WO2015108835A1 - 3,3-disubstituted-1-hydroxytriaz-1-ene 2-oxides and wound-healing compositions using them - Google Patents

3,3-disubstituted-1-hydroxytriaz-1-ene 2-oxides and wound-healing compositions using them Download PDF

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WO2015108835A1
WO2015108835A1 PCT/US2015/011100 US2015011100W WO2015108835A1 WO 2015108835 A1 WO2015108835 A1 WO 2015108835A1 US 2015011100 W US2015011100 W US 2015011100W WO 2015108835 A1 WO2015108835 A1 WO 2015108835A1
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oxide
ene
ethoxy
oxo
diazene
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PCT/US2015/011100
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French (fr)
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Eduardo J. Martinez
John J. Talley
Kevin D. Jerome
Terri L. Boehm
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Sarmont Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D205/00—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom
    • C07D205/02—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D205/04—Heterocyclic compounds containing four-membered rings with one nitrogen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/36—Oxygen or sulfur atoms
    • C07D207/40—2,5-Pyrrolidine-diones
    • C07D207/404—2,5-Pyrrolidine-diones with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention relates to the use of esters, carbonates and imides of 3,3-disubstituted- 1-hydroxytriaz-l-ene 2-oxides that release nitric oxide (NO) under physiologic conditions to aid in wound healing.
  • NO nitric oxide
  • Nitric oxide is an important endogenous signaling molecule and vasodilator. NO is synthesized from L-arginine by the enzyme NO synthase (NOS), which exists in three distinct isoforms, namely the constitutively expressed endothelial (eNOS) and neuronal (nNOS) forms, and the mainly inducible form (iNOS). NOS exhibits a number of important pharmacological actions including vascular relaxation (vasodilatation) and inhibition of platelet aggregation and adhesion. Inhibition of NO synthesis leads to an increase in systemic blood pressure.
  • NOS NO synthase
  • nNOS neuronal
  • iNOS mainly inducible form
  • NO also prevents atherogenesis by inhibiting vascular smooth muscle cell proliferation and preventing low-density lipoprotein oxidation and macrophage activation.
  • Vascular NO generation is important in controlling blood pressure, and a growing body of evidence indicates that NO signaling is a key factor in counteracting the onset and development of several CV diseases including hypertension, myocardial infarction and stroke.
  • Compositions that release NO under physiologic conditions have been the subject of numerous articles and patents. For example US patent 5,814,666 discloses so-called nonoate salts for the treatment of infectious diseases caused by pathogenic microbes.
  • the invention relates to compounds of formula:
  • R is H or (Ci-C 6 )alkyl
  • R is chosen from
  • R may additionally be:
  • R 3 and R 4 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
  • R 3 and R 4 form an optionally substituted 4- to 7-membered ring;
  • R 5 and R 6 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
  • R 10 is (Ci-C 6 )alkyl.
  • the invention in another aspect, relates to methods for treating cutaneous injuries comprising bringing a cutaneous injury into contact with a compound as described herein.
  • the invention relates to pharmaceutical compositions containing a pharmaceutically acceptable carrier and a compound as described herein.
  • compositions that release NO under physiologic conditions have been the subject of numerous articles and patents.
  • the instant invention relates to a novel genus of chemical compounds that release NO under physiologic conditions. They have the generic structure I:
  • R is:
  • R 3 is saturated (Ci-Cio)hydrocarbyl and R 4 is (Ci- Cio)hydrocarbyl substituted with-OH or -COOH; in others, R 3 and R 4 taken together with the nitrogen to which they are attached, form an optionally substituted 4- to 7-membered ring, for example a hydroxy-substituted 4- to 7-membered ring or an unsubstituted, saturated heterocycle.
  • Ci to C 2 o hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthylethyl. Hydrocarbyl refers to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched.
  • aliphatic hydrocarbyl substituents examples include isopropyl, 2-butenyl, 2- butynyl, cyclopentyl, norbornyl, etc.
  • Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
  • alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof.
  • Alkyl refers to alkyl groups from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
  • Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms.
  • Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, norbornyl and the like.
  • carbocycle is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state.
  • C 3 -C 10 carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene;
  • C 8 -C 12 carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene.
  • Carbocycle if not otherwise limited, refers to monocycles, bicycles and polycycles.
  • Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S.
  • the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized.
  • a heterocycle may be non-aromatic (heteroaliphatic) or aromatic (heteroaryl). Examples of heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline,
  • tetrahydroisoquinoline benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like.
  • heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and tetrahydroquinolinyl.
  • Alkoxy or alkoxyl refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms of a straight or branched configuration attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower-alkoxy refers to groups containing one to four carbons. For the purpose of this application, alkoxy and lower alkoxy include
  • halogen means fluorine, chlorine, bromine or iodine atoms. In one embodiment, halogen may be a fluorine or chlorine atom.
  • substituted refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical.
  • Oxo is also included among the substituents referred to in "optionally substituted”; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl).
  • 1, 2, or 3 hydrogen atoms are replaced with a specified radical.
  • more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine.
  • substituents are halogen, haloalkyl, alkyl, acyl, hydroxyalkyl, hydroxy, alkoxy, haloalkoxy, aminocarbonyl oxaalkyl, carboxy, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino arylsulfonyl, arylsulfonylamino, and benzyloxy.
  • halogen (Ci-C 4 )alkyl, halo(Ci-C 4 )alkyl, (Ci-C 4 )alkoxy, halo(Ci-C 4 )alkoxy, and aminocarbonyl.
  • the compounds described herein may contain, in a substituent R x , double bonds and may also contain other centers of geometric asymmetry; unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included.
  • the compounds may also contain, in a substituent R x , one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-.
  • the present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms.
  • Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
  • pharmaceutically acceptable salt refers to salts whose counter ion derives from pharmaceutically acceptable non-toxic acids and bases. Suitable
  • acids for salts of the amino-substituted compounds of the present invention include, for example, acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic,
  • Suitable pharmaceutically acceptable base addition salts for the carboxylate-substituted compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, ⁇ , ⁇ '- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.
  • the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • a plurality of molecules of a single structure may include at least one atom that occurs in an isotopic ratio that is different from the isotopic ratio found in nature.
  • Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include H, 3 H, U C, 13 C, 14 C, 15 N, 35 S, 18 F, 36 C1, 125 1, 124 I and 131 I respectively.
  • Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention.
  • Tritiated, i.e. 3 H, and carbon- 14, i.e., 14 C, radioisotopes are particularly preferred for their ease in preparation and detectability. Persons skilled in the art recognize that deuterium has been used to improve metabolic stability of compounds, and that principle can be applied to these compounds.
  • Radiolabeled compounds of formulae I and II of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent. [0028] Although this invention is susceptible to embodiment in many different forms, preferred embodiments of the invention are shown. It should be understood, however, that the present disclosure is to be considered as an exemplification of the principles of this invention and is not intended to limit the invention to the embodiments illustrated.
  • treatment or “treating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit.
  • Therapeutic benefit includes accelerating the process of wound-healing. Inasmuch as NO is known to possess antimicrobial properties, a medicament that liberates therapeutically useful concentrations of NO would both decrease pathogen load and stimulate one or more of the overlapping phases of wound healing: clotting, inflammation, cell recruitment and proliferation, revascularization, epithelium formation including collagen deposition, and tissue remodeling.
  • Criteria for assessing the suitability of nitric oxide releasing pro-drug derivatives include: (1) thermal stability to accommodate common environments, (2) sufficient nitric oxide release to achieve in vivo efficacy, and (3) non-spontaneous activation to improve effectiveness of nitric oxide administration. It will be noted that nitric oxide release can vary among chemical species and subgenera in the genus described herein. The more rapid release species are better suited for bolus delivery of medicament, whereas the species whose activation is slower are better suited to sustained-release formulations. Molecules that are activated by endogenous enzymes released by the wound provide one approach to activation, but it is also possible to add stable exogenous enzymes to a topical formulation to modulate nitric oxide release and performance.
  • the simplest embodiment of a medicament utilizes the esterases and amidases that are already present at the wound to activate the nitric oxide releasing pro-drugs. Diffusion of the compounds described herein from the bandage or salve to the wound provide the base-line nitric oxide release kinetics. Under circumstances in which endogenous enzymes in the wound are not sufficient to elicit efficacious release of nitric oxide from compounds in a bandage or salve, exogenous stable enzymes, often lyophilized, may be mixed into a bandage, such that wetting initiates the nitric oxide release process.
  • pro-drugs can be activated by spraying or wetting the bandage with an enzyme solution similar to products used in the eye care industry for cleaning contact lenses.
  • a precursor of NO would exhibit stability at 40 °C for more than 4 months.
  • Compounds with lower stability are nonetheless useful, but greater reliance would be placed on formulation and on controlling storage conditions. Assuming inherent stability is good, NO release per mole of test compound greater than 50% in a 90-minute test is advantageous for rapid-release or bolus type medication, whereas low percent release of NO in the 90 minute test indicates potential utility for extended duration medicaments.
  • the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants that are in themselves known, but are not mentioned here.
  • the starting materials are either commercially available, synthesized as described in the examples or may be obtained by the methods well known to persons of skill in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field.
  • a protecting group refers to a group, which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable.
  • the protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or "deprotection” occurs after the completion of the reaction or reactions in which the functionality would interfere.
  • Silyl groups e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl(TOM) and triisopropylsilyl (TIPS)
  • TMS trimethylsilyl
  • TDMS tert-butyldimethylsilyl
  • TOM tri-iso- propylsilyloxymethyl
  • TIPS triisopropylsilyl
  • Trityl (triphenylmethyl, Tr)
  • N-Boc-protected amino alcohols to N-alkylamino alcohols: (S)-2- Boc-alaninol, (S)-2-Boc-leucinol, (S)-2-(Boc-amino)-3-phenyl-l-propanol, (S)-2-Boc-3- methyl- 1-butanol, and (S)-2-Boc-isoleucinol are converted to (5)-2-N-methyl-alaninol, (S)- 2-N-methyl-leucinol, (5)-2-(N-methylamino)-3 -phenyl- 1 -propanol, (S)-2-N-methyl-3- methyl- 1-butanol, and (5)-2-N-methyl-isoleucinol) respectively using established procedures (See also Reddy, G.V., Rao, G.V., Sreevani, V.
  • the N-protected oxazoline (1 eq.) is dissolved in dry ACN under nitrogen atmosphere and treated with sodium cyanoborohydride (1 eq.) followed by trimethylsilyl chloride (1 eq.).
  • the reaction mixture is stirred at room temperature for 30 min and the solvent is evaporated under reduced pressure.
  • the residue is suspended in ethyl acetate and washed with a solution of 0.5 N sodium hydroxide.
  • the organic layer is separated and the aqueous layer is extracted again with ethyl acetate.
  • the combined organic layers are washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure.
  • the residue is purified by silica gel column chromatography to afford pure Boc-N-methyl amino alcohols.
  • Boc-Deprotection is accomplished using standard conditions stirring at room temperature in 5 : 1 - dichloromethane(dichloromethane)/trifluoroacetic acid (TFA)(v/v). After 4 h the reaction is evaporated, the residue is dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The organic layers are pooled, dried over sodium sulfate, filtered to remove solids, and evaporated to dryness resulting in N-methylamino alcohols.
  • numerous aldehydes such as paraldehyde, benzaldehyde,
  • isobutyraldehyde trimethylacetaldehyde, 2-methylbutyraldehyde, 2-ethylbutyraldehyde, etc. are commercially available and can be used in this reaction to generate starting materials (See Table 1).
  • Sodium NONOate (SNO) Salts (see also Velazquez, C.A., Chen, Q., Citro, M ., Keefer, L.K. and Knaus, E.E. J Med Chem 2008, 51, 1954-1961): A N-methylamino alcohol (See Table 1) (10 g, 0.13 mol) is added to a solution of sodium methoxide (7.2 g, 0.13 mol, 30.5 mL of a 25% w/v solution in MeOH) and ether (150 mL) with stirring at 25 °C. The flask is evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 72 hrs. The product is isolated by filtration and then suspended in ether (100 mL) with stirring for 15 min. The suspension is filtered, collected and dried under reduced pressure until a constant weight is achieved. Starting materials and corresponding sodium NONOate salts are listed in Table 1.
  • a NONOate sodium salt from Table 1 (1.0 eq.) is suspended in ACN and a solution of electrophile (e.g. chloromethylphthalimide, chloromethyl acetate, 1-chloroethyl ethylcarbonate) is added at room temperature in ACN followed by solid sodium iodide (1.4 eq). The resulting mixture is stirred at room temperature for 12 h and then evaporated under reduced pressure. The residue is suspended in ethyl acetate and washed with a solution of 0.2 N sodium
  • aldehydes such as paraldehyde, benzaldehyde, isobutyraldehyde, trimethylacetaldehyde, 2- methylbutyraldehyde, 2-ethylbutyraldehyde, etc. are commercially available and can be used in this reaction.
  • the capping reagent R ⁇ CHCl in Scheme 1 may be made by reacting a 1-chloroalkylchloroformate with an alcohol in the presence of base:
  • reaction mixture is diluted with ethyl acetate and filtered through Celite.
  • the filtrate is extracted with 5 % aqueous sodium bicarbonate and washed with dichloromethane.
  • the aqueous layer is acidified with dilute hydrochloric acid and washed with dichloromethane.
  • the combined organic layers are separated, dried, filtered and concentrated under reduced pressure.
  • the residue is purified by silica gel column chromatography to afford O-capped NONOates shown in Tables 3, 5, and 7.
  • N-Methylethanolamine (2.0 g, 26.63 mmol) was added to a solution of sodium methoxide (1.44 g, 26.63 mmol, 6.0 mL of a 25% w/v solution in methanol) and ether (30 mL) with stirring at 25 °C.
  • the flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) and stirred at 25 °C for 24 hrs.
  • the product was isolated by filtration and then suspended in ether (30 mL) and stirred for 15 min. The suspension was filtered, collected and dried at 25 °C under reduced pressure to give (NS-06) as a white fine powder (2.72 g, 65% yield).
  • 1H NMR 400 MHz, D 2 0) ⁇ 3.42-3.40 (m, 2H), 2.95-2.92 (m, 2H), 2.63 (s, 3H).
  • N-Methylamino-l-propanol (4.75 g, 53.29 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (2.80 g, 31% yield).
  • (Z)-l-Hydroxy-3-(6-hydroxyhexyl)-3-methyltriaz-l-ene 2-oxide sodium salt (NS- 12)
  • Azetidine hydrochloride (25.0 g, 267.2 mmol) was added to a solution of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) and diethyl ether (75 mL) with stirring at 25 °C. After 5 minutes, the resulting sodium chloride precipitate was filtered and the filtrate was placed in a pressure vessel. Another aliquot of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) was added and the flask was evacuated with vacuum and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 2 days.
  • NO nitric oxide
  • N-Vinyl phthalimide (0.100 g, 0.577 mol) was treated with 4N HCl/dioxane (0.433 mL, 1.73 mmol) at 25 °C. After 3 h the reaction mixture was concentrated under reduced pressure to provide an off white solid (112 g, 93% yield).
  • Example 1 (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-((5)-l-hydroxypropan-2- yl)-3-methyltriaz-l-ene 2-oxide
  • NONOate salt NS-01 (0.350 g, 2.05 mmol) suspended in acetonitrile (2.5 mL), pre- cooled in an ice-bath, was treated with a solution of l-(chloroethyl) phthalimide (0.323 g, 1.54 mmol) in acetonitrile (2.5 mL) and then sodium iodide (0.231 g, 1.54 mmol) was added. The resulting mixture was stirred for 30 minutes then the ice-bath was removed and the mixture was stirred for an additional hour. The acetonitrile was removed under reduced pressure.
  • Example 2 (5',Z)-l-((l,3-Dioxoisoindolin-2-yl)methoxy)-3-(l-hydroxypropan-2-yl)- 3-methyltriaz-l-ene 2-oxide
  • NONOate salt NS-01 (0.270 g, 1.58 mmol) suspended in acetonitrile (3.0 mL) was treated with a solution of N-(chloromethyl)phthalimide (0.280 g, 1.43 mmol) in acetonitrile (3.0 mL) and then sodium iodide (0.215 g, 1.43 mmol) was added. The resulting mixture was stirred at 25 °C overnight. N,N-dimethylformamide (1.0 mL) was added to the reaction mixture which was then heated to 60 °C for 1 hour. The acetonitrile was removed under reduced pressure.
  • Example 3 (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)methoxy)-3-ethyl-3-(2- hydroxyethyl)triaz-l-ene 2-oxide
  • N-(Chloromethyl) phthalimide (503 mg, 2.57 mmol) and NS-04 (400 mg, 2.34 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (452 mg, 63% yield).
  • LC t r 2.68 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • Example 4 (Z)-l-((l,3-Dioxoisoindolin-2-yl)methoxy)-3-(2-hydroxyethyl)-3- isopropyltriaz-l-ene 2-oxide
  • N-(Chloromethyl) phthalimide (197 mg, 1.19 mmol) and NS-05 (200 mg, 1.08 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (177 mg, 51% yield).
  • LC t r 2.84 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • Example 5 (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-(2-hydroxyethyl)-3- methyltriaz-l-ene 2-oxide
  • NS-06 (0.312 g, 1.99 mmol) and l-(chloroethyl) phthalimide (0.500 g, 2.39 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (227 mg, 31% yield).
  • LC t r 2.68 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
  • N-(Chloromethyl) phthalimide (259 mg, 1.33 mmol) and NS-06 (250 mg, 1.59 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (257 mg, 65% yield).
  • Example 7 (Z)-3-(tert-Butyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-(2- hydroxyethyl)triaz-l-ene 2-oxide
  • N-(Chloromethyl) phthalimide (216 mg, 1.10 mmol) and NS-07 (200 mg, 1.00 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (223 mg, 66% yield).
  • 1H NMR 400 MHz, CDC1 3 ) ⁇ 7.98-7.74 (m, 4H), 5.84 (s, 2H), 3.51-3.48 (m, 2H), 3.24-3.20 (m, 2H), 1.58 (s, 9H).
  • LC t r 3.06 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • Example 8 (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-(3-hydroxypropyl)-3- methyltriaz-l-ene 2-oxide
  • NS-09 (0.395 g, 2.31 mmol) and l-(chloroethyl) phthalimide (0.441 g, 210 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (90 mg, 13% yield).
  • LC t r 2.80 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
  • Example 9 (2)-2-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-l-(3-hydroxyazetidin-l- yl)diazene oxide
  • Example 10 (Z)-2-(l-(l,3-Dioxoisoindolin-2-yl)methoxy)-l-(3-hydroxyazetidin-l- yl)diazene oxide
  • N-(Chloromethyl) phthalimide (1.05 g, 5.38 mmol) and NS-13 (1.0 g, 6.45 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (614 mg, 39% yield).
  • Example 1 1 ( ⁇ ,Z)-2-((l ,3-Dioxoisoindolin-2-yl)methoxy)-l-(2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
  • N-(Chloromethyl) phthalimide (587 mg, 3.00 mmol) and NS-16 (500 mg, 2.73 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (307 mg, 35% yield).
  • 1H NMR 400 MHz, d-DMSO
  • LC t r 2.73 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • Example 51 (Z)-3-(2-Carboxyethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
  • Example 8 Sodium metaperiodate (0.225 g, 1.05 mmol) was added to a mixture of Example 8 (0.081 g, 0.251 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a colorless oil (82 mg, 97% yield).
  • Example 52 (5',Z)-l-(2-Carboxypyrrolidin-l-yl)-2-((l,3-dioxoisoindolin-2- yl)methoxy)diazene oxide
  • Example 11 (200 mg, 0.62 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (165 mg, 80%> yield).
  • 1H NMR 400 MHz, CDCI3) ⁇ 7.97-7.77 (m, 4H), 5.74 (s, 2H), 4.59-4.53 (m, 1H), 3.84- 3.79 (m, 2H), 3.69-3.62 (m, 2H), 2.31-2.25 (m, 2H).
  • LC t r 2.71 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C).
  • ES(pos)MS m/z 357 (M+Na calcd for Ci 4 Hi 4 N 4 0 6 requires 357).
  • Example 53 (Z)-3-(Carboxymethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
  • Example 5 (0.227 g, 0.736 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (224 mg, 95% yield).
  • LC t r 2.80 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
  • Example 54 (Z)-3-(Carboxymethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3- methyltriaz-l-ene 2-oxide
  • Example 6 (328 mg, 1.11 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (253 mg, 74% yield). ⁇
  • Example 54 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 54 as a solid.
  • Carboxylic acids listed in Table 3 are converted to the alkaline or ammonium salts by similar methods (i.e.
  • CARBONATE-CAPPED ALCOHOL NONOATES [00101] 1 -Chloroethyl tert -butyl carbonate
  • tert-Butyl alcohol (3.34 mL, 35 mmol) and pyridine (3.39 mL, 42 mmol) were dissolved in 90 mL dichloromethane and cooled to -78 °C.
  • 1-Chloroethyl chloroformate (5.0 g, 35 mmol) was added dropwise and the reaction was slowly allowed to warm to room temperature, then stirred for 3 days. The rxn was evaporated, dissolved in ethyl acetate, then washed with water and brine, dried over magnesium sulfate and evaporated. It was noted that product evaporated under high vacuum.
  • Example 91 (25,Z)-1-Hydroxy-2,3,7,11,1 l-pentamethyl-9-oxo-6,8,10-trioxa- 3,4,5-triazadodec-4-ene 4-oxide
  • NS-01 (0.350 g, 2.05 mmol) suspended in acetonitrile (2.0 mL) was treated with a solution of 1-chloroethyl tert -butyl carbonate (0.336 g, 1.86 mmol) in acetonitrile (1.0 mL) and then sodium iodide (1.25 g, 8.37 mmol) was added. The resulting mixture was heated to 60 °C for 3 hours. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate.
  • Example 92 (Z)-3-Ethyl-l-hydroxy-7,l 1,1 l-trimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • Example 93 (Z)-l-Hydroxy-3-isopropyl-7,l 1,1 l-trimethyl-9-oxo-6,8,10-trioxa- 3,4,5-triazadodec-4-ene 4-oxide
  • NS-05 500 mg, 2.7 mmol
  • 1-chloroethyl t-butyl carbonate 488 mg, 2.7 mmol
  • LC t r 3.40 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • Example 94 (Z)-l-Hydroxy-3,7-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec- 4-ene 4-oxide
  • NS-06 (0.250 g, 1.59 mmol) was suspended in acetonitrile (3.0 mL) and to this was added a solution of 1 -chloroethyl ethyl carbonate (0.291 g, 1.91 mmol) in acetonitrile (2.0 mL) and sodium iodide (0.286 g, 1.91 mmol) and the resulting mixture was stirred at 25 °C overnight. The acetonitrile was removed under reduced pressure and the residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate.
  • Example 95 (Z)-l-Hydroxy-3,7,l 1,1 l-tetramethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • Example 96 (Z)-3-(2-Hydroxyethyl)-2,2,7,l 1,1 l-pentamethyl-9-oxo-6,8,10- trioxa-3,4,5-triazadodec-4-ene 4-oxide
  • NS-07 500 mg, 2.51 mmol
  • 1-chloroethyl tert -butyl carbonate (454 mg, 2.51 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (240 mg, 30% yield).
  • Example 97 (Z)-13-Hydroxy-2,2,6,10-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10- triazatridec-8-ene 9-oxide
  • NS-09 (2.30 g, 13.44 mmol) suspended in acetonitrile (10.0 mL) was treated with a solution of 1 -chloroethyl tert -butyl carbonate (1.87 g, 10.35 mmol) in acetonitrile (10.0 mL) and then sodium iodide (1.55 g, 10.35 mmol) was added. The resulting mixture was stirred at ambient temperature for 4 days then heated to 60 °C for 32.5 hours. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate.
  • Example 100 (Z)-2-(l-((Ethoxycarbonyl)oxy)ethoxy)-l-((5)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
  • NS- 16 (2.07 g, 11.3 mmol) and freshly made 1-iodoethyl ethyl carbonate (4.12 g, 16.9 mmol) were dissolved in 15 mL acetonitrile and stirred at room temperature overnight. The reaction was evaporated, diluted with ethyl acetate, washed with 0.2N Na 2 S 2 0 3 and brine, dried over magnesium sulfate and evaporated. Chromatography was performed using 50%> ethyl acetate/hexanes to yield a colorless oil (890 mg, 28% yield).
  • Example 101 (Z)-2-(l-((tert-Butoxycarbonyl)oxy)ethoxy)-l-((5)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
  • Example 103 (Z)-l-Hydroxy-3,7,l l-trimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • NS-06 (0.250 g, 1.59 mmol) and 1-iodoethyl isopropyl carbonate (0.492 g, 1.91 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 100, with the exception no sodium iodide was added: (141 mg, 33% yield).
  • Example 104 (Z)-3 -Ethyl- 1 -hydroxy- 1 l-methyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • Example 105 (Z)-3-Ethyl-l-hydroxy-7,l l-dimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • Example 170 (Z)-l-Carboxy-2,6-dimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec-3- ene 3 -oxide
  • Example 94 0.130 g, 0.52 mmol
  • ruthenium(III)chloride cat
  • acetonitrile 1.0 mL
  • ethyl acetate 1.0 mL
  • water 1.5 mL
  • the resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®.
  • the aqueous reaction mixture was then extracted into ethyl acetate.
  • the combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a colorless oil (111 mg, 80% yield).
  • Example 171 (Z)-l-Carboxy-2,6,10,10-tetramethyl-8-oxo-5,7,9-trioxa-2,3,4- triazaundec-3-ene 3 -oxide [00140]
  • Example 95 400 mg, 1.44 mmol was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (352 mg, 83% yield).
  • LC t r 5.04 minutes (C-18 column, 5 to 95%
  • Example 172 (Z)-l-Carboxy-3,7, l 1 ,1 l -tetramethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
  • Example 173 (Z)-l-(( l S)-2-Carboxypyrrolidin-l-yl)-2-(l - ((ethoxycarbonyl)oxy)ethoxy)diazene oxide
  • Example 100 (150 mg, 0.54 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170, step 2: (146 mg, 93%> yield).
  • 1H NMR 400 MHz, d-DMSO
  • Example 174 (Z)-2-(l-((tert-Butoxycarbonyl)oxy)ethoxy)-l-((5)-2- carboxypyrrolidin- 1 -yl)diazene oxide
  • Example 101 (150 mg, 0.49 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (74 mg, 47% yield). ⁇
  • Example 175 (Z)-l-Carboxy-2,10-dimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec- 3-ene 3 -oxide
  • Example 102 (250 mg, 1.0 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (238 mg, 90%> yield).
  • LC t r 2.84 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
  • ES(pos)MS m/z 288 (M+Na calcd for C8H15N3O7 requires 288).
  • Examplel75 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 175 as a solid.
  • Carboxylic acids listed in Table 5 are converted to the alkaline or ammonium salts by similar methods. [00150] Table 5. Carbonate-Capped Carboxylic Acid Nonoates.
  • Example 322 (Z)-l -(3-Hydroxyazetidin-l -yl)-2-((pivaloyloxy)methoxy)diazene oxide
  • NS-13 300 mg, 2.27 mmol
  • chloromethyl pivalate (244 mg, 1.62 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 94: (56 mg, 10% yield).
  • Example 520 (Z)-l-(Azetidin-l-yl)-2-((pivaloyloxy)methoxy)diazene oxide
  • NS-26 (2.00 g, 14.38 mmol) and chloromethyl pivalate (1.55 g, 10.27 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 94: (132 mg, 5.6% yield).
  • 1H NMR 400 MHz, CDC1 3 ) ⁇ 5.78 (s, 2H), 4.14-4.10 (m, 4H), 2.25-2.17 (m, 2H), 1.24 (s, 9H).
  • LC t r 3.72 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C).
  • Example 352 (Z)-2-(l -Acetoxyethoxy)- l-((5)-2-carboxyazetidin-l-yl)diazene oxide
  • Example 341 Sodium metaperiodate (2.0 mmol) is added to a mixture of Example 341 (0.50 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the carboxylic acid.
  • Example 352 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 352 as a solid.
  • Carboxylic acids listed in Table 7 are converted to the alkaline or ammonium salts by similar methods.
  • Example 377 (Z)-l -((2,5-dioxopyrrolidin-l -yl)methoxy)-3-(2-hydroxyethyl)-3- methyltriaz-l-ene 2-oxide
  • NS-06 (1.0 mmol) is suspended in N,N-dimethylformamide (1.0 mL) and the solution is treated with a solution of N-(chloromethyl)succinimide (1.0 mmol, prepared according to J Org Chem 1993, 55(18), 4913-18) in N,N-dimethylformamide (1.0 mL). Sodium iodide (1.0 mmol) is added and the mixture is heated to 60 °C for 1 hour.
  • Example 377 The mixture is poured into water (25 mL), extracted with ethyl acetate (25 mL), the organic layer is washed with a 0.2 N solution of sodium thiosulfate and brine, dried over sodium sulfate, filtered, and concentrated. The residue is purified by silica gel chromatography using ethyl acetate in hexane to afford Example 377.
  • Example 377 Sodium metaperiodate (1.0 mmol) is added to a mixture of Example 377 (0.25 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The mixture is stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous mixture is extracted into ethyl acetate, the combined organic layers are washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide Example 426.
  • Example 426 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 426 as a solid.
  • Carboxylic acids listed in Table 9 are converted to the alkaline or ammonium salts by similar methods.
  • Reference wells were diluted with 150 ⁇ _, of sodium nitrite stock solutions in PBS. Remaining wells were diluted with 150 ⁇ _, of PBS and then every well received 20 ⁇ _, of Griess Reagent (Promega Cat. # G2930). Wells were mixed, the plates were incubated for 30 minutes at room temperature, and then absorbance was measured at 562 nm using a microplate reader. Reference standards final concentrations were 100, 33.3, 11.1, 3.7, 1.23, and 0.41 ⁇ nitrite and final test concentrations for all compounds was 30 ⁇ . The average DMSO blank readings were subtracted from test readings and a standard curve was generated from the reference standard wells. Nitrite levels were determined, and percent release of nitric oxide was calculated, relative to theoretical maximum (60 ⁇ ), for each compound.
  • PK Pharmacokinetics of nitric oxide release is measured by administering a single oral (PO) gavage dose to Sprague Dawley rats.
  • test compound two (2) Sprague Dawley (CD® IGS) male rats are used. Animals are fasted before the study and fed only after the 8-h blood draw. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are administered oral (PO) in 2% DMSO/0.5% methylcellulose/0.1% Tween 20 vehicle at 30 mg/kg using 10 mL/kg volume per animal. Compounds are formulated by making a 150 mg/mL DMSO compound stock and adding to warm 0.5 % methylcellulose/0.1% Tween-20 at 35-40°C to make a clear solution or fine suspension. Animals found in severe distress or a moribund condition are euthanized.
  • PO oral
  • 2% DMSO/0.5% methylcellulose/0.1% Tween 20 vehicle at 30 mg/kg using 10 mL/kg volume per animal.
  • Compounds are formulated by making a 150 mg/mL DMSO compound stock and adding to warm 0.5 % methylcellulose/0.1
  • Peripheral blood collections are done primarily through venipuncture of the tail or saphenous veins or by jugular catheter at various times, (e.g. pre-dose, 15min, 30min, lh, 2h, 4h, 8h, and 24h).
  • Whole blood samples are collected in K 2 EDTA microtainer (Fisher # 02-669-38), processed to plasma by centrifugation, and the plasma is frozen at -80°C.
  • Thawed plasma samples (30 ⁇ ) are diluted with PBS (70 ⁇ ) along with control rat plasma. Samples are spun at 2000x g for 10 minutes and then 80 ⁇ of 30% PBS-diluted plasma samples are transferred into the appropriate well of a 96-well plate. Sodium nitrate is used in standard curve wells at 100, 33.3, 1 1.1 , 3.7, 1.23, and 0.41 ⁇ . To each well is added ⁇ ⁇ of the nitrate reductase solution and ⁇ ⁇ of the enzyme co-factors solution to convert nitrate to nitrite (Sigma #06239 Nitrite/Nitrate Assay Kit).
  • the plate is incubated at room temperature for 2 h and then 50 ⁇ ⁇ of Griess Reagent A is added to each well, and mixed. After 5 min., 50 ⁇ ⁇ of Griess Reagent B is added to each well, and mixed. The plate is incubated for 10 minutes at room temperature, and the absorbance is measured at 540nm with a microplate reader. A standard curve is generated from the reference standard wells and nitrate/nitrite (NO x ) levels are determined ( ⁇ ) and standard deviations (+/-S.D.) for each blood draw and plot against time (h) of blood draw.
  • NO x nitrate/nitrite
  • Table 1 Nitric oxide release in vivo after oral administration in 2 rats at 30 mpk.
  • Phthalimide-capped compounds 9 and 1 showed very similar activity before and after heating, indicating good stability. Carbonate-capped compounds 92, 95 and 96 showed useful stability with some variation among species. Finally, compound 500, a compound not of the invention,
  • MAP mean arterial blood pressure
  • Celecoxib is used as the positive control for these studies.
  • Monolayer Wound Healing Assay Cell proliferation in confluent A549 monolayers is blocked by a 30 minute pre-incubation in the presence of mitomycin C (3 ⁇ g/mL). Test compounds, in cell culture buffer, are added to confluent monolayers 30 minutes before wound induction. A549 monolayers are subsequently scratched with a pipette tip. Wound areas are evaluated with phase contrast microscopy on an inverted microscope. Images of the same areas are obtained at intervals from zero to 96 hours. Cell migration rate through wound healing is evaluated from the images using Paint.Net v.3.10 software. Cell migration is expressed as the fold change in the migration area, relative to untreated control cells at the same time period.
  • Excisional In Vivo Wound Healing Model Experiments using endothelial NO synthase (eNOS) gene knockout (KO) mice have been conducted to determine the absolute requirement of nitric oxide for wound healing in vivo and the contribution of eNOS to this effect (Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H1600-H1608, 1999). This model is applied to drug testing by administering compounds topically directly to the wound, or systemically via oral dosing.
  • eNOS endothelial NO synthase
  • Wild-type (WT) and eNOS KO mice (2-3 mo of age, weighing 20-26 g each, and evenly distributed across both genders) are anesthetized, their dorsum is clipped free of hair, and the skin is prepped with povidone-iodine.
  • a full-thickness wound (1.5 x 1.5 cm), including the panniculus carnosus, is created sharply with fine scissors. After the wound edges are retracted, the wound outline is traced onto an acetate sheet. To keep the area clean, the wounds are dressed with a clear, bioocclusive dressing (Op-Site; Owens Minor, Greensburg, PA).
  • Compounds are administered every-other day topically by completely covering the wound with compound treated cream, or daily (q.d.) by systemic administration via oral gavage (2% DMSO/0.5% methylcellulose/0.1% Tween 20 vehicle at 30 mg/kg using 10 mL/kg volume per animal).
  • Compounds are formulated for topical administration by dissolving/suspending the compound (50 mg) into a mixture of the liquid components (373 mg of 2-propanol, 93 mg of transcutol, 93 mg of isopropyl myristate and 3 mg of labrasol), followed by mixing with melted PEG 4000 (388 mg), which upon cooling semi-solidifies into a cream.
  • Compounds are formulated for oral administration by making a 150 mg/mL DMSO compound stock and adding to warm 0.5 %
  • the tracings are digitized, and the areas are calculated in a blinded fashion using a computerized algorithm (Sigma Scan; Jandel Scientific, San Raphael, CA).
  • the rate of excisional wound closure in compound-treated mice is compared to vehicle treated eNOS KO mice and WT mice to determine the effect of compounds on wound healing.
  • the present invention relates to pharmaceutical compositions comprising a compound of formula I together with one or more
  • compositions will usually be formulated for topical application.
  • topical application there are employed as non-sprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water.
  • Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc.
  • sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon.
  • the formulation may be impregnated into a fiber dressing or bandage and provided to the patient in that form.
  • Formulations for topical application are well known to those skilled in the art, and general methods for their preparation are found in any standard pharmacy school textbook, for example Remington: The Science and Practice of Pharmacy. Chapter 90 of the 19th edition of Remington entitled " Medicated
  • the topical pharmaceutical carrier may include any substance capable of dispersing and maintaining contact between the active ingredients and the skin.
  • the vehicle may be glycerin, alcohol or water based.
  • Water based compositions may, if desired, be thickened with a suitable gel to provide a less mobile composition.
  • Such compositions are well known in the art. Examples of such vehicles include aloe vera, which is a gel base, together with ethanol, isopropyl alcohol, water, propylene glycol and a non-ionic surfactant such as laureth-4.
  • aloe vera which is a gel base
  • ethanol isopropyl alcohol
  • water propylene glycol
  • a non-ionic surfactant such as laureth-4.
  • Other water-based alcohol/ glycerin vehicles and carriers are within the scope of the present invention.
  • a typical water-based lotion will contain from 45 to 50 parts of glycerin, one to three parts Tween 80TM, from 45 to 50 parts of water and from 1
  • ointments emulsions or dispersions in which water, if present, is a minor constituent.
  • Typical ointment formulation comprises from 90 to 98 parts of a mixture of petrolatum, mineral oil, mineral wax and wool wax alcohol, from 0.5 to 3 parts of a mixture of polyoxyethylene and sorbitan monooleate (Tween 80TM), from 1 to 5 parts of water, and from 1 to 50 parts of the compound of the invention.
  • Another suitable non-aqueous ointment can be prepared from 95 parts of liquid petrolatum USP, 5 parts polyethylene and from 1 to 50 parts of the compound of the invention. The resulting ointment spreads easily and has an even consistency over wide temperature extremes. It is, in addition, non-irritating and non-sensitizing.
  • Formulations of the compounds of the invention may also be prepared containing from 0 to 25% by weight of urea.
  • the water content will vary from 5 to 50% by weight of the composition.
  • Any suitable ointment carrier may be used such as lanolin, ethylene glycol polymers and the like.
  • borate salts may often be added to stabilize the pharmaceutical composition (see U.S. patent 2,917,433,).
  • Optional additional therapeutic ingredients that may be added to the compositions include compounds known in the art to be effective topical antibiotics, such as nystatin, clindamycin, erythromycin, metronidazole, silver sulfadiazine, chlorhexidine gluconate and sodium fusidate.

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Abstract

Esters, carbonates and imides of 3,3-disubstituted-1-hydroxytriaz-1-ene 2-oxides of the formula I are disclosed. The compounds release nitric oxide (NO) under physiologic conditions, and pharmaceutical compositions containing them are useful to aid in wound healing.

Description

3,3-DISUBSTITUTED-l-HYDROXYTRIAZ-l-ENE 2-OXIDES AND WOUND-HEALING COMPOSITIONS USING THEM
STATEMENT AS TO RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH
[0001] This invention was made with Government support under Contract No.
1R43GM099569-01, awarded by the National Institutes of General Medical Sciences.
Accordingly, the U.S. Government has certain rights in this invention.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims priority from US provisional application 61/927,150, filed January 13, 2014. The entire contents of 61/927,150 are incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to the use of esters, carbonates and imides of 3,3-disubstituted- 1-hydroxytriaz-l-ene 2-oxides that release nitric oxide (NO) under physiologic conditions to aid in wound healing.
BACKGROUND OF THE INVENTION
[0004] Nitric oxide (NO) is an important endogenous signaling molecule and vasodilator. NO is synthesized from L-arginine by the enzyme NO synthase (NOS), which exists in three distinct isoforms, namely the constitutively expressed endothelial (eNOS) and neuronal (nNOS) forms, and the mainly inducible form (iNOS). Nitric oxide exhibits a number of important pharmacological actions including vascular relaxation (vasodilatation) and inhibition of platelet aggregation and adhesion. Inhibition of NO synthesis leads to an increase in systemic blood pressure. NO also prevents atherogenesis by inhibiting vascular smooth muscle cell proliferation and preventing low-density lipoprotein oxidation and macrophage activation. Vascular NO generation is important in controlling blood pressure, and a growing body of evidence indicates that NO signaling is a key factor in counteracting the onset and development of several CV diseases including hypertension, myocardial infarction and stroke. [0005] Compositions that release NO under physiologic conditions have been the subject of numerous articles and patents. For example US patent 5,814,666 discloses so-called nonoate salts for the treatment of infectious diseases caused by pathogenic microbes.
Similarly, Ghaffari et al [Nitric Oxide 14, 21-29 (2006)] reported that gaseous NO has antibacterial properties that can be beneficial in reducing bacterial burden in infected wound in burn injuries or non-healing ulcers. Shekhter et al. [Nitric Oxide 12, 210-219 (2005)] reported that gaseous NO improves healing of skin wounds in rats, and Dashti et al. [PakJ Med Sci, 20, 211-214 (2004)] reported that diethylenetriamine NONOate is useful in enhancing wound healing in diabetic rats. Diethylenetriamine NONOate is, however, an inherently unstable compound and quickly breaks down spontaneously at room temperature. As a consequence, it is usually stored at -80°C, which limits its utility as a practical wound- healing medicament.
SUMMARY OF THE INVENTION
[0006] In one aspect, the invention relates to compounds of formula:
Figure imgf000003_0001
wherein
R is H or (Ci-C6)alkyl;
2
R is chosen from
Figure imgf000003_0002
(b) -OC(=0)OR 7 ; and, when R 3 and R 4 , taken together with the nitrogen to which they are attached, form an optionally substituted 4-membered ring, R may additionally be:
(c) -OC(=0)R9;
R3 and R4 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
8 9 substituted with from one to three groups chosen from -OH, -COOH, -OR°, -COOR" and -OC(=0)R9; or taken together with the nitrogen to which they are attached, R3 and R4 form an optionally substituted 4- to 7-membered ring;
R5 and R6 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
8 9 substituted with from one to three groups chosen from -OH, -COOH, -OR°, -COOR" and -OC(=0)R9; or R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a 5- to 7-membered cyclic imide, said cyclic imide optionally fused to a second six-membered ring, which six-membered ring may be substituted with from one to
Q
three groups chosen from (Ci-Ce)alkyl, halogen, cyano, amino, hydroxy, -COOH, -OR , -COOR9 and -OC(=0)R9 ;
7 8 9
R', R° and R" are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl substituted with from one to three groups chosen from -OH, -COOH, -OR10, -COOR10 and -OC(=0)R10; and
R10 is (Ci-C6)alkyl.
[0007] In another aspect, the invention relates to methods for treating cutaneous injuries comprising bringing a cutaneous injury into contact with a compound as described herein.
[0008] In another aspect, the invention relates to pharmaceutical compositions containing a pharmaceutically acceptable carrier and a compound as described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0009] As discussed above, compositions that release NO under physiologic conditions have been the subject of numerous articles and patents. The instant invention relates to a novel genus of chemical compounds that release NO under physiologic conditions. They have the generic structure I:
Figure imgf000005_0001
2
[0010] In some embodiments of the invention, R is:
Figure imgf000005_0002
. In some of these embodiments, R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a 5- to 7-membered cyclic imide. The cyclic imide may be fused to a second six-membered ring, which six-membered ring may be substituted with from one to three groups chosen from (Ci-Ce)alkyl, halogen, cyano, amino, hydroxy, -COOH, -OR8, -COOR9 and -OC(=0)R9. In some embodiments R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a phthalimide.
[0011] In some embodiments, R2 is -OC(=0)OR7, and in some of these embodiments, R7 is (Ci-Cio)hydrocarbyl.
[0012] In some embodiments, R3 is saturated (Ci-Cio)hydrocarbyl and R4 is (Ci- Cio)hydrocarbyl substituted with-OH or -COOH; in others, R3 and R4 taken together with the nitrogen to which they are attached, form an optionally substituted 4- to 7-membered ring, for example a hydroxy-substituted 4- to 7-membered ring or an unsubstituted, saturated heterocycle. In further embodiments, the ring is an optionally substituted azetidine and R2 is -OC(=0)R9, in which R9 may be (Ci-Cio)hydrocarbyl.
[0013] Throughout this specification the terms and substituents retain their definitions. [0014] For convenience and clarity certain terms employed in the specification, examples and claims are described herein.
[0015] Ci to C2o hydrocarbon includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthylethyl. Hydrocarbyl refers to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched. Examples of aliphatic hydrocarbyl substituents include isopropyl, 2-butenyl, 2- butynyl, cyclopentyl, norbornyl, etc. Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
[0016] Unless otherwise specified, alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof. Alkyl refers to alkyl groups from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
[0017] Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, norbornyl and the like.
[0018] Unless otherwise specified, the term "carbocycle" is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state. Thus (C3-C10) carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene; (C8-C12) carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene. Carbocycle, if not otherwise limited, refers to monocycles, bicycles and polycycles.
[0019] Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Unless otherwise specified, a heterocycle may be non-aromatic (heteroaliphatic) or aromatic (heteroaryl). Examples of heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline,
tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like. Examples of heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and tetrahydroquinolinyl.
[0020] Alkoxy or alkoxyl refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms of a straight or branched configuration attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower-alkoxy refers to groups containing one to four carbons. For the purpose of this application, alkoxy and lower alkoxy include
methylenedioxy and ethylenedioxy.
[0021] The term "halogen" means fluorine, chlorine, bromine or iodine atoms. In one embodiment, halogen may be a fluorine or chlorine atom.
[0022] As used herein, the term "optionally substituted" may be used interchangeably with "unsubstituted or substituted". The term "substituted" refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl, aryl, cycloalkyl, or heterocyclyl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxy lower alkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, lower alkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [- C(=0)0-alkyl], alkoxycarbonylamino [ FiNC(=0)0-alkyl], aminocarbonyl (also known as carboxamido) [-C(=0)NH2], alkylaminocarbonyl [-C(=0)NH-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl (including cycloalkylaminoalkyl), dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfinyl, alkylsulfonyl, acylaminoalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryloxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyloxyphenyl, and benzyloxy. "Oxo" is also included among the substituents referred to in "optionally substituted"; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl). In one embodiment, 1, 2, or 3 hydrogen atoms are replaced with a specified radical. In the case of alkyl and cycloalkyl, more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine. In preferred embodiments, substituents are halogen, haloalkyl, alkyl, acyl, hydroxyalkyl, hydroxy, alkoxy, haloalkoxy, aminocarbonyl oxaalkyl, carboxy, cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, alkylthio, alkylsulfinyl, alkylsulfonyl, alkylsulfonylamino arylsulfonyl, arylsulfonylamino, and benzyloxy. Most preferred are halogen, (Ci-C4)alkyl, halo(Ci-C4)alkyl, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, and aminocarbonyl.
[0023] Substituents Rn are generally defined when introduced and retain that definition throughout the specification and in all independent claims.
[0024] The compounds described herein may contain, in a substituent Rx, double bonds and may also contain other centers of geometric asymmetry; unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. The compounds may also contain, in a substituent Rx, one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (R)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques.
[0025] As used herein, and as would be understood by the person of skill in the art, the recitation of "a compound" - unless expressly further limited - is intended to include salts of that compound. In a particular embodiment, the term "compound of formula I" refers to the compound or a pharmaceutically acceptable salt thereof. For example, R3 or R4 may contain -COOH and these compounds may exist as salts, -COO" M+, wherein M is any counterion.
[0026] The term "pharmaceutically acceptable salt" refers to salts whose counter ion derives from pharmaceutically acceptable non-toxic acids and bases. Suitable
pharmaceutically acceptable acids for salts of the amino-substituted compounds of the present invention include, for example, acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic,
hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. Suitable pharmaceutically acceptable base addition salts for the carboxylate-substituted compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, Ν,Ν'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.
[0027] It will be recognized that the compounds of this invention can exist in radiolabeled form, i.e., the compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
Alternatively, a plurality of molecules of a single structure may include at least one atom that occurs in an isotopic ratio that is different from the isotopic ratio found in nature.
Radioisotopes of hydrogen, carbon, phosphorous, fluorine, chlorine and iodine include H, 3H, UC, 13C, 14C, 15N, 35S, 18F, 36C1, 1251, 124I and 131I respectively. Compounds that contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e. 3H, and carbon- 14, i.e., 14C, radioisotopes are particularly preferred for their ease in preparation and detectability. Persons skilled in the art recognize that deuterium has been used to improve metabolic stability of compounds, and that principle can be applied to these compounds. Compounds that contain isotopes UC, 13N, 150, 124I and
18
F are well suited for positron emission tomography. Radiolabeled compounds of formulae I and II of this invention and prodrugs thereof can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed in the Examples and Schemes by substituting a readily available radiolabeled reagent for a non-radiolabeled reagent. [0028] Although this invention is susceptible to embodiment in many different forms, preferred embodiments of the invention are shown. It should be understood, however, that the present disclosure is to be considered as an exemplification of the principles of this invention and is not intended to limit the invention to the embodiments illustrated. It may be found upon examination that certain members of the claimed genus are not patentable to the inventors in this application. In this event, subsequent exclusions of species from the compass of applicants' claims are to be considered artifacts of patent prosecution and not reflective of the inventors' concept or description of their invention; the invention encompasses all of the members of the genus I that are not already in the possession of the public.
[0029] As used herein, the terms "treatment" or "treating" are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including, but not limited to, therapeutic benefit. Therapeutic benefit includes accelerating the process of wound-healing. Inasmuch as NO is known to possess antimicrobial properties, a medicament that liberates therapeutically useful concentrations of NO would both decrease pathogen load and stimulate one or more of the overlapping phases of wound healing: clotting, inflammation, cell recruitment and proliferation, revascularization, epithelium formation including collagen deposition, and tissue remodeling.
[0030] Criteria for assessing the suitability of nitric oxide releasing pro-drug derivatives include: (1) thermal stability to accommodate common environments, (2) sufficient nitric oxide release to achieve in vivo efficacy, and (3) non-spontaneous activation to improve effectiveness of nitric oxide administration. It will be noted that nitric oxide release can vary among chemical species and subgenera in the genus described herein. The more rapid release species are better suited for bolus delivery of medicament, whereas the species whose activation is slower are better suited to sustained-release formulations. Molecules that are activated by endogenous enzymes released by the wound provide one approach to activation, but it is also possible to add stable exogenous enzymes to a topical formulation to modulate nitric oxide release and performance.
[0031] The simplest embodiment of a medicament utilizes the esterases and amidases that are already present at the wound to activate the nitric oxide releasing pro-drugs. Diffusion of the compounds described herein from the bandage or salve to the wound provide the base-line nitric oxide release kinetics. Under circumstances in which endogenous enzymes in the wound are not sufficient to elicit efficacious release of nitric oxide from compounds in a bandage or salve, exogenous stable enzymes, often lyophilized, may be mixed into a bandage, such that wetting initiates the nitric oxide release process. For example, lyophilized fetal calf serum or porcine esterases can be mixed with the compounds described herein, packaged into a bandage, and wetted with pH=7.4 buffered saline solution to activate. Different amounts of enzyme or pro-drug allow for tunable nitric oxide administration. Alternatively, pro-drugs can be activated by spraying or wetting the bandage with an enzyme solution similar to products used in the eye care industry for cleaning contact lenses.
[0032] Optimally, a precursor of NO would exhibit stability at 40 °C for more than 4 months. Compounds with lower stability are nonetheless useful, but greater reliance would be placed on formulation and on controlling storage conditions. Assuming inherent stability is good, NO release per mole of test compound greater than 50% in a 90-minute test is advantageous for rapid-release or bolus type medication, whereas low percent release of NO in the 90 minute test indicates potential utility for extended duration medicaments.
[0033] In general, the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants that are in themselves known, but are not mentioned here. The starting materials are either commercially available, synthesized as described in the examples or may be obtained by the methods well known to persons of skill in the art. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations can be readily obtained from the relevant scientific literature or from standard textbooks in the field. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions can vary with the particular reactants or solvent used. Those skilled in the art will recognize that the nature and order of the synthetic steps presented can be varied for the purpose of optimizing the formation of the compounds described.
[0034] A comprehensive list of abbreviations utilized by organic chemists appears in the first issue of each volume of the Journal of Organic Chemistry. The list, which is typically presented in a table entitled "Standard List of Abbreviations", provides definitions for abbreviations not found in the following list of abbreviations:
ACN acetonitrile
AcOH acetic acid
DCM dichloromethane or methylene chloride
DIPEA diisopropylethylamine
DMAP 4-dimethylaminopyridine or N,N-dimethylaminopyridine
DME 1 ,2-dimethoxyethane
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
eq. equivalents
EtOAc ethyl acetate
EtOH ethanol
H+ protic acid
HPLC high performance liquid chromatography
h hour
INT intermediate
LC/MS liquid chromatography mass spectrometry
LDA lithium diisopropylamide
MeOH methanol
min minute(s)
n L milliliter
mmol millimole
NaHMDS sodium bis(trimethylsilyl)amide
NIS N-iodosuccinimide
NMMO 4-methylmorpholine N-oxide
NONOate 1-hydroxytriaz-l-ene 2-oxide derivative
OXONE® potassium peroxomonosulfate
Pd(dppf)Cl2 [1,1 '-bis(diphenylphosphino)ferrocene]dichloropalladium(II)
Pd/C palladium on carbon
Ph phenyl
Psi pounds per square inch
TEA triethylamine
TEMPO (2,2,6,6-tetramethylpiperidin-l -yl)oxy
TFA trifluoroacetic acid
THF tetrahydrofuran TLC thin layer chromatography
TPP triphenyl phosphine
TsOH / toluenesulfonic acid
[0035] Terminology related to "protecting", "deprotecting" and "protected" functionalities occurs throughout this application. Such terminology is well understood by persons of skill in the art and is used in the context of processes that involve sequential treatment with a series of reagents. In that context, a protecting group refers to a group, which is used to mask a functionality during a process step in which it would otherwise react, but in which reaction is undesirable. The protecting group prevents reaction at that step, but may be subsequently removed to expose the original functionality. The removal or "deprotection" occurs after the completion of the reaction or reactions in which the functionality would interfere. Thus, when a sequence of reagents is specified, as it is in the processes described herein, the person of ordinary skill can readily envision those groups that would be suitable as "protecting groups". Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T.W.Greene and P.G.M.Wuts [John Wiley & Sons, New York, 1999].
List of protecting groups (abbreviations):
Acetyl (Ac)
Acylals
Carboallyloxy (Alloc)
Benzoyl (Bz)
Benzyl (Bn, Bnl)
Benzyl esters
Carbamate
Carbobenzyloxy (Cbz)
Dimethoxytrityl, [¾z's-(4-methoxyphenyl)phenylmethyl] (DMT)
Dithianes
Ethoxyethyl ethers (EE)
Fluorenylmethyloxycarbonyl (Fmoc)
Methoxymethyl ether (MOM)
Methoxytrityl [(4-methoxyphenyl)diphenylmethyl], MMT)
Methyl Ethers
Methyl (Me) Methyl esters
Methylthiomethyl ether
Orthoesters
Oxazoline
Pivaloyl (Piv)
Phthalimido
/?-Methoxybenzyl carbonyl (Moz or MeOZ)
/?-Methoxybenzyl (PMB)
Propargyl alcohols
Silyl groups (e.g., trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso- propylsilyloxymethyl(TOM) and triisopropylsilyl (TIPS))
Silyl esters
tert- utyl esters
tert-Butyloxycarbonyl (Boc or tBoc)
Tetrahydropyranyl (THP)
Tosyl (Ts or Tos)
Trimethylsilylethoxymethyl (SEM)
Trityl (triphenylmethyl, Tr)
β-Methoxyethoxymethyl ether (MEM)
(4-nitrophenyl)sulfonyl or (4-nitrophenyl)(dioxido)-lambda(6)-sulfanyl) (Nosyl) 2-cyanoethyl
2-nitrophenylsulfenyl (Nps)
3,4-Dimethoxybenzyl (DMPM)
2,2,4, 6,7-Pentamethyldihydrobenzofuran-5 -sulfonyl (Pbf)
[0036] Compounds of the present invention can be prepared using methods illustrated in general synthetic schemes and experimental procedures detailed below. These general synthetic schemes and experimental procedures are presented for purposes of illustration and are not intended to be limiting. Starting materials used to prepare compounds of the present invention are commercially available or can be prepared using routine methods known in the art. Scheme 1 illustrates synthesis of Capped-NONOate Alcohols and Carboxylic Acids. [0037] Conversion of N-Boc-protected amino alcohols to N-alkylamino alcohols: (S)-2- Boc-alaninol, (S)-2-Boc-leucinol, (S)-2-(Boc-amino)-3-phenyl-l-propanol, (S)-2-Boc-3- methyl- 1-butanol, and (S)-2-Boc-isoleucinol are converted to (5)-2-N-methyl-alaninol, (S)- 2-N-methyl-leucinol, (5)-2-(N-methylamino)-3 -phenyl- 1 -propanol, (S)-2-N-methyl-3- methyl- 1-butanol, and (5)-2-N-methyl-isoleucinol) respectively using established procedures (See also Reddy, G.V., Rao, G.V., Sreevani, V. and Iyengar, D.S. Tetrahedron Letters 2000, 41, 949-951). A N-protected amino alcohol (1.0 eq.), paraformaldehyde (3 eq.), and /?ara-toluenesulfonic acid (0.1 eq.) are dissolved in toluene and refluxed for 30 min using a Dean Stark trap. The solvent is evaporated under reduced pressure to give a crude residue that is purified by silica gel column chromatography to give afford pure N- protected oxazolines. The N-protected oxazoline (1 eq.) is dissolved in dry ACN under nitrogen atmosphere and treated with sodium cyanoborohydride (1 eq.) followed by trimethylsilyl chloride (1 eq.). The reaction mixture is stirred at room temperature for 30 min and the solvent is evaporated under reduced pressure. The residue is suspended in ethyl acetate and washed with a solution of 0.5 N sodium hydroxide. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The combined organic layers are washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue is purified by silica gel column chromatography to afford pure Boc-N-methyl amino alcohols. Boc-Deprotection is accomplished using standard conditions stirring at room temperature in 5 : 1 - dichloromethane(dichloromethane)/trifluoroacetic acid (TFA)(v/v). After 4 h the reaction is evaporated, the residue is dissolved in ethyl acetate and washed with saturated sodium bicarbonate solution. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The organic layers are pooled, dried over sodium sulfate, filtered to remove solids, and evaporated to dryness resulting in N-methylamino alcohols. In addition to paraformaldehyde, numerous aldehydes such as paraldehyde, benzaldehyde,
isobutyraldehyde, trimethylacetaldehyde, 2-methylbutyraldehyde, 2-ethylbutyraldehyde, etc. are commercially available and can be used in this reaction to generate starting materials (See Table 1).
[0038] Sodium NONOate (SNO) Salts (see also Velazquez, C.A., Chen, Q., Citro, M ., Keefer, L.K. and Knaus, E.E. J Med Chem 2008, 51, 1954-1961): A N-methylamino alcohol (See Table 1) (10 g, 0.13 mol) is added to a solution of sodium methoxide (7.2 g, 0.13 mol, 30.5 mL of a 25% w/v solution in MeOH) and ether (150 mL) with stirring at 25 °C. The flask is evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 72 hrs. The product is isolated by filtration and then suspended in ether (100 mL) with stirring for 15 min. The suspension is filtered, collected and dried under reduced pressure until a constant weight is achieved. Starting materials and corresponding sodium NONOate salts are listed in Table 1.
[0039] Scheme 1 : Synthesis of O-Capped-NONOate Alcohols and Carboxylic Acid
Figure imgf000016_0001
a sodium NONOate salt
Figure imgf000016_0002
[0040] Capping Sodium NONOates with Electrophiles {see also Velazquez, C.A., Chen, Q., Citro, M.L., Keefer, L.K. and Knaus, E.E. J Med Chem 2008, 51, 1954-1961): A NONOate sodium salt from Table 1 (1.0 eq.) is suspended in ACN and a solution of electrophile (e.g. chloromethylphthalimide, chloromethyl acetate, 1-chloroethyl ethylcarbonate) is added at room temperature in ACN followed by solid sodium iodide (1.4 eq). The resulting mixture is stirred at room temperature for 12 h and then evaporated under reduced pressure. The residue is suspended in ethyl acetate and washed with a solution of 0.2 N sodium
thiosulfate. The organic layer is separated and the aqueous layer is extracted again with ethyl acetate. The combined organic layers are washed with brine, dried over sodium sulfate, filtered, and the filtrate is evaporated under reduced pressure. The residue is purified by silica gel column chromatography to afford O-capped NONOates shown in Tables 2, 4, and 6. [0041] When R2 is -OC(=0)OR7, the capping reagent R1R2CHC1 in Scheme 1 may be made by the method of Ulich, L.H. and Adams, R. [J. Am. Chem. Soc. 1921, 43, 660-667)]: An acyl chloride (1.0 eq) and aldehyde (1.2 eq) are mixed in a round-bottomed flask equipped with a reflux water condenser. A minute quantity of anhydrous zinc chloride is added resulting in considerable heat evolution (in many cases causing the reaction mixture to boil gently). The reaction is heated further to 90 °C for 3 to 4 h and at the end of this time the reaction mixture is distilled with a fractionating column preferably under diminished pressure. The distillate is then redistilled either under atmospheric or diminished pressure to yield halogenated alkyl esters. In addition to paraformaldehyde, numerous aldehydes such as paraldehyde, benzaldehyde, isobutyraldehyde, trimethylacetaldehyde, 2- methylbutyraldehyde, 2-ethylbutyraldehyde, etc. are commercially available and can be used in this reaction.
Figure imgf000017_0001
[0042] When R2 is -OC(=0)OR7 or -OC(=0)R9, the capping reagent R^CHCl in Scheme 1 may be made by reacting a 1-chloroalkylchloroformate with an alcohol in the presence of base:
Figure imgf000018_0001
[0043] When R3 and R4 individually, or taken together form a 4- to 7-membered ring, are substituted with -CH2OH, the OH may be oxidized to -COOH by the method of Chakrapani et al. [Org Lett 2008, 10, 5155-5158]: Capped NONOate (1.0 eq.) is dissolved in ACN and water (2:3 v/v) and treated with sodium periodate (NaI04) (4.0 eq.) and ruthenium trichloride hydrate (RuCl3) (0.04 eq). The reaction mixture is diluted with ethyl acetate (1 volume) and stirred overnight. The reaction mixture is diluted with ethyl acetate and filtered through Celite. The filtrate is extracted with 5 % aqueous sodium bicarbonate and washed with dichloromethane. The aqueous layer is acidified with dilute hydrochloric acid and washed with dichloromethane. The combined organic layers are separated, dried, filtered and concentrated under reduced pressure. The residue is purified by silica gel column chromatography to afford O-capped NONOates shown in Tables 3, 5, and 7. Compounds in which R3 and R4 are substituted with -OR8, -COOR9 and -OC(=0)R9; may be made from the alcohol and acid by methods well known in the art.
[0044] (5',Z)-l-Hydroxy-3-(l-hydroxypropan-2-yl)-3-methyltriaz-l-ene 2-oxide sodium salt (NS-01)
Figure imgf000018_0002
[0045] A solution of iodine (4.92 g, 19.39 mmol) in tetrahydrofuran (10 mL) was slowly added dropwise to a suspension of N-methyl-L-alanine (2.00 g, 10.39 mmol) and sodium borohydride (1.83 g, 48.49 mmol) in tetrahydrofuran (50 mL), pre-cooled in an ice-bath. Once the addition was complete the reaction mixture was heated to reflux. After 18 h the reaction was allowed to cool to ambient temperature and then methanol (5 mL) was slowly added. The mixture was stirred at ambient temperature for 30 minutes and then concentrated under reduced pressure. The residue was dissolved in 20% KOH (w/w) (30 mL) and then stirred at ambient temperature for 4 hours. The aqueous reaction mixture was extracted with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a clear oil (2.07 g, 100% yield).
[0046] Crude alaninol (19.39 mmol) was added to a solution of sodium methoxide (1.05 g, 19.39 mmol, 4.5 mL of a 25% w/v solution in MeOH) and ether (25 mL) with stirring at 25 °C. The flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 2 days. The product was isolated by filtration and the filtrate was concentrated under reduced pressure. The residue was treated with acetonitrile and the precipitate that developed was collected by filtration. This process was repeated a second time and the solids collect were combined with the original product to give an off- white solid (0.935 g, 28% yield).
[0047] (Z)-3 -Ethyl- l-hydroxy-3-(2-hydroxyethyl)triaz-l-ene 2-oxide sodium salt (NS-04) o-
HO ^\ N .ONa
N N
[0048] 2-(Ethylamino) ethanol (5.0 g, 56.1 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (9.58 g, 50%> yield). 1H NMR (400 MHz, D20) δ 3.41 (t, 5.6 Hz, 2H), 2.94 (t, J=5.7 Hz, 2H), 2.84 (q, J=7.1 Hz, 2H), 0.83 (t, 7.1 Hz, 3H).
[0049] (Z)-l-Hydroxy-3-(2-hydroxyethyl)-3-isopropyltriaz-l-ene 2-oxide sodium salt (NS- 05) o-
HO ^\ N¼. ^ONa
N N
A
[0050] 2-(Isopropylamino) ethanol (13.0 g, 126 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (4.02 g, 17%> yield). 1H NMR (400 MHz, D20) δ 3.44 (t, 5.8 Hz, 2H), 3.21-3.15 (m, 1H), 3.08 (t, J=5.9 Hz, 2H), 1.00 (d, J=6.3 Hz, 6H). [0051] (Z)-l-Hydroxy-3-(2-hydroxyethyl)-3-methyltriaz-l-ene 2-oxide sodium salt (NS-06)
Figure imgf000020_0001
[0052] N-Methylethanolamine (2.0 g, 26.63 mmol) was added to a solution of sodium methoxide (1.44 g, 26.63 mmol, 6.0 mL of a 25% w/v solution in methanol) and ether (30 mL) with stirring at 25 °C. The flask was evacuated and then charged with nitric oxide (NO) (40 psi internal pressure) and stirred at 25 °C for 24 hrs. The product was isolated by filtration and then suspended in ether (30 mL) and stirred for 15 min. The suspension was filtered, collected and dried at 25 °C under reduced pressure to give (NS-06) as a white fine powder (2.72 g, 65% yield). 1H NMR (400 MHz, D20) δ 3.42-3.40 (m, 2H), 2.95-2.92 (m, 2H), 2.63 (s, 3H).
[0053] (Z)-3-(fert-Butyl)-l-hydroxy-3-(2-hydroxyethyl)triaz-l-ene 2-oxide sodium salt
(NS-07) ^ONa
Figure imgf000020_0002
[0054] 2-(t-Butylamino) ethanol (10.0 g, 85.6 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (3.07 g, 18% yield). 1H NMR (400 MHz, D20) δ 8.38 (s, 1H), 3.41 (t, 5.9 Hz, 2H), 3.12 (t, J=5.9 Hz, 2H), 1.11 (s, 9H).
[0055] (Z)-l-Hydroxy-3-(3-hydroxypropyl)-3-methyltriaz-l-ene 2-oxide sodium salt (NS- 09)
Figure imgf000020_0003
[0056] N-Methylamino-l-propanol (4.75 g, 53.29 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (2.80 g, 31% yield). [0057] (Z)-l-Hydroxy-3-(6-hydroxyhexyl)-3-methyltriaz-l-ene 2-oxide sodium salt (NS- 12)
Figure imgf000021_0001
[0058] 6-(Methylamino)hexan-l -ol (1.0 g, 7.62 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (1.67 g as colorless oil, 100% yield. 1H NMR (400 MHz, D20) δ 3.54-3.48 (m, 2H), 2.84-2.79 (m, 2H), 2.63 (s, 3H), 1.51-1.42 (m, 4H), 1.31-1.21 (m, 4H).
[0059] (Z)-2-Hydroxy-l-(3-hydroxyazetidin-l-yl)diazene oxide sodium salt (NS-13)
Figure imgf000021_0002
[0060] 3-Hydroxyazetidine hydrochloride (5.0 g, 46.1 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (8.98 g, >100% yield (contaminated with -25% NaCl)). 1H NMR (400 MHz, D20) δ 4.43-4.38 (m, 1H), 4.04-4.00 (m, 2H), 3.88-3.84 (m, 2H).
[0061] (5',Z)-2-Hydroxy-l-(2-(hydroxymethyl)pyrrolidin-l-yl)diazene oxide sodium salt (NS-16)
Figure imgf000021_0003
[0062] (5)-(+)-2-Pyrrolidinemethanol (5.0 g, 49.4 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of NS-01 : (4.36 g, 48%> yield). 1H NMR (400 MHz, d-DMSO) δ 3.37-3.19 (br m, 4H), 2.97-2.90 (br m, 2H), 1.87- 1.50 (m, 4H). LC tr=0.90 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
[0063] (Z)- 1 -(Azetidin- 1 -yl)-2-hydroxydiazene oxide sodium salt (NS-26)
Figure imgf000022_0001
[0064] Azetidine hydrochloride (25.0 g, 267.2 mmol) was added to a solution of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) and diethyl ether (75 mL) with stirring at 25 °C. After 5 minutes, the resulting sodium chloride precipitate was filtered and the filtrate was placed in a pressure vessel. Another aliquot of sodium methoxide (61 mL of a 25% w/v solution in methanol, 267.2 mmol) was added and the flask was evacuated with vacuum and then charged with nitric oxide (NO) (40 psi internal pressure) with stirring at 25 °C for 2 days. Diethyl ether (100 mL) was added to the resulting suspension to fully precipitate the product. The precipitate was isolated by filtration to give a white solid (13.05 g, 35% yield). 1H NMR (400 MHz, D20) δ 3.87 (t, J=7.5Hz, 4H), 2.07 (pent, J=7.5Hz, 2H).
[0065] Table 1. Sodium NONOates.
Figure imgf000022_0002
Amine NONOate Salt NONOate Salt Name
(Z)-\ -hydroxy-3 -(4-hydroxybutyl)-3 -
4-(methylamino)butan- 1 -ol NS-10
methyltriaz-l -ene 2-oxide sodium salt
(Z)-l -hydroxy-3-(5-hydroxypentyl)-3-
5-(methylamino)pentan-l -ol NS-1 1
methyltriaz-l -ene 2-oxide sodium salt
(Z)-\ -hydroxy-3 -(6-hydroxyhexyl)-3 -
6-(methylamino)hexan-l -ol NS-12
methyltriaz-l -ene 2-oxide sodium salt
(Z) -2 -hydroxy- 1 -(3-hydroxyazetidin-l - azetidin-3-ol NS-13
yl)diazene oxide sodium salt
(Z) -2 -hydroxy- 1 -(3-(hydroxymethyl)azetidin-l - azetidine-3 -methanol NS-14
yl)diazene oxide sodium salt
(Z)-\ -hydroxy-3, 3-bis(2 -hydroxyethyl)triaz-l - diethanolamine NS-15
ene 2-oxide sodium salt
(S,Z)-2-hydroxy-l -(2-
L-prolinol NS-16 (hydroxymethyl)pyrrolidin-l -yl)diazene oxide sodium salt
(Z)-l -hydroxy-3-((2S,3R)-l -hydroxy-3-
N-methyl L-isoleucinol NS-17 methylpentan-2-yl)-3-methyltriaz-l -ene 2-oxide sodium salt
(S, )-l -hydroxy-3-(l -hydroxy-4-methylpentan-
N-methyl L-leucinol NS-18
2-yl)-3-methyltriaz-l -ene 2-oxide sodium salt
(S,Z)- 1 -hydroxy-3 -( 1 -hydroxy-3 -phenylpropan-
N-methyl L-phenylalaninol NS-19
2-yl)-3-methyltriaz-l -ene 2-oxide sodium salt
(Z)-2-hydroxy-l -(3-hydroxypiperidin-l - piperidin-3-ol NS-20
yl)diazene oxide sodium salt
(Z)-2-hydroxy-l -(4-hydroxypiperidin-l - piperidin-4-ol NS-21
yl)diazene oxide sodium salt
(Z)-2-hydroxy-l -(3-(hydroxymethyl)piperidin- piperidine-3 -methanol NS-22
1 -yl)diazene oxide sodium salt
(Z)-2-hydroxy-l -(4-(hydroxymethyl)piperidin- piperidine-4-methanol NS-23
1 -yl)diazene oxide sodium salt
(Z) -2 -hydroxy- 1 -(3-hydroxypyrrolidin-l - pyrrolidin-3-ol NS-24
yl)diazene oxide sodium salt
(Z)-2-hydroxy-l -(3-(hydroxymethyl)pyrrolidin- pyrrolidine-3 -methanol NS-25
1 -yl)diazene oxide sodium salt
(Z)-\ -(azetidin-1 -yl)-2-hydroxydiazene oxide azetidine NS-26
sodium salt PHTHALIMIDE-CAPPED ALCOHOL NONOates
[0066] l-(Chloroethyl) phthalimide
Figure imgf000024_0001
[0067] N-Vinyl phthalimide (0.100 g, 0.577 mol) was treated with 4N HCl/dioxane (0.433 mL, 1.73 mmol) at 25 °C. After 3 h the reaction mixture was concentrated under reduced pressure to provide an off white solid (112 g, 93% yield). 1H NMR (400 MHz, CDC13) δ 7.95-7.89 (m, 2H), 7.83-7.77 (m, 2H), 6.32 (q, J=6.8 Hz, 1H), 2.19 (d, J=6.8 Hz, 2H), LC tr=3.05 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
[0068] Example 1 : (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-((5)-l-hydroxypropan-2- yl)-3-methyltriaz-l-ene 2-oxide
Figure imgf000024_0002
[0069] NONOate salt NS-01 (0.350 g, 2.05 mmol) suspended in acetonitrile (2.5 mL), pre- cooled in an ice-bath, was treated with a solution of l-(chloroethyl) phthalimide (0.323 g, 1.54 mmol) in acetonitrile (2.5 mL) and then sodium iodide (0.231 g, 1.54 mmol) was added. The resulting mixture was stirred for 30 minutes then the ice-bath was removed and the mixture was stirred for an additional hour. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was subjected to chromatography using ethyl acetate in hexanes to provide a tan oily solid (42 mg, 8.5% yield). 1H NMR (400 MHz, CDC13) δ
7.94-7.88 (m, 2H), 7.81-7.76 (m, 2H), 6.29 (dq, J=2.3, 6.6 Hz, 1H), 4.09-3.90 (m, 1H), 3.63-3.49 (m, 2H), 2.88 (d, J=5.6 Hz, 2H), 2.05 (d, J=6.6 Hz, 3H), 1.63-1.54 (br s, 1H), 1.04 (d, J=6.8 Hz, 3H). LC tr=2.86 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). ES(pos)MS m/z 345 (M+Na calcd for C14H18N4O5 requires 345).
[0070] Example 2: (5',Z)-l-((l,3-Dioxoisoindolin-2-yl)methoxy)-3-(l-hydroxypropan-2-yl)- 3-methyltriaz-l-ene 2-oxide
Figure imgf000025_0001
[0071] NONOate salt NS-01 (0.270 g, 1.58 mmol) suspended in acetonitrile (3.0 mL) was treated with a solution of N-(chloromethyl)phthalimide (0.280 g, 1.43 mmol) in acetonitrile (3.0 mL) and then sodium iodide (0.215 g, 1.43 mmol) was added. The resulting mixture was stirred at 25 °C overnight. N,N-dimethylformamide (1.0 mL) was added to the reaction mixture which was then heated to 60 °C for 1 hour. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was subjected to prep-chromatography using ethyl acetate in hexane to provide a tan oily solid (14 mg, 3.0% yield). 1H NMR (400 MHz, CDCI3) δ 7.92-7.87 (m, 2H), 7.78-7.73 (m, 2H), 5.67 (s, 2H), 2.97 (s, 1H), 2.19 (s, 2H), 1.59 (s, 6H). LC tr=2.65 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). ES(pos)MS m/z 331 (M+Na calcd for C13H16N4O5 requires 331).
[0072] Example 3: (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)methoxy)-3-ethyl-3-(2- hydroxyethyl)triaz-l-ene 2-oxide
Figure imgf000025_0002
[0073] N-(Chloromethyl) phthalimide (503 mg, 2.57 mmol) and NS-04 (400 mg, 2.34 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (452 mg, 63% yield). 1H NMR (400 MHz, CDC13) δ 7.97-7.78 (m, 4H), 5.80 (s, 2H), 3.68-3.66 (m, 2H), 3.36-3.31 (m, 4H), 1.12 (t, J=7.1 Hz, 3H). LC tr=2.68 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
[0074] Example 4: (Z)-l-((l,3-Dioxoisoindolin-2-yl)methoxy)-3-(2-hydroxyethyl)-3- isopropyltriaz-l-ene 2-oxide
Figure imgf000026_0001
[0075] N-(Chloromethyl) phthalimide (197 mg, 1.19 mmol) and NS-05 (200 mg, 1.08 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (177 mg, 51% yield). 1H NMR (400 MHz, CDC13) δ 7.95-7.77 (m, 4H), 5.80 (s, 2H), 3.95-3.87 (m, 1H), 3.63-3.60 (m, 2H), 3.30 (t, J=5.2 Hz, 2H), 1.15 (d, J=6.5 Hz, 6H). LC tr=2.84 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
[0076] Example 5: (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-(2-hydroxyethyl)-3- methyltriaz-l-ene 2-oxide
Figure imgf000026_0002
[0077] NS-06 (0.312 g, 1.99 mmol) and l-(chloroethyl) phthalimide (0.500 g, 2.39 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (227 mg, 31% yield). 1H NMR (400 MHz, CDC13) δ 7.94-7.88 (m, 2H), 7.82-7.76 (m, 2H), 6.27 (q, J=6.6 Hz, 1H), 3.76 (br q, J=5.1 Hz, 2H), 3.43 (t, J=5.4 Hz, 2H), 3.01 (s, 3H), 2.04 (d, J=6.6 Hz, 3H). LC tr=2.68 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
ES(pos)MS m/z 331 (M+Na calcd for Ci3Hi6N405 requires 331). [0078] Example 6: (Z)-l-((l,3-Dioxoisoindolin-2-yl)methoxy)-3-(2-hydroxyethyl)-3- methyltriaz-l-ene 2-oxide
Figure imgf000027_0001
[0079] N-(Chloromethyl) phthalimide (259 mg, 1.33 mmol) and NS-06 (250 mg, 1.59 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (257 mg, 65% yield). 1H NMR (400 MHz, CDC13) δ 7.95-7.89 (m, 2H), 7.81-7.75 (m, 2H), 5.74 (s, 2H), 3.74 (t, J=5.1 Hz, 2H), 3.47 (t, J=5.2 Hz, 2H), 3.07 (s, 3H), 2.08 (br s, 1H). LC tr=2.78 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). ES(pos)MS m/z 317 (M+Na calcd for C12H14N4O5 requires 317).
[0080] Example 7: (Z)-3-(tert-Butyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-(2- hydroxyethyl)triaz-l-ene 2-oxide
Figure imgf000027_0002
[0081] N-(Chloromethyl) phthalimide (216 mg, 1.10 mmol) and NS-07 (200 mg, 1.00 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (223 mg, 66% yield). 1H NMR (400 MHz, CDC13) δ 7.98-7.74 (m, 4H), 5.84 (s, 2H), 3.51-3.48 (m, 2H), 3.24-3.20 (m, 2H), 1.58 (s, 9H). LC tr=3.06 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
[0082] Example 8: (Z)-l-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-3-(3-hydroxypropyl)-3- methyltriaz-l-ene 2-oxide
Figure imgf000027_0003
[0083] NS-09 (0.395 g, 2.31 mmol) and l-(chloroethyl) phthalimide (0.441 g, 210 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (90 mg, 13% yield). LC tr=2.80 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
ES(pos)MS m/z 345 (M+Na calcd for Ci4Hi8N405 requires 345).
[0084] Example 9: (2)-2-(l-(l,3-Dioxoisoindolin-2-yl)ethoxy)-l-(3-hydroxyazetidin-l- yl)diazene oxide
Figure imgf000028_0001
[0085] l-(Chloroethyl) phthalimide (500 mg, 2.39 mmol) and NS-13 (444 mg, 2.86 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 1 : (89 mg, 12% yield). 1H NMR (400 MHz, CDC13) δ 7.92-7.89 (m, 2H), 7.80-7.78 (m, 2H), 5.72 (q, J=6.6 Hz, 1H), 4.48-4.42 (m, 1H), 4.30-4.24 (m, 2H), 4.01- 3.92 (m, 2H), 2.02 (d, J=6.6 Hz, 3H). LC tr=2.71 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C).
ES(pos)MS m/z 329 (M+Na calcd for Ci3Hi4N405 requires 329).
[0086] Example 10: (Z)-2-(l-(l,3-Dioxoisoindolin-2-yl)methoxy)-l-(3-hydroxyazetidin-l- yl)diazene oxide
Figure imgf000028_0002
[0087] N-(Chloromethyl) phthalimide (1.05 g, 5.38 mmol) and NS-13 (1.0 g, 6.45 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (614 mg, 39% yield). 1H NMR (400 MHz, CDC13) δ 7.95-7.92 (m, 2H), 7.82-7.79 (m, 2H), 5.72 (s, 2H), 4.58-4.49 (m, 1H), 4.36-4.29 (m, 2H), 4.06-3.99 (m, 2H), 2.53 (d, J=6.3 Hz, 1H). LC tr=2.45 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C). ES(pos)MS m/z 315 (M+Na calcd for Ci2Hi2N405 requires 315). [0088] Example 1 1 : (^,Z)-2-((l ,3-Dioxoisoindolin-2-yl)methoxy)-l-(2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
Figure imgf000029_0001
[0089] N-(Chloromethyl) phthalimide (587 mg, 3.00 mmol) and NS-16 (500 mg, 2.73 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 2: (307 mg, 35% yield). 1H NMR (400 MHz, d-DMSO) δ 7.97-7.94 (m, 2H), 7.83-7.80 (m, 2H), 5.74 (s, 2H), 4.17-4.06 (m, 1H), 3.77-3.57 (m, 4H), 2.81 (t, J=5.9 Hz, 1H), 2.1 1-2.04 (m, 1H), 1.98-1.91 (m, 1H), 1.85-1.78 (m, 2H). LC tr=2.73 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
[0090] Table 2. Phthalimide-Capped Alcohol NONOates.
Figure imgf000029_0002
(¾- 1 -( 1 -( 1 ,3 -dioxoisoindolin-2-yl)meth^
1-ene 2-oxide
(Z)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-l-((S)-2-(hydroxymethyl)azetidin-l-yl)diazene oxide
(S,Z)-2-((l,3-dioxoisoindolin-2-yl)methoxy)-l-(2-(hydroxymethyl)azetidin-l-yl)diazene oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-ethyl-3-(2-hydroxyethyl)triaz-l-ene 2-oxide
(Z)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(2-hydroxyethyl)-3-isopropyltriaz-l-ene 2-oxide
(Z)-3-(tert-butyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(2-hydroxyethyl)triaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(l-hydroxy-2-methylpropan-2-yl)-3-methyltriaz-l- ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-(l-hydroxy-2-methylpropan-2-yl)-3-methyltriaz-l- ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-(3-hydroxypropyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(4-hydroxybutyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-(4-hydroxybutyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(5-hydroxypentyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-(5-hydroxypentyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-(6-hydroxyhexyl)-3-methyltriaz-l-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-(6-hydroxyhexyl)-3-methyltriaz-l-ene 2-oxide
( )-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-l-(3-(hydroxymethyl)azetidin-l-yl)diazene oxide
( )-2-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-l-(3-(hydroxymethyl)azetidin-l-yl)diazene oxide
(Z)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3,3-bis(2-hydroxyethyl)triaz-l-ene 2-oxide
(Z)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3,3-bis(2-hydroxyethyl)triaz-l-ene 2-oxide
(Z)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-l-((S)-2-(hydroxymethyl)pyrrolidin-l-yl)diazene oxide
(Z)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-((2S,3R)-l-hydroxy-3-methylpentan-2-yl)-3- methyltriaz- 1-ene 2-oxide
(Z)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-((2S,3R)-l-hydroxy-3-methylpentan-2-yl)-3- methyltriaz- 1-ene 2-oxide
( )-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-((S)-l-hydroxy-4-methylpentan-2-yl)-3-methyltriaz- 1-ene 2-oxide
(S, )-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-(l-hydroxy-4-methylpentan-2-yl)-3-methyltriaz-l- ene 2-oxide 37 (Z)-l -(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-3-((S)-l -hydroxy-3-phenylpropan-2-yl)-3-methyltriaz- 1 -ene 2-oxide
38 (S,Z)- 1 -(( 1 ,3 -dioxoisoindolin-2-yl)methoxy)-3 -( 1 -hydroxy-3 -phenylpropan-2-yl)-3 -methyltriaz- 1 - ene 2-oxide
39 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(3-hydroxypiperidin-l -yl)diazene oxide
40 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(3 -hydroxypiperidin- 1 -yl)diazene oxide
41 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(4-hydroxypiperidin-l -yl)diazene oxide
42 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(4-hydroxypiperidin- 1 -yl)diazene oxide
43 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(3-(hydroxymethyl)piperidin-l -yl)diazene oxide
44 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(3 -(hydroxymethyl)piperidin- 1 -yl)diazene oxide
45 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(4-(hydroxymethyl)piperidin-l -yl)diazene oxide
46 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(4-(hydroxymethyl)piperidin- 1 -yl)diazene oxide
47 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(3-hydroxypyrrolidin-l -yl)diazene oxide
48 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(3 -hydroxypyrrolidin- 1 -yl)diazene oxide
49 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)ethoxy)-l -(3-(hydroxymethyl)pyrrolidin-l -yl)diazene oxide
50 (Z)-2-( 1 -(1 ,3-dioxoisoindolin-2-yl)methoxy)- 1 -(3 -(hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
501 (Z)-2-(l -( 1 ,3-dioxoisoindolin-2-yl)ethoxy)- 1 -(azetidin- 1 -yl)diazene oxide
502 (Z)-2-(l -(l ,3-dioxoisoindolin-2-yl)methoxy)-l -(azetidin-l -yl)diazene oxide
PHTHALIMIDE-CAPPED CARBOXYLIC ACID NONOATES
[0091] Example 51 : (Z)-3-(2-Carboxyethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
Figure imgf000031_0001
[0092] Sodium metaperiodate (0.225 g, 1.05 mmol) was added to a mixture of Example 8 (0.081 g, 0.251 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a colorless oil (82 mg, 97% yield). 1H NMR (400 MHz, CDCls) δ 7.91 (d, J=5.5 Hz, 2H), 7.90 (d, J=5.5 Hz, 2H), 7.78 (d, J=5.5 Hz, 1H), 7.76 (d, J=5.5 Hz, 2H), 6.27 (q, J=6.6 Hz, 1H), 3.61-3.55 (dt, J=2.2,6.8 Hz, 2H), 2.99 (s, 3H), 2.61- 2.55 (dt, J=l .5,6.8 Hz, 2H), 2.04 (d, J=6.6 Hz, 3H). LC tr=2.85 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
[0093] Example 52: (5',Z)-l-(2-Carboxypyrrolidin-l-yl)-2-((l,3-dioxoisoindolin-2- yl)methoxy)diazene oxide
Figure imgf000032_0001
[0094] Example 11 (200 mg, 0.62 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (165 mg, 80%> yield). 1H NMR (400 MHz, CDCI3) δ 7.97-7.77 (m, 4H), 5.74 (s, 2H), 4.59-4.53 (m, 1H), 3.84- 3.79 (m, 2H), 3.69-3.62 (m, 2H), 2.31-2.25 (m, 2H). LC tr=2.71 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C). ES(pos)MS m/z 357 (M+Na calcd for Ci4Hi4N406 requires 357).
[0095] Example 53: (Z)-3-(Carboxymethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
Figure imgf000032_0002
[0096] Example 5 (0.227 g, 0.736 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (224 mg, 95% yield). 1H NMR (400 MHz, CDCI3) δ 7.95-7.89 (m, 4H), 7.88-7.83 (m, 4H), 6.23 (q, J=6.6 Hz, 1H), 4.27-4.13 (m, 2H), 3.15 (s, 3H), 1.97 (d, J=6.6 Hz, 3H). LC tr=2.80 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). [0097] Example 54: (Z)-3-(Carboxymethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3- methyltriaz-l-ene 2-oxide
Figure imgf000033_0001
[0098] Example 6 (328 mg, 1.11 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 51 : (253 mg, 74% yield). Ή
NMR (400 MHz, CD30D) δ 7.97-7.87 (m, 4H), 5.71 (s, 2H), 4.27 (s, 2H), 3.23 (s, 3H). LC tr=2.59 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 331 (M+Na calcd for C12H12N4O6 requires 331).
[0099] Example 54 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 54 as a solid. Carboxylic acids listed in Table 3 are converted to the alkaline or ammonium salts by similar methods (i.e. lithium, sodium, potassium, calcium, ammonium, diethanolamine, diethylamine, diethylaminoethanol, epolamine, ethanolamine, ethylenediamine, lysine, meglumine, morpholineethanol, niacinamide, piperazine, tert-butylamine (erbumine), thiamine, trolamine, etc.).
[00100] Table 3. Phthalimide-Capped Carboxylic Acid NONOates.
Figure imgf000033_0002
oxide
( )-3-(2-carboxyethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-ene 2-oxide
(Z)-3 -(3-carboxypropyl)- 1 -( 1 -( 1 ,3 -dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz- 1 -ene 2-oxide
( )-3-(3-carboxypropyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-ene 2-oxide
(Z)-3 -(4-carboxybutyl)-l -( 1 -( 1 ,3 -dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz- 1 -ene 2-oxide
( )-3-(4-carboxybutyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-ene 2-oxide
( )-3-(5-carboxypentyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz-l-ene 2-oxide
(Z)-3 -(5-carboxypentyl)- 1 -(( 1 ,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz- 1 -ene 2-oxide
( )-l-(3-carboxyazetidin-l-yl)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)diazene oxide
( )-l-(3-carboxyazetidin-l-yl)-2-((l,3-dioxoisoindolin-2-yl)methoxy)diazene oxide
(Z)-l-((S)-2-carboxypyrrolidin-l-yl)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)diazene oxide
( )-3-(carboxymethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-ethyltriaz-l-ene 2-oxide
( )-3-(carboxymethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-ethyltriaz-l-ene 2-oxide
(Z)-3 -(carboxymethyl)- 1 -(1 -( 1 ,3 -dioxoisoindolin-2-yl)ethoxy)-3-isopropyltriaz- 1 -ene 2-oxide
(Z)-3 -(carboxymethyl)- 1 -(( 1 ,3-dioxoisoindolin-2-yl)methoxy)-3-isopropyltriaz- 1 -ene 2-oxide
( )-3-((S)-l-carboxyethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz-l-ene 2-oxide
(S, )-3-(l-carboxyethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-ene 2-oxide
(Z)-3-((lS,2R)-l-carboxy-2-methylbutyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
(Z)-3-((lS,2R)-l-carboxy-2-methylbutyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3- methyltriaz-l-ene 2-oxide
(Z)-3 -((S)-\ -carboxy-3 -methylbutyl)- 1 -(1 -( 1 ,3 -dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz- 1 - ene 2-oxide
(S,Z)-3 -( 1 -carboxy-3 -methylbutyl)- 1 -((1 ,3 -dioxoisoindolin-2-yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
( )-3-((S)-l-carboxy-2-phenylethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz-l- ene 2-oxide
(S, )-3-(l-carboxy-2-phenylethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-ene 2-oxide
( )-3-((S)-l-carboxy-2-methylpropyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)-3-methyltriaz-l- ene 2-oxide (S, )-3-(l-carboxy-2-methylpropyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)-3-methyltriaz-l-
82 ene 2-oxide
83 (Z)-3-(tert43utyl)-3-(carboxymethyl)-l-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)triaz-l-ene 2-oxide
84 ( )-3-(tert43utyl)-3-(carboxymethyl)-l-((l,3-dioxoisoindolin-2-yl)methoxy)triaz-l-ene 2-oxide
85 (Z)-l-(3-carboxypiperidin-l-yl)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)diazene oxide
86 (Z)-l-(3-carboxypiperidin-l-yl)-2-((l,3-dioxoisoindolin-2-yl)methoxy)diazene oxide
87 (Z)-l-(4-carboxypiperidin-l-yl)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)diazene oxide
88 (Z)-l-(4-carboxypiperidin-l-yl)-2-((l,3-dioxoisoindolin-2-yl)methoxy)diazene oxide
89 (Z)-l-(3-carboxypyrrolidin-l-yl)-2-(l-(l,3-dioxoisoindolin-2-yl)ethoxy)diazene oxide
90 (Z)-\ -(3-carboxypyrrolidin-l -yl)-2-((l ,3-dioxoisoindolin-2-yl)methoxy)diazene oxide
CARBONATE-CAPPED ALCOHOL NONOATES [00101] 1 -Chloroethyl tert -butyl carbonate
CI XJ <
[00102] tert-Butyl alcohol (3.34 mL, 35 mmol) and pyridine (3.39 mL, 42 mmol) were dissolved in 90 mL dichloromethane and cooled to -78 °C. 1-Chloroethyl chloroformate (5.0 g, 35 mmol) was added dropwise and the reaction was slowly allowed to warm to room temperature, then stirred for 3 days. The rxn was evaporated, dissolved in ethyl acetate, then washed with water and brine, dried over magnesium sulfate and evaporated. It was noted that product evaporated under high vacuum. The product was distilled to yield 1 -chloroethyl tert-butyl carbonate as a colorless oil (2.46 g, 39% yield). ). 1H NMR (400 MHz, CDC13) δ 6.41 (d, J=5.6 Hz, 1H), 1.82 (d, J=5.6 Hz, 3H), 1.53 (s, 9H).
[00103] Example 91 : (25,Z)-1-Hydroxy-2,3,7,11,1 l-pentamethyl-9-oxo-6,8,10-trioxa- 3,4,5-triazadodec-4-ene 4-oxide
T A- [00104] NS-01 (0.350 g, 2.05 mmol) suspended in acetonitrile (2.0 mL) was treated with a solution of 1-chloroethyl tert -butyl carbonate (0.336 g, 1.86 mmol) in acetonitrile (1.0 mL) and then sodium iodide (1.25 g, 8.37 mmol) was added. The resulting mixture was heated to 60 °C for 3 hours. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was subjected to prep-chromatography using ethyl acetate in hexanes to provide an amber oil (29 mg, 5.3% yield). 1H NMR (400 MHz, CDC13) δ 6.45 (dq, J=2.5, 5.6 Hz, 1H), 4.21-3.75 (m, 1H), 3.71-3.54 (m, 2H), 2.98 (d, J=3.5 Hz, 3H), 1.65 (d, J=5.6 Hz, 3H), 1.52 (s, 9H), 1.15-1.11 (m, 3H). LC tr=3.20 minutes (C-18 column, 5 to 95%
acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
ES(pos)MS m/z 316 (M+Na calcd for CnH23N306 requires 316).
[00105] Example 92: (Z)-3-Ethyl-l-hydroxy-7,l 1,1 l-trimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000036_0001
[00106] 1-Chloroethyl tert -butyl carbonate (500 mg, 2.77 mmol) and NS-04 (521 mg, 3.05 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (421 mg, 52% yield). 1H NMR (400 MHz, CDC13) δ 6.47 (q, J=5.6 Hz, 1H), 3.72-3.70 (m, 2H), 3.38-3.13 (m, 4H), 1.66 (d, J=5.6 Hz, 3H), 1.52 (s, 9H), 1.16 (t, J=7.1 Hz, 3H). LC tr=3.19 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 316 (M+Na calcd for CiiH23N306 requires 316).
[00107] Example 93: (Z)-l-Hydroxy-3-isopropyl-7,l 1,1 l-trimethyl-9-oxo-6,8,10-trioxa- 3,4,5-triazadodec-4-ene 4-oxide
Figure imgf000036_0002
[00108] NS-05 (500 mg, 2.7 mmol) and 1-chloroethyl t-butyl carbonate (488 mg, 2.7 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (465 mg, 56% yield). 1H NMR (400 MHz, CDC13) δ 6.46 (q, J=5.6 Hz, 1H), 3.85-3.79 (m, 1H), 3.68-3.65 (m, 2H), 3.38-3.23 (m, 2H), 1.65 (d, J=5.6 Hz, 3H), 1.52 (s, 9H), 1.18 (dd, J=5.6, 3.8 Hz, 6H). LC tr=3.40 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
ES(pos)MS m/z 330 (M+Na calcd for C12H25N3O6 requires 330).
[00109] Example 94: (Z)-l-Hydroxy-3,7-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec- 4-ene 4-oxide
Figure imgf000037_0001
[00110] NS-06 (0.250 g, 1.59 mmol) was suspended in acetonitrile (3.0 mL) and to this was added a solution of 1 -chloroethyl ethyl carbonate (0.291 g, 1.91 mmol) in acetonitrile (2.0 mL) and sodium iodide (0.286 g, 1.91 mmol) and the resulting mixture was stirred at 25 °C overnight. The acetonitrile was removed under reduced pressure and the residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was chromatographed using 50% ethyl acetate in hexanes to give an oily residue (61 mg, 15% yield). 1H NMR (400 MHz, CDC13) δ 6.44 (q, J=5.6 Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 3.76 (t, J=5.1 Hz, 2H), 3.53-3.42 (m, 2H), 3.08 (s, 3H), 2.19-1.76 (br s, 1H), 1.64 (d, J=5.6 Hz, 3H), 1.32 (t, J=7.1 Hz, 3H). LC tr=2.60 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
ES(pos)MS m/z 274 (M+Na calcd for C8Hi7N306 requires 274).
[00111] Example 95: (Z)-l-Hydroxy-3,7,l 1,1 l-tetramethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000037_0002
[00112] 1-Chloroethyl tert -butyl carbonate (2.0 g, 11.07 mmol) and NS-06 (1.4 g, 7.26 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (1.75 g, 51% yield). 1H NMR (400 MHz, CDCI3) δ 6.44 (q, J=5.6 Hz, 1H), 3.80 (t, J=5.0 Hz, 2H), 3.50 (dt, J=5.0, 1.2 Hz, 2H), 3.10 (s, 3H), 1.64 (d, J=5.6 Hz, 3H), 1.52 (s, 9H). LC tr=3.02 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 302 (M+Na calcd for C10H21N3O6 requires 302).
[00113] Example 96: (Z)-3-(2-Hydroxyethyl)-2,2,7,l 1,1 l-pentamethyl-9-oxo-6,8,10- trioxa-3,4,5-triazadodec-4-ene 4-oxide
Figure imgf000038_0001
[00114] NS-07 (500 mg, 2.51 mmol) and 1-chloroethyl tert -butyl carbonate (454 mg, 2.51 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (240 mg, 30% yield). 1H NMR (400 MHz, CDC13) δ 6.47 (q, J=5.6 Hz, 1H), 3.56 (t, J=4.9 Hz, 2H), 3.30-3.20 (m, 2H), 1.65 (d, J=5.6 Hz, 3H), 1.51 (s, 9H), 1.26 (s, 9H). LC tr=3.61 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 344 (M+Na calcd for C13H27N3O6 requires 344).
[00115] Example 97: (Z)-13-Hydroxy-2,2,6,10-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10- triazatridec-8-ene 9-oxide
Figure imgf000038_0002
[00116] NS-09 (2.30 g, 13.44 mmol) suspended in acetonitrile (10.0 mL) was treated with a solution of 1 -chloroethyl tert -butyl carbonate (1.87 g, 10.35 mmol) in acetonitrile (10.0 mL) and then sodium iodide (1.55 g, 10.35 mmol) was added. The resulting mixture was stirred at ambient temperature for 4 days then heated to 60 °C for 32.5 hours. The acetonitrile was removed under reduced pressure. The residue was suspended in ethyl acetate and then washed with a 0.2 N solution of sodium thiosulfate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was subjected to chromatography using ethyl acetate in hexanes to provide a yellow oil (390 mg, 13% yield). 1H NMR (400 MHz, CDCI3) δ 6.44 (q, J=5.6 Hz, 1H), 3.78-3.71 (dt, J=2.8,6.8 Hz, 2H), 3.04 (s, 3H), 2.03-1.94 (br t, 1H), 1.85-1.76 (m, 2H), 1.65 (d, J=5.6 Hz, 3H), 1.52 (s, 9H). LC tr=3.12 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). ES(pos)MS m/z 316 (M+Na calcd for
C11H23N3O6 requires 316).
[001 17] Example 98: (Z)-16-Hydroxy-2,2,6,10-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10- triazahexadec-8-ene 9-oxide
Figure imgf000039_0001
[001 18] 1-Chloroethyl tert -butyl carbonate (192 mg, 1.17 mmol) and NS-12 (250 mg, 1.17 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (614 mg, 39% yield). 1H NMR (400 MHz, CDC13) δ 6.42 (q, J=5.6 Hz, 1H), 3.66 (t, J=6.5 Hz, 2H), 3.36-3.32 (m, 2H), 3.00 (s, 3H), 1.63 (d, J= 5.6 Hz, 3H), 1.60-1.53 (m, 4H), 1.51 (s, 9H), 1.42-1.37 (m, 4H). LC tr=3.62 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C). ES(pos)MS m/z 358 (M+Na calcd for Ci4H29N306 requires 358).
[001 19] Example 99: (Z)-2-(l-((tert-Butoxycarbonyl)oxy)ethoxy)-l-(3-hydroxyazetidin-l- yl)diazene oxide
Figure imgf000039_0002
[00120] 1-Chloroethyl tert -butyl carbonate (430 mg, 2.38 mmol) and NS-13 (444 mg, 2.86 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (121 mg, 18% yield). 1H NMR (400 MHz, CDC13) δ 6.44-6.35 (bm, 1H), 4.63-4.49 (bm, 1H), 4.42-4.29 (bm, 2H). 4.10-3.99 (bm, 2H), 2.43-2.34 (bs, 1H), 1.69-1.59 (bs, 3H), 1.52 (bs, 9H). LC tr=3.03 minutes (C-18 column, 5 to 95%
acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C).
ES(pos)MS m/z 300 (M+Na calcd for Ci0Hi9N3O6 requires 300).
[00121] Example 100: (Z)-2-(l-((Ethoxycarbonyl)oxy)ethoxy)-l-((5)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
Figure imgf000040_0001
[00122] 1-Chloroethyl ethyl carbonate (5.0 g, 32.8 mmol) was dissolved in 125 mL acetonitrile. Sodium iodide (22.1 g, 147.5 mmol) was added and stirred at 60 °C for 1.5 hours. The reaction was evaporated, diluted with ether and filtered. The filtrate was evaporated to a dark red oil, to afford 1-iodoethyl ethyl carbonate (4.12 g, 51% yield). NS- 16 (2.07 g, 11.3 mmol) and freshly made 1-iodoethyl ethyl carbonate (4.12 g, 16.9 mmol) were dissolved in 15 mL acetonitrile and stirred at room temperature overnight. The reaction was evaporated, diluted with ethyl acetate, washed with 0.2N Na2S203 and brine, dried over magnesium sulfate and evaporated. Chromatography was performed using 50%> ethyl acetate/hexanes to yield a colorless oil (890 mg, 28% yield). 1H NMR (400 MHz, d- DMSO) δ 6.28 (dq, J=2.6, 5.6 Hz, IH), 4.76 (dt, J=2.9, 5.7 Hz, IH), 4.12 (q, J=7.1 Hz, 2H), 3.97-3.86 (m, IH), 3.55-3.31 (m, 4H), 1.96-1.77 (m, 4H), 1.47 (d, J=5.5 Hz, 3H), 1.17 (dt, J=18.4, 7.1 Hz, 3H). LC tr=2.98 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 300 (M+Na calcd for C10H19N3O6 requires 300).
[00123] Example 101 : (Z)-2-(l-((tert-Butoxycarbonyl)oxy)ethoxy)-l-((5)-2- (hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
Figure imgf000040_0002
[00124] 1-Chloroethyl tert -butyl carbonate (448 mg, 2.48 mmol) and NS-16 (500 mg, 2.73 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (223 mg, 29% yield). 1H NMR (400 MHz, d-DMSO) δ 6.43 (dq, J=1.0, 5.6 Hz, IH), 4.19-4.07 (m, IH), 3.82-3.56 (m, 3H), 2.91-2.79 (m, IH), 2.13-2.05 (m, 2H), 2.00-1.93 (m, 2H), 1.87-1.79 (m, 2H), 1.64 (d, J=5.6 Hz, 3H), 1.52 (d, J=1.4, 9H). LC tr=3.30 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). [00125] Example 102: (Z)-l-Hydroxy-3,l l-dimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000041_0001
[00126] Chloromethyl isopropyl carbonate (1.75 g, 11.47 mmol) and NS-06 (1.98 g, 12.62 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (1.9 g, 66% yield). 1H NMR (400 MHz, CDC13) δ 5.81 (s, 1H), 4.95 (dq, J=6.3, 6.3 Hz, 1H), 3.83-3.80 (m, 2H), 3.57-3.53 (m, 2H), 3.14 (s, 3H), 1.30 (d, J=6.3 Hz, 6H). LC tr=2.61 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 274 (M+Na calcd for C8Hi7N306 requires 274).
[00127] 1 -Iodoethyl isopropyl carbonate
Figure imgf000041_0002
[00128] To a solution of 2-propanol (2.66 mL, 34.97 mmol) and pyridine (3.39 mL, 41.96 mmol), pre-cooled in a dry-ice/acetone bath was slowly added 1 -chloroethyl chloro formate. The resulting mixture was allowed to slowly warm to 25 °C overnight, with stirring. After concentration under reduced pressure the mixture was dissolved in ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to give 1 -chloroethyl isopropyl carbonate as a pink/orange oil (5.11 g, 88% yield).
[00129] Sodium iodide (20.69 g, 138.0 mmol) was added to the crude solution of chloroethyl isopropyl carbonate (5.11 g, 30.67 mmol) in acetonitrile (50 mL). The resulting mixture was heated to 60 °C for 2 h and then allowed to cool to 25 °C. The reaction mixture was concentrated under reduced pressure and the resulting mixture was treated with diethyl ether. The solids were removed by filtration and the filtrate was concentrated under reduced pressure to obtain 1 -iodoethyl isopropyl carbonate as a dark orange oil (5.43 g, 69%> yield).
[00130] Example 103: (Z)-l-Hydroxy-3,7,l l-trimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000042_0001
[00131] NS-06 (0.250 g, 1.59 mmol) and 1-iodoethyl isopropyl carbonate (0.492 g, 1.91 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 100, with the exception no sodium iodide was added: (141 mg, 33% yield). 1H NMR (400 MHz, CDC13) δ 6.45 (q, J=5.6 Hz, IH), 4.89 (sep, J=6.3 Hz, IH), 3.77 (t, J=5.2 Hz, 2H), 3.53-3.42 (m, 2H), 3.08 (s, 3H), 1.64 (d, J=5.6 Hz, 3H), 1.31 (d, J=6.3 Hz, 3H). LC tr=2.96 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C). ES(pos)MS m/z 288 (M+Na calcd for C9H19N3O6 requires 288).
[00132] Example 104: (Z)-3 -Ethyl- 1 -hydroxy- 1 l-methyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000042_0002
[00133] 1-Chloromethyl isopropyl carbonate (1.0 g, 6.55 mmol) and NS-04 (1.23 g, 7.21 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 91 : (312 mg, 18% yield). 1H NMR (400 MHz, CDCI3) δ 5.83 (s, 2H), 4.97-4.92 (m, IH), 3.74-3.71 (m, 2H), 3.39-3.32 (m, 4H), 1.33 (d, J=6.3 Hz, 6H), 1.16 (t, J=7.1 Hz, 3H). LC tr=2.77 minutes (C-18 column, 5 to 95%> acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 288 (M+Na calcd for C H1 N3O6 requires 288).
[00134] Example 105: (Z)-3-Ethyl-l-hydroxy-7,l l-dimethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000042_0003
[00135] 1-Chloroethyl isopropyl carbonate (1.0 g, 6.0 mmol) and NS-04 (1.13 g, 6.6 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 100: (898 mg, 53% yield). 1H NMR (400 MHz, CDCI3) δ 6.50 (q, J=5.6 Hz, 1H), 4.95-4.90 (m, 1H), 3.72-3.68 (m, 2H), 3.40-3.31 (m, 2H), 3.31-3.22 (m, 2H), 1.67 (d, J=5.6 Hz, 3H), 1.33 (dd, J=6.3, 1.6 Hz, 6H), 1.14 (t, J=7.1 Hz, 3H). LC tr=3.90 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 302 (M+Na calcd for CioH2iN306 requires 302).
[00136] Table 4. Carbonate-Capped Alcohol NONOates.
Figure imgf000043_0001
(S,Z)-l l-(hydroxymethyl)-2,10,12-trimethyl-4-oxo-3,5 ,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
(S,Z)-l l-(hydroxymethyl)-2,2, 10, 12-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
(Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -((iS)-2-(hydroxymethyl)azetidin- 1 -yl)diazene oxide
(Z)-2-( 1 -((fert-butoxycarbonyl)oxy)ethoxy) - 1 -((iS)-2-(hydroxymethyl)azetidin- 1 -yl)diazene oxide
(Z)-3-ethyl-l -hydroxy-7-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
(Z)-l-hydroxy-3-isopropyl-7-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
(Z)-3-(2-hydroxyethyl)-2,2,7-trimethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
(Z)-l-hydroxy-2,2,3,7-tetramethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
(Z)-l-hydroxy-2,2,3,7,l 1,1 l -hexamethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
(Z)-13-hydroxy-6,10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatridec-8-ene 9-oxide
(Z)-14-hydroxy-6,10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
(Z)-14-hydroxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
(Z)-14-hydroxy-2,2,6, 10-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
(Z)-15-hydroxy-6,10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
(Z)-15-hydroxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazapentadec-8-ene 9-oxide
(Z)-15-hydroxy-2,2,6, 10-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
(Z)-15-hydroxy-2,10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
(Z)-15-hydroxy-2,2,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazapentadec-8-ene 9-oxide
(Z)-16-hydroxy-6,10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazahexadec-8-ene 9-oxide
(Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -hydro xyazetidin- 1 -yl)diazene oxide
(Z)- 1 -(3 -hydro xyazetidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
(Z)- 1 -(3 -hydro xyazetidin- 1 -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
(Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(3 -hydro xyazetidin- 1 -yl)diazene oxide
(Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -(hydro xymethyl)azetidin- 1 -yl)diazene oxide
(Z)- 1 -(3 -(hydro xymethyl)azetidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
(Z)-2-( 1 -((tert-butoxycarbonyl)oxy)ethoxy) - 1 -(3 -(hydroxymethyl)azetidin- 1 -yl)diazene oxide
(Z)- 1 -(3 -(hydro xymethyl)azetidin- 1 -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
(Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(3 -(hydroxymethyl)azetidin- 1 -yl)diazene oxide
(Z)-l-hydroxy-3-(2-hydroxyethyl)-7-methyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide 142 (Z)-l-hydroxy-3-(2-hydroxyethyl)-7, l l-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
143 (Z)-l-hydroxy-3-(2-hydroxyethyl)-l l-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
144 (Z)-l -hydro xy-3 -(2-hydroxyethyl)- 11,11 -dimethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
145 (Z)- 1 -((5)-2-(hydroxymethyl)pyrrolidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
146 (iS)-l-(2-(hydroxymethyl)pyrrolidin-l -yl)-2-(((isopropoxycarbonyl)oxy)rnethoxy)diazene oxide
147 (iS',Z)-2-(((tert-butoxycarbonyl)oxy)methoxy) - 1 -(2-(hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
148 (1 \S,\2R,Z)- \ l-(hydroxymethyl)-6,10,12-trirnethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
149 (1 \S,\2R,Z)- \ l-(hydroxymethyl)-2,10,12-trirnethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
150 (1 \S,Z)-\ l-(hydroxymethyl)-6,10,13-trirnethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
151 (S,Z)-l l-(hydroxymethyl)-2,10,13-trirnethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
152 (2iS,z)-2-benzyl-l -hydroxy-3,7-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
153 (S,Z)-2 -benzyl- 1 -hydro xy-3, 1 l-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
154 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -hydro xypiperidin- 1 -yl)diazene oxide
155 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(4-hydro xypiperidin- 1 -yl)diazene oxide
156 (Z)- 1 -(4-hydro xypiperidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
157 (Z)- 1 -(4-hydro xypiperidin- 1 -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
158 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -(hydro xymethyl)pyrrolidin- 1 -yl)diazene oxide
159 (Z)- 1 -(3 -(hydro xymethyl)pyrrolidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
160 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(4-(hydroxymethyl)piperidin- 1 -yl)diazene oxide
161 (Z)- 1 -(4-(hydroxymethyl)piperidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
162 (Z)-2-( 1 -((fert-butoxycarbonyl)oxy)ethoxy) - 1 -(4-(hydroxymethyl)piperidin- 1 -yl)diazene oxide
163 (Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(4-(hydroxymethyl)piperidin- 1 -yl)diazene oxide
164 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -hydro xypyrrolidin- 1 -yl)diazene oxide
165 (Z)-2-( 1 -((fert-butoxycarbonyl)oxy)ethoxy) - 1 -(3 -hydro xypyrrolidin- 1 -yl)diazene oxide
166 (Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(3 -hydro xypyrrolidin- 1 -yl)diazene oxide
167 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy) - 1 -(3 -(hydro xymethyl)pyrrolidin- 1 -yl)diazene oxide
168 (Z)-2-( 1 -((fert-butoxycarbonyl)oxy)ethoxy) - 1 -(3 -(hydroxymethyl)pyrrolidin- 1 -yl)diazene oxide
169 (Z)- 1 -(3 -(hydro xymethyl)pyrrolidin- 1 -yl)-2-((isobutyryloxy)methoxy)diazene oxide
510 (Z)-2-( 1 -((tert-butoxycarbonyl)oxy)ethoxy) - 1 -(azetidin- 1 -yl)diazene oxide 511 (Z)-2-( 1 -((ethoxycarbonyl)oxy)ethoxy)- 1 -(azetidin- 1 -yl)diazene oxide
512 (Z)- 1 -(azetidin- 1 -yl)-2-( 1 -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
513 (Z)- 1 -(azetidin- 1 -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
514 (Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(azetidin- 1 -yl)diazene oxide
CARBONATE-CAPPED CARBOXYLIC ACID NONOATES
[00137] Example 170: (Z)-l-Carboxy-2,6-dimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec-3- ene 3 -oxide
Figure imgf000046_0001
[00138] Sodium metaperiodate (0.467 g, 2.18 mmol) was added to a mixture of Example 94 (0.130 g, 0.52 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a colorless oil (111 mg, 80% yield). 1H NMR (400 MHz, CDC13) δ 6.46 (q, J=5.6 Hz, 1H), 4.28 (q, J=18.1 Hz, 2H), 4.25 (q, J=7.1 Hz, 2H), 3.29 (s, 3H), 1.65 (d, J=5.6 Hz, 3H), 1.34 (t, J=7.1 Hz, 3H). LC tr=2.74 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 288 (M+Na calcd for CgHisNsO? requires 288), ES(neg)MS m/z 264 (M-H calcd for C8H15N3O7 requires 264).
[00139] Example 171 : (Z)-l-Carboxy-2,6,10,10-tetramethyl-8-oxo-5,7,9-trioxa-2,3,4- triazaundec-3-ene 3 -oxide
Figure imgf000046_0002
[00140] Example 95 (400 mg, 1.44 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (352 mg, 83% yield). 1H NMR (400 MHz, CDC13) δ 6.44 (q, J=5.6 Hz, IH), 4.29 (q, J=18.1 Hz, 2H), 3.28 (s, 3H), 1.63 (d, J=5.6 Hz, 3H), 1.52 (s, 9H). LC tr=5.04 minutes (C-18 column, 5 to 95%
acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C).
ES(pos)MS m/z 316 (M+Na calcd for Ci0Hi9N3O7 requires 316).
[00141] Example 172: (Z)-l-Carboxy-3,7, l 1 ,1 l -tetramethyl-9-oxo-6,8,10-trioxa-3,4,5- triazadodec-4-ene 4-oxide
Figure imgf000047_0001
[00142] Sodium metaperiodate (0.983 g, 4.60 mmol) was added to a mixture of Example 97 (0.321 g, 1.09 mmol) and ruthenium(III)chloride (cat) in acetonitrile (2.5 mL), ethyl acetate (2.5 mL) and water (4.0 mL). The resulting mixture was stirred at ambient temperature for 5.0 h and then diluted with water and filtered through a pad of celite. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide a brown solid (335 mg, 100% yield). 1H NMR (400 MHz, CDC13) δ 6.43 (q, J=5.6 Hz, IH), 3.72-3.60 (dt, J=3.2,6.8 Hz, 2H), 3.08 (s, 3H), 2.69 (t, J=6.8 Hz, 2H), 1.64 (d, J=5.6 Hz, 3H), 1.52 (s, 9H). LC tr=3.21 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23 °C).
[00143] Example 173 : (Z)-l-((lS)-2-Carboxypyrrolidin-l-yl)-2-(l - ((ethoxycarbonyl)oxy)ethoxy)diazene oxide
Figure imgf000047_0002
[00144] Example 100 (150 mg, 0.54 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170, step 2: (146 mg, 93%> yield). 1H NMR (400 MHz, d-DMSO) δ 6.29-6.23 (m, IH), 4.39-4.33 (m, IH), 4.12 (dq, J=1.8, 7.1 Hz, 2H), 3.67-3.49 (m, 2H), 2.28-2.20 (m, IH), 1.95-1.86 (m, 4H), 1.46 (dd, J=2.9, 5.5 Hz, 3H), 1.17 (ddt, J=19.3, 14.2, 2.3 Hz, 3H). LC tr=2.92 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 314 (M+Na calcd for C10H17 3O7 requires 314).
[00145] Example 174: (Z)-2-(l-((tert-Butoxycarbonyl)oxy)ethoxy)-l-((5)-2- carboxypyrrolidin- 1 -yl)diazene oxide
Figure imgf000048_0001
[00146] Example 101 (150 mg, 0.49 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (74 mg, 47% yield). Ή
NMR (400 MHz, CDC13) δ 6.41 (q, J=5.6 Hz, 1H), 4.65-4.58 (m, 1H), 3.87-3.76 (m, 1H), 3.72-3.61 (m, 1H), 2.33-2.77 (m, 2H), 2.13-2.03 (m, 2H), 1.63 (d, J=5.6 Hz, 3H), 1.52 (s, 9H). LC tr=3.28 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 342 (M+Na calcd for
C12H21N3O7 requires 342).
[00147] Example 175: (Z)-l-Carboxy-2,10-dimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec- 3-ene 3 -oxide
Figure imgf000048_0002
[00148] Example 102 (250 mg, 1.0 mmol) was converted to the title compound by a procedure similar to that described in the synthesis of Example 170: (238 mg, 90%> yield). 1H NMR (400 MHz, CDC13) δ 5.80 (s, 2H), 4.98-4.92 (m, 1H), 4.31 (s, 2H), 3.32 (s, 3H), 1.34 (d, J=5.6 Hz, 6H). LC tr=2.84 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 mL/min with detection 254 nm, at 23° C). ES(pos)MS m/z 288 (M+Na calcd for C8H15N3O7 requires 288).
[00149] Examplel75 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 175 as a solid. Carboxylic acids listed in Table 5 are converted to the alkaline or ammonium salts by similar methods. [00150] Table 5. Carbonate-Capped Carboxylic Acid Nonoates.
Figure imgf000049_0001
194 (Z)-2-carboxy-2,3,7-trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
195 (Z)-2-carboxy -2,3,7, 1 1 ,1 l -pentamethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
196 (Z)-l -carboxy-3,7-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
197 (Z)-13-carboxy-6, 10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatridec-8-ene 9-oxide
198 (Z)-13-carboxy -2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
199 (Z)-\ 3 -carboxy -2,2,6, 10-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
200 (Z)-14-carboxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
201 (Z)-14-carboxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
202 (Z)-14-carboxy-2,2,6, 10-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
203 (Z)-14-carboxy-2, 10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
204 (Z)-14-carboxy-2,2,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
205 (Z)-15-carboxy-6, 10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
206 (Z)-\ 5-carboxy -2,2,6, 10-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazapentadec-8-ene 9-oxide
207 ( )-l -(3-carboxyazetidin-l -yl)-2-(l -((ethoxycarbonyl)oxy)ethoxy)diazene oxide
208 ( )-l -(3-carboxyazetidin-l -yl)-2-(l -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
209 ( )-2-(l -((tert-butoxycarbonyl)oxy)ethoxy)-l -(3-carboxyazetidin-l -yl)diazene oxide
210 ( )-l -(3-carboxyazetidin-l -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
21 1 ( )-2-(((tert-butoxycarbonyl)oxy)methoxy)- 1 -(3 -carboxyazetidin- 1 -yl)diazene oxide
212 (Z)-l -((S)-2-carboxypyrrolidin-l -yl)-2-(l -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
213 (S,Z)-l -(2-carboxypyrrolidin-l -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
214 (S,Z)-2-(((tert-butoxycarbonyl)oxy)methoxy)-l -(2-carboxypyrrolidin-l -yl)diazene oxide
215 (l lS,12R,Z)-l l -carboxy-6,10, 12-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
216 (l lS,12R,Z)-l l -carboxy-2,10, 12-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
217 (1 \S,Z)-\ l -carboxy-6, 10,13-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
218 (S,Z)-l l -carboxy-2, 10,13-trimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
219 (2S,Z)-2-carboxy-3,7-dimethyl-9-oxo-l -phenyl-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
220 (S,Z)-2-carboxy-3, l l -dimethyl-9-oxo-l -phenyl-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide 221 ( )-l -(3-carboxypiperidin-l -yl)-2-(l -((ethoxycarbonyl)oxy)ethoxy)diazene oxide
222 (Z)-l -(3-carboxypiperidin-l -yl)-2-(l -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
223 ( )-l -(4-carboxypiperidin-l -yl)-2-(l -((ethoxycarbonyl)oxy)ethoxy)diazene oxide
224 ( )-l -(4-carboxypiperidin-l -yl)-2-(l -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
225 ( )-2-(l -((tert-butoxycarbonyl)oxy)ethoxy)-l -(4-carboxypiperidin-l -yl)diazene oxide
226 ( )-2-(((tert-butoxycarbonyl)oxy)methoxy)-l -(4-carboxypiperidin-l -yl)diazene oxide
227 ( )-l -(3-carboxypyrrolidin-l -yl)-2-(l -((ethoxycarbonyl)oxy)ethoxy)diazene oxide
228 (Z)-2-(l -((tert-butoxycarbonyl)oxy)ethoxy)-l -(3-carboxypiperidin-l -yl)diazene oxide
229 (Z)-\ -(3-carboxypyrrolidin-l -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
230 (Z)-\ -((S)-2-carboxyazetidin-l -yl)-2-(l -((isopropoxycarbonyl)oxy)ethoxy)diazene oxide
231 (Z)-\ -((S)-2-carboxyazetidin-l -yl)-2-(l -((methoxycarbonyl)oxy)ethoxy)diazene oxide
232 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-(((ethoxycarbonyl)oxy)methoxy)diazene oxide
233 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
234 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-(((methoxycarbonyl)oxy)methoxy)diazene oxide
235 (S, )-2-(((tert-butoxycarbonyl)oxy)methoxy) - 1 -(2-carboxyazetidin- 1 -yl)diazene oxide
236 (Z)-2-carboxy-2,3,7, l l -tetramethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
237 (Z)-10-carboxy-5,9,10-trimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
238 (Z)-2-carboxy-2,3-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
239 (Z)-2-carboxy-2,3,l l -trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
240 (Z)-10-carboxy-9, 10-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
241 (Z)-2-carboxy-2,3,l 1 , 1 l -tetramethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
242 (Z)-l -carboxy-3,7,l l -trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
243 (Z)-\ 1 -carboxy-5,9-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
244 (Z)-l -carboxy-3-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
245 (Z)-l -carboxy-3, l l -dimethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
246 (Z)-\ 1 -carboxy-9-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
247 (Z)-l -carboxy-3, l 1 ,1 l -trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide 248 (Z)-12-carboxy-5,9-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazadodec-7-ene 8-oxide
249 (Z)-13-carboxy-10-methyl-4-oxo-3,5,7-trioxa-8,9,10-triazatridec-8-ene 9-oxide
250 (Z)-13-carboxy-2, 10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatridec-8-ene 9-oxide
251 (Z)-12-carboxy-9-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazadodec-7-ene 8-oxide
252 (Z)-13-carboxy -2,2,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
253 (Z)-14-carboxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
254 ( )-14-carboxy-10-methyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
255 ( )-13-carboxy-9-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazatridec-7-ene 8-oxide
256 (Z)-15-carboxy -2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazapentadec-8-ene 9-oxide
257 (Z)-14-carboxy-2,6,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
258 (Z)-15-carboxy-10-methyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
259 (Z)-15-carboxy-2, 10-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazapentadec-8-ene 9-oxide
260 ( )-14-carboxy-9-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazatetradec-7-ene 8-oxide
261 (Z)-15-carboxy -2,2,10-trimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazapentadec-8-ene 9-oxide
262 ( )-l -(3-carboxyazetidin-l -yl)-2-(l -((methoxycarbonyl)oxy)ethoxy)diazene oxide
263 ( )-l -(3-carboxyazetidin-l -yl)-2-(((ethoxycarbonyl)oxy)methoxy)diazene oxide
264 ( )-l -(3-carboxyazetidin-l -yl)-2-(((methoxycarbonyl)oxy)methoxy)diazene oxide
265 ( )-l -((S)-2-carboxypyrrolidin-l -yl)-2-(l -((methoxycarbonyl)oxy)ethoxy)diazene oxide
266 (S, )-l -(2-carboxypyrrolidin-l -yl)-2-(((ethoxycarbonyl)oxy)methoxy)diazene oxide
267 (S, )-l -(2-carboxypyrrolidin-l -yl)-2-(((methoxycarbonyl)oxy)methoxy)diazene oxide
268 (Z)-3-(carboxymethyl)-7,l l -dimethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
269 (Z)-9-(carboxymethyl)-5-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
270 (Z)-3-(carboxymethyl)-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
271 (Z)-3-(carboxymethyl)-l l -methyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
272 (Z)-9-(carboxymethyl)-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
273 (Z)-3-(carboxymethyl)-l 1 ,1 l -dimethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
274 (Z)-3-(carboxymethyl)-2,7, l l -trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide 275 (Z)-9-(carboxymethyl)-5,10-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
276 ( )-3-(carboxymethyl)-2-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
277 (Z)-3-(carboxymethyl)-2,l l -dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
278 ( )-9-(carboxymethyl)-10-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
279 (Z)-3-(carboxymethyl)-2,l 1 ,1 l-trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
280 (Z)-l -carboxy -2,6,10-trimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec-3-ene 3-oxide
281 (Z)-l -carboxy-2,6-dimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazadec-3-ene 3-oxide
282 (Z)-l -carboxy-2-methyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec-3-ene 3-oxide
283 (Z)-l -carboxy-2-methyl-8-oxo-5,7,9-trioxa-2,3,4-triazadec-3-ene 3-oxide
284 (Z)-l -carboxy-2,10,10-trimethyl-8-oxo-5,7,9-trioxa-2,3,4-triazaundec-3-ene 3-oxide
285 (10S,Z)-10-carboxy-5,9-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
286 (S,Z)-2-carboxy-3-methyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
287 (S,Z)-10-carboxy-9-methyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
(1 \S,\2R,Z)-\ l-carboxy-2,6,10,12-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-
288
oxide
289 (10S,1 lR,Z)-10-carboxy-5,9,l l-trimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazatridec-7-ene 8-oxide
290 (1 \S,\2R,Z)-\ l-carboxy-10,12-dimethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-oxide
291 (10S,1 lR,Z)-10-carboxy-9,l l-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazatridec-7-ene 8-oxide
(1 \S,\2R,Z)-\ l-carboxy-2,2,10,12-tetramethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatetradec-8-ene 9-
292
oxide
293 (1 \S,Z)-\ 1 -carboxy -2,6,10, 13-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
294 (10S,Z)-10-carboxy-5,9,12-trimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazatridec-7-ene 8-oxide
295 (S,Z)-\ 1 -carboxy-10, 13-dimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
296 (S,Z)-10-carboxy-9,12-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazatridec-7-ene 8-oxide
297 (S,Z)- 11 -carboxy-2,2, 10, 13-tetramethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatetradec-8-ene 9-oxide
298 (2S,Z)-2-carboxy-3,7,l l -trimethyl-9-oxo-l -phenyl-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
299 (10S,Z)-10-carboxy-5,9-dimethyl-3-oxo-l l-phenyl-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
(2S,Z)-2-carboxy-3,7,l 1,1 l-tetramethyl-9-oxo-l-phenyl-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-
300
oxide 301 (S,Z)-2-carboxy-3-methyl-9-oxo-l -phenyl-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
302 (S,Z)-10-carboxy-9-methyl-3-oxo-l l -phenyl-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
303 (S,Z)-2-carboxy-3, 1 1 ,1 1 -trimethyl-9-oxo-l -phenyl-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
304 (10S,Z)-10-carboxy-5,9,l l -trimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazadodec-7-ene 8-oxide
305 (1 \S,Z)-\ l -carboxy-2,2,6, 10,12-pentamethyl-4-oxo-3,5,7-trioxa-8,9,10-triazatridec-8-ene 9-oxide
306 (S,Z)- 1 1 -carboxy-10, 12-dimethyl-4-oxo-3,5,7-trioxa-8,9, 10-triazatridec-8-ene 9-oxide
307 (S,Z)-10-carboxy-9, l l -dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazadodec-7-ene 8-oxide
308 (Z)-3-(carboxymethyl)-2,2,7, 1 1 -tetramethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
309 (Z)-9-(carboxymethyl)-5,10,10-trimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
(Z)-3-(carboxymethyl)-2,2,7, l 1 ,1 l -pentamethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-
310
oxide
31 1 (Z)-3-(carboxymethyl)-2,2-dimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
312 (Z)-3-(carboxymethyl)-2,2, l l -trimethyl-9-oxo-6,8,10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
313 (Z)-9-(carboxymethyl)-10,10-dimethyl-3-oxo-2,4,6-trioxa-7,8,9-triazaundec-7-ene 8-oxide
314 (Z)-3-(carboxymethyl)-2,2, 1 1 , 1 1 -tetramethyl-9-oxo-6,8, 10-trioxa-3,4,5-triazadodec-4-ene 4-oxide
315 ( )-l -(3-carboxypiperidin-l -yl)-2-(l -((methoxycarbonyl)oxy)ethoxy)diazene oxide
316 ( )-l -(3-carboxypiperidin-l -yl)-2-(((ethoxycarbonyl)oxy)methoxy)diazene oxide
317 (Z)-l -(3-carboxypiperidin-l -yl)-2-(((isopropoxycarbonyl)oxy)methoxy)diazene oxide
318 ( )-l -(3-carboxypiperidin-l -yl)-2-(((methoxycarbonyl)oxy)methoxy)diazene oxide
319 ( )-2-(((tert-butoxycarbonyl)oxy)methoxy)-l -(3-carboxypiperidin-l -yl)diazene oxide
320 ( )-l -(4-carboxypiperidin-l -yl)-2-(l -((methoxycarbonyl)oxy)ethoxy)diazene oxide
321 ( )-l -(4-carboxypiperidin-l -yl)-2-(((ethoxycarbonyl)oxy)methoxy)diazene oxide
ESTER-CAPPED ALCOHOL NONOATES
[00151 ] Example 322: (Z)-l -(3-Hydroxyazetidin-l -yl)-2-((pivaloyloxy)methoxy)diazene oxide
Figure imgf000054_0001
[00152] NS-13 (300 mg, 2.27 mmol) and chloromethyl pivalate (244 mg, 1.62 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 94: (56 mg, 10% yield). 1H NMR (400 MHz, CDC13) δ 5.78 (d, J=1.6Hz, 2H), 5.15-5.09 (m, 1H), 4.90-4.69 (m, 1H). 4.60-4.42 (m, 1H), 4.39-4.34 (m, 1H), 4.09-4.03 (m, 1H), 1.23 (s, 9H). LC tr=2.86 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C). ES(pos)MS m/z 270 (M+Na calcd for C9Hi7N305 requires 270).
[00153] Example 520: (Z)-l-(Azetidin-l-yl)-2-((pivaloyloxy)methoxy)diazene oxide
Figure imgf000055_0001
[00154] NS-26 (2.00 g, 14.38 mmol) and chloromethyl pivalate (1.55 g, 10.27 mmol) were converted to the title compound by a procedure similar to that described in the synthesis of Example 94: (132 mg, 5.6% yield). 1H NMR (400 MHz, CDC13) δ 5.78 (s, 2H), 4.14-4.10 (m, 4H), 2.25-2.17 (m, 2H), 1.24 (s, 9H). LC tr=3.72 minutes (C-18 column, 5 to 95% acetonitrile/water over 6 minutes at 1.7 ml/min with detection 254 nm, at 23° C).
ES(pos)MS m/z 254 (M+Na calcd for C9Hi7N304 requires 254).
[00155] Table 6. Ester-Capped Alcohol NONOates.
Figure imgf000055_0002
332 ( )-l -((S)-2-(hydroxymethyl)azetidin-l -yl)-2-((propionyloxy)methoxy)diazene oxide
333 (S, )-l -(2-(hydroxymethyl)azetidin-l -yl)-2-((pivaloyloxy)methoxy)diazene oxide
334 ( )-l -((S)-2-(hydroxymethyl)azetidin-l -yl)-2-((isobutyryloxy)methoxy)diazene oxide
335 ( )-2-((benzoyloxy)methoxy)-l -((S)-2-(hydroxymethyl)azetidin-l -yl)diazene oxide
336 (Z) -2 -(acetoxymethoxy) - 1 -((S) -2 -(hydroxymethyl) azetidin- 1 -yl) diazene oxide
337 (Z)-l -((S)-2-(hydroxymethyl)azetidin-l -yl)-2-(l -(propionyloxy)ethoxy)diazene oxide
338 (Z)-l -((S)-2-(hydroxymethyl)azetidin-l -yl)-2-(l -(pivaloyloxy)ethoxy)diazene oxide
339 (Z)-l -((S)-2-(hydroxymethyl)azetidin-l -yl)-2-(l -(isobutyryloxy)ethoxy)diazene oxide
340 (Z) -2 -( 1 -(benzoyloxy) ethoxy) - 1 -((S) -2 -(hydroxymethyl)azetidin- 1 -yl) diazene oxide
341 (Z)-2-( 1 -acetoxyethoxy)- 1 -((S)-2-(hydroxymethyl)azetidin-l -yl)diazene oxide
342 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-((propionyloxy)methoxy)diazene oxide
343 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-((pivaloyloxy)methoxy)diazene oxide
344 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-((isobutyryloxy)methoxy)diazene oxide
345 (Z)-2-((benzoyloxy)methoxy)- 1 -(3 -(hydroxymethyl) azetidin- 1 -yl)diazene oxide
346 (Z)-2-(acetoxymethoxy)- 1 -(3-(hydroxymethyl)azetidin- 1 -yl)diazene oxide
347 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-(l -(propionyloxy)ethoxy)diazene oxide
348 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-(l -(pivaloyloxy)ethoxy)diazene oxide
349 (Z)-l -(3-(hydroxymethyl)azetidin-l -yl)-2-(l -(isobutyryloxy)ethoxy)diazene oxide
350 (Z)-2-(l -(benzoyloxy)ethoxy)-l -(3-(hydroxymethyl)azetidin-l -yl)diazene oxide
351 (Z)-2-( 1 -acetoxyethoxy)- 1 -(3-(hydroxymethyl)azetidin- 1 -yl)diazene oxide
520 (Z)-l -(azetidin-1 -yl)-2-((pivaloyloxy)methoxy)diazene oxide
521 (Z)-l -(azetidin-1 -yl)-2-((propionyloxy)methoxy)diazene oxide
522 (Z)-l -(azetidin-1 -yl)-2-((isobutyryloxy)methoxy)diazene oxide
523 (Z)-2-((benzoyloxy)methoxy)- 1 -(azetidin- 1 -yl)diazene oxide
524 (Z)-2-(acetoxymethoxy)- 1 -(azetidin- 1 -yl)diazene oxide
525 (Z)-l -(azetidin-1 -yl)-2-(l -(propionyloxy)ethoxy)diazene oxide
526 (Z) - 1 -(azetidin- 1 -yl) -2 -( 1 -(pivaloyloxy) ethoxy) diazene oxide 527 ( )-l -(azetidin-l -yl)-2-(l -(isobutyryloxy)ethoxy)diazene oxide
528 (Z) -2 -( 1 -(benzoyloxy) ethoxy) - 1 -(azetidin- 1 -yl) diazene oxide
529 (Z)-2-( 1 -acetoxyethoxy)- 1 -(azetidin- 1 -yl)diazene oxide
ESTER-CAPPED CARBOXYLIC ACID NONOATES
Example 352: (Z)-2-(l -Acetoxyethoxy)- l-((5)-2-carboxyazetidin-l-yl)diazene oxide
Figure imgf000057_0001
[00156] Sodium metaperiodate (2.0 mmol) is added to a mixture of Example 341 (0.50 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The resulting mixture was stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous reaction mixture was then extracted into ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure to afford the carboxylic acid.
[00157] Example 352 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 352 as a solid. Carboxylic acids listed in Table 7 are converted to the alkaline or ammonium salts by similar methods.
[00158] Table 7. Ester-Capped Carboxylic Acid NONOates.
Figure imgf000057_0002
357 (S, )-2-(acetoxymethoxy)-l-(2-carboxyazetidin-l -yl)diazene oxide
358 (S, )-2-((benzoyloxy)methoxy)-l -(2-carboxyazetidin- l-yl)diazene oxide
359 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-((isobutyryloxy)methoxy)diazene oxide
360 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-((pivaloyloxy)methoxy)diazene oxide
361 (S,Z)- 1 -(2-carboxyazetidin- 1 -yl)-2-((propionyloxy)methoxy)diazene oxide
362 (Z)-2-(l-acetoxyethoxy)-l-(3-carboxyazetidin-l -yl)diazene oxide
363 (Z)-2-(l-(benzoyloxy)ethoxy)-l -(3-carboxyazetidin-l-yl)diazene oxide
364 ( )-l -(3-carboxyazetidin-l-yl)-2-(l -(isobutyryloxy)ethoxy)diazene oxide
365 ( )-l -(3-carboxyazetidin-l-yl)-2-(l -(pivaloyloxy)ethoxy)diazene oxide
366 ( )-l -(3-carboxyazetidin-l-yl)-2-(l -(propionyloxy)ethoxy)diazene oxide
367 (Z)-2-(acetoxymethoxy)- 1 -(3-carboxyazetidin- 1 -yl)diazene oxide
368 (Z)-2-((benzoyloxy)methoxy)- 1 -(3 -carboxyazetidin- 1 -yl)diazene oxide
369 ( )-l -(3-carboxyazetidin-l-yl)-2-((isobutyryloxy)methoxy)diazene oxide
370 ( )-l -(3-carboxyazetidin-l-yl)-2-((pivaloyloxy)methoxy)diazene oxide
371 ( )-l -(3-carboxyazetidin-l-yl)-2-((propionyloxy)methoxy)diazene oxide
SUCCINIMIDE-CAPPED ALCOHOL NONOATES
[00159] Example 377: (Z)-l -((2,5-dioxopyrrolidin-l -yl)methoxy)-3-(2-hydroxyethyl)-3- methyltriaz-l-ene 2-oxide
Figure imgf000058_0001
[00160] NS-06 (1.0 mmol) is suspended in N,N-dimethylformamide (1.0 mL) and the solution is treated with a solution of N-(chloromethyl)succinimide (1.0 mmol, prepared according to J Org Chem 1993, 55(18), 4913-18) in N,N-dimethylformamide (1.0 mL). Sodium iodide (1.0 mmol) is added and the mixture is heated to 60 °C for 1 hour. The mixture is poured into water (25 mL), extracted with ethyl acetate (25 mL), the organic layer is washed with a 0.2 N solution of sodium thiosulfate and brine, dried over sodium sulfate, filtered, and concentrated. The residue is purified by silica gel chromatography using ethyl acetate in hexane to afford Example 377.
[00161 ] Table 8. Succinimide-Capped Alcohol NONOates.
Figure imgf000059_0001
oxide
388 ( *)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-(2-hydroxyethyl)-3-isopropyltriaz-l -ene 2-oxide
389 (2^-3-(tert-butyl)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-(2-hydroxyethyl)triaz-l - ene 2-oxide
390 ( *)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-(l -hydroxy-2-methylpropan-2-yl)-3- methyltriaz-l -ene 2-oxide
391 (2*)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -( 1 -hydroxy-2-methylpropan-2-yl)-3 - methyltriaz- 1 -ene 2-oxide
392 (2*)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -(3 -hydroxypropyl)-3 -methyltriaz- 1 -ene 2-oxide
393 (2*)- 1 -( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3 -(4-hydroxybutyl)-3 -methyltriaz- 1 -ene 2-oxide
394 (2*)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -(4-hydroxybutyl)-3 -methyltriaz- 1 -ene 2- oxide
395 (Z)- 1 -( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3 -(5 -hydroxypentyl)-3 -methyltriaz- 1 -ene 2-oxide
396 (Z)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -(5-hydroxypentyl)-3-methyltriaz- 1 -ene 2-oxide
397 (2)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-(6-hydroxyhexyl)-3-methyltriaz-l -ene 2-oxide
398 (2)-l -((2,5-dioxopyrrolidin-l -yl)methoxy)-3-(6-hydroxyhexyl)-3-methyltriaz-l -ene 2-oxide
399 (2")-2-( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)- 1 -(3 -(hydroxymethyl)azetidin- 1 - yl)diazene oxide
400 (2)-2-((2,5-dioxopyrrolidin-l -yl)methoxy)-l -(3-(hydroxymethyl)azetidin-l - yl)diazene oxide
401 (2)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3,3-bis(2-hydroxyethyl)triaz-l -ene 2- oxide
402 (Z)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3,3-bis(2-hydroxyethyl)triaz- 1 -ene 2- oxide
403 (2)-2-(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-l -((S)-2-(hydroxymethyl)pyrrolidin-l - yl)diazene oxide
404 (2)-l -(l -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-((2S,3R)-l -hydroxy-3-methylpentan-2- yl)-3 -methyltriaz- 1 -ene 2-oxide
405
(2)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -((2S,3R)- 1 -hydroxy-3 -methylpentan-2- yl)-3-methyltriaz-l-ene 2-oxide
406 (Z) -(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-3-((S)-l-hydroxy-4-methylpentan-2-yl)-3- methyltriaz-l-ene 2-oxide
407 (S,Z)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)-3 -( 1 -hydroxy-4-methylpentan-2-yl)-3 - methyltriaz-l-ene 2-oxide
408 (Z)-l-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-3-((S)-l-hydroxy-3-phenylpropan-2-yl)-3- methyltriaz-l-ene 2-oxide
409 (S,Z)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)-3 -( 1 -hydroxy-3 -phenylpropan-2-yl)-3 - methyltriaz-l-ene 2-oxide
410 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(3-hydroxypiperidin-l-yl)diazene oxide
411 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(3-hydroxypiperidin-l-yl)diazene oxide
412 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(4-hydroxypiperidin-l-yl)diazene oxide
413 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(4-hydroxypiperidin-l-yl)diazene oxide
414 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(3-(hydroxymethyl)piperidin-l- yl)diazene oxide
415 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(3-(hydroxymethyl)piperidin-l- yl)diazene oxide
416 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(4-(hydroxymethyl)piperidin-l- yl)diazene oxide
417 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(4-(hydroxymethyl)piperidin-l- yl)diazene oxide
418 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(3-hydroxypyrrolidin-l-yl)diazene oxide
420 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(3-hydroxypyrrolidin-l-yl)diazene oxide
421 (Z)-2-( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)- 1 -(3 -(hydroxymethyl)pyrrolidin- 1 - yl)diazene oxide
422 (Z)-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(3-(hydroxymethyl)pyrrolidin-l- yl)diazene oxide
560 (Z)-2-(l-(2,5-dioxopyrrolidin-l-yl)ethoxy)-l-(azetidin-l-yl)diazene oxide
561 ( )-2-((2,5-dioxopyrrolidin-l-yl)methoxy)-l-(azetidin-l-yl)diazene oxide
SUCCINIMIDE-CAPPED CARBOXYLIC ACID NONOATES [00162] Example 426: (Z)-3-(carboxymethyl)-l-((2,5-dioxopyrrolidin-l -yl)methoxy)-3- methyltriaz-l-ene 2-oxide
Figure imgf000062_0001
[00163] Sodium metaperiodate (1.0 mmol) is added to a mixture of Example 377 (0.25 mmol) and ruthenium(III)chloride (cat) in acetonitrile (1.0 mL), ethyl acetate (1.0 mL) and water (1.5 mL). The mixture is stirred at ambient temperature for 3.5 h and then diluted with water and filtered through a pad of celite®. The aqueous mixture is extracted into ethyl acetate, the combined organic layers are washed with water and brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure to provide Example 426.
[00164] Example 426 (1.0 eq.) is treated with 0.5 M NaOH solution (1.0 eq.) in ethanol and the solution is evaporated. The resulting solid is washed twice with diethyl ether and dried to provide the sodium salt of Example 426 as a solid. Carboxylic acids listed in Table 9 are converted to the alkaline or ammonium salts by similar methods.
[00165] Table 9. Succinimide-Capped Carboxylic Acid NONOates.
Figure imgf000062_0002
430 (Z)-3-(2-carboxypropan-2-yl)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3-methyltriaz- 1-ene 2-oxide
431 (Z)-3 -(2-carboxyethyl)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
432 (Z)-3-(3-carboxypropyl)- 1 -(1 -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3 -methyltriaz- 1 -ene 2-oxide
433 (Z)-3-(3-carboxypropyl)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
434 (Z)-3-(4-carboxybutyl)- 1 -( 1 -(2,5 -dioxopyrrolidm- 1 -yl)ethoxy)-3 -methyltriaz- 1 -ene 2-oxide
435 (Z)-3-(4-carboxybutyl)-l -((2,5-dioxopyrrolidin-l -yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
436 (Z)-3-(5-carboxypentyl)- 1 -( 1 -(2,5 -dioxopyrrolidm- 1 -yl)ethoxy)-3 -methyltriaz- 1 -ene 2-oxide
437 (Z)-3-(5-carboxypentyl)- 1 -((2,5-dioxopyrrolidin-l -yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
438 (Z)-l -(3 -carboxyazetidin- 1 -yl)-2-(l -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)diazene oxide
439 (Z)-l -(3 -carboxyazetidin- 1 -yl)-2-((2,5 -dioxopyrrolidm- 1 -yl)methoxy)diazene oxide
440 (Z)- 1 -((5)-2-carboxypyrrolidin- 1 -yl)-2-( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)diazene oxide
441 (Z)-3-(carboxymethyl)- 1 -(1 -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-ethyltriaz- 1 -ene 2- oxide
442 (Z)-3-(carboxymethyl)- 1 -((2,5-dioxopyrrolidin-l -yl)methoxy)-3-ethyltriaz- 1 -ene 2- oxide
443 (Z)-3-(carboxymethyl)- 1 -(1 -(2,5-dioxopyrrolidin-l -yl)ethoxy)-3-isopropyltriaz- 1 - ene 2-oxide
444 (Z)-3-(carboxymethyl)- 1 -((2,5-dioxopyrrolidin-l -yl)methoxy)-3-isopropyltriaz-l - ene 2-oxide
445 (Z)-3-((S)- 1 -carboxyethyl)- 1 -( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3 -methyltriaz- 1 - ene 2-oxide
446 (S,Z)-3 -( 1 -carboxyethyl)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3 -methyltriaz- 1 -ene 2-oxide
447 (Z)-3-((15',2R)-l -carboxy-2-methylbutyl)-l -(1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3- methyltriaz-l-ene 2-oxide 448 (Z)-3 -(( 1 S,2R)- 1 -carboxy-2-methylbutyl)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)-3 - methyltriaz-l-ene 2-oxide
449 (Z)-3-((S)- 1 -carboxy-3 -methylbutyl)- 1 -( 1 -(2,5 -dioxopyrrolidin- 1 -yl)ethoxy)-3 - methyltriaz-l-ene 2-oxide
450 (S,Z)-3 -( 1 -carboxy-3 -methylbutyl)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)-3 - methyltriaz-l-ene 2-oxide
451 (Z)-3-((S)- 1 -carboxy-2-phenyl ethyl)- 1 -( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
452 (S,Z)-3 -( 1 -carboxy-2-phenylethyl)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)-3 - methyltriaz-l-ene 2-oxide
453 (Z)-3 -((S)- 1 -carboxy-2-methylpropyl)-l -(1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)-3- methyltriaz-l-ene 2-oxide
454 {S,Z)-3-{\ -carboxy-2-methylpropyl)- 1 -((2,5-dioxopyrrolidin- 1 -yl)methoxy)-3- methyltriaz-l-ene 2-oxide
455 (Z)-3-(tert-butyl)-3-(carboxymethyl)- 1 -(1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)triaz-l - ene 2-oxide
456 (Z)-3 -(tert-butyl)-3 -(carboxymethyl)- 1 -((2,5 -dioxopyrrolidin- 1 -yl)methoxy)triaz- 1 - ene 2-oxide
457 (Z)- 1 -(3 -carboxypiperidin- 1 -yl)-2-( 1 -(2,5 -dioxopyrrolidin- 1 -yl)ethoxy)diazene oxide
458 (Z)-l -(3-carboxypiperidin- 1 -yl)-2-((2,5-dioxopyrrolidin- 1 -yl)methoxy)diazene oxide
459 (X)- 1 -(4-carboxypiperidin- 1 -yl)-2-( 1 -(2,5 -dioxopyrrolidin- 1 -yl)ethoxy)diazene oxide
460 (X)- 1 -(4-carboxypiperidin- 1 -yl)-2-((2,5-dioxopyrrolidin- 1 -yl)methoxy)diazene oxide
461 (Z)-l -(3 -carboxypyrrolidin- 1 -yl)-2-( 1 -(2,5-dioxopyrrolidin- 1 -yl)ethoxy)diazene oxide
462 (X)- 1 -(3 -carboxypyrrolidin- 1 -yl)-2-((2,5-dioxopyrrolidin- 1 -yl)methoxy)diazene oxide
[00166] In Vitro Measurement of Nitric Oxide Release in Serum: Test compounds were dissolved into DMSO to make 3 mM stocks and stored at -20 deg C. Solutions of 5% rat serum (Sigma Cat. # S7648) or 15% human serum (Bioreclaimation, Cat. # HMSRM) were made in PBS (% v/v). In a 96-well plate, 3
Figure imgf000064_0001
of DMSO in 127 PBS were placed in blank wells and 3 μΙ_, of DMSO in 127 μΙ_, of diluted serum were placed in wells for reference standards. Test wells were charged with 127 μΙ_, of diluted serum followed by 3 μΙ_, of 3 mM compound DMSO stocks tested in quadruplicate. Plates were incubated at 37 °C for 90 minutes, removed from the incubator, and placed on ice. Reference wells were diluted with 150 μΙ_, of sodium nitrite stock solutions in PBS. Remaining wells were diluted with 150 μΙ_, of PBS and then every well received 20 μΙ_, of Griess Reagent (Promega Cat. # G2930). Wells were mixed, the plates were incubated for 30 minutes at room temperature, and then absorbance was measured at 562 nm using a microplate reader. Reference standards final concentrations were 100, 33.3, 11.1, 3.7, 1.23, and 0.41 μΜ nitrite and final test concentrations for all compounds was 30 μΜ. The average DMSO blank readings were subtracted from test readings and a standard curve was generated from the reference standard wells. Nitrite levels were determined, and percent release of nitric oxide was calculated, relative to theoretical maximum (60 μΜ), for each compound.
[00167] Table 10. Nitric oxide release in vitro.
Ex. % NO (Rat Serum 90 rain) % NO (Humars Serum 90 mirt)
1 16% 22%
2 4% 7%
21 % 29%
4 20% 27%
5 18% 26%
5% 9%
8 18% 24%
9 15% 19%
1 0 31% 40%
51 8% 13%
54 15% 28%
91 26% 16%
92 41 % 26%
93 37% 21 %
94 76% 1 9%
95 39% 26%
96 26% 25% 97 44% 24%
98 63% 14%
99 33% 20%
100 63% 10%
101 49% 23%
102 64% 12%
104 74% 13%
105 70% 2%
170 8% 2%
171 29% 18%
172 32% 19%
173 5% <!%»
174 22% 10%
175 <1% <1 %
78%. 8%
520
[0170] Measurement of Nitric Oxide Release In Vivo: Pharmacokinetics (PK) of nitric oxide release is measured by administering a single oral (PO) gavage dose to Sprague Dawley rats.
[0171] For each test compound, two (2) Sprague Dawley (CD® IGS) male rats are used. Animals are fasted before the study and fed only after the 8-h blood draw. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are administered oral (PO) in 2% DMSO/0.5% methylcellulose/0.1% Tween 20 vehicle at 30 mg/kg using 10 mL/kg volume per animal. Compounds are formulated by making a 150 mg/mL DMSO compound stock and adding to warm 0.5 % methylcellulose/0.1% Tween-20 at 35-40°C to make a clear solution or fine suspension. Animals found in severe distress or a moribund condition are euthanized. Peripheral blood collections are done primarily through venipuncture of the tail or saphenous veins or by jugular catheter at various times, (e.g. pre-dose, 15min, 30min, lh, 2h, 4h, 8h, and 24h). Whole blood samples are collected in K2EDTA microtainer (Fisher # 02-669-38), processed to plasma by centrifugation, and the plasma is frozen at -80°C.
[00168] Thawed plasma samples (30μί) are diluted with PBS (70μί) along with control rat plasma. Samples are spun at 2000x g for 10 minutes and then 80μί of 30% PBS-diluted plasma samples are transferred into the appropriate well of a 96-well plate. Sodium nitrate is used in standard curve wells at 100, 33.3, 1 1.1 , 3.7, 1.23, and 0.41 μΜ. To each well is added ΙΟμΙ^ of the nitrate reductase solution and ΙΟμΙ^ of the enzyme co-factors solution to convert nitrate to nitrite (Sigma #06239 Nitrite/Nitrate Assay Kit). The plate is incubated at room temperature for 2 h and then 50μΙ^ of Griess Reagent A is added to each well, and mixed. After 5 min., 50μΙ^ of Griess Reagent B is added to each well, and mixed. The plate is incubated for 10 minutes at room temperature, and the absorbance is measured at 540nm with a microplate reader. A standard curve is generated from the reference standard wells and nitrate/nitrite (NOx) levels are determined (μΜ) and standard deviations (+/-S.D.) for each blood draw and plot against time (h) of blood draw.
[00169] Table 1 1. Nitric oxide release in vivo after oral administration in 2 rats at 30 mpk.
Ex. Ex. 94 Ex. 520
Pre-Dose NO (μΜ) 9.5 (+/-0.6) 10.4 (+/-3.8)
2 1ι ΝΟχ (μΜ) 155.4 (+/-5.3) 1 12.4 (+/-17.5)
4 1ι ΝΟχ (μΜ) 128.2 (+/-5.5) 95.6 (+/-6.1)
8 b ΝΟχ (μ'Μ) 64.1 (+/-2.7) 52.5 (+/-5.3)
[00170] Shelf-life and Compound Stability: Several compounds were checked for NO- release efficiency after 3 months at 40 °C compared to samples stored at -20 °C.
Phthalimide-capped compounds 9 and 1 showed very similar activity before and after heating, indicating good stability. Carbonate-capped compounds 92, 95 and 96 showed useful stability with some variation among species. Finally, compound 500, a compound not of the invention,
Figure imgf000067_0001
500 did not show any NO release after 3 months at 40 °C. This is consistent with the HPLC results of the stability test in which the amount of test compound remaining after 2 months at 40 °C was below the level of detection.
[00171] Compound Effects on Blood Pressure: Increased systemic levels of nitric oxide are expected to effect blood pressure in an subject or animal, compounds of the present invention are evaluated for blood pressure effects in spontaneously hypertensive rats (SHR).
[00172] Thirty-two male, spontaneously hypertensive rats (SHR), 12-weeks old (four groups of eight) are used in this study. Initially, mean arterial blood pressure (MAP) is measured through tail-cuff daily, throughout the study. Animals undergo 2 days of blood pressure training and 1 day of baseline blood pressure measurements. Animals are weighed and dosed individually by body weight on the day of treatment. Compounds are
administered orally (PO) or by intraperitoneal (IP) injection once on Day 1 at 10 mg/kg using 10 mL/kg volume per animal. Blood pressures are monitored for 6 days post-dose. A total of 7 time points are measured: Day 0 for baseline and Days 1, 2, 3, 4, 5, and 6 of the study. Animals found in severe distress or in a moribund condition are euthanized.
Celecoxib is used as the positive control for these studies.
[00173] Monolayer Wound Healing Assay: Cell proliferation in confluent A549 monolayers is blocked by a 30 minute pre-incubation in the presence of mitomycin C (3 μg/mL). Test compounds, in cell culture buffer, are added to confluent monolayers 30 minutes before wound induction. A549 monolayers are subsequently scratched with a pipette tip. Wound areas are evaluated with phase contrast microscopy on an inverted microscope. Images of the same areas are obtained at intervals from zero to 96 hours. Cell migration rate through wound healing is evaluated from the images using Paint.Net v.3.10 software. Cell migration is expressed as the fold change in the migration area, relative to untreated control cells at the same time period.
[00174] Excisional In Vivo Wound Healing Model: Experiments using endothelial NO synthase (eNOS) gene knockout (KO) mice have been conducted to determine the absolute requirement of nitric oxide for wound healing in vivo and the contribution of eNOS to this effect (Am. J. Physiol. 277 (Heart Circ. Physiol. 46): H1600-H1608, 1999). This model is applied to drug testing by administering compounds topically directly to the wound, or systemically via oral dosing.
[00175] Briefly, wild-type (WT) and eNOS KO mice (2-3 mo of age, weighing 20-26 g each, and evenly distributed across both genders) are anesthetized, their dorsum is clipped free of hair, and the skin is prepped with povidone-iodine. A full-thickness wound (1.5 x 1.5 cm), including the panniculus carnosus, is created sharply with fine scissors. After the wound edges are retracted, the wound outline is traced onto an acetate sheet. To keep the area clean, the wounds are dressed with a clear, bioocclusive dressing (Op-Site; Owens Minor, Greensburg, PA). Compounds are administered every-other day topically by completely covering the wound with compound treated cream, or daily (q.d.) by systemic administration via oral gavage (2% DMSO/0.5% methylcellulose/0.1% Tween 20 vehicle at 30 mg/kg using 10 mL/kg volume per animal). Compounds are formulated for topical administration by dissolving/suspending the compound (50 mg) into a mixture of the liquid components (373 mg of 2-propanol, 93 mg of transcutol, 93 mg of isopropyl myristate and 3 mg of labrasol), followed by mixing with melted PEG 4000 (388 mg), which upon cooling semi-solidifies into a cream. Compounds are formulated for oral administration by making a 150 mg/mL DMSO compound stock and adding to warm 0.5 %
methylcellulose/0.1% Tween-20 at 35-40°C to make a clear solution or fine suspension. Wound closure is monitored by tracing the wound area every other day onto acetate sheets. The occlusive dressing is changed at the time of tracing and topical compound
administration. The tracings are digitized, and the areas are calculated in a blinded fashion using a computerized algorithm (Sigma Scan; Jandel Scientific, San Raphael, CA). The rate of excisional wound closure in compound-treated mice is compared to vehicle treated eNOS KO mice and WT mice to determine the effect of compounds on wound healing.
[00176] According to a further aspect, the present invention relates to pharmaceutical compositions comprising a compound of formula I together with one or more
pharmaceutically carriers and, optionally, one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. For the treatment of cutaneous wounds, the pharmaceutical compositions will usually be formulated for topical application. [00177] For topical application, there are employed as non-sprayable forms, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon. The formulation may be impregnated into a fiber dressing or bandage and provided to the patient in that form. Formulations for topical application are well known to those skilled in the art, and general methods for their preparation are found in any standard pharmacy school textbook, for example Remington: The Science and Practice of Pharmacy. Chapter 90 of the 19th edition of Remington entitled " Medicated
Applications" describes the more common types of topical dosage forms (pages 1577- 1597.)
[00178] The topical pharmaceutical carrier may include any substance capable of dispersing and maintaining contact between the active ingredients and the skin. The vehicle may be glycerin, alcohol or water based. Water based compositions may, if desired, be thickened with a suitable gel to provide a less mobile composition. Such compositions are well known in the art. Examples of such vehicles include aloe vera, which is a gel base, together with ethanol, isopropyl alcohol, water, propylene glycol and a non-ionic surfactant such as laureth-4. Other water-based alcohol/ glycerin vehicles and carriers are within the scope of the present invention. A typical water-based lotion will contain from 45 to 50 parts of glycerin, one to three parts Tween 80TM, from 45 to 50 parts of water and from 1 to 50 parts of the compound of the invention.
[00179] Also included in the scope of the invention are ointments, emulsions or dispersions in which water, if present, is a minor constituent. Typical ointment formulation comprises from 90 to 98 parts of a mixture of petrolatum, mineral oil, mineral wax and wool wax alcohol, from 0.5 to 3 parts of a mixture of polyoxyethylene and sorbitan monooleate (Tween 80TM), from 1 to 5 parts of water, and from 1 to 50 parts of the compound of the invention. Another suitable non-aqueous ointment can be prepared from 95 parts of liquid petrolatum USP, 5 parts polyethylene and from 1 to 50 parts of the compound of the invention. The resulting ointment spreads easily and has an even consistency over wide temperature extremes. It is, in addition, non-irritating and non-sensitizing.
[00180] Formulations of the compounds of the invention may also be prepared containing from 0 to 25% by weight of urea. In general, in such urea containing ointments, the water content will vary from 5 to 50% by weight of the composition. Any suitable ointment carrier may be used such as lanolin, ethylene glycol polymers and the like. In the case of formulations containing urea, it is known in the art that borate salts may often be added to stabilize the pharmaceutical composition (see U.S. patent 2,917,433,).
[00181] Optional additional therapeutic ingredients that may be added to the compositions include compounds known in the art to be effective topical antibiotics, such as nystatin, clindamycin, erythromycin, metronidazole, silver sulfadiazine, chlorhexidine gluconate and sodium fusidate.

Claims

1. A compound of formula:
Figure imgf000072_0001
wherein
R1 is H or (Ci-C6)alkyl;
R is chosen from
Figure imgf000072_0002
(b) -OC(=0)OR 7 ; and, when R 3 and R 4 , taken together with the nitrogen to which they are attached, form an optionally substituted 4-membered ring, R may additionally be:
(c) -OC(=0)R9;
R3 and R4 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
8 9 substituted with from one to three groups chosen from -OH, -COOH, -OR°, -COOR" and -OC(=0)R9; or taken together with the nitrogen to which they are attached, R3 and R4 form an optionally substituted 4- to 7-membered ring;
R5 and R6 are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl
8 9 substituted with from one to three groups chosen from -OH, -COOH, -OR°, -COOR" and -OC(=0)R9; or R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a 5- to 7-membered cyclic imide, said cyclic imide optionally fused to a second six-membered ring, which six-membered ring may be substituted with from one to
Q
three groups chosen from (Ci-Ce)alkyl, halogen, cyano, amino, hydroxy, -COOH, -OR , -COOR9 and -OC(=0)R9 ; 7 8 9
R', R° and R" are independently chosen from (Ci-Cio)hydrocarbyl and (Ci-Cio)hydrocarbyl substituted with from one to three groups chosen from -OH, -COOH, -OR10, -COOR10 and -OC(=0)R10; and
R10 is (Ci-C6)alkyl.
2
2. A compound according to claim 1 wherein R is
Figure imgf000073_0001
3. A compound according to claim 2 wherein R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a 5- to 7-membered cyclic imide, said cyclic imide optionally fused to a second six-membered ring, which six-membered ring may be substituted with from one to three groups chosen from (Ci-Ce)alkyl, halogen, cyano, amino, hydroxy, -COOH, -OR8, -COOR9 and -OC(=0)R9.
4. A compound according to claim 3 wherein R5 and R6 taken together with the -C(=0)NC(=0)- to which they are attached, form a phthalimide.
5. A compound according to claim 1 wherein R 2 is -OC(=0)OR 7
6. A compound according to claim 5 wherein R7 is (Ci-Cio)hydrocarbyl.
7. A compound according to any one of claims 1 to 6, wherein R is saturated (Ci- Cio)hydrocarbyl and R4 is (Ci-Cio)hydrocarbyl substituted with-OH or -COOH.
8. A compound according to any one of claims 1 to 6, wherein R3 and R4 taken together with the nitrogen to which they are attached, form an optionally substituted 4- to 7-membered ring.
9. A compound according to claim 8 wherein R3 and R4 taken together with the nitrogen to which they are attached, form a hydroxy-substituted 4- to 7-membered ring.
10. A compound according to claim 8 wherein R3 and R4 taken together with the nitrogen to which they are attached, form an unsubstituted, saturated heterocycle.
11. A compound according to claim 1 wherein R3 and R4, taken together with the nitrogen to which they are attached, form an optionally substituted azetidine and R is -OC(=0)R9.
12. A compound according to claim 11 wherein R9 is (Ci-Cio)hydrocarbyl.
13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to any of claims 1-6, 11 or 12.
14. A pharmaceutical composition according to claim 13 wherein said
pharmaceutically acceptable carrier is a carrier for topical administration.
15. A method for treating cutaneous injuries comprising bringing a cutaneous injury into contact with a compound according to any of claims 1-6, 11 or 12.
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US20060046967A1 (en) * 2004-08-26 2006-03-02 Apparao Satyam Prodrugs containing novel bio-cleavable linkers
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US20060046967A1 (en) * 2004-08-26 2006-03-02 Apparao Satyam Prodrugs containing novel bio-cleavable linkers
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