1-B inhibited the enzyme reaction and to what extent, and the reaction was followed for 15 min. The results showed that glutathione alone produced minimal conversion of CDNB, relative to the rate of conversion in the additional presence of GST-Tt, whereas 20 uM 1-B caused a rapid and extensive inhibition of the GST- enzymatic activity.

Example 4: [0084] This example illustrates the preparation of a compound of the invention according to the following reaction: Preparation of 4-A [0085] To a solution of quinizarin and sodium carbonate in 1,4-dioxane was added an equivalent molar amount of 1, 5-difluoro-2,4-dinitrobenzene. The reaction was stirred at reflux for one week. Dioxane was then removed on a rotary evaporator and the remaining red solid was extracted with dichloromethane. The mixture was dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a brown oil.

Purification was carried out on a Flash 40 system using a Kp-Sil column equipped with a SIM attachment, utilizing a 5: 1 dichloromethane: hexane solvent system. The desired product, 4-A, was obtained as an orange solid NMR (DSMO-d6) 8 6. 33 (d, 1H, J=12. 5 Hz), 6.82 (q, 2H), 7.05-7.20 (m, 3H), 7.39-7.42 (m, 1H), 8.13 (d, 1H, J=7. 7 Hz), 12.07 (s, 1H).

Preparation of 4-B [0086] DMA/NO and compound 4-A were dissolved in an equivalent molar ratio in acetone and stirred at room temperature for 24 h. Solvent was removed on a rotary evaporator and the remaining brown solid was extracted with dichloromethane. The mixture was filtered and the filtrate was evaporated to a yellow solid. Recrystallization was carried out in ethanol: mp 268-270°C (following discoloration at 183-185°C) ; 1H NMR (CDC13) 8 2.93 (s, 6H), 6.89 (s, 1H), 7.72 (q, 2H), 7.92-7.95 (m, 2H), 8.04-8.07 (m,'H), 8. 23-8.26 (m, 1H), 8.96 (s, 1H), 12.91 (s, 1H) ; UV (EtOH) kax (E) 220 nm (1.44 mM-1 cm-1), 254 nm (1.57 mM~lcm~l). Anal. Calcd. for C22Hl5NsOlo (with 1 mole MeOH present): C, 51.02; H, 3.54; N, 12.94. Found: C, 51.39; H, 3.15; N, 12.7.

Example 5: [0087] This example illustrates the preparation of a compound of the invention according to the following reaction: 0 0 Benzoyl Chloride Triethylamine OH O OH 0 0 OH 5-A FaF 02N N02 0 N02 + O O I N NN-O-Na 5_B I O 'NOS N02 N02 O O O O 5-C 5-C N02 011-1N '0"N -o N Preparation of 5-A [0088] To a solution of 866 mg (3.6 mmol) of 1,8-dihydroxyanthraquinone in dichloromethane was added 1 equivalent of triethylamine (0.560 mL; 3.6 mmol) followed by 0.42 mL (3.6 mmol) of benzoyl chloride. The resulting solution was stirred at room temperature for 1 h, washed with water, dried over sodium sulfate, filtered through a layer of magnesium sulfate and concentrated under vacuum to give 1.50 g of an orange solid. The crude material was chromatographed on silica gel using 2: 1 dichloromethane: hexane as the solvent to give 1 g of a yellow solid: mp 179-180°C ; NMR (CDCl3) 6 7.56-7.62 (m, 5H), 7.64-7.71 (m, 1H), 7.80-7.93 (m, 2H), 8.2.8-8.39 (m, 3H). Anal. C, H. Calcd. for C2lHl2Os : C, 73. 25; H, 3.51. Found: C, 73.00; H, 3.53.

Preparation of 5-B [0089] The benzoyl protected anthraquinone compound, 5-A, was dissolved in acetone and stirred at room temperature. An equimolar amount of potassium carbonate was added, followed by an equimolar amount of 1, 5-difluoro-2,4-dinitrobenzene. The reaction was stirred for 24 h, at which time it had become a clear, yellow solution.

Acetone was then removed on a rotary evaporator and the remaining yellow solid was extracted with dichloromethane. The mixture was dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a yellow, waxy solid. Purification was carried out on a Kp-Sil column using a Flash 40 system utilizing 100% dichloromethane as solvent. The desired product was obtained as a yellow solid: mp 199-201 C ;'H NMR (CDC13) 8 8.48 (d, 1H, J=7. 7), 8.18-8.26 (m, 2H), 8.05 (t, 1H), 7.98 (t, 1H), 7.82- 7.88 (m, 3H), 7.76-7.80 (m, 1H), 7.73-7.75 (m, 1H), 7.55 (t, 2H), 7.14 (d, 1H, J=12. 2); 13C NMR (CDCl3) 8 106.40,106.62,124.66,124.75,124.94,125.44,125.82,128.59, 128.79,129.48,129.71,130.13,130.20,130.48,133.92,134.11,134. 23,134.25,135.31, 135.41,136.41,149.39,150.89,156.37,156.46,156.96,159.12,164. 51,180.40,181.43; UV (acetonitrile) ax (s) 252 nm (11.1 mM'cm~'). Anal. Calcd. for C27HI3FN2Og 1/2 H20 : C, 60.34; H, 2.63; N, 5.21. Found: C, 60.63; H, 2.61; N, 5.21.

Preparation of 5-C [0090] An equimolar amount of DMA/NO was added to a solution of the 5-B in acetone with stirring. The reaction immediately turned a pale green and stirring continued at room temperature for 24 h. Solvent was removed on a rotary evaporator and the remaining brown solid was extracted with dichloromethane, dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a green oil. Purification was carried out using a Kp-Sil column in a Flash 40 Kp-Sil column in a system initially utilizing a 10: 1 dichloromethane: hexane solvent system that was changed following the collection of 120 15-mL fractions to 100% ethyl acetate. The desired product was obtained as a green solid. Recrystallization was carried out in ethanol: mp: 182-183°C ; 'H NMR (DMSO-d6) 8 2.02 (s, 6H), 5.99 (s, 1H), 6.72 (t, 2H), 6.90-7.44 (m, 11H), 7.73 (s, 1H).

Example 6: [0091] This example illustrates the preparation of a compound of the invention according to the following reaction: Preparation of 6-A [0092] In an Erlenmeyer flask was placed 4.43 g (0.013 mol) of 4- (methylamino) phenol sulfate. This was treated with 50 mL of an aqueous solution containing 3 g of sodium bicarbonate and the mixture was shaken to liberate the free base. The aqueous mixture was partitioned with 50 mL of dichloromethane and the entire two-phase system was added slowly to a solution of 2.65 g (0.013 mol) of 1,5- difluoro-2,4-dinitrobenzene in 20 mL of dichloromethane. The mixture was stirred overnight, whereupon the organic layer was separated and the aqueous portion was extracted with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered through magnesium sulfate, and concentrated on a rotary evaporator to give 3.9 g of product. The residue exhibited an NMR spectrum consistent with the structure 6-A; it was used for the next step without further purification : NMR (CDC13) 8 3.38 (s, 3 H), 6.75 (s, 1 H), 6.84 (d, 2 H), 7.02 (d, 2 H), 8.57 (d, 1 H).

Preparation of 6-B [0093] To a slurry of 1.6 g (0.0052 mol) of compound 6-A and 5 mL (0.05 mol) of acetic anhydride was added a drop of concentrated sulfuric acid. The mixture was stirred until it became a homogeneous solution (about 5 min). The solution was allowed to stand for 10 min and carefully poured over crushed ice. Aqueous 10% sodium hydroxide was added until pH 7 was reached, then the solution was made slightly basic with aqueous sodium bicarbonate. The mustard colored precipitate that formed was collected by filtration, washed with water and dried under vacuum to give 1.76 g of product that needed no further purification: mp 133-134 °C ; NMR (CDCl3) 6 2.31 (s, 3H), 3.43 (s, 3H), 6.89 (d, 1H), 7.13 (s, 4H), 8.6 (d, 1H). Anal. Calcd for ClsHl2N3o6F : C, 51.58; H, 3.46; N, 12.03. Found: C 51.64; H, 3.53; N, 11.79.

Preparation of 6-C [0094] To a slurry of 400 mg (3.15 mmol) of DMA/NO in 10 mL of acetone was added a solution of 796 mg (2.28 mmol) of 6-B in 50 mL of acetone. After stirring at room temperature for 72 h, the mixture was filtered, evaporated and chromatographed twice on a Kp-Sil column in a Flash 40 system using dichloromethane and 5: 1 dichloromethane: ethyl acetate as the eluting solvents to give 117 mg of product: mp 170-171 °C ; NMR (CDCl3) 6 2.30 (s, 3 H), 3.17 (s, 6 H), 3.40 (s, 3 H), 7.05 (s, 1H), 7.11 (m, 4 H), 8.64 (s, 1 H); UV (ethanol) Xmax (g) 256 nm (6.85 mM~l cm~l) and 366 nm (6.46 mM-1 cm-1). Anal. Calcd for C17H18N6O18#H2O) : C, 46.05; H, 4.32; N, 18.95.

Found: C, 46.50; H, 4.34; N, 18. 70.

Example 7: [0095] This example illustrates the preparation of a compound of the invention according to the following reaction: Preparation of 7-A [0096] A solution of 2.5 g (0.0123 mol) of 1, 5-difluoro-2,4-dinitrobenzene in 25 mL of tetrahydrofuran was placed in a 100 mL round bottom flask. To this was added 2.6 g (0.025 mol) of anhydrous sodium carbonate. To the stirred mixture was added a solution of 1.86 g (0.0123 mol) of 4-methylaminobenzoic acid in 12 mL of tetrahydrofuran. The reaction mixture was stirred for 48 h at room temperature. The tetrahydrofuran was removed on a rotary evaporator and the residue was extracted with dichloromethane and washed with water. The solution was dried over sodium sulfate, filtered through magnesium sulfate and evaporated under vacuum to give 3.6 g of an orange solid. This was recrystallized from ethanol : mp 133-134°C ; NMR (CDC13) 8 2.95 (s, 3 H), 6.62 (d, 2 H), 7.49 (d, 1 H), 7.99 (d, 2 H), 8.93 (d, 1 H). Anal. Calcd for C14HION306F : C, 50.16; H, 3.01; N, 12.53. Found: C, 50.16; H, 3.02; N, 12.46.

Preparation of 7-B [0097] To a partial solution of 2.6 g (0.0077 mol) of 7-A in 10 mL of tetrahydrofuran and 10 mL of t-butyl alcohol was added 10 mL of 5% aqueous sodium bicarbonate. A solution of 1.4 g (0.016 mol) of sodium (Z)-1- (N, N- dimethylamino) diazen-1-ium-1, 2-diolate (DMA/NO) in 10 mL of 5% sodium carbonate was added dropwise and the mixture was stirred at room temperature for 72 h, concentrated under vacuum and extracted with aqueous sodium bicarbonate. The aqueous portion was washed with dichloromethane then acidified with 10% aqueous hydrochloric acid and extracted with dichloromethane. The solution was dried as described above and evaporated under vacuum to give 1.4 g of an orange solid. The solid was purified by recrystallization from ethanol to give orange plates: mp 146- 148°C ; NMR (CDC13) 6 2.94 (s, 3 H), 3.28 (s, 6 H), 6.61 (d, 2 H), 7.55 (s, 1 H), 8.01 (d, 2 H), 8.92 (s, 1 H); UV (ethanol) (e) 220 nm (15.5 mM'cm'') and 316 nm (22.2 mM-1 cm-1). Anal. Calcd. for C16H16N6O8#1/3(C2H5OH) : C, 45.95; H, 4.16; N, 19.29.

Found C, 45.80; H, 3.86, N, 18.91.

Example 8: [0098] This example illustrates the preparation of a compound of the invention according to the following reaction: Preparation of 8-A [0099] A heterogeneous mixture of 94 mg (0.22 mmol) of compound 1-A of Example 1 and 507 mg of linear polyethylenimine (MW 25 kDa, Polysciences Inc., Warrington, PA: C, 54.7; H, 11. 1 ; N, 32.5) was heated at reflux for 6 h. The mixture was allowed to cool to room temperature and the dark solid was collected by filtration, washed with acetone and dried under vacuum to give 471 mg of an amorphous brown solid (C, 51.84; H, 8.62; N, 21.28).

Preparation of 8-B [00100] A slurry of 317 mg of compound 8-A in methanol and 0.1 mL of methanolic sodium methoxide was charged with 40 psi of nitric oxide and stirred at room temperature for 48 h. The pressure was released and the reaction mixture was filtered and rinsed with first acetone then copious amounts of water. The brown polymer was dried under vacuum (C, 42.02; H, 6.26; N, 25.89; Na, 1.73).

NO Release Measurements: [00101] 2.67 mg of compound 8-B was suspended in 2 mL of phosphate buffer and incubated at 37°C. NO release was measured over a 100 h period to give an integrated signal indicating an equivalent weight of 3000 g/mole of NO.

[00102] In regards to compound 8-B, n is an integer from 1 to 500,000, preferably from 1 to 300,000, most preferably from 1 to 250,000.

Example 9: [00103] This example illustrates the incorporation of the diazeniumdiolate functionality and a superoxide donor moiety into antifungal agents: of m vo ? o C ci cl NEZ Ketoconazole \ Ketoconazole/NO/Oz O 0 'N 0 0 N N o 1-A 0 O /\/=\\CI ci ci 0 O Deacyl-Ketoconazole 7 N=N N 0 //U NO'O-N N O KetoconazolelNO CL N Preparation ol deacyl-ketoconazole [00104] Ketoconazole (ICN Biomedicals Inc., Akron, OH), 500 mg (0.94 mmol) was deacylated in refluxing 10% aqueous sodium hydroxide as described by Whitehouse et al. (J. Pharm. Biomed. Anal., 8: 603-606 (1990)) to give deacyl-ketoconazole in near quantitative yield.

Reaction of deacyl-ketoconazole with NO [00105] A solution of 287 mg (0.59 mmol) of the deacylated compound in 5 mL of 1: 1 tetrahydrofuran: dichloromethane was placed in a micro Pan bottle, treated with 0.136 mL (0.63 mmol) of methanolic sodium methoxide, charged with 60 psi of NO and stirred for 72 h. The pressure was released and the resulting white precipitate was collected by filtration, washed with ether and dried under vacuum to give 53 mg of product: mp 169-170°C ; UV (0.01 MNaOH) xmas (s) 226 nm (21.9 mM-'cm-'), 248 nm (16.6 mM'cm-1), 294 nm (1.7 mM~lcm~l) ; t/2 17 min at 25°C ; NMR (D20) 6 2.95 (b, 4H), 3.11 (b, 4H), 3.36 (m, 3H), 3.58 (m, 1H), 4.07 (m, 3H), 6.59 (b, 2H), 6.78 (b, 4H), 7.23 (b, 1H), 7. 35 (b, 2H), 7.56 (b, 1H).

Preparation of ketoconazole/NO/O [00106] The methodology developed for the preparation of compound 6-C of Example 6 and subsequent examples may be used to link deacyl-ketoconazole to compound 1-A of Example 1.

Example 10: [00107] This example illustrates a zwitterionic derivative of mitoxantrone bis- diazeniumdiolate that is susceptible to diazeniumdiolation, with the functional groups at which diazeniumdiolate moieties can be attached being emphasized.

Example 11: [00108] This example illustrates the preparation of PROLI/NO derivatives 11-A and 11-B according to the following reactions: Preparation of 11-A [00109] A solution of PROLI/NO (1.44 g, 5.7 mmol) in 5 % sodium bicarbonate was cooled to 0°C under argon. To this solution was added 2,4-dinitrofluorobenzene (725 , ut, 5.7 mmol) in t-butanol, dropwise with stirring. The reaction was allowed to warm to room temperature and stirred for 24 h. The yellow solution was then made acidic by the addition of 10 % aqueous HCI, extracted with methylene chloride, washed with water, dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a yellow solid (1.8 g).

Preparation of 11-B [00110] In a 100 mL round bottom flask were placed the crude 11-A (1.54 g) and 50 mL of 1: 1 cyclohexane/dichloromethane. Amberlyst-15 (1.5 g) was added to the solution and the flask was transferred to a N2 bubbler system fitted with an inlet tube for the 2-methylpropene gas. The contents of the flask were then placed under an N2 atmosphere using the bubbler system and the 2-methylpropene was bubbled into the solution. The reaction was allowed to proceed with stirring over 24 h.

[00111] After 24 h, the introduction of the gases was suspended and the reaction mixture was filtered through cotton pulp using an upside-down filter adapter. The filtrate was then evaporated to dryness at room temperature on a rotary evaporator. The resulting yellow oil was further dried for 4 h at 1 mm Hg on an oil pump to give about 1.5 g of crude product.

[00112] To purify 11-B, a Kp-Sil Flash 40 System was utilized with a 4x14 cm column and an initial 2: 1 methylene chloride: hexane solvent system. One hundred ten 15 mL fractions were collected. Two compounds were isolated, neither of which was the desired product. Twenty additional fractions were collected with a 5: 1 methylene chloride: hexane solvent system, and were found to be blank. Twenty further fractions were collected with a straight ethyl acetate solvent system and tubes 8-15 were combined and evaporated to yield a yellow film, which crystallized upon standing. The product was recrystallized in ethanol: mp 153-154°C ;'H NMR (CDC13) 8 1.47 (s, 9H), 2.06-2.27 (m, 3H), 2.32-2.47 (m, 1H), 3.85-3.93 (m, 1H), 3.98-4.06 (m, 1H), 4.59-4.63 (m, 1H), 7.63 (d, 1H, J=9. 3), 8.43 (ABq, 1H), 8.89 (d, 1 H, J=2. 7 Hz); 13C NMR (CDCl3) 8 22.07,27.95,28.17,50.34,62.68,82.83,117.18,122.18,129.12,141 .74, 154.53,169.59; UV (EtOH) Xmax (g) 306 nm (1.40 mM~l cm~l). Anal. Calcd for C15Hl9N508 : C, 45.34; H, 4.82; N, 17.63; Found: C, 45.35; H, 4.89; N, 17.61.

[00113] It will be appreciated by those skilled in the art that a superoxide-generating moiety can be attached to the compound of 11-B. For example, the t-butyl ester of the carboxyl moiety of the compound of 11-B can be replaced with a superoxide-generating moiety by conversion to an appropriately Q-substituted amide or alternate ester group.

Example 12: [00114] This example illustrates a superoxide-and nitric oxide-releasing quinizarin derivative prepared according to the following reaction schemes: Quinizarin O-Benzoylquinizarin Preparation of 12-A [00115] Quinizarin was dissolved in acetone and stirred at room temperature.

Triethylamine was first added dropwise, followed by the slow addition of benzoyl chloride. The reaction was stirred at room temperature for 48 hours. Acetone was removed and the remaining red solid was taken up in methylene chloride, washed with water, dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a bright orange solid. A Flash 40 purification system equipped with a Sample Injection Module (SIM) attachment and a 2: 1 methylene chloride: hexane solvent system was used for purification of this sample. O-Benzoylquinizarin, 12-A, was obtained as an orange solid: mp: 202-204°C ;'H NMR (DMSO-d6) 6 7.64 (d, 1H, J=9. 1), 7.75-7.81 (m, 2H), 7.86 (d, 1H, J=9. 1), 7.89-7.95 (m, 1H), 7.99-8.08 (m, 2H), 8.15-8.20 (m, 1H), 8.27- 8.33 (m, 2H), 8.33-8.37 (m, 1H) ; 13C NMR (DMSO-d6) 8 115.80,123.36,125.78, 126.43,126.85,128.95,129.11,130.03,132.08,133.48,134.03,134. 12,134.57,135.31, 142.76,160.14,164.66,180.80,188.08; UV (acetonitrile) a,", ax (E) 223 mn (20.3 mM- 1 cm-1), 254 nm (23.8 mM-1 cm-1), 406 nm (4.2 mm-lcrn-1). Anal. Calcd. for C2lHlzOs: C, 73.25; H, 3.51. Found: C, 73.43; H, 3.89.

Preparation of 12-B [00116] O-Benzoylquinizarin, 12-A, was dissolved in acetone with gentle warming.

Potassium carbonate was added, followed by the 1, 5-difluoro-2, 4-dinitrobenzene. The reaction was stirred at reflux for 24 hours. Acetone was removed and the remaining green solid was extracted with methylene chloride, dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a green solid. A Flash 40 silica gel chromatography system equipped with a SIM column and a 5: 1 methylene chloride: hexane solvent system was used for purification. A pure product, 12-B, was obtained in good yield: mp: 274-276°C ;'H NMR (DMSO-d6) 6 7.29 (d, 1H, J=12. 4), 7.67-7.72 (m, 2H), 7.80-7.89 (m, 3H), 7.94-8.06 (m, 4H), 8.21-8.23 (m, 2H), 9.02 (d, 1H, J=7. 7); 13C NMR (DMSO-d6) 8 106.91,107.26,125.03,126.38,126.46,128.94,129.01,130.00, 130.58,130.70,130.91,132.69,132. 90,132.98,134.48,134.51,148.42,148.95,156.07, 156.28,156.43,159.65,164.33,180.61,181.26; UV (acetonitrile) Smax (g) 254 nm (39.0 mM-lcrri 1). Anal. Calcd. for C27HI4N209 (calculated with 0.5 mol H2O) : C, 60.34; H, 2.63; N, 5.21. Found: C, 60.54; H, 2.56; N, 5.25.

Preparation of 12-C [00117] The nitric oxide-releasing compound (DMA/NO) and sodium carbonate were cooled in acetone to 0°C under a steady stream of argon. A solution of quinone 12-B in acetone was added, and the reaction was allowed to warm to room temperature and stirred for 72 hours. Acetone was removed and the remaining brown solid was extracted with methylene chloride, dried over sodium sulfate, filtered through magnesium sulfate, and evaporated to a green oil. This was purified with a Kp-Sil Flash 40 system and an original solvent system of straight methylene chloride, which was changed to 100% ethyl acetate following the collection of 3 L of impurities. The desired product, 12-C, was obtained as a green crystalline solid: mp: 184-186°C ; 1H NMR (DMSO-d6) 8 2.98 (s, 6H), 6.94 (s, 1H), 7.67-7.72 (m, 2H), 7.80-7.88 (m, 3H), 8.01-8.04 (m, 4H), 8.21- 8.24 (m, 2H), 8.99 (s, 1H) ; 13C NMR (DMSO-d6) 6 40.84,104.61,125.49,126.03, 126.39,126.48,126.77,128.92,128.98,130.08,130.47,130.86,132. 57,132.71,132.92, 133.06,134.15,134.48,134.52,148.21,149.29,153. 54,164.44,180.62,181.08; UV (acetonitrile)#max(#) 254 nm (54.9 mM~Icm~l). Anal. Calcd. for C 9Hl9N501, (calculated with 0.5 mol MeOH) : C, 56.30; H, 3.07; N, 10.55. Found: C, 56.28; H, 3.36; N, 11.13.

Example 13: [00118] This example illustrates some nucleophilic anti-tumor agents that are susceptible to diazeniumdiolation, with the functional groups at which diazeniumdiolate moieties can be attached being emphasized: Daunorubicin O OH O CH OH 3 'OH OCH3 O OH b Aclacinomycin A r/0 OCHg OCH3 cl3 0 Hz OH Doxorubicin OH O a OH S lu CHg O j? P j? N (0) 2 0 IOH 1°l r N (CH3) 2 0 0 c OH CH20H H3C OH 0 OC30 OH O O C N ¢ O<=l O OH Streptonigrin 0 HO I N COOH <= HZN II ./ HAN H2N Ho HO OCH3 OCH [00119] These examples confirm the tremendous versatility of the compounds of the invention. For instance, it is believed that NO by itself is unable to modify the active site structure of GST (an enzyme expressed in, for example, tumor cells) in aerobic medium. To overcome this difficulty, compound 1-B was synthesized, in which the quinone ring is attached to the R3 substituent. Quinones are widely known to undergo one-electron reduction under a variety of physiologically relevant conditions to produce semiquinone radicals which in turn pass the electron to molecular oxygen, regenerating the quinone and producing a superoxide ion (see, e. g., Powis, G., Free Radic. Biol.

Med., 6: 63-101 (1989)), with many turnovers of this process being possible as long as there is sufficient 02 and reductant. When compound 1-B was examined for its suitability as a substrate for GST-n :, no reaction could be detected. To determine whether this was the result of extra-tight versus negligible interaction between the drug candidate and the enzyme, 1-B and the protein were mixed and the normal substrate used in assessing GST activity (1-chloro-2, 4-dinitrobenzene, or CDNB) was subsequently added; the CDNB also failed to react, confirming that compound 1-B had effectively inactivated the enzyme. Evidence that the cosubstrate GSH can serve as both the nucleophile needed to initiate NO generation and the reductant that drives concurrent superoxide production was achieved as in Examples 2 and 3-A. The former confirms the rapid generation of nitric oxide obtained on mixing GSH with compound 1-B, while Example 3-A confirms the nearly instantaneous reduction of ferricytochrome c in the presence of GSH when compound 1-B was added. The ICso value for the effect of this compound on both the 71 : and a isoforms of the enzyme was in the range of 0.4- 0.7 uM, apparently the result of non-competitive inhibition, suggesting tight inhibitory binding at an allosteric site.

[00120] The toxicity of compound 1-B to a series of three variants of the liver cell line HepG2 was characterized by an LCso of about 30 uM for each, but the agent proved much more toxic to the NO-sensitive leukemia cell lines HL60 and U937, in which LCSo's of 1-2 uM were registered. This suggests that such compounds may have anti- leukemic activity in their own right, over and above their potential ability to decrease drug resistance in NO-insensitive tumor cells.

[00121] There are many other potential therapeutic applications for the superoxide- generating diazeniumdiolates of the invention. The fact that parasites responsible for schistosomiasis rely on a functioning GST unique to that organism allows for similarly designed inhibition of that isoform based on the availability of detailed structural data on the parasites'GST (McTigue et al., J. Mol. Biol., 246: 21-27 (1995)). Recent evidence that simple NO generators such as compound 1-B may react with the nucleophilic sulfur atoms in the zinc finger structures of the HIV nucleocapsid p7 protein (which is essential for viral replication and infectivity) (see, e. g., Saavedra et al., J. Org. Chem. (2001)) to eject the zinc and incapacitate the protein is evidence of antiviral activities as well. Finally, insoluble polymers of Formula I (see Example 8) have useful biocidal properties, for example, when used as coatings on materials susceptible to fouling by biofilms.

[00122] As mentioned previously, many pharmacologically active compounds of Formula I are accessible through this novel technology. Three 3-ring analogues of the naphthoquinone derivative of Example 1 have been prepared (Examples 4,5, and 12), and diazeniumdiolation of numerous quinone-type anticancer drugs should produce hybrid molecules with improved activity; examples are shown in Example 13. Because the semiquinone radicals that reduce 02 to 02-can be prepared by 1-electron oxidation of hydroquinones and catechols as well as by 1-electron reduction of 1,4- and 1,2- quinones, molecules containing both a diazeniumdiolate function and 1,4- or 1,2- dihydroxyaromatic rings can also be used for the purposes outlined above ; this concept can also be extended to o-and p-aminophenol derivatives such as those shown in Examples 6 and 7, compounds whose 1-electron oxidation would give similar radicals.

Azo compounds can similarly support superoxide formation (see, e. g., Ingold et al., Patent Application CA 96-2193457 (1998)), as can aromatic amines including o- toluidine and o-anisidine (see, e. g., Munday et al., Free Radic. Res., 21: 67-73 (1994)), 1,2,3-triaminobenzene and 2,3,6-triaminopyridine (see, e. g., Munday et al., Free Radic.

Res., 21: 67-73 (1994)), and N, N, N', N'-tetramethyl-p-phenylenediamine (see, e. g., Munday, R., Chem. Biol. Interact., 65: 133-143 (1988). Superoxide-generating aromatic nitro compounds proposed as antitrypanisomal agents (see Blumenstiel et al., Biochem.

Pharmacol., 58: 1791-1799 (1999)) can be potentiated by diazeniumdiolation. An example in which the superoxide-generating moiety is attached to X is that of the mitoxantrone derivative of Example 10. Other superoxide-generating functional units can be attached to X besides quinone derivatives; for example, paraquat (see, e. g., Day et al., Proc. Natl. Acad. Sci. U. S. A., 96: 12760-12765 (1999)) and other bipyridyl bis- onium species (see, e. g., Jones et al., J. Toxicol. Clin. Toxicol., 38: 123-128 (2000)) are well known to exert their toxic effects through generation of such reactive oxygen species. Additionally, certain Amadori compounds have been shown to serve as redox cyclers capable of generating superoxide ion in a similar process (Mossine et al., Chem.

Res. Toxicol., 12: 230-236 (1999)). Many other examples are listed above.

[00123] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[00124] The use of the terms"a"and"an"and"the"and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e. g.,"such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[00125] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.