JP5015903B2 - O2-glycosylated 1-substituted diazene-1-ium-1,2-diolates and compositions containing such diolates - Google Patents

Description

[Technical Field of the Invention]
The present invention relates to O ² -aryl 1-substituted diazene-1-ium-1,2-diolates (O ² -aryl diazenium diolates), O ² -glycosylated 1-substituted diazenium diols. And O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates, and compositions containing such diazeniumdiolates, such dia The present invention relates to a method for using zenium diolates and a method for producing O ² -aryldiazenium diolates.

[Background of the invention]
Nitric oxide (NO) is involved in a wide variety of bioregulatory processes, and compounds containing or capable of releasing nitric oxide are useful for modulating these processes. Has been proven. A wide variety of nitric oxide-containing and / or release adducts such as trinitroglycerin, nitroprusside, etc. are known in the art (US Pat. No. 5,405,919 (Keefer et al., Restricting Their Use in Biological Applications). Is outlined in)). The limited utility of such compounds has led in part to the development of another biologically useful nitric oxide generating compound, namely diazeniumdiolates.

N ₂ O ₂ as diazeniumdiolates ^- compounds comprising a functional group, and is distinguished from structurally and functionally nitrosamines (e.g., Reilly, U.S. Pat. No. 3,153,094). Known diazeniumdiolates are disclosed in recently issued patents. US Pat. Nos. 5,039,705 (Keefer et al.) And 5,208,233 (Keefer et al.) Disclose secondary amine-nitric oxide adducts and salts thereof. US Pat. Nos. 5,155,137 (Keefer et al.) And 5,250,550 (Keefer et al.) Disclose nitric oxide and polyamine complexes. US Pat. Nos. 5,389,675 (Christodoulou et al.) Disclose mixed ligand metal complexes of nitric oxide-nucleophile adducts, US Pat. Nos. 5,525,357 (Keefer et al.) And 5,405,919 (Keefer et al.) A combined nitric oxide / nucleophile adduct composition is disclosed. US Pat. Nos. 4,954,526 (Keefer et al .; '526 patent) and 5,212,204 (Keefer et al.) Disclose the use of ionic diazeniumdiolates as cardiovascular agents. In addition, '526 patent, O 2 ^- discloses substituted and metal binding diazeniumdiolates. Keefer et al., US Pat. No. 5,366,997 ('997) has the formula:

_Wherein, N _{2 O} 2 ^- group ^{O 2} - oxygen is bound to the functional group ^{R 3.} ]
Diazeniumdiolates having the following are disclosed: When the R ³ group is cleaved from O ² -oxygen, NO can be released spontaneously.

Keefer et al ('997) (i) R ¹ and R ² together with the nitrogen atom to which they are attached may form a pyrrolidinyl, piperazino or other heterocyclic group, (ii) R ³ is a C _1-12 straight chain or C _3-12 branched chain alkyl, C _1-12 unsubstituted / substituted acyl, which may be olefinic and / or optionally substituted with hydroxy, halo, acyloxy or alkoxy , Sulfonyl, carboxamide, sulfinyl, sulfenyl, carbonate-derived group or carbamate-derived group, and (iii) pyrrolidinyl group has the structure:

[Wherein w = 4 and R ⁴ = hydrogen, C _1-8 linear or branched alkyl, C _3-8 cycloalkyl, or substituted or unsubstituted aryl. Keefer et al. ('997) discloses that R ³ is aryl or substituted aryl, and that the pyrrolidino group can be substituted with a substituted or unsubstituted carboxyl group at the 2-position. (See also Example 1 of US Pat. No. 5,632,981). Similarly, Keefer et al. ( '997) is, O ² of diazeniumdiolates - does not disclose glycosylation.

Heretofore, it was not known that there could be O ² -aryl substituents of diazeniumdiolates. Furthermore, chemical studies of previously disclosed diazeniumdiolates have shown that they are generally at least stable at high as well as low pH, and certain other “nitro vasodilators” “Unlike drugs, their NO release rates led to the result that they were not affected by the presence of nucleophilic thiols.

Accordingly, there is a need for a class of diazeniumdiolates that provides advantages over other currently available diazeniumdiolates. In this regard, O ² -aryl substituted diazeniumdiolates are advantageous in that they can spontaneously release NO under alkaline conditions or after nucleophilic attack. O ² -aryl substituted diazeniumdiolates can also spontaneously release NO after a combination of oxidative or electrophilic activation and nucleophilic attack.

Therefore, a primary object of the present invention, in order to protect the diazeniumdiolate from spontaneous release of NO, N ₂ O ₂ ^- group O ² - oxygen is induced by aryl or substituted aryl group, It is to provide a nitric oxide / nucleophile adduct. Another object of the present invention is to develop a new class of diazeniumdiolates that resist release of nitric oxide under neutral or acidic solutions, but release NO upon nucleophilic attack or pH increase. Is to provide. Yet another object of the present invention is to provide O ² -glycosylated 1-substituted diazene-1-ium-1,2-diolates and O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazenes. It is to provide -1-ium-1,2-diolates. It is a further object of the present invention to provide compositions containing such compounds, including compositions containing nitric oxide / nucleophile adducts containing novel targeting moieties. A related object is according to the biological tissue targeting strategy that gives greater flexibility and specificity by the targeting NO release, O 2 ^- is to provide an aryl-substituted diazeniumdiolates. It is a further object of the present invention to provide methods for using such compounds. These and other objects of the invention, as well as additional inventive features, will be apparent from the description of the invention given below.

  The present invention has the formula:

[Wherein, X is an inorganic group or an organic group, and Q is an aryl group. ]
Provides an O ² -aryl substituted diazeniumdiolate (ie, O ² -aryldiazeniumdiolate). In the compounds of this novel class, atoms of the aryl group Q N ₂ O ₂ ^- attached to an oxygen - O ² functional group. The diazeniumdiolates of formula (I) are stable in terms of the generation of nitric oxide hydrolyzable in neutral to acidic solutions. Surprisingly, these new compounds, or products resulting from the oxidative or electrophilic activation of these compounds, have a basic or nucleophilic environment in which the aryl group is separated from the diazeniumdiolate residue. Below it has been demonstrated that nitric oxide can be generated.

The present invention also provides O ² -glycosylated 1-substituted diazene-1-ium-1,2-diolates and O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazene-1-ium. -1,2-diolates are also provided, both of the formula:

[Wherein X and R are organic and / or inorganic groups as defined herein, but for O ² -glycosylated 1-substituted diazeniumdiolates, R must be a sugar. . ].

Furthermore, with respect to O ² -glycosylated 1-substituted diazene-1-ium-1,2-diolates, the group X can be any organic or inorganic group. Preferably, X contains an atom different from carbon and hydrogen and is bonded to the nitrogen of the diazeniumdiolate via an atom different from carbon. Most preferably, X is an amino group and is bonded to the nitrogen of the diazeniumdiolate via a nitrogen atom.

For O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolates, X in formula Ia is

The [1- (2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates are represented by the following formula: :

[Wherein R ²² is hydrogen, hydroxy, OM (where M is a cation), halo or X ¹ R ²³ R ²⁴ (where X ¹ is oxygen, nitrogen or sulfur, and R ²³ and R ²⁴ are independently substituted or unsubstituted C _1-24 alkyl, substituted or unsubstituted C _3-24 cycloalkyl, substituted or unsubstituted C _2-24 olefin group, substituted or unsubstituted aryl (eg, acridine , Anthracene, benzene, benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline, naphthalene, oxazole, phenanthrene The Enanthridine, phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine, porphine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrocholine, pyrrole, quinolizinium ion, quinoline, quinoxaline, quinazoline, sidone, tetrazole, thiazole, thiophene , Thyroxine, triazine and triazole), or a heterocyclic group (for example, glycosyl and the like, and when X ¹ is O or S, R ²⁴ is absent). ]. Alternatively, when X ¹ is nitrogen, R ²³ and R ²⁴ together with X ¹ are heterocycles, for example

Wherein A is N, O or S, w is 1-12, y is 1 or 2, z is 1-5, and R ⁸ , R ⁹ , R ²⁵ and R ²⁶ are , Hydrogen, C _1-8 straight chain alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl or aryl. A heterocyclic ring selected from the group consisting of The above R ²³ and R ²⁴ groups may be unsubstituted or appropriately substituted. For example, the R ²³ and R ²⁴ groups may be suitably substituted with acyloxy, acylthio, hydroxy, amino, carboxy, mercapto, halo, amide, sulfonyl, sulfoxy, sulfenyl, phosphono, phosphato, and the like.

In addition, with respect to O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolates, the group R of formula Ia is any organic or inorganic group. Which may be covalently bonded to the terminal oxygen of the diazeniumdiolate, as shown, but different from hydrogen and substituted or unsubstituted C _1-12 linear or C _3-12 branched alkyl A substituted or unsubstituted C _2-12 linear or C _3-12 branched olefin group, a substituted or unsubstituted C _1-12 acyl, sulfonyl, carboxamide, glycosyl group, aryl group, or the formula — (CH ₂ ) _n — ^{ON = n (O) NR 28} R 29 ( wherein, n is an integer of 2-8, and ^{R 28} and ^{R 29} are independently _{C 1-12} straight chain _{alkyl, C 3-12} branched chain Al _Le, or a _{C 2-12} straight-chain or _{C 3-12} branched chain olefinic group, or ^{R 28} and ^{R 29,} together with the nitrogen atom to which they are attached, a heterocyclic group, preferably pyrrolidino , A piperidino, piperazino or morpholino group). The above R groups may be unsubstituted or substituted. Preferred substitutions include substitution with hydroxy, halo, acyloxy, alkoxy, acylthio or benzyl.

From another aspect, the present invention includes compositions, including pharmaceutical compositions, containing the diazeniumdiolate of the present invention. The pharmaceutical composition preferably additionally contains a pharmaceutically acceptable carrier.
From yet another aspect, the present invention provides a method of using a compound according to the present invention.
From yet another aspect, the present invention provides a method for producing O ² -aryl diazeniumdiolates.

FIG. 1 is a graph of Trp37 fluorescence (RFU) versus time (minutes) showing the release of zinc from the HIV-1 nuclear capsid p7 protein by O ² -aryldiazeniumdiolates. In the graph, ○ indicates a negative control, that is, no drug, □ indicates a positive control, that is, 624151 (see Rice et al . Antimicrob. Agents Chemother. 41: 419426 (1997)), and ■ indicates the compound of Example 1 (LK1) ♦ indicates the compound (LK2) of Example 8, ▲ indicates the compound (LK3) of Example 5, ● indicates the compound (LK4) of Example 10, and × indicates the compound of Example 11 (LK4). LK5). FIG. 2 is a graph of relative NO release rate versus time (minutes) showing the catalytic action of NO release from DNP-PYRRO / NO by glutathione S-transferase (GST).

[Detailed Description of the Invention]
O ² -arylated diazeniumdiolates The present invention has the formula:

[Wherein, X is an organic group or an inorganic group, and Q is an aryl group. ]
O ² -aryl 1-substituted diazeniumdiolates (ie, O ² -aryl 1-substituted diazene-1-ium-1,2-diolates) are provided.
According to the present invention, N ^₂ O ₂ ^- O ₂ radicals - oxygen is directly bonded to atom ring aryl group. In other words, there are no spacer atoms (eg, methylene) that separate O ² -oxygen from the aryl ring. If, aryl group comprises two or polycyclic group, if all the rings of the aryl group is not necessarily aromatic, O 2 ^- bond between the oxygen and the aryl group is a part of an aromatic ring Through atoms. Furthermore, O ² -oxygen can be bonded to any aromatic ring atom of the aryl group that can be bonded to the O ² -oxygen of the N ₂ O ₂ ^- group. N ^₂ O ₂ ^- O ₂ radicals - atoms of the aromatic ring capable of binding oxygen, but may also other bonds, typically carbon or nitrogen.

Any special but not think lead to theory, O 2 ^- bond with oxygen atoms of the aryl ring are presently considered to be achieved by binding to activated atoms of the ring. Activation can be achieved via any suitable mechanism. In this regard, the preferred mechanism for activating the aryl ring is to react the diazeniumdiolate via a partially positively charged aryl ring atom or, more particularly, an amino substituent of the ring structure. By replacing.

In the first preferred reaction mechanism, the aryl ring is substituted with a suitable electron withdrawing group, which is part of the ring and reacts with diazeniumdiolate, as in Example 12. The former may be a “leaving group”. One skilled in the art will recognize that the electron withdrawing group and the leaving group may in some cases be the same group. The leaving group replaces the diazeniumdiolate to form the O ² -aryldiazeniumdiolate of the present invention. Suitable leaving groups include, but are not limited to, F, Cl, Br, I, NO ₂ , OSO ₂ R, and OSO ₃ R (where R is an organic group, a metal center, etc.), The composition is well understood by those skilled in the art. For purposes of illustration, suitable R groups include, but are not limited to, H, alkyl, alkenyl, or aryl. This reaction is based on the well-known S _N Ar mechanism. See, for example, Nucleophilic Aromatic Displacement: The Influence of the Nitro Group , Francois Terrier, VCH Publishing, Inc., New York, New York, pages 1-11 (1991). Preferably, these S _N Ar reactions are carried out on an electron deficient aryl ring containing at least one electron withdrawing group.

In a second preferred reaction mechanism, the aryl reactant is substituted with an appropriate amino group to allow direct derivatization of the ring atom of the aryl group attached to the substituted amino group (eg, diazo of the amino group). After conversion). After incorporation into the compounds of the present invention, O ² to be activated - no requirement for atoms of the aryl ring oxygen bond. However, if this atom is activated after incorporation into a compound of the present invention, the diazeniumdiolate group to which it is attached can be replaced via subsequent nucleophilic substitution (eg, suitable strong Under base). In other words, as a result of oxidative or electrophilic activation, the compounds of the present invention are altered to activate the aryl ring atom attached to the O ² -oxygen, and thus, as described above, the compound is Undergoes nucleophilic substitution.

Advantageously, the compounds of the present invention have novel and useful properties that are absent from other nitric oxide / nucleophile adducts previously known in the art. In general, the compounds of the present invention are stable under neutral or acidic pH (that is, compounds of formula I do not generate NO under neutral or acidic pH). Another advantageous property of the compounds of the present invention is that the O ² -aryl bond is well susceptible to cleavage by nucleophiles containing hydroxide ions. When aryl diazeniumdiolate, placed under basic or nucleophilic environment - specific O ² - aryl O ² diazeniumdiolate or oxidative or electrophilic activated the invention The aryl bond to O ² -oxygen can be broken. The resulting diazeniumdiolate ions spontaneously decrease through a predictable first stage mechanism and generate NO. The resulting aryl group is replaced with a nucleophile provided by its environment. If the nucleophile provided by the environment is part of the enzyme, the enzyme can be inactivated. Due to the susceptibility of O ² -aryl diazeniumdiolates to nucleophilic attack, the compounds are also prodrugs for targeting nitric oxide to nucleophilic tissue components, biological sites and biological microenvironments. Especially follow the design.

When thiols are present in a mixture, the compounds of the present invention are also useful for identifying and measuring individual thiols (organic-SH containing compounds). For example, a sample expected to consist of C ₄ -C ₈ linear thiols should have the product of Example 1 dissolved in tetrahydrofuran or other inert solvent and then assayed for an excess molar resulting solution. It can be analyzed by mixing with the sample. After the subsequent reaction is complete, a portion is HPLC analyzed using a UV detection system. It is seen in the resulting chromatogram peaks, and their retention times were identified by a reliable induced separately, comparing the retention times of the standards of the individual C ₄ -C ₈ linear thiol , And the amount can be measured by converting the peak area into a concentration via an individual standard curve.

With respect to O ² -aryldiazeniumdiolates, an “aryl group” as used herein, whether or not it is part of a (homo) cyclic, heterocyclic or polycyclic structure. Without mentioning any aromatic group. A standard understanding of “fragrance” is used here (see, eg, LG Wade, Jr., Organic Chemistry, 2d Edition, Prentice Hall, Englewood Cliffs, New Jersey, 682-683 (1991)). The aryl group used here may also have a wide variety of substituents. Any suitable aryl substituent can be used provided that the substituent does not break the aromaticity of the aryl ring.

Turning back to the aryl group Q of formula I, Q is meant to include all aryl groups that follow (or can be followed) the reaction of the diazeniumdiolate with the O ² -oxygen atom. Such groups Q include homocyclic, heterocyclic and polycyclic aromatic structures, and derivatives thereof. Examples of the aryl group Q include acridine, anthracene, benzene, benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole. , Isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine, porphine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrocholine, pyrrole, quinolidinium ion, quinoline, Quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine, Triazine and triazole.

Consistent with the present invention, each of these aromatic compounds Q is indefinitely derivatized with a number of substituents well known in the art that can be substituted for the aromatic ring as long as the aromaticity of the ring is maintained. obtain. For example, as a substituent for the aryl group Q, X [N (O) NO] ⁻ (wherein X is defined hereinafter and is the same as X in formula I), halo, hydroxy, alkylthio, arylthio, alkoxy , aryloxy, amino, mono- - or di - substituted amino, ammonio or substituted ammonio, nitroso, cyano, sulfonato, mercapto, nitro, _oxo, C 1 _{-C 24} aliphatic _radical, C 3 _{-C 12} olefinic groups, _{C 3} -C _{24 cycloalkyl,} C 3 _{-C 24} heterocycloalkyl, benzyl, phenyl, substituted benzyl, substituted phenyl, benzylcarbonyl, phenylcarbonyl, saccharides, substituted benzylcarbonyl, and a substituted phenylcarbonyl and phosphorus derivative group. Examples of phosphorus-derived groups include phosphate and phosphono groups. Examples of phosphate groups include (OH) ₂ P (O) O— and substituted (OH) ₂ P (O) O— groups, where one or more oxygen atoms are independently S or NR (Wherein R 'is understood to be a C ₁ -C ₁₀ aliphatic group, cycloalkyl or aryl group). Examples of substituents for C ₁ -C ₂₄ aliphatic groups include C ₁ -C ₂₄ acyl, and

[Wherein R is hydrogen, substituted or unsubstituted C ₁ -C ₂₃ aliphatic group, substituted or unsubstituted C ₃ -C ₂₃ cycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ olefin group, benzyl, phenyl, a substituted benzyl or substituted phenyl, said substituted benzyl or substituted phenyl, nitro, halo, _hydroxy, C 1 _{-C 24 alkyl,} C 1 _{-C 24} alkoxy, amino, _mono--C 1 _{-C 24} alkylamino, di - C ₁ -C ₂₄ alkylamino, cyano, substituted with 1-5 substituents selected from the group consisting of phenyl and phenoxy. ]. Suitable saccharides include ribose, glucose, deoxyribose, dextran, starch, glycogen, lactose, fucose, galactose, fructose, glucosamine, galactosamine, heparin, mannose, maltose, sucrose, sialic acid and cellulose. Other suitable saccharides are phosphorylated, 3,5-cyclophosphorylated and polyphosphorylated hexoses and pentoses.

Examples of substituted aryl compounds of the present invention that can be bonded to a diazeniumdiolate group include dinitrophenol (benzene), hypoxanthine (purine), uridine (pyrimidine), vitamin K ₅ (naphthalene) and ribosyluridine (nucleoside). Can be mentioned.

In another specific embodiment of the invention, the aryl group is identical or structurally similar to a molecule normally found in living organisms or a substitution thereof. These biologically relevant groups include the group consisting of nucleotides, nucleosides, nucleic acids, peptides including peptide hormones, non-peptide hormones, vitamins and other enzyme cofactors (such as porphyrins). More can be selected. Examples of biologically suitable aryl groups, thyroxine, NAD (or NADH), chlorophyll a ₅ hypoxanthine, uridine and vitamin K.

The following reaction schematic illustrates the process for producing the O ² -aryl diazeniumdiolates of the present invention. In these illustrated reaction schematics, generally a solution of diazeniumdiolate (X— [N ₂ O ₂ ⁻ ]) in 5% aqueous sodium bicarbonate solution (which is weakly basic) is preferably Cool to 0 ° C. under a blanket of inert gas such as nitrogen. Then a solution of one equivalent of activated fragrance reagent in a solvent such as t-butyl alcohol, dimethyl sulfoxide or N, N-dimethylformamide is slowly added. Without being bound to any particular theory, it is believed that polar aprotic solvents are preferred. The reaction temperature is increased gradually, for example, to room temperature or higher for smaller reactive aryl groups. Generally, a precipitate is formed upon addition. The mixture is then gradually warmed to room temperature and stirred overnight. The product is extracted with a suitable extractant such as dichloromethane and subsequently washed with dilute hydrochloric acid and then with sodium bicarbonate solution. The organic layer is dried with a suitable desiccant such as sodium sulfate, preferably filtered through a layer of anhydrous magnesium sulfate, and the solvent is distilled off under vacuum to obtain the crude product. Usually the product is a solid. Recrystallization from ethanol or other suitable solvent is a preferred method of purification of the product.

Those skilled in the art will appreciate that these conditions can be modified to suit the particular application of those skilled in the art.
Chlorinated quinoline and isoquinoline can react with diazeniumdiolate, Cl substituents, as shown below, O ² diazeniumdiolate - replaces oxygen.

Moreover, quinazoline can be incorporated as shown below.

  Phthalazines are also incorporated as shown below in accordance with the present invention.

  Acridine can be incorporated as shown below.

  Cinnoline can also be incorporated as shown below.

  Quinoxaline can also be incorporated as shown below.

Oxygen - and nitrogen - also containing heteroaromatic compounds also according to the present invention, O ² diazeniumdiolate - can be used as aromatic reagents for oxygen displacement. For example, oxazole and benzoxazole can be derivatized at the 2-position as shown below.

  Similarly, thiazole and benzothiazole can also be derivatized at the 2-position.

Derivatized vitamin K ₇ may also be prepared as shown below.

The O ² -diazeniumdiolated atom of the aryl ring in the rightmost (immediately above) structure is not activated. Thus, the rightmost structure will resist nucleophilic attacks that regenerate X—N ₂ O ₂ ^— , in other words, the production of NO will be reduced spontaneously. Therefore, in order to generate NO, the rightmost structure needs to undergo oxidative preactivation prior to nucleophilic attack. This oxidative property targets certain cell or organ types that can independently perform the required oxidation, thus limiting NO exposure to the desired tissue while avoiding exposure at other NO sensitive sites in the tissue. Pre-activation requirements may also be advantageous.

  Examples of classes of compounds that require electrophilic preactivation include the compounds shown below.

Triazines can also be aromatic reagents that form the O ² -aryl substituted diazeniumdiolates of the present invention, as shown below. The synthesis of such compounds improves the effectiveness of existing triazine-derived herbicides.

Nucleic acids, and also bases resulting nitro they contain (including ribosylation bases) Further, O ² of the present invention - may be useful as aromatic reagent to form an aryl substituted diazeniumdiolates. This is illustrated in Example 13.

Another interesting O ² -arylated diazeniumdiolate is shown as the product of the following reaction; it can generate both NO and allopurinol upon hydrolysis.

Advantageously, allopurinol is already known to be pharmaceutically useful. Thus, by converting known pharmaceutically useful compounds containing appropriate aryl groups to the O ² -aryl diazeniumdiolates of the present invention, the present invention enhances existing drugs by releasing NO. Let

  Similarly, derivatives of biopterin diazeniumdiolate can be prepared from substituted pteridines as shown below.

  An example of a suitable aryl substitution utilizing a bond through a heteroatom is shown in the reaction scheme below and can be performed by reacting with BuONO or other suitable nitrosating agent.

  A structural analog of Bendazac, a well-known anti-inflammatory agent, can be prepared as follows.

According to the present invention, any of the compounds defined as diazeniumdiolates can undergo O ² -aryl substitution. Thus, X of the compound having Formula I can be any organic or inorganic group. Preferably, X contains an atom different from carbon and hydrogen, and is bonded to the nitrogen of the N ₂ O ₂ ^— group through an atom different from carbon. Most preferably, it is an amine of X, which is bonded to the nitrogen of the N ₂ O ₂ ^— group through a nitrogen atom. Suitable X groups also include, but are not limited to, C ₁ -C ₂₄ aliphatic groups, aryl, and non-aromatic ring groups. “Aliphatic group” means an acyclic group containing carbon and hydrogen and which may contain nitrogen, oxygen, sulfur, phosphorus and halogen. “Aryl” means a group comprising at least one aromatic ring, as described above. Preferably, the aryl group is a _C 3 _{-C 30} containing radical. A non-aromatic ring group means a group containing at least one ring structure and no aromatic ring. Preferably, the non-aromatic ring is a _C 3 _{-C 30} containing radical.

The group X of formula I may be unsubstituted or substituted with a suitable additional group, for example — [N (NO) O ⁻ ], halo, hydroxy, alkylthio, alkoxy, aryloxy, amino, mono - or di - substituted amino, cyano, sulfonato, mercapto, nitro, substituted or unsubstituted _C 1 _{-C 12} aliphatic group, a substituted or unsubstituted _C 3 -C ₈ cycloalkyl, substituted or unsubstituted _C 3 -C ₈ heterocycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ olefinic group, benzyl, phenyl, substituted benzyl, substituted phenyl, benzylcarbonyl, phenylcarbonyl, saccharides, substituted benzylcarbonyl, substituted phenylcarbonyl and phosphorus derivative group. Examples of phosphorus-derived groups include phosphato and phosphono groups. Examples of phosphato groups include (OH) ₂ P (O) O— and substituted (OH) ₂ P (O) O— groups, wherein one or more oxygen atoms are independently S or NR (Wherein R 'is understood to be a C ₁ -C ₈ containing aliphatic group, cycloalkyl or aryl group). Preferred substituents for C ₁ -C ₁₂ aliphatic groups include C ₁ -C ₁₂ acyl, and

[Wherein, _R, C 1 _{-C 10} substituted or unsubstituted aliphatic _group, C 3 _{-C 11} olefinic _groups, C 3 -C ₈ substituted or unsubstituted cycloalkyl, benzyl, phenyl, substituted benzyl or substituted phenyl There, the substituted benzyl or substituted phenyl, halogen, _hydroxy, C 1 -C _{4 alkyl,} C 1 -C ₄ alkoxy, amino, _mono--C 1 -C ₄ alkylamino, di _-C 1 -C ₄ alkylamino, Substituted with 1 or 2 selected from the group consisting of phenyl and phenoxy. ]. Suitable saccharides and polysaccharides include ribose, glucose, deoxyribose, dextran, starch, glycogen, lactose, galactose, fructose, glucosamine, galactosamine, heparin, mannose, maltose, sucrose, sialic acid and cellulose. Other suitable saccharides are phosphorylated, 3,5-cyclophosphorylated and polyphosphorylated pentoses and hexoses.

In one embodiment of the invention, X is an inorganic group disclosed in US Pat. No. 5,212,204. Preferred embodiments of formula I X is an inorganic ^group, - _O 3 S- (sulfite), and - ^- which is O (oxide).

In another embodiment of the invention, X is a polyamine disclosed in US Pat. No. 5,155,137. Thus, the polyamine substituted O ² -aryl diazeniumdiolates have the following formula:

[Wherein Q is the same as Q in formula I and is defined as above; b and d may be the same or different and are 0 or 1, and R ¹ , R ² , R ³ , R ^4] And R ⁵ , the same or different, hydrogen, substituted or unsubstituted C ₃ -C ₈ cycloalkyl, substituted or unsubstituted C ₁ -C ₁₂ linear or branched alkyl, substituted or unsubstituted benzyl, substituted or unsubstituted benzoyl, substituted or unsubstituted _C 3 _{-C 12} olefinic group, phthaloyl, acetyl, trifluoroethyl acetyl, p- toluyl, include t- butoxycarbonyl or 2,2,2-trihalo -t- butoxycarbonyl. The values of i, j and k in Formula II may be the same or different and are integers from 2 to 12. ]

In a preferred embodiment of the present invention, O ² -aryl diazeniumdiolates are derived from the compounds disclosed in US Pat. Nos. 5,039,705 (Keefer et al.) And 4,954,526 (Keefer et al.) Have.

[Wherein R ⁶ and R ⁷ may be the same or different and may be H, C ₁ -C ₁₂ linear alkyl, C ₁ -C ₁₂ alkoxy or acyloxy substituted linear alkyl, C ₂ -C ₁₂ hydroxy or halo. substituted straight chain _alkyl, C 3 _{-C 12} branched _alkyl, C 3 _{-C 12} hydroxy, halo, alkoxy or acyloxy substituted branched chain _alkyl, C 2 _{-C 12} straight chain olefinic groups, and _C 3 _{-C 12} branched olefins Selected from the group consisting of groups, unsubstituted or substituted with hydroxy, alkoxy, acyloxy, halo or benzyl, provided that both R ⁶ and R ⁷ are not H; or R ⁶ and R ⁷ together with the nitrogen atom to which they are attached,

(Wherein A is N, O or S, w is 1 to 12, y is 1 or 2, z is 1 to 5, R ⁸ is hydrogen, C ₁ -C _8. linear _alkyl, C 3 -C ₈ branched _alkyl, C 3 -C ₈ cycloalkyl, a substituted or unsubstituted aryl (e.g., phenyl, tolyl, etc.), and ^{R 9} is _hydrogen, C 1 -C ₆ straight A heterocyclic alkyl selected from the group consisting of a chain alkyl or a C ₃ -C ₆ branched chain alkyl). ]. Typical azacrown groups are 1-aza-12-crown-4, 1-aza-15-crown-5 and 1-aza-18-crown-6. When A is nitrogen, the nitrogen atom itself may be substituted, for example as disclosed in US application Ser. No. 08 / 475,732, incorporated herein by reference.

Further examples include O ² -aryl substituted diazeniumdiolates derived from compounds disclosed in US Pat. No. 5,250,550, previously incorporated by reference, fully incorporated by reference, It has the following formula:

[Wherein D is

Where R ¹⁰ and R ¹¹ are the same or different. The substituents R ¹⁰ and R ¹¹ may be any suitable substituent, examples being hydrogen, C _3-8 cycloalkyl, C _1-12 linear or branched alkyl, benzyl, benzoyl, phthaloyl, Acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl and 2,2,2-trihalo-t-butoxycarbonyl. In Formula IV, f is an integer of 0-12. ]

Suitable O ² -aryl substituted diazeniumdiolates also include the compound of Example 14.

An alternative preparation of O ² -arylated diazeniumdiolates is possible through the application of the following literature reaction (Stevens, J. Org. Chem. 29: 311-315 (1964)).

By replacing the methoxide Stevens reaction, O ² of various structures - ^- aryloxy anion ArO it is possible to obtain the aryl diazeniumdiolates. Similarly, it is possible to obtain derivatives corresponding to ArS ⁻ species.

O ² -Glycosylated diazeniumdiolates and 1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates The present invention also provides two other new classes of diazeniumdiolates Provide a kind. This one class of diazeniumdiolates contains hydrolytically labile groups (R) and is cleaved to free diazeniumdiolates (NO donors) X—NO═NO 2 ^— . Sometimes, non-toxic and possibly beneficial sugars are released, and sugar-based receptor-mediated phenomena can be utilized. Other classes of diazeniumdiolates are inter alia ultrafast NO donors that have proven to be effective as antithrombotic and vasodilators, but are inherently derivatized by their instability. Provides a prodrug of 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate disodium salt (PROLI / NO), which is extremely difficult (Saavedra et al., J. Med. Chem. 31: 4361-4365 (1996); and US Pat. No. 5,632,981 (Saavedra et al.)). The newly discovered ability to produce a prodrug of the ultra-fast NO donor PROLI / NO indicates that the PROLI / NO prodrug is the desired organ for metabolic removal of the stabilized O ² -protecting group. Or allows free movement through the circulation system until it reaches the cell type, thereby providing rapid release of NO at a specific or preferred site and at a controlled rate near the targeted tissue The need for administration by infusion is eliminated. Furthermore, the corresponding nitrosamine (N-nitrosoproline) is unlikely to be carcinogenic when formed in biological media, unlike other nitrosamines.

Accordingly, the present invention is, ^{O 2} - glycosylated 1-substituted diazen-ium-1,2-diolate acids ^{(O 2} - glycosylated diazeniumdiolates) and ^{O 2} - substituted 1 - [(2- Carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolates (1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates) are provided. Both of these can be represented by the following formula:

[In Formula Ia, X and R are organic and / or inorganic groups as defined herein. ].

O ² -glycosylated diazeniumdiolates With respect to O ² -glycosylated diazeniumdiolates, a class of compounds defined as diazeniumdiolates (eg, US Pat. Nos. 5,039,705, 5,208,233, (See 5,155,137, 5,250,550, 5,389,675, 5,525,357, 5,405,919, and related patents and patent applications.) Diazeniumdiolate O ² is used for glycosylation If possible, it can undergo O ² -glycosylation. The group R of formula Ia can be any sugar, which is linked to the O ^{2 of the} diazeniumdiolate by the 2-position of the pyranose or furanose ring. This sugar may be functionalized. Desirably, the sugar and its derivatives are hydrolyzable at physiological pH. The sugar can be a monosaccharide, disaccharide (eg, sucrose or maltose), oligosaccharide or polysaccharide. Preferred sugars and polysaccharides include, among others, ribose, glucose, deoxyribose, fucose, lactose, galactose, fructose, glucosamine, galactosamine, mannose, maltose, sucrose, and many sugars that serve as recognition sequences in receptor-mediated cell-cell interactions. And oligosaccharide units. Other preferred saccharides include phosphorylated, 3,5-cyclophosphorylated and polyphosphorylated pentoses and hexoses.

  By way of example, this sugar residue (shown linked to diazeniumdiolate for illustration) may be an amino sugar (eg, glucosamine or substituted glucosamine having the following structure):

[Wherein R ¹² and R ¹³ may be the same or different and are hydrogen, C _1-6 alkyl, acyl, phosphate, sulfate, peptide or protein. ]. The sugar residue may be, for example, glucuronic acid or a derivative thereof represented below:

[Wherein R ¹⁴ is X ¹ R ¹⁵ R ¹⁶ , wherein X ¹ is N, O or S, and when X ¹ is N, R ¹⁵ and R ¹⁶ are independently hydrogen, substituted Or unsubstituted C _1-24 alkyl, C _3-24 cycloalkyl, C _2-24 olefin group, aryl (eg, acridine, anthracene, benzene, benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll) , Cinnoline, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine, porphine, Pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrocholine, pyrrole, quinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole) or heterocyclic groups (e.g. When X ¹ is O or S, there is no R ¹⁶ group. ].

Alternatively, when X ¹ is nitrogen, R ¹⁵ and R ¹⁶ form a heterocyclic ring selected from the group consisting of the following groups:

[Wherein, A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R ⁸ is hydrogen, C _1-8 straight Chain alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl, aryl (eg, phenyl, tolyl, etc.) or carboxylate and derivatives thereof described below, and R ⁹ is hydrogen, C _1-6 Linear alkyl or C _3-6 branched alkyl. ]. The above groups may be unsubstituted or appropriately substituted.

  Examples of azacrown groups (ie when A is N) include 1-aza-12-crown-4, 1-aza-15-crown-5 and 1-aza-18-crown-6. . When A is nitrogen, the nitrogen atom itself may be substituted, for example as described in US patent application Ser. No. 08 / 475,732.

Further with respect to O ² -glycosylated diazeniumdiolates, the group attached to the carbonyl group via X ¹ may be any that does not prevent cleavage to the diazeniumdiolate anion.

Further with respect to O ² -glycosylated diazeniumdiolates, the group attached to the carbonyl group via X ¹ may be any that does not prevent cleavage to the diazeniumdiolate anion.

Preferably, the group X contains an atom different from carbon and hydrogen and is bonded to the nitrogen of the N ₂ O ₂ ^— group via an atom different from carbon. Most preferably, X is an amino group and is bonded to the nitrogen of the N ₂ O ₂ ^— group through a nitrogen atom. Suitable groups for X include, but are not limited to, C _1-24 aliphatic groups, aryl and non-aromatic ring groups. “Aliphatic group” means an acyclic group containing carbon and hydrogen and which may contain nitrogen, oxygen, sulfur, phosphorus or halogen. “Aryl” means a group comprising at least one aromatic ring. Preferably, the aryl group is a C _6-30 group. “Non-aromatic ring group” means a group containing at least one ring structure and no aromatic ring. Preferably, the non-aromatic ring group is a C _6-30 group. Furthermore, X is unsubstituted Any or suitable addition group (e.g., - [N (NO) O ^-], halo, hydroxy, alkylthio, alkoxy, aryloxy, amino, mono- - or di - substituted amino, cyano, sulfonato , Mercapto, nitro, substituted or unsubstituted C _1-12 aliphatic group, substituted or unsubstituted C _3-8 cycloalkyl, substituted or unsubstituted C ₃ -C ₁₂ olefin group, substituted or unsubstituted C _3-8 heterocycle Alkyl, benzyl, phenyl, substituted benzyl, substituted phenyl, benzylcarbonyl, phenylcarbonyl, sugar, substituted benzylcarbonyl, substituted phenylcarbonyl and phosphorus-derived groups). Examples of phosphorus-derived groups include phosphato and phosphono groups. Examples of phosphato groups include (OH) ₂ P (O) O— and substituted (OH) ₂ P (O) O— groups, wherein one or more oxygen atoms are independently S or NR ¹⁷ (wherein R ¹⁷ is a C _1-8 aliphatic group,

(Wherein R ¹⁸ is a C _1-10 unsubstituted or substituted aliphatic group), C _3-8 unsubstituted or substituted cycloalkyl, benzyl, phenyl, substituted benzyl or substituted phenyl. . ) May be substituted. When benzyl or phenyl is substituted, preferably it is halogen, hydroxy, C _1-4 alkyl, C _1-4 alkoxy, amino, mono-C _1-4 alkylamino, di-C _1-4 alkylamino, phenyl And substituted with one or two substituents selected from the group consisting of phenoxy.

In one embodiment of the invention, X of formula Ia is an inorganic group as described in US Pat. No. 5,212,204. A preferred embodiment of Formula Ia X is an inorganic group ^is - (oxide) ^- _O 3 S- (sulfite) and -O.

In another embodiment of the invention, X in formula Ia is a polyamine as defined in US Pat. No. 5,250,550. Thus, polyamine O ² -glycosylated diazeniumdiolates have the following formula:

[Wherein Q is the same as defined above for R in formula Ia, b and d may be the same or different and are 0 or 1, and R ¹ , R ² , R ³ , R ⁴ and R ⁵ are the same or different and each represents hydrogen, substituted or unsubstituted C _3-8 cycloalkyl, substituted or unsubstituted C _1-12 linear or branched alkyl, substituted or unsubstituted benzyl, substituted or unsubstituted benzoyl , substituted or unsubstituted _C 3- _{C 12} olefin group, phthaloyl, acetyl, trifluoroacetyl, a p- toluyl, t-butoxycarbonyl or 2,2,2-trihalo -t- butoxycarbonyl. ]. The values of i, j, and k in Formula II are the same or different and are integers of 2 to 12.

  In a preferred embodiment of the invention, the diazeniumdiolates are derived from the compounds disclosed in US Pat. Nos. 5,039,705 (Keefer et al.) And 4,954,526 (Keefer et al.) And have the following formula:

Wherein R is the same as defined above for R in formula Ia and R ¹⁹ and R ²⁰ are the same or different and are hydrogen, C _1-12 linear alkyl, C _3-12 A branched alkyl, or a C _2-12 linear or C _3-12 branched olefin group. However, R ¹⁹ and R ²⁰ are not both hydrogen. ]. Any of the above substituents may be unsubstituted or substituted with an alkoxy, acyloxy, acylthio, hydroxy, halo or benzyl group.

Alternatively, R ¹⁹ and R ²⁰ together with the nitrogen atom to which they are attached form a heterocycle selected from the group consisting of the following groups:

[Wherein, A is N, O, or S, w is 1-12, y is 1 or 2, z is 1-5, R ⁸ is hydrogen, C _1-8 straight Chain alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl, substituted or unsubstituted aryl (eg, phenyl, tolyl, etc.) or carboxylate and derivatives thereof described below, and R ⁹ is hydrogen, C _1-6 straight chain alkyl or C _3-6 branched chain alkyl. ]. The above groups may be unsubstituted or appropriately substituted.

  Examples of azacrown groups (ie when A is N) include 1-aza-12-crown-4, 1-aza-15-crown-5 and 1-aza-18-crown-6. . When A is nitrogen, the nitrogen atom itself may be substituted, for example as described in US patent application Ser. No. 08 / 475,732.

Further examples include O ² -glycosylated diazeniumdiolates derived from the compounds disclosed in US Pat. No. 5,250,550, having the following formula:

[Wherein D is

Where R ²¹ is the same as defined above for R in the sugar of formula Ia and R ¹⁰ and R ¹¹ may be the same or different and may be any suitable group (for example , Hydrogen, C _3-8 cycloalkyl, C _1-12 linear or branched alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl and 2,2,2-trihalo- t-butoxycarbonyl may be mentioned). ]. In Formula IV, f is an integer of 0-12.

Preferred O ² -glycosylated diazeniumdiolates are those compounds with respect to formula Ia where X is N (CH ₂ CH ₂ NH ₂ ) ₂ and R is fucose or mannose.

  The above compounds can be produced according to methods known to those skilled in the art. Glycopyranosylation reagents include acetobromo-α-galactose and acetobromoglucosamine. Glycofuranosylation reagents include tribenzyl-α-arabinofuranosyl bromide and bromoacetylxylose.

Oligosaccharides can be purchased from, for example, Sigma Chemical Co. (St. Louis, MO) and Carbomer Specialty Biochemicals and Polymers (Westborough, Mass.). In addition, oligosaccharides are described in Preparative Carbohydrate Chemistry, Stephen Hanessian, ed., Marcel Dekker, New York, NY (1997) and Polysaccharides in Medicinal Applications, Severian Dumitriu, ed., Marcel Dekker, New York, NY (1996). Can be synthesized according to well-established procedures, including chemical and enzymatic production methods such as

A protected linear or branched polysaccharide may be activated by halogenation at the anomeric end for subsequent reaction with a diazeniumdiolate ion, followed by glycosylation of the diazeniumdiolate. . Activated disaccharides for the production of O ² -glycosylated diazeniumdiolates include acetobromo-α-maltose and acetobromo-α-lactose.

O ² -glycosylated diazeniumdiolates are useful when molecular signaling and recognition processes, including cell adhesion, involve carbohydrates. For example, O ² -glycosylated diazeniumdiolates are considered useful for the treatment of infections such as those caused by parasites (eg, the genus Lyshmania), viruses or bacteria, and inflammation and metastasis. It has been. In this regard, O ² -glycosylated diazeniumdiolate can be made to be directed to the mannose-fucose receptor, as exemplified in Example 36. Sugar residues (in this case mannose) are thought to protect diazeniumdiolate. This mannose binds to the mannose-fucose receptor on macrophages and NO is released when O ² -mannosylated diazeniumdiolate enters the cell where the sugar residues are cleaved.

1-[(2-Carboxylato) pyrrolidin-1-yl] diazeniumdiolates With respect to 1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates, the group X of formula Ia is ,

1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates can be structurally represented by the following formula:

[Wherein R ²² is hydrogen, hydroxyl, OM (where M is a cation), halo or X ¹ R ²³ R ²⁴ (wherein X ¹ is N, O or S, and X ¹ is When N, R ²³ and R ²⁴ are independently substituted or unsubstituted C _1-24 alkyl, C _3-24 cycloalkyl, C _2-24 olefin group, aryl (eg, acridine, anthracene, benzene, benzofuran, Benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline, naphthalene, oxazole, phenanthrene, phenanthridine, phenothiazine, Phenoxazine, fu Luimide, phthalazine, phthalocyanine, porphine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrocholine, pyrrole, quinolizinium ion, quinoline, quinoxaline, quinazoline, sydnone, tetrazole, thiazole, thiophene, thyroxine, triazine, and triazole Or a heterocyclic group (for example, glycosyl etc.) and when X ¹ is O or S, R ²⁴ is absent.). ]. Alternatively, when X ¹ is nitrogen, R ²³ and R ²⁴ together with the nitrogen atom to which they are attached form a heterocycle, for example a heterocycle selected from the group consisting of the following groups:

Wherein, A is N, O or S,, w is 1 to 12, y is 1 or 2, z is 1-5, and ^{R 8,} R ^{9, R 25} and R ²⁶ is hydrogen, C _1-8 straight chain alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl or aryl. ]. The above groups may be unsubstituted or appropriately substituted.

The R ²⁶ substituent on nitrogen (N-4) is hydrogen, a C _1-8 alkyl group, an aryl group or C (O) —YR ²⁷ , where Y is sulfur, oxygen or nitrogen, and R ²⁷ Is CH ₂ OCH ₃ , vinyl, C _1-9 straight chain alkyl, C _3-6 branched chain alkyl, C _3-8 cycloalkyl, polyethylene glycol, polysaccharide, or other polymer, peptide or protein). There may be. ]. YR ²⁷ is activated to bind to proteins, peptides, phospholipids, polysaccharides, oligosaccharides, purines, pyrimidines and biocompatible polymers (ie polyethylene glycol, polylactides and polycaprolactone). It may be a linker (for example, a hydroxysuccinimidyl group). YR ²⁷ may be an activating group for the carbonyl group that makes the carbonyl group an electrophilic site that reacts with nucleophilic functional groups of oligopeptides, polyamines and proteins. YR ²⁷ can react carbonyl groups with many nucleophiles and can react with polymers (eg, polyethylene glycol) to form polymer-linked compounds.

Further with respect to 1-[(2-carboxylato) pyrrolidin-1-yl] diazeniumdiolates, the group R of formula Ia can be any covalently bonded organic or inorganic group, unlike hydrogen and C _1-12 straight chain or C _3-12 branched chain alkyl, C _2-12 straight chain or C _3-12 branched chain olefin group, C _1-12 acyl, sulfonyl, C _3-12 cycloalkyl, carboxamide, glycosyl mentioned above A group, an aryl group as described above, or a formula — (CH ₂ ) _n —ON═N (O) NR ²⁸ R ²⁹ , wherein n is an integer of 2-8, and R ²⁸ and R ²⁹ are independently C _1-12 straight chain alkyl, C _3-12 branched chain alkyl, C _1-12 straight chain or C _3-12 branched chain olefin group, or R ²⁸ and R ²⁹ are Together with the nitrogen atom in this form a heterocyclic ring, preferably a pyrrolidino, piperidino, piperazino or morpholino group. The above R groups may be unsubstituted or appropriately substituted. Preferred substitutions include substitution with hydroxy, halo, acyloxy, alkoxy, acylthio or benzyl.

The above compounds can be produced according to methods known to those skilled in the art. See, for example, Sanger, Biochem. J. 39: 507-515 (1945).

O ² -substituted 1-[(2-carboxylato) pyrrolidin-1-yl] diazenium diolates are more stable than O ² -unsubstituted anions in aqueous solution, and often It provides an advantage over other diazeniumdiolates in that they can be activated for NO release by enzymatic action. Furthermore, the N-nitroso derivative is formed by the net formal cleavage of the N—N double bond of 1-[(2-carboxylato) pyrrolin-1-yl] diazen-1-ium-1,2-diolate. This N-nitroso compound is non-carcinogenic. Such compounds are believed to be particularly useful in the treatment of fulminant liver failure, malaria, respiratory disorders, impotence, and various cardiovascular / blood disorders.

Another particularly useful embodiment of the polymeric binding diazeniumdiolates present invention, O ² of the formula I - including glycosylation diazeniumdiolates - aryl diazeniumdiolates or O ² of the formula Ia Where X is a polymer or any O ² -aryl diazeniumdiolate or O ² -glycosylated diazeniumdiolate of the present invention is incorporated into the polymer matrix. PROLI / NO may also be a polymer bond through R ²⁰ and R. Both of these embodiments, "bound to have the polymer" N ₂ O ₂ ^- results in a functional group. By "bound to the polymer", N ₂ O ₂ ^- functional group is, is associated with some physical or chemical polymer matrix (the Associated), are incorporated into the polymer matrix (Incorporated) or Means contained.

N ₂ O ₂ ^- physical ties functional group to the polymer (association) or binding (Bonding), by co-precipitation and nitric oxide / nucleophile complex of the polymer, and N ₂ O ₂ ^- group Can be achieved by covalent bonding of to the polymer. N ₂ O ₂ ^- chemical bonding to the base polymer, for example, a nucleophilic group of nitric oxide / nucleophile adduct covalently attached to the polymer, N ₂ O ₂ ^- are groups bonded The nucleophile residue may form part of the polymer itself (ie, in the polymer backbone or attached to a pendant group on the polymer backbone). Nitric oxide releasing N ₂ O ₂ ^- functional group is associated with a portion of the polymer, contained within or polymers are incorporated into the polymer, i.e., a manner that is "bound" to the polymer in the present invention Not critical, all means of association, incorporation and bonding are contemplated by the present invention.

Site-specific application of polymer-bound adduct composition, nitric oxide releasing N ₂ O ₂ ^- enhance the selectivity of action of the functional groups. If N ₂ O ₂ are bonded to the polymer ^- if functional groups are necessarily localized, the effect of their nitric oxide release will be concentrated in the tissues in which they are in contact. If the polymer is soluble, the selectivity of action can be further arranged, for example, by binding to an antibody specific for the targeting tissue or by derivatization of such an antibody. Similarly, N ₂ O to small peptides that mimic the ligand recognition sequence for key receptors, such as binding to oligonucleotides capable of site-specific interaction with targeting sequences in nucleic acids. _{The 2} ^- group linkage provides localized nitric oxide release.

The O ² -diazenium diolates of the present invention are disclosed in US Pat. Nos. 5,525,357 (Keefer et al.) And 5,405,919 (Keefer et al.), And US Patent Application No. 08 / 419,424 (Smith et al.) Incorporated herein by reference) can be derived from the materials disclosed. Any of a wide variety of polymers can be used in connection with the present invention. The selected polymer need only be biologically acceptable. Examples of polymers suitable for use in the present invention include polyolefins (eg, polystyrene, polypropylene, polyethylene, polytetrafluoroethylene, polyvinyl chloride, poly (vinylidene fluoride)), polyethers (eg, polyethylene glycol), polysaccharides (eg, Dextran), polyesters (eg, poly (lactide / glycolide)), polyamides (eg, nylon), polyurethanes, polyethyleneimines, biopolymers (eg, peptides, proteins, oligonucleotides, antibodies) And nucleic acids), star blast dendrimers, polysaccharides and the like.

In this regard, polymers containing diazeniumdiolate can react with the sugar, sugar N ₂ O ₂ ^- is bonded to a functional group.

Formation of biopolymer-derived diazeniumdiolate provides a biopolymer-bound diazeniumdiolate composition that can be specifically applied to the biological site of interest. Site-specific application of biopolymers bound diazeniumdiolate, the action of the nitric oxide releasing diazeniumdiolate (which in PROLI / NO, O 2 ^- aryl or O ² - glycosylated bond or O-R Selectivity that occurs following bond cleavage (see page 31)). For the other polymers disclosed above, if the diazeniumdiolate bound to the biopolymer is localized due to the intrinsic properties of the molecule, its nitric oxide releasing effect Will concentrate on the organization that is. If the biopolymer is soluble, the selectivity of action can be further arranged, for example, by binding to an antibody specific for the targeting tissue or by derivatization of such an antibody. Similarly, diazenium to small peptides that mimic the ligand recognition sequence for key receptors, such as binding to oligonucleotides capable of site-specific interaction with targeting sequences in nucleic acids. The attachment of the diolate group provides localized nitric oxide release. Other proteins, peptides, polypeptides, nucleic acids and polysaccharides can be used as well. US Pat. No. 5,405,919 (Keefer et al.) And US Pat. No. 5,632,981 (Saavedra et al.), Fully incorporated herein by reference, disclose similar compounds and products useful in the manufacture of diazeniumdiolates. .

By way of example, O ² -arylated piperazine diazeniumdiolate can be covalently linked to a polypeptide comprising an IKVAV recognition sequence important for cancer cell chemotaxis. Metastasis by maintaining both the ability to regenerate NO as an anti-adhesive agent and the affinity of IKVAV sequences for cancer cells and / or vascular and lymphoid sites to which cancer cells are likely to attach Can be reduced or even prevented. Furthermore, the aryl group may be selected such that it provides additional anticancer cell activity. Substitution at N ⁴ position of piperazine, peptides, polypeptides, proteins such, it can be used to bind the glycosylated diazeniumdiolate to polysaccharides and nucleotides.

  The diazeniumdiolates of the present invention can be used as a means to reduce the risk of developing solid cancers associated with medical implants such as prostheses, stents and breast implants prior to their surgical attachment to the body. It is believed that it can be used to coat. In addition, the prostheses and implants can be manufactured using diazeniumdiolate as an essential component of the starting material. A medical device incorporating diazeniumdiolate provides a useful medical structure that also favors local release of NO, and is a very valuable bifurcated to the treatment of many biological disorders. Provide a two-pronged approach.

As is well known in the composition the art, nitric oxide and N ₂ O ₂ ^- compounds containing functional groups, in part, the role of the various fields of nitric oxide in the organism adjustment process (multifaceted role ) Can have broad utility. Accordingly, the present invention also provides compositions, including pharmaceutical compositions, containing the diazeniumdiolate of the present invention. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

  Although any suitable method of administering the diazeniumdiolate composition of the present invention to an animal (eg, a mammal) can be used, and more than one route of administration can be used to administer a particular composition. Those skilled in the art will appreciate that one particular route of administration provides a more rapid and effective response than another route of administration. Pharmaceutically acceptable carriers are also well known to those skilled in the art. The choice of carrier will be determined in part by both the particular composition as well as the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

  Formulations suitable for oral administration are predetermined as (a) an effective amount of a liquid solution such as diazeniumdiolate dissolved in a diluent such as water or saline, and (b) a solid or granule. Capsules, sachets or tablets each containing a certain amount of the active ingredient, (c) a suspension in a suitable liquid, and (d) a suitable emulsion. Tablets may contain one or more lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, gum arabic, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other supplements. Forms, colorants, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmaceutically compatible carriers may be included. Lozenges contain active ingredients in flavoring agents (usually sucrose and gum arabic or tragacanth), and perfumes in inert bases (gelatin and glycerin or sucrose and gum arabic emulsions, gels, etc.) It contains the active ingredient and contains carriers known in the art in addition to the active ingredient.

  The diazeniumdiolates of the present invention may be prepared alone or in combination with other suitable ingredients into aerosol formulations to be administered by inhalation. These aerosol formulations are placed in a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen and the like.

  Formulations suitable for parenteral administration may contain aqueous and non-aqueous solutions, ie, antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the recipient. Isotonic sterile injectable solutions and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. The formulation is given in single-dose or multiple-dose sealed containers such as ampoules and vials, and for injection, just add a sterile liquid carrier such as water just before use. It may be stored in a lyophilized state. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

In the present invention, the amount administered to an animal (particularly a human) should be sufficient to exert a therapeutic effect on the animal over an appropriate time frame. Dosage depends on the strength of the particular composition used (considering at least the rate of NO generation, the range of NO generation and the biological activity of degradation products from diazeniumdiolates), the animal to be treated Will be determined by the condition and weight. The dosage will also be determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of a particular composition. A suitable dosage for internal administration is 0.01-100 mg / kg / day. A preferred dosage is 0.01 to 35 mg / kg / day. A more preferable dose is 0.05 to 5 mg / kg / day. In a typical pharmaceutical composition for administration, O 2 ^- appropriate concentration aryl diazeniumdiolates is 0.05-15% (per weight). A preferred concentration is 0.02 to 5%. A more preferable concentration is 0.1 to 3%.

Methods of Use In view of the above, the present invention provides methods of using the diazeniumdiolates of the present invention. In one embodiment, a method of treating an animal (eg, a mammal) having a biological disorder treatable with nitric oxide is provided. This method comprises administering to an animal (eg a mammal) a diazeniumdiolate according to the present invention in an amount sufficient to treat a biological disorder in the animal. In this embodiment, a “biological disorder” is a biological disorder resulting from a genetic defect or infection by an infectious agent (eg, a virus, bacterium, or parasite) as long as the disorder is treatable with nitric oxide. Any biological disorder, including

  In another embodiment of the method of use, a method is provided for treating an animal (eg, a mammal), eg, for infection by a virus, bacterium, or parasite (eg, Leishmania). The method includes administering to the animal (eg, a mammal) a sufficient amount of diazeniumdiolate to treat the infection in the animal.

In one aspect of this embodiment of the invention, for example, a virus (eg, retrovirus, particularly HIV, more particularly HIV-1), a bacterium (eg, Gram-positive bacteria) or a parasite (eg, Giardia genus). ) (none of these also, O 2 ^- for infection with aryl by diazeniumdiolate comprising a zinc finger protein that can be inactivated), animal (e.g., a method of treating a mammal) is provided. `` Zinc finger protein '' means a protein containing a short amino acid domain containing cysteines alone or containing 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), including the contents of all references cited therein, by reference. Fully incorporated here.) “Inactivated” means that the activity of the zinc finger protein that is inactivated partially or completely disappears. Such inactivation should itself lead to inactivation of the animal's biologically important zinc finger proteins to such an extent that they endanger the animal's health and good living conditions. Absent. This method provides an animal (eg, a mammal) with an O ² -aryl diazeniumdiolate in an amount sufficient to inactivate a zinc finger protein in the infectious agent to treat infection in the animal. Administration.

The method can also be applied as a means of treating plants, plant cells or cultured tissues thereof against infection by infectious agents such as viruses (eg tobacco streak virus (TSV) or alfalfa mosaic virus (AIMV)). (South and Summers (1990) above; and Sehnke et al., Virology 168: 48 (1989)).

The method described here is a C-X2-C-X4-H-X4-C motif (where "C" represents cysteine, "H" represents histidine, "X" represents any amino acid, And “2” and “4” represent the number of “X” amino acids) (see, for example, Wain-Hobson et al., Cell 40 (1): 9-17 (1985)). is there. Such motifs characterize retroviruses, particularly the retrovirus gag protein. Thus, the methods herein include retroviruses such as HIV, in particular HIV-1, which include nucleocapsid p7 protein (NCp7 protein) containing two zinc binding domains (Rice et al., Nature Medicine 3 (3): 341-345 (1997); and Rice et al., Reviews in Medical Virology 6: 187-199 (1986)). Actual and / or potential zinc fingers may also include, inter alia, gene products of the EIA genomic region of adenovirus, large T antigen from Simon virus 40 (SV40) and polyoma virus, 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 T4 derived gene 32 protein (G32P) (Giedroc et al., Biochemistry 28: 2410 (1989)) and has been identified in a bacteriophage protein (Berg, Science 232: 484 (1986)). Such proteins can be isolated according to methods known in the art (see references cited in South and Summers (1990) above) and O ² capable of inactivating such zinc finger proteins. -Aryl diazeniumdiolates can be identified, for example, according to the zinc finger assay described herein and the description of Rice et al., J. Med. Chem. 39: 3606-3616 (1996).

Only for steroid hormone receptors containing zinc fingers with a motif containing 4 or 5 cysteines, O ² -aryldiazeniumdiolates are used to modulate steroid hormone activity in animals (eg mammals). obtain. Thus, the present invention also provides for an animal (eg, a mammal) comprising a steroid hormone receptor protein comprising a zinc finger that requires modulation of steroid hormone activity and can be inactivated by O ² -aryldiazeniumdiolate. Provide a method of modulating steroid hormone activity. This method administers an animal (eg, a mammal) with an O ² -aryl diazeniumdiolate sufficient to inactivate a steroid hormone receptor protein to modulate steroid hormone activity in the animal. Including that.

  In yet another embodiment, a method of treating an animal (eg, a mammal) for cancer and its metastases is provided. The method includes administering to the animal (eg, a mammal) a sufficient amount of diazeniumdiolate to prevent cancer growth or metastasis in the animal.

In one aspect of this embodiment, for cancers due at least in part, directly or indirectly, to the activity of zinc finger proteins that can be inactivated by O ² -aryldiazeniumdiolates. Methods of treating (eg, mammals) are provided. This method inactivates zinc finger proteins to treat cancer in animals (Rice et al., PNAS 89: 7703-7707 (1992)), ie to prevent cancer growth or metastasis in animals Administration of a sufficient amount of O ² -aryldiazeniumdiolate to an animal (eg, a mammal).

In still yet another embodiment, chemotherapeutic agents (eg, Kelley et al., Biochem. J. 304: 843-848 (1994) due to the action of enzymes that adversely affect the activity of chemotherapeutic agents, for example. ), In particular, methods for treating animals (eg, mammals) for cancer resistant to treatment with DNA damaging agents (eg, alkylating or oxidizing agents). . The method includes administering to the animal (eg, a mammal) an O ² -aryl diazeniumdiolate sufficient to make the cancer in the animal susceptible to treatment with a chemotherapeutic agent. Thus, such methods can be used as an additional therapy to chemotherapy as needed.

For example, certain O ² -aryl diazeniumdiolates are glutathione S-transferases, particularly isozymes π (eg, Ji et al., Biochemistry 32 (49): 12949-12954 (1993); and Ji et al., Biochemistry. 36: 9690-9702 (1997)). Therefore, the reversible consumption of glutathione from the active site of glutathione S-transferase π by enzyme binding of this compound with glutathione using O ² -aryldiazeniumdiolate is the Xenobiotic compounds such as chemotherapeutic agents, especially alkylating agents (eg chlorambucil, melphalan and hepsulfam) and other DNA damaging agents (eg agents that induce electrophilic attack or oxidation) Detoxification can be prevented (see, eg, Morgan et al., Cancer Chemother. Pharmacol. 37: 363-370 (1996)). The method is also applicable for screening drug resistant cancer cell lines in vitro.

  In another embodiment, a method of treating an inanimate object for the presence of a potentially infectious virus, bacterium or parasite is provided. This method involves contacting an inanimate object with a sufficient amount of the diazeniumdiolate of the present invention to reduce the presence of a potentially infectious virus, bacterium or parasite. “Latent infectivity” means the ability to infect an animal (eg, a mammal).

In one aspect of this embodiment, a potential infectious agent (eg, a virus, bacterium, or parasite) on an inanimate object, any of which can be inactivated by O ² -aryl diazeniumdiolate Methods for reducing the presence of finger proteins (including finger proteins) are provided. This method involves a sufficient amount of O ² -aryl to inactivate the zinc finger protein so as to reduce the presence of potential infectious agents (eg, viruses, bacteria or parasites) on the inanimate object. Including contacting a diazeniumdiolate with an inanimate object. “Latent infectivity” means the ability to infect an animal (eg, a mammal) directly or indirectly.

The invention is further illustrated by the following examples, which, of course, do not limit the scope of the invention in any way. For the following examples, NO is obtained from Matheson Gas Products (Montgomeryville, PA), β- and α-glycosidase and porcine liver esterase are obtained from Sigma Chemical Co. (St. Louis, MO) and polyurethane (Tecoflex). Was obtained from Thermedics Inc. (Woburn, Mass.), And glucose and mannose were obtained from Aldrich Chemical Co. (Milwaukee, Wis.). Proton NMR spectra were recorded using a 300 MHz Varian Unity Plus or Varian XL-200 NMR spectrometer. Spectra were obtained in deuterated chloroform for covalent compounds and in D ₂ 0 for salts. Chemical shifts were recorded in parts per million (ppm) at low magnetic fields from TMS. Low resolution and high resolution mass spectral (MS) measurements were performed on a VG-Micromass Model 7070 spectrometer. Unless otherwise indicated, MS data was sampled through a direct probe and accumulated in electron impact mode. Ultraviolet (UV) spectra were performed on an HP 8451A Diode Array spectrophotometer as an aqueous solution or 0.01 M NaOH solution. Glutathione S-transferase kinetics was monitored by measuring changes in UV absorption at 380 nm using a Beckman DU 640 spectrophotometer. Chemiluminescence measurements were performed on a Thermal Energy Analyzer Model 502A instrument (Thermedics, Inc., Woburn, MA). Elemental analysis was performed by Atlantic Microlab Inc.

Example 1
In this example, the preparation of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate is described.
A solution of 1.67 g (11 mmol) of sodium diethylaminodiazeniumdiolate in 20 ml of 5% aqueous sodium bicarbonate solution was cooled to 0 ° C. under nitrogen and 1.3 ml of 2,4-dinitrofluorobenzene in 10 ml of t-butyl alcohol. (0.01 mol) of solution was added slowly. A precipitate formed upon addition. The mixture was allowed to warm slowly to room temperature and then stirred overnight. The product was extracted with dichloromethane and then washed with cold dilute hydrochloric acid and then sodium bicarbonate solution. The organic layer was dried over sodium sulfate, filtered through a magnesium sulfate layer, and the solvent was distilled off under vacuum to obtain 1.3 g of a red oily product, which was left to crystallize. Recrystallization from ethanol gave yellow-orange needles: mp 76-7 ° C; NMR δ 1.25 (t, 6H), 3.58 (q, 4H), 7.68 (d, 1H), 8.44 (m, 2H ), 8.89 (m, 1H); UV (ethanol) λ _max (ε) 218 (17.4 mM ⁻¹ cm ¹ ) and 302 (15.6 mM ⁻¹ cm ⁻¹ ) nm; MS, exact mass: C ₁₀ H Calculated as ₁₃ N ₅ O ₆ (M +) 299.0865; Found M + 299.08658; C, H, N analysis: Calculated as C ₁₀ H ₁₃ N ₅ O ₆ : C 40.13%, H 4.35%, N 23.41% Found: C 40.21%, H 4.43%, N 23.37%.

Example 2
In this example, an anionic diazeniumdiolate, its O ² -aryl substituted (O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1, Reproduction from 2-Georate will be described.
O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate 85 mg (0.28 mmol) prepared as in Example 1 in 1 ml of ether The solution of was cooled to −4 ° C. and treated with 1 ml of diethylamine. The solution was kept at -4 ° C for 1 hour to obtain a precipitate. The solid was collected by filtration. The filtrate was concentrated and analyzed by NMR. The residue was found to be identical to an authentic sample of 2,4-dinitro-N, N-diethylaniline. The precipitate was washed with petroleum ether and dried under N ₂ , λ _max 250 nm; NMR (D ₂ O) δ 0.96 (t, 6H), 1.28 (t, 6H), 2.94 (q, 4H), 5.4 mg of product with 3.08 (q, 4H) was obtained. This product was found to be identical to an authentic sample of diethylammonium 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate.

Example 3
In this embodiment, mediated by sodium methoxide, O 2 ^- aryl diazeniumdiolate O ² - is described chemical cleavage of aryl bond.
A solution of 16 mg (0.064 mmol) of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate in 1 ml of ether in 0.14 mmol of methanol Was treated with 29 μl of 25% sodium methoxide and allowed to stand at −4 ° C. for 2 hours. The solid was filtered off, washed with ether and dried in vacuo to give 4 mg of the same solid as an authentic sample of 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate sodium salt. Obtained.

Example 4
In this example, the reaction of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate with sodium methoxide in methanol. Tick will be explained. The kinetics of this reaction is that of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate in an alkaline or nucleophilic environment. The conversion rate to-(N, N-diethylamino) diazen-1-ium-1,2-diolate ion is shown.
Excess NaOMe was used in the reaction. Aliquots were collected at intervals and quenched with 0.1 N HCl in methanol. The disappearance of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate was monitored by HPLC and found to meet the first-order rate equation. It was. This means that log [O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2 is found to find k _{obs ′} in four different concentrations of NaOMe. Georate was determined by plotting against time. Similarly, a second order rate constant (7.87 M ⁻¹ min ⁻¹ ) was determined by plotting log k _obs against log [NaOMe].

Example 5
In this example, the preparation of O ^2- (2,4-dinitrophenyl) 1- (N-isopropylamino) diazen-1-ium-1,2-diolate is described.
A solution of 84 mg (0.597 mmol) of sodium 1- (N-isopropylamino) diazen-1-ium-1,2-diolate in 1 ml of 5% sodium bicarbonate was cooled to 0 ° C. and 2,4-dinitrofluoro Benzene 69 mg (0.55 mmol) was added. The ice bath was removed and the mixture was stirred overnight at room temperature, then the mixture was extracted with dichloromethane. The extract was dried over sodium sulfate, filtered and evaporated in vacuo to give 86 mg of a film which crystallized upon standing: mp 92-93 ° C; NMR δ 1.39 (d, 6H), 3.99 (septet, lH), 6.93 (d, 1 H), 8.27 (dd, 1H), 8.5 (b, 1H), 9.15 (d, lH).

Example 6
In this example, the synthesis of pyrrolidinium 1- [pyrrolidin-1-yl] diazen-1-ium-1,2-diolate is described.
A solution of 36 g (0.507 mol) pyrrolidine in 50 ml ether and 25 ml acetonitrile was placed in a 500 ml Parr bottle, degassed and charged with 40 psi nitric oxide. The reactor was cooled to -80 ° C. The pressure was maintained at 40 psi. After 4 hours, the pressure was released and the crystalline product was filtered off in a fritted glass funnel and then washed with cold ether under a nitrogen atmosphere. The material was dried in a vacuum desiccator at 1 mm Hg and 25 ° C. for 3 hours to give 23 g (45%) of white needles: mp 68-70 ° C. C, H, N analysis: C ₈ H ₁₈ N ₄ Calculated as _{O 2: C 47.51%, H} 8.97%, N 27.70%; Found: C 47.62%, H 9.04% , N 27.46%.
For subsequent O ² -arylation, the pyrrolidinium salt was converted to the more stable sodium salt by treatment with 10 N NaOH to facilitate cation exchange. It was then filled with a large amount of ether and the product was filtered off.

Example 7
This example provides an alternative method for the preparation of the sodium salt of 1- (pyrrolidin-1-yl) diazen-1-ium-1,2-diol shown in Example 6.
A solution of 28.2 g (0.397 mol) pyrrolidine in 100 ml acetonitrile and 100 ml ether was mixed with 94 ml (0.4 mol) 25% sodium methoxide in methanol. The resulting solution was flushed with nitrogen and then charged with 40 psi NO and stirred at room temperature for 2 days to form a cloudy precipitate (this precipitate formed within 1 hour after exposure to NO) Began to do.) The product was filtered off by releasing the pressure. The product was washed with ether and dried in vacuo to give 32.1 g (54%) of a white powder: UV (0.01 N NaOH) λ _max (ε), 252 nm (8.84 mM ⁻¹ cm ⁻¹ ); t _1/2 8.5 ° C. at 25 ° C. and 2.8 seconds at 37 ° C. (in phosphate buffer pH 7.4); NMR (D ₂ 0) δ 1.91 (m, 4H), 3.22 (m, 4H).

Example 8
In this example, the preparation of O ^2- (2,4-dinitrophenyl) 1- (pyrrolidin-1-yl) diazen-1-ium-1,2-diolate is described.
A solution of 556 mg (3.63 mmol) of sodium 1- (pyrrolidin-1-yl) diazen-1-ium-1,2-diolate in 10 ml of 5% aqueous sodium bicarbonate solution was cooled to 0 ° C. A solution of 456 μl (3.63 mmol) of 2,4-dinitrofluorobenzene in 2 ml of t-butyl alcohol was added and the resulting mixture was stirred at room temperature overnight. The yellow-orange precipitate was collected by filtration, washed with water and dried to give 758 mg of product, which was recrystallized from ethanol: mp 94-95 ° C; NMR, δ 2.04 (m, 4H), 3.35 ( m, 4H), 6.90 (d, 1H), 8.20 (dd, 1H), 8.67 (d, 1H); MS, m / z (%), 297 (M ⁺ , 1), 220 (100), 237 ( 30), 190 (94), 180 (15), 162 (10), 149 (26), 130 (20), 100 (95), 70 (24), 63 (35), 56 (18); Exact mass: C ₁₀ H ₁₁ N ₅ calculated for O ₆ ^{(M +)} 297.0708; Found 297.0709.

Example 9
In this example, the preparation of sodium 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate is described.
A solution of 20 g (0.126 mol) of N-carboethoxypiperazine in 60 ml of methanol was placed in a Parr bottle. The solution was treated with 27.4 ml (0.126 mol) of a 25% sodium methoxide solution in methanol. The system was degassed and charged with 40 psi nitric oxide and held at 25 ° C. for 48 hours. The white crystalline product was filtered off and washed with cold methanol and copious amounts of ether. The product was dried in vacuo to yield 14.5 g (48%) of sodium 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazene-1-ium-1,2-diolate, mp 184- 5 ° C; UV (0.01 N NaOH) λ _max (ε) 252 nm (10.4 mM ^-1 cm ^-1 ); NMR (D ₂ 0) δ 1.25 (t, 3H), 2.15 (q, 2H), 3.11 (m , 4H), 3.68 (m, 4H). Analysis: Calculated as C ₆ H ₁₃ N ₄ O ₄ Na: C 35.00%, H 5.42%, N 23.33%, Na 9.58%; Found: C 34.87%, H 5.53%, N 23.26%, Na 9.69% .
The half-life of this compound at pH 7 and 25 ° C. was estimated to be 5 minutes. This measurement was based on the disappearance of the 252 nm chromophore in the ultraviolet spectrum.

Example 10
In this ^embodiment, O 2 - (2,4-dinitrophenyl) 1 - [(4-ethoxycarbonyl) piperazin-1-yl] diazen-ium-1,2 for the preparation of dioleate be described.
A solution of 1.073 g (0.0045 mol) of sodium 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazene-1-ium-1,2-diolate in 10 ml of 5% sodium bicarbonate at 0 ° C. under nitrogen. Cooled down. A portion of a solution of 0.89 ml (0.0044 mol) of 2,4-dinitrofluorobenzene in 10 ml of t-butyl alcohol was added. A precipitate formed upon addition. The mixture was stirred at room temperature for 4 hours. The product was extracted with dichloromethane. The extract was washed with water, dried over sodium sulfate and filtered through an anhydrous magnesium sulfate layer. The solvent was distilled off to obtain an orange glass, which was allowed to stand for crystallization. The product was recrystallized from ethanol: dichloromethane to give 1.3 g (76%) of analytically pure material: mp 140-141 ° C .; NMR δ 1.32 (t, 3H), 3.63 (m, 4H) , 3.74 (m, 4H), 4.19 (q, 2H), 7.66 (d, 1 H), 8.48 (q, 1H), 8.88 (d, 1 H); UV (H ₂ 0) λ _max (ε) 210 nm (13.3 mM ⁻¹ cm ⁻¹ ), 300 nm (12 mM ⁻¹ cm ⁻¹ ). Analysis: Calculated as C ₁₃ H ₁₆ N ₆ O ₈ C 40.61%, H 4.20%, N 21.87%;
Found: C 40.74%, H 4.13%, N 21.98%.

Example 11
In this ^embodiment, O 2 - (-4- 2- chloro-yl) 1- (N, N- diethylamino) describing diazen-ium-1,2 for the preparation of dioleate.
A solution of 2,4-dichloropyrimidine 600 mg (4 mmol) in 2 ml of dimethyl sulfoxide and 5 ml of tetrahydrofuran is syringed with sodium 1- (N, N-diethylamino) in 5 ml of tetrahydrofuran at room temperature under nitrogen. Diazin-1-ium-1,2-diolate was added to a slurry of 678 mg (4.37 mmol) and the resulting mixture was stirred for 72 hours. To the mixture was added 5 ml of ether. After washing with water, the organic layer was dried over sodium sulfate, filtered through a magnesium sulfate layer, and the solvent was distilled off to obtain 679 mg of an oily product, which was crystallized at -20 ° C. This material was recrystallized from ether-petroleum ether: mp 37-38 ° C; NMR δ 1.25 (t, 6H), 3.56 (q, 4H), 7.00 (d, lH), 8.50 (d, lH); UV, λ _max (ε) 268 nm (9.3 mM ⁻¹ cm ⁻¹ ). C, H, N Analysis: Calculated for C ₈ H ₁₂ N ₅ O ₂ Cl: C 39.11%, H 4.92%, N 28.51%, Cl 14.43%; Found: C 38.96%, H 4.96%, N 28.35 %, Cl 14.60%.

Example 12
In this ^embodiment, O 2 - (2-chloro-pyrimidin-1-yl) 1 - [(4-ethoxycarbonyl) piperazin-1-yl] diazen-ium-1,2 for the preparation of dioleate be described.
A solution of 262 mg (1.76 mmol) of 2,4-dichloropyrimidine in 3 ml of dimethyl sulfoxide was added to sodium 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazene in 10 ml of tetrahydrofuran at room temperature under nitrogen. The slurry was added to a slurry of -1-ium-1,2-diolate 424 mg (1.76 mmol) and stirred for 72 hours. The resulting homogeneous solution was treated with 100 ml of water. The precipitate was filtered off and dried in vacuo to give 300 mg of product: mp 136-137 ° C; NMR δ 1.29 (t, 3H), 3.69 (m, 4H), 3.71 (m, 4H), 4.18 (q, 2H), 6.99 (d, 1H), 8.52 (d, 1H); (UV) λ _max (ε) 270 nm (4.1 mM ⁻¹ cm ⁻¹ ).
This compound is converted to 2,4-dimethoxypyrimidine by nucleophilic substitution using methoxide to replace the chlorine atom at the C2 position and the diazeniumdiolate at the C4 position.

Example 13
This example describes the synthesis of the following compounds.

General synthesis of compounds 1 to 5 : A 1M solution of sodium 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate in dimethyl sulfoxide was stirred at 5 ° C. under nitrogen. 0.95 molar equivalent of an arylating agent-containing tetrahydrofuran 1M solution was injected through a septum. The reaction mixture was warmed to room temperature, stirred overnight, quenched with ice water and extracted with ether. The ether was washed with water, dried over sodium sulfate, filtered through a magnesium sulfate layer and concentrated on a rotary evaporator. Although the purification method was changed in each preparation, it is described together with the following individual compounds (Note: Compounds 1 to 5 are O ² -aryl compound libraries constructed in parallel using a liquid phase synthesis method. More selected product). NMR spectra were measured in CDCl _3.

O ² - (2-nitro-4-trifluoromethylphenyl) 1-(N, N-diethylamino) diazen-ium-1,2-diolate, 1: 4-fluoro-3-nitro benzotrifluoride employed And arylated. The product was purified by preparative HPLC using a 1 inch C-18 column and eluting with a solvent gradient from 20% aqueous acetonitrile to 50% acetonitrile: 50% water. A 42% yield of the product was obtained as an oil: NMR δ 1.23 (t, 6H), 3.50 (q, 4H), 7.66 (d, 1H), 7.82 (d, 1H), 8.28 (s, 1H) .

O ² - (2-nitro-4-carboxyphenyl Lato phenyl) 1-(N, N-diethylamino) diazen-ium-1,2-diolate, 2: In this preparation of 4-fluoro-3-nitrobenzoic acid Using. The product was purified on a Biotage Flash 40 system using a 4.0 × 15.0 cm KP-Sil column. The system was eluted with 5: 1 dichloromethane: ethyl acetate at 15 psi pressure and an elution rate of 25 ml / min to give the product in 22% yield: mp 115-6 ° C; NMR δ 1.22 (t, 6H), 3.33 (q, 4H), 7.06 (d, 1H), 8.03 (dd, 1H), 8.37 (m, 1H).

O ² - (5-nitropyridin-2-yl) 1-(N, N-diethyl) diazen-ium-1,2-diolate, 3: 2-bromo-5 reaction product of a nitropyridine Ether: Recrystallized from ethanol to give pure 3 in 62% yield: mp 77-8 ° C; NMR δ 1.24 (t, 6H), 3.53 (q, 4H), 7.21 (dd, 1H), 8.52 (dd, 1H), 9.17 (dd, 1H). C, H, N analysis: calculated for _{C 9 H 13 N 5 O 4} : C 42.35%, H 5.13%, N 27.44%; Found: C 42.46%, H 5.14% , N 27.52%.

O ² - (3,5-dinitro-2-yl) 1-(N, N-diethyl) diazen-ium-1,2-diolate, 4: 2-chloro-3,5-nitropyridine using Annealation as described in general procedure. The crude product was recrystallized from ether: petroleum ether to give 4 in 33% yield: mp 56-7 ° C .; NMR δ 1.28 (t 6H), 3.57 (q, 4H), 8.81 (d, 1H ), 9.10 (d, 1H).

O ² - (3- nitropyridin-2-yl) 1-(N, N-diethyl) diazen-ium-1,2-diolate, 5: 2-chloro-3-nitropyridine was used in the reaction . The crude product was purified on a Flash 40 system using a 4.0 x 7.0 cm KP-Sil column eluting with 100% dichloromethane to give the product as a viscous oil in 52% yield: NMR δ 1.25 (t , 6H), 3.55 (q, 4H), 7.26 (m, 1H), 8.48 (m, 2H).

Example 14
This example illustrates the preparation of O ² -vinyl 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate (V-PROLI / NO).
O ² - (2-bromoethyl) 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate 2-bromoethyl ester 3.56 g (9.2 mmol) in 10 N hydroxide 10 ml of sodium solution was added.
When the biphasic mixture was stirred at 25 ° C., the compound gradually dissolved in the aqueous layer. After stirring overnight, the UV of the reaction mixture showed maximum absorption at 266 nm (starting material absorbed at 252 nm), indicating the formation of vinyl groups.
The solution was cooled to 0 ° C. and carefully acidified to pH 4 by slow addition of 10% hydrochloric acid. Care must be taken to keep the solution cold while adding the acid. The acidic solution was extracted with ethyl acetate, dried over sodium sulfate, and filtered through a magnesium sulfate layer. The solvent was distilled off to obtain 1.4 g of an oily product. Purification was done on a Flash 40 system with 4.0 x 7.0 cm KP-Sil column and 2: 1 ethyl acetate: cyclohexane as eluent: ir (film) 3163, 2987, 1734, 1630, 1490 cm ^-1 ;
NMR (CDCl ₃ ) δ 2.06-2.3 (m, 4H), 3.62 (m, 2H), 4.47 (q, 1H), 4.77 (ABq,
1H), 5.02 (ABq, 1H), 6.75 (q, 1H); UV λ _max (ε) 266 nm (6.3 mM ⁻¹ cm ⁻¹ ); MS, m / z (%) 201 (M ⁺ , 5) , 176 (10), 150 (49), 145 (27), 114 (9), 99 (45), 70 (99.9), 69 (57), 68 (45).

Example 15
In this example, NO is regenerated from O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate in the presence of glutathione, but is absent In the following, I will explain that it will not play.
A 10 mM phosphate buffer containing 1 mM glutathione (GSH) was purged with argon for 10 minutes and degassed, and then a 3 ml aliquot was O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino). ) Diazene-1-ium-1,2-diolate was mixed with 3 μl of a 2 mM dioxane solution. The release of NO was monitored by chemiluminescence while maintaining the mixture at 37 ° C. After a short lag time, a peak of nitric oxide evolution was observed about 15 minutes after the start of the reaction, followed by an easily detectable level for about 100 minutes. Total NO generation during the first 112 minutes was about 9 nmol. Assuming that 2 nmol of NO per mol of O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate is generated, this 9 nmol is This corresponds to about 75% of the total yield.
Generation of NO was not observed when the reaction was repeated as described above except that GSH was removed. Nucleophilic glutathione reacted with O ^2- (2,4-dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate to produce NO according to the following formula.

This embodiment, O ² of the present invention - that several aryl diazeniumdiolate compounds, often found in the active site of the enzyme, which may be a nucleophilic substitution by nucleophilic side chains of amino acids such as cysteine Is shown. As a result of such nucleophilic substitution, an aryl derivative substituted with an amino acid residue and diazeniumdiolate capable of producing NO is generated through a predictable primary reaction.
O ^2- (2,4-Dinitrophenyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate and glutathione were assayed in the presence and absence of glutathione S-transferase. . The assay was performed in a final volume of 3 ml using 0.1 M phosphate buffer (pH 7.4) in a cell compartment temperature controlled at 25 ° C. The enzyme concentration was 0.7 μg / ml, while the glutathione concentration was 1.4 mM. The diazeniumdiolate concentration was varied from 50-100 μM. Using the integral type of the Henri-Michaelis-Menten equation, it was found that K _m was 46.3 μM and V _max was 0.89 μM min ⁻¹ .

Example 16
This example describes a synthetic route useful for the production of diazeniumdioled nucleotides, nucleosides and nucleic acids, and further converts the diazonium group to a diazeniumdiolate after converting the amino group to a diazonium group. A synthesis route of O ² -aryl diazeniumdiolates including reacting with azobenzene will be described.
2'-deoxycytidine is reacted with nitric oxide in the presence of a suitable one-electron oxidant that results in the conversion of the cytidine amino group to a diazonium group (the oxidant is reduced and hydroxylated by the conversion). Product ions are generated). The resulting diazotized (ie, diazonium derivative) pyrimidine is then reacted with 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate ion as described in the previous examples, This produces a diazeniumdiolate 2'-deoxyuridine derivative. This diazeniumdiolate 2′-deoxyuridine derivative can react with strong nucleophiles (eg, hydroxide ions). This will regenerate 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate ion and 2′-deoxyuridine. This regenerated 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate ion will generate NO through a predictable primary reaction. This example demonstrates the principle of a mechanism that is suitable for targeting nitric oxide to specific biological sites in mammals and increasing the specificity of NO action.

Example 17
This example demonstrates that O ² -aryl diazeniumdiolate can inactivate zinc finger proteins by the release of zinc.
Samples of recombinant nucleocapsid protein p7 (p7NC) from HIV-1 (LO Arthur, AIDS Vaccine Program, NCI-FCRDC, Frederick, MD) in μg / ml in 10 mM sodium phosphate buffer (pH = 7.0) And treated with 25 μmol O ² -aryldiazeniumdiolate (in a total volume of 1.0 ml). As shown in FIG. 1, which is a graph of Trp37 fluorescence (RFU) versus time (minutes), the sample was washed with 10 mM sodium phosphate buffer at various time intervals to prevent the introduction of any artificial quenching effect. Fluorescence intensity of tryptophan residue (Trp37) in the C-terminal zinc finger of p7NC in each sample was measured as previously described (Rice et al., Int Antiviral News 3: 87-89 (1995)). The excitation and emission wavelengths utilized in the Shimadzu RF500O fluorescence spectrometer were 280 and 351 nm, respectively. The results are shown in FIG. In the figure, ○ represents a negative control, that is, no drug, □ represents a positive control, that is, 642151 (Rice et al. (1997), see above), ■ represents the compound of Example 1 (LK1), and ◆ represents The compound (LK2) of Example 8 is represented, ▲ represents the compound (Lk3) of Example 5, ● represents the compound (Lk4) of Example 10, and x represents the compound of Example 11 (LK5). Results O ² - aryl diazeniumdiolate indicating that can release zinc from zinc finger proteins.

Example 18
This example demonstrates the anti-HIV activity of O ² -aryl diazeniumdiolates.
A cancer cell line of T4 lymphocytes called CEM-SS was grown in a synthetic medium containing fetal calf serum (Rice et al., Advances in Pharmacol. 33: 389-438 (1995)). According to the National Cancer Institute's XTT-based cell viability assay (see, for example, Rice et al. (1995), supra), O ² -aryl diazeniumdiolates were infected and uninfected with HIV-1 CEM-SS cells were administered at a concentration range of 10 ^−3.5 to 10 ^−7.0 M.
After exposing CEM-SS cells to the compounds, the percent T cell survival was examined. HIV-1-infected CEM-SS cells contacted with any of the above toxic concentrations of the above O ² -aryl diazeniumdiolates also substantially increased viability compared to untreated cells. Compounds 1-3 from Example 13 were particularly effective.

Example 19
This example describes the preparation of disodium 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate.
A solution of 10 g (0.087 mol) of L-proline in 39 ml (0.18 mol) of 25% sodium methoxide (in methanol), 20 ml of methanol and 40 ml of ether is degassed to 20 psi of nitric oxide at 40 psi. Time exposure. The pressure was released and the solid residue was collected by filtration, washed with ether and dried in vacuo to give 17 g of white solid: mp 250 ° C. (decomposition); UV (0.01 N NaOH) λ _max (ε) 252 nm (8.4 mM ⁻¹ cm ⁻¹ ); NMR (D ₂ 0) δ 1.71 (m, 1H), 1.91 (m, 2H), 2.27 (m, 1H), 3.27-3.43 (m, 2H), 4.04 (m, 1H (A singlet of methanol at 3.34 is also observed); ¹³ C NMR, 24.45 ppm, 30.97, 48.73 (methanol), 54.95, 67.70, 182.75.
C, H, N analysis: Calculated as C ₅ H ₇ N ₃ O ₄ Na ₂ · CH ₃ OH: C 28.69%, H 4.41%, N 16.73%, Na 18.30%; Found: C 28.65%, H 3.99%, N 16.74%, Na 18.04%.

Example 20
This example describes the preparation of O ² -methyl 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate methyl ester.
Disodium 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate (methanol solvate, FW 251; 6.8 g; 0.027 mol) in a 300 ml 3-neck flask And cooled to -20 ° C. Cold methanol (−20 ° C .; 200 ml) was added to the solid with stirring to obtain a homogeneous solution, which was further cooled to −35 ° C. A solution of 9.5 ml (0.1 mol) of dimethyl sulfate in 25 ml of ether was added dropwise over 15 minutes. The reaction mixture was then gradually warmed to room temperature and stirred for an additional 4 hours. The progress of the reaction was monitored by silica gel TLC using 10: 1 dichloromethane: ether as the developing solution. The reaction mixture was filtered, methanol was removed on a rotary evaporator and the residue was extracted with dichloromethane. The solution was washed with aqueous sodium bicarbonate, dried over sodium sulfate, and filtered through a magnesium sulfate layer. The solvent was distilled off to obtain an oily substance, which was allowed to stand for crystallization. Ether: Recrystallized from petroleum ether to give 945 mg (18%) of analytically pure sample: mp 62-63 ° C; UV (0.01 N NaOH), λ _max (ε) 252 nm (6.79 mM ^{− 1} cm ⁻¹ ); NMR δ2.05 (m, 3H), 2.30 (m, 1H), 3.65 (m, 1H), 3.75 (s, 3H), 3.83 (m, 1H), 3.96 (s, 3H) , 4.55 (m, 1H); MS m / z (%) 203 (M ⁺ , 6), 188 (20), 58 (35), 120 (22), 99 (100), 95 (34), 69 ( 36), 59 (24); exact mass calculated as C ₇ H ₁₃ N ₃ 0 ₄ (M ⁺ ) 203.0906, found (M ⁺ ) 203.0906.
C, H, N analysis: Calculated as C ₇ H ₁₃ N ₃ 0 ₄ C 41.38%, H 6.45%, N 20.68%: Found C 41.48%, H 6.43%, N 20.59%.

Example 21
This example describes the preparation of O ^2- (N, N-dimethylsulfamoyl) 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate.
A solution of 1.08 ml (0.01 mol) of N, N-dimethyl-sulfamoyl chloride in 5 ml of tetrahydrofuran was added to disodium 1-[(2-carboxylato) pyrrolidine-1 in 25 ml of 0.1 N NaOH (saline solution). -Il] diazen-1-ium-1,2-diolate was added to a cold (0 ° C.) solution of 1.57 g (0.0062 mol). The reaction mixture was warmed to room temperature and stirred overnight. The aqueous layer was extracted with dichloromethane, and the organic layer was dried over anhydrous sodium sulfate. After extraction, the aqueous layer did not show significant UV absorption, thus indicating that the extract was free of diazeniumdiolate. The organic layer was filtered through a magnesium sulfate layer, and the solvent was removed by a rotary evaporator to obtain 989 mg of a pale yellow oily substance, which was subjected to silica gel chromatography using 5: 1 dichloromethane: ethyl acetate as an eluent. Fractions containing the desired product were combined and concentrated in vacuo to give a solid which was recrystallized from ether: petroleum ether: mp 97-98 ° C; UV (0.01 N NaOH) λ _max (ε) 266 nm (8.05 mM ^-1 cm ^-1 ); NMR δ 2.16 (m, 3H), 2.40 (m, 1H), 3.01 (s, 6H), 3.83 (m, 1H), 3.94 (m, 1H), 4.69 ( q, 1H), 6.80 (b, 1H).
C, H, N, S analysis: Calculated as C ₇ H ₁₄ N ₄ S0 ₆ : C 29.79%, H 5.00%, N 19.85%, S 11.36%: Actual measurement C 29.93%, H 5.09%, N 19.76 %, S 11.27%.

Example 22
This example describes the preparation of O ² -methoxymethyl 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate methoxymethyl ester.
A slurry of disodium 1-[(2-carboxylato) pyrrolidin-1-yl] diazene-1-ium-1,2-diolate 485 mg (1.93 mmol) in 20 ml of anhydrous tetrahydrofuran was cooled to 0 ° C. under a nitrogen atmosphere. . After adding triethylamine (0.5 ml) to the cold solution, 360 mg (4.45 mmol) of chloromethyl methyl ether was slowly added, followed by dropwise addition of 0.5 ml of methanol. The solution was then stirred for 1.5 hours with cooling. The reaction mixture was warmed to room temperature and stirred for an additional 1.5 hours under nitrogen. After stopping the reaction with crushed ice, the solvent was removed on a rotary evaporator and the residue was extracted with dichloromethane. The organic layer was washed with water, dried over sodium sulfate, filtered through magnesium sulfate and evaporated in vacuo to give 330 mg of a yellow oil, which was silica gel with 5: 1 dichloromethane: ethyl acetate as eluent. Purified on column: UV (H ₂ 0) λ _max (ε) 250 nm (8.58 mM ⁻¹ cm ⁻¹ ); NMR δ 2.09 (m, 3H), 2.35 (m, 1H), 3.48 (s, H) , 3.71 (m, 2H), 3.90 (m, 1H), 4.61 (dd, 1H), 5.17 (ab q, 2H), 5.31 (ab q, 2H).
C, H, N analysis: Calculated as C ₉ H ₁₇ N ₃ 0 ₆ : C 41.06%, H 6.51%, N 15.96%: found C 40.87%, H 6.53%, N 15.76%.

Example 23
In this ^embodiment, O 2 - (2-bromoethyl) 1 - describes [(2-carboxylato) pyrrolidin-1-yl] Preparation of diazen-ium-1,2-diolate 2-bromoethyl ester.
A solution of 20 ml (0.28 mol) of bromoethanol in 50 ml of dichloromethane was cooled to 0 ° C. and a solution of 11.25 ml (0.28 mol) of sulfuryl chloride in 50 ml of dichloromethane was added dropwise to the solution. The resulting solution was kept at 4 ° C. for 72 hours. The solution was washed with cold 10% NaOH until the wash was clearly basic as a result of the test. The organic layer was dried over sodium sulfate, filtered through a magnesium sulfate layer and concentrated on a rotary evaporator. The resulting crude product (2-bromoethoxysulfonyl chloride, BrCH ₂ CH ₂ OSO ₂ Cl) was vacuum distilled to give 35 g (56%) of a colorless oil: boiling point (at 1.5 mmHg) 73-75 ° C; NMR δ 3.64 (t, 2H), 4.752 (t, 2H); MS m / z (%) 221 (M ⁺ , 1), 143 (10), 129 (25), 106 (100), 93 ( 62). C, H, N, S, X Analysis: C ₂ H ₄ SO ₃ calculated for ClBr: C 10.75%, H 1.80 %, S 14.35%, 31.72% as 71.52% and Cl the total halogen as Br; Found : C 10.82% H 1.80%, S 14.35%, 71.63% of total halogen as Br and 31.78% as Cl.
Disodium 1-[(2-carboxylato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate (4.86 g; 0.0194 mol) is placed in a 100 ml round bottom flask with 2.2 g of anhydrous sodium carbonate. It was. The flask was immersed in a dry ice-acetonitrile bath (−40 ° C.) and 50 ml of cold (−20 ° C.) ethanol was added. The mixture was then stirred and stabilized at −40 ° C. under a nitrogen atmosphere. To the cold slurry, 9.45 g (0.0422 mol) of 2-bromoethoxysulfonyl chloride was added by syringe over 10 minutes. After stirring for 2 hours, the reaction mixture was warmed to 15 ° C. and stirred for an additional 2 hours. The reaction mixture was poured into 250 ml of ice water and extracted with dichloromethane. The organic layer was washed with an aqueous sodium hydrogen sulfite solution, dried over sodium sulfate, filtered through a magnesium sulfate layer, and then the solvent was removed with a rotary evaporator. The crude product was subjected to silica gel column chromatography using 1: 1 cyclohexane: ethyl acetate as eluent to give 2.7 g (36%) of yellow oil: NMR δ 2.11 (m, 3H), 2.35 ( m, 1H), 3.55 (m, 4H), 3.68 (m, 1H), 3.86 (m, 1H), 4.46 (m, 4H), 4.59 (m, 1H); UV (H ² 0) λ _max (ε ) 252 nm (6.6 mM ⁻¹ cm ⁻¹ ).

Example 24
In this ^embodiment, O 2 - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl The preparation of the-(2-mercaptoethyl)] ester is described.
1,8-diazabicyclo [5.4.0] undec-7-ene (DBU, 1.03 g; 0.0068 mol ) and, ^O in tetrahydrofuran 35 ml 2 - (2-bromoethyl) 1 - [(2-carboxylato) Pyrrolidin-1-yl] diazen-1-ium-1,2-diolate 2-bromoethyl ester was added to a solution of 1.33 g (0.0034 mol) and the resulting solution was stirred at room temperature under nitrogen. Two equivalents of thiolacetic acid (0.479 ml, 0.0068 mol) were added and the mixture was stirred at room temperature for 2 hours. The mixture was filtered and the solid residue was washed with ether. The filtrate was evaporated and dried under reduced pressure, and the residue was extracted with methylene chloride. The organic layer was washed successively with ice-cooled 5 N HCl, aqueous sodium bicarbonate and water. The solution was dried over sodium sulfate, filtered through a magnesium sulfate layer and evaporated in vacuo to give 710 mg of a yellow oil. Chromatography was performed on a silica gel column, eluting with 1: 1 cyclohexane: ethyl acetate: UV (H ₂ 0) λ _max (ε) 232 nm (7.0 mM ⁻¹ cm ⁻¹ ); NMR δ 2.09 (m, 3H ), 2.36 (m, 1H), 2.38 (s, 6H), 3.09 (m, 4H), 3.78 (m, 2H), 4.27 (m, 4H), 4.55 (m, 1H).

Example 25
This example describes the determination of the half-life of the compound prepared in Example 24 at 25 ° C. and pH 7.4 in the absence and presence of porcine liver esterase.
0.009 M O ² - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl - (2 -Mercapto-ethyl)] ester in ethanol stock solution was prepared. The decay of this compound was monitored at 25 ° C. as a 1.5 × 10 ⁻⁴ M solution in a 4 ml quartz cuvette containing 3 ml phosphate buffer (pH 7.4) and 50 ml stock solution. The disappearance of the 232 nm chromophore was monitored with an ultraviolet spectrophotometer. The half-life was estimated to be 3.2 hours.
A second set of experiments was performed using the above parameters, and the decay after adding 5 ml of porcine liver esterase suspension was measured. The half-life for the esterase reaction was 8 minutes at 25 ° C.

Example 26
In this ^embodiment, O 2 - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl The preparation of a nitric oxide releasing polymer mixture of-(2-mercaptoethyl)] ester is described.
^O 2 in tetrahydrofuran 1 ml - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl A solution of 50 mg (0.132 mmol) of-(2-mercaptoethyl)] ester was dissolved in a solution of 498 mg of polyurethane in 10 ml of tetrahydrofuran. After concentrating the uniform lacquer dry nitrogen gas flow, and further dried under high vacuum, the polymer composite 1 mg per O ² - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxy A solid containing 0.091 mg (0.24 mmol) of (lato) pyrrolidin-1-yl] diazen-1-ium-1,2-diolate [S-acetyl- (2-mercaptoethyl)] ester was obtained. The rate of NO release was measured at 37 ° C. using a chemiluminescence detector as a function of time after immersion of a 32 mg aliquot of diazeniumdiolate (in 2 ml phosphate buffer, pH 7.4). One set of experiments was performed in a buffer free of contaminants, while the other set was performed in the presence of porcine liver esterase. In the absence of enzyme, a very small amount of NO was released over 200 hours, but NO production was observed at a significant rate when the enzyme was present in the buffer. This indicates that as diazeniumdiolate leaks from the polymer composite, it is hydrolyzed enzymatically and further cleaved to NO.

Example 27
In this embodiment, beta-nitric oxide-releasing cyclodextrin ^O 2 - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen- The introduction of ium-1,2-diolate [S-acetyl- (2-mercaptoethyl)] ester is described.
β-cyclodextrin (228 mg, 0.201 mmol) was mixed with 2 ml of water and heated to 65 ° C. to obtain a homogeneous solution. The temperature ^solution, O 2 - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl -(2-Mercaptoethyl)] ester 76 mg (0.201 mmol) was added. A white precipitate formed upon mixing. The mixture was cooled to room temperature and the product was collected by filtration, washed with water and dried in vacuo to give 170 mg of product. 33 mg of ^O 2 - [S- acetyl - (2-mercaptoethyl)] 1 - [(2-carboxylato) pyrrolidin-1-yl] diazen-ium-1,2-diolate [S- acetyl - ( 2-mercaptoethyl)] ester: An aqueous solution containing a β-cyclodextrin mixture showed a maximum absorption at 232 nm and a molar extinction coefficient (ε) of 10.8 mM ⁻¹ cm ⁻¹ . The NO release rate was measured at 37 ° C. using a chemiluminescence detector as a function of time after immersion of a 13 mg aliquot of the encapsulated material (in 4 ml phosphate buffer, pH 7.4). One set of experiments was performed in a buffer free of contaminants, while the other set was performed in the presence of porcine liver esterase. In the absence of enzyme, very little NO was released over 400 hours, but when the enzyme was present in the buffer, NO production was observed at a significant rate.

Example 28
This example describes the general procedure for the preparation of O ² -glycosylated diazeniumdiolates.
2,3,4,6-Tetraacetyl-α-D-glucopyranosyl bromide (acetobromoglucose) was prepared as described in Redemann et al . , Org. Syn. Coll. Vol. III: 11-14 (1955). Prepared. 2,3,4,6-Tetraacetyl-α-D-mannopyranosyl bromide (acetobromomannose) was prepared as described in Levene et al., J. Biol. Chem. 90: 247-250 (1931) . Prepared. A slurry of 1 equivalent of diazeniumdiolate in dimethyl sulfoxide (DMSO) (0.5 mmol solid / 1 ml DMSO) was then stirred at room temperature under nitrogen with 0.03 equivalent of silver oxide. 1.2 equivalents of acetobromomannose or 0.5M solution of acetobromoglucose in DMSO was added dropwise and the mixture was stirred for 3 days. The obtained homogeneous solution was poured into 100 ml of ice water and extracted with ether. The ether layer was washed with water, dried over sodium sulfate and treated with charcoal. The solution was filtered through magnesium sulfate, concentrated on a rotary evaporator and dried in vacuo. The glucose derivative was purified by recrystallization, but the glassy mannose addition product required column chromatography.

Example 29
This example describes the preparation of sodium 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate ("DEA / NO").
A solution of 119 g (1.63 mol) diethylamine in 100 ml 1: 1 ether: acetonitrile was placed in a 500 ml Parr bottle. The solution was degassed and charged with 40 psi nitric oxide and left at room temperature overnight. The crystalline product was collected by filtration under reduced pressure and dried under nitrogen to give 13 g of diethylammonium 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate. This salt was treated with 10 ml of 10 M sodium hydroxide solution and the resulting paste was treated with 200 ml of ether to obtain the sodium salt. This sodium salt ("DEA / NO") was collected by vacuum filtration, washed with ether and dried in vacuo to give 7.1 g of product: UV (in 0.01 N NaOH) λ _max (ε) 250 (6.88 mM ⁻¹ cm ⁻¹ ); NMR (D ₂ 0) δ 0.96 (t, 3H), 2.94 (q, 2 H); δ 0.84 (t, 3 H) and 2.75 (q, 2 H) in DMSO-d ₆ .

Example 30
In this example, O ^2- (2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate The preparation is described.
DEA / NO (2.98 g; 0.019 mol) in DMSO was reacted with acetobromoglucose 6.9 g; 0.017 mol) as described in the general procedure of Example 28. The product was recrystallized from petroleum ether to give 108 mg of 5.7 g (72%) crystalline solid: mp 107-108 ° C .; UV λ _max (ε) 228 nm (6.92 mM ⁻¹ cm ⁻¹ ); NMR δ 1.11 (t, 6H, J = 7.11), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H), 2.07 (s, 3H), 3.21 (q, 4H, J = 7.12 ), 3.81 (m, 1H), 4.20 (m, 2H), 5.14 (m, 1H), 5.33 (m, 3H). Analysis: calculated for _{C 18 H 29 N 3 O 11} : C, 46.65; H, 6.31; N, 9.07. Found: C, 46.73; H, 6.26; N, 9.01.

Example 31
In this example, O ^2- (2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl) 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate ( The deacylation of Example 30) is described.
A solution of 253 mg (0.55 mmol) of the above compound in 5 ml of methanol was stirred with 10 μl of 25% methanolic sodium methoxide. The progress of the reaction was monitored by TLC using 5: 1 CH ₂ Cl ₂ : ethyl acetate. The reaction was complete within 1 hour at 25 ° C.
Dowex-50W-H ⁺ resin (1 g) was added to the stirring methanol solution. The mixture was filtered to remove the resin, the methanol solution was evaporated in vacuo and O ² -glucopyranosyl 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate 122 mg (75 %): UV λ _max (ε) 228 nm (6.4 mM ⁻¹ cm ⁻¹ ); NMR (CDCl ₃ ) δ 1.08 (t, 6H), 3.23 (9, 4H), 5.59 (m, 4H) , 3.88 (m, 2H), 5.29 (m, 1H)
Surprisingly, the deacylated product, despite its acetal-like structure, was very slowly at pH 3 1- (N, N-diethylamino) diazen-1-ium-1,2-diolate (DEA / NO) anion and then only cleavage to NO. Even more surprising, this cleavage proceeded very rapidly at pH 13.

Example 32
This example describes the preparation of sodium 1-[(1-ethoxycarbonyl) piperazin-4-yl] diazen-1-ium-1,2-diolate.
A solution of 20 g (0.126 mol) of carboethoxypiperazine in 60 ml of methanol was placed in a Parr bottle. The solution was treated with 27.4 ml (0.126 mol) of 25% sodium methoxide in methanol. The system was degassed and charged with 40 psi nitric oxide and held at 25 ° C. for 48 hours. The white crystalline product was filtered off and washed with cold methanol and copious amounts of ether. The product was dried in vacuo to give 14.5 g of sodium 1-[(1-ethoxycarbonyl) piperazin-4-yl] diazen-1-ium-1,2-diolate (48% yield): mp: 184 UV (0.01 N NaOH) λ _max (ε) 252 nm (10 mM ⁻¹ cm ⁻¹ ); NMR (D ₂ 0) δ 1.25 (t, 3H), 3.11 (m, 2H), 3.68 (m , 2H), 2.15 (q, 2H). _{Analysis: C 6 H 13 N 4 O} 4 calculated for Na: C 35.00%, H 5.42 %, N 23.33%, Na 9.58%. Found: C 34.87%, H 5.53%, N 23.26%, Na 9.69%. The half-life of this compound at pH 7 and 25 ° C. was estimated to be 5 minutes. This measurement was based on the disappearance of the 252 nm chromophore in the ultraviolet spectrum.

Example 33
In this ^{embodiment, O} 2 - (glucopyranose-2-yl) 1 - [(1-ethoxycarbonyl) piperazin-4-yl] describes the preparation of diazen-ium-1,2-dioleate tetraacetate ester To do.
Acetobromoglucose (2.055 g; 0.005 mol) and sodium 1-[(1-ethoxycarbonyl) piperazin-4-yl] diazen-1-ium-1,2-diolate 1.11 g (0.00466 mol) as described above. It reacted with ^{O 2} - (glucopyranose-2-yl) 1 - [(1-ethoxycarbonyl) piperazin-4-yl] diazen-ium-1,2-dioleate tetraacetate ester 624 mg (25%) Obtained: UV λ _max (ε) 228 nm (7.20 mM ⁻¹ cm ⁻¹ ); NMR δ 1.26 (t, 3H), 2.02 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H ), 2.09 (s, 3H), 3.46 (m, 4H), 3.68 (m, 4H), 3.82 (m, 1H), 4.17 (q, 2H), 4.25 (m, 3H), 5.27 (m, 3H) .

Example 34
In this ^{embodiment, O} 2 - (mannopyranose-2-yl) 1 - [(1-ethoxycarbonyl) piperazin-4-yl] describes the preparation of diazen-ium-1,2-dioleate tetraacetate To do.
Acetobromomannose (10.2 g; 0.025 mol) and sodium 1-[(1-ethoxycarbonyl) piperazin-4-yl] diazen-1-ium-1,2-diolate 5.28 g (0.022 mol) as described above. Reaction gave 6.4 g (53%) of glassy material: UV λ _max (ε) 238 nm (7.5 mM ⁻¹ cm ⁻¹ ); NMR δ 1.29 (t, 3H), 2.01 (s, 3H) , 2.05 (s, 3H), 2.11 (s, 3H), 2.17 (s, 3H), 3.13 (m, 1H), 3.50 (m, 4H), 3.78 (m, 5H), 4.19 (q, 2H), 4.27 (m, 3H), 5.28 (m, 3H), 5.42 (m, 1H).

Example 35
This example describes the preparation of O ² -glycosylated diazeniumdiolate directed to the mannose-fucose receptor.
Bis- [2- (N-ethoxycarbonylamino) ethyl] amine: A three-necked flask equipped with two dropping funnels was immersed in an ice-water bath. Diethylenetriamine (10.7 g, 0.104 mol) was placed in the cold flask and dissolved in 100 ml of 95% ethanol. To the cold solution was added dropwise 10 ml (0.205 mol) of ethyl chloroformate. A solution of 10.6 g (0.1 mol) sodium carbonate in 100 ml distilled water was added simultaneously with 10 ml (0.205 mol) ethyl chloroformate. The reaction mixture was stirred at room temperature overnight. The ethanol was removed on a rotary evaporator and the aqueous portion was extracted with dichloromethane. The organic layer was washed with water and then extracted with 5% hydrochloric acid. Separate the organic layer containing the neutral product. The aqueous layer was extracted with dichloromethane and made basic with sodium hydroxide. The product was extracted into dichloromethane, dried over sodium sulfate, filtered through magnesium sulfate and evaporated to give 4 g of a colorless oil: NMR (CDCl ₃ ) δ 1.25 (t, 6H), 2.78 (m, 4H), 3.36 (m, 4H), 4.14 (q, 4H), 5,13 (b, 2H).
Sodium 1- [bis- {2- (N-ethoxycarbonylamino) ethyl} amino] diazen-1-ium-1,2-diolate: bis- [2- (N-ethoxy in 20 ml ether and 5 ml methanol) Carbonylamino) ethyl] amine 2.6 g (0.011 mol) in 50 ml Parr bottle, treated with 25% methanolic sodium methoxide 2.4 ml (0.011 mol), degassed and cooled at -80 ° C. Charged with 50 psi of nitric oxide. A cloudy precipitate was observed after stirring for 3 hours. The mixture was exposed to NO for 24 hours, the pressure was released and the product was filtered. The solid was washed with ether and dried in vacuo to give 1.26 g (35%) of diazeniumdiolate: mp 170-2 ° C .; UV λ _max (ε) 252 nm (7.6 mM ⁻¹ cm ⁻¹ ); NMR δ 1.24 (t, 6H), 3.19 (m, 8H), 4.11 (q, 4H).
O ² - (mannose-2-yl) 1- [bis - {2- (N- ethoxycarbonyl amino) ethyl} amino] diazen-ium-1,2-dioleate tetraacetate: Dimethyl sulfoxide (DMSO) 2 A portion of a solution of sodium 1- [bis- {2- (N-ethoxycarbonylamino) ethyl} amino] diazen-1-ium-1,2-diolate 251 mg (0.763 mmol) in ml was added at 0 ° C. under nitrogen. Cooled to. To this was added 10 mg (0.06 mmol) of silver acetate, and then 1 ml of a 0.82 M acetobromomannose solution in tetrahydrofuran was slowly added. The reaction mixture was stirred for 48 hours at room temperature, poured into ice water and extracted with ether. The ether solution was dried over sodium sulfate, filtered through a magnesium sulfate layer and evaporated in vacuo to give 307 mg of an oil: UV λ _max 240 nm.
O ² - (mannose-2-yl) 1- [bis (2-aminoethyl) amino] diazen-ium-1,2-diolate]:
10 N NaOH 2 ml, ethanol 2 ml and water a 2 ml mixture of ^{O 2} - (mannose-2-yl) 1- [bis - {2- (N- ethoxycarbonyl amino) ethyl} amino] diazen- A solution of 145 mg (0.23 mmol) of ium-1,2-diolate tetraacetate was heated to reflux for 15 hours. The solution was concentrated under vacuum and the remaining aqueous solution was extracted with dichloromethane. The aqueous solution was evaporated to dryness under vacuum. The residue was taken up in methanol and passed through a 10 g, 60 cc Prepack C-18 column and eluted with methanol. Fractions showing maximum absorption at 236 nm were collected and the solvent was distilled off to obtain 32 mg of white powder: NMR (CD ₃ OD) δ 2.74 (t, 4H), 3.02 (t, 4H), 3.74 (m , 4H), 4.2 (m, 3H); UV λ _max 238 nm.

Example 36
This example describes the preparation of a combinatorial library using disodium 1- (2-carboxylato) pyrrolidin-1-yl diazene-1-ium-1,2-diolate (PROLI / NO) as a starting material. .
Piperazine trityl resin 1 available from Calbiochem-Novabiochem Int'l. (San Diego, Calif.) Is treated with sulfuryl chloride to form chlorosulfonamide 2. This resin is reacted with PROLI / NO to give compound 3. Free carboxylic acid can be activated to 4 by reaction with dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide. Nucleophilic addition of R ³⁰ XH (X═O, N, S) to resin-bound diazeniumdiolate provides a potentially large library of compound 5 substituted with the carboxylate group of the molecule . 5 base hydrolysis liberates anionic diazeniumdiolate 6 from the resin. This library 6 can be reacted with an electrophile ³¹ X to form a new set of compounds having structure 7.

  The publications, patents and patent applications cited herein are individually and specifically stated for each publication to be incorporated by reference, and are hereby incorporated by reference to the same extent as fully disclosed. It is to be incorporated.

  While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. It is contemplated that the present invention may be practiced other than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.

Claims (18)

Formula Ia:

[In the formula, _{^{X, - O 3 S-, - O-}} , amino, _polyamino, C 1 _{-C 24} aliphatic _group, from the group consisting of C 6 _{-C 30} aryl and _C 6 _{-C 30} non-aromatic ring group And R is a sugar that is attached to O ^{2 of the} diazeniumdiolate by the 2-position of the pyranose or furanose ring. However, when X is a sugar, it is not bonded to N ^{1 of the} diazeniumdiolate by the 2-position of the pyranose ring or furanose ring. ]
O ² -glycosylated 1-substituted diazene-1-ium-1,2-diolate.   The diazeniumdiolate of claim 1, wherein the sugar is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides.   The diazeniumdiolate of claim 2, wherein the disaccharide is sucrose or maltose.   The diazeniumdiolate of claim 2, wherein the monosaccharide is mannose, fucose or glucose.   The diazeniumdiolate of claim 1, wherein the sugar is a recognition sequence for receptor-mediated cellular events.   The diazeniumdiolate of claim 1, wherein X is an amino group attached to the nitrogen of the diazeniumdiolate through a nitrogen atom. X is, - [N (NO) O -], halo, hydroxy, alkylthio, alkoxy, aryloxy, amino, cyano, sulfonato, mercapto, _nitro, C 1 _{-C 12} aliphatic _radical, C 3 -C ₈ cycloalkyl C ₃ -C ₈ heterocycloalkyl, C ₃ -C ₁₂ olefinic group, benzyl, phenyl, benzylcarbonyl, phenylcarbonyl, sugar, phosphono, phosphato, and one or more oxygen atoms are independently S or NR ¹ (wherein R ¹ is a C ₁ -C ₈ aliphatic group, C ₃ -C ₈ cycloalkyl, benzyl, phenyl or R ¹⁸ C═N (OH) (wherein R ¹⁸ is a C ₁ -C ₁₀ aliphatic) A diazo group of any one of claims 1-6 substituted with a group selected from the group consisting of phosphato Bromide Geo rate. formula
[Wherein b and d may be the same or different and may be 0 or 1, and R ¹ , R ² , R ³ , R ⁴ and R ⁵ may be the same or different and represent hydrogen, C ₃ -C _{Composed of 8} cycloalkyl, C _1-12 linear or branched alkyl, benzyl, benzoyl, phthaloyl, acetyl, trifluoroacetyl, p-toluyl, t-butoxycarbonyl and 2,2,2-trihalo-t-butoxycarbonyl Selected from the group and i, j and k are the same or different and are integers of 2 to 12. ]
The diazeniumdiolate of any one of claims 1-7. X is R ¹⁹ R ²⁰ N— and R ¹⁹ and R ²⁰ are the same or different and are hydrogen, C _1-12 linear alkyl, C _3-12 branched alkyl, or C _2-12 linear Or a C _3-12 branched olefin group, wherein R ¹⁹ and R ²⁰ may be substituted with an alkoxy, acyloxy, acylthio, hydroxy, halo or benzyl group, or
R ¹⁹ and R ²⁰ together with the nitrogen atom to which they are attached,
Wherein A is NH, O or S, w is 1-12, y is 1 or 2, z is 1-5, R ⁸ is hydrogen, C _1-8 straight Chain alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl, aryl or carboxylato, and R ⁹ is hydrogen, C _1-6 straight chain alkyl or C _3-6 branched chain alkyl. The diazeniumdiolate according to any one of claims 1 to 7, which forms a heterocyclic ring selected from the group consisting of: R ¹⁹ R ²⁰ N-is _{N (CH 2 CH 2 NH 2} ) 2, and the sugar is fucose or mannose, diazeniumdiolate of claim 9.   Sugar, pentose or hexose, ribose, deoxyribose, lactose, galactose, fructose, glucosamine, glucose, mannose, fucose, galactosamine or glucuronic acid, one of which is phosphorylated, 3,5-cyclophosphorylated or polyphosphorylated The diazeniumdiolate of claim 1, wherein Glucosamine has the structure:
Or
Wherein R ¹² and R ¹³ may be the same or different and are hydrogen, C _1-6 alkyl, acyl, phosphate, sulfate, peptide or protein;
R ¹⁴ is X ¹ R ¹⁵ R ¹⁶ (wherein X ¹ is N, O or S, and when X ¹ is N, R ¹⁵ and R ¹⁶ are independently hydrogen, C _1-24 alkyl, C _{3-24 cycloalkyl, C 2-24} olefinic group, the aryl or R ¹⁵ and ^{R 16,} are,

Wherein A is NH, O or S, w is 1-12, y is 1 or 2, z is 1-5, R ⁸ is hydrogen, C _1-8 linear Alkyl, C _3-8 branched chain alkyl, C _3-8 cycloalkyl, aryl or carboxylato, and R ⁹ is hydrogen, C _1-6 straight chain alkyl or C _3-6 branched chain alkyl.) form a heterocyclic ring selected from the group consisting of, when X ¹ is O or S, not present R ¹⁶ groups. ). ]
The diazeniumdiolate of claim 11 having   Aryl is acridine, anthracene, benzene, benzofuran, benzothiophene, benzoxazole, benzopyrazole, benzothiazole, carbazole, chlorophyll, cinnoline, furan, imidazole, indole, isobenzofuran, isoindole, isoxazole, isothiazole, isoquinoline, naphthalene , Oxazole, phenanthrene, phenanthridine, phenothiazine, phenoxazine, phthalimide, phthalazine, phthalocyanine, porphine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrocholine, pyrrole, quinolizinium ion, quinoline, quinoxaline, quinazoline, Sidnone, tetrazole, thiazole, thiophene, thyroxine, triazine It is selected from the group consisting of fine-triazole, diazeniumdiolate of claim 12. formula
[Wherein D is
(Wherein R ²¹ is a sugar bonded to O ^{2 of the} diazeniumdiolate by the 2-position of the pyranose ring or furanose ring), f is an integer of 0-12, and R ¹⁰ and R ¹¹ may be the same or different and are hydrogen, C ₃ -C ₈ cycloalkyl, C ₁ -C ₁₂ linear or C ₃ -C ₁₂ branched alkyl, benzyl, benzoyl, phthaloyl, acetyl, tolu It can be fluoroacetyl, p-toluyl, t-butoxycarbonyl or 2,2,2-trihalo-t-butoxycarbonyl. ]
O ² -glycosylated 1-substituted diazene-1-ium-1,2-diolate. (I) any of the diazeniumdiolates of claims 1-4 , or (ii) formula Ia:
[Wherein X and R are sugars bonded to O ^{2 of} diazeniumdiolate by the 2-position of the pyranose ring or furanose ring. ]
A composition for releasing nitric oxide under physiological conditions, comprising a diazeniumdiolate, and a carrier. A composition for treating or preventing a biological disorder in an animal that can be treated or prevented with nitric oxide,
(I) any of the diazeniumdiolates of claims 1-4 , or (ii) formula Ia:
[Wherein X and R are sugars bonded to O ^{2 of} diazeniumdiolate by the 2-position of the pyranose ring or furanose ring. ]
A composition comprising a diazeniumdiolate, and a carrier. 17. The composition of claim 16 , wherein the biological disorder is selected from the group consisting of infection, inflammation, metastasis, fulminant liver failure, malaria, respiratory disorder, impotence, cardiovascular disorder, and blood disorder. A composition for inactivating or inhibiting a protein in an amount sufficient to inactivate or inhibit the protein,
(I) any of the diazeniumdiolates of claims 1-4 , or (ii) formula Ia:
[Wherein X and R are sugars bonded to O ^{2 of} diazeniumdiolate by the 2-position of the pyranose ring or furanose ring. ]
A composition comprising a diazeniumdiolate, and a carrier.